PMID- 1703539 TI - Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption. AB - The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity. PMID- 1703540 TI - Analysis of the primary sequence and microtubule-binding region of the Drosophila 205K MAP. AB - We have sequenced cDNA clones encoding the Drosophila 205K microtubule-associated protein (MAP), a protein that may be the species specific homologue of mammalian MAP4. The peptide sequence deduced from the longest open-reading frame reveals a hydrophilic protein, which has basic and acidic regions that are similar in organization to mammalian MAP2. Using truncated forms of the 205K MAP, a 232 amino acid region could be defined that is necessary for microtubule binding. The amino acid sequence of this region shares no similarity with the binding motif of MAP2 or tau. We also analyzed several embryonic cDNA clones, which show the existence of differentially spliced mRNAs. Finally, we identified several potential protein kinase target sequences. One of these is distal to the microtubule-binding site and fits the phosphorylation consensus sequence of proteins phosphorylated by the mitosis specific protein kinase cdc2. Our data suggest that the 205K MAP uses a microtubule-binding motif unlike that found in other MAPs, and also raise the possibility that the activities of the 205K MAP may be regulated by alternative splicing and phosphorylation. PMID- 1703541 TI - Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles. AB - The major histological components of the hair follicle are the hair cortex and cuticle. The hair cuticle cells encase and protect the cortex and undergo a different developmental program to that of the cortex. We report the molecular characterization of a set of evolutionarily conserved hair genes which are transcribed in the hair cuticle late in follicle development. Two genes were isolated and characterized, one expressed in the human follicle and one in the sheep follicle. Each gene encodes a small protein of 16 kD, containing greater than 50 cysteine residues, ranging from 31 to 36 mol% cysteine. Their high cysteine content and in vitro expression data identify them as ultra-high-sulfur (UHS) keratin proteins. The predicted proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Genomic blots reveal that the UHS keratin proteins are encoded by related multigene families in both the human and sheep genomes. Tissue in situ hybridization demonstrates that the expression of both genes is localized to the hair fiber cuticle and occurs at a late stage in fiber morphogenesis. PMID- 1703542 TI - The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation. AB - The myelin-associated glycoproteins (MAG) are members of the immunoglobulin gene superfamily that function in the cell interactions of myelinating glial cells with axons. In this paper, we have characterized the structural features of these proteins. The disposition of MAG in the bilayer as a type 1 integral membrane protein (with an extracellularly disposed amino terminus, single transmembrane segment, and cytoplasmic carboxy terminus) was demonstrated in protease protection studies of MAG cotranslationally inserted into microsomes in vitro and in immunofluorescent studies with site specific antibodies. A genetically engineered MAG cDNA, which lacks the putative membrane spanning segment, was constructed and shown to encode a secreted protein. These results confirm the identify of this hydrophobic sequence as the transmembrane segment. Sequencing of the secreted protein demonstrated the presence of a cleaved signal sequence and the site of signal peptidase cleavage. To characterize the disulfide linkage pattern of the ectodomain, we cleaved MAG with cyanogen bromide and used a panel of antibodies to coprecipitate specific fragments under nonreducing conditions. These studies provide support for a novel disulfide linkage between two of the immunoglobulin domains of the extracellular segment. Finally, we report that MAG is posttranslationally palmitylated via an intramembranous thioester linkage. Based on these studies, we propose a model for the conformation of MAG, including its RGD sequence, which is considered with regard to its function as a cell adhesion molecule. PMID- 1703543 TI - The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins. AB - The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells. CD44 has also been implicated in lymphocyte binding to high endothelial venules during lymphocyte homing. Interestingly, the monoclonal antibody Hermes-3, which blocks lymphocyte binding to the high endothelial venules of mucosal lymphoid tissue, had no effect on the binding of labeled hyaluronate. Furthermore, the binding of lymphocytes to high endothelial cells of lymph nodes and mucosal lymphoid tissue was not significantly affected by treatment with agents that block the binding of hyaluronate (hyaluronidase, excess hyaluronate and specific antibodies). Thus, CD44 appears to have at least two distinct functional domains, one for binding hyaluronate and another involved in interactions with mucosal high endothelial venules. PMID- 1703544 TI - Early expression of the gene for interphotoreceptor retinol-binding protein during photoreceptor differentiation suggests a critical role for the interphotoreceptor matrix in retinal development. AB - Interphotoreceptor retinol-binding protein (IRBP), the major protein component of the subretinal space, is in a strategic position to mediate cellular interactions between the retinal pigmented epithelium (RPE) and the neural retina. While IRBP appears to be involved in vitamin A transport during the visual cycle in the adult, the role of this protein during eye development has not been determined. As a first step to understanding the role of IRBP during retinal development, we have studied the expression of the mRNA for this glycolipoprotein during photoreceptor differentiation in the rat. A rat neural retina cDNA library was prepared from which an IRBP clone was isolated. The clone contains an open reading frame followed by a 3' noncoding sequence ending in 10 adenosine residues. The coding region has an identity of 83.9 and 82.5% with the nucleotide sequence of human and bovine IRBP, respectively. Rats (Sprague-Dawley, Wistar, and Royal College of Surgeon pink-eyed controls) have a 6.4 and a 5.2-kb mRNA for IRBP which are present in a 1:4 ratio and thus are the only vertebrate known to definitely have more than one major form of the IRBP message. Genomic Southern blots are consistent with the hypothesis that there is only one allele of the IRBP gene, suggesting that the two forms are produced by alternative processing of the mRNA. To generate an antisense RNA probe for use in molecular titration assays and Northern blots, an Eco RI-Bam HI fragment from the coding region was subcloned in between flanking Sp6 and T7 promoters. Total RNA was prepared from undissected rat globes from postnatal days p0-p22. The expression of the mRNA for IRBP was studied by Northern blots and the level of the transcripts determined by solution hybridization assays. Approximately 10(5) IRBP mRNA transcripts/micrograms total eye RNA are present at birth. This increases to a final level of 3.1 X 10(6) transcripts/micrograms total RNA by p9. The one-half maximal level of the mRNA occurs at p4.2 which is 2 wk before the one-half maximal level of IRBP is reached in the subretinal space (Gonzalez-Fernandez, F., R. A. Landers, P. A. Glazebrook, S.-L. Fong, G. I. Liou, D. M. K. Lam, and C. D. B. Bridges. 1984. J. Cell Biol. 99:2092-2098). The expression of the mRNA for IRBP reflects the developmental emergence of the interphotoreceptor matrix as an important structure within the retina.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1703546 TI - Role of growth factors in the contraction and maintenance of collagen lattices made with arterial smooth muscle cells. AB - The contraction of collagen lattices made with arterial smooth muscle cells was studied in medium MCDB 107 without serum or supplemented with 1% fetal bovine serum, plus insulin, transferrin, and low-density lipoprotein. Under these conditions, smooth muscle cell mitogens including HBGF-1 (aFGF), PDGF, and EGF stimulated contraction. Stimulation by HBGF-1 was more profound than with other factors tested. HBGF-1 stimulation of lattice contraction was blocked by protein synthesis inhibitors, but not inhibitors of DNA synthesis. Histological observations indicated that HBGF-1 also enhanced the maintenance of healthy cells in the lattice. Taken together, these observations suggest that HBGF-1 stimulates lattice contraction, not by a mitogenic effect, but by stimulating synthesis of specific cellular proteins. Since the greatest effects of HBGF-1 on lattice contraction were seen during the first 72 h following casting, the effects on maintenance of cell viability are probably less important in promoting lattice contraction. PMID- 1703545 TI - The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin. AB - The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation-dependent manner. Binding was inhibited by soluble vitronectin, by RGD-containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins. PMID- 1703547 TI - Differential effects of interferon gamma and alpha on in vitro model of angiogenesis. AB - The formation of blood vessels in vivo (angiogenesis) is an important process and is usually initiated in response to injury, tumor growth, or normal tissue development. We have studied the effect of human interferon (IFN) alpha (alpha) and gamma (gamma) on the capillary-like network formation in an in vitro model of angiogenesis using human umbilical vein endothelial cells (HUVEC). When HUVEC cells are plated on Matrigel (reconstituted basement membrane matrix enriched in laminin), a network of capillary like structures (endotubes) rapidly forms. IFN alpha enhanced the tube formation in a dose-dependent manner, whereas IFN-gamma significantly inhibited the tube formation. In addition, both the enhancement and inhibition of angiogenesis by IFN-alpha and gamma was found to be greater if the cells were pretreated with IFN for 12 hr before plating on the Matrigel. These results suggest that IFN may play an important role in several vascular processes including early stages of wound healing, recanalization of thrombi, tumor growth, metastasis, normal growth, and development. PMID- 1703548 TI - Chondrocytes inhibit endothelial sprout formation in vitro: evidence for involvement of a transforming growth factor-beta. AB - Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti-TGF-beta antibodies to the cocultures significantly reduced the inhibitory effect of chondrocytes on sprout formation. Chondrocyte-conditioned medium or exogenously added TGF-beta 1 have a similar albeit transient inhibitory effect. Depletion of TGF-beta from chondrocyte conditioned medium with anti-TGF-beta antibodies and solid-phase protein-A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte-derived TGF-beta-like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF-beta. PMID- 1703549 TI - Differential effects of myosin-antibody complexes on contractile rings and circumferential belts in epitheloid cells. AB - The role of myosin filaments during assembly and activity of microfilament rings was analyzed by microinjecting epitheloid cells (PtK2 and LLC-PK1 kidney cell lines) with specific anti-myosins. Six monoclonal antibodies directed against the light meromyosin (LMM) region of the myosin molecule were characterized with respect to epitope location, and their effects on actin-activated MgATPase as well as on assembly, structural integrity and stability of myosin filaments. All of these antibodies recognized LLC-PK1 myosin, but only three reacted with PtK2 myosin. The remaining three served as matching controls in experiments with this cell line. When injected in amounts sufficient to yield an excess of antibody over myosin, the reactive antibodies significantly delayed formation and constriction of the contractile ring in mitotic cells. These rings contained less myosin, but not less actin, than the controls. This indicates that the recruitment and alignment of actin in the cleavage furrow can occur independently of other components of the contractile ring. After completion of cytokinesis, the majority of the injected cells was unable to assemble a normal circumferential belt. This resulted in defective epitheloid sheets. Approximately one third of these cells showed grossly distorted cell shapes and an increase in locomotory activity. All these changes were fully reversible with time, suggesting that the effects of the antibodies were overcome by protein synthesis. The differential sensitivity seen between contractile rings and peripheral belts is discussed with respect to differences in their architecture, stability and proposed function. PMID- 1703550 TI - Tailless keratins assemble into regular intermediate filaments in vitro. AB - To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs. PMID- 1703551 TI - Removal of endotoxins using macroporous spherical poly(gamma-methyl L-glutamate) beads and their derivatives. PMID- 1703552 TI - Determination of a novel potent immunosuppressant (FK-506) in rat serum and lymph by high-performance liquid chromatography with chemiluminescence detection. PMID- 1703553 TI - Action of staphylococcal exfoliative toxins on epidermal cell cultures and organotypic skin. AB - In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB. PMID- 1703554 TI - Expression of interleukin 1 in burn scars. AB - A histochemical study using a Biotin-Streptavidin procedure for demonstrating interleukin 1 (IL-1) in burn scar specimens is described. Among seventeen scar specimens, five positive reactions to IL-1 were observed in the epidermis. No positive reactions in the dermal tissue, however, were detected. The twenty specimens of normal skin used as controls showed no positive reaction in either the dermis or the dermis. PMID- 1703555 TI - [Clinical studies on tumor markers for monitoring prostate cancer patients; the evaluation of prostate-specific antigen and comparison with prostatic acid phosphatase and gamma-seminoprotein]. AB - To evaluate the usefulness of tumor markers in monitoring the patients with prostate cancer, serial measurements of serum prostate-specific antigen (PA), prostatic acid phosphatase (PAP) and gamma-seminoprotein (gamma-Sm) were performed in 78 stage C or D patients. Positive rates of each marker prior to the treatment were as follows; PA; 75%, PAP; 56% and gamma-Sm; 62% in stage C, and PA; 95%, PAP; 79% and gamma-Sm; 91% in stage D. In most cases showing PR (partial response) and S (stable) in clinical responses, these three markers decreased their serum titers corresponding to clinical course if the markers were elevated at the start of the treatment. But the usefulness of PAP was lessened because of its lower positive rate than those of PA and gamma-Sm. In 33 PD (progressive disease) cases, positive rates of each marker at time of clinical diagnosis as PD were found to be 85% in PA, 55% in PAP and 76% in gamma-Sm. And with the combination assays of these three tumor markers, positive rate was elevated to 88%. Moreover, elevation of serum values of these three markers at 3 months before the progression event were observed in 50% of PA, 39% of PAP and 46% of gamma-Sm. Then the prognostic significance of each marker was examined. In PA and PAP, there were statistical differences in non-relapsing rates between patients whose reduction rates from the pretreatment value on 7th day were more than and less than 50%. But in gamma-Sm, a statistical difference between each group was firstly observed on 14th day. As a result, in monitoring patients with prostate cancer, PA and gamma-Sm are more useful than PAP and, in prediction of patients' prognosis, PA is more useful than gamma-Sm. PMID- 1703556 TI - The influence of intra-arterial infusion of cisplatinum and bleomycin on the cell cycle in uterine cervical carcinoma. AB - We examine the influence of CDDP and BLM on the cell cycle in the patients with uterine cervical cancer. Specimens were obtained from 8 cases of local recurrent uterine cervical cancer which were treated with CDDP and BLM bolus infusions into bilateral internal iliac arteries. Except for two patients who showed no response according to the criteria of the National Cancer Institute (NCI), the other patients exhibited partial response (PR). The concentrations of BLM and CDDP were 0.83 +/- 0.05 microgram/g and 2.9 +/- 0.54 microgram/g in the tumor tissues 24 hours after treatment. The peak values of the drug concentrations in serum, which were observed at 30 min for both drugs, were 0.92 +/- 0.12 microgram/ml BLM and 1.02 +/- 0.65 microgram/ml CDDP and the concentrations of the drugs decreased rapidly in the serum. The higher levels of CDDP and BLM were retained in the tumor tissue 24 hours after treatment, than in serum. Changes in DNA content determined by flow cytometry, revealed that proliferation indices (PI) increased after treatment only in PR cases. The mean increase in the ratio was 0.10 +/- 0.015. The BrdU staining showed that cells in S phase increased in tumor tissues. Labeling indices increased from 0.10 +/- 0.04 to 0.18 +/- 0.05. These results suggested that cell synchronization was induced by CDDP and BLM in vivo. PI seemed a useful parameter for measuring drug sensitivity. PMID- 1703557 TI - A patch-clamp study of acetylcholine-activated ion channels in Ascaris suum muscle. AB - Acetylcholine-activated single-channel currents were recorded from cell-attached and inside-out patches of isolated muscle vesicles from Ascaris suum. Acetylcholine (1-10 mumols l-1) activated cation-selective channels of two amplitudes: 40-50 pS and 25-35 pS. Both channels had linear I/V relationships and mean open durations independent of voltage. The larger conductance was analysed in detail to determine its open-, closed- and burst-time kinetics; the open and burst durations were composed of two components (short and long), while closed durations had at least three components (short, intermediate and long). The data were then corrected to allow for missing short events in order to estimate various parameters including corrected mean open time (1.26 + 0.11 ms, mean +/- S.E.). Values were also derived for the efficacy (beta/alpha = 4.9) and affinity [1/KD = 147 x 10(3) (mol l-1) -1] of acetylcholine at this receptor. Larger concentrations of acetylcholine (25-100 mumols l-1) were shown to produce desensitization and caused single-channel currents to occur in clusters with long closed times (mean 171 s) between clusters. It was concluded that the extrasynaptic muscle of Ascaris suum contains two types of acetylcholine activated ion channel and these are possible sites of action of various anthelmintic drugs. This paper is the first to describe acetylcholine-activated single-channel currents in invertebrate muscle. PMID- 1703558 TI - Analysis of the newly identified neutralization epitopes on VP7 of human rotavirus serotype 1. AB - Neutralizing monoclonal antibodies (MAbs) directed to the VP7 protein and neutralization-resistant mutants were used to analyse the antigenic structure of VP7 of human rotavirus serotype 1. Cross-neutralization tests using the MAbs and the resistant mutants indicated the existence of two functionally independent neutralization epitope regions (S1 and S2) on VP7. Region S1 corresponds to a single epitope domain of VP7 which has been detected previously. Two MAbs prepared in this study recognized the S1 region, and the resistant mutants they selected had amino acid substitutions at positions 94 or 213. On the other hand, region S2 is considered to be a novel epitope. Single or double amino acid substitutions were detected in the variable regions (amino acid positions 145, 217 and 221) and in the constant regions (positions 104, 201 and 291) of the VP7 protein of mutants selected by MAbs directed to the S2 region. It was suggested that the variable region E (amino acids 208 to 221) includes two independent neutralization sites, and that amino acid substitutions in the constant region of VP7 also affect serotype-specific neutralization epitopes. Neutralization epitopes on VP7 are considered to be highly dependent on the conformation of VP7. PMID- 1703559 TI - In vivo protective effect of tumour necrosis factor alpha against experimental infection with herpes simplex virus type 1. AB - C57BL/6 mice, which differ genetically from other strains by their resistance to herpes simplex virus type 1 (HSV-1) infection, were inoculated intraperitoneally with different doses of tumour necrosis factor alpha (TNF-alpha). Mice pretreated with 100 ng, or even 10 ng, of TNF-alpha showed prolonged survival compared to control mice that were infected with 10(7) p.f.u. of HSV-1. Significant protection was observed in mice injected 4 or 8 h prior to or after HSV-1 inoculation, respectively. Protection was also observed when mice which differed at their H-2 locus were treated with TNF-alpha after infection with HSV-1. Interferon could not be detected in the sera of mice at different time points after infection with HSV-1 or injection of TNF-alpha and there was no enhanced interferon titre in mice treated with both TNF-alpha and HSV-I, suggesting some interferon-independent protection. However, mice treated with TNF-alpha showed a marked activation of natural killer (NK) cells compared to untreated control mice or mice that were treated with HSV-1 alone. To test whether enhanced NK cell activity is responsible for TNF-alpha-induced protection, mice were injected with the NK cell-specific antibody anti-asialo Gm-1. In this experimental protocol the survival rate was almost unaffected, indicating that the observed protection was not due to activation of NK cells and that TNF-alpha is involved in the regulation of antiviral mechanisms other than the activation of interferons. Although additional production of interferon induced by TNF-alpha cannot be excluded, an antiviral effect of TNF-alpha on the course of HSV-1 infection may be postulated from our data. PMID- 1703560 TI - Sequence of the gene encoding the major neutralization antigen (VP7) of serotype 10 rotavirus. AB - The sequence of the gene encoding the major neutralization antigen (VP7) of the type member (bovine virus strain B223) of the proposed serotype 10 of rotavirus has been determined. This was done using a rapid new strategy involving direct sequencing by primer extension of cDNA generated from the relevant virus gene (gene 8) using a combined reverse transcription/polymerase chain reaction. The sequence obtained is 1062 bp in length and contains a single long open reading frame capable of encoding a protein of 326 amino acids. Comparison of the sequence to that of the corresponding gene of the other major virus serotype found in cattle, serotype 6, showed that it was approximately 25% divergent at the nucleotide level and 18% divergent at the amino acid level. PMID- 1703561 TI - Comparison and differentiation of potyvirus isolates and identification of strain , virus-, subgroup-specific and potyvirus group-common epitopes using monoclonal antibodies. AB - A panel of monoclonal antibodies (MAbs) generated against an admixture of 12 potyvirus isolates was used to compare and differentiate diverse potyviruses. Both native and denatured virions of strains of bean yellow mosaic (BYMV), potato virus Y, tobacco etch, pea seed-borne mosaic, iris severe mosaic, iris mild mosaic and asparagus virus-1 potyviruses were used as immunogen and as antigen for screening of the hybridoma cell lines. Thirty cell lines secreting potyvirus specific antibodies reactive in indirect antigen-coated plate (ACP-) ELISA were selected for detailed analysis. All 30 MAbs reacted with at least one strain of BYMV; 11 MAbs reacted with between one and eight of the nine BYMV strains and an additional three MAbs reacted only with isolates within the BYMV subgroup (BYMV, pea mosaic virus and clover yellow vein virus). The remaining 16 MAbs reacted with a BYMV isolate and with at least one of the other 43 potyvirus isolates tested. MAb PTY 1 reacted with all 55 potyvirus isolates tested (representing at least 33 different and distinct aphid-transmissible potyviruses). The potyvirus cross-reactive MAbs generally gave higher reactivity values in ACP-ELISA with dissociated virus than with polyclonal antibody-trapped intact virions in triple antibody sandwich ELISA (i.e. were cryptotope-specific). The BYMV strain- and virus-specific MAbs reacted strongly with both types of antigens (i.e. were metatope-specific). At least 25 distinct epitopes (12 cryptotopes and 13 metatopes) could be identified from the MAb-antigen reactivity patterns. The distribution of these epitopes between virus isolates can be used to detect and differentiate potyviruses in infected plant extracts and to examine virus architectures. Some of these epitopes are shared by potyvirus isolates not previously shown to be serologically related. The broad spectrum-reacting MAb PTY 1 recognizes a cryptotope conserved on all of the aphid-transmissible potyviruses examined and should be a valuable tool for the detection and assay of these potyviruses. PMID- 1703562 TI - Monoclonal idiotypic and anti-idiotypic antibodies to human immunodeficiency virus type 1 envelope glycoprotein. AB - Murine monoclonal antibodies (MAbs) gp41-1 (IgG2a) and gp41-2 (IgG1), directed against the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), were produced and characterized. These MAbs recognized both gp160 and gp41 and reacted with divergent HIV-1 isolates. Surface binding assays using viable HIV infected cells indicated that these MAbs were directed against surface-exposed epitopes. Both MAbs caused a reduction in reverse transcriptase activity. Syngeneic monoclonal antiidiotypic antibodies (anti-ids) against gp41-1 were also generated. Six anti-ids (agp41-11 to agp41-16) were selected by ELISA using F(ab')2 fragments of gp41-1; no reaction was observed when fragments from an irrelevant IgG2a MAb were used. Anti-ids were recognized by both gp41-1 and gp41 2 biotinylated MAbs. Competitive ELISA studies suggested that anti-ids were directed against at least three distinct idiotopes on gp41-1. All anti-ids reacted with idiotopes associated with both heavy and light chains and not with separated chains. The binding of MAbs gp41-1 and gp41-2 to HIV-infected cells was inhibited by each anti-id, except for the binding of gp41-2 which was not affected by the presence of agp41-12. Immunization of rabbits with agp41-11 and agp41-13 resulted in an antibody response against recombinant gp160. These studies indicated that these two anti-ids contain a surrogate image of the antigen recognized by gp41-1. PMID- 1703563 TI - Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity. AB - The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539-- -Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1. PMID- 1703564 TI - Epitope changes on the haemagglutinin molecule of recently isolated H1N1 influenza viruses. AB - We have studied changes of epitopes on the haemagglutinin molecule (HA) of H1N1 influenza viruses isolated between 1977 and 1986. For this purpose monoclonal antibodies (MAbs) were raised against the HA of the influenza A/England/333/80 and A/Yamagata/120/86 strain viruses. In order to define the amino acid residues responsible for the change of epitopes, we prepared several HA cDNAs modified by site-directed mutagenesis and cloned them into a simian virus 40 expression vector (SVHA). The substitution of glycine with serine at position 125c (suffix indicates presence in H1 but not in H3 subtype HAs) on the HA of the influenza A/USSR/90/77 strain virus resulted in the loss of epitope 110 (epitopes were named after MAbs) and created new epitopes 139 and 15, which were observed on the HA of A/England/333/80 and a few isolates from 1983. These new epitopes disappeared from the HA in some of the isolates in 1983 and most of the isolates in 1984 and 1986. The disappearance of epitopes 139 and 15 seems to be associated with the loss of epitope W18, which was identified on the HA of A/USSR/90/77. We suggested previously that amino acid residue 189 was involved in epitope W18. We therefore expressed an HA protein with two amino acid substitutions at positions 189 and 125c and found that the conversion of glutamine to lysine at position 189 in SVHA-67 prevented the expression of epitopes 139 and 15. PMID- 1703565 TI - Comparison of the cervicovaginal gram stain and rapid latex agglutination slide test for identification of group B streptococci. PMID- 1703566 TI - Internal derangement of the temporomandibular joint: a histochemical study. AB - The purpose of this study was to correlate histologic findings in temporomandibular joint (TMJ) condyles and discs with their macroscopic appearance at surgery. The 24 patients with internal derangement of the joint included 20 women and 4 men (mean age, 37 years; range, 18 to 61 years). The tissue lesions varied in degree from mild soft-tissue fraying and bone remodeling to extensive resorption and new cartilage and bone formation with high phosphatase enzyme activities, and even to loss of articular soft tissue and breakdown of cortical bone. Reactions may arise in the hard tissues before they occur in the articular surface layers. PMID- 1703567 TI - Burring technique in modern podiatric palliative care. AB - A review of the literature reveals sparse references regarding the application of burs in the conservative treatment of soft tissues and nails. The most recent publications were in 1946. The authors present an update on the technology and practical application of burs in podiatric medicine, their metallurgy, types, and recommended bur selection for reduction, remodeling, and burnishing of hyperkeratotic tissue and thickened nails. PMID- 1703568 TI - Identification of feeding and nutrition problems in young children with neuromotor involvement: a self-assessment. AB - The purpose of this article is to provide information needed to identify feeding and nutrition problems in children from birth to twenty-four months. Oral-motor and self-feeding skill development should be viewed within the framework of overall development and changing nutrition needs. Neuromotor dysfunction affects feeding and nutrition through changes in muscle tone, reflexes and the response to sensory stimulation. Nutrient compromises, including delays in texture progression, decreased fluid intake, and problems associated with self-feeding and food selection must be considered when assessing the nutrition/feeding needs of children. Successful completion of the self-assessment can be used to check the reader's basic understanding of the subject. PMID- 1703569 TI - Modulation of calcium-activated non-specific cation currents by cyclic AMP dependent phosphorylation in neurones of Helix. AB - 1. Currents through calcium-activated non-specific cation (CAN) channels were studied in the fast burster neurone of Helix aspersa and Helix pomatia. CAN currents were activated by reproducible intracellular injections of small quantities of Ca2+ utilizing a fast, quantitative pressure injection technique. 2. External application of forskolin (10-25 microM), an activator of adenylate cyclase, caused the endogenous bursting activity of the cells to be replaced by beating activity. These same concentrations of forskolin reduced CAN currents reversibly to about 50%. 3. External application of IBMX (3-isobutyl-1 methylxanthine, 100 microM), an inhibitor of phosphodiesterase, the enzyme which breaks down cyclic AMP, reduced CAN currents reversibly to about 40%. 4. External application of the membrane-permeable cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl-cyclic AMP (100 microM) caused almost complete block of the CAN current. A marked reduction in the CAN current was also observed following quantitative injections of cyclic AMP (internal concentrations up to 50 microM) directly into the cells from a second pressure injection pipette. 5. Similar results were obtained with quantitative injections of the catalytic subunit (C subunit) of the cyclic AMP-dependent protein kinase (internal concentrations 10( 4) units of enzyme) directly into the cells from a second pressure injection pipette. 6. Injection of the non-hydrolysable GTP analogue, GTP-gamma-S (internal concentrations 100 microM), which stimulates G-proteins, produced a prolonged increase in CAN current amplitude by as much as 300%. 7. External application of serotonin (100-200 microM) caused a transition from bursting to beating activity of the neurones and mimicked cyclic AMP's effects on CAN currents. Two other neurotransmitters, dopamine and acetylcholine, were not significantly effective in reducing CAN currents. 8. Injection of a peptide inhibitor of cyclic AMP dependent protein kinase suppressed serotonin's action on bursting and on CAN current. 9. Our results indicate that CAN currents in Helix burster neurones are modulated by cyclic AMP-dependent membrane phosphorylation. They suggest that the physiological transmitter that induces this second messenger action is serotonin. The dual control of CAN channels by two second messengers, namely Ca2+ and cyclic AMP, has important functional implications. While Ca2+ activates these channels which generate the pacemaker current in these neurones, cyclic AMP-dependent phosphorylation down-regulates them, thereby resulting in modulation of neuronal bursting activity. PMID- 1703570 TI - Cholinergic stimulation activates a non-selective cation current in canine pyloric circular muscle cells. AB - 1. Cholinergic stimulation of circular muscle from the canine pyloric sphincter results in excitatory junction potentials and an increase in slow-wave frequency. Experiments were performed on isolated pyloric muscle cells to determine the effects of acetylcholine on membrane conductance and voltage-dependent ionic currents. 2. Acetylcholine depolarized circular muscle cells and increased membrane conductance. Under voltage clamp, these effects were associated with the development of an inward current. 3. The ACh-dependent current (IACh) reversed at about -20 mV and was about equally selective for potassium and sodium. Changes in the chloride gradient had no effect on the reversal potential of IACh. 4. The response to ACh was blocked by atropine suggesting that the response was mediated by muscarinic receptors. IACh could not be elicited in the presence of ions normally used to block potassium currents (e.g. bath-applied TEA+ and replacement of Ki+ with Csi+. 5. In some cells single-channel openings could be resolved in response to ACh. These channels had a slope conductance of 30 pS, and open probability increased with depolarization. 6. Acetylcholine had little or no effect on voltage-dependent Ca2+ currents, and increased voltage-dependent outward currents. The latter effect may have been due to increased release of Ca2+ from internal stores. 7. The non-selective cationic current elicited by ACh can explain the excitatory junction potentials in pyloric muscle cells that are generated by transmural nerve stimulation and may also explain the chronotropic effects of ACh on slow waves. PMID- 1703571 TI - Intracellular recording from neurones of the guinea-pig gall-bladder. AB - 1. Intracellular recordings were made from neurones of the guinea-pig gall bladder in vitro. Intracellular injection of horseradish peroxidase revealed a simple structure, consisting of a soma and a single process, but no discernible dendritic arborization. 2. The resting membrane potential was -50.5 +/- 0.4 mV and the input resistance was 80 M omega. 3. Gall-bladder neurones spiked only once at the onset of depolarizing current pulses. Action potentials were blocked by tetrodotoxin, but a Ca2(+)-dependent spike could be elicited in the presence of tetrodotoxin and tetraethylammonium. 4. Action potential after hyperpolarizations had a duration of 172 +/- 3.7 ms and reversed at a membrane potential of -93 mV; this reversal potential was linearly related to the logarithm of the external potassium concentration. The initial phase of the after hyperpolarization was inhibited by tetraethylammonium (1-10 mM) and was not affected by 3,4-diaminopyridine. The late phase of the after-hyperpolarization was blocked by apamin (10 nM) or curare (500 microM). Both the early and late phases of the after-hyperpolarization were inhibited when the preparation was perfused with a calcium-free, high-magnesium solution. The calcium-free, high magnesium solution had no effect on the membrane potential or input resistance of these cells. 5. Fast excitatory synaptic responses and antidromic responses were elicited in gall-bladder neurones by focal stimulation of fibre tracts. High frequency fibre tract stimulation often resulted in prolonged, calcium-dependent, depolarizations that were associated with a decrease in input resistance. 6. 5 Hydroxytryptamine and substance P caused depolarizations that were associated with a decrease in input resistance. Bethanechol caused hyperpolarizations that were associated with a decrease in input resistance and which were blocked by atropine. PMID- 1703572 TI - The inhibition of single N-methyl-D-aspartate-activated channels by zinc ions on cultured rat neurones. AB - 1. Single channels activated by N-methyl-D-aspartate (NMDA) were studied in outside-out patches of cultured hippocampal neurones in the presence of glycine and absence of magnesium. The effects of the transition metal ions zinc and cadmium on NMDA channels were tested by placing the membrane patch at the mouth of one of an array of large barrelled flow pipes. 2. Amplitude histograms revealed several conductance levels between 5 and 45 pS with the majority of NMDA activated openings greater than 25 pS. Zinc (5-100 microM) and cadmium (30-100 microM) reduced the number of large conductance events in a voltage-independent manner. Zinc (30 microM) reduced the large conductance openings by approximately 70-80%. The small number of events under 20 pS precluded quantitative assessment of the effects of zinc and cadmium on these conductance levels. Zinc inhibition of the calculated macroscopic current due to NMDA-activated channels could be fitted with a single binding site isotherm with an IC50 of 12 microM. 3. Zinc and cadmium also reduced the mean open time of the two largest conductance events of 38 and 43 pS; this reduction was voltage independent. Open-time histograms were fitted with the sum of two exponentials. In the presence of 5 microM-NMDA at -60 mV, tau o2 = 10.49 ms and tau o1 = 1.47 ms; in 30 microM-zinc, tau o2 = 3.49 and tau o1 = 0.8 ms. The 'blocking' rate constant calculated at a membrane potential of +40 mV from the slope of 1/tau o2 vs. [zinc]o was 4 x 10(6) M-1 S-1. 4. Closed time analysis revealed brief (tau c = 0.4-1.0 ms) zinc-insensitive gaps; longer closed-time intervals were not analysed since all patches contained more than one channel. Both burst duration and the number of bursts were reduced in the presence of zinc. 5. At holding potentials negative to -40 mV in magnesium-free solutions, zinc also induced high-frequency flickering of the open channel which included complete channel closures at 4 kHz filtering. No zinc-induced flickering was seen at positive membrane potentials. The flickering was dose dependent, becoming prominent at zinc concentrations above 30 microM. Cadmium did not induce flickering at concentrations up to 100 microM.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1703573 TI - Single-channel measurement from the cyclic GMP-activated conductance of catfish retinal cones. AB - 1. A patch of plasma membrane was excised, in the inside-out configuration, from the outer segment tip of a catfish cone and recorded electrically with a patch pipette. A solution of 118 mM-NaCl was present on both sides of the membrane. 2. With the solution outside the pipette containing a low concentration (typically several micromoles per litre) of cyclic GMP and the membrane potential held at a non-zero level, brief steps of current indicative of the openings of single ion channels could be detected. There was no sign of desensitization to the ligand over a period of tens of seconds. 3. The prominent openings were associated with a conductance near 50 pS and an open-time constant of 0.5 ms or less. There was also an indication of sub-state openings. 4. The conductance of the large openings appeared to be invariant between -50 mV and +50 mV. However, the macroscopic current-voltage relation measured at a saturating concentration of cyclic GMP showed a slight upward curvature, which we attribute to a voltage dependence in the open probability of the fully liganded channel. 5. The relation between mean current and cyclic GMP concentration had an average Hill coefficient of about 2.4. The Hill coefficient was not affected by membrane voltage, but the conductance was activated by cyclic GMP slightly more readily at depolarizations; this could be adequately explained by a higher open probability of the fully liganded channel at positive voltages. 6. In several experiments, the membrane patch apparently contained a single cyclic GMP-activated channel, in that the measured current never rose above that for a single channel even at high concentrations of cyclic GMP. In these cases, a high concentration of the ligand simply engaged the channel in a literally continuous burst of openings, with an open probability of 0.8-0.9 at between -30 mV and +30 mV. The amplitude distribution of the burst under these conditions could be described by a beta distribution, consistent with the channel switching predominantly between a single closed state and a single open state when fully liganded. 7. Estimates of channel density on the cone membrane ranged from about 2 to 130 microns -2, with an average of 20 microns -2. This observed density is about ten times lower than the density of the homologous channel on rod membrane, being roughly in inverse relation to the tenfold larger surface membrane area of the cone outer segment.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1703574 TI - Calcium currents in hair cells isolated from the cochlea of the chick. AB - 1. Calcium currents were characterized in tall hair cells isolated from the chick's cochlea to determine what types of Ca2+ channels existed and if these varied in cells with differing voltage responses to current injection. 2. Whole cell, tight-seal recordings showed that the current-voltage relation of cochlear hair cells of the chick was dominated by K+ current. However, when outward K+ current was blocked it was found that all hair cells had a smaller, maintained inward current. 3. This inward current was a Ca2+ current since it required Ca2+ in the external medium, could also be carried by Ba2+, and was blocked reversibly by 5 mM-Co2+ and by Ni2+ and Cd2+ at micromolar concentrations. The Ca2+ channels were opened at membrane potentials positive to -50 mV, and the current was maximal near 0 mV. 4. The dihydropyridine BayK8644 (0.5 microM) produced a voltage-dependent increase of inward current. Ten micromolar nifedipine partially blocked the inward current. The outward Ca2(+)-activated K+ current was also reduced in the presence of 10 microM-nifedipine. These effects of dihydropyridines were completely reversible. 5. The Ca2+ current had rapid activation kinetics, reaching steady-state levels within 1 ms. If all outward currents were completely blocked the Ca2+ current showed no inactivation during depolarization lasting 200 ms. 6. No differences in voltage activation range, pharmacology, or kinetics of the Ca2+ current were found in tall hair cells from apical and basal regions of the cochlea. This is in contrast to the marked differences in K+ currents amongst cells from these two widely separated regions of the cochlea. PMID- 1703575 TI - Modulation of the [Ca2+] sensitivity of myosin phosphorylation in intact swine arterial smooth muscle. AB - 1. The [Ca2+] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulus-specific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media. 2. The relative [Ca2+] sensitivity of phosphorylation depended on the type of stimulus (ranked high to low sensitivity): contractile agonists (histamine, phenylephrine) = endothelin (sustained contraction) = combination of histamine and NaF greater than NaF alone = endothelin (initial contraction) = combination of histamine and depolarization = combination of NaF and depolarization greater than depolarization = Bay K 8644 = combination of depolarization and low-dose phorbol diester. 3. Activation of L-type Ca2+ channels with Bay K 8644 induced a [Ca2+] sensitivity of phosphorylation similar to depolarization, suggesting that any other effects of high KCl (such as cellular swelling) were not responsible for the low [Ca2+] sensitivity of phosphorylation. 4. The addition of either histamine or NaF (an activator of G proteins) to depolarized tissues produced similar increases in the [Ca2+] sensitivity of phosphorylation, suggesting that NaF (possibly by activation of a G protein) can mimic contractile agonist-induced increases in the [Ca2+] sensitivity of phosphorylation. 5. Phorbol dibutyrate enhanced the contractile effect of depolarization, and this enhancement was primarily caused by increases in [Ca2+] rather than an alteration in the [Ca2+] sensitivity of phosphorylation. 6. These data suggest that the [Ca2+] sensitivity of phosphorylation in smooth muscle may be regulated by agonists (possible by G protein activation); however, the role of protein kinase C activation or depolarization induced Ca2+ compartmentalization requires further study. PMID- 1703576 TI - Characterization of voltage-dependent calcium channels expressed in Xenopus oocytes injected with mRNA from rat heart. AB - 1. The properties of voltage dependent cardiac Ca channels expressed in Xenopus laevis oocytes after injection of mRNA from rat heart were investigated using the double-microelectrode voltage-clamp technique. 2. Endogenous Ba current (IBa,E) and expressed cardiac Ba current (IBa,C) were studied at various external concentrations of barium (Ba2+). These two entities could be distinguished by their amplitude and their pharmacology. IBa,C was more sensitive to the inorganic Ca channel blocker manganese (Mn2+). The contaminant IBa,E presented properties of voltage dependence identical to IBa,C, but was negligible in the presence of a low external Ba2+ concentration (2 mM). 3. In 2 mM-Ba2+, IBa,C activated at -35 mV, peaked at -14 mV, and reversed at +26 mV. Steady-state inactivation properties, in consideration of the half-inactivation potential of -35 mV, were also typical of L-type Ba currents. However, the decay of IBa,C was very slow (time constant of inactivation near 600 ms). No evidence for the expression of cardiac transient Ca channels (T-type) was found. 4. IBa,C was enhanced after exposure to the 1,4-dihydropyridine (DHP) agonist Bay K 8644. The enhancement of IBa,C was voltage dependent (maximum at -30 +/- 5 mV) and associated with a slowing in current decay. Current-voltage and concentration-response curves obtained for various Ba2+ concentrations revealed an antagonism between external Ba2+ and the 1,4-DHP agonist Bay K 8644. Similar results were found using the ( )Bay K 8644 pure agonist isomer. 5. We conclude that oocytes injected with mRNA from rat heart expressed only the high threshold, long-lasting or L-type Ca channels. The availability of expressed L-type Ca channels for quantitative pharmacological studies using low Ba2+ concentration has been demonstrated. PMID- 1703577 TI - Vulvar Paget's disease. Is immunocytochemistry helpful in assessing the surgical margins? AB - From January 1977 to December 1988, 19 patients with biopsy-proven Paget's disease of the vulva underwent simple or radical vulvectomy at the University of Miami/Jackson Memorial Medical Center. All vulvectomy specimens were evaluated immunocytochemically for the expression of carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and low-molecular-weight keratins 8 and 18 (LMK), both in areas containing neoplastic cells and in histologically negative surgical margins. Neoplastic Paget's cells stained positively for CEA in all cases; they were positive for EMA and LMK in 18 and 17 cases, respectively. In all eight cases with underlying in situ or invasive carcinomas, CEA, EMA and LMK were localized in the underlying tumors as well. None of the histologically proven negative margins reacted for CEA, EMA or LMK on immunocytochemistry. CEA appears to be a valuable immunocytochemical marker for extramammary Paget's disease; EMA and LMK are also expressed by the majority of such cases. None of these markers, however, is of added value in identifying Paget's cells in surgical margins if those margins appear negative on routine hematoxylin-and eosin staining. PMID- 1703578 TI - Identification of a balanced translocation carrier by spouse's low maternal serum alpha fetoprotein associated with an aneuploid fetus. PMID- 1703579 TI - Protein- and RNA-synthesis independent bactericidal activity of ciprofloxacin that involves the A subunit of DNA gyrase. AB - Ciprofloxacin, unlike nalidixic acid, can kill Escherichia coli cells in the absence of synthesis of protein or RNA. Hence, chloramphenicol or rifampicin do not abolish the bactericidal activity of ciprofloxacin against wild-type E. coli. Protein and RNA synthesis were not required for the bactericidal activity of ciprofloxacin against nalB, nalC and nalD mutants of E. coli. However, the addition of chloramphenicol or rifampicin abolished the bactericidal activity of ciprofloxacin against a nalA mutant in nutrient broth. It is concluded that the ability of ciprofloxacin to kill E. coli in the absence of protein or RNA synthesis involves the A subunit of DNA gyrase. PMID- 1703580 TI - The role of chest computed tomography in the diagnosis of drug-related reactions. AB - Computed tomography (CT) of the chest provides important information toward the diagnosis of drug-induced lung disease. CT's ability to demonstrate subtle parenchymal and pleural changes, small nodules, and adenopathy is valuable in the early detection of drug-related reactions. CT is also of value in monitoring the appearance, progression, and resolution of pulmonary damage in patients receiving potentially toxic drugs. The CT appearances of specific drug reactions are reviewed, including the spectrum of CT findings in bleomycin toxicity and amiodarone-induced lung disease. PMID- 1703581 TI - Evaluation of ventricular tachycardia with respect to syncope in patients with old myocardial infarction, dilated cardiomyopathy and no overt heart disease. AB - The incidence and the direct cause of syncope in ventricular tachycardia (VT) among patients with old myocardial infarction (OMI, n = 48), dilated cardiomyopathy (DCM, n = 18) and no evidence of heart disease (IVT, n = 43) were compared. The presence or absence of syncope in each patient was surveyed by a standardized questionnaire and a variety of electrocardiographic parameters for aggravating arrhythmias were measured. Syncope occurred in 19 of 43 OMI patients (40%), in 5 of 18 DCM patients (28%) and 6 of 43 IVT patients (14%) and significantly more often in OMI than IVT (p less than 0.01). Ventricular fibrillation (VF) was confirmed in 14 of the 19 OMI patients with syncope, in 3 of the 5 DCM patients with syncope and 1 of 6 IVT patients with syncope. The incidence of VF was significantly higher in OMI than in IVT (p less than 0.01). Mean VT cycle lengths (VTRR'm) in OMI patients with and without syncope were 0.35 +/- 0.07 sec and 0.42 +/- 0.10 sec, respectively (p less than 0.05). VTRR'ms in DCM patients with and without syncope were 0.43 +/- 0.10 sec and 0.42 +/- 0.10 sec, respectively (NS). VTRR'ms in IVT patients with and without syncope were 0.27 +/- 0.04 sec and 0.41 +/- 0.10 sec, respectively (p less than 0.01). The results show that the high frequency of VT rate was the main cause of syncope in IVT, while VF was the main cause of syncope in OMI.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703582 TI - [Changes of lysosomal enzymes in pancreatic juice following hepatectomy in rats]. AB - To explore the changes in the exocrine pancreas and the possible stimulation by gut hormones of secretion of lysosomal enzyme into the pancreatic juice, we measured the caerulein-stimulated amylase output, the cathepsin B output into the pancreatic juice and the pancreatic tissue amylase and cathepsin B contents, as well as the distribution of cathepsin B in acinar cells in both the regenerating (4 days) and recovery (8 days) stages after about 70% hepatectomy in rats. The amylase output increased significantly after hepatectomy. Cathepsin B was secreted into the pancreatic juice after stimulation by caerulein in both the control and hepatectomized groups. Four days after hepatectomy, the cathepsin B output was significantly greater than that of the control group, and redistribution of cathepsin B in acinar cells was noted. These changes in cathepsin B disappeared in the recovery stage (8 days) after hepatectomy. In addition, the pancreatic amylase content was significantly higher than in the control group, but the pancreatic cathepsin B content did not change significantly. These results suggest that caerulein stimulated the secretion of lysosomal enzyme into the pancreatic juice in the same way as digestive enzymes and that lysosomal enzymes in the pancreatic juice play an important physiological role. Furthermore, these results indicate the increased synthesis and secretion of amylase in and from acinar cells as well as the fragility of acinar cells. PMID- 1703583 TI - [Effect of intraduodenal oligopeptide on rat pancreatic exocrine secretion and release of secretin and CCK]. AB - We investigated whether intraduodenal (id) oligopeptide with three or four amino acids residues (pH 7.0) stimulates pancreatic exocrine secretion and release of endogenous plasma secretin and CCK in anesthetized rats. Id administration of oligopeptides in three doses (25, 100, 400 mg/hr) at a speed of 4 ml/hr resulted in dose-related increases in pancreatic secretion of pancreatic juice volume, bicarbonate, and amylase outputs (r = 0.598, 0.673, and 0.426, P less than 0.05 - 0.001), and plasma concentrations of secretin and CCK (r = 0.743, 0.425, P less than 0.001 and 0.05). Intravenous administration of CCK-antagonist, CR1505 (5 mg/kg.hr) markedly inhibited oligopeptide-stimulated amylase output, but did not affect pancreatic juice volume and bicarbonate output. These results suggest that id oligopeptide increases pancreatic exocrine secretion and releases endogenous secretin and CCK. PMID- 1703584 TI - Combined use of in situ hybridization and unlabeled antibody peroxidase anti peroxidase methods: simultaneous detection of type I procollagen mRNAs and factor VIII-related antigen epitopes in keloid tissue. AB - In this study, we developed methodology that allows the combined use of in situ hybridization and peroxidase anti-peroxidase techniques on the same tissue section. A human pro alpha 1(I) collagen cDNA and antibodies to factor VIII related antigen were used on keloid tissue sections as a model for a fibrotic reaction. The basic protocols of the techniques were modified to obtain optimal results. The feasibility of this new method was demonstrated by elucidation of type I procollagen gene expression in the cells of blood vessel wall and the adjacent fibroblasts. In the case of capillaries, pro alpha 1(I) collagen mRNAs were detected within endothelial cells identified by the presence of factor VIII related antigen. Pro alpha 1(I) collagen mRNAs were also found in close proximity of medium-size blood vessels, but in this context clearly outside the vessel wall. These results may contribute to the understanding of pathogenetic aspects of keloids and other fibrotic conditions. Thus, the combination of in situ hybridization and peroxidase anti-peroxidase techniques provides a useful tool to examine gene expression simultaneously both at mRNA and protein levels in fibrotic tissues. This methodology is also applicable to a variety of other biologic and pathologic situations. PMID- 1703585 TI - Constitutive expression of endothelin gene in cultured human mesangial cells and its modulation by transforming growth factor-beta, thrombin, and a thromboxane A2 analogue. AB - The present study was designed to assess whether human glomerular mesangial cells in culture express preproendothelin gene and whether endothelin gene expression in the mesangium is regulated by factors potentially released by inflammatory cells and platelets infiltrating the glomerular tuft during the course of various types of glomerulonephritis. For this purpose mesangial cells were incubated for 6 hours in the presence of absence of interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TBF-beta), the thromboxane A2 analogue U-46619, and thrombin. Resting mesangial cells expressed a 2.3-kilobase mRNA on blot hybridization analysis with a human cDNA preproendothelin probe, indicating that this type of cells, in addition to glomerular endothelial cells, constitutively expresses endothelin gene. IL-1 beta did not change endothelin mRNA levels in respect to unstimulated mesangial cells. At variance, TGF-beta, U-46619, and thrombin had a marked effect on endothelin mRNA, stimulating a 3- to 8-fold increase over basal levels. Quantification of actin mRNA and analysis of the autoradiographic signals provided validation of the difference in the endothelin mRNA levels. Expression of preproendothelin mRNA in either resting or stimulated mesangial cells was associated with synthesis and release of the corresponding peptide in the cell supernatant as determined by a specific radioimmunoassay for endothelin. Endothelin production from IL-1 beta stimulated mesangial cells was not different from that of unstimulated cells, whereas a significant (p less than 0.01) increase in endothelin production was observed after cell stimulation with TGF-beta, U-46619, and thrombin. The demonstration that mesangial cells constitutively express mRNA for preproendothelin and release endothelin into culture medium, together with the finding that endothelin gene expression and production in mesangial cells are regulated by molecules potentially released at glomerular level during an inflammatory reaction may suggest that endothelin participates in the complex process of glomerular disease progression. PMID- 1703586 TI - Neuron-associated class III beta-tubulin isotype, microtubule-associated protein 2, and synaptophysin in human retinoblastomas in situ. Further immunohistochemical observations on the Flexner-Wintersteiner rosettes. AB - We studied by immunohistochemistry 26 retinoblastomas in situ using monoclonal antibodies specific for the neuron-associated class III beta-tubulin isotype (h beta 4), microtubule-associated protein 2 (MAP2), and synaptophysin. Anti-h beta 4 and anti-MAP2 immunostaining was consistently obtained in the Flexner Wintersteiner rosettes, in fleurettes, in Homer Wright (neuroblastic) rosettes, and also variably among poorly differentiated tumor cells. A similar pattern of antisynaptophysin immunopositivity was seen, but was especially pronounced in the adluminal borders of cells forming the Flexner-Wintersteiner rosettes. The demonstration of h beta 4, MAP2, and synaptophysin epitopes in poorly differentiated and maturing neoplastic phenotypes in retinoblastomas attests to the neuronal character of this embryonal tumor. Immunoreactivity toward h beta 4 and MAP2 epitopes by poorly differentiated neoplastic cells may indicate early neuronal commitment in retinoblastoma. The consistent immunostaining of Flexner Wintersteiner rosettes with monoclonal antibodies to h beta 4 and MAP2 is in keeping with the previous ultrastructural documentation of microtubules with a neuronal-like spatial organization present in the cells of these structures. PMID- 1703587 TI - Localization of type I and III collagen and fibronectin production in injured gastrocnemius muscle. AB - The healing of muscle rupture consists of two simultaneous processes, regeneration of disrupted muscle fibers and production of connective tissue scar. These two processes are at the same time supportive to and competitive with each other. Their balanced progression is necessary for optimal healing. Synthesis of three connective tissue proteins, collagen types I and III and fibronectin, was analyzed during the regeneration process from 2 days to 3 weeks by Northern blot and in situ hybridization and immunohistochemistry. For this purpose a partial standard rupture of the gastrocnemius muscle was induced in 56 rats by a strike with a blunt spring-loaded hammer. Northern blot analysis of the specific mRNAs during the healing process revealed distinctly different expression patterns for fibronection and type I and III collagens. During the early stages (days 2 and 3) fibronectin, derived mainly from plasma, was abundant in the traumatized area, but local production of fibronectin mRNA by fibroblasts had also already started by day 2, closely followed by that of type III collagen. This early active synthesis of type III collagen and fibronectin was followed by a decrease after 1 week. The production of type I collagen mRNAs was activated somewhat later and remained elevated for at least 3 weeks. The muscle cells did not contain procollagen mRNAs. The observed sequence of connective tissue proteins reflects the particular function that each carries out during muscle wound healing (e.g., fibronectin in fibroblast trapping, type III collagen in plasticity/flexibility, and type I collagen in tensile strength). PMID- 1703588 TI - Role of quinidine in the mexiletine-quinidine interaction: electrophysiologic correlates of enhanced antiarrhythmic efficacy. AB - Quinidine has multiple electrophysiologic effects, including prolongation of ventricular conduction time, repolarization, and refractoriness. The purpose of this study was to address the relative contributions of these electrophysiologic effects to the enhanced anti-arrhythmic activity observed when quinidine is combined with mexiletine. We compared antiarrhythmic and electrophysiologic effects observed when quinidine or its stereoisomer quinine were combined with mexiletine. Quinine and quinidine both prolong conduction time; however, these agents have divergent effects on ventricular repolarization time and refractoriness. The modest prolongation of conduction time observed with quinine and mexiletine-quinine in the absence of change of ventricular refractoriness was not associated with antiarrhythmic efficacy. The antiarrhythmic efficacy of mexiletine-quinidine exceeds that of mexiletine-quinine, suggesting that the ability of quinidine to prolong refractoriness and repolarization contributes to the antiarrhythmic efficacy of mexiletine-quinidine. Although, both the mexiletine-quinidine combination and quinidine monotherapy prolonged refractoriness to a similar extent, the mexiletine-quinidine combination produced greater antiarrhythmic efficacy and prolonged interventricular conduction within the periinfarct zone to an extent greater than did quinidine alone. We concluded that the role of quinidine in producing enhanced antiarrhythmic activity when combined with mexiletine includes both prolongation of refractoriness and conduction time in the periinfarct zone. PMID- 1703589 TI - Comparative effects of antidepressants, amitriptyline, and maprotiline on intraventricular conduction, effective refractory period, and incidence of ventricular arrhythmias induced by programmed stimulation in dog hearts after myocardial infarction. AB - The effects of amitriptyline and maprotiline, standard tricyclic and tetracyclic antidepressants, on intraventricular conduction, the effective refractory period (ERP), and the incidence of ventricular arrhythmias induced by programmed stimulation were studied and compared in dog hearts after myocardial infarction. Amitriptyline at doses of 1-3 mg/kg significantly slowed ventricular conduction of the infarcted zones in a frequency-dependent and dose-dependent manner. Amitriptyline at doses of 2 and 3 mg/kg slowed conduction slightly in normal zones. The ERP was prolonged by amitriptyline at a dose of 2 mg/kg. Amitriptyline increased the incidence of ventricular arrhythmias induced by programmed stimulation. Maprotiline at doses of 1-3 mg/kg slowed conduction in infarcted zones to a lesser extent as compared with amitriptyline, although severely depressed conduction in the infarcted zone was obviously slowed by maprotiline. Maprotiline did not increase the incidence of ventricular arrhythmias significantly. From the present results, maprotiline appears to have less cardiac toxicity than amitriptyline, although maprotiline produces a slight decrease in conduction of infarcted zones. PMID- 1703590 TI - Altered beta-adrenergic sensitivity and protein binding to 1-propranolol in the elderly. AB - The elderly are reported to be less sensitive to the beta-blocking effects of propranolol. However, age-related changes in the stereoselective pharmacokinetics or protein binding of propranolol enantiomers could have confounded the results of previous studies because only 1-propranolol contributes significantly to the beta-blocking effects of the racemate. To avoid these confounding variables, we studied 10 young (mean 28 years) and 10 elderly (mean 64 years) subjects, and determined the cardiac beta-receptor sensitivity in terms of unbound, active 1 propranolol. The doses of isoproterenol required to increase heart rate (HR) by 25 beats/min were determined before and during a continuous infusion of propranolol. The serum concentration of 1-propranolol was determined by enantioselective high-performance liquid chromatography (HPLC), and the unbound fraction was determined by equilibrium dialysis. The apparent in vivo receptor dissociation constant for unbound 1-propranolol increased from 0.066 +/- 0.047 ng/ml in the young to 0.218 +/- 0.264 ng/ml in the older group (p less than 0.05). The unbound fraction was decreased in the older subjects (0.141 +/- 0.023 vs. 0.121 +/- 0.025, p less than 0.05) because of an increase in alpha 1-acid glycoprotein concentration (55 +/- 11 mg/dl vs. 72 +/- 19 mg/dl, p less than 0.05). Advancing age was associated with a decreased sensitivity to isoproterenol (rs = 0.76, p less than 0.05) and to unbound 1-propranolol (rs = 0.45, p less than 0.05). We conclude that the older subjects have (a) decreased sensitivity to the beta-blocking effects of 1-propranolol and to the agonist effects of isoproterenol, and (b) a lower unbound fraction of 1-propranolol. PMID- 1703591 TI - Pacing-induced heart failure in the dog: evaluation of peripheral vascular alpha adrenoceptor subtypes. AB - Postjunctional alpha-adrenoceptor characteristics were evaluated in canine dorsal pedal arterial and saphenous vein rings studied before and after development of severe pacing-induced heart failure (CHF). Before CHF, all agonists produced concentration-dependent increases in tension of both blood vessels. After development of CHF, the responsiveness and sensitivity of the vessels to the alpha 1-agonists and the mixed agonists were significantly increased as compared with control. The maximum responses to BHT 920 and BHT 933 remained unaltered after CHF, but both vessels showed decreased sensitivity to BHT 920. Before CHF, the rank order of potency with respect to norepinephrine (NE) for the dorsal pedal artery was as follows: NE greater than epinephrine greater than methoxamine greater than BHT 933 greater than BHT 920, and for the saphenous vein was epinephrine greater than NE greater than BHT 933 greater than methoxamine greater than BHT 920. At peak CHF, the rank order of potency for the artery was epinephrine greater than NE greater than methoxamine greater than BHT 933 greater than BHT 920, whereas in the vein BHT 920 was approximately 80 times less potent than NE (as compared with being only five times less potent before CHF). Prazosin was a potent, competitive antagonist (pA2 values of 9.2 and 9.0 for the artery and the vein) of methoxamine-induced contractions before development of CHF. Prazosin had a 10-fold lower potency against epinephrine-induced contractions in the dorsal pedal artery, whereas it was not competitive against epinephrine in the saphenous vein. Against the selective alpha 2-agonists, prazosin either showed no antagonism or was not competitive. After CHF, prazosin was non competitive against all agonists tested. Yohimbine was a potent, competitive antagonist against BHT 920 both before and at CHF. Yohimbine had intermediate antagonism against epinephrine and produced no antagonism of methoxamine-induced contractions. We conclude that increased reactivity and sensitivity of the peripheral vasculature to alpha 1-agonists occurs at CHF. PMID- 1703592 TI - Renal versus hindquarter hemodynamic responses to vasopressin in conscious rats. AB - Experiments were performed on conscious rats to (a) compare the responsiveness of the renal and hindquarter vascular beds to infusions of exogenous arginine vasopressin (AVP), and (b) determine whether either bed demonstrates V2 vasopressinergic vasodilation when the vasoconstrictor properties of AVP are blocked. Rats were chronically instrumented with pulsed Doppler flow probes on either the left renal artery or the distal abdominal aorta as well as with femoral arterial and venous catheters. One series of experiments examined the vascular responses of these two beds to exogenous AVP infused intravenously (i.v.) at 0.2, 2.0, or 5.0 ng/min. The lowest infusion rate was associated with no detectable changes in mean arterial blood pressure (MAP), heart rate (HR), renal or hindquarter blood flow (RBF or HQBF), or vascular resistance in these beds. In contrast, the higher infusion rates caused a marked increase in MAP, a decrease in HR, and a reduction in HQBF; RBF was unaffected, however. A second series of experiments tested for the presence of a V2-vasodilatory influence during infusion of AVP at 5 ng/min by selectively blocking V1-vasopressinergic receptors or both V1- and V2-receptor types. Little evidence for V2-mediated vasodilation was found in either vascular bed, however. We conclude that although the renal vasculature appears relatively insensitive to exogenous AVP, this insensitivity probably is not due to vasodilation mediated by activation of V2 receptors. PMID- 1703593 TI - Production and biologic interactions of prostacyclin and platelet-activating factor in acute myocardial ischemia in the perfused rabbit heart. AB - This study showed that the 1-O-hexadecyl form of platelet-activating factor (PAF) is released along with 6-keto-prostacyclin1 alpha (6-keto-PGF1 alpha) in the perfusates of ischemic-isolated rabbit heart. During the early phase of the reperfusion, the release of PAF and 6-keto-PGF1 alpha was particularly increased (PAF, from 5.4 +/- 1.2 to 19.7 +/- 2.0 ng/min; 6-keto-PGF1 alpha, from 2.3 +/- 0.1 to 27.4 +/- 1.8 ng/min) and this event was coupled with the characteristic mechanical alterations of myocardial performance and perfusion pressure (PP). These changes were effectively antagonized by pretreatment of the hearts with both prostacyclin (PGI2, 20 ng/ml) and the PGI2-releaser defibrotide (400 micrograms/ml). The protecting activity observed with PGI2 was paralleled by a reduction in the rate of PAF release during reperfusion (from 19.7 +/- 2.0 to 6.8 +/- 0.2 ng/min). In defibrotide-treated preparations, inhibition of PAF formation was associated with a pronounced stimulation of 6-keto-PGF1 alpha generation, which was particularly marked on reperfusion (from 27.4 +/- 1.8 to 197.5 +/- 8.2 ng/min). Indomethacin (1 microgram/ml) ablated the antiischemic effect of defibrotide but did not potentiate the rate of formation of PAF despite inhibition of 6-keto-PGF1 alpha biosynthesis. From these results, we conclude that in a model of severe myocardial ischemia, in which the major determinants of cardiac performance are under control, generation of PAF and PGI2 appears to play an important biologic role in determination of the severity of myocardial ischemic damage. PMID- 1703594 TI - Inhibitory effects of a novel antiplatelet aggregating agent, E-5510, on cyclic flow variations in electrically stimulated coronary arteries of the pig. AB - We examined the inhibitory effects of a novel antiplatelet aggregating agent, E 5510, on cyclic flow variations (CFVs) of coronary blood flow (CBF) in anesthetized open-chest pigs. These CFVs, which are characterized by progressive declines in CBF followed by sudden restoration of flow, were initiated by electrical stimulation of the intimal surface of the left circumflex coronary artery (LCX). A reduction in CBF to zero during CFVs was accompanied by ischemic changes in the surface electrocardiogram and regional segment shortening of the left ventricular wall. Occlusive thrombi were detected postmortem in the coronary arteries of the animals in which CFVs had occurred. After CFVs had been observed for 1 h, E-5510 (0.01 or 0.1 mg/kg) or saline was administered intravenously. Once CFVs were initiated, both the frequency and the severity (the mean of the three lowest nadirs of CBF) were unchanged by the administration of saline. E 5510 at 0.01 mg/kg decreased the frequency of CFVs from 7.7 +/- 0.9 to 4.6 +/- 1.1 CFV s/h (n = 7, p less than 0.05), and increased the mean lowest nadir from 13.5 +/- 5.2% of the CBF level before electrical stimulation to 54.3 +/- 11.1% (n = 7, p less than 0.01). E-5510 at 0.1 mg/kg further decreased the frequency from 8.9 +/- 0.7 to 2.4 +/- 0.5 CFVs/h (n = 9, p less than 0.01), and increased the mean lowest nadir from 14.3 +/- 3.2% to 53.6 +/- 10.8% (n = 9, p less than 0.01). E-5510, however, showed no ameliorative effect on ischemia-induced myocardial dysfunction, as expressed by the decrease in regional myocardial shortening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703595 TI - Hemodynamic effects of intraatrial administration of deferoxamine or deferoxamine pentafraction conjugate to conscious dogs. AB - Deferoxamine (DFX) is a specific Fe3+ chelator that is used to manage iron overload, and is being evaluated as an agent to reduce ischemic organ damage that involves iron-mediated OH formation. However, high intravascular doses cause significant hemodynamic changes that may limit or counteract beneficial effects. We used conscious, closed-chest dogs to test the hypothesis that conjugating DFX to pentafraction, a high molecular weight fraction of pentastarch, could reduce such hemodynamic changes. We infused 50 mg/kg of body weight of native DFX, or an equivalent dose as DFX-pentafraction, intraatrially over 15 min. Within 10 min of starting the infusion. DFX increased heart rate from pre-drug values of 105 +/- 11 (mean +/- SEM; N = 9) to 158 +/- 13 beats/min, and reduced left ventricular (LV) systolic pressure from 131 +/- 3 to 99 +/- 16 mm Hg, LV end-diastolic pressure from 12 +/- 3 to 3 +/- 3 mm Hg, and mean arterial pressure (MABP) from 101 +/- 5 to 74 +/- 13 mm Hg. In two dogs, MABP decreased to less than or equal to 35 mm Hg. These parameters returned to predrug values by 60 min after infusion. All of these changes were statistically significant (p less than 0.05). In contrast, infusing DFX-pentafraction (N = 9) caused no significant cardiac or hemodynamic changes other than a transient and slight (approximately 7%) increase in systolic arterial pressures. This conjugate, which prolongs the plasma half life and does not alter the iron-chelating activity of native DFX, eliminates many undesirable hemodynamic actions. It may be a useful therapeutic alternative to native DFX in some settings. PMID- 1703596 TI - Effects of platelet-activating factor on myocardial contraction and myocardial relaxation of isolated perfused guinea pig hearts. AB - Platelet-activating factor (PAF) is an important mediator of cardiovascular shock owing to immunologic reactions, including anaphylaxis and endotoxaemia. Previous studies have shown that PAF is a potent cardio-depressive agent causing a marked coronary constriction and a sustained impairment of myocardial contractility. In this study, we attempted to characterize further the prolonged PAF effects on coronary circulation and myocardial contractile force in isolated guinea pig hearts perfused at constant pressure (60 cm H2O) or constant flow which was adjusted to a level of 100% above basal flow. In addition, the PAF-induced changes of ventricular systolic and diastolic function were distinguished. In the hearts perfused at constant pressure, PAF induced a dose-dependent (0.57, 5.7, and 57 pmol/min) decrease of coronary flow rates, left ventricular pressure (LVP), LV contraction (peak positive dP/dt) and LV relaxation (peak negative dP/dt). The decrement of peak negative dP/dt was more pronounced than that of peak positive dP/dt. Maintenance of coronary flow rates only attenuated, but did not suppress, the PAF-induced ventricular malfunction, and it improved ventricular relaxation less than it did ventricular contraction. Pretreatment with the PAF antagonist WEB 2086 (19.7 nmol/min) almost completely abolished the effects of the highest PAF dose on coronary circulation and ventricular contractile parameters. We conclude that the cardiodepressive effects of PAF are due to coronary constriction and direct contractile events. Furthermore, PAF impairs ventricular diastolic function more than ventricular systolic function. PMID- 1703597 TI - Effects of propranolol on premature action potentials in canine Purkinje and ventricular muscle. AB - We compared the effects of a low (0.09 microgram/ml) concentration of propranolol expected to produce only beta-adrenoceptor blockade and a high concentration (0.9 microgram/ml) expected to produce additional direct local anesthetic-like electrophysiological effects on basic and premature action potentials. Both isolated dog cardiac Purkinje and ventricular muscle fibers were examined using conventional microelectrode techniques. The low concentration of propranolol produced no significant electrophysiological change in either fiber type. The high concentration of propranolol shortened the action potential duration and refractoriness while decreasing the maximal upstroke velocity (Vmax) in both Purkinje and ventricular muscle fibers at a constant basic cycle length. In Purkinje fibers, the high concentration also slowed the kinetics of restitution of the action potential duration (tau c from 124.6 +/- 6.5 to 201.4 +/- 16.0 ms, p less than 0.01, n = 7), slowed the recovery kinetics of Vmax, and shifted the early portion of the normalized restitution curve toward longer action potential duration values in both fiber types. The range of premature action potential durations, defined as the difference between action potential durations during the first 100 ms of restitution, was decreased by the high concentration of propranolol in both Purkinje and ventricular muscle fibers by 39.5% and 33.9%, respectively. These findings indicate that (a) low concentrations of propranolol produced no direct electrophysiological effect, and (b) high concentrations of propranolol produced several potential antiarrhythmic effects in addition to the previously reported effects on Vmax and the action potential duration. PMID- 1703598 TI - Antiischemic and hemodynamic effects of intravenous isradipine, a new calcium antagonist, in coronary heart disease: a comparative double-blind cross-over study with nifedipine. AB - In a double-blind cross-over study, 10 patients with stable angina pectoris owing to coronary heart disease were investigated in supine position during rest and bicycle exercise for the effect of 0.4 mg of intravenous (i.v.) isradipine in comparison to 2 mg i.v. nifedipine on cardiac hemodynamics and myocardial ischemia. At rest, both drugs significantly decreased total peripheral resistance (TPR) and mean arterial blood pressure (MAP), whereas heart rate (HR) increased. The pressures and resistance of the pulmonary circulation remained uninfluenced at rest. During symptom limited-exercise, both medications reduced TPR despite an unchanged MAP. Mean pulmonary artery pressure decreased significantly after both medications, whereas right atrial pressure (RAP), pulmonary capillary wedge pressure (PCWP), and pulmonary vascular resistance (PVR) decreased significantly only after nifedipine. The improvement of mean ischemic ST-segment depression averaged 44 +/- 6% (mean +/- SEM, p less than or equal to 0.01) after nifedipine and 45 +/- 7% (p less than or equal to 0.01) after isradipine. The time until angina appeared increased after isradipine by 89 +/- 28% (p less than or equal to 0.05) and after nifedipine by 105 +/- 42% (p less than or equal to 0.01). Significant differences between the two medications appeared only for cardiac output (CO) at rest (p less than or equal to 0.05), during which state the increase after isradipine was higher than after nifedipine, and for exercise HR (p less than or equal to 0.01), during which state only nifedipine induced a significant increase in frequency. We conclude that at the chosen dosages the hemodynamic and antiischemic effects of isradipine are similar to the effects that occur after nifedipine. PMID- 1703599 TI - Electrophysiological properties of SD-3211, a novel putative Ca2+ antagonist, in isolated guinea pig and rabbit hearts. AB - The cardiac effects of SD-3211, a novel non-dihydropyridine type of Ca2+ antagonist, were examined in isolated guinea pig and rabbit hearts using an electrophysiological technique. SD-3211 (10(-6)-10(-5) M) shortened the action potential duration of guinea pig papillary muscles in a concentration-dependent manner without affecting the resting potential or the maximum upstroke velocity (Vmax). The Vmax of slow responses induced by high extracellular K+ and isoproterenol was inhibited by SD-3211 at concentrations of greater than 10(-6) M. Elevation of extracellular Ca2+ by 2 mM reversed this inhibited response. The inhibitory effect of SD-3211 on the slow response was enhanced as the stimulation frequency was increased. In Langendorff-perfused rabbit hearts electrically driven at 2.0 Hz, SD-3211 (10(-8)-10(-6) M) produced a concentration-dependent prolongation of the atrium-His bundle conduction time (A-H interval) as well as a reduction in the developed tension of ventricular muscle, whereas SD-3211 did not affect the His bundle-ventricular conduction time (H-V interval) significantly. The potency of SD-3211 in A-H prolongation was greater than those of diltiazem and bepridil, but weaker than those of nicardipine, nifedipine, and verapamil. The effect of SD-3211 on the A-H interval was more pronounced at higher stimulation frequencies. SD-3211 was intermediate between nicardipine and verapamil in its intensity of frequency-dependent effects on the A-H interval. These results suggest that SD-3211 has a preferential and frequency-dependent inhibitory action on cardiac slow Ca2+ channels. PMID- 1703600 TI - Blunted renal response to atrial natriuretic peptide in congestive heart failure rats is reversed by the alpha 2-adrenergic agonist clonidine. AB - We wished to determine whether pharmacologic inhibition of the exaggerated sympathetic nerve activity in congestive heart failure (CHF) could restore the renal response to exogenous atrial natriuretic peptide (ANP) administration. Left ventricular (LV) myocardial infarction was induced in Sprague-Dawley rats (n = 16) by coronary artery ligature. Four to 6 weeks postoperatively, an isotonic saline (controls) or clonidine 5 micrograms/h infusion was given. Four hours later, all animals received incremental doses of rat ANP (99-126) (0.25, 0.5 and 1.0 microgram/kg/min). The continuous clonidine infusion transiently increased urinary volume (UV) as compared with the saline controls. Mean arterial pressure (MAP), heart rate (HR), and plasma norepinephrine (NE) were significantly decreased by clonidine. The graded ANP infusions significantly increased UV (saline 39.13 +/- 12.45 and clonidine 90.25 +/- 13.69 microliters/min, p less than 0.05) and UNaV (saline 4.26 +/- 1.10 and clonidine 8.81 +/- 1.59 mumol/min, p less than 0.05) in clonidine-pretreated rats as compared with saline-pretreated rats. We conclude that the diuretic and natriuretic responses to ANP are significantly increased in CHF after presynaptic inhibition of NE release by low dose clonidine. PMID- 1703601 TI - Ruthenium red improves postischemic contractile function in isolated rat hearts. AB - The effect of ruthenium red (inhibitor of mitochondrial calcium entry) on reperfusion contractile function and enzyme release was determined in isolated perfused rat hearts and compared with that of diltiazem. The hearts were made ischemic for 25 min and reperfused for 30 min. They were pretreated with 1-10 microM ruthenium red, 1 microM diltiazem, or vehicle. All concentrations of ruthenium red significantly improved reperfusion contractile function without affecting lactate dehydrogenase (LDH) release or contracture. Diltiazem significantly improved reperfusion function and reduced LDH release and contracture formation. Ruthenium red still improved function even when given only during reperfusion. Diltiazem increased reperfusion oxygen consumption and efficiency of oxygen utilization whereas ruthenium red improved efficiency without an increase in oxygen consumption. Diltiazem significantly increased reperfusion functional reserve in these hearts, although ruthenium red did not. Ruthenium red reduced coronary flow and contractile function in nonischemic myocardial tissue and the reduced function appeared to be secondary to the reduced coronary flow as well as to a direct negative inotropic effect. Thus, ruthenium red improved reperfusion contractile function and oxygen efficiency; this may be related to its ability to block mitochondrial calcium uptake. PMID- 1703602 TI - Clinical studies with the potassium channel activator cromakalim in normotensive and hypertensive subjects. AB - Eight normotensive subjects received single and multiple doses of cromakalim (1 mg) and placebo in a randomised double-blind cross-over study to examine general tolerance to cromakalim and its effects on blood pressure (BP), heart rate (HR), and pressor responses to norepinephrine (NE) and angiotensin II (AII). In a second study, 10 hypertensive patients whose BP control was unsatisfactory with atenolol 50-100 mg received additional treatment with placebo followed by cromakalim 1 mg daily for 4 weeks. Assessments were made of BP, HR, apparent hepatic blood flow and renal blood flow (RBF), pulmonary function, and the pharmacokinetics of atenolol. Cromakalim was generally well tolerated in both normotensive and hypertensive subjects. In the normotensive group, cromakalim produced a reflex increase in HR without any detectable decrease in BP: average (placebo-subtracted) increases in HR at 4 h were 16 beats/min with subjects in an erect position after the single dose and 14 beats/min after 7 days. Cromakalim had no effect on pressor responses to NE and AII. Addition of cromakalim to atenolol was associated with modest further reductions in BP between 0.5 and 3 h after drug administration, with maximal reductions of 21/14 mm Hg (subjects in supine position) 2 h after the first dose. Cromakalim had no effect on apparent liver blood flow and RBF, pulmonary function, and the steady-state pharmacokinetics of atenolol. Single and multiple 1-mg doses of cromakalim are well tolerated but are associated with only modest vasodilator activity. PMID- 1703603 TI - Involvement of rolipram-sensitive cyclic AMP phosphodiesterase in the regulation of cardiac contraction. AB - The involvement of rolipram-sensitive phosphodiesterase (PDE IV) in regulation of cardiac contraction was investigated by studying the effect of selective inhibitors (rolipram, denbufylline, Ro 20-1724) on guinea pig left atria contraction. In contrast to milrinone and SK&F 94120 (inhibitors of the cyclic GMP-inhibited PDE, PDE III), (+/-)-rolipram and denbufylline (0.1-30 microM) did not produce any positive inotropic effect in normal (2.5 mM) or elevated (3-3.2 mM) external CaCl2 concentration. In these conditions, Ro 20-1724 produced only a slight but significant increase of contraction over control levels. In the presence of forskolin (an adenylate cyclase activator) or SK&F 94120 (a PDE III inhibitor), which produced an increase of the response to electrical stimulation of approximately 10%, (+/-)-rolipram, denbufylline, and Ro 20-1724 all exerted concentration-dependent positive inotropic effects (mean EC50 values were 20, 25, and 125 nM, respectively, in the presence of forskolin). Rolipram exhibited stereospecificity: the (-)-enantiomer was 10 times more potent than the (+) enantiomer. Neither preincubation of the atria with atenolol nor pretreatment of the guinea pigs with reserpine significantly modified the effect of PDE IV inhibitors obtained in the presence of forskolin. These data show that in the presence of cyclic AMP-dependent positive inotropic agents, PDE IV inhibitors exert a positive inotropic effect which probably does not involve enhanced catecholamine release from sympathetic nerve endings. This suggests that PDE IV may play a role in regulation of cardiac contraction in physiologic conditions in which the sympathetic outflow produces a stimulation of adenylate cyclase in cardiac cells. PMID- 1703604 TI - Effects of endothelin-1 in the isolated heart under ischemic and cardioplegic conditions. AB - We examined the effects of the vasoconstrictor peptide endothelin-1 in isolated hearts under ischemic and cardioplegic conditions. Isolated isovolumic rat hearts were perfused with Krebs-Henseleit buffer at constant pressure. Cumulative dose response curves were obtained for endothelin-1 boluses of 0.04-400 pmol in four groups of hearts. Coronary flow decreased with increasing dosages and was almost abolished at 400 pmol in control hearts perfused at a constant pressure of 100 mm Hg. In hearts made ischemic by reducing coronary perfusion pressure to 35 mm Hg, thus reducing coronary flow by 76%, the constrictor effect of endothelin-1 was well preserved. The endothelin-1 dose-response curve was unaltered when hearts were perfused with buffer containing 30 mM KCl to abolish mechanical activity without reducing extracellular Ca2+ concentration. A fourth group of hearts was perfused with Ca2(+)-free buffer, thus eliminating the source of extracellular Ca2+ as well as mechanical activity. In this group, the constrictor response to endothelin-1 was largely, but not completely, abolished, with a maximal constrictor effect of only 19% as opposed to 87% in control hearts. We conclude that in isolated rat heart endothelin-1 is a potent coronary constrictor under ischemic perfusion conditions and that absence of mechanical activity does not affect the action of endothelin-1, for which the presence of extracellular Ca2+ is essential. The small residual constrictor response with Ca2(+)-free perfusion is probably due to release of Ca2+ from intracellular stores. PMID- 1703605 TI - Efficacy of epicardial controlled-release lidocaine for ventricular tachycardia induced by rapid ventricular pacing in dogs. AB - The effects of epicardial lidocaine on ventricular tachycardia (VT) induced by rapid ventricular pacing (50 Hz) were studied in dogs. Lidocaine-polyurethane (28% wt/wt) matrixes (40-50 mg, 5 x 5 mm) were placed proximal to bipolar left ventricular epicardial electrodes in open-chest anesthetized dogs (n = 9) after VT was established by rapid ventricular pacing. In the first set of experiments, matrices were removed immediately after VT had converted to sinus rhythm. Control animals underwent VT induction protocol with a nondrug-containing matrix positioned next to the epicardial electrode. In the lidocaine-treated animals, VT conversion was noted in all animals and occurred after 51.7 +/- 8.9 s (mean +/- SE), with a VT threshold current elevation of 73.5 +/- 11.2% above baseline at the time of conversion which progressed to 259 +/- 44% of the initial value by 5.4 +/- 0.5 min post-matrix placement. Lidocaine 1.4 +/- 0.1 mg was delivered to the myocardium at the time of VT conversion (0.11 +/- 0.01 mg/kg). In comparison, accelerated VT persisted for 5 min in three of five control animals, and progressed to ventricular fibrillation (VF) in the other two animals. In a separate series of eight dogs, the lidocaine-polyurethane matrixes were left in palce for 4 h so that we could study the sustained antiarrhythmic action of controlled-release lidocaine on VT induction. The results of these experiments demonstrated a maintenance of the VT threshold elevation at a level of 53.9 +/- 10.8% after 4 h, with a net lidocaine dose of 0.52 +/- 0.06 mg/kg after 4 h of controlled release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703606 TI - Quinidine/quinine: stereospecific electrophysiologic and antiarrhythmic effects in a canine model of ventricular tachycardia. AB - The two major electrophysiologic effects of quinidine are prolongation of refractoriness and prolongation of conduction time. To determine which of these effects contributes to its antiarrhythmic effect, we compared the electrophysiologic effects of quinidine and its stereoisomer quinine (which was expected to prolong conduction time but not refractoriness) in 24 dogs with inducible sustained ventricular tachyarrhythmia late after ischemic injury. Conscious but sedated animals were randomly assigned to receive infusions of saline, quinidine, or quinine. Serum concentrations of quinidine and quinine were 18 +/- 9 and 23 +/- 8 microM, respectively. Both drugs prolonged conduction times to a similar extent, but quinidine prolonged local repolarization times and refractoriness much more than quinine. Sustained ventricular tachyarrhythmia was consistently inducible during placebo (saline) studies. Antiarrhythmic efficacy was observed with quinidine (3 of 12) but not quinine (0 of 15) or saline (0 of 13) (p less than 0.05, Chi-square test). Quinidine also significantly prolonged monomorphic ventricular tachycardia (VT) cycle length (157 +/- 33 ms on quinidine vs. 129 +/- 26 ms at baseline, p less than 0.001) whereas quinine had no significant effect. Thus, prolonging refractoriness is important in preventing the induction of ventricular tachyarrhythmias and in prolonging VT cycle length. PMID- 1703607 TI - UK-52,046 (a novel alpha 1-adrenoceptor antagonist) and the role of alpha adrenoceptor stimulation and blockade on atrioventricular conduction. AB - The effects of increasing intravenous (i.v.) doses of prazosin, phenylephrine, flecainide, and UK-52,046 on blood pressure (BP), heart rate (HR) and the specialized conduction system (AH and HV intervals) were investigated in anaesthetized dogs. Results indicated that UK-52,046 (1-8 micrograms/kg) had no effect on BP or HR, but reduced (p less than 0.05) BP at doses of 16 and 32 micrograms/kg; no change occurred in HR. During sinus rhythm (SR) the AH or HV intervals did not change as compared with placebo; on pacing (PA) UK-52,046 (4-16 micrograms/kg) decreased the AH interval. After prazosin BP decreased (p less than 0.05) after 20-40 micrograms/kg; HR increased after all doses (5-40 micrograms/kg). The HV interval was unaltered, but the AH interval decreased after 40 micrograms/kg during SR and PA. After phenylephrine (continuous infusion, 50 micrograms/ml/min) BP increased at 33 min and HR was decreased at 23 and 33 min. The AH interval lengthened during PA (p less than 0.05), but there was no effect on the HV interval. Administration of flecainide (0.5-2.0 mg/kg) had no effect on BP or HR but increased the HV interval during SR and PA (1 and 2 mg/kg, p less than 0.05). The results indicate that the alpha 1-adrenoceptor agonist phenylephrine and the alpha 1-adrenoceptor antagonist prazosin, on PA altered the AH (but not the HV) intervals in opposite directions in association with changes in HR and BP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703608 TI - Double-blind study of dilevalol and captopril, both in combination with hydrochlorothiazide, in patients with moderate to severe hypertension. AB - Sixty-one patients (41 men, 20 women) aged 29-73 years, with moderate to severe hypertension, were enrolled in a multicentre study to compare the efficacy, safety, and tolerability of dilevalol (D) and captopril (C). At the end of the baseline period, supine diastolic blood pressure (SuDBP) was 105-140 mm Hg on hydrochlorothiazide (HCTZ) 25 mg once daily and placebo t.i.d. Patients were randomly assigned to D + HCTZ (n = 29) or C + HCTZ (n = 32) and entered phase II titration of D (100-800 mg b.i.d.) or C (12.5 mg b.i.d. to 50 mg t.i.d.). If SuDBP was greater than 99 mm Hg, hydralazine was added (25 mg once daily to 50 mg b.i.d.). If SuDBP was less than or equal to 99 mm Hg, patients entered phase III, a 3-month maintenance period. Demographic profiles were not significantly different between the two groups. Baseline supine BP (mean +/- SEM) was similar in the two groups (D + HCTZ: 182 +/- 3/112 +/- 1; C + HCTZ: 179 +/- 4/113 +/- 1 mm Hg), as was baseline standing BP (D + HCTZ: 175 +/- 3/114 +/- 2; C +/- HCTZ: 173 +/- 4/113 +/- 1 mm Hg). At the end of phase II, there were no significant differences between treatments with respect to the changes in BP from baseline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703609 TI - Beta 1- and beta 2-receptors are differentially desensitized in an experimental model of heart failure. AB - Recent reports suggested that a complex alteration in beta-receptor function occurs in failing human myocardium. We evaluated beta-receptor-subtype activity in an experimental model of monocrotaline (MCT)-induced cardiomyopathy in the rat. Through pulmonary hypertension, MCT causes right ventricular hypertrophy (RVH), either associated with heart failure or not, beta-Receptor function was evaluated in both failing-hypertrophic and hypertrophic hearts in binding studies with [125I]iodocyanopindolol (ICYP) and by measuring adenylate cyclase (AC) activity. In the right failing ventricle, beta 1- but not beta 2-receptor density was decreased. Lesion-associated modifications in the adenylate cyclase system were also observed: isoproterenol- and guanosine 5' [beta, gamma imido]triphosphate [Gpp(NH)p]-stimulated cyclic AMP formation was reduced in the right failing ventricle, while the cyclic AMP responses to NaF and forskolin were unchanged. On the other hand, no changes in either beta-receptor density or function were found in hypertrophic ventricles. MCT-induced heart failure in the rat is thus associated with a selective decrease of beta 1-receptor density and function. These results suggest that MCT-induced cardiac failure may be an appropriate model in which to investigate heart insufficiency further. PMID- 1703610 TI - Sulfhydryl group in angiotensin converting enzyme inhibitors and superoxide radical formation. AB - The superoxide radical scavenging effects of the SH group in the captopril molecule has been proposed to be the basis of the "cadioprotective" effect of this angiotensin converting enzyme (ACE) inhibitor in animal models of myocardial injury. We determined the effects of captopril, another ACE inhibitor with an SH group (SQ 26,703), its stereoisomer without ACE inhibitory effect but with an SH group (SQ 14,534), another ACE inhibitor without a SH group (enalaprilat), and N acetylcysteine on superoxide radical generation by human neutrophils and by the purine-xanthine oxidase reaction. None of the compounds examined decreased superoxide radicals in therapeutic concentrations; however, SH-containing agents directly reduced spectrophotometric absorbance of ferricytochrome C. Thus, SH containing agents with or without ACE inhibitory effects do not scavenge superoxide radicals. PMID- 1703611 TI - Melatonin response to atrial natriuretic peptide administration in healthy volunteers. AB - Recent observations have demonstrated that the pineal hormone melatonin (MLT) plays a role in the neuroendocrine control of the cardiovascular system. On the other hand, it has been observed that the cardiac hormone alpha-atrial natriuretic peptide (ANP) may regulate the neuroendocrine functions. The present study was carried out to investigate the possible relationship between cardiac and pineal endocrine functions. Six healthy volunteers were treated on two different occasions with placebo or ANP at a dose of 0.1 mg i.v. as a bolus. An increase of greater than 100% in MLT serum levels was seen in 2/6 subjects. These preliminary results would suggest that ANP may play a role in the regulation of MLT secretion. Further studies will be needed to define better the cardiac-pineal interactions. PMID- 1703612 TI - Comparative observations on the curative results of the treatment of central aphasia by puncturing the yumen point versus conventional acupuncture methods. PMID- 1703613 TI - An improved silver staining technique as an alternative nuclear or combined nuclear nerve-fiber impregnation for comparative light-, secondary and backscattered electron scanning microscopy. AB - Slices of rat brain were stained by a new silver impregnation technique. This method takes into consideration the pH-dependent differences of silver stain affinity of nerve tissues and can be used alternatively as a stain for nuclei or as a method for combined demonstration of nuclei nerves fibers. The slices were studied at the light microscopical (LM) level and subsequently with a scanning electron microscope, using secondary (SSEM = classical SEM), and backscattered electron detectors (BSEM). This new silver staining technique offers the opportunity of comparative studies with regard to different information acquired with LM, SSEM and BSEM. The described method allows to distinguish between nervous and glial tissue without necessarily damaging the glial tissue surrounding the nerve fibers. Specifically, scanning electron microscopy with backscattered electron detector of in situ preparations provides a higher contrast of stained and unstained tissue and increased depth of focus as compared to secondary electron detectors. PMID- 1703614 TI - Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold. AB - Fluoro-Gold has been a very popular fluorescent tracer used recently for retrogradely labeling projection neurons. In this study we described the advantages of using antibodies to Fluoro-Gold in conventional immunohistochemical reaction protocols to further extend the usefulness of this tracer for both light and electron microscopic neuroanatomical studies. PMID- 1703615 TI - Uptake of Phaseolus vulgaris leucoagglutinin (PHA-L) by axons of passage. AB - Iontophoretic injection of Phaseolus vulgaris leucoagglutinin (PHA-L) into the spinal trigeminal tract, the trapezoid body or the inferior cerebellar peduncle produced extensive labelling of axons of passage. Anterograde transport labelled axon terminals more than 13 mm distant and retrograde transport labelled cells more than 3 mm distant. Axons of passage were labelled with both PHA-L and biotinylated PHA-L, and were observed following injection with different micropipette tip sizes, different durations of iontophoresis, and with continuous or pulsed iontophoretic current. The results suggest that while different injection parameters may influence the number of axons that are labelled, the possibility of labelling axons of passage should be considered whenever PHA-L is used. PMID- 1703616 TI - Effect of Triton X-100 in the Golgi-Kopsch method. AB - Application of a detergent, Triton X-100, dramatically increased the number of impregnated neurons in the Golgi-Kopsch method. This modification is particularly useful for obtaining a panorama of the neuronal field in adult brains. PMID- 1703617 TI - Automatic quantification of fast axonal transport in neuronal cell cultures. AB - A method is presented which allows the automatic quantification of the fast axonal transport of endogenous organelles in neurites of cultured neuronal cells. Stretches of videotape recordings from Allen video enhanced contrast (AVEC) microscopy are digitized by currently available image processor hardware and analysed off-line on a MicroVAX II. Movements along the axon are calculated in great detail, allowing statistically significant changes to be detected. Interaction from the operator is minimised, thereby bypassing tedious manual analysis. This paper further reports the application of this system to the effect of vanadate treatment on axonal transport in cultures of rat embryonic hippocampal neurons. PMID- 1703618 TI - Improved detection of rotavirus shedding by polymerase chain reaction. AB - To improve identification of children excreting rotavirus a method for the amplification of rotavirus RNA by the polymerase chain reaction (PCR) was developed. The assay was compared with a solid-phase enzyme immunoassay in the detection of rotavirus shedding by infants in hospital during the winter peak of rotavirus infections. Forty children were studied in an intermediate care unit after transfer from intensive care units. Only two were admitted primarily because of diarrhoea; the other thirty-eight were admitted for management of various other disorders. Rotavirus shedding was detected by enzyme immunoassay in twenty of the infants, and nine of these (aged 1 week to 8 months) remained in hospital for more than 5 days after the initial detection of rotavirus and could be studied long term. Of 103 faecal samples from the nine infants, 60 (58%) contained rotavirus RNA detected by reverse-transcriptase (RT)/PCR, whereas only 37 (36%) were positive for rotavirus antigen by the immunoassay (chi 2 = 10.3, p less than 0.002). The geometric mean time of rotavirus shedding was 9.5 (range 1 19) days as detected by RT/PCR and 5.7 (range 1-17) days by the immunoassay (p less than 0.018). In five of the nine children, RT/PCR detected rotavirus shedding for 2-7 days longer than the immunoassay and in four children RT/PCR was positive 1 or more days before rotavirus antigen was detected. Further studies should attempt to find out whether infected infants are capable of spreading wild type virus during periods when they are not shedding antigen as detectable by enzyme immunoassay. PMID- 1703619 TI - Anaphylactoid reaction to BCG vaccination. PMID- 1703620 TI - The utility of 2-hydroxypropyl-beta-cyclodextrin as a vehicle for the intracerebral and intrathecal administration of drugs. AB - The substituted glucopyranose ring structure 2-hydroxypropyl-beta-cyclodextrin (CDEX) increases the solubility of molecules by inclusion of the agent in the lipophilic interior of the ring. This property is of particular use for the administration of molecules by the intracerebral (ICV) or intrathecal (IT) routes. In concentrations up to 40% w/v (isotonic), this agent (10 microliters) effect upon nociceptive or motor function after IT injection or on EEG and general behavior after ICV injection in rats. Using 20% CDEX, there is no change in the ED50 as compared to saline on the hot plate (HP) after IT injection of morphine, D-Ala2-D-Leu5 enkephalin or Tyr-Aib-Gly-gPhe-mAib-NH2, (Aib: alpha aminoisobutyric acid) although there is an increase in their respective durations of effect. Cyclic peptide opioids: Tyr-c[D-A2bu-Gly-D-beta Nal(1)-D-Leu] (A2bu: alpha, gamma-diaminobutyric acid; beta-Nal(1): beta-naphthylalanine(1)) or Tyr c[DA2bu-Gly-beta Nal(1)-D-Leu] are insoluble in saline but are readily dissolved in CDEX, and display a naloxone-sensitive antinociception following spinal administration. In other studies, saline insoluble capsaicin is administered in 25% dimethylsulfoxide (DMSO) or 20% CDEX (15 microliters; 5 mg/ml) which result in a significant reduction in the spinal levels of substance P and calcitonin gene related peptide and an increase in the HP latency. DMSO alone, but not CDEX alone, reduces the levels of the two peptides. These data emphasize the utility of complexation with CDEX for intracerebral drug delivery and compatibility with brain and spinal tissue. PMID- 1703621 TI - Health care financing for severe developmental disabilities. AB - The 1985-86 data from 308 children and young adults under age 25 with autism and from 326 with severe or profound mental retardation can be compared to national data from the 1980 MNCUES and the 1987 NMES because the methods are similar. These data provide detailed answers to the questions, what health care services are used? what are the expenses? Who pays them? Until now, the absence of comprehensive national data had hindered the development of new approaches to financing the care of children with serious, lifelong conditions. These data permit policymakers to take into account the needs and expenditures for severely developmentally disabled children when reforming the health care financing system. None of the children or young adults had expenditures in excess of $50,000, and very few reached the upper $20,000s. For children with autism the average annual health care expenditure was about $1,000 and about $1,700 for young adults, compared to the $414 average for all American children. They received an average of four physician visits annually, slightly above the U.S. average for children. Their hospitalization rate was twice the average for children. Hospitalization accounted for one-third the health care expenditures among children with autism, but for two-thirds among young adults. For children and young adults with severe retardation the average expenditure on health care was about $4,000, due to the physical impairments in two thirds of the children. They averaged about 12 physician visits annually, falling to 8 among young adults. Children were hospitalized about eight times the national rate, and young adults about twice. Among severely retarded children and young adults living at home, hospitalization accounted for over half the health care expenses, but for only one third for those in residential placement. Unfortunately, preventive and habilitative services were but a tiny fraction of health care expenditures and were demonstrably underutilized. Only 60% of these children had routine dental examinations within the last 12 months, a worse record than the average child. For the individuals whose primary physicians judged that they would benefit from physical or speech therapy, less than one quarter were receiving them. Care for seriously, chronically disabled children places great burdens on immediate family members. Only 20% of the severely retarded youngsters from age 10 to 24 could be left alone at home, even for a few minutes, and only 30% of the autistic ones. These developmental disabilities create needs for personal care and family support that traditionally have not been considered health services.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1703622 TI - Induction of an immune response to the porin of Haemophilus influenzae type b by monoclonal anti-idiotypic antibodies. AB - Monoclonal anti-idiotypes were generated against monoclonal antibody (mAb) Hb-2 which recognized a highly conserved epitope on the outer membrane porin protein from Haemophilus influenzae type b (Hib). Four hybridomas reacting with F(ab') 2 fragments of Hb-2 were selected and characterized. Inhibition studies using syngeneic anti-anti-idiotypic antisera suggested that at least three different antigenic determinants on Hb-2 were recognized by these monoclonal anti idiotypes. The binding of each anti-idiotype to Hb-2 was inhibited by Hb-2 whereas the reaction was not affected by any other anti-Hib mAb. Complete inhibition of the binding of anti-idiotype to the idiotype could be achieved with 10 micrograms of total outer membrane protein (OMP) from Hib suggesting that the anti-idiotypes might be directed against paratope-associated idiotypes. Outer membrane antigens not recognized by mAb Hb-2 did not inhibit the reaction. Furthermore, the pre-incubation of Hb-2 with each anti-idiotype specifically prevented the reaction of Hb-2 with its antigen. Antibodies with specificity for the porin were generated in guinea pigs immunized with anti-idiotypes AHb-22 and AHb-23. This study indicates that these particular monoclonal anti-idiotypes may be used as an antigen substitute for the porin of Hib in a xenogeneic species. PMID- 1703623 TI - Neovascularisation of the epiphysis. AB - Two experiments in the rabbit model have been carried out to see if a vascular pedicle placed in a growing epiphysis would establish new capillaries. In one, the vascular pedicle of peroneus longus muscle was placed in the proximal fibula epiphysis. In the other, the proximal fibula was excised and the epiphysis placed across the saphenous artery and vein in the groin. After 3 months, histological examination of the epiphyses in both experiments showed that no new vessels had grown from the vascular pedicle. Cartilage is known to inhibit vascularisation, and these results suggest that attempts to revascularise the epiphysis, for example, following Perthes' disease, may fail. For the same reason, attempting to supplement the epiphyseal vessels by implanting a new vessel before or after epiphyseal transfer may fail. PMID- 1703624 TI - Renal cell carcinoma in children: histology, immunohistochemistry, and follow-up of 10 cases. AB - Ten renal cell carcinomas in children under 15 years were investigated. The average age was 122.5 months and the girls predominated in our cases (7 girls, 3 boys). By using the classification of Thoenes et al., Pathol Res Pract 181: 125 143, 1986 a predominance of clear cell-eosinophilic tumor cell type and of the tubulopapillary growth pattern was found. Immunohistochemistry revealed a heterogeneity of cytokeratin expression. By using the monoclonal antibodies Cam 5.2 and KL 1, cytokeratins were found in 7 cases each. The other 4 cytokeratin antibodies used were less sensitive. The expression of cytokeratin 13 in 3 cases suggested a more complex histogenesis than assumed. Vimentin was found in 3 tumors, but an association to a higher grade (G) of malignancy was not found in these cases. One tumor expressed the Tamm-Horsfall-protein, which is predominantly found in the distal tubule of the normal kidney. In summary the results of immunohistochemistry characterized the great heterogeneity of these tumors. Follow-up information was available in 9 cases. All patients with G I- and G II-tumors were free of disease after an average time of 39.6 months (mean 27 months). Two of the 3 cases with G III-tumors died after 9 and 15 months, despite additional chemo- or radiotherapy. Therefore tumors of grade I and II of the Thoenes classification seem to have a good prognosis. PMID- 1703625 TI - [Relationship between perineural invasion and local recurrence of rectal carcinoma: a preliminary study with immunohistochemical staining with anti-NCAM: preliminary report]. PMID- 1703626 TI - One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose. AB - The key regulatory step in starch biosynthesis is catalyzed by the tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase). In leaf and storage tissue, the enzyme catalyzes the synthesis of ADP-glucose from glucose-1-phosphate and ATP. Using heterologous probes from maize, two sets (B and S) of cDNA clones encoding potato AGPase were isolated from a tuberspecific cDNA library. Sequence analysis revealed homology to other plant and bacterial sequences. Transcript sizes are 1.9 kb (AGPase B) and 2.1 kb (AGPase S). Northern blot experiments show that the two genes differ in their expression patterns in different organs. Furthermore, one of the genes (AGPase S) is strongly inducible by metabolizable carbohydrates (e.g. sucrose) at the RNA level. The accumulation of AGPase S mRNA was always found to be accompanied by an increase in starch content. This suggests a link between AGPase S expression and the status of a tissue as either a sink for or a source of carbohydrates. By contrast, expression of AGPase B is much less variable under various experimental conditions. PMID- 1703627 TI - Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene. AB - The gene coding for starch phosphorylase (EC 2.4.1.1) was isolated from a potato genomic library constructed in lambda EMBL3. It is an unusually long plant gene (16.4 kb) which encodes a preprotein of 966 amino acids. The phosphorylase coding sequence is interrupted by 14 introns whose positions do not match those of the introns in the human glycogen phosphorylase gene. A 78 amino acid central peptide unique to plant plastidial phosphorylases is hypothesized to have arisen through the mis-splicing of an intron-exon junction site in an ancestral gene. The fifth intron of the phosphorylase is very large (approximately 7 kb) and contains a copia-like transposable element inserted in the opposite orientation to that of the phosphorylase gene. This element has been named Tst1; it is bordered on the 5' and 3' sides by long terminal repeats of 285 and 283 bp respectively, which define an internal domain of 4492 bp. Tst1 contains 4 open reading frames (ORFs) that encode protein domains for a reverse transcriptase, an integrase, an RNA binding site and a protease. Transcription of the phosphorylase gene appears to proceed unimpaired through the copia element. PMID- 1703628 TI - A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. AB - Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor. PMID- 1703629 TI - mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities. AB - The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes. PMID- 1703630 TI - A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase. AB - Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells. PMID- 1703631 TI - Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells. AB - The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways. PMID- 1703632 TI - The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins. AB - The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses. PMID- 1703634 TI - Clinical application of stable xenon CT-CBF studies without denitrogenation. AB - Noninvasive and simplified methods for estimating regional cerebral blood flow (CBF) and regional partition coefficient (lambda) using the inhalation of stable xenon (Xes) and computed tomographic (CT) scanning are described. Thirty percent Xes in 70% oxygen was inhaled for 240 seconds and exhaled for 160 seconds during serial CT scanning without denitrogenation in 26 patients with cerebrovascular diseases and four volunteer controls. During the investigation, the end-tidal Xes concentration was continuously monitored with a thermoconductivity analyzer to determine the build-up range (A value) and build-up rate constant (K value) of the artery by the curve fitting method. Calculated A and K values were corrected by the following formulae reported previously: for patients aged 0-20 years, Ae = 0.75Aa + 2.15, Ke = 0.67Ka + 0.69; 21-40 years, Ae = 0.56Aa + 3.24, Ke = 0.38Ka + 1.12; 41-60 years, Ae = 0.91Aa + 1.95, Ke = 0.38Ka + 1.32; over 61 years, Ae = 0.52Aa + 3.81, Ke = 0.31Ka + 1.55 (Ae and Ke were calculated with end-tidal Xes concentration, Aa and Ka were calculated by direct sampling of arterial blood). A CBF map (f map) and lambda map made with corrected A and K values demonstrated reliable distribution. The CBF was high in the gray matter, low in the white matter, and much lower in the infarcted area. lambda was high in the white matter, low in the gray matter, and much lower in the infarcted area. Eight patients were examined with and without denitrogenation. Both the f map and lambda map with denitrogenation were compatible with those without denitrogenation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703633 TI - Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein. AB - GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway. PMID- 1703635 TI - New screening system for unruptured cerebral aneurysms--combination of an expert system and DSA examination. AB - We have designed a screening system to diagnose unruptured aneurysms, including the use of digital subtraction angiography (DSA). We surveyed 115 patients who had undergone clipping procedures after subarachnoid hemorrhage (SAH) and questioned them with regard to the subjective symptoms. Sixty-eight of 92 patients who returned the questionnaire reported, prior to rupture, headache, eye pain, and neck pain most frequently, and also impairment of extraocular movements, ptosis, visual field defects, and motor and sensory disturbances. Nineteen (47.5%) of 40 patients who had complete pain relief after surgery complained of headache from 1 week to 1 month before SAH. In addition, nine patients (22.5%) complained of headache for several years, and were also pain free after surgery. For the indication of DSA, we employed an expert system based on fuzzy set theory. Seven groups of parameters are: Group 1, a basic questionnaire concerning age, sex, and past and family histories; Group 2, 15 warning signs selected on the basis of retrospective study; and Groups 3-7, detailed questions concerning each sign. Scoring weights assigned to each condition based on the results of the retrospective study, and threshold values were determined by several neurosurgeons. The certainty factors for intermediate hypotheses were calculated from these weights and threshold values and summed up, from which the conclusion was obtained. Twelve new cases of unruptured cerebral aneurysm were diagnosed using this screening system. This system may improve the ability to diagnose cerebral aneurysms before rupture. PMID- 1703636 TI - Intra-arterial ACNU chemotherapy employing 20% mannitol osmotic blood-brain barrier disruption for malignant brain tumors. AB - The clinical effects and problems of intra-arterial water-soluble antitumor nitrosourea (ACNU) therapy following osmotic blood-brain barrier modification are discussed. Twenty-one patients with malignant brain tumors were divided into two groups. Group 1 consisted of 16 patients treated by operation, irradiation, and two or more courses of intracarotid infusion of ACNU 100 mg/body (1.7-2.2 mg/kg) following 20% mannitol 200 ml (1.3-1.6 ml/sec) (7 grade 4 astrocytomas, 5 grade 3 astrocytomas, and 4 others). Group 2 consisted of five patients treated by operation, irradiation, and repeated intracarotid infusion of ACNU 100 mg/body alone (grade 4 astrocytoma). The 2-year survival rate in Group 1 was 79% (11 of 14 cases followed up for longer than 2 years) and the 3-year survival rate was 67%. Five of seven grade 4 astrocytoma patients (71%) in Group 1 survived for more than 1 year 6 months, whereas four of five grade 4 astrocytoma in Group 2 died within 1 year 6 months. The measurement of the ACNU concentration in tumor tissues and blood in 11 brain tumors, after intracarotid infusion of ACNU with blood-brain barrier disruption, showed peak values in the tumor tissues of 3.02 32.53 micrograms/gm (mean, 9.67 micrograms/gm), about three to five times as high as that in blood in most cases. This method used in Group 1 appears to be relatively safe without permanent neurological deficits and offers a potential therapeutic effect when used in combination with appropriate premedication in suitable patients. PMID- 1703637 TI - Follow-up study on metastatic cerebellar tumor surgery--characteristic problems of surgical treatment. AB - Recently, the incidence of metastatic cerebellar tumors has increased. The authors operated on 12 cases of metastatic cerebellar tumors, with total or subtotal removal of nodules in brain metastases. Surgical complications observed as a result of postoperative investigation are presented. 1) Some cases developed carcinomatous meningitis within a short period. During removal of a tumor on the superior cerebellar surface attention should be paid to the prevention of dissemination to the cerebral cisterns adjacent to the tumor. 2) Some cases demonstrated peritoneal metastasis probably due to dissemination via the ventriculoperitoneal shunt tube, suggesting that great care should be taken during a shunt operation after removal. 3) Both carcinomatous meningitis occurring after removal and remote metastasis via the shunt tube were related to recurrence after removal of the cerebellar metastatic lesion, raising the issue of whether or not total macroscopic removal should be included in the indications for surgical treatment of cerebellar metastasis. Those cases in which surgery is indicated should also be routinely treated by postoperative irradiation. PMID- 1703638 TI - Venous angioma: follow-up study and therapeutic considerations. AB - Ten patients with cerebral venous angioma (VA) were followed up for 12 to 106 months. Seven VAs were found as a result of intracerebral hemorrhage and the others were found incidentally. Among three VAs cauterized or partially excised, one disappeared but two were unchanged on follow-up angiography. Another VA, treated by irradiation following evacuation of the hematoma, gradually reduced in size on angiography. In the remaining six VAs treated conservatively, follow-up angiography demonstrated no visible change. During the follow-up period, bleeding from VA was encountered in one patient who had previously suffered from intracerebral hemorrhage. Prevention of bleeding from VA is considered important; however, complete extirpation of VA is difficult since the resectable area of normal brain parenchyma including the VA is very limited. From our experience, radiation therapy is believed to be useful when VA is considered to carry the risk of hemorrhage. PMID- 1703639 TI - Clinicopathological study of multiple gliomas--report of three cases. AB - Three cases of multiple gliomas with postmortem findings including a rare case of multicentric glioma are presented. A 59-year-old female was hospitalized with decreased mental activity and gait disturbance. Computed tomographic (CT) scans and magnetic resonance (MR) images showed two independent mass lesions in the left frontal and the right temporal lobes, shown by postmortem to have no communication. Histologically, they were a gemistocytic astrocytoma and an anaplastic astrocytoma, respectively. Therefore, multicentric glioma was diagnosed. A 66-year-old male was admitted with slow mentation and gait disturbance. CT scans and MR images demonstrated two mass lesions; one overriding the bilateral frontal lobes through the corpus callosum and the other in the left temporal lobe. Postmortem examination showed that both lesions were glioblastoma and the left temporal tumor was accompanied by subarachnoid dissemination. A 29 year-old male was hospitalized with gustatory hallucination and convulsions of the right upper extremity. CT scans revealed two mass lesions in the right frontal and the left temporal lobes. MR images demonstrated communication between the two lesions through the corpus callosum. The left temporal tumor developed into the occipital lobe and another new lesion appeared in the right temporal lobe despite chemotherapy and irradiation. Postmortem examination revealed communication between the three masses through the corpus callosum. Histologically, all three tumors were glioblastoma. Multicentric gliomas have been reported at various incidences from 2.3 to 9.1%. However, multicentric gliomas with multiple tumors of different histologies are very rare and only 16 cases have been reported. MR imaging is more valuable than CT scanning to detect communication between two or more lesions. PMID- 1703640 TI - Intracranial hemorrhage associated with nontraumatic disseminated intravascular coagulation--report of four cases. AB - The authors report four cases of intracranial hemorrhage associated with nontraumatic disseminated intravascular coagulation (DIC). Two cases demonstrated a sudden onset of intracerebral hemorrhage. The other two showed chronic subdural hematoma initially, followed by acute multiple intracranial hemorrhages or general hemorrhagic diathesis. The underlying disorders were glioblastoma multiforme, thoracoabdominal aortic aneurysm, acute promyelocytic leukemia, and stomach cancer associated with disseminated carcinomatosis of the bone marrow. All patients died eventually. When the underlying disorder has a rare incidence of DIC as in glioblastoma multiforme or thoracoabdominal aortic aneurysm, the possibility of DIC and the need for immediate initiation of replacement therapy should be recognized, although the mortality is very high because the underlying disorder cannot be eliminated quickly. When the underlying disorder has a high incidence of DIC as in acute promyelocytic leukemia or disseminated carcinomatosis of the bone marrow, it is mandatory to start replacement therapy and treatment for the underlying disorder simultaneously. DIC can be controlled when the treatment for the underlying disorder is effective. PMID- 1703641 TI - Aspergillosis of the central nervous system causing subarachnoid hemorrhage from mycotic aneurysm of the basilar artery--case report. AB - The authors present an extremely rare case of aspergillosis of the central nervous system (CNS) causing subarachnoid hemorrhage (SAH). A 78-year-old female developed facial pain, progressive deterioration in left visual acuity, and left total ophthalmoplegia. Computed tomography demonstrated a heterogeneously enhanced mass extending from the sphenoid sinus to the left cavernous sinus and left orbit, and angiography showed luminal narrowing and irregularity of the left internal carotid artery at its siphon. Biopsy of the left orbital and sphenoid sinus mass resulted in the diagnosis of Aspergillus granuloma. Despite combined administration of amphotericin-B and 5-FC, she became comatose from brainstem infarction and finally, suddenly died. Postmortem examination revealed massive SAH due to a ruptured mycotic aneurysm of the basilar artery. Aspergillosis of the CNS is a growing problem with the wider use of immunosuppressants and antibiotics. To the authors' knowledge, however, only 13 cases of CNS aspergillosis causing SAH have been reported. The prognosis is absolutely bad, with all patients dying from rupture of major intracranial arteries such as the internal carotid artery and basilar artery. Early diagnosis and vigorous chemotherapy are important. PMID- 1703642 TI - Multiple intracerebral arteriovenous malformations in deep structure--case report. AB - The authors describe a case of multiple intracerebral arteriovenous malformations (AVMs) in a 23-year-old male with two distinct, deep-seated AVMs. One was located in the left basal ganglia, which had bled twice, and the other in the splenium. They were removed separately. He recovered satisfactorily with only a mild dysphasia. The authors emphasize that the therapeutic principle for multiple AVMs is the same as that for a solitary AVM. Multiplicity alone does not dictate the operability. Dissection just adjacent to the nidus and direct coagulation of an AVM are the indicated techniques, especially in cases of deep-seated AVMs in order to reduce postoperative neurological deficit. PMID- 1703643 TI - Mixed pituitary adenoma and gangliocytoma associated with acromegaly--case report. AB - A gangliocytoma in the sellar region is very rare. We report a case of an intrasellar gangliocytoma complicated by pituitary adenoma presenting with acromegaly. A 52-year-old female was admitted to our hospital with headache, mild acromegaly, and bitemporal hemianopsia, and endocrinological study found a high serum level of growth hormone (GH). A computed tomographic scan revealed a tumor in the sellar region, which was almost totally removed by trans-sphenoidal surgery. Histological examination of the resected specimen showed diffuse, chromophobe-type pituitary adenoma, partially containing cholesterin clefts. Areas of clusters of dysmorphic neurons, adjacent to or mixed with pituitary adenoma, were diagnosed as gangliocytoma. The immunohistochemical examination showed GH-releasing hormone (GRH)-positive dysmorphic neurons and GH-positive pituitary adenoma. We consider that the trophic effect of GRH secreted by the neurons of GRH-producing intrasellar gangliocytoma probably caused the GH producing pituitary adenoma. PMID- 1703644 TI - Immunochemical identification of vasopressin in neurons and granule-containing cells of blood vessels of the human brain. PMID- 1703645 TI - The participation of mediator and peptidergic systems of the brain in conditioned reflex mechanisms. AB - New data on the significance of mediator and peptidergic systems of the striatal level in the organization of alimentary conditioned reflexes are presented in this report. The role of acetylcholine-, dopamine-, GABA-and P-ergic systems of the caudate nucleus and the amygdala in the realization of positive and negative conditioned reflexes was investigated. The experiments were carried out on dogs with chemotrodes and microelectrodes implanted in subcortical structures. The results of the experiments with microinjections of the relevant substances into individual subcortical structures showed that activation of the same mediator system in various structures may lead to both unidirectional and multidirectional behavioral effects. On the other hand, the activation of various subcortical mediator systems can lead to identical changes in conditioned reflex activity. The effect of the administration of activators or blockers of a subcortical mediator system depends in many ways on the functional state of the nervous system at the moment of administration and on the localization of the microinjection. It is difficult to predict beforehand the role of various subcortical structures in the organization of integrated behavioral acts. The question of the necessity of studying mediator and peptidergic systems of each subcortical structure in order to understand their significance in the mechanisms of the conditioned reflex is raised. PMID- 1703646 TI - Management of unresectable malignant esophageal obstruction. AB - Malignant esophageal obstruction (MEO), especially with esophago-respiratory fistula (ERF), requires efforts to achieve a meaningful degree of palliation. Laser vaporization (LV) of the esophageal tumor and placement of an endoesophageal prosthesis (EEP) represents a new combination for palliation of MEO. The purpose of the present study was to evaluate the neodymium-yttrium aluminum-garnet (Nd:Yag) laser in reopening the esophageal channel to permit both swallowing and insertion of an EEP. Twenty-three consecutive patients with MEO were evaluated, and ERF was documented by preoperative contrast study in eight patients. All 23 patients underwent laser vaporization, dilation, and surgical placement of EEP. Adequate swallowing was attained in 21 patients; one patient with an ERF experienced recurrent aspiration from failure of the EEP to occlude the fistulous tract. Operative morbidity was 17% (4/23), which included: wound infection, 2; persistence of ERF, 1; esophageal perforation, 1; and food impaction, 1. Thirty-day operative mortality was 9% (2/23). Palliation was excellent in 87% (20/23), with discharge from the hospital by the seventh postoperative day. Mean survival was 3.3 months. We conclude that laser vaporization followed by placement of an EEP represents a major advance in the palliation of MEO. PMID- 1703647 TI - Cortical laminar distribution of rat thalamic ventrolateral fibers demonstrated by the PHA-L anterograde labeling method. AB - The cortical innervation pattern of rat thalamic ventrolateral (VL) fibers was investigated with the Phaseolus vulgaris-leukoagglutinin (PHA-L) anterograde labeling method. The PHA-L-labeled terminals were distributed in the sensorimotor cortex where the cerebello-cerebral (C-C) evoked responses were recognized. The labeled terminals were detected in all layers except layer VI. In the upper part of layer I, the most densely packed terminals were formed and moderately labeled terminals were seen in layers IV and V. Such a laminar distribution of PHA-L labeled VL terminals is consistent with the profiles of the C-C responses according to laminar field potential analysis. PMID- 1703648 TI - A rating scale to evaluate research posters. AB - Many nursing conferences include research reports in the form of oral reports, symposia discussions, and poster presentations. To assist faculty, students, and clinicians in the systematic preparation and critique of poster presentations, the author discusses her 30-item research-poster appraisal tool (R-PAT). The tool assists nurses in preparing nursing research posters, guides nursing faculty in grading research posters that are a class assignment, and lets viewers systematically critique posters. PMID- 1703649 TI - Correlation between histology and nerve excitability after reinnervation of paralyzed strap muscles in the rabbit. AB - We have recently shown that the mean muscle chronaxie for nerve pedicle implanted into denervated rabbit strap muscle is comparable to that of normal nerve. This study correlates excitability with histologic characteristics of muscles reinnervated via nerve-muscle pedicles (NMP) and direct nerve implants (DNI). Strength duration curves were measured in 13 rabbits 3.5 to 5 months after reinnervation by NMP (n = 6) and DNI (n = 7). Following this, control (n = 5) and reinnervated straps were harvested immediately before the animals were killed and frozen in liquid nitrogen. The material was submitted for hematoxylin-eosin stains as well as trichrome stains for general morphology, myofibrillar ATPase and NADH for fiber typing, and cholinesterase for determination of denervated fibers. In all animals with low chronaxie, expected type grouping from reinnervation was noted (n = 10). By contrast, the three animals in which chronaxie was abnormally elevated demonstrated fibrosis, inflammation, and absence of or poor type grouping. This suggests that type grouping is necessary for excitability after reinnervation of paralyzed striated muscles. PMID- 1703650 TI - Distribution of neuropeptide-like immunoreactive nerve fibers in the canine larynx. AB - The distribution of neuropeptide immunoreactive nerve fibers in the canine larynx was examined. In the epithelium of supra- and subglottic regions, a dense distribution of substance P (SP)- and calcitonin gene-related polypeptide (CGRP) immunoreactive (IR) nerve fibers was observed. Some vasoactive intestinal polypeptide (VIP)-IR intraepithelial nerve fibers were also seen in the subglottic region. In the laryngeal glands, a dense distribution of VIP-IR nerve fibers with a few SP- and enkephalin (ENK)-IR nerve fibers were found around the acini. In the walls of arteries in the lamina propria, many VIP-, SP-, and CGRP IR nerve fibers were seen, whereas neuropeptide Y-, ENK-, and VIP-IR nerve fibers were predominantly distributed around the arteries in the vocal muscle. In the free edge of the vocal cord, few immunoreactive nerve fibers were detected within the epithelium and around the arteries in the lamina propria. These results suggest that there are regional differences in the occurrence of peptides in nerve fibers innervating the epithelium and the blood vessels in the larynx and that the perception mechanism of the epithelium and the regulatory system of local blood flow are varied according to their location in the larynx. PMID- 1703651 TI - Decreased local toxicity with subcutaneous diamorphine (heroin): a preliminary report. AB - We report the cases of 5 patients who developed severe local toxicity during the subcutaneous administration of morphine sulphate and hydromorphone hydrochloride. All patients required site changes more frequently than once every 24 h due to redness, swelling, or pain while receiving morphine or hydromorphone. All patients showed prolongation in the duration of sites of infusion once an equianalgesic dose of diamorphine hydrochloride (heroin) was started. No change in pain control or systemic toxicity was detected with diamorphine. These findings suggest that diamorphine could be a useful alternative for patients who develop severe toxicity to subcutaneous morphine or hydromorphone. PMID- 1703652 TI - Biologic and immunomodulating factors in the treatment of pediatric acquired immunodeficiency syndrome. PMID- 1703653 TI - [Diagnostic usefulness and prognostic value of inhibitory capacity and other biochemical parameters of plasma in acute pancreatitis in humans]. AB - Assessment of the degree of severity of acute pancreatitis by means of biochemical parameters is still a subject of extensive studies. The purpose of the present study was a trial of evaluation of the diagnostic usefulness and prognostic value of certain tests for proteinase-anti-proteinase equilibrium and acute phase indices in acute pancreatitis in 52 patients (36 women and 16 men aged 22 to 83 years). The control group compared 29 healthy volunteers. The patients were classified according to the aetiology of the disease: pancreatitis connected with bile duct disease, alcoholic and non-classifiable, and another classification was based on the clinical course (medium severe, severe). Significantly higher concentration of immunoreactive trypsin (TLI) was found in the first two weeks in the group of severe disease as compared to the group with medium severe pancreatitis (p less than 0.01). TLI was significantly higher than in controls for 2 months after clinical recovery. The serum inhibitory capacity was significantly reduced in severe cases in relation to medium severe ones, particularly between 3 and 7 days of the disease (p less than 0.001). Similarly as trypsin concentration, reduced inhibitory capacity in relation to controls persisted for up to 2 months after pancreatitis. No significant differences were noted in the concentration of alpha 1 protease inhibitor and C3 and C4 complement components, in the studied groups. The serum alpha 2 macroglobulin level was significantly decreased between days 3 and 7 (p less than 0.05). The values of alpha 2 macroglobulin were correlated in that time with the values of the inhibitory capacity. PMID- 1703654 TI - [Verification of therapeutic levels of procainamide and N-acetyl- procainamide in ventricular arrhythmia]. AB - Therapeutical efficacy was clinically evaluated in 21 patients with ventricular cardiac arrhythmias. The drug was given orally with preceded intramuscular dose. Therapeutic effect was verified by the measurements of procainamide and N acetylprocainamide concentrations in blood serum to determine the minimal effective concentration of the drug required to obtain satisfactory antiarrhythmic effect. Procainamide proved effective in cardiac arrhythmias in 14 patients (66.7%) with statistical significance in the acute myocardial infarctions; blood serum procainamide plus N-acetylprocainamide levels being were below the therapeutical range. The poor correlation of the dose of the drug and respective procainamide, N-acetylprocainamide concentrations in blood was observed. Relationship of the therapeutical effects blood serum level of the drug should be estimated basing of the assays of both procainamide and N acetylprocainamide . PMID- 1703655 TI - [Evaluation of biological availability and anti-arrhythmic effect of a new drug Phenytoinum "Polfa"]. AB - Studies were performed in 15 patients with ventricular arrhythmia. During the first day, the patients received 1000 mg of a new micronised form of Phenytoinum "Polfa" or adequate dose of a foreign drug in 3 doses every 3 hours and subsequently during 10 days alternatively native or foreign drug in a daily dose 300 mg. Twenty-four EKG Holter monitoring and determination of serum drug level were carried out after a 10-day treatment; area under the curve (AUC) in one 8 h dose interval was determined. Studies have shown usefulness of a new form of Phenytoinum (Polfa). Blood serum drug levels near to the therapeutic ones were observed. Steady-state Phenytoinum concentration was 11.1 +/- 5.9 micrograms/ml and after foreign drug it was 11.7 +/- 6.1 micrograms/ml, AUC0-8 was 90.4 and 105.3 micrograms/ml/h respectively. In 9/15 patients (60%) Phenytoinum (Polfa) produced substantial improvement in the cardiac arrhythmia. PMID- 1703656 TI - Development factors in the contractile response of the rabbit bladder to both autonomic and non-autonomic agents. AB - Previous work in this laboratory has demonstrated that the bladders of 1-day-old and 1-week-old rabbits generate higher pressures in whole-bladder preparations than bladders from mature 8-week-old rabbits. In addition, the density of cholinergic receptors does not change during this maturation period. The present study was designed to determine if the increased responsiveness of the neonatal bladder was specific for cholinergic stimulation. Using bladder strips, we have demonstrated that the newborn bladders generated much greater tension in response to ATP, serotonin, histamine, and substance P. The response of the 1-day-old bladder smooth muscle to these contractile agents was at least double the response of the 8-week-old bladders. However, the response of all age groups to bethanechol was approximately the same, and the response to KCl was only 40% greater in the 1-day-old bladders as compared to the adult. These current studies indicate that the newborn bladder responds to a variety of nonadrenergic, noncholinergic agonists to a significantly higher degree than the adult bladder, and that maturation is accompanied by substantial changes in the pharmacology of the bladder. PMID- 1703657 TI - [The results of hypofractionated irradiation in patients with bronchogenic carcinoma]. AB - Resulting from the declared palliative aim and from time economic and tumor affecting high dosed single fraction meanwhile a hypofractionated irradiation rhythm was applied to more than 500 patients with lung carcinoma and 5 x 5 Gy were given in daily succession if possible. A hospitalization was necessary in a third of the patients. In spite of histology (NSCLC: 46%, SCLC: 35%) a mean survival of 12 months was attained, in which the fate is determined for 75% of the patients within the first year after therapy. Detrimental effects were not provoked by the applied irradiation mode. PMID- 1703658 TI - Comparative analysis of West Nile virus strains isolated from human and animal hosts using monoclonal antibodies and cDNA restriction digest profiles. AB - Three West Nile (WN) virus strains isolated in Bangui, Central African Republic (CAR), from patients with hepatitis were analysed comparatively with the prototype WN virus strain and 7 WN strains previously isolated from birds (2 strains), mosquitoes (3 strains) and ticks (2 strains) in CAR. The comparison was based on two techniques: an epitopic analysis by indirect immunofluorescence assay using a panel of 9 monoclonal antibodies to WN virus, and an analysis of HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA. Similar results were obtained with both techniques: the 3 human strains were found to be identical to each other and identical or very close to mosquito and tick strains, whereas prototype WN virus and bird strains were significantly different from the human strains. As "classical" infections due to WN virus without hepatic involvement were also reported during the period of isolation of the arthropod strains, we concluded that the same virus subtype may have been the cause of different infection patterns. A new definition of the disease spectrum of WN virus, including the possibility of liver involvement, should be established. Clearly, the Egyptian prototype WN virus represents a different topotype. Bird strains also appear to be different from human and arthropod strains, raising the question of their transmissibility and pathogenicity for man, and of the role of birds in the natural cycle of WN virus. PMID- 1703659 TI - [An endodermal sinus tumor (yolk sac tumor) of the anterior mediastinum]. AB - An own case of a primary mediastinal endodermal sinus tumour (yolk sac tumour) is presented that was transformed under therapy into an AFP-negative process and occurred combined with an arcus aortae dexter. This is followed by a description of the clinical and diagnostic patterns of signs and symptoms and of the treatment of this very rare dysontogenetic tumour. PMID- 1703660 TI - Cystic fibrosis transmembrane conductance regulator: nucleotide binding to a synthetic peptide. AB - Multiple mutations in the gene responsible for cystic fibrosis are located within a region predicted to encode a nucleotide-binding fold in the amino terminal half of the cystic fibrosis transmembrane conductance regulator protein. A 67-amino acid peptide (P-67) that corresponds to the central region of this putative nucleotide binding site was chemically synthesized and purified. This peptide bound adenine nucleotides. The apparent dissociation constants (Kd's) for the trinitrophenyl (TNP) adenine nucleotides, TNP-adenosine triphosphate, TNP adenosine diphosphate, and TNP-adenosine monophosphate, were 300 nanomolar, 200 nanomolar, and greater than 1 micromolar, respectively. The Kd for adenosine triphosphate was 300 micromolar. Circular dichroism spectroscopy was used to show that P-67 assumes a predominantly beta sheet structure in solution, a finding that is consistent with secondary structure predictions. On the basis of this information, the phenylalanine at position 508, which is deleted in approximately 70 percent of individuals with cystic fibrosis, was localized to a beta strand within the nucleotide binding peptide. Deletion of this residue is predicted to induce a significant structural change in the beta strand and altered nucleotide binding. PMID- 1703661 TI - ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43 kildalton protein. AB - Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein. PMID- 1703662 TI - [AIDS. The final challenge]. PMID- 1703663 TI - [Effect of capsaicin on the release of substance P from spinal cord and blood pressure]. AB - The role of substance P (SP) in the regulation of sympathetic outflow to the cardiovascular system was assessed. Intrathecal injection(ith) of capsaicin caused a release of SP from the spinal cord and resulted in an increase in blood pressure and heart rate accompanied by an elevation of plasma adrenaline and noradrenaline. This pressor response was blocked by ith SP antagonist D-Pro2, D Phe7, D-Trp9-SP or antiserum against SP. The immunohistochemical study showed that the SP-like immunoreactivity in T-8 of the spinal cord was decreased as the amount of capsaicin administrated was increased. Spinal cord transection had no effect on pressor responses caused by ith 10 micrograms capsaicin. These results suggest that SP transmits excitatory information to the cardiovascular system via spinal sympathetic pathway. PMID- 1703664 TI - [Studies on the mechanism of gastric mucosal injury induced by water-immersion stress in rats]. AB - Changes in the degree of gastric mucosal injury, gastric acid output, secretion of gastric barrier mucus and gastric motility were made in 5 groups of rat: I, Restraint alone plus saline under room temperature; II, Water-immersion plus saline; III, Water-immersion plus atropine (0.5 mg/kg); IV, Water-immersion plus phenoxybenzamine (10 mg/kg); V, Water-immersion plus pentobarbital (30 mg/kg). The results were as follows: (1) Water-immersion stress resulted in severe gastric mucosal lesions, an increase in the gastric acid output, a decrease in the secretion of gastric barrier mucus and an increase in gastric motility. (2) Atropine attenuated gastric mucosal injury significantly, inhibited gastric acid output and gastric motility, while gastric barrier mucus was increased. (3) Sodium pentobarbital also significantly attenuated gastric mucosal injury and inhibited gastric motility, but had no effect on gastric acid output. (4) Phenoxybenzamine failed to affect on gastric mucosal injury, gastric acid output, gastric barrier mucus secretion and gastric motility. These results suggest that the gastric hypercontractility, the reduction of gastric barrier mucus and the increase in gastric acid output participate in varying degrees in the formation of gastric mucosal injury induced by water-immersion stress, but when gastric motility is inhibited and gastric barrier mucus increases, only the existence of gastric acid can not induce severe gastric mucosal injury. PMID- 1703665 TI - Percutaneous transcatheter arterial embolization of bone and soft tissue tumors. AB - Arterial embolization was performed in 36 patients with tumors of bone and soft tissue. Embolization was the only treatment in seven patients with benign lesions. Fourteen patients underwent embolization before surgery to obtain hemostasis and/or reduce tumor size. Fifteen patients with inoperable primary bone tumors or skeletal metastases underwent palliative embolization. The best results were obtained in aneurysmal bone cysts. PMID- 1703666 TI - Meniscal repair following meniscectomy: mechanism and protective effect. Experimental study in the dog. AB - Meniscal repair was studied to evaluate the mechanism and its potential protective effects on the articular cartilage in an experimental model consisting of 68 knees of adult dogs on which five different types of medial meniscectomy were performed. The results were assessed by macroscopic, microangiographic, and histological methods, after a sequential follow-up period of 10-450 days. Two different mechanisms of meniscal repair were observed, depending on whether meniscal section had been performed in vascular (total meniscectomy) or avascular (subtotal or partial meniscectomy) zones. It was also observed that the repaired meniscal tissue does not prevent articular cartilage degeneration. This is more closely related to the size of the meniscal fragment preserved at meniscectomy. Due to the biomechanical importance of the meniscus and the lack of functional relevance of the repaired meniscal tissue, the most conservative approach possible to meniscectomy is recommended. PMID- 1703667 TI - Human papillomavirus in prostatic cancer: no evidence found by in situ DNA hybridization. AB - Human papillomavirus has been associated with benign squamous tumors, intraepithelial neoplasia, and invasive squamous cancer. The role of human papillomavirus as the most likely precursor of cervical dysplasia is well studied. We know of no available information as to the possible role of human papillomavirus in prostatic hyperplasia and cancer. We studied formalin-fixed paraffin-embedded tissues of 20 cases of glandular hyperplasia and 20 cases of prostatic cancer by in situ DNA hybridization for human papillomavirus using commercially available biotinylated DNA probes detected by an avidin-biotin peroxidase technique. We found no evidence of DNA hybridization to human papillomavirus-6, -11, -16, -18, -31, -33, or -35 in prostate tissue. Our results show no association between prostatic cancer or hyperplasia and the human papillomavirus genomes that were studied. PMID- 1703668 TI - Characterization of cytoadhesion molecules on human monocytes and tissue macrophages. AB - The presence of proteins antigenically related to the GPIIb/IIIa complex expressed on platelets have been investigated on tissue macrophages recovered by bronchoalveolar lavage (lung alveolar macrophages, LAM) or peritoneal lavage (peritoneal macrophages, PM) as well as on monocytes. Polyclonal antibodies (pab) directed against human platelet GPIIb/IIIa and the vitronectin receptor (VnR), and mouse monoclonal antibodies (mab) against human GPIIb, GPIIIa or the GPIIb/IIIa-complex were used. Triton X-100 extracts of bronchoalveolar cells (BAC) (containing 94% LAM) and the ultrasedimentable fraction of cell-free bronchoalveolar lavage (US) reacted with the polyclonal antibodies against the GPIIb/IIIa-complex and the VnR, but only with one (P4) of the mabs. Cell microscopy after immunogold labelling and alkaline phosphatase immunostaining, as well as immunofluorescence using the P4 mab and the polyclonal anti-GPIIb/IIIa clearly demonstrated positive membrane staining of LAM, PM and monocytes. Both BAC and US extracts gave rise to immunoprecipitates in crossed and rocket immunoelectrophoresis using anti-GPIIb/IIIa and anti-VnR. Our data indicate that monocytes and their monocyte-derived tissue counterparts constitutively express GPIIb/IIIa-like antigen(s) on their membrane. The presence of such antigen(s) on tissue macrophages makes it unlikely that platelet contamination is responsible for these findings. PMID- 1703669 TI - Mode of action of PGI2 and of its stable derivative iloprost on platelets and leukocytes. PMID- 1703670 TI - New stains for blood and bone marrow cells. AB - Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells. PMID- 1703671 TI - A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC. AB - The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C, followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, IIA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs. PMID- 1703672 TI - Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer. AB - The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative. PMID- 1703673 TI - Vital fluorescent staining technique for microspores of Brassica napus. AB - The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development. PMID- 1703674 TI - Recognition of dominant T cell-stimulating epitopes from the circumsporozoite protein of Plasmodium falciparum and relationship to malaria morbidity in Gambian children. AB - Cellular immune responses to the Plasmodium falciparum circumsporozoite (CS) protein were measured by proliferation and interferon-gamma production in a cohort of children aged 3 to 8 years, living in The Gambia. Anti-CS antibody titres, malariometric indices and sickle cell status were also determined. Malaria morbidity in the ensuing malaria transmission season was monitored by weekly health questionnaire, axillary temperature measurements and examination of blood films. Exposure to malaria was inferred from entomological data collected during the transmission season. Immunological and parasitological measurements were repeated at the end of the rainy season. Immunological findings were compared between children who experienced clinical malaria or asymptomatic infection and children who had no evidence of infection. No association was found between cellular immune responses to the CS protein at the beginning of the transmission season and subsequent susceptibility to infection except among children with high titres of antibody to (NANP)40. Seropositive children who did not become infected had a higher mean proliferative response to the Th3R epitope than seropositive children who did become infected. High titres of anti-(NANP)40 antibodies alone were not protective. Responses to the Th2R epitope were significantly higher at the end of the rainy season than at the beginning in children who experienced an asymptomatic infection. Responses to variant sequences of the 2 epitopes were highly correlated at an individual level but there was no correlation between proliferative and interferon responses to a particular epitope. PMID- 1703675 TI - Inter-alpha-trypsin inhibitor: emergence of a family within the Kunitz-type protease inhibitor superfamily. AB - Plasma inter-alpha-trypsin inhibitor (I alpha TI) is a major representative of the superfamily of Kunitz-type protease inhibitors in mammals. Formerly, I alpha TI was considered to be a single polypeptide, but recent molecular genetic studies have demonstrated an unexpected multipolypeptide chain structure. The newly discovered genes and gene products form the basis of a new family of I alpha TI-related protease inhibitors. PMID- 1703676 TI - A GABA transporter cDNA has been cloned. PMID- 1703677 TI - Corticospinal, rubrospinal and rubro-olivary projections: a unifying hypothesis. AB - There has been a dispute about the corticospinal and rubrospinal tracts for about 100 years. Both are descending motor pathways and have remarkably similar functional properties. It has been proposed previously that each system is primarily active in different movement contexts. The corticospinal tract is most involved when a new movements is being learnt, while the rubrospinal tract is preferentially active when automated movements are being executed. However, what structure decides which system should be in use? In this article Philip Kennedy discusses the evidence that the rubro-olivary tract switches between the two systems depending on the context of the movement. PMID- 1703678 TI - Keeping cool: a hypothesis about the mechanisms and functions of slow-wave sleep. AB - Current evidence supports a hypothesis that slow-wave sleep (SWS) in mammals and birds is controlled by thermoregulatory mechanisms, and provides brain and body cooling as a primary homeostatic feedback process. Recent work has identified a medial preoptic area anterior hypothalamic and basal forebrain neuronal network which integrates thermoregulatory and hypnogenic controls. This network induces EEG and behavioral deactivation, in part, through suppression of the reticular activating system. Studies have shown that SWS, like other heat loss processes, is facilitated when brain temperature exceeds a threshold level. This threshold is hypothesized to be determined by responses of POAH thermosensitive neurons and to be regulated by both circadian and homeostatic processes. Many known chemomodulators of SWS appear to act on this hypnogenic thermoregulatory system. At a functional level, SWS-induced brain and body cooling would provide several adaptations including lower energy utilization, reduced cerebral metabolism, protection of the brain against the sustained high temperatures of wakefulness, facilitation of immune defense processes and regulation of the timing of behavioral activity relative to the circadian light-dark cycle. This concept provides a comprehensive model for analysis of sleep homeostasis. PMID- 1703679 TI - Is the cerebral cortex modular? AB - Two types of modular subunit, differing in size, have been hypothesized to exist in the cerebral cortex. The first, known as a mini-column, consists of a group of 110 +/- 10 cells which form a fascicle about 30 micrograms in diameter oriented perpendicular to the cortical surface. Mini-columns are believed to be organized into larger modular groupings, referred to here as macro-columns, with a diameter of about a millimetre or less. Nicholas Swindale argues in this article that there is very little real evidence in favour of either type of module. As an alternative, he suggests that the diversity of types of columnar organization, both within and between different cortical areas, may reflect the diversity of types of information stored in the cortex. Consequently, columnar organization can be expected to vary within and between species, and even between different individuals of the same species. This new interpretation is in line with current neural network theories, which do not demand the existence of structural modularity, but show how complex forms of organization can result from the existence of simple processing rules between the elements of a structure given complex structured inputs. PMID- 1703680 TI - Genes required for specifying cell fates in Drosophila embryonic sensory nervous system. AB - The nervous system contains a diverse group of cells. Specification of the correct cell fate is obviously important for the proper function of the nervous system, yet how are the fates of different neurons determined during development? Very little is known about the underlying molecular mechanisms used in the mammalian nervous system. How, for example, are certain cells directed to form pyramidal cells rather than local interneurons? In the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans, some progress has been made in studying neuronal fate determination. For instance, in Drosophila, a number of genes acting at different levels have been found to control this process. They function to (1) endow a subset of the ectodermal cells in the early embryo with the potential to become neuronal precursors, (2) commit some of these cells to the fate of neuronal precursors, (3) specify the identity of these neuronal precursors, and (4) specify the identity of the individual progeny cells of a neuronal precursor. In this review, we discuss first the rationale of the genetic approach, then outline the working hypothesis and, finally, briefly describe the genes known to be involved in the formation of the sensory nervous system in Drosophila. We also discuss the prospects for extrapolating these molecular mechanisms and principles to vertebrate and invertebrate neural development. PMID- 1703681 TI - Presynaptic inhibition of muscle spindle and tendon organ afferents in the mammalian spinal cord. AB - More than 30 years ago, Frank and Fuortes proposed that the synaptic effectiveness of muscle spindle afferents associated with spinal motoneurones could be diminished by the activation of nerves from flexor muscles. Since that time, research has focused on disclosing the mode of operation and the spinal pathways involved in this presynaptic inhibitory control. Initially, it was assumed that the same last-order interneurones mediated presynaptic inhibition of both muscle spindle and tendon organ afferent fibres. More recent evidence indicates that the synaptic effectiveness of these two groups of afferents is controlled by separate sets of GABAergic interneurones synapsing directly with the intraspinal terminals of the afferent fibres. This unique arrangement allows for selective control of the information on muscle length or muscle tension, despite the convergence of muscle spindle and tendon organ afferents on second order interneurones. PMID- 1703682 TI - Conversion of liver allograft recipients from cyclosporine to FK 506-based immunosuppression: benefits and pitfalls. PMID- 1703683 TI - Comparative immunoregulatory effects of rapamycin, FK 506 and cyclosporine on mitogen-induced cytokine production and lymphoproliferation. PMID- 1703684 TI - In vivo regulation of cytokine expression: effects of cycloheximide and cyclosporine (CyA) on IFN-gamma and TNF-alpha expression. PMID- 1703685 TI - Memory T cells express high levels of a novel leukocyte common antigen epitope identified by the A6 MAb. PMID- 1703686 TI - A comparative study between cyclosporine and FK 506 effects on renal microsomal P 450. PMID- 1703687 TI - Mechanism of unresponsiveness in rats induced by a short course of FK 506 or CyA. PMID- 1703688 TI - Effect of the immunosuppressant FK 506 on insulin release from adult rat islets of Langerhans. PMID- 1703689 TI - Protective effect of FK 506 against hepatic ischemia in rats. PMID- 1703690 TI - Immunosuppressive effects of FK 506 and 15-deoxyspergualin in rat lung transplantation. PMID- 1703691 TI - Positive crossmatch in primary human liver allografts under cyclosporine or FK 506 therapy. PMID- 1703692 TI - Structural relationship between HLA-A2-restricted and anti-A2 allospecific T-cell recognition: analysis by mutation of the codons for polymorphic and conserved residues. PMID- 1703693 TI - Basic principles of three rapid fluorochrome assays for DNA quantification and cell counting. PMID- 1703694 TI - FK 506: short- and long-term treatment after cardiac transplantation in nonhuman primates. PMID- 1703695 TI - Effect of FK 506 on glucose metabolism in the cynomolgus monkey: studies in pancreatic transplant recipients and nontransplanted animals. PMID- 1703696 TI - FK 506-induced nephrotoxicity in rats. PMID- 1703697 TI - Morphological and immunological analysis of rats with long-term-surviving limb allografts induced by a short course of FK 506 or cyclosporine. PMID- 1703698 TI - Use of rapamycin for the suppression of alloimmune reactions in vivo: schedule dependence, tolerance induction, synergy with cyclosporine and FK 506, and effect on host-versus-graft and graft-versus-host reactions. PMID- 1703699 TI - Accelerated atherosclerosis in heart transplants in the rat simulating chronic vascular rejection: effects of prostacyclin and angiopeptin. PMID- 1703700 TI - Effect of FK 506 and cyclosporine on heart allograft survival in rats. PMID- 1703701 TI - Initial experience with FK 506 as an immunosuppressant for nonhuman primate recipients of cardiac allografts. PMID- 1703702 TI - Effect of FK 506 on heart allograft survival in highly sensitized recipient rats in comparison with cyclosporine. PMID- 1703703 TI - Stimulation of liver regeneration by prostacyclin. PMID- 1703704 TI - Effect of splenectomy in combination with FK 506 and 15-deoxyspergualin on cardiac xenograft survival. PMID- 1703706 TI - Donor bone marrow cell facilitates induction of tolerance to kidney allografts in dogs treated with fractionated lymphoid irradiation and FK 506. PMID- 1703705 TI - FK 506 prevents spontaneous diabetes in the BB rat. AB - The BB rat is the experimental analogue of human juvenile diabetes mellitus. From 30 through 120 days after birth, 59 BB rats were treated with water (n = 20), or FK 506 in daily intragastric doses of 1 mg/kg (n = 19) or 2 mg/kg (n = 20). Diabetes developed in 75%, 15%, and 0% of the three groups. Animals protected from diabetes by FK 506, and killed in the nondiabetic state at 120 days had normal intraperitoneal glucose tolerance tests, virtual absence histopathologically of autoimmune insulitis, normal pancreatic insulin content, and immunocytochemical confirmation of islet insulin and glucagon content. Forty five to 75 days after stopping FK 506, about 3/4 of the animals who were diabetes free at 120 days have remained so. These results provide support for a clinical trial of FK 506 for recent onset diabetes. PMID- 1703707 TI - Significance of pancreatic allograft rejection in swine abdominal organ cluster transplantation. PMID- 1703708 TI - Dextran 40 successfully replaces the non-essential hydroxyethylstarch in the University of Wisconsin solution for 72-hour simple cold storage of the canine kidney. PMID- 1703709 TI - The effect of FK 506 on canine pancreatic islet cell function in beagle dogs. PMID- 1703710 TI - Prevention of immune rejection in rat islet allografts by continuous SC administration of FK 506 using a mini-osmotic pump. PMID- 1703711 TI - Sequential multiple donor islet transplantation: a model for repeated transplantation into the portal system in dogs. PMID- 1703712 TI - Combined liver-islet allotransplantation in man under FK 506. PMID- 1703713 TI - Interleukin-7 (IL-7) and its effect on the expression of the intercellular adhesion molecule (ICAM-1) in T cells. PMID- 1703714 TI - Antibodies against T-cell receptor peptide arrest autoimmune rejection of normal central nervous system myelin. PMID- 1703715 TI - Isolation of a murine renal cell population which expresses a truncated T-cell receptor-alpha mRNA. PMID- 1703716 TI - The mechanism of tolerance induced by a perioperative injection of bone marrow cells and a brief course of FK 506. PMID- 1703717 TI - Effects of the new and highly active immunosuppressant, rapamycin, on lymphoid tissues and cells in vivo. PMID- 1703718 TI - Prominent prolongation of islet xenograft survival in combination therapy with FK 506 and 15-deoxyspergualin. PMID- 1703719 TI - [The improvement of the tolerance of the kidney for ischemia with the prostacyclin analog iloprost]. AB - Operative trauma, ischemia, release of catecholamines and thermal stimuli in consequence of harvesting, preservation and transplantation deteriorate the renal capillary circulation. By crossing of tolerance limits arises an irreversible transplant damage. The conditions of our examinations were placed in this field. By pre- and postischemic application of Iloprost was demonstrable an improvement of the ischemic tolerance of the kidney. Correction of renal hemodynamics, antiplatelet action and cytoprotection are the reasons of the protective influence of Iloprost in our opinion. PMID- 1703720 TI - [Cisplatin-induced nephrotoxic side effects of cytostatic chemotherapy of testicular tumors]. AB - The nephrotoxic side effect of Cis-platin was investigated in 38 patients with testicular tumors. The serum creatinine and the creatinine clearance served as function parameters. A temporary or permanent restriction of the creatinine clearance developed 31 of 38 patients during the therapy period. Increased creatinine levels were found in 6 patients. Two of them got into a chronic renal insufficiency. A pretherapeutic risk group of patients or a precarious dose of Cis-platin could not be found out. PMID- 1703721 TI - [Surgical treatment of spinal metastases]. AB - An analysis of indications, techniques, results of decompression and stabilization in 164 spinal metastasis was carried out. Actual possibilities of rigid stabilization from the anterior and posterior approach are demonstrated. PMID- 1703722 TI - [Surgical treatment of gastric carcinoma: complications and results]. AB - Between 1974 and 1989 468 patients with gastric cancer were operated upon. The correlations between age, tumour staging, operability, postoperative complications, and perioperative mortality are analyzed. The further course of the disease was followed carefully in all patients. Therefore it is possible to correlated survival with tumour stage and operability. PMID- 1703723 TI - [Soupault-Couinaud intrahepatic biliodigestive anastomosis: an effective palliative treatment of liver hilus tumors]. PMID- 1703724 TI - The complications of newer transplant antirejection drugs: treatment with cyclosporin A, OKT3, and FK506. PMID- 1703725 TI - Stability of bleomycin sulfate reconstituted in 5% dextrose injection or 0.9% sodium chloride injection stored in glass vials or polyvinyl chloride containers. PMID- 1703726 TI - Nutrient modulation of inflammatory and immune function. AB - The metabolic response to injury occurs after a diverse group of surgical injuries including major surgical intervention, shock, infection, and sources of inflammation such as pancreatitis. The response is mediated by the macroendocrine system, the autonomic nervous system, and the cell-cell communication system. The clinical manifestations include now well-described clinical, physiologic, and metabolic characteristics. The approach of aggressive source control, invasive circulatory resuscitation, and nutrition/metabolic support has been associated with an overall reduction in morbidity and mortality. In those patients who do not respond to this approach, the disease process progresses to multiple organ failure syndrome with its associated high mortality. Altering the route of feeding, preventing single nutrient and generalized nutrient deficiency, and reducing nosocomial infections with selective gut decontamination have not significantly altered the course or outcome of the disease process in this latter group of patients with persistent hypermetabolism. The available data support the position that this persistent hypermetabolism represents abnormal metabolic regulation resulting in persistence of the inflammatory response with associated suppression of the immune defenses. A number of research approaches are being taken to understand and modulate this abnormal state of regulation. Because of the role of specific nutrients in these regulatory processes, beyond their role in classic nutrition support, nutrients such as arginine n-3 polyunsaturated fatty acids, and RNA are being evaluated for their ability to modulate inflammation and to improve immune function. Preliminary results are encouraging. PMID- 1703727 TI - Anaphylactic anaesthetic reactions. The value of paper radioallergosorbent tests for IgE antibodies to muscle relaxants and thiopentone. AB - The three currently available paper radioallergosorbent tests ('suxamethonium', alcuronium and thiopentone) were evaluated. 'Suxamethonium' radioallergosorbent test (which employs choline conjugated to paper discs) proved to be reliable in the detection of allergy to neuromuscular blockers, which were confirmed as the most common cause of anaphylactic reaction during general anaesthesia. Thiopentone radioallergosorbent test may also be useful, and is recommended in conjunction with 'suxamethonium' radioallergosorbent test in the preliminary investigation of reactions. Patients with positive 'suxamethonium' radioallergosorbent test usually require further testing, including alcuronium radioallergosorbent test, skin testing with a wide range of drug concentrations or leucocyte histamine release test. PMID- 1703728 TI - Severe anaphylactoid reaction to hydroxyethyl starch. AB - A case is presented of a severe anaphylactoid reaction to hydroxyethyl starch solution that occurred peri-operatively and required extended intensive management of the resultant bronchospasm and hypotension. Subsequent intradermal injection of hetastarch produced a delayed positive response, suggestive of a complement-mediated mechanism. PMID- 1703729 TI - [Plasma levels of atrial natriuretic peptide, cyclic guanosine monophosphate and renin following 7.5% NaCl + 6% hydroxyethyl starch or Ringer's lactate. A comparative study of 6 normal subjects]. AB - Small-volume resuscitation with hypertonic saline in combination with dextran appears to be very successful in experimental animals, where better results are achieved than in animals treated with a traditional infusion regime. This effect is apparently related to improved organ blood flow due to reflex vasodilatation. This reflex is based on the arrival of hypertonic solution in the pulmonary circulation. The expansion of intravascular volume would seem to be of secondary importance. Atrial natriuretic peptide (ANP) is released from secretory granules located in atrial cardiocytes. Atrial distention appears to be the predominant stimulus triggering ANP production. In addition to the natriuretic and diuretic effects, ANP leads to vasodilation, especially when vascular tone is elevated; the sympathetic reflex seems to be attenuated. Cyclic Guanosine Monophosphate (cGMP) is an intracellular messenger and is partly released by ANP in the membrane-bound form. Renin excretion is highly influenced by ANP. The object of this study was to evaluate the influence of a hypertonic solution on this hormonal regulatory system. METHOD. This study compared a hypertonic sodium chloride solution (7.5%) in combination with hydroxyethyl starch (6%) (HH) to Ringer's lactate (RL). Six healthy volunteers received 4 ml/kg HH and 1 week later 500 ml RL. The infusion was administered in 20 minutes via a central venous catheter 70 cm in length. Blood pressure, heart rate, hemoglobin (Hb), hematocrit (Hk), colloid osmotic pressure (COP), sodium (Na+), chloride (Cl-), and plasma osmolarity were measured before starting and 5 and 30 min following infusion. At the same times ANP, cGMP, and plasma renin were also determined. RESULTS. Both groups showed no change in blood pressure or heart rate. The decrease of Hb, Hk, and COP in the HH and RL groups indicated the expansion of circulating plasma volume. HH infusion caused significant increases in ANP and cGMP, whereas plasma renin declined significantly. After RL infusion, ANP and renin values were very similar to the HH group except in one volunteer, who showed an extreme increase in ANP (760 pg/ml) 5 min after HH infusion. cGMP did not increase significantly in the RL group. On comparison of the two groups, only a significant difference in plasma osmolarity and in sodium and chloride levels was noted. CONCLUSION. We found that hypertonic NaCl (7.5%) with HH was well tolerated. Release of ANP and cGMP after HH infusion in healthy volunteers was not as high as expected, and the vasodilatory effect of hypertonic solutions was not explained by ANP or cGMP release in this investigation. PMID- 1703730 TI - A general procedure for evaluation of immunological relevance of synthetic peptides: peptides synthesized on paper in enzyme-linked immunosorbent assay. AB - Multiple continuous-flow solid-phase peptide synthesis has been adapted for synthesis of peptides on a cellulose carrier (Whatman 3MM paper). Paper-bound synthetic peptides that represent antigenic determinants of particular proteins detected antibodies against the respective proteins in an enzyme-linked immunosorbent assay. The method is applied to the synthesis, and use in site directed serology, of four peptides derived from the gp41 glycoprotein of HIV, the Epstein-Barr virus-determined nuclear antigen-1 and VCA proteins of the Epstein-Barr virus, and the early region of human papillomavirus type 11. PMID- 1703731 TI - A microtiter plate-based assay for phosphoenolpyruvate carboxylase. AB - A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography. PMID- 1703732 TI - [Biophysical approach to the antigen-antibody reaction in solid phase immunoassay]. AB - The main properties of solid/liquid interfaces are briefly considered before the weak chemical bonds involved in Ag-Ab interactions (hydrogen, electrostatic, Van Der Waals and hydrophobic bonds) are related in detail. The reversible nature of these non covalent bonds allows the discussion of the Ag-Ab reaction in the usual terms of chemical equilibrium the thermodynamic values associated with this equilibrium can be described. However, the complexity of the Ag-Ab reaction is strongly increased if Ag or Ab are bound to a solid phase. The modifications brought about by the solid phase are debated with the intention of understanding the physical and chemical parameters of the Ag-Ab reaction in solid phase immunoassays. PMID- 1703733 TI - [Solid phase hybridization]. AB - Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The possibility of using them in clinical biology has been considered for several years. The transfer from basic to applied research can now take place due to the new technology of non-radioactive probes (cold probes). Hybridization features the use of a probe nucleic acid molecule and of a target nucleic acid molecule. It leads to the formation of a double-stranded molecule, also called duplex, which can be detected with great sensitivity. For this to be done, the probe can be tagged with a variety of labels such as a radio-isotope or a non isotopic marker which can be detected for its fluorescence, luminescence or enzyme activity or by immuno-reaction(s). Hybridization can be performed either in a liquid solution or on a solid support. In the first case, hybridization is generally followed by a separation step which makes use of solid phase to isolate the hybrid (duplex) formed. This separation can be achieved by biochemical means (adsorption chromatography, differential precipitation, electrophoresis, etc.) or by immunological means (affinity chromatography, immuno-capture). In the second case, the support can be used in different ways according to the assay format employed to perform hybridization. The target DNA can be immobilized by contacting the sample to be tested with the support: simple specimen deposit with a pipette (for low volumes), or by aspiration under vacuum or by (capillary or electrophoretic) transfer from an agarose gel. In the sandwich assay format, probe DNA is immobilized onto the support to specifically extract the target DNA from the medium under assay. Support to be employed for hybridization purpose can be selected from the group consisting of: nylon (N), nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes; N+NC mixed membranes; cellulose and its derivatives; polyoside supports (Sephacryl, Sepharose, etc.), latex particles, polystyrene microplates. The coupling procedures and capacity vary according to the type of support used. PMID- 1703734 TI - [Pharmacokinetics and changes in the physical properties of blood and urine after administration of dextran 60000]. AB - A study was carried out to assess the changes induced by an infusion of dextran, molecular weight 60,000 daltons, in blood and urine. Plasma and urine dextran and serum protein concentrations, haematocrit, blood and urine viscosities, and blood oncotic pressure were measured in 10 consecutive male patients. Fifteen min after administration of 20 ml dextran 1000 (Promit), they were each given 500 ml (30 g) dextran 60 (Hemodex) over 30 min for plasma volume expansion. The measurements were carried out at the end of the infusion, and then at regular intervals over a 48 h period. The highest dextran blood concentrations were found at the end of the infusion, decreasing thereafter with a distribution half-life of 1.83 +/- 0.64 h, and an elimination half-life of 25.5 +/- 7.6 h. Haematocrit values decreased by 12%, and serum protein concentrations by 9.5%, after the end of the infusion. These changes remained significant for 9 h; they were probably due to the dilution effect of 500 ml of dextran. Colloid osmotic pressure was not significantly altered (20.7 +/- 4.7 mmHg vs. 23.1 +/- 5.1 mmHg 48 h after the end of the infusion). The colloid osmotic pressure due to dextran 60 compensated for the fall in protein concentration. A decrease in blood viscosity was found at different shear rates, despite dextran 60 being highly viscous. This could also be explained by a dilution effect. The highest degree of urinary excretion occurred 30 min after the end of the infusion, and lasted for 3 h. Forty-five percent of the total dextran dose had been excreted by the 48th hour.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703735 TI - Early changes in lung tissue hyaluronan (hyaluronic acid) and hyaluronidase in bleomycin-induced alveolitis in hamsters. AB - Intratracheal instillation of bleomycin in hamsters initiates a series of events that mimic human interstitial pulmonary fibrosis. Because glycosaminoglycans and particularly hyaluronan (hyaluronic acid, HA), may play an important role in the extracellular matrix response to early injury and subsequent fibrosis, this study was undertaken to define the early time course of changes in HA and hyaluronidase. Hamsters were given either 1 unit bleomycin sulfate in 0.2 ml saline or 0.2 ml saline (control), and randomly selected animals from both groups were killed at Days 3, 5, 6, 7, 9, and 17. Glycosaminoglycan fractions prepared from lung tissue of individual animals were analyzed for HA. The maximal HA content was reached 6 days after instillation of bleomycin and was 14.6-fold the normal value. The weight of injured lungs was 2.3-fold the control value. Thus, the increase in HA content was 30-fold. By Day 7 the HA content had dropped sharply. It then declined gradually to approximately double control values at Day 17. The specific activity of lysosomal hyaluronidase was the same in bleomycin treated lungs and control lungs. Total units of the enzyme were increased in injured lungs, even at the time of maximal HA content, indicating active turnover of HA. The maximal HA content occurs prior to the rise in collagen and elastin biosynthesis. This observation in addition to the magnitude of the increase and its abrupt decline suggest that HA may be an important initiating factor for pathologic changes in lung extracellular matrix components. PMID- 1703736 TI - [University by radio and television in Great Britain. The Open University]. AB - There are some 20 years since Great Britain was given a new and important University (established by a Royal Charter in 1969), especially intended for students wanting to achieve their education at home. The whole teaching, guided by tutors, is supported by various materials: booklets, audio- and videotapes which are completed by television and radio broadcasts for many courses and informations. After successful examinations are obtained the usual degrees and certificates delivered by the British Universities. Furthermore, this kind of education begins to be available in a few other countries (Belgium, Luxembourg, The Netherlands, Cyprus, Hong Kong). PMID- 1703737 TI - Characterization of peripheral blood and salivary gland lymphocytes in secondary Sjogren's syndrome. AB - Secondary Sjogren's syndrome (SS) is defined as a condition of patients with sicca symptoms in association with a connective tissue disease such as rheumatoid arthritis. This study was designed to investigate the peripheral blood and affected minor salivary gland (SG) tissue lymphocytes with monoclonal antibodies in patients with secondary SS having rheumatoid arthritis. Minor SG lymphocytes of the patients and normals were determined in the fresh-frozen sections of the minor SG biopsy samples using monoclonal antibodies with immunoperoxidase technique. Peripheral blood of secondary SS patients revealed significant reduction in CD3+ and CD8+ cells. CD4/CD8 radio, HLA-DR+ cells, and B-cells were unchanged. SG biopsies showed varying degrees of lymphocytic infiltration with predominance of CD3+CD4+ cells located at the periductal areas. CD8+ cells were found to be in low numbers within the infiltrates. IgG- and IgA-producing plasma cells were both numerous in the biopsy samples. Our findings suggest that there is an alteration of lymphocyte subpopulations at the local site of inflammation in the salivary glands without, however, a corresponding alteration in the peripheral blood. PMID- 1703738 TI - Clinical features and complications of epidemic group A meningococcal disease in Sudanese children. AB - The clinical presentation and laboratory features in relation to short-term outcome in 118 prospectively studied Sudanese children who were admitted with meningococcal (MC) meningitis and/or septicaemia during the 1988 group A MC epidemic in Greater Khartoum are described. Their ages ranged from 25 days to 15 years (mean: 78 months) and 42% were less than 5 years old. The male:female ratio was 1.6:1. Forty (34%) came from one of the peri-urban shanty towns encircling Greater Khartoum. A history of MC immunization (A and C vaccine) was obtained in 22%, but only five children (4.8%) had the vaccine between 4 weeks and 1 year before their illness. The commonest symptoms on admission were vomiting, neck rigidity and diarrhoea. Convulsions were significantly more frequent in children under 5 years old (p = 0.0005). Fifty-six (47%) had evidence of malnutrition. In descending order, fever, neck stiffness and Kernig's sign were the most commonly observed signs, the latter two being significantly more often detected in children older than 1 year. Twenty-four patients 20%) had disturbed consciousness. The case fatality rate was 6.3% and this was significantly higher in those presenting with meningococcal septicaemia (p = 0.0006). Other significant associations with mortality were short duration (less than 1 day) of symptoms (p = 0.0006) and clinical shock detected on admission p = 0.003). Transient complications were infrequent and permanent neurological sequelae were confined to bilateral profound sensorineural hearing loss in three children (2.9%) and hemiplegia in two 1.9%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703739 TI - Mortality and immediate morbidity in term babies with low Apgar scores (Zimbabwe). AB - A retrospective study was undertaken to document immediate morbidity and mortality associated with low Apgar scores (5 or less) at 5 min in singleton term babies (37 weeks or more). From October 1986 to February 1987, 84 term babies had low Apgar scores, giving an incidence of 15/1000 live births. Overall mortality in these patients was 42%, and in those in whom Apgar scores remained 0-3 at 5 min, mortality was 77%. Evidence of severe-to-moderate hypoxic-ischaemic encephalopathy was noted in 44 (52%) and 17 (20%) failed to establish spontaneous respiration within the 1st few hours of life. Meconium aspiration syndrome was diagnosed in 29 (35%). A control group of 141 term babies with 5-min Apgar scores of 7 or more were identified and the presence of possible risk factors associated with low Apgar scores was assessed. Primiparity and prolonged second stages of labour in all parities were found to be significantly associated with low Apgar scores. Improved obstetric care and appropriate management of the newborn at delivery and in the neonatal period may help to reduce mortality and morbidity in some term babies. PMID- 1703740 TI - Rotavirus studies in Indian (Asian) South African infants with acute gastro enteritis: II. Clinical aspects and outcome. AB - In a prospective study of rotavirus (RV) diarrhoea in Indian (Asian) infants in South Africa, it was found that in common with findings elsewhere in the world, vomiting and dehydration are prominent features of this disease. The dehydration was usually isotonic, though both hypo- and hypernatraemia did occur. Hypokalaemia was found to be much less common in RV than in non-RV diarrhoea. Unlike findings elsewhere, no definite 'rotavirus syndrome' associated with pyrexia and respiratory symptoms could be identified in RV diarrhoea as these occurred with equal frequency in non-RV patients. The mean total duration of RV diarrhoea (i.e. before admission plus during the hospital stay) was 5 days. The RV patients took significantly longer to recover from their diarrhoea than the non-RV ones, and mixed RV-bacterial infections prolonged the illness even more. Breast-feeding was associated with milder disease. Less than 2% of both RV and non-RV cases developed persistent diarrhoea of longer than 14 days' duration and this was most frequent in patients under 6 months of age with poor nutritional status. PMID- 1703741 TI - Epidemiology of juvenile chronic arthritis and other connective tissue diseases among children in Kuwait. AB - An 8-year hospital-based retrospective study on the epidemiology of juvenile chronic arthritis (JCA), systemic lupus erythematosus (SLE) and other connective tissue diseases among children in Kuwait is described. There were 108 children with JCA, 20 children with SLE, 23 children with other connective tissue diseases and 24 children with arthritis of familial Mediterranean fever (FMF). The average annual incidence of JCA was 2.84 cases/10(5) children under the age of 12 years and the 1988 prevalence was 18.7/10(5). The polyarticular, systemic and oligoarticular onset types were observed in 42, 29 and 29%, respectively. The incidence and prevalence of SLE were 0.53 and 3.37/10(5), respectively. The findings are compared with those from other countries. PMID- 1703742 TI - Hypernatraemic dehydration in the Hail region of Saudi Arabia. AB - Infants presenting with hypernatraemic dehydration were studied prospectively in order to describe its incidence and the predisposing factors. Five hundred and twenty children with gastroenteritis were admitted to the Paediatric Unit of Hail General Hospital over a 1-year period from 1 June 1985 to 1 June 1986. Twenty five children (4.8%) had hypernatraemia (Na+ greater than 150 mmol/l) and all 25 were under 1 year of age, 23 (92%) being under 6 months. Twenty (80%) came from families living in the villages and had a poor educational background. All the babies were bottle-fed. The majority of the mothers did not know how to prepare food hygienically and with the appropriate water/milk proportions. Most of the infants presented with high fever (+39 degrees C) and the majority were underweight for their age. Two babies died and one had evidence of neurological damage. This study indicates that the incidence of hypernatraemic dehydration is significant in this region and causes serious morbidity and mortality. It confirms the importance of breast-feeding and the need to educate the public in the proper preparation of bottle feeds when breast-feeding is not possible. PMID- 1703743 TI - Iron and folate status in Gambian children with malaria. AB - Iron and folate status were evaluated in a group of 106 Gambian children with malaria and variable degrees of anaemia. In children with malaria, normal or increased levels of red cell folate were found in 75 patients at presentation and in 15 patients 1-2 weeks after treatment with anti-malarials alone, despite the presence of giant metamyelocytes and megaloblasts in the bone marrow in some cases. Twenty-eight per cent of patients were found to have deficient bone marrow iron stores but malaria could not be directly implicated as the cause of this deficiency. Iron deficiency could also not be implicated as the sole cause of dyserythropoiesis in patients with malarial anaemia. Excess storage iron and the presence of ring sideroblasts were found in the bone marrow in some cases. It is concluded that the morphological changes including dyserythropoiesis, occasional megaloblasts, giant metamyelocytes and ring sideroblasts seen in the bone marrows of these children are manifestations of disturbed marrow function in malaria and are not related to haematinic deficiency. Because of the high rate of iron deficiency found in these patients it is recommended that Gambian children with severe anaemia should receive iron therapy after adequate treatment of malaria. PMID- 1703744 TI - Verrucous lesions in children in the tropics. AB - A wide variety of skin disorders in children are encountered by doctors practising in tropical countries. While some of them are common and pose little difficulty in their management, a few are uncommon, run a protracted course and cause errors in diagnosis. Two patients--one with cutaneous tuberculosis and the other with chromomycosis--are described and illustrate the importance of early and prompt detection of disease in children. PMID- 1703745 TI - Henoch-Schoenlein syndrome in Qatar: the effects of steroid therapy and paucity of renal involvement. AB - This is a retrospective study of 40 patients admitted to Hamad General Hospital in the state of Qatar between January 1983 and December 1987 with the diagnosis of Henoch-Schoenlein syndrome. Of the 40 patients, 25 were boys and 15 were girls, with a ratio of 1.6:1. Ages ranged from 2 years 3 months to 13 years, with a mean of 6 years. There were six episodes of recurrence in four patients. There was a clustering of cases in late summer and early winter. About half of the patients had a history of preceding upper respiratory infection. All of them had the typical skin rash. The percentages of joint, gastro-intestinal and renal manifestations were 80%, 65% and 17.39%, respectively. One patient had penile swelling which has not been reported before. Steroid therapy seemed to enhance early resolution of abdominal pain but did not affect the course of the syndrome. Sixty-seven per cent of the patients were followed up for from 4 weeks to 5 years, with a mean of 8 months. Only one patient with renal involvement continued to have proteinuria with microscopic haematuria and hypertension. The rest were normal within about 2 months. The remarkably low incidence of renal involvement in our study may be related to local variations in causative factors. Henoch Schoenlein syndrome is a milder disease in Qatar than in other countries. PMID- 1703746 TI - Initial treatment of bacterial meningitis in Yaounde, Cameroon: theoretical benefits of the ampicillin-chloramphenicol combination versus chloramphenicol alone. AB - A prospective 6-month study in Yaounde evaluated 49 children aged from 2 months to 8 years, hospitalized with bacterial meningitis. They were randomly assigned to one of two initial treatment groups, either an ampicillin-chloramphenicol combination (group A) or chloramphenicol alone (group B). The majority of patients were infected with Haemophilus influenzae, and the majority of deaths were caused by Streptococcus pneumoniae. Altogether, 17.9% of Haemophilus influenzae isolates were ampicillin-resistant and 3.6% chloramphenicol-resistant. We found no isolate resistant to both antibiotics. Response to both treatments was similar in both groups. The theoretical risk of treatment failure with ampicillin was higher than with the ampicillin-chloramphenicol combination (p less than 0.05). There was no statistically significant difference between the risk of treatment failure with the ampicillin-chloramphenicol combination and the risk with chloramphenicol alone (p less than 0.05), but the latter was increased by the occurrence of chloramphenicol-resistant isolates of Streptococcus pneumoniae (11.1%). Although treatment with an ampicillin-chloramphenicol combination is four times more expensive than treatment with chloramphenicol alone, costwise it is also one-quarter the price of a third-generation cephalosporin (moxalactam). At present, the ampicillin-chloramphenicol combination can be suggested as the first choice for initial treatment considering both the epidemiological data and the cost/efficiency ratio in the area of Yaounde. PMID- 1703747 TI - Schistosomiasis associated with a mediastinal mass: case report and review of the literature. AB - A 13-year-old girl presented with fever, night sweat, weight loss, abdominal pain, haematuria and hepatosplenomegaly. Urinalysis revealed many Schistosoma haematobium ova, but rectal snip examination was negative for schistosomal ova. X ray and CT scan of the chest revealed enlargement of the anterior, superior, mediastinal and left suprahilar lymph node with an adjacent left pulmonary parenchymal opacity and small peripheral lesions on the right side. A bone marrow aspiration and biopsy was normal. The patient was treated with Praziquantel for her urinary schistosomiasis. Because of her clinical and radiological chest findings, the possibilities of lymphoma and tuberculosis were considered. Therefore, she underwent a thoracotomy and biopsy of her thoracic lesions. The histopathology revealed pulmonary granulomas surrounding schistosoma ova with reactive mediastinal lymph adenitis. PMID- 1703748 TI - Neonatal necrotizing fasciitis--a complication of poor cord hygiene: report of three cases. AB - Necrotizing fasciitis is a potentially lethal syndrome which rarely affects Occurrence in the neonatal period is uncommon, although it has been estimated that up to 50% of reported paediatric cases involve the neonate. Two previous reports have associated the syndrome with neonatal omphalitis. Three cases seen in the University of Benin Teaching Hospital are presented, with poor umbilical cord stump hygiene being the immediate predisposing/associated condition. Two of the infants had Fourier's gangrene and the third developed gangrene of the anterior abdominal wall. PMID- 1703749 TI - Probable breast-milk borne brucellosis in a young infant. AB - A young infant with acute brucellosis is reported. He presented with a septicaemia-like picture. Diagnosis was based on a fourfold rise of Brucella agglutination titres and a positive blood culture. He had been exclusively breast fed when his mother developed brucellosis 4 weeks after delivery. It is strongly suspected that the transmission of Brucella melitensis to this infant was through the maternal breast-milk. PMID- 1703750 TI - Urinary tract infection caused by Shigella sonnei: a case report. AB - We report a case of severe urinary tract infection caused by Shigella sonnei in a 3-year-old girl with vesico-ureteric reflux and no history of dysentery. Treatment with co-trimoxazole in a dose of 48 mg/kg for 10 days was given and the infection was eradicated. Possible sources of infection are discussed. PMID- 1703751 TI - Mebendazole poisoning in infancy. AB - Accidental mebendazole poisoning in an 8-week-old infant and respiratory arrest with tachyarrhythmia associated with continuous seizures is reported. Exchange transfusion was undertaken as a life-saving measure. Mebendazole, like piperazine citrate, has considerable neurotoxicity, especially in infancy, and we propose the use of exchange transfusion as a means of mebendazole elimination in infants. PMID- 1703752 TI - Intradural lipoma in a child. AB - We report the case of a 5-year-old Nigerian boy in whom conus medullaris and cauda enquina compression was due to an intradural lipoma, a rare tumour of the spinal canal in the tropics. The literature on the subject is briefly reviewed and the differential diagnoses are discussed. The occurrence of the lesion in this child, without other features of spinal dysraphism, is noteworthy. PMID- 1703753 TI - Burkitt's lymphoma presenting with blindness: a case report. AB - A 10-year-old schoolboy was referred to the Ophthalmic Unit of Ahmadu Bello University Teaching Hospital because of sudden loss of sight following 5 days of severe frontal headache. The child had bilateral ptosis with internal and external ophthalmoplegia and fixed and dilated pupils. There was no papilloedema. Eight days later, a jaw tumour and a rapidly enlarging abdominal tumour appeared. A fine needle aspiration biopsy of the jaw tumour confirmed Burkitt's lymphoma. Combination chemotherapy with cyclophosphamide, vincristine and methotrexate (COM) led to a rapid resolution of the jaw and abdominal tumour but the child never regained his sight. Cerebrospinal fluid examination was not helpful in reaching a diagnosis. PMID- 1703754 TI - Cryptococcal meningitis in a child with systemic lupus erythematosus. AB - This is a case report of fatal cryptococcal meningitis in a child with systemic lupus erythematosus being treated with prednisolone and azathioprine. It is believed to be the first case of cryptococcal meningitis recorded in a child in Saudi Arabia. PMID- 1703755 TI - cDNA and derived amino acid sequence of rabbit nasal cytochrome P450NMb (P450IIG1), a unique isozyme possibly involved in olfaction. AB - Olfactory-specific cytochrome P450NMb was previously purified to electrophoretic homogeneity from microsomes of rabbit nasal mucosa in this laboratory. In the present study, a cDNA library made from poly(A)+ RNA from rabbit nasal mucosa was screened with antibodies to this P450, and eight immunopositive clones were isolated and characterized. The sequence determined from two overlapping clones contained an open reading frame of 1446 nucleotides, with the predicted first 39 amino acids corresponding to residues 12 to 50 of purified NMb, except for position 46, where Leu was encoded instead of the Glu residue that was found earlier by Edman degradation analysis. The complete polypeptide, including residues 1 to 11, contains 494 amino acid residues and has a molecular weight of 56,640. Sequence comparisons indicated that NMb is more than 50% identical to members of the rabbit P450 gene II family, including IIB4, IIC3, IIC5, IIE1, and IIE2, and 83% identical to rat P450olf1 (IIG1). Hybridization of NMb to electrophoretically fractionated rabbit nasal poly(A)+ RNA revealed 3.6- and 2.1 kb species, but with a probe derived from the 3'-nontranslated portion of the cDNA only the 3.6-kb band was observed, suggesting the use of alternate polyadenylation sites or splicing. In agreement with the known tissue-specific distribution of NMb protein, NMb transcripts were found in olfactory mucosa, but not in liver, lung, intestine, or kidney. Genomic hybridization analysis indicated that there may be only one copy of the NMb gene present in the rabbit genome. PMID- 1703756 TI - Intralesional bleomycin sulfate therapy for warts. A novel bifurcated needle puncture technique. AB - A multiple puncture technique using a bifurcated vaccination needle to introduce bleomycin sulfate (1 U/mL sterile saline solution) into warts resulted in elimination of 92% of a random series of 258 warts after a single treatment. Recurrence was not observed during a 6-month follow-up period. Six of the 66 patients required two to seven treatments for wart eradication, and four patients requested alternative therapy after initial failure with a single bleomycin treatment. PMID- 1703757 TI - [Prospects of studying the antiviral properties of cytokines]. PMID- 1703758 TI - Isolate B12, which harbours a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov. AB - The Sulfolobus isolate B12 and its endogenous virus-like element SSV1 have provided a fruitful system for detailed analysis of certain aspects of archaebacterial molecular biology, especially those concerning gene expression. In the course of clarifying this isolate's taxonomic position, we determined DNA base composition, ability to grow autotrophically, nucleotide sequence of 16S ribosomal RNA, and level of total genomic homology to other Sulfolobus strains. Although the results generally demonstrate a similarity to S. solfataricus, DNA DNA hybridisation and 16S rRNA sequence data indicate that isolate B12 in fact represents a distinct species. PMID- 1703759 TI - Telephone use to elicit voice or speech in brain injured subjects. AB - The initiation of speech is often delayed in the early stages of recovery from a serious brain injury. We have found a high percentage of patients with both speech and swallowing problems. This makes bedside assessment of swallowing safety difficult because one cannot listen for the sound of aspirated material on the vocal cords when a patient is at high risk for silent aspiration and is often unable to cooperate with a videofluoroscopic study. The use of the telephone has been described several times for aphasia treatment, but not to elicit speech or assess swallowing safety early after brain injury. This study, therefore, recruited subjects who had brain injuries and (1) were referred early for swallowing and other evaluations, (2) were out of coma and able to follow some commands, and (3) did not initiate voice or speak when asked to. Subjects were asked three questions under two different conditions: face to face and after ringing the telephone from another room. The results were recorded on videotape and analyzed by another investigator for quantifiable differences. Six of the seven subjects responded better with the telephone stimulus than without. This technique may elicit voice or speech early after brain injury in some patients and may be useful in bedside assessment of swallowing safety. It may also serve as an example of appropriate stimulation of brain injured subjects coming out of coma. PMID- 1703760 TI - Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins. AB - Two polyclonal antisera were raised in rabbits to the phospholipase C-solubilized forms of pig renal dipeptidase (EC 3.4.13.11) and pig aminopeptidase P (EC 3.4.11.9). These antisera were purified and shown to cross-react with other glycosyl-phosphatidylinositol (G-PI)-anchored proteins isolated from pig, human and trypanosomes. The epitopes involved in this cross-reactivity were characterized by Western-blot analysis after mild acid or nitrous acid treatment of the G-PI-anchored proteins and by a competitive e.l.i.s.a. with other G-PI anchored proteins and individual components of the anchor structure. These studies revealed that the primary epitope for both antisera is the inositol 1.2 (cyclic)monophosphate that is formed on phospholipase C cleavage of the intact G PI anchor. Other minor epitopes, such as phosphoethanolamine, probably involve side-chain modifications to the core anchor structure that may be species specific. PMID- 1703761 TI - 1-C-6 epitope in cartilage proteoglycan G2 domain is masked by keratan sulphate. AB - The presence of the protein epitope recognized by monoclonal antibody 1-C-6 was investigated on the globular G1 and G2 domains of pig cartilage proteoglycan core protein. After reduction of disulphide bonds and removal of keratan sulphate chains with keratanase, both G1 and G2 domains were shown to contain the epitope. However, without keratanase digestion the epitope on the G2 domain was poorly detected. The results suggest that a keratan sulphate chain substituted close to the epitope sequence in the G2 domain prevents antibody access to the epitope and thus masks its detection. This shows the 1-C-6 epitope to be a conserved protein sequence in the G2 domain of proteoglycans from different species, but its detection may be masked by glycosylation. PMID- 1703763 TI - Use of anti-idiotypic antibodies as probes for in vitro and in vivo identification of substance P receptor. AB - In order to develop immunological tools for studying the receptor of the neuropeptide substance P (SP), anti-idiotypic antibodies (anti-Id abs) were produced by immunization with anti-SP antibodies whose specificity was close to that of the SP receptor. Immunological studies revealed structural similarity between some anti-Id abs and SP itself. As a consequence, these anti-Id abs were able to bind to mammalian SP receptors. These antibodies were used in immunocytochemistry to label SP receptors both in the rat spinal cord and in rat and guinea pig peripheral tissues (parotid gland and trachea, respectively). Like SP, anti-Id abs were able to trigger protein secretion by isolated rat parotid gland cells. Finally, it was shown that anti-Id abs in vivo modulated reactivity to chemical stimuli. These antibodies therefore appear to be promising tools for further biochemical, cytochemical, and pharmacological characterization of SP receptors. PMID- 1703762 TI - Antibodies as probes for ligand gating of single sarcoplasmic reticulum Ca2(+) release channels. AB - A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single channel level. PMID- 1703764 TI - Factors influencing the expression of endogenous retrovirus-like sequences in Rat 6 cells. AB - Previous studies from this laboratory have demonstrated increased levels of expression of endogenous rat leukemia virus (RaLV) and 30S retrovirus-like sequences in liver and colon tumors induced by chemical carcinogens in rats. During the process of normal liver regeneration, the levels of RaLV RNA were dramatically increased, whereas the levels of 30S RNA did not change. The present study examined several factors that might influence the expression of these sequences in the Rat 6 embryo fibroblast cell line. Rat 6 cells in either log phase or confluent cultures were treated with cycloheximide, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), or the activated carcinogen benzo[a]pyrene diol epoxide (BPDE) for various periods of time up to 48 h. Northern blot analyses of total RNA indicated that cells in untreated cultures in log phase had higher levels of RaLV and 30S RNAs than did confluent cells. Within 10 h cycloheximide (2 or 10 micrograms/mL) markedly increased the levels of RaLV and 30S RNAs in both log-phase and confluent cells. BPDE (100 ng/mL) induced a marked increase in the levels of RaLV RNA at 4 to 10 h, which returned to the basal level by 24 h in the log-phase cells; but no significant change was seen in the confluent cells. The level of 30S RNA also increased moderately in the BPDE treated log-phase cells and was maximal at 24 h; but no change was seen in confluent cells. Treatment with TPA (100 ng/mL) induced no significant increase in either RaLV or 30S RNA levels in the log-phase or confluent cells. The exposure of Rat 6 cells to 5-azacytidine (3 microM for 24 h) led to a marked increase in the levels of both RaLV and 30S RNAs, which persisted during at least 15 subsequent passages in the absence of the drug. Thus, inhibition of protein synthesis, DNA damage, or hypomethylation can increase the expression of certain endogenous retrovirus-like sequences, but an activator of protein kinase C does not. PMID- 1703765 TI - The v-ras oncogene inhibits the expression of differentiation markers and facilitates expression of cytokeratins 8 and 18 in mouse keratinocytes. AB - Cultured mouse keratinocytes can be initiated in vitro by the introduction of a v rasHa gene by viral transduction. Previous studies indicated that v-rasHa transduced keratinocytes have a high proliferation rate in medium with 0.05 mM Ca2+ and resist terminal differentiation in medium with greater than 0.1 mM Ca2+, a culture condition in which normal cells mature into squames. The current studies demonstrate that v-rasHa keratinocytes do not express transcripts or protein for epidermal early differentiation markers keratins 1 and 10 when cells are challenged with 0.12 mM Ca2+, which is a signal for expression of these genes in normal cells. Both transcript and protein for the late differentiation marker loricrin are also diminished in v-ras keratinocytes, but filaggrin, also a late differentiation-related gene product, is expressed in nearly normal amounts but at a different Ca2+ optimum. Modification of intracellular Ca2+ with ionomycin failed to restore the expression of any suprabasal keratinocyte markers. In contrast to the effects on normal products of keratinocyte differentiation, the introduction of the v-rasHa gene facilitated the expression of keratins 8 (K8) and 18 (K18). These keratins are characteristic of embryonic cells and cells of simple adult epithelia but not stratified squamous epithelia such as skin. Like normal differentiation markers, the expression of K8 and K18 was dependent both on the v-ras oncogene and the Ca2+ concentration of the culture medium, with greater than 0.1 mM Ca2+ being optimal. At the optimal Ca2+ level, the majority of v-ras keratinocytes expressed K8 and K18 after 96 h, and many cells had reduced amounts of the normal keratinocyte cytokeratin K14. These studies indicate that the v-ras gene causes substantial reprogramming of epidermal physiology, producing an unusual phenotype devoid of early suprabasal markers but at least partially permissive for late marker expression. Furthermore, the Ca2(+) dependent expression of K8 and K18 suggests that a normal signalling pathway used in keratinocyte differentiation is diverted to an abnormal endpoint. PMID- 1703766 TI - Endogenous retroviral expression in the human placenta. PMID- 1703767 TI - Spontaneous release of granulocyte colony-stimulating factor (G-CSF) by alveolar macrophages in the course of bacterial pneumonia and sarcoidosis: endotoxin dependent and endotoxin-independent G-CSF release by cells recovered by bronchoalveolar lavage. AB - Because granulocyte colony-stimulating factor (G-CSF) is known to induce granulopoiesis and activate mature neutrophils, this factor could be important in determining the number and functional activity of neutrophils at sites of lung disease. The purpose of this study was to evaluate the ability of lung immune and inflammatory cells to produce G-CSF, and to seek evidence for the spontaneous production of this factor by cells recovered by lavage from controls and patients with lung diseases in which neutrophils may play a pathogenetic role. Lavage cells from controls produced little G-CSF spontaneously. Alveolar macrophages (AM), but not lymphocytes, produced large amounts following endotoxin stimulation. Lavage cells from patients with respiratory failure associated with bacterial pneumonia, but not those with respiratory failure from noninfectious causes, spontaneously released G-CSF (32 +/- 24 and less than 1 U/10(6) AM, respectively). Lavage cells from five of 15 patients with sarcoidosis and one of five patients with diffuse pulmonary fibrosis also spontaneously released G-CSF, which could not be explained by endotoxin exposure. The release of G-CSF by endotoxin-dependent and -independent mechanisms could play a role in the recruitment and activation of neutrophils in bacterial pneumonia and participate in the pathogenesis of some interstitial lung diseases. PMID- 1703768 TI - Role of ultrasonic tissue characterization to distinguish reversible from irreversible myocardial injury. AB - Tissue characterization reflects structural and functional integrity of tissues. Inasmuch as reversible ischemia causes no structural damage and irreversible ischemia results in persistent structural myocardial damage, we postulated that ultrasonic tissue characterization can distinguish the two types of injuries. Anesthetized open chest dogs underwent 15 minutes (group 1, n = 5) and 90 minutes (group 2, n = 8) of acute total occlusion of the left anterior descending coronary artery, followed by 3 hours of reperfusion. Myocardial ischemia infarction was confirmed with segment shortening, electronmicroscopic examination, and triphenyl tetrazolium chloride staining. Integrated backscatter Rayleigh 5 (IBR5), a measure of ultrasonic backscatter, and Fourier coefficient of amplitude modulation (FAM), an index of cardiac cycle dependent variation in backscatter, were measured at baseline, during ischemia, and after reperfusion. Group 1 (reversible ischemia) showed an increase in IBR5 from -48 +/- 1.2 dB at control to -45 +/- 1.0 dB (p less than 0.01) during ischemia, which returned to baseline after reperfusion (-47 +/- 1.3 dB). FAM was blunted during ischemia (6.2 +/- 1.0 dB during control versus 1.2 +/- 1.0 dB during ischemia, p less than 0.01) and recovered completely during reperfusion. Segment shortening was abolished during ischemia (18% +/- 3% during control versus -12% +/- 5% during ischemia, p less than 0.01) and recovered partially during reperfusion (4% +/- 5%). The group 2 animals with irreversible myocardial injury showed an increase in IBR5, from -49 +/- 1.2 dB during control to -44 +/- 1.0 dB during ischemia (p less than 0.01) and paradoxical bulging of the ischemic region (17% +/- 3% to -7% +/- 3%, p less than 0.01) during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703769 TI - Phase III trial of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) versus cisplatin, etoposide, bleomycin and prednisone (CisEBP) for the treatment of advanced non-Hodgkin's lymphoma of high grade malignancy. The Danish Lymphoma Study Group. AB - The trial included 85 previously untreated patients (median age 61 years) with stage III or IV non-Hodgkin's lymphoma (NHL) of the subtypes centrocytic lymphoma, diffuse centroblastic lymphoma, immunocytoma, immunoblastic lymphoma, or unclassified lymphoma of high grade malignancy. The patients were randomized to 9 monthly treatment cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or CisEBP (cisplatin, bleomycin, etoposide, prednisone). Patients who had failed to achieve even a partial response (PR) after the completion of 2 cycles were switched to the alternative regimen. Complete response (CR) on primary treatment was obtained in 70% (55-83%) of CHOP-treated patients and in 25% (13-41%) of CisEBP-treated patients (p = 0.0004). Secondary CHOP treatment produced CR in 7 (30%) of 24 patients and secondary CisEBP treatment led to CR in 2 (15%) of 14 patients. The median survival was 3.4 years in the CHOP arm and 2.6 years in the CisEBP arm (p = 0.78). Hematologic toxicity was mainly leukocytopenia and anemia in both treatment arms. Non-hematological toxicity was slight, and late toxicity was insignificant. Three treatment-related deaths were noted. We conclude that CHOP induces more remissions than CisEBP in advanced lymphomas of high grade malignancy. PMID- 1703770 TI - The specificity of the binding of platelet activating factor (PAF) to anti-PAF antibodies. AB - Quantitative hapten inhibition experiments employing sheep anti-PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C16- and C18:1 PAF proved to be the most potent inhibitors and more active than C18-PAF while phospholipids with a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The 1-acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2. PMID- 1703771 TI - What can we expect from myeloid growth factors? PMID- 1703772 TI - Alpha-fetoprotein-lectin binding as a marker of tumour activity or liver damage. AB - To establish whether alpha-fetoprotein (AFP) produced in the early post-treatment phase of a patient with a germ cell tumour of the testis or the ovary originates from the tumour or is due to an underlying disturbance in liver function, the binding of AFP to concanavalin A (Con A) was investigated as a discriminative variable. A two-step assay is described that can distinguish the type of AFP produced at levels as low as 10 ng/ml. A Con A-binding ratio of 12-43% was found in the patients with disseminated germ cell tumours and in patients with AFP positive gastrointestinal carcinomas. AFP from the liver gives ratios below 10%. PMID- 1703773 TI - Post-extrasystolic potentiation without a compensatory pause in normal and diseased hearts. AB - Variables derived from left ventricular volume were used to study post extrasystolic potentiation. Left ventriculograms were obtained from 11 healthy individuals and 49 patients with coronary heart disease (30 with a previous myocardial infarction and 19 without any signs of myocardial damage). Post extrasystolic potentiation was induced by a regularly driven right atrial rhythm that was interrupted by one atrial extrasystole in such a way that the post extrasystolic RR interval was kept equal to the basic RR interval. The left ventricular end diastolic volumes of the pre-extrasystolic and post-extrasystolic beats were equal. In all groups there was evidence of post-extrasystolic potentiation in one or more of the indices of left ventricular function (ejection fraction, mean normalised systolic ejection rate, and systolic volume, and stroke volume). Potentiation was especially evident in patients with left ventricular damage; this suggests that a compensating mechanism is an intrinsic property of the myocardium. The Frank-Starling mechanism does not contribute to the increased performance of the post-extrasystolic beat in normal individuals or in patients with coronary artery disease. PMID- 1703774 TI - Keratinization of rat vaginal epithelium--V. Modulation of intracellular calcium by estradiol. AB - Changes in the calcium levels under the influence of estradiol were investigated in rat vaginal epithelial cells (VEC). After single estradiol injection, the immature rats showed 1.5-fold increase in Ca2+ levels within 15 min when compared to control animals. Progesterone priming brought calcium levels well below control values throughout the experimental period (up to 12 h). Ca2+ levels in serum did not show any appreciable change. Localization of calcium in VEC with electron microscopy showed aggregates of calcium oxalate on the inner nuclear membrane, nucleolus, mitochondria and keratohyaline granules. After 15 min of estradiol priming, maximum electron density was seen on all these cell organelles mentioned above, however, by 30 min the electron density was reduced considerably and did not increase during the experimental period (up to 12 h). PMID- 1703775 TI - Multiple synergistic signal transduction pathways regulate c-fos expression in Swiss 3T3 cells: the role of cyclic AMP. AB - The promoter region of the c-fos gene contains several upstream enhancer elements which dictate the transcriptional response to specific intracellular signals. Among these is the cyclic AMP (cAMP)-responsive element, which is required for c fos expression by cAMP in vitro. However, we have previously shown that cAMP elevating agents cause only a slight increase in c-fos mRNA levels in intact Swiss 3T3 cells. Here, we show that cAMP can potentiate the induction of c-fos by other second messengers. A combination of forskolin and either phorbol-12,13 dibutyrate or epidermal growth factor leads to an increase in c-fos mRNA levels comparable to those induced by bombesin or serum. Furthermore, cAMP-elevating agents can act in synergy with calcium ionophores (which alone do not induce c fos) to increase c-fos expression by up to 60% of the bombesin response, although, in parallel cultures, this combination does not stimulate the phosphorylation of 80K, an Mr 80,000 protein, which is a major substrate for protein kinase C. These results suggest that, in intact cells, cAMP acts synergistically with distinct intracellular signals to stimulate c-fos transcription through protein kinase C-dependent and -independent pathways. PMID- 1703776 TI - Regulation of mdr RNA levels in response to cytotoxic drugs in rodent cells. AB - The mdr gene, which encodes an energy-dependent multidrug efflux pump termed P glycoprotein, is expressed in some normal human and rodent tissues, including the adrenal gland, kidney, liver, colon, small intestine, and brain and testis capillary endothelial cells. Because of the important role played by the multidrug transporter in determining sensitivity of normal tissues and resistance of cancers to chemotherapeutic drugs, we and others have been determining the environmental factors which regulate expression of the mdr gene. In previous studies, expression of the human MDR1 gene has been shown to be regulated by heat shock, arsenite, and cadmium in a kidney carcinoma cell line, and mdr RNA is dramatically elevated in rat liver after partial hepatectomy or treatment of the animals with cytotoxic agents. We have now investigated the genetic response of the mdr gene to acute cytotoxic insults in rodent and human tissue culture cells. Following exposure to several drugs, most of which are known to be substrates for the multidrug transporter, mdr RNA levels were found to increase substantially in the rodent cells, but not the human cells. Furthermore, RNA levels for topoisomerase II, an intracellular target for these drugs, decreased in the rodent cells. These results suggest a complex pattern of regulation of mdr RNA levels, depending on animal species and cell type, and possible coordinate regulation with topoisomerase II RNA levels. PMID- 1703777 TI - Potentiation of MyoD1 activity by 5-aza-2'-deoxycytidine. AB - A mouse myogenic determination gene, MyoD1, was transfected into the human osteogenic sarcoma cell line TE85. Several stably transfected clones were isolated which, at low frequencies, formed multinucleated cells with the appearance of skeletal myotubes. Southern blot analysis confirmed the integration of multiple copies of the mouse MyoD1 gene, and Northern analysis and immunofluorescence confirmed its expression in the transfectants. Characterization of the transfectants showed that they expressed immunologically detectable myosin, desmin, mRNA for myogenin, and the delta subunit of the acetylcholine receptor. The cells assembled a functional contractile apparatus since they contracted in response to acetylcholine added to the culture medium. The presence of MyoD1 protein did not abrogate the expression of two genes active in bone cells but not in muscle cells. The transfected cells therefore displayed a chimeric phenotype by expressing simultaneously bone and muscle genes. Interestingly, treatment of the MyoD1 transfected cells with 5-aza-2' deoxycytidine resulted in a substantial increase in the frequency of myogenic conversion. Thus, the methylation inhibitor increased the ability of MyoD1 to function as a trans-acting factor and activate the muscle phenotype. PMID- 1703778 TI - Use of a centromere-specific DNA probe (p82H) in nonisotopic in situ hybridization for classification of micronuclei. AB - The DNA probe p82H was used to visualize centromeric DNA in micronuclei (MN) of human cells. Slides prepared from cultures treated by the aneugen (causing aneuploidy) colcemid showed significantly more MN with centromeric signals than those treated by the clastogen (causing chromosome breakage) bleomycin. These results indicate that in situ hybridization with the alphoid p82H DNA probe is a suitable method with which to distinguish between MN containing whole chromosomes and acentric fragments, and hence allows one to discriminate between the clastogenic and aneugenic effects in MN formation. PMID- 1703779 TI - Localization of DNA adducts induced by N-acetoxy-N-2-acetylaminofluorene in Chinese hamster ovary cells using electron microscopy and colloidal gold. AB - DNA adduct induction by N-acetoxy-N-2-acetylaminofluorene (N-Ac-AAF) has been investigated in Chinese hamster ovary (CHO) cells using immunoelectron microscopy. The major RNA and DNA adducts, N-(guanosin-8-yl)-2-aminofluorene (G C8-AF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), were localized with a rabbit anti-G-C8-AF antiserum and colloidal gold cytochemistry. Appropriate controls, including incubation of untreated cells with normal rabbit serum and immunogen-absorbed serum, demonstrated that colloidal gold deposits were indicative of the presence of adducts. The localization of gold particles in close association with nuclear chromatin revealed high concentration of adducts in DNA and RNA of nuclei. Morphometric evaluation of adduct formation in organelles of from different carcinogen exposures showed that 85-88% of total adducts were concentrated in nuclei. DNA adducts remaining in nuclei after RNAse treatment appeared to concentrate in heterochromatic areas, and these areas contained 59% of bound gold particles by morphometry. A total of 137-178 particles were found in nuclei of treated cells vs. 15-26 in the surrounding cytoplasm. Treated cells incubated with normal rabbit serum or specific adduct absorbed serum showed 19-34 particles for all cellular compartments. PMID- 1703780 TI - The relationship between clinical and biochemical changes following neuroleptic treatment in schizophrenia. AB - Clinical improvement in psychotic symptoms is not immediate when neuroleptic treatment is commenced. Previous studies have demonstrated the development of biochemical tolerance to the effects of neuroleptics on the dopamine system. This study demonstrates a relationship between this biochemical change and the clinical changes occurring in the patients. The results can be explained in terms of dopamine receptor changes in a way that is compatible with the Dopamine Hypothesis for schizophrenia. PMID- 1703781 TI - Reactivity of cloned, expressed human Fc gamma RIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of Fc gamma RIII. AB - We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies. PMID- 1703782 TI - Monoclonal antibodies to epitope of CD45R (B220) inhibit interleukin 4-mediated B cell proliferation and differentiation. AB - A mAb, ALP-2, produced by immunizing rats with proliferating lymph node cells from MRL/Mp-lpr/lpr mice, had an inhibitory effect on recombinant interleukin 4 (rIL-4)-mediated proliferation and differentiation of murine B cells. Kinetic analysis revealed that the ALP-2 exerted these inhibitory effects at an early phase of B cell proliferation and differentiation. Nevertheless, the proliferative response of thymocytes to rIL-4 was not inhibited by ALP-2. In addition, ALP-2 inhibited neither the B cell proliferation induced by interleukin 2 nor the B cell differentiation by interleukin 5. Cross-inhibition experiments, together with immunoblotting analysis, revealed that the LP-2 recognized by ALP-2 appears to be an epitope of CD45R (B220) molecule(s), the isoform(s) of the Ly-5 antigen system. Epitope mapping analysis using CD45 gene transfectants showed that Lp-2 epitope is dependent upon the expression of the first alternatively spliced exon of CD45 gene. Functional studies showed that the mAb to an epitope of CD45R, RA3-2C2, but not RA3-6B2, had similar inhibitory effects on the IL-4 mediated proliferative response of B cells. These findings suggest that the CD45R molecules associated with Lp-2 and RA3-2C2 epitopes are probably related to a signal transduction provided by IL-4 in B cells. The possibility that different pathways are operative for IL-4-mediated signaling between B and T cells has to be considered. PMID- 1703783 TI - Surface immunoglobulin ligands and cytokines differentially affect proliferation and antibody production by human CD5+ and CD5- B lymphocytes. AB - Normal human peripheral blood B lymphocytes were separated into CD19+ CD5+ and CD19+ CD5- subsets by dual-color FACS sorting. In most experiments the cells were activated with Staphylococcus aureus Cowan I (SAC) and cultured in the absence or presence of recombinant human IL-1 alpha, IL-2, or IL-6, or combinations of these cytokines. Unstimulated CD5+ and CD5- B cells showed a comparable, low level of incorporation of [3H]thymidine into DNA. SAC stimulated proliferation of CD5+ and CD5- B cells, and this proliferation was augmented by IL-2 in the case of CD5- B cells. Anti-mu beads stimulated some proliferation of the CD5- subset and augmented SAC-induced proliferation of these cells. In contrast, anti-mu beads did not stimulate proliferation of the CD5+ subset and had no effect on SAC induced proliferation of these cells. CD5+ B cells activated by anti-mu beads were stimulated to proliferate in the presence of IL-4, but not in the presence of IL-2. These observations support the interpretation that two signals are required for proliferation of CD5+ B cells. Using a two-step culture system, SAC activation itself did not induce Ig production by either subset of purified B cells. However, it primed the cells for antibody production in the presence of IL 2. IL-1 and IL-6 by themselves augmented antibody formation by these cells slightly, if at all. However, IL-6, and to a lesser extent IL-1, augmented antibody production in the presence of IL-2. Under the culture conditions used CD5- B cells produced IgM, IgG, and IgA whereas the CD5+ B cells produced almost exclusively IgM. The expression on B cells of surface activation markers was analyzed after culture for 2 days with SAC or anti-mu beads. In both subsets expression of Leu-23 and Leu-21 was increased, with some differences in intensity (Leu-23 greater in CD5+ cells, Leu-21 greater in CD5- cells). SAC increased IL-2R expression to a greater extent than anti-mu beads. In neither subset was expression of CD23 increased. These observations are discussed in the context of the possible role of the CD5+ subset of B lymphocytes as components of a system of natural immunity. PMID- 1703784 TI - Characterization of murine complement receptor type 2 and its immunological cross reactivity with type 1 receptor. AB - We have previously demonstrated that some mAbs prepared against mouse complement receptor type 1 (CR1) bind a 150,000 Mr protein in addition to the 190,000 Mr CR1 protein. We now identify the 150,000 Mr murine protein as complement receptor type 2 (CR2), since: (i) one of the monoclonal antibodies that bind this protein inhibits rosette formation between mouse B cells and C3d-bearing sheep erythrocytes; (ii) as is known for human CR2, this protein is present on B lymphocytes but not T lymphocytes; and (iii) this protein must have affinity for C3b, since it has weak factor I cofactor activity. In addition, this protein resembles the 145,000 Mr human CR2 molecule in size. Since four of the five mAbs that were produced by immunization with CR1 also bound CR2, and they bind to different CR1 epitopes, it seems that murine CR1 and CR2 share multiple epitopes. Injection of mice with one of the CR1-CR2 cross-reactive mAbs almost eliminated both CR1 and CR2 expression, but did not decrease B cell numbers or the expression of B cell IgM, Ia, or B220 antigens. In contrast, injection of mice with a non-cross-reactive anti-CR1 antibody only modulated CR1 expression. These antibodies should thus provide useful tools for the study of the in vivo roles of B cell complement receptors. PMID- 1703785 TI - Production of cytokines by mouse B cells: B lymphomas and normal B cells produce interleukin 10. AB - We have examined a panel of murine Ly-1+ B lymphomas and purified normal murine peritoneal B cells separated into subsets on the basis of expression of the Ly-1 surface antigen, for their ability to produce cytokines. Where possible, we have used a combination of cytokine detection methods in order to compensate for differences in sensitivity and specificity, and the possibility of inhibitors masking an activity. All the lymphomas tested were shown to constitutively express TGF-beta and CSIF/IL-10. In addition, varying levels of IL-6, TNF-alpha and TNF-beta, and G-CSF, were demonstrable in most of the lymphomas, and variants of one lymphoma (CH12) additionally produced varying levels of IL-3, IL-4, and GM CSF. FACS purified normal Ly-1+ and Ly-1- peritoneal B cells, were also shown to express RNA encoding CSIF/IL-10, IL-6, TNF-alpha and TNF-beta, and very low levels of G-CSF, following stimulation with LPS. These data were supported by the detection of IL-6 and CSIF/IL-10 in supernatants from LPS-stimulated Ly-1+ and Ly 1- B cells using specific immunoassays. None of the lymphomas or B cell preparations produced IL-1 alpha, IL-2, IL-5, IL-7, or IFN-gamma. The purity of our normal B cell populations was assessed by phenotypic analysis on the FACS and also by the disappearance of certain mRNA transcripts after purification, e.g. CD4, c-fms, GM-CSF, and IFN-gamma, most of which could be detected in LPS stimulated total peritoneal cell populations. This suggested that our B cell purification method had reduced, to a level undetectable in our assays, contaminating T cells (CD4), macrophages (c-fms, GM-CSF), and NK cells (IFN gamma). Absence of IL-3, IL-4, IL-5, and GM-CSF expression by LPS-stimulated Ly 1+ and Ly-1- B cells reduced the concern that contaminating peritoneal mast cells could account for the observed cytokine production. We therefore believe our data provide strong support for production of a subset of cytokines by LPS-stimulated normal B cells. Both the Ly-1+ B lymphomas and normal Ly-1+ and Ly-1- B cells appear capable of expressing IL-6, TNF-alpha, TNF-beta, and CSIF/IL-10.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1703786 TI - Release of macromolecules and daunomycin from hydrophilic gels containing enzymatically degradable bonds. AB - The synthesis of hydrophilic gels based on N-(2-hydroxypropyl)methacrylamide copolymers crosslinked via degradable oligopeptide sequences is described. The influence of the conditions of preparation and of the gel structure on the equilibrium degree of swelling (network density) was determined. To evaluate the potential of such gels for controlled delivery of macromolecules and drugs, the release of FITC-dextrans of different molecular weights was studied and the rate of release was found to depend mainly on the equilibrium degree of swelling and not on the structure of the crosslinks. However, the degradation of the gels by a mixture of lysosomal enzymes isolated from rat liver (Tritosomes) or chymotrypsin was dependent on both the equilibrium degree of swelling and the structure of the crosslinks (length of the oligopeptide sequence and structure of the amino acid residues). The release of the anticancer drug daunomycin imbibed in gels was pH dependent, the rate of release being higher at lower pH. In addition, a gel was synthesized which contained a pentapeptide in the crosslinks and daunomycin bound via a tetrapeptide side-chain, and in this case, incubation with Tritosomes led to degradation with simultaneous release of the drug. PMID- 1703787 TI - Alu interspersed repeats: selfish DNA or a functional gene family? AB - Within the genomes of higher eukaryotic cells, short interspersed repetitive sequences appear to be ubiquitous, but also remarkably varied with respect to copy number and position. Many of these repeat families, including the human Alu family, can be transcribed by RNA polymerase III, and evidence has accumulated from a variety of sources that levels of repeat transcripts whose transcription is dependent on RNA polymerase III are sensitive to cellular transformation as well as changes in differentiation state. Although interspersed repetitive sequences have in the past been dismissed as nonfunctional, the discovery of the linkage to differentiation state, as well as other recent developments, suggest that the question of repeat sequence functionality should be reexamined. PMID- 1703788 TI - Acidic residues at the carboxyl terminus of p60c-src are required for regulation of tyrosine kinase activity and transformation. AB - Protein phosphorylation sites act to transduce signals into changes in enzymatic activity, representing a point of interaction within a regulatory pathway. The amino acid sequence surrounding a phosphorylation site may well have several functions, including recognition by an appropriate kinase. By generating random mutations in its immediate vicinity, we have examined the sequence requirements of a regulatory tyrosine phosphorylation site, Tyr527, in the proto-oncogene product, p60c-src. The transforming and kinase activities of p60c-src are repressed by phosphorylation of Tyr527. Mutations were made around Tyr527 without changing Tyr527 or the kinase domain. Twenty-nine mutants were sequenced and classified as transforming or nontransforming for Rat-2 cells. Nontransforming mutants contained a surprising variety of COOH-terminal mutations, although acidic residues were present at positions 518 and 524 in all nontransforming mutants. Transforming mutants that contained single-residue changes at Asp518 and Ser522 demonstrated the importance of these residues. Other transforming mutants contained two or more substitutions, but the results are most simply explained if residues Glu524 and Thr523 are also important for normal regulation. Transforming mutations reduced the phosphorylation of Tyr527. We conclude that only a few of the residues in the COOH terminus other than Tyr527 are required to ensure normal phosphorylation and repression of activity in fibroblasts. Other residues may have been conserved during evolution to permit normal function and regulation in other cell types. PMID- 1703789 TI - Lectin binding sites in normal, scarred, and lattice dystrophy corneas. AB - Normal, scarred, and dystrophic corneas were histochemically probed with a panel of 16 lectins by means of an avidin-biotin revealing system. Normal corneal epithelial cells, keratocytes, and endothelial cells expressed at least two distinct N-linked oligosaccharide subsets, of the non-bisected, biantennary and bisected, bi-/triantennary types. Corneal scars stained variably with the lectin subsets described above, and with Maclura pomifera agglutinin. Lattice dystrophy corneas showed a loss of the oligosaccharide expression observed on the plasma membranes of normal epithelial cells, and there was concurrent deposition of extracellular glycoprotein within the corneal stroma, which was of the same oligosaccharide subsets as were lost from the epithelial cell plasma membranes. This extracellular stromal glycoprotein was far more widely deposited than the amyloid and extended well beyond the stromal scarring. We propose that these observations are related and that in lattice corneal dystrophy a glycoprotein(s) is shed from the plasma membranes of epithelial cells and sequestrated within the corneal stroma, where it subsequently stimulates amyloid deposition. PMID- 1703790 TI - Interactions of intracellular mediators of amylase secretion in permeabilized pancreatic acini. AB - Mouse pancreatic acini were permeabilized with streptolysin O to investigate amylase secretion stimulated by various intracellular mediators and the kinetics of secretion as a function of temperature. Amylase secretion was temperature dependent in that the initial rate of Ca2(+)-stimulated secretion increased with increasing temperature. In addition, there was no enhancement of Ca2(+) stimulated secretion by GTP[gamma S] at 14 degrees C, while enhancement was maximal at 30 degrees C. GTP[gamma S]-mediated enhancement of secretion at a given temperature was mostly due to sustained secretion with a small increase in secretory rate. At 30 degrees C Ca2(+)-stimulated secretion was also enhanced by cAMP and phorbol ester (TPA) to similar extents as by GTP[gamma S]. The maximally effective concentration of cAMP was 1-10 microM in the presence of 0.1 mM isobutylmethylxanthine. The enhancements of Ca2(+)-stimulated amylase secretion by all combinations of cAMP (100 microM plus 0.1 mM isobutylmethylxanthine), TPA (1 microM), and GTP[gamma S] (30 microM) were fully additive. In Ca2(+)-free buffer, cAMP, TPA or GTP[gamma S] individually had no effect on amylase secretion. Together, TPA and GTP[gamma S] stimulated Ca2(+)-independent secretion, which was 187 +/- 38% of basal. Cyclic AMP together with TPA and GTP[gamma S] in the absence of Ca2+ stimulated 329 +/- 30% of basal secretion. Ca2(+)-stimulated amylase secretion was decreased about 50% by metabolic inhibition, while the enhancement by cAMP, TPA or GTP[gamma S] was totally blocked by metabolic inhibitors. These data demonstrate that amylase secretion in the acinar cell is mediated by multiple intracellular pathways which act in parallel and probably converge at a distal step in the exocytotic process. PMID- 1703791 TI - Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy. AB - The human androgen receptor (hAR) is an important regulatory protein particularly in male sexual differentiation. The investigation of hAR functionality has been hampered by the lack of AR specific monoclonal antibodies recognizing the functional domains of the receptor. Therefore production of high affinity mono specific polyclonal (PAbs) and monoclonal antibodies (MAbs) directed to the hAR was initiated following the synthetic peptide (SP) strategy. Five hAR specific peptides were selected on the basis of their predicted antigenic properties avoiding homology with other steroid hormone receptors. Peptide specific polyclonal antisera were obtained following selected immunization protocols. Mono specific polyclonal antibody responses were elicited to all peptides in mice and rabbits. Crossreactivity of the peptide specific antisera with the native hAR in various biochemical assays was observed with two out of five peptides. Peptide SP61 (hAR residues 301-320) was used for the generation site-directed MAbs specific for the hAR. Specificity for the hAR was established by immunoprecipitation, immune-complex density gradient centrifugation and immunohistochemistry on human prostate tissue sections. The multi-assay performance of the selected high affinity antibodies proved the usefulness of the straight forward peptide approach and opens a wide field of possible biochemical and physiological investigations into questions related to androgen action. PMID- 1703792 TI - Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients. AB - Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U 14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U 14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells. PMID- 1703793 TI - [Activity of human natural killer cells against target cells possessing different sensitivity to interferon]. AB - The cytotoxic activity of peripheral blood natural killers (NK) against target cells (TC) J-96 and L-929 with high sensitivity to interferon (IFN) action, J-41 and MCB resistant to IFN action and line K-562 labelled by H3-uridine was studied in 14 hrs cytotoxic test. It has been shown that human TC J-96 didn't differ from the J-41 in their sensitivity to NK cytotoxicity and they are strongly resistant to NK than TC K-562. The murine TC L-929 as the human TC didn't differ from the MCB in their sensitivity to NK lysis and had also the same sensitivity to NK as the K-562 cells. PMID- 1703794 TI - [Effects of lymphokines on DNA structure of in vitro cultured human lymphocytes. Connection with cAMP and oligoadenylate synthetase systems]. AB - We investigated the influence of recombinant interferons (INF) alpha 2 and gamma and interleukin (IL) 2 and natural purified IL 1 on the activity of oligoadenylate synthetase proliferation level and the DNA structure of cultured in vitro human peripheral blood lymphocytes. It was shown that the proliferation of mitogen-stimulated lymphocytes increased in the presence of IL 1, IL 2 and INF gamma, but there was no proliferation in the presence of INF alpha 2. Oligoadenylate synthetase activity was increased after 18 hours incubation in the presence of all these lymphokines, but after 48 and 72 hours it was increased only in the presence of INF alpha 2 or ConA or INF alpha 2 with ConA together. INF alpha 2, INF gamma and IL 1 stabilized the DNA structure of intact and mitogen-stimulated lymphocytes. cAMP and oligoA synthetase are the specific second messengers of the interferon system and they stabilized the lymphocytes DNA structure too. Cultivation of lymphocytes in the presence of RNA and protein biosynthesis inhibitors--actinomycin D and cycloheximide was followed by accumulation of alkali-labile sites in their DNA. That means that some short lived proteins are needed for the stabilization of native DNA structure. PMID- 1703795 TI - Differential and synergistic effects of human granulocyte-macrophage colony stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures. AB - The ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM-CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo. PMID- 1703796 TI - IgE-mediated anaphylactic degranulation of isolated human skin mast cells. AB - Isolated human skin mast cells (HSMC) were prepared and cultured overnight before functional and electron microscopic studies. Mast cell suspensions were examined after stimulation with anti-IgE to produce anaphylactic degranulation or examined in buffer-incubated controls. Histamine release was measured in replicate samples. Control, isolated HSMC studied by electron microscopy were well preserved and fully granulated. Although all granule patterns reported for human mast cells were found, crystal granules were the most prevalent, as is true for HSMC in situ. Individual mast cells containing both crystal and scroll granules occurred. Lipid bodies were rare, as in HSMC in situ. Control, isolated mast cells did not express granule changes associated with either piecemeal degranulation or recovery during wound healing in situ; nor were morphologic changes of anaphylactic degranulation present. Spontaneous histamine release was 0% in control samples. Anaphylactic degranulation of isolated HSMC was accompanied by 24% maximum histamine release and characteristically showed extrusion of altered, membrane-free granules through multiple pores in the plasma membrane to the exterior of the cell. Other morphologic aspects of anaphylactic degranulation, as expressed in isolated human lung mast cells, were also present. These events included granule swelling, fusion, alteration of matrix contents, degranulation channel formation, pore formation, and shedding of granules, membranes, and surface processes. The ultrastructural morphology of isolated HSMC and their IgE-mediated degranulation shows some differences from similar studies of isolated human lung mast cells and of human lung and gut mast cells in biopsy samples. These differences include crystal granules as the predominant granule pattern, minor numbers of lipid bodies, and extrusion of granules during anaphylactic degranulation as characteristic for HSMC. By contrast, isolated human lung and gut mast cells have more scroll granules and particle granules, respectively, and more lipid bodies. In isolated human lung mast cells, anaphylactic degranulation is almost exclusively an intracellular fusion event characterized by the formation of complex degranulation channels within which altered granule matrix materials solubilize. In addition to morphologic differences between mast cells of skin, lung, or gut origin, functional differences have also been reported among mast cells of these organs. The ultrastructural morphology of isolated HSMC is identical to that of skin mast cells in biopsy samples, thereby validating the usefulness of this new source of HSMC for correlative functional and morphologic studies. PMID- 1703797 TI - T-cell translocation gene 1 (Ttg-1) encodes a nuclear protein normally expressed in neural lineage cells. AB - We previously identified and cloned T-cell translocation gene 1 (Ttg-1), a putative zinc finger protein, as a result of its deregulated expression in a T cell acute lymphoblastic leukemia cell line (RPMI 8402) with a t(11;14)(p15;q11). We have now characterized its genomic organization and identified the major transcriptional start site to lie within an initiator-like motif. Ttg-1 is normally expressed in mouse brain and not in thymus. The mouse neuroblastoma cell line, N2a, also expresses Ttg-1. Antibodies raised against a TrpE-Ttg-1 fusion protein precipitate an 18-Kd nuclear protein from metabolically labeled 8402 cells. Immunofluorescence of N2a cells shows a nuclear pattern. The two potential zinc finger domains in Ttg-1 are highly homologous to similar regions in lin-11, mec-3, and lsl-1. This data suggests that Ttg-1 may be involved in gene regulation. PMID- 1703798 TI - Neutrophil activation through high-affinity Fc gamma receptor using a monomeric antibody with unique properties. AB - The high-affinity, type I Ig Fc receptor (Fc gamma RI) for human IgG1, human IgG3, murine IgG2a, and murine IgG3 is highly expressed on monocytes, neutrophils (PMN) in certain disease states, and phagocytes treated with interferon-gamma (IFN-gamma). We studied the activation of the human PMN oxidative burst and stimulated fluid pinocytosis induced by three monoclonal antibodies (MoAbs) directed against Fc gamma RI (CD64) to study the role of this receptor in Fc mediated cellular activation. All three MoAbs were capable of triggering PMN activation from IFN-gamma-treated PMN when cross-linked with goat antimurine Ig reagents. However, MoAb 197 alone demonstrated a concentration-dependent activation of the oxidative burst without the use of a second cross-linking antibody. The oxidative burst and stimulated fluid pinocytosis responses induced by monomeric MoAb 197 could be elicited only after the IFN-gamma induction of approximately 8,000 Fc gamma RI receptor equivalents and did not occur in freshly isolated or control-cultured PMN. Competitive blocking of Fc binding of MoAb 197 by human IgG or purified Fc fragments inhibited cellular activation. We believe the ability of MoAb 197 to activate these oxidative burst and fluid pinocytic responses was because of its murine IgG2a subclass, which allowed it to function as a trivalent anti-Fc gamma RI antibody binding through the combination of its two FAB regions and the Fc domain. This study demonstrates that the cross-linking of CD64 can activate PMN oxidative and endocytic responses and supports a role for Fc gamma RI in the human neutrophil inflammatory response. PMID- 1703800 TI - Estimation of human blood plasma 5-hydroxyindoleacetic acid and homovanillic acid. AB - A method for simultaneous quantification of plasma homovanillic acid and 5 hydroxyindoleacetic acid has been developed, permitting more efficient neurochemical examinations of these often interrelated biogenic amine systems. Zinc sulphate and sodium hydroxide solutions were used for precipitating the protein in plasma prior to injection on the column. This technique allows for cleaner chromatography, greater sensitivity and high precision. The method uses high performance liquid chromatographic separations of these compounds on C18 reversed phase columns with electrochemical detection. The detailed results from controls and untreated parasuicide patients are given. PMID- 1703799 TI - Increased urinary excretion of 2-thiobarbituric acid reactants in rats exposed to diesel engine exhaust. PMID- 1703801 TI - [Transfusion medicine at present and in the future]. PMID- 1703802 TI - [HIV study of the German Red Cross of West Germany 1985-1989: prevalence/incidence and mode of infection, retrospective studies]. AB - All German Red Cross Blood Banks in the FRG cooperate in a study on prevalence and epidemiology of HIV-antibodies in blood donors. The prevalences of 1 to 2 Western blot positive donors per 100,000 donors/donations quarterly since 1987 are constantly low. The measures undertaken to defer persons at risk seem to be effective. The "look back" data confirm the "rest risk" of HIV-infections by blood products in the FRG which is estimated to be about 1:1 million on the basis of the epidemiologic data. More than 90% of the donors infected are members of risk groups of heterosexual partners to persons at risk. Until today, in blood donors there is no indication of an increasing number of unaware heterosexually acquired HIV-infections. PMID- 1703803 TI - [Medical aspects of blood transfusion 1989/90. 23rd Congress of the German Society for Blood Transfusion and Immunohematology. Kiel, 5-9 September 1989. Proceedings]. PMID- 1703804 TI - [Production of leukocyte-poor thrombocyte concentrates: results with a new kind of filter system]. AB - A newly developed filter-system (Sepacell PL) for the removal of leukocytes from single donor platelet concentrates was tested. It removes contaminating leukocytes by 99.9%, the platelet loss was calculated as less than 1%. Platelet ultrastructure and function do not seem to be altered by the filtration. Therefore, the tested filter system seems to be a suitable device for leukocyte depletion of single donor platelet concentrates. Further clinical studies, however, are necessary to prove the efficiency of the filter system in preventing alloimmunization in vivo. PMID- 1703805 TI - [Effect of erythrocyte contamination on thrombocyte storage]. AB - The influence of red cells on the storage of platelet concentrates (PC) is still questionable. Therefore, we investigated 40 PCs with 4 different red cell concentrations (less than or equal to 5000, 10,000, 50,000 and 100,000/microliters), but equal leukocyte contamination, in a paired study during 5 day storage. In correlation to the red cell contamination, there was a significant reduction of platelets, discoids and especially "functional active" platelets already after addition of the red cells, which was due to a spontaneous platelet aggregation. The difference increased during the storage (p less than 0.05 and p less than 0.001). The other parameters investigated (pH, aggregation, adherence, beta-TG) showed the well-known storage alterations, but no special influence of the red cells. Therefore, PCs should contain the least red cell contamination for storage purposes. PMID- 1703806 TI - [Changes in the thrombocyte count and density in thrombocyte concentrates during storage with reference to thrombocyte concentration and composition of the bags]. AB - 25 platelet concentrates prepared from fresh whole blood are stored for 5 days in different bags: smooth inside surface: Poli-Olefine PL 732 (Fenwal) n = 11; PVC F 702 (Biotest) n = 5; rhombic inside surface: PVC BBC 1030 (Terumo) n = 9. Samples are separated with a Perkoll gradient of densities (1.053, 1.069, 1.079 and 1.095). The platelet count of each sample of the density fractions are determinated in a Neubauer counting chamber. Moreover, pH and pCO2 are estimated. The platelet count is reduced during storage in bags with rhombic inside structure. The analysis of pH and pCO2 do not show significant differences between the groups. In platelet concentrates with counts greater than 1200 x 10(9)/L, stored in bags with a rhombic inside surface, the highest portion of light (density 1.053) and the lowest portion of heavy (density 1.079) platelets are found at the end of storage time. PMID- 1703807 TI - [Bacterial growth of thrombocyte preparations during storage time]. AB - Standard practice of platelet storage at room temperature may contribute to the risk of sepsis after platelet transfusion. Platelets in vitro inoculated with 10(3) or more different microorganisms showed logarithmic bacterial growth throughout the 5 days of storage. These data support the hypothesis that bacterial contamination of platelets might become clinically significant during the 5 days of storage. PMID- 1703808 TI - Plateletapheresis with the new cell separator AS-104. AB - Summarizing several studies, it can be shown that the new cell separator AS-104 (Fresenius), in comparison to the older systems, provides a real advantage concerning the separation efficiency, the purity of the platelet concentrates (PC), the platelet function and the donor safety. The results of 5 different separation protocols demonstrate the technical flexibility and developmental potential of this system. From the experience of 580 plateletapheresis runs the successful improvement of the cell separator, and the disposables can be documented as the frequency of disturbances and discontinuations are reduced considerably. The preliminary results of an uncontrolled, not randomized transfusion study of 244 transfusions in 49 patients show comparable posttransfusion increments as obtained with PC of the CS-3000 (Baxter). PMID- 1703809 TI - [Screening for HIV-1 antibodies in recipients of potentially HIV infected preserved blood]. AB - On 7/1/85 the screening of HIV-1-antibody as marker of HIV infectivity of blood has been introduced in our institute. Since then 76,244 donations have been screened. 15 of long-term donors proved to be seropositive. All their blood preparations before seropositivity were traced back for at least 2 years. The look back of the infectivity of these components is given in the table. The conclusion seems warranted that safety of blood transfusion has clearly improved. The time-interval of seronegative but infectious donations of later seropositive donors, however, remains to be determined. PMID- 1703810 TI - [Comparison of 3 types of cell separators]. AB - The aim of the present investigation was to compare the efficiency of three commercially available cell separators with respect to thrombocyte yields. In addition, advantages and disadvantages of the different cell separator types were studied. There was no difference in the total number of platelets harvested by the different cell separators. In contrast the thrombocyte count of packages from the V 50 device was significantly lower as compared with the others (p = 0.05). We conclude that the cell separators tested are comparable with respect to the efficiency of processing platelet concentrates. However, the larger amount of plasma of the V 50 might be a disadvantage in some circumstances. The AS 104 has advantages in the handling and the duration of cell separation. In addition it offers technical improvements such as control of the ACD flow by a separate adjustable pump and a hemolysis detector. PMID- 1703811 TI - [Comparison of thrombocyte concentrates of the cell separator AS 104 with preparations of the cell separator CS 3000 (Fenwal)]. AB - Platelet concentrates of the cell separator AS 104 (Fresenius) are compared with those of the cell separator CS 3000 (Fenwal). At each cell separator two platelet preparation protocols and at CS 3000 additionally a WBC preparation protocol was analysed. Platelet counts before and after cellapheresis as well as separation efficiencies show no significant differences between the separation protocols (A, B) and the two cell separators. Platelets of varying volumes show different separation efficiencies. While in WBC concentrates the separation efficiencies of platelets continuously increase from small (2fl) to large (20 fl) platelets, the efficiencies in platelet concentrates decrease in volume range 2 fl-8 fl, and increase in range 8 fl-20 fl. There are no differences between the cell separators. The efficiencies of platelets with 2 fl and 4 fl volumes differ significantly (p less than 0.001) between the protocols A and B of AS 104. After WBC-donation, platelet loss of donors corresponds with platelet yields of the concentrates for the platelet volume range 2 fl-20 fl. Compared with the calculated platelet loss of donors after platelet donation more small platelets with 2 fl-8 fl volumes are found in platelet concentrates. The yields of platelets with 10 fl-20 fl volumes are in accordance with donor's platelet loss. PMID- 1703812 TI - [Comparison of 4 procedures in plasmapheresis: studies of modifying the hemostasis potential]. AB - The separation of plasma using the systems "Plasmapur Monitor" (Fa. Organon), "Autopheresis-C" (Fa. Baxter) and "PCS" (Fa. Haemonetics) was compared with the conventional blood bag centrifugation. In 16 apheresis per method, several parameters with the main focus on blood coagulation were examined in addition to other criteria. Compared to conventional centrifugation of blood bags, the plasma separation machines led to an only slight activation of the coagulation system, which seems to be negligible for donors as well as recipients. Furthermore, a decrease of coagulation factors and inhibitors in the collected plasma was most pronounced using bag centrifugation. Beyond this, particularly in apheresis systems with plasma filtration the low number of remaining cells meets the aim to reach a high quality of transfused plasma. PMID- 1703813 TI - [Subcutaneous injection of desmopressin (DDAVP) for increasing factor VIII and von Willebrand factor in plasmapheresis]. AB - Desmopressin (DDAVP) is an effective tool for increasing factor VIII (FVIII)/von Willebrand factor (vWF) in normal subjects and in patients with haemophilia A and von Willebrand's disease. There are a few studies utilizing DDAVP to increase the FVIII/vWF yield in plasmapheresis donors. In these studies, DDAVP was given either by intranasal administration or intravenous infusion. The aim of our pilot study was to investigate the subcutaneous (s.c.) injection of DDAVP in combination with double bag plasmapheresis. 8 donors with blood group A and 5 with O were given 0.4 micrograms/kg body weight DDAVP as a s.c. injection 30 min prior to the first donation. FVIII increased 3.7 and ristocetin cofactor 2.5 the initial value in the donors. In the plasma bags, a 3 (1st bag) and 3.5 (2nd bag) fold higher FVIII level was found, when compared with baseline levels. There was only a mild decrease of blood pressure, and serious adverse effects were not observed. Our data suggest that s.c. DDAVP may be more effective and especially suited for increasing FVIII/vWF yield in plasmapheresis donors. PMID- 1703814 TI - [An in vitro bleeding test--a sensitive method for detection of disorders of platelet function in thrombocyte donors]. AB - The in-vitro-bleeding-test (IVBT) is able to detect sensitively the disorders of platelet function. They are frequently caused by von-Willebrand-syndrome or aspirin. In 7.4% of our thrombocytapheresesis donors the IVBT was abnormal. The plasmatic coagulation has no influence on the IVBT. PMID- 1703815 TI - [10 years plasma exchange with the IBM (Cobe) 2997]. AB - From 1979 to 1989 fifty patients, representing 18 different diagnoses/indications, were treated with 385 non-selective therapeutic plasma exchanges (TPE). In the first five years of the survey, the neurological diseases predominated; since 1984, these cases were treated by the Dept. of Neurology. Five % human albumin solution, fresh-frozen-plasma and saline solution were used as replacement fluids. In the beginning, the exchange volume was 1.5-2 times that of the patient's plasma volume, now it is less and varies with the disorder. ACD A was used as anticoagulant. 11% of the TPE treatments side effects, and in another 3% technical or operator mistakes were noticed. As a supportive therapy, the plasma exchange was effective in the hyperviscosity syndrome, myasthenia gravis, thrombotic thrombocytopenic syndrome, myasthenia gravis, thrombotic thrombocytopenic purpura (TTP), hypercholesterolaemia, Guillain-Barre Syndrome, haemolytic crisis of a homozygous sickle-cell anaemia. There was, however, no definite convincing success in acute liver failure. PMID- 1703816 TI - [Screening of blood donors for anti-HIV-1]. AB - Repeatedly reactive results (also with reproducibility from donation to donation) are found in normal blood donor screening. They are obviously not HIV-1- (and/or HIV-2) specific. In some cases it cannot be clarified if we have to face a seroconversion. The cause of some antibodies (e.g. isolated p17 and/or p24 and/or p55) is not yet known. Therefore we cannot decide whether these results could be neglected or have to be considered a serious risk of transfusion associated infection. PMID- 1703817 TI - [Thrombocyte crossmatching: clinical experience with the Capture P-test]. AB - A commercially available solid phase red cell adherence test (Capture P) for platelet crossmatching was tested. The specificity of the test is 94.8%, the sensitivity 47.9%. Whereas the predictive value for negative results is low (52.9%), the predictive value for positive results was calculated as 93.8%. Therefore, the Capture P test seems to be useful for the exclusion of incompatible single donor platelet concentrates from transfusion. When negative results are obtained, however, no precise prediction of the transfusion success can be made. PMID- 1703819 TI - [ABO blood groups and platelet transfusion]. AB - Platelet transfusion without regard for AB0 compatibility is controversially discussed. Therefore, we studied the success of 136 AB0 compatible and 52 incompatible platelet applications in 37 patients. Our results suggest, that AB0 matching can improve the response of platelet transfusion. PMID- 1703818 TI - [Comparison of 4 crossmatching methods for predicting the success of platelet transfusion]. AB - Long term platelet transfusion support often results in alloimmunization of the recipients and refractoriness to further platelet transfusions. Crossmatch tests between the recipients' serum and the donor platelets offer a potential solution to this problem. In the present study, 207 donor-recipient pairs were studied in 65 patients. We compared four assays (LCT, PAIFT, ELISA and MAIPA) in their ability to predict the response in patients receiving multiple platelet transfusions. All four techniques showed a similar predictability of transfusion outcome (79-80%). Of the four assays, the LCT had the highest specificity (100%) and the PAIFT had the highest sensitivity (70%). In comparison to the three other techniques, the MAIPA assay, a glycoproteinspecific immunoassay, offers the possibility to distinguish between HLA-specific (cytotoxic and noncytotoxic) and platelet-specific antibodies in the same serum specimens. Our results demonstrate that HLA antibodies are the major cause of platelet transfusion refractoriness. Platelet specific (i.e. anti-Zwb) and ABO antibodies are involved in approximately 10%. PMID- 1703820 TI - [Can thrombocyte crossmatching improve the efficiency of platelet transfusion?]. AB - We did crossmatching in 220 transfusions of single donor platelet concentrates using the purchaseable solid phase immunoassay Capture-P (Immucor, Rodermark, FRG), checking posttransfusion response by calculating the corrected count increment (CCI). Our results show that posttransfusion response in transfusions of ABO major incompatible platelet concentrates is virtually reduced only in those transfusions accompanied by a positive crossmatch in Capture-P. As Capture P can detect IgG antibodies only, positive test results could well be due to a reaction of recipient immune isoagglutinins with ABH antigens expressed on donor platelets, therefore. PMID- 1703821 TI - [Selection of HLA compatible thrombocyte donors using electronic data processing with a newly developed score]. AB - We developed a program for personal computers which selects platelet donors for a given patient according to the following criteria: 1. Anti-CMV-status, 2. ABO compatibility of various degrees, and 3. HLA-incompatibility. For evaluation of HLA-incompatibility, we created a score which presently provides the following classification: 0 = identical antigen, 5 = same supertypic antigen or blank in the donor, 50 = very frequently cross-reactive antigen, 75 = frequently cross reactive antigen, very rare antigen or antigen poorly expressed on platelets in the donor, 100 = mismatch. PMID- 1703822 TI - [Component therapy with cryo-poor plasma (CPP)]. AB - National blood programs are means of strategy in the hands of the transfusiologists. Because of the dramatically increase of FFP use new ways in component therapy have to be gone. The use of CPP instead of FFP in operative medicine is one possibility to support an increasing demand of factor VIII. The first results of a clinical trial with special attention to factors I and VIII are presented in this article. PMID- 1703823 TI - [Inhibitor hemophilia A--diagnostic and therapeutic significance of controlled substitutions with factor VIII]. AB - 10 haemophiliacs with constant low inhibitor levels to factor VIII (0.5-2.5 Bethesda units (BU) per ml) and 4 patients without antibodies were examined by a controlled substitution with factor VIII. We investigated the factor VIII in-vivo recovery, the half disappearance time and the biological half-life. 4 different reaction patterns could be observed: 6 patients with antibodies showed the same recovery as those haemophiliacs without inhibitors but the half life was shorter. 2 inhibitor patients did not achieve the expected increments of factor VIII, but had normal half life. One patient had reduced recovery and shortened half life. Only in one patient (inhibitor levels 0.8-1.0 BU/ml) all parameters were in the normal range. PMID- 1703824 TI - [Hemostatic therapy in a patient with acquired von Willebrand disease and Waldenstrom disease with plasmapheresis and DDAVP]. AB - A 73-year old patient suffering from IgM-paraproteinemia, plasma hyperviscosity, and acquired v. Willebrand's disease developed two consecutive bleeding episodes. He was treated by double-bag plasmapheresis and double-bag plasmapheresis combined with DDAVP. This combined therapy was superior to plasmapheresis alone, since bleeding ultimately stopped and did not reoccur. Thus, labour and expense of large volume plasma exchange and the costs of F VIII therapy could be prevented. PMID- 1703825 TI - [Use of phospholipids (Fibraccel) for hemostasis in thrombocytopenic thrombocytopathic patients]. AB - Eight thrombocytopenic/pathic patients received an intravenous infusion of phospholipids. In-vitro-bleeding test, thrombelastography and resonance thrombography were performed in order to show the hemostatic effect. There was no case in which phospholipids had an efficacy comparable to the transfusion of platelets. Two patients suffered from a severe anaphylactoid reaction after the administration of phospholipids. PMID- 1703826 TI - [Quality control of prothrombin complex preparations: in vivo and in vitro findings]. AB - Prothrombin complex concentrates (PCC) are frequently used for the treatment of acquired coagulation factor deficiencies. However, a major problem when using these preparations is their thrombogenic potential. Thus, the potency and tests for thrombogenicity were studied prospectively in 7 different PCC's. Human albumine and a factor IX concentrate served as controls. The potency of coagulation factors and inhibitors varied considerably, especially for proteins S and C. Two preparations exhibited high amidolytic activities in vitro. These activities could be quenched in part by the addition of hirudin or antithrombin III. Additionally, these two preparations caused more severe cardiopulmonary reactions in rabbits and an increase of fibrin split products. We conclude that the use of these preparations in patients, in whom an acquired protein C or S defect, or a hypercoagulable state, can be suspected, cannot be recommended. PMID- 1703827 TI - [Experiences with transfusion of CMV tested blood preparations after bone marrow transplantation]. AB - Cytomegalovirus infections are frequent and often life-threatening in bone marrow transplant recipients. Among 112 of our patients after bone marrow transplantation (BMT), 28 developed clinical symptoms or laboratory test results of CMV infection, and 15 of them died. In our experience blood cell substitution with products from CMV negative blood donors does not completely prevent CMV infections. Other factors like disturbances of immunoreconstitution after BMT are relevant in promoting the development of CMV disease. PMID- 1703828 TI - [Use of blood components in 30 liver transplantations at the Hamburg Eppendorf University Clinic 1984 to 1989]. AB - Among all major organ transplantations, orthotopic liver transplantation (LTX) requires the most extensive blood component support. From 1984 till 1989 we performed 30 LTX procedures on 27 patients. The number of blood components used totalled 1,000 units of packed red cells (RBC), 966 units of fresh-frozen plasma, 246 units of whole blood, 170 units of random-donor platelet-rich plasma and 71 units of platelet concentrates from cell separator. Three retransplantations were performed within 2 days after the first LTX. The lowest use of blood components was registered in patients with hepatoma, the highest in alcoholtoxic cirrhosis. Ten of the 27 patients are still alive. PMID- 1703829 TI - [Stem cell concentrates in autologous transplantation]. AB - For some years, there has been an increasing success in transplanting PBSC instead of autologous bone marrow in patients suffering from leukemic diseases. In healthy cytapheresis donors, we achieved peripheral blood mononuclear cell (PBM) recoveries of 74%, using a discontinuous flow separation technique (Haemonetics V50, Lymphosurge). At the moment, we have collected PBSC from 4 patients (3 AML, 1 ALL) in 28 cytapheresis procedures ranging from 12.9% to 80.1% (mean: 40.1%), whereas stem cell recoveries, defined by CFU-GM, were significantly better (mean: 66%). At present, one of these patients has been transplanted with PBSC alone, receiving 2.11 x 10(8) PBM/kg with 2.8 x 10(4) CFU GM/kg. His posttransplantation cytopenia was shorter compared to other patients undergoing autologous bone marrow transplantation. The period of disease-free survival is now more than eight months while he is completely reintegrated into social and working life. PMID- 1703830 TI - [Erythrocyte substitution and isoagglutinin titer following ABO-incompatible bone marrow transplantation]. AB - About 2 or 3 weeks after transplantation, even ABO-Incompatible bone marrow shows a successful graft. Haemopoiesis is not always free of problems. Suppression of erythropoiesis is caused by persistently incompatible agglutinins. Patient's well being is limited by longer periods of low levels of haemoglobin concentration. Long-lasting need for transfusions is related to the well-known risk of infections. A procedure to eliminate the residual titers of alloantibodies should be discussed in time with the staff of the transfusion department. PMID- 1703831 TI - [ABO-major incompatible bone marrow transplantation. Experiences with a simple method for the preparation of erythrocyte-poor bone marrow]. AB - The special preparative technique presented here, used in ABO-major-incompatible BMTs, enables an excellent stem cell yield (mean: 94%), reducing the number of incompatible RBCs to a minimum (mean: 3.5 ml packed RBCs). This easy procedure is performed by simple mechanical means, using a pair of opposed, specially formed yaws and a modification of a common blood-collecting bag. PMID- 1703832 TI - [Techniques for bone marrow preparation--a comparison of various systems]. AB - Marrow cell concentration is a fundamental requirement when it becomes necessary to remove red blood cells prior to freezing for autologous or ABH-incompatible allogenous marrow transplantation. We compared 5 different separation techniques and two different cell separators (a: Haemonetics V50 and b: IBM/COBE 2997) routinely used for platelet and granulocyte production. PMID- 1703833 TI - [Status of data of HLA-typed donors for unrelated bone marrow transplantation in Hannover: organization and results]. AB - The pool of HLA-A, B and C-typed blood donors in Hannover for cell-donation increased from 2162 in 1988 to 2406 in 1989. 1032 blood donors declared their readiness to donate thrombocytes; 667 would like to donate bone marrow, 453 of them have undergone complete HLA-A, B, C and DR-typing. Until August 31, 1989 we looked in our data bank for an HLA-identical bone marrow donor for 84 patients from other transplantation centers and for 19 patients who are being looked after in Hannover and in national and international data banks. PMID- 1703834 TI - [Search for bone marrow donors for patients with hematologic-oncologic diseases]. AB - In recent years, bone marrow transplantation (BMT) has been used as a curative treatment for patients with intractable hematopoietic disorders, such as leukemia. However, lacking other options, only 30 percent of patients will have an HLA-identical family-member bone marrow donor. For patients without HLA identical family members, HLA-identical, MLC non-reactive unrelated donors have been used. However it is very difficult to find HLA-compatible donors from an unrelated donor registry, due to the highly polymorphic HLA-system. Therefore, we need approximately 6-12 months to find an HLA-matched donor who would agree to donate bone marrow. PMID- 1703835 TI - [Organization of a bone marrow donor database]. AB - We report on the organisation of developing a bone marrow donor file. Emphasis was placed on adequate information for and informed consent and on adherence to legal requirements for maintaining confidentiality. Nearly 7500 blood donors received a three part information, including the possibility of registration as a potential bone marrow donor. About 430 donors are now registered in this bone marrow donor file. PMID- 1703836 TI - [Viral safety in hemotherapy. Current aspects of hepatitis C and parvovirus B19 detection for plasma component therapy]. AB - A major risk of plasma component therapy was the transmission of infectious diseases. Heat inactivation, TNBP/detergent- or beta-propiolactone/UV-treatment were introduced to reduce the risk of virus transmission, most notably those that cause hepatitis or AIDS. Therefore we discuss topical problems of virus inactivation, particularly for the recently discovered hepatitis C-virus and the well-known parvovirus B19. PMID- 1703837 TI - [Personal computer-assisted "acceptable hemoglobin concentration". An example of preoperative autologous blood donation]. AB - At rest, the actual cardiac output (CO) exceeds the CO required to cover the oxygen consumption VO2, provided the hemoglobin level and the non-Hb parameters impacting on cellular oxygen supply (e.g. paO2, pH and body temperature) are normal. The size of this hemodynamic buffer as a function of Hb levels and the non-Hb parameters can be quantified for any clinically conceivable combination including VO2. As Hb levels decrease, the patient's condition is progressively destabilized in the sense that the gradual vanishing of the buffer makes him increasingly sensitive to abnormalities of the non-Hb parameters, such as hypermetabolism, arterial hypoxemia, and alkalosis. This concept is used to illustrate the course of patients participating in an autologous blood predeposit program. PMID- 1703838 TI - [Development and results of autologous blood transfusion in Magdeburg]. AB - The autologous blood transfusion is an important part of the modern hemotherapy. Especially in orthopedics presurgical blood donation of the patients is possible since the operations are exactly planable close. Cooperation between the clinic and the transfusion service is necessary. The results of autologous transfusions in 1,086 patients (1,428 donations) of an orthopedic clinic over a period of six years were critically analyzed. PMID- 1703839 TI - [Autologous blood transfusion in elective mouth, jaw, and facial surgery. Experience with 53 patients]. AB - From June 1987 to July 1989 53 patients with elective operations donated their own blood for a later possible retransfusion. Although autologous transfusion was time-consuming nearly all patients accepted this method because they received the safest of all possible transfusions. So preoperative psychological anxieties could be reduced and the decision for the operation was made easier. PMID- 1703840 TI - [Preoperative autologous blood donation]. AB - This presentation shows our experiences with the preoperative autologous blood donation existing since 1987, 246 patients of the cardiothoracic surgery participated in this program. The preoperative concentration of hemoglobin was above 12g/dl 76.8% of the patients despite the frequent donations, 36.5% of the participants could be transfused with their own blood products. Further reduction of homologous blood transfusion could be achieved with a second preoperative plasmapheresis and the donation of erythropoietin. PMID- 1703841 TI - [Differentiated blood replacement therapy in endoprosthesis surgery of the hip joint]. AB - In a prospective clinical study with 37 patients (41-84 years) undergoing a first implantation of hip joint endoprothesis, a total blood loss of two liters in average was registrated including the first 24 h postoperative wound drainage blood. Sufficient volume replacement was achieved without any homologous blood components by consequently using a comprehensive concept of autologous transfusion including acute preoperative hemodilution, intra and postoperative blood salvage and preoperative plasma predeposit (ATU = Autologous Transfusion Concept of Ulm). Plasma and blood substitution was managed mainly by polygelin and autologous fresh frozen plasma completed by autologous warm blood and processed autologous washed packed red cells. Through this any organization troubles or disadvantages of whole blood predeposit may be avoided. To increase the net gain of washed packed red cells time limited use of blood salvage disposables should be extended. PMID- 1703842 TI - [A new model for optimizing autologous transfusion by retrospective analysis of the transfusion requirement]. AB - To determine the efficacy of an autologous predeposit blood program in a 2000-bed university hospital, we studied a total of 38,500 homologous transfusions administered to 8,806 patients in 1988. Only 4,170 patients were transfused; 18% of those received greater than 12 units and required 61% of all blood products, while patients with 1 to 6 units (65%) used only 22%. Patients were categorized according to similar diagnoses and therapy. For patients with elective surgery and possible predeposit blood we calculated a) the fraction of patients which presumably would not require homologous blood and b) how many of 1 to 10 predonated autologous units would not be outdated. The estimates of utilization are presented as "benefit-disadvantage-diagrams". With increasing numbers of predonations, the fraction of patients with additional needs of homologous blood decreases and the wastage rate of autologous blood increases: Favourable autologous/homologous proportions were found in orthopedic and cardiovascular surgery patients and some women with reduction mammaplasty. The "break-even" point for eligible diagnosis is set on 70 to 90% utilisation rate in patients without prospective need of homologous blood. The optimal number of predonations ranges from 3 to 8 units; depending on diagnosis, surgical patients should predeposit only in selected cases. An estimated maximum of 18 percent of all transfused patients in our hospital may thus be efficiently enrolled in a preoperative autologous blood program. PMID- 1703843 TI - [Is preservation of erythrocyte concentrates with added solutions an alternative to deep freezing in heart surgery autologous blood donation?]. AB - In patients undergoing cardiovascular operations the deep freezing of autologous red blood cells (RBC) was compared with the liquid storage in PAGGS-Sorbitol. On an average 3.9 units of blood were predeposited in the group with deep freezing and 3.4 in the group with liquid storage. The number of complications was the same in both groups despite the shorter intervals between the donations in the group with liquid storage. 58 out of 74 patients (= 78%) with deep frozen autologous RBC and 42 out of 65 patients (= 65%) with liquid stored RBC need no homologous blood. 12% of the liquid stored RBC were discared due to unforseen delaying of the operation. PMID- 1703844 TI - [Preoperative autologous blood bank: communication, documentation, informed consent, iron supplement, testing, blood bank reports, cost control, transfusion]. AB - Preoperative deposition auf autologous blood requires a strenuous effort for its initiation and operation. K.R.A.F.T.A.K.T. (literal translation: strenuous effort) became therefore the acronym for the program realized at this institution; it stands for: K = communication between patient and his physicians (primary care, surgeon, blood banker) R = direction of the program through the primary care physician (who does what, when, where, how and how much of it) A = informed consent of the patient prior to the first donation F = iron supplement (100mg Fe++ daily beginning 2 weeks prior) T = collection, processing, labelling, storage, pretransfusion testing, and release in accordance with GMP and legal requirements A = blood bank reports on the available units to all concerned K = cost control (limited to operations in need of transfusion) T = transfusion of autologous prior to any homologous unit. We report initial experiences and a cost assessment. PMID- 1703845 TI - [Experience with a preoperative autologous blood donation program at a university clinic]. AB - We started our preoperative autologous blood donation program in January 1987, comprising orthopedic, cardiological and gynecological patients with prospective perioperative homologous blood requirements. The safety and practicability of our program could be improved by a precise definition of organizational procedures and the development of a special EDP program. The number of patients/units of autologous blood increased from 35/85 in 1987 to 68/260 in 1988 and will reach approximately 120/500 in 1989. Severe side effects did not occur during that period, 72% of the autologous units were transfused, 80% of the patients did not need additional homologous blood transfusions. Considering unsolved problems of homologous blood transfusions, further development of preoperative autologous blood programs is necessary. PMID- 1703846 TI - [Hematologic parameters in repeated autologous blood donation]. AB - Up to six units of autologous blood can be provided for patients with heart surgery, hip joint replacement or scoliosis. This study was undertaken to evaluate hematological parameters in juvenile and elderly patients and the tolerance of 6 weeks preoperative autologous blood donations. We furthermore investigated the approximate "net blood gain" of the autologous procedure. For an optimal stimulation of erythropoiesis, under vigorous substitution of ferrous sulfate, the autologous donations should start as early as 4 to 6 weeks instead of 2-3 weeks prior to the scheduled surgery, even if only 3 units are required prospectively. The net Hb gain of the autologous procedure in 12-68 years old patients reached a mean of 141 g and 231 g Hb at 4 and 6 donations, respectively. This is equivalent to 2.5 and 4.1 homologous units of RBC (approximately 56g Hb each). Up to 6 units of autologous blood can easily be provided by employing "additive solutions" (PAGGS mannitol), avoiding tedious alternatives like "leap frog-techniques" or freezing of blood. PMID- 1703847 TI - [Comparison of the suitability of first time and related blood donors]. AB - In order to lessen the fear of transfusion-transmitted AIDS, formalized autologous and directed donation programs have been established. The advantages of directed donations, however, have been questioned. A comparison of the qualification of first time homologous and directed donations showed in our groups significant differences for HBsAg positivity, ESR and hemoglobin. The high rate of HBsAg positivity had to be attributed to directed donors of Turkish nationality due to the different epidemiological situation. It seems that directed donations do not improve transfusion safety. PMID- 1703848 TI - [Hepatitis C virus antibodies (HCV) in patients treated with chronic hemodialysis]. AB - Patients undergoing chronic hemodialysis are frequently affected by nosocomial viral infections, as indicated by the high prevalence of HBV antibodies. The question, to what extent HCV is transmitted by blood transfusions (NANB-PTH), or acquired in a nosocomial manner, is still unanswered. We therefore evaluated the HCV antibody rate of 387 sera of dialysis patients on the "Eurotransplant" waiting list. The HCV antibody reactivity ranged from 5.4 to 12.0% between different dialysis centers. In highly immunized (HLA antibody positive) long-term dialysis patients 31.3% (vs. 8.3% in non-immunized patients) were HCV antibody positive. PMID- 1703849 TI - [Experience with ambulatory, preoperative autologous blood donation in heart surgery patients with special reference to hemodynamic parameters]. AB - Our study is based on 843 autologous blood donations that we collected from 379 patients. The coronary angiogram, the diagnosis of the ventricles and the ergometry served to judge the patients suffering from a coronary insufficiency. The complication rate could be reduced to 1% with the help of volume substitution during the donation and the contraindications we ascertained. The exercise electrocardiogram proved to be a significant parameter of the complication rate. It was not the number and the extent of stenoses, but the interpretation of the myocardial oxygen supply that proved to be helpful for the diagnosis based on the evaluation of the coronary angiogram. Our technique of collecting blood and the assessment of the patients' ability as donors give even patients with highly affected coronary vessels the possibility of a preoperative autologous blood donation. PMID- 1703850 TI - [Procedure for producing a resuspended erythrocyte concentrate during technical plasmapheresis: infrequent donation with the same erythrocyte concentrate quality]. AB - This procedure enables gaining an additional red cell concentrate (RCC) during a plasmapheresis (Autopheresis C, Baxter) by using a 3-blood-bag system and the additive solution Sag-M. The quality of the RCC and the fresh frozen plasma (FFP) is equivalent to those prepared from whole blood. This method is adapted to the ambulatory autologous blood donation in cases when more FFP than RCC is needed. PMID- 1703851 TI - [Normal immune function in centroblastic-centrocytic and centroblastic lymphoma]. AB - We tested in vitro lymphocyte reactivity using 5 mitogens and 9 antigens, measured lymphocyte and lymphocyte subpopulation counts, granulocyte chemotaxis and adherence in 10 patients with centroblastic-centrocytic lymphoma (CB-CC), 7 patients with centroblastoma (CB) and 88 healthy controls. The control group could be divided into 3 clusters with significantly different lymphocyte reactivity by means of cluster and discrimination analysis (BMDP2N): 21 high-, 18 medium- and 49 low responders. Comparing lymphocyte reactivity of CB-CC and CB patients to each of the 3 immunofunctional different control groups yielded the surprising result that both malignant lymphomas are equivalent to the low responding control group and show a low, but still normal in vitro immune function. PMID- 1703852 TI - [Normal immune function in highly malignant non-Hodgkin's lymphomas]. AB - We performed lymphocyte transformation tests using 14 different mitogens and antigens, determined T-cell- and T-cell subpopulation counts (CD3, CD4, CD8), the CD4/8 ratio, granulocyte chemotaxis and granulocyte adherence in 88 healthy controls, 8 patients with immunoblastoma (IB) and 6 patients with lymphoblastoma (LB). Using cluster and discrimination analyses (F-test; t-test) according to the programs of the UCLA (BMDP2N) the control group (n = 88) could be divided into 3 groups with significantly different lymphocyte responses to mitogenic and antigenic stimulation (21 high-, 18 medium, and 49 low responders). Comparing the lymphocyte reactivity of IB and LB patients to these immunofunctional different clusters we found a complete accordance of the lymphoma patients to the healthy low responder group. PMID- 1703853 TI - HLA antigen frequencies in familial Crohn's disease (CD). AB - A possible association of Crohn's disease (CD) with MHC (major histocompatibility complex) markers was investigated in families with more than one affected member. HLA-A, B, C, DR and DQ typing was performed in 21 CD families with two or more CD patients. The following HLA-antigens showed increased relative risk (RR) values for CD: B44 (RR = 2.43; B15 (Bw62, Bw63) (RR = 2.03); DR7 (RR = 1.85); DR4 (RR = 1.06). Three of 44 patients were DR4- and four DR7-homozygous. The risk haplotype B44/DR7 was observed in four and Bw62/DR4 in three CD patients, respectively. CD affected family members (female greater than male) shared HLA haplotypes more frequently than expected by mendelian laws. None of the differences reached statistical significance. PMID- 1703854 TI - HLA-antigen frequencies in patients with progressive systemic sclerosis and morphea. AB - A possible HLA disease association was investigated in 40 patients (38 female, 2 male) with progressive systemic sclerosis (PSS) and 42 patients (32 female, 10 male) with morphea. The HLA A, B, C, DR phenotypes of patients were compared with 193 healthy controls. The following relative risk (RR) values were determined in PSS: A1 (1.38), A2 (1.39), B8 (1.67), B15 (3.22), DRw8 (6.30) and in morphea patients: A3 (1.43), B7 (1.39), B40 (1.81), Bw60 (2.49), DR2 (2.38) and DRw8 (2.55), indication a relatively weak, HLA-linked genetic predisposition for the manifestation of the disease. The HLA "risk" antigens for the two clinically different subtypes of the disease are also different: raised A1/B8 frequencies, like in our PSS group, correlate with a high, pathological immune response (autoimmune disorders). In contrast, A3/B7/DR2 prevalence, like in our morphea group, correlates with a low immune response. HLA typing may contribute to clinical differential diagnosis and possibly also prognosis. PMID- 1703855 TI - [Results of treatment of habitual abortion by immune stimulation with partner and/or foreign donor lymphocytes]. AB - 47 couples with repeated spontaneous first trimester abortions were referred for immunotherapy with lymphocytes from spouse and/or unrelated donor towards stimulation of blocking antibody, protecting the next pregnancy. 40 couples were treated, 38% did not yet conceive again. 69% of the 29 observed pregnancies were successful, as were 3 among the 7 untreated women. A prospective, controlled trial is necessary to determine the value of active immunotherapy recurrent spontaneous first trimester abortion. PMID- 1703856 TI - [Immune hematologic diagnosis in women with habitual abortion]. AB - It has been proposed that unexplained recurrent miscarriages are caused by maternal immune rejection of the semi-allogenic fetus. The problem has been treated by immunization with paternal leukocytes to induce maternal antipaternal antibodies that may block immune rejection of the fetus. It has been suggested that excessive sharing of HLA antigens predisposes couples to recurrent abortion. However, we did not find evidence for excessive HLA-sharing. The degree of sharing did not differ significantly between the couples with recurrent miscarriages and controls with successful pregnancies. PMID- 1703857 TI - [Passive immunotherapy with polyvalent immunoglobulins in women with habitual abortions]. AB - Intravenous immunoglobulins (IVIG) were used in an alternative approach to immunotherapy with allogeneic leukocytes to prevent recurrent spontaneous abortions (RSA). Twenty five women with a history of greater than or equal to 3 unexplained RSA were treated with IVIG during their next pregnancy. An initial dose of 30 g (0.5-0.6 g/kg body weight) was administered at gestational week 5-6. Treatment was continued every 3 weeks with 20 g (0.3-0.4 g/kg) and terminated by week 25. In primary RSA the success rate was 82-85%, which is in the same range as for leukocyte therapy. In contrast, IVIG was successful in only 40% of secondary aborters. We conclude that IVIG can be an effective treatment for prevention of primary RSA. Compared to leukocyte therapy the risks of transmission of infections and of HLA immunization can be avoided. PMID- 1703858 TI - [Hepatitis C antibodies in non-A, non-B hepatitis patients and members of HIV risk groups (pilot study)]. AB - In a multi-center study sera from NANB-hepatitis (NANBH) patients and members of so-called HIV-risk groups (homosexuals, i.v.-drug abusers, hemophiliacs) were investigated by the recombinant-based HCV-antibody EIA, 74.4% of chronic NANBH patients and 20% of acute NANBH patients were anti-HCV reactive, 33.3% of HIV-1 positive homosexuals, 43.5% of i.v.-drug abusers and 73.5% of hemophiliacs. The true prevalence of infection remains to be determined by a second, independent (confirmatory) test. PMID- 1703859 TI - [Plasmapheresis and/or intermediate high dosage immunoglobulin therapy--effective measures in threatened premature labor and intrauterine hemolysis in anti-PP1Pk ( Tja)?]. AB - Anti-PP1Pk developed by women has been associated with abortion early in pregnancy and hemolytic disease of the newborn. The case of a 19-year-old woman who had had 2 spontaneous abortions in the first trimester is presented. When treated with plasma exchange and substituted with 5% albumin and intravenous immunoglobulin begun at six weeks' gestation and continued until the 30th week, she delivered a viable female infant without anemia. According to our data and review of literature, the effectiveness of therapy by plasmapheresis and/or immunoglobulin is discussed. PMID- 1703860 TI - [Decreased survival in colorectal cancer. Surprising results of a multivariate analysis]. AB - The survival rate after curative surgery of colorectal carcinoma was reduced by the application of fresh frozen plasma. This result was verified using multivariance analysis. In contrast, the transfusion of erythrocytes with and without buffy-coat was of no influence on postoperative morbidity. PMID- 1703861 TI - Interaction of erythrocytes, lymphocytes and thrombocytes with macrophages after treatment with sialidase. PMID- 1703862 TI - [The effect of endo-beta-galactosidase on lactosamine sialoantigens Gd, Fl, Vo, Li]. AB - Human red cells (RBC) were treated with endo-beta-galactosidase from Bacteroides fragilis removing type 2 polylactosamine chains [Galssl-4GlcNAc]n from the RBC surface by cleaving internal Galssl-4Glc(NAc) bonds of linear lactosamine sequences. I and i built up by unsubstituted branched and linear chains were inactivated. The sialo-antigen Vo resembling i antigen expression on adult, newborn and i adult RBC was also inactivated, demonstrating that the Vo determinant is created by sialylation of linear chains. Fl and Gd antigens known to be sialylated polylactosamine chains were not inactivated. Because Gd antigenicity is increased by increasing the length of sugar sequences and Vo determinants are obviously short chain sequences, the action of endo-beta galactosidase could depend on the length of sialylated sequences on the RBC membrane. PMID- 1703863 TI - [Trials in mathematical standardization of immune hematologic methods]. AB - Immunofunctional methods are characterized by a great range of variation. Our results suggest, that multivariate experiments and mathematics may improve interpretation. PMID- 1703864 TI - [Human blood groups in non-human primates--their significance for immune genetics and biology of evolution]. AB - We studied human blood groups in different nonhuman primates for observing how they were transformed during the different evolutionary stages that eventually led to man. Our results underline that comparative studies of these systems in primates offer a unique tool for the investigation of the development of the principle systems today encountered in man. PMID- 1703865 TI - [Evaluation of the immunomagnetic beads separation technique as a routine method for HLA typing]. AB - We analyzed the HLA data of 1,427 patients and healthy blood donors from Hamburg and Hessen, to evaluate the reliability of the immunomagnetic beads method (IMB), compared to the standard NIH and the twocolor fluorescence (TCF) typing method. Concerning HLA class I, the IMB method and the NIH-method yielded almost identical results, whereas the determination of class II HLA-DR/DQ antigens required a higher rate of retyping (20%), compared the TCF method. Hence, the IMB method should not replace the standard typing procedures, particularly in organ donor crossmatching, unless quality controls, e.g. of HLA-genotyped families have rendered unanimous results. PMID- 1703866 TI - [Importance of screening for antibodies to cytomegalovirus in organ transplantation]. AB - The influence of screening blood donors for CMV antibodies on the incidence of CMV infection after transplantation was examined in 81 heart and 81 liver transplant recipients. All heart recipients received CMV-seronegative blood, while the liver recipients were given both CMV-positive and CMV-negative blood, due to the large number of units of blood required. In CMV seropositive organ recipients CMV reactivations occurred at about the same rate in heart and liver recipients (60.4% to 63.4%), independently from the CMV status of the organ donor. In CMV seronegative organ recipients a CMV infection rate of 60-70% was recorded for the recipients of CMV-positive organs. The recipients of hearts from CMV-IgG-negative donors remained CMV-negative. By contrast, 5 CMV infections occurred in recipients of CMV-negative livers. These CMV infections were probably caused by transfusion of CMV-IgG-positive blood. Our results suggest that CMV negative recipients should be given exclusively CMV negative blood, if possible. PMID- 1703867 TI - [Photometric detection of erythrocyte phagocytosis in the monocyte-macrophage assay]. AB - The relative rate of red blood cell (RBC) in vitro phagocytosis by monocytes was measured photometrically by the diaminobenzidinehemoglobin reaction. Our results demonstrate, that the in vitro monocyte-macrophage assay (MMA) allows a specific and sensitive estimation of low RBC-phagocytosis rates. Therefore, the MMA is a useful tool for further characterisation of antibody-monocyte interaction and is of clinical importance in blood group antibody analysis. PMID- 1703868 TI - [Monoclonal antibodies to antigens of the Rh system]. AB - For the development of monoclonal antibodies, which are useful for typing of Rh system antigens, human monoclonal antibodies are needed. We have established a method for raising human monoclonal antibodies using EBV-transformation and subsequent stabilisation by fusing with mouse myeloma or heteromyeloma cell lines. Using this method, we have produced several clones reacting with the D antigen which can be used for typing purposes as well as for research purposes to differentiate epitopes and categories. PMID- 1703869 TI - [Detection of erythrocyte antibodies using solid phase assays]. AB - The use of solid-phase assays for the detection of irregular antibodies facilitates automatization and rationalization in blood group serology. We compared the solid screen assay, (SP-assay, Biotest AG, Frankfurt, FRG) with conventional assays for antibody screening. The SP assay was as sensitive as the conventional indirect antibody test (IAT). Whereas only 4% of cold-reactive antibodies detected in enzyme tests showed positive results in the SP-assay, this test produced unspecific positive reactions in 7% of all tested sera. We found two additional antibodies with the SP-assay, not detectable in conventional techniques (anti-D, immune anti-A). PMID- 1703870 TI - Advances in solid phase red cell adherence methods and transfusion serology. PMID- 1703871 TI - [The gel centrifugation test in serologic compatibility testing]. AB - The gel centrifugation test is described in compatibility testing. The conventional method was modified resulting in easy handling for technical assistant. 2560 of compatibility testings were performed. 5% of more relevant antibodies could be detected compared with conventional tube test. PMID- 1703872 TI - [3 new antibody detection tests. A comparative study]. AB - Erythrocyte antibodies are of clinical importance since they may cause decreased red cell survival as the result of hemolytic transfusion reactions, hemolytic disease of the newborn or autoimmune hemolytic anemia. A variety of in vitro antibody screening tests are employed to detect their presence in donor and patient sera. Three new methods now available for pretransfusion testing--two solid phase antiglobulin tests and a gel centrifugation test--are compared in parallel with a conventional tube test. PMID- 1703873 TI - [Limits of routine blood group serology in perinatal diagnosis]. AB - We report on a multigravida with high-titer Rh-incompatibility who was admitted for delivery at term. Multiple intrauterine transfusions had resulted in nearly total replacement of fetal cells with donor cells. Routine serologic methods were unable to determine the blood group of the newborn and resulted in a falsely negative direct antiglobulin test. Four weeks later the newborn developed severe anemia in need of transfusion. The anemia was explained by a) suppression of erythropoesis because of early optimization of tissue oxygenization with the prenatally transfused HbA-containing donor cells, b) their limited survival in the infant and c) the accelerated elimination of the baby's own red cells by the maternal antibody. PMID- 1703874 TI - [Cytomegalovirus diagnosis in blood donors and risk patients]. AB - The transfusion transmitted CMV is a possibly dangerous complication of transfusion therapy in immunodeficient patients. We therefore investigated blood donors and patients at increased risk of CMV-infection for CMV-IgG-and-IgM antibodies. The following CMV IgG and CMV IgM prevalence rates were observed: blood donors 63.7% (0.98%), organ donors 76.9% (0%), patients on hemodialysis 83.0% (17.0%) and drug abusers 57.7% (1.3%). We conclude from our results that CMV IgG testing is useful at every blood donation to provide a large pool of CMV negative blood donors. PMID- 1703875 TI - [The gel centrifugation test--principles of the method]. AB - A new procedure for the detection of the antigen-antibody reactions in the immunohematology is described. The new method showed clear and stable reactions improving interpretation of test results. PMID- 1703876 TI - [The "Eilbek capillary"--a new immune hematologic technic]. AB - Studying RBC sedimentation in hematocrit capillaries filled with a variety of separation media (Sephacryl S-300, Ultrogel AcA22 and others) and spinned down for a short time we found some striking advantages in comparison to standard hemagglutination techniques. The scope of diagnostic procedures may be improved and broadened as far as anemias caused by autoantibodies and infections as well as hemolytic disease of the newborn--especially AB0-erythroblastosis--and pretransfusion testing are concerned. PMID- 1703877 TI - Patients with anti-Vel--immunohematological characterization and transfusion management. AB - As reported by other authors we can confirm that the frequency of the blood group Vel negative in Lower Saxony is 1:4000. We report on a patient whose serological characteristics (IgM- and IgA-anti-Vel) made the transfusion of Vel positive blood impossible. Since it was not possible to obtain sufficient Vel negative red cell units in time, the patient was convinced to donate blood for autologous transfusions. This should be the procedure of first choice in the transfusion management of such patients. PMID- 1703878 TI - [Use of a microtest plate method in pregnancy thrombocyte serology]. AB - In search of a time saving concept for verifying a neonatal allo-immune thrombocytopenia (NATP) we tested the suitability of a solid phase platelet assay for antibodies (ab) which might induce NATP. Compared to PSIFT the test is equally appropriate for the detection of platelet specific and HLA ab. Using maternal, paternal, P1A1 neg. and random platelets the test gives quick answers to: a) is one dealing with a NATP? b) is anti-P1A1 involved? PMID- 1703879 TI - [The Sebastian platelet syndrome. A new form of hereditary thrombocytopenia with giant thrombocytes and inclusion bodies in granulocytes]. AB - A new variant of hereditary thrombocytopenia combined with giant platelets and inclusion bodies in the leukocytes different from those found in the May Hegglin anomaly and from Dohle bodies is presented. Investigations of platelet function, platelet nucleotides and clotting studies revealed normal results. Beside their size and spherical shape platelets did not show morphologic abnormalities. The inclusion bodies were similar to those found in the Fechtner syndrome, a variant of Alport's syndrome. However, the feature of Alport's syndrome, including high frequency deafness, congenital cataracts, and chronic interstitial nephritis are absent in the family pedigree. Thus the Sebastian platelet syndrome represents a unique syndrome with giant platelets and inclusion bodies in the leukocytes. PMID- 1703880 TI - [The Br(a)/Br(b) alloantigen system in thrombocytes]. AB - Platelet specific alloantibodies cause neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura (PTP) and may be found in patients who are refractory to HLA-matched platelet transfusion. Most platelet alloantigen systems comprise two alleles: Zw(a)/Zw(b), Ko(a)/Ko(b), Bak(a)/Bak(b), Yuk(a)/Yuk/b). The corresponding antibodies are detected with the platelet immunofluorescence test, radioimmunoprecipitation, immunoblot, and with a glycoprotein-specific immunoassay. Epitopes of the Zw-, Bak-, and Yuk-alloantigen systems are localized on the GPIIb/IIIa complex. Recently, we characterized antibodies against two alleles of a new alloantigen system (Bra/Brb). Anti-Bra is the second most important antibody to cause NAIT, following Anti-Zwa. Anti-Brb was found in three polytransfused patients. The Br-antibodies may be detected using a glycoprotein specific enzyme immunoassay (MAIPA assay) and radioimmunoprecipitation. The frequency of the Br-phenotypes in our population is Br(a+b-) 1%, Br(a+b+) 20%, Br(a-b+) 79%. The number of binding sites for Anti-Bra is 2000/platelet on homozygous and 1000/platelet on heterozygous platelets. The Br-alloantigens are localized on GPIa, which is identical with the alpha chain of VLA-2 on activated T-lymphocytes. PMID- 1703881 TI - [Sr(a), a "private" thrombocyte alloantigen as a cause of neonatal alloimmune thrombocytopenia]. AB - Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal immunization against fetal platelet antigens. In the serum of a mother who gave birth to a child with the typical clinical picture of NAIT we found an antibody directed against the new platelet antigen Sra. Anti-Sra was found to react in the immunofluorescence test and in a glycoprotein (GP) specific immunoassay (MAIPA) using a monoclonal antibody against GP IIb/IIIa for antigen immobilization with the child's and father's platelets. Radioimmunoprecipitation and immunoblot allowed to assign the Sra-antigen to GP IIIa. The phenotype frequency was estimated less than 1%, characterising the Sra-antigen as the first 'private' alloantigen on platelets. PMID- 1703882 TI - [Serologic characterization of platelet-associated IgG and its significance for the diagnosis of thrombocytopenias]. AB - Detection of platelet autoantibodies in patients with autoimmune thrombocytopenic purpura (AITP) is a notoriously difficult task. The quantitative measurement of PAIgG is rather unreliable as a diagnostic procedure because it tends to yield false positive results in thrombocytopenic patients. In this study two different techniques allowing discrimination of platelet autoantibodies and unspecific IgG bound to the patients' autologous platelets were compared to the quantitative measurement of PAIgG in thrombocytopenic patients. These two techniques are: 1. investigation in the platelet adhesion immunofluorescence test of eluates prepared from the patients' platelets and 2. detection of platelet autoantibodies on the isolated glycoprotein complexes IIb/IIIa, Ib/IX, and Ia/IIa. Results show that positive results with these two techniques are much more specific for autoimmune thrombocytopenia than elevated amounts of PAIgG measured on whole platelets. PMID- 1703883 TI - [219 Zw(a) positive mothers of children with clinically suspected neonatal alloimmune thrombocytopenia]. AB - The sera of 219 Zwa-positive mothers who gave birth to children with clinically suspected neonatal alloimmune thrombocytopenia (NAIT) were tested for platelet reactive antibodies using the platelet adhesion immunofluorescence test and a glycoprotein-specific immunoassay (MAIPA). In 102 families a serological crossmatch of maternal serum against paternal platelets was performed. 12 sera contained platelet specific antibodies: Zwb (1), Bra (4), Bra and HLA (5), Baka and HLA (1), Sra (1); three further sera contained antibodies against blood group A (1), blood group B (1), blood group B and HLA (1). Anti-Bra was the most important antibody in this cohort. There is evidence that in certain cases antibodies against blood group antigens A or B may cause NAIT. Clinical data of NAIT duo to anti-Bra suggest a less severe bleeding tendency than in NAIT due to anti-Zwa. The HLA-DRw6 antigen was identified as an immunogenetic marker for the immunization against the Bra-antigen. PMID- 1703884 TI - [CMV antibody determination with two enzyme immunoassays in each case]. AB - In each of the two present studies (Frankfurt blood donor center and blood bank of the university of Hamburg) two commercial enzyme immunoassay (EIA) test kits for detecting cytomegalovirus (CMV) antibodies were compared according to their concordance. In the Frankfurt study, 448 sera of blood donors and patients were tested for IgG- and IgM-specific antibodies by CMV-ELA Test (Medac); IgG-specific antibodies were tested in 3 serum dilutions (1:10, 1:100, 1:1000) of each sample, 221 sera were additionally assayed using the EIA of ABBOTT. Both methods showed the same results in 214/221 (96.8%) sera (p less than 0.001). The determination of CMV-IgG-specific antibody levels were as follows: Out of 294 seropositive individuals, titers greater than or equal to 1:1000 were seen in 81% (238). IgM antibodies to CMV were determined in 22/448 serum specimen, which all had IgG antibodies too; there were 18/22 (81.8%) samples with elevated titers for CMV-IgG (greater than or equal to 1:1000). In the second study, 500 sera of blood donors from Hamburg were tested for CMV-IgG antibodies by two enzyme immunoassays, CMV IgG-ELA (Medac) and Anti-CMV-IgG-ELISA (Biotest). The results were concordant in 418 (83.6%) of the 500 samples (p less than 0.01). All EIA procedures were found to be convenient test methods for CMV antibody screening and the results showed significant correlations. Furthermore, the determination of CMV-IgG-specific antibodies seems to be sufficient for routine CMV screening in blood donor centers. PMID- 1703885 TI - [Serologic characterization of granulocyte-specific antibodies in neonatal alloimmune neutropenias]. AB - Serological properties of nine granulocyte specific alloantibodies in maternal sera and sera of the newborns were investigated by granulocyte immunofluorescence, granulocyte agglutination and granulocyte cytotoxicity. In the immunofluorescence assay the reactions were more prominent than in the agglutination assay. Only one antibody yielded a positive reaction in the cytotoxicity assay. Four NA2-, two NA1- and one NB1-antibody could be identified. The immunofluorescence assay showed a mixed pattern with the NB1-antibody, indicating a heterogenous distribution of the NB1-antigen on granulocytes of some donors. In titration studies with heterozygous and homozygous donors a gene-doses effect was demonstrable. PMID- 1703886 TI - [Autoimmune hemolytic anemia with temporary weakening of Gerbich antigens]. AB - A third patient with autoimmune hemolytic anemia due to autoantibodies against Gerbich antigens is described. The patient's serum contained strong hemagglutinating antibodies of the IgA plus IgG classes which reacted with all red blood cells (RBC) tested, but not with Gerbich-negative cells. Although the patient was typed as Gerbich-positive, his serum failed to react with his own RBC, and the sensitization of his erythrocytes with autoantibodies was only demonstrable if eluates of his RBC were used. The failure of the autoantibodies to react with autologous RBC at the peak of hemolysis most likely reflects a weakening of Gerbich antigens during the course of autoimmune hemolytic anemia. PMID- 1703887 TI - T and Tk transformation in hemolytic uremic syndromes. AB - In two cases of hemolytic uremic syndrome (HUS) the bacterial pathogenesis of the disease could be elucidated by lectin red cell agglutination tests. The possible role of anti-T and anti-Tk antibodies is discussed. Transfusions of fresh plasma had no adverse effects. The fatal outcome in one case was caused by disseminated intravascular coagulation (DIC). PMID- 1703888 TI - [Fatal course of immune hemolytic anemia following administration of diclofenac]. AB - We report of a case of a 56 years old female patient who died of severe haemolytic anaemia induced by an injection of a single dose of diclofenac. We suppose that an immune complex reaction caused the haemolytic crisis. PMID- 1703889 TI - [Analysis of irregular antibodies in the department of transfusion medicine 1984 1988]. AB - In the University Hospital of Hamburg we performed approximate 80,000 screening tests for irregular RBC antibodies in the years 1984-1988. Among 1,815 (2.27%) positive tests, Rhesus and concomitant antibodies occurred in 0.77% (615/80,000), other clinically important antibodies in 0.37% (293 cases). We found significantly less Rh antibodies than in the previous period of the mid seventies (Rh prophylaxis, completely Rh-compatible transfusions). PMID- 1703890 TI - [Guidelines for electronic-data-processing-controlled serial diagnosis of donor blood samples]. AB - Increasing performance figures and the necessity to save expenses oblige transfusion services to automatize their donors' laboratory examination. Sufficient hard- and software for sample distribution and processing is now available. Following aspects should be regarded when switching to automatic serial screening: SAFETY: The identity of blood-donor, donation and laboratory result will be achieved by machine readable labeling and on-line communication between working-stations and central administration. Flexibility: Easy automatic selective laboratory screening will be possible using special barcodes including sample identification and working orders. A modular hardware concept with easily accessible programming control allows it to implement new devices or methods. Ergonomy: Automatic sample processing including selective screening and simultaneous operating robotic sample processors increase working quality, sample output and time benefits. Economy: Improved working conditions will result in saving reagents and compensating staff limitations. PMID- 1703891 TI - [Standardization of bar code information on blood unit labels for automation of blood unit exchange between blood donor and transfusion services and blood banks]. AB - Exchange of blood units between transfusion services and blood depots, using EDP systems requires standardisation of unit identification and bar-codes. The ISBT Working Party on Data Processing defined recommendations for blood-unit identification by Codabar. Our group (German Society of Transfusion Medicine and Immunohematology, Section Data Processing and Standardization) extended and adapted these standards to the German blood bank settings. This paper describes the basic definitions and standards. A complete outline will be published in the next future. PMID- 1703892 TI - [CMV screening with the latex agglutination test in blood donors. Comparison between EIA and latex agglutination]. AB - For CMV-Screening of blood donors the latex agglutination test (LAT) can be used as a complementary rapid analysis to the enzyme linked immunosorbent assay (EIA). In 98.56% there was no discrepancy between LAT and EIA in the serum, in 1.44% there was a negative LAT and a positive EIA. PMID- 1703893 TI - [Transfusion commission and service recommendations for increasing efficiency in transfusion medicine]. AB - Establishing a University Hospital Transfusion Committee has improved the communication between all the hospitals and enhanced the aspects of security and efficiency in transfusion medicine. Moreover, the field of transfusion medicine has gained more recognition through the university hospital management. PMID- 1703894 TI - [Experience with a microcomputer-controlled blood bank administration system in the blood bank of the Hannover Medical University]. AB - During the last three years two components of a modular administration system were implemented in the blood bank of the Medical University of Hannover (MHH). Considering the integration of the blood bank system to the central hospital information system of the clinic the aspect of independence and self-controlling by the blood bank was the most important point. This goal was reached by a couple of PC's which are compounded by a local area network (LAN). This LAN can communicate with the host of the computer center of the MHH. By this way the short staff capacity was not stressed more than absolute necessary by taking over all parameters of the central data base. On the other hand all functions for administrating the blood bank were in self-responsibility of the department. This concept of different modules has the possibility of using single parts like donor or storage-system in other blood banks without implementing the whole system. PMID- 1703895 TI - [Automated serial diagnosis of donor blood samples. Combined pipette robot systems]. AB - Combined robotic sample processors allow economic and ergonomic laboratory screening of blood donors without mismatching of samples. The modular configuration of the whole system serves as basis for easily achievable adaptation to future performance claims or installation at other institutions. PMID- 1703896 TI - [Automated serial diagnosis of donor blood samples. Ergonomic and economic organization structure]. AB - A comprehensive computer-aided administration-system for blood-donors is presented. Ciphered informations of barcode-labels allow the automatic and nevertheless selective pipetting of samples by pipetting-robots. Self-acting analysis-results are transferred to a host-computer in order to actualize a donor data-base. PMID- 1703897 TI - [Blood bank dictionary, definition of the concept and blood unit status for blood recipients--an electronic data processing system]. AB - The definition of terms using the blood bank data dictionary results in a number of advantages in practice. Previously vague terms could be clearly defined when they are broken down into their constituent parts. The relationships within a blood bank can be more precisely documented by working with standard terms. The descriptions within the specification are thereby unambiguous and in this way a precise program specification can be achieved. Standardized terms facilitate the functional comparison of programs from different authors or different software sources. The use of defined terms within wards and the blood bank area results in more transparency even before the introduction of data processing. PMID- 1703898 TI - [Blood component therapy with additives]. AB - Since 1988, 96% of all blood donations in Sweden are given in a multiple blood bag system using a SAGMAN-System. This system has many advantages over conventional blood bags without additive solution. We routinely eliminate 50-80% of the leucocytes and more than 80% of the platelets by separation of buffy-coat from red cells using such a system. We have compared the preparation results of a SAGMAN-System with a similar system containing top and bottom outlets (BAT) using 2 different semiautomatic separation systems. With these new bag systems and separation devices we received similar results. However by optimizing the procedure it should be possible to obtain a higher reduction of leucocytes and platelets at comparable red cell yield using the BAT-System. PMID- 1703899 TI - [Analysis of donor self exclusion in repeat blood donors]. AB - A confidential self-exclusion (self-exclusion means that the donor doesn't want his blood to be used for transfusion purposes) questionnaire was given during 12 months to 19,108 periodical blood donors in the College of Medicine in Hannover. 237 donors stated that their blood was not to be used for transfusion purposes. 70% of them were males, 30% females. 53% of these self-exclusion donors were between 21 and 30 years old. Among these self-exclusion donor, all the HIV antibody-test showed negative results. The blood units from these donors were destroyed. The financial loss was DM 48,000. From among donors who thrice wished self-exclusion, one third declared promiscuity as the motive for self-exclusion, one third did not understand the questionnaire and one third could not or did not want to explain the self-exclusion. The question about the efficiency of the confidential donor self-exclusion process may be answered positively compared to the results of Nusbacher's study, which evaluated five times as many blood donations. PMID- 1703900 TI - [Blood component therapy after conversion to erythrocyte concentrate with added solutions]. AB - Increased national quality requirements for blood products have led to the replacement of the conventional double-bag system with CPDA-1 anticoagulant by a 4-bag system with additive solution, buffy-coat free. Simultaneously the production of fresh blood was stopped for logistical reasons. Acceptance and possible drawbacks of these important changes in blood supply were analyzed in a postmarketing surveillance study involving some 30 hospitals. Generally the imposed changes have found good acceptance. No major problems have been recorded with regard to transfusion technique, albumin replacement, bleeding tendency etc. Pediatric stations experienced some difficulties with low protein levels in exchange transfusion. Some hospitals did regret the disappearance of fresh blood, but its replacement by the combination of red blood cells, fresh frozen plasma and platelet concentrates was well accepted. PMID- 1703902 TI - [A new multiple blood bag system with top and bottom drainage. Comparison with the conventional multiple bag system]. AB - Blood components were investigated following manual preparation in a conventional 4-bag system (A) or automatic preparation in a top and bottom system (B). To overcome inter donor differences two units of whole blood were pooled and subsequently split up into the original bags. Leukocyte-poor platelet concentrates (PC) were manufactured from both types of buffy-coat (A and B). The volumes of the various blood components were similar, whereas the residual leukocytes and platelets in the red cell concentrates from the top and bottom system were significantly lower (p less than 0.01). Both types of PC contained less than 10(7) leukocytes and could be stored for 7 days maintaining a pH above 6.5. We conclude that the top and bottom system enables automatic preparation of red cell concentrates and plasma with a significantly better buffy-coat removal than with manual processing. PMID- 1703901 TI - [49 day storage of erythrocyte concentrates in blood bags with the PAGGS-mannitol solution]. AB - The red cell preservation solution PAGGS-Mannitol differs from the well documented PAGGS-Sorbitol only by the exchange from Sorbitol in Mannitol. In a clinical investigation with volunteers, up to 21 of the pure PAGGS-Mannitol solution were infused within 4 h. Blood and urine parameters were determined. The solution was well tolerated, no unexpected change of blood and urine parameters was found. The 24 h red cell in vivo survival rate of PAGGS-Mannitol was found to be 74.5 +/- 4.4%, after 49 days storage, a value which was described for PAGGS Sorbitol before. In vitro data on storage of red cell concentrates with and without buffy coat were determined for hemolysis, 2,3-DPG and ATP using a blood bag system with DEHP and TOTM plastisized PVC. If 1% hemolysis is regarded as acceptable, red cell concentrates can be stored in PAGGS-Mannitol with a TOTM plastisized blood bag system up to 42 days. Under these conditions the amount of plastisizer in the red cell concentrate was found to be only 1% of the amount determined in a standard DEHP plastisized PVC blood bag system. PMID- 1703903 TI - Separation of platelet concentrates (PC) from buffy coat (BC) using the bottom and top drainage (BAT) system. AB - By the preparation of platelet concentrates (PC) from buffy coat (BC), the blood component separation may be improved considerably. One can use new bag systems with bottom and top drainage (BAT) which allow partial automation of the separation procedure. Thus, it is possible to produce very pure red cell concentrates (RCC) and FFP, as well as BC, by one separation step under well standardized conditions. Several preparative factors influence the quality of the PCs, especially the storage of the whole blood and the BC. Optimal yields (0.72 0.78 x 10(11) platelets), separation efficiency (67.7-70.8%) and lowest cell contamination (leukocytes 0.1-0.2 x 10(8), red cells 0.2-0.3 x 10(8) per concentrate) were obtained with whole blood stored for 1 h and BC for 2-4 h before resuspension by 30 min face-over-face-rotation. In respect to the release of leukocyte proteases during the storage, the BC should not rest for more than 2 3 h before separation of the platelets in order to prevent potentially functional impairment of the platelets by leukocyte proteases. PMID- 1703904 TI - [Production of leukocyte-reduced, neocyte-rich erythrocyte concentrate]. AB - With a new method which is easy to handle, leukocyte depleted neocyte enrichment can be obtained from ordinary whole blood units using centrifugation. In comparison to alternative methods less time for preparation will be needed without significant changes in enzyme activities during storage indicating red cell lesion. PMID- 1703905 TI - [Leukocyte contamination in standard erythrocyte concentrates, prepared with the Biopack-U-bag and Biotrans separator]. PMID- 1703906 TI - [P-31 NMR studies of in vivo regeneration of preservative-induced changes in 2,3 diphosphoglycerate content of erythrocytes]. AB - In this study, we analyzed the regeneration kinetics and the reversibility of the 2.3-DPG reduction in stored red cells after transfusion. Within 3 h after transfusion the 2.3-DPG levels raised up to 40% of the patients' prior 2.3-DPG concentration, although the 2.3-DPG content of the transfused red cells was less than 10% of before. The complete regeneration of the 2.3-DPG concentration occurred after 36 to 48 h after transfusion. There was a close correlation of the results obtained by P-31-NMR and enzymatic determination of DPG. PMID- 1703907 TI - [Complement activation during storage of preserved blood]. AB - We have provided evidence that during storage of CPD-Al stabilized whole blood factors of the complement cascade get activated. We prospectively measured C3a desArg, C4a-desArg, and C42C3-activatorcomplex in 30 units of whole blood in the absence and presence of the proteinase inhibitors aprotinin and eglin over a storage period of 28 days. C3a-desArg increased from basic levels of 276 +/- 35 ng/ml to maximal levels of 1922 +/- 218 ng/ml after 2 weeks of storage. Similarly there was an increase in C4a-desArg levels from 172 +/- 16 ng/ml (day 0) to 1483 +/- 221 (day 14), but C4a-desArg levels further increased with time. By contrast C42 C3-activatorcomplex levels increased only moderately from 170 +/- 18 micrograms/ml (day 0) to 207 +/- 13 micrograms/ml (day 28). The addition of aprotinin and eglin could not inhibit the increase in activated complement factors. Our observations might be of clinical importance, especially during massive transfusions, since C3a-desArg in concentrations less than 1 ng/ml enhances the activation of platelets. PMID- 1703908 TI - [Rapid and sensitive determination of the adenylate energy charge in stored erythrocytes using high pressure liquid chromatography]. AB - Using reversed-phase high-performance liquid chromatography (HPLC), we developed a fast method for the determination of the adenine-nucleotides ATP, ADP, and AMP in human red blood cells (RBC). The method is sensitive (10(-9)-10(-10) M), specific (greater than 95% peak-homogeneity), and the results are well reproducible. The three substances are assayed in a single step, which is essential for the exact determination of the adenylate-energy-charge (AEC). We applied the technique to fresh and stored RBC's. After six weeks' storage in PAGGS-sorbitol we found only a slight decrease in ATP content, but marked increases in ADP and AMP contents. Accordingly, there was a decrease in the AEC, whereas the total nucleotide pool (TNP) did not change significantly. As AEC and TNP reflect the state of the cellular energy metabolism, they are related to the viability of stored RBC. Thus, we consider the described method to be helpful for the quality control of RBC stored in new or modified media. PMID- 1703909 TI - [Recombinant human erythropoietin (rh-EPO) in chronic, dialysis-dependent renal failure: effects on macro- and microcirculation and hematologic parameters]. AB - Recombinant human erythropoietin (rh-EPO) has been shown to be effective in the treatment of renal anemia. Additionally, rh-EPO improves the hemostatic defect of uremia. On the other hand, a hypertensinogen effect and an increased risk for thrombosis have been reported in hemodialysis (HD) patients with rh-EPO. 20 HD patients in Homburg were recruited for a multicenter, placebo-controlled study (MF 3981), aiming to assess the risk of rh-EPO. Initially, 10 patients received rh-EPO at a dose of 3 x 80 U/kg body weight and week which was subsequently adjusted according to the hematocrit. After 6 months, the patients receiving placebo were changed to rh-EPO therapy. Clinical and laboratory data were obtained before, as well as 1, 3, 6 and 12 months after beginning of the study. Erythrocyte counts increased significantly in the rh-EPO group. Also, an increase of platelet count, fibrinogen and plasma viscosity was observed during rh-EPO. Tissue type plasminogen activator and plasminogen activator inhibitor as well as von-Willebrand-factor remained unchanged, although a shortening of the bleeding time was observed. Blood pressure and arterial blood flow were not influenced by rh-EPO. PMID- 1703910 TI - [Initial results of the anti-HIV-1/2 combined ELISA]. AB - Between March 30 and July 31, 1989, 117,874 blood donations were tested for anti HIV-1 and in a combined ELISA for anti-HIV-1/HIV-2 (both Abbott). On each repeat reactive (14 = 0.01% for anti-HIV-1 and 400 = 0.34% for anti-HIV-1/HIV-2), a Western blot (WB) was performed. Two donors, who tested repeatedly reactive in both ELISAs, were confirmed anti-HIV-1 positive by WB. As the specificity of anti HIV-1/HIV-2 ELISA is considerably lower (99.66%) than of anti-HIV-1 (99.99%), introduction of a different HIV-2 specific ELISA should be discussed to prevent loss of too many blood donations and donors through anti-HIV screening. PMID- 1703911 TI - [Preparation of leukocyte-poor thrombocyte concentrates from buffy coat by using a new quadruple bag system]. AB - It has repeatedly been shown that platelet concentrates (PC) can be prepared from buffy coat in guadruple Saline(S)-Adenin(A)-Glucose(G)-Mannitol(M) (SAG-M) systems. Their in vivo and in vitro quality was reported to be excellent and their contamination with leucocytes was 10-20 times lower than in PC prepared from platelet rich plasma. We have confirmed these observations (unpublished results). One particular technical problem which we encountered was the appearance of folds in the satellite bag during the second centrifugation step rendering the purity of the PC quite unpredictable. In collaboration with Biotrans, we have developed a modified quadruple bag (733861100) including a special insert (Heraeus Christ, Osterode, FRG) for the centrifugation rotor which has proved advantageous over existing systems and will be briefly described. PMID- 1703912 TI - [Quality of thrombocyte preparations from buffy coat or platelet-rich plasma]. AB - One hundred platelet concentrates, fifty prepared from buffy coat and fifty prepared from platelet-rich plasma were examined. In platelet concentrates from buffy coat a lower leukocyte contamination (1.4 +/- 1.2 x 10(7) vs 13.2 +/- 15 x 10(7], a significant higher ATP content (6.3 +/- 1.7 mmol/10(14) platelets vs 3.9 +/- 1.9 mmol/10(14) platelets) and a better response in ADP- or collagen-induced aggregation could be determined during storage of five days. Our results indicate that preparation of platelet concentrates from buffy coat is preferable to their preparation from platelet-rich plasma. PMID- 1703913 TI - Diffuse proliferative glomerulonephritis in Behcet's syndrome. AB - Renal involvement in Behcet's syndrome is infrequent and the reported cases of glomerulonephritis consisted mainly of patients with focal glomerulonephritis. A patient with Behcet's syndrome and diffuse proliferative sclerosing glomerulonephritis with predominant disposition of IgM is described. PMID- 1703914 TI - Leiomyosarcoma of bone. A clinicopathologic, immunohistochemical, and ultrastructural study of five cases. AB - The authors identified five leiomyosarcomas (LMS) in a review of 13 nonmatrix producing spindle cell sarcomas of bone. Only two were initially recognized as LMS; the others had been diagnosed as malignant fibrous histiocytoma (two) and fibrosarcoma (one). The patients, four of whom were women, ranged in age from 32 to 70 years. Sites included proximal humerus (two), distal femur (two), and rib (one). All tumors presented with clinical and radiographic features consistent with a diagnosis of primary bone neoplasms, although one probably represented a solitary metastasis from a primary uterine LMS. Radiographs showed lytic bone destruction with a moth-eaten appearance, and three cases had soft tissue extension. Histologically, all tumors showed broad, interlacing fascicles of spindle cells with pleomorphic nuclei, frequent mitoses, and necrosis. Two cases had a focal storiform pattern and bizarre multinucleated cells, and two other cases had focally prominent osteoclast-like giant cells. Extensive immunoreactivity for muscle actin was seen in all cases and for desmin in three. In each case, electron microscopy showed definite smooth muscle differentiation including cytoplasmic filaments with densities. At this writing, two patients are free of disease (including the patient with a presumed metastasis), one is alive with locally recurrent disease, and two are dead of disease. Experience suggests that LMS of bone is a distinct clinicopathologic entity that may be more common than previously recognized. Application of immunohistochemistry and electron microscopy to nonmatrix-producing bone sarcomas should facilitate diagnosis of additional cases. PMID- 1703915 TI - Multimodality cisplatin treatment in nonresectable alpha-fetoprotein-positive hepatoma. AB - Twenty-eight patients with alpha-fetoprotein-positive (AFP+) nonresectable hepatoma have been enrolled in a new multimodality Phase I, II program. Induction therapy consisted of 50 mg/m2 intravenous cisplatin followed by 2100 cGy irradiation to the tumor volume in seven fractions over 10 days. Hepatic arterial infusion of 50 mg/m2 cisplatin (IA-CDDP) was then administered at monthly intervals. Twenty-one patients have completed induction and at least two cycles of IA-CDDP. Twelve-month cumulative survival was 52% for all 28 patients and 69% for the 21 patients completing induction and IA-CDDP. Median survival has not yet been reached. Response rate (complete and partial) was 36% overall and 48% among the 21 patients who completed treatment. The improved survival of the present series of patients as well as the minimal hematologic toxicity suggests possible further integration of new modalities for therapy. PMID- 1703916 TI - Combined chemotherapy and radiation therapy in advanced inoperable squamous cell carcinoma of the head and neck. The final report of a randomized trial. AB - Between 1983 and 1986, the National Institute for Cancer Research in Genoa and affiliated institutions conducted a randomized study to compare two different ways of combining chemotherapy (CT) and radiation therapy (RT). One hundred sixteen patients were randomized to receive neoadjuvant CT followed by definitive RT (treatment arm A) or alternating CT and RT. In treatment arm A, RT consisted of 70 Gy to the involved areas and 50 Gy to the uninvolved neck at 2 Gy/fraction, five fractions per week. In treatment arm B, RT consisted of 60 Gy to involved areas and 50 Gy to the uninvolved neck in three courses of 20 Gy each, 2 Gy/fraction, ten fractions/2 weeks alternated with four courses of CT. CT consisted of vinblastine 6 mg/m2 intravenously followed 6 hours later by bleomycin 30 IU intramuscularly, day 1; methotrexate 200 mg intravenously, day 2; leucovorin rescue, day 3. CT was repeated every 2 weeks up to four courses. The same CT was used in both treatment arms of the study. Fifty-five patients were entered in treatment arm A and 61 in treatment arm B. Complete responses were 7/48 and 19/57 in treatment arms A and B, respectively (P less than 0.03). Four year progression-free survival was 4% in treatment arm A and 12% in treatment arm B (P less than 0.02), and four-year survival was 10% in A and 22% in B (P less than 0.02). Mucosal tolerance was significantly worse in treatment arm B (P less than 0.00004). The subgroup analysis shows the major improvement of alternating CT and RT in patients with the worst prognostic characteristics. PMID- 1703917 TI - Salvage chemotherapy for patients with germ cell tumors. The Memorial Sloan Kettering Cancer Center experience (1979-1989). AB - Twenty-eight of 124 (23%) advanced germ cell tumor (GCT) patients who were treated on four successive platin-based induction regimens and who failed to achieve a durable complete response (CR) remain alive (median follow-up, 50 months). An analysis of prognostic factors for response and survival was conducted on the 94 patients who received salvage chemotherapy. Survival and/or response to salvage therapy were significantly enhanced for patients with a prior CR to induction chemotherapy, treatment with a cisplatin-based salvage regimen, a testis primary site, a normal serum human chorionic gonadotropin level, a normal serum lactate dehydrogenase level, one site of metastasis, and an Indiana Class of 6 or less. Patients with a prior incomplete response (IR) had a particularly poor prognosis (P = 0.00007) with only 4 of 52 (9%) patients alive (median follow up, 37 months) compared with 15 of 42 (36%) patients with a prior best response of a CR (median follow-up, 35 months). The poor survival of patients who fail to achieve a durable CR to induction chemotherapy warrants the continued investigation of new salvage therapy. The identification of prognostic features may direct salvage therapy and aid in the interpretation of clinical trials of salvage regimens. PMID- 1703918 TI - The Lewis X antigen. A new paraffin section marker for Reed-Sternberg cells. AB - Using a monoclonal antibody specific to the Lewis X antigen (anti-Lex), the authors studied 103 cases of Hodgkin's disease (HD) in comparison with 57 cases of non-Hodgkin's lymphoma (NHL); three cases of granulocytic sarcoma (GS); two cases of malignant histiocytosis (MH); one case of monoblastic leukemia (ML); one case of interdigitating reticulum cell sarcoma (IRCS); six cases of histiocytosis X (HX); one case of reticulohistiocytoma (RH); 44 various reactive conditions of the lymph node (LN). Reed-Sternberg and related (R-S) cells stained selectively in 80 of 92 cases of HD (87.0%), excluding 11 cases of lymphocyte predominance type. The stain was better in B-5-fixed specimens than in formalin-fixed specimens, showing a dense deposit of reaction products at a paranuclear site and on the cell surface. The staining results were compared with those of Leu-M1 and found to be superior both qualitatively and quantitatively (detection rate of R-S cells: 87.0% versus 68.5% of Leu-M1). Granulocytes, rare epithelioid histiocytes, and some endothelial and/or erythrocytes also stained with anti-Lex. The stain had positive results in three cases of GS showing a diffuse cytoplasmic staining pattern. Of NHL, two of 29 peripheral T-cell lymphomas stained to show rare paranuclear deposits without cell surface staining. The stain had negative results in MH, ML, IRCS, HX, and RH. Of 45 reactive LN, minute subcapsular collections of Lewis X+, altered-appearing Langerhans'-like cells, were observed in all ten LN from human immunodeficiency virus (HIV)-associated persistent generalized lymphadenopathy (PGL). The stain had negative results in all other various reactive conditions of LN. In conclusion, Lewis X staining is useful as a marker for R-S cells in paraffin sections with staining results superior to those of Leu-M1. Lewis X staining also detects subcapsular clustering of altered appearing Langerhans'-like cells in PGL, which has not been described previously and warrants additional study. PMID- 1703919 TI - Fibrin formation on vessel walls in hyperplastic and malignant prostate tissue. AB - To explore mechanisms of coagulation activation in adenocarcinoma of the prostate, the occurrence and distribution of components of coagulation and fibrinolysis pathways in situ were studied by means of immunohistochemical techniques applied to frozen sections of fresh malignant and benign hyperplastic prostatic tissue obtained at transurethral resection. Fibrinogen was distributed throughout the perivascular and tumor connective tissue in both malignant and benign disease but was not present in adjacent areas of normal prostate. Antibodies specific for fibrin and D-dimer crosslink sites stained vascular endothelium focally in both malignant and benign tissues. Both neoplastic cells and benign hyperplastic glandular epithelial cells stained weakly and in a patchy distribution for tissue factor and focally for low-molecular-weight urokinase type plasminogen activator. Focal staining of vascular endothelium was also observed for tissue plasminogen activator and plasmin-antiplasmin complex neoantigen. By contrast, no tissue staining was observed for factor VII, factor X, factor XIII "a" subunit, high-molecular-weight urokinase-type plasminogen activator, plasminogen activator inhibitors 1 to 3, protein C, and protein S. Thus, the similarity in findings between benign hyperplastic and neoplastic prostate tissue, the lack of either an intact tumor cell-associated coagulation pathway or fibrin formation, and the presence of fibrin on vascular endothelium are consistent with the concept that coagulation activation in prostatic cancer may not be due to a direct effect of the tumor cells on the clotting mechanism. Rather, such activation may be induced by a soluble tumor product that activates procoagulant activity on certain host (for example, vascular endothelial) cells. These findings, together with the lack of effect of warfarin anticoagulation on the clinical course of patients with prostatic cancer, contrast with findings in certain other tumor types and suggest that coagulation activation may not contribute to progression of adenocarcinoma of the prostate. PMID- 1703920 TI - Hemodynamic comparison of combined therapy by nitroglycerin tape and ibopamine with combination of nitroglycerin tape and nifedipine. AB - Fifteen congestive heart failure patients (NYHA: class III or IV) were enrolled in the study and were classified into two groups. Six patients (group I) received combined therapy by nitroglycerin tape (5 mg) and ibopamine (100 mg), while the remaining 9 patients (group II) were given nitroglycerin tape (5 mg) and nifedipine (10 mg). The effects of the combined treatments on hemodynamics were compared between the two groups using Swan-Ganz catheter method. No significant differences were noted in the hemodynamic baseline values of the two groups before treatment. In group I mean pulmonary arterial pressure (mPA) and systemic vascular resistance (SVR) decreased and the cardiac index (CI) increased, while the heart rate (HR) and mean blood pressure (mBP) remained unchanged. In group II mBP, mPA and SVR were lowered, whereas CI and HR were augmented. There were no significant differences between the two groups with respect to mPA and CI. However, mBP decreased in group II, while it remained unchanged in group I, with significant difference between the two groups (p less than 0.01). Preload and afterload, on the base of mPA and SVR, respectively, decreased in both groups, while cardiac output increased, suggesting that both treatments were useful for the improvement of cardiac output. Mean BP decreased in group II, although it remained unchanged in group I. These results suggest that the combination of nitroglycerin and ibopamine may be more useful in hypotensive patients with heart failure. PMID- 1703921 TI - Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism. AB - Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers. PMID- 1703922 TI - Characterization and purification of DNA-RNA complexes related with 1731 and copia-like transposable elements in a Drosophila cell line. AB - DNA-RNA complexes were characterized and purified in a Drosophila melanogaster cell line. Such duplexes were shown to be specific of 1731 and other "copia-like" transposable elements. DNA-RNA complexes were purified through a Sephadex G-75 column from a global nucleic acid preparation or from a total RNA fraction prior to DNA-A and RNA-A treatment. They incorporated both labelled thymidine and uridine and their resistance or sensibility to enzymes or chemicals was consistent with that being expected with such hybrid molecules. From that intermediate form of reverse transcription, the resulting labelled cDNAs were obtained and were shown to be homologous to different drosophila "copia-like" transposable elements. These results suggest that most of the "copia-like" transposable elements were amplified through a reverse transcription pathway in Drosophila melanogaster. PMID- 1703923 TI - A consensus motif common to all Rho-dependent prokaryotic transcription terminators. AB - We have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far. PMID- 1703924 TI - Ontogeny of thymic B cells in normal mice. AB - Ontogeny of thymic B cells and their surface characteristics were analyzed using monoclonal antibodies (mAbs) against B220 molecules (CD45, CD45R). A small number of B cells were detected in fetal thymus on Gestation Day 14 (approximately 3.5% of the low-density fraction). Similarly, the percentage of B cells in the low density fraction was 3.2% on Gestation Day 18, and 3.5% on Day 1 after birth. These were the same level as that of adult mice. CD5+ B cells, which form the major population of thymic B cells, were also found in the fetal life (0.5% on Day 14 and 2.2% on Day 16 in the low-density cells). The percentage of CD5+ B cells in B cell-enriched fraction was about 65% on Day 1 after birth, which is the same level as that in adult mice. These results indicate that a small number of B cells or cells in the B-cell lineage are present in the fetal thymus and also suggest the importance of these thymic B cells in the negative selection of T cells during early developmental stages. PMID- 1703925 TI - Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules. AB - Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear. PMID- 1703926 TI - Immobilized anti-CD3-induced T cell growth: comparison of the frequency of responding cells within various T cell subsets. AB - Human T cells can be divided into subsets based on the expression of CD29, CD45RA, CD45RO, LFA-3, or CD11a. It has been suggested that the subset of CD4+ T cells that expresses high densities of CD29, CD11a, CD45RO, and LFA-3 contains "memory" T cells, whereas the subset of cells that expresses CD45RA contains "naive" T cells. In order to obtain a more complete picture of the functional capacities of human naive and memory CD4+ and CD8+ T cell subsets, highly purified T cells were activated with a uniform stimulus and responses were examined in bulk cultures and under limiting dilution conditions. T cell activation was achieved with an immobilized mAb to the CD3 molecular complex, 64.1. In bulk cultures, immobilized 64.1 stimulated a vigorous response. Moreover, the number of cells entering the cell cycle, the magnitude of the [3H]thymidine incorporation, and the growth of the cells over 6 days in culture by naive and memory CD4+ T cells was comparable. To delineate the frequency of responsive cells in each subset more precisely, cells were cultured with immobilized 64.1 at limiting dilution and the precursor frequency of responding cells was assessed by examining wells microscopically for visible growth. Immobilized 64.1 was able to induce some T cells from each subset to grow in the complete absence of AC, when exogenous IL2 was present. The number of responding CD4+ and CD8+ cells was comparable. The percentage of naive cells responding in each population was approximately three times greater than the frequency of memory cells. IL4 could also support the growth of immobilized 64.1-activated CD4+ T cells, but the frequency of responding cells was much lower than that supported by IL2. The vast majority of the IL-4 responsive CD4+ cells resided within the naive cell subset. The data indicate that the response of CD4+ and CD8+ naive and memory T cell subsets to immobilized anti-CD3 depends on the density of responding cells. Naive T cells have an enhanced capacity to grow when cultured in the absence of other T cells or accessory cells. This ability may facilitate their expansion during primary immune responses. PMID- 1703927 TI - Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness. AB - We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells. PMID- 1703928 TI - Spectroscopic analysis of charge transfer complexes between morpholine and iodine. AB - It has been demonstrated spectroscopically that many nitrogen-containing heterocyclic compounds can form charge transfer complexes with iodine. The complexes of morpholine with iodine were shown to be of the n-sigma type with a 1:1 stoichiometry. A strong donor-acceptor interaction was found (Kc = 1261 +/- 12 mol-1 at 20 degrees C in CCl4), considerably higher than those of complexes of aromatic compounds with iodine. The high value of the formation constant for this complex indicated that morpholine could serve as a starting point for the synthesis of novel anti-thyroid drugs. PMID- 1703929 TI - [Histological and immunohistochemical study on 8 cases of medullary carcinoma of the thyroid]. AB - Histological review and immunohistochemical studies of 8 cases of medullary carcinoma were carried out by using ABC technique. The results showed 8 calcitonin positive cases, 3 Somatostatin positive cases, 7 NSE positive cases, 5 CEA positive cases and 8 keratin positive cases. In addition, histogenesis, histological characteristics and the evaluation of immunohistochemistry in diagnosis of thyroid medullary carcinoma are discussed. PMID- 1703930 TI - [Pathology and immunohistochemical demonstration of beta-HCG in intracranial primary germinoma]. AB - Clinical pathologic observation and immunohistochemical demonstration on beta- HCG are reported in 13 cases of intracranial primary germinomas, including 7 cases of germinomas in the region of the pineal gland and 6 cases in the hypothalamic area. Among them, there were 11 cases of simple type and 2 cases of beta--HCG--producing tumors with a character of differentiation of trophoblasts in the latter. This paper supports the concept that intracranial primary germinoma is an atypical teratoma arising from the germ cells. The significance of beta--HCG immunohistochemical demonstration in the pathologic diagnosis of germinomas and the mechanism of endocrine disturbance are discussed. PMID- 1703931 TI - Load dependence of left ventricular diastolic pressure-volume relations during short-term coronary artery occlusion. AB - We evaluated the effect of altered loading conditions on left ventricular (LV) diastolic pressure-volume relations during acute coronary artery occlusion that was produced by inflation of an intracoronary balloon. Open-chest anesthetized dogs (n = 18) were instrumented so that LV pressure (micromanometer) and LV volume (conductance) could be measured without disturbing the pericardium. The effects of brief periods of occlusion (1-2 minutes) were assessed under steady state conditions before and after dextran infusion with the pericardium present and absent and during vena caval occlusion. Under steady-state conditions before dextran infusion with the pericardium removed, at an LV end-diastolic pressure (EDP) of 8.4 +/- 1.4 mm Hg, occlusion resulted in a rightward shift in the diastolic portion of the LV pressure-volume loop (delta LVEDP, 2.7 +/- 2.3 mm Hg; delta LVEDV, 6.3 +/- 4.7 ml, both p less than 0.05 versus control). After dextran infusion (LVEDP, 20.9 +/- 6.0 mm Hg), occlusion resulted in a rightward and upward shift in the diastolic portion of the LV pressure-volume loop (delta LVEDP, 5.8 +/- 4.4 mm Hg; delta LVEDV, 4.2 +/- 3.0 ml, both p less than 0.05 versus control). At low cardiac volumes before dextran infusion, the intact pericardium did not affect the response to occlusion. By contrast, after dextran infusion in the presence of an intact pericardium, LVEDP significantly increased (delta, 6.4 +/- 3.6 mm Hg, p less than 0.05) but LVDEV did not (delta, 0.7 +/- 1.5 ml, p = NS). There was a parallel upward shift in the diastolic portion of the LV pressure-volume loop that was eliminated by removal of the pericardium. Thus, the change in LV diastolic pressure and volume during occlusion varied and depended on the baseline cardiac volume and presence of the pericardium. Before dextran infusion with the pericardium present and absent, coronary artery occlusion did not alter the LV diastolic chamber stiffness parameter, which was calculated from the diastolic interval of an averaged steady-state beat (0.040 +/ 0.019 versus 0.036 +/- 0.015 mm Hg/ml, p = NS). After dextran infusion with the pericardium present and absent, coronary artery occlusion increased the LV diastolic chamber stiffness parameter (0.057 +/- 0.034 and 0.074 +/- 0.034 mm Hg/ml, both p less than 0.05 versus controls, respectively). Vena caval occlusion eliminated the shifts in the diastolic portion of the LV pressure-volume loop with the pericardium present and absent.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1703932 TI - Tangential intracortical pathways and the development of iso-orientation bands in cat striate cortex. AB - Evidence is accumulating that tangential connections are a prominent feature of cortical organization. In the mammalian visual cortex, these connections appear to be related to columnar systems and theoretical considerations have suggested that they contribute to the development of regularly spaced orientation columns. We tested this assumption and examined whether early cortical lesions affected the formation and layout of the pattern of iso-orientation bands. In the striate cortex of 2-week-old kittens, tangential fiber paths were disrupted unilaterally along the representation of the horizontal meridian either by cuts, or by suction or by implanting pieces of Teflon. At this age, tangential fibers are still growing, orientation selectivity is only poorly developed and 2-deoxyglucose mapping does not yet reveal orientation columns. When the kittens were 7-10 weeks old, the organization of orientation bands was studied with the 2-deoxyglucose technique in flat-mounts of both visual cortices. In addition, kittens from the same litters as the experimental animals, having received the same lesions at the same postnatal day were subjected to neuroanatomical experiments to assess the effect of the lesion on the tangential fibers. These investigations revealed that a small fraction of tangential fibers had grown across the lesion when no mechanical barrier was implanted while disruption of tangential connections was complete in cases who had received Teflon implants. Apart from minor irregularities that were confined to the vicinity of the lesion, the 2 deoxyglucose experiments showed no differences in the pattern of orientation bands between the lesioned and intact hemispheres. In both, the bands extended throughout all cortical layers and their main trajectories were orthogonal to the representation of the vertical meridian. We conclude that at least from two weeks of age onwards, intracortical tangential connections are not necessary for the development of the regular pattern of iso-orientation bands in the striate cortex of cats. PMID- 1703933 TI - Tannic-acid staining material on high endothelial venules and lymphocytes in skin and peripheral lymph nodes in Staphylococcus aureus-associated erythroderma. AB - The recognition and binding of glycoprotein receptors on lymphocytes to specific antigens present on high endothelial venules (HEV) precedes the egress of lymphocytes from the blood stream into the tissues. In this paper, we report the presence of HEVs with tannic-acid staining material (TASM+ HEVs) in Staphylococcus aureus-associated erythroderma, which allow the migration of CD8+ lymphocytes from the bloodstream into the epidermis. TASM positivity is also expressed on lymphocytes within the regional lymph nodes, and by intravascular lymphocytes prior to leaving the TASM+ HEV. It is proposed that TASM positivity may represent a molecule, which may function in binding lymphocytes to HEVs prior to egress from the HEV. (TASM is lost from lymphocytes after leaving the HEVs). The expression of TASM positivity may form an essential part of the CD8+ lymphocyte-HEV recognition system, and may be the means whereby CD8+ lymphocytes generated in the regional lymph nodes by various mitogens (in this case by staphylococcal mitogens) may 'home' to specific sites within the epidermis. TASM positivity on both the HEVs and lymphocytes may serve as a convenient marker of such a system. PMID- 1703934 TI - Substance-P immunoreactivity in active edges of psoriatic plaques. PMID- 1703935 TI - Selective inhibition of responses to carbachol and McN-A-343 in the rabbit vas deferens. AB - 1. The effect of several selective muscarine receptor antagonists were evaluated on the responses of carbachol (CCh) and McN-A-343 (McN) during sympathetic nerve stimulation in the rabbit vas deferens. 2. The muscarine M1 receptor antagonist pirenzepine exhibited similar apparent pKB values for antagonism of the prejunctional inhibitory response of either CCh (pKB, 8.2) or McN (pKB, 8.5) on sympathetic nerve stimulation. 3. The muscarine M2 receptor antagonists, pancuronium and the bisalkyl ammonium compound 'C7/3-phth' were selective inhibitors of the postjunctional facilitatory response produced by CCh on sympathetic nerve stimulation. They were also 17- and three-fold, respectively, less potent against the inhibitory responses of McN on sympathetic nerve stimulation. The apparent pKB value of pancuronium on the inhibitory response produced by CCh did not differ significantly (P greater than 0.05) from that using McN. A similar finding was made for C7/3-phth. 4. Selective blockade of the inhibitory response to CCh with pirenzepine (0.03 or 0.5 mumol/L) did not significantly (P greater than 0.05) modify the apparent pKB value for pancuronium on the facilitatory response of CCh. 5. Selective blockade of the facilitatory response to CCh with a low concentration of pancuronium (0.5 mumol/L) did not significantly (P greater than 0.05) modify the apparent pKB value for pancuronium (30 mumol/L) on the inhibitory response of CCh. 6. It is suggested that CCh and McN activate the same prejunctional M1 muscarine receptor and that pancuronium is the most selective of the muscarine M2 receptor antagonists presently tested in this preparation for distinguishing between muscarine M1 and M2 receptors. PMID- 1703936 TI - [Pentose phosphate pathway in neuromuscular diseases--evaluation of muscular glucose 6-phosphate dehydrogenase activity and RNA content]. AB - There have been several reports concerning elevated glucose 6 phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of pentose phosphate pathway (PPP), in experimental muscle disturbances. PPP produces ribose, a substrate of RNA, and NADPH which is a cofactor of fatty acid synthesis. PPP also has a role of by-path pathway of glycolysis. Then, we evaluated G6PDH activity and RNA content in biopsied quadriceps muscle. The subjects were muscles from 23 neurogenic amyotrophy, 54 myopathy including 19 progressive muscular dystrophy (PMD), and 10 controls whose muscle was obtained at orthopedic surgery. Neurogenic amyotrophy consisted of 12 amyotrophic lateral sclerosis (ALS), 4 spinal muscular atrophy and 7 peripheral nerve disorders. Myopathy were 3 Duchenne dystrophy, 2 congenital muscular dystrophy, 8 limb-girdle type dystrophy, 6 facio-scapular +-humeral muscular dystrophy, 6 myotonic dystrophy, 6 mitochondrial myopathy, 5 endocrinological myopathy, 3 hypokalemic myopathy, 8 polymyositis and 4 other inflammatory myopathy. The assays of G6PDH and RNA were performed after Glock's and Fleck's methods, respectively. The control values were 3.6 +/- 0.8 nmol formed NADPH/mg protein/min (M +/- SD) in G6PDH and 0.69 +/ 0.17 micrograms/mg non-collagen protein in RNA. Most cases of PMD, as well as some cases of ALS, hyperthyroidism, mitochondria hypokalemic myopathy, inflammatory myopathy showed increased values (beyond M + 2SD of control) both in G6PDH and RNA. There were significant positive correlations between G6PDH activity and RNA content in PMD and motor neuron disease. Myotonic dystrophy showed normal values in both G6PDH and RNA. Half number of cases of mitochondrial myopathy demonstrated increased G6PDH alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703937 TI - Determination of dependence of spin-lattice relaxation rate in serum upon concentration of added iron by magnetic resonance imaging. AB - Dependence of spin-lattice relaxation rate (1/T1) in serum upon concentration of added iron was studied in the concentration range 0.0179-0.179 mmol l-1 for each of ferrous and ferric iron. In conjunction with the serum study, 1/T1 in solutions of transferrin and a mixture of albumin and gamma globulin was also studied as a function of added iron concentration. At low concentrations 1/T1 in serum increases linearly with increasing amounts of iron for each ion, and then reaches saturation for ferrous iron, whereas it shows an inflection for ferric iron. To explain the partition of added iron between various serum components, the effect of iron on 1/T1 in serum was compared with those of transferrin and the mixture. This effect can be defined as relaxivity or the incremental increase in relaxation rate per millimolar of added iron. At low concentrations the relaxivities of iron in serum, about 0.91 mmol-1 l s-1 for ferric and 0.95 mmol-1 l s-1 for ferrous ion, approximate well to the relaxivity of iron in transferrin solutions, which was measured to be about 0.92 mmol-1 l s-1. Furthermore, at high concentrations the relaxivity of ferric iron in serum, 0.44 mmol-1 l s-1, becomes similar to that of the mixture which is about 0.39 mmol-1 l s-1. These findings imply that iron added to serum first satisfies the binding requirements of transferrin, and the binding of iron to the other serum proteins occurs at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703938 TI - Direct gram stain and urease test to detect Helicobacter pylori. AB - Antral biopsies were obtained by gastrointestinal endoscopy on 143 adult patients with dyspeptic symptoms of gastritis or peptic ulcer disease. A direct Gram stain and a direct urease test were performed on each biopsy in addition to culture. Forty-three biopsies (30%) were considered positive for Helicobacter pylori based on culture or histologic examination, or both. Thirty-one biopsies (72% sensitivity) were positive for both direct tests, whereas 95 of 100 negative cultures were negative for both tests. Thirty-eight of the 43 positive biopsies were Gram stain positive (sensitivity, 88%; specificity, 100%). The direct urease test alone was positive at 4 hr for 29 biopsies (sensitivity, 67%; specificity, 100%) and at 24 hr for 38 biopsies (sensitivity, 74%; specificity, 95%). Rapid presumptive diagnosis of H. pylori in antral biopsies was obtained when at least one direct test, Gram stain or urease, was positive. PMID- 1703939 TI - Production and characterization of murine monoclonal antibodies specific for serogroups E1 and E4 Salmonella. AB - Two anti-Salmonella serogroup E-specific monoclonal antibodies (MAbs) are described. Neither antibody reacted with any of the 58 strains of serogroups A-D Salmonella tested by enzyme immunoassays nor did they react with any of the 21 other species of enterobacteria, 15 species of other Gram-negative bacteria, and 6 species of Gram-positive bacteria. In contrast, all 14 strains of serogroups E1 and E4 Salmonella reacted with both antibodies. Ascitic fluids of these two antibodies agglutinated all 42 strains of serogroups E1 and E4 Salmonella tested by slide agglutination method but did not agglutinate any of the 107 strains of other serogroups of Salmonella. Lysogenic conversion of serogroup E1 Salmonella strains by phages epsilon 15 and epsilon 34 resulted in loss of reactivities of these strains with the MAbs. PMID- 1703940 TI - Direct detection and amplification of Helicobacter pylori ribosomal 16S gene segments from gastric endoscopic biopsies. AB - Helicobacter pylori is an organism thought to play an important causative role in gastritis and peptic ulcer diseases. We have designed an RNA dot blot assay for the detection of H. pylori, using as probe a synthetic oligonucleotide complementary to its 16S rRNA. We have also used oligonucleotide primers, complementary to conserved sequences within bacterial ribosomal 16S genes, to amplify a H. pylori ribosomal 16S DNA fragment via the polymerase chain reaction (PCR). After determining the DNA sequence of this amplified H. pylori fragment, primers were designed for specific PCR amplification of H. pylori ribosomal 16S DNA sequences. Samples from clinical endoscopic biopsies were PCR amplified with universal 16S ribosomal primers to detect the presence of bacteria and with H. pylori-specific primers to uniquely detect H. pylori. Finally, by comparing the H. pylori-specific PCR assay to commonly used diagnostic tests, we demonstrate that the molecular technique of PCR amplification shows promising applications for the clinical detection of H. pylori. PMID- 1703941 TI - Angiotensin-converting enzyme inhibition, kinins, and stress ulceration. PMID- 1703942 TI - Further validation of FETAX: evaluation of the developmental toxicity of five known mammalian teratogens and non-teratogens. AB - The developmental toxicity of five compounds was evaluated with the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to 5-azacytidine, methotrexate, pseudoephedrine, aspartame, and amaranth for 96 h. Three separate static-renewal assays were conducted for each compound. Based on Teratogenic Index [LC50/EC50 (malformation)] values, types and severity of induced malformations, and embryo growth, 5-azacytidine and methotrexate tested as having strong teratogenic potential. Pseudoephedrine scored as having moderate teratogenic potential, but amaranth and aspartame had little or no teratogenic potential. Results support the use of FETAX for the screening of developmental toxicants. PMID- 1703943 TI - N70 and P100 can be independently affected in multiple sclerosis. AB - We have studied the relationship between N70 and P100 of the pattern visual evoked potential in 98 patients with multiple sclerosis and in 59 controls. In patients with multiple sclerosis, P100 was either absent or had prolonged latency in 121 eyes (61.7%), while N70 was absent or prolonged in 97 eyes (49.5%). The total number of eyes with either N70 and/or P100 abnormalities was 137 (69.9%). Eighty eyes (40.8%) had abnormal latency of both P100 and N70, while 41 eyes showed P100 delays without corresponding N70 changes. Seventeen eyes had abnormal N70, but normal P100 latency. N70 and P100 appear to be more often absent in the definite rather than in the possible multiple sclerosis group. These data show that N70 and P100 can be independently affected in patients with MS. PMID- 1703944 TI - Pre-stimulus spectral EEG patterns and the visual evoked response. AB - The relationship between the latencies and amplitudes of the N1 and P2 components of the visual evoked potential (VEP) and the psychophysiological state of the brain immediately preceding the time of the stimulus has been investigated in 7 male subjects. Power spectral measures in the delta, theta, alpha and beta bands of the 1 sec pre-stimulus EEG were used to assess the brain state, and low intensity flashes, delivered randomly between 2 and 6 whole seconds, were used as the stimuli. Trials were ranked separately according to the relative amounts of pre-stimulus power in each EEG band and were partitioned into groups by an equal pre-stimulus spectral power criterion. Averaged EPs were computed from these groups and multiple regression analysis was used to relate pre-stimulus spectral power values to EP features. Five of the 7 subjects displayed consistent increases in N1-P2 amplitude as a function of increasing pre-stimulus relative alpha power. The between-subjects effect of pre-stimulus EEG on N1 latency was small, but was moderate for P2 latency (both significant). Both N1 and P2 latency were found to decrease with increasing amounts of pre-stimulus relative delta and theta power. PMID- 1703945 TI - Somatosensory evoked potential recovery (SEP-R) in myoclonic patients. AB - Recovery of somatosensory evoked potentials (SEPs) was studied by paired stimulation of the median nerve in patients with various kinds of myoclonus. This technique revealed the hyperexcitability of the central nervous system (CNS) which could not be detected by the conventional SEP technique using a single stimulus. This technique would be useful for studying the excitability of the CNS. PMID- 1703946 TI - Interactions of somatosensory evoked potentials: simultaneous stimulation of two nerves. AB - To analyse the mechanism by which sensory inputs are integrated, interactions of somatosensory evoked potentials (SEPs) in response to simultaneous stimulation of two nerves were examined in 12 healthy subjects. Right, left and bilateral median nerves were stimulated in random order so that a precise comparison could be made among the SEPs. The arithmetical sum of the independent right and left median nerve SEPs was almost equal within 40 msec of stimulus onset to that evoked by the simultaneous stimulation of bilateral median nerves. However, a difference emerged after 40 msec. The greatest difference was recorded after 100 msec. Sensory information from right and left median nerves may interact in the late phase of sensory processing. Left median, left ulnar, and both nerves together were stimulated. The sum of the SEPs of left median and ulnar nerves was not equal to that evoked by the simultaneous stimulation of the two nerves even at early latencies. Differences between them were first recorded at 14-18 msec and became greater after 30-40 msec. It is suggested that the neural interactions between impulses in the median and ulnar nerves begin below the thalamic level. PMID- 1703947 TI - Cortical somatosensory potentials evoked by magnetic stimulation: effect of body height, age, and stimulus intensity. AB - Cortical somatosensory evoked potentials elicited by non-invasive magnetic stimulation at Th10, Th12, and L5 vertebral levels and at mid-gluteus and ankle were studied in 45 normal controls varying in body height and age. Peak latencies of P2 evoked by ankle, mid-gluteus and L5 stimulation were correlated with body height. However, P2 latencies evoked by Th10 and Th12 stimulation were independent of body height. In contrast, all N2 latencies evoked by Th10, Th12, L5, mid-gluteus and ankle were correlated with body height. There were no correlations between latencies of P3 and N3 and body height. No peak latencies of cortical components were affected by age within the age group studied (15-67 years). In the intensity study, N2 showed double peaks when low to moderate stimulus intensities were applied to Th12 vertebra. Furthermore, in 2 patients with mild somatosensory dysfunction of lumbar roots or cord, N2 evoked by L5 stimulation revealed double peaks with prolonged interpeak latencies between the first peak of N2 and P3, which might be related to the temporal dispersion of the afferent fibers. These data suggest that the N2 component is induced by both fast and slow afferent fibers. PMID- 1703948 TI - Estimation of conduction velocity of the spino-thalamic tract in man. AB - This is the first report of estimating conduction velocity (CV) of the slowly conducting somatosensory spinal tracts or the spino-thalamic tract (STT) in man. The CV of the STT was measured by recording somatosensory evoked potentials (SEPs) following CO2 laser stimulation of the hand and foot, which was previously shown to cause pain or heat sensation by activating cutaneous nociceptors and by its ascending signals through A delta fibers and probably STT. When the CV of A delta fibers was assumed to be 10-15 m/sec, the CV of STT was found to be approximately 8-10 m/sec in normal young subjects. It was slightly slower in subjects over 60 years of age. In contrast, the CV of the posterior column, which was calculated based on SEPs following electrical stimulation of the median and posterior tibial nerves, was approximately 50-60 m/sec. PMID- 1703949 TI - Late pain-related magnetic fields and electric potentials evoked by intracutaneous electric finger stimulation. AB - Surface magnetic and electric recordings were used to localize the sources of late pain-related magnetic fields and electric potentials, evoked by painful intracutaneous electric finger stimulation. We find that the source of the P90m component of the evoked magnetic field lies in the finger area of the primary somatosensory cortex; the sources of the N150m and P250m are found to reside in the frontal operculum. These findings are unexpected from the evoked electric potential data, which suggest a central location for these sources. We also note that the interpretation of the electric data was confounded by the presence of an alpha-like oscillation, which overlapped many components of the evoked potential. PMID- 1703950 TI - Gender differences in source location for the N100 auditory evoked magnetic field. AB - Auditory evoked magnetic fields were recorded in response to contralateral stimulation over the right hemisphere in 6 adult males and 6 adult females. The data were fit to a model of a current-dipole source in a homogeneous sphere and 5 parameters of the dipole were computed--3 spatial coordinates, orientation, and strength. When average values for the dipole parameters were compared between sexes, it was found that the current source for the N100m is located more than 1 cm posterior in females and is oriented pointing more downward. These findings were replicated in separate measurement sessions. Viewing of individual magnetic resonance images did not reveal a corresponding anatomical disparity in the location of the primary auditory cortex which is assumed to produce the N100m. Therefore, functional organization of the auditory cortex may be different for the sexes. PMID- 1703951 TI - The effect of click rate on latency and interpeak interval of the brain-stem auditory evoked potentials in children from birth to 6 years. AB - Latency and interpeak interval of the brain-stem auditory evoked potentials at different click rates were measured in 80 healthy children from birth to 6 years, and 21 adults. Clicks were presented at 10, 30, 50, 70 and 90/sec, and 70, 40 and 20 dB HL. At high stimulus intensity (70 dB SL), all latencies of waves I, III and V and the I-V, I-III and III-V intervals showed a progressive prolongation with increasing repetition rate. The latency- and the interval-rate functions were similar for all age groups but their slopes were slightly steeper in younger than in older. As click rate increased from 10/sec to 90/sec, the latencies of waves I, III and V at different age groups were prolonged by 4-10%, 9-13% and 12 15% respectively, and the intervals of I-V, I-III and III-V were prolonged by 15 16%, 8-16% and 14-24% respectively. The mean increments of wave V latency and I-V interval in different age groups were 0.404-0.575 and 0.332-0.526 msec respectively with increasing click rate from 10 to 50/sec, and 0.697-1.009 and 0.629-0.776 msec respectively with increasing click rate from 10 to 90/sec. The younger the age the larger the absolute increments for all these BAEP parameters, but the increasing rates for a BAEP measure were similar among different age groups, exhibiting no age-dependent differences.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1703952 TI - Visual 'cognitive' evoked potentials in the behaving monkey. AB - Using a visual 'oddball' paradigm we studied ERPs in monkeys trained in a 'go' 'no-go' discrimination task. The stimuli were 2.5 cpd sinusoidal gratings differing only in orientation (0 degrees or 25 degrees). Monkeys released a lever during 1 of 2 response windows (RW), 480-1762 or 740-1672 msec, following target stimulus onset. Target stimulus presentation probabilities were 1.0, 0.5 and 0.3. The primary evoked potentials recorded to either the target or non-target stimulus were similar in all monkeys. P3 signals progressively emerged in the monkeys only to the target stimulus. P3 recorded at Cz, P3, and P4 had similar mean latencies and amplitudes. Eye movements showed no relationship to P3 potentials. Neither the primary visual potentials nor P3 changed significantly as a function of RW. P3 amplitude was inversely related to target probability. When the target stimulus was presented 100% of the time (P = 1.0) P3 disappeared over 4-5 blocks of trials, while the primary evoked potentials remained consistent. PMID- 1703953 TI - Evoked potentials recorded from brain-stem nuclei in awake cat: interaction of tone bursts and continuous tone. AB - Previous work indicated that components of the auditory thalamocortical potential evoked by a brief binaural tone burst could be enhanced by certain stimulus combinations, e.g., a brief tone burst in the presence of a continuous tone. The principal questions of the present study were whether enhanced components could be obtained caudal to thalamocortex and whether monaural stimuli would be effective in producing enhancement. Eight cats received permanent electrodes in cochlear nucleus and the nucleus of the inferior colliculus. Custom earmolds were made for each ear of each animal. The median attenuation produced by the earmolds was 35 dB and the use of a single earmold approximated monaural stimulation. Auditory evoked potentials were recorded from the electrodes while the animals were unanesthetized but comfortably restrained. Brief 6.25 kHz tone bursts were presented against a background of silence or of a 4.96 kHz continuous tone. In the presence of the continuous tone, enhanced components were obtained from a majority of the electrodes in inferior colliculus but from none of the electrodes in cochlear nucleus. The late negative component in the colliculus potential was increased in amplitude while other components were reduced in amplitude by the continuous tone. The latencies of all components from all electrodes were increased by the presence of the continuous tone. It was concluded that enhancement effects could be obtained at the level of inferior colliculus, and that binaural stimulation does not appear to be necessary to produce enhanced components. PMID- 1703954 TI - The development of auditory and visual evoked potentials in early treated phenylketonuric children. AB - Brain-stem auditory evoked potentials (BAEPs) and flash visual evoked potentials (F-VEPs) were gathered from 8 early treated phenylketonuric (PKU) children in a prospective longitudinal investigation during the 1st to the 12th months after birth. No consistent differences were found in the wave morphology of evoked potentials in PKU children from that of age-matched controls. Studying the latency of some components showed that in BAEPs, wave I latency was similar to control values for the whole year, but that the I-V interpeak mean latency (I-V IPL) was always significantly longer than in controls. In F-VEPs wave N1 latency was significantly longer than in controls only at 1-2 months of age, but returned to control values at 3-4 months (when all children were on dietary therapy) and remained in this range up to the 12th month. The mean latency of the P2 wave of flash VEPs was always significantly longer in PKU children than in controls. These results show that relevant alterations in evoked potentials may be found in PKU children several months after starting dietary therapy. This suggests that information processing in the brain may be impaired for a long time, due to abnormal metabolic conditions between birth and the onset of dietary therapy. PMID- 1703955 TI - DNA damage and repair induced by bleomycin in mammalian and insect cells. AB - We assessed the response of mosquito (ATC-15) and mammalian (CHO) cells to bleomycin (BLM). Comparison of the data obtained in both cell lines indicates that DNA in the chromatin of mosquito cells is 10 to 20 times more resistant to BLM, and that the DNA damage induced by this antibiotic is better repaired in mosquito than in mammalian cells. Permeability of the cell membrane for BLM was found to be the same for both cell lines. Moreover, the time-kinetics of BLM damage to nuclear DNA was similar for ATC-15 and CHO cells. The low sensitivity of mosquito cells to BLM is reflected in better growth efficiency. These cells exhibit a satisfactory growth at BLM doses that produce a permanent arrest of growth in CHO cells. It is proposed that variations in the chromatin structure and in the intracellular free amino acid pool may play an important role in the differential response of insect and mammalian cells to BLM. PMID- 1703956 TI - Reverse transcriptase in archaebacteria. Purification and characterization of a primase-reverse-transcriptase complex from Halobacterium halobium. AB - A primase-reverse-transcriptase of Halobacterium halobium was purified by column chromatography on DEAE-cellulose, hydroxyapatite and carboxymethyl-cellulose, followed by sedimentation on a glycerol gradient. The enzyme is a multifunctional enzyme containing reverse transcriptase. DNA polymerase and RNase H activities and does not require a performed primer to initiate DNA synthesis. Using a single stranded DNA as template, this enzyme synthesizes oligonucleotides (8-12 bases) that can be used a primer by Escherichia coli DNA nucleotidyltransferase I (DNA polymerase I, Klenow fragment). Two polypeptides of 67 and 57 kDa were found after 14750-fold purification of the enzyme. PMID- 1703957 TI - The major serum protein of Drosophila larvae, larval serum protein 1, is dispensable. AB - Null alleles of all three genes (alpha, beta and gamma) coding for the major serum protein of Drosophila larvae, larval serum protein 1, have been characterized at the protein level and by analysis of their RNA and DNA. Each allele completely lacks one polypeptide chain. beta shows an unaltered gene but reduced levels of RNA, alpha shows restriction-fragment-length polymorphism and shows low levels of transcript, while gamma is a deletion. The three null alleles have been combined to give a strain without the gene for larval serum protein 1 which survives. PMID- 1703958 TI - Activation of the double-stranded RNA-dependent eIF-2 alpha kinase by cellular RNA from 3T3-F442A cells. AB - The interferon induced double-stranded-RNA-dependent eIF-2 alpha kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3 F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2 alpha kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2 alpha kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2 alpha kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2 alpha kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF 2 alpha kinase by regulatory RNA was prevented by addition of a high concentration of poly(I).poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2 alpha kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2 alpha kinase, are consistent with a physiological role for the dsRNA dependent eIF-2 alpha kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed. PMID- 1703959 TI - T helper cell epitope of rabies virus nucleoprotein defined by tri- and tetrapeptides. AB - T cell clones and subsequently hybridomas were generated from rabies virus-immune C3H/He mice to an immunodominant epitope of the viral nucleoprotein, termed 31D, that had previously been identified by a 15-amino acid-long synthetic peptide. T cells to this epitope that by phenotypical and functional characteristics belonged to the T helper cell subset were shown to respond to most rabies and rabies-related viruses. In order to define the minimal sequence needed to elicit a response from 31D-specific T cell clones or hybridomas, a number of peptides of varied lengths, i.e. 3-32 amino acids long, were tested. The ability of the peptides to induce a response was inversely correlated in their lengths, i.e., short peptides (3-5 amino acids long) had to be used at 10(6) times higher concentrations as compared to long peptides (15 or 32 amino acids long). Conversely, the specificity of the T cell response was directly correlated to the length of the peptides, i.e., while the response to 15-amino acid-long peptides exhibited a high degree of specificity, the response to 3- to 5-amino acid-long peptides showed a high degree of flexibility. The long as well as the short peptides had to be presented in association with I-Ek. We speculate that in this system the T cell receptor interacts predominantly with a peptide-induced modification of the I-Ek molecule. PMID- 1703960 TI - Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent on epitope patchiness, antibody affinity and complement concentration. AB - The relative complement-mediated lytic capability of the IgG subclass isotypes was studied using a matched set of mouse-human chimeric anti-(4-hydroxy-3 nitrophenyl)acetyl (NP) antibodies. The subclass pattern was shown to be highly dependent on variations in antigen concentration and to lesser extent on variation in epitope patchiness, antibody binding affinity and complement concentration. In general, the IgG3 subclass was most effective in inducing cytolysis at the different conditions used and only at high antigen concentration did the IgG1 subclass mediated more efficient cytolysis than IgG3. The IgG2 isotype required a relative high antigen concentration to be cytolytic while the IgG4 isotype was not cytolytic at any of the conditions tested. These individual characters of each of the IgG subclasses makes it conceivable that a subtle system of immunoregulation exists among the subclasses. PMID- 1703961 TI - Signal transduction by HLA class II antigens expressed on activated T cells. AB - Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross-linking of HLA-DR molecules expressed on allospecific, CD4+ T clones and cell lines can function as transduction elements that trigger rapid cellular responses including tyrosine phosphorylation of cellular proteins and mobilization of Ca2+ from internal stores. The proteins phosphorylated on tyrosine were distinct from those observed after cross-linking CD4. Ligation of CD4 and class II molecules generated a synergistic effect of the intracellular free Ca2+ concentration response that required an interaction between the molecules on the cell surface. Since class II is the natural ligand for CD4, the present data suggest that class II is induced on activated T cells to regulate CD4 function, possibly by specific interaction with the CD4 associated p56lck protein tyrosine kinase. PMID- 1703962 TI - Parameters affecting the immunogenicity of a liposome-associated synthetic hexapeptide antigen. AB - The effect of liposome association on the immunogenicity of the hexapeptide IRGERA was investigated. When administered in the absence of a carrier and adjuvant this peptide, which corresponds to a linear epitope located at the C terminus of histone H3, was not immunogenic. When mice were immunized with the peptide covalently linked to the surface of small unilamellar vesicles containing monophosphoryl lipid A as adjuvant, a relatively long-lasting response with memory cell induction was observed. The anti-peptide antibodies raised in this way reacted with the cognate sequence in the native histone. In contrast, coupling of the peptide to the surface of large vesicles yielded both an IgM and IgG response of short duration whereas encapsulation of the free peptide in large vesicles was ineffective. These results indicate that with short synthetic peptides, liposomes provide a substitute for a carrier protein. However, an adjuvant has to be incorporated in the vesicles in order to obtain an efficient immune response. Such an approach may be useful for designing synthetic vaccines. PMID- 1703963 TI - Fibronectin facilitates the migration of human natural killer cells. AB - The interaction of lymphocytes with the extracellular matrix plays an important role in the immune defence against tumor cells and virus-infected cells. We have examined the effect of matrix proteins on the migration of large granular lymphocytes (LGL) through 3-microns pores in Nuclepore filters in a Boyden invasion chamber. Fibronectin bound on the filter surface significantly increased (p less than 0.001) the capacity of LGL to migrate, whereas soluble fibronectin did not. In addition, a significantly higher (p less than 0.001) percentage of LGL was capable of migration through fibronectin-coated filters than through untreated filters. With fibronectin-coated filters, a strong enrichment of CD16+ and CD56+CD3- cells with LGL morphology and reduction of CD3+ cells was found among migrating cells when the incubation time was 4 h or less. Later agranular lymphocytes, mainly CD3+ T lymphocytes, also started to migrate. Laminin coating of filters also facilitated migration, and when filters were coated with both fibronectin and laminin the increase in migration was equal to the sum of the increases induced by each protein alone. Interactions between cell surface and the Arg-Gly-Asp (RGD) peptide sequence of many matrix proteins had no role in the LGL migration through untreated filters. However, when filters were coated with either fibronectin or laminin, or with both, peptide containing the RGD sequence reduced migration to the level of untreated filters, whereas an Arg-Gly-Glu control peptide had no effect. Our results show that unstimulated LGL/natural killer cells are capable of rapid migration through matrix-coated porous membranes, and that interactions between cell surface receptors and the RGD sequence of fibronectin and probably laminin are utilized in this process. PMID- 1703964 TI - Identification of a major antigenic epitope on CNBr-fragment 11 of type II collagen recognized by murine autoreactive B cells. AB - Immunization of certain strains of mice with native type II collagen (CII) induces both development of arthritis and an antibody response to autologous CII. The autoantibody response in a high-responder strain, the DBA/1 mouse, has been described earlier, and a number of monoclonal antibodies have been characterized for arthritogenicity and autoreactive binding to cartilage in vivo and in vitro. Here we map the antigenic epitope of one of these arthritogenic monoclonal antibodies (CII-C1). It belongs to a group of antibodies recognizing the CNBr fragment alpha 1(II)-CB11 of CII. Using the enzyme-linked immunosorbent assay technique, we show that the antibody reacts only with native, triplehelical CII, but not with other collagens. The antibody is able to stain specifically the CB11 fragment by immunoblotting, suggesting some partial renaturation of the CNBr fragment into triple-helical structures after blotting. The binding site of CII C1 on CB11 was further focused by rotary shadowing of antibody-labeled CII to a site 89 +/- 8 nm from the amino end of CII, corresponding to the middle of CB11. This location was confirmed by cleavage of CB11 with trypsin, separation of the tryptic peptides by high-performance liquid chromatography and dot-blot analysis of the antigenic peptides with the CII-C1 antibody. Sequencing of the single positive peptide located the antigenic epitope within the sequence GFAGQAGPAGATGAPGRP (residues 316-333). Assuming 0.29 nm per residue, this corresponds to a position within 92-96.5 nm from NH2 terminal end of CII. Apart from glycine residues, which are not exposed on the triple-helical structure, only two amino acid residues (F-x-y-Q) are conserved in CII from different species but are not found in the triple-helix of other collagens except type IV collagen. Therefore, this structure is likely to be of critical importance for the binding of the CII-C1 antibody. Of potential importance is that this structure is also found in certain other arthritogenic proteins such as 65-kDa mycobacterial protein, in CMV and EBV. PMID- 1703965 TI - Role of bone marrow cells in autoantibody production and lymphoproliferation in the novel mutant strain of mice, CBA/KlJms-lprcg/lprcg. AB - The novel mutant gene, lprcg, is allelic with lpr, but complements the gld gene in induction of lymphoproliferation. Mice with this autosomal recessive mutation, CBA/KlJms-lprcg/lprcg (CBA-lprcg), served in this study to analyze the abnormalities of bone marrow (BM) stem cells responsible for autoantibody production and lymphoproliferation by BM transfer experiments. Transferred CBA lprcg BM cells might have differentiated into so-called double-negative, anomalous lymphoid cells and caused production of autoantibodies such as anti-DNA antibodies in the environment of normal CBA/KlJms-(+)/+ (CBA-(+)) mice. Macroscopic graft-vs.-host-like disease as reported in lpr----non-lpr BM transfer was not observed in these recipients. In this BM chimera, however, lymphoproliferation did not ensue and the host's lymph nodes became atrophic. The lymphoproliferation required the coexistence of lprcg BM cells and lprcg lymph nodes in CBA-(+) mice. The results indicate that the functions of the lprcg gene are expressed at both BM and lymph node levels. Thus, this mutant strain of mice should provide an excellent model for analyzing aberrant lymphocyte differentiation from the BM cells leading to autoimmunity and lymphoproliferative disorder. PMID- 1703966 TI - The identification of the beta 2-microglobulin binding antigen encoded by the human CD1D gene. AB - Human cluster of differentiation (CD1) is a family of cell surface glycoproteins composed of a 43-49-kDa heavy chain non-covalently associated with beta 2 microglobulin. Five human CD1 genes have been detected and cloned. Three genes (CD1A, -B and -C) encode the serologically defined CD1a, -b and -c antigens. Thus two genes remain, CD1D and CD1E, whose protein products have not been characterized so far. This report describes how a beta-galactosidase-CD1D fusion protein was used to raise specific antisera and a monoclonal antibody against the CD1D gene product. The monoclonal antibody defines a cell surface molecule expressed on a cortical thymocyte cell line and is composed of a 49-kDa heavy chain associated with beta 2-microglobulin, which is serologically distinct from CD1a. PMID- 1703967 TI - [Carcinogenic risk of automobile exhaust: a review]. AB - The increasing number of motor vehicles is associated with diffuse exposure to their exhausts, formed by mixtures of hundreds of chemical compounds. Some of these compounds cause chronic irritation of the respiratory mucosa, while others are carcinogenic in experimental animals, particularly polycyclic aromatic hydrocarbons and nitroarenes. This review aims to describe epidemiologic evidences of carcinogenicity of engine exhausts. The study of the effects in humans of the exposure at issue is impaired by several difficulties: widespread exposure of a large part of the population, with low levels of exposure; aspecific effects, shared with other exposures; concurrent exposures acting on the same targets, particularly the respiratory mucosa (smoking, occupational exposures); difficulties in the quantitation of exposure to exhaust. We review the epidemiologic studies concerning occupational exposures published up to June, 1988. Overall, excess risks for lung and bladder cancers and for hematolymphopoietic malignancies have been reported, particularly in people exposed to diesel exhaust. Excesses are modest, and their interpretation is often impaired by lacking or inadequate consideration of the effects of potential confounders. The review considers, for each study, enrollment procedures, information on exposure, and inclusion of potential confounders. In addition, aspects concerning internal coherence, such as a dose-response relationship, are considered. Particularly relevant are two studies on USA railroad workers, showing a relative risk of 1.4 among those exposed to diesel exhaust. The authors used, in the estimation of exposure, actual measurement of dose in recent years for some jobs and workplaces. Levels of exposure to respirable dusts were around 82-330 micrograms/m3, after allowing for cigarette smoking. According to the International Agency for Research on Cancer, these is limited evidence of carcinogenicity in humans of diesel exhaust, and inadequate evidence for gasoline exhaust. PMID- 1703968 TI - Influence of murine mast cell growth factor (c-kit ligand) on colony formation by mouse marrow hematopoietic progenitor cells. AB - Purified natural and recombinant murine mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with other cytokines for effects in vitro on colony formation by multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells from BDF1 mouse bone marrow. Both preparations stimulated Epo-dependent CFU-GEMM and enhanced Epo-dependent BFU-E colony numbers and size. MGF had some stimulating activity for CFU-GM. When used in combination with plateau concentrations of pokeweed mitogen mouse spleen cell conditioned medium or granulocyte-macrophage colony stimulating factor (CSF), MGF enhanced in greater than additive fashion colony formation by CFU-GM. MGF also enhanced the size of colonies formed, an enhancement greatest for colonies containing granulocytes and macrophages. MGF did not enhance Macrophage CSF stimulated colony numbers or size. MGF seems to be an early acting cytokine with preferential effects on the growth of more immature hematopoietic progenitor cells. PMID- 1703969 TI - Improved automated differential counts of leukocytes from newborn infants using pre-dilution of blood samples. AB - A method for automated differential leukocyte counting using peroxidase staining and light scatter (H1) failed to present results in more than 25% of blood samples from newborn infants. These failures were mostly seen at or below 2 weeks of age, and with high concentrations of haemoglobin F (HbF). Dilution of the blood with equal amounts of isotonic saline made the problem disappear almost completely. The results of differential counts on such diluted blood samples also compared more favourably with traditional manual counts of blood smears than did the results obtained with undiluted blood. The present method with blood diluted 1/2 is therefore recommended for routine use in newborn infants. PMID- 1703970 TI - Standardization and harmonization of the blood count: the role of International Committee for Standardization in Haematology (ICSH). AB - Scientific principles of standardization were first applied in haematology in 1963 when the International Committee for Standardization in Haematology was established with a primary objective to improve the measurement of haemoglobin. Subsequently, ICSH has established Expert Panels on a wide range of haematological topics, including especially a Panel on Cytometry. The purpose of haematological standardization is to obtain precision, accuracy, specificity and harmonization of results between different laboratories in all countries and also between different instruments or methods in the same laboratory. To achieve these objectives ICSH sponsors collaborative studies by scientists from academic centres and from industry and uses a consensus procedure for establishing standards on the basis of the scientific data, followed by an educational programme to ensure that the standards are adopted worldwide. ICSH defines material standards and standardized methods. Material standards are classified as primary international standards, certified reference materials, secondary standards and calibrators. These must be distinguished from control preparations which are intended exclusively for quality control. Standardization of methods must also be considered at four levels: definitive, reference, selected and routine. Each has a place in practice but their roles must be clearly defined. ICSH has an established protocol for evaluation of automated blood cell counters. This defines the levels of precision and accuracy of instrument performance. It is also necessary to assess "clinical utility". The main requirement of the practising haematologist is clinical reliability and harmonization of results for comparability. One of the major functions of ICSH is to provide an interface for collaboration between the manufacturers who develop the instruments and the users in order to achieve this goal. PMID- 1703971 TI - Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes. AB - 1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably. PMID- 1703972 TI - Fibronectin, hyaluronan, and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration. AB - "Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo. PMID- 1703973 TI - Altered expression of diabetes in BB/Wor rats by exposure to viral pathogens. AB - Autoimmune diabetes mellitus affects greater than 50% of diabetes-prone BB (DP BB) rats but less than 1% of diabetes-resistant BB (DR BB) rats. We report an outbreak of spontaneous diabetes among DR BB rats that coincided with serologic evidence of the onset of viral infection. This apparent link between a change in the environment and the expression of diabetes then led us to study the interaction of environmental exposure to viral pathogens in this disorder with virally seropositive and seronegative populations of BB rats and polyinosinic polycytidylic acid (poly I:C), an interferon inducer known to accelerate diabetes onset in DP rats. We administered a cytotoxic anti-RT6 monoclonal antibody, poly I:C, or both to DR rats. Depletion of the RT6.1+T-lymphocyte population has previously been shown to induce diabetes and thyroiditis in DR rats. RT6 alone did not induce diabetes in seronegative DR rats, and poly I:C was only weakly effective, but nearly all animals given both reagents became diabetic. When given to seropositive DR rats, either reagent alone induced diabetes; when given to non BB rats, neither agent was effective. Poly I:C also accelerated the onset of DP diabetes to a greater extent in seropositive than in seronegative rats. We conclude that expression of the genetic predisposition to diabetes present in all BB rats depends on cellular factors that include the presence or absence of regulatory (RT6+) T lymphocytes and modulatory environmental factors including exposure to viral pathogens. PMID- 1703974 TI - Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. AB - The cofactor requirements of macrophage nitric oxide (NO.) synthase suggest involvement of an NADPH-dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di-2 thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (IC50's 50-150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5'-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5 diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO. synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO.-generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and endothelial cells depends on an NADPH-utilizing flavoprotein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO(.). PMID- 1703975 TI - Prospective trial for early detection of pancreatic cancer by elevated serum immunoreactive elastase. AB - Early detection of pancreatic cancer was prospectively evaluated by measuring serum immunoreactive elastase (IRE) in 722 patients in two hospitals during the past 18 months. Patients included in the study were over 40 years of age and had symptoms suggestive of pancreatic disease such as upper abdominal pain, discomfort or mass, jaundice, weight loss, or diabetes. Among the 722 patients, 171 exhibited elevation of serum IRE. Pancreatic diseases were subsequently found in 42% of the 171 patients. Pancreatic cancer was found in 22 patients, among which 17 had elevated serum IRE. Among the 17 pancreatic cancer patients with elevated IRE, 10 underwent radical resection of the cancer but in none of the five patients with normal serum IRE could radical resection be carried out. Three of the 10 patients had a small cancer less than 2 cm in diameter and two of them survived for more than three years. Patients over 40 or 45 years of age complaining of upper abdominal pain of recent onset that cannot be explained by diseases other than that of the pancreas would be candidates for measurement of serum elastase, and this is an effective way to detect pancreatic cancer at an early stages. PMID- 1703976 TI - A case of gallbladder cancer producing granulocyte-colony-stimulating factor. AB - A patient with pleomorphic giant cell carcinoma of the gallbladder also had mild neutrophilia, and this tumor was found to be human granulocyte colony-stimulating factor (G-CSF)-positive on immunohistochemical staining. CSF activity in urine and serum was not examined, but leukocytosis disappeared immediately after cholecystectomy. These findings suggest that this was a G-CSF-producing tumor. PMID- 1703977 TI - Gastrosto-biliary irrigation for maintenance of stent patency. PMID- 1703978 TI - Characteristics of activation of rat mast cells by polyethylenimines and a polyallylamine: effects on the release of histamine and of arachidonate and its metabolites. AB - 1. Three polyethylenimines and one polyallylamine released radioactivity from rat peritoneal mast cells labeled with [1-14C]arachidonic acid and concomitantly released histamine from the cells. 2. This enhancement of the release of radioactivity was inhibited by phospholipase A2 inhibitors, quinacrine (1 mM) and p-bromophenacyl bromide (10 microM), suggesting that polyethylenimine and polyallylamine activates phospholipase A2 to generate arachidonate and its metabolites. 3. The effects of H-7 or K-252a, general kinase inhibitors for the release of histamine and of arachidonate and its metabolites induced by the polycations, were different from those of W-7, a calmodulin inhibitor. The mechanisms to generate arachidonate and its metabolites seemed to differ from those to release histamine; activation of phospholipase A2 by the polycations was calmodulin-dependent. 4. p-Bromophenacyl bromide inhibited the histamine release induced by polyethylenimines and a polyallylamine, suggesting that arachidonate production by means of phospholipase A2 activation by polycations is an important process in the release of histamine from mast cells. PMID- 1703979 TI - Residual secretion of amylase induced by isoproterenol from rat parotid gland slices. AB - 1. During pretreatment with isoproterenol (ISP) for 2 min the accumulation of cyclic AMP increased in a dose-dependent manner but amylase release did not increase even in the highest dose of ISP used. 2. After a brief pretreatment with ISP, the following 10-min incubations with fresh medium without ISP caused increase in amylase secretion (residual secretion). However, cyclic AMP accumulation returned to the non-treated level during the residual secretion of amylase. 3. Both residual amylase release and cyclic AMP accumulation after pretreatment with ISP were enhanced in the presence of isobutyl-methylxanthine. Residual amylase release was not affected in the absence of extracellular calcium ion. 4. We suggest that there may be another pathway than cyclic AMP to cause residual amylase secretion induced by brief pretreatment with ISP in rat parotid acinar cells. PMID- 1703980 TI - The efficiency of RNA 3'-end formation is determined by the distance between the cap site and the poly(A) site in spleen necrosis virus. AB - The efficiency of RNA 3'-end formation of spleen necrosis virus (SNV) is determined by the distance between the cap site and the poly(A) site. When the distance between the cap site and the poly(A) site was shorter than 500 bases, only 3-9% of the RNA was polyadenylated at the SNV poly(A) site. However, when the distance between the cap site and the poly(A) site was 1400 bases or more, 70% of the total RNA was polyadenylated at the SNV poly(A) site. In contrast, the poly(A) signal sequences of the thymidine kinase (tk) and SV40 late genes functioned at high efficiency, even with a distance between the cap site and the poly(A) site that was short enough to inactivate the SNV poly(A) signal. Therefore, this distance-dependent inactivation of RNA 3'-end formation is specific for SNV sequences and perhaps for related retroviruses. This finding explains the difference between the 5' and 3' poly(A) sites in many retrovirus RNAs. PMID- 1703981 TI - Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations. AB - Differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF) is a glycoprotein that controls differentiation of pluripotential stem cells. Alternative transcription generates both diffusible and matrix-associated forms of DIA/LIF. Transcriptional analysis using a sensitive ribonuclease protection assay revealed that the two messages are expressed independently, consistent with the proposition that the two forms of DIA/LIF have distinct biological roles. DIA/LIF expression was found to be activated early during differentiation of embryonic stem (ES) cells, providing a mechanism for feedback regulation of stem cell renewal. Expression of DIA/LIF by mesenchymal cells was shown to be controlled in a paracrine manner by polypeptide regulatory factors. Specific expression of the two forms of DIA/LIF was also demonstrated in the egg cylinder stage mouse embryo. The combination of cell type-specific and signal-specific regulation enables very precise control over DIA/LIF expression and may represent an important component of the regulatory networks that govern stem cell proliferation and differentiation during mammalian development. PMID- 1703982 TI - SH2 mutants of c-src that are host dependent for transformation are trans dominant inhibitors of mouse cell transformation by activated c-src. AB - The c-src gene encodes a membrane-associated protein-tyrosine kinase, pp60c-src, whose substrates and regulators have not been identified. In an effort to obtain mutants that might assist the search for proteins that interact with pp60c-src, we have generated by site-directed mutagenesis two alleles of chicken c-src that are host dependent for transformation and inhibit transformation of mouse cells by activated c-src in a trans-dominant manner. These alleles, named M6 and M9, encode nonconservative changes within the highly conserved FLVRES sequence in the src homology-2 (SH2) region of pp60c-src, as well as an activating change, Y527F, near the carboxyl terminus. M6 and M9 transform chicken embryo fibroblasts (CEF) more efficiently than the parental allele (Y527F c-src), but fail to transform mouse NIH-3T3 cells. The product of M6-src is less stable than activated pp60c src in NIH-3T3 cells and shows decreased protein tyrosine kinase activity on all tested substrates; the product of M9-src, however, is stable, has a novel pattern of substrate preference for tyrosine phosphorylation in vitro, and induces a pattern of phosphotyrosine-containing proteins in mouse cells that is similar to that induced by Y527F c-src, even though it fails to transform these cells. Both M6 and M9 inhibit transformation of NIH-3T3 cells by activated c-src in a dose dependent manner, as assayed by resistance of M6- or M9-expressing cells to transformation by Y527F c-src or by morphological reversion of cells previously transformed by activated c-src following introduction of M6 or M9. When reversion occurs, the concentration of protein encoded by the active allele declines, without change in level of src mRNA or rate of src protein synthesis, implying that the products of M6 and M9, when present at adequate levels, can destabilize transformation-competent src protein. These alleles offer new opportunities for interfering with the actions of src-related genes and for isolating cellular factors required for the functions of those genes. PMID- 1703983 TI - Clinical management of the terminally ill. AB - Physicians who provide primary care for the elderly are spending more time caring for terminally ill patients. Although curing these patients' illnesses is impossible, it is often possible to improve their quality of life and give them more control over their illness. Communication with the patient and family members, advance directives, and planning for death are important. Symptoms such as pain, dyspnea, and nausea can usually be controlled. Other health care professionals and hospice care when appropriate can also be helpful. PMID- 1703984 TI - [Participation of the sensory nerves in the inflammatory response induced by irritants]. AB - Participation of the sensory neurons, especially substance P (SP)-containing neurons, in the inflammatory response induced by formalin (FOR), croton oil (CRO), mustard oil (MUS), carrageenin (CAR), dextran (DEX) and egg white (EGG) was studied in mice and rats. FOR-induced foot edema was significantly inhibited by denervation of the sciatic nerve of rats, but it was slightly facilitated by that of the saphenous nerve. CAR-induced edema was not influenced by denervation of both nerves. In mice pretreated with capsaicin of the sciatic nerve, the early phase of foot edema induced by FOR, CRO or MUS was significantly inhibited. Edema induced by CAR, DEX or EGG was not inhibited by the capsaicin treatment. Spantide (an SP antagonist) at 0.1 mg/kg showed the same result as the capsaicin treatment. FOR, CRO or MUS caused a marked plasma extravasation, which was inhibited by spantide. The weak plasma extravasation elicited by CAR, DEX or EGG was not inhibited by spantide. FOR, CRO or MUS caused an intense biphasic nociceptive response; and the early phase of the response was inhibited by spantide. CAR, DEX or EGG caused little or no nociception. These findings suggest that SP antidromically released from the primary sensory neurons may induce potent vascular responses such as vasodilatation and plasma extravasation in the early phase of inflammation induced by FOR, CRO and MUS, whereas inflammatory responses induced by CAR, DEX and EGG may progress through different processes. PMID- 1703985 TI - Immunocytochemical determination of ABH and MN antigens on dried blood traces in the nanoliter range. AB - The absorption-elution test and the mixed cell agglutination reaction are both ultimately based on the ability of indicator cells to agglutinate. This agglutination reaction requires a relatively large amount of antigenic epitopes, and, in addition, a relatively high volume of blood traces. The immunocytochemical demonstration of epitopes requires a lower volume, which, however must be fixed for investigation, thus possibly causing damage to the epitopes and thereby preventing detection. An immunocytochemical method is presented which permits ABH and MN antigen determination on dried blood traces of nanoliter quantities without special fixation. This method is based on immunocytochemical demonstration of antigens directly on the cell membrane in combination with the use of a coated glass slide that ensures maximum economy of epitopes. PMID- 1703986 TI - Identification of T-cell epitopes of autoantigens using recombinant proteins; studies on experimental autoimmune myasthenia gravis. AB - In the Lewis rat, T-cell lines from animals immunized with native or denatured Torpedo nAChR recognize the Torpedo-derived recombinant protein T alpha X1 omega (alpha-2-200) but not the equivalent mouse- or chick-derived recombinant proteins X4 omega or C alpha X1 omega (alpha 6-216 and alpha 35-216, respectively). T-cell lines derived from animals immunized with T alpha X1 omega, X4 omega or C alpha X1 omega are specific for the homologous protein. This lack of cross-species reactivity suggests caution in the use of Torpedo nAChR-selected lines generated from human patients. Proteolysis and fractionation of the products by reverse phase HPLC was effective in localization of a T-cell epitope of X4 omega, a mouse derived recombinant protein. With Lewis rats, the major epitope of T alpha X1 omega is alpha 97-112. However, the major epitope of the mouse-derived protein, X4 omega, as determined by proteolytic digestion and fractionation of the products by reverse-phase HPLC, is alpha 14-22. This shift in T-cell epitope between closely related proteins may result from the conservation of sequence of alpha 97-112 between mammalian species. PMID- 1703987 TI - Antigen presentation and HLA-DR expression by FK-506-treated human monocytes. AB - The novel macrolide immunosuppressant FK-506 demonstrated superior potency to cyclosporin A (CsA) in the inhibition of purified protein derivative (PPD) induced human T-cell proliferation. Pulsing of monocytes with PPD for 24 hr in the presence of FK-506 did not impair their capacity to subsequently induced the proliferation of purified autologous T cells. In contrast, FK-506 profoundly inhibited the proliferative response of T cells to antigen-pulsed monocytes. Recombinant IL-2, but not IL-1, partially restored the proliferative response to PPD in the presence of the drug. FK-506 had no effect on basal or rIFN-gamma induced expression of HLA-DR on monocytic cell line cells. These findings provide the first evidence that FK-506 can profoundly inhibit soluble antigen-induced human T-cell proliferation at concentrations which do not significantly impair the accessory function of mononuclear phagocytes. PMID- 1703988 TI - Equine monoclonal antibodies recognize common epitopes on variants of equine infectious anaemia virus. AB - Equine-murine xenohybridoma cells were produced using SP2/0 murine myeloma cells and splenic lymph node cells obtained from horses infected with 10(6) TCID50 of single cloned variants of equine infectious anaemia virus (EIAV). The xenohybridomas secreted equine IgG monoclonal antibodies reactive with EIAV in enzyme immunoassays employing purified virus. Seven antibodies were studied in detail. They bound to viral glycoproteins (gp90 or gp45) in radioimmunoprecipitation assays, and reacted with homologous EIAV as well as five other cloned variants of EIAV. When evaluated against a single cloned variant of EIAV (EIAV-WSU5), two antibodies bound to different epitopes on gp90. The five remaining antibodies reacted with the same or overlapping epitopes on gp45. None of the antibodies exhibited viral neutralizing activity. PMID- 1703989 TI - Reduced activity of DAF on complement enzymes bound to alternative pathway activators. Similarity with Factor H. AB - Attachment of C3b to activators of the alternative pathway of complement results in a decrease in regulatory activity expressed by Factor H. Decay-accelerating factor (DAF) and Factor H were found to exhibit quantitatively similar decreases in regulatory activity toward the C3 convertase (C3b,Bb) bound to activators, such as zymosan (Zym) and rabbit erythrocytes (ER), compared to non-activators, such as sheep (ES) and bovine (EB) erythrocytes. Purified DAF and Factor H, in 0.1% NP-40, were assayed by measuring the amount required to release 50% of the radiolabelled Bb in 10 min from C3b,Bb on Zym or cross-linked erythrocytes. The relative effectiveness (i.e. the restriction index, RI) of DAF for accelerating the decay of C3b,Bb on the various particles was: ES (1.0), ER (0.04) and Zym (0.03). The RI for Factor H was: ES (1.0), ER (0.04) and Zym (0.07). The rate of decay of C3b,Bb induced by DAF and Factor H showed similar restriction. The results suggest that the regulatory properties of DAF are reduced if the cells on which it resides become activators of the alternative pathway as a result of transformation, virus infection or surface alteration. These findings may explain reports of dysfunctional DAF on alternative pathway-activating cells. PMID- 1703990 TI - Inflammatory responses induced by substance P in rat paw. AB - Substance P (SP) injection in the plantar region of rat hind paw caused a dose related inflammation, which reached a peak within 10 min of injection and declined after 60 min. Low doses (0.25-0.063 mg/kg) of SP-antagonists like (D Pro2, D-Trp7,9)-SP and (D-Pro2, D-Phe7, D-Trp9)-SP pretreatment significantly inhibited the SP induced paw oedema, while higher doses (0.5-1 mg/kg) showed agonistic effects. Pretreatment with diphenhydramine alone or along with low doses of SP-antagonists was highly significant in blocking this inflammation, the latter combination being more effective than the former. Pretreatment with acute capsaicin produced a synergestic effect on SP induced paw oedema, while pretreatment with chronic capsaicin significantly inhibited this SP induced paw oedema. The results indicate involvement of histamine and possible therapeutic importance of capsaicin in SP mediated inflammatory type of responses. PMID- 1703991 TI - A longitudinal follow up of neurodevelopment of high risk newborns--a comparison of Amiel-Tison's method with Bayley Scales of Infant Development. AB - The neurodevelopment of 42 high risk babies and 7 control babies was assessed longitudinally till the age of 12 months by using two different methods. The method of neurological evaluation described by Amiel-Tison was used, and the results compared with those of a standard developmental test, the Bayley Scales of Infant Development. The Amiel-Tison method was found to be a sensitive test for picking up abnormalities till the age of 9 months, but lost its advantage over the Bayley Scales at 12 months. Besides, the test was quick, simple to learn and did not need a special kit or a trained psychologist and was hence found to be a good screening method. PMID- 1703992 TI - Electrical instability in patients undergoing surgery for atrioventricular septal defect. AB - Postoperative 24-hour Holter monitoring was performed in 106 patients with atrioventricular septal defect in order to identify the incidence of atrial and ventricular arrhythmias. Of the patients, 72 had separate atrioventricular orifices, including 13 with a small ventricular component to the defect, and 34 patients had a common atrioventricular orifice. Two groups of abnormal patients were found. First, patients with good electrical stability characterized by isolated atrial (9 patients) and ventricular (25 patients) extrasystoles falling in classes I and II of Lown. Second, patients with marked electrical instability characterized in one patient by repetitive atrial extrasystoles, in another by atrial flutter, in 2 by polymorphic ventricular extrasystole and in 8 by couplets or triplets. Electrical instability in individual patients was then compared, by means of logistic regression analysis, with operative, surgical and postoperative variables. There was no incidence of sudden death in our series. After surgical repair, ventricular arrhythmias were more frequent than atrial arrhythmias (33% vs. 10%) and were unrelated to the type of atrioventricular septal defect. Cardiac electrical instability after operation was significantly related to larger operative body size, higher postoperative end diastolic diameter of the right ventricle, larger size of the ventricular septal defect, coexistence of postoperative right bundle branch block and left anterior hemiblock. Conversely, the risk of arrhythmias was reduced by more recent operative data and by greater shortening fraction of the left ventricle. PMID- 1703993 TI - Increased angiogenesis during wound healing in rats with streptozotocin-diabetes. AB - The neovascularization that occurs during wound healing in the perforated mesenteric membrane was quantitatively studied in rats with streptozotocin induced diabetes. The virtually avascular mesenteric windows in both controls and diabetic rats were perforated with a scalpel during laparatomy. At designated intervals during days 1-63 postoperatively, the mesenteries were excised, fixed, and cut perpendicularly through the wound margin or the central part of a closed wound. An unperforated window from each animal served as an internal control. The numerical microvascular density of the healing tissue and the unperforated control mesentery was assessed by morphometry on photomicrographs with regard to number of vascular profiles per mm length of mesentery or mm2 sectioned tissue area. The vascularity in unperforated windows of both diabetics and age-matched healthy controls was unaffected by laparotomy whereas perforation significantly increased the numerical microvascular density of the mesentery. The onset of angiogenesis in the perforated mesenteries occurred on days 5-7 and in both diabetics and controls, wound areas retained an increased numerical microvascular density throughout the observation period. The numerical microvascular density of the wound areas was greater in diabetics than controls following closure of the wounds. A number of wounds healed without being vascularized. The number of vascularized wounds was significantly higher in diabetic animals than controls. PMID- 1703994 TI - Gamma endorphin and HLA class I related immune functions. Preliminary observations. PMID- 1703995 TI - Neuropeptide modulation of interferon gamma production. PMID- 1703996 TI - Modulation of thymic endocrine function, cytokeratin expression and cell proliferation, by hormones and neuropeptides. PMID- 1703997 TI - Neuroendocrine markers expressed by human natural killer (NK) cells are also detectable on human small cell carcinomas, neuroblastomas, and sea urchin coelomocytes. PMID- 1703998 TI - Interaction between nerve growth factor and lysophosphatidylserine in rat peritoneal mast cells. PMID- 1703999 TI - Human immunodeficiency virus antibody responses determined by site-directed serology. AB - In this brief review, the use of synthetic peptides representing linear antigenic sites in human immunodeficiency virus (HIV) structural proteins for detection of antibodies in sera from HIV-infected individuals is discussed. It has been demonstrated that peptide antigens offer unique advantages for determination of HIV-specific antibodies. In particular, various peptides representing the region of amino acids 580-620 in the transmembranous glycoprotein have been effectively used. Of primary importance is the fact that properly designed site-specific serological tests allow a distinction between type-specific antibodies, a quality not provided by any other currently available test. Furthermore, synthetic peptide antigens allow the design of highly simplified and effectively standardized assays. Hereby, they lend themselves to use for screening purposes or confirmatory testings not only in industrialized countries, but also in developing countries. It is also possible that tests with selected peptides may measure antibodies which have value in predicting the risk for infection in children born to seropositive mothers and for progression of disease in infected individuals. PMID- 1704000 TI - Map of sequential B cell epitopes of the HIV-1 transmembrane protein using human antibodies as probe. AB - Antibodies of individuals infected with the human immunodeficiency virus type 1 (HIV-1) were used to probe the antigenicity of the HIV-1 transmembrane protein of 41 kD (gp41) by antibody-reactive peptide scanning (Pepscan). Eleven distinct sequential antibody-binding sites were defined by testing reactivity to 339 overlapping nonapeptides spanning the complete gp41 amino acid sequence. Such analysis only maps continuous antibody-binding sites of nine amino acids in length and does not identify putative discontinuous or assembled epitopes. Three B cell epitopes (aa 609-622; aa 655-699; aa 664-681) at the amino-terminal border of the putative transmembrane anchor and two (aa 732-748; aa 744-762) at the carboxyl-terminal border of this domain were the most antigenic. One antibody binding domain (aa 834-852) with four amino acids homologous to the beta-1 domain of HLA class II beta-chain was recognized by the serum in 1 of 4 AIDS patients tested and not by any of the eight sera from symptom-free individuals. Although functional domains of gp41 involved in virus replication, cytopathicity and possibly immunosuppression were shown to bind antibodies of HIV-1-infected individuals, no relationship between antibody recognition patterns and disease progression was apparent. PMID- 1704001 TI - Early increase in CGRP- and VIP-immunoreactive nerves in the skin of streptozotocin-induced diabetic rats. AB - We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes. PMID- 1704002 TI - Effect of vincristine or bleomycin on radiation-induced cell killing of mice spermatogonial stem cells: the importance of sequence and time interval. AB - The effect of single doses of vincristine (VCR) or bleomycin (BLM) on mice spermatogonia was investigated, and the influence of either of these drugs on the radiation response of murine spermatogonial stem cells was examined. When assessed by flow cytometry, VCR (1.0 mg/kg) or BLM (100 mg/kg) reduced the survival in the differentiated spermatogonia to 4% and 37% of controls, respectively (p less than 0.05). VCR reduced the stem cells to 79% of controls (p less than 0.05), whereas BLM had no apparent effect on the stem cells (p greater than 0.05). Drugs were administered intraperitoneally up to 28 days before or after local irradiation with 9 Gy. VCR produced significant enhancement of radiation-induced damage to spermatogonial stem cells, which was most prominent when administered 6 or 12 hr after irradiation. BLM administered before irradiation or 1 hr after radiotherapy produced significant enhancement. PMID- 1704003 TI - An unusual correlation between ppGpp pool size and rate of ribosome synthesis during partial pyrimidine starvation of Escherichia coli. AB - Escherichia coli was exposed to partial pyrimidine starvation by feeding a pyrBI strain orotate as the only pyrimidine source. Subsequently, differential rates of synthesis of rRNA and of a few ribosome-associated proteins as well as the pool sizes of nucleoside triphosphates and ppGpp were measured. As the orotate concentration in the medium was reduced, the growth rate decreased and the pools of pyrimidine nucleotides, particularly UTP, declined. We did not observe the normal inverse relation between concentration of ppGpp and growth rate; rather, we observed that the ppGpp pool was low at slow growth rates. Upshifts in growth rate were made by adding uracil to a culture growing slowly on orotate. Downshifts could be provoked by adding aspartate plus glutamate to a culture growing at a high concentration of orotate. Following the upshift, both the rates of synthesis of the ribosomal components and the pool of ppGpp increased rapidly, while they all decreased after the downshift. These results are discussed in relation to the role of ppGpp in the growth rate control and the stringent response. PMID- 1704004 TI - Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella. AB - Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen. PMID- 1704005 TI - Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase. AB - In Escherichia coli K-12, expression of zwf, the gene for glucose 6-phosphate dehydrogenase, is coordinated with the cellular growth rate and induced by superoxide-generating agents. To initiate the study of the molecular mechanisms regulating its expression, the gene was cloned and its DNA sequence was determined. The 5' ends of zwf mRNA isolated from cells growing in glucose and acetate minimal media were mapped. The map was complex in that transcripts mapped to -45, -52, and -62, with respect to the beginning of the coding sequence. Three analytical methods were used to search the DNA sequence for putative promoters. Only one sequence for a promoter recognized by the sigma 70 form of RNA polymerase was found by all three search routines that could be aligned with a mapped transcript, indicating that the other transcripts arise by processing of the mRNA. A computer-assisted search did not reveal a thermodynamically stable long-range mRNA secondary structure that is capable of sequestering the translation initiation region, which suggests that growth-rate-dependent regulation of glucose 6-phosphate dehydrogenase level may not be carried out by a mechanism similar to the one for the gene (gnd) for 6-phosphogluconate dehydrogenase. The DNA segment between the -10 hexamer and the start point of transcription resembles the discriminator sequence of stable RNA genes, which has been implicated in stringent control and growth-rate-dependent regulation. PMID- 1704007 TI - Monoclonal antibody mapping of structural and functional plectin epitopes. AB - To map structural and functional epitopes of the cytomatrix protein plectin, a set of mAbs was prepared by immunization of mice. Using immunoblot analysis of plectin fragments obtained after limited digestion with various proteases, two groups of mAbs were distinguished. The epitopes of one group (1) were located on a 130-kD terminal segment of the plectin 300-kD polypeptide chain, whereas those of the other group (2) bound within a 40kD segment confined to a central domain of the polypeptide chain. Domains containing the epitopes of group 2 mAbs were shown to include in vitro phosphorylation sites for kinase A, whereas kinase C phosphorylation sites were found on the same terminal segment that contained group 1 mAb epitopes. Rotary shadowing EM of mAb (Fab fragment) -decorated plectin molecules at various states of aggregation, ranging from characteristic dumbbell-shaped single molecules to highly complex multimeric structures, revealed that the epitopes of group 1 as well as those of group 2 mAbs were located on plectin's roughly 200-nm long rod domain interlinking its two globular end domains. Epitopes of group 1 mAbs were localized within a region near the center of the rod, those of group 2 in more peripheral sections near the globular end domains. Solid-phase binding assays carried out in the presence of Fab fragments of mAbs demonstrated an interference of certain group 1 mAbs in the interactions of plectin with vimentin and lamin B. On the other hand, plectin's self-interaction was inhibited mainly by Fab fragments with epitopes in the peripheral rod domain (group 2 mAbs). Together, these results suggested that the molecular binding sites of plectin for vimentin and lamin B, as well as the phosphorylation sites for kinase C, were confined to a defined central section of plectin's rod domain. In addition, they suggest an involvement of peripheral rod sections in plectin self-association. PMID- 1704006 TI - Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase). AB - The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus. PMID- 1704008 TI - The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP-, N-CAM-, NGF receptor-) depends on growth inhibition. AB - The present experiments were designed to clarify the relationship between cAMP elevation, proliferation and differentiation in Schwann cells. They were carried out on short-term cultures of cells obtained from neonatal rat sciatic nerves. It was found that the myelin-related phenotype was expressed in response to agents that elevate or mimic intracellular cAMP (forskolin, cholera toxin, cAMP analogues), provided cell division was absent. This phenotype included upregulation of the major myelin protein P0 and downregulation of GFAP, N-CAM, A5E3, and NGF receptor. In contrast, when cells were cultured in conditions where cell division occurred, elevation of intracellular cAMP produced an alternative response, characterized by DNA synthesis and absence of myelin-related differentiation. The cAMP mediated induction of an early Schwann cell antigen, 04, followed a different pattern since it was induced equally in dividing and nondividing cells. These observations are consistent with the proposal that during development of the rat sciatic nerve: (a) cAMP elevation, possibly induced by axon-associated factors, is a primary signal responsible for the induction of 04 expression in proliferating Schwann cells during the premyelination period; (b) subsequent withdrawal of cells associated with the larger axons from the cell cycle acts as a permissive secondary signal for induction of myelination, since in quiescent cells the ongoing cAMP elevation will trigger myelination. PMID- 1704009 TI - GMP-140 binds to a glycoprotein receptor on human neutrophils: evidence for a lectin-like interaction. AB - GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP 140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP 140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism. PMID- 1704010 TI - Hyperthyroidism due to a pituitary adenoma composed of two different cell types, one secreting alpha-subunit alone and another cosecreting alpha-subunit and thyrotropin. AB - A 37-yr-old female presented with clinical signs and symptoms of mild hyperthyroidism, high serum levels of free T4 (24.2 pmol/L), free T3 (11.7 pmol/L), and sex hormone-binding globulin (157 nmol/L) as well as measurable (by immunofluorometric assay) serum TSH concentrations (1.9 mU/L) in the absence of any known methodological interference. The above finding indicated the presence of hyperthyroidism due to inappropriate secretion of TSH, whose neoplastic origin was documented by computed tomographic scan showing a 1-cm pituitary adenoma. The diagnosis was confirmed by elevated alpha-subunit levels (9.2 micrograms/L) and alpha-subunit/TSH molar ratio (25.2) as well as absent TSH suppression after T3 administration. TRH injection (200 microgram, iv) caused impaired TSH (from 3.0 to 4.8 mU/L) and unexpectedly exaggerated alpha-subunit (from 8.8 to 18.2 micrograms/L) responses. Such a discrepancy was also observed after other dynamic tests. Double gold particle immunostaining of the adenomatous tissue removed at surgery showed that all of the cells contained secretory granules positive for alpha-subunit, while very few cells were positive for TSH beta and alpha-subunit. In conclusion, the present study demonstrates the existence of TSH-induced hyperthyroidism due to a pituitary adenoma composed of two different cell types: one secreting alpha-subunit alone and another cosecreting alpha-subunit and TSH. PMID- 1704011 TI - Thyrotropin-secreting pituitary adenomas: report of seven cases. AB - Seven patients with hyperthyroidism due to a TSH-secreting pituitary macroadenoma have been observed of a total of 800 patients with pituitary tumors over a period of 15 yr. Serum TSH levels varied between 1.1-36.3 mU/L. The serum alpha-subunit level was low in 1 case, while in 4 other cases the concentration was elevated and varied between 3.7-7.8 micrograms/L. Serum TSH beta levels were normal in the 4 cases in which it was determined. Serum GH or PRL levels were elevated in 5 cases. In 1 patient the cosecretion of TSH, GH, and PRL was confirmed by immunocytochemical examination. Serum TSH and alpha-subunit responses to TRH, GnRH, CRF, GRF, dexamethasone, methimazole, T3, and bromocriptine administration were variable when studied. Serum TSH and alpha-subunit circadian rhythms were absent in 1 case and inverted in another. A serum alpha-subunit pulsatility without TSH pulses was observed in 1 patient. Five patients underwent transsphenoidal adenomectomy. Three of 4 patients operated on in our center were cured, but a recurrence of the adenoma was found in 1 of them after 5 yr. The fifth patient was not cured. Treatment with octreotide in 3 patients resulted in normalization of serum TSH, GH, and thyroid hormones levels. Cosecretion of PRL in 1 case and alpha-subunit in 2 cases was also inhibited. Partial tachyphylaxis occurred in 1 patient. In summary, heterogeneity in clinical presentation, hormonal expression, and therapeutic response appears to characterize these TSH secreting adenomas. PMID- 1704012 TI - Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. AB - A silver staining method used routinely for detecting bacterial lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gels (C. Tsai and E. Frasch, Anal. Biochem. 119:115-119, 1982) appeared to be inappropriate for visualizing certain LPS preparations. It did not stain S-form fractions of polyagglutinable Pseudomonas aeruginosa LPS or several partly deacylated (alkali treated) S-form LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, these LPS preparations could be detected by anti-LPS sera after electroblotting onto nitrocellulose, thereby confirming their integrity and presence in the polyacrylamide gel. This is because LPS fractions containing a low number of fatty acids are washed out of the gel during the initial fixing step (40% ethanol-4% acetic acid, overnight). By omitting this fixing step, which was originally developed for detecting proteins, and by increasing the LPS oxidation time (from 5 to 20 min), we restored the ability to detect LPS fractions that otherwise would not be stained. These modifications did not affect the detection of other S- and R-form LPSs. Thus, differences in the number of fatty acids present in polyagglutinable P. aeruginosa LPS may result in a selective loss of fatty acid-deficient S-form LPS in these apparent R-form LPS preparations. This modified procedure provides a fast, simple, and sensitive way to analyze LPS in polyacrylamide gels despite the number of acyl groups present. PMID- 1704013 TI - Detection and identification of Treponema hyodysenteriae by using oligodeoxynucleotide probes complementary to 16S rRNA. AB - Oligodeoxynucleotide probes (17 and 28 bases long) complementary to a unique region of Treponema hyodysenteriae 16S rRNA were developed. These probes bound specifically to partially purified rRNA and whole-cell rRNA of T. hyodysenteriae. No binding to partially purified rRNA or whole-cell rRNA of Treponema innocens, Treponema succinifaciens, Treponema bryantii, or Escherichia coli occurred under stringent conditions. The 28-base probe was 5 to 10 times more sensitive than the 17-base probe when hybridized with T. hyodysenteriae rRNA. The 28-base probe detected T. hyodysenteriae in the feces of experimentally inoculated pigs exhibiting clinical signs of swine dysentery. PMID- 1704014 TI - Assessment of antigenic determinants for the human T cell response against myelin basic protein using overlapping synthetic peptides. AB - Immunization of experimental animals with myelin basic protein (MBP) or with specific MBP encephalitogenic determinants induces an autoimmune central nervous system (CNS) disease, experimental allergic encephalomyelitis, often studied as a model for human demyelinating disorders. This study examines the antigenic determinants of MBP recognized by human T cells using overlapping, synthetic peptides and T cell lines and clones isolated from four HLA-typed, neurologically normal subjects. T cell lines and clones isolated from individual subjects recognized at least one and as many as five distinct T cell determinants. In some instances the peptides recognized included determinants previously shown to induce experimental allergic encephalomyelitis (EAE) in experimental animals. In this group of four subjects, some determinants of MBP, including residues 5-25, 35-47, 65-75, and 81-100, were recognized by T cells derived from more than one individual suggesting that these regions may be particularly immunogenic for humans. PMID- 1704015 TI - Breakdown of blood-brain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific CD8+ T cells. AB - Intracerebral inoculation of lymphocytic choriomeningitis virus (LCMV) generally results in a fatal T cell-mediated meningitis. In a previous study we have demonstrated a compromised blood-brain barrier (BBB) under such conditions. Using semi-quantitative radiography and the low molecular tracer 2-amino-[1 14C]isobutyric acid we now demonstrate an uncompromised BBB in i.c. infected T cell-deficient nu/nu mice, but serious dysfunction in heterozygous littermates. Transfer experiments were used to characterize and compare the cell subset(s) involved in inducing BBB dysfunction and fatal disease. It was demonstrated that Thy-1+, CD8+ class I-restricted T cells were mandatory for the increase in BBB permeability as well as for mortality. In addition, depletion of class II restricted CD4+ cells significantly weakened both effects of cell transfer. These results support the idea of a causal relationship between virus-specific T cell activity, BBB dysfunction, and fatal disease. Furthermore, the data indicate that T helper cells augment the response of LCMV-specific CD8+ effector cells. PMID- 1704016 TI - T cell immunity and interferon-gamma secretion during experimental allergic encephalomyelitis in Lewis rats. AB - An immunospot assay that detects single secretory cells was used to enumerate interferon-gamma secreting cells (IFN-gamma-sc) in mononuclear cell suspensions from the central nervous system (CNS) and peripheral lymphoid organs after actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats. In the CNS compartment there was a significant increase in the number of IFN-gamma sc preceding the onset of the clinical signs of EAE. Both in rats with EAE and rats immunized with Freund's complete adjuvant (FCA) the number of IFN-gamma-sc increased in peripheral lymphoid organs, as compared to non-immunized controls. In view of the potent immunoregulatory effects of IFN-gamma, its intra-CNS secretion may play a crucial role for clinicopathological events in EAE. To study the numbers of primed T cells that in response to myelin antigens produced IFN gamma, mononuclear cell suspensions from peripheral lymphoid organs were precultured to allow for antigen uptake, presentation and T cell triggering, followed by enumeration of IFN-gamma-sc. T cells responding to a peptide of myelin basic protein (MBP) that previously have been shown encephalitogenic in Lewis rats, appeared initially and were quantitatively dominant over the course of EAE. Later, T cell reactivities to multiple regions of MBP appeared, showing that the concept of immunodominance in EAE is non-absolute and time dependent. Splenocyte cultures from EAE rats exposed to the different antigens showed a reduced number of IFN-gamma-sc compared to cultures not exposed to antigen, suggesting an antigen-induced suppression of T cell effector molecules. PMID- 1704017 TI - Antigenic cross-reaction between beta-galactoside binding lectin and myelin basic protein. PMID- 1704018 TI - Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. AB - Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641 mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor. PMID- 1704019 TI - Chronic neutropenia. A new canine model induced by human granulocyte colony stimulating factor. AB - Normal dogs were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 10 micrograms/kg/day for 30 d, which caused an initial neutrophilia, followed by a prolonged period of chronic neutropenia. A control dog treated with recombinant canine G-CSF (rcG-CSF) showed persistent neutrophilia over 3 mo. Serum from dogs during neutropenia contained an antibody to rhG-CSF, which neutralized the stimulatory effects of both rhG-CSF and rcG-CSF on dog marrow neutrophilic progenitor cell growth and on NFS-60 cell proliferation. 4 mo after discontinuation of rhG-CSF, the dogs' neutrophil counts returned to the normal range. Rechallenge with the rhG-CSF re-induced severe neutropenia in 1 wk. Neutropenia was transferred by plasma infusion from a neutropenic dog to a previously normal dog. These data suggest that human rhG-CSF immunizes normal dogs and thereby induces neutralization of endogenous canine G CSF and neutropenia. This model system should allow more precise definition of the in vivo role of G-CSF. PMID- 1704020 TI - Immunohistochemical localization of the matrix glycoproteins--tenascin and the ED sequence-containing form of cellular fibronectin--in human permanent teeth and periodontal ligament. AB - The expression of two matrix glycoproteins, tenascin and cellular fibronectin (cFN), has been studied in fully developed human permanent teeth, periodontal ligament, and alveolar bone, in both frozen and paraffin-processed material. Polyclonal antibodies to tenascin and a monoclonal antibody recognizing the ED sequence specific to at least some forms of cFN were used. Staining for both tenascin and cFN was positive in the dental pulp, odontoblastic layer, cementoblast-pre-cementum zone, and on the periosteal as well as endosteal surfaces of the alveolar bone. In the periodontal ligament, cFN was evenly distributed, whereas tenascin was accumulated in the attachment zones. Pre-dentin stained for tenascin but not for cFN. Mineralized dentin and cementum were tenascin- and cFN-negative. The relative staining intensity for tenascin was greater than that for cFN in the cementoblast-pre-cementum layer and in the attachment zones of the periodontal ligament, whereas cFN stained more intensely in the pulp. In frozen material, antigenicities were well-preserved. Paraffin processing facilitated precise recognition of tissue morphology, but the antigenicity of cFN was lost. The co-expression of tenascin and cFN in the dental pulp, cementogenic zone, and on the surfaces of the alveolar bone may reflect the ability of the cells to deposit mineralized tissue matrices. The pronounced expression of tenascin in the interfaces between mineralized and non-mineralized tissues suggests that it is functionally associated with mechanical stress and may thus have at least two distinct functions. The relative amounts of the two matrix glycoproteins may contribute to regulation of tissue structure. PMID- 1704021 TI - Effects of hydroprene exposure on the physiology and insecticide susceptibility of German cockroaches (Orthoptera: Blattellidae). AB - We determined nonlethal physiological effects of hydroprene on the German cockroach, Blattella germanica L., and the effects of hydroprene exposure on German cockroach susceptibility to traditional insecticides. Live and dry weights of German cockroach adults exposed as nymphs to the juvenile hormone analog hydroprene increased significantly (6-35%) in both sexes. The change in weight was affected by hydroprene dose and time after adult emergence. Hydroprene treatment did not affect total body lipids and carbohydrates consistently. However, total body urates of males and females increased significantly. The susceptible and multiresistant German cockroach strains were significantly more susceptible to propoxur at the lower hydroprene application rate (0.697 mg per tub) than the untreated cockroaches. Hydroprene application also significantly lowered the LD50 for chlorpyrifos of the multiresistant strain at the lower hydroprene application rate. However, hydroprene failed to affect chlorpyrifos susceptibility of the susceptible strain. PMID- 1704022 TI - Immunoglobulin deficiency syndromes and therapy. PMID- 1704023 TI - Mast cell numbers and staining characteristics in the normal and allergic human conjunctiva. AB - In this study we report the numbers and metachromatic dye-staining characteristics of mast cells (MCs) in the conjunctiva of normal subjects and patients with seasonal allergic conjunctivitis caused by pollenosis. In addition, we have used a monoclonal antibody to the MC-specific enzyme, tryptase, to enumerate tryptase-positive cells immunohistochemically. Tarsal conjunctival MCs were found to be present in increased numbers in the allergic compared to the nonallergic subjects. Most of the MCs exhibited staining that was formaldehyde resistant, compatible with their identification as connective tissue MCs or basophils. The high positive staining for tryptase confirmed that the metachromatic cells stained with toluidine blue were MCs and not basophils, both in normal and allergic subjects. PMID- 1704024 TI - Modification of the fluorescent allergosorbent test as an inhibition assay for determination of cross-reactivity among aeroallergens. AB - The fluorescent allergosorbent test was adapted as an inhibition assay to determine cross-reactivity between aeroallergens. With this method, similar antigenic determinants were found between short ragweed and giant ragweed, cocklebur, lamb's-quarter, rough pigweed, marsh elder, and goldenrod. Cocklebur and giant ragweed were highly potent in their ability to competitively bind to short ragweed IgE. The other pollens demonstrated lower potency of cross-reacting antigens. The fluorescent allergosorbent test-inhibition assay appears to be a useful method to determine cross-reactivity among aeroallergens. PMID- 1704025 TI - A dietary education program for hypercholesterolemic children and their parents. AB - A parent-child autotutorial dietary education program for 4- to 10-year-old, hypercholesterolemic children and their families was developed and pilot tested. The 10-lesson program, designed for weekly use at home, uses a "talking-book" approach (audiotapes with accompanying picture booklet) for the child. Parents are provided with information on ways to make recommended dietary changes, along with hands-on activities to do with the children. To help match the instructional approach to the wide developmental range within the children's age span, materials are divided into three program levels that use different story characters and concept presentations. During program development, evaluation by two children (and their parents) for each of the program levels guided the design and refinement of the lessons. A pilot test among 22 hypercholesterolemic children (whose treatment was limited to diet modification) revealed that children within the 4- to 10-year age range liked the "talking-book" approach and identified positively with the story characters. Parents indicated that their materials were clear and helpful. Between the baseline and 3-month follow-up visits, the children exhibited a significant increase in knowledge of heart healthy foods, a decrease in total fat consumption that approached significance, and a significant decrease in plasma low-density-lipoprotein cholesterol values. PMID- 1704026 TI - The influence of food temperature on postprandial blood pressure reduction and its relation to substance-P in healthy elderly subjects. AB - Blood pressure (BP) in the elderly may decrease after a meal or oral glucose loading. The mechanism of this phenomenon is still unclear. In addition, the effect of the temperature of a meal on postprandial BP is unknown. However, it has been suggested that vasoactive gastrointestinal peptides are involved in the etiology of postprandial BP reduction. Therefore, we studied the effects of a cold and a warm glucose solution on BP, heart rate, plasma glucose, insulin, and substance-P levels in 15 healthy elderly subjects with a mean age of 74 +/- 3 (SD) years. With an interval of at least 2 days, a warm (50 degrees C) and a cold (5 degrees C) solution (75 g glucose/300 mL water) were given in random order. After the cold glucose loading mean arterial pressure increased by a maximum of 3.9 +/- 1.3 mmHg (P less than 0.01). In contrast, BP decreased after the warm solution by a maximum of 8.0 +/- 1.1 mmHg (P less than 0.001). Neither test had an influence on plasma substance-P levels. Our data suggest that postprandial blood pressure reduction in the elderly is dependent on food temperature. Substance-P does not seem to play a role in this phenomenon. PMID- 1704027 TI - Management of sinusitis. PMID- 1704028 TI - Application of stains-all staining to the analysis of axonemal tubulins: identification of beta-tubulin and beta-isotubulins. AB - The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins. PMID- 1704029 TI - Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines. AB - Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL 5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells. PMID- 1704030 TI - Expression and tyrosine phosphorylation of the T cell receptor zeta-subunit in human thymocytes. AB - Recent evidence suggests that the zeta-subunit of the TCR complex plays a critical role in transducing signals initiated by the Ag receptor heterodimer. Because thymic maturation involves specific interactions between the TCR complex and thymic stromal cells, the zeta-subunit has been postulated to also play a role in this process. To assess the potential for zeta to contribute to thymocyte maturation, we have used an anti-zeta mAb (TIA-2) to quantitate its expression in mature (CD3bright) and immature (CD3dim and CD3-) populations of human thymocytes. Using both flow cytometric and immunoblotting analysis, we found that the relative expression of TCR-zeta varied directly with the surface expression of CD3. Importantly, TCR-zeta was detected in the majority of CD3- thymocytes, indicating that its expression precedes the surface appearance of CD3:TCR. In thymocytes, TCR-zeta was found to be constitutively phosphorylated on tyrosine residues. The relative expression of phospho-zeta varied directly with the maturational stage of the thymocyte, with the mature (CD3bright), single positive cells accounting for most of the phospho-zeta found in the human thymus. The expression of phospho-zeta could be significantly increased by activating thymocytes with mAb reactive with either CD3 or CD2. These results suggest that TCR-zeta is functionally linked to the major thymocyte activation receptors. PMID- 1704031 TI - Endothelial cell adhesiveness for human T lymphocytes is inhibited by transforming growth factor-beta 1. AB - Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier. PMID- 1704032 TI - Induction of CD4+, human T lymphotropic virus type-1-specific cytotoxic T lymphocytes from patients with HAM/TSP. Recognition of an immunogenic region of the gp46 envelope glycoprotein of human T lymphotropic virus type-1. AB - Although the humoral response to human T lymphotropic virus type-1 (HTLV-I) has been well characterized in patients with HTLV-I-associated neurologic disease (HAM/TSP), little is known about a functional HTLV-I-specific human T cell response, such as CTL, in these patients. To define both the phenotype of the responding CTL and the fine specificity of this response, long term T cell lines were generated from two HAM/TSP patients who were from two different countries. Patient's peripheral blood lymphocytes were repeatedly stimulated in vitro with an HTLV-I expressing autologous T cell line. The resultant long term T cell culture was shown to be CD4+ and cytotoxic for targets expressing HTLV-I Ag. Using a panel of synthetic peptides that span hydrophilic regions of the HTLV-I gp46 envelope glycoprotein, the CTL lines generated from both patients were shown to recognize the same region of the HTLV-I envelope between amino acids 196-209 as defined by the synthetic peptide sp4a1. Interestingly, this sequence overlaps a region of HTLV-I envelope that had also been shown to elicit a strong B cell response in HAM/TSP patients (amino acids 190-203). One CTL line recognized this HTLV-I epitope in the context of HLA DQ5 whereas the other CTL line was restricted by HLA DRw16. The generation of two independent CTL lines from two HAM/TSP patients from different geographic areas that recognize the same region of the HTLV-I envelope glycoprotein highlights the immunogenic nature of this envelope region. PMID- 1704033 TI - Influenza A-specific, HLA-A2.1-restricted cytotoxic T lymphocytes from HLA-A2.1 transgenic mice recognize fragments of the M1 protein. AB - Previous studies have indicated that in transgenic mice expressing human class I MHC molecules, it is difficult to demonstrate a significant CTL response to a viral Ag in the context of the transgenic molecule. In this paper, a procedure is reported for the isolation of influenza-specific murine CTL restricted by the human class I molecule HLA-A2.1. The principal specificity of such CTL is for a fragment of the influenza M1 protein that has been previously shown to be immunodominant for human HLA-A2.1-restricted CTL. CTL of this specificity were also established through the use of peptide-pulsed rather than virus-infected stimulators. The dependence of murine CTL recognition upon peptide length and HLA A2 structure was established to be similar to that previously reported for human CTL. However, the fine specificity of CTL maintained on virus-infected stimulators was somewhat different from that of CTL maintained with M1 peptide. This suggests that differences in surface density or peptide structure between peptide-pulsed and virus-infected stimulators may result in the outgrowth of T cells with different receptor structures. The immunodominance of the M1 peptide determinant in both mice and humans suggests that species-specific differences in TCR structure, Ag-processing systems, and self-tolerance are of less importance than limitations on the ability of antigenic peptides to bind to appropriate class I molecules. These results thus establish the utility of the transgenic system for the identification of human class I MHC-restricted T cell epitopes. PMID- 1704034 TI - Truncation analysis of several DR binding epitopes. AB - Peptide regions crucial for binding to four different DR alleles (DR1, DR2, DR5, and DR52a) have been localized in five unrelated DR binding peptides (dynorphin 1 13, sperm whale myoglobin 132-153, influenza hemagglutinin 307-319, pigeon cytochrome c 88-104, and tetanus toxoid 830-843) by testing panels of truncated analogs for DR binding. It was found that in most cases, different DR alleles recognize almost identical, albeit distinct, core regions, suggesting that different DR alleles may recognize similar structures on their peptide ligands. Furthermore, it was found that these core regions, notwithstanding their derivation from unrelated sequences, share a common structural pattern. When the sequences of several other unrelated determinants were scrutinized, the structural motif identified was present in some, but absent in other good DR binders, suggesting that good DR binding capacity of peptide molecules may be compatible with more than one single sequence pattern. PMID- 1704035 TI - The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site directed mutagenesis. AB - The analysis of the antigenic surface of staphylococcal nuclease was begun by generating and characterizing a panel of mAb. Twelve mAb were selected from a large number of anti-nuclease mAb and characterized for affinity and isotype, by their ability to block enzyme activity, and by complementation and competitive inhibition assays for the relative location of epitopes. The mAb were placed in complementation groups based on their distinct binding patterns. These groups define a series of eight overlapping epitopes that are estimated to cover a large portion of the nuclease surface. Four mAb blocked the enzyme activity of nuclease. The epitopes defined by two of these four mAb were localized on the surface of nuclease using single amino acid variant Ag generated by site-directed mutagenesis of the cloned nuclease coding sequence. mAb-25 maps to residue 46 which is located at the edge of the enzyme active site consistent with its ability to inhibit enzyme activity. mAb-19, which also blocks enzyme activity and belongs to the same complementation group as mAb-25, was unaffected by the substitution at position 46. This suggests that mAb-19 and mAb-25, if they do react with the same epitope, have differences in fine specificity. mAb-22 blocks enzyme activity and belongs to an overlapping complementation group. The fourth mAb, mAb-1, which belongs to a distinct, nonoverlapping, complementation group, does not blocks enzyme activity, and is directed to a region of nuclease that includes the amino acid at position 133. This residue is located a short distance from the active site in a region that has been suggested to participate in binding of DNA, a substrate for nuclease. Therefore, the four epitopes defined by these mAb are localized at or near the enzyme active site. PMID- 1704036 TI - The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis. AB - Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid. PMID- 1704037 TI - Functional comparison of the upstream regulatory DNA sequences of four human epidermal keratin genes. AB - The promoters of epidermal keratin genes, K5, K6, and K10 were cloned and their functions compared with that of the previously described promoter of the K14 keratin gene in non-epithelial and transformed epithelial cell lines, as well as in primary cultures of cells derived from simple and stratified epithelia. The four promoters were functional only in epithelial cells. Although the promoter for the basal cell-specific, acidic-type K14 gene was active in all epithelial cells tested, its basic-type partner, K5, and the promoter for the hyper proliferation-associated K6 were active only in primary cultures of stratified epithelia. The promoter for the epidermal differentiation-specific K10 keratin gene was active at a low level in primary cultures of stratified epithelial cells on non-epidermal origin. Thus, the K14 gene promoter is functional in all epithelial cells, but the upstream regions of the K5 and K6 keratin genes restrict their expression to stratified epithelia, whereas the epidermal determinants of the K10 gene are not in the proximal upstream sequences. PMID- 1704038 TI - Distribution of proteoglycans during the hair growth cycle in human skin. AB - The involvement of proteoglycans in hair growth has been recognized through the observation of increased hair growth in diseases such as the mucopolysaccharidoses and pre-tibial myxedema, which involve an increase in skin proteoglycan content. In an attempt to understand this, we have examined the distribution of chondroitin 6 sulphate (C6S), unsulphated chondroitin (COS), dermatan sulphate (DS), and heparan sulphate proteoglycans (HSPG) in frozen tissue sections of normal scalp by immunostaining. Results show that during anagen, the thick connective tissue sheath around the follicle strains strongly for C6S, COS, and DS. COS is uniquely associated with this region and is not found beneath the epidermis or infundibular epithelium. HSPG is, however, localized in the basement membrane zone adjacent to the outer root sheath. In addition, all of these proteoglycans are localized in the dermal papilla. In mid catagen, we observed significant loss of C6S and COS staining from both the dermal papilla and the connective tissue sheath, but no decrease in staining for HSPG. In late catagen, very little staining of C6S and COS was observed. In early anagen, we observed that C6S was again present in the connective tissue sheath and dermal papilla; however, COS staining appeared to be weaker and less closely associated with the follicle. HSPG staining was observed in early anagen in a pattern very similar to that found for other basement membrane components. Results for DS were not obtained for catagen or early anagen. These results provide further evidence that hair growth is associated with the presence of chondroitin proteoglycans in the follicle environment and that the cessation of growth is associated with their removal. Further studies are underway to characterize the relationship between hair growth and proteoglycans. PMID- 1704039 TI - Isolation of cDNA for human epidermal type I transglutaminase. AB - Keratinocytes of stratified epithelia, including the epidermis, express two distinct forms of transglutaminase, type I and type II. Type I transglutaminase activity is responsible for cell envelope formation in terminally differentiating cultured keratinocytes. Transglutaminase enzymatic activity has been associated with several proteins that are differentially expressed in vivo and in vitro. To elucidate the relationship between the epidermally expressed transglutaminases, cDNA for type I transglutaminase was cloned from a human high-calcium keratinocyte lambda gt11 library. cDNA fragments, generated by PCR primed with a mixture of oligonucleotides coding for five invariant amino acids in the active site, were used as a screening probe. Based on the sequence analysis of 1653 nt contained in the lambda 1-126a clone and on the pattern of expression of a complementary approximately 3-kb transcript, we report cloning of the epidermal type I transglutaminase gene. The expression of this gene is regulated by calcium ions and retinoic acid in cultured human keratinocytes. There are highly conserved regions near the active site cysteine residues that may be important for the enzyme's specialized functions. PMID- 1704040 TI - Cochlear otosclerosis: statistical analysis of relationship of spiral ligament hyalinization to hearing loss. AB - This paper presents an analysis of the relationship of the amount of hyalinization of the spiral ligament, secondary to cochlear otosclerosis (spongiosis), to the amount of hearing loss. This relationship was previously studied by Parahy and Linthicum. It was found that the larger the amount of hyalinization, the greater the hearing loss. This hyalinization is a measure of the amount of toxic enzymes being excreted into the inner ear fluid. These enzymes are suspected to affect the metabolic function of the hair cells. PMID- 1704041 TI - Insulin-like growth factor-I (IGF-I) and IGF-binding proteins both decline in the rat during late pregnancy. AB - Insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding proteins (IGFBP) were examined in rat serum during pregnancy and lactation. IGF-I concentrations determined after acid column chromatography of serum were low during the last third of pregnancy. IGF-II was undetectable in pregnant and non-pregnant rats. IGF-binding protein (IGFBP) concentrations, measured as high molecular mass activity in the IGF-I RIA and the IGF-II RRA of acid column fractions, paralleled the changes observed with IGF-I. Western ligand blot analysis of serum from non pregnant rats revealed a 40-50 kDa IGFBP aligning with IGFBP-3, a smaller 28-30 kDa doublet and 24 kDa IGFBP. Serum from rats in late pregnancy lacked IGFBP-3, whereas the smaller IGFBP persisted during late pregnancy. IGFBP-3 reappeared in postpartum animals. The fall in serum IGF-I is consistent with a maternal catabolic state during late pregnancy which may maximize substrate availability for the developing fetus. PMID- 1704042 TI - Characterization of insulin-like growth factor-binding proteins in rat serum, lymph, cerebrospinal and amniotic fluids, and in media conditioned by liver, bone and muscle cells. AB - Insulin-like growth factor-binding proteins (IGFBPs) in rat serum, lymph, amniotic fluid and cerebrospinal fluid (CSF), and in rat cell-conditioned media were characterized using a combination of gel-permeation chromatography, Western immunoblots and Western-ligand analysis. Adult serum and abdominal lymph contained a 200 kDa IGFBP (the putative type-II IGF receptor) and a 150 kDa IGFBP that contained subunits of 40-50 kDa aligning with porcine IGFBP-3 on Western ligand blots. In addition, both fluids contained the smaller IGFBPs: a 30 kDa IGFBP which was immunoreactive with IGFBP-2 antiserum, a 28 kDa IGFBP which electrophoresed with human IGFBP-1, and a 24 kDa IGFBP. In contrast, fetal serum and amniotic fluid lacked the 150 kDa and the 28 kDa IGFBPs. CSF contained only a 30 kDa IGFBP, but this was not IGFBP-2. Several IGFBPs were detected in media conditioned by liver, bone and muscle cells. Liver-derived cells and some hepatoma cell lines produced similar patterns upon ligand blot analysis, i.e. IGFBPs of 30 kDa (which reacted with IGFBP-2 antiserum), 28 kDa and 24 kDa. A hepatoma cell line, HTC, and a smooth muscle cell line contained only an IGFBP of 26 kDa. Skeletal muscle-derived cells (L6 myoblasts) produced a 28 kDa, a 26 kDa and a 24 kDa IGFBP. Both calvarial osteoblasts and osteogenic sarcoma cells produced an IGFBP of 30 kDa that cross-reacted with IGFBP-2 antisera. In addition, osteogenic sarcoma cells produced a 28 kDa and a 24 kDa IGFBP. These results allow us partially to classify and to compare the IGFBPs in rat fluids and those produced by cultured cells. PMID- 1704043 TI - The influence of adrenal hormone status on neuroendocrine peptides in the rat anterior pituitary gland. AB - Neuropeptide Y (NPY), neurotensin (NT), substance P (SP) and vasoactive intestinal peptide (VIP) are four structurally unrelated neuroendocrine peptides which affect anterior pituitary function. All four peptides appear to be locally synthesized in the anterior pituitary gland and have been shown to be regulated by thyroid and/or sex hormone status. We show here that NT, SP and VIP but not NPY are influenced by adrenal hormone status in the male rat pituitary gland. Adrenalectomy increased the content of VIP (35.4 +/- 4.0 (S.E.M.) vs control 11.9 +/- 1.1 pmol/g wet weight) but decreased that of SP (18.8 +/- 2.3 vs control 36.7 +/- 3.5 pmol/g wet weight). Adrenalectomy combined with castration decreased the content of SP (14.6 +/- 3.5 vs control 36.7 +/- 3.9 pmol/g wet weight) but had no effect on VIP content. Treatment with dexamethasone produced significant decreases in NT, SP and VIP contents (17.8 +/- 2.3 vs control 32.6 +/- 3.4 pmol/g wet weight, 5.5 +/- 0.9 vs control 36.7 +/- 3.9 pmol/g wet weight and 4.2 +/- 0.6 vs control 11.9 +/- 1.1 pmol/g wet weight respectively). The changes in pituitary peptide contents occurred in parallel with changes in mRNA levels, suggesting that alterations in glucocorticoid hormone status can alter the synthesis of these peptides. These results, together with the known effects of these neuroendocrine peptides suggest possible functions for locally produced SP and VIP in regulating the secretion of adrenocorticotrophin and/or other pro opiomelanocortin-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704044 TI - Influence of steroid hormone treatments or pregnancy on [3H]prazosin and [3H]rauwolscine binding to myometrial alpha-adrenoceptors in the ewe. AB - The adrenergic antagonists [3H]prazosin and [3H] rauwolscine were used to identify alpha 1- and alpha 2-adrenoceptors respectively in the ovine myometrium. Ewes were allocated to four groups according to steroid hormone treatments or physiological status, namely ovariectomized ewes either as untreated controls, treated with oestradiol-17 beta or progestagen plus oestradiol-17 beta, and pregnant ewes at mid-gestation. Binding of both [3H]prazosin and [3H]rauwolscine to membrane preparations from the ovine myometrium was saturable, of high affinity and rapidly reversed by phentolamine (10 mumol/l). Based on the relative order of potency of selected adrenergic agonists and antagonists, the myometrial binding sites labelled by [3H]prazosin and [3H]rauwolscine were characterized as alpha 1- and alpha 2-adrenoceptors respectively. Saturation binding studies with [3H]prazosin showed that the number of alpha 1-adrenoceptors was low (maximal binding capacity, Bmax, between 19 and 24 fmol/mg protein) and there were no noticeable differences between the animal groups. Moreover, the equilibrium dissociation constant (Kd) did not vary significantly between groups (Kd between 0.10 and 0.17 nmol/l). In contrast, saturation binding studies with [3H]rauwolscine revealed the presence of a high number of alpha 2-adrenoceptors. Values of Bmax were far higher in the pregnant ewes (1096 +/- 241 fmol/mg protein; means +/- S.D.) than in any of the non-pregnant ovariectomized ewes. For these latter groups, the highest Bmax values were found in the group treated with both progestagen and oestrogen (382 +/- 77 fmol/mg protein) compared with treatment with oestrogen alone (101 +/- 8 fmol/mg protein) or with controls (82 +/- 12 fmol/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704045 TI - Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance. AB - In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A. PMID- 1704046 TI - Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Single-channel analysis. AB - Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PA65 fragment of anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N+ induces a flickery block of the PA65 channel when present on either side of the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (greater than 2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants tau b and tau u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [tau u/(tau u + tau b)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of blocking, thus demonstrating that these single channels mirror the behavior seen with many (greater than 10,000) channels in the membrane. In going from large negative to large positive voltages (-100 to +160 mV) on the cis (PA65 containing) side of the membrane, one sees the mean blocked time (tau b) increase to a maximum at +60 mV and then steadily decline for voltages greater than +60 mV, thereby clearly demonstrating that Bu4N+ is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N+. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N+ present on the cis side of the membrane, plateaus at large positive cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited. PMID- 1704047 TI - Diffusion limitation in the block by symmetric tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer membranes. AB - Current flow through the channel formed in planar phospholipid bilayer membranes by the PA65 fragment of anthrax toxin is blocked, in a voltage-dependent manner, by tetraalkylammonium ions (at micromolar concentrations), which bind to a blocking site within the channel lumen. We have presented evidence that diffusion plays a significant role in the kinetics of blocking by tetrabutylammonium ion (Bu4N+) from the cis (toxin-containing) side of the membrane (Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942); in this paper we examine the implications and consequences of diffusion control for binding kinetics. As expected for a diffusion-affected reaction, both the entry rate constant (kcis1) of Bu4N+ from the cis solution to the blocking site and the exit rate constant (kcis-1) of Bu4N+ from the blocking site to the cis solution are reduced if the viscosity of that medium is increased by the addition of dextran. In conformity with both thermodynamics and kinetic arguments, however, the voltage-dependent equilibrium binding constant, Keq (= kcis-1/kcis1), is not altered by the dextran-induced viscosity increase of the cis solution. The entry rate constants (kcis1) for tetrapentylammonium (Pe4N+), tetrahexylammonium (Hx4N+), and tetraheptylammonium (Hp4N+) are also diffusion controlled, and all of them, including that for Bu4N+, attain a voltage-independent plateau value at large positive cis voltages consistent with diffusion limitation. Although the plateau value of kcis1 for Hx4N+ is only a factor of 3 less than that for Bu4N+, the plateau value for Hp4N+ is a factor of 35 less. This precipitous fall in value indicates, from diffusion-limitation theory, that the diameter of the channel entrance facing the cis solution is not much larger than the diameter of Hp4N+, i.e., approximately 12 A. PMID- 1704048 TI - Antigenic relatedness among the Norwalk-like agents by serum antibody rises. AB - The Norwalk, Snow Mountain (SMA), and Hawaii agents are etiologically associated with separate outbreaks of acute viral gastroenteritis. Previous cross-challenge of volunteers, immune electron microscopy, and/or enzyme-immunoassay analysis suggested that these agents are antigenically distinct. We examined paired sera from human volunteers challenged with these agents for the presence of homologous and heterologous serum antibody titer rises to the agents. Two-way cross reactions occurred between Hawaii agent and SMA. A one-way cross-reaction occurred between Norwalk agent and SMA, as volunteers challenged with Norwalk agent had heterologous serum antibody titer rises to SMA, but the reverse did not occur. The Norwalk and Hawaii agents had minimal cross-reaction, with only one volunteer challenged with Hawaii agents having a heterologous rise to Norwalk agent. These observations indicate varying degrees of antigenic relatedness among these agents. PMID- 1704049 TI - Major antigenic domain recognized by monoclonal antibodies maps within the carboxy-terminal moiety of a recombinant human immunodeficiency virus-1 p24 protein. AB - Antigenicity in mice of a recombinant polypeptide including the complete amino acid sequence of mature human immunodeficiency virus type 1 p24 protein was studied by induction of monoclonal antibodies (MAbs). A panel of nine recloned hybridomas secreting MAbs with anti-p24 reactivity was isolated and further characterized. Competitive inhibition experiments suggested that the MAbs could be grouped into four epitopic classes corresponding to at least two distinct determinants. Analysis of reactivity to recombinant p24 deletion variants indicated that all the recognized epitopes are localized within a carboxy terminal domain (amino acids 168-208) which should be largely exposed in recombinant as well as authentic antigen. Lack of response to N-terminal and central portions of p24 suggests that the antigenicity of those regions in the natural polypeptide is strongly conformation-dependent. PMID- 1704050 TI - Dentistry in the Soviet Union. Russian impressions. PMID- 1704051 TI - CDA Code of Ethics. PMID- 1704052 TI - A growth factor from neuronal cell lines stimulates myelin protein synthesis in mammalian brain. AB - Oligodendroglia growth factor (OGF) is a 16-kDa soluble protein produced by neuronal cell lines. This factor, when incubated with brain glia in culture, selectively stimulates growth of oligodendroglia, the myelin-producing cells of the CNS. OGF infused into the cerebral cortex of the adult rat accelerates the production of myelin proteins as shown by increased specific activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase), by stimulated synthesis of myelin basic protein, and by elevations in levels of myelin proteolipid protein RNA. The ability of OGF to induce myelin protein production in vivo suggests that neuron-secreted growth factors help to regulate myelin formation within the CNS. PMID- 1704053 TI - NGF rescues substance P expression but not neurofilament or tubulin gene expression in axotomized sensory neurons. AB - A reduction in the supply of retrogradely transported NGF has been proposed as a possible signal for the axotomy response in dorsal root ganglion (DRG) neurons. Components of the axotomy response that have previously been well characterized in axotomized DRG cells include changes in cytoskeletal gene expression and changes in the expression of neurotransmitters/neuromodulators such as substance P. In this study, we examined the role of NGF in the axotomy response by examining protein synthesis and mRNA levels of the low-MW neurofilament protein (NF-L) and beta-tubulin in DRG cells at 1, 7, and 12 d after axotomy with and without continuous administration of exogenous NGF. We also examined substance P levels in the DRG by immunocytochemistry under the same experimental conditions. Sciatic nerves of adult male rats were unilaterally transected at the midthigh level, and the proximal nerve stumps were placed into Silastic tubes connected to osmotic minipumps that were filled with biologically active NGF. NGF (0.5 mg/ml in saline) was continuously infused (0.5 microliter/hr) onto the proximal stumps of transected sciatic nerves for 1-12 d. Control animals were prepared in an identical fashion except that the nerves were treated with saline alone. At death, DRGs were removed from the animals; the L4 experimental DRGs (axotomized) and contralateral L4 DRGs (uninjured) were used immediately for protein synthesis experiments, while the experimental and contralateral L5 DRGs were fixed in 4% paraformaldehyde and subsequently used for in situ hybridization and immunocytochemistry. From another set of experimental animals, the L4 and L5 DRGs were harvested and used for total RNA isolation and RNA blotting experiments. Immunocytochemical studies using a polyclonal antibody to substance P showed that the immunodetectable levels of this peptide decreased to undetectable levels in DRG neurons after axotomy and saline administration. However, in axotomized neurons treated with NGF, the level of immunodetectable substance P did not decrease, but instead, increased over even that present in normal DRG neurons. Pulse labeling of DRGs with 35S-methionine:cysteine followed by 2-dimensional (2D) gel electrophoresis and fluorography revealed that the synthesis of neurofilament (NF) proteins was decreased, while that of tubulin was increased, 12 d after sciatic nerve transection. NGF administration to axotomized neurons did not alter this pattern. Quantitative analysis of in situ hybridizations of DRG neurons and RNA blot analysis with cDNA probes specific for NF-L and beta tubulin mRNAs showed that NGF treatment of axotomized DRGs did not significantly affect cytoskeletal gene expression at the mRNA level.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1704054 TI - Localization of technetium-99m-glucarate in zones of acute cerebral injury. AB - The potential structural similarity of technetium-99m-labeled glucaric acid (99mTc-glucarate) to that of fructose suggests that this agent may enter cells by a sugar transport system. Studies with LLC-PK1 cells demonstrated inhibition of 99mTc-glucarate uptake by fructose, confirming this potential relationship. Since anaerobic metabolism can use either glucose or fructose, we hypothesized that 99mTc-glucarate may concentrate in areas of acute ischemic injury. To test this hypothesis, 63 adult rats with middle cerebral artery (MCA) occlusion followed by reperfusion were injected with 99mTc-glucarate and in vivo and ex vivo images were acquired. Seven animals were also studied with 18FDG and high resolution PET imaging. The radionuclide images were compared to the results of triphenyl tetrazolium chloride (TTC) staining and conventional histopathology. Thirty-five rats had significant accumulation of 99mTc-glucarate and no TTC staining (indicating infarction) in the involved hemisphere. Of the remaining 28 rats with TTC staining (suggesting viability) of the involved hemisphere, 16 (57%) had 99mTc-glucarate accumulation. In the seven rats that were studied with both 99mTc glucarate and 18FDG, 99mTc-glucarate accumulated at the center of the occluded MCA territory while 18FDG activity was decreased in this region. These results suggest that 99mTc-glucarate is a sensitive marker of acute severe cerebral injury, but its mechanism of localization is probably different from that of 18FDG. PMID- 1704055 TI - Changes in the composition of mammary tissue, liver and muscle of rat dams during lactation and after weaning. AB - This investigation was conducted to evaluate the changes in the total content of protein and RNA in liver and muscles of rat dams before and after acute separation from their litters. Groups of 8-12 rats fed ad libitum were killed on the 12th (group L12) and 20th d (group L20) of lactation and on the 1st (group W1) and 7th d (group W7) after weaning. Nonpregnant, nonlactating rats paired for age served as controls. Dry matter, protein, DNA and RNA levels of the mammary gland, liver and muscles of the right hindlimb were determined. Wet weight and total organ or tissue protein, DNA and RNA were higher in mammary glands of L12 and L20 than in age-matched controls. These values were lower in groups W1 and W7 than in the lactating groups. No changes were noted in the total liver protein or DNA content, but total liver RNA was greater in groups L12 and L20 than in controls or group W1. Total muscle dry matter, DNA and RNA were significantly lower in groups L12 and L20 than in groups W1 and W7. Muscle protein content increased progressively from the 12th to 20th d of lactation to a peak in group W1, and it decreased to values found in age-matched controls in group W7. Although the muscle protein mass of the hindlimb during peak lactation (group L12) was only 63% of that in nonlactating control rats, within 1 d of weaning it was significantly higher than in nonlactating rats. Similar changes in RNA suggest that these changes in protein content are related to an adaptative mechanism designed to handle the surplus of plasma amino acids not used by the mammary gland after weaning. PMID- 1704056 TI - Cell patterns in the surface of rabbit articular cartilage revealed by the backscatter mode of scanning electron microscopy. AB - To study the distribution of cells in the surface layer of articular cartilage, rabbit hip and knee specimens were stained with silver and studied by scanning electron microscopy (SEM). The cartilage was treated en bloc using the Gomori methenamine silver technique, which stains the nuclei of exposed cells with reduced silver. The intact surface was then studied with a binocular microscope and SEM in the backscatter mode Only those cells within 30 microns of the surface stained, permitting that population to be imaged selectively. Depressions in the surface were related to groups of cells in clusters or rows bounded by collagen fibers. This study demonstrates the effectiveness of backscatter imaging in the study of chondrocytes. The relationship between surface contours and underlying cells is more complex than previously described. PMID- 1704057 TI - [Double immunoenzymatic and two-color flow cytometric analyses of tonsillar T cell subsets]. AB - Distribution of various T-cell subsets in the palatine tonsil was investigated by two-color flow cytometry and double immunoenzymatic stain. Tonsillar lymphocytes contained many (about 20%) helper T (CD4+ Leu8-) cells and few (only 1%) suppressor T (CD8+ CD11b+) cells. In interfollicular area, each T-cell subset, i.e., helper, helper-inducer (CD4+ CD29+), suppressor-inducer (CD4+ CD45RA+), cytotoxic (CD8+ CD11b-), and suppressor, was identified by double immunoenzymatic labeling technique using alkaline phosphatase and peroxidase. On the other hand, only two T-cell subsets, helper, and cytotoxic T-cell subpopulations, were recognized in germial center. These results indicate that double immunoenzymatic stain as well as two-color flow cytometry gives us useful informations in terms of tonsillar T-cell subsets. PMID- 1704058 TI - Species and stage specificity of Leishmania donovani antigens. AB - Monoclonal antibodies D2 and D13 were produced in mice using Leishmania donovani promastigote membrane fractions. To study the species and stage specificity of the antigens recognized by these antibodies, we examined amastigotes prepared in vitro and cultured promastigotes by indirect immunofluorescence with monoclonal antibodies D2 and D13. Monoclonal antibody D2 showed weak reactivity for 9 of 9 strains of L. donovani complex promastigotes and 8 of 9 amastigotes. In contrast, only 2 of 22 strains from other complexes yielded equivocal reactions. Monoclonal antibody D13, however, had much broader reactivity. D13 reacted with all the promastigotes and amastigotes of L. donovani complex isolates as well as with 10 of 22 promastigotes and 8 of 13 amastigotes from other complexes. The high degree of species specificity seen with monoclonal antibody D2 provides a rationale for further study of this antibody and its purified antigen for parasite identification and serodiagnosis of visceral leishmaniasis. The strong fluorescent signal noted with D13 and the presence of the D13 epitope on all L. donovani complex parasites supports studies on its role as an antigen in immunoprophylaxis of visceral leishmaniasis. PMID- 1704059 TI - Basic approaches to anti-retroviral treatment. PMID- 1704060 TI - HIV-1 propagates in human neuroblastoma cells. AB - A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS. PMID- 1704061 TI - Pneumocystis carinii pneumonia in patients with AIDS: evaluation of lavage and staining techniques in diagnosis. AB - The diagnostic yield of unilateral vs. bilateral bronchoalveolar lavage (BAL) was prospectively evaluated in 65 consecutive patients suspected of having Pneumocystis carinii pneumonia (PCP) complicating acquired immune deficiency syndrome (AIDS). Gram-Weigert (GW), Papanicolaou (PAP), and Gomori's methenamine silver (GMS) stains were used for identification of P. carinii in all cases. Forty-eight patients had PCP that was identified by GW staining of BAL in 47/48 patients followed by PAP/GMS staining of BAL in 44/48 patients and PAP/GMS staining of bronchial washings in 40/48 patients. In patients with bilateral interstitial infiltrates, unilateral lavage was sufficient for diagnosis of PCP when GW stain was utilized. In patients with PCP complicating AIDS, the diagnostic yield of BAL may be increased by use of both GW and GMS stains. PMID- 1704063 TI - Determination of iloprost in 5% dextrose in water solution by reversed-phase high performance liquid chromatography. AB - A high-performance liquid chromatographic (HPLC) method utilizing absorbance detection was developed for the rapid and precise determination of the stable prostacyclin analogue iloprost in 5% dextrose in water solution (D5W). Samples were prepared for chromatographic analysis by extracting the drug into chloroform, evaporating the solvent, and solubilizing the residue in methanol. The resulting solutions were chromatographed on a Shandon ODS Hypersil column using 0.02 M potassium phosphate (pH 3.0), methanol, and acetonitrile (456:144:400, v/v) at a flow rate of 1.8 mL/min. 2-Naphthoic acid was employed as an internal standard. The detector response at 207 nm was linear (correlation coefficient greater than 0.9998) for concentrations of iloprost from 10 ng on column, the lower limit of quantitation, up to 7 micrograms on-column. The precision of the method was approximately 5% based on peak area, with a recovery of iloprost from D5W of 87%. Liquid-liquid extraction was found to be superior to solid-phase sorbent extraction as a sample preparation method due to incompatibility of dextrose with the sorbent bed. PMID- 1704062 TI - Effects of long-term zidovudine treatment on cell-mediated immune response and lymphokine production. AB - The effects of long-term zidovudine treatment on functional parameters of cell mediated immunity were investigated in 15 symptomatic HIV-antibody-positive patients with clinical evidence of opportunistic infections. Mononuclear leukocytes were obtained before administering the drug, and after 3 and 6 months of treatment. The cells were stimulated with lectins in order to assess variations of mitogen-induced lymphocyte proliferation and production of gamma interferon (IFN) and interleukin-2 (IL-2), and with Newcastle disease virus (NDV) to assess variations of alpha-IFN production. Mean proliferative responses to PHA and Con-A did not show any significant change between baseline, 3 months, and 6 months values (25,158 +/- 11,763, 24,662 +/- 8,955, and 34,924 +/- 16,283 D-cpm, respectively, for PHA, p greater than 0.05; and 5,470 +/- 1,890, 4,953 +/- 2,518, and 4,539 +/- 3,286 D-cpm, respectively, for Con-A, p less than 0.05). Mean response to PWM (9,707 +/- 4,429 D-cpm at entry) increased significantly after 3 months, but returned to baseline values at 6 months (17,039 +/- 5,123 and 10,314 +/- 3,855 D-cpm, respectively, p = 0.016). IL-2 production (5.51 +/- 4.0 I.U. at entry) rose particularly at 3 months and persisted at the same levels at 6 months (9.6 +/- 4.8 and 9.5 +/- 6 I.U., respectively, p less than 0.05). By contrast, a moderate but significant decrease in gamma- and alpha-IFN production was observed (65 +/- 2.2, 35.1 +/- 1.6, and 22 +/- 2 I.U., respectively, for gamma-IFN, p less than 0.05; 60 +/- 3, 64 +/- 2.6, and 12 +/- 1.5 I.U., respectively for alpha-IFN, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704064 TI - Potential anti-AIDS agents. Synthesis and antiviral activity of naphthalenesulfonic acid derivatives against HIV-1 and HIV-2. AB - Certain naphthalenesulfonic acid analogues have been synthesized and evaluated for their inhibitory effects on HIV-1- and HIV-2-induced cytopathogenicity, HIV-1 giant cell formation, and HIV-1 reverse transcriptase (RT) activity. A bis(naphthalenedisulfonic acid) derivative having a biphenyl spacer emerged as the most potent and selective inhibitor of virus-induced cytopathogenicity in MT 4 cells. The ED50 values for this compound were 7.6 and 36 microM for HIV-1 and HIV-2, respectively. No toxicity to the host cells was detected at 98 microM. This compound also inhibited giant cell formation and was superseded in potency by a bis(naphthalenedisulfonic acid) derivative having a flexible decamethylene spacer. In the cell-free RT assay, a long-chain amide derivative exhibited the most inhibition of RT. All the compounds that achieved complete inhibition of virus-induced cytopathogenicity at concentrations not toxic to host cells were derivatives of 4-amino-5-hydroxy-2,7- naphthalenedisulfonic acid. These analogues represent new leads for the development of anti-HIV agents. PMID- 1704065 TI - Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: direct correlation of antiviral potency with molecular weight. AB - Aurintricarboxylic acid (ATA) was fractionated by a combination of dialysis, ultrafiltration, and gel permeation chromatography. The number average and weight average molecular weights of the ATA fractions were determined by the universal calibration method. The sulfonic acid analogue of ATA was prepared and separated in high and low molecular weight fractions. The phosphonic acid analogue of ATA was also synthesized. All of the ATA fractions were tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture as well as against HIV 1 in CEM cell culture. The abilities of the fractions and analogues to inhibit syncytium formation between HIV-1- and HIV-2-infected HUT-78 cells and uninfected MOLT-4 cells were evaluated. In addition, the fractions and analogues were tested for cytotoxicity in mock-infected MT-4 cells, prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor, inhibition of the binding of anti gp120 monoclonal antibody to gp120, inhibition of attachment of HIV-1 virions to MT-4 cells, and inhibition of HIV-1 reverse transcriptase. In all of these assays except cytotoxicity, there was a correlation of potency with molecular weight. The higher the molecular weight, the higher the activity. Several of the lower molecular weight fractions of ATA, which bound to gp120 but not to CD4, prevented HIV-1 and HIV-2 cytopathicity. A similar profile was observed for the phosphonic acid analogue of ATA and the lower molecular weight fraction of the sulfonic acid analogue. The results on the ATA fractions indicate that the binding of ATA to gp120 in the absence of CD4 binding is sufficient for anti-HIV activity. The active compounds bind more avidly to gp120 than to CD4. The anti-HIV activity of the ATA fractions is due to inhibition of virus binding due to an interference with the gp120-CD4 interaction. PMID- 1704066 TI - Synthesis and anti-HIV activities of low molecular weight aurintricarboxylic acid fragments and related compounds. AB - Several compounds corresponding to fragments of the schematic representation of the polymeric structure of aurintricarboxylic acid (ATA) have been prepared and tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture and HIV-1 in CEM cell culture. Both the triphenylcarbinol 3 as well as the triphenylmethane 5 were found to afford protection against the cytopathogenicity of HIV-2 in MT-4 cells and HIV-1 in CEM cells, but they were inactive against HIV-1 in MT-4 cells. Both substances were also found to inhibit syncytium formation when MOLT-4 cells were cocultured with HIV-2-infected HUT-78 cells, but were inactive in this assay against HIV-1-infected cells. When observed, the activity is generally moderate in degree of protection and requires concentrations in the 10(-4) molar range. In contrast to ATA, both of these substances were inactive when tested for prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor and also for inhibition of HIV-1 reverse transcriptase. These substances therefore appear act by a mechanism that is distinct from that of polymeric ATA. Several active and inactive structural analogues of 3 and 5 were also synthesized. The anti-HIV activity in this series seems to depend on the presence of anionic carboxylate groups, since the methyl esters 4, 6, and 12 were uniformly inactive. The diphenylmethanes 8, 14, 18, and 19 also reproducibly inhibited the cytopathic effect of HIV-1 in CEM cell culture. PMID- 1704067 TI - Decay of mRNA encoding ribosomal protein S15 of Escherichia coli is initiated by an RNase E-dependent endonucleolytic cleavage that removes the 3' stabilizing stem and loop structure. AB - The transcripts of the rpsO-pnp operon of Escherichia coli, coding for ribosomal protein S15 and polynucleotide phosphorylase, are processed at four sites in the 249 nucleotides of the intercistronic region. The initial processing step in the decay of the pnp mRNA is made by RNase III, which cuts at two sites upstream from the pnp gene. The other two cleavages are dependent on the wild-type allele of the rne gene, which encodes the endonucleolytic enzyme RNase E. The cuts are made 37 nucleotides apart at the base of the stem-loop structure of the rho independent attenuator located downstream from rpsO. The cleavage downstream from the attenuator generates an rpsO mRNA.nearly identical with the monocistronic attenuated transcript, while the cleavage upstream from the transcription attenuator gives rise to an rpsO mesage lacking the terminal 3' hairpin structure. The rapid degradation of the processed mRNA in an rne+ strain, compared to the slow degradation of the transcript that accumulates in an rne- strain, suggests that RNase E initiates the decay of the rpsO message by removing the stabilizing stem-loop at the 3' end of the RNA. PMID- 1704068 TI - Rest of world ready to follow this hemisphere's approach to eliminating polio in near future. PMID- 1704069 TI - Immune system boosters set for FDA go-ahead. PMID- 1704070 TI - [Recent progress in antiviral agent]. PMID- 1704071 TI - [Drug therapy of infections in hematologic disorders ]. PMID- 1704072 TI - [Countermeasure against infections in malignant diseases]. PMID- 1704073 TI - [Treatment of infections in primary immunodeficiency syndromes]. PMID- 1704075 TI - Serotyping of O and pilus antigens of Escherichia coli strains isolated from chickens with coli-septicemia. AB - One hundred and fifty one Escherichia coli strains were isolated from broiler chickens with coli-septicemia in Aichi (63 strains), Shizuoka (58 strains), and Kagoshima (30 strains) prefectures from 1980 to 1987, and their O and pilus antigens were serologically typed. One hundred and twenty five strains (82.8%) were typed into 23 O serogroups, and twenty six strains (17.2%) remained untypable. The predominant O serogroups were O2 (35 strains, 23.2%) and O78 (24 strains, 15.9%). Distribution of O serogroup was different, depending on prefectures where they are isolated. In total, 109 strains (72.2%) possessed Type 1 and/or Fmsha pili (Type 1; 41 strains, Fmsha; 22 strains, and Type 1 and Fmsha; 46 strains), and 42 strains (27.2%) were non-piliated. All the strains lacked K88, K99, 987P, F41, and Att25 pili. The ratios of piliated strains to non piliated ones were almost the same among the three prefectures. Strains possessing Type 1 pili showed variety of O antigens, but most of the strains with Fmsha pili belonged to O2 serogroup. PMID- 1704074 TI - [Current practice and future prospects of AIDS-therapy]. PMID- 1704076 TI - Immunological features in hairless descendants derived from Mexican hairless dogs. PMID- 1704077 TI - Aldosterone-regulated ion transporters in the kidney. AB - Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of the renal collecting duct. In these cultured epithelial cells aldosterone activates apical Na+/H+ exchange, initiating a cascade of intracellular events such as cell growth, epithelial cell polarity, and stimulation of transepithelial ion transport. Transepithelial K+ secretion is triggered by the insertion of new ion channels and the activation of previously quiescent channels with increasing cytoplasmic pH. Aldosterone supplies the cell with ion transporters necessary for adequate function of the renal collecting duct when the organism is metabolically challenged. PMID- 1704078 TI - Neuroendocrine carcinoid tumours of the breast: a variant of carcinoma with neuroendocrine differentiation. AB - Carcinoid tumours most frequently develop in the gastrointestinal tract but have been described in many organs of the body. In 1977 the first cases were reported in the mamma, followed by descriptions of argyrophilic carcinoid-like, neuroendocrine mammary tumours by many investigators who performed immunohistochemical and ultrastructural examinations. The existence of true carcinoids in the mamma is still a controversial issue. Eight mammary neoplasms with monomorphous cytonuclear features, five of the small cell carcinoid-like variety and three composed of larger cells, were examined by immunohistochemical and ultrastructural examination. We believe this kind of tumours are ductal or lobular carcinomas with focal or more extensive neuroendocrine features and are the result of a dual differentiation of neoplastic precursor stem cells along epithelial and endocrine lines. Consequently, we consider that treatment of such cases should not be different from that of the ordinary type of mammary carcinomas. PMID- 1704079 TI - Subrenal capsule assay as a chemosensitivity test for primary esophageal squamous cell carcinoma. AB - The efficiency of the subrenal capsule assay (SRCA) was studied with fresh tissue of esophageal squamous cell carcinoma. The day-to-day changes in 10 carcinoma cases were evaluated for 9 days. The cancer cells continued to proliferate from the 3rd to the 7th day after the implantation and then decreased. The host reaction was recognized histologically from the 3rd or 4th day to the 9th day. However, the immune reaction did not significantly influence the evaluation of SRCA until the 7th day. The immunohistochemical staining with anti bromodeoxyuridine monoclonal antibody revealed the existence of cancer cells at the DNA synthesizing stage (S stage) in the graft until the 7th day. In chemosensitivity test by SRCA, 21 patients were studied, and all were evaluable. 5-FU administration produced a response in 8/21 cases (38.1%), VDS in 8/21 (38.1%), and CDDP in 3/21 (14.3%). Used in combination, CDDP + VDS was effective in 7/18 cases (38.9%) and CDDP + BLM in 6/18 cases (33.3%). PMID- 1704081 TI - Abstracts, 20th annual meetings. Keystone Symposia on Molecular and Cellular Biology. Molecular evolution of introns and other RNA elements. February 2-8, 1991. PMID- 1704080 TI - Squamous carcinoma of the distal esophagus: a survival study. AB - A survival study for squamous carcinomas of the distal esophagus treated by the Southern California Permanente Medical Group in the interval of 1954 to 1988 was undertaken. We found radiation therapy and surgery equally efficacious in terms of cure for patients without distant disease and performance status sufficient to tolerate treatment. We did not find survival benefit for patients treated with palliative surgery, and plan less invasive endoscopic means along with chemotherapy and radiation for palliation, reserving surgery for special circumstances. PMID- 1704082 TI - Thromboexclusion of the right ventricle in children with pulmonary atresia and intact ventricular septum. AB - Twelve children with pulmonary atresia and intact ventricular septum underwent closure of the tricuspid valve as a part of a new surgical procedure. In two cases a concomitant Fontan operation was performed. In each patient the right ventricle was very small and right ventricular pressure was higher than systemic pressure. Ventricle-coronary connections provided flow of desaturated blood from the right ventricle into the coronary arteries in 11 of 12 cases. Five of the 12 children did not survive operation and postmortem examination of each revealed severe acute and chronic myocardial ischemic damage and high-grade obstruction or interruption of the proximal left anterior descending coronary artery. Preoperative angiography demonstrated occlusive changes in the coronary arteries, resulting in right ventricular dependent circulation, in all five children who died and in one child who survived operation. Seven children who survived operation are well 4 months to 3.5 years later. Two have undergone subsequent successful Fontan operation and two others are considered suitable candidates for this operation. Tricuspid valve closure is recommended for a carefully selected group of infants with pulmonary atresia and intact ventricular septum provided a right ventricular-dependent coronary circulation can be excluded on the basis of preoperative coronary cineangiography. PMID- 1704083 TI - The combined effects of Il-3, GM-CSF and G-CSF on the in vitro growth of myelodysplastic myeloid progenitor cells. AB - The decreased or absent in vitro colony formation in response to single recombinant haematopoietic growth factors has been reported previously. Here we report on the effects of the combination of interleukin 3 (Il-3), granulocyte macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) and the effect of the conditioned medium of the giant tumour cell line (GCT-CM) on the proliferation of myelodysplastic (MDS) marrow myeloid progenitor cells and normal bone marrow (NBM) controls. Colony growth was most effectively sustained by GCT CM and G-CSF in normal bone marrow (NBM) cultures. GM-CSF and Il-3 were less effective in inducing myeloid granulocytic colony growth, whereas the effects of Il-3 and GM-CSF were found to be approximately additive. The number of NBM granulocytic colonies induced by G-CSF and GCT-CM stimulation were comparable, whereas this granulocyte colony stimulating activity could be neutralized by anti G-CSF antibodies. In addition GCT-CM was found to contain burst promoting activity, which could be neutralized by anti-Il-3 antibodies. Il-3 did not enhance the G-CSF activity in NBM cultures. No additive effect of stimulation with the combination of Il-3 and GM-CSF was observed in MDS marrow cultures, suggesting that these growth factors act on an identical progenitor cell population in MDS. G-CSF stimulated the growth of significantly lower colony numbers than GCT-CM, in contrast to NBM cultures. The decreased granulocytic colony formation of MDS marrow cells could clearly be enhanced by co-stimulation with Il-3. These results suggest that MDS myeloid progenitor cells require the exposure to both a pluripotent colony stimulating factor, like Il-3, and a lineage specific factor, like G-CSF, for optimal proliferation. PMID- 1704085 TI - [The treatment of Guillain-Barre syndrome with high gamma-globulin doses]. PMID- 1704084 TI - Gamma interferon augments functional and phenotypic characteristics of vitamin D3 induced monocytoid differentiation in the U937 human leukaemic cell line. AB - We studied the effect of gamma interferon on 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced differentiation of the human leukaemic cell line U937. Both phenotypic and functional aspects of 1,25(OH)2D3-induced differentiation were significantly augmented by gamma interferon (IFN). Gamma interferon had little effect alone but increased butyrate esterase staining and expression of CD14 antigen and the 40 kD Fc receptor (FcRII) in response to 1,25(OH)2D3. Ability to phagocytose IgG-opsonized bacteria, and superoxide burst in response to both IgG opsonized bacteria and phorbol ester, was greater after incubation with both IFN and 1,25(OH)2D3 than with either agent alone. The degree of functional activation of cells showed a positive correlation with FcRII expression. In addition, IgG induced generation of superoxide by differentiated cells was considerably reduced by pre-incubation with the anti-Fc receptor antibody IV3. We conclude that gamma interferon augments 1,25(OH)2D3-induced differentiation and functional activation of the U937 cell line. Increased functional activation may, in part, be due to up regulation of surface FcR11. PMID- 1704086 TI - [Intravenous administration of immunoglobulin speeds up improvement in Kawasaki disease]. PMID- 1704087 TI - Increased maternal serum alpha fetoprotein in congenital hypothyroidism. PMID- 1704088 TI - Mechanism of action of FK 506 and cyclosporin. PMID- 1704089 TI - Comparison of continuous subcutaneous and intravenous hydromorphone infusions for management of cancer pain. AB - To compare the safety and efficacy of subcutaneous and intravenous infusion of opioid analgesics, a randomised, double-blind, crossover trial was carried out in inpatients. 15 patients with severe cancer pain received two 48 h infusions of hydromorphone--one subcutaneously and one intravenously in randomly allocated order. The study was made double-blind by the use of two infusion pumps throughout; during the active subcutaneous infusion the intravenous pump delivered saline and vice versa. Serial measurements of pain intensity, pain relief, mood, and sedation by means of visual analogue scales showed no clinically or statistically significant difference between the two infusion routes. Side-effects were slight, and the mean number of morphine injections for breakthrough pain did not differ significantly between the routes (4.8 [SD 4.5] for intravenous vs 5.3 [5.6] for subcutaneous). Plasma hydromorphone concentrations measured at 24 h and 48 h of infusion showed stable steady-state pharmacokinetics; the mean bioavailability from subcutaneous infusion was 78% of that with intravenous infusion. Because of the simplicity, technical advantages, and cost-effectiveness of continuous subcutaneous opioid infusion into the chest wall or trunk, intravenous opioid infusion for the management of severe cancer pain should be abandoned. PMID- 1704090 TI - FK 506. PMID- 1704091 TI - Plasma expanders as cause of paraproteinuria-like artifact. PMID- 1704092 TI - Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA. PMID- 1704093 TI - The use of bromodeoxyuridine cytokinetic studies as a prognostic indicator of cancer of the head and neck. AB - Traditional measures of head and neck tumors often fail to predict patient outcome or clinical course, particularly in nonadvanced disease. This problem of unpredictable tumor behavior has been one focus of cell proliferation studies. Such studies, however, have been limited by difficult methodology. A newer method of quantifying tumor cell proliferation using bromodeoxyuridine is applicable for head and neck squamous cell carcinomas, as shown in the present study. The relative ease with which cell proliferation can be evaluated using this technique will allow large numbers of head and neck tumors to be studied, enabling correlations with tumor behavior to be made. PMID- 1704094 TI - Acid-fast and H&E stainings can be combined better than in the TRIFF method. PMID- 1704095 TI - The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure-function relationships in the TraT protein. AB - The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed. PMID- 1704096 TI - Purification and characterization of RNase P from Clostridium sporogenes. AB - RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl. PMID- 1704098 TI - Regulation of drosophila folylpolyglutamates during development. AB - The polyglutamate status of reduced folates during the larval, pupal and adult stages of Drosophila melanogaster development was investigated. The chain length distribution is very similar and is predominantly pentaglutamate. Half-life estimates of the hydrolytic degradation to the monoglutamate showed larva less than pupa less than adult. This raises the possibility that polyglutamate hydrolase may have a role in regulating the total intracellular reduced folate content of the different developmental stages. PMID- 1704097 TI - Neurokinin A and B: potent vasodilators in the canine forelimb. AB - Neurokinin A and neurokinin B may play a role in control of the peripheral circulation in either physiological or pathophysiological conditions. We have infused these peptides intra-arterially at three infusion rates each to assess their actions on vascular pressures, blood flows and total and segmental resistances in skin and skeletal muscle in the canine forelimb. Neurokinin A infusions (.01, .1, and 1 micrograms/min) decreased total forelimb resistance; transiently, 26% and 57%, respectively. The decrease in resistance was equally distributed between the skin and skeletal muscle circulations and was manifest in both large artery and small vessel resistances. Systemic and forelimb arterial pressures were decreased in a dose-dependent manner. Neurokinin B infusions (.5, 1 and 5 micrograms/min) decreased total forelimb resistance 29%, 31%, and 52%, respectively. The decrease in resistance was equally distributed between the skin and skeletal muscle circulations and was the result of decreases in both large artery and small vessel resistances. Systemic and forelimb arterial pressures were decreased in a dose-dependent manner. The potent effect of neurokinins on vascular resistance and their concentration in perivascular nerves innervating the resistance vessels of the circulation suggests a potential role for these neuropeptides in circulatory control. PMID- 1704099 TI - Decreased levels of guanyl nucleotide-binding regulatory protein alpha-subunits in Y1 adrenocortical tumor cell mutants resistant to forskolin. AB - Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'-yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the alpha-subunits of Gs and Gi-2 were reduced by 70-80% relative to the levels in parental Y1 cells. The levels of the beta 36-subunit were much less affected, and the levels of the alpha i-3 and beta 35-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs alpha and Gi alpha RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs alpha and Gi alpha-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G alpha subunits or their incorporation into the plasma membrane. PMID- 1704100 TI - Differential regulation of rat T-kininogen by tumor necrosis factor and interleukin-6. AB - Rat T-kininogen (T-KG), a cysteine protease inhibitor, is an acute phase reactant which is induced to high levels in response to inflammation. Both hormones and cytokines participate in this regulation. To investigate the cis-acting elements responsible for the induction of gene expression, various 5'-fragments of the rat T-KG gene were fused to a chloramphenicol acetyltransferase marker gene. These constructs were transfected into a rat hepatoma cell line which was then treated with tumor necrosis factor or interleukin-6 or both cytokines. Expression of the chloramphenicol acetyltransferase gene was induced with interleukin-6 treatment, but suppressed by tumor necrosis factor. The 5'-region of the T-KG gene responsible for conferring both of these effects was localized between nucleotides -404 to -210 upstream of the transcription start site. Fragments containing this region were found to be effective in either orientation, and could also regulate a heterologous promoter. PMID- 1704101 TI - Amplification of the transcriptional signal mediated by the tandem cAMP response elements of the glycoprotein hormone alpha-subunit gene occurs through several distinct mechanisms. AB - cAMP stimulates transcription of the human glycoprotein hormone alpha-subunit gene in choriocarcinoma cells. Combined treatment with phorbol esters potentiates this effect. Tandem cAMP response elements (CREs) in the proximal 5'-flanking sequence mediate the effect of cAMP. In this report, we show that the CREs can also mediate the synergistic effect of phorbol esters. In addition to serving as an inducible cis-acting element, the two CREs act synergistically to increase basal transcription. We now provide direct evidence via equilibrium binding studies that tandem CREs bind their trans-acting factors cooperatively. However, the level of cooperativity is insufficient to explain the high degree of transcriptional synergism, suggesting that another element in the alpha-subunit promoter may be required for complete synergism. In support of this hypothesis, we show that synergism is drastically reduced when the CREs are removed from the context of their native promoter and linked to a heterologous promoter. Thus, amplification of the transcriptional signal mediated by the tandem CREs occurs through at least three distinct mechanisms. First, at the level of signal transduction by convergence of the A- and C-kinase pathways; second, through homotropic interactions of trans-acting factors binding to tandem CREs; and finally through heterotropic interaction of the two CREs with another, as yet, undefined cis-acting element(s) in the human alpha-subunit promoter. PMID- 1704104 TI - Effects of growth phase and antibiotics on quantitative DNA/RNA hybridization. AB - Synthetic probes complementary to ribosomal RNA are increasingly used in the detection of bacteria. Many applications, however, require the quantitation of bacteria. We therefore tested the influence of growth phase and representative antibiotics (ampicillin, chloramphenicol and gentamycin) on the outcome of DNA/RNA filter hybridization using radiolabelled probes and a multisample digital autoradiograph for quantitative monitoring. Hybridization efficiency seemed influenced by the binding capacity of the membrane, availability of target molecules and physiological growth control. For chloramphenicol the absolute hybridization signal remained constant over the experimental period. Only a slight decrease was found in experiments with gentamycin whereas viable counts dropped 10,000-fold. For ampicillin a decrease in viable counts was paralleled by diminishing signal strengths. Relative signal strengths (counts per viable cell) increased in all experiments with antibiotics. In conclusion; (i) RNA probes seem to detect bacteria even after onset of antimicrobial therapy; (ii) DNA/RNA filter hybridization appears not suitable for accurate quantitation of bacteria. PMID- 1704102 TI - An alpha-subunit-secreting cell line derived from a mouse thyrotrope tumor. AB - The anterior pituitary contains multiple distinct endocrine cell types that secrete individual hormones. To derive a pure cell culture population in which to study the regulation of the alpha-subunit of TSH free of other hormones and cell types, we have developed a clonal continuous cell line from the transplantable thyrotrope tumor MGH101A. This cell line expresses alpha-subunit mRNA, secretes alpha-subunit protein, and has maintained a stable phenotype for over 3 yr in culture. However, as is the case for the transplantable tumor from which they are derived, these cells do not express the beta-subunit of TSH or respond to TRH or thyroid hormone. We have used this cell line to investigate regulation of the alpha-subunit mRNA by the second messengers, cAMP and phorbol esters, and by glucocorticoids. Phorbol esters increase alpha-subunit mRNA levels significantly (3.5-fold), as does cAMP (1.8-fold). In contrast, glucocorticoids decrease mRNA levels from cAMP-induced or basal levels (2-fold). These cells should prove valuable for study of alpha-subunit gene expression in an isolated renewable clonal cell culture system. PMID- 1704103 TI - Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. AB - Study of the molecular and cellular biology of the gonadotropin hormones would be greatly facilitated by the availability of immortalized anterior pituitary gonadotrope cell lines. We directed expression of the simian virus-40 (SV40) T antigen (Tag) oncogene to specific cells in the anterior pituitary of transgenic mice using the promoter/enhancer region from the human glycoprotein hormone alpha subunit gene. Transgenic mice carrying this fusion gene developed anterior pituitary tumors. Clonal cell lines established from these tumors express the endogenous mouse alpha-subunit gene and synthesize and secrete alpha-subunit protein. However, they do not express beta-subunit genes. Alpha-subunit mRNA is induced by GnRH in a dose- and time-dependent manner, but is not regulated by TRH. Thus, we have targeted tumorigenesis in transgenic mice to anterior pituitary cells of the gonadotrope lineage to immortalize this specific endocrine cell while maintaining several highly differentiated functions unique to gonadotropes. PMID- 1704105 TI - The low KM-phosphodiesterase inhibitor denbufylline enhances neuronal excitability in guinea pig hippocampus in vitro. AB - The actions of the phosphodiesterase inhibitor denbufylline on the excitability of hippocampal neurons were investigated by means of extracellular and intracellular recordings. Denbufylline, which has been shown to selectively inhibit a low KM, Ca2+/calmodulin-independent phosphodiesterase isozyme, concentration-dependently increased the amplitude of the extracellularly recorded CA1 population spike evoked by electrical stimulation of the Schaffer collateral/commissural pathway. Concentration-response-curves yielded an EC50 for denbufylline of 0.76 microM. In comparison, the non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) also produced an increase in the amplitude of the population spike. From the concentration-response-curve, which was steeper than that of denbufylline, an EC50 for IBMX of 1.04 microM was obtained. However, despite their similar EC50 values, denbufylline was found to be significantly more potent at lower concentrations (less than or equal to 300 nM) than IBMX. Intracellular recordings from CA1 pyramidal cells revealed postsynaptic actions of denbufylline (300 nM) as indicated by a small drug induced depolarization (2-5 mV) associated with an increase in membrane input resistance by 10-20%. In addition, denbufylline blocked the accommodation of trains of action potentials evoked by the injection of depolarizing current pulses. The results suggest i) that accumulation of adenosine-3',5'-monophosphate (cAMP) in the postsynaptic cell and/or in the presynaptic terminal produced by blockade of phosphodiesterases leads to enhanced synaptic transmission in the CA1 area of the hippocampus and ii) that a low KM, Ca2+/calmodulin-independent cAMP phosphodiesterase is an important component involved in the regulation of the intracellular cAMP level at synapses of central nervous system neurons. PMID- 1704106 TI - Cortical senile plaques in coronary artery disease, aging and Alzheimer's disease. AB - Mild alterations in cognitive function are present in normal aging and severe cognitive alterations are a hallmark of Alzheimer's disease (AD). The cognitive change in AD has been correlated to the characteristic pathologic lesions in the brain, senile plaques (SP) and neurofibrillary tangles. Senile plaques are the most consistent correlative marker in AD. We present preliminary data indicating that abundant SP are found in the brains of nondemented patients dying with or as a result of critical coronary artery disease (cCAD) compared to nonheart disease (non-HD) subjects; 15 of 20 cCAD patients contained SP and only two of 16 non-HD patients contained SP. PMID- 1704107 TI - Tracing neuronal connections in postmortem human hippocampal complex with the carbocyanine dye DiI. AB - This report describes the ability of the carbocyanine dye DiI to trace hippocampal complex connections in a paraformaldehyde immersion-fixed human postmortem brain. Six months after the placement of DiI crystals into the hilus of the dentate gyrus, the CA1 hippocampal subfield and the lateral entorhinal cortex, 50-microns thick, vibratome cut sections were examined using an epifluorescence microscope with a rhodamine filter. In association with DiI labeled granule, pyramidal and multipolar type neurons, we observed dendrites containing dendritic spines and axons. DiI-labeled fibers were observed coursing within classically described hippocampal pathways for at least 8 mm distal to the injection site. Photoconversion of diaminobenzidine (DAB)-treated DiI sections produced a stable record of labeled profiles. These findings indicate that DiI is a useful method for investigating intrinsic local circuit connections in normal aldehyde-fixed postmortem human brain and suggests that DiI could be a powerful tool to examine altered neural connectivity in humans with neurological disease. PMID- 1704108 TI - Thyrotropin-releasing-hormone-immunoreactive innervation of thyrotropin-releasing hormone-tuberoinfundibular neurons in rat hypothalamus: anatomical basis to suggest ultrashort feedback regulation. AB - Thyrotropin-releasing-hormone (TRH)-synthesizing neurons in the medial and periventricular parvocellular subdivisions of the rat hypothalamic paraventricular nucleus (PVN) are involved in regulation of the anterior pituitary. Since ultrashort feedback regulation of TRH in the hypothalamus has been suggested by physiological studies, we sought to identify the presence of TRH synaptic contacts containing TRH on TRH tuberoinfundibular neurons in the PVN. An immunocytochemical study was performed at light- and electron-microscopic levels using antiserum directed to the N-terminal cryptic sequence of the TRH precursor, preproTRH 25-50. At the light-microscopic level, contacts between TRH immunoreactive (IR) fibers and the perikarya and processes of TRH-IR neurons were observed in medial and periventricular subdivisions of the PVN. At the ultrastructural level, TRH-neurons appeared either tightly juxtaposed to TRH immunopositive perikarya and dendrites or to establish axodendritic and axosomatic contacts suggestive of synaptic associations. These data provide a morphologic basis to support a neuroendocrine role for TRH or processed forms of proTRH in the PVN and in particular suggest their involvement as neuromodulators in an ultrashort feedback regulation of TRH tuberoinfundibular neurons. PMID- 1704109 TI - The effects of two FMRFamide related peptides (A-18-F-amide and F-8-F-amide; 'morphine modulating peptides') on the endocrine and exocrine rat pancreas. AB - The effects of two recently isolated mammalian FMRFamide related peptides (A-18-F amide and F-8-F-amide) on the encocrine and exocrine rat pancreas were investigated. A-18-F-amide (10, 100, 1000 pM) inhibited concentration dependently glucose (10 mM)- and arginine (10 mM)-induced insulin secretion from the isolated perfused rat pancreas during the first (controls: 100%; 10 pM: 114%; 100 pM: 63%, p less than 0.05; 1000 pM: 31%, p less than 0.05) and the second secretion phase (controls: 100%; 10 pM: 102%; 100 pM: 78%; 1000 pM: 27%, p less than 0.05). The inhibitory actions of A-18-F-amide on pancreatic D-cell secretion were more pronounced during the first than the second phase (first phase: controls: 100%; 10 pM: 95%; 100 pM: 37%, p less than 0.05; 1000 pM: 39%, p less than 0.05%; second phase: controls: 100%; 10 pM: 113%; 100 pM: 72%; 1000 pM: 59%, p less than 0.05). F-8-F-amide (at 1000 pM) inhibited stimulated insulin (controls: 100%; first phase: 26%, p less than 0.05%; second phase: 20%, p less than 0.05) and somatostatin release (controls: 100%; first phase: 14%, p less than 0.05; second phase: 29%, p less than 0.05). Both peptides were without effect on basal and CCK 8-stimulated amylase release from isolated incubated rat pancreatic acini. PMID- 1704110 TI - Constant neurofibrillary changes in the neocortex in progressive supranuclear palsy. Basic differences with Alzheimer's disease and aging. AB - Neocortical neurofibrillary tangles (NFT) revealed by Bodian technique and anti tau immunolabelling were seen in 5/5 cases of progressive supranuclear palsy (PSP) aged 58-76 years. These lesions differed from Alzheimer's disease or age related changes: (1) they were most frequent in the precentral gyrus (Brodmann's area 4) whereas associative areas are predominantly lesioned in Alzheimer's disease; (2) they affected mainly large pyramidal neurons and small cells, relatively sparing the cell population selectively involved in Alzheimer's disease; (3) they predominated in layers V and VI of area 4, whereas NFT are most dense in layers III and V in Alzheimer's disease; (4) mature senile plaques (1/5 cases) and beta-amyloid diffuse deposits (3/5 cases), which usually precede or go together with NFT in Alzheimer's disease were rare or absent (2/5) in PSP. Neuropil threads and tufts of abnormal fibres were also seen. In addition, NFT and neuropil threads were found in the hippocampus. PSP is thus another example of abnormal storage of tau developing in the neocortex in the absence of beta amyloid deposits. It might prove a useful model for the understanding of the mechanisms of localization and spreading of tau storage in the brain. PMID- 1704111 TI - The subserosal ganglia of the human taenia. AB - Specimens of the taenia from the sigmoid colon of female patients undergoing surgery for carcinoma of the rectum were studied histochemically and immunohistochemically for acetylcholinesterase (AChE) and for vasoactive intestinal polypeptide (VIP)-, substance P (SP)-, somatostatin (SOM)-, neuropeptide Y (NPY)-, calcitonin gene-related peptide (CGRP)- and Met-enkephalin (mENK)-immunoreactivity. Autonomic ganglia were observed on the serosal surface of the longitudinal muscle of the taenia. The subserosal ganglia contained SP-, mENK-, NPY-, SOM-, but not CGRP- or VIP-immunoreactive nerve fibres. In addition, they contained SP-, mENK- and NPY-, but not CGRP-, SOM- and VIP-immunoreactive nerve cell bodies (although CGRP- and VIP-immunoreactive nerve fibres were observed in the longitudinal muscle of the taenia). AChE-activity was found both in nerve fibres and nerve cell bodies in these ganglia. The greatest numbers of nerve cell bodies contained AChE, followed in decreasing order by SP, mENK and NPY. The possible function of the subserosal ganglia of the human taenia is discussed. PMID- 1704112 TI - Proteolytic enzymes do not destroy the N-methyl-D-aspartate (NMDA) sensitivity of acutely isolated hippocampal CA1 and CA3 neurons from postnatal rats. AB - Using whole cell recordings in combination with the concentration clamp technique it was shown that even enzymatically isolated hippocampal CA1 and CA3 neurons exhibit N-methyl-D-aspartate (NMDA) and L-aspartate currents. These currents were completely blocked by 0.5 mM Mg2+ or partially blocked (72.7 +/- 2.4%) by DL-2 amino-5-phosphonovalerate (50 microM) whereas 10 microM glycine (Gly) potentiated the NMDA responses (3.1 +/- 1.6-fold). A half-maximum dose of 55 microM was calculated from the sigmoid NMDA dose-response curve in the presence of 10 microM Gly. The amplitudes of these currents did not depend on the type of the proteolytic enzyme used for cell isolation. PMID- 1704113 TI - Hypoxic suppression of K+ currents in type I carotid body cells: selective effect on the Ca2(+)-activated K+ current. AB - Whole-cell K+ currents were recorded in isolated type I carotid body cells using the patch-clamp technique. Hypoxia (pO2 25 torr) reversibly suppressed K+ currents in a voltage-dependent manner: maximal effects were seen at low, positive test potentials, where the Ca2(+)-activated component of K+ currents was greatest. Enhancing this component with 5 microM BAY K 8644 exaggerated the effects of hypoxia, and when the component was inhibited (100 microM Cd2+ or 5 microM nifedipine) hypoxic effects were abolished. As hypoxia does not affect Ca2+ currents directly, these data indicate the suppressive effect of hypoxia is selective for the Ca2(+)-activated component of K+ currents in type I cells. PMID- 1704114 TI - Fetal olfactory bulb transplants send projections to host olfactory cortex in the rat. AB - We are using the rat olfactory system to study developmental details of neurotransplantation. Tritiated [3H]thymidine-labeled fetal olfactory bulbs (OBs), were transplanted immediately into sites from which the neonatal host OB was removed. Subsequently, a small lesion was placed in the region of the transplanted OB and the tissue studied, using degeneration methods and autoradiography. Only OB's with extensive [3H]-label and precise lesions confined to the labeled areas were used. Degeneration was found mainly in the ipsilateral piriform cortex with lesser amounts at other nearby sites. The results demonstrate successfully transplanted donor OBs that send axons to specific and appropriate target areas of the host brain. PMID- 1704115 TI - Midbrain periaqueductal gray neurons with substance P- or enkephalin-like immunoreactivity send projection fibers to the nucleus accumbens in the rat. AB - Employing a combination of retrograde tracing and immunocytochemistry, midbrain periaqueductal gray (PAG) neurons with substance P- or leucine-enkephalin-like immunoreactivity in the rat were found to send projection fibers to the nucleus accumbens bilaterally with an ipsilateral dominance. These neurons were most frequently seen in the ventrolateral and ventral parts of the medial PAG subdivisions at all rostrocaudal levels of PAG. PMID- 1704116 TI - [The characteristics of the engorgement of the eye vessels in the dynamics of the burn process]. AB - Peculiarities of eye blood filling in dynamics of a burn process of different severity have been studied in 29 patients with mild burns, in 19--with moderate, in 56--with severe, and in 32--with extremely severe. It is found that changes in ocular vessels blood filling correlate with severity of the burn, the degree of the development of the burn process. The usage of rheopolyglucine and cavinton in a complex treatment of eye burns positively influences the course of the burn disease. The degree of the increase of eye blood filling is in inverse dependence on the degree of destructive changes in eye tissues. Restoration of blood filling as a result of the usage of rheopolyglucin and cavinton in the shortest terms after burn stimulates the stoppage of inflammatory-destructive changes and acceleration of regenerative processes. PMID- 1704117 TI - [Methodological approaches to detecting mRNA in histological sections]. AB - This paper reviews methodological approaches to detection of mRNA on histological sections using in situ hybridization. Alongside with the review of literature we present a detailed description of our own modification of this technique, based on the use of paraffin sections of the material fixed by replacement in the frozen state. Particular attention is given to methodological problems facing the researcher who is using in situ hybridization for the first time. PMID- 1704118 TI - Expression of the c-kit proto-oncogene in the murine male germ cells. AB - The proto-oncogene c-kit encodes a transmembrane protein tyrosine kinase receptor. The c-kit gene has recently been shown to be allelic with the W locus. Mutations at the white spotting locus (W) affect various aspects of hematopoiesis, melanogenesis and gametogenesis during development and in the adult animal. We have investigated the expression of the proto-oncogene c-kit in mouse testicular cell populations. The c-kit mRNA was found to be expressed at high levels in spermatogonia, and at lower levels in meiotic pachytene spermatocytes. Moreover, two novel testis-specific c-kit transcripts of 3.5 and 2.3 kb are present in postmeiotic haploid germ cells. These results suggest a role of c-kit not only during testis development in the embryo, but also throughout all stages of male germ cell development after birth. PMID- 1704119 TI - Identification and characterization of a human homolog of the Schizosaccharomyces pombe ras-like gene YPT-3. AB - The Polymerase Chain Reaction was used to amplify ras and ras-like sequences from two human cDNA libraries. Members corresponding to each of the three major ras subfamilies (ras, rho, and rab/YPT) were identified. The one homologous to rab/YPT, referred to here as YL8, appears to be the human homolog of the recently reported Schizosaccharomyces pombe YPT3 gene. The YL8 gene could encode a guanine nucleotide binding protein of 216 amino acids with about 70% amino acid sequence identity to S. pombe YPT3, and is transcriptionally active in a variety of human cell lines. PMID- 1704121 TI - T-lymphocyte subgroups and the activity of human natural killer (HNK) cells in low-grade and high-grade malignant cases of non-Hodgkin lymphoma. AB - T-lymphocyte subgroups and the percentage and activity of Human Natural Killer (HNK) cells were investigated in 24 patients suffering from low-grade and 24 patients with high-grade malignancies of non-Hodgkin lymphoma (NHL). The ratio of CD3, CD4, Leu-7, and HNK cells as well as the release by HNK cells of the 51Cr bound to target cells were found decreased, depending on the pathological stage. The tests were performed with OKT-monoclonal sera. A significant change was observed in the reactivity of bone marrow cells to monoclonal sera; the change was identical in character with that observed when lymphocytes isolated from the peripheral blood were used in the same tests. No significant changes could be observed in the quantitative relations of immunoglobulins. Such changes could by no means be expected, on the basis of the unchanged number of T-suppressor lymphocytes (CDB). As to the supposed immunological relation in detail, the role of a plasma factor that reduces T-lymphocyte formation is assumed to have primary importance in this phenomenon. PMID- 1704120 TI - Overexpression of c-fos in a human pre-B cell acute lymphocytic leukemia derived cell line, SMS-SB. AB - The c-fos proto-oncogene is found to be overexpressed at least 30-fold in SMS-SB, a pre-B leukemic cell line compared to other cell types. No gross alteration of the c-fos gene structure in SMS-SB cells can be detected by karyotypic or Southern analyses. C-fos in SMS-SB cells can still be induced by serum, TPA and the calcium ionophore, A23187, and superinduced by the combination of serum and cycloheximide. The elevated levels of c-fos transcripts are not due merely to an increased life span of mRNA, as the half-life of steady state c-fos mRNA in SMS SB cells is about 40 min. Nuclear run-on transcription assays demonstrate that the transcription rate of c-fos in SMS-SB cells is up-regulated about 30-fold as compared to another pre-B leukemic cell line, NALM-6. In order to determine whether this is a cis- or transacting effect, we sequenced the 550 base pairs upstream of the SMS-SB c-fos coding region and found no mutations upstream of the mRNA CAP site. However, two point mutations at positions +23 and +98, respectively, were found in the 5' noncoding region of the first exon. Consistent with the overexpression of c-fos mRNA, the 55 kD c-fos protein is present in SMS SB cells through detection by immunoprecipitation, but could not be detected in NALM-6 cells. SMS-SB cells however, do not appear to contain more abundant AP-1 DNA binding activity than NALM-6 cells. PMID- 1704122 TI - Endogenous interferon (IFN) in patients with acute hepatitis B. AB - The occurrence of serum interferon was studied in 39 patients with acute viral hepatitis B. Antiviral activity of interferon in serum was determined by measuring the inhibition of the CPE of vesicular stomatitis virus on bovine kidney cells (MDBK). Among these patients only 5 (12.8%) had detectable serum interferon level during the first week of hospitalization. The antiviral activity of the interferon-positive sera was low (5-10 IU/ml). PMID- 1704124 TI - Pancreatic secretory responses to long-term infusions of secretin and cerulein in humans. AB - We measured pancreatic enzyme and bicarbonate responses to graded doses of intravenous secretin or cerulein alone or together in healthy human subjects. Bicarbonate responses were steady and well maintained during the last 3.5 h of the 4 h of infusions of secretagogues, giving evidence for a constant pancreatic flow rate. Potentiation (more-than-additive response) was observed between secretin and cerulein for bicarbonate secretion, but not for enzyme secretion. Secretin stimulated pancreatic enzyme secretion. The effect was most pronounced with amylase secretion and less prominent with lipase, trypsin, and chymotrypsin secretion. Changes in the proportion of enzymes were seen over time, with trypsin and chymotrypsin output declining towards the end of cerulein infusion. We conclude that in humans the effects of secretin on pancreatic enzyme secretion are complex and include time-dependent changes in the enzyme mixture, but potentiation between secretin and cerulein does not occur for enzyme output. PMID- 1704123 TI - Heterogeneity in the specificity of the islet cell cytoplasmic antibody response in insulin-dependent diabetes mellitus. AB - We assessed the heterogeneity in the islet cell cytoplasmic antibody (ICA) response of insulin-dependent diabetes mellitus (IDDM) patients via indirect immunofluorescence on frozen sections of human, bovine, and porcine pancreas. The three substrates detected comparable frequencies of ICA positives among the IDDM sera tested, whereas control sera were ICA negative on all three substrates. However, individual IDDM serum samples showed heterogeneity in ICA binding on the three pancreata. Of 28 sera tested on all three substrates, 22 were ICA positive on human pancreas, three were ICA positive on bovine pancreas, and two were ICA positive on porcine pancreas. Sensitivity of ICA epitopes to neuraminidase treatment and periodate oxidation suggests that glycoconjugates are recognized by serum ICA. Cholera toxin blocked ICA binding. However, the functional cholera toxin receptor ganglioside Gm1 is resistant to neuraminidase treatment and periodate oxidation. Therefore, it is unlikely that Gm1 is the ICA determinant. These data suggest that not all ICA antigens are equivalently expressed on islets from different pancreata and/or that each individual responds to a hierarchy of islet antigens such that restricted patterns of specific ICA binding are found. PMID- 1704125 TI - Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase. AB - Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy. In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20. The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods. Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay. The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment. Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive. PMID- 1704126 TI - Cloning and expression of a rat brain alpha 2B-adrenergic receptor. AB - We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors. PMID- 1704127 TI - FKB1 encodes a nonessential FK 506-binding protein in Saccharomyces cerevisiae and contains regions suggesting homology to the cyclophilins. AB - FK 506, a powerful immunosuppressant that blocks allograft rejection by preventing T-cell activation, binds to an 11-kDa protein called the FK 506 binding protein (FKBP). Like cyclophilin, a cytosolic protein that binds another immunosuppressant, cyclosporin A, FKBP possesses peptidylprolyl cis-trans isomerase activity. We have isolated a genomic clone encoding the yeast FKBP (FKB1). The gene encodes a protein of 114 amino acids having a calculated Mr of 12,158. Disruption of the gene shows that FKB1 is not essential for growth. A search of translated nucleic acid data bases revealed bacterial FKBP homologs in Neisseria meningiditis and Pseudomonas aeruginosa. Comparison of the conserved amino acids in FKBP homologs with the conserved amino acids in the cyclophilins has revealed a region of similarity that we speculate to be a homologous domain related to the functional similarities of the two proteins. PMID- 1704129 TI - Precerebellin is a cerebellum-specific protein with similarity to the globular domain of complement C1q B chain. AB - The cerebellum contains a hexadecapeptide, termed cerebellin, that is conserved in sequence from human to chicken. Three independent, overlapping cDNA clones have been isolated from a human cerebellum cDNA library that encode the cerebellin sequence. The longest clone codes for a protein of 193 amino acids that we term precerebellin. This protein has a significant similarity (31.3% identity, 52.2% similarity) to the globular (non-collagen-like) region of the B chain of human complement component C1q. The region of relatedness extends over approximately 145 amino acids located in the carboxyl terminus of both proteins. Unlike C1q B chain, no collagen-like motifs are present in the amino-terminal regions of precerebellin. The amino terminus of precerebellin contains three possible N-linked glycosylation sites. Although hydrophobic amino acids are clustered at the amino terminus, they do not conform to the classical signal peptide motif, and no other obvious membrane-spanning domains are predicted from the cDNA sequence. The cDNA predicts that the cerebellin peptide is flanked by Val-Arg and Glu-Pro residues. Therefore, cerebellin is not liberated from precerebellin by the classical dibasic amino acid proteolytic-cleavage mechanism seen in many neuropeptide precursors. In Northern (RNA) blots, precerebellin transcripts, with four distinct sizes (1.8, 2.3, 2.7, and 3.0 kilobases), are abundant in cerebellum. These transcripts are present at either very low or undetectable levels in other brain areas and extraneural structures. A similar pattern of cerebellin precursor transcripts are seen in rat, mouse, and human cerebellum. Furthermore, a partial genomic fragment from mouse shows the same bands in Northern blots as the human cDNA clone. During rat development, precerebellin transcripts mirror the level of cerebellin peptide. Low levels of precerebellin mRNA are seen at birth. Levels increase modestly from postpartum day 1 to 8, then increase more dramatically between day 5 and 15, and eventually reach peak values between day 21 and 56. Because cerebellin-like immunoreactivity is associated with Purkinje cell postsynaptic structures, these data raise interesting possibilities concerning the function of the cerebellin precursor in synaptic physiology. PMID- 1704128 TI - Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. AB - Fertilization of Xenopus laevis eggs triggers a period of rapid cell division comprising 12 nearly synchronous mitoses. Protein synthesis is required for these divisions, and new proteins appear after fertilization. Others proteins however, which are synthesized in the unfertilized egg, are no longer made in the early embryo. To identify such proteins, a differential screen of an egg cDNA library gave nine clones corresponding to mRNAs that are deadenylylated soon after fertilization. The sequence of one of these clones (Eg1) revealed a high homology to p34cdc2, the kinase subunit of maturation-promoting factor. Only 12 amino acids in the deduced amino acid sequence were unique to Eg1 when its sequence was compared to all other known examples of cdc2. Despite this strong similarity, however, Eg1 was unable to complement a yeast cdc2- mutant in Schizosaccharomyces pombe or a cdc28 mutant of Saccharomyces cerevisiae. Four Eg1 transcripts, two major and two minor, were found in Xenopus oocytes and early embryos. These RNAs appeared very early (stage I) in oogenesis and their level remained constant until the midblastula transition, at which time they declined. Eg1 RNA is found in the poly(A)+ fraction of oocytes only between the time of meiotic maturation and fertilization--that is to say, in the unfertilized egg. At fertilization the RNA loses its poly(A) tail and at the same time leaves the polyribosomes. PMID- 1704130 TI - Effects of binocular deprivation on the development of clustered horizontal connections in cat striate cortex. AB - Intrinsic horizontal axon collaterals in the striate cortex of adult cats specifically link columns having the same preferred orientation; consequently, retrograde tracer injections result in intrinsic labeling that is sharply clustered. We have previously shown that the normal development of this circuitry involves the emergence of crude clusters from an unclustered pattern during the second postnatal week. Crude clusters are later refined to the adult level of specificity by the selective rearrangement of axonal arbors that initially project to incorrect orientation columns. Here we report that depriving animals of patterned visual experience by binocular lid suture prior to natural eye opening had no discernible effect on the emergence of crude clusters. In contrast, cluster refinement was dramatically affected by binocular deprivation. Injections of retrograde tracers in the striate cortex of animals binocularly deprived for greater than 1 month revealed only crude clusters, indicating that horizontal axon collaterals projecting to incorrect orientation columns were retained well past the age when they normally would have been eliminated. Layer 2/3 pyramidal cells from 6-week-old binocularly deprived animals had abnormal distributions of intrinsic horizontal axon collaterals that mirrored the lack of cluster refinement. The radial clustering of their horizontal collaterals was considerably less precise than normal. These cells, nevertheless, developed many of the features of normal mature arbors, including the distal axonal branches not seen in arbors from younger animals with normal visual experience. Together, these results indicate that axonal rearrangements occurred, but with reduced specificity. Thus, binocular deprivation did not simply arrest the development of this orientation-specific circuit at an immature state but limited the accuracy with which axon collaterals were added or eliminated. We suggest that development of this orientation-specific circuitry, like ocular dominance column segregation, may depend on temporal correlation of activity for regulation of axonal rearrangement. The specificity of rearrangement may be degraded in binocularly deprived cats because they do not experience sharply oriented visual stimuli necessary for concurrent activation of same-orientation columns. PMID- 1704131 TI - GTPase-activating protein interactions with the viral and cellular Src kinases. AB - GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src. PMID- 1704132 TI - Cardiac fibroblasts are predisposed to convert into myocyte phenotype: specific effect of transforming growth factor beta. AB - Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor beta 1 (TGF-beta 1), acquire certain myocyte specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-beta 1 (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-beta 1 for 24 hr expressed mRNA corresponding to sarcomeric actin mRNA. Immunofluorescence staining and light microscopy showed that cultured cardiac fibroblasts treated with TGF-beta 1 became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-beta 1, cell proliferation (as measured by [3H]thymidine incorporation) was moderately increased (1.5-fold, P less than 0.001). NIH 3T3 cells and human skin fibroblasts, treated with TGF-beta 1, did not express sarcomeric actin mRNA. Treatment of cardiac fibroblasts with the mitogenic agent phorbol 12-myristate 13-acetate or with norepinephrine, angiotensin II, or interleukin 1 beta did not induce myocyte-specific actin mRNA. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-beta 1 in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. Our findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-beta 1 seems to be a specific inducer of such conversion. PMID- 1704134 TI - Cloning of murine ferrochelatase. AB - Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The activity of ferrochelatase is deficient in the inherited disease protoporphyria. In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe. The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences. Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined. The ferrochelatase protein and mRNAs have different relative concentrations in different tissues. The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria. PMID- 1704133 TI - Rapid induction of the c-jun protooncogene in the avian oviduct by the antiestrogen tamoxifen. AB - This report describes a rapid regulation of the expression of the c-jun protooncogene by the antiestrogen tamoxifen (Tam). The c-jun protooncogene codes for an important component of the AP-1 transcription factor complex, which regulates the expression of many unlinked genes. Repeated experiments have shown that Tam rapidly increases the steady-state c-jun mRNA levels in the avian oviduct but decreases the levels in the liver. The Tam effects are time- and dose dependent. These results are supported by other studies that have demonstrated that 17 beta-estradiol decreases steady-state levels of c-jun protooncogene mRNA in oviducts of animals fully withdrawn from estradiol. The effect of Tam in the avian oviduct is in contrast to the reported effects of Tam on the expression of practically all other genes in the avian oviduct and other animal tissues. Transcription analyses using nuclear runoff experiments with oviduct nuclei demonstrate a decrease in the c-jun gene transcription within minutes after Tam treatment with a return to 75% of control values by 4 hr. The fact that Tam transiently decreases the transcription of the c-jun gene but increases the steady-state c-jun mRNA levels suggests that Tam must alter both transcriptional and post-transcriptional events. The results support a role of the c-jun protooncogene as a regulatory gene in the cascade model for steroid action whereby steroids rapidly regulate the regulatory genes, which in turn regulate many other structural genes. PMID- 1704135 TI - Molecular cloning and chromosomal localization of the murine homolog of the human helix-loop-helix gene SCL. AB - The human SCL gene is a member of the family of genes that encode the helix-loop helix (HLH) class of DNA-binding proteins. A murine SCL cDNA was isolated from a normal macrophage cDNA library by using HLH-specific oligonucleotides as hybridization probes. The coding region is 987 base pairs and encodes a predicted protein of 34 kDa. The nucleotide sequence of the coding region shows 88% identity to the human SCL gene, and the amino acid sequence is 94% identical. The HLH motif and upstream hydrophilic region are entirely conserved in the murine and human proteins. The identity between the mouse and human sequences was less marked in the 5' and 3' untranslated regions. Two murine SCL transcripts that differ in the 3' noncoding region have been detected in fetal liver and various cell lines. Variation was also observed in the 5' untranslated region. Interestingly, immediately downstream of the protein-termination codon, both the human SCL sequence and the murine homolog share an E-box element--the suggested target site for DNA binding of HLH proteins. The murine SCL homolog was mapped to the central part of chromosome 4. PMID- 1704136 TI - A 45-kDa protein antigenically related to band 3 is selectively expressed in kidney mitochondria. AB - Anion exchange similar to that catalyzed by erythrocyte band 3 occurs across many nonerythroid cell membranes. To identify anion-exchange proteins structurally related to band 3, we immunoblotted rabbit kidney medullary membrane fractions with anti-band 3 antibodies. Immunoblots using antibodies to the cytoplasmic domain of band 3 revealed cross-reactive proteins in the plasma membrane fraction only. In contrast, two monoclonal antibodies against band 3 membrane domain labeled a 45-kDa protein; further immunoblotting and immunogold studies of membrane fractions and kidney sections using one of the anti-membrane domain antibodies showed that labeling was strongest in mitochondria of H(+)-secreting collecting duct cells. Tissue-to-tissue expression of the 45-kDa mitochondrial protein was variable: kidney medulla greater than heart greater than kidney cortex much greater than liver. We conclude that a 45-kDa protein with immunological cross-reactivity to the erythrocyte band 3 membrane domain is expressed in mitochondria in a highly cell-specific fashion and speculate that the protein may play a role in mitochondrial anion transport. PMID- 1704138 TI - Single-channel data and missed events: analysis of a two-state Markov model. AB - Patch-clamp recording permits investigation of the gating kinetics of single ion channels. Careful statistical analysis of kinetic data can yield clues as to the molecular events underlying channel gating. However, it is important that such analysis should take full account of the limitations that arise from the finite time resolution of patch-clamp recording techniques. Single-ion-channel data are generally interpreted in terms of Markov process models of channel gating mechanisms. Experimental channel records suffer from time interval omission, i.e. failure to detect brief channel openings and closings. This leads to an identifiability problem when analysing single-channel data, i.e. different gating mechanisms provide equally convincing descriptions of the same experimental data. We consider a two-state Markov model of receptor-channel gating in which the channel opening rate is proportional to the agonist concentration, C in equilibrium with OA. By using computer-simulated data, the approximate likelihood of the data is maximized to yield parameter estimates for the model. At a single agonist concentration there is an identifiability problem in that two pairs of parameter estimates are obtained. The 'true' parameter estimates cannot be distinguished from the 'false' ones. By considering data corresponding to a range of agonist concentrations one may identify the 'true' parameter estimates as those that do not change as the agonist concentration is increased. Alternatively, one may identify the 'true' parameter estimates directly by maximizing a global likelihood, the latter being obtained by simultaneous consideration of data obtained at several different agonist concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704139 TI - Radioimmunodetection (RID) with 131 I-labeled monoclonal anti-beta HCG-antibodies in testicular tumors. PMID- 1704140 TI - In-111-labeled platelet kinetic studies identify those patients with chronic ITP who will respond to intravenous gammaglobulin. PMID- 1704137 TI - Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine. AB - The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13 acetate (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-alpha, and IL-6, in the absence of cytotoxic or cytostatic effects. Reverse transcriptase activity, inducible by PMA, TNF-alpha, or IL-6, was decreased by 80-90% after pretreatment with GSH, GSE, or NAC. The induction of total HIV protein synthesis was also decreased appreciably after pretreatment with GSH, GSE, or NAC. The accumulation of HIV mRNA was substantially suppressed after pretreatment with NAC but to a lesser extent after pretreatment with GSH or GSE. Although PMA induces the expression of TNF-alpha in U1 cells, the suppressive effect of GSH, GSE, and NAC on PMA-induced HIV expression in U1 cells was not associated with the inhibition of TNF-alpha expression. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection. PMID- 1704141 TI - The effects of prostacyclin analogue ZK 36374 and thromboxane synthetase inhibitor UK 38485 on mesenteric ischemia in guinea pigs. AB - The effects of ZK 36374, a prostacyclin analogue and UK 38485, a thromboxane synthetase inhibitor were studied in guinea pigs after performing mesenteric arterial occlusion. In this study, while ZK 36374 significantly lowered the alkaline phosphatase and creatine phosphokinase values two hours after mesenteric arterial occlusion when compared with the control group (p less than 0.005), UK 38485 did not induce any change. In guinea pigs, when given together, ZK 36374 and UK 38485 lowered the enzyme levels to preligation values and the difference was nonsignificant (p greater than 0.1). The histopathologic investigation of the small intestine after giving ZK 36374 and UK 38345 together revealed minimal changes. These findings stress the importance of preserving the PGI2 levels in the PGI2/TXA2 ratio in preventing the increase of lysosomal enzyme levels and histopathologic changes after mesenteric arterial occlusion in guinea pigs. PMID- 1704146 TI - Comparative effect of distal and proximal intestinal resection and bypass on the rat exocrine pancreas. AB - We studied the effects of small-bowel resection and bypass on pancreatic function in rats subjected to a 50% distal resection (DR), a 50% proximal resection (PR), a 50% jejunal bypass (BP) or an intestinal transection (SH) (controls). Duodenal contents were collected after cannulation (under basal conditions). Afterwards, an in vivo duodenal perfusion was made using a glucose/saline solution and perfusate was collected for 1 h. Following this, a cholecystokinin (CCK) solution was injected into the jugular vein (1 U/kg body wt.) and perfusion continued for another 1 h. Basal duodenal volume only increased in rats with a PR, and no significant changes occurred in protein content. In basal conditions, no decreases in amylase, lipase, trypsin, or chymotrypsin activities after DR, PR or BP were detected. When animals were subjected to a perfusion and CCK stimulation, no significant changes occurred in animals with BP; the volume was maintained in rats with PR and DR but a decrease in protein and enzymatic contents was found. We concluded that, in basal conditions, the lack (resections) or exclusion (BP) of 50% of the small bowel does not negatively affect the digestive function. When however, a sustained activity is required, the extirpation of intestinal surface provokes a fall in enzymatic activities and is not modified if only the intestinal transit is suppressed, as occurs in the cases of BP. PMID- 1704142 TI - A prostacyclin analogue, iloprost, augments the ovulatory response of the LH stimulated in vitro perfused rat ovary. AB - Ovaries from immature rats, primed with pregnant mare's serum gonadotropin (PMSG; 20 IU, on day 28), were perfused in vitro in a recirculating system for 21 h from the morning of day 30 of age. Stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) in vitro at 0 h of perfusion resulted in 2.4 +/- 0.75 (mean +/- SEM) ovulations per treated ovary, whereas no ovulations occurred in the unstimulated group. When the addition of LH was supplemented hourly for 10 h with a stable prostacyclin analogue, Iloprost, at concentrations of 0.01 microM or 0.1 microM, the ovulation rate increased significantly (p less than 0.05) to 6.6 +/- 1.3 and 10.2 +/- 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 microM) did not cause any follicular ruptures when added by itself at every hour up to 10 h. The addition of Iloprost did not affect the release of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone or estradiol from unstimulated or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positive modulatory role of prostacyclin in the LH-induced ovulatory process for the rat. PMID- 1704143 TI - Insulinlike growth factor-binding proteins. PMID- 1704144 TI - The plasmid-encoded arsenical resistance pump: an anion-translocating ATPase. AB - An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyses extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance. PMID- 1704145 TI - Methylcholanthrene-induced murine fibrosarcoma cell line BMT-11 secretes granulocyte colony-stimulating factor. AB - Murine fibrosarcoma cell line BMT-11 was induced with 3-methylcholanthrene and maintained in culture. Transplantation of BMT-11 into syngeneic C57BL/6 mice produced leukocytosis consisting of marked increments of neutrophils and monocytes associated with massive splenomegaly. In order to elucidate the mechanisms of this leukemoid reaction, we studied the changes occurring in hematopoietic progenitor cells in BMT-11-transplanted mice. The numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid colony-forming units (CFU-E), erythroid burst-forming units (BFU-E), and mixed colony-forming units (CFU-Mix) in the spleen showed dramatic 216-fold, 18-fold, 64-fold, and 80 fold increases, respectively, relative to the value in the control mice 5 weeks after the BMT-11 implantation. In contrast, the levels of progenitor cells in the bone marrow remained within normal limits. The nature of the colony-stimulating factor (CSF) secreted from BMT-11 tumor cells was also studied. BMT-11 conditioned medium (BMT-11-CM), BMT-11 tumor extract, and sera from the mice bearing transplanted BMT-11 tumor contained CSF that stimulated mainly granulocyte and macrophage lineages. Furthermore, the expression of the granulocyte colony-stimulating factor (G-CSF) gene in BMT-11 cells were detected by Northern blot analysis. PMID- 1704148 TI - [Kawasaki disease]. AB - First described in Japan in 1967, Kawasaki disease is now observed in all countries. Despite extensive research, the aetiology of this infectious disease which affects mainly infants and young children remains mysterious. The six main clinical presentations of Kawasaki disease are: prolonged fever, conjunctivitis, lesions of the oral and pharyngeal mucosa, inflammatory reddening and swelling of the hands and feet, skin rash and cervical lymph node enlargement. Laboratory data show an inflammatory syndrome and thrombocytosis. The disease usually resolves within a few weeks. The major risk is cardiovascular involvement with pericarditis, myocarditis and, chiefly, coronary artery aneurysms which usually regress but are the main cause of mortality (1 to 2 p. 100 of the cases). Treatment consists of gammaglobulins and aspirin in high doses; when administered at an early stage, it prevents the formation of coronary aneurysms. PMID- 1704147 TI - [Treatment of extrauterine pregnancy with RU 486. A case report]. AB - At a time when the medical treatment of extrauterine pregnancy is developing, the authors report on one case which regressed after the administration of a single 3 x 200 mg dose of RU 486. The discussion touches upon the methods of administration, the indication, and the progressive aspects which open up the field to further studies. PMID- 1704149 TI - A spinal transcommissural connection for symmetrical sympathetic reflex response. Intra-axonal tracing study in the rat. AB - Wheatgerm agglutinin conjugated horseradish peroxidase was injected into the ganglion cervicale superior of the sympathetic trunk of seven adult rats. Labelled neurons were found in the ipsilateral anterior commissural nucleus of the spinal cord at the C1 level. Transcommissural crossing of labelled fibres and symmetrical labelling of neurons in the contralateral nucleus were also found. Labelled fibres could then be followed contralaterally into the superior cervical ganglion where labelled neurons were also found. PMID- 1704152 TI - Langerhans' cell histiocytosis pathobiology and pathogenesis. PMID- 1704151 TI - Generation of cAMP-activated chloride currents by expression of CFTR. AB - Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3',5'-monophosphate (cAMP) increased anion permeability and chloride currents in cells expressing CFTR, but not in cells expressing a mutant CFTR (delta F508) or in nontransfected cells. The simplest interpretation of these observations is that CFTR is itself a cAMP-activated chloride channel. The alternative interpretation, that CFTR directly or indirectly regulates chloride channels, requires that these cells have endogenous cryptic, chloride channels that are stimulated by cAMP only in the presence of CFTR. PMID- 1704150 TI - Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence. AB - Saimiri monkeys immunized with a recombinant protein containing 20 copies of the nine amino acid repeat of the Plasmodium vivax circumsporozoite (CS) protein developed high concentrations of antibodies to the repeat sequence and to sporozoites, but were not protected against challenge. After intravenous injection of an immunoglobulin G3 monoclonal antibody (NVS3) against irradiated P. vivax sporozoites, four of six monkeys were protected against sporozoite induced malaria, and the remaining two animals took significantly longer to become parasitemic. Epitope mapping demonstrated that NVS3 recognizes only four (AGDR) of the nine amino acids within the repeat region of the P. vivax CS protein. The monkeys immunized with (DRAADGQPAG)20 did not produce antibodies to the protective epitope AGDR. Thus, determination of the fine specificity of protective immune responses may be critical to the construction of successful subunit vaccines. PMID- 1704154 TI - [Acute phase proteins as diagnostic aid in veterinary medicine]. AB - The acute phase response is the general nonspecific defence mechanism induced by noxious stimuli, which precedes the specific defence mechanism of the immune response. In addition to other changing physiological parameters, the concentration of a number of proteins changes significantly during the acute phase response. These acute phase proteins were studied in detail in human subjects. However, the knowledge of acute phase proteins in domestic animals, is limited. This knowledge is reviewed in the present paper. Attention is directed to C-reactive protein (CRP), serum amyloid-A (SAA) and haptoglobin (Hp). These three proteins promise to be the most useful ones in the routine diagnosis of diseases of animals, which is discussed at the end of the paper. PMID- 1704153 TI - Malignant pleural effusion. AB - Malignancy is the most common cause of exudative pleural effusion in patients over the age of 60 years. Control of the effusion significantly reduces morbidity and improves quality of life. Tube thoracostomy with subsequent chemical pleurodesis is the treatment of choice for patients with tumors that are relatively insensitive to systemic chemotherapy. The agents most commonly used for chemical pleurodesis are tetracycline and bleomycin. A 13-center randomized trial compared tetracycline 1 g and bleomycin 60 U. Median time to recurrence or progression of the malignant effusion was 32 days for tetracycline and more than 46 days for bleomycin (P = .037). The recurrence rate within 30 days of instillation was 36% for bleomycin (10 of 28 patients) and 67% (18 of 27 patients) for tetracycline (P = .023). At 90 days, the recurrence rate was 30% (11 of 37) for bleomycin, and 53% (19 of 36) for tetracycline (P = .047). From this study, the authors concluded that intrapleural bleomycin appears superior to tetracycline for controlling malignant pleural effusions. Selected patients who fail tube thoracostomy and chemical pleurodesis should be considered for pleuroperitoneal shunting or pleurectomy. PMID- 1704156 TI - Ultrastructural analysis of apoptosis induced by the monoclonal antibody anti-APO 1 on a lymphoblastoid B cell line. AB - Apoptosis (programmed cell death) is the most common form of death in eukaryotic cells. We recently described a monoclonal antibody, anti-APO-1, which induces apoptosis of cells from the human B-lymphoblastoid line SKW 6.4 and of cells from a variety of other human lymphoid cell lines. This model of apoptosis was now studied ultrastructurally. SKW 6.4 cells undergoing apoptosis showed the following morphological changes: condensation of the cytoplasm and karyoplasm, formation of large electron-opaque aggregates of the chromatin lining the nuclear membrane, "blebbing" of the cell membrane at an early stage of apoptosis, and dilatation of the mitochondria. Two hours after adding anti-APO-1, the nuclear membrane was ruptured. Occasionally, large vesicular enlargement of endoplasmic reticulum or Golgi appeared in the cytoplasm. Finally, total breakdown of all cell membranes and cellular disintegration was observed. PMID- 1704155 TI - Carbaryl-induced changes in indoleamine synthesis in the pineal gland and its effects on nighttime serum melatonin concentrations. AB - The effects of different doses of chronically administered carbaryl on rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during darkness (2300 h and 0100 h) when pineal melatonin synthesis is high were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Carbaryl was administered at total doses (over 6 days) of either 50, 125 or 250 mg/kg by gastric gavage. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light/dark cycles of 14:10 with lights off at 2100 h. Pineal NAT and HIOMT activities and pineal melatonin were increased at 0100 h following carbaryl administration at all three doses. Conversely, serum melatonin was increased at 2300 h after the 250 mg/kg dose of carbaryl while all three doses of the pesticide reduced serum melatonin levels at 0100 h. Pineal 5-HTP, 5-HT and 5-HIAA levels were usually increased at 2300 h but unaffected at 0100 h. The results indicate that carbaryl has significant effects on pineal melatonin synthesis and secretion. PMID- 1704157 TI - Rapid contrasting of extracellular elements in thin sections. AB - Standard methods for contrasting ultrathin sections generally have their greatest effect on cells and cellular components, whereas extracellular elements remain relatively electron-lucent. Occasionally, some extracellular elements even fail completely to react with the staining solutions. We describe a method for rendering a uniformly high contrast to extracellular tissue components. This consists of a brief prestaining of grids with diluted tannic acid in distilled water. Simple, rapid, and versatile, this procedure can be routinely applied to all tissue samples examined by electron microscopy. As an additional advantage, the method greatly enhances the electron density of intracellular glycogen. Higher concentrations of tannic acid give increased electron density, especially to elastin, and can therefore be used as an elastin stain. PMID- 1704158 TI - Histological analysis of ultrasonic images of the prostate: an accurate technique. AB - Ultrasonography of the prostate furnishes images which still cannot be fully interpreted morphologically. In a cadaver study, the ultrasound images of two groups (21 and 19) prostates, obtained in a water bath, were compared with histology slides taken at corresponding levels. In the first part of the study, using a 4 MHz probe, there was a correlation between hyperechoic lesions and stone formations in 9 out of 15 cases. A relation between hypoechoic lesions and the existence of a carcinoma could also be established in 4 out of 12 cases. In the second part of the study, using a 7 MHz probe, there was a correlation between hyperechoic lesions and stone formations in all cases. Hypoechoic lesions correlated with the presence of a carcinoma in 1 out of 8 cases. The technique used appears to be well suited for the comparative study of ultrasound images and histology. Application of the 7 MHz probe is preferable as, because of a better resolution, smaller lesions can be detected. The results of this study are not very encouraging for the use of transrectal ultrasound for the detection of small prostatic carcinomas. PMID- 1704159 TI - Hemibody irradiation in advanced prostatic carcinoma. AB - In summary, hemibody irradiation has developed as a safe, efficient technique for palliating multiple sites of symptomatic osseous metastases, which occur so often in advanced prostatic carcinoma. The rapidity, frequency, and duration of pain relief, as well as the convenience to the patient of a solitary treatment to multiple symptomatic areas simultaneously, make this type of treatment especially appealing. By following premedication and radiation dose guidelines, both acute and delayed side effects can be kept tolerable or at a minimal incidence. Although sequential hemibody radiation has also been explored as "systemic" therapy, the results in prostatic carcinoma have not proved dramatic, and complications have been considerable. Hormonal therapy would certainly seem to be less life-threatening and equally beneficial according to present data. As a palliative treatment, however, hemibody irradiation is a pragmatic option for relieving prostatic cancer pain. PMID- 1704160 TI - Prostatic urethroplasty with balloon catheter as a nonsurgical alternative for prostatic hyperplasia. AB - Prostatic urethroplasty with balloon catheter is a new interventional uroradiological procedure that appears promising in treating benign prostatic hyperplasia in selected patients. As with many of the interventional radiographic procedures, it reduces the cost of therapy, it is associated with very low morbidity in an outpatient setting, and it can be performed in almost any patient, regardless of their medical condition. The recovery period is minimal. The procedure is easy to perform, but certain guidelines have to be followed to avoid complications. The workup of these patients is relatively the same as a patient undergoing surgical intervention. The patient needs to be thoroughly assessed to exclude the possibility of prostatic malignancy. PMID- 1704162 TI - [Use of contrykal in the prevention of recurrences and metastases of cancer of the upper respiratory tract]. AB - The analysis of data on 96 patients with cancer of the upper respiratory tract showed contrical to improve results of radiotherapy in terms of frequency of recurrence and dissemination. The regimen of administration of contrical is given. PMID- 1704164 TI - [The immunotherapy of type-1 diabetes mellitus--experiences, results and prospects]. PMID- 1704161 TI - Effect of LH-RH analogue in patients with benign prostatic hyperplasia. AB - Twelve prostatic hyperplasia patients with total urine retention and, consequently, a permanent catheter, were treated for six months with luteinizing hormone-releasing hormone (LH-RH) analogue having long-term effect. Seven of the 12 patients no longer needed a permanent catheter after the six-month treatment period. Transurethral resection of the prostate was performed for 3 patients. The remaining 2 patients continued to have a catheter. The mean prostatic volume, which was 83 g at the start of the study, fell by 51 percent, to 41 g. Treatment with LH-RH proved to be safe even for patients in poor physical condition, and it also seemed to improve symptoms of prostatic hyperplasia. PMID- 1704163 TI - [Effects of cytostatic agents on proliferative activity and growth of human tumor heterografts in subrenal capsule in mice]. AB - The initial and final proliferative activity (PA) of heterografts of human lung and breast tumors were assayed in cultures grown under the renal capsule in mice with and without cytotoxic drugs. The PA values in the control group proved similar. Chemotherapy-induced changes in PA did not correlate with those in heterograft growth rate. PMID- 1704165 TI - [Euthanasia--limits of therapy in oncology]. AB - Nowadays progress in medicine keeps human beings alive more frequently in hopeless and depressing situations associated with physical discomfort and pain for a long time. This situation, which is at least partly caused by medical treatment, should be generally reviewed, in particular with respect to the treatment of cancer patients. This principles of action or non-action should be re-examined at intervals retrospectively and continuously adapted to new developments. PMID- 1704166 TI - [Percutaneous transvascular embolization as a palliative measure in bleeding gynecological malignancies of the pelvis]. AB - Between 1981 and 1983 48 patients with an average age of 54 years have had an arterial embolization of internal iliac artery. In 7 patients this was done preoperatively within a clinical study, in 41 ones because of profuse vaginal bleedings. In 6 patients there was a vital indication. In all cases survival time was 152 days. In two cases a re-embolization was necessary. In these two patients survival time was on average 338 days. The manoeuvre was done with particles of gelatin of different size suspended in contrast medium. One complication could be observed, but was not caused directly by embolization. In cases of profuse or vital bleedings which conservatively cannot be stopped embolization is method of first choice with no contraindication. PMID- 1704167 TI - Antiarrhythmic treatment with flecainide (Tambocor). Clinical experience from 107 patients. AB - The long-term clinical effect of oral flecainide treatment was evaluated in 107 pts (10-82 yrs). Indications for treatment were: atrial fibrillation 38%, atrial flutter 16%, ventricular tachycardia 24%, ventricular ectopic beats 10% and supraventricular tachycardia 12%. Daily flecainide dosage was 200 (100-400) mg. Follow-up period 3 mths (15 days-15 mths). Based on the history and ECG flecainide had been effective in 51 pts. The improvement was most pronounced in pts suffering from supraventricular tachycardia involving an accessory bypass tract (84-92%). Flecainide had been discontinued in 50 pts due to: insufficient effect in 28, side effects in 17 and for other reasons in 5. The side effects indicating flecainide withdrawal (pts) were: cerebral symptoms (4), gastrointestinal complaints (2), bradyarrhythmias (2), heart failure (3) and suspected pro-arrhythmia (4). (Ventricular tachycardia in 3, 1:1 AV-conducting during atrial flutter in 1). PMID- 1704168 TI - Propafenone versus quinidine slow-release for the treatment of chronic ventricular arrhythmias. AB - The efficacy and side-effects of oral propafenone 300 mg b.i.d. were compared to those of quinidine slow-release 800 mg b.i.d. in a randomized double-blind placebo controlled cross-over study in 12 patients with symptomatic premature ventricular complexes (PVCs). Furthermore during steady-state the plasma levels of propafenone and quinidine were measured repeatedly over an 8-hour period and correlated to the numbers of PVCs. In 6 patients both drugs reduced PVCs by 80%. In 2 patients this effect was obtained by propafenone and not by quinidine, while the reverse was found in another 2 patients. In 2 patients neither of the drugs was able to reduce PVCs by 80%. During treatment with quinidine 4 patients experienced diarrhoea and 1 patient suffered headaches taking propafenone. The plasma levels showed great variation. No correlation between the plasma levels expressed as area under the concentration-time curve and the reduction of PVCs was found. PMID- 1704169 TI - Long-term storage effects on canine osteochondral allografts. AB - We have studied long-term (to 60 days) effects of 4 degrees C storage in culture media on the histologic, mechanical, and chemical properties of the cartilage from osteochondral shell allografts from the dog. The structural integrity of the cartilage matrix was intact up to 60 days of storage, for the mechanical properties represented by the aggregate modulus and apparent permeability remained normal. These data are supported by normal safranin-O staining as well as normal glycosaminoglycan content and total collagen concentration. However, chondrocyte viability, as assessed by 35SO4 uptake and hematoxylin and eosin preparations, decreased dramatically with time. We believe that the longer storage to 60 days is not indicated, unless conditions can be modified to maintain cell viability. PMID- 1704170 TI - [Critical analysis of the new TNM staging (UICC, 1987) of cancer of the cervical esophagus in relation to therapeutic decisions]. AB - 66 consecutive patients with a tumor confined to the cervical esophagus underwent surgical resection. The comparison between clinical and pathological TNM stage showed a clinical understaging in 30 patients. 25 of the 56 patients who had undergone curative resection had lymph node metastases: positive mediastinal and abdominal nodes were found in 8 (32%) and 0 cases, respectively. The mean survival after curative resection of the 10 evaluable patients with metastatic periesophageal, recurrent and/or paratracheal nodes was 22.4 months; of the 6 evaluable patients with positive mediastinal nodes it was 10.3 months; and of the 5 patients with positive deep latero-cervical nodes it was 5.8 months. The 2-year actuarial survival after curative resection (in the 53 operative survivors) was as follows (according to pathologic TNM staging): Stage I (n = 3) 100%, Stage IIA (n = 17) 30%, Stage IIB (n = 3) 33%, and Stage III (n = 30) 22%. The exact location of neoplastic recurrence after curative resection was documented in 13 cases; it was in the neck in 8 cases (61%); both neck and at a distance in 3 cases (23%) and only at a distance in 2 (16%). The clinical TNM staging of cervical esophageal cancer was not in agreement with the pathological findings in nearly 50% of the cases and is, therefore, inaccurate and unreliable both for therapeutic decision-making and for prognostic evaluations. Endoscopic ultrasound, which was not used in most of the patients studied here, may improve the accuracy of clinical TNM staging. The N classification, which defines only the cervical nodes as regional nodes, appears to be arbitrary since the pathological staging showed metastatic mediastinal nodes in 32% of the N + cases, with a survival comparable to that of patients with metastatic nodes only in the neck. The prognostic value of pathological TNM staging was not confirmed in the present study since only Stage I patients had a significantly better prognosis than patients in the other stages. This may be due to the small number of patients considered or to lymph node understaging caused by the fact that most patients did not undergo mediastinal lymphadenectomy through a thoracotomy or a sternum splitting. PMID- 1704171 TI - Effect of estradiol on RNA synthesis and nuclear protein phosphorylation in rat liver. AB - Experiments were made to study the effect of 17, beta-estradiol on the RNA synthesis and nuclear protein phosphorylation in the liver of sexually mature female albino rats: sham-operated, ovariectomized and hormone-treated after the ovariectomy (20 micrograms estradioldipropionate/100 g body mass). The rate of RNA synthesis is determined in vivo by the 3H-uridine incorporation in the acid insoluble fraction, whereas the nuclear protein phosphorylation is determined in vitro by the incorporation of gamma-32 P-ATP in isolated cell nuclei. Increased RNA synthesis is found to occur under the effect of the hormone, which corresponds to the hormone's effect on the uterine cells. The hormone stimulates the nuclear protein phosphorylation--an effect which could be associated with the gene expression. PMID- 1704172 TI - Studies on brain monoamine and neuropeptide systems after neonatal intracerebroventricular 6-hydroxydopamine treatment. AB - In order to study the effects of a neonatal dopamine lesion on dopaminergic, serotonergic and peptidergic systems, Sprague-Dawley rats were treated by intracerebroventricular administration of 6-hydroxydopamine (100 micrograms, days 3 and 6) following desipramine pretreatment (25 mg/kg s.c.). At 60-70 days postnatally a profound reduction of dopamine- and 3,4-dihydroxyphenylacetic acid levels was found in striatal and limbic forebrain regions concomitant with an extensive loss of tyrosine hydroxylase-immunoreactive fibers, while no significant alteration in noradrenaline levels was seen. A marked loss of tyrosine hydroxylase-immunoreactive cell profiles was also observed in the substantia nigra and ventral tegmental area in mesencephalon. In striatum, but not in other regions analysed, an almost 100% increase in serotonin levels and serotonin-immunoreactive fiber density was observed following 6-hydroxydopamine treatment. However, the number of serotonin-immunoreactive cell profiles in the median and dorsal raphe nuclei was not altered. The 6-hydroxydopamine treatment also led to reductions in substance P levels in striatum, nucleus accumbens and ventral mesencephalon. The cholecystokinin level in nucleus accumbens and neurotensin level in ventral mesencephalon were also reduced. A neonatal intracerebroventricular 6-hydroxydopamine treatment thus leads to a lesion of dopamine neurons in the mesencephalon with extensive loss of dopamine fibers in several forebrain areas, while localized serotonin fiber sprouting is induced in striatum. Furthermore, concomitant reductions of the levels of peptides related to the dopamine system occur following the 6-hydroxydopamine treatment. Behavioral disturbances such as hyperactivity and cognitive deficiencies occurring after a dopamine lesion early in life might therefore be due to plastic alterations in several different transmitter/neuromodulator systems as a direct or indirect consequence of the lesion. PMID- 1704173 TI - Cellular responses to mechanical loading in vitro. AB - A technique has been established in which cancellous bone biopsies may be simultaneously perfused and subjected to mechanical load bearing. Assessments of cell viability over a period of 24 h were based on the cAMP response to parathyroid hormone, intracellular lactate dehydrogenase activity, and electron micrograph morphology. Two cellular responses to mechanical loading were demonstrated similar to those that follow "osteogenic" loading in vivo, as reported previously. These were (1) a rise in intracellular G6PD in lining cells immediately after loading, and (2) an increase in RNA synthesis measured in osteocytes 6 h after loading. In vivo the osteogenic response to loading was modulated by indomethacin. In these in vitro experiments, addition of indomethacin inhibited both the loading-related G6PD and the RNA responses. PMID- 1704174 TI - Acute cerebral ischemia in rabbits: correlation between MR and histopathology. AB - The histologic description of cerebral ischemia is complex, and within most lesions there are regional variations in degrees of neuronal cell injury, edema, and neuropil disruption. These parameters of tissue injury were analyzed histopathologically in transient and permanent experimental cerebral ischemia in 15 rabbits and the results were spatially correlated with MR images of pre- and postmortem (formalin-fixed) brains. MR was performed at 1.5 T (eight animals) and at 0.38 T (seven animals). Areas of high signal on T2-weighted MR images were closely correlated with histologic signs of cytotoxic glial edema and with disruption of the neuropil (widening of the interstitial spaces in the background matrix of glial and neuronal cellular processes), but MR tended to underestimate the extent of ischemic neuronal injury, especially low-grade histologic changes (mild neuronal shrinkage and nuclear basophilia). Low-grade ischemic neuronal changes were often found in the penumbra zone of ischemic lesions in areas that appeared normal on T2-weighted MR. High-grade neuronal injury was also seen occasionally in areas of normal signal on MR, especially in the striatum. No significant differences were seen on T2-weighted MR between the experimental groups with respect to transient vs permanent occlusion, in vivo vs in vitro MR, and low vs high magnetic field. In the setting of suspected acute cerebral ischemia, an abnormal T2-weighted MR study often underestimates the extent of neuronal ischemic injury, especially potentially reversible injury; and a normal MR study does not completely exclude significant neuronal ischemic injury. PMID- 1704175 TI - Cytologic criteria to distinguish hepatocellular carcinoma from nonneoplastic liver. AB - The authors reviewed a series of fine-needle aspiration biopsy (FNAB) specimens of the liver to identify useful cytologic criteria to distinguish hepatocellular carcinoma (HCC) from nonneoplastic liver. Ten cytologic features were examined in this study: high cellularity, acinar pattern, trabecular pattern, hyperchromasia, pleomorphism, irregularly granular chromatin, uniformly prominent nucleoli, multiple nucleoli, increased nuclear/cytoplasmic ratio, and atypical naked hepatocytic nuclei. These features were examined in a series of 82 FNAB specimens from 52 patients with HCC and 30 patients with nonneoplastic lesions. With the use of a step-wise logistic regression analysis, three features were identified as predictive of HCC: increased nuclear/cytoplasmic ratio (P = 0.001), trabecular pattern (P = 0.002), and atypical naked hepatocytic nuclei (P = 0.03). When these three criteria were used, the sensitivity of diagnosing HCC by FNAB was 100%, and the specificity was 87%. PMID- 1704176 TI - The use of cytokeratin as a sensitive and reliable marker for trophoblastic tissue. AB - The use of human chorionic gonadotropin (hCG), human placental lactogen (hPL), and pregnancy-specific beta-1-glycoprotein (SP1) as markers for trophoblastic tissue is well documented in the literature. However, it is not widely recognized that cytokeratin is a very sensitive and reliable marker for all types of trophoblastic tissues. The authors have studied 100 cases of human placental tissue ranging in age from 2 to 40 weeks. Unlike hCG and hPL, which stain only the syncytiotrophoblast and intermediate trophoblast, cytokeratin (AE1/AE3) stains all three types of trophoblastic tissue. The staining of placental tissue for cytokeratin is strong and very consistent throughout pregnancy. Because of its high sensitivity and ability to stain cytotrophoblast, it is believed that it could be very useful in the study of the pathologic process of implantation sites, especially in tissue obtained from patients who present with missed and habitual abortions. PMID- 1704177 TI - Ossifying fibromyxoid tumor of soft parts. Additional observations of a distinctive soft tissue tumor. AB - The author studied four subcutaneous soft tissue tumors, similar to those recently described by Enzinger and associates (Am J Surg Pathol 1989;13:817) by the name "ossifying fibromyxoid tumor," by immunohistochemistry and electron microscopy to further understand the cellular nature of this lesion. The four tumors were composed of uniform round cells often surrounded by a lacunar space. The tumors often contained a peripheral zone of metaplastic bone. The cellularity was high, but the mitotic rate was low, suggesting a benign or borderline nature of the lesion. Longer follow-up was available for three cases, showing recurrence free survival times of 11, 8, and 3 years. Immunohistochemistry studies revealed that all tumors were strongly positive for S-100 protein and focally positive for Leu-7, whereas melanoma-specific marker HMB45 was negative. Vimentin was the main type of intermediate filament protein, and one case also contained scattered glial fibrillary acidic protein-positive cells. Epithelial markers (keratins, epithelial membrane antigen), desmin, and muscle actins were negative. Electron microscopic examination showed partial, sometimes reduplicated, basal lamina surrounding many cells. Complex cell processes were also present. No myofilaments were found. The immunohistochemical and electron microscopic results may suggest that this tumor has Schwann's cell differentiation. PMID- 1704178 TI - Glycol methacrylate embedding in diagnostic pathology. A standardized method for processing and embedding human tissue biopsy specimens. AB - A standardized "low-cost" processing and embedding procedure is described for the preparation of semithin glycol methacrylate (GMA) sections of human tissue biopsy specimens for use in routine diagnostic pathology. The method is highly reliable and reproducible and is based on many years of experience in fundamental GMA research. The procedure combines the application of new, less toxic, plasticizers and the preparation of high-quality GMA mixtures. Microwave irradiation was used to improve staining qualities and to reduce staining times. An improved reticulin staining procedure is presented as well. PMID- 1704179 TI - Primary hepatic carcinoid tumor. An electron microscopic and immunohistochemical study. AB - A case of primary carcinoid tumor of the liver with striking morphologic and electron microscopic features is reported. Conventional histologic examination showed a prominent paranuclear clear zone in numerous tumor cells. By electron microscopic examination, this clear zone corresponded to a paranuclear mass of intermediate filaments admixed with neurosecretory granules and other cytoplasmic organelles. PMID- 1704180 TI - Antisperm antibody detection using concurrent cytofluorometry and indirect immunofluorescence microscopy. AB - Anti-sperm antibodies from serum and seminal plasma were detected by concurrent flow cytometry and epifluorescence microscopy using fluorescein-conjugated antihuman immunoglobulins. Experimental conditions were designed, taking advantage of several monoclonal antisperm antibodies, to test aspects of the assay before clinical application. Perturbation of membrane integrity altered both the localization of binding and the number of sperm cells positive for bound antibodies. In specimens from selected infertility patients, 21.6% of the females and 40.8% of the males had significant levels of antisperm antibodies. Differences in the incidence of isoimmunity between female partners of antibody positive or antibody-negative males and differences in the localization of antigens targeted by serum versus seminal plasma antibodies in men support the idea that, in some cases, immunity to sperm cells may be the result of altered sperm antigens. PMID- 1704181 TI - Fine structure of active and healed duodenal ulcer. AB - In order to characterize the fine structure of active and healed duodenal ulcers, we examined tissue specimens of patients with active duodenal ulcer disease (n = 30) before and after treatment with either antacids (n = 16) or H2-receptor antagonists (n = 14), by light microscopy and various electron microscopic techniques, e.g., scanning and transmission electron microscopy. The characteristic histological feature of both the active and healed duodenal ulcer was the appearance of periodic acid-Schiff (PAS)-positive epithelial cells at the edge of the ulcers. Electron microscopy revealed that these cells were similar to a special type of mucus-secreting cell in the antrum (surface mucous cell). Their mucus granules contained mainly neutral glycoproteins. Helicobacter pylori were found attached to these cells in tissue specimens from 12 of 30 patients (40%). The mucous structure destroyed during the ulcerative phase regained its normal net-like structure after treatment. The ultrastructural healing process of duodenal ulcer was characterized by the presence of gastric metaplasia, by stunted microvilli of the duodenal epithelium (p less than 0.001 vs. control group), and an increased number of lysosome-like bodies (p less than 0.001 vs. control group) of the epithelial cells. These results were independent of the type of treatment, and showed that the repair mechanisms were incomplete after a 4-wk period of treatment. PMID- 1704182 TI - Exocrine pancreatic function in chronic liver diseases. AB - To confirm the respective influence of chronic alcoholism and liver disease on exocrine pancreatic function in cholecystokinin secretin (CS), tests were performed on patients with chronic liver cirrhosis (LC) and non-cirrhotic (nLC) disease of alcoholic (A) and nonalcoholic (nA) etiology. Results were compared in four subgroups (ALC, N = 26; AnLC, N = 45; nALC, N = 18; and nAnLC, N = 43). Volume of duodenal juice and bicarbonate output (BO) were increased and maximal bicarbonate concentration was decreased in ALC, compared with those in normal controls. Comparison of LC and nLC indicated that the volume, BO, and amylase output (AO) were greater in LC than in nLC of alcoholic etiology, but not in those of nonalcoholic etiology. The initial disappearance rate (KICG) of indocyanine green (ICG) excretion correlated with a parameter of CS test in alcoholic liver disease (vs. volume: r = -0.51, p less than 0.01 vs BO: r = 0.40, p less than 0.01), but not in nonalcoholic liver disease. Concurrent chronic pancreatitis with pain and definite exocrine insufficiency was observed in only one ALC patient and in four AnLC patients, but in none of the nonalcoholics. In alcoholic liver disease, exocrine pancreatic secretion tends to increase with severity of liver damage, but concurrence of definite chronic pancreatitis is not correlated with the severity. PMID- 1704183 TI - Endodermal sinus tumor of the ovary during pregnancy: a case report. AB - Serum alpha-fetoprotein screening led to the detection of an endodermal sinus tumor of the ovary in a 24-year-old female in week 17 of pregnancy. After surgery, chemotherapy was postponed. In week 28 levels of serum alpha-fetoprotein increased, but delivery was delayed until 33 weeks' gestation. After delivery, the patient received four chemotherapy courses (cisplatin, etoposide, and bleomycin). Mother (24 months after last chemotherapy) and child are doing well. PMID- 1704184 TI - Analysis of antigen expression at multiple tumor sites in epithelial ovarian cancer. AB - The question of whether the antigenic phenotype of human epithelial ovarian cancer varies in a given patient between the primary tumor and metastatic sites or among metastatic sites themselves is an important issue in planning potential therapeutic strategies for ovarian cancer. We have obtained tumor specimens from at least two separate sites during operations on 12 patients with epithelial ovarian cancer, and we have typed these specimens with a group of 18 monoclonal antibodies that react with cell-surface glycoprotein and carbohydrate antigens, including blood group antigens. Antibodies with relative specificity for malignant cells as well as those that detect more widely distributed epithelial antigens were used. A total of 31 specimens from 12 patients with advanced adenocarcinoma (8 serous, 3 undifferentiated, 1 endometrioid) of the ovary were studied, including fresh ascites cells in two patients. Frozen sections of tumor specimens were stained with the antibodies by the indirect immunoperoxidase technique and graded semiquantitatively. Little difference was seen in antigenic expression of tumors that were obtained from various sites in the same patient for either the epithelial cell markers or blood group markers. Intratumoral antigenic heterogeneity was seen, but this was generally quite consistent within a given patient's specimens. As anticipated, variations in antigen expression were seen among specimens from different patients. The antigenic phenotype of the tumor specimens in a given patient, as determined immunohistochemically by our group of antibodies, showed only minor variation among primary and metastatic sites. PMID- 1704185 TI - Disk neovascularization in chronic anterior uveitis. PMID- 1704186 TI - Binding of tumor necrosis factor alpha to activated forms of human plasma alpha 2 macroglobulin. AB - We tested the hypothesis that human plasma alpha 2 macroglobulin (alpha 2M) is a latent binding glycoprotein for human tumor necrosis factor alpha (TNF-alpha). Human recombinant 125I-TNF-alpha was incubated for 2 hours (37 degrees C) with purified native alpha 2M and with alpha 2M that was modified by reaction with methylamine or various proteinases. 125I-TNF-alpha/alpha 2M complexes were detected by nondenaturing polyacrylamide gel electrophoresis after autoradiography or by liquid chromatography on Superose-6. 125I-TNF-alpha bound strongly but noncovalently to alpha 2M-plasmin and alpha 2M-methylamine. There was minimal binding of 125I-TNF-alpha to native alpha 2M, alpha 2M-trypsin, or alpha 2M-thrombin. A 10(6) molar excess of porcine heparin did not reduce the binding of 125I-TNF-alpha to alpha 2M-methylamine or alpha 2M-plasmin. alpha 2M plasmin or alpha 2M-methylamine added to human plasma or serum preferentially bound 125I-TNF-alpha in the presence of native alpha 2M. 125I-TNF-alpha also bound to 'fast' alpha-macroglobulins in methylamine-reacted human, rat, mouse, swine, equine, and bovine plasma. However, TNF-alpha, preincubated with either alpha 2M-plasmin or alpha 2M-methylamine, remained a potent necrogen for cultured L929 cells. Purified 125I-TNF-alpha/alpha 2M-plasmin complex injected intravenously in CD-1 mice rapidly cleared from the circulation, unless the alpha 2M-receptor pathway was blocked by coinjection of excess alpha 2M-trypsin. These findings demonstrate that alpha 2M is a latent plasmin-activated binding glycoprotein for TNF-alpha and that TNF-alpha/alpha 2M-plasmin complexes can be removed from the circulation by the alpha 2M-receptor pathway. This suggests that alpha 2M may be an important regulator of the activity and distribution of TNF alpha in vivo. PMID- 1704187 TI - Hybridohistochemical demonstration of alternative splicing of the CALC-I gene. AB - Precursor RNA is processed to mature mRNA by excision of the noncoding introns. This process is called splicing and usually is studied by in vitro transcription experiments and by Northern blotting. The pre-mRNA of the first calcitonin gene (CALC-I) can be spliced alternatively into two different mRNAs encoding either for calcitonin or for calcitonin gene-related peptide (alpha-CGRP). The expression of CALC-I was studied by in situ hybridization in the nervous system of the rat with calcitonin-specific and CGRP-specific probes. We found evidence for alternative splicing of the pre-mRNA of CALC-I in the motor neurons of the spinal cord. alpha-CGRP mRNA was found in the nuclei and the cytoplasm of the spinal motor neurons, whereas hybridization with a calcitonin-specific probe was located strictly in the nuclei. Because no calcitonin RNA and protein was detected in the cytoplasm of the cells, we concluded that the hybridization obtained in the nucleus was the pre-mRNA encoding for alpha-CGRP. PMID- 1704188 TI - Immunoreactivity of anti-streptococcal monoclonal antibodies to human heart valves. Evidence for multiple cross-reactive epitopes. AB - Association of group A streptococci with acute rheumatic fever and valvular heart disease is well established; however the basis of valve injury remains unclear. In this study, anti-streptococcal monoclonal antibodies (MAbs) cross-reactive with myocardium were reacted with sections from 22 rheumatic valves, nine normal, five endocarditic, one 'floppy,' and one Marfan valve. In immunohistochemical studies, MAb reactivity was observed with cardiac myocytes, smooth muscle cells, cell surface and cytoplasm of endothelial cells lining valves, and valvular interstitial cells. Endothelial basement membrane and elastin fibrils reacted with the MAbs, whereas collagen was unreactive. Similar reactivity was seen with sera from acute rheumatic fever patients. The anti-streptococcal MAbs reacted with intravalvular myosin and vimentin in Western blots, and purified elastin competitively inhibited the binding of the anti-streptococcal MAbs to whole group A streptococci. The data show that human heart valves have numerous sites of immunoreactivity with anti-streptococcal MAbs and acute rheumatic fever sera of potential importance in the pathogenesis of rheumatic valvular injury. PMID- 1704189 TI - Platelet-complement interactions in mesangial proliferative nephritis in the rat. AB - Complement has been reported to mediate mesangiolysis and glomerular hypercellularity in the rat in a model of glomerulonephritis (GN) induced with anti-Thy 1 antibody. To investigate the mechanism for the complement-mediated hypercellularity, the authors first determined if the effect of complement depletion was to inhibit cell proliferation or whether the effect was primarily to inhibit leukocyte infiltration. Rats depleted of complement with cobra venom factor (CVF) had 1) significantly less mesangiolysis than controls at day 5 (0.6 +/- 0.1 versus 3.4 +/- 0.4, scale 0-4+, P less than 0.001); 2) less cell proliferation, as assessed by immunostaining for the proliferating cell nuclear antigen (PCNA)/cyclin, a cell-cycle-dependent antigen (0.5 +/- 0.1 versus 2.4 +/- 0.7 cells/glomerular cross-section, P less than 0.01); and 3) less leukocyte infiltration as assessed by immunohistochemical labeling (0.6 +/- 0.1 versus 1.9 +/- 0.3 cells/glomerular cross-section, P less than 0.01). Because it was reported recently that platelets also mediate glomerular cell proliferation in this model, this study examined whether the mechanism for complement-mediated cell proliferation involved an effect on glomerular platelet localization. The glomerular uptake of 111In-labeled platelets was quantitated in normal and CVF treated rats at 1, 4, 12, and 24 hours after induction of GN. Rats with anti-Thy 1 GN had substantial glomerular accumulation of platelets at all times studied, peaking at 4 hours (608 +/- 171 platelets per glomerulus). Complement depletion profoundly reduced glomerular platelet localization in anti-Thy 1 GN (mean less than 35 platelets per glomerulus at all times studied, P less than 0.05). Thus these studies demonstrate an important role for complement in mediating platelet localization in anti-Thy 1 GN, an effect that may account for the complement dependent, neutrophil-independent glomerular hypercellularity in this model. PMID- 1704190 TI - Antibodies to non-beta regions of the beta-amyloid precursor protein detect a subset of senile plaques. AB - A central unresolved issue in Alzheimer's disease is the origin of the extracellular amyloid beta protein (A beta P) found in senile plaques and its relationship to the dystrophic neurites that intimately surround it. Here the presence and distribution within senile plaques of various epitopes of the beta amyloid precursor protein (APP) are compared with the distribution of A beta P itself and markers for plaque neurites. Several principal findings emerge: 1) antibodies to regions of APP outside of A beta P ('APP antibodies') recognize only a subgroup of senile plaques; 2) within these plaques, APP antibodies label discrete globular and granular structures morphologically resembling neurites; 3) virtually all of the plaques labeled by APP antibodies also contain neurites reactive with antibodies to tau; 4) double labeling with anti-tau and an APP antibody shows that the neuritelike profiles stained by the APP antibody are always closely associated with tau-positive neurites within the same plaque and that a minority of profiles appear to be labeled by both antibodies; and 5) antibodies to different regions throughout APP label the same profiles within plaques, suggesting the presence of the full-length precursor. The authors conclude that only a subgroup of senile plaques contain APP epitopes and that the immunostained structures are neurites. Because many A beta P-containing plaques in neocortex, cerebellum, and striatum were found to be devoid of any APP labeling, as were vascular A beta P deposits, it is unlikely that the extracellular A beta P is principally derived from the APP found within dystrophic neurites. The immunodetection of apparently full-length APP, an axonally transported protein, in selected plaque neurites provides yet another protein marker of neuritic dystrophy, possibly indicative of an aberrant regenerative response. PMID- 1704191 TI - Vascular and nonvascular expression of INCAM-110. A target for mononuclear leukocyte adhesion in normal and inflamed human tissues. AB - Inducible cell adhesion molecule 110 (INCAM-110), is a 110-kd adhesion receptor for lymphocytes and monocytes identified on cytokine-activated endothelium. Using immunoperoxidase techniques, little or no INCAM-110 was detected on endothelium in normal human tissues. In contrast, INCAM-110 was expressed in postcapillary venules in a variety of active inflammatory processes. In acute appendicitis, INCAM-110 was found coincident with strong expression of endothelial leukocyte adhesion molecule 1 (ELAM-1), a cytokine-inducible molecule that functions in neutrophil adhesion. However, in certain chronic inflammatory processes (eg, sarcoidosis), INCAM-110 was observed without simultaneous ELAM-1 expression. Anti INCAM-110 antibody E1/6 also marked several extravascular cell types, including lymphoid dendritic cells, some tissue macrophages, synovial lining cells, and reactive mesothelial cells. These data suggest a role for endothelial INCAM-110 in the pathophysiology of both acute and chronic inflammatory reactions. Furthermore INCAM-110 may function as an adhesion molecule for mononuclear leukocytes in a variety of extravascular sites. PMID- 1704192 TI - Enhanced expression of neural cell adhesion molecules and tenascin (cytotactin) during wound healing. AB - Both neural cell adhesion molecules (N-CAM) and tenascin (cytotactin) are important in embryonic morphogenesis but their expression is reduced greatly in adults. This study examined whether they are induced during wound healing. The spatial and temporal expression patterns of these two and other adhesion molecules in the healing of skin, cartilage, and tendon were compared. Neural cell adhesion molecules, tenascin, and fibronectin are induced in the granulation tissue but the order of prevalence is fibronectin, tenascin, N-CAM. The order of appearance is N-CAM and fibronectin, and then tenascin. The order of disappearance is N-CAM, tenascin, fibronectin. Liver cell adhesion molecules are present in the epidermis undergoing re-epithelization. Explant cultures showed that N-CAM and tenascin are synthesized by wound fibroblasts. These results suggest that N-CAM and tenascin, widely used in embryonic morphogenesis, are induced in a variety of connective tissues during wound healing. PMID- 1704193 TI - Early cellular events in evolving cutaneous delayed hypersensitivity in humans. AB - The delayed-type hypersensitivity reaction (DHR) in human skin is prototypic for many inflammatory dermatoses. However the cellular events that precede gross lesion formation are unknown. In this study, inflammatory cell populations and adhesion molecule expression in early phases of DHR elicited by 2,4 dinitrochlorobenzene were evaluated. The first discernible event (at 1 hour) was mast cell degranulation, followed by induction of endothelial leukocyte adhesion molecule (ELAM-1) expression on dermal postcapillary venules at 2 hours. Endothelial leukocyte adhesion molecule expression peaked at 24 hours and declined by 48 hours. In contrast, endothelial expression of intercellular adhesion molecule-1 (ICAM-1) remained at constitutive levels. Intrafollicular T cell migration occurred independent of ICAM-1 expression and commenced as early as 4 hours after challenge. Mature, activated CD4-positive lymphocytes that expressed a helper-inducer/memory phenotype predominated in early lesions. These results demonstrate in vivo that mast cell degranulation, ELAM-1 expression, and memory T-cell-follicular interactions are key events in subclinical evolutionary stages of cutaneous DHR. PMID- 1704194 TI - Keratin subsets in spindle cell sarcomas. Keratins are widespread but synovial sarcoma contains a distinctive keratin polypeptide pattern and desmoplakins. AB - The presence of individual keratin polypeptides and desmoplakins was immunohistochemically studied in 25 spindle cell sarcomas of different types using acetone-fixed frozen sections. Results revealed that keratins 8 and 18 were present in a high number of tumors: 9 of 9 synovial sarcomas, 5 of 7 leiomyosarcomas, 5 of 5 malignant schwannomas, and 1 of 4 undifferentiated spindle cell sarcomas. In addition to keratins 8 and 18, the glandular component of synovial sarcoma showed prominent reactivity with antibodies to keratins 7 and 19. Also the glandular epithelial cells in synovial sarcoma showed desmoplakin immunoreactivity preferentially in a luminal distribution, but desmoplakin was absent in other spindle cell sarcomas. Furthermore keratin 13 was seen focally in 4 of 9 synovial sarcomas. In contrast, keratins 7, 13, and 19 were practically absent in leiomyosarcomas, malignant schwannomas, and undifferentiated spindle cell sarcomas. The widespread presence of keratins 8 and 18 in various spindle cell sarcomas may reflect aberrant keratin expression in mesenchymal cells, previously described in cultured transformed fibroblasts. The presence of keratins 7 and 19 and desmoplakin is highly associated with morphologically observable epithelial differentiation restricted to synovial sarcoma among spindle cell sarcomas. PMID- 1704195 TI - Acetylcholine-induced vasodilatation in rabbit hindlimb in vivo is not inhibited by analogues of L-arginine. AB - Nitric oxide (NO) or related nitroso compounds are an endothelium-derived relaxing factor (EDRF), originating from metabolism of L-arginine, L-Arginine analogues with chemically altered guanidino moity are potent and specific inhibitors of EDRF(NO) release. We evaluated effects of two L-arginine analogues, NG-monomethyl-L-arginine (L-NMMA, 100 microM) and N omega-nitro-L-arginine (L NARG, 30 microM), on acetylcholine-, substance P-, and nitroglycerin-induced relaxation in the blood-perfused rabbit hindlimb in vivo and femoral arteries in vitro. L-NMMA and L-NARG selectively inhibited the vasodilator response to acetylcholine in rabbit femoral arteries in vitro, whereas endothelium independent response to nitroprusside increased. L-NMMA (1.6 mg/min ia) in the blood-perfused rabbit hindlimb in vivo increased vascular resistance in the hindlimb by 23 +/- 3% (means +/- SE; n = 10) but did not inhibit the vasodilator responses to acetylcholine or substance P. L-NARG (10 mg/kg iv) increased systemic blood pressure by 26 +/- 3% (n = 7) and vascular hindlimb resistance by 22 +/- 9% (n = 8), and blood flow to hindlimb musculature, measured with microspheres, decreased by 46 +/- 5% (n = 6). Pretreatment with L-NARG, however, did not impair vasodilator responses to acetylcholine and substance P. These findings are consistent with the view that basal tone in resistance vessels in the rabbit hindlimb may be mediated by nitroso compounds, whereas agonist stimulated vasodilation may be mediated by other mechanisms that do not involve the NO-synthesizing enzyme. PMID- 1704197 TI - Inhibitory renorenal reflexes: a role for substance P or other capsaicin sensitive neurons. AB - In anesthetized rats, we examined whether inhibitory renorenal reflex responses to renal pelvic mechanoreceptor (MR) and chemoreceptor (CR) stimulation were mediated by substance P (SP)-containing neurons. Capsaicin (0.5 ng to 5 micrograms) injected into the renal pelvis increased afferent renal nerve activity (ARNA) dose dependently, from 60 +/- 19 to 333 +/- 105%. For a given ARNA response, a 100-fold higher dose was required when capsaicin was injected into the renal interstitium compared with the renal pelvis. Renal pelvic administration of SP (25 ng) increased ipsilateral ARNA by 126 +/- 34% and contralateral urine flow rate and urinary sodium excretion by 21 +/- 4 and 28 +/- 7%, respectively, a response similar to that produced by renal MR and CR stimulation. Mean arterial pressure was unaffected. Ipsilateral renal denervation abolished the contralateral diuresis and natriuresis produced by SP. In rats treated with capsaicin (950 mg/kg subcutaneously over 1 wk) to deplete sensory neurons of SP, renal MR and CR stimulation failed to elicit a renorenal reflex response. The data suggest that the renorenal reflex responses to renal MR and CR stimulation are mediated at least, in part, by SP neurons or other sensory neurons susceptible to depletion by capsaicin. PMID- 1704196 TI - Chemoreception for an insoluble nonvolatile substance: starch taste? AB - Substances that are insoluble in both water and lipids, such as starch, are commonly assumed to be tasteless. Starch was suspended in water with a viscous gum. Rats given a choice of fluid containing starch and the same fluid without starch consistently preferred fluids containing starch. Rats were able to detect as little as 0.5% starch from several species of plants (corn, rice, wheat, and potato). In contrast, rats ignored comparable concentrations of cellulose suspended in water. Rats were also capable of choosing the fluid containing higher levels of starch when given a choice of 1 vs. 2% starch or 0.5 vs. 1% starch. This ability to detect starch did not appear to be mediated by salivary alpha-amylase because 1) raw starch is highly resistant to hydrolysis by salivary amylase, 2) starch preference was not correlated with the susceptibility of the starch to hydrolysis by salivary amylase, and 3) starch preference was not blocked by partial or extensive desalivation. Attempts to extract impurities with either organic solvents or water did not provide any evidence that such impurities contribute to starch preference. These experiments point to a seemingly novel form of chemoreception that could be useful to animals that need to identify starch-rich foods. PMID- 1704198 TI - Single-stranded RNA molecular weight and shape determination by differential pressure capillary viscometry, sedimentation velocity, and gel electrophoresis. AB - Determination of the size of a population of nucleic acids can be achieved by several distinct methods. Most of these methods are cumbersome and require complicated equipment or techniques. We demonstrate here the use of a differential pressure capillary viscometer for the rapid and simple determination of RNA molecular weight. This highly sensitive viscometer allowed single viscosity determinations on dilute solutions of RNA, providing a direct measure of the intrinsic viscosity without the need to extrapolate from several concentrations. The molecular weights and conformations of the linear single stranded RNA homopolymer poly(inosinic acid) (poly(I] and the single-stranded RNA (ssRNA) copolymer poly(cytidylic acid:uridylic acid, 12:1) (poly(C12,U], were determined. The ssRNAs were synthesized in a range of sizes (100 to 100,000 bases). They were widely polydisperse. The Mandelkern-Flory equation (1952, J. Chem. Phys. 20, 212-214), which requires both the intrinsic viscosity and sedimentation coefficient of a macromolecule, was used to calculate the molecular weights. The molecular weights determined by agarose gel electrophoresis were compared to those determined by intrinsic viscosity plus sedimentation coefficient. The correlation between the molecular weights determined by these two methods was good, at R2 greater than or equal to 0.92. The conformations of the RNAs were determined by application of the Mark-Houwink equation. The Mark Houwink exponents for poly(I) and poly(C12,U) intrinsic viscosities were 0.90 and 0.84, respectively. When compared to other nucleic acid polymers, for which the conformations have been established by several methods, poly(I) and poly(C12,U) are rigid, extended random coils, in a low-salt buffer (15 mM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704199 TI - A quantitative assessment for transcriptional pausing of DNA-dependent RNA polymerases in vitro. AB - A simple definition for pause strength (tau i) has been given, quantitatively describing transcriptional pausing of RNA polymerases. It permits derivation of practical assessments, based on single-round transcription reactions, which measure the average time a polymerase stops in vitro at certain sites during transcription elongation. We demonstrate that pause strengths can be determined with high accuracy when transcription elongation is started simultaneously from radioactively labeled and purified ternary complexes and transcripts are separated on sequencing gels. Our concept is exemplified by measuring pause strengths on supercoiled templates in the leader region of the Escherichia coli rrnB operon in the presence and the absence of the transcription factor NusA. PMID- 1704200 TI - Separation of interstitial and tubular cells of human testis by means of an anti substance P-antiserum. Androstane-3 beta, 17 beta-diol is exclusively formed by seminiferous tubules. AB - Leydig cells and seminiferous tubules of human testicular tissue were successfully separated by means of an anti-substance P-antiserum. On incubation with (14C)-testosterone or (14C)-dihydrotestosterone (DHT) it could be shown that besides DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) also 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) originated in the tubular compartment. The ratio 3 alpha/3 beta-diol was found to be higher (0.7) here compared to incubation with whole testicular tissue (0.2), indicating that the testicular interstitium influences the metabolism of DHT by the seminiferous tubules. PMID- 1704201 TI - [The clumsy child]. PMID- 1704202 TI - Laboratory investigation of cerebrospinal fluid proteins. AB - Analysis of CSF proteins is useful in the diagnosis and management of neurological diseases in the following situations: 1. In inflammatory conditions when there is breakdown of blood-CSF barrier integrity. Meningitis is a medical emergency, with CSF total protein measurement being only a screening test. 2. In the detection of immune responses within the CNS. This is by far the most important application in a routine clinical setting, as it is now a firmly established criterion in the diagnosis of multiple sclerosis. Oligoclonal bands restricted to the CSF are the only reliable indicators of intrathecal immunoglobulin G synthesis and are practically always associated with inflammatory disease of the CNS. The method fo choice for detecting oligoclonal bands is isoelectric focusing with immunofixation. Quantitative measurement of IgG in the CSF is of no value in diagnostic pathology. 3. In destructive brain diseases when brain-specific proteins are released into the CSF, measurement of these proteins can give prognostic information. PMID- 1704203 TI - Influence of inhaled cadmium on the immune response to influenza virus. AB - Cadmium may exacerbate pulmonary infections. In a previous study, however, cadmium appeared to enhance mouse resistance to influenza pneumonia. We report herein on the influence of cadmium intoxication in mice on different factors of anti-influenza immunity, e.g., antibody response, local production of interferon, pulmonary cellular response, and the interaction between pulmonary alveolar macrophages and the influenza virus. Cadmium inhalation did not affect production of antibodies or interferon. The protective effect appeared to be related to an enhanced supply to phagocytic cells into the lung. PMID- 1704204 TI - A comparison study of two methods of peanut agglutinin staining with S100 immunostaining in 29 cases of histiocytosis X. PMID- 1704205 TI - Parathyroid hormone secretion and target organ response in experimental acute pancreatitis. AB - To determine changes in parathyroid hormone secretion and target organ response caused by acute pancreatitis before the development of systemic toxic conditions, experimental acute pancreatitis was induced in rats with a choline-deficient, ethionine-supplemented diet. After 7 days, the rats were weighed and bled, and one kidney was assayed for 25-hydroxyvitamin D1 hydroxylase activity. Several manifestations of pancreatitis were observed in rats given the diet: weight loss (from 29.6 to 26.3 g vs that for control rats, from 29 to 52.8 g) and lower dietary intake (15.5 vs 47 g per rat per 7 days). Serum amylase levels fell from 1794 to 350 U/L in rats given the choline-deficient, ethionine-supplemented diet compared with levels of 1800 to 2100 U/L in control rats. The pancreases of rats given the choline-deficient, ethionine-supplemented diet showed degeneration, necrosis, and hemorrhaging. Serum levels of calcium, phosphorus, chloride, and parathyroid hormone did not change significantly throughout the experiment. Renal 25-hydroxyvitamin D1 hydroxylase activity was higher than in control rats (8.9 +/ 0.8 vs 7.6 +/- 0.6 fmol/mg of kidney per minute). Acute pancreatitis in this experimental animal model does not alter serum levels of calcium and parathyroid hormone or reduce target organ responsiveness to the hormone. PMID- 1704206 TI - [The immunohistochemical heterogeneity of mesangioproliferative glomerulonephritis]. AB - Immunohistochemical examination of puncture biopsies of the kidneys taken from 172 patients with mesangioproliferative glomerulonephritis (MPGN) showed its heterogeneity. MPGN with IgA deposits, MPGN with IgG deposits, MPGN with IgM deposits, MPGN with C3 deposits and immunonegative MPGN are separated. Clinically, nephrotic syndrome prevails in IgG and IgM, MPGN, various clinical forms occur in MPGN with C3 and immunonegative variants, hematuric and latent forms occur in MPGN with IgA. MPGN with IgM deposits has its own morphological and clinical features, this permitting to consider it as an independent form of glomerulonephritis. PMID- 1704207 TI - [Changes in the neural apparatus of the cerebral arteries in atherosclerosis]. AB - Nervous apparatus of the brain arteries (450-60 microns in diameter) in 55-64 year-old patients with cerebral atherosclerosis was studied. Alterations in the afferent innervation were manifested in the increased sinuosity of fibers, areas of hyper- and hypoimpregnation, appearance of thickenings in the fibers. Changes in the vasomotor innervation consisted of the disturbances of the nerve network structure, an increase of nerve fibers concentration and varicosity in the cholinergic plexuses and their decrease in the adrenergic ones. Restructurings of the nerve apparatus were more pronounced in the arteries of large caliber. PMID- 1704208 TI - [A poorly differentiated apudoma of the gallbladder]. AB - Apudoma was found in the gall bladder removed in a 76-year-old woman because of the chronic calculous cholecystitis exacerbation. Carcinoid syndrome was absent clinically. Histologically, the tumour was a poorly differentiated carcinoid with areas of small cell and polymorphic carcinoma. Argyrophilic Pasquale reaction in the tumour cells was negative, electron microscopically small neurosecretory granules were found. Numerous ACTH-reactive cells and single serotonin-reactive cells were revealed in the tumour parenchyma by means of immunohistochemical PAP method using antibodies against ACTH, serotonin, calcitonin, somatostatin, insulin, glucagon, P-substance. Focal hyperplasia and intestinal metaplasia of epithelium with the increase of the number of argyrophilic, ACTH-reactive cells were observed outside the tumour. PMID- 1704209 TI - ["Fenaf"--a new histochemical stain]. AB - The authors have synthesized a new dye which by its histochemical properties is analogous to the imported aldehyde-fuchsin but much cheaper. The production of this dye under the trade name "Fenaf" is organized at Shostka's factory of chemicals. The dye reveals neurosecretion, thyrothrophocytes of the adenohypophysis, tissue mast cells and other structures that are stained with aldehyde-fuchsin. PMID- 1704210 TI - A 3-dimensional model to describe the relation between prism directions, parazones and diazones, and the Hunter-Schreger bands in human tooth enamel. AB - Differences in the shapes of the Hunter-Schreger bands in different teeth and in different areas of a tooth have never been explained. They are almost certainly related directly to differences in the courses and organization of prisms. A computerized model was used to construct vertical sheets of enamel prisms whose courses could be varied in 3 dimensions. The output was viewed as a 3-dimensional picture of the prisms, or as a 2-dimensional picture of the Hunter-Schreger bands, or as a "longitudinal" section showing prism profiles (parazones and diazones). The effects on the shapes of the bands of changing the parameters describing prism courses were often unexpected. The following appeared to be important for human enamel: (a) the shapes of the enamel-dentine junction and the growth lines, (b) the thickness of the enamel, (c) the amount by which prisms widen as they pass to the surace, (d) prism courses in the longitudinal plane and (e) the amount by which the oscillations of prisms in the transverse plane were progressively dampened. It was concluded that modelling prisms in 3 dimensions, and checking the appearance of cut surfaces of the model, provides the only sure way of understanding the 3-dimensional arrangement of prisms in most enamels that contain Hunter-Schreger bands. PMID- 1704211 TI - Composition of whole unstimulated saliva of healthy children: changes with age. AB - Whole unstimulated saliva was collected from 136 healthy subjects divided into 5 groups according to age: (1) 25 infants, 7-11 months old; (2) 28 toddlers, 2-3 yr old; (3) 28 children, 6-8 yr old; (4) 28 adolescents, 12-14 yr old; (5) 27 adults, 25-63 yr old. The concentrations of Na, K, total protein, IgA and amylase activity were measured. A significant ascending linear correlation with age was found for concentrations of Na, total protein, IgA and amylase activity. There were significant differences between age groups in K and IgA concentrations. Salivary amylase activity was very variable, and a significant difference was found between infants and toddlers only. Salivary composition thus changes significantly during childhood, implying a process of development and maturation of the salivary glands and indicating the need of age-matched controls for the clinical use of saliva. PMID- 1704212 TI - Loculated fluid. A previously undescribed fluorescein angiographic finding in choroidal neovascularization associated with macular degeneration. Macular Photocoagulation Study Reading Center. AB - The Foveal Photocoagulation Study, a component of the Macular Photocoagulation Study, is designed to evaluate whether laser treatment can reduce the risk of severe visual loss in eyes with well-defined choroidal neovascular membranes associated with macular degeneration that extend through the foveal center. On one third of the 554 baseline angiograms of study patients enrolled in and whose eyes were graded in the study as of January 31, 1990, the Reading Center staff has noted an unusual pattern of hyperfluorescence in the late-transit frames that has not been described previously. This pattern, which we call "loculated fluid," consists of a well-demarcated area of hyperfluorescence that appears to represent pooling of fluorescein in a compartmentalized space anterior to the choroidal neovascular leakage. Although the loculated fluid may conform to a pattern of typical cystoid macular edema, it can also pool within an area deep to the sensory retina in a shape that does not bear any resemblance to cystoid macular edema. This pattern is important to recognize because it (1) should not be confused with the angiographic pattern or extent of choroidal neovascularization and (2) should be differentiated from a serous detachment or tear of the retinal pigment epithelium. PMID- 1704213 TI - Characterization of circulating antibodies against Chlamydia psittaci in turkeys. AB - Plasma and joint fluids from turkeys experimentally inoculated with Chlamydia psittaci strain TT3 were evaluated by immunoblotting to identify antibodies elicited by chlamydial antigens during the course of infection. Protein antigens from elementary bodies of TT3 were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose before being probed with plasma or synovial fluid from TT3 inoculated birds. The major outer-membrane protein (MOMP), the 60,000-molecular weight proteins, and a 97,400-molecular-weight protein were the predominant antigens recognized by IgG in the plasma and joint fluids. Plasma IgG specific for the 97,400 protein band was first detectable at day 10 postinoculation (PI). Antibodies to the 60,000-molecular-weight protein and MOMP were first detected at days 14-17 PI and at days 7-10 PI, respectively, in some birds, and as late as days 36-42 PI and days 42-70 PI in others. The antibodies were still present at day 142 PI. Immunoblotting techniques indicated that the antigens to which these antibodies were reacting were protein. These observations may have implications for the development of serodiagnostic assays as well as the identification of potential proteins for subunit immunogens in birds. PMID- 1704214 TI - Damage specific mammalian endonucleases. PMID- 1704215 TI - Peripheral blood leukocytes as a surrogate marker for cisplatin drug resistance: studies of adduct levels and the repair gene ERCC1. PMID- 1704216 TI - Inhibition of nucleic acid synthesis in P388 lymphocytic leukemia cells in culture by cis-platinum derivatives. AB - Cis-diaminedichloroplatinum(II) [cDDP] and three related derivatives Pt(mal)(NH3)2, PtCl2(dach) and Pt(mal) (dach) have been observed to possess cytotoxicity against the growth of P388 lymphocytic leukemia cells. DNA synthesis in P388 cells was inhibited by the agents in a manner which was consistent with their ED50 values for cytotoxicity. When P388 cells were treated with these platinum complexes in vitro at doses which caused more than 80% inhibition of DNA synthesis, no significant inhibition was observed for thymidine, kinase, thymidine monophosphate kinase, carbamoyl phosphate synthetase, or aspartate transcarbamoylase activities. Thus, there was no evidence that these agents inhibited de novo purine, pyrmidine, or deoxynucleotide synthesis. All of the agents did inhibit the nuclear DNA polymerase activity, but the extent of inhibition was 20% or less at doses which caused greater than 70% inhibition of DNA synthesis. Thus, the inhibition of DNA synthesis appeared to be due to cisplatinum(II) drug binding to the DNA bases. This was estimated to be 1 atom of platinum per 1500-3000 DNA base pairs which is consistent with other studies. The platinum complexes with chloro leaving ligands caused considerable DNA strand scission by 24 h at 10 times the ED50 dose, most likely a measure of impending cell death. In contrast, the platinum complexes with malonato leaving ligands did not cause significant strand scission by 24 h at similar doses. They also exhibited a significant delay in the inhibition of DNA synthesis. These data were interpreted as resulting from slower monoadduct to diadduct conversion, but it is not possible to eliminate the possibility of a different mode of interaction with DNA or a different mechanism of cytotoxicity for the malonato compounds. PMID- 1704217 TI - Increased gene expression of matrix metalloproteinase-3 (stromelysin) in skin fibroblasts from patients with severe recessive dystrophic epidermolysis bullosa. AB - Gene expression of matrix metalloproteinase 3 (MMP-3 = stromelysin) was examined in the skin fibroblasts obtained from patients with severe recessive dystrophic epidermolysis bullosa (RDEB). Steady-state mRNA level of MMP-3 was selectively increased in the unstimulated RDEB cells by a post-transcriptional mechanism. A parallel study on the susceptibility of type VII collagen to MMPs revealed that this type of collagen is degraded by MMP-3, but not by MMP-1 (collagenase). These data suggest that MMP-3 may play an important role in the blister formation fo the skin in RDEB patients by the degradation of anchoring fibrils consisting of type VII collagen. PMID- 1704218 TI - Demonstration of an active component of inter-alpha-trypsin inhibitor in the brains of Alzheimer type dementia. AB - The putative precursor of A4 amyloid protein associated with Alzheimer's disease is known to have a domain with an amino acid sequence characteristic of a Kunitz type serine protease inhibitor. Human serum inter-alpha-trypsin inhibitor (ITI) is the most similar inhibitor. We screened brain tissues with senile dementia of the Alzheimer type in an attempt to detect ITI immunoreactivity employing immunohistochemical methods. For this purpose, we used the antibody raised against acid-stable proteinase inhibitor (ASPI) which is an active component of ITI. ASPI immunoreactivity was found to be localized in diffuse type senile plaques, the perivascular area and subpial layer. Reactive astrocytes with intense ASPI immunoreactivity were present in the pyramidal layer of the parahippocampus, where loss of neurons was observed. These findings suggest that ITI may be related to the pathogenesis of Alzheimer type dementia. PMID- 1704219 TI - Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis. AB - Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development. PMID- 1704220 TI - Developmental and tissue-specific expression of rat T-kininogen. AB - The ontogeny of T-Kininogen expression in the rat liver was examined. Levels approximately 6 fold higher than seen in the adult liver are present during the perinatal period. Elevated levels are also seen in the maternal liver, beginning 5 days prior to parturition. The timing of induction in the fetal and maternal liver is distinct, suggesting independent regulation. While T-Kininogen mRNA is mainly synthesized in the liver, low but significant expression is seen in other tissues, including lung and kidney. PMID- 1704221 TI - Signal transduction through the vasoactive intestinal peptide receptor stimulates phosphorylation of the tyrosine kinase pp60c-src. AB - The present study demonstrates that signal transduction through a receptor lacking intrinsic tyrosine protein kinase activity involves a rapid and potent phosphorylation of a non-receptor tyrosine protein kinase in the membranes. Vasoactive intestinal peptide (VIP) stimulates phosphorylation of a membrane protein with a M.W. of 56 KD (pp60) in the cultured chick embryonic retinal pigment epithelium. VIP stimulates phosphorylation of the pp60 with such efficiency and potency that the maximal phosphorylation has been observed at the earliest time (3 minutes at 1 x 10(-6)M VIP) and the lowest concentration (1 x 10(-11)M for 20 minutes) examined. Western blot analysis with a monoclonal antibody anti-pp60src (GD11, Parsons et al., J. Virol. 51, 272-282, 1984) indicates that the pp60 is the pp60c-src, a normal cell oncogene product with intrinsic tyrosine protein kinase activity. PMID- 1704222 TI - Vitronectin in the substratum of endothelial cells is cross-linked and phosphorylated. AB - Vitronectin (VN), previously shown to be a substrate for purified transglutaminases, was demonstrated in this study to be cross-linked when incubated with HUVEC and EAhy926 cells. The cross-linking was calcium-dependent and required that VN be plated at the substratum of the cells. These cells also phosphorylated VN, but in contrast to a previous study demonstrating a cAMP dependent protein kinase in platelets, the phosphorylation of VN by was decreased with the addition of 1mM cAMP. The cross-linking of VN by endothelial cells demonstrates that the adhesion of these cells to VN is a dynamic process in which the substratum may be enzymatically altered. Furthermore, the modifications of VN by cross-linking and phosphorylation could modulate the functions of VN and influence events such as endothelial cell proliferation and angiogenesis. PMID- 1704223 TI - Tissue distribution and species difference of the brain type glucose transporter (GLUT3). AB - The complementary DNA for the human brain type glucose transporter (GLUT3) was used to determine its tissue specific expression in human, monkey, rabbit, rat, and mouse. Under high stringent conditions, 4.1 and 3.2 kilobase (kb) GLUT3 transcripts in monkey and a single 4.1 kb GLUT3 mRNA in rabbit, rat, and mouse were detected by RNA blot analysis. Although the GLUT3 transcripts were widely distributed, as are the erythrocyte type glucose transporter (GLUT1) transcripts, this mRNA is most abundant in the brain. However, the relative abundance of GLUT3 mRNA in the various regions of the monkey brain shows a different pattern from that of GLUT1 mRNA: GLUT3 is most highly expressed in the frontal lobe of the cerebrum, whereas GLUT1 is most abundant in the basal ganglia and the thalamus. Moderately higher GLUT3 mRNA levels were detected in the parietal lobe of the cerebrum, hippocampus, and cerebellum than the levels of GLUT1 transcripts. We also detected GLUT3 mRNA in adult human psoas major muscle, although it has been reported that the GLUT3 gene is scarcely expressed in adult human skeletal muscle of the thigh. In addition, in the rat and the mouse, no transcripts of the GLUT3 gene were detected in liver, kidney, small intestine, skeletal muscle, or fat besides in brain. Thus, the expression of the GLUT3 gene seems to be restricted to the brain in rodents. These results suggest that the expression of GLUT1 and GLUT3 genes might be regulated by different mechanisms. PMID- 1704224 TI - Expression of protein disulfide isomerase in cultured rat liver epithelial cells is unrelated to the growth inhibitory effect of TGF-beta 1. AB - The effect of TGF-beta 1 treatment on the level of protein disulfide isomerase (PDI) mRNA in normal and chemically or spontaneously transformed rat liver epithelial cell lines was investigated. TGF-beta 1 at 1 or 10 ng/ml concentrations did not significantly decrease the mRNA level of PDI at 4 or 24 hours after exposure to TGF-beta 1, irrespective whether the cell line was sensitive or resistant to the growth-inhibitory effect of TGF-beta 1 at these concentrations. The results indicate that in normal or neoplastic rat liver epithelial cells, the expression of PDI is unrelated to the growth inhibitory effect of TGF-beta 1. PMID- 1704225 TI - N-carboxymethylchitosan-N,O-sulfate as an anti-HIV-1 agent. AB - N-carboxymethylchitosan-N-O-sulfate (NCMCS), a sulfated polysaccharide derivative of chitin, inhibited the propagation of the human immunodeficiency virus type 1 (HIV-1) in human CD4+ cells and that of Rauscher murine leukemia virus (RLV) in murine fibroblasts. A dose-dependent inhibition of both viruses was observed without significant cytotoxicity. NCMCS blocked the binding of HIV-1 to human CD4+ target cells and competitively inhibited HIV-1 reverse transcriptase. Thus, NCMCS may prevent HIV-1 infection by inhibiting viral adsorption to the CD4 receptor and reverse transcription of the viral genome. PMID- 1704226 TI - Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines. AB - Rat monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned. A cDNA library was constructed from mRNA extracted from Con A-stimulated rat spleen cells. After 2 rounds of screening with a radiolabeled oligonucleotide probe and DNA sequencing from plasmid DNAs, one clone which encoded for MCP-1 was obtained. The cDNA clone comprises 665 base pairs with an open reading frame which encodes for 148 amino acid protein. Spleen cells expressed a significant amount of MCP-1 mRNA without stimulus. But higher expression was observed when cells were stimulated with Con A. MCP-1 mRNA was also expressed in tumor cell lines, although the amount of expressed MCP-1 mRNA was different among cell lines. PMID- 1704227 TI - Cell populations synthesizing cartilage proteoglycan core protein in the early chick limb bud. AB - Cartilage specific macromolecules are known to be synthesized in the mesenchyme of the embryonic chick limb bud, especially in areas of prechondrogenic condensations (Shinomura et al, 1984). Even though the mesenchyme seems homogeneous according to histological criteria, studies in the past have suggested the presence of different cell populations with different chondrogenic potential (Solursh et al, 1982; Swalla et al, 1984). In this study we have investigated by means of flow cytometry, the synthesis of proteoglycan core protein during early development of the chick limb bud in order to identify the different chondrocyte progenitor cells. We were able to identify by virtue of different size and density a cell population which synthesizes core protein extensively at stage 24 and stage 25 of development. This cell population synthesizes core protein predominantly at the proximal half of the limb bud at stage 24. However at stage 25 the same population synthesizes core protein predominantly at the distal half of the limb bud. These observations indicate that the distal half of stage 25 limb bud is mostly homogeneous with prechondrogenic cells and is in agreement with in vitro experiments that show high chondrogenic potential of the mesenchymal cells from this stage. PMID- 1704228 TI - Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase. AB - Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using [32P]rA12-18.dT10 as a representative template-primer and [alpha-32P]dTTP as a representative 2'-deoxynucleoside-5' triphosphate. UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero-(p66/p51) and homodimeric (p66/p66, p51/p51) forms of the enzyme. In the case of the p66/p51 heterodimer, the form of the enzyme believed to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit. These results suggest that the polymerase activity of the heterodimer residues on p66. PMID- 1704229 TI - Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells. AB - Hepatocyte growth factor (HGF), which is a potent growth factor of adult rat hepatocytes in primary culture, also strongly stimulated DNA synthesis of rabbit renal tubular epithelial cells in secondary culture. Its mitogenic activity was dose-dependent, being detectable at 3 ng/ml and maximal at 30 ng/ml. Over 20% of the cells were shifted to the S-phase by HGF alone, judging by the labeling index. HGF had additive effects with EGF, acidic fibroblast growth factor (a FGF), and insulin. Transforming growth factor-beta 1 (TGF-beta 1) strongly inhibited DNA synthesis of renal tubular cells stimulated by HGF. The growth of renal tubular epithelial cells was also regulated by cell density: DNA synthesis stimulated by HGF was high at lower cell density and was strongly suppressed at high cell density. These results suggest that HGF may act as a renotropic factor in compensatory renal growth or renal regeneration in vivo. PMID- 1704230 TI - Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-gamma by human hepatoma cells and its inhibitory effect on hepatitis B virus replication. AB - Asialofetuin-tacked liposomes (AF-liposomes) encapsulating interferon (IFN)-gamma were bound and internalized into a human hepatoma cell line, HepG2 cells, selectively through asialoglycoprotein receptor, but not non-tacked liposomes (N liposomes). AF-liposomal IFN-gamma was more effective for inhibition of viral DNA replication in hepatitis B virus (HBV)-producing clone from HepG2 cells transfected with HBV-DNA than N-liposomal IFN-gamma. AF-liposomes may increase the therapeutic potential of IFN-gamma through asialoglycoprotein receptor in treating HBV-infected hepatocytes. PMID- 1704231 TI - Ionophore A23187-induced protein-tyrosine phosphorylation of human platelets: possible synergism between Ca2+ mobilization and protein kinase C activation. AB - Addition of ionophore A23187 to washed human platelets caused a time- and dose dependent increase in the phosphotyrosyl content of 135, 124 and 76 kDa proteins. Platelets loaded with intracellular Ca2+ chelator 5,5'-dimethyl-bis-(o aminophenoxy)-ethane-N, N, N', N'-tetraacetic acid before addition of A23187 exhibited no protein-tyrosine phosphorylation. Replenishment of such platelets with extracellular CaCl2 restored A23187-induced protein-tyrosine phosphorylation. Upon stimulation with A23187, both aspirin and ADP scavengers treated platelets exhibited protein-tyrosine phosphorylation without phosphoinositide hydrolysis and protein kinase C activation. These data show (a) that A23187 stimulates protein-tyrosine phosphorylation by the elevation of intracellular Ca2+, and (b) that A23187-induced protein-tyrosine phosphorylation is independent of formation of endoperoxides/thromboxane A2, released ADP, phosphoinositide hydrolysis and protein kinase C activation. Furthermore, a synergistic effect of A23187 and protein kinase C activators in stimulating protein-tyrosine phosphorylation is suggested. PMID- 1704232 TI - Beta-subunits of equine chorionic gonadotropin and lutenizing hormone with an identical amino acid sequence have different asparagine-linked oligosaccharide chains. AB - The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a beta-subunit with an identical amino acid sequence. The Asn-linked oligosaccharide chains of eCG beta and eLH beta were quantitatively liberated as tritium-labeled oligosaccharides by hydrazinolysis followed by N acetylation and NaB3H4-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCG beta contained neutral and sialylated oligosaccharides, while eLH beta contained neutral, sialylated, sulfated, and both sialylated and sulfated oligosaccharides. In addition, elution profiles on a Bio-Gel P-4 column of the neutralized oligosaccharide mixtures of eCG beta and eLH beta were different, indicating that the molecular masses of oligosaccharides of the two glycoproteins are different. Therefore, this suggests that the structures of the Asn-linked oligosaccharide chains of eCG beta and eLH beta are different although they have an identical amino acid sequence. PMID- 1704233 TI - Purification and immunochemical interrelationship of insulin receptors from rat brain and liver. AB - Insulin receptors from rat brain and liver were purified. Brain purified receptor exhibited protein bands of apparent Mr = 135,000 and 95,000 molecular weight corresponding to alpha- and beta-subunits, retained a tyrosine specific protein kinase activity and demonstrated phosphorylation that is hormonally sensitive. Antisera were raised against both insulin receptor preparations and enzyme-linked immunosorbent assay was developed. The comparison of two insulin receptors was based on a displacement enzyme-linked immunosorbent assay where antisera were interchanged on predetermined optimal dilutions. This indicated that both insulin receptors possess some unique antigenic determinants thereby implying a structural difference. PMID- 1704234 TI - Selective cytotoxic effects of immunotoxin--monoclonal anti-AFP-abrin-A chain conjugate on several human hepatoma cell lines. AB - A highly specific monoclonal antibody (anti-AFP) against alpha-fetoprotein (AFP) was linked to N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) to form conjugates which were purified with a protein A-sepharose CL-4B affinity column. The conjugate, PDP-Anti-AFP was then covalently coupled to the toxic abrin-A chain to synthesize immunotoxins. The immunotoxin, anti-AFP-abrin-A conjugate, which was also purified with a protein A-sepharose CL-4B affinity column, had a molecular weight of 180,000 and had 80% antigen-binding activity that of anti-AFP activity and 92% toxicity of abrin-A chain. The immunotoxin showed selective cytotoxicities toward the AFP secreting human hepatoma cell lines, such HepG2 and Hep3B, but not toward AFP non-secreting human hepatoma cell line, PLC/PRF/5. PMID- 1704235 TI - Hyaluronic acid-like substance from mouse ovaries with angiogenic activity. AB - Glycosaminoglycans prepared from extracts of non-luteal mouse ovaries (JCL-ICR strain) were assayed for neovascularization by implanting Elvax films, impregnated with test samples, on the lateral wall of the sheath of m. rectus abdominis in adult mice of the same strain. Neovascularization occurred in a dose dependent manner. When purified by chromatography on Dowex 1-x2 and DEAE Sephadex columns, fractions eluted with 0.5 M NaCl showed strong neovascularizing activity. On further purification by high performance liquid chromatography using TSK gel DEAE 2 SW column, the fraction with a retention time nearly coincident with that of hyaluronic acid possessed high neovascularizing activity. The activity of this fraction was markedly reduced when treated with streptococcal hyaluronidase. The present results suggest that glycosaminoglycans, especially a hyaluronic acid-like substance, are involved in ovarian neovascularization. PMID- 1704236 TI - Polypeptide growth factors and angiogenesis. AB - Angiogenesis is the term used to describe the formation and development of blood vessels. The renewed interest in regulation and mechanistic aspects of angiogenesis depends on advances in the comprehension of metastatic dissemination of cancers, ischaemic heart disease and blood-brain barrier formation. Recently, many poly-peptide growth factors have been discovered which regulate the angiogenic process, most of them are stimulators and few inhibitors have been described. There is some evidence that many polypeptide growth factors employ prostanoids as second messengers. If this evidence will be extended to angiogenic factors, it will be possible to use inhibitors of prostaglandin H synthase and/or prostanoid receptor blockers in the control of tumour induced angiogenesis. PMID- 1704237 TI - Effects of selenium on RNA synthesis and content in rat pancreas. AB - In order to investigate the effect of selenium supplementation on RNA in the rat pancreas, the rate of in vitro incorporation of [3H]uridine into RNA by pancreas slices derived from two groups of rats fed either a low-selenium diet or a diet supplemented with 0.25 mg/kg selenium as selenite was examined. The RNA and lipid peroxide contents and glutathione peroxidase activity in homogenates from the pancreas were also determined. After feeding for 12-14 weeks, the rates of [3H]uridine incorporation were significantly higher in the pancreatic tissue from the selenium-supplemented diet group. Concomitantly, an increase in glutathione peroxidase activities and RNA content, and a reduction of lipid peroxides, were also found in the pancreatic tissue of the selenium-supplemented group. The results suggest that selenium supplementation at a level of 0.25 mg/kg selenium could promote RNA synthesis with an increase in glutathione peroxidase activity and a decrease of lipid peroxides. PMID- 1704238 TI - Interleukins and interferons: yin-yang modulators of PGH synthase in human macrophages. AB - Prostaglandin H synthase (PGHs) not only is an unstable enzyme, mainly when it is challenged with substrate, but its mRNA is one of the shortest lived species so far identified in mammalian cells. Therefore, signals regulating its level are critical for the role it plays in many cells. The expression of genes coding for PGHs appears to be under the control of polypeptide growth factors. This is in accordance with our previous data showing that a colony stimulating factor-1 (CSF 1)-like factor induces the de novo synthesis of PGHs in human monocytes, as demonstrated by immunoblotting. Here we extend this concept by showing that interleukin (IL)-1 alpha behaves as a potent inducer of PGHs in human macrophages, as indicated by the block in its action due to the addition of RNA and protein synthesis inhibitors. Interferons (IFNs) alpha and beta, however, inhibit prostanoid production in a dose-dependent fashion mainly when macrophages activated by serum are tested. Thus, the PGHs system appears to be under a fine control, CSF-1 being the main regulator during the differentiation from pro monocyte to monocyte and from monocyte to macrophage, and IL-1 (and perhaps IL-2) as well as IFN alpha and beta, the regulators during differentiation and/or proliferation of human macrophages. PMID- 1704239 TI - Two-layer gradient separation of granulocytes for functional tests. AB - Two gradient separation techniques were compared with the dextran sedimentation method for the separation of granulocytes from blood. Both techniques gave adequate yield and excellent purity, and flow cytometric analysis of surface membrane markers gave no indication of subset selection during the procedure. Cells separated by the three methods behaved comparably in functional tests including random migration, chemotaxis and chemiluminescence. The gradient separation techniques are rapid and efficient methods for the preparation of near 100% pure granulocyte suspensions for functional studies. PMID- 1704240 TI - Expression of p21RAS in odontogenic tumors. AB - Using an immunohistochemical assay 10 benign odontogenic tumors were evaluated for expression of the HRAS- and KRAS-encoded gene products p21RAS. Overexpression of p21RAS was found in ameloblastomas, ameloblastic fibromas and odontogenic myxomas compared with normal human developing teeth. The highest expression was noted in a recurrent plexiform ameloblastoma in which almost 100% of the tumor cells were brightly reactive. In general, p21RAS was preferentially expressed in ectodermal cells of odontogenic tumors, consistent with the findings in the tooth germs. The significance of p21RAS expression is considered in relation to the biological behavior of ameloblastomas. PMID- 1704241 TI - Two new Escherichia coli O groups: O172 from "Shiga-like" toxin II-producing strains (EHEC) and O173 from enteroinvasive E. coli (EIEC). AB - Two Escherichia coli strains were established as antigenic test strains for two new O groups, O172 and O173. The O172 strain (EHEC) which produces "Shiga-like" toxin II (verocytotoxin 2) was isolated from a case of haemorrhagic colitis while the enteroinvasive O173 strain (EIEC) originated from a child with diarrhoea. PMID- 1704242 TI - Interleukin-6 and tumour necrosis factor alpha are expressed by keratinocytes but not by Langerhans cells. AB - The presence of human cytokines was examined in parallel skin biopsies and epidermal single cell preparations obtained from normal individuals. Using biotin avidin-peroxidase and immunofluorescence techniques and antibodies against recombinant cytokines, a granular intercellular/membrane-associated staining for interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha), but not IL-1 alpha or beta, was observed. An epidermal cytoplasmic staining pattern was also detected, which was most pronounced using the anti-rIL-6 antiserum. In the epidermal single cell preparations, membrane-associated staining was detected for both IL-6 and TNF alpha. Double staining revealed that CD1-positive Langerhans cells (LC) failed to express any of the examined cytokines. In vitro binding of rIL-6 or rTNF alpha to skin sections and epidermal single cell preparations indicated that the cell surface-associated IL-6 and TNF alpha originally demonstrated on keratinocytes were truly membrane-bound. Finally, co-cultivation of epidermal cells with an IL-6 responsive cell line, B9, and testing of epidermal cell supernatants in this assay, indicated that the in vivo membrane bound IL-6 had biological activity. PMID- 1704243 TI - Migratory paths and phenotypic choices of clonally related cells in the avian optic tectum. AB - We used retrovirus-mediated gene transfer to study the migration of clonally related cells in the developing chicken optic tectum. Clonal cohorts initially form radial arrays in the ventricular zone (approximately E5), but eventually divide into three separate migratory streams. In the first migration, a minor population of cells migrates tangentially along axon fascicles in medio-laterally directed files (approximately E6-E7); these eventually differentiate into multipolar efferent cells. After E7, the majority of cells in each clone migrate radially along fascicles of radial glia to form the tectal plate, wherein they differentiate into neurons and astrocytes. Around E9, a set of small cells leaves the radial arrays in superficial layers to form a second tangential migration; at least some of these differentiate into astrocytes. Thus, as the tectum develops, cells derived from a single multipotential precursor migrate along three separate pathways, follow separate guidance cues, and adopt distinct phenotypes. PMID- 1704244 TI - Glutamate-induced increases in intracellular Ca2+ in cultured frog tectal cells mediated by direct activation of NMDA receptor channels. AB - Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels. PMID- 1704245 TI - Self-help materials for anxiety: a randomized controlled trial in general practice. AB - The efficacy of a self-help package in treating chronic anxiety was evaluated in a randomized controlled trial in which the intervention group received self-help materials in the form of an audiotape and booklet, in addition to their current treatment. The intervention was successful in terms of mean depression scores (P = 0.01), anxiety scores (P = 0.04) and general health questionnaire scores (P = 0.02) which were significantly lower for the intervention group than for the controls. In addition, the depression scores fell faster for the intervention group than for the controls. The overall mean reduction in three months in adjusted depression scores was approximately two points greater for the intervention group than for the controls (P = 0.02). Clinicians welcomed the package as a valuable addition to the therapies available for managing chronic anxiety problems. Further studies should include larger sample sizes, taking into account the non-response to postal questionnaires over time. PMID- 1704246 TI - Prognostic value of reduced heart rate variability after myocardial infarction: clinical evaluation of a new analysis method. AB - The relation between heart rate variability, measured from standard 24 hour electrocardiogram recordings in patients convalescent after a myocardial infarction, and the occurrence of sudden death and spontaneous, symptomatic, sustained ventricular tachycardia were assessed in a consecutive series of 177 patients admitted with acute myocardial infarction and surviving to 7 days. In addition to the analysis of heart rate variability, the occurrence of non sustained arrhythmias on 24 hour electrocardiographic monitoring, and the results of clinical assessment, signal averaged electrocardiography and ejection fraction were analysed and were related to outcome. During a median of 16 months of follow up (range 10-30 months) there were 17 end point events (11 (6.2%) sudden deaths) and six (3.4%) episodes of sustained ventricular tachycardia. An index of the width of the frequency distribution curve for the duration of individual RR intervals was used to measure heart rate variability. This mean (SD) index was significantly smaller in those with end point events (16.8 (8.0)) than in those without events (29.0 (11.2)). The relative risk of the occurrence of an end point event in those with a heart rate variability index less than 25 was 7.0. Multivariate analysis showed that of all the variables examined a reduced heart rate variability index was the single most powerful predictor of end point events. Measurement of heart rate variability by this simple, automated, operator independent method provided useful information on the arrhythmic propensity in patients convalescent after myocardial infarction. PMID- 1704247 TI - src-related tyrosine protein kinases as signaling components in hematopoietic cells. AB - The src-related tyrosine protein kinases are thought to be involved in the transduction of signals controlling cell growth as well as in specialized functions in fully differentiated, nonproliferating cells. The association of one of these kinases, p56lck, with the CD4 and CD8 cell-surface receptors in T lymphocytes has provided a model system through which the first function for a src-related tyrosine kinase has been defined. These initial observations in T lymphocytes have led investigators to explore the potential association of the src-related tyrosine kinases with other receptor complexes. Evidence that these kinases may be involved in mediating signaling events through such diverse cellular receptors as those found in B lymphocytes and basophils is currently being pursued. PMID- 1704248 TI - [Primary cancers of the gallbladder]. PMID- 1704249 TI - Oncology: haematopoietic growth factors. PMID- 1704250 TI - Tenascin expression in hyperproliferative skin diseases. AB - The expression of tenascin, a recently discovered extracellular matrix glycoprotein, was studied by immunohistochemistry in normal human skin and in a number of skin diseases with epidermal hyperproliferation such as psoriasis, basal cell carcinoma, Bowen's disease and solar keratosis. Tenascin expression in the upper dermis of normal skin was found to vary from almost absent to patchy along the basal membrane. Staining was continuous and intense around blood vessels, hair follicles and eccrine sweat ducts. In basal cell carcinoma a marked expression of tenascin was found in the tumour stroma, especially adjacent to the basal membrane surrounding the tumour cell nests. In Bowen's disease and solar keratosis, tenascin expression was found in the dermis next to the keratinocytes. In psoriasis the dermal papillae of clinically involved skin were intensely stained and a continuous band of tenascin was present in the upper dermis along the basal membrane. The distribution of tenascin differed from other known extracellular matrix components. PMID- 1704251 TI - The value of anticarcinoembryonic antigen, human milk factor globulin, and antikeratin antibodies in differentiating mesothelioma from lung carcinoma. AB - Monoclonal anticarcinoembryonic antigen (antiCEA), human milk factor globulin (HMFG2), and antikeratin antibodies were assessed for their value in the differential diagnosis of pleural mesothelioma (53 cases) and carcinoma of the lung (60 cases) in material from necropsies. In 40 of the cases pleural biopsies were also studied in the same manner. AntiCEA was found to be the best discriminating antibody for most types of mesothelioma; HMFG2 was slightly less valuable but a useful additional tool. Antikeratin was the least useful. For both antiCEA and HMFG2 antibodies, however, the proportion of carcinomas staining was smaller than in previous studies and this, combined with the positive staining of some mesotheliomas, reduces the value of the reactions in the individual case. Medical panels adjudicating compensation claims should not use these reactions as the sole criteria in deciding the origin of the tumours in these cases. PMID- 1704252 TI - Greetings from the RNA world. RNA Processing sponsored by the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, May 16-20, 1990. PMID- 1704253 TI - Overexpression of wild-type and dominant negative mutant vimentin subunits in developing Xenopus embryos. AB - Vimentin belongs to the diverse multigene family of intermediate filament proteins, each member of which is expressed in a tissue-specific and developmentally regulated pattern. The existence of vimentin filaments has been documented in oocytes, eggs, and early embryos of Xenopus laevis, but the role of these cytoskeletal components remains unknown. To investigate the functions of vimentin during early development in Xenopus, we induced the overexpression of wild-type and deletion mutant subunits in most of the cells of embryos by injecting synthetic RNA into fertilized eggs. Wild-type vimentin subunits, as well as subunits lacking most of the amino-terminal head piece, assembled into normal appearing filaments in vivo. Deletion mutants of the fourth alpha-helical rod domain were assembly incompetent and dominantly inhibited the polymerization of wild-type subunits when both types of subunit were co-expressed in cells. Expression of at least a tenfold excess of wild-type or mutant subunits within cells of embryos did not lead to any detectable morphological or developmental abnormalities, suggesting that the presence and proper regulation of vimentin expression is not essential during the initial stages of embryogenesis in Xenopus. PMID- 1704254 TI - Evidence for allelic exclusion in Chinese hamster ovary cells. AB - Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704255 TI - A novel cell-permeable cromoglycate derivative inhibits type I Fc epsilon receptor mediated Ca2+ influx and mediator secretion in rat mucosal mast cells. AB - Type I Fc epsilon receptor (Fc epsilon RI) mediated Ca2+ uptake and secretion of rat serosal mast cells have been shown to be inhibited by disodium 1,3-bis [(2' carboxylatochromon-5'-yl) oxy]-2-hydroxypropane (disodium cromoglycate, DSCG), which is widely employed in the treatment of allergic asthma [Foreman et al. (1977) Br. J. Pharmacol. 59, 473P-474P; Cox (1967) Nature (London) 216, 1328 1329]. This drug was also found to modify the protein phosphorylation pattern of these mast cells. [Theoharides et al. (1980) Science 207, 80-82]. We have isolated by affinity chromatography on a water-insoluble cromoglycate-carrying matrix a cytosolic enzyme recently identified as a nucleoside 5'-diphosphate kinase. In order to examine a possible intracellular activity of the drug, a cell permeant cromoglycate derivative, 1,3-bis [[2' [[(acetoxymethyl)oxy]carbonyl]chromon-5'- yl]oxy]-2-hydroxypropane [bis(acetoxymethyl) cromoglycate, CG/AM], has been synthesized, and its uptake and effect on the Fc epsilon RI-mediated exocytosis of mast cells was investigated. A tritium-labeled CG/AM derivative, used as radioactive tracer, was found to permeate mucosal mast cells of the rat line RBL-2H3 and accumulate intracellularly up to 40-fold its extracellular concentration following hydrolysis by cytoplasmic hydrolases. A CG/AM dose dependent inhibition of the Fc epsilon RI-induced mediator secretion was observed in RBL-2H3 cells loaded with this compound (I50 approximately 40 microM extracellular CG/AM). A similar dose dependent inhibition was observed for both the Fc epsilon RI-mediated transient rise in the concentration of cytosolic free Ca2+ ions [( Ca2+]i) and the net Ca2+ influx, as monitored by the fluorescent indicator Quin2 and the radioactive tracer 45Ca2+, respectively. These results clearly show that cell-permeant cromoglycate inhibits the Fc epsilon RI-mediated Ca2+ influx into the cell and further underscore the dominant role of this process in the coupling of stimulus to secretion in RBL cells. Furthermore, with the identification of nucleoside 5' diphosphate kinase as a potential intracellular target for CG activity, distinct mechanisms of action may be inferred for cell-permeant and nonpermeant forms of CG. PMID- 1704256 TI - Characterization of human alpha 2-macroglobulin monomers obtained by reduction with dithiothreitol. AB - We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1 inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3. PMID- 1704257 TI - Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding. AB - In recent years, many studies have suggested a direct role for alpha 2 macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M complexes, together with results of beta aminopropionitrile competition, confirmed that insulin incorporation occurs via the same gamma-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to alpha 2M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha 2M. Phenyl isothiocyanate derivatization of the N terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha 2M. PMID- 1704258 TI - Analysis of the plasma elimination kinetics and conformational stabilities of native, proteinase-complexed, and reactive site cleaved serpins: comparison of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, antithrombin III, alpha 2 antiplasmin, angiotensinogen, and ovalbumin. AB - Proteinase inhibitors of the serpin superfamily may exist in one of three distinct conformations: the native form, a fully active protein with the reactive site loop intact; the proteolytically modified form in which inhibitory capacity is abolished; and the proteinase-complexed form, a stable equimolar complex between the inhibitor and a target proteinase. Here, the specificity and kinetics of the plasma elimination of different serpin conformations are compared. Proteinase-complexed serpins were rapidly cleared from the circulation. However, the native and modified forms were not cleared rapidly, indicating that the receptor-mediated pathways which recognize the complexes fail to recognize the native and modified forms. This result suggests that significant structural differences exist between modified and proteinase-complexed serpins. The structural differences were probed by using transverse urea gradient gel electrophoresis, a technique that allows comparisons of the conformational stabilities of proteins. With the exception of the noninhibitory serpins ovalbumin and angiotensinogen, the modified and proteinase-complexed serpins were both stabilized thermodynamically compared to the native forms. In addition, the proteinase component of the serpin-proteinase complex was usually thermodynamically stabilized. These data are used to compare the conformations of serpin-proteinase complexes with those of native and modified serpins; they are discussed in terms of a model whereby serpins inhibit proteinases in a manner similar to that described for other types of protein inhibitors of serine proteinases. PMID- 1704259 TI - Identification of subunit contact sites on the alpha-subunit of lutropin. AB - Peptides corresponding to the entire sequence of the alpha-subunit of the human glycoprotein hormones were synthesized by using standard solid-phase procedures. Purified peptides were incubated in the presence of alpha- and beta-subunits of bovine lutropin, and subunit recombination was monitored by difference spectroscopy, reverse-phase high-pressure liquid chromatography, and gel filtration chromatography. Although the binding of alpha-peptides to either subunit could not be detected by these techniques, it was possible to demonstrate that some peptides could inhibit the recombination of alpha- and beta-subunits. Specifically, alpha-peptide 33-58 allowed only 0-11% of subunit recombination in 24 h (38-56% after 48 h), while alpha-peptide 51-65 allowed 10-60% of subunits to recombine in 24 h (65-94% in 48 h). Peptides 1-15, 11-27, 22-39, 61-78, and 73-92 of the alpha-subunit could not inhibit subunit recombination at any time or at any concentration tested. The data suggest that at least a portion of the alpha subunit contact site has been identified, and results are discussed in terms of protein structure assessment tools. PMID- 1704260 TI - Three-dimensional structure and antigen binding specificity of antibodies. AB - A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site. PMID- 1704261 TI - Nobel lecture. Retroviruses and oncogenes. I. PMID- 1704262 TI - Adoptive immunotherapy with high-dose interleukin-2: kinetics of circulating progenitors correlate with interleukin-6, granulocyte colony-stimulating factor level. AB - Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells results in significant tumor regression in patients with advanced cancer. We have investigated the kinetics of circulating erythroid (BFU-E) and granulocytic-macrophage (CFU-GM) progenitors after IL-2 therapy in 11 cancer patients, mainly affected by metastatic melanoma and renal cell carcinoma. Administration of IL-2 from day 1 through day 5 constantly induced a dramatic decrease of the number of circulating BFU-E and CFU-GM, which then showed a striking rebound (up to values fourfold and sevenfold higher, respectively, than the pretherapy levels) on discontinuation of IL-2, ie, from day 5 through day 10. A similar kinetic pattern was observed during and after the second cycle of IL-2 administration. 3[H]-thymidine killing experiments showed that the cycling activity of the progenitors was virtually unmodified in the rebound phases. To explore the mechanism(s) underlying this kinetic pattern, we have analyzed the plasma concentration of several hematopoietic growth factors, including IL-1 beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM CSF), G-CSF, and erythropoietin (Ep). No modifications in the levels of IL-3, GM CSF, or IL-1 beta were observed, whereas a pronounced increase of IL-6 and G-CSF concentration was monitored, starting at day 3 and peaking at day 5 of treatment (a parallel, but modest, increase of Ep level was also observed). The elevation of IL-6 and G-CSF concentration is directly correlated with and may, at least in part, underlie the subsequent rebound of circulating hematopoietic progenitors. Furthermore, the increase in IL-4 level observed at day 10 of therapy may mediate the eosinophilia gradually starting at this stage of treatment. PMID- 1704263 TI - Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound. AB - In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion. PMID- 1704264 TI - Loss of interferon antibodies during prolonged continuous interferon-alpha 2a therapy in hairy cell leukemia. AB - Although highly active in hairy cell leukemia (HCL), interferons (IFN) are not curative in this disease; current data indicate that prolonged IFN therapy will be necessary to control disease in the majority of patients. We previously observed acquired IFN resistance in association with neutralizing IFN-alpha 2a antibodies in small numbers of patients with HCL. This finding suggests that the requisite long-term therapy may be compromised if there is an increasing incidence over time of neutralizing antibodies. We performed a follow-up study of IFN antibodies in our patients receiving continuous IFN therapy. All 16 patients who were previously antibody negative remained so. Surprisingly, all nine patients who previously had non-neutralizing IFN antibodies became antibody negative after a median of 14.5 months. Moreover, 3 of 10 patients who had neutralizing antibodies became antibody negative and five had only non neutralizing antibodies a median of 10 months from the time neutralizing antibody had first been detected. Only two patients had persisting neutralizing antibodies. Inhibition of neopterin synthesis, inhibition of generation of 2', 5' oligoadenylate synthetase activity, and inability to detect IFN in serum after subcutaneous injection of IFN-alpha 2a was observed only in the one patient tested with neutralizing IFN antibodies confirming that these antibodies have functional significance in vivo. We conclude that, although neutralizing IFN antibodies inhibit the effectiveness of IFN in vivo, these antibodies are produced only transiently during long-term therapy. The long-term effectiveness of this drug will not likely be affected in most patients by neutralizing antibody. PMID- 1704265 TI - Ki-B5: a monoclonal antibody unrelated to CD45 recognizes normal and neoplastic human B cells in routine paraffin sections. AB - In the search for immunoreagents appropriate for the histopathologic diagnosis of malignant B-cell lymphomas in routinely processed paraffin sections, a new monoclonal antibody, Ki-B5, was generated using a high-grade B-cell lymphoma as the immunogene. Ki-B5 is a mouse IgG1/kappa that recognizes five protein fractions of about 84, 82, 55, 48, and 27 Kd after biosynthetic radiolabeling and immunoprecipitation. Protein fractions with the molecular weights of approximately 84 and 82 Kd were expressed on the cell surface and show that Ki-B5 is probably unrelated to CD45. It was possible through electron microscopy to visualize the membrane-bound portion of Ki-B5. Extensive immunohistologic studies on normal human tissue and various neoplasias demonstrated the high specificity of Ki-B5 to normal human B cells and a minor subgroup of plasma cells. Except for ML-2, which is a myelomonocytic human cell line, Ki-B5 exclusively recognized the B-cell lineage, including EB-3, BALL-1, and NALM-1. All carcinomas, sarcomas, and malignant melanomas tested with Ki-B5 were negative. Although normal granulocytes and monocytes were constantly negative, three of eight myelomonocytic leukemias coreacted with this antibody. Eight of the 57 T-cell lymphomas studied were positive to Ki-B5. Five were classified as lymphoblastic, two represented T8-CLL, and one was classified as immunoblastic T-cell lymphoma. Only 3 of 126 cases of B cell lymphoma, including rare types not considered in the current classifications, were negative to Ki-B5. Plasmacytomas were also negative, except for one case. Irrespective of the cases of lymphoblastic lymphoma and plasmacytoma, Ki-B5 represents a new monoclonal antibody appropriate for the diagnosis and immunophenotyping of malignant lymphomas in routinely processed paraffin sections. PMID- 1704266 TI - An epitope on the transferrin receptor preferentially exposed during tumor progression in human lymphoma is close to the ligand binding site. AB - We have previously reported an anti-transferrin receptor antibody, Trump, which was originally selected for its ability to discriminate low- and high-grade lymphomas. This feature was distinct from the other anti-transferrin receptor antibodies such as OKT9. In the present study, further immunochemical analysis was performed to define the nature of the antigenic site recognized by the Trump antibody. Trump was found to block the binding of transferrin both to solubilized and to surface transferrin receptors; conversely, transferrin could block the binding of Trump only to surface transferrin receptors. Therefore, the epitope recognized by Trump is near but not identical to the transferrin binding site. Stimulation of peripheral blood lymphocytes with phytohemagglutinin induced both the OKT9 epitope and the Trump epitope, but 12-phorbol 13 myristate acetate induced only the OKT9 epitope. Growth of some cell lines was inhibited by Trump but not by OKT9. No structural difference was found between transferrin receptor molecules reactive with Trump and those reactive with OKT9. In support of these results, Trump was able to immunoprecipitate transferrin receptor molecules solubilized from low-grade follicular lymphoma cells even though it did not bind to the receptors exposed on the surface of these cells. These findings imply that low-grade lymphoma cells differ from high-grade lymphoma cells not in the structures of their transferrin receptors but in their exposure of the molecule on the cell surface. PMID- 1704267 TI - Interaction of two different disorders in the beta-globin gene cluster associated with an increased hemoglobin F production: a novel deletion type of (G) gamma + ((A) gamma delta beta)(0)-thalassemia and a delta(0)-hereditary persistence of fetal hemoglobin determinant. AB - We report two different disorders of the beta-globin gene cluster segregating in a Belgian family: a novel deletion that results in (G) gamma + ((A) gamma delta beta)(0)-thalassemia (thal) and a heterocellular hereditary persistence of foetal hemoglobin of the Swiss type linked to a delta(0)-thal gene (delta (0)-HPFH). Heterozygosity for the heterocellular HPFH brings about a moderate (3.4% to 8.24%) increase of hemoglobin (Hb) F having a G gamma/A gamma ratio of 4:1, whereas carriers of the G gamma + ((A) gamma delta beta)(0)-thal deletion show in their peripheral blood a considerably higher (15%) percentage of Hb F. Both defects interact in the compound heterozygotes for G gamma + ((A) gamma delta beta)(0)-thal and delta(0)-HPFH producing a further increase (up to 24%) of fetal Hb consisting entirely of G gamma chains. Molecular characterization of the (G) gamma + ((A) gamma delta beta)(0)-thal by means of Southern analysis showed that the deletion spans about 50 kb, removing the 3' end of the A gamma-gene, the psi beta-, delta-, and beta-genes. A number of possible mechanisms leading to the overproduction of Hb F in HPFH and (G) gamma + ((A) gamma delta beta)(0)-thal will be discussed. PMID- 1704268 TI - Vesiculation of platelets during in vitro aging. AB - Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall. PMID- 1704269 TI - Studies of the antithrombotic effects of dextran 40 following microarterial trauma. AB - The effects of dextran 40 on platelet function and thrombus formation have been studied in vivo in arteries of the rabbit ear. Intra-aortic infusions of 32P labelled platelets were followed by infusions of 1.7 g dextran 40 in 17 ml saline/kg b w. Treated and untreated groups were studied using as trauma either end-to-end anastomosis or arteriotomy (7 mm)/intimectomy (5 mm). After restoring blood flow, bleeding times at the sites of anastomosis and arteriotomy/intimectomy were recorded. Accumulations of labelled platelets were followed in vivo for 2 hours, after which patencies were determined and amounts of red thrombotic material evaluated. In a separate series of measurements, haematocrit levels after dextran infusion were studied. Nearly all vessels in the end-to-end anastomosis groups were patent. In the arteriotomy/intimectomy groups, there was a significant increase in patency following dextran infusion. Dextran infusion did not alter platelet accumulation following end-to-end anastomosis, but following arteriotomy/intimectomy median values were somewhat reduced. Its antithrombotic effects must therefore arise mainly at stages of thrombus formation subsequent to platelet deposition. PMID- 1704270 TI - Studies of receptor-mediated inhibition of 45Ca accumulation into synaptosomes. AB - 1. The effects of alpha 2-adrenoceptor and kappa-opiate receptor activation on 45Ca accumulation into rat cortical synaptosomes were examined. 2. Clonidine (1 microM) and U50488H (1 microM) significantly reduced 45Ca accumulation under both resting (5 mM K+) and depolarizing (15-30 mM K+) conditions. 3. The inhibitory effects of the agonists on 45Ca accumulation into synaptosomes were enhanced in the presence of the Na(+)-Ca2+ exchange inhibitor sodium orthovanadate (vanadate, 2 mM), and were not present in mitochondrial preparations. 4. When the agonists were used together, their inhibitory effects were not additive but were, in fact, attenuated. 5. In the presence of the alpha 2-adrenoceptor antagonist idazoxan (1 microM), the inhibitory effect of U50488H on 45Ca accumulation was enhanced. A similar increase in the inhibitory effectiveness of clonidine was observed in the presence of naloxone (20 microM). 6. When synaptosomes were pretreated with 3 isobutyl-1-methylxanthine (IBMX, 0.5 mM), dibutyrylcyclic AMP (db-cyclic AMP, 10 microM) or 8-bromo-cyclic AMP (8Br-cyclic AMP, 10 microM), the inhibitory effects of clonidine and U50488H were abolished, suggesting that a decrease in cyclic AMP production is part of the receptor-effector coupling mechanism of both receptor systems. 7. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.05 microM) increased 45Ca accumulation but did not alter the inhibitory effects of clonidine or U50488H, thus showing that the effects of the agonists are not mediated by protein kinase C. 8. We conclude that alpha 2-adrenoceptor and Kappa opiate receptor activation dramatically reduce 45Ca influx through Ca21 channels (e.g., by 50%), that there is a functional antagonism between the two receptor systems and that in both cases, the receptor effector mechanism involves a decrease in cyclic AMP production. PMID- 1704271 TI - Bay K 8644, modifier of calcium transport and energy metabolism in rat heart mitochondria: a new intracellular site of action. AB - 1. The dihydropyridine Ca2+ channel agonist Bay K 8644 (10-200 microM) produced a concentration-dependent increase in State 4 respiration in the rat heart mitochondria with the highest concentration (200 microM) increasing the rate from 33.1 +/- 0.7 to 187.0 +/- 13.3 ng atoms O2 consumed min-1 mg-1 protein. 2. Bay K 8644 (200 microM) reduced State 3 respiration from 247.2 +/- 11.7 to 174.4 +/- 0.06 ng atoms O2 min-1 mg-1 protein, reduced the respiratory control index (RCI) from 5.3 +/- 0.45 to 1.1 +/- 0.03 and reduced the ADP:O ratio from 2.75 +/- 0.03 to 1.3 +/- 0.15. 3. A similar, but smaller, stimulation of State 4 respiration was seen with nitrendipine (25-200 microM), the rate increasing from 22.6 +/- 1.0 to 33.1 +/- 1.8 ng atoms O2 consumed min-1 mg-1 protein in the presence of 200 microM nitrendipine. 4. Bay K 8644 (10-60 microM) increased the total Ca2+ uptake into rat heart mitochondria, the total increasing from 248.8 +/- 8.4 to 406.9 +/- 17.6 ng Ca2+ mg-1 protein at 60 microM Bay K 8644 (EC50 = 18.9 +/- 1.4 microM). 5. Bay K 8644 (10-60 microM) produced a concentration-dependent reduction in the Ca2+ influx rate (IC50 = 52.5 +/- 2.8 microM). Similar effects were seen with (+) Bay K 8644 and (-)-Bay K 8644. 6. Nitrendipine (40-120 microM) stimulated Ca2+ efflux from mitochondria preloaded with the ion; the efflux rate increasing from 2.9 +/- 0.05 to 114.2 +/- 6.2 nmol Ca2+ min-1 mg-1 protein (EC50 = 57.3 +/- 1.3 microM). 7. These data indicate dihydropyridine-induced changes in the activity of the mitochondrial Na+/Ca2 . antiporter pathway; nitrendipine causing stimulation and Bay K 8644 causing inhibition. PMID- 1704272 TI - Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla. AB - 1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H] nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H] nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H] nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown. PMID- 1704273 TI - Dihydropyridines alter adenosine sensitivity in the rat hippocampal slice. AB - 1. The effects of adenosine and a range of adenosine analogues, which are resistant to uptake processes, were studied in the presence of dihydropyridines and verapamil on the population spike potential recorded from the CA1 area of the hippocampal slice. 2. Nifedipine and Bay K 8644, a calcium channel antagonist and activator respectively, enhanced the inhibitory action of adenosine in a concentration-dependent manner. This was in contrast to their effect on adenosine analogues where the inhibition of the population potential was significantly attenuated. Similar interactions between the adenosine compounds and the dihydropyridines were also displayed in studies on spontaneous epileptiform activity in the CA3 region. 3. This effect of nifedipine and Bay K 8644 was not shown by the dihydropyridines, nimodipine or nitrendipine, or by the phenylalkylamine, verapamil. 4. Addition of the adenosine uptake blocker dipyridamole reversed the action of nifedipine on adenosine, so that inhibition by adenosine was now attenuated by nifedipine in a similar manner to that observed with the adenosine analogues. 5. These results can be explained with reference to binding studies that show displacement of adenosine analogues from the adenosine receptor by dihydropyridines. An action at the adenosine uptake site by the dihydropyridines explains the enhancement of adenosine inhibition. 6. The possible sites for this interaction are discussed. PMID- 1704274 TI - Genetics of RA: the HLA shared epitope hypothesis and its implications. PMID- 1704275 TI - Prostatic specific antigen. PMID- 1704276 TI - Can histological assessment predict the outcome in interstitial cystitis? AB - In a retrospective study we compared the outcome of 39 patients with interstitial cystitis to the histological findings at initial diagnostic bladder biopsy. The degree of inflammation and fibrosis and the mast cell counts were assessed on each biopsy. The prognostic relevance of the clinical features of age, duration of symptoms, frequency, nocturia, pain and bladder capacity was assessed. The study showed no statistical correlation between the severity of histological findings at diagnosis and the eventual outcome of the disease. Over 50% of patients with severe histological abnormalities responded to conservative treatment. Although the majority of patients with mild pathological changes responded to conservative treatment, some did require surgical intervention. The clinical features of pain, nocturia and bladder capacity showed significant differences between the 2 patient groups. However, the former 2 features are subject to many variables and the latter probably has too wide a range to be useful as a prognostic indicator. Only the response of patients to conservative treatment will indicate those who are not being helped and who may eventually require surgical treatment. PMID- 1704278 TI - Haemospermia: a prospective study. AB - A prospective study of 74 men with haemospermia is presented. Most (76%) had experienced 1 or 2 episodes only and 9 (12%) were over 40 years old. Simple investigations led to a diagnosis in all 9 of these men and of those under 40, no pathology could be detected in 48%. A diagnosis was made in 32 of the remaining 34 men by simple investigations. In patients over 40 years of age with haemospermia, a potentially treatable cause will normally be found by routine investigation which should include cystoscopy. In younger men, non-invasive investigation alone should identify any pathology. Invasive investigations should be reserved for those patients in whom the problem is prolonged, excessive or in association with other symptoms. PMID- 1704277 TI - Short-term radiotherapy as palliative treatment in patients with transitional cell bladder cancer. AB - We report the results and complications of treatment with palliative, short-term radiotherapy in 162 elderly or disabled patients. Improvement in tumour associated symptoms was noted in 75 of these patients and 72 survived for more than a year. Those who responded to radiotherapy had a 5-year cancer-free survival rate of 58% compared with 4% in patients who did not respond to treatment. Survival was also affected by stage and indications for radiotherapy. In 85 patients without severe symptoms, where the tumour was judged curable but the patient was unsuitable for a full course of radiotherapy, the 5-year cancer free survival rate was 21%, which is in accordance with what can be achieved with full-course radiotherapy; 42% had various minor acute side effects. The 5-year late complication rate was 7%. PMID- 1704279 TI - Epitopes of gap junctional proteins localized to neuronal subsurface cisterns. AB - Several lines of evidence indicate the existence of channels that mediate the movement of calcium from the extracellular space directly into some intracellular calcium storage compartment and from one intracellular membrane-bounded compartment to another. The possibility that such channels resemble intercellular communication pathways formed by gap junction proteins (connexins) was investigated in rat brain. Antibodies against a rat liver gap junction protein (connexin32) were found to recognize several distinct proteins on Western blots of brain homogenates. In motoneurons these antibodies immunohistochemically labelled portions of neuronal endoplasmic reticulum membranes that form subsurface cisterns (SSCs) adjacent to the plasma membrane. These results suggest that SSCs and connexin-like proteins may be involved in the process of calcium mobilization in neurons. PMID- 1704280 TI - Alpha 2-macroglobulin is an astroglia-derived neurite-promoting factor for cultured neurons from rat central nervous system. AB - The neurite promoting factors in the astroglial conditioned medium (As-CM) were characterized by using primary cultures of embryonic rat neocortical neurons. The factors in the As-CM bind to lectins such as wheat germ agglutinin (WGA), suggesting that they contain sugar moieties. When the WGA-bound fractions were applied on a Superose 6 column, the activity was recovered mainly in two fractions, peak I and peak II. The peak II fraction was further purified by Mono Q anion exchange chromatography. A single protein band of 180 kDa was detected in the final Mono Q fraction by sodium dodecylsulfate polyacrylamide gel electrophoresis. The molecular weight coincided with that of alpha 2 macroglobulin (alpha 2M). Western blotting showed that the single protein band was reacted with anti-alpha 2M antibody but not with anti-fibronectin and anti laminin antisera. The neurite-promoting activity of the Mono Q fraction was inhibited by anti-alpha 2M antibody. Furthermore, commercially available alpha 2M also promotes neurite outgrowth in our assay system. These results strongly suggested that alpha 2M is one of the neurite-promoting factors in the As-CM. PMID- 1704281 TI - The postsubicular cortex in the rat: characterization of the fourth region of the subicular cortex and its connections. AB - The hippocampal formation contributes importantly to many cognitive functions, and therefore has been a focus of intense anatomical and physiological research. Most of this research has focused on the hippocampus proper and the fascia dentata, and much less attention has been given to the subicular cortex, the origin of most extrinsic projections from the hippocampal formation. The present experiments demonstrate that the postsubiculum is a distinct area of the subicular cortex. The major projections to the postsubiculum originate in the hippocampal formation, the cingulate cortex, and the thalamus (primarily from the anterodorsal (AD) nucleus and to a lesser extent from the anteroventral (AV) and lateral dorsal (LD) nuclei). These projections differ from the thalamic projections to presubiculum and parasubiculum. Efferent projections from the postsubiculum terminate in both cortical and subcortical areas. The cortical projections terminate in the subicular and retrosplenial cortices and in the caudal lateral entorhinal and perirhinal cortices. Subcortical projections primarily end in the AD and the LD nuclei of the thalamus. These thalamic projections end in areas that are distinct from those to which the presubiculum and parasubiculum project. For instance, the postsubiculum has a dense terminal field in the AD nucleus, but presubicular axons terminate predominantly in the AV nucleus. The cortical projections also distinguish postsubiculum. All subicular areas project to the entorhinal cortex, but the postsubicular projection ends in the deep layers (i.e. IV-VI), whereas presubiculum projects to layers I and III, and parasubiculum projects to layer II. Postsubiculum projects to retrosplenial granular b cortex and only incidentally to retrosplenial granular a cortex. In contrast presubiculum projects to the retrosplenial granular a cortex but not to the retrosplenial granular b cortex. These differences clearly mark the postsubiculum, the presubiculum, and the parasubiculum as distinct regions within the subicular cortex and suggest that they subserve different roles in the processing and integration of limbic system information. PMID- 1704282 TI - Release of immunoreactive substance P in the spinal cord during development of acute arthritis in the knee joint of the cat: a study with antibody microprobes. AB - In anaesthetized spinal cats, the release of immunoreactive substance P in the spinal cord during development of an acute inflammation in one knee joint was studied with antibody microprobes. The microprobes bore antibodies directed to the C- or N-terminus of substance P. With the normal knee joint, innocuous mechanical stimuli (flexion, pressure) did not result in spinal release of immunoreactive substance P. Following injection of kaolin and carrageenan into a knee, evidence for release of substance P following joint stimulation was found in 7 of 10 cats. Such release did not occur for several hours after joint injection and was detected predominantly in the superficial dorsal horn, the dorsal columns and at the dorsal surface of the spinal cord. In some experiments release was detected in the deep dorsal horn and upper ventral horn. Release of immunoreactive substance P required periods of mechanical stimulation such as flexion of, or pressure to, the inflamed joint. The failure to detect central release of substance P from stimulation of normal joints, and the release of substance P, after a delay, from inflamed joints, suggest that the fibres releasing this compound require sensitization by inflammatory mediators before they are excited by joint stimuli. PMID- 1704283 TI - Effects of fluoxetine or D-fenfluramine on serotonin release from, and levels in, rat frontal cortex. AB - Using in vivo microdialysis of frontal cortex in anesthetized rats, as well as analysis of frontal cortex homogenates, we examined the effects of chronic administration of fluoxetine (30 mg/kg, i.p.) or D-fenfluramine (7.5 mg/kg, i.p.), administered daily for 3 days, on serotonin and 5-HIAA levels a day later. Measurements were also taken after 3-, 7- , and 21-day recovery periods. Neither chronic fluoxetine nor D-fenfluramine changed basal serotonin release. Both treatments, however, transiently decreased the release of serotonin evoked by an acute dose of D-fenfluramine (10 mg/kg, i.p.). Release initially was completely suppressed in fluoxetine-pretreated animals but returned to normal by the 21st day of washout; following D-fenfluramine pretreatment, normal release was attained by the 7th day of washout. Both fluoxetine and D-fenfluramine transiently decreased 5-HIAA levels in the dialysates and tissues. Both drugs also caused prolonged changes in frontal cortex serotonin levels, D-fenfluramine lowering them but fluoxetine elevating them. These results suggest that, at comparable dosage levels relative to their ED50s, fluoxetine and D-fenfluramine cause comparable reversible effects on brain serotonin release. The drugs also cause prolonged but opposite changes in brain serotonin levels, probably reflecting differences in the extents to which they or their principal metabolites release serotonin and block its reuptake. PMID- 1704284 TI - GABA-immunoreactive boutons make synapses with inspiratory neurons of the dorsal respiratory group. AB - Intracellular labelling with horseradish peroxidase (HRP) combined with gamma aminobutyric (GABA) immunocytochemistry was used to assess the GABAergic input to inspiratory bulbospinal neurons of the dorsal respiratory group in the cat. The relationship between GABA-immunoreactive (GABA-IR) boutons and intracellularly labelled neurons was examined at the light microscopic and ultrastructural levels. At the light microscopic level, GABA-IR boutons were frequently found in close apposition to dendrites and cell bodies of labelled neurons. The presence of synapses was confirmed with electron microscopy. In addition, synaptic specializations were observed between immunoreactive boutons and unlabelled terminals which in turn formed synaptic contacts with HRP-labelled dendrites, a finding consistent with presynaptic inhibition. These results demonstrate a direct GABAergic input to a functionally defined population of medullary respiratory neurons, and suggest involvement of this neurotransmitter in the control of these neurons. PMID- 1704285 TI - Structure-function relationships of insulin receptor interactions in cultured mouse astrocytes. AB - Insulin receptor binding and effects upon uridine incorporation into nucleic acid were studied and compared to 3 insulin analogues, DPI, DPI-amide and DOI, in primary cultures of mouse astrocytes. From half-maximal inhibition of [125I]monoiodoinsulin binding (0.14 nM), the relative binding affinities of DPI, DPI-amide and DOI were 20%, 80% and 4% as compared with the native insulin, respectively. IC50 values were 158.5 nM for insulin, 199.5 nM for DPI-amide, 794.3 nM for DPI and 3980 nM for DOI. The corresponding relative potencies in stimulating [14C]uridine incorporation into TCA-insoluble material were 9% for DPI, 100% for DPI-amide and 1.1% for DOI. These findings are commensurate with data for these analogues in other insulin-sensitive tissues. It is concluded that the astrocyte insulin receptor has similar structural requirements to receptor in tissues outside the CNS, at least in terms of the involvement of the C-terminus of the insulin B-chain. PMID- 1704286 TI - Relays from the spinal cord and solitary nucleus through the parabrachial nucleus to the forebrain in the cat. AB - Projections from the spinal cord and solitary nucleus to the lateral parabrachial nucleus (PBN1) in the cat were directly compared using double anterograde tracing methods. The two inputs were found to overlap within a well-circumscribed zone in the rostral 2/3 of PBN1. This zone was flanked ventrally by a zone receiving only solitary nucleus input and dorsally by a zone receiving only spinal input. Other authors have shown that neurons within these three recipient zones (overlap area, solitary nucleus and spinal cord) project to different forebrain targets (hypothalamus, amygdala and thalamus, respectively). This orderly input-output organization is likely to provide part of the framework for PBN's complex involvement in the coordination of respiratory and cardiovascular activities and their association with pain, visceral sensation and emotion. PMID- 1704287 TI - The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat. AB - In order to examine the synaptic input to dopaminergic neurones in the substantia nigra from GABAergic terminals and terminals that contain substance P, double and triple immunocytochemical studies were carried out at the light and electron microscopic levels in the rat. In a first series of experiments sections of the substantia nigra were incubated to reveal axon terminals containing either substance P or glutamate decarboxylase and then incubated to reveal dopaminergic neurones using tyrosine hydroxylase immunocytochemistry. Examination of this material in the light microscope revealed that many substance P- and glutamate decarboxylase-immunoreactive boutons were associated with the dopaminergic cells. In the electron microscope it was found that the perikarya and dendrites of the dopaminergic neurons received symmetrical synaptic input from terminals that displayed immunoreactivity for substance P or glutamate decarboxylase. A small proportion of the substance P-positive boutons formed asymmetrical synapses. In a second series of experiments sections of the substantia nigra were processed by the pre-embedding immunocytochemical technique for tyrosine hydroxylase and then the post-embedding immunogold technique for gamma-aminobutyric acid (GABA). Examination in the electron microscope revealed that the tyrosine hydroxylase positive neurons received symmetrical synaptic input from many GABA-positive terminals. Quantitative analyses demonstrated that a minimum of 50-70% of all boutons afferent to the dopaminergic neurones display glutamate decarboxylase or GABA immunoreactivity. Triple immunocytochemical studies i.e. pre-embedding immunocytochemistry for tyrosine hydroxylase and substance P, combined with post embedding immunogold staining for GABA, revealed that some of the substance P immunoreactive boutons that were in contact with the dopaminergic neurones also displayed GABA immunoreactivity. In a third series of experiments the combination of anterograde transport of lectin-conjugated horseradish peroxidase or biocytin with post-embedding GABA immunocytochemistry demonstrated that at least one of the sources of GABA-containing terminals in the substantia nigra is the striatum. The results of the present study: (1) demonstrate that dopaminergic neurones in the substantia nigra receive symmetrical synaptic input from GABAergic and substance P-containing terminals, (2) show that a proportion of these terminals contain both substance P and GABA and (3) suggest that the major synaptic input to dopaminergic neurones is from GABAergic terminals and that a part of this innervation is derived from the striatum. PMID- 1704288 TI - Inhibition of calcium currents in cultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel coupling. AB - Single channel recordings from rat myenteric plexus neurons demonstrated the presence of two categories of Ca2+ channels. One type of Ca channel had a slope conductance of 27 pS and was sensitive to dihydropyridines while the other channel type had a conductance of 14 pS and was dihydropyridine-insensitive. The 14 pS channel was mostly inactivated at a holding potential of -40 mV, while the 27 pS channel was much more resistant to depolarized holding potentials. A majority of whole-cell current was reprimed by the use of negative holding (-90 mV) potentials, when compared to that obtained at a holding potential of -40 mV. These properties are consistent with N- and L-type Ca channels previously described. In general, the inactivating part of the whole-cell Ca2+ current, selectively reprimed by negative holding potentials, was inhibited by neuropeptide Y (NPY). Depolarization-induced [Ca2+]i transients assessed using fura-2 showed that the inhibitory effects of nitrendipine and NPY were additive. The effects of NPY were abolished by pertussis toxin pretreatment. Single-channel experiments showed that neither the 14 nor the 27 pS Ca channel currents were inhibited by the addition of NPY outside the patch pipette. These results suggest that NPY modulates N-type Ca2+ channels selectively in these neurons and that an easily diffusible second messenger does not appear to participate in receptor/channel coupling. PMID- 1704289 TI - Intranigral substance P stimulation of striatal dopamine release is inhibited by spantide II: a new tachykinin antagonist without apparent neurotoxicity. AB - The effects of intranigral injections of Spantide II, a novel tachykinin antagonist, on extracellular dopamine, and dihydroxyphenylacetic acid (DOPAC) levels in the rat striatum were studied using in vivo microdialysis. The ability of Spantide II to inhibit intranigral substance P or neurokinin A stimulation of striatal dopamine levels was also studied. A unilateral injection (all substances were injected in a volume of 0.2 microliter) of Spantide II (0.7 nmol) into the substantia nigra, pars reticulata (SNR) of halothane anaesthetized rats produced a short-lasting decrease in dopamine levels in the ipsilateral striatum. Striatal DOPAC levels showed no change after Spantide II. A unilateral injection of substance P (0.07 nmol) into the SNR produced an increase in ipsilateral striatal dopamine levels, which was prevented when substance P was co-administered with Spantide II (0.7 nmol). A unilateral injection of neurokinin A (0.09 nmol) into the SNR produced an increase in ipsilateral striatal dopamine levels, which was not modified when neurokinin A was co-administered with Spantide II (0.7 nmol). Immunohistochemical analysis using antisera to tyrosine hydroxylase and neuropeptide K, as well as Cresyl violet staining, revealed that intranigral injections of Spantide II (0.7 nmol) did not produce significant damage in the substantia nigra. The results indicate that Spantide II is not 'neurotoxic' when injected intranigrally, and that it is a selective antagonist of substance P in the substantia nigra. Furthermore, the reduction of striatal dopamine levels after intranigral Spantide II injections suggests that the nigrostriatal dopamine projection is tonically stimulated by striatonigral substance P. PMID- 1704290 TI - Serotonin release varies with brain tryptophan levels. AB - This study examines directly the effects on serotonin release of varying brain tryptophan levels within the physiologic range. It also addresses possible interactions between tryptophan availability and frequency of membrane depolarization in controlling serotonin release. We demonstrate that reducing tryptophan levels in rat hypothalamic slices (by superfusing them with medium supplemented with 100 microM leucine) decreases tissue serotonin levels as well as both spontaneous and electrically-evoked serotonin release. Conversely, elevating tissue tryptophan levels (by superfusing slices with medium supplemented with 2 microM tryptophan) increases both tissue serotonin levels and serotonin release. Serotonin release was found to be affected independently by tryptophan availability and frequency of electrical field-stimulation (1-5 Hz), since increasing both variables produced nearly additive increases in release. These observations demonstrate for the first time that both precursor-dependent elevations and reductions in brain serotonin levels produce proportionate changes in serotonin release, and that the magnitude of the tryptophan effect is unrelated to neuronal firing frequency. The data support the hypothesis that serotonin release is proportionate to intracellular serotonin levels. PMID- 1704291 TI - Projections of substance P-immunoreactive neurons located in the ventromedial medulla to the A7 noradrenergic nucleus of the rat demonstrated using retrograde tracing combined with immunocytochemistry. AB - Stimulation of neurons located in the ventromedial medulla (VMM), including the nucleus raphe magnus (RMg), produces antinociception which appears to be mediated in part by activation of spinally-projecting noradrenergic neurons located in the A7 catecholamine nucleus. Although the identity of the VMM neurons that project to the A7 nucleus is not known, there is indirect evidence that these neurons contain substance P. This possibility was examined by injecting the retrograde tracer Fluoro-Gold into the A7 nucleus and determining whether substance P immunoreactive neurons in the VMM were labeled with Fluoro-Gold. The results of these experiments demonstrated that numerous substance P-immunoreactive cells in the RMg, gigantocellular reticular nucleus pars alpha and the paragigantocellular reticular nucleus were retrogradely labeled by an injection of Fluoro-Gold into the A7 nucleus. These observations indicate that substance P-containing neurons in these areas of the VMM project to the A7 nucleus. Thus, the antinociception induced by stimulation of the VMM may be mediated by activation of substance P containing neurons that project to and activate spinally projecting noradrenergic neurons in the A7 nucleus. PMID- 1704292 TI - Hepatocellular carcinoma coexisted with second malignancy--a study of 13 cases from a consecutive 440 autopsy cases of HCC. AB - In a consecutive 440 autopsy cases of hepatocellular carcinoma (HCC), 13 patients (2.95%) were found to have a second primary malignant tumor. All of the patients were male. The age ranged from 40 to 69 years old. (mean: 56.5) Peak incidence occurred in the seventh decade. The associated neoplasms included 4 cases of colorectal adenocarcinoma, 2 cases of thyroid cancer, 2 cases of retroperitoneal sarcomas, 1 case of pancreatic adenocarcinoma, 1 case of esophageal squamous cell carcinoma, 1 case of common bile duct adenocarcinoma, 1 case of renal cell carcinoma, and 1 case of prostatic adenocarcinoma. The organ most commonly involved was large bowel (4 cases). Epithelial origin neoplasms comprised the vast majority (84.6%). Of the 13 cases, 2 associated malignancies existed metachronously, 4 and 5 years before HCC. The others were found at the same time as HCC. The clinical and pathological observations included age, sex, serum alpha fetoprotein (AFP), serum hepatitis B surface antigen (HBsAg), cirrhosis, gross and histologic appearance. The above presentations were similar in cases with and without second malignancy. We failed to find any factor that was possibly related to the etiology of the second neoplasm. Much more such cases are needed for further evaluation. PMID- 1704293 TI - Psychiatric considerations in premature birth. AB - Five percent to ten percent of all live births are premature. Modern neonatology has dramatically improved survival rates. Morbidity rates, however, are as high as 60%, according to some studies. In this paper, outcome and intervention findings are described. Experiential, familial and socioeconomic factors interact synergistically with preterm birth and biological vulnerability to create cognitive, behavioural and interpersonal disorders. Principles of intervention are suggested, based on promoting organization within the infant and a responsive, active approach in the caregivers. PMID- 1704294 TI - Absence of reverse transcriptase activity in virus-like particles released by a breast carcinoma cell line (8701-BC) PMID- 1704295 TI - Chromosome aberrations and oncogene alterations in two new breast tumor cell lines. AB - Two new breast tumor cell lines (UISO-BC-1 and UISO-BC-2) have been established from pleural effusions obtained from patients with confirmed diagnosis of breast cancer. Cytogenetic investigation shows several numerical and structural aberrations in both cell lines. Each cell line appears to have distinctive karyotypic aberrations. Although a common marker chromosome was not found in both cell lines, several breakpoints (i.e., 1q11, 3q11, 7p11, 9q11, and 13q11) were commonly involved in the marker chromosomes of both lines. Double minute (dmin) chromosomes were also observed in these two cell lines. Sixteen oncogene probes were used to study the oncogene amplification and overexpression; among these, only neu and c-myc probes detected multiple gene copies. A 10-fold amplification and a 20-fold overexpression of the neu were observed in the UISO-BC-1 line, whereas a threefold and a fivefold amplification of c-myc were found in UISO-BC-1 and UISO-BC-2, respectively. Moderately enhanced expression (sixfold) of c-myc was also observed in the UISO-BS-2 line. No gross rearrangement of these genes or aberrant RNAs was detected in these tumor cell lines. PMID- 1704296 TI - The complement-inhibiting protein, protectin (CD59 antigen), is present and functionally active on glomerular epithelial cells. AB - Protectin (CD59 antigen) is a 20-kD phosphatidyl-inositol-linked membrane protein that inhibits formation of the membrane attack complex (MAC) of complement on homologous cells. Although the antigen has been identified in a number of human tissues, until recently a functional role had been demonstrated only in circulating cells. Using immunofluorescence techniques we have shown the presence of protectin on human glomerular epithelial cells (GEC) in culture and on GEC, tubular epithelial cells and endothelial cells in frozen sections of normal human renal cortex. In addition, we present evidence that this protein functions in protection of GEC from homologous complement: cultured cells incubated with the Fab2 fragment of a monoclonal anti-protein antibody were markedly more susceptible to killing by homologous serum than were cells in the absence of Fab2 anti-protectin. These findings suggest that this protein may be important in the maintenance of glomerular integrity in vivo, and may be of relevance in certain renal diseases. PMID- 1704297 TI - Increased CD5-positive B lymphocytes in type I diabetes. AB - CD5+ B lymphocytes have been implicated in the production of polyspecific and monospecific antibodies that bind self-antigens, and increased proportions of this B cell subset occur in patients with some autoimmune diseases. We investigated the proportion of peripheral blood CD5+ B lymphocytes in type I diabetic patients. Compared with 18 age-matched healthy subjects, 11 out of 28 (39.2%) type I diabetic patients had increased proportions of circulating CD5. B lymphocytes with no alterations in the numbers of circulating B and T lymphocytes. Although all patients with increased CD5 B lymphocytes also had serum islet cell antibodies and/or insulin autoantibodies, the occurrence of increased proportions of CD5+ B lymphocytes and serum autoantibodies was not significantly correlated. Increased proportions of CD5+ B lymphocytes was not related to the time elapsed since the clinical onset of diabetes. In addition, regardless of being increased or normal, the proportion of CD5+ B lymphocytes appeared as a relatively constant phenotype after 1 year of follow-up studies at 3-month intervals in eight patients. Although the significance of these findings remains to be established, the possibility exists that CD5+ cells play a role in the pathogenesis of type I diabetes. PMID- 1704298 TI - Biological activity of a bombesin-like peptide extracted from the intestine of the ratfish, Hydrolagus colliei. AB - 1. Ratfish (Hydrolagus colliei) intestines were boiled in water to inactivate proteases and then treated with cold 4% trifluoroacetic acid to extract bombesin like peptides. 2. The extract was fractionated in several steps using reverse phase and ion exchange HPLC, and bombesin-like immunoreactive peptides were detected by radioimmunoassay using an antiserum specific for the bioactive C terminal region of bombesin. 3. A highly purified bombesin-like peptide containing fraction stimulated amylase release in a dose-responsive fashion from rat pancreatic acini; the dose-response curve was parallel to a bombesin standard, and the ratfish peptide stimulated the same maximal rate of amylase secretion as the bombesin standard. 4. A potent, highly selective bombesin receptor antagonist completely abolished the stimulation of amylase release caused by the ratfish peptide, demonstrating the specificity of the response. 5. Estimates of the bombesin-like peptide concentration of this fraction by radioimmunoassay and by bioassay were nearly identical, indicating that ratfish bombesin is very similar biologically and antigenically to frog skin bombesin. PMID- 1704299 TI - The influence of substance P and its fragments on endogenous phosphorylation of synaptosomal membrane protein (synapsin) from cerebral cortex of rat brain. AB - 1. The effects of substance P and its fragments and analogue of a C-terminal fragment on cyclic AMP-dependent phosphorylation of synapsin I in synaptosomal membranes (SM) from cerebral cortex were investigated. 2. SP(I-II) and SP(1-4) at 10(-3) M caused a marked stimulation of synapsin I phosphorylation. 3. A C terminal fragment of SP (SP6-11) had no effect on phosphorylation of synapsin 1. 4. Analogue of C-terminal fragment [(Tyr8)SP6-11] at 10(-3) M distinctly inhibits phosphorylation of synapsin I. 5. These data suggest that SPI-II and its C- and N terminal fragments have a modulator function against the phosphorylation of some rat brain proteins. PMID- 1704300 TI - Allergic contact dermatitis from topical and systemic steroids. PMID- 1704301 TI - The Goodpasture antigen. PMID- 1704302 TI - A comparison of different methods of extraction of the M-protein from Streptococcus equi. AB - The molecular weights of the proteins produced in different extracts of Streptococcus equi were compared on immunoblots with antisera against acid extracted and mutanolysin extracted M-protein. Acid and alkaline extracts of S. equi contained some peptides of similar molecular weight that reacted with antiserum against an acid extracted 41,000 m.w. fragment suggesting that these fragments contained common epitopes. Comparison of the amino acid compositions of the 35,000 m.w. fragment of the alkaline extract and the 41,000 m.w. fragment of the acid extract suggest that these immunologically reactive fragments were probably derived from the same protein. Little cross-reactivity was observed between antisera against S. equi acid extracted protein and the native 58,000 m.w. M-protein. This suggests that conformational epitopes on the native M molecule are not present after acid treatment. PMID- 1704303 TI - [Abnormal platelet function and ultrastructure in patients with severe viral hepatitis]. AB - A study of aggregation and release function of platelet and its ultrastructure in 27 patients with severe viral hepatitis (SVH) was carried out. The results showed that a) the maximum aggregation rate (MAR) decreased markedly; b) platelet 5-HT decreased strikingly and plasma 5-HIs increased significantly; c) the change of MAR was in positive correlation with that of platelet 5-HT and platelet 5 HT/plasma 5-HIs and the change of platelet 5-HT was in negative correlation with that of plasma 5-HIs; d) the ultrastructural abnormalities of platelet suggested its renewal, denaturation and activation in vivo. These results indicate that the abnormal platelet function and ultrastructure in patients with SVH are mainly related to the activation of platelet in vivo. PMID- 1704304 TI - [The application of 5-HT and 5-HIAA in the diagnosis of apudomas]. AB - 5-HT apudomas are rare. In our country, so far there has been no detailed report about using whole blood 5-HT and urinary 5-HIAA in the diagnosis of apudomas. Measurement of whole blood 5-HT in 87 and urinary 5-HIAA in 44 apudoma patients has been carried out in our laboratory since 1980. The results showed that the values of whole blood 5-HT and urinary 5-HIAA were significantly higher in apudoma patients than those in non-apudoma, postoperative apudoma and normal groups. The value of whole blood 5-HT over 130 micrograms/ml and urinary 5-HIAA over 30 mg/24 h were preliminarily recommended as diagnostic for apudomas. PMID- 1704305 TI - [Quantitative determination of 7 proteins in csf of patients with Guillain Barre syndrome]. AB - Quantitative determinations were done for seven protein components namely albumin, IgG, transferrin, alpha-2 macroglobulin, IgA, IgM and ceruloplasmin in the CSF and sera from patients with Guillain Barre syndrome. The results showed that each of the protein significantly increased. All of these correlated remarkably with the increased values of total proteins in CSF. It seemed that the concentrations of the protein in CSF were influenced by those of the proteins in serum and the size of the corresponding protein molecule. PMID- 1704306 TI - Anatomic nature and surgical significance of anal sinus and anal intramuscular glands. AB - The nature of anal sinus and anal intramuscular glands is well known, but their origin is still discussed. In the past years a surprising new theory about the invagination of the proctodeum into the hindgut was put forward. Evidence supporting this theory would be the existence of an "anorectal band" and "epithelial debris" in the subanoderm formed during total or partial obliteration of the anal sinuses. To characterize these histological structures, the authors examined 62 autopsy specimens with conventional and special immunohistologic staining methods. In none of the examined specimens could the structures mentioned above be detected. Nearly 90 percent of our specimens contained the well known anal sinuses. In fetuses, neonatal deaths, and children, more than one half of the anal sinuses were accompanied by anal intramuscular glands penetrating the internal anal sphincter, whereas in adult specimens the anal intramuscular glands were rare. Eight postoperative, idiopathic chronic anal fissures and three postoperative anal fistulas examined with the same staining procedures showed epithelial cells at the base of the fissures or fistulas in two cases (25 percent) and three cases (100 percent), respectively. The results reinforce the theory that anal sinuses and anal intramuscular glands are separate anatomic entities and indicate a new theory of anal development. For idiopathic, chronic anal diseases anal sinuses have little surgical significance. Anal intramuscular glands should be the anatomic correlate of anal fistulas. PMID- 1704307 TI - [Extrasystoles during extracorporeal biliary shockwave lithotripsy. Their incidence and clinical significance]. AB - Incidence and clinical significance of cardiac side effects of extracorporeal shock-wave lithotripsy (ESWL) were prospectively analysed for 85 patients (26 men, 59 women; mean age 44 [17-81] years) with cholecystolithiasis (n = 70) or choledocholithiasis (n = 15). 24-hour ECG monitoring was undertaken on the day of treatment. Additionally, during ESWL cardiac rhythm and blood pressure were monitored. ESWL was performed with an electromagnetic lithotriptor under light anaesthesia with intravenous diazepam (10 mg) and pethidine (75-100 mg). There were no superventricular premature systoles in any of the patients during treatment. In 15 patients with occasional ventricular premature systoles (VPS) (6 81 per 23 hours) in the 24-hour ECG the number of VPS increased during the one hour ESWL procedure significantly to 6-55 (P less than 0.05). 14 of these patients had an unremarkable cardiac history. Changing the lithotriptor coupling angle failed to suppress the VPS in only two patients. In these two it was necessary to trigger the shock wave with the ECG. Blood pressure rose markedly (up to 220 mm Hg systolic) during ESWL in only three patients, known hypertensives. But this rise was easily controlled with nifedipine, 10 mg sublingually. These data demonstrate that ESWL is a safe alternative to operative treatment, even in the presence of existing cardiac disease. Nonetheless, precautions should be taken in case there are complications. PMID- 1704308 TI - Rheumatic pneumonia in a 10-year old Ethiopian child. AB - A 10-year-old female child who presented with worsening respiratory distress and acute rheumatic fever is discussed. Patient had also right side consolidation and a diagnosis of rheumatic pneumonia by post-morterm lung biopsy. A literature review on rheumatic pneumonia is made. PMID- 1704309 TI - Effect of gamma-butyrolactone on the retrograde axonal transport of neurotensin in dopaminergic neurones. AB - gamma-Butyrolactone is a drug known to inhibit the flow of electrical impulses in dopaminergic neurones and to prevent dopamine release in the striatum. In this study, we investigated the effects of this compound on the retrograde transport of neurotensin in the nigrostriatal dopaminergic pathway. The amount of radioactivity measured in the substantia nigra after injection of iodinated neurotensin into the striatum and the time course of its accumulation were not found to be modified in gamma-butyrolactone-treated rats. We therefore conclude that the retrograde axonal transport of neurotensin in the nigrostriatal pathway is not affected by reduced dopaminergic firing or by inhibition of the striatal release of dopamine. PMID- 1704310 TI - The discriminative stimulus properties of cocaine: effects of BAY K 8644 and nimodipine. AB - Calcium channel blockers appear to reduce the cardiac toxicity of cocaine and some stimulant-induced behaviors. The present experiment was designed to test whether the internal state induced by cocaine is altered by the calcium antagonist nimodipine. Substitution tests with the calcium agonist BAY K 8644 were also conducted in rats (N = 8) trained to discriminate cocaine (10 mg/kg) from saline in a two-lever, water-reinforced drug discrimination paradigm. Cocaine (0.625-10 mg/kg) produced a dose-related increase in drug-lever responding while BAY K 8644 (0.25-2 mg/kg) and nimodipine (0.2-0.8 mg/kg) engendered primarily saline responding. In combination with cocaine (2.5-10 mg/kg), nimodipine shifted the cocaine dose-response curve to the right at doses of 0.2 and 0.4 mg/kg; this attenuation did not increase with higher doses of nimodipine (0.8 and 1.6 mg/kg). The present results suggest that nimodipine may partially block the discriminative stimulus properties of cocaine, however, this reduction is neither robust nor dose-related. Thus, nimodipine might be expected to only marginally alter the subjective cocaine state in humans. PMID- 1704311 TI - Effects of piperine on the motility of the isolated guinea-pig ileum: comparison with capsaicin. AB - Piperine (1 microM), a congener of capsaicin, produced an initial contraction blocked the capsaicin-sensitive contractile response to mesenteric nerve stimulation and inhibited the twitch response induced by field stimulation in the isolated guinea-pig ileum. These three effects of piperine (1 microM) were rapidly desensitized and significantly antagonized by ruthenium red (0.5-1 microM), an inorganic dye known to antagonize the effects of capsaicin. The contractile effect of piperine was abolished by application of tetrodotoxin plus desensitization with substance P or by extrinsic denervation. The inhibitory effect of piperine (1 microM) on the twitch response was antagonized by desensitization with calcitonin gene-related peptide (CGRP). Moreover, cross tachyphylaxis between piperine and capsaicin was observed, suggesting that a similar mechanism may be involved in the effects of these agents. The contractile effects induced by piperine (10 microM) and the subsequent inhibitory effects on the twitch response were not desensitized and largely persisted after extrinsic denervation. The contractile effects of piperine (10 microM) were not strongly inhibited by tetrodotoxin plus desensitization with substance P. It was concluded that the lower concentration of piperine caused contraction and inhibited the twitch responses by releasing substance P and CGRP, respectively, from sensory nerves, and blocked the response to mesenteric nerve stimulation by a mechanism similar to that of capsaicin. At higher concentrations, piperine had non-specific direct actions on the smooth muscle. PMID- 1704312 TI - Molecular cloning and sequence analysis of the cDNA encoding the human acrosin trypsin inhibitor (HUSI-II). AB - A complete cDNA clone encoding the human acrosin-trypsin inhibitor HUSI-II has been isolated from a cDNA library of human testis and completely sequenced. The cDNA of 594 bp contained an open reading frame of 252 base pairs. The deduced amino acid sequence comprised the complete amino acid sequence of HUSI-II and a putative signal peptide. Northern blotting analysis revealed that HUSI-II is synthesized in testis, epididymis and seminal vesicle, but not in the prostate gland. PMID- 1704313 TI - ompC mutants which allow growth on maltodextrins show increased channel size and greater voltage sensitivity. AB - Misra and Benson [(1988) J. Bacteriol. 170, 3611-3617] showed that point mutations in the ompC gene can allow Escherichia coli to grow on maltotriose in the absence of LamB. This report shows that these mutants produce OmpC porins with increased single channel conductance compared to the wild type. The mutants showed similar voltage dependence to each other and to PhoE by being totally closed at 200 mV. The wild type from various sources was largely insensitive to voltages below 200 mV and thus 6 point mutations at 3 sites appear to increase the voltage dependence of OmpC channels. PMID- 1704314 TI - The rat alpha 2-C4 adrenergic receptor gene encodes a novel pharmacological subtype. AB - A rat gene and brain cDNA (pA2d) encoding the homologue of the human alpha-C4 adrenergic receptor subtype were isolated and characterized. RNA blots indicate that this gene is expressed in brain, heart and kidney but not in lung, liver or pancreas. Yohimbine, WB-4101 and prasozin all exhibited high affinity for this receptor in binding studies. Clonidine was more potent and efficacious than norepinephrine in inhibiting forskolin-stimulated cAMP production in CHO cells expressing pA2d. Together, these data suggest that the alpha 2-C4 gene product defines a previously undescribed pharmacological subtype of alpha 2-adrenergic receptor. PMID- 1704315 TI - Nucleolar organiser regions in iris melanocytic tumours: an accurate predictor? AB - A silver staining technique which demonstrates the nucleolar organiser region (NOR) was used in paraffin sections of iris naevi and melanomas. The technique shows argyrophilic NOR associated proteins (AgNORs) which are seen in nuclei as black dots. In nine iris naevi the AgNOR count ranged from 1.54 to 3.82 (mean 2.73), in 21 melanomas from 1.89 to 8.31 (mean 4.67). Mean AgNOR counts greater than four dots per nucleus were only seen in malignant lesions, thereby differentiating between benign and malignant tumours, whenever high AgNOR counts were found. We subsequently examined three tumours in the intermediate group of aggressive naevi, Jakobiec group 6: there were counts averaging 4.08 but with a wide standard deviation of counts indicating that the behaviour of this group of aggressive tumours is likely to depend on the percentage of cells bearing the higher numbers of NORs, which may represent mitotic potential or increased metabolic rate. PMID- 1704316 TI - cAMP analogs and selective inhibitors used to study low Km Mucor rouxii cAMP phosphodiesterase. AB - 1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed. PMID- 1704317 TI - Inter-alpha-trypsin inhibitor: a plasma proteinase inhibitor with a unique chemical structure. PMID- 1704318 TI - Phenobarbital-induced cytoprotective mechanisms in menadione metabolism: the role of glutathione reductase and DT-diaphorase. AB - 1. Treatment with N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU) (80 microM) led to decreases in cell viability in both naive and sodium phenobarbital (PB) induced hepatocytes. 2. Dicumarol (30 microM) selectively increased the cytotoxicity of menadione in hepatocytes isolated from naive vs PB-pretreated rats. 3. Inclusion of both BCNU and dicumarol to the incubation medium abolished the characteristic concentration-response curves of the hepatocytes for menadione. 4. A greater proportion of menadione was metabolized by DT-diaphorase in the hepatocytes isolated from PB-pretreated rats. 5. The role of glutathione reductase vs DT diaphorase in mitigating menadione-cytotoxicity in the naive vs PB-induced hepatocyte is discussed. PMID- 1704319 TI - Properties and distribution of cyclic AMP phosphodiesterase from rat liver. AB - 1. Phosphodiesterase activity in rat liver supernatant and solubilized rat liver particulate fractions was chromatographed on Q Sepharose and several characteristics of each peak determined. 2. Rat liver supernatant contained four peaks of activity. The first two of these corresponded to type I and II phosphodiesterases. The fourth peaks was similar to a type V activity and the third peak could not be definitely classified. 3. Particulate activity solubilized by mild protease treatment also contained four peaks of activity. The first two corresponded to the first two from the supernatant, the fourth was a type IV enzyme which is the insulin activated phosphodiesterase. The third peak could not be definitely characterized. 4. Particulate activity solubilised by Triton X-100 contained three peaks. Two had the properties of a type IV enzyme but only one of these was immunologically identified as the insulin sensitive enzyme. The remaining activity was similar to the chymotrypsin peak 3 activity. 5. Most of the particulate phosphodiesterase of rat liver is found in a microsomal fraction, and most is the insulin sensitive type IV enzyme. PMID- 1704320 TI - Forskolin down-regulates type-1 plasminogen activator inhibitor and tissue-type plasminogen activator and their mRNAs in human fibrosarcoma cells. AB - We have studied the effect of the adenylate cyclase-stimulating agent forskolin on expression of components of the plasminogen activation system in the human fibrosarcoma cell line HT-1080. By enzyme-linked immunosorbent assays, forskolin was found to cause a 2 to 4-fold decrease in intracellular and culture medium levels of type-1 inhibitor of plasminogen activators (PAI-1) and tissue-type plasminogen activator (t-PA). This was true for cells not treated with other agents and for cells, in which the PAI-1 and t-PA levels had been increased 5 to 10-fold by treatment with dexamethasone. This down-regulation could be traced back to corresponding decreases in the cellular levels of PAI-1 and t-PA mRNAs. Of the two PAI-1 mRNAs, the 2.4 kb species was 5-fold decreased by forskolin in cells treated with dexamethasone, while the 3.4 kb transcript was unaffected; in cells not treated with dexamethasone, forskolin affected the two PAI-1 transcripts in parallel. These studies show that in addition to the many inducers of PAI-1, PAI-1 gene expression is also subject to negative modulation by cyclic AMP. They also show that t-PA gene expression, in contrast to the induction by cyclic AMP observed in many other cell lines, may also be subject to negative regulation by cyclic AMP. Thus, hormonal agents acting with cyclic AMP as a second messenger may be involved in down-regulating PAI-1 and t-PA expression in vivo. PMID- 1704321 TI - Down-regulation of tyrosinase mRNA levels in melanoma cells by tumor promoters and by insulin. AB - Mouse melanoma cells in culture respond to melanocyte-stimulating hormone (MSH) or to cyclic AMP analogues by demonstrating an increase in tyrosinase activity. In this study the effect of the tumor promoter, 12-O-tetradecanoylphorbol 13 acetate (TPA), on the hormonal induction of tyrosinase was examined. TPA was found to lower basal levels of tyrosinase activity in melanoma cells and to reduce tyrosinase levels in cells treated with either MSH (10(-7) M), dibutyryl cAMP (10(-4) M), isobutylmethylxanthine (IBMX, 10(-4) M), or with the potent MSH analogue, [Nle4,D-phe7]-alpha-MSH. The phorbol ester, phorbol 12,13-dibutyrate was also effective in lowering tyrosinase activity levels, while 4 alpha-phorbol 12,13-didecanoate, which does not bind protein kinase C, was ineffective. In order to determine how TPA may reduce tyrosinase activity in melanoma cells, the levels of tyrosinase mRNA in untreated or TPA-treated cells were determined by Northern blot analysis. A marked down-regulation of constitutive levels of tyrosinase mRNA was observed in cells treated with the tumor promoter. Tyrosinase mRNA levels in cultures exposed to TPA for 48 h were only 7% of control levels. Tyrosinase mRNA levels in cells treated with both MSH and TPA were also lower than in cells treated with MSH alone. Previous studies from this laboratory have shown that insulin both lowers basal tyrosinase activity in melanoma cells and antagonizes the MSH stimulation of the enzyme. We have now determined that this inhibition is also due to reduced levels of tyrosinase mRNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704322 TI - Porcine retrovirus reverse transcriptase: optimal conditions for its determination. PMID- 1704323 TI - Characterization of EIAV immunogenicity during persistent infections: humoral responses and antigen targets. PMID- 1704324 TI - Prospects for a human immunodeficiency virus/AIDS vaccine. PMID- 1704325 TI - Development of a specific serological test and an efficient subunit vaccine to control bovine leukemia virus infection. AB - Study of the antigenic structure of the Bovine Leukemia Virus (BLV) envelope glycoprotein gp51 with a panel of mouse monoclonal antibodies (MAbs) has allowed the identification of biologically important determinants directly involved in the infectivity of BLV. Considering the various facts reported in this paper, it follows that diagnostic and vaccination procedures that make use of gp51 in a native configuration constitute a prerequisite for the design of an efficient BLV eradication program. To improve the efficacy of a serological detection test, MAbs have been selected as reagents of choice to develop a competition enzyme linked immunosorbent assay (cELISA). Recombinant vaccinia virus expressing gp51 and gp30 was indicated as a very promising protective vaccine against BLV infection. PMID- 1704326 TI - Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites. AB - In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from foreskin and trunk skin. When transplanted onto nude mice, keratinocytes reformed a regular epithelium with expression of the differentiation markers, keratins K1 and K10, involucrin and filaggrin. Tissue homeostasis improved in later transplants, as made apparent by coexpression and regular distribution of K1 and K10. Since this was achieved in transplants, whether in contact with mesenchyme or separated by collagen matrix, renormalization was obviously mediated by diffusible factors. In vitro, the host mesenchymal influence could largely be mimicked by recombining organotypic cultures (keratinocytes on lifted collagen gels) with de-epidermized dermis, but tissue homeostasis was apparently not achieved. Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in isolated cells and reconstituted tissues. The hyperproliferative character of foreskin epidermis, with its less-pronounced stratum granulosum, was maintained in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme. Thus, we could demonstrate with these model systems that: (a) the regulation of keratinocyte growth and differentiation is mesenchyme-dependent; (b) it is mediated by diffusible factors; but that (c) differences between epidermis of different body sites are also controlled by intrinsic programs. PMID- 1704327 TI - Effects of galanin on the opossum internal anal sphincter: structure-activity relationship. AB - The present study was carried out to investigate the effects of porcine galanin (1-29), N-terminal fragment galanin-(1-10), C-terminal fragment galanin-(15-29), and the middle fragment galanin-(7-16) on the spontaneous tension of the opossum internal anal sphincter and on the decrease in the resting internal anal sphincter tension in response to neural stimulation by electrical field stimulation. Galanin and galanin-(1-10) caused a concentration-dependent decrease in the resting tension of internal anal sphincter and an augmentation of the percent decrease in the resting tension with electrical field stimulation. Galanin-(15-29), on the other hand, produced an increase in the resting tension of the internal anal sphincter and had no effect on the electrical field stimulation-induced decrease in the resting tension. Galanin-(7-16) produced no significant effect on the internal anal sphincter. The decrease in the internal anal sphincter tension by galanin and galanin-(1-10) was partially antagonized by tetrodotoxin, whereas the increase in the internal anal sphincter tension caused by galanin-(15-29) was not modified by tetrodotoxin. In contrast to its effect in the internal anal sphincter, galanin caused an increase in the resting tension and suppressed a decrease in the lower esophageal sphincter tension in response to electrical field stimulation. From these findings we conclude that (a) galanin exerts an inhibitory effect on the internal anal sphincter by activating galanin receptors both at the intramural inhibitory neurons and at the internal anal sphincter smooth muscle and that the N-terminal portion of galanin may be responsible for these actions; (b) the contractile action of galanin is produced by its action on the smooth muscle; and (c) the actions of galanin on the gastrointestinal tract are tissue specific. PMID- 1704328 TI - Expression of leukocyte adhesion molecules in the liver of patients with chronic hepatitis B virus infection. AB - Virus-specific T-cell responses are believed to be involved in the pathogenesis of liver cell injury secondary to hepatitis B virus infection. In this study, liver biopsy specimens from patients with chronic hepatitis B virus infection were analyzed for expression of two major pathways of adhesion used by cytotoxic T cells to interact with target cells. The lymphocyte function-associated antigen 3 was found preferentially expressed on hepatocytes of patients with active hepatitis B virus replication, whereas the expression of the intercellular adhesion molecule 1 on hepatocytes seemed more closely related with inflammatory activity. Adhesion molecules were also highly expressed on T lymphocytes found in areas of piecemeal and spotty necrosis, indicating the presence of antigen specific "memory" T cells at the site of hepatocellular injury. This study suggests that the expression of the lymphocyte function-associated antigen 3 on hepatocytes may be important for viral elimination. The coordinate expression of the intercellular adhesion molecule 1 may regulate inflammatory response and enhance viral antigen presentation to T cells. Conversely, the absence of hepatocyte adhesion molecules might be a favorable factor for viral persistence. PMID- 1704329 TI - Characterization of a rat pancreatic secretory protein associated with pancreatitis. AB - A new protein was purified from the pancreatic juice of rats with acute pancreatitis. That protein, not detectable in control animals, was called "pancreatitis-associated protein." It was first observed 6 hours after induction of experimental pancreatitis with taurocholate or cerulein, reached maximal levels of 45 micrograms/mg protein in zymogen granules and 1.8 micrograms/mg protein in pancreatic tissue during the acute phase (48 hours), and disappeared during recovery (day 5). It was never detected in spleen, liver, kidney, heart, or lung. The detection limit of the assay system was 12 ng/mg protein, so that pancreatitis-associated protein levels increased at least 100-fold in pancreatic tissue during the acute phase. The molecular weight (12,000) and isoelectric point (8.2) were determined by two-dimensional gel electrophoresis. Subcellular fractionation and immunoelectron microscopy showed that the protein was synthesized on the rough endoplasmic reticulum and stored in zymogen granules before being secreted, similar to other pancreatic secretory proteins. Immunoblotting and two-dimensional gel electrophoresis revealed that the same protein was synthesized upon induction of pancreatitis by cerulein infusion, by retrograde injection of bile acids, or pancreatitis induced by pancreatic surgery. The pancreatitis-associated protein is therefore an acute-phase protein that differs from other proteins of that family because of its exocrine nature. PMID- 1704330 TI - More news on the cystic fibrosis gene. PMID- 1704331 TI - [Ultrastructural organization and argentophilic areas of the lymphoblastic nucleoli in patients with acute lymphoblastic leukemia]. AB - The ultrastructural organization and the state of silver-staining nucleoli (Ag NOR) of peripheral blood and bone marrow lymphoblasts of 10 patients with acute lymphoblastic leukemia (ALL) were studied in the complex with the biochemical assay of the ribosomal levels in the cellular cytoplasm of these patients. In the majority of the patients investigated the morphological picture of the nucleolus and the character of Ag-NOR have evidenced the high functional activity of pRNA synthesizing apparatus and correlated with the high ribosomal level in the cytoplasm of the cells studied. Significant accumulation of granular components (RNP-particles) in the nucleoli of the lymphoblasts, reflecting disorders in their transport from nucleus to cytoplasm, were detected only in 2 patients. PMID- 1704332 TI - [Morpho-functional characteristics of normal and pathological human megakaryocytes studied by selective silver staining of cell nucleoli]. AB - The activity of nucleolar organizer (NO) in megakaryocytes (MG) from 8 donors, 10 patients with immune thrombocytopenias (IT), 17 patients with chronic myelocytic leukemia (CML) and 14 patients with multiple myeloma (MM) was studied by silver staining. The average number of nucleoli in MG of normal donors comprised 21.8 per nucleus with a range from 16.6 to 33.7. It was significantly lower in MG of CML patients, and, on the contrary, it was higher in MG of IT patients. The average number of Ag grains per nucleus reflecting their activity in relation to ribosomal RNA synthesis was found to be the highest (127 +/- 32.1) in MG of IT patients but rather low (43.2 +/- 7.2) in CML patients as compared to those of the control (76.5 +/- 11.1) and MM patients (86.0 +/- 5.6). The differences in the functional state of MG in varying diseases as well as possibilities of using this new approach in hematology have been discussed. PMID- 1704333 TI - [Evaluation of Ag-positive chromosomal nucleolus organizer regions in malignant cell interphase]. PMID- 1704334 TI - [Various activities of the nucleolus organizer region in normal and leukemic bone marrow cells: semiquantitative data and computer- assisted image analysis by silver staining]. AB - A cytochemical technique with silver nitrate staining was used to study the nucleolar organizer activity in metaphase spread of bone marrow cells from 13 patients with acute lymphocytic leukemia (ALL), 11 patients with chronic myelogenous leukemia (CML), 7 patients with acute myelogenous leukemia (AML), and 4 normal persons. Additionally, computer-assisted image analysis was used to quantitate the amount of silver staining in interphase nuclei of bone marrow cells from 6 untreated ALL patients, 3 normal subjects and 1 bone-marrow transplant recipient. The results obtained have indicated that the nucleolar organizer reactivity (NOR) is significantly lower in the control group than that in ALL patients. NOR activity level is significantly lower in both CML patients in chronic phase, and AML patients than in the ALL group, and is similar to that in the control group. When the data obtained for the interphase nuclei were compared with those obtained for the metaphase spread, a strong correlation was recorded between the fraction of bone-marrow metaphases stained positively with silver, the average number of silver-positive nucleolar organizer regions per metaphase, and the amount of silver staining in the interphase nuclei. Silver staining used for the detection of these disease-related differences in NOR activity can serve as a diagnostic procedure in evaluating human leukemias. The computer-assisted image analysis of bone marrow cell interphase nuclei would be useful for more accurate resolving biological and medical problems. PMID- 1704335 TI - Monoclonal antibodies against serotype specific and conserved epitopes in morphotype E Escherichia coli flagellins. AB - Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype strains belonging to morphotype E. Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45. Several mAbs which recognised conserved epitopes were also examined. mAb 7-56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filament surface. Similarly, mAb 1-5.1 recognised an internal epitope shared only by serotypes H1 and H12. Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments. PMID- 1704336 TI - Development of sensitive methods for the detection of mycobacteria by DNA probes. AB - A sensitive method allowing the detection of mycobacteria by DNA probing has been developed. Besides the choice of a relevant probe encoding for part of the ribosomal RNA genes, the critical step allowing this high sensitivity was the method by which mycobacteria were lysed. Sonication of mycobacteria in the presence of chloroform followed by dot blot by this lysate gave the highest sensitivity in the detection of sequences homologous to the DNA probes. PMID- 1704337 TI - A new defective phage containing a randomly selected 8 kilobase-pairs fragment of host chromosomal DNA inducible in a strain of Bacillus natto. AB - A new defective phage, designated PBND8, was induced in Bacillus natto strain IAM1207 with bleomycin and mitomycin C. PBND8 particles contained a randomly selected 8 kilobase-pairs (kbp) fragment of the host chromosomal DNA. Electron microscopy showed that PBND8 has a small head with a complex tail structure like PBSX, a defective phage of Bacillus subtilis 168. The PBND8 head, however, is clearly smaller than that of PBSX which contains 13-kbp fragments of the host chromosomal DNA. SDS-polyacrylamide gel electrophoretic analysis revealed that the structural proteins of PBND8 are distinct from those of PBSX and PBSY (PBSZ) of B. subtilis W23. PBND8 exhibited a bacteriocin-like killing activity to the other Bacillus cells. PMID- 1704338 TI - [Transcription of Drosophila MDG4 mobile element under hyperthermic conditions]. AB - The Drosophila melanogaster cultured cells were subjected to stable transformation by cotransfection with two plasmids, one of which conferred G418 resistance and the second contained the Drosophila retrotransposon MDG4 (gypsy) under control of different promoter fragments of the heat shock protein gene hsp70. Transcription of these constructs as well as of the endogenous gypsy was examined in the conditions of heat shock. Active degradation of preexisting MDG4 transcripts is observed after heat shock. Transcription of MDG4 is restored during recovery but its termination and/or 3' end processing becomes aberrant. PMID- 1704339 TI - Effects of free radicals on the electrophysiological function of cardiac membranes. AB - Abnormal electrical activity in heart cells can result in irregular heart rhythms or arrhythmias. Any form of pathological or toxicological damage to the sarcolemmal membrane presents the risk of precipitating arrhythmias and compromise of the heart's function as a pump. An array of cardiovascular conditions from coronary artery disease and myocardial infarction to cardiomyopathies and hypertrophy, can induce arrhythmias. Many of these conditions recently have been linked to increases in free radical production. Early studies suggesting a role for free radicals in the abnormal function of ischemic and reperfused hearts use anti-free radical interventions to reduce arrhythmias. More recent works have taken advantage of different free radical generating systems to show a reproducible sequence of changes in the cellular action potential; these data suggest changes in the transmembrane movement of ions through membrane channels. Biochemical evidence supports a possible involvement of ion exchange mechanisms in the cardiac sarcolemma. All the evidence indicate that free radical injury may have profound effects on the electrical function of myocardial cells. PMID- 1704340 TI - Auditory brainstem responses in children with developmental speech disorders. PMID- 1704341 TI - Antigen processing and presentation. AB - An overview of the various aspects of antigen degradation and presentation is given with special emphasis on the possible occurrence of variation in the enzymatic machinery present in different cells or individuals. Different procedures for epitope mapping are also presented as well as the characterization of universal epitopes in humans. The latter finding is of considerable importance for the development of subunit vaccines. Finally, the structure of T cell epitopes is discussed. PMID- 1704342 TI - Characterization of regulatory T cell responses to defined immunodominant T cell epitopes of the Plasmodium falciparum antigen Pf155/RESA. AB - Several immunodominant B and T cell epitopes of the P. falciparum blood stage antigen Pf155/RESA, a vaccine candidate, are located in the central (5') and C terminal (3') invariant repeat regions of the molecule. Here we have attempted to functionally analyze human T cell responses to some of the T cell epitopes. For this purpose short synthetic peptides corresponding to these epitopes were used to study the induction of in vitro expression of IL-4 mRNA, IFN-gamma secretion, proliferation and B cell help for antibody production. In individual malaria immune donors these different T cell activities were not correlated. The findings emphasize the importance of examining multiple parameters of T cell activation when estimating the total proportion of individuals responding to a defined antigen. IL-4 mRNA was expressed in activated T cells of donors who had elevated serum concentrations of antibodies to the peptide used for T cell activation. These results suggest the involvement of IL-4 producing T helper cells in the induction of Pf155/RESA specific antibody production in individuals in which immunity has been induced by natural infection. Taken together, these findings also suggest that functionally distinct CD4+ T cells occur in humans similarly to what has been described in mice. In further experiments, we have also attempted to establish MHC class II restriction of the immune response to these epitopes at the level of the donor populations. When studying monozygotic twins, antibody responses to Pf155/RESA derived peptides and some of the T cell responses could be paired within the twin pairs, indicating a genetic regulation of their B cell responses. Whether or not this regulation reflects MHC class II restriction, or other factors needs to be elucidated. PMID- 1704343 TI - The T cell reactivity against the major merozoite protein of Plasmodium falciparum. AB - We have undertaken a systematic search for T cell epitopes within the sequence of the major merozoite surface antigen (GP190) of Plasmodium falciparum. Recombinant polypeptides expressed in E. coli were used to evaluate the reactivity of peripheral blood mononuclear cells (PBMC) from both inhabitants of a rural community of West Africa exposed to P. falciparum transmission and from German patients with diagnosis of acute malaria. Although the proliferative response of the PBMC was in most cases very low, several T cell clones could be established. Deletion analysis of each gp190-derived polypeptide allowed the identification of six different T cell epitopes. Epitopes could be mapped within the dimorphic region of gp190, which also contains the sequences most frequently recognized by sera from adult individuals living in endemic areas. PMID- 1704344 TI - Responses of T cells from sensitized donors to recombinant and synthetic peptides corresponding to sequences of the Plasmodium falciparum SERP antigen. AB - In the present work, we intend to determine the capacity of human lymphocytes to recognize subfragments of the serine-stretch protein SERP, a blood-stage antigen from Plasmodium falciparum. Individuals sensitized by a previous P. falciparum infection were studied. Some recombinant proteins (RP) including RP7 and RP10 (amino acids 631-684 and 631-892 of SERP, respectively), were recognized in proliferation assays by lymphocytes from 28 sensitized individuals and not by lymphocytes from control, non-sensitized, donors. Synthetic peptides covering predefined zones of particular interest were tested and appeared to induce proliferative responses of lymphocytes from sensitized donors, allowing identification of putative T cell epitopes. PMID- 1704345 TI - Amino acid sequences recognized by T cells: studies on a merozoite surface antigen from the FCQ-27/PNG isolate of Plasmodium falciparum. AB - Twenty-six overlapping peptides, spanning the entire FCQ-27/PNG sequence of the Plasmodium falciparum antigen known as merozoite surface antigen 2 were screened for their ability to induce the proliferation of peripheral blood lymphocytes (PBL) obtained from 12 donors living in Honiara, Solomon Islands where P. falciparum is endemic. A recombinant (r) form of MSA2, known as Ag 1609 was also screened in these assays and tetanus toxoid (TT) antigen was included as a control. The location of the predicted T cell determinants within MSA2 was examined using the algorithm, AMPHI and by scanning MSA2 for amino acid sequences showing the Rothbard motif. There were 13 predicted amphipathic helical sites and five examples of Rothbard sequences in the antigen. The location of these with regard to the peptides tested is shown. Nine of the 12 individuals responded to TT with high stimulation indices (greater than 4) being obtained in the majority of donors. Only three individuals responded to r-MSA2 with the stimulation indices (SI) in the range of 2.4-4.1. Peptides from both the constant and variable regions of MSA2 were recognized in the proliferative assays. However, the majority of the positive proliferative responses were to peptides which spanned the central variable region which included the two copies of the 32-amino acid repeat occurring in the antigen. High SI comparable to those obtained to TT were seen in some individuals with some peptides. There was considerable variation between donors in number and nature of the peptides recognised and two donors did not respond to any of the antigens tested. The significance of these findings to vaccine development is discussed. PMID- 1704346 TI - Variation of T cell epitopes of the Plasmodium falciparum CS protein: its occurrence, extent and relevance. PMID- 1704347 TI - Longitudinal study of the cellular response to Pf155/RESA and circumsporozoite protein in Madagascar. AB - In a community follow-up conducted in the Central Highlands of Madagascar, the cellular responses to synthetic peptides from the immunodominant regions of Pf155/RESA and the repeat region of the circumsporozoite protein were studied. Seasonal variations of the T cell response were measured at the individual level; the relationship between this response and the presence of parasites in blood was assessed; the question of the possible protective value of the lymphocyte proliferation in presence of a specific antigen was addressed. PMID- 1704348 TI - Isolation and characterization of protective cytolytic T cells in a rodent malaria model system. AB - Protective immunity against malaria is induced by immunization with irradiation attenuated sporozoites. Here we report the isolation of cytolytic T-cell (CTL) clones from BALB/c (H-2d) mice immunized with either Plasmodium berghei or Plasmodium yoelii sporozoites. The epitopes recognized by these CTL can be mimicked by synthetic peptides corresponding to a homologous region in the CS proteins of both rodent malaria species. Both peptides are recognized by the CTL in the context of the same MHC class I molecule, H-2 Kd. In vivo adoptive transfer of the CTL clones into non-immune syngeneic mice protected them from a lethal challenge of infectious sporozoites. PMID- 1704349 TI - Protective anti-sporozoite antibodies induced by a chemically defined synthetic vaccine. AB - Chemically defined synthetic polymers, known as multiple antigen peptide systems (MAPs) represent an effective and novel approach for engineering peptide-based vaccines. Ten different mono and diepitope MAP models, containing different arrangements and stoichiometry of functional B and/or T helper epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize mice. High titers of antibody and protective immunity against sporozoite challenge were elicited by MAPs containing T and B epitopes arranged in tandem and in equimolar amounts. These results indicate that MAPs may serve as a basis for developing subunit vaccines to induce high levels of antibodies against sporozoites. PMID- 1704350 TI - Peptide-primed CD4+ cells and malaria sporozoites. AB - We have mapped a T cell epitope in the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-mer synthetic peptide corresponding to the amino acid positions 59-79 (referred to as Py1), induced specific proliferation in BALB/c and C57BL/6 mice, and provided help for the production of antibodies to peptides from the repetitive region, (QGPGAP)n, of the same CS protein, when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Long-term CD3+CD4+CD8-TCR alpha beta+ T cell lines and clones were derived from both strains of mice. These lines and clones, that proliferated in an MHC-restricted fashion, did not recognize peptides from the homologous region of another murine malaria parasite, P. berghei. About 50% of these clones produced detectable amounts of IFN-gamma and IL-2, whereas the remaining produced IL-4, IL-5, and IL-6. In preliminary experiments, some of these clones specifically inhibited P. yoelii sporozoite development in vitro and conferred protection in vivo in passive transfer experiments. These findings show that heterogenous T cell populations are activated in mice upon immunization with a short peptide from the P. yoelii CS protein and that some of these cells could be active in the effector arm of the immune response against malaria sporozoites. PMID- 1704351 TI - Plasmodium falciparum sexual stage antigens: immunogenicity and cell-mediated responses. AB - Antibody and cell-mediated immune responses to the transmission-blocking target antigens of Plasmodium falciparum, Pfs 48/45, were determined in infected non immune patients and in immune individuals from an endemic area. Characterization of the B cell epitopes with monoclonal antibodies showed that there were five regions identifiable but there could be interactions between them causing either competitive or enhancing effects. Sera from infected non-immune patients contained antibodies that would compete with one or more of the mAbs to the different epitopes. Immune responsiveness to purified Pfs 48/45 in P. falciparum immune adults measured as lymphoproliferation, production of interferon-gamma, or as Pfs 48/45-specific antibody was very limited. This did not appear to be due to MHC class II restriction, to diversity in structure of the parasite antigens or to a failure of immunological memory. The antibody-response data were more consistent with down-regulation of immunity as a result of prolonged exposure to infection. PMID- 1704352 TI - Immunogenicity of Plasmodium falciparum sexual stage antigens: implications for the design of a transmission blocking vaccine. AB - Immunogenicity of sexual stage antigens and boosting of transmission blocking antibodies following a natural infection are two critical factors in the design of an effective, subunit vaccine to block the transmission of malaria from man to mosquito. Immunogenicity and boosting are both T cell-dependent. Antigens, such as the 230-kDa, the 48/45-kDa, and the 40/10-kDa, expressed early in the extracellular forms of the sexual stage of Plasmodium falciparum, have limited immunogenicity in humans and in mice. In contrast, Pfs25, expressed predominantly in zygotes and ookinetes, has widespread immunogenicity in mice. Pfs25 expressed by a recombinant vaccinia virus (vSIDK) is also widely immunogenic in mice, and induces transmission blocking antibodies following multiple inoculations with vSIDK. The implications of these immunogenicity data are discussed relative to the design of an effective transmission blocking vaccine. PMID- 1704353 TI - Histomorphologic and immunophenotypic spectrum of primary gastro-intestinal B cell lymphomas. AB - In order to compare primary gastro-intestinal (GI) B-cell lymphomas histomorphologically and immunophenotypically with orthologous steps of B-cell differentiation within the mucosa-associated lymphoid tissue (MALT) of the GI tract, a comprehensive panel of well characterized leucocyte differentiation antigens was composed. It comprised immunoglobulin constituents CD5, CD10, CD11c, CD20, CD23, CD24, CD30, CDw32, CD38, CD39, CDw75, CD76, and vimentin. These antigens yield characteristic immunoprofiles for the following B-cell compartments of the MALT, per se closely linked to cytologically distinct B-cell phenotypes: mantle zone (MZ), extrafollicular compartment (EF), follicle center (FC), and plasma-cell compartment (PC). An unselected series of 31 MALT B lymphomas (13 of low and 18 of high grade malignancy) was classified histologically in routine preparations and subsequently characterized immunohistochemically using fresh frozen tissue, monoclonal antibodies (MAbs) against the antigen panel listed above, and an indirect immunoperoxidase method. The final classification considered both morphology and immunoprofile of tumor cells. Ten tumors were "typical" in both respects: 2 closely corresponded to MZ, 5 to EF, 2 to FC and 1 to PC. The remaining 21 cases were characterized as "atypical" because of anaplastic cytology and/or abnormal co-expression and/or loss of antigens. A hybrid EF/FC phenotype was most frequently observed together with centrocyte-like or centrocytic anaplastic cytology of tumor cells. We conclude that MALT B-cell neoplasia comprises a broad spectrum of histo- and immunophenotypes ranging from well differentiated forms closely mimicking normal B-cell development to highly abnormal tumors which cannot be subclassified. PMID- 1704354 TI - Squamous-cell carcinoma antigen (SCC-A) values related to clinical outcome of pre invasive and invasive cervical carcinoma. AB - The serum concentration of squamous-cell carcinoma antigen (SCC-A), a subfraction of tumour antigen, was determined by RIA from healthy donors (control group) and from patients with malignant cervical disease. Ninety-six percent (173/180) of the healthy patients had squamous-cell carcinoma antigen serum levels below 2 ng/ml. Ten of 70 (14.3%) patients with CIN III, 53.8% (34/62) of patients with invasive squamous-cell carcinoma stage I, 85.8% (30/35) with stage II and 96.5% (27/28) with stage III/IV had squamous-cell carcinoma antigen serum levels above 2 ng/ml. We observed that 22.5% (11/49) of patients with a tumour volume below 10 ml and 92.6% of patients with a tumour volume greater than 10 ml had squamous cell carcinoma antigen levels above 2 ng/ml (p less than 0.005). SCC-A was correlated with recurrence or progressive disease in 90.0% of cases. Other risk factors such as depth of invasion, microscopic parametrial involvement, lymphatic and/or vascular space permeation and histological grade were not correlated with squamous-cell carcinoma antigen. Furthermore, this marker increased 4.3 +/- 2.7 months before clinical evidence of recurrence or progressive disease. We conclude that serial serum levels of squamous-cell carcinoma antigen provide a means for early detection of recurrence or progressive disease. This tumour marker might also be useful for monitoring the treatment effects and has some prognostic value. PMID- 1704355 TI - Binding of epidermal growth factor-dextran conjugates to cultured glioma cells. AB - Some gliomas, melanomas and squamous carcinomas have large numbers of EGF receptors which could, in these cases, be used for targeting with toxic agents. We investigated whether EGF could be conjugated to dextran, which is a suitable carrier for toxic agents, without losing its ability to bind to the receptor. Dextran of 20 kDa molecular weight was activated with I-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) yielding highly active pyridinium-isourea derivatives. EGF was coupled to the activated dextran through the amino terminus and glycine was added to block residual activity. The EGF-dextran conjugate was, after purification on Sephadex G25 and Sephacryl 200 columns, tested for its receptor binding properties on human malignant glioma, U343MGaC12:6, cells. The conjugate inhibited binding of 125I-EGF in a competitive assay, showing that the binding was receptor-specific. Dextran conjugated with glycine, without EGF, had no inhibitory effect. The conjugate was radio-labelled either on the EGF part with 125I or on the dextran part with 3H-glycine, and the internalization patterns were compared to the internalization of 125I-EGF. The radioactivity of the conjugates remained cell-associated for more than 20 hr, regardless of whether the radioactivity was on the EGF or on the dextran part, while the radioactivity of unconjugated EGF rapidly disappeared from the cells. Most of the cell-associated radioactivity was, at all analysed time intervals, located intracellularly. Thus, it seems promising to use dextran, conjugated with EGF, as a carrier of, for example, toxic radioactive nuclides. PMID- 1704356 TI - Target lysis by human LAK cells is critically dependent upon target binding properties, but LFA-1, LFA-3 and ICAM-1 are not the major adhesion ligands on targets. AB - The cytotoxicity mediated by the CD2+ CD3- lymphocyte subset, either NK or LAK, is puzzling since no specific antigen recognition structures, equivalent to the CD3-associated heterodimer T-cell receptor, have been recognized on these cells so far. The possibility exists that the CD3- cytotoxic effectors recognize their targets through non-specific adhesion mechanisms. The goal of this study was: (a) to examine the correlation between binding properties and susceptibility to lysis of 6 informative target cell lines; (b) to evaluate the role, as ligands on these targets, of adhesion molecules such as LFA-1, LFA-3 and ICAM-1. The effectors used in this study were IL-2-activated LGL, predominantly CD3-, or highly purified CD3- lymphocytes from normal human donors. The 6 target lines studied included 2 pairs of EBV-transformed B-cell lines (721 LCL vs. 721.134, and MM vs. MM-10F2) in which the parental lines were resistant to lysis while HLA variants were susceptible. A third pair was the Daudi Burkitt cell line, susceptible to LAK lysis, and an HLA-positive transfected Daudi line which was more resistant to lysis. The binding properties of these targets to LAK effectors (conjugate formation) were evaluated using a sensitive double fluorescence flow cytometry method. In each pair examined, the susceptible targets formed more conjugates and were surrounded by more cytotoxic LAK effectors than their resistant counterparts, indicating that the conjugation properties of targets are closely correlated with their susceptibility to LAK lysis. The expression of adhesion molecules on the informative targets was examined by indirect immunofluorescence and their role was evaluated by inhibition of lysis after pre-coating the targets with the relevant antibodies. The differences in the expression of the classical cell-cell adhesion molecules LFA-1, LFA-3 and ICAM-1 on the target surfaces were only marginal, insufficient to explain the striking differences in susceptibility to lysis and in binding properties. Coating the target cells with antibodies directed against these adhesion determinants had no effects on the lysis of susceptible target cells. The same antibodies reacting with the LAK effectors did inhibit lysis. Taken together, these results suggest that, on the targets, presently undefined membrane adhesion structures may have a major role in conjugate formation between target and CD3- effectors and determine the susceptibility of the targets to lysis. PMID- 1704357 TI - The role of nitric oxide in mediating endothelium dependent relaxations in the human epicardial coronary artery. AB - We have examined the ability of the endothelium of human epicardial coronary arteries to secrete vasorelaxant substances in response to pharmacological stimulation and under basal conditions. In addition, we have attempted to characterise the chemical identity and biochemical pathway for the synthesis of endothelial derived relaxing factor. Human epicardial coronary arteries were removed from patients who were undergoing heart transplantation for reasons other than ischaemic heart disease. Arteries were cut into segments and suspended in 5 ml organ baths containing a modified Tyrodes solution at 37 degrees C, and gassed with a mixture of 95% oxygen and 5% carbon dioxide. Substance P (10(-10) - 10(-7) M) elicited a dose-dependent relaxation of the coronary segments but this action of substance P was dependent upon an intact endothelium. The maximum response of substance P was equivalent to 89 +/- 8.5% of the maximum effect induced by 1 microgram/ml glyceryl trinitrate. This vasorelaxant effect of substance P was unaffected by the presence of 10(-6) M indomethacin. L-NG-monomethyl-arginine (10(-4) M), a specific inhibitor of formation of nitric oxide from L-arginine, antagonised the relaxations induced by substance P, decreasing the maximum response of substance P to 34 +/- 10.5% of the response to glyceryl trinitrate. Upon application, L-NG-monomethyl-arginine caused a further 23.1 +/- 3.0 increase in tension on preconstricted vessels. This increase in tension was reversed with the addition of L-arginine, but was unaffected by D-arginine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704358 TI - Duodenal obstruction in advanced pancreatic carcinoma: how effective is gastroenterostomy in palliation? AB - Gastro-enterostomy produces variable and often disappointing results in the management of duodenal obstruction due to advanced pancreatic carcinoma. In a series of 51 patients who had palliative surgery for carcinoma of the pancreas five required a gastro-enterostomy. Three of these subsequently had difficulty with gastric emptying. The problem of gastro-enterostomy failure in palliation of pancreatic carcinoma is discussed. PMID- 1704359 TI - Beta-thalassemia due to frameshifts at codons 5, 6, 8, and 8/9; hematological observations in heterozygotes. PMID- 1704360 TI - Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen. AB - Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development. PMID- 1704361 TI - BMA031, a monoclonal antibody suited to identify the T-cell receptor alpha beta/CD3 complex on viable human T lymphocytes in normal and disease states. AB - Two types of T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) alpha beta or the more recently identified TCR gamma delta. Whereas TCR alpha beta + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibility complex molecules by proliferation, lymphokine secretion, and/or cytotoxicity, the potential ligand specificities and functions of TCR gamma delta + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR gamma delta + cells play in the immune system. They can be used to quantify TCR gamma delta + cells and TCR alpha beta + cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functional assays. Only few antibodies reactive with common determinants on either TCR alpha beta or TCR gamma delta are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR alpha beta + cells. However, WT31 has recently been shown to cross react with TCR gamma delta. We describe an mAb, BMA031, that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR alpha beta/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR gamma delta mAbs, using TCR alpha beta and TCR gamma delta expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients. PMID- 1704362 TI - Long-term results of infusional 5-FU, mitomycin-C and radiation as primary management of esophageal carcinoma. AB - An analysis of the results of 90 patients with esophageal cancer treated prospectively with combined chemotherapy and radiation without surgery and with a median follow-up of 45 months is presented. Fifty-seven patients with Stage I or II disease received definitive treatment consisting of 6,000 cGy in 6 to 7 weeks and 5-FU (1,000 mg/m2/24 hr) as a continuous intravenous (IV) infusion for 96 hours, starting on days 2 and 29. Mitomycin C (10 mg/m2) was administered as a bolus injection on day 2. Thirty-three patients received palliative treatment (5,000 cGy plus above chemotherapy) for Stage III, IV, or otherwise advanced disease (extraesophageal spread, distant metastases, multiple primary tumors). Follow-up ranged from 1 month to 96 months. Overall median survival of Stage I and II patients was 18 months with 3- and 5-year actuarial survival of 29% and 18%, respectively, while the median disease specific survival was 20 months with an actuarial disease specific survival of 41% and 30% at 3 and 5 years, respectively. A multivariate analysis of sex, histology, tumor location, and tumor size on survival revealed that the effect of stage was highly significant (Stage I versus II, 73% versus 33% at 3 years, p = .01), whereas the effect of sex approached significance (females versus males, 57% versus 34% at 3 years, p = less than .1). The actuarially determined local relapse-free rate for Stage I and II patients at both 3 and 5 years was 70%. Multivariate analysis again indicated stage to be highly significant (Stage I versus II, 100% versus 60% at 3 years, p = less than .01), whereas sex approached significance (female versus male, 75% versus 66% at 3 years, p = .07). The pattern of failure may be altered with this treatment regimen from local to one dominated by distant metastases. Of 29 patients who have failed, 14 (48%) had any component of local failure, whereas 21 (72%) had a distant failure as a component of failure. The median survival of patients with Stage III or IV disease was 9 months and 7 months, respectively. Palliation in this group of patients with advanced disease was good as 77% were rendered free of dysphagia post-treatment, and 60% were without dysphagia until death with a median dysphagia-free duration of 5 months. Severe toxicities were uncommon and nearly all were transient. Eleven of 90 patients (12.2%) had severe acute toxicities, whereas only 3 patients (3.3%) developed significant late treatment-related complications requiring hospitalization for management. PMID- 1704363 TI - Aminoacylation of alanine minihelices. "Discriminator" base modulates transition state of single turnover reaction. AB - RNA hairpin minihelices that recreate the acceptor-T psi C stem of Escherichia coli alanine tRNA are charged specifically with alanine, provided that they encode the critical G3:U70 base pair that is the major determinant for the identity of an alanine tRNA. These model substrates were used to investigate the role in charging of N73, the unpaired nucleotide that is just three positions removed from the amino acid attachment site and which is sometimes referred to as the "discriminator" base. Previous work showed that, while substrates which encode G3:U70 are all charged by alanine tRNA synthetase regardless of the base at position 73, catalytic efficiency is substantially higher with substrates that have the wild-type A73. To identify a specific step in aminoacylation that is affected by substitutions of A73, we studied the single turnover charging of A73, U73, C73, and G73 minihelices, using preformed, enzyme-bound alanyl adenylate and saturating concentrations of the respective minihelices. Compared to the A73 substrate, the transfer of activated amino acid to bound RNA is sharply reduced for the substituted N73 minihelices. The low efficiency of transfer is not due to an abortive reaction in which the adenylate or a transiently charged RNA is hydrolyzed. Instead, under the conditions used, the active adenylate remains on the enzyme for extended periods and simply reacts slowly with bound N73 RNA. The results suggest that the nature of the discriminator base is a critical determinant of the transition state for the reaction of bound alanyl adenylate with RNA on the surface of the enzyme. PMID- 1704364 TI - Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase. AB - Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase. PMID- 1704365 TI - The complete cDNA sequence of human hexabrachion (Tenascin). A multidomain protein containing unique epidermal growth factor repeats. AB - Hexabrachion (Tenascin) is a large glycoprotein that appears in extracellular matrices as a disulfide-linked multimer. It is synthesized in an ordered fashion at particular sites during development, is made in large amounts by certain tumors, and is found in restricted tissue locations in the adult. In this report, we describe the sequence of a full length cDNA of human hexabrachion. The encoded protein contains a total of 2203 amino acids and is a linear array of discrete reiterated domains. At the 5' end are encoded hydrophobic residues and 8 flanking cysteines predicted to be responsible for assembly of hexabrachion polypeptides into a radially arranged, six-armed complex. Following this region are 14 1/2 contiguous 31-amino acid epidermal growth factor-like repeats that have a unique structure with respect to the known examples of this type of domain. Immediately adjacent to these repeats lie 15 uninterrupted segments of approximately 90 amino acids which are similar to the Type III units found in fibronectin. At the carboxyl terminus of the protein is a 210-amino acid domain that is similar to fibrinogen. The domain structure of this protein is consistent with the potential for interaction with multiple ligands and for roles in cell adhesion and/or signaling. PMID- 1704366 TI - Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin. AB - The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI 1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule. PMID- 1704367 TI - Analysis of the altered mRNA stability (ams) gene from Escherichia coli. Nucleotide sequence, transcriptional analysis, and homology of its product to MRP3, a mitochondrial ribosomal protein from Neurospora crassa. AB - The product of the altered mRNA stability (ams) gene of Escherichia coli is involved in decay of mRNA. The complete nucleotide sequence of a 4-kilobase BamHI restriction fragment containing the ams coding sequence was determined. Transcription of the ams gene was analyzed by high resolution S1 mapping. A promoter was found with a homology score of 58% 361 nucleotides upstream from the start codon of ams. The ams structural gene consists of an open reading frame of 2,445 nucleotides. The protein predicted from this open reading frame has a molecular mass of 91,327 Da, which is significantly smaller than that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Confirmation of the ams coding sequence was obtained by comparing the predicted amino acid sequence with that derived by amino-terminal analysis of gel-purified Ams protein. The predicted protein sequence of the ams gene was screened against translations of the GenBank DNA sequence data base. A homology of 18% over a region of 315 amino acids of the carboxyl terminus of the Ams product was found to MRP3, a mitochondrial ribosomal protein from Neurospora crassa. A smaller region of homology (29% in 86 residues) was found to the human U1 small nuclear ribonucleoparticle 70,000-Da protein. PMID- 1704368 TI - Orientation of non-blue cupric complexes on DNA fibers. AB - Three different orientations of non-blue, type 2 cupric complexes on DNA fibers are obtained from EPR data. The cupric complex of bleomycin, CuBlm, binds as described previously (Shields, H., McGlumphy,C., and Hamrick, P., J., Jr. (1982) Biochim. Biophys. Acta 697, 113-120), except possibly with more restricted motion. The square plane of CuBlm makes an angle of about 65 degrees with the fiber axis. The tridentate complex 2-formylpyridine monothiosemicarbazonato Cu2+ binds with its planar structure perpendicular to the fiber axis. In contrast, other tridentate cupric complexes of tripeptides, CuGHK and CuGHG, bind with the square plane parallel to the fiber axis. The bound forms of Cu(GHK) and Cu(GHG) are determined mostly by the GH moiety in the complex; the contribution of lysine in defining the orientation of the copper moiety is minimal. Thus, the structure of the ligand determines the orientation of these complexes on DNA. PMID- 1704369 TI - Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9-39) amide, a specific exendin receptor antagonist. AB - Exendin-3 increased cellular cAMP levels and amylase release from dispersed acini from guinea pig pancreas. Low concentrations (0.1-3 nM) caused a 12-fold increase in cAMP, whereas higher concentrations (0.3-3 microM) caused an additional 24 fold increase in cAMP. Maximal cAMP with the highest concentration tested was the same as the maximal response with secretin, vasoactive intestinal peptide (VIP), peptide histidine isoleucine, helodermin, or helospectin-I. In terms of amylase release, exendin-3 had the same efficacy but was the least potent of these peptides. Exendin-3-induced increases in amylase release were inhibited by VIP receptor antagonists and the new peptide (greater than 0.1 microM) competed with radiolabeled VIP for binding sites on dispersed acini. Increasing concentrations of an exendin-3 fragment, exendin-3(9-39) amide, did not increase cAMP or amylase release but inhibited the increase in cAMP observed with 0.1-3 nM exendin-3. The fragment did not alter the effects of other peptides that are known to increase acinar cAMP. We conclude that exendin-3 interacts with at least two receptors on guinea pig pancreatic acini; at high concentrations (greater than 100 nM) the peptide interacts with VIP receptors, thereby causing a large increase in cAMP and stimulating amylase release; at lower concentrations (0.1-3 nM) the peptide interacts with a putative exendin receptor, thereby causing a smaller increase in cAMP of undetermined function. Exendin-3(9-39) amide is a specific exendin receptor antagonist. PMID- 1704370 TI - Secretion incompetence of bovine pancreatic trypsin inhibitor expressed in Escherichia coli. AB - There is increasing evidence that protein folding and protein export are competing processes in prokaryotic cells. Virtually all secretion studies reported to date, however, have employed proteins that are relatively uncharacterized in terms of their folding behavior and three-dimensional structure. In contrast, the structural and biochemical parameters governing the folding of bovine pancreatic trypsin inhibitor (BPTI) and several of its mutants have been studied intensively. We therefore undertook a study of the secretion behavior in Escherichia coli of recombinant BPTI and its mutants. Wild-type BPTI and two well-characterized folding mutants (C14A, C38A)BPTI and (C30A, C51A)BPTI (missing the 14-38 and 30-51 disulfide bonds, respectively), were investigated by analyzing their expression fused to an E. coli signal sequence or to two synthetic IgG-binding domains of staphylococcal protein A. Both disulfide mutants are destabilized relative to wild-type BPTI and exhibit markedly altered folding kinetics: one (C14A, C38A) folds more slowly than wild-type BPTI and the other (C30A, C51A) unfolds more rapidly. Both mutants were observed to be exported 3-10 times more efficiently than the wild-type molecule. Moreover, the levels of unprocessed preprotein in the cytoplasm were severalfold higher for the wild-type fusion than for the fusion to the two folding mutants. Intracellular degradation of the BPTI moiety was also observed. These results are consistent with traffic of intracellular BPTI preproteins on at least three routes along the secretory pathway: (a) facile secretion of unfolded material, (b) intracellular folding leading to secretion blockage, and (c) degradation followed by export of truncated molecules. A novel feature of these findings is the implication that disulfide bonds can form in the bacterial cytoplasm and lead to secretion incompetence. PMID- 1704371 TI - Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180 kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities. AB - The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8 12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit. PMID- 1704372 TI - Modulation of HeLa cell growth by transfected 7SL RNA and Alu gene sequences. AB - Alu and 7SL RNA gene sequences were tested for the potential to regulate mammalian cell growth by introducing these sequences into HeLa cells in a coupled DEAE-dextran transfection/affinity cell sorting system. Both Alu and 7SL RNA genes mediated inhibition of [3H]thymidine and [35S]methionine incorporation in recipient cells. In addition, short term growth curves were performed on calcium phosphate/DNA cotransfected, affinity-purified cells. This second assay revealed that transfected Alu and 7SL RNA can also cause suppression of HeLa cell proliferation. To investigate whether transcription or polymerase III (pol III) transcription factor binding was required for inhibitory activity, mutations were introduced into RNA pol III B block promoter elements in each of these genes. Suppression of [3H]thymidine incorporation was dependent on the presence of this pol III element in both genes; likewise, 7SL RNA-mediated growth suppression required the presence of the pol III B block promoter element. The results of this study indicate that Alu and 7SL RNA gene sequences interact with cellular factors that are important for HeLa cell proliferation and suggest that these pol III-transcribed elements may be involved in the regulation of cellular growth. PMID- 1704373 TI - The genes for the trophoblast interferons and the related interferon-alpha II possess distinct 5'-promoter and 3'-flanking sequences. AB - The genes for trophoblast interferons (IFN) bovine trophoblast protein-1 (bTP-1) and ovine trophoblast protein-1 (oTP-1) are expressed massively in the trophectoderm of preimplantation bovine and ovine concepti during the period of maternal recognition of pregnancy. These 172-amino acid IFN are closely related to the IFN-alpha II, a family of "long" IFN expressed in virus-induced leukocytes. Genomic Southern blotting with a full-length bTP-1 cDNA revealed about 15 genes that bind the probe with varying intensities. By using more specific probes for the 3'-ends of the cDNA, we have shown that only four to five of these represent bTP-1 genes, whereas no more than another four are IFN-alpha II. Genes for bTP-1 and IFN-alpha II, all of which are intronless, have been isolated from bovine genomic libraries, and their nucleotide sequences were compared. Additional bTP-1 genes and two distinct oTP-1 genes have been isolated from bovine and ovine genomic DNA by using the polymerase chain reaction procedure in conjunction with specific 3'- and 5'- primers (derived from the bTP 1 gene sequences determined above). The promoter region up to 100 bases upstream from the transcription start sites of the trophoblast IFN are almost completely conserved across different genes and across species, yet show only limited sequence identity with IFN-alpha II. Two putative binding regions for interferon regulatory factor-1 as well as several GAAANN motifs (where N is any nucleotide) that have been implicated in viral responsiveness of alpha 1- and beta-IFN genes are retained at identical positions in all of the trophoblast genes. Putative interferon regulatory factor-1-binding nucleotide hexamers and GAAANN motifs are also present in the IFN-alpha II genes, but these are organized very differently than in the trophoblast IFN genes. A GAAATG motif is present in IFN-alpha II promoters but is absent in trophoblast IFN genes. Based on the evidence presented, it is proposed that the trophoblast interferons constitute a separate subclass of IFN-alpha distinct from IFN-alpha II and IFN-alpha I. PMID- 1704374 TI - Platelet adhesion onto the Langmuir-Blodgett film of poly(gamma-benzyl L glutamate)-poly(ethylene oxide)-poly(gamma-benzyl L-glutamate) block copolymer. PMID- 1704375 TI - High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. AB - The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4 methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities. PMID- 1704376 TI - Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils. AB - The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence. PMID- 1704377 TI - Studies on the mechanism of scatter factor. Effects of agents that modulate intracellular signal transduction, macromolecule synthesis and cytoskeleton assembly. AB - Scatter factor (SF) is a cytokine that causes cohesive epithelial colonies to 'scatter' into isolated cells and stimulates epithelial cell migration. To investigate SF's mechanism(s), we screened agents that modulate various intracellular processes for effects on scattering of Madin-Darby canine kidney (MDCK) cells. Selected agents were studied in quantitative migration assays using microcarrier beads. Agents that activate the adenylate cyclase (AC) pathway caused mild to moderate inhibition of scattering and migration, while modulators of Ca2+/calmodulin pathways had little effect on scattering. In contrast, phorbol esters (PMA, PDD) and protein kinase C (PKC) inhibitors (staurosporine, H-7, 7,8 dihydroxychlorpromazine) markedly enhanced and accelerated scattering; PMA and staurosporine also stimulated migration. Diacylglycerol analogues (e.g. diC8), naphthalenesulfonamide PKC activators (SC-9, SC-10) and inactive phorbol esters (e.g. 4a-PDD) did not potentiate scattering, while PKC depletion by 48 h pre incubation with PMA markedly stimulated scattering. Thus, PMA-enhanced scattering may be related to down-modulation of PKC. Scattering was blocked by inhibitors of protein and RNA but not DNA synthesis; SF- and agent-stimulated migration were ablated by cycloheximide. Scattering and migration were inhibited by an anti microfilament (cytochalasin B) but not anti-microtubule (e.g. colcemid) agents. These findings suggest that SF-induced epithelial mobility may be mediated, in part, by protein synthesis, alterations in protein phosphorylation (?inhibition of PKC), and actin filament reorganization. They indicate directions for further studies. PMID- 1704378 TI - Temporal expression, polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis. AB - A novel domain of epitopes is expressed by a family of high-Mr proteins at the anterior pole of the germ cell nucleus during spermiogenesis, and later by two low-Mr proteins at the anterior and posterior poles of the nucleus during sperm maturation in the epididymis. Initially, monoclonal antibodies (mAbs) PNT-1 (IgG2b) and PNT-2 (IgG2a) bound to antigens present in a cap-like configuration at the apical pole of the spermatid nucleus at step 5 of spermiogenesis. The distribution of epitopes on the nucleus expanded posteriorly until, in testicular sperm they covered the anterior pole down to the distal limits of the subacrosomal perforatorium. By contrast, sperm from the epididymis and vas deferens bound both mAbs in two distinct regions on the nucleus, one on the dorsal margin of the anterior pole, and the other in a ventral zone at the posterior pole. On SDS-PAGE and isoelectric focusing (IEF) immunoblots, both mAbs bound three major proteins with Mr of approximately 80,000, 77,000 and 75,000 from spermatids and testicular sperm, and proteins of Mr 50,000 and 48,000 in epididymal and vas deferens sperm. Both the high- and low-Mr protein families were recovered in germ cell nuclear/perinuclear matrices. Their mobilities on SDS PAGE were not altered by exo- or endoglycosidases or by aminoethylation in denaturing conditions. mAb PNT-1 bound to the sperm proteins with a Ka of 3.53 x 10(12) M-1 and mAb PNT-2 with a Ka of 2.08 x 10(12) M-1. From competition binding data, mAbs PNT-1 to -10 appeared to recognize six adjacent or overlapping epitopes on the same proteins. These data suggest the high-Mr proteins, the thecins, present at the anterior pole of haploid germ cells are processed at the onset of sperm maturation to yield two low-Mr proteins that then occupy two distinct domains at the anterior and posterior poles of the nucleus. PMID- 1704379 TI - Detection and identification modes for the highly sensitive and simultaneous determination of various biogenic amines by coulometric high-performance liquid chromatography. AB - Detection and identification modes for the rapid, selective, highly sensitive and simultaneous determination of catecholamines, indoleamines and related metabolites by high-performance liquid chromatography (HPLC) with series of coulometric working electrodes (CWE) were investigated. Five detection modes were examined: (1) oxidative single mode using a single CWE, (2) oxidative screen mode using a series of two CWE, (3) redox mode using a series of two CWE, (4) redox reductive screen mode using a series of three CWE and (5) redox-reductive screen mode using a series of four CWE. For the highly sensitive detection of catechol compounds, oxidative single, redox and redox-reductive screen modes were suitable. Oxidative single and oxidative screen modes were better than the other modes for indole and o-methylated catechol compounds. For the selective detection of these compounds, however, the redox-reductive screen mode was best. The specific ratio obtained in HPLC with the redox or redox-reductive screen mode is useful as an index for identification purposes. These findings suggest that HPLC with the redox-reductive screen mode of detection is applicable to neuroscience studies. PMID- 1704380 TI - Separation of high-molecular mass RNAs by high-performance liquid chromatography on hydroxyapatite. AB - High-molecular-mass RNAs [transfer-(t-), 5S-, 18S- and 28S-RNA] in 25 mM sodium acetate buffer (pH 6.0) were separated by high-performance liquid chromatography (HPLC) on hydroxyapatite using a linear gradient (120 min-duration) from 0.03 to 0.147 M of phosphate buffer (pH 7.0) containing 0.3 M potassium chloride and 1 mM sodium azide with a slope of 2 mM/ml at a flow-rate of 0.5 ml/min. When the RNAs were dissolved in 4 M guanidine isothiocyanate-25 mM sodium acetate buffer (pH 6.0)-0.1 M beta-mercaptoethanol (4 M GIT), t-, 5S- and 18S- or 28S-RNAs but not 18S- and 28S-RNAs were separated. RNAs extracted from rat superior cervical ganglia with 4 M GIT could be separated. Thus, HPLC on hydroxyapatite is a rapid and accurate means of quantifying and/or preparing high-molecular-mass RNAs such as t- and ribosomal RNAs. PMID- 1704381 TI - Analysis of low molecular weight hydrocarbons including 1,3-butadiene in engine exhaust gases using an aluminum oxide porous-layer open-tubular fused-silica column. AB - A method for the quantitative analysis of individual hydrocarbons in the C1-C8 range emitted in engine exhaust gases is described. The procedure provides base line or near base-line resolution of C4 components including 1,3-butadiene. With a run time of less than 50 min, the light aromatics (benzene, toluene, ethyl benzene, p- and m-xylene, and o-xylene) are resolved during the same analysis as aliphatic hydrocarbons in the C1-C8 range. It is shown that typical 1,3-butadiene levels in engine exhaust are about 5 ppm at each of two engine conditions. Aromatic hydrocarbon levels show a dependence on engine operating conditions, benzene being about 20 ppm at high speed and about 40 ppm at idle. PMID- 1704382 TI - Comparison of two O-serogrouping systems for mesophilic Aeromonas spp. AB - Two O-serogrouping systems for mesophilic Aeromonas spp., National Institute of Health, Tokyo, Japan, and National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands, containing 44 and 30 reference strains, respectively, were compared. Nine strains in each system were considered identical. Thirty-four unique strains were identified, and 22 strains of the two systems were in an a,b-a,c type of relationship to each corresponding O group. PMID- 1704383 TI - Purification and characterization of Giardia lamblia antigens in the feces of Mongolian gerbils. AB - In a recent study, we identified Giardia lamblia 65- and 70-kDa antigens in the feces of infected Mongolian gerbils. The 65-kDa antigen was from a strain isolated from a human with symptoms of giardiasis, and the 70-kDa antigen was from a strain isolated from a human with no symptoms of giardiasis. In this study, we used preparative electrophoresis and electroelution techniques to purify these antigens to a degree which showed a single discrete protein band on silver-stained polyacrylamide gels. By enzyme-linked immunoelectrotransfer blot, common epitopes on the 65- and 70-kDa antigens were indicated by their cross reactivity with rabbit anti-65-kDa and anti-70-kDa sera. By indirect immunofluorescence assay, the cysts and trophozoites of the two strains cross reacted with these sera. Of seven lectins tested, only concanavalin A bound to the 70-kDa antigen, suggesting a glycoprotein, and it possessed a low isoelectric point as assessed by preparative isoelectric focusing. Molecular mass estimations of these antigens by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis were similar to the 65- and 70-kDa estimations obtained by native polyacrylamide gradient gel electrophoresis. Although the 65- and 70-kDa antigens proved to be resistant to 100 degrees C heat and stable in storage for up to 25 months at -20 degrees C, neither appeared to be the same as a fecal G. lamblia antigen with similar molecular mass found by other investigators. This suggests that variable G. lamblia antigens may be found in the feces of infected humans. PMID- 1704384 TI - Binding of S protein by Neisseria gonorrhoeae and potential role in invasion. AB - An agglutination assay was used to examine the binding of purified human S protein (vitronectin, serum spreading factor) to 201 clinical isolates of Neisseria gonorrhoeae. Strains belonging to the protein IA serovars were significantly (P less than 0.001) more reactive in agglutination tests with human S protein and were more serum resistant than strains belonging to the protein IB serovars. The strains from patients with disseminated infections belonged predominantly to the IA serovar (19 of 23) and, with the exception of IA-4 and certain IB serovars, avidly agglutinated with S protein. The serovar IA-4 and IB strains isolated from joint or cerebrospinal fluid failed to agglutinate with S protein and appeared to be less serum resistant than most other IA isolates. Cysteine hydrochloride or 2-mercaptoethanol inhibited agglutination of S protein and a more than twofold increase in resistance to killing by fresh human serum following preincubation with S protein; the serum-sensitive parent strain did not agglutinate S protein, and serum resistance was not increased following preincubation with this protein. Binding of S protein by gonococci may represent a novel pathogenic mechanism that can contribute to serum resistance. PMID- 1704385 TI - Use of rapid, nonradioactive DNA probes in culture confirmation tests to detect Streptococcus agalactiae, Haemophilus influenzae, and Enterococcus spp. from pediatric patients with significant infections. AB - Acridinium ester-labeled, chemiluminescent DNA probe tests (Accuprobe; Gen-Probe, Inc.) for culture confirmation of Streptococcus agalactiae, Haemophilus influenzae, and Enterococcus spp. were compared with conventional identification techniques. The probe is a DNA oligomer that is a complementary to the RNA of the target. The DNA-RNA hybrids are measured in a luminometer. The 40-min assay requires one reaction tube and the addition of three reagents. When two colonies were used to add a sample of the reaction tube, 325 of 327 isolates were detected by the probe. Isolates of 404 nonprobe target organisms did not hybridize with the probe. PMID- 1704386 TI - Radioimmunoassay for insulin-like growth factor (IGF) II: interference by pure IGF-binding proteins. AB - A new radioimmunoassay for insulin-like growth factor-II (IGF-II) is described. Compared to recombinant DNA-derived IGF-II standard, the cross-reactivity of natural or recombinant IGF-I was less than 1%. The ED50 for IGF-II standard was 1.0 ng/ml, and the mean IGF-II level in acid-ethanol-extracted serum from healthy adults was 525 +/- 87 ng/ml (SD, n = 30). Addition of the IGF binding protein IGFBP-1 (BP-28, PP12) caused dose-dependent inhibition of IGF-II tracer binding to antiserum, increasing to greater than 90% inhibition at 400 ng/ml IGFBP-1. In contrast, the IGF binding protein IGFBP-3 (BP-53) caused approximately 30% inhibition of tracer binding at 20 ng/ml IGFBP-3, with no further inhibition up to 400 ng/ml IGFBP-3. The influence of added IGF binding proteins on IGF-II displacement curves varied depending on both the type and concentration of binding protein added. It is concluded that interference in IGF radioimmunoassays by IGF binding proteins depends both on the types of binding proteins present, and on the IGF concentration, in the test samples. PMID- 1704387 TI - Radioimmunoassay for human type II collagen. AB - Human articular cartilage type II collagen (h coll.II) was purified and used to develop a radioimmunoassay. The sequential saturation procedure allowed a sensitivity of 3 ng/tube. The intra and between assay coefficients of variation were less than 10 and 20% respectively in the linear part of the curve. The assay was highly specific for native human articular type II collagen. There was no cross-reactivity with other constituents of cartilage: human proteoglycans, fibronectin, laminin and hyaluronic acid did not interfere with the assay. No cross-reactivity existed with bovine collagen types I, III, IV. However, native collagens from human placenta (I, III, IV, V, VI), rat and calf skin type I collagens and bovine type II collagen produced a weak cross-reaction only at high doses. Concerning the latter, inhibition curves were not parallel. Parallelism of inhibition curves were observed for dilution of type II collagen, produced by human chondrocytes in three-dimensional culture. All of these characteristics indicate that radioimmunoassy of type II collagen is a very sensitive and specific method available for the study and quantification of type II collagen in in vitro experimental conditions. PMID- 1704388 TI - Airway effects of inhaled bradykinin, substance P, and neurokinin A in sheep. AB - The effects of inhaled bradykinin (BK), substance P (SP), and neurokinin A (NKA) on pulmonary resistance and airway responsiveness to carbachol were studied in conscious allergic sheep. Inhaled BK (20 breaths, 0.1 to 5.0 mg.ml-1) caused dose dependent increases in pulmonary resistance. Neither inhaled SP nor NKA (20 breaths, 0.1 to 1.0 mg.ml-1) produced significant bronchoconstriction in allergic sheep. However, the response to SP could be enhanced (p less than 0.05) by pretreatment with the neutral endopeptidase inhibitor, thiorphan (40 breaths, 1 mg.ml-1). Sheep that were allergic to Ascaris suum antigen were 5.9 times (p less than 0.05) more sensitive to the constrictor effects of BK than nonallergic sheep. BK-induced bronchoconstriction was blocked in a dose-dependent fashion by the BK beta 2-receptor antagonist, NPC 567 (D-arginine[hydroxyproline3,D phenylalanine7]BK). Atropine (0.2 mg.kg-1, intravenously) and nedocromil sodium (1 mg.kg-1 in 3 ml of saline, aerosolized) significantly inhibited the BK-induced bronchoconstriction by 97% and 43%, respectively. Chlorpheniramine (2 mg.kg-1, intravenously) had no effect. NKA caused a transient increase in airway responsiveness in allergic sheep, producing a mean 1.9-fold leftward shift in dose-response curves to aerosolized carbachol (p less than 0.05). This hyperresponsiveness was not evident 24 hours after NKA challenge. Neither SP nor BK changed airway responsiveness. Thus, in allergic sheep, inhaled BK caused a more pronounced bronchoconstriction than that observed in nonallergic sheep. The bronchoconstriction was blocked by a BK-receptor antagonist and appeared to be partially mediated via cholinergic reflexes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704389 TI - Development of the sympathetic nervous system response to endotoxicosis in the rat: importance of non-baroreflex mechanisms in pre-weanlings and adults. AB - The sympathetic nervous system response to endotoxicosis was studied in the rat at ages before (11-12 days) and after (19-20 days) maturation of the baroreflex and in adults by recording preganglionic impulses during i.v. infusions of endotoxin (S. enteriditis). At all ages, the discharge rate increased before there was any decrease in arterial blood pressure. The magnitude of the increase (65%) was the same in 11-12 days-old and adult rats while 19-20 days-old rats were hyperactive (278% increase). Subsequently, in the hypotensive phase of the endotoxicosis (diastolic pressure decrease 50-60%) there was an additional increase in discharge in the 19-20 days- old rats (43% and in adults (70%) but not in 11-12 days-old rats. The hypotensive discharge rate of the 11-12 days-old rat reached only 20% of the maximum; it reached 80% in the hyperactive 19-20 days old rat and 65% in adults. At all ages, the elevated hypotensive discharge rate persisted after steady state blood pressure was raised by infusing dextran. The discharge rate was diminished transiently during pressor responses to accelerated infusion or bolus injections of dextran. The conclusions are: (i) endotoxic stimulation of sympathetic outflow is initiated by a non-baroreflex mechanism in adult as well as in pre-weaning rats; (ii) there is added stimulation during hypotension after the baroreflex is mature, but (iii) the non-baroreflex stimulation continues to excite the preganglionic neurons and obtunds baroreflex feedback inhibition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704390 TI - Monoclonal antibodies against bovine type IX collagen (LMW fragment): production, characterization, and use for immunohistochemical localization studies. AB - Four high-affinity monoclonal antibodies (MAb) which react specifically with the low molecular weight (LMW) fragment of bovine type IX collagen (BIX) have been produced in mice. On the basis of the ability of these MAb to cross-react with type IX collagen purified from human, rat, and chick cartilage and to inhibit one another in a competitive inhibition assay, we conclude that the MAb D1-9, B3-1, and B2-7 recognize unique epitopes, whereas MAb B4-5 recognizes the same epitope as B3-1. None of the MAb reacted with bovine type I, II, and XI collagen. MAb D1 9 and B3-1 were tested for their ability to bind to tissue antigen, using an immunohistochemical assay system. Positive immunoperoxidase reactions were observed in the perichondrocytic regions of human and rat costochondral cartilage. Positive responses were also detected in rat auricular cartilage, as well as in tissue obtained from the middle and inner ears of rats and mice. This report demonstrates the relative ease of producing MAb to heterologous type IX collagen and the utility of these MAb for localizing type IX collagen in cartilage and cartilage-like tissues. PMID- 1704391 TI - Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation. AB - Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids. PMID- 1704392 TI - Polyethyleneimine as a contrast agent for ultrastructural localization and characterization of proteoglycans in the matrix of cartilage and bone. AB - We examined the presence of proteoglycans in the extracellular matrix of cartilage and bone in fetal mouse radii at the ultrastructural level, using the cationic dye polyethyleneimine (PEI). After staining with this dye, the proteoglycans appeared as granules in the uncalcified bone matrix and as extended winding structures in the cartilage matrix. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. Inhibition of the proteoglycan synthesis by beta-D-xyloside resulted in smaller PEI-positive windings in the cartilage matrix. These observations suggest that the winding, PEI-positive structures represent proteoglycan aggregates. No loss of PEI positive material in the calcified cartilage matrix was seen, suggesting that proteoglycans do not need to be removed to make the matrix calcifiable. PMID- 1704393 TI - Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. AB - Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material. PMID- 1704394 TI - Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells. AB - It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells. PMID- 1704395 TI - Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells. AB - A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV 1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains. PMID- 1704396 TI - A human CD5+ B cell clone that secretes an idiotype-specific high affinity IgM monoclonal antibody. AB - We previously demonstrated the occurrence of a naturally arisen human anti idiotypic B cell clone, that we transformed with EBV (EBV383). We show evidence that EBV383 not only expresses the CD5 surface Ag, but also contains the 2.7-kb mRNA transcript encoding this protein. In addition, we show the presence of the 3.6-kb mRNA precursor. Most Ig produced by CD5+ B cells are polyreactive natural IgM antibodies encoded by unmutated copies of germline VH genes. However, in this study we present data demonstrating the monoreactive high affinity character of the anti-idiotypic antibody (mAb383) produced by EBV383. These data are in agreement with our previous observations, showing that the VH chain of mAb383 is encoded by an extensive somatically mutated VHV gene in a way that is consistent with an Ag-driven immune response. A possible role for this remarkable anti idiotypic antibody in the maintenance of B cell memory is discussed. PMID- 1704397 TI - Six epitopes reacting with human cytotoxic CD8+ T cells in the central region of the HIV-1 NEF protein. AB - In order to identify the target epitopes recognized by specific CTL in the NEF protein of HIV-1, 33 peptides derived from the HIV-BRU sequence were tested with NEF-specific CTL generated from HIV-seropositive donors. Six different epitopes were identified and several points were remarkable: 1) They were all located in two regions of the central part of the NEF protein corresponding to residues 73 to 94 and 113 to 147, respectively. 2) The CTL issued from a single donor could recognize several peptides of the NEF protein. 3) Some of these peptides could be recognized in association with at least two or three different HLA class I molecules. 4) Two different overlapping epitopes were present in a relatively short sequence of 15 amino acids. These results suggest that multiple epitopes corresponding to different HLA restrictions could coexist in a relatively small region of the NEF protein. The implications of these results in vaccine strategies using synthetic peptides bearing CTL epitopes are discussed. PMID- 1704398 TI - A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype. AB - C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC. PMID- 1704399 TI - Activation of phospholipase D in a rat mast (RBL 2H3) cell line. A possible unifying mechanism for IgE-dependent degranulation and arachidonic acid metabolite release. AB - RBL 2H3 cells (a model of mast cell function) were sensitized with anti-TNP IgE (0.5 micrograms/ml) and triggered to secrete both histamine and arachidonic acid (AA) metabolites by the addition of TNP-OVA (0 to 100 ng/ml). After a 3-min delay, the release of both groups of mediators proceeded in a parallel manner. In cells labeled with [14C]-AA, TNP-OVA produced a rapid increase in phosphatidic acid (PA), and subsequently, 1,2-diacylglycerol (DAG) and intracellular AA levels. Concurrently, there was a decrease in [14C]-AA labeled phosphatidylcholine. The release of labeled AA from phosphatidylcholine in response to TNP-OVA was paralleled by a liberation of free choline but no evidence of liberation of phosphorylcholine. When ethanol (0.05 to 2% v/v) was included in the culture medium, phosphatidylethanol was synthesized at the expense of PA and DAG, with a resulting inhibition of secretion. D,1 propranolol, an inhibitor of PA phosphohydrolase, inhibited the IgE-dependent production of [14C]-DAG, and [14C]-free fatty acid but not [14C]-PA. The IgE-dependent release of both histamine and AA metabolites was completely inhibited by pretreatment with propranolol. Taken together, the above results suggest that phospholipase D is activated upon cross-bridging of IgE receptors on the surface of RBL 2H3 cells and that this may be a pivotal step in the signal transduction cascade leading to the release of both presynthesized and de novo synthesized mediators. PMID- 1704400 TI - Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. AB - rIL-1 beta treatment of cultured human endothelial cells (HEC) promotes polymorphonuclear leukocyte (PMN) adhesion and transmigration. Using in vitro quantitative monolayer adhesion and videomicroscopic transmigration assays, we have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and the leukocyte adhesion complex, CD11/CD18, to these processes. Maximal enhancement of PMN adhesion and transmigration were observed after 4 h of rIL-1 beta treatment, when surface expression of ELAM-1 had peaked and ICAM-1 was modestly increased. Blocking mAb directed to either ELAM-1 or ICAM-1 inhibited greater than 90% of the up regulated PMN transmigration. Blocking mAb directed to either CD11a/CD18 (LFA-1, a ICAM-1 counter-receptor), CD11b/CD18 (Mo-1), or CD18 (common beta 2-integrin) also blocked greater than 90% of PMN transmigration. At later time points (24 or 48 h), ELAM-1 surface expression was markedly decreased, whereas ICAM-1 expression was increased over the 4-h level; PMN adhesion remained elevated (approximately 50 to 60% of 4 h level), but transmigration returned to levels seen with unactivated HEC. These data indicate that PMN interaction with at least two distinct HEC adhesion molecules is necessary for transendothelial migration and suggests that PMN adhesion and transmigration, although interrelated, are mechanistically distinct processes. PMID- 1704401 TI - Priming with T helper cell epitope peptides enhances the antibody response to the envelope glycoprotein of HIV-1 in primates. AB - The induction of a memory immune response to HIV, mediated by any kind of effector mechanism, requires the induction of T cell help. In previous studies performed in different murine MHC haplotypes, three immunodominant T cell epitopes (T1, T2, and TH4.1) had been identified in the HIV envelope glycoprotein. Moreover, these peptides were proliferative T cell epitopes in humans. In this study, rhesus monkeys, Macaca mulatta, were primed with these three peptides either in combination or given separately. Half of the monkeys had a proliferative response to one or more of the priming peptide(s). Those monkeys who had a T cell proliferative response also had a high antibody response after one boost with a suboptimal dose of the native protein gp 160, whereas three of four control monkeys who had received only the native protein immunization gave no detectable antibody response, and one displayed a very weak response. For reasons that are unclear, antibodies only to the gp41 portion of gp 160 could be detected in the sera. Thus, the peptides can prime Th cells in primates for an enhanced antibody response on first exposure to the whole protein. The three peptides belong to highly conserved and nonglycosylated regions of the envelope protein. The fact that the peptides acted as immunogenic T cell proliferative and helper epitopes in nonhuman primates is very encouraging for including them in future vaccine studies in humans. PMID- 1704402 TI - T cell epitopes of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-immunized chimpanzee. AB - The humoral and cellular antisporozoite immune responses of a laboratory-born chimpanzee were measured following multiple exposures to the bites of Plasmodium vivax-infected mosquitoes. T cell lines and clones derived from the chimpanzee's PBL were used to identify T cell epitopes of the P. vivax circumsporozoite (CS) protein. Two independently obtained cell lines, established by culturing the PBL with either a recombinant P. vivax circumsporozoite (rPvCS) protein or a pool of synthetic peptides spanning the rPvCS sequence, recognized a 20-mer peptide from a nonpolymorphic region of the carboxyl terminus of the CS protein. This peptide overlaps a sequence homologous to region II of the Plasmodium falciparum CS protein. A third T cell line recognized an epitope within the central repeat domain, which has recently been found to be a polymorphic region of the P. vivax CS protein. The CD4+ clones derived from this third T cell line secreted IFN gamma and IL-2 when stimulated with either the P. vivax repeat peptide (DRAAGQPAG)2 or the rPvCS protein. PMID- 1704403 TI - Fibroblast stimulation in schistosomiasis. XI. Purification to apparent homogeneity of fibroblast-stimulating factor-1, an acidic heparin-binding growth factor produced by schistosomal egg granulomas. AB - Liver fibrosis is the most serious complication of infection with Schistosoma mansoni and Schistosoma japonicum and is responsible for severe morbidity and mortality in hundreds of thousands of patients in many Third World nations. The pathogenesis of this condition remains to be elucidated. We proposed that certain cytokines produced by cells that comprise the chronic granulomas that surround the helminth eggs within the liver initiate hepatic fibrogenesis. We now report our successful purification to apparent homogeneity of the egg granuloma-derived fibroblast mitogen. The high affinity of this factor for heparin (elutes from heparin-Sepharose with 1.5 M NaCl) facilitates its purification by a two-step procedure, and identifies the cytokine as a heparin-binding growth factor (HBGF). Furthermore, because it has an isoelectric point approximately equal to 6.2, it has one of the characteristics of a class 1 (acidic) HBGF. We immunized rabbits with the purified factor and observed that the resulting antibodies (IgG) detected the factor but not acidic fibroblast growth factor (the prototypic class 1 HBGF) either by dot-blot ELISA or neutralization of biologic activity. The granuloma product and fibroblast growth factor also differ in target-cell specificity and amino acid composition. On the basis of these distinctions, we have designated the granuloma-derived mitogen fibroblast-stimulating factor-1. With the availability of purified fibroblast-stimulating factor-1 and the future analysis of its amino acid sequence, its structural relationship to other mesenchymal growth factors can be determined. PMID- 1704404 TI - Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells. AB - CTL clones were developed from tumor infiltrating lymphocytes (TIL) from the ascites of a patient with ovarian carcinoma by coculture of TIL with autologous tumor cells and subsequent cloning in the presence of autologous tumor cells. These CTL clones expressed preferential cytolytic activity against autologous tumor cells but not against allogeneic ovarian tumor cells and the NK-sensitive cell line K562. The cytolytic activity of these CTL against autologous tumors was inhibited by anti-TCR (WT31 mAb), anti-HLA class I, and anti-CD3 mAb but not by the NK function antibody Leu 11b. Cloning of the autologous tumor cells in vitro revealed that the CTL clones of the ovarian TIL expressed differential abilities to lyse autologous tumor cell clones. The specificity analysis of these autologous tumor specific CTL suggested that they recognize several antigenic determinants present on the ovarian tumor cells. Our results indicate the presence of at least three antigenic epitopes on the tumor cells (designated OVA 1A, OVA-1B, and OVA-1C), one of which (OVA-1C) is unstable. These determinants are present either simultaneously or separately, and six types of ovarian clones can be distinguished on the basis of their expression. These results indicate that CTL of the TIL detect intratumor antigenic heterogeneity. The novel heterogeneity identified within the ovarian tumor cells in this report may be of significance for understanding cellular immunity in ovarian cancer and developing adoptive specific immunotherapeutic approaches in ovarian cancer. PMID- 1704405 TI - Tumor markers in patients with chronic renal failure. AB - In order to evaluate the specificity of tumor markers in chronic renal failure, we have determined serum levels of carcinoembryonic antigen (CEA), carbohydrate antigen 19.9 (CA 19.9), carbohydrate antigen 50 (CA 50), alphafetoprotein (AFP), neuron-specific enolase (NSE), prostatic acid phosphatase (PAP), prostatic specific antigen (PSA), squamous cell carcinoma antigen (SCC), carbohydrate antigen 15.3 (CA 15.3) and carbohydrate antigen 125 (CA 125) in 30 patients with chronic renal failure and in 36 hemodialyzed patients without clinical evidence of neoplasia. CEA, CA 50, NSE and SCC frequently show increased serum levels, suggesting a renal metabolism, while others remain, generally, within the normal levels. PMID- 1704406 TI - Aging is associated with reduced liver regeneration and diminished thymidine kinase mRNA content and enzyme activity in the rat. AB - We studied the effect of aging on rat liver regeneration. We compared the time course of total hepatic mass, DNA and RNA accumulation, and thymidine kinase (TK) messenger RNA (mRNA) content and enzyme activity, after two-thirds partial hepatectomy in 6-week-old (young adult) and 1-year-old rats. Whereas 6-week-old rats had completely regenerated all liver mass, DNA, and RNA by 7 days, the regenerating 1-year-old rat livers at 7 days contained only 60% to 70% of the mass, DNA, and RNA in the normal 1-year-old rat liver. However, rates of tissue regeneration during this time were very similar in both ages. At 28 days the weight and RNA content of the 1-year-old rat livers were 93% of normal, but total DNA was still reduced, at 78% of normal. In the younger rats TK mRNA content and enzyme activity increased substantially after partial hepatectomy and were observed to peak at 24 hours. In the 1-year-old rats TK mRNA was less abundant in normal liver and the peak level observed was lower and delayed until 48 hours. Nonetheless, the incremental increase from baseline to peak was greater in the 1 year-old than in the 6-week-old rats. In contrast to the mRNA, TK activity peaked at 24 hours, but at substantially lower levels than in the 6-week-old rats. Rates of disappearance of TK mRNA and enzyme activity after peak levels were not significantly different by age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704407 TI - Development of an immunoenzymometric assay for alpha 1-microglobulin and measurement of its serum concentration in normal and HIV-infected persons. AB - alpha 1-Microglobulin was purified from urine to a purity of 97.7% in a yield of 25.8%, and was used to produce antibodies in sheep. These antibodies, purified by affinity chromatography, were used to develop a rapid one-step and a two-step immunoenzymometric assay (IEMA). The equilibrium in the reaction between solid phase-adsorbed antibodies and antigen and between the antigen and enzyme-labelled antibodies was attained within 30 and 100 min, respectively. The one-step IEMA permits a good differentiation of low alpha 1-microglobulin concentrations after 30 min reaction time. Its detection limit is 0.35 micrograms/l, and its measurement range is between 0.5 and 100 micrograms/l. The IEMA correlates well with radial immunodiffusion (r = 0.973). The mean alpha 1-microglobulin serum concentration in women is insignificantly lower (33.2 mg/l) than in men (36.1 mg/l). In both sexes the alpha 1-microglobulin concentration increases with age. HIV-infected symptomless men have a significantly lower (15.9 mg/l) alpha 1 microglobulin concentration in serum than normal persons, whereas in AIDS patients it is significantly higher (45.5 mg/l). PMID- 1704409 TI - Purification of El Tor cholera enterotoxins and comparisons with classical toxin. AB - In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml 1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross neutralizing activity. PMID- 1704408 TI - Regeneration of adsorbed and covalently immobilized antibodies on solid phases for immunoassay. AB - A technique for the reproducible re-use of antibody-coated solid phases for immunoassays is described. The method based on the dissociation of the antigen antibody complexes. Two different procedures, using glycine buffer (pH 2.3) or ethanolamine, were developed. More than ten immunoassay cycles can be realized with the same antibody-coated microtitre plates. These procedures were tested with competitive and sandwich immunoassays, monoclonal and polyclonal antibodies, and a commercial immunoassay kit. PMID- 1704410 TI - Inhibition of mouse retroviral disease by bioactive glutaminase-asparaginase. AB - Glutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viraemic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compared favourably with those of azidothymidine given intraperitoneally at 30 mg/kg/day. Western blots performed on splenic tissue from control and treated animals indicated that glutamine depletion prevented readthrough of an amber codon at the gag-pol junction, stopping translation of viral mRNA at that point. Treatment of RLV-infected animals with PGA resulted in nearly a 200% increase in mean survival time even when therapy was initiated late in the course of the disease. To our knowledge, this is the first demonstration that a nutrient required for viral replication can be enzymically depleted in vivo to inhibit viral replication. PMID- 1704411 TI - Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. AB - Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5' end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [alpha-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase. PMID- 1704412 TI - Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus. AB - Passive administration of neutralizing monoclonal antibody (MAb) to glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) was found to protect mice from an HSV-1 strain SC16 challenge infection. To investigate further the protective potential of gH, recombinant vaccinia viruses were constructed which expressed the HSV-1 gH open reading frame under the control of the vaccinia virus 7.5K early/late promoter or the 4b late promoter. Immunization with recombinant viruses, however, did not induce the production of neutralizing antisera and the mice were not protected from zosteriform spread or the establishment of latent infection following viral challenge. The gH produced by the recombinant vaccinia viruses differed in electrophoretic mobility and antigenicity from authentic HSV 1 gH. Only one of three neutralizing MAbs specific for conformational epitopes on gH was able to immunoprecipitate gH synthesized in recombinant vaccinia virus infected cells. In addition cell surface expression of gH was not detected in cells infected with the recombinant vaccinia viruses. PMID- 1704413 TI - Cytotoxic T lymphocyte discrimination between type A Epstein-Barr virus transformants is mapped to an immunodominant epitope in EBNA 3. AB - An immunodominant Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) epitope, represented by peptide 68, has been mapped to the EBV nuclear antigen, EBNA 3. The epitope is recognized by class I-restricted CTLs through HLA-B8 and is functionally active on type A but not type B lymphoblastoid cell lines (LCLs). Herein we show that peptide 68 is not expressed as a functional CTL epitope by type A LCLs infected with an EBV B95-8 isolate. CTLs from cultures stimulated with autologous type A IARC-BL74 or QIMR-WIL LCLs lysed autologous cells stimulated with phytohaemagglutinin (PHA blasts) and coated with exogenous peptide 68. No peptide 68-specific CTLs were generated in cultures stimulated with autologous type A B95-8 or type B AG876 LCLs. However, the B95-8 LCL coated with peptide 68 was effective in the induction of a peptide-specific CTL response. A peptide 68-specific CTL clone failed to lyse the B95-8 LCL, type B AG876 LCL and PHA blasts, although such targets were lysed when coated with peptide 68. PMID- 1704414 TI - Chemoprophylaxis of scrapie in mice. AB - Three applications of the polyanion pentosanpolysulphate about 2 months before infection of mice with scrapie completely protected animals infected with up to 100 LD50, and considerably prolonged the lifespan of those infected with 100 to 10,000 LD50. The clinical diagnosis was confirmed by immunoblot analysis for the protein of scrapie-associated fibrils. PMID- 1704415 TI - The effect of some platinum compounds on the biosynthesis of RNA and its precursors. AB - The effect of cis-DDP (cis-diamminedichloroplatinum(II)), trans-DDP (trans diamminedichloroplatinum(II)), SPC (spermine-platinum(II) complex), and K2PtCl4 on the ribomononucleotide and RNA metabolism was studied. When Ehrlich ascites tumor cells were preincubated with the aforementioned compounds and then labeled with [C14]uridine a clear-cut suppression of the radioactive labeling of RNA was observed. As radioactivity incorporated into the pool of the free uridine nucleotides in the cells treated with platinum compounds was even higher in comparison with that of the non-treated cells a conclusion may be drawn with certainty that the platinum compounds studied inhibit RNA biosynthesis. It was also found that under the effect of these compounds in the in vivo-assessed rate of the conversion of uridine nucleotides into cytidine nucleotides was considerably diminished. Using NaH14CO3 as a radioactive precursor it was shown that platinum compounds also inhibited purine biosynthesis de novo, in particular the conversion of IMP into GMP and AMP. The pronounced inhibitory effect of the platinum compounds on essential steps of the pyrimidine and purine biosynthesis de novo may be at least partly responsible for the firmly established inhibition in the present study of RNA biosynthesis by platinum compounds. The inhibition of the synthesis of the mononucleotides and RNA by the platinum compounds may be closely related to their cytostatic and cytotoxic activities. PMID- 1704416 TI - L-3,4-dihydroxyphenylalanine synthesis by genetically modified Schwann cells. AB - We have investigated whether Schwann cells can be modified by gene transfer to synthesize L-3,4-dihydroxyphenylalanine (L-DOPA), the immediate precursor in the formation of dopamine. By using a retrovirus containing a rat tyrosine hydroxylase (TH) cDNA, we established an immortalized rodent Schwann cell line that stably expressed high levels of TH and secreted L-DOPA in vitro when supplied with tyrosine and the essential cofactor biopterin. We also infected primary Schwann cells and demonstrated that cells expressing TH secreted L-DOPA while maintaining their capacity to myelinate neurons in vitro. This study indicate that it may be feasible to utilize autotransplantation of genetically modified Schwann cells to alleviate the movement disorders in Parkinson's disease. PMID- 1704417 TI - Brain tryptophan hydroxylation in the portacaval shunted rat: a hypothesis for the regulation of serotonin turnover in vivo. AB - Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5 hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo. PMID- 1704418 TI - Galanin reduces carbachol stimulation of phosphoinositide turnover in rat ventral hippocampus by lowering Ca2+ influx through voltage-sensitive Ca2+ channels. AB - The 29-amino-acid peptide galanin (GAL) caused concentration-dependent inhibition of the accumulation of 3H-inositol phosphates (3H-InsPs) induced by the muscarinic agonist carbachol (CARB; 10(-3)-10(-5) M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono-, bis-, tris-, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 microM) did not affect alpha-adrenergic or serotonergic type 2 receptor-mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H-InsPs nor affected muscarinic receptors in binding studies. Blockade of the T-, N-, and L-types of voltage-sensitive calcium channel (VSCC) with 200 microM Cd2+ reduced muscarinic receptor-mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 microM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 microM abolished the GAL inhibition of CARB-stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 microM Bay K 8644, a selective agonist of the L-type VSCC, prevented the inhibitory effect of GAL. Blockade of the L-type VSCC with nifedipine (1 microM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704419 TI - Characterization of histamine release from the rat hypothalamus as measured by in vivo microdialysis. AB - The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringer's solution was perfused at a rate of 1 microliter/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)-containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, caused an approximately twofold increase in the HA output 30-60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by approximately 70% 4-5 h after treatment with alpha fluoromethylhistidine (alpha-FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in alpha-FMH-treated rats. These results suggest that both HA-N-methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS. PMID- 1704420 TI - Purification and characterization of neuron-specific surface antigen defined by monoclonal antibody BM88. AB - Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain. PMID- 1704421 TI - Dihydropyridine modulation of voltage-activated calcium channels in PC12 cells: effect of pertussis toxin pretreatment. AB - In this study, we report the effect of pertussis toxin pretreatment on dihydropyridine modulation of voltage-sensitive calcium channels in PC12 cells. The rise in intracellular calcium concentration caused by potassium depolarization is not affected significantly by pertussis toxin pretreatment. Nicardipine, a dihydropyridine derivative, added either before or after potassium induced depolarization, reduces the resultant elevation in cytosolic calcium level both in control and in pertussis toxin-treated cells. The dihydropyridine agonist Bay K 8644, when added before potassium, is able to enhance the potassium induced spike of cytosolic calcium levels, an effect significantly reduced by pertussis toxin pretreatment. Moreover, the addition of Bay K 8644 after potassium holds the intracellular calcium concentration at a cytosolic sustained level during the slow inactivating phase of depolarization. This effect of Bay K 8644 is inhibited by nicardipine. Pertussis toxin pretreatment slightly weakens the effect of Bay K 8644 when added after potassium-induced depolarization, whereas it significantly reduces the nicardipine inhibition of cytosolic calcium rise stimulated by potassium and Bay K 8644, but not by potassium alone. In conclusion, our findings suggest that a pertussis toxin-sensitive guanine nucleotide regulatory protein could be involved in the interaction between dihydropyridine derivatives and voltage-dependent calcium channels. PMID- 1704422 TI - Developmental expression of myelin protein genes in dysmyelinating mutant mice: analysis by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry. AB - Gene expression for myelin proteolipid protein (PLP) and myelin basic protein (MBP) in the dysmyelinating mutant mice shiverer and jimpy was analyzed by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry. The level of PLP transcription in shiverer brains was lower than that in controls at postnatal day 18 but relatively higher at later stages. In spite of the considerable amount of hybridization with PLP cDNA, immunoreaction for PLP was greatly reduced in shiverer mice throughout their lives, probably owing to a defect in the assembly of PLP into myelin. Abnormal deposition of PLP in oligodendroglial cell bodies suggested that transport of PLP to myelin is delayed in shiverer brains. The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mice. MBP mRNA in jimpy mice is localized preferentially in oligodendroglial cell bodies, a result suggesting that oligodendrocytes in jimpy are mostly the immature type. Although transcriptional activity of the MBP gene in jimpy was greatly reduced, a finding reflecting the decrease in the number of mature oligodendrocytes, that of the PLP gene remained high at early stages. The discrepancy of the two gene expressions is discussed relative to the role of PLP transcripts at early stages of myelination. PMID- 1704423 TI - Developmental changes in distribution of acidic fibroblast growth factor in rat brain evaluated by a sensitive two-site enzyme immunoassay. AB - We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period. PMID- 1704424 TI - Fatty acid composition of human myelin proteolipid protein in peroxisomal disorders. AB - Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester-bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP-acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders. PMID- 1704425 TI - Further demonstration that [Pro9]-substance P is a potent and selective ligand of NK-1 tachykinin receptors. AB - Previous studies have indicated that [Pro9]-substance P ([Pro9]-SP) possesses very good affinity for NK-1 binding sites and that, in contrast to substance P, it interacts selectively with these sites. Therefore, [3H][Pro9]-SP (75 Ci/mmol) was synthesized in order to study its binding to membranes of the rat brain. Specific binding of [3H][Pro9]-SP (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analysis and Hill plots revealed the existence of a single population of noninteracting binding sites (KD and Bmax values: 1.48 nM and 29.7 fmol/mg of protein, respectively). Competition studies with several tachykinins and analogues indicated that the pharmacological profile of [3H][Pro9]-SP binding sites is identical to that of NK-1 binding sites. Rat brain sections labeled with either [3H][Pro9]-SP or [3H]SP, revealed a close similarity in the topographical distribution of [3H][Pro9]-SP and [3H]SP binding sites. Biochemical, pharmacological, and autoradiographic data obtained with [3H][Pro9]-SP did not provide any evidence for the existence of subtypes of NK-1 binding sites. [Pro9]-SP had neither agonist nor antagonist properties on NK-2 and NK-3 receptors. Indeed, it did not stimulate phosphoinositide turnover on the hamster urinary bladder (NK-2 assay) and was devoid of activity on the contraction of the rabbit pulmonary artery (NK-2 assay) and of the rat portal vein (NK-3 assay). As a result of its high selectivity, [Pro9]-SP thus appears an excellent tool for investigating the functional properties of NK-1 receptors. PMID- 1704426 TI - Selective increase of iron in substantia nigra zona compacta of parkinsonian brains. AB - Histochemical and biochemical determinations of total iron, iron (II), and iron (III) contents in brain regions from Parkinson's and Alzheimer's diseases have demonstrated a selective increase of total iron content in parkinsonian substantia nigra zona compacta but not in the zona reticulata. The increase of iron content is mainly in iron (III). The ratio of iron (II):iron (III) in zona compacta changes from almost 2:1 to 1:2. This change is thought to be relevant and may contribute to the selective elevation of basal lipid peroxidation in substantia nigra reported previously. Iron may be available in a free state and thus can participate in autooxidation of dopamine with the resultant generation of H2O2 and oxygen free radicals. PMID- 1704427 TI - Enhanced NAD(P)H:quinone reductase activity prevents glutamate toxicity produced by oxidative stress. AB - Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18 RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease. PMID- 1704429 TI - Ionic currents in crustacean neurosecretory cells. AB - 1. The patterns of electrical activity and membrane characteristics of a population of neurosecretory-cell somata in the X-organ of the crayfish were investigated with microelectrodes and whole-cell, voltage-clamp techniques. Some neurons (56%) were silent but could be excited by intracellular current injection: other cells showed spontaneous tonic activity (35%), and some had spontaneous bursting activity (9%). The spiking activity was abolished by tetrodotoxin (TTX) exposure and by severing the axon near the cell body. After axotomy, only a small, slow, regenerative depolarization remained that could be blocked by Cd2+. 2. Under voltage clamp the steady-state I-V curve in low [Ca2+]i (9 X 10(-9) M) showed a slope conductance of 16.7 +/- 3.9 (SD) nS (n = 10) at -50 mV and zero current potential of -50.1 +/- 7.7 mV. In current-clamp mode these neurons were either silent or fired tonically. With high [Ca2+]i (1.7 X 10(-6) M) both the slope conductance and inward and outward currents were reduced. In some neurons high [Ca2+]i reveals a negative slope resistance in the range of -46 to 41 mV. It could be supressed by removing [Na+]o, but it was TTX insensitive. These are the neurons that under current clamp showed bursting activity. 3. The main inward current in cell somata was a Ca2+ current of 2 +/- 0.6 nA (n = 18), activated at -40 mV and peaking at 20 mV. It showed relaxation with prolonged pulses. No Na(+)-dependent, TTX-sensitive inward currents were recorded with short (100-ms) pulses in axotomized neurons. 4. Two outward currents could be distinguished.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704428 TI - Slowly progressive aphasia: three cases with language, memory, CT and PET data. AB - Three cases of slowly progressive speech and language disturbance were studied at various points post onset (three, five and 15 years respectively). Language, neuropsychological and brain imaging (computer tomography and positron emission tomography) evaluations were completed on all three patients. The data suggest that the syndrome of "progressive aphasia": 1) does not involve a uniform symptom complex; 2) does not necessarily develop into a full blown dementia syndrome; 3) varies greatly in rate of progression from case to case; 4) is associated with normal brain structure (on computer tomography); and 5) is associated with abnormal left temporal lobe metabolism as measured by fluorodeoxyglucose (FDG) positron emission tomography (PET). One patient had histological findings consistent with Alzheimer's disease at necropsy. PMID- 1704430 TI - Identical IgM antibodies recognizing a glycine-alanine epitope are induced during acute infection with Epstein-Barr virus and cytomegalovirus. AB - We studied antibody production in serial serum samples from patients with acute Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections. Sera were analyzed both by enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide (P62) derived from the glycine-alanine repeating region of the Epstein-Barr nuclear antigen (EBNA-1) and by immunoblotting. In prior studies, we have shown that patients with acute EBV infection make IgM antibodies that react with this peptide, that recognize a viral-specific protein (EBNA-1), and that bind with a number of proteins present in uninfected cells; however, antibody binding to these autoantigens was inhibited by the peptide. IgG antibodies reactive with the peptide did not appear until months after the disease and were specific for the EBNA-1 protein. We now find that patients with acute CMV infection but not those with acute infections from a variety of other nonherpes organisms also produce IgM antibodies that recognize the EBV-derived peptide P62. These antibodies also appear to recognize the same cellular proteins as the EBV-induced IgM antibodies. The IgM antibodies appeared in all acutely infected CMV patients studied and occurred both in patients with previous EBV infections and in one patient studied who had not previously been exposed to EBV. It appears that infection with EBV or CMV can induce the synthesis of a very similar or identical set of IgM antibodies. PMID- 1704432 TI - Biphasic reduction and concanavalin A binding properties of serum alpha fetoprotein in preterm and term infants. AB - Reference values for postnatal serum alpha-fetoprotein (AFP) and concanavalin A (ConA) binding subfractions of AFP in preterm and term infants are presented. Preterm infants had 10-fold higher serum concentrations of AFP than did term infants at birth. The reduction of serum values of AFP was biphasic in both groups and differed significantly between the two groups. The first declining phase continued for approximately 4 months in preterm and for 2 months in term infants, and was related to the degree of prematurity. The AFP values reached adult levels by 12 months in preterm and by 9 months in term infants. The developmental pattern of the carbohydrate moiety of AFP was determined by ConA fractioning. The proportion of the ConA nonreactive subfraction of AFP in preterm and term infants at birth was 6% and 13%, respectively; it increased more rapidly in term than in preterm infants but reached 85% to 95% by the age of 6 months in both infant groups. Our results indicate that the postnatal maturation of AFP synthesis is dependent on gestational age. Malignant recurrences of neonatal sacrococcygeal teratomas were associated with an increase in serum concentration of AFP and a decrease in the proportion of the ConA nonreactive subfraction of AFP. PMID- 1704431 TI - Clinical and biologic effects of granulocyte colony stimulating factor in the treatment of myelokathexis. AB - Successful treatment of a patient with myelokathexis, a rare form of chronic neutropenia associated with recurrent infections, is described. Rapid mobilization of bone marrow neutrophils and improved myeloid morphologic features were observed after treatment with human granulocyte colony stimulating factor. Transient thrombocytopenia and bone pain were observed during treatment. Although neutrophil chemotaxis, superoxide production, and FcRIII surface expression were reduced, the patient improved clinically after restoration of a normal neutrophil count. PMID- 1704433 TI - Effects of pirenzepine and AF-DX 116 on ganglionic transmission in the cardiac sympathetic nerves of the dog: interaction of M1 and M2 receptors with nicotinic receptors. AB - Effects of the selective M1 receptor antagonist pirenzepine and the selective M2 receptor antagonist AF-DX 116 on ganglionic transmission were examined in anesthetized dogs, in order to elucidate a functional role of M1 and M2 receptors. Preganglionic or postganglionic stimulation of the cardiac sympathetic nerves (SNS, 0.5-16 Hz) produced frequency-dependent increases in heart rate. Pirenzepine (3-100 microgram/kg) caused dose-dependent and significant inhibition of positive chronotropic response to preganglionic SNS but not to postganglionic SNS. AF-DX 116 (10-100 micrograms/kg) had no effect on the preganglionic SNS induced tachycardia. The simultaneous administration of pirenzepine (30 micrograms/kg) and hexamethonium (C6, 1 mg/kg), and the subsequent administration of 10 mg/kg of C6, inhibited more potently the tachycardic responses to preganglionic SNS than each dose of C6 did by itself. The enhancement by pirenzepine of the C6-induced inhibition was evident at high frequencies (8 and 16 Hz) of SNS. In contrast, the blocking effect of C6 (1 and 10 mg/kg) on ganglionic transmission was significantly attenuated by AF-DX 116 (30 micrograms/kg). The attenuation by AF-DX 116 was observed at a wide range of stimulation frequency (0.5-8 Hz). These results suggest that M1 receptors play a facilitatory role in ganglionic transmission but M2 receptors do not contribute to the transmission when nicotinic pathway is intact. However, the activation of M2 receptors would further suppress ganglionic transmission when nicotinic transmission is inhibited. Under these conditions, activation of M1 receptors would mediate the transmission elicited by high frequency of stimulation. PMID- 1704434 TI - Pharmacologic comparison of selected agonists for the M1 muscarinic receptor in transfected murine fibroblast cells (B82). AB - The radioligand binding and functional properties of 10 muscarinic agonists for the M1 muscarinic receptors were characterized on the murine fibroblast B82 cells, which have been transfected with the m1 gene. All of the muscarinic agonists completely inhibited [3H](-)methyl-3-quinuclidinyl benzilate binding to the M1 muscarinic receptor in the transfected B82 cells. Their apparent inhibition constant values for agonist/[3H](-)methyl-3-quinuclidinyl benzilate inhibition experiments correlate well with their EC50 values in stimulating phosphatidylinositide hydrolysis. Based on the maximal functional effects: (+) cismethyl-dioxolane, oxotremorine-M, acetylcholine, carbachol and methacholine are most efficacious, McN-A-343 and arecoline are least efficacious, whereas the efficacies of oxotremorine and pilocarpine are intermediate. In addition, McN-A 343 inhibited carbachol-stimulated phosphatidylinositide hydrolysis. Spare receptors were detected for oxotremorine-M, methacholine and carbachol, but not the rest of the agonists, by comparing the receptor-occupancy curves with the concentration-response curves. These results suggest that the presence of a quaternary nitrogen (trimethylammonium group) within the structure of the agonist may be important for the expression of full agonist activity. PMID- 1704435 TI - CCK-8, CCK-4 and gastrin-induced contractions in guinea pig ileum: evidence for differential release of acetylcholine and substance P by CCK-A and CCK-B receptors. AB - The cholecystokinin (CCK) receptor involved in contraction of guinea pig ileal longitudinal muscle to cholecystokinin is poorly understood; some studies have suggested that contraction was mediated via a CCK-A receptor whereas other studies have implicated CCK-B receptors in ileal contraction to CCK. To clarify this, we compared the effects of CCK-8 sulfate, CCK-4 and gastrin in radioligand binding studies and longitudinal ileal contractility in vitro. Contraction to all three peptides was abolished by tetrodotoxin (3 x 10(-7)M), confirming the neuronal nature of the CCK receptors mediating contraction to all three peptides. Maximal CCK-8S contractions were inhibited by 80% in the presence of atropine (10(-6)M), and entirely by the combination of atropine and a substance P receptor antagonist (3 x 10(-5)M). CCK-4 and gastrin-induced contractions were unaffected by substance P receptor blockade, but were abolished by atropine. Two selective CCK-A and CCK-B receptor antagonists, L-364,718 and L-365,260, respectively, were used to probe further the receptors involved in ileal contraction to this peptide family. Radioligand binding studies in mouse brain, rat pancreas and guinea pig stomach confirmed the selectivity of these antagonists. The CCK-A selective antagonist, L-364,718, potently inhibited ileal contractions to CCK-8S (-log KB = 9.35) with 10-fold lower affinity at receptors mediating contraction to CCK-4 ( log KB = 8.25). In contrast, the CCK-B receptor antagonist, L-365,260, did not affect contraction to CCK-8S (-log KB less than 7) but potently inhibited contraction to CCK-4 (-log KB = 9.24).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704436 TI - Differential regulation of human basophil and lung mast cell function by adenosine. AB - Adenosine was found to modulate the activity of the human basophil and lung mast cell (HLMC) differently. In the basophil, adenosine inhibited the anti-IgE stimulated release of histamine and leukotriene C4 (LTC4) and increased total cell cyclic AMP (cAMP) levels. Substituted adenosine analogs had a rank order potency of: N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than R-phenylisopropyladenosine for the inhibition of immunoglobulin E triggered mediator release from the basophil and increases in cAMP levels. The adenosine receptor antagonist, 8-phenyltheophylline, antagonized both the NECA induced inhibition of mediator release and elevations in cyclic nucleotide. The purinergic transport inhibitor, dipyridamole, reversed the inhibition by adenosine of histamine release but not LTC4 generation, suggesting that these two actions are mechanistically separable. Dipyridamole failed to modify the adenosine-induced elevation in cAMP. In contrast to the findings in the basophil, the response to adenosine in the HLMC was biphasic in nature. Thus, at low concentrations of the nucleoside, adenosine potentiated the release of histamine and LTC4 from immunologically activated HLMC, whereas at higher concentrations a counteractive inhibitory process was observed. Analogs of adenosine had the same effects on HLMC; NECA was more potent than R-phenylisopropyladenosine for both the potentiating and inhibitory components of the biphasic response. Low concentrations of adenosine analogs, which potentiated secretion, initiated modest elevations in cAMP levels, whereas higher concentrations, which inhibited secretion, significantly augmented cAMP levels. Although R phenylisopropyladenosine was almost as potent as NECA at elevating cAMP in HLMC, it was not as efficacious. The NECA-induced modulation of HLMC mediator release and elevations in cAMP were antagonized by 8-phenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704437 TI - Therapeutic defibrination with ancrod does not protect canine myocardium from reperfusion injury. AB - The purpose of this study was to determine whether normal fibrinogen contributes to the development of myocardial reperfusion injury by acting as a substrate in vivo for neutrophil adhesion. This was tested in a dog model of acute myocardial infarction that used pentobarbital anesthetized dogs subjected to 90 min regional myocardial ischemia and 5 h reperfusion. Dogs were treated with 1 unit/kg Ancrod (venom from the Malayan pit viper, Agkistrodon rhodostoma) or vehicle i.v. 60 min after left circumflex coronary artery occlusion. Therapeutic defibrination was verified in Ancrod-treated dogs by measurements of clottable fibrinogen, alpha-2 antiplasmin and plasminogen, by the activated partial thromboplastin time and by immunoelectrophoresis. Fibrinogen was depleted to below detectable limits of the assay (less than 0.05 mg/ml) after treatment with Ancrod. The defibrination effect was accomplished by the expected activation of the fibrinolytic system: alpha-2 antiplasmin was decreased by 10% and plasminogen activity was decreased by 30% with Ancrod treatment. There were no measureable differences between the two treatment groups in heart rate, mean arterial blood pressure, rate pressure product or circumflex coronary blood flow throughout the 90 min of regional ischemia or during the 5 h of reperfusion. The relative severity of ischemia between the two treatment groups was similar when assessed with radiolabeled microsphere measurement of myocardial blood flow. The accumulation of neutrophils (measured by myeloperoxidase activity) within the myocardium after reperfusion was not reduced by prior depletion of fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704438 TI - Purification of the microsomal Ca2(+)-ATPase from rat liver. AB - The Ca2(+)-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficoll-sucrose treatment, column chromatography with agarose-hexane adenosine 5'-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca2(+)-ATPase was stable for at least two weeks when stored at -70 degrees C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca2(+) ATPase. Further characterization of the ER Ca2(+)-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca2(+)-ATPase cross-reacted with the purified Ca2(+)-ATPase from rat liver ER membranes. PMID- 1704439 TI - Chloride channels in human platelets: evidence for activation by internal calcium. AB - Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single channel currents were measured when the pipette contained isotonic BaCl2 or MgCl2 saline; over 30 sec -5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at -35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl-. Erev was shifted 30 mV/10-fold change in external Cl- (replaced by gluconate), was similar with BaCl2 or MgCl2 in the pipette and was not significantly shifted by replacing external Na+ with K+. Addition of 1 mM BAPTA to the MgCl2 pipette saline prevented activation of Cl- currents; with isotonic CaCl2 internal saline, current appeared immediately upon patch rupture, suggesting that the Cl- channels are dependent on internal Ca2+. 5 nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl- conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl- currents in human platelets. PMID- 1704440 TI - Individual domains of colicins confer specificity in colicin uptake, in pore properties and in immunity requirement. AB - Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C terminal domains of colicin A and colicin E1 by their respective immunity proteins. PMID- 1704441 TI - The effects of Bay K 8644 on diastolic function in the dog heart. AB - The effects of increased cytosolic calcium on cardiac mechanics were studied in open chest dogs instrumented with ultrasonic crystals and a miniature pressure transducer. Calcium was increased either by promoting calcium influx with Bay K 8644 (Bay K) or by increasing extracellular calcium concentration. A single dose of Bay K (10 micrograms/kg/min) was administered to each dog. Bay K increased LV systolic pressure, maximal rate of rise of LV pressure (LV + dP/dt), mean velocity of circumferential fiber shortening (Vcf), and calculated LV end systolic wall stress. The time constant of isovolumic pressure decay (T) was calculated following two different methods: (a) a semilogarithmic method (Tz), and (b) using the linear relation between LV - dP/dt vs LV pressure (T1). Whereas Tz decreased from 31.7 +/- 2.6 to 26.7 +/- 1.7 ms (P less than 0.05), no changes were detected in T1 (46.3 +/- 4.4 vs 50.9 +/- 4.0 ms N.S.) The asymptote value (PB) decreased after Bay K from -9 +/- 2.8 to -22.5 +/- 4.2 mmHg (P less than 0.05). The same results were obtained when the changes in the loading conditions of the heart produced by Bay K were controlled by mechanical manoeuvers or after beta-blockade with propranolol. When calcium chloride was administered in amounts that will produce equal contractile changes as Bay K, a decrease in PB was also observed (from -14.7 +/- 1.6 to -27.7 +/- 6.1 mmHg (P less than 0.05]. Tz decreased from 29.6 +/- 3.6 to 22.5 +/- 2.9 ms (P less than 0.05) and no changes in T1 (52.5 +/- 5 vs 52.4 +/- 7.3 ms, N.S.) were detected. The decrease in the asymptote reported herein could induce a false decrease in the time constant if the altered values of PB are not considered, or another method of calculation of the time constant is used. Neither Bay K nor elevated extracellular calcium concentration modified the diastolic compliance. Changes in loading conditions or a cAMP pathway can be ruled out as a cause of the decrease in PB, since the results were reproduced under controlled loading conditions and beta blockade. These data suggest that increasing cytosolic calcium does not alter either the relaxation rate or the diastolic compliance but does decrease the value toward left ventricular pressure decays. PMID- 1704443 TI - Clonal heterogeneity of a human functioning tumor which produces granulocyte colony-stimulating factor. AB - Two different clonal strains have been isolated from a human squamous cell carcinoma which produces granulocyte colony-stimulating factor (G-CSF). The clones differed in morphology, growth characteristics, karyotype, production of G CSF, histology of the tumors produced by inoculating the cultured cells, and in the development of granulocytosis in host mice transplanted with the cultured cells. A well-differentiated squamous cell carcinoma was developed by inoculation with clone 1 cells into athymic nude mice. No CSF activity was detected in the cultured medium of the cells. The host mice with the transplanted cells showed no increase in peripheral blood neutrophils. Clone 2, which formed a poorly differentiated squamous cell carcinoma in nude mice, produced a large amount of CSF in vitro and developed a marked neutrophilia in host nude mice. Clone 1 cells were more sensitive to bleomycin than were clone 2 cells in vitro. The results suggested the G-CSF producing tumor to be heterologous and the cells with different functional properties, including G-CSF production, sensitivity to bleomycin, and keratinization, to pre-exist in the parental cell population. PMID- 1704442 TI - Biological and clinicopathological significance of endocrine differentiation of gastric adenocarcinoma evaluated by double immunohistochemical labeling for chromogranin A and bromodeoxyuridine. AB - To elucidate the biological and clinicopathological significance of endocrine differentiation in gastric adenocarcinoma, an immunohistochemical study was made of 127 cases with ascertained five-year survivals, and of 45 recent cases of bromodeoxyuridine (BrdU) labeling. Endocrine differentiated cancer cells were demonstrated in 37 out of the 127 cases (29.1%) evaluated by chromogranin A (CGA) immunoreactivity, and all CGA-positive tumors were classified as advanced gastric cancer. Analysis of retrospective five-year survival rates revealed the adenocarcinomas with endocrine differentiation to have had significantly longer survival times than those without endocrine immunoreactivity in stage II, but not in stages III or IV. Double immunolabeling for CGA and BrdU in the other 45 adenocarcinoma cases showed only a single CGA-positive cancer cell with BrdU incorporation among a total of 454 CGA-positive cells examined. There was no significant difference between the labeling indices of the general cancer population and the cancer cells adjacent to CGA-positive cells. In conclusion, endocrine differentiation of gastric cancer is not uncommon, particularly in advancing cancer, and it would be a useful marker for a better prognosis in stage II. Probably, endocrine differentiated cancer cells are almost dormant with virtually no DNA synthesizing activity, and their paracrine effect is most unlikely to work in vivo. PMID- 1704444 TI - [Hepatectomy in repeated operations on patients with parasitic diseases of the liver]. AB - The results of resection of the liver in repeated operations on patients with parasitic disease of the liver are discussed. Operations were conducted on 22 patients with alveococcosis and 7 patients with echinococcosis. By means of the method of atypical liver resection radical operations were performed on 20 patients (69%) and palliative resections of the liver in 9 patients (31%). The palliative resections were combined with cryodestruction of the remaining parasitic tissue, which improved the therapeutic effect significantly and prolonged the patients' survival. The volume of the resection ranged from a segment to one half of the liver. The fatality rate was 6.8%. PMID- 1704445 TI - [Obturation ileus in cancer of the large intestine]. AB - The analysis of treatment and diagnosis is presented for 168 colon cancer patients with occlusive ileus. Late diagnosis is responsible for high percentage of palliative operations (61.9%) with a 26% mortality. Radical surgery involves, as a rule, delayed primary one-stage resection and establishment of a single- or doubled-barreled anus. The mortality in this case reaches 19%. Advances in colon cancer treatment results and ways to reduce mortality in this conditions lie in early diagnosis and better knowledge by general practitioners of occlusive ileus pathogenesis and clinical appearance. PMID- 1704446 TI - [An immunoenzyme method of detecting natural antibodies to thrombin and alpha 2 macroglobulin]. AB - An enzyme immunoassay technique was developed for the detection of natural IgG and IgM antibodies to thrombin and alpha 2-macroglobulin in normal donor plasma and serum. The optimal conditions for the detection of IgG antibodies to alpha 2 macroglobulin are the same for the blood plasma and serum. IgG antithrombin antibodies are detectable in the plasma within a small range of the sorbed antigen concentrations, and if the serum is analyzed, this range is lowered by two orders. Natural antibody levels to the studied antigens were found to be constant in a person. PMID- 1704447 TI - [The rapid diagnosis of chronic nonspecific lung diseases]. AB - Rapid methods for the detection of viral antigens and immunoglobulins in nasal and bronchial washings from patients with chronic nonspecific pulmonary diseases (CNPD) are described. These methods are based on viral antigen and immunoglobulin agglutination with cellulose particles sensitized with specific sera and gamma globulin fractions. The investigation takes just 3-5 min. Respiratory syncytial virus, influenza A and B, parainfluenza viruses, adenoviruses, immunoglobulins G and M, and immunoglobulin A in low levels are detectable with the use of the described methods. Studies carried out over the course of treatment helped detect low titers of viral antigens and IgG and IgM and a high titer of IgA. PMID- 1704448 TI - [Detection in the bile of antigens to microorganisms]. PMID- 1704449 TI - [Methods of assessing the activity of the monooxygenase enzyme system (review of the literature)]. PMID- 1704450 TI - [The potentiometric determination of cholinesterase activity in the blood and tissues]. PMID- 1704451 TI - [Determination of the phenotype of N-acetyltransferase activity]. AB - The authors suggest a simple easily reproducible micromethod for determination of genetically determined activity of N-acetyltransferase (EC 2.3.1.5) and acetylator phenotype. The method consists in oral intake of sulfadimezine as an acetylation substrate, followed by measurement of the enzyme activity from the degree of sulfadimezine acetylation in blood or urine samples. Examinations of 121 children with acute respiratory viral infections (ARVI) and 36 healthy children have shown that children with a slow acetylation type are more liable to ARVI. This was confirmed by the fact that complicated ARVI forms in young children occurred as a rule only in 'slow acetylators', mostly in children with the lowest N-acetyltransferase activity. PMID- 1704452 TI - [Beta 2-microglobulin in the diagnosis of specific involvement of the kidneys in acute leukemias]. AB - Measurements of the serum and urinary beta 2-microglobulin (beta 2-Mg) concentrations in 138 adult patients with various forms of acute leukemia, which were carried out in various periods of the disease, have revealed that serum beta 2-Mg levels were two times higher in the patients with leukemic involvement of the kidneys (5.54 +/- 3.50 mg/l) than in those without renal involvement (2.77 +/ 1.07 mg/l). Consequently, urinary beta 2-Mg excretion was much higher in the patients with renal involvement than in those with intact kidneys (1.512 +/- 3.222 mg/l vs. 0.388 +/- 1.175 mg/l). Mathematical analysis has shown that the risk of leukemic involvement of the kidneys makes up 93.3 percent in acute leukemia patients with serum beta 2-Mg levels surpassing 6.0 mg/l. This permitted a conclusion on the diagnostic value of beta 2-Mg measurements for the detection of specific involvement of the kidneys in acute leukemia. PMID- 1704453 TI - [Thyroid status of the body in leukemias]. AB - Disordered thyroid status was detected in patients with various clinical and morphological forms of leukemia and with hypoplastic anemia. These disorders present as low triiodothyronine blood concentration and a deficit of this hormone in the body. A certain correlation has been detected between the degree of the thyroid status disorders and the leukemic process malignancy. PMID- 1704454 TI - [An aniline method of determining glucose in biological fluids]. AB - A simple method for glucose measurements in biologic fluids has been developed, making use of aniline chromogen. Using this method, one may do without carcinogenic ortho-toluidine and work with less concentrated (and less acid) acetic acid solutions. Introduction of the aniline method of glucose measurement will help improve the working conditions of laboratory assistants at laboratories employing the ortho-toluidine method at present. PMID- 1704455 TI - [Interleukins: functional role as mediators of the immune system (review of the literature)]. PMID- 1704456 TI - [Glucosignal test strips for the rapid determination of blood glucose]. PMID- 1704457 TI - [The determination of hydroxyproline in daily urine]. AB - Daily urine hydroxyproline was measured using Stegemann's modified technique. The modified procedure makes use of some elements of the technique developed by Firschein et al. (hydrolysis, filtration, and neutralization of filtrate). The suggested modification is highly sensitive and well reproducible. PMID- 1704458 TI - [The determination of free hemoglobin in blood plasma using the hemiglobincyanide method]. AB - The authors have proved the possibility of using the hemiglobincyanide method for measurements of blood plasma free hemoglobin: free hemoglobin was measured in autoblood reinfused from the operation field by various methods (45-benzidine method, 3 parallel analyses in every study, and 45-hemiglobincyanide method, modified by increasing the amount of the tested material from 0.02 to 2.0 ml, also 3 parallel analyses per sample); a correction coefficient equal to 71.7 is suggested, and correction of the data according to a formula. PMID- 1704459 TI - [The permeability of the blood-cerebrospinal fluid barrier in the first 24 hours after cranio-cerebral injury]. AB - The permeability of the blood-liquor barrier (BLB) for blood proteins was studied in 30 patients within 24 hours after craniocerebral injuries of various severity and 21 patients whose condition was critical within 24 hours after different neurosurgical interventions. BLB permeability was assessed from the ratio of albumin/IgG in the cerebrospinal fluid and blood. A direct though not always statistically significant relationship between BLB permeability and the severity of injury was revealed. The authors suppose that this methodological approach, combined with other methods of investigation, may be useful in assessing the course of brain edema. Local IgG synthesis in the central nervous system was detected in half of the injured patients and in half of those in the critical condition. Local immune humoral reaction of the central nervous system appears to be largely related to the trauma severity and not influencing the outcome. This phenomenon is to be verified by specific immunologic methods. PMID- 1704460 TI - [Functional characteristics of the erythrocytes in patients with lung cancer]. AB - Peripheral blood red cells were studied in 35 newly detected (untreated) male patients with Stages III-IV lung cancer by cytophotometric (sulfhydryl groups, lipoproteins) and interferometric (dry mass) techniques. The reference groups included patients with chronic nonspecific pulmonary diseases (CNPD) and healthy volunteers. The findings evidence that sulfhydryl group, lipoprotein concentrations and dry mass mean values of red cells are significantly lower in lung cancer patients than those in both reference groups. The changes detected in CNPD patients are similar to those in lung cancer patients but are less manifest. PMID- 1704461 TI - [A method of determining the permeability of erythrocyte membranes for monovalent ions]. AB - A modified method for the determination of red cell membrane permeability for sodium and potassium monovalent ions is described. The described modification essentially cuts down the time of investigation and accelerates potassium ion transport. Instead of ouabain, an imported reagent, strophanthin K glycoside manufactured in the USSR is used in the test. PMID- 1704462 TI - [A method of determining erythrocyte deformability]. AB - A simple method for determining red cell deformability is suggested, based on red cell packing during centrifugation. This method was used to study red cell deformability in patients with essential hypertension and angina of effort and found it reduced. PMID- 1704463 TI - [Determination of the leukocyte count in oral smears]. AB - The routine method for estimation of the count of leukocytes that migrated to the oral cavity, making use of a special chamber, is difficult. The author suggests leukocyte scintillation in 1 microliter of oral washing in Goryayev's chamber. The data of 83 analyses of oral washings carried out in patients with different patterns of inflammatory processes in periodontal tissues are presented. PMID- 1704464 TI - [The functional activity of blood neutrophils in chronic myeloleukemia]. PMID- 1704465 TI - [A bioluminescent method of determining the sensitivity of microflora to antibiotics]. AB - A bioluminescent rapid method (a three-hour test) has been developed to assess the susceptibility of microflora to antibiotics. It is based on comparison of intracellular ATP content in a sample (pus) after 3 h incubation at 37 degrees C in liquid culture medium (dilution: sample/culture medium--1:9) on a shaker in the presence and absence (control) of a single antibiotic concentration. A criterion has been offered for quantitative estimation of antibiotic susceptibility of microorganisms. The authors have optimized the measurements of intracellular ATP by the rapid bioluminescent method using immobilized firefly luciferase-based ATP reagent. The results of the method are in good correlation with those of the standard agar diffusion technique. PMID- 1704466 TI - [A diagnostic serum with antibodies to virulent Yersinia]. AB - A diagnostic agglutinating adsorbed rabbit serum with antibodies to Yersinia strains containing virulence plasmid with molecular mass of 40-50 mD was prepared. Trials of this serum in agglutination test on the glass with 69 Yersinia strains (Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pseudotuberculosis) and with 42 other Enterobacteriaceae strains have confirmed the specificity and sufficiently high activity of the agent in respect of virulent Yersinia strains. Experiments have demonstrated the possibility of using this serum in tests for the detection of yersiniasis agents and for rapid assessment of individual Yersinia clones in respect of the presence of plasmid with a molecular mass of 40-50 mD. PMID- 1704467 TI - [Blood hydrolysates for microbiological culture media]. AB - Blood hydrolysates contain two times less nucleic acids than meat hydrolysates, the levels of vitamins B2, B3, B6, and B9 are lower in them either. The authors suggest that this is the cause of a poorer growth of microorganisms in culture media with blood hydrolysates, repeatedly reported in literature. They have demonstrated that addition of nucleic acids and vitamins to blood hydrolysates improves the microorganism growth. PMID- 1704468 TI - [The determination of C. pylori in patients with gastroduodenal diseases]. AB - A total of 46 gastric mucosa biopsy specimens obtained during surgery and 90 gastric juice samples from patients with gastroduodenal diseases were examined. Campylobacter-like microorganisms were detected in 84.6 percent of biopsy specimens and in 73.9 percent of gastric juice samples (in gram-stained smears). Urease activity was detected in 75.9 and 13.3 percent, respectively. C. pylori growth in solid nutrient medium in microaerophilic conditions was observed in 44.2 and 5.7 percent of the examined samples, respectively. The studies have demonstrated the efficacy of using disks with urea from standard kits manufactured by the Research Institute for Epidemiology and Microbiology in the city of Gorky in the urease test. PMID- 1704469 TI - [An effective method of detecting intestinal Balantidium]. PMID- 1704470 TI - [The use of basic fuchsin to stain smears for gonococci and Trichomonas]. PMID- 1704471 TI - A novel non-perfusion Timm method for human brain tissue. AB - A simple modification of the Timm sulphide-silver method for unfixed brain tissue is described. Instead of perfusing animals with a sodium sulphide solution, sulphide treatment was performed by exposure to mounted frozen sections to H2S gas. The staining pattern in the rat brain obtained with this modification is similar to that With the Neo-Timm method, primarily yielding staining of the neuropil of telencephalic structures. Because perfusion is not required, the modified Timm method can also be applied to postmortem human tissue. Application of the modified Timm stain to the human tissue is exemplified on the hippocampus with special attention to the non-mossy fiber staining. PMID- 1704472 TI - Translingual approach of the basal surface of the diencephalon of the rat and retrograde labeling from the median eminence. AB - A unique surgical procedure is described by which the basal surface of the diencephalon can be exposed without cannulation of the trachea. The basal surface of the diencephalon is exposed through a midsagittal incision by splitting the oral diaphragm, tongue, and soft palate respectively. Then a small hole is drilled in the base of the skull. After manipulation on the base of the brain is completed, the hole in the skull is plugged with Histoacryl and the soft palate and tongue are sutured. Rats having such interventions survive in excellent condition for days without the need of intensive medical care. PMID- 1704473 TI - Oxygen-exacerbated bleomycin pulmonary toxicity. AB - Bleomycin is an antineoplastic agent with potential for producing pulmonary toxicity, attributed in part to its free radical-promoting ability. Clinical and research experiences have suggested that the risk of bleomycin-induced pulmonary injury is increased with the administration of oxygen. We report a case in which the intraoperative administration of oxygen in the setting of previous bleomycin therapy contributed to postoperative ventilatory failure. Our patient recovered with corticosteroid therapy. Physician awareness of a potential interaction between oxygen and bleomycin may help reduce the morbidity and mortality related to bleomycin therapy. PMID- 1704474 TI - [Amylase determination in pleural effusions caused by neoplasms]. PMID- 1704475 TI - Priming effects of mammalian tachykinins on human neutrophils. AB - The undecapeptide substance P (SP) is known to activate different cell types involved in inflammatory and immune processes. By evaluating primed stimulation of human neutrophils, we now demonstrate that SP (10 nM-0.1 mM) dose-dependently enhances superoxide anion production from cells stimulated by the phospholipid mediator Platelet Activating Factor (PAF). We also provide evidence that neurokinin A (NKA), which is released, as well as SP, from C fibers of sensory nerves, potentiates PAF-evoked superoxide anion generation, while neurokinin B (NKB) is ineffective. PMID- 1704476 TI - Substance P induces migration of capillary endothelial cells: a novel NK-1 selective receptor mediated activity. AB - Substance P (SP) has been indicated as a main mediator of neurogenic inflammation, leading to vasodilation, increase in vascular permeability and modulation of immune cell function. Certain vascular effects produced by SP are endothelium mediated. We have studied the effect of SP and of selective NK-1, NK 2 and NK-3 receptor agonists on migration of cultured capillary endothelial cells of bovine origin. Our results indicate that SP (10(-14)-10(-6) M) induces a concentration-dependent migration of endothelial cells with maximal activity at 10(-10) M. This effect was mimicked by the selective NK-1 receptor agonist which showed a similar concentration-dependent curve, while selective NK-2 and NK-3 receptor agonists were ineffective. Our conclusions are that endothelial cells possess specific receptors for SP of the NK-1 type which affect mobilization of capillary endothelial cells. PMID- 1704477 TI - Protective effect of recombinant human granulocyte colony-stimulating factor (rG CSF) against various microbial infections in neutropenic mice. AB - Protective effect of recombinant human granulocyte colony-stimulating factor (rG CSF) on microbial infections was studied in cyclophosphamide (CPA)-induced neutropenic mice. The neutropenic mice showed severely decreased resistance against systemic infections of Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Staphylococcus aureus, and Candida albicans. When such mice were injected subcutaneously with rG-CSF on four consecutive days beginning the day after CPA injection, the decreased anti-microbial resistance of the mice was restored to the level of that in normal mice. The anti-infective effect of rG-CSF was dose-dependent and the 50% effective doses (ED50) in various microbial infections tested were 1-10 micrograms/kg/day. The results suggest that rG-CSF is useful for protection of neutropenic patients from microbial infections. PMID- 1704478 TI - Idiotypical identity of IgG myeloma protein with monoclonal IgM, Bence Jones protein, and Fv derived from one patient. AB - Serum from a patient (KK) with IgG2-lambda myeloma was shown to contain multiple paraproteins corresponding to an IgM-lambda monoclonal protein (MMP), a lambda type Bence Jones protein (BJP), and a 30 kDa component in addition to the IgG2 myeloma protein (GMP). These proteins possessed common idiotypic determinants, as judged by their monoclonal reactivity with rabbit anti-GMP idiotype antibody (aId) in the immunofixation electrophoresis. Analysis with aId absorbed with either H or L chain of GMP revealed that the 30 kDa component shared both VH and VL with GMP and MMP, while BJP carried only the VL idiotype. The 30 kDa component, however, failed to react with antibody to either the mu, gamma, alpha, kappa, or lambda isotype, indicating that it had an Fv-like molecular composition. These results suggest that myeloma cells of KK had diverged from the same precursor B cell clone to produce MPs of different isotypes and altered molecular constructions. PMID- 1704479 TI - [The antigenic relationship between Brucella abortus, Brucella melitensis and Yersinia enterocolitica serotype 0:3 and 0:9]. AB - Antigenic interrelationship between B. abortus, B. melitensis, Y. enterocolitica serotype 0:3 and serotype 0:9 were investigated in hyperimmun sera obtained from rabbits, by using agglutination and agglutinin absorbtion tests. Agglutination was determined at 1/2560 titer of B. abortus and B. melitensis hyperimmun sera when their own antigens were used. On the other hand, these sera agglutinated Y. enterocolitica serotype 0:9 antigen at 1/1280 and 1/320 titer respectively. As it was seen that high level antigenic relation was found between B. abortus and Y. enterocolitica serotype 0:9 while antigenic resemblance was found at lower level between B. melitensis and Y. enterocolitica serotype 0:9. There wasn't any significant interrelationship between B. abortus, B. melitensis and Y. enterocolitica serotype 0:3. In conclusion it is though that it is impossible to distinguish B. abortus and Y. enterocolitica serotype 0:9 infections from each other by using agglutination tests when 0 antigens are used. PMID- 1704480 TI - Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors. AB - We have synthesized two peptides that correspond to unique regions of the amino terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function. PMID- 1704481 TI - Molecular cloning of the cDNAs encoding a novel insulin-like growth factor binding protein from rat and human. AB - cDNA clones encoding a novel insulin-like growth factor-binding protein (IGFBP) purified from rat serum and human bone cell-conditioned medium have been isolated from rat liver and human placenta, liver, and ovary cDNA libraries. The deduced amino acid sequences of the cDNAs revealed a mature polypeptide consisting of 233 amino acids for the rat, while the human structure contains an additional four amino acid sequence in the middle region of the molecule. This protein, now proposed to be named IGFBP-4, contains two extra cysteines compared with the previously characterized IGFBP-1, -2, and -3, but the alignment of the remaining 18 cysteines is conserved across the four IGFBPs. Amino acid sequence comparison among the four binding proteins within the rat species demonstrated that both the amino- and carboxy-terminal one thirds of the molecules are highly conserved, while the middle one third region, where no cysteines are present except for the two that exist in IGFBP-4, is the most divergent. The overall sequence homology among the four rat IGFBPs is very similar (53-59%), suggesting that their individual genes diverged from a single ancestral gene at about the same evolutionary time point. Northern analysis of the IGFBP-4 mRNA in rat tissue demonstrated that transcription of the IGFBP-4 gene is highly active in the liver, although a single 2.6-kilobase IGFBP-4 mRNA band was detectable in all tissues examined, including adrenal, testis, spleen, heart, lung, kidney, liver, stomach, hypothalamus, and brain cortex. PMID- 1704482 TI - Different combinations of regulatory elements may account for expression of the glycoprotein hormone alpha-subunit gene in primate and horse placenta. AB - Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses. PMID- 1704483 TI - Thyroid hormone regulation of peptidylglycine alpha-amidating monooxygenase expression in anterior pituitary gland. AB - Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a copper-, molecular oxygen-, and ascorbate-dependent enzyme which catalyzes the COOH terminal amidation of bioactive peptides. Expression of PAM in the adult male rat anterior pituitary was evaluated after experimental manipulation of thyroid status. Levels of PAM mRNA increased 4- to 7-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil or thyroidectomy and were not diminished below control levels in animals made hyperthyroid by treatment with T4. Treatment of thyroidectomized animals with T4 prevented the increase in PAM mRNA levels; similar doses of T4 returned serum TSH and anterior pituitary PAM mRNA to euthyroid values. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by reverse transcription and the polymerase chain reaction, thyroid status did not affect the distribution of PAM mRNA among its various alternatively spliced forms. The specific activity of PAM in the anterior pituitary was increased slightly in both the soluble and particulate fractions from chemically hypothyroid rats; the majority of the PAM activity in the rat anterior pituitary was soluble, and increased secretion of enzyme may account for the lesser effect of chemical thyroidectomy on specific activity compared to mRNA levels. Western blot analysis demonstrated a 104-kDa PAM protein in particulate fractions prepared from control, PTU-treated, and T4-treated animals. The soluble fraction contained major PAM proteins of 95 and 75 kDa, and PTU treatment brought about an increase in the prevalence of the 75-kDa form of PAM protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704484 TI - Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver. AB - Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained. PMID- 1704485 TI - Dexamethasone stimulates transcription of the insulin-like growth factor-binding protein-1 gene in H4-II-E rat hepatoma cells. AB - Binding proteins for the insulin-like growth factors (IGFBP) are important modulators of the biological actions of IGF-I and IGF-II. Concentrations of one of these proteins, IGFBP-1, in human plasma and IGFBP-1 mRNA in rat liver are markedly altered in diabetes and fasting. We now examine the regulation of IGFBP 1 and IGFBP-I mRNA in H4-II-E cells, a rat cell line derived from the minimal deviation H35 Reuber hepatoma previously reported to synthesize IGFBP-1 as its predominant IGF-binding protein. Confluent H4-II-E cells in serum-free medium were incubated with different hormones for 48 h, and the conditioned medium was analyzed by ligand blotting. Dexamethasone (10(-6) M) increased levels of 30-kDa IGFBP-1 approximately 10-fold; stimulation was half-maximal at 6 x 10(-9) M dexamethasone. No stimulation was seen with progesterone, testosterone, IGF-I, or rat GH, whereas insulin gave a small inhibition. Immunoblot analysis using a monoclonal antibody to human IGFBP-1 confirmed that the 30-kDa IGFBP induced by dexamethasone was IGFBP-1. IGFBP-1 mRNA was increased to a similar extent (7 fold), as determined by Northern blot hybridization using human or rat IGFBP-1 cDNA probes. The stimulation of IGFBP-1 mRNA was observed within 3 h after the addition of dexamethasone; IGFBP-1 in the medium increased more slowly. After withdrawal of dexamethasone from stimulated cells, IGFBP-1 mRNA decreased by 80% after 48 h; IGFBP-1 decreased more slowly. The increased abundance of IGFBP-1 mRNA in dexamethasone-treated cells primarily reflected increased transcription rather than increased mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704486 TI - Identification of a novel member (GDF-1) of the transforming growth factor-beta superfamily. AB - A cDNA clone encoding a new member (designated GDF-1) of the transforming growth factor-beta (TGF beta) superfamily was isolated from a library prepared from day 8.5 mouse embryos. The nucleotide sequence of GDF-1 predicts a protein of 357 amino acids with a mol wt of 38,600. The sequence contains a pair of arginine residues at positions 236-237, which is likely to represent a site for proteolytic processing. The C-terminus following the presumed dibasic cleavage site shows significant homology with the known members of the TGF beta superfamily, matching the other family members at all of the invariant positions, including the seven cysteine residues with their characteristic spacing. GDF-1 is most homologous to Xenopus Vg-1 (52%), but is not likely to be the murine homolog of Vg-1. In vitro translation experiments were consistent with GDF-1 being a secreted glycoprotein. Genomic Southern analysis indicated that GDF-1 may be highly conserved across species. These results suggest that GDF-1 is most likely an extracellular factor mediating cell differentiation events during embryonic development. PMID- 1704487 TI - [The effect of hormones on the rate of axonal transport in the ventral spinal nerve roots of rats]. AB - The Wistar male rats in the age of 8-12 months were injected 7-8 microliter of aqueous solution of L-leucine-14C (specific activity 12543 megaBq/mmol) into the area of the ventral horn at the level of L5,6 segment of the spinal cord. The study of radioactivity in various sections of the respective frontal root was performed after one hour. It was found that estradiol dipropionate, testosterone propionate, insulin and small doses of thyroxin increased the axonal transport of the labelled material, while hydrocortisone, large doses of thyroxin, castration and thyroidectomy caused its delay. It is concluded that the axonal transport is under a pronounced hormonal control. PMID- 1704488 TI - [The divergence of the axonal collaterals of the thalamic neurons to the visual and associative cortexes in cats]. AB - Projections of the thalamic neurons to the visual (area 17) and the parietal association (area 7) cortices were examined by retrograde axonal transport of fluorescent dyes. It was found that the pulvinar neurons can be divided into three groups with respect to their connections with these cortical areas: 1- projecting to area 7 (the largest cell group); 2--projecting to area 17 (a smaller cell group) and 3--sending their axons to the both cortical areas (only few cells). Neurons belonging only to first two groups were found in the lateral posterior nucleus. Divergence of axonal collaterals of pulvinar neurons can secure the existence of parallel pathways transmitting information to the visual and associative cortices. PMID- 1704489 TI - [The ultrastructural characteristics of the pyramidal callosal neurons of layer III in the primary auditory area (A1) of the cat cortex]. AB - An electron microscope study of retrogradely labelled pyramidal neurons in layer III of the primary auditory cortex (AI) after HRP injections into the contralateral AI has been carried out in cats. From 4 to 10 synapses were usually revealed on somatic profiles of these callosal neurons. Synapses occupied 20.0% of the somatic surface of these neurons. All of the revealed synapses on the somata of callosal neurons had symmetric contacts and were formed by axon terminals with small elongated synaptic vesicles. Average length of these synaptic contacts was 1.6 microns. In layer III anterogradely labelled terminals of callosal fibres were also revealed. The majority of them contained large round synaptic vesicles and formed asymmetric contacts on spines. Three labelled axon terminals with small elongated vesicles were found to form symmetric axo-somatic synapses on callosal neurons of layer III. PMID- 1704490 TI - [The extrasynaptic effect of the neuropeptides vasopressin, oxytocin and vasotocin on the electrically controlled ion channels of the membrane in mollusk neurons]. AB - Extrasynaptic effects of vasopressin, oxytocin, vasotocin on the membrane electrosensitive ionic channels were studied in model experiments using isolated somata from the CNS of mollusc Lymnaea stagnalis. The neuropeptide in concentrations of 1.10(-16)-1.10(-6) mol/l either blocked or induced biphasic dose-dependent effect on amplitudes of the membrane ionic currents. PMID- 1704491 TI - Suppression of the cerebral vasospastic actions of oxyhemoglobin by ascorbic acid. AB - Oxyhemoglobin (Oxy-Hb) produced a concentration-dependent contraction of monkey, dog, and bovine cerebral artery strips. Treatment of Oxy-Hb with ascorbic acid suppressed the ability of Oxy-Hb to contract the arteries, especially in the monkey arteries. The ability of intracisternally applied Oxy-Hb to constrict the basilar artery in anesthetized dogs was diminished when Oxy-Hb was treated previously with ascorbic acid (AsA-Hb). The contraction caused by Oxy-Hb was suppressed by treatment with indomethacin and aspirin in isolated bovine cerebral arteries. Endothelium-dependent relaxations elicited by substance P and relaxations induced by stimulation of the vasodilator nerves with nicotine were suppressed by treatment with Oxy-Hb and AsA-Hb; however, the inhibitory effect of AsA-Hb was markedly less. Oxy-Hb attenuated nitroglycerin-induced relaxations in a dose-dependent fashion, whereas AsA-Hb in concentrations up to 1.6 x 10(-5) M did not significantly influence the relaxations. It is concluded that incubation of Oxy-Hb with ascorbic acid alters markedly the biological activity of Oxy-Hb; the vasoconstrictor activity is suppressed, and the ability to diminish vasodilator actions is minimized. These findings provide a rationale for the use of ascorbic acid in cisternal irrigation to prevent the development of cerebral vasospasm after a subarachnoid hemorrhage. PMID- 1704492 TI - Spinal cord stimulation: a contemporary series. AB - Forty-three patients with chronic pain disorders of different causes were selected for spinal cord stimulation. All underwent implantation of a ribbon electrode through a small laminotomy, under general anesthesia. Thirteen patients (30%) failed to obtain significant pain relief during a period of trial stimulation, and their electrodes were removed. The remainder underwent a definitive implant and were followed for a mean of 13 months (range, 3-33 months). Nineteen of them (63%) continued to experience pain relief. A detailed analysis of this series, as well as a literature review, is presented. PMID- 1704493 TI - Serotonergic changes following proestrous treatment with p,p'-DDT. AB - The effects of 25 and 75 mg/kg p,p'-DDT on the CNS serotonergic system were examined in proestrous female rats. Females were treated with p,p'-DDT on the morning of proestrus and were sacrificed that evening. Levels of serotonin (5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were examined in cortex, hippocampus, hypothalamus and preoptic areas. The binding of 3'-8-OH-DPAT [2-hydroxy-2-N, N-(di-propylamino)-tetralin], an agonist for 5-HT1A receptors, was examined in hippocampus and frontal cortex. P,p'-DDT decreased the level of 5 HT in frontal cortex and hippocampus. Elevations in 5-HIAA were present in the hypothalamus but only at the higher dose of p,p'-DDT. The dose of 25 mg/kg p,p' DDT produced an increase in the Bmax for 3H-8-OH-DPAT binding to frontal cortical and hippocampal membranes. Membrane preparations from females given 75 mg/kg p,p' DDT fell into two categories. Some were similar to the control but with a slightly higher Kd; others could not be analyzed by traditional linear or nonlinear regression procedures because they showed a constant proportion of bound label, independent of the concentration of 3H-ligand in the reaction. In vitro, p,p'-DDT did not compete with 3H-8-OH-DPAT for binding to cortical membranes so it is unlikely that residual pesticide in the membrane preparation accounted for the binding results. These binding results are particularly interesting because, in previous studies, the dose of 25 mg/kg p,p'-DDT was shown to be more potent than 75 mg/kg p,p'-DDT in reducing female rodent lordosis behavior.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704494 TI - Maxillofacial considerations in orthotopic liver transplantation. AB - Orthotopic liver transplantation is now a widely used treatment for patients with end-stage liver disease. Lifelong pharmacologic immunosuppression renders these patients susceptible to many infections. The purpose of this article is to provide guidelines for treating the patient with end-stage liver disease, both before and after transplantation. In addition, we shall discuss some of the medical implications associated with end-stage liver disease and their clinical presentations and appropriate presurgical management. Adverse side effects of long-term immunosuppression and their effect on the oral and maxillofacial region shall also be discussed. Last, a brief discussion of FK506, the latest immunosuppressant, will be presented together with the implications of its use on our surgical treatment of these patients. PMID- 1704495 TI - HIV infection: clinical features and treatment of thirty-three homosexual men with Kaposi's sarcoma. AB - The clinical findings of patients with oral Kaposi's sarcoma are reviewed. These oral findings commonly included candidiasis, hairy leukoplakia, gingivitis associated with human immunodeficiency virus (HIV), periodontitis, and other symptoms, including xerostomia. The other common symptoms of HIV disease that may be of importance in leading to a diagnosis are reviewed in this patient group. Treatment by local radiotherapy or by intralesional vinblastine of these oral Kaposi's sarcomas resulted in successful palliation, with more than 50% regression of the lesions in 80% of the patients treated. PMID- 1704496 TI - Partial transformation of human thyroid epithelial cells by mutant Ha-ras oncogene. AB - We have previously shown that activation of ras oncogenes by mutation is a frequent early event in human thyroid neoplasia. Using amphotropic retroviral vectors to achieve gene transfer, we demonstrate here that human primary thyroid epithelial cells can be partially transformed by an activated cellular or viral Ha-ras oncogene, in the absence of a cooperating oncogene. The transformation event induced by ras involves temporary rescue from senescence for up to 20 rounds of cell division together with morphological alteration, growth factor independence and anchorage independence. It has therefore been possible to reconstruct in vitro a key early event in the genesis of human epithelial neoplasia. PMID- 1704497 TI - AgNOR counts in endometrial and endocervical carcinomas. AB - The histologic differentiation between endometrial and endocervical adenocarcinomas is a common diagnostic problem. In the present study, the distribution of nucleolar organizer regions (NOR) is evaluated in the two groups of neoplasm, but it is concluded that NOR counts do not offer any histological discriminant between endometrial and endocervical carcinomas. PMID- 1704498 TI - [Evaluation of sensitivity and specificity of various staining methods for the detection of myocardial fibrosis]. AB - Fifty bioptic endomyocardial samples from 15 patients with idiopathic dilatative cardiomyopathy have been stained with PicroSirius, Masson-Goldner, Van Gieson Verhoeff, AFOG. The extension of stained areas has been evaluated by morphometry using a videoscan. As control sections samples have been taken from lower third of the right ventricle at autopsies of healthy subjects dying in road accidents. Morphometric analysis of subendocardial interstitial and perivascular fibrosis has been performed. For the authors the most useful stain in the evidentiation of fibrosis was PicroSirius both in the bioptic samples and the autoptic ones. PMID- 1704499 TI - Thyroid hormone receptor and receptor-related RNA levels in developing rat brain. AB - The proto-oncogene c-erbA and its analogues are genes that encode thyroid hormone receptors. In rats, at least two loci have been identified by their homology to c erbA. Each locus can produce at least two distinct mRNA species via RNA processing. However, one of those transcripts, r-erbA alpha-2, does not yield a triiodothyronine (T3)-binding protein when translated in vitro. Proper development of the rat brain is thyroid hormone-dependent during the perinatal period and there is a documented increase in brin nuclear T3-binding capacity during that time. By hybridizing cDNA probes specific to various rat erbA-like transcripts with RNA extracted from developing rat brain, we sought changes in thyroid hormone receptor mRNA levels that correspond with changes in T3-binding capacity in rat brain during the perinatal period. Three mRNA of the erbA family showed variations in relative abundance during brain development. Oddly, r-erbA alpha-2, a variant that does not code for a T3-binding protein, was the most abundant erbA-like RNA to correlate with the reported changes in T3-binding capacity. The message for rat r-erbA alpha-1 that encodes a functional T3 receptor also varies with T3-binding capacity but at a level that is at least 8 fold lower than the r-erbA alpha-2 message. The level of rat erbA beta-1 message also varies in rat brain during the perinatal period but not in a way that correlates with changes in T3-binding capacity. PMID- 1704500 TI - Murine hybridoma antibodies enhance bactericidal activity of human cord blood against K1 Escherichia coli strains. AB - Murine hybridoma antibodies directed against the capsule and O-side chain determinants of the Escherichia coli strain Bort (018ac:K1:H7) were evaluated for their ability to enhance bactericidal activity of cord blood against K1 E. coli strains possessing O antigens common in neonatal E. coli infection, i.e. 018, 07, and 01. The antibodies to the K1 capsule and O-side chain efficiently enhanced cord polymorphonuclear leukocyte-mediated killing of K1 encapsulated E. coli strain possessing a homologous O antigen, but the IgM antibody to the K1 capsule exhibited approximately 10-fold greater activity than did the IgG3 antibody to O side chain (weight basis). Both antibodies required complement for their opsonic activities. Our findings indicate that antibodies directed against the capsule and O-lipopolysaccharides are able to restore the opsonic activity of cord blood against K1 E. coli, suggesting that these antibodies may be useful in the prevention and therapy of neonatal E. coli infection. PMID- 1704502 TI - Progress and development of cardiac pacing electrodes. PMID- 1704501 TI - [Indicators of immunologic reactivity and the alpha fetoprotein level in newborn infants with thymomegaly and bronchopneumonia]. AB - Forty-two newborns with bronchopneumonia were under observation. Of these, 22 also presented with thymomegaly . The acute disease period was characterized by higher concentrations of alpha-fetoprotein and the decrease of the transforming activity of lymphocytes in response to phytohemagglutinin in newborn patients with thymomegaly as compared to the same parameters in newborn patients without thymomegaly . Study into correlative interrelations has revealed a significant reverse relation between the content of alpha-fetoprotein and IgG. PMID- 1704503 TI - Alignment of P waves for signal averaging. AB - Signal averaged atrial activity recorded from the body surface is finding increased use in assessing atrial arrhythmias. The process of signal averaging is critically dependent upon precise alignment of the waveform of interest. The alignment accuracy of wide (0.01-300 Hz) and narrow (10-300 Hz) bandpass filtered P waves recorded from the body surface were compared to atrial electrograms recorded from temporary pacing wires. The locations of the filtered P waves relative to the atrial electrogram were calculated beat-by-beat for 100 beats in 15 patients aged 1 month to 71 years. The standard deviation of the alignment error for the wide filtered surface P wave averaged 1.7 +/- 0.8 msec and for the narrow filtered P waves was 0.8 +/- 0.4 ms. These results demonstrate that high pass filtering will improve alignment accuracy, and therefore, improve frequency resolution for the analysis of signal averaged P waves. PMID- 1704504 TI - Coexistence of complete infra-Hisian block, WPW syndrome and Mobitz type II Kent Bundle block. AB - A 65-year-old patient with a chief complaint of syncope had an ECG with ventricular preexcitation and intermittent second-degree atrioventricular (AV) block. AV conduction was maintained by the accessory pathway only, with no evidence of AV nodal conduction. Electrophysiological study demonstrated that the QRS duration and morphology did not increase with atrial pacing; however, A-H prolongation occurred with increased pacing rates. PMID- 1704505 TI - Ventricular pacing from the atrial channel of a DDD pacemaker: a consequence of pacemaker twiddling? AB - The breakdown of pacemaker lead insulation under conditions of mechanical stress leading to failure of pacing is well recognized. We present a case where adjacent breakdown of insulation in two unipolar pacing leads resulted in inappropriate ventricular pacing. Replacement of the leads rectified the problem. PMID- 1704506 TI - Return ventricular rhythm: new mechanism for tachyarrhythmia. AB - A case study is presented in which the electrocardiographic patterns during tachycardia showed ventricular premature depolarizations with retrograde conduction to the atria probably using a left-sided anomalous pathway and reentry to the ventricles with normal ventricular activation and duplication of the heart rate. This type of tachycardia is being labeled "return ventricular rhythm" and, as far as it is known, this is the first description of its mechanism. PMID- 1704507 TI - Pacemaker mediated tachycardias in single chamber rate responsive pacing. AB - Although pacemaker mediated tachycardias are classically associated with dual chamber pacemakers, single chamber rate responsive pacemakers are also susceptible to such tachycardias under special circumstances. A unipolar activity sensing rate responsive pacemaker (Activitrax 8403) was implanted in an 83-year old man with complete atrioventricular block. The pacemaker was programmed at an output of 5 V, activity threshold medium, rate response 5, and lower and upper rates of 70 and 125 beats/min, respectively. He presented with palpitations at rest and muscle twitching of the pacemaker pocket 4 months after implantation. Examination confirmed that the pacemaker had flipped over, resulting in pocket pacing which in turn activated the activity sensor, resulting in a rate response. The increase in pacing rate lead to a higher frequency of pocket pacing, thus leading to positive feedback increase in rate. With the patient at rest, pacemaker mediated rates were 106, 91, and 74 beats/min at low, medium and high thresholds, respectively. Decreasing the output to 2.5 V eliminated pocket pacing and the tachycardia. As a result of the reversal of the pacemaker, a similar rate response during exercise could only be achieved at a more sensitive rate responsive setting. Thus, pacemaker mediated tachycardia can complicate pacemaker "flipping" in single chamber activity sensing rate responsive pacemakers. Methods for the avoidance and treatment of pacemaker flipping are discussed. A review of other sensor mediated tachycardias is also presented. PMID- 1704508 TI - The effect of multiple shocks on canine cardiac defibrillation. AB - To determine if multiple shocks adversely affect the success of later shocks compared with early shocks, we analyzed the success rates of initial shocks (defibrillation attempts 1-5), first half shocks (defibrillation attempts 1-20) and second half shocks (defibrillation attempts 21-40) in a canine model. Epicardial patches were placed on the right and left ventricle in 28 dogs. Ventricular fibrillation was induced by a 60-Hz shock. After 30 seconds, defibrillation was attempted using 7, 12, 13, or 18 joules with either a uniphasic or biphasic rectangular waveform. The uniphasic waveform was 5 msec in duration; the biphasic waveform was 10 msec, with the lagging 5-msec pulse one half the amplitude of the leading 5-msec pulse. For uniphasic shocks, the right ventricular patch was positive; for biphasic shocks, the right ventricular patch was positive during the leading 5 msec of the shock and negative during the lagging milliseconds. A total of 960 fibrillation episodes were evaluated; no dog was involved in more than 40 fibrillation episodes. The success rates of defibrillation attempts 1-5, defibrillation attempts 1-20, and defibrillation attempts 21-40 were similar at 12, 13, and 18 joules. This information supports the continued use of up to 40 fibrillation trials in canine cardiac defibrillation. However, at 7 joules defibrillation attempts 21-40 were more successful than defibrillation attempts 1-5, and 1-20. With our methodology, these data are consistent with the hypothesis that low energy shocks create a "sensitizing" effect on cardiac tissue, allowing more successful defibrillation with repeated shocks. PMID- 1704509 TI - Physical determinants of the endocardial P wave. AB - Reliable atrial sensing of intrinsic P wave activity is important to ensure optimal atrial or dual chamber pacemaker function. Various physical factors (e.g., posture, respiration, exercise) may influence P wave characteristics and impair adequate sensing. To investigate this phenomenon, we measured the average of three P wave amplitudes (PWA) and calculated slew rates from telemetered printouts acquired from Pacesetter pacemakers in 32 patients. These measurements were performed in various body positions, with upright exercise and in varying stages of respiration. RESULTS: the mean supine PWA increased on full inspiration (3.56 +/- 1.3 mV versus 3.25 +/- 1.2 mV during quiet respiration, P less than 0.001), and also increased significantly with full expiration. The mean PWA increased on assuming the erect position (3.25 +/- 1.2 mV increasing to 3.49 +/- 1.3 mV, P less than 0.001); in the upright position, the mean erect PWA during quiet respiration was not significantly influenced by the stage of respiration. The mean upright exercise PWA did not differ significantly from the preexercise erect PWA (3.50 +/- 1.2 with exercise, and 3.47 +/- 1.5 before exercise; P = NS). Calculated slew rates were not different lying versus standing. CONCLUSIONS: the mean supine PWA increases significantly at the extremes of respiration and on assuming the erect body position; upright exercise results in no appreciable change in the erect PWA. Atrial sensitivity adjustments based on standard supine testing should be adequate for all body positions. PMID- 1704510 TI - Directional variability of stimulation threshold measurements in isolated guinea pig cardiomyocytes: relationship with orthogonal sequential defibrillating pulses. AB - Reports on delivery of separated orthogonal pulses markedly improving cardiac defibrillation have suggested that the stimulation threshold of heart fibers varies in accordance with their orientation within the electric field. The present work was aimed at investigating the directional variability of stimulation thresholds in isolated guinea pig cardiomyocytes. This variability was measured in 48 single myocytes by rotating each one through a theta (theta) angle between two-fixed parallel electrodes 1.1 cm apart, thus making theta vary between the electric field and the myocyte axis. For theta = 0 degrees, the mean longitudinal current stimulation threshold was 16.92 +/- 4.20 mA (n = 48). When theta was increased by increments of 10 degrees up to 90 degrees, the stimulation threshold increased in an exponential way. For theta = 90 degrees, the mean transverse stimulation threshold was 63.13 +/- 13.30 mA. These results clearly demonstrate the dependence of isolated cardiomyocyte stimulation thresholds on their orientation within the electric field and may account for the improved efficacy of defibrillation previously observed after delivery of orthogonal pulses. PMID- 1704511 TI - Intraventricular electrogram analysis for ventricular tachycardia detection: statistical validation. AB - Time-domain analysis of intraventricular electrogram morphology during ventricular tachycardia (VT) and sinus rhythm or atrial fibrillation (SR/AF) has been proposed as a method for increasing the specificity of pathological tachycardia detection by antitachycardia devices. However, few studies have validated the use of such analysis with statistical methods. When statistical methods have been utilized, it has been assumed that the distribution of the values derived from analysis of the intracardiac electrograms have had a normal (gaussian) distribution. In this study, we sought to determine whether: (1) the distribution of values derived from analysis of intracardiac electrogram during SR/AF and VT is gaussian or nongaussian; and (2) the discrimination of monomorphic VT from SR/AF using SR/AF templates can be validated statistically. Two previously proposed time-domain methods--correlation waveform analysis (CWA) and area of difference (AD)--were selected for evaluation of 29 patients with 33 distinct, sustained monomorphic VTs. An initial SR/AF template was used to analyze subsequent SR/AF and VT passages with a minimum of 50 consecutive depolarizations using a "best-fit" alignment. The values derived from each analysis were examined subsequently for skewness (asymmetry) and kurtosis (shape) using two-tailed tests (P less than 0.02). For passages of SR/AF, a normal (gaussian) distribution was present in only 24% (CWA), and 45% (AD); for passages of VT, normal distribution was present in only 58% for both CWA and AD. Using appropriate statistical testing with nonparametric tolerance intervals, CWA and AD discriminated VT from SR/AF in 29 out of 33 (88%), and 30 out of 33 (91%) instances, respectively, with 95% confidence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704512 TI - Stability of pacing threshold, impedance, and R wave amplitude at rest and during exercise. AB - The pacing threshold of the human heart may be altered by physiological factors such as physical exercise. These changes may influence the individual programming of a pacemaker, since pacemakers can be programmed at pulse amplitudes of 2.5 volts and less. We investigated 22 patients with a multiprogrammable ventricular demand pacemaker 3 months after implantation; 16 patients had received a steroid eluting lead and six patients had an Elgiloy lead. Parameters measured at rest and immediately after exercise were: Voltage threshold at pulse durations between 0.05 and 0.6 ms, impedance, R wave amplitude and energy consumption for the pacing threshold at 0.5 ms pulse duration. All patients performed a symptom limited supine bicycle exercise test. None of the investigated parameters showed a significant difference between rest and exercise, neither for the steroid eluting lead nor for the Elgiloy lead. The data suggest that the individual programming of a pacemaker adapted to the measurements at rest is also reliable and safe during exercise. PMID- 1704513 TI - Catheter ablation of accessory pathways: technique and results in 248 patients. AB - Two hundred and forty-eight patients with refractory arrhythmias related to an accessory pathway underwent catheter ablation. Cathodal shocks (160 to 240 joules) were delivered through the distal electrode of a standard catheter (usually a quadripolar electrode catheter with 5-mm interelectrode distances). A paddle electrode positioned opposite to the catheter served as the anode. Ablation of 24 right anteroseptal, 16 right parietal, 86 posteroseptal, 120 left parietal and four Mahaim pathways was clinically successful in eliminating symptomatic tachycardia in 236 patients (greater than 96%) over a follow-up of 3 to 64 months. There was no procedure-related death but two patients developed a ventricular fibrillation at the fifth and seventh day, respectively. The latter led to a sudden death since this side effect occurred after discharge. There were no instances of systemic embolus but one pericardial effusion required subxiphoid needle drainage 6 weeks after the procedure. Other complications included: AV block in four patients with posteroseptal and in one with a right anterior septal pathway. In conclusion, a successful clinical outcome may be achieved in most patients. Catheter ablation is an important alternative to cardiac surgery and in our opinion represents first-line treatment when therapy is required. PMID- 1704514 TI - Initial clinical experience with rate adaptive cardiac pacing using two sensors simultaneously. AB - In the rate adaptive pacemakers, all presently available sensors show one or more drawbacks. Combining two sensors in a single pacemaker, we tried to optimize its rate responsive characteristics. In this study, we present the rate adaptive behavior of a two sensor pacemaker system, using both QT interval and activity sensing. In addition, we compared the rate response with that of each sensor alone. Nine patients with an implanted QT interval sensing pacemaker, and an externally attached activity sensing pacemaker performed three exercise stress tests on treadmill. The QT interval, measured by the implanted pacemaker, and the activity level, were transmitted to an external computer. This computer contained the two sensor rate adaptive algorithm, and reprogrammed the implanted pacemaker on beat-to-beat basis. CONCLUSION: In the two sensor mode the rate increases immediately at the onset of exercise, caused by the prompt response of the activity sensor. Further rate increase is driven by the QT interval sensor and therefore proportional to the level of exercise. Furthermore, the rate decay during the recovery phase is more physiological. PMID- 1704515 TI - Longitudinal dissociation within the reentry pathway of ventricular tachycardia. AB - Two cases of nonsustained, repetitive ventricular tachycardia are analyzed. In both, the episodes of tachycardia do not contain random numbers of beats, but the complexes in each phase of tachycardia are either always in even numbers (case 1) or always in odd numbers (case 2). This indicates longitudinal dissociation within the reentry circuit: i.e., there are two functionally separate pathways in some part of the reentry circuit, and the reciprocating impulse runs alternatively through the two pathways. Tachycardia ends due to block of the impulse always in the same pathway, thus, the number of beats in each episode of tachycardia is always either in odd or even numbers. PMID- 1704516 TI - Linking: a mechanism of intermittent preexcitation in the Wolff-Parkinson-White syndrome. AB - Intermittent preexcitation in the Wolff-Parkinson-White syndrome has been equated with a long accessory pathway refractory period and long R-R interval between preexcited beats in atrial fibrillation and therefore a low risk for sudden death. A case of Wolff-Parkinson-White syndrome in which preexcitation became intermittent following procainamide infusion, with only moderate prolongation of the accessory pathway refractory period but marked prolongation of the shortest preexcited R-R interval in atrial fibrillation, is described. Programmed ventricular and atrial stimulation demonstrated that intermittent preexcitation was caused by concealed conduction producing a linking phenomenon, facilitated by the antiarrhythmic drug. Linking due to concealed retrograde penetration of a propagated impulse into the accessory pathway may contribute to the disparity between accessory pathway refractory period and shortest preexcited R-R interval in atrial fibrillation in some patients and may be a confounding factor in the interpretation of noninvasive tests of accessory pathway conduction. PMID- 1704517 TI - The effects of percutaneous catheter ablation on preexisting permanent pacemakers. AB - STUDY OBJECTIVE: Determine the effect of percutaneous catheter ablation (CA) on permanent pacemakers. MEASUREMENTS AND RESULTS: Twenty-three patients who underwent CA at The Cleveland Clinic Foundation from September 1983 to January 1990, and had a previously implanted pacemaker were studied. Electrocardiographic data during the CA procedure and clinic data including pacemaker evaluations were analyzed. Fifty-two percent (12/23) of the pacemakers malfunctioned: five developed transient ventricular loss of capture; two undersensing; one oversensing; three could not be interrogated or programmed, and one did not respond to the magnet test. Four patients developed syncopal episodes and two severe dizziness after the procedure. All had their pacemakers replaced. In total, seven were explanted. Destructive analysis by the individual manufacturer identified pacemaker circuitry failure in five. Unipolar pacemakers and anodal ablation procedures had more frequent and severe malfunctions, but the difference was not statistically significant. CONCLUSIONS: Pacemaker malfunction is frequent during CA. It may be prevented by programming the pacemaker, when possible, to the nonfunctioning mode (000 mode). Temporarily disconnecting the pacemaker during ablation requires further evaluation as an alternative approach. Close follow-up can detect pacemaker malfunction and prevent complications. PMID- 1704518 TI - Dynamic electrophysiology of ventriculoatrial conduction: implications for DDD and DDDR pacing. AB - The behavior of ventriculoatrial conduction (VAC) during exercise remains unknown. In order to determine its characteristics and the consequences it might have on dual chamber pacemaker technology and programming, 17 patients underwent an electrophysiological study (EPS) of atrioventricular conduction (AVC) and of VAC during a protocol including three steps: supine rest, upright position, and finally during cycloergometric exercise; the measurements were done at progressively increasing pacing rates. During a preimplantation EPS, Wenckebach points AVC-W and VAC-W and conduction times, AVCT and VACT (as a function of pacing rate), were measured in ten consecutive patients using temporary leads and an external device. In another study, AVCT, VACT, AVC-W, and VAC-W were measured by telemetric recordings under identical conditions in seven patients implanted earlier with a DDD pacemaker. A 1/1 VAC was observed in 7/17 patients (41%) at rest, and in 13/17 patients (76%) at the end of the protocol; VAC was never observed in 4/17 patients (23%), but occurred in six of the ten patients initially free, three standing at rest and three on exercise. For all patients, the VAC behavior remained of "nodal" type, indicated by a progressive increase in VACT as pacing rate rose up to the VAC-W point. Neither the existence of exercise induced VAC nor the maximal VACT-W could be predicted from AVC or VAC data obtained at rest. However, at the same pacing rates, standing up and exercise induced a shortening effect on VACT, and improved the VAC-W by an average of 33%. These results suggest that the electrophysiological behavior of VAC does not obey any general rule and cannot be predicted individually. It would thus appear unwise to base pacemaker mediated tachycardia (PMT) protection solely on long postventricular atrial refractory period (PVARP) programming in DDD patients. This work also revealed the potential risks of a rate responsive auto-adaptive PVARP algorithm as proposed in certain new devices. PMID- 1704519 TI - The A-R interval as exercise indicator: a new option for rate adaptation in single and dual chamber pacing. AB - We investigated the possibility to use the interval from an atrial stimulus to the ventricular R wave (A-R interval) as an indicator of physical stress, in 16 patients with pacemakers implanted for severe atrial bradycardia but with intact AV conduction. The A-R interval was studied during incremental atrial pacing at rest and during exercise with a constant workload. In addition, the atrial pacing rate was kept constant just above spontaneous sinus rate and the dynamics of the A-R interval were studied during exercise with a low constant workload and during a maximal exercise test with increasing workload. Incremental atrial pacing prolonged the A-R interval and this response was blunted during exercise (P less than 0.003). Atrial pacing at a constant rate and during a constant workload resulted in an almost direct shortening of the A-R interval. When the workload was increased but the atrial rate kept constant, a pronounced shortening of the A R interval was noted (P less than 0.0001). It is concluded that changes of the A R interval during different kinds of exercise were prompt and predictable in patients with sinus node dysfunction but intact AV conduction. In these patients the shortening of the A-R interval during exercise may be a suitable indicator for rate adaptive atrial pacing. PMID- 1704520 TI - A reappraisal of atrioventricular nodal excitability during functional 2:1 block: what should be the gauge? AB - By using extrastimulation technique the effective refractory period (ERP) of the atrioventricular (AV) node is conventionally defined as the longest A1A2 interval at which conduction of A2 is blocked within the node. With the prolongation of the AV nodal transit time that is associated with this technique, the delayed AV nodal arrival of the A1 impulse will cause an overestimation of the true interval if the A1A2 interval is used as the gauge. Under this condition, the conventional approach will not accurately delineate the ERP of the AV node. However, this prolongation of the nodal transit time can be calculated by subtracting the basal conduction time from the conducted A-H interval. By subtracting out the prolongation from the longest A1A2 at which A2 impulse is blocked, not only an interval more indicative of the true AV nodal ERP can be obtained, but also the apparent discrepancies between the in vitro and human AV nodal recovery curves disappear. PMID- 1704521 TI - Quantitating AV nodal function: has A1A2 outlived its usefulness? PMID- 1704522 TI - NASPE young investigator awardee-1990. Complex rhythms resulting from overdrive suppression in electrically stimulated heart cell aggregates. AB - Spontaneously beating embryonic chick heart cell aggregates were stimulated with current pulses delivered either as periodic trains, or at a fixed delay after each action potential. Following stimulation at fixed rates faster than the intrinsic rate, there was a transient slowing of the spontaneous rhythm. This response, called overdrive suppression, can lead to a complex evolution of rhythms. During periodic stimulation there is a continuum of dropped beat patterns, and during fixed delay stimulation bursting activity appears. This study provides a conceptual basis for understanding analogous rhythms in the intact heart. PMID- 1704523 TI - Incompatibility of different pacemaker connectors. PMID- 1704524 TI - Single-lead VDD pacing system. PMID- 1704525 TI - [The Ventak AICD Model 1550]. PMID- 1704526 TI - Cardiac pacing and electrophysiology. Cardiostim '90. June 20-23, 1990, Nice, France. Proceedings. PMID- 1704527 TI - Automatic adjustment of pacing parameters based on intracardiac impedance measurements. AB - The selection of the parameters used for rate control is determined not only by technical feasibility, but also by patient considerations. Technical feasibility considerations include long-term stability and reliability of the sensor, as well as susceptibility to interference. The patient considerations relate to the patient's physical state, including the various physiological and pathologic conditions. The rate response must be in proportion to circulatory demand. The specificity of the rate response is of particular importance for the patient with low cardiac reserve. The development of future pacemakers aims at providing additional patient benefits, with a reduction of the effort associated with initial parameter selection and patient follow-up. This will be achieved by utilizing a better understanding of the integration of the control mechanisms for the entire cardiovascular system under physiological and pathological conditions. To support the development of the future pacemakers, we must utilize realistic multiparametric models of the cardiovascular system. These models will assist the evaluation of potential algorithms for integrating multisensor signals into a single pacing rate. The parameterization and validation of these models are important issues to be addressed. Intracardiac impedance measurements in conjunction with microprocessor controlled signal processing and improved lead technology provide a great variety of practical applications for physiological control of the pacing rate and the automatic adjustment of pacemaker parameters. The concepts of an "intelligent" pacemaker capable of automatic control in response to changes in the pacing requirements under a variety of physiological and clinical conditions are presented. PMID- 1704528 TI - Endless-loop tachycardias: description and first clinical results of a new fully automatic protection algorithm. AB - Endless-loop tachycardia (ELT) is one of the most common pacemaker mediated tachycardia. An innovative ELT protection algorithm has proven to be clinically effective. A new improved version that will eliminate the need to program any parameter is now under clinical evaluation. Nine patients entered the study: six men and three women, aged 52 +/- 22 years. This automatic algorithm needs only 10 cycles to detect and confirm an ELT. Three hundred thirty-three ELTs lasting more than 9 cycles have been induced and analyzed. The total results are the following: mean duration: 6.7 sec +/- 3.1; mean ELT rate: 137 +/- 21.9 bpm, mean programmed upper rate limit (URL): 142.5 +/- 26.5 bpm (Only 70% of ELTs presented rates equal to programmed URL). (1) ELTs reduced by postventricular atrial refractory period (PVARP) extension on one cycle: 291 ELTs (87%). ELT rate: 128.5 +/- 18.2 bpm. (2) Retrograde block: algorithm operation may induce a retrograde block due to a short atrioventricular delay (AVD) applied during the confirmation phase to discriminate an ELT from a stable sinus rhythm. Thirty-two ELTs (10%) have been reduced and detected on a retrograde block occurrence. (3) Algorithm failure due to an unstable ventriculoatrial conduction time (VACT) even at fixed rate or to a retrograde Wenckebach behavior on AVD reduction during the confirmation phase. A total of 10 algorithms failed to detect or confirm an ELT have been recorded (3%). Mean duration: 8.2 +/- 4.2 sec, mean ELT rate: 148.9 +/- 14.3 bpm. This new fully automatic algorithm has reduced 97% of ELTs, including high rate episodes (100-175 bpm).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704529 TI - PR interval behavior during exercise: implications for physiological pacemakers. AB - The relationship between heart rate response and the dynamic changes in the PR interval was assessed in 631 patients undergoing routine cardiac exercise tests for a variety of clinical indications. Patients were stratified into four subsets: nonmedicated normals (n = 437), patients on beta-antagonist agents (n = 118), those on antiarrhythmic agents alone (n = 61) and those with a clinical diagnosis of advanced (New York Heart Association [NYHA] Class III or IV) congestive heart failure. All patients were in stable sinus rhythm throughout the test. PR intervals were measured at rest, at mid-exercise and at peak exercise. Mean PR intervals shortened to a statistically significant degree in most subgroups. This effect was predominantly observed in the earlier stages of exercise. In patients with advanced heart failure, there was no statistically significant shortening of exercise PR intervals later in exercise, demonstrating a parallel with their relatively blunted heart rate response. These changes in exercise PR intervals suggest that implanted pacemaker algorithms may be constructed to maximize hemodynamic benefit in patients requiring physiological pacemakers. PMID- 1704530 TI - AV delay and exercise stress tests: behavior in normal subjects. AB - In normal subjects the atrioventricular (AV) conduction is accelerated during exertion. The relationship between heart rate and AV delay is usually described as "linear". Looking at the increasing importance given to an appropriate AV synchrony in permanent dual chamber and P synchronous pacing we present the results of an investigation performed to study the correlation between AV conduction time and heart rate under stress conditions, and disclose some new aspects of this matter, which will possibly be useful for a further improvement of pacemaker technology. PMID- 1704531 TI - Effect of short atrioventricular delay on cardiac output. AB - Short atrioventricular (AV) delay modifies late diastolic filling dynamics. The effect of this change on cardiac output (CO) was studied in closed chest, AV blocked canine preparations (N:10), during AV sequential pacing (80 bpm). CO (thermodilution technique) and transmitral flow velocity (TMFV, pulsed-wave Doppler) were measured and compared (paired t-test) on the basis of TMFV pattern, when atrial contraction (A wave) started just after early diastolic transmitral flow deceleration (PR: 219 +/- 25 ms, mean +/- SD) and when A wave occurred at the end of late diastole and shortened due to the next ventricular contraction (PR: 56 +/- 11 ms). The short AV delay resulted in 12.0 +/- 5.9% decrease of CO, reflecting the interrupted late diastolic atrial transport. Properly timed atrial contraction is necessary for optimal AV sequential pacing. PMID- 1704532 TI - Assistant programming software: a new tool for an improved programming of pacemakers. AB - Programming the new DDD pacemakers is becoming increasingly difficult. One must take into account the pacemaker's complexity, the fact that some parameters are linked to others, and the clinical profile of the patient. This difficult problem will lead to the design of software to assist programming, which will help the implanting physicians in choosing adequate programmed settings adapted to the functioning of the device and to the physiology and pathology of the patient. These programming aides should meet certain basic requirements to make them safe, efficient, and easy to use. One such system designed by ELA Medical, "Programming Assistant" is herein described. The preliminary results of an initial study on the acceptance of this programming aide among physicians involved in cardiac pacing are given and discussed. PMID- 1704533 TI - Left atrial size and wall motion in patients with permanent ventricular and atrial pacing. AB - It is well known that during permanent ventricular pacing atrial arrhythmias and embolic complications occur much more frequently in comparison to permanent atrial or sequential pacing. Hemodynamic disturbances caused by ventriculoatrial conduction (VAC) are thought to be responsible for those complications. The aim of this study was to compare the left atrial size and its wall motion in three groups of patients with sick sinus syndrome. Group 1: 58 patients with VVI pacing and VAC observed (22 males, 36 females, aged 31-86, mean 62.3). Group 2: 43 patients with primary AAI pacing (13 males, 30 females, aged 27-74, mean 57.8). Group 3: 13 patients with AAI or DDD replacing the primary VVI mode due to pacemaker syndrome and/or heart failure, all with VAC present during VVI pacing (7 males, 6 females, aged 26-80, mean 59.8). Two-dimensional/M-mode echocardiography was performed in all these patients. In group 1 mean diastolic as well as mean systolic atrial diameters were significantly greater (P less than 0.005) and wall motion significantly smaller (P less than 0.005) in comparison to the other groups. Left atrial wall motion amounted to only 7.4% of the mean diastolic diameter in this group. Mean left atrial diastolic and systolic diameters and wall motion in patients with pacemakers preserving atrioventricular synchrony (group 2 and group 3) were almost identical and wall motion amounted to about 22% of the diastolic diameter in both these groups. We conclude that ventriculoatrial conduction leads to significant enlargement of left atrium and to the atrial wall-motion decrease. This predisposes to arrhythmias and embolic complications.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704534 TI - True incidence of pacemaker syndrome. AB - Although the purported incidence of pacemaker syndrome according to the literature is only 5%-15%, this is based on a series of patients with VVI pacing. Increasing numbers of studies are being reported in which patients prefer the dual chamber mode despite little benefit being demonstrated on objective testing, suggesting that pacemaker syndrome may be more common than is generally reported. This study was designed to evaluate the reported symptoms in a series of patients programmed to both the VVI and one or more dual chamber modes. Forty unselected patients with dual chamber pacemakers were entered into a blind, randomized trial comparing the symptoms associated with VVI pacing to those associated with dual chamber pacing. Patients were randomized to either VVI or dual chamber pacing. At the end of 1 week, questionnaires rating 16 different symptoms were completed. Blood pressure, LV function, presence of ventriculoatrial conduction, and ability to override the pacemaker were evaluated. The pacemaker was then programmed to the other mode. Overall, 12 of 16 symptoms were significantly worse in the VVI as compared to dual chamber mode. The most highly significant (P less than 0.005) were shortness of breath, dizziness, fatigue, pulsations in the neck or abdomen, cough, and apprehension. Pacemaker syndrome was clinically recognized in 83% of patients paced in the VVI mode with 65% of patients experiencing moderate to severe symptoms. There were no readily identified clinical, hemodynamic, or electrophysiological parameters that predicted which patients would develop pacemaker syndrome. Thus, when patients have an opportunity to experience both pacing modes in close proximity to one another, there is a high incidence of pacemaker syndrome in the VVI mode. PMID- 1704535 TI - Usefulness of 1-hour and 24-hour heart rate Holter inbuilt in new TX* rate adaptive pacemakers. AB - The rate adaptive TX* pacemaker uses the evoked QT interval as an indicator of physiological demand. In order to obtain a rate adaptation close to physiological patterns we used in the past, in each patient, on the slope value and/or the T wave sensing window, controlling via exercise stress testing and Holter the results achieved. It was an expensive method, but the system produced effective rate responsive pacing. The new series of TX* pacemakers (Quintech 919 and Rhythmyx), beside the dynamic slope feature, are equipped with a 1-hour heart rate Holter (HRH) that can be used during effort without the need for manually recording the heart rate. In this mode TX* pacemakers calculate the average heart rate over 20-second periods and stores the values continuously for 1 hour. In addition, a 24-hour HRH is available, which calculates the average heart rate over 7.5-minute periods, showing heart rate trend during the last day prior to interrogation. Each HRH can be accessed by the programmer and printed out. Using four Quintech 919* and five Rhythmyx units, the inbuilt HRH proved its utility by making the heart rate adaptation checking procedure easier, faster, and more economic. PMID- 1704536 TI - Evaluation of rate-responsive pacemakers by transesophageal Holter monitoring of spontaneous atrial rate. AB - One of the most important problems in rate responsive (RR) pacing is the clinical experimental evaluation of the reliability of various sensors. In particular, it is difficult to test their sensitivity and specificity during daily activity of the patients. Atrial rate, when present and normal, is the most physiological marker of metabolic requirements, but sometimes it is impossible to analyze the P wave in ventricular paced rhythm during routinely performed tests (e.g., ergometric test and 24-hour Holter monitoring). During various physical activities, we monitored atrial electrograms on an esophageal lead on the first channel of a standard Holter tape recorder; on the second channel a surface ECG lead was recorded. We selected 10 patients with high grade heart block and normal sinus node function paced in RR-VVI mode. RR pacing was obtained using various sensors (body activity, blood temperature, spike-T interval, minute ventilation). The good quality of recording allowed an easy evaluation of atrial and ventricular rates. In four cases an appropriate increase in heart rate was documented; sensitivity threshold and/or rate response slope were reprogrammed when indicated. The pacing rate of one patient did not parallel the atrial rate during walking only. In three cases, we observed a delay in the ventricular rate increase, with ventricular rate decreasing at peak exercise despite further atrial rate increase. In the last two patients, we observed inappropriate pacing response; pacing rate increased later and to a lower level than the atrial one. This new method is applied easily and appears reliable to evaluate the response of RR pacemakers to individual metabolic needs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704537 TI - The six-minute walk--an adequate exercise test for pacemaker patients? AB - In many pacemaker patients bicycle and treadmill ergometry are not practicable. As an alternative, we performed a 6-minute walk on a 20-m corridor in 97 pacemaker patients, who were asked to walk as far as possible determining their speed by themselves. Results were compared with those of bicycle ergometry in 42 of these patients and with treadmill exercise of a group of 92 other pacemaker patients. In the 6-minute walk, performance and maximal heart rate were slightly lower (49 +/- 18 W; 96 +/- 23 beats/min) than in bicycle (57 +/- 16 W; 110 +/- 26 beats/min) and treadmill ergometry (50 +/- 37 W; 102 +/- 35 beats/min). A good correlation was found between walking and bicycling (r = 0.74) and in subgroups of patients with different pacemaker indications. All patients preferred the walk to bicycle ergometry considering it to be more related to daily physical activity. In conclusion, a 6-minute walk is a simple and physiological exercise test for nearly all pacemaker patients with good correlation to other types of exercise. It seems to be preferable to other tests because of its better acceptance and practicability. PMID- 1704538 TI - Sympathetic nervous system response to dynamic exercise in complete AV block patients treated with AV synchronous pacing with fixed AV delay or with auto-AV delay. AB - To investigate the sympathetic nervous system (SNS) responses and circulatory responses to exercise in eight patients (five male and three female) with complete atrioventricular block (CAVB) treated with atrioventricular (AV) synchronous pacing, a symptom-limited, multistaged treadmill stress test was performed, and plasma norepinephrine (NE) and circulatory parameters were measured at rest, at peak exercise, and in the recovery period. The eight patients were tested using the fixed AV interval (150 or 156 msec). Their exercise tolerance was generally poor. In all measured points, plasma NE levels were significantly higher in the eight study patients than those in the 12 normal subjects (eight male and four female). Systolic blood pressure (SBP) of CAVB patients elevated significantly after exercise compared to that at peak exercise. Heart rate (HR) responses of CAVB patients were characterized by their poor increase at peak exercise. These results suggest that some latent cardiac dysfunction continues in the CAVB patients however satisfactorily the AV synchronous pacing might perform. AV synchronous pacing with three different kinds of auto-atrioventricular delay (auto-AVD) was applied to three of the eight patients. In each AVD mode, a treadmill stress test was performed repeatedly according to the same protocol. Plasma NE concentrations under the condition with fixed AVD at peak exercise increased compared to those under the other two conditions with auto-AVD. These findings suggest that AV synchronous pacing with auto-AVD was better than that with fixed AVD during exercise. Plasma NE response to exercise seems to be a useful indicator for evaluating the condition of patients treated with DDD pacemakers and their adaptation for cardiac function. PMID- 1704540 TI - Computerized pacemaker patient analysis. AB - We have developed computer hardware and software that imports analog waveforms and other measured data from a patient into the PaceBase database system supported by any IBM PC/AT compatible. The programmable A/D converter has the capacity to acquire the pacemaker artifact from the surface ECG leads. Analysis of the pacemaker artifact permits confirmation of pulse width as well as programmability and facilitates discovery of pacemaker hardware failures otherwise undetectable. Continuous recording of real-time surface ECG can be made as other measurements or storing functions are being performed. In this way, sporadic or infrequent intrinsic events are automatically recorded and can be selected to be played back and reviewed or stored on the permanent record. Pacemaker spike detection enhances identification of paced and intrinsic complexes by emphasizing the paced artifact. Even bipolar atrial spikes with pulse widths as short as 0.05 ms are easily identified; myopotential muscle noise is rejected. Enhancement of pacemaker spikes takes the guesswork out of interpretation of ECGs, especially for bipolar systems and when testing or troubleshooting for myopotential tracking or inhibition. PMID- 1704539 TI - c-AMP and ANP levels in VVI and DDD pacing with different AV delays during daily activity and exercise. AB - Nine patients (three males) mean age 68 +/- 8 years, having complete heart block, and paced in the DDD mode were examined in VVI and DDD pacing with 100 and 150 ms atrioventricular delays (AVD) during rest and exercise. Plasma atrial natriuretic peptide (ANP) and cyclic AMP (c-AMP) were measured at rest and at peak exercise test. ANP plasma levels at rest were significantly higher in VVI pacing compared to 150 AVD (P less than 0.03). On exercise, ANP release was statistically increased only in DDD with 150 ms AVD, while in VVI it remained in high levels at exercise but no significant change was found (p:ns). c-AMP during rest was unchanged in any pacing mode or AVD, but on exercise DDD pacing with short AVD (100 ms) released lower c-AMP plasma levels, than at rest (p:ns). DDD pacing with long AVD (150 ms) during exercise produced statistically higher c-AMP plasma levels (P less than 0.05) than at rest. Also in VVI pacing the c-AMP plasma levels were statistically higher than at rest (P less than 0.02). Adrenergic activity seems to be lower during exercise in DDD pacing with shorter AVD (100 ms) than in DDD with 150 ms AVD or VVI pacing. No difference was found in c-AMP plasma levels at rest. ANP release was also found to be lower at exercise in DDD pacing with short AVD (100 ms) than in DDD with 150 ms AVD. ANP plasma levels at rest were statistically higher in VVI pacing. PMID- 1704541 TI - A new system for follow-up patients with permanent pacemakers. AB - The use of a new computerized system for coordinating technical and clinical data and especially for filing the parameters found during out-patient checkups enables us to rationally carry out follow-up of approximately 900 patients with pacemakers in our electrostimulation laboratory without wasting time or data. The system is composed of a personal Olivetti M240 computer, with a 20 Bbyte and 640 Kram hard disk, an analog-digital interface and a personalized data base for coordinating and filing the parameters found. The type of pacemaker implanted (monocameral, dual chamber or rate responsive) is of no consequence to the system that is based on the acquisition of the surface electrocardiogram. A comparison of the filed data on printouts or graphs allows us to note every type of misfunction or anomaly in the stimulation system in real time. PMID- 1704542 TI - "Pacecare"--a computerized database for pacemaker follow-up. AB - A computerized database for pacemaker follow-up has been designed to run on IBM compatible hardware and to accept pulse generator and lead models of all manufacturers. Stored data includes patient, physician and implant details, indications for pacing, underlying rhythm, complications and management, program settings, and follow-up measurements. Typing is minimized by the use of "pop-up" lists and prepared pulse generator template displays. At each follow-up visit a patient's file is retrieved by surname or number, a visit record created, and measurements documented. As the template of the previous visit is used, recording of the clinic visit takes less than 1 minute. Changes in pacing rates (base or magnet), pulse widths, lead thresholds, lead impedance, and battery cell impedance can be displayed graphically for immediate recognition of end-of-life parameters or suspected malfunction. The program will print patient, implantation and clinic visit summary reports, clinic appointment lists, letters to patients, and annual reports. Two Melbourne hospitals have now entered over 3,600 patients into the database. Valuable information has been obtained regarding implantation details and trends with pulse generator and lead usage. Pacecare is a sophisticated, yet user friendly, computerized database for pacemaker follow-up. Recording of clinic visits is fast and changes in testing parameters can be recognized immediately. PMID- 1704543 TI - An artificial neural network to localize atrioventricular accessory pathways in patients suffering from the Wolff-Parkinson-White syndrome. AB - The electrocardiographic localization of atrioventricular accessory pathways has been extensively described in the literature by a number of well-known electrophysiologists and surgeons. These descriptions, often represented as decision trees, are useful, but do not apply in all cases. To formalize the process of determining the proper localization, this expert human knowledge could be represented in an expert system. But since reasoning is partly based on the use of heuristic knowledge, and are often not represented in the written description of the human expert, the results will be suboptimal. On the other hand, by using a self-learning neural network approach, the causal relations between input (polarity of the delta waves) and output (the correct localization) do not have to be defined by the expert. It is derived by the neural network, by analyzing a learning set of cases consisting of the ECG plus the corresponding correct localization. In our set of 60 cases, 2 hours of training were required to learn how to localize all cases correctly. From a control set of 25 cases, 23 were interpreted by the system satisfactorily. CONCLUSION: the neural network approach can be useful in situations where causal relations between the electrocardiogram and underlying mechanism are partly undefined. PMID- 1704544 TI - Circadian rate variation in rate-adaptive pacing systems. AB - By mimicking the natural rate slowdown during sleep, a pacing system can enhance patient comfort while improving device longevity. A new family of rate-adaptive pacemakers accomplishes this circadian rate variation by modeling the patient's sleep-wake cycle using a time-of-day clock inside the device. Furthermore, to account for minor variations in the patient's sleep-wake cycle, sensor data can be used to automatically adapt the starting and stopping points of this change in rate. Using the model, the device begins to gradually reduce the pacing rate beginning one-half hour before the selected bed time. Similarly, the rate begins to increase one-half hour before the selected waking time. Sensor data indicating the presence or absence of patient activity are used to adapt the selected bed and wake times, enabling the system to maintain an appropriate schedule despite changes in the patient's actual schedule. The model approximates natural human adaptation times for time zone or work shift changes. This circadian rate adaptation does not preclude the system's rate response to patient activity; even during sleep time the response to moderate or heavy exercise is essentially unchanged. PMID- 1704545 TI - A randomized double-blind, cross-over study of the linear and nonlinear algorithms for the QT sensing rate adaptive pacemaker. AB - We have compared the pacing rate responses during cardiopulmonary exercise testing in 11 patients (mean 59 years, six female) with implanted QT sensing rate adaptive pacemakers who were randomly programmed to 1-month periods in the linear and nonlinear algorithms using a double-blind, cross-over design. Exercise testing was performed at the end of each month block and symptoms were scored with the MacMaster questionnaire. With exercise, the time to a 10 beats/min increment in rate was significantly less with the nonlinear compared to the linear algorithm (126 sec vs 255 sec, P = 0.02) but there were no significant differences in exercise duration, the peak pacing rate, the peak VO2, the VO2 at the anaerobic threshold or the mean correlation coefficients of the pacing rate VO2 relationship. Rate oscillation occurred in seven patients in the linear algorithm and in two patients in the nonlinear setting. Initial deceleration of the pacing rate at the onset of exercise occurred in seven patients in the linear algorithm and in four patients in the nonlinear setting. The nonlinear algorithm is associated with a faster response time during exercise and fewer instances of rate instability. However, it has not overcome the problem of a dip in the pacing rate at the beginning of exercise. The major difference in the function of the two algorithms is faster initial acceleration with the nonlinear algorithm. This is explained by the significantly higher values of the slope setting at the lower rate limit for the nonlinear versus the linear algorithm (6.3 ms/ms vs 5.1 ms/ms). PMID- 1704546 TI - Relationship between right atrial and mixed venous oxygen saturation and heart rate during exercise in normal subjects and patients with cardiac disease. AB - An ideal sensing variable for use in rate responsive pacemakers should measure a physiological parameter that closely correlates with heart rate during various activities in a diverse group of subjects. Nineteen patients, 12 normal and 7 patients with heart disease, were studied to assess the relationship between mixed venous oxygen saturation and heart rate. In patients with heart disease right atrial oxygen saturation and heart rate were also compared. Each subject underwent pulmonary artery catheterization and performed seated cycle ergometer exercise. Gas exchange and heart rate were measured continuously and blood sampled at frequent intervals. Normal patients were studied at rest and during steady-state exercise (mean work rate 149 watts). Patients were studied at rest, steady-state exercise (mean work rate 37 watts), and during incremental exercise (5-10 watts/min) to tolerance. There were 248 paired right atrial or mixed venous oxygen saturation/heart rate observations obtained. Changes in mixed venous oxygen saturation and heart rate were not substantially altered by fitness or cardiac disease. Rate responsive pacemakers sensing changes in oxygen saturation may be a superior sensing variable for both normal and patients with heart disease. PMID- 1704547 TI - Intrapatient comparison between chronic VVIR and DDD pacing in patients affected by high degree AV block without heart failure. AB - In patients affected by high degree AV block without preexisting congestive heart failure there is no definite demonstration that DDD pacing gives real clinical advantages in respect to VVIR pacing. We performed an intrapatient, long-term study between the two pacing modes in 14 high degree AV block patients, using the Medtronic Synergyst 7027 dual chamber pacemaker, who could be programmed alternatively in DDD or VVIR mode. After a 4-week run-in period following the pacemaker implant, patients completed a randomized, double-blind, cross-over study to compare the effect of 6-week period VVIR and DDD pacing on symptoms and cardiovascular parameters. A semiquantitative score scale was used to quantify the symptoms of general well-being, palpitations, dizziness, pulsating sensation in the neck or abdomen, shortness of breath at rest and during effort, chest pain, and NYHA classification. The sum of symptom scores was 10.4 +/- 6.7 in VVIR period and 4.6 +/- 2.7 in DDD period (P less than 0.001); five patients (36%) crossed over early from VVIR to DDD because of intolerable symptoms; overall, eight patients preferred the DDD mode and no one preferred the VVIR. Cardiac output at rest (echo-Doppler method) was 4.7 +/- 1.4 versus 5.7 +/- 1.6 liter/min (P less than 0.01), body weight was 65.9 +/- 6.6 versus 64.9 +/- 6.1 kg (P less than 0.02), atrial natriuretic peptide was 236 +/- 112 versus 198 +/- 110 pg/mL (P less than 0.01), respectively, during VVIR and DDD modes. Effort tolerance was similar with the two modes of pacing (68 +/- 15 vs 70 +/- 18 watts/min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704548 TI - Pacing in atrial arrhythmias. AB - The history of pacing to control the atrial arrhythmias of sinoatrial node disease (SND) is reviewed and is demonstrated to have become more physiological in recent years. The importance of atrial stimulation is emphasized especially in the context of the natural history of SND. The role of single and dual chamber rate responsive pacing for correction of chronotropic incompetence is outlined and guidelines are proposed for the management of the different types of SND presentation. PMID- 1704549 TI - Clinical predictors and natural history of atrial fibrillation in patients with DDD pacemakers. AB - Effective DDD pacing requires that patients remain free of atrial fibrillation (AF). Four hundred eighty-nine consecutive patients undergoing initial transvenous DDD implants were reviewed to determine the incidence of postimplant AF in this population and to assess what factors, known at implant, predicted the later development of AF. The variables analyzed included age, sex, indication for implant (dominant SA or AV node disease), history of AF, atrial electrogram characteristics and pacing threshold, and the status of retrograde conduction. Forty-eight patients (9.8%) developed AF a mean of 23 months postimplant, and 11 of these patients returned to sinus rhythm and were managed once again in DDD for significant periods. A prior history of AF and the presence of dominant sinoatrial disease were far more prevalent in the patients who developed AF (P less than 0.001) though the vast majority of patients with these two independent risk factors remained in sinus rhythm through much or all of their follow-up period. We conclude that the incidence of AF is not of a magnitude to preclude DDD pacing in the vast majority of patients in sinus rhythm at implant. PMID- 1704550 TI - Superior cardiac hemodynamics of atrioventricular synchrony over rate responsive pacing at submaximal exercise: observations in activity sensing DDDR pacemakers. AB - The relative hemodynamic profile between dual chamber pacing (DDD) and activity sensing rate responsive pacing (VVIR) was compared in ten patients with dual chamber rate responsive pacemakers (Synergist II). With a double blind, randomized exercise protocol, DDDR pacemakers were programmed into VVI, VVIR, and DDD (AV interval 150 msec) modes and in seven patients the test in the DDD mode was repeated with the AV interval programmed at 75 msec. A treadmill exercise test of 6-minutes duration (2 stages, Stage I at 2 mph, 0% gradient and Stage II at 2 mph, 15% gradient) was performed at each of the programmed settings, with a rest period of 30 minutes in between tests. Cardiac output was assessed using continuous-wave Doppler sampling ascending aortic flow and expressed as a percentage of the value achieved during VVI pacing. During exercise, pacing rate between DDD and VVIR pacing was similar but was higher with DDD at the first minute of recovery (91 +/- 4 vs 81 +/- 3 beats/min, respectively). Cardiac output was significantly higher at rest, during low level exercise, and recovery with DDD pacing compared with VVIR pacing (resting: 21 +/- 14 vs -2 +/- 7%; Stage I: 36 +/- 6 vs 16 +/- 7%; Stage II: 25 +/- 15 vs 10 +/- 8%; recovery: 26 +/- 12 vs 4 +/- 9%; P less than 0.05 in all cases). Systolic blood pressure was significantly higher during low level of exercise in the DDD mode. Shortening of the AV interval to 75 msec did not significantly affect cardiac output during exercise, but cardiac output after exercise was reduced (2 +/- 6 vs 23 +/- 6% at an AV interval of 150 msec, P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704552 TI - Rate augmentation and atrial arrhythmias in DDDR pacing. AB - Dual chamber, rate-modulated pacemakers provide the capability of augmenting the heart rate of patients with chronotropic incompetence but also may cause atrial arrhythmias because of high rate, competitive atrial pacing. We studied ten patients with two consecutive 24-hour Holter monitors during which they were alternately programmed to either DDD or DDDR pacing in random order. Maximum heart rates (max HR) were measured at every 15-minute interval during each 24 hour period. DDDR pacing showed rate augmentation, 80 +/- 7 average max HR when compared with DDD pacing, average max HR 76 +/- 5. These results were even more striking when waking hours (7 am to 10 pm) were compared: average max HR 86 +/- 7 DDDR versus 78 +/- 4 average max HR DDD. Several patients showed marked rate augmentation. Seven of ten patients preferred DDDR pacing over DDD pacing. In the entire population, DDDR pacing did not result in an increased number of atrial arrhythmias (1.25 atrial events/24 hour) when compared to DDD pacing (1.75 atrial events/24 hour). We conclude that DDDR pacing provides heart rate augmentation during daily life in a clinical population while not resulting in a significant increase in atrial arrhythmias. PMID- 1704551 TI - Comparative evaluation of rate modulated dual chamber and VVIR pacing. AB - While dual chamber pacing is considered superior to VVI pacing at rest, there is a continuing debate as to the relative benefit of AV synchrony versus rate increase with exercise. To evaluate this question and to correlate different methods of evaluation, 14 patients with DDDR pacemakers were studied using serial treadmill exercise test with a CAEP protocol. Patients were exercised in DDD, DDDR, and VVIR modes. Echo-Doppler cardiac outputs were determined and pulmonary gas exchange was measured during exercise. There was a significant improvement in cardiac output with exercise in the DDDR versus VVIR modes, and in DDDR versus DDD modes in patients with chronotropic incompetence. There were small increases in exercise duration in DDDR versus VVIR modes, and small but consistent increases in VO2 at all levels of exercise, though not statistically significant. In this group of patients, DDDR pacing was superior to VVIR pacing, and superior to DDD pacing when chronotropic incompetence was present. PMID- 1704553 TI - A new feature for control of inappropriate high rate tracking in DDDR pacemakers. AB - A limitation of current DDD and DDDR pacemakers is the inability to distinguish between inappropriate high rate atrial sensed events that are physiologically appropriate to track (e.g., elevated sinus rates resulting from exercise, emotional responses, etc.) and those that are physiologically inappropriate to track (e.g., paroxysmal atrial dysrhythmias, myopotentials, retrograde conduction, etc.). The sophistication of sensing circuitry is not yet sufficiently advanced to permit a pacemaker to distinguish appropriate atrial events by morphology. The addition of an independent sensor to a DDD pacemaker (i.e., DDDR) gives more information about the patient's condition, especially with respect to exercise. This information can be used to judge the appropriateness of a high sensed atrial rate, and to modulate the pacemaker's response. If the sensor input is below a specified level, indicating lack of exercise, the DDDR can track sensed atrial events only to a tolerably low limit the conditional ventricular tracking limit (CVTL). Wenckebach-type behavior ensues at the CVTL until the sensor input increases, indicating that exercise is occurring, or until the sensed atrial rate decreases. If the sensor input indicates exercise, the DDDR pacemaker can track up to the programmed maximum rate. Two DDDR systems have been developed that incorporate this feature; one based on temperature, the other on activity. Currently the CVTL is set at a value about 30 ppm above the pacing rate, as a compromise to support emotional needs not seen by the sensor. Improved sensors could cause the decision to raise the tracking limit (i.e., recognition of physiological need for higher rates) to be more accurate, perhaps making the CVTL proportional to the sensor signal. PMID- 1704554 TI - Transatrial implantation of transvenous pacing leads as an alternative to implantation of epicardial leads. AB - State-of-the-art pacing modalities are not readily utilized with conventional epicardial pacing lead implantation techniques. A transatrial implantation technique was developed combining a limited surgical approach with transvenous leads. Six patients who were poor candidates for transvenous implants have received DDD or DDDR pacemakers by this approach. The limited surgical approach includes resection of the third or fourth costal cartilage through a small skin incision, reflection of the pleura, and opening of the pericardium. The introducer and transvenous leads are inserted through a right atrial pursestring suture. The leads are positioned in the right ventricle and right atrium using standard fluoroscopic techniques. Through the incision, the subcutaneous tissue pocket is constructed on the right anterior chest wall. The leads are connected to the pacemaker without the need for adaptors or tunneling. There were no procedure-related complications. The magnitude of the surgery and postoperative morbidity are significantly less than for a standard thoracotomy, median sternotomy, and transdiaphragmatic epigastric or subcostal approach. The utility of the transatrial implantation technique is that it allows the use of state-of the- art bipolar dual chamber pacemakers restoring access to all pacing modalities for those patients not candidates for transvenous implantation. PMID- 1704555 TI - A method for estimating endocardial electrode stability. AB - Assessment of cardiac pacing electrode stability following implantation is of major importance. We describe a method of testing electrode stability using a provocative test. The method includes synchronized electrical pacing of the left phrenic nerve by an especially designed external pacemaker. A positive response is abrupt, a diaphragmatic contraction, which in turn induces an abrupt heart movement in its diastole, its relaxation phase. Fifty-six various endocardial electrodes were tested in this fashion. Seven electrodes, one with flange tip, two tined, and four had no fixation dislodged after this pacing test. Insignificant elevation of acute ventricular pacing threshold was observed. While this method appears to be advantageous, additional clinical data is necessary. PMID- 1704556 TI - Intravascular lead extraction using locking stylets, sheaths, and other techniques. AB - Septicemia necessitates extraction of chronic pacemaker leads. Using locking stylets and sheaths to extract leads via the implantation vein (subclavian, cephalic, or jugular) and maneuvering devices, sheaths, and retrieval baskets via the femoral approach, extraction of 228 leads implanted 5 days to 240 months (mean 55 months) was attempted in 136 patients (mean 62 years) at 34 institutions. In addition to septicemia (9%) and infection (39%), total 48%, indications included prophylaxis/replacement (40%), and other (12%). Seventy seven leads were atrial, 151 ventricular; 147 were unipolar, 81 bipolar; 96 had silicone insulation, 127 polyurethane, 1 poly/silicone, and 2 undetermined. Fixation included tines or fins (160), screw (40), flange (12), and other (16). One hundred and ninety-four leads were completely extracted, 19 partly extracted, and 15 not extracted. Procedural complications were: torn atrium requiring open heart repair (1), hemothorax requiring a chest tube and blood transfusions (1), subacute hemothorax requiring drainage 18 days after discharge (1), thrombosis treated by drugs (1), and myocardial avulsion without sequela (1). Important observations included the significant training required due to the large number of possible clinical variables, and the need to be prepared for life-threatening cardiovascular complications. With training, procedures done at higher volume and lower volume institutions met with similar success. CONCLUSION: Intravascular lead extraction is a viable technique whose benefits outweigh the risks, given the proper intensive training and open heart surgical backup, and may obviate the need for open heart surgery for lead extraction. PMID- 1704557 TI - Intravascular lead extraction using locking stylets and sheaths. AB - Chronic lead extraction using intravascular countertraction techniques was studied in patients with over 65 different lead models including passive and active fixation devices. Indications for removal of 115 leads implanted 5 days to 264 months (mean 58 months) in 62 patients (mean 65 years) included septicemia, subcutaneous tissue infection, preerosion, free-floating lead, lead trapped in valve, too many leads, pain, and vein thrombosis. The superior vena cava (SVC) approach was attempted in 101 leads and was successful in 82 attempts (71% of total leads). The inferior vena cava (IVC) approach via the femoral vein was required to extract 14 (12%) leads inaccessible to the SVC approach and the 19 leads that failed the SVC approach (29% of total leads). The SVC procedure includes a sized stylet locked at the tip and telescoping sheaths advanced over the lead to the heart. An IVC procedure includes placement of a 16 F sheath workstation via a femoral vein into the right atrium. A deflection catheter and Dotter snare in an 11 F sheath were advanced through the workstation into the right atrium. The lead was maneuvered into position, snared, and pulled into the workstation. For both the SVC and IVC approaches, the leads were removed by applying traction on the lead and countertraction with the sheaths. In experienced hands, these techniques have proven safe and effective for removing chronic transvenous leads. PMID- 1704558 TI - Drug-eluting collar--a new approach to reducing threshold. AB - This study evaluated the clinical performance of leads with a drug-eluting (less than 0.5 mg of dexamethasone sodium phosphate) collar (DEC) placed adjacent to a 4-mm2 Pt/Ir coated electrode (Telectronics model 030-368). Ten trailing tined ventricular silicone DEC leads and ten control leads (model 030-359) were implanted transvenously in patients of comparable age, sex, and indication for pacing. Early threshold rise, chronic thresholds (Vario), and lead impedances were monitored by pacer telemetry. The DEC lead had substantially lower thresholds in the first few weeks (P less than 0.0005) as well as chronically (P less than 0.025). The study has shown that a drug dose of less than 0.5 mg, released from a drug-eluting collar (DEC), is effective in reducing thresholds. PMID- 1704559 TI - Chronic ventricular electrograms: do steroid-eluting leads differ from conventional leads? AB - The aim of steroid-eluting leads is to reduce chronic pacing thresholds. Whether steroid-eluting leads also modify acute and chronic R wave amplitudes as well as R wave sensing of the pulse generator was investigated in 31 patients with a unipolar ventricular pacemaker. Four different leads were implanted: Two steroid eluting leads with different electrode surface areas (8 mm2 and 5.8 mm2) and two conventional leads (Target Tip, Elgiloy lead). At implantation filtered R wave amplitudes, peak-to-peak values, and slew rates were measured by a pacing system analyzer. One year after implantation R wave amplitudes were directly determined from intracardiac electrograms and compared to the peak-to-peak data at implantation. Additionally, R wave inhibition was evaluated at a sensitivity setting of 5 mV. There were no differences among the four leads in respect to any of the parameters studied at implantation. At follow-up, no differences in R wave amplitudes were found leading to an appropriate sensing in all leads. Steroid eluting leads did not differ from conventional leads and a smaller electrode surface area of 5.8 mm2 had no influence on ventricular electrogram. Together with a pacemaker with an input impedance of 37 kohms R wave sensing was a correct setting of 5 mV. PMID- 1704560 TI - In vivo elution rate of drug eluting ceramic leads with a reduced dose of dexamethasone sodium phosphate. AB - We have evaluated the in vivo elution rate and the threshold voltage performance of a new lead incorporating a controlled delivery device based on a porous ceramic collar. The drug, dexamethasone sodium phosphate (DSP less than 0.2 mg), was contained within the pores of a ceramic collar that was positioned externally and adjacent to a 4 mm2 Pt/Ir coated electrode. Thirty-three leads comprising a porous ceramic drug eluting collar (DEC) were implanted in the right ventricle of 12 sheep. In vivo elution was determined by analyzing the drug remaining in the collar after 1, 3, 11, and 28 days. Voltage thresholds were measured at implant and then weekly for 28 days on three sheep. Results were compared to leads with identical electrodes but with silicone DEC (DSP less than 0.5 mg). The in vivo elution rate of the ceramic DEC leads was fast with approximately 50% of the drug content on the first day. Although the drug content and elution rates were different for the ceramic and silicone DEC leads, the threshold performance of the leads was similar. For ceramic and silicone DEC leads, threshold voltages at implant and at 4 weeks were 0.29 +/- 0.09 compared to 0.37 +/- 0.08 and 0.42 +/- 0.08 compared to 0.44 +/- 0.13, respectively. The results show that a relatively rapid release of a reduced dose of DSP from a DEC is still effective in reducing threshold peaking. PMID- 1704561 TI - Steroid-tipped leads versus porous platinum permanent pacemaker leads: a controlled study. AB - There is little data directly comparing steroid-tipped permanent pacemaker leads to otherwise state-of-the-art porous platinum leads. Eighteen patients receiving unipolar generators capable of low voltage outputs were randomized at the time of implant to receive either steroid-tipped leads or porous platinum leads. All leads were unipolar, tined, passive fixation, and placed in the right ventricular apex or atrial appendage. This study is single center. At implant, threshold pulse width was determined at 3 voltages (2.5, 1.5, and 0.8 V). Follow-up thresholds were determined at weeks 1, 2, 3, and 4, and at 3 and 6 months. There was no difference in implant thresholds or amplitudes for sensing. By 2 weeks postimplant, lower thresholds were noted for the steroid leads, and this discrepancy grew more significant with time. There was no significant postimplant rise in threshold for steroid-tipped leads. At 6 months, the average threshold pulse width for ventricular steroid leads at 0.8 V was 0.3 +/- 0.1 msec. In contrast, five patients with standard leads did not capture at maximum pulse width at 0.8 V (P less than 0.0001). There was no significant difference in the amplitude of the chronic atrial electrogram. This study shows steroid-tipped leads to offer a significant advantage in reducing thresholds early postimplant and chronically. PMID- 1704562 TI - Sputter-deposited TiN electrode coatings for superior sensing and pacing performance. AB - The sensing and pacing performance of pacemaker electrodes is characterized by the electrochemical properties of the electrode/tissue interface affecting tissue reactions and the kinetics of the ionic exchange. The usually smooth metallic electrode surface results in a high pass filter characteristic. To better match the electrode's filter characteristic to the spectral content of the depolarization signal, various combinations of electrode shape, material and surface structure have been researched. The electrode with sputter-deposited TiN coating presented in this report has been designed to meet the demand for low acute as well as chronic thresholds and superior sensing performance not only with respect to spontaneous activity but also regarding the detection of the evoked response. The clinical results obtained with this electrode prove the excellent pacing and sensing properties resulting from minimized polarization losses and optimized filtering of the signal to be detected, respectively. The acute and chronic clinical advantages over previous concepts are attributed mainly to the biocompatibility of the material used and the microcrystalline surface structure achieved by the coating process. The design concept of the new electrode is presented together with the clinical results obtained. While the advancements in microelectronics and battery technology have certainly formed the basis for the development of pulse generators featuring an ever increasing versatility of functions at the same or even smaller pacemaker dimensions, from a point of view of pacing system performance the development of improved electrode concepts as the one presented must be regarded as equally indispensable. PMID- 1704563 TI - Chronic animal testing of new cardiac pacing electrodes. AB - To evaluate the electrical performance of new electrode technologies, 24 leads containing either carbon coated porous titanium (BIOPORE, (Intermedics, Inc., Freeport, TX], iridium oxide (IROX), or iridium oxide coated with polyethylene glycol (IROX-PEG) electrodes (eight of each) were implanted into the ventricles of 12 canines. Stimulation threshold data was measured at regular intervals for 24 weeks. Low acute values were observed for all leads (0.32 +/- .13 V at 0.6 msec pulse width), but the IROX-PEG electrode demonstrated lower subchronic, peak, and chronic values. Compared to implant, the IROX-PEG electrodes' stimulation thresholds rose only 0.23 V when chronic conditions occurred. There were no significant differences between the electrodes in pacing impedance or R wave amplitude measurements. We conclude that both IROX and IROX-PEG technologies represent a promising approach to the design of more efficient cardiac pacing leads. PMID- 1704564 TI - A new efficient NanoTip lead. AB - The ideal lead has low, stable acute and chronic thresholds, high pacing impedance, and good sensing. Leads with low, stable thresholds have been developed, but pacing impedance has been in the 600 omega region. One way to increase pacing impedance is to decrease the electrode's surface area. The threshold performance and sensing ability of less than 5 mm2 electrodes have been considered questionable, up to now. We have developed a 1.5 mm2 porous, platinized, steroid-eluting electrode and have demonstrated in canine studies that it has excellent performance. Chronic thresholds are low at about 0.65 +/- 0.28 V (ventricular) and 0.42 +/- 0.12 V (atrial) at 0.5 msec. Chronic pacing impedance is in the 1200-1300 omega region, but mean chronic R and P wave source impedance is less than or equal to 1500 omega. Sensing is excellent, with almost double the P wave amplitudes usually measured in the canine. PMID- 1704566 TI - Survival of implantable pacemaker leads. The Implantable Lead Registry. AB - Early polyurethane leads were reported to have a high incidence of materials failure. In 1979 a six-center registry was begun. By October 31, 1989, 7,311 leads had been registered and lead longevity was calculated by individual manufacturer, cumulatively for all manufacturers and for individual leads. Each lead was registered at implant and at removal from service as having been removed for materials failure or for unrelated reasons. Calculations were made for 23 models and for a total of three manufacturers with more than 100 leads implanted. Four thousand seven hundred and sixty three leads (65.1%) were from Medtronic; 2,177 (29.8%) from Cordis; 297 (4.1%) from Intermedics; and 77 (1.0%) were from all others. The 10-year cumulative survival of Medtronic leads was 96.6 +/- 0.4%; for Cordis leads it was 99.9 +/- 0.1% and for Intermediates leads 97.7 +/- 0.9%. Three lead models registered over 100 units and had poorer survival at 5 years than the remainder of all manufacturers' experience. They were #6972 (n = 107) with 78.6 +/- 4.5%; #6991U (n = 294) with 90.6 +/- 1.9%; and #4012 (n = 288) with 97.3 +/- 1.5%. Other possibly failed models did not achieve statistical significance. It can be anticipated that a lead which survives to the seventh year will have prolonged longevity as thereafter additional failures are uncommon. PMID- 1704565 TI - A new single lead VDD pacing system. AB - In 24 patients with advanced heart block and normal sinus node function, a new single lead VDD pacing system was implanted. At implantation, the endoatrial, bipolar electrogram was recorded in all patients. The lead position was checked by means of chest X-ray. At discharge and after 1, 3, and 6 months, testing for myopotential inhibition, telemetric evaluation of the endoatrial potential, and Holter recordings were made. After discharge, 18 patients performed two cardiopulmonary exercise tests at two different rate-matched AV intervals. All investigations showed good AV synchrony and a lack of interferences by myopotentials. The maximum rate-matched AV interval provided a significantly improved exercise capacity, which was more evident in patients with signs of myocardial failure. PMID- 1704567 TI - Stiffness of the distal tip of bipolar pacing leads. AB - The stiffness of a bipolar pacing lead, particularly between anode and cathode, may be responsible for myocardial penetration and perforation. Following an unprecedented 7% incidence of high threshold exit block with a single model bipolar ventricular endocardial lead, a study was undertaken to compare pacing lead stiffness between anode and cathode of six models of bipolar leads from two manufacturers; Telectronics (T) and Medtronics (M). Four leads had polyurethane insulation; T 030-284 (Laser Dish), T 329-259 (Cordis, Encor), M 4012 (Target Tip), and M 4004 (Capsure). Two leads had silicone rubber insulation; M 5026 (Capsure) and M 5024 (Capsure SP). All leads were subjected to two stiffness tests. The Tip Deflection Test involved securing the lead at 45 degrees at the indifferent electrode and applying a force to deflect the tip 5 mm. The three point bending test involved placing the lead over two fixed bars in contact with the anode and cathode. Midway a third bar was pushed onto the lead and the force to deflect the lead 2 mm was recorded. The results showed that pacing leads with polyurethane insulation were much stiffer than those with silicone rubber insulation. The T 030-284 because of its construction was found to be the stiffnest. The next stiffnest was the M 4012. Both these leads had an unacceptable incidence of high threshold exit block; 7% with the T 030-284 (89 implants) and 3% with the M 4012 (102 implants). No cases of high threshold exit block were documented with the other four pacing leads and in particular the silicone rubber M 5026 (344 implants).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704568 TI - Appraisal of pacing lead performance using transtelephonic follow-up data. AB - Reliable performance statistics for implantable pacing leads are not readily available because of difficulties involved in data collection. An alternate source of data exists in the form of commercial monitoring service data. The example considered here involves transtelephonic follow-up data collected by CardioCare, a cardiac monitoring service. This presentation describes a procedure used to identify complications arising from pacing leads, and also presents a method for proper analysis of the data. Analyses comparing silicone leads and polyurethane leads were performed on leads manufactured by five major manufacturers in the United States. The results indicate that the performance of polyurethane unipolar leads is similar to that of the silicone unipolar leads. However, the polyurethane bipolar leads did not perform as well as the silicone bipolar leads. PMID- 1704569 TI - Variability of the intracardiac electrogram: effect on specificity of tachycardia detection. AB - Correlation has been described as a method of high sensitivity to distinguish ventricular arrhythmias from sinus rhythm but the specificity of this algorithm has not been assessed. Ten patients with a history of chronic ventricular tachycardia were studied. The ventricular endocardial electrogram was recorded during sinus rhythm at rest immediately following exercise and during their clinical ventricular tachycardia. Each complex recorded during these sample periods was correlated with a template constructed during sinus rhythm at rest. Although for each patient the range of correlation values obtained at rest were clearly separated from those obtained during ventricular tachycardia, in 69% of cases there was overlap of the range in sinus tachycardia and ventricular tachycardia. PMID- 1704570 TI - Detection of ventricular tachycardia using scanning correlation analysis. AB - Cross correlation is an accurate method for distinguishing normal sinus rhythm (NSR) from ventricular arrhythmias. The computational demands of the method, however, have prohibited development of an implantable device using correlation. In this study, temporal data compression prior to correlation analysis was used to reduce the total number of computations. Unipolar and bipolar intracardiac electrograms of NSR and 23 episodes of ventricular tachycardia (VT) from 23 patients were obtained from a right ventricular apex electrode catheter during routine electrophysiology studies. The data were filtered (1-11 Hz), digitized (250 samples/sec) and temporally compressed to 50 samples/sec. Data compression removed four out of every five samples by only saving the sample with the maximum excursion from the last saved sample. The average squared correlation coefficient (r2) was computed for the NSR and VT episodes using each patient's NSR waveform as a template. In all 23 patients, the r2 values showed large separation between NSR versus VT in both unipolar (0.93 +/- 0.05 vs 0.20 +/- 0.16, P less than 0.005) and bipolar (0.91 +/- 0.07 vs 0.17 +/- 0.11, P less than 0.005) electrode configurations using template lengths of 80% the intrinsic interval (avg +/- SD). Narrow templates (40% intrinsic interval or less) often resulted in multiple r2 peaks during each heart cycle and degraded the r2 separation (n = 10, P less than 0.005). High pass filtering at 3 Hz also degraded the r2 separation (n = 10, P less than 0.05). Standard noncompressed correlations indicated that data compression had negligible effects on the results. Thus, a computationally efficient cross correlation method was found to be a reliable detector of VT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704571 TI - Nonhomogeneous local atrial activity during acute atrial fibrillation: spectral and dynamic analysis. AB - Atrial fibrillation (A Fib) has been categorized into four different types (I-IV) based on the morphology of the epicardial bipolar electrogram. In the present study, we hypothesized that these same types of A Fib also exist at endocardial sites. Simultaneous high, mid, and low right atrial endocardial bipolar electrograms were analyzed during acute A Fib induced by a rapid train of stimuli (20-40 Hz) for 1-3 seconds in anesthetized closed-chest dogs (N = 7, total of 72 episodes). A Fib lasted between 3 seconds and a few minutes (22.3 +/- 22.8 sec). During A Fib, bipolar electrograms (0.5-500 Hz) were both discrete (types I and II) on electrograms recorded at one site and at the same time irregular (type III) on electrograms recorded at another site. The three simultaneously recorded electrograms encompassed all combinations of the four types of A Fib. When A Fib had a discrete electrogram morphology (types I and/or II), the mean rate of the A Fib was 494 +/- 93 beats/min. At a given site, electrogram morphology also changed type over time. Fast Fourier transform (FFT) of the digitized electrograms (8-10 sec, 800 Hz digitization) showed peaks mostly below 15 Hz (range 0-30 Hz), that were either discrete (narrow band) with clear harmonic components, or had continuous (broad band) spectra, that changed in a time and site dependent manner. Phase plane plots (PPP), a plot of voltage versus rate of change of voltage, varied with respect to time and location. However, the morphology of these PPP often inscribed well defined structure suggesting dynamics compatible with deterministic chaos, rather than random dynamics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704572 TI - Temporal electrogram analysis: algorithm development. AB - The automatic discrimination of physiological from pathological tachycardias by rate criteria alone lacks adequate specificity. Tachycardia detection algorithms based upon morphological analysis of the endocardial electrogram have been attributed high specificity although their specificity has not been proven. A previous study had shown temporal electrogram analysis (TEA) to be an algorithm of high sensitivity in the detection of ventricular arrhythmias despite low computational demands. In this study, the specificity and potential for automatic implementation have been assessed. Manual adjustment of thresholds for individual patients gave a maximum potential sensitivity of 97% (26/27 arrhythmias correctly recognized as non-sinus). The use of automatic setting of thresholds reduced sensitivity to 81%. The specificity of the algorithm, as assessed by exercise testing, was only 60%. PMID- 1704573 TI - Atrial antitachycardia pacing in patients with supraventricular tachycardia: clinical experience with the Intertach pacemaker. AB - During a 3-year period, 22 patients with recurrent supraventricular tachycardia have been treated with antitachycardia pacemakers (Intermedics Intertach, 262-12, n = 17, and Intertach II, 262-16, n = 5). Eighty-two percent were female, the mean age was 44 +/- 14 years; 86% had atrioventricular node reentrant tachycardia. Symptoms had occurred over 11.8 +/- 7.1 years, with 3.6 hospital admissions per patient, despite 4.7 +/- 2.1 antiarrhythmic drugs. Following pacemaker implantation, during a follow-up of 14.8 +/- 11.5 months, only two patients have been readmitted to a hospital because of supraventricular tachycardia (mean 0.1 per patient). One patient is taking an antiarrhythmic agent, and four are taking beta adrenergic blocking agents. Thus, 23% are taking cardioactive drugs (it was anticipated that two patients would continue on drugs after pacemaker implantation). There have been no serious complications. Atrial antitachycardia is thus an effective therapy in carefully selected patients with recurrent supraventricular tachycardia, reducing hospital admissions for supraventricular tachycardia and reducing the need for antiarrhythmic drugs. PMID- 1704574 TI - Clinical experience with the Intertach 262-12 pulse generator in patients with recurrent supraventricular and ventricular tachycardia. AB - An antitachycardia pulse generator, the Intermedics Intertach 262-12 was implanted in 16 patients (14 patients with supraventricular tachycardia of various origins and two patients with recurrent ventricular tachycardia), who were not responsive to various antiarrhythmic drug regimens. The follow-up was from 6-49 months (mean 30.9 +/- 13.8). Five patients had a follow-up of over 3 years. The device was used in all patients. One patient with ventricular tachycardia died from a nonarrhythmic cause. Loss of responsiveness to burst pacing was observed in 1/14 patients with supraventricular tachycardia and nontolerance of antitachycardia pacing in one patient. Overall clinical success of pacing was observed in 13/16 patients = 81%. The pacemaker proved to be a versatile system with reliable tachycardia detection and termination functions. PMID- 1704575 TI - Supraventricular tachycardia control with Tachylog II: long-term follow-up. AB - Ten patients aged 16-63 years (mean 36.3) had the Siemens P56T "Tachylog II" pacemaker implanted for treatment of drug refractory supraventricular tachycardia. The pacemaker incorporates a noninvasive electrophysiological study (EPS) facility and a sophisticated Holter function combined with a unique "learning" self-search antitachycardia algorithm. The Holter reveals that new tachycardias arise that are not previously detected at conventional EPS. The number of stimuli in the initiation sequences during noninvasive EPS proved highly variable, however, termination sequences remained constant in the long term. There was variability of timing of stimuli in the long term that was significantly greater for "new" tachycardias than for "original" arrhythmias. Long-term follow-up (at 1 month, 6 months, and 1 year) have shown that 90% of patients have good tachycardia control without the need for drugs. All patients confirm the acceptability of this pacemaker for control of their arrhythmias in the long term. PMID- 1704576 TI - Computerized methods of pace-mapping for topical diagnosis of ventricular arrhythmias. AB - Surgical treatment of ventricular arrhythmias (VAs) is an effective method of solving sudden cardiac death. The important problem of surgical treatment of VAs is the topical diagnosis of the origin of arrhythmia. The precise localization of this area is extremely important for effectiveness of operation. The method of epicardial mapping of the heart during the induced paroxysm of VA is widely used for solving this problem. This problem is difficult when it is impossible to induce tachycardia during the operation. The method of pace-mapping was developed for such cases. This method includes sequential electrical stimulation of ventricles with ECG recording and comparison between intraoperative and preoperative ECG. The goal is the identification of the area of ventricles closed to preoperative ECG with VA. Manual processing of these data is difficult and takes time. The results of such processing have a low precision. That is why we propose using computerized methods. The goal of this investigation is to develop computerized methods of topical ECG diagnosis of VA. We propose a method of computer comparative analysis of ECG morphology for spontaneous and stimulated ECG. The goal is the determination with stimulated ECG the point of ventricles with ECG closest to spontaneous ECG during ventricular tachycardia. Nine patients (15-57 years old) with VA were studied. The main result of this investigation is that computerized pace-mapping method is an effective tool for topical diagnostics of different forms of VAs, which is impossible to induce by electrical programmed stimulation. PMID- 1704577 TI - Differentiation of sinus rhythms from supraventricular tachydysrhythmias by activation sequence and timing. AB - Implantable device detection of tachydysrhythmias remains unreliable and inexact. False responses may occur because of misinterpretation of sinus tachycardia (ST) as a supraventricular tachydysrhythmia (SVTD). Timing of atrioventricular (AV) activation and ventricular dispersion identified and discriminated between ST and SVTDs in 11 dogs. Three bipolar epicardial electrodes recorded left atrial and left and right ventricular depolarizations simultaneously during normal sinus rhythm (NSR) (mean of 5 beats in 11/11 dogs), ST produced by phlebotomy (50 beats in 10 episodes in 6/11) or isoproterenol infusion (105 beats in 21 episodes in 10/11), sinus bradycardia (SB) produced by vagal stimulation (140 beats in 29 episodes in 10/11), and during atrial flutter (AFL) (15 beats in 3 episodes in 3/11) and atrial fibrillation (AF) (152 beats in 31 episodes in 9/11) induced by programmed electrical stimulation. During lidocaine infusion, NSR (55 beats in 11 episodes in 10/11 dogs), SB (84 beats in 17 episodes in 7/11), AFL (10 beats in 2 episodes in 1/11), and AF (103 beats in 21 episodes in 7/11) were recorded. During isoproterenol infusion, SB (45 beats in 9 episodes in 5/11), AFL (15 beats in 3 episodes in 2/11), and AF (64 beats in 13 episodes in 5/11) were recorded in addition to ST. The interval between the left atrial and left ventricular intrinsic deflections (A-V1) and between the left and right ventricular intrinsic deflections (V1-V2) of each beat was measured. The mean value (msec) of A-V1 and V1-V2 in each episode was compared to NSR in the same dogs. A difference of greater than or equal to 16 ms was used for differentiation. In all cases except SB with first-degree AV block, V1-V2 in each episode was insignificant (0-14 msec), categorizing the rhythms as supraventricular. During NSR, ST and SB without AV block, delta A-V1 was small (0-15 msec). In contrast delta A-V1 was greater than or equal to 16 ms in 6/8 episodes of AFL. The remaining two episodes could be differentiated by the greater number of atrial versus ventricular beats. AF could be detected by the variability of A-V1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704578 TI - Surgery of tachyarrhythmia: intracardiac closed heart cryoablation. AB - Closed heart surgery without the use of cardiopulmonary bypass (CPB) is one of the trends of surgical treatment of tachyarrhythmias. Having rich experience of epicardial cryoablation, the authors introduced the original technique of intracardiac cryoablation. They have demonstrated the feasibility of creation of complete AV block in patients with supraventricular tachycardias by AV node-His bundle cryoablation, elimination of AV junctional ("nodal") tachycardia by perinodal cryoablation, cryoablation of septal and paraseptal left posterior AV accessory pathways and ectopic foci in atrial septum, complete or partial cryoisolation or cryofragmentation of the atria in patients with atrial flutter and/or fibrillation, and cryoablation or arrhythmogenic zones in ventricles. Good results (arrhythmia-free patients) were obtained in 82%-100%. Cryoablation on the closed heart without the use of CPB has the following advantages: (a) the possibility of continuous monitoring of cardiac electrical activity during the operation; (b) ablation efficacy control; and (c) diminished trauma and little risk of surgical intervention. PMID- 1704579 TI - Perioperative mapping of parahisian accessory pathways. AB - In 1989, two patients were operated for deep septal "parahisian" pathways in our institution. Three different mapping techniques were used. (1) Epicardial activation mapping with a belt of 21 bipolar electrodes positioned around the heart. This belt was positioned either on the atrial or on the ventricular side of the atrioventricular annulus in order to localize both the atrial and the ventricular insertion of the bypass tract. (2) Right intra-atrial activation mapping on the normothermic beating heart with a bipolar hand-held probe. (3) Right intra-atrial cryomapping at 0 degrees C. The "parahisian" pathways are remote from the epicardium and the pattern of epicardial activation is different from that of the free-wall pathways. Case 1: The electrophysiological study showed a concealed anteroseptal bypass tract. The peroperative atrial epicardial mapping during orthodromic tachycardia (OT) showed simultaneous activation of the posteroseptal area and of the basis of the right appendage. Right intra-atrial mapping during OT showed an anteroseptal "parahisian" pathway. Case 2: The ECG and electrophysiological study showed a right posterior pathway. The first site of epicardial ventricular activation during atrial stimulation was the right posterior area, 30 ms after the onset of the delta wave. The first site of epicardial atrial activation during OT was the posteroseptal area. The right intra-atrial mapping showed a posteroseptal "parahisian" bypass tract. This localization was confirmed with cryomapping. CONCLUSIONS: Some patterns of epicardial mapping may suggest the presence of a deep septal "parahisian" bypass tract: retrograde atrial activation at different sites (mimicking activation among multiple pathways); delay between the delta wave and the first epicardial electrogram. Right intra-atrial activation and cryomapping are useful to confirm the diagnosis. PMID- 1704580 TI - The results of surgery for tachyarrhythmias in children. AB - Tachycardia in children is generally considered harmful and frequently is transformed into so-called arrhythmogenic cardiomyopathy. The goal of this report was the investigation of the result of surgical treatment and how it was dependent on the type of tachycardia, the presence of combined, or concomitant heart pathology. We have operated on 146 patients at an age of 8 months to 16 years (mean 9.6 +/- 2.7 yrs) from 1982 until April 1, 1990. Surface mapping was performed in patients with WPW syndrome. All patients underwent electrophysiological study. The duration of the disease was 8.4 +/- 1.9 years. 89% of patients suffered from syncopal episodes. The heart rate during tachycardia exceeded 200 beats/min in 95% of children. In 98% of patients palpitation lasted more than 3 hours. Seven types of tachycardia were seen in operated children. All patients were divided into three groups depending on the absence or presence of CHD or several types of arrhythmia. Sixty-seven patients (45.8%) with so-called noncomplicated tachycardias (without additional heart disease) were included in group I. Forty-seven patients (32.2%) with tachycardia and CHD were in group II and 52 patients (21.9%) with multiple tachycardias that had life-threatening prognosis were in group III. The total efficacy of surgical treatment in group I was 97%. The worst results were in group II patients. The total positive results in this group was 81%. In group III patients with life threatening arrhythmias, total efficacy was 93.8%. PMID- 1704581 TI - Surgery for atrial tachycardia. AB - Atrial flutter is associated with a macro-reentrant loop including an area of slow conduction cryoablation of which prevents atrial flutter to occur. Three patients underwent such intervention. Atrial fibrillation is associated with multiple reentrant circuits (leading circle of Allessie) that requires a critical surface area to perpetuate. We have designed an operation, the corridor operation, which isolate the sinus node and the AV node within a small segment of atrial tissue, to restore the chronotropic function of the sinus node. Nine patients underwent the corridor operation at our institution. There were eight men and one woman. Five had incessant atrial fibrillation and four paroxysmal. One patient had associated mitral valve stenosis and one cardiomyopathy. There were no perioperative complications. Six patients had normal sinus node function postoperatively including all the four patients with documented normal sinus node function preoperatively. Three patients required implantation of an AAI pacemaker. Two patients had recurrence of atrial fibrillation within the corridor. Our experience suggests that the corridor operation should be restricted to patients with documented good sinus node function and without structural heart disease. Our experience with five patients with paroxysmal sinus node tachycardia has been disappointing. Only one patient had long-term success although better series have been published. PMID- 1704582 TI - Fulguration for AV nodal tachycardia: results in 42 patients with a mean follow up of 23 months. AB - This report describes a catheter ablation technique to treat atrioventricular nodal reentrant tachycardia while preserving anterograde conduction, and its application in 42 patients with drug-refractory repetitive episodes of tachycardia. One of these patients had common and reverse forms of tachycardia. Using atrial activation in the His-bundle lead as a reference, the optimal ablation site was selected by positioning an electrode catheter to obtain a synchronous or earlier atrial activation than the reference during tachycardia. At this site, His-bundle deflection was completely absent, or was present at a low amplitude (less than 0.1 mV). In the majority of patients, these criteria were found in the immediate vicinity of the site of proximal His-bundle recording (adjacent to the reference catheter). Shocks of 160 or 240 joules (J) were delivered at this site (mean +/- SD = 518 +/- 392 J/session) with a resulting preferential abolition of impairment of fast retrograde conduction. Anterograde conduction, though modified, was preserved in all patients, except for four (10%) patients who remained in complete heart block. Thirty patients (70%) remained free of arrhythmia without medication or pacemaker for a mean follow-up period of 23 +/- 13 (2-63) months. Six other patients (15%) were controlled with a previously ineffective medication. PMID- 1704583 TI - Better predictors of successful His-bundle ablation analysis of first shocks. AB - We have reported here that a longer HV interval in association with a larger His amplitude yields a high rate of success when used to position the ablating catheter for His-bundle ablation. Additionally, we have shown that double discharge shocks are more effective than single discharge shocks, and that negative polarity is more effective than positive polarity. The use of bipolar or tripolar, and not quadripolar catheters, was also associated with a higher success rate. In our institution, using a bipolar catheter, we attempt to record an HV interval greater than 55 msec and a His amplitude greater than 0.35 mV. When both of these criteria are fulfilled, we use 3 to 4 joules per kg, and a single discharge shock. When one or the other of these criteria are not fulfilled, we use the double discharge shock method. Using these techniques, we have achieved successful His-bundle ablation with only one shock in all but one of the most recent 21 consecutive patients. PMID- 1704584 TI - Noninvasive cardiac stimulation revisited. PMID- 1704585 TI - Transcutaneous cardiac pacing: evaluation of cardiac activation. AB - The effects of transcutaneous cardiac pacing (TCP) on cardiac activation were evaluated by endocavitary recording (HRA, RVA) in eight patients, in order to test the possibility to obtain a simultaneous atrial and ventricular stimulation. The transcutaneous pacemaker used was the Pace Aid 52 (pacing rate 50-160 ppm, current output 10-150 mA, pulse width 20 sec). The two skin electrodes (surface area 50 cm2) were placed on the chest in anteroposterior position. Ventricular capture was observed in all patients (threshold = 74 +/- 14 mA), simultaneous atrial capture was obtained in only four cases (threshold = 138 +/- 25 mA). In conclusion, our data show that four-chamber simultaneous stimulation by TCP is possible, but only with pacing energies much higher than those usually required to capture the ventricle. The ability of TCP to simultaneously pace the atria and ventricles, though not relevant in the emergency cardiac stimulation for symptomatic severe bradyarrhythmias, could be useful in the treatment of reentrant supraventricular tachycardias. PMID- 1704586 TI - The effect of suppression of the distortion artifact during transcutaneous pacing on the shape of the QRS complex. AB - The quality of the ECG recording during transcutaneous pacing was evaluated in six healthy volunteers. The transcutaneous pacing stimulator was an NP4D special unit to which was attached an electronic suppressor of artifact generated by the transcutaneous stimulating impulse. The relationship between this suppression of artifact distortion and the resulting QRS complex was analyzed. The results revealed that the suppression time (described as a 2-mm oscillation from 100-Hz frequency) was required for ECG distortion elimination and that this is dependent on the threshold of ventricular pacing. The width of the resulting QRS complex diminishes as the suppression is extended over 110 ms. These results suggest the necessity of individual adjustment of the suppression time so that the efficacy of transcutaneous pacing is adequately assessed. PMID- 1704587 TI - Transcutaneous cardiac pacing for termination of tachyarrhythmias. AB - Transcutaneous cardiac pacing (TCP) was used for interruption of tachyarrhythmias in 31 patients: 20 with ventricular tachycardia (VT); eight with atrioventricular reentrant tachycardia (AVRT) and three had atrioventricular nodal tachycardia (AVNT). The stimulators used (Pace Aid 50/52) allow pacing at programmable rates (50-160 ppm) and output (10-200 mA at 20-msec pulse duration), when possible overdrive pacing was used. Short bursts of stimuli were delivered with increasing current intensity until interruption of the arrhythmia or to the maximum energy tolerated by the patient. VTs were interrupted in eight of the 20 patients: four of the six (67%) treated by overdrive pacing and four of the 14 (29%) were treated by underdrive pacing. Supraventricular tachycardias (SVT) were terminated in eight of the 11 patients: seven out of eight (88%) AVT, and one out of three AVNT (33%). We observed two cases of arrhythmia worsening: a VT acceleration and induction of ventricular fibrillation in a patient with AVNT. TCP was well tolerated by the majority of the patients. We conclude that TCP is an effective method for interruption of ventricular and supraventricular reentrant tachycardias, but the risk of arrhythmia worsening must be considered. PMID- 1704588 TI - Description of the strength-interval relation with external noninvasive pacing. AB - The strength-interval relationship obtained by external pacing (EXP) was compared to curves obtained by endocardial pacing from a right ventricular site. There were 17 patients, age 45 +/- 17 years. Effective and functional ventricular refractory periods (ERP, FRP) were determined during a ventricular drive train of 8 stimuli at a cycle length 500 msec. Endocardial pacing threshold current averaged 0.5 +/- 0.3 mA (range 0.2-1.0 mA) and EXP threshold current averaged 64 +/- 14 mA (range 40-80 mA). At twice threshold, endocardial ventricular ERP was 235 +/- 32 msec and FRP was 262 +/- 29 msec. At 10 mA above threshold, EXP ventricular ERP was 276 +/- 29 msec and was significantly longer than endocardial ERP at twice threshold (P less than 0.001). EXP ventricular FRP shortened to 237 +/- 39 msec at twice threshold and was similar to endocardial ERP (P = 0.55). EXP ventricular FRP was significantly longer than endocardial ERP (280 +/- 29 vs 262 +/- 29 msec, P less than 0.001) at twice threshold. EXP strength-interval curves were similar to endocardial curves in 14 of 17 (82%) patients. We conclude, that at twice threshold, similar coupling intervals can be obtained with both endocardial and EXP. Therefore, EXP can provide critically coupled extrastimuli for programmed ventricular stimulation. PMID- 1704589 TI - Emergency cardiac pacing for severe bradycardia. AB - Our study included the treatment of transcutaneous cardiac pacing (TCP) in 32 patients: (A) 19 patients were treated in the emergency area for complete symptomatic AV block before endocavitary pacing; (B) five patients were in asystole following DC shock or out-of-hospital cardiac arrest; and (C) eight patients were affected by bifascicular block undergoing emergency surgery and were treated in order to prevent complete AV block. Two transcutaneous stimulators were used. PaceAid-CRC model 50/52 with 20-msec pulse width; the electrodes were positioned on the V3 ECG position and on the back. RESULTS: in all but two patients, it was possible to obtain stable cardiac capture; in one patient arrived in hospital in asystole after prolonged cardiac arrest and in the other one was affected by complete AV block, TCP was ineffective. In groups A and B, TCP was maintained for a mean time of 15 minutes; in group C, TCP was tested in all patients, but performed in only one patient during surgery. Mean threshold was 81 mA. Stimulation was well tolerated in all but five patients. TCP is a reliable, noninvasive method that offers the possibility to initiate pacing within seconds and can be used by medical staff. In our opinion, it should be considered as the first choice emergency treatment of severe symptomatic bradycardia. In asystole, beneficial effects can be obtained only if TCP is performed early enough after the onset of arrhythmia. PMID- 1704590 TI - Electrophysiological evaluation of tachycardias using transesophageal pacing and recording. AB - Programmed electrical stimulation of the heart to initiate and terminate tachycardia has been useful in the evaluation of supraventricular and ventricular tachyarrhythmias. A wide use of these procedures, however, failed because of the expense of the invasive approach as well as the lack of physician experience in smaller hospitals. These disadvantages of the invasive proceeding can be abolished by transesophageal pacing. In our study, supraventricular tachycardias were initiated by programmed transesophageal atrial stimulation in 251 patients (AV node reentry in 75 patients, orthodromic AV reciprocating tachycardia using accessory pathway in 97 patients, antidromic AV reciprocating tachycardia in 11 patients, and atrial reentry in 39 patients). The stimulation protocol included one and two extrastimuli during sinus rhythm and after a pacing drive at different cycle lengths. The electrophysiological mechanism of tachycardias was determined by surface ECG, VA interval (esophageal lead), initiation mode at programmed transesophageal stimulation and by behavior of AV conduction and refractoriness. In 29 patients the mechanism of tachycardia was not clear. Invasive electrophysiological study was done in 219 of these 251 patients. In only nine patients, the supposed mechanism of tachycardia was not confirmed by invasive investigation. In 11 patients, the electrophysiological mechanism remained uncertain. In conclusion, the noninvasive transesophageal pacing is an appropriate method for evaluation of supraventricular tachycardia. It allows serial drug testing in a simple manner for finding an effective antiarrhythmic treatment. PMID- 1704591 TI - Transesophageal atrial pacing complications in patients suspected of tachy-brady syndrome. AB - The clinical effects of transesophageal atrial pacing (TAP) were assessed in 308 patients. Indications for TAP included evaluation for pacemaker implantation in patients suspected of sinus node dysfunction and determination of the suitable type of pacemaker. Most patients underwent program stimulation including rapid as well as burst stimulation. In one patient, following the study, cerebral arterial embolism occurred, most likely secondary to an induced arrhythmia. That was the only single case of permanent consequences following TAP. Additionally, one patient was accidentally stimulated in the ventricle using low voltage electric current that induced ventricular fibrillation. This was promptly reversed with defibrillation. Twenty-six patients in whom an arrhythmia was previously induced, required medical therapy, two of whom required cardioversion, and 24 required drug therapy, subsequent to clinical intolerance of the arrhythmia. No lethal complications occurred. PMID- 1704592 TI - Atrioventricular Wenckebach point and progression to atrioventricular block in sinoatrial disease. AB - The value of measurement of the atrioventricular (AV) Wenckebach point at rest as a predictor of progression to AV block was investigated prospectively. Twenty four patients with sinoatrial disease without evidence of conduction disturbance on 12-lead ECG or 24-hour ambulatory monitoring were paced with Medtronic Activitrax II, Medtronic Legend, or Telectronics Meta MV systems in AAI or AAIR modes. Patients were monitored for symptoms and evidence of AV block on 24-hour tapes. The mean age of the patients was 67 years (range: 42-88). There were 11 males and 13 females. The mean follow-up time was 10.7 +/- 5 months. Four patients required revision of pacing system as a result of development of AV block during follow-up. One other patient manifested intermittent second-degree AV block and remains in AAI. The AV Wenckebach points measured at 1 month postimplantation in the four patients who developed AV block requiring revision of system were 140, 125, 165, and 60 (mean 123 +/- 4). The mean AV Wenckebach point at first assessment in the remaining 20 patients was 153 +/- 24. The mean age of those requiring revision of system was 71 +/- 7 compared with 67 +/- 14 in those who did not. In this small series the frequency of development of significant AV block was 17%. This is markedly higher than in other recently reported series. The study demonstrates that an AV Wenckebach point above 120/min does not confer immunity from progression to AV block. PMID- 1704593 TI - Improved dual chamber pacing mode in paroxysmal atrioventricular conduction disorders. AB - Dual chamber pacing may sometimes be directly indicated for carotid sinus hypersensitivity, vasovagal syndrome, and certain cases of sinoatrial block and intermittent atrioventricular (AV) block, although AV conduction is dominantly normal. At times of normal AV conduction, competition between ventricular pacing and spontaneous ventricular depolarization may occur, with its adverse hemodynamic effects on ventricular function and unnecessary drainage of pacemaker battery energy. A new mode of stimulation is described, called automatic DDD mode, which functions in 'pseudo-AAI' mode during normal AV conduction and reverts to classical DDD function during episodes of AV blocks. Furthermore, during pseudo-AAI function, the pacemaker measures certain physiological parameters that serve to automatically program certain parameters used in DDD mode. Preliminary clinical evaluation has shown that this new mode functions satisfactorily. PMID- 1704594 TI - Diagnostic value of carotid sinus hypersensitivity. AB - In order to evaluate the diagnostic value of carotid sinus hypersensitivity (CSH) we have investigated 163 asymptomatic patients (88 male, 75 female, mean age 57.9 +/- 22.7 years) and 210 symptomatic patients (108 males, 102 females, mean age 61.1 +/- 28.1 years) with syncopes or dizziness. Thirty two of the 163 asymptomatic patients (20%) and 87 of the 210 symptomatic patients (41%) showed CSH (asystole greater than or equal to 3 sec during carotid sinus pressure). Male patients had a higher number of CSH than female (28% vs 10% in the asymptomatic group, 48% vs 34% in the symptomatic group). Electrophysiological investigations were performed in all 210 symptomatic patients. Normal electrophysiological results had 94 of the 210 patients. Thirty seven of these 94 patients showed CSH (39%). Prolonged sinus node recovery time (SNRT) and/or prolonged sinoatrial conduction time (SACT) were evaluated in 38 patients. Seventeen of the 38 patients had CSH (45%). Disorders of atrioventricular (AV) conduction were evaluated in 43 patients. Seventeen of the 43 patients showed CSH (40%). Thirty five patients had both AV conduction disorders and prolonged SNRT or SACT. Sixteen of these 35 patients showed CSH (46%). In conclusion, no significant difference was found between patients with and without pathological electrophysiological results. The CSH is without value for predicting sinus node dysfunction and AV conduction disorder. PMID- 1704595 TI - Pacing for carotid sinus syndrome and sick sinus syndrome. AB - The real incidence of pacemaker implants for carotid sinus syndrome (CSS) and the relation between CSS and sick sinus syndrome (SSS) is not precisely known. Patients who needed pacing therapy because of atrial bradyarrhythmias were investigated by means of carotid sinus massage, dynamic ECG, and invasive electrophysiological sinus node evaluation. Of 298 consecutive patients receiving a pacemaker implant, 36 (12%) had a severe cardioinhibitory carotid sinus reflex with reproducible spontaneous symptoms (CSS), 33 (11%) had sinus bradycardia less than 50 beats/min or an abnormal electrophysiological evaluation (SSS) and 24 (8%) had both (CSS + SSS). The annual incidence was 40, 37, and 26, respectively, implants per year/million of inhabitants (total incidence 325). Patients affected by CSS, if compared with those affected by SSS, showed: a higher prevalence of syncope (97% vs 42%); more syncopal episodes per patient (2.9 +/- 2 vs 1.8 +/- 0.9); a lower prevalence of associated cardiac diseases (53% vs 100%); cardiac enlargement (36% vs 88%); heart failure (6% vs 36%) and paroxysmal atrial fibrillation (0% vs 42%); and a more frequent indication for VVI pacing (75% vs 3%). In patients with CSS + SSS, intermediate characteristics were present. In conclusion, CSS is as frequent an indication to cardiac pacing as SSS; clinical differences justify a distinction between them, even if they are associated in 26% of cases. PMID- 1704596 TI - Morbidity and mortality of patients with sinus node disease: comparative effects of atrial and ventricular pacing. AB - In patients with sinus node disease (SND), VVI pacing seems an inappropriate method of cardiac stimulation because of its potential adverse hemodynamic and arrhythmic effects. AAI-DDD pacing, preferred because of lower morbidity, may also determine a higher survival rate. We examined retrospectively two groups of patients with SND. Stimulated respectively with VVI pacing (group 1 = 57 patients) and AAI pacing (group 2 = 53 patients). The mean duration of the follow up interval was 40.1 months for group 1 and 45 months for group 2. Ten patients (17.5%) in the VVI group and five (9.4%) in the AAI died. During the follow-up, in the VVI group three patients developed congestive heart failure and ten developed chronic atrial fibrillation, whereas only one case of heart failure and two with atrial fibrillation have been recorded in the AAI group. Moreover, four patients had embolic complications in group 1. Five patients (9.4%) with AAI pacing were converted to sequential pacing due to the occurrence of second-degree heart block. The statistical analysis was developed by the X2 test for the comparison of the proportion of the events (atrial fibrillation, congestive heart failure, embolic accidents) in the two groups: a significantly higher morbidity (P less than 0.01) was recorded in the AAI group. Survival is also higher in AAI patients, but the survival rate difference, calculated using the Mantel-Cox method, is not statistically significant. The findings of our study show that in SND the superiority of AAI pacing over VVI is statistically significant as far as morbidity is concerned, and we have also noticed an evident but not statistically significant superiority regarding mortality. PMID- 1704597 TI - Differences between atrial single chamber pacing (AAI) and ventricular single chamber pacing (VVI) with respect to prognosis and antiarrhythmic effect in patients with sick sinus syndrome. AB - Several studies suggest different effects of atrial (AAI) and ventricular single chamber pacing (VVI) for sick sinus syndrome with respect to the suppression of atrial tachycardias and to the prognosis. With this aspect in mind, we studied 222 patients with sick sinus syndrome, 110 of whom had been supplied with AAI systems, and 112 with VVI systems, in the period from January 1978 to December 1986. The mean observation period was 53 +/- 28 months. The cumulative 5-year survival rate was not significantly different in the two groups. After subgroups with comparable underlying diseases had been differentiated, patients with coronary heart disease showed a significantly higher survival rate (P less than 0.05) under AAI pacing, and the same was shown for patients with no underlying heart disease (P less than 0.02). The incidence of chronic atrial fibrillation was 6% in the AAI group and 19% in the VVI group. Patients with preexisting atrial tachyarrhythmias showed the lowest incidence of chronic atrial fibrillation under AAI pacing. Under VVI pacing this incidence was a function of the basic rate of the pacemaker systems. In conclusion, the pacing mode seems to have a prognostic importance in spite of all methodological difficulties. A suppressive effect of AAI pacing on atrial dysrhythmias can also be assumed. PMID- 1704598 TI - Symptoms, cardiovascular risk profile and spontaneous ECG in paced patients: a five-year follow-up study. AB - Only few data are available about the course of symptoms, cardiac diseases, and spontaneous rhythm in pacemaker patients. Therefore, we followed the course of 308 paced patients (age 72 +/- 11 years) with a mean implantation time of 63 +/- 45 months. RESULTS: The symptom triad of syncope, dizziness, and dyspnea improved remarkably in 93% of patients. Thirty-nine percent suffered from coronary heart disease. The risk factors of hypertension (47%), nicotine (37%), and diabetes mellitus (25%) were found significantly more often than in a normal population with the same age and sex profile. In VVI paced patients with sick sinus syndrome (SSS, n = 67) atrial fibrillation (AF) occurred significantly more often (42%) than in patients with AV block (n = 80, 23%, P less than 0.05). Only one out of 41 DDD paced patients showed AF at follow-up. VVI stimulation seems to favor AF due to retrograde conduction in SSS. Only 3% of patients with SSS developed second- or third-degree AV block. Therefore, atrial pacing is preferable in most patients with SSS. PMID- 1704599 TI - Evolution of P wave characteristics after pacemaker implantation. AB - This study is an investigation of the long-term effects of VVI pacing on the atrium as derived from the evolution of P wave characteristics of 285 patients. The occurrence of left and right atrial disease is demonstrated as well as the evolution of left atrial hypertrophy in some cases. A comparison is made with DDD pacing and special attention is given to the progression to atrial fibrillation. PMID- 1704600 TI - Heart rate variability and sudden infant death syndrome. AB - The sudden infant death syndrome (SIDS) is the most common cause of death in infancy. The pathophysiological mechanism leading to SIDS is still obscure. In the QT hypothesis, the mechanism must be an arrhythmogenic sympathetic imbalance: the infants die suddenly of cardiac arrhythmia. Recently, it has been suggested that analysis of heart rate variability (HRV), expressed as standard deviation or variance analysis, can provide adequate information on sympathovagal interaction. We studied 150 newborns enrolled in a previous prospective electrocardiographic study to evaluate the predictive value of QT interval for SIDS. We analyzed the ECGs recorded with infants alert on the fourth day of life and after 2 months. For each ECG, the HRV was calculated using the first standard deviation of of RR intervals (ms) measured for 1 minute. The average RR interval was 441 +/- 71 ms at the fourth day and 410 +/- 39 ms at the second month. The QTc and HRV mean values were 396 +/- 23 and 23 +/- 12 ms at the fourth day, 412 +/- 19 and 15 +/- 7 msec at the second month. Therefore, the SD values of heart rate were correlated with QTc in order to assess a possible relationship between the two variables. The correlation coefficient and regression equation were: -0.639 and y = 423.67 - 1.18*X (P less than 0.001) at the fourth day, -0.146 and y = 418.09 - 0.37*X (NS) at the second month. In conclusion, our data seems to confirm a delayed maturation or impaired functioning of the autonomic nervous system in the first weeks of life, reflecting a direct correlation with QT prolongation. PMID- 1704601 TI - Permanent transvenous pacing after a Mustard procedure. AB - We report the case of a 20-year-old man born with transposition of the great arteries who underwent emergency balloon septostomy and subsequently a Mustard procedure. When aged 20 years, he had several syncopal attacks due to sinoatrial disease for which he was simply and successfully paced transvenously in VVI mode. PMID- 1704602 TI - Is activity sensored pacing in children and young adults a feasible option? AB - Activity sensing pacemakers are being utilized with increasing frequency in adults, but less frequently in the pediatric and young adult age group. We evaluated 12 young patients with activity sensing devices. Seven of the implanted devices were VVIRO, four were DDDRO and one was AAIRO. Patients ranged in age from 1-21 years, mean 9.3 years, median 8 years. Weights ranged from 8.25 to 80 kgs, mean 35 kgs, median 27.6 kgs. Anatomical diagnoses revealed six normal hearts, two P/O tetralogy, one P/O transposition, two P/O ablations, and one P/O Fontan. Rhythm diagnoses included congenital complete atrioventricular block in five, acquired complete atrioventricular block in three, sinus bradycardia in two, and His-bundle catheter ablation in one patient. Rate responsive settings of medium and 7 were used in 10 out of 12 patients. Treadmill exercise testing showed that 5 out of 7 patients' rates were controlled by the activity sensor at peak exercise while two were tracking sinus rhythm (average rate increase was 76.8 ppm in the five patients). Ambulatory monitoring revealed that 10 out of 12 patients used their activity sensor to control their heart rate (average rate increase was 54 ppm). Follow-up has ranged from 1-26 months, mean 6.8 months, median 5.5 months. There was one death. Effective rate modulated, activity sensing pacing was achieved in 7 out of 9 young patients. It should be considered when the atrium itself is not a feasible sensor. PMID- 1704603 TI - Rate-adaptive cardiac pacing in children using a minute ventilation biosensor. AB - Chronotropic integrity is required for a normal cardiac output response to exercise. We evaluated a rate-adaptive ventricular demand pacemaker (Telectronics, META-MV) which uses minute ventilation as the sensed physiological variable for adjusting pacing rate, in seven young patients with a mean age of 11.4 years. All patients had clinically significant bradycardia related to complete heart block (n = 4) or sinus node dysfunction (n = 3). For the entire group, paced heart rates increased from 70 +/- 10 beats/min to 151 +/- 19 beats/min with exercise testing. The onset of rate adaptation took less than 30 seconds. Changes in paced rate were linearly related to workload, VO2 (5.9 to 20.7 mL/min/kg) and minute ventilation (8-65 L/min). The decline in pacing rate after exercise was related directly to the gradual decrease in minute ventilation and VO2. Our data show that minute ventilation closely and accurately reflects the metabolic demands of varying workloads in children and can be used to achieve physiological, rate-adaptive pacing. PMID- 1704604 TI - Nonlinear dynamic analysis of temporally heterogeneous action potential characteristics. AB - Rate-dependent bifurcations and aperiodic changes in action potential duration and amplitude were observed in periodically stimulated cardiac Purkinje and ventricular muscle cells isolated from dogs with quinidine-induced ventricular fibrillation and ventricular tachycardia. The slope of the action potential duration restitution curve was higher in the quinidine intoxicated fibers than in normal untreated fibers. Aperiodicity and bifurcations in action potential duration (APD) could not be observed in normal untreated cardiac fibers. The data suggest that induction of reentrant ventricular fibrillation and ventricular tachycardia could be brought about not by fixed but rather by changing alterations in cellular electrical activity. Theory based on nonlinear dynamics seems to provide a quantitative basis for such an analysis. This could have important implications in the issue of sudden cardiac death, a major problem in cardiology. PMID- 1704605 TI - Distinctive response of arrhythmogenic right ventricular disease to high dose isoproterenol. AB - Arrhythmogenic right ventricular disease is a potential cause of ventricular arrhythmias. Diagnosis is important due to the risk of sudden death, particularly as first symptom. Diagnosis is based on the angiographic demonstration of abnormal right ventricular morphology and function, while the sensitivity of noninvasive tests is relatively low. Following a particular observation studied in 1984, we prospectively assessed the diagnostic value of high dose isoproterenol infusion in 44 patients with an angiographically determined arrhythmogenic right ventricle. A continuous infusion of isoproterenol (8-30 micrograms/min) was administered during a 3-minute period, regardless of the obtained heart rate. In a control group of 50 patients without structural heart disease, isoproterenol induced a monomorphic ventricular tachycardia salvo in only one patient (2%). In patients with an arrhythmogenic right ventricle, isoproterenol induced one or more ventricular tachycardia runs in 39/44 cases (88%): one triplet in three patients, several runs in 23 patients and a sustained ventricular tachycardia in 13 patients. Arrhythmia was polymorphous in 85% of cases, but left bundle branch block morphology was the predominant pattern. In conclusion, high dose isoproterenol is a highly sensitive test for the diagnosis of arrhythmogenic right ventricular disease. PMID- 1704606 TI - Predictors of ventricular tachycardia inducibility in programmed electrical stimulation and the effectiveness of serial drug testing: Polish multicenter study. AB - In 100 patients with IHD and complex ventricular arrhythmias, programmed electrical stimulation was performed using up to three extrastimuli at sinus rhythm, and paced 100, 120 and 140 beats/min delivered from the RV apex, outflow tract or the LV with ventricular mapping to evaluate late potentials (LP) in 41 patients. Sustained monomorphic VT (SMVT) was provoked in 91% of 42 patients with a history of VT/VF, P less than 0.001, all five patients had SMVT in 24-hour ECG, P less than 0.005, and 91% of 21 patients with LV dyskinesis, P less than 0.01. After depolarizations were found in 62% of 21 patients with a history of VT, in 58% of 31 patients with inducible VT, P less than 0.01 and in five of six patients with LV dyskinesis. In patients with inducible VT, LP had a higher amplitude (105 +/- 35 vs 60 +/- 47 microV) and were more delayed (202 +/- 96 vs 133 +/- 75 msec) than in noninducible patients. In 17 patients, serial drug testing was performed after oral administration using mexilitene, disopyramide, chinidine, propafenone, sotalol, and amiodarone. If one drug was tested, the therapy efficacy was 25%, if two drugs-60%, and if three drugs-75%. In eight patients, VT was inducible in all tests, but in only one of these patients chronic antiarrhythmic therapy was not effective. We conclude that the most important predictors of VT inducibility are a history of VT or 24-hour ECG, and LV dyskinesis. Serial drug testing is efficient only when many drugs are tested, but even if VT is inducible, it does not exclude the possibility of a good clinical outcome in chronic therapy. PMID- 1704607 TI - Reentrant junctional tachycardias. AB - Most of the tachycardia arising in the atrioventricular (AV) junction are reentrant in nature. The two most common variety are AV nodal reentry and AV reentry utilizing an accessory pathway of the Kent bundle type. Typically these tachycardias have narrow QRS complex and are regular but an associated right or left bundle branch block could result in a wide QRS complex. Other mechanisms for wide QRS in AV junctional tachycardia include: (a) antidromic reentry; (b) preexcited tachycardia using two accessory pathways; (c) AV nodal reentry with incidental accessory pathway participation; and (d) atriofascicular (nodoventricular)e Mahaim participation. A variety of surface ECG and intracardiac electrophysiological methods are used to delineate the precise mechanisms which is essential for successful nonpharmacological therapy in these patients. PMID- 1704608 TI - Electrophysiological basis of ventricular late potentials. AB - The presence of late potentials on the body surface recording was correlated with ventricular activation maps of reentrant circuits in the postinfarction canine model of reentrant excitation. Late potentials were found to correlate with delayed myocardial activation. However, during a reentrant rhythm complete diastolic activity on the body surface could not be detected if the mass of electrically active cells was too small and/or if very slow conduction in part of the reentrant circuit generated low amplitude extracellular potentials. Myocardial zones responsible for late potentials during a basic rhythm (e.g., sinus rhythm) may not necessarily be part of the critical zone of slow conduction during reentrant activation. Dynamic changes in late potentials are not amenable to temporal signal averaging techniques but could be detected by a high resolution beat-to-beat recording. A thorough understanding of the electrophysiological limitations of late potentials in the signal-averaged ECG could result in better utilization of the technique in clinical practice as well as in the development of new approaches for the detection of the arrhythmogenic substrate. PMID- 1704609 TI - Application of beat-to-beat techniques. AB - The focus of this report is to describe a system for recording surface His Purkinje and ventricular late potentials on a beat-by-beat basis outside of a shielded environment. An AC magnetic field monitoring device was designed for improved site selection, orientation, and quality control of the acquisition. His Purkinje signals are detected utilizing spatial averaging and specific channel selection algorithms applied to discriminate random noise from signal. Beat-by beat vectormagnitude complexes were generated from pairs of X, Y, and Z leads. Both infinite impulse response (IIR) filters, modified for beat-by-beat approaches, and finite impulse response (FIR) filters were utilized. Using the IIR filter, beat-by-beat recordings from test subjects were compared to the signal averaged electrocardiogram (SAECG). Measurement parameters from normal test subjects fell within the previously specified normal range for the SAECG. The IIR filter applied to beat-by-beat recordings exhibited sharp frequency response and a precisely defined cutoff frequency allowing maximal attenuation of the low frequency components in the ST segment. While filter ringing was eliminated, discontinuity and distortion of the filtered waveform resulted. The FIR filter with linear phase response retained the integrity and morphology of the complex but because of its flat frequency response, the ST segment was not as well attenuated and it was more difficult to isolate late potentials. A high order FIR filter should be used if the desire is to match the frequency response of the four-pole IIR filter, since the frequency response of the FIR filter is primarily determined by the order of the filter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704610 TI - The signal averaged electrocardiogram and programmed stimulation in patients with complex ventricular arrhythmias. AB - The signal averaged electrocardiogram (SA-ECG), programmed electrical stimulation (PES), and left ventricular ejection fraction (EF) studies were utilized for risk stratification and management of patients with complex ventricular arrhythmias and nonsustained ventricular tachycardia (VT). The study population included 90 patients (63 with coronary artery disease and 27 with dilated cardiomyopathy). Sustained monomorphic VT was induced in 22 cases (24%), ventricular fibrillation (VF) in 10 (11%), and no sustained VT/VF in 58 (64%). An abnormal SA-ECG was recorded in 23 patients (26%) and was more common in patients with than in those without induced sustained VT (68% vs 12%, P less than 0.0001). None of 33 patients with normal SA-ECG and EF greater than or equal to 40% had induced VT. Patients were followed-up for 2.5 +/- 0.8 years off antiarrhythmic therapy, unless they had induced sustained VT. The 3-year sudden death rate was 19% in the group with induced sustained VT, 0 in that with induced VF, and 9% in that without induced VT/VF (P = NS). The 3-year total cardiac mortality was higher in patients with than in those without EF less than 40% (27% vs 7%, P less than 0.05). It is concluded that patients with organic heart disease and spontaneous nonsustained VT may not need PES or antiarrhythmic therapy if SA-ECG is normal and EF is greater than or equal to 40%, since their risk of induced VT and sudden death is low. On the other hand, patients with abnormal SA-ECG and/or EF less than 40% may require PES, since their risk for induced VT is high. Antiarrhythmic therapy may also be considered in these patients. PMID- 1704611 TI - Atrial peptides induce mast cell histamine release. AB - Human atrial natriuretic peptide [ANF(1-28)] contains five arginine residues and carries an overall positive change of four. It was hypothesized that atrial peptides may induce mast cell histamine release. In vitro, three atrial peptides [ANF(1-28), (3-28) and (5-28)] were demonstrated to induce dose-dependent histamine release from isolated rat peritoneal mast cells. In vivo, ANF(3-28) produced a dose-dependent increase in rat skin permeability which was blocked by antagonists of histamine and serotonin. The results indicate atrial peptides are capable of inducing mast cell degranulation in a manner similar to that described for other positively charged peptides. PMID- 1704612 TI - Gastrointestinal neuropeptides suppress human colonic lamina propria lymphocyte DNA synthesis. AB - Gastrointestinal neuropeptides have been shown to modulate the circulatory immune system, but their effect on the mucosal immune system is not well defined. We studied the effect of VIP, SOM, S-P and Bomb on thymidine incorporation into human colonic lamina propria lymphocyte (LPL) DNA. Physiologic concentrations of VIP, SOM, S-P and Bomb significantly suppressed thymidine incorporation into Con A-stimulated human LPL. These neuropeptides did not affect DNA synthesis when LPL were induced with phorbol ester (PDB) and calcium ionophore (ionomycin). Our data suggest that a) VIP, SOM, S-P, and Bomb may have a regulatory role in the human mucosal immune system, and b) Bomb should be added to the list of neuropeptides which affect the gut immune system. PMID- 1704613 TI - Pharmacological characterization of CGRP1 receptor subtype in the vascular system of the rat: studies with hCGRP fragments and analogs. AB - In order to examine whether the truncated fragments of hCGRP, hCGRP(8-37) or hCGRP(12-37), behave as competitive CGRP receptor antagonists in the vascular system of the rat, systemic blood pressure was continually monitored in pentobarbital-anesthetized Sprague-Dawley rats. The IV administration of 7.9-527 pmol hCGRP/rat caused dose-related reductions in mean arterial blood pressure that lasted, depending on the dose, about 3-10 min. In contrast, hCGRP fragments 8-37 or 12-37 proved inactive up to 60,000 pmol/rat. Pretreatment with either 10 or 30 nmol hCGRP(8-37) or 20 or 90 nmol hCGRP(12-37)/rat reduced the magnitude of the CGRP-induced hypotensive responses caused by 79 pmol hCGRP/rat; pretreatment with 10 nmol of the hCGRP fragments displaced about 3-fold the hCGRP as well as the [Cys(ACM)2.7]hCGRP dose-response curve to the right in a parallel fashion. The specificity of hCGRP(8-37) as a CGRP receptor antagonist was documented by the finding that it did not antagonize the hypotensive responses induced with bradykinin, histamine or substance P. PMID- 1704614 TI - Influence of substance P on carotid sinus nerve baro- and chemoreceptor activity in rabbits. AB - Substance P (SP) is abundant in the carotid sinus nerve (CSN) and has been implicated in baro- and chemoreceptor reflexes. We examined the effect of SP on blood pressure, heart rate, phrenic nerve activity, hindlimb perfusion pressure, and cardiac contractile strength in urethane-anesthetized rabbits with bilaterally cut cervical sympathetic, vagus, and aortic depressor nerves. Retrograde simultaneous injection of SP (0.5-2.7 micrograms/kg in 0.2-0.3 ml saline) into both carotid sinus areas via the internal carotid arteries decreased blood pressure (by 56%), heart rate (by 13%), cardiac contractility (by 25%) and phrenic nerve activity (by 77%). The effect on hindlimb perfusion pressure was variable. There was both a reflex effect and direct hindlimb vasodilation. In another group of rabbits, the carotid sinus areas were vascularly isolated and perfused with SP (0.19 micrograms/min dissolved in Locke's solution) or Locke's solution alone for 5 min. While carotid sinus perfusion pressure was maintained in the range of 80-120 mmHg, mean arterial blood pressure, heart rate, and unit activity from the CSN were recorded. SP increased the activity of 11 of 18 baroreceptor fibers and inhibited all of 20 chemoreceptor fibers. SP decreased mean arterial blood pressure and heart rate, but the changes were less than those obtained with injection of SP into nonisolated carotid sinus arteries because systemic effects of SP, which in some cases counteracted the reflex effects, were eliminated. PMID- 1704615 TI - Galanin in porcine vagal sensory nerves: immunohistochemical and immunochemical study. AB - In this work, the presence of galanin was examined by immunohistochemistry, radioimmunoassay and high performance liquid chromatography (HPLC) in porcine nodose ganglia, mainly constituted of cell bodies from the vagal sensory neurons. Galanin-like immunoreactivity (Gal-LI) was revealed in 10 to 15% of the total cell bodies by the indirect immunofluorescent technique of Coons. For comparison, a positive staining was revealed in a few cell bodies of the submucous plexus and in fibers located in the different layers of the ileum. The extractable Gal-LI content in nodose ganglia was 7.2 +/- 0.8 pmol/g wet tissue, which represents a concentration about nine times lower than that found in the ileum. HPLC of extractable material revealed a predominant peak which coeluted with the synthetic peptide. We propose that, in pigs, galanin may play a role in the transmission of visceral information through the vagal afferences. PMID- 1704616 TI - Stimulation of feeding by galanin: anatomical localization and behavioral specificity of this peptide's effects in the brain. AB - The neuropeptide galanin (GAL) has been found to elicit eating after injection into the hypothalamic paraventricular nucleus (PVN). To determine whether GAL's effect in the brain is anatomically specific, this peptide (0.1 or 0.3 nmol) was microinjected into one of 14 different brain areas of rats, and its impact on subsequent food intake was measured. Among the hypothalamic sites tested, only the PVN and the adjacent periventricular region yielded a significant eating response to GAL. With injection into the PVN, a feeding response was observed without apparent changes in other food-associated behaviors, e.g., drinking, grooming, resting and sleeping, or low and high levels of activity. All other hypothalamic and extrahypothalamic sites tested were unresponsive to GAL, with the exception of the amygdala where a significant eating response was observed. These findings suggest that central GAL elicits feeding by acting in an anatomically localized and behaviorally specific manner. In light of other pharmacological and anatomical evidence, it is suggested the PVN GAL, in modulating feeding behavior, may work in association with the catecholamine norepinephrine (NE) which is known to coexist with GAL in PVN neurons. PMID- 1704617 TI - Influence of monensin on adriamycin transport and histamine release in rat peritoneal mast cells. PMID- 1704618 TI - Dose-dependent miosis by substance P eyedrops in humans. PMID- 1704619 TI - GABA and neuropeptides affect anaphylaxis in guinea-pig airways. PMID- 1704620 TI - Platelet aggregation and histamine release. PMID- 1704621 TI - Effects of risperidone on motor and sexual activity in the rat. PMID- 1704622 TI - Influence of DHP ligands on PCP-induced effects in rats. PMID- 1704623 TI - Enhancement of GABA-dependent chloride channel by "in vivo" administration of ethanol. PMID- 1704624 TI - Effect of human granulocyte colony stimulating factor (G-CSF) and reduced glutathione (GSH) on drug-induced leukopenia in golden Syrian hamsters. PMID- 1704625 TI - Pain control. PMID- 1704626 TI - Chorioangioma: a new inclusion in the prospective and retrospective evaluation of elevated maternal serum alpha-fetoprotein. AB - Two cases are presented which illustrate the association of elevated maternal serum alpha fetoprotein (MSAFP) levels and chorioangiomas. These cases emphasize the importance of ultrasound study of the placenta in MSAFP elevation evaluation. In addition, placentas from pregnancies with otherwise unexplained MSAFP elevations should be submitted for pathologic study. A definitive retrospective diagnosis may thus be provided. PMID- 1704627 TI - Reduced fetal hepatic alpha-fetoprotein levels in Down's syndrome. AB - In the majority of pregnancies involving a Down's syndrome (DS) fetus, the level of alpha-fetoprotein (AFP) measured in maternal serum and amniotic fluid is reduced to about 70 per cent of the level attained in normal pregnancies. Causes of this decrease may include the production of an altered AFP molecule with modified turnover or transport properties, or a reduction in the level of AFP synthesis. We examined hepatic AFP mRNA transcripts and compared AFP polypeptide isoforms in liver tissue samples obtained from a group of DS and normal abortuses. No differences was detected in the structure of the AFP mRNA transcript or in the charge or mass of AFP polypeptides in the two sample groups. However, the hepatic AFP level, expressed as microgram AFP/mg protein, was significantly lower in a group of 28 DS cases relative to a group of 47 normal controls (p = 0.04). This difference in hepatic AFP concentration did not appear to be the result of a general reduction in the level of total protein or total RNA production. The greatest difference between the AFP levels of the DS and normal groups was observed in the earliest samples examined (i.e., at 17-19 weeks of age) where the median AFP levels differed by about 20 per cent. PMID- 1704628 TI - Antigenic differences between Trichinella spiralis and T. pseudospiralis detected by monoclonal antibodies. AB - Antigenic differences between Trichinella spiralis and T. pseudospiralis were established using two monoclonal antibodies (mAbs) that show different specificities to muscle larvae of the two variants. Enzyme-linked immunosorbent assay (ELISA) revealed that mAb 3G6 reacts positively against T. spiralis, T. nelsoni, T. nativa and T. pseudospiralis, whereas mAb 3E10 does not react with T. pseudospiralis under the same experimental conditions. These antigenic differences were confirmed after preabsorption of the antibodies with serial dilutions of extracts of T. spiralis or T. pseudospiralis muscle larvae. The indirect immunofluorescence technique showed that the antigen corresponding to mAb 3G6 is located in the stichosomes and the cuticle surface of both T. spiralis and T. pseudospiralis. In contrast, mAb 3E10 positively stained cryostat sections of T. spiralis, forming a dense reaction product on the surface of the whole larvae and the surrounding capsule. This antibody can be quite useful as a specific probe for distinguishing T. spiralis from T. pseudospiralis in taxonomic studies. Using an avidin-biotin system, we could prove that mAb 3G6 recognizes an excretory/secretory-type antigen. PMID- 1704629 TI - Intrinsic pancreatic nerves after mechanical denervation of the extrinsic pancreatic nerves in dogs. AB - Little is known about the influence of cutting the extrinsic pancreatic nerves on the morphology and function of the intrapancreatic nerves in dogs. For this reason, intrapancreatic nerves of mongrel dogs were studied, using electron microscopy and immunohistochemistry, after truncal vagotomy, after celiac and superior mesenteric ganglionectomy, and after a combination of both operations, i.e., removing all extrinsic nerves of the pancreas. Dogs with intact extrinsic and intrinsic pancreatic nerves served as controls. Studies were performed 1-2 weeks and up to 5 months after one or both denervation procedures. For immunohistochemical and electron microscopic studies the animals were perfused with glutaraldehyde-formaldehyde-picric acid solution and the tissue was embedded in Epon or paraffin. Both immunohistochemical and electron microscopic studies revealed that signs of degenerating intrapancreatic nerves occurred only in the early phase (up to 30 days) after operation. After 60 days, hypertrophy of pancreatic nerve fibers was observed. The most striking finding was that the integrity of the intrapancreatic ganglia and nerves was almost preserved after complete extrinsic denervation. In controls there was a strong intrapancreatic innervation with vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI), substance P (SP), and neuropeptide Y (NPY) nerves. SP and NPY nerves significantly decreased after the different denervation procedures, but the other peptidergic nerves were not altered by truncal vagotomy, ganglionectomy, or the combination of both procedures. We conclude that the dog pancreas contains extensive intrinsic peptidergic nerves, which, with the exception of SP and NPY-nerves, are greatly independent of the integrity of the extrinsic nerves. PMID- 1704630 TI - Effects of H7 and staurosporine on cytosolic free calcium and amylase secretion in rat pancreatic acini. AB - Pretreatment of rat pancreatic acini with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 5 or 10 min reduced cytosolic free calcium and amylase secretion stimulated by submaximal concentration (10(-6) M) of carbachol in a dose dependent manner. 10(-7) M TPA inhibited initial amylase secretion and had no effect on sustained secretion stimulated by 10(-6) M carbachol. 1-(5 Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), a protein kinase C inhibitor, partially blocked these inhibitory effects of TPA. Cytosolic calcium concentration and initial amylase secretion were recovered with 10-100 microM H7 in TPA-treated acini. H7 was more effective than N-(2-guanidinoethyl)-5 isoquinoline-sulfonamide hydrochloride in increasing cytosolic free calcium in TPA-treated acini. TPA completely blocked an increase in cytosolic free calcium by 10 mM NaF. These findings indicated that TPA caused the inhibitory effects by means of activating protein kinase C, and suggested that protein kinase C might regulate enzyme secretion by inhibiting calcium mobilization, probably through a postreceptor-mediated mechanism. PMID- 1704631 TI - Somatostatin analog, SMS 201-995, inhibits pancreatic exocrine secretion and release of secretin and cholecystokinin in rats. AB - We studied the effect of a synthetic octapeptide somatostatin analog, SMS 201-995 (sandostatin), on pancreatic exocrine secretion and on plasma secretin and cholecystokinin (CCK) levels in vivo in anesthetized rats. The exocrine pancreas was stimulated by either intravenous infusion of both secretin (0.06 CU/kg/h) and cholecystokinin octapeptide (CCK-8) (0.03 micrograms/kg/h) or intraduodenal infusion of oleic acid (pH 6.5) in a dose of 0.25 mmol/h. Intravenous administration of SMS 201-995 in three different doses of 100, 200, and 400 ng/kg/h resulted in dose-related inhibition of pancreatic secretion in terms of volume, bicarbonate, and amylase stimulated by exogenous secretin and CCK. Intraduodenal oleic acid stimulated pancreatic secretion, including volume, bicarbonate, and amylase, and this was accompanied by a significant elevation in the plasma concentrations of secretin and CCK. Intravenous administration of SMS 201-995 in the three different doses described above caused dose-dependent suppression of the increase in pancreatic exocrine secretion as well as the plasma concentration of secretin and CCK induced by intraduodenal infusion of oleic acid. It is concluded that SMS 201-995 inhibits pancreatic exocrine secretion and the release of endogenous hormones, such as secretin and CCK, in rats. PMID- 1704632 TI - Pancreatic elastase 1 after pancreatic transplantation. AB - Human pancreatic elastase 1 (E1) is a novel pancreas-specific proteinase that has not yet been investigated after pancreatic transplantation (PTx). Using a recently developed E1 ELISA, we studied the E1 serum curves in 36 type I diabetic patients subjected to PTx with enteric exocrine diversion from the pretransplant value up to 8 years after transplantation (n = 731 samples). A characteristical pattern was observed: following PTx, E1 rose above the normal range to a peak within 6 days and then gradually fell to stabilize after 4-6 weeks at an elevated level (10 ng/ml) for approximately 1 year. Two to 8 years after PTx, E1 levels were still slightly elevated (1-6 ng/ml) in 14/20 patients. During 24 acute rejection episodes, E1 was found not be a sensitive rejection marker during the early postoperative period because of its slow decline from the peak level. However, increasing E1 levels in seven patients more than 2 months after PTx were associated with a variety of lesions to the pancreatic graft, thus suggesting a useful marker indicating exocrine graft damage late after PTx. The slightly elevated levels even years after PTx are most probably due to the non-portal venous drainage of the pancreatic graft. PMID- 1704633 TI - Fecal isoamylase activity in patients with pancreatic diseases. AB - Fecal isoamylase activity was studied in 93 consecutive patients (26 in the recovery stage of acute pancreatitis, 24 with chronic pancreatitis, 13 with pancreatic cancer, and 30 with other gastrointestinal diseases) and compared with fecal chymotrypsin activity and the results of the secretin test. Seventy-six healthy subjects were studied as controls. Both pancreatic (p)-type and salivary (s)-type isoamylase activities in stool were determined by inhibitor assay as well as cellulose acetate electrophoresis. The mean fecal amylase activity in healthy subjects was 757 +/- 88 IU/g (p-type isoamylase: 77 +/- 2%, s-type isoamylase: 23 +/- 2%). There was a good correlation between fecal p-type isoamylase and chymotrypsin activities (r = 0.625, p less than 0.001). Fecal p type isoamylase activity in patients with chronic pancreatitis and pancreatic cancer was significantly lower than in healthy subjects (p less than 0.001). Patients with moderate and severe exocrine pancreatic insufficiency as determined by the secretin test had significantly lower fecal p-type isoamylase activity. Daily fat intake did not affect fecal amylase or isoamylase activities. Fecal s type isoamylase activity in patients with hypoacidity was significantly higher than in patients with hyperacidity, but no difference in fecal p-type isoamylase activity was observed. It is concluded that analysis of fecal isoamylase activity is useful in the assessment of pancreatic function. PMID- 1704634 TI - Tumour markers in monitoring the treatment of advanced prostate cancer. PMID- 1704635 TI - [Granulocytopoietic growth factors. Biological properties]. PMID- 1704636 TI - [Intravenous gamma globulin in newborns]. AB - Eight newborns with a mean gestational age of 36.1 weeks and a mean weight of 2863 grams were treated intravenously with 3% gammaglobulin (Sandoglobulina). We used two different infusion rates: one, of 0.5-1.5 mg/kg/min., and a second one, of 7-9 mg/kg/min. Close cardiopulmonary monitoring was carried out by bioelectronic means. The changes of heart rate, mean blood pressure and respiratory rate before and during the gammaglobulin infusion were recorded. We did not find any significant variation between the values recorded before and during the infusion. These values were all within tolerable physiological ranges except for those for respiratory rate, where 7 out of 8 patients had mild tachypnea. Stable sick newborn because of bacterial infections tolerated well the infusion of intravenous gammaglobulin at rates of 0.5-9.0 mg/kg/min., when the doses used were between 0.25 mg/kg/day and 1.0 gm/kg/day, for 4-6 days. PMID- 1704637 TI - Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. AB - A phagocytic assay performed with human peripheral mononuclear cells and malaria infected erythrocytes enabled the study of opsonizing antibodies in human serum from donors presenting different levels of protective immunity. Opsonizing activity was found in sera from individuals who could be considered immune, i.e. asymptomatic parasite carriers and subjects residing in endemic regions who presented neither symptoms nor parasites. This contrasted with subjects showing an absence of symptoms or who had experienced only a single malarial infection. All sera contained high levels of antimalarial antibodies, as shown by immunofluorescence assay (IFA). IgG of different subclasses were immunopurified from these sera. All subclasses were shown to bind to the surface of infected erythrocytes by FACS analysis, but only IgG1 and IgG3 were able to mediate opsonization. IgG2 and IgG4 did not opsonize and inhibited the opsonizing activity of IgG1 and IgG3 in competition experiments. These results suggest the existence of a correlation between immune protection, the ability of serum to mediate opsonization of infected erythrocytes and the predominance, in this serum, of IgG1 and IgG3 over IgG2 and IgG4 directed against the surface of the infected erythrocytes. PMID- 1704638 TI - Polysaccharide nature of O antigen in protective ribosomal preparations from Shigella: experimental evidence and implications for the ribosomal vaccine concept. AB - Shigella ribosomal vaccine (SRV) was previously shown to be highly active in induction of mucosal and systemic O-antibody response and protection against Shigella infection in guinea pigs and monkeys. In this study, the O-specific component (OSC) was isolated from the SRV by affinity chromatography using rabbit O antibodies coupled to CNBr-Sepharose. The results of the reaction with carbocyanine dye as well as chemical data show that ribosomal OSC is devoid of lipid A and KDO, which are characteristic of classical LPS. The comparison of OSC with various LPS-related substances led to the conclusion that ribosomal OSC is similar to and probably identical with cytoplasmic O polysaccharide (L hapten), an O-side-chain polymer which accumulates in cytoplasm. It is hypothesized that the extremely high immunogenicity of SRV depends on a cooperative action of OSC, representing an epitope-specific part of the vaccine, and a ribosomal particle which serves as a vector, providing amplification of the immunogenic effect. The data obtained indicate the presence of a non-covalent link between the two components of the ribosomal vaccine. PMID- 1704639 TI - Effect of endogenous tachykinins on neuro-effector transmission of vagal nerve in guinea-pig tracheal tissue. AB - To elucidate the effect of endogenous tachykinins on neuro-effector transmission of vagal nerves, we performed in vitro experiments using guinea-pig tracheal smooth muscle. The subthreshold dose (the highest dose which did not induce any smooth muscle contraction) of capsaicin (10(-8) to 10(-7) M) increased the amplitudes of contractions evoked by electrical field stimulation (EFS) significantly, but not those by acetylcholine (ACh). The inhibitor of neutral endopeptidase, phosphoramidon (10(-7) to 10(-6) M), increased the contractions evoked by EFS significantly. The inhibitor of cholinesterase, physostigmine (10( 6) to 10(-5) M), induced smooth muscle contractions, but such contractions were inhibited by atropine, suggesting the spontaneous release of ACh from the vagal nerve terminals. The subthreshold dose of substance P or capsaicin increased the contractions evoked by physostigmine. These results indicated that endogenous tachykinins increase the spontaneous ACh release as well as the ACh release in response to vagal stimulation from the nerve terminals. Furthermore, it is suggested that the excitatory effects of the tachykinins on the vagal neuro effector transmission may be modulated by neutral endopeptidase in the guinea pig. PMID- 1704640 TI - Isoprinosine in the treatment of chronic active hepatitis type B. AB - 21 patients with chronic active hepatitis B (CAH-B) were treated for 1-2 years with isoprinosine, while another 18 patients served as control group. All patients were initially DNA polymerase (DNAp) and HBeAg positive. Nine (43%) treated patients became persistently negative for DNAp, seroconverted to anti-HBe and showed histological remission on follow-up biopsy. Among simultaneously followed controls 5 (28%) lost DNAp and 4 (22%) also lost their HBeAg. However, only 2 (11%) seroconverted to anti-HBe. Histological improvement was seen in 5 (28%) controls. Thus, it seems that isoprinosine may exert a beneficial effect on the course and outcome of CAH-B. PMID- 1704641 TI - Homelessness and cognitive performance in children: a possible link. AB - The developmental impact of homelessness on children was assessed by using a screening battery to measure cognitive and language delays as well as emotional status. The results of testing 88 children in a family shelter program indicate a possible significant impact of homelessness on both the cognitive and language development of these children, with language more severely affected. Emotional pathology, however, was not detected with the screening instruments used. Most service providers who deal with homeless children fail to recognize their special programming needs. It is important for the social worker to advocate with the educational system for these children's special needs. PMID- 1704642 TI - Carcinoma of the head of the pancreas. AB - Optimal treatment for unresectable carcinoma of the pancreas remains controversial. This study was done to examine the relationship between perioperative jaundice and postoperative morbidity, and type of palliative biliary bypass and postoperative morbidity and jaundice clearance. Seventy-six patients with obstructive jaundice secondary to carcinoma of the head of the pancreas were studied. Forty-nine patients underwent one of four different types of palliative bypass: 1, cholecystojejunostomy (n = 22); 2, choledochojejunostomy (n = 11); 3, choledochoduodenostomy (n = 9), and 4, cholecystoduodenostomy (n = 7). Age, sex and preoperative health status were similar for all operative groups, as well as for those with and without postoperative morbidity. The postoperative complication rate was 33 per cent and there was one postoperative death. Length of preoperative jaundice and peak preoperative bilirubin levels were independent of morbidity. Postoperative morbidity was similar for each type of bypass used and no significant difference was found when cholecystoenteric (1 and 4) and choledochoenteric (2 and 3) bypass were compared. The results of this study support the view that postoperative morbidity is not directly related to the presence of jaundice preoperatively. Furthermore, the rate of jaundice clearance and the occurrence of postoperative complications are not dependent on the type of bypass used. PMID- 1704643 TI - Stapled bypass for nonresectable carcinoma located in the upper part of the stomach. PMID- 1704644 TI - Characteristics of chemical binding to alpha 2u-globulin in vitro--evaluating structure-activity relationships. AB - alpha 2u-Globulin (alpha 2u) has been shown to accumulate in the kidneys of male rats treated with 2,2,4-trimethylpentane (TMP). 2,4,4-Trimethyl-2-pentanol (TMP-2 OH), a metabolite of TMP, is found reversibly bound to alpha 2u isolated from the kidneys of these treated rats. The objectives of the following study were to characterize the ability of [3H]TMP-2-OH to bind to alpha 2u in vitro and to determine whether other compounds that cause this protein to accumulate have the same binding characteristics. Although compounds that have been shown to cause the accumulation of alpha 2u in male rat kidneys compete in vitro with [3H]TMP-2 OH for binding to alpha 2u, they do so to varying degrees. The binding affinity (Kd) of the [3H]TMP-2-OH-alpha 2u complex was calculated to be on the order of 10(-7) M. The inhibition constant values (Ki) determined for d-limonene, 1,4 dichlorobenzene, and 2,5-dichlorophenol were all in the range 10(-4) M, whereas the Ki values for isophorone, 2,4,4- or 2,2,4-trimethyl-1-pentanol, and d limonene oxide were determined to be in the range 10(-6) and 10(-7) M, respectively. TMP and 2,4,4- and 2,2,4-trimethylpentanoic acid did not compete for binding. This suggests that other factors, besides binding, are involved in the accumulation of alpha 2u. In this study the ability of a chemical to bind to alpha 2u was used as a measure of biological activity to assess structure activity relationships among the chemicals tested and known to cause the accumulation of alpha 2u. The results so far suggest that binding is dependent on both hydrophobic interactions and hydrogen bonding. PMID- 1704645 TI - Long term pulmonary impairment following a single exposure to methyl isocyanate. AB - Groups of guinea pigs were exposed for 3 hr to 6, 13, 19, 27, or 37 ppm of methyl isocyanate (MIC). Pulmonary performance was evaluated immediately postexposure and for a period of 1 year in the animals surviving 19 or 37 ppm of MIC. At 6 and 13 ppm deterioration of pulmonary performance was observed but complete recovery occurred within a few weeks. In those animals surviving 19 or 37 ppm some recovery occurred but a long lasting impairment of pulmonary performance was observed. One year after a single exposure to MIC these animals presented a condition best described as chronic obstructive lung disease. Using flow-volume measurements during air breathing and during CO2 challenge, animals surviving at 19 or 37 ppm presented typical abnormalities associated with airflow limitation along the conducting airways. One year after exposure lung hyperinflation was also present. At this time the poor ventilatory response to CO2 was due to mechanical limitation of lung expansion during inspiration and airflow limitation during expiration. The findings were similar to humans with severe obstructive lung disease. Microscopic examination substantiated the flow-volume measurements in that the main bronchi, small bronchi, and bronchioles were found to have an increase in dense fibrous tissue, while the alveolar level was characterized by destruction of alveolar walls and an increase in septal thickness. Thus a single exposure to MIC, if the concentration is high enough, is sufficient to induce permanent pulmonary toxicity. PMID- 1704646 TI - [Results of an inquiry with patients on the effect of show cases and display cases in the stomatological waiting room]. AB - The results of interviews in 530 patients of two dental clinics (Schwerin and Berlin) are demonstrated. The subject was effectiveness of dental health education aids, as there are displays and boxes. Both educational possibilities appeared to be useful and psychological favourable. PMID- 1704647 TI - [Plastic skull for practical education in stomatological radiology]. AB - A plasticmodell of a skull manufactured by Deutsches Hygienemuseum Dresden, German Democratic Republic, was produced with Ba2SO4, natural teeth and special constructions for the practical training of students in Stomatological Radiology. Examples show the radiographed x-ray-photograms. PMID- 1704648 TI - Effects of BWH-4 anti-CD4 monoclonal antibody on rat vascularized cardiac allografts before and after engraftment. AB - We studied the effects and mechanism of action of BWH-4, an IgG2a mouse antirat CD4 monoclonal antibody that recognizes a distinct epitope on the CD4 molecule, in LEW recipients of (LEW x BN)F1 vascularized heterotopic cardiac allografts. Ten animals received daily injections of 700 micrograms BWH-4 for ten days after engraftment. Median actuarial allograft survival was 37 days for the treated animals compared with 7.5 days for controls (n = 6). There was depletion of peripheral blood CD4+ lymphocytes to 4 +/- 1% one week posttransplant (normal = 48 +/- 4%, n = 6). Six additional animals were treated with a lower dose (100 micrograms) of BWH-4 daily for ten days. Median actuarial allograft survival was 10 days and the circulating CD4+ cells decreased to 30 +/- 6% at one week posttransplant. All six low-dose-treated animals mounted an anti-BN alloantibody response by 3 weeks, while only one of the ten high-dose-treated animals had positive alloantibodies two weeks posttransplant. Lymphocyte-mediated cytotoxicity against donor lymphoblasts was completely abolished in the high-dose treated animals when compared with acutely rejecting controls. We also used low dose (100 micrograms) BWH-4 to pretreat eleven experimental animals for 7 days prior to engraftment. The circulating CD4+ cells decreased to only 12 +/- 2% on the day of transplantation and 26 +/- 9% one week posttransplant. However, six (55%) pretreated animals survived more than 55 days. There was a 66% decrease in lymphocyte-mediated cytotoxicity against donor lymphoblasts when compared with acutely rejecting controls. We conclude that the anti-CD4 mAb, BWH-4, prevents acute rejection of vascularized heterotopic rat cardiac allografts; this effect is mediated by depletion of the CD4+ T cell subset, suppression of alloantibody production, and inhibition of lymphocyte-mediated cytotoxicity. PMID- 1704650 TI - Fatal intraperitoneal perforation of the bladder: 2 case reports. PMID- 1704649 TI - Induced expression of endothelial-leukocyte adhesion molecules in human cardiac allografts. PMID- 1704651 TI - In situ Hpa II endonuclease digestion on fixed chromatin of solid tumor cells. AB - Neoplastic cells from different tumors (lung, colon, rectal, and pancreatic carcinoma, synovial sarcoma, and Wilm's tumor) were fixed on slides and in situ digested with Hpa II and Msp I restriction enzymes. Staining of samples with the DNA specific fluorochrome ethidium bromide showed a clearcut decreased fluorescence after Hpa II digestion in neoplastic cells as compared to normal controls, whereas Msp I digestion produced the same pattern in neoplastic and in normal cells. The authors hypothesize that the altered state of methylation in neoplastic cells could affect the Hpa II activity on fixed chromatin. PMID- 1704652 TI - Serum amylase and isoamylase values in dogs with pancreatic disease. AB - Serum amylase and isoamylase values were determined in three groups of dogs. The first group contained control dogs while the other groups contained dogs with confirmed exocrine pancreatic insufficiency and diabetes mellitus respectively. The trypsin-like immunoreactivity test was also carried out on sera from dogs with exocrine pancreatic disease (EPI). A significant difference was detected in the serum amylase values between the three groups which may be of limited diagnostic value. Dogs with EPI had values lower than normal while those with diabetes mellitus had values higher than control dogs. No evidence of exocrine pancreatic insufficiency was found in dogs with diabetes mellitus. PMID- 1704653 TI - Characterization of Mengo virus neutralization epitopes. AB - A set of four monoclonal antibodies which neutralized the infectivity of Mengo virus was used to select 20 non-neutralizable (escape) mutants. Altered amino acids were identified by sequence analyses of the capsid-coding regions of the mutant virus genomes. Mutations were found predominantly in proteins VP2 and VP3, while mutations in VP1 were detected only as second mutations. The Mengo virus VP2 mutations at amino acid residues 2144, 2145, 2147, and 2148 align with site Nlm II in human rhinovirus-14 and site 2 in polioviruses 1 and 3. The mutation at 2075 as well as those at 3057, 3061, and 3068 in VP3 correspond to site 3 in poliovirus. These alignments notwithstanding, the results of cross-neutralization experiments indicate the existence of a single composite neutralization site on the Mengo virion. Considering the three-dimensional structure of the Mengo capsid, the amino acids which are altered in the escape mutants are all exposed on the outer surface and none are found in the "pit," the probable site for binding of a cellular receptor. The VP3 mutations are located in the VP3 "knob" and the VP2 mutations on a nearby ridge. Together these mutations define a set of epitopes within a single composite antigenic determinant which forms a crescent shaped area around the three-fold icosahedral axes of the Mengo virion. PMID- 1704654 TI - Peptide mapping of neutralizing and nonneutralizing epitopes of duck hepatitis B virus pre-S polypeptide. AB - Antibodies to the envelope proteins of duck hepatitis B virus neutralize viral infection in vitro. Using a library of murine monoclonal antibodies (Mabs) against the envelope proteins, we previously identified four neutralizing and two non-neutralizing epitopes on the pre-S region of the large envelope proteins. In this study we report the localization of all but one of these epitopes at the amino acid level. All but 28 nucleotides of the pre-S and S genes were cloned in pUC vectors and expressed in Escherichia coli. All Mabs in this study reacted with the expressed gene products in Western blots. Deletion mutants of the pre-S region were generated and their expressed products tested on Western blots for reactivity with the Mabs. Of the three epitopes involved in neutralization, the epitope found to be immunodominant in convalescent ducks was localized to nine amino acids of the middle portion of the pre-S gene product, while a second epitope was mapped to nine amino acids upstream of the immunodominant epitope and the third epitope to seven amino acids adjacent to the S gene. One of the two non neutralizing epitopes was located between the two groups of neutralizing epitopes while the other mapped to the same region as one of the neutralizing epitopes. Our data indicate that several regions of the pre-S polypeptide may play a role in neutralization of hepadnaviruses. PMID- 1704655 TI - Productive infection of CD4+ cells by selected HIV strains is not inhibited by anti-CD4 monoclonal antibodies. AB - Differential susceptibility of four diverse HIV strains to inhibition of infection of CD4+ CEM cells by anti-CD4 monoclonal antibodies was studied. The highly cytopathic HIV-1 246 and NDK strains were able to infect CEM cells and undergo several cycles of replication at saturating doses of LEU3-A, OKT4-A, and 13B8-2 monoclonal antibodies, whereas propagation of reference HIV-1 BRU and weakly cytopathic strain HIV-1 PAS was inhibited. Postadsorption treatment by anti-CD4 antibodies had stronger inhibitory effect than did treatment during the virus adsorption period. In parallel experiments, the same monoclonal antibodies successfully blocked syncytium formation between uninfected MT4 cells and CEM cells infected by all four HIV-1 virus strains tested. To explain these seemingly contradictory data we have postulated that anti-CD4 antibodies efficiently inhibit cell-to-cell but not virus-to-cell infection. PMID- 1704656 TI - Inhibition of HIV and SIV infectivity by blockade of alpha-glucosidase activity. AB - Processing of HIV and SIV envelope oligosaccharides is critical for proper intracellular trafficking and function. An inhibitor of alpha-glucosidases I and II, N-butyl deoxynojirimycin (N-BuDNJ), retards HIV-1 and SIVmac spread in lymphocytes and monocytes by diminishing virus infectivity, and also causes a reduction in syncytia formation between infected cells and uninfected lymphocytes. N-BuDNJ retards envelope processing from the precursor form to the mature surface (SU) and transmembrane proteins in HIV-1- and SIVmac-infected cells, as well as in cells infected with vaccinia-HIV-1 envelope recombinant virus. However, no significant reduction is seen in the amount of SU in released virus particles, though the virus particle-associated SU from N-BuDNJ-treated cells has an altered electrophoretic mobility. In contrast, N-BuDNJ had no effect on GAG protein synthesis and processing. These findings demonstrate a critical requirement for oligosaccharide processing by alpha-glucosidases I and II for HIV 1 and SIVmac envelope processing and fusogenicity. PMID- 1704657 TI - Human immunodeficiency virus infection: association with altered intracellular levels of cAMP and cGMP in MT-4 cells. AB - T-cells from human immunodeficiency virus (HIV)-infected patients are characterized by a number of qualitative deficiencies including defective T-cell activation. The latter has previously been shown to be normally regulated by cAMP. In this study the patterns of cAMP and cGMP induction in MT-4 cells following HIV infection were investigated. The MT-4 cells were infected with HIV (strain IIIb) and at selected times postinfection (p.i.), culture supernatants were tested for HIV replication by reverse transcriptase activity or HIV P24 Ag. The cells were also examined for their intracellular levels of cAMP and cGMP by radioimmunoassay. HIV infection was associated with an increase in intracellular levels of cAMP and cGMP. The cAMP was increased 40-fold by Day 8 and cGMP 4-fold by Day 4 Pl. The increase in intracellular levels of the cyclic nucleotides (CN) were virus specific, dependent on virus dosage, genetically conserved among the two fresh patient isolates tested, and were abolished by uv inactivation. An increase in cAMP and cGMP was also observed in other cell lines infected with HIV. The sustained elevation in CN level observed could certainly influence cell activation and HIV replication and may potentially have clinical relevance. PMID- 1704658 TI - Endogenous origin of defective retroviruslike particles from a recombinant Chinese hamster ovary cell line. AB - The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. Continuous-flow ultracentrifugation has been used to concentrate extracellular particles from culture fluid of a recombinant CHO cell subclone for molecular characterization. Particles exhibiting reverse transcriptase activity and associated with mammalian C-type retrovirus structural proteins banded in sucrose gradients at a density characteristic of retroviruses. Examination of gradient-purified particles by electron microscopy revealed morphology and size similar to other retroviruses. Double-gradient-purified particles contained RNA which hybridized to probes for murine leukemia virus, and endogenous Chinese hamster intracisternal A-particle elements. DNA sequence analysis of a cDNA clone isolated from purified particles revealed multiple interruptions of the endonuclease reading frame, providing one possible explanation for the noninfectious nature of the observed particles. Sequences present as RNA in purified particles were also present as conserved, repetitive, provirus sequences in genomic DNA of all CHO cell lines examined and in Chinese hamster liver DNA. The observed particles are therefore likely to be the products of endogenous retroviruslike elements present in the germline of Chinese hamsters. PMID- 1704659 TI - Differential replication in zucchini squash of a cucumber mosaic virus satellite RNA maps to RNA 1 of the helper virus. AB - Cucumber mosaic virus (CMV) supports the replication and encapsidation of its satellite RNA, both in solanaceous and cucurbit host plants; however, different strains of CMV support the replication of satellite RNAs with different efficiency. In addition, replication of satellite RNA is very efficient in solanaceous host plants and generally poor in cucurbit host plants. The WL1 satellite (WL1-sat) RNA is an exception, and replicates to high levels in both solanaceous and curcubit host plants with most CMV strains as the helper virus. Two strains of CMV were used in this study: Fny-CMV, which replicates the WL1-sat RNA efficiently in all hosts tested; and Sny-CMV, which does not replicate the WL1-sat RNA to detectable levels in zucchini squash (Cucurbita pepo), but does replicate WL1-sat RNA efficiently in other hosts. Using pseudorecombinants constructed between Fny-CMV and Sny-CMV we have mapped to RNA 1 the ability to support the efficient replication of WL1-sat RNA in zucchini squash. PMID- 1704660 TI - Biological characterization of infectious molecular clones derived from a human immunodeficiency virus type-1 isolate with rapid/high replicative capacity. AB - In order to molecularly characterize rapidly and slowly replicating HIV-1 variants, molecular clones were obtained from a rapid/high virus isolate. This isolate, 4803, had only been passaged in peripheral blood mononuclear cells (PBMC) prior to cloning. Molecular cloning was done in bacteriophage lambda-dash using high molecular weight DNA of isolate 4803 infected PBMC. Seven recombinant phages were identified. The clones were found to be related to each other and differed only at 1 or 2 restriction sites (out of 28). The molecular clones were transfected into various cell types by electroporation. The phenotype of progeny viruses was found to be dependent on the cell type used for transfection. Progeny viruses produced by PBMC cultures differed from the parental isolate in that they did not form syncytia and lacked the capacity to replicate in cell lines. Since transfection of PBMC yielded progeny viruses within 1 week, this phenotype is considered to be the true phenotype of the clones. Transfection of the T-lymphoid HUT-78 cell line and of the monocytoid U937-2 cell line yielded progeny viruses after considerable delay (more than 1 month). Progeny viruses from HUT-78 cells were similar to the parental isolate in that they formed syncytia in PBMC and replicated in all cell lines tested. Progeny viruses from U937-2 cells showed an intermediate phenotype in that they replicated in U937-2 but not in T-lymphoid cell lines. These results indicate that molecular clones of a rapid/high virus may have a restricted replicative capacity compared to the parental, genetically heterogenous virus isolate. PMID- 1704661 TI - Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants. AB - The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program. PMID- 1704662 TI - Identification of an epitope encoded in the env gene of Friend murine leukemia virus recognized by anti-Friend virus cytotoxic T lymphocytes. AB - We have previously shown that strong epitopes recognized by anti-Friend virus (FV) cytotoxic T lymphocytes (CTL) in H-2b mice are encoded in both the env and gag/pol regions of the helper friend leukemia virus genome. Two approaches have been used to identify these epitopes. At the nucleic acid level, we have constructed env genes with either of two in-frame deletions: pKR2, an env gene with a 681-bp deletion in the gp70 region and inserted into the pSV2-gpt-1 expression vector; and pKR1, an env gene with an 81-bp deletion in the p15E region and inserted into pSV2-gpt-1. Cell clones were established by transfecting Fisher rat embryo cells with pDb (the H-2Db restriction element), pNEO (for G418 selection) and either pKR1 or pKR2. Db and env gene expression was monitored by immunoprecipitation with polyclonal antibodies or by detection of viral RNA on Northern blots. Expressor cell clones were tested for susceptibility to lysis by polyclonal anti-FV/Db CTL in 51Cr-release assays. Whereas cells expressing pKR1 were lysed to the same extent as cells expressing the intact env gene, cells expressing pKR2 were resistant to lysis, suggesting that all detectable env epitopes are encoded within the 681-bp deletion. Polypeptides representing the two most likely candidate epitopes encoded in this segment were synthesized and tested for their abilities to sensitize FRE cells expressing Db alone for lysis by the CTL. One 17-mer polypeptide, AGTGDRLLNLVQGAYQA [corrected], functioned as a strong CTL epitope in this assay, but the other 18-mer polypeptide was inactive. Studies of the role of this epitope in the immune response to candidate viral vaccines are in progress. PMID- 1704663 TI - [The efficacy of recombinant alfa-2 interferon in viral hepatitis B]. PMID- 1704664 TI - [A case of diphtheria in an adult]. PMID- 1704665 TI - [Treatment with antihistaminic preparations]. PMID- 1704666 TI - [Interferon and its use for treating hairy cell leukemia]. PMID- 1704668 TI - Defects of the external nose following treatment of malignant tumours and methods of their removal. AB - Defects of the nose after oncological operations were classified on the basis of clinical material and indications to primary and delayed rhinoplasty were pointed out. The problems of choosing the method of plasty were considered depending on the localization of the defect, its size and previous treatment. The advantages of arterialized flaps and free revascularized transplants were discussed. Examples were given. PMID- 1704667 TI - [The effect of thyrostatic treatment on circulating thyrotropin-receptor antibodies in Basedow's disease]. AB - In 80 patients with Basedow's disease (20 nontreated and 60 under treatment with methimazole) were examined. The antibodies inhibiting the binding of TSH (TBII) by the radioreceptor method (Thyrotropin-Receptor-Anti-Korper Assay = TRAK), TRH test, T3, T4, FT4, FT. TRAK-positive) values above 15 u/l) were 80% of the nontreated and 20% of the treated patients. In the course of 3 up to 6 months from the start of the treatment 60% of the examined patients were negative and at the end of the 18th month--92% were negative. A significant difference in the frequency of the recurrences was found between the TRAK-positive and the TRAK negative patients. Between the TRAK, FT3, FT4, the size of the struma, the presence of ophthalmopathy [correction of ophthalmycthere] are positive relations. The following conclusions are made: 1. The determination of TBII and TRAK is not only of diagnostic but also of prognostic importance. 2. Methimazole exerts a specific immunosuppressive action and can lead to an immunologic remission in a comparatively short time. PMID- 1704669 TI - Urgent repair of electrical injuries: analysis of 40 cases. AB - 40 patients suffering from electrical injuries were rehabilitated using a new system of urgent conservative debridement and flap coverage in the period from October 1984 to July 1988. For this purpose, a 1-4 year follow-up was needed. Good appearance and functions of the extremities or organs were obtained and the amputation rate was lower (7.5%) than that used in patients treated with traditionally conservative methods (47.7% in the same hospital. PMID- 1704670 TI - Regulation of traumatic skin wound healing by influencing the process in the neighbouring zone of the wound. AB - After the burn, the authors immediately applied the albuminous hydrolysate Hydropot, a propolis-urea ointment and Trypsin compresses (3,500 E) to the burned area and its vicinity, and repeated this procedure for several days. Samples for histological examination were taken after the animals' decapitation every 2 hrs. and on the 3rd, 5th and 14th post-injury days. The morphological picture showed a difference in rate and quality of regeneration. The authors are of the opinion that the processes occurring in the neighbourhood of the traumatic skin wound can be influenced and that regeneration can be regulated. PMID- 1704671 TI - Method of Converse for correction of prominent ears: comparison of results. AB - We compared the objective and subjective evaluations of the results after correction of the earposition by the method of Converse with the results by the method of Reichert and Mustarde. The mean values obtained by the method of Converse correspond to the mean normal values. The patients are very satisfied with their results. A comparison of the results of these three methods do not show any aggravating differences. PMID- 1704672 TI - The free full thickness lid graft in the eyelid margin reconstruction. AB - An operating method for the eyelid margin reconstruction is described. Using a free full thickness lid graft obtained from the opposite lid in 46 cases of lid margin basal cell carcinoma always in one intervention without tarsorhaphy, satisfactory final cosmetic as well functional success resulted. PMID- 1704673 TI - Classification of the syndromes of branchial arches 1 and 2. AB - On the basis of observations of 393 patients over many years, the author considers it possible to distinguish 5 types among the various forms of the syndrome of branchial arches 1 and 2. Two types are characteristic of the syndrome of branchial arch 1: the mandibular type and the mandibuloauricular type. The former is unilateral and occurs very rarely, the latter is encountered more frequently and can be unilateral or bilateral. For the syndrome of branchial arches 1 and 2, three types are characteristic: the auricular, the mandibulofacial and auricular, and the craniofacial, articular and auricular. The auricular type is mainly characterized by marked anomalies of the cochlea and the sense of hearing, occurs most frequently of all and is also bilateral. The mandibulofacial and auricular type exhibits more pronounced affections of both the cochlea and hearing and the bones of the facial skeleton. It occurs frequently and is unilateral. The craniofacial, articular and auricular type shows the most serious combinations of defects of the cochlea, the base of skull, the temporal bone, the facial skeleton, and the absence of the branch and the condylar process of mandible as well as hypoplasia of the soft tissues of the face. Systemic dysplasias of the face, the jaws and the cochlea are combined with defects of other, frequently distant, systems. PMID- 1704674 TI - Effects of galanin and calcitonin gene-related peptide on insulin and glucagon secretion in man. AB - To study the influence of the pancreatic neuropeptides, galanin and calcitonin gene-related peptide, on insulin and glucagon secretion in man, synthetic porcine galanin (80 pmol.kg-1.min-1; N = 6) or synthetic human calcitonin gen-related peptide (10 pmol.kg-1.min-1; N = 5) was infused intravenously in human volunteers. Following 5 min of infusion, arginine (5 g bolus + 10 mg.kg-1.min-1) was given. Galanin did not affect basal or arginine-stimulated insulin secretion judged from determinations of plasma insulin and C-peptide. Similarly, galanin did not affect arginine-stimulated glucagon secretion. Calcitonin gene-related peptide did not affect basal or arginine-stimulated insulin or glucagon secretion. However, calcitonin gene-related peptide slightly potentiated the arginine-induced hyperglycemia (p less than 0.01). Thus, in man, galanin has no influence on insulin or glucagon secretion when infused at 80 pmol.kg-1.min-1, whereas CGRP at 10 pmol.kg-1.min-1 induces slight hyperglycemia. PMID- 1704675 TI - Cholesteatoma debris as an activator of human monocytes. Potentiation of the production of tumor necrosis factor. AB - Tumor necrosis factor (TNF) is a cytokine which stimulates osteoclastic bone resorption and inhibits collagen synthesis in vitro. In this study the effect of human cholesteatoma debris and its constituents on the production of TNF-alpha by human monocytes in vitro was studied. Cultured human peripheral monocytes secreted TNF into the culture medium when exposed to cholesteatoma debris in a dose-dependent manner. The TNF production, however, was partially inhibited by the treatment of the debris with polymyxin B which inhibits biological activities of lipopolysaccharide (LPS). When individual constituents of cholesteatoma debris, i.e. keratin, cholesterol, lauric acid and LPS, were added to the cultured monocytes at concentrations equivalent to those in the debris, significant production of TNF was observed only with the keratin and LPS. These data suggest that cholesteatoma debris is a potent activator of the TNF production of human monocytes in vitro, and that LPS and keratin are responsible for the production. PMID- 1704676 TI - The relationship between nasalance and nasal resistance to airflow. AB - The relationship between nasal airway resistance and nasalance in healthy volunteers and in subjects suffering from symptoms of acute rhinitis was investigated. A non-invasive objective measure of nasalance using the Nasometer (Kay Electronics) was used and an inverse correlation between airway resistance and nasalance was found (r = 0.67, r2 = 0.46, p less than 0.0001). The effect of a topical nasal decongestant on nasal airway resistance and nasalance was investigated, and significant changes were seen both in resistance and nasalance (p less than 0.0001) with a correlation in the changes seen in both parameters (r = 0.82, r2 = 0.66, p less than 0.0001). The measure of nasalance may be useful in assessing various forms of nasal treatment including nasal, adenoidal and palatal surgery. The measurement of nasalance is well tolerated by subjects of all ages and is particularly useful in subjects with high nasal resistance where rhinomanometry tends to be unstable. PMID- 1704677 TI - Studies of peripheral blood toxic neutrophils as a predictor of coronary risk in Kawasaki disease--the pathogenetic role of hematopoietic colony-stimulating factors (GM-CSF, G-CSF). AB - Many toxic neutrophils, characterized by cytoplasmic swelling, vacuolization and toxic granulation, can be seen in the acute phase of Kawasaki Disease (KD; mucocutaneous lymph node syndrome). Polymorphonuclear neutrophils (PMNs) of 36 patients with acute and convalescent phase KD were studied with reference to their morphological characteristics. The percentage of toxic neutrophils in the acute phase tended to be higher in the group with coronary artery lesions (CAL) than in the group without CAL. The results suggest that observation of toxic neutrophils in the acute phase of KD may be useful in predicting a high risk of CAL. In vitro cultivation studies of PMNs from healthy persons, in the presence of diverse hematopoietic colony-stimulating factors (CSF), revealed that GM-CSF (granulocyte-macrophage-CSF) and G-CSF (granulocyte-CSF) participated in the induction of cytoplasmic vacuolization of PMNs, and that the healthy PMNs morphologically mimicked peripheral PMNs seen in the acute phase of KD. PMID- 1704678 TI - Developmental dysphasia: clinical importance and underlying neurological causes. AB - This survey deals with two aspects of developmental dysphasia which are relevant to child psychiatry; the early diagnosis and treatment of children with developmental dysphasia, which may prevent the progression of learning and behaviour disorders, and the underlying biological causes of this neuro developmental disorder. The pathophysiology of developmental dysphasia is complex and age-related. In the pre-verbal and early verbal stage, the severity of the clinical picture is primarily determined by concomitant motor pathology (motor dysfunction, dysarthria, general and oral dyspraxia) and by receptive pathology (hearing and auditory perception). In the verbal period, linguistic problems start to play a role, and often combine with oral motor symptoms to present a mixed picture. The various language syndromes do not become clear until some time later. After the kindergarten period, the oral motor and perceptual problems decrease and the language disorders continue to play a role and influence the child's conversation, internal speech and learning a school. In a relatively small number of children without oral motor, perceptual or memory problems, there can be a basic syndrome of "pure dysphasia" without any other neurological signs. These children are very likely to have a genetically determined developmental disorder on a limited neuronal level (no cerebral damage of any kind!) such as an abnormal asymmetry of the hemispheres. In somewhat more than half the patients, this basic syndrome is accompanied by other neurological signs, most of which are indicative of functional disorders of the left hemisphere. There can also be symptoms of the right hemisphere, of the corpus callosum and of the afferent pathway systems for auditory perception. The nature and causes of these anomalies can be multifarious, so that it is unfeasible to speak of THE substrate or THE pathogenesis. Treatment should not be confined to speech therapy techniques, but should also take into consideration the existence of abnormal motor and affective development and can thus only be optimally given by a highly trained team whose expertise also extends to the schooling aspect. PMID- 1704679 TI - [Influence of the active compounds isolated from pilose antler on syntheses of protein and RNA in mouse liver]. AB - The polyamines of pilose antler (PASPA) consist of putrescine (PU, 70.9%), spermidine (SPD, 26.3%) and spermine (SP, 2.8%). The incorporations of [3H] leucine into protein and [3H] uridine into RNA in mouse liver tissue were increased when PASPA was given orally to mice at the dose of 30 mg/kg for 4 successive days. The incorporations of [3H] leucine into liver protein and [3H] uridine into the cytosolic and nuclear RNA were also increased by treatment with PU (21 mg/kg). In addition, the RNA polymerase activity in the solubilized liver nuclear fraction of PU (21 mg/kg)-treated mice was increased. SPD only promoted the synthesis of protein in mouse liver tissue at the dose of 8 mg/kg. However, SP showed no effect on the synthesis of protein and RNA polymerase activity under the used dose (1 mg/kg). The results suggest that PASPA is the main active substance responsible for the promotion of the synthesis of protein and RNA in mouse liver. PMID- 1704680 TI - Interferon immunomodulation in domestic food animals. PMID- 1704681 TI - Dysphasia and dementia in residential homes. PMID- 1704682 TI - Effect of bacterial endotoxin on plasma concentration of haptoglobin and fibrinogen in rats treated with metopyrone. AB - During the acute phase of the inflammatory process there is a characteristic increase in some plasma proteins called collectively acute phase reactants (APR) as well as in the levels of corticosteroids. A bacterial endotoxin (LPS) that induces a strong acute phase response, indicated by high levels of fibrinogen and haptoglobin, did not show this effect when administered to rats treated previously with metopyrone, a specific inhibitor of corticosteroid hormone synthesis. These results suggest that adequate levels of these hormones are important for the production of acute phase reactants. PMID- 1704683 TI - Mechanism of action of H2-antagonists on histamine- or dimaprit-stimulated H2 receptors of spontaneously beating guinea-pig atrium. AB - Cimetidine, ranitidine and famotidine are antagonists of the histamine H2 receptors on the spontaneously beating right atrium of the guinea pig. When analyzed by the classical Schild method their pA2-values are respectively: 6.3, 6.8 and 7.7 with dimaprit as agonist and 5.8, 6.5 and 7.7 with histamine as agonist. Radioligand-displacement studies with [3H]-tiotidine as radioligand resulted in pKd values for cimetidine, ranitidine and famotidine of 6.3; 6.9 and 8.2 respectively. In dimaprit-stimulated atria all antagonists added at concentrations above their Kd values depressed the maximal increase in frequency. In the presence of histamine this effect was much less pronounced and only visible at concentrations of ranitidine and famotidine around 10.Kd. The rightward shift of the curves as well as the decrease in Emax are reversible but the dissociation constants of the antagonists are small (less than 10(-3) s-1). The spontaneously beating right atrium showed receptor reserve for histamine and virtually no receptor reserve for dimaprit. The results have been interpreted in a model in which H2-antagonists act mainly by competing with the agonist for the histamine receptor site but have in addition a distinct affinity for a secondary site on the receptor. Occupation of this site by the antagonist prevents building up of the stimulus elicited by the agonist and thus decreases the Emax. In systems with receptor reserve (histamine) the effect of antagonist binding to the secondary binding site is seen only at high concentration of antagonist while in absence of receptor reserve (dimaprit) the depression of Emax is directly visible. Simulations of the model show that the affinity of this secondary binding site is 50-(famotidine) to 100-(cimetidine and ranitidine) fold lower than for the agonist binding site. PMID- 1704684 TI - Isolation and immunological analysis of Trypanosoma cruzi glycolipids. AB - A water-soluble glycolipidic fraction from Trypanosoma cruzi was isolated using a mixture of hexane and isopropanol. Analysis by SDS-polyacrylamide gel electrophoresis, after staining by silver and Sudan Black B, showed that the fraction contained one band with a relatively high mobility. Its reactivity and specificity with human chagasic sera and T. cruzi infected mouse sera or with sera from patients with several other pathologies was determined by an immunoradiometric assay. The glycolipid-based radioimmunoassay for the detection of T. cruzi antigens provided a sensitive measure of its activity. However, cross reactivity with several sera from patients with visceral and cutaneous leishmaniasis was detected. PMID- 1704685 TI - No safe place: parental alcoholism and adolescent suicide. AB - A child growing up in an alcoholic home develops either little self-consolidation (I-ness) and efficacy (I can) or a distorted self (I am insignificant). This results in a desperate search for a soothing-object (We-ness). The sadomasochistic behaviors, which a youth witnesses and is subjected to, become internalized as survival skills, but ultimately fail. These factors set the stage for a destructive modus operandi. When there is peer group attachment pressure, this teen does not find security when questioning, "Who am I?" because there is no "I" and no "We". Instead, this adolescent experiences fear, anxiety, and range, and wonders, "What's going to happen to me?" This propels the youth into frantic behaviors that are meant to confirm a sense of "We-ness" and competence. The result, however, is greater frustration and a mirroring of the opposite. Also, since there is a diminutive capacity for trust and an exiguous chance to reach out or respond to significant others, external soothingness becomes unobtainable. When the adolescent is confronted with aloneness, helplessness, and hopelessness, desperation results and a search for a safe place ensues. Suicide holds such an illusion. It is the embodiment of sadomasochism and permits the cognition "I am capable." A case study illustrates the problems. PMID- 1704686 TI - Differences in immunoglobulin preparations for intravenous use: a comparison of six products. AB - The use of intravenous gamma globulin products in both children and adults has increased markedly over the past 5 years, since these agents were licensed in the United States. Product competition has become fierce, with each supplier touting the merits of its own product. A summary of the six products currently available in the United States is presented. Comparisons are made according to method of product manufacture, product specifications, antibody titers, opsonization data, IgG subclass quantification, ease of administration, and side effects. PMID- 1704687 TI - Neonatal uses of intravenous immunoglobulin. AB - Intravenous gamma-globulin (IVGG) has several potential uses in neonates. A considerable amount of study is going into the evaluation of its role in neonatal sepsis. Preliminary results from two large controlled trials suggest that there may be a reduction in nosocomial sepsis following infusion of intravenous immunoglobulin (IVIG) in small premature infants, but the data are still incomplete. It is important to distinguish the two types of neonatal immune thrombocytopenia. In maternal autoimmune disease, neonatal intracranial hemorrhage has an incidence of only 1-2%, and treatment can be initiated postnatally in the thrombocytopenic neonate. Since antenatal hemorrhage is very rare, treatment of the mother for the sake of the fetus seems unnecessary. In alloimmune thrombocytopenia, on the other hand, intracranial hemorrhages occur in approximately 20% of all identified cases, and as many as one-half of these occur antenatally. Pilot studies of maternally administered IVGG have indicated that it may elevate the fetal platelet count and prevent intracranial hemorrhage. PMID- 1704688 TI - Direct quantitation of biotin-labeled nucleotide analogs in RNA transcripts. AB - We have developed a method to determine directly the number of biotinylated (Bio) nucleotide analogs incorporated into RNA transcripts. Transcripts synthesized in vitro in the presence of [alpha 32-P]CTP and varying concentrations of Bio-4-UTP were subjected to alkaline hydrolysis and the resulting 2' and 3' nucleoside monophosphates separated by reverse-phase HPLC. The amount of 32P transferred to each monophosphate was indicative of the frequency of their incorporation into the transcript. Transcripts synthesized in the presence of equimolar concentrations of Bio-4-UTP and UTP resulted in 70 out of the 125 possible UTP sites occupied by Bio-4-UMP. This study agrees with kinetic data in suggesting that T7 RNA polymerase does not significantly discriminate between the natural and the biotinylated nucleotide. Therefore, the number of biotinylated residues that are incorporated into a transcript can be controlled by varying the ratio of Bio-4-UTP to UTP in the transcription reaction. We have shown that as few as 10 Bio-4-UMP residues per 486 nucleotide transcript still results in greater than 90% binding efficiency on a streptavidin/biotin-cellulose affinity column. PMID- 1704689 TI - Elastin in human, baboon, and mouse liver: an immunohistochemical and immunoelectron microscopic study. AB - Light microscope histochemistry and immunohistochemistry, and routine electron microscopy techniques were performed to analyse elastin distribution and structure in the human liver compared with that in baboon and mouse. In man and baboon, elastic fibers stained by iron hematoxylin or orcinolnew fuchsin seemed to be solitary and were few in number; in the mouse they were thinner but abundant, both in the portal tract and in hepatic veins. Orcein or resorcin fuchsin stains, employed after oxidation of tissue sections, revealed a network comprising elastic, elaunin, and oxytalan fibers, which was also demonstrated by immunofluorescence with anti-elastin antibody in man and baboon. At the ultrastructural level, the elastic fibers of the human portal tract corresponded to discontinuous patches of amorphous material intermingled with few microfibrils. These contrasted with the thinner elastic fibers of baboon and mouse liver which had a core of amorphous material. In man and baboon, these fibers meshed into slender bundles of microfibrils often exhibiting small spots of amorphous material (elaunin fibers) and terminated as isolated microfibrils (oxytalan fibers). Immunoelectron microscopy of elastin carried out on baboon liver tissue labelled the amorphous material and also its microfibrillar component. Immunoperoxidase deposits were also associated with isolated bundles of microfibrils in the baboon portal stroma. Immunolabelling and elastic stains disclosed an important elastin portal network located around vascular, biliary structures and interspaced with collagen bundles. The structural polymorphism of elastin, assembling different relative amounts of amorphous material and microfibrils, might have a relationship with the required elasticity in a given species. PMID- 1704690 TI - Multiple-dose evaluation of intravenous hydromorphone pharmacokinetics in normal human subjects. AB - We measured the pharmacokinetics of hydromorphone in normal volunteers given three doses of the drug (10, 20, and 40 micrograms/kg) as intravenous 45-s injections on different days. Concentrations of hydromorphone in plasma from serial blood samples were measured by a high-performance liquid chromatography method specific for hydromorphone with a detection limit of 0.1 ng/mL. In all cases, plasma hydromorphone concentration versus time data for individual subjects were best described by a triexponential (instead of mono- or biexponential) function. Furthermore, we found that the pharmacokinetics of hydromorphone was independent of dose across the range studied. Averaged across doses, the distribution and terminal elimination half-lives were 1.27 min (t1/2 pi), 14.7 min (t1/2 alpha), and 184 min (t1/2 beta), respectively. Average values for systemic clearance, initial dilution volume, and steady-state volume of distribution were 1.66 L/min (Cl), 24.4 L (Vc), and 295 L (Vdss). Our results indicate that hydromorphone pharmacokinetic parameters are linear across a fourfold range of doses that are usually employed clinically and that previously reported pharmacokinetic values for hydromorphone (based on radioimmunoassay measurements) deserve reconsideration. PMID- 1704691 TI - Substance p-induced cutaneous and bronchial reactions in children with bronchial asthma. AB - We studied substance P-induced cutaneous and bronchial reactions in children with allergic disorders. Furthermore, inhibitory effects of lidocaine on these responses elicited by substance P were also studied. In the intradermal test, children with moderate or severe bronchial asthma showed significantly stronger reactions than controls. In bronchial challenge with substance P, children with bronchial asthma showed an increasing fall in FEV1 and V50 in proportion to the severity of their asthma. Concomitant use of lidocaine significantly inhibited not only substance P-induced intradermal erythema and wheal reactions but air flow limitations. It also inhibited histamine-induced erythema and house dust induced wheal and erythema reactions. No adverse reactions to lidocaine were observed. These results suggest that substance P might play an important role in cutaneous and bronchial hypersensitivity reactions in humans and that lidocaine has an inhibitory effect on substance P-induced reactions. PMID- 1704692 TI - [Alpha fetoprotein and type I tyrosinemia]. PMID- 1704693 TI - Isolation and characterization of a human monoclonal antibody which reacts with breast and colorectal carcinoma. AB - A human monoclonal antibody with specificity for breast and colorectal carcinoma has been isolated after hybridoma formation between regional lymph node lymphocytes from a patient with breast carcinoma and a mouse x human heteromyeloma (SPAZ-4). The monoclonal, MAB 15.2.3, is an IgM, kappa, which binds to an epitope found on at least four glycoproteins with approximate molecular weights of 37, 39, 43 and 56 kilodaltons (kD). These antigens are expressed intracellularly and on the surface of breast and colorectal carcinoma cells and their level of expression is increased by treatment with recombinant human leukocyte (IFN-alpha A) or immune (IFN-gamma) interferon. Analysis of formalin fixed tumor cell lines indicates specificity for carcinomas. Similarly, immunostaining of paraffin-embedded tissue indicates both cell membrane and intracytoplasmic reactivity with breast (8 of 12), colorectal (3 of 4) and prostate (1 of 3) carcinomas. By employing a series of enzymes which specifically cleave sugar residues on proteins, it was demonstrated that the binding of MAB 15.2.3 could be increased. These observations suggest that the epitope recognized by MAB 15.2.3. is protein in nature and expressed on a series of glycoproteins. Pretreatment of Colo 205 (human colorectal carcinoma) cells with MAB 15.2.3 prior to implantation into nude mice, results in a reduction in the size and weight of tumors. With appropriate genetic engineering, resulting in the conversion of this antibody to an IgG, MAB 15.2.3. could prove of value for the diagnosis and ultimately the therapy of human breast and colorectal carcinomas. PMID- 1704694 TI - Two-wavelength mercury arc lamp excitation for flow cytometric DNA-protein analyses. AB - Correlated DNA- and protein-determinations were performed using flow cytometry and DAPI-sulforhodamine 101 (SR101) staining. Two-wavelength UV-green excitation using a single HBO lamp was found to be superior to single wavelength UV excitation, since it resulted in a higher specificity in the SR101 measurements. The effects of overlapping in the emission spectra and of energy transfer from DAPI to SR101 were minimised. Different staining conditions and the specificity of SR101 staining were examined. No binding of SR101 to DNA and RNA could be detected. Good reproducibility was achieved in the protein measurements using human lymphocytes as an internal reference. Examples are given of the analysis of mixed cell populations with different cellular protein contents and different growth and metabolic activities. Correlated measurements of DNA and protein appear to be useful for further analysis of tumor cell populations. PMID- 1704695 TI - Effect of lysosomotropic and membrane active substances on adriamycin uptake and histamine release. AB - It has recently been shown that adriamycin is accumulated in mast cells by an active transport system, which closely resembles the outward transport system of multidrug resistant (MDR) cells. The present study was undertaken in order to test the effect of substances which are known to limit or reverse resistance in MDR cells on adriamycin uptake and histamine release in rat peritoneal mast cells. The lysosomotropic amines chloroquine, nicotine, propranolol, atropine, methylamine, ammonium chloride and quinacrine were only slightly effective at very high concentrations; no effect could be observed with the lysosomotropic amine amantadine. The carboxylic ionophores monensin and nigericin were, on the contrary, extremely efficacious; in particular, the effect of monensin was more evident when mast cells were preincubated with the ionophore for 10 or 30 minutes while only a slight inhibition was evident when the two substances were added simultaneously. Removal of monensin from the incubation medium before challenge with adriamycin did not abolish the inhibitory action of the ionophore. Among the tested membrane active agents, Tween 80 and Triton WR-1339 were able to limit adriamycin uptake and histamine release. The microscopical examination showed that in mast cells treated with adriamycin, an intense fluorescence was present in cytoplasmic granules; on the contrary, mast cells preincubated with monensin showed hardly any fluorescence. PMID- 1704696 TI - Charting OR use. A practical graph to visualize use. PMID- 1704697 TI - Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir. AB - Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3 (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment. PMID- 1704699 TI - [Effect of non-steroidal anti-inflammatory agents on interferon induction]. AB - Nonsteroid antiinflammatory agents (NAIA) such as antipyrine, butadion, acetylsalicylic acid, sodium salicylate, stampyrine and 4-iodantipyrine are not interferonogenic. Still, they stimulated interferonogenic action of poly(G).poly(C) in studies on animals. Relation between the interferon stimulating action of the NAIA and their effect on activity of prostaglandin and the influence on the immune system was suggested. PMID- 1704698 TI - C reactive protein and immunoglobulin G in synovial fluid and serum in joint disease. AB - C reactive protein (CRP) and immunoglobulin G (IgG) were measured in synovial fluid and serum of 72 patients (29 with rheumatoid arthritis (RA), 17 with osteoarthritis, 11 with crystal synovitis, seven with undifferentiated arthritis, and eight with seronegative arthritis). The synovial fluid:serum (SF:S) ratios were compared with those calculated from the SF:S ratios of transferrin, caeruloplasmin, and alpha 2 macroglobulin, using the binomial test within groups and the Mann-Whitney test between groups. In RA synovial fluid CRP concentrations were lower than expected and IgG concentrations higher than expected. In osteoarthritis CRP concentrations were higher than expected. In seronegative arthritis IgG concentrations were raised. The ratio of CRP:IgG was depressed in RA. These findings are consistent with a role for CRP in the inflammatory process of RA, while the CRP:IgG ratio may be of value in the differential diagnosis of joint disease. PMID- 1704700 TI - [Inhibition of Chlamydia trachomatis multiplication by interferon inducer larifan in mice with experimental infection]. AB - Interferon (IF)-inducing capacity of C. trachomatis was shown in experiments on mice CBA. The levels of IF production in the parenchymatous organs correlated with accumulation of the pathogen in them. The use of larifan, a natural double stranded IF inductor, according to the treatment scheme provided high levels of endogenic IF in infected mice. It inhibited multiplication of Chlamydiae in the lungs and lymph nodes detected cytoscopically by light and fluorescence microscopy and with infection titration. The effect of the inductor combined with tetracycline was of additive nature. Double intraperitoneal administration of larifan with an account of the hyporeactivity phase and daily administration of tetracycline proved to be the most efficient. It is possible to successfully use IF inductors in accordance with the treatment schemes in infection caused by C. trachomatis which makes promising their clinical application in therapy of chlamydiosis. PMID- 1704701 TI - [A carcinoid tumor in a mature thymic teratoma]. AB - A description of a carcinoid in the mature thymic teratoma in a 43-year-old woman. Teratoma was represented by the skin with appendages, intestinal and respiratory epithelium, salivary gland, fat tissue, islands of cartilage and ependyma. A carcinoid of alveolo-trabecular type consisting of cells with argyrophilic and argentaffinic properties was found in the area of submucous glands of a respiratory tube. Serotonin and neurotensin were found by means of PAP method in carcinoid cells. PMID- 1704702 TI - Tracheobronchial lasers, brachytherapy and stents. PMID- 1704703 TI - [Quantitative determination of the carbohydrate determinants of O-antigens of Yersinia pseudotuberculosis and Pseudomonas fluorescens]. AB - Interactions of O-specific polysaccharides, LPS and LPS-protein complex with specific antibodies isolated on a polysaccharide-substituted immunoadsorbent gel and monovalent Fab-fragments of the antibodies were investigated. The association constants and quantity of Fab-fragments bound to these antigens were established. The interaction of the LPS and LPS-protein complexes was shown to depend considerably on concentration of antigens, and only 10-15% of carbohydrate determinants are accessible for binding with antibodies. PMID- 1704704 TI - Expression of class II major histocompatibility complex antigen on mouse sperm and its roles in fertilization. AB - The expression of class II major histocompatibility complex (MHC) antigen on the membrane of mouse sperm head was clearly demonstrated by indirect immunofluorescence (IIF) test, enzyme immunoassay (EIA), and mixed lymphocyte sperm reaction (MLSR) as well as the examination of its expression at transcriptional levels by northern dot blotting. Furthermore, the roles of class II MHC antigen of sperm in fertilization were investigated with an in vitro fertilization (IVF) system. The successful ratio of IVF was significantly decreased by treatment of sperm with anticross-reactive region of class II MHC antigen monoclonal antibody (MAb) but not with antiprivate region MAb. These results were not due to disturbances of sperm mobility by these MAbs. It was strongly suggested that the monomorphic or its related region of class II MHC antigen on sperm played an important role in the recognition between sperm and egg in fertilization. PMID- 1704705 TI - T lymphocyte activation: a biological model of signal transduction. PMID- 1704706 TI - Silver staining of DNA restriction fragments for the ultrarapid identification of pseudorabies virus strains. AB - Restriction endonuclease analysis of pseudorabies virus DNA has been used to study various virus strains. To make use of a rapid technique for the identification of viral strains, studies have been undertaken to facilitate purification of the DNA from viral particles present in cytoplasmic fractions. The ultrasensitive photochemical silver staining of nucleic acid, described by Beidler et al. (Analytic Biochemistry 1982: 126) has been adapted and applied to the detection of pseudorabies virus restriction fragments. In a period of 5 h more than 1 microgram of DNA can be extracted from a 25 cm2 plastic flask containing infected cells and purified without ultra centrifugation. Low molecular weight DNA was separated from high molecular weight DNA by polyacrylamide gel electrophoresis. The restriction fragments were selectively visualized by silver staining which can detect 0.025 micrograms of total pseudorabies virus DNA. The electrophoretic DNA pattern of vaccine and wild strains has been studied using these techniques and the results agree with previously published data. PMID- 1704707 TI - A short history of nuclear activation analysis. AB - The major developments in the field of nuclear activation analysis, from 1936 to 1989, are discussed. The developments are grouped into five consecutive time periods. The impact of various scientists on the development of the field in the first 35 years is also discussed. PMID- 1704708 TI - Proceedings of the International Conference on Nuclear Analytical Methods in the Life Sciences. Gaithersburg, Maryland, 1989. PMID- 1704709 TI - Analysis of biological samples using prompt gamma radiations induced by 14.7-MeV neutrons. AB - We investigate a method for determining the elemental composition of biological samples that uses prompt gamma rays induced by 14.7-MeV neutrons. Alpha particles are produced simultaneously with the neutrons, which exit opposite the alpha detector through the vacuum chamber wall. The sample under investigation is irradiated and emits gamma radiations in a spectral energy distribution characteristic of the material. Barium-fluoride (BaF2) and high-purity germanium (HPGe) gamma detectors view the sample and record the spectrum of gamma radiation. PMID- 1704710 TI - A new technique for the study of erythrocyte survival by double labeling and use of a well-type Ge detector. AB - A double label procedure with 57Co and 58Co has been developed for detailed in vivo studies of erythrocyte survival. A well-type Ge detector is used in the measurements. The activities necessary for these experiments are very low, and the associated dose received by the test persons can be neglected. PMID- 1704711 TI - Charged-particle activation analysis of biological material. AB - Charged-particle activation analysis offers a number of interesting possibilities for the determination of trace elements in biological material. It allows the determination of those elements that are difficult or impossible to determine by neutron activation, such as Li, B, Al, Si, V, Cr, Ni, Cd, Sn, Tl, and Pb. Up to now, protons have been successfully applied to samples of both vegetal and human origin. A number of difficulties have to be overcome, one of which is excessive heating of the samples owing to the limited range of the charged particles, thus giving rise to a high energy deposition in a small volume. Moreover, the sample composition has to be known to allow the calculation of the range of the particles. An interesting alternative has been proposed using an internal standard together with a standard additions procedure. Proton activation analysis was tested on a wide variety of reference materials, giving evidence that accurate results can be obtained for many trace elements, even when applying a purely instrumental method. Thus, the method can also be applied in the certification of reference materials, since nuclear methods are independent of chemical properties of the sample. PMID- 1704712 TI - An X-ray microprobe facility using synchrotron radiation. AB - An X-ray microprobe for trace elemental analysis at micrometer spatial resolutions, using synchrotron radiation (SR), is under development. The facility consists of two beamlines, one including a 1:1 focusing mirror and the other an 8:1 ellipsoidal mirror. At present, "white light" is used for excitation of the characteristic X-ray fluorescence lines. Sensitivities in thin biological samples are in the range of 2-20 fg in 100 microns2 areas in 5 min irradiation times. Scanning techniques, as well as microtomography and chemical speciation, are discussed. Application to a specific biomedical study is included. PMID- 1704713 TI - Radioisotope induced X-ray emission (RIXE) studies in life sciences. AB - The inherent advantages of radioisotope induced X-ray emission (RIXE) are briefly presented, with emphasis on their applications to analysis in the life sciences. The following selected applications by RIXE are discussed in general: (1) In vivo analysis: the determination of stable iodine in the thyroid and heavy metals in human bone; (2) the analysis of small tissue samples (about 10 mg) for the simultaneous determination of trace elements between 25 less than or equal to Z less than or equal to 40; data are given from a time study of the disease progress of Lewis lung tumor on a rat's liver over a 24-d period; and (3) a homogeneity study of iron, strontium, and zirconium in IAEA's SL-3 Lake sediment standard reference material is presented. PMID- 1704714 TI - Nuclear analytical methods for trace element studies in calcified tissues. AB - Various nuclear analytical methods have been developed and applied to determine the elemental composition of calcified tissues (teeth and bones). Fluorine was determined by prompt gamma activation analysis through the 19F(p, alpha gamma) 16O reaction. Carbon was measured by activation analysis with He-3 ions, and the technique of Proton-Induced X-ray Emission (PIXE) was applied to simultaneously determine Ca, P, and trace elements in well-documented teeth. Dental hard tissues: enamel, dentine, cementum, and their junctions, as well as different parts of the same tissue, were examined separately. Furthermore, using a Proton Microprobe, we measured the surface distribution of F and other elements on and around carious lesions on the enamel. The depth profiles of F, and other elements, were also measured right up to the amelodentin junction. PMID- 1704715 TI - Fluorine concentrations in bone biopsy samples determined by proton-induced gamma ray emission and cyclic neutron activation. AB - Fluorine concentrations in bone biopsy samples taken from the iliac crest of subjects, divided into four groups depending on the length of dialysis treatment, and aluminium levels in blood and bone pathology, in terms of osteoporosis, were determined by two instrumental methods. Proton-induced gamma-ray emission (PIGE), making use of the resonance reaction of 19F(p, alpha gamma)16O at 872 keV, and cyclic neutron activation analysis (CNAA), using the 19F(n, gamma)20F reaction in a reactor irradiation facility, were employed. Rutherford backscattering (RBS) was used to calculate the volume, and, hence, mass of the sample excited in PIGE by determining the major element composition of the samples in order to express results in terms of concentration. From this preliminary investigation, a relationship is suggested between fluorine concentrations in bone and aluminium levels in the system. PMID- 1704716 TI - Study of physiopathological phenomena in dental enamel by neutron activation analysis. AB - An epithermal neutron activation method is used to determine the concentration of mineral elements in human dental enamel. A large number (252) of samples from ancient and modern origins are analyzed. The analytical results are mathematically processed using a statistical multivariant method. This allows to differentiate deciduous from permanent teeth and decayed from sound enamel. It is also possible to distinguish the teeth coming from two different necropoles. The origin and the localization of determined elements in the mineralized part, or in the aqueous-organic part, of enamel is suggested. Their role, as witnessed in the physiopathological phenomena of dental enamel, is discussed. PMID- 1704717 TI - Neutron activation analysis of biological samples with a preirradiation chemical separation. AB - A preirradiation separation procedure has been developed to separate Al, Cu, Mn, and V from biological materials. Chelex-100 resin is used as the separation medium, and the resin is irradiated directly. Three NIST biological Standard Reference Materials and five samples of human blood serum, obtained under carefully controlled conditions, have been analyzed by NAA following this separation. PMID- 1704718 TI - Trace elemental content of biological materials. A comparison of NAA and ICP-MS analysis. AB - The advantages and disadvantages of neutron activation analysis (NAA) and inductively coupled plasma-source mass spectrometry (ICP-MS) for the analysis of biological materials is reviewed. Comparison is made between NAA (instrumental) and ICP-MS (conventional pneumatic solution nebulization and laser ablation) analysis of the biological reference material National Bureau of Standards (NBS) SRM 1577 Bovine Liver. Relatively good agreement is achieved between the results for the 18 elements analyzed by both techniques and those either certified or reported in the literature. Elemental concentrations for Li, Mg, Al, Ca, Cr, Mn, Fe, Cu, Zn, Br, Rb, and Cs are also reported for IAEA Mixed Human Diet (H9), NBS SRM 909 Human Serum, and NBS SRM 1577a Bovine Liver, analyzed by solution nebulization ICP-MS. PMID- 1704719 TI - Instrumental neutron activation analysis of geological and biological reference materials using the ko-standardization method. AB - The ko-standardization method used in instrumental neutron activation analysis (INAA) was applied to the determination of some elements in geological and biological reference materials. We analyzed NBS SRM 1572 Citrus Leaves and SRM 1645 River Sediment and the CRM materials, IAEA Soil-7, SL-1, and MA-A-2. Comparison is made with reference values whenever available. Good agreement is found. The potential of the ko-standardization method in reactor INAA is discussed. PMID- 1704720 TI - Soluble metals in the atmosphere and their biological implications. A study to identify important aerosol components by statistical analysis of PIXE data. AB - Multivariate statistical analysis has been applied to time series measurements of aerosol elemental composition from PIXE analysis of filter samples, and principal components have been resolved that represent distinct particle types in an external mixture in the atmosphere. In this study, it is argued that a combination of chemical and statistical analyses of the data may be more powerful in determining chemical species in atmospheric aerosols than studied that employ mainly direct chemical analysis of chemical species in unresolved mixtures of aerosol particle samples. Sulfur is generally associated with mineral dust elements. It is reasoned that the association may represent sulfuric acid coatings on particles that can lead to mineral dissolution and solubilization of significant amounts of aluminum, iron, and other metals. Upon wet or dry deposition to the surface, the fluxes of these metals in biologically-available form may be sufficient to affect primary productivity in the world ocean and cause ecological damage in lakes. As a consequence, the fluxes of biogenic trace gases to the atmosphere may be changed, possibly leading to changes in the tropospheric concentration of ozone. The inputs to lakes of soluble aluminum, which is toxic to fish, may be partly by deposition directly from the atmosphere, thus not limited to leaching of soils by acid deposition. Human inhalation of soluble aluminum and other solubilized mineral metals may account, in part, for the observed geographic pattern of deaths attributed to chronic obstructive pulmonary disease (COPD) that show high rates in cities of the Western US and the southeast region, but low in most of the midwest and northeast. PMID- 1704721 TI - Major and trace elements in spruce needles by NAA. AB - Concentrations of 23 elements in needles of Norway spruce (P. abies) have been determined at 47 sites. It is shown that a thorough removal of the aerosols sitting on the needles surface is necessary in order to get the inherent needle concentrations. Neutron activation was used to determine concentrations from 10( 9) to 10(-2) g/g. Irradiation and counting conditions are given. The essential elements, Ca, Cl, Cu, Fe, K, Mg, Mn, P, Zn, and the nonessential elements, Al, As, Ba, Br, Co, Cs, Hg, La, Na, Rb, Sb, Sc, Sr, and V, could be determined. The concentrations of most elements are about a factor of 6 smaller than the mean concentrations in land plants. Analytical reproducibility was much better than the variation among individual trees, and the variation within sites is smaller than among sites. In general, essential elements have smaller variations than nonessential elements. For some elements, variations between sites are owing to differences in the soil pH or the emission situation. PMID- 1704722 TI - Transfer of toxic elements from the atmosphere to cattle and game animals. AB - Neutron activation analysis was used for determining trace element content in hair of herbivorous animals. Pollutant deposition from the air onto foliage must be considered as the main cause of contamination of the animals. PMID- 1704723 TI - Determination of trace elements in aerosol samples by instrumental neutron activation analysis. AB - Two nuclear techniques, Energy-Dispersive X-Ray Fluorescence Analysis (EDXRF) and Instrumental Neutron Activation Analysis (INAA), were used to analyze aerosol samples collected in the city of Sao Paulo, Brazil. Na, Cl, Mn, V, Al, Sm, Mo, W, La, As, Br, Sb, K, Ba, Se, Th, Cr, Rb, Ca, Fe, Ce, and Sc were determined by INAA, and Al, Si, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Ga, As, Se, Br, Rb, Sr, Hg, and Pb were determined by EDXRF. A preliminary identification of the main source of the atmospheric aerosol was performed based on enrichment factor and correlation coefficient calculations. PMID- 1704724 TI - Production of 134Cs thermo-generated aerosols. Study of their behavior after deposition on spruce trees. AB - This paper presents observations made on the behavior of radioactive cesium in laboratory conditions. 134Cs-loaded aerosols are generated from UO2 pellets in an electric oven and allowed to deposit on small spruce trees. The transfer of radioactivity to the plants has been studied. In another series of experiments, trees are submitted to artificial rain contaminated by the radioactive aerosols. The two modes of contamination are compared. PMID- 1704725 TI - Whole body content and turnover of Cs and K. AB - In persons who had accumulated radiocesium from the fallout of the Chernobyl nuclear reactor accident, the amount of radiocesium and radiopotassium was measured in the total body and in urine. The stable Cs content in the urine was determined at the same time by instrumental nuclear activation analysis. From the data obtained, the total body Cs pool was calculated to be 1500 micrograms, that of K 110 g. The Cs turnover rate was found to be 0.64%/d for men and 0.81 for women; the K turnover 2.4 and 2.7, respectively. The Cs:K ratio in the total body was 4 times higher than that in urine, demonstrating that, in relation to the intake, the body is able to accumulate more Cs than K. PMID- 1704726 TI - Studies of cadmium uptake in bone and its environmental distribution. AB - Rat experiments indicate that oral ingestion of cadmium through drinking water leads to an accumulation of cadmium in bone, in addition to liver and kidney. After five weeks of cadmium intake in drinking water (50 to 100 mg/L), the bone cadmium levels increased in proportion to the intake concentration. Bone and kidney histology showed no signs of bone or kidney damage up to 5 wk of cadmium ingestion. Cadmium accumulation in bone was a primary phenomenon and not secondary to renal failure. In addition, cadmium levels have been estimated in a variety of sources, e.g., foodstuff, fertilizer, and sewage sludge, using neutron and proton activation analyses and atomic absorption spectrophotometry. Cadmium levels of Canadian foods are in the range of 0.002-0.07 mg/kg, and soils are in the range of 0.55 to 1.72 mg/kg. Fertilizers contain cadmium from 0.3 to 1.25 mg/kg, whereas sewage sludge contains up to 122 mg/kg. PMID- 1704727 TI - Elemental concentrations in hair of inhabitants of a cadmium-polluted area. AB - To investigate the present situation of inhabitants living in the cadmium polluted area of Toyama Prefecture, 95 hair samples were analyzed instrumentally by the neutron activation method. The concentrations were compared with nonpolluted samples. The concentrations of Cd, Cu, Zn, and Cu/Zn in hair were compared with those in some reported organs. The values of Cu/Zn were significantly lower (p less than 0.001) in hair of both female groups, but only somewhat lower (p less than 0.05) in hair of the male group. Causes of these facts are discussed. Results obtained from principal component analysis are also mentioned. PMID- 1704728 TI - Determination of manganese in airborne particulates in a dry-cell battery factory using X-ray fluorescence technique. AB - Feasibility of using X-ray fluorescence techniques for the determination of airborne manganese in dry-cell battery factories was investigated. Both Energy Dispersive (EDX) and Wavelength Dispersive (WDX) X-ray fluorescence techniques of analysis were used. The minimum detectable quantity of Mn by the EDX method was found to be 50 micrograms for membrane filter (AA type) and 100 micrograms for cellulose filter (pore size 2.5 microns), respectively, with a counting time of 2000 s. The most suitable exciting source was 1.11 GBq (30 mCi) Pu-238. The minimum detectable quantity of Mn by the WDX method was 10 micrograms, with a counting time of 100 s. Results of the X-ray fluorescence and the AA methods were found to be in good agreement. Field measurements were carried out in a dry-cell battery factory. Concentrations of airborne manganese in the factory were found to be 0.02-41.1 mg/m3. PMID- 1704729 TI - Nondestructive determination of arsenic in urine by epithermal neutron activation analysis and Compton suppression. AB - Epithermal neutron activation analysis, in conjunction with Compton suppression, has been employed to determine arsenic levels in artificially doped urine samples. Typical detection limits were of the order of 10 ng/g. Replicate determinations gave precision values between 2 and 12%, whereas accuracy measurements were between +/- 1 and +/- 20%. Biological and geological reference materials from the National Institute of Standards and Technology (NIST) were also analyzed for arsenic content. Typically, the precision achieved again was between 2 and 12%, whereas the accuracy measurements were in excellent agreement with the certified values. PMID- 1704730 TI - Environmental impact of the gold mining industry in Ghana. AB - X-ray Fluorescence (XRF) Analysis and Atomic Absorption Spectrophotometry (AAS) have been used in assessing heavy metal pollution from some gold mines in Ghana. The presence and levels of heavy metals in gold ore, gold tailings, inland waters, and river sediments have been determined. Using these techniques, the heavy metals: Cr, Mn, Fe, Cu, Zn, As, Pb, Rb, Sr, Y, Zr, and Nb were identified in some of the solid samples within a concentration range of 0.08 ppm--4.9%. However, the inland waters showed the presence of only Fe and Zn at levels of 0.08-2.4 micrograms/mL. PMID- 1704731 TI - Trace element determination in scalp hair of people working at a copper smelter. AB - Instrumental neutron activation analysis was performed to assess the exposure degree of a worker group from a copper smelter. The results were compared with data of inhabitants of Kinshasa and Bandaka in Zaire. Excessive concentrations of Mg, Ca, V, Co, Cu, As, and Cd were observed in the investigated group. The distribution patterns of trace elements are shown. The plots of Cu concentration relations to selected elements, i.e., As, Co, and Zn, showed a plateau at about 300 ppm of Cu. PMID- 1704732 TI - Air quality status in Kinshasa as determined by instrumental neutron activation analysis, atomic absorption spectrometry, and ion-exchange chromatography. AB - Three independent analytical techniques: instrumental neutron activation analysis, atomic absorption spectrometry, and ion-exchange chromatography were applied to airborne particulates collected on filters, and to atmospheric acid gases collected in carbonate buffer solutions. Twenty trace elements and seven acid gases and acid aerosols were determined. Results were compared with those observed elsewhere and showed that air pollution is low in Kinshasa and should not cause anxiety. The main known sources of pollutants are vehicle exhaust and aeolian processes on stripped soils. PMID- 1704733 TI - Mapping technique based on elemental hair composition data. AB - The content of 24 elements has been determined in 2000 hair samples of the inhabitants of Uzbekistan. INAA was used for analysis. Reference material IAEA HH 1 and laboratory standards were used. Measurements were done using a Ge(Li) detector and a multi-channel analyzer. No correlation was found between the element content and hair color and ethnic group, whereas the content of some elements depended on sex, hemoglobin content, blood group, and occupation. Arithmetical and geometrical means and medians were calculated. Cumulative histograms show mainly lognormal distributions. Maps of selected regions, based on the elemental hair composition data, were made. The element content was shown to depend on the location of large cities and biogeochemical anomalies. The relationship between the death rate and the element content in hair in some countries has been shown. PMID- 1704734 TI - Environmental specimen banking. A complement to environmental monitoring. AB - The risk of environmental damage from the countless chemicals and chemical combinations is estimated by monitoring the air, water, soil, and biota and comparing the findings to known risks. Environmental monitoring involves the collection and chemical analysis of selected samples to determine pollutant trends, forecast damage to the environment, and develop control strategies. However, past experience has taught us that there is always a level of uncertainty associated with the chemical analysis of complex environmental samples. This uncertainty arises from insufficient analytical sensitivity to detect trace levels of damaging pollutants, unsuspected analytical inaccuracies associated with unknown interferences, and limited resolution to identify and quantify all pollutants of interest. Environmental specimen banking can serve as a complement to environmental monitoring by allowing future chemists to analyze preserved samples retrospectively with emerging analytical methodologies. In other words, specimen banking will give later environmentalists the opportunity to characterize chemicals whose hazards were not recognized or for which sufficiently accurate analytical methods were not available. This will enable us to determine whether a newly recognized pollutant is truly a new environmental threat or whether it has always been with us. Although environmental specimen banking is still in its infancy, we now have many experiences that clearly demonstrate that valuable information can be produced. In all environmental monitoring programs, some consideration should be given to preserving selected samples. Ideally, specimen banking will involve; (1) a stable funding commitment, (2) a cryogenic storage facility, (3) development of selection criteria to collect the most valuable environmental specimens, and (4) a continuing research program to develop optimal procedures for collecting, handling, and preserving specimens. PMID- 1704735 TI - Alaskan marine mammal tissue archival project. AB - A project to establish an archive of Alaskan marine mammal tissues was conceived in 1987 to be a part of the National Biomonitoring Specimen Bank (NBSB). Protocols and field collection of marine mammals, long-term storage, and analysis are summarized in this paper. Instrumental neutron activation analysis has been used for an initial evaluation of trace element content in samples of northern fur seal (Callorhinus ursinus) from the Pribilof Islands. The findings agree with previously observed trace element levels in northern fur seals. The archived specimens can be used in future studies when comparison of past and present pollution levels are needed. PMID- 1704736 TI - CADEIA and REJECT. Codes to compute relevant gamma-gamma and gamma-X true coincidence lines in absolute counting of gamma rays with a LEPD. AB - CADEIA and REJECT are computer codes for generating gamma-gamma and gamma-X true coincidence lines for calculation of correction factors for absolute counting of gamma rays in a low energy photon detector. They are written in FORTRAN 77. True coincidence lines are generated by CADEIA; they are submitted to selection by REJECT according to the rejection criteria defined by L. Moens et al. and F. De Corte. Final relevant lines for true-coincidence correction factors are given as output of REJECT. PMID- 1704737 TI - A comprehensive study on the contents and leaching of trace elements from fly-ash originating from Polish hard coal by NAA and AAS methods. AB - In order to assess the environmental risks associated with the emission of fly ash into the atmosphere and its storage on waste heaps, the trace element contents of fly-ashes from burning Polish hard coal were determined by a newly developed INAA method. Leaching of trace elements from the fly-ash by water and H2SO4 solution (pH approximately 2.5) simulating acid rain, respectively, was studied using AAS and spectrophotometric methods. Analogous experiments were done with neutron-irradiated fly-ash, following the composition of the eluate gamma spectrometrically. The new fine fly-ash (CTA-FFA-1) candidate reference material was prepared, and the certification was undertaken on the basis of an international intercomparison run. Preliminary evaluation of results shows that at least 38 elements will be certified and, in addition, the "information values" for at least 12 elements will be given. PMID- 1704738 TI - An environmental research on trace elements in freshwater fish by neutron activation analysis. AB - For a case study of environmental contamination, radiochemical activation analysis has been applied to the crucians collected in the Han River. Sixteen trace elements (Hg, Cd, As, Br, Cu, Na, K, Se, Cr, Hf, Rb, Fe, Zn, Co, La, and Cs) were separated into three groups using distillation and diethyldithiocarbamate extraction methods, and their contents were determined by a single comparator method. Compared with the values 15 years ago, the values for mercury and cadmium have been drastically decreased at the middle and lower part of the river, but no typical change is found in other elements. PMID- 1704739 TI - Identifying sources of groundwater pollution using trace element signatures. AB - A simple receptor modeling approach has been applied to groundwater pollution studies and has shown that marker trace elements can be used effectively in source identification and apportionment. Groundwater and source materials from one coal-fired and five oil-fired power plants, and one coal-tar deposit site have been analyzed by instrumental neutron activation analysis for more than 20 minor and trace elements. In one of the oil-fired power plants, trace element patterns indicated a leak from the hazardous waste surface impoundments owing to the failure of a hypolon liner. Also, the extent and spatial distribution of groundwater contamination have been determined in a coal-tar deposit site. PMID- 1704740 TI - An application of INAA and PIXE on the analysis of nutritional and toxicological elements in samples of drinkable water. AB - Multielemental detection using Instrumental Neutron Activation Analysis (INAA) and Proton-Induced X-Ray Emission (PIXE) of drinkable water of Florence (Italy) is described. The concentrations of 52 elements were measured. No chemical treatment was performed on the samples, before or after irradiation. On the basis of an appropriate combination of the two analytical techniques, a procedure for aluminum determination was developed. Results are briefly discussed from a methodological point of view. PMID- 1704741 TI - Plastics from household waste as a source of heavy metal pollution. An inventory study using INAA as the analytical technique. AB - An inventory study to the levels of cadmium in the plastic component of household waste was carried out utilizing INAA as the analytical technique. In a 2-h irradiation, 2-d decay, and 1-h measurement, protocol adequate sensitivities could be obtained for Cd, but also for a group of other metals: Cr, Co, Ni, Cu, Sr, Zn, As, Se, Mo, Sn, Sb, Ba, and Hg. Red-, orange-, and yellow-colored plastics either contain Cd at high levels (over 1000 mg/kg) or have relatively low Cd concentrations (less than 50 mg/kg). High concentrations were also occasionally found for Sr, Se, Ba, Sb, and Hg. INAA appeared very well to be routinely usable for such analysis because of the absence of a destruction step, adequate sensitivity, high accuracy, and multielement results. PMID- 1704742 TI - Reference man and woman more fully characterized. Variations on the basis of body size, age, sex, and race. AB - Total body neutron activation analysis, prompt-gamma neutron activation analysis, and whole body counting have been used to determine the elemental composition of the human body. The total body elements measured were potassium, nitrogen, calcium, sodium, chlorine, and phosphorus. Total body water was also determined by the dilution principle using tritiated water. Observations were made in an adult US population that totaled 1374 and ranged in age from 20 to 90 yr. The dataset for the white population consisted of 175 males and 1134 females observations; for the black population, it consisted of 30 male and 35 female observations. The variation in the elemental composition of both males and females in any 5-yr age group was large and ranged up to 20% (SD). Age-, race-, sex-, and size-specific differences were evident. When equations were developed that predicted the elemental composition of the adult on the basis of age, weight, and height, the variation in the age groups was reduced approximately in half. Age-specific mean values for the 20- to 29-yr-old white population were also compared with values for the International Commission on Radiological Protection (ICRP)-23 Reference Man. The "average" young adult male was larger than Reference Man; the in vivo data also indicated a larger skeletal mass, more lean tissues and body water, but lower body sodium. When in vivo prediction equations were used to adjust for size differences, good agreement was found between the expected values and those for Reference Man. The ICRP-23 does not contain elemental data for Reference Woman; therefore, the in vivo data in the present study provide the first estimates of body composition for Reference Woman. PMID- 1704743 TI - In vivo neutron activation analysis of organ cadmium burdens. Referent levels in liver and kidney and the impact of smoking. AB - In vivo neutron activation measurements of liver and kidney cadmium have been made in 77 exposed workers and 101 referents. Cadmium levels were higher in exposed workers than in referents; both in liver, 25.7 cf. 0.6 micrograms/g, and kidney, 17.9 cf. 2.7 mg. The 19 referents who never smoked had lower mean organ cadmium burdens than the other referents, the difference achieving statistical significance in the kidney, p less than .01. Cigarette smoking was estimated to increase cadmium body burden by 370 +/- 140 micrograms/pack year. These referent cadmium levels are similar to, although slightly below, previous in vivo and autopsy data. PMID- 1704744 TI - In vivo measurements of cadmium and lead in occupationally-exposed workers and an urban population. AB - This paper reports the preliminary findings of a survey of lead and cadmium body burdens in a nonoccupationally exposed population in Swansea, Wales, using the techniques of in vivo neutron activation and X-ray fluorescence analysis. Some measurements on an occupationally cadmium-exposed group are also included. The results confirm the association between cadmium and smoking and bone lead and age. The in vivo measurements demonstrate a degree of comparability with other data, which supports the further detailed analysis of the relationships between body burden and exposure, on the one hand, and possible health effects on the other. PMID- 1704745 TI - Recent developments in in vivo neutron activation analysis facilities. AB - Two new facilities for in vivo activation analysis of patients have been designed, developed, and constructed at Toronto General Hospital. One of these is for the determination of body calcium for the diagnosis of osteoporosis and other diseases associated with bone loss. The other is for the measurement of total body nitrogen for the determination of protein status. These facilities replace old university facilities and take into account the comfort and management of patients. In addition, in the case of the calcium facility, the precision of the measurements has been improved because of larger detector volume and increased neutron source strength. Both the facilities are now in routine hospital clinical use. PMID- 1704746 TI - A proposed three-phase counting system for the in vivo measurement of the major elements using pulsed 14 MeV neutrons. AB - It is proposed to employ a pulsed source of 14 MeV neutrons for in vivo activation analysis. This would permit the differentiation, with time, of the resulting gamma ray emission into three separate spectra, according to the type of nuclear reaction and mode of decay. The three-phase counting has been divided into approximately 10 microseconds during "beam-on," and 200 and 800 microseconds during "beam-off." Measurements of the major elements, C, N, O, Cl, and P, to give nutritionally-important body compartments of total body fat, protein, water, minerals, and extracellular water, thus is expected with a single scan. PMID- 1704747 TI - Measurements of total body calcium by prompt-gamma neutron activation analysis using a 252Cf source. AB - A clinical neutron activation instrument has been developed for in vivo elemental analysis. Utilizing the prompt-capture gamma ray technique, simultaneous total body (TB) measurements of primarily Ca, but also Cl, N, C, and H are routinely performed. This paper describes a technique for the measurement of TBCa (g) that relies on the use of TBCl as an internal standard. The method has been tested with four anthropomorphic phantoms covering a range of body habitus. The mean discrepancy between the measured and known Ca contents was 3.6%. The technique has been applied to two patient groups, and encouraging results were obtained. PMID- 1704748 TI - Determination of trace elements in standard reference materials by the ko standardization method. AB - The ko-standardization method is suitable for routine multielement determinations by reactor neutron activation analysis (NAA). Investigation of NIST standard reference materials SRM 1571 Orchard Leaves, SRM 1572 Citrus Leaves, and SRM 1573 Tomato Leaves showed the systematic error of 12 certified elements determined to be less than 8%. Thirty-four elements were determined in NIST proposed SRM 1515 Apple Leaves. PMID- 1704749 TI - Combination of neutron activation analysis, tracer techniques, and biochemical methods in the investigation of selenium metabolism. AB - In several studies on rats, the metabolism of selenium was investigated. The quantitative determination of the element was carried out by instrumental neutron activation analysis. For in vivo tracer experiments, 75Se-labeled selenium compounds were used. In addition to these methods, procedures for the measurement of the selenoenzyme glutathione peroxidase, and for the investigation of other selenoproteins, were applied. In this way, information on the specific pools and sites of action of the element, on biologically important selenoproteins and the regulation of the selenium metabolism, was obtained. PMID- 1704750 TI - Speciation of chromium in plasma and liver tissue of endstage renal failure patients on continuous ambulatory peritoneal dialysis. AB - This work is a first attempt to determine the speciation of Cr in human plasma. With the aid of in vitro and in vivo 51Cr-labeled experiments, it was possible to develop the necessary biochemical techniques for the separation of the plasma proteins. Further work will use real samples, taking care to avoid contamination of the various fractions and to preserve the original binding of the Cr to the specific plasma compounds. In a first attempt on the distribution of Cr over the different organelles of liver tissue, work will be restricted to in vivo labeled experiments with rats. The procedure to do the speciation work seems so elaborate that it may be impossible ever to achieve the contemplated speciation of Cr in human liver tissue by subcellular fractionation. PMID- 1704751 TI - A search for longitudinal variations in trace element levels in nails of Alzheimer's disease patients. AB - Levels of Br, Hg, K, and Zn were determined by INAA in the nails of Alzheimer's disease (AD) patients at 6-mo intervals for up to 3 yr. These elements have been shown to be imbalanced in AD nail. Bromine showed no trends. Mercury tended to decrease in nail with increasing age of patient, and with the duration and severity of the dementia. Potassium and Zn tended to increase with these same factors. Hence, progressive changes in trace-element levels do occur in AD nail, although imbalances are detected even in the earliest sampled stages of the disease. PMID- 1704752 TI - Whole body elimination routes of gold in humans after a single-dose application of the antirheumatic auranofin. AB - After removal of the gallbladder (cholecystectomy) 7 patients were administered the antirheumatic agent Auranofin in its commercially available tablet form (Ridaura) 3 d postoperative. The single dose consisted of 5 tablets (4.35 mg of gold). Gold was determined in samples of blood, plasma, urine, bile, and feces (1 2 mL and 2.5 g, respectively). The specimens were drawn 10 min to 8 d p.a. and on d 14 p.a. The determinations were performed by instrumental neutron activation analysis (INAA). The limit of detection was less than 1 ng of gold. The courses of the mean gold concentrations show maxima after 1.9 h in blood and plasma, and after 16 h in urine and bile. The mean half-lives (terminal phases) are 7.6 d (blood), 23 d (plasma), and 6.5 d (bile). PMID- 1704753 TI - Trace element distribution in the hair of some sickle cell anemia patients and controls. AB - Hair samples of some young sickle cell anemia (SCA) and control patients in Nigeria were analyzed for 12 elements, viz, Se, Hg, Cr, Fe, Zn, Co, Cu, Br, As, Sb, Na, and Sc, using Instrumental Neutron Activation Analysis (INAA). With the exception of Cu, which was found to be significantly higher in the hair of SCA patients (at the 0.05 level of the t-test), there were generally no significant differences in elemental concentrations within the two groups. A preliminary study of the elemental contents of the fingernails of the same subjects showed a higher abundance of most of the elements in nail than in hair. These preliminary results were compared with similar studies from some other parts of the world. PMID- 1704754 TI - Minor and trace elemental contents of cancerous breast tissue measured by instrumental and radiochemical neutron activation analysis. AB - Many minor and trace elements influence the permeability of cell membranes by competing for binding sites, and exert direct or indirect action on the carcinogenic process. Instrumental and radiochemical neutron activation analysis has been employed for the determination of more than 20 elements in normal and cancerous breast tissues of 6 patients. Most trace elements, viz., Zn, Cu, Mn, Co, Se, Br, As, Sb and Cd, are elevated in cancerous tissue, whereas lower levels are observed for Fe, Cs, I, and Sr. Similarly, concentrations of minor constituents, such as Na, K, P, Cl, and Mg, are enhanced compared to normal tissue. Several elements incorporate into the normal cell, change its enzymatic activity, and accelerate the growth of tumor. PMID- 1704755 TI - Instrumental neutron activation analysis of kidney stones. AB - Kidney stone samples of the types calcium oxalate, uric acid, and xanthine were analyzed for their elemental contents by neutron activation analysis to study both the elemental correlation and influence of element on stone precipitation processes. Elements, such as Al, Au, Br, Ca, Cl, Co, Cr, Fe, Hg, I, K, Mg, Na, Sb, Se, Sr, and Zn, were determined quantitatively. Calcium oxalate stones contained higher concentration of all the elements analyzed compared to uric acid or xanthine stones. The concentrations of Cl, Fe, K, Na, Sr, and Zn were relatively higher than Au, Co, Cr, and Sb. A positive correlation exists between Ca and Zn, whereas a negative correlation exists between Sr and Ca. Zinc may play an important role in the formation of calcium oxalate stone. PMID- 1704756 TI - Studies on zinc- and cadmium-bound proteins in bovine kidneys by biochemical and neutron activation techniques. AB - Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd protein complexes were observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results showed the presence of at least four Zn-binding proteins with mol wt greater than 300,000, 260,000, 89,000, and 27,000 and at least three Cd-binding proteins of mol wt greater than 300,000, 32,000, and 13,000. PMID- 1704757 TI - Metabolic deposition of selenium and cadmium into the hair and other tissues of the guinea pig. AB - To better understand the significance of hair trace-element measurements and their relationships with the trace-element levels in body organs and fluids, a series of controlled animal experiments were conducted in which several trace elements were periodically measured during a 90-day chronic exposure to selenium and cadmium. Chronic selenium exposure appeared to be reflected by elevated selenium levels in the hair, kidneys, and liver. Chronic cadmium exposure, although reflected by kidney and liver elevation, appeared not to be reflected by corresponding increases in its concentration in the hair. PMID- 1704758 TI - Carbon determination in human teeth by activation with He-3 ions. AB - A nuclear analytical method, involving activation with 3He ions, was developed to determine carbon content in human teeth with well-documented histories. The tooth samples were irradiated with 2.7-MeV3He particles at 50 nA intensity, and the activity of 14O induced through the reaction 12C(3He, n)14O, determined by counting the 2.31-MeV gammas. Different dental hard tissues were studied separately. A solid piece of silver steel, the carbon content of which was accurately determined by chemical means, was used as the standard. The carbon content in different teeth varied from 4-7%. The overall experimental accuracy was better than 4.5%. PMID- 1704759 TI - Vanadium determination at the ultra-trace level in biological reference materials and serum by radiochemical neutron activation analysis. AB - In order to help resolve present inconsistencies of two orders of magnitude or more in reported levels of vanadium in human serum and blood, a totally postirradiation radiochemical neutron activation analysis (NAA) method was further developed and applied to some pertinent nanogram and subnanogram reference materials. In particular, the second generation human serum reference material of Versieck was found to contain a value of 0.67 +/- 0.05 ng/g dry wt., corresponding to 0.061 +/- 0.005 /4/ ng/mL original fresh serum. Results are also reported for some other appropriate CRMs. Additionally, a small-scale study in 10 normal subjects (5 m, 5 f) revealed levels similar to those in the serum reference material and in agreement with the lowest data reported in the literature. Discussion of pitfalls of vanadium determination and the use of reference materials is included. PMID- 1704760 TI - A new microcomputer-controlled neutron activation and analysis system. Description and applications for the analysis of bioenvironmental specimens. AB - A microcomputer-controlled irradiation and measurement system and a microprocessor-controlled sample changer have been installed at the SLOWPOKE-2 Facility at the Royal Military College of Canada (RMC). These systems can provide the gamut of instrumental neutron activation analysis (INAA) techniques for the analyst. Custom software has been created for system control, data acquisition, and off-line spectral analysis using programs that incorporate Gaussian peak fitting methods of analysis. The design and use of the equipment is discussed, and the performance is illustrated with results obtained from the analysis of marine sediment and biological reference materials. PMID- 1704761 TI - Factors affecting the levels reported for vanadium in human serum. AB - Chemometric techniques may be applied to extract significant analytical information from a series of publications that present methods and results for determining trace elements in biological material. This approach was applied to the total of 28 papers published in 1971-1988 that reported determination of vanadium in normal human serum or plasma; the levels spanned four orders of magnitude. The most important factors affecting the analytical results were found to be the choice of analytical method and the experience of the laboratory in trace-element research. Results from the most experienced laboratories with the best analytical methods were found to be correlated with the precision of the data, indicating that the correct concentration of vanadium would be less than 1 mg/m3. This is in agreement with results subsequently obtained by radiochemical neutron activation analysis of eight samples of serum from Danish colleagues. PMID- 1704762 TI - Determination of trace elements in human serum by inductively coupled plasma-mass spectrometry. Comparison with nuclear analytical techniques. AB - The determination of trace and ultratrace elements in human serum by ICP-MS is described. The accuracy of the method is tested using a "second generation" human serum reference material. Elements determined include Fe, Co, Cu, Zn, Br, Rb, Sr, Mo, and Cs. The method is compared to nuclear analytical methods (NAA, PIXE). Perspectives for the future are also discussed. PMID- 1704763 TI - Aspects of accuracy and precision in the determination of As and Sb in biological materials by neutron activation analysis. AB - Aspects of accuracy and precision of the determination of As and Sb in biological materials, using neutron activation with hydride generation, are discussed. It proves necessary to perform a yield determination for each sample. Both radiotracers and reactivation have been applied, and their practical use for yield correction is discussed. Results for several RMs are presented. PMID- 1704764 TI - Homogeneity and evaluation of the new NIST leaf certified reference materials. AB - The NIST has produced and is in the process of certifying two new leaf CRMs, SRM1515 Apple Leaves and SRM 1547 Peach Leaves, as replacements for the no longer available NBS Orchard Leaves and the almost depleted Citrus Leaves. These two new materials have been processed and are being thoroughly evaluated and should provide the most advanced natural matrix botanical trace-element reference materials available. Caution should be used in determining a basis weight (drying) for these CRMs because of their very fine particle size. Homogeneity has been established by instrumental neutron activation analysis on both leaf materials for five elements, to date, to better than 1.5% (1 s) for 100-mg sample sizes. PMID- 1704765 TI - Instrumental neutron activation analysis of Standard Reference Material 1941, Organics in Marine Sediment. Element content and homogeneity. AB - The National Institute of Standards and Technology has developed a new Standard Reference Material 1941, "Organics in Marine Sediment." In addition to the organic constituents, over 30 elements have been determined by instrumental neutron activation analysis and prompt-gamma activation analysis. The homogeneity of the material was investigated and relative standard deviations of single element concentrations in 250-mg samples were found to be 1% or less with regard to major inorganic constituents and rare earth elements. A slightly higher relative SD was found for elements that may stem from biological or anthropogenic input. The element concentrations determined in this work are discussed in comparison to concentrations in other similar reference materials. Concentrations for 31 elements will be included for information on the certificate. PMID- 1704766 TI - Applicability of microwave acid digestion to sample preparation of biological materials for analysis by particle-induced X-ray emission (PIXE). AB - A microwave acid digestion method for the preparation of biological samples for PIXE analysis is presented. The precision and accuracy of the entire PIXE analytical procedure, including the microwave digestion step, were evaluated by analyzing eight certified reference materials. For elements heavier than K, and for concentration levels from 2 micrograms/g upward, the total random error of a single analysis is in the range of 2-5%. The accuracy is better than 5%. The detection limits are down to 0.3 micrograms/g. PMID- 1704767 TI - New essential trace elements for the life sciences. AB - The possible importance of some new essential trace elements in nutrition is discussed. Most likely, insufficient intake of a specific trace element becomes obvious only when the body is stressed in some way that enhances the need for that element; this has been supported by recent findings with selenium. The trace elements boron and copper may be of nutritional significance in a manner similar to selenium. When the diets of animals and humans are manipulated to cause possible changes in cellular integrity or in hormone responsiveness, a large number of responses to dietary boron occur. The findings indicate that boron is important for optimal calcium and, thus, bone metabolism. High dietary cystine and fructose exacerbate the signs of copper deficiency in rats; this indicates that the response to copper deficiency by humans would vary with the amino acid and carbohydrate composition of the diet. There is some evidence that chromium, molybdenum, nickel, arsenic, and vanadium may also be of nutritional significance under stress conditions. In other words, an increasing number of studies have been performed that have examined the importance of trace element nutriture in various forms of nutritional, metabolic, hormonal, or physiologic stress in animals and humans. These studies indicate that situations will be found in which a trace element is of nutritional significance. It is likely that some of the trace elements are more important in human nutrition than is now generally acknowledged. PMID- 1704768 TI - Neutron capture prompt-gamma activation analysis of foods. AB - The suitability of neutron capture prompt-gamma activation analysis (PGAA) for multielement analysis of foods was investigated. A total of 22 elements was observed in 40 food and mineral supplements. Hydrogen, B, C, N, Na, S, Cl, and K concentrations were determined in NIST RM 8431a Mixed Diet and in a wet diet composite made from FDA Total Diet Study collections. Because the neutron flux is so low for PGAA, the method is nondestructive and reanalysis of analytical portions is possible. Both diet materials were analyzed before and after freeze drying to check for element losses during this process. No losses were found for RM 8431a, but significant losses of B and Na were observed for the wet composite. The measured loss of hydrogen for the wet composite was not consistent with the assumption that the lost mass was water only. PMID- 1704769 TI - Activation analysis of human diet samples with epithermal neutrons. AB - Activation of diet samples by epithermal reactor neutrons is a very sensitive method for the determination of iodine. The method was developed and optimized for its determination. However, it turned out that with the same measurement valid results were also obtained for Br, Cl, K, Na, and P. The procedure involves the irradiation of the sample in a boron nitride (BN) container for 1 min, followed by gamma-counting for 20 min after a decay time of 7-15 min. The necessary sample weight is 0.5 g (dry), and no chemical operations have to be performed. The capacity is 15 samples per working day, and results are available immediately. PMID- 1704770 TI - Determination of selenium in duplicate diets of residents of Pinhel, Portugal, by neutron activation. AB - A rapid cyclic instrumental neutron activation analysis (CINAA) method has been used to determine the selenium content of 27 duplicate diet samples from each of the 27 districts surrounding Pinhel, Portugal. The accuracy and precision of the CINAA method have been evaluated by analyzing certified reference materials and observed to be within +/- 5-10% for samples containing at least 40 ppb of selenium. The detection limit has been found to vary between 26-42 ppb selenium depending on the sample composition. The average daily dietary intake has been calculated as 37 micrograms of selenium per day. PMID- 1704771 TI - The use of Compton suppression spectrometers for trace element studies in biological materials. AB - A straightforward method for demonstrating the powerful background reduction of Compton suppression spectrometers for neutron activation purposes is presented. The shorter acquisition time needed in Anti-Compton mode (A/C on) for peaks of appropriate counting statistics, compared to normal gamma counting (A/C off), allows a much higher sample throughput, thus compensating for the higher cost of the instrument. Two examples of artificial mixtures of radionuclides demonstrate the drastic time saving for measurement of monoenergetic decaying isotopes. The comparison of results from three different instruments proves the general usefulness of Compton suppression spectrometers for Neutron Activation Analysis of biological samples. PMID- 1704772 TI - Multielement determination in rice, wheat, and barley by instrumental neutron activation analysis. AB - INAA has been used for the determination of Na, Mg, Al, Cl, K, Sc, Cr, Mn, Fe, Co, Cu, Zn, As, Se, Br, Rb, Sr, Mo, and W in grains of rice, wheat, and barley, which were collected from different plant fields in Iraq. Samples and standards were irradiated in the IRT-5000 reactor, at neutron fluxes of 2 x 10(13) cm-2.s-1 and 3.2 x 10(11) cm-2.s-1. Interferences of photopeaks with each other were considered, and reaction interferences were calculated and determined experimentally. Accuracy of our method was assessed by the analysis of IAEA standards Wheat Flour and Bovine liver. A good agreement has been achieved between the present results and recommended values. The precision and detection limit were determined for all elements in all types of grain. PMID- 1704773 TI - Nuclear and atomic analytical techniques in environmental studies in South America. AB - The use of nuclear analytical techniques for environmental studies in South America is selectively reviewed since the time of earlier works of Lattes with cosmic rays until the recent applications of the PIXE (particle-induced X-ray emission) technique to study air pollution problems in large cities, such as Sao Paulo and Rio de Janeiro. The studies on natural radioactivity and fallout from nuclear weapons in South America are briefly examined. PMID- 1704774 TI - Use of nuclear physics methods in life sciences in the USSR. AB - Analytical nuclear physics methods, as well as radioanalytical, X-ray, and radiometric techniques are widely used in the Soviet Union in life sciences studies. Main approaches to the study of natural materials by activation analysis, including bulk analyses, localized analyses, speciation, and in vivo experiments are described. Examples of applications in agriculture to increase protein content of wheat, to improve crop yield, and to enhance resistivity of cotton plants to diseases are given. Activation analysis is used in medicine for prognosis and study of processes of pathogenesis. Detailed information on trace elements is valuable in environmental studies, and general regularities in biogeochemistry may be clarified with activation analysis. PMID- 1704775 TI - Applications of nuclear analytical methods in the life sciences as exemplified by recent research programs of the IAEA. AB - More than 300 research nuclear reactors are presently in operation in 56 countries, most of which are suitable for neutron activation analysis (NAA). Typical applications in the life sciences are exemplified by recent research programs of the IAEA involving the participation of institutes in 47 countries. Topics of interest include applications of NAA (together with XRF and PIXE) in occupational health, nutrition, and environmental studies, including analyses of solid wastes and aerosols. The IAEA supports such programs not only by providing funding, but also through quality assurance and information services. PMID- 1704776 TI - Determination of mercury in human serum and packed blood cells by neutron activation analysis. AB - A method is described for the determination of mercury in human blood serum and packed blood cells employing neutron activation analysis. Great attention was devoted to the collection and manipulation of the samples. The accuracy and precision of the method were tested by analyzing biological reference materials and by comparing the concentrations measured in a number of serum samples to those obtained by another, independent technique (cold vapor atomic absorption spectrometry) in the same samples. The article reports the levels measured in blood serum and packed blood cells samples from 15 adult volunteers, as well as the figures determined in a "second-generation" biological reference material (freeze-dried human serum), prepared and conditioned at the University of Ghent. PMID- 1704777 TI - Determination of main and trace element contents in human brain by NAA and ICP AES methods. AB - A study was undertaken to determine the average values for elements in normal human brain (11 individuals, age group 65-75). Twelve brain parts were selected from both hemispheres. Determinations were carried out by NAA and ICP-AES. The main elements (Na, K, Mg, Ca, Fe, P, S) and trace elements (Al, B, Co, Cr, Cu, Mn, Ni, Pb, Zn) were investigated. Quality control was ensured by using NBS Bovine Liver SRM. The results obtained with independent methods were compared, and the data show a good correlation. On the basis of these investigations, the regional distribution of elements can be given. PMID- 1704778 TI - Levels of some trace elements in selected autopsy organs, and in hair and blood samples from adult subjects of the Italian population. AB - This study, which is part of a research program for the determination of trace element reference levels in various human tissues for the Italian population, presents the concentrations of Se, Hg, Cr, Cs, Sc, Rb, Zn, Fe, Co, and Sb in lung, liver, spleen, and kidney autopsy samples taken from 14 adult subjects of the Italian population who died from accidental causes. Concentrations of the same trace elements are given also for blood and hair samples taken from subjects of the general Italian population and of a population group living in a small coastal town that has an average annual fish consumption well above the national average. The analytical method used was Instrumental Neutron Activation Analysis. The levels and distribution of the trace elements in the various human organs examined are analyzed and discussed. PMID- 1704779 TI - Trace element distribution in human eye. AB - A hundred samples of four types of eye tissues and hair from a set of 10 donors from the city of Prague were analyzed using INAA. The hair samples of the set of donors were taken as the indicator of the environmental conditions. Within the framework of the set of results obtained for 30 chosen elements, the differences between various types of tissues and sex were tested. The lowest trace element deposition was found for the lens tissue. The iris differs predominantly by the higher contents, particularly of metal elements. Slightly higher contents of practically all trace elements under study were found in all eye tissues taken from male donors. PMID- 1704780 TI - Pigmentation and temporal effects on trace elements in hair. AB - Variations in trace element concentration in the head and facial hair of five individuals with ever-increasing amounts of white hair were examined. Hair was collected from the scalp, cheeks, and chin from one donor on a regular basis since 1984. Samples were separated into white and pigmented fractions, and analyzed at SLOWPOKE-Toronto by INAA for the short-lived, isotope-producing elements Br, Ca, Cl, Cu, I, Mg, Mn, Na, S, and Zn. Temporal concentration variations of these elements over time, and variations of the elemental concentrations in pigmented and white hair were established. PMID- 1704781 TI - Preliminary study of the distribution of the toxic elements As, Cd, and Hg in human hair and tissues by RNAA. AB - In order to study the relationships between trace element concentrations of hair and internal body burdens, a radiochemical NAA technique has been used for determination of the elements As, Cd, and Hg in autopsy samples of liver, kidney cortex, lung, and hair from 24 male persons who died by accident. High significant positive correlations were observed between the As concentration in hair and in kidney-cortex, and between Cd and Zn concentrations in kidney-cortex. The contents of Cd, both for lung and kidney-cortex, were related to the smoking habits of the subjects. PMID- 1704782 TI - Study of correlation of Se content in human hair and internal organs by INAA. AB - The correlations of essential element Se between human hair and kidney-cortex, liver, and lung from the same subjects were investigated by instrumental neutron activation analysis, using the reaction 76Se (n, gamma) 77mSe, for 24 Chinese autopsies. The concentration of Se is higher in kidney-cortex (2.04-5.36 mg/kg) than in liver (0.73-2.29), lung (0.50-1.85), and hair (0.37-1.43). It is important to know that there are significant relationships of Se concentration between hair and the other three internal organs. The correlation coefficient by linear regression analysis are 0.639, 0.570, and 0.635 for liver, lung, and kidney-cortex, respectively; and the P values are all less than 0.01 for the three tissues. PMID- 1704783 TI - Determination of inorganic components in Brazilian medicinal plants by neutron activation analysis. AB - Instrumental neutron activation analysis (INAA) has been applied to multielemental determinations of medicinal extracts obtained from the plants. Cordia Verbenacea DC, Folidago Microglossa DC, and Petiveria Alliacea. Concentrations of the elements Al, Br, Ca, Cl, Co, Cs, Fe, K, La, Mg, Mn, Na, Rb, Sb, and Zn have been determined in dried extracts of these herbs by short and long irradiations under a thermal neutron flux of 10(11)-10(13) n/cm2s in the IEA R1 nuclear reactor. The NBS Tea Leaves (1572) and NIES Pepperbush (1) reference materials were analyzed simultaneously with the plant extracts. The results obtained in these analyses have shown a good accuracy and reproducibility of the method. The relative errors and the relative standard deviations were less than 10% for most of the elements analyzed. PMID- 1704784 TI - Tomography and elemental analysis of biological systems. AB - The various forms of tomography, expressed as transmission and emission modes using gamma rays and neutrons, are generally discussed in terms of providing information nondestructively about the distribution of elemental composition in a plane through an object. The combination of the principles of tomography with neutron activation analysis is the basis for neutron-induced gamma-ray emission tomography. The concept of detection limit in induced gamma-ray emission tomography, as proposed, incorporates a further factor that is a measure of the quality of the image produced. A specific example is given for the elemental analysis and imaging of a bone specimen. PMID- 1704785 TI - PIXE determination of essential trace elements in some traditional Chinese medicines. AB - The essential trace elements in 30 traditional Chinese medicines, (24 tonics and 6 nontonics) were determined by proton-induced X-ray emission. The authors' previous suggestion that traditional Chinese medicines may be classified by the order of magnitude of their essential trace elements, thus indicating their pharmacological effects, is not justified. The pharmacological effect of a trace element or its essentiality may be dependent on some ligand that can be chelated with it. A nonlinear mapping algorithm, however, shows that the 30 traditional Chinese medicines are nearly separated into two groups, indicating their tonic or nontonic pharmacological effects. PMID- 1704786 TI - Inductively coupled atomic emission spectrometry and neutron activation analysis for the determination of element reference values in human lung tissue. AB - An investigation was undertaken in order to assess the performance of neutron activation analysis and inductively coupled plasma atomic emission spectrometry techniques for determining reference values for minor and trace elements in human lungs of urban subjects. Results show that in both instances experimental conditions must be carefully optimized to guarantee reliability of experimental data. Strict criteria for tissue sampling and pretreatment also had to be set. Provisional reference values for ca. 50 elements could thus be established. PMID- 1704787 TI - Application of polyacrylamide gel electrophoresis/neutron activation analysis for protein quantification. AB - A combination of two methods, polyacrylamide gel electrophoresis (PAGE) and neutron activation analysis (NAA), has been applied to solutions containing phosphoproteins for the purpose of protein quantification. The proteins were separated by molecular weight using PAGE, and then the whole gel was activated by neutron bombardment. Densitometric measurements of the developed bands from 32P, taken from autoradiographs of the activated gels, resulted in quantification of the phosphorus, and then the related protein. This PAGE/NAA method was applied to several phosphoprotein-containing materials, including commercial milk products and reference materials, i.e., IAEA A-11, milk powder, and SRM 1845, Cholesterol in Egg Powder. PMID- 1704788 TI - Instrumental neutron activation analysis of biological samples. AB - The elemental compositions of 18 biological reference materials have been processed, for 14 stepped combinations of irradiation/decay/counting times, by the INAA Advance Prediction Computer Program. The 18 materials studied include 11 plant materials, 5 animal materials, and 2 other biological materials. Of these 18 materials, 14 are NBS Standard Reference Materials and four are IAEA reference materials. Overall, the results show that a mean of 52% of the input elements can be determined to a relative standard deviation of +/- 10% or better by reactor flux (thermal plus epithermal) INAA. PMID- 1704789 TI - Location and quantification of metal ions in enzymes combining polyacrylamide gel electrophoresis and particle-induced X-ray emission. AB - A method is presented to identify and determine the relative amounts of protein bound metal ions in situ. Proteins or their subunits are separated by SDS-PAGE, the appropriately dried gel sections are directly scanned by a collimated proton beam of 3 MeV energy, and the characteristic X-rays produced are detected. The determination of Fe content of an iron-sulfur protein (HiPiP), as well as the Fe and Ni analysis of the hydrogenase from Thiocapsa reseopersicina, have shown the feasibility of this technique. PMID- 1704790 TI - Expanded programme on immunization. Progress towards eradicating poliomyelitis from the Americas. PMID- 1704791 TI - Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria. AB - Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis. PMID- 1704792 TI - Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov. AB - The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (less than or equal to 5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (greater than or equal to 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704793 TI - Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. AB - Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and "Campylobacter upsaliensis." Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus "Flexispira," with "Flexispira rappini" as the only species, is closely related to the genus Helicobacter. The free-living, sulfur reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI. PMID- 1704794 TI - Impaired uptake of 5 hydroxytryptamine platelet in essential hypertension: clinical relevance. AB - Serotonin (5-hydroxytryptamine; 5HT) kinetics and platelet activation by 5HT were studied in patients with essential hypertension (n = 45), and in matched normotensive subjects (n = 45). Platelet response to 5HT and plasma beta thromboglobulin increased with age in men, both normotensives and hypertensives. Beta-thromboglobulin and 5-hydroxyindoleacetic acid (5HIAA) excretion were higher in hypertensive men than in women. In women, no changes in platelet activity or 5HIAA excretion were found. 5HT plasma concentrations increased with blood pressure. Platelet 5HT uptake (Vmax and KM) were the lowest in hypertensive men greater than or equal to 60 years of age. This may indicate that 5HT uptake in vivo in normotensives is far below maximum (VNT much less than Vmax), whereas in hypertensive men it may be close to maximum (VHT approximately Vmax). This could reflect significantly higher 5HT plasma concentrations in vivo hypertensives than in normotensives. The reduced uptake (which was found only in hypertensive men) may indicate an insufficient compensation of the enhanced 5HT release from aggregating platelets in older men, in whom platelet activity is enhanced in vivo. It is concluded that the defect in platelet 5HT uptake in hypertensives- along with the enhanced platelet aggregation--may contribute to a critical increase in 5HT plasma concentrations locally. An increase in 5HT concentrations leads to biochemical changes (higher 5HIAA excretion) as well as to an enhanced stimulation by 5HT. This may be of clinical relevance especially in older men, in whom 5HT2-receptor mediated responses are enhanced. PMID- 1704795 TI - Malignant fibrous histiocytoma of the conjunctiva. AB - Malignant fibrous histiocytoma (MFH) of the conjunctiva is an extremely rare tumour, and only three previous cases have been reported. We describe two patients with MFH of the conjunctiva: a 58-year-old white male with epibulbar tumour who had exenteration and is alive after five years' follow-up, and a 3 1/2 year-old African girl with xeroderma pigmentosum and an MFH of her right eye conjunctiva, the first reported case of this association. The characteristics and the methods of diagnosis of MFH are discussed. PMID- 1704796 TI - A microscopic electrostatic model for the amphotericin B channel. AB - A microscopic model of an amphotericin B channel is proposed. The structure of the pores is generated using the atomic coordinates of the molecule in the structure determined experimentally by X-ray diffraction. The net charges of the atoms are determined by Mulliken analysis. With these charges the electrostatic energy profiles are calculated for a monovalent ion passing through the channels formed by different number of antibiotic molecules having different radii. The water inside the channel was considered through a continuum medium using the dielectric constant of the bulk, and the membrane contribution was included using the virtual images of the pore in a dielectric slab of epsilon = 3. The model satisfactorily explains the permeability and selectivity characteristics as well as other observations yet unexplained. The electrostatic profiles obtained reinforce the hypothesis of the existence of channels formed by a variable number of units. PMID- 1704797 TI - Hyaluronan-binding proteins on cultured J 774 macrophages. AB - Cultivated macrophages of murine cell-line J 774 were found to bind high molecular-weight (molecular weight average approx. 5.10(6) [3H]hyaluronan (HA) by a saturable mechanism at 4 degrees C. Half-maximal binding was observed at 7-8 microgram/ml (1.4-1.6 nM) and the maximal binding was reached at 30-40 microgram/ml. Scatchard plot analysis revealed that approx. 20,000 molecules could bind to each cell with a Kd of 1.5 nM. The binding could be effectively inhibited by unlabeled HA. Also chondroitin sulphate inhibited the binding, but only to about 50%. At 37 degrees C the J 774 cells took up and degraded the polysaccharide effectively. Affinity chromatography on HA coupled to agarose of solubilized surface-iodinated J 774 cells, revealed that a protein of approx. 60 kDa, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography, could be specifically eluted with HA-oligosaccharides. Our results suggest that J 774 macrophages can bind HA by a mechanism compatible with receptor-binding, and carry a 60 kDa HA-binding protein on their surface. This receptor-binding may mediate uptake and degradation of the polysaccharide and influence the levels and turnover of HA in interstitial fluid as well as the release of HA into the bloodstream. PMID- 1704798 TI - Metabolic and secretory response of parotid cells to cationic amino acids. Uptake and catabolism of L-arginine and L-ornithine. AB - L-Arginine and L-ornithine, which stimulate amylase release, are taken up by rat parotid cells. L-Arginine is converted, in an NADPH-dependent manner and to a limited extent to L-citrulline in parotid cell homogenates, despite the absence of ornithine transcarbamylase activity. L-Arginine is largely converted to urea and L-ornithine. The generation of putrescine and polyamines from L-ornithine occurs at a very low rate, relative to the cell content in performed amines. The major fate of exogenous or arginine-derived ornithine consists in its conversion to L-glutamate, which is then further metabolized. These findings raise several hypotheses for the secretory response of the parotid cells to cationic amino acids, including their accumulation as positively charged molecules inside the cell and the generation of either NO, amines, substrates for a transglutaminase catalyzed reaction, or ATP through oxidative catabolism. However, each of these hypotheses meets with objections, the modality for the stimulation of amylase release by cationic amino acids being eventually considered as an unsettled matter. PMID- 1704799 TI - Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2. AB - The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo. PMID- 1704800 TI - Changes in intracellular free calcium activity in Xenopus eggs following imposed intracellular pH changes using weak acids and weak bases. AB - The aim of this work was to determine the potential relationships between rises in intracellular pH (pHi) and intracellular free calcium activity (Cai2+) during cell activation in Xenopus eggs. To do this, we used two weak bases, NH4Cl and procaine, and a weak acid, CO2, and measured Cai2+ variations in response to these imposed pHi variations. NH4Cl and procaine increased Cai2+ in both unactivated and activated eggs. Procaine was found to alkalinize the egg cytoplasm, whereas the other weak base, NH4Cl, acidified the egg cytoplasm. On the other hand, CO2 was found to acidify the cytoplasm and to substantially decrease Cai2+, also in unactivated and activated eggs. In addition, CO2 triggered an increase in the conductance of the plasma membrane to Cl- ions, similarly to what had been found previously with weak bases (Charbonneau, M. (1989) Cell Differ. Develop. 26, 39-52). These Cl- channels, similarly to the sperm-triggered Cl- channels during the fertilization potential, are supposed to be Ca2(+)-sensitive. Therefore, the changes in Ca2+ observed in response to CO2 do not seem to be responsible for the opening of these Cl- channels, which would rather be triggered by an increase in Cai2+ localized near the plasma membrane. We conclude therefore that weak acids and bases represent appropriate tools for studying cytosolic Ca2+ homeostasis, but not for dissecting the complex pathways involved in signal transduction. PMID- 1704801 TI - Thermodynamic analysis of the transcription cycle in E. coli. AB - The E. coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination. In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex. The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process. This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete. PMID- 1704802 TI - Identification of functionally distinct domains of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction. PMID- 1704803 TI - The Georgia type of nondeletional hereditary persistence of fetal hemoglobin has a C---T mutation at nucleotide-114 of the A gamma-globin gene. PMID- 1704804 TI - Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. AB - Juvenile chronic myelogenous leukemia (JCML) is a good model for the study of myeloproliferation because JCML hematopoietic progenitor cells grow in vitro at very low cell densities without the addition of exogenous stimulus. Previous studies have demonstrated that this proliferation is dependent on granulocyte macrophage colony-stimulating factor (GM-CSF), and that removal of monocytes from the cell population before culture eliminates this "spontaneous" myeloproliferation, suggesting a paracrine role of monocyte stimulation. However, subsequent studies have shown that increased GM-CSF production from the JCML monocytes is not a consistent finding and therefore not a plausible sole mechanism. In examining hematopoietic growth factor dose-response curves, both JCML GM and erythroid nonadherent progenitor cell populations displayed a marked and selective hypersensitivity to GM-CSF. Responses to interleukin-3 and G-CSF were identical to control dose-response curves. This is the first demonstration of a myeloid leukemia in which hypersensitivity to a specific growth factor appears to be involved in the pathogenesis of the disease. PMID- 1704805 TI - Stages I and II diffuse large cell lymphomas: prognostic factors and long-term results with CHOP-bleo and radiotherapy. AB - One hundred forty-seven patients with Ann Arbor stages I and II diffuse large cell lymphoma (DLCL) were treated with combination chemotherapy consisting of cyclophosphamide, doxyrubicin, prednisone, and low-dose bleomycin (CHOP-Bleo) and involved-field radiation (IF XRT) between 1974 and 1984. A complete remission (CR) was attained by 54 of 57 patients with stage I disease and by 78 of 90 patients with stage II disease. Thirty-five patients had relapsing disease that occurred within 3 years in 31. The overall 10-year survival rate, counting all deaths, for patients with stage I was 72% as compared with 43% for patients with stage II (P less than .01). Determinate survival rates, censoring eight unrelated deaths, were similar to the overall survival rates: 77% and 51%, respectively. A multivariate analysis identified three independent prognostic factors: age, tumor extent, and serum lactic dehydrogenase (LDH) level. When the combined effect of tumor extent and LDH level were taken into consideration in the analysis, three risk groups for survival were identified. The best group, which consists of patients with minimum tumor and normal LDH levels, had a 10-year determinate survival of 79%. Patients with extensive tumors and elevated LDH levels had the poorest survival rate of 44%. An intermediate-risk group with a determinant survival of 62% was composed of patients with either extensive tumors or elevated LDH levels. These differences demonstrate the need to develop different treatment strategies based on risk factors for survival for patients with apparently localized Ann Arbor stages I/II DLCL. PMID- 1704806 TI - B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors. AB - We have recently described a reproducible method whereby colonies containing cells that secrete immunoglobulin (Ig) can be grown from normal, human, adult bone marrow samples. The present report characterizes the cells that initiate these colonies. It is shown that all clonogenic cells express the CD19 surface antigen, as removal of these cells before plating in the B-cell colony assay abolished the subsequent growth of plaque-forming, B-lineage colonies. Cells from both the CD10+ and CD20+ B-lineage subpopulations initiated the growth of B-cell colonies, as removal of either subset resulted in a 50% reduction in the number of resulting B-cell colonies. The removal of activated B cells (CD23+), plasma cells (PCA-1+), or myeloid cells (CD13+) did not lead to a significant depletion in B-cell colony formation. Pre-B cells that were not yet committed to Ig light chain expression were also able to differentiate and proliferate into Ig secreting colonies under the culture conditions used. Colonies initiated by these light chain uncommitted cells were distinguished using a replicate protein immunoblotting technique, which detects the simultaneous secretion of Ig kappa and Ig lambda from single colonies. These experiments provide evidence that the CD10 antigen is expressed on B-lineage cells before Ig light chain commitment, whereas CD20 is not. In conclusion, this B-cell colony assay provides a system for studying the differentiation of bone marrow-derived B cells and their precursors into Ig-secreting cells. PMID- 1704807 TI - Hepatitis C, D and E virus infection. PMID- 1704808 TI - Detecting diabetic retinopathy. PMID- 1704809 TI - Management of lung cancer. PMID- 1704810 TI - Crossed aphasia in Chinese: a clinical survey. AB - According to our clinical observations from various aspects of stroke patients, such as the total incidence of aphasia, the incidence of aphasia after left brain damage of the dextrals, the aphasia that occurs in patients without hemiplegia, and the types of aphasia, a much higher incidence of crossed aphasia is seen among the stroke patients of the Han (the largest ethnic group in China) as compared with the Uighur-Kazaks (U-K) in China and the Occidentals documented in the literature. Motor aphasia is most common and pure sensory or posterior aphasia is rarely seen in Han patients. The distinct features of the Chinese language is a possible explanation for this difference. We suspect that language function of the Han is not localized in the left brain but in the right or both hemispheres. There is no definite Wernicke's area in the left brain of the Chinese people and the neural pathway of the language function in the brain of the Chinese people is not similar to people who speak phonetic languages. Consequently the universal applicability of the theories of cerebral laterality of the language function and dominant hemisphere established by Dax and Broca are questioned in this paper. PMID- 1704811 TI - Effects of word-onset cuing on picture naming in aphasia: a reconsideration. AB - When an aphasic is unable to name an object, giving the patient the opening sounds of the target name will often trigger the correct response. Eighteen aphasic subjects were tested using a gating paradigm to compare word onset durations necessary to elicit correct names after an initial naming failure with those necessary for recognizing the same words when spoken in isolation with no picture present. Prerecognition errors were also examined. Results suggested that the facilitation of naming found when examiners supply word-onset sound cues may be due in part to a two-stage process consisting of stem-completion followed by matching the picture with the potential name as generated. PMID- 1704812 TI - [Heart arrest during spinal anesthesia for transurethral resection of the prostate. Apropos of a case]. AB - We report a cardiac arrest in a 66 years healthy patient during a spinal anesthesia for a transurethral resection of prostate. The accident occurred one hour and fifteen minutes after the subarachnoidal injection of hyperbaric lidocaine 80 mg, at the end of surgery, but before any postural change. We attempt to elucidate the surgical or anesthesiologic precipitating factors that lead to the cardiac arrest in this patient. However, there was no real etiology that formally explained the genesis of the accident. This case is to add to a series of recently published accidents that occurred during spinal anesthesia in healthy patients. PMID- 1704813 TI - Depletion flocculation and depletion stabilization of erythrocytes. AB - At dextran (Mw approximately 500,000) concentrations from 2 to approximately 10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations greater than 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations greater than 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration. PMID- 1704814 TI - The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic beta-cells. AB - In recent years, the electrical burst activity of the insulin releasing pancreatic beta-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K+ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca2+ level in the pancreatic beta-cell. PMID- 1704815 TI - Gas supersaturation tolerances in amoeboid cells before and after ingestion of bubble-promoting particles. AB - Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macrophages and macrophage-like tumor cells, and for cells of the slime mold Dictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogen-supersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 x 10(7) Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed. PMID- 1704816 TI - Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. AB - The first successful freezing of early embryos to -196 degrees C in 1972 required that they be cooled slowly at approximately 1 degree C/min to about -70 degrees C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to -70 degrees C, the results is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well. PMID- 1704817 TI - A comparison of thermodynamic approaches to predict the adhesion of dairy microorganisms to solid substrata. AB - Four different thermodynamic approaches were compared on their usefulness to predict correctly the adhesion of two fouling microogranisms from dairy processing to various solid substrata. The surface free energies of the interacting surfaces were derived from measured contact angles according to: 1. The equation of state; 2. The geometric-mean equation using dispersion and polar components neglecting spreading pressures; 3. The geometric-mean equation using dispersion and polar components while accounting for spreading pressures; and 4. The Lifshitz-van der Waals/Acid-Base approach. All approaches yielded similar surface free energies for the low energy surfaces. Application of approach 1 with different liquids did not give consistent values for the high surface free energy substrata. The dispersion or Lifshiftz-van der Waals components were nearly equal for approaches 2, 3, and 4; however, the polar or acid-base components differed greatly according to the approach followed. Approaches 1 and 2 correctly predicted that adhesion should occur, although the trend with respect to the various solid substrata was opposite the one experimentally observed, as was also the trend predicted by approach 4. Only approach 3 correctly predicted the observed bacterial adhesion with respect to the various solid substrata. In approach 3 and 4, adhesion was frequently found, despite a positive free energy of adhesion. This was attributed to either possible local attractive electrostatic interactions, inadequate weighing of surface free energy components in the calculation of free energies of adhesion, or to additional forces arising from structured interfacial water. PMID- 1704818 TI - Effect of 2,4-dihydro-3H-1,2,4-triazole-3-thiones and thiosemicarbazones on iodide uptake by the mouse thyroid: the relationship between their structure and anti-thyroid activity. AB - Antithyroid activity of 2,4-dihydro-3H-1,2,4-triazole-3-thiones and thiosemicarbazones was tested by measuring the uptake ratio of thyroid: serum (T/S) of 125I through the mouse thyroid. Substitution with an alkyl group at the 5-position of the triazole nucleus remarkably increased the activity but substitution at the N-2 and/or N-4 positions caused a significant decrease in the activity, indicating the necessity of unsubstituted thioureylene moiety for the antithyroid activity. Thiosemicarbazone derivatives which are an open ring structure of triazoles showed comparable antithyroid activities to those in a ring form, but one thiosemicarbazone showed a much higher toxicity than the corresponding ring form compound. This suggests that the ring structure is not essential for the activity but is necessary to reduce toxic effect. Of fourteen compounds tested, 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione was the most potent antithyroid compound with low toxicity, with a potency tenfold that of propylthiouracil, a drug currently used. PMID- 1704819 TI - Synthesis and anti-human immunodeficiency virus (HIV-1) activity of 3'-deoxy-3' (triazol-1-yl)thymidines and 2',3'-dideoxy-3'-(triazol-1-yl)uridines and inhibition of reverse transcriptase by their 5'-triphosphates. AB - 3'-Deoxy-3'-(1,2,3-triazol-1-yl)thymidines (5a, 6a, 8a, 11a, and 12a) and 2',3' dideoxy-3'-(1,2,3-triazol-1-yl)uridines (5b, 6b, 8b, 11b, and 12b) were synthesized as cyclic analogues of 3'-azido-3'-deoxythymidine (AZT) and 3'-azido 2',3'-dideoxyuridine (CS-87) by the cyclization of 5'-trityl derivatives (1a, b) of AZT and CS-87 using alpha-ketophosphorus ylides and with acetylenic compounds followed by deprotection of the 5'-trityl group. It was hypothesized that the triazole nitrogen atoms could mimic and distorted azido group. However, no significant activity against human immunodeficiency virus type 1 (HIV-1) was observed with any of these compounds. 5'-Triphosphates (17a and 18a, b), prepared from 5a and 6a, b, were inactive against HIV-1 and Rauscher murine leukemia virus (RLV) reverse transcriptases. PMID- 1704820 TI - Enzyme histochemical and immunohistochemical characterization of oval and parenchymal cells proliferating in livers of rats fed a choline-deficient/DL ethionine-supplemented diet. AB - Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes. PMID- 1704821 TI - Construction of a recombinant adenovirus containing the denV gene from bacteriophage T4 which can partially restore the DNA repair deficiency in xeroderma pigmentosum fibroblasts. AB - The denV gene from bacteriophage T4 encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Cells from excision repair-deficient xeroderma pigmentosum (XP) patients are able to carry out excision repair initiated by the denV gene product and introduction of the denV gene into XP cells results in the partial restoration of colony-forming ability after irradiation with UV light. In this work we have constructed a helper-independent recombinant human adenovirus, Ad5denV, which contains the denV gene. A 1.9 kb cartridge consisting of the denV gene flanked by the long terminal repeat (LTR) promoter from Rous sarcoma virus (RSV) and the simian virus 40 (SV40) polyadenylation (poly A) splice signals, was inserted into the E3 region of an E3 deletion mutant (Ad5d1E3) of adenovirus type 5. Infection of human fibroblasts and other permissive human cells with Ad5denV resulted in lytic infection and expression of the denV gene was confirmed by primer extension of infected cell RNA. The ability of the denV gene to restore the DNA repair deficiency in XP fibroblasts was examined using host cell reactivation of viral structural antigen formation for UV-irradiated adenovirus. The control virus, Ad5VSV, was also a recombinant which contained the gene for vesicular stomatitis virus glycoprotein G inserted into the E3 region of Ad5d1E3. UV survival of Ad5denV was similar to that of Ad5VSV following infection of two normal fibroblast strains and a Cockayne syndrome fibroblast strain, CS7SE, from complementation group B. In contrast, UV survival of Ad5denV was significantly greater than that for Ad5VSV after infection of three unrelated XP fibroblast strains from complementation groups A, C and E. However, UV survival of Ad5denV in the XP fibroblasts did not reach levels obtained in normal fibroblasts, indicating that restoration of the XP defect was partial. PMID- 1704822 TI - In vivo formation and repair of O6-methylguanine in human leukocyte DNA after intravenous exposure to dacarbazine. AB - Blood leukocyte DNA obtained from 11 Hodgkin's disease patients undergoing ABVD chemotherapy was analysed for the presence of the precarcinogenic adduct O6 methylguanine (O6-meG) at various times (1-2 h up to 49 h) after i.v. treatment with the methylating drug dacarbazine. Adduct formation was detected in all but one of the patients examined at levels ranging up to 0.45 fmol/micrograms DNA (7.2 x 10(-7) mol/mol guanine). The levels of the adduct decreased by approximately 30% over the 24 h following exposure and were usually not detectable 49 h after exposure. In five out of seven individuals examined after more than one treatment, consistent methylation responses were noted, while in the remaining two cases the responses were mixed. No correlation between the extent of adduct formation and lymphocyte levels of the repair enzyme O6 alkylguanine-DNA alkyltransferase was observed. The average extent of O6-meG formation 1 h after dacarbazine treatment was (4.3 +/- 3.1) x 10(-2) fmol/micrograms DNA per mg/kg dose [( 1.2 +/- 0.8) x 10(-3) fmol/micrograms DNA per mg/m2 dose)]. Following exposure of rats to similar doses of dacarbazine, the corresponding levels of adduct in blood leukocyte DNA were 1.1 x 10(-2) fmol/micrograms DNA per mg/kg dose (2.6 x 10(-3) fmol/micrograms DNA per mg/m2 dose). PMID- 1704823 TI - Alterations of the nucleolar organizer regions during 4-nitroquinoline 1-oxide induced tongue carcinogenesis in rats. AB - The number of silver-stained nucleolar proteins (AgNORs) was enumerated in preneoplastic and neoplastic rat tongue lesions induced by 4-nitroquinoline 1 oxide (4-NQO). Male ACI/N rats were given 0 to 10 p.p.m. 4-NQO for 12, 20 or 36 weeks to induce hyperplasia, dysplasia and neoplasm in tongue. The mean numbers of AgNORs stained by a modified one-step silver colloid method in various epithelial lesions were as follows: normal squamous epithelium (n = 5), 1.52 +/- 0.03; non-lesional squamous epithelium (n = 5), 1.58 +/- 0.04; hyperplasia (n = 20), 1.84 +/- 0.15; dysplasia (n = 20), 2.32 +/- 0.12; papilloma (n = 6), 2.23 +/ 0.10; and squamous cell carcinoma (n = 4), 3.06 +/- 0.26. Thus, the mean number of AgNORs showed a stepwise increase from untreated and treated, histologically normal squamous epithelium through hyperplasia and dysplasia to squamous cell papilloma and carcinoma, although the value of severe dysplasia was between those of papilloma and carcinoma. These results indicate that the mean number of AgNORs may reflect the proliferative nature of tongue lesions, as suggested in carcinogenesis of other organs, and also suggest that severe dysplasia is a direct precursor lesion for squamous cell carcinoma of the tongue induced by 4 NQO. PMID- 1704824 TI - Effect of phenylalanine on pancreatic amylase secretion in chicks (Gallus domesticus). AB - 1. Effect of phenylalanine (Phe) on pancreatic amylase secretion in growing chicks was investigated in four experiments. 2. In Experiment 1, birds were injected through a wing vein with 0.25 ml Phe at 0, 0.1, 0.5, 2.5 and 12.5 mM in physiological saline. No significant difference was observed in amylase secretion among treatments. 3. Effect of various concentrations of Phe with cholecystokinin (CCK, 0.31 Crick unit) on amylase secretion was investigated in Experiment 2. Amylase secretion increased with time, although no significant effect was detected in Phe treatment. 4. Efficacy of Phe and tyrosine (Tyr) injection with CCK on amylase secretion was compared. There was no significant difference between Phe and Tyr treatments. 5. Birds were injected intraperitoneally with dl p-chlorophenylalanine (p-CP), which is an inhibitor of phenylalanine hydroxylase, or saline 1 day before the collection of pancreatic amylase in Experiment 4. Both chicks showed increased amylase secretion with CCK (0.31 Crick unit), whereas the response was at a drastically reduced rate in chicks with the p-CP treatment. PMID- 1704825 TI - Thrombolysis: go with the flow. PMID- 1704826 TI - Cardiotoxicity of interferon. A review of 44 cases. AB - Cardiovascular complications have occurred in clinical trials of interferon. We review herein experience to date of cardiotoxicity with all types of interferons in cancer patients. The most common presentations of cardiotoxicity were cardiac arrhythmia, dilated cardiomyopathy, and symptoms of ischemic heart disease, including myocardial infarction and sudden death. The cardiac effects were not related to the daily dose, cumulative total dose, or period of therapy. Some of the patients in whom interferon has caused cardiovascular sequelae have had a history of coronary heart disease or have previously been given chemotherapy with drugs known to be cardiotoxic. In most of the patients, cardiac toxicity was reversible following the cessation of the drug therapy. PMID- 1704827 TI - Active vs passive rhythms as an explanation of bigeminal rhythm with similar P waves. AB - We describe the criteria for differential diagnosis between 3:2 sinoatrial block from atrial bigeminy due to an ectopic focus in the sinus or parasinus zone. In the 3:2 sinoatrial block the RR interval of the basic rhythm is similar to the short R-R interval of the paired rhythm. In atrial bigeminy, the R-R interval of the basic rhythm is similar to the long R-R interval of the paired rhythm. PMID- 1704828 TI - Acute porphyria presenting with hyperamylasemia. AB - An elevation of serum amylase and lipase has not been reported previously to occur with porphyria. In this report, we describe a patient who presented with the clinical and laboratory picture of pancreatitis: elevated amylase, lipase, amylase-creatinine clearance ratio, and with abdominal pain. Only after extensive evaluation, was the patient found to have porphyria. On two separate occasions, with hematin therapy, her serum amylase decreased, as did her clinical symptoms of porphyria and her urinary quantitative porphyrins. This suggests an association between elevation of the serum amylase and lipase with acute porphyria. Moreover, this association can lead to delay in establishing the diagnosis of acute porphyria. PMID- 1704829 TI - Palliation for esophageal and cardial cancer. PMID- 1704830 TI - [Venus' poison. History of the effect of syphilis]. PMID- 1704831 TI - [Human papillomaviruses--association with malignant and semimalignant tumors of the skin and skin-connected mucous membranes]. PMID- 1704832 TI - Estimation of time delay between EEG signals for epileptic focus localization: statistical error considerations. AB - A theoretical analysis of the variance for the time delay estimate between two EEG signals, obtained via the phase spectrum method, is presented. Explicit theoretical formulae for the variance are obtained and compared via simulations to experimentally derived results for estimate variability. The variance of the time delay estimate is inversely proportional to the frequency range of interest, to the number of data segments utilized for spectral estimation, and to the coherence between the two EEG signals. The simulations indicate that the formulae can be used even with non-gaussian and relatively narrow-band EEG-like data. A minimum-variance estimate for the time delay is also presented. PMID- 1704833 TI - The cause of increased pupillary light reflex latencies in diabetic patients: the relationship between pupillary light reflex and visual evoked potential latencies. AB - In 42 diabetic patients the relationship between the latency of the pupillary light reflex and the pattern reversal visual evoked potential (P100) was examined. Fifty-five percent of diabetic patients had pupillary light reflex latencies above the normal range. In 19% the visual evoked potentials were prolonged when compared to the normal range. Latencies of pupillary light reflexes and VEPs showed no correlation. There was a minimal correlation between the presence of retinopathy and prolongation of both the pupillary light reflex and the visual evoked response latency (kappa coefficients respectively: 0.31, P less than 0.01 and 0.36, P less than 0.02). The presence of an increased pupillary light reflex latency was positively correlated with a reduced respiratory sinus arrhythmia (kappa coefficient: 0.58, P less than 0.0001). Increased VEP latencies showed no correlation with signs of cardiovascular autonomic neuropathy. We conclude that the afferent optic pathway can be affected in diabetic patients. However, prolongation of pupillary light reflex latency in diabetic patients is primarily due to an efferent pupillary defect and represents parasympathetic dysfunction. PMID- 1704834 TI - Recovery functions of fast frequency potentials in the initial negative wave of median SEP. AB - Following far-field potential of P14 after median nerve stimulation, we identified several small wavelets, designated here as fast frequency potentials or FFP, over the ascending and descending phases of the major negative wave of 'N20.' In 10 normal subjects who had well defined FFP, namely N16, N17, N18 and N19, we studied recovery functions of FFP by applying paired (condition and test) stimuli of various interstimulus intervals (ISIs) ranging from 3 to 150 msec. Recovery of FFP was determined by the difference wave forms, i.e., subtracting the response of conditioning stimuli from the response to paired stimuli. Measuring latencies and amplitude of FFP and also of the far-field potential of P14, we found recoveries of P14 and each FFP to be different; P14 recovered with the shortest ISI while progressively longer ISIs were required for full recovery of the later FFP. This finding suggests the existence of progressively increased number of interspersed synapses from the early to late FFP. It is unlikely that FFP represents passage of the afferent volley from one anatomical site to the next, but rather that they are cortically generated via a closely situated polysynaptic network. The polysynaptic nature of FFP is consistent with the observation that they disappear in sleep and also during anesthesia. The study indicates that FFPs represent different physioanatomical systems from the primary negative wave of 'N20' that results from specific sensory input to the primary sensory cortex. PMID- 1704835 TI - The N400 component of event-related potentials in schizophrenic patients: a preliminary study. AB - ERPs were recorded during a word recognition task to investigate cognitive dysfunction in schizophrenia. Thirteen medicated schizophrenics and 26 normal controls were tested. In each trial a pair of stimuli, S1 (a word) and S2 (a word or a non-word), were presented. The subjects were required to discriminate between a word and a non-word for S2 (lexical decision task). In a related (R) condition, S2 was the antonym of S1 (e.g., brother-sister); in an unrelated (U) condition, S1 and S2 were semantically unrelated (e.g., brother-drive); in the non-word (N) condition, S2 was a non-word (e.g., brother-grofe). The ERPs for S2 were analyzed, and the contextual effects on the ERPs for S2 observed for both the patients and controls. For both groups, in the U and N conditions S2 elicited a large negative-trending deflection (N370). In contrast, in the R condition it elicited only a small negative-trending notch. There was no difference in the amplitude of N370 between the groups, but its latency was more prolonged or its wave shape more extended for the schizophrenics than for the controls. The N400 amplitude is concluded to remain unchanged in schizophrenics. PMID- 1704836 TI - Effects of performance feedback on P300 and reaction time in closed head-injured outpatients. AB - In an earlier study, we have demonstrated that the choice reaction times (RTs) of closed head-injured patients may be reduced significantly by instructions which emphasize speed, thus indicating that this portion of the RT delay was not due to a motor deficit. Despite the reduction in patient RTs, however, they remained significantly longer than those of controls. Furthermore, the RTs of patients were 56 msec longer than their P300 latencies, whereas those of controls occurred at about the same time as P300. The delay which remained in patient RTs was thus not entirely due to longer stimulus evaluation times. The goal of the present study was to further reduce patient RTs using feedback (FB) on response speed and time windows within which subjects were required to respond. When FB on speed and a 'narrow' time window were used, patient RTs occurred at about the same time as P300 latency (as was also the case with controls) indicating that the longer RTs of these patients in our earlier study, and in the other conditions of the present study, could not have been exaggerated beyond P300 latency due to a motor deficit. It was suggested that CHI patients may not be able to internally monitor their responses to the same degree as controls. The provision of external cues may have compensated for this deficit. PMID- 1704837 TI - Effects of the anticonvulsant benzodiazepine clonazepam on event-related brain potentials in humans. AB - The effects of the benzodiazepine clonazepam (a drug used as anticonvulsant) on event-related brain potentials were investigated in healthy human subjects. Thirty-six male student volunteers (mean age 30 years) received clonazepam or a placebo in a double-blind setting. VEPs (visual evoked potentials) were obtained from the standard checkerboard reversal procedure; AEPs (auditory evoked potentials) and slow cortical potentials (CNV) were measured during a 2-stimulus reaction time paradigm, in which the quality of the acoustic S1 signalled whether the acoustic S2 would follow after 2 sec or after 6 sec. Each S2 requested a speeded button press. Compared to placebo, clonazepam significantly reduced P100 amplitude of the VEP and the amplitudes of the AEP components N1 and P3. On the other hand, clonazepam boosted the development of a distinct N2 which was not apparent in placebo subjects. The CNV was significantly reduced and reaction time increased under clonazepam compared to placebo. Specific versus non-specific damping effects of the benzodiazepine are discussed, comparing the present result with the pattern of ERP effects of the anticonvulsant carbamazepine that had been obtained using the same experimental paradigms. PMID- 1704838 TI - Long-lasting depression of central synaptic transmission following prolonged high frequency stimulation of cutaneous afferents: a mechanism for post-vibratory hypaesthesia. AB - High-frequency vibration or electrical stimulation of cutaneous afferents may produce long-lasting hypaesthesia. Such stimulation alters the excitability of axons in the peripheral nerve but there is evidence that this does not completely explain the hypaesthesia. The present study was undertaken to determine whether a prolonged afferent barrage results in depression of synaptic transmission at a central site. Changes in central excitability to cutaneous inputs were examined in normal subjects by measuring the cerebral evoked potential at different stages after high-frequency conditioning stimulation of the digital nerves. Changes in peripheral excitability were eliminated by adjusting the stimulus intensity so that a constant afferent volley entered the central nervous system. Following the conditioning stimulation (4-5 T, 200 Hz, 10 min), the cortical potential evoked by constant submaximal test volleys was depressed by up to 50% for 25 min. The attenuation was less profound (10-20%) but more prolonged (greater than 45 min) when maximal test volleys were used, and occurred regardless of whether the high frequency stimulation was applied to the test digit or to adjacent digits. It is concluded that prolonged activation of cutaneous afferents causes a depression in central excitability independent of and additional to peripheral changes, and it is suggested that this mechanism contributes to the associated perceptual disturbances. By analogy it is suggested that the hypaesthesia associated with prolonged vibration may be of central rather than peripheral origin. PMID- 1704839 TI - Quantitative EEG changes under various conditions of hyperventilation in the sensorimotor cortex of the anaesthetized cat. AB - The effects on the EEG rhythms recorded from the sensorimotor cortex (post sigmoid gyrus) of anaesthetized cats were studied under 4 conditions of artificial mechanical hyperventilation (HV) before and after cervical bilateral vagotomy. In animals with intact vagus nerves, using visual examination, EEG changes were only observed within the 2nd min during HV produced by increased stroke volume (delta V) with associated hypocapnia. Quantitative EEG (qEEG) showed that, for the same increase in minute ventilation and the same degree of hypocapnia, delta V induced a greater and earlier relative decrease (2nd min) in the power density of delta, theta and alpha bands, than increased pump frequency (delta F). The delta F tests produced a fall only in the theta band and within the 3rd min. With constant paCO2, transient modifications occurred only with delta V and were limited to the first 30 sec. In bivagotomized cats, moderate EEG responses to delta V plus associated hypocapnia persisted partly in the alpha band. Finally, no changes appeared with delta V or delta F when the vagus nerves were cut and paCO2 was maintained constant. The present data suggest strongly that, in anaesthetized cats, peripheral vagal afferents from the respiratory system play a major role in the EEG changes caused by artificial hyperventilation. PMID- 1704840 TI - The cholinergic system and EEG slow waves. PMID- 1704841 TI - Electroencephalogram and seizures in chronic alcoholism. AB - CNS complications of chronic alcoholism are frequently difficult to assess due to the variety of direct and secondary conditions which can result from alcoholic drinking and lifestyles. The influence of alcoholism and alcohol-related factors on the EEG of patients with chronic alcoholism was studied in 213 patients (15.4% of all adults who had EEGs) using visual analysis. The influence of a variety of alcohol-related factors - drinking history, clinical complications, traumatic head injuries, head CT findings and laboratory results - on the EEG and alcohol related seizures was studied. The effect of EEG results on the decision to treat alcohol-related seizures was also assessed. 152 of the patients had seizures, mostly (90% of those with defined seizure types) generalized tonic-clonic seizures. 53% of all seizures occurred in the early withdrawal period (8 h to 7 days abstinence). A history of partial seizures was significantly associated with findings of focal EEG abnormalities, a history of head injuries and structural lesions on CT. The clinical significance of these findings was unclear, however, as the majority of patients who had focal EEG abnormalities or structural brain lesions still appeared to have generalized withdrawal seizures. The EEG and CT appeared to be complementary tests: for most patients, focal abnormalities were demonstrated on only one of the two tests. The majority of patients (56%) with normal EEGs had predominantly low voltage recordings (less than 25 muV), compared with 13.9% of 1167 patients without a history of alcoholism (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704842 TI - CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes. AB - We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d [3H]Urd into DNA. This was compared with similar liposomes targeted to MHC encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4 targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly. PMID- 1704843 TI - Predictive value of colony-forming unit assays for engraftment and leukemia-free survival after transplantation of chemopurged syngeneic bone marrow in rats. AB - We evaluated the efficacy of in vitro clonogenic assays for acute myeloid leukemia (AML) (CFU-Leuk) and granulocyte-macrophage progenitor cells derived from normal bone marrow (BM) (CFU-GM) to predict hematopoietic engraftment, median survival time (MST) and leukemia-free survival (LFS) in LBN rats that received injections of untreated or drug-treated AML and/or normal BM cells. Injection of untreated AML cells resulted in a log-linear relationship between AML cell dose and time of death from leukemia; LBN rats given 10(6) cells died with AML (MST, 24 days; range, 19-28) after injection. A minimum of 0.5-1.0 X 10(6) untreated normal BM cells was needed to insure satisfactory hematopoietic reconstitution in at least 50% of lethally irradiated LBN rats. After ex vivo incubation with graded concentrations of 4-hydroperoxycyclophosphamide (4HC) or bleomycin (BLEO), LBN AML or normal BM cells were cultured for CFU-Leuk or CFU-GM and injected into untreated or lethally irradiated syngeneic recipients. Over a variety of drug concentrations (4HC, 3-30 micrograms/ml; BLEO, 100-10,000 mU/ml) and cell doses (10(6)-10(7)/animal) examined, the log-kill estimates derived from in vitro CFU-Leuk assays correlated with the observed MST or LFS. Recovery of greater than 1% CFU-GM from 4HC- or BLEO-treated suspensions of normal BM was associated with satisfactory engraftment in lethally irradiated LBN rats. Clonogenic assays also predicted for engraftment and LFS in animals that received mixtures of AML and normal BM cells (1:10) treated with 4HC and/or BLEO. We conclude that CFU-Leuk and CFU-GM assays are useful screening techniques to develop and evaluate strategies for ex vivo purging with chemotherapeutic agents in this preclinical model of autologous marrow transplantation for AML. PMID- 1704844 TI - Survival and retrovirus infection of murine hematopoietic stem cells in vitro: effects of 5-FU and method of infection. AB - Gene replacement therapy for diseases of the hematopoietic system requires efficient gene transfer to pluripotent hematopoietic stem cells. We have systematically compared a number of protocols for retrovirus-mediated gene transfer into murine repopulating hematopoietic stem cells. Recipients of infected bone marrow cells were analyzed for the presence of the transduced provirus 4 months after transplantation. Our results show that 5-fluorouracil (5 FU) pretreatment of donor animals was required for efficient gene transfer and that 5-FU-treated bone marrow retained more repopulating activity in culture than untreated bone marrow. A comparison of retrovirus-mediated gene transfer by co cultivation of bone marrow cells with retrovirus producer cells as opposed to gene transfer by culturing bone marrow cells in retrovirus-containing supernatant revealed that gene transfer by cocultivation was more efficient than supernatant infection. However, the repopulating ability of bone marrow cells cocultured with retrovirus producer cells was reduced compared to bone marrow cells cultured in virus-containing medium. PMID- 1704845 TI - Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages. AB - The cDNA for human stem cell factor (hSCF) has been cloned and expressed in mammalian and bacterial hosts and recombinant protein purified. We have examined the stimulatory effect of recombinant human SCF (rhSCF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors (CSFs) and erythropoietin (rhEpo). RhSCF alone resulted in no significant colony formation, however, in the presence of rhGM-CSF, rhG-CSF or rhIL-3, rhSCF stimulated a synergistic increase in colony numbers. In addition, increased colony size was stimulated by all combinations. The morphology of cells in the colonies obtained with the CSFs plus rhSCF was identical to the morphology obtained with rhGM-CSF, rhG-CSF or rhIL-3 alone. RhEpo also synergised with rhSCF to stimulate the formation of large compact hemoglobinized colonies which stained positive for spectrin and transferrin receptor and had a morphological appearance consistent with normoblasts. RhSCF stimulation of low density non-adherent, antibody depleted, CD34+ cells suggests that rhSCF directly stimulates progenitor cells capable of myeloid and erythroid differentiation. PMID- 1704846 TI - Reciprocal heterotopic callosal connections between the two striate areas in Tupaia. AB - WGA-HRP injections were placed into area 17 close to the border with area 18 of Tupaia belangeri in order to study the callosal connections of the striate area in this animal. Most callosal neurons were found in the striate cortex (57.6 86.9%), some in the extrastriate area 18 (10.6-28.1%), and a few in even more temporal regions (2.5-14.3%). Concerning only the area 17, reciprocal homotopic connections could be observed as a strip along the area 17/18 border. Additionally, heterotopic callosal connections could be seen in regions representing the binocular visual field, especially the lower part. The area 17 cells were mostly located in the supragranular layers II and III (94.1-97.2%). But neurons could also be found in the infragranular layers, especially layer VI (2.6-5.2%) and in layer IV (0.2-1.1%). Homotopic projections were mostly seen in layers IIIc and V. The majority of the supragranular and infragranular neurons are pyramidal cells. However, a newly defined subpopulation of neurons, most probably stellate cells, were discovered forming a band in sublayer IIIc, very close to the layer III/IV border. PMID- 1704848 TI - Immunocytochemical study of the subcommissural organ of rats with induced postnatal hydrocephalus. AB - The subcommissural organ (SCO)-Reissner's fiber (RF) complex of rats suffering from postnatal hydrocephalus was investigated immunocytochemically (peroxidase antiperoxidase technique) by use of an anti-serum against bovine RF. Hydrocephalus was induced by injecting kaolin into the cisterna magna or by intracerebral infection with Borna disease virus. The kaolin-injected, hydrocephalic male rats were divided into two groups: (1) possessing an open communication between the fourth ventricle and the central canal of the spinal cord; (2) enduring an obliteration of this communication. In the latter group of rats the dilation of the ventricular cavities was far greater than in the former group. The Borna disease virus-infected female rats developed a severe hydrocephalus although in these animals all ventricular cavities and the central canal were in fully open communication. All rats belonging to the above-mentioned three groups displayed essentially the same alterations of their SCO-RF complex: (i) A reduction in the size of SCO and in the height of the ependymal secretory cells. (ii) A progressive disappearance of the immunoreactive hypendymal cells. (iii) The amount of AFRU-immunoreactive secretory material located in the rough endoplasmic reticulum was reduced. (iv) In contrast, the amount, location and immunoreactivity of the apical secretory granules did not undergo variations in comparison to sham-operated rats. (v) In the area of the SCO the layer of pre-RF material was thin or missing and a RF was not formed, and thus the central canal was also free of such secretory products. (vi) Clusters of AFRU-immunoreactive material were found attached to the wall of the Sylvian aqueduct.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704847 TI - Striatonigral GABA, dynorphin, substance P and neurokinin A modulation of nigrostriatal dopamine release: evidence for direct regulatory mechanisms. AB - The striatonigral pathway contains several neurotransmitters which may regulate the activity of the nigrostriatal dopamine projection in the rat. This was investigated by measuring extracellular dopamine levels in the striatum, using microdialysis, after injections of GABA (300 nmol/0.2 microliters), dynorphin A (0.5 nmol/0.2 microliters), substance P (0.07 mnol/0.2 microliters) or neurokinin A (0.09 nmol/0.2 microliters) into the ipsilateral substantia nigra, pars reticulata (SNR). Intranigral injections of GABA or dynorphin A inhibited, while intranigral injections of substance P or neurokinin A stimulated dopamine levels in the ipsilateral striatum. In rats with ibotenic acid lesions (2.5 micrograms/0.5 microliters) in the SNR, intranigral injections of GABA or dynorphin A inhibited, while intranigral injections of substance P or neurokinin A stimulated dopamine levels in the ipsilateral striatum. These responses were not significantly different than those in unlesioned rats. Analysis of the intranigral lesion with in situ hybridization revealed a heavy loss of glutamic acid decarboxylase mRNA expression in the SNR and a significant loss of tyrosine hydroxylase (TH) mRNA expression in the SNC. Immunohistochemical analysis revealed a disappearance of TH-Like immunoreactivity (LI) im dendrites in the SNR, a considerable loss of TH-LI cell bodies in the SNC and a restricted loss of neuropeptide K-LI in the SNR around the tip of the injection cannula. Furthermore, lesioned rats rotated ipsilateral to the lesion after apomorphine (1 mg/kg, s.c.), indicating that the basal ganglia output mediated via the SNR GABA neurons was impaired on the lesioned side. Analysis of the striatum revealed that a dense TH-LI fiber network could still be seen on the lesioned side. Furthermore, basal and amphetamine stimulated extracellular dopamine levels in the striatum on the lesioned side were not significantly depleted. This indicates that the ascending nigrostriatal dopamine projection was functionally intact on the lesioned side. These findings indicate that intranigral GABA, dynorphin A, substance P and neurokinin A modulation of ipsilateral striatal dopamine release is mediated via direct action on the nigrostriatal projection. Thus, it is suggested that the striatonigral pathway, which contains GABA, dynorphin, substance P and neurokinin A, exerts a direct regulatory effect on the activity of the nigrostriatal dopamine projection. PMID- 1704849 TI - Grafts of dissociated cerebellar cells containing Purkinje cell precursors organize into zebrin I defined compartments. AB - A prominent feature of the mammalian cerebellum is its organization into parasagittal compartments. One marker of such compartments is the zebrin I molecule that is expressed by bands of Purkinje cells (PC). In order to understand better the basis for the development of this organization, we have transplanted dissociated rat cerebellar anlage, taken during the period of proliferation of PC precursors, into kainic acid lesioned adult rat cerebellum. As previously observed, the resultant grafts exhibited trilaminar structures reminiscent of the normal cerebellum. In every case, the PC in the resultant grafts were organized into zebrin I+ and - compartments. In one case, most of the grafted PC were integrated into a region of PC deficient host molecular layer that was induced by pretreatment with kainic acid. Clear bands defined by zebrin I reactivity were seen where groups of the grafted PC had entered the host molecular layer. These bands did not correlate in distribution or size with host bands. Hypotheses compatible with these findings that involve specific and non specific aggregation of PC are discussed. PMID- 1704850 TI - The topographic relationship between shifting binocular maps in the developing dorsal lateral geniculate nucleus. AB - The major mammalian subcortical visual structures receive topographically ordered projections from both eyes. In the adult dorsal lateral geniculate nucleus (dLGN) each projection terminates in separate restricted regions of the nucleus. This pattern is different during development. Initially in ferrets the projections from each eye to the dLGN overlap throughout this structure. Although the projections do not occupy regions that are appropriate given the adult pattern, they are both retinotopically organised. Consequently, the formation of the adult pattern requires that the two retinotopic projections shift in relation to one another. The experiments undertaken here on the newborn ferret demonstrate the relationship between the two unsegregated projections in terms of their retinal origin and relative pattern of projection to the dLGN. By establishing the relationship between the projections at this stage of development it is possible to determine the relative changes that must be made between them in order to bring about the adult pattern of registration. By mapping the two unsegregated projections with a combination of retinal lesions and anterograde tracing methods it is demonstrated that at birth the ipsilateral projection arises from the temporal retina, and the contralateral projection arises from the entire retina. Because of the significant contralateral projection from the temporal retina the relatively sharp nasotemporal division found in the adult is not present at this stage. This element of the contralateral projection maps in continuity with the rest of this projection and terminates at the caudal pole of the nucleus. However, it is probably lost before the adult pattern has clearly started to develop.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704851 TI - Regulation of mRNA levels of alkaline phosphatase gene in neutrophilic granulocytes by granulocyte colony-stimulating factor and retinoic acid. AB - We examined steady-state levels of mRNA for alkaline phosphatase in neutrophils (NAP) treated with granulocyte colony-stimulating factor (G-CSF). The amount of mRNA for NAP was shown to increase after 6 hours of culture with G-CSF when no increase in NAP activity was yet observed, and the transcript was the greatest after 20-24 h of culture with G-CSF. Treatment of neutrophils with both G-CSF and retinoic acid augmented the amount of mRNA for NAP over the amount obtained by G CSF alone, which was most marked at 24 h. These results show that both G-CSF mediated NAP induction and its potentiation by retinoic acid are due to the increased levels of mRNA for NAP. PMID- 1704852 TI - In vitro functions of neutrophils induced by treatment with rhG-CSF in severe congenital neutropenia. AB - Neutrophils (and monocytes) from 5 patients suffering from severe congenital neutropenia (SCN) were investigated in vitro after induction of this cell type by treatment with recombinant human granulocyte colony-stimulating factor (rh G-CSF) in vivo. Some abnormal morphological features were seen, such as anomalies of nuclei of phagocytes. No limitations were found 1) in the ability of neutrophils and monocytes to produce reactive oxygen intermediates (ROI) following stimulation with phorbol-myristate-acetate (PMA), 2) in the ability of neutrophils to phagocytose particles of zymosan A and to produce ROI simultaneously and 3) in the ability to kill bacteria of the species staphylococcus aureus. However the specific migration of neutrophils in a gradient of FMLP under soft agar was decreased to approximately 50% in all patients tested as compared to healthy controls. In addition, spontaneous motility was decreased in one patient. Nevertheless, the good clinical improvements of patients suffering from SCN after treatment with rh G-CSF appeared to be due to induction of neutrophils displaying overall good functional activities with respect to natural defense. PMID- 1704853 TI - A liquid culture method for the in vitro growth of hemopoietic progenitor cells from normal human adult peripheral blood allowing for analysis by multiparameter flow-cytometry. AB - A liquid culture method has been developed allowing for the in vitro growth of peripheral blood-derived hemopoietic progenitor cells of myeloid, erythroid, monocytic and megakaryocytic lineages. Adherent cell- and CD21-positive cell depleted PBMC from normal subjects have been cultured in the presence of rhEPO, rhGM-CSF or rhIl-3. Culturing cells in liquid cultures and in plasma clots, a similar dose-response was observed for granulocytic cells/liquid culture and granulocytic colonies/plasma clot with rhGM-CSF, and also for erythroid cells/liquid culture and erythroid colonies/plasma clot with rhEPO. Comparing serum- liquid cultures to serum+ liquid cultures, the ratio of CD13+ cells to CD15+ cells was higher in serum- cultures, indicating a maturation arrest of myecloid cells with serum deprivation. Using dual-colour flow-cytometry, cell cycle analysis of CD13+ cells, comparing the effects of rhGM-CSF to those of rhIl 3, have been performed. The liquid culture method promises to be a useful tool for the study of in vitro differentiation and proliferation of hemopoietic progenitor cells. PMID- 1704854 TI - Why families of children with biological deficits require a systems approach. AB - A number of assumptions related to the systemic therapists' view of family functioning in the case of biological deficit in the child are presented and challenged. The families' behavior, it is argued, may not be primarily responsible for the difficulties of their biologically impaired offspring. Severity of handicap need not be directly linked to stress in these families, nor is avoiding an individual diagnosis helpful to them. To further clarify the relevant issues, the family's "reactions" to a dysfunctional member, along with the professionals' previously unhelpful approaches to dealing with the impairment of these children, are presented. Finally, the therapeutic implications of the position presented in this article are drawn, and specific recommendations for working with these families are offered. PMID- 1704855 TI - Induction of morphological change by tyrosine kinase inhibitors in Rous sarcoma virus-transformed rat kidney cells. AB - Erbstatin and methyl 2,5-dihydroxycinnamate, related tyrosine kinase inhibitors, induced a morphological change in temperature-sensitive Rous sarcoma virus transformed rat kidney (RSVts-NRK) that brought the cells close to the morphology of their normal counterpart. Erbstatin did not change the morphology of normal or Kirsten sarcoma virus-transformed rat kidney cells. Erbstatin also inhibited morphological transformation of RSVts-NRK cells induced by a shifting in temperature. Actin stress fibres were observed only in normal cells and not in transformed cells. Erbstatin induced stress fibre organization in transformed cells. Erbstatin and methyl 2,5-dihydroxycinnamate increased fibronectin gene expression in RSV-transformed cells. Thus, tyrosine kinase inhibitors induced normal phenotypes specifically in v-src-expressing cells. PMID- 1704856 TI - Identification of the protein and cDNA of the cardiac Na+/H+ exchanger. AB - We examined the myocardial form of the Na+/H+ exchanger. A partial length cDNA clone was isolated from a rabbit cardiac library and it encoded for a Na+/H+ exchange protein. In comparison with the human Na+/H+ exchanger, the sequence of the 5' end of the cDNA was highly conserved, much more than the 3' region, while the deduced amino acid sequence was also highly conserved. To further characterize the myocardial Na+/H+ exchange protein, we examined Western blots of isolated sarcolemma with antibody produced against a fusion protein of the Na+/H+ exchanger. The antibodies reacted with a sarcolemma protein of 50 kDa and with a protein of 70 kDa. The results show that the rabbit myocardium does possess a Na+/H+ exchanger protein homologous to the known human Na+/H+ exchanger. PMID- 1704857 TI - Effects of the general anaesthetic Propofol on the Ca2(+)-induced permeabilization of rat liver mitochondria. AB - The molecular mechanism of the Ca2(+)-induced permeabilization of rat liver mitochondria was evaluated by studying a new effect of the commonly used general anaesthetic Propofol (2,6-diisopropylphenol). The compound was found to induce an apparent uptake of Ca2+ at steady-state in the Ca2(+)-distribution between the medium and the mitochondria, and to inhibit swelling and release of accumulated Ca2+ induced by inorganic phosphate, t-butyl hydroperoxide, diamide or FCCP plus Ruthenium red. The compound did not stimulate the activity of the Ca2(+) uniporter and it is concluded that the effects seen are due to the inhibition of the Ca2(+)-dependent, unspecific permeability increase. The results suggest two mechanisms whereby Propofol stabilizes the mitochondrial membrane in the presence of Ca2+: (i) by interaction with the putative pore, thus causing its closure; and (ii) by scavenging of free radicals thus inhibiting its opening during oxidative stress. PMID- 1704858 TI - The 5-55 single-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor. AB - An analogue of the BPT1 folding intermediate that contains only the disulphide bond between Cys-5 and Cys-55 has been prepared by mutation of the other four Cys residues to Ser. On the basis of its circular dichroism and 1H-nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding. PMID- 1704859 TI - Characterization of a chicken polyubiquitin gene preferentially expressed during spermatogenesis. AB - We have previously reported that a chicken polyubiquitin gene (Ub II) not expressed under normal or heat shock conditions in chick fibroblasts is transcribed during spermatogenesis [(1987) Nucleic Acids Res. 15, 9604]. The level of Ub II mRNA is several-fold higher in testis cells than in somatic tissues. The gene Ub II possesses characteristic features not seen in the polyubiquitin gene expressed in heat shock conditions (Ub I). The 5' noncoding region of Ub II shows the consensus cAMP regulatory element (CRE) followed immediately downstream by a CA dinucleotide. It has been proposed that this extended CRE may be involved in the coordinate expression of various genes during spermatogenesis. PMID- 1704860 TI - Carcinosarcoma of the lung. A case-history of disseminated disease and review of the literature. AB - A case of disseminated pulmonary carcinoma is presented. Metastases of both carcinoma and sarcoma were confirmed via light microscopy and immunohistological examination. Our observations on this rare tumour are compared with a review of the literature. PMID- 1704861 TI - Alternate expression of the HNK-1 epitope in rhombomeres of the chick embryo. AB - Rhombomeres are regarded as the manifestation of innate segmentation within the vertebrate CNS. To investigate developmental changes occurring in the CNS and PNS, a series of chick embryos were immunostained with several monoclonal antibodies. The HNK-1-immunoreactivity (IR) appeared in rhombomeres (r) 3 and r5 around stage 15, when r2 and r4 were not stained. This alternate pattern is similar to the Krox-20 gene expression in the mouse embryo. At levels of r2 and r4, HNK-1+ neural crest cell masses were attached to the CNS forming cranial sensory ganglia. In these rhombomeres, an accumulation of neuroepithelial cells near the cranial nerve root and early development of neuroblasts in the basal plate were observed. The above observations seem to suggest that the alternate HNK-1-IR in rhombomeres might be related to the expression of cell adhesion molecules, and therefore also to the adhesion of the cranial ganglion precursors to the CNS, which takes place every other rhombomere in the preotic region. Thus, the alternate pattern of the HNK-1-IR seems to be related to the morphogenesis of preotic branchial nerves. PMID- 1704862 TI - Expression of heat-shock locus hsr-omega in nonstressed cells during development in Drosophila melanogaster. AB - The hsr-omega locus forms one of the largest Drosophila heat-shock puffs and produces three major transcripts. These three transcripts are also produced constitutively, at lower levels, in almost all tissues and developmental stages. The amounts of the transcripts in nonstressed cells are modulated during development. The hormone ecdysone leads to increased levels of hsr-omega transcripts in cultured cells, suggesting that changing ecdysone titers may play a role in the developmental changes of hsr-omega transcript levels. By in situ hybridization to RNA in tissue sections, we detect only two cell types that lack hsr-omega transcripts--the preblastoderm embryo and the primary spermatocyte. There are no maternal transcripts of hsr-omega in the embryo. Transcripts appear abruptly at the time that the zygotic genome becomes transcriptionally active, shortly before the formation of the cellular blastoderm. No constitutive hsr omega transcripts are found in primary spermatocytes. The spermatocytes cannot respond to heat shock by transcribing either hsr-omega or hsp70 RNA. Constitutive hsr-omega transcription is resumed later in spermatogenesis and hsr-omega RNA is detected in differentiating spermatids. These spermatids are also capable of mounting a heat-shock response, as measured by increases in hsr-omega and hsp70 RNA. PMID- 1704863 TI - Magnetic resonance imaging in children with spastic diplegia: correlation with the severity of their motor and mental abnormality. AB - Magnetic resonance imaging (MRI) findings for 34 children with spastic diplegia, examined between two and 10 years of age, were analysed. Dilatation of the trigone, atrophy of the peritrigonal white matter and prominent deep cortical sulci were seen. On T2-weighted images, periventricular high-intensity areas in the white matter adjacent to the trigones and bodies of the lateral ventricles were seen in many children. These MRI features may reflect the pathological changes of periventricular leukomalacia in children with spastic diplegia. Among the MRI findings, only the amount of white matter correlated with severity of disability: white matter reduction corresponded to the more severe motor disabilities. PMID- 1704864 TI - Clumsiness in children--do they grow out of it? A 10-year follow-up study. AB - The question of whether problems of motor co-ordination in early childhood recede with age has rarely been addressed. This paper reports the findings from a follow up study of 17 children, identified by their teachers as having poor motor co ordination at age six. Now age 16, these children and their matched controls completed a battery of assessments. The results suggest that the majority of children still have difficulties with motor co-ordination, have poor self-concept and are experiencing problems of various kinds in school. However, there are individual differences in the extent to which the children have learned to cope with their continuing difficulties over the years. PMID- 1704865 TI - [Anti-androgen therapy in the female]. AB - Besides the various androgenic activities actions in women, signs of androgenisation such as acne, seborrhoe, hirsutism and alopecia, play an important clinical role and require a proper diagnostic evaluation and individualised medical treatment. After development and thorough clinical evaluation of antiandrogen, a variety of therapeutic possibilities have become available. The main antiandrogens are: Cyproterone acetate, chlormadinone acetate and spironolactone. They are used in mono- or combination-therapy regimens. Means of application (oral, parenteral), dosages used and different forms of combination therapies are presented under practical aspects. The effects of such treatment modalities are outlined. PMID- 1704866 TI - [Giant condyloma (Buschke-Lowenstein) of the vulva]. AB - Diagnosis, therapy and biological behaviour of the Buschke-Lowenstein tumour of the vulva are described. Surgical treatment is accompanied by an antiviral therapy with interferon. Viral genesis of HPV 6 and/or HPV 11 is probably the cause of this intermediate tumour a type between condyloma and invasive squamous cell carcinoma. PMID- 1704867 TI - Carbon tetrachloride-induced alterations of hepatic calmodulin and free calcium levels in rats pretreated with chlordecone. AB - Calmodulin, a low molecular weight Ca2+ binding protein, regulates a large number of cell activities including cell division. Previous studies from our laboratory indicated excessive accumulation of Ca2+ in hepatocytes succeeded by rapid glycogen breakdown and suppressed cell division in rats receiving CCl4 after previous dietary exposure to 10 ppm chlordecone. Since calmodulin plays a major role in Ca2(+)-regulated events and has been reported to be localized in mitotic apparatus during cell division, we have assessed subcellular distribution of calmodulin and estimated cytosolic phosphorylase a to indicate cytosolic free Ca2+ levels in livers of rats fed 0 ppm or 10 ppm (chlordecone) in the diet for 15 days before CCl4 (100 microliters/kg) administration to understand the role of Ca2(+)-calmodulin in chlordecone + CCl4 toxicity. Hepatotoxicity was assessed by determining serum AST and ALT succeeded by histopathological observations of liver sections. Serum aminotransferases were significantly elevated 6 hr after CCl4 administration to normal rats and returned to control level by 24 hr. However, serum AST and ALT elevations were severalfold higher, and progressive increase was observed starting 4 hr after CCl4 administration to chlordecone rats. Histopathological observations of liver sections for necrotic, swollen and lipid-laden cells provided findings commensurate with the serum enzyme data. These data indicate that normal rats do recover from CCl4 hepatotoxicity. However, the CCl4 hepatotoxicity is progressive in chlordecone rats without recovery. In normal rats, CCl4 administration resulted in a slight increase in phosphorylase a starting at 6 hr.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704868 TI - Recognition of major histocompatibility complex antigens on cultured human biliary epithelial cells by alloreactive lymphocytes. AB - We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-gamma-interferon monoclonal antibody. In addition, recombinant human gamma-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell-induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody-blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID- 1704869 TI - CAVBP/DEP alternating chemotherapy for the treatment of intermediate and high grade non Hodgkin's lymphoma: final results of a pilot study. AB - Between May 1984 and September 1986, 40 patients with intermediate or high grade non Hodgkin's lymphoma were treated with a novel protocol, which alternated a conventional regimen consisting of cyclophosphamide, doxorubicin, vincristine, bleomycin, and prednisone (CAVBP) with a second chemotherapy regimen, including two drugs with newly discovered activity against lymphomas, such as cis-platin, etoposide, and prednisone (DEP). Twenty-one patients (52.5 per cent) achieved a complete response, 11 patients (27.5 per cent) had a partial response. Eight of the 21 complete responders (38 per cent) relapsed 5 to 24 months after completion of treatment. With a median follow-up of over 40 months, 22 patients are alive, six with disease and three in a second complete response after salvage chemotherapy. Factors negatively associated with response included 'B' symptoms, advanced stage of disease, bulky tumour, poor performance status, number of extranodal sites of disease. 'B' symptoms, bulky tumour, and poor performance status were also negatively associated with survival. Toxicity was modest, with no treatment-related deaths and only six cases of severe leukopenia. The results of this pilot study do not justify comparison of CAVBP/DEP with more efficacious regimens in prospective, randomized trials. PMID- 1704871 TI - BHC induced testicular impairments in rats. AB - Benzene hexachloride (BHC) was fed to mature male rats weighing 160 g at dosages of 3 and 6 mg/kg body weight over a period of 180 days. Significant decrease in testicular weight and degeneration of seminiferous tubules with deformed spermatogenic cells were noted at a dose of 6 mg/kg BHC. Marked increase in BHC residue in testis revealed that the drug was able to cross blood-testis barrier. PMID- 1704870 TI - Protein tyrosine phosphatase domains from the protochordate Styela plicata. AB - Protein tyrosine phosphorylation is an important regulatory mechanism in cell physiology. While the protein tyrosine kinase (PTKase) family has been extensively studied, only six protein tyrosine phosphatases (PTPases) have been described. By Southern blot analysis, genomic DNA from several different phyla were found to cross-hybridize with a cDNA probe encoding the human leukocyte common antigen (LCA; CD45) PTPase domains. To pursue this observation further, total mRNA from the protochordate Styela plicata was used as a template to copy and amplify, using polymerase chain reaction (PCR) technology, PTPase domains. Twenty-seven distinct sequences were identified that contain hallmark residues of PTPases; two of these are similar to described mammalian PTPases. Southern blot analysis indicates that at least one other Styela sequence is highly conserved in a variety of phyla. Seven of the Styela domains have significant similarity to each other, indicating a subfamily of PTPases. However, most of the sequences are disparate. A comparison of the 27 Styela sequences with the ten known PTPase domain sequences reveals that only three residues are absolutely conserved and identifies regions that are highly divergent. The data indicate that the PTPase family will be equally as large and diverse as the PTKases. The extent and diversity of the PTPase family suggests that these enzymes are, in their own right, important regulators of cell behavior. PMID- 1704872 TI - Characterization by somatic cell genetics of a monoclonal antibody to the MDR1 gene product (P-glycoprotein): determination of P-glycoprotein expression in multi-drug-resistant KB and CEM cell variants. AB - We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells. PMID- 1704873 TI - Isolation and characterization of a serologically defined tumour membrane antigen from a chemically-induced rat sarcoma, HSN. AB - Polypeptides containing the tumour antigenic determinant present on the external domain of a membrane antigen of the 3-4 benzpyrene-induced rat fibrosarcoma HSN have been isolated and purified. Following cleavage from intact cells with trypsin, the peptides were purified by immunoaffinity chromatography and SDS PAGE. Three polypeptides of molecular weight 120, 45 and 42 kDa were obtained that bound the specific rat monoclonal antibody (MAb) 11/160 in Western blots. Chemical analyses of the 45-kDa fragment yielded the following sequence: NH2-I V F P H G S L M V I L E H T Q K P All 14 of the syngeneic MAbs prepared from rats bearing the HSN tumour competed with each other to bind to the specific antigen. Western blots of the purified tryptic fragments probed with 125I-AbI revealed that, while some of the MAbs (11/160, ALN/12/17 and ALN/9/94) recognized a sequential determinant, others (ALN/11/53, ALN/16/53, and AL/3/12) bound to a conformational epitope. It is concluded that the AbI bind to distinct but overlapping epitopes. PMID- 1704874 TI - Modifying effects of preexisting pulmonary fibrosis on biological responses of rats to inhaled 239PuO2. AB - We investigated the modifying effects of preexisting, bleomycin-induced pulmonary fibrosis on the deposition, retention, and biological effects of inhaled 239PuO2 in the rat. Among rats exposed to similar airborne concentrations of 239PuO2, initial lung burdens of 239Pu per kilogram body mass were similar whether or not pulmonary fibrosis was present. However, clearance of 239Pu from the lungs was significantly decreased in the rats with preexisting pulmonary fibrosis. The incidence of lung lesions (epithelial hyperplasia, diffuse macrophage increases and aggregation, and loose and dense connective tissue) was significantly greater among rats with preexisting pulmonary fibrosis than among the exposed controls. Rats with preexisting fibrosis had shorter life spans than 239PuO2-exposed control rats. When groups of rats with similar alpha doses to the lungs were compared, the incidences of neoplastic lesions in the lung, the times to death of rats with lung neoplasms, and the risk of lung tumors per unit of alpha dose to the lungs in rats with or without pulmonary fibrosis were similar. The results of this study suggest that humans with uncomplicated pulmonary fibrosis may not be more sensitive to the carcinogenic effects of inhaled 239PuO2 than are individuals with normal lungs, assuming that the total alpha doses to the lungs are similar. PMID- 1704875 TI - In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA. AB - A specific method for in situ-hybridization of slow myosin heavy chain MHCI (beta cardiac MHC) mRNA was established with the use of a nonradioactively labeled cRNA probe. The digoxigenin-labeled probe was the T7-RNA polymerase transcript from a 350 bp SacI fragment of a rabbit beta-cardiac MHC cDNA. Northern blot analyses of RNA preparations from skeletal and cardiac muscles with homologous and complementary RNA proved the specificity of the hybridization. The in situ hybridization was applied for studying the distribution of MHCI mRNA in normal fast- and slow-twitch muscles, as well as in muscles undergoing fast-to-slow transformation by chronic low-frequency stimulation. The majority of soleus muscle fibers was intensely stained, whereas fast-twitch muscles contained only a few positive fibers. The intracellular distribution of the hybridization product showed a clear relationship to the nuclei with intense staining of the perinuclear regions within the subsarcolemmal space. The more intensely stained fibers of transforming muscle displayed hybridization product also within the nuclei. As revealed by inspection of longitudinal sections at high magnification and polarized light, MHCI mRNA was also detectable in the myofibrils in a cross striational pattern resulting from staining of the I-bands. PMID- 1704876 TI - Capillary area in early low-dose streptozocin-treated mice. AB - It has been reported that vasoconstriction of intra-islet capillaries plays an important role in the initiation of the insulitis seen in the islets of Langerhans of diabetic animals. Nevertheless, only a few studies have concentrated on islet vessels. This led us to perform an experiment with the aim to compare the islet capillary area of normal untreated and multiple low-dose streptozocin (LDS) (40 mg/kg b.wt. i.p./5 days)-treated mice. In order to identify endothelial cells a method devised by Gomori, based on the fact that these cells present alkaline phosphatases on their surface, was used. Results revealed that in LDS-treated animals the capillary area per islet is significantly reduced when compared to the vascular area of controls (p less than 0.05). This could be due to a vasoconstriction phenomenon that occurs in the islet capillaries after the streptozocin administration and before the appearance of any inflammation. Our findings could demonstrate that vasoconstriction events are involved in initiation of the diabetic disease. PMID- 1704877 TI - Myofibrillar M-band proteins in rat skeletal muscles during development. AB - The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units. PMID- 1704878 TI - Bleomycin chemotherapy for metastatic squamous cell carcinoma in a ferret. AB - Bleomycin, an antitumor antibiotic, was effective in temporarily reducing the size of a metastatic squamous cell carcinoma in a ferret. The tumor had recurred after previous excision and had metastasized to the right submandibular lymph node. The ferret was treated without effect at a dosage of 10 U/m2, sc, once a week. The dosage was increased to 20 U/m2 once a week, and reduction of tumor size was observed. PMID- 1704879 TI - Chemotherapy for stage IV non-small cell lung cancer. AB - Chemotherapy regimens with consistent activities in NSCLC have been identified. A modest but statistically significant increase in survival has been shown in a single large randomized trial, with three smaller trials showing either similar results or a trend toward an improved survival. However, chemotherapy-related toxicity remains significant and seems exacerbated in patients with a poor performance status at the time of diagnosis. Identification of chemotherapy regimens with higher activity in NSCLC remains a necessity, and the treatment of stage IV patients on clinical trials should be encouraged. Outside the context of a clinical trial, chemotherapy is indicated in selected stage IV patients with good performance status in an attempt to palliate their symptoms and increase their survival. PMID- 1704880 TI - Palliative radiotherapy. AB - We have reviewed the role of radiation therapy in the palliative treatment of patients with non-small cell lung cancer. The use of radiation treatment results in effective palliation of chest symptoms such as dyspnea, cough, hemoptysis, and chest pain. In addition, the pain and suffering associated with skeletal and hepatic metastases are effectively alleviated by radiation therapy with minimal morbidity. Devastating neurologic complications can be avoided or alleviated in a great proportion of patients undergoing radiation therapy for cerebral metastases and spinal cord compression. Therefore, radiation therapy is a potent modality in relieving or reducing the suffering of patients with lung cancer. This is also a modality that has wide applicability; very few patients are not suitable candidates for that has wide applicability; very few patients are not suitable candidates for treatment regardless of their performance status. The aim of the treatments should always be prompt intervention using radiation therapy schedules that will minimize treatment time yet produce the desired results in a high proportion of patients. Protracted radiation schedules are not warranted in such patients except in special clinical situations. Palliation with radiation therapy is achieved quite promptly, with minimal side effects and a very small risk of any long-term consequences in patients who have a limited life expectancy. PMID- 1704881 TI - Supportive care of the lung cancer patients. AB - The care of lung cancer patients involves a specialized team approach. Complications arising from the underlying illness or its treatment are common occurrences that must be anticipated and treated properly. Psychosocial issues commonly arise in patients and their families during the course of the illness. Caregivers skilled in helping a patient and family members come to terms with this frequently fatal disease are integral members of a multidisciplinary team of cancer specialists. PMID- 1704882 TI - RNase T affects Escherichia coli growth and recovery from metabolic stress. AB - To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed. PMID- 1704883 TI - Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1. AB - The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster. PMID- 1704884 TI - Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product. AB - The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. PMID- 1704885 TI - Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus. AB - Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal. During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases. A DNA probe of the omega 4521 gene was constructed. Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal. A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation. To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated. All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521. Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg). sasA and sasB mutants differ phenotypically during growth and development. Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels. sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation. In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally. The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A signal-dependent regulation of omega 4521. PMID- 1704886 TI - Sequence analysis and expression of the Salmonella typhimurium asr operon encoding production of hydrogen sulfide from sulfite. AB - A chromosomal locus of Salmonella typhimurium which complements S. typhimurium asr (anaerobic sulfite reduction) mutants and confers on Escherichia coli the ability to produce hydrogen sulfide from sulfite was recently cloned (C. J. Huang and E. L. Barrett, J. Bacteriol. 172:4100-4102, 1990). The DNA sequence and the transcription start site have been determined. Analysis of the sequence and gene products revealed a functional operon containing three genes which have been designated asrA, asrB, and asrC, encoding peptides of 40, 31, and 37 kDa, respectively. The predicted amino acid sequences of both asrA and asrC contained arrangements of cysteines characteristic of [4Fe-4S] ferredoxins. The sequence of asrB contained a typical nucleotide-binding region. The sequence of asrC contained, in addition to the ferredoxinlike cysteine clusters, two other cysteine clusters closely resembling the proposed siroheme-binding site in biosynthetic sulfite reductase. Expression of lacZ fused to the asr promoter was repressed by oxygen and induced by sulfite. Analysis of promoter deletions revealed a region specific for sulfite regulation and a second region required for anaerobic expression. Computer-assisted DNA sequence analysis revealed a site just upstream of the first open reading frame which had significant homology to the FNR protein-binding site of E. coli NADH-linked nitrite reductase. However, asr expression by the fusion plasmid was not affected by site-specific mutations within the apparent FNR-binding site. PMID- 1704887 TI - Characterization of the late-gene regulatory region of phage 21. AB - A segment of Escherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages lambda and 82. The leader RNA is about 45 nucleotides long and consists essentially entirely of sequences encoding the p independent terminator that is the putative target of the antitermination activity of Q21. Like the corresponding regions of lambda and 82, the 21 late gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase. The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in trans, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of lambda and 82. There is little evident similarity in the primary sequences of the three Q genes. PMID- 1704888 TI - Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. AB - Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682 685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase. PMID- 1704889 TI - Molecular cloning of a novel human cdc2/CDC28-like protein kinase. AB - A homology probing approach was utilized to isolate a new human protein kinase. Deoxyoligonucleotide probes recognizing a conserved subdomain in the COOH terminal portion of protein kinases identified a cDNA clone encoding a putative kinase with predicted serine/threonine phosphorylation specificity. The full length, 1.7-kilobase pair cDNA hybridizes to 1.7- and 3.4-kilobase mRNA transcripts in a number of tissues. The size of the encoded protein is 454 amino acids and consists of an NH2-terminal 130-residue segment, which may represent a regulatory region, followed by a 324-residue catalytic domain. Comparisons and alignments of the primary sequence and predicted secondary structure of the catalytic region to other known kinases reveal that the new kinase, denoted "CLK" (for CDC-like kinase), represents a prototype for a new family of human protein kinases bearing significant homology to the yeast cdc2/CDC28 kinases that regulate the cell cycle. PMID- 1704890 TI - The epitopes of apolipoprotein A-I define distinct structural domains including a mobile middle region. AB - The definition of epitopes on apoA-I provides evidence for a very dense packing of the peptide chain and supports the proposed supersecondary structure, made of repetitive antiparallel helices. Screening with overlapping synthetic hexapeptides shows that only a few epitopes are continuous and that all continuous contact sequences identified within the epitopes coincide or are contiguous to a putative beta-turn, such as residues 1-8, 48-55, 98-104, 118-123, and 135-140. On the N-terminal half of apoA-I we identified 6 overlapping tertiary discontinuous epitopes, uniquely constituted by amino acids and discontinuous sequences on helical segments that are far apart and which define a particular region with a complex tertiary structure. Among these are the epitopes for antibody 5G6, which reacts with residues 45-51, 83-92, 119-126, and 136-143; for A16, which reacts with 14-19, 23-28, and 60-82; and for r-FC1, which reacts with 1-8, 29-35, 78-83, and 98-121. The very far apart discontinuous sequences included in these epitopes can be explained by the predicted turns and coiled domains, and thus provide evidence for such a tertiary structure. Alternatively, these results could also be explained by intermolecular epitopes involving the N terminal region. In contrast, in the middle of apoA-I, all identified epitopes are shorter and discontinuous within the secondary structure and are constituted by residues forming a beta-turn with all or part of an adjacent alpha-helix. We hypothesize that these multiple epitopes, that are mostly limited to a single helix, reflect the existence of a very mobile domain, possibly with hinged pairs of adjacent alpha-helices. On the C-terminal half of apoA-I, several monoclonal antibodies react with overlapping epitopes located between residues 149 and 186, which probably reflects 2 antiparallel alpha-helices interrupted by a beta-turn. PMID- 1704891 TI - Characterization of human glucocerebrosidase from different mutant alleles. AB - Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120--- Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme. PMID- 1704892 TI - Characterization of high affinity binding sites for charybdotoxin in human T lymphocytes. Evidence for association with the voltage-gated K+ channel. AB - Charybdotoxin (ChTX) inhibits with high affinity a voltage-gated K+ channel that is present in human T lymphocytes. In this system, 125I-ChTX binds specifically and reversibly to a single class of sites which display a Kd of 8-14 pM, as measured by either equilibrium or kinetic binding protocols. The maximum density of sites, 542 sites/cell, correlates well with the density of K+ channel as determined by electrophysiological experiments. Binding of 125I-ChTX is modulated by the ionic strength of the incubation media and by Ca2+. Increasing concentrations of either K+, Na+, or Ca2+ cause inhibition of toxin binding. Inhibition of binding by Ca2+ is due, primarily, to an effect on toxin dissociation rates. Increasing the pH of the external media from 6.8 to 8.5 enhances toxin binding, due to an increase in affinity with no significant effect on the maximum density of receptor sites. Different agents that block the voltage gated K+ channel in human T lymphocytes, inhibit toxin binding. Mitogen stimulated T cells display 2.5-3-fold increase in toxin binding as compared with unstimulated control cells. These data, taken together, suggest that 125I-ChTX binding sites identified in this study, represent the predominant voltage-gated K+ channel present in peripheral human T lymphocytes. Therefore, 125I-ChTX is a useful probe for elucidating the physiological role of this type of K+ channel. PMID- 1704893 TI - Positive and negative regulations of plasmid CoLIb-P9 repZ gene expression at the translational level. AB - Expression of the repZ gene involved in DNA replication of the ColIb-P9 plasmid depends on translation of a transcribed repZ leader sequence (repY) and is negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. To further understand the regulatory loop of repZ expression, we isolated and characterized replication-defective ColIb-P9 mutants that affected the level of repZ expression. Here we report that mutations occurring in two complementary sequences, one (5'GGCG3') in the inc region and one in the repY region, reduce the level of repZ expression without affecting transcription. The mutations in one complementary sequence were suppressed by compensatory base changes in the other sequence, restoring the ability of repZ expression. These results indicated that interaction by base pairing between the two complementary sequences of RepZ mRNA was essential for repZ translation. The two sequences, separated by 107 bases from each other, have a potential to form a novel pseudoknot in the RepZ mRNA leader. We also found that some mutations in the 5'GGCG3' sequence altered the specificity of Inc RNA, thereby reducing significantly its regulatory activity. Thus, this single specific sequence is involved in both positive and negative regulations for repZ expression. Possible regulatory mechanisms of repZ expression are discussed. PMID- 1704895 TI - Thermodynamics of A:G mismatch poly(dG) synthesis by human immunodeficiency virus 1 reverse transcriptase. AB - Human immunodeficiency virus 1 (HIV-1) reverse transcriptase has been found to conduct error-prone synthesis on DNA and RNA templates. We find here that tolerance of an A:G mispair with poly(rA) as template is particularly strong, such that extensive poly(dG) synthesis is conducted. This type of extensive misincorporation is not observed with several reference DNA polymerases. Surprisingly, HIV reverse transcriptase processivity and kcat for dGMP misincorporation and normal dTMP incorporation are about the same. However, the Km value for dGTP in poly(dG) synthesis is approximately 1000-fold higher than the Km for dTTP in poly(dT) synthesis. Comparison of thermodynamic parameters for dGMP misincorporation and normal dNMP incorporation indicates a lower energy of activation for dGMP misincorporation than for normal dNMP incorporation. Entropy of activation (delta S*) for normal dTMP incorporation is positive (approximately 10 cal/kmol), whereas delta S* for dGMP misincorporation is negative (-36 cal/kmol). Since differences in delta S* are usually considered to reflect differences in solvation for the transition state complex, these results are consistent with the interpretation that the active site of HIV reverse transcriptase is flexible enough to misincorporate dGMP without the usual dispersion of water molecules. PMID- 1704894 TI - Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump. AB - A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump. PMID- 1704896 TI - Capillary electrophoresis of proteins under alkaline conditions. AB - Successful separations of proteins by capillary electrophoresis in uncoated fused silica capillaries is limited by adsorption and variable rates of electroendosmosis, which can compromise quantitative accuracy and precision. Operation at extremes of pH to minimize these problems is useful in special cases but is not a general strategy for protein separations. Three alternative strategies are described: use of capillaries coated with a linear hydrophilic polymer, the use of acidic solutions to wash the capillary between runs, and the incorporation of additives into the electrophoresis buffer to minimize adsorption during analysis. Applications of these techniques to protein samples is demonstrated. PMID- 1704897 TI - Identification of aprotinin degradation products by the use of high-performance capillary electrophoresis, high-pressure liquid chromatography and mass spectrometry. AB - A preparation of bovine aprotinin, bovine pancreatic trypsin inhibitor, was subjected to high-performance capillary electrophoresis (HPCE) analysis and the purity was calculated to be approximately 80%. The two dominating contaminants were integrated to approximately 7% each as compared to the intact molecule. Characterization by high-pressure liquid chromatographic (HPLC) and mass spectrometric analysis was carried out on digests of the reduced and alkylated molecules. The contaminants were identified as truncated aprotinin, missing one and two amino acids, respectively, at the C-terminus. No such structures were identified in similar amounts in preparations of recombinant aprotinin by HPLC or HPCE. PMID- 1704898 TI - Large-scale purification of prosomes from calf's liver. AB - Prosomes, cytoplasmatic ribonucleoprotein complexes containing small ribonucleic acid (19S small cytoplasmic RNPs), are ubiquitous in eukaryotic organisms. A new method for the preparation of prosomes in large amounts, starting with ca. 2 kg of calf's liver, is described. A combination of centrifugation and low- and high pressure chromatography was used to purify intact particles. An alternative purification of prosomes with Solanum tuberosum agglutinin bound to divinyl sulphone-activated agarose is discussed. Calf's liver prosomes have a similar protein composition and RNA content to prosomes isolated from other tissues. PMID- 1704899 TI - Disappearance of labelled IGF-2 from plasma of the ovine fetus in late gestation. AB - The role of IGF-2 in the fetus and its possible influence on fetal growth remains speculative. We investigated the size distribution of unsaturated binding sites for labelled oIGF-2 in ovine fetal plasma. In addition, the disappearance of each form of protein bound IGF-2 in the late gestation ovine fetus (125-135 days, n = 5) was estimated. One minute after injection into the fetal femoral vein, 125IoIGF- circulated in the fetal femoral artery bound primarily to a 50 kDa binding protein. Only a small amount of binding to a 150 kDa binding protein was seen with little to no free IGF-2 present. IGF-2 also circulated in association with a large molecular weight complex (ca. 250 kDa) presumed to be circulating receptor bound IGF-2. The half life of the 250 kDa form of IGF-2 was 385.9 +/- 65.4 min, for the 150 kDa form 308.0 +/- 65.0 min, for the 50 kDa form was 35.5 + 2.6 min and for the free form of IGF-2 was 1.6 +/- 0.6 min. There was no apparent movement of intact IGF-2 out of the fetal circulation into any of the fetal fluids or into the maternal circulation. Similarly there was no consistent placental uptake of IGF-2 from the fetal circulation. PMID- 1704900 TI - Immunomagnetic isolation of NK and LAK cells. AB - The present study describes the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells. Antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles (Dynabeads M-450) were used for the positive isolation of CD56+ cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56+ cells (less than or equal to 3% contaminating cells) was obtained with this method. The cellular yield of CD56+ cells was high (5.3% of the unseparated PBMC). The CD56+ cells remained unactivated after separation and preserved their functional characteristics, as measured by cytotoxic activity against the NK sensitive K562 cells. Incubating the CD56+ cells with IL-2 resulted in high LAK activity, as measured by cytotoxic activity against Daudi cells. Large numbers of functionally active CD56+ cells were obtained from IL-2 stimulated lymphocytes using anti-CD56 coated Dynabeads 450. A further enrichment of effector cells with LAK activity was accomplished by depleting the CD56+ cells for T-cells by anti-CD3 coated Dynabeads M450. The immunomagnetic isolation technique described was easy to perform, did not require expensive equipment and yielded NK and LAK cells of satisfactory purity. PMID- 1704901 TI - An improved method for the detection of DNA fragmentation. AB - An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling. PMID- 1704902 TI - The production of antibodies to pentosanpolysulfate (ELMIRON, SP-54). AB - Pentosanpolysulfate (PPS) represents the product obtained after sulfation of xylan and is composed of beta 1----4-D-xylopyranose residues sulfated at C2 and C3. Studies have shown that this compound can often be effective in relieving the symptoms of interstitial cystitis (IC). In order to elucidate the mode of action of PPS in IC, a sensitive and reliable assay was needed. To this end we prepared an immunogenic form of PPS by coupling it to methylated bovine serum albumin (MBSA). This complex was used to immunize NZW rabbits (1 mg, IM). Four of five animals responded with anti-PPS antibodies, three of which had high titer (greater than 1/2000) as measured by an enzyme-linked immunosorbent assay (ELISA). All sera were routinely absorbed with an MBSA-Sepharose immunoadsorbent to remove anti-MBSA antibodies. ELISA inhibition tests were used to determine the sensitivity and specificity of the sera. At least 50 ng/ml of PPS could be routinely detected by this assay. A number of naturally occurring proteoglycans, polysaccharides, monosaccharides and disaccharides were examined for reactivity with the antibodies but only heparin was an effective inhibitor. Absorption with heparin immunoadsorbents reduced, but did not eliminate, the ability of heparin to inhibit anti-PPS binding. This activity could be destroyed by treatment with heparinase without affecting PPS inhibition. Normal urine did not affect the ELISA or ELISA inhibition tests and thus allowed the determination of PPS levels in IC patient urines. Initial analysis of seven IC patients receiving oral PPS revealed urine concentration of 0.8-16.0 micrograms/ml. No inhibition could be detected in pre-treatment urine samples. PMID- 1704903 TI - Granulocyte colony-stimulating factor enhances the phagocytic and bactericidal activity of normal and defective human neutrophils. AB - Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation of myeloid cells and may be a valuable adjunct in prevention and treatment of neutropenia associated infections. Neutrophil (PMNL) phagocytic and microbicidal functions against Staphylococcus aureus and Candida albicans blastoconidia were therefore evaluated. Bacterial phagocytosis and bactericidal activity were significantly enhanced by approximately 50%-70% after preincubation of normal PMNL with G-CSF in concentrations of 1000-4000 units/ml for 10 min at 37 degrees C. G-CSF in similar concentrations enhanced the defective bactericidal activity of PMNL from HIV-1-infected patients by approximately 70%-150% and reached the baseline control PMNL killing. However, G-CSF enhanced neither phagocytosis nor fungicidal activity of normal PMNL against C. albicans blastoconidia. These data demonstrate that G-CSF enhances the antibacterial but not the antifungal activities of human PMNL in vitro and also improves the defective PMNL bactericidal activity of HIV-1 infected patients. PMID- 1704904 TI - Production of monoclonal antibodies against a new carcinoma-associated marker in view of developing a serological test. AB - By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 kDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen. PMID- 1704905 TI - AFP, CEA, CA 19-9 and TPA in hepatocellular carcinoma. AB - The present study is based on the assay of four markers (AFP, CEA, TPA, Ca 19-9) using IRMA methods in 36 normal subjects, 44 cirrhosis and 66 HCC patients. Parametric and non parametric tests were used to test differences and correlations. ROC curves and discriminant functions were also elaborated. Normal 95% "cut-off" was determined by the "boostrap" method yielding: CEA 3.4 ng/ml; Ca 19-9 55 U/ml; TPA 58U/l and AFP 5.2 ng/ml. In HCC patients the values of the four markers were, on average, significantly different from those of normal subjects. However, only AFP and TPA exhibited high diagnostic accuracy (90%) for detection of the tumor. Higher than normal mean values for all markers were, also observed in cirrhotic patients. Only AFP yielded effective discrimination between HCC and cirrhosis. The positive prediction for the presence of the tumor on cirrhotic ground was 95% for AFP values higher than 18.5 ng/ml, with a 78% negative predictive value with a 6 ng/ml threshold. Association of AFP with TPA showed only a marginal diagnostic improvement. Results were not improved at all by combining CEA and Ca 19-9 with AFP and/or TPA. In conclusion, AFP is and remains the best marker for HCC and the only one effective in discriminating of HCC from cirrhosis. TPA may be considered a valid alternative if cirrhosis is not present. CEA and Ca19-9 are of no use. PMID- 1704906 TI - Expression of thermotolerance following microinjection of poly(A)RNA isolated from thermotolerant CHO cells. AB - Poly(A)RNA was isolated from thermotolerant cells and microinjected into recipient non-tolerant Chinese hamster ovary (CHO) cells. The injected cells expressed thermotolerance to a subsequent test heat treatment both in terms of the end-points of colony formation (cell survival) and resumption of protein synthesis after test heating (translational labelling). The magnitude of thermotolerance expression was dependent on the experimental end-point (increase up to 3.8-fold for translational labelling and approximately 2-fold for survival) and on the time between microinjection and the test heat treatment. Control experiments showed that poly(A)RNA from non-tolerant cells did not alter the heat response of microinjected cells. Proteins corresponding to the poly(A)RNA from thermotolerant cells were analysed by in vitro translation and by labelling of microinjected cells, followed by SDS-PAGE. In vitro translations showed high levels of transcripts for classical heat-shock proteins (HSP 70/72, 89, 110) in poly(A)RNA from thermotolerant versus control cells. However, proteins synthesized in intact cells showed no detectable differences when cells were microinjected with poly(A)RNA from thermotolerant versus control cells, or not injected at all. In principle the data show that microinjection of specific poly(A)RNA fractions can be used for defining the contribution of individual gene products to the cellular heat response. PMID- 1704907 TI - The impact of breakthrough clinical trials on survival in population based tumor registries. AB - Three statistical models are developed to study the impact that two breakthrough clinical trials (MOPP for Hodgkin's disease and PVB for disseminated testicular cancer) had on survival in the Connecticut tumor registry and the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) registry program. A segmented regression model is used in conjunction with the Cox semi-parametric proportional hazards model, as well as the parametric Weibull and exponential cure models. These models allow us to determine approximately when survival first began to improve dramatically, indicating that improved treatments had become available, and how long it took for survival to level off again indicating that the full population survival impact had been realized. In addition, the degree to which the parametric models fit allows us to determine if the survival improvements occur within a parametric family. Results of the modelling indicate that dissemination took approximately 11 years in Hodgkin's disease while only 3 years in disseminated testicular cancer. In both disease sites survival first broke with prior trends between the time that the breakthrough trial started and its publication, indicating that earlier moderately successful 'precursor' trials with combination chemotherapy may have initiated the improved population survival trends. Reasons for the difference in dissemination time in the two cancer sites are examined in order to understand what factors may be responsible for the speed of dissemination and effective utilization of new therapies. PMID- 1704908 TI - Immunohistochemical demonstration of keratin in ameloblastoma as an indication of tumor differentiation. AB - Keratin expression was studied immunohistochemically in 27 ameloblastomas using polyclonal antibody against wide-spectrum keratins (TTL) and monoclonal antibodies against lower- and higher-molecular-weight keratins (PKK1 and KL1), respectively, to clarify the tumor differentiation. Reactions with TTL and KL1 antibodies were generally positive in the stellate cells of the follicular or acanthomatous ameloblastomas. Cell nests of the basal cell type were positive for PKK1. On the other hand, the reactions with TTL or KL1 in the plexiform type were generally weak or absent. From these facts, it was concluded that the follicular, as well as acanthomatous, ameloblastoma is liable to undergo squamous differentiation, whereas the plexiform ameloblastoma remains in primitive stage of tumor differentiation. PMID- 1704910 TI - The physician as biographer. PMID- 1704909 TI - Social and demographic characteristics of patients admitted to a palliative care unit. AB - In this retrospective study we reviewed the charts of 169 consecutive admissions to the Palliative Care Unit, Edmonton General Hospital. Demographic and social characteristics of patients were assessed in order to determine the likelihood that the patients could be managed at home according to currently available services. The mean age of the population was 65 +/- 12 years, 97 (57%) were women, they had a variety of cancers with major prevalence of the most frequent adult tumors, and 72% of patients were coming from acute care hospitals. Each patient had an average of 2.7 +/- 1.8 children (median = 3), a mean of 0.18 +/- 0.6 dependents (median = 0), a mean of 1 +/- 0.9 support persons at home (median = 1), and a mean of 2.6 +/- 1 support persons outside the household (median = 2). Of a total of 119 main caregivers who lived in the same household as the patient, 69 (58%) were not able to take care of the patient. Only 27 patients (16%) considered that there were major financial problems, and all 169 patients had universal health care available (95 patients had additional private health care coverage). Of 125 patients who were asked where they preferred to die, 112 (90%) stated that they did not want to die at home. Our data suggest that the lack of family support and lack of intensive home care services are the main obstacles to home care of terminally ill cancer patients in our province. Home care services as currently available are not able to care for a large number of patients with terminal cancer. More prospective research into this subject is badly needed. PMID- 1704911 TI - Nursing the remarried family in a palliative care setting. PMID- 1704912 TI - Overcoming professional opposition. PMID- 1704913 TI - Head and neck carcinomas. PMID- 1704914 TI - Palliative care in Canada: 1990. AB - While there has been little change in numbers of programs across the country in the past four years, there does appear to have been an increase in the size of teams and in the variety of professional representation on teams, as well an increased number of programs with volunteers. The information suggests a certain consolidation of earlier efforts. Diversification of programs continues, with an increasing number of programs now in long-term care settings. Heidemann in 1986 raised the questions: What is quality palliative care? How do we know we are delivering quality palliative care? The questions continue to challenge us in 1990, as we continue to lack a set of standards against which to measure our efforts. PMID- 1704915 TI - There is a palliative Europe to build/1st Congress of the European Association of Palliative Care. PMID- 1704916 TI - Home palliative care: the challenge in Palermo. PMID- 1704917 TI - The last 48 hours of life. AB - Though patients usually die peacefully, problems may arise in the last period of a terminal illness. In the final days new symptoms may arise or there may be exacerbation or recurrence of symptoms previously well controlled. Two hundred consecutive hospice patients were studied. The incidence was noted of pain, dyspnea, moist breathing, nausea and vomiting, confusion, restlessness, jerking and twitching, difficulty in swallowing, incontinence and retention of urine, sweating, moaning and groaning, and loss of consciousness. Each symptom is considered and the results of the management employed are noted. Many of the features appearing in the last days of a terminal illness, especially cancer, can be attributed to organic brain disease consequent to metabolic disorder associated with multi-organ failure. An awareness of the nature of the problems that may arise in the last 48 hours of life makes it possible to keep the patient comfortable to the end. PMID- 1704918 TI - Immunohistochemical distribution of keratin proteins in human gingival heterotransplants in nude mice. AB - Clinically healthy human gingivae from deciduous molar regions were transplanted to subcutaneous sites of nude mice (nu/nu NC). Transplants were harvested after posttransplantation periods of 5, 6, 7, 8.5, 10.5 and 12 weeks and examined histologically after staining with hematoxylin-eosin (H.E.), bisbenzimide, and a panel of mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. Central parts of transplants contained human connective tissue covered by human stratified squamous epithelium which were unkeratinized in 5- to 7-wk old transplants and most frequently (75%) parakeratinized in 8.5-wk to 12-wk transplants. Comparison of keratin expression before and after transplantation revealed a progressive keratin reconstitution, i.e., keratin markers of basal/suprabasal cells preceded those of suprabasal/spinous cell layers and immunohistochemical markers of keratinization preceded routine histologically observed parakeratinization. Original keratin staining and essential features of histodifferentiation were reconstituted and maintained after 8.5 wk but graft recovery rate decreased drastically 12 wk after transplantation. This study shows that the human gingiva/nude mouse model is useful in experimental studies of the gingival keratin profile in the period 8.5 to 10.5 wk after transplantation. PMID- 1704919 TI - Scanning electron microscopic studies and x-ray microanalysis of hyaline bodies in odontogenic cysts. AB - 70 odontogenic cysts with hyaline bodies (HB) were examined by light and scanning electron microscopy. The scanning electron microscopic studies were performed on dried tissue material, which had been previously examined by light microscopy and energy dispersive x-ray analysis of the histologic sections. The form and basic structure of both the early forms of HB as well as the HB Type I and II could be identified more closely. The early forms, the outer component of the HB Type II and the inner and outer component of the HB Type I consisted of a fine-grained substance, which presumably goes through varying "partial homogenization", thus giving the HB an increased firmness and elasticity. The HB are a product of the epithelium of odontogenic cysts and have direct contact to the outer layer of the adjacent cyst epithelium via its intercellular bridges. PMID- 1704920 TI - Cellular immunosuppression in oral lichen planus. AB - Functions of peripheral blood lymphocytes and neutrophils from 30 oral lichen planus (OLP) patients were examined using healthy persons as controls. Two-color flow cytometry of lymphocytes revealed no proportional difference in CD3 or CD4 cells between OLP and controls. CD8CD11b (suppressor T) and CD3HLA-DR+ cell populations increased significantly in OLP when compared with controls, and CD4/CD8 cell ratio decreased in OLP. Mitogenic response of patients' CD8 and CD4Leu8- cells was similar to that in controls. However, weaker blastogenesis of CD4Leu8+ cells, the most excellent responders in T cell subsets, was observed in OLP. Serum IFN beta level in OLP (8.4 +/- 4.8 IU/ml) was significantly lower than in controls (13.7 +/- 5.0 IU/ml) whereas no difference between the two groups could be found in IFN alpha or gamma. As for in vitro cytokine production by IL-2 stimulated lymphocytes, there was no difference in GM-CSF generation between the two groups, but, IFN gamma and IL1 beta production of patients' lymphocytes was less than that in healthy donors (57.6 +/- 50.7 VS 78.7 +/- 39.6 u/ml, 152.3 +/- 93.5 VS 258.7 +/- 65.4 pg/ml, respectively). Moreover, superoxide generation of patients' neutrophils by PMA stimulation was significantly insufficient as compared with controls' (84.9 +/- 30.9 VS 110.8 +/- 24.1 pmol/min/10(4) cells). Nevertheless, natural killer cell activities of both groups distributed in the same range. These results suggest that OLP patients' lymphocyte and neutrophil functions are impaired, and that cellular immunosuppression is a pathologic characteristic of OLP. PMID- 1704921 TI - Quantitative structure-activity relationship analysis of cation-substituted polyaromatic compounds as potentiators (amplifiers) of bleomycin-mediated degradation of DNA. AB - A set of 21 polyheteroaromatic compounds substituted with flexible cationic groups and of similar molecular size has been analyzed for binding with DNA and for effects of the bleomycin-mediated degradation of the DNA double helix. Increases in apparent rates of the DNA digestion were observed in all cases under the experimental conditions of noncompetitive binding of these compounds and bleomycin to DNA. Surprisingly, the quantitative structure-activity relationship analysis revealed two distinct correlations despite close structural similarities for the set of bleomycin amplifiers. These unusual results are explained in terms of the formation of two stereochemically different ternary complexes of activated bleomycin-DNA-amplifier. The relevance of this finding for the design of new bleomycin amplifiers is discussed. PMID- 1704922 TI - Southeast Asian countries tackle cancer control. PMID- 1704923 TI - Routine screening for cancer of the prostate. AB - The value of screening for prostate cancer remains unclear. Although digital rectal examination, transrectal ultrasonography, and determination of serum prostate-specific antigen levels may lead to early detection of a malignancy, these procedures have never been shown to reduce disease-specific mortality from prostate cancer. Unfortunately, several potential errors found in uncontrolled trials may suggest benefit from screening where none exists. Only a large, randomized, controlled clinical study demonstrating decreased mortality from prostate cancer can prove that screening is beneficial. Until such a study is performed, patients should be informed of both the potential benefits and the risks of screening and treatment. PMID- 1704924 TI - Characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18. AB - We have previously reported that the most common human serum immunoglobulin G antibody reactivities to human papillomavirus type 16 and type 18 (HPV16 and HPV18)-encoded proteins are directed against the minor capsid proteins (HPV16 L2 and HPV18 L2) and to the E7 protein of HPV16 (S. A. Jenison, X.-P. Yu, J. M. Valentine, L. A. Koutsky, A. E. Christiansen, A. M. Beckmann, and D. A. Galloway, J. Infect. Dis. 162:60-69, 1990). In this study, the antibody-reactive segments of the HPV16 E7, HPV16 L2, and HPV18 L2 polypeptides were mapped by using nested sets of deleted recombinant proteins. A single major immunoreactive region was identified in the HPV16 E7 polypeptide between amino acids (aa) 21 and 34 (DLYCYE QLNDSSEE). In contrast, three distinct immunoreactive regions of the HPV16 L2 polypeptide were present in the segment between aa149 and aa204, and three distinct immunoreactive regions of the HPV18 L2 polypeptide were present in the segment between aa110 and aa211. With the exception of one serum sample, serum immunoglobulin G antibodies which reacted with HPV16 L2 polypeptides or with HPV18 L2 polypeptides were not cross-reactive. PMID- 1704925 TI - Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. AB - Hepadnaviruses replicate their circular DNA genomes via reverse transcription of an RNA intermediate. The initial product of reverse transcription, minus-strand DNA, contains two copies of a short direct repeat (DR) sequence, termed DR1 and DR2. Plus-strand DNA synthesis initiates at DR2 on minus-strand DNA, using as a primer a short, DR1-containing oligoribonucleotide derived by cleavage and translocation from the 5' end of pregenomic RNA. To clarify the sequence requirements for plus-strand primer cleavage and translocation, we have constructed mutants of the duck hepatitis B virus bearing base changes in or around the DR1 sequence in the primer. A point mutation at the terminal nucleotide of DR1 has a striking phenotype: normal levels of duplex viral DNA are produced, but nearly all of the DNA is linear rather than circular. Mapping of the 5' end of plus-strand DNA reveals that primer cleavage occurs with normal efficiency and accuracy, but the primer is not translocated to DR2; rather, it is extended in situ to generate duplex linear DNA. Other mutations just 3' to DR1 similarly affect primer translocation, although with differing efficiencies. Linear DNA found in wild-type virus preparations has the same fine structure as the mutant linears described above. These results indicate that (i) plus-strand primer cleavage and translocation are distinct steps that can be dissociated by mutation, (ii) lesions in sequences not included in the primer can severely inhibit primer translocation, and (iii) elongation of such untranslocated primers is responsible for the variable quantities of linear DNA that are found in all hepadnaviral stocks. PMID- 1704926 TI - Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis. AB - Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established. PMID- 1704927 TI - Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes. AB - Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp41 (amino acids 598 through 609) reacted with astrocytes found in human and rodent central nervous system tissue. The monoclonal antibodies bound to a 43-kDa protein found in central nervous system tissue preparations. These results indicate that human immunodeficiency virus type 1 gp41 contains a common epitope with astrocytes and that an immune response to human immunodeficiency virus type 1 gp41 could generate antibodies that are cross-reactive to astrocytes. Furthermore, anti astrocyte antibodies, which were directed at a common epitope with the gp41 sequence, were found to be present in cerebrospinal fluid from some AIDS patients with central nervous system complications. Astrocytes regulate the environment for appropriate neuronal function, and astrocyte hyperactivity (astrocytosis) is known to be the common and early pathologic event in brains from patients with central nervous system AIDS. We suggest that antibody-induced effect(s) on astrocytes could lead to the physiologic neuronal dysfunctions observed in AIDS patients. PMID- 1704928 TI - Anti-adenovirus type 5 cytotoxic T lymphocytes: immunodominant epitopes are encoded by the E1A gene. AB - Virus specific, major histocompatibility complex-restricted, cytotoxic T lymphocytes (CTL) generated in Fischer strain rats infected with human adenovirus type 5 (Ad5) were found to recognize antigenic determinants encoded within the Ad5 early region 1A (E1A) gene. Preliminary mapping studies suggest that the E1A CTL epitopes are encoded within the regions between bp 625 to 810 and 916 to 974 in the first exon of this gene. These epitope-coding regions occur within subregions of E1A that are conserved functionally, and to some extent structurally (approximately 50% sequence homology), among adenoviruses of different groups. Nevertheless, Ad5-specific CTL lysed only targets infected with adenoviruses of the same group (group C; e.g., Ad2) and not targets infected with adenoviruses of different groups (groups A, B, and E). These results suggest that virus-specific CTL may limit adenoviral dissemination by destroying virus infected cells at an early stage in the viral replicative cycle, during E1A gene expression. Expression of other adenovirus genes does not appear to be required to target infected cells for elimination by CTL. PMID- 1704930 TI - Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli. AB - A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine. PMID- 1704929 TI - Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells. AB - A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus. The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter. Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4 chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting. In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA. Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth arrested and log phase cells, respectively. PMID- 1704931 TI - Biochemical alterations in the levels of DNA, RNA and protein in discrete areas of rat brain following Nuvacron toxicity. AB - The levels of DNA, RNA and Protein were estimated in cerebral hemisphere, cerebellum and brain stem of male albino rats with Nuvacron (0, 0, dimethyl-0-1 methyl 3-methylamino-3-oxe-1-propenyl phosphate) 4 mg/kg body weight intraperitoneally (i.p.) daily for 10 days. The daily i.p. dose of Nuvacron depleted the level of DNA and protein in all brain regions. Increased level of RNA was observed in cerebral hemisphere, cerebellum and brain stem. PMID- 1704932 TI - [Detection of antigenic determinants of autoantibodies in idiopathic thrombocytopenic purpura using anti-platelet monoclonal antibodies and flow cytometry in whole blood]. AB - To detect antigenic sites of platelet-bound autoantibodies in chronic ITP, whole blood or platelet-rich plasma from patients was stained with monoclonal antibodies (MoAbs) directed against platelet membrane glycoproteins (GPs) and with FITC-conjugated anti-mice IgG in an unwashed system followed by flow cytometric analysis. Platelets were identified by light-scattering profile and the amount of anti GP MoAb bound to platelets was measured and expressed as mean fluorescence intensity. This system enabled to quantitate the amount of intact GPs on platelet surface, regardless of activation of platelet. As a result, decreased amount of platelet-bound anti GPIIb/IIIa MoAb was noted in 5 of 22 patients with chronic ITP comparing to those with normal subjects. On the contrary, none of these patients revealed any demonstrable decrease in the amount of anti GPIb MoAb binding. From these observations, we suggest that the decreased MoAb binding to platelets in these five patients indicates the binding of autoantibody directed toward antigenic determinants, which are on or close to the epitope of this MoAb. PMID- 1704933 TI - [Assessment of the antitumor effect after remission induction therapy for non Hodgkin's lymphoma--an approach to a response-adapted therapy]. AB - We assessed the response of various tumors, following each of three consecutive courses of CHOP or CHOP-Bleo therapy in 32 untreated patients with intermediate or high-grade non-Hodgkin's lymphoma (NHL), and also retrospectively compared these results to those for a group in whom complete remission (CR) had been obtained within five courses and a non-CR group. A low CR rate was observed in these patients who showed no or only temporary antitumor effects (persistent increase, no change, or increase after a transient decrease) after two courses of therapy. Furthermore, we measured total tumor volume and tumor regression rate after each course of therapy in 21 patients who had measurable tumors, and determined their cut off values for distinguishing between CR and non-CR. When the cut off values were applied to the effects observed in the 21 patients, positive predictive values after each course of therapy were high. False negative values after the second course of the therapy were low, and showed 0% when the cut off value determined from tumor regression rate was used. These results suggest that we can accurately predict the probability of CR after the second course of CHOP or CHOP-Bleo therapy, although this should be further confirmed by prospective study. In addition, such an analysis may prove useful in setting up individualized response-adapted therapy for NHL. PMID- 1704934 TI - [Relapsing polychondritis in a patient with myelodysplastic syndrome]. AB - Relapsing polychondritis is a rare disorder of uncertain origin characterized by recurrent inflammation of cartilage. A case of myelodysplastic syndrome (MDS) associated with relapsing polychondritis is reported. A 60-year-old man who had been diagnosed as MDS was admitted because of pain and swelling in the bilateral preauricular regions and cheek. A diagnosis of relapsing polychondritis was made by coexistence of auricular chondritis, arthropathy, ocular inflammation and audio-vestibular disturbance. He also developed ocular palsies and optic neuritis. He was treated with prednisolone, azathioprine, dapsone, and then with steroid pulse therapy. Moreover, plasmapheresis and high dose gamma-globulin therapy were undertaken. However, all these treatments were unsuccessful and he died of respiratory failure. PMID- 1704935 TI - [Structure and function of the serous membrane--with special reference to mesothelial cells]. PMID- 1704937 TI - [Giemsa stain and significant stains for effusion cytology in body cavities]. PMID- 1704936 TI - [Techniques and results used in staining PAS and alcian blue]. PMID- 1704939 TI - [Immunohistochemical studies on bladder tumours. Immunohistochemical evaluation on intravesical therapy for bladder tumours]. AB - The focus of my study was the immunohistochemical studies of tumour cells induced by ADM intravesical instillation therapy when stained by several antigens, those being; Carcinoembryonic antigen (CEA), Ferritin, Beta 2 Microglobulin (beta 2 MG), Keratin and Glycogen before and after the therapy. Then I discussed as to their value in indicating therapeutic efficacy. There were 18 cases of bladder cancer studied using the Peroxidase-antiperoxidase technique (PAP) of staining. Data indicated a 44.4% response rate following therapy with intravesical ADM. Immunohistochemical evaluation showed improvement as indicated by not staining with CEA. Also observed was no change and slightly diffuse staining with Keratin: and a decrease in all layers of Glycogen expression: However this could not be observed with the Ferritin or beta 2-MG. It is believed this demonstrates that immunohistochemical expressions of staining with CEA, Keratin, and Glycogen may be a sensitive indicator of a therapeutic response to intravesical ADM. PMID- 1704938 TI - [Immunohistochemical studies on bladder tumours. Evaluation by CEA, ferritin, beta 2-MG, keratin and glycogen]. AB - The focus of my study was the immunohistological expressions of bladder tumour cells when stained by several antigens, those being; Carcinoembryonic antigen (CEA), Ferritin, Beta 2 Microglobulin (beta 2-MG), Keratin and Glycogen. There were 59 cases of bladder cancer studied using the Peroxidase-antiperoxidase technique (PAP) of staining. Specimens were formalin-fixed, paraffin embedded, and then sectioned. Some correlation was found between histological grade, stage and CEA incidence but was not found in the Ferritin or beta 2-MG. Keratin and Glycogen were detected in all cases. These staining patterns were mosaic according to higher grade and stage. It is believed this demonstrates that immunohistochemical expressions of staining with CEA, Keratin, and Glycogen may be a sensitive indicator of histological grade, stage. PMID- 1704940 TI - Granulocyte-colony stimulating factor enhances the cytotoxic effects of methotrexate to bladder cancer cells in vitro. AB - It is possible that a strategy designed to stimulate cancer cells in the active cell cycle may increase the effectiveness of S-phase specific anti-cancer agents such as methotrexate. In this study, the effects of granulocyte-colony stimulating factor (G-CSF) on the proliferation of cultured human bladder cancer cells and on the cytotoxicity of anti-cancer drugs to bladder cancer cells were studied in vitro. The 3H-thymidine uptake of cultured human bladder cancer cells, KU-1 and NBT-2, was significantly higher when the cells were treated with 10 ng/ml G-CSF than without G-CSF after 24- and 48-hour incubation. However, the cell numbers of KU-1 and NBT-2 were not significantly affected by 72-hour treatment with 10 ng/ml G-CSF. The binding of 125I-labeled KW-2228, a muteins of G-CSF, to KU-1 and NBT-2 was inhibited by unlabeled KW-2228 in a concentration dependent manner, which demonstrated the presence of G-CSF receptors on both cells. Scatchard analysis showed that the receptor densities of KU-1 and NBT-2 were 1770 and 3070 per cell, respectively. The combination treatment with methotrexate and G-CSF resulted in a significant increase in cytotoxic effects, when compared with methotrexate treatment alone. This study supports the possibility that the combination therapy of methotrexate and G-CSF increases clinical response in the treatment of advanced bladder cancer. PMID- 1704941 TI - [Results of early education of developmentally delayed infants and young children by parents with a clinical rehabilitation program]. AB - Early registration of disturbances in the development of infants was based on neurological examination. Since 1984 exists the possibility of psychological evaluation of infants and children in early childhood in the Department of Paediatrics of the University of Rostock. Parents of children suffering from disturbances of development receive additionally to the concept of physiotherapy an early intervention program to furtherance of children. In this way best conditions are prepared in development and evaluation of admission in special institutions (special kindergarten, day-nursery). PMID- 1704943 TI - [The creation of a national reference laboratory]. PMID- 1704942 TI - [A national reference laboratory (the opinion of a chief specialist in the USSR)]. PMID- 1704944 TI - [The preparation of blood samples for the automatic measurement of erythrocyte size]. AB - The following procedure is proposed: fixation for 12 h in low ionic strength solution (4 percent formaldehyde in 50 mM sodium phosphate buffer), drying of the suspension drop on the slide, gallocyanin staining. All red cells were contrast stained without light central spot. The sizes of different red cell groups on the slide differed by less than 5 percent. PMID- 1704945 TI - [The determination of erythrocyte deformability using a filtration method]. AB - Red cell deformability was assessed in donors, patients with chronic renal insufficiency, and with thermal injuries by filtration through special filters. Filtration of donor red cells decreased their count but insignificantly; in patients with chronic renal insufficiency this characteristic was lowered, particularly so before hemoperfusion, whereas after hemoperfusion red cell deformability improved. Changes of red cell deformability in patients with thermal injuries is an objective criterion reflecting the status of the erythron peripheral component in connection with the severity of injury and course of its healing. PMID- 1704946 TI - [pH-metric study of ion equilibrium in erythrocyte suspensions exposed to heat]. AB - A method of differentiated measurements of pH is suggested to be used in study of the time course of ionic equilibrium in red cell suspensions exposed to heating. The described method yields more accurate and complete data on the type and distribution of red cells as regards their membrane characteristics. PMID- 1704947 TI - [The relation between the phagocytic function and the cytochemical indices of blood neutrophils]. PMID- 1704948 TI - [The use of parietal pH-metry in the diagnosis of intestinal diseases]. AB - Parietal measurements of pH values in patients with various intestinal diseases and in normal subjects have revealed that alkaline medium with gradual elevation of the values in the caudal direction is characteristics of the normal large intestine; acid pH values were registered in patients with lactase insufficiency, nonspecific ulcerative colitis, in contrast to those with post-dysentery colitis and tumors of the large intestine. Lactulose and sodium sulfate were found the factors that significantly influence the large intestine parietal pH values, the effects of wheat bran were lower; magnesium sulfate and sodium hydrocarbonate mineral water with medium mineral content had no effect on parietal pH values. PMID- 1704949 TI - [New diagnostic possibilities of the pH-metry of gastric juice]. AB - Based on theoretical estimates, a nomogram has been derived, permitting estimation of free, total, and true acidity, bicarbonate alkalinity, acid/alkaline balance from the data of gastric juice pH-metry. A more accurate demarcation between hypo-and anacid states is suggested. PMID- 1704950 TI - [The gastrin-producing function of the stomach in patients with chronic alcoholism]. AB - Basal and maximum gastrin levels were measured in 81 patients with various stages of chronic alcoholism and different periods of alcohol intake, in 23 patients with chronic gastritis of nonalcohol etiology, and in 12 normal subjects. The findings permit a conclusion on the depressive effect of alcohol on the function of gastrin-producing G-cells, this resulting in lowered levels of both basal and maximal gastrin. A direct correlation between the degree of alcohol depression of gastric gastrin production and the length of alcohol consumption was revealed. PMID- 1704951 TI - [The determination of the volume of circulating plasma using the indicator T 1824]. AB - A method for measuring circulating plasma volume (CPV) by diluting T-1824 indicator is described, making allowance for the exponential pattern of labeled serum albumin elimination from the vascular bed. The method consists in double collection of blood samples after intravenous injection of the indicator, computation of the constant of its extravasation rate, and extrapolation to the zero time. Analysis of the results obtained by this method and CPV values derived from the hematocrit and circulating blood volume--(CBV) estimated with the help of Albert table (1971), as well as with CPV value derived from the hematocrit and CBV measured by impedance method in 22 patients with slight uncomplicated ventral and diaphragmal hernias has demonstrated high linear correlation coefficients, +0.77 and +0.78 (p less than 0.01), respectively. PMID- 1704952 TI - [Determination of the free radical processes in condensates of exhaled air in children with recurrent bronchitis]. AB - Free-radical processes were assessed in exhaled air condensates of healthy children and of those suffering from recurrent bronchitis during remission and at the onset of exacerbation. 3 percent hydrogen peroxide-induced chemiluminescence technique was employed with the results recorded by chemiluminometer, manufactured in this country. The data evidence an increase of the total induced chemiluminescence in the patients during exacerbation vs. that in healthy children; such measurements may become a valuable test in the diagnosis of chronic and recurrent pulmonary disease in children. PMID- 1704953 TI - [The acid-base status of the blood of normal subjects with different ABO blood group systems in different seasons of the year]. AB - A total of 180 normal subjects with all AB0 blood groups, aged 18 to 50, were examined with the use of Astrup's micromethod in various seasons--in spring, summer, fall, and winter. The blood was found more acid in summer than in the fall and more alkaline in spring than in winter. Hypocapnia was detected in all the examinees over the year. Therefore the results of blood acid-base measurements in the patients, made at a certain season, should be compared with the reference values for the corresponding season. Differences in blood acid-base parameters of various subjects in the same seasons were negligible. PMID- 1704954 TI - [Determination of the oligobiopolymerized sialic acids in the blood]. AB - A method for measurement of the blood serum (plasma) oligobiopolymerized sialic acids (OSA) has been developed, making use of the known procedures for measuring these amino sugars. Blood OSA levels of normal subjects and intact rats have made up 59.6 +/- 2.3 and 79.0 +/- 3.4 mg/l, respectively. OSA levels were shown to rise in immobilization stress (by 36 percent), aseptic granulomas (by 28 percent), and active phase of rheumatic fever (by 128 percent). The method is recommended for clinical diagnostic studies as an additional diagnostic test. PMID- 1704955 TI - [Aims and tasks of a national reference laboratory]. PMID- 1704956 TI - [Microdot immunoenzyme analysis of chorionic gonadotropin in pregnancy]. AB - A microdot enzyme immunoassay test system has been developed for measuring chorionic gonadotropin levels in urine samples, on the basis of affine purified antibodies to hormonal beta-subunit and antibody complex with horse radish peroxidase. The results are assessed visually with the use of a color indicator scale obtained in experiments with reference preparations containing 12.5, 50, and 200 mMU of the hormone per ml. Comparison of the intensity of microdot color permits characterizing the level of hormone secretion and diagnose pregnancy 4-5 days before the first day of expected menstruation (50 mMU/ml). The specificity, reproducibility, and sensitivity of the developed test system are not inferior to common enzyme immunoassay on the plates; the method does not involve the use of sophisticated equipment and is reagent-saving. The diagnostic accuracy of the test system is 100 percent. PMID- 1704957 TI - [The determination of immunoreactive gastrin in gastric juice]. AB - A number of conditions should be observed when pretreating gastric juice for gastrin measurements, in order to obtain reliable results. The authors compare the results of gastric juice pretreatment by various methods and the results obtained with the use of different radioimmunoassay kits. Calculation coefficients are suggested, based on the results of computer processing of standard curves. Gastrin concentrations in the blood serum and gastric juice (pretreated and untreated) are compared in the same patients. PMID- 1704958 TI - [The daily dynamics of blood lipids in elderly subjects with hypertension]. AB - Three groups of elderly subjects were examined: patients with sclerotic systolic hypertension (SSH), with essential hypertension (EH), and normal subjects. The blood levels of total lipids, cholesterol, triglycerides, low and very low density lipoproteins, phospholipids, and nonesterified fatty acids were significantly higher in the patients than in normal subjects. In contrast to those in normal subjects, acrophases of atherogenic lipid fractions (cholesterol, triglycerides, low and very low density lipoproteins) in the patients were synchronized and occurred in the day time (at 2-4 p.m.). Diverse directions of circadian changes in lipid fractions in the patients and normal subjects may contribute to alteration in the rhythmicity of other metabolisms and in the blood coagulation system, thus leading to various complications. This fact necessitates development of methodologic approaches to chronotherapy of the afore-said disorders. To detect latent atherogenic shifts in the lipid transport system in elderly patients with SSH and EH, studies of lipid metabolism parameters are recommended to be carried out between 2 and 4 o'clock p.m. PMID- 1704959 TI - [The effect of sorbitol on the determination of keto amines using a colorimetric method]. AB - The presence of sorbitol in a concentration 11 mg/ml in a sample reduces by 68 percent the values of fructose glycine measured by colorimetry with thiobarbituric acid. Such sorbitol concentration has no effect on furfural production and on 5-hydroxymethylfurfural reaction with thiobarbituric acid. Measurements of glycolyzed hemoglobin have demonstrated that sorbitol in concentration 11 mg/ml completely inhibits hydrolysis. This fact should be borne in mind when measuring blood glycosylated hemoglobin by colorimetry with thiobarbituric acid and exclude sorbitol from patients' rations before measurements. PMID- 1704960 TI - [Determination of the dispersity of suspensions using photoelectric colorimeters of different makes]. AB - Different conditions of measuring the suspension dispersity of photoelectric colorimeters of two brands,KFK-2 and FEK-56, result in distorted data: the dispersity index (DI) measured by KFK according to the method developed for FEK is inaccurate. The presence of 2 photoreceptors in the KFK apparatus, in contrast to the FEK one, results in elevated values of optic density (D) when the apparatus sensitivity is changed, starting from 590 nm wavelength (this fact is mentioned in the operation instructions) and eventually breaks the inversely proportional relationships between the D value and wavelength. DI estimation is based on the proportional relationships between wavelengths and D values obtained with a selected light filter pair. The wavelength selection mode had to be changed in the procedure of DI measurement by KFK-2 (or by KFK-MP) so that the light filter pair was placed in the same sensitivity zone; the distance between the cuvette and photo element was also changed (the cuvette with the sample was placed not in the cuvette holder but at a maximum distance from the photo element, closer to the source of light). Such modifications helped obtain compatible results of optic density measurements and DI values with photoelectric colorimeters of various brands. This is true only for suspensions rather than for transparent solutions, which fact is explained by the theory of light dispersion by suspension particles. PMID- 1704961 TI - [An immunoenzyme method of determining pregnancy-associated alpha 2 glycoprotein]. AB - Methods for purification of pregnancy-associated alpha 2-glycoprotein, obtained antibodies to it, and enzyme immunoassay of this protein are described. Enzyme immunoassay permits measurement of this protein in blood serum and mononuclear cell cultures in a wide range of concentrations. The method sensitivity may be improved by removing the antibodies cross-reacting with animal proteins. PMID- 1704963 TI - [An automated rapid method of complement determination]. PMID- 1704962 TI - [Determination of the sorption properties of microplates for immunoenzyme analysis]. AB - The authors have examined the possibility of using horse radish peroxidase for assessment of the sorption characteristics of microplates for enzyme immunoassay; the results obtained by the technique modified by the authors and by one of the routine methods (the direct method) were in high correlation. Sorption capacities and homogeneity of the plates manufactured in Leningrad and abroad were compared; the plates made in this country were found inferior to foreign ones in respect of both parameters almost twofold. Sorption capacity of the microplates may be improved by gamma-irradiation; the optimal irradiation dose was defined: 1.5 mrad. PMID- 1704964 TI - [The application of Rebuck's skin window technique]. PMID- 1704965 TI - [Serological diagnosis of syphilis and AIDS in a dermato-venereal clinic]. PMID- 1704966 TI - [Determination of complement in an automatic mode]. AB - A method for the complement determination is suggested by a 50 percent hemolysis microtest with the use of ELISA processor II for automated procedure and registration. The results obtained with this technique virtually do not differ from the data of the routine macromethod. The method is suggested for mass immunologic screenings. PMID- 1704967 TI - [The etiology of salmonellosis]. AB - Dynamic changes in the etiologic structure of salmonelloses on the territory of the town resulted in alteration of the predominant Salmonella strains. Many-year studies of the etiologic structure of salmonelloses in humans and animals and of serologic variants of Salmonella strains isolated from the environment helped establish the regularities that characterize the ecology of these agents. PMID- 1704968 TI - [Preservation of microorganisms by deep freezing]. AB - Meningococci, pneumococci, and hemophilic bacilli may be stored at -70 degrees C. Only half (54.4 percent) of Meningococcal cultures were viable after 11 months storage, 41.6 percent retained their serologic properties. Conditions for stable long storage of meningococci at -70 degrees C are to be searched for. Pneumococci retained their initial parameters for 8 months, this permitting recommendation of such storage for these bacteria. No data on the possibility of hemophilic bacilli storage is available yet. PMID- 1704969 TI - [Preparation of immune sera to group A streptococcus lipoteichoic acid]. AB - A method is described for the preparation of sera highly active in reactions with Group A Streptococcus lipoteichoic acid. Ultrasound-destroyed biomass of Streptomyces levoris K-3053 strain is used as the antigen. This strain was isolated in studies of soil Streptomyces; it contains sufficient amounts of nonglycosylated glycerolteichoic acid of the first-third types, that is identical in structure to polyglycerophosphate skeleton of Group A Streptococcus lipoteichoic acid. PMID- 1704970 TI - [The spectrum of extracellular proteins of staphylococci--the passport of a strain]. AB - The possibility of detecting identical strains was analyzed by comparing extracellular protein spectra of Staphylococcus aureus strains from the collection of the L. A. Tarasevich Institute for Standardization and Control of Medical Preparations. Analysis of the findings confirms that S. aureus and other Staphylococci should be characterized by their protein spectra and this marker should be present in the certificate of the strain in collections. Protein spectrum determination is obligatory for production strains. PMID- 1704971 TI - [The bactericidal activity of aqueous solutions of pervomur]. PMID- 1704973 TI - [Do we need a reference laboratory?]. PMID- 1704972 TI - [Results of interlaboratory control of photometers]. PMID- 1704974 TI - Direct coronary vasodilation induced by intracoronary vasoactive intestinal peptide. AB - Vasoactive intestinal peptide (VIP) is a neurotransmitter that has been identified in epicardial coronary arteries. To evaluate the direct effect of VIP on coronary hemodynamics and blood flow, graded doses of VIP (0.01, 0.03, 0.10, and 0.30 micrograms/min) were infused into the left coronary artery of 7 patients at the time of diagnostic cardiac catheterization for chest pain syndromes. None of the patients had coronary stenoses greater than 50% during subsequent angiography. Coronary sinus VIP concentrations increased during each infusion (22 +/- 28 pg/ml at baseline to 109 +/- 22 pg/ml at 0.30 micrograms/min; p less than 0.05), but arterial VIP was elevated (39 +/- 29 pg/ml) only at the maximal dose of 0.30 micrograms/min. During all dosages of VIP, heart rate, right atrial and left ventricular end-diastolic pressure, and the heart rate x blood pressure product did not change. Moreover, neither mean aortic pressure nor left ventricular peak + dP/dt changed significantly at doses less than 0.30 micrograms/min; at 0.30 micrograms/min, mean aortic pressure decreased (97 +/- 15 to 90 +/- 15 mm Hg; p less than 0.05) and LV peak + dP/dt increased (1,621 +/- 230 to 1,801 +/- 226 mm Hg/s; p less than 0.05). Compared to baseline, the arterial-coronary sinus O2 content difference and myocardial O2 extraction diminished progressively at the 0.03, 0.10, and 0.30 micrograms/min doses of VIP (118 +/- 12 ml O2/L vs. 94 +/- 15, 70 +/- 9, and 61 +/- 26 ml O2/L, respectively, and 0.64 +/- 0.05 vs. 0.53 +/- 0.10, 0.38 +/- 0.06, and 0.34 +/- 0.15, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704975 TI - The acute effect of captopril on cerebral blood flow, its CO2 reactivity, and cerebral oxygen metabolism in human volunteers. AB - The present study was undertaken to examine the effect of captopril on cerebral blood flow (CBF), cerebral metabolic rate of oxygen (CMRO2) and CO2 reactivity in eight young healthy volunteers. The mean arterial blood pressure (MABP) was measured intraarterially, and CBF was measured by inhaled xenon-1233. The CO2 reactivity was defined as the relative change in CBF per 0.1 kPa change in arterial CO2. During the CO2 reactivity tests, the CBF changes were estimated by the arteriovenous-oxygen-difference method (CBFsat). Median CBF was 52 ml/100 g/min (50-56) and without any significant regional side-to-side asymmetries. No significant change was observed after captopril. The median resting MABP was 90 mm Hg (89-93), and was slightly reduced by 5 mm Hg (4-6) 1 h after an oral dose of captopril (50 mg). CMRO2 was 3.7 ml/100 g/min (3.5-3.8) and was unchanged after captopril. For the entire range of PaCO2 the median slope of the ln(CBFsat) versus PaCO2 regression line was 1.4 (1.3-1.6) at baseline compared to 1.4 (1.3 1.7) after captopril (p = 0.23). Considering the PaCO2 values between 4.0 and 6.5, the CO2 reactivity was 2.4%/0.1 kPa (2.1-2.4) at baseline and increased significantly to 2.9%/0.1 kPa (2.1-3.2) after captopril (p less than 0.05). No side effects were observed. The present study shows an overall unchanged CO2 reactivity; however, with an increased CO2 reactivity of the cerebral vessels for small changes in PaCO2 around the resting value of PaCO2 and a maintained coupling of CBF and CMRO2 after peroral captopril. PMID- 1704976 TI - Pharmacologic profile of cromakalim in the treatment of myocardial ischemia in isolated rat hearts and anesthetized dogs. AB - The detailed antiischemic pharmacology of the potassium channel activator cromakalim was determined in isolated globally ischemic rat hearts and a canine model of coronary occlusion and reperfusion. Cromakalim significantly improved reperfusion function in rat hearts starting at a concentration of 1 microM; this effect peaked at 7 microM. No cardiodepressant effects were observed in nonischemic tissue with cromakalim until a concentration of 100 microM was achieved, and this effect was reversed by glyburide. The antiischemic effect of 7 microM cromakalim was also completely reversed by glyburide and the novel ATP sensitive potassium channel blocker sodium 5-hydroxydecanoate (5-HD). Glyburide did not reverse the antiischemic effects of 1 microM diltiazem. Cromakalim not only improved reperfusion contractile function in rat hearts, but improved the functional reserve and efficiency of O2 utilization. In anesthetized dogs, intracoronary cromakalim (0.1 micrograms/kg/min given throughout ischemia and reperfusion) significantly reduced infarct size in hearts subjected to 90-min coronary occlusion and 5-h reperfusion. Along with this reduced infarct size, the frequency of ectopic beats and the proportion of animals fibrillating during reperfusion were significantly reduced by cromakalim. In isolated globally ischemic and reperfused rat hearts, cromakalim was significantly profibrillatory. Thus, cromakalim is significantly cardioprotective, and may have the propensity for profibrillatory activity, although this is not true under all conditions. PMID- 1704977 TI - Atrial natriuretic factor blocks the pressor action of endothelin. AB - The present study was designed to determine the pharmacologic effects of rat atrial natriuretic factor (r-ANF; 250 ng/kg/min), endothelin-3 (ET-3; 340 ng/kg/min), and combined r-ANF and ET-3 infusion on cardiac hemodynamics and renal function in anesthetized rats. The change in mean arterial pressure (delta MAP) was 13 +/- 2 mm Hg during ET-3 infusion alone. Delta MAP was -9 +/- 2 mm Hg during r-ANF infusion alone. Combined infusion of ET-3 and r-ANF resulted in a change in MAP of -7 +/- 3 mm Hg. The decrease in MAP during combined infusion of ET-3 and r-ANF occurred due to a decrease in cardiac output. Infusion of r-ANF did not block the cardiac output. Infusion of r-ANF did not block the ET-3 induced increase in total peripheral resistance (TPR). Infusion of r-ANF alone resulted in an 11-fold increase in urinary sodium excretion. ET-3 infusion completely blocked the ANF-induced natriuresis in part by markedly decreasing the glomerular filtration rate (GFR). ET-3 or r-ANF infusion alone resulted in two- or eightfold increases, respectively, in circulating r-ANF levels compared to vehicle alone. However, combined infusion of r-ANF and ET-3 resulted in a dramatic 27-fold increase in circulating r-ANF when compared to levels obtained during vehicle infusion alone. The present study demonstrates that at pharmacologic levels, r-ANF blocks the pressor action of ET-3 by decreasing cardiac output rather than TPR. Furthermore, ET-3 blocks the natriuretic action of r-ANF, partly by decreasing GFR. PMID- 1704978 TI - Reentrant tachycardia in a thin layer of ventricular subepicardium: effects of d sotalol and lidocaine. AB - Ventricular tachycardia (VT) was induced by premature stimulation or fast pacing in 14 Langendorff-perfused rabbit hearts whose left ventricular endocardial and intramural cells had been selectively killed by freezing. VTs were caused by apparent reentrant excitation in the surviving thin (1 mm thick) subepicardial layer of anisotropically oriented cells having ostensibly normal membrane characteristics. During VT, 100 microM d-sotalol (seven hearts) or 30 microM lidocaine (seven hearts) was added to the perfusate. Electrophysiological variables were measured before and during drug exposure at both slow (S1 = 300 ms) and fast (S1 = 150-180 ms) pacing rates. Sotalol prevented VT reinduction in six of seven preparations, compared to only two of seven with lidocaine. Lidocaine prolonged the functional refractory period (FRP) and slowed conduction velocity (CV). Lidocaine prolonged the wavelength of the cardiac impulse (= FRP x CV) by 18% at slow rates but reduced it by 21% at fast rates. Sotalol, however, since it increased the FRP without reducing CV, caused wavelength prolongation at both rates (43% at slow rates, 26% at fast rates). Thus, this VT model may provide an important contrast of class I and class III drug action, with the drug effects on wavelength predicting susceptibility to VT induction. PMID- 1704979 TI - Atrial natriuretic factor attenuates sympathetic reflexes during lower body negative pressure in normal men. AB - To assess further the effects of atrial natriuretic factor on autonomic nervous system reflexes in normal humans, the hemodynamic and neurohormonal responses to lower body negative pressure were measured at control and during infusions of atrial natriuretic factor and nitroglycerin in nine normal male subjects. The control -20 mm Hg lower body negative pressure was characterized by significant reductions in right atrial and pulmonary wedge pressures, as well as stroke volume and cardiac output. This was associated with a reflex increase in forearm vascular resistance and plasma norepinephrine. During the infusion of atrial natriuretic factor, the same -20 mm Hg lower body negative pressure produced a larger decrease in mean arterial pressure of 7.9 +/- 3.9 mm Hg (p less than 0.05), as well as a larger decrease in stroke volume (41.3 +/- 4.2 ml/beat) and cardiac output (2.0 +/- 0.3 L/min). Atrial natriuretic factor infusion did not affect the increase in forearm vascular resistance during lower body negative pressure, but did attenuate the increase in plasma norepinephrine. To control for nonspecific vasodilator actions, lower body negative pressure was also repeated during nitroglycerin infusion. Nitroglycerin infusion did not significantly change the responses of blood pressure, cardiac output, stroke volume, forearm vascular resistance, or plasma norepinephrine during lower body negative pressure. Thus, these data demonstrate that atrial natriuretic factor infusion can attenuate sympathetic nervous system reflexes evoked during lower body negative pressure. These inhibitory effects on the sympathetic nervous system may contribute to many of the observed hemodynamic actions of atrial natriuretic factor. PMID- 1704980 TI - Effect of pinacidil on myocardial blood flow in the chronically pressure overloaded hypertrophied left ventricle. AB - This study examined the effect of pinacidil on the transmural distribution of myocardial blood flow in the chronically pressure overloaded hypertrophied left ventricle. Studies were performed in six dogs in which banding of the ascending aorta had resulted in an 88% increase in left ventricular mass, as well as in six normal control animals. Two doses of pinacidil were administered to decrease mean arterial pressure by approximately 10 mm Hg (low dose) and 20 mm Hg (high dose). Animals with hypertrophy required significantly smaller drug doses to achieve the desired reductions in arterial pressure. During control conditions mean myocardial blood flow was significantly higher in animals with hypertrophy (1.90 +/- 0.21 ml/min/g) than in normal animals (1.12 +/- 0.08 ml/min/g; p less than 0.05). Subendocardial flow (endo) exceeded subepicardial flow (epi) in normal dogs during control conditions (endo/epi = 1.41 +/- 0.13), but not in animals with hypertrophy (endo/epi = 1.06 +/- 0.06; p less than 0.05). Pinacidil caused coronary vasodilation with similar relative increases in blood flow in both normal and hypertrophied hearts, so that after pinacidil, absolute blood flow rates remained higher than normal in animals with hypertrophy. Pinacidil caused a redistribution of blood flow away from the subendocardium in normal hearts (endo/epi = 0.90 +/- 0.11 during high-dose pinacidil) and in hearts with hypertrophy (endo/epi = 0.81 +/- 0.13 during high-dose pinacidil). The endo/epi ratios during high-dose pinacidil were not significantly different between the two groups. This study demonstrates that pinacidil is a potent coronary vasodilator in both normal and hypertrophied hearts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704981 TI - Effects of amiodarone on cardiac electrophysiology and inducibility of arrhythmias in chronically infarcted dogs: late arrhythmias, haemodynamics, and sympatholytic actions. AB - The electrophysiological and antiarrhythmic effects of acute (2 and 10 mg/kg i.v.) and chronic (400 mg/day p.o. for 28 days) amiodarone (AM) treatment were compared in anaesthetised dogs with 5-6-day-old myocardial infarcts. Intravenous AM prolonged the RR interval, sinus node recovery time, the PR interval, and atrial to His conduction time by 36, 33, 25, and 36%, respectively. Corresponding increases after oral amiodarone were 50, 57, 12, and 26%. Atrial and His-Purkinje conduction times were unchanged. Atrial and ventricular refractory periods were increased especially after oral treatment. Oral AM additionally prolonged QRS, QT, and paced QT (by 4, 34, and 19%, respectively). Effects of oral AM on ventricular repolarisation and on the fast inward sodium current were confirmed in vitro. Both modes of AM administration protected against inducible arrhythmias, an effect that was more marked during normal sinus rhythm than during pacing in orally treated dogs. Oral amiodarone failed to protect against spontaneous late arrhythmias 24 h after infarction whilst both modes of administration noncompetitively inhibited isoprenaline-induced tachycardia. Oral AM reduced blood pressure (13%) and LV dP/dt/P (24%) whereas cardiac output was maintained by an increase in stroke volume. It was concluded that oral AM is haemodynamically well tolerated and that prolonged ventricular repolarisation enhanced by bradycardia together with sympatholytic actions may be important mechanisms for antiarrhythmic efficacy, whereas the mechanisms involved in i.v. efficacy are less clear but may depend, at least partly, on sympatholytic actions and perhaps (tentatively) on sodium channel block in Purkinje tissue. PMID- 1704982 TI - Glucose and lipid metabolism during long-term lisinopril therapy in hypertensive patients. AB - The effects of long-term lisinopril therapy on glucose tolerance and serum lipid profiles were investigated prospectively in 30 hypertensive patients: 15 with normal glucose tolerance and the remaining 15 with glucose intolerance. The levels of plasma glucose, serum lipids, and plasma glycosylated hemoglobin A1c were determined before and during long-term (6 months) therapy with lisinopril. A 75 g oral glucose tolerance test was performed before and during long-term lisinopril therapy. Lisinopril produced significant falls in both systolic and diastolic blood pressure in both patient groups during the long-term therapy. Neither fasting nor post-glucose-load venous plasma glucose levels were altered in either group of patients, and no patients with normal glucose tolerance developed diabetes mellitus during the study. There was no significant change in the insulinogenic index (delta IRI/delta BS at 30 min post-glucose load) in both patient groups. No significant changes in fasting levels of serum cholesterol, triglycerides, and HDL-cholesterol were observed in either group during long-term lisinopril therapy. Long-term lisinopril therapy does not compromise glucose or lipid metabolism in hypertensive patients. Lisinopril may have an advantage as an antihypertensive agent that can be given to hypertensive patients without concern that it might alter their serum lipid concentrations or impair glucose tolerance. PMID- 1704983 TI - Antihypertensive efficacy and tolerability of a fixed combination of metoprolol and felodipine in comparison with the individual substances in monotherapy. The Swedish/United Kingdom Study Group. AB - In this double-blind, randomized, parallel-group study, the aim was to compare the efficacy and tolerability of a new fixed combination of felodipine and metoprolol with the individual components in monotherapy. After a placebo period of 4 weeks, 159 patients with mild to moderate essential hypertension were randomized to extended-release formulations of either felodipine plus metoprolol 10 + 100 mg (FM), felodipine 10 mg (F), or metoprolol 100 mg (M) once daily if supine diastolic blood pressure greater than 95 mm Hg. After 12 weeks of active treatment, the reductions in supine blood pressure (24 h after dosing) were 20/14, 13/10, and 11/8 mm Hg for FM, F, and M, respectively. The difference in change was 7/4 mm Hg (p = 0.004/p = 0.006) and 8/5 mm Hg (p = 0.0002/p less than 0.0001) for the fixed combination and F or M, respectively. Blood pressure control (diastolic blood pressure less than 90 mm Hg after 12 weeks) was significantly better for the combination than for F and M, i.e., 71%, 49% (p = 0.008), and 34% (p = 0.004), respectively. Adverse experiences were those to be expected from previous studies with felodipine and metoprolol and did not differ in frequency between groups. It can be concluded that a fixed combination of metoprolol and felodipine has a clinically relevant and significantly better blood pressure reduction 24 h postdose than the individual substances in monotherapy, without decreased tolerability. PMID- 1704984 TI - Effects of MDL 11,939 on action potential and contractile force in cardiac tissues: a comparison with bretylium, clofilium, and sotalol. AB - The effect of MDL 11,939 hydrochloride [alpha-phenyl-1-(2-phenylethyl)-4 piperidinemethanol HCl], a novel antiarrhythmic agent, was studied using microelectrode recording techniques. MDL 11,939 (10(-7) - 10(-6) M increased the action potential (AP) duration in both guinea pig papillary muscle and dog Purkinje fiber with a maximum effective concentration of 10(-6) to 3 x 10(-6) M. MDL 11,939 up to and including 10(-6) M did not alter upstroke velocity (Vmax) of the AP in either tissue but 10(-5) M decreased the Vmax in Purkinje fibers. Compared to the other class III antiarrhythmic agents (bretylium, clofilium, and d-sotalol), MDL 11,939 was among the most potent in increasing AP duration in the papillary muscle, while being relatively less effective in increasing AP duration in Purkinje fiber. Bretylium did not increase the AP duration in papillary muscle unless it was reserpinized. MDL 11,939, clofilium, and d-sotalol all produced negative inotropic effects in papillary muscle, whereas bretylium produced a positive inotropic effect. The positive inotropic effect of bretylium was abolished by reserpinization. In papillary muscle preparations depolarized with 22 mM K+, MDL 11,939 at concentrations greater than or equal to 10(-5) M depressed Vmax of the slow AP, suggesting that it inhibited the inward Ca2+ current, and such an effect may contribute to the negative inotropic effect of this agent. In the depolarized preparations, the slow AP duration was still increased by concentrations of MDL 11,939 greater than or equal to 10(-6) M. In conclusion, MDL 11,939 has cellular electrophysiological properties that suggest a class III antiarrhythmic activity. PMID- 1704986 TI - Relationship between Vmax and conduction velocity in uniform anisotropic canine ventricular muscle: differences between the effects of lidocaine and amiodarone. AB - The purpose of these experiments was to determine if Vmax in anisotropic myocardium varies approximately as the square of the conduction velocity (theta) after the addition of lidocaine (6.5 micrograms/ml) and amiodarone (20 micrograms/ml). We measured Vmax and theta in 16 epicardial strips of uniform anisotropic ventricular muscles, over a wide range of stimulation frequencies. The relationship of Vmax to theta 2 was evaluated by linear regression analysis. We found that the decrease in Vmax was proportional to the square of the decrease in theta in the presence of lidocaine both during longitudinal (LP) and transverse (TP) propagation (mean slope +/- SEM: 0.961 +/- 0.047 and 0.918 +/- 0.068, respectively). The changes in Vmax, in the presence of amiodarone, were not predicted by the quadratic changes in theta during TP. However, during LP, the changes in Vmax and theta were well fitted by the predicted relationship. The slope was significantly different from that of lidocaine (2.399 +/- 0.673 vs. 0.961 +/- 0.047, p less than 0.05). On the other hand, the predicted values of theta, assuming theta = square root of Vmax x k, were significantly more depressed than the measured values. We conclude that in uniform anisotropic ventricular muscle, when the changes in Vmax and theta are solely due to a decrease in sodium conductance, a quadratic relationship between the changes in both variables is seen and the slope of the regression line should be 1, such as we have shown for lidocaine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1704985 TI - Effects of nitrendipine and cilazapril on renal hemodynamics and albuminuria in hypertensive patients with chronic renal failure. AB - In 11 hypertensive patients with chronic renal failure we studied the short-term effects of the calcium antagonist nitrendipine, the angiotensin-converting enzyme inhibitor cilazapril, and the combination of both drugs on blood pressure, renal hemodynamics, and proteinuria in a randomized, double-blind, placebo-controlled way. After one week of treatment, blood pressure at 2-5 h after drug administration amounted to 159 +/- 5/101 +/- 3 mm Hg (means +/- SEM) during placebo. Nitrendipine, cilazapril, and the combination lowered mean arterial pressure by 1.4 +/- 1.6 (NS), 6.0 +/- 1.7 (p less than 0.10), and 10.3 +/- 2.1% (p less than 0.01), respectively. Glomerular filtration rate did not change. As compared to placebo, renal blood flow increased and renal vascular resistance decreased significantly during the combination. Filtration fraction amounted to 22.7 +/- 1.2% during placebo and was 22.0 +/- 1.4 (NS), 20.4 +/- 1.2 (p less than 0.01), and 20.5 +/- 1.4% (p less than 0.05) during nitrendipine, cilazapril, and the combination, respectively. During nitrendipine, albuminuria was slightly higher than during placebo: 0.86 +/- 0.39 vs. 0.58 +/- 0.25 mg/min (NS). During cilazapril alone and during the combination of both drugs, albuminuria was lower as compared to nitrendipine: 0.38 +/- 0.14 mg/min (p less than 0.01) and 0.44 +/- 0.18 mg/min (p less than 0.01), respectively. The data suggest that the combination of nitrendipine and cilazapril is an effective treatment in renal hypertension. In addition, cilazapril alone as well as the combination with nitrendipine reduced albuminuria, possibly by decreasing filtration fraction and/or reduction of blood pressure. PMID- 1704987 TI - Effects of endothelin on arterial pressure and venous tone in intact and hexamethonium-treated conscious rats. AB - The dose-response effects of the potent vasoconstrictor peptide endothelin on mean arterial pressure (MAP), heart rate (HR), and mean circulatory filling pressure (MCFP), an index of body venous tone, were investigated in conscious and unrestrained, intact rats as well as in rats continuously infused with the ganglionic blocker hexamethonium. The dose of hexamethonium selected was that which reduced the reflex tachycardia induced by i.v. injections of acetylcholine by 50%. In intact rats and rats pretreated with hexamethonium, i.v. injections of endothelin caused dose-dependent increases in MAP and decreases in HR. Low doses of endothelin did not affect MCFP while the highest dose in the intact rat, and the two highest doses in hexamethonium-treated rats, caused small but significant increases in MCFP. Our results suggest that endothelin has small effects on body venous tone in contrast to its effectiveness in raising arterial pressure. PMID- 1704988 TI - Effects of xamoterol on the reversible cycling of cardiac beta-adrenoceptors. AB - We have examined the effects of xamoterol, a partial beta 1-adrenoceptor agonist, on cardiac beta-adrenoceptors using isolated myocytes and cell-free preparations. Xamoterol was considerably less effective than isoproterenol in stimulating adenylate cyclase activity but the difference was narrowed by 1 microM forskolin, presumably by inducing more efficient coupling to the catalytic subunit of the enzyme. Xamoterol mediated a time- and concentration-dependent loss of beta adrenoceptors from the cell surface of cardiac myocytes through a process of internalization sensitive to the cytoskeleton inhibitors, colchicine and cytochalasine. In cell-free preparations, loss of membrane-bound beta adrenoceptors induced by xamoterol, but not that induced by 1 microM isoproterenol, was prevented by the inhibitor of protein kinase A. In contrast, 100 nM heparin (an inhibitor of beta-receptor kinase) prevented the isoproterenol but not the xamoterol-mediated decline of beta-receptors. In addition, 1 microM xamoterol attenuated the isoproterenol-mediated internalization of beta adrenoceptors in cardiac myocytes over a wide range of isoproterenol concentrations. This attenuation required activation of protein kinase A. These results suggest that the influence of xamoterol on the cycling of cardiac beta adrenoceptors involves different pathways than those utilized by isoproterenol. PMID- 1704989 TI - Influence of metoprolol and cilazapril on blood pressure and on sleep apnea activity. AB - Up to 50% of hypertensive men are subject to sleep apnea (SA). With a prevalence in men of up to 10%, SA is a common illness and hypertension (HT) one of its early symptoms. It is important to have available a drug treatment that will effectively control blood pressure (BP) without exacerbating symptoms of SA. Twelve patients with SA and HT were investigated in a double-blind, comparative trial. Patients were randomly allocated to either metoprolol (M) 100 mg daily or cilazapril (C) 2.5 mg daily. Polysomnographic measurements under standardized conditions including intraarterial BP monitoring were taken on two consecutive nights each before and after the 1-week treatment. Values in the M group were (mean +/- 95% CI) systolic BP 161 +/- 2.1 vs. 148 +/- 2.2 mm Hg (p less than 0.01); diastolic BP 98 +/- 1.8 vs. 93 +/- 1.8 mm Hg (p less than 0.01); and HR 73 +/- 1.2 vs 65 +/- 1.1 beats/min (p less than 0.01). Corresponding figures for the C group were systolic BP 140 +/- 2.1 vs. 127 +/- 2.1 mm Hg (p less than 0.01); diastolic BP 95 +/- 1.7 vs. 78 +/- 1.7 mm Hg (p less than 0.01); and HR 82 +/- 1.1 vs. 79 +/- 1.2 beats/min (p less than 0.01). Whereas C reduced both BP and HR in all sleep phases, M produced no changes during REM sleep. SA activity was 45 (range 15-91) vs. 34 (range 2-57) apneas per hour of sleep in the M group and 54 (range 21-84) vs. 40 (range 8-72) apneas per hour in the C group (p less than 0.01). There were no changes in total sleep time or in the proportions of non-REM to REM sleep. Both M and C reduce nocturnal BP in SA patients, but the effect of C is seen in all sleep phases. C has a more favorable effect on the disturbed nocturnal blood pressure of SA patients. PMID- 1704990 TI - Nicotine enhances angina pectoris-like chest pain and atrioventricular blockade provoked by intravenous bolus of adenosine in healthy volunteers. AB - An attempt was made to study possible interaction between neuromodulation by adenosine and nicotine stimulatory effects. Dose-effect curves were made double blind in 7 nonsmoking, nonsnuffing healthy volunteers (25-49 years) before and during exposition to nicotine roughly corresponding to the nicotine of one cigarette, 2 mg ingested from a chewing gum (800 chews during 20 min). Chest pain was estimated by the Borg CR-10 scale. ECG was followed, and respiration was recorded continuously by spirometry. Maximal tolerable dose of adenosine was 12.7 +/- 3.0 mg. Chest pain increased dose dependently to 5.7 +/- 1.7 units. Nicotine increased the pain response by 20 +/- 15%, (p less than 0.02). The total time with atrioventricular (AV) block provoked by adenosine increased with nicotine (7 +/- 12%, p less than 0.03) while increased ventilation provoked by adenosine was unaffected by nicotine. In conclusion, interaction between adenosine and nicotine was demonstrated. Nicotine enhanced both stimulatory (chest pain) and inhibitory actions (AV-block) of adenosine. PMID- 1704991 TI - Effects of a novel Ca2+ entry blocker, CD-349, and TMB-8 on renal vasoconstriction induced by angiotensin II and vasopressin in dogs. AB - The effects of a Ca2+ entry blocker CD-349 and an intracellular Ca2+ release inhibitor TMB-8 on renal vasoconstriction induced by angiotensin II (ANG II) and arg-vasopressin (AVP) were examined in anesthetized dogs. Intrarenal bolus injection of ANG II (3-10 ng/kg), AVP (5-20 ng/kg) or a Ca2+ entry promotor Bay K 8644 (0.1-0.4 micrograms/kg) produced a dose-dependent decrease in renal blood flow (RBF). Intrarenal infusion of CD-349 (0.03-0.3 micrograms/kg/min) suppressed the RBF responses to ANG II, AVP, and Bay K 8644. The RBF responses to ANG II and AVP were augmented slightly by intrarenal infusion of Bay K 8644 (0.3 micrograms/kg/min). Intrarenal infusion of TMB-8 (0.03-0.1 mg/kg/min) also suppressed the RBF responses to ANG II and AVP, whereas it did not affect the RBF response to Bay K 8644. These results suggest that vasoconstriction induced by ANG II or AVP is mediated both by the influx of Ca2+ through dihydropyridine sensitive Ca2+ channels and the release of Ca2+ from TMB-8-sensitive Ca2+ pools in the in vivo dog kidney. PMID- 1704992 TI - Development of hypertension in WKY rats after transplantation of parathyroid glands from SHR/SP. AB - We have investigated the role of the parathyroid gland (PTG) in the long-term development of blood pressure (BP) in stroke prone spontaneously hypertensive rats (SHR/SP) and Wistar Kyoto (WKY) rats. After ablation of their own PTGs, SHR/SP animals received PTGs from WKY rats and vice versa. Transplantation resulted in a normal calcium and parathyroid hormone status without signs of hypoparathyroidism. All animals received a high salt diet (8% NaCl) for 4 weeks after transplantation of PTGs. In SHR/SP, which received PTGs from WKY, development of high BP was clearly attenuated when compared to sham-operated SHR/SP rats. WKY rats with PTGs from SHR/SP rats became hypertensive, while WKY sham-operated animals remained normotensive. PTGs from SHR/SP rats are able to induce hypertension in normotensive WKY rats. PMID- 1704993 TI - Electrophysiologic and antiarrhythmic actions of pirmenol on rabbit and guinea pig cardiac preparations. AB - The electrophysiological and antiarrhythmic effects of pirmenol HCl were examined using the microelectrode technique applied to multicellular preparations and the suction-pipette whole-cell clamp method applied to ventricular myocytes from rabbit and guinea pig hearts. Pirmenol at 5 microM and higher doses suppressed the sinus node automaticity by depressing the slow diastolic depolarization without changing the maximum diastolic potential. Pirmenol at 1 microM and higher doses depressed the maximum upstroke velocity (Vmax) of action potentials and prolonged the action potential duration at 90% repolarization in atrial muscles and Purkinje fibers without affecting resting membrane potentials. Pirmenol at 5 microM depressed the early part of the plateau and lengthened the final repolarization of the action potentials in ventricular myocytes, of which effects were attributed to the depression of the calcium current and the delayed outward K+ current. Triggered tachyarrhythmias arising from delayed afterdepolarizations in papillary muscles and ventricular myocytes were markedly inhibited by 1-5 microM pirmenol. The drug changed the amplitude and appearance of the transient inward current in ventricular myocytes. These results suggest that pirmenol has electrophysiologic properties that could provide an antiarrhythmic action on various types of arrhythmias. PMID- 1704994 TI - Effects of pretreatment with 2-O-octadecylascorbic acid, a novel free radical scavenger, on reperfusion-induced arrhythmias in isolated perfused rat hearts. AB - The effects of pretreatment with 2-O-octadecylascorbic acid (CV-3611), a novel liposoluble free radical scavenger, on reperfusion-induced arrhythmias were studied in isolated perfused rat hearts (n = 15 per group). The hearts were subjected to 10 min of coronary artery occlusion and 3 min of reperfusion. Pretreatment with CV-3611 (5 and 20 mg/kg) reduced the incidence of ventricular fibrillation (VF; reversible plus sustained) from its control value of 93% to 47% (p less than 0.05). Furthermore, CV-3611 reduced the incidence of sustained VF in a dose-dependent manner, from 67% in the control group to 13% in the CV-3611, 20 mg/kg treated group (p less than 0.01). CV-3611 (5 and 20 mg/kg) reduced the incidence of ventricular tachycardia (VT) from its control value of 93% to 73%. Pretreatment with ascorbic acid (5 mg/kg) had no effect on VF and VT. The myocardial content of CV-3611 was proportional to the dosage. We concluded that CV-3611 could reduce significantly the susceptibility to reperfusion-induced arrhythmias, especially VF, and that its effect may be due to the elimination of oxygen-derived free radicals by CV-3611 present in the membrane and the capture of lipid radicals, thereby inhibiting lipid peroxidation. PMID- 1704995 TI - Prevention of myocardial reperfusion injury in experimental coronary revascularization following ischemic arrest by a novel antiinflammatory drug, ONO 3144. AB - The cardioprotective effects of a novel antiinflammatory drug, ONO-3144 (ONO), on ischemic-reperfused myocardium were investigated using an in situ pig heart model. Heart was subjected to 2 h of regional ischemia, with the final 1 h having superimposed global cardioplegic arrest followed by 1 h of reperfusion. ONO (20 microM) was administered after the arrest at the onset of reperfusion. Left ventricular developed pressure (LVDP), maximum rate of rise of left ventricular pressure (LV dp/dt), and left ventricular end-diastolic pressure (LVEDP) were measured under isovolumic conditions to assess cardiac contractility and compliance. ONO improved LVDP and LV dp/dt, and reduced LVEDP after 60 min of reperfusion compared to control. This drug also improved segment shortening and end-diastolic length significantly after 15 and 60 min of reperfusion. Slight improvements in oxygen consumption and creatine kinase (CK) release were also noted. In addition, ONO reduced lipid peroxidation and thromboxane formation but enhanced the production of prostaglandins. In vitro studied demonstrated ONO to be effective scavengers for both hydroxyl (OH.) and hypohalite (OCL.) radicals. The results suggest that myocardial reperfusion injury that developed after ischemic arrest was reduced significantly by ONO. This drug inhibited such injury, probably by directly scavenging potentially harmful radicals such as OH. and OCI., which are generated in ischemic-reperfused myocardium. PMID- 1704996 TI - [Disorders of the colloid-osmotic state of blood and methods of their correction during angiography in children]. PMID- 1704997 TI - Triple post-transcriptional control. AB - The ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host. Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin. Regulation of ermC gene expression occurs at the post-transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization. PMID- 1704998 TI - Fatal carbon monoxide poisoning in a camper-truck--Georgia. PMID- 1704999 TI - Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines. AB - A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5 fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD 1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU. PMID- 1705000 TI - Prolonged ethanol inhalation decreases gamma-aminobutyric acidA receptor alpha subunit mRNAs in the rat cerebral cortex. AB - Ethanol administration to rats by ethanol vapor inhalation (14 days) results in a 40-50% reduction in the level of gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit mRNAs [4.4 and 4.8 kilobases (kb)] in the cerebral cortex. The level of alpha 2 subunit mRNA (8.0 kb) was also reduced by 29%, whereas there was no effect of prolonged ethanol exposure on the level of alpha 3 subunit mRNA (3.1 kb). Ethanol exposure did not alter the steady state levels of cerebral cortical glutamic acid decarboxylase or beta-actin mRNAs. Moreover, no alterations in the levels of total RNA, poly(A)+ RNA, or rRNA were observed, suggesting that the ethanol-induced reductions in GABAA receptor alpha 1 and alpha 2 subunit mRNAs were not the result of a generalized effect of ethanol administration on transcription or mRNA turnover. These ethanol-induced reductions in GABAA receptor alpha subunit mRNAs may underlie alterations in GABAA receptor function or number observed following prolonged ethanol exposure in rats. PMID- 1705001 TI - Induction of deoxycytidine kinase by 5-azacytidine in an HL-60 cell line resistant to arabinosylcytosine. AB - Induction of 2'-deoxycytidine kinase (dCK) by 5-azacytidine (5-Aza-C) in a dCK deficient HL-60 cell line resistant to 1-beta-D-arabinofuranosylcytosine (Ara-C) (HL-60/Ara-C) was examined by measurement of the incorporation of [3H]deoxycytidine ([3H] dCyd) into cellular DNA, the kinetic properties of purified dCK, the cytotoxic potency (IC50 values), and the DNA methylation patterns of 5-Aza-C-treated and untreated cells. Following a 72-hr exposure to 1 microM 5-Aza-C, the incorporation of [3H]dCyd into DNA was increased 6-fold and the total dCK activity was increased 12-fold, with a peak for both on day 6. The onset of dCK induction was dependent on the length of exposure time. The IC50 values for cell growth inhibition by Ara-C on day 3 were 0.08 microM for HL-60 cells, 12.5 microM for HL-60/Ara-C cells, and 0.55 microM for HL-60/Ara-C cells pretreated with 5-Aza-C for 40 hr. The Km and Vmax of dCyd for HL-60 dCK were similar to those for 5-Aza-C-induced HL-60/Ara-C dCK. The restriction enzymes Hpall, which cleaves CCGG sequences but cannot cleave at sites methylated at the internal cytosines (5'-CMeCGG), and Mspl, which cleaves sequences with internal methylated cytosine but cannot cleave at sites methylated at external cytosines (5'-MeCCGG), were used for DNA methylation pattern determination. The newly synthesized DNA of HL-60 wild-type cells was cleaved by Mspl to a greater extent than that of HL-60/Ara-C cells. After exposure to 1 microM 5-Aza-C for 40 hr, methylation patterns of newly synthesized DNA reverted in HL-60/Ara-C cells to a clevage pattern similar to that in HL-60 wild-type cells. When compared with untreated control, DNAs from 5-Aza-C-treated resistant cells were cleaved to a greater extent by Mspl than by Hpall, suggesting that internal cytosine-residue methylation was relatively uninhibited.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705002 TI - Deletions in the SH2 domain of p60v-src prevent association with the detergent insoluble cellular matrix. AB - p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association. PMID- 1705003 TI - Regulated expression of human alpha- and beta-globin genes in transient heterokaryons. AB - We have examined the expression of human alpha- and beta-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic beta-globin genes are activated. In both the parental MEL cells and the heterokaryons, the alpha-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult beta-globin genes. These results suggest that kinetic differences in the activation of alpha- and beta-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic beta-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult beta-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult beta-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes. PMID- 1705004 TI - The gene encoding rat insulinlike growth factor-binding protein 1 is rapidly and highly induced in regenerating liver. AB - The liver is an epithelioid organ that can regenerate following partial hepatectomy. Although it is composed mainly of hepatocytes, it has a complex, multicellular architecture, implying that intercellular communications must exist during regeneration. As in other mitogen-stimulated cells, immediate-early growth response genes induced in the absence of prior protein synthesis are likely to play an important regulatory role in the regenerative process. Through differential screening of regenerating liver cDNA libraries, we found that one of the most highly expressed immediate-early genes in liver regeneration encodes the rat homolog of the low-molecular-weight insulinlike growth factor (IGF)-binding protein (IGFBP-1). This protein has been implicated in enhancing the mitogenic effect of IGF on tissues. IGFBP-1 gene induction is transcriptionally mediated and specific to regenerating liver, as the gene is not expressed in mitogen stimulated fibroblasts. IGFBP-1 expression has been shown to increase under low insulin conditions such as diabetes, and the complex regulation of expression is indicated by our finding that insulin treatment of H35 rat hepatoma cells, which induces proliferation, also causes a rapid decrease in transcription and expression of the IGFBP-1 gene. Of note, IGFBP-1 mRNA is abundant in fetal rat liver, implying that it participates in normal liver growth and development. Although regenerating liver cells continue to produce IGF-I, we did not detect IGF-I receptor mRNA during the first 24 h after hepatectomy. However, some IGFBPs may act to enhance the activity of IGF-I independently of IGF-I receptors. Thus, IGF-1 and IGFBPs may interact with hepatocytes or nonparenchymal liver cells, through either IGF-I or novel receptors. In this way, IGFBP-I and IGF-I could act in a paracrine and/or autocrine fashion in maintaining normal liver architecture during regeneration. PMID- 1705005 TI - Interleukin-6 signals activating junB and TIS11 gene transcription in a B-cell hybridoma. AB - The events in interleukin-6 (IL-6) signal transduction leading to primary response gene activation were analyzed in murine B-cell hybridoma and plasmacytoma cells which require IL-6 for growth. IL-6 stimulation of IL-6 deprived cells resulted in the rapid and transient tyrosine phosphorylation of a 160-kDa cellular protein (p160). This was followed by the highly selective induction of two primary response genes, junB/AP-1 transcription factor and TIS11. junB and TIS11 inductions were unaffected by cycloheximide, suggesting that posttranslational modifications accounted for their activation. Activation of junB and TIS11 transcription required rapid tyrosine kinase activity as well as a different protein kinase activity sensitive to the potent kinase inhibitor, H7, and activated following p160 tyrosine phosphorylation. This H7-sensitive kinase appears to be distinct from any well-characterized protein kinase-second messenger system. On the basis of these findings, we propose that IL-6-induced signal transduction proceeds through a novel protein kinase cascade which activates junB and TIS11 gene transcription. PMID- 1705006 TI - A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization. AB - Recent studies provide evidence that defective receptors can function as a dominant negative mutation suppressing the action of wild-type receptors. This causes various diminished responses in cell culture and developmental disorders in murine embryogenesis. Here, we describe a model system and a potential mechanism underlying the dominant suppressing response caused by defective epidermal growth factor (EGF) receptors. We used cultured 3T3 cells coexpressing human wild-type receptors and an inactive deletion mutant lacking most of the cytoplasmic domain. When expressed alone, EGF was able to stimulate the dimerization of either wild-type or mutant receptors in living cells as revealed by chemical covalent cross-linking experiments. In response to EGF, heterodimers and homodimers of wild-type and mutant receptors were observed in cells coexpressing both receptor species. However, only homodimers of wild-type EGF receptors underwent EGF-induced tyrosine autophosphorylation in living cells. These results indicate that the integrity of both receptor moieties within receptor dimers is essential for kinase activation and autophosphorylation. Moreover, the presence of mutant receptors in cells expressing wild-type receptors diminished the number of high-affinity binding sites for EGF, reduced the rate of receptor endocytosis and degradation, and diminished biological signalling via EGF receptors. We propose that heterodimerization with defective EGF receptors functions as a dominant negative mutation suppressing the activation and response of normal receptors by formation of unproductive heterodimers. PMID- 1705007 TI - RNA polymerase II elongation complexes paused after the synthesis of 15- or 35 base transcripts have different structures. AB - We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation. PMID- 1705009 TI - The tumor suppressor p53 is bound to RNA by a stable covalent linkage. AB - We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40) transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism. PMID- 1705008 TI - Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins. AB - Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis. PMID- 1705010 TI - Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins. AB - The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr) dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins. PMID- 1705011 TI - A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA. AB - We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme. PMID- 1705012 TI - The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase. AB - The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world. PMID- 1705014 TI - Ion channels. Making a submicroscopic hole in one. PMID- 1705013 TI - Tst-1, a member of the POU domain gene family, binds the promoter of the gene encoding the cell surface adhesion molecule P0. AB - Tst-1, a member of the POU domain gene family, is expressed in specific neurons and in myelinating glia in the mammalian nervous system. Bacterially expressed Tst-1 binds specifically to the promoter of the gene encoding myelin protein P0, a Schwann cell surface adhesion molecule. In cotransfection assays, Tst-1 can specifically repress the P0 promoter. The N-terminal part of Tst-1 protein is highly glycine- and alanine-rich, a structural feature shared by the helix-loop helix protein TFEB. PMID- 1705015 TI - Regulation of leukocyte migration by activation of the leukocyte adhesion molecule-1 (LAM-1) selectin. AB - A central feature of host defence is the ability of leukocytes to enter tissues in response to immune or inflammatory stimuli. The leukocyte adhesion molecule-1 (LAM-1) regulates the migration of human leukocytes by mediating the binding both of lymphocytes to high endothelial venules of peripheral lymph nodes and of neutrophils to endothelium at inflammatory sites. As lymphocytes and neutrophils express the same LAM-1 protein, it is not clear how lineage-specific differences in leukocyte migration are controlled. We now report that the affinity of LAM-1 for a carbohydrate-based ligand, PPME, is dramatically increased following lymphocyte and neutrophil activation by lineage-specific stimuli. In addition, activation of lymphocytes by physiological stimuli enhanced LAM-1-dependent binding to high endothelial venules. Thus, transient changes in LAM-1 affinity after leukocyte stimulation probably directly influence leukocyte migration. PMID- 1705016 TI - Identifying antigenic T-cell sites. AB - Computer algorithms that have been used successfully on protein sequences for the prediction of antigenic T-cell sites have been collected into a single computer software package called TSites. PMID- 1705017 TI - University/community poster sessions. AB - Graduate nursing students created posters to illustrate their practicum projects at the end of a field course. Posters were presented at a poster session on a university campus in Philadelphia, open to preceptors, other nurse colleagues, and the university community. Students recognized the advantages of the poster session, including learning how to conduct a professional presentation in public and how to construct an attractive, literature-based poster to hold the attention of colleagues attending the session. PMID- 1705018 TI - Facilitation of memory by peripheral administration of substance P and naloxone using avoidance and habituation learning tasks. AB - This paper summarizes results of a series of experiments dealing with the effects of the neuropeptide substance P (SP) on avoidance learning and habituation. Several doses of SP (0.5, 5, 50, 100, 250, 500 micrograms/kg) were administered posttrial intraperitoneally (IP). Three inhibitory one-trial avoidance tasks were used; uphill, step-down and step-through (alcove). Habituation was measured in an open field by recording the number of rearings. The posttrial injection of SP facilitated avoidance responses as well as reduced rearing in a dose- and time dependent way. Pretraining and pretest injections (IP) of naloxone facilitated avoidance behavior and potentiated the action of SP, also in a dose-dependent manner. These results demonstrate that: a) peripheral posttraining administration of SP enhances memory; b) SP facilitates not only aversive or positively motivated learning tasks, but also habituation, which is a form of learning that involves neither positive nor negative reinforces; c) SP does not exert its effect by a long-lasting proactive action on performance during the testing trial; d) naloxone potentiates the SP posttraining effect. These data, therefore, suggest that memory-enhancing effects of SP are, at least in part, mediated via interactions between this peptide and endogenous opioid systems. PMID- 1705019 TI - [The importance of diesel engine exhaust in public health environmental protection/ecological health protection]. AB - Ecological health protection considers itself to be mainly devoted to the principle of prevention, contrary to the still predominant principle of defence against an existing threat. The importance of diesel engine exhaust gases is discussed against this background and in view of the increasing relevance of emissions by traffic vehicles in respect of environmental hygiene, especially in highly congested areas, The relevant exhaust gas components are demonstrated and the present-day legal framework is critically appraised. PMID- 1705020 TI - [Primary and secondary inoperable teratoid hepatoblastoma--multidisciplinary management]. AB - Hepatoblastomas are the most frequent malignant liver tumors of childhood. Prognosis mostly depends on their resectability. Unresectable tumors are almost invariably fatal, unless their size can be decreased by chemotherapy and/or irradiation to permit surgical removal. In the present case report we discuss the clinical course of a 15 months old boy, who was admitted with primary inoperable teratoid hepatoblastoma. After alternating intravenous and intraarterial chemotherapy as well as radiation therapy, the tumor could be successfully resected. Specific problems of histological diagnosis and of the therapeutic management are discussed, and an overview of the recent literature in behalf of the histological basis, therapeutic possibilities and prognostic factors of hepatoblastomas is given. PMID- 1705021 TI - [Gentamicin level in the prostate and its pharmacokinetics in patients with benign prostatic hypertrophy]. AB - The study involved 15 male patients with periurethral prostatic adenoma without complete anuresis. The patients were given 80 mg of gentamicin intramuscularly one day before surgery and 80 mg in a one-hour infusion immediately before an operation. Gentamicin blood concentrations were measured. Pharmacokinetic parameters were calculated and dosage schemes for each patient basing on the antibiotic blood levels. Gentamicin levels in removed adenomas were also determined. Adenomas weighed between 18.0 and 45.8 grams while gentamicin concentration ranged from 1.31 to 3.8 micrograms/mL. It was found that gentamicin concentration in adenomas depend upon their weight. Moreover, pharmacokinetic parameters of this antibiotic exert negligible effect on its levels in adenoma. PMID- 1705022 TI - Immunohistochemical staining of infiltrates in oral lichen planus. AB - Immunohistochemical examination of lymphocyte and accessory cell infiltrates was performed in 24 cases of oral lichen planus using a double staining technique. The major population of the superficial stromal lymphocytes was T cells which were mostly composed of dominant CD 4+Leu8- (helper T cells) and lesser numbers of CD 8+11b- cells (cytotoxic T cells). Contrarily, many more CD 8+11b- cells than CD 4+Leu8- cells had infiltrated the epithelium. Some infiltrated T cells expressed interleukin-2 receptor and about half of cytotoxic T cells expressed class II antigen. HLA-DR-positive monocytes were also observed in both the superficial stroma and the epithelium. A number of HLA-DR-bearing CD 1+ cells (Langerhans/dendritic cells) and CD 11c+ cells (macrophages) were observed in the lower layers of the epithelium which were sometimes degenerative. Having significant correlation with the infiltration intensities of subepithelial macrophages and epithelial CD 8+ cells (cytotoxic/suppressor T cells), epithelial Langerhans cells variably infiltrated. Superficial stromal CD 8+ cells correlated with epidermal CD 8+ and CD 4+ cell (helper/inducer T cells) infiltrates. These findings are consistent with the notion that Langerhans cells and macrophages play an important role in antigen presentation, and suggest that cellular immunity, mediated by cytotoxic T cells with helper T cells, may be related to the pathogenesis of oral lichen planus. PMID- 1705023 TI - The application of immunocytochemical techniques to routinely-fixed and stained cytologic specimens. An aid in the differential diagnosis of undifferentiated malignant neoplasms. AB - Routinely-fixed and Papanicolaou stained smears with the cytologic diagnosis of undifferentiated malignant neoplasm that had been prepared with cells obtained by fine needle aspiration biopsy (n = 7), pulmonary lavage (n = 5), or thoracentesis (n = 3) from 15 unselected patients were stained by an immunocytochemical technique to evaluate the presence of keratin proteins and the leukocyte common antigen (LCA). Commercially available, well-characterized monoclonal antibodies with specificities for keratin proteins and the leukocyte common antigen, and a streptavidin-biotin-horseradish peroxidase labelling method were used. Evaluation of the stained smears revealed the presence of one of the two antigens in material obtained from each patient, thus indicating the probable cell-lineage of the neoplastic cells. The specificity of the monoclonal antibody reagents used was further evaluated in routinely-fixed and stained cytologic material from 24 histologically confirmed carcinomas and 12 lymphomas. In conclusion, immunocytochemical techniques may be successfully applied to routinely processed archival cytologic smears to determine the antigenic profile of morphologically undifferentiated cells and therefore aid in the differential diagnosis of undifferentiated malignant neoplasms. PMID- 1705024 TI - A method for detection of human papillomavirus DNA in stored slides of stained cervical smears. AB - A simple method was developed for extraction of DNA from stored slides of Papanicolaou stained cervical smears. HPV type 16 DNA was detected by DNA hybridization in 7/10 samples from known HPV-16 positive patients, whereas the 10 samples from negative patients were also negative with our assay. Using our method for obtaining data from stained cervical smears, it will be possible to design and perform historical cohort studies on stored material. PMID- 1705025 TI - Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae. AB - We have previously described a virulence gene in Vibrio cholerae (irgA) that is more than 850-fold regulated in response to iron. Negative regulation of irgA by iron occurred at the transcriptional level, and there was a dyad symmetric nucleotide sequence in the vicinity of the irgA promoter homologous to the Fur binding site in Escherichia coli. When irgA was cloned into E. coli, we showed that transcription of irgA required 900 base pairs of DNA upstream of the irgA promoter that contained an open reading frame in inverse orientation to irgA. In the present study, we show that this upstream region of DNA encodes a gene in inverse orientation to irgA (named irgB) that is also negatively regulated by iron. Insertional inactivation of irgB on the V. cholerae chromosome leads to loss of expression of a chromosomal irgA'-'phoA fusion (in which the primes indicate truncated genes), which is restored to normal by provision of irgB on a plasmid in trans. DNA sequencing of irgB shows that the protein product (IrgB) is homologous to the LysR family of positive transcriptional activators, and secondary structure analysis of IrgB predicts a helix-turn-helix DNA binding motif. The promoters of irgB and irgA are divergent but overlap each other and the previously defined Fur-binding site. We propose a model for iron regulation of irgA expression in V. cholerae. In the presence of sufficient iron, transcription of both irgA and irgB is negatively regulated by a Fur-like protein. In low iron conditions, negative regulation of transcription is removed, and production of IrgB leads to positive transcriptional activation of irgA. It seems likely that the high induction ratio of irgA expression under low- and high iron conditions (850-fold) relates to the fact that its cognate positive transcriptional activator (irgB) is itself negatively regulated by iron. PMID- 1705026 TI - Carbohydrate ligands for endothelial-leukocyte adhesion molecule 1. AB - The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury. Several receptors have been implicated in this interaction, including a family of putative carbohydrate binding proteins. We report here the identification of an endogenous carbohydrate ligand for one of these receptors, endothelial-leukocyte adhesion molecule 1 (ELAM-1). Radiolabeled COS cells transfected with a plasmid containing the cDNA for ELAM-1 were used as probes to screen glycolipids extracted from human leukocytes. COS cells transfected with this plasmid adhered to a subset of sialylated glycolipids resolved on TLC plates or adsorbed on polyvinyl chloride microtiter wells. Adhesion to these glycolipids required calcium but was not inhibited by heparin, chondroitin sulfate, keratan sulfate, or yeast phosphomannan. Monosaccharide composition, linkage analysis, and fast atom bombardment mass spectrometry of the glycolipids indicate that the ligands for ELAM-1 are terminally sialylated lactosylceramides with a variable number of N acetyllactosamine repeats and at least one fucosylated N-acetylglucosamine residue. PMID- 1705027 TI - Reconstitution in vitro of RNase H activity by using purified N-terminal and C terminal domains of human immunodeficiency virus type 1 reverse transcriptase. AB - Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus. PMID- 1705028 TI - Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. AB - The plasmid-encoded YopH protein is a protein-tyrosine phosphatase (PTPase; EC 3.1.3.48) that is essential for Yersinia virulence. We have investigated the molecular basis for the role of PTPase activity in Yersinia pathogenesis. Allelic recombination was employed to introduce a defined mutation into the yopH plasmid gene. Conversion of the essential Cys-403 to Ala in the catalytic domain of the protein abolished YopH PTPase activity and significantly reduced the virulence of Yersinia pseudotuberculosis in a murine infection model. 32P-labeled phosphotyrosine-containing proteins were immunoprecipitated from extracts of Y. pseudotuberculosis-infected cell monolayers and analyzed by SDS/PAGE to assess the impact of YopH on host protein phosphorylation. Major proteins of 200, 120, and 60 kDa were dephosphorylated in macrophages associated with wild-type Y. pseudotuberculosis. Selective removal of phosphate from the 120- and 60-kDa proteins was shown to be specific to the YopH PTPase activity. Phagocytosis of the bacteria was not required for this dephosphorylation activity, suggesting that YopH is functionally expressed by extracellular bacteria. These observations indicate that the essential function of YopH in Yersinia pathogenesis is host protein dephosphorylation. PMID- 1705029 TI - The use of sarkosyl in generating soluble protein after bacterial expression. AB - Actin, like many other proteins, is highly insoluble after expression in Escherichia coli. In order to understand the origin of insoluble aggregates, we asked whether morphological inclusions were always correlated with insolubility. The strain expressing actin was compared to one that expresses part of the myosin tail; the latter strain yields soluble protein after various cell lysis or disruption procedures. Morphological inclusions were observed in both strains, indicating there is no obligate relationship between solubility and inclusions. Studies presented here suggest that extreme insolubility results from coaggregation of the actin with bacterial outer membrane components upon bacterial lysis. The properties of the outer membrane have been exploited in the development of nondenaturing procedures that yield soluble actin. One procedure involves the disruption of coaggregates with sarkosyl detergent (N laurylsarcosine); another prevents the formation of coaggregates by lysing in the presence of sarkosyl. These methods may be useful for other proteins that become insoluble after bacterial expression. PMID- 1705030 TI - Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. AB - Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function. PMID- 1705031 TI - Sexually dimorphic distribution of substance P in specific anterior pituitary cell populations. AB - Substance P (SP) immunoreactivity is detectable in the rat pituitary by RIA; however, immunolocalization has been difficult. We used a sensitive immunogold silver-enhancement staining technique to cytochemically locate SP in the gland. SP-immunoreactive (SP-ir) cells were seen in anterior pituitary (AP), and occasional SP-ir fibers and terminals were seen in both AP and posterior pituitary. Colocalization studies showed the vast majority of SP-ir cells in the male AP to be also immunoreactive for growth hormone (GH). These GH/SP-ir cells represent approximately 23% of the somatotroph population in the male. SP-ir cells did not colocalize with lactotrophs, gonadotrophs, or corticotrophs; however, rare thyroid-stimulating hormone/SP-ir cells were found in the male AP. Comparisons of pituitaries from males and females revealed that females have 70% fewer SP-ir cells and that only approximately 6% of the somatotrophs in the female express SP. This sexual dimorphism is diminished in 6-day ovariectomized rats because this treatment increases the GH/SP-ir cell population 3-fold. This result suggests that the previously reported estrogen-induced decrease in SP gene and peptide expression in the pituitary occurs, at least in part, in a subpopulation of somatotrophs. To test this hypothesis, distribution of SP-ir cells was examined in pituitaries from estrogen- and oil-treated ovariectomized rats. Estrogen reduced the percentage of somatotrophs with SP immunoreactivity by 70% compared with ovariectomized oil-treated controls, indicating that estrogen most likely regulates SP levels in the pituitary by acting on a subpopulation of somatotrophs to suppress SP expression. Estrogen does not appear to alter SP immunoreactivity that is detected in the additional population of SP cells that colocalize with thyroid-stimulating hormone. These SP-expressing thyrotrophs were seen 6-fold more frequently in the female than in the male pituitary, regardless of steroid status. These studies reveal that males have more total SP-ir cells in the AP than do females and that there is a sexually dimorphic pattern of SP distribution in the gland. Males have a higher percentage of SP-ir GH cells, whereas females have more SP-ir thyrotrophs than do males. Identification of independently regulated SP-ir somatotroph and thyrotroph populations provides a basis for investigating the roles of SP in autocrine or paracrine regulation of pituitary hormone secretion. PMID- 1705032 TI - Anti-reovirus receptor antibody accelerates expression of the optic nerve oligodendrocyte developmental program. AB - Previous studies showed that the cell-surface receptor for reovirus serotype 3 (Reo3R) appears at an early stage of oligodendrocyte differentiation and that anti-Reo3R antibodies and Reo3R-binding peptides induce galactocerebroside expression by developing oligodendrocytes. In the present studies, anti-Reo3R antibodies are shown to stimulate additional features of the program of oligodendrocyte development, including the loss of the A2B5 marker and expression of myelin basic protein. In anti-Reo3R antibody-treated cultures, galactocerebroside was expressed by cells having the morphology of immature oligodendrocyte precursors. Reo3R binding did not appear directly to inhibit or stimulate proliferation of glial progenitor cells or to affect their lineage commitment. Cell-surface structures utilized as a receptor by reovirus type 3 appear to play a role in the regulation of the initiation or rate of execution of the oligodendrocyte developmental program. PMID- 1705033 TI - Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase C. AB - It has been found that the principal biochemical pathway activated in B cells stimulated by antigen- or anti-immunoglobulin-mediated crosslinking of surface immunoglobulin is that resulting in hydrolysis of phosphatidylinositol bisphosphate with generation of diacylglycerol and inositol trisphosphate. Recent evidence suggests that surface immunoglobulin-mediated B-cell activation can proceed without detectable increases in the concentration of either diacylglycerol or intracellular Ca2+ concentration, implicating involvement of other non-protein-kinase-C/Ca2(+)-dependent signal-transduction pathways. Therefore, we sought evidence for activation of a signaling pathway that is associated with growth regulation in other cell types--i.e., the protein-tyrosine kinases. We now show that crosslinking of membrane immunoglobulin by mitogenic antibodies leads to rapid tyrosine phosphorylation of several cellular substrates, consistent with the induction of a tyrosine kinase activity. This increase in tyrosine phosphorylation is weakly (if at all) stimulated by other B cell mitogens, including phorbol esters and ionophores, and does not require the presence of detectable protein kinase C. Furthermore, inhibition of anti immunoglobulin-stimulated phosphatidylinositol bisphosphate hydrolysis does not inhibit activation of this tyrosine kinase-dependent pathway. These findings suggest that occupancy of the membrane immunoglobulin receptor may induce multiple pathways of activation. PMID- 1705034 TI - Evidence for separate pathways within the tecto-geniculate projection in the tree shrew. AB - Two layers (3 and 6) in the dorsal lateral geniculate nucleus (GLd) of the tree shrew (Tupaia belangeri) receive projections from the superficial layers of the superior colliculus. The goal of this study was to determine whether the same or different cells in the superior colliculus give rise to the projections to layers 3 and 6 by following individual axons labeled with biocytin from the superior colliculus to the GLd. The results show that the terminal fields differ in the two layers--those in layer 3 are restricted to a line of projection, whereas those in layer 6 are elongated along the dimension orthogonal to a line of projection. Another important difference between axons that project to GLd layers 3 and 6 is that those that project to layer 6 give off collaterals to the posterior pretectal nucleus, whereas at least some axons that project to layer 3 send a collateral to the ventral lateral geniculate nucleus (GLv). These results suggest that the superior colliculus exerts separate influences on these two GLd layers, both of which project to separate targets above layer IV in the striate cortex. The biocytin method has proved useful by showing the dendritic trees of the superior colliculus cells of origin, the pathways taken by the axons (including the presence of collaterals), and the terminal fields both within and outside the GLd. PMID- 1705035 TI - Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. AB - Myogenin and MyoD are proteins that bind to the regulatory regions of a battery of skeletal muscle genes and can activate their transcription during muscle differentiation. We have recently found that both proteins interact with the enhancer of the nicotinic acetylcholine receptor (nAChR) alpha subunit, a gene that is regulated by innervation. This observation prompted us to study if myogenin and MyoD transcript levels are also regulated by skeletal muscle innervation. Using Northern blot analysis, we found that MyoD and myogenin mRNA levels begin to decline at embryonic day 17 and attain adult levels in muscle of newborn and 3-week-old mice, respectively. In contrast, nAChR mRNAs are highest in newborn and 1-week-old mouse muscle and decline thereafter to reach adult levels in 3-week-old mice. To determine if the down-regulation of myogenin and MyoD mRNA levels during development is due to innervation, we quantitated message levels in adult calf muscles after denervation. We found that in denervated muscle myogenin and MyoD mRNAs reach levels that are approximately 40- and 15 fold higher than those found in innervated muscle. Myogenin mRNAs begin to accumulate rapidly between 8 and 16 hr after denervation, and MyoD transcripts levels begin to increase sharply between 16 hr and 1 day after denervation. The increases in myogenin and MyoD mRNA levels precede the rapid accumulation of nAChR alpha-subunit transcripts; receptor mRNAs begin to accumulate significantly after 1 day of denervation. The effects of denervation are specific because skeletal alpha-actin mRNA levels are not affected by denervation. In addition, we found that the repression of myogenin and MyoD expression by innervation is due, at least in part, to "electrical activity." Direct stimulation of soleus muscle with extracellular electrodes repressed the increase of myogenin and MyoD transcripts after denervation by 4- to 3-fold, respectively. In view of these results, it is interesting to speculate that myogenin and/or MyoD may regulate a repertoire of skeletal muscle genes that are down-regulated by electrical activity. PMID- 1705036 TI - Early phenotype expression of cortical neurons: evidence that a subclass of migrating neurons have callosal axons. AB - The use of [3H]thymidine labeling in combination with various axonal transport tracers has revealed that a subset of migrating neurons in the fetal monkey cerebrum issue axons to the opposite cerebral hemisphere while still migrating to their final positions in the cortical plate. Other cortical neurons with the same "birthdate" (i.e., that underwent their last round of DNA synthesis on the same day) are not retrogradely labeled by tracer injections of the opposite hemisphere. These findings suggest that the cardinal distinction between projection and local circuit neurons may be specified in postmitotic neurons before they acquire their final positions in the cortex. PMID- 1705037 TI - Multimerization of AAGTGA and GAAAGT generates sequences that mediate virus inducibility by mimicking an interferon promoter element. AB - Multimeric AAGTGA and GAAAGT, when inserted before a minimal promoter, mediate virus-inducible transcription. We have determined that the active sequence within these multimers is TGAAAGTGAAAGT, which is structurally similar to GAGAAGTGAAAGT, a positive response element delineated in the beta-interferon gene promoter. Both sequences behave like protoenhancers and are similar as regards induction by virus or interferon regulatory factor 1 when supported by a simian virus 40 enhancer. PMID- 1705038 TI - An antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed by tetrahydroimidazo-[4,5,1-jk] [1,4]benzodiazepin-2 (1H)-one and thione derivatives. AB - Screening of pharmacologically acceptable prototype compounds has recently led to the discovery of a series of ultraselective inhibitors of human immunodeficiency virus (HIV)-1 replication, the tetrahydroimidazo[4,5,1-jk] [1,4]-benzodiazepin 2(1H)-one and -thione (TIBO) derivatives. The TIBO compounds completely suppress the formation of proviral DNA in acutely infected cells, as revealed by polymerase chain reaction (PCR) analysis. TIBO derivatives are inhibitory to the reverse transcriptase (RT) of HIV-1 but not that of HIV-2 or other retroviruses. The inhibition is most effective with poly(C)-oligo(dG) as the template/primer, and it is selectively directed against the RNA-dependent DNA polymerase activity and not the accompanying DNA-dependent DNA polymerase and ribonuclease H activity of HIV-1 RT. Kinetic studies point to an uncompetitive inhibition with regard to the template/primer. TIBO compounds are active against HIV-1 replication through a unique interaction with HIV-1 RT. The experimental data indicate the existence of a target on HIV-1 RT that is responsible for the inhibition of replication and a mode of action unrelated to that of previously studied RT inhibitors. PMID- 1705039 TI - Intracellular metabolism and mechanism of anti-retrovirus action of 9-(2 phosphonylmethoxyethyl)adenine, a potent anti-human immunodeficiency virus compound. AB - 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of retrovirus (i.e., human immunodeficiency virus) replication in vitro and in vivo. Uptake of PMEA by human MT-4 cells and subsequent conversion to the mono- and diphosphorylated metabolites (PMEAp and PMEApp) are dose-dependent and occur proportionally with the initial extracellular PMEA concentrations. Adenylate kinase is unable to phosphorylate PMEA. However, 5-phosphoribosyl-1-pyrophosphate synthetase directly converts PMEA to PMEApp with a Km of 1.47 mM and a Vmax that is 150-fold lower than the Vmax for AMP. ATPase, 5'-phosphodiesterase, and nucleoside diphosphate kinase are able to dephosphorylate PMEApp to PMEAp, albeit to a much lower extent than the dephosphorylation of ATP. PMEApp has a relatively long intracellular half-life (16-18 hr) and has a much higher affinity for the human immunodeficiency virus-specified reverse transcriptase than for the cellular DNA polymerase alpha (Ki/Km: 0.01 and 0.60, respectively). PMEApp is at least as potent an inhibitor of human immunodeficiency virus reverse transcriptase as 2',3'-dideoxyadenosine 5'-triphosphate. Being an alternative substrate to dATP, PMEApp acts as a potent DNA chain terminator, and this may explain its anti-retrovirus activity. PMID- 1705041 TI - cDNA analysis predicts a cornea-specific collagen. AB - In the development of chicken corneal stroma, two or more collagens often interact, either as constituents of a single heterotypic fibril or as components of the fibril surface. The latter, fibril-associated collagens, may facilitate interactions between fibrils and the surrounding extracellular matrix or between fibrils themselves. In an effort to isolate putative nonfibrillar collagens that may have such a function, we screened a 13-day embryonic cornea cDNA library under reduced stringency conditions, using a cDNA probe for a collagenous domain of type XII collagen. We isolated a 4.2-kilobase (kb) cDNA that predicts a "collagenous" protein that has three unusual, if not unique, features. (i) The putative polypeptide encoded by this cDNA has a structural arrangement in which numerous stretches of Gly-Xaa-Yaa triplets, typical of collagens, are interrupted by non-Gly-Xaa-Yaa regions. One of the potential triple-helical domains is 246 amino acids long, but most are much smaller, consisting of 15-36 amino acids. Many are very rich in the helix-stabilizing imino acid proline. (ii) Northern blot analyses demonstrated strong cDNA hybridization to a 6.8-kb mRNA whose expression is restricted to the cornea. No hybridization was observed to mRNAs from the nine other tissues used in these analyses, even with extended exposure of the film. (iii) The cDNA contains a short (less than or equal to 425-base pair) sequence in the 3' untranslated region of the 6.8-kb mRNA that hybridizes to a 7.8-kb mRNA that has a wide tissue distribution. PMID- 1705040 TI - Stimulation of cAMP and phosphomonoester production by melanotropin in melanoma cells: 31P NMR studies. AB - A major part of the present understanding of the molecular basis of signal transduction has been gained from in vitro studies using classical biochemical methods. In this study, we used 31P NMR spectroscopy to investigate the response of live M2R mouse melanoma cells to stimulation by melanocyte-stimulating hormone (MSH; melanotropin). In the presence of 3-isobutyl-1-methylxanthine and a synergistic dose of forskolin (1.67 microM), MSH induced a transient (approximately 60-min) rise in the cellular concentration of 3',5'-cyclic adenosine monophosphate (cAMP), which coincided in time with an equivalent decrease (approximately 40%) in ATP. However, no detectable change in phosphocreatine concentration was observed. Concomitantly, MSH induced a striking and unexpected increase in the concentration of three phosphomonoester (PME) metabolites (approximately 2-fold increase in total PME signal area); one signal has been assigned to phosphoethanolamine. The levels of the PMEs remained high for 2-4 hr and declined slowly (approximately 10 hr) to basal level, following perfusion with fresh culture medium. The increase in PME was also observed after stimulation with MSH alone. In contrast, stimulation with a high dose of forskolin (50 microM) and isobutylmethylxanthine (0.2 mM), although effective in stimulating the production of cAMP, did not induce the PME response. Evaluation of the cells' energetics indicated that the enhanced production of phosphoethanolamine is probably not due to ethanolamine phosphorylation. Therefore, it is likely to result from hydrolysis of phosphatidylethanolamine by a specific phospholipase C. The response of the PMEs appears to be regulated by a cAMP-independent process, suggesting the existence of an alternative transduction pathway controlled by MSH. PMID- 1705042 TI - Cell-cycle dependence of heat-induced interphase death in mouse L5178Y cells. AB - Cell lysis and eosin staining were observed in L5178Y cells within the first 3 h of post-hyperthermia incubation at 37 degrees C, after which both leveled to a plateau. Lysis and eosin staining were proportional to the severity of heat in asynchronous cells, whereas it was maximum in the most heat-sensitive M phase, intermediate in S, and least in heat-resistant G1 for the same heat treatment. Further, leakage of labeled [3H]thymidine and a decrease in radioactivity retained within heated cells coincided with an increase in eosin staining, indicating that the dye uptake was due to membrane damage. It was presumed that the eosin-stained fraction represented dead cells. The percentage eosin-stained cells reached a plateau, and this level was used to determine survival; when the results were compared with those obtained by the colony formation method, they were identical. By comparing the two survival assay methods we concluded that cell death after hyperthermia in L5178Y cells is mainly by interphase death in all phases of the cell cycle. The reasons for this conclusion are that a reduction in survival could be detected within one generation of L5178Y cells by the eosin staining method, and the survival values obtained by this method were identical to those obtained by the colony formation method. PMID- 1705043 TI - [Analysis of RNA by using the polymerase chain reaction]. PMID- 1705044 TI - [Tetralogy of Fallot in children--management and results of treatment]. AB - The authors present the results of surgical treatment obtained in 288 children with tetralogy of Fallot. In 127 children palliative procedures (pulmonary systemic anastomoses) were employed, whereas 161 patients were subjected to total surgical corrections of the defect, performed in cardiopulmonary bypass. In 127 (79%) belonging to the latter group the correction was a single stage procedure, and in 34 (21%), it followed pulmonary-systemic anastomoses. Perioperative mortality rate in children subjected to single stage procedures was 12%, being lightly lower than the rate observed in patients subjected to two stage procedures. The analysis of operative results corrected for age revealed a higher mortality rate in children operated when less then two years of age (17%) in comparison to older patients (11%). PMID- 1705045 TI - [Non-surgical treatment of prostatic hyperplasia]. PMID- 1705046 TI - Immunohistochemical assessment of ER-P31, a mouse anti-oestrogen receptor protein monoclonal antibody in human breast cancers: comparison with ER-ICA (Abbott) and radioligand assays. AB - A new mouse anti-oestrogen receptor (ER) monoclonal antibody, ER-P31, has been developed. Comparisons of immunohistochemical detection of ER in human breast cancers using ER-P31 with anti-ER from ER-immunocytochemical assay (ER-ICA; Abbott Diagnostic Division) and radioligand dextran-coated charcoal (DCC) assays were carried out. A total of 63 breast cancers, both ER-negative and -positive, were tested. A highly significant degree of correlation between immunostaining for ER-P31 and both ER-ICA and DCC assays was obtained. It is hoped that once ER P31 is widely available commercially, determination of ER status in breast cancers should be routinely and economically available to all women with breast cancer. Moreover, with the introduction and implementation of a screening programme for detecting breast cancers, immunocytochemical determination of ER status in unequivocal and equivocal positive fine-needle aspirates of breast lesions can be readily performed. PMID- 1705047 TI - Different behaviors in the production and release of SCC antigen in squamous-cell carcinoma. AB - Squamous-cell carcinoma antigen (SCC antigen), formerly referred to as TA-4, is closely related to the grade of differentiation. Immunohistochemical studies have demonstrated that SCC antigen is also increased in the keratinizing or large-cell nonkeratinizing type of squamous-cell carcinoma but not in the small cell type. On the other hand, the appearance rate of SCC antigen in the blood circulation is almost the same in these three types of squamous-cell carcinoma. The present study was conducted to clarify the discrepancy in the production and release of this tumor marker using a squamous-cell carcinoma cell line, SKG-IIIa. SKG-IIIa cells were treated with 10 microM 5-azacytidine, a potent hypomethylating agent, to obtain several sublines with different behavior in the production of SCC antigen, and cloned by a limiting dilution technique. One subline (B-5) released significantly greater amounts of SCC antigen into the incubation medium as compared with other sublines (A-5). In in vivo studies, groups of nude mice received subcutaneous injections of the A-5 or B-5 subline, and the serum SCC antigen levels were determined after the animals exhibited palpable tumors. The serum levels of SCC antigen were significantly higher in the animals inoculated with the B-5 cells than in those inoculated with the A-5 cells. On the other hand, flow-cytometric analysis and immunohistochemical studies using a polyclonal antibody revealed that the intracellular contents of SCC antigen were greater in the A-5 cells than in the B-5 cells. Radioautography was performed using a 125I labeled monoclonal antibody specific against the acidic fraction of this marker, a dominant form released outside the cells, which revealed that the production of the acidic fraction was somewhat greater in the B-5 cells than in the A-5 cells. These results suggest that the production and release of SCC antigen are different phenomena in squamous-cell carcinoma and that the release of SCC antigen is likely influenced by the production of the acidic fraction. PMID- 1705048 TI - Fetal hemoglobin screening in whole blood and in plasma of cancer patients. AB - Fetal hemoglobin (HbF) screening was performed on 296 cancer patients. In almost 80% of the patients with active disease (n = 197) and 35-60% of those with inactive disease (n = 99), the concentration of HbF in the blood was above the normal range. Among patients with metastatic breast carcinoma (n = 27), 89% had a high HbF concentration. The HbF level was significantly higher (p less than 0.001) in patients with active disease than in those with inactive disease. There is evidence of an ectopic production of HbF into the plasma of patients, but it will be necessary to develop a method for the prevention of hemolysis in order to establish this claim. PMID- 1705049 TI - Epitope group specificity of six immunoassays for carcinoembryonic antigen. AB - We have mapped the epitope group specificity of the monoclonal antibodies employed in six commercial sandwich immunometric assays for carcinoembryonic antigen (CEA), using cross-inhibition experiments with monoclonal antibodies previously classified in an International Workshop on the Epitope Reactivity of Monoclonal Antibodies against CEA. In this workshop it was shown that most monoclonal antibodies could be classified into one of five epitope groups, designated Gold groups 1-5. We found that of the six assay kits tested, five used solid-phase antibody of Gold group 4. Three of these (CIS ELSA 2-CEA, Hybritech Tandem-R CEA, Roche CEA EIA Duomab 60) used labeled antibody of Gold group 1, the remaining two (Abbott CEA-RIA Monoclonal, Wallac/LKB DELFIA CEA) used antibodies of Gold group 5. The sixth assay kit (Behringwerke Enzygnost CEA) used a solid phase antibody of Gold group 1, and a polyclonal labeled antibody. PMID- 1705050 TI - Multiple sclerosis: cells secreting antibodies against myelin-associated glycoprotein are present in cerebrospinal fluid. AB - We evaluated the B-cell response in cerebrospinal fluid (CSF) and blood by enumerating cells secreting antibodies to myelin-associated glycoprotein (MAG) and, for reference, to myelin basic protein (MBP), two myelin components which may constitute targets for autoimmune attack in multiple sclerosis (MS). Among 25 untreated MS patients, 12 had cells in CSF secreting anti-MAG IgG antibodies (mean value 1 per 1429 CSF cells) and three also had cells secreting anti-MAG antibodies of the IgM isotype but at lower levels. In CSF from 2 out of 10 MS patients examined, anti-MAG and anti-MBP IgG antibody-secreting cells were present concurrently. Antibody-secreting cells were less frequent in blood and bone marrow, reflecting compartmentalization to CSF. Anti-MAG antibody-secreting cells were found in CSF from only 1 out of 27 control patients. The intrathecal production of anti-MAG and anti-MBP antibodies may be important in the pathogenesis of MS. PMID- 1705051 TI - Properties of four acute phase proteins: C-reactive protein, serum amyloid A protein, alpha 1-acid glycoprotein, and fibrinogen. AB - Four plasma proteins, referred to as positive acute phase proteins because of increases in concentration following inflammatory stimuli, are reviewed: C reactive protein (CRP), serum amyloid A protein (SAA), alpha 1-acid glycoprotein (AAG), and fibrinogen. The CRP and SAA may increase in concentration as much as 1000-fold, the AAG and fibrinogen approximately twofold to fourfold. All are synthesized mainly in the liver, but each may be produced in a number of extrahepatic sites. The role of cytokines in induction of the acute phase proteins is discussed, particularly the multiple functional capabilities of interleukin-6 (IL-6). Other cytokines that regulate acute phase gene expression and protein synthesis include IL-1, tumor necrosis factor alpha, interferon gamma, as well as other stimulatory factors and cofactors. The physicochemical characteristics of each protein are reviewed together with the molecular biology. For each protein, the known biological effects are detailed. The following functions for CRP have been described: reaction with cell surface receptors resulting in opsonization, enhanced phagocytosis, and passive protection; activation of the classical complement pathway; scavenger for chromatin fragments; inhibition of growth and/or metastases of tumor cells; modulation of polymorphonuclear function; and a few additional diverse activities. The role of plasma SAA is described as a precursor of protein AA in secondary amyloidosis; other functions are speculative. AAG may play an immunoregulatory role as well as a role in binding a number of diverse drugs. In addition to clot formation, new data are described for binding of fibrinogen and fibrin to complement receptor type 3. Finally, the concentration of each protein is discussed in a wide variety of noninfectious and infectious disease states, particularly in connective tissue diseases. The quantification of the proteins during the course of various acute and chronic inflammatory disorders is useful in diagnosis, therapy, and in some cases, prognosis. PMID- 1705052 TI - Pharmacologic modulation of fetal globin gene expression in the perinatal period. PMID- 1705053 TI - A study on the vascular supply of the supraspinatus tendon. AB - The vascular supply of the rotator cuff in 22 adult shoulders was studied by way of mixture infusion of gelatin and India ink and vascular cast, in combination with Scanning Electronic Microscopy of the vascular pattern in the supraspinatus tendon. It was found that the vessels of the supraspinatus tendon mainly derive from the anterior circumflex humeral and suprascapular arteries. Macroscopically, an avascular zone or critical zone could be seen on the surface of supraspinatus, and the mean distance of the external edge from the osteo-tendinous attachment was 7.8 mm. The area of the avascular zone increased with age. PMID- 1705054 TI - Lymphatic drainage of the middle lobe of the adult lung. PMID- 1705055 TI - Peptide coexistence in axon terminals within the superficial dorsal horn of the rat spinal cord. AB - The somata of primary sensory neurons have been shown to contain up to four (and possibly more) neuroactive peptides. Although each of these peptides has been separately located in axon terminals within the superficial dorsal horn of the spinal cord, it is not clear whether multiple peptide coexistence is also a feature of terminal varicosities. The aim of this study was to determine whether the peptides substance P (SP) and calcitonin gene-related peptide (CGRP), which are colocalized in the somata of a large number of primary sensory neurons, coexist in the central terminals of these neurons in the spinal cord. The protein A-gold technique of antigen localization was used to screen single boutons in laminae I and II of the rats spinal cord for SP- and CGRP-like immunoreactivity at the ultrastructural level. The results show that SP and CGRP are colocalized within a large number of synaptic boutons in the superficial dorsal horn. Furthermore, evidence was obtained to suggest that both SP and CGRP may be found in the same synaptic vesicle within these boutons. These findings indicate that both SP and CGRP may be coreleased from single terminals in the superficial dorsal horn. This is of considerable interest in view of the reported interaction between SP and CGRP in nociceptive behavioral responses in the rat. PMID- 1705056 TI - Tyrosine hydroxylase and galanin mRNA levels in locus coeruleus neurons are increased following reserpine administration. AB - The neuropeptide galanin coexists in 80-90% of the norepinephrine-containing neurons in the locus coeruleus. In situ hybridization histochemistry was used to examine the effects of reserpine treatment or swim stress on tyrosine hydroxylase and galanin mRNA concentrations in locus coeruleus neurons. Reserpine administration significantly increased tyrosine hydroxylase and galanin mRNA levels in the locus coeruleus. The reserpine-induced increase in tyrosine hydroxylase mRNA was significantly correlated with the reserpine-induced increase in galanin mRNA. Three consecutive days of swim stress did not significantly alter either tyrosine hydroxylase or galanin mRNA concentrations in the locus coeruleus. These data suggest that both tyrosine hydroxylase and galanin gene expression in locus coeruleus neurons may be regulated by a reserpine-sensitive mechanism. PMID- 1705057 TI - Synaptic interactions between GABAergic neurons and trigeminothalamic cells in the rat trigeminal nucleus caudalis. AB - Synaptic interactions between GABAergic neurons and thalamic projecting cells within the trigeminal nucleus caudalis were examined using a combined method of GABA immunohistochemistry and retrograde WGA-HRP labeling of the trigeminothalamic pathway. Results showed that GABA-positive neurons and projecting cells were separate but closely intermingled within the spinal trigeminal nucleus. GABAergic axon terminals formed symmetric synaptic connections with the cell bodies and dendrites of trigeminothalamic neurons. In turn, some WGA-HRP filled axon terminals, presumed to originate from axon collaterals of the projecting neurons, formed synaptic connections with GABA containing neurons. The results suggest that in the spinal trigeminal nucleus there is a reciprocal modulation between GABA neurons and trigeminothalamic cells. PMID- 1705058 TI - Differential effects of intracerebroventricular colchicine administration on the expression of mRNAs for neuropeptides and neurotransmitter enzymes, with special emphasis on galanin: an in situ hybridization study. AB - The axonal transport blocker colchicine has been extensively used in immunohistochemical studies to induce accumulation of neuroactive compounds, especially neuropeptides, in neuronal somata and thus improve their visualization. To assess whether colchicine might, in addition, influence the synthesis of such compounds, we have now used in situ hybridization to examine the levels of mRNAs encoding for several neuropeptides (galanin [GAL], cholecystokinin [CCK], somatostatin [SOM], neuropeptide Y [NPY]) and neurotransmitter-synthesizing enzymes (choline acetyltransferase [ChAT], tyrosine hydroxylase [TH], amino acid decarboxylase [AADC], and glutamic acid decarboxylase [GAD]) after intraventricular administration of the drug. The results show that colchicine differentially modifies the levels of several mRNA species in different brain areas. Thus GAL mRNA levels increase in virtually all regions examined, including the basal forebrain, hypothalamus, dorsal raphe nucleus, locus coeruleus, and nucleus tractus solitarii. In addition, after colchicine treatment, GAL mRNA appears to be induced in the ipsilateral hemisphere in regions such as the cortex, hippocampus, striatum, lateral septum, and some nuclei of the thalamus as well as within white matter, where it cannot be detected in control animals. Although GAL mRNA in the vast majority of cases is neuronal, some findings indicate a possible glial localization. In parallel, colchicine depletes ChAT mRNA and increases GAD mRNA in the basal forebrain and striatum and decreases AADC mRNA in the dorsal raphe nucleus and locus coeruleus. In the latter nucleus, NPY and TH mRNA levels are increased by colchicine. In contrast, TH mRNA and also CCK mRNA levels decrease in the substantia nigra. In the cortex, hippocampus, and thalamus ipsilateral to colchicine injection CCK mRNA levels are markedly decreased, whereas SOM mRNA is decreased and NPY mRNA increased in the hippocampus but unchanged in the cortex. The results are discussed with reference to the possible artifacts that the use of colchicine might induce in immunohistochemical mapping studies and in relation to possible neurotoxic actions of colchicine, in some cases perhaps related to impaired retrograde transport of growth factor(s). PMID- 1705059 TI - Redistribution of fibronectin and keratin localization patterns in early wounded confluent PtK2 cells. AB - Single and double-label immunofluorescence were used to study the fibronectin (FN) and keratins (Ks) localization patterns in early wounded confluent PtK2 cells. A time-course study (0 hr, 2 hr, 6 hr and 24 hr) gives the following results: before wounding, the FN localizations of confluent cells are composed of curved and sometimes branched strands or fibrils. The Ks network is formed by radial fluorescent filaments connecting the Ks centers near the nuclei with a linear fluorescence underlying the cell membrane. Two hr after, the FN localizations are redistributed at the cell-cell contact areas. The radial Ks filaments are compacted around the nuclei, some of them delineate the cytoplasmic periphery of the wounded cells. Six hr later, the method shows redistributed FN localizations at the cell-cell contact areas. An alveolar pattern is formed enclosing each of the adjacent cells. The codetected Ks filaments are retracted around the nuclei. The underlying cell-cell contact areas are also well demonstrated. It may be noted that these areas are FN-labelled. Twenty-four hr after wounding, the FN alveolar pattern persists. The redistributed Ks filaments have some similarity to those seen before wounding. PMID- 1705060 TI - Morphological behavior of cultured bovine adrenal medulla capillary endothelial cells. AB - Bovine adrenal medulla capillary endothelial cells were isolated and cloned, and their morphological behaviors in vitro were examined. In the culture of primary or early passage, one type of colony formed intracellular lumina both on the dish and in the three dimensional collagen gel. Another type proliferated well and showed morphology ranging from slender-shape to cobblestone shape, and were easily cloned. Cloned cells which showed slender-shapes formed tubular network on plastic dish after addition of PMA, OAG or vanadate, and these cells also formed multicellular tubules in the three dimensional collagen gel. However, the formation of diaphragmed fenestrae by these slender-shape clones was rare. One clone which showed cobblestone shape formed diaphragmed fenestrae, when cultured on collagen gel for more than one month. Isolated colonies or clones showed heterogeneity of cell shape, angiogenic behaviors and fenestrae formation. PMID- 1705061 TI - Suppression of adrenocortical function in female mice by lindane (gamma-HCH). AB - Lindane (gamma-HCH) given to adult female mice orally at various doses and over varying periods adversely affected adrenocortical function. Adrenal weights decreased and both fasciculata and reticularis zones markedly regressed. Histopathological lesions were also noted and plasma and glandular glucocorticoid contents were significantly reduced. Simultaneously an increase in cholesterol and a decrease in ascorbic acid content of the adrenal glands were noticed. The adrenotoxic effect of this chlorinated insecticide possibly results from depressed corticoid production in situ and/or suppressed activity of enzyme(s) that catalyze the peripheral transformation of steroids. PMID- 1705062 TI - Value of prostate-specific antigen measurements in predicting lymph node involvement in prostatic cancer. AB - The preoperative serum levels of prostate-specific antigen (PA) were determined in 35 consecutive patients who had clinically localized prostatic cancer and underwent bilateral staging pelvic lymphadenectomy. When 10.0 ng/ml of PA was used as the cutoff value for lymph node staging the specificity of an elevated PA level in revealing lymph node involvement was 77% with a sensitivity of 85% and an accuracy of 80%. Only 1 of 18 (6%) patients with negative PA levels (below 10 ng/ml) and a preoperative Gleason score 2-7 tumor had metastatic lymph node disease. Among the 17 patients with positive PA levels (above 10 ng/ml) or a Gleason score 8-10 tumor, 12 (71%) had lymph node involvement. The preoperative PA level and the extent of local tumor invasion correlated strongly with each other. Our study suggests that the PA level could be a simple and objective parameter with which to predict metastatic lymph node disease if used in conjunction with the Gleason histological grade. PMID- 1705063 TI - [Ways to improve the results of surgical treatment of necrotizing pancreatitis]. AB - An analysis of surgical treatment of 152 patients withnecrotizing pancreatitis has been made. Lethality after resection of the pancreas made up 29%, after palliative operations-49%. A removal of the greater part of the necrotized gland and Wirsung's duct surgery against the background of intensive conservative therapy was found to prevent the development of complications, to improve ++cardio-hemodynamics and outcomes of the disease. PMID- 1705064 TI - Interdigitating reticulum cells in human renal grafts. AB - Seventeen human renal graft biopsies taken 1 h to 50 days after transplantation and 3 human renal non-graft biopsies (2 minimal change and 1 non-tumour portion of angiomyolipoma) were investigated with immunoelectron microscopy in order to identify interdigitating reticulum cells (IDC) or dendritic cells (DC) in renal tissues. The antibodies used consisted of a rabbit polyclonal antibody of antihuman S100 beta protein, mouse monoclonal antibodies of antihuman HLA-DR, anti-CD3, and anti-CD1a. IDC or DC were identified in 11 renal grafts. They were found both in the glomerular and interstitial (peritubular) capillary lumens but not in the interstitium of 1 case: both were present in the interstitial capillary lumens and interstitium of another case, and in the interstitium only of 9 cases. In the remaining 6 grafts and 3 non-grafts they were not detected. These 6 grafts and 3 non-grafts did not show any pathological change except for foot process fusion of the glomerular epithelia in 2 cases of minimal change. These findings suggest that IDC or DC are not normally present in human renal tissues. The presence of the cell in the glomerular and peritubular capillary lumens of a biopsy taken after 1 h and their presence in the interstitial capillary lumens of another graft biopsy, suggest that the IDC or DC in human renal grafts are derived from recipients, not donors, and that they migrate from the circulating blood toward the interstitium. PMID- 1705065 TI - Co-expression of cytokeratin and vimentin filaments in rete testis and epididymis. An immunohistochemical study. AB - In 11 testes of different developmental stages (from 10-week-old embryos to adult) the cytokeratin and vimentin expression patterns of rete testis and epididymis were investigated immunohistochemically in formaldehyde-fixed paraffin embedded material. In addition, immunofluorescence microscopy including double immunofluorescence was performed on frozen sections of 3 of these 11 cases. Rete testis and epididymis cells displayed a heterogeneous co-expression of cytokeratin and vimentin. In double immunohistochemistry, differences in distribution of keratin and vimentin intermediate filaments with predominance of cytokeratins in the apical cytoplasmic regions and of vimentin filaments in the basal portions of the cells were found. Cytokeratin expression preceded the appearance of vimentin: cytokeratin was already detectable in 10-week-old embryos, while weak vimentin immunoreactivity was first seen in 12-week-old embryos and became conspicuous in testes around the perinatal period. In testes of children up to 2 years of age the cytoplasmic distribution of cytokeratin and vimentin was more homogeneous. Predominance of the basal cell portions for vimentin and the apical regions for cytokeratin staining were less pronounced than in adult testes. In the proximal and distal parts of the epididymis a different intermediate filament expression pattern was found with a clear predominance of cytokeratin near the rete. PMID- 1705066 TI - So-called embryonal hyperplasia of Bowman's capsular epithelium: an immunohistochemical and ultrastructural study. AB - The so-called embryonal hyperplasia of Bowman's capsular epithelium (EHBCE) is a rather specific lesion occurring in kidneys of patients maintained on chronic dialysis. It consists of poorly differentiated cells proliferating around sclerosed or obsolescent glomeruli. In this study, immunohistochemical and ultrastructural characterization of EHBCE was performed. The poorly differentiated cells in the lesion exhibited a positive reaction for vimentin and a negative one for cytokeratin (PKK 1) and epithelial membrane antigen. On ultrastructural examination, specialized junctions between adjoining cells, microvilli-like structures on their surfaces, and immature basal folds were observed. These observations suggest that the cells of EHBCE may be associated with the anlage of glomerular epithelium. The background in which neoplasms like renal cell carcinoma or atypical epithelium of cyst wall develop in end-stage kidneys of adult patients on long-term dialysis may cause such a proliferation of poorly differentiated cells in young or paediatric age group patients. PMID- 1705067 TI - Solid-cystic (papillary-cystic) tumours within and outside the pancreas in men: report of two patients. AB - Solid-cystic (papillary-cystic) tumours (SCT) of the pancreas are distinctive neoplasms with a predilection for young female patients. This is the first detailed report describing the occurrence of SCT in two young male patients. Except for the extapancreatic occurrence of one of the tumours (in the retroperitoneal region behind the head of the pancreas), all other clinicopathological features were identical to those characterizing the SCT in women. Immunostaining was (at least focally) positive for Lu 5 (broad spectrum keratin marker), vimentin and alpha-1-antitrypsin. The tumours were negative for neuroendocrine markers (except for neuron-specific enolase), pancreatic hormones and enzymes, pancreatic stone protein, carcinoembryonic antigen, CA 19-9 and nuclear oestrogen and progesterone receptors. This report does not support the suggested female sex hormone dependence of SCT. PMID- 1705068 TI - [Diagnosis and surgical treatment of cancer of the gallbladder and extrahepatic bile ducts]. AB - Data on 148 cases of cancer of the gallbladder and extrahepatic bile ducts were studied. Jaundice proved the cardinal symptom. No clear-cut clinical picture of the disease was identified. Diagnostic procedure should start with ultrasonography. Cholangiectasia and the enlarged pancreatic head make the case for fiber bronchoscopy and hypotonic duodenography. Cancer-negative patients should further undergo transcutaneous transhepatic cholangiography and, if proving still negative, retrograde cholangiopancreatography. Resection of bile ducts with simultaneous lymphadenectomy is considered radical. The authors suggest a surgical procedure for cancer of the gallbladder which includes resection of the liver, hepatico-choledoctomy and cholecystectomy with formation of cholangio-jejuno-anastomosis using disposable transhepatic drains. Recanalization of bile ducts by transhepatic drain is considered optimal for palliation. Survival depends upon extent of surgery and level of bile duct obstruction. PMID- 1705069 TI - [Diagnosis and surgical treatment of liver tumors]. AB - The authors' experience showed that a complex of ultrasonography, computer tomography, laparoscopy and angiography assures reliable diagnosis of hepatic tumor and allows to evaluate its nature and operability. Resection of the liver is the surgical procedure of choice for hepatic tumors. Resection of varying extent was performed for 22 benign tumors yielding good immediate and end results. It was carried out in 14 patients with hepatic cancer. End results proved satisfactory, particularly, in combined application of surgery and chemotherapy. PMID- 1705070 TI - [Arachnoid cysts (pathomorphological studies)]. AB - Based on a macro- and microscopic analysis of the material of 2 section cases and 29 biopsies of persons operated on for arachnoidal cysts two types of cysts were distinguished: 1) arachnoidal cysts that occurred as a result of isolated congenital pathology of the leptomeninx; 2) cysts representing a manifestation of the combined developmental abnormality of the CNS occurring in the form of arachnoidal cysts coupled with intracerebral ones and with pathology of the adjacent cortical parts in the form of gross impairment of its architectonics and developmental abnormality of cerebral vessels. PMID- 1705071 TI - [Pathological structure of blood vessels of cerebral cortex in Alzheimer's disease in comparison with various other types of mental retardation (oligophrenia, Down's syndrome)]. AB - The characteristic features of the brain cortex architectonics were studied and compared in Alzheimer's disease, oligophrenia and Down's disease. Some analogous alterations were established to be characteristic of all the three forms of pathology. On this basis the role is discussed of congenital disturbances of the angioarchitectonics in both mental retardation and dementia development. PMID- 1705072 TI - From unfractionated heparins to low molecular weight heparins. AB - Interest in low molecular weight heparins as potential antithrombotic agents has been stimulated by two observations. These were that low molecular weight heparins have a different anticoagulant profile from unfractionated heparin and that some low molecular weight heparins are less haemorrhagic in animal models than unfractionated heparins for equivalent antithrombotic effects. Subsequently, it was shown that low molecular weight heparins inhibit platelet function and impair vascular permeability less than unfractionated heparin and that low molecular weight heparins have a longer biological half-life than unfractionated heparin. A number of low molecular weight heparins have been evaluated in clinical trials in general surgery, orthopaedic surgery and in the treatment of venous thrombosis. Low molecular weight heparins are highly effective in orthopaedic surgery where they appear to be more effective than unfractionated heparin. Low molecular weight heparins have also been shown to be either as effective or more effective than unfractionated heparin in preventing post operative thrombosis following general surgery. In preliminary studies, low molecular weight heparins appear to be as effective as unfractionated heparin in the treatment of venous thrombosis but larger studies are required using clinically relevant outcome measures. PMID- 1705073 TI - Developing testicular microvasculature in the golden hamster, Mesocricetus auratus: a model for angiogenesis under physiological conditions. AB - The ultrastructure of the developing testicular microvasculature in the testes of immature (3, 5, 8, 10, 12, 16, 20, 25, 30 and 35 days old) golden hamsters was examined and compared to the testicular microvasculature of adult (3 months old) hamsters. In addition, in 16- to 35-day-old hamsters vascular permeability was studied after localization of injected horseradish peroxidase (HRP). Angiogenic processes were present in the testes of all examined immature hamsters and were most conspicuous between 8 and 25 days of age. These processes were absent in the testes of 3-month-old hamsters. On days 3 and 5, few undifferentiated blood vessels with activated endothelium were present in the interstitial spaces. Endothelial cell migration started from these 'mother vessels' and led to invasion of intertubular spaces by vascular sprouts, before vascularization of peritubular spaces occurred (after day 12). Sprouting endothelial cells were identified by the presence of a basal lamina and characterized by abundant cytoplasm and cell organelles. HRP-positive slits were seen in developing vessels, which opened to form the vascular lumen. HRP exited the vascular lumen through unspecialized endothelial contacts and micropinocytotic vesicles. By day 16, the blood-testis barrier prevented HRP from entering the seminiferous tubules beyond the basal compartment. By days 30 and 35 most testicular microvessels and at the age of 3 months all testicular microvessels were of the mature type, with narrow inactive endothelium and specialized cell contacts (including tight junctions). These results demonstrate that the postnatal vascularization of the testis in the golden hamster is a timed complex process. Due to high permeability, vascular sprouts are likely to influence the metabolic situation and thus the maturation processes of the testis. Angiogenesis in the golden hamster testis shares typical morphological features with angiogenic processes in other organs and species under various pathological and physiological conditions. We therefore conclude that the postnatal testis can be viewed as a physiological model of angiogenesis. PMID- 1705074 TI - Salivary defense mechanisms in juvenile periodontitis. AB - The local, saliva-associated defense mechanisms of 28 juvenile periodontitis (JP) patients and their age- and sex-matched controls were studied. Lysozyme, lactoferrin, salivary peroxidase, myeloperoxidase, and thiocyanate concentrations were determined from both whole saliva and parotid saliva. The total concentrations of salivary IgA, IgG, and IgM were assayed. The periodontal condition and the salivary flow rates were registered. Among the JP patients, a significantly elevated concentration of IgG was found in parotid saliva but not in whole saliva. Salivary peroxidase activities were significantly low both in the whole and in the parotid saliva samples of the JP patients, and leukocyte derived myeloperoxidase was present in significantly low amounts in whole saliva of these patients. Because both glandular (salivary peroxidase) and polymorphonuclear-cell-derived (myeloperoxidase) enzyme activities were low among the JP patients, suppressed peroxidase-mediated host defense mechanisms could be characteristic of JP. PMID- 1705075 TI - Patient acceptance of prenatal alpha-fetoprotein screening: a preliminary study. AB - Forty-five obstetrical patients who received an educational session about maternal serum alpha-fetoprotein (MSAFP) screening were followed until the 20th to 26th week of gestation to determine what percentage would decide to have MSAFP testing. These patients were then given a knowledge test and questioned about the factors that led to their decision about the test. Fourteen of the 45 study patients (31%) elected to have MSAFP testing. The patient's decision to have MSAFP testing was found to be significantly associated with 1) reporting a longer length of time discussing the test with the physician, 2) reporting that the opinion of a significant other about the test was important, and 3) older age. There is a need for further study of patients' acceptance of MSAFP testing to see if the relatively low acceptance rate found in this study is also found in other practices. PMID- 1705076 TI - [Effects of transurethral resection on the serum concentration of prostate specific antigen and prostatic acid phosphatase]. AB - Serum concentration of PSA and PAP in 30 patients (25 prostate benign hyperplasias and 5 carcinomas) are evaluated prior to transurethral resection, right at the end, after 24 hours and 5 days later. Following TUR a significant increase in BHP is detected, this being less severe in the case of carcinomas. Subsequently, after 24 hours, PSA and PAP levels in BHPs are similar to baseline values, while there is a significant decrease in carcinomas after 5 days. Thus, determination of these markers in prostatic carcinomas should never be postponed after a TUR. PMID- 1705077 TI - Antiviral activities of a human monoclonal antibody against human cytomegalovirus. PMID- 1705078 TI - Antigenic and structural properties of mutants in herpes simplex virus 1 glycoprotein B. PMID- 1705079 TI - Herpes simplex virus type 1 infection in mice with severe combined immunodeficiency (SCID). PMID- 1705080 TI - Definition of platelet-derived histamine releasing factor (PDHRF) and histaminergic receptors modulating platelet aggregation. PMID- 1705081 TI - Iloprost, a stable analogue of PGI2: clinical results and pathophysiological considerations. PMID- 1705082 TI - Involvement of eicosanoids in angiogenesis. PMID- 1705083 TI - Eicosanoid synthesis by spinal cord astrocytes is evoked by substance P; possible implications for nociception and pain. AB - Prostaglandin synthesis by astrocytes in culture has been shown to be stimulated by a range of mediators including ATP, interleukin-1 and the neuropeptide substance P. In this paper we present evidence that astrocytes from rat spinal cord, but not other CNS regions, release prostaglandins in response to treatment with sub-micromolar concentrations of the neuropeptide substance P, a neuromodulator that may be involved in regulating the input of nociceptive information into the spinal cord. This in vitro phenomenon, if representative of physiological responses, suggests that astrocytes may play a role in central processing of noxious input. The fact that astrocytes from rat cortex do not exhibit substance P-evoked prostanoid release provides further evidence for regional astrocyte heterogeneity. PMID- 1705084 TI - Effects of prostacyclin analogues in in vivo tumor models. AB - Much attention has recently focused on the role of tumor cell-platelet interaction in the metastatic cascade. Prostacyclin and stable prostacyclin analogues have been shown to inhibit specifically the formation of metastases in experimental tumor models. This action is based on their ability to reduce the attachment of tumor cells to platelets and to inhibit adhesion of tumor cells platelet aggregates to the endothelial lining. To investigate the antimetastatic potential of two prostacyclin analogues (Iloprost and Eptaloprost, Schering AG), we have tested these compounds in the spontaneously metastasizing R 3327 MAT Lu prostate carcinoma of the Cop rat in two types of experiments. Treatment was performed for 33 days, starting one day before s.c. implantation of the tumor. The primary s.c.-implanted tumor remained in situ throughout the experiment. In the first test, Iloprost (0.3 micrograms/kg/min) and Eptaloprost (0.1 micrograms/kg/min) were administered via Alzet mini pumps s.c.. There was a considerable reduction of the number of visible lung metastases by Eptaloprost. In the second test, Eptaloprost was administered p.o. in doses of 0.1 and 0.5 mg/kg daily. The number of lung metastases was significantly reduced. Both compounds had no effect on the growth of the primary tumor in the first as well as in the second test. These data show that the prostacyclin analogue Eptaloprost has a significant antimetastatic activity in a spontaneously metastasizing tumor model and thus merits further investigation. PMID- 1705085 TI - Endothelial cell 13-HODE synthesis and tumor cell endothelial cell adhesion. PMID- 1705086 TI - Antimetastatic action of stable prostacyclin analogs in mice. PMID- 1705087 TI - Mechanisms of the antimetastatic activity of stable prostacyclin analogues: modulation of host immunocompetence. PMID- 1705088 TI - Tumour cell proliferation by thromboxane A2: a receptor-mediated event. PMID- 1705089 TI - [A study on local administration of thrombin following transurethral resection of the prostate--clinical investigation with four-way balloon catheter]. AB - The effect of local administration of thrombin via a newly devised four-way balloon indwelling catheter was investigated on 89 patients who underwent transurethral resection of the prostate (TURP). The catheter was introduced into the bladder immediately after TURP, the balloon was inflated with sterile water and mild moist sponge traction was applied to seal the bladder neck for 15 minutes. At the same time, the thrombin solution, 5,000 U in 5 ml of saline, was then injected into the prostatic fossa via the newly added infusion channel to promote early hemostasis. The results were compared with those of 36 randomized control patients, who were treated with the conventional three-way balloon catheter of the same size. The results obtained with this new device were favorable, showing significantly less postoperative hemorrhage in the thrombin infusion group than in the control group. In 7 of 89 thrombin infused patients, serum FDP revealed mild elevation for 2 hours after TURP. In 2 of these 7 patients FDP was closely correlated with thrombin infusion. However, no adverse reactions were observed in any patient in the thrombin infusion group. In conclusion, our new device to administer locally the thrombin solution is effective and safe for management of bleeding after TURP. PMID- 1705090 TI - The use of monoclonal antibody 44-3A6 in cell blocks in the diagnosis of lung carcinoma, carcinomas metastatic to lung and pleura, and pleural malignant mesothelioma. AB - Monoclonal antibody (MoAb) 44-3A6 recognizes a glandular differentiation associated antigen and has been used to identify exocrine differentiation in pulmonary carcinomas. The authors assessed its value in the diagnosis of lung carcinomas metastatic to lung/pleura and pleural malignant mesothelioma (MM), using cell blocks derived from cytologic specimens. Sixty-three primary lung carcinomas, 31 metastatic adenocarcinomas (ACs) (from breast, gastrointestinal tract, or genitourinary tract), and 36 MMs were immunostained with 44-3A6, Leu M1, and anti-carcinoembryonic antigen (CEA). The results confirm the value of 44 3A6 in identifying ACs but do not allow distinction between those of pulmonary, breast, GIT, or ovarian mucinous derivation. Endometrial, ovarian serous, and renal ACs are essentially nonreactive, as are almost all MMs. The occurrence of one positive MM predicates caution in interpreting 44-3A6 positivity in isolation, but, judiciously used with other discriminating antibodies such as Leu M1 and anti-CEA, 44-3A6 is of value in the differential diagnosis of ACs and MMs. Further, its applicability to cytologic specimens may obviate the need for more invasive diagnostic procedures and lead to rapid, accurate diagnosis. PMID- 1705091 TI - Automated immunohistochemical estrogen receptor in fixed embedded breast carcinomas. AB - The authors immunohistochemically assessed the presence of estrogen receptor (ER) in formalin-fixed, paraffin-embedded tissue sections of 68 breast carcinomas by an automated method using Pronase (CalBiochem, La Jolla, CA) predigestion and alkaline phosphatase detection (Method 1). These results were compared with those obtained by an automated peroxidase-antiperoxidase method with DNAse pretreatment of fixed embedded sections (Method 2), with ER immunostain on frozen sections (Method 3), and with biochemical results (dextran-coated charcoal cytosolic [DCC] assay). Compared with the DCC assay, Methods 1, 2, and 3 gave sensitivities of 54%, 25%, and 89%, respectively. The sensitivity for Method 1 was increased to 74% in those cases with DCC results showing greater than 50 fmol/mg protein. These findings indicate that ER immunohistochemical studies on formalin-fixed paraffin-embedded tissues (as assayed by Method 1) provide useful clinical information when the results are positive. A negative result, especially if surrounding normal elements are not positive, may indicate no receptors, receptor levels less than 50 fmol/mg protein, or improper tissue preservation. In the absence of fresh tissue for ER assay by DCC assay or of frozen sections for immunostaining, and with an understanding of its limitations, this method may be useful. PMID- 1705092 TI - My4 antibody staining of non-Hodgkin's lymphomas. AB - The My4 antibody, one of a number of monoclonal antibodies that react with the CD14 antigen, was originally reported to weakly stain monocytes, macrophages, and granulocytes. However, recent studies have shown that the My4 antibody also stains normal peripheral blood B lymphocytes and some subtypes of B-cell non Hodgkin's lymphoma. Thus, the authors have studied a large series of non Hodgkin's lymphomas stained with the My4 antibody. In frozen sections of reactive lymph node biopsy specimens, the My4 antibody strongly stained mantle zone B lymphocytes and weakly reacted with dendritic reticulum cells and histiocytes. In a series of 245 non-Hodgkin's lymphomas, the My4 antibody stained 111 (45%) cases: 108 of 189 (57%) B-cell lymphomas, 3 of 50 (6%) T-cell lymphomas, and 0 of 6 null cell lymphomas. My4-positive B-cell lymphomas occurred in all histologic subtypes with the exception of small noncleaved cell lymphomas. Follicular lymphomas were most often My4 positive (82%). My4 antibody staining showed no correlation with Working Formulation grade. All three My4-positive T-cell lymphomas had a mature T-cell phenotype. Seventy-six of the 111 (68%) My4 positive lymphomas were also analyzed with at least one other anti-CD14 antibody, either Mo2 and/or Leu-M3. In all cases the antigens that react with Mo2 and Leu M3 were not expressed. Thus, the staining of reactive and neoplastic B cells by My4 appears to be unique to this antibody and is not a feature of all anti-CD14 antibodies. PMID- 1705093 TI - Mycobacteremia in acquired immune deficiency syndrome. Rapid diagnosis based on inclusions in the peripheral blood smear. AB - In 16 cases of human immunodeficiency virus-associated Mycobacterium avium intracellulare complex (MAC) infection, 7 were diagnosed after finding intracytoplasmic negatively staining linear inclusions within histiocytes using Romanowsky-stained bone marrow aspirate smears. Four patients had inclusions within monocytes and neutrophils in the peripheral blood smear. The authors believe these cases represent the first reported examples of MAC inclusions observed within leukocytes in Wright's-stained peripheral blood smears. Inclusions usually were found in the setting of prominent toxic changes in leukocytes such as large Dohle bodies, marked granulation, and vacuolation. These inclusions are characteristic of mycobacteria and can be confirmed by acid fast stains and mycobacteriologic culture. The authors present the clinical and laboratory setting in which identification of inclusions in peripheral blood smears may be a rapid, minimally invasive, and cost-effective method of diagnosing mycobacterial infection. PMID- 1705094 TI - Effect of reversible androgen deprivation on hemoglobin and serum immunoreactive erythropoietin in men. AB - To examine the role of testosterone in the maintenance of hemoglobin levels, we studied the effect of reversible androgen deprivation on hemoglobin, serum immunoreactive erythropoietin, and serum testosterone in seven men treated with a luteinizing hormone-releasing factor (LHRH) agonist for 6 months and then followed for an additional 6 months. The mean serum testosterone level was 4.35 +/- 1.05 ng/ml initially and it decreased to castrate levels in all patients by 6 months. After stopping therapy, there was a rapid increase in serum testosterone such that by 12 months the mean concentration was normal. The pretreatment hemoglobin was 15.2 +/- 0.9 g/dl (mean +/- SD); after 6 months of androgen deprivation it had fallen to 14.1 +/- 0.4 g/dl (P less than 0.05). Six months after stopping therapy, the hemoglobin rose to pre-treatment levels. Before treatment, serum immunoreactive erythropoietin was 9.5 +/- 4.6 mu/ml (mean +/- SD) and did not change significantly during or after the 6 month period of androgen deprivation. No significant inhibition of burst-forming unit-erythroid (BFU-E) or colony-forming unit-granulocyte macrophage (CFU-GM) was observed at the serum level of nafarelin acetate obtainable in vivo. These data suggest that, within the normal range of hemoglobin in men, androgens are a determinant of the red cell mass. PMID- 1705095 TI - Severe and fatal anthracycline cardiotoxicity at cumulative doses below 400 mg/m2: evidence for enhanced toxicity with multiagent chemotherapy. AB - Three cases of severe, progressive, and in two cases, fetal cardiomyopathy secondary to anthracycline chemotherapy are reported. All of the patients were receiving multiagent chemotherapy for extremity sarcomas consisting of doxorubicin, high-dose methotrexate, bleomycin, cyclophosphamide, dactinomycin, and cisplatinum at the onset of their congestive heart failure. Cardiomyopathy developed in each patient at cumulative anthracycline doses less than 400 mg/m2. These cases suggest that enhanced cardiotoxicity may occur when anthracyclines are used in combination with other agents such as cyclophosphamide, bleomycin, cisplatinum, and methotrexate and that cumulative anthracycline doses considered to be "safe" may need to be lowered in these circumstances. PMID- 1705096 TI - NaF-induced amylase release from rat parotid cells is mediated by PI breakdown leading to Ca2+ mobilization. AB - We have examined the effects of sodium fluoride (NaF) on amylase release, cellular adenosine 3',5'-cyclic monophosphate (cAMP) level, inositol phosphate formation, and cytosolic free Ca2+ concentration ([Ca2+]i) in dispersed rat parotid acini or cells. At concentrations greater than 1 mM, NaF significantly increased amylase release. The maximum response was observed at 10 mM NaF and was comparable to that of the muscarinic-cholinergic agonist carbachol. Removal of extracellular Ca2+ with EGTA markedly suppressed the NaF-induced secretory response. At concentrations up to 10 mM, NaF did not increase the cellular level of cAMP, indicating that the NaF-induced amylase release is not mediated by cAMP. NaF (1-20 mM) caused a slow increase in [Ca2+]i in a concentration-dependent manner, as monitored with the fluorescent Ca2+ indicator fura-2, and the increased [Ca2+]i did not decline for at least 10 min after addition of NaF. In the absence of extracellular Ca2+, NaF evoked only a small and transient increase in [Ca2+]i. The addition of 10 mM NaF produced a significant accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. These results suggest that the NaF-induced amylase release is mediated by a breakdown of phosphoinositides leading to Ca2+ mobilization. The effects of fluoride may be through the action of F- on the GTP-binding protein(s) coupled to phospholipase C. PMID- 1705097 TI - Stimulation by cGMP of apical Na channels and cation channels in toad urinary bladder. AB - The effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on apical membrane cation conductances in the toad urinary bladder were investigated. 8 BrcGMP (1 mM) added to the serosal solution increased the amiloride-sensitive short-circuit current (INa) after a delay of 5 min to a steady-state value 1.8 times that of controls achieved after 30 min. Similar effects were seen when the bladders were bathed on the serosal side with a normal NaCl Ringer solution and with a high-K sucrose solution to depolarize the basolateral membrane. Under the latter conditions, the amiloride-sensitive transepithelial conductance increased in parallel with the short-circuit current, indicating stimulation of apical membrane Na channels. The threshold concentration for observing the stimulation of INa was 100 microM, 10-100 times larger than the concentration of 8 bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) required to elicit an increase in INa. Currents through an outwardly rectifying Ca-sensitive cation conductance (Iout) were also increased by 1.8-fold relative to controls. This stimulatory effect occurred after a delay of 15 min and reached maximal levels 90 120 min after addition of the nucleotide. The effects of cGMP on INa were not additive with those of 8-BrcAMP or with antidiuretic hormone, an agent known to act by increasing cAMP within the cell. Addition of 1 mM 3-isobutyl-1 methylxanthine to the serosal side of the bladders stimulated INa by 1.3-fold and Iout by 2.4-fold. In both cases, subsequent addition of cGMP produced no further activation of either conductance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705098 TI - Modulation of Gs activity by phorbol myristate acetate in rat hepatocytes. AB - Activation of protein kinase C promotes heterologous desensitization of hepatic adenylate cyclase. The basis for this desensitization was explored by use of a strategy with several independent approaches. Although not influencing the amount of forskolin-stimulated adenylate cyclase activity (catalyst), treatment with phorbol 12-myristate 13-acetate (PMA) decreased adenylate cyclase activation in response to either sodium fluoride or guanylyl imidodiphosphate [Gpp(NH)p]. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cholera toxin-treated hepatocytes and both the basal and GTP-stimulated adenylate cyclase activity of membranes from toxin-treated cells displayed a marked reduction in response to PMA. The ability of cholate extracts of hepatocyte membranes to reconstitute beta adrenergic-stimulated adenylate cyclase activity of membrane of S49 mouse lymphoma cyc- cells was reduced by treatment with PMA. Cholera toxin-catalyzed labeling of Gs alpha-subunits was likewise diminished by phorbol ester treatment. Immunoblots of membranes from control or PMA-treated hepatocytes showed no difference in the amount of Gs alpha. Immunoprecipitation studies failed to detect phosphorylation of this G protein alpha-subunit. The data demonstrate that PMA induces an alteration in the functional status of Gs without altering the amount of this transmembrane signaling element. The alteration in Gs function may play a significant role in heterologous desensitization. PMID- 1705099 TI - Distinct epitopes on amiloride. II. Variably restricted epitopes defined by monoclonal anti-amiloride antibodies. AB - Specific regions of amiloride appear to participate in binding to receptors on amiloride-sensitive transport proteins. Previous studies characterizing epitopes on amiloride recognized by anti-amiloride antibodies have demonstrated that antibodies recognize specific domains on amiloride and that these epitopes are determined, in part, by the site on amiloride used to couple to carrier protein. The 3,5-diaminopyrazinyl and guanidinocarbonyl moieties were identified as distinct epitopes. Since Na(+)-selective transport proteins are sensitive to changes of the halide on the amiloride molecule, additional monoclonal anti amiloride antibodies were raised to determine whether the C-6 halo group of amiloride could be identified as an important site for drug-antibody binding. The epitopes recognized by a series of three monoclonal antibodies raised against amiloride coupled to rabbit serum albumin through its C-5 NH2-group were defined. Two antibodies recognize extensive regions on the amiloride molecule, including both the acylguanidino and pyrazinyl groups. In addition, both antibodies are sensitive to changes in the C-6 halo group on amiloride. A third antibody was relatively insensitive to changes in the halide in the C-6 position of the pyrazine ring of amiloride and recognized a more restricted epitope on amiloride. PMID- 1705100 TI - Voltage gating of the mitochondrial outer membrane channel VDAC is regulated by a very conserved protein. AB - Soluble protein preparations obtained from the mitochondrial fractions of three very different organisms, Neurospora crassa, rat, and potato, were discovered to greatly enhance the voltage sensitivity of the mitochondrial outer membrane channel, VDAC. The active ingredient, referred to as the VDAC modulator, increased the rate of voltage-dependent channel closure by approximately 10-fold. The modulator from one species increased the closing rate of VDAC channels from all three species. The activity is pronase sensitive and not mimicked by another negatively charged protein, BSA. The highly conserved property of this modulator suggests an important physiological role in regulating mitochondrial function. PMID- 1705101 TI - Neuroimmune regulation of colonic secretion in guinea pigs. AB - The role of submucosal neurons in anaphylactic-like responses in colonic epithelium from immunized guinea pigs was examined 6-8 wk after inoculation with 2 x 10(3) infective Trichinella spiralis larvae. Serosal addition of T. spiralis antigen (20 micrograms/ml) to muscle-stripped segments of colon set up in flux chambers evoked a maximum increase in short-circuit current within 5 min in immune, but not nonimmune, guinea pigs. Quercetin, a membrane-stabilizing drug, and pyrilamine, a histamine H1 receptor antagonist, attenuated epithelial responses evoked by T. spiralis antigen. Antigen-induced epithelial responses were reduced by neural blockade with tetrodotoxin and by the muscarinic receptor antagonist atropine but not by blockade of nicotinic receptors with mecamylamine. Antigenic challenge of colonic mucosa from immune guinea pigs enhanced the secretory responses to endogenously released neurotransmitters evoked by electrical field stimulation and substance P. In the presence of antigen, the tetrodotoxin-insensitive component of the carbachol response was enhanced and was reversed by quercetin but not pyrilamine. The results suggest that submucosal cholinergic nerves play a role in mediating the rapid epithelial responses evoked by worm antigen in the colonic mucosa of T. spiralis-immune guinea pigs. Interaction of immunological mediators with neurotransmitters in the submucosal plexus augments the secretory mucosal response triggered by T. spiralis in immunized hosts. PMID- 1705102 TI - Use of antibodies in the study of protein structure and function in lung diseases. AB - Proteins normally fold in a variety of three-dimensional structures. This variety of forms accounts for a multiplicity of functions. One of the major undertakings of modern biochemistry is to determine the structure of a given protein and in doing so learn about its function. Much of the effort in the field of protein chemistry is currently focused on defining areas or domains within the molecule that are responsible for its function. Antibodies specific for functional domains on a protein have been powerful tools in these studies and have provided a great deal of the current knowledge related to the location and function of well defined structural areas within the protein molecules. The use of antibodies in the study of proteins has considerably advanced our knowledge of both the structure of proteins and their interaction with other molecules. In reviewing the use of antibodies for structure and/or function analysis, we have tried not only to review the techniques and applications involving antibodies but also to show how useful antibody reagents may be designed and prepared. In this review, some of the newer uses of antibodies in modern biology will be described, and a few illustrations of each will be provided. PMID- 1705103 TI - Osteosarcomas of the heart. AB - We reviewed nine primary cardiac sarcomas with osteosarcomatous differentiation. The patients' ages ranged from 24 to 67 (mean 38 years). All tumors were surgical specimens from the left atrium; many were clinically diagnosed as atypical myxomas. In eight cases complete excisions were attempted, one requiring reconstruction with grafting; one tumor was biopsied only. Two tumors extended into the pulmonary veins. Three patients died within 2 weeks after the initial surgery from postoperative complications; five patients had metastatic disease or died from disease; and one patient was lost to follow-up. Metastatic sites included lungs, thyroid, and skin. In addition to osteosarcoma, four tumors showed chondroid differentiation, three had osteoclastic cells, four had a prominent spindle cell component, and one had myxoid areas. All tumors showed immunohistochemical positivity for vimentin; stains for cytokeratin and desmin were negative. S-100 positivity was demonstrated in chondrosarcomatous areas of one tumor. We conclude that most cardiac osteosarcomas are clinically mistaken for myxomas because of location in the left atrium. They are larger, tend to infiltrate, and are very aggressive neoplasms. Histologically a variety of patterns may be encountered in addition to the osteosarcoma. PMID- 1705104 TI - Characterization of stage-specific antigens of Paragonimus westermani. AB - In order to develop a serodiagnostic assay to detect antigens of Paragonimus westermani in biological specimens, we generated monoclonal antibodies to antigens of metacercariae and adult worms, and partially characterized the epitopes recognized by representative Mabs. Metacercarial stage-specific determinants were periodate-sensitive and protease-resistant, indicating that they are carbohydrate epitopes. In contrast, adult worm-specific determinants resisted periodate treatment but were sensitive to proteases, indicating that they are polypeptides. The majority of cross-reactive Mabs studied were directed against phosphorylcholine determinants. When used in a dot-ELISA, Paragonimus specific Mabs could detect 0.3-7 ng/ml parasite antigen in human sera. PMID- 1705106 TI - Time-course studies of immediate and delayed immune reactivity in patients with atopic dermatitis treated with herbal drugs. AB - We followed a number of in vitro parameters in nine adult patients with atopic dermatitis for a period of 3 weeks during which the patients took herbal drugs that are claimed to improve chronic eczema. The following investigations were performed weekly: blood leucocytes, circulating eosinophils, serum IgE, mitogen responsiveness of lymphocytes, interleukin 1 release, soluble interleukin 2 receptor levels in serum, and in vitro histamine release from basophils in blood. The study demonstrated increased levels of IgE in six patients, increased levels of soluble interleukin 2 receptor in six patients, and a significant correlation between the amount of IgE in serum and the maximal release of histamine from basophils in blood. In this open study five patients experienced a clinical worsening of their disease during the intake of herbal medicine. All other parameters were within normal levels. There was no significant change in the in vitro parameters during the observation period. PMID- 1705105 TI - Clinical and immunohistochemical studies of subependymal giant cell astrocytomas associated with tuberous sclerosis. AB - Two cases of TS associated with brain tumors had severe psychomotor retardation and early onset of long-term intractable convulsions, compared with cases without tumors. In one case, the tumor was partially cystic and progressed rapidly. Immunohistochemical studies of neuron specific enolase, glial fibrillary acidic protein and myelin basic protein revealed differences in positivity between cell types and between cases. These results suggested that the origin of the tumor cells could be variably differentiated cells. PMID- 1705107 TI - [Prenatal diagnosis of trisomy 21]. AB - Trisomy 21 is the most frequent chromosome anomaly found in the living newborns. Prenatal diagnosis by amniocentesis was limited until recently to older pregnant women. Maternal blood biochemical markers (alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin) and ultrasonographic signs (nuchal fold thickening, femur length to biparietal diameter ratio) can discriminate a group of higher-risk women even though they are not aged. Numerous factors need to be considered when establishing such a screening program including pre-analytical variables (gestational age, diabetes, smoking, race), analytical variables (choice of reagents, quality control) or post-analytical (result reporting, follow-up of abnormal results). Until now the study of these markers has been restricted to the second trimester but they could become useful earlier during pregnancy. PMID- 1705109 TI - Nerve growth factor prevents toxic neuropathy in mice. AB - Taxol is a promising new antitumor drug with therapeutic use that is limited by a toxic sensory neuropathy. Taxol is also cytotoxic to dorsal root ganglion neurons in vitro, but this effect is prevented by cotreatment with the trophic protein, nerve growth factor. We sought to develop an animal model and then to determine whether nerve growth factor can prevent taxol neuropathy in vivo. Administration of taxol to mice resulted in a profound sensory neuropathy characterized by decreases in dorsal root ganglion content of the peptide neurotransmitter, substance P, elevated threshold to thermally induced pain, and diminished amplitude of the compound action potential in the caudal nerve. Coadministration of nerve growth factor prevented all of these signs of neurotoxicity. These findings suggest that administration of nerve growth factor may prevent certain toxic sensory neuropathies. PMID- 1705108 TI - Squamous cell carcinoma of the oesophagus: 10 years on. PMID- 1705110 TI - [Histologic characterization of late asthmatic response in guinea pigs]. AB - We examined lung tissues in a guinea pig model actively sensitized by inhalation of aerosolized ovalbumin (OA) and found reproducible late bronchial responses (LBR) 7 h and 24 h after OA challenge. Light microscopic examination revealed bronchoconstriction, damage to the epithelium, eosinophil infiltration in both the epithelium and subepithelium, retention of mucous in the pulmonary bronchial lumens and a decrease in the mucous content of Periodic Acid-Schiff (PAS) stain positive cells in the bronchial epithelium in LBR. An increase in the number of PAS-stain positive cells of in the bronchial epithelium was also observed in LBR. Electron microscopic examination revealed eosinophil migration around mast cell at 7 hs after OA challenge. Two different mechanisms of degranulation of eosinophil specific granules were observed in LBR. In one mechanism, the granule core changes after its matrix has changed. In the other mechanism, the granules were exocytosed as a whole, accompanied by the cell membrane and densely clumped chromatin, suggesting burst of eosinophil. PMID- 1705111 TI - The use of lot-feeding to enhance the reduction of benzene hexachloride residues in cattle. PMID- 1705112 TI - A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model. AB - Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue. PMID- 1705113 TI - Relationship between the calcium-mobilizing action of inositol 1,4,5 trisphosphate in permeable AR4-2J cells and the estimated levels of inositol 1,4,5-trisphosphate in intact AR4-2J cells. AB - Various experimental strategies were employed in an effort to explain the previously reported [Horstman, Takemura & Putney (1988) J. Biol. Chem. 263, 15297 15303] paradoxically high levels of inositol 1,4,5-trisphosphate [(1,4,5)IP3] in resting and substance-P-stimulated AR4-2J cells. The concentration-effect curves for substance-P-induced [3H](1,4,5)IP3 formation in [3H]inositol-labelled cells and substance-P-induced increase in intracellular [Ca2+] were essentially superimposable, suggesting that formation of (1,4,5)IP3 is limiting for cellular Ca2+ mobilization. In electrically permeabilized AR4-2J cells, (1,4,5)IP3 and other inositol polyphosphates stimulated Ca2+ release with potencies similar to those reported for other cell types, including the parent pancreatic acinar cell. Compartmentalization of basal (1,4,5)IP3 was suggested by the fact that this material was stable in the presence of antimycin A, although this toxin completely blocked agonist stimulation of phospholipase C. However, subcellular fractionation as well as permeabilization of the cells with Staphylococcus aureus alpha-toxin failed to provide evidence for binding or sequestration of [3H](1,4,5)IP3 in AR4-2J cells. The density of (1,4,5)IP3 receptors in AR4-2J cells was not sufficiently large to impose non-linearity in the relationship between (1,4,5)IP3 concentration and (1,4,5)IP3-induced Ca2+ release. Thus the apparent high concentrations of (1,4,5)IP3 in resting and stimulated AR4-2J cells are not indicative of atypically low sensitivity or high concentration of (1,4,5)IP3 receptors, nor is there evidence for compartmentalization of (1,4,5)IP3 outside of the cytoplasm in these cells. It is possible that soluble factors in the cytoplasm of AR4-2J cells regulate the free concentration of (1,4,5)IP3 or the sensitivity of receptors to (1,4,5)IP3. PMID- 1705114 TI - Proteoglycans of human articular cartilage. Identification of several populations of large and small proteoglycans and of hyaluronic acid-binding proteins in successive cartilage extracts. AB - Two specimens of human articulage were successively extracted with solutions of phosphate-buffered saline (PBS), 7 M-urea and 4 M-guanidine hydrochloride (Gdn HCl). Proteoglycans from individual extracts were fractionated by DEAE-Sephacel chromatography and gel chromatography on Sephacryl S-400. The presence of three populations of large proteoglycans was demonstrated in all three extracts by composite agarose/polyacrylamide-gel electrophoresis (CAPAGE). The population corresponding to the fastest CAPAGE band of aggregating proteoglycans was shown to be extremely polydisperse, having Mr (as estimated by SDS/PAGE) decreasing continuously from more than 300,000 to the size corresponding to 'free' hyaluronic acid-binding region (HABR) (about 70,000). A rather polydisperse set of HABR-containing fragments which spanned a broad range of sizes, and also differed in their keratan sulphate contents, was isolated from both 7 M-urea and 4 M-Gdn-HCl extracts. PBS and 7 M-urea extracts, but not the Gdn-HCl extract, further contained small proteoglycans, identified as fast-migrating bands on CAPAGE electrophoretograms. One of those small species was recognized with an antibody against the small proteoglycan PG II; the other two remain to be positively identified. However, the glycosaminoglycan of the small species which was present exclusively in the PBS extract was identified as keratan sulphate; this species may thus belong to the family of small keratan sulphate-containing proteolygans. PMID- 1705115 TI - Further studies on the topography of human platelet glycoprotein IIb. Localization of monoclonal antibody epitopes and the putative glycoprotein IIa- and fibrinogen-binding regions. AB - Glycoprotein IIb (GPIIb) is a major glycoprotein of the human platelet plasma membrane, which together with glycoprotein IIIa (GPIIIa) forms a Ca2(+)-dependent heterodimer, GPIIb/IIIa, which serves as the major fibrinogen receptor in activated platelets. The precise localization of the epitopes for six anti-GPIIb monoclonal antibodies (M1-M6) has been determined by a combination of enzymic and chemical cleavage procedures, peptide isolation, N-terminal sequence analysis, peptide synthesis and enzyme immunoassay. The following localizations were found: M1, beta 1-16-36, beta 2-4-24; M2, alpha 747-755; M alpha 2, alpha 837-843; M3, alpha 849-857; M4, alpha 143-151; M5, alpha 550-558; M6, alpha 657-665. Besides considerations of the degree of exposure of these epitopes, several remarkable features are readily apparent. The earliest and main chymotryptic cleavage site of GPIIb in whole platelets is between alpha cysteine-545 and alpha phenylalanine 551. The epitope for M3 was located within the same sequence (alpha 842-857) as is the epitope for PMI-1 [Loftus, Plow, Frelinger, D'Souza, Dixon, Lacy, Sorge & Ginsberg (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7114-7118] in spite of the fact that the exposure of the latter in whole platelets is EDTA-dependent whereas that in the former is not. The epitope for M5 shares full homology with the 540-548 peptide stretch of the alpha-subunit of the vitronectin receptor, and this antibody cross-reacts with endothelial cells. The M6 epitope is located in the 25 kDa membrane-bound fragment of GPIIb, which is most epitope is destroyed at an early stage of chymotrypic digestion. This suggests that this region of GPIIb, somewhere between the epitope for M5 (alpha 550-558) and the epitope for M2 (alpha 747-755), may carry the surface of interaction of GPIIb with GPIIIa in the GPIIb/IIIa heterodimer. Finally, the sequence where the epitope for M6 has been located (alpha 657-667) was the only one found to be hydropathically complementary to the gamma 402-411 peptide of fibrinogen within the amino acid sequence of both GPIIb and GPIIIa. This complementariness, the EDTA- or thrombin dependence of the exposure of the alpha 657-665 stretch in whole platelets to M6 and the ability of this antibody to inhibit platelet aggregation led us to postulate that this peptide stretch is a putative binding site for fibrinogen in the platelet receptor.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1705116 TI - The use of thioglycollate to demonstrate DNA AP (apurinic/apyrimidinic-site) lyase activities. Biological consequences of thiol addition to the 5' product of a beta-elimination reaction at an AP site in DNA. AB - Thioglycollate reacts with the 5' product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA. The 3'-terminal thioglycollate unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography. In contrast, the mammalian AP endonuclease is unable to excise a 3'-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences. PMID- 1705117 TI - Inhibition of a receptor-operated calcium channel in pig aortic microsomes by cyclic GMP-dependent protein kinase. AB - We have further characterized a putative receptor-operated Ca2+ channel that is activated by histamine and guanosine 5'-[beta gamma-imido]triphosphate. Insensitivity to verapamil, diltiazem or nicardipine, but inhibition by Ni2+ and SK&F 96365, further identify the channel with receptor-mediated Ca2+ entry in intact cells. Inhibition of the channel by cyclic-GMP-dependent protein kinase may contribute to vascular relaxation in response to nitrovasodilators. PMID- 1705118 TI - Potent anti-angiogenic action of AGM-1470: comparison to the fumagillin parent. AB - The anti-angiogenic activity of AGM-1470, a new synthetic analog of fumagillin isolated from Aspergillus fumigatus, was extensively examined both in vitro and in vivo using four different types of assay and compared to that of the fumagillin parent. Locally administered AGM-1470 inhibited the angiogenesis in the chick embryo chorioallantoic membrane assay and the rat corneal assay. In the rat sponge implantation assay, systemically administered AGM-1470 inhibited angiogenesis induced by basic fibroblast growth factor. Furthermore, in the rat blood vessel organ culture assay, AGM-1470 (1-1,000 ng/ml) was found to selectively inhibit the capillary-like tube formation of endothelial cells with a minimal effect on the non-endothelial cell growth. AGM-1470 showed more potent anti-angiogenic activity and less toxicity than the fumagillin parent. Therefore, AGM-1470 is much better than the fumagillin parent as anti-angiogenic compound. PMID- 1705119 TI - Activation of alpha-myosin heavy chain gene expression by cAMP in cultured fetal rat heart myocytes. AB - The effect of cAMP on cardiac myosin heavy chain (MHC) gene expression in primary cultures of 18-day-old fetal rat heart myocytes was investigated. When myocytes were treated with either 10 microM forskolin or 1 mM 8-bromo-cAMP for 48 h, the relative amount of the V1 to -V3 myosin isoform ratio increased 3-fold. The abundance of alpha-MHC mRNA was also increased 3-to-4-fold in forskolin treated vs control cells. However, no appreciable change was observed in the level of beta-MHC mRNA. In addition, a 70% increase in the transcription rate of the cardiac MHC gene was observed by nuclear run-on assay following treatment of cells with 10 microM forskolin for 12 h. These results demonstrate the preferential induction of alpha-MHC mRNA by cAMP which is, in part, mediated by transcriptional activation of the gene. PMID- 1705120 TI - Incorporation of tRNA into normal and mutant HIV-1. AB - During retroviral assembly, tRNAs are incorporated into the virion, one of which serves as a primer for the reverse transcription reaction. Using two dimensional polyacrylamide gel electrophoresis, we have studied the patterns of tRNAs incorporated into HIV-1 (3B) produced either in the lymphoid cell line H-9 or in the monocytic cell line U937. We have also examined viral tRNA patterns incorporated in a non-infectious, mutant virion which lacks pol gene products and processed gag protein. Our results lead to the following conclusions: 1) tRNA incorporated into HIV-1 is a select subpopulation of the host-cell's tRNA. 2) The type of tRNA incorporated into the virion is dependent upon cell type. 3) There can be multiple species of tRNA of similar mobilities tightly associated to the viral genome. 4) The packaging of putative primer tRNA into virions requires either the synthesis of pol gene products, the processing of gag proteins, or both, while the incorporation of non-primer tRNAs does not. PMID- 1705121 TI - Transforming growth factor beta 1 differentially regulates alpha-fetoprotein and albumin in HuH-7 human hepatoma cells. AB - Transforming growth factor beta 1 (TGF-beta 1) is known to inhibit hepatocyte growth in vitro and in vivo. In this study, we analyzed the effect of TGF-beta 1 on alpha-fetoprotein (AFP) and albumin gene expression in HuH-7 human hepatoma cells. TGF-beta 1 inhibited cell growth in a dose dependent manner. The cellular secretion rate of AFP but not albumin was suppressed significantly by TGF-beta 1. TGF-beta 1 caused a significant reduction in the level of AFP mRNA. In contrast, the levels of albumin mRNA or beta-actin mRNA were not changed by TGF-beta 1. In transient transfection experiments, TGF-beta 1 resulted in selective repression of AFP promoter activity. These results suggest that TGF-beta 1 is one of the key factors involved in the differential regulation of the AFP gene and the albumin gene. PMID- 1705122 TI - Endothelin-1 stimulation of noradrenaline and adrenaline release from adrenal chromaffin cells. AB - Endothelin-1 (ET-1) stimulated release of both noradrenaline and adrenaline from cultured bovine adrenal chromaffin cells; stimulated release was small compared to that elicited by 50 mM potassium. Sarafotoxin-6b stimulated release to a similar extent as ET-1. The ET-1 stimulated release had an EC50 of about 1 nM. This calcium-dependent release was partially inhibited by nitrendipine (1 microM), but there was no synergistic interaction with the calcium channel agonist BAY K 8644 (1 microM). There was also no synergistic release seen when submaximal stimulation with potassium was combined with ET-1. Stimulation of fura 2 loaded cells with ET-1 produced an unusual timecourse of response which rose slowly to a maximum which was sustained. These results show that ET-1 may stimulate both noradrenaline and adrenaline containing chromaffin cells by a mechanism which, while partially dependent on dihydropyridine sensitive calcium channels, is distinct from the calcium channel agonist or membrane depolarization. PMID- 1705123 TI - Fluorescent markers for hypoxic cells. A study of novel heterocyclic compounds that undergo bio-reductive binding. AB - The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells. Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc. as a diagnostic method. In this paper, the synthesis of some heteroaromatic nitro compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours. Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry. Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic:oxic differential fluorescence was observed. The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo. PMID- 1705124 TI - Anti-HIV-1 and HIV-2 activity of naphthalenedisulfonic acid derivatives. Inhibition of cytopathogenesis, giant cell formation, and reverse transcriptase activity. PMID- 1705125 TI - [The coexpression of keratin and vimentin in untreated squamous cell carcinoma of the ENT tract]. AB - Forty-four untreated squamous-cell carcinomas of the upper aerodigestive tract were examined immunohistochemically with antibodies against keratin and vimentin. Surprisingly, in 18% of cases, a coexpression of both intermediate filaments was found both in undifferentiated carcinomas and in highly differentiated carcinomas, where it was only possible to observe this phenomenon in the basal cell layer. The tumor-biological relevance of these findings must be the subject of further investigations. PMID- 1705126 TI - Circulating immune complexes detected by a direct nephelometric assay and acute phase proteins in malignant tumors. AB - Circulating immune complexes (CIC) were previously reported to be higher in extended cancer rather than in localized one, by a few groups of investigators as well as by ourselves. The techniques employed were quite different in each study. We described a "direct" laser nephelometric assay. At the same time we determined the serum levels of 4 acute phase proteins (AFP)--alpha 2 macroglobulin, C reactive protein, haptoglobin, alpha 1 acid glycoprotein--by conventional light scattering method, in order to investigate on other possible "immune-modulating" factors. The possible correlations either with prognosis or clinical stage are looked for; the hypothetical behaviour of CIC as one of the serum "blocking factors" and of acute phase reactant as "immune modulating" proteins are discussed. The data concerning the techniques able to remove CIC from the blood stream of the cancer patients were also showed (plasmapheresis and immunoadsorption). PMID- 1705127 TI - [Analysis of receptor expression on astrocytic cells]. AB - Astrocytes are regarded as matrix of the neuron in central nervous system (CNS) and involve nutritional and supporting function of neuron. It was clarified that human and murine cultured astrocytes had Fc receptor (FcR) on their cell surface from the study of EA rosette assay, reverse ADCC (antibody dependent cellular cytotoxicity) and flow cytometric analysis with anti-FcR monoclonal antibodies (mAb) in this study. Human glioma cells express FcR III recognized by mAb MG 12 and mouse astrocytes express FcR II recognized by mAb 2.4 G 2. Expression of FcR on human astrocytes is compatible with FcR-mediated human immunodeficiency virus (HIV)-1 infection in CNS. Expression of adhesion molecules engaged in T and natural killer cell cytotoxicity was also investigated for human glioma cells. CD 56 (NKH-1 or Leu 19), which is an isoform of N-CAM (neural cell adhesion molecule) mainly distributed on human NK cells and a subset of T cells, was also expressed in neuroglial cells. LFA-3, a ligand for CD 2, but not ICAM-1, a ligand for LFA-1, was, expressed on glioma cells. So, CD 56 was suggested to be a new adhesion molecule in NK cell mediated lysis of glioma cells by their homotypic adhesive character. PMID- 1705128 TI - Carpal tunnel syndrome: some views on its management. AB - Despite its high incidence and its reputation for simplicity and efficacy, carpal tunnel release does not invariably produce good results and dissatisfied patients are not infrequently encountered. Unsatisfactory results are due to inaccurate diagnosis and, all too frequently, iatrogenic surgical complications. Surgical technique plays an important role in the achievement of good quality results. Various technical points are controversial and are here discussed: the incision, the division of the retinaculum, neurolysis, repair of the transverse carpal ligament and postoperative management. Our views on the management of recurrence, postoperative sequelae and complications are outlined. PMID- 1705129 TI - The role of thoracic outlet syndrome in the double crush syndrome. AB - The association between thoracic outlet syndrome (TOS) and carpal tunnel syndrome (CTS) (40 cases), ulnar neuropathy (UN) (19 cases) and radial tunnel syndrome (29 cases) is investigated. The possibility of a double crush syndrome is considered with reference to the difficulties in diagnosis. It is demonstrated that in approximately half of all cases the proximal neuropathy precedes the distal one. Despite the fact that surgical treatment of the thoracic outlet syndrome appears to improve the distal neuropathy it is still difficult to decide, in a given case, which decompression takes priority with the exception of carpal tunnel release which is generally to be performed first. The historical background and theoretical basis of the management of double crush syndrome is outlined and arguments for and against the association of the various neuropathies are presented. PMID- 1705130 TI - Restoration of elbow flexion and wrist extension in brachial plexus paralyses by means of free muscle transplantation innervated by intercostal nerve. AB - Traumatic lesions of the brachial plexus with nerve root avulsion pose extremely complex therapeutic problems. Since 1965, the authors have performed neurotization of the musculocutaneous nerve by intercostal nerves in order to restore elbow flexion. The results of this operation are considerably poorer when the interval between the trauma and the neurotization exceeds 6 months. In these late cases, the authors have used, since 1978, a free muscle transfer reinnervated by a transposed intercostal nerve. 17 patients have been operated by means of this technique; 8 of the 11 patients with a follow-up exceeding one year recovered elbow flexion force evaluated to be M3 or more. Encouraged by these results, the authors have extended this procedure to restoration of wrist extension by free musculocutaneous transfer of the gracilis muscle innervated by intercostal nerves. 17 of the 29 patients operated have a follow-up of more than one year and 9 these cases have regained wrist extension with a score of M3 or more. PMID- 1705131 TI - Traumatic brachial plexus palsy in children. AB - We have reviewed 25 children with traumatic brachial plexus palsy during the last fifteen years. Motor vehicle injury was responsible for 17 of these cases. Associated lesions were noted in 68%. All lesions were supraclavicular, including C5-C6 in 5 cases, C5-C7 in 5 cases and C5-T1 in 13 cases. Precise data was not available for two patients. Root avulsion was noted in 63% of our patients. Neurotization was performed in eight of the sixteen patients who had surgical repair. Tendon transfers were performed in 9 patients, 5 of which have had a previous surgical plexus repair. Elbow flexion was restored in all but one, and protective sensation of the hand in 40% of the patients undergoing plexus surgery. We conclude that surgery should be performed on children who have no evidence of nerve regeneration three months following traumatic brachial plexus palsy. PMID- 1705132 TI - Comparison of the results of trapeziometacarpal arthrodesis and arthroplasty in men with osteoarthritis of the trapeziometacarpal joint. AB - Thirty-five men with disabling osteoarthritis at the base of the thumb had 47 thumb reconstructions, including 16 arthrodeses, 12 cemented metal-plastic implants, 6 Swanson condylar implants, 1 Kessler implant, 5 Swanson trapezium implants, and 7 tendon interposition arthroplasties. The mean age was 55 years, the minimum follow-up was 2 years, and the mean follow-up was 52.6 months. Mean pinch strength improved from 6.5 kg preoperatively to 7.2 kg postoperatively. Two thirds of the patients were completely satisfied with the results of the operation. Results were poorer in patients who did heavy work. Choice of procedure had little effect on strength and satisfaction. PMID- 1705133 TI - A radiological technique for measurement of the height of the trapezial cavity. Applications in pre- and post-operative assessment in osteoarthritis of the base of the thumb. AB - The authors present an original radiological technique which allows a precise measurement of the height of the trapezial cavity. The measurement is performed on an antero-posterior radiological projection of the trapezio-metacarpal joint in a neutral position, as described by Kapandji. The first point of reference is fixed and consists of a line projected through the radial articular surface of the second metacarpal with the trapezium. Two perpendicular lines to this first point of reference, are then projected respectively from the distal extremity of the scaphoid and the base of the first metacarpal, so defining a space which corresponds to the height of the trapezial cavity. We have used this measurement for assessment of the column of the thumb, in a series of 33 patients operated on for trapezio-metacarpal osteoarthritis (18 patients were treated by trapezectomy, ligamentous stabilisation and tendon interposition following Burton's or Jones technique and 15 patients underwent insertion of a Swanson trapezium implant. Radiologic measurement pre- and post-operatively provided information relative to the change of the height of the trapezial cavity after the two different surgical techniques, and to assess the kinetics of collapse of the trapezial gap following trapezectomy. PMID- 1705134 TI - Transfer of a composite island homodigital distal interphalangeal joint to replace the proximal interphalangeal joint. Technique and case report. AB - The authors present an original technique of PIP joint reconstruction by an homo digital island compound DIP joint transfer. The transfer consists of joint, capsule, volar plate, and ligaments. The distal joint is fused and the islanded joint interposed at the PIP level. In our clinical case, a flexion of 80 degrees was obtained with an extension lack of 15 degrees. This salvage procedure can be useful in selected well motivated patients. It provides rapid bone healing, good lateral stability, potential for growth in children, long term preservation of cartilage and acceptable range of motion. PMID- 1705135 TI - Occurrence of Dupuytren's disease beneath a full thickness skin graft: a semantic reappraisal. AB - Recurrence beneath a Wolfe graft is reported and discussed. While the dermofasciectomy appears able to inhibit myofibroblast proliferation where the graft is in direct contact with the graft bed, it is possible that, in the finger, the narrow strip behind the neurovascular bundle may, very rarely, escape this control and proceed to extension in depth. PMID- 1705136 TI - Dupuytren's contracture with associated changes in the plantar aponeuroses and in the auricular conchae. AB - The author reports a forty year old man presenting with Dupuytren's contracture affecting both hands, feet and auricular conchae. The patient is epileptic having been treated for twenty eight years with phenobarbitone and phenytoin. He had histologically proven Dupuytren's disease on both hands, but unfortunately no material was available from the ears. The follow up to date suggests that the auricular lesions have progressed slowly. PMID- 1705137 TI - Comparison of the monoamine oxidase inhibiting properties of two reversible and selective monoamine oxidase-A inhibitors moclobemide and toloxatone, and assessment of their effect on psychometric performance in healthy subjects. AB - 1. The effects of two reversible, predominantly monoamine oxidase-A (MAO-A) inhibitors, moclobemide (150 mg three times daily) and toloxatone (400-200-400 mg day-1) on monoamine metabolites and psychometric performance were compared in a double-blind placebo controlled crossover study in 12 healthy subjects. 2. After 7 days of moclobemide/toloxatone/placebo administration subjects were hospitalized for 24 h on day 8. Blood samples were drawn every 2 h for determination of plasma noradrenaline (NA), 3,4-dihydroxyphenylglycol (DHPG), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA). Urine was collected for measurements of normetanephrine and 3-methoxytyramine excretion. Psychometric performance (short- and long-term memory, critical flicker fusion frequency, choice reaction time) and subjective feelings were assessed before each drug intake (in the morning, at noon, in the evening). 3. Compared with placebo, both reversible monoamine oxidase inhibitors decreased the plasma concentration of DHPG and HVA. The overall fall in DHPG (AUC from 0 to 24 h) was 44% during moclobemide and 12% during toloxatone (P less than 0.001) and the overall decrease in HVA was 38% and 20% (P less than 0.005) on moclobemide and toloxatone, respectively. 4. Before the next drug intake, MAO-A inhibition, as judged by the decrease of plasma DHPG concentration, was significantly different from placebo with moclobemide but not with toloxatone. 5. Moclobemide, but not toloxatone, exerted a moderate, but significant inhibition of the deamination of 5-hydroxytryptamine (5-HT) as judged by the fall in plasma 5-HIAA concentration. Neither drug influenced plasma NA concentration. 6. A significant rise in urinary excretion of normetanephrine was observed on moclobemide and to a lesser extent on toloxatone. The urinary excretion of 3-methoxytyramine was significantly raised by moclobemide but not by toloxatone. 7. Neither moclobemide nor toloxatone altered memory function, vigilance, subjective feelings or sleep characteristics of the subjects. PMID- 1705138 TI - Differences in metabolite content between intact pancreases and their perchloric acid extracts. A 2D 1H/31P correlation NMR study. AB - Perchloric and hydrochloric acid extracts of intact pancreases from healthy rats and from rats with experimental acute pancreatitis were analysed using 2D 1H/31P correlation NMR spectroscopy. Major differences were found in the 2D maps between the extracts and the intact tissues. In the case of the intact diseased pancreas the prominent 31P signal at the phosphodiester region has been assigned in our previous work as lecithin/taurocholate complex. However, the signal found in the same chemical shift region in extracts of diseased pancreases as well as healthy ones, is identified here as the phosphodiester residue of oligoribonucleotides. In these extracts additional 31P signals were found and assigned as phosphomonoester and phosphodiester hydrolysis products of RNA. The amounts of these compounds, as a function of the acid concentrations were determined, and conditions for their minimization were defined. PMID- 1705139 TI - Effect of vitamin A on wound epidermis during forelimb regeneration in adult newts. AB - The effects of vitamin A on blastemal epidermis were studied during the early postamputational period of forelimb regeneration in Triturus alpestris. Vitamin A was administered through oral intubation at a dose of 250 IU per gram of body weight per day. The results were evaluated by morphometry, histology, and autoradiography. After 7, 11 and 14 days of treatment, several alterations were observed in the wound epidermis: a) reversal of keratinization; fewer keratinized cells were counted in sections from vitamin A-treated limbs; b) decrease in the incorporation of tritiated thymidine, as judged by estimation of labeling indices; c) increased mitotic activity in the cells of the stratum germinativum, and in the middle layer of the epithelial cells, as well. The significance of these cellular effects is discussed against the relevant literature. PMID- 1705140 TI - Inoperable non-small-cell lung cancer (NSCLC): a Medical Research Council randomised trial of palliative radiotherapy with two fractions or ten fractions. Report to the Medical Research Council by its Lung Cancer Working Party. AB - Two policies of palliative thoracic radiotherapy for non-small-cell lung cancer have been compared in a randomised multicentre controlled trial. A total of 369 patients with inoperable, histologically or cytologically confirmed disease, too advanced for radical 'curative' radiotherapy, and with their main symptoms related to the primary intrathoracic tumour even if metastases were present, were studied. They were allocated at random either to a regimen of 17 Gy given in two fractions of 8.5 Gy 1 week apart (F2 regimen), or to a conventional multifractionated regimen of either 30 Gy in ten fractions or 27 Gy in six fractions (a biologically equivalent dose), given daily except at weekends (FM regimen). On admission, 93% of the patients had cough, 47% haemoptysis, 57% chest pain, 58% anorexia, and 11% dysphagia. As assessed by the clinicians, palliation of the main symptoms was achieved in high proportions of patients ranging in the F2 group from 65% for cough to 81% for haemoptysis and in the FM group from 56% for cough to 86% for haemoptysis. Haemoptysis, chest pain, and anorexia disappeared for a time in well over half the patients with these symptoms, and cough in 37%. For all the main symptoms, the median duration of palliation was 50% or more of survival. Performance status improved in approximately half of the patients with a poor status on admission. All these results were similar in the two treatment groups. As assessed daily by the patients using a diary card, the quality of life deteriorated slightly during treatment but then improved steadily during the next 5 weeks. The proportion of patients with dysphagia increased considerably during treatment, but fell to the pretreatment level during the next 2 weeks. The results were similar in the two groups. Radiation myelopathy was suspected in one (F2) patient. There was no difference in survival between the two groups (log-rank test), the median survival time from the date of allocation being 179 days in the F2 and 177 days in the FM group. In the light of all the findings, the regimen of two fractions of 8.5 Gy given 1 week apart is recommended. PMID- 1705141 TI - Indole and porphyrin content of the Syrian hamster harderian glands during the proestrous and estrous phases of the estrous cycle. AB - Porphyrin and indole metabolism was studied in the Harderian glands of Syrian hamsters during the proestrous and estrous stages of the estrous cycle. Porphyrins remained unaltered during these stages, but levels of different indoles (5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, and 5-hydroxyindole acetic acid) exhibited pronounced changes during the dark:light period in both proestrous and estrous. There was a strong parallelism between 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine and 5-hydroxyindole acetic acid levels. Hydroxytryptophan rhythms appeared slightly shifted from those of the other indoles. Immunoreactive melatonin present in the Harderian glands did not show a significant day-night change during the stages studied. PMID- 1705142 TI - Partial purification of human prostatic 5 alpha-reductase (3-oxo-5 alpha steroid:NADP+ 4-ene-oxido-reductase; EC 1.3.1.22) in a stable and active form. AB - Human hyperplastic prostate tissue was homogenised in high ionic strength buffer and the post nuclear homogenate was incubated with 0.8% octyl glucoside and bovine brain lipids. Dialysis of the resulting liposome suspension yielded a preparation in which 5 alpha-reductase was active and stable for at least three weeks and showed an increase in specific activity (Vmax +/- SD = 48.9 +/- 7.4 pmol DHT/mg protein/ml) over that of the starting homogenate (Vmax +/- SD = 5.6 +/- 1.5 pmol DHT/mg protein/min) of 8.7 times. PMID- 1705143 TI - Putting the finger on DNA. Zinc Finger Gene Meeting sponsored by the Imperical Cancer Research Fund, London, UK, February 15-16, 1990. PMID- 1705144 TI - Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs. AB - The keratins 8 and 18 of simple epithelia differ from stratified epithelial keratins in tissue expression and regulation. To examine the specific properties of human keratin 8, we cloned and sequenced the cDNA from a placental mRNA expression library and defined the optimum state of such clones for expression in bacterial plasmid vectors. Using the polymerase chain reaction we identified and sequenced three introns and located the single active gene for keratin 8, out of a background of 9 to 24 pseudogenes, on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is thus far unique among the keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral keratin gene than the keratins of more highly differentiated epithelia. PMID- 1705145 TI - Rat small intestinal morphology and tissue regulatory peptides: effects of high dietary fat. AB - Sprague-Dawley rats (3 weeks old) were fed on isoenergetic diets in which 40% of the total energy was provided as fat either in the form of butter (high saturated fat), olive oil (high monounsaturated fat) or maize oil (high polyunsaturated fat), with one group on low-fat (10% of total energy) standard diet as a control. Animals were killed after 8.4 (se 0.8) weeks by cardiac puncture. Similar pieces of jejunum and ileum were prepared for morphometric studies. Extracts of tissue from the proximal and distal segments of the whole small intestine from four animals per group were assayed using established techniques for enteroglucagon, motilin, neurotensin, somatostatin, substance P and vasoactive intestinal peptide (VIP). We found that maize oil and olive oil increased villus height: crypt depth ratio in both jejunum and ileum. Maize oil increased tissue concentrations of somatostatin (P less than 0.05) and substance P (P less than 0.005) in the proximal segment. Both maize oil and olive oil increased tissue concentrations of neurotensin and substance P (P less than 0.005) in the distal segments. These observations may explain the improvement of intestinal absorption of fluid following supplementation with polyunsaturated fat. PMID- 1705146 TI - [Internalization and degradation of latrotoxin bound to rat brain synaptosomes]. AB - The accessibility to trypsin of 125I-labeled latrotoxin bound to rat brain synaptosomes was investigated. It was shown that latrotoxin bound to synaptosomes in the cold can be practically completely removed by trypsin treatment. The resistance of latrotoxin to proteolysis increases during its incubation with synaptosomes (37 degrees C). Concanavalin A (10(-6) M) decreases toxin binding by 30%, but fully prevents internalization (incorporation). Moreover, latrotoxin is not incorporated into synaptosomal membrane fragments irrespective of duration and temperature of incubation. Latrotoxin incorporated into synaptosomal membranes undergoes degradation by endogenous proteases resulting in the formation of TCA-soluble products. PMID- 1705147 TI - [N-tris-(beta-D-galactopyranosyloxymethyl)glycine methylamide as an antigenic determinant of neoglycoproteins]. AB - A chemical synthesis of N-tris (beta-D-galactopyranosyloxymethyl) glycine methylamide (trisgalactosylglycine) has been carried out. Trisgalactosylglicine derivatives of bovine serum albumin, ovalbumin and amyloglucosidase from Aspergillus have been obtained. The binding of trisgalactosylglycine residues to bovine serum albumin and ovalbumin was performed by the carbodiimide method; amyloglucosidase galactosylation was performed by using the reductive amination method. The latter technique seems to be the most mild one because it does not interfere with the peptide structure of the protein being analyzed. The antiserum specifically raised against the trisgalactosylglycine derivative of bovine serum albumin as well as the monospecific antibodies isolated from it can interact with both the antigen and the trisgalactosylglycine derivatives of ovalbumin and amyloglucosidase. Native proteins are not precipitated with this antiserum. This suggests that the trigalactosylglycine residues (bovine serum albumin, ovalbumin, amyloglucosidase) covalently bound to various proteins act as immunologic determinants regardless of the mode of their binding. PMID- 1705148 TI - Alpha-fetoprotein in congenital hypothyroidism. AB - alpha-Fetoprotein (alpha-FP) was measured in dried blood spots from normal, congenital hypothyroid (CH) and transient hyperthyrotropinemic (TH) newborns as well as in serum from CH and TH babies together with thyroxine, triiodothyronine and thyrotropin. The half-life of alpha-FP had a median value of 12 days in the CH cases and 4.9 days in the TH cases. alpha-FP was significantly higher in the CH group before treatment and showed a significant rise after discontinuation of thyroxine therapy. It would appear that thyroid hormones influence alpha-FP metabolism and that a hypothyroid environment results in increased alpha-FP levels. PMID- 1705149 TI - [Primary trigemino-cerebellar afferents demonstrated by the method of fluorescent retrograde double labeling in rats]. AB - Two axonally transported fluorescent substances, Granular Blue and Nuclear Yellow, were applied in the mandibular nerve and in the entire half of ipsilateral cerebellar cortex respectively. Observations of serial slices by using fluorescent photonic microscope showed double marked cells in the caudal part of homolateral trigemino-mesencephalic nucleus. Cells body had a 28-35 microns diameter and showed an oval-shaped and a pseudo-unipolar type. Numbers of neurons were no more than 10-15 per animal. There was no indication of double coloured cells either in the trigeminal ganglion or in the adjacent bulbo reticulo-pontic regions. It is concluded that identified neurons have their origin into periodontical structures. They bring the proof of non-vestibular direct cerebellar pathways. PMID- 1705150 TI - Enzyme degradation, high performance liquid chromatography and liquid secondary ion mass spectrometry in the analysis of glycoproteins. AB - Analysis of small amounts of glycoproteins by high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS) together with enzyme digestion has been investigated using fetuin as a model. Preliminary data indicates that 71% of the expected peptides were detected by LSIMS analysis of 200 pmol total digest. HPLC profiles of peptides and glycopeptides were obtained from 2 nmol of digest using a reversed phase (C18) column eluted in a solvent system containing TFA, water and acetonitrile. This has provided glycopeptides for subsequent oligosaccharide analysis. Strategies are reviewed for the chromatographic characterization of oligosaccharides following their release from glycopeptides by chemical and enzymatic procedures. PMID- 1705151 TI - The clinical efficacy of citalopram in treatment of emotional disturbances in dementia disorders. A Nordic multicentre study. AB - In this multicenter study, the clinical efficacy of citalopram was investigated in 98 patients with moderate AD/SDAT or VD using a combined double-blind and open technique with placebo and citalopram. Analyses were made for each diagnosis after four weeks of double-blind treatment. Patients with AD/SDAT treated with citalopram showed a significant improvement in emotional bluntness, confusion, irritability, anxiety, fear/panic, depressed mood and restlessness. Those improvements were not found after treatment with placebo. There were no significant improvements in patients with VD. No improvements were recorded in motor or cognitive impairment. Citalopram provoked few and comparatively mild side-effects. None of the changes observed during the double-blind withdrawal period were identified as withdrawal symptoms or rebound phenomena. PMID- 1705152 TI - The role of serotonin in non-cholinergic excitatory transmission in the guinea pig inferior mesenteric ganglion. AB - The non-cholinergic late slow excitatory postsynaptic potential (ls-EPSP) of the guinea pig inferior mesenteric ganglion (IMG) was previously believed to be mediated by substance P (SP) or several other neuropeptides. Yet, the pharmacological evidence presented here indicates that serotonin (5-HT) may be another transmitter for the ls-EPSP in the guinea pig IMG. Repetitive stimulation of the presynaptic nerves elicited ls-EPSP in about half of the IMG neurons. Application of 5-HT or SP caused, in a portion of the IMG neurons, a slow depolarization similar to ls-EPSP. Fifty-six out of 88 (63.6%) neurons with ls EPSP and 13 out of 35 (37.1%) neurons with ls-EPSP were sensitive to 5-HT and SP, respectively. Superfusion of the ganglia with 5-HT markedly suppressed the ls EPSP evoked in 5-HT sensitive neurons. Similarly, exogenously applied SP attenuated the ls-EPSP of SP-sensitive neurons. However, prolonged superfusion of 5-HT or SP had no effect on the ls-EPSP elicited in 5-HT or SP-insensitive neurons, respectively. Furthermore, the ls-EPSPs elicited in 5-HT-sensitive neurons as well as the 5-HT-induced depolarization were reversibly suppressed by cyproheptadine, a 5-HT antagonist, and enhanced by fluoxetine, a 5-HT reuptake inhibitor. In contrast, the ls-EPSP of 5-HT insensitive neurons and SP-induced depolarization were not appreciably changed by those two drugs. Pretreatment with p-chlorophenylalanine, a 5-HT biosynthesis inhibitor, did not change the general electrophysiological characteristics of the neurons and did not suppress nicotinic neurotransmission, but markedly reduced the occurrence rate of ls-EPSP from 53.8% to 15.1% (P less than 0.005). Collectively, our results indicate that, besides SP, 5-HT may be involved in mediating the ls-EPSP in a subpopulation of neurons in the guinea pig IMG. The type of transmitter mediating ls-EPSP is apparently not limited to 5-HT and SP, as about 30% of the neurons with ls-EPSP were found to be insensitive to both 5-HT and SP and prolonged superfusion with both did not affect appreciably the ls-EPSP elicited in these neurons. PMID- 1705153 TI - Exposure to excess glucocorticoids alters dendritic morphology of adult hippocampal pyramidal neurons. AB - We have used Golgi-impregnated tissue to demonstrate that exposure to excess glucocorticoids alters dendritic morphology in a specific population of neurons in the adult rat hippocampus. Daily injection of 10 mg of corticosterone for 21 days resulted in decreased numbers of apical dendritic branch points and decreased total apical dendritic length measured in a 100-microns-thick section in CA3 pyramidal cells compared to sham-injected and non-injected controls. In contrast, no changes were observed in CA3 pyramidal cell basal dendritic morphology. Furthermore, no changes were observed in the dendritic morphology of CA1 pyramidal cells or granule cells of the dentate gyrus. Cross-sectional cell body area of any of the 3 cell types examined in this study was unaffected by corticosterone treatment. Finally, qualitative analysis of Nissl-stained tissue from the same brains revealed increased numbers of darkly staining, apparently shrunken CA3 pyramidal cells in corticosterone treated compared to control brains. The changes in dendritic morphology we have observed may be indicative of neurons in the early stages of degeneration, as prolonged exposure to high levels of corticosterone has been shown by others to result in a loss of CA3 pyramidal cells. Additionally, these results suggest possible structural alterations which may occur under physiological conditions in which corticosterone levels are chronically elevated such as in aged animals. PMID- 1705154 TI - Autoradiographic distribution of brain neurokinin-1/substance P receptors using a highly selective ligand [3H]-[Sar9,Met(O2)11]-substance P. AB - The autoradiographic distribution of neurokinin (NK)-1 receptors was visualized in the rat brain using the highly selective ligand, [3H]-[Sar9,Met(O2)11] substance P. This ligand apparently binds to a single class of high affinity (Kd = 1.4 +/- 0.5 nM), low capacity (Bmax = 160 +/- 3.0 fmol/mg protein) sites in rat brain membrane preparations. The ligand selectivity profile reveals that substance P (SP) and unlabeled [Sar9,Met(O2)11]-SP are potent competitors of [3H] [Sar9,Met(O2)11]-SP binding while NK-2 and NK-3 analogues are virtually inactive demonstrating the selectivity of this radioligand for the NK-1 receptor class. Autoradiographic data show that [3H]-[Sar9,Met(O2)11]-SP binding sites are broadly but discretely distributed in rat brain, the highest densities of sites being located in the external plexiform layer of the olfactory bulb, striatum, olfactory tubercule, amygdala-hippocampal area, endopiriform and entorhinal cortices, superior colliculus, locus coeruleus and substantia gelatinosa of the spinal cord. This distribution is similar, but not identical, to that previously reported for NK-1 sites using less selective ligands such as [125I]Bolton-Hunter SP. For example, some difference in labelling patterns are observed in the hippocampal formation. This could be explained by the existence of NK-1 receptor subtypes, only one of them being recognized by [3H]-[Sar9,Met(O2)11]-SP or by the greater selectivity of this radioligand for NK-1 over NK-2 and NK-3 receptor classes. PMID- 1705155 TI - n-hexane induces parkinsonism in rodents. AB - A case of human parkinsonism, due to n-hexane exposure, was recently described. On the basis of this observation, we treated mice and rats with n-hexane and its principle toxic metabolite 2,5-hexanedione. The mice underwent a chronic treatment intraperitoneum, the rats were treated stereotaxically into the substantia nigra. At biochemical analysis of the striata, dopamine and homovanillic acid levels were significantly lower compared with control animals; norepinephrine, serotonin, 5-hydroxindolacetic acid levels were unchanged. The rats treated with 2,5-hexanedione showed an apomorphine-induced rotational behavior significantly higher compared to controls. Since n-hexane and its metabolites are environmental contaminants and by-products of endogenous metabolic pathways, we propose that they may play a role in inducing parkinsonism in humans. PMID- 1705156 TI - Repeated electroconvulsive shock increases glial fibrillary acidic protein, ornithine decarboxylase, somatostatin and cholecystokinin immunoreactivities in the hippocampal formation of the rat. AB - Rats were submitted to single or repeated (7 days, one session for each day) sessions of electroconvulsive shock. A computer-assisted morphometric and microdensitometric analysis of glial fibrillary acidic protein-, ornithine decarboxylase-, somatostatin- and cholecystokinin-like immunoreactivities was performed in the hippocampal formation and other brain areas. The results of the study showed a significant increase of the intensity of the immunostaining for glial fibrillary acidic protein, ornithine decarboxylase, somatostatin and cholecystokinin in the hippocampal formation and distinctively in the dentate gyrus following repeated, but not single, electroconvulsive shock. No significant change was found in the number of somatostatin- and cholecystokinin-like immunoreactive cell bodies in any hippocampal subregion and in the number of glial cells in the hilus of dentate gyrus in rats treated with single or repeated electroconvulsive shock. It is a distinct possibility that the observed increase in the content of the neuropeptides in the hippocampal formation reflects a compensatory response of the brain to seizure-inducing stimuli and that such an increase may play a role in the therapeutic effect of electroconvulsive shock. PMID- 1705157 TI - Evidence that Fluoro-Gold can be transported avidly through fibers of passage. AB - Small iontophoretic injections of the retrograde tracer Fluoro-Gold were restricted to the dorsal columns in the cervical enlargement of 6 rats. Large numbers of neurons were labeled in the lumbosacral dorsal horn in each rat. In the most effective case, more than 1800 neurons were labeled in alternate sections through nine examined segments. Many neurons were also labeled in lumbosacral dorsal root ganglia of all cases. This study, in contrast to previous reports, indicates that Fluoro-Gold can be transported avidly by axons passing through, but not terminating in, injection sites. PMID- 1705158 TI - Immunoreactivity for glutamic acid decarboxylase and several neuropeptides in the spinal cord of the raccoon. AB - The peroxidase-antiperoxidase method was used to examine major immunohistochemical features of the spinal cord of adult raccoons. The lateral portions of the ventral horn contained many large multipolar neurons that showed cholecystokinin-like immunoreactivity, suggesting the coexistence of cholecystokinin with acetylcholine in a subset of motoneurons. The dorsal horn revealed unique but overlapping patterns of immunoreactivity for glutamic acid decarboxylase, somatostatin, substance P, vasoactive intestinal polypeptide and cholecystokinin. The data imply that some of the peptides may coexist within the same dorsal root ganglion cells and their spinal cord processes. PMID- 1705159 TI - A substance P projection from the VMH to the dorsal midbrain central gray: implication for lordosis. AB - Substance P has been implicated in the modulation of lordosis behavior at the level of the dorsal midbrain central gray (dMCG). Bilateral injections of substance P into the dMCG facilitate estrogen-induced lordosis behavior in ovariectomized female rats. Input from the ventromedial nucleus of the hypothalamus (VMH) to the dMCG is a vital link in the central nervous system control that mediates the expression of lordosis behavior. Substance P-containing cells have been localized in the VMH and substance P binding sites are localized in the dMCG; this suggested to us that substance P neurons originating in the VMH may terminate in the dMCG. The present study examined the projection of substance P-immunoreactive neurons (SP-IR) in the VMH to the dMCG. The present study examined the projection of substance P-immunoreactive neurons (SP-IR) in the VMH to the dMCG. The retrograde tract tracer fluorogold revealed cell bodies throughout the extent of the VMH and sP immunofluorescence labelled a subpopulation of these cells particularly in the ventrolateral part of the VMH. The majority of sP-projection cells was localized in the caudal two-thirds of the VMH. Thirteen percent of the sP-IR cells were observed to project to the dMCG, while approximately 17% of the sP-IR cells of the ventrolateral part of the VMH projected to the dMCG. These results provide morphological evidence for a substance P projection from the VMH to an area where substance P has been demonstrated to facilitate lordosis behavior.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705160 TI - Peptide displacement of [3H]5-hydroxytryptamine binding to bovine cortical membranes. AB - Chemical studies have demonstrated that peptides such as the encephalitogenic (EAE) peptide of myelin basic protein (MBP) and luteinizing hormone-releasing hormone (LHRH) can bind serotonin (5-hydroxytryptamine, 5-HT) in vitro. The present research was undertaken to determine whether such binding interferes with 5-HT binding to its 5-HT1 receptors on bovine cerebral cortical membranes. EAE peptide and LHRH displaced [3H]5-HT with IC50s of 4.0 x 10(-4) and 1.8 x 10(-3) M respectively. MBP itself also showed apparent displacing ability with an IC50 of 6.0 x 10(-5) M, though it also caused aggregation of cortical membranes that might have interfered with normal receptor binding. These results support previous suggestions that the tryptophan peptide region of MBP may act as a 5-HT receptor in the neural system. We also tested the effects of muramyl dipeptide (N acetyl-muramyl-L-Ala-D-isoGln, MD), a bacterial cell-wall breakdown product that acts as a slow-wave sleep promoter, binds to LHRH and EAE peptide, and competes for 5-HT binding sites on macrophages. It showed no significant displacement of 5 HT binding to cortical membranes (IC50 greater than 10(-1) M), but its D-Ala analogue did (IC50 = 1.7 x 10(-3) M). Thus, it seems likely that the 5-HT-related effects of naturally occurring muramyl peptides are physiologically limited by receptor types. PMID- 1705161 TI - Diurnal variations in the feeding responses to norepinephrine, neuropeptide Y and galanin in the PVN. AB - The feeding responses elicited by injection of norepinephrine (NE), neuropeptide Y (NPY) and galanin (GAL) into the paraventricular nucleus (PVN) were studied at two different times of the dark (active) cycle in male Sprague-Dawley rats maintained ad lib on pure nutrient diets. The feeding response elicited by NE in the PVN, characterized by a potent and selective stimulatory effect on ingestion of the carbohydrate diet, was significantly stronger during the early dark period (+11.7 kcal over vehicle baseline) relative to the late dark period (+7.6 kcal). A similar pattern of effects was observed with NPY in the PVN, which also selectively potentiated carbohydrate ingestion. The effects of GAL were different from those observed with NE and NPY. Whereas the total amount of food consumed after PVN GAL injection was similar in the early and late dark periods, the macronutrient selection patterns exhibited at these two times were different. During the early dark period, PVN GAL had a small stimulatory effect on carbohydrate, in addition to a strong enhancement of fat intake; in the late dark period, in contrast, GAL stimulated intake only of the fat diet. These findings may reflect differential functions of these hypothalamic neurotransmitters in controlling nutrient ingestion at different periods of the circadian cycle. PMID- 1705162 TI - Serotonin binding sites. II. Muramyl dipeptide binds to serotonin binding sites on myelin basic protein, LHRH, and MSH-ACTH 4-10. AB - Previously, we reported the existence of structurally similar serotonin binding sites on myelin basic protein, LHRH, and MSH-ACTH 4-10. We now report that the adjuvant peptide, muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln) also binds to these sites. This observation may help to explain previous observations of serotonin-like activity by muramyl peptides, including the promotion of slow-wave sleep and fever induction. The observation may also provide an important link between the immune system and the nervous system that may explain the role of muramyl dipeptide adjuvants in causing autoimmune diseases to serotonin-regulated proteins and their receptors, as well as the alterations in serotonin levels that are often observed in autoimmune diseases. The observation provides concrete evidence for a dual-antigen hypothesis for the induction of autoimmune diseases by an adjuvant-peptide complex. Application of such a mechanism for induction of autoimmunity may be of importance in understanding a number of postinfectious and postvaccinal neuropathies, and suggests a possible etiology for autism, in which many patients have high blood serotonin levels, autoimmune reactions to myelin basic protein, and antibodies to serotonin binding sites. Finally, the observation suggests that glycopeptides may act as neurotransmitters. PMID- 1705163 TI - Cholecystokinin-induced variations in hypothalamic serotonergic system of the "cafeteria" rat. AB - Cholecystokinin octapeptide sulfate (CCK 8 S) appears to act via a serotonergic mechanism on several behavioral paradigms, including satiety. In the present study, CCK 8 S was found to induce slight nonsignificant serotonergic changes in hypothalamic nuclei of the normal rat. In the "cafeteria" rat, however, it increased both 5-HT and 5-HIAA levels in the ventromedial hypothalamus (VMH), 5 HT levels in the paraventricular nucleus (PVN) and decreased 5-HIAA levels in the dorsomedial hypothalamus (DMH). These data suggest that the primary site of action of CCK 8 S is in the median hypothalamus, contrasting with an absence of effect in the lateral hypothalamus (LH). The involvement of 5-HT in the effect of CCK 8 S is further suggested. However, the relationship between these neurochemical changes and CCK 8 S-induced satiety is not clear. Nonetheless, a special sensitivity to CCK 8 S of the obese "cafeteria" rat is evidenced, which contrasts with a reduced response of genetic obesity models. PMID- 1705164 TI - Beta 1 integrin expression on human small cell lung cancer cells. AB - The integrins are a supergene family of cell surface glycoproteins that promote cellular adhesion. Each member of the family is an alpha/beta heterodimer composed of a distinct alpha subunit noncovalently linked to one of at least six common beta subunits. These include the six beta 1 integrins (alpha 1-6/beta 1) which represent receptors for extracellular matrix proteins and the three beta 2 integrins (alpha L, alpha M, alpha X/beta 2) that are expressed by leukocytes and which bind to C3bi and/or endothelial ligands. Recently, it was reported that certain human tumor cells express the beta 1 integrins and that small cell lung cancer (SCLC) cell lines express the beta 2 integrin Mo1 (alpha M/beta 2). To extend these initial observations, we examined SCLC cell lines for integrin expression at the glycoprotein and mRNA levels and assessed the potential function of these integrins in promoting SCLC adhesion. An indirect immunofluorescence analysis of five SCLC cell lines (NCI-H187, H345, H146, H209, and N417) using alpha and beta subunit-specific monoclonal antibodies demonstrated the uniform expression of beta 1 (beta 1 much greater than beta 2 greater than or equal to beta 3 congruent to beta 4). Among the beta 1-associated alpha subunits, alpha 3 was uniformly expressed at high surface density by all five cell lines (as confirmed in H345 cells by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of anti-beta 1 and anti-alpha 3 immunoprecipitates), while alpha 5 was not detected. The leukocyte (beta 2 associated) alpha M and alpha L subunits were also variably expressed by the five lines. Consistent with the surface expression of beta 1 integrin gene products, beta 1 (but not beta 2) mRNA was detected in SCLC cells by Northern blot analysis. That beta 1 integrin expression was involved in SCLC adhesion was suggested by the adherence of H345 cells to laminin, a known ligand for the alpha 3 beta 1 integrin. Moreover, an antibody specific for the beta 1 subunit inhibited this adhesion, indicating that the beta 1 subunit promotes adhesion to laminin. We conclude that beta 1 integrin molecules are expressed by human SCLC cells (with uniform expression of alpha 3/beta 1) and promote their adhesion to laminin. PMID- 1705165 TI - Phase I clinical trial of fazarabine as a twenty-four-hour continuous infusion. AB - A phase I trial of fazarabine (ara-AC, 1-beta-D-arabinofuranosyl-5-azacytosine, NSC 281272) administered as a 24-h continuous infusion was performed in 24 adults with solid tumor malignancies. The majority of patients had received prior marrow suppressive therapy. Level 7 (54.5 mg/m2/h for 24 h) was the maximum tolerated dose since during 6 evaluable first courses, 2 episodes of grade 4 granulocytopenia and 3 episodes of grade 3 occurred. Moderate thrombocytopenia also occurred at level 7 with 3 episodes of grade 1 and 1 episode of grade 4 thrombocytopenia during 6 first course treatments. Minimal myelosuppression, principally leukopenia, was seen prior to level 7. The nadir WBC through 47 courses had a linear relationship with plasma steady-state concentrations of ara AC. The only other toxicity noted was moderate nausea/vomiting, which did not appear to be dose related. Plasma steady-state concentrations of ara-AC were reached in all patients within 4-6 h and ranged from 1.1 microM (11 mg/m2/h for 24 h) to 7.5 microM (54.5 mg/m2/h for 24 h). The mean total body clearance of ara AC for 47 courses, levels 1-7, was 592 +/- 147 (SD) ml/min/m2 which is similar to prior pharmacokinetic data from the 24-h and 72-h infusion trials of the Pediatric and Medicine Branches, respectively. There were no objective disease responses during the trial. The recommended adult phase II dose for a 24-h infusion of ara-AC is 45-50 mg/m2/h. PMID- 1705166 TI - Role of natural killer and T-cells in interferon induced inhibition of spontaneous metastases of the B16F10L murine melanoma. AB - We have previously demonstrated that interferon (IFN) treatment of mice bearing the spontaneously metastasizing B16F10L murine melanoma on days -5 to -1 prior to surgical removal (day 0) of the primary tumor resulted in survival of greater than 50% of treated mice. The antitumor effect was correlated with an early increase of natural killer (NK) cell cytotoxicity followed by a later developing specific cytolytic T-cell response. The purpose of this study was to establish definitively the roles of NK, CD4, and CD8 cells as mediators of the antitumor/antimetastatic effects of IFN treatment by administration of anti asialo-GM1, anti-L3T4, and/or anti-Lyt-2 antisera. Depletion of NK cells, alone or in combination with T-cells, eliminated the protective effect of IFN treatment. Depletion of both CD4 and CD8 cells, however, did not significantly alter the therapeutic effect of IFN therapy. Collectively, these data conclusively demonstrated the importance of NK cells as mediators of the IFN induced antitumor state. However, in mice depleted of CD4 cells alone, the protective effect of IFN was eliminated, in spite of the presence of intact NK cells. In vitro analysis of NK cytotoxicity on day 1 after surgery demonstrated (a) a lack of IFN induced stimulation of NK activity in CD4 depleted mice and (b) a significant increase in both baseline and IFN induced NK cytotoxicity in CD8 depleted mice. These data suggested a CD8 cell mediated inhibition of NK activity/stimulation in CD4 depleted mice, possibly responsible for the lack of response to IFN therapy in that group. These results demonstrate the importance of not only individual components of the immune system but also the interaction of these components in both the natural and IFN induced control of spontaneous B16F10L metastases. PMID- 1705167 TI - Antagonism between camptothecin and topoisomerase II-directed chemotherapeutic agents in a human leukemia cell line. AB - To search for possible synergy between topoisomerase (topo) II-directed chemotherapeutic agents and topo I-directed agents, IL-60 human progranulocytic leukemia cells were incubated with etoposide in the absence or presence of camptothecin (CPT). Treatment of HL-60 cells for 1 h with 15-20 microM etoposide resulted in the death of 99-99.9% of the cells as assessed by colony formation in soft agar. Unexpectedly, simultaneous incubation with 1 microM CPT increased the survival of etoposide-treated cells as much as 30-fold. Inhibition of etoposide cytotoxicity was observed at CPT concentrations as low as 0.01 microM and was one half maximal at 0.1 microM. CPT also antagonized the cytotoxicity of 4'-(9 acridinylamino)methanesulfon-M-anisidide and daunorubicin, two structurally unrelated topo II-directed agents. Topotecan, a CPT analogue currently undergoing Phase I clinical trials, had a similar effect. Studies using an alkaline unwinding assay (to measure DNA strand breaks) and Western blotting (to assess formation of covalent adducts involving topo II) revealed that CPT did not alter the ability of etoposide to stabilize topo II-DNA adducts. CPT is a potent inhibitor of both DNA and RNA synthesis. To further assess the mechanism by which CPT diminished the cytotoxicity of topo II-directed agents, inhibitors of DNA synthesis or RNA synthesis were substituted for CPT. Aphidicolin, an inhibitor of replicative DNA polymerases, enhanced the survival of etoposide-treated HL-60 cells less than 3-fold. In contrast, inhibitors of RNA synthesis (cordycepin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) enhanced the survival of etoposide-treated HL-60 cells as much as 20-fold. The potential biological and therapeutic implications of these results are discussed. PMID- 1705168 TI - Testing for induction of DNA synthesis in human hepatocyte primary cultures by rat liver tumor promoters. AB - Parenchymal liver cells were isolated from human liver pieces of surgical waste as well as from rat livers. DNA synthetic activity was measured after different times in primary culture by [3H]thymidine incorporation and autoradiography. Labeling of control cultures of human hepatocytes at densities between 8,000 and 15,000 cells/cm2 was very low (0.4 to 1.3%). Human recombinant epidermal growth factor increased labeling 2- to 4-fold (P less than 0.01). Treatment with known inducers of liver growth in rats, namely, cyproterone acetate, alpha hexachlorocyclohexane, nafenopin, phenobarbital, and rifampicin did not increase the number of labeled human liver cells. In some of the experiments, a 24-h exposure to the chemicals of rat or human hepatocytes was followed by a 24-h treatment with epidermal growth factor (EGF). In rat hepatocytes, incorporation rates were significantly increased. Cyproterone acetate and EGF acted in an additive manner, alpha-hexachlorocyclohexane and EGF were clearly overadditive, and phenobarbital had little effect. In human hepatocytes, little alteration in labeling indices was found; in some cases labeling was, rather, found to be lower than in cultures treated with EGF alone. These results show that human hepatocytes cultured in vitro are sensitive to stimulation of DNA synthesis by EGF; they differ from rat hepatocytes in their response to some drugs which show liver growth-promoting activity in rodents. PMID- 1705169 TI - Differential glucocorticoid regulation of glucagon gene expression in cell lines derived from rat and hamster islet cell tumors. AB - Glucocorticoid regulation of peptide hormone gene expression was studied in two cell lines derived from rodent islet cell tumors. In rat RIN1056A cells, dexamethasone reduced the levels of glucagon mRNA transcripts while markedly inducing the expression of the angiotensinogen gene. In contrast, dexamethasone had no effect on the regulation of glucagon gene expression in hamster InR1-G9 cells. Wild type InR1-G9 cells did not support the induction of the murine mammary tumor virus promoter by glucocorticoids, suggesting that these cells lacked the necessary cellular factor(s) for glucocorticoid responsiveness. Introduction of the glucocorticoid receptor into wild type InR1-G9 cells restored glucocorticoid induction of the murine mammary tumor virus promoter, but not glucocorticoid regulation of glucagon gene expression. Dexamethasone treatment of Sprague-Dawley rats had no effect on the levels of pancreatic glucagon mRNA transcripts. The results of these studies demonstrate that glucocorticoid regulation of glucagon gene expression is restricted to the immortalized RIN1056A cell line, providing additional evidence for cell-specific diversity in the regulation of peptide hormone gene expression in neuroendocrine tumors. PMID- 1705170 TI - Morphometric and colorimetric analyses of human tumor cell line growth and drug sensitivity in soft agar culture. AB - Previous studies have demonstrated the suitability of image analysis of tetrazolium-stained colonies to assess growth and drug sensitivity of human tumor cells cultivated in soft agar culture. In the present study, the potential utility of colorimetric analysis to expedite experimental drug evaluations using human tumor cell lines was investigated. The same culture dishes were assessed by image analysis and by formazan colorimetry for purposes of comparing multiple methods of measuring growth as well as growth inhibition. Replicate cultures treated with 2-(p-iodonitrophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide exhibited nearly identical colony count and volume indices as well as excellent correlation in colorimetric end points. Colony-forming unit volume analysis versus colorimetric assessment of the same cultures following dimethyl sulfoxide extraction of protamine sulfate-rinsed, dried soft agar cultures exhibited excellent linear correlation for both growth (Pearson r ranging from 0.95 to 1.00) and drug sensitivity (Pearson r ranging from 0.90 to 0.99, and Spearman r ranging from 0.82 to 0.97) and similar drug sensitivity profiles. Results of the current investigation indicate that end points of soft agar culture remain stable for a period of at least 2 weeks following assay termination. In addition, a colorimetric detection range of 1.3-2.2 log units permits determinations of survival levels ranging from 100 to 5% of respective control levels. Colorimetric analysis is anticipated to expedite soft agar colony formation assay evaluations (a) by reducing the need to use the more rigorous and time-consuming image analysis procedures to measure activity in preliminary drug sensitivity assays and (b) by permitting the determination of effective concentration ranges of new experimental agents for subsequent, more detailed investigations. PMID- 1705171 TI - Expression of the natural killer cell-associated antigens CD56 and CD57 in human neural and striated muscle cells and in their tumors. AB - The expression of the natural killer cell-associated antigens CD56 and CD57 which are known to show sequence homologies to the neural cell adhesion molecule N-CAM was examined immunohistochemically in normal and regenerative human neural and straited muscle cells and in their tumors using monoclonal antibodies Leu-19 and Leu-7. The pattern of expression of CD56 and CD57 in neural tissue was assessed in comparison with that of Mr 68,000 neurofilament. Dendritic interstitial cells were discriminated from neural cells by application of the pan leukocyte antigen CD53. In normal tissue, CD56 was expressed in thin nerve fibers, fine varicose and sensory nerve endings, cell membranes of ganglion cells, and fetal striated muscle cells. Thick nerve fibers, perikarya of ganglion cells, and adult striated muscle fibers were CD56 negative. In the normal state, CD57 was restricted to thick nerve fibers. Enhanced expression or reexpression of CD56 was found in regenerative neural cells which, in part, were also CD57 positive and in regenerative, CD57-negative striated muscle cells. In neural tumors, CD56 was detectable in 3 of 3 benign and 8 of 13 malignant schwannomas, 1 of 4 peripheral neuroepitheliomas, 4 of 4 ganglioneuromas, and 8 of 8 (ganglio-)neuroblastomas, whereas CD57 was restricted to one benign and one malignant schwannoma. Furthermore, CD56, but not CD57, could be found in 8 of 8 rhabdomyosarcomas. All in all, the pattern of expression of CD56 is comparable to that of N-CAM. CD57 exhibits a very restricted binding pattern which, in most instances, is complementary to that of CD56. The absence of CD56 in some neural tumors might reflect alterations in cell-cell interactions. Its reexpression in regenerative striated muscle cells and in rhabdomyosarcomas suggests a role of CD56 as an oncodevelopmental antigen. PMID- 1705172 TI - Monoclonal antibody PD41 recognizes an antigen restricted to prostate adenocarcinomas. AB - A monoclonal antibody (MAb) designated PD41 (IgG1k) was generated by hyperimmunizing BALB/c mice with a membrane preparation prepared from a moderately to poorly differentiated prostate carcinoma surgical specimen. The immunohistochemical reactivity of MAb PD41 was shown to be highly restricted to the ductal epithelia and secretions of prostate adenocarcinoma tissues. Sixty five % of the prostate tumor specimens were stained with MAb PD41, whereas no staining of the fetal or benign prostate specimens was observed. PD41 reacted minimally with normal prostate tissues, with less than 1% of the epithelial cells staining. This MAb did not react with nonprostate carcinomas or to a variety of normal human tissues. Using both radioimmunoassay and immunofluorescent procedures, several cultured human tumor cell lines, human blood cells, and purified antigens to prostate-specific antigen and prostatic acid phosphatase also were found not to express the PD41 antigen. MAb PD41 also was shown to bind to the target antigen present in seminal plasma obtained from prostate carcinoma patients but not to seminal plasma from normal donors. Immunoblots of gel separated components of prostate carcinoma tissue extracts indicate that the molecular weight of the proteins carrying the PD41 antigenic determinant can differ among individual tumors, ranging from Mr 90,000 to greater than 400,000. However, in seminal plasma from prostate cancer patients, the predominant component recognized by PD41 is the diffuse Mr greater than 400,000 band. It appears that this monoclonal antibody may recognize a prostate carcinoma associated mucin-like antigen, which is preferentially expressed on prostate carcinomas, and therefore, may be a useful marker to distinguish benign prostate hyperplasia from prostate carcinoma. PMID- 1705173 TI - A quantitative in vivo mouse model used to assay inhibitors of tumor-induced angiogenesis. AB - An in vivo model of tumor-induced angiogenesis was used to monitor two known inhibitors of angiogenesis, protamine sulfate and the steroid tetrahydro S. Tumor cells entrapped in alginate beads were injected s.c. into mice. Blood vessel induction was measured by two quantitative methods: measurement of hemoglobin at the alginate pellet site, and pooling of radiolabeled RBC to the alginate pellet site. The two methods gave parallel results. Tetrahydro S with or without heparin inhibited blood vessel growth by 50%, and protamine sulfate inhibited blood vessel growth by 85%. These results were supported by gross morphology and histological analysis of the alginate pellet site. PMID- 1705174 TI - Expression of angiogenic growth factor genes in primary human astrocytomas may contribute to their growth and progression. AB - Astrocytomas are highly malignant brain tumors and are among the most neovascularized solid tumors. We have investigated the expression of the angiogenic growth factors acidic fibroblast growth factor and transforming growth factor-alpha, together with its receptor epidermal growth factor receptor, in 30 primary astrocytomas. Both acidic fibroblast growth factor and transforming growth factor-alpha, together with epidermal growth factor receptor, are found to be greatly overexpressed in these tumors when compared with normal brain. This overexpression of angiogenic growth factors may underlie the intense neovascularization characteristic of astrocytomas. PMID- 1705175 TI - Covalent binding of human alpha 2-macroglobulin to deglycosylated ricin A chain and its immunotoxins. AB - In this report we demonstrated that human alpha 2-macroglobulin (alpha 2M) reacts with deglycosylated ricin A chain (dgA) and its immunotoxins to form high molecular weight complexes (molecular mass approximately 800 kDa). This interaction has a t1/2 at 37 degrees C of 5 h and reaches completion at 24 h. Complexes of alpha 2M-dgA cannot be dissociated by guanidine, sodium dodecyl sulfate, or low pH, but can be partially dissociated by reducing agents, such as 2-mercaptoethanol in the presence of sodium dodecyl sulfate. This indicates that dgA or dgA-containing immunotoxins are bound to alpha 2M by disulfide bonds. The dgA-binding site on alpha 2M and the mechanism underlying its interaction with dgA are different from those described for proteases or methylamine. alpha 2M complexes do not bind to Blue-Sepharose 4B or anti-A chain-Sepharose, suggesting that the sites on dgA which bind Cibacron Blue or polyclonal anti-A chain antibodies are sterically blocked or modified by interaction with alpha 2M. The interaction of alpha 2M with dgA or its immunotoxins results in a 2- to 3-fold decrease in the activity of the dgA in both cell-free assays and cytotoxic assays. However 12 h after injection into mice, only 11% of immunotoxin was bound to alpha 2M because of the slow kinetics of the interaction versus the more rapid t1/2 of the immunotoxin in the circulation. PMID- 1705176 TI - Interleukin-2-induced lymphoproliferative responses. AB - Interleukin-2 (IL-2) is capable of both stimulating an in vitro lymphoproliferative response and augmenting non-major-histocompatibility-complex (MHC)-restricted cytotoxicity. However, there are conflicting reports about the phenotypes of responding cells. In the present studies, we determined phenotypes of Ficoll/Hypaque-separated peripheral blood mononuclear cells stimulated with 50, 100 or 1000 U/ml IL-2; analyses were performed after 1, 3 and 5 weeks. With all concentrations, there was a progressive increase in CD3+ cells: after 3-5 weeks more than 90% of the cells reacted with this antibody. However, the proportions of CD4+ and CD8+ cells proved to be a function of the IL-2 concentration. Cultures containing 50 U/ml or 100 U/ml favored the expansion of the CD4+ subset. By contrast, in cultures stimulated with 1000 U/ml. CD8+ cells predominated. At baseline, CD8+ cells comprised 28 +/- 2%; after 3 weeks, this value increased to 51 +/- 5%. In addition, the proportion of CD56+ (Leu19, NKH-1) cells depended on the amount of IL-2. At 50 U/ml, there was no appreciable change in CD56+ cells. However, at 1000 U/ml, CD56+ cells increased from 17 +/- 1% (day 0) to 39 +/- 4% (3 weeks). This increase was primarily due to an expansion of the CD3+ CD56+ subset (non-NMC restricted cytotoxic T cells). By contrast, natural killer (NK) cells, as measured by the CD16 antibody, steadily declined at all IL 2 concentrations. PMID- 1705177 TI - Analysis of natural killer activity and natural killer cell subsets in patients with bladder cancer. AB - In order to analyze the state of the natural resistance system of bladder cancer patients in vivo, we measured natural killer (NK) activity and NK cell subsets of peripheral blood lymphocytes (PBL) from 46 patients with bladder cancer and 25 age- and sex-matched healthy volunteers. The mean NK activity in patients with low-stage bladder cancer was similar to that in the controls, while NK activity in patients with high-stage bladder cancer was significantly depressed. The mean proportions of Leu7+ cells in patients with both low-stage and high-stage bladder cancer were significantly higher than that in the controls. The mean proportion of Leu11 a+ cells in patients with low-stage bladder cancer was similar to that in the controls, while in patients with high-stage bladder cancer it was significantly higher. This study demonstrates the abnormal immunological state of bladder cancer patients: namely, abnormalities exist not only in NK activity but also in the proportions of circulating NK cell subsets. PMID- 1705178 TI - A combined radiologic and endoscopic approach to removal of occluded double mushroom biliary endoprosthesis: technical note. AB - A combined radiologic and endoscopic approach to the removal of an occluded double-mushroom biliary endoprosthesis was successfully performed in a patient with terminal pancreatic carcinoma. The present technique is beneficial when there is limited percutaneous and endoscopic access to the biliary system. PMID- 1705180 TI - [The immune status in children with recurrent otitis media]. AB - The results of the research task revealed that the cause of relapsing otitis were in one third of the children various disorders of the immune system (primary and secondary). In the youngest children without disorders of immunity detectable by laboratory methods most probably retarded maturation of the specific antibody response is involved. In cases of confirmed immunity disorders, immunotherapy proved useful. PMID- 1705179 TI - Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. AB - The nature of involvement of the cystic fibrosis gene product (CFTR) in epithelial anion transport is not yet understood. We have expressed CFTR in Sf9 insect cells using the baculovirus expression vector system. Reactivity with antibodies against 12 different epitopes spanning the entire sequence suggested that the complete polypeptide chain was synthesized. Immunogold labeling showed localization to both cell-surface and intracellular membranes. Concomitant with CFTR expression, these cells exhibited a new cAMP-stimulated anion permeability. This conductance, monitored both by radioiodide efflux and patch clamping, strongly resembled that present in several CFTR-expressing human epithelial cells. These findings demonstrate that CFTR can function in heterologous nonepithelial cells and lend support to the possibility that CFTR may itself be a regulated anion channel. PMID- 1705181 TI - [Alpha-fetoprotein in umbilical cord blood and hyperbilirubinemia in normal neonates]. AB - In a group of 154 mature normal neonates the authors recorded significantly higher mean alpha-fetoprotein values in children with hyperbilirubinaemia (higher than 205 mumol/l) than in other neonates. The differences were observed also in children of equal gestation age. Development of hyperbilirubinaemia is more probable and indication for phototherapy is significantly higher in neonates with alpha-fetoprotein levels in umbilical blood above 100 mg/l and these infants must be carefully observed. When the concentration is above 130 mg/l, the development of hyperbilirubinaemia may be assumed with certainty. Examination of alpha fetoprotein in umbilical blood is an useful indicator of the functional maturity of the liver, but does not ensure reliable prediction of hyperbilirubinaemia in individual neonates. A concentration of alpha-fetoprotein above 100 mg/l is exceptional among neonates without hyperbilirubinaemia, while lower values do not rule out the development of hyperbilirubinaemia. PMID- 1705182 TI - [Monitoring developmental risks in children from families with first degree consanguinity (inbreeding)]. PMID- 1705183 TI - Screening for high-risk pregnancies with maternal serum alpha-fetoprotein (MSAFP). The Canadian Society of Clinical Chemists' Task Force on MSAFP Screening. PMID- 1705184 TI - Elevated G gamma:A gamma globin chain ratio in homozygous beta thalassemia. AB - G gamma:A gamma chain ratios were determined in homozygous beta thalassemia and cord blood samples using triton-urea polyacrylamide gel electrophoresis. The mean G gamma/G gamma + A gamma proportion in the two groups were 0.62 +/- 0.10 and 0.72 +/- 0.03, respectively. There was no significant correlation of the gamma chain composition in either fetal hemoglobin or total hemoglobin levels; this suggests that these two factors do not influence gamma chain ratios. There was also no marked variation in the G gamma:A gamma ratios in beta thalassemia patients when they were divided into higher fetal hemoglobin (greater than 50%) and lower fetal hemoglobin (less than 50%) groups. These observations are consistent with the finding of a selective advantage of G gamma chains over A gamma chains in disorders where the erythropoietic stress is higher than normal, and may be inherited as a specific genetic entity in haplotypic polymorphism. PMID- 1705185 TI - Glyceryl ethers in peroxisomal disease. AB - 1-O-Alkyl and 1-O-alk-1-enyl (plasmalogens) glyceryl ether lipid levels were measured in post-mortem brain and/or liver biopsies from 7 patients with ultrastructural and biochemical evidence of a defect in peroxisomal biogenesis and/or enzymological evidence of a disturbance in ether lipid synthesis. Near normal levels of both species of glyceryl ether lipids were found in neonatal adrenoleukodystrophy and infantile Refsum's disease but marked deficiencies were found in Zellweger's syndrome and rhizomelic chondrodysplasia punctata, the latter manifesting the most profound reduction in ether lipid levels. These observations suggest that little ether lipid biosynthesis occurs in vivo in rhizomelic chondrodysplasia punctata or Zellweger's syndrome. However, in some phenotypes with apparently gross reductions in peroxisomal numbers, e.g. neonatal adrenoleukodystrophy and infantile Refsom's disease, there is significant ether lipid synthesis in liver and brain. PMID- 1705186 TI - Elastolytic activity of human blood monocytes characterized by a new monoclonal antibody against human leucocyte elastase. Relationship to rheumatoid arthritis. AB - The leucocyte elastase of human blood monocytes was investigated by applying a new monoclonal antibody which did not block the enzyme activity against elastin. In a fixed population of mononuclear cells (MNC) and using fluorescence activated cell sorting (FACS), the human leucocyte elastase (HLE) antibody identified a subgroup of CD14+ cells which contained all the elastase activity and which could be blocked by a specific chloromethylketone elastase inhibitor. By anti-CD14 labelling the HLE positive cells were identified as monocytes and amounted to 88% of this cell type (median: range 72-96%). In a parallel study of patients with active rheumatoid arthritis (RA) and control donors the elastolytic capacity of PMA-stimulated live MNC was higher for RA patients than for controls (p less than 0.02). For control donors the number of HLE+ cells correlated well to the elastolysis in the same MNC sample (Spearman's rho:0.83, p less than 0.05); in the samples from RA patients no correlation was found between the normal number of HLE+ cells and the increased elastolysis (rho: 0.54, p greater than 0.20). PMID- 1705187 TI - Correlation of metal-binding proteins and proteinase inhibitors with immunological parameters in rheumatoid synovial fluids. AB - Metal-binding proteins (ceruloplasmin, transferrin, ferritin, and lactoferrin), proteinase inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha trypsin inhibitors), and albumin were assayed in synovial fluid obtained from 20 patients with rheumatoid arthritis (RA) and 15 with osteoarthritis (OA). The levels of proteinase inhibitors and metal-binding proteins, except transferrin, were significantly increased in synovial fluid from RA patients as compared with synovial fluid from OA patients. Metal-binding proteins significantly correlated with rheumatoid factor and immune complexes in synovial fluid from RA patients. Proteinase inhibitor levels also significantly correlated with C-reactive protein, and complement components. These results suggest that the raised level of metal-binding proteins and proteinase inhibitors in synovial fluid from RA patients reflect inflammatory activity, and hence may play an important role in the pathogenesis of inflammatory joint diseases. PMID- 1705188 TI - Analysis of polyethylene glycol precipitates from SLE sera: antibody enrichment in association with disease activity. AB - Twenty two systemic lupus erythematosus patients were assessed clinically for periods of up to six years. Polyethylene glycol precipitates of serial serum samples were analysed for the presence of autoantibodies by specific enzyme linked immunoabsorbent assays. Increased enrichment of anti-Ro(SSA) was found to correlate with disease activity. Longitudinal studies of individual patients showed an association between disease exacerbation and enrichment of anti Ro(SSA), anti-La(SSB) and anti-ssDNA. Circulating immune complexes isolated by anti-C3 affinity chromatography were also found to contain anti-Ro(SSA), anti La(SSB) and anti-ss DNA and fixation of complement by complexes could be demonstrated. Enrichment of these autoantibodies in renal eluates of two patients with diffuse proliferative glomerulonephritis provided further evidence for a pathogenic role of these immune complexes. PMID- 1705189 TI - Local venomotor response to intravenous infusion of substance P and glyceryl trinitrate in systemic sclerosis. AB - Endothelial injury has been shown in ultrastructural studies in systemic sclerosis (SSc). In the present study, we tested the functional response of the endothelium in the early phase of the disease and at a more advanced stage. Substance P (SP) (25 and 50 ng) and glyceryl trinitrate (GT) (100 and 200 ng) were infused intravenously in 6 control subjects and in 6 female patients affected with early systemic sclerosis (SSc) to verify whether or not a lack of endothelial function could be detected. Venous compliance was assessed with a linear variable differential technique. Endothelial injury was evaluated by the von Willebrand Factor Antigen (vWF:Ag) plasma level and platelet aggregation by the platelet aggregate ratio (PAR). Endothelial-mediated response to SP was deficient in SSc patients in comparison with controls, whereas endothelium independent vasodilation was detected after the infusion of GT both in patients and controls. All vWF:Ag levels were in the normal range while in vivo platelet aggregation was increased as demonstrated by the PAR levels in the SSc subjects. The experiment was repeated in the same patients when the clinical status worsened and the levels of vWF:Ag were noted to rise: the endothelium dependent vasodilatory response to SP was still deficient as before and a decrease in vasodilatory response to GT was noted. vWF:Ag levels were significantly increased and the PAR still demonstrated an increased platelet aggregation. These data clearly indicate that an early functional deficit of the endothelial function is present before the onset of extensive visceral and skin involvement. The mechanism that provokes the impairment of the endothelial function need further investigation. PMID- 1705190 TI - Histochemical localization of calcium with potassium pyroantimonate in the articular tissues in calcium pyrophosphate dihydrate crystal deposition disease. AB - The ultrastructural localization of calcium in the articular cartilage, meniscus, and synovium of patients with calcium pyrophosphate dihydrate (CPPD) crystal deposition disease was examined by using potassium pyroantimonate. Focal areas with deposition of CPPD crystals in each tissue showed electron-dense precipitates of the antimony-calcium complex: (1) in the cytoplasm of hypertrophic chondrocytes around CPPD crystals, especially on the margins of intracellular lipid droplets or within electron-dense amorphous material or both; and (2) on the margins of lipid droplets and within electron-dense amorphous material in the degenerated matrix surrounding the hypertrophic chondrocytes. The slender rodlike structures consisting of electron-dense precipitates of the antimony-calcium complex, suggestive of the precursor to rodlike CPPD crystals, were also observed in the degenerated matrix. These findings suggest that the hypertrophic chondrocytes may contribute to the formation of CPPD crystals by intracellular accumulation of calcium and subsequent release of high concentration of calcium into their surrounding matrix. PMID- 1705191 TI - Elution of vancomycin, daptomycin, and amikacin from acrylic bone cement. AB - Increasing antibiotic resistance of bacteria that infect prosthetic joints has stimulated interest in the incorporation of more effective antimicrobial agents into polymethylmethacrylate (PMMA). Vancomycin and daptomycin are effective against nearly all staphylococci and streptococci, and amikacin has a broader spectrum against gram-negative bacilli than do other aminoglycosides such as gentamicin. These three antibiotics maintained bioactivity after incorporation into several commonly used preparations of PMMA and eluted readily into the surrounding medium. Preparing PMMA under negative atmospheric pressure, which decreases porosity, caused a 50% reduction in antibiotic release; the addition of 25% dextran, which increases porosity, greatly facilitated elution of these antibiotics. Based on their broad antibacterial effect against gram-positive and gram-negative bacteria, inclusion of vancomycin and amikacin in PMMA merits clinical study. The addition of these antibiotics to PMMA, together with dextran, may be applicable when structural integrity is unimportant but a substantial local antimicrobial effect is desired, such as in the use of antibiotic containing beads to treat osteomyelitis. PMID- 1705192 TI - The skin hyperaemic response to local injection of substance P and capsaicin in diabetes mellitus. AB - Recent studies have demonstrated impaired skin hyperaemia to local injury in diabetes mellitus. In order to gain insight into the mechanisms of impaired hyperaemia, dose-response curves to intradermal substance P (25, 50, 100 pmol) and capsaicin (1.0, 2.5, 5.0 nmol) were examined before and after histamine blockade with chlorpheniramine, in 6 patients with uncomplicated Type 1 diabetes and 9 matched control subjects. Skin hyperaemia was measured indirectly as the peak laser Doppler flow in proximity to the area of hyperaemia. The response to the three doses of substance P was significantly lower in diabetic patients (0.37 +/- 0.12 (+/- SD), 0.51 +/- 0.12, 0.67 +/- 0.09 V) than in control subjects (0.57 +/- 0.15, 0.70 +/- 0.19, 0.84 +/- 0.21 V; p less than 0.01). In contrast there was no significant difference in skin hyperaemia to capsaicin between diabetic patients (0.41 +/- 0.07, 0.50 +/- 0.09, 0.59 +/- 0.09 V) and control subjects (0.41 +/- 0.06, 0.52 +/- 0.08, 0.63 +/- 0.07 V). Following chlorpheniramine, the response to capsaicin remained unaltered (0.39 +/- 0.07, 0.51 +/- 0.05, 0.60 +/- 0.07 in diabetic patients and 0.43 +/- 0.08, 0.50 +/- 0.10, 0.63 +/- 0.07 V in control subjects), but there was a significant reduction in hyperaemia to substance P in both patients (20.4 +/- 12.3% reduction, p less than 0.05) and control subjects (20.6 +/- 14.1% reduction, p less than 0.05). It is suggested that impaired skin hyperaemia may represent decreased vascular reactivity to locally released substance P from peripheral nerve fibres. PMID- 1705193 TI - Changes in metabolism induced by oral contraceptives containing desogestrel and gestodene in older women. AB - Forty women aged 35-45 years were investigated to determine changes in haemostasis, lipids and lipoproteins whilst taking combined contraceptive pills containing the new third generation progestogens, desogestrel and gestodene. There was no statistically significant difference between the two preparations in any of the parameters studied. Women taking the combined pill showed increases in fibrinogen and factor X and a reduction in antithrombin III when compared with their control values. There were also small but significant increases in triglycerides and triglyceride-rich lipoproteins. Total high density lipoprotein cholesterol (HDL), high density lipoprotein-2 cholesterol (HDL2), high density lipoprotein-3 cholesterol (HDL3) and apolipoprotein A-1 were all increased at some stages of the treatment cycle, whereas low density lipoprotein cholesterol (LDL) showed a reduction in the first cycle of treatment. The changes in lipids and lipoproteins would not appear to increase the risk of cardiovascular disease, however the effects of the increase in the pro-coagulant factors are uncertain. PMID- 1705194 TI - Cytoskeletal and contractile protein distribution in lung development and injury. PMID- 1705195 TI - Response of rat type II pneumocyte Na,K-ATPase to hyperoxic injury. PMID- 1705196 TI - Control of capillary growth and differentiation by extracellular matrix. Use of a tensegrity (tensional integrity) mechanism for signal processing. PMID- 1705197 TI - Mechanisms of alveolar fibrosis following acute lung injury. Presence of angiogenesis bioactivity in the lower respiratory tract. PMID- 1705198 TI - Macrophage vitronectin receptor, CD36, and thrombospondin cooperate in recognition of neutrophils undergoing programmed cell death. PMID- 1705199 TI - Bleomycin regulation of transforming growth factor-beta mRNA in rat lung fibroblasts and subpopulations. PMID- 1705200 TI - Bleomycin stimulates production of transforming growth factor-beta by rat pulmonary artery endothelial cells. PMID- 1705201 TI - Regulation of neutrophil function by acute phase reactants. Implications for resolution of the adult respiratory distress syndrome. PMID- 1705202 TI - Hydroxylation of collagen by lungs of rats administered bleomycin. PMID- 1705203 TI - Insulin-like growth factor-1 (IGF-1) mRNA expression in bone marrow derived macrophages is stimulated by chrysotile asbestos and bleomycin. A potential marker for a reparative macrophage phenotype. PMID- 1705204 TI - Transforming growth factor-beta production by lung macrophages and fibroblasts. PMID- 1705205 TI - [The region of the replication initiation located at the 5' termination of the domain of chicken alpha-globulin genes contains a transcriptional enhancer]. PMID- 1705206 TI - [The structural determinants of the myelin basic protein that define sensitivity to viral infections. The cloning and determination of the primary mRNA structure of the myelin basic protein in adults]. PMID- 1705207 TI - Developmental expression of proteolipid protein and DM20 mRNAs and proteins in the rat brain. AB - Proteolipid protein (PLP) and DM20, two abundant proteins of myelin, are produced from alternatively spliced mRNAs from the primary PLP gene transcript. Recent studies on the mouse, bovine and human central nervous systems have found that DM20 protein is expressed prior to PLP during development. As development proceeds, however, PLP becomes the predominant protein. This implies that there must be some form of regulation controlling the ratio of these proteins during development. If this regulation occurs during transcription or splicing, the PLP/DM20 mRNA ratio should mimic the PLP/DM20 protein ratio during development. In order to study these closely related mRNAs, we developed a method that used oligonucleotide probes to specifically identify PLP or DM20 mRNA. In this study, we established that (1) as with other species studied, the DM20 protein is present at higher or equivalent levels to PLP during early rat brain development, and (2) PLP and DM20 mRNAs have essentially identical developmental profiles in the rat, although the DM20 mRNA is expressed at lower levels than PLP mRNA. Since the PLP/DM20 protein ratio in young rats does not reflect the PLP/DM20 mRNA ratio, the mechanism of regulation responsible for altering the PLP/DM20 protein ratio during development must occur after transcription and splicing. Possible posttranscriptional mechanisms controlling the ratio of these proteins during development are discussed. PMID- 1705208 TI - Spatial distribution of mRNAs for myelin proteins in primary cultures of mouse brain. AB - A nonradioactive in situ hybridization procedure was employed to study the distribution of mRNAs for myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP) in oligodendrocytes in primary cultures of mouse brain. This procedure provided good cellular localization and allowed rapid detection of the mRNAs with low backgrounds. Gradual movement of MBP mRNAs from oligodendrocyte somas into processes was observed with time in culture. The MBP mRNAs were observed to be distributed in an asymmetric fashion within the somas and cell processes. Antigalactocerebroside staining indicated the presence of oligodendrocyte processes prior to movement of MBP mRNA, suggesting that the presence of processes alone was insufficient for translocation of MBP mRNAs. The mRNAs for PLP and MAG remained confined to the oligodendrocyte somas at all times in culture at least up to 28 days. While most of the CNP mRNAs were observed to be associated with the perikarya of oligodendrocytes, in less than 1% of these cells the presence of CNP mRNA in the processes was evident. This suggests that there may exist a subset of oligodendrocytes in which the translocation of these messages occurs. PMID- 1705209 TI - Tyrosine phosphorylated proteins decrease during differentiation of neuronal and glial cells. AB - Tyrosine kinases have been implicated in the development of the nervous system. To investigate their role, immunoblotting with phosphotyrosine antibodies was used to identify substrates of tyrosine kinases during glial and neuronal differentiation in the rat and mouse. Fourteen prominent phosphotyrosine containing proteins were detected in oligodendrocyte-type 2 astrocyte (O2A) progenitor cells. When O2A cells differentiated into type 2 astrocytes, a phosphotyrosine-modified protein of 74 kilodaltons (kDa) decreased 15-fold in abundance, and phosphotyrosine-containing proteins of 36-40 kDa declined. When O2A cells differentiated into oligodendrocytes, a prominent 71-kDa phosphotyrosine-modified protein became undetectable. During retinoic acid induced neuronal differentiation of the mouse embryonal carcinoma cell line P19S101A1 (P19), an 80-kDa phosphotyrosine-containing protein decreased from high levels in the undifferentiated state to undetectable levels after 96 h in aggregation. Retinoic acid treatment also caused a rapid decrease in levels of a 96-kDa phosphotyrosine-containing protein. Cell-cell contact occurring as a result of aggregation resulted in decreases in 130- and 90-kDa phosphotyrosine containing proteins in both retinoic acid-induced and control cultures. Cultured rat central nervous system cerebral cortical neurons and peripheral nervous system dorsal root ganglion neurons exhibited prominent phosphotyrosine-modified proteins of 90 and 46 kDa the same sizes as those in P19 neurons. The phosphotyrosine-containing proteins involved in the retinoic acid-induced differentiation of P19 cells to neurons were different from those altered in the glial differentiation of O2A cells to astrocytes or oligodendrocytes, indicating that the tyrosine kinase substrates modified during nervous system differentiation may be cell-type-specific. PMID- 1705210 TI - Two isoforms of myelin-associated glycoprotein accumulate in quaking mice: only the large polypeptide is phosphorylated. AB - We have shown that the developmentally regulated appearance of the two myelin associated glycoprotein (MAG) polypeptides in normal mouse brain myelin does not reflect the developmental pattern of differential splicing of primary gene transcripts as determined earlier by RNase protection assays. Contrary to expectation, the large (L-MAG) and small (S-MAG) polypeptides are present in about equal amounts at a relatively early stage of myelination, day 24 or earlier. In quaking (qk) mutant mouse brain myelin, both MAG polypeptides are evident at all ages examined; the relatively greater abundance of S-MAG compared to L-MAG at early ages (days 18 and 22) confirms our earlier observation on in vitro translations of mRNA. At later ages (day 27 and beyond) both isoform are present in approximately equal amounts. The L-MAG but not the S-MAG polypeptide can be phosphorylated by kinases that are endogenous to isolated qk myelin, analogous to the phosphorylation we have observed in vivo in normal mice and in their isolated myelin. PMID- 1705211 TI - Death of individual oligodendrocytes in jimpy brain precedes expression of proteolipid protein. AB - Immunocytochemistry and thymidine autoradiography were combined to determine the time elapsed between cell division and the expression of proteolipid protein (PLP) in individual oligodendrocytes in normal mouse brain. In jimpy (jp) brains, autoradiography was used to determine the time elapsed between cell division in an individual oligodendrocyte and evidence of cell death. Oligodendrocytes in normal mouse brain do not express PLP until 72 h after a single injection of [3H] thymidine. In contrast, oligodendrocytes in jp brains begin to die within 9-11 h after an injection of thymidine. The jp mouse is one of several X-linked, hypomyelinated mutants in which a defect has been demonstrated in the gene coding for PLP. It has been presumed that the lack of this protein in the myelin sheath is responsible for the jp phenotype. However, the present study shows that individual jp oligodendrocytes begin to die long before they would normally have synthesized detectable levels of PLP. Therefore, it seems unlikely that the death of jp oligodendrocytes is due to the absence of PLP in myelin sheaths. Oligodendrocyte death and other early jp abnormalities may be due to the presence of abnormal PLP message which may interfere with glial differentiation. Alternatively, the PLP message may code for another protein which is important for normal development of neuroglia. PMID- 1705212 TI - Transcriptional regulation studies of myelin-associated genes in myelin-deficient mutant rats. AB - To identify and assess the consequences of the mutation in myelin-deficient (md) rats, the myelin proteolipid protein (PLP) gene and its expression were studied in md rats. Southern blots of the PLP gene demonstrated that no major deletions or insertions have occurred in this gene. In addition, the mutation in this gene does not result in a splicing defect in the RNAs, since all exons are represented in md PLP RNAs. These data are consistent with results in another laboratory indicating that a point mutation in the PLP gene in md rats results in a single amino acid alteration in the protein. To elucidate the molecular mechanisms producing reduced levels of PLP, myelin basic protein (MBP) and glycerol phosphate dehydrogenase (GPDH) mRNAs, and their corresponding proteins in md rats, in vitro transcription assays were performed. Transcription of the PLP gene in nuclei isolated from 23-day-old md rat brains was dramatically reduced relative to normal tissue. Thus, the single amino acid alteration in this protein alters the regulation of transcription of this gene. In contrast, the transcriptional activities of the MBP and GPDH genes in md rats were indistinguishable from normal animals. Thus, the lower level of MBP and GPDH mRNA and protein in md rats relative to normal results from a posttranscriptional event. PMID- 1705214 TI - EMG and motor control. PMID- 1705213 TI - Turns-amplitude analysis at different sampling frequencies. AB - In the EMG interference pattern the number of potential reversals of more than 100 microV (i.e., turns) measured may be low if a low sampling frequency is used. This might be more pronounced in a myopathic muscle with small short potentials than in a control muscle, resulting in a decreased diagnostic yield. The influence of sampling frequencies from 6 to 200 kHz on the turns, mean amplitude and ratio of turns to mean amplitude analysed on the interference pattern at a force of 30% of maximum was examined at 10 sites in each of 5 control muscles, 5 myopathic muscles and 5 neurogenic muscles (i.e., 150 sites in all). In this small group of muscles low sampling frequency did not tend to reduce the diagnostic yield of the measurements. Proper reproducibility was obtained with sampling frequencies down to 17 kHz. However, due to systematic errors, measurements obtained with sampling frequencies below 25 kHz require comparable control values or compensations for errors. As time intervals between turns (not studied here) have a skew distribution with many small time intervals a sampling frequency of 50 kHz or higher is probably required. PMID- 1705215 TI - Orthodromic sensory conduction along the ring finger in normal subjects and in patients with a carpal tunnel syndrome. AB - The purpose of the present study was to examine the value of measuring sensory conduction along the median and ulnar nerves of the fourth finger in the diagnosis of a carpal tunnel syndrome (CTS). In 23 controls, sensory conductions along median and ulnar nerves were identical. In 28 of 38 patients with CTS, stimulation of the ring finger revealed a reduced conduction velocity along sensory median nerve fibres in contrast to normal conduction along ulnar sensory nerve fibres. In 5 patients, a sensory action potential was absent over the median nerve and in another 5 sensory conduction was normal along both nerves. We conclude that testing of sensory conduction along the ring finger is useful in about 74% of patients with CTS, while in the remaining 26% other fingers must be examined to establish the diagnosis. PMID- 1705216 TI - Masseter inhibitory reflex in movement disorders. Huntington's chorea, Parkinson's disease, dystonia, and unilateral masticatory spasm. AB - Evoked by electrical stimulation of the mental nerve, the masseter inhibitory reflex consists of an early and a late silent period (SP1 and SP2), which interrupt the voluntary electromyographic (EMG) activity in the masseter muscle. We recorded the masseter inhibitory reflex and measured its latency, depth of suppression, duration and recovery cycle to paired stimuli, in patients with Huntington's chorea. Parkinson's disease, dystonia, or unilateral masticatory spasm. In patients with Huntington's chorea the reflex data and recovery cycle were normal. In patients with Parkinson's disease or dystonia, although the reflex data were normal, SP2 recovered far more rapidly than it did in control subjects. This is possibly due to hypoactivity of an inhibitory control of the polysynaptic chain of ponto-medullary interneurons that mediate SP2. In patients with unilateral masticatory spasm, both SP1 and SP2 were absent. Suppression is probably absent because this involuntary movement originates at a point along the peripheral course of the nerve. PMID- 1705217 TI - Scalp topography of giant SEP and pre-myoclonus spike in cortical reflex myoclonus. AB - Scalp topography of the giant SEP and the pre-myoclonus spike demonstrated by jerk-locked back averaging was studied by using a computer-assisted evoked potential mapping technique in 5 patients with cortical reflex myoclonus. The initial positive peak of giant SEP was localized to the postcentral region contralateral to the stimulus and was associated with a negative potential field of the same latency at the frontal region in all cases. The main positive peak of pre-myoclonus spike was localized to the postcentral region contralateral to the myoclonus in 4 cases, and maximal at the midline postcentral region extending contralaterally with respect to the myoclonus in 1 case. The postcentral positive peak was associated with a frontal negativity in 2 of the 5 cases. In those 2 cases, the main components of giant SEP and the pre-myoclonus spike showed a similar scalp distribution with respect to the hand which was stimulated or myoclonic jerks were recorded from, although the latter was much smaller and less sharp than the former. These findings support our previous hypothesis that those 2 activities might be generated, at least in part, by common physiological mechanisms. In 3 other cases, however, the postcentral positive peak of the pre myoclonus spike was not associated with a frontal negativity. PMID- 1705218 TI - Coil placement in magnetic brain stimulation related to skull and brain anatomy. AB - The influence of coil position on the size of electromyographic responses evoked by transcranial magnetic brain stimulation was systematically evaluated. The position of the stimulation coil (11.6 cm outer diameter, Novametrix) was recorded by constructing individual grids on the skull surface using extracranial bony landmarks and was then related to underlying cerebral sulci by analysis of magnetic resonance images of the brain and skull. The largest responses in the right first dorsal interosseus (FDI) muscle, when using anticlockwise coil currents as viewed from above, were found, on average (5 subjects), to occur with the coil centred 2 cm behind and left of the vertex. For the right tibialis anterior (TA) muscle the largest responses were obtained, on average, with the coil centred around a point 4 cm anterior to and 4 cm left of the vertex. Between individuals, the optimal coil position for responses in a particular muscle varied up to 2 cm. This variability could be explained by the variability in the relationship between the central sulcus and the extracranial bony landmarks of each individual. Responses could be obtained whenever the windings of the coil passed over the appropriate region of the motor cortex, providing evidence that magnetically evoked responses do originate from activation of the precentral gyrus. The size of the cortically evoked responses for a given stimulus intensity depended on the direction of the coil currents passing over the motor area. For FDI muscles, the largest responses were obtained when the coil currents passed over the lateral part of the motor strip from forwards behind and transversely to the central sulcus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705219 TI - A theoretical calculation of the electric field induced in the cortex during magnetic stimulation. AB - We present a mathematical model for calculating the electric field induced in the head during magnetic stimulation of the cortex. The electric field arises from 2 sources: (1) the changing magnetic field creates an electric field in the tissue by electromagnetic induction, and (2) a charge distribution arises on the surface of the head and produces its own electrostatic field. A 3-sphere model is used to represent the brain, skull and scalp. The electric field as a function of the coil position, shape and orientation is computed numerically. The charge distribution partially shields the brain from the stimulus. The electric field is insensitive to the skull conductivity, in contrast with electrical stimulation using surface electrodes. Different coil shapes and orientations are considered, and a figure-of-eight coil is shown to deliver the largest and most focal stimulus. PMID- 1705220 TI - Motor evoked potentials and central motor conduction: studies of transcranial magnetic stimulation with recording from the leg. AB - To determine central conduction times in the corticospinal pathways of humans using magnetic stimulation, we have developed a method for consistently recording conduction times between the motor cortex and the L4-5 level of the spinal cord. In 30 subjects, motor evoked potentials (MEPs) were recorded from the tibialis anterior muscle following contralateral motor cortex and peroneal nerve stimulation. In 18 of these subjects, the L4-5 intervertebral space was stimulated. The stimuli consisted of single, painless, short-duration magnetic pulses. In 12 subjects, measurements were made during voluntary ankle dorsiflexion, and during vibration of the TA tendon at rest. All subjects had measureable MEP latencies of 30.3 +/- 2.2 msec (mean +/- S.D.). The central motor conduction time (CMCT) was calculated using both a direct as well as an indirect method. The direct method in 18 subjects had a mean value of 16.2 +/- 1.7 msec, while the indirect method in all 30 subjects was 13.8 +/- 1.8 msec. No significant correlation of the CMCT was found with either age or height in these subjects. Ankle dorsiflexion significantly reduced the MEP latency and increased the amplitude, whereas vibration of the TA tendon significantly increased the amplitude alone. We conclude that MEPs may be consistently and painlessly measured in the lower extremity using magnetic stimulation in adults. Facilitation of the MEPs was produced more consistently by voluntary contraction than by vibratory stimulation of the tibialis anterior muscle tendon. Finally, CMCT was independent of both age and height in our study population. PMID- 1705221 TI - Magnetic stimulation of motor cortex and nerve roots in children. Maturation of cortico-motoneuronal projections. AB - Magnetoelectrical stimulation of motor cortex and peripheral nerve roots was used to establish the maturational profiles of central and peripheral nervous system conduction times in normal children in upper and lower extremity muscles. To obtain true central conduction times, not contaminated by variation due to different preinnervational levels, a protocol using stimulation under full muscular relaxation was used. With this protocol clear responses of the upper extremity could be obtained after the first year of life, whereas in the lower extremity they could not be obtained before the fourth year of life. The developmental profile of central conduction times to upper and lower extremity muscles showed an age-dependent acceleration with adult values not being reached before the age of about 10 years. In contrast, peripheral conduction times obtained after cervical or lumbar root stimulation to upper and lower extremities showed a much faster maturational profile, with adult values being reached at about the age of 3 years. The data are discussed in relation to the available literature on neuromorphological development and provide normative values for the understanding of normal development of motor control in children as well as for application of this technique in children with motor disturbances. PMID- 1705222 TI - Influence of posture and voluntary background contraction upon compound muscle action potentials from anterior tibial and soleus muscle following transcranial magnetic stimulation. AB - Compound muscle action potentials (CMAPs) were recorded from anterior tibial (TA) and soleus (SOL) muscles following transcranial magnetic stimulation of motor cortex in 10 healthy subjects: (1) while standing upright without support, (2) while sitting, and (3) while lying supine. The results of this study demonstrate a significant influence of posture upon the amplitudes of CMAPs. Postural facilitation presented itself, firstly, in terms of a higher incidence of bilateral activation of distal leg muscles during stance and, secondly, as significant enhancement of the amplitude of CMAPs while standing as compared to lying supine. The onset latency, however, did not disclose a significant shortening during stance. To assess the effects of preinnervation subjects voluntarily adjusted the level of TA activity to 5%, 10% and 20% of maximum isometric force respectively before cortex stimulation. Voluntary background contraction resulted in a significant increase of amplitude of CMAPs but, in contrast to postural facilitation, concomitant with a significant decrease in onset latency. These results point to a somewhat different mechanism of facilitation during stance as compared to voluntary preinnervation. But it cannot be decided whether cortical mechanisms, different descending systems, the spinal circuitry or a combination of these factors is responsible for the observed effects. PMID- 1705223 TI - A comparison of turns analysis and motor unit analysis in electromyography. AB - We compared the results of turns analysis and motor unit analysis on 4056 electromyographic interference patterns (IPs) from normal subjects and patients with neuromuscular disorders. The motor unit analysis involved decomposing the IPs into their component motor unit action potentials (MUAPs) using automatic decomposition electromyography (ADEMG). We checked the accuracy of the decompositions by attempting to reconstruct some of the IPs from their identified MUAPs using computer simulations. The simulations revealed that ADEMG typically identified more than 60% (but not all) of the MUAPs in a given IP. Both turns and MUAP properties showed regular and related changes with force, age, muscle, and recording electrode type. The number of turns in each IP was highly correlated with the number of active MUAPs (r = 0.65), the mean MUAP firing rate (r = 0.72), the mean number of turns per MUAP (r = 0.34), and the product of these 3 properties (r = 0.83). The mean amplitude change per turn was highly correlated with the mean MUAP amplitude (r = 0.82), but also depended on the number of turns per MUAP. Due to the lack of a one-to-one relationship between the turns analysis properties and the MUAP properties, the turns analysis properties by themselves did not provide sufficient information to infer unambiguous physiological information about motor unit morphology or firing behavior. PMID- 1705224 TI - Electrophoretic transport of solutes in aqueous two-phase systems. AB - Electrophoretic transport of proteins across the interface between the phases of an aqueous polymer two-phase system can be greatly impeded in comparison with transport within the individual phase. This effect can be controlled by modifying the affinity of the protein for a phase by suitable manipulations of such variables as pH. The effect is not caused by differences in the electrophoretic velocity between the two phases, nor by large changes in pH at the interface. An analogy exists between this phenomenon and the related subject of diffusion of electrolytes across the phase interface. PMID- 1705225 TI - Pharmacological evidence indicating a role of GABAergic systems in termination of limbic seizures. AB - The role of inhibitory systems in the initiation and termination of seizures in limbic circuits of the rat was tested by measuring the time to onset and duration of maximal dentate activation before and after the administration of GABAergic agents. Both 0.1 and 0.3 mg/kg of the GABAA receptor antagonist bicuculline lengthened maximal dentate activation while 0.3 mg/kg was needed to reversibly decrease the time to onset. Picrotoxin, an antagonist at the GABAA receptor/channel complex, lengthened maximal dentate activation, but did not alter the time to onset. Muscimol, a GABAA receptor agonist, shortened maximal dentate activation, but did not lengthen the time to onset. Baclofen, a GABAB receptor agonist (3 and 10 mg/kg), had no effect on the time to onset of maximal dentate activation, while 10 mg/kg baclofen shortened maximal dentate activation. These results demonstrate that agents that have selective action at both GABAA and GABAB synapses alter the duration of maximal dentate activation more than they influence the time to onset of maximal dentate activation and suggest that GABAergic mechanisms are critical in the termination of seizures. PMID- 1705226 TI - Germ cell tumours of testis: prognostic factors and results. AB - Prognostic features regarding the outcome of 232 patients with testicular germ cell tumours treated from 1980 to 1987 in Sheffield are reviewed. Delay in diagnosis was a constant feature. The majority had a swelling for over 3 months. After 6 months mortality doubled, compared with patients with a shorter history. Initial serum levels of alpha-fetoprotein and beta-human chorionic gondadotropin, particularly if both were elevated over 100, and more so 500 IU/l, indicated a subsequent mortality in non-seminomatous tumours of 24 and 37%, respectively, while only 1.5% died if both values were below 100. The overall survival rate was 97% in seminomas and 78% in non-seminomas; no patients with well or moderately differentiated tumours died. There were no deaths after the 3rd year. Surveillance treatment in stage-I tumours was safe and mortality-free. PMID- 1705227 TI - A simple, self-retaining intraurethral catheter for treatment of prostatic obstruction. AB - An intraurethral catheter was successfully used in 38 of 47 (80.9%) patients with benign prostatic hyperplasia who were awaiting surgery or in whom surgery constituted a high risk. All patients had indwelling catheters due to urinary retention. The device was inserted via a cystoscope or a specially designed insertion set. The insertion was performed in the outpatient clinic under local anesthesia and did not require more time than the cystoscopy itself. The intraurethral catheter remained in place for up to 41 weeks. None of the patients developed clinically evident urinary tract infections. Voiding and continence were satisfactory in all, although 8 (21%) suffered some degree of frequency of micturition. There are apparently no limitations in the use of the intraurethral catheter. Compared to other techniques and devices for treatment of urinary retention, the intraurethral catheter appears to be more physiological, easier to insert and remove, and more economical. We recommend intraurethral catheter insertion for up to 6 months in patients who are awaiting surgery and as an alternative for high-risk patients. In the latter, the intraurethral catheter should be changed after 6 months. PMID- 1705228 TI - Effects of high energy shock wave exposure on renal function during extracorporeal shock wave lithotripsy for kidney stones. AB - In order to study the effects of high energy shock wave exposure on the kidney in extracorporeal shock wave lithotripsy (ESWL) using Dornier HM3, renal hemodynamics and renal function before and after ESWL were analyzed by 99mTc-DTPA renoscintigraphy. Various urinary enzyme activities (LDH, GOT, GPT, NAG, gamma GTP) and low molecular protein concentrations (alpha 1-microglobulin, beta 2 microglobulin) before and after ESWL were also compared. In the early phase of the renoscinitgram obtained in the 1st min after injection of 99mTc-DTPA, the time required to reach maximum radioactivity was significantly prolonged after ESWL in both the affected and contralateral kidney. This indicated that renal blood flow decreased in both the affected and contralateral kidney immediately after ESWL. An analysis of the 30-min renoscintigram showed that urinary clearance was delayed in the affected kidney in spite of no overt obstruction due to stone fragments. As for urinary enzyme activities and low molecular protein concentrations, they were standardized by urinary creatinine concentration measured at the same time. Urinary LDH, GOT, GPT and NAG activities remarkably increased on the day of ESWL followed by a decrease close to pretreatment levels on the 4th day, though these levels were still significantly higher than pretreatment levels. Urinary gamma-GPT activity was significantly higher than the pretreatment level only on the day of ESWL. Urinary alpha 1-microglobulin and beta 2-microglobulin concentrations significantly increased on the day of ESWL and were still high on the 4th day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705229 TI - Capsaicin desensitization in vivo is inhibited by ruthenium red. AB - The effect of systemic administration of Ruthenium Red on the excitatory and desensitizing effect of capsaicin was investigated in rats. Ruthenium Red was injected s.c. 30 min before capsaicin was administered. The excitatory effect of capsaicin on corneal, perivascular and visceral afferents was not influenced by treatment with Ruthenium Red. However, determination of the neuropeptide content and evoked neuropeptide release in peripheral organs and dorsal spinal cord 48 h after treatment showed that Ruthenium Red attenuated the 'desensitizing' effect of capsaicin at peripheral, but not at central, endings of primary afferents. On the other hand, a capsaicin-elicited autonomic reflex mediated by visceral afferents was still obtained in 9 of 14 rats that had received Ruthenium Red and capsaicin. The results indicate that a single dose of Ruthenium Red, which does not reduce the acute excitatory effect of capsaicin, reduces the desensitizing effect of capsaicin on peripheral endings of primary afferents in vivo. This long lasting protective effect of Ruthenium Red suggests that it is possible to pharmacologically differentiate between the acute and chronic effects of capsaicin. PMID- 1705230 TI - Pharmacological activity of cholecystokinin analogues modified in the Met28-Gly29 region. AB - Analogues of the C-terminal octapeptide of cholecystokinin (CCK) modified in the Met28-Gly29 region, were tested for their ability to interact with peripheral cholecystokinin receptors on rat pancreatic acini and to stimulate amylase secretion. These analogues were further evaluated for their ability to recognize central CCK receptors on guinea pig brain membranes. The behavioral effect of these analogues was also tested after intrastriatal injection into mice. It appeared that these analogues were full CCK agonists in the peripheral system. Although some induced dopaminomimetic effects after intrastriatal injection into mice, being as potent as the C-terminal octapeptide of cholecystokinin (CCK-8), others did not have any effect and were able to antagonize CCK-8 actions in the striatum. The results of this study confirm that one can obtain very potent CCK analogues by modifying the peptide bond between Met28 and Gly29, and that this modification can produce either CCK agonists or antagonists of CCK-induced dopamine transmission in the striatum. PMID- 1705231 TI - Tachykinins stimulate lyso-PAF:acetyl-CoA acetyltransferase activity in neutrophils. PMID- 1705232 TI - Vimentin, carcinoembryonic antigen and keratin in the diagnosis of mesothelioma, adenocarcinoma and reactive pleural lesions. AB - An immunohistochemical study of reactive pleural lesions, adenocarcinomas and mesotheliomas using carcinoembryonic antigen (CEA), cytokeratin and vimentin was carried out. All the specimens were obtained at surgery except for 11 mesotheliomas found at necropsy. Vimentin was positive in 23 of 27 mesotheliomas and negative in all the adenocarcinomas and 4 of 17 reactive mesothelial lesions. Conversely, CEA was positive in all the adenocarcinomas but negative in all mesotheliomas. Immunoreactivity for vimentin was seen in only 3 of 11 post-mortem mesotheliomas. Vimentin is a useful adjunct to the tissue diagnosis of mesothelioma especially when CEA is negative and cytokeratin positive. Its use appears largely confirmed to well fixed surgically derived tissues. PMID- 1705233 TI - Development and validation of a competitive enzyme immunoassay for chum salmon prolactin: a comparison to radioimmunoassay. AB - An enzyme immunoassay (EIA), based on a competitive assay system, for the measurement of prolactin (PRL) in the pituitary of salmonid fishes and of hormone released in medium from incubated pituitary was developed using a rabbit antiserum to chum salmon PRL (PRL, a combination of PRL I and PRL II). Chum salmon PRL was coupled to horseradish peroxidase (HRP). The incubation procedure for the antigen-antibody reaction was analogous to that in the radioimmunoassay (RIA) for PRL. The antibody-bound HRP-PRL was separated by a double antibody method. The enzyme activity in the precipitate was followed by a colorimetric method, in which 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o phenylenediamine were used as substrates. PRL, PRL I, and PRL II showed exactly the same competitive curves in the EIA system. PRL (127-158) showed the highest cross-reactivity among the fragments of PRL examined. Low cross-reactivity was seen with other hormones isolated from chum salmon pituitary. The displacement curves for pituitary extracts from several salmonids, including chum salmon, coho salmon, and rainbow trout, were parallel to that of the PRL standard, whereas those from the carp and tilapia showed negligible cross-reactivity. A parallel displacement curve to the PRL standard was also seen with incubation medium of the pars distalis of the chum salmon pituitary. Plasma from chum salmon, coho salmon, and rainbow trout gave nonspecific HRP activity in the EIA. The values of PRL-EIA were significantly correlated (y = 0.99x + 1.06, r = 0.942, P less than 0.05, n = 24) with those obtained in PRL-RIA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705234 TI - The action of hydrogen peroxide on the formation of thiobarbituric acid-reactive material from microsomes, liposomes or from DNA damaged by bleomycin or phenanthroline. Artefacts in the thiobarbituric acid test. AB - Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H2O2 caused a loss of cytochrome P-450 but not of cytochrome b5. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H2O2 induces lipid peroxidation, but this was found to be due largely or completely to an effect of H2O2 on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H2O2 inhibited ascorbate/Fe(III)-induced microsomal lipid peroxidation, but part of this effect was dues to an action of H2O2 in the TBA test itself. H2O2 also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu2+/o phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-ion iron/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H2O2. PMID- 1705235 TI - The coming-out process for homosexuals. AB - Coming out is a core developmental process for homosexual persons that spans many years. It usually begins in childhood with feelings of being different and progresses through various stages, including acknowledgment of homosexuality, disclosure to others, acceptance of a homosexual identity, experimentation and exploration, and intimacy. Ideally, the process ends in consolidation, a stage in which homosexuals no longer view themselves primarily in terms of sexual orientation. The author describes the various stages of the coming-out process and discusses the clinical implications for therapy with homosexual patients. PMID- 1705236 TI - The antibody response in BALB/c mice to the Plasmodium falciparum circumsporozoite repetitive epitope covalently coupled to synthetic lipopeptide adjuvant. AB - The repetitive epitope of Plasmodium falciparum circumsporozoite protein (Asn-Ala Asn-Pro)3 [(NANP)3] was coupled to tripalmitoyl-S-glyceryl-cysteine (P3C) and tripalmitoyl-S-glyceryl-cysteinyl-serine (P3CS). The lipopeptide P3CS is a potent B-cell and macrophage activator. The resulting immunogenic lipopeptides were used for immunization of the low responder mouse strain BALB/c. These low molecular weight conjugates induced specific anti-(NANP)3 IgG and IgM levels without any carrier proteins or admixed adjuvants after a single administration. PMID- 1705237 TI - Manipulation of intestinal immune responses against ovalbumin by cholera toxin and its B subunit in mice. AB - We studied the effect of mucosal presentation of ovalbumin (OVA) conjugated to cholera toxin (CT) or cholera toxin B subunit (CTB) on the intestinal immune responses against OVA. Mice were primed intraperitoneally (i.p.) with OVA in a water-in-oil emulsion and boosted intraduodenally (i.d.) with OVA conjugated to CT or CTB in various molar ratios. Responses were evaluated by testing intestinal secretions for OVA-specific antibodies and by quantifying the OVA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine. OVA CT conjugates were tested in a molar ratio ranging from 1.8:1 to 4500:1. OVA-CTB conjugates were tested in a molar ratio ranging from 0.25:1 to 500:1. The optimum intestinal immune response was reached at a molar ratio of 1.8:1 for OVA-CT and 5:1 for OVA-CTB. The binding capacity of OVA-CTB, but not of OVA-CT, to GM1 ganglioside corresponded with the capacity to enhance the intestinal immune response. The effect of conjugating CTB or CT to OVA on the immune response against OVA was more striking when mice were not only boosted i.d., but also primed i.d. Both OVA-CT and OVA-CTB induced detectable immune responses, whereas free OVA did not. Therefore, the carrier effect of CT or CTB is essential to trigger a mucosal immune response against OVA when presented mucosally only. We conclude that enhancing antigen uptake greatly facilitates mucosal immune responses. PMID- 1705238 TI - Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum. AB - An immunoglobulin A monoclonal antibody (MAb5C3) was developed against a 15-kDa surface glycoprotein (GP15) of Cryptosporidium parvum sporozoites. Indirect immunofluorescence and colloidal gold immunoelectron microscopy revealed that the antibody reacted with both the sporozoite and merozoite surface plasma membranes. On Western immunoblots, MAb5C3 binding was found to be strongly inhibited when 200 mM N-acetylglucosamine was used as a competing sugar. N-Acetylgalactosamine inhibited binding of the antibody only slightly, whereas glucose, mannose, and galactose failed to inhibit binding. MAb5C3 was found to recognize a similar 15 kDa epitope associated with a Cryptosporidium sp. isolated from guinea pigs. However, MAb5C3 failed to react with any proteins or glycoproteins associated with C. baileyi from chickens, Cryptosporidium sp. (= bovine C. muris) from cattle, C. serpentis from a rat snake, bradyzoites of Besnoitia darlingi from an opossum, sporozoite/oocyst extracts of Caryospora bigenetica from an eastern diamondback rattlesnake, sporozoites of Eimeria nieschulzi and E. papillata from rats and mice, or tachyzoites of Toxoplasma gondii (RH strain). When hybridoma supernatants containing MAb5C3 were administered orally to suckling mice experimentally infected with C. parvum, a 75% reduction in developmental stages was seen histologically at 72 h postinfection and a 67.5% reduction in mean oocyst output was found at 6 days postinfection. PMID- 1705239 TI - Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases. AB - The region flanking the transposon in a Tn1545-induced lecithinase-negative mutant of Listeria monocytogenes EGD was cloned and sequenced. The transposon had inserted in ORF D, the open reading frame previously identified downstream from hlyA, the gene encoding listeriolysin O. The complete sequence of ORF D from strain EGD has been determined as well as that of two other strains: LO28, a clinical isolate; and LM8, an epidemic strain. ORF D is 1,533 bp long and encodes a protein highly homologous to metalloproteases of bacilli, Serratia sp., Legionella pneumophila, and Pseudomonas aeruginosa. It was renamed prtA. Northern RNA blot analysis indicated that prtA is the first gene of a 6-kb operon, suggesting that the lecithinase-negative phenotype of the mutant might be due to a polar effect of the transposon insertion. PMID- 1705240 TI - Localization of immunogenic regions on the flagellin proteins of Campylobacter jejuni 81116. AB - The purpose of this study was to localize antigenic regions on the flagellin protein of Campylobacter jejuni 81116. This strain has two flagellin genes, flaA and flaB, which are 95% identical; only flaA seems to be expressed in motile C. jejuni 81116 cells. Fragments of flaA and flaB were cloned in the bacterial expression vector pEX, and the expression products were incubated with flagellin specific antibodies. Monoclonal antibodies to broadly cross-reactive epitopes recognized fragments that are located in the termini (CF16 and CF17) and in the center (CF15) of both flagellin A and B proteins. Most of the serotype-specific monoclonal antibodies (CF1, CF2, CD3, CF4, and CF13) reacted with only the center of flagellin A in an area where flagellin A and B differ in 6 amino acid residues. The epitopes in this area were further characterized by competitive binding experiments. The charge and molecular weight microheterogeneity of flagellin, as determined by two-dimensional gel electrophoresis, were unrelated to the expression of both flagellin genes or parts thereof. PMID- 1705241 TI - Antigenic determinants of the chlamydial major outer membrane protein resolved at a single amino acid level. AB - Antigenic determinants were identified from seven chlamydial major outer membrane proteins by using overlapping hexapeptides and polyclonal antisera. Sixty-one determinants were detected, and 30 were surface exposed on the native organisms. The two negatively charged residues, aspartic acid and glutamic acid, were found most often in determinants. Thirteen antigenic sites were further characterized by alanine substitution. Differences in fine specificities of these linear determinants were observed in alanine substitution profiles. Five determinants had adjacent critical residues, while eight had critical residues alternated with noncritical residues. Complete replacement analysis of two antigenic determinants provided more detailed information for elucidating the structural basis of the specificity of antigen-antibody interaction and suggested a correlation between sequence conservation and tolerance to amino acid substitution for antigenic sites subject to intense immune selection pressure. PMID- 1705242 TI - Serological responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin. AB - The B subunit of Shiga toxin and Shiga-like toxin (SLT-1) and its fragments are potentially immunogenic and may generate protective humoral responses against the action of these toxins. We have analyzed the antibody response of rabbits immunized with pure B subunit of SLT-1 or synthetic fragments of the subunit. The immune response to the native B subunit was found to be largely directed at conformational epitopes. More importantly, rabbits immunized with the B subunit were protected from a lethal challenge with SLT-1, indicating that the B subunit represents an excellent vaccine candidate to counter the effects of Shiga toxin and SLT-1 in humans. Polyclonal antibodies against a synthetic peptide corresponding to residues 28 to 40 of the B subunit neutralized the cytotoxicity of SLT-1 towards Vero cells. This region is thus exposed in the native state of the B subunit. The sequence specificity of other antipeptide antisera also provides clues to the state of folding and assembly of the B subunit. Antisera to synthetic peptides representing the N- and C-terminal regions of the SLT-1 B subunit did not cross-react with native B subunit but strongly recognized denatured forms of the protein. Finally, the monoclonal antibody 13C4 was shown to bind to a discontinuous epitope expressed only on the native form of the protein. These immunological reagents can be used to probe the conformational state of the B subunit and the holotoxin as it relates to their functional properties. PMID- 1705243 TI - Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects. AB - Most juvenile periodontitis patients respond to infection by Actinobacillus actinomycetemcomitans by producing serum antibodies. Specific antigens inducing the humoral immune response have not been identified, nor has the role of the resulting antibodies in disease progression been determined. Adsorbed and unadsorbed sera from juvenile periodontitis patients and normal subjects were analyzed by enzyme-linked immunosorbent assay and Western blots (immunoblots), using digested and undigested bacterial sonicates and French pressure cell fractions to determine the biochemical class, cross-reactivity, and cellular location of the antigens in different A. actinomycetemcomitans serotypes. Antigens detected by using high-titer sera included the following: (i) serotype specific nonprotein material located on the cell surface, (ii) soluble-fraction proteins showing highly variable antibody binding, (iii) cross-reactive proteins, and (iv) a protein present in soluble and cell wall fractions and immunopositive for all sera tested. In addition, one apparently nonprotein component that was enriched in the cell wall fraction was observed. Sera with high immunoglobulin G titers to one, two, three, or none of the three A. actinomycetemcomitans serotypes were observed. There was a high degree of variation from one patient to another in the humoral immune response to serotype-specific and cross-reactive antigens. As demonstrated by whole-cell adsorption experiments, the serotype specific surface antigen accounted for approximately 72 to 90% of the total antibody-binding activity for sera with titers greater than 100-fold above background, while cross-reactive antigen accounted for less than 28%. Antibody binding the whole-cell sonicate for high-titer sera was inhibited 90% by lipopolysaccharide from the same serotype, strongly suggesting that lipopolysaccharide is the immunodominant antigen class. PMID- 1705244 TI - Resolution of chlamydial genital infection with antigen-specific T-lymphocyte lines. AB - To determine cell-mediated immune mechanisms involved in the resolution of chlamydial genital infection of mice, we utilized an established murine model in which it has been demonstrated that resolution of infection occurs independently of the antibody response. Splenic T lymphocytes were obtained from mice that had previously been immunized with viable elementary bodies of the mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. Antigen-reactive T lymphocytes were maintained and expanded in vitro by frequent restimulation with UV light inactivated MoPn in the presence of antigen-presenting cells and recombinant interleukin-2 (rIL-2). Flow cytometry indicated that this cell line was at least 92% positive for the pan-specific T-cell marker Thy1.2. Stimulation of the cells in the presence of syngeneic antigen-presenting cells plus MoPn antigen and in the absence of exogenous IL-2 induced the cells to produce IL-2 activity in culture supernatants. Following adoptive transfer, this T-lymphocyte line was effective in inducing resolution of an ongoing MoPn genital infection in congenitally athymic nude mice which otherwise maintain chronic unresolved infections. The line was less efficient in resolving the infection after longer periods in culture. An additional T-lymphocyte line was derived from the spleens of athymic mice that had received the first line and had resolved the infection. These T cells were also capable of inducing resolution of the infection. Lastly, this cell line was treated with specific antibody and complement to delete either CD4+ or CD8+ T lymphocytes in an attempt to enrich for T-cell subpopulations prior to transfer into infected athymic mice. The anti-CD4-treated line was essentially depleted of CD4 cells, while the anti-CD8-treated line was only partially enriched for CD4 cells, with a large proportion of CD8 cells still present. Nude mice that received either of the treated T-cell lines or the parental cell line were capable of resolving the infection, although the line with increased numbers of CD4 cells was more efficient than either the parental line or the CD8 line. PMID- 1705245 TI - Identification of a surface-exposed immunodominant epitope on outer membrane protein P1 of Haemophilus influenzae type b. AB - Eight murine monoclonal antibodies (MAbs) directed against outer membrane protein P1 of Haemophilus influenzae type b were generated and characterized. Seven of the eight MAbs reacted with recombinant P1 and purified P1 protein from H. influenzae type b strains MinnA and 1613; MAb P1.8 was specific for the latter strain. A panel of 32 nontypeable and 140 encapsulated Haemophilus strains recovered worldwide representing the major clonal families of serotypes a, b, and d was used to evaluate the distribution among Haemophilus strains of the epitopes identified by the P1-specific MAbs. The epitope reactive with the seven MAbs which recognized P1 from strains MinnA and 1613 was shared by 92% of the encapsulated Haemophilus isolates tested. The epitope is present in the H. influenzae type b strains from clonal families commonly recovered from cases of invasive disease in North America and Europe. A series of nested 5' and 3' deletions of the P1 gene were constructed and analyzed to localize the determinants on P1 recognized by the MAbs. MAbs P1.2, P1.4, P1.5, P1.6, and P1.7 recognized an epitope localized to the carboxy-terminal portion of P1. Murine MAbs P1.1 and P1.3 and two human MAbs, HiH-7 and HiH-10, recognized a complex epitope which was partially localized to the carboxy-terminal portion of the P1 protein. These data indicate that an immunodominant surface-exposed epitope is present on the carboxy-terminal portion of the P1 protein of type b Haemophilus isolates responsible for the majority of invasive disease in North America. PMID- 1705247 TI - [Effect of hemodilution on systemic and capillary hematocrit]. AB - In a cross-over study including 10 clinically healthy male volunteers, the hematocrit in the macro- and microcirculation was investigated prior to and 1 h, 3 h, 6 h and 24 h after iso- or (3 months later) hypervolemic hemodilution using 500 ml hydroxyethyl starch. The dilution effect observed after isovolemic hemodilution is more pronounced than after hypervolemic hemodilution. While isovolaemic hemodilution with 500 ml medium molecular weight hydroxyethyl starch leads to a reduction in the systemic hematocrit by 8.5% by volume, the capillary hematocrit at the time of the most marked dilution (1 h after hemodilution) decreases by only 1.3 hematocrit points in the cutaneous capillaries. After hypervolemic hemodilution, the systemic hematocrit decreases significantly by maximally 5.4% by volume, while the capillary hematocrit does not change significantly. PMID- 1705246 TI - New model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin A from hybridoma tumors protects against Vibrio cholerae infection. AB - Secretory immunoglobulin A (sIgA) plays a role in defense against Vibrio cholerae and other microorganisms that infect mucosal surfaces, but it is not established whether sIgA alone can prevent disease. We report here a strategy for identifying the antigen specificities of monoclonal sIgA antibodies that are capable of providing such protection. IgA hybridomas were generated from Peyer's patch lymphocytes after oral immunization with V. cholerae Ogawa 395. A clone was selected that produced dimeric monoclonal IgA antibodies directed against an Ogawa-specific lipopolysaccharide carbohydrate antigen exposed on the bacterial surface. Hybridoma cells were used to produce subcutaneous "backpack" tumors in syngeneic mice, resulting in secretion of monoclonal sIgA onto mucosal surfaces. Neonatal mice bearing anti-lipopolysaccharide hybridoma backpack tumors were specifically protected against oral challenge with 100 50% lethal doses of virulent Ogawa 395 organisms. Thus, the IgA hybridoma backpack tumor method identifies protective epitopes in the mucosal system and demonstrates that a single monoclonal sIgA can be sufficient to protect against intestinal disease. PMID- 1705248 TI - [Preoperative autologous blood donation, effect of hydroxyethyl starch on the reticuloendothelial system and opsonins]. AB - The effect of 6% low molecular weight (LMW) HES (MW 270,000) on reticuloendothelial function was studied in 9 male patients, median of age 50 years, who were scheduled for vascular surgery. An erythrocyte clearance test (Anti Rh) labeled with 99 mTc, evaluating primarily the splenic section of the RES, showed a considerable variation (37-87%) prior to hemodilution. 24 hours after replacement of 15 ml/kg body weight of blood, there was no significant change of the clearance rate, a follow up weeks later approached the base line values. Plasma opsonins like fibronectin, complement fraction 3, complement fraction 4 and immunoglobulin G showed a proportional decrease following hemodilution, and after 24 hours approached control levels. Changes at this time seemed to be independent from the course of the hematocrit. It is concluded that LMW HES does not adversely influence the spleen dependent phagocytic capacity of the RES system. PMID- 1705249 TI - Substance P increases and prolongs increased output of T4 (CD4) lymphocytes from lymph nodes of sheep in vivo: is it a mediator of immunological memory? AB - There are receptors on lymphocytes for substance P which are found both on small recirculating and on blast lymphocytes. The principal effect of substance P on lymphocytes appears to be a stimulating one, both in vitro and in vivo. The in vivo administration of substance P to sheep by acute infusion into cannulated afferent lymphatics of peripheral lymph nodes has been found to stimulate efferent lymph flow and the output into efferent lymph of both small recirculating and blast lymphocytes. We here report that substance P both enhances and prolongs the enhancement of the output of T4 (CD4) lymphocytes from lymph nodes of sheep in vivo. This output-stimulating effect appears to be specific to T4 (CD4) lymphocytes and is associated with a depressant effect on the output of T8 (CD8) and B lymphocytes. The output-stimulating effect on small T4 (CD4) lymphocytes is quite prolonged, lasting in excess of 96 h after a single 50 micrograms acute infusion. A brief post-infusion depression in T4 (CD4) lymphocyte output is associated with an equally brief, but marked, elevation in the output into efferent lymph of the arachidonic acid metabolite, thromboxane B2. The output-stimulating effect of substance P on blast T lymphocytes is confined to the T4 (CD4) blast lymphocytes. Substance P or a similar molecule may be of value when a specific T4 (CD4) lymphocyte output stimulant effect is desired. A single prior (6 days) acute infusion of substance P into a popliteal lymph node via its cannulated afferent lymphatic produced profound changes in the response to nodal drainage area immunization with killed S. muenchen bacteria. The latent period prior to increased antibody production was abolished, as was the standard post-immunization 'shutdown' period of decreased output of lymphocytes into efferent lymph. These changes were accompanied by a marked and progressive increase in antibody production. The findings reported here suggest substance P-induced long-term potentiation (LTP) of the immune response and raise the question of an involvement of substance P as a major mediator of immunological memory. PMID- 1705251 TI - Keratin 19 expression in the adult and developing human mammary gland. AB - In the adult human mammary gland, most of the luminal epithelial cells express keratin 19 (K19+). However, in some small ducts and terminal ductal lobular units where branching would be expected to occur during pregnancy, the pattern of expression of this keratin is heterogeneous. While the keratin 19 negative cells (K19-) appear to have a high proliferative potential in vitro and in vivo, they have a lower secretory activity than the K19+ cells as monitored by expression of secretory component in the resting breast or casein in the pregnant gland. That the K19- cells form a separate proliferative compartment in the luminal cell lineage is suggested by the fact that they are absent in the prepubertal breast and only appear at puberty associated with branching ducts, and newly formed lobules. Our observations are consistent with the hypothesis that the K19- luminal cell is less differentiated than and may be precursor to the K19+ luminal cell, which represents the fully differentiated phenotype able to produce milk in response to a hormonal stimulus. PMID- 1705250 TI - Double and triple immunocytochemical labelling at the light microscope level in histopathology. AB - Double and triple immunocytochemical detection methods for routine use in histopathology were investigated. For double immunostaining, the combinations of immunogold-silver staining (IGSS, black) with an immunoperoxidase method (3-amino 9-ethylcarbazole, red-brown) or with an immunoalkaline phosphatase method (Fast Red TR, red) proved very useful. These were followed by a Haematoxylin counterstain. An alternative approach using immunoperoxidase (red-brown) and immunoalkaline phosphatase (Fast Blue, BB, blue) methods was also successful, particularly for frozen sections of unfixed tissue and for the establishment of intermediate filament coexpression in tumours. The coexistence of desmin with vimentin in rhabdomyosarcoma, and of glial fibrillary acidic protein with vimentin in ependymoma, could be demonstrated directly by means of non crossreacting murine and rabbit antibodies in the above combinations. The black (IGSS), red-brown (immunoperoxidase) and blue (immunoalkaline phosphatase) colours gave excellent contrast in triple immunostaining. The side-by-side demonstration of helper and suppressor T lymphocytes during renal allograft rejection, of kappa and lambda light chains in B-immunoblastic lymphoma, and of T and B lymphocyte populations in non-Hodgkin's lymphomas provided immediate information on the topography and cellular organization of the tissues. PMID- 1705252 TI - Distribution of substance p-like immunoreactivity in nerves of the gastrointestinal tract of the frog Rana esculenta L. AB - Substance P-like immunoreactivity in the alimentary canal of the frog Rana esculenta L. was studied by means of the indirect immunoperoxidase method. In all segments of the gastrointestinal tract, immunoreactivity was revealed in both the myenteric and the submucosa plexus. Stained nerve cells were observed in the myenteric plexus but not in the submucous plexus. The proximal part of the oesophagus and hindgut were free of immunoreactive perkarya. The stained nerve cells were of the Dogiel type I category in the foregut, and type II in the midgut. Both the musculature and gastrointestinal glands were supplied with immune-positive fibres. These results indicate that substance P may play similar roles in the frog gut, as described previously in mammals and fish. PMID- 1705253 TI - The effect of alpha-adrenergic receptor blockers prazosin and yohimbine on cerebral metabolism and biogenic amine content of traumatized brain. AB - Widespread decrease in local cerebral glucose utilization (LCGU) previously shown to occur 3 days after a local freezing lesion was interpreted as reflecting a depression of functional activity in the affected areas. In parallel experiments, cortical norepinephrine (NE) content of traumatized brain was found to be decreased. The effects of prazosin (PZ), an alpha 1-adrenergic receptor blocker, and yohimbine (YOH), an alpha 2-blocker, on glucose use and biogenic amine content of lesioned rat brain were studied to determine if the changes in the noradrenergic system associated with injury are of functional importance, to identify the receptors that may be involved in mediating the action of NE in injured brain, and to look for evidence of interaction between the noradrenergic and the serotonergic systems in traumatized brain. PZ (1 mg/kg) given 30 min before the lesion ameliorated the subsequent metabolic cortical depression seen in untreated animals. PZ given for 3 days starting before the lesion (3 mg/kg/day) was also effective in normalizing LCGU in areas where it was depressed by lesioning, despite the fact that this regimen induced significant global decrease in LCGU in normal animals. Once cortical metabolic depression had developed 3 days after the lesion, it could not be modified by PZ. YOH was less effective than PZ and was so only when given for 3 days (22.5 mg/kg/day in three divided doses). PZ (3 mg/kg/day in three divided doses) slightly but significantly decreased the accumulation of the serotonin (5-HT) metabolite 5 hydroxyindoleacetic acid in the traumatized hemisphere. These results provide evidence that blockage of alpha 1-adrenergic receptors prevents the development of cortical dysfunction associated with brain trauma. This implies that the noradrenergic system plays a role in the functional consequences of injury and that this effect is, at least in part, mediated by alpha 1-adrenergic receptors. Furthermore, alpha 1-adrenergic receptor blockage appears to modulate cortical turnover of 5-HT, previously also implicated in functional consequences of brain injury. The data are compatible with inhibitory effects of NE in the cortex and suggest a potential of alpha 1-adrenergic blockage in development of novel therapeutic approaches to brain injury. PMID- 1705254 TI - Chronic trigeminal ganglionectomy or topical capsaicin application to pial vessels attenuates postocclusive cortical hyperemia but does not influence postischemic hypoperfusion. AB - Marked hyperemia accompanies reperfusion after ischemia in the brain, and may account for the propensity of cerebral hemorrhage to follow embolic stroke or carotid endarterectomy, and for the morbidity that follows head injury or the ligation of large arteriovenous malformations. To evaluate the contribution of trigeminal sensory fibers to the hyperemic response, CBF was determined in 12 symmetrical brain regions, using microspheres with up to five different isotopic labels, in four groups of cats. Measurements were made at 15-min intervals for up to 2 h of reperfusion after global cerebral ischemia induced by four-vessel occlusion combined with systemic hypotension of either 10- or 20-min duration. In normal animals, hyperemia in cortical gray matter 30 min after reperfusion was significantly greater after 20 min (n = 10) than after 10 min (n = 7) of ischemia (312 ml/100 g/min versus 245 ml/100 g/min; p less than 0.01). CBF returned to preischemic levels approximately 45 min after reperfusion and was reduced to approximately 65% of basal CBF for the remaining 75 min. In cats subjected to chronic trigeminal ganglionectomy (n = 15), postocclusive hyperemia in cortical gray matter was attenuated by up to 48% on the denervated side (249 versus 150 ml/100 g/min; p less than 0.01) after 10 min of ischemia. This effect was maximal in the middle cerebral artery (MCA) territory, and was confined to regions known to receive a trigeminal innervation. In these animals, substance P (SP) levels in the MCA were reduced by 64% (p less than 0.01), and the density of nerve fibers containing calcitonin gene-related peptide (but not vasoactive intestinal polypeptide or neuropeptide Y) was decreased markedly on the lesioned side. Topical application of capsaicin (100 nM; 50 microliters) to the middle or posterior temporal branch of the MCA 10-14 days before ischemia decreased SP levels by 36%. Postocclusive hyperemia in cortical gray matter was attenuated throughout the ipsilateral hemisphere by up to 58%, but the cerebral vascular response to hypercapnia (PaCO2 = 60 mm Hg) was unimpaired. The duration of hyperemia and the severity of the delayed hypoperfusion were not influenced by trigeminalectomy, capsaicin application, or the intravenous administration of ATP. These data demonstrate the importance of neurogenic mechanisms in the development of postischemic hyperperfusion, and suggest the potential utility of strategies aimed at blocking axon reflex-like mechanisms to reduce severe cortical hyperemia. PMID- 1705255 TI - Effect of subarachnoid hemorrhage on serotonin uptake and metabolism in rabbit basilar artery. AB - The effects of subarachnoid hemorrhage (SAH) on neuronal uptake and metabolism of serotonin (5-HT) in the rabbit basilar artery were examined. Extracted 3H-amines from the isolated arteries after incubation with [3H]5-HT were separated by column chromatography. Radioactivity of 5-HT and 5-hydroxyindoleacetic acid was, respectively, 52.7 +/- 13.9 and 22.9 +/- 5.4 x 10(2) dpm/mg tissue in the control group (n = 8); 32 and 18% of control after denervation (n = 6); 99 and 12% of control after treatment with pargyline (n = 7); and 65 and 76% of control after SAH (n = 7). These results suggest that the neuronal uptake of 5-HT is impaired by SAH, although monoamine oxidase activity is relatively preserved. PMID- 1705256 TI - Gastrointestinal symptoms II. PMID- 1705257 TI - Physical symptoms. I--Respiratory and central nervous system. PMID- 1705258 TI - Short-term changes in serum insulin-like growth factors (IGF) and IGF binding protein 3 after different modes of intravenous growth hormone (GH) exposure in GH deficient patients. AB - Virtually all circulating insulin-like growth factors I and II (IGF-I and IGF-II) are bound to specific binding proteins (IGFBP), of which IGFBP-3 is the quantitatively most important. The mechanisms regulating the close coordination between serum levels of IGFs and IGFBP-3 is poorly understood. We therefore evaluated the temporal association of serum IGF-I, IGF-II, and IGFBP-3 measured by RIAs after well defined short-term GH exposure in GH-deficient patients. Six patients (mean +/- SE age: 20.5 +/- 1.1 yr) each underwent three GH study protocols in random order. Each study was preceded by 4 weeks without GH therapy. Two units of GH were administered iv as either: 1) two boluses, 2) eight boluses, or 3) a constant infusion. The duration of each study was 44 h including at least 16 h after termination of GH administration. Increments in serum IGF-I occurred 4 6 h after initiated GH exposure in all studies. In the two-bolus study the IGF-I increase was modest with mean +/- SE peak values of 12.4 +/- 2.1 nmol x L-1 after GH administration. In the eight bolus and constant infusion studies significantly higher IGF-I levels were generated: 17.0 +/- 2.2 nmol x L-1 (8 bolus) and 18.8 +/ 1.1 h nmol x L-1 (infusion). In contrast the time course change in serum IGF-II did not differ in the three studies, and it was characterised by a sluggish increase of approximately 30% evidenced after 16-20 h. The changes in IGFBP-3 were almost identical in the three studies. After a lag phase of approximately 18 20 h a gradual increase of approximately 40%, which had not ceased at the end of the study period, was observed. The molar ratio of serum IGF-I plus IGF-II:serum IGFBP-3 remained constant with values between 0.8-0.9 except in the constant infusion experiment, in which the ratio increased significantly with time reaching a mean peak value, which exceeded 1.0, after 24 h. Our data suggest that a pulsatile GH pattern is not superior to constant GH levels as regards generation of IGFs and IGFBP. The earlier increase in serum IGF-I compared to IGF II and IGFBP-3 suggests that IGF-I may be the main regulator of IGFBP-3 production. Accordingly, the slow increase in serum IGF-II, which paralleled that of IGFBP-3, could indicate that serum IGF-II levels mainly depend on the concentration or binding site availability of IGFBP-3. PMID- 1705259 TI - PEER's review: refining the early detection of developmental-behavioral murmurs. PMID- 1705260 TI - Hepatocyte expression of HBcAg and serum HBeAg in hepatitis B: comparison of polyclonal and monoclonal antibodies during a trial of interferon. AB - The distribution and quantitative expression of HBcAg in relation to serum HBeAg and liver histology before and after a trial of interferon in 50 patients with chronic type B hepatitis were evaluated using polyclonal and monoclonal antibodies. In general, both antisera showed a similar pattern in terms of the distribution of HBcAg, with predominant localisation of HBcAg in the cytoplasm in HBeAg positive patients with chronic active hepatitis. Semiquantitative analysis showed, however, that there was a higher degree of cytoplasmic expression of HBcAg with polyclonal than with monoclonal anti-HBc. Some of the HBeAg positive patients with only a focal expression of HBcAg in the cytoplasm by polyclonal anti-HBc showed no expression of HBcAg with monoclonal anti-HBc. The expression of HBcAg with polyclonal anti-HBc correlated better with the histological features of chronic active hepatitis or the persistence of serum HBeAg on follow up, suggesting that it did not result from non-specific or false positive staining. All of the HBeAg negative patients with minimal histological changes or inactive cirrhosis were HBcAg negative with both antisera. In conclusion, though both polyclonal and monoclonal antibodies produced a quite similar distribution of HBcAg in patients with chronic type B hepatitis, polyclonal antibody seemed to be more sensitive in detecting HBcAg in the cytoplasm than did monoclonal anti HBc, and the expression of HBcAg with polyclonal anti-HBc correlated better with the clinical and histological outcome. PMID- 1705262 TI - Immunoassay of P2 protein in cerebrospinal fluid in neurological disorders. AB - Cerebrospinal fluid samples were obtained at lumbar puncture from 53 patients with a wide variety of neurological disorders. Cerebrospinal fluid samples were tested for the presence of P2 protein, a constituent of myelin, with an enzyme linked immunosorbent assay technique using a specific polyclonal antibody. High concentrations of P2 in the cerebrospinal fluid paralleled a raised IgG index (clearance ratio), the presence of oligoclonal bands, as well as raised white cell counts or depressed albumin:IgG ratios. Twenty one patients had been diagnosed as having definite or probable multiple sclerosis and the remaining 32 had other conditions. Of the 13 patients with high positive P2, 12 (92%) were in the multiple sclerosis category; of the 40 patients with low (12) or undetectable (28) P2 concentrations, only nine (23%) were diagnosed as having multiple sclerosis. In this patient population the presence of high immunoreactive P2 concentrations in cerebrospinal fluid was closely associated with evidence of intrathecal immunoglobulin synthesis and with the clinical diagnosis of multiple sclerosis. On this basis it is suggested that immunoassay of P2 concentration in the cerebrospinal fluid may be of potential value in the investigation of patients with demyelinating disorders. PMID- 1705261 TI - Endothelial markers in malignant vascular tumours of the liver: superiority of QB END/10 over von Willebrand factor and Ulex europaeus agglutinin 1. AB - A new monoclonal antibody, QB-END/10, raised against the CD34 antigen in human endothelial cell membranes and haemopoietic progenitor cells, was studied for its usefulness as a marker of neoplastic vascular cells in 21 angiosarcomas and seven malignant haemangioendotheliomas of the liver. QB-END/10 was both more sensitive and more specific than Von Willebrand factor (VWF) and Ulex europaeus 1 agglutinin (UEA-1) in labelling endothelial cells and it did not cross react with epithelia as UEA-1 often does. Staining was uniformly strong and clear in all histological variants of these two tumours. QB-END/10 should prove particularly useful in the differential diagnosis of malignant vascular tumours of the liver. PMID- 1705263 TI - Monoclonal immunofluorescence compared with silver stain for investigating Pneumocystis carinii pneumonia. AB - Two hundred and eighty two specimens from 220 patients positive for HIV with respiratory tract symptoms, or febrile illness, or both, were examined for the presence of Pneumocystis carinii. Specimens were either induced sputum samples or bronchoalveolar lavage fluids. To establish the optimal method for laboratory diagnosis a comparison was made of detection of the organism by use of monoclonal antibody and immunofluorescence with conventional silver staining methods. Three commercially available reagents for immunofluorescence were also compared. Immunofluorescence was significantly more sensitive than the silver stain and the best results for immunofluorescence were obtained using. Northumbria Biologicals Ltd reagents. PMID- 1705264 TI - CD5 positive B cells in peripheral blood and lymph nodes in rheumatoid arthritis. PMID- 1705265 TI - Phosphorylation-dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord. AB - The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons. PMID- 1705266 TI - Columnar organisation of the inferior olive projection to the posterior lobe of the rat cerebellum. AB - The organisation of the olivocerebellar projection to lobules VI, VIII, and IX of the posterior lobe of the rat cerebellum was investigated in detail by using the retrograde tracer wheat germ agglutinin-horseradish peroxidase. Small, well defined rostro-caudally orientated columns of olive cells were found to project to different parasagittal areas in the posterior lobe. A column of olive cells about 2,000 microns in rostro-caudal length in subnucleus "c" and nucleus beta of the caudal medial accessory olive (MAO) provides climbing fibre input to the most medial part of lobules VI and IX, but this projection is displaced laterally in lobule VIII by a projection from a column of cells about 600 microns in rostro caudal length in lateral caudal MAO (subnucleus "a"). It is possible that each of these columns of olivary neurones may be further subdivided in the rostro-caudal axis so that different sections project to different medio-lateral parts of the cortex. A fine-grain 'sublobular' localisation may also exist: the projection to midline lobule VIc arises at caudal levels of the olive from a band of cells in the transition region between subnucleus "c" and nucleus beta, whilst by comparison the projection from caudal levels of the olive to lobules VIa and VIb arises from cells located more ventrally in nucleus beta. Evidence is also presented to confirm that the posterior lobe vermis in the rat extends further laterally than in other mammals and that part of it receives a projection from a column of olive cells, 1,000 microns in rostro-caudal length, in a newly defined region of caudal MAO, termed subnucleus "b1." PMID- 1705267 TI - Central distribution of afferent pathways from the uterus of the cat. AB - Afferent pathways from the uterus of the cat were labeled by injections of horseradish peroxidase (HRP), wheat germ agglutinin-HRP, or fluorescent dyes into the uterine cervix and uterine horns. Afferent input to the uterus arises from small to medium size neurons (average size 31 x 28 microns) in dorsal root ganglia at many levels of the spinal cord (T12-S3). The segmental origin correlates with the location of the afferent terminal field in the uterus. Eighty seven percent of the dorsal root ganglion cells (average, 822 on one side) innervating the cervix are located in sacral ganglia, whereas 97% of the cells innervating the uterine horn (average 479 on one side) are located in lumbar ganglia. Double dye labeling experiments indicate that a small percentage (average 15%) of lumbar neurons innervating the uterine cervix also innervate the uterine horn. The majority (70-80%) of afferent input to the uterine cervix passes through the pelvic nerve and the remainder through the pudendal nerve, whereas afferent input to the uterine horn must travel in sympathetic nerves. Ovariectomy (10-14 days) did not change significantly the number, sizes, or segmental distribution of uterine afferent neurons. In some cats (25%) injections of WGA-HRP into the uterine cervix labeled neurons (90-125 per animal) in lamina VII in the S2 spinal segment in the region of the sacral parasympathetic nucleus. Central projections of uterine horn afferent neurons were not labeled; however, afferent projections from the cervix were detected in the sacral spinal cord. The most prominent labeling was present in Lissauer's tract and in lamina I and outer lamina II on the lateral edge of the dorsal horn. From this region some labeled axons extended through lamina V into the dorsal gray commissure. Very few afferents were labeled on the medial side of the dorsal horn. These results are discussed in regard to the physiological function of uterine afferents and the possible transmitter role of vasoactive intestinal polypeptide, which is present in a large percentage (70%) of cervical afferent neurons. PMID- 1705268 TI - Afferent and efferent connections of the oculomotor region of the fastigial nucleus in the macaque monkey. AB - Afferent and efferent connections of the fastigial oculomotor region (FOR) were studied in macaque monkeys by using axonal transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP). When injected HRP is confined to the FOR, retrogradely labeled cells appear in lobules VIc and VII of the ipsilateral vermis and in group b of the contralateral medial accessory olive (MAO). In reference to the maps of topographical organization, the extent of the effective site in the fastigial nucleus (FN) could be assessed from the distributions of labeled Purkinje cells (P cells) in the vermis and labeled olivary neurons in the MAO. In contrast to the unilateral nature of the P-cell and climbing-fiber projections, those from the other brainstem regions to the FOR were bilateral. Following the injection of HRP into the FOR, the largest number of retrogradely labeled cells appeared in the pontine nuclei. Although the number of labeled cells was greater on the contralateral side in both the peduncular and dorsomedial pontine nuclei (DMPN), the number of each side was virtually identical in the dorsolateral pontine nucleus (DLPN). In the nucleus reticularis tegmenti pontis (NRTP), labeled cells were located only in its medial and dorsolateral portions bilaterally. In the vestibular complex, labeled cells appeared in the superior (SVN), medial (MVN), and inferior vestibular nuclei (IVN) bilaterally. The lateral vestibular nucleus (LVN), including y group and the ventrolateral vestibular nucleus, were free of labeled cells. Labeled cells appeared also in the perihypoglossal nucleus (PHN) bilaterally. In the pontine raphe (PR) and paramedian pontine reticular formation (PPRF), labeled cells appeared bilaterally in the caudal third of the area between the oculomotor and abducens nuclei. Labeled cells appeared also in the mesencephalic and medullary reticular formation. Tracing of anterogradely labeled axons demonstrated that most fibers from the FOR decussated within the cerebellum and entered the brainstem via the contralateral uncinate fasciculus. Some crossed fibers ascended with the contralateral brachium conjunctivum and terminated in the midbrain tegmentum. A small contingent of fibers advanced further to the thalamus. In the mesodiencephalic junction, labeled terminals were found contralaterally in the rostral interstitial nucleus of medial longitudinal fasciculus (riMLF) and a medial portion of FOrel's H Field. They appeared also in the central mesencephalic reticular formation (cMRF), the periaqueductal gray (PAG), the posterior commissure nucleus, and the superior colliculus. The oculomotor and trochlear nuclei, the red nucleus, and the interstitial nucleus of Cajal were free of labeled terminals.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1705269 TI - Substance P- and opioid-immunoreactive structures in olfactory centers of the cat: adult pattern and postnatal development. AB - Substance P (SP)-ir and opioid-ir structures were studied in the cat main olfactory bulb (MOB), accessory olfactory bulb (AOB), and olfactory peduncle. In the MOB, the opioid-ir and the majority of the SP-ir neurons belong to the granule cell type. SP-ir granule cells reside in the deeper granule cell layer, whereas opioid-ir granule cells reside in the superficial granule cell layer, internal plexiform, and mitral cell layer. Many granule cells are observed in the external plexiform and glomerular layer. Other granule cells were found in the bulbar/peduncular white matter, the taenia tecta, and the genu of the corpus callosum. A new substance P-ir cell type was identified in the glomerular layer. This cell type was also identified by using the technique of intracellular injection of Lucifer Yellow. The cell type corresponds neither to the external tufted type nor to the short axon cell types described so far. The AOB resembles the MOB with respect to large numbers of SP-ir and opioid-ir granule cells. In addition, a few opioid-ir neurons, probably superficial mitral cells, were found in the glomerular layer. The AOB is surrounded by islands of immunoreactive granule cells, which connect to the granule cell layer by extremely long processes. Opioid-ir and SP-ir beaded axons pass through the olfactory peduncle terminating on granule cells, and ascend as far as the glomerular layer. All subdivisions of the anterior olfactory nucleus (AON) contain immunoreactive terminal fields. Afferent fibers and terminal plexuses derive from a population of immunoreactive neurons located predominantly in the region of the septo olfactory junction. They have large somata. Their axons form recurrent collaterals, some of which run rostrally in the peduncular white matter. Others ascend caudally towards the septal region. The fibers seem to remain ipsilaterally, since the olfactory limb of the anterior commissure and the commissure proper are devoid of SP-ir and opioid-ir fibers. During development SP and opioid immunoreactivity were found only in differentiated granule cells. The peptides were not detectable in migrating or immature granule cells, as identified in Golgi-impregnated material. The granule cell population largely develops during postnatal life. The number of opioid-ir granule cells increases slowly and continuously, reaching the adult level not before the sixth postnatal month. Strikingly, SP-ir granule cell number increases fast and reaches a transient peak during the second month. Thereafter it declines (40% decrease) to the adult density, which is similar to that of opioid-ir granule cells. PMID- 1705270 TI - Macaque accessory optic system: II. Connections with the pretectum. AB - Connections of the accessory optic system (AOS) with the pretectum are described in the macaque monkey. Injections of tritiated amino acids in the pretectum demonstrate a major contralateral projection to the dorsal (DTN), lateral (LTN), and medial (MTN) terminal nuclei of the AOS and a sparser projection to the ipsilateral LTN. Injections of retrograde tracers, Fast Blue (FB), or wheat germ agglutinin horseradish peroxidase (WGA-HRP) plus nonconjugated horseradish peroxidase (HRP) in the LTN show that the pretectal-LTN projection originates from two nuclei. The main source of pretectal efferents to the LTN is from the pretectal olivary nucleus (OPN) and is entirely contralateral. This projection, which appears unique to primates, originates from the large multipolar cells of the OPN. In addition to this projection, the nucleus of the optic tract (NOT) projects to the ipsilateral LTN, as in nonprimates. Injection of WGA-HRP in the pretectum shows a reciprocal predominantely ipsilateral projection from the LTN to the pretectum. Retinas were observed after injection of FB in the LTN. The retinal ganglion cells projecting to the AOS are mainly distributed near the fovea and in the nasal region of the contralateral eye, suggesting a nasotemporal pattern of decussation. The demonstration of a direct connection between LTN and OPN forces to a reconsideration of the functional role of the AOS. Previous descriptions of luminance responsive cells in the LTN support a possible participation of this nucleus in the control of the pupillary light reflex. PMID- 1705271 TI - Sublaminar organization within layer VI of the striate cortex in Galago. AB - In this study we examined the organization of projections from the striate cortex to the dorsal lateral geniculate (GL) and pulvinar (PUL) nuclei in the prosimian Galago by using retrograde transport methods. Injections of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the PUL labeled two bands of cells in the striate cortex: the first consisted of large pyramidal cells in the upper half of layer V; the second consisted of small and medium-size pyramidal cells located in the deepest part of layer VI. The location of cells within layer VI coincided with a clear cytoarchitectonic sublayer, VIb, which contains fewer and paler staining cells than VIa. Injections of WGA-HRP involving all layers of the GL produced an uninterrupted band of pyramidal cells distributed throughout layer VI (a and b), including the region labeled after injections into the PUL. Thus as a first approximation, layer VI can be divided into an upper tier (VIa) that projects only to the GL and a lower tier (VIb) that projects to both the GL and PUL. Injections of WGA-HRP that were restricted to one or a few GL layers revealed a further refinement of the subdivisions within layer VI. Injections into the parvicellular and intercalated (or koniocellular) layers of the GL labeled neurons predominantly in the upper half of layer VIa, whereas injections restricted to the magnocellular layers labeled neurons in the lower half of layer VIa and in layer VIb. In order to determine whether individual neurons in layer VIb send axon collaterals to both the GL and PUL, we injected WGA-HRP into one nucleus and fluorescent rhodamine latex beads into the other. In three experiments, we found only one double-labeled cell. In sum, the results provide evidence that layer VI is divided into at least three sublayers: upper VIa, which projects to the intercalated and parvicellular GL layers; lower VIa, which projects to the magnocellular GL layers; and VIb, which sends separate projections to the magnocellular layers of the GL and to the PUL. The segregation observed is sufficiently discrete to propose the existence of multiple, descending pathways from layer VI of the striate cortex that complement those ascending from the GL and PUL. PMID- 1705272 TI - Galanin immunoreactivity in the mudpuppy cardiac ganglion. AB - The source of galanin-immunoreactive fibers in the cardiac ganglion and on cardiac muscle in mudpuppy (Necturus maculosus) has been determined utilizing immunohistochemical techniques. The galanin-immunoreactive fibers are not processes of afferent fibers originating in either the rostral four dorsal root ganglia or vagal sensory ganglia. Following colchicine treatment, all of the postganglionic parasympathetic neurons and a subpopulation of the small intrinsic neurons in the cardiac ganglion exhibit galanin immunoreactivity. The majority of the galanin-immunoreactive fibers that form complexes on the parasympathetic postganglionic neurons are derived from galanin-immunoreactive small intrinsic neurons, although some of these connections may represent collateral processes from other parasympathetic postganglionic neurons. All of the galanin immunoreactive processes that innervate cardiac muscle are derived from postganglionic parasympathetic neurons in the cardiac ganglion. PMID- 1705273 TI - Immunohistochemical localization of 5-hydroxytryptamine, histamine and histidine decarboxylase in the rat major pelvic and coeliac-superior mesenteric ganglion. AB - The localization of 5-hydroxytryptamine (5-HT), histamine and histidine decarboxylase (HDC), the enzyme synthesizing histamine, was studied in the rat major pelvic and coeliac-superior mesenteric ganglia by an indirect immunofluorescence technique. Small cells (10-20 microns in diameter) exhibiting 5-HT, histamine or HDC immunoreactivities were observed in clusters or occurred as solitary cells in both ganglia. In the major pelvic ganglia, solitary histamine-immunoreactive principal neurons were also observed. Colocalization studies indicated that all 5-HT-, histamine- and HDC-immunoreactive small cells in these ganglia were labelled with tyrosine hydroxylase (TH), suggesting that they are small intensely fluorescent (SIF) cells. In the coeliac-superior mesenteric ganglia, all TH-immunoreactive SIF cells were also intensely immunoreactive for 5-HT and HDC. In the major pelvic ganglia, all TH immunoreactive SIF cells contained 5-HT immunoreactivity, and the majority of them were also intensely immunoreactive for HDC. In both ganglia, however, only a subpopulation of TH-immunoreactive SIF cells displayed histamine immunoreactivity. The results indicate that in the rat major pelvic and coeliac superior mesenteric ganglia, a population of catecholamine-containing SIF cells contain 5-HT and histamine suggesting a diverse role SIF cells may have in so far as modulation of ganglion transmission is concerned. PMID- 1705274 TI - Unresectable hepatocellular carcinoma: a prospective controlled trial with tamoxifen. AB - The liver is an estrogen responsive organ. Clinically, estrogens may play a role in the induction of liver tumors and, experimentally, estrogens are involved in the control of hepatocyte proliferation. The results of a prospective controlled clinical trial using an anti-estrogen, tamoxifen, in patients with unresectable hepatocellular carcinoma (HCC) are presented below. Thirty-eight consecutive cirrhotics with HCC were allocated to either 30 mg/day tamoxifen or no treatment. The two groups of patients were matched for mean age, male/female ratio, Child Pugh risk group, approximate tumor volume (US and/or CT scan) and etiology of the underlying cirrhosis. The drug appeared to have no side effects. Survival was significantly prolonged in tamoxifen-treated patients with 22% (vs. 5%) survival at 12 months. No differences were observed between males and females or alcoholic and non-alcoholic cirrhosis. In 53% of tamoxifen-treated patients the levels of alpha-fetoprotein dropped and, in this subgroup, survival was further prolonged. Tumor volume, lactate dehydrogenase (LDH) and alkaline phosphatase slowly increased, suggesting a slower, but continuous, progression of the disease. In conclusion, anti-estrogen treatment appears effective in the palliation of unresectable or otherwise untreatable HCC. A reduction in alpha-fetoprotein levels appears to be a favorable prognostic index. PMID- 1705275 TI - Intraarterial adriamycin and lipiodol for inoperable hepatocellular carcinoma: a comparison with intravenous adriamycin. AB - The technique of 'targeting' cytotoxic drugs by mixing them with the contrast medium lipiodol is now widely used in Japan and the Far East where it has been reported to enhance response rates in patients with hepatocellular carcinoma. In the present study 19 patients with this tumour were treated with intra-(hepatic) arterial adriamycin (60 mg/m2), at least one course of which was combined with lipiodol (10-20 ml). Two patients (11%) had a remission as indicated by a significant fall in serum alphafetoprotein and there was a reduction of tumour size in one of these. The median survival period was 3 months (range 1-18) with the two responding patients surviving 8 and 12 months. This response rate was no better than the figure of 14% seen in 31 consecutive patients treated with intravenous adriamycin at the same dose, and the survival curves of the two groups of patients were not significantly different. Lipiodol in combination with adriamycin is not superior to intravenous adriamycin administered alone. PMID- 1705276 TI - [Microheterogeneity of alpha-fetoprotein in the amniotic fluid--developmental changes in the molecular structure of carbohydrate chain]. AB - By means of lectin affinity crossed-line immunoelectrophoresis, microheterogeneity of alpha-fetoprotein (AFP) was studied in 41 amniotic fluid samples between 41 to 287 days of gestation. Microheterogeneity was assessed by the structural differences in the carbohydrate chain identified by the specific binding between oligosaccharide molecule(s) and certain lectins such as concanavalin A, Lens culinaris hemagglutinin, phytohemagglutinin E or Ricinus communis agglutinin I. It was found that four carbohydrate chains were identifiable at an early stage of gestation; (1) mannose (Man).core type, (2) bisecting N-acetylglucosamine (GlcNAc) type, (3) fucose (Fuc) type, and fucosyl bisecting GlcNAc type. A carbohydrate chain with both bisecting GlcNAc and Fuc was found only at an early stage of of gestation. A carbohydrate chain with either bisecting GlcNAc or Fuc also decreased with fetal growth, and AFP at the end stage of gestation was composed mainly of Man.core type carbohydrate chain. The addition or removal of the oligosaccharide molecule takes place at the Goldi complex subjected to various modifications by the glycosylation enzyme, and the present findings indicate that a certain enzyme (s) for the processing of oligosaccharide differs according to the developmental change in AFP-producing sites or the maturation of AFP-producing cells, and such is responsible for the different AFP carbohydrate chains in the amniotic fluid at different gestational stages. PMID- 1705277 TI - Immunosuppression by gramicidin S of experimental autoimmune uveoretinitis, pinealitis and autoimmune encephalomyelitis. AB - Using an in vitro lymphocyte proliferation assay we screened several cyclic peptide antibiotics (bacitracin, oleandomycin, capreomycin, colistin, virginiamycin, and gramicidin S) for their immunosuppressive activity. Gramicidin S (GrS) was found to inhibit [3H]-thymidine incorporation into concanavalin A stimulated and E coli lipopolysaccharide-stimulated lymphocytes. In vivo studies, experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in female Lewis rats by immunization with bovine S-antigen and experimental autoimmune encephalomyelitis (EAE) were induced by immunization of rats with rat brain homogenates. GrS suppressed the onset of these inflammatory diseases at nontoxic concentrations. Evidence was obtained that GrS inhibits [3H]-thymidine incorporation into lymphocytes by preventing transport of the compound across the membrane. Since GrS binds to various cell membranes, GrS would suppress the proliferation of not only lymphocytes but also of other immune cells by modifying cell membrane properties. The present study indicates that a search for compounds which cause proper cell membrane modification should be a worthwhile strategy for development of immunosuppressive drugs. PMID- 1705278 TI - Transforming growth factor beta suppresses human immunodeficiency virus expression and replication in infected cells of the monocyte/macrophage lineage. AB - The pleiotropic immunoregulatory cytokine transforming growth factor beta (TGF beta) potently suppresses production of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome, in the chronically infected promonocytic cell line U1. TGF-beta significantly (50-90%) inhibited HIV reverse transcriptase production and synthesis of viral proteins in U1 cells stimulated with phorbol myristate acetate (PMA) or interleukin 6 (IL-6). Furthermore, TGF-beta suppressed PMA induction of HIV transcription in U1 cells. In contrast, TGF-beta did not significantly affect the expression of HIV induced by tumor necrosis factor alpha (TNF-alpha). These suppressive effects were not mediated via the induction of interferon alpha (IFN-alpha). TGF-beta also suppressed HIV replication in primary monocyte-derived macrophages infected in vitro, both in the absence of exogenous cytokines and in IL-6-stimulated cultures. In contrast, no significant effects of TGF-beta were observed in either a chronically infected T cell line (ACH-2) or in primary T cell blasts infected in vitro. Therefore, TGF-beta may play a potentially important role as a negative regulator of HIV expression in infected monocytes or tissue macrophages in infected individuals. PMID- 1705279 TI - T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes. AB - T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined. PMID- 1705280 TI - Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response. AB - Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes. PMID- 1705281 TI - T cell-T cell killing is induced by specific epitopes: evidence for an apoptotic mechanism. AB - Epstein-Barr virus-specific cytotoxic T lymphocyte clones were shown to be an effective target for their own lysis when incubated in the presence of their specific epitopes but not in the presence of irrelevant epitopes. The mode of cell killing appeared to be by apoptosis and was prevented by previously described inhibitors of the process. Degranulation, as measured by serine esterase activity, was involved in this form of T cell-T cell killing. This is the first report of T cell-T cell killing by apoptosis and is only observed in the presence of a specific epitope. This result may be of significance in the use of peptide-based vaccines. PMID- 1705282 TI - Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigen. AB - Human resting B lymphocytes enter a state of sustained proliferation when incubated with both mouse fibroblastic L cells stably expressing Fc gamma RII/CDw32 and anti-CD40 antibodies. We have explored the effects of 11 recombinant human cytokines (CKs) on induced cell proliferation and immunoglobulin (Ig) production. Interleukin 4 (IL-4) was the only CK able to enhance anti-CD40-induced B cell multiplication as measured by enumeration of viable cells, and interferon gamma (IFN-gamma) further stimulated this induced proliferation. IL-4 enhanced the production of IgM and IgG by B cells and induced them to produce IgE. Combinations of IL-4 and IL-2 resulted in the production of large amounts of IgM and IgA. Interestingly, IFN-gamma did not inhibit the production of IgE by cells stimulated with anti-CD40 and IL-4. None of the tested CK combinations resulted in the production of large quantities of IgG. Therefore, this new culture system represents a unique model to study isotype regulation in highly purified human B lymphocytes, in addition to allowing the generation of long-term factor-dependent human B cell lines. PMID- 1705283 TI - Human recombinant interferon gamma enhances neonatal polymorphonuclear leukocyte activation and movement, and increases free intracellular calcium. AB - In previous studies, we have reported that after chemotactic factor stimulation, PMNs from neonates fail to undergo certain critical activation steps. Furthermore, the concentration of free intracellular calcium reached is significantly below that of PMNs from adults. Interferon-gamma (IFN-gamma) is a lymphokine that has been shown to activate phagocytic cells, and IFN-gamma messenger RNA production by neonatal mononuclear leukocytes has been reported to be depressed. In the present studies, we found that recombinant human IFN-gamma markedly enhanced the chemotactic responses of PMNs from neonates to levels that were not different from that of PMNs from adults. Furthermore, preincubation of the neonatal cells with this recombinant human lymphokine also corrected the abnormality in intracellular calcium metabolism. These results suggest that this developmental defect in phagocytic cell movement may be the result of an intrinsic defect in IFN-gamma production resulting in deficiency of this critical phagocyte-activating lymphokine. PMID- 1705284 TI - In situ and slot hybridization analysis of RNA in colorectal tumours and normal colon shows distinct distributions of mitochondrial sequences. AB - cDNA clones of mRNAs with an abnormal abundance in familial adenomatous polyposis were used to examine the levels and distribution of the mRNAs in tissues from 15 patients with colorectal cancer. Of 12 cloned sequences studied by slot hybridization, one was substantially reduced in tumours compared with normal tissue. Sequence analysis showed this to code for IgA. In situ hybridization was consistent with slot hybridization and immunohistochemistry. Two mitochondrially encoded sequences had distinct distributions detected by in situ hybridization but did not have detectable quantitative differences in whole tumour or mucosa extracts. PMID- 1705285 TI - Adenomatous hyperplasia of the rete testis in the undescended testis. AB - Multiple foci of micronodular or tubulopapillary structures were noted in the rete testis of 13 cases of undescended testes. These structures were lined by low columnar to cuboidal epithelium, showed back-to-back crowding, and were supported by a thin lamina propria. These changes, referred to as adenomatous hyperplasia of the rete testis, appear to be a frequent finding in the undescended testis. An age-matched control group did not show any of these features. PMID- 1705286 TI - The fate of vacuolation in liver cells with special reference to hyaline globules. AB - This study was undertaken in rats to clarify the mechanisms and time necessary for recovery from vacuolation in liver cells. Vacuoles were produced by congestion of the liver due to constriction of the inferior vena cava just below the diaphragm, and changes in vacuoles were examined quantitatively and qualitatively until 24 h after release of the constriction. Vacuoles in liver cells decreased in number by half within 5 min after recovery from congestion. The remaining vacuoles metamorphosed to hyaline globules by condensation of the contents. The number of hyaline globules increased with a peak occurring at 3-6 h after recovery from congestion, although the number of vacuoles decreased gradually. Only a few small vacuoles and hyaline globules were found in liver cells in pericentral areas at 24 h after recovery from congestion. These data indicate that vacuoles may be discharged promptly from the liver cell cytoplasm after recovery from congestion, and the remaining vacuoles may metamorphose to hyaline globules by condensation of the contents and finally fade into the cytoplasm. PMID- 1705287 TI - Oestrogen receptor staining of breast carcinomas: a comment. PMID- 1705288 TI - Allergic rhinitis model with Brown Norway rat and evaluation of antiallergic drugs. AB - An animal disease model of allergic rhinitis was developed with Brown Norway (BN) rat, a high immunoglobulin E responder strain. BN rats were immunized with ovalbumin (OA) and made to suffer from allergic rhinitis. The severity of allergic rhinitis was assessed by determining the extent of the three kinds of markers, Evan's blue, histamine and N-acetyl-beta-D-glucosaminidase, released into the nasal perfusate following the OA challenge to the nasal cavity of OA sensitized BN rats. This experimental system was appreciated by antiallergic drugs; chlorpheniramine maleate inhibited the release of Evan's blue and elevated that of histamine, but did not affect the N-acetyl-beta-D-glucosaminidase level. Halopredone acetate inhibited the releases of all the three markers. The estimation of the released markers using allergic rhinitis brought about in BN rats was found to be a useful experimental system for evaluating the effect of drugs on allergic rhinitis. PMID- 1705289 TI - Biliary excretion of mitomycin C dextran conjugates in relation to physicochemical characteristics of carrier dextran. AB - Biliary excretion characteristics of polymeric prodrugs of mitomycin C (MMC), mitomycin C-dextran conjugates with cationic or anionic charge (MMC-Dcat, MMC Dan) and with different molecular weights of dextran (10,000, 70,000, and 500,000) were studied in rat. Following intravenous injection, bile was periodically collected and concentrations of free and dextran-conjugated MMC in it were determined by bioassay. MMC administered as a free form was excreted rapidly into bile and 1.8% of the dose was recovered within 2 h. A small amount of MMC was gradually excreted into bile after administration of all MMC-Ds and total recovery at 8 h was less than 1% of dose. In this case, a major part of excreted MMC was recovered as a conjugated form. MMC-Dcat gave a larger total excretion of MMC than MMC-Dan and excretion was also affected by the molecular weight of carrier dextran. The biliary recovery of MMC-Dcat labeled with 14C at a spacer moiety was significantly higher than that of conjugated MMC determined by bioassay suggesting release and/or inactivation of MMC in MMC-D during the circulation in the body. These results were compared with biliary excretion of model macromolecules with the same molecular weight but different electric charge in order to clarify the effect of electric charge on the biliary excretion of macromolecules. Cationic macromolecules exhibited higher biliary excretion in relation to greater hepatic uptake. PMID- 1705290 TI - Channels formed in phospholipid bilayer membranes by diphtheria, tetanus, botulinum and anthrax toxin. AB - Diphtheria, tetanus, botulinum, and anthrax toxin are multipartate toxins, one of the domains of which is (or is presumed to be) an enzyme. Cell intoxication requires that the enzymatic portion gain access to the cytosol via endocytosis into an acidic vesicle compartment of the cell. Translocation of the enzyme across the vesicular membrane is dependent on the low pH of the vesicle and involves another domain of the toxin; for each of these toxins, that domain is capable of forming channels in phospholipid bilayer membranes. These channels are large (greater than 12 A diameter) and voltage-gated, and the pH conditions required for their formation in lipid bilayers are similar to those existing in acidic vesicles and required for cell intoxication. PMID- 1705291 TI - Microheterogeneity of acute phase proteins in the differentiation of polymyalgia rheumatica from polymyositis. AB - We studied an alpha-1-acid glycoprotein (AGP) and an alpha-1-antichymotrypsin (ACHT) microheterogeneity in sera of patients with polymyalgia rheumatica (PMR), giant cell arteritis (GCA/PMR), polymyositis/dermatomyositis (PM/DM) and healthy individuals by affinity electrophoresis with concanavalin A (Con-A) as the ligand. Our results are expressed as reactivity coefficients. The mean of AGP reactivity coefficients (AG-RC +/- SD) in PMR (0.92 +/- 0.17) and GCA/PMR (0.91 +/- 0.12) were significantly lower compared with the mean AG-RC in patients with PM/DM (1.48 +/- 0.52) as well as in healthy individuals (1.34 +/- 0.9). Moreover, an additional microheterogeneous form of AGP was noted in patients with PM/DM. In parallel, we also found that the mean of ACHT reactivity coefficients (AC-RC +/- SD) were lower in patients with PMR (2.94 +/- 1.24) and GCA/PMR (1.66 +/- 0.16) compared with healthy individuals (3.92 +/- 1.17). The mean of AC-RC in patients with PM/DM (6.74 +/- 4.35) was significantly higher than in patients with PMR and GCA/PMR as well as in healthy individuals. Our results show that the changes in reactivity of AGP and ACHT with Con-A are useful diagnostic markers for the differentiation of PMR and GCA/PMR from PM/DM. PMID- 1705292 TI - Human chorionic gonadotropin expression by bladder cancers: biology and clinical potential. PMID- 1705293 TI - Correlation of ultrasound guided and digitally directed transrectal biopsies of palpable prostatic abnormalities. AB - The authors evaluated 51 patients with palpable prostatic abnormalities detected during digital rectal examination. These findings consisted of a nodule or an area of induration. Each palpable abnormality was confined to 1 prostatic lobe and there was no suggestion of extracapsular extension of neoplasm or systemic metastatic disease. All patients underwent 7.0 MHz. sagittal ultrasound guided transrectal biopsy followed by digitally directed transrectal biopsy. Biopsies were obtained only from the area of interest. The procedure was performed in the outpatient clinic without use of sedation or anesthesia. Digitally directed biopsies were positive for adenocarcinoma in 9 lesions. Ultrasound guided biopsies detected adenocarcinoma in 23 lesions, including all those detected by the blind digitally directed technique. This study demonstrates greater diagnostic accuracy using 7.0 MHz. ultrasound guided techniques and its routine use is warranted in the evaluation of palpable prostatic abnormalities. PMID- 1705294 TI - Adjuvant radiation therapy in patients with detectable prostate specific antigen following radical prostatectomy. AB - Adjuvant radiation therapy following radical prostatectomy for adenocarcinoma of the prostate was given to 25 patients. Of these patients 8 had microscopic lymph node metastasis, 8 had seminal vesicle invasion without positive lymph nodes, 6 had positive surgical margins and 3 had only capsule penetration. Their only evidence of residual disease was detectable serum prostate specific antigen (PSA) by the Yang assay. A total of 15 patients (60%) had a subsequent decrease in PSA to less than 0.3 ng./ml. and an additional 5 (20%) had a decrease in PSA by more than 50%. Currently 8 patients have no detectable PSA after a median followup of 18 months (17 to 38 months) since initiating radiation therapy. Only 1 of 12 patients with detectable PSA immediately after radical prostatectomy has had a durable response to adjuvant radiation therapy. In contrast 7 of 13 patients with a delayed increase in PSA had a durable response. The ability of adjuvant radiation therapy to eliminate serum PSA in patients with a delayed increase in PSA after radical prostatectomy is encouraging. However, longer followup, including the use of nonradiated control subjects, is needed to assess the ability of adjuvant radiation therapy to control local disease and prolong patient survival. PMID- 1705295 TI - Patients with lower motor spinal cord lesion: a decrease of vasoactive intestinal polypeptide, calcitonin gene-related peptide and substance P, but not neuropeptide Y and somatostatin-immunoreactive nerves in the detrusor muscle of the bladder. AB - Specimens of the detrusor muscle of the bladder from four patients with lower motor neurone lesion and three patients with carcinoma of the bladder used as "controls", were studied immunohistochemically for vasoactive intestinal polypeptide, neuropeptide Y, calcitonin-gene related peptide, substance P and somatostatin. The greatest density of nerves in the bladder from "control" patients contained neuropeptide Y, followed in a decreasing order by vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P and somatostatin. Neuropeptide Y- and vasoactive intestinal polypeptide immunoreactive nerves were found throughout the smooth muscle and the base of the mucosa, while calcitonin gene-related peptide-, substance P- and somatostatin immunoreactive nerves were found predominantly in nerve bundles with a few single fibres at the base of the mucosa. Vasoactive intestinal polypeptide-, neuropeptide Y- and calcitonin gene-related peptide-immunoreactive nerves were also located around blood vessels. In patients with lower motor neurone lesion, there was a decrease in the density of vasoactive intestinal polypeptide-, calcitonin gene-related peptide- and substance P-immunoreactive nerves, but there was little change in neuropeptide Y- or somatostatin-immunoreactive nerves. Urinary retention, bladder areflexia and deficient sensation may be directly linked to neuropeptide neuropathy in patients with lower motor neurone lesion. PMID- 1705296 TI - Cytostatic effects of suramin on prostate cancer cells cultured from primary tumors. AB - Suramin is currently undergoing clinical trials as a chemotherapeutic agent for prostate cancer. The effects of suramin on cultured human epithelial cells derived from normal, benign hyperplastic, and malignant prostate tissues were examined. In serum-free medium, suramin inhibited the clonal growth of prostate cells at a half-maximal dose of approximately 10 micrograms/ml. Growth inhibition by suramin was completely reversible even after 24 hours of exposure. In conjunction, suramin did not alter cellular phenotype with regard to expression of keratins and prostate-specific antigens. Although suramin is reportedly an antagonist of growth factor-mediated mitogenesis, ten-fold excesses of growth factors did not appreciably suppress the cytostatic activity of suramin. In comparison to the activities of other possible chemotherapeutic agents, suramin would appear suboptimal because its inhibitory effects are reversible and it does not induce a terminally differentiated cellular phenotype. PMID- 1705297 TI - [Indomethacin inhalation therapy]. AB - We studied the effect indomethacin (IND) on electrical properties of airway epithelial cells and on sputum production in patients with chronic respiratory infection. Addition of IND (3 x 10(-6) M) to the mucosal side of Ussing chamber reduced short-circuit current from 5.9 +/- 1.2 to 1.7 +/- 0.5 microA/cm2 (p less than 0.001, n = 12) in canine cultured tracheal epithelium, an effect that was dependent on CI. Inhalation of IND for two weeks resulted in a decrease in the amount of sputum from 208 +/- 25 to 102 +/- 19 ml/day, (p less than 0.01, n = 13), and the concentrations of cyclooxygenase products in the sputum without alteration of inflammatory parameters. These results indicate that IND inhalation is a promising therapy in improving excessive sputum production and that this effect may be attributable to inhibition of Cl secretion and the subsequent reduction in water movement toward the airway lumen. PMID- 1705298 TI - Treatment of male impotence: a new option. PMID- 1705299 TI - [A new concept for keratoprosthesis]. AB - Although keratoprosthesis (after Strampelli and Cardona) is successful in selected cases, it remains doubtful whether the apparent problems of these procedures can be solved. The Cardona-Strampelli concept was therefore abandoned; a new design was evolved and new alloplastic materials were used--silicone for the optical zone and carbon fibers for the haptic part of the prosthesis. The interaction of living tissue with these alloplastic materials was investigated in tissue-culture experiments and in vivo, and indicated that the materials had excellent properties for prosthetic purposes. Stable anchorage of the implants was assured by the strong adhesion of the fibroplasts to the carbon fibers. In more than a year of observation no granulomatous reaction was observed. In animal experiments, the special design of the prosthesis-tissue interface prevented epithelial downgrowth, one of the major problems in keratoprosthesis. Moreover, a fluid-tight interaction of the alloplastic material and the tissue was achieved. PMID- 1705300 TI - Differential expression of extracellular proteins is correlated with angiogenesis in vitro. AB - Strains of bovine aortic endothelial cells, grown on plastic under conventional culture conditions and in the absence of growth factor supplementation, exhibited a sprouting phenotype and a predisposition toward the formation of cords and tubular structures. We examined endothelial cells at different stages of tube formation. Analysis of metabolically labeled proteins showed that the synthesis of type I collagen was initiated in sprouting cells and during the formation of tubular structures. SPARC (secreted protein, acidic and rich in cysteine) a Ca2(+)-binding protein associated with cellular shape change and morphogenetic processes (Sage H, Vernon RB, Funk SE, Everitt EA, Angello J: J Cell Biol 109:341, 1989), was upregulated during spontaneous tube formation. Levels of messenger RNA for type I collagen and SPARC corroborated the stage-specific increases observed for these proteins. Differential levels of transcription were apparent in multilayered cells directly involved in tube formation, in comparison with cells comprising either the tubes or the confluent monolayers at a distance from the tubes. Analysis of DNA synthesis indicated that multilayered sprouting cells in the proximity of the endothelial tubes were actively proliferating, whereas cells that had been incorporated into tubes showed low levels of DNA synthesis. Immunolabeling studies revealed a dense accumulation of SPARC and type I collagen in the cytoplasm of cells that were situated near the growing tubes. Two other secreted proteins, type III collagen and thrombospondin, were expressed constitutively by subconfluent cultures and were increased in those cells contributing to tube formation. We propose that type I collagen and SPARC are specifically related to the angiogenesis-like phenomenon displayed by bovine aortic endothelial cells in vitro. Type I collagen might facilitate the active migration of endothelial cells, or the stabilization of the resulting tubes, with SPARC directing the re-organization and dynamic assembly of the tubular network. PMID- 1705301 TI - High molecular weight components are main constituents of Mallory bodies isolated with a fluorescence activated cell sorter. AB - Mallory bodies (MBs) are cytoplasmic filamentous aggregates containing cytokeratin (CK) material. They occur in hepatocytes of patients with alcoholic liver disease (i.e., alcoholic hepatitis) and can also be induced experimentally in mice by chronic griseofulvin intoxication. To further investigate components and mechanisms involved in MB formation, a new method for MB purification was established. MBs present in a liver homogenate of griseofulvin-fed mice were labeled with a murine monoclonal antibody specific for MBs and a second fluorescein isothiocyanate-conjugated (anti-mouse IgG and IgM) antibody and subsequently isolated by two sequential sorting procedures using a fluorescence activated cell sorter (FACS). Purity of MB isolates was over 90% as revealed by computer analysis of sorting signals and fluorescence and electron microscopy. Electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gels revealed three MB-related polypeptides with apparent molecular masses of 48, 55, and 65 kilodaltons but most of the highly purified MB material did not enter the gel or remained at the interphase between stacking and resolving gels. Western blotting with CK-specific antibodies showed the presence of CK epitopes in the high molecular weight MB material, which has a similar amino acid composition as normal liver CKs. These results establish that very high molecular weight material is the main constituent of MBs and suggest that a post-translational modification of CKs by covalent crosslinks is a principal mechanism of MB pathogenesis. PMID- 1705302 TI - F-met peptide-induced degranulation of human basophils. AB - We examined the kinetics of morphologic change induced by stimulation of human basophils with f-Met peptide using partially purified cells from normal donors. Supernatants were collected at 30 and 60 minutes and assayed for histamine with an automated fluorometric technique. Samples of basophils were prepared for electron microscopy at 0, 10, 20, 30 seconds and 1, 2, 5 and 10 minutes poststimulation with f-Met peptide. We found that f-Met peptide, a bacterial peptide, induced a unique sequence of morphologic events that included morphologies we have previously identified and termed piecemeal degranulation in human basophils in situ as well as those induced by IgE mechanisms ex vivo and termed anaphylactic degranulation, thus supporting a general degranulation model for basophils and mast cells (Dvorak HF, Dvorak AM: In Clinics of Haematology, Granulocyte and Monocyte Abnormalities, Vol 4, edited by Lichtman MA, p 651. London, WB Saunders Co, Ltd, 1975). In addition to this degranulation continuum, we found that chambers of releasing granules underwent extraordinary increases in size as they emptied their contents and before their resolution by extrusion. The enlarging granule chambers accumulated numerous concentric dense membranes, vesicles, and Charcot-Leyden crystals. These early changes generally preceded 1/2 maximum histamine release, whereas the later extrusion of full granules, emptied granules and their membranous contents coincided with 1/2-maximum histamine release (Warner JA, Peters SP, Lichtenstein LM, Hubbard W, Yancey KB, Stevenson HC, Miller PJ, MacGlashan DW Jr. J Leukocyte Biol 45:558, 1989). Shedding of membranes from several sources accompanied extrusion of granules and intragranular Charcot-Leyden crystals. These sources included the expanded granule membranes from empty granules, granule membranes from full granules, collections of intragranular concentric dense membranes and vesicles, and surface membranes and processes. These extraordinary membrane shifts were generated and persisted over the 10-minute period examined and coincided with the later time frame within which leukotriene C4 was generated and released from human basophils stimulated by f-Met peptide (Warner JA, Peters SP, Lichtenstein LM, Hubbard W, Yancey KB, Stevenson HC, Miller PJ, MacGlashan DW Jr: J Leukocyte Biol 45:558, 1989). Viable basophils, completely free of both full and empty granules, showed some morphologic evidence of recovery of granule products by 10 minutes after stimulation with f-Met peptide. The unique morphology of f-Met peptide-induced degranulation of human basophils is supported by the uniqueness of the biochemical events associated with this trigger.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1705303 TI - Acute pulmonary and renal injury after administration of heterologous anti-lung antibodies in the rat. Characterization of ultrastructural binding sites, basement membrane epitopes, and inflammatory mediation systems. AB - Male Sprague-Dawley rats developed acute lung and renal injury after administration of heterologous anti-lung antibody. Both organs demonstrated an increase in protein permeability after antibody binding to basement membrane (BM) antigens (lung permeability index 0.342 +/- 0.009 versus control 0.214 +/- 0.011: p less than 0.05. Urinary protein excretion 5.112 +/- 0.899 mg/hour versus control 0.402 +/- 0.008 mg/hour: p less than 0.01). The threshold value for the development of lung injury was 27.2 +/- 4.8 micrograms of antibody globulin/g of tissue (micrograms/gm). Immunoblot analysis probing with the anti-lung antibody revealed at least one common antigenic determinant (82 to 84 kilodaltons) bound within collagenase-solubilized pulmonary and glomerular BMs. Increasing doses of antibody produced hemorrhagic pneumonitis and diffuse alveolar damage. Immunofluorescence microscopy confirmed linear alveolar and glomerular BM antibody binding. Immunogold electron microscopy allowed precise identification, in intense linear patterns, of BM binding sites within lung and glomeruli. Functional lung injury was prevented by either leukocyte-depletion or complement depletion (lung permeability index antibody-treated, complement-depleted 0.235 +/ 0.034: both p greater than 0.05 compared with controls). Injury mediation in this acute humoral model of lung damage is both complement- and leukocyte dependent, as previously described for the renal component of heterologous anti glomerular BM antibody-induced inflammatory disease. PMID- 1705304 TI - Fractionated irradiation and early changes in salivary glands. Different effects on potassium efflux, exocytotic amylase release and gland morphology. AB - Irradiation is a potent treatment modality of head and neck cancer. However, the irradiation is usually associated with an influence on salivary glands with ensuing dryness and discomfort for the patients. In the present study we used different in vitro secretory models and morphologic characterization of rat parotid gland. Radiation was given to one gland on a 5-day schedule with 6 MV photons (total dose 20, 30, 35, 40, 45 Gy). The contralateral gland served as control, and the analysis of glands were performed 10 days after the last irradiation treatment. The noradrenaline stimulated electrolyte secretion (86rubidium tracer for potassium) was decreased in relation to the irradiation dose and in comparison to contralateral control glands. Noradrenaline stimulated exocytotic amylase release was not affected by irradiation and, there were no signs of obvious quantitative morphologic alterations after irradiation compared with controls. The results suggest that there are differences in the sensitivity to radiation for the two different secretory processes in salivary glands, and, thus, the structures regulating electrolyte and fluid secretion seem to be more vulnerable to irradiation than the process of exocytosis. The results, however, do not allow discrimination between temporary cellular impairment and irreversible damage leading to cell death. PMID- 1705305 TI - Recruitment of neutrophils in the local endotoxin response: association with de novo endothelial expression of endothelial leukocyte adhesion molecule-1. AB - Endothelium is central to the cellular infiltration that develops during inflammation, and a prominent feature of its involvement is the expression of adhesion molecules for circulating leukocytes. In the present study, we assessed the kinetics of endothelial adhesion molecule expression during the cutaneous endotoxin response in baboons. Immunostained cryostat sections and hematoxylin and eosin-stained paraffin sections of skin biopsies were examined using set scoring systems to provide semiquantitative data on the changes in endothelial phenotype and induced polymorphonuclear leucocyte (PMN) accumulation. Endothelium in control skin did not express endothelial leukocyte adhesion molecule (ELAM)-1 but did show a relatively weak expression of intercellular adhesion molecule (ICAM)-1. By 2 hours after injection of endotoxin (500 mcg of Escherichia coli derived lipopolysaccharide), a marked expression of ELAM-1 developed that was associated with concurrent extensive adhesion and extravasation of PMN. The ELAM 1 expression subsequently decreased and was virtually absent by 9 hours. Mean scores for endothelial expression of ICAM-1 changed comparatively little over this time course, and mononuclear cell accumulation was minimal. The response to endotoxin differs from that to tumor necrosis factor injection; the latter causes sustained ELAM-1 expression, and delayed but pronounced increases in ICAM-1, with accompanying mononuclear cell extravasation. Thus, local endotoxin administration provides a model of acute inflammation in which PMN accumulation is associated with striking endothelial expression of ELAM-1. In this model, appreciable elevations in ICAM-1 expression are unnecessary for PMN infiltration. PMID- 1705306 TI - In vitro effects of recombinant hemopoietic growth factors on progenitor cells from patients with myelodysplastic syndromes. AB - The effects of four recombinant hemopoietic growth factors (HGF) and of the impure factor HTB9 on proliferation and maturation of marrow myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells were studied in 22 cases of myelodysplastic syndromes (MDS). In most cases, IL-3, GM-CSF and G-CSF increased significantly the number of myeloid colonies, the best combination being IL-3 + GM-CSF. A significant increase in the myeloid colony/cluster ratio was also noted, but cytological examination of colony cells showed little maturation. The analysis of myeloid colony surface markers with four monoclonal antibodies (to CD13, CD15, CD33 and CD34) showed minor modifications with an increase of CD13 and CD15 in about one third of cases when compared to control without HGF. Erythroid colonies were obtained in one case with erythropoietin alone, and in 19 cases with the addition of GM-CSF and/or IL-3. In short-term liquid cultures, IL-3, GM-CSF and G CSF increased 3H-thymidine incorporation. We conclude that progenitor cells of most MDS are able to proliferate in the presence of HGF, with wide case-to-case variations. However, the pattern of growth remains abnormal when compared to normal marrow. Although the combination of IL-3 and GM-CSF is the most efficient, there is a large overlap in the stimulating effects of all factors studied. PMID- 1705307 TI - [5-alpha reductase inhibitor in prostatic adenoma?]. PMID- 1705308 TI - [Cross-reacting antigens of amebas and rat intestinal mucosa]. AB - The experiments have shown the presence of cross-reacting systems in Entamoeba histolytica and in intestinal tissues of random noninbred albino rats. The antigenic affinity between E. histolytica and E. moshkovskii was expressed very weakly. PMID- 1705309 TI - Control of excessive lead exposure in radiator repair workers. AB - In 1988, 83 automotive repair workers with blood lead levels (BLLs) greater than 25 micrograms/dL were reported to state health departments in the seven states that collaborated with CDC's National Institute for Occupational Safety and Health (NIOSH) in maintaining registries of elevated BLLs in adults. In 18 (22%) of these 83 persons, BLLs were greater than 50 micrograms/dL. Among automotive repair workers for whom a job category was specified, radiator repair work was the principal source of lead exposure. The major sources of exposure for radiator repair workers are lead fumes generated during soldering and lead dust produced during radiator cleaning. This report summarizes current BLL surveillance data for radiator repair workers and describes three control technologies that are effective in reducing lead exposures in radiator repair shops. PMID- 1705310 TI - Structural and functional identification of GP57/51 antigen of Trypanosoma cruzi as a cysteine proteinase. AB - Purified GP57/51, a Trypanosoma cruzi glycoprotein earlier identified as a major antigen in infected humans, was subjected to N-terminal sequence analysis. Alignment of the first 30 amino acids revealed that its N-terminal region is virtually identical to that reported for a cysteine-proteinase isolated from the Tulahuen strain, including the presence of active site cysteine at position 25. The finding of serine at position 24 of GP57/51 (Y strain) has further increased the homology between this protozoan antigen with other members of the eukaryotic family of cysteine proteases, including human cathepsin L. Functional analysis of GP57/51 indicated that the antigen is indeed an active thiol proteinase, which is active across a wide pH range (5-7.5). This was shown using either human IgG or gelatin substrates co-polymerized into polyacrylamide gels prepared for electrophoresis, and also by enzyme assays peformed with the synthetic substrate Z-phe-arg-NMec. The enzyme was activated by thiol containing reagents, and was strongly inhibited by low concentrations of E-64 (IC50 0.1 microM), cystatin (IC50 1 microM), leupeptin (IC50 0.1 microM) and antipain (IC50 0.1 microM). Monoclonal antibodies directed against distinct epitopes of GP57/51 absorbed the hydrolytic activity from purified preparations, demonstrating that the antigenic and enzymatic activities were indeed expressed by the same molecular entities. The subcellular localization of immunoreactive molecules was investigated by electron microscopy; immunogold staining was conspicuously found in vesicles belonging to the endosomal-lysosomal system, in tissue culture trypomastigotes as well as in epimastigotes. The possibility that this highly antigenic protease is actively secreted and/or leaked out of damaged parasites is under investigation; its release to tissues and to the circulation may contribute to pathology, considering that it (i) can degrade proteins across a wide pH range and (ii) stimulates immune T cells from chronic chagasic patients. PMID- 1705311 TI - Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin. AB - Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which may be applicable to both epidemiological research and clinical studies. PMID- 1705312 TI - Sensitivity of ultrasound in detecting spina bifida. PMID- 1705313 TI - The disturbance of nonverbal functions in dysphasia. AB - Various modalities of six neuropsychological functions (graphia, calculia, finger gnosis, right-left orientation, praxia and constructive praxia) referred to as parietal or nonverbal have been investigated in the light of speech disorders. We examined 20 patients with brain lesion of vascular origin, who met the diagnostic criteria of mild and moderate dysphagia, 13 patients with Wernicke's and 7 with Broca's dysphasia. Verbal and nonverbal functions in patients with ischemic focuses of the speech area of the left hemisphere were investigated the Boston Diagnostic Aphasia Examination (BDAE). The investigation revealed that the presence and the type of mild and moderate dysphasia had a noteworthy role in pathoplasticity of correlated signs, thus implying in clinical practice a parietal lesion. Generally, poorer and at the same time more heterogeneous results were obtained in patients with Wernicke's dysphasia, mostly on calculia and right-left orientation. Finger agnosia was not considered as an authentic parietal sign, while each modality of graphia was impaired to a varying extent in speech disorders caused by presylvian and retrosylvian lesions. The paper also deals with the significance of lobulus parietalis inferior in speech. PMID- 1705314 TI - [Experimental hemorrhagic shock: 6% dextran in solution with 7.5% NaCl versus 6% dextran in solution with 0.9% NaCl]. PMID- 1705315 TI - Time-course variations induced by pargyline on the 5-hydroxyindole compounds measured in the nucleus raphe dorsalis and in blood: a voltammetric and HPLC approach in the rat. AB - Ninety min after pargyline (Pargy, 75 mg/kg) injection, the +300 mV voltammetric signal, measured in the nucleus raphe dorsalis (nRD) of freely moving rats, disappeared completely. This effect was also followed (2-7 h post-injection) by the reappearance of a peak (post-Pargy-peak) at the same +300 mV potential. The height of this post-Pargy signal was still decreased by Pargy (30 or 75 mg/kg). Endogenous 5-hydroxyindoleacetic acid (5-HIAA) contents, measured with HPLC in the nRD of Pargy-treated rats, exhibited analogous variations. Inhibition of monoamine oxidases by Pargy seems thus to be effective in blocking 5-HIAA production over only 2 h. The 15-fold increase of 5-HT endogenous content in the nRD (3 h after injection) was not reflected in the voltammetric extracellular measurements performed 90 min to 7 h after Pargy injection; this increase is suspected to be mainly intracellular. PMID- 1705316 TI - Differential organization of crossed and uncrossed projections from the superior olive to the inferior colliculus in the mole. AB - Projections from the medial (MSO) and lateral (LSO) superior olivary nuclei to the inferior colliculus (IC) were examined in the mole. In each mole, Fluoro-Gold was injected into one IC and wheat germ-agglutinated horseradish peroxidase (WGA HRP) into the other IC; most MSO neurons were double-labeled bilaterally, while most LSO neurons were single-labeled bilaterally. The results indicate that the bilateral projections from the MSO and LSO to the IC are mainly established by single MSO neurons with axon collaterals to the IC on both sides, and by two separate populations of LSO neurons with axons to the contralateral or ipsilateral IC. PMID- 1705317 TI - Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones. AB - The whole-cell patch-clamp technique was used to examine the effect of substance P (SP) on glutamate-induced currents in freshly dissociated rat spinal dorsal horn neurons (LI-III). In 48% of examined cells SP (10(-10)-10(-6) M) at -70 mV, induced in inward current that desensitized in the continued presence of SP. When applied simultaneously with, or prior to L-glutamate, SP caused a potentiation of L-glutamate-induced current in 65% of the tested cells. Since glutamate activates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in rat dorsal horn neurons, selective agonists, kainate, quisqualate, alpha-amino-3-hydroxy-5-methyl 4-isoxazole-propionic acid (AMPA) and NMDA were used to determine which subtype of excitatory amino acid receptors interacted with SP. We found that the responses to quisqualate, kainate, and AMPA were not significantly affected by SP (less than 20% increase). In contrast, the inward currents induced by NMDA (30 300 microM) appear to be reduced and potentiated after the administration of 2 200 nM of SP. These results suggest that post-synaptic mechanisms of action of tachykinins may contribute to the regulation of the strength of glutamate mediated excitatory transmission in the rat spinal dorsal horn. PMID- 1705318 TI - Interleukin-3 and phorbol esters induce different patterns of immediate-early gene expression in an interleukin-3 dependent cell line. AB - The myeloid interleukin-3 (IL-3) dependent cell line, FDC-P1, enters the G0 stage of the cell cycle after IL-3 deprivation for 24 hr. The expression of 13 protooncogenes and immediate-early genes was compared with 4 "control" genes after the addition of either IL-3 or phorbol myristate acetate (PMA) to IL-3 deprived cells. mRNA transcripts encoding c-myc and the T-cell receptor c-gamma gene were induced to high levels only after IL-3 addition, whereas c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were induced transiently only after PMA addition. The remaining genes (fra-1, p53, jun-D, c-Ha-ras, c-Ki-ras, c-raf, beta actin, ornithine decarboxylase, and histone 2B) were detected after culture with either IL-3 or PMA. When cells were serum- and IL-3-deprived, c-fos, fos-B, c jun, jun-B, Krox-20, and Krox-24 were detected after exposure to either serum or PMA. Moreover, culture with cycloheximide and PMA resulted in superinduction of these genes, whereas cycloheximide and IL-3 addition did not. mRNAs encoding these genes had half-lives of 10-20 min after PMA treatment, whereas that of beta actin was longer (greater than 2 hr), suggesting that short mRNA half-lives contributed to the transient nature of these genes. Although c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 expression can be detected in IL-3-dependent cells after exposure to either PMA or serum, these genes were not detected after IL-3 addition, which allows cell-cycle progression. These results document the existence of IL-3 and PMA-responsive genes and demonstrate that IL-3 and protein kinase C agonists can induce distinct patterns of gene expression. PMID- 1705319 TI - Introduction of an activated RAS oncogene into murine bone marrow lymphoid progenitors via retroviral gene transfer results in thymic lymphomas. AB - Activating mutations within the various RAS protooncogenes have been detected in many human and murine hematopoietic neoplasms. Their causal significance has been difficult to assess, partly due to the lack of an animal model directly linking these mutations to hematopoietic neoplasms. A high-titer, helper-free recombinant retrovirus was used to introduce an activated Harvey RAS gene under the transcriptional control of the Moloney leukemia virus LTR into murine bone marrow cells. Eleven of fifteen mice reconstituted with these bone marrow cells developed fatal thymic lymphomas 10-12 week post-transplant. Analysis of DNA and RNA from tumor cells revealed the integrated proviral genome and provirally encoded RAS mRNA respectively. Immunophenotyping and T-cell receptor rearrangement analysis of fresh tumor cells and of cell lines derived from these tumors showed them to be T-cells arrested midway through thymic development. Despite evidence of proviral integration in marrow cells of mice with thymic tumors, no other hematologic abnormalities were detected. The short latency and reproducibility of thymic lymphoma development in mice transplanted with marrow transduced with this retrovirus suggests a direct causal effect of expression of an activated RAS gene in the transformation of a bone-marrow-derived lymphoid progenitor. PMID- 1705320 TI - Characterization of Int-5, a locus associated with early events in mammary carcinogenesis. AB - Three chemically-induced precancerous mammary hyperplasias, independently isolated in BALB/c mice, all contained mouse mammary tumor virus (MMTV) proviral DNA integrated into a common region in chromosomal DNA, designated Int-5 (Formerly Int-H, Gray et al., 1986). This site was cloned from a hyperplastic outgrowth (D2) into lambda phage. A 1.7 Kb Hind III DNA fragment, which flanks the 5' end of the MMTV insert, was generated from the cloned Int-5 region. This fragment was used as probe (IH-2) to localize Int-5 to mouse chromosome 9. The IH 2 sequence was highly conserved in DNA of several mammalian species including man, in three other widely divergent vertebrate phyla, and in C. elegans. The Int 5 region, containing 5.6 Kb 5' and 12.8 Kb 3' to the MMTV integration site in D2, was cloned in EMBL-3 from a BALB/c genomic DNA library. cDNA complementary to poly A+ lactating mammary gland RNA, annealed with Sst-1 fragments spanning most of the BALB/c Int-5 clone. The highest level of Int-5 specific poly A+ mRNA was detected in D2 tumor. Lactating mammary gland and D2 hyperplastic alveolar nodule contained 5-fold less Int-5 RNA while liver contained 8-fold less Int-5 RNA. Int 5 cDNA (IH-3) annealed with two RNA species of approximately 3.3 and 4.0 Kb. These data are consistent with the hypothesis that Int-5 contains an oncogene, different from any other previously described, involved in early events in some models of chemical carcinogenesis. PMID- 1705321 TI - Preparation of a monoclonal antibody, SZ-51, that recognizes an alpha-granule membrane protein (GMP-140) on the surface of activated human platelets. AB - To identify the changes on the platelet plasma membrane after platelet activation, we prepared monoclonal antibodies specific for activated platelets. Hybridoma cell line SZ-51 was screened for antibodies which bound to thrombin activated platelets but not to resting platelets. 125I-SZ-51, and IgG1, bound 780 molecules/platelet on resting platelets. However, it bound 11,000 molecules/platelet on thrombin activated platelets with high affinity (4.2 nmol/L). The isolated protein using affinity chromatography showed a single band in both periodic acid-Schiff and Coomassie blue staining. Immunoblot analysis revealed that SZ-51 reacted with a 140,000 molecular weight protein which was identified to be alpha-granule membrane protein (GMP-140) in radioimmunoassay. These results demonstrate that the antigen recognized by SZ-51 is GMP-140, which is expressed on the plasma membrane of activated platelets. PMID- 1705322 TI - Hematologic data in 825 cases of beta-thalassemia trait in Spain. AB - We present the hematologic data of 825 cases of beta-thalassemia trait (687 cases of beta-thalassemia trait and 138 cases of delta beta-thalassemia trait). There were no significant differences between the red cell indices of the patients with beta and delta beta-thalassemia trait. In patients with beta-thalassemia trait, MCV was significantly reduced in 97% of the males and 99% of the females. All the patients with delta beta-thalassemia trait showed low MCV values. Red cell morphology was altered in the vast majority of cases, with basophilic stippling in 96% of the patients. Most patients came from provinces with the highest incidence of malaria in the past. PMID- 1705323 TI - The secret sharer as a guide to compassion. AB - Joseph Conrad's novella demonstrates the ways in which over-identification with a stranger influences and clouds the critical judgment of a young professional. It provides rich material for discussion on the caution needed by young nurses to make decisions based on critical judgment and not personal feelings. PMID- 1705324 TI - Vagal stimulation for control of complex partial seizures in medically refractory epileptic patients. AB - Chronic intermittent stimulation of the vagus nerve is a new method currently being tested for the treatment of medically intractable complex partial seizures (CPS). We have studied the effects of vagal stimulation in nine patients with CPS for 4-16 months to determine its safety and efficacy. With the patients maintained on constant dosages of antiepileptic drugs, we recorded the electroencephalogram and electrocardiogram, and performed clinical laboratory tests and gastric analysis over a 6-week baseline period. The neurocybernetic prosthesis (NCP) was then implanted and connected to two spiral electrodes wound around the left vagus nerve. After a 4-week placebo period, vagal stimulation was started. Stimulation parameters were increased stepwise at monthly intervals until patients were being stimulated for 30-second periods at 20-50 Hz with 1-2 mA of current at 250-500 microseconds pulses. A second 4-week placebo period was added 3 months after the implantation. Thereafter, vagal stimulation was resumed and self-stimulation with magnetic activation was allowed for a 1-minute period at the onset of an aura. Six patients had a significant reduction in the frequency, intensity, or duration of seizures. All patients tolerated the implantation and stimulation well and none reported pain, discomfort, or important changes in their daily activities, sleep habits, eating, swallowing, or breathing. There were no remarkable changes in blood pressure or heart rate. PMID- 1705325 TI - Characterization and modification of brain activity with deep brain stimulation in patients in a persistent vegetative state: pain-related late positive component of cerebral evoked potential. AB - A series of eight patients in a persistent vegetative state (PVS) were subjected to chronic deep brain stimulation (DBS) for the purpose of promoting recovery from the PVS. The characteristics of the brain activity in these patients were evaluated from the late positive component of the cerebral evoked potential in response to painful stimuli (pain-related P250). While any neurological scoring system for the comatose state includes evaluations of motor reactions to painful stimuli, the pain-related P250 is unique in terms of its ability to assess the cortical responsiveness to painful stimuli directly and quantitatively without involving functions of the motor system. It was found that the pain-related P250 was more or less depressed in patients in a PVS. It was repeatedly demonstrated in four patients, however, that the pain-related P250 could be transiently increased by preceding stimulation of the mesencephalic reticular formation. Furthermore, a persistent increase in the pain-related P250 was produced in these four patients following chronic DBS of the mesencephalic reticular formation or nonspecific thalamic nuclei for more than 6 months, and this was correlated with the clinical improvements. These results imply that responsiveness at the cortical level to pain is depressed in the PVS. It also appears that some fraction of the depression may, however, be functionally produced and potentially reversible. PMID- 1705326 TI - Treatment of cerebral ischemia with electrical stimulation of the cervical spinal cord. AB - We observed an increase in cerebral blood flow (CBF) for control of pain but were otherwise normal. Based on that observation, we implanted stimulators for cervical spinal cord stimulation (cSCS) in three patients who had symptomatic cerebral ischemia. Two had severe basivertebral occlusive disease and one had bilateral carotid occlusive disease. In all three cases, cSCS alleviated the symptoms of ischemia. Xenon-CBF studies or single-photon emission computer tomography (SPECT) showed increased CBF in response to cSCS. Although no mechanism clearly responsible for this remarkable therapeutic efficacy can be proposed yet, further clinical trials of cSCS for inoperable cerebral ischemia may be justified. PMID- 1705327 TI - Cerebral hemodynamics during spinal cord stimulation. AB - An increase of cerebral blood flow (CBF) during spinal cord stimulation (SCS) has been shown in man using the 133Xenon inhalation technique. We report the effects of SCS on cerebral hemodynamics studied with transcranial Doppler sonography (TCD). Twenty-six patients with epidural electrodes implanted at the cervical level (11 patients) and at the thoracic level (15 patients) were investigated with recordings of CBF velocity in the middle cerebral arteries before and during SCS. Sixty-three and two-thirds percent of patients with cervical stimulation and 29.4% of patients with thoracic stimulation showed a decrease of cerebral vascular resistance simultaneously with an increase of the velocity of CBF. PMID- 1705328 TI - Dual chamber pacing aborts vasovagal syncope induced by head-up 60 degrees tilt. AB - To determine if pacing might prevent syncope in cardioinhibitory 'Malignant Vasovagal Syndrome' (also known as 'Neurally-Mediated Bradycardia/Hypotension'), a study of dual chamber pacing during head-up 60 degrees tilt was undertaken. Paired invasive tilts were performed in 10 patients who had a history of recurrent syncope, normal routine investigations including electrophysiological study and prior tilt-induced vasovagal syncope. Vasovagal reactions of identical severity were produced by prolonged 60 degrees head-up tilt on consecutive days in seven out of 10 patients. On day 2, without pacing, seven patients had tilt induced vasovagal reactions and six became syncopal during the reaction. On day 3, with temporary DVI pacing with rate hysteresis, seven patients had tilt induced vasovagal reactions and 1 patient was syncopal. Syncope was aborted in the other five patients. DVI pacing significantly improved cardiac index (CI) (one +/- 0.2 to 1.6 +/- 0.3 L/min/m2, P less than 0.01) and mean arterial blood pressure (MABP) (30 +/- 11 to 48 +/- 12 mmHg, P less than 0.01) during vasovagal reactions on day 3 compared with day 2. The mean period of time that patients could tolerate in the tilted position after the onset of the tilt-induced vasovagal reaction was significantly prolonged by pacing from 0.9 +/- 1.2 to 3.2 +/- 1.6 min (P less than 0.01). Dual chamber pacing may abort syncope in 85% of patients with cardioinhibitory malignant vasovagal syndrome. Pacing may prolong consciousness sufficiently during a vasovagal reaction to allow injury to be avoided. PMID- 1705329 TI - Treatment of thalamic pain by chronic motor cortex stimulation. AB - All forms of therapy, including chronic stimulation of the thalamic relay nucleus, can provide satisfactory pain control in only 20%-30% of cases of thalamic pain syndrome. In order to develop a more effective treatment for thalamic pain syndrome, we investigated the effects of stimulation of various brain regions on the burst hyperactivity of thalamic neurons recorded in cats after deafferentiation of the spinothalamic pathway. Complete, long-term inhibition of the burst hyperactivity was induced by stimulation of the motor cortex. Based on this experimental finding, we treated seven cases of thalamic pain syndrome by chronic motor cortex stimulation employing epidural plate electrodes. Excellent or good pain control was obtained in all cases without any complications or side effects. During the stimulation, an increase in regional blood flow of the cerebral cortex and thalamus, a marked rise in temperature of the painful skin regions, and improved movements of the painful limbs were observed. These results suggest that thalamic pain syndrome can be most effectively treated by chronic motor cortex stimulation. PMID- 1705330 TI - Implantable electrical stimulator-alloprosthesis in repair of postoperative hernias. AB - An implantable electrical stimulator-alloprosthesis produced better regeneration of connective tissue for prevention of recurrences in patients after surgery for postoperative hernias. The experiments demonstrated that the use of the electrical stimulator-alloprosthesis resulted in significant improvement in connective tissue regeneration, tissue proliferation through the prosthesis and in the speed of post-operative suture healing. The device was successfully used in 30 patients. The period of treatment decreased by an average of 10 days. The method is recommended for wide clinical use, especially in cases of postoperative recurrent hernias and in patients with suppressed regenerative ability of connective tissue. PMID- 1705331 TI - A simultaneous, noninvasive comparison with sinus rhythm, of two activity sensing, rate adaptive pacemakers, in an elderly population. AB - We compared the rate response to low level work, arm exercise, step testing, and treadmill exercise between the Siemens Sensolog 703 S (Sg) and the Medtronic Activitrax (Ax) pacemakers, and simultaneous sinus rhythm (SR) in an elderly population. In ten subjects mean age 70.6 years, range 49-81 years, the pacemaker responses were noninvasively compared by strapping the units to the chest wall under constant and equal pressure applied by an adjustable belt. Pacemaker rates and SR were recorded simultaneously on a three-channel ECG recorder. The units were programmed in situ to give an increase in rate from 70 beats/min sitting, to 100 beats/min after walking at a normal pace for 30 seconds. Programming took three times longer for Sg (P less than 0.02). The response to standing and bending was poor for both units (Ax mean 75.7 beats/min, Sg mean 77.6 beats/min), when compared to SR (mean 90.3 beats/min). A 20-step test resulted in a greater rate response from Ax (mean 105.3 beats/min) than from Sg (mean 96.3 beats/min [P = 0.09]), though both were still less than SR (116.3). There were significant differences between the two pacemakers on treadmill testing, at peak rate achieved (Ax mean 112.5 beats/min; Sg mean 102.7 beats/min [P less than 0.005], SR mean 122.5 beats/min) and at end exercise (Ax mean 112.5 beats/min; Sg mean 92.9 beats/min [P less than 0.002], SR mean 121.3 beats/min). Arm exercise, however, resulted in a significantly greater response from Sg than Ax (105.1 and 88.5 beats/min, respectively; P less than 0.01, SR 98.7 beats/min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705332 TI - Advances in applications of radiofrequency current to catheter ablation therapy. AB - In the past 5 years, numerous reports have appeared of the use of radiofrequency (RF) current as an energy source for catheter ablation therapy of cardiac arrhythmias. Laboratory investigations using in vitro and in vivo animal models to study the biophysical and histologic effects as well as to assess the efficacy and safety of this technique in ablating various cardiac structures have been extensively performed and have led to successful applications in humans. Clinical experience with RF catheter ablation of the AV junction and accessory pathway has proliferated each year. However, experience with RF ablation of the VT focus remains quite limited. With further advances in catheter electrode design for mapping and ablation, a widespread application of RF catheter ablation to the treatment of cardiac arrhythmias is anticipated. PMID- 1705333 TI - The right ventricular outflow tract as an alternative permanent pacing site: long term follow-up. AB - The long-term characteristics of the right ventricular outflow tract have been assessed as an alternative permanent pacing site to the right ventricular apex. Thirty-three consecutive patients requiring ventricular pacing were randomized to be paced from one of the two sites. Pacing was performed using a screw-in lead, and a programmable pacemaker was used to facilitate threshold testing. There was no significant difference in the lead positioning time or any acute implant measurement (e.g., threshold at 0.5 msec 0.4 +/- 0.2 V for both sites, P = 0.99). Chronic measurements were also comparable during follow-up (mean 73 months) with a mean threshold at most recent follow-up of 0.15 +/- 0.2 msec (apex) and 0.13 +/ 0.21 msec (outflow tract) at 5 V, P = 0.81. There was only one pacing related complication, a lead dislodgment (outflow tract) in a pacemaker twiddler. Overall, both sites were highly satisfactory. PMID- 1705334 TI - A technique for intraoperative arrhythmia induction during automatic implantable defibrillator placement. PMID- 1705336 TI - Biostim 1990. PMID- 1705335 TI - Performance of implantable cardiac rhythm management devices. PMID- 1705337 TI - Autonomic stimulation. AB - Therapeutic stimulation of the autonomic nervous system has been limited by lack of qualitative or quantitative evaluation of autonomic mechanisms. This article provides an historical review of knowledge about autonomic pathways and critical evaluation of available tests of autonomic function. Recent developments in evaluation of autonomic dysfunction and improvement in techniques of neurostimulation have facilitated the development of a number of clinically useful treatments for bladder control, sexual problems, peripheral vascular disease, angina pectoris, and seizure disorders. Future therapeutic measures may allow specific control of hypertension, pain, cardiac arrhythmias, trophic disorders and balance. PMID- 1705338 TI - Safety of external cardioversion/defibrillation in patients with internal defibrillation patches and no device. AB - Placement of prophylactic epicardial defibrillation patches at time of open-heart surgery in patients at risk for postoperative arrhythmias has been strongly questioned. Concern has centered on the ability to safely perform subsequent external defibrillation if needed. From 61 patients who were treated with a two stage strategy we identified 17 who, while wearing epicardial patches and no generator, received external cardioversion/defibrillation for 20 episodes of hemodynamically unstable ventricular arrhythmias. All the patients had one small and one large patch. Eighteen of the episodes were induced during electrophysiological testing (with transthoracic shocks delivered via pad electrodes oriented in an apex-posterior configuration) and two were spontaneous. The episodes occurred at 21 +/- 27 days from patch implant. Thirteen episodes (65%) were converted with one shock at an energy level of 185 +/- 65 J. Seven (35%) required a second shock at 351 +/- 22 J. The accumulated energy requirement was 286 +/- 205 J. No adverse outcomes were noted. The number of episodes requiring more than one shock and the energy requirements were not different from those in a control group of 20 similar arrhythmias treated with the same equipment. Under these conditions, external cardioversion/defibrillation in patients with one large and one small epicardial defibrillation patch was uniformly successful. Further data is needed in the out-of-hospital setting and on the results of external defibrillation in patients with two large patches. PMID- 1705339 TI - Neurophysiological effects of left vagal stimulation in man. PMID- 1705341 TI - The implantable neurocybernetic prosthesis system. AB - The neurocybernetic prosthesis system (NCP) is an implantable, multiprogrammable pulse generator that delivers constant current electrical signals to the vagus nerve for the purpose of reducing the frequency and severity of epileptic seizures. The signals are delivered on a predetermined schedule, or may be initiated by the patient with an external magnet. The device is implanted in a subcutaneous pocket in the chest just below the clavicle, similar to pacemaker placement. The stimulation signal is transmitted from the prosthesis to the vagus nerve through a lead connected to an electrode which is a multi-turn silicone helix, with a platinum band on the inner turn of one helix. The prosthesis can be programmed with any IBM- compatible personal computer using NCP software and a programming wand. PMID- 1705340 TI - Balance and cognitive impairment in two epileptic patients before and after vagal nerve stimulation. AB - Balance and cognition were assessed in two patients with uncontrolled complex partial seizures. The patients were on anticonvulsant medications and were treated with left vagal stimulation. Balance and cognition were assessed before and after vagal stimulation, and the results were compared with age matched controls and older patients with Parkinson's disease. Severe impairments of function were found in the epileptic patients, and such negative effects of medication make vagal stimulation a potentially practical alternative treatment for uncontrolled epilepsy. PMID- 1705342 TI - Vagal stimulation reduces the severity of maximal electroshock seizures in intact rats: use of a cuff electrode for stimulating and recording. AB - J. Zabara showed that repetitive vagal stimulation (VS) prevents or ameliorates convulsive seizures in dogs. We have studied the effects of VS on maximal electroshock seizures (MES) in intact rats: (1) A 5 wire cuff electrode was developed for stimulating and recording from the vagus. Compound action potentials (AP) were recorded and strength-duration curves obtained for A and C fibers. There is a monotonic relationship with a negative slope between heart rate (HR) and AP amplitude. C fibers remain excitable for 25 days after cuff implant. (2) The anticonvulsant efficacy of VS is directly related to the fraction of vagal C fibers stimulated and the frequency of stimulation. (3) The anticonvulsant efficacy of VS has been established using two rat models of human epilepsy. VS abolishes the extensor component of the tonic phase of a MES and shortens or prevents tonic seizures induced by pentylenetetrazol (PTZ). (4) VS appears to act via release of large quantities of the inhibitory mediators GABA and glycine throughout large volumes of the brain. (5) It is rational to test VS in man as a treatment for intractable seizures. PMID- 1705343 TI - Aortic regurgitation during systole: color flow mapping and Doppler interrogation following the Damus-Kaye-Stansel procedure. AB - Echocardiographic evidence of systolic aortic regurgitation following a Damus Kaye-Stansel procedure for palliation of complex double-outlet right ventricle is presented. This procedure directs left ventricular output to the aorta through a proximal main pulmonary artery-aortic anastomosis and utilizes a valved conduit between the right ventricle and distal pulmonary artery. Postoperative Doppler and color flow echocardiographic findings revealed systolic and diastolic regurgitation from the native aorta to the right ventricle. Aortic valve closure at the time of the original Damus-Kaye-Stansel procedure would eliminate regurgitant flow and circumvent subsequent closure of this valve due to increased systolic aortic regurgitation. PMID- 1705344 TI - Effective use of patient education illustrations. AB - Effective illustrations can greatly enhance patient education materials, yet many illustrations do not aid instruction as much as they could. By following the above recommendations, patient education materials developers and illustrators can together accomplish their objectives. PMID- 1705345 TI - Case study: development and evaluation of a model of patient education in surgical wards. AB - In this contribution we offer a description of a research project in which an attempt is undertaken to improve patient education by nurses at surgical wards in hospitals. In a pilot study it was found that the current practice of patient education is inconsistent and often does not meet patients' demands. Based on this pilot, measures are developed to (a) standardise the information supplied to patients and (b) promote patients' opportunities to collect information by themselves, in order to meet individual demands. The effectiveness of the measures is evaluated by collecting data on physical recovery and emotional well being among patients undergoing cholecystectomy or herniorraphy. PMID- 1705346 TI - Enhancing surgical nurses' patient education: development and evaluation of an intervention. AB - This research tested whether staff nurses could provide enhanced patient education and whether increases in education improved surgical patient outcomes. A protocol for patient education was developed from earlier research. Then a multifocal intervention was implemented to motivate and teach staff nurses and to increase structural support for patient education. Following the intervention, patients reported receiving more preoperative information and psychosocial support, but not skills training. These increases occurred without measurable opportunity costs in other areas of nurses' work and generalized to nontargeted patient groups. Concomitantly, patients experienced shortened postoperative hospital stays and decreased use of anti-emetics/sedatives and hypnotics, demonstrating the clinical effectiveness of the increased education. PMID- 1705347 TI - Hospital-based case management for medically fragile infants: program design. AB - A hospital-based multidisciplinary team is demonstrating services for medically fragile infants which facilitate transition from the hospital to the community and home. The team provides education and counseling for communities and families to overcome barriers to services. With individualized case management, the team facilitates family access to the community services they need in infant health, infant development and family functioning. The team plans the infant's discharge from the hospital and collaborates on the initial Individualized Family Service Plan (IFSP) with the family and local community providers. The initial IFSP details potential outcomes in the areas of infant health, infant development and family functioning for the first year the infant is home. Systematic home visits with the team's nurse or developmental consultant, the family and the local provider continue the education and counseling process during the infant's first year of life. This describes the educational aspect of case management services for medically fragile infants. PMID- 1705348 TI - Hospital-based case management for medically fragile infants: results of a randomized trial. AB - A recent federal law has expanded the eligibility for multidisciplinary evaluation and assessment of infants suspected of developmental delay. At the same time, modern neonatal care has increased infant survival. These two developments have created a need for family education regarding the need for health and developmental intervention, as well as counseling to maintain family participation in these services. This study compares the education and counseling services of a hospital-based case management team with the traditional discharge and follow-up services of a neonatal intensive care unit (NICU). Subjects were the 10% most medically severe infants discharged from a neonatal intensive care unit over a 16-month period. Preliminary data suggests a significantly greater number of families accessed community-based, coordinated, comprehensive health and developmental services when they received case management services. The data suggest that hospital professionals generally refer the most medically severe 3% of this group for services under a traditional model. However, the additional 7% referred under the transition model showed greater developmental benefits from the services during the first 6 months of life. These data support current federal initiatives for early intervention services which are family-centered, community-based and coordinated. PMID- 1705349 TI - Ionic requirements for catecholamine aggregating actions on the teleost Poecilia reticulata melanophores. AB - Norepinephrine (NE) and phenylephrine (Phe) were employed to study the ionic requirements for alpha adrenoceptor activation in the teleost Poecilia reticulata melanophores. As expected the beta adrenoceptor blocker, propranolol, increased the sensitivity of the preparation to NE (5.8 times), and was therefore employed in all the experimental procedures. Neither cocaine (a neuronal uptake blocker) nor dexamethasone (an extraneuronal uptake blocker) enhanced the sensitivity of the preparation to NE, suggesting that these inactivating mechanisms would not play a role in P. reticulata pigmentary system. However, in the absence of calcium, the dose-response curve (DRC) to NE was displaced to the left about 3.5 times, whereas the DRC to Phe was not affected. These results indicate that a neuronal uptake is active, but was not demonstrated by the classical pharmacological tools, probably due to an assymmetric display of the nervous endings. The DRC to NE was rightward displaced (14.1 times) in the presence of the calcium channel blocker Verapamil, whereas the DRC to Phe was not affected. These data suggest that P. reticulata melanophores possess a mixed population of alpha 1 and alpha 2 adrenoceptors, the activation of the latter eliciting an extracellular calcium influx. In sodium-free saline, the DRC to NE was rightward shifted (6.6 times) and the response to Phe was impaired in such a way that the maximal response was not achieved. The DRC to both NE and Phe were rightward displaced (7.9 and 2.7 times respectively) in the presence of the sodium channel blocker tetrodotoxin (TTX) 10(-7)M.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705350 TI - Effects of varying frequency of sympathetic stimulation on chloride and amylase levels of saliva elicited from rat parotid gland with electrical stimulation of both autonomic nerves. AB - Simultaneous stimulation of the parasympathetic and sympathetic nerves to the parotid gland of rats elicited saliva at a rate dependent on the frequency of sympathetic stimulation when parasympathetic frequency was maintained at 16 Hz. The flow rate was lowest at 2 Hz (sympathetic), moderate at 5 Hz, and highest at 16 Hz. Cl concentration of the saliva evoked with stimulation of both nerves was highest at the highest frequency and flow rate (maintained at the level of 102 mEq/liter, for 35 min) and lowest at 2 Hz (declining from 40 mEq/liter initially to 28 mEq/liter). With sympathetic nerve stimulation alone, Cl concentration ranged from 27 to 58 mEq/liter when frequency was varied from 2 to 16 Hz, and with parasympathetic stimulation alone (16 Hz), it ranged from 132 to 124 mEq/liter. Amylase concentration of sympathetically elicited saliva was, in contrast, highest at 2 Hz (1.5 times the level at 5 Hz, and twice the level at 16 Hz), and nearly 18-38 times that seen with parasympathetic stimulation alone. The same pattern was found when both nerves were stimulated, and at 2 Hz (sympathetic), amylase concentration was 1.6 times the level at 5 Hz and 2.6 times the level at 16 Hz. When the two nerves were simultaneously stimulated, the total amount of amylase secreted over 35 min was twice as high as that observed with sympathetic nerve stimulation alone, at any frequency. The relation of frequency to norepinephrine concentration was examined. There was no consistent difference in norepinephrine concentration related to variation in frequency of sympathetic stimulation. Only when both nerves were stimulated at 16 Hz was there a statistically significant reduction in norepinephrine concentration of 46%. A relation between frequency of sympathetic stimulation, flow rate, amylase concentration, and Cl concentration was established, but these changes could not be directly correlated with quantitative differences in norepinephrine concentration. PMID- 1705351 TI - Bleomycin: a pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis. AB - Bleomycin is a unique DNA-interactive antitumor agent that has become a popular tool in studies of the pathogenesis of interstitial pulmonary fibrosis. The biochemical and morphological changes seen in the lungs of many species after bleomycin simulate those seen in humans. The availability of these animal models of interstitial pulmonary fibrosis also provides the opportunity to investigate novel pharmacological approaches to preventing this disease. PMID- 1705352 TI - Microvascular A-V shunts and the growth of autologous tissue flaps in Millipore chambers. AB - An animal model was developed using the male Sprague-Dawley rat to establish a protocol and design criteria for the growth of autologous tissue based on a microvascular pedicle. It became apparent that growth within the chamber depended heavily on membrane porosity and the presence of the microarteriovenous shunt. Different membrane porosities ranging from 0.0, 0.25, 1.2, to 8.0 microns were evaluated (n = 48). Optimal growth occurred with the 1.2-microns and the 8.0 microns micropore Millipore. Growth within the chamber consisted of a radial deposition of collagen and neovascularization originating from the arteriovenous (A-V) anastomosis. In contrast, control chambers (no A-V anastomosis), with the preceding range in membrane porosity, experienced little to no cell growth. In addition, the majority of A-V shunts did not remain patent in chambers having 0.0 microns porosity or 0.25-microns porosity. Thus it is apparent that a strong relationship exists between membrane porosity, patency, and in situ vascularization allowing for the proliferation of cells and collagen. PMID- 1705353 TI - Immunological rejection of grafted tissue in xenogeneic neural transplantation. PMID- 1705354 TI - Restoration of function to the denervated spinal cord after implantation of embryonic 5HT- and substance P-containing raphe neurones. PMID- 1705355 TI - Extensive efferent projections of intra-striatally transplanted striatal neurons as revealed by a species-specific neurofilament marker and anterograde axonal tracing. PMID- 1705356 TI - Behavioral recovery from MPTP-induced parkinsonism in monkeys after intracerebral tissue implants is not related to CSF concentrations of dopamine metabolites. PMID- 1705358 TI - Development of substance P (SP)-containing cells in the central nervous system: consequences of neurotransmitter co-localization. PMID- 1705357 TI - Angiogenesis and the blood-brain barrier in solid and dissociated cell grafts within the CNS. AB - Available evidence suggests that blood vessels indigenous to solid CNS and peripheral tissues grafted to the brain are sustained and maintain the morphological and permeability characteristics they manifest in normal life. Furthermore, these vessels of graft origin anastomose (albeit not rapidly) with vessels of the surrounding host tissue predominantly at the host-graft interface and less so, or not at all, within the graft itself. For these reasons, blood brain and brain-blood barriers, evident in the late fetal and neonatal CNS, can be expected to exist within CNS grafts placed intracerebrally or extracerebrally, providing the graft remains viable. Peripheral neural and non-neural tissues not possessing cellular barriers to circulating macromolecules do not acquire such barriers subsequent to their transplantation within the CNS. The absence of a blood-brain barrier in the adrenal gland grafted intracerebrally may be relevant for the treatment of Parkinson's disease with blood-borne therapeutics. Compared to solid tissue grafts, cell suspension grafts have the potential of becoming vascularized rapidly. That cell suspensions of neurons and of glia are supplied with BBB vessels of host origin and that the permeability characteristics of host BBB vessels are altered by a tumor cell suspension reaffirm the belief that the type of transplanted cell/tissue indeed determines the permeability characteristics of the blood vessels supplying it. The suspected immunologic privilege of the CNS is not absolute. Eventual host rejection of allografts placed within the third ventricle may be a dual consequence of the absence of a BBB at the level of the host median eminence and involvement of the minor histocompatibility complex. PMID- 1705359 TI - [Anemia-induced focal cerebral symptoms in carotid stenoses. Observations of pathophysiology]. AB - Four patients with cerebral ischemic events related to anemia or therapeutic hemodilution were observed within a 10-month period. Upper gastro-intestinal hemorrhage was responsible in 2 patients. The 3rd suffered from chronic polyarthritis and erosive gastritis, and in the 4th instance, the decrease of hematocrit was the result of a therapeutic hemodilution for polycythemia. A significant carotid stenosis corresponding to the symptoms was diagnosed in all patients. Passage of emboli in the middle cerebral or ophthalmic artery respectively was detected in two patients during the transcranial Doppler exam. Analysis of the effect of a decrease of hematocrit on arterial stenoses suggests that cerebral ischemic events related to blood loss are partially due to induced embolism. PMID- 1705360 TI - [Alzheimer's dementia and zinc]. AB - In Alzheimer's dementia (AD) the Primum Movens is Amyloid (AM) production on precapillaries: Dyshoric Angiopathy, and capillaries: Senile Plaques (SP) producing Blood-Brain-Barrier (BBB) disturbances, entry in the brain of toxic metals which displace the zinc. Cerebral AM alone may be asymptomatic. Clinical symptoms (Amnesia, Instrumental Disorders) appear when AM induces Neighbouring neuritic alterations: Paired Hellical Filaments (PHF) and Distant neuronal body lesions: Neurofibrillary Tangles (NFT). The AM is coded by a locus on the chromosome 21 and a duplication of this locus should be the etiology of cerebral AM in AD. In AD cerebral zinc decreases particularly in the hippocampus. The zinc enzyme Superoxyde-Dismutase (SOD) is coded by a locus also on the chromosome 21 near AM and the plasma level of SOD is high in AD. Zinc deposits observed in capillary AM-SP, result probably from the excess of plasmatic SOD. Other metals: Iron, aluminium are also observed in the AM-SP and their excess in the brain may be related to the decrease of zinc by metal to metal displacement. The decrease of functional zinc in the brain may interfere in the pathogenesis of PHF-NFT by metalotoxicity, neighbouring and distant to AM. Without AM, NFT are produced also by metalotoxicity and therefore brain zinc displacement. a) by lead: Encephalopathia saturnica b) by many metals: Guam Encephalopathy c) by aluminium d) by BBB disturbances leading probably to an abnormal entry of metals in the brain (Dementia Pugilistica, viral encephalitides). NFT may be produced by the deficiency of the following zinc enzymes: 1. Those of DNA metabolism, indicating abnormal DNA and therefore abnormal protein synthesis: PHF-NFT. 2. Those of neuronal detoxication: SOD, Carbonic Anhydrase, Lactate Dehydrogenase leading to neuronal toxicity particularly in the hippocampus normally rich in SOD. 3. Of Glutamate (GLU) Dehydrogenase (GDH) resulting in an excitotoxic increase of GLU. 4. Those of the metabolism of neurotransmitters (NT): neuropeptides, Histamine, GABA, Acetylcholine. Therapeutic proposition: a zinc complex crossing the BBB should be useful a) to prevent that the AM produces PHF-NFT by Neighbouring and Distant metalotoxicity and DNA changes; b) to regularise zinc-enzymes of neuronal detoxification and of neurotransmitters metabolisms. Preliminary trials by zinc Aspartate give yet promising results. PMID- 1705361 TI - [Multiple psychotherapy--how many therapists are needed?]. AB - For a variety of reasons it seems worthwhile to consider Multiple Psychotherapy: 1. A multicausal understanding of psychic and mental illnesses has already led to installing more and more multiple therapeutic settings. 2. Patients suffering from so-called early disturbances show a tendency to split their transference between different members of a therapeutic team. By intentionally employing several team members one may therapeutically make use of these split transferences. 3. As a consequence of the enormous general interest in the psychiatric and therapeutic professions a great number of helpers are at hand, who often do not have enough work and could usefully be brought into action in multiple treatment. This paper presents an overview over multiple Therapy literature and discusses it. PMID- 1705362 TI - Enhancement of HIV-1 cytocidal effects in CD4+ lymphocytes by the AIDS-associated mycoplasma. AB - Coinfection with Mycoplasma fermentans (incognitus strain) enhances the ability of human immunodeficiency virus type-1 (HIV-1) to induce cytopathic effects on human T lymphocytes in vitro. Syncytium formation of HIV-infected T cells was essentially eliminated in the presence of M. fermentans (incognitus strain), despite prominent cell death. However, replication and production of HIV-1 particles continued during the coinfection. Furthermore, the supernatant from cultures coinfected with HIV-1 and the mycoplasma contained a factor that inhibited the standard reverse transcriptase enzyme assay. The modification of the biological properties of HIV-1 by coinfection with mycoplasma may be involved in the pathogenesis of acquired immunodeficiency syndrome (AIDS). PMID- 1705363 TI - Major chromatin changes accompany extinction of alpha-fetoprotein gene in hepatoma x fibroblast hybrids. AB - The sensitivity of the alpha-fetoprotein (AFP) gene to digestion by the enzyme DNaseI, and the presence of hypersensitive sites in the 5' region of this gene, were examined in hepatoma x fibroblast hybrid cells that exhibit extinction of AFP gene expression. Major changes occur in the extinguished gene, i.e., loss of long-range sensitivity to DNase digestion and of the hypersensitive sites. In this respect, the extinguished gene resembles the corresponding silent gene present in fibroblasts, but differs from the silent gene present in normal adult hepatocytes. These observations suggest that extinguisher factors acting on the AFP gene alter its conformation. PMID- 1705364 TI - Chromosomal localization of Cd1d genes in the mouse. AB - Southern blot hybridization of DNA from Chinese hamster x mouse somatic cell hybrids was used to assign the mouse Cd1d genes to chromosome 3. Analysis of the progeny of an intersubspecies backcross was used to position these genes near the gene for glucocerebrosidase, Gba. PMID- 1705365 TI - [Hodgkin's disease treated with chemotherapy at reduced dosage]. AB - Fifty-six patients with Hodgkin's disease in advanced stages (III-IV) were entered in a study aimed to compare standard doses (28 cases) with low doses (28 cases) ABVD regimen. Both groups had similar clinico-pathological features. The group receiving standard doses chemotherapy achieved complete remission rate of 89% versus 85% for the low-doses group. The number of relapses was, respectively, 8 and 9, and the duration of remission was 66 and 57 months, respectively. Presently, 24 patients have died, 11 of the standard doses group and 13 of the reduced doses group. Toxicity was notably higher in the group receiving standard dosage, where fatal myelosuppression was present in two instances. Those data appear to suggest that chemotherapy with reduced doses for Hodgkin's disease can attain similar response rate and survival than those achieved with standard treatment, with the advantages of a lower toxicity. PMID- 1705366 TI - Esophageal tumors--treatment by endoscopic intubation. AB - Esophageal tumors are often inoperable, because of the poor general condition of patients or the coexistence of metastatic disease. Endoscopic intubation is a safe method of palliation in these patients which can restore esophageal patency and lead to an improved nutritional status. At the Endoscopy Division of the National Cancer Institute in Milan, 141 patients with inoperable esophageal cancer underwent endoscopic intubation between 1978 and 1989. The 7-day mortality was 9/141 (6.3%) patients. In 114/132 surviving patients there was an improvement in the nutritional status and general condition. The complication rate was 17.7% (25/141 patients) and the mean survival was 5.9 months. PMID- 1705367 TI - [The mitochondrium--a fascinating organelle]. PMID- 1705368 TI - [Prognosis of inoperable non-small cell bronchial cancer]. AB - The article describes a retrospective study of the prognosis for 450 patients with inoperable non-small cell lung cancer discharged during the period 1970 to 1979. 81% of the patients were males. The predominant cell type was squamous cell carcinoma. One out of seven patients was inoperable because of poor cardiorespiratory function, six out of ten because of mediastinal metastases. Median survival was six months and was related both to staging of the tumor and to cell type. The prognosis was worst for patients with adenocarcinoma. Only 8% of the 450 patients were alive after two years. Specific and palliative therapy had a minor effect on median survival. Controlled clinical trials can probably settle whether an extension of the criteria for surgical treatment can improve the prognosis of this large group of patients. PMID- 1705369 TI - Effects and levels of coenzyme Q10 in lung and liver in bleomycin-hamster model of lung fibrosis. AB - As part of an ongoing study to search for potential antifibrotic agents which may reduce bleomycin-induced fibrosis of lung, hamsters were treated with 25 mg/kg coenzyme Q10 (CoQ10) for 2 days prior to and daily for 14 days after intratracheal instillation of bleomycin (5 U/kg body wt.). CoQ10 was found not to attenuate bleomycin-induced lung fibrosis. Treatment with CoQ10 did not raise the lung and liver content of CoQ10. On the other hand, bleomycin treatment alone tended to increase the lung and liver content of CoQ10. At the higher dose (7.5 U/kg), bleomycin almost doubled the hepatic CoQ10 content. Treatment with the lower dose (5 U/kg) of bleomycin and CoQ10 was always associated with higher levels of CoQ10 in both liver and lungs than treatment with either of them alone. PMID- 1705370 TI - [Changes in the content of nucleic acids and total protein in exocrine pancreatocytes and the composition of their population in experimental acute pancreatitis in rats]. AB - Using cytophotometry, contents of DNA, RNA and total protein were measured in the rat's exocrine pancreatocytes (EP) in normal conditions and at different stages of pancreatitis induced by cooling the spleen part of the pancreas with chlorethyl. In the duodenal (not damaged) part of the pancreas some drastic changes in the EP ploidy distribution were shown to occur. They led to the formation of a qualitatively new population pattern with 4c and 2c + 2c cells prevailing (more than 60% of the total content), which are by 1.5-3 times more active in RNA and protein synthesis and accumulation than the normal cells. The population structure rearrangement in the EP and their functional activity rise took place at two stages. At the first one the intracellular defense mechanisms of the EP in response to acute necrobiotic processes in the pancreas tissue were activated, at the second one the supracellular mechanisms regulating synthetic processes leading to a rapid adaptation of viable EP to new conditions were switched on. PMID- 1705371 TI - Communicating nursing research through poster presentations. PMID- 1705372 TI - [Increased expression of the OKM5 antigen in blood monocytes in vitiligo]. AB - In 25 patients suffering from vitiligo, we determined the T-cell profile (OKT3, 4 and 8; anti-Leu7) as well as the peripheral monocytes (OKM5) by means of monoclonal antibodies. Comparing the patients with a control group (n = 8), we found no differences regarding the T-cell count, whereas the expression of OKM5 antigens on peripheral monocytes was significantly elevated in patients with vitiligo. PMID- 1705373 TI - Hemagglutinin-neuraminidase (HN) amino acid alterations in neutralization escape mutants of Kilham mumps virus. AB - The hemagglutinin-neuraminidase genes of the Kilham strain of mumps virus and three neutralization escape mutants (M11, M12 and M13) of this strain (Love et al., 1985a) were sequenced using their genomes as template. The predicted amino acid sequences were compared. While one mutant had only one amino acid substitution the other two mutants had four and five respectively. A putative region for the epitope of the selected neutralizing monoclonal antibody was identified in a hydrophilic region encompassing amino acids 352-360, since the single amino acid substitution of one mutant occurred in this region and the other two mutants showed non-conserved amino acid changes in this part of the protein. The previously sequenced prototype strain RW, which lacks capacity to react with the selected neutralizing monoclonal antibody also has one non conserved amino acid change in the region of the proposed neutralizing epitope. The three mutants showed different biological characteristics. These particular characteristics were therefore interpreted to be primarily associated with strain specific amino acid changes outside the region of the presumed neutralizing epitope. The decrease in molecular weight in one mutant (M11) was shown to be due to a substitution in position 329 of an asparagine for an aspartic acid, leading to abolishment of a potential N-linked glycosylation site. In the other mutants, one substitution in position 239 of a lysine for a methionine was correlated with an increased neuraminidase activity of strain M12, while a substitution in position 360 of an arginine for a cysteine appeared to represent the most likely explanation for the reduced neurovirulence of strain M13. PMID- 1705374 TI - [Common channel syndrome--2 case reports]. AB - The common channel syndrome is a disease mostly of young girls. To demonstrate the problems of diagnosis and therapy we report on 2 cases operated on in 1987 and 1989. The long delay of several years between the onset of symptoms and the diagnosis might be shortened by improved diagnostic possibilities (Ultrasound, ERCP). In any case of hyperamylasemia or chronic pancreatitis in childhood an obstruction of the biliary tract should be excluded. The common channel syndrome can be treated by means of sphincteroplasty or a bilidigestive anastomosis. PMID- 1705375 TI - Chromogranin B-like immunoreactivity in the mouse submandibular salivary gland during postnatal development. AB - Presence and distribution, developmental changes, and molecular features of chromogranin B proteins within the submandibular salivary glands of male mice were investigated by 2-dimensional electrophoresis and PAP immunostaining. Chromogranin B-like immunoreactivity was detected in the juxta-acinar (JA)) cells of the submandibular salivary gland during the first week of postnatal development. During the 2nd and 3rd weeks, chromogranin B-like immunoreactivity reached the plateau level, while its reduction seemed to be occurred in correspondence to the appearance of the granular convoluted tubule cells, in which they did not show any chromogranin B-like immunoreactivity. Up to 7 weeks of age, chromogranin B-like immunoreactivity was substantially decreased, but not completely. A few JA cells, chromogranin B-like immunoreactive, retained even in the adult morphologically matured submandibular salivary gland of mice. A predominant protein which can be revealed by 2-dimensional electrophoresis and by immunocytochemistry, was chromogranin A. Chromogranin C was not detected in any cell type of the submandibular salivary gland of mice from early postnatal period up to 35 d of age. Physiological significance and possible function as well as an application of these findings were discussed. PMID- 1705376 TI - Participation of collagen types I, III, IV, V, and fibronectin in the formation of villi fibrosis in human term placenta. AB - The indirect immunofluorescence method was used to study the human term placenta in pathological pregnancy for the distribution of collagen types I, III, IV, V, and fibronectin in fibrosis stromatis villi. All collagen types and fibronectin were shown to participate in fibrosis villorum formation. Fibronectin was also detected in the fibrinoid that surrounded villi at stroma. The presence of free cytotrophoblast cells in the fibrinoid was accompanied by a noticeable increase in fibronectin fluorescence. A significant amount of collagen types IV and V and a less amount of collagen types I and III were identified. PMID- 1705377 TI - Morphological, developmental and immunohistochemical observations on the opossum pituitary with emphasis on the pars intermedia. AB - Development of the pituitary in Didelphis follows the general pattern of that described for most mammalian species. The dorsal region of a multichambered pituitary vesicle, which forms from Rathke's pouch, comes to lie adjacent to the presumptive infundibulum by the 10 1/2 d of gestation. The epithelial wall of this vesicle consists of spindle-shaped cells. The dorsal wall of the upper chamber of the pituitary vesicle forms the pars intermedia; the ventral wall of this chamber gives rise to cells of the pars distalis. Corticotropes, somatotropes, and lactotropes are seen in the presumptive adenohypophysis of the 11 1/2 d embryo. Gonadotropes and thyrotropes appear about 1 d later, shortly after birth. By the 2 postnatal week, the adult distribution of all 5 cell types within the pars distalis appears to have been established. The wall bounding the pituitary cleft in the adult represents an epithelial continuum limited by a basal lamina and corresponds to the upper chamber of the original pituitary vesicle. Ultrastructurally, the limiting walls of the pituitary cleft consists of stellate (marginal) cells, large, bulbous cells, and granulated cells. The latter correspond to the various endocrine cell types normally associated with the pars distalis. Non-granular folliculo-stellate cells also are observed within the epithelial cords of the pars distalis. PMID- 1705378 TI - [Immunohistochemistry of cytokeratin expression in human blood vessel endothelia with special reference to synovial connective tissue]. AB - 41 tissue samples derived from the synovia, epidermis, and other organs were studied immunohistochemically using monoclonal antibodies against intermediate filaments. We observed a coexpression of cytokeratin and vimentin in endothelia of small blood vessels preferably enriched in locations with enhanced fluid transport and processes of ultrafiltration via endothelial cells (synovial tissue, ganglion). The presence of cytokeratin 18 in endothelial cells has been confirmed by gel-electrophoresis with immunoblotting. In many cases, the coexpression of cytokeratin and vimentin reveals a correlation to the secretion of simply and epithelially differentiated cells as could be demonstrated for other tissue structures in previous studies. The increased expression of cytokeratins in endothelia of ganglion walls is discussed in relation to synovia like misfunction of the joint connective tissue. PMID- 1705379 TI - [Effect of dextran 40,000 on collagen-induced platelet aggregation]. AB - In blood samples of 12 healthy male volunteers under 50 years old the collagen induced platelet aggregation and the influence of a new 10% dextran 40.000 (Dextran Ebewe) was studied. Dextran was added to the samples with a concentration from 10 microliters up to 50 microliters/ml blood. In a concentration of 20 microliters/ml blood a significantly reduced collagen induced platelet aggregation was be observed. Higher dosages did not differ in a significant manner from this effect. PMID- 1705380 TI - Formation of nerve twig-like nests and schwannoma in an unusual case of neurofibroma. AB - A case of unusual neurofibroma in an 18-year-old Japanese male is reported. The histology of the tumor was characterized by nerve twig-like nests intermingled with fascicular bundles. In the central portion, the tumor also contained a lobular lesion showing features characteristic of schwannoma. Immunohistochemically, the tumor cells in both the nests and the lobular lesion demonstrated a mostly positive reaction for S-100 protein. S-100 protein-positive and -negative cells were observed in equal numbers in the fascicular bundles surrounding the nests. This unusual nerve sheath tumor in which the S-100 protein positive cells form both nerve twig-like nests and lobular schwannoma has not been reported previously. The origin of the S-100 protein-positive cells in the two lesions is also discussed. PMID- 1705381 TI - Eosinophilic neuronal inclusion bodies in the temporal lobe. AB - Intracytoplasmic neuronal inclusion bodies were found in the temporal lobe of an elderly woman. The oval or rod-shaped inclusion bodies were eosinophilic, showed bright red staining with azan, and were about half the size of the nucleus of a large neuron. They were non-argyrophilic and non-congophilic. Ultrastructurally, the inclusion bodies consisted of aggregates of filamentous materials showing partial periodicity. Among inclusion bodies reported up to now, the present ones resembled some described previously as "thalamic inclusions", but were different with regard to their partial filament periodicity, and unusual in that they were located in the deep layer of the temporal lobe and not in the thalamic nuclei. PMID- 1705382 TI - Biosynthesis of P450 products of arachidonic acid in humans: increased formation in cardiovascular disease. PMID- 1705383 TI - Activation of group II phospholipase A2 gene via two distinct mechanisms in rat vascular smooth muscle cells. PMID- 1705384 TI - Signal transduction coupled to prostaglandin D2. PMID- 1705385 TI - Modulation of platelet activation by prostaglandin E2 mimics. PMID- 1705386 TI - Usefulness of d, I sotalol for suppression of chronic ventricular arrhythmias. AB - Sotalol is a unique beta-blocking drug, possessing significant class III antiarrhythmic activity. The efficacy and safety of 2 doses of sotalol (320 and 640 mg/day, divided in 2 doses) were compared to placebo in a 6-week randomized, double-blind, multicenter study of 114 patients with chronic ventricular premature complexes (VPCs) at frequencies of greater than or equal to 30/hour. Sotalol significantly reduced VPCs in patients receiving both low (n = 38) and high (n = 39) doses, compared with patients (n = 37) receiving placebo (by 75 and 88%, respectively, vs 10%; p less than 0.001, sotalol vs placebo; p less than 0.05, high vs low dose). The individual efficacy criterion (greater than or equal to 75% VPC reduction) was achieved in 34% of low-dose and 71% of high-dose sotalol versus 6% of placebo-treated patients (p less than 0.003, sotalol vs placebo; p = 0.007, high vs low dose). Repetitive beats were suppressed 25% by placebo (difference not significant), 80% by low-dose (p less than 0.003) and 78% by high-dose sotalol (p less than 0.005). Sotalol decreased heart rate (by 24 to 25%, p less than 0.001) and increased PR (by 4 to 6%, p less than 0.001) and corrected JT intervals (by 12 to 13%, p less than 0.001), but did not change ejection fraction. Proarrhythmia (nonfatal) occurred in 3 sotalol and in 2 placebo patients. Nine discontinued therapy because of adverse effects (1 low dose and 8 high dose, p less than 0.02). In summary, sotalol is an efficacious antiarrhythmic drug for VPC suppression; in lower doses, it is somewhat less effective but better tolerated. PMID- 1705387 TI - Interleukin 1 beta and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro. AB - Cytokines are local mediators released by cells of the immune system in response to stimulation by a variety of agents. These polypeptides may interact directly or indirectly with bone cells. The objectives of this study were (1) to localize prostaglandin E (PGE) and the cytokine interleukin-1 beta (IL-1 beta) in the periodontal ligament after the application of mechanical force to teeth in vivo and (2) to determine the effects of mechanical stress or IL-1 beta (or the two in combination) on PGE synthesis and bone resorption by fibroblasts in the human periodontal ligament (PDL). In 24 female cats, one maxillary canine was tipped distally by 80 gm force for 12 hours, 24 hours, or 7 days. PGE and IL-1 beta were localized immunohistochemically in serial jaw sections, and semiquantitation of cellular-staining intensity was done by microphotometry. Unstressed periodontal ligament cells stained mildly for PGE and IL-1 beta, but the staining intensity increased significantly in sites of tension. Human periodontal ligament fibroblasts were preincubated with mechanical stress and/or IL-1 beta in the presence or absence of indomethacin for 1 hour. Then the media were replaced by BGJb (Fitton-Jackson modification) medium (GIBCO), and incubation was continued for 4, 8, or 24 hours in conditioned media. PGE concentrations in conditioned media were determined by radioimmunoassay, and bone-resorbing activity in conditioned media was assessed by 45Ca release from prelabeled neonatal mouse calvaria. The conditioned media derived from cells stimulated by mechanical stress plus IL-1 beta caused significantly more bone resorption than the conditioned media obtained from cells that had been treated by each factor alone. The addition of indomethacin did not inhibit bone resorption completely. These results demonstrate that periodontal ligament cells respond to mechanical stress by increased production of PGE, and that IL-1 beta enhances this response. PMID- 1705388 TI - Undetected fatal acute pancreatitis: why is the disease so frequently overlooked? AB - An analysis of postmortem investigations between 1980 and 1985 revealed 43 patients with acute pancreatitis. In 13 (30.2%) of them, the diagnosis was first established at autopsy. In eight of the latter patients, the diagnosis could have been present on admission. The etiology was alcoholism in three patients, hypothermia in one, biliary tract disease in one, and unknown in three patients. In five patients, acute pancreatitis developed after gastric, pancreatic, or biliary tract surgery. Abdominal pain was present in only one patient. Amylase levels had been estimated in 11 patients, but the level was in the diagnostic range (greater than or equal to 3 times of upper normal level) in only four. Consequently, ultrasound examination was performed in only two of the latter four patients, but failed to show the pancreas because of intestinal gas. To diagnose acute pancreatitis at an earlier stage and to improve therapy and prognosis, we recommend that serum amylase levels be measured and ultrasound examination be performed. If the results are inconclusive, this should be followed by computed tomography for all abdominal emergency cases and for patients who have undergone cardiopulmonary or upper abdominal surgery, especially when the patients deteriorate or fail to improve postoperatively. PMID- 1705389 TI - The effects of vitamin K on the generation of des-gamma-carboxy prothrombin (PIVKA-II) in patients with hepatocellular carcinoma. AB - The clinical significance of des-gamma-carboxy prothrombin (PIVKA-II) in hepatocellular carcinoma (HCC) was investigated in 112 patients with and without vitamin K administration. The positivity rate of PIVKA-II was significantly decreased in patients receiving vitamin K (28.5%), compared with those without vitamin K administration (54.5%, p less than 0.05). The plasma levels of vitamin K derivatives [phylloquinone (VK1), menaquinone-4 (MK4), and menaquinone-7 (MK7)] measured were not decreased in patients with HCC, but were significantly increased in MK4 and VK1 + MK4 + MK7. The amount of PIVKA-II in plasma did not correlate with the plasma levels of vitamin K derivatives. However, PIVKA-II was decreased by the administration of vitamin K, and all of the six patients with more than 5.0 ng/ml of VK1 + MK4 + MK7 were within normal limits, whereas half of 32 patients with less than that had abnormal levels of PIVKA-II. Thus, it was suggested that PIVKA-II was not elevated due to vitamin K deficiency, but might result from the impaired metabolism or availability of vitamin K in the tumor. Therefore, PIVKA-II should be measured without vitamin K administration. PMID- 1705390 TI - Giardia lamblia infestation reveals underlying Whipple's disease in a patient with longstanding constipation. AB - Whipple's disease is an uncommon disorder, generally associated with gastrointestinal symptoms; of these, diarrhea is a common feature. We report a case of Whipple's disease associated with chronic constipation which was not diagnosed until after Giardia lamblia infestation had caused diarrhea. To the best of our knowledge this association has not previously been reported. The clinical, laboratory, endoscopic, and manometric aspects are described and discussed. PMID- 1705391 TI - Direct carbohydrate analysis of glycoproteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel. AB - A procedure for the carbohydrate analysis of glycoproteins electrotransferred to a polyvinylidene difluoride membrane is described. The glycoproteins (plant lectins, transferrin, and vitronectin) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a membrane. Each of the glycoprotein bands visualized by staining with Coomassie brilliant blue R-250 was excised from the membrane and subjected to direct hydrolysis either in 2.5 M trifluoroacetic acid at 100 degrees C for 6 h for neutral sugars and hexosamines, or in 0.05 M H2SO4 at 80 degrees C for 1 h for sialic acids. The hydrolysate obtained was analyzed for neutral sugars, hexosamines, and sialic acids independently by three different systems of high performance liquid chromatography. The analytical values were reproducible with reasonable accuracy and agreed with those expected with recoveries of 57-66%. The method was successfully applied to a mannose-specific lectin of Sophora japonica bark, which is composed of four different subunits that aggregate sugar specifically. Because the four subunits could be separated by SDS-PAGE alone, the method proved useful for determining their carbohydrate compositions. Three of them were shown to contain carbohydrates typical of N-linked oligosaccharides of plant origin, which agreed well with the results of the binding assay carried out on a membrane using various horseradish peroxidase-labeled lectins. PMID- 1705392 TI - A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription. AB - A small-scale plasmid preparation is described that is useful for a variety of procedures from double-stranded sequencing to in vitro transcription. No specialized equipment or reagents are required. The preparation of plasmid DNA does not require the use of RNase; instead the larger RNAs are precipitated with 2.5 M ammonium acetate. The resulting plasmid DNA is used routinely for double stranded sequencing with the Klenow fragment of DNA polymerase and has been used for generating deletions with exonuclease III. In addition, the plasmid DNA has been used to generate transcripts with T7 RNA polymerase that translate well in reticulocyte lysate. PMID- 1705393 TI - An alternative method for a fast separation of phosphotyrosine. AB - A simple and rapid procedure is described for fully separating phosphotyrosine from phosphoserine and phosphothreonine through one-dimensional thin-layer chromatography. The migration properties of these phosphoamino acids are compared with those of CMP, UMP, ATP, ribose phosphate, and inorganic orthophosphate, considered the most frequent comigrating products derived from 32P-labeling experiments. We demonstrate that Rf values for the three phosphoamino acids differ from those displayed by the mentioned contaminating compounds. One of the most relevant advantages of this procedure is that a complete separation of phosphotyrosine can be achieved in only 90 min. PMID- 1705394 TI - Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions. AB - Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium. PMID- 1705395 TI - Staining of free sulfhydryl groups of proteins after separation by isoelectric focusing of Bio-Rad standards and lens crystallins. AB - In this study, we propose a new staining method for free sulfhydryl groups of proteins after having separated native samples by thin-layer isoelectric focusing (IEF) in absence of detergents. A comparison was made between proteins stained purple for free sulfhydryl groups (SH) and proteins stained blue by Coomassie blue (CB). A stainability factor, F = %SH/%CB, was calculated for each protein. The Bio-Rad IEF standards containing seven marker proteins for pH scale determination were stained purple, in the same way as they were designed for CB staining. To prove the validity of the currently proposed staining method for a defined protein system such as the eye lens crystallins, these proteins were also stained after IEF as described above. The factor F was calculated for all alpha-, beta-, and gamma-crystallin components that stained in both methods. We discovered that alpha-crystallins contained comparatively high amounts of free SH groups, while some beta- and gamma-crystallin components also contained considerable amounts of free SH groups. The SH staining procedure with 2,2' dihydroxy-6,6'-dinaphthyl disulfide applied after IEF appeared to be useful, specific, and reproducible. PMID- 1705396 TI - A microtiter-based assay for the detection of protein tyrosine kinase activity. AB - A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation. PMID- 1705397 TI - A multiple-staining procedure for the detection of different DNA fragments on a single blot. AB - We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4 chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705398 TI - The preparation of highly enriched fractions of binucleated rat hepatocytes by centrifugal elutriation and flow cytometry. AB - A procedure is described for the isolation of highly enriched fractions of binucleated hepatocytes from rat liver. Liver cells isolated by EGTA and collagenase perfusion were initially subjected to centrifugal elutriation and second to flow cytometry coupled with Hoechst 33342 staining. The elutriation step yielded hepatocyte fractions which contained almost entirely mononuclear diploid cells and fractions enriched in binucleate hepatocytes. The fractions with the highest proportion of binucleated hepatocytes contained between 50 and 56% of these cells. Subsequent flow cytometric cell sorting yielded fractions which contained greater than 80% binucleated cells. These cells were viable in culture as demonstrated by the immunohistochemical detection of bromodeoxyuridine incorporation. PMID- 1705399 TI - Synthesis of phosphotyrosyl-containing phosphopeptides by solid-phase peptide synthesis. AB - The synthesis of phosphotyrosine-containing phosphopeptides using solid-phase peptide synthesis (SPPS) techniques is described. We present the synthesis of a Boc-phosphotyrosine derivative, which when used with modifications of the conventional SPPS protocol permits the incorporation of phosphotyrosine into synthetic peptides. The resulting phosphopeptides were authenticated by fast atom bombardment mass spectrometry, amino acid analysis, and phosphate assay. Alkaline phosphatase was found to dephosphorylate synthetic phosphopeptides at different rates, supporting the potential use of these synthetic substrates for studies of phosphoprotein phosphatases. Synthesis of a phosphopeptide using the described protocol has several advantages over the preparation of phosphopeptides via enzymatic phosphorylation. PMID- 1705400 TI - Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung. AB - The development of the cardio-pulmonary innervation was studied in whole-mount specimens of chick embryos stained with the anti-neurofilament protein (NFP) antibody. From the morphological point of view, vagal branches could be classified into two categories, i.e., the branchial branches primarily related to the pharyngeal arches, and intestinal arborization derivatives which are associated primarily with the primitive gut. The former consisted of the superior cardiac branch innervating the truncus arteriosus of the heart, and the latter, the sinal branch, pulmonary branches as well as recurrent nerve and the other intestinal branches. The superior cardiac branch at first develops as a pair of branchial branches which passes into the truncus arteriosus at stage 25, and later rotates along the aortic arch 6, thus making an asymmetrical configuration by stage 27. The sinal branch is a medial branch which first develops at stage 24. It arises from the junction of each intestinal arborization in close association with the pulmonary branch. PMID- 1705401 TI - [Cancer in megaesophagus: our experience]. PMID- 1705402 TI - Elevation of [Ca2+]i and the activation of ion channels and fluxes by extracellular ATP and phospholipase C-linked agonists in rat parotid acinar cells. AB - Extracellular ATP initiates a variety of changes in the parotid acinar cell. The initial effect appears to be the entry of Ca2+ (and perhaps Na+), and a series of ion transport events result from the subsequent elevation of [Ca2+]i. Agonists of phospholipase C-linked receptors elevate [Ca2+]i by a different pathway, involving the generation of inositol polyphosphate compounds, but share in the subsequent initiation of the ion transport events. Although the maintenance of the physiological changes may depend on specific inositol polyphosphate intermediates, the critical initiating factor is the elevation of [Ca2+]i. Fluid secretion by the parotid gland is triggered by the action of neurotransmitters, which alter the membrane permeability of the acinar cell. The similarities between the two receptor-mediated activation pathways suggests that ATP may act as a neurotransmitter and play a role in the control of fluid secretion. Basing our analysis on the purinoceptor characteristics outlined by Gordon, we suggest that the parotid receptor belongs to the P2Z class, which is highly sensitive to ATP4-. Basing his analysis on the earlier report by Gallacher of the effects of ATP on mouse parotid cells, Gordon placed the parotid purinoceptor in a different P2 subclass (P2Y). However, our findings of an increased potency of ATP in the absence of Mg2+, as well as the potency order of different nucleotides, indicate that the P2Z class is a more appropriate category. PMID- 1705403 TI - Gene defects in beta-thalassemia and their prenatal diagnosis. PMID- 1705404 TI - Analysis of human gamma-to-beta switching in transgenic mice. PMID- 1705405 TI - Stopping the biologic clock for globin gene switching. AB - The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if expression of the adult (beta) globin genes is defective and produces little or no protein, as in the beta-thalassemias. Preventing or reversing the globin gene switch could provide a way of keeping the abnormal globin genes "silent" and maintaining expression of the fetal globin gene. We have identified a class of agents which, when present in elevated plasma concentrations during gestation, inhibits the gamma----beta-globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma-globin and decrease beta-globin expression in cultured erythroid cells of patients with beta thalassemia. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited and even reversed in some fetal lambs. Histone hyperacetylation, which maintains active chromatin structure, and an effect on the gamma-globin promoter appear to be mechanisms of action involved. These data suggest that inhibiting expression of abnormal beta-globin genes by pharmacologic means may in the future be possible for treatment of individuals with beta-globin disorders. PMID- 1705406 TI - Pharmacologic manipulation of fetal hemoglobin in the hemoglobinopathies. PMID- 1705407 TI - Pharmacologic manipulation of fetal hemoglobin. Update on clinical trials with hydroxyurea. PMID- 1705408 TI - Regulation of gamma-globin expression in hereditary persistence of fetal hemoglobin. PMID- 1705409 TI - A model for reactivation of hemoglobin F synthesis in normal adult erythropoiesis. PMID- 1705410 TI - Beta-thalassemia in Algeria. PMID- 1705411 TI - Molecular studies of beta-thalassemia in Israel. Mutational analysis and expression studies. PMID- 1705413 TI - Epidermolysis bullosa simplex (Koebner) is a keratin disorder. Ultrastructural and immunohistochemical study. AB - A skin biopsy specimen was obtained from a 1-month-old female with epidermolysis bullosa simplex (Koebner). Histologically, an intraepidermal separation was seen and considered to be formed by cytolysis of the epidermal basal cells. Ultrastructurally, the basal cells were lacking in cytoplasmic tonofilaments, and the initial change of the cytolysis seemed to be cleavages of the cytoplasm. Immunohistochemically, a basal cell keratin was expressed in a suprabasal cell layer but not in the basal cell layer, and a panepithelial keratin was not detected in the basal cell layer. These findings suggest that keratin production of the epidermal cells may be delayed, resulting in a weakness of the basal cells against minor trauma to the skin. PMID- 1705412 TI - Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase. AB - We studied the effect of the natural marine substance illimaquinone on the catalytic activities of reverse transcriptase from human immunodeficiency virus type 1. Illimaquinone inhibited the RNase H activity of the enzyme at concentrations of 5 to 10 microgram/ml, whereas RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities were considerably less susceptible to this inhibition. Two synthetic derivatives of illimaquinone, in which the 6' hydroxyl group at the ortho position to one of carbonyl groups of the quinone ring was modified, proved ineffective in inhibiting the human immunodeficiency virus type 1 reverse transcriptase RNase H function, suggesting involvement of the 6'-hydroxyl group in blocking the enzymatic activity. PMID- 1705414 TI - [Diclofenac sodium therapy in irritating micturition syndrome following TUR for benign prostatic hyperplasia]. AB - The irritative micturition syndrome is commonly observed following endoscopic surgery for benign prostatic hyperplasia. This condition almost consistently presents following removal of the bladder catheter and may last from 15 to 45 days. The present study was undertaken to determine the efficacy of treatment with diclofenac sodium. Fifty patients submitted to endoscopic resection for benign prostatic hyperplasia received the agent IM a few hours prior to catheter removal and posteriorly via the rectal route for a total treatment period of 13 days. Good results were achieved in 95.5% of the cases. Mild side effects were observed in 14% of the patients. PMID- 1705415 TI - [Partial parietal cystectomy and cystoplasty using a lyophilized human dura mater patch as an alternative in palliative surgery for bladder cancer]. AB - Fifteen patients with infiltrating bladder carcinoma underwent partial cystectomy and cystoplasty with lyophilized human dura at our hospital from 1983 to 1988. The 5-year survival rate was 75% for stage B1, 60% for B2, 40% for C and 0% for D1. There were no intra- or post-operative deaths and the post-operative complications were few. This procedure may be useful in selected patients with bladder tumors who cannot be submitted to more radical procedures due to a coexisting pathological condition representing a high surgical risk or in elderly patients. PMID- 1705416 TI - Hypoxia and inflammatory synovitis: observations and speculation. PMID- 1705417 TI - Surgical management of 552 carcinomas of the extrahepatic bile ducts (gallbladder and periampullary tumors excluded). Results of the French Surgical Association Survey. AB - Five hundred fifty-two cases of primary carcinoma of the extrahepatic bile ducts (gallbladder and periampullary tumors excluded) collected from 55 surgical centers were reviewed retrospectively. Three hundred seven patients (56%) had upper-third lesions (proximal carcinoma), whereas 71 (13%) and 101 (18%), respectively, had middle-third and lower-third bile duct carcinomas. The remaining patients had diffuse lesions. Resectability rates were 32% for upper third localization compared to 47% and 51% for middle-third and lower-third localization, respectively. The operative mortality rate for proximal carcinomas was significantly lower with resection (16%) compared with palliative surgery (31%) (p less than 0.05). Overall 1-year survival (operative deaths excluded) was 68% after tumor resection compared to 31% after palliative surgery (p less than 0.001). Long-term results after surgical resection correlated with local and regional extension of the disease. The results of this study show that resection of extrahepatic bile duct carcinomas, particularly in an upper-third localization, often is associated with worthwhile long-term survival. PMID- 1705418 TI - Progress of patients with pulmonary atresia after systemic to pulmonary arterial shunts. AB - Between February 1980 and June 1987, 42 shunts were placed in 39 infants with pulmonary atresia: 33 were modified Blalock-Taussig shunts with polytetrafluoroethylene (PTFE) and 9 were classic Blalock-Taussig shunts. There were four hospital deaths not related to the shunts. The remaining 35 patients were followed up for 1.6 months to 6.3 years (mean, 24.7 +/- 18 months). Repeat cineangiocardiographic studies revealed stenosis or distortion of the pulmonary arteries related to the site of the shunt in 11/22 patients (50%) with PTFE shunts and in 1/6 (17%) with classic Blalock-Taussig shunts; the stenosis was severe in only 1 patient. Mean increase in the pulmonary arterial index in the group with classic Blalock-Taussig shunts was 117 +/- 52 mm2/m2 (not significant) and in the group with PTFE shunts, 158 +/- 21 mm2/m2 (p less than 0.001). Late shunt occlusion occurred in 1 patient 23 months postoperatively. Thereafter, shunt patency rate remained at 94% +/- 6%. At the end of 1 year 81% +/- 7% of patients were judged to have adequate palliation, but between 2 and 3 years, only 60% +/- 10%. Univariate analysis showed that after 2 years the ranking order for successful palliation was classic Blalock-Taussig, 5-mm PTFE, and 4-mm PTFE shunts, but differences did not achieve statistical significance. PMID- 1705419 TI - Identification of the endocrine cells detected by the monoclonal antibody HISL-19 in human tissues. AB - The human endocrine cells reacting with the monoclonal antibody HISL-19 were identified with hormone antisera of proven specificity using a double immunostaining procedure. The epitope for HISL-19 was found in all types of pituitary cells except ACTH cells, in thyroid C cells, in all types of adrenal medullary and pancreatic islet cells and in somatostatin and pancreatic polypeptide cells of the gastrointestinal mucosa. No staining was found in parathyroid cells and in most gastrointestinal endocrine cells. Either paranuclear focal accumulation or diffuse cytoplasmic distribution of immunoreactive material were found. The spectrum of HISL-19 immunoreactive cells was found to be only in part complementary to that of cells immunoreactive for chromogranin A. Thus, it is concluded that the monoclonal antibody HISL-19 is a useful addition to other immunohistochemical markers for the detection of cells showing neuroendocrine features. PMID- 1705420 TI - Variation of tyrosine aminotransferase expression during the day in rats of different ages. AB - The activity of the enzyme tyrosine aminotransferase and the synthesis of its specific mRNA were evaluated at different hours of the day in the liver of 3-, 12 and 24-month old BN rats. The enzyme activity has a circadian rhythm with a peak at midnight in 3- and 12-month old, which shifts to 03.00 hrs in 24-month old animals, in agreement with previous results. The expression of TATmRNA also changes during the day indicating circadian fluctuations which change with age. In 3-month old rats the TATmRNA peak is at 19.00 hrs, preceding that of the enzyme activity. In 12-month old rats the TATmRNA synthesis reaches a maximum at midnight and in 24-month old rats at 03.00 hrs. The results show that the circadian rhythm of tyrosine aminotransferase activity is due to a different gene expression throughout the day, which is influenced by age. PMID- 1705421 TI - Establishment of monoclonal antibodies against human acidic fibroblast growth factor. AB - Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44 132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product. PMID- 1705422 TI - Patch clamp analysis of a partially purified ion channel from rat liver mitochondria. AB - A protein fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine was reconstituted into phospholipid vesicles by detergent dialysis. Vesicles were fused to a diameter of 10 microns or larger by dehydration and rehydration. Patch clamp recordings carried out in detached mode with a symmetrical solution of 150 mM KCl, 5 mM HEPES, and 0.1 mM CaCl2 revealed conductance increments of 140 pS. Transitions of 40 pS were less frequently observed. Control vesicles which lacked protein showed no channel activity. The probability for the 140 pS channel to be open increased with increasing voltage in the range from 20 to 80 mV (positive potentials relative to what was the vesicle interior prior to excision), while the single channel conductance remained essentially constant. The 140 pS channel did not open at negative voltages. The voltage dependence suggests asymmetric incorporation of the 140 pS channel into vesicle membranes during reconstitution. PMID- 1705423 TI - Involvement of retinoic acid nuclear receptors in retinoic acid-induced tissue transglutaminase gene expression in rat tracheal 2C5 cells. AB - The involvement of retinoic acid nuclear receptors (RARs) in the induction of tissue transglutaminase (TG) by retinoic acid in rat tracheal 2C5 cells was determined. The levels of RAR alpha and RAR beta were altered in 2C5 cells by transfection with RAR expression vectors. Increased expression of RAR alpha increased the induction of tissue TG by retinoic acid. In contrast, decreased RAR alpha expression, using an antisense RAR alpha expression vector, diminished the normal level of tissue TG induction caused by retinoic acid. Transfectants overexpressing RAR beta were also more responsive to retinoic acid for the induction of tissue TG, although the magnitude of TG induction was not as great as resulted from RAR alpha overexpression. These results indicate that the levels of the RAR alpha and RAR beta dictate the magnitude of tissue TG induction by retinoic acid. PMID- 1705424 TI - Physico-chemical properties of the new antibronchospastic agent isbufylline. AB - Physico-chemical properties, i.e. elemental analysis, spectra (NMR, MS, IR, UV), X-ray crystal structure, solubilities, partition coefficient, melting point, HPLC, GC, and data about purity and stability of 1,3-dimethyl-7-isobutyl xanthine (isbufylline, TE/06, CAS 90162-60-0), a new drug with antibronchospastic properties, are reported. PMID- 1705425 TI - Anti-platelet activating factor, anti-leukotriene D4 and some other antiallergic activities of mequitazine. AB - The bronchoconstrictions of guinea pigs elicited in vivo by leukotriene D4 (LTD4) and platelet activating factor (PAF) were inhibited by pretreatment with mequitazine (CAS 29216-28-2) (p.o.) in a dose-dependent manner at doses of 5-20 mg/kg. Mequitazine (0.5-50 nmol/l) also inhibited LTD4- and PAF- induced contractions of guinea pig tracheal chain. These results suggest that mequitazine possesses antagonistic activity for LTs and PAF; the effects of mequitazine against these two agonists were much more potent than those of ketotifen. The histamine release induced by either of compound 48/80, concanavalin A or A23187 was inhibited by mequitazine at concentrations ranging from 1 to 20 mumols/l. In the inhibition process, mequitazine may act not only to inhibit the Ca2+ release from intracellular Ca store of mast cells but also to stabilize the lipid bilayer of the cell membrane as shown in the order parameter and hypotonic hemolysis. From the present study, it was assumed that mequitazine may exert antiallergic activity by antagonizing LTs and PAF as well as by inhibiting histamine release from mast cells. PMID- 1705426 TI - Effects of the new phosphodiesterase inhibitor griseolic acid on insulin release in rat pancreatic islets. AB - A new cyclic AMP phosphodiesterase inhibitor, griseolic acid (CAS 79030-08-3), in a dose-dependent manner increased insulin release and cyclic AMP level in rat pancreatic islets in the presence of 5.5 mmol/l glucose. Griseolic acid (0.26 mmol/l) or 3-isobutyl-1-methylxanthine (IBMX) (1 mmol/l) enhanced insulin release and cyclic AMP level both in the presence of 5.5 and 16.7 mmol/l glucose (no significant difference). In perifusion system, 45Ca++ efflux and insulin release showed a monophasic increase when the islets were exposed to 1.3 mmol/l griseolic acid or 1 mmol/l IBMX in the presence of 5.5 mmol/l glucose (no significant difference). In a cell-free system, griseolic acid had a stronger inhibitory effect on cyclic AMP phosphodiesterase activity than IBMX, has a stimulatory effect on insulin release through an increase of cyclic AMP by inhibiting phosphodiesterase in pancreatic islets, but it might not cross the plasma membrane easily. PMID- 1705427 TI - Interactions of saruplase with acetylsalicylic acid, heparin, glyceryl trinitrate, tranexamic acid and aprotinin in a rabbit pulmonary thrombosis model. AB - In anesthetized rabbits with pulmonary embolized thrombi the interactions of saruplase (recombinant unglycosylated single-chain urokinase-type plasminogen activator, CAS 99149-95-8) with acetylsalicylic acid (ASA), glyceryl trinitrate (nitroglycerin, GTN), heparin, and the antifibrinolytics tranexamic acid and aprotinin have been studied. Lysis rates were evaluated as reduction of the weight and as reduction of the incorporated 125J-fibrin content of the embolized thrombi. In untreated controls the spontaneous 125J-fibrinolysis was 8.3 +/- 0.7% and the thrombus weight was reduced by 55.3 +/- 4.5% (mainly due to loss of H2O) at 195 min after the thromboembolization. Infusion of saruplase (21.5 micrograms/kg.min) for 60 min significantly increased the 125J-fibrinolysis to 36.8 +/- 3.7% and the reduction of the thrombus weight to 69.9 +/- 2.1%. ASA (50 mg/kg p.o.) by itself did not exert a lytic effect; in combination with saruplase ASA insignificantly augmented the 125J-fibrinolysis rate and significantly further increased the thrombus weight reduction. GTN (5.0 micrograms/kg.min i.v.) neither influenced the spontaneous nor the saruplase-mediated lysis rates. Treatment with heparin (200 IU/kg i.v.-bolus plus 50 IU/kg.min i.v.-infusion) resulted in significant greater thrombus weight reduction than observed in untreated controls; in combination with saruplase heparin significantly intensified the lytic effect. Tranexamic acid (30 mg/kg i.v.) and aprotinin (30.10(3) KIU/kg i.v.) completely abrogated the lytic effect of saruplase. Treatment with saruplase alone produced an insignificant decrease of the plasma level of fibrinogen by 23% to 200 +/- 20 mg/dl. ASA, GTN and aprotinin did not influence this slight fibrinogenolysis in saruplase-treated animals. A slight decrease of plasma fibrinogen levels was observed by heparin, whereas the decrease by tranexamic acid was significant. PMID- 1705428 TI - On the propeller structure of isolated Watson-Crick base pairs. PMID- 1705429 TI - Involvement of voltage-dependent calcium channels (VDCC) in the action of GnRH on GtH release in common carp (Cyprinus carpio L): comparison with K+ action. AB - The involvement of different types of voltage-dependent calcium channels (VDCC) in the stimulatory action of GnRH (in comparison with K+) on maturational gonadotropin (GtH) release was investigated using superfused carp pituitary cells. The action of these 2 stimulants was not modified either by D600 or nifedipine (drugs blocking L-type of VDCC). Cadmium (Cd2+), which blocks all types of VDCC indifferently, provoked a dose-dependent stimulation of GtH secretion. Cd2+ action was not altered by addition of sGnRH in any of the doses. Similar results were obtained using K+ as a secretagogue, but only the highest dose of Cd2+ (200 mumol/l) was able to completely block K+ action. Low doses (0.1 and 1 mumol/l) of the L-type VDCC activator BAY-K8644 did not change basal GtH secretion and had no effect on sGnRH-stimulated GtH secretion. Surprisingly, doses (10 mumol/l and higher) of BAY-K8644 evoked dose-dependent inhibition of GtH secretion. On the other hand, a higher concentration (20 mumol/l) of nifedipine provoked a stimulation of GtH release. Our results indicate that the stimulatory action of GnRH and K+ involves activation of a certain type of cadmium-sensitive VDCC (probably T- or N-type VDCC) whereas dihydropyridine and diphenylalkylamine sensitive VDCC (L-type VDCC) does not participate in this phenomenon. The inhibitory action of BAY-K8644 and, on the other hand, the stimulatory action of nifedipine indicate that L-type VDCC probably play a role in other physiological pathways regulating GtH release in carp. PMID- 1705430 TI - Crossed immunoelectrophoresis does not allow accurate determination of inter alpha-trypsin inhibitor and its derivatives in plasma. AB - By crossed-immunoelectrophoresis (CIE) of plasma, using anti-inter-alpha-trypsin inhibitor (ITI) immunoglobulins, beside native ITI, related components are visualized as an heterogeneous peak migrating farther than ITI. The area corresponding to this peak is largely increased in case of inflammatory disease. So, quantitative CIE has been previously proposed for discrete evaluation of ITI and its derivatives. We herein present evidence that CIE does not allow a clear separation of ITI from its derivatives: in this system, they are to some extent coprecipitated. Therefore the resulting overestimation of ITI may explain the previously reported absence of inverse relation between relative contents of ITI and derivatives in case of inflammatory disorders. Our data confirm the view that ITI acts as a precursor of smaller immunologically related inhibitors. PMID- 1705431 TI - Characterization of the proctolinergic and GABAergic systems in the terminal ganglion of the male and female cockroach, Periplaneta americana, and effects of insecticide treatment. Immunohistochemistry and computer-assisted image analysis. AB - The distribution of proctolin- and gamma-aminobutyric acid (GABA)-containing neurons in males and females of the American cockroach, Periplaneta americana, was evaluated by means of immunohistochemistry. Sex-related differences in the localization of proctolinergic cells are restricted to the median region of the terminal ganglion. No coexistence was found for GABA and proctolin. The high specificity of the GABA staining was confirmed by labelling of identical neurons with both anti-GABA and anti-glutamic acid decarboxylase (GAD) antisera. The volume fractions occupied by GABA- and proctolin-immunoreactive elements were evaluated using a computer-based method of planimetric area percentage. The GAD staining was markedly decreased after carbaryl insecticide treatment and the volume fraction of proctolin-immunoreactive neurons was significantly reduced after application of another insecticide, dieldrin. PMID- 1705432 TI - [Effect of cerulein on human salivary secretion]. AB - To evaluate the effect of cerulein on salivary secretion in man, 25 healthy volunteers have been studied. In the morning, before breakfast, saline solution (125 ml/h) has been infused at a constant flow, for one hour. Saline solution has been followed, for other 60', by saline solution + Cerulein at doses of 0.05, 0.1, 0.2, 0.3, 0.5 microgram/Kg/h. These different doses have been administered in different days according to a randomized order. A wash out by saline solution, at same velocity as before, has been carried out after Cerulein . Samples of saliva were collected every 15'. On salivary specimens the amylase activity (U/l) has been dosed. Each dose of Cerulein induced a reduction of salivary flow. By blocking the administration of Cerulein the salivary secretion returned to basal value in about one hour. No inhibitory effect has been noted on amylase secretion using different doses of Cerulein . PMID- 1705433 TI - Granulocyte colony-stimulating factor (G-CSF) receptors on acute myeloblastic leukaemia cells and their relationship with the proliferative response to G-CSF in clonogenic assay. AB - The number and the affinity of granulocyte colony-stimulating factor (G-CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabelled recombinant human G-CSF (rhG-CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15-130 pM) G-CSF receptors on the AML blasts. The number of G-CSF receptors varied from 55 to 1,200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G-CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross-linking study revealed that the G-CSF receptor has an approximate molecular weight of 140,000. Autoradiography showed heterogeneity of the distribution of G-CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG-CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G-CSF receptor number expressed by blasts that formed colonies on stimulation with rhG CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G-CSF may be predicted when the blasts express a large number of G-CSF receptors. Accordingly, it may be safer to restrict the clinical use of G-CSF to AML patients who have blasts with a low G-CSF receptor expression and no response to G-CSF in blast colony assay. PMID- 1705434 TI - Ratio of immunochemically determined amniotic fluid acetylcholinesterase to butyrylcholinesterase in the differential diagnosis of fetal abnormalities. AB - A total of 111 amniotic fluid samples, clear or blood stained, with elevated levels of alpha-fetoprotein and acetylcholinesterase was analysed by immunoassays specific for acetylcholinesterase and butyrylcholinesterase and the acetylcholinesterase/butyrylcholinesterase-ratios determined. Samples from 40 pregnancies associated with anencephaly, 47 pregnancies associated with open spina bifida or encephalocele and six pregnancies with fetal intrauterine death or miscarriage all had ratios of greater than 0.14. All 11 pregnancies with fetal ventral wall defects had ratios less than 0.14 as had four pregnancies with normal outcome and elevated levels of alpha-fetoprotein and acetylcholinesterase. Three fetuses with both open spina bifida and ventral wall defects were associated with ratios above 0.14. These results suggest that immunochemical determination of acetylcholinesterase and butyrylcholinesterase can be used to distinguish pregnancies complicated by anencephaly, open spina bifida, encephalocele and miscarriage from those with ventral wall defects and samples with false positive elevated levels of alpha-fetoprotein and acetylcholinesterase. The procedure is accurate and simple to carry out and well suited to routine use in a clinical chemistry laboratory. PMID- 1705435 TI - The immunoperoxidase localization of tumour markers in ovarian cancer: the value of CEA, EMA, cytokeratin and DD9. AB - Primary tumours from 40 patients with epithelial ovarian cancer, treated at St Thomas's Hospital over a 10-year period, were studied for the immunocytochemical expression of the following tumour markers in formalin-fixed paraffin embedded material: carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), cytokeratin (CAM 5.2), and DD9. An indirect immunoperoxidase staining technique was used. All of the tumours were positive for EMA and CAM 5.2, and 30% of them were positive for both CEA and DD9. The absence of CEA and DD9 may be of value in differentiating between metastatic abdominal adenocarcinomas of ovarian origin and those of gastrointestinal origin, but no indication of prognosis was obtained using these epithelial markers. The strong and widespread staining of all the tumours for EMA suggests that this may be a useful marker for detecting metastatic or recurrent disease by immunoscintigraphy. PMID- 1705436 TI - A novel dipyridodiazepinone inhibitor of HIV-1 reverse transcriptase acts through a nonsubstrate binding site. AB - A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3 b:2',3'-e]- [1,4]diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1). An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation. BI-RG-587 and close structural analogues competitively protected RT 1 from inactivation by the photoaffinity label. A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds. Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation. A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1. PMID- 1705437 TI - Cordycepin analogues of 2',5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase. AB - Analogues of 2',5'-oligoadenylates (2-5A), the cordycepin (3'-deoxyadenosine) core trimer (Co3) and its 5'-monophosphate derivative (pCo3), were shown to display pronounced anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. Treatment of HIV-1 infected H9 cells with 1 microM Co3 or pCo3 resulted in an almost 100% inhibition of virus production. The compounds were encapsulated in liposomes targeted by antibodies specific for the T-cell receptor molecule CD3. Substitution of one or two cordycepin units in Co3 or pCo3 decreased the antiviral activity of the compounds. pCo3 did not stimulate 2-5A-dependent ribonuclease L activity and displayed no effect on the amount of cellular RNA and protein. At a concentration of 10 microM the cellular DNA polymerases alpha, beta, and gamma were almost insensitive toward Co3 or pCo3. In contrast, these compounds reduced the activity of HIV-1 reverse transcriptase (RT) by 90% at a concentration of 10 microM if the viral RNA genome and the cellular tRNALys.3 was used as template/primer system; if the synthetic poly(A).(dT)10 was used as template/primer, no marked inhibition was observed. Dot-blot, gel-retardation, and cross-linking assays showed that Co3 or pCo3 interfere with the binding site of tRNALys.3 to RT. These results indicate that inhibition of RT at the level of initiation of the enzymic reaction is a novel approach to inhibit HIV-1 replication. PMID- 1705438 TI - Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody. AB - Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross reactivity by inducing nonspecific protein aggregation. PMID- 1705439 TI - Photo-modulated ion channels based on covalently linked gramicidins. AB - The covalent coupling of two gramicidin A monomers proved to be a useful tool for the rational design of ion channels with predictable electrophysiological properties (Stankovic, C.J., Heinemann, S.H., Delfino, J.M., Sigworth, F.J. and Schreiber, S.L. (1989) Science 244, 813-817; Stankovic, C.J., Heinemann, S.H. and Schreiber, S.L. (1990) J. Am. Chem. Soc. 112, 3702-3704). Herein we report on our first efforts to equip such channels with an artificial gating mechanism. Gramicidin monomers were covalently linked with 3,3'-azobis(benzeneacetic acid). Based on computer modeling of the beta-helix channel motif, this linker in its dark-adapted (trans) form does not allow for the formation of unimolecular ion channels, while the photo-activated (cis) form was expected to provide this possibility. The electrophysiological assays showed that (A) the trans-isomer does form characteristic ion channels, and (B) irradiation transforms these channels into a new distinct, flickering channel type in a reversible manner. The results are discussed in the framework of intermolecular gramicidin aggregates. PMID- 1705440 TI - Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria? AB - A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim. Biophys. Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla. A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes. The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl. Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa). Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin. No cross-reactivity was observed with antibodies against yeast porin. The peptide maps of the two bands showed slight differences. The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed. An extensive immunological comparison of different mitochondrial porins is presented. PMID- 1705441 TI - Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter. AB - The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner. PMID- 1705442 TI - A model for the extracellular release of PAF: the influence of plasma membrane phospholipid asymmetry. AB - Recent studies suggesting that cellular activation leads to enhanced transbilayer movement of phospholipids and loss of plasma membrane phospholipid asymmetry lead us to hypothesize that such events may govern the release of PAF, a potent, but variably release, lipid mediator synthesized by numerous inflammatory cells. To model these membrane events, we studied the transbilayer movement of PAF across the human erythrocyte and erythrocyte ghost plasma membrane, membranes with documented phospholipid asymmetry which can be deliberately manipulated. Utilizing albumin to extract outer leaflet PAF, transbilayer movement of PAF was shown to be significantly enhanced in erythrocytes and ghosts altered to lose membrane asymmetry when compared to movement in those with native membrane asymmetry. Verification of membrane changes was demonstrated using merocyanine 540 (MC540), a dye which preferentially stains loosely packed or hydrophobic membranes, and acceleration of the modified Russell's viper venom clotting assay by externalized anionic phospholipids. Utilizing the erythrocyte ghost loaded with PAF in either the outer or the inner leaflet, enhanced transbilayer movement to the opposite leaflet was seen to accompany loss of membrane asymmetry. Studies utilizing ghosts loaded with albumin intracellularly demonstrated that 'acceptor' molecules binding PAF further influence the disposition of PAF across the plasma membrane. Taken together, these findings suggest that the net release of PAF from activated inflammatory cells will depend on localization of PAF to the plasma membrane, transbilayer movement, which is facilitated by alteration of membrane phospholipid asymmetry, and removal from the membrane by extracellular and intracellular 'acceptor' molecules. PMID- 1705443 TI - Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies. AB - Epitopes on human brain creatine kinase (B-CK) recognized by three monoclonal antibodies have been located by chemical cleavage methods, followed by peptide synthesis or analysis of specificity for natural variants (isoforms). One antibody, CK-HTB, recognizes a conformational, or assembled, surface epitope on native CK which is also present on partially unfolded forms. It requires an Asn residue at position 300 in the amino acid sequence and will not recognize variants with Lys or His in this position. This results in a striking specificity of the antibody, which binds to B-CK only in chicken and man, but to muscle-form (M-CK) only in the rat. The results suggest that Asn-300 is exposed on the enzyme surface as part of a relatively denaturation-resistant region. Two monoclonal antibodies, CK-END1 and CK-END2, recognise epitopes within 53 amino acids of the C-terminus and bind to a synthetic hexapeptide representing the last six amino acids of human B-CK (Leu-375-Lys-380). The two antibodies show overlapping, but distinct, specificities in their binding to CK variants. CK-END1 requires Met-376 and will not tolerate Ile in this position, whereas CK-END2 requires Leu-375 and will not tolerate Met. Neither antibody binds to native CK, though both will bind to a folding intermediate and to partially unfolded states. This shows that the C terminus of CK becomes inaccessible to the antibodies during those later stages of protein folding associated with recovery of enzyme activity and suggests that the protein may 'tuck in its tail' during one of the final steps. PMID- 1705444 TI - The phosphorylation of a putative sperm microtubule-associated protein 2 (MAP2) is uniquely sensitive to regulation. AB - We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes. PMID- 1705445 TI - Differential expression of extracellular matrix components in rat Sertoli cells. AB - We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages. PMID- 1705446 TI - Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum. AB - The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules. PMID- 1705447 TI - Interleukin-1 beta is a potent stimulator of prostaglandin synthesis in bovine luteal cells. AB - Interleukin-1 (IL-1) is a polypeptide that has both local and systemic effects on numerous tissues, including endocrine cells. To evaluate the effect of IL-1 on luteal function, bovine luteal cells were cultured for 5 days with increasing concentrations (0.1, 0.5, 1.0, 2.5, 5.0, 10.0 ng/ml) of recombinant bovine interleukin-1 beta (rbIL-1 beta). IL-1 beta increased the production of luteal 6 keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2), and prostaglandin F2 alpha (PGF2 alpha) in a dose-dependent manner, but had no effect on progesterone (P4) production. Treatment with the cyclooxygenase inhibitor, indomethacin (5 micrograms/ml), inhibited basal, as well as rbIL-1 beta stimulated prostaglandin production. Addition of Iloprost (a synthetic analogue of prostacyclin, 5 ng/ml) suppressed basal production of PGF2 alpha and PGE2, but did not reduce the stimulatory effect of rbIL-1 beta. Similarly, PGF2 alpha suppressed basal, but not IL-1 beta-stimulated, production of 6-keto-PGF1 alpha. PGE2 had no effect on the synthesis of either PGF2 alpha or 6-keto-PGF1 alpha. P4 (1.75 micrograms/ml) reduced basal as well as rbIL-1 beta-stimulated production of 6-keto-PGF1 alpha, PGE2, and PGF2 alpha. These results indicate that IL-1 beta could serve as an endogenous regulator of luteal prostaglandin production. It appears that IL-1 beta action is not modified by exogenous prostaglandins, but is at least partially regulated by elevated P4. It is possible that the role of IL-1 beta in stimulation of luteal prostaglandin production may be confined to a period characterized by low P4 levels, such as during luteal development or regression. PMID- 1705448 TI - Ion-water and ion-polypeptide correlations in a gramicidin-like channel. A molecular dynamics study. AB - This work describes a molecular dynamics study of ion-water and ion-polypeptide correlation in a model gramicidin-like channel (the polyglycine analogue) based upon interaction between polarizable, multipolar groups. The model suggests that the vicinity of the dimer junction and of the ethanolamine tail are regions of unusual flexibility. Cs+ binds weakly in the mouth of the channel: there it coordinates five water molecules and the #11CO group with which it interacts strongly and is ideally aligned. In the channel interior it is generally pentacoordinate; at the dimer junction, because of increased channel flexibility, it again becomes essentially hexacoordinate. The ion is also strongly coupled to the #13 CO but not to either #9 or #15, consistent with 13C NMR data. Water in the channel interior is strikingly different from bulk water; it has a much lower mean dipole moment. This correlates with our observation (which differs from that of previous studies) that water-water angular correlations do not persist within the channel, a result independent of ion occupancy or ionic polarity. In agreement with streaming potential measurements, there are seven single file water molecules associated with Cs+ permeation; one of these is always in direct contact with bulk water. At the mouth of an ion-free channel, there is a pattern of dipole moment alteration among the polar groups. Due to differential interaction with water, exo-carbonyls have unusually large dipole moments whereas those of the endo-carbonyls are low. The computed potential of mean force for CS+ translocation is qualitatively reasonable. However, it only exhibits a weakly articulated binding site and it does not quantitatively account for channel energetics. Correction for membrane polarization reduces, but does not eliminate, these problems. PMID- 1705449 TI - Gramicidins A, B, and C form structurally equivalent ion channels. AB - The membrane structure of the naturally occurring gramicidins A, B, and C was investigated using circular dichroism (CD) spectroscopy and single-channel recording techniques. All three gramicidins form channels with fairly similar properties (Bamberg, E., K. Noda, E. Gross, and P. Lauger. 1976. Biochim. Biophys. Acta. 419:223-228.). When incorporated into lysophosphatidylcholine micelles, however, the CD spectrum of gramicidin B is different from that of gramicidin A or C (cf. Prasad, K. U., T. L. Trapane, D. Busath, G. Szabo, and D. W. Urry. 1983. Int. J. Pept. Protein Res. 22:341-347.). The structural identity of the channels formed by gramicidin B has, therefore, been uncertain. We find that when gramicidins A and B are incorporated into dipalmitoylphosphatidylcholine vesicles, their CD spectra are fairly similar, suggesting that the two channel structures could be similar. In planar bilayers, gramicidins A, B, and C all form hybrid channels with each other. The properties of the hybrid channels are intermediate to those of the symmetric channels, and the appearance rates of the hybrid channels (relative to the symmetric channels) corresponds to what would be predicted if all three gramicidin molecules were to form structurally equivalent channels. These results allow us to interpret the different behavior of channels formed by the three gramicidins solely on the basis of the amino acid substitution at position 11. PMID- 1705450 TI - Osmotic compression and stiffness changes in relaxed skinned cardiac myocytes in PVP-40 and dextran T-500. AB - Sarcomere lengths, cell widths, indices of stiffness, and striation pattern uniformity were determined from radially compressed isolated adult cardiac myocytes from the rat. Single cells were bathed in a series of relaxing solutions containing 0-15% concentrations of nonpenetrating long chain polymers PVP-40 and dextran T-500. There were no significant changes observed in average sarcomere lengths or in striation pattern uniformity at any concentration. But cell widths decreased and stiffness increased in both polymers in a concentration-osmotic pressure-dependent relationship. Changes in cell width and stiffness were repeatable in either polymer, but only after an initial compression with a 10 or 15% concentration solution. The observed reduction in cell width after initial compression correlates well with known myofilament lattice spacing compression in rat cardiac muscle and is qualitatively similar to compressions seen in skeletal muscle preparations. But the cardiac myofilament lattice may not be as compressible as the skeletal lattice. Like skeletal muscle, stiffness exhibits a two-phase relationship where most of the increase occurs at solution osmotic pressures greater than 20 Torr. Finally, the inherently greater passive stiffness length relationship of cardiac muscle is maintained at higher osmotic pressures such that the passive elastic modulus is strongly length dependent. PMID- 1705451 TI - Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644. AB - Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents. PMID- 1705452 TI - [Effects of tumor necrosis factor on the tonus of cerebral arteries]. AB - It was determined that tumor necrosis factor (TNF) is capable of decreasing the local brain's blood flow on 45.6% (in the concentration of 6 micrograms/kg); to make a spasm of the pial arteries on 39.6%. In vitro experiments TNF increased the amplitude of the rhythmical and the tonic contractions of the brain's arteries smooth muscles (3.6 X 10(-8) M). The direct action of the TNF in the vascular wall is endothelium-dependent. PMID- 1705453 TI - [Effects of an epiphyseal peptide preparation epithalamin on serotonin metabolism in the pineal gland of rats]. AB - 5-day morning injections to pubertal male rats of polypeptide epithalamin preparation obtained from cattle epiphysis, in dose of 0.25 mg/100 g body mass induced the increase of serotonin epiphyseal concentration night peak, N acetylserotonin and melatonin and didn't produce any essential influence on 5 methoxytryptamine, 5-oxy- and 5-methoxyindoleacetic acid level. It has been concluded, that epiphyseal peptides and indoles interact according to ultrashort connection, epiphyseal peptides point of application is the reaction of tryptophan transformation into serotonin and its further metabolism in N acetylserotonin and melatonin. It has been suggested that the increase of epiphyseal melatonin production is on the basis of epithalamin therapeutic action. PMID- 1705454 TI - [Dynamics of alkaline hydrolysis of RNA oligoadenylates in hydrolysis of homogenates of the rat cerebral cortex and liver]. PMID- 1705455 TI - [Suppression of cytotoxic cellular reactions by the production of antibodies to rhamnose determinants of streptococcal group A polysaccharide cross-reacting with antigens of the skin epithelium]. AB - By the BALB/c mice after different periods of immunization with the streptococci group A, treated with pepsin, antibodies belonging to autoantibodies to the determinants (DT) of polysaccharide (A-PS), cross-reactive (CR) with the epithelial skin cells, were investigated. In one of the mice groups, in the autologous system, on the target cells--macrophages of lymph nodes, the suppression of cytotoxic (CT) reactions was obtained. The CR are bound with the delayed type hypersensitivity appearing after the sensibilization with BCG. The suppression effect correlate (z-0.95) with the presence in the sera antibodies to the rhamnose DT'S of A-PS, which cross-react with the cells of basal and superbasal layers of skin epithelium. Antibodies to the group specific of the A PS, cross-react only with the basal skin layer and not produce the suppression of CT reactions. It is possible that they also prevent the suppression of CT reactions, bound with the CR antibodies to the rhamnose DT-S of A-PS. The obtained data corroborate the earlier supposition that the autoantibodies to the CR DT'S of A-PS reacting with the skin epithelial cells as a rule common the thymus epithelial cells. It is possible that different IRD'S can prevent or stimulate the development of autoimmune processes by the infections with the streptococci group A. PMID- 1705456 TI - [Cellular mechanisms of suppression of T-lymphocyte proliferation by lung cells in experimental tuberculosis]. AB - The ability of interstitial lung cells from mice, infected with Mycobacterium tuberculosis H37Rv, to suppress proliferative responses of immune lymphocytes to mycobacterial (PPD) and unrelated (Staphylococcus aureus cytoplasm) antigens was studied. Two types of suppression were observed: the specific one, which was characteristic of the PPD-response only; and non-specific. The latter was mediated mainly by prostaglandins, since it could be abolished by indomethacin. Both types of suppression depended on the presence of plastic and nylon wool adherent phagocytes from infected lung. Though the depletion of T or B lymphocytes from the lung cell population have not abrogated the suppressive effect, some intercellular interactions were required for antigen-specific suppression, since the presence of nylon wool adherent cells in the population of responder lymph node cells was necessary for its development. PMID- 1705457 TI - [Level of serotonin and 5-hydroxyindoleacetic acid in the brain and immunocompetent organs after immunization]. AB - Twenty minutes after immunization the activation of the serotoninergic system was observed. High level of serotonin metabolism was retained for 24 h after immunization. Change of serotonin level in immunocompetent organs and adrenals took place later than in the brain. PMID- 1705458 TI - [Fast direct method of obtaining metaphase and prometaphase chromosomes from chorion biopsy cells and human embryos during the lst semester of pregnancy]. AB - Samples of chorionic villi and embryonic tissues (brain, brain--sheaths) are thoroughly washed with Hank's solution, immediately subjected to hypotonic treatment (0.9% sodium citrate plus few drops of 0.01% colchicine) 37 degrees C, 30 min, prefixed 20 min with equal amount of standard fixative mixture, twice fixed in standard fixative solution (1 hour, -10 degrees C), hydrated with equal volume of distilled water (5-10 min), dried, macerated directly on the slide with 60% acetic acid. The cell suspension is then evenly spread on the slide surface, dried, postfixed and stained. The method provides sufficient amount of metaphase and prometaphase mitotic plates suitable for differentiating staining in 1.5-2 hours after sampling and might be recommended for routine chromosomal analysis in prenatal diagnosis of inherited diseases during early pregnancy. PMID- 1705459 TI - Graft failure after T cell-depleted bone marrow transplantation: clinical and immunological characteristics and response to immunosuppressive therapy. AB - We have investigated the clinical and immunological features of 10 cases of graft failure after T cell-depleted marrow transplantation. In addition, the hypothesis that the process of graft failure can be reversed by immunosuppressive therapy with cyclosporin + steroids +/- monoclonal antibodies was tested in seven patients. Early graft failures (before day 50) presented a uniform clinical syndrome with a host T lymphocytosis preceding the loss of the graft. In the majority of cases of late graft failure (after day 50) a syndrome comprising delayed granulopoietic regeneration, fever of unknown origin and abdominal symptoms was observed. Surface marker analysis of peripheral blood and bone marrow lymphocytes implicated a population of CD3+, CD8+, DR+ host T lymphocytes with a frequent co-expression of the Leu7+ antigen in the pathogenesis of graft failure. Immunosuppressive therapy reversed graft failure in the three cases of incomplete graft failure (i.e. with residual reticulocytes) and failed in the four cases of complete graft failure (i.e. no residual reticulocytes). PMID- 1705460 TI - Tumour markers in screening. PMID- 1705461 TI - Role of the protease-antiprotease balance in peritoneal exudate during acute pancreatitis. AB - The peritoneal exudate formed during experimental pancreatitis is toxic when administered intraperitoneally or intravenously to other animals. Overwhelming of the peritoneal antiprotease defences by proteolytic enzymes released from the pancreas may be a key factor responsible for this toxicity and is examined in the current study. Human pancreatitis exudates possessed tryptic amidase activity indicating trypsinogen activation. The trypsin inhibitory capacities of exudates were reduced indicating a degree of consumption of the peritoneal antiproteases. Of 21 exudates examined, three showed marked reduction of their trypsin inhibitory capacity indicating almost complete consumption of their antiproteases. All three patients were shocked at the time of sampling, two dying of fulminant pancreatitis within 24 h. Overwhelming of the peritoneal antiproteases was not confirmed, but may occur in a few instances where proteolytic enzyme release or zymogen activation continues. Intraperitoneal administration of exogenous antiproteases prolongs survival in rats with pancreatitis and has been suggested as a therapy in man. The current data suggests that few patients are likely to benefit from such an approach. PMID- 1705462 TI - Changes in fast axonal transport of glycoproteins in optic nerves of mice infected with Semliki Forest virus. AB - [3H]fucose incorporation into mouse retinae and subsequent axonal transport of glycoproteins to optic nerve terminals was studied before and during optic nerve demyelination induced by Semliki Forest virus (SFV) infection. As was previously found for [3H]proline-labelled protein, axonally transported glycoprotein was increased before demyelination. Fluorographic analysis of the increased glycoproteins and proteins, after separation by gel electrophoresis, showed particularly large increases in labelling of 2 glycoproteins (38.1 kDa and 45.0 kDa) and 3 protein (15.9 kDa, 23.8 kDa and 27.7 kDa) bands. These increases were due to host-cell rather than viral components. At the time of demyelination, however, [3H]fucose incorporation into retinae of SFV-infected mice was significantly depressed, resulting in reduced label incorporation into axonally transported glycoproteins. Since glycoproteins play a role in axon-glia recognition and adhesion, the early changes in their axonal transport may contribute to the mechanism of demyelination. PMID- 1705463 TI - Effects of anticonvulsant treatment and low levels of folate and thiamine on amine metabolites in cerebrospinal fluid. AB - A total of 157 epileptic patients were studied with respect to (1) biogenic amine precursors and metabolites in the CSF, (2) levels of folate and thiamine in the blood and CSF, (3) length of treatment with phenytoin (PHT), (4) PHT intoxication, (5) CNS atrophy. Alterations in CSF amine metabolite levels were related primarily to PHT intoxication, and low CSF folate and thiamine levels, but not to length of treatment or CNS atrophy. PHT intoxication increased CSF 5 hydroxyindoleacetic acid (5HIAA). Low folate levels were associated with decreased CSF 5HIAA and homovanillic acid, while low thiamine levels were associated with decreased CSF 5HIAA and 3-methyoxy-4-hydroxyphenylethylene glycol. It remains to be seen to what extent these alterations in biogenic amine metabolism, mediated by low CNS vitamin levels, also lead to deficits in cerebral function. PMID- 1705464 TI - Neurotransmitter-specific uptake and retrograde transport of [3H]glycine from the inferior colliculus by ipsilateral projections of the superior olivary complex and nuclei of the lateral lemniscus. AB - Neurotransmitter-specific uptake and retrograde axonal transport of [3H]glycine were used to identify glycinergic projections to the inferior colliculus in chinchillas and guinea pigs. Six h after injection of [3H]glycine in the inferior colliculus, autoradiographically labeled cells were found ipsilaterally in the ventral nucleus of the lateral lemniscus, the lateral superior olive and the dorsomedial periolivary nucleus. These 3 regions accounted for 95% of the labeled projection neurons, with the remainder scattered elsewhere in the ipsilateral superior olivary complex. No labeled cells were found contralaterally even after survival times as long as 24 h. Retrograde transport of HRP from the inferior colliculus in these same cases confirmed the presence of additional projections that did not accumulate [3H]glycine. These included ipsilateral projections from the medial superior olive and cochlear nucleus and contralateral projections from the inferior colliculus, dorsal nucleus of the lateral lemniscus, lateral superior olive, periolivary nuclei and cochlear nucleus. The results implicate uncrossed projections from the ventral nucleus of the lateral lemniscus, lateral superior olive, and dorsomedial periolivary nucleus as the principal sources of inhibitory glycinergic inputs to the inferior colliculus. PMID- 1705465 TI - 125I-BH[Sar9, Met(O2)11]-SP, a new selective ligand for the NK-1 receptor in the central nervous system. AB - The selective agonist [Sar9,Met(O2)11]-SP was radioiodinated with 125I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with 125I-BH.SP. Specific binding of 125I-BH[Sar9,Met(O2)11]-SP was temperature-dependent, saturable and reversible. In brain homogenates, 125I BH[Sar9,Met(O2)11]-SP interacted with a single class of high affinity (kd = 1.0 nM) non-interacting binding sites (Bmax of 15 fmol/mg protein). In the central nervous system, 125I-BH-[Sar9,Met(O2)11]-SP apparently labeled the same number of binding sites as 125I-BH.SP (19 fmol/mg proteins). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by 125I-BH[Sar9,Met(O2)11]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar9,Met(O2)11)]-SP, an excellent correlation (r = 0.96) was found for the Ki values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (Ki) of various neurokinin-related peptides for the brain site were compared with their biological activities on various isolated organs (dog carotid artery, guinea-pig ileum, rat portal vein). NK-1 binding sites characterized in rat brain homogenates appear to be identical with those present on the dog carotid artery, a preparation known to possess exclusively the NK-1 receptor type. PMID- 1705466 TI - Long-term retrograde labelling of neurons. AB - The intensity of labelling of neuronal perikarya with Fluoro-gold or rhodamine microspheres appeared unchanged in rats surviving one year after surgery. These tracers may be used for sequential labelling with long intervals and to study brain connections in precious specimens. PMID- 1705467 TI - Monosynaptic projection from the pedunculopontine tegmental nuclear region to the reticulospinal neurons of the medulla oblongata. An electron microscope study in the cat. AB - The magnocellular reticular nucleus (MRN) of the cat was observed electron microscopically following the combined application of kainic acid into the pedunculopontine tegmental nuclear region (PPN region) and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) into the spinal cord. Some degenerating axon terminals arising from the PPN region were found to make synaptic contacts with retrogradely HRP-labeled reticulospinal neurons in the MRN. PMID- 1705468 TI - Nimodipine enhances spontaneous activity of hippocampal pyramidal neurons in aging rabbits at a dose that facilitates associative learning. AB - The functional activity of hippocampal neurons is strongly correlated with behavioral performance in a vertebrate model learning system, rabbit eyeblink conditioning. Using this system, we have previously shown that (a) complete removal of the hippocampus blocks acquisition of the conditioned response; (b) a calcium-dependent postsynaptic afterhyperpolarization is reduced in pyramidal cells recorded intracellularly in hippocampal slices taken from conditioned rabbits; and (c) nimodipine, a 1,4-dihydropyridine calcium-channel antagonist, facilitates acquisition of the conditioned response in aging rabbits. Although calcium-channel antagonists directly block neuronal calcium currents in vitro, they also alter cerebral blood flow in vivo. Thus, the effects of nimodipine on hippocampal neuronal activity in awake animals were examined, with controls for cerebrovascular changes. A total of 457 pyramidal cells and 160 theta cells were studied. During infusion of nimodipine, pyramidal cell firing activity was enhanced and theta interneuron activity was suppressed at all doses tested in aging animals. This effect was rapidly reversed when infusion of the drug ceased. The greatest enhancement of neuronal firing was seen at the most behaviorally effective dose of nimodipine. The enhancement of pyramidal cell firing was age dependent, with greater increases in firing activity seen in aging than in young animals, but with a similar dose-dependent pattern of effects in the two age groups. Two other calcium-channel antagonists, nifedipine and flunarizine, did not significantly alter spontaneous firing rates of hippocampal neurons. A calcium-channel agonist, BAY-K-8644, produced less easily interpretable results. BAY-K-8644 enhanced interneuron activity at one dose, but enhanced pyramidal cell activity at a dose one log unit higher. The calcium-channel agonist's enhancement of pyramidal cell activity at the highest dose was sustained up to 1 h after drug infusion. Nimodipine's enhancement of the activity of hippocampal pyramidal cells is consistent with the hypothesis that these neurons, which play a necessary role in some forms of learning, may mediate the calcium-channel antagonist's behavioral effects. PMID- 1705469 TI - Substantia nigra pars reticulata projects to the reticular thalamic nucleus of the cat: a morphological and electrophysiological study. AB - The projections of substantia nigra pars reticulata (SNr) toward the reticular (RE) thalamic complex of cat were studied morphologically and electrophysiologically. Numerous SNr cells were retrogradely labeled following injections of horseradish peroxidase conjugated to wheat-germ agglutinin (WGA HRP) in the rostral and rostrolateral part of the RE thalamic nucleus. Iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHA-L) in the SNr confirmed that this retrograde labeling was not a consequence of tracer diffusion in neighboring structures, but reflected a genuine SNr projection to the RE thalamic nucleus. Indeed, widely branched and varicose PHA-L-positive fibers were found in the rostral and rostrolateral pole of the RE thalamic nucleus following PHA-L injections in the SNr. Furthermore, in agreement with previous data indicating that nigrothalamic cells are GABAergic, electrical stimulation of the SNr evoked a short-latency inhibitory effect acting on both spontaneous and cortically-evoked discharges of most RE thalamic neurons. These results are discussed in light of the possible role of the SNr and RE thalamic complex in attentional processes. PMID- 1705470 TI - A distinct type of displaced ganglion cell in a mammalian retina. AB - The morphology, dendritic stratification and laminal position of the soma of retinal ganglion cells were analyzed in Golgi preparations and in other rabbit retinas containing cells backfilled from the superior colliculus. Only one type, among 40 Golgi-impregnated types identified, always had its cell body displaced to the amacrine cell sublayer of the inner nuclear layer. The displaced ganglion cell of rabbit retina has a small cell body, very wide dendritic field with sometimes unbranched dendrites extending up to a millimeter from the cell body. The dendritic tree is narrowly stratified just under the amacrine cell bodies in stratum 1, and therefore does not co-stratify with starburst (cholinergic) amacrine cells, but rather with dopaminergic amacrine cells. Its correlate among ganglion cells backfilled from tectum is apparently a very sparse population of small-bodied cells mixed with a variable population of misplaced ganglion cells of varying size and type. The authentic displaced ganglion cell of rabbit retina, unlike the large displaced ganglion cell of birds, is apparently not a directionally selective ganglion cell, and its functional role in vision is presently unknown. PMID- 1705471 TI - Tissue culture tube contaminant inhibits excitatory synaptic channels. AB - Nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus oocytes. Large, slowly desensitizing macroscopic currents were induced by rapidly infusing an electrolyte solution containing 50 microM ACh in the presence of atropine. The N-methyl-D-aspartate (NMDA) subset of glutamate receptors was studied in CA1 hippocampal neurons. Macroscopic currents were induced by rapidly applying 30 microM NMDA and 10 microM glycine in the presence of picrotoxin, strychnine and tetrodotoxin. Exposing the electrolyte solutions to Falcon brand polypropylene tissue culture tubes (Becton Dickinson Labware) decreased the current through the nAChR channels or through the NMDA receptor channels (1). Purified water shaken in the Falcon brand tubes showed a broad absorbance peak near 200 nm. Before exposing the water to the Falcon tubes, the purified water gave no absorbance signal. The results indicate that a substance released from the Falcon tubes inhibits the currents through nAChR and NMDA receptor channels. Our experiments were with 50-ml Falcon 2098 tubes from lot numbers 72180188 and 80290188 and with 15-ml Falcon 2097 tubes from lot number 83560283. These were the only Falcon tubes we tested. PMID- 1705472 TI - Cryostat sectioning of large brains made easy. PMID- 1705473 TI - Nailfold capillary microscopy and bleomycin-induced vascular toxicity. PMID- 1705474 TI - Cell motility in angiogenesis and tumor metastasis. PMID- 1705475 TI - Alpha 2-HS glycoprotein phenotypes and quantitative hormone and bone measures in postmenopausal women. AB - It has been suggested that inherited traits play a role in the development of osteoporosis by providing a background for the modulation of gene expression. In this study, we examine the influence of the different alleles of alpha 2-HS glycoprotein (AHSG), a protein of the bone matrix, on quantitative estrogens, estrone and estradiol, and bone measures, bone area and density. Estrogens provide a protective effect against fractures in older women and were thus included in the analyses. Isoelectric focusing of AHSG from sera followed by immunoblotting was used to type 163 white postmenopausal women participating in a clinical trial of the effects of walking on bone loss. Plasma hormones were measured by a combination of extraction, column chromatography, and radioimmunoassay; bone measures on the dominant radius were determined with computerized tomography. Analysis of variance was done on estrogen and bone measures after controlling for the effects of age and body mass index. The two major alleles of AHSG result in three phenotypes, designated AHSG 1-1, AHSG 2-1, and AHSG 2-2. The AHSG 1-1 homozygote showed a decreased concentration of estradiol, the AHSG 2-2 homozygote showed an increased concentration, and the AHSG 2-1 heterozygote was intermediate (P = 0.001). Estrone demonstrated a similar pattern in residual analysis although it did not reach statistical significance. PMID- 1705476 TI - Doxorubicin stimulates transcription from the human immunodeficiency virus long terminal repeat sequences. AB - A recombinant plasmid carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1) linked to the reporter chloramphenicol acetyl transferase (CAT) gene was stably introduced into rat liver cells. The transfectant cells expressed CAT activity from the HIV LTR. The response to doxorubicin was studied and it was found that at the optimum concentration of 20 micrograms/ml doxorubicin, the expression of CAT from the HIV LTR was stimulated by 65-fold. Our results suggest caution against therapy including doxorubicin in the treatment of AIDS patients. PMID- 1705477 TI - Human carcinoma cells express receptors for distinct domains of thrombospondin. AB - Thrombospondin (TSP), an adhesive glycoprotein, is incorporated into the extracellular matrix, mediates cell attachment and spreading, chemotaxis, haptotaxis, and may participate in the directed movement of cells in metastasis. Evidence from several model systems suggests that these functions may be mediated by different domains within the TSP molecule. Radioligand binding assays on 11B squamous carcinoma cells with 125I-Radioligand binding assays on 11B squamous carcinoma cells with 125I-TSP demonstrated the presence of 1.2 x 10(6) sites/cell with an apparent Kd of 74 nM. Binding studies using TSP fragments demonstrated that both the NH2 terminal heparin-binding domain (HBD) and the COOH terminal fragment with a molecular weight of 140,000 (140K) retained the ability to bind 11B cells in a time-dependent, dose-dependent, saturable, and specific manner. The HBD bound to 11B cells with an apparent Kd of 1.2 microM at 1.4 x 10(6) sites/cell. Binding of 140K to cells demonstrated half-maximal binding at 36 nM and a Bmax of 1.9 x 10(5) sites/cell. The binding of 140K also showed a high degree of positive cooperativity with a Hill slope of +3.5, suggesting that binding one 140K molecule to cells leads to increased binding of additional 140K molecules. In addition, the HBD and 140K showed no cross-competition in binding assays. Therefore, it appears likely that these distinct TSP domains bind to separate sites on the cell surface. Neither vitronectin or the peptide RGDS were able to inhibit the binding of TSP or 140K to 11B cells. Based on these data, there appears to be more than one distinct receptor on 11B cells for TSP; one receptor class which mediates the binding of the HBD and a second receptor class which mediates the binding of the Mr 140,000 fragment. PMID- 1705478 TI - Galanin stimulates Ca2+ mobilization, inositol phosphate accumulation, and clonal growth in small cell lung cancer cells. AB - Addition of the neuropeptide galanin to small cell lung cancer (SCLC) cells loaded with the fluorescent Ca2+ indicator fura-2-tetraacetoxymethylester causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i) followed by homologous desensitization. Galanin increased [Ca2+]i in a concentration-dependent fashion with half-maximum effect (EC50) at 20-22 nM in H69 and H510 SCLC cells. Galanin mobilized Ca2+ from intracellular stores since its effects on [Ca2+]i were not blocked by chelation of extracellular Ca2+. Pretreatment with pertussis toxin (200 ng/ml for 4 h) did not prevent galanin induced Ca2+ mobilization. In contrast, direct activation of protein kinase C with phorbol esters attenuated the Ca2+ response induced by galanin. The effects of galanin could be dissociated from changes in membrane potential: galanin did not increase membrane potential in SCLC cells loaded with bis(1,3 diethyltiobarbiturate)-trimethineoxonol and induced Ca2+ mobilization in depolarized SCLC cells, i.e., in cells suspended in a solution containing 145 mM K+ instead of Na+. Galanin also caused an increase in the formation of inositol phosphates in a time- and dose-dependent manner (EC50 10 nM). A rapid increase in the inositol trisphosphate fraction was followed by a slower increase in the inositol monophosphate fraction. Galanin stimulated clonal growth of both H69 and H510 cells in semisolid (agarose-containing) medium. This growth-promoting effect was sharply dependent on galanin concentration (EC50 20 nM) and markedly inhibited by [Arg6,D-Trp7,9,MePhe8]substance P, a recently identified broad spectrum neuropeptide antagonist. The results show for the first time that galanin receptors are coupled to inositol phosphate and [Ca2+]i responses in SCLC cells and, in particular, that this neuropeptide can act as a direct growth factor for these human cancer cells. PMID- 1705479 TI - Leukocyte biophysics. An invited review. AB - The biophysical properties of leukocytes in the passive and active state are discussed. In the passive unstressed state, leukocytes are spherical with numerous membrane folds. Passive leukocytes exhibit viscoelastic properties, and the stress is carried largely by the cell cytoplasm and the nucleus. The membrane is highly deformable in shearing and bending, but resists area expansion. Membrane tension can usually be neglected but plays a role in cases of large deformation when the membrane becomes unfolded. The constant membrane area constraint is a determinant of phagocytic capacity, spreading of cells, and passage through narrow pores. In the active state, leukocytes undergo large internal cytoplasmic deformation, pseudopod projection, and granule redistribution. Several different measurements for assessment of biophysical properties and the internal cytoplasmic deformation in form of strain and strain rate tensors are presented. The current theoretical models for active cytoplasmic motion in leukocytes are discussed in terms of specific macromolecular reactions. PMID- 1705480 TI - The surface free energy of Leishmania mexicana amazonensis. AB - Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively. PMID- 1705481 TI - The influence of different flow velocities on tumor cell survival in micropore filters. AB - Earlier studies have shown a lower degree of lodgement and early survival of tumor cells in muscle than in liver after infusion via the femoral artery and portal vein, respectively. A possible explanation to this difference might be that the tumor cells are mechanically destroyed, and thus die more rapidly in muscle because they enter this capillary network at a much higher flow velocity. In the present study, the effect on early tumor cell (rat fibrosarcoma) survival of a high and a low flow velocity/deformation rate was evaluated in micropore (5 microns) filters, using isotope (Cr51) technique. These experiments were combined with scanning electron microscopic (SEM) analyses. The filter experiments showed no significant differences in the rate of cell death in the filters between tumor cells subjected to high or low deformation rates, and there were no qualitative differences in tumor cell appearance in the SEM study. It is, therefore, concluded that the difference in tumor cell lodgement and survival between muscle and liver is not primarily caused by differences in the rate of cell deformation upon entry of the organ capillary network. PMID- 1705482 TI - Splitting cell adhesiveness into independent measurable parameters by comparing ten human melanoma cell lines. AB - The concept of cell adhesiveness was analyzed by looking for correlations between the adhesive behavior and measurable biological properties of different cell populations. Ten established lines of melanoma cells were assayed for passive deformability (by micropipet aspiration), active spreading (by measuring the height/diameter ratio after incubation on different surfaces), density and mobility of concanavalin A binding sites (by quantitative analysis of fluorescence microscopic images), spontaneous and concanavalin A-mediated agglutination (by measuring the number of cell conjugates resisting calibrated shearing forces), and binding to glass capillary tubes (with a quantitative assay of binding strength). Forty-four different parameters were thus measured, and each set of determinations was repeated 2 or 3 t at different days on each cell line. Analysis of variance was performed to assess the capacity of each parameter to discriminate between different lines. Correlations between different parameters were studied in order to understand a possible influence of cell intrinsic properties on the behavior of individual cells. The following conclusions were suggested by experimental data 1. Cell spreading ability, resistance to slow deformation within a micropipette and ability to form shear resistant bonds, are independent properties. It is therefore suggested that different mechanisms rule the cell deformations on time scales of several minutes, tens of seconds, and fractions of a second. 2. Cell spreading ability may effectively influence binding strength only when adhesive stimuli are low, since in this case, cell stiffness is likely to impair the formation of extensive contact areas. 3. Individual cells may display marked heterogeneity within a given population, that emphasizes the danger of using averaged parameters to predict rare events (such as metastasis formation). 4. The most useful parameters to discriminate between different cell lines were, spreading ability and shear resistant lectin agglutination, and substrate adhesion. It is concluded that cell adhesion is influenced by several measurable cellular properties that may display independent variations. The importance of a given parameter depends on the conditions of bond formation and rupture. PMID- 1705484 TI - [Clinical analysis of 200 cases of necrotic thromboangiitis obliterans]. AB - Two hundred cases of necrosis thromboangiitis obliterans (199 males, 1 females; age 17 approximately 63 years) were observed. The illness course averaged more than 5 years. Focuses of upper limbs were 6 cases and those of lower limbs 194 cases. 98 cases and 102 cases suffered from ulcer and gangrene respectively. 100 cases belonged to the first degree, 62 cases to the second degree, 38 cases to the third degree of III stage. 171 cases were treated by the combination of TCM and WM except 3 cases which were actively discharged and 26 cases which were to be given amputation. RESULTS: 90 cases within 171 cases were cured, 52 cases were improved obviously, 7 cases improved, 5 cases ineffective and 17 cases to be given amputation. The writers regard three principles should be followed in the treatment of necrosis thromboangiitis obliterans by the combination of TCM and WM. (1) The main importance was to improve blood circulation to remove blood stasis. (2) Controlling infection was the key point, effective antibiotics and hormone should be added besides these herbal medicines for clearing away heat and toxic materials. (3) General protecting therapy, performing various local operations on right period on the basis of improvement of blood circulation of the involved limbs, the wound could heal successfully. The writers suggest that the amputation should not be given above the level of the main artery obliterans. PMID- 1705483 TI - Fusion of enveloped viruses with cells and liposomes. Activity and inactivation. AB - The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion. Influenza virus and Sendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case of Sendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a "fresh" batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via "inactive" sites. Details of the low pH inactivation of fusion capacity of influenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA2, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes. PMID- 1705485 TI - Endothelial cells and angiogenic growth factors in cancer growth and metastasis. Introduction. PMID- 1705486 TI - Extracellular matrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis. AB - Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a solid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM produced in vitro and in basement membranes of the cornea and blood vessels in vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase. PMID- 1705487 TI - Gangliosides, copper ions and angiogenic capacity of adult tissues. AB - The report summarizes the work of our laboratory aimed at improving the understanding of the angiogenic response of adult tissues, an event that transforms a micro-embolus of neoplastic cells into a growing metastasis. Attention has been focused on tumor-induced angiogenesis. The following aspects of the subject are discussed: (a) relationship between size of vascular network and tumor growth rate or tumor cell population; (b) angiogenic capacity of tumors and role that prostaglandin E1 may have as an angiogenesis factor; (c) relationship between acquisition of angiogenic capacity and neoplastic transformation of a cell population; (d) modification of tissue composition at the onset of angiogenesis; (e) behaviour of copper ions and copper carriers in the course of the angiogenic response; (f) the influence of gangliosides on endothelial cell motility, survival and growth in vitro; (g) modulation of the angiogenic response by gangliosides (GM1, GT1b) in vivo. PMID- 1705488 TI - Effect of recombinant granulocyte colony-stimulating factor (rG-CSF) on chemotherapy-induced neutropenia in patients with urogenital cancer. AB - The effects of recombinant granulocyte colony-stimulating factor (rG-CSF) on the myelosuppression, especially neutropenia, induced by cancer chemotherapy in patients with urogenital cancer were investigated in a randomized, controlled clinical study. In this study, rG-CSF was given subcutaneously at a dose of 2 micrograms/kg per day for 14 consecutive days. Changes in neutrophil counts were compared between the first (no rG-CSF) and second cycles (rG-CSF treatment period) of chemotherapy. rG-CSF administration was found to be effective in reducing the duration of neutropenia, in elevating the neutrophil nadir, and in reducing recovery time. Based on comparisons between the randomized rG-CSF treatment group (with rG-CSF) and the control group, treatment with rG-CSF resulted in the moderation or prevention of neutropenia and the acceleration of recovery. These results demonstrate that in chemotherapy of patients with urogenital cancer, in which neutropenia is a dose- or schedule-limiting factor, the concomitant use of rG-CSF may enable an increase in the dose (higher single dose or increased dose per unit of time) or shorten the chemotherapy period. PMID- 1705489 TI - Pharmacokinetics of thio-TEPA and TEPA in the conventional dose-range and its correlation to myelosuppressive effects. AB - A total of 13 patients with ovarian cancer were studied during the initial two courses of i.v. thio-TEPA treatment they underwent after primary surgery. Following an increase in the dose from 60 to 80 mg for the second course, no sign of saturation of thio-TEPA elimination processes or of formation of the metabolite TEPA occurred, indicating dose-independent pharmacokinetics. Myelosuppression after courses was registered by serial measurements of platelets and leukocytes. The time to platelet nadir was quite uniformly 3 weeks and tended to be longer than that of leukocytes, which averaged 2 weeks but showed greater interindividual variation. Linear regression analyses of pharmacokinetic parameters versus myelosuppression revealed statistically significant correlations between thio-TEPA pharmacokinetics and the percentage of reductions in leukocytes and platelets at their mean nadirs. In contrast, no such correlation could be demonstrated for TEPA despite its greater exposure to the body in terms of AUC. We advocate further investigation of this pharmacokinetic pharmacodynamic relationship so as to establish individualized dosing of thio TEPA. PMID- 1705490 TI - Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio). AB - 1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle. 2. On a Sephadex G25 column (3 x 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530. 3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A. 4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A. 5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P. 6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb. PMID- 1705491 TI - Small-volume resuscitation from hemorrhagic shock in dogs: effects on systemic hemodynamics and systemic blood flow. AB - BACKGROUND AND METHODS: This study compared canine systemic hemodynamics and organ blood flow (radioactive microsphere technique) after resuscitation with 0.8% saline (Na+ 137 mEq/L), 7.2% hypertonic saline (Na+ 1233 mEq/L), 20% hydroxyethyl starch in 0.8% saline, or 20% hydroxyethyl starch in 7.2% saline, each in a volume approximating 15% of shed blood volume. Twenty-four endotracheally intubated mongrel dogs (18 to 24 kg) underwent a 30-min period of hemorrhagic shock, from time 0 to 30 min into the shock period, followed by fluid resuscitation. Data were collected at baseline, 15 min into the shock period, immediately after fluid infusion, 5 min after the beginning of resuscitation, and at 60-min intervals for 2 hr, (65 min after the beginning of resuscitation, and 125 min after the beginning of resuscitation). The animals received one of four randomly assigned iv resuscitation fluids: saline (54 mL/kg), hypertonic saline (6.0 mL/kg), hydroxyethel starch (6.0 mL/kg) or hypertonic saline/hydroxyethyl starch (6.0 mL/kg). RESULTS: Mean arterial pressure increased in all groups after resuscitation. Cardiac output increased with resuscitation in all groups, exceeding baseline in the saline and hypertonic saline/hydroxyethyl starch groups (p less than .05 compared with hypertonic saline or hydroxyethyl starch). Sixty five minutes after the beginning of resuscitation, cardiac output was significantly (p less than .05) greater in either of the two colloid-containing groups than in the hypertonic saline group. After resuscitation, hypertonic saline and hydroxyethyl starch produced minimal improvements in hepatic arterial flow, hypertonic saline/hydroxyethyl starch increased hepatic arterial flow to near baseline levels, and saline markedly increased hepatic arterial flow to levels exceeding baseline (p less than .05, saline vs. hydroxyethyl starch). One hundred twenty-five minutes after the beginning of resuscitation, hepatic arterial flow had decreased in all groups; hepatic arterial flow in the hypertonic saline group had decreased to levels comparable with those during shock. Myocardial, renal, and brain blood flow were not significantly different between groups. CONCLUSIONS: Small-volume resuscitation with the combination of hypertonic saline/hydroxyethyl starch is comparable with much larger volumes of 0.8% saline, and is equal to hypertonic saline or hydroxyethyl starch in the ability to restore and sustain BP and improve organ blood flow after resuscitation from hemorrhagic shock. PMID- 1705492 TI - Advantages of a narrow-range, medium molecular weight hydroxyethyl starch for volume maintenance in a porcine model of fecal peritonitis. AB - OBJECTIVE: To compare the effectiveness of two hydroxyethyl starch solutions of different molecular weight ranges for volume maintenance in a porcine model of fecal peritonitis. DESIGN: Randomized prospective trial. SETTING: Laboratory investigation. SUBJECTS: Adolescent female pigs weighing approximately 30 kg. INTERVENTIONS: We compared diafiltered 6% pentastarch with 6% high molecular weight hetastarch for volume maintenance in a porcine model of fecal peritonitis. The number average molecular weight of pentastarch is higher than hetastarch, although the weight average molecular weight is lower, i.e., a narrow range of medium weight molecules. The infusion rate of each agent was adjusted to maintain baseline arterial Hct for less than or equal to 7 hr after instrumentation and induction of fecal peritonitis. MAIN OUTCOME MEASUREMENTS: The volume of fluid required to maintain arterial Hct was compared along with comparisons of hemodynamic and histologic responses associated with the two agents. RESULTS: Significantly less pentastarch was required to prevent hemoconcentration than hetastarch (109 +/- 22.8 vs. 150 +/- 10.3 mL/kg; p less than .05) while hemodynamics, colloid osmotic pressure, and oxygen transport responses were similar. Capillary patency was greater (21.99 +/- 3.68 vs. 10.09 +/- 1.17%; p less than .05) and mean alveolar capillary barrier thickness was less (2.36 +/- 0.13 vs. 3.06 +/- 0.17 microns; p less than .05) with pentastarch than with hetastarch, as judged by electron microscopy. CONCLUSIONS: These data suggest that pentastarch is better retained in the circulation in capillary leak syndromes compared with hetastarch. PMID- 1705493 TI - Chromosome structure and condensation in relation to DNA integrity. AB - Single strand DNA breaks were photo-induced in Chinese hamster chromosomes. An electron microscope analysis indicated dispersion or loss of chromatin, and the linear continuity of the metaphase chromosome was mainly represented by longitudinal chromatin fibres. The degree of chromosome damage was related directly to the photo-exposure, and inversely to the condensation level reached by the chromosome prior to exposure. Photo-irradiation of G2 or early prophase chromatin induced chromosome pulverization. However, if the chromatin had reached the maximum metaphase condensation then DNA breakdown only partially disorganized the chromosome general morphology, suggesting that DNA integrity is a factor in chromosome condensation and structural stability at mitosis. PMID- 1705494 TI - Contextual analysis and intermediate cell markers enhance high-resolution cell image analysis for automated cervical smear diagnosis. AB - Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone. PMID- 1705495 TI - Immunocytochemical evaluation of central nervous system tumors obtained by the Cavitron ultrasonic surgical aspirator. AB - The Cavitron ultrasonic surgical aspirator (CUSA) is a dissecting system that allows quick and effective removal of CNS tumors without traction or excessive manipulation of normal tissue. In this article, the immunoperoxidase staining patterns of cytology specimens obtained with the CUSA are compared with those from their corresponding resected surgical specimens employing a battery of monoclonal and polyclonal antibodies. Eleven cases of meningioma, three cases of glioblastoma multiforme, one astrocytoma, and two schwannomas were evaluated. In both CUSA cytologic biopsies and surgical biopsies, all the meningiomas showed strong staining for vimentin and epithelial membrane antigen, while two showed focal staining for cytokeratins. The glioblastoma multiforme and astrocytoma cases showed positivity for vimentin, S-100 protein, and glial fibrillary acidic protein, while the schwannomas stained positively for vimentin and S-100 protein. With only rare exceptions, the immunocytochemistry of the CUSA and surgical specimens correlated well in all of these cases in terms of strength of reaction and localization. There were no false-positive staining reactions in the CUSA material. This study suggests that reliable morphologic and immunoperoxidase studies can be performed on cytologic material obtained by the CUSA, which could aid in making an accurate and specific diagnosis of a variety of CNS tumors. PMID- 1705496 TI - Fine-needle aspiration cytology of osteoclastic giant-cell tumor of the pancreas. AB - Osteoclastic giant-cell tumor (OGCT) of the pancreas is a rare tumor. We present the fine-needle aspiration (FNA) and bile cytology findings of an OGCT arising in the head of the pancreas in a 72-yr-old male, along with immunocytochemical studies that were done on the cytologic material. The smears showed numerous giant cells with clustered, overlapping, uniform, bland-appearing nuclei with prominent nucleoli consistent with osteoclastic-type multinucleated giant cells. A second population of mononucleated cells appearing singly or in groups having similar nuclear features was also present. Immunocytochemical studies performed on the FNA and bile duct fluid material demonstrated positive staining of the malignant cells for vimentin, alpha-1 antichymotrypsin, and alpha-1 antitrypsin and negative staining for high- and low-molecular-weight cytokeratin, pooled monoclonal cytokeratin, epithelial membrane antigen, and carcinoembryonic antigen. Although not definitive, these studies are supportive of a mesenchymal stromal histogenesis of this unusual pancreatic malignancy. PMID- 1705497 TI - Aspiration cytology of hemangiopericytoma: a report on two cases. AB - The cytological features of fine-needle aspiration in two cases of hemangiopericytoma are described. The diagnosis was confirmed by cell block and the silver impregnation method, demonstrating reticulin fibers on smears as well as on the cell block. Histopathological correlation was done in both cases. PMID- 1705498 TI - Cytology of papillary adenoma of the nipple: a case diagnosed on fine-needle aspiration. AB - A 45-yr-old woman being treated for carcinoma of the uterine cervix was found to have a right nipple nodule. Fine-needle aspiration of the nodule showed a very cellular smear with clusters of ductal cells and many scattered naked nuclei. Papillary clusters with and without a vascular core also were present and enabled a cytological diagnosis of papillary adenoma. Excision of the lesion demonstrated the histopathologic features of a papillary adenoma of the nipple. PMID- 1705499 TI - Recurrent multifocal adult rhabdomyoma diagnosed by fine-needle aspiration cytology: report of a case and review of the literature. AB - The fine needle aspiration (FNA) cytology of a recurrent multifocal extracardiac adult rhabdomyoma is described, and the literature is reviewed. The patient presented with dysphagia and bilateral palpable neck masses 21 yr after resection of a rhabdomyoma of the tongue. The clinical differential diagnoses included ptotic submandibular glands and lymphadenopathy. The aspiration smears and cytospin preparations contained large polygonal cells with abundant granular cytoplasm with indistinct borders and uniform, peripherally located nuclei. Cross striations were identified within the cytoplasm of some cells on Papanicolaou and modified Wright-Giemsa stains. This case represents only the fourth description of the cytology of this entity and the first reported case of a recurrence diagnosed by FNA. The characteristic cytomorphologic features enabled a definitive diagnosis to be made 21 yr after the original resection, sparing a poor-risk patient a debilitating surgical procedure for a benign, slow-growing neoplasm. PMID- 1705500 TI - The use of the diff-quik stain in the immediate interpretation of fine-needle aspiration biopsies. PMID- 1705501 TI - Matrix crystals in cytologic urine specimens. AB - Three cytologic urine specimens from separate patients seen over a period of 3 mo were prepared by the Papanicolaou method. They contained crystals (uric acid type in two and magnesium ammonium phosphate in one) that incorporated variable amounts of organic (mucoprotein) matrix; many appeared by light microscopy to be made exclusively of matrix. Scanning electron microscopy performed in one of the specimens containing uric acid crystals suggested that the matrix forms were in progressive stages of mineralization. These cases, plus a similar one reported recently by us, demonstrate that the detection by urine cytology of organic matrix incorporated in the structure of urinary crystals is not rare and that the Papanicolaou staining method facilitates such detection. PMID- 1705502 TI - Lipid-rich metastatic balloon-cell melanoma: diagnosis by a multimodal approach to aspiration biopsy cytology. AB - Fine needle aspirates from one of multiple liver nodules revealed a large number of discohesive malignant cells with abundant vacuolated cytoplasm. The patient had had left eye enucleation the year before, for a melanoma with focal areas of clear cell change. Pap stained preparations from the liver FNA displayed well the nuclear features of balloon cell melanoma, including anisonucleosis, prominent nucleoli and intranuclear inclusions. Diff-Quik stain demonstrated best the cytoplasmic features such as distinct cell margins and finely dispersed and sharply delineated clear vacuoles. No pigmentation was noted but melanoma was suspected after the history prompted comparison with the enucleated specimen resected a year previously. A multimodal battery of ancillary methods including electron microscopy (EM) and immunocytochemistry (ICC) allowed the confirmation of balloon-cell melanoma, a rare variant not previously described in the eye. By EM, abundant lipids were identified along with melanin-containing structures resembling melanosomes. S-100 positivity along with negativity for epithelial, lymphohistiocyte and germ cell markers was compatible with melanoma. These findings are consistent with the view that cytologic detection of poorly cohesive, hypervacuolated cells does not exclude the possibility of melanoma. This rare example of a lipid-containing melanoma stresses the value of obtaining good clinical history, comparing FNAs to any pre-existing material and utilizing a multimodal approach to cytologic diagnosis in select cases. PMID- 1705503 TI - Elevation of growth hormone (GH) and prolactin receptors in transgenic mice expressing ovine GH. AB - The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein 1(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors. PMID- 1705504 TI - Development and characterization of a new, highly specific antibody to the human chorionic gonadotropin-beta fragment. AB - In addition to high concentrations of hCG, pregnancy urine contains even higher concentrations of a fragment of the hCG beta-subunit. This biologically inactive material complicates immunological measurement of hCG, since it cross-reacts with many polyclonal and monoclonal antibodies to the hCG beta-subunit that are employed for assays of hCG in urine. Although we and others have developed antibodies to this fragment, specific measurement of the fragment in the presence of free hCG beta has remained difficult due to intrinsic cross-reactivity of these antibodies with the intact hCG beta. Rather than attempt to increase specificity by assay optimization, we developed a new, highly specific monoclonal antibody, designated B210, which cross-reacts less than 0.1% with the free hCG beta-subunit in both liquid and solid phase immunoassay formats. We have used this new monoclonal antibody in immunoradiometric assays to measure specifically the hCG beta fragment in urine throughout pregnancy as well as in the sera of two individuals with cancers producing the hCG beta-subunit. We discovered that the hCG beta fragment can bind three monoclonal antibodies simultaneously, indicating that although the epitope for antibody B210 is a new determinant exposed on the hCG beta fragment and not on intact hCG or on free hCG beta-subunit, the hCG beta fragment retains at least two other hCG beta-related epitopes intact, i.e. those that bind monoclonal antibodies B108 and B201. PMID- 1705505 TI - Transforming growth factor-beta stimulates production of insulin-like growth factor-binding protein-3 by human skin fibroblasts. AB - Human neonatal fibroblasts in monolayer culture produce insulin-like growth factor-binding protein-3 (IGFBP-3), the IGF-binding subunit of the circulating 140-kDa IGFBP complex. We now report that transforming growth factor-beta (TGF beta) is a potent stimulator of IGFBP-3 production by fibroblasts. After 72-h incubation with 1 ng/ml TGF beta, the levels of IGFBP-3 in conditioned medium were increased 5.8 +/- 1.2-fold (mean +/- SE; n = 9). Half-maximal stimulation of IGFBP-3 production was seen at 0.4 +/- 0.05 ng/ml TGF beta (n = 4). Coincubation of fibroblasts with TGF beta and either IGF-I or IGF-II at 50 ng/ml enhanced IGFBP-3 production 1.5- to 2-fold compared to TGF beta alone. As previously reported, fetal calf serum (FCS) stimulated IGFBP-3 production 5- to 6-fold; 1 ng/ml TGF beta increased the stimulated production of IGFBP-3 by FCS a further 2.5- to 3.5-fold. Acidification of FCS before addition enhanced the stimulation of IGFBP-3 compared to that caused by untreated FCS, but decreased further potentiation by TGF beta. This effect of acidified FCS was reversed by a neutralizing antibody to TGF beta. Similarly, the stimulation of IGFBP-3 levels by human serum or conditioned serum-free fibroblast medium was significantly increased by acidification of serum or medium before addition and was reversed by TGF beta antibody. These observations are consistent with acid-mediated activation of latent TGF beta added in serum or secreted by fibroblasts. Since IGFBP-3 is known to regulate IGF activity in fibroblasts, these results raise the possibility that TGF beta may modulate IGF actions in these cells by stimulating the production of IGFBP-3. PMID- 1705506 TI - Epitope mapping of human follicle stimulating hormone-alpha using monoclonal antibody 3A identifies a potential receptor binding sequence. AB - Five monoclonal antibodies (mabs) were generated (3A, 4B, 5F, 2E, 1E) by immunizing BALB/c mice with human (h) FSH. The mabs were used to relate antigenic structures (epitopes) to function (receptor binding). All five mabs could immunoneutralize (inhibit binding to receptor) hFSH and could be placed into two groups based on potency (degree of neutralization). Group I mabs (5F, 2E, 1E) were less potent than group II mabs (3A, 4B) even though group I mabs had a 2 fold higher average affinity constant than group II. Those data suggested that group II mabs recognize an epitope near or in the receptor binding site of hFSH. Immunoradiometric epitope cross-matching demonstrated that group I and group II mabs recognize different epitopes. Further characterization of 5F and 3A (representative of group I and group II, respectively) utilized an enzyme-linked immunosorbent assay (ELISA) and a RIA. In the ELISA, both mabs bound hFSH and hFSH alpha but not hFSH beta. In the RIA, 3A bound [125I]hFSH and [125I]hFSH alpha but not [125I]hFSH beta. In contrast, 5F bound only [125I]hFSH. hFSH effectively competed with [125I]hFSH for 5F and 3A. In contrast, hFSH alpha competed with [125I]hFSH for 5F but not for 3A even though 3A could bind hFSH alpha in the ELISA and the RIA. These results suggest that 3A and 5F recognize different epitopes. The epitope recognized by 3A is unique in that its conformation appears to be dependent on association with hFSH beta. Since 3A was a more potent inhibitor of receptor binding than 5F, its epitope specificity was characterized further by epitope mapping. This was accomplished utilizing a peptide ELISA and by affinity chromatography. The results from epitope mapping demonstrated that 3A recognizes sequences 61-78 and 73-92 with binding to 73-92 being 4-fold greater than to 61-78. Thus, the epitope comprised of sequence 73-92 (and to a lesser extent 61-78) appears to be important for receptor binding. PMID- 1705507 TI - Hormonal responsiveness by immature rabbit uterine epithelial cells polarized in vitro. AB - This paper reports the development of an in vitro cell culture system of polarized uterine epithelial (UE) cells from the immature rabbit. UE cells from immature rabbit were cultured on EHS (Engelbreth-Holm-Swarm) matrix-coated semipermeable filter inserts in a serum-free, phenol red-free defined medium. The cells in primary culture attached, proliferated, formed monolayers, and exhibited structural and functional polarity. Structural differentiation was validated by ultrastructural features, such as apical microvilli, intercellular junctions, desmosomes, and polar organization of apical vs. basal membrane domains. Trans monolayer epithelial resistance, a reflection of functional tight junctions, preferential basal uptake of [35S]methionine, polarized distribution of labeled secretory proteins, and increased secretory activity marked by preferential apical secretion (greater than 90%) of proteins were indices of functional polarity. With the development and maintenance of polarized state by the UE cells, some of the newly synthesized proteins were sorted and vectorially secreted into their distinct compartments. De novo synthesis and exclusive apical secretion of uteroglobin (a progesterone-induced protein in vivo) by the polarized UE cells occurred in response to progesterone treatment in vitro. The hormone responsiveness was maintained for a prolonged period under these conditions. A culture system in which the UE cells maintain functional and morphological polarity and sustain their hormonal responsiveness facilitates the study of regulation of specialized functions by the UE cells that are regulated by steroid hormones and their interactions with the other uterine cell types. PMID- 1705508 TI - Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini. AB - Treatment of rabbit pancreatic acini with the phorbol ester, 12-O tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid. PMID- 1705509 TI - Immobilized anti-CD5 together with prolonged activation of protein kinase C induce interleukin 2-dependent T cell growth: evidence for signal transduction through CD5. AB - Monoclonal antibodies (mAb) identifying the CD5 antigen were used to stimulate human peripheral blood T lymphocytes. Three out of three anti-CD5 mAb, 10.2, OKT1 and anti-Leu-1 induced vigorous proliferation of purified T cells in the presence of 1.6 nM phorbol 12-myristate 13-acetate (PMA). Immobilization of anti-CD5 mAb on a solid support was necessary for the induction of a proliferative response. Neither 1.6 nM PMA, nor immobilized anti-CD5 mAb were mitogenic as a sole stimulus. mAb identifying CD4, CD7, CD11a, CD18, and major histocompatibility complex class I molecules were not comitogenic with PMA. Anti-CD5/PMA-induced cell proliferation proceeded by an interleukin 2 (IL 2)-dependent mechanism, as was demonstrated by the cell surface expression of the p55 chain of the IL 2 receptor (IL 2R), the production of IL 2 and the inhibition of the proliferative response by anti-IL 2R mAb anti-Tac. There was no strict requirement for detectable numbers of monocytes, although cell proliferation could be enhanced by the monocyte-derived cytokines IL 1 and IL 6. Phorbol 12,13-dibutyrate and mezerein could substitute for PMA in this activation pathway, but synthetic diacylglycerols and phorbol esters that do not activate protein kinase C (PKC) could not, indicating a need for prolonged activation of PKC. T cells activated by anti-CD5/PMA are sensitive to inhibition by cyclosporin A (CsA) and by prostaglandin E2 (PGE2). This contrasts with anti-CD28/PMA-induced T cell proliferation, which is resistant to CsA and PGE2. Cell surface expression of CD5 was strongly up-regulated by PMA, whereas CD3 expression was down-regulated. We conclude that T cell activation can be triggered by engagement of CD5 by immobilized anti-CD5 mAb, combined with prolonged activation of PKC. These data support a role for CD5 as an independent signal transducing molecule. PMID- 1705510 TI - Cross-linking of VLA/CD29 molecule has a co-mitogenic effect with anti-CD3 on CD4 cell activation in serum-free culture system. AB - In this study, we demonstrated that antibodies reactive with a common beta 1 subunit (CD29) of the VLA family of antigens could synergize with CD3 but not CD2 in inducing CD4 cell proliferation in a serum-free culture system. The extent of activation was similar to that obtained when CD4 cells were cultured with anti CD3 plus fibronectin. The proliferative response induced via the CD3-CD29 pathway was associated with the interleukin (IL) 2 autocrine pathway, including IL 2 production and IL 2 receptor expression. Specifically, cross-linking of VLA-5, but not other members of the VLA family of antigens with the CD3 molecule induced strong CD4 cell activation. However, cross-linking of CD3 with other cell surface antigens such as CD4 did not induce CD4 cell activation in this serum-free culture system. The above results strongly suggest that the VLA/CD29 family of antigens may play an important role in regulating CD4 T cell activation via the CD3-T cell receptor pathway. PMID- 1705511 TI - Evidence for differential responsiveness of human CD5+ and CD5- B cell subsets to T cell-independent mitogens. AB - Tonsillar resting B cells were separated into CD5+ and CD5- cell subsets and stimulated with the thymus-independent mitogens, Staphylococcus aureus Cowan strain I (SAC) or insolubilized anti-mu monoclonal antibodies (a mu Ab). CD5+ cells incorporated [3H]thymidine more efficiently than unfractionated cells when stimulated with SAC and their response was augmented by the addition of interleukin (IL) 2 to the cultures. CD5+ cells also proliferated in response to a mu Ab provided that IL 2 was present, SAC-, but not a mu Ab-stimulated CD5+ cells produced IgM and IgG molecules when IL 2 was added to the cultures and also secreted autoantibodies with rheumatoid factor activity and sometimes also with anti-single-stranded, but not double-stranded, DNA activity. The efficient response of CD5+ cells was not explained by the fact that they contained cells already activated in vivo. Thus, they did not express the CD23, CD69, CD71 and CD39 activation markers, failed to incorporated [3H]thymidine and to secrete Ig spontaneously or in response to IL 2 and were found to be in a quiescent state by cell cycle flow cytometric analysis. In contrast to CD5+ cells, CD5- cells displayed very little or no [3H]thymidine incorporation in response to SAC or to a mu Ab and their poor responsiveness was not altered by changing either the doses of the stimulants, the timing of the cultures, by co-culturing the cells together with CD5+ cells, or by adding IL 2 or IL 4. Immunofluorescence studies showed that freshly prepared CD5- cells did not have surface activation markers but that they expressed them following SAC stimulation. Thus, unlike that observed for CD5+ cells, SAC seems to be capable of activating CD5- cells but does not appear to be a sufficient stimulus for driving the cells into the subsequent phases of the cell cycle. The above findings, that demonstrate marked differences in the response to CD5+ and CD5- cells to thymus-independent stimuli, may bear relevance for the understanding of the normal clonal expansion of CD5+ cells as well as for the pathogenesis of autoimmune diseases. PMID- 1705512 TI - The effect of interleukin 3 upon IgE-dependent and IgE-independent basophil degranulation and leukotriene generation. AB - Recent investigations have shown that the hematopoietic growth factor interleukin 3 (IL 3) enhances histamine release of mature human basophils. Furthermore, basophils exposed to IL 3 generate large amounts of leukotriene C4 in response to C5a, a basophil agonist which by itself is unable to promote lipid mediator formation. Also IL 3 renders the cells responsive to factors which do not otherwise induce basophil mediator release. Here we show in more detail how IL 3 affects the release of preformed and newly synthesized inflammatory mediators by basophils in response to antigen, anti-IgE, N-formyl-methionyl-leucyl phenylalanine (FMLP) and C5a. In cells triggered by maximally effective concentrations of these agonists, IL 3 enhances histamine release, although it more profoundly affects leukotriene generation, in particular in response to stimuli which by themselves are inefficient or poor inducers of lipid mediator formation. This change in the mediator-release reaction occurs at low IL 3 concentrations, over the same concentration range of IL 3 of 0.01-1.0 U/ml regardless of which triggering agent is used as a second signal. Pretreatment of basophils with IL 3 results in a left shift of the dose-response curves for mediator release of both IgE-dependent and IgE-independent agonists by approximately one order of magnitude. IL 3 affects only the extent and not the time course of IgE-independent peptide-induced basophil degranulation. By contrast, histamine and leukotrienes are released more rapidly in response to IgE dependent stimulation after IL 3 priming. IL 3 also shortens the lag time and increases the rate of leukotriene generation in basophils triggered by FMLP. The priming process induced by IL 3 does not require extracellular calcium. Basophils exposed to IL 3 release significant amount of mediators in response to C5a, even in EDTA buffers without addition of Ca2+/Mg2+, indicating that in the presence of IL 3 the Ca2(+)-dependent mediator release induced by C5a becomes partially Ca2+ independent. Thus, we find that IL 3 strongly affects the mediator profile, the amounts of mediators released, the dose-response curves and the kinetic of the release reaction in basophils stimulated with diverse agonists. The data further support the hypothesis that IL 3 plays an important role in inflammatory processes, in particular in hypersensitivity reactions. PMID- 1705513 TI - The effect of the immunosuppressant FK-506 on alternate pathways of T cell activation. AB - Structurally unrelated, FK-506 and cyclosporin (CsA) bind to and inhibit the action of distinct cytoplasmic receptors, FK-506-binding protein (FKBP) and cyclophilin (CyP), respectively. These receptors, termed immunophilins, share no sequence similarity, and yet both have been demonstrated to be capable of catalyzing the cis-trans isomerization of peptidyl-prolyl bonds (rotamase activity). Because FK-506 and CsA bind to different intracellular target structures, we investigated the spectrum of action of FK-506, in comparison to CsA, on T cell activation. We have shown that FK-506, like CsA, is able to inhibit T cell activation mediated not only by the T cell receptor-CD3 complex, but also via another surface molecule, CD2. T cell proliferation, stimulation of interleukin 2 production, and induction of apoptosis were all sensitive to inhibition by both FK-506 and CsA. With each parameter of activation, FK-506 is approximately 10-100-fold more effective than CsA. In contrast, FK-506 did not affect T cell proliferation induced by anti-CD28 monoclonal antibody in the presence of phorbol 12-myristate 13-acetate. This CD28 pathway, however, was inhibited by a structural homology of FK-506, rapamycin, demonstrating that the mechanism of action of FK-506 has specificity. These data suggest that immunophilins or the complex of drug coupled to immunophilin (i.e. FK-506/FKBP, CsA/CyP) are involved in and regulate selective pathways of T cell stimulation. PMID- 1705514 TI - Conserved V(D)J junctional sequence of cross-reactive cytotoxic T cell receptor idiotype and the effect of a single amino acid substitution. AB - A dominant T cell population bearing the cross-reactive idiotype of T cell antigen receptor (TcR) has been obtained using an anti-TcR monoclonal antibody (mAb) developed in syngeneic mice. Forty-four cytotoxic T cell (CTL) clones with reactivity to a mAb (N9-127) were selected out of 396 H-2Db-restricted CTL clones specific for FBL-3 tumor antigen from C57BL/6 mice. These CTL clones were divided into two groups according to the blocking pattern of cytotoxic activities with mAb N9-127. All eight CTL clones chosen from both groups expressed TcR with a specific combination of alpha and beta chains (V alpha 1J alpha 112-2/V beta 10D beta 2.1J beta 2.7), and the difference in the blocking susceptibility resided in a single amino acid substitution (Gly to Asp) in the D-J joint of beta chain. This provides direct evidence for the molecular basis of cross-reactive idiotypes of TcR recognized by mAb. PMID- 1705515 TI - The distribution pattern of the sympathetic nerve fibers to the cerebral arterial system in rat as revealed by anterograde labeling with WGA-HRP. AB - To clarify the projection route and the expansion of the terminal plexus of the sympathetic nerve fibers innervating the cerebral arterial system in rat, we labeled the postganglionic fibers originating in the superior cervical ganglion and traced their entire course by anterograde labeling with wheat germ agglutinin horseradish peroxidase. Sympathetic innervation of the internal cerebral artery by labeled fibers actually began just at the portion where it enters the intradural space, and innervated it up to the small pial arteries located in the subarachnoid space, but not the intracerebral arterioles. On the main arteries in the circle of Willis, bundles of nerve fibers ran parallel to the long axis of the vessels and branched perpendicularly their terminal twigs with regular intervals to form a rib-structure pattern. On the arterial branches derived from the circle of Willis, a fine nerve bundle and delicate terminal axons formed a meshwork instead of a rib-structure pattern. These observations confirmed the existence of differences in the distribution pattern of the nerve plexus, which strongly affects the strength and quality of vasoconstriction by sympathetic activation in each level of the cerebral arterial system. PMID- 1705517 TI - A double-label study of efferent projections to the cochlea of the chicken, Gallus domesticus. AB - Intracochlear bilateral injection of the fluorescent retrograde markers fast blue and diamidino yellow was used to identify the brainstem location of the olivocochlear efferents in the domestic chicken. The overall distribution pattern of neurones was similar to that of recent studies using horseradish peroxidase as the retrograde tracer, although the number of labelled neurones was significantly greater than previously reported. The average number of labelled neurones projecting to any one cochlea was 242, with roughly equal numbers located ipsilaterally or contralaterally. After bilateral injection of the two tracers, no double-labelled neurones were detected. PMID- 1705516 TI - Monoclonal antibody HNK-1 selectively stains a subpopulation of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat cerebral cortex. AB - Monoclonal antibody HNK-1 is shown to outline selectively a subpopulation of GABAergic neurons containing a specific calcium-binding protein parvalbumin (PV) in the adult rat parietal cortex, using pre- and postembedding immunocytochemistry at light microscopic level. About 98% of HNK-1 stained cells in the rat parietal cortex were PV immunoreactive. About 95% of HNK-1 immunoreactive cells were also shown to be stained with a lectin, Vicia villosa agglutinin (VVA), with a specific affinity for terminal N-acetylgalactosamine, which has been previously shown to stain selectively a subpopulation of PV containing GABAergic neurons in this region. Furthermore almost all HNK-1 immunoreactive cells were also stained with a monoclonal antibody, 3B3, which is specific for chondroitin sulfate proteoglycan. 3B3 was shown in the present study to stain selectively a subpopulation of PV-immunoreactive neurons in the adult rat parietal cortex. In addition, a direct comparison of two monoclonal antibodies HNK-1 and VC1.1 revealed that these two were identical in their staining properties and that they defined the same subset of PV-containing GABAergic neurons in the rat parietal cortex. PMID- 1705518 TI - Axonal projection of descending pathways responsible for eliciting forelimb stepping into the cat cervical spinal cord. AB - The descending pathways responsible for eliciting forelimb stepping are located in the lateral funiculus (Yamaguchi 1986). In order to determine into which spinal segments the descending pathways project and to know the projections and functions of the other descending system, the ventral funicular pathways, we placed various lesions in the cervical spinal cord of decerebrate cats with the lower thoracic cord transected and studied their effects on forelimb stepping evoked by stimulation of the midbrain locomotor region. (1) The lateral funiculus was transected on one side. The operation removes descending input to all the segments caudal to the lesion. Experiments with serial transections from the caudal to rostral segment revealed that stepping activity of the limb on the lesioned side is reduced when the lesion is placed at the level between the C6 and C7 segment and then between C5 and C6. A slight reduction of activity was also observed after a lesion placed between C7 and C8. (2) Consistently, bilateral transection of the lateral funiculus at the level between C5 and C6 abolished stepping movements of both forelimbs. (3) The cervical cord was split in the parasagittal plane through the dorsal root entry. The operation removes the descending input to the segment in which the lesion is placed. The parasagittal lesions from the C1 to C6 did not abolish stepping activity, although a lesion placed between C5 and C6 could slightly affect stepping. The results, (1)-(3) suggest that the lateral funicular pathways project into the spinal segments mainly at the C6-C7 level with some rostrocaudal extension into C5 and C8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705519 TI - Hippocampal input to a "visceral motor" corticobulbar pathway: an anatomical and electrophysiological study in the rat. AB - The hippocampus has previously been shown to influence cardiovascular function, and this effect appears to be mediated by the connection the hippocampus has with the infralimbic area of the medial frontal cortex (MFC), a region which projects directly to the nucleus of the solitary tract (NTS) in the dorsal medulla. In the present study, anatomical and electrophysiological techniques were utilized to determine the degree of convergence of hippocampal input to the MFC on neurons in the MFC which project to the NTS. Injections of the anterograde and retrograde neuroanatomical tracer wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) into the NTS retrogradely labelled cells in the infralimbic and prelimbic regions of the MFC. Injections of WGA-HRP into the ventral hippocampus anterogradely labelled terminals in the MFC which, at the light microscopic level, closely overlapped the origin of the descending projection from the MFC to the brainstem. Electron microscopic analysis revealed that anterogradely labelled terminals make synaptic contact primarily on dendritic processes in the neuropil adjacent to retrogradely labelled cells. In addition, anterogradely labelled terminals did, in some cases, make synaptic contact on the somas of retrogradely labelled cells. Electrical stimulation of the NTS antidromically activated cells in the infralimbic and prelimbic areas of the MFC. The average latency of antidromic activation was 30 msec, corresponding to a conduction velocity of approximately 0.7 m/s. Electrical stimulation of the ventral hippocampus orthodromically activated cells in the MFC. With an appropriate delay between the hippocampal and NTS stimuli, the orthodromic and antidromic potentials could be made to collide. The results of this study establish a structural as well as functional link between the hippocampus and NTS-projection neurons in the MFC. PMID- 1705520 TI - Physiological properties of projection neurons in the monkey striatum to the globus pallidus. AB - In order to study neuronal information transfer from the striatum to the globus pallidus (GP) during voluntary movement, we recorded activity of electrophysiologically identified projection neurons in the putamen to the GP while the monkey was performing learned movement tasks. Two categories of putamen neurons were recorded: one with tonic spontaneous discharges at about 2-7 impulses/s responded to external sensory stimuli (type I); the other with very low spontaneous discharge rates less than 0.5 impulses/s showed phasic burst discharges which were time-locked to limb or orofacial movements (type II). All of the putamen neurons identified as projecting to the GP (external and/or internal segment) by an antidromic activation from electrical stimulation of the GP were type II cells. It was concluded that the movement-related activity of type II putamen neurons is transferred to GP and/or SN during voluntary movement but tonically active type I cells do not project to the GP. PMID- 1705521 TI - Difference in central projection of primary afferents innervating facial and intraoral structures in the rat. AB - Transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate was used to study the central projection of primary afferent neurons innervating facial and intraoral structures. The examined primary neurons innervating the facial structures were those comprising the frontal and zygomaticofacial nerves and those innervating the cornea, while the primary neurons innervating the intraoral structures included those innervating the mandibular incisor and molar tooth pulps and those comprising the palatine nerve. The primary afferents innervating the facial structures project to the lateral or ventral parts of the trigeminal principal, oral and interpolar subnuclei, and to the rostral cervical spinal dorsal horn across laminae I through V, with a greater proportion being directed to the spinal dorsal horn. The primary afferents innervating the intraoral structures terminate in the dorsomedial subdivisions of the trigeminal principal, oral and interpolar subnuclei, and in laminae I, II, and V of the medial medullary dorsal horn, with a much denser projection being distributed to the rostral subnuclei. In addition to the above brain stem trigeminal sensory nuclear complex, they project to the supratrigeminal nucleus, caudal solitary tract nucleus, and paratrigeminal nucleus. These observations agree with previously reported data that the central projection of trigeminal nerve is organized in different manners for the facial and intraoral structures. Furthermore, the present findings in conjunction with our previous studies clarify that the central projection of primary afferents from the facial skin is organized in a clear somatotopic fashion and that the terminal fields of primary afferents from the intraoral structures extensively overlap in the brain stem trigeminal nuclear complex particularly in its rostral subdivisions. The central mechanism of trigeminal nociception is discussed with particular respect to its difference between the facial and intraoral structures. PMID- 1705522 TI - Accuracy of reinnervation by peripheral nerve axons regenerating across a 10-mm gap within an impermeable chamber. AB - The axon regeneration following a peripheral nerve injury often fails to restore a complete functional recovery. One of the causes of this unsatisfactory result has been attributed to regrowth of regenerating fibers to inappropriate peripheral targets. The accuracy of reinnervation by axons regenerating across a 10-mm gap within an impermeable chamber has been studied by using a sequential retrograde double-labeling technique. Despite the long gap between the nerve stumps, at 4 weeks a mean of 30.5% of the regenerating axons can reinnervate the original muscular area. These data confirm previous studies in which a preferential reinnervation is reported not to be absolutely dependent on the axon's mechanical alignment. PMID- 1705523 TI - Colloidal gold as a permanent marker of cells. AB - We have demonstrated that colloidal gold-labelled serum proteins are taken up by a number of cells in cultures established from the postnatal rodent neopallium. The colloidal gold enters and remains within secondary lysosomes over extended periods of time and, as well, persists after the subculture of these cells. The cell types that readily take up the label in our culture system are type-1 astrocytes, glial precursor cells and macrophages, whereas, only a small number of oligodendrocytes take up the label. The use of serum proteins to introduce colloidal gold into cells therefore seems to be a convenient and easy way to permanently mark cells. PMID- 1705524 TI - The reaction of ferric- and ferrous salts with bleomycin. AB - 1. A comparative study shows that ferrous ions give a much better yield of Fe(III)-bleomycin than ferric ions, when iron salt is added to bleomycin in a buffer solution (pH 7.2). 2. The amount of Fe(III)-bleomycin formed after addition of ferric ions was markedly increased in the presence of ferric ion binding compounds (BSA, citrate) or reducing agents (ascorbate, cysteine). PMID- 1705525 TI - Carbohydrate-dependent epitope mapping of human thyrotropin. AB - To probe possible effects of carbohydrate chains in the conformation of pituitary glycoprotein hormones, two radiolabeled derivatives of human thyroid-stimulating hormone (hTSH), either partially deglycosylated in the beta-subunit or fully deglycosylated in both the alpha- and beta-subunits, were compared to the native hormone for binding to monoclonal as well as polyclonal antibodies. Monoclonal antibodies were screened for their ability to bind the intact hormone (anti hTSH), hTSH and its free alpha-subunit (anti-alpha) or its free beta-subunit (anti-beta). A panel of 14 monoclonal antibodies directed against at least eight out of the 12 epitopes known to be present in the hormone was tested in solid phase assays for their capacity to bind intact and deglycosylated forms of hTSH. All of them displayed identical recognition of native and partially deglycosylated 125I-hTSH. In contrast, binding of fully deglycosylated 125I-hTSH to anti-hTSH and anti-beta antibodies was dramatically lost while that of anti alpha was preserved. This clearly indicates that most of the epitopes specific for subunit association as well as those present on the beta-subunit are glycosylation dependent. No alteration was found in antibody recognition following deglycosylation of free individual subunits, indicating that the carbohydrate effect can only occur in the combined dimer. Using polyclonal antisera raised against the International Reference Preparations, we found that the deglycosylated hormone could be bound by the anti-beta antiserum although at a much lower dilution than the native antigen, suggesting the presence of at least one glycosylation-independent epitope in the beta-subunit. Competitive binding assays revealed that deglycosylated hTSH is 5 times less immunoreactive toward the anti-beta compared to the anti-alpha antiserum. The current data thus demonstrate the presence of the glycosylation-independent epitopes in the alpha subunit of hTSH and the localization of most of the glycosylation-dependent domains in the beta-subunit. PMID- 1705526 TI - Coordinate reduction of rat pancreatic islet glucokinase and proinsulin mRNA by exercise training. AB - Exercise training results not only in enhanced insulin sensitivity but also in a reduction in insulin secretion. In this study, we examined the effects of exercise training on the expression of genes potentially related to insulin synthesis and glucose-stimulated insulin release by measuring pancreatic islet proinsulin, glucose-transporter (GLUT2), and glucokinase mRNAs. Female Wistar rats were subjected to 100 min of running at 25 m.min-1 up a 15% incline for 90 min/day for 6 days/wk for 3 wk. Pancreatic mRNA was evaluated by Northern- and dot-blot analysis with [32P]cRNA probes. We found no change in the pancreatic content of GLUT2 mRNA but found marked decreases in the content of proinsulin mRNA (78%, P less than 0.005) and glucokinase mRNA (65%, P less than 0.001). These results suggest that exercise modulates both islet glucose metabolism and insulin synthesis at the level of gene expression. Furthermore, there was a significant correlation between the decreases in glucokinase and proinsulin mRNA concentrations (r = 0.95, P less than 0.001), suggesting that expression of these genes is regulated in parallel. PMID- 1705527 TI - Effects of cimetidine and omeprazole on angiogenesis in granulation tissue of acetic acid-induced gastric ulcers in rats. AB - We investigated the effects of cimetidine and omeprazole on angiogenesis in granulation tissue and on the healing of gastric ulcers induced by acetic acid in rats. Either cimetidine (50 or 100 mg/kg) or omeprazole (10 or 20 mg/kg) was orally administered once daily for 9 consecutive days from the day following ulcer production. The ulcer index on the 10th and 30th days after ulcer production, and the extent of angiogenesis on the 10th day were examined. Cimetidine dose-dependently decreased the extent of angiogenesis on the 10th day, whereas the ulcer index on the 10th days was not significantly different between cimetidine-treated and control rats. The ulcer index of the groups treated with cimetidine during the initial 9-day period was increased compared with the control group on the 30th day. In contrast, oral omeprazole did not affect angiogenesis on the 10th day and decreased the ulcer index on both the 10th and 30th days. These results suggest that oral cimetidine may inhibit angiogenesis in ulcer granulation tissue possibly via the blocking of histamine H2 receptors and this may be one cause of delayed ulcer healing. PMID- 1705528 TI - Generation of a functional cDNA encoding the LdH2 class-I molecule by using a single-LTR retroviral shuttle vector. AB - The use of a new single long-terminal-repeat retroviral shuttle vector has allowed us to obtain copies of the Ld gene with the first seven exons spliced correctly, as well as many other partially spliced or aberrantly recombined copies. Nucleotide sequencing performed on double-stranded DNA with primers specific for the vector and for the coding region of the gene, allowed rapid screening of the recovered plasmids. Synthetic oligodeoxyribonucleotides were then used to link the 5 nt of the last exon, and the functionality of the cDNA copy was verified by expression in transfected L(TK-) cells. Cells that produced the Ld antigen were detected by immunofluorescence and were shown to synthesize an immunoprecipitable molecule of the expected size. In addition, Ld-producing cells were susceptible to killing by Ld-restricted cytotoxic T-lymphocytes. This material should prove useful for mutagenesis and for expression in cell types in which expression of the genes of the major histocompatibility complex appears to be highly regulated. PMID- 1705529 TI - [The effect of pesticides on the formation of antigen-specific suppressor cells]. PMID- 1705530 TI - Oxygen radicals in lung pathology. AB - Pulmonary tissue can be damaged in different ways, for instance by xenobiotics (paraquat, butylated hydroxytoluene, bleomycin), during inflammation, ischemia reperfusion, or exposure to mineral dust or to normobaric pure oxygen levels. Reactive oxygen species are partly responsible for the observed pulmonary tissue damage. Several mechanisms leading to toxicity are described in this review. The reactive oxygen species induce bronchoconstriction, elevate mucus secretion, and cause microvascular leakage, which leads to edema formation. Reactive oxygen species even induce an autonomic imbalance between muscarinic receptor-mediated contraction and the beta-adrenergic-mediated relaxation of the pulmonary smooth muscle. Vitamin E and selenium have a regulatory role in this balance between these two receptor responses. The autonomic imbalance might be involved in the development of bronchial hyperresponsiveness, occurring in lung inflammation. Finally, several antioxidants are discussed which may be beneficial as therapeutics in several lung diseases. PMID- 1705531 TI - Development and application of an in vitro model for screening anti-hepatitis B virus therapeutics. AB - The development of effective anti-hepatitis B virus agents has been hampered by the lack of reliable in vitro systems for the screening of new therapeutics. In an effort to circumvent this problem, we have developed an in vitro system for screening anti-hepatitis B virus drugs using hepatitis B virus DNA-transfected Hep G2 cells. The cell line designated 2.2.15 produces replicative viral DNA intermediates, mature Dane particles and high levels of viral antigens. Subconfluent 2.2.15 cells were treated with a variety of commonly used anti hepatitis B virus therapeutics, and their efficacy was determined by analyzing changes in the replicative cellular or extracellular hepatitis B virus DNA content by Southern blotting or slot-blot hybridization. The slot-blot method was sensitive, reproducible and rapid and correlated well with Southern blotting. Analysis of the media for hepatitis B virus DNA was indicative of changes in intracellular, replicative hepatitis B virus DNA, permitting sampling of the media. Therefore 2.2.15 cells may provide a valuable method for identifying and monitoring effective anti-hepatitis B virus therapeutics. Using this system to test various agents, we confirm that 2'-deoxyguanosine strongly inhibited viral replication, whereas others tested were less effective. Correlation with in vivo systems is now needed. PMID- 1705532 TI - Alterations in nuclear scaffold constituents during carbon tetrachloride-induced liver regeneration. AB - Liver regeneration was induced in rats by treatment with CCl4, which results in substantial regenerative activity with a sharp mitotic response 2 days after intoxication. Closely paralleling the mitotic index, we observed fourfold increases in nuclear scaffold nucleoside triphosphatase, an activity thought to participate in nucleocytoplasmic RNA transport and in the 46 kD putative enzyme and its selective photolabeling. Because previous work has indicated that the 46 kD protein may be proteolytically derived from lamins A/C by cleavage at a tyrosine residue at aa376, we investigated the response of lamin A/C transcripts during this regeneration. Surprisingly, Northern blot analyses after CCl4 administration showed low levels of lamin A/C transcripts (which appeared to be predominantly poly[A]-), and we found a decrease in immunoprecipitable lamins A/C from in vitro translation of poly(A)- selected RNA. To circumvent potential problems with such analyses, we used reverse transcription/polymerase chain reaction amplification of lamin A/C transcripts from total cytoplasmic RNA. These assays showed a transient, comparatively minor increase in lamin A/C transcripts 1 day after treatment, but levels rapidly declined from 1 to 3 days and were decreased at 3 to 5 days. However, nuclear scaffold protease activity, which shows a considerable selectivity for lamins A/C and may be involved in derivation of the 46 kD protein, increased in parallel to the mitotic response and increases in nucleoside triphosphatase, as assessed using a nonspecific (Azocoll) protease assay. Assays with a specific tyrosine-containing substrate (Z-Y-Sbenzyl) showed an increase that mirrored that observed with the nonspecific substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705533 TI - A novel human multi-locus DNA family detected by pJU78 (DF31). AB - pJU78 is a 2.9-kb cloned human DNA segment derived from Xq24-q26. When used as a hybridization probe, it detects some 25 related sequences dispersed over the genome, including several autosomes and the X and Y chromosomes. Several pJU78 related sequences were chromosomally allocated and five different restriction fragment length polymorphisms detected and partially characterized in population and family studies. This sequence family was not found in non-primate species. The sequence appears to lack CpG-island character and is not detectably expressed in a variety of human tissues. PMID- 1705534 TI - The dilemma of withholding or withdrawing nutrition. AB - Recently, societal concerns have developed about the artificial prolongation of dying. Life extension is not always viewed as humanitarian. Artificial nutrition and hydration are seen frequently as necessary comfort measures and not merely life extenders. Now debate is rising about whether or not even these basic life support measures are not merely prolonging dying. In this paper, the issue of withholding or withdrawing nutrition and hydration from certain patients is discussed, including the competing ethical arguments, the nursing perspective and the implications of this dilemma for nursing, health care and society. PMID- 1705535 TI - Muroctasin [MDP-Lys(18)] augments the production of granulocyte colony stimulating factor (G-CSF) from human peripheral blood mononuclear cells in vitro. AB - N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-Lysine (MDP Lys(L18), muroctasin) is an immunopotentiating substance. Neutrophilia and elevated levels of colony-stimulating factor (CSF) in peripheral blood were previously found after the administration of this compound in both mice and humans. To specify the type of CSF and to elucidate the mechanisms of the neutrophilia, we cultured human peripheral blood mononuclear cells (PBMC) in the presence of muroctasin and measured the levels of granulocyte CSF (G-CSF) in the culture supernatants using our sensitive enzyme-linked immunosorbent assay. G-CSF is an active hematopoietic growth factor specific for cells of a neutrophilic lineage, and muroctasin was found to significantly augment the G-CSF production from PBMC in vitro (P less than 0.01). Furthermore, production of G-CSF from human PBMC in the presence of muroctasin was also supported by the Northern blot analysis using cDNA encoding G-CSF as a probe. PMID- 1705536 TI - Enzymatic and histologic investigations into the course of pancreatic alterations induced by anti-acinar-cell-antiserum. AB - A model of acute pancreatitis (AP) was developed by application of anti-acinar cell-antiserum in rats. Within 24 h postoperatively the binding of antibodies to the pancreas, histological findings, and the activities of lipase and a-amylase in serum and pancreas were analyzed. While after intraaortic administration morphological alterations could not be observed, after single dose intraductal injection, typical macroscopical and histological signs of AP were found. This pancreatic injury was characterized by a mild and protracted course favoring investigations of the early phase of pathogenesis. As soon as 2 h after intraductal application of antiserum, serum amylase and lipase were already increased and microscopic examination of the pancreatic tissue revealed pancreatic edema, inflammatory infiltration and parenchymal necrosis. Intra- and extrapancreatic fat necrosis occurred at 16 h post injectionem. The results suggest that parenchymal damage plays an essential role in the manifestation of this experimental AP and that fat necrosis seems to be a secondary event. PMID- 1705537 TI - Characterization of heterogeneous distribution of tumor blood flow in the rat. AB - Angioarchitectures of ascites hepatoma AH109A and Sato lung carcinoma (SLC) were quantitatively compared by measuring the following morphometric parameters: vascular density, vascular length, distance between tissues and their nearest blood vessel, and total length of microvascular network per unit area. When the vascular networks in these two types of tumors were compared in the initial stage, the morphological parameters were almost identical. Correlations between tumor size and the number of starting vessels and between enlargement of the tumor and the ensuing increase in pressure of the starting vessel were also evaluated with a microcomputer and an apparatus for measuring microvascular pressure. The total length of tumor vascular network to which one starting vessel supplied blood increased exponentially as the tumor increased in size exponentially. There was a positive correlation between tumor size and the number of starting vessels. The range of the blood supply from one starting vessel was evidently limited. The pressure of the starting vessel increased with enlargement of the tumor size. As soon as the pressure of the starting vessel reached a plateau, however, there was a rapid increase in low-flow or no-flow areas in regions within the tumor. From the results obtained, we consider that low-flow or no-flow areas, resistant to delivery of anticancer drugs, inevitably appear with the progression of tumor growth. PMID- 1705538 TI - A pulmonary large cell carcinoma cell line expressing neuroendocrine cell markers and human chorionic gonadotropin alpha-subunit. AB - A cell line producing the neuroendocrine cell surface antigen and human chorionic gonadotropin (hCG) alpha-subunit, designated as KTA7, was established from human large cell carcinoma using a serum-free medium, ACL-3. KTA7 continued to grow in the ACL-3 medium, showing the morphological characteristics of large cell undifferentiated carcinoma. The KTA7 cells reacted with antibodies such as 6H7 and MOC1 directed against the cell surface antigens and PGP9.5 directed against a cytoplasmic protein of neuroendocrine cells but did not possess either most epithelial markers other than low-molecular-weight keratin (Cytokeratin) or neuron-specific enolase. The KTA7 cells, by immunostaining with anti-hCG subunit antibodies, were shown to produce hCG alpha- but not beta-subunit. Northern blot analysis showed KTA7 RNA to synthesize hCG alpha-subunit mRNA but not that of the hCG beta-subunit. Thus, the hCG alpha-subunit alone was independently expressed in KTA7. Chromosome analysis showed loss of alleles of chromosome 3p and 17 in KTA7 cells but not loss of 13q. KTA7 was considered to be derived from large cell undifferentiated carcinoma with neuroendocrine differentiation (large cell neuroendocrine tumor) and thus may fine use in studies on the pathobiology of large cell-type neuroendocrine tumors since it expresses at the same time marker substances of neuroendocrine differentiation and the hCG alpha-subunit. PMID- 1705539 TI - Patterns of neurofilament stain in the spiral ganglion of the developing and adult mouse. AB - The objective of the study was to identify neurofilament-positive cells and their projections in the intact spiral ganglia of the mouse. One polyclonal and three monoclonal antibodies against neurofilament triplet subunits NF 68 K, 160 K and 200 K were used. In the newborn mouse most of the spiral neurons and their processes stain positively, although the perikaryal stain is very light. During early postnatal development, some cells show a selective intense stain. The progressive myelination of the neuronal processes further restricts the stain to a small neuronal population of positive perikarya and to their nonmyelinated fibers. This pattern of stainability implies that the neurofilament-positive cells are compatible with the type II spiral neurons. The stain reveals two populations of spiral neurons: 1) the cells which are scattered within the ganglion and show a bipolar distribution of fibers; and 2) the cells that form an interrupted chain along the intraganglionic bundle. The latter cells are also bipolar, but their peripheral processes join the intraganglionic bundle for varying distances before reaching the radial bundles. The identification of selective groupings of filamentous nonmyelinated cells in the corresponding location in different mammals is discussed. In conclusion, the use of neurofilament antibodies in staining of the intact spiral neurons permitted us to identify a distinct cell population of neurofilament-positive nonmyelinated nerve cells located along and projecting (at least partly) into the intraganglionic bundle. PMID- 1705540 TI - Effects of chronic cochlear de-efferentation on auditory-nerve response. AB - The olivocochlear bundle was sectioned at the floor of the fourth ventricle in a series of cats. From three to thirty weeks post-operatively, recordings were made from single auditory-nerve fibers. Tuning curves, spontaneous discharge rates, and rate-level functions for tones at the characteristic frequency were measured and compared to normal data. Light- and electron-microscopic analysis of the cochleas suggested the lesions were complete, for both classes of cochlear efferents, in three cases. Electrophysiological data from these cases showed normal thresholds, tuning curves and rate-level functions; however, the distributions of spontaneous activity suggested significant decreases in average rates in the de-efferented cases. PMID- 1705542 TI - Nucleotide sequence and molecular characterization of pnlA, the structural gene for damage-inducible pectin lyase of Erwinia carotovora subsp. carotovora 71. AB - In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins. PMID- 1705541 TI - Analysis of the V antigen lcrGVH-yopBD operon of Yersinia pseudotuberculosis: evidence for a regulatory role of LcrH and LcrV. AB - Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca2(+)-regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order lcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the lcrV and lcrH gene products were found to be regulatory proteins having important roles in the Ca2(+)-controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+. PMID- 1705543 TI - Precise mapping of the rnpB gene encoding the RNA component of RNase P in Escherichia coli K-12. AB - In Kohara's library derived from Escherichia coli K-12 W3110 (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), multiple copies of chromosomal sequence are found at 68 and at 64 to 65 min (M. Umeda and E. Ohtsubo, J. Mol. Biol. 213:229-237, 1990). We have determined that the rnpB gene (previously mapped at 70 min [B. J. Bachmann, Microbiol. Rev. 54:130-197, 1990]) is located within these segments of repeated sequences as five separate copies, together with tdcA, B, C, and R (mapped at 68 min [Bachmann, 1990]) and six unidentified open reading frames. Since close linkage of rnpB and tdc is found in various strains of E. coli K-12, the rnpB gene should be mapped at 68 min rather than 70 min. PMID- 1705544 TI - The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans. AB - The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form. PMID- 1705545 TI - Monoclonal antibody (VII-M31) to bovine factor VII: a specific epitope in the gamma-carboxyglutamic acid domain. AB - A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla) domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla domain constituting the amino-terminal portion of bovine factor VII. PMID- 1705546 TI - Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis. AB - Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3. PMID- 1705547 TI - The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum. AB - Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure. PMID- 1705548 TI - Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase. AB - Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade. PMID- 1705549 TI - A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain. AB - A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5 trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein. PMID- 1705550 TI - Regulation of reverse uniport activity in mitochondria by extramitochondrial divalent cations. Dependence on a soluble intermembrane space component. AB - We previously reported that uncoupling Ca2(+)-loaded mitochondria in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) produces a partial expression of the permeability transition. From this and related observations, it was proposed that the absence of external free Ca2+ is inhibitory to reverse activity of the Ca2+ uniporter (Igbavboa, U., and Pfeiffer, D.R. (1988) J. Biol. Chem. 263, 1405-1412). By using Sr2(+)-instead of Ca2(+)-loaded mitochondria, the transition is avoided upon treatment with EGTA plus uncoupler, and inhibition of reverse uniport activity can be observed directly. In the presence of physiological Mg2+ concentrations, reverse uniport of Sr2+ is eliminated by external EGTA following a brief period of rapid activity. It is proposed that binding of Mg2+ rather than Sr2+ (Ca2+) at an external site is responsible for the inhibition. Regulation at the external site is modified by the size of the Sr2+ load. EGTA, in the presence of Mg2+, does not inhibit the reverse uniport dependent release of Sr2+ from mitoplasts. The inhibitory effect can be recovered by adding back the soluble components obtained as the intermembrane space fraction following removal of the outer membrane. The soluble factor could be a regulatory subunit which contains the external cation binding site. Adjustments to uniporter activity due to regulation by the binding site and/or the soluble factor may be slow and may be significant in determining how mitochondria respond to rapid Ca2+ transients in vivo. PMID- 1705551 TI - Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin. AB - Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators. PMID- 1705552 TI - Organization, structure, and expression of the gene encoding the rat substance P receptor. AB - The gene for the rat substance P receptor has been cloned, its genomic structure determined, and the patterns of mRNA expression extensively analyzed. Unlike many genes encoding G protein-coupled receptors, the protein-coding region of this gene is divided into five exons consisting of 965, 195, 151, 197, and 2,010 base pairs. The substance P receptor gene extends more than 45 kilobases in length, and the splice sites for the exons occur at the borders of the sequences encoding putative membrane-spanning domains. The transcription initiation site has been defined by solution hybridization-nuclease protection and nucleotide sequence analyses, and lies downstream of a conventional TATA sequence. Substance P receptor mRNA levels in various tissues have been quantitated using solution hybridization-nuclease protection assays and were found to comprise from 0.00008 to 0.0016% of total RNA levels. Relatively high levels of substance P receptor mRNA are seen in the urinary bladder and the sublingual salivary gland, whereas moderate levels are observed for the submandibular salivary gland, striatum, hippocampus, midbrain, and olfactory bulb with lower levels in the remainder of the central nervous system and alimentary canal. These results are discussed in relation to the evolutionary role of multiple exons for a G protein-coupled receptor and with regard to the locations and mechanisms of substance P receptor gene expression. PMID- 1705553 TI - Calcium as a potential physiological regulator of integrin-mediated cell adhesion. AB - alpha v beta 1 and alpha v beta 3 are two related members of the integrin family of cell surface receptors both of which interact with their ligands through the Arg-Gly-Asp recognition sequence, alpha v beta 1 and alpha v beta 3 share the same cation-binding subunit, alpha v, suggesting a similar cation requirement for both integrins. Instead, we observed that Ca2+ exerts different effects on their binding function. The attachment of alpha v beta 3-loaded liposomes to vitronectin and the alpha v beta 3-mediated adhesion of U 251 cells to an Arg-Gly Asp-containing peptide was supported equally well by Ca2+ and Mg2+. However, IMR 32 cells which bind to Arg-Gly-Asp-containing peptides through alpha v beta 1 adhered in Mg2+ but not in Ca2+. In agreement, Ca2+ did not support the attachment of alpha v beta 1-loaded liposomes to the macromolecular ligand fibronectin or the binding of alpha v beta 1 to Gly-Arg-Gly-Asp-Ser-Pro-Lys Sepharose in affinity chromatography experiments. Furthermore, in the presence of a constant Mg2+ concentration, Ca2+ had opposite effects on the two receptors in that it inhibited the alpha v beta 1-mediated adhesion of IMR 32 cells to the peptide substrate while enhancing alpha v beta 3-mediated adhesion of U251 cells. The Ca2+ effects occurred at physiological cation concentrations and therefore, our data suggest a physiological role for Ca2+ as a regulator of integrin function and indicate a possible involvement of the beta subunits in cation binding. PMID- 1705554 TI - Cellular differentiation regulates expression of Cl- transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells. AB - The gene defective in cystic fibrosis has recently been shown to code for a membrane protein designated the "cystic fibrosis transmembrane conductance regulator" (CFTR) protein. While it has been shown that detectable levels of the mRNA for the normal CFTR protein are present in epithelial cells from different tissues, factors which regulate CFTR expression have not been identified. A clonal cell line originating from a human colon adenocarcinoma (HT29-18) differentiates to multiple epithelial cell types when deprived of glucose in the culture medium. In these studies, mRNA isolated from these cells was examined by hybridization to a 1.45-kilobase cDNA probe which encodes transmembrane portions of the CFTR protein between exons 13 and 19. Cellular differentiation of HT29-18 causes a 9-18-fold increase in CFTR mRNA abundance versus the mRNA for the structural proteins actin and tubulin. Cellular differentiation also causes a 5 fold increase in second messenger-regulated Cl- transport which is sensitive to a Cl- channel blocker (diphenylamine 2-carboxylate). Subclones of HT29-18 which are committed to differentiate to either a mucin-secreting (HT29-18-N2) or an "enterocyte-like" (HT29-18-C1) phenotype have also been examined. In both subclones, elevated levels of CFTR mRNA are observed when compared with undifferentiated HT29-18 cells. However, during cellular differentiation, the regulation of CFTR mRNA abundance and membrane enzyme expression by the subclones is different from HT29-18. The results show that elevated CFTR mRNA occurs in multiple differentiated intestinal epithelial cell types, despite a phenotype specific regulation of membrane protein expression. This suggests that CFTR expression plays a role in the differentiated functions of multiple epithelial phenotypes and that both cellular differentiation and cellular phenotypes are factors which regulate CFTR expression. PMID- 1705555 TI - Higher order structure of the ribosomal 5 S RNA. AB - The structure of the ribosomal 5 S RNA was examined using Fe(II)-EDTA, a solvent based reagent that cleaves the phosphodiester backbone of both double- and single stranded RNA but is restricted by the three-dimensional structure. In the yeast 5 S RNA, cleavages were significantly restricted in six specific regions of the molecule; restrictions in only two of these regions were clearly dependent on a high salt/magnesium ion environment. A comparison of four RNAs of diverse origin revealed strong similarities in the cleavage profiles supporting a highly conserved higher order structure. Taken together with previous studies these data provide a more detailed modeling of the three-dimensional structure. PMID- 1705556 TI - Variable O-glycosylation of CD13 (aminopeptidase N). AB - The monoclonal antibody AG6 was raised against HUVE cells and was confirmed as recognizing CD13 (aminopeptidase N) by its reactivity with the CD13 cDNA transfectant pipz "4". However, in sequential immunoprecipitation studies with another anti-CD13 monoclonal, WM15, neither was able to completely remove the molecules recognized by the other. As both monoclonals recognize the same cDNA transfectant, the differences cannot be in the amino acid sequence, suggesting that the CD13 subpopulations have glycosylation differences. Neither neuraminidase and endoglycosidase H treatment of the immunoprecipitates, nor tunicamycin and monensin treatment of proliferating cells differentiated the molecular subpopulations. The epitopes are protein and not carbohydrate as both monoclonals were able to precipitate deglycosylated CD13 from tunicamycin- and monensin-treated cells and o-glycanase-treated purified CD13. Sequential immunoprecipitation studies with WM15 and AG6 of pipz "4" and purified o glycanase-treated CD13 showed that the subpopulations were due to the O-linked oligosaccharides. Sequential immunoprecipitations with seven other CD13 monoclonals show that there are at least five subpopulations of CD13. It is postulated that the differences between the subpopulations are due to differential utilization of glycosylation sites or subtle differences in oligosaccharide composition causing variable masking of protein epitopes. Similar observations with the integrin group of molecules suggests that this may be an example of a more general phenomenon among glycoproteins. PMID- 1705557 TI - Modulation of antiviral activity of interferon and 2',5'-oligoadenylate synthetase gene expression by mild hyperthermia (39.5 degrees C) in cultured human cells. AB - The antiviral/antiproliferative/antitumor properties of interferon (IFN) are potentiated by a febrile temperature (Heron, I., and Berg, K. (1978) Nature 274, 508-510; Fleischmann, W. R., Fleischmann, C. M., Jr., and Gindhart, T. D. (1986) Cancer Res. 46, 1722-1726; Groveman, D. S., Borden, E. C., Merritt, J. A., Robins, H. I., Steeves, R., and Bryan, G. T. (1984) Cancer Res. 44, 5517-5521). To investigate the role of 2',5'-oligoadenylate (2-5A) synthetase in linking temperature with these biological functions of IFN, antiviral activities and expression of the 2-5A synthetase gene were measured simultaneously in control and type I IFN-treated HL-60 and WISH cells at the selected elevated temperature of 39.5 degrees C +/- 0.5 degrees C (herein referred to as hyperthermia). In both cell lines, the IFN-mediated antiviral effect was enhanced 3-10-fold. Concurrently, enzymatic assays and immunoblot analyses with an anti-40-kDa synthetase antibody clearly gave a 2-3-fold increase of synthetase above that observed at the normal cell culture temperature (37 degrees C). These results suggest that potentiation of 2-5A synthetase must partially account for the enhanced antiviral activity of IFN at the higher cell culture temperature. The supranormal elevation of 2-5A synthetase was accompanied by a parallel increase in the steady-state concentration of 2-5A synthetase mRNA, which is likely to contribute to the observed increase in enzyme level. Transient reporter gene expression studies using plasmid constructs carrying 2-5A synthetase gene promoter linked in tandem with chloramphenicol acetyltransferase showed that IFN inducible chloramphenicol acetyltransferase activity was not influenced by hyperthermia, suggesting that transcription activation is an unlikely explanation for the observed 2-5A synthetase mRNA level increases. Messenger RNA stability assays showed that the half-life (t1/2) of 2-5A synthetase mRNA was extended from 2 to 4 h at 39.5 degrees C. Under identical conditions, the t1/2 of poly(A)+ RNA remained unchanged whereas the t1/2 of beta-actin mRNA was reduced. Taken together, these results are consistent with the interpretation that selective stabilization of 2-5A synthetase mRNA at the elevated temperature is a major factor contributing to the potentiation of antiviral activity of IFN by hyperthermia. PMID- 1705559 TI - Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells. AB - Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state. PMID- 1705558 TI - Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily. AB - The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions. PMID- 1705560 TI - Binding of actin to liver cell membranes: the state of membrane-bound actin. AB - Previous work has shown that actin binds specifically and saturably to liver membranes stripped of endogenous actin (Tranter, M. P., S. P. Sugrue, and M. A. Schwartz. 1989. J. Cell Biol. 109:2833-2840). Scatchard plots of equilibrium binding data were linear, indicating that binding is not cooperative, as would be expected for F- or G-actin. To determine the state of membrane-bound actin, we have analyzed the binding of F- and G-actin to liver cell membranes. G-actin in low salt depolymerization buffer and EF-actin, a derivative that polymerizes very poorly in solution, bind to liver cell membranes as well as untreated actin in polymerization buffer. Phalloidin-stabilized F-actin binds, but to a lesser extent. The binding of F- and G-actins are mutually competitive and are inhibited by ATP, suggesting that both forms of actin bind to the same sites. For untreated actin in polymerization buffer, the time course of binding is biphasic, with an initial rapid component which is followed by a plateau phase, then a second, slower component. The binding kinetics of pure F-actin and pure G-actin are both monophasic and match the fast and slower components, respectively, of untreated actin. In the reconstituted system, membrane-bound actin does not stain with rhodamine-phalloidin, nor are actin filaments detected by EM. Distinct regions of amorphous material, however, are visible, which stain with an anti-actin antibody. The exact nature of this material has yet to be determined. A model of actin binding is presented. PMID- 1705561 TI - Monoclonal antibodies show that kinase C phosphorylation of GAP-43 during axonogenesis is both spatially and temporally restricted in vivo. AB - To study the role of kinase C phosphorylation in the distribution and function of GAP-43 we have generated a panel of mAbs that distinguish between GAP-43 that has been phosphorylated by kinase C and forms that have not. One class of antibodies, typified by 2G12/C7, reacts with only the phosphorylated form of GAP-43; it recognizes the peptide IQAS(PO4)FR equivalent to residues 38-43 that includes the single kinase C phosphorylation site at serine. Another, exemplified by 10E8/E7, reacts with both phosphorylated and nonphosphorylated forms. We have used the antibodies to study the distribution of kinase C-phosphorylated GAP-43 during axonogenesis and in the adult nervous system. Two major findings emerge. First, there is a lag between the initiation of axon outgrowth and the phosphorylation of GAP-43 by kinase C. The extent of this lag period varies between the different structures studied. In some cases, e.g., the trigeminal nerve, our result suggest that kinase C phosphorylation may be correlated with proximity of the growing axon to its target. Second, kinase C-phosphorylated GAP-43 is always spatially restricted to the distal axon. It is never seen either proximally or in cell bodies, even those with high levels of GAP-43 protein. This result also implies that GAP-43 is axonally transported in the non-kinase C phosphorylated form. Thus, kinase C phosphorylation of GAP-43 is not required for axon outgrowth or growth cone function per se and may be more related to interactions of the growth cone with its environment. PMID- 1705562 TI - Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. AB - Biochemical fractionation procedures previously shown to remove 95% of cellular protein, DNA, and phospholipid, were combined with fluorescence in situ hybridization to provide a critical evaluation of the retention and spatial preservation of specific primary transcripts within the chromatin-depleted nuclear substructure, operationally defined as the nuclear "matrix." This unique approach made it possible to directly address whether nuclear extraction procedures preserve, create, or destroy ribonucleoprotein filament structures. Comparison of nuclei before and after fractionation demonstrated that localized foci or "tracks" of specific nRNA are unambiguously retained in the nuclear matrix preparation. Two well-characterized nuclear fractionation procedures were used and three Epstein-Barr virus-infected cell types investigated, including latently and permissively infected cells carrying integrated or episomal genomes. The EBV primary transcripts as well as nucleolar RNA were preserved within the remaining nuclear substructure with unambiguous spatial and quantitative fidelity. Image processing and quantitative microfluorimetry, together with [3H]thymidine labeling of DNA, show that essentially 100% of the RNA signal intensity remained after removal of 85% of the DNA. That the native RNA distribution was unchanged was shown in other experiments in which the same individual nRNA tracks were examined before and after fractionation. Results conclusively demonstrate that the tight restriction of RNA to highly localized sites is independent of bulk DNA removal and of extensive extraction of proteins and phospholipids. Hence, this work provides direct visual evidence that the primary transcripts studied are localized via their binding to, or comprising part of, non-chromatin nuclear substructure. PMID- 1705563 TI - Voltage-dependent calcium channels regulate GH4 pituitary cell proliferation at two stages of the cell cycle. AB - Calcium is an intracellular signal implicated in the regulation of cell proliferation. We have examined the growth regulatory role of voltage-dependent calcium channels (VDCC) in a rat pituitary cell line (GH4C1) that expresses two well-characterized VDCC subtypes (L and T) and is growth-inhibited by several agents known to enhance calcium entry. Thyrotropin-releasing hormone (TRH), tetradecanoylphorbol acetate (TPA), and epidermal growth factor (EGF), each known to enhance calcium entry in GH4 cells, decrease GH4 cell number and incorporation of [3H]-thymidine. The growth inhibitory action of these agents is cytostatic with a predominant effect to block G1 cells from entering S-phase. We next examined the growth regulatory action of pharmacologic agents that interact directly and specifically with type L VDCC. Activation of type L VDCC with the dihydropyridine BAY K8644 inhibits GH4 proliferation as measured by cell number and [3H]-thymidine incorporation. This action of BAY K8644 is enhanced by a submaximal K(+)-maintained depolarization, and the growth inhibitory action of these agents is also cytostatic as evident by the block of G1 cells from entering S-phase. Nimodipine, an antagonist specific for type L VDCC blocks (IC50 = 30 nM) BAY K8644-inhibited cell proliferation by substantially reducing the S-phase block. Taken together these findings indicate that calcium entry through type L VDCC inhibits GH4 cell proliferation by blocking entry into S-phase. By contrast, nimodipine caused only a small reversal of the TRH-induced S-phase block, suggesting that TRH inhibits proliferation by a mechanism that differs at least in part from L-channel activation. Unexpectedly, nimodipine, given alone, caused a substantial inhibition of GH4 cell proliferation. This action of nimodipine was cytostatic, yet differed from calcium channel activators in that the percentage of S-phase cells was unchanged whereas G2-M-phase cells increased with a parallel decrease in G1-phase cells. Similar effects were also observed with other classes of calcium channel blockers. Taken together these results indicate that calcium entry through VDCC regulates GH4 cell proliferation differently depending on the stage of the cell cycle. In G1-phase cells, sustained entry of calcium through type L VDCC blocks entry into S-phase. In G2-M-phase cells entry of calcium promotes progression through mitosis. PMID- 1705564 TI - 1,25-dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. AB - The hormone, 1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3), inhibits lymphocyte activation in vitro. We studied the ability of the vitamin D metabolite to interfere in vivo with a primary T cell-mediated model of autoimmunity, murine experimental autoimmune encephalomyelitis (EAE). Within 2 wk of antigenic challenge, immunized animals will develop acute paralysis with central nervous tissue inflammation. If mice survive, a rise in antibody titer develops within a month. The administration of 0.1 microgram 1,25-(OH)2-D3 i.p. given every other day for 15 d, starting 3 d before immunization, significantly prevented the development of EAE. The rise in antibody titer to myelin basic protein was also abrogated. Histopathologic lesions of EAE were inhibited by treatment with the sterol. These results suggest a potent immunosuppressive role for 1,25-(OH)2-D3 in vivo in the modulation of a cell-mediated model of autoimmunity. PMID- 1705565 TI - Activation of phospholipase C in human B cells is dependent on tyrosine phosphorylation. AB - Cross-linking of the surface antigen receptor on B lymphocytes has been demonstrated to lead to activation of phospholipase C (PLC) with subsequent increases in production of inositol phosphates and diacylglycerol. In turn, these second messengers increase cytosolic free calcium [( Ca2+]i) and activate the serine threonine phosphotransferase protein kinase C (PKC). These processes are thought to play a major role in B cell activation and proliferation. However, the mechanism linking the B lymphocyte antigen receptor to phospholipase C remains to be identified. We demonstrate herein that activation of the antigen receptor on human lymphocytes, in addition to activation of PLC, increases tyrosine phosphorylation of specific substrates. Tyrphostins, a new class of tyrosine kinase inhibitors which compete for substrate binding site of specific tyrosine kinases have recently been synthesized. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced proliferation, oncogene expression, tyrosine phosphorylation, increases in [Ca2+]i, and production of inositol phosphates. The same inhibitors were without effect on B cell proliferation induced by phorbol esters and cation ionophores which directly activate PKC and increase [Ca2+]i thus bypassing PLC. These findings strongly indicate that tyrphostins do not exhibit significant nonspecific toxicity and suggest that they act proximal to PLC. The ability of the tyrphostins to block increases in [Ca2+]i and inositol phosphate production, after activation of the B cell antigen receptor, indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor and PLC. PMID- 1705566 TI - Tumor necrosis factor downregulates granulocyte-colony-stimulating factor receptor expression on human acute myeloid leukemia cells and granulocytes. AB - Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G CSF receptor expression becomes evident within 10 min after incubation of the cells with TNF at 37 degrees C and is not associated with an apparent change of the dissociation constant (Kd). The TNF effect does not occur at 0 degrees C and cannot be induced by IL-2, IL-6, or GM-CSF. TNF probably exerts its effect through activation of protein kinase C (PKC) as the TNF effect on G-CSF receptor levels can be mimicked by 12-O-tetradecanoylphorbol-13- acetate. The PKC inhibitor Staurosporine (Sigma Chemical Co., St. Louis, MO) as well as protease inhibitors can completely prevent G-CSF receptor downmodulation. Thus, it appears TNF may act as a regulator of G-CSF receptor expression in myeloid cells and shut off G-CSF dependent hematopoiesis. The regulatory ability of TNF may explain the antagonism between TNF and G-CSF stimulation. PMID- 1705567 TI - Apical secretion of lysosomal enzymes in rabbit pancreas occurs via a secretagogue regulated pathway and is increased after pancreatic duct obstruction. AB - Lysosomal hydrolases such as cathepsin B are apically secreted from rabbit pancreatic acinar cells via a regulated as opposed to a constitutive pathway. Intravenous infusion of the cholecystokinin analogue caerulein results in highly correlated apical secretion of digestive and lysosomal enzymes, suggesting that they are discharged from the same presecretory compartment (zymogen granules). Lysosomal enzymes appear to enter that compartment as a result of missorting. After 7 h of duct obstruction is relieved, caerulein-stimulated apical secretion of cathepsin B and amylase is increased, but the ratio of cathepsin B to amylase secretion is not different than that following caerulein stimulation of animals never obstructed. These findings indicate that duct obstruction causes an increased amount of both lysosomal and digestive enzymes to accumulate within the secretagogue releasable compartment but that duct obstruction does not increase the degree of lysosomal enzyme missorting into that compartment. Pancreatic duct obstruction causes lysosomal hydrolases to become colocalized with digestive enzymes in organelles that, in size and distribution, resemble zymogen granules but that are not subject to secretion in response to secretagogue stimulation. These organelles may be of importance in the development of pancreatitis. PMID- 1705568 TI - Decreased content and surface expression of alpha-granule membrane protein GMP 140 in one of two types of platelet alpha delta storage pool deficiency. AB - To determine whether alpha-granule membranes are present in platelets of patients with storage pool deficiencies of both alpha and dense granules (alpha delta SPD), we examined the content and surface expression of the alpha-granule membrane protein GMP-140 in one patient (J.C.) with a severe alpha-granule deficiency and in three members of a family (family C) with milder alpha-granule deficiencies. Surface expression of GMP-140 in stimulated platelets, assessed by flow cytometric measurements of the binding of two anti-GMP-140 monoclonal antibodies, was 24-38% of normal values in platelets from patient J.C., vs. 60 95% of normal values in family C. Total platelet content of GMP-140, determined in platelet lysates by antigen-capture ELISA, was 49% of normal in patient J.C., but normal in the members of family C. Platelets of patient J.C. were found to be heterogeneous with respect to GMP-140 content and surface expression by both flow cytometry and immunogold electron microscopy. Approximately 80% of her platelets expressed little or no GMP-140 after stimulation, whereas the remaining 20% expressed normal amounts of GMP-140 and showed extensive immunogold labeling of typical alpha-granules and clear vacuoles. No such heterogeneity was found in platelets from family C. These findings in the severe alpha delta-SPD patient are in clear contrast to the observations of normal GMP-140 content in the three other alpha delta-SPD patients, and in patients with the gray platelet syndrome, reported previously by others. These results illustrate the phenotypic heterogeneity of alpha-granule deficiencies in human platelets, and suggest that a defect in granule formation in the megakaryocytes may account for the alpha granule defect in at least one form of alpha delta-SPD. PMID- 1705569 TI - In vitro and in vivo activation of endothelial cells by colony-stimulating factors. AB - This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation/differentiation program (including proliferation and migration) related to angiogenesis. PMID- 1705570 TI - Staining of in vivo subsurface degradation in dental composites with silver nitrate. AB - A previously reported technique for staining areas of degradation in dental composite restorations was evaluated in 51 removed restorations. The staining reagent was silver nitrate, which penetrated the degraded subsurface as ionic silver and was subsequently developed into colored deposits of metallic silver. Several artefacts were recognized that resulted in an apparent image of subsurface stain. Most importantly, the presence of a layer of adsorbed silver on the edge of the specimen exaggerated the extent of staining. In order for the true depth of stain to be determined, thin sections of the materials should first be examined with a stereomicroscope to distinguish any contribution from adsorbed silver on the specimen edge. With this regimen, no stain was present in 41% of the restorations, and in a further 30%, the depth of stain was less than 50 microns. In two composites, the depth of stain was greater than 900 microns, and in a number of specimens, localized stain was found in association with attrition scars. Energy-dispersive x-ray analysis indicated that the amount of silver present in the degraded layers was very small. Overall, the results indicated that the staining technique is useful in the study of composite degradation. PMID- 1705571 TI - Production of a pokeweed antiviral protein (PAP)-containing immunotoxin, B43-PAP, directed against the CD19 human B lineage lymphoid differentiation antigen in highly purified form for human clinical trials. AB - We describe a standardized method for the preparation and purification of a potent immunotoxin against B-lineage leukemia/lymphoma cells, constructed with the ribosome inhibitory single chain plant toxin pokeweed antiviral protein (PAP) and a murine IgG1 monoclonal antibody (MoAb) specific for the human B lineage differentiation antigen CD19 for human clinical trials. PAP was prepared from spring leaves of Phytolacca americana plants by ammonium sulfate precipitation and purified to homogeneity by successive steps of ion exchange chromatography. B43 MoAb was produced in vitro by hollow fiber technology and purified to homogeneity by affinity chromatography. PAP toxin and B43 MoAb were modified via their free amino groups prior to their intermolecular conjugation. 2 iminothiolane was used to introduce reactive sulfhydryl groups into PAP and N succinimidyl 3-(2-pyridyldithio) propionate was used to introduce 2-pyridyl disulfide bonds into B43 MoAb. Modified PAP was reacted with modified B43 MoAb resulting in a sulfhydryl-disulfide exchange reaction and yielding disulfide linked PAP-B43 MoAb conjugates, which we refer to as B43-PAP immunotoxin. B43-PAP immunotoxin was subjected to preparative gel filtration chromatography and cation exchange chromatography to obtain a highly purified, sterile, and pyrogen-free immunotoxin preparation with less than 5% free antibody contamination and less than 0.5% free PAP contamination. The final product displayed a high affinity for and a very potent anti-leukemic activity against B lineage leukemia cells. With slight modifications, the procedures detailed in this report should be generally applicable to preparation of other PAP-MoAb conjugates for treatment of cancer or AIDS. PMID- 1705572 TI - Purification and assay of insulin-like growth factor-binding protein-1: measurement of circulating levels throughout pregnancy. AB - Insulin-like growth factor-binding protein-1 (IGFBP-1) has been purified from amniotic fluid by anion exchange, hydrophobic interaction and gel filtration chromatography. The overall recovery of the purification process was 12.2%. The purified IGFBP-1 yielded a single band on SDS-PAGE gel but showed two bands (34 kDa and 68 kDa) on Western blot under non-reducing conditions. Polyclonal antisera were raised by immunization of sheep using the purified IGFBP-1. The best antiserum bound 50% of 125I-labelled IGFBP-1 at a final dilution of 1:500,000. A radioimmunoassay for IGFBP-1 was developed. This assay had a minimum detection limit of 5 micrograms/l, and was used to determine serum levels in non pregnant and pregnant women. There was no cross-reaction with a wide variety of materials tested. Serum IGFBP-1 levels in non-pregnant individuals (33 +/- 16 (S.D.) micrograms/l) were found to be significantly lower than those in the second (96 +/- 64 micrograms/l) and third trimesters (95 +/- 60 micrograms/l) of pregnant women. During pregnancy, circulating IGFBP-1 levels increased rapidly in the first trimester and reached a peak at 12-13 weeks of gestation (107 +/- 75 micrograms/l). The level then remained at 80 +/- 53 to 103 +/- 70 micrograms/l until term. PMID- 1705573 TI - The cyclic voltammetric study of iron-tallysomycin in the absence and presence of DNA. AB - Cyclic voltammetric studies on iron-tallysomycin complexes have been conducted with and without the presence of calf thymus DNA. Fe(II)-TLM samples exhibit a cyclic voltammogram with only a reduction peak at -230 +/- 5 mV vs Ag/AgCl. The addition of DNA substrate causes the shift of this reduction peak to -140 +/- 10 mV vs Ag/AgCl. This large shift in the positive direction implies that the regeneration of Fe(II)-TLM through the reduction of Fe(III)-TLM is facilitated with the aid of DNA. It also implies that the metal-binding/oxygen-activation domain may be directly involved in the formation of iron-tallysomycin-DNA ternary complex. Air oxidation of Fe(II)-TLM produces an activated intermediate with the following CV characteristics, Ipc/Ipa = 0.90; delta E = 65 mV; Ereduction peak = 100 mV vs Ag/AgCl. Addition of DNA abolishes the redox peaks of this voltammogram, signifying inactivation of the activated species through reaction with substrate. Air oxidation of preformed Fe(II)-TLM-DNA complex did not give a discernable cyclic voltammogram, nor did preformed Fe(III)-TLM and Fe(III)-TLM DNA samples. PMID- 1705574 TI - Differential inhibition of reverse transcriptase and various DNA polymerases by digallic acid and its derivatives. AB - Digallic acid (gallic acid 5,6-dihydroxy-3-carboxyphenyl ester) [4] was found to be a potent inhibitor of the activities of the reverse transcriptases from murine leukemia virus (MLV) and human immunodeficiency virus (HIV). Under the reaction conditions specified for each of MLV and HIV reverse transcriptases, both enzymes were inhibited by approximately 90% in the presence of 0.5 micrograms/ml digallic acid. Under the same conditions, however, gallic acid had no effect on the reverse transcriptase activity. The mode of the inhibition by digallic acid was partially competitive with respect to the template.primer, (rA)n.(dT)12-18', and noncompetitive to the triphosphate substrate, dTTP. The Ki value of digallic acid for HIV-reverse transcriptase was determined to be 0.58 microM. Examination of several derivatives of digallic acid have shown that all three hydroxyl groups at the 3, 4, and 5 positions seem to be required for the inhibitory activity of these compounds. Besides reverse transcriptase, DNA polymerases alpha and beta were moderately inhibited by digallic acid, whereas DNA polymerase gamma, terminal deoxynucleotidyltransferase, and E. coli DNA polymerase I were virtually insensitive to inhibition by this compound. PMID- 1705575 TI - Multiple system degeneration with glutamate dehydrogenase deficiency: pathology and biochemistry. AB - The neuropathological findings in a patient with antemortem diagnosis of olivopontocerebellar atrophy (OPCA) and reduced leucocytic glutamate dehydrogenase (GDH) activity included cerebellar cortical degeneration, most marked in the superior vermis, mild atrophy of the pons and the inferior olivary nucleus, marked reduction of anterior horn cells at all levels and gliosis in both lateral columns. GDH activities and their thermolability in "soluble" and "particulate" fractions in the cerebral cortex, cerebellar hemisphere and vermis were not significantly different from the values in two control brains. GDH mRNA in the patient's brain was not altered in size or amount. PMID- 1705576 TI - Retention of J1/tenascin and the polysialylated form of the neural cell adhesion molecule (N-CAM) in the adult olfactory bulb. AB - To gain insight into the cellular and molecular mechanisms underlying neurogenesis in adult mouse olfactory bulb, several adhesion molecules expressed by glial cells and neurons were investigated. In the germinal zone of the olfactory bulb, the subependymal layer of the rostral region of the lateral ventricles, two adhesion molecules are detectable that are characteristic of early morphogenetic events: J1/tenascin and the polysialylated form, the so called embryonic form, of N-CAM. The polysialylated form of N-CAM is expressed by most cells in the subependymal layer, and by some astrocytes and neurons in the granular layer adjacent to the subependymal layer. This suggests that bipotential precursor cells retain expression of the embryonic form during their migration from the subependymal layer and during the first stages of differentiation into neurons and glia. Expression of the polysialylated form of N-CAM is also retained in monolayer cultures of six-day-old olfactory bulbs, 55 days after seeding in vitro. J1/tenascin was detectable in the subependymal layer using two monoclonal antibodies. The immunostaining pattern was different between the two antibodies and more restricted to the subependymal layer than when staining with polyclonal J1 antibodies was performed, indicating that J1/tenascin exists in distinct isoforms. Finally, our observations suggest that, in the adult olfactory bulb, L1 is not only a neuron-neuron adhesion molecule, but it may also be involved in neuron-glia interactions, since it is found at contact sites between these two cell types. L1, therefore, may be a neuron-glia adhesion molecule in some parts of the CNS, while it is not in others. PMID- 1705577 TI - Heterogeneity in spinal radial glia demonstrated by intermediate filament expression and HRP labelling. AB - Considerable evidence indicates that radial glial cells play an active role in guiding growing neurites during development of the vertebrate CNS. In this paper we describe subpopulations of radial glial in the spinal cord of the axolotl. Amphibians maintain radial glia throughout life, and subpopulations are described using anatomical criteria following filling of individual cells with horseradish peroxidase and immunocytochemical staining with a range of intermediate filament antibodies. Radial glial cells in specific regions of the spinal cord stain with a range of antibodies specific to human keratins 8 and 18, and to glial fibrillary acid protein (GFAP). Some of these antibodies show selective localized to specific regions of individual glial cell processes. Immunoblotting analysis indicates that two keratins are present in the axolotl CNS corresponding to the two earliest embryonic keratins of vertebrates, keratin 8 and 18. Comparisons of molecular weight indicate that these may correspond to keratins identified in Xenopus laevis, the genes of which have been cloned. Axolotl GFAP is also identified in Western blots and may be present in two forms of differing molecular weight. These results are discussed in terms of the likely role of radial glial cells, and comparisons are drawn between the keratin and GFAP types seen in the axolotl spinal cord and of those in other vertebrate groups. PMID- 1705578 TI - Anti-GABA antibodies label a subpopulation of chemical synapses which modulate an electrical synapse in crayfish. AB - Antibodies raised against gamma-aminobutyric acid (GABA) were used to stain sections from the crayfish abdominal nervous system, and the sections were examined under the electron microscope using a protein-A/gold conjugate secondary label. Sections were taken through the third ganglionic root, and through the interganglionic connective at the base of the third root posterior to the ganglia. The third root contains two very large motor axons, a non-GABAergic excitor (Motor Giant; MoG), and a GABAergic inhibitor (Flexor Inhibitor; FI). Only one of the two large axons stained positively for GABA, confirming that the antibody has high specificity for GABAergic neurones. The MoG is driven by powerful electrical synapses from the giant fibres, but also receives inhibitory chemical synaptic input which can gate the excitatory input. There is no physiological evidence for any other form of chemical input. However, at the ultrastructural level, the MoG is postsynaptic to three types of chemical profiles; SE-type containing round agranular vesicles, SI-type containing pleomorphic vesicles, and SM-type containing a mixture of round agranular and dense-cored vesicles. There is a highly differentiated staining pattern of these three synaptic types. Only the SI-type profiles stain positively with the GABA antibody, while the SE- and SM-type do not show significant staining. This suggests that the MoG can under some circumstances receive chemical input other than GABAergic inhibitory input. These other types of input have yet to be physiologically identified. PMID- 1705579 TI - Lysosomal activity at nodes of Ranvier during retrograde axonal transport of horseradish peroxidase in alpha-motor neurons of the cat. AB - Lysosomal activity at nodes of Ranvier of feline hindlimb alpha-motor neurons was examined by light and electron microscopical acid phosphatase (AcPase) histochemistry during retrograde axonal transport of intramuscularly injected horseradish peroxidase (HRP). Several nodes along the PNS parts of the alpha motor axons of the HRP-injected side showed accumulations of AcPase-positive bodies in the constricted nodal axon segment and the adjacent paranodal axoplasm. Such lysosomal accumulations were most prominent in the ventral root and differed in number and intensity depending on survival time after the HRP injection. At nodes showing high AcPase activity the axoplasm proximal to the nodal midlevel was occupied by many small, AcPase-positive, vesiculotubular profiles. Larger AcPase-positive bodies were mainly situated distal to the nodal midlevel. Double incubation for demonstration of both HRP and AcPase activity showed similar accumulations of AcPase-positive bodies at some of the HRP-transporting nodes. The AcPase activity differed considerably between nodes exhibiting comparable levels of HRP-positivity. Many of the AcPase-positive bodies also contained HRP reaction product. At some HRP-positive nodes the number of AcPase-positive bodies situated in the paranodal axon-Schwann cell network was elevated when compared to nodes of the contralateral, control side. In contrast to the PNS nodes, the nodal occurrence and distribution of lysosomes in the CNS part of alpha-motor axons seemed not to be affected by HRP transport. These observations support our previous proposal that nodes of Ranvier in the PNS parts of alpha-motor axons, in contrast to their CNS nodes, possess an ability to control passage of and initiate lysosomal degradation of axonally transported substances. Such an ability may provide a protective function to the motor neuron by restricting the intraneuronal transport of materials imbibed by the axon terminals outside the CNS. PMID- 1705580 TI - A cost-minimization study of cancer patients requiring a narcotic infusion in hospital and at home. AB - We conducted a retrospective, non-randomized, cost-minimization study, from the perspective of the Ministry of Health, to compare the cost of managing cancer patients who required narcotic infusions, in hospital and at home. Our medical costs averaged $369.72 per inpatient day and $150.24 per outpatient day (saving $219.48 per diem, 1988 Canadian dollars), while narcotic costs were the same for any given patient in both settings. Sensitivity analysis showed that no reasonable changes in the quantity and cost of services reduced our savings by more than 50%. During incremental analysis, savings increased as more outpatient days were managed by our centre, from $0.00 for 318 days, to more than $500,000 for over 2000 days per annum. As this program has been extremely cost effective and preferred by our patients, other hospitals and central funding agencies might consider establishing a regional outpatient narcotic infusion program to reduce their costs. PMID- 1705581 TI - Bone metastases: pathophysiology and management policy. AB - The pathophysiology and options for management of bone metastases as well as criteria for determining response to therapy are reviewed. Bone metastases are frequently one of the first signs of disseminated disease in cancer patients. In the majority of patients, the primary tumor is in the breast, prostate, or lungs. Although almost all patients will die of their disease, a proportion of the patients will survive for several years. Treatment is primarily palliative: the intention is to relieve pain, prevent fractures, maintain activity and mobility, and, if possible, to prolong survival. Therapeutic options include local treatment with radiotherapy and/or surgery, and systemic treatment using chemotherapy, endocrine therapy, radioisotopes, agents such as diphosphonates, which inhibit resorption of bone, as well as analgesic and antiinflammatory drugs. The mechanisms by which pain is relieved by several of these therapies remain unclear but actions beyond a simple tumoricidal effect appear to be important. There have been few randomized trials comparing the therapeutic options, and the criteria for assessing response to therapy have, in general, been poorly defined. There is a need for rigorous clinical investigations that assess the efficacy of the various therapeutic possibilities by using well defined and validated criteria of response. PMID- 1705582 TI - The identification of prostate-specific antigen. PMID- 1705583 TI - Detection of infection with human immunodeficiency virus (HIV) type 1 in infants by an anti-HIV immunoglobulin A assay using recombinant proteins. AB - To diagnose infection with the human immunodeficiency virus (HIV) soon after birth in infants born to HIV type 1-infected women, we developed antiviral IgA Western blot and dot blot assays with recombinant HIV-1 proteins. Thirty-three infants born to HIV-1-seropositive mothers and nine infants born to HIV-1 seronegative intravenous drug-abusing mothers were followed prospectively. Infection was documented by positive virus culture. Results with the polymerase chain reaction were used for comparison. Twelve infants were found infected with HIV-1; the earliest age at which cultures became positive ranged from birth to 31 weeks of age. Of the 12 culture-positive infants, 10 had anti-HIV IgA antibodies detectable initially between birth (cord blood) and 27 weeks of age. Anti-HIV IgA was not present in the uninfected infants or in the control subjects, either by Western blot or dot blot assays. Testing for anti-HIV IgA antibodies with recombinant HIV-1 proteins is an effective method for detecting viral infection in newborn and young infants. PMID- 1705584 TI - Clinical use of the Kleihauer-Betke test. AB - Results of all Kleihauer-Betke (KB) tests performed in 1988, at a center with 4,201 deliveries, were reviewed. Two hundred and twenty-seven tests were performed on maternal specimens from 205 patients. Eighteen (8.8%) of the 205 patients had positive test results. Medical records were available for 147 (71.7%) of the patients, including 17 of the 18 patients with a positive result. Indications for testing were: vaginal bleeding (33%), maternal trauma (31%), unexplained fetal death (5%), Rh incompatibility (3%), fetal distress (3%), and miscellaneous (24%). Most of the tests were performed antepartum. In only one case, and without clear benefit, did the KB test prompt a clinical intervention. At least two of the 18 patients with positive test results had probable false positive results due to maternal hemoglobin F. Such false positive KB test results may be misleading. Further evaluation of the role of the KB test in obstetrical management is needed. PMID- 1705585 TI - A comparative study of the effect of arterial and venous occlusion after various periods of ischemia. AB - The author attempted to quantify any additional tissue damage resulting from one hour of venous occlusion after controlled periods of ischemia in skeletal muscle, compared to equal time periods of ischemia. The rat quadriceps muscle was used. pH and temperature were monitored during the experiment and the damage was evaluated on histochemical sections. Of the other two parameters monitored in this study, the pH recovery rate during reperfusion best reflected tissue perfusion and served as a predictor for the extent of tissue damage, when global ischemia or the combination of ischemia and postischemic venous stasis did not exceed five hours. Apart from the nonstained lethally damaged areas, interstitial tissue edema and cellular infiltration were constant findings in all muscles that sustained ischemia or ischemia and subsequent venous occlusion. However, both findings were more pronounced in the muscles that underwent venous obstruction. Muscle fibers were more edematous after venous occlusion, compared to ischemic muscles. Thrombosed veins and collapsed arteries, surrounded by excessive edema and inflammatory reaction, were common in muscles after ischemia and venous occlusion. Comparing the difference in percent of lethal damage from one hour of postischemic venous stasis versus the damage from additional ischemia of equal duration, findings demonstrated comparable damage when reperfusion was established in less than five hours. PMID- 1705586 TI - Revascularization of an ischemic limb by use of a muscle pedicle flap: a rabbit model. AB - A rabbit model of hind limb ischemia was designed to demonstrate that new, hemodynamically significant arterial connections will develop between ischemic skeletal muscle and an independently perfused muscle pedicle flap. The right common iliac artery was divided in 15 rabbits. In eight rabbits a muscle flap based on the left deep inferior epigastric artery was transposed to the right thigh (flap group). In seven rabbits a sham operation was performed where the flap was sutured to the abdominal wall (sham group). After 7 days angiography demonstrated arterial connections between the flap and the native limb circulation in all of the flap group animals. The flap increased muscle perfusion in the ischemic limb (2.99 ml/100 gm muscle/minute in the flap group, vs 2.06 ml/100 gm muscle/minute in the sham group, p less than 0.005). Hemodynamically significant vascular connections will develop between a well-perfused muscle flap and an ischemic limb. The augmentation in perfusion provided by these connections can be quantified. PMID- 1705587 TI - Azacitidine. PMID- 1705588 TI - [Discriminant analysis of quantification theory on prognostic factors of chemotherapeutic effects on advanced testicular cancer. East Japan Testicular Tumor Study Group]. AB - Prognostic factors of chemotherapy against advanced testicular cancer were analysed in 33 cases studied by East Japan Testicular Tumor Study Group. In this study a discriminant analysis of quantification theory (a multivariate analysis) was adapted, taking 7 factors as comparative variables. The significant factors for complete tumor response were clinical stage, histology, and tumor markers (HCG-beta, AFP). Prognostic score was calculated in each case by quantification theory, which correctly discriminated the group with CR from that without CR, at a probability of 90.1%. The results of our study indicate that the outcome of chemotherapy on advanced testicular cancer may be predicted with probability, by patients' status and adopted treatment. It may enable one to make an adequate treatment schedule for each patient. PMID- 1705589 TI - [Mass screening for prostatic cancer in Hisayama town, Fukuoka]. AB - A mass screening for prostatic cancer was conducted in Hisayama town, Fukuoka Prefecture, Japan from 1988 to 1989. As the primary screening questionnaire survey about urination and measurement of serum prostate specific antigen (PSA) were performed in 789 of the 1025 male residents over 50 years old (77.0%). For the second screening with digital rectal examination and transrectal ultrasonography, 184 subjects (23.3%) were picked up. Prostatic cancer was suspected in 33 (4.2%) of the 134 subjects who actually underwent the second screening examination. Prostatic biopsy and other examinations such as measurement of prostatic acid phosphatase and gamma-seminoprotein, urinalysis and radiographic examination were performed in 26 as the third screening, and prostatic cancer was detected in 5 (0.63%); 3 in stage B and 2 in stage C. Four of the 5 subjects with prostatic cancer had complained no urinary symptoms on questionnaire. Serum PSA was useful for detecting early stage prostatic cancer. PMID- 1705590 TI - [State of the rabbit aorta after orthostatic tests]. AB - Morphometric examinations of endothelium film preparations and histological investigations of aortic sections were performed 1 to 50 days after exposure of rabbits to orthostatic tests. Immediately and 1.5 months after exposure the intima showed a significant increase of endotheliocytes, vacuolization of the cytoplasm and nuclei, development of karyopyknosis and karyolysis as well as increase of monocytes and lymphocytes. Beginning with day 4 the inner surface of the intima displayed microthrombi and beginning with day 7 the media showed necrotic changes, which may cause wall deformation due to the destruction of the collagen and elastic layers. At the same time the endothelium exhibited adaptive compensatory processes, which included increased mitotic activity of endotheliocytes and their intracellular regeneration. The identification of the coefficient of variation of centered ordinates helped to distinguish an early phase of orthostatic responses (days 1-2), phase of adaptive and compensatory changes (3-15), and phase of remote effects and stabilization of the aortic endothelium. These observations should be taken into consideration when determining optimal time periods of recovery after lesions caused by acute fluid shifts. PMID- 1705591 TI - [Biochemical diagnosis of lysosomal storage diseases in medico-genetic centers. I. Mucopolysaccharidoses, mucolipidoses (review of the literature)]. PMID- 1705592 TI - [The activity of the glutathione-dependent enzymes of erythrocytes in chronic liver diseases in children]. AB - Activities of red cell glutathione-dependent enzymes, glutathione peroxidase (GP), glutathione reductase, and glutathione transferase (GT), were measured in 70 children suffering from chronic hepatitis and liver cirrhosis with various forms and activities of the conditions. Manifest changes in GP and GT activities were revealed. Measurements of GT activities are recommended for assessment of the liver process severity and for early detection of the liver detoxifying function stress. PMID- 1705593 TI - [Assessment of the role of the loop of Henle and the distal tubule in the formation of concentrated urine]. PMID- 1705595 TI - [Nuclear fragmentation of neutrophilic granulocytes in patients with angina]. AB - The informative value of the neutrophilic segmentation index (NSI) was estimated in 77 patients with primary streptococcal tonsillitis and in 40 normal young subjects. This index was found reduced at the peak of the disease in all the patients, this decrease depending on the disease severity. During convalescence the sooner NSI recovered, the milder was the disease course. This permits recommending NSI as an objective laboratory criterion permitting assessment of the severity of bacterial tonsillitis and the efficacy of its treatment. PMID- 1705594 TI - [Determination of the rate of utilization of glucose in a suspension of erythrocytes]. AB - The authors suggest a new method for measuring the rate of glucose utilization in a cell (red cell) suspension, making use of interferometry. This method allows an easy examination of glucose utilization kinetics in time with but 1000 cells. Thermostatic control of the sample in the cuvette of the device permits detecting the temperature effects on glucose metabolism kinetics and thus define the temperature optimal for the process. A correlation was revealed between the point of structure phase transition in the red cell membrane and the optimal temperature for glucose utilization within the range of physiologic temperatures. This method may be useful in medical, biochemical, physiologic, and hematologic studies. The relative error of the technique is 3 percent. PMID- 1705596 TI - [Determination of the erythrocyte count using a microcolorimeter]. AB - Red cell count is estimated with the use of a MKMF-1 microcolorimeter. 10 ml of Gower's solution are used; 0.02 ml of blood are added to this solution. Photometry of red cell suspension is carried out in a cuvette with a 0.5 cm optic route at lambda 610 nm. Microampermeter data are read from the optic density D scale. The D value is substituted into the formula: red cell count (RC) = K.D.10(6), where K coefficient is found experimentally by measuring the D and by estimating red cell count in microscopy, and is equal to 33.3. This formula permits estimating red cell count in 1 microliter of blood. PMID- 1705597 TI - [Lactoferrin and leukocytes in the blood of oncologic patients]. AB - Peripheral blood lactoferrin level and leukocyte count were measured by enzyme immunoassay in the sera of 288 normal subjects and 247 patients with malignant tumors of various localization. Normal lactoferrin level has been found 1055 +/- 191.0 ng/ml. In the patients it was changed either higher or lower, irrespective of the tumor site. This has led the authors to a conclusion on the acute phase pattern of the time course of lactoferrin level in oncologic patients. Elevation of lactoferrin level anticipated changes in leukocyte count. PMID- 1705598 TI - [Lyophilized plasma for use in calculating the activity of the prothrombin complex factors]. AB - Soviet lyophilized donor reference plasma (a reagent), intended for plotting a calibration graph or for calculating the activities of prothrombin factors (index) according to a formula, is stable for 12-14 months. It is fit for the aforesaid purposes with the blood plasma of both donors and patients suffering from various degrees of the studied procoagulant deficits. PMID- 1705599 TI - [Evaluation of single-stage methods of studying the activity of factor VIII]. AB - Accurate diagnosis of coagulopathies related to changed factor VIII activity, studies of specific activities of antihemophilic cryoprecipitate globulin used for hemostatic correction in patients, and monitoring of therapy efficacy are impossible without a reproducible, sensitive, standard, and available method. The authors have compared two single-stage methods developed by Soviet scientists: that by V. P. Baluda et al. and that by L. P. Papayan and P. V. Khrolova. Both methods have proved sufficiently sensitive and capable to detect factor VIII deep deficiency, but L. P. Papayan and P. V. Khrolova's modification was found higher reproducible, and therefore chosen for further investigation in order to standardize it. Besides the conditions of reproducing the method, reference plasma, substrate plasma deficient for factor VIII, and partial thromboplastin are to be standardized. PMID- 1705600 TI - [Methodologic and diagnostic aspects of the study of aminotransferase activity (review of the literature)]. PMID- 1705601 TI - [A photometric method of determining the activity of antithrombin III using a peptide coumarylamide substrate]. AB - Toz-Gly-Pro-Arg-4-methylcoumaryl-7-amide is recommended for antithrombin-III (AT III) photometry as a substrate. This substrate is cleaved by thrombin at 366 nm. AT-III photometry with this substrate is in accord with the requirements to methods used in clinical laboratory diagnosis, and the results of such measurement are in good correlation with the data of AT-III analysis with Chromozym-TH substrate. Use of photometry with methylcoumaryl substrates for measuring protease activities is more available than the traditional fluorometry. PMID- 1705602 TI - [Cytologic diagnosis of adenocarcinomas and rare forms of endometrial cancer]. AB - The paper sums up studies on cytomorphologic features of endometrial carcinoma and on possibility of its cytologic diagnosis and verification of histologic forms. 83 cases are analyzed. Cytologic studies have revealed endometrial carcinoma in 70 (84.3 percent) cases. In 63 (75.9 percent) cases the histologic form of the condition was correctly identified, including 19 with differentiated adenocarcinoma, 25 with moderately-differentiated adenocarcinoma, 5 of the 7 cases with poorly-differentiated adenocarcinoma, and 4 of the 5 cases with glandular-squamous-cell carcinoma. PMID- 1705603 TI - [Development of a bank of morphometric data on breast diseases using the Integral 2MT analyzer]. PMID- 1705604 TI - [Evaluation of allergy to conditionally pathogenic fungi]. AB - Sensitization to Candida albicans, Mucor, Cladosporium, Penicillium, Alternaria, Rhizopus nigricans was studied in vitro and in vivo in patients suffering from allergic diseases. The overwhelming majority of the positive responses to skin tests were of the immediate type. In parallel with the immediate responses, delayed ones were recorded in some patients. Positive responses in radioallergosorbent test and in estimation of the neutrophil injury index according to V. A. Fradkin did not always correlate with the skin test results. High titers of total IgE in some cases did not coincide with the titers of specific antibodies. PMID- 1705605 TI - [A comparative analysis of the proliferative response of blood mononuclears to mitogens]. AB - Comparison of two parameters of the peripheral blood mononuclear proliferative response to mitogens (stimulation index, absolute incorporation of radioactive label into proliferating cultures) has shown a discrepancy between the results obtained with the use of these parameters. PMID- 1705606 TI - [Screening by the method of radioimmunologic analysis]. AB - To simplify the technique of radioimmunoassay, rubber holders were used that held the test tubes when the solid phase was washed with flow water. Instead of hermetically sealing the test tubes during incubation of the test samples and labeled antiserum, the authors have placed the samples in a polyethylene bath with wet cloth. PMID- 1705607 TI - [The value of determining antibodies to DNA in patients with purulent diseases of the lungs]. AB - Anti-DNA antibody titers are elevated in the patients with purulent diseases of the lungs and antibodies to nDNA appear that are undetectable in normal subjects. These data correlate with the inflammatory process activity in the lungs, with the diameter of the decayed cavity, disease duration, and efficacy of therapy. Dynamic titration of antibodies to DNA (particularly nDNA) over the course of the disease appears to be an important prognostic test reflecting the stability of autoimmune impairments and the efficacy of the therapeutic scheme used in the treatment of patients with purulent diseases of the lungs. PMID- 1705608 TI - [The determination of neuraminidases in vibrios of the 01 and non-01 groups]. AB - Vibrio cholerae 01 and non 01 neuraminidase may be rapidly detected with the use of a synthetic 4-methylumbelliferyl-neuraminic acid substrate. Other vibrios and Enterobacteriaceae show no neuraminidase activity against this substrate. The method may be used with microorganism cultures grown in solid and liquid nutrient media. The time of analysis is 15-20 min. PMID- 1705609 TI - [Pseudomonas APS nutrient medium for the isolation and identification of Pseudomonas aeruginosa and Pseudomonas putida]. AB - Pseudomonas APS selective medium has been developed on the basis of a newly detected selective antibacterial action of oxaphenamide (p oxyphenylsalicylamide), a cholagogue. This medium permits a single-stage combined isolation and identification of P. aeruginosa after 16-24 hrs incubation of inoculated material at 42 degrees C. If the material is incubated at 35-37 degrees C, isolation of P. putida and P. aeruginosa is possible, that are differentiated by a nitroreductase microtest within 3 hrs. The sensitivity and selective ability of the developed medium are superior to other media manufactured in this country and commercial media with cetrimide and irgasan, produced abroad. PMID- 1705610 TI - [Immunodiagnosis of drug-resistant Mycobacterium tuberculosis]. AB - Comparative analysis of the results of studying M. tuberculosis drug resistance in the material from 223 patients with pulmonary tuberculosis by the bacteriologic method and of antibody production studied in 3 serologic tests (passive hemolysis, complement consumption, and indirect hemagglutination--PH, CC, and IHA, respectively) has shown the highest number of positive results in CC in the patients excreting streptomycin-resistant strains, whereas patients excreting isoniazid-resistant strains had the highest titers in IHA. The levels of complement-fixing and hemagglutinating antibodies accumulation depended on the type and degree of drug resistance and were not related to the form or phase of tuberculous process. PMID- 1705611 TI - [The identification of lactose-negative enterobacteria]. AB - Study of the morphologic and enzymatic characteristics of 10 strains of lactose negative enterobacteria, sent to the L. A. Tarasevich State Institute for Control and Standardization as B. paracoli in order to determine their taxonomic status in accordance with the 1974 and 1984 Bergey classifications has referred 9 of these strains to Hafnia alvei. PMID- 1705612 TI - [The use of Tetrahymena pyriformis infusoria to determine the level of toxemia in patients]. AB - Blood toxicity was estimated with the use of Tetrahymena pyriformis strain GL in patients with diffuse peritonitis. The time of the infusoria survival in the blood serum was a criterion of toxemia level. Infusoria life span was found to depend on peritonitis severity and serum concentration of medium-molecular peptides. PMID- 1705613 TI - [Diagnosis of pneumococcal otitis in children]. AB - The pneumococcal origin of purulent otitis was diagnosed in 74 (35 percent) of 211 children, in whom acute respiratory viral diseases or purulent otitis were previously diagnosed, by a combination of indirect immunofluorescence, cytobacterioscopic and culture methods. Use of indirect immunofluorescence and of special medium for isolation of atypical forms of the agent (L-forms and unbalanced growth forms) helped diagnose pneumococcal otitis in 47 (22.3 percent) more patients. PMID- 1705614 TI - [Optimization of bacteriologic studies of cholera]. AB - A colored medium is described, consisting of dry nutrient agar, 1 percent mannose, and 0.02 percent bromothymol blue. Use of this medium helps accelerate the process of vibrio growth and differentiate mannose-positive vibrios from mannose-negative ones. The described medium is simple to make and use, thus recommending it for bacteriologic laboratories. PMID- 1705615 TI - [Teratoblastoma of the lung in a child]. AB - A rare case of teratoblastoma located in the lungs is described. Pleural fluid was examined; its principal morphologic component was vascular tissue, which permitted a tentative diagnosis of a vascular tumor (possibly perithelioma); the presence of connective tissue and oxyphilic interstitial substance elements necessitated a differential diagnosis to rule out chemodectoma. The final diagnosis of teratoblastoma was made in pathohistologic examination. PMID- 1705616 TI - [Preparation of slides for work with adhering cells]. PMID- 1705617 TI - [Use of Soviet makes of chromatographic paper and paper backing for rapid tests in programs of screening for hereditary metabolic defects]. PMID- 1705618 TI - Somatostatin analogues inhibit angiogenesis in the chick chorioallantoic membrane. AB - The mechanism responsible for alterations in tumor growth following administration of somatostatin analogues is unknown. Somatostatin analogues, SMS 201-995 and RC-160, have demonstrated the potential to inhibit both tumor growth and vascularity, in vivo and in vitro. We hypothesized that SMS and RC-160 inhibit angiogenesis and this inhibition may alter tumor growth. To test this hypothesis, 2 mm methylcellulose disks containing concentrations of SMS 201-995 and RC-160 at 0, 0.5, 2.5, or 50 micrograms per disk, were implanted on the chorioallantoic membrane (CAM) of 6- to 7-day-old shell-less chick embryos. Inhibition of blood vessel growth in the region of the disk was visually assessed 24-36 hr following disk implantation and graded (0-4) based on the radius of the zone of inhibition from the center of the disk. The overall incidence of inhibition for the somatostatin analogues at concentrations of 0.5, 2.5, and 50 micrograms per disk was 13, 56, and 61% for SMS and 27, 49, and 68% for RC-160, respectively. Overall incidence of inhibition for the positive (inhibitory) control was 70.5% and those for buffer (negative) controls were 3-14%. Somatostatin analogues were associated in a dose-related fashion with both a greater percentage of inhibition of blood vessel growth and an increased grade of inhibition. Inhibition of angiogenesis may be a mechanism responsible for the tumor regression observed in vivo following SMS or RC-160 therapy. PMID- 1705619 TI - Molecular mechanisms of renin release. AB - Renin secretion from renal juxtaglomerular cells is controlled by the intrarenal blood pressure, sodium chloride load of the organism, sympathetic nerve activity, and a number of hormones. The mechanisms by which these parameters exert their effects are not well understood. However, it is reasonable to assume that they act via signal transduction systems, implying the role of second messenger molecules in the control of renin secretion. This article summarizes present knowledge about the cellular mechanisms of renin secretion. Moreover, current concepts about the mechanisms by which the blood pressure and the sodium chloride load could influence renin secretion are discussed. PMID- 1705620 TI - The renin-angiotensin system as a therapeutic target. Proceedings of an international symposium. Basel, Switzerland, October 29-30, 1989. PMID- 1705621 TI - Expression of the human angiotensinogen gene in human cell lines. AB - The human angiotensinogen gene consists of five exons interrupted by four introns and spans 12 kilobases. The gene is expressed in liver and HepG2 cells derived from human hepatoma. To examine whether the angiotensinogen gene is expressed in extrahepatic cells, RNAs from kidney and several human cell lines have been isolated and analyzed by Northern blot hybridization with the cloned gene as a probe. The mRNA for angiotensinogen was detected in human kidney and human glioblastoma (A-172) cells. To investigate the endogenous regulation of angiotensinogen gene expression, HepG2 cells were cultured in the presence of 3 aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase. The expression of the angiotensinogen gene was demonstrated to be completely suppressed by 3-aminobenzamide (10 mM). PMID- 1705622 TI - Angiotensin-converting enzyme: structural relationship of the testicular and the pulmonary forms. AB - Angiotensin-converting enzyme exists in two isozymic forms that are encoded by two mRNAs. Recently, the complete primary structures of the testicular isozymes from rabbit and human and of the pulmonary isozymes from mouse and human have been determined. We report here the cloning of a portion of the cDNA for the rabbit pulmonary isozyme by using the polymerase chain reaction. The sequence of this cDNA was identical to the sequence of the corresponding portion of rabbit testicular cDNA. This observation suggests that the two mRNAs are transcribed from the same gene. Comparison of the structures of the enzyme isolated from different species is useful for identifying the functional constraints on the evolutionary changes of its structure. PMID- 1705623 TI - Angiotensin-[1-7]: evidence for novel actions in the brain. AB - Structure-activity studies of angiotensin II (Ang II) have clearly established that the carboxyl-terminal phenylalanine is crucial for activation of angiotensin receptors associated with a broad range of effects including vasoconstriction, aldosterone release, and dipsogenesis. Thus, the heptapeptide angiotensin-[1-7] (Ang-[1-7]) has been classified as an inactive metabolite of the hormone Ang II. This assumption was seriously questioned in light of the report from our laboratory that Ang-[1-7] is the major metabolite of dog brainstem homogenates both in the absence and in the presence of angiotensin-converting enzyme inhibitors. These data raised the possibility of a functional role for Ang-[1-7] in the brain. Subsequent studies in the in vitro hypothalamoneurohypophysial system of the rat demonstrated that Ang-[1-7] is equipotent with Ang II in its activation of AVP release. Other Ang II-like actions have since been identified for Ang-[1-7] in the brain. For example, microinjection of Ang-[1-7] into brainstem nuclei of the rat promotes bradycardia and hypotension; in addition, Ang-[1-7] mimics the effect of Ang II in augmenting the intrinsic discharge rate of neurons within the vagal-solitary complex of the dog. Our laboratory has also provided evidence for synthesis and storage of Ang-[1-7] in the brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705624 TI - The angiotensin II receptor and the actions of angiotensin II. AB - Angiotensin II (Ang II) is a potent effector peptide of the renin-angiotensin system that exerts a wide variety of physiological actions on the cardiovascular, renal, endocrine, and central and peripheral nervous systems. Angiotensin exerts its actions by binding to specific receptors in the plasma membrane of various tissues. Structure-activity relationship studies and competition-binding experiments have identified a potency series of angiotensin analogues. Such studies have demonstrated that target organs display different preferences for Ang II and homologues such as Ang III and des-[Phe8] angiotensin II. Similarly, agents that normally are considered to be pure receptor antagonists for a given response (tissue) are full agonists in other tissues. Indirect evidence obtained from the above studies have led to the speculation that there are multiple angiotensin receptor subtypes among various tissues as well as within single cell types. Multiple mechanisms of signal transduction have been demonstrated for angiotensin. For example, depending on the effector organ, angiotensin stimulates phosphoinositide turnover and release of internal calcium, modulates voltage dependent calcium channels, directly activates calcium channels, and inhibits adenylate cyclase activity. Recently, the identification of selective, high affinity peptide and nonpeptide antagonists has resulted in further characterization of angiotensin receptors into distinct subtypes. In addition, dithiothreitol, an agent that reduces disulfide bridges, has been a useful tool in the characterization of angiotensin receptors as the subtypes apparently are not affected equally by this agent. However, further work needs to be performed to characterize angiotensin receptors with respect to heterogeneity, structure, transducing mechanisms, and physiological function. PMID- 1705625 TI - Biochemical characterization of two angiotensin II receptor subtypes in the rat. AB - Two angiotensin II receptor subtypes, A and B, have been described by means of specific and selective ligands (Biochem Biophys Res Commun 1989; 163:284-91). The present report describes the binding characteristics and the distribution of these subtypes in various rat tissues. Adrenal and uterus expressed both subtypes but in different proportions. Subtype B predominated in the adrenal glomerulosa (60%), whereas there was a greater proportion of subtype A in the medulla (70%). In uterus, subtype B was preferentially expressed (70%), and there was no difference in receptor distribution between muscle layers and serosa. Liver, kidney, and cultured aortic smooth muscle cells expressed only subtype B. In all of the tissues tested, the Ki values of various competing ligands were similar for each subtype expressed. It is proposed that subtype B is the vascular receptor. The function of subtype A, however, is still to be determined. PMID- 1705626 TI - Modulation of extracellular matrix by angiotensin II: stimulated glycoconjugate synthesis and growth in vascular smooth muscle cells. AB - A role for angiotensin II (Ang II) in the pathogenesis of hypertension and atherosclerosis was studied using cultured vascular smooth muscle cells from spontaneously hypertensive rats. Chronic exposure of vascular smooth muscle cells, cultured in the presence of 1% plasma-derived serum, to Ang II resulted in a dose-dependent stimulation in growth and incorporation of radiolabeled matrix precursors into extracellular matrix-associated glycoconjugate material. The hormone also stimulated the incorporation of [3H]glycine into extracellular matrix glycoproteins and proteoglycans synthesized by cultures rendered quiescent by maintenance on serum-free medium for 48 h prior to exposure to Ang II. This was negated in the presence of saralasin. In quiescent cultures, a single exposure to angiotensin induced a rapid induction of mRNA coding for the extracellular matrix glycoprotein thrombospondin. Similar results were obtained with cells maintained on medium containing 1% plasma-derived serum; however, the levels of induction were reduced by this procedure. This study demonstrated that Ang II was capable of stimulating both growth and matrix elaboration by cultured vascular smooth muscle cells. These observations are indicative of a pathophysiological role for the vasoconstrictor peptide, which may contribute significantly to the development of hypertension. PMID- 1705627 TI - The proliferative response to vascular injury is suppressed by angiotensin converting enzyme inhibition. AB - Smooth muscle cell (SMC) proliferation and formation of extracellular matrix in the intima of muscular arteries are major processes that can lead to vascular stenosis in arteriosclerosis or after coronary angioplasty. These processes are also seen in the proliferative response to balloon catheter-induced vascular injury of the rat carotid artery, and result in marked neointima formation by 14 days after catheterization. We have shown recently that the angiotensin converting enzyme (ACE) inhibitor cilazapril strongly suppressed this development of neointima. In this report, we show that the beneficial effects on neointima formation persist for at least 8 weeks after stopping treatment with cilazapril, and that continuous treatment may have additional inhibitory effects during the late phases of vascular remodeling after injury. To investigate further the possible mechanisms, we examined several vasoactive compounds in this model. Another ACE inhibitor of a different chemical class, captopril, reduced neointima formation as strongly as cilazapril (67 and 78%, respectively), but the calcium antagonist verapamil was not active as an inhibitor of neointima formation, despite similar lowering of blood pressure. Hydralazine and a new calcium antagonist, Ro 40-5967, partially suppressed neointima formation (36%, p less than 0.005 and 33%, p less than 0.05, respectively). In vitro, neither cilazapril nor its active metabolite, cilazaprilate, had any effect on SMC proliferation in response to serum or PDGF. To characterize further the role of angiotensin II (Ang II), we tested in cell culture the effects of Ang II and cilazaprilate on mRNA levels of several proteins potentially involved in regulating the SMC response.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705628 TI - An angiotensin with prolonged action and blood pressure--lowering properties. AB - [Sar1,Phe(Br5)8] angiotensin II (Br5Ang II) is a specific, quasi-irreversible antagonist of angiotensin II (Ang II) in vitro. In vivo, this compound is a very potent, Ang II-specific antagonist with a very long duration of action against Ang II-induced blood pressure (BP) increases. In the "low-sodium" dog, this compound induces a prolonged BP reduction during and after intravenous infusion at doses comparable to cilazapril, a potent ACE inhibitor. The physicochemical and pharmacokinetic behavior of this peptide was therefore assessed to understand and interpret the prolonged antagonistic and antihypertensive activity of this peptide. Binding studies using beef adrenocortical membranes indicated specific binding of Br5Ang and related analogues to Ang II receptors with a Kd of 1.41 x 10(-9) M against iodinated [Sar1, D-Phe8]Ang II, a standard radioligand antagonist. Iodinated Br5AngII exhibited a very high degree of nonspecific binding to the membranes. It had an octanol-water partitioning coefficient of log P of + 0.903, a coefficient 84-fold higher than for [125I][Sar1, D-Phe8]Ang II. Association kinetics of [125I]Br5Ang II were similar to the standard ligand [125I] [Sar1, D-Phe8]Ang II, but the half-life of dissociation was four times higher (60 vs. 15 min at 20 degrees C). Molecular modeling indicates a practically identical conformational behavior of both peptides, Br5Ang II and [Sar1, D-Phe8]Ang II but with an expanded hydration shell over the Br5 residue. It is concluded that the prolonged duration of action is due to the increased hydrophobicity of the peptide, which leads to a slow dissociation from the Ang II receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705629 TI - Binding characteristics and vascular effects of various angiotensin II antagonists. AB - Subtypes of angiotensin II (Ang II) receptors have been recently identified using specific ligands (see Whitebread et al. Biochem Biophys Res Comm 1989; 163:284 291). The present study compares the binding characteristics of different structural classes of Ang II receptor ligands in rat aortic smooth muscle cells (Ang IIB subtype) and in human uterus (Ang IIA subtype) and their effects on the constrictor response to Ang II in isolated rabbit aortic rings. Saralasin and [Sar1 Ile8]-Ang II displayed similar affinity for the two subtypes. In contrast, CGP 42112A bound with high affinity to the uterus (Ki 0.24 nM), but showed a low affinity for the aortic receptor (Ki 1,760 nM). Compound 89 displayed affinity for the aortic receptor only (Ki 26 nM) whereas Ex 169 recognized specifically the uterus receptor (Ki 310 nM). In rabbit aortic rings, saralasin, [Sar1 Ile8]Ang II, CGP42112A and compound 89 inhibited Ang II-induced contractions at concentrations similar to those required to bind to the Ang IIB receptor subtype. IC50s were 3, 0.7, 1,850, and 23 nM respectively. Ex 169 was ineffective in concentrations up to 100 microM. There was a highly significant correlation between inhibition of Ang II-induced contraction in aortic rings and binding to smooth muscle cells. This correlation does not exist with human uterus. Our results indicate that the Ang IIB receptor subtype is responsible for vascular contractions. Antagonists of this vascular receptor subtype are potential antihypertensive agents. PMID- 1705630 TI - Comparison of the acute hypotensive effects of renin inhibition, converting enzyme inhibition, and angiotensin II antagonism in rats. AB - The purpose of this study was to compare the acute hypotensive efficacy of different types of inhibitor of the renin-angiotensin system. A renin inhibitor (RI), CGP 44,099 A, a converting enzyme inhibitor (CEI), enalaprilat, a peptidic angiotensin II (Ang II) antagonist, [Sar1, Ile8]Ang II (P-Ang IIA), and a nonpeptidic Ang II antagonist devoid of agonistic properties, 2-butyl-4-chloro-1- ([2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl)-5- (hydroxymethyl)imidazol (NP-Ang IIA), were administered intravenously to sodium-depleted rats (SDRs), renal hypertensive rats (two-kidney, one-clip) (RHRs), and spontaneously hypertensive rats (SHRs). The four compounds were all effective in lowering blood pressure (BP) in SDRs and RHRs. The maximum hypotensive response observed within 30 min of administration was similar for all four compounds (approximately 30 mm Hg in SDRs and 60 mm Hg in RHRs). In SHRs, the P-Ang IIA induced a pressor response whereas the RI, CEI, and NP-Ang IIA lowered BP to a similar extent (approximately 15 mm Hg). Pretreatment with the CEI completely prevented the hypotensive response to RI in SHRs, and vice versa. These observations indicate that the principal mechanism by which converting enzyme inhibitors lower BP after acute administration in these rat models is by inhibition of the formation of Ang II. The pressor response to the P-Ang IIA in SHRs is probably a consequence of its partial agonistic properties. Renin inhibitors and nonpeptidic Ang II antagonists, devoid of agonistic properties, promise to be effective antihypertensive agents similar to the CEIs. PMID- 1705631 TI - Control of renal function by intrarenal angiotensin II in the dog. AB - The purpose of this study was to determine if conversion of angiotensin I to angiotensin II (Ang II) within the kidney is important in the control of renal function following sodium depletion. Infusion of a short-acting angiotensin I converting enzyme (ACE) inhibitor, BPP5a, into the left kidney at a dose of 30 micrograms/kg/min failed to alter significantly the pressor response to Ang I administered in a femoral vein. Mean arterial pressure and renal function in the contralateral noninfused kidney also remained unchanged to intrarenal infusion of the ACE inhibitor into the left kidney. Thus, the ACE inhibitor used in these studies can be effectively localized to the renal circulation when infused intrarenally at 30 micrograms/kg/min. Intrarenal infusion of the ACE inhibitor at 30 micrograms/kg/min into sodium-restricted dogs failed to alter renal blood flow (RBF) significantly. However, the glomerular filtration rate (GFR), urine volume, and sodium excretion all increased significantly in response to intrarenal ACE inhibition. Intrarenal Ang II generation therefore appears to play a physiologically important role in the control of GFR, urine volume, and sodium excretion but not RBF following sodium depletion. The increase in GFR following intrarenal infusion of the ACE inhibitor may suggest that Ang II is formed mainly at glomerular or preglomerular sites. PMID- 1705632 TI - Detrimental and beneficial effects of converting enzyme inhibitors on the kidney. AB - It has been recognized recently that even minor elevations in blood pressure contribute to progression of renal failure and that antihypertensive treatment retards progression. Despite suggestive experimental data, no unequivocal clinical evidence is available to date to document that converting enzyme inhibitors (CEIs) are superior to alternative antihypertensive agents with respect to halting progression. CEIs undoubtedly reduce albuminuria, independent of their effect on systemic blood pressure, and this is related to alteration of glomerular permselectivity. Recent experimental data suggest that growth processes in damaged kidneys are an important aspect of progression. It is a fascinating perspective, but yet unproven, that CEIs interfere with these processes. PMID- 1705633 TI - Clinical pharmacology of enalkiren, a novel, dipeptide renin inhibitor. AB - Enalkiren (A-64662), a potent, dipeptide renin inhibitor, mimics the transition state of the human renin substrate, angiotensinogen. Enalkiren has been shown to produce dose-related suppression of plasma renin activity (PRA) and angiotensin II when administered intravenously. Doses of enalkiren of less than 0.1 mg/kg induced little hemodynamic response in normotensive and hypertensive volunteers despite marked suppression of PRA. However, at doses of 0.3 and 1.2 mg/kg, enalkiren produced significant, dose-related decreases in systolic and diastolic blood pressure (BP) in hypertensive patients, and the BP response was enhanced by pretreatment with hydrochlorothiazide. The effects of enalkiren on PRA and BP are prolonged despite its relatively short elimination phase plasma half-life (1.6 h). Persistent pharmacologic activity without evidence of tachyphylaxis was demonstrated during 1 week of treatment in hypertensive patients. The observed dissociation between suppression of PRA and BP response and the recruitment of dose-related BP decrements, despite complete suppression of PRA, are unexplained phenomena. The results of clinical trials with enalkiren are encouraging, and suggest that renin inhibitors may be safe, useful therapeutic agents in the management of hypertension. PMID- 1705634 TI - The human renin gene in transgenic mice. AB - We have obtained three transgenic mouse lines carrying the human renin gene. All three lines have been shown to transmit the introduced human gene to progeny. In the present study, we have demonstrated that the human renin gene was expressed and correctly spliced in the kidneys of both transgenic parents and progeny. PMID- 1705635 TI - In vivo and in vitro expression of myeloid antigens on B-lineage acute lymphoblastic leukemia cells. AB - The expression of myeloid antigens has been extensively examined using two-color analysis in 43 children with B-lineage acute lymphoblastic leukemia (ALL). On pre culture cells, CD33 expression was frequently observed in CD19+, CD10- B precursor ALL, and CD14 was expressed only on the cells from B-precursor ALL expressing CD19, CD10 and CD20, and B-ALL. After 2 or 3 days of culture without TPA, CD13 emerged on the cells from 21 of 29 patients irrespective of the presence or the absence of fetal calf serum in the culture. Of four patients with CD10+ B-precursor ALL, which showed no expression of CD13 after culture, two had T-cell associated antigens. Whereas the addition of TPA to the culture enhanced the expression of CD13 on the cells from acute non-lymphocytic leukemia (ANLL), TPA reduced the expression of this antigen on B-precursor cells. These findings suggest that the regulatory mechanism of CD13 expression may be different between B-precursor ALL and ANLL. Co-culture with cycloheximide mostly abrogated the induction of CD13, suggesting that CD13 expression was mainly dependent on de novo protein synthesis. PMID- 1705636 TI - Granulocyte colony-stimulating factor, not granulocyte-macrophage colony stimulating factor, co-operates with retinoic acid on the induction of functional N-formyl-methionyl-phenylalanine receptors in HL-60 cells. AB - The interaction of colony-stimulating factors (CSF) and retinoic acid (RA) in the proliferation and differentiation of HL-60 cells was examined. Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulated the proliferation of HL 60 cells in a dose-dependent manner at concentrations of 0.01-100 ng/ml; however, the proliferation due to GM-CSF was suppressed by 100 nM RA. Granulocyte colony stimulating factor (G-CSF) slightly stimulated the proliferation of HL-60 cells at concentrations above 10 ng/ml. Neither G-CSF nor GM-CSF alone induced 12-o tetra-decanoyl-phorbol-13-acetate (TPA)- or N-formyl-methionyl-phenylalanine (FMLP)-stimulated nitro-blue tetrazolium (NBT) reduction at concentrations of 0.01-100 ng/ml. G-CSF induced TPA- and FMLP-stimulated NBT reduction in the presence of 100 nM RA, but GM-CSF induced only TPA-stimulated NBT reduction. RA in addition to G-CSF synergistically increased FMLP binding to HL-60 cells, accompanied by increased NBT reduction in response to FMLP. RA in addition to GM CSF markedly increased FMLP binding to HL-60 cells more than that induced by RA alone, but the combined treatment with RA and GM-CSF did not increase FMLP stimulated NBT reduction more than that induced by RA alone. The results suggest that G-CSF stimulates RA-induced morphological and functional differentiation of HL-60 cells, but the differentiation-enhancing effects of GM-CSF are limited, whereas the growth-stimulating effect of GM-CSF on HL-60 cells is greater than that of G-CSF. PMID- 1705637 TI - c-myc gene expression in a leukemic T-cell line bearing a t(8;14) (q24;q11) translocation. AB - We have examined the expression of the c-myc protooncogene in human T-cell leukemic KE-37R cells carrying a t(8;14) (q24;q11) translocation. The breakpoint on chromosome 8 is located at 2.2 kb downstream of c-myc exon 3 and the 3' part of the TcR-alpha gene (14q11) has been juxtaposed to c-myc. Our results showed that the steady-state levels of c-myc RNA transcripts were increased and the P1/P2 ratio of c-myc promoter utilization did not change, indicating that preferential utilization of P2 was maintained in the rearranged gene. High levels of electrophoretically normal p64 and 67 c-myc proteins were detected and both products kept their instability. In addition, transcription from promoter P0 was not detectable. Our results suggest that the activation of the gene is likely to result from its juxtaposition to the enhancer element of the TcR-alpha gene located downstream of the Ca region which stimulates constitutive synthesis of normal c-myc transcripts from the rearranged allele. PMID- 1705638 TI - Positive and negative effects of tumor necrosis factor on colony growth from highly purified normal marrow progenitors. AB - The effects of recombinant human tumor necrosis factor alpha (TNF alpha) on colony growth were studied using highly enriched progenitor cells from normal human bone marrow. Supplementation of TNF to culture resulted in a dose-dependent suppression of granulocyte colony-stimulating factor (G-CSF) induced granulocytic colony formation and also erythropoietin (Epo) induced erythroid burst formation. However, the number of erythroid bursts, stimulated by interleukin-3 (IL-3) plus Epo, increased when TNF was added at comparable concentrations. Further, TNF enhanced eosinophilic colony growth induced by IL-3 or granulocytic-macrophage colony-stimulating factor (GM-CSF). In GM-CSF cultures TNF (100-1000 U/ml) also induced granulocytic and macrophage colonies. The addition of neutralizing antibodies against G-CSF, GM-CSF, or interleukin-6 (IL-6) to culture did not abrogate the observed effects of TNF, so that stimulation of myeloid colony growth was unlikely to result from the secondary induction of G-CSF or GM-CSF. TNF therefore exerts favourable effects on hematopoietic progenitors responsive to the more primitive colony-stimulating factors (IL-3, GM-CSF) and potent negative effects on precursors reactive to the single lineage G-CSF and Epo. These contrasting effects of TNF suggest that TNF, when available to marrow progenitors at similar tissue concentrations, may drive hematopoiesis within the progenitor cell compartment into selected directions. PMID- 1705639 TI - Reduced response to granulocyte colony-stimulating factor in W/Wv and Sl/Sld mice. AB - The response of W/Wv and Sl/Sld mice to recombinant granulocyte colony stimulating factor (rG-CSF) was investigated. Purified rG-CSF was injected every day for 1 week in doses up to 1000 micrograms/kg. Both untreated Sl/Sld and W/Wv mice initially showed an ordinary number of neutrophils and then an increase in neutrophils in response to rG-CSF injections. However, the effective dose of rG CSF was much higher than that for normal mice. An increase in splenic CFU-GM was observed in both types of mice receiving 1000 micrograms/kg of rG-CSF, regardless of the reported defects in their hemopoietic system. These results indicate that W/Wv and Sl/Sld mice show a reduced response to exogenous rG-CSF and that a large amount of exogenous rG-CSF may allow an increase in neutrophils in certain hemopoietic defects. PMID- 1705640 TI - [Corticosteroid therapy in polymyalgia rheumatica and temporal arteritis is only suppressive]. PMID- 1705641 TI - [Which nasal spray is preferred by patients?]. PMID- 1705642 TI - Point mutation in meningococcal por A gene associated with increased endemic disease. AB - The por A gene, which encodes expression of meningococcal class 1 outer membrane protein, responsible for antigenic subtype specificity, has been cloned and sequenced in an isolate of Neisseria meningitidis (B:15:P1.7,16) from a patient in the Gloucester area with meningococcal meningitis. Comparison of the sequence with that of the equivalent gene from the P1.7,16 reference strain reveals a point mutation which generates a single aminoacid change in the epitope responsible for P1.16 specificity. Monoclonal antibodies with P1.16 specificity do not react with synthetic peptides that correspond to the altered epitope, and do not promote complement-mediated bactericidal killing of the isolate. Analysis of other strains shows widespread distribution of infections due to B:15:P1.7,16 meningococci with the altered epitope (P1.16b) in England and Wales. PMID- 1705643 TI - Biological therapy for metastatic renal cell cancer. PMID- 1705644 TI - Interstitial lymphocytic myocarditis in Whipple's disease. PMID- 1705645 TI - Exploiting angiogenesis. PMID- 1705646 TI - Rapid diagnosis of malaria by fluorescence microscopy. PMID- 1705647 TI - Cutaneous hypersensitivity reactions due to thiacetazone in HIV-1 seropositive patients treated for tuberculosis. AB - The effects of the human immunodeficiency virus (HIV) on tuberculosis management was investigated in 227 patients initially treated with a regimen containing streptomycin, isoniazid, and thiacetazone (STH). 93 of these 227 were HIV seropositive. 60 patients, of whom 18 were HIV-seropositive, received a regimen consisting of streptomycin, isoniazid, rifampicin, and pyrazinamide (SHRZ) in the initial phase, and thiacetazone and isoniazid (TH) in the continuation phase. Cutaneous hypersensitivity reactions occurred in 22 of 111 (20%) HIV-seropositive patients, and in 2 of 176 (1%) HIV-seronegative patients (RR = 18, 95% CI 4.4-76, p less than 10(-7]. During the first 8 weeks of treatment 18 reactions occurred among the 93 HIV-seropositive patients on STH, whereas no reaction occurred in 17 HIV-seropositive patients during the initial phase of SHRZ/TH (p = 0.04). None of the 18 HIV-seropositive patients with cutaneous reactions who were subsequently challenged with isoniazid reacted, nor did any of the 10 tested with streptomycin, but 6 of the 7 challenged with thiacetazone reacted. 3 patients (all HIV-positive and with toxic epidermal necrolysis) died as a result of the cutaneous reaction. These results have major implications for tuberculosis control programmes in Africa. PMID- 1705648 TI - Antibodies to endothelial cells. PMID- 1705649 TI - Low-dose aprotinin for reduction of blood loss after cardiopulmonary bypass. PMID- 1705650 TI - Isoelectric focusing of amniotic fluid alpha-fetoprotein to improve diagnosis of neural tube defects. PMID- 1705651 TI - Haloperidol treatment increases D2 dopamine receptor protein independently of RNA levels in mice. AB - Haloperidol, administered to mice in their drinking water, produced a 21% increase in striatal D2 dopamine receptor density after seven days of continuous exposure. The steady-state D2 receptor RNA prevalence was unaffected by this treatment, yet the RNA coding for preproenkephalin was elevated, as expected. These data indicate that the homologous up-regulation of dopamine receptor density by antipsychotic drugs proceeds by mechanisms other than changes in RNA abundance. PMID- 1705652 TI - Nuclear retinoic acid receptors: cloning, analysis, and function. PMID- 1705653 TI - The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome. AB - In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain anar-14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination. PMID- 1705654 TI - DNA sequence of the Doc retroposon in the white-one mutant of Drosophila melanogaster and of secondary insertions in the phenotypically altered derivatives white-honey and white-eosin. AB - We analysed the structure of the white locus of Drosophila melanogaster in a family of related white mutants. The white-one mutant has bleach white eyes, and a Doc transposable element is inserted into the promotor region of the white locus. The DNA sequence of this Doc insertion was determined, and showed it to be closely related to other Drosophila melanogaster retroposons such as the I factor and the F, G and jockey elements. There are two long open reading frames, which encode a putative nucleic acid binding protein and a putative reverse transcriptase, respectively. Two independent, partially pigmented derivatives were analysed by cloning sequences from this region. In white-honey a transposable element of the retroviral class, B104, is inserted within the Doc element. In white-eosin there is an insertion within the Doc element of a 190 bp sequence that appears to be a member of a novel family of transposable elements. This pogo element is of the same structural class as the Drosophila melanogaster P and hobo elements. These data are consistent with the hypothesis that the Doc retroposon cannot excise, and that, for the white-one mutation, flies with altered phenotypes are most often generated by the insertion of additional transposable elements. PMID- 1705655 TI - cis modification of the steroid 21-hydroxylase gene prevents its expression in the Y1 mouse adrenocortical tumor cell line. AB - The Y1 mouse adrenocortical tumor cell line retains the ability to synthesize and secrete steroids, but does not express steroid 21-hydroxylase (C21) and, therefore, does not produce 21-hydroxylated steroids. In this investigation the mechanisms underlying the loss of C21 activity in the Y1 cell line were explored. A 9-kilobase BglII fragment containing the C21 gene was cloned from the Y1 genome. This genomic clone directed the synthesis of C21 transcripts and 21 hydroxylated steroid products when transfected back into the Y1 cell line. As determined by restriction endonuclease digestions with MspI and HpaII, enzymes that distinguish between unmethylated and methylated CCGG sites, the endogenous C21 gene was extensively methylated in Y1 adrenal cells and in cells from other mouse tissues that do not normally express this gene. In contrast, the C21 gene was hypomethylated in primary cultures of mouse adrenal cells which normally synthesize large amounts of C21. The cloned C21 gene transfected into Y1 cells initially was unmethylated, but became extensively methylated with prolonged culture of the cells; prolonged culture of these transfectants also resulted in a loss of C21 expression. Loss of C21 expression in Y1 transfectants, however, temporally preceded the extensive methylation of the transfected C21 gene. Furthermore, treatment of Y1 cells with 5-azacytidine caused a demethylation of the endogenous C21 gene, but did not result in the recovery of C21 expression. These results indicate that Y1 cells contain a functional C21 gene that has been silenced by a reversible cis-modification event.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705656 TI - Transcriptional regulation of rat insulin-like growth factor-binding protein-1 expression by growth hormone. AB - The low mol wt insulin-like growth factor-binding protein (IGFBP-1) originally isolated from amniotic fluid has been considered to be GH independent. In this report we have examined the effect of GH on hepatic IGFBP-1 expression in the hypophysectomized rat. Using a rat IGFBP-1 cDNA probe we now demonstrated that hepatic IGFBP-1 expression is up-regulated in the GH-deficient rat. In addition, using ligand blotting, an increase in the abundance of a 30-kDa [125I]IGF-I-BP was detected in both hepatic extracts and serum from hypophysectomized rats. After a single ip injection of GH, the IGFBP-1 transcription rate was reduced within 30 min to the levels seen in the sham-operated control rats. Similarly, hepatic IGFBP-1 mRNA abundance was reduced after both acute GH injection and chronic GH treatment for 8 days. These data demonstrate that IGFBP-1 expression is inversely regulated by GH. PMID- 1705657 TI - Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus. AB - The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus. PMID- 1705658 TI - The expression pattern of an insulin-like growth factor (IGF)-binding protein gene is distinct from IGF-II in the midgestational rat embryo. AB - Insulin-like growth factor-II (IGF-II), the predominant form of IGF in fetal and neonatal serum and tissues, is found in vivo complexed with IGF-binding proteins. One of these binding proteins, IGFBP-2, is present at high levels in fetal rat plasma and binds both IGF-I and IGF-II with high affinity. We here have used in situ hybridization to compare the distribution of IGFBP-2 mRNA with that of IGF II mRNA in embryonic day 13.5-15 rat embryos. The spatial patterns of IGF-II and IGFBP-2 expression in the fetal trunk were distinct and, in general, nonoverlapping. Most mesoderm derivatives that express IGF-II at high levels contained little, if any, IGFBP-2 mRNA. Instead, IGFBP-2 mRNA was expressed at high levels in many cell types derived from ectoderm and endoderm. The expression of IGFBP-2 mRNA in the central nervous system (CNS) during this developmental period was examined in particular detail. The three most prominent sites of IGFBP 2 expression in the CNS were comprised of cells with nonneuronal phenotypes: 1) the epithelium of the choroid plexus, a tissue that produces cerebrospinal fluid; 2) the floor plate, an area that can guide axonal outgrowth from commissural neurons of the spinal cord in vitro; and 3) the infundibulum, the progenitor of the posterior pituitary that is believed to influence differentiation of the adjacent intermediate pituitary.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705659 TI - Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. AB - Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B1 to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment. PMID- 1705660 TI - Insusceptibility of Cockayne syndrome-derived lymphocytes to plasminogen activator-like protease induction by ultraviolet rays and its abolition by interferon. AB - Protease induced by ultraviolet rays (UV) has been extensively investigated in human cells. Plasminogen activator-like protease (PA) activity was induced soon after UV irradiation in peripheral lymphocytes derived from healthy donors. In contrast, UV-irradiated lymphocytes derived from Cockayne syndrome (CS) cases did not show marked protease inducibility. Epstein-Barr (EB) virus-transformed CS lymphoblastoid cells were also characterized by insusceptibility to UV-induction of PA. However, when CS-derived cells were treated with a human interferon (HuIFN) preparation prior to irradiation, induction of PA activity was detected, irrespective of the kind of HuIFN (alpha or gamma). The results indicate the possibility of abnormal PA metabolism in CS-derived cells. PMID- 1705661 TI - In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites. AB - We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene. PMID- 1705662 TI - Polymyositis mediated by T lymphocytes that express the gamma/delta receptor. AB - BACKGROUND: The invasion and destruction of nonnecrotic muscle fibers by CD8+ cytotoxic T cells is considered a hallmark of polymyositis. In the cases of polymyositis reported so far, the autoinvasive CD8+ T cells expressed the common form of T-cell receptor for the recognition of antigen, the so-called alpha/beta T-cell receptor. We describe a 69-year-old man with polymyositis mediated by CD4 , CD8- T cells expressing the recently discovered, uncommon gamma/delta T-cell receptor. METHODS: We used immunofluorescence or immunoperoxidase techniques to study frozen sections of muscle from our patient, who had mild weakness of cervical and proximal limb muscles, and from control patients with polymyositis, inclusion-body myositis, dermatomyositis, or granulomatous myopathy with monoclonal antibodies against T-cell-related antigens (CD2, CD3, CD4, CD8, and gamma/delta T-cell receptor), B cells (CD22), major histocompatibility complex (MHC) and MHC-related antigens (MHC Class I, CD1a, CD1b, and CD1c), and the 65-kd heat-shock protein. The membrane contacts between the autoinvasive cells and the sarcolemma were investigated by electron microscopy. RESULTS: In the patient described here, but not in 28 others with inflammatory myopathies, myriad gamma/delta T cells surrounded and invaded nonnecrotic muscle fibers. All muscle fibers were highly reactive for MHC Class I antigen and the 65-kd heat-shock protein. Treatment with prednisone improved the clinical and histologic findings. CONCLUSIONS: Polymyositis can be mediated by gamma/delta T cells. This new form of polymyositis appears to be highly responsive to steroids. PMID- 1705663 TI - Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. AB - Epstein-Barr virus (EBV), a human herpesvirus, establishes a persistent asymptomatic infection of the circulating B-lymphocyte pool. The mechanism of virus persistence is not understood but, given the limited lifespan of most B cells in vivo, it seems most likely that EBV-infected cells must gain access to the long-lived memory B-cell pool. Here we show in an in vitro system that EBV, through expression of the full set of eight virus-coded 'latent' proteins, can protect human B cells from programmed cell death (apoptosis), the deletion mechanism which normally restricts entry into memory. We have found that EBV positive Burkitt's lymphoma (BL) cell clones retaining the original tumour cell phenotype and expressing only one of the virus latent proteins, the nuclear antigen EBNA 1, are extremely sensitive to apoptosis; in this respect they resemble the tumour's normal cell of origin found in the germinal centres of lymphoid tissue. By contrast, isogenic BL cell clones which have activated expression of all eight EBV latent proteins are resistant to the induction of apoptosis. The EBV latent proteins should therefore be seen not just as activators of B-cell proliferation but, perhaps more importantly, as mediators of enhanced B-cell survival. PMID- 1705664 TI - Lymphocyte homing. Skin-seeking memory T cells. PMID- 1705665 TI - Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase. AB - Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis. PMID- 1705666 TI - ELAM-1 is an adhesion molecule for skin-homing T cells. AB - Endothelial cell leukocyte adhesion molecule-1 (ELAM-1) has been described as an inducible endothelial cell-adhesion molecule for neutrophils, and is believed to have a key role in the extravasation of these cells at sites of acute inflammation. Here we report that ELAM-1-transfected COS cells also bind a unique skin-associated subset of circulating memory T cells defined by the expression of the cutaneous lymphocyte-associated antigen. T cells expressing this antigen bind at least as well as neutrophils to expressed ELAM-1, whereas other lymphocytes in the peripheral blood bind poorly, or not at all. Immunohistological survey of chronically inflamed tissue specimens revealed that vascular expression of ELAM-1 occurs at cutaneous sites in preference to noncutaneous sites. We conclude that at sites of chronic inflammation, ELAM-1 may function as a skin vascular addressin, a tissue-selective endothelial cell-adhesion molecule for skin-homing memory T lymphocytes. PMID- 1705667 TI - Activation-independent binding of human memory T cells to adhesion molecule ELAM 1. AB - The induction of an ensemble of adhesion molecules on endothelial cells by inflammatory cytokines is likely to be crucial to the differential migration of T lymphocyte subsets into inflammatory sites. Two molecular pathways involving the VLA-4 and LFA-1 integrins are known to mediate T-cell adhesion to activated endothelium. Here we show that a third pathway involving the rapidly inducible endothelial cell-surface adhesion molecule ELAM-1 contributes to the binding of resting CD4+ T cells to IL-1-induced human endothelial cells. All three pathways contribute to the greater adhesion to endothelium of memory T cells than naive T cells. There are two unique features of T-cell adhesion to purified ELAM-1: first, ELAM-1 exclusively mediates adhesion of memory T cells; second, memory T cell binding to ELAM-1 is independent of acute activation events that regulate integrin-mediated adhesion. Thus, ELAM-1 may be of primary importance in the initial attachment of memory T cells to inflamed endothelium in vivo and to the preferential migration of memory T cells into tissue and inflammatory sites. PMID- 1705668 TI - [Intramedullary tumors--surgical factors determining the outcome]. AB - The recent advances in the surgery of intramedullary tumors are due to the strategy of a total removal by microsurgical techniques and the early diagnosis by MRT. 84 patients with intramedullary tumors with a long-term follow-up up of more than 20 years were retrospectively analysed so that subgroups e.g. macroscopic versus microscopic, palliative procedures versus whether or not new technology like the ultrasonic aspirator and the CO2-Laser were used were included. PMID- 1705669 TI - [A case of cranial and intracranial metastasis from testicular seminoma]. AB - The case presented is of a 44-year-old man with skull and intracranial metastasis of seminoma. He was operated on for a testicular tumor at 41 years of age. Pathologically, it was pure seminoma. Coronal CT scan showed tumor invasion of the subcutaneous vault, and of the epidural and intraparenchymal region of the right parietal region. We treated him with a combination of surgical excision, radiation and PVB chemotherapy. He was neurologically disease-free for ten months after being discharged. However, he then succumbed to liver metastasis of seminoma. Seminoma mainly metastasizes via the lymph stream. CNS metastasis of seminoma is only 0.7% in Japan. We would like to stress that prophylactic chemotherapy would be essential even after patients get remission from CNS metastasis of seminoma. PMID- 1705670 TI - Serotonin-, substance P- or leucine-enkephalin-containing neurons in the midbrain periaqueductal gray and nucleus raphe dorsalis send projection fibers to the central amygdaloid nucleus in the rat. AB - By a double-labeling method combining the retrograde tracing of horseradish peroxidase and the immunocytochemical technique, serotonin-, substance P- or leucine-enkephalin-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) and the nucleus raphe dorsalis (DR) of the rat were found to send projection fibers to the central amygdaloid nucleus bilaterally with an ipsilateral dominance. These PAG neurons were chiefly distributed in the ventrolateral PAG subdivision and the ventral parts of medial PAG subdivision at the middle and caudal levels of PAG. PMID- 1705671 TI - Localization of neuropeptide receptor mRNA in rat brain: initial observations using probes for neurotensin and substance P receptors. AB - The expression of receptors for neurotensin and substance P was examined in rat brain and spinal cord using in situ hybridization with synthetic oligonucleotide probes. Strong hybridization signals for neurotensin receptor mRNA were observed over neurons i.a. in the diagonal band, medial septal nucleus, nucleus basalis magnocellularis, suprachiasmatic nucleus, supramammillary area, substantia nigra and ventral tegmental area. Strong hybridization signals for substance P receptor mRNA were observed over scattered, large neurons in the striatum, and in the spinal cord over neurons in the dorsal horn, the area around the central canal and preganglionic autonomic neurons. Thus, discrete neurons in several brain regions express a G-protein-coupled receptor with which endogenous neurotensin and substance P may interact. PMID- 1705672 TI - Auditory projections to the inferior colliculus of the rat are present by birth. AB - Despite extensive literature on the anatomical organization of the adult mammalian auditory system, the ontogeny of internuclear connections is still obscure. We studied the postnatal development of afferent brainstem connections to the inferior colliculus in rat pups using WGA-HRP and DiI as tracers. At birth, connections to the inferior colliculus from the cochlear nuclei and nuclei of the superior olivary complexes and the lateral lemnisci are present. During successive development, there are no obvious changes in the quantity of labeled neurons or the basic labeling pattern. These results provide evidence that all input connections from auditory brainstem nuclei to the rat's inferior colliculus are established prenatally. PMID- 1705673 TI - Development of substance P, Leu-enkephalin and serotonin profiles in the lateral geniculate nuclear complex of albino rat. AB - Immunohistochemical studies for analysing the development of the profile of two peptides--substance P (SP) and Leu-enkephalin (Leu-ENK), and serotonin (SER)- have been conducted on the lateral geniculate nuclear (LGN) complex of albino rats at gestation day 18 and various postnatal age periods. SP immunoreactivity is found to increase from 1 day postnatal (DPN) up to 20 DPN and decrease thereafter, whereas the SER and Leu-ENK-immunoreactive fibres and terminals seen as occasional fibres at 1, 5, and 10 DPN are better visualized from 20 DPN and gradually increase up to 40 DPN. The possible role and significance of the changes seen in these putative neurotransmitters/neuromodulators with development are discussed. PMID- 1705674 TI - The neurotoxicity of zinc in the rat hippocampus. AB - Intrahippocampal injections of zinc chloride (5-10 nmol) caused a discrete lesion in the rat hippocampus, involving all neuronal perikarya. In addition to the necrosis, the lesion was also characterized by a decrease in staining of the neuropil, the presence of pyknotic neurons, and occasionally infarction. Pathological changes occurred within 8 h of an injection, and neuronal loss, as judged by the loss of Nissl staining, was complete within 24 h. On the other hand, the loss of acidophilic staining of the neurons was more gradual, as acid fuchsin staining was still present in neurons in the periphery of the damaged area 4 days later. In comparison with an excitotoxic lesion, glial infiltration into the damaged area was minimal, even up to 3 weeks later, suggesting that some glial cell toxicity also occurred. PMID- 1705675 TI - The neurotoxicity of ouabain, a sodium-potassium ATPase inhibitor, in the rat hippocampus. AB - Intrahippocampal injection of 1 nmol ouabain, a sodium/potassium (Na+,K(+) )ATPase inhibitor, produced a necrotic lesion within 4 days, characterised by a massive invasion by foaming macrophages. A lower dose of ouabain (0.1 nmol) produced a more discrete lesion of all groups of neuronal perikarya in the hippocampus, with only a minimal degree of glial infiltration. The neuronal perikaryal death produced in the subicular, CA1 and CA2 regions was only partially decreased by intraperitoneal injections of the anticonvulsants diazepam and MK-801; these drugs were without effect in the CA3 or hilar interneuronal regions. At neither dose of ouabain was there any indication of neuronal loss in brain regions outside the hippocampus, typically produced by prolonged seizure activity. It is suggested that ouabain has a two-fold action, a release of toxic acidic amino acids and a prolonged depolarization of neurons leading to osmolysis or calcium necrosis. PMID- 1705676 TI - Differential occurrence and distribution of galanin in adrenal nerve fibres and medullary cells in rodent and avian species. AB - The presence and distribution of galanin (GAL) in adrenal glands of rodent and avian species was investigated by light microscopic immunohistochemistry. GAL immunoreactivity was found in all medullary cells of guinea pig, duck and chicken adrenals. In contrast, only a subpopulation of medullary cells stained for GAL in Phodopus (Djungarian hamster) while the neuropeptide was completely missing in chromaffin cells of rat and pigeon. In rat, guinea pig and pigeon, GAL immunoreactive nerve fibres were frequent in subcapsular regions and sparse in deeper cortical layers and in the chromaffin tissue. In contrast, only very few GAL fibres were found in Phodopus and no GAL fibres were observed in the adrenal glands of duck. In the chicken adrenal gland, fibres containing GAL were numerous throughout the organ and occurred in close vicinity to both steroidogenic as well as catecholaminogenic cells. The striking differences in the presence of GAL positive cells and fibres are more pronounced between species within the rodent or avian group, respectively, than between the different vertebrate orders. The hitherto unknown and surprising variability of GAL expression and distribution in adrenal glands of various species suggests species-dependent functional (autocrine, paracrine and/or endocrine) roles of GAL in the neuroadrenal axis. PMID- 1705677 TI - High- and low-threshold calcium currents in neurons acutely isolated from rat sensorimotor cortex. AB - Neurons were isolated by papain treatment and trituration of the frontoparietal cortex of 14 to 28-day-old rats. Whole cell voltage clamp revealed a slowly inactivating high-threshold Ca2+ current, activated positive to -45 mV, and a transient low-threshold Ca2+ current, activated positive to -65 mV. The high threshold current was more sensitive to block by Cd2+ and the low-threshold current was more sensitive to block by Ni2+. Replacement of Ca2+ by Ba2+ increased the high-threshold current and reduced the low-threshold current. The high-threshold current was enhanced by Bay K 8644 and reduced by nimodipine and omega-conotoxin. The low-threshold current was also reduced by nimodipine but was insensitive to Bay K 8644 and omega-conotoxin. The properties of the currents were consistent with different underlying Ca2+ channel types. PMID- 1705678 TI - Galanin receptors in the post-mortem human brain. Regional distribution of 125I galanin binding sites using the method of in vitro receptor autoradiography. AB - The distribution of putative receptors for the peptide galanin was studied in the normal post-mortem human brain by using 125I-galanin (0.5 nM) in combination with in vitro receptor autoradiography. Specific binding of 125I-galanin was found in a large number of brain areas throughout the neuraxis. Highest binding densities occurred in the basal forebrain and hypothalamus, while the basal ganglia, major parts of the thalamus and the tectum were found to be poor in binding sites. All cortical areas harboured 125I-galanin binding, and in the visual cortex a laminated pattern was present. In the hippocampus, 125I-galanin binding occurred in layer 2 of the entorhinal cortex, in the uncus and in the hippocampal-amygdala area. In the brain-stem, 125I-galanin binding was found in serotoninergic noradrenergic cell groups as well as in the reticular formation and in the parabrachial nuclei. Galanin receptors may, thus, mediate the response of galanin in numerous structures in the human brain. PMID- 1705679 TI - Chemoreceptor responses to substance P, physalaemin and eledoisin: evidence for neurokinin-1 receptors in the cat carotid body. AB - Substance P (SP) belongs to a group of peptides called tachykinins. Biological effects of SP are mediated by tachykinin receptors that have been classified as neurokinin-1 (NK-1), NK-2 and NK-3 subtypes. The aim of the present study is to elucidate the tachykinin receptor subtype(s) that mediate the excitatory effects of SP in the carotid body. For this purpose, we compared the carotid body responses elicited by SP with that of physalaemin and eledoisin. In other tissues, physalaemin exhibits equi or greater potency at NK-1 receptors and eledoisin exerts its effects more on NK-2 and NK-3 subtypes compared to SP. Experiments were performed on eight cats that were anaesthetized, paralyzed and artificially ventilated with room air. Close carotid body administration of SP and physalaemin produced dose-dependent augmentation of the chemoreceptor afferent activity. Chemoreceptor discharge, however, was unaffected by eledoisin. Compared to that by SP, the magnitude of excitation produced by physalaemin was the same at lower doses but significantly greater with the highest dose (100 nmol). The time course of the response induced by physalaemin, however, was the same as that by SP. The present results demonstrate that in the carotid body physalaemin is also either equi or relatively more potent than SP, whereas eledoisin has no effect on the chemoreceptor discharge. It is suggested that stimulation of the carotid body by SP is mediated by NK-1 but not NK-2 or NK-3 receptors. PMID- 1705680 TI - Interactions between emotional stress due to fear and hypovolemic stimuli in the control of vasopressin secretion in rats. AB - Interactions between emotional stress due to fear and hypovolemic stimuli on vasopressin secretion were studied in rats. Intraperitoneally injected dextran did not significantly change plasma osmolality and arterial blood pressure but increased blood hemoglobin and plasma vasopressin level. An i.v. infused physiological solution reversed these changes. Emotional stress due to fear acquired by learning suppressed plasma vasopressin level in dextran-injected rats. Emotional stress due to fear produced by low-frequency footshocks also suppressed the increased plasma vasopressin level. These results suggest that emotional stress due to fear interacts with afferent neural signals originating from cardio-vascular volume receptors in the control of vasopressin secretion. PMID- 1705681 TI - Substance P-like immunoreactive neurons in the nucleus tractus solitarii of the rat send their axons to the nucleus accumbens. PMID- 1705682 TI - Changes in expression of mRNA specific for cell adhesion molecules (L1 and NCAM) in the transected peripheral nerve of the adult rat. AB - The relative amounts of transcripts specific for the adhesive molecule L1 and the neural cell adhesion molecule (NCAM) in the distal stump of transected rat sciatic nerves were determined by quantitative Northern blot analysis during the first four weeks after injury. Two days after operation, transcript levels were approximately 50% lower in the lesioned than in the unsevered control nerve reaching maximum values 2 and 3 times over control for L1 and NCAM mRNAs, respectively, after 3 weeks. In contrast, expression of the mRNAs specific for the myelin-associated glycoprotein (MAG) and for the major myelin protein of the peripheral nervous system, P0, dropped to 10% or less of the control after nerve injury and did not exceed 30 and 20%, respectively, 3 weeks after the lesion. The up-regulation of L1- and NCAM-specific mRNAs in distal nerve stump parallels the induction of L1 and NCAM protein expression by Schwann cells of lesioned nerves as revealed by previous immunocytochemical studies and may reflect the Schwann cells' capacity to recapitulate development and to promote axonal regrowth. PMID- 1705683 TI - Frontal cortical projections from the suprageniculate nucleus in the rat, as demonstrated with the PHA-L method. AB - Frontal cortical projections from the rat suprageniculate nucleus (SG) were investigated by an anterograde tracing using Phaseolus vulgaris-leucoagglutinin (PHA-L). After PHA-L injection into the SG, labeled terminals were found in the frontal and temporal cortical regions. Labeled terminals in the frontal cortex were almost exclusively localized in the medial agranular area, and those in the temporal cortex were mainly localized in the primary and associational auditory areas. The labeled terminals in cortices were distributed predominantly in layers I, III and IV. The results suggest that ascending information through the SG projects to the medial agranular area in the frontal cortex as well as to the temporal cortex. PMID- 1705684 TI - Retrograde neuronal labelling by E. coli enterotoxin subunit B. AB - The present communication reports that subunit B of Escherichia coli heat-labile enterotoxin (LTB) can be utilized as a powerful tracer in neuroanatomical studies. LTB was injected into the limb muscle of the chick, or into the superior cervical ganglion of the rat. Sections were processed with an immunohistochemical technique using an antibody against LTB. After the LTB injection into the muscle, retrogradely labelled motoneurons were found: the entire extent of dendritic arborizations appeared labeled. After the LTB injection into the ganglion, virtually all of the preganglionic neurons were retrogradely labelled in the rat spinal cord. PMID- 1705685 TI - Effects of promoting patients' participation in self-care on postoperative recovery and satisfaction with care. AB - The effectiveness of patients' participation in self-care aimed at expediting the rate of recovery from surgery and increasing satisfaction with care received was tested with adult patients undergoing pyelolithotomy and nephrolithotomy. Forty subjects participating in the study were randomly assigned to either an experimental (n = 20) or a control group (n = 20). Patients in the experimental group participated in their self-care through nurse-patient interaction in addition to the usual care received in the setting. Results of the study indicated that patients in the experimental group had significantly less pain sensation and distress, used fewer analgesics, ambulated more, had fewer complications, and had higher satisfaction with care than patients in the control group. Since the experimental intervention was based on Orem's and King's theories, these findings support the value of application of these two nursing theories in practice. PMID- 1705686 TI - Identification of the antigenic proteins of Cowdria ruminantium. AB - Immunoblotting of Cowdria ruminantium proteins with sheep or bovine antiserum identified 2 antigenically conserved proteins, one being an immunodominant 31 kDa and the other a minor 27 kDa protein. These proteins are present in the electrophoretic profiles of the Welgevonden, Ball 3 and Kwanyanga stocks and are recognized by sheep antiserum to the Welgevonden, Ball 3, Kwanyanga, Mali, Comoro, Breed, Germishuys, Kumm and Mara stocks and by bovine antiserum to the Welgevonden stock of C. ruminantium. The stocks did not reveal identical or unique antigenic properties which could explain differences in pathogenicity and cross-immunity observed amongst the various stocks of C. ruminantium. PMID- 1705687 TI - The v-src oncogene blocks the differentiation of a murine myeloid progenitor cell line and induces a tumorigenic phenotype. AB - We have investigated the ability of the v-src oncogene to block the differentiation of the murine myeloid progenitor cell line 32D cl3. In response to granulocyte-colony stimulating factor (G-CSF), 32D cl3 cells are induced to differentiate into mature granulocytes (Valtieri et al., 1987). In contrast, no differentiation was observed following G-CSF treatment of 32D cl3 cells infected with a murine retrovirus carrying the wild-type v-src oncogene. Furthermore, cells infected with a v-src temperature-sensitive (ts) mutant did not differentiate at the permissive temperature, however, at the nonpermissive temperature G-CSF induced granulocytic differentiation. Differentiation of 32D cl3 cells infected with ts WP31A (ts LA31A src gene inserted into amphotropic murine leukemia virus 4070A; Anderson et al., 1987) occurred with the same kinetics as uninfected 32D cl3 cells. Temperature-shift experiments indicate that after 72 hours of treatment with G-CSF at the nonpermissive temperature, approximately half of the 32D cl3 cells infected with ts WP31A virus become committed to differentiation. Prior to that time, activation of v-src by shifting the cells to the permissive temperature resulted in the presence of only undifferentiated blast cells after six days in culture. In contrast to normal 32D cl3 cells, cells infected with the wild-type v-src were tumorigenic when injected into nu/nu Swiss mice. Lesions appeared in the spleen, liver, kidney, lungs and lymph nodes following subcutaneous injection. Growth factor-independent cells were recovered from the tumor, spleen, bone marrow and a lymph node of tumor bearing nude mouse. Analysis of the proviral integration site by inverse polymerase chain reaction (PCR) demonstrated that the tumor cells were of donor cell origin. PMID- 1705688 TI - Expression of the chronic myelogenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 dependent myeloid cell line. AB - We have studied the effect of a replication-defective murine retroviral vector expressing the chronic myelogenous leukemia associated oncoprotein p210bcr/abl in murine IL-3 dependent myeloid 32D C13(G) cells. This cell line can be induced to differentiate along either the granulocytic or monocytic lineages thus permitting an independent assessment of the effect of p210bcr/abl on growth and differentiation. Cells expressing p210bcr/abl displayed a complete non-autocrine abrogation of IL-3 dependence and an enhanced response to an activity in FBS which is not IGF-I or IGF-II. During the first few generations following infection with the bcr/abl vector, cells became larger with an increased fraction of cells in G2/M and monocyte/macrophage markers were expressed. Four cytoplasmic proteins phosphorylated in response to IL-3 in the parental cell line with apparent molecular weights of 98, 70, 62, and 52 Kd were amongst those constitutively phosphorylated in p210bcr/abl expressing cells. These results suggest that the functional substitution of IL-3 by p210bcr/abl is due to constitutive activation of proteins involved in IL-3 signal transduction. Alterations of cell differentiation, cell cycle and growth which cannot be attributed to IL-3 like effects indicate that p210bcr/abl has pleiotropic effects involving several other pathways of cellular regulation. PMID- 1705689 TI - Structure and expression of the murine hck gene. AB - The hck gene encodes a src-like protein-tyrosine kinase that is expressed predominantly in cells of myeloid origin. To examine the molecular basis of hck expression, we have cloned and characterized the murine hck gene and defined a set of transcriptional regulatory sequences. Transcripts from the hck gene have heterogeneous 5' ends, with one major and several minor transcriptional start sites. The promoter lacks a TATA-like sequence, but has three Sp-1 and two AP-2 binding sites. Constructs containing 2.5Kb or 0.6Kb of sequence 5' to the major transcription start site were capable of directing expression of a reporter gene transfected into fibroblasts. Expression of the former construct, but not the latter, was inducible with LPS. We conclude that LPS induction of hck transcripts, previously demonstrated in myeloid cells, results from the presence of an LPS-responsive element located within the hck promoter. PMID- 1705690 TI - Transgenic mice carrying a murine amylase 2.2/SV40 T antigen fusion gene develop pancreatic acinar cell and stomach carcinomas. AB - The mouse pancreatic amylase Amy-2.2 gene was fused to the structural gene for SV40 T antigen, and 51 independent transgenic founder mice carrying the fusion gene were generated. The majority of the founders and 100% of their offspring in the derived transgenic lines developed pancreatic acinar cell carcinomas and stomach carcinomas. Transgenic animals also had a high incidence of metastatic carcinomas in other tissues. The development of stomach carcinomas was unexpected because the Amy-2.2 promoter was not previously known to be expressed in stomach. Northern blot analyses and ribonuclease protection assays showed that Amy-2.2 is expressed in stomach, at approximately 0.05% of the level in pancreas. Expression of the fusion gene in stomach, therefore, appears to represent a previously unrecognized activity of the Amy-2.2 promoter. Examination of young transgenic mice demonstrated that preneoplastic lesions were present in pancreas and stomach before the development of neoplastic lesions in either tissue, consistent with the notion that stomach neoplasms are primary neoplasms and not metastases from the pancreas. Ribonuclease protection assays demonstrated that properly initiated large T and small t antigen transcripts were present in pancreas and stomach during tumorigenesis. T antigen protein was also detected in pancreas and stomach by immunohistochemistry. A time course for tumorigenesis was established for several transgenic mouse lines in which distinct types of lesions appeared at predictable times. This study provides the basis for future analysis of the role of SV40 T antigen in the progression and maintenance of pancreatic and stomach carcinomas. PMID- 1705691 TI - Neuropathic pain. PMID- 1705692 TI - The nature of opioid responsiveness and its implications for neuropathic pain: new hypotheses derived from studies of opioid infusions. AB - In recent years, the observation that the response of patients to opioid drugs may be influenced by properties inherent in the pain or pain syndrome, such as its pathophysiology, has evolved into the belief that certain types of pain, e.g., neuropathic pains, may be unresponsive to these drugs. This concept has important implications for both clinical practice and basic understanding of opioid mechanisms. We critically evaluate opioid responsiveness, particularly as it relates to neuropathic pain, and propose a clinically relevant definition and a paradigm for its investigation. The paradigm is illustrated by analgesic responses to opioid infusion in 28 patients with neuropathic pains and by a detailed presentation of the pharmacokinetic and pharmacodynamic relationships in one of these patients, whose central pain responded promptly to an infusion of hydromorphone. From this analysis, we hypothesize that (1) opioid responsiveness in man can be defined by the degree of analgesia achieved during dose escalation to either intolerable side effects or the occurrence of 'complete' or 'adequate' analgesia; (2) opioid responsiveness is a continuum, rather than a quantal phenomenon; (3) opioid responsiveness is determined by a diverse group of patient characteristics and pain-related factors, as well as drug-selective effects; and (4) a neuropathic mechanism may reduce opioid responsiveness, but does not result in an inherent resistance to these drugs. Given the complexity of factors contributing to opioid responsiveness and the observation that outcome cannot be reliably predicted, opioids should not be withheld on the assumption that pain mechanism, or any other factor, precludes a favorable response. Both the clinical use of opioids and paradigms to investigate opioid responsiveness should include dose escalation to maximally tolerated levels and repeated monitoring of analgesia and other effects. PMID- 1705693 TI - Prolonged relief of neuralgia after regional anesthetic blocks. A call for further experimental and systematic clinical studies. AB - Thirty-eight consecutive patients with neuralgia after peripheral nerve injury were treated with one or two series of peripheral local anesthetic blocks. All patients experienced an initial total relief of ongoing pain for 4-12 h. Evoked pain (hyperalgesia or allodynia), which occurred in 17 patients, was blocked simultaneously with the spontaneous pain. In 18 patients the analgesia outlasted the conduction block and there was a period of complete pain relief of 12-48 h in 13 patients and of 2-6 days in the other 5. In 8 patients there was a second phase of analgesia of 4 h to 6 days duration occurring within 12 h of pain recurrence. Thus, mono- or biphasic prolonged complete analgesia occurred in 25 out of 38 patients. A prolonged analgesia may be the result of a central action of the local anesthetic at the spinal level after intra-axonal incorporation and centripetal axoplasmic transport. To test this hypothesis, an experimental study with [3H]lidocaine was performed in 6 rats. The radioactive local anesthetic was injected into one hind limb foot with the other side serving as a control. Tissue samples from the peripheral nerve, nerve root and the lumbosacral spinal cord segment were analyzed for radioactivity using a scintillation counter technique at various time intervals after the [3H]lidocaine injection. There was a low grade of activity in all samples and no difference between the test side and the control side. Thus these experiments provided no evidence in support of this hypothesis. Various alternative peripheral and central mechanisms are discussed. Further studies specifically directed to these alternatives and with longitudinal controls are prompted. PMID- 1705694 TI - A brief psychotic reaction during the administration of intravenous 2 chloroprocaine for the treatment of chronic pain. PMID- 1705695 TI - Developmental changes in agonist-mediated colonic smooth muscle contraction in the rabbit. AB - We studied smooth muscle strips from rabbit distal colon to determine age-related changes in length-tension properties and agonist-mediated contraction. Strips from newborn (1-d-old) and weanling (11-wk-old) rabbits were oriented to measure isometric tension in longitudinal muscle. Active tension comprised 47 +/- 4 and 75 +/- 5% of the total tension in the newborn and weanling, respectively. Total and active tensions in the weanling were greater than in the newborn (p less than 0.001). Although the potencies for bethanechol were similar, the maximal response was nearly 9-fold greater in weanlings (6900 +/- 292 mN/cm2) versus newborns (753 +/- 112 mN/cm2), p less than 0.001. Maximal stress increased with age for bethanechol, high extracellular potassium, substance P, neurokinin A, cholecystokinin octapeptide, bombesin, and serotonin. ED50 for bethanechol, substance P, neurokinin A, and bombesin did not change with age. Serotonin was 12 times more potent in newborns versus weanlings (p less than 0.05). In contrast, cholecystokinin octapeptide was five times less potent in newborns (18.6 nM versus 3.4 nM, respectively, p less than 0.05). Substance P-induced contractions were inhibited partially by atropine. We conclude that length-tension properties of longitudinal colonic smooth muscle differ, and responses to agonists increase with age. PMID- 1705696 TI - Insulin-like growth factors I and II and their binding proteins in rat milk. AB - IGF-I and -II are peptide growth factors that may be important contributors to the growth-promoting properties associated with milk. IGF in extracellular fluids, including serum and milk, are carried by specific high-affinity binding proteins (IGFBP). In this study, the levels of IGF-I and -II in rat serum and milk were quantified by specific RIA, and the IGFBP were characterized using Western ligand blotting and autoradiography throughout lactation. The levels of IGF-I in both milk and maternal serum decreased during lactation. Serum IGF-I decreased from 743 +/- 187 micrograms/L on d 1 to 391 +/- 106 (mean +/- SD) on d 21 of lactation, and milk IGF-I levels fell from 30 +/- 10 to 13 +/- 8 micrograms/L. Levels of IGF-II in serum and milk were much lower than IGF-I, and were unaffected by lactation. In maternal serum, several IGFBP were identified: IGFBP-3, which migrates as four glycosylated bands with apparent Mr from 38 to 42 kD and one to two nonglycosylated bands with apparent Mr of 28 to 29 kD, and an IGFBP with an apparent Mr of 24 kD. In milk, IGFBP-3, the 24-kD binding protein, and a third IGFBP with an apparent Mr of 29 kD were identified. Treatment of milk and serum with Endoglycosidase F reduced the four glycosylated IGFBP-3 bands (38 42 kD) to two bands with apparent Mr of 35 and 32 kD. In rat milk, but not adult rat serum, the IGFBP with an apparent Mr of 29 kD was immunoprecipitable by an antibody that recognizes IGFBP-2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705697 TI - Passive tactile stimulation effects on the muscle tone of hypotonic, developmentally delayed young children. AB - A pilot study was performed with 13 children, 1 to 4 yr. of age, to explore an heretofore uninvestigated area. The effect of passive tactile stimulation was an increase in muscle activation and hence the muscle tone of 13 hypotonic, developmentally delayed children. PMID- 1705698 TI - Cellular and segmental distribution of Ca2(+)-pump epitopes in rat intestine. AB - We used a monoclonal antibody (5F10) specific for the human erythrocyte plasma membrane Ca(++)-pump to demonstrate the presence and distribution of Ca(++)-pump epitopes in rat intestine. In paraffin embedded tissue sections, antibody 5F10 binds to epitopes in the basolateral membranes of absorptive cells in rat duodenum and portions of jejunum but not ileum. Western blot analysis of intestinal mucosal proteins with antibody 5F10 shows binding of antibody to major bands of Mr approximately 135,000 and Mr approximately 72,000, and to lesser bands of Mr approximately 125,000 and Mr approximately 27,000. This pattern was seen in mucosal homogenates of rat duodenal and jejunal cells and to a lesser extent in ileal cells. The Mr approximately 135,000 band corresponds to the molecular weight of Ca(++)-pumps in other tissues. The other bands correspond in size to known proteolytic fragments of the Ca(++)-pump. Slot-blot analysis of nitrocellulose immobilized mucosal homogenates shows binding of 5F10 to be greatest in duodenum and least in ileum. Ca(++)-transport studies by the everted gut sac technique show a correlation between vitamin D induction of active Ca(++) transport and the segmental distribution of Ca(++)-pump epitopes. PMID- 1705699 TI - Cyclic-AMP-dependent phosphorylation modulates the stereospecific activation of cardiac Ca channels by Bay K 8644. AB - Voltage-gated Ca channels have been reported to be regulated by membrane potential, phosphorylation and binding of specific agonists or antagonists such as dihydropyridines. We report here evidence that cyclic AMP (cAMP) modulates the activation of Ca-channel current by the dihydropyridine agonist Bay K 8644. Bay K 8644 (racemate) alone induces a primary voltage-dependent, potentiating effect on peak current amplitude and accelerates the current decay. In contrast, in the presence of cAMP activators, we observed a striking slowing of the decay in addition to the increase in peak current. The agonist (-)-Bay K 8644, but not the antagonist (+)-Bay K 8644, when applied in combination with cAMP, forskolin or isoproterenol, mimics the effect of the racemate. We have interpreted the results presented here in respect of a cAMP-dependent modulation of Bay K 8644 effects on cardiac Ca-channel currents. It may open the new perspective that dephosphorylated and phosphorylated Ca channels have distinct pharmacology. PMID- 1705700 TI - The role of blood flow and/or muscle hypoxia in capillary growth in chronically stimulated fast muscles. AB - Capillary supply, the proportion of oxidative fibres and blood flow were studied in fast rat muscles (tibialis anterior, TA, and extensor digitorum, EDL) made ischaemic by ligation of the common iliac artery, in chronically stimulated muscles and in ischaemic chronically stimulated muscles. Stimulation was carried out for 6 h/day at 10 Hz (three periods of 2 h with 90-120-min intervals between stimulations) for 10-12 days using electrodes implanted in the vicinity of the lateral popliteal nerve. Blood flow (measured by radioactive microspheres) was 3.62 +/- 0.52 ml.100 g-1.min-1 at rest and 78.4 +/- 14.6 ml.100 g-1.min-1 (mean +/- SEM) during isometric contractions at 4 Hz. Ischaemic muscles had significantly lower blood flow at rest as well as during contractions (72 +/- 14% and 25 +/- 4% of the values in contralateral muscles respectively). Stimulated muscles had significantly higher flow than contralateral control muscles during contractions; stimulated ischaemic muscles had normal blood flow at rest, but the increase in flow during contractions was limited to a similar extent to that in ischaemic muscles alone. Of all anatomically present capillaries (staining for alkaline phosphatase in frozen sections) the capillary/fibre ratio increased by 36% in stimulated tibialis anterior, but was not significantly different from control muscles in stimulated ischaemic TA and was even lower than in control muscles in stimulated ischaemic EDL. The proportion of fast oxidative fibres (estimated on the basis of histochemical staining for myosin ATPase and succinate dehydrogenase) increased from 53.2 +/- 3.2% in normal EDL to 82.0 +/- 2.3% in chronically stimulated EDL and to 100% in chronically stimulated ischaemic muscles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705701 TI - [Kaposi's sarcoma in AIDS. 70 cases]. AB - Although Kaposi's sarcoma is the most frequent of malignant diseases associated with AIDS and one of the first recognized manifestations of the syndrome, its management has not yet been standardized. This study concerns 70 AIDS patients with Kaposi's sarcoma followed up in the author's hospital department between june 1985 and december 1989. Most of these patients were homosexual or bisexual males. The mean time elapsed between the finding of a positive HIV test and the discovery of Kaposi's sarcoma was 15 +/- 3.2 months. Systematic evaluation disclosed visceral lesions in 50 percent of the patients. Chemotherapy was used in 55 cases, interferon-alpha in 23 cases and radiotherapy in 13 cases. The presence of visceral lesions clearly reduced survival time, although deaths were mainly due to opportunistic infections. Atypical forms of Kaposi's sarcoma sometimes make its diagnosis difficult, but the main problem is that of treatment, in view of the underlying immunodepression. The authors advocate the setting up of multicentre trials aimed at determining the best therapeutic approach in these patients. PMID- 1705702 TI - Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene. AB - The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. We have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, two dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45 degrees C and their thermal survivals were determined by colony-formation assay, we found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress. PMID- 1705703 TI - High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine. AB - The whey acidic protein (WAP) is a major milk protein in mice, rats, and rabbits but has not been found in milk of livestock including swine. To determine whether mammary gland regulatory elements from the WAP gene function across species boundaries and whether it is possible to qualitatively alter milk protein composition, we introduced the mouse WAP gene into the genome of swine. Three lines of transgenic swine were analyzed, and mouse WAP was detected in milk from all lactating females at concentrations of about 1 g/liter; these levels are similar to those found in mouse milk. Expression of the corresponding RNA was specific to the mammary gland. Our results suggest that the molecular basis of mammary-specific gene expression is conserved between swine and mouse. In addition the WAP gene must share, with other milk protein genes, elements that target gene expression to the mammary gland. Mouse WAP accounted for about 3% of the total milk proteins in transgenic pigs, thus demonstrating that it is possible to produce high levels of a foreign protein in milk of farm animals. that it is possible to produce high levels of a foreign protein in PMID- 1705704 TI - Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end. AB - We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses of these sequences show (i) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical origin, (ii) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (iii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. These data differentiate HCV from the flaviviruses and indicate a closer evolutionary relationship of HCV with the pestiviruses. However, HCV also appears to be substantially different from other known pestiviruses. These data are consistent with the assignment of HCV to a separate viral genus. PMID- 1705705 TI - Heritable retroviral transgenes are highly expressed in chickens. AB - This report describes expression of heritable reticuloendotheliosis virus (REV) vector ME111 in 20 independent lines of transgenic chickens. The results are strikingly different from studies of Moloney virus in transgenic mice, where restricted expression of inherited proviruses has led to their use primarily as insertional mutagens rather than general agents for gene transfer. In contrast, the REV ME111 provirus is actively transcribed in a variety of tissues from transgenic chickens, is expressed from transcriptional control elements present in the long terminal repeat of the provirus, and codes for active neomycin phosphotransferase II. The REV vector system as applied to the chicken represents a departure from the long-established paradigm of retroviral transgenes in mice and provides a new approach to the study of avian biology. PMID- 1705706 TI - Endonucleolytic degradation of puf mRNA in Rhodobacter capsulatus is influenced by oxygen. AB - The formation of pigment-protein complexes in facultatively photosynthetic bacteria is regulated by the oxygen tension in the culture. It is shown that the degradation of some mRNA species encoding components of the photosynthetic apparatus is affected by oxygen. The puf mRNA segment, encoding the pigment binding proteins of the reaction center, and the 0.5-kb puc mRNA species, encoding pigment-binding proteins of the light-harvesting LHII antenna complex of Rhodobacter capsulatus were degraded more rapidly under high oxygen tension than under low oxygen tension. Studies on strains having deletions or insertions in the puf operon indicate that rate-limiting endonucleolytic cleavage in the reaction center coding region of the polycistronic puf mRNA was influenced by growth conditions. However, other mRNA segments, for which exonucleolytic degradation was postulated to be rate-limiting, decayed with the same rate under either high or low oxygen tension. Likewise, the degradation of the puhA mRNA, the cycA mRNA, and the cytfbc mRNA was found to be independent of the oxygen tension in the culture. The data strongly suggest that specific mRNA sequences or structures are responsible for the observed oxygen effect on mRNA stability. PMID- 1705707 TI - Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport. AB - We find that half of the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in the squid (Loligo pealei) giant axon is cytoplasmic and that this species of G protein is intermediate in size between the two forms present in axolemma. This G protein is transported toward synaptic terminals at 44 mm/day. Moreover, these data are consistent with there being two additional steps leading to the maturation of G proteins: (i) association with and transport on intracellular organelles and (ii) modification at the time of transfer to the plasmalemma resulting in a molecular weight shift. Since the other two components of G protein-mediated signal transduction pathways, receptors and effector enzymes, are known to be delivered to the synaptic terminals by fast axonal transport, our findings introduce the possibility that these three macromolecules are assembled as a complex in the cell body and delivered together to the plasma membrane of the axon and synaptic terminals. PMID- 1705708 TI - Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. AB - Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthesized by bovine aortic endothelial cells and subcellular fractions thereof was assayed by its stimulating effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL 6 cells). The release of EDRF/NO by intact endothelial cells could be stimulated with bradykinin, thrombin, or ADP and was abolished in Ca2(+)-free medium. When subcellular fractions were analyzed, some EDRF/NO-synthesizing activity was found in the cytosolic fraction, but most of the activity was associated with the particulate fraction. Both enzyme activities required L-arginine and NADPH for EDRF/NO synthesis, both were inhibited by NG-nitro-L-arginine and NG-methyl-L arginine, and hemoglobin or methylene blue abolished the effect of the EDRF/NO produced by both enzymes. Both enzymes were highly sensitive to Ca2+; the major increase in activity occurred between 100 and 500 nM free Ca2+. Exposure of the particulate enzyme activity to 1 M KCl removed 39% of the protein and reduced total activity by 46%, but the activity was restored when exogenous calmodulin (CaM) was added. Further KCl washes caused little further loss of protein or EDRF/NO synthase activity. The KCl-washed particulate enzyme could be solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The CaM antagonists calmidazolium and trifluoperazine as well as the CaM-binding protein calcineurin inhibited the EDRF/NO synthesis by both the cytosolic and the particulate enzyme. These effects were partially reversed with exogenous CaM. Partial purification of the cytosolic and solubilized particulate enzymes by affinity chromatography on adenosine 2',5'-bisphosphate-Sepharose resulted in EDRF/NO synthase activities dependent on exogenous CaM. We conclude that endothelial cells contain both cytosolic and particulate enzymes that synthesize EDRF/NO. Both enzymes are regulated by free Ca2+ and, at least in part, by CaM. PMID- 1705709 TI - Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart. AB - Five distinct K+ channel cDNA molecules (RK1 to RK5) were cloned from either rat heart or rat aorta cDNA libraries. Four of the channels, RK1 to RK4, are similar or identical to Shaker-like K+ channels previously identified in rat brain cDNA libraries. Major differences among RK1 to RK4 exist in the amino- and carboxyl terminal regions and in amino acids representing potential extracellular sequence between the S1 and S2 hydrophobic domains. RK5 encodes a unique channel of 490 amino acids having six hydrophobic domains but only five basic residues in the putative voltage-sensing domain. Unlike RK1 to RK4, RK5 is a rat homologue of the Drosophila Shal family of K+ channels, which have not been previously described in mammals. Although RK5 mRNA is present in cardiac atrium and ventricle, it is most abundant in brain. RK1, RK2, and RK3 transcripts are predominantly found in brain but are present also at lower levels in other tissues, such as heart and aorta. RK2 is absent from skeletal muscle whereas RK1 and RK3 are present in this tissue. RK4 mRNA is ubiquitous in electrically excitable tissue, being present at comparable levels in atrium, ventricle, aorta, brain, and skeletal muscle. The cloning of RK5 confirms the presence in mammals of all four Drosophila K+ channel families: Shaker, Shab, Shaw, and Shal. PMID- 1705710 TI - Expressional potency of mRNAs encoding receptors and voltage-activated channels in the postmortem rat brain. AB - The stability and integrity of mRNAs encoding neurotransmitter receptors and voltage-activated channels in the postmortem rat brain was investigated by isolating poly(A)+ mRNA, injecting it into Xenopus oocytes, and then examining the expression of functional neurotransmitter receptors and voltage-activated channels in the oocyte membrane by electrophysiological recording. This approach was also used to assess the stability of mRNAs in brains that were incubated in oxygenated mammalian Ringer's solution for various lengths of time and from brains that were freshly frozen and then thawed at room temperature. Oocytes injected with mRNA from up to 21-hr postmortem brains gave large agonist- and voltage-activated responses, indicating that mRNAs encoding neurotransmitter receptors and voltage-activated channels are relatively stable in postmortem brain tissue. In contrast, oocytes injected with mRNA from brains incubated in Ringer's solution exhibited smaller responses, and oocytes injected with mRNA from tissue that was frozen and then thawed displayed very small or undetectable responses. Northern blot analysis using a nucleic acid probe for rat brain Na(+) channel mRNA indicated that the size of the Na+ currents in injected oocytes reflected the levels of mRNA for Na+ channels in the different mRNA preparations. Thus, the expressional potency of mRNAs encoding neurotransmitter receptors and voltage-activated channels is quite stable in postmortem brains in situ, but it is reduced if the brains are kept in oxygenated saline, and freezing and thawing of tissue results in rapid degeneration of mRNA. PMID- 1705711 TI - Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP. AB - Glucose uptake by adipose tissue is mediated by two glucose transporters: GLUT4, which is most abundant, and GLUT1. While GLUT1 is expressed in many tissues, GLUT4 is unique to tissues that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue). In the diabetic state and during starvation, insulin-stimulated glucose uptake and GLUT4 expression are decreased in tissue adipocytes. Using 3T3-L1 adipocytes in culture, we investigated the possibility that these effects are mediated by elevated cellular cAMP. When 3T3 L1 adipocytes were treated for 16 hr with forskolin or 8-Br-cAMP, GLUT4 mRNA and protein were decreased by approximately 70%, while expression of GLUT1 mRNA and protein was increased 3-fold. These changes were accompanied by an increased basal rate of 2-deoxyglucose uptake and a loss of acute responsiveness of hexose uptake to insulin. The magnitude of GLUT4 mRNA depletion/GLUT1 mRNA accumulation was dependent upon the concentration of 8-Br-cAMP. The decrease of GLUT4 mRNA caused by 8-Br-cAMP was the result of a decreased transcription rate, while the half-life of the message was unaffected. The increase in GLUT1 mRNA caused by 8 Br-cAMP was the result of both transient transcriptional activation and mRNA stabilization. We suggest that down-regulation of GLUT4 mRNA in adipose tissue in the diabetic state and during starvation is the result of repression of transcription of the GLUT4 gene caused by cAMP. PMID- 1705712 TI - Construction of a uniform-abundance (normalized) cDNA library. AB - We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in lambda phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and self-annealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue. PMID- 1705713 TI - FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. AB - FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506. PMID- 1705714 TI - Growth suppression induced by wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression. AB - The p53 gene is a frequent target of mutation in a wide variety of human cancers. Previously, it was reported that conditional expression of wild-type p53 protein in a cell line (GM47.23) derived from a human glioblastoma multiform tumor had a negative effect on cell proliferation. We have now investigated the effect that induction of wild-type p53 protein in this cell line has on the expression of the proliferating-cell nuclear antigen gene. The proliferating-cell nuclear antigen gene encodes a nuclear protein that is an auxiliary factor of DNA polymerase delta and part of the DNA replication machinery of the cell. We show that inhibition of cell cycle progression into S-phase after induction of wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen mRNA and protein expression. PMID- 1705716 TI - A method for the selective measurement of the radiosensitivity of quiescent cells in solid tumors--combination of immunofluorescence staining to BrdU and micronucleus assay. AB - C3H/He mice bearing the SCC VII tumor were irradiated after being given 10 injections of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells in the tumors, and the tumors were then excised and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (which blocks cytokinesis), and the micronucleus frequency in unlabeled cells was determined using immunofluorescence staining to BrdU. The micronucleus frequency was then used to calculate the surviving fraction of the unlabeled cells, using the regression line relating the micronucleus frequency to the surviving fraction determined separately for the total tumor cell population. Using this technique, a cell survival curve could be determined for the unlabeled cells, which were regarded as the quiescent cells. Assays performed both immediately after and 24 h after irradiation of normally-aerated tumors showed that unlabeled cells were more radioresistant and had a greater capacity for repair of potentially lethal damage than the tumor cell population as a whole. Moreover, when the assay was performed immediately after the irradiation of both normally-aerated and hypoxic tumors, it was found that unlabeled cells had a much higher hypoxic fraction than the tumor cell population as a whole. This appears to be a useful method for determining the responses of quiescent cells in solid tumors to various treatments. PMID- 1705715 TI - Macrophage heterogeneity occurs through a developmental mechanism. AB - The versatility and importance of macrophages in host defense and homeostasis have long been recognized. Anatomically, macrophages isolated from various tissues manifest extreme differences in shape, in metabolic and functional activities, and in the expression of macrophage-specific markers. To determine the mechanisms responsible for generating macrophage heterogeneity, we have employed the reverse transcription-polymerase chain reaction to molecularly phenotype colonies of bone marrow-derived macrophages during differentiation in vitro. By utilizing this method, results have revealed a hierarchal expression of macrophage-associated genes. Tumor necrosis factor alpha was expressed in all colonies analyzed suggesting an important role for this molecule during macrophage differentiation. Predominant colony phenotypes observed were unique for (i) the period of differentiation and (ii) the growth factor with which they were derived (either colony-stimulating factor 1 or granulocyte-macrophage colony stimulating factor). Exogenous stimulation of the cultures with either bacterial lipopolysaccharide or interferon-gamma led to predictable phenotypic transitions. These results suggest that macrophage heterogeneity is generated through differentiation-related mechanisms and that generated macrophage phenotypes are then maintained by systemic environmental constraints. PMID- 1705717 TI - Antidepressant-anxiolytic interactions: involvement of the benzodiazepine-GABA and serotonin systems. AB - 1. Recent studies have demonstrated that antidepressant drugs are actually more effective than BZ's in the treatment of anxiety states. The role of two major neurochemical substrates that may be implicated in the anxiolytic activity of antidepressants, the benzodiazepine (BZ)-GABA receptor chloride ionophore complex and central serotonergic pathways, are focused on in this review. 2. A wide range of antidepressants elicit a reduction in BZ receptors and display anxiolytic effects within a conflict paradigm. 3. The anxiolytic activity of antidepressants, however, does not appear to be mediated via the BZ receptor, but possibly via another component of the complex such as the chloride channel associated with the GABAA receptor. 4. Additionally, as possible candidates for the mechanism of anxiolytic activity of these compounds, results of pharmacological, behavioral and clinical studies point to the importance of serotonin (5-HT)1A receptors and 5-HT transporter sites as targets for the action of antidepressants, triazolobenzodiazepines and anxioselective piperazine derivatives. PMID- 1705718 TI - Alpha-methylserotonin, a substitute transmitter for serotonergic neurons. AB - 1. Administration to rats of alpha-methyltryptophan (AMTP) gives rise to alpha (AM5HT) in the brain along with a decrease of cerebral 5HT. 2. Analysis of fractions prepared from brains of AMTP-injected rats shows that AM5HT occurs mainly in the synaptosomes. 3. The synaptosomal content of AM5HT in proportion to the total AM5HT in the brain represents the same ratio as for the corresponding fractions of 5HT. PMID- 1705719 TI - ASA-induced release of histamine from nasal mucous membranes in analgesic intolerance and polyposis nasi. AB - Tissue samples from the polypous mucous membrane and the inferior nasal concha were taken from 13 patients with polyposis nasi and from 12 other patients with an additional intolerance to analgesics. The tissue of the inferior nasal concha from patients without polyposis nasi served as a control. The relative histamine content of the samples (in ng/mg dry weight) and the relative histamine release (in %) after addition of acetylsalicylic acid (ASA) were determined. A significantly higher relative histamine content in the tissue samples of polyp patients without an intolerance to analgesics was seen in comparison to the other two groups. The relative histamine release of both patient groups with nasal polyposis was comparable. The control group exhibited both an increased spontaneous release of histamine as well as a higher relative histamine release from the tissue of the inferior nasal concha. PMID- 1705720 TI - The role of some activators and inhibitors of calcium channels on the dynamics of serum magnesium. AB - Magnesemia was determined in male rats weighting 120 +/- 10 g after intravenous administration of nifedipine, an antagonist of calcium channels, and BAY-K 8644, an activator of calcium channels. Nifedipine does not alter the basal level of serum Mg 30 minutes after administration in normal animals or in animals in which chemical sympathectomy was induced by administration of 6 OH-dopamine. On the other hand, BAY-K 8644 induces a significant rise of basal magnesemia from 2.1 +/ 0.2 mg% to 2.7 +/- 0.1 mg% in normal animals. In animals sympathectomized with 6 OH-dopamine, their rise is maintained at about the same level from 2.0 +/- 0.1 mg% after administration). Propranolol previously administered inhibits the stimulating action induced by the calcium channel agonist, BAY-K 8644. PMID- 1705721 TI - Transmission electron microscope observations on enamel following silver methenamine staining. AB - Transmission electron microscope observations on porcine enamel and secretory ameloblasts showed that silver methenamine material was located inside secretory vesicles in secretory ameloblasts and along the enamel crystals inside immature enamel. It is concluded that silver methenamine is able to stain enamel proteins selectively inside these tissues. PMID- 1705722 TI - [The recognition of rat epididymal epithelial cells]. AB - The antibodies of keratin and vimentin were used as the histochemical probes determined by the immuno-fluorescent technique to recognize the rat epididymal epithelial cells in the different ages from the connective tissue both in intact epididymides and in isolated cultured cells. It also showed that an enriched suspension of epididymal epithelial cells could be obtained by sequential digestion with 0.05% trypsin and 0.1% collagenase. The morphological characteristics were appeared during the cells in culture. Therefore the epididymal epithelial cells isolated and cultured by present methods could be used as a research model to study their functions. PMID- 1705723 TI - Titrated intravenous barbiturates in the control of symptoms in patients with terminal cancer. AB - Patients with terminal cancer may have a series of severe and dehumanizing physical and psychologic symptoms. To improve symptom control in the final days and hours of life, we administer intravenous barbiturates continuously to provide heavy sedation or continuous somnolence. Titrated dosage is then reduced to a minimum, after a desired steady-state has been achieved. Improved symptom control is provided, and the patient's dignity is maintained until death. PMID- 1705724 TI - [Therapeutic tactics in mechanical jaundice]. PMID- 1705725 TI - [The cobalt-60 irradiation of skeletal metastases--conventional fractionation versus accelerated hyperfractionation]. AB - Purpose of this study was to compare the results of two different modalities of palliative radiation i.e. conventional fractionated (group I: 35 patients) vs. hyperfractionated radiation (group II: 20 patients). Group I received 1.8 to 2.3 Gy one time a day (total dose 30 to 40 Gy), with an average treatment duration of 20 days. Group II received 1.8 to 2 Gy two times a day (total dose 25 to 35 Gy), with an average treatment duration of ten days. Regression of complaints occurred in 80% of group I, with an average of twelve days, and in 95% of group II, with an average onset of four days after beginning of treatment. Neither acute nor long term complications did occur in any group. The advantages of the hyperfractionated radiation modality therefore are on one hand a higher regression-rate of subjective complaints, with equal good recovery of clinical and radiological findings and lack of side-effects especially those of the myelon, and on the other hand are resulting in a shortening of hospitalisation. PMID- 1705726 TI - The role of cholecystokinin in the pathogenesis of acute pancreatitis in the isolated pancreas preparation. AB - In a variety of animal models of acute pancreatitis, cholecystokinin-receptor antagonists have ameliorated the injury response. These results suggest that cholecystokinin may play a primary role in the pathogenesis of pancreatitis initiated by multiple stimuli. In an effort to test this theory, a sensitive and high affinity cholecystokinin-receptor antagonist L364,718 was administered to four different models of acute pancreatitis that were produced in the ex vivo perfused canine pancreas preparation. The four models of pancreatitis were initiated by cerulein infusion, partial duct obstruction with secretin stimulation, oleic acid infusion, and a 2-hour period of ischemia. In each model, pancreatitis was manifest by edema formation, weight gain, and hyperamylasemia during a 4-hour perfusion. In cerulein infusion-induced pancreatitis L364,718 inhibited edema formation and weight gain (31 +/- 5 gm versus 7 +/- 6 gm; p less than 0.05) and significantly decreased plasma amylase activity (36,605 +/- 21,216 U/dl versus 9421 +/- 5149 U/dl; p less than 0.05). The acute pancreatitis induced by the other three stimuli was not ameliorated by L364,718 treatment. We conclude that in the ex vivo-perfused canine pancreas preparation cerulein-induced pancreatitis is mediated at least in part by the cholecystokinin receptor. Early blockade of the cholecystokinin receptor was of no benefit in treating the other models of pancreatitis, suggesting that cholecystokinin is not involved in the early pathogenesis. PMID- 1705727 TI - Malignant melanoma of the biliary tract: a case report. AB - A 58-year-old man was seen with obstructive jaundice and discomfort in the upper abdomen. Computed tomographic and ultrasound examinations revealed a soft-tissue mass in the gallbladder. Cholecystectomy and choledochotomy revealed a soft black mass in the gallbladder and a second one in the intrapancreatic portion of the common bile duct. Each was diagnosed as malignant melanoma. Subsequently, a Whipple resection of the pancreas, duodenum, and distal bile duct revealed a melanoma circumferentially invading and obstructing the distal common duct. No lymph node or distant metastasis was identified. Repetitive searches for another primary site have been negative. The tumor apparently originated in the biliary tract. The patient remains almost well 2 years after diagnosis. PMID- 1705728 TI - Lymphoma of the gastric stump: report of a case. AB - We report a case of primary lymphoma on a previously resected stomach in a 62 year-old man. The patient was treated 22 years earlier with a partial gastrectomy and Billroth II reconstruction for a benign gastric ulcer. The rarity of this entity and its possible relationship with pseudolymphoma or lymphoid nodular hyperplasia is discussed, and the literature is reviewed. PMID- 1705729 TI - Hyaline droplet nephropathy resulting from exposure to 3,5,5 trimethylhexanoyloxybenzene sulfonate. AB - Acute oral dosing of 3,5,5-trimethylhexanoyloxybenzene sulfonate (THBS) to adult male and female rats causes a male rat-specific nephrotoxicity manifested as exacerbation of hyaline droplet formation. This chemical is structurally distinct from the volatile hydrocarbons known to cause male rat-specific kidney lesions. Therefore, to classify THBS as a hyaline droplet-inducing agent, experiments were conducted to determine whether [14C]THBS equivalents bound to alpha 2 mu-globulin and caused the protein to accumulate in male rat kidney cortex. Two-dimensional gel electrophoretic separation of male rat kidney proteins indicated that alpha 2u-globulin levels in kidney increased 24 hr after a single oral dose of THBS (500 mg/kg). Furthermore, a sex-dependent retention THBS was noted as there was approximately 10 times more THBS equivalents in male rat kidney than in female rat kidney. Equilibrium dialysis experiments indicated that 40% of THBS equivalents bound reversibly to male rat kidney proteins, whereas no interaction between THBS and female rat kidney proteins was detected. Specific binding of THBS to alpha 2mu-globulin was determined by anion-exchange HPLC after which metabolites in the alpha 2u-globulin fraction were identified by gas chromatography with parallel radioactivity-mass spectrometry and mass spectrometry-matrix isolation Fourier-transform infrared analysis. Four metabolites of THBS were found in this protein fraction, and the major component (approximately 70%) was identified as the cis gamma-lactone of 3,5,5 trimethylhexanoic acid. Experiments were also conducted in mice to determine whether THBS bound to any mouse kidney proteins, particularly mouse urinary protein. The results indicated that there was no interaction between THBS and mouse urinary protein, a protein which shares significant homology with alpha 2u globulin. These results indicate that THBS treatment exacerbates hyaline droplet formation in male rat kidneys by binding to alpha 2mu-globulin, thereby causing the protein to accumulate in the renal cortex. The interaction between THBS and alpha 2mu-globulin appears to be unique to this male rat-specific protein as THBS does not interact with a very similar protein found in mice. PMID- 1705730 TI - Effect of alpha thalassaemia, G-6-PD deficiency and Hb F on the nature of sickle cell anaemia in south-western Saudi Arabia. AB - This study was conducted on sickle cell anaemia (SCA) patients from the south western province (SWP) of Saudi Arabia to determine the effect of thalassaemias, glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and Hb F level on the clinical presentation of sickle cell anaemia. The results showed that associated alpha-thalassaemia improved the haematological parameter values while associated G-6-PD deficiency and high Hb F level did not play a significant role in amelioration of the disease in these patients. Hb S/beta(0) -thalassaemia cases showed a severe anaemia similar to the SCA patients without alpha-thalassaemia. However, considerable improvement of the haematological parameters were found in patients with S/beta(0)-thalassaemia and associated alpha-thalassaemia. This paper reveals that alpha-thalassaemia may partially ameliorate the clinical manifestations of SCA in Saudi patients from the SWP, while high Hb F level and G 6-PD deficiency do not modify SCA to any statistically significant extent. PMID- 1705731 TI - Thyrotoxicosis in Nigeria. Analysis of a five year experience. AB - Fourty cases of thyrotoxicosis seen over a 5-year period in the endocrinology service of a teaching hospital in Ibadan, Nigeria have been reviewed. The clinical manifestations are not different in any way from the pattern described in other parts of the world and there was a female preponderance in a ratio of 4:1. The ratio of diffuse toxic goiter to toxic multinodular goiter was only 3:2, underscoring the relative uncommonness of Grave's disease, an autoimmune disorder, in Nigerian Africans. The study also demonstrated an increase in the hospital prevalence of thyrotoxicosis in Ibadan in the last 10-15 years; however, the incidence rate of 8 cases per year is still low when compared to experiences in other parts of the world. The presence of a goiter, ocular changes, high sleeping pulse rate, fine silky-smooth skin and thin, short and scanty hair have been identified as important clinical clues to look for to aid in the diagnosis of a difficult case in an environment where there are no facilities for thyroid function tests. Surgery was the most dependable form of treatment because of the cost and scarcity of drugs and complete absence of radiotherapy. PMID- 1705732 TI - Early detection program for prostate cancer: results and identification of high risk patient population. AB - Three hundred sixty-two men underwent transrectal ultrasound of the prostate (TRUS), digital rectal examination (DRE), and serum prostate-specific antigen (PSA) determination as part of an early detection program for prostate cancer. Thirty-seven (10%) cancers were detected. DRE had the highest sensitivity and specificity, 89 percent and 84 percent, respectively. TRUS and PSA had comparable sensitivities (84% and 81%) and specificities (82% and 82%). The positive predictive values of DRE, TRUS, and PSA determination were 39 percent, 35 percent, and 33 percent, respectively. We found a cancer detection rate of 16 percent among patients with symptoms of bladder outlet obstruction and 5 percent in patients without these symptoms. The detection rate was 36 percent for physician-referred patients and 3 percent for self-referred patients. This suggests to us that at the present time the best utilization of medical resources to increase prostate cancer detection is to educate men to have annual medical evaluations by primary-care physicians who are encouraged to incorporate risk assessment and screening DRE as part of their routine practice. Any man with either abnormal findings on examination or increased risk should be referred to a urologist for further evaluation. PMID- 1705733 TI - Expression of the gene of the alpha-smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells. AB - Fat storing cells (FSCs) in the liver represent the main site of vitamin A deposition in the body. These cells are considered to play an important role during scar formation and fibrogenesis in the liver. The putative descent of FSCs from the fibroblastic or from the myofibroblastic system have not been determined yet by morphological or immunohistochemical studies. To further define the origin of these liver cells, we analysed the pattern of expression of three structural proteins: vimentin, desmin and the alpha-smooth muscle (SM)-actin isoform in FSCs of the rat liver, in smooth muscle cells (SMCs) from the aorta and in rat skin fibroblasts. FSCs were studied by immunohistochemical methods immediately after isolation, at days 3 and 7 after plating. FSC-gene-expression was also analysed by Northern blot analysis of total RNA extracted from cells in culture at days 3 and 7 after isolation. Arterial SMCs and skin fibroblasts were studied in a similar way. For comparison, isolated rat hepatocytes and Kupffer cells (Kc) were studied. Of freshly isolated FSCs, 100% were vimentin-positive, 50% were desmin positive, but all were alpha-SM-actin negative. Three days after isolation, FSCs were clearly positive for vimentin and desmin and weakly alpha-SM-actin-positive, as demonstrated by indirect immunofluorescence as well as by the immunoperoxidase technique. Desmin, alpha-SM-actin and vimentin staining was further increased at day 7 after isolation, and alpha-actin specific transcripts in FSC-RNA were clearly detectable at day 7 after isolation. Passaged arterial SMCs were vimentin and alpha-SM-actin-positive, but desmin-negative and fibroblasts were only vimentin-positive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705734 TI - Management of a severe transfusional problem in a patient with alloantibody to Kpb (K4). AB - The case of a 58-year-old male with severe anemia after hemicolectomy is described. The patient proved to be Kp(b-), and the serum contained anti-Kpb. Because no Kp(b-) donors were available, two incompatible units are administered after intravenous gammaglobulin (400 mg/kg/day) and hydrocortisone (500 mg). The tolerance was good, without signs of increased red cell destruction. Pending the arrival of compatible blood from the American Red Cross, the hematocrit reached 18%, and the direct antiglobulin test remained negative. PMID- 1705735 TI - Immune hemolytic anemia associated with biclonal cold autoagglutinins. AB - A 65-year-old man with bladder outlet obstruction due to prostatic hypertrophy was incidentally discovered to have cold-antibody autoimmune hemolytic anemia (cold-aggluthinin syndrome; CAS) due to autoanti-I (titer 1,024 at 4 degrees C and 64 at 30 degrees C), and a biclonal gammopathy. Immunofixation electrophoresis of serum and a red blood cell eluate revealed the patient's autoantibody to be biclonal IgM kappa and IgA kappa. No underlying cause could be determined to explain the development of either the biclonal gammopathy or the CAS. To our knowledge, this is the first reported case of CAS associated with a biclonal gammopathy and biclonal cold autoagglutinins of the IgM kappa, IgA kappa type. PMID- 1705736 TI - Effects of hypertonic saline (7.5%)/dextran 70 on human red cell typing, lysis, and metabolism in vitro. AB - The introduction of a 7.5% hypertonic saline/6% dextran 70 (HSD) solution into clinical trials for the treatment of hypovolemic states, and the past concerns regarding the possible interference of dextran with blood serology, prompted us to investigate the effects of HSD on human red-cell typing and stability. HSD was evaluated with fresh and 35-day stored CPDA-1 red cells from 12 healthy donors. A 1:5 mixture of HSD to blood in vitro had no effect on ABO, Rh, and MN typing in both fresh and stored blood. HSD produced no significant lysis with fresh cells and a minimal level with stored blood. No evidence of metabolic or morphologic changes was seen after HSD treatment. The results of this study suggest that the clinical use of HSD for the treatment of hemorrhagic shock will not affect blood group determinations or red-cell stability from stored blood which may be infused after the HSD-treated patient is transported to a hospital. PMID- 1705738 TI - [The age-dependent phenomenon of root dentin transparency and the consequences deriving therefrom for gangrene treatment]. PMID- 1705739 TI - [Oncogenes in neuro-oncology]. PMID- 1705737 TI - G-CSF and GM-CSF in clinical trials. AB - Hematopoietic growth factors have now been purified, cloned, and produced in bacteria and yeast. Those that are currently in clinical study include erythropoietin, GM-CSF, G-CSF, M-CSF (also called CSF-1), and multi-CSF (also called interleukin 3). Growth factor appear likely to enhance the recovery and function of circulating white cells after standard-dose cancer therapy and high bone-dose cancer therapy with marrow transplant and to restore leukocyte numbers and competence in the acquired immune deficiency syndromes and myelodysplastic syndromes. Phase I, II trials in AIDS, in cancer patients receiving chemotherapy, in cases of myeloproliferative disease, and after bone marrow transplant have been published. The results of phase III studies are just becoming available. PMID- 1705740 TI - Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents. Effects on overall RNA synthesis and synthesis of the initiating dinucleotide. AB - A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system. Escherichia coli DNA dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses. By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes. PMID- 1705741 TI - [Changes of intermediate filament during mitosis in two epithelial cell lines]. AB - Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis. PMID- 1705742 TI - [A novel monoclonal antibody against human keratins]. AB - In this paper, we reported a novel monoclonal antibody against human keratins, R 6-2-14. The antigen used for immunization was derived from human callus, keratins in which traditionally are classified as "Soft" keratins. However, when we studied the tissue specificity of this antibody, it was found that it only reacted strongly with "Hard" keratins of various mammalian species, but no detectable cross-reactivity with any of the "Soft" keratins. This antibody may provide a useful tool for the study of hair regeneration, nail regeneration, corn pathology and differentiation of mammalian epidermal derivatives. PMID- 1705743 TI - Punctate porokeratotic keratoderma: some pathogenetic analyses of hyperproliferation and parakeratosis. AB - An 82-year-old Japanese woman had numerous palmoplantar keratotic plugs and pits, resembling 'music box spines'. Histological examination revealed compact columns of parakeratosis in the horny layer. Ultrastructually, the affected stratum corneum contained numberous variable-sized pyknotic nuclei, and cells in the stratum granulosum contained fewer keratohyalin granules. Autoradiographic analysis by [3H]thymidine [3H]TdR incorporation into epidermal cells of affected skin slices in organ culture revealed that only basal cells below the keratotic plug were stimulated to proliferate. Two-dimensional gel electrophoresis revealed that palmar keratotic plugs contained the keratin filaments that are specifically present in the plantar viable epidermal layer, or other hyperproliferative epithelial cells. PMID- 1705744 TI - Lack of increase in granulocyte colony-stimulating factor in psoriatic skin. AB - Previously, we showed an elevated level of pro-inflammatory cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) in psoriatic skin. Granulocyte (G) CSF, which is also released from the infiltrating cells and epidermal keratinocytes, profoundly influences the biological activities of terminally differentiated neutrophils, in addition to its supporting effects on the proliferation and differentiation of progenitor cells of neutrophil lineage. We have carried out enzyme immunoassay for G-CSF in suction blister fluids and horny tissue extracts from psoriatic skin. Although some samples of the blister fluids and stratum corneum extracts showed G-CSF, there were no significant differences between the concentration in normal and psoriatic skin. These results suggest that, among CSFs, GM-CSF plays a more important role than G-CSF in the local immune responses in psoriasis. PMID- 1705745 TI - Vasoactive intestinal polypeptide stimulates parathyroid hormone release by interaction with cyclic adenosine monophosphate production of bovine parathyroid cells. AB - Influence of vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene related peptide, and substance P was investigated on dispersed parathyroid cells of adult cattle. At a physiological concentration of extracellular calcium, vasoactive intestinal polypeptide stimulated the parathyroid hormone release in a dose-dependent manner, whereas no effects were noted for the other peptides. The dependency of PTH secretion upon extracellular calcium was shifted to the right by vasoactive intestinal polypeptide at 10(-6) mol/l, with a tendency for greater effects at low (0.5 mmol/l) than high concentrations (2.0-3.0 mmol/l) of the cation. Vasoactive intestinal polypeptide significantly enhanced cAMP release of the parathyroid cells, whereas no influence was noted on cytoplasmic calcium or pH within the cells. The results suggest that vasoactive intestinal polypeptide stimulates the PTH release by interaction with cAMP production of the parathyroid cells. This effect may contribute to the development of hypercalcemia in patients with neuroendocrine tumours secreting vasoactive intestinal polypeptide. PMID- 1705746 TI - Coxiella burnetii antigens may induce resistance to tumour growth. AB - Coxiella burnetii antigens stimulate the defence against growth of hepatoma 22a cells. The antigen-stimulated mice survived longer, they considerably later developed palpable tumours and showed a retarded tumour growth. The enhanced resistance to tumour growth may be explained by at least 2 interrelated phenomena; namely by the induction of interferon-like activity and an increased NK cell activity. PMID- 1705747 TI - Determination of serotypes of red clover necrotic mosaic virus by enzyme-linked immunosorbent assay. AB - The direct double antibody sandwich (DAS) type of enzyme-linked immunosorbent assay (ELISA) was used to determine the degree of serological and antigenic differences, among the three serotypes (A, B, C) of red clover necrotic mosaic virus (RCNMV). Homologous and heterologous antibody titres in the used IgGs to isolates TpM34 (serotype A), TpM48 (serotype B) and isolate No. 6 (serotype C) as determined by ELISA were 100- to 200-fold higher than by ring precipitation test. Intensity of homologous and heterologous reactions in ELISA depended on the concentration of antigen, of the IgG used for coating and of the labelled IgG. The IgG preparations used contained 50 to 100 times higher concentration of homologous (serotype-specific) than heterologous (interserotype-specific) antibodies. Such a great difference between the two antibody types accounts for a comparatively high degree of selectivity of the homologous reactions. PMID- 1705748 TI - Presence of antibody reactive with synthetic peptide derived from L2 open reading frame of human papillomavirus types 6b and 11 in human sera. AB - Nine oligopeptides corresponding to segments of different open reading frame (ORF) proteins of human papillomavirus (HPV) 6b and HPV-16 were prepared and tested for reactivity with human sera in enzyme-immunoassay (ELISA). Of these only heptadecapeptide derived from L2 ORF of HPV-6b, and encoded also by L2 ORF of HPV 11, was reactive with some human sera. Over 400 human sera of different origin were tested for the presence of antibody to this antigen. While less than 15% of sera from healthy subjects or cervical carcinoma patients were found antibody positive, sera from the majority of condylomata accuminata (CA) patients were reactive. The antibody titres varied from 1:10 (initial serum dilution) to 1:80; in this respect there was no marked difference between sera from CA patients and the other subjects. The prevalence of antibody was higher among promiscuous than nonpromiscuous women. This is in line with the assumption that sexual intercourse is the most important route of HPV 6 and 11 transmission. PMID- 1705749 TI - Rapid regression of coronary dilatation in Kawasaki disease with intravenous gamma-globulin. PMID- 1705750 TI - Electrocardiographic, enzymatic and echocardiographic evidence of myocardial damage after Tityus serrulatus scorpion poisoning. PMID- 1705751 TI - Intercellular adhesion molecule-1 in human corneal endothelium. Modulation and function. AB - The endothelium lining the posterior corneal surface performs physiologic pump functions essential to corneal clarity and integrity. A hallmark of keratitis, anterior ocular inflammation, and corneal allograft rejection is leukocyte adherence to the corneal endothelium (CE) forming keratitic precipitates. To elucidate mechanisms governing cornea-leukocyte interactions, cultured human CE cells and intact corneas were examined for expression of intercellular adhesion molecule-1 (ICAM-1), which binds the lymphocyte function-associated antigen-1 (LFA-1) on all leukocytes and enhances delayed-type hypersensitivity mediated by class II major histocompatibility complex antigens. Immunohistochemistry on culture CE cells using monoclonal anti-ICAM-1 antibody yield positive staining that increased after exposure to interleukin-1-beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (gamma-IFN). Standard leukocyte adherence assays demonstrated ICAM-1-mediated CE-neutrophil binding, which was specifically blocked by antibody to ICAM-1 or antibodies to LFA-1 on neutrophils. In whole human corneas, gamma-IFN increased CE and stromal keratocyte ICAM-1 immunoreactivity and enhanced CE-neutrophil adherence. As in CE cell cultures, antibody to ICAM-1 effectively blocked neutrophil binding to the CE cells of whole corneas. These results are the first to demonstrate ICAM-1 in ocular tissue. They indicate that CE cells express functional ICAM-1, which may be modulated by inflammatory cytokines, ICAM-1 provides mechanisms for keratitic precipitate formation, regulation of corneal leukocyte trafficking and the generation of immune responses that may be crucial to allograft rejection. PMID- 1705752 TI - Early events in liver allograft rejection. Delineation of sites of simultaneous intragraft and recipient lymphoid tissue sensitization. AB - The early events of liver allograft rejection in untreated rats were studied in the DA to BN rejection strain combination and compared with DA and BN liver isograft recipients. In the liver allografts, T-cell infiltration first occurred at 2 days after transplantation and localized to the portal triads and subjacent to the terminal hepatic venules (THV), regions rich in intensely Ia + spindle and dendritic-shaped interstitial cells. Double staining showed distinct 'clustering' between donor Ia-positive dendritic-shaped cells and W3/25+ infiltrating lymphocytes, or to a lesser extent, OX8+ cells. The infiltrating mononuclear cells underwent blastogenesis and proliferated in both the triads and THV regions at 3 and 4 days. Donor Ia-positive cells were also noted in the W3/25+ periarterial lymphatic sheath and marginal zone of the recipient spleen 1 day after transplantation. The number of these cells in the spleen peaked at 3 to 4 days, but were no longer detectable by 10 to 12 days. Mitotic activity became evident in these same regions by days 3 and 4. Paracortical blastogenesis (day 2) and proliferation (days 3 and 4) were also noted in the regional lymph nodes of liver allograft recipients, but no donor Ia+ cells were found in the mesenteric nodes or thymus of the allograft recipients. These results demonstrate that sensitization of the recipient lymphoid tissue to liver allografts can occur both peripherally (intragraft) and centrally (spleen and lymph nodes). Passenger leukocytes (donor dendritic cells) are likely the primary stimulators of the rejection reaction. Still, it is probable that other pathways of sensitization exist. PMID- 1705753 TI - Secondary deposition of beta amyloid within extracellular neurofibrillary tangles in Alzheimer-type dementia. AB - The hippocampal areas of 34 autopsy specimen brains from aged demented and nondemented subjects were examined using double staining of Bodian and beta protein. In 18 cases (75.5 +/- 7.4 years old), none of the extracellular neurofibrillary tangles (E-NFTs) were immunoreactive with beta protein. In 16 cases (82.9 +/- 5.4 years old), the minority of E-NFTs were immunoreactive with beta-protein antiserum. These beta-immunoreactive E-NFTs frequently appeared in the areas having senile plaques, while they were not observed in the area lacking beta-immunoreactive senile plaques. The ultrastructure of beta-immunoreactive E NFTs revealed that they consisted of extracellular amyloid fibrils, extracellularly located bundles of paired helical filaments, astroglial processes and degenerating neurites. These findings suggest that the beta immunoreactivity of E-NFTs comes from secondary deposition of amyloid fibrils. PMID- 1705754 TI - Basal cell-specific and hyperproliferation-related keratins in human breast cancer. AB - In normal breast tissue and in noninvasive breast carcinomas, various keratin-14 antibodies were reactive predominantly with the basal/myoepithelial cell layer, although mainly in terminal and larger ducts luminal cells sometimes also were stained. A similar reaction pattern was found with an antibody directed against keratin 17, although this antibody was more often found negative than keratin 14 in the pre-existing myoepithelial cells in intraductal carcinomas. Furthermore antibodies reactive with hyperproliferation-related keratins 6 and 16 were used. One of these (LL025) was completely negative in normal breast tissue and noninvasive breast carcinomas. However 10% of the invasive carcinomas were diffusely or focally positive with this latter antibody, while in 18 of 115 cases of invasive breast carcinomas studied, a basal cell phenotype was detected. A relatively high concordance was found between the carcinomas immunostaining with the basal cell and the hyperproliferation-related keratins, but not between these markers and the proliferation marker Ki-67. This supports the conclusion that basal cells in breast cancer may show extensive proliferation, and that absence of Ki-67 staining does not mean that (tumor) cells are not proliferating. PMID- 1705755 TI - Resistance of ascending vasa recta to transport of water. AB - A study was undertaken to determine the effect of increasing capillary pressure on volume flux in ascending vasa recta (AVR). In one experiment (group I), AVR were blocked by a single injection of paraffin wax and subjected to free-flow microperfusion at 10 nl/min. Collected fluid was obtained from the perfused vessels by micropuncture. In a second experiment (group II), AVR segments were isolated between two paraffin blocks and perfused at 10 nl/min. In group II, the collection pipette was pressurized to 0, 10, or 20 mmHg. Transmembrane volume flux was determined by measuring the change in concentration of fluorescein isothiocyanate-labeled dextran (2 x 10(6) mol wt) from perfusate to collected fluid. In group I, measurements revealed a capillary pressure of 10.3 +/- 0.5 (SE) mmHg and volume flux of 4.3 +/- 1.0 nl.mm-1.min-1. In group II, volume flux was 1.8 +/- 1.3, 5.9 +/- 1.0, and 11.2 +/- 1.1 nl.mm-1.min-1 at collection pressures of 0, 10, or 20 mmHg, respectively. Based on these data and an AVR diameter of 20 microns, AVR hydraulic conductivity is between 12.5 x 10(-6) and 18.7 x 10(-6) cm.s-1.mmHg-1. The papillary AVR have a high hydraulic conductivity. This is consistent with their role as the sole conduit for removal of water from the papillary interstitium. PMID- 1705756 TI - Carbonic anhydrase in turtle bladder mitochondrial-rich luminal and subluminal cells. AB - Bladders from March-April turtles were processed for carbonic anhydrase (CA) cytochemically using the method of D.A. Riley, S. Ellis, and J. Bain (Neuroscience 13: 189, 1984). CA-positive cells comprised 11.1 +/- 0.7% of mucosal epithelial cells. Microplicated (MP) cells comprised 47.2 +/- 1.8% of CA positive cells and displayed at least two distinct staining patterns: the first was characterized by reaction product that filled the luminal one-third, including the terminal web and microplicae. These cells possessed extensive microplicae, a morphological feature of ongoing H+ secretion. The second was characterized by reaction product distributed throughout cells, excluding the terminal web and microplicae, with greatest intensity in the luminal one-third below the terminal web. These cells possessed flattened microplicae, a morphological feature of diminished H+ secretion. Microvillated (MV) cells comprised 6.0 +/- 1.0% of CA-reactive cells. The basal layer was occupied by 46.8 +/- 1.7% of CA-positive cells, which were termed subluminal (SL) cells. SL cells were mitochondrial rich and did not contact the lumen. Extracellular CA staining was common between the lateral margins of contiguous mitochondrial-rich or non mitochondrial-rich cells. PMID- 1705757 TI - Carbonic anhydrase and proton secretion in turtle bladder mitochondrial-rich cells. AB - Bladders from actively feeding turtles were processed for carbonic anhydrase (CA) cytochemically. CA-positive cells were identified as microplicated (MP) cells, microvillated (MV) cells, and subluminal (SL) cells. After acute enhancement of H+ secretion with 5% CO2, MP cells displayed extensive microplicae and a reduced density of apical subplasmalemmal vesicles, and they were CA reactive throughout a large part of the cytoplasm including the microplicae. After acute inhibition of H+ secretion with a pH 4.5 mucosal bath, CA staining was excluded from the microplicae and apical subplasmalemmal region of most MP cells, whereas microplicae varied from extensive to reduced, and subapical vesicle density remained elevated. MV cells were characterized by basolateral staining with sparing of the MV and apical subplasmalemmal region in all settings except 1) after 5% CO2 and 2) when MV cells were found in areas in which MP cells were stained to the lumen. These results indicate that CA is active at the site of H+ secretion in MP cells and is correlated with the acute acid-base status of the bladder. PMID- 1705758 TI - Pharmacological evidence for two types of myocardial sarcoplasmic reticulum Ca2+ release. AB - Contractions of guinea pig papillary muscles were studied at 37 degrees C under a variety of conditions and stimulation rates that markedly alter the pattern of tension development. When rested-state contractions (RSCs) were enhanced by treatments that increase intracellular adenosine 3',5'-cyclic monophosphate (0.1 1 microM isoproterenol, 1-10 microM forskolin), a markedly enhanced late peak tension developed after a 100-ms delay. Such late peak tension was selectively depressed by local anesthetics (200-400 microM procaine, 4-10 microM tetracaine, or 0.5-1 mM ethyl aminobenzoate). In contrast, 0.1-1 microM ryanodine had little effect on late peak tension, whereas 5 mM caffeine reduced the delay before tension development. Inotropic interventions such as increased external Ca2+ concentration or the Ca2+ channel agonist BAY K 8644 did not elicit such distinct late peaking RSCs. Rapid initial tension development observed under a variety of situations (short cycle lengths, stimulation rates of 0.25 Hz plus isoproterenol, decreased external Na+ concentration) was markedly depressed by 0.01-1 microM ryanodine and by caffeine, whereas local anesthetics had little effect. These results suggest two pharmacologically distinct types of sarcoplasmic reticulum Ca2+ release: 1) Ca2+ that accumulates during prior depolarizations is released immediately upon depolarization and decreased by ryanodine and caffeine; 2) extracellular Ca2+ that enters the myocyte is accumulated and released after an initial delay and is selectively depressed by low concentrations of local anesthetics. PMID- 1705759 TI - Kinetics of plasma fibronectin: increased lung tissue incorporation after postoperative bacteremia. AB - Fibronectin is an adhesive glycoprotein that may influence the permeability of the vascular barrier. We compared the plasma disappearance of purified human fibronectin (hFn) and its incorporation into lung tissue in control nonbacteremic and bacteremic sheep after surgery to determine the influence of postoperative bacteremia on the plasma clearance and lung deposition of hFn. Lymph fistulas were surgically prepared 48 h before either saline or bacterial challenge to allow for collection of timed samples of plasma, lung lymph, and peripheral lymph. On the day of the study, the sheep were anesthetized and given either 5 X 10(8) Pseudomonas aeruginosa in saline or saline alone. In parallel, they were infused intravenously with 100 mg of hFn, and the hFn concentration in plasma, lymph, and tissue extracts was subsequently determined by enzyme-linked immunosorbent assay over an 8-h experimental interval. Localization of endogenous sheep fibronectin (sFn) and the injected hFn in serial lung tissue samples was determined by dual-label immunofluorescence. In nonbacteremic control sheep, plasma hFn levels declined with an initial rapid slope (t1/2 = 0.53 +/- 0.19 min), followed by a second, gradual slope (t1/2 = 21.7 +/- 2.5 h), whereas the concentration of hFn in lung lymph and peripheral lymph rose exponentially with time. In bacteremic sheep, the early plasma disappearance of hFn was similar, but the second phase of plasma clearance was faster (t1/2 = 7.4 +/- 0.3 h). Lung tissue from control and bacteremic sheep contained the same level of extractable hFn. However, tissue extraction followed by immunofluorescence microscopy indicated that lung tissue from bacteremic sheep contained more nonextractable hFn than lung tissue from nonbacteremic sheep. Thus postoperative bacteremia that elicits acute lung vascular injury will increase the plasma disappearance of hFn and its incorporation into the lung tissue. PMID- 1705760 TI - Prolongation of cocaine effect. PMID- 1705761 TI - Effects of capsaicin on mechanical, cellular, and mediator responses to antigen in sensitized guinea pigs. AB - To assess the role of tachykinins (TKs) in immediate hypersensitivity allergic reactions in guinea pigs (GPs), we compared airway mechanics and bronchoalveolar lavage (BAL) cell and inflammatory mediator profiles in three groups of GPs after ovalbumin aerosol challenge: (1) saline-sensitized, noncapsaicinized (control) (n = 9); (2) ovalbumin-sensitized, noncapsaicinized (OS) (n = 9); (3) ovalbumin sensitized capsaicinized (OSC) (n = 9). Lung resistance (RL), dynamic elastance (EL), BAL cell counts, histamine, and eicosanoid mediator levels were measured at baseline on Day 1, and then on Day 14 after aerosolized antigen challenge. We found significant increases on Day 14 compared with Day 1 in the following: (1) postchallenge RL and EL in OS and OSC GPs, but not in control GPs; (2) BAL total cells and red cells in OS and OSC GPs; (3) BAL prostaglandin D2 (PGD2) thromboxane B2 (TxB2), sulfidopeptide leukotrienes (LTC4/D4/E4), and histamine in OS and OSC animals. Further, when data from all GPs were considered in distributed fashion, we noted positive linear correlations between peak postchallenge RL versus BAL concentrations of each of the following: PGD2, PGF2 alpha, TxB2, LTC4/D4/E4, leukotriene B4 (LTB4), and histamine. We found no significant differences in mediator or cellular responses between OS and OSC GPs. To verify that our method of capsaicinization resulted in TK depletion from the lungs of OSC GPs, substance P (SP) and neurokinin A (NKA) lung tissue levels were measured by ELISA in seven other animals, four treated with capsaicin using the same protocol and three treated with diluent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705762 TI - Iloprost for scleroderma. PMID- 1705763 TI - Interferon inhibitors. PMID- 1705764 TI - [Contribution of monoclonal antibody HMB45 in the histopathologic diagnosis of melanoma]. AB - We have tested the diagnostic value in malignant melanoma of HMB45, a monoclonal antibody available for use on paraffin-embedded tissue. MATERIAL AND METHOD. Tissues tested. The following pathological tissues were tested: 10 intradermal and 11 compound naevi; 6 spitz naevi; 20 dysplastic naevi; 10 blue naevi; 2 Bednar's tumours; 6 Sutton naevi; 15 melanonychias; 21 cutaneous and 11 ocular malignant melanomas (MM), and 3 achromic metastases. Control tissues were: vitiligo (20), carcinoma (5), malignant schwannoma of the orbit (1), soft tissue sarcoma (5) and malignant lymphoma (5). Antibodies. The antibodies used were antiprotein S100, antivimentin, anticytokeratin (KL1), monoclonal antileucocyte (CD45) antibodies and HMB45, a monoclonal antibody of the IgG 1 type obtained from lymph node metastases from pigmented malignant melanomas. RESULTS. None of the control tissues were stained by the HMB Ab. Intradermal naevi did not react positively. Compound naevi: the juntional cells were stained by HMB45 in 2/10 cases. Dysplastic naevi: HMB45 showed heterogeneous reactivity of junctional cells in 15/20 cases, and this correlated with the degree of atypia. Blue naevi: HMB45 stained the superficial and deep cells in 3/10 cases. Bednar's tumour: no cell was stained by HMB45. Spitz naevi: HMB45 gave an intensely positive reaction of junctional cells in 4/5 cases and a weaker reaction of dermal cells. Sutton naevi: the naevus cells were not stained by HMB45 in 5/6 cases. In simple melanocytic hyperplasia of the nail bed, only a few atypical cells were stained. In superficially spreading melanoma (SSM) all neoplastic cells were stained by HMB45 in proportion to their degree of atypia. Residual naevus cells were negative. The anti S100 and the antivimentin antibodies stained all neoplastic and naevus cells. In nodular melanoma (NM), HMB45 stained all neoplastic cells in proportion to their degree of atypia. The antivimentin Ab stained the neoplastic cells, and so did the anti-S100 Ab which also stained inflammatory cells. In acral-lentiginous melanoma (ALM), HMB stained the dermal tumoral cells moderately and the junctional cells more strongly. In ocular melanoma, HMB45 strongly stained the fusiform cells and less strongly the epithelioid cells. In achromic metastases from cutaneous malignant melanomas, HMB45 strongly stained the neoplastic cells but did not stain the peritumoral cells. DISCUSSION. The purpose of this study was to compare the value of HMB45 with that of other immunohistochemical staining methods A. Main data from the literature. (ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1705765 TI - [Loose anagen hair syndrome]. AB - In 1984, Zaun described a new type of pilary dysplasia characterized by easily pluckable hair. We were able to observe three patients with this anomaly, and this paper is an attempt at reviewing the subject on the basis of these new cases compared with those previously published. Clinical features. The major sign of the anomaly is that hairs can be pulled off in tufts easily and painlessly, and promptly grow again (our cases). In all cases reported so far the hair was blond or dark-blond; in some patients it is described as scintillating (our cases). The hair shaft is usually thin (one of our cases), but it may be of normal caliber (two of our patients). In the occipital region the hairs are entangled, dry and short (our cases). They are implanted wide apart or at normal intervals (our patients); areas of alopecia are sometimes encountered (our cases). Hair growth may be slow (one of our cases) or of normal speed (two of our cases). The eyelashes and eyebrows, as well as other body hairs, are normal in all patients (fig. 1). Microscopy. The trichogram is exclusively composed of anagenic and dystrophic roots (our cases). Pathological examination by biopsy of the scalp is characteristic: transverse and oblique sections of the follicles show that the hairs are oval, triangular or trapezoidal in shape (fig. 7). Alterations of the inner epithelial sheath are also present, including keratinization and early decomposition (fig. 8), lack of complementation (fig. 7) and fissures between the cuticle of the inner epithelial sheath and of the hair, in the keratogenic area (fig. 9). (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705766 TI - Biochemical measurement in cord blood as an indicator of neonatal maturity. AB - Neonates were categorized by gestational scoring (G. Score) using physical and neurological signs, into two groups: TERM and preterm (PRE) using a score corresponding to approximately 38 weeks of gestation as the dividing point. Cord blood concentrations of alphafetoprotein (AFP), fetal hemoglobin (HGF), prealbumin (PALB), creatinine (CRE), and pseudocholinesterase (PC) were determined. The CRE, AFP, and HGF were divided by birthweight in grams (WT) to give three additional markers: CRE/WT, AFP/WT and HGF/WT. Strong Pearsonian correlations between CRE/WT, AFP/WT, and HGF/WT, and G. Scores in the PRE group were obtained. Histograms of AFP/WT and HGF/WT values gave non-Gaussian distributions and resulted in the clear separation of a group of extremely premature infants from the rest of the population. The plots also revealed substantial separation between the TERM and PRE groups. The CRE/WT was predominantly Gaussian, with separation of a group of neonates including the six very preterm infants. It is concluded that CRE/WT, AFP/WT, and HGF/WT have the greatest potential as maturity markers, while PC and PALB have little if any potential. PMID- 1705768 TI - Effects of angiotensin II and bilateral nephrectomy on norepinephrine catabolism in central nervous system. AB - Effects of angiotensin II (AII) on norepinephrine (NE) catabolism in hypothalamus and medulla oblongata of male rats were studied. 3H-NE uptake, 3H-NE/3H-NE metabolites ratio (NE/MET) and monoamineoxidase (MAO) activity were measured in vitro in both organs. Lack of circulating AII was elicited by means of 48 h bilateral nephrectomy. Pargyline and bilateral nephrectomy increased NE uptake and NE/MET ratio, while in nephrectomized plus pargyline treated groups and additive effect on these results was observed in both organs. All decreased the NE/MET ratio. Pargyline reversed the latter effects of AII. The peptide increased MAO activity in both organs, while bilateral nephrectomy decreased the activity of the enzyme. The results showed that AII modulates NE catabolism by means of MAO activity, eventually at the presynaptic noradrenergic ending sites in the central nervous system. PMID- 1705767 TI - Correlation of serum antigen and antibody concentration with clinical features in HIV infection. AB - Serum antigen and antibody values were studied in 164 infants and children infected perinatally with HIV. HIV antigens p17, p24, gp41, and gp120 were determined in sera by immunoblot and antigen capture assays. Lymphocyte blast transformation, serum immunoglobulins, and circulating immune complexes were also evaluated. Altogether 50 patients had HIV antigens measured: 31 (62%) patients had p17 antigen in the serum and 29 (58%) had p24 antigen present. In 19 (38%) and nine (18%) patients, respectively, gp120 and gp41 were detected. All four HIV antigens were detected in seven (14%) patients. There was a positive correlation between the concentration of each HIV sequential specimens were outcome. When sequential specimens were analysed, 120 (73%) patients had p24 antigen present. Patients with stage P2B and P2D (Centers for Disease Control classification) had the highest concentrations of p24 antigen with a mean of approximately 200 pg/ml. Altogether 70% of patients with a p24 antigen concentration of greater than 30 pg/ml eventually died or had severe clinical disease within six to 24 months. Infants under 15 months of age with a p24 antigen concentration as low as 5 pg/ml also did poorly. Increased immunoglobulins and decreases in mitogenic responses and absolute CD4+ lymphocyte counts were more prevalent in patients with raised p24 antigen. Raised concentrations of circulating immune complexes were seen in the symptomatic phase of the disease whereas in the terminal stage of the disease raised serum antigen and a decrease in circulating immune complexes and absolute CD4+ lymphocyte count were evident. Loss of p24 and/or p17 antibody as well as a decreasing ELISA optical density for HIV antibody also signalled progression of the disease. PMID- 1705769 TI - Electrical parameters of the toad skin: effects of forskolin. AB - Forskolin stimulated short-circuit current (SCC) and transepitelial electrical conductance (G) in the isolated skin of the toad Bufo arenarum in a concentration dependent manner, between 1.0 x 10(-6) and 2.4 x 10(-5) M. At the latter concentration, glandular secretion appeared to be stimulated also. The increase in G was considerably greater in skins bathed in Ringer solution than in solutions containing no chloride. The increased SCC was abolished by amiloride, a specific blocker of sodium transport in amphibian membranes, irrespective of the anion present in the solution bathing the skin. G was also decreased by amiloride to control values in skins bathed in solutions without chloride, but remained elevated in the presence of Cl-. The increase in SCC following exposure to forskolin, 4.4 x 10(-6) M, was not altered when furosemide, a specific blocker of chloride transport, was present in the Ringer solution bathing the dermal side of the skin. The response to forskolin, 2.4 x 10(-5) M, however, was significantly decreased by dermal furosemide; the inhibitor was ineffective in the absence of chloride. The data indicate that forskolin acts on at least two sites: stratum granulosum cells (the main pathway for sodium transport, and an alternate site, responsible for the increase in permeability to chloride. In addition, at high concentration of the agent, glandular secretion is also stimulated. The data suggest that the adenylate cyclase-cyclic AMP system is involved in the regulation of the permeability of the toad skin to sodium and chloride, probably by separate cell types. PMID- 1705770 TI - Interaction of some limbic structures which exert inhibitory effect on corticosterone secretion. AB - The interaction between limbic structures which exert inhibitory influence on corticosterone secretion was investigated in the rat. The following experiments were performed: 1) electrical stimulation at mammillary medial nucleus (MMN) in rats with lesioned anterodrosal thalami nucleus (ADTN) or intermediate tegmental area; 2) electrical stimulation at ADTN in rats with lesioned retrosplenial cortex (RC). Bilateral stimulation at MMN in ADTN or RC-lesioned rats produces an increase in plasma corticosterone concentration. In animals with lesioned RC, values of plasma corticosterone after stimulation at ADTN were higher than before stimulation. Taking into consideration that electrical stimulation of MMN or ADTN in intact rats produces a decrease in plasma corticosterone concentration, these studies demonstrate that MMN and ADTN exert inhibitory influence on corticoadrenal activity only when their projection areas remain intact. PMID- 1705771 TI - Studies on human placental glutathione S-transferase. Multiplicity and mother age influence. AB - Human placental glutathione S-transferase was purified to apparent homogeneity by direct application of the crude homogenate into glutathione linked sepharose affinity chromatography. Chromatofocusing analysis in the presence of reduced glutathione resolved the enzyme into three acidic peaks eluted at pH 6.0, 5.7 and 5.5. About 36% of the initial activity was recovered in the isozyme fraction eluted at pH 6.0 whereas the isozymes eluted at pH 5.7 and 5.5 accounted for 20% and 25% of the activity respectively. Disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a single protein band in all the three separated isozymes. These isozymes were homodimers with an apparent relative molecular mass of 44.000 and subunit molecular mass of 21.000. The isozymes were immunologically related to each other and to the enzyme from goat and sheep placentae. Mother age had no influence in the placental glutathione S transferase activity, albeit the activity was slightly higher in placenta obtained from younger women. PMID- 1705772 TI - Age-related differences in the cardiovascular responses to haemorrhagic shock in rats. AB - The cardiovascular responses to haemorrhagic shock were studied in male Sprague Dawley rats of different age groups, ranging from 6-15 weeks (body weight 250-460 g). Haemorrhagic shock was induced by bleeding (2% body weight), under urethane anaesthesia, from the cannulated femoral artery at a rate of 1 ml/min. It was found that the younger rats had significantly smaller values of left ventricular pressure and dLVP/dtmax following haemorrhage and a greater mortality rate. Older animals exhibited significantly greater falls in blood pressure and pulse rate during the bleeding procedure, and slower recovery in these parameters after the bleeding was stopped. However, these rats had a significantly higher left ventricular pressure and dLVP/dtmax following haemorrhage, and a markedly lower mortality rate. The findings demonstrate the existence of age-related cardiovascular responses to haemorrhagic shock in rats. PMID- 1705773 TI - Toxic impact of phosphamidon on protein degradation in phasic and tonic muscles of marine prawn, Penaeus indicus (H. Milne Edwards). AB - Levels of various protein fractions, (sarcoplasmic, myosin, actin, non-collagen and collagen) and the rate of their degradation by proteases were studied in phasic and tonic muscles of marine prawn, Penaeus indicus following acute (2 d) and chronic (15 d) exposure to sublethal concentration of phosphamidon. During exposure, greater decrease in sarcoplasmic protein fraction was observed in phasic muscle as compared to other myofibrillar proteins. But the sarcoplasmic protein content showed an elevation in tonic muscle. The changes in protein fractions were more pronounced during acute exposure than chronic exposure both in phasic and tonic muscles. These changes were correlated with the elevation of the acidic, neutral and basic protease activities during acute and chronic exposure. Free amino acids were increased during acute exposure, while they showed a significant decrease during chronic exposure in both the muscles. These results indicate that protein metabolism in both phasic and tonic muscles was significantly altered following phosphamidon exposure. These differential responses observed at acute and chronic exposure indicate the operation of compensatory mechanisms to mitigate the phosphamidon toxic stress. PMID- 1705774 TI - Action of cholecystokinin on the dog sphincter of Oddi: influence of anti cholinergic agents. AB - Effects of cholecystokinin (CCK) on bile flow through the sphincter of Oddi (SO) were studied in anaesthetized dogs. Intravenous injection of CCK (0.25, 0.5, 1 and 2 IDU/Kg) elicited a dose-dependent reduction in flow through the SO in the first minutes after CCK administration. Pirenzepine and atropine decreased significantly (P less than 0.05) by 29% and a 40% respectively the inhibitory effect induced by 1 IDU/Kg of CCK, whereas hexamethonium elicited an increase in the inhibitory effect induced by 0.5 IDU/Kg of CCK (P less than 0.05). Intravenous infusion of cummulative doses of CCK had different effects according to the dose infused. Lower doses (0.025 and 0.05 IDU/Kg/min) increased transphincteric flow, however, high doses (0.1, 0.2 and 0.4 IDU/Kg/min) were inhibitory. These finding indicated that CCK had two effects on the SO : firstly, a contractile effect, probably mediated through a direct myogenic action and neuronal release of ACh, and secondly a relaxant effect, probably mediated by stimulation of inhibitory postganglionic neurons. PMID- 1705775 TI - Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat. AB - The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism. PMID- 1705776 TI - Alterations in the physicochemical properties of renal cortical membranes in rats with experimental cirrhosis of the liver. AB - Changes in the major component of renal cortical membranes as well as membrane fluidity and Na+, K+, ATPase activity have been studied in membranes from the renal cortex of rats with experimental liver cirrhosis, which show renal sodium and water retention, and in normal animals. Rats with cirrhosis of the liver show a decrease in cholesterol, phospholipid and protein content, without changes in cholesterol/phospholipid molar ratio. In addition there is a small decrease in 14:0 and 18:2 and an increase in 20:4 content, without differences in unsaturation degree. Membrane fluidity was decreased in renal membranes from cirrhotic rats when compared with normal ones. Na+, K+, ATPase activity was higher in cirrhotic than in normal renal membranes could be related with the changes in renal water and electrolyte changes shown by cirrhotic rats. PMID- 1705777 TI - Erucic acid metabolism in rat heart. A combined biochemical and radioautographical study. AB - Metabolism of Erucic Acid was studied in rat heart in comparison with that of oleic acid, particularly in relation with diet lipids. Rats were fed for 3 or 60 days a diet containing 30% of the calories of either Rapessed Oil, rich in erucic acid or sunflower seed oil rich in linoleic acid. They were I.V. injected with tritiated erucic or oleic acid. After 1 or 15 min the radioactivity recovered in heart lipids was very low whatever the diet (1 to 2%). One minute after injection of erucic acid the radioactivity was mainly recovered in the free fatty acid fraction and as untransformed erucic acid. After 15 min the major part of radioactivity was recovered in the triacylglycerol fraction which contained a high proportion of labelled oleic acid formed by shortening of erucic acid. When oleic was injected, the radioactivity was principally recovered in triacylglycerols as untransformed oleic acid whatever the experimental conditions. Electron microscopy showed that a much higher proportion of peroxisomes, was present in heart cells, following sunflower seed oil diet as compared to rapeseed oil diet. In all cases mitochondria supported the greater part of radioactivity, especially when erucic acid was injected in rats fed rapeseed oil. After sunflower seed oil, a noticeable radioactivity was observed in peroxisomes, most of them containing silver grains, especially when oleic acid was injected. According to the data reported, peroxisomes do not seem more implicated than mitochondria in the metabolism of erucic acid in myocardium. PMID- 1705778 TI - Inotropic effect of hyperosmotic NaCl solutions on the isolated rat cardiac tissue. AB - The inotropic effect of Krebs-Henseleit solution rendered hyperosmotic by addition of NaCl or sucrose (increments of 50, 100, 150 and 200 mOsm/l) on myocardial contractile activity was studied in rat isolated left atria paced at 4, 16 and 64 stim/min. The solutions did not affect the peak tension (Tp) at 4 stim/min, whereas sucrose caused a dose-dependent increase in Tp at 16 stim/min and NaCl decreased Tp at 64 stim/min. The total time duration of the contraction was increased in a dose-dependent fashion by both solutes, but the effect of NaCl was attenuated at 64 stim/min. The results showed that, in the isolated rat atrial tissue exposed to hyperosmotic NaCl solutions, the negative inotropic effect of increased Na+ concentration overcomes the positive influence of hyperosmolality only at higher pacing rates (about 1 Hz). PMID- 1705779 TI - [Modification of precocious evoked auditory potential amplitudes observed in tinnitus]. AB - Brainstem evoked response audiometry has been performed in 139 patients complaining of tinnitus, unilateral or bilateral. Amplitudes of I, III, V waves and the amplitude relations I/III, I/V, III/V have been compared with those obtained in a normal population (n = 20). Amplitudes of waves I and III decreased according to their localization: decrease of left I and III for left tinnitus, decrease of left I and right III for right tinnitus, and for I and III both left and right for bilateral tinnitus. Recording of brainstem evoked response before any therapeutical treatment, especially study of amplitudes, could therefore provide information an localization of tinnitus. PMID- 1705780 TI - Purification and properties of liver arginase from teleostean fish Clarias batrachus (L.). AB - Liver arginase of Clarias batrachus has been purified to 56.3-fold employing ammonium sulphate fraction, DEAE-cellulose and CM-cellulose chromatography. Bidirectional polyacrylamide gel electrophoresis shows the presence of two isoenzymes of arginase. The enzyme has a molecular weight of about 87,000 and Km 15.38 mM for L-arginine, optimum pH 9.5 and temperature 37 degrees C. Ornithine and leucine as competitive whereas valine and isoleucine act as non-competitive inhibitors with respect to L-arginine as substrate. PMID- 1705781 TI - Slight differences between adenosine deaminases from different species an immunochemical study. AB - IgGs against adenosine deaminase from rat brain, rat liver, mouse duodenum and human erythrocyte were purified from rabbit antisera with yields of 82-87%. The inhibition of adenosine deaminase by the antienzyme is studied, and it is demonstrated that rat and mouse antibodies are tight-binding inhibitors. These antibodies inhibit either the rat or the mouse enzymes and do not inhibit the human erythrocytes enzyme. The human antibody does not inhibit either the human or the rat or mouse enzyme. These results indicate that some differences in antigenic behaviour near the active site must be encountered among species. Comparing the sequenced of the two products corresponding to two adenosine deaminase genes recently sequenced (human and murine) a hypothesis concerning the localization of the adenosine deaminase active site is proposed. PMID- 1705782 TI - 6-OHDA sympathectomy and exercise performance in the rat. AB - 6-hydroxydopamine (6-OHDA) was utilised for the study of the sympathetic nervous system in the resting rats and rats submitted to prolonged exercise. In order to reduce the acute physiological stress associated with an injection of 6-OHDA, beta-1 and alpha-1 adrenoceptors were blocked before the treatment leading to sympathectomy. Sympathectomised rats were divided in two groups: one sacrificed at rest, 24 hours after the treatment. The other group was sacrificed after a treadmill exercise to exhaustion. Running time to exhaustion was 36.0 +/- 4.5 min (mean +/- S.E.M.). This group ran significantly less than a control group brought to exhaustion in 73.7 +/- 10.0 min of exercise (P < 0.05). In order to make appropriate comparisons, another control group was run for 36 min. Some differences were observed between corresponding control and sympathectomized groups. At rest: 1) a lower plasma level of insulin, and 2) a higher plasma free fatty acid concentration were observed in sympathectomized rats. After 36 min of exercise: 1) a lower plasma concentration of norepinephrine, 2) no decrease of the plasma level of insulin, 3) no increase in the plasma glucagon concentration, and 4) a higher plasma glucose level were observed in sympathectomised rats when compared to control rats running for the same time. The lower plasma norepinephrine concentration in exercised sympathectomised rats suggests a lower sympathetic nervous activity in these animals than in control rats. The absence of a decrease of plasma insulin concentration and of an increase in glucagon can be attributed to this lower sympathetic activity in sympathectomised rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705783 TI - Effect of extracellular volume expansion on erythrocyte sodium transport in rats. AB - The effects of extracellular volume expansion (EVE) on the major sodium transport systems and sodium and potassium contents in rat erythrocytes have been examined in the present study. Study has been performed in anesthetized Wistar rat weighing about 300 g. Acute extracellular volume expansion (EVE) was induced by a constant intravenous saline infusion (3% body wt, 3 hours). Rats anaesthetized and catheterized but not expanded were used as controls. Arterial blood samples from control and expanded rats were obtained at the same time, and assayed immediately. Intracellular sodium and potassium concentration and ouabain sensitive (Na(+)-K(+)-pump) and bumetanide sensitive (Na(+)-K(+)-cotransport system) outward Na+ fluxes in erythrocytes were measured. The effect of plasma on erythrocyte transport was also analyzed by measuring 86Rb uptake. Neither of two plasma cations (Na+ and K+) were modified by the EVE. Also intracellular Na+ and K+ levels remained unvariable. Total Na+ efflux was not modified by EVE, but pump mediated Na+ efflux was smaller after than before EVE. The ouabain-inhibible Na+ efflux rate constant decreased after EVE (from 687 +/- 81 to 525 +/- 29 h-1 x 10( 3); P less than 0.05). Both Na(+)-K(+)cotransport-mediated Na+ efflux and passive permeability increased significantly after EVE. The incubation with plasma from saline-infused animals induced a significant decrease in Rb uptake rate constant, that was not observed after incubation with plasma from non-expanded rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705784 TI - [Alpha 2 adrenergic control of ventilation in the rat]. AB - In anaesthetized rats, ventilatory stimulation induced by phentolamine, an alpha sympatholytic agent, emphasizes the role of some adrenergic mechanisms in the control of the respiratory centres activity. Phentolamine (5 and 10 mg.kg-1, iv) stimulates ventilation after a 4 s latency, tidal volume and respiratory rate being both increased. A same response can also be provoked 10 min later, by a second identical iv administration, systemic blood pressure remaining then stable at its previous low level. Hyperventilation is also observed when phentolamine is injected in totally denervated rats, without any remaining baro- or chemosensitivity. Stimulation is thus due to a central activity in relation with the release of inhibitory influences. Phentolamine also causes hyperventilation after prazosin pretreatment indicating that the alpha 1 adrenergic blockade is not involved in the post-phentolamine stimulation. This is an alpha 2 adrenergic transmission dependent mechanism. Variation of the systemic blood pressure is not the main mechanism involved in the hyperventilation induced by phentolamine. Meanwhile, baroreceptor activity modulates the central response to the drug, as shown by the negative influence of the post-vasopressin arterial hypertension. Hyperoxia is also a modulating factor acting by two ways: an inhibition of the peripheral chemoreceptors activity is added to an arterial hypertension. On the other side, activation of these chemoreceptors by almitrine bismesilate increases the respiratory responses to phentolamine. As already shown by one of us (Lagneuax, 1986), phentolamine pretreated rats are more responsive to hypoxia and to almitrine. Moreover, these phentolamine pretreated rats are protected against cardiovascular collapses and against apnea, frequently observed during hypoxia without CO2 compensation. PMID- 1705785 TI - Production of superoxide anion and hydrogen peroxide by KB cells in an anoxia reoxygenation model, and role of allopurinol. PMID- 1705786 TI - Striated muscle heteroplasia in the uterine round ligament. A report of 30 cases. AB - The presence of nonneoplastic mature striated muscle, ie, striated muscle heteroplasia, is described in the proximal stumps of the round ligaments of the uterus. It was an incidental finding in 30 (2.8%) of 1064 surgical specimens studied without clinical or pathologic significance. It is deemed to represent aberrant persistence, differentiation, and maturation of gubernacular rhabdomyoblasts, which normally involute in female fetuses. The reasons for its sporadic occurrence in normal adult women are unknown. PMID- 1705787 TI - Relationships between structure and induction of hyaline droplet accumulation in the renal cortex of male rats by aliphatic and alicyclic hydrocarbons. AB - A rapidly growing list of hydrocarbons has been reported to induce morphological changes in the kidney of adult male rats, beginning with hyaline droplet accumulation (HDA) followed by the development of granular casts, later on chronic nephrosis as sequela, and finally renal adenomas and carcinomas. The present study focuses on identifying structure-based properties common to HDA inducing aliphatics and cycloaliphatics. On the basis of rank-ordered activities reported in the literature, a calculated n-octanol-water partition coefficient above 3.5 and the presence of an isopentyl structural moiety appear to be associated with HDA-inducing activity in aliphatics. A binding site model for highly active aliphatics has been derived by superimposing their minimum energy conformations along the common isopentyl substructure and calculating the union volume of their respective van der Waal (VDW) volumes. Generalization of this model to include cycloaliphatics has been achieved by maximizing the steric overlap of the VDW volumes of the compounds with their binding site union volume. HDA-inducing cycloaliphatics are correctly identified on the basis of their negligible excess volume. This approach has been used to predict the HDA-inducing activity of previously untested compounds. Eighteen aliphatic/cycloaliphatic hydrocarbons were screened in a study on adult male Wistar rats treated with 250 mg/kg per day for 5 days. Azan-stained kidney sections were semiquantitatively evaluated for the presence of HDA. The predicted and observed HDA activities were in very good agreement. PMID- 1705788 TI - VP4 relationships between porcine and other rotavirus serotypes. AB - VP4 relationship of Australian porcine rotaviruses were identified using genetic reassortants and MAbs. All porcine virus isolates except BEN-144 appeared to share VP4 antigenicity with OSU virus. VP4 and BEN-144 virus (Gottfried-like virus) showed some antigenic relationships with the human neonatal viruses ST-3 and RV-3. In addition, VP4 of porcine CRW-8 showed antigenic relationships with simian SA-11. RRV and also canine K9 viruses, while that of porcine TFR-41 showed at least one way VP4 antigenic relatedness with UK bovine rotavirus. Furthermore, BMI-1 virus which is antigenically similar to an American virus SB1-A (a naturally occurring reassortant) may have arisen similarly by gene reassortment in nature in Australia. PMID- 1705789 TI - Neutralizing determinants of canine herpesvirus as defined by monoclonal antibodies. AB - Monoclonal antibodies (MoAbs) against canine herpesvirus (CHV) were produced to identify the immunogenic proteins of the virus carrying neutralizing determinants. A panel of 24 MoAbs showing neutralizing activities was obtained and tentatively classified into 3 different groups based on their reactivity patterns in immunoblotting analysis. Group I consisting of 10 clones was specific for 145/112 kDa; Group II of 9 clones, for 80 kDa; and Group III of 5 clones, for 41 kDa glycoproteins (gps). Complement-requirement for neutralizing activities of the MoAbs suggests that gp 145/112 and gp 80 elicit mainly complement-requiring and -enhanced neutralizing antibodies, while gp 41 elicits complement-independent ones. In addition, these MoAbs were used in ELISA additivity tests for functional and topographical mapping of epitopes in each of the CHV gp. The results indicated that antigenic reactivities of gp 145/112 and gp 80 were, respectively, localized on at least 5 and 7 overlapping epitopes. On the other hand, 4 epitopes were identified on gp 41. PMID- 1705790 TI - Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes. AB - Monoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents. PMID- 1705791 TI - [Inhibitory effect of FK-506 on the development of late asthmatic response and on the increased bronchial responsiveness]. AB - We examined the effects of FK-506, a potent immunosuppressive agent, on the development of late asthmatic response (LAR) and on the increased bronchial responsiveness to acetylcholine following LAR in guinea pig model of asthma. Guinea pigs sensitized by repeated inhalation of ovalbumin (OA) were intravenously given metopiron 24 hours before and 30 minutes before antigen challenge and to prevent death from immediate severe bronchoconstriction, chlorpheniramine maleate was also injected. When we defined LAR as the responses with a two-fold increase in respiratory resistance during the late phase of antigen challenge, twelve out of fifteen control animals demonstrated apparent LAR. However, when guinea pigs were treated with FK-506 from the beginning of immunization period, the development of LAR was completely inhibited, although similar magnitude of immediate bronchoconstriction was observed, and a subsequent increase in bronchial responsiveness was significantly blocked. We also measured bronchial responsiveness to acetylcholine before, 24 and 72 hours after antigen challenge. FK-506-treated animals inhibited an increase in bronchial responsiveness to acetylcholine. These results suggest that the involvement of cell-mediated immunity may be important in the development of LAR and an increase in bronchial responsiveness. PMID- 1705792 TI - [Autotomy of sensory nerve endings in the area of microvessels]. AB - The phenomenon of autotomy of peripheral receptor terminals, in the area of the intestinal microvessel bed has been analysed. This phenomenon is accompanied with a real separation of the terminal patches or large fragments of receptors with their successive degeneration. The effect of the terminals autotomy is succeeded in reproducing and analysing in dynamics in tissue culture. The computer analysis of the image demonstrates that autotomy is observed not only in developing structures during early period of ontogenesis, but in well developed, as regards all morphological criteria, tissue receptors of mature animals. The investigations have been performed by means of silver nitrate impregnation after Bielschowsky--Gros, as well as using vital microscopy. An idea has been formed that autotomy is a part of the cyclic process of autotomy--regeneration of the terminals. This process is connected with retractile cytopoiesis, apocrine secretion. A high concentration of proteolytic enzymes in the amputated degrading fragment of the receptor can serve as a trophic factor, activating the local metabolic process in the area of microvessels. PMID- 1705793 TI - [Vascularization of the myenteric plexus in the small intestine of pigeons]. AB - By means of light and electron microscopy vascularization of the myenteric plexus has been studied in the pigeon small intestine. Ganglia of the plexus, their cell composition, ultrastructure of neurons have been described. Links of the microcirculatory bed of the intramural ganglia are characterized, interrelations of capillaries with neurons are described, quantitative estimation of microhemovessels, surrounding the microcirculatory bed of the myenteric plexus in the intestinal wall in birds and mammalia. PMID- 1705794 TI - [Intraspinal organ in man]. AB - In the ependymal zone of the spinal cord at the LI-SIII level an ependymal glandular organ has been described. Its highest secretory activity coincides with the period of the greatest functional activity of the human reproductive system functioning. Certain stages in development of the organ, its cell composition, blood supply, afferent and efferent innervation have been studied. In secretory cells of the organ peptide with cardio- and vasotonic properties has been identified immunochemically. The action of the organ is considered in connection with role, which the ependymal glands of the brain play in regulation of the organism's function. PMID- 1705795 TI - [Hypoplastic left heart syndrome. Clinical evaluation of 40 patients]. AB - PURPOSE: To study the hypoplastic left heart syndrome (HLHS) emphasizing clinical aspects and therapeutic measures. PATIENTS AND METHODS: Forty patients, 24 male, ages ranging between one to 120 days (mean six days). Diagnosis was established by clinical elements, eletrocardiogram, chest X-ray and Doppler echocardiogram. Eight patients underwent cardiac catheterization. In 21 patients anatomical diagnosis was confirmed by necropsy. RESULTS: Twenty-two patients (55%) died, in spite of all possible clinical measures. In 16 (40%) palliative surgery was performed, Norwood technique in 14 and pulmonary artery banding with Dacron graft interposition between pulmonary trunk and brachiocephalic trunk in two. Immediate postoperative results were favorable in three patients (Norwood technique). Both patients underwent the other described technique died at 12th and 30th postoperative day due to infection. CONCLUSION: HLHS is a severe cardiac anomaly with a fast downhill evolution mostly at first week of life. The good results depend basically of early diagnosis as well as of an adequate clinical management for an appropriate time in indication of the palliative surgical technique. PMID- 1705796 TI - Comparison of postmortem magnetic resonance imaging and neuropathologic findings in the cerebral white matter. AB - Two types of high-signal intensity abnormalities are frequently found bilaterally in the cerebral white matter of brains of elderly patients on T2-weighted magnetic resonance imaging (MRI) scans. One is located in the immediate periventricular region; the other, in the deep subcortical white matter (centrum semiovale). The diagnostic implications of this second type continue to be uncertain. To determine the neuropathologic correlates of these lesions, the brains from seven elderly patients were fixed in buffered formaldehyde solution, subjected to MRI scanning, and examined neuropathologically. Variable degrees of bilateral periventricular (subependymal) sharply defined areas of high-signal intensity were found in all the brains, and the larger of these showed corresponding areas of myelin pallor with gliosis and dilated perivascular spaces. Discrete bilateral patches of high-signal intensity were found in the centrum semiovale in five patients. Myelin and axon stains showed varying degrees of diffuse white matter pallor in many areas examined, both with and without these areas of high-signal intensity on MRI scans. Neither the myelin nor the axon stains showed discrete white matter abnormalities that corresponded to the MRI findings. We believe that these changes, so commonly found on MRI scans in the elderly, reflect actual changes in the white matter but that their nature and clinical significance need to be elucidated. PMID- 1705797 TI - Tumour necrosis factor-alpha immunodetection in blood monocytes and serum: preliminary findings in weight-losing cancer patients. AB - The peptide tumour necrosis factor-alpha (TNF-alpha) is a central mediator of the host response. Identifying where and when TNF-alpha is produced may give insights into its potential role in various pathophysiological states. This paper describes a quantitative analysis of TNF-alpha expression in peripheral blood monocytes (PBM) at the single cell level. A pilot study has been undertaken, using this method to assess TNF-alpha expression in PBM from healthy volunteers and cancer patients. We also report mildly elevated serum TNF-alpha levels in the cancer patients, using an immunoradiometric assay (IRMA) sensitive to 1 pg/mL of recombinant TNF-alpha. The results of this preliminary investigation suggest that TNF-alpha production may be altered in cancer patients. PMID- 1705798 TI - Extracellular ATP causes lysis of mouse thymocytes and activates a plasma membrane ion channel. AB - Extracellular ATP (ATPo) caused a concentration-dependent lysis of mouse thymocytes. Lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase, was preceded by depolarization of the plasma membrane and by Ca2+ influx. Both Na+ uptake (which sustained plasma membrane depolarization) and Ca2+ influx showed (1) the same dependence on the ATPo concentration; (2) the same nucleotide specificity; and (3) the same Hill coefficient. However, whereas the rise in the cytosolic free Ca2+ concentration ([Ca2+]i) was fully inhibited by the known Ca2+ blocker verapamil, plasma membrane depolarization was enhanced under these conditions. Plasma membrane depolarization was greater and was shifted to lower ATPo concentrations in the absence of extracellular Ca2+ (Ca2+o), whereas the rise in [Ca2+]i was greater in Na(+)-free media. Plasma membrane depolarization also occurred in Na(+)-free choline- or methylglucamine containing media, and was potentiated by chelation of free divalent ions with EDTA, supporting previous reports pointing to ATP4-as the active species. Among a number of purine and pyrimidine nucleotides, only adenosine 5'-[gamma thio]triphosphate and ADP were partially effective. Furthermore, ethidium bromide (Mr 380), Lucifer Yellow (Mr 463) and Eosin Yellowish (Mr 692) did not permeate through the ATPo-activated channel. These findings suggest that lytic effects of ATPo in mouse thymocytes depend on the activation of a membrane channel with low selectivity for cations and an Mr cut-off of 200. PMID- 1705799 TI - Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy. AB - Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are 'cryptic' and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes. PMID- 1705800 TI - Wide cavum septum pellucidum: a marker of disturbed brain development. AB - A wide cavum septum pellucidum defined as a separation of greater than 1 cm of the leaves occurs uncommonly. Nine children with wide cavum septum pellucidum were studied; 8 were abnormal. Observed abnormalities included cognitive impairment (8), seizures (4), hypoplasia of the corpus callosum (4), optic nerve hypoplasia (2), and growth failure (4). The incidence of intellectual dysfunction, the association with midline anomalies of the brain, and growth failure all suggest that wide cavum septum pellucidum may represent part of a spectrum of midline brain anomalies. PMID- 1705801 TI - Structure in lissencephaly determined by immunohistochemical staining. AB - The brains of patients with lissencephaly were examined by peroxidase antiperoxidase immunohistochemical staining of synaptophysin, myelin basic protein, and glial fibrillary acidic protein. In contrast to the normal cortical pattern, the cortex, with a smooth surface, demonstrated quite different staining patterns in the molecular, superficial cellular, sparsely cellular, and deep cellular layers. The molecular layer was abnormally positive with synaptophysin staining. The superficial cellular layer was also diffusely stained for synaptophysin; there was a positive reaction in the linearly arranged myelin sheaths. The sparsely cellular layer revealed less staining for synaptophysin, but was perivascularly positive for glial fibrillary acidic protein. In the deep cellular layer, synaptophysin staining had multiple neuronal columns and myelin basic protein-staining had a reticular pattern around neuronal columns. These results suggest that the sparsely cellular layer may correspond to the molecular layer and white matter in normal brain; neurons with forming myelin sheaths in the superficial cellular layer regularly penetrate the surface of the molecular layer, forming arrested cortical columns in the deep cellular layer. PMID- 1705802 TI - Interactions between cytokines and alpha 2 macroglobulin. PMID- 1705803 TI - Electron field within an electron field treatment of the scalp or other curved surfaces. AB - The treatment of a curved surface is a challenge for radiation therapy. This patient had previous treatment with electrons to the "skull cap" area for malignant melanoma. The current task was to design a treatment schema to include the rest of the scalp while avoiding the skull cap. Opposing lateral 6-MeV electron fields were used, as well as two anterior 6-MeV electron fields, one field within the other, to produce an acceptable radiation dose to the tumor volume. PMID- 1705804 TI - First trimester maternal serum alpha-fetoprotein screening for Down syndrome and other aneuploidies. AB - Low maternal serum AFP (MSAFP) values in the first trimester of pregnancy have been associated with an increased risk for chromosome disorders. In our own first trimester chorionic villus sampling (CVS) series, MSAFP determinations were carried out in 1,448 singleton pregnancies. Aneuploidies were detected in 26 of these. The pre-CVS MSAFP values in these pregnancies were compared to those in pregnancies with normal outcome. Statistical analysis did not show a diagnostically useful correlation between low first trimester MSAFP values and aneuploidy in our cohort. PMID- 1705805 TI - Prevention of hepatitis C in partners of anti-HCV positive subjects by periodic gammaglobulin administration. AB - Since hepatitis C is sexually transmitted, this poses the problem of protecting the anti-HCV negative partners of anti-HCV positive subjects. Since no specific prophylaxis exists for hepatitis C, I suggest that 4 ml of normal human gammaglobulin should be given to the anti-HCV negative partners intramuscularly every two months until a vaccine against hepatitis C virus is available. PMID- 1705806 TI - Phenotypic changes in hepatic mast cells accumulating around the metacestodes of Taenia taeniaeformis in rats. AB - Staining properties, kinetics and degranulation of the hepatic mast cells (HMC) accumulating around the metacestodes of Taenia taeniaeformis in the liver of rats were studied. Two different types of HMC, designated as Type I and Type II, could be classified according to histochemical properties and response to compound 48/80. Type I cells resembled mucosal mast cells (MMC), whereas Type II did not. HMC, mostly Type I, were observed from day 14 postinfection (PI), while Type II were seen only from day 28 PI. On day 28 PI, Type II represented a transitional staining pattern between MMC and connective tissue mast cells (CTMC). The ratio of Type II to Type I increased gradually with the course of infection and was about 1 to 1 on day 70 PI. At this time, most of the HMC that constituted Type II as well as CTMC could be stained with berberine sulfate. While the phenotypic change of HMC to CTMC was found in the middle and inner capsular layers, most of the HMC in the outer capsular layer maintained the phenotype of MMC. The present results suggest that hepatic mast cells increased as the MMC phenotype, then showed the heterogeneity in which the transitional form of mast cells emerged followed by the appearance of the CTMC type. PMID- 1705807 TI - Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins. AB - Extracellular matrix (ECM) glycoproteins regulate neuronal development and axonal growth. In this paper, the ECM glycoprotein vitronectin was identified and localized in the embryonic chick neuroretina. To identify potentially important neurite outgrowth-promoting molecules, responses of embryonic chick retinal neurons to vitronectin and thrombospondin, another retinal ECM constituent, were examined. These neurons were shown to attach and extend neurites on either glycoprotein. Integrins containing the alpha v or beta 1 subunits mediate both responses to vitronectin and neurite outgrowth on thrombospondin. Attachment to thrombospondin was inhibited by heparin, suggesting that neurons also utilize a proteoglycan or sulfated glycolipid as a receptor for this glycoprotein. Thus, retinal neurons use specific receptors to interact with vitronectin and thrombospondin, two glycoproteins present in the embryonic neuroretina, suggesting roles for these ligands and their receptors in retinal development. PMID- 1705808 TI - Expression of acidic fibroblast growth factor mRNA in the developing and adult rat brain. AB - We have localized acidic fibroblast growth factor (aFGF) mRNA in the developing and adult rat brain using in situ hybridization histochemistry. Prenatally, hybridization to aFGF mRNA was observed throughout the brain, with the strongest signal associated with cells of the developing cortical plate. Postnatally, labeling was localized to specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer and dentate granule cells was observed and became progressively more intense with maturation. Labeling was also observed in both the external and internal granule cell layers of the developing cerebellum. Pyramidal cells of the neocortex as well as neurons of the substantia nigra and locus ceruleus also express aFGF. This pattern persists into adulthood, although the intensity of the labeling is significantly reduced in the adult brain. These patterns of hybridization correlate with specific developmental events and suggest that aFGF plays a significant role in both central nervous system development and neuronal viability in the adult brain. PMID- 1705809 TI - Substance P and neurokinin A in human nasal mucosa. AB - The tachykinins substance P (SP) and neurokinin A (NKA) were studied in human inferior turbinate nasal mucosa by radioimmunoassay, immunohistochemistry, and autoradiography and for their effect upon mucus release in an in vitro culture system in order to infer their potential functions in the upper respiratory tract. Similar amounts of SP (1.03 +/- 0.12 pmol/g wet weight; mean +/- SEM; n = 26) and NKA (0.76 +/- 0.23; n = 7) were found. NKA and SP immunoreactive nerve fibers were found in the walls of arterioles, venules, and sinusoids and as individual fibers in gland acini, near the basement membrane, and in the epithelium. [125I]SP bound to arterioles, venules, and glands. [125I]NKA bound only to arterioles. In short-term explant culture of fragments of human nasal mucosa, both 1 microM SP and 1 microM NKA stimulated release of [3H]glucosamine labeled respiratory glycoconjugates. These results indicate that SP and NKA have similar distributions in nociceptive sensory nerves in human nasal mucosa. The distribution of [125I]SP binding sites is consistent with a role for SP as a vasodilator and mucous secretagogue. The presence of [125I] NKA binding sites on vessels suggests a primary role for NKA in regulating vasomotor tone. PMID- 1705810 TI - Monocyte-macrophage differentiation induced by human upper airway epithelial cells. AB - We examined the ability of conditioned medium (CM) generated by human upper airway epithelial (Ep) cells from normal (NN) and inflamed, allergic rhinitis (AR) and nasal polyp (NP) tissues to induce monocytic differentiation of hemopoietic progenitors of the HL-60 myeloid leukemia cell line in vitro. In HL 60 cells cultured in RPMI with 10% FBS, there was differentiation to 0.4 +/- 0.4% monocytic cells. NN-, AR-, and NP-EpCM induced differentiation to 23 +/- 6%, 42 +/- 11%, and 71 +/- 10% monocytic cells, respectively. EpCM also induced isolated peripheral blood nonadherent mononuclear cells to express monocyte/macrophage specific antigens as detected by immunohistochemistry using FMC-32 monoclonal antibodies (anti-CD14). We also examined the cytokine content of these EpCMs and found that they contained granulocyte/macrophage colony-stimulating factor (GM CSF): 126 +/- 35, 198 +/- 22, and 489 +/- 118 pg/ml for NN-, AR-, and NP-EpCM, respectively. These CMs also contained granulocyte-CSF (G-CSF) and interleukin-6 (IL-6), but there were no significant differences between normal and inflamed tissue-derived cell supernatants. No macrophage-CSF (M-CSF) was detected in these EpCMs. Recombinant human GM-CSF, G-CSF, and IL-6, alone and in combinations, at doses similar to or greater than those found in the EpCMs, did not induce comparable monocytic differentiation of HL-60 cells. Preincubation of the EpCM with neutralizing anti-GM-CSF, anti-G-CSF, or anti-IL-6 antibodies did not significantly inhibit the monocytic differentiation induced by the EpCM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705811 TI - Cyclic AMP-mediated inhibition of cell growth by prostaglandin D2 in a fibroblastic cell line (EBTr). AB - We have recently reported that prostaglandin D2 (PGD2) specifically elevates the intracellular cyclic AMP (cAMP) level in a fibroblastic cell line derived from bovine embryonic trachea (EBTr) (K. Sugama, T. Tanaka, H. Yokohama, M. Negishi, H. Hayashi, S. Ito, and O. Hayaishi, Biochim. Biophys. Acta, 1011: 75-80, 1989). Since the in vitro and in vivo inhibitory effects of PGD2 on cell growth are attributed to the dehydrate product 9-deoxy-delta 9,12-13,14-dihydro-PGD2 (delta 12-PGJ2), the role of cAMP as a mediator of PGD2-induced cell growth has been questioned. In this study we investigated the effects of PGD2 on cAMP level, DNA synthesis, and c-myc expression using EBTr cells growth-arrested by serum starvation. Quiescent EBTr cells synchronously resumed [3H]thymidine incorporation at 12 h after the addition of serum, an incorporation which ended at 21 h. PGD2 at 1 microM inhibited approximately 30% incorporation without any delay of initiation, and it also caused a rapid increase in the cAMP level at a maximum of 10 min. The inhibition of [3H]thymidine incorporation was increased 40 45% by 5 microM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The expression of c-myc mRNA induced by serum at 1-4 h was also inhibited by PGD2. Time-course studies support the association between the reduction of c-myc expression and the successive inhibition of DNA synthesis by PGD2. The dose dependency of PGD2 for these three processes correlated well with each other, and the effects were evident at as low as 10 nM and specific for PGD2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705812 TI - Tiazofurin down-regulates expression of c-Ki-ras oncogene in a leukemic patient. AB - The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis. PMID- 1705813 TI - Tenascin in mammary gland development: from embryogenesis to carcinogenesis. PMID- 1705814 TI - [The prevention and prenatal diagnosis of neural tube defects]. AB - The AA. provide with the schemes for the study of Neural Tube Defects (D.T.N.) yet the continuation and the prevention of NTD'S clinical cases. Also, the AA. show a protocol of action. It is indispensable for all the Spina Bifida Sections to work together by means of an Orthogenesis and Familiar Planning Unit. PMID- 1705815 TI - The treatment of squamous cell carcinoma of the paranasal sinuses. AB - Of 72 cases of paranasal sinus malignancy that were identified, 47 were biopsy proven squamous cell carcinomas. Actuarial analysis of these patients receiving both radical and palliative treatment revealed an overall 5-year survival of 37.4%, and a relapse-free survival of 31.6%. For patients receiving radical treatments (i.e. radiotherapy alone, or preoperative radiotherapy followed by elective surgery), the 5-year survival results were identical (46%). In this series radiotherapy alone is an effective treatment, and subsequent planned surgery did not improve the survival. PMID- 1705816 TI - Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. AB - The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct. PMID- 1705817 TI - Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro. AB - A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells. PMID- 1705818 TI - Bleomycin-dependent damage to the bases in DNA is a minor side reaction. AB - The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation. PMID- 1705819 TI - Epitope mapping of antibodies to acetylcholine receptor alpha subunits using peptides synthesized on polypropylene pegs. AB - Concurrent synthesis of overlapping octameric peptides corresponding to the sequence of the Torpedo acetylcholine receptor (AChR) alpha subunit has been carried out on polypropylene supports functionalized with primary amino groups according to a method developed by M. Geysen [(1987) J. Immunol. Methods 102, 259 274]. The peptides on the solid supports have been used in an enzyme-linked immunosorbent assay. Interactions of the synthetic peptides with antibodies are then detected without removing them from the solid support. By this procedure, epitopes of both antisera and monoclonal antibodies to the Torpedo acetylcholine receptor, its subunits, and synthetic peptide fragments have been mapped. Both rat and rabbit antisera to the alpha subunit show major epitopes spanning the residues 150-165, 338-345, and 355-366 on the Torpedo AChR alpha subunit. Epitopes of monoclonal antibodies to these major epitopes and to others have been rather precisely mapped by using this technique with peptides of varying lengths. The specificity of several of these mAbs are of interest because they have been used in mapping the transmembrane orientation of the AChR alpha-subunit polypeptide chain. PMID- 1705820 TI - Alterations in mitochondrial Ca2+ flux by the antibiotic X-537A (lasalocid-A). AB - A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results. PMID- 1705821 TI - An analysis of the topography of molecular forms 1 and 3 of rat liver biliverdin reductase using polyclonal antibodies. AB - Rat liver biliverdin reductase exists in two molecular forms. The major one (molecular form 1) is transformed, under conditions of oxidative stress into another molecular form (molecular form 3) which is an S-S bridged dimer of form 1. The chemical modifications of the thiol, arginine and lysine residues of molecular form 1 which resulted in an inhibition of its catalytic activity did not affect the activity of molecular form 3. Rabbit polyclonal antibodies raised against form 1 did not recognize form 3. This lack of recognition persisted even when the dimer (form 3) was denatured with SDS or urea under non-reductive conditions. Reduction of form 3 with reduced thioredoxin gave the monomeric form 1, which was fully recognized by the antibodies. The latter recognized the biliverdin reductases from rat spleen and kidney to the same extent as they did with form 1. Molecular form 1 was completely inhibited by the addition of the antibodies. This inhibition was prevented by preincubation of the enzyme with either the substrate (biliverdin) or the cosubstrate (NADPH). Preincubation with the latter or with NADP+ (but not with bilirubin) strongly impaired the recognition of form 1 by the antibodies. Modification of the lysine or arginine residues of form 1 which were involved in substrate binding, impaired the interaction of the enzyme with the antibodies. The antisera blocked the enzymatic conversion of form 1 to form 3, but alkylation of the thiol residue involved in this dimerization had no effect on the interaction of form 1 with the antibodies. The lack of recognition of form 3 by the antibodies suggest that the antigenic site of the former becomes buried upon dimerization. PMID- 1705822 TI - Prenatal and postnatal expression of mRNA coding for rat prothrombin. AB - The levels of prothrombin mRNA in prenatal and postnatal rat tissues were analyzed in order to determine tissue distribution of prothrombin expression and to determine if increases in liver prothrombin mRNA during development correlated with previously documented developmental increases in plasma prothrombin levels. Maternal tissues were also analyzed in order to determine if prothrombin mRNA levels varied due to gestational or postpartum influences. Northern analysis demonstrated that rat liver prothrombin mRNA levels increased several-fold late in gestation and reached maximal levels by 13 days after birth. Prothrombin mRNA was also expressed in diaphragm, stomach, intestine, kidney, spleen and adrenal tissues during development. In maternal tissues during pregnancy, prothrombin mRNA was expressed in liver, diaphragm, stomach, uterus and placenta. Prothrombin mRNA levels in each of these tissues that were positive by Northern analysis were quantitated by solution hybridization analysis. Between gestational day 18 and postnatal day 13, liver prothrombin mRNA levels increased from approx. 600 to 2100 molecules per cell (a 3.5-fold increase). In maternal liver during pregnancy, between day 18 and day 22, prothrombin mRNA levels increased from approx. 1800 to 2100 molecules per cell. Immediately after delivery, maternal liver prothrombin mRNA levels decreased to approx. 50% of preparturition levels. Prothrombin mRNA levels in placental tissue ranged from approx. 100 to 250 molecules per cell. In other fetal, postnatal and maternal tissues, prothrombin mRNA expression was less than 100 molecules per cell. These results demonstrate that the level and tissue-type expression of prothrombin mRNA varies in response to prenatal and postnatal influences. PMID- 1705823 TI - Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas. AB - The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK 8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP 1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system. PMID- 1705824 TI - Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha. AB - The mitogenic-adipogenic effect exerted by arachidonic acid, which leads to terminal differentiation of Ob1771 mouse preadipocytes, has been shown to be (i) blocked by cyclooxygenase inhibitors, (ii) mimicked by a stable analogue of prostacyclin (carbaprostacyclin) and (iii) potentiated by PGF2 alpha. Since these prostanoids are known to be synthesized and secreted by preadipocytes, we have proposed that both prostacyclin as the key mediator and PGF2 alpha as a modulator control the expression of terminal events of adipose conversion by means of an autocrine mechanism (Gaillard, D. et al. and Negrel, R. et al. Biochem. J. (1989) 257, 389-397 and 399-405). In order to test this hypothesis, the release of prostacyclin, characterized under the form of its stable degradation product 6 keto-PGF1 alpha, and that of PGF2 alpha have been studied in the culture medium of Ob1771 cells. A striking increase in the release of 6-keto-PGF1 alpha and to a minor degree of PGF2 alpha was observed when cells were exposed to arachidonic acid as shown by using [3H]arachidonic acid prelabelled cells or by radio immunoassays. Since antagonists of PGF2 alpha and PGI2 receptors were not available, specific antibodies directed against PGF2 alpha and 6 beta-PGI1, another stable analogue of prostacyclin, were added as neutralizing agents in the culture medium. These antibodies were able to counteract the mitogenic-adipogenic effect of arachidonic acid. Prostacyclin and PGF2 alpha thus appear as autocrine mediators in the process of adipose conversion. PMID- 1705825 TI - Effect of pentosan polysulphate, standard heparin and related compounds on protein kinase C activity. AB - Ca2+/phospholipid-dependent protein kinase (PKC) was inhibited by sulphated polysaccharides. Pentosan polysulphate (PPS) and heparin were 8-10-times more potent than dextran sulphate or heparan sulphate. Steady-state studies revealed that PPS was a competitive inhibitor with respect to ATP with an apparent Ki value of 0.32 micrograms/ml and a non-competitive inhibitor with respect to histones. In contrast, the inhibition of PKC by heparin was competitive with substrate and non-competitive with respect to ATP. The interaction of sulphated polysaccharides with the catalytic domain of PKC was further demonstrated by the absence of effect on [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC. Furthermore, PPS and heparin inhibited equally cAMP-dependent protein kinase and tyrosine protein kinase. Structure-function relationships indicated that the Inhibition of protein kinases by PPS and heparin fractions was highly dependent on molecular weight. Additionally, PKC-affinity chromatography revealed that a high-molecular-weight heparin fraction with strong anti-PKC activity was eluted. We set out to demonstrate that heparin and PPS, which are potent antiproliferative agents on vascular smooth muscle cells (SMC), alter intracellular PKC activity (both membrane and cytosolic). Therefore, it is suggested that the mechanism by which sulphated polysaccharides inhibit SMC growth may be by direct inhibition of PKC in SMC. PMID- 1705826 TI - Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase. AB - Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5' dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol. PMID- 1705827 TI - [The spatial reorganization of the perinucleolar area of hematopoietic cells in human leukemias]. AB - The structure of perinucleolar zone in nuclei of peripheral blood hemopoietic cells has been studied. The cells of human patient with chronic lympholeukemia, chronic myeloleukemia, various forms of acute leucosis have been investigated. The phenomenon of radial strings and border structures has been found in these cells. The features have been revealed by visualization of ribonucleoproteides and lipoproteins. The same process takes place in human lymphocytes after 6-24 h of 3-phosphoglyceric aldehyde stimulation in vitro. A comparative study of morphologic peculiarities of hemopoietic cell nucleolar apparatus depending on cell cycle phase has been performed. The action of cytostatics upon the structure of nucleus perinucleolar zone has been investigated. PMID- 1705828 TI - The pharmacology of hemoglobin switching: of mice and men. PMID- 1705829 TI - Mutations of the p53 gene in lymphoid leukemia. AB - p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase-polymerase chain reaction, single strand conformation polymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemias were examined and two with acute lymphoblastic leukemia and one with Waldenstrom's macroglobulinemia were identified to have mutations in the coding region of the p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore, expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias. PMID- 1705830 TI - Serum hepatitis C virus sequences in posttransfusion non-A, non-B hepatitis. AB - We investigated 17 patients (12 males and 5 females, ages 2 to 57 years old) with posttransfusion non-A, non-B hepatitis to determine relationships between clinical courses and hepatitis C virus (HCV) markers. The patients were grouped according to time course of abnormal serum alanine aminotransferase (ALT) levels into three categories (chronic biochemical disease, biochemically resolved chronic disease, and acute disease). Latest serum samples (1.3 to 10.8 years after blood transfusion) were used to detect antibodies against C100-3 antigen (anti-HCV) by enzyme-linked immunosorbent assay and HCV sequences by polymerase chain reaction (PCR) assay. Of the 17 patients, 13 patients (76.5%) were anti-HCV positive and 8 patients (47.1%), including one anti-HCV negative case, were positive for HCV RNA. In total, 14 patients (82.4%) were positive for either HCV markers. With respect to clinical course, HCV RNA was detected in six of eight patients (75%) with chronic biochemical disease, and in two of five patients (40%) with biochemically resolved chronic disease. HCV RNA was not detectable in convalescent sera from four patients with acute disease. These results show that there is a relationship between clinical status and HCV viremia, but that normal liver function tests do not always represent the clearance of the virus. Viremia in two patients with normal ALT level suggests that hepatitis is not only caused by viral cytopathic effects, but also by immunologic reactions against virus infected cells. Thus, PCR is useful in determining the persistence of HCV infection as well as to diagnose anti-HCV negative HCV infection. PMID- 1705831 TI - Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha. AB - These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic fetal liver (FL) were treated with either c-myb antisense oligomers or 3H thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited (73.2% +/- 10.4% and 74.2% +/- 12.7%). Using highly purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of highly purified adult PB and FL BFU E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase. Under the same experimental conditions a progressive increase of the mRNA level of DNA polymerase alpha was observed. These observations suggest that in early erythroid differentiation c-myb activation is associated with the progression of progenitors into the S phase of the cell cycle, as well as to the synthesis of DNA polymerase alpha. PMID- 1705832 TI - Evidence for the location of the receptor-binding site of human erythropoietin at the carboxyl-terminal domain. AB - Five different peptides (P1: 84-95; P2: 152-166; P3: 52-63; P4: 7-23; P5: 110 123) homologous to relatively hydrophilic regions of human erythropoietin (huEpo) have been synthesized to identify biologically active domains of the hormone. All peptides were able to induce high titers of peptide-specific antibodies in rabbits. Antisera from rabbits induced by recombinant huEpo (rhuEpo) contained a relatively high amount of antibodies preferentially directed against three peptides (P2, P4, and P5), of which P4 comprised the amino-terminal region, P2 the carboxyl-terminus, and P5 an interior region previously described as the receptor-binding site. The same three peptides were able to induce rhuEpo specific antibodies, whereas P1 and P3 lacked this activity. Only peptide-P2 induced antisera inhibited the biologic activity of rhuEpo in a cell proliferation assay, indicating that the carboxyl-terminal region of the molecule is essentially involved in the biologic function of rhuEpo. PMID- 1705833 TI - Sequential generations of hematopoietic colonies derived from single nonlineage committed CD34+CD38- progenitor cells. AB - Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL 3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization. PMID- 1705834 TI - Erythropoietin is both a mitogen and a survival factor. AB - Erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells, but whether it functions solely as a survival factor or also acts as a mitogen is unresolved. Because late erythroid progenitor cells (CFU-E) are largely in cell cycle, we examined this issue by using an Ep-dependent, murine erythroleukemia cell line, HCD-57. In the presence of human Ep and fetal calf serum, HCD-57 cells had a doubling time of approximately 24 hours, and during log-phase growth approximately 36% of the cells were in G1, 45% in S, and 19% in G2/M. With Ep deprivation, there was a gradual loss of viability and an arrest of proliferation with a 44% increase in the G0/G1 population, which could be reversed by reexposure to Ep even after 72 hours of hormone withdrawal. As little as 2 hours of exposure to Ep was sufficient to stimulate DNA synthesis, and the lag time for initiation of DNA synthesis after exposure to the hormone was approximately 10 hours as measured by either incorporation of labeled thymidine into DNA or cell cycle analysis by flow cytometry. RNA synthesis, by contrast, was initiated within 2 hours after exposure to Ep and did not require DNA synthesis. Total cell DNA content increased after exposure to Ep, indicating that it was acting as mitogen in HCD-57 cells. Ep was also able to stimulate DNA synthesis in the absence of serum as well as in its presence, indicating that the hormone could act as both a competence and a progression factor. Qualitative analysis of the integrity of HCD-57 DNA by electrophoresis in agar as well as direct measurement of DNA fragmentation after metabolic labeling with radioactive thymidine indicated that programmed cell death was occurring that could be reduced but not completely prevented by Ep. These data indicate that Ep acts as both a mitogen and a survival factor for HCD-57 cells. PMID- 1705835 TI - Blood mononuclear cells from patients with severe congenital neutropenia are capable of producing granulocyte colony-stimulating factor. AB - Severe congenital neutropenia (SCN) is a disorder of myelopoiesis characterized by severe neutropenia or absence of blood neutrophils secondary to a maturational arrest at the level of promyelocytes. We examined peripheral blood mononuclear cells (PBMC) of SCN patients who demonstrated normalization of their blood neutrophil counts in a phase II clinical study with recombinant human granulocyte colony-stimulating factor (rhG-CSF). When stimulated in vitro with bacterial lipopolysaccharides (LPS), PBMC of those SCN patients produced G-CSF activity, as judged by proliferation induction of the murine leukemia cell line, NFS-60. Western and Northern blot analysis showed G-CSF protein and G-CSF-mRNA indistinguishable in size from those of normal controls. We conclude that PBMC of the SCN patients tested are capable of synthesizing and secreting biologically active G-CSF in vitro. PMID- 1705836 TI - 1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells. AB - 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes. PMID- 1705837 TI - Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin. AB - We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF 1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF 1/heparin. These data establish that growing primary cultures of HUVEC with HBGF 1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis. PMID- 1705838 TI - Locus control region-A gamma transgenic mice: a new model for studying the induction of fetal hemoglobin in the adult. AB - All pharmacologic agents that induce fetal hemoglobin (Hb) have been discovered with in vivo studies of humans, macaques, and baboons. We tested whether transgenic mice carrying human fetal (gamma) globin genes provide a model for studying the pharmacologic induction of HbF in the adult. In initial studies, phenylhydrazine-induced hemolytic anemia, 5-azacytidine, butyrate, or combinations of these treatments failed to activate the human gamma-globin gene in a transgenic mouse line carrying a 4.4-kb G gamma globin gene construct that is expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human A gamma gene linked to the locus control region (LCR) regulatory sequences and expressing heterocellularly HbF (about 25%, gamma positive cells) were used. Treatments with erythropoietin, 5-azacytidine, hydroxyurea, or butyrate resulted in induction of gamma gene expression as documented by measurement of F-reticulocytes, the gamma/gamma + beta biosynthetic ratio and the level of steady state gamma mRNA. Administration of erythropoietin or butyrate to transgenic mice carrying a muLCR-beta (human) globin construct, failed to increase human beta-globin expression. These results suggest that the muLCR-A gamma transgenic mice provide a new model for studying the induction of fetal Hb in the adult. PMID- 1705840 TI - CD8/CD57 lymphocytosis in common variable immunodeficiency. PMID- 1705839 TI - In vivo administration of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF, interleukin-1 (IL-1), and IL-4, alone and in combination, after allogeneic murine hematopoietic stem cell transplantation. AB - BALB/c mice (H-2d) given 10 Gy total body irradiation (TBI) followed by 10(7) bone marrow (BM) and 10(6) spleen cells from C57BL/6 (H-2b) donor mice received recombinant cytokines intraperitoneally (IP) twice daily. The effect on neutrophil recovery rate, graft-v-host disease (GVHD), and survival was assessed. Four reagents were used: granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-1 (IL-1) and IL-4, both alone and in combination. The most effective combination for increasing the circulating absolute neutrophil account (ANC) above the control value at day 7 posttransplant was the combination of G-CSF and IL-1 (mean ANC 2.4 +/- 1.6 x 10(9)/L as compared with control value of 0.07 +/- 0.05, P less than .02), followed by G-CSF alone (mean ANC 1.1 +/- 0.2, P less than .0001), the combination of GM-CSF plus IL-1 (mean ANC 0.8 +/- 0.3, P less than .002), and the combination of G-CSF plus GM CSF (mean ANC 0.8 +/- 0.3, p less than .005). At day 10 posttransplant, the most effective combination in raising the ANC was the combination of G-CSF plus GM-CSF (mean ANC 7.5 +/- 2.3 as compared with control value of 3.5 +/- 1.1, P less than .01), followed by G-CSF alone (mean ANC 6.9 +/- 2.1, P less than .02). At the doses used, neither G-CSF nor GM-CSF had a deleterious effect on the incidence or severity of GVHD; indeed, GM-CSF was associated with improved survival. In contrast, IL-1 at doses greater than or equal to 100 ng twice daily caused marked early mortality, and there was a suggestion that IL-4 at doses of 500 ng twice daily resulted in increased late mortality, possibly owing to exacerbation of GVHD. This model appears to be of value for exploring the use of hematopoietic growth factors before they are used clinically in marrow allograft recipients. PMID- 1705841 TI - Beta-adrenergic receptor stimulates L-type calcium current in adult smooth muscle cells. AB - The hormonal regulation of L-type calcium current was investigated in freshly isolated tracheal smooth muscle cells using the whole-cell configuration of the patch-clamp technique. Isoproterenol stimulated the L-type calcium current 2.6 fold through beta-adrenoceptors. Dialysis of these cells with cyclic AMP, cyclic AMP analogues or the catalytic subunit of cyclic AMP kinase had no effect on basal or isoproterenol-stimulated calcium current. The calcium current was stimulated and inhibited by dialysis of the cells with GTP gamma S and GDP beta S, respectively. Evidently, in some smooth muscle cells the beta-adrenoceptor couples directly to L-type calcium channels via a G protein. PMID- 1705842 TI - Dynamics of capillary perfusion in the brain. AB - The present study investigates the question of capillary recruitment and reserve capillaries in the brains of awake rats. Perfused capillaries were marked by intravenous globulin-coupled fluorescein isothiocyanate (FITC) and cerebral blood flow was measured autoradiographically. During hypercapnia, the density of perfused capillaries was unchanged compared to normocapnia, although blood flow was markedly increased. This shows the lack of capillary recruitment in the brain during the high flow that occurs during hypercapnia. In additional studies using fluorescent staining both of morphologically existing and of perfused capillaries, perfusion of all capillaries during normal, normocapnic conditions was found. These experiments show the lack of any capillary reserve in the rat brain. PMID- 1705843 TI - Lindane pollution near an industrial source in northeast Spain. PMID- 1705844 TI - Reversibility of the inhibitory effect of atrazine and lindane on cytosol 5 alpha dihydrotestosterone receptor complex formation in rat prostate. PMID- 1705845 TI - Cancer services. PMID- 1705846 TI - Autoradiographic visualization of NK-3 tachykinin binding sites in the rat brain, utilizing [3H]senktide. AB - The autoradiographic distribution of the selective NK-3 tachykinin agonist [3H]senktide was investigated in rat brain. [3H]Senktide bound with high affinity (KD less than 2.5 nM) and high specificity (greater than 75%) to cerebral cortex and numerous subcortical sites, including the substantia nigra pars compacta. In addition, moderately dense binding was seen in the median but not the dorsal raphe nucleus, and this was disrupted by 5,7-dihydroxytryptamine (5,7-DHT) induced destruction of 5-HT neurons. 5,7-DHT lesions did not affect the binding of [3H]senktide to forebrain regions, suggesting that 5-HT terminals are devoid of NK-3 receptors. PMID- 1705847 TI - Muscarinic regulation of cyclic AMP metabolism in rat neostriatal cultures. AB - Muscarinic receptor expression and function were investigated in cultured rat neostriatum. Muscarinic receptor levels were determined from saturation binding experiments performed on intact cultures using [3]N-methylscopolamine. In cultures maintained for 3, 7 and 12-14 days in vitro, the Bmax was 2.3, 5.4 and 10.9 fmol/culture. The average number of receptors per neuron increased during the 2nd week in vitro. Carbachol (100 microM) had no significant effect on basal cAMP levels but reduced cAMP levels elevated by forskolin. Carbachol significantly reduced cAMP levels stimulated with dopamine only in cultures untreated with a phosphodiesterase inhibitor. Comparing equimolar doses, the carbachol response was more sensitive to the M1 selective antagonist pirenzepine than the cardioselective M2 antagonist AF-DX 116. These results suggest that the muscarinic receptors regulate cAMP levels in neostriatal neurons and, in so doing, provide a post-synaptic substrate for the interaction of dopamine and acetylcholine. PMID- 1705848 TI - Targeted neuronal lesion induced by photosensitizing dyes. AB - Free radical-induced phototoxicity mediated by laser irradiation was investigated in the rabbit facial nerve. Azure-C, mesoporphyrin, or the dye conjugated to the protein carrier horseradish peroxidase were injected into the levator alae nasi muscle. Two to 7 days after uptake and laser exposure, nerve sections showed varying degrees of cellular modifications including: severe membrane degradation and associated lipid peroxide granules, distended mitochondria, and mitochondrial loss. Immunoblots of homogenates from treated nerves revealed specific changes in neurofilament and myelin basic protein. The site specific damage produced in vivo by photosensitizing dye resembles abnormalities in aging neurons and in Batten's disease, both hypothesized to be cases of free radical-peroxidation reactions. These reactions differ from those found in transection and crush lesions. PMID- 1705849 TI - CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study. AB - The retrograde transneuronal viral cell body labeling method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons which project to the pterygopalatine ganglion. Small injections of a suspension of pseudorabies virus (PRV) were made in the pterygopalatine ganglion of rats and after 4 days their brains wer e processed for immunohistochemical detection of PRV. Some of the tissues were stained with a dual immunofluoresence method that permitted the visualization of PRV and neurotransmitter enzyme or serotonin immunoreactivity in the same cell. Retrograde cell body labeling was detected in the ipsilateral ventrolateral medulla oblongata in the region that has been termed the superior salivatory nucleus. This area was the same region that was retrogradely labeled after Fluoro-Gold dye injections in the pterygopalatine ganglion. Retrograde transneuronally infected cell bodies that provide putative afferent inputs to the pytergopalatine parasympathetic preganglionic neurons were mapped throughout the brain. In the medulla oblongata, transneuronally labeled neurons were seen in the nucleus tractus solitarii, dorsomedial part of the spinal trigeminal nucleus and gigantocellular reticular nucleus. In most experiments, some A1 catecholamine cells and serotonin neurons of the raphe magnus, raphe pallidus, raphe obscurus, and parapyramidal nuclei were labeled. In the pons, labeled cells were found in the parabrachial nucleus. A5 catecholamine cell group, and non-catecholamine part of the subcoeruleus region. In the midbrain, cell body labeling was located in the central gray matter and retrorubral field. In the diencephalon, labeling was found mainly in the hypothalamus. The areas included the lateral hypothalamic area, lateral preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and ventral zona incerta. Contralateral second order cell body labeling was seen in the tuberomammillary nucleus of the hypothalamus. Some of these cells were histidine decarboxylase-immunoreactive. In the forebrain, the bed nucleus of the stria terminalis, substantia innominata, and an area of the cerebral cortex called the amygdalopiriform transition zone were labeled. PMID- 1705850 TI - Hair cell tufts and afferent innervation of the bullfrog crista ampullaris. AB - Within the bullfrog semicircular canal crista, hair cell tuft types were defined and mapped with the aid of scanning electron microscopy. Intracellular recording and Lucifer Yellow labeling techniques were used to study afferent responses and arborization patterns. Dye-filled planar afferent axons had mean distal axonal diameters of 1.6-4.9 microns, highly branched arbors, and contacted 11-24 hair cells. Dye-filled isthmus afferent axons had mean distal axonal diameters of 1.8 7.9 microns, with either small or large field arbors contacting 4-9 or 25-31 hair cells. The estimated mean number of contacts per innervated hair cell was 2.2 for planar and 1.3 for isthmus afferent neurons. Data on evoked afferent responses were available only for isthmus units that were observed to respond to our microrotational stimuli (less than 3 degrees/s peak rotational velocity). Of 21 such afferent neurons, 8 were successfully dye-filled. Within this small sample, high-gain units had large field arbors and lower-gain units had small field arbors. The sensitivity of each afferent neuron was analyzed in terms of noise equivalent input (NEI), the stimulus amplitude for which the afferent response amplitude is just equivalent to the RMS deviation of the instantaneous spike rate. NEI for isthmus units varied from 0.63 to 8.2 degrees/s; the mean was 3.2 degrees/s. PMID- 1705851 TI - Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons. AB - In this report, we demonstrate that cholera-toxin B subunit (CTb) is a very sensitive retrograde tracer in the central nervous system when recognized by streptavidin-peroxidase immunohistochemistry. We further show that: (1) injection of a small volume of CTb gives rise to small sharply defined injection sites limited to the cell group of interest associated with the labeling of all the known afferent projections, (2) CTb is taken up, and anterogradely as well as retrogradely transported in damaged but not intact fibers of passage, (3) CTb can be applied iontophoretically, allowing us to study the afferents to small cell groups without any evidence of tissue necrosis in the sites and therefore without artefactual labeling due to uptake by damaged fibers of passage, (4) the use of 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens, the addition of a 48 h colchicine treatment and the development of a double immunohistochemical method allow the biochemical characterization of the cell of origin of particular pathways in the CNS, (5) CTb is also anterogradely transported with an extensive filling of axons and axon terminals and thereby opens up the possibility of identifying simultaneously the afferents as well as the efferents of the group of cells studied and finally (6) the very long conservation of the preparation, the possibility of counterstaining it and of making camera lucida drawings allow easy and precise localization of the retrogradely labeled cells. PMID- 1705852 TI - Intraventricular infusion of HBGF-2 promotes cerebral angiogenesis in Wistar rat. AB - The angiogenic effects of chronic intraventricular infusion of basic fibroblastic growth factor (HBGF-2) were assessed in the Wistar rat cerebral cortex. HBGF-2 increased both capillary density and area of perfused vasculature without altering capillary diameters in periventricular cortex adjacent to the site of infusion. Although it has been demonstrated that growth factors can cause brain endothelial proliferation in vitro, this is the first report of in vivo cerebral angiogenesis. PMID- 1705853 TI - The isolation of cultured oligodendrocytes from rat optic nerve. AB - Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat optic nerve employing a modified method of McCarthy and DeVellis, and cultured oligodendrocytes were analyzed for characteristic morphology and maturation events by phase contrast, MBP immunofluorescent and scanning electron microscopy. Two types of oligodendrocytes, type 1 OLs and type 2 OLs, on poly-L-lysine-coated dishes were obtained. Type 1 OLs showed extensive networks of processes and type 2 OLs elaborated membranous expansion along extensive networks, and it was demonstrated that type 2 OLs developed from type 1 OLs by phase contrast-microscopic observation. By scanning electron-microscopic observation, membrane structure in type 2 OLs clearly corresponded to membranous expansion. Our results suggest that type 1 OLs and type 2 OLs are in the different states of the myelinogenesis, and that membranous expansion and membrane structure in type 2 OLs correspond to myelin membrane. PMID- 1705854 TI - Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina. AB - The distribution of anionic sites on the cell membranes and basal laminae of vascular endothelial cells in the rat sciatic nerve was investigated using cationic ferritin (CF) and cationic colloidal gold (CCG). Nerves fixed by perfusion followed by immersion were chopped into 400 microns thick slices and incubated in CF or embedded in LR White resin for staining with CCG. Using electron microscopy, the distribution of these tracers was investigated. The results indicated that microdomains of various charge densities exist. Diaphragms of caveolae and transendothelial channels, and luminal endothelial processes are highly anionic, the basal laminae of endothelial cells and pericytes and luminal membranes are medium and abluminal membranes least anionic. Inter-endothelial tight junctions were unlabelled and not penetrated by CF. These structures are thought to represent charge and size filters that control permeability of the vasa nervorum. The distribution of these charge-size filters is discussed in terms of the blood-nerve barrier, a physiological property present in the endo- but absent in the peri- and epineurial vessels. PMID- 1705855 TI - Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy. AB - The distribution of granule cells in the dentate gyrus of the hippocampal formation was studied in control autopsy and temporal lobe epilepsy (TLE) specimens. In control tissue, the granule cell somata were closely approximated and formed a narrow lamina with a distinct, regular border with the molecular layer. In 11 of 15 TLE specimens, the granule cell somata were dispersed and formed a wider than normal granule cell layer. The granule cell somata extended into the molecular layer to varying extents, creating an irregular boundary between the lamina. The dispersed granule cells were frequently aligned in columns, and many of these neurons displayed elongated bipolar forms. The extent of granule cell dispersion appeared to be related to the amount of cell loss in the polymorph layer of the dentate gyrus. Granule cell dispersion was not consistently associated with granule cell loss although 5 of the 11 specimens with granule cell dispersion also showed moderate to marked granule cell loss. The most common features in the histories of the TLE cases with granule cell dispersion were severe febrile seizures or seizures associated with meningitis or encephalitis during the first 4 years of life. The dispersion of the granule cells suggests that there has been some alteration in the patterns of cell migration in a subpopulation of cases with severe TLE. The resultant ectopic positions of the granule cells could lead to changes in both the afferent and efferent connections of these neurons and, thus, contribute to the altered circuitry of the hippocampal formation in TLE. PMID- 1705856 TI - In vivo release of serotonin in cat dorsal vagal complex and cervical ventral horn induced by electrical stimulation of the medullary raphe nuclei. AB - Extracellular levels of serotonin (5-hydroxytryptamine; 5-HT) were monitored by microdialysis in the dorsal vagal complex (DVC) and the ventral horn of the spinal cord at the level of the phrenic motor nucleus in decerebrated cats. A selective serotonin uptake inhibitor, alaproclate (10(-4) M) was included in the dialysis probe perfusion fluid to increase basal and stimulated levels of 5-HT. Electrical stimulation (30 Hz, 10 V, 0.5 ms) in the nucleus raphe obscurus, containing neurons projecting to the DVC and to the ventral horn, induced a 2-3 fold increase of the 5-HT release in both these regions. After termination of the stimulation, the release gradually decreased during the following 60 min. Substance P, which coexists with 5-HT in descending neurons, did not significantly affect the 5-HT release when it was added (100 microM) to the probe perfusion fluid. The present findings are in accordance with the hypothesis that prolonged release of 5-HT is responsible for the previously demonstrated long lasting facilitation of phrenic activity following raphe obscurus stimulation. PMID- 1705857 TI - Organelle flux in intact and transected crayfish giant axons. AB - The flux of organelles moving by fast axonal transport in distal segments of severed crayfish medial giant axons (MGAs) and lateral giant axons (LGAs) was measured for survival times of up to 35 days (MGAs) or 60 days (LGAs). The response to transection occurred in 4 phases: (1) Organelle fluxes remained nearly normal for the first 24 h. (2) Fluxes then declined continuously until day 6 or 7. (3) A rebound toward normal levels lasted until day 21 (MGAs) or longer (LGAs). (4) During the final phase, fluxes declined either to zero (MGAs) or plateaued at a level which was a significant percentage of normal flux (LGAs). Changes in anterograde and retrograde flux were identical. The distribution of various size classes of translocating vesicles in distal segments of these axons was normal until day 4, with small and medium size, rapidly moving vesicles predominating. Afterwards, larger, slower vesicles predominated. During long-term survival, the axons remained physiologically intact, and cytoskeletons appeared to be normal, retaining intact microtubules which remained normally oriented with positive ends pointing distally. The evidence suggests that the two initial phases of the response to transection represent clearance from distal segments of organelle traffic which normally moves between axon and cell body. The rebound phase may be trauma induced, possibly a transient phase of cytoplasmic degeneration resulting from the loss of trophic support from the cell body. Differences between LGAs and MGAs with respect to organelle flux during prolonged survival, i.e. during the 4th phase of the response to transection, are consistent with different mechanisms of long-term survival which have been proposed for these axons. PMID- 1705858 TI - Nystatin-perforated patch recordings disclose NMDA-induced outward currents in cultured neocortical neurons. AB - Excitatory amino acid-induced responses were studied in cultured rat neocortical neurons using two types of whole-cell patch-clamp recordings. Conventional recording methods, using either KCl or CsCl in the patch pipet, showed N-methyl-D aspartate (NMDA) currents to be biphasic, consisting of peak and steady-state currents with similar reversal potentials. Recordings obtained with the nystatin perforated patch method and KCl as the principal intracellular cation disclosed an NMDA-evoked outward current. Outward currents were not seen with either recording method in response to kainate or quisqualate, nor in response to NMDA when CsCl was the major intracellular cation. The NMDA-evoked outward current is attributed to activation of a potassium current by calcium entering through the NMDA channel. This outward current may serve to limit neuronal depolarization during excessive NMDA-mediated excitation. PMID- 1705859 TI - An immunohistochemically distinct population of cat ciliary ganglion cells. AB - In the cat ciliary ganglion, 16% and 23% respectively of the neurons are surrounded by nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) and somatostatin (SOM). One-third of the ganglion cell perikarya are immunoreactive to neuropeptide Y (NPY). Analysis of the coincidence of immunoreactivities shows a striking correlation. Practically all of the ganglion cells surrounded by CGRP-like immunoreactive (LI) nerve fibers also are surrounded by SOM-LI nerve processes and demonstrate NPY-LI perikarya. These observations define a subset of NPY-LI ciliary ganglion cells with a particular peptidergic input and perhaps a specific physiologic function. PMID- 1705860 TI - Change of circulating thyroid autoantibody titers in Graves' hyperthyroidism after antithyroid drugs therapy. AB - Parallel measurements of circulating thyroglobulin (Tg), microsomal [TPO(mic)] and TSH-receptor antibodies [TSH-R(rr)] were performed in 30 cases of Graves' hyperthyroid patients who received antithyroid drug (ATD) therapy for 2.8 +/- 1.7 years (range: 0.5-5.0 years). Before ATD therapy, positive Tg, TPO(mic) and TSH R(rr) antibodies were found in 33.3%, 83.3% and 86.7% of our patients with Graves' disease, respectively. The titers of Tg and TPO(mic) antibodies remained unchanged in most patients after ATD treatment. However, the TSH-R(rr) antibody titers decreased in 46.7%, elevated in 26.7% and unchanged in 26.7% after ATD therapy. Six cases got long-term remission. Measurements of Tg and TPO antibodies have no value in forecasting the outcome of the disease. The change of circulating TSH-R(rr) antibody from positive to negative is more predictive of remission after ATD therapy than the decrease of thyrotropin-binding inhibiting immunoglobulin index only. However, the former change indicating a persistent remission is not certain. PMID- 1705861 TI - A brief-duration combination chemotherapy for elderly patients with poor prognosis non-Hodgkin's lymphoma. AB - Curative combination chemotherapy is available for many patients with aggressive non-Hodgkin's lymphoma (NHL); however, treatment of elderly patients with these regimens is difficult due to excessive toxicity. From 1983 to 1988 the authors treated 26 patients 65 years and older with aggressive NHL with a novel 8-week chemotherapy regimen containing bleomycin, etoposide, cyclophosphamide, doxorubicin, methotrexate with leucovorin, and prednisone (BECALM), designed to preserve dose intensity and minimize toxicity. Median age was 75 years. Histologic types included the following: 20 intermediate grade (16 large noncleaved cell; two large cleaved cell; one intermediate grade, unspecified); six high grade (four small noncleaved cell; one immunoblastic sarcoma B-cell; one high grade, unspecified). Twenty-one patients were Stage III or IV. Twenty-two of 26 patients had one or more of the following: tumor greater than 10 cm; multiple extranodal sites; lactate dehydrogenase (LDH) 400 IU/l or greater; small noncleaved cell histologic type. Chemotherapy consisted of bleomycin 20 U intravenously (IV) weeks 1 and 7; etoposide 75 mg/m2 IV every day x 3 days on week 4; cyclophosphamide 600 mg/m2 IV weeks 1, 4, 7; doxorubicin 40 mg/m2 IV weeks 1, 7; methotrexate 50 mg/m2 IV weeks 1, 2, 4, 5, 7, 8 with oral leucovorin rescue; prednisone 60 mg orally for 10 days on weeks 1, 4, 7. Eighteen patients completed the 8-week treatment course. There were 13 complete responses (CR); seven patients remain in continuous CR at a median follow-up of 37.5 months. There have been five relapses, including one late relapse; and one patient died of an intercurrent illness in CR. Overall and actual event-free survivals are 38% and 27%, respectively. The major toxicities were neutropenic fever and mucositis. There were four treatment-related deaths. The authors conclude that BECALM chemotherapy can be administered to elderly patients with aggressive NHL. Although neurotoxicity and cumulative toxicity from bleomycin and anthracycline are avoided, the regimen remains moderately toxic, particularly with respect to myelosuppression. Treatment results compare favorably with other reported regimens in this group of patients with multiple poor prognostic features. PMID- 1705862 TI - CD34 antigen expression in children with Philadelphia chromosome-positive acute lymphoblastic leukemia. AB - One characteristic of Philadelphia chromosome (Ph')-positive acute leukemia is the occasional presence of both lymphoid and myeloid features in the same leukemia. This phenomenon supports the theory that this subtype of acute leukemia arises from lymphoid-myeloid stem cell, pluripotent progenitors. Very few reports, however, describe the immunophenotype, especially CD34 antigen, of Ph' positive acute lymphoblastic leukemia (ALL). It has been shown that CD34, the human progenitor cell antigen, is found on 1% or less of normal human bone marrow cells, approximately 30% of acute leukemias, and multipotent progenitor cells; CD34 is not found on normal peripheral blood cells. A high frequency of CD34 expression was found in children with Ph'-positive ALL: CD34 was positive for all six patients tested, and one had an acute mixed-lineage leukemia. These findings suggest the involvement of a pluripotent stem cell in Ph'-positive ALL. PMID- 1705863 TI - Synovial sarcoma in children and adolescents. A report from the Kiel Pediatric Tumor Registry. AB - Of 49 cases of synovial sarcoma, which represent 5.8% of all soft tissue sarcomas with confirmed diagnosis in the files of the Kiel Pediatric Tumor Registry (Kiel, Germany), 35 occurred in patients up to the age of 18 years. The lower extremities were the most common. The 35 cases included 21 biphasic and 14 monophasic fibrous synovial sarcomas. The different cell types constituting synovial sarcoma could be demonstrated by conventional light microscopic study, but more readily so by immunohistochemical study, particularly when antibodies against cytoskeletal components were applied. Aberrant antigen expression was noticed for the neural markers, protein S-100, and neuron-specific enolase. Moreover, four tumors were positive for Ki M7. Collagen type IV was found in all tumors tested. For the 20 patients enrolled in the Cooperative Soft Tissue Sarcoma Study of the German Society of Pediatric Oncology (GPO) the survival rate at 7 years is 63%. When five patients with initial recurrence are excluded, the survival rate is 72%. It is concluded that immunohistochemical study is useful in the diagnosis and differential diagnosis of synovial sarcomas despite certain limitations. Multimodality treatment approach has improved the overall prognosis. There is no relationship between histologic subtype and prognosis according to the classification scheme employed in this study. PMID- 1705864 TI - Cessation of treatment in advanced cancer. AB - A major responsibility for all physicians, but particularly for oncologists, is to recognize the time when active antitumor treatment ceases to have a rational basis. The decision to treat or not to treat at any stage of disease requires an analysis of "the legitimate aims of therapy." We have acquired the ability to cure or prolong survival in an increasing proportion of patients with several types of cancer. For patients who fail to achieve those results and for those with tumors that are rarely amenable to specific therapy, the choice of less surely effective therapy is an option; the patient must participate in the decision, armed with as much information and insight as possible, for conventional and experimental treatment. A distinction must be made between specific antitumor therapy and palliative measures for which cessation is never an option. Happily, cessation of treatment must also be considered when therapy has been so successful that the patient has achieved complete remission. At what point may treatment be discontinued without the danger of relapse? These issues are rarely crisply defined, but primary concern for the patient and careful analysis of the available data can lead to appropriate value judgements. PMID- 1705865 TI - Study of RNA synthesis in the livers of aging mice by means of electron microscopic radioautography. AB - The application of 3H-uridine radioautography results in labeling of the liver cells in which RNA is synthesized at various ages of the mouse. Quantitative changes of RNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains were mainly located in the nucleoli and nuclei and a few in the mitochondria and rough surfaced endoplasmic reticulum of almost all of the cell populations at various ages. The number of silver grains in the hepatocyte gradually increased after birth, reached the maximum at 14 days of postnatal age, then decreased to 24 months with aging. The number of silver grains of the euchromatin was more than those of the heterochromatin of the hepatocyte nuclei at various ages. The number of silver grains of the granular components was more than those of the fibrillar components of the hepatocyte nucleoli at various ages. However, the ratio of silver grains among euchromatin, heterochromatin, granular components and fibrillar components remained approximately constant. PMID- 1705866 TI - Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant. AB - The ligand (KL) for the c-kit receptor is a growth factor encoded at the mouse steel (Sl) locus. KL exists in both cell surface and soluble forms, though little is known of the regulation and functional significance of these forms. We show here that tissue-specific alternative splicing gives two types of KL mRNA. Both encode a transmembrane domain, but in transfected cells one produced the soluble form of KL at relatively high levels, whereas the other preferentially gave the cell surface form. Cell surface KL not only stimulated proliferation, but also mediated cell-cell adhesion. The SId allele, which impairs development of hematopoietic cells, melanocytes, and germ cells, has a deletion in the KL gene removing the transmembrane and intracellular domains. Expression of a corresponding cDNA gave a soluble protein that stimulated cellular proliferation but was not associated with the cell surface. These results provide evidence that cell surface KL has a critical role in the intact organism. PMID- 1705867 TI - The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. AB - The function of the T cell antigen receptor (TCR) invariant chains, CD3 gamma, delta, epsilon, and zeta, is poorly understood. Evidence suggests that CD3 couples receptor ligand binding to intracellular signaling events. To examine the role of the CD3 zeta chain in TCR-mediated signal transduction, a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain was constructed. The CD8/zeta chimera is expressed independently of the TCR and is capable of transducing signals that, by criteria of early and late activation, are indistinguishable from those generated by the intact TCR. These data indicate that CD8/zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggest that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms. PMID- 1705868 TI - Ontogeny of substance P-immunoreactive structures in the primate cerebral neocortex. AB - The distribution and the ontogeny of substance P (SP)-immunoreactive structures were investigated in the various cortical areas of macaque monkey cerebrum at embryonic day 120 (E120), embryonic day 140 (E140), newborn (Nb), postnatal day 30, postnatal day 60 (P60) and adult stages, using an immunohistochemical method. SP-immunoreactive cell bodies and fibers were detectable at E120 and the cell number increased until Nb stage. At E140, many immunoreactive cells were present in the upper part of layer V. Some of them seemed to be developing pyramidal cells which ascended their fibers toward layer I. After Nb stage, the number of immunoreactive structures decreased. By P60, the distribution patterns of SP immunoreactive structures reached the adult level. Between Nb and P60, we occasionally observed structures which were presumably degenerated neurons and fibers. The distribution and developmental ontogeny of immunoreactivities were different among the various cortical areas. In areas OC and FA (von Bonin and Bailey), we observed the high densities of immunoreactive fibers and terminals, in spite of low numbers of cell somatas. While, in the association areas (areas FD, PE, TA and TE), there existed larger numbers of immunoreactive cells at E140 and newborn stages, following the decrease of cell number until P60. Our present study shows the transient increase and the following decrease of the numbers of SP-immunoreactive cells. Since we observed SP-immunoreactive pyramidal cells and degenerating cells during development, the decrease of immunoreactivities may be due to both cell death and change in phenotype. PMID- 1705869 TI - Postnatal development of opioid regulation of micturition in the kitten. AB - Endogenous opioids tonically regulate micturition in adult mammals. The present study sought to determine if opioids regulate micturition in neonatal kittens. Naloxone (up to 2 mg/kg given i.p. or i.v. to unanesthetized/ketamine anesthetized or chloralose-anesthetized kittens, respectively), an opioid receptor antagonist, produced no effects in unanesthetized, ketamine anesthetized, or chloralose-anesthetized kittens that had been prepared for bladder pressure recording, until 3 weeks of age. This indicates that endogenous opioids are not tonically regulating micturition in neonatal kittens. From 3 weeks up to at least 6 weeks of age, naloxone (100 micrograms/kg i.p. or i.v.) weakly facilitated bladder activity by transiently increasing the amplitude and/or duration of bladder contractions, but no effects on frequency of contractions was recorded. Morphine (up to 2 mg/kg given i.p. or i.v. to unanesthetized/ketamine-anesthetized or chloralose-anesthetized kittens, respectively), an opioid agonist, did not inhibit bladder contractions in unanesthetized or ketamine-anesthetized neonatal kittens, but it did inhibit (at a threshold dose of 100 micrograms/kg) and completely abolished (at a dose of 300 micrograms/kg) bladder activity in chloralose-anesthetized kittens in a dose dependent, naloxone-reversible manner. Surprisingly, following morphine administration to unanesthetized or ketamine-anesthetized neonatal kittens, naloxone now produced an adult-like enhancement of bladder activity. These latter results indicate that opioid receptors, whose inhibitory effects are anesthetic dependent, are present along the micturition reflex pathway in neonates. Immunohistochemical studies of the sacral spinal cord revealed that opioid peptides are distributed similarly in neonatal and adult cats. PMID- 1705870 TI - Presence of reverse transcriptase in human leukemias and lymphomas. AB - Reverse transcriptase (RT) transcribes viral RNA into DNA to be integrated into the host genome. To study epidemiological aspects of human leukemias and lymphomas which are known to express retroviruses, clinical specimens in this report were assayed for divalent cation-dependent viral-specific RT. The assay was carried out with cells solubilized with a detergent to release RT enzyme. RT was purified with poly(U)-Sepharose which fixed all DNA polymerases and assayed with 4 synthetic homopolymers, oligonucleotide primed-templates, poly(rA) oligo(dT)12-18 or poly(dA)-oligo(dT)12-18 with Mg2+, poly(rC)-oligo(dG)12-18 or poly(rCm)-oligo(dG)12-18 with Mn2+ as divalent cation and [methyl-3H]thymidine 5' triphosphate or deoxy[8-3H]guanosine 5-triphosphate respectively. Radioactivity incorporation of the precipitate allows quantitation of RT activity. One Hodgkin's disease, one out of 2 B lymphomas, one out of 2 T lymphomas, eight out of 12 leukemias were found to be positive for RT activity as well as acquired immunodeficiency syndrome (AIDS) patients, known to express RT. The obtained RT activity in hematological malignancies was found to be comparable to positive controls such as RT enzymes purified from avian myeloblastosis and Moloney murine leukemia viruses. PMID- 1705871 TI - Stable isotope dilution analysis of very long chain fatty acids in plasma, urine and amniotic fluid by electron capture negative ion mass fragmentography. AB - A sensitive and selective stable isotope dilution electron capture negative ion chemical ionization mass fragmentography method applying pentafluorobenzyl derivatives was developed for the accurate quantitation of very long chain fatty acids. This technique allowed detection of 1-5 pg of each compound and was applied to plasma (100 microliters), amniotic fluid (1 ml) and urine (1 ml). Normal concentrations were established and the concentrations in samples of selected patients with classified peroxisomal disorders were determined. In plasma samples of all patients the C26:0/C22:0 ratios were elevated (range 0.03 0.43), compared to the control ratios (range 0.003-0.021). The ratio C26:0/C22:0 was elevated in four of five amniotic fluid samples from fetuses with peroxisomal disorders (range 0.18-0.54) when compared with controls (range 0.05-0.25). An elevation of the ratio C26:1/C22:0 was observed in all five amniotic fluid samples (range 0.22-0.60 vs. 0-0.08 in controls). Urinary C26:0 concentrations were lower than in plasma and amniotic fluid and diagnostic ratios were not elevated in patients with peroxisomal disorders. PMID- 1705872 TI - Measurement of beta 2-microglobulin, retinol-binding protein, alpha 1 microglobulin and urine protein 1 in healthy children using enzyme-linked immunosorbent assay. AB - Enzyme-linked immunosorbent assays (ELISA) have been developed for the measurement of beta 2-microglobulin (B2M), retinol-binding protein (RBP), alpha 1 microglobulin (A1M) and urine protein 1 (UP1) in children. Results from random urine samples in 43 children (31 for B2M) are, when corrected for urine creatinine (geometric mean (range)): B2M 9.8 (6.0-40.7) micrograms/mmol, RBP 8.1 (less than 1-24.5) micrograms/mmol, A1M 0.4 (0.1-2.2) mg/mmol and UP1 17.8 (less than 2-309.4) micrograms/mmol. Fractional excretions (FE) in 23 children (14 for B2M) are (geometric mean (range)): FEB2M 0.04% (0.02-0.10%) and FEUP1 0.10% (0.01 1.21%). Results in overnight urine collections are also presented. Our results extend existing data for normal ranges in adults to include children and provide data on UP1 concentrations. PMID- 1705873 TI - Differential diagnosis of an hyperamylasemia in an acute abdominal syndrome. PMID- 1705874 TI - Spontaneous lymphocyte proliferation in HTLV-I/II infection reflects preferential activation of CD8 and CD16/56 cell subsets. AB - Previous studies have shown that lymphocytes from HTLV-infected persons spontaneously proliferate when cultured in vitro. We investigated which cell subsets become activated in this response. Mononuclear cells from 16 HTLV seropositive former blood donors and 9 seronegative controls were cultured for 7 days; activation was then assessed by measuring DNA synthesis in cultured cells and by monitoring CD25 expression by CD3, CD4, CD8, CD19, and CD16/56 lymphocyte subsets. Of the 16 cultures of HTLV + donor cells, 10 showed spontaneous proliferation (Prol + group) and 6 did not (Prol - group). Cytofluorometric analysis revealed a significant increase in the fractions of CD8 cells and CD16/56 cells expressing CD25 for the Prol + group, compared to the Prol- and control groups. Similarly, the fractions of CD25 cells expressing CD8 or CD16/56 were significantly increased in the Prol + group. Although neither the fraction of CD4 cells expressing CD25 nor the fraction of CD25 cells expressing CD4 were increased for the Prol + group, the modal fluorescence intensity value for CD25 expression by CD4 cells was increased, suggesting some CD4 cell activation occurred as well. Blastoid cells were, on average, 79% CD25 +, whereas the sum of CD4 + CD25 + (27%), CD8 + CD25 + (30%), CD19 + CD25 + (3%), and CD16/56 + CD25 + (35%) subsets was 95%; the presence of 17% CD8 + CD16/56 + cells accounted for most of this discrepancy. These findings indicate that spontaneous lymphocyte proliferation in HTLV infection reflects preferential activation of CD8 and CD16/56 cell subsets, apparently including the minor CD8 + CD16/56 + subset. PMID- 1705875 TI - [Severe ventricular arrhythmia secondary to indapamide-induced hypopotassemia]. AB - We report on two patients, treated with indapamide for mild hypertension, who developed life-threatening ventricular arrhythmias. The former showed severe hypokalemia, QT interval prolongation and "torsade de pointes": the latter, who suffered from ischemic heart disease, had slightly decreased serum potassium and ventricular fibrillation. In both cases no other cause accounting for hypokalemia and ventricular arrhythmia was found. Therefore we stress that serum potassium and ECG must be carefully monitored during indapamide therapy, mainly in patients with cardiac disease. PMID- 1705876 TI - Interleukin-6 is a major myeloma cell growth factor in vitro and in vivo especially in patients with terminal disease. PMID- 1705877 TI - The isolated glomerulus in culture. Cellular reactions of isolated glomeruli in vitro and identification of glomerular cellular outgrowth. PMID- 1705878 TI - Status at four years of age in 280 children weighing 2,300 g or less at birth. AB - To assess the functional ability in low birth weight children at age 4-5, 114 survivors with very low birth weight (VLBW) less than or equal to 1,500 g, 166 survivors with birth weight 1,501-2,300g (LBW), and 115 comparison children with normal birth weight (NBW) were enrolled in a follow-up study. Twenty-four (21%) VLBW and 11 (6.6%) LBW-children had major clinical abnormalities compared to 1 (0.9%) of the NBW children. Twenty-two percent of the VLBW-children were below the 3rd-centile for height and weight. Fewer VLBW than LBW-children were neurologically and ophthalmologically normal. Even after exclusion of the handicapped children, the LBW as well as the VLBW children scored significantly lower than the NBW children in the McCarthy Scales of Children's Abilities, most markedly in the motor and perceptual performance scales. A simple test using grooved pegs clearly demonstrated the poor visual-motor integration in VLBW and LBW children. A marked difference between the NBW and the two LBW groups was seen in a qualitative evaluation of motor performance. Only 5% of VLBW children scored at or above the median for the NBW in all of three fields: general cognitive, motor performance, and pegboard, as opposed to 11% of LBW and 18% of NBW children. Neither sex, age corrected for prematurity, nor psycho-social background factors explained the differences between the groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705879 TI - [The immunotherapy of advanced renal cell carcinoma]. PMID- 1705880 TI - Kainate activated single channel currents as revealed by domoic acid. AB - We have studied the properties of kainic acid receptor-activated channels using domoic acid as an agonist. Similarities of the electrophysiological, pharmacological and noise properties of domoic acid and kainic acid-evoked currents confirm that domoate is a potent and specific agonist of the kainate receptor. Single-channel properties of domoic acid-evoked currents were directly determined from outside-out membrane patches for the first time, and results were compared with those obtained by fluctuation analysis of macroscopic currents. Small conductance cationic-selective channels of approximately 4 pS and a mean open time of 2 to 3 ms were detected using both methods. PMID- 1705881 TI - Identification of a cationic channel in synaptosomal membranes. AB - Synaptosomal membranes were fused with liposomes using the 'hydration technique' to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters. PMID- 1705882 TI - Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity. AB - The amphibian Xenopus laevis expresses several types of immunoglobulin light chain (IgL). cDNA clones for two IgL isotypes, C sigma 1 and C sigma 2, were analysed. C sigma is expressed in spleen and mitogen-stimulated B cells, like another Xenopus IgL type, termed C rho. C sigma shares less than 33% residues with C rho or with CL regions of shark, chicken and mammals. This suggests that C sigma diverged from a common ancestor of CL regions before or at the emergence of amphibians. Two families of VL elements, V sigma 1 and V sigma 2 are associated with C sigma (but not with C rho). They rearrange to their own set of JL elements, J sigma 1 and J sigma 2, which are poorly related to other J elements of the Ig gene family. The Xenopus genome contains a few V sigma 2 and multiple V sigma 1 elements (comparable with mammalian V kappa), but only two C sigma genes. Thus, the organization and expression of Xenopus IgL loci are apparently similar to mammalian IgL loci but different from shark and chicken IgL loci. Only a few VL elements are expressed, since cDNA clones show extensive sharing of CDR1 and CDR2 sequences; some clones differ only in CDR3. Rearranging VL and JL elements increases CDR3 diversity in both V sigma families, but abortive rearrangements are frequent in V sigma 1 regions. The very poor heterogeneity of expressed VL elements therefore appears to limit antibody diversity in Xenopus. PMID- 1705883 TI - Different patterns of receptor-activated cytoplasmic Ca2+ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2+ buffering. AB - Agonist-specific cytosolic Ca2+ oscillation patterns can be observed in individual cells and these have been explained by the co-existence of separate oscillatory mechanisms. In pancreatic acinar cells activation of muscarinic receptors typically evokes sinusoidal oscillations whereas stimulation of cholecystokinin (CCK) receptors evokes transient oscillations consisting of Ca2+ waves with long intervals between them. We have monitored changes in the cytosolic Ca2+ concentration ([Ca2+]i) by measuring Ca2(+)-activated Cl- currents in single internally perfused mouse pancreatic acinar cells. With minimal intracellular Ca2+ buffering we found that low concentrations of both ACh (50 nM) and CCK (10 pM) evoked repetitive short-lasting Ca2+ spikes of the same duration and frequency, but the probability of a spike being followed by a longer and larger Ca2+ wave was low for ACh and high for CCK. The probability that the receptor-evoked shortlasting Ca2+ spikes would initiate more substantial Ca2+ waves was dramatically increased by intracellular perfusion with solutions containing high concentrations of the mobile low affinity Ca2+ buffers citrate (10-40 mM) or ATP (10-20 mM). The different Ca2+ oscillation patterns normally induced by ACh and CCK would therefore appear not to be caused by separate mechanisms. We propose that specific receptor-controlled modulation of Ca2+ signal spreading, either by regulation of Ca2+ uptake into organelles and/or cellular Ca2+ extrusion, or by changing the sensitivity of the Ca2(+)-induced Ca2+ release mechanism, can be mimicked experimentally by different degrees of cytosolic Ca2+ buffering and can account for the various cytosolic Ca2+ spike patterns. PMID- 1705884 TI - Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid. AB - The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene-encoded precursor poly-proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M-PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport-defective mutant-infected cells, suggesting that transport of M-PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein-lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding-defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell. PMID- 1705885 TI - A specific combination of substrates is involved in signal transduction by the kit-encoded receptor. AB - The kit protooncogene encodes a transmembrane tyrosine kinase related to the receptors for the platelet derived growth factor (PDGF-R) and the macrophage growth factor (CSF1-R), and was very recently shown to bind a stem cell factor. To compare signal transduction by the kit kinase with signaling by homologous receptors we constructed a chimeric protein composed of the extracellular domain of the epidermal growth factor receptor (EGF-R) and the transmembrane and cytoplasmic domains of kit. We have previously shown that the chimeric receptor transmits potent mitogenic and transforming signals in response to the heterologous ligand. Here we demonstrate that upon ligand binding, the ligand receptor complex undergoes endocytosis and degradation and induces short- and long-term cellular effects. Examination of the signal transduction pathway revealed that the activated kit kinase strongly associates with phosphatidylinositol 3'-kinase activity and a phosphoprotein of 85 kd. In addition, the ligand-stimulated kit kinase is coupled to modifications of phospholipase C gamma and the Raf1 protein kinase. However, it does not lead to a significant change in the production of inositol phosphate. Comparison of our results with the known signaling pathways of PDGF-R and CSF1-R suggests that each receptor is coupled to a specific combination of signal transducers. PMID- 1705886 TI - Three-dimensional structure of natural charybdotoxin in aqueous solution by 1H NMR. Charybdotoxin possesses a structural motif found in other scorpion toxins. AB - A 600-MHz proton NMR study of natural charybdotoxin, a toxin acting on K+ channels, is reported. The unambiguous sequential assignment of all the protons of the toxin was achieved. The analysis of NOEs and of backbone coupling constants showed the existence of an alpha-helix (residues 10-19) and of an antiparallel beta-sheet in the 26-35 part. Three-dimensional structures were generated by distance geometry, using a set of 114 interresidual calibrated constraints (63 sequential, 47 medium and long range, 4 hydrogen bonds) and 29 phi angles. These structures show that charybdotoxin is composed of a beta-sheet linked to an alpha-helix by two disulphide bridges and to an extended fragment by the third disulphide bridge. Comparison with the other known structures of long and short scorpion toxins shows that this structural motif is common to all these proteins. PMID- 1705887 TI - A peptide-sensitive channel of large conductance is localized on mitochondrial outer membrane. AB - Applying the technique of 'tip-dip' to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel. PMID- 1705888 TI - Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope. AB - A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats. PMID- 1705889 TI - Effects of N-acetylprocainamide and sotalol on ion currents in isolated guinea pig ventricular myocytes. AB - The effects of N-acetylprocainamide (NAPA) and sotalol on membrane current systems of guinea-pig ventricular myocytes were examined and compared with those of quinidine using patch-clamp techniques. All of the drugs prolonged the action potential duration (i.e. Class III effect) in isolated guinea-pig papillary muscles. In isolated ventricular cells. NAPA (300 microM) and sotalol (100 microM) produced a decrease in the delayed outward potassium current (IK) concomitantly with a slight decrease in the calcium current (ICa), which was similar to quinidine (10 microM). NAPA also slightly depressed the inward rectifier potassium current (IKrect). Thus, NAPA and sotalol both inhibited IK, and this action appears to be mainly responsible for their Class III effect. PMID- 1705890 TI - Differential effects of indolepyruvic acid and 5-hydroxytryptophan on indole metabolism in the pineal gland of the rat during the light-dark cycle. AB - The effect of two serotonin precursors, 5-hydroxytryptophan (5-OH-TRP) and indolepyruvic acid (IPA), a tryptophan ketoanalogue, on rat pineal indole metabolism during the light-dark cycle was investigated. 5-OH-TRP drastically increased the production of 5-hydroxyindoleacetic acid at a dose of only 10 mg/kg, whereas 50-100 mg/kg was needed to reach higher serotonin levels. It had no effect on the pathway leading to the production of N-acetylserotonin and melatonin. IPA, on the other hand, led to a marked dose-related increase in tryptophan, 5-OH-TRP, serotonin and 5-OH-indoleacetic acid, and was also active on N-acetylserotonin and melatonin synthesis in both phases. The different behaviour of these two substances with regard to melatonin synthesis was also confirmed by their effects on N-acetyltransferase, since IPA increased, whereas 5 OH-TRP decreased its activity. These data suggest that an increase in serotonin does not necessarily lead to an increase in melatonin, and that IPA may in fact induce this effect by altering the activity on N-acetyltransferase, which is regarded as a key enzyme in pineal hormone synthesis. PMID- 1705891 TI - Anti-substance P anti-idiotypic antibodies modulate the secretory process in the rat parotid gland in vitro. AB - Anti-idiotypic antibodies (anti-Id) obtained in rabbits in response to immunization with polyclonal anti-substance P antibodies (anti-SP) were shown to bind specifically and with high affinity to membranes from rat parotid gland cells. Whereas substance P (SP) was unable to displace anti-Id from membrane binding sites, anti-Id partly inhibited the binding of radiolabelled substance P. Like substance P, anti-Id were able to trigger protein secretion by parotid cells i.e. to behave as physiological agonists of the neuropeptide. Under our experimental conditions, the biological effects of both ligands appear to be additive. Unlike substance P, however, anti-Id did not potentiate the secretory response induced by a beta-adrenoceptor agonist. Taken together, the present results might indicate that anti-Id interact with epitope(s) at or/and near the peptide-combining site on the substance P receptor. These data demonstrate further the possibility of raising pharmacologically active anti-receptor antibodies through the immunological anti-idiotypic approach. PMID- 1705892 TI - Calcium dependence of the contraction produced by endothelin (ET-1) in isolated guinea-pig trachea. AB - Endothelin (ET-1, 1 pM to 0.1 microM) produced a concentration-dependent contraction of isolated guinea-pig trachea. BAY K 8644 (1 microM) did not significantly alter the concentration-response curve for ET-1. Incubation with nicardipine (10 microM) partly inhibited responses to low concentrations (10 pM to 1 nM) of ET-1 while verapamil (10 microM) and diltiazem (10 microM) were ineffective. La3+ (10 microM) and Cd2+ (10 microM) preferentially depressed the responses evoked by high concentrations (30 nM-0.1 microM) of ET-1 without affecting the responses evoked by low concentrations of the peptide. Incubation in Ca2(+)-free (with EDTA, 1 mM) medium resulted in suppression of the responses elicited by low concentrations of ET-1 and partial inhibition of the responses elicited by high concentrations of the peptide. It is concluded that responses to ET-1 are dependent on extracellular Ca2+. The promotion of Ca2+ entry by ET-1 is not confined to a particular class of Ca2+ channel. An intracellular source of Ca2+ is also mobilized by high concentrations (greater than 10(-9) M) of ET-1. PMID- 1705893 TI - Neuropeptide Y and JO 1784, a selective sigma ligand, alter intestinal ion transport through a common, haloperidol-sensitive site. PMID- 1705895 TI - Epitope mapping of human thyroglobulin reveals a central immunodominant region. AB - Thyroglobulin is the thyroid hormone precursor and the major antigen frequently involved in autoimmune diseases. The primary structure of human thyroglobulin is known but the spatial structure remains largely undetermined. By using fusion protein produced in prokaryotic system we have characterized seven short immunoreactive peptides carrying at least one epitope. None of them includes hormonogenic sites, but five are concentrated in the central part of the monomeric molecule, which thus emerges as the major immunogenic region of this protein. PMID- 1705894 TI - Schistosome vaccines. AB - Schistosomiasis control currently relies primarily on chemotherapy which is both expensive and temporary. There is an urgent need for an effective vaccine. Studies in animal models and man have demonstrated the existence of protective immunity. Antibody-dependent cell-mediated cytotoxicity mechanisms involving eosinophils and macrophages have been implemented in destruction of the parasites. Antigens expressed on the surface of the schistosomulum are among the targets of protective immune responses. Vaccines comprising recombinant antigens are now being tested in vivo for their capacity to evoke protective responses. Live oral vaccines based on attenuated Salmonella expressing schistosomular surface antigens are being developed. PMID- 1705896 TI - Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel. AB - The reversible acetylcholine esterase inhibitor (-)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorata electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maelicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable albeit only from other than the transmitter binding site(s). PMID- 1705897 TI - Proglycogen: a low-molecular-weight form of muscle glycogen. AB - We recently reported that muscle contains a trichloroacetic acid-precipitable component having Mr approx. 400 kDa that can be glucosylated by an endogenous enzyme acting on UDPglucose. This component contains within itself the autocatalytic, self-glucosylating protein glycogenin, the primer for glycogen synthesis. We now report that this substance, to which we give the name proglycogen, is a glycogen-like molecule constituting about 15% of total glycogen. It acts as a very efficient acceptor of glucose residues added from UDPglucose. Further, that the endogenous enzyme that adds the glucose to proglycogen is not the autocatalytic protein but a glycogen synthase-like enzyme. Proglycogen may be an intermediate in the synthesis and degradation of macromolecular glycogen and may exist and be metabolized as a separate entity. Consideration should now be given to the revival of the concept that tissue contains two forms of glycogen. One is proglycogen. The other is the 'classical', macromolecular glycogen. Additionally, proglycogen and glycogen may be glucosylated by different forms of synthase. PMID- 1705898 TI - Phosphotyrosine as a specificity determinant for casein kinase-2, a growth related Ser/Thr-specific protein kinase. AB - The motif Ser-Ser-Ser-Glu-Glu is readily phosphorylated by casein kinase-2 (CK 2), a growth-related protein kinase whose consensus sequence is Ser(Thr)-Xaa-Xaa Glu(Asp) [(1990) Biochim. Biophys. Acta 1054, 267-283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK-2 phosphorylation, the phosphotyrosyl peptide Ser-Ser-Ser-TyrP-TyrP actually being a substrate more efficient than Ser-Ser-Ser-Glu-Glu itself both in terms of Km (0.69 vs 2.43 mM) and Vmax. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK-2. While Ser-Ser-Ser-TyrP TyrP is a better substrate than Ser-Ser-Ser-SerP-SerP, which in turn is better than Ser-Ser-Ser-Glu-Glu, Ser-Ser-Ser-ThrP-ThrP is a less efficient substrate than Ser-Ser-Ser-Glu-Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK-2 appears to be TyrP greater than SerP much greater than ThrP. PMID- 1705899 TI - Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells. AB - We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity. PMID- 1705900 TI - Phosphorylation and activation of hormone-sensitive lipase in isolated macrophages. AB - Hormone-sensitive lipase (HSL) is responsible for the neutral cholesterol ester hydrolase activity in macrophages. Incubation of intact WEHI macrophages or mouse peritoneal macrophages leads to phosphorylation of HSL, which is increased by incubation with either dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine or okadaic acid. Correspondingly, these agents also activate neutral cholesterol ester hydrolase activity in intact WEHI cells. Regulation of mobilisation of esterified cholesterol in macrophages may be of antiatherogenic value, which this model system now allows us to investigate further. PMID- 1705901 TI - Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein. AB - Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP. PMID- 1705902 TI - Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line. AB - We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system. PMID- 1705903 TI - Co-purification of a small RNA species with multicatalytic proteinase (proteasome) from rat liver. AB - Previous studies have come to different conclusions about the presence of RNA in particles known variously as prosomes, proteasomes or multicatalytic proteinase (MCP). To determine the reason for this, MCP was isolated from rat liver by 4 different purification protocols. One major band of RNA, about 80 nucleotides in length, co-purified in all preparations. The amount of RNA detected was less than one molecule per MCP particle suggesting that there may be more than one population of MCP in rat liver cells. PMID- 1705904 TI - PUVA increases ornithine decarboxylase gene expression in mouse skin. AB - The influence of topical PUVA on the activity of ornithine decarboxylase (ODC) and its gene expression was investigated in the skin of hairless mouse. After 1 h of application of 0.3% 8-methoxypsoralen, irradiation of 3 J/cm2 of ultraviolet A (UV-A) was administered. The ODC activity markedly increased and peaked at 24 h following UV-A irradiation. The ODC mRNA level, analyzed in dot-blot analysis, elevated to about 5 times that of control at 24 h after treatment. These results show that the PUVA-induced ODC activity is due in part to an increase in ODC gene expression. PMID- 1705905 TI - Contributions of basic immunology to human health. AB - The sixth symposium in the series "Contemporary Topics in Immunology" was held in New Orleans on June 3, 1990, at the joint meeting of The American Association of Immunologists and the American Society of Biochemistry and Molecular Biology. The symposium was sponsored jointly by The American Association of Immunologists, the Clinical Immunology Society, and and the National Institute of Allergy and Infectious Diseases, and was titled "The Contributions of Basic Immunology to Human Health." Five speakers, whose research has clear relevance to the treatment and prevention of major human diseases, discussed topics of great current interest: hematopoietic stem cells, cell adhesion and lymphocyte homing; the complexities of autoimmunity and approaches to diverting or depressing autoaggressive immunity; structure and functions of the interferons and the construction of designer and chimeric interferons; the varied functions of transforming growth factors and molecular events that regulate the synthesis of TGF beta; and the roles of cytokines in the expression of human immunodeficiency virus and the prospects for controlling HIV infections by regulating selected cytokines. This symposium will be remembered for the exceptional clarity with which each speaker illustrated how fundamental knowledge in immunology fuels advances in the treatment and prevention of those human disorders that involve the immune system. PMID- 1705906 TI - The nature of noncholinergic membrane potential responses to transmural stimulation in guinea pig ileum. AB - The effect of substance P antagonism on membrane potential responses to transmural nerve stimulation in the presence of atropine was examined in circular smooth muscle of the guinea pig ileum. Intracellular recordings of membrane potential responses recorded 3-5 mm oral to the transmural stimulus consisted of an inhibitory junction potential followed by two distinct depolarizations referred to as early and late excitatory junction potentials. Substance P antagonism was achieved by desensitization with high doses of substance P or use of the antagonist Spantide (Sigma Chemical Co., St. Louis, MO). Substance P antagonism had no effect on the amplitude of the inhibitory junction potential, caused an increase in the latter portion of the early excitatory junction potential, and abolished the late excitatory junction potential. The excitatory junction potential potentiated by substance P receptor antagonism was associated with a decrease in membrane resistance, increased in amplitude with conditioning hyperpolarizations to the estimated equilibrium potential for K+, and was blocked by the Cl-/HCO3- exchange inhibitor DIDS or prolonged perfusion with low-chloride solution. These studies suggest that a noncholinergic, non-substance P neurotransmitter is released from enteric motoneurons that produces excitation through an increase in smooth muscle chloride conductance. PMID- 1705907 TI - Inhibition of anaphylaxis-evoked intestinal fluid secretion by the dual application of an H1 antagonist and cyclooxygenase inhibitor. AB - The regulation of anaphylaxis-mediated fluid secretion by the small intestine was examined in rats immunized by infection with Trichinella spiralis and reinfected by intraduodenal injection with L1 larvae. Net fluid secretion, which was measured as the volume of fluid present in the intestine 30 minutes after the challenge infection, was significantly greater in both actively and passively immunized rats than in nonimmune rats. The amount of fluid recovered from the immune host was equivalent to that secreted in response to 50 micrograms/kg of prostaglandin E2 or 250 micrograms/kg of cholera toxin. Worm-induced fluid secretion in immune hosts was reduced by treatment with diphenhydramine and inhibited by the dual application of diphenhydramine and indomethacin. Indomethacin alone had no effect despite inhibiting mucosal prostaglandin synthesis. Fluid secretion was unaltered by prior treatment of immune rats with a 5-lipoxygenase inhibitor, L-651,392, and only slightly reduced when L-651,392 was used in combination with indomethacin. After a challenge infection, more histamine was released into intestinal loops of immune rats than those of nonimmune rats. Prechallenge treatment of immune rats with indomethacin caused a twofold increase in histamine release. In summary, anaphylaxis-induced fluid secretion in the small intestine is mediated largely by histamine and cyclooxygenase products. This secretion can be lowered by treatment with diphenhydramine and further reduced by diphenhydramine in combination with indomethacin. The paradoxical effects of indomethacin when used alone and in combination with diphenhydramine are explained by the downregulation of histamine release by products of the cyclooxygenase pathway. PMID- 1705908 TI - Stimulation of chicken growth hormone release by phorbol esters. AB - Synergism between thyrotropin-releasing hormone (TRH) and human pancreatic growth hormone-releasing factor (hpGRF) has been shown in a primary (48 hr) culture of chicken adenohypophyseal cells established in this laboratory. The purpose of the present study was to determine if phorbol esters acting alone or in concert with TRH or hpGRF affect chicken GH release. Collagenase-dissociated chicken adenohypophyseal cells were treated (2 hr) with combinations of TRH, hpGRF, phorbol esters (activators of protein kinase C; PKC), and pharmacologic agents that increase cAMP. Phorbol myristate acetate (PMA) or phorbol dibutyrate (PDBu) alone stimulated GH release in a dose-dependent manner; either phorbol ester (10( 6) M) increased GH release from 100 to 390% over the value obtained in the absence of test agents (control). Similarly, hpGRF (10(-9) M), 8 Br-cAMP (10(-3) M), forskolin (10(-6) M), or isobutylmethylxanthine (IBMX, 10(-3) M) alone elevated GH release by at least 60% over the control value. The combined effects of phorbol esters (either PMA or PDBu) and hpGRF, 8 Br-cAMP, or forskolin on GH release were additive. Only one combination, phorbol esters with IBMX, exerted synergistic effects on GH release. No synergy was shown between TRH (1.3 x 10(-9) M) and either phorbol ester. These findings are the first to implicate PKC in chicken GH release in vitro. In addition, these studies, together with previous results, suggest that TRH and hpGRF synergy occurs via a pathway that arises prior to activation of PKC. PMID- 1705909 TI - The adenohypophysis of the Australian lungfish, Neoceratodus forsteri--an immunocytological study. AB - The cell types in the adenohypophysis of Neoceratodus resemble closely those already described for Lepidosiren and Protopterus. Four of these were immunocytochemically identified as prolactin cells, gonadotropes, corticotropes, and melanotropes. Antiserum to bullfrog growth hormone could not distinguish between prolactin cells and somatotropes. Anti-bullfrog prolactin, however, did selectively stain the prolactin cells, which allowed the identification of the somatotropes. The presumptive thyrotropes, as the only remaining cell type in the pars distalis, can then be tentatively identified by default. Likewise a PAS positive cell type in the pars intermedia had no immunoreactivity to any of the antisera used. The functional significance of this cell remains to be demonstrated. One of the more unexpected findings was the presence of large numbers of cells immunoreactive to alpha-MSH in the proximal pars distalis. The implications of the presence of these cells in adult lungfish are discussed. The distribution of cell types within the pituitary of Neoceratodus showed more regionalization than is present in the other lungfish and corresponded more closely to that described for primitive actinopterygian fish. The general structure of the pituitary of Neoceratodus also resembled primitive actinopterygian fish more closely than it did amphibians, unlike the pituitaries of Lepidosiren and Protopterus. The evolutionary significance of this is also discussed. PMID- 1705910 TI - [Hairy-cell leukemia as a model for studying the effects of interferon on the immune system]. PMID- 1705911 TI - [The interferon system and interferon receptor binding]. PMID- 1705912 TI - [Interferon in the treatment of Philadelphia-positive chronic myeloid leukemia]. PMID- 1705913 TI - [Interferon: a new weapon in the therapeutic strategy of multiple myeloma]. PMID- 1705914 TI - Nerve fibers in human myocardial scars. AB - The relationships between ischemic heart disease, myocardial scars, ventricular nerve fibers, and ventricular arrhythmias have not been established despite considerable evidence suggesting important correlations. We recently described the reactions of nerve fibers in necrotic, healing, and healed rat myocardium. Prompted by these studies and by the lack of similar information for humans, we studied the structural relationships between nerve fibers and human myocardial scars. Hearts were obtained from transplant surgery and autopsy. Nerve fibers were labeled with antibody to S-100 protein. Light and electron microscopy of left ventricular scars revealed (1) fiber densities greater than those in adjacent intact myocardium, (2) fiber aggregates concentrated irregularly along the periphery of lesions, (3) fibers few in number or absent in the deeper aspects of scars, and (4) axonal enlargements containing clear and dense storage granules within the fiber aggregates. Like all other elements of the scars, the nerve fibers appeared to be oriented predominantly in the long axis of myocytes located at the edges of the lesions. Based on our experimental findings in rat hearts, these studies suggest that human myocardial nerve fibers regenerate after necrotizing injuries and that at least some of the resulting scar-associated fibers have structural features differing from those in uninjured myocardium. We suspect that these structural differences might be associated with functional alterations that could affect the triggering of ventricular arrhythmias. PMID- 1705915 TI - Prostitutes and public health services cooperate on AIDS--prevention in Brazil. PMID- 1705916 TI - Approaches to AIDS education for the grassroots in Nigeria. AB - The most important aspect of reaching people at the grassroots level is to ensure their understanding by using the most appropriate language, and by ensuring that the largely illiterate population will, nevertheless, be well served by print and electronic mass media. The article also emphasizes the need to adapt educational aids to the audience they address, as well as the preference of offering interactive education. PMID- 1705917 TI - Characterization of IL-6 production by B cells. AB - IL-6 production by antigen-specific and antigen-non-specific B cells was studied. It was found that low concentrations of antigen (0.001 micrograms/ml) could stimulate IL-6 production by antigen-specific B cells (10(3)-10(4) fold lower concentration of antigen when compared with that of antigen-nonspecific B cells). A kinetic study showed that IL-6 activity in supernatants from antigen-specific B cells was detectable as early as 2 hours. The production of IL-6 by non-specific B cells and by monocytes was distinctly different. These results suggest that antigen-specific B cells may play a critical role in early T cell activation by antigen. PMID- 1705918 TI - 5'-3' and 3'-5' translation of the same RNA results in hydropathically similar peptides that are antigenically related. AB - When a single RNA sequence is read in either the 5'-3' or 3'-5 direction, the translated peptides often are hydropathically similar even though their sequences may be different. To investigate whether hydropathically similar peptides might also be antigenically related, two peptides were synthesized from the substance P anti-sense RNA transcript: CAU CAA UCC AAA GAA CUG CUG AGG CUU GGG UCG. Translation of this RNA in the 5'-3' direction and in the 3'-5' direction resulted in two different peptides. HQSKELLRLGS and AGFGVVKKPNY, respectively. As anticipated, both peptides shared similar hydropathic profiles but were quite different with respect to their sequences. To examine their antigenic relatedness, mice were immunized with either peptide, and monoclonal antibodies were produced. Using an enzyme-linked immunosorbent assay, it was possible to demonstrate that the majority of monoclonal antibodies, selected for reactivity against the original immunogen, also reacted with the other peptide. The observed binding was determined to be specific since reactivity could be blocked with either soluble peptide. Thus, we demonstrate that hydropathically similar peptides obtained from the same RNA but translated in opposite directions are antigenically related despite difference in amino acid sequences. PMID- 1705919 TI - Regulation of tumor necrosis factor secretion in leukocytes from alpha-1 antitrypsin deficient humans. AB - Alpha-1-antitrypsin (AT) is one of several alpha-globulins which have been shown to be inhibitors of human peripheral blood monocyte TNF secretion in vitro. AT deficiency states exist, within which individuals of either the PiSS or PiZZ phenotype have reduced hepatocyte and mononuclear phagocyte AT secretion when compared to normal PiMM subjects. Here we have compared the capacity of peripheral blood monocytes of all three phenotypes to respond to both enhancers and inhibitors of TNF secretion. All monocytes exposed to lipopolysaccharide (LPS), interferon-gamma (IFN-gamma) and endotoxin, PGE2, transforming growth factor-beta 1, whole plasma alpha-globulins, purified AT and IL-6 responded equally with respect to the secretion of TNF. Our findings show that the regulation of TNF secretion in leukocytes from AT deficient humans is normal and suggest that defective AT secretion alone does not result in the aberrant regulation of TNF secretion. PMID- 1705920 TI - Interferon-gamma can remain on the cell surface during the induction of the antiviral state. AB - The antiviral activity of cell-associated, non-elutable recombinant human gamma interferon (rHuIFN-gamma) was neutralized by antibody. The neutralization of cell associated rHuIFN-gamma was maximal through 2 h (60-100%) and declined through 8 h (20-40%). Concomitantly, the antiviral activity of cell-associated [Met-Gln-Asp Pro]-rHuIFN-gamma was sensitive to trypsin digestion over the same time period. However, the cell-associated antiviral activity of [Cys-Tyr-Cys]-rHuIFN-gamma remained sensitive to trypsin through 8 h. Neutralization of cell-associated rHuIFN-gamma by antibodies to the N-terminal end of HuIFN-gamma suggests that the N-terminal end(s) of cell-associated rHuIFN-gamma is directed outward from the receptor. Further, immunoprecipitation of radio-labelled rHuIFN-gamma by antibody alone suggests that biologically active rHuIFN-gamma is an oligomer. Taken together, these studies suggest that neutralization of cell-associated rHuIFN gamma is probably due to divalent binding of antibody to or between rHuIFN-gamma in receptors on the cell surface. Also, our studies indicate that rHuIFN-gamma can remain associated with the cell surface during the induction of the antiviral state (AVS) and that binding of antibody to cell-associated rHuIFN-gamma inhibits the molecular events responsible for induction of the AVS. PMID- 1705921 TI - Autogenic training and future oriented hypnotic imagery in the treatment of tension headache: outcome and process. AB - The aim of the present study was (a) to investigate the relative efficacy of autogenic training and future oriented hypnotic imagery in the treatment of tension headache and (b) to explore the extent to which therapy factors such as relaxation, imagery skills, and hypnotizability mediate therapy outcome. Patients were randomly assigned to the 2 therapy conditions and therapists. 55 patients (28 in the autogenic therapy condition and 27 in the future oriented hypnotic imagery condition) completed the 4 therapy sessions and 2 assessment sessions. No significant main effect or interaction effects for treatment condition or therapist was revealed. A significant effect for time in analyzing scores for headache pain, pain medication usage, depression, and state anxiety was found. In the self-hypnosis condition, pain reduction proved to be associated with depth of relaxation during home practice (as assessed with diaries) and capacity to involve in imagery (as assessed with the Dutch version [van der Velden & Spinhoven, 1984] of the Creative Imagination Scale [Barber & Wilson, 1978/79; Wilson & Barber, 1978]). After statistically controlling for relaxation and imagery, hypnotizability scores (as assessed with the Dutch version [Oyen & Spinhoven, 1983] of the Stanford Hypnotic Clinical Scale [Morgan & J.R. Hilgard, 1975, 1978/79]) were significantly correlated with ratings of pain reduction. Results are discussed in the context of the neo-dissociation and social-cognitive model of hypnoanalgesia. The clinical relevance and the methodological shortcomings of the present study are also critically assessed. PMID- 1705922 TI - Reduced mature size in progeny of swine severely restricted in protein intake during pregnancy. AB - The objective was to determine the effects of maternal gestation diet on mature body and organ size and composition of progeny. Female progeny of swine fed daily 1.8 kg (6,000 kcal DE) of a semipurified diet containing less than 0.5% protein (PR), 1.8 kg (6,000 kcal) of a standard maize-soybean meal-based gestation diet (C) or 0.6 kg (2,000 kcal DE) of C diet to d 70 of gestation and 1.8 kg (6,000 kcal DE) from d 71 to parturition (R) were used. They were fed ad libitum a standard maize-soybean meal-based growing diet to age 25 wk, then continued on limited feed intake to age 62 wk, then returned to ad libitum intake to age 119 wk. Body weight and ultrasonically determined subcutaneous backfat depth were recorded at 62, 79, 97, 105 and 119 wk of age and indices of body composition and organ weights were recorded on carcasses of animals killed at age 119 wk. Progeny of PR dams were permanently stunted in mature body weight and in weights of eviscerated carcass, heart, liver, kidneys, stomach and small intestine, but not in cerebrum, cerebellum weights or RNA and DNA content. It is concluded that severe protein restriction during gestation in swine resulted in permanent whole body weight stunting of progeny but that weights and total protein, RNA and DNA content of cerebrum, cerebellum and RNA and DNA concentration of longissimus muscle were unaffected. PMID- 1705923 TI - Lens beta-adrenergic receptors. Functional coupling to adenylate cyclase and photoaffinity labeling. AB - Beta-adrenergic drugs affect lens epithelial and fiber cells. The regulation and cellular integration of lens beta-adrenergic responses are largely unknown. These studies further characterize beta-adrenergic receptors in lens cells with respect to cyclic adenosine monophosphate (cAMP) production and identification of receptor polypeptides. Stimulation of beta-adrenergic receptors in organ-cultured chick lenses resulted in dose-dependent increases in intracellular cAMP levels. Isoproterenol-elicited cAMP accumulation was found in both epithelial/superficial fiber cells and cortical fiber cells. Hormonal stimulation also apparently initiated additional mechanisms involved in the regulation of cAMP levels (ie, phosphodiesterase activation/receptor desensitization). Individual receptor polypeptides were identified in epithelial and fiber membranes with the photoaffinity probe 125I-iodocyanopindolol diazarine. The probe specifically labeled distinct populations of receptor polypeptides in the two cell types. Lens beta-adrenergic receptors were also shown to bind (-) stereoisomers of adrenergic ligands preferentially. These results indicate that differentiating fiber cells are hormonally sensitive to beta-adrenergic stimulation and that epithelial and fiber cells may respond differentially to beta-adrenergic drugs, at least in part, because of their distinct receptor polypeptides. PMID- 1705924 TI - Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation. AB - The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5 negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing. PMID- 1705926 TI - Silver stains for identification of neuroendocrine cells. A study of the chemical background. AB - The chemical background of silver stains used for visualization and characterization of peripheral neuroendocrine cells in the gastrointestinal tract and pancreas, and of their corresponding tumours, was studied in tissue sections and by a dot-blot technique. Sequential staining of pancreatic islets with an immunohistochemical procedure and silver staining of the same tissue section revealed that chromogranin A immunostained cells also displayed an argyrophil reaction with the Grimelius method, but no argentaffin reaction with the Masson technique. Accordingly, purified chromogranin A (15 micrograms or less) treated in formalin and applied to nitrocellulose did not show any argentaffin reaction but displayed a dose-related argyrophil reaction. Equal quantities of other polypeptide components did not give rise to any silver reaction. Further dot-blot studies showed that the tryptophan and tyrosine metabolites, dopamine, norepinephrine, 5-hydroxytryptamine and 5-hydroxinodole caused strongly argentaffin and argyrophil reactions while epinephrine, 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophan gave only the former reaction. Among other chemical components studied, only guanine displayed weak silver staining. The results indicate that the reaction products between aldehydes and the granular content of biogenic amines synthesized from tryptophan and tyrosine display an argentaffin reaction and that the granular chromogranin A caused an argyrophil but no argentaffin reaction. PMID- 1705925 TI - Difference in the ability of blood group-specific lectins and monoclonal antibodies to recognize the ABH antigens in human tissues. AB - Twelve different kinds of blood group-specific lectins have been used along with monoclonal anti-A, -B and -H antibodies for detecting the corresponding antigens in selected human tissues. Although most of the lectins recognized the antigens in the tissue sections examined, they displayed marked differences in their recognition patterns in certain tissues. Helix asparsa agglutinin (HAA), Helix pomatia agglutinin (HPA) and monoclonal anti-A antibody recognized A antigens in the mucous cells of salivary glands from blood group A or AB nonsecretor as well as secretor individuals, whereas Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin-I (GSA-I), Sophora japonica agglutinin (SJA) and Vicia villosa agglutinin (VVA) did not bind to them from nonsecretors. A antigens in endothelial cells, lateral membrane of pancreatic acinar cells and small mucouslike cells of submandibular glands from some individuals were likewise recognized by HAA and HPA but not by other blood group A-specific lections. In contrast, both HAA and HPA did not recognize the A antigens in mucous cells of Brunner's glands while other A-specific lectins and monoclonal anti-A antibody reacted specifically with the antigens. Such a difference was not observed with lectins specific for blood group B. However, the B antigens in Brunner's glands were recognized by these lectins but not with monoclonal anti-B antibody. The difference in labelling ability was also noted among the blood group H-specific lectins and monoclonal anti-H antibody in endothelial cells of blood vessels. Ulex europaeus agglutinin-I reacted with these cells irrespective of ABO and the secretor status of the individuals, while Anguilla anguilla agglutinin and monoclonal anti-H antibody reacted only with those cells from blood group O individuals. No reaction was observed with Lotus tetragonolobus agglutinin in these tissue sites. These results suggest a great diversity of blood group antigens in different human tissues. PMID- 1705927 TI - Alloreactive T-cell clones identify multiple HLA-DQw3 variants. AB - HLA-DQw3 is a broadly defined alloantigen that has been subdivided by serological, biochemical, and molecular methods into three distinct specificities: DQw7, DQw8, and DQw9. In order to characterize functionally relevant structural polymorphisms within this family of alloantigens, we generated a series of DQw3-reactive T-cell clones that together recognize six different variants of DQw3. T-cell clones IG11 and IG9 were found to recognize three distinct functional variants associated with a majority of DQw3+ cells, while clones 21J, IE6, 64B, and IC3 recognized four more narrowly distributed functional variants associated with unique DQw7, DQw8, and DQw9 subsets. Comparison of known DQB gene sequences suggested candidate recognition sites for clones IG11 and 64B in the region of amino acid residues 66 to 71 and residue 57 of the DQ beta chain. In contrast, no unique DQB or DQA sequences were found that individually corresponded to the reactivity patterns of clones 21J, IE6, IG9, or IC3, suggesting that an interaction between DQ alpha and DQ beta chains determines allo-recognition. These data are consistent with the hypothesis that T cells recognize specific alloepitopes on HLA class II molecules, either as distinct structural elements that trigger an alloresponse or, more indirectly, as contact elements that influence alloreactivity by governing the binding of foreign peptide. The results illustrate the diversity of possible T cell responses directed toward HLA-DQ molecules and suggest that T cell recognition of the DQ heterodimer alone, or a peptide antigen bound to the DQ heterodimer, can be affected either by the individual DQ alpha and beta chains, or by a more complex interaction between the two. PMID- 1705928 TI - Synthesis and activity of butirosin derivatives with 5''-amidino and 5'' guanidino substituents. AB - The preparation and antibacterial activity of the 5''-guanidino (6) and 5'' amidino (7) derivatives of 4'-deoxybutirosin A (1) as well as the 5''-guanidino derivative (8) of butirosin A are described. The key intermediates, tetra-N benzyloxycarbonyl-5''-azido derivatives were selectively reduced with NiCl2-NaBH4 to give the corresponding 5'-amino derivatives. Subsequent guanidination or amidination followed by deblocking afforded the final compounds 6, 7 and 8. The 5''-guanidino derivatives (6 and 8) were more active against Gram-positive and Gram-negative bacteria than the corresponding 5''-hydroxy derivatives (1 and butirosin A). Compound 6 was also active against a variety of methicillin resistant Staphylococcus aureus (MRSA). PMID- 1705929 TI - Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved. AB - The genes coding for the lactose permease and beta-galactosidase, two proteins involved in the metabolism of lactose by Lactobacillus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli. The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs). One of these ORFs codes for the lactose permease gene, and the other codes for the beta galactosidase gene. The lactose permease gene is located in front of the beta galactosidase gene, with 3 bp in the intergenic region. The two genes are probably transcribed as one operon. Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the beta-galactosidase gene. This promoter is similar to those found in E. coli with general characteristics of GC-rich organisms. In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L. bulgaricus genome, which is rich in GC (GC content of 54%). The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus. The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E. coli. Little homology was found between the lactose permease of L. bulgaricus and E. coli, but the residues which are involved in the binding and the transport of lactose are conserved. The carboxy terminus is similar to that of the enzyme III of several phosphoenolpyruvate-dependent phosphotransferase systems. PMID- 1705930 TI - Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum. AB - The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase. PMID- 1705931 TI - A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK. AB - A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control. PMID- 1705932 TI - Brain-specific small RNA transcript of the identifier sequences is present as a 10 S ribonucleoprotein particle. AB - BC-1 RNA is a small RNA transcript of the identifier repetitive sequences present in rodent genomes. The RNA has been reported to be specific to the brain and confined to the cytoplasm. The RNA level increases during the 1st month after birth. To understand its cytoplasmic function, it seems important to examine whether BC-1 RNA is present as an RNP. It is believed that the protein component may govern the functions of BC-1 RNA in the brain cells. In the present report, we have demonstrated that BC-1 RNA is not free but complexed with proteins to form a 10 S RNP in the cytoplasm. We have also shown that the 10 S RNP is not associated with cytoplasmic structures such as polysomes/ribosomes or microsomes. The buoyant density of the RNP was 1.26 g/cm3 in metrizamide. Furthermore, some of the protein components were shown to be in direct contact with RNA, since photo-cross-linking adducts of protein to BC-1 RNA were identified upon UV irradiation of the 10 S BC-1 RNP. PMID- 1705933 TI - Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes. AB - Secretion of chloride ions via apically located anion-selective channels in epithelia regulates fluid formation and cytosolic Cl- homeostasis. In order to understand the biochemical basis of Cl- channel function, we attempted to isolate this transporter from bovine tracheal apical membranes. Initially, peripheral polypeptides were removed from apically enriched vesicles by washing with alkaline buffer (pH 10.8) containing 2 mM CHAPS. The resulting pellet contained 50-60% of the original protein and displayed 2-fold enhanced Cl- channel activity compared to untreated vesicles. The pellet was treated with Triton X-100, and the solubilized proteins were separated on the cationic exchanger CM-cellufine. Washing the resin with a pH 8.0-8.3 buffer eluted a fraction with enriched Cl- channel activity. This fraction contained less than 5% of the total solubilized protein. A subsequent separation was performed using the anionic exchanger AM cellufine. The highest activity was found in the fractions eluted by 80-120 mM KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a major 38,000-Da protein band. This band was electroeluted from the gel under nondenaturing and nonreducing conditions and reconstituted into phosphatidylcholine liposomes. KCl-loaded vesicles containing the purified 38-kDa protein transported up to 5 nmol of 125I-/mg of protein/5 min. This value was 15 fold higher than the uptake measured in vesicles reconstituted with total solubilized membrane proteins and 4-fold higher compared to the CM-cellufine enriched fraction. The observed 125I- uptake was 90% inhibited by 100 microM 4,4 bis(isothiocyano)-2,2'-stilbenedisulfonate or 10 microM valinomycin. In summary, we have developed a biochemical protocol for the isolation of a 38 kDa protein mediating potential-dependent and 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate sensitive Cl- channel activity. PMID- 1705934 TI - Synthesis of the photoaffinity probe 3-(p-azidobenzyl)-4-hydroxycoumarin and identification of the dicoumarol binding site in rat liver NAD(P)H:quinone reductase (EC 1.6.99.2). AB - A photoaffinity analog of 4-hydroxycoumarin containing an azidobenzyl group at the 3-position and, if desired, carbon-14 or tritium radionuclides has been synthesized and characterized. This compound, 3-(p-azidobenzyl)-4 hydroxycoumarin, serves as an effective competitive inhibitor of the dicoumarol sensitive NAD(P)H:quinone reductase (EC 1.6.99.2; DT-diaphorase) from rat liver, having an apparent inhibition constant of 6.6 x 10(-8) M, a value comparable to that observed for dicoumarol (1.7 x 10(-9) M), significantly lower than for Warfarin (3.5 x 10(-5) M) and well within the range required of an effective photoaffinity reagent. Irradiation of the reductase with ultraviolet light in the presence of the photoprobe resulted in the covalent labeling of up to 10% of the protein. Greater than 99% of the covalent incorporation is precluded by the addition of 15 microM dicoumarol, consistent with the specific labeling of the 4 hydroxycoumarin binding site of this enzyme by this photoaffinity reagent. Further evidence of a high degree of specificity is provided by the isolation and sequence analysis of the peptides covalently modified by this reagent. A single region within the protein was found to be labeled, with threonine 127 and tyrosine 128 being the only amino acid residues that were observed to be modified. These results, for the first time, define a portion of the 4 hydroxycoumarin binding site within a protein that has a well established sensitivity to this type of anticoagulant and, because dicoumarol serves as a competitive inhibitor for pyridine nucleotides in this enzyme, may also define a portion of this unusual pyridine nucleotide binding site. In addition, these results suggest that this reagent may be effective as a highly specific photoaffinity probe in the identification of other proteins that are similarly inhibited by 4-hydroxycoumarin derivatives, such as the microsomal enzymes associated with the vitamin K-dependent carboxylation system. PMID- 1705935 TI - Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression. AB - A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica. PMID- 1705936 TI - Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant. AB - This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate. PMID- 1705937 TI - Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene. AB - Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193--- Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme. PMID- 1705938 TI - Spinal narcotics for postoperative analgesia in total joint arthroplasty. A prospective study. AB - Sixty patients who were scheduled to have an elective total hip or knee arthroplasty were randomly assigned to one of three groups of twenty patients each before operation with spinal anesthesia. A double-blind technique was used throughout the study. The patients in Group I (control group) received hyperbaric 1 per cent tetracaine with epinephrine as the subarachnoid spinal anesthetic; the patients in Group II (morphine group), hyperbaric 1 per cent tetracaine with epinephrine and a single subarachnoid dose of Duramorph (morphine sulphate), 0.5 milligram; and those in Group III (Dilaudid group), hyperbaric 1 per cent tetracaine with epinephrine and a single subarachnoid dose of Dilaudid (hydromorphone hydrochloride), 0.002 milligram per kilogram of body weight. During the first twenty-four hours after the operation, the patients in Group II and Group III had significantly less pain compared with those in Group I. This was shown by the use of a visual linear-analog pain scale (p less than 0.05), the patients' ratings of the quality of relief of pain (p less than 0.02), and comparative measurements of the pain-altering medications that were used (p less than 0.05). The patients in Group II and Group III did not have any more complications or side effects than those in Group I. There was no significant difference in the quality and duration of analgesia between Group II and Group III. PMID- 1705939 TI - Modulation of native chondroitin sulphate structure in tissue development and in disease. AB - Chondroitin sulphate proteoglycans are synthesised by different tissues and cell types, and the chondroitin sulphate chains are variably sulphated. Three monoclonal antibodies 3B3, 7D4 and 6C3 that recognise different native chondroitin sulphate epitopes have been used to investigate changes in structure during embryonic tissue development in the chick and in the response of mature canine articular cartilage during experimental osteoarthritis. Strong focal expression of the epitopes was seen during development of chick bursa, which was different for the three epitopes and which changed during 5 days of development. In embryonic chick limb, although chondroitin sulphate is present throughout the cartilage, the 3B3 epitope, which is at the non-reducing terminus of chains, was only expressed on chondroitin sulphate within one region of the sub-articular cartilage. In mature canine articular cartilage the expression of this epitope on proteoglycans was very low, but when determined 3 or 6 months after induction of experimental osteoarthritis the level was greatly increased in all joints tested (23/23). The abundance of the other two native chondroitin sulphate epitopes was also increased in this experimental disease. The results show that expression of the chondroitin sulphate epitopes detected by the monoclonal antibodies changes during cellular differentiation and development and suggests that it is closely controlled by the cells synthesising chondroitin sulphate chains. PMID- 1705940 TI - Assessing the differentiation state of cultured bovine urothelial cells: elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I. AB - Although significant progress has recently been made in culturing mammalian urothelial cells, relatively little is known about their biochemical differentiation. In this paper, we assessed the differentiation state of cultured bovine urothelial cells by analyzing their keratins and a cell surface marker, uroplakin I. Urothelial cells were serially cultured either in a serum-free medium, or in a serum-containing medium in the presence of 3T3 feeder cells, with similar results. Despite their stratified appearance, both normal urothelium and cultured urothelial cells synthesize mainly K8, K18 and K19, keratins that are typically seen in simple epithelia. However, cultured urothelial cells synthesize a greatly increased amount of K5 and K6 keratins, which are usually expressed by stratified epithelia but present only in trace amounts in normal urothelium. These data indicate that, as far as keratin synthesis is concerned, cultured urothelial cells undergo an altered pattern of differentiation towards a more 'stratified phenotype'; this unusual finding has interesting implications for urothelial evolution. In the meantime, many superficial cells in cultured urothelial colonies make uroplakin I, a 27 x 10(3) Mr protein subunit of the asymmetrical unit membrane (AUM) characteristic of urothelial (superficial) umbrella cells. These results indicate that cultured urothelial cells undergo, at least in part, AUM biogenesis. Cultured urothelial cells thus provide a useful experimental model system for studying certain early steps of AUM formation. PMID- 1705941 TI - Human hair growth in vitro. AB - We report for the first time the successful maintenance and growth of human hair follicles in vitro. Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watchmakers' forceps. Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinised hair shaft, and was not associated with the loss of hair follicle morphology. [methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labelling of keratins showed that their patterns of synthesis did not change with maintenance. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth-regulatory effect on hair follicles in vitro and that EGF mimics the in vivo depilatory effects that have been reported in sheep and mice. PMID- 1705942 TI - Selective digestion of nuclear envelopes from Xenopus oocyte germinal vesicles: possible structural role for the nuclear lamina. AB - We have used enzymic digestion as a structural probe to investigate components of the nuclear envelope of germinal vesicles from Xenopus oocytes. Previous studies have shown that these envelopes are composed of a double membrane in which nuclear pore complexes are embedded. The nuclear pore complexes are linked to a fibrous lamina that underlies the nucleoplasmic face of the envelope. The pores are also linked by pore-connecting fibrils that attach near their cytoplasmic face. Xenopus oocyte nuclear envelopes were remarkably resistant to extraction with salt solutions and, even after treatment with 1 M NaCl or 3 M MgCl2, pores, lamina and pore-connecting fibrils remained intact. However, mild proteolysis with trypsin selectively removed the lamina fibres from Triton-extracted nuclear envelopes to leave only the pore complexes and connecting fibrils. This observation confirmed that the pore-connecting fibrils were different from the lamina fibres and were probably constructed from different proteins. Trypsin digestion followed by Triton treatment resulted in the complete disintegration of the nuclear envelope, providing direct evidence for a structural role for the lamina in maintaining envelope integrity. Digestion with ribonuclease did not produce any marked change in the structure of Triton-extracted nuclear envelopes, indicating that probably neither the pore-connecting fibrils nor the cytoplasmic granules on the pore complexes contained a substantial proportion of RNA that was vital for their structural integrity. PMID- 1705943 TI - Protection of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific T cell clones. AB - The protective effect of a mouse hepatitis virus type-4 (MHV-4)-specific CD8+ cytotoxic T cell clone and a CD4+ helper T cell clone was examined by the adoptive transfer into brains of mice lethally infected with MHV-4. Mice survived acute encephalitis if more than 5 x 10(5) cells of either type of the virus specific T cell clones had been transferred into H-2-matched recipients by 1 day post-infection. Although the adoptive transfer of both types of the T cell clones suppressed viral growth and viral antigen-positive cells in the brains, a significant inhibition of virus replication by the cytotoxic T cell clone was detected prior to that induced by the helper T cell clone. Histologically, cell destruction was prominent in the brains of mice which received the cytotoxic T cell clone. These results demonstrate that both the CD8+ cytotoxic T cell and the CD4+ helper T cell can protect mice from a lethal MHV-4 infection in the central nervous system. PMID- 1705944 TI - Tachykinin-mediated modulation of the primary antibody response in rats: evidence for mediation by an NK-2 receptor. AB - Evidence for the involvement of primary afferent nerves and their associated neuropeptides in in vivo immunologic responses has been based on experiments in rats in which destruction of primary afferent nerves by the sensory neurotoxin capsaicin results in a diminished ability of the animal to mount a primary antibody response to sheep red blood cell (SRBC) antigen. This effect was shown to be reversed by substance P infusion immediately following antigenic stimulation. In this report we show that neurokinin A (NKA) is 12 times more potent than substance P in its capacity to reverse the effects of neonatal capsaicin pretreatment on the antibody response. Neurokinin A has a pD2 of 6.65 compared to 5.98 for substance P. In addition, NKA was more potent than substance P in reversing the effects of surgical lesions 2 days prior to antigenic stimulation. The effects of the D- and L-Pro9 analogues of [Glp6, Pro9]-SP6-11 on the plaque-forming cell response in capsaicin-treated rats provide further support for the hypothesis that the tachykinin receptor modulating the primary antibody response is an NK-2 receptor. These results demonstrate, for the first time, a role for NKA in in vivo immunomodulation. PMID- 1705945 TI - Immunohistochemical localization of vitronectin, its receptor and beta-3 integrin in Alzheimer brain tissue. AB - The vitronectin receptor (VNR) is an integrin which consists of an alpha-subunit which can associate with multiple beta-subunits. A polyclonal antibody to this integrin weakly stained resting microglia in white matter of control brain and strongly stained reactive microglia in both gray and white matter of Alzheimer brain. This antibody, as well as a monoclonal antibody to beta 3, stained some platelets in capillaries of both control and Alzheimer tissue. When the antiserum was immunoabsorbed with a preparation enriched in the alpha-chain of the vitronectin receptor, it failed to stain microglial cells, but continued to stain platelets. When it was immunoabsorbed with a peripheral blood platelet preparation, all immunostaining was abolished. These results indicate that the vitronectin receptor of microglia is associated with a beta-chain different from beta 3, but that beta 3 is expressed by some platelets in brain capillaries. An antibody to vitronectin itself stained senile plaques and neurofibrillary tangles in Alzheimer entorhinal cortex, but only residual plasma in control tissue. Senile plaques positive for vitronectin had microglial cores strongly positive for the vitronectin receptor. The high levels of vitronectin receptor on reactive microglia in areas containing extracellular vitronectin suggest the possibility that vitronectin is serving an opsonizing function for microglial phagocytosis. PMID- 1705946 TI - Cytotoxic effects of myelin basic protein-reactive T cell hybridoma cells on oligodendrocytes. AB - Cells of a rat/mouse T cell hybridoma reactive to the encephalitogenic peptide of myelin basic protein (MBP) was found to be cytotoxic to 51Cr-labelled rat oligodendrocytes (oligos) inducing 52 +/- 5% 51Cr release vs. 28 +/- 2% spontaneous 51Cr release from replicate oligos. The hybridoma cells were not toxic for rat astrocytes or concanavalin A-stimulated lymphoblasts. Hybridoma T cells reactive to an experimental allergic encephalomyelitis-irrelevant antigen (ovalbumin) were not cytotoxic to oligos. The cytotoxic reaction required cell cell contact but did not require the in vitro presence of antigen-presenting cells MBP. The target antigen on the oligos is not yet defined. These studies suggest that MBP-reactive T cells can be directly cytotoxic to oligos in the absence of other cell populations. PMID- 1705947 TI - Immunohistochemical characterization of palisaded, encapsulated neuroma. AB - Eleven cases of palisaded, encapsulated neuroma (PEN) were studied by routine light microscopic and immunohistochemical methods. PEN showed the following staining reactions: strong, diffuse for S-100 protein (11/11) and collagen Type IV (Col. IV) (11/11); moderately strong, focal for myelin basic protein (MBP) (9/11) and Leu-7 (9/11); weak, focal for MBP (2/11), Leu-7 (2/11), neuron specific enolase (NSE) (4/11), neural filaments (NF) (5/11), epithelial membrane antigen (EMA) (6/11), and negative for glial fibrillary acidic protein (GFAP). Bielschowsky silver stain was positive in 11/11, but only four cases contained numerous axons. Col. IV and EMA stains showed a complete capsule in 1/11, incomplete capsules in 5/11 and no discernible capsules in the remainder. We concluded that (1) the immunologic profile of PEN is not specific; (2) conventional silver stain remains a suitable method to demonstrate axon-like structures; (3) the ratio of axons to Schwann cells is variable and generally less than 1:1, as previously assumed; (4) MBP and Leu-7 coexpression in Schwann cells suggests myelinization of some of the nerve fibers; (5) the pattern of EMA reaction supports perineurial cell involvement in PEN; and (6) despite the current definition of PEN, they are usually not completely encapsulated as evidenced by Col. IV and EMA strains. PMID- 1705948 TI - Host-dependent variation of bluetongue virus neutralization. AB - To determine if neutralizing epitopes of Bluetongue virus (BTV) 17 are host dependent, e.g., that monoclonal antibodies (mAb) to Bluetongue virus 17 (BTV 17) differ in their ability to neutralize BTV infectivity in insect versus mammalian cells, a panel of neutralizing mAb was developed. The relative neutralizing titer of eight mAb for BTV 17 infectivity in mammalian versus insect target cells was determined. Four mAb differed in their relative neutralization titer when assayed on mammalian target cells as compared to insect target cells. These findings suggest that different epitopes involved in neutralization might be important in virus infectivity and neutralization in mammalian versus insect target cells. To determine which viral protein(s) these mAb bind, the specificity of the mAb was determined by radioimmunoprecipitations. Five BTV 17 neutralizing mAb bound to the major outer coat protein P2, a 98-kDa protein, whereas the BTV protein(s) bound by the other three neutralizing mAb could not be determined. The potential role of the two BTV outer coat proteins in infection of mammalian and insect host cells is discussed. PMID- 1705949 TI - Pediatric mastocytosis. AB - The onset of mastocytosis occurs between birth and 2 years of age in approximately 55% of all cases; an additional 10% develop the disease before the age of 15 years. Mastocytosis in these age groups differs in many respects from mastocytosis that has its onset in adulthood. The typical presentation of pediatric-onset mastocytosis consists of cutaneous manifestations: either a solitary mastocytoma, urticaria pigmentosa, or, less commonly, diffuse cutaneous mastocytosis. Particularly in infants, bullous eruptions may occur. Mastocytosis in infants and children may involve internal organs, including the bone marrow and the gastrointestinal tract, although such manifestations appear to be less common in children than in adults. Plasma histamine levels may be elevated in pediatric-onset mastocytosis. Treatment usually involves the use of H1 and H2 antihistamines to control itching and to control the hypersecretion of gastric acid that may occur. The prognosis for children with mast cell disease is variable; approximately half of the children with urticaria pigmentosa may experience resolution of lesions and symptoms by adolescence. PMID- 1705950 TI - Epitope mapping of the laminin molecule in murine skin basement membrane zone: demonstration of spatial differences in ultrastructural localization. AB - Results of studies performed to date with polyclonal antilaminin antibodies have been conflicting as to the ultrastructural localization of this glycoprotein in skin basement membrane zone (BMZ). Whereas initial reports suggested its presence solely within the lamina lucida (LL), others have suggested that laminin is instead an exclusive component of the lamina densa (LD). In an attempt to more critically address this issue, we have examined both intact and partially separated (via 1 M NaCl) murine skin BMZ by indirect immunoelectron microscopy via a two-step immunoperoxidase technique on unfixed cryopreserved tissue, utilizing nine well-characterized monoclonal antibodies with binding specificity for laminin. Localization of the sites of the epitopes recognized by these antibodies on isolated laminin molecules was previously determined by rotary shadowing and by biochemical analyses on enzymatic fragments of laminin. Whereas at least faint immunoreactants were detected in both regions with eight of nine antibodies, predominant staining was noted within the LL with three of eight and within (and even sparsely below) the LD in three of eight. One antibody bound solely to the LL; another bound equally within both regions. Although some overlap was noted, it appears that the epitope on the distal portion of the long arm of the laminin molecule resides primarily within the skin LD, whereas epitopes on more central portions of the short arms are present within the LL or within both LL and LD. The findings of stratification of laminin epitopes within skin BMZ supports a similar recent observation in mouse kidney and suggests that portions of the laminin molecule span both LD and LL, and that there may be a non random spatial orientation for the laminin molecule within murine skin BMZ. PMID- 1705951 TI - Characterization of human sebaceous cells in vitro. AB - Human sebaceous cells, isolated from adult human skin, were cultured on either bovine type I collagen or mitomycin-C-treated 3T3 fibroblasts. Sebaceous cells, termed "sebocytes", were determined to be epithelial in nature by positive staining with monoclonal antikeratin antibodies BG2 and BG12. However, sebocyte colonies were also negative for keratins found in differentiated cells of keratinocyte colonies, as defined by monoclonal antikeratin antibodies CC2 and CC6. Sebocytes did not produce cornified envelopes in vitro and could only be induced to produce small quantities (less than 5%) of envelopes with a calcium ionophore. Sebocyte growth characteristics in a variety of serum, dexamethasone, and hydrocortisone combinations were significantly different from those of human facial keratinocytes. Sebocytes also displayed a growth curve and plating efficiency that were different from those of keratinocytes. Large lipid droplets within growing sebocytes could be visualized with oil red o staining. Additionally, squalene and wax/cholesterol esters were made by sebocytes in vitro in greater amounts than by facial keratinocytes, as determined by thin-layer chromatography of organic extracts of 3H2O-labeled sebocytes. Sebocytes synthesized greater quantities of lipid, on a per-cell and protein basis, than did keratinocytes. PMID- 1705952 TI - Cross-antigenicity between the scabies mite, Sarcoptes scabiei, and the house dust mite, Dermatophagoides pteronyssinus. AB - This study demonstrated that antigens of the parasitic mite Sarcoptes scabiei (SS) cross-react with antigens of the house dust mite Dermatophagoides pteronyssinus (DP). Crossed immunoelectrophoresis (CIE) reaction of SS extract with rabbit anti-DP serum resulted in multiple immunoprecipitates. Reciprocal CIE reactions gave similar results. Immunoprecipitates from both reactions bound IgE in the sera of dust-mite-sensitive patients who had no history of scabies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved protein/peptide bands of both SS and DP also bound serum IgE from dust-mite-allergic patients following immunoblotting. Non-allergic control sera gave no IgE binding to either SS or DP antigens. These results indicate that patients with atopy to dust mites exhibit circulating antibodies built to DP but that recognize determinants on SS antigens. It is highly probable that scabietic patients build antibodies to SS antigens that also recognize DP antigens. These results raise questions concerning the reported isotypic antibody responses to SS because the sensitivity of scabietic patients to house dust mites has not been previously evaluated. This cross-reactivity may play an important role in the susceptibility to scabies and its clinical manifestations. PMID- 1705953 TI - Increased number of dendritic cells in draining lymph nodes accompanies the generation of contact photosensitivity. AB - Draining lymph node cells isolated from mice 48 h after topical exposure to tetrachlorosalicylanilide (TCSA) + UVA radiation (TCSA + UVA) demonstrated a two fivefold increase in the number of dendritic cells (DC) compared with control mice treated with vehicle + UVA. The increase in number of DC was both time and dose dependent, with the peak DC accumulation occurring at 48 h post application and at a TCSA dose of 1.0%. Photospecificity was evident in that mice irradiated prior to treatment with TCSA (UVA/TCSA) demonstrated no significant increase in DC accumulation. The accumulation of DC was followed by a significant increase in total lymph node cellularity. An in situ 3H-thymidine incorporation assay showed a significant increase in proliferative activity of cells isolated from the draining lymph nodes of mice treated with TCSA + UVA as compared to naive, vehicle, or UVA/TCSA-treated mice. Dendritic cells isolated from mice treated 24 h earlier with TCSA + UVA, but not those from naive mice or mice treated with UVA/TCSA, were capable of TCSA-specific antigen presentation. Responder lymphocytes from untreated mice or mice photosensitized with musk ambrette showed a much lower response to DC isolated from TCSA + UVA-treated mice, demonstrating the specificity of the reaction. DC-depleted lymph node cells were unable to stimulate this blastogenesis response. These results suggest that application and photoactivation of TCSA induces cellular and functional changes in the lymph node DC indicative of their involvement in the induction phase of a contact photoallergic reaction. PMID- 1705954 TI - Resilient children: individual differences in developmental outcome of children born to drug abusers. AB - Since 1977, we have been following the neurobehavioral development of two groups of children: a group born to women on methadone maintenance and a drug-free comparison group. This study used the data on the children evaluated at 36 months of age to determine whether distinct patterns of developmental outcome can be identified, and which medical, familial, or environmental characteristics are associated with developmental differences. The children were clustered on four measures at 36 months: head circumference percentile, Merrill-Palmer Scale score, neurological evaluation, and referrals for developmental problems. Three distinct clusters emerged, with methadone children disproportionately frequent in Cluster 3, the group showing the poorest development. Comparisons of the clusters on a wide range of variables revealed consistent differences between Cluster 1 and Cluster 3 children in maternal responsiveness and incidence of neglect and family violence. These findings indicate that distinct developmental patterns do occur within this predominantly lower-class ghetto population; further, that children born to methadone-maintained women are more likely to show poor development. However, when the environment provides nurturance and stability, methadone children can show resilience and develop well. PMID- 1705955 TI - A method to measure simultaneously cyclic AMP and inositol phosphate accumulation in rat brain slices. AB - The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion-exchange chromatography and their concentrations were determined by means of a double labeling technique, the substrates adenine and inositol being labeled with 14C and 3H, respectively. The recoveries were 70-80% for inositol phosphates and 40 50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with alpha- and beta-adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of 3H-inositol phosphates and [14C]cyclic AMP in a concentration-dependent process. EC50 values were similar for the two, 8.5 +/- 3.9 microM for 3H-inositol phosphates and 20.2 +/- 6.3 microM for [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of 3H-inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two receptor-coupled effectors. PMID- 1705956 TI - Altered protein tyrosine phosphorylation in Alzheimer's disease. AB - The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease. PMID- 1705957 TI - Identification of the new isoforms of mouse myelin basic protein: the existence of exon 5a. AB - Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet. PMID- 1705958 TI - Expression of the myelin-associated glycoprotein in cultures of immortalized Schwann cells. AB - Although the myelin-associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S-16, showed a remarkably high MAG concentration of about 1 ng/microgram of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S-16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galactocerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S-MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with [3H]borohydride reduction after periodate oxidation, demonstrated that most of the MAG expressed by the S 16 cells is located on the cell surface. This line of immortalized Schwann cells expressing a remarkably high level of MAG should be useful for investigating the cell biology and function of this glycoprotein. PMID- 1705959 TI - Characterization of the carbohydrate chain on the substance P receptor in the rat brain cortex: effect of lectins on [3H]substance P binding. AB - The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of alpha-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system. PMID- 1705960 TI - Interferon protects normal human granulocyte/macrophage colony-forming cells from Ara-C cytotoxicity. AB - Interferons (IFN) have clinical efficacy in certain hematologic malignancies. Combining IFN with conventional cytotoxic agents has been proposed as a means of improving therapy for diseases such as chronic myelogenous leukemia (CML). In this study, we examined the effect of recombinant interferons alone and in combination with Ara-C on normal and leukemic human hematopoietic progenitor cells (CFU-GM) in vitro. Mononuclear cells from normal bone marrow, peripheral blood of patients with CML, or the acute nonlymphocytic leukemia cell line HL-60 were incubated with alpha-, beta-, or gamma-IFN (0-1,000 units/ml) followed by the addition of Ara-C. The survival of normal CFU-GM was significantly increased if cells were treated with IFN 1 h before 3 h of Ara-C exposure. Similar IFN pretreatment of CML and HL-60 progenitors failed to protect leukemic CFU-GM from Ara-C-induced toxicity. This selective protection of normal CFU-GM may have clinical application. PMID- 1705961 TI - Effects of granulocyte colony-stimulating factor on hematopoietic injury induced by anticancer drugs in mice. AB - The in vivo effects of the subcutaneous administration of the granulocyte colony stimulating factor (G-CSF) on chemotherapy-induced hematopoietic injury were evaluated in BALB/c mice. Mice treated with chemotherapeutic drugs were injected once a day for up to 12 days with 2 micrograms of recombinant human G-CSF, and following this, bone marrow cellularity, spleen weight, and peripheral blood cell counts were measured 24 h after cessation of the G-CSF treatment. Treatment of normal mice had minimal effect on the elevation of the white blood cell (WBC) count or on the hosts' resistance to K. pneumoniae infection. Spleen weight was significantly higher in normal mice treated with G-CSF, and the platelet counts were slightly lower. In mice treated with cyclophosphamide (150 mg/kg), G-CSF treatment caused an elevation of WBC and an enhancement of antibacterial resistance. Variable amounts of an accelerated recovery of neutrophils by G-CSF treatment was also observed in nimustine hydrochloride (50 mg/kg)-, mitomycin c (MMC) (8 mg/kg)-, or vindesine sulfate (VDS) (4 mg/kg)-treated mice. A significant decrease in PLT counts was observed in MMC- or VDS-treated hosts given G-CSF. These results indicate that administration of G-CSF may facilitate hematopoietic recovery in chemotherapy-treated cancer patients and that it may help them to increase their resistance to life-threatening infection. Conversely, treatment with G-CSF and chemotherapeutic drugs may cause a more severe thrombocytopenia than is observed with only chemotherapy treatment. PMID- 1705962 TI - A chromatic horizontal cell in the Xenopus retina: intracellular staining and synaptic pharmacology. AB - 1. We identified a chromatic-type horizontal cell (C-cell) in the Xenopus retina by intracellular dye injection with Lucifer yellow or horseradish peroxidase (HRP). C-cells hyperpolarized in response to blue light and depolarized in response to red light. 2. In either photopic or mesopic states, moderate intensity blue and red stimuli evoked responses that were inverted with respect to each other but of similar waveform and latency. In the presence of a bright green adapting field, the maximal voltage (Vmax) of the hyperpolarizing and depolarizing response component approached 30 mV; the kinetics of both waveforms were fast, and the hyperpolarizing response was followed by a small depolarizing overshoot at light OFF. Thus the blue-sensitive photoreceptor is capable of initiating large visual signals under photopic conditions when transmission from green-sensitive rods is suppressed. Under mesopic conditions (no adapting field) the kinetics of both waveforms were slower. The Vmax of the hyperpolarizing response reached 30-40 mV, whereas the cone-mediated depolarization saturated at 15 mV. 3. Both response components of the C-cell showed large receptive fields with no center-surround antagonism. 4. The C-cell perikaryon was located in the distal inner nuclear layer. It emitted four to seven long, tapering processes that ran horizontally for 90-100 microns. Two kinds of terminal dendrites, short and long, extended from the tapering processes toward the layer of photoreceptor bases. 5. Glycine (5-10 mM) completely eliminated the depolarizing response of the C-cell, whereas the hyperpolarizing component was unaffected. In contrast, gamma-aminobutyric acid (GABA; 5-10 mM) had no obvious effect on either component. 6. The C-cell light response was modified in two stages by cis-2,3 piperidine dicarboxylic acid (cis-PDA; 0.5-5 mM): first the depolarizing response disappeared; then the membrane potential hyperpolarized concomitant with a large reduction or elimination of the hyperpolarizing light response. In contrast, DL-2 amino-4-phosphonobutyric acid (APB) had no obvious effect on either response component or the membrane potential of the cell. 7. Our pharmacological findings are consistent with the view that the hyperpolarizing response in the C-cell is mediated by direct synaptic input from a blue-sensitive photoreceptor. The depolarizing response mediated by the red-sensitive cone could be explained by a direct synapse from the red cone or an indirect pathway involving luminosity (L type) horizontal cells. PMID- 1705963 TI - Cation current activated by hyperpolarization in a subset of rat nucleus accumbens neurons. AB - 1. Intracellular recordings were made from neurons in slices of rat nucleus accumbens in vitro. Membrane currents were measured in the potential range -60 to -120 mV with the use of a single-electrode, voltage-clamp amplifier. 2. A minority of neurons (28/285) was identified that had resting membrane potentials almost 20 mV less negative than the majority of the cells. These cells, but not the majority, had an inward current that activated slowly when the cells were hyperpolarized from -60 to -120 mV. The time constant of activation was approximately 3 s at -70 mV and 100 ms at -120 mV. 3. This inward current was completely blocked by external cesium (2 mM) but unaffected by barium. The current was reduced in solutions containing low-sodium concentration and increased in solutions with high-potassium concentration; its reversal potential was estimated to be -36 mV. 4. It is concluded that two types of neuron can be distinguished in the rat nucleus accumbens on the basis of the presence or absence of a cation current activated by hyperpolarization. This current (IH, also called If and IQ) causes the neurons to have less-polarized resting potentials than the majority of nucleus accumbens neurons. PMID- 1705964 TI - Inward rectification in the inner segment of single retinal cone photoreceptors. AB - 1. Single cone photoreceptors were dissociated from the retina of a lizard with the aid of papain. The majority of the cells lost their outer segments but had well-preserved, large synaptic pedicles. Electrical properties of the cells were studied with tight-seal electrodes in the whole cell configuration. On the average, cone inner segments had a resting potential of -55 mV, and at this potential their input resistance was 2.6 G omega and their capacitance was 8 pF. 2. Under current clamp the cones exhibited a pronounced anomalous voltage rectification in response to hyperpolarizing currents. The voltage rectification was eliminated by external Cs+. 3. The Cs(+)-sensitive current underlying voltage rectification was isolated by blocking other currents present in the cone. Co2+ blocked a voltage-dependent Ca2+ current and a Ca2(+)-dependent Cl- current, and tetraethylammonium (TEA)+ blocked a delayed-rectifier K+ current. 4. The Cs(+) sensitive current was activated by hyperpolarization to potentials more negative than -50 mV, and its current-voltage (I-V) relationship exhibited inward rectification. 5. The inward-rectifying current was selective for K+, but not exclusively. Increasing external K+ concentration 10-fold shifted the reversal potential by 13 mV. If Na ions also permeate through the inward-rectifying channels, the ratio of permeabilities (PK+/PNa+) in normal solution is approximately 3.9. 6. The kinetics of the inward-rectifying current were described by the sum of two exponentials, the amplitudes and time constants of which were voltage dependent. 7. The voltage dependence of the inward-rectifying current was described by Boltzmann's function, with half-maximum activation at 79 mV and a steepness parameter of 7.5 mV. 8. The voltage dependence and kinetics of the inward-rectifying current suggest that it is inactive in a cone photoreceptor in the dark. However, it becomes activated in the course of large hyperpolarizations generated by bright-light illumination. This activity will modify the waveform of the photovoltage--the current will generate a depolarizing component that opposes the light-generated hyperpolarization. PMID- 1705965 TI - The origin of corticospinal projections from the premotor areas in the frontal lobe. AB - We determined the origin of corticospinal neurons in the frontal lobe. These neurons were labeled by retrograde transport of tracers after injections into either the dorsolateral funiculus at the second cervical segment or the gray matter of the spinal cord throughout the cervical enlargement. Using retrograde transport of tracer from the arm area of the primary motor cortex, we defined the arm representation in each premotor area in another set of animals. We found that corticospinal projections to cervical segments of the spinal cord originate from the primary motor cortex and from the 6 premotor areas in the frontal lobe. These are the same premotor areas that project directly to the arm area of the primary motor cortex. The premotor areas are located in parts of cytoarchitectonic area 6 on the lateral surface and medial wall of the hemisphere, as well as in subfields of areas 23 and 24 in the cingulate sulcus. The total number of corticospinal neurons in the arm representations of the premotor areas equals or exceeds the total number in the arm representation of the primary motor cortex. The premotor areas collectively comprise more than 60% of the cortical area in the frontal lobe that projects to the spinal cord. Like the primary motor cortex, each of the premotor areas contains local regions that have a high density of corticospinal neurons. These observations indicate that a substantial component of the corticospinal system originates from the premotor areas in the frontal lobe. Each of the premotor areas has direct access to the spinal cord, and as a consequence, each has the potential to influence the generation and control of movement independently of the primary motor cortex. These findings raise serious questions about the utility of viewing the primary motor cortex as the "upper motoneuron" or "final common pathway" for the central control of movement. PMID- 1705966 TI - Brain-specific polyA- transcripts are detected in polyA+ RNA: do complex polyA- brain RNAs really exist? AB - Transcripts encoded by 2 different rat genomic clones, rg13 and rg100, appear to be typical brain-specific polyA- RNAs, as defined by previous criteria (rare, polysomal, and postnatally expressed from single-copy genes). However, we have found by using a sensitive nuclease protection assay that low levels of these transcripts (10% and 3%, respectively) are detected in polyA+ RNA. To determine if rg transcripts that fractionate as polyA- could have resulted from nicking of polyA+ RNA, we assessed the integrity of 2 known polyA+ RNAs, those of tyrosine hydroxylase, a 2-kilobase (kb) mRNA, and sodium channel, a 9.5-kb RNA. Using RNA prepared by several different procedures, including LiCl-urea and guanidine thiocyanate followed by CsCl centrifugation, the shorter message fractionated as polyA+ after 2 cycles over oligodeoxythymidine (oligo-dT) cellulose, whereas the majority of the longer sodium channel RNA fractionated as polyA, as assayed by nuclease protection using probes from the 5' end of the 2 genes. However, on Northern blots, the same RNA preparations showed an intact 9.5-kb sodium channel band only in polyA+ RNA, suggesting that the polyA- RNAs were randomly cleaved, resulting in a smear of sizes that could not be detected as a discrete band. These data imply that long messages may be nicked during standard isolation procedures and that this would not be detected by Northern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1705967 TI - Channeling of developing rat corticospinal tract axons by myelin-associated neurite growth inhibitors. AB - CNS myelin contains 2 membrane proteins that are potent inhibitors of neurite growth (NI-35 and NI-250). Because myelin formation starts at different times in different regions and tracts of the CNS, this inhibitory property of myelin could serve boundary and guidance functions for late-growing fiber tracts. In the rat, the corticospinal tract (CST) grows into and down the spinal cord during the first 10 postnatal days, in close proximity to the sensory tracts fasciculus cuneatus and gracilis. Immunofluorescence for myelin constituents showed that, in the rostral half of the spinal cord, the myelinating tissue of these ascending tracts surrounds the growing, myelin-free CST in a channellike fashion. Elimination of oligodendrocytes by x-irradiation of the newborn rats, or application of antibody IN-1, which neutralizes the inhibitory substrate property of CNS myelin, resulted in significant anatomical aberration of CST fibers. In particular, the tract was larger in cross-section, and aberrant CST fibers and fascicles intermixed with the neighboring sensory ascending tracts. These results assign an important channeling and "guard-rail" function to the oligodendrocyte associated neurite growth inhibitors for the developing CST in the rat spinal cord. PMID- 1705968 TI - Modular organization of projection neurons in the matrix compartment of the primate striatum. AB - It is well known that the striatum has a chemical architecture dividing it into striosomes and matrix, and that these compartments have different input-output connections. However, striatal afferent-fiber systems also form vividly patchy terminal fields in the matrix, and studies in the past year have uncovered instances of nonstriosomal clustering of striatal output neurons. In the experiments reported here, we systematically investigated this output-neuron clustering in the primate, using the striatopallidal system as a model. Our goals were to determine whether the modular organization is a general characteristic of projection neurons in the striatal matrix, whether the modularity occurs independent of striosomal boundaries, and whether the output modules are systematically organized. We studied the distribution of striatopallidal projection neurons in 9 adult squirrel monkeys by centering deposits of the retrograde tracer HRP-WGA in either the external segment or the internal segment of the globus pallidus. Following injections of each type, many retrogradely labeled neurons appeared in the striatal matrix in clusters and bands having cross-sectional diameters of 0.2-0.8 mm. Comparisons with adjoining sections stained to demonstrate striosomes established that the local groups of striatal output neurons sometimes abutted striosomes but often did not. The retrogradely labeled clusters and bands appeared both in the caudate nucleus and in the putamen. Their arrangements were regular and often periodic. These findings suggest that the large matrix compartment of the primate striatum, which is the primary site of origin of striatal outputs to the pallidum and the reticular part of the substantia nigra, contains systematic mosaics of projection neurons. We propose that this output-neuron modularity of the striatal matrix in the primate could serve as the template for redistribution of the massive afferent-fiber systems of the striatum into specialized striatopallidal output channels. PMID- 1705969 TI - Development of serotonin-induced ion currents in identified embryonic Retzius cells from the medicinal leech (Hirudo medicinalis). AB - Retzius cells and the cutaneous baroreceptive P-cells of adult medicinal leeches (Hirudo medicinalis) respond to 5-HT by Cl- as well as by monovalent cation conductances (Drapeau et al., 1989). However, chemical synaptic connections between Retzius cells (Liu and Nicholls, 1989) as well as Retzius cells and P cells in culture (Drapeau and Sanchez-Armass, 1988) are mediated by postsynaptic Cl- currents in response to 5-HT release from the presynaptic cell. It was the aim of the present study to find out whether the response, which has been shown to be involved in synaptic transmission or whether the cation current, which has so far only been observed upon extrasynaptic 5-HT application, dominates during embryogenesis. Currents induced by transmitter application with a fast perfusion system were measured with a single-electrode voltage clamp and patch pipettes in whole-cell configuration. Control experiments, performed on Retzius cells from adult leeches after 1 d in culture, showed Cl- currents in response to 5-HT application in all cells investigated. Ninety percent of the cells tested showed additional 5-HT-elicited Na+, K+, or both currents. However, the conductance of cation currents amounted only to 10% of the Cl- currents. Embryonic Retzius cells were identified in dissociated cultures by their violet-blue fluorescence acquired by preincubation of intact ganglia or embryos in culture medium containing the autofluorescent 5-HT analog 5,7-dihydroxytryptamine. The first responses to 5-HT were measured at embryonic day 10 (E10), after voltage activated Ca2+ and Na+ currents had already developed. 5-HT application induced exclusively Cl- currents until E16. This suggests that the receptor-channel complex, which also mediates synaptic responses, is expressed first in measurable quantities. Hence, there is currently no indication that extrasynaptic receptors dominate in immature cells in this preparation. PMID- 1705970 TI - Development of striatal compartmentalization following pre- or postnatal dopamine depletion. AB - Nigrostriatal dopamine (DA) projections terminate in distinct patches during the late prenatal and early postnatal period in the rat. During the first postnatal week, patches of DA fibers overlap with clusters of striatal neurons that share several identified characteristics. The early segregation of striatal cell types into either these patches or the surrounding matrix becomes a permanent organizational feature of the striatum. In order to determine whether the heterogeneous distribution of DA influences the formation of cellular patches, the developmental organization of chemically identifiable cell types was examined in normal rats and in rats DA depleted as infants (0 or 3 d) or in utero (embryonic days 17-18). During the first postnatal week, corresponding patches of DA afferents and substance P (SP)-immunoreactive neurons existed in the striatum of normal animals, and AChE-positive zones overlapped these patches in the lateral striatum. Injection of 6-hydroxydopamine into the lateral ventricles of fetal or infant rats produced a dramatic loss of striatal DA terminals. Neither the patchy distribution of SP-immunoreactive neurons nor the distinctive pattern of AChE staining present during the first 2 postnatal weeks was disrupted. During the third postnatal week, cells immunoreactive for leu-enkephalin or calbindin D28k were confined to the matrix compartment, and this compartmentalization was also not noticeably changed by pre- or postnatal DA depletion. In adult animals, overlapping patches of leu-enkephalin- and SP-immunoreactive fibers were observed, regardless of whether any DA terminals remained. Thus, the basic organization of the striatal patch and matrix compartments develops normally in the absence of DA innervation through much of the formative period. Although these observations do not completely dismiss the possibility that the first DA afferents to appear in the striatal primordia influence contracted striatal cells to develop the patch phenotype, they suggest that the patchy distribution of DA afferents may be secondary to the early clustering of striatal neurons forming the patch compartment. PMID- 1705971 TI - Both alpha- and beta-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors. AB - A family of genes has been identified that encodes subunits of nicotinic acetylcholine receptors (nAChRs) and is expressed in the nervous system. Functional neuronal nAChRs can be expressed in Xenopus oocytes by injection of RNA encoding 1 of 2 different beta-subunits (beta 2, beta 4) in pairwise combination with RNA encoding 1 of 3 different alpha-subunits (alpha 2, alpha 3, alpha 4). We examined the sensitivity of these 6 different alpha- beta-subunit combinations to the nicotinic agonists ACh, nicotine, cytisine, and 1,1-dimethyl 4-phenylpiperazinium (DMPP). Each subunit combination displayed a distinct pattern of sensitivity to these 4 agonists. The alpha 2 beta 2 combination was 5 fold more sensitive to nicotine than to acetylcholine, while the alpha 3 beta 2 combination was 17-fold less sensitive to nicotine than to ACh, and the alpha 3 beta 4 combination was equally sensitive to both nicotine and ACh. nAChRs composed of alpha 2, alpha 3, or alpha 4 in combination with beta 2 were 14-100 fold less sensitive to cytisine than to ACh. In contrast, nAChRs composed of alpha 2, alpha 3, or alpha 4 in combination with beta 4 were 3-17-fold more sensitive to cytisine than to ACh. The alpha 2 beta 2, alpha 3 beta 2, and alpha 3 beta 4 combinations were each equally sensitive to DMPP and ACh, while the alpha 2 beta 4, alpha 4 beta 2, and alpha 4 beta 4 combinations were 4-24-fold less sensitive to DMPP than to ACh. We also demonstrated that these differences are neither a consequence of variation in the relative amounts of RNA injected nor an artifact of oocyte expression. The oocyte system can accurately express ligand-gated ion channels because mouse muscle nAChRs expressed in oocytes display pharmacological properties similar to those reported for these receptors expressed on BC3H-1 cells. We conclude that both the alpha- and the beta-subunits contribute to the pharmacological characteristics of neuronal nAChRs. PMID- 1705972 TI - Distributions of spinothalamic, spinohypothalamic, and spinotelencephalic fibers revealed by anterograde transport of PHA-L in rats. AB - Fibers projecting from several levels of the spinal cord to the diencephalon and telencephalon were labeled anterogradely with Phaseolus vulgaris leucoagglutinin injected iontophoretically. Labeled fibers in the thalamus confirmed projections previously observed. In addition, many labeled fibers were seen in the hypothalamus and in telencephalic areas not generally recognized previously as receiving such projections. In the hypothalamus, these areas included the lateral hypothalamus (including the medial forebrain bundle), the posterior hypothalamic area, the dorsal hypothalamic area, the dorsomedial nucleus, the paraventricular nucleus, the periventricular area, the suprachiasmatic nucleus, and the lateral and medial preoptic areas. In the telencephalon, areas with labeled fibers included the ventral pallidum, the globus pallidus, the substantia innominata, the basal nucleus of Meynert, the amygdala (central nucleus), the horizontal and vertical limbs of the diagonal band of Broca, the medial and lateral septal nuclei, the bed nucleus of the stria terminalis, the nucleus accumbens, infralimbic cortex, and medial orbital cortex. These results suggest that somatosensory, possibly including visceral sensory, information is carried directly from the spinal cord to areas in the brain involved in autonomic regulation, motivation, emotion, attention, arousal, learning, memory, and sensory-motor integration. Many of these areas are associated with the limbic system. PMID- 1705973 TI - The standardized assessment of argyrophilic nucleolar organizer regions in meningeal tumors. AB - Tissue markers of cellular proliferation have been recently utilized as prognostic indicators in tumors of the central nervous system. Nucleolar organizer regions represent transcriptionally active sites of ribosomal deoxyribonucleic acid (DNA) and can be identified by a simple argyrophilic technique. The authors describe a standardized approach to the assessment of these argyrophilic nucleolar organizer regions in meningeal tumors. Twenty-five meningiomas were classified histologically into benign, atypical, or malignant groups. In addition, two hemangiopericytomas and one leptomeningeal melanoma were examined. Appropriate sections were silver stained and argyrophilic nucleolar organizer regions were counted in 200 nuclei. The mean argyrophilic nucleolar organizer region count was statistically different (p less than 0.001) between benign tumors (245 +/- 156, 1.23/cell), atypical tumors (497 +/- 135, 2.49/cell), and malignant tumors (921 +/- 59, 4.61/cell). The count for recurrent meningiomas (544 +/- 76) was also statistically different (p less than 0.02) from non recurrent tumors (329 +/- 183). The standardized assessment of argyrophilic nucleolar organizer regions can be easily performed by any surgical pathology laboratory without specialized equipment and, in meningeal tumors, may be useful as an independent indicator of biological behavior. PMID- 1705974 TI - Flow cytometry detection and evaluation of bladder tumors. AB - The diagnosis and classification of bladder cancer are based primarily on histologic and cytologic light microscopy. The significant cytologic alternations are abnormal (increased) DNA content and structural changes in chromatin. Measurements of DNA content carried out by flow cytometry on suspensions of tumor cells and bladder irrigation specimens correlate well with clinical and biopsy findings. Badalament et al (Badalament RA, Kimmel M, Gay H, et al. Cancer 1987;59:2078-2085) reported that a single bladder wash flow cytometry correctly detected 83% of bladder cancers. The technique is most sensitive in detecting early, in situ, and superficially invasive carcinoma. In 100 urologic patients with non-neoplastic disease of the bladder, Klein et al (Klein FA, Herr HW, Sogani PC, et al. J Urol. 1982;127:946-948) reported only two false-positive examinations. Flow cytometry appears to be at least as valuable as conventional urinary cytology, without the need of an experienced cytopathologist. However, as specimens must be collected by bladder irrigation via catheter or cytoscope, the technique is most suitable not for population screening, but in monitoring high risk populations: industrial workers or others exposed to carcinogens, persons with a history of urothelial tumors, and adult patients referred for urologic examinations because of hematuria, unexplained, recurrent cystitis, or other urologic symptoms. DNA flow cytometry also has been valuable in monitoring the conservative treatment of bladder cancer and of predictive value in the intravesical bacille Calmette-Guerin treatment of superficial carcinomas of the bladder. Dual parameter measurements of DNA content and antigen expression are now under evaluation, as are measurements of chromatin structure alteration, metabolism, and proliferative rate. These promise to discriminate subsets of bladder cancer that may be predictive of clinical behavior. PMID- 1705975 TI - Basaloid carcinoma of salivary glands, a variety of undifferentiated adenocarcinoma. Immunohistochemical study of intermediate filament proteins in 24 cases. AB - Among adenoid cystic carcinomas of salivary glands (ACCs), the solid basaloid type has a poor prognosis similar to that of undifferentiated adenocarcinomas. We studied 24 cases in immunohistochemistry using antibodies reactive with keratins of various molecular weights, vimentin, S-100 protein, and its A and B subunits. Our findings were correlated with the histological pattern and with the variable degree of differentiation of these carcinomas. In comparison with other types of ACC, intermediate filament proteins in this group were weakly expressed. The co expression of cytokeratin and vimentin was noted in some cases. Additional features noted were the presence of cribriform cavities associated with solid lobules and areas of necrosis giving a comedocarcinomatous pattern. In these two variants, cells characterized by the dual expression of cytokeratin and S-100 protein were seen. In the highly malignant anaplastic variety, only a few cells were weakly positive with antisera to cytokeratin and vimentin. This group shows similarities to undifferentiated adenocarcinomas of salivary glands. Such similarities could be explained by the common origin of these tumours from intercalated ducts. PMID- 1705976 TI - Cellular H- and M-type lactate dehydrogenase (LDH) isoenzymes and tumour diagnosis--an immunohistochemical assessment. AB - Cellular H- and M-type lactate dehydrogenase (LDH) isoenzymes were determined immunohistochemically in a wide range of normal, inflammatory, benign neoplastic, and malignant tissues to assess the possible value of the isoenzymes in tumour diagnosis. Both types of LDH were detected almost ubiquitously in normal tissues. Increased staining intensity of LDH, especially M-type LDH, was demonstrated in most types of malignant tumours studied. Similar increased staining intensity of the isoenzymes was also noted to parallel the proliferative activity of the cells in non-neoplastic diseases and benign neoplasms. The results suggest that changes of cellular H- and M-type LDH isoenzymes are not specific for any type of malignant tumours. The increase in cellular H- and M-type LDH isoenzymes appears to be related to proliferation of cells in general. PMID- 1705977 TI - The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis. AB - RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven. PMID- 1705978 TI - [Neuropeptide-mediated transmission of nociceptive information and its regulation. Novel mechanisms of analgesics]. AB - Substance P and somatostatin may be transmitters of nociceptive information, which are involved in the transmission of pressure and heat nociceptive information, respectively, in the spinal dorsal horn. Calcitonin gene-related peptide, which is present in the primary sensory neurons having substance P or somatostatin, may function as a pain-promoting substance and be involved in the production of inflammation-induced hyperalgesia. The descending noradrenergic system plays a role in inhibiting nociceptive transmission in the spinal dorsal horn, and inhibits the release of substance P evoked by noxious mechanical stimulation. Persistent noxious stimuli increase the release of Met-enkephalin from the nucleus reticularis gigantocellularis, which promotes the activity of the descending noradrenergic system. Morphine activates the descending noradrenergic system, acting on the nucleus reticularis gigantocellularis. Morphine also activates the descending serotonergic system, which inhibits the release of somatostatin evoked by thermal noxious stimulation. PMID- 1705979 TI - [Studies on anti-allergic agent. I. 1,2,3-trisubstituted-2-propen-1-one derivatives, 3,4-disubstituted-4-oxo-2-butenoic acids and the related compounds]. AB - A new series of 1,2,3-trisubstituted-2-propen-1-one derivatives (9a-v, 10a-j, 13a c, 14a-j) and 3,4-disubstituted-4-oxo-2-butenoic acids (6a-i, 7a-n) with azole compounds were synthesized. Inhibitory activities against rat passive cutaneous anaphylaxis (PCA) reaction and histamine release from rat mast cells were tested. The ester derivatives (7a-n, 14a-j) exhibited a more potent inhibitory activity against histamine release compared with alkylamine (9a-v), beta-hydroxyethoxy (10a-j) and carboxylic acid (6a-i, 13a-c) derivatives, but somewhat weaker in their anti-PCA activity. Structure-activity relationships were discussed. PMID- 1705980 TI - Translocation of diphtheria toxin to the cytosol and formation of cation selective channels. AB - A number of protein toxins act by translocating an enzymatically active polypeptide to the cytosol. The translocation process is best understood in the case of diphtheria toxin which binds to cell surface receptors, is then taken up by endocytosis and is subsequently translocated to the cytosol, where it inactivates elongation factor 2. The translocation of the enzymatically active part of the toxin can be induced at the level of the plasma membrane upon exposure to low pH of cells with surface-bound toxin. Receptor molecules appear to be involved in the translocation process, which also requires an inward directed H(+)-gradient and permeant anions. Cation-selective channels are formed in the membrane upon toxin entry. The B-fragment alone is much more efficient in inducing channels than the whole toxin. The current model of the translocation process is discussed. PMID- 1705981 TI - Cytokeratin pattern in normal and HPV infected oral mucosa in women with genital HPV infections. AB - The distribution of cytokeratins Nos. 19 (CK 19), 14, 16 and 17 (CK2-27), and 8 and 18 (CK 60-61) in 96 oral mucosal biopsies taken from women with genital HPV infections were studied by immunohistochemistry, using polyclonal antibody CK 19, as well as monoclonal antibodies CK 2-27 and CK 60-61. White staining of the buccal mucosa after acetic acid application, which recently was shown to be affected mostly by smoking and age, could not be explained by differences in cytokeratin pattern. In HPV DNA-positive biopsies, the staining with CK 19 antibody in the basal cell layer was more intense than in HPV DNA-negative biopsies. The staining with CK 2-27 antibody was seen in 76% and 91% of the basal and superficial layers, respectively, even though these low molecular weight cytokeratins should be found mainly from the basal and parabasal cells. CK 60-61 staining was almost similar to that seen recently in normal genital mucosa. When trying to distinguish oral HPV infections from normal mucosa, CK 2-27 and CK 60 61 stainings were of no diagnostic value. The more efficient expression of CK 19 in HPV DNA-positive samples suggests that viral infection might accelerate the production of low molecular weight cytoskeletal protein. This could be interpreted as evidence that HPV might disturb the keratinocyte differentiation in the basal cells. As a result of the present study, CK 19 staining in oral mucosa needs to be further studied in regard to viral infections, because it may help to better understand the interaction between a virus and a host cell. PMID- 1705982 TI - Cell kinetics and response to primary intra-arterial chemotherapy in patients with advanced oral cavity tumors. AB - The relationship was analyzed between cell kinetics, defined as 3H-thymidine labeling index (3H-TdR LI), and clinical responses for 35 previously untreated patients with locally advanced oral cavity squamous cell carcinomas. Patients were treated with three cycles of vincristine and bleomycin (VB) or of VB plus methotrexate (VBM), given by intra-arterial infusion followed by surgery. The objective clinical response rate was higher after VBM treatment than after VB, whereas overall clinical response rates were similar for patients with slowly or rapidly proliferating tumors. The probability of relapse-free survival at 4 yr and overall survival was significantly higher for patients with high 3H-TdR LI tumors given VBM than for those given VB. For patients with slowly proliferating tumors, there were no differences in relapse-free survival and overall survival between the two treatments. These data suggest that patients with rapidly proliferating tumors benefit from primary intensive chemotherapy treatment including methotrexate and that cell kinetics can be used to formulate rational clinical protocols for oral cavity cancers. PMID- 1705983 TI - Light microscopic, cytochemical and ultrastructural studies of a rat odontogenic epithelial cell line. AB - An odontogenic epithelial cell line, ROE-2B, was established by propagating disaggregated immature, unmineralized maxillary third molar tooth germs from 11 day old Sprague-Dawley rats on a feeder layer of Mitomycin-C treated NIH 3T3 embryonic mouse fibroblasts. The cell line has been maintained for more than 6 months and through 7 passages. Light microscopic examination of cells revealed colonies with epithelial morphology. Electron microscopic examination confirmed the epithelial nature by the demonstration of tonofilaments, desmosomes and basal lamina. Cells were also shown to have secretory vacuoles, an abundance of granular endoplasmic reticulum, free ribosomes, Golgi complex and mitochondria. Surface activity in the form of pseudopodia-like projections and micropinocytosis were noted. Epithelial cells forming keratin were demonstrated by a positive histochemical reaction with Rhodanile Blue. Immunohistochemical studies showed a positive reaction for CAM 5.2 indicating that the ROE-2B cells express the cytokeratins of simple or glandular epithelia. The ROE-2B cell line will be useful for studies on in vitro biological behaviour of odontogenic epithelial cells, and may allow the establishment of in vitro models of odontogenic tumours. PMID- 1705984 TI - Papilliferous keratoameloblastoma. AB - A case of papilliferous keratoameloblastoma is reported which is only the second ever documented. The patient was a 76-yr-old black woman with a large expansile multilocular radiolucency of the body, angle and ramus of the mandible. Histologically the lesion consisted of sheets of cystic follicles filled with necrotic debris and sometimes parakeratin. The vast majority of the follicles were lined by a papilliferous epithelium consisting of large rounded cells with centrally placed nuclei. True papillary projections with cores of connective tissue were also present. The remainder of the follicles were lined by a thin parakeratinising stratified squamous epithelium. Histological features characteristic of ameloblastoma were absent. Final classification of these lesions will have to await the reporting of further cases. PMID- 1705985 TI - Influence of mRNA determinants on translation initiation in Escherichia coli. AB - We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli. Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation. We measured these differences using extension inhibition analysis, a technique we have called "toeprinting". The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely. Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation. PMID- 1705986 TI - Amplification of CCl4 toxicity by chlordecone: destruction of rat hepatic microsomal cytochrome P-450 subpopulation. AB - Previous work has established marked amplification of CCl4 hepatotoxicity by prior exposure to chlordecone (CD). Since CCl4 is toxic by virtue of its bioactivation by the hepatomicrosomal cytochrome P-450 (cyt P-450) system, which is in turn destroyed, our first interest was to determine if cyt P-450 isozymes were selectively destroyed in this interaction. CoCl2 also decreased hepatic P 450 contents, so our other interest was to observe whether CoCl2 selectively decreased or spared CCl4 metabolizing P-450 enzymes. Solubilized hepatic microsomes from variously treated rats were used. The treatment protocol was dietary CD (10 ppm, for 15 d), and CCl4 (100 microliters/kg, ip). The treatments were CD alone, CCl4 alone, CD + CCl4 and with or without CoCl2 (60 mg/kg/d, sc for 2 d) treatment on d 13 and 14 of the dietary protocol. The control group received normal diet and corn oil vehicle. The key mixed-function oxidase (MFO) parameters measured were microsomal protein, cyt P-450 content, and aminopyrine demethylase (APD). Decrease of P-450 levels ranged from 2.2-fold (CD + CCl4) to 1.3-fold (CD + CoCl2). APD activity decreased by 48 and 26.6% in CD + CCl4 and CD + CoCl2 treatments, respectively. Using an anion-exchange high-performance liquid chromatography (HPLC) column, solubilized microsomal hemoproteins were resolved into five peaks. The P-450 content associated with each peak was determined. In CD rats there was slight increase in peak heights, whereas peak heights in CCl4 and control treatments were similar. CoCl2 decreased all peaks, the decrease of peak I being maximal. In CD + CCl4 treatment, absence of peaks II and III was noted. Microsomal proteins stained for heme showed decreased staining intensity of hemo-protein bands, particularly band 4 (MW 52,000), which was absent in CD + CCl4 interaction. These findings suggest that (1) CoCl2 does not selectively decrease or spare any P-450 isozymes and (2) CD + CCl4 interaction does destroy specific P-450 isozymes. PMID- 1705987 TI - Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19. AB - In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies. PMID- 1705988 TI - Characterization and genetic analysis of alternatively spliced transcripts of hepatitis B virus in infected human liver tissues and transfected HepG2 cells. AB - The transcriptional map of hepatitis B virus (HBV) has been expanded recently by the discovery of a singly spliced transcript in hepatoma cell lines transfected with cloned viral DNA and a doubly spliced one in naturally infected human liver tissues. By the use of reverse transcription and a subsequent polymerase chain reaction, the two spliced HBV RNAs were shown to be present in both types of cells. As further evidence, an HBV mutant was constructed and found to exclusively express the singly spliced RNA. This mutant was also used to quantitate the two spliced species in transfected HepG2 cells; they were found to be equally abundant, and each represented about 30% of the pregenomic RNA. The HBV mutant could still produce replication-competent HBV virions when transfected into HepG2 cells, indicating that the doubly spliced transcript, just like the singly spliced one, was not essential for HBV replication. However, the two abundant spliced HBV transcripts were detected in most naturally infected human liver tissues, suggesting that they may have biologic functions in vivo. PMID- 1705989 TI - Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus. AB - The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2' 5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2' 5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA. PMID- 1705990 TI - Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones. AB - Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes. PMID- 1705991 TI - Down regulation of poliovirus receptor RNA in HeLa cells resistant to poliovirus infection. AB - A line of HeLa cells (SOFIA) was previously isolated that is resistant to poliovirus infection and does not express functional virus binding sites at the cell surface. The expression of the poliovirus receptor (PVR) gene in SOFIA cells was examined to determine the molecular basis for the failure of these cells to express PVRs. Southern blot analysis of genomic DNA revealed that the PVR gene in SOFIA cells did not contain gross alterations. However, PVR transcripts were not detected in Northern (RNA) blot analysis of SOFIA cell RNA. In vitro nuclear run on analysis showed that transcription of PVR-specific RNA was reduced in SOFIA cells. Treatment of SOFIA cells with 5-azacytidine restored susceptibility to poliovirus infection, which correlated with the appearance of PVRs at the cell surface, as detected with anti-PVR monoclonal antibody D171. PVR RNA was detected in clones derived from 5-azacytidine-treated SOFIA cells. SOFIA cells were converted to poliovirus sensitivity at a rate of 5 to 7%, suggesting that down regulation of PVR expression involved few cellular targets. Resistance of SOFIA cells to poliovirus infection therefore appears to result from down regulation of PVR RNA, leading to lack of PVR expression at the cell surface. Methylation may play a role in regulating the expression of the PVR gene, which is not essential for survival of HeLa cells. PMID- 1705992 TI - Direct evidence of a role for amino acid 101 of VP-1 in central nervous system disease in Theiler's murine encephalomyelitis virus infection. AB - The DA virus, a member of the TO subgroup of Theiler's virus, invokes a chronic demyelinating disease in its natural host, the mouse, RNA transcripts from a cDNA clone, pDAFL3, are infectious, and the resulting virus, DAFL3, produces in mice a disease indistinguishable from that caused by the DA virus. Using oligonucleotide directed site-specific mutagenesis, a single nucleotide, cytosine at position 3305 (viral genome), was changed in this infectious cDNA to a thymine. The mutated nucleotide is located in an area coding for a neutralizing epitope on loop II of VP-1. Virus OSM101, produced from the mutagenized plasmid pDA101, had the same growth characteristics and plaque phenotype in vitro as the virus DAFL3 produced from clone pDAFL3. However, in vivo in the mouse, virus OSM101 was markedly less neurovirulent than DAFL3. Central nervous system tissues from mice infected 4 to 6 weeks previously with the OSM101 virus contained less infectious virus and fewer infected cells than central nervous system tissues from animals infected with the control virus, DAFL3. Thus, we demonstrated that the single nucleotide change resulting in an amino acid substitution at position 101 (threonine to isoleucine) of VP-1 determines one aspect of Theiler's virus persistence and disease in mice. PMID- 1705993 TI - Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction. AB - The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecular-weight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription. PMID- 1705994 TI - Strain-specific neutralizing determinant in the transmembrane protein of simian immunodeficiency virus. AB - Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn Asp) determined the strain specificity of the monoclonal antibody. This strain specific neutralizing determinant is located within a variable region of SIVmac and human immunodeficiency virus type 2 (HIV-2) which includes conserved, clustered sites for N-linked glycosylation. The determinant corresponds exactly to a variable, weak neutralizing epitope in HIV-1 TMP which also includes conserved, clustered sites for N-linked glycosylation. Thus, the location of at least one neutralizing epitope appears to be common to both SIVmac and HIV-1. Our results suggest a role for this determinant in the viral entry process. Genetic variation was observed in this neutralizing determinant following infection of a rhesus monkey with molecularly cloned SIVmac239; variant forms of the strain specific, neutralizing determinant accumulated during persistent infection in vivo. Selective pressure from the host immune response in vivo may result in sequence variation in this neutralizing determinant. PMID- 1705995 TI - Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination. AB - Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype. PMID- 1705996 TI - Stable T-p53 complexes are not required for replication of simian virus 40 in culture or for enhanced phosphorylation of T antigen and p53. AB - We generated a number of simian virus 40 (SV40) mutants with single amino acid substitutions in T antigen between residues 388 and 411. All but one mutant (398LV) replicated like wild-type SV40 and gave rise to normal-size plaques. Three different mutations at residue 402 (Asp to Glu, Asn, or His) totally prevented the formation of stable complexes with the cellular protein p53 in monkey cells but had no effect on virus replication. Only one other mutation in this region, involving residue 401 (Met to Thr), slightly inhibited the formation of T-monkey p53 complexes. The three mutant T antigens with substitutions at residue 402 also formed no stable complexes with human p53 but generated low levels of complexes with mouse p53. These results indicate that residue 402 is critical for binding to monkey and human p53 proteins and is important for binding to mouse p53. We suggest that it is one of several points of contact. In cells infected with any one of the three residue 402 mutant viruses. T antigen and p53 became increasingly phosphorylated, as they were in cells infected with wild-type virus. Our data therefore show that stable T-p53 complexes are not required for replication of SV40 in culture or for enhanced phosphorylation of either protein. PMID- 1705997 TI - Protein binding to the interferon response enhancer correlates with interferon induction of 2'-5'-oligoadenylate synthetase in normal and interferon-resistant Friend cells. AB - The induction of transcription of the 2'-5'-oligoadenylate (2-5A) synthetase gene by type I (alpha/beta) and type II (gamma) interferons (IFNs) has been studied in wild-type (w.t.) and IFN-resistant Friend leukemia cells (FLC). Following IFN treatment, new complexes are formed in vitro between the IFN-responsive sequence (IRS) of the 2-5A synthetase gene and cellular proteins. Within minutes after IFN alpha/beta addition to w.t. FLC, an IRS-protein complex, designated F1, is detected, as already observed in several human cell lines. In response to IFN gamma, a novel complex, designated Fg, is observed in w.t. FLC. The Fg complex appears within 3 h, while an F1-like complex is faintly visible 10 to 24 h later. In the IFN-alpha/beta-resistant FLC, IFN-gamma induces only the Fg complex and fails to induce F1. Fg formation is correlated with the IFN-gamma-induced transcription of the 2-5A synthetase gene and the appearance of the corresponding enzymatic activity in both w.t. and IFN-alpha/beta-resistant FLC. These findings suggest that F1 and Fg represent two distinct effector complexes by which type I and type II IFNs, respectively, induce 2-5A synthetase. PMID- 1705998 TI - Human transformed trophoblast-derived cells lacking CD4 receptor exhibit restricted permissiveness for human immunodeficiency virus type 1. AB - We investigated the nature of interaction of the malignantly transformed cell lines of trophoblast origin BeWo, JAR, and JEG-3 with three different human immunodeficiency virus type 1 (HIV-1) isolates (RF, 3B, and NDK). After inoculation with cell-free virus, the persistence of infection was determined for 1 month by monitoring the presence of viral DNA in the cells by the polymerase chain reaction (PCR). Furthermore, the infectious virus in the culture supernatant was assayed with CEM-SS cells, and attempts to rescue the virus by cocultivation with CEM-SS cells were made. Appraised on the basis of the relative amount of viral DNA and the frequency of positive cocultivation. JEG-3 was the most permissive and BeWo was the least permissive cell line. However, when the cells were transfected with two biologically active molecular clones of HIV-1, the BRU and NDK isolates, all three cell lines turned out to support the production of mature virus progeny to the same extent. The abundance of viral DNA sequences in the infected cells varied with the isolate, showing an overall decline from RF to NDK. The amount of viral DNA in the cells and its expression decreased during the period of observation; this decrease was mirrored in an erosion of the virus recovery rate at cocultivation from 71% recovery on day 8 to failure of isolation on day 32. None of the cell lines expressed detectable amounts of cell surface CD4 molecules when assayed by flow microfluorometry and direct radioimmunoassay. Northern (RNA) blot hybridization analysis of both the total RNA and the mRNA did not reveal any CD4-specific message: nonetheless, by using the PCR, sequences specifically related to the CD4 gene were uncovered. The data demonstrate that the trophoblast-derived cell lines are susceptible to infection with HIV and that they support transient viral replication in the initial phases of infection. However, the latent form of infection may persist over a period of several weeks. PMID- 1705999 TI - The threshold of pain and neurotransmitter's change on pain in Parkinson's disease. AB - Among Parkinson's disease (PD) patients complaining of pain, 10 with pain not associated with a motor fluctuation or L-dopa therapy were evaluated. The controls were 14 PD without pain and eight with thalamic pain syndrome. The threshold of pain and neurotransmitters in CSF were measured in the three groups. In PD with pain, the maximum tolerance level and tourniquet pain ratio decreased significantly. In PD with pain, the score on the self-depression scale increased significantly and 5-hydroxy-indole acetic acid (5-HIAA) among the neurotransmitters decreased significantly. These results suggest that decreases in the threshold of pain and changes of serotonin in CSF are involved in the development of specific pain in PD who do not respond to L-dopa. PMID- 1706000 TI - Secretion of insulin-like growth factor I and its binding proteins by collecting duct cells. AB - Insulin-like growth factor I (IGF-I) has been found in the kidney, particularly in the collecting duct in the rat. Since cultured rabbit collecting duct cells constitute a convenient system for in vitro studies, we have examined whether these cells secrete IGF-I. Culture medium conditioned by collecting duct cells was concentrated by reverse phase chromatography and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent molecular weights of 7.5 and greater than 25 kilodaltons (kD) were identified by IGF-I RIA. A smaller amount of 10 kD species was also observed. Further characterization of 7.5 kD IGF-I immunoreactive species by reverse phase HPLC showed that it eluted in a single peak. To determine whether the higher molecular weight species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I (thr59) for two hours at 30 degrees C and applied to a Sephadex G100 column equilibrated in a non-dissociating buffer. The major peak of radioactivity was confined to a high molecular weight region; there was no radioactivity in the fractions corresponding to 7.5 kD. Western ligand analysis of unreduced conditioned medium identified two IGF-I binding species of 25 and 30 kD, similar in size to species observed in normal rabbit serum. 125I IGF-I binding as assessed in a charcoal adsorption assay could be displaced by IGF-I and IGF-II but not by insulin. Further characterization of the 10 kD peak of IGF-I immunoreactivity indicated that it did not possess IGF-I binding activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706001 TI - Role of bone in regulation of systemic acid-base balance. PMID- 1706002 TI - Lymphocyte adhesion molecules in T cell-mediated lysis of human kidney cells. AB - The complementary adhesion molecules LFA-1 (CD11a, 18)/ICAM-1 (CD54) and LFA-2 (CD2)/LFA-3 (CD58) have been shown to be important in T cell interaction with lymphoid target cells. The role of these ligand pairs in cytotoxicity against somatic cells is less well established. While LFA-3 is expressed by all cells in the kidney, ICAM-1 expression is low in normal kidneys but is increased in allograft rejection. An in vitro cytotoxicity assay was used to examine the relative importance of the two adhesion ligands in immune damage against kidney cells in rejection. HLA-A2 specific cytotoxic T lymphocyte (CTL) recognition of cultured human kidney cells (HKC), of predominantly renal tubular cell origin, was studied. Immunofluorescence studies showed that both induced and uninduced HKC target cells expressed ICAM-1, MHC class I and LFA-3, but only MHC class I and class II antigens and ICAM-1 were significantly upregulated by cytokine induction. Effector cells expressed LFA-1 and LFA-2 but little or no ICAM-1 and LFA-3. Cytokine induction of ICAM-1 expression on HKC target cells increased their susceptibility to lysis. Monoclonal antibody against ICAM-1 or LFA-1 produced the greatest inhibition of HKC lysis, and their effects were not additive. Antibody against LFA-2 (CD2) or LFA-3 also produced significant inhibition, but to a lesser degree, and no additive effect was found.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706003 TI - Immunolocalization of endothelial and leukocyte adhesion molecules in human rheumatoid and osteoarthritic synovial tissues. AB - Leukocyte adhesion to endothelium plays an important role in the development and perpetuation of chronic inflammatory diseases such as rheumatoid arthritis (RA). In order to help define the role of adhesion molecules in arthritic disorders, we have studied the expression of CD11c, endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 in synovial tissues from patients with RA and osteoarthritis (OA) by immunohistochemistry. CD11c is expressed predominantly on macrophages deep within RA and OA synovial tissues, as well as on some synovial tissue lining cells. ELAM 1 has endothelial reactivity, being present mainly on venules and capillaries and staining more blood vessels in RA than OA. VCAM-1 is present predominantly on synovial tissue macrophages and, to a lesser degree, on synovial tissue endothelial cells of venules, capillaries, and arterioles in both RA and OA. Like ELAM-1, VCAM-1 appears to be present more often on endothelial cells in RA than in OA tissues. VCAM-1 is present on macrophages isolated from RA synovium as well as macrophages in situ. Intercellular adhesion molecule-1 is more broadly distributed than the other adhesion molecules, being found on endothelium, macrophages, some fibroblasts, and some lymphocytes in both RA and OA tissues. This study shows that ELAM-1, a molecule that was previously thought to be important mainly in acute inflammatory reactions, is also found in RA, a chronic inflammatory disease, as well as in OA. Thus, ELAM-1 as well as VCAM-1 and intercellular adhesion molecule-1 may be involved in mediating the leukocyte traffic into RA and OA synovium. PMID- 1706004 TI - Comparative epitope analysis of neuronal cytoskeletal proteins in Alzheimer's disease senile plaque neurites and neuropil threads. AB - Dystrophic neurites in senile plaque (SP) coronas and neuropil threads (NTs) massively accumulate in Alzheimer's disease (AD) cortex. Hence, they may contribute to the cognitive deficits in AD. However, the composition of these neuritic lesions is poorly understood, and it is not known if they derive from axons, dendrites or both. To gain insights into the composition and derivation of SP neurites and NTs in AD, we undertook an in situ epitope mapping study wherein we probed these lesions using 278 monoclonal antibodies specific for spatially distinct epitopes in each neurofilament (NF) subunit, or in microtubule associated proteins, i.e., tau and microtubule-associated protein 2. The middle molecular weight NF subunit (NF-M) and tau were extensively represented in SP neurites, i.e., epitopes extending from the NH2 to COOH domains of NF-M and tau were present. In contrast, microtubule-associated protein 2 was not present in any SPs, and only epitopes in the core domain of the low (NF-L), and in the tail piece of the high molecular weight subunit were detected in SP neurites. SP cores never stained with these antibodies. NTs were similar to SP neurites in that they contained the same complement of tau epitopes, and were devoid of any microtubule associated protein 2 immunoreactivity, but they were also distinct because they rarely contained any NF determinants. These antigenic dissimilarities between SP neurites and NTs suggest that NTs and SP neurites are distinctly separate lesions that reflect widespread disruption of the neuronal cytoskeleton in AD. PMID- 1706005 TI - Effects of dietary fish oil on the induction of experimental membranous nephropathy in the rat. AB - We examined the effect of a fish oil-enriched diet on the development of experimental membranous nephropathy in the rat induced by administration of cationic bovine gamma globulin (CBGG). Rats were placed on a fish oil-enriched diet and control rats received a diet containing an equivalent amount of beef tallow. After 6 weeks on either diet, rats were pre-immunized and injected with CBGG. Proteinuria was significantly reduced in the fish oil-fed group as compared to the control group (160 +/- 40 mg/24 hours, n = 15, versus 280 +/- 36 mg/24 hours, n = 17, p less than 0.02). Glomerular filtration rate was also significantly higher in the fish oil-fed rats than in controls (0.91 +/- 0.07 ml/minute, n = 11, versus 0.60 +/- 0.05 ml/minute, n = 10, p less than 0.005). Glomerular production of prostaglandin E2 and thromboxane B2 the stable product of thromboxane A2, were inhibited by 68% and 70%, respectively, by the fish oil enriched diet (n = 8, p less than 0.01 versus control). Glomerular leukotriene B4 was also inhibited by 50% in the fish oil-treated rats (n = 6, p less than 0.01), but inhibition of leukotriene B4 by the specific inhibitor L-663,536 in control rats did not ameliorate proteinuria. There was no difference in the amount of distribution of glomerular immune deposits as demonstrated by immunofluorescence and electron microscopy between the experimental and control groups. Moreover, comparable amounts of glomerular IgG deposits were present in the two groups. The specific immune response, assessed by measuring anti-BGG antibody levels, was not different between the two dietary groups, while more than 85% suppression of the splenic T- and B-cell mitogenic response to concanavalin-A and lipopolysaccharide was noted in rats fed the fish oil-enriched diet. We conclude that a fish oil enriched diet reduces proteinuria and preserves the glomerular filtration rate in rats with CBGG-induced membranous nephropathy. Its mechanism of action remains to be established. PMID- 1706006 TI - Prevention of cardiovascular risk and arterial hypertension. Introduction. PMID- 1706007 TI - Enhanced cell proliferation in essential hypertension. AB - In order to define the molecular mechanism involved in enhancement of spontaneously hypertensive rat (SHR) cell proliferation, we have compared the actions of fetal calf serum (FCS) and angiotensin II on both SHR and Wistar-Kyoto (WKY) rat aortic smooth muscle cells. Both compounds are more mitogenic in SHR cells than in controls. However, phospholipase C (PLC) hyperresponsiveness can be seen only under angiotensin stimulation, as are the expressions of c-jun, c-fos, and c-myc. Oncogene overexpression therefore appears to be more strongly related to PLC hyperreactivity than to enhanced proliferation of SHR aortic smooth muscle cells. PMID- 1706008 TI - Calcium channel blockers and atherosclerosis. AB - There is evidence that calcium antagonists (calcium channel blockers) may suppress atheroma formation in animals fed high-fat diets. Studies on the antiatherosclerotic effects of calcium blockers have suggested a variety of possible mechanisms: (a) lowering of arterial pressure, (b) decrease in atherogenic plasma lipoproteins, (c) suppression of accumulation of intracellular lipids, (d) suppression of atherogenic platelet dysfunction, (e) prevention of dyslipidemic endothelial injury, (f) inhibition of chemotaxis and cell migration, (g) inhibition of cell proliferation, (h) inhibition of deposition of matrix proteins, (i) suppression of tissue mineralization, and (j) retardation of cell necrosis. Although it is tempting to ascribe the antiatherosclerotic effects of calcium blockers to a blockade of calcium channels, other possible common mechanisms of action involving low-affinity drug-binding sites must be considered. Recently, two randomized, prospective clinical trials designed to determine the effects of calcium channel blockers on the progression of coronary artery disease have been completed. Results of the trials suggest that calcium channel blockers suppressed the progression of coronary atherosclerosis. The utility of calcium channel blockers for the treatment of atherosclerosis will require further evaluation. PMID- 1706009 TI - Calcium antagonists in myocardial infarction. AB - Calcium antagonists are effective cardioprotective agents in experimental models of myocardial infarction. However, clinical trials in acute myocardial infarction and in postinfarction secondary prevention led to conflicting results related to the small size of the majority of the trials and possible differences among individual agents with distinct ancillary properties. Furthermore, one has to consider separately the trials in patients with Q-wave infarction and in others with non-Q-wave infarction. Q-wave patients do not seem to benefit from therapy with calcium antagonists. However, the efficacy of early administration of verapamil or diltiazem cannot be ruled out as the available data are not conclusive, mainly because of the small size of the trials and the delay in administering the drugs. We have shown encouraging results with diltiazem, which, compared to placebo, decreased the infarct size measured with serial thallium SPECT defect scores, and increased left ventricular ejection fraction in acute Q wave infarctions. Non-Q-wave infarction is another area where evidence of positive beneficial effects of diltiazem is strong, as shown by a recent trial conducted by Gibson et al. Diltiazem reduced the early reinfarction rate and postinfarction angina and 1-year cardiac mortality and nonfatal reinfarction rate. Consistent with these findings are results from subgroup analysis of the multicenter diltiazem postinfarction trial. Prophylactic use of diltiazem may be useful and should be considered in patients with non-Q-wave infarction along with aspirin, as no other treatment is yet available for this condition, at least for the time being and until the thrombolysis TIMI phase III trial is terminated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706010 TI - Sustained-release diltiazem and prevention of cardiovascular risk in hypertensive patients. AB - The prevention of coronary disease in hypertensive patients will see progress in the years to come. It is clearly too much, however, to expect this progress to come exclusively from the application of new therapies, as the incidence of coronary disease in hypertensive patients depends on several factors, which are, essentially, the persistence and severity of hypertension and the major cardiovascular risk factors associated with arterial hypertension, including hypercholesterolemia, diabetes, and nicotine abuse. Which new solutions to this problem can a novel therapy for arterial hypertension using calcium antagonists in general and diltiazem in particular thus provide? The majority of patients consulting their doctor for arterial hypertension also present with other risk factors associated with increased blood pressure, mostly hypercholesterolemia and diabetes. The antihypertensive efficacy of diltiazem can no longer be doubted; moreover, diltiazem monotherapy with single daily doses is of great advantage for compliance in hypertensive patients. As diltiazem has been in use for more than 10 years and its dosage has been gradually diminished, the risk of a new long term iatrogenic pathological process is becoming less and less conceivable. Sustained-release diltiazem is effective as monotherapy at single daily doses of 300 mg as evidenced by an effect/dose study in 105 patients: DBP = -17 mm Hg with 300 mg (72% of the patients responded to this dosage).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706011 TI - Development and pharmacokinetics of a new sustained-release formulation of diltiazem. AB - Among the antihypertensive agents, calcium antagonists and in particular diltiazem play a leading role. To improve the conditions of diltiazem administration in the treatment of hypertensive patients a galenic formulation allowing administration of a single daily dose has been developed. The studies discussed in the following are concerned with the pharmacotechnical and pharmacokinetic development of sustained-release microgranules (sustained-release diltiazem). Bioavailability studies were performed after single-dose administration and after repeated-dose administration which were compared to the conventional formulation of diltiazem, Tildiem. After single-dose administration the following results were achieved: a relative bioavailability of 1.06 +/- 0.35; a prolongation of tmax of 7.16 +/- 2.66 vs. 2.46 +/- 0.80 h; and a longer final half-life of 11.8 +/- 5.3 vs. 5.6 +/- 1.1. After repeated administration of sustained-release diltiazem at a single daily dose of 240 mg compared to repeated administration of the conventional formulation of diltiazem at doses of 120 mg every 12 h over a period of 6 days, the following results were obtained: a relative bioavailability of 0.90 +/- 0.17; a lowering effect on Cmax of 191 +/- 65 vs. 230 +/- 95 ng/ml; statistically equivalent minimum concentrations of 62 +/ 21 ng/ml vs. 74 +/- 33 ng/ml. In an additional study, the effect of concurrent food intake on the bioavailability of diltiazem was confirmed. An increase in bioavailability of 28% in combination with an increase in interindividual variability was observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706012 TI - Effects of sustained-release diltiazem on blood pressure and serum lipids: a multicenter, randomized, placebo-controlled study. AB - A randomized, double-blind dose-response study on the antihypertensive action of sustained-release diltiazem was performed in four parallel groups. The aim of the trial was to evaluate the antihypertensive efficacy of sustained-release diltiazem and its dose-dependent clinical and biological tolerance. The four homogeneous groups consisted of 25 patients each who had presented with mild to moderate hypertension (diastolic blood pressure of 95-115 mm Hg) in the supine position. The study protocol comprised three successive periods: a placebo period lasting 14 days to verify the persistence of arterial hypertension under placebo; a first therapeutic period during which each of the 25 patients of the four groups received a single daily dose every morning at 9 a.m. of a capsule containing 0.240, 300, or 360 mg of sustained-release diltiazem over a period of 28 days; and a second therapeutic period during which the patients of the four groups received 240, 300, 360, and 300 mg, respectively. All other antihypertensive treatment had been suspended at least 15 days before the initial period under placebo. The results were evaluated with respect to clinical parameters such as systolic blood pressure, diastolic blood pressure, and heart rate, which were measured at 9 a.m. 24 h after the last administration of the drug on days 14, 28, and 56. The plasma serum levels of diltiazem were measured on days 28 and 56, the biological parameters, including measurements of hepatic and renal function as well as lipid and glucose levels, on days 14 and 56, and electrocardiography was performed on days 14, 28, and 56.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706013 TI - Pathophysiological role of the vascular smooth muscle cell. AB - The vascular smooth muscle cell of the arterial media plays a predominant role in functional and structural alterations of the arterial wall in pathophysiological processes such as arterial hypertension, atheroma, or normal aging. The observed alterations are related to the three activities of the vascular smooth muscle cell, namely contractility, secretion of proteins from the extracellular matrix, and proliferation and migration. In arterial hypertension, vascular smooth muscle cells are functionally more contracted and structurally hypertrophic, and more collagen is secreted than under normal conditions. Similar structural changes are observed in the normal aging process. With respect to vascular smooth muscle cells, atheroma is characterized by their subintimal migration and proliferation, and by excessive excretion of collagen associated with other phenotypic modifications that are expressed in their regression to a myofibroblastic state. Regardless of the pathophysiological context, these phenotypic modifications of the vascular smooth muscle cell are always linked to an activation of the phosphoinositol pathways and to calcium accumulation. The activation of the phosphoinositol pathways seems to be a common feature of the different types of arterial hypertension. This activation can be associated with an increase in vasoactive peptides such as angiotensin II, vasopressin, or endothelin as in the secondary types of hypertension or directly related to an increase in vasoconstriction; or, as an exception, it can be spontaneously active in vivo and in vitro, as in the model of cultured vascular smooth muscle cells of the spontaneously hypertensive rat (SHR).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706014 TI - Comparative study on monotherapy with sustained-release diltiazem 300 mg and enalapril 20 mg in mild to moderate arterial hypertension. AB - The antihypertensive efficacy of sustained-release diltiazem 300 mg at a single daily dose was evaluated and compared to that of enalapril 20 mg at a single daily dose, and to a combination therapy of enalapril 20 mg with sustained release diltiazem 300 mg in patients with mild to moderate arterial hypertension (supine diastolic blood pressure between 95 and 115 mm Hg). After a washout period and a placebo period, each lasting 2 weeks, 96 patients were randomized in this double-blind study to three parallel groups that were treated for 28 days. In the groups treated with diltiazem, enalapril, or the combination of both, the drop in arterial blood pressure was 20, 11, and 19 mm Hg for supine diastolic blood pressure (supine DBP): 18, 12, and 19 mm Hg for standing diastolic blood pressure (standing DBP); 22, 20, and 23 mm Hg for supine systolic blood pressure (supine SBP); and 21, 19, and 24 mm Hg for standing systolic blood pressure (standing SBP), respectively. The reduction in DBP was significantly more pronounced in the group treated with sustained-release diltiazem 300 mg than in the group treated with enalapril 20 mg. Cardiac tolerance was good for patients treated with sustained-release diltiazem 300 mg alone or the combination therapy: no orthostatic hypotension or lengthening of the PR interval in the electrocardiogram was observed. Although side effects of sustained-release diltiazem 300 mg occurred more frequently, they were not severe and disappeared immediately when treatment was suspended. PMID- 1706015 TI - Efficacy and tolerance of sustained-release diltiazem 300 mg and a diuretic in the elderly. AB - A randomized, double-blind, parallel study has been performed to compare the antihypertensive action and clinical and biological tolerance of monotherapy with two different substances: sustained-release diltiazem 300 mg and a diuretic combining 25 mg of hydrochlorothiazide and 50 mg of triamterene (HCT-T). Antihypertensive treatment was suspended at least 15 days before initiation of a placebo period that lasted 14 days and allowed confirmation of persisting hypertension. Eighty-five patients who were at least 65 years old were included in the study: 42 in the group treated with sustained-release diltiazem 300 mg and 43 in the group treated with a diuretic. All of them presented with mild to moderate arterial hypertension, characterized by supine diastolic blood pressures (supine DBP) of 95-115 mm Hg. Duration of treatment was 3 months. After 1 and 3 months, efficacy and tolerance of the antihypertensive treatment were evaluated. On day 30, a significant decrease (p less than 0.0001) in the main parameter, i.e., supine DBP, as well as other parameters such as standing diastolic blood pressure (standing DBP), supine systolic blood pressure (supine SBP), and standing systolic blood pressure (standing SBP) was observed. Blood pressure was reduced by 18, 16, 28, and 26 mm Hg, respectively, in the sustained-release diltiazem 300 mg group and by 13, 12, 21, and 18 mm Hg, respectively, in the diuretic group. Moreover, on day 90, blood pressure values were maintained in the diuretic-treated group, whereas an additional significant reduction in supine DBP (p less than 0.002) was noted in the sustained-release diltiazem 300 mg-treated group (-4.8 mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706016 TI - Lifestyle for preventing cardiovascular disease. AB - The World Health Organization (WHO) Expert Committee recommends dietary changes together with the smoking-control programs for prevention of cardiovascular diseases in the population. Promoting habitual physical activity is an important part of the population strategy. The lifestyle changes proposed in the population strategy require professional education, community leader education, public education, mass media education, community organization, and environmental changes. Effective population prevention of cardiovascular diseases needs national commitment, policy, planning, and development. PMID- 1706017 TI - New vistas on nonpharmacological approaches to hypertension. Proceedings of the satellite symposium to the 13th scientific meeting of the International Society of Hypertension. June 23, 1990, St. John's, Newfoundland, Canada. PMID- 1706018 TI - Cardiovascular diseases and alimentary comparison study: preliminary analysis from Brazil. AB - The Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study (1) was designed to study the relationship of dietary factors to blood pressure (BP) and other major cardiovascular disease (CVD) in widely different populations of both industralized and developing countries. The primary aim of the research was to test specific hypotheses linking the intake of certain dietary constituents, e.g., sodium (Na), potassium (K), calcium (Ca), and protein, to BP (core study). The final aim was to contribute to the scientific information base required to guide the formulation of dietary goals for the primary prevention of CVD. The results of the preliminary analysis of data from Brazil in 57 inhabitants (22 men and 35 women) suggest a nonsignificant statistical correlation of Na intake estimated by urinary Na excretion and diastolic BP (DBP) and systolic BP (SBP) (p greater than 0.05), K intake estimated by urinary K excretion for DBP and SBP (p greater than 0.05), and taurine intake estimated by taurine urinary excretion for DBP and SBP (p greater than 0.05). A positive correlation was found between body mass index (BMI) and BP (p less than 0.01), for both DBP and SBP. PMID- 1706019 TI - Cardiovascular risk factors and health behavior: some preliminary findings from the Cardiovascular Diseases and Alimentary Comparison Study. AB - This paper presents some preliminary findings from one particular Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study data collection center. The population and the environment of the Western Isles, Scotland, from where the subjects were drawn, are described. The methodology was as according to the CARDIAC Study protocol. The results show that in this population there is a high mean serum total cholesterol level, a high prevalence of smokers, and a high mean body mass index. However, knowledge, attitudes, and reported behavior change regarding diet were encouraging. Much further data processing work remains to be done. PMID- 1706020 TI - Cardiovascular Diseases and Alimentary Comparison Study: preliminary analysis of data from Western Australia. AB - Data from 49 men and 48 women included in the Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study in Perth, Western Australia, were analyzed. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were positively correlated (p less than 0.01) with urine sodium, creatine, taurine, histidine, and 3-methyl histidine but not with calcium, magnesium, or potassium. SBP was related (p less than 0.01) with body mass index (BMI). Urine nitrogen, creatinine, and amino acids correlated (p less than 0.001) with each other and with urine sodium, potassium, calcium, and magnesium. Urine magnesium correlated (p less than 0.001) with urine calcium and potassium; urine calcium was not related significantly to urine sodium or potassium. In backwards multiple regression with data from urine collections, SBP was significantly related only to urine sodium (11.9% of variance explained). If alcohol was included as an independent variable, reducing the number of valid cases because of missing values, both alcohol and urine sodium were significant in regression (19.9% of variance explained). In men, DBP was significantly related to BMI and the ratio of 3-methylhistidine to creatine (23.7% of variance explained). For DBP in women, urine sodium was the only variable needed in regression (58.4% of variance explained). Interpretation must be cautious, because these analyses are based on relatively few cases and on single 24-h urine samples. The data are in keeping with suggestions that obesity, alcohol consumption, a meat diet, and sodium intake are important factors predisposing to elevation of blood pressure. PMID- 1706021 TI - Preliminary report of the Cardiovascular Diseases and Alimentary Comparison Study: the Israeli experience. AB - The relationship between blood pressure and dietary constituents including potassium, sodium, calcium, and protein was studied in 183 randomly selected men and women, 50-54 years of age. Twenty-five of the subjects were drug-controlled hypertensive patients. Subjects were investigated by automated BP measurements, 24-h urine collection, and blood sampling. Mean systolic BP (SBP) was 119 +/- 17 mm Hg, placing the sample between Shanghai and Sweden on the distribution chart of the Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study. Salt intake estimated by 24-h sodium excretion was 8.5 +/- 3.4 g/day, between Australia and Okinawa on the distribution chart. Potassium was 48.55 +/- 20 20 mEq/day, between Urumqi (China), and Beppu and Ohda (Japan). Calcium was 162.3 +/ 89.9 mg/day. Urea nitrogen, which might reflect protein intake, was 9.5 +/- 3.1 g/day. Cholesterol was 195.1 +/- 38.1 mg/dl, between Brazil and Hirosaki. The Israeli results, as well as the data on other countries participating in the CARDIAC Study, show wide variability in the profiles generated by the investigated parameters. Each parameter placed Israel with a different CARDIAC Study group. PMID- 1706022 TI - Cardiovascular risk factors in two Ecuadorian urban and rural populations. The Ecuadorian-Japan Cooperative CARDIAC Study Group. AB - We examined the specific hypotheses linking the intake of sodium, potassium, calcium, magnesium, and protein to blood pressure (BP) and the relationship between dietary factors and mortality from the major cardiovascular diseases (CVD) in the Ecuadorian populations. Two Ecuadorian populations, the urban and the rural, were selected from Quito and Vilcabamba, respectively. From Quito: 87 men and 83 women; from Vilcabamba: 71 men and 91 women aged 50-54 were randomly selected for BP measurement, 24-h urine collection, and blood sampling according to the Cardiovascular Disease and Alimentary Comparison (CARDIAC) Study protocol. Samples were analyzed at CARDIAC center in Izumo, Japan. Mean systolic blood pressure (SBP) was not much different in the two populations, but mean diastolic blood pressure (DBP) and body mass index (BMI) were significantly lower in Vilcabamba (p less than 0.001). Mortality from stroke was higher in Vilcabamba, whereas coronary death rate was higher in Quito. Both sodium intake and sodium/potassium ratio were higher in Vilcabamba (p less than 0.001). Protein intake and serum cholesterol were higher in Quito (p less than 0.001). Urinary taurine excretion was higher in Quito. There was no difference in W3/W6 fatty acids ratio between the two populations. Multiple regression analyses of intracommunity correlation indicated that both SBP and DBP were highly significantly related with BMI in Quito and that urinary excretions were inversely related to SBP. Serum cholesterol was positively related to coronary death rate. Mortality from stroke was inversely related to both serum cholesterol and protein and was positively related to salt consumption. PMID- 1706023 TI - Correlation between objective automatic and auscultatory mercury manometer blood pressure measurements. AB - The automatic blood pressure (BP) measurement device (A) developed for the World Health Organization (WHO) Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study was compared in Spain with the standard auscultatory-mercury manometer (M) in 440 randomly selected subjects, 50-54 years old (50% men and 50% women). BPs were recorded simultaneously in the same arm. Systolic BP (SBP), analyzed in 1,440 readings, was 127.5 +/- 18.9 mm Hg by A system and 127.6 +/- 18.6 mm Hg by M system (NS). Diastolic BP (DBP) was 71.3 +/- 11.7 mm Hg by A and 79.3 +/- 11 mm Hg by M (p less than 0.001). We confirmed good correlations in SBP and DBP measured by both methods (r = 0.95 and r = 0.88, respectively; both p less than 0.001). Nearly complete consistency was noted between SBP readings by both methods but not between DBP readings; the readings by M were significantly higher than those by A. Since A's consistency with SBP and DBP readings obtained by the intra-arterial method (the most commonly used method) previously had been proven to be satisfactory, DBP obtained by clinical practice is possibly overestimated in adults. PMID- 1706024 TI - Estimation of protein intake: comparison of dietary assessment and urinary excretion. AB - Estimates of dietary intake of nutrients have many problems, and confirmation of these estimates by chemical measurements of excretory products helps to establish their validity. The correlation between urinary amino acid excretion and dietary history of protein intake are reported herein. Diet diaries were kept by and 24-h urine collected from participants in the Trial of Antihypertensive Therapy and Management, a multicenter cooperative trial comparing the effects of drugs and diet on blood pressure control, side effects, and quality of life. The nutritional information was coded and the data stored by the nutritional analyses facility at Albert Einstein College of Medicine. The chemical determinations were done in the laboratories of the Department of Pathology, Izumo, Japan. The correlation between total protein and 3-methylhistidine was r = 0.42 (n = 96). The correlation was similar for animal protein (r = 0.4). These correlations are of the same order of magnitude as those between dietary sodium and urine sodium, and dietary potassium and urine potassium. We conclude that measurement of urinary protein excretion products may be a useful way to estimate dietary protein intake. PMID- 1706025 TI - Diet and hypertension in Tanzania. AB - A survey was conducted on an urban population in the city of Dar es Salaam and on a rural community in both Handeni and the pastoral Masai in Monduli to investigate the relationship between diet and hypertension in Tanzania. Blood pressure (BP) was measured using an automatic BP-measuring machine. Biological markers of dietary intake were measured in 24-h urine and in blood. Hypertension was noted to be a bigger problem in the capital city, where the rate of obesity and salt intake were higher whereas potassium, protein, and polyunsaturated fatty acid intake were lower. Therefore, attention to dietary habits may reduce the growing problem of hypertension in Tanzania. PMID- 1706026 TI - Relationship between urinary amino acids and diets in Spanish Cardiovascular Diseases and Alimentary Comparison Study. AB - A randomized, cross-sectional study was carried out to examine dietary pattern and urinary amino acids excretion in two Spanish free-living populations. A total of 288 adults (50-54 years old; 50% men and 50% women), randomly selected from the census, took part in the study; of these, 146 were living in an urban district of Madrid and 142 in a rural area. Dietary intakes by 7-day recall questionnaires, smoking habits, and alcohol intake were examined in each participant, and 24-h urine was collected in aliquot cups designed for the Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study. All amino acid excretions averaged higher in men than in women in both areas, and higher in the urban than in the rural population. Main dietary differences between urban and rural populations were higher consumption of fish in urban than in rural populations and higher consumption of bread in rural than in urban populations. Urinary taurine excretion as well as 3-methylhistidine or histidine were significantly associated with fish and meat consumptions, respectively, estimated by 7-day dietary recall questionnaires. PMID- 1706027 TI - Alcohol consumption as a risk factor for high blood pressure from the Cardiovascular Diseases and Alimentary Comparison Study. CARDIAC Cooperative Research Group). AB - The relationship between alcohol consumption and blood pressure (BP) was studied in 29 centers in 12 countries as part of the International Cooperative Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study. From each population, 100 men and 100 women aged 50-54 were randomly selected for BP measurement by automated system and for a standardized interview to complete the questionnaire relating to alcohol consumption. In cross-center simple linear regression analysis, mean alcohol consumption calculated from a previous week's drinking did not show a linear association to BP. In within-center multiple linear regression analysis, relationships between high alcohol intake (much greater than 300 g/week) and BP were assessed among 26 centers with high alcohol drinkers after being adjusted for other confounding variables. Positive associations of high alcohol intake with systolic BP (SBP) and diastolic BP (DBP) were noted in 17 and 16 centers, respectively. Thus, high alcohol intake was positively and independently associated with BP in individual subjects. PMID- 1706028 TI - Comparative studies on the relation between nutritional conditions and blood pressure levels of two rural populations with lower incidences of ischemic heart diseases in Japan and Spain. AB - Comparative studies on nutritional constituents and blood pressure (BP) levels were performed in two rural areas--in Japan (Ohda) and Spain (Navas)--with low incidences of ischemic heart disease. BP levels of both populations were relatively low, probably resulting from potassium- and magnesium-rich Mediterranean diets in Navas and from fish protein-rich Japanese diets in Ohda assessed by 24-h urine analyses according to the World Health Organization (WHO) Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study. PMID- 1706029 TI - Relationship between dietary factors and blood pressure in China. The Sino-Japan CARDIAC Cooperative Research Group. AB - As part of the international cooperative Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study, we carried out surveys for the relationship of dietary factors to blood pressure (BP) in 10 areas in China, (Altai, Beijing, Guangzhou, Guiyang, Hetian, Lhasa, Shanghai, Shijiazhuang, Tulufan, and Urumqi). Systolic BP and diastolic BP were significantly positively associated with salt excretion and body mass index. However, 3-methylhistidine divided by creatinine, and taurine divided by urea nitrogen in 24-h urine were significantly negatively associated with both BPs. These results suggest that meat protein intake may beneficially influence BP, whereas salt may adversely affect BP. PMID- 1706030 TI - International cooperative study on the relationship between dietary factors and blood pressure: a report from the Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study. AB - To investigate the epidemiological relationship of dietary factors to blood pressure (BP) and major cardiovascular diseases, we carried out the International Cooperative Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study, which so far involves 45 centers in 20 countries. From each population, 100 men and 100 women aged 50-54 years were randomly selected for BP measurement, 24-h urine collection, and blood test. Various biological markers of diets from urine and blood were analyzed centrally in the Izumo CARDIAC center. In within-center multiple regression analyses, body mass index (BMI) was strongly positively and independently associated with BP. Urine magnesium (Mg) excretion was negatively and independently associated with BP. In cross-center simple regression analyses, systolic and diastolic BP showed significant correlations with BMI (p less than 0.01) and 24-h urinary sodium (Na) excretion (p less than 0.005) in men and significant inverse correlations with urinary 3-methylhistidine (3-MH) divided by creatinine (p less than 0.01 and p less than 0.005, respectively) in men, and were inversely correlated with urinary Mg in women (p less than 0.05). Therefore, Na and BMI adversely affect BP, whereas animal protein and Mg intakes may have beneficial influence on BP. PMID- 1706031 TI - Canadian Consensus Conference on Nonpharmacological Approaches to the Management of High Blood Pressure: recommendations. AB - The issue of nondrug treatment, either as a sole or an adjunct therapy in combination with a drug treatment for high blood pressure (BP), is controversial. In an attempt to resolve controversies and to arrive at a consensus, the Canadian Consensus Conference on Nonpharmacological Approaches to the Management of High Blood Pressure was convened in March 1989 in Halifax, Nova Scotia. State-of-the art information on seven key nonpharmacological issues about body weight, alcohol, salt, potassium and calcium intake, physical exercise, and relaxation were reviewed, and a multidisciplinary consensus panel arrived at recommendations aimed at those members of the general public who are normotensive, those with high BP, health professionals, and, in some cases, the government and the food industry. This panel also suggested further studies in each of the topic areas. PMID- 1706032 TI - Canadian Consensus Conference on Nonpharmacological Approaches to the Management of High Blood Pressure: postconference initiatives. AB - Following the Canadian Consensus Conference on Nonpharmacological Approaches to the Management of High Blood Pressure. March 1989, the Coalition through its member organizations, launched a number of initiatives stemming from the conference. The Coalition achieved a unified document acceptable to all levels of the health profession for nonpharmacological approaches to hypertension, which will be the very first approach prior to treatment with drugs. The information dissemination was successfully carried out. With the cooperation of all member organizations the Coalition is hopeful of executing its future planned strategies for successful intervention of nonpharmacological approaches to the treatment of high blood pressure. PMID- 1706033 TI - Cardiovascular risk factors in Newfoundland population and modification of their level through nonpharmacological intervention. AB - In the rural area of Newfoundland, 522 residents volunteered for a cardiovascular risk factor evaluation. Medical history, blood pressure (BP), blood cholesterol, smoking, and body mass index were assessed. Seventy-five percent of the population had at least one of these risk factors. The most frequent condition had been hypercholesterolemia in 61% of respondents. An intensive 6-month educational program aimed at a group of 41 high-risk individuals led to a significant decline in systolic BP, diastolic BP, body mass index, and serum cholesterol. Although the risk factor levels increased again after termination of the intervention program 6 months later, they have remained below the original values. The results of this pilot study led to a design of a major random clinical trial on nonpharmacological therapy, to be implemented in the years 1990 1993. PMID- 1706034 TI - Blood pressure levels in the elderly with or without nutritional intervention. AB - Blood pressure (BP) examination and urine analyses for the assessment of dietary Na, K, and protein intakes were carried out according to the standardized methods for the World Health Organization Cardiovascular Diseases and Alimentary Comparison Study in aged people with or without a longlasting nutritional intervention. Nutritional intervention to reduce salt intake and increase K and protein intakes was effective for BP reduction even in aged people over 65 years. PMID- 1706035 TI - Salt sensitivity of blood pressure in patients with primary hypertension. AB - Objective measures of blood pressure (BP) sensitivity to 72-h salt depletion were evaluated. Salt sensitivity is defined as a measurable decrease of diastolic BP (DBP) after depletion. Changes in office auscultatory and oscillometric DBP were compared with oscillometric ambulatory DBP. In 35 women and men with mild hypertension, 24-h ambulatory DBP; sodium, potassium, albumin, and creatinine in 24-h urine; serum-creatinine; and body weight were measured before and at the end of the salt-free period. The oscillometric method detected larger and more uniform decreases in DBP compared to the auscultatory method. The salt depletion induced changes in auscultatory DBP but not in ambulatory DBP were positively related to its baseline level. The salt sensitivity was positively related to the age and negatively related to the number of hypertensive symptoms. It was not related to body mass index and body weight decrease after salt depletion. The changes in ambulatory DBP were correlated to changes in office DBP (r = 0.46 for the auscultatory method; r = 0.58 for the oscillometric method). In only half the cases, the direction and size of pressure changes were reflected similarly in all three methods. Although the correlation between the methods points to the biological soundness of the salt sensitivity concept, the individual classification is prone to variation. PMID- 1706036 TI - Trends of diet and blood pressure in Guangzhou, South China. AB - As part of the International Cooperative Cardiovascular Diseases and Alimentary Comparison (CAR-DIAC) Study surveys to determine the relationship of dietary factors to blood pressure were carried out in 1985 (pilot study) and 1989 (core study). Thirteen men and 16 women, and 102 men and 115 women aged 50-54 in the rural population of Panyu county in Guangzhou were randomly selected for the pilot and core studies, respectively. Blood pressure was measured by an automatic system and 24-h urine collection by aliquot cups. All urinary specimens were analyzed in the WHO Collaborating Center (Izumo, Japan). Seven hypertensive cases and 17 borderline cases of hypertension were found in 1989 but none in 1985. Mean systolic blood pressure (SBP) and diastolic blood pressures (DBP) were increased, and the intake of dietary sodium (Na), sodium chloride (NaCl), and the ratio of sodium to potassium (Na/K) were increased markedly. The intake of magnesium (Mg) was decreased in 1989. Correlation analyses showed that body mass index was positively related to SBP and DBP (p less than 0.05), Na and NaCl were positively related only to DBP (p less than 0.05), and Mg was inversely related to SBP but with no statistical significance. These results indicate that trends of rise of prevalence rate of hypertension and mean values of SBP and DBP in association with increased dietary Na and decreased Mg intake may be due to rapid changes in dietary habits, changes in lifestyle, and the differing socioeconomic status in the area and may highlight the importance of dietary factors in the prevention of hypertension. PMID- 1706037 TI - Phosphate as a factor in sodium sensitivity in normal and high renin hypertension. AB - Disturbed phosphate (PO4) metabolism has been documented in spontaneously hypertensive rats but poorly studied in humans. Twenty-seven drug-free hypertensive subjects were studied on both a 10- and 100-mmol sodium diet. The response of mean arterial pressure to sodium repletion was directly correlated to the response of plasma renin activity (r = 0.540, p = 0.004) and inversely related to the percent response of serum PO4 concentrations. In the sodium replete state serum PO4 concentration correlated inversely with plasma vitamin D concentration (r = 0.419, p = 0.026), consistent with PO4 acting as a determinate of vitamin D production. The response of serum calcium and plasma vitamin D concentrations to sodium repletion were correlated (r = 0.392, p = 0.043), consistent with serum calcium levels being a dependent variable, but the responses of serum PO4 and vitamin D concentrations were not. This study suggests that PO4 metabolism may be a determinate of blood pressure response to sodium repletion. PMID- 1706038 TI - Urinary cations and blood pressure in the Chinese population. AB - Three collaborative studies on the relationship between blood pressure (BP) and urinary electrolytes confirmed a positive intrapopulation relationship between sodium (Na) intake and BP, especially in the northern districts of China. In the present article, data of overnight urinary electrolytes and BP of 3,251 subjects from 16 districts of China were pooled and analyzed. A consistent significantly positive correlation was shown between BP and UNa/UK ratio. Both men and women with a UCa less than 10 mmol/day showed a significant correlation of BP with UNa/UK. After age, body mass index adjusted, the correlation between BP and UNa or UNa/UCr showed consistently in males. BP correlated negatively with UCa only in men, whereas women in the age 50-59 group were found most sensitive to UNa/UK and a negative correlation of diastolic BP with UK was only found in women. PMID- 1706039 TI - The effect of sodium intake on ventricular performance in healthy men. AB - We analyzed the effects of acute sodium intake on left ventricular performance in 11 healthy normotensive men using Doppler echocardiography. For 2 weeks, they took 2,700-kcal diets containing 6 g and 24 g sodium chloride per day in the low sodium phase (5 days) and in the high sodium phase (5 days), respectively. Blood pressure, urine, hematocrit, and echocardiographic values were measured every day throughout this study. Although a decrease in the serum norepinephrine concentration and an increase in the ejection fraction were noticed in the low sodium phase, there was no significant change in the relation between end systolic stress and mean velocity of circumference shortening. All subjects also performed a cold pressor test in both phases, and we analyzed the end-systolic stress-volume relation (modified Emax). Modified Emax of the high sodium phase was significantly larger than that of the low sodium phase. As published in previous research on dogs, we consider that this discrepancy at rest may be caused by a dysfunction at the cardiac adrenergic neuroeffector junction in humans, too, and that sodium intake may enhance the change in ventricular contractility by a cold pressor test. PMID- 1706040 TI - Systemic arterial hypertension: results of the change from pharmacological to nonpharmacological treatment. AB - The effect of nonpharmacological measures--diet (restriction to salt), weight reduction, stress avoidance, stopping smoking, and exercise--are analyzed after being applied to 145 hypertensive individuals (average age 60.3 years) using antihypertensive drugs for 6 months without nonpharmacological therapy. The initial systolic arterial pressure was 177 mm Hg; at the end of 4 weeks of treatment, it was 151.3 mm Hg. The initial diastolic arterial pressure was 98.5 mm Hg; at the end of 4 weeks it was 89.8 mm Hg (p less than 0.001). The proposal for treating hypertension with nonpharmacological measures represents a challenge and opens a new horizon to scientific research. PMID- 1706041 TI - Excerpts from the WHO CARDIAC Study Protocol. PMID- 1706042 TI - Epidemiological study on hypertension in northern Japan. AB - Aging for 5 years, from 50 to 55 years of age, influenced the various markers in a normotensive group but a similar correlation could not be seen in a hypertensive group. This suggests an impaired control of physiological homeostasis in hypertensive subjects. An increase in body mass index (BMI) is an important predictor in understanding the pathogenesis of hypertension, although the mechanism is uncertain. A reduction in BMI may reduce the risk of high blood pressure. PMID- 1706043 TI - [Vascular architecture and neovascularization is surveyed with a new casting technique]. AB - Tubular, alveolar and caval systems in the body can be replicated by injecting a solidifying mass, surrounding tissue then being removed and the corroded cast of the cavity analyzed. Long recognized, this approach has gained impetus with the advent of the scanning electron microscope and acrylic polymers. Microvascular corrosion casting combined with analysis in the scanning electron microscope is now a powerful tool for the investigation of e.g. vascular architecture and angiogenesis in different organs and species (including man) and under both physiological, experimental and pathological conditions. Different questions at issue and the morphology of the microcirculation are presented briefly with some key references. PMID- 1706044 TI - Endothelin immunoreactivity of airway epithelium in asthmatic patients. AB - There is extensive pharmacological and physiological evidence that endothelin-1 influences airway calibre. In mammals, endothelin receptor occur on airway smooth muscle, local storage and release of the peptide have been demonstrated, and inhalation of endothelin-1 induces bronchoconstriction. To investigate the relation between endothelins and asthma the expression of this peptide in endobronchial biopsy specimens was examined immunohistochemically with an antiserum against endothelin-1. Biopsy specimens from 17 asthmatic patients and 11 atopic and non-atopic healthy controls revealed striking differences, with endothelin expression being evident in airways epithelium and vascular endothelium in 11 of the 17 asthmatic patients but in only 1 of 11 controls. These results suggest that endothelins may play a part in the exaggerated bronchomotor tone of asthma. PMID- 1706045 TI - One explanation of the asthma paradox: inhibition of natural anti-inflammatory mechanism by beta 2-agonists. AB - Our understanding of the mechanisms contributing to the pathogenesis of bronchial asthma has increased substantially over the past decade. This has been accompanied by the introduction of a range of new drugs for the treatment of this disorder, and the usage of anti-asthma drugs is increasing. Despite these changes and an increased awareness of the disease, asthma remains the only "preventable" disease where the morbidity and mortality are still increasing in most parts of the world. This "asthma paradox" requires explanation, and this article is an attempt to provide a plausible scientific one. The hypothesis expresses concern that a long recognised, but little publicised, pharmacological property of the drug class most widely prescribed for the treatment of asthma, the beta 2 adrenoceptor agonists--namely, the inhibition of mast-cell degranulation--may be contributing to the world wide increase in morbidity and mortality from asthma via the inhibition of a natural anti-inflammatory mechanism. PMID- 1706046 TI - Potential molecular competitor for HIV. PMID- 1706047 TI - Colony-stimulating factors for neutropenia in glycogen storage disease Ib. PMID- 1706048 TI - Terminal dehydration and intravenous fluids. PMID- 1706049 TI - Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used. AB - The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used. PMID- 1706050 TI - Calcium staining by the glyoxal-bis-(2-hydroxyanil)-method in the livers of rats treated with CCl4, diltiazem, and with both agents together. AB - We studied the histochemistry of Ca in livers treated with CCl4, diltiazem (one of the Ca antagonists), and both agents together to determine whether hepatocytes or other parts of the liver lesions show Ca staining and whether the grade or location of Ca in these injuries varies. For Ca staining, cryostat sections were treated by the glyoxal-bis-(2-hydroxyanil) (GBHA)-method using O.C.T. imbedding compound instead of paraffin. Diltiazem-treated rats showed Ca granules in the bile canaliculi around the terminal hepatic veins and Kupffer cells 6 h after intragastric injection. Rats treated with CCl4 showed fine red granules in the cytoplasm of the hepatocytes around the terminal hepatic veins as soon as 5 min mildly and 2 h moderately after intraperitoneal injection. Hepatocytes around the terminal hepatic veins showed positive Ca granules 6 to 30 h after intragastric injection of CCl4. Hepatocytes stained by Ca showed acidophilic degeneration and coagulative necrosis. The hepatocytes of rats treated with both diltiazem and CCl4 revealed fewer Ca granules than those treated with CCl4 alone. In summary, Ca was stained by the GBHA method from the early stage of liver injury by CCl4 and was closely involved in acidophilic degeneration and coagulative necrosis of hepatocytes. The Ca staining in liver cells in CCl4-treated rats was decreased by diltiazem. PMID- 1706051 TI - Purification and reconstitution of epithelial chloride channels. PMID- 1706052 TI - Receptor identification. PMID- 1706053 TI - Chloride channel blockers. PMID- 1706054 TI - Stimulation of secretion by secretagogues. PMID- 1706055 TI - Electrophysiology of pancreatic acinar cells. PMID- 1706056 TI - Electrogenic and electroneutral ion transporters and their regulation in tracheal epithelium. PMID- 1706057 TI - Turtle colon: keeping track of transporters in the apical and basolateral membranes. PMID- 1706058 TI - Gastric glands and cells: preparation and in vitro methods. PMID- 1706059 TI - Collision-induced dissociation. PMID- 1706060 TI - Linkage positions in glycoconjugates by periodate oxidation and fast atom bombardment mass spectrometry. PMID- 1706061 TI - Constituents of nucleic acids: overview and strategy. PMID- 1706062 TI - Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. PMID- 1706063 TI - Preparation of trimethylsilyl derivatives of nucleic acid components for analysis by mass spectrometry. PMID- 1706064 TI - Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry. PMID- 1706065 TI - Electron ionization mass spectra of trimethylsilyl derivatives of nucleosides. PMID- 1706066 TI - [Features of mutated changes of genomic RNA of cold-adapted and hr-variants of influenza group A virus, detected by RNA:RNA hybridization]. AB - The presence of mutations in the majority of the genes of cold-adapted strains A/Leningrad/134/17/57 (H2N2), A/Leningrad/134/47/57 (H2N2) and A/PR/8/59/1 (H1N1) of influenza A virus has been demonstrated by the RNA-RNA hybridization with the subsequent electrophoresis of double-stranded RNA in 7.5% polyacrylamide gel. The strains were cultivated 17, 47 and 59 passages in the chicken embryos at 25 degrees C. In the genomes of variants passaged in chicken embryos at optimal temperature of incubation 36 degrees C (hr-variants) the used technique permits identification of a single mutant gene. The obtained data suppose the attenuation of cold-adapted vaccine strains of influenza A virus and their high genetic stability to be a result of selection of the variants obtaining multiple mutations in the genome during passaging of the virions at cold temperature. The attenuation of hr-variants is defined by 1-2 mutations (first of all in HA-gene) that makes understandable their inability to serve as donors for recombinant live influenza vaccines construction. PMID- 1706067 TI - [Differences in the structure of the hemagglutinin gene in variants of the influenza A (H3N2) virus, differing in immunogenic activity]. PMID- 1706068 TI - Sister-chromatid exchange in human B- and T-lymphocytes exposed to bleomycin, cyclophosphamide, and ethyl methanesulfonate. AB - Sister-chromatid exchange (SCE) frequencies were investigated in mitogen stimulated cultures of highly purified human peripheral blood B- and T lymphocytes exposed to bleomycin (BM), cyclophosphamide (CP), or ethyl methanesulfonate (EMS). In untreated controls, T-lymphocytes showed twice as many SCEs as B-lymphocytes. CP (with metabolic activation) and EMS significantly increased the SCE frequencies. EMS induced a similar, dose-dependent SCE increase in both cell populations, whereas CP induced more SCEs in T- than in B lymphocytes. No clear SCE increase was found in B- and T-lymphocytes treated with BM. PMID- 1706069 TI - Structural determination of a directly mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2',4'-tetranitrobiphenyl in Salmonella typhimurium TA98. AB - In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2',4'-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO2 and Al2O3 column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2',4'-tetranitrobiphenyl with S9 mix at 37 degrees C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4'-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4'-diamino-2',4 dinitrobiphenyl by its chemical and physico-chemical properties. PMID- 1706070 TI - Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. AB - Lymphocyte-specific tyrosine protein kinase p56lck is physically associated with CD4 and CD8 T-cell surface molecules, suggesting that it may transduce CD4/CD8 triggered tyrosine phosphorylation signals during antigen stimulation. Indeed, antibody-mediated aggregation of CD4 (to mimic interaction with its ligand, major histocompatibility complex (MHC) class II molecules), rapidly elevates the kinase activity of p56lck and is associated with marked changes in tyrosine protein phosphorylation. Genetic analyses suggest that the interaction of CD4/CD8 with p56lck results in a positive signal during antigen-induced T-cell activation. To evaluate directly the role of p56lck in T-cell activation, we introduced a constitutively activated form of Lck protein (tyrosine 505 to phenylalanine 505 mutant); in a CD4-negative, MHC-class II restricted mouse T-cell hybridoma. We report here that, as for transfection of CD4, expression of the Lck mutant enhanced T-lymphocyte responsiveness. This finding provides direct evidence that p56lck can positively regulate T-cell functions and that it mediates at least some of the effects of CD4 and CD8 on T-cell activation. PMID- 1706071 TI - Cell-surface regulation of beta 1-integrin activity on developing retinal neurons. AB - Integrins are a family of alpha beta heterodimeric receptors that mediate cell cell and cell-substratum interactions. Integrin binding to extracellular ligands regulates cell adhesion, shape, motility, intracellular signalling and gene expression. Mechanisms that regulate integrin function are, therefore, central to the participation of integrins in a diverse set of cellular events. Here we report the identification of TASC, a monoclonal antibody to a novel epitope on the integrin beta 1 subunit, which inhibits cell adhesion to vitronectin but promotes adhesion to laminin and collagen types I and IV. We show that developing retinal neurons that have lost responsiveness to laminin regain the ability to bind laminin in the presence of TASC. Thus, beta 1-class integrins are likely to occupy multiple affinity states that can be modulated at the cell surface. PMID- 1706072 TI - Nucleic acid sequence-based amplification. AB - Nucleic acid sequence-based amplification (NASBA) is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature. PMID- 1706073 TI - [Pain treatment in patients with cancer; experiences with an outpatient clinic 'Integrated Pain Treatment']. PMID- 1706074 TI - [Pheochromocytoma; current viewpoints on diagnosis and therapy]. PMID- 1706075 TI - [Relationship between the use of postgraduate educational material on human immunodeficiency virus and the number of HIV consultations in family practice]. AB - An enquiry using a structured questionnaire was conducted among all 6300 GPs in the Netherlands in order to assess the distribution of HIV-related problems over general practices in the Netherlands and the influence on it of the use of the 'HIV-wijzer voor de huisarts', a loose-leaf handbook on HIV, distributed since 1988 among all Dutch general practitioners. The enquiry was conducted one year after its publication. The 2156 respondents (34%) appeared to be reasonably representative of all GPs. The results show minor imperfections because a small proportion of the questionnaires was filled out incompletely. Almost 90% of respondents mentioned HIV-related consultations, 24% had HIV-seropositive patients and 18% had AIDS patients. These numbers were correlated mainly with municipality size, less with region. The use of the 'HIV-wijzer' was related to occurrence of HIV-related consultations and AIDS patients in a practice. GPs who do not encounter these problems are little motivated to read the 'HIV-wijzer'. For them, other means of education have to be developed or HIV has to be included in existing education programmes on other subjects. PMID- 1706076 TI - Testicular cancer with enlarged mediastinal lymph nodes: a diagnostic pitfall. AB - A patient with embryonal cell carcinoma restricted to the left testicle, without retroperitoneal but with mediastinal lymph node enlargement and highly elevated serum alpha-fetoprotein levels, is presented (T1N4M0). Because stage III of the disease was presumed, he received chemotherapy, which was unfortunately complicated by a bleomycin-induced pneumonitis. At re-evaluation after chemotherapeutic treatment, it appeared that the tumor marker level had decreased exponentially after the operation and that the mediastinal lymph node enlargement was due not to metastatic disease but to sarcoidosis. The necessity of calculating the half-life of tumor markers after operation and histological examination of the mediastinal lymph nodes prior to chemotherapy in such cases, is discussed. PMID- 1706077 TI - [Inflammation and rejection]. AB - The inflammation represents an important cause of allograft tissue lesions during ongoing rejection episodes. Such inflammation is mediated, at least in part, via the release of different soluble mediators. Some of these endogenous inflammation mediators are well known and include prostaglandins, leukotriens, histamine or histaminergic compounds and factors induced by the activation of the complement cascade (C3a, C5a). More recently, different molecules have been characterized, namely cytokines that are potent mediators of both immune and inflammatory reactions. Cytokines are produced by a wide variety of cells types (i.e., inducing immune and non immune cells) and expressing pleiotropic effects. The synergism between these various inflammation mediators finally provoke the anatomopathological lesions characteristic of a rejection process that are vascular lesions, edema formation and white blood cell chemotaxis leading to massive cellular infiltration. The aim of this brief review is to analyze the more recent data of the literature concerning cytokines that, applied to the field of organ transplantation, may provide further insights onto the relationships existing between inflammation and allograft rejection. PMID- 1706078 TI - Development and distribution of opsin-like immunoreactivity in the dystrophic retinas of rdle mutant mice. AB - The opsin-like immunoreactivity in the retinas of C57BL/6J rdle mutant mice has been studied by light-microscopic immunocytochemistry. Positively labeled cells were found in the normal heterozygous and homozygous mutant mouse outer nuclear layer (ONL) as early as postnatal day 3 (PN3). Beginning at PN10, in the retinas of the homozygous mutant mice, labeled photoreceptor cells rapidly decreased in number and disappeared after PN42. In the decreased ONL, remaining opsin-positive cells were labeled at higher density than those of controls. The retinal pigment epithelium was also moderately labeled during the loss of opsin-positive photoreceptor cells. In addition, sparse opsin-immunoreactive cells were demonstrated in the inner nuclear layer (INL) in the retinas of both the mutant and non-dystrophic mice as early as PN10 and are presumed to be ectopic photoreceptor cells. However, these displaced photoreceptor cells disappeared by PN28 in mutants along the same time course as those in the ONL but were still present in the PN28 retina of controls and seemed to be more abundant at later adult ages. There was no difference in the developmental regulation of opsin in the heterozygous and normal controls. PMID- 1706079 TI - Prenatal diagnosis in diabetic gravidas: utility of ultrasound and maternal serum alpha-fetoprotein screening. AB - The differences in both the biology of pregnancy and the content of routine care between gravidas with and without diabetes mellitus lead to important differences in the potential utility of both ultrasound examination and maternal serum alpha fetoprotein (MSAFP) screening. However, both diagnostic methods have become standards of care for these patients, without critical evaluation. This study examines the utility of both ultrasound and MSAFP in diabetic women. Four hundred thirty-two women with diabetes mellitus antedating pregnancy were examined sonographically between 12-23 weeks' gestation. Of these, 393 were also screened with MSAFP determinations. At delivery, 32 of these fetuses were found to have 38 major congenital malformations. All fatal or potentially life-threatening defects had been diagnosed in utero by sonography before 24 weeks' gestation. Ultrasound had a positive predictive value of 90% and a negative predictive value of 97% for identification of major birth defects before 24 weeks' gestation. There were 14 MSAFP values greater than 2.0 multiples of the median, and nine of these patients elected to undergo amniocentesis. Maternal serum alpha-fetoprotein screening had a positive predictive value of 17% and a negative predictive value of 94%. No malformations were detected through MSAFP screening that had not been diagnosed by sonography. No malformations missed sonographically were detected by MSAFP screening, and none of the amniocenteses were helpful diagnostically. We conclude that MSAFP screening is of minimal utility for diagnosing major congenital malformations in a high-risk population examined universally by an experienced sonographer. PMID- 1706080 TI - Using videography to teach retropubic space anatomy and surgical technique. AB - This report describes an innovative use of videography in teaching retropubic space anatomy and surgery. After the standard abdominal incision has been made, the second surgical assistant places a gynecologic laparoscope, fitted with an endoscopic camera, into the retropubic space and guides the laparoscope documenting the anatomical landmarks and surgical repair as they are shown by the surgeon. All members of the operating team can see the procedures on a color monitor in real time. The videotapes can be edited later to produce teaching films. Since 1986, this technique has been used in 30 patients with no intraoperative complications and no postoperative retropubic space infections. PMID- 1706081 TI - Immunoprofile of mucoepidermoid carcinomas of minor salivary glands. AB - Because the data on the antigenic phenotype of mucoepidermoid carcinoma (MEC) are incomplete and somewhat disparate, 45 MECs were evaluated immunohistochemically for low- and high-molecular-weight keratins, vimentin, glial fibrillary acidic protein, smooth muscle actin, and S-100 protein. Tumors stained uniformly for keratins and, on occasion, focally for vimentin. Tumors were nonreactive with antibodies to glial fibrillary acidic protein and, with few exceptions, to muscle specific actins and S-100 protein. Clear cell and papillary histologic variants were seen as potential diagnostic pitfalls. If used with hematoxylin-and-eosin stained sections, limited potential is seen for this antibody panel in surgical pathology. Myoepithelial cell-associated antigens are expressed to a very limited extent in MECs. PMID- 1706082 TI - Malignant cyst of the lateral aspect of the neck: branchial cleft carcinoma or metastasis? AB - A 58-year-old man had a left jugulodigastric mass, which was found to be cystic by computed tomography, and no evidence of other lesions. Grossly and histologically, the surgical specimen consisted of a thin-walled, fluid-filled cyst lined by squamous epithelium that varied in appearance from benign to invasive squamous cell carcinoma. The findings supported a differential diagnosis of branchial cleft carcinoma (BCCA) versus cystic growth of a lymph node metastasis from an occult malignancy. On this basis, guided biopsies of the upper aerodigestive tract were performed, with strong suspicion of a tonsillar bed lesion. Microscopic examination revealed the primary tumor within tissue excised from the left tonsillar fossa. Comparison of the current case with cases of BCCA and cystic tonsillar metastases from the literature illustrated the potential pitfalls in rendering a diagnosis of BCCA. Recognition of this lesion as a distinctive clinical variant of oropharyngeal carcinoma is warranted. PMID- 1706083 TI - Production of granulocyte colony stimulating factor in decidual tissue and its significance in pregnancy. AB - The increase of the number of polymorphonuclear leukocytes (PMN) in peripheral blood during pregnancy is well known. Granulocyte colony stimulating factor (G CSF) has been found to act as a hemopoietic factor for PMN. This study analyzed G CSF concentrations in the peripheral blood of pregnant women and examined whether decidual tissue acted as source of G-CSF production. The proliferation of chorionic cells measured by [3H]-thymidine uptake and its association with G-CSF were also examined. The results revealed that the concentration of G-CSF increased during the course of pregnancy and was positively correlated with the number of PMN. Large amounts of G-CSF was produced in cultures of decidual cells. We also found that G-CSF stimulates the proliferation of the chorionic cells. It was concluded that the decidual cells have a G-CSF producing activity associated with the elevation of PMN and play some role in the proliferation of chorionic cells during pregnancy. PMID- 1706084 TI - [Lethal midline granuloma (LMG)]. PMID- 1706085 TI - Management of labyrinthine fistulas caused by cholesteatoma. AB - The surgical management of labyrinthine fistulas caused by cholesteatoma remains controversial. Forty cases (41 ears) of labyrinthine fistulas were reviewed. This represented 10% of our total series of cholesteatomas in adults and children (426 ears). Clinical presentation, extent of disease, results of fistula testing and audiometric studies, and radiographic findings were analyzed. A canal wall-down procedure was performed in all but one patient. Generally an attempt was made to completely remove the cholesteatoma, to graft the fistulous area, and to reconstruct the middle ear mechanism in one stage. The matrix was preserved in patients with large fistulas where the involved ear was the only hearing one, when the matrix was adherent to the underlying optic duct, and in selected elderly persons. Long-term followup did not reveal a significant difference in hearing, degree of vertigo, or incidence of recidivism when those patients in whom the matrix was removed were compared with those in whom the matrix was preserved. The importance of recognizing the presence of a labyrinthine fistula preoperatively is stressed, along with the need to be prepared for an unexpected fistula. Operative management is described. PMID- 1706086 TI - Trophoblast proteins and maternal recognition of pregnancy. AB - IFNs are produced by conceptus and/or placental tissues in several mammalian species. Of these IFNs, the trophoblast interferons, oTP-1 and bTP-1, are clearly the most well characterized and have been found to be members of an unusual 172 amino-acid-long IFN-alpha subfamily. Although classified as IFN-alpha IIs, they are unique in two respects. First, the 3' non-coding regions of their mRNAs differ from those of other IFN-alpha s and, secondly, oTP-1 and bTP-1 are expressed in extraordinarily large amounts during a defined period of early pregnancy. oTP-1 and bTP-1 have physiological actions which are clearly anti luteolytic, although it is suspected that they are not the only conceptus products required to maintain the corpus luteum of pregnancy. A role for trophoblast interferons in local regulation of the uterine immune system is also anticipated. Because IFNs are known to exhibit other activities, including effects on cell proliferation, cell differentiation, and the induction of specific gene transcription in target cells, the trophoblast and/or placental interferons may also be influencing uterine function by such mechanisms. However, this question remains largely unexplored. Finally, the regulation of expression of the trophoblast interferons represents an important area of research that has the potential to lead to significant reductions in the incidence of embryonic loss in mammals. PMID- 1706087 TI - Developmental and behavioral disorders in children with congenital hypothyroidism. AB - Congenital hypothyroidism (CHT) causes severe physical and mental retardation if untreated. If treatment is delayed sequelae of brain damage will persist. The best results will be attained if treatment is started in the first weeks of postnatal life. Most of our patients came to our clinic beyond the age of one year, 45% were older than 5 years. Almost all of them had disturbances in growth and development at the time of diagnosis. Treatment with thyroid hormones resulted in improvement in physical growth, but psycho-social achievements were often very disappointing. In this paper a description of developmental and behavioral disorders of children with CHT before and after years of treatment has been made. Persistent inabilities in speaking, writing and reading, arithmetic and behavioral disorders were found. Some recommendations are given. PMID- 1706088 TI - The role of the primary health care provider in fostering the development of young children. PMID- 1706089 TI - Gastrin-releasing peptide gene products in midtrimester human fetal lung with and without maternal smoking history during pregnancy. AB - A preliminary morphological study on human fetal lungs with positive maternal smoking history demonstrated alterations of the neuroepithelial bodies (NEBs). We studied human fetal lung tissue between the gestational ages of 12 weeks and 19 weeks, comprising 12 cases with a smoking history during pregnancy (Group 1) and eight cases without a smoking history during pregnancy (Group 2). We demonstrated, by immunocytochemistry, the presence of gastrin-releasing peptide (GRP) gene products: GRP 14-27 in all 20 cases, and C terminal peptide of pro-GRP (C-flanking peptide) in 17 cases. Quantification of the neuroepithelial cells (NECs) was made by computer-enhanced image analysis using the Context Vision system, expressing 1) the total stained areas of the NECs per unit area of section and 2) the total staining areas of the NECs per unit area of airway epithelium, measured as the area of cytokeratin immunoreactivity in an adjacent section. The results show no statistically significant difference between groups 1 and 2 for either GRP 14-27 or C-flanking peptides. The apparent lack of influence of maternal smoking during pregnancy on the expression of GRP gene products in the NECs could be a reflection of inherently reduced reactivity of the cells during the gestation period studied. However, a larger series is needed before any conclusions can be made. Alternatively, the adverse effects of smoking might be reflected during the canalicular phase of lung development; an increased immunoreactivity appears to be present during that period. The expression of pro GRP gene products in the pulmonary NECs of older fetuses and neonates with maternal smoking history during pregnancy requires further study. PMID- 1706090 TI - Morphometric and biochemical changes in lungs of growing rats treated with a calmodulin antagonist. AB - To determine the role of calmodulin in postnatal lung growth and development, 4 week-old rats were injected intraperitoneally on consecutive days with trifluoperazine (TFP), a potent and specific calmodulin antagonist, for a period fo 3 weeks and studied in comparison with normal controls and undernourished weight-matched animals. TFP treatment resulted in stunting of lung growth such that observed normal increments in morphometrically determined total number of alveoli and alveolar surface area and in biochemically determined DNA, elastin, and collagen contents of the lungs were diminished in comparison with age-matched normal controls. However, the TFP treatment also resulted in reduced daily food intake and body weight gain. In the TFP group, lung weight and lung volume were also reduced compared with the weight-matched control group. This resulted in reduced alveolar surface area, total number of alveoli, DNA, collagen, and elastin in the TFP group compared with values in the weight-matched controls. Thus the TFP-induced lung changes were not due to inanition and/or reduced somatic growth. The TFP treatment resulted in reduced activities of calmodulin and cyclic adenosine monophosphate (cAMP)-phosphodiesterase in the lungs of the animals, independent of their nutritional status. Based on these findings, we suggest that calmodulin may be an important regulatory component of postnatal lung growth and development. PMID- 1706091 TI - Absence of interferon in sera of patients with Kawasaki syndrome. AB - We measured serum interferon concentrations in 42 patients with Kawasaki syndrome. The children ranged in age from 7 months to 6 years. All acute sera were obtained within 12 days of the onset of fever. Convalescent sera (illness day 19 to 56) were available from 25 of 42 patients. Sera were also obtained from 40 controls ranging in age from 2 months to 12 years. Control sera included healthy children (n = 14), children with bacterial infection (n = 10) and children with viral infection (n = 16). Sera were coded and interferon concentrations were measured blindly using human diploid fibroblast cell monolayers challenged with 10(4) plaque-forming units of vesicular stomatitis virus. Specimens from 10 of 16 patients with viral infection were positive for interferon. Three of 10 patients with bacterial infection had detectable serum interferon. No interferon was detected in specimens from the 14 healthy control children or the 42 children with Kawasaki syndrome. Despite the use of a sensitive assay we were unable to detect interferon in the sera of patients with Kawasaki syndrome. PMID- 1706092 TI - Comparison of computed tomography and 57Co-bleomycin scintigraphy in staging the mediastinal lymph nodes of patients with non-small-cell lung cancer. AB - The value of computed tomography (CT) and of 57Co-bleomycin scintigraphy (57Co BLM) in staging the mediastinal lymph nodes was compared in 28 patients suffering from non-small-cell lung cancer. The results were assessed against the pathological findings obtained during thoracotomy or mediastinoscopy. CT staging of the mediastinum had a sensitivity of 75%, a specificity of 80%, an accuracy of 79%, a positive predictive index of 60% and a negative predictive index of 89%. 57Co-BLM scintigraphic staging had a sensitivity of 43%, a specificity of 94%, an accuracy of 80%, a positive predictive index of 75% and a negative predictive index of 81%. In this small series these differences were not statistically significant; it thus appears that CT and 57Co-BLM are of equal value in staging the mediastinum. Mediastinoscopy is not contributory in case of a negative CT or 57Co-BLM. A positive CT or 57Co-BLM, however, indicates the need for histological verification of the mediastinal findings. PMID- 1706093 TI - [The effect of salbutamol on histamine release from basophils in vivo and in vitro]. AB - In vivo and in vitro basophil histamine release inhibition by salbutamol was investigated in patients with bronchial asthma. The study involved 14 patients in stable period of the disease: FEV1 = 66-84% of the normal values. Histamine release was determined following an incubation of the isolated basophils with concanavalin A (Sigma Co., USA) or specific allergens (dust, grass pollens, mites; Bencard, UK). Histamine was assayed with isotope-enzymatic technique according to Shaff and Beaven with histamine N-methyltransferase. Salbutamol administered intravenously in the dose of 1 mg inhibited allergen-induced histamine release from the basophils isolated from patient's blood within 30 minutes. Salbutamol in the concentration of 8 X 10(-6) M inhibited in vitro histamine release induced by an allergen and concanavalin A. PMID- 1706094 TI - Discriminant analysis for assessing the value of amniotic fluid microvillar enzymes in the prenatal diagnosis of cystic fibrosis. AB - We have analysed the sensitivity, specificity, and reliability of biochemical diagnosis based on microvillar membrane enzyme assay and using discriminant analysis in amniotic fluid samples obtained from 54 pregnancies at high risk for cystic fibrosis and 125 normal pregnancies. Our results show that amniotic fluid trehalase, alkaline phosphatase, alkaline phosphatase isoenzymes and gamma glutamyltransferase enzyme activities measured during 16-20 gestational weeks, in spite of their non-specificity for cystic fibrosis, have a very good predictive value for fetal cystic fibrosis or exclude the possibility of the disease. Overall enzyme activity analysis provided over 90 per cent reliability of the method. PMID- 1706095 TI - Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding. AB - Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti (NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis. PMID- 1706096 TI - Expression of cloned receptor subunits produces multiple receptors. AB - Expression in Xenopus oocytes of cloned nicotinic acetylcholine receptors (alpha, beta, gamma and delta subunits of BC3H1 receptor), produced more than one sort of functional receptor. This heterogeneity was detectable neither as heterogeneity in single channel conductance, nor as heterogeneity in the burst length. It was seen most obviously as differences from patch to patch in the maximum fraction of time for which the channels are held open at high acetylcholine concentrations, and it was also detectable as differences in the shut time distributions at low acetylcholine concentrations. PMID- 1706097 TI - Glutamate receptors of rod bipolar cells are linked to a cyclic GMP cascade via a G-protein. AB - Whole-cell patch-clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish. Inclusion of the G-protein activator, GTP gamma S, in the intracellular patch solution mimicked the action of glutamate, inducing an increase in net outward current (interpreted as a decrease in inward current), a decrease in membrane conductance and block of light responses. Cyclic GMP (cGMP) in the patch pipette increased inward current and membrane conductance, and blocked light responses. Cyclic AMP had no effect. IBMX, a phosphodiesterase inhibitor, produced the same effect as cGMP, suggesting the presence of a cGMP phosphodiesterase in rod bipolar cells. These results indicate that the glutamate receptors of on-bipolar cells are coupled via a G-protein to regulate intracellular cGMP, which, in turn, results in the opening of sub synaptic membrane channels. The similarity to phototransduction is striking, and the proposed scheme would account for the high gain in transmission of rod signals to on-bipolar cells. PMID- 1706098 TI - Dynorphin A and substance P in the cerebrospinal fluid of schizophrenic patients. AB - Radioimmunoassay procedures were used to assay levels of dynorphin A (DYN A) and substance P-like activity (SP) in cerebrospinal fluid (CSF) obtained from 10 schizophrenic patients before and after neuroleptic treatment, from 10 matched patients with other psychiatric disorders before and after treatment, and from 10 nonpsychiatric surgical controls. The highest mean concentration of CSF DYN A was found in the schizophrenic group on admission (significant vs. nonpsychiatric controls). The concentration remained almost unaltered after 4 weeks of zuclopenthixol treatment despite a highly significant decrease of overt psychopathology assessed by the Brief Psychiatric Rating Scale (BPRS). There was no significant difference between the mean CSF DYN A levels of the nonschizophrenic psychiatric patients and the surgical controls. When all psychiatric patients were pooled together, there was a significant correlation between the level of CSF DYN A and the BPRS total score. With regard to CSF SP levels, no statistically significant differences were observed within or between the groups studied. Neither was there a significant correlation between the concentration of CSF SP and overt psychopathology. Nevertheless, the mean CSF SP concentration of three patients with major depression was clearly higher than the corresponding mean concentration of the other patients in the nonschizophrenic group. PMID- 1706099 TI - [Emotional experience and subjective illness perception in chronic aphasia--a comparison of patients with their family members]. AB - The results of a pilot study are demonstrated. 10 chronically aphasic patients and their relatives were interviewed by a semi-structured questionnaire. Main themes are the subjective perception and the emotional experience of psychosocial changes, psychological disturbances, communicative and neuropsychological impairments, as well as the perception of restraints caused by symptoms of disease. Methodological difficulties which arise from linguistic and communicative restrictions of aphasic patients are discussed. Data show clear emotional restraints of aphasics and their relatives in occupational, social, psychological and communicative areas. The results indicate independence of subjectively perceived alteration and its emotional experience. The differences of the emotional experience of aphasics and their relatives are discussed. While aphasics are more burdened by restrictions of independence, their relatives gave more importance to emotional changes and familiar problems. PMID- 1706100 TI - Electrophysiological identification of cell types in cortical collecting duct monolayers. AB - Double-barrel microelectrodes were used to determine membrane voltages and the intracellular pH (pHi) in primary cultures of cortical collecting duct cells (CCD) grown in the absence of aldosterone. Electrophysiologically, two main cell types were identified. In cell type 1, the apical membrane voltage (Va) was -60 +/- 5 mV. The fractional resistance of the apical membrane (fRa) was 0.40 +/- 0.03, and pHi was 7.21 +/- 0.04. Exposure to 50 mM K+ on the apical side depolarized Va by 21 +/- 4 mV. When Cl- was replaced by cyclamate two types of responses were observed: (a) depolarization of Va by 26 +/- 3 mV while pHi remained unchanged, and (b) no change in Va. In cell type 2, Va was -36 +/- 5 mV, fRa was 0.91 +/- 0.03 and increasing apical [K+] from 5 to 50 mM did not change Va. Two subpopulations were distinguished by the response of pHi to lowering apical [Cl-]. In one of them pHi increased from 6.99 +/- 0.05 to 7.11 +/- 0.07. In the other, pHi was significantly decreased from 7.16 +/- 0.08 to 7.03 +/- 0.07. These results are compatible with the conclusion that about 50% of the impaled cells type 2 have a Cl-/HCO-3 exchanger at the apical membrane. In summary, two different cell types can be identified electrophysiologically in CCD monolayers. Cell type 1 has the electrical characteristics of principal cells. Cell type 2 resembles the intercalated cells. The cell alkalinization observed in approximately 50% of the cells type 2 in response to Cl- removal suggests the presence of an apical Cl-/HCO-3 exchanger. Thus, these cells should be the bicarbonate-secreting cells. The remaining cells should correspond to the acid secreting cells. PMID- 1706101 TI - Intracellular chloride activity in cultured mesangial cells. AB - Glomerular mesangial cells require Cl ions for the development of a variety of metabolic and functional properties. In the present study the electrochemical distribution for Cl- was examined in cultured rat mesangial cells with Cl(-) sensitive intracellular microelectrodes. It was determined that the intracellular Cl activity exceeded the levels predicted for a passively distributed ion. This was further substantiated by exposing mesangial cells to 10(-5) M bumetanide which drove intracellular Cl to a value close to electrochemical equilibrium. We conclude that Cl accumulates in mesangial cells, against its electrochemical gradient, through a transport pathway that is highly sensitive to bumetanide. PMID- 1706102 TI - Na:K pump abundance and function in MDCK cells: effect of low ambient potassium. AB - Side-specific expression and activity of Na:K pump was studied in Madin-Darby canine kidney (MDCK) cells, a tissue culture model of distal renal tubular epithelium, exposed to low ambient potassium. Confluent monolayers grown on teflon filters in dual chambers were treated with a low K+ medium from 45 min to 72 h. After both acute (45 min) and longer-term (24-72 h) exposure to low K+ (0.7 mM), cation cycling rate of existing pump units increased substantially, while there was no significant change in total cell Na-K-ATPase activity or in basolateral surface pump density. Although a small quantity of Na:K pumps (less than 10%) was consistently present apically, it also did not increase after exposure to low K+, or when the monolayers were provided K+ only from the apical side. In MDCK monolayers low K+ enhances the rate of K+ uptake by the existing pump units but does not increase the total number of pumps or their deployment on either cell surface. PMID- 1706103 TI - Metabolism of lactate by a suspension of dog thick ascending limbs: relations with transport. AB - The addition of substrate in the form of lactate (L), but not glucose (G), increases the respiration of canine thick ascending limb (TAL) segments in a saturable (above 2 mM) fashion. More than 60% of this stimulation is ouabain sensitive (1 mM ouabain) even if L and G transport are both sodium-insensitive processes in TAL. Thus L, but not G, specifically stimulates Na+ entry in TAL cells and its subsequent transport by the Na+,K(+)-ATPase. If chloride is substituted for by gluconate, no significant substrate-induced stimulation of ouabain-sensitive respiration is observed. SITS (4-acetamino-4' isothiocyanostilbene-2,2'-disulfonic acid) also interferes with the L-induced stimulation of respiration. Thus L entry in TAL appears to be directly or indirectly coupled to the transepithelial flux of Cl-. Furosemide (F), but not amiloride, also inhibits this stimulation suggesting that the accelerated Na+ entry triggered by the application of L occurs through the F-sensitive carrier or that lactate transport is F-sensitive in TAL cells. In accord, F specifically impairs the metabolism of L (as compared to G). These data suggest that in intact TAL tubules both lactate uptake and oxidation are directly or indirectly influenced by the transcellular flux of NaCl. This organization may participate to maintain a stoichiometry between the transport of NaCl and the availability of L to support the energetic needs of TAL cells. PMID- 1706104 TI - Effect of hippurate on glucose utilization in rat kidney cortex slices. AB - Hippurate action on glucose utilization was evaluated in rat kidney cortex slices. Studies have shown the following. (1) Hippurate inhibits markedly basal as well as insulin-stimulated glucose utilization and basal gluconeogenesis. (2) Ca deficiency and specific Ca channel blockers diltiazem and isradipine abolish the hippurate inhibition of glucose utilization. (3) K+ channel blockers, i.e. the increased K+ concentration in incubation medium, procaine and sulfonylurea drugs also abolish the hippurate inhibition of glucose utilization. It is concluded that hippurate and benzoate operate through the ATP-dependent K+ channel. PMID- 1706105 TI - Metabolic heterogeneity of isolated cortical and juxtamedullary glomeruli in adriamycin nephrotoxicity. AB - The anticancer drug adriamycin (ADR) is selectively toxic to glomerular cells when administered intravenously (5 mg/kg b.w.) to female MWF/Ztm rats. Recent data have shown that the proteinuria associated with the lesion does not occur in cortical glomeruli, suggesting the selective injury of juxtamedullary glomeruli. In the present study, the effect of ADR on glomerular metabolism was studied with special reference to possible differences between cortical and juxtamedullary glomeruli. On day 7 after ADR treatment, cortical and juxtamedullary glomeruli were separately isolated by the sieving method and 14C glucose oxidation to 14CO2 and the incorporation of 3H proline into macromolecules were measured in vitro and used to study target selective injury in ADR-treated rats compared to control rats. The investigations revealed differences in the response of cortical and juxtamedullary glomeruli to ADR. ADR treatment increased proline incorporation over a 4-hour incubation period in both glomerular populations compared to controls, but the effect was significantly (p less than 0.05) more pronounced in juxtamedullary glomeruli (juxtamedullary: 187 +/- 8% of control; cortical: 167 +/ 4% of control). Glucose oxidation was enhanced after 4 h only in juxtamedullary glomeruli (juxtamedullary: 132 +/- 3% of control; cortical: 82 +/- 10% of control). These data show that glomerular damage caused by ADR is associated with a stimulating effect on glomerular metabolism which is more marked in juxtamedullary than in cortical glomeruli, thus indicating a heterogenous response of different glomerular populations and supporting the concept that the selective damage of juxtamedullary glomeruli accounts for the proteinuria. PMID- 1706106 TI - Two patterns of dopa decarboxylase immunoreactivity in sympathetic axons supplying rat renal cortex. AB - Three ultrastructural cytochemical methods have been used to classify the innervation of the rat renal cortex. Every axon seen contained chromaffin reactive, dense core vesicles and stained for tyrosine hydroxylase, indicating that they were all catecholaminergic. About 10% of the axons associated with smooth muscle and juxtaglomerular cells of the arteriolar vessels also contained dopa decarboxylase, but this enzyme was not present in any of the peritubular axons. Our results are compatible with the possibility that, in the rat, the juxtaglomerular blood vessels, but not the renal tubules, are supplied by dopaminergic as well as by noradrenergic nerves. PMID- 1706107 TI - Tubular function by lithium clearance, plasma amino acids and hormones following a meat meal in childhood. AB - Tubular function was measured by lithium clearance (CLi) and by its derived formulae before and after the transient increase (lasting 90 min) in glomerular filtration rate (GFR) following a meat meal (2g protein/kg body weight) in 12 normal children. Three baseline and 4 clearances after the meal were obtained, each lasting 30 min. The mean baseline CLi was 23.1 +/- 1.64 ml/min/1.73 m2. At peak GFR response (60 min from starting the meal), CLi averaged 27.6 +/- 2.4 ml/min/1.73 m2 (p less than 0.025 vs. baseline) and it was further increased (32.2 +/- 5.04 ml/min/1.73 m2, p less than 0.01 vs. baseline) 120 min after starting the meal, while GFR returned to baseline values. Fractional lithium excretion averaged 0.23 +/- 0.04 at baseline and increased continuously after the meat meal and, at completion of the study, it averaged 0.38 +/- 0.07 (p less than 0.025 vs. baseline). The distal absolute and fractional sodium reabsorption increased throughout the studies following the meal and peaked at 120 min. The functional changes were associated with a statistically significant increase in the plasma concentration of insulin, glucagon, and total amino acids after the meal. The latter at the end of the study was almost doubled (5,600 +/- 780 versus 3,200 microM at baseline, p less than 0.01). The data indicate that the tubulo glomerular feedback mechanism operates normally after a meat meal. The finding on increased distal sodium reabsorption might point to the existence of an insulin dependent mechanism. PMID- 1706108 TI - Circadian variation in renal function of the obese rat. AB - The influence of food and water intake on renal function was assessed by comparisons between the hyperphagic Zucker obese rat and its lean littermate, which demonstrates nocturnal dominance in activity. Serum creatinine and cortisol levels, creatine kinase activities, creatinine and urine clearances, and sodium and potassium excretion rates were measured over a 24-hour period in both lean and obese rats (n = 24 each). Six rats in each group were studied every 8 h to permit characterization over a 12-hour light/dark cycle at 2-hour intervals. Urine and creatinine clearances were increased in lean rats during the dark phase coincident with onset of eating. Similarly, renal sodium and potassium excretion rates were markedly increased during the dark cycle, despite relatively constant serum potassium and sodium levels over the 24-hour period. In contrast, no circadian patterns in urine and creatinine clearances were found in the obese rat, which exhibits continuous feeding habits throughout the 24-hour period. Moreover, renal electrolyte excretion in the obese rat was modestly increased during the dark cycle, unlike the significant differences over time observed in lean rats. Serum creatinine levels were increased during the dark cycle in both rat groups. Creatine kinase activity, a measure of ambulatory activity, was constant in lean rats during the study period. Although creatine kinase activity was increased in obese rats during the dark cycle, no correlations with renal functional parameters were found. These results indicate that differences in food and water intake are significant determinants in diurnal cyclic changes in renal function. PMID- 1706109 TI - Hormone receptors and cellular signal transduction. PMID- 1706110 TI - Cytokines and cell growth factors, their role in the regulation of acute phase reactants. PMID- 1706111 TI - Management problems of the adult with cystic fibrosis. AB - Aggressive palliative therapy which includes antibiotics, physiotherapy, exercise and adequate nutrition remain the basis for treatment and account for improved survival into adulthood. However, although mean actuarial survival into the third decade of life is to be expected in well organised cystic fibrosis centres, a plateau has probably been reached and we need innovative forms of treatment before we can expect further improvements. Indeed, as patients have matured into adulthood new clinical problems have arisen; diabetes is more common, liver disease can progress, and vasculitis may affect joints, skin and brain. Furthermore, social and psychological problems are magnified by patient realization of a deteriorating lethal disease. This article discusses the recommended provision of care for adults with cystic fibrosis, the management of medical and social problems, and finally, how the introduction of heart lung transplantation has affected patient management in the terminal phase of the disease. PMID- 1706112 TI - [Cystic fibrosis in changing times. Clinical aspects, basic defect and molecular biology aspects]. AB - Life quality of patients suffering from cystic fibrosis (CF) has been significantly improved by early diagnosis and advanced therapy during the past three decades. Today, an increasing number of severely affected patients reach adult life no longer requiring the care of a pediatrician but of a specialist for internal diseases. The CF gene has recently been cloned and the most common defect defined, thus providing prenatal diagnosis and carrier detection in CF families. Although the identification of the CF gene is expected to have important clinical consequences, including new therapeutic perspectives, in the distant future, the present therapeutic concept must aim at early treatment of lung disease and pancreatic insufficiency. Intensive therapy, however, is for the patient's lifetime and requires adequate individual control. PMID- 1706113 TI - [Antibody deficiency syndrome: new facts]. PMID- 1706114 TI - Characterization of a Plasmodium falciparum epitope recognized by a monoclonal antibody with broad isolate and species specificity. AB - Monoclonal antibody (MAb) 7H8 raised against Plasmodium yoelii reacted with a series of proteins from P. falciparum that range in molecular weight from 46 to 194 kDa. By immunofluorescence assay, this MAb reacted with all isolates of P. falciparum tested. MAb 7H8 was used to screen a genomic expression library of asexual blood stage antigens of P. falciparum, Malayan Camp K+ and 7 independent clones were identified. These 7 clones were sequenced and the epitope recognized by MAb 7H8 in the recombinant protein of one of these clones was mapped. This epitope contained Lys Tyr Pro as core amino acids. However, similar sequences were not found in the other clones, indicating that this MAb binds to a structural epitope formed by different amino acids. The variable composition of the epitope may account for the number of P. falciparum malarial proteins recognized by MAb 7H8. PMID- 1706115 TI - [Acute phase proteins in the diagnosis of suppurative-infectious diseases after cesarean section]. PMID- 1706116 TI - Serum of patients with prostatic cancer or benign prostatic hypertrophy contains nonpolar testosterone. AB - We have previously described a nonpolar form of radioimmunoassayable serum testosterone (NPT) not measured by available antitestosterone antibodies. It is detected by mild alkaline hydrolysis of the petroleum ether extract of serum and subsequent radioimmunoassay. The properties of NPT are consistent with that of a fatty acid ester of testosterone or dihydrotestosterone. The serum of young males contains 1 to 3 ng/ml NPT, but it is not detected in female serum. We measured serum testosterone and NPT levels in 36 men between 58 and 87 years of age. Seventeen subjects with advanced prostatic cancer (NPT 1.70 +/- 1.44 ng/ml) were compared with a control group consisting of six patients with benign prostatic hypertrophy (BPH) and 13 patients with no prostatic disease (NPT 0.72 +/- 0.46, P = 0.017). There was no significant difference between BPH patients and patients with no prostatic disease; the results were pooled. The concentration of NPT in prostatic cancer patients but not in controls was inversely correlated with that of testosterone. Immunoassayable testosterone was present in the serum of two orchiectomized patients and, therefore, cannot derive solely from the testes. PMID- 1706117 TI - Pathways of parasympathetic and sensory cerebrovascular nerves in monkeys. AB - Using immunohistochemistry, we studied the origins and pathways of parasympathetic and sensory nerve fibers to the pial arteries in four squirrel monkeys. Following its application to the surface of the middle cerebral artery, the retrograde axonal tracer True Blue accumulated in parasympathetic neurons of the sphenopalatine ganglion and the internal carotid ganglion. The latter is strategically located where the internal carotid artery enters the cranium. Fibers from the sphenopalatine ganglion reach the internal carotid artery in the cavernous sinus region after running as rami orbitales. Before reaching the internal carotid artery, the fibers bypass aberrant sphenopalatine ganglia, with the most distant, the cavernous ganglion, being located in the cavernous sinus region. True Blue also accumulated in sensory neurons of the ophthalmic and maxillary divisions of the trigeminal ganglion and in sensory neurons of the internal carotid ganglion. Fibers from the ophthalmic division of the trigeminal ganglion reach the internal carotid artery as a branch through the cavernous sinus, bypassing the cavernous ganglion. Fibers from the maxillary division also bypass the cavernous ganglion after reaching it via a recurrent branch of the orbitociliary nerve. Thus, the cavernous ganglion forms a confluence zone for parasympathetic and sensory fibers in the region. In addition, parasympathetic and sensory fibers leave the confluence zone to follow the abducent and trochlear nerves backward to the basilar artery and tentorium cerebelli, respectively. Clinical implications are discussed. PMID- 1706118 TI - In vivo and in vitro binding of C4 molecules on red cells: a correlation of numbers of molecules and agglutination. AB - The fourth component of human complement (C4) is one that is essential to the antibody-mediated classical activation pathway. C4d, present on all normal and most patient red cells (RBCs), may be detected by the human antisera anti-Rodgers (Rg) and -Chido (Ch). A study has been made of the Rg/Ch antigens on normal and patient RBCs in an attempt to understand the mechanism by which C4 is bound to normal RBCs in the absence of RBC antibodies (Abs). Because RBCs from C1q deficient patients express Rg/Ch, it seems that C1q is not essential for C4 binding. Treatment of normal RBCs with proteolytic enzymes, including trypsin, eliminated positive reactions with anti-Rg/Ch even though the C4d fragment is considered to be resistant to cleavage by trypsin. By correlating agglutination reactions with numbers of bound C4d and C3d molecules, it is evident that both C4d and C3d were affected by trypsin treatment and that anti-Rg/Ch were not capable of agglutinating RBCs with less than 50 molecules of bound C4d. It is concluded that trypsin-sensitive and -insensitive RBC membrane structures may both act as acceptors for C4. RBCs with null phenotypes of the major blood group systems all expressed Rg/Ch antigens, so none of the structures that carry these antigens act preferentially as acceptors for C4. PMID- 1706119 TI - Analysis of a seal and a porpoise morbillivirus using monoclonal antibodies. PMID- 1706120 TI - Characterization of monoclonal antibodies to feline T lymphocytes and their use in the analysis of lymphocyte tissue distribution in the cat. AB - We describe the development of three monoclonal antibodies to feline T lymphocytes. Antibody 1.572 stains 93% of feline thymocytes, 49% of lymph node, and 65% of spleen lymphocytes. Two-color analysis shows 1.572 does not stain Ig bearing cells, and 1.572-positive lymphocytes plus Ig-positive lymphocytes make up approximately 90% of peripheral blood lymphocytes (PBL), suggesting that 1.572 is a pan-T cell marker. The other two monoclonal antibodies, 3.357 and CAT30A, stain a smaller population of thymocytes (59%) of which 40% express both antigens. The 3.357 antigen is found on 23% of lymph node and 47% of spleen lymphocytes, while the CAT30A antigen is found on 29% of lymph node and 19% of spleen lymphocytes. Two-color analysis shows that 3.357 and CAT30A stain mutually exclusive subpopulations of 1.572-positive cells. Using thymocytes as an antigen source, antibody 3.357 precipitated a molecule of 66,000 molecular weight (Mw) under nonreducing conditions and a heterodimer of 32,000 and 34,000 under reducing conditions, suggesting that 3.357 recognizes the feline CD8 homologue. Antibody CAT30A precipitated a molecule of 55,000 Mw under both reducing and nonreducing conditions, which suggests it recognizes the feline CD4 homologue. Analysis of PBL profiles of 35 normal cats using the three monoclonal antibodies indicates that the distribution of feline PBL subpopulations is similar to man, including the CAT30A:3.357 ratio (1.74), which is identical to reported CD4:CD8 ratios in man. Based on these data, the feline CD4 and CD8 homologues are similar to those reported in other species. PMID- 1706121 TI - [Antitrypsin effects of alpha-2-macroglobulin]. AB - Purified preparations of alpha 2-macroglobulin were preincubated with bovine trypsin at various molar ratios. Effects of the preincubation were measured before and after addition of soybean trypsin inhibitor in excess if N-benz-L-arg p-nitroanilide was used as a substrate. Two types of interaction between alpha 2 macroglobulin and trypsin were detected. One of them was carried out depending on the "trap" hypothesis, another type of the interaction led to the enzyme loss of both proteolytic activity and its ability to hydrolyze the low molecular substrate. The data obtained suggest that various molecular forms of native alpha 2-macroglobulin were responsible for these types of interaction. PMID- 1706122 TI - A methylsulphonyl metabolite of a polychlorinated biphenyl can serve as a ligand for alpha 2mu-globulin in rat and major-urinary-protein in mice. AB - 1. Kidneys from rats given an intraperitoneal dose of 4,4'-bis[( 3H]methylsulphonyl)-2,2',5,5'- tetrachlorobiphenyl[(CT3SO2)2TCB)] contained (CT3SO2)2TCB associated with a protein which has been isolated and characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot and immunodiffusion analysis to be alpha 2 mu-globulin (alpha 2 mu). 2. The same radioactive alpha 2 mu-globulin complex was isolated from urine of rats given an i.p. dose of (CT3SO2)2TCB. This complex represented 12.3% (0.27% dose) and 9.3% (0.06% dose) of the radioactivity excreted in 0-24 h and 24-48 h urine, respectively. 3. The radioactivity was extractable from alpha 2 mu and characterized by t.l.c. (co-chromatography in two solvent systems) to be (CT3SO2)2TCB. 4. A radioactive-protein complex was isolated from urine of mice given an i.p. dose of (CT3SO2)2 TCB. The radioactive-protein complex was characterized to be mouse major urinary protein (MUP) by SDS-PAGE, immunoblot and immunodiffusion analysis. This complex was not detected in mouse kidney. 5. Urinary excretion of radioactive-MUP complex represented 51.7% (2.3% dose) and 28% (1.3% dose) of the radioactivity excreted in 0-24 h and 24-48 h urine, respectively. PMID- 1706123 TI - [Cross-reacting Candida albicans antigens in the pathogenesis of candidiasis]. AB - Organ- and tissue-specific antigens with affinity to C. albicans have been detected in some organs and tissues of the body. The occurrence of C. albicans colonization of different organs correlates with the presence of cross-reacting antigens in these organs. PMID- 1706124 TI - Prevention of adhesions by high molecular weight dextran in rats. Re-evaluation in nine experiments. AB - The efficacy of 32% dextran 70 (Hyskon) in the prevention of postoperative peritoneal adhesions was evaluated in nine different experiments in a total of 320 rats under standardised conditions. Hyskon reduced adhesion formation, only in rats that were also subjected to abrasion of the caecum. In the remaining eight experiments Hyskon was ineffective. We conclude that more extensive experimental and clinical studies are desirable before this agent is recommended for prophylaxis against adhesions; any prophylactic regimen should be tested in a wide range of experiments. In addition, analysis of the control data suggested that the stimulus for inducing adhesions in some experiments was so strong that it could not be countered by any prophylactic regimen. A new microsurgical method that resulted in a 50 per cent response and an equal number of low grade and high grade adhesions was used. This method is well suited for investigation of both the stimulatory and the inhibitory effects of a test agent. PMID- 1706125 TI - Relevant factors in the prognosis of ductal pancreatic carcinoma. AB - Because it is important to assess relevant prognostic factors when deciding which patients will profit from pancreatic resection and which will not, 484 patients with ductal pancreatic carcinoma who were operated on between 1 January 1978 and 31 December 1987 in the Department of General and Abdominal Surgery of Mainz Medical School were studied retrospectively. Prognostically favourable factors were: a history of 8 weeks or less, the presence of jaundice, the absence of back pain, a well to moderately differentiated carcinoma, a tumour less than 3 cm in diameter, a preoperative CA19-19 value of less than 400 U/ml, and no sign of lymph node metastases or other metastatic spread. Only 19 patients (3.9%) satisfied these criteria. Our objective is to now check these results in a prospective study with a larger number of patients and, in addition, to compare the quality of life of patients who were treated by resection with that of those who received palliative treatment. PMID- 1706126 TI - Evidence that neuronally released vasoactive intestinal polypeptide inhibits the release of serotonin from enterochromaffin cells of the guinea pig small intestine. AB - Isolated small intestinal segments of the guinea pig were arterially perfused and the release of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid into the portal venous effluent was determined by HPLC with electrochemical detection. Test substances were intra-arterially applied. The muscarine receptor agonist oxotremorine (1 mumol/l) inhibited the release of 5-hydroxytryptamine by about 50%. In the presence of the neurotoxin tetrodotoxin, oxotremorine enhanced the release of 5-hydroxytryptamine by 145%, indicating that the inhibitory effect of oxotremorine was mediated by the release of a neurotransmitter. Exogenous vasoactive intestinal polypeptide (1-100 pmol/l) inhibited the release of 5 hydroxytryptamine by about 50%, an effect antagonized by a specific antibody to vasoactive intestinal polypeptide. This antibody to vasoactive intestinal polypeptide, on its own, had no effect on the release of 5-hydroxytryptamine. However, it prevented the inhibitory effect of oxotremorine. In the presence of the antibody to vasoactive intestinal polypeptide, unlike in the presence of tetrodotoxin, oxotremorine did not stimulate the release of 5-hydroxytryptamine. In conclusion, activation of neuronal muscarine receptors in the guinea pig small intestine enhances the release of several neurotransmitters which can inhibit the release of 5-hydroxytryptamine. The present experiments provide good evidence that vasoactive intestinal polypeptide is one of them. PMID- 1706127 TI - Response of hamster to the antifertility effect of gossypol. AB - One hundred and five sexually mature male hamsters were divided in different groups. In the first experiment hamsters were administered gossypol, 10 mg/kg and 20 mg/kg/body weight/day, for twenty and thirty days. In the second experiment hamsters were administered gossypol, 5, 10 and 20 mg/kg/body weight/day, for sixty days. In the third experiment, hamsters were administered gossypol 5 mg, 10 mg, 20 mg and 40 mg/kg body weight/day for 45 days. Animals in all the groups were given gossypol by oral intubation every day. No significant effect on the body weight of hamsters following gossypol treatment was observed. At low doses the weights of testis and accessory sex organs were not statistically different from those of the controls. A significant decrease in testis and epididymis weight was however observed following high doses of gossypol. Low doses of gossypol treatment did not affect the motility of the vas deferens spermatozoa. The vas deferens spermatozoa were however immotile after 40 mg/kg/day gossypol treatment. Gossypol treatment induced a series of histological changes in the seminiferous epithelium of the hamster testis. The earliest sign of drug effect was seen in spermatids and with the increase in doses the effects became more pronounced and extended to the spermatocytes. At 40 mg/kg dose an almost complete arrest of spermatogenesis was observed. Quantitatively, the ratio of pachytene spermatocytes: resting spermatocytes and step 7 spermatids: pachytene spermatocytes decreased significantly. The step 7 spermatids did not mature to step 19 spermatids at all. Histochemically activities of ATPase, SDH and LDH decreased with the increasing doses of gossypol, the activity of 3B hydroxysteroid dehydrogenase was not affected by gossypol treatment. In testis the glucose-6-phosphatase activity was not affected significantly but the activities of fructose 1, 6-diphosphatase and glucose-6-phosphate isomerase decreased significantly with the increasing doses of gossypol. Amylase activity rose significantly at higher doses. Marked changes in LDH and LDH-X were however observed with the increase in gossypol dose. In liver the activity of glucose-6 phosphatase increased significantly while the activities of fructose 1, 6 diphosphatase, glucose-6-phosphate isomerase and amylase were not affected following gossypol treatment. The glycogen contents however increased significantly following high doses of gossypol. No changes in testosterone production and plasma levels of testosterone were observed following gossypol treatment. PMID- 1706128 TI - Effect of gossypol on the fertility of male rats. AB - Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6 diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706129 TI - Distribution of elastic system fibres in the rat tail tendon and its associated sheaths. AB - The distribution of elastic, elaunin and oxytalan fibres in tendons and tendon sheaths was studied by means of light and electron microscopy. The different fibres were characterised by comparing their typical ultrastructural pictures with a combination of selective staining techniques for light microscopy with the appropriate enzymatic digestions. Since it was observed that the rat tail tendon and its associated sheaths are not homogeneous structures from the point of view of the distribution of the elastic system fibres, a systematic description of the differential distribution of the oxytalan, elaunin and elastic fibres in the tendon and in the endo-, peri-, epi- and paratendineum is presented. Mature elastic fibres are present in the tendon and in the para-, epi- and endotendineum, whereas these fibres could not be detected in the peritendineum. Very many elaunin fibres are characteristically recognised in the endotendineum; these fibres are present in the peritendineum and in the tendon fascicles too, while the epi- and paratendineum are devoid of elaunin fibres. The peri- and endotendineum contain numerous oxytalan fibres, whereas these fibres are absent from the tendon fascicles and the epi- and paratendineum. The functional implications of the foregoing findings are discussed. PMID- 1706130 TI - Effects of space flight on bone formation and resorption. AB - Samples of femurs and tibiae of male Wistar rats subjected to a 13 day space flight on the biosatellite Cosmos 1887--were investigated and compared with vivarium and synchronous controls or immobilized rats, using histological and histomorphometric methods. 1. After flight in the metaphysis of bones the density and volume of the spongious trabeculae diminished significantly indicated by the Sv and Vv histomorphometric values and histological data comparing to the controls. In the diaphysis, the density of trabeculae decreased too. 2. In the flight group significant suppression of bone formation was determined by histological and histomorphometric (decrease of the OS, OB and OBI values) methods. 3. In the flight group according to the histological pictures the signs of bone resorption (increase of Hoswship's lacunae, osteoclastic activity, structural rarefication of spongious and cortical bones, osteon disintegration, osteocytic osteolysis) were revealed, which was substantiated by the histomorphometric results (increase of osteoclastic index: OCI). 4. Significant differences between flight and immobilized groups were not determined, except the osteoid value, which was increased in the case of immobilization. 5. Some histomorphometric values related to bone formation of synchronous control group showed close relationship rather to the flight group than to the vivarium control group. PMID- 1706131 TI - Effect of emetine on the plasma cell population of chicken's gland of Harder. AB - The emetine effectively abolished the plasma cell population in the chicken's gland of Harder by day 3 of treatment. The plasma cell content regenerated by day 5 following emetine injection, possibly from a metabolically inactive, resting B cell population which was resistant to the emetine treatment. By day 7 extracellular substance in a very large quantity appeared among the plasma cells and epithelial cells which might represent a hyperactive plasma cell secretion. The changes in the circulating antibodies measured by hemagglutination well correlated with the plasma cell content in the gland of Harder. The gland of Harder (GH) is an accessory lacrimal gland. Its main function is to lubricate the nictitating membrane and keep the surface of the eyeball wet. The presence and regulatory function of cAMP dependent histone kinase was showed. Since it has been published that in chicken the interstitium of this gland contains a proper amount of plasma cells, this observation called the attention of many investigators to study the role of GH in the immune response. The B cell maturation in the chicken GH has been studied. The surface marker studies have proved that beside the B cells the gland contains functionally adequate number of T cell which exert stimulatory effect on B cells to promote their transformation to plasma cells. In addition to T and B cells small number of macrophages also occur. In the 9 H the number of plasma cells is age dependent. At hatch only a few plasma cells occur in the interstitium of the gland but by 3 weeks of age they become predominant. Different isotypes of immunoglobulins are secreted by these plasma cells. PMID- 1706132 TI - Lysozyme synthesis in osteoclasts. AB - Osteoclasts may or may not be directly related to monocytes and macrophages, but it is well established that these cell types share a number of features in common. In the present study we sought to extend this comparison by assessing lysozyme synthesis in osteoclasts, an enzyme known to be produced and secreted in large amounts by monocytes and macrophages. Our data show that freshly isolated chicken osteoclasts and osteoclasts in situ contain an abundant amount of lysozyme and correspondingly high steady-state levels of the enzyme's messenger RNA. Marrow macrophages, at various stages of in vitro maturation, also possess lysozyme mRNA but in amounts approximately two to four times lower than osteoclasts. These observations reaffirm the monocyte-macrophage nature of the osteoclast but raise questions about the function of the lysozyme in this cell. At present, the role of the lysozyme in osteoclast activity remains unexplained. PMID- 1706133 TI - [Is there a surprise factor in the evaluation of suicide risk in depression?]. AB - The suicidal risk evaluation seems to be difficult. The authors think that it could be created for a surprising factor identified with a low control of impulsiveness added to low levels of 5-hydroxyindoleacetic acid in cerebrospinal fluid. PMID- 1706134 TI - Modulation of ATP sensitive K+ channels: a novel strategy to reduce the deleterious effects of anoxia. PMID- 1706135 TI - [Combined effects of alpha-interferon and anticancer drugs against renal cell carcinoma]. AB - The direct antitumor effects of combined administration of alpha-interferon and chemotherapeutic agents against the human tumor cell line derived from renal cell carcinoma were examined in vivo as xenograft in nude mice. The administration regimen was as follows: Human lymphoblastoid interferon (HLBI) was injected intramuscularly (1 x 10(5) IU/mouse/day) every day for 14 days. Peplomycin (PEP), adriamycin (ADM), or 5-fluorouracil (5-FU) was also administered at a dose of one third of their LD50. We evaluated the effect 20 days after initial administration using the ratio of mean tumor weight. Combined administration of HLBI and PEP or ADM was determined effective and inhibited tumor growth more strongly than HLBI alone or control. Furthermore, the histopathological examination suggested that the effect of combined administration was cytostatic rather than cytolytic. PMID- 1706136 TI - [Evaluation of anti-androgen therapy for benign prostatic hypertrophy by using transrectal prostatic ultrasonography]. AB - We evaluated the effect of anti-androgen therapy for benign prostatic hypertrophy on 33 patients who had undergone transrectal prostatic ultrasonography both before and after administration of anti-androgenic drugs in our clinic. Patients treated with chlormadinone acetate (CMA) orally, or TSAA-291 intramuscularly showed a significant reduction in the prostatic weight. However, this reduction was not correlated with the degree of symptomatic improvement. In addition, patients who showed no symptomatic improvement despite a reduction in the prostatic weight often had prostatic stones or small inflammatory cell infiltrations in their prostatic tissue. Therefore, the coexistence of prostatitis or bladder neck obstruction with prostatic hypertrophy may contribute to the lack of symptomatic improvement in such patients. PMID- 1706137 TI - Benign polyp with prostatic-type epithelium of the urinary bladder: a case report. AB - We report a case of benign polyp with prostatic-type epithelium of the urinary bladder. A 58-year-old male presented with gross hematuria. Cystoscopic examination revealed a 2-mm polypoid lesion in the mid trigone. Immunohistochemical demonstration of prostatic acid phosphatase and prostatic specific antigen in the urothelial cells as well as the prostatic-type epithelial cells suggested the histogenesis of this polyp to be metaplasia of the urothelium. PMID- 1706138 TI - Prostatic urethra dilatation with the Gianturco self-expanding metallic stent: a feasibility study in cadaver specimens and dogs. AB - In an effort to develop a transcatheter technique for dilatation of the prostatic urethra without the use of balloons, the feasibility of using Gianturco self expanding stents was evaluated. Initially, eight human cadaveric prostatic urethras were stented to evaluate the ability of the stent to dilate the lumen. In all cases, the device attained its unconstrained diameter immediately on placement. Subsequently, stents were placed in the prostatic urethra of 12 dogs and followed up for 1 month (four dogs), 3 months (one dog), and 6 months (seven dogs). Five bare straight-end prostheses, one nylon-covered straight-end device, and six flared-end stents were used. Three of the bare straight-end stents migrated during the follow-up, whereas the nylon-covered and flared-end stents did not. Stent diameters greater than 1.3 times the urethral diameter caused moderate to marked edema and inflammation. After 6 months, white deposits were found on the solder points, presumably from electrolysis. Our experience suggests that placement of Gianturco self-expanding stents may be a useful method of dilating and maintaining the luminal diameter of the prostatic urethra, although care must be taken to select the proper stent size. PMID- 1706139 TI - Eight-month longitudinal study of urinary excretion of albumin and tubular proteins in diabetic subjects. AB - To evaluate the importance of tubular proteinuria in diabetic nephropathy, we studied the serial changes of micro-albuminuria, microproteinuria and protein patterns by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in 38 diabetic patients over 8 months. There was a significant correlation between the amount of micro-albuminuria measured by radio-immunoassay and the amount of microproteinuria quantitated by the Coomassie brilliant blue dye binding method (r = 0.976; p less than 0.0001). Micro-albuminuria of 50 mg/day was equivalent to microproteinuria of 190 mg/day. Among the 38 diabetic patients, 26 had micro-albuminuria above 50 mg/day, while 12 had micro-albuminuria below this level. There was a significant correlation between the amount of microproteinuria and haemoglobin A1, showing that the quantity of microproteinuria was affected by metabolic control. Diabetic patients with micro albuminuria of above 50 mg/day have a significantly higher diastolic blood pressure than those below this level. Among the diabetic patients with micro albuminuria of less than 50 mg/day, the amount of micro-albuminuria and microproteinuria remained constant, whereas progressive increases in micro albuminuria and microproteinuria were observed among the 12 diabetic subjects with micro-albuminuria above 50 mg/day. These support the prognostic importance of this quantity of micro-albuminuria. The protein patterns as revealed by SDS PAGE with Coomassie blue staining show a significant loss of low-molecular-weight proteins in 7 patients, which may therefore suggest tubular damage. The loss of tubular proteins persisted over a period of 8 months in all 7 subjects, and the amount gradually increased over this period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706140 TI - Maternal serum alpha-fetoprotein screening: further consideration of low-volume testing. AB - Unrecognized assay drift that may occur during low-volume (fewer than 500 specimens per week) maternal serum alpha-fetoprotein testing could result in either underestimation or overestimation of the number of pregnant women who are at increased risk of fetal malformations and genetic anomalies. Quality control software programs that incorporate the use of a multirule Shewhart chart are designed to detect assay drift. Careful selection of quality control sera for inclusion in analytic assays and appropriate application of a multirule quality control procedure to values that are obtained on these control materials should detect assay drift, regardless of the volume of patients' specimens in the run. PMID- 1706141 TI - Does unexplained second-trimester (15 to 20 weeks' gestation) maternal serum alpha-fetoprotein elevation presage adverse perinatal outcome? Pitfalls and preliminary studies with late second- and third-trimester maternal serum alpha fetoprotein. AB - Several reports have suggested that persons with an unexplained maternal serum alpha-fetoprotein elevation at 15 to 20 weeks' gestation are at an increased risk for a variety of other pregnancy complications (e.g., preeclampsia) and adverse perinatal outcomes (e.g., fetal death, low-birth-weight infants). However, ascertainment biases could explain some of these reported findings, and predictive value of unexplained elevated maternal serum alpha-fetoprotein levels in the prediction of pregnancy complications seems limited. If elevated second trimester levels were truly predictive of pregnancy complications, we reason that third-trimester levels could prove even more useful. We thus studied late second trimester and early third-trimester (24 to 36 weeks' gestation) maternal serum alpha-fetoprotein levels with the same enzyme immunoassay we use to evaluate routine second-trimester (15 to 20 weeks' gestation) levels. Values rose up to 32 weeks and fell slightly thereafter. Variance was greater than at 15 to 20 weeks but not so great as to preclude clinical usefulness in the third trimester. Of 279 women with a normal (0.4 to 2.49 multiples of the median) maternal serum alpha-fetoprotein value at 15 to 20 weeks' gestation, 270 (96.8%) showed levels in the same range later in gestation; however, none of six singleton pregnancies with unexplained maternal serum alpha-fetoprotein levels greater than 2.50 multiples of the median at 15 to 20 weeks' gestation showed maternal serum alpha fetoprotein levels in this range at 24 to 36 weeks' gestation. The relationship between second- and third-trimester maternal serum alpha-fetoprotein levels in abnormal pregnancies remains to be elucidated in a large sample. Thus we are conducting not only cohort but also cross-sectional studies. Preliminary findings suggest that women with preterm premature rupture of membranes or with premature labor show elevated late second-trimester and early third-trimester maternal serum alpha-fetoprotein levels; however, larger sample sizes are necessary. PMID- 1706142 TI - Effect of diabetes and insulin treatment of diabetic rats on total RNA, poly(A)+ RNA, and mRNA in skeletal muscle. AB - We have assessed the time course of alterations in several biochemical parameters and expression of specific mRNAs in gastrocnemius muscle following both the induction of diabetes and the administration of insulin to diabetic rats. Muscle mass, total RNA, and total protein were reduced, whereas poly(A)+ RNA relative to total RNA was increased following the induction of diabetes. All the above parameters, with the exception of poly(A)+ RNA, were reciprocally and rapidly altered following administration of insulin to 3-day diabetic animals. These changes suggest that during the induction of diabetes 1) total cellular protein is reduced at a rate that is less than the reduction in gastrocnemius mass, whereas RNA is reduced at a rate 1.5 times the reduction in tissue mass, and 2) poly(A)+ RNA is elevated relative to total RNA. After insulin administration, there appears to be coordinate synthesis of both poly(A)+ RNA and ribosomal RNA, assuming 85% of total RNA is ribosomal. Therefore, we conclude that poly(A)+ RNA is more stable than ribosomal RNA during diabetes, whereas the amounts of poly(A)+ RNA and ribosomal RNA are increased at the same rates following insulin administration to diabetic animals. Analysis of expression of specific gene products over the same time course, as assessed by in vitro translation of total RNA followed by two-dimensional gel analysis, suggests that there are a few mRNAs that are very rapidly altered in response to insulin administration. The mRNAs that are altered demonstrate variable temporal patterns of either repression or full or transient expression. These rapid, but limited, alterations in gene expression may prove important in the development of the defects that occur in skeletal muscle in response to diabetes. PMID- 1706143 TI - Characterization of ANF receptors in cultured renal cortical vascular smooth muscle cells. AB - Because atrial natriuretic factor (ANF) has been demonstrated to decrease resistances in cortical renal vessels in vivo, we studied 125I-ANF binding and ANF-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in subcultured vascular smooth muscle cells (VSMC) prepared from the rabbit renal cortex. 125I-ANF specific binding at 4 degrees C represented 70% of total binding and reached a plateau at 30-60 min. Equilibrium saturation binding curves suggested one group of high-affinity receptor sites (KD = 78 +/- 16 pM, Bmax = 45 +/- 11 fmol/mg) but were compatible with several groups exhibiting close binding parameters. ANF, [Ala7,Ala23]ANF (a linear analogue), and C-ANF-(4-23) (a ligand of C receptors) inhibited 125I-ANF binding at 37 degrees C with nearly similar potencies. In contrast, at 4 degrees C, complete or nearly complete inhibition of binding was obtained with ANF and linear ANF, the latter exhibiting the weakest potency, whereas C-ANF-(4-23) displaced only 35% of the tracer. ANF markedly stimulated cGMP accumulation, with a threshold concentration of 10 pM and a stimulation of 115 times basal value at 0.1 microM. Linear ANF was also stimulatory with a much weaker potency. Around 25% of 125I-ANF bound to cell surface was internalized at 37 degrees C. Phenylarsine oxide partially inhibited internalization as well as the inhibitory potency of C-ANF-(4-23) on 125I-ANF binding. As shown by high-performance liquid chromatography extracellular 125I ANF was rapidly degraded at 37 degrees C into its 125I-COOH-terminal tripeptide and 125I-Tyr.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706145 TI - Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver. AB - Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to "oval" cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture. PMID- 1706144 TI - Regulation of endothelin-mediated calcium mobilization in vascular smooth muscle cells by isoproterenol. AB - We have investigated the effect of isoproterenol on endothelin-induced Ca2+ mobilization in A10 vascular smooth muscle cells. Endothelin (ET) stimulates a rapid and sustained elevation of intracellular Ca2+ mediated by production of inositol phosphates, release of intracellular Ca2+, and activation of a plasmalemmal Ca2+ influx pathway. This influx pathway appears to be a L-type channel because it is inhibited by nicardipine and activated by BAY K 8644. Depolarization of the cells, by elevating extracellular K+, activated a pharmacologically similar channel and produced a similar change in intracellular Ca2+ concentration. Preincubation of cells with isoproterenol reduced the peak Ca2+ response to endothelin and blocked the sustained elevation. However, isoproterenol did not alter K(+)-induced Ca2+ entry. Thus it appears that ET induced entry is mediated by intracellular signals and not by depolarization. With the use of cells incubated in Ca2(+)-free medium containing 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, isoproterenol was shown to inhibit Ca2+ release from intracellular pools by 36 +/- 3%. Furthermore, isoproterenol pretreatment or addition of adenosine 3',5'-cyclic monophosphate (cAMP) to saponin-permeabilized cells inhibited inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-induced Ca2+ release from intracellular sites. Similar effects were seen with forskolin. Propranolol reversed the inhibitory effects of isoproterenol. Isoproterenol pretreatment also inhibited the rapid formation of Ins(1,4,5)P3 and [2-3H]inositol 1,3,4,5-tetrakisphosphate stimulated by endothelin and reduced the sustained formation of these compounds. Finally, isoproterenol and forskolin led to a greater than 10-fold increase in intracellular cAMP levels. This stimulation of adenylate cyclase by isoproterenol was completely blocked by propranolol. It appears then that the beta-agonist isoproterenol interacts with a beta-adrenergic receptor, elevates cAMP, and thereby alters endothelin-induced Ca2+ mobilization. Inhibition of Ins(1,4,5)P3 formation, reduction in the responsiveness of the Ins(1,4,5)P3 intracellular receptor, and perhaps inhibition of ET-induced Ca2+ entry appear to be involved. PMID- 1706146 TI - cAMP-activated C1 conductance is expressed in Xenopus oocytes by injection of shark rectal gland mRNA. AB - Development of reliable expression systems for use in identification and functional characterization of proteins required for secretory Cl channel activity is key to understanding the molecular basis of cystic fibrosis (CF). Until now, heterologous expression of epithelial Cl channels had not been accomplished. We show here that Xenopus oocytes express an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl conductance after injection of mRNA from shark rectal gland. Current through this conductance was rapidly activated by intracellular application of cAMP, reversed near the chloride equilibrium potential (ECl), blocked by the Cl channel inhibitor 5-nitro-2-(3 phenylpropylamino) benzoate, and was not affected by preincubation with the intracellular calcium buffer bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N' tetraacetic acid tetraacetoxymethyl ester, a condition that prohibits activation of the endogenous Ca-activated Cl conductance. PMID- 1706147 TI - Separate K+ and Cl- transport pathways are activated for regulatory volume decrease in jejunal villus cells. AB - We assessed ion transport mechanisms operative during regulatory volume decrease (RVD) in jejunal villus enterocytes, isolated in suspension from guinea pig jejunum and examined with electronic cell sizing. Immediately after reduction of osmolarity (153 mosmol/kg medium) enterocytes swelled, but within 5 min they shrank by 50%. This RVD, which was complete by 20 min, was unaffected by Li+ substitution for Na+ or by Na(+)-free (N-methyl-D-glucose, NMDG+) medium. Passive loss of K+ is required for RVD because both the magnitude and direction of RVD changed when external [K+] varied. Increasing K+ permeability with gramicidin (0.5 microM) accelerated RVD in NMDG+ medium (10.0 +/- 0.8 vs. 6.2 +/- 0.4% min 1, P less than 0.01) suggesting that K+ loss is rate limiting for RVD. Inhibition of K(+)- and Ca2(+)-activated K+ conductance with Ba2+ (5 mM, P less than 0.005), quinine (100 microM, P less than 0.005), or apamin (1 microM, P less than 0.005) prevented RVD. Inhibition of Cl- conductance with 9-anthracenecarboxylic acid (100 microM, P less than 0.005) or dipyridamole (75 microM, P less than 0.005) also prevented RVD. In isotonic HCO3(-)-buffered medium, the addition of gramicidin to cells generated conditions in which anion permeability was rate limiting for cell swelling. This swelling was inhibited 97% by 100 microM 4,4' diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In K(+)-free, HCO3(-) buffered medium containing DIDS and gramicidin hypotonic swelling resulted in continued (secondary) swelling (rel vol 1.19 +/- 0.01 vs. 1.25 +/- 0.02, P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706148 TI - Dual pathways for agonist-stimulated arachidonic acid release in pancreatic acini: roles in secretion. AB - The present experiments were performed to determine pathways responsible for arachidonic acid release stimulated by cholecystokinin (CCK) and phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), and the roles of pathways in the secretory response in dispersed acini from guinea pig pancreas. Both CCK octapeptide (CCK-OP) and PMA increased intracellular arachidonic acid. To determine the source of released arachidonic acid, we measured the effects of PMA and CCK-OP on cellular 1,2-diacylglycerol and lysophosphatidylcholine (LPC) and of diglyceride lipase inhibitor RHC 80267 on [3H]arachidonic acid release. Both PMA and CCK-OP increased 1,2-diacylglycerol and LPC. RHC 80267 had no effect on LPC but inhibited the increase in [3H]arachidonic acid release with a concentration of CCK-OP that was maximal for enzyme secretion. The increase in [3H]arachidonic acid release with PMA or a supramaximal concentration of CCK-OP was not inhibited by RHC 80267. In parallel fashion, RHC 80267 inhibited amylase release caused by maximally effective concentrations of CCK-OP but not that caused by PMA or by supramaximally effective concentrations of CCK-OP. Arachidonic acid stimulated amylase release. Exogenous addition of phospholipase A2 caused increases in [3H]arachidonic acid release, LPC formation, and amylase release. The results indicate that there are at least two pathways responsible for the increase in free cellular arachidonic acid stimulated by pancreatic agonists. One is sequential action of phospholipase C and diglyceride lipase on phosphatidylinositol. The other is a phospholipase A action on phosphatidylcholine. The results also suggest a stimulatory role for both pathways in the secretory response. PMID- 1706149 TI - Effect of a purified somatostatin monoclonal antibody and its Fab fragments on gastrin release. AB - The effect of a purified somatostatin monoclonal antibody, SOMA 10, and its Fab fragments on gastrin release have been examined in the isolated perfused rat stomach. SOMA 10 was purified from ascites fluid by ammonium sulfate precipitation followed by hydroxylapatite (HAP) chromatography. This gave a preparation of greater than 90% purity, as assessed by fast affinity and size exclusion high-performance liquid chromatography (HPLC). HAP-purified SOMA 10 had a binding capacity of 760 mmol somatostatin/mol antibody, a dissociation constant of 2.2 nM, and recognized amino acid residues 5-12 of the somatostatin molecule. Fab fragments of SOMA 10 were prepared and purified by protein A affinity chromatography, and the purity assessed by HPLC and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Single passage perfusion of SOMA 10 (100 micrograms/ml) into the gastric vasculature caused a paradoxical decrease in basal gastrin release. However, recirculation of the antibody (20 micrograms/ml) caused an increase in cumulated gastrin release. Perfusion of Fab fragments of SOMA 10 (66 micrograms/ml) also increased gastrin secretion. These results support the suggestion that somatostatin exerts a tonic restraint on gastrin release and that penetration of the antibody to the site of somatostatin release is necessary for it to have an effect. PMID- 1706150 TI - Leu-7 immunoreactivity in human and rat embryonic hearts, with special reference to the development of the conduction tissue. AB - The distribution pattern of Leu-7 (HNK-1) in developing human embryonic hearts and rat hearts was studied by immunohistochemistry. Human and rat embryos at Streeter's stages XIII approximately XX and fetus stage I were used. Leu-7, which is absent in the newborn rat heart, is expressed transiently in the embryo and fetus I stages. The earliest embryonic heart shows two incomplete circular structures with immunoreactivity in the myocardium along the primitive atrioventricular cushion and bulboventricular canal. These two structures become localized topographically in the definitive atrioventricular node and atrioventricular bundle after rearrangement and partial disappearance during embryonic development. At Streeter's stages XVIII approximately XX, Leu-7 immunoreactivity appears to localize topographically in almost all the pathways of the conduction system, although some discontinuities are observed in the atrioventricular junction and atrial internodal tracts. Thereafter, immunoreactivity decreases gradually and differentially by site and stage. The precise nature of Leu-7 immunoreactive cells, that is, whether or not they are neurogenic or myogenic, is not revealed by this study. The present observations are discussed in connection with the hypothesis that specialized ring tissue is the primordium of the conduction system. PMID- 1706151 TI - Neuropeptides and airway submucosal gland secretion. AB - Our study using feline tracheal isolated submucosal gland preparation has revealed that substance P (SP) produces an increase in submucosal gland secretion through the actions of both mucus ejection by glandular contraction and macromolecule secretion from secretory cells, and that the two actions are both mediated by a peripheral cholinergic action. In contrast, SP has no significant effect on macromolecule secretion from secretory cells in tracheal explants, probably because of epithelial suppression. Our study using an isolated gland preparation has also indicated that VIP potentiates mucous glycoprotein secretion induced by cholinergic stimulation through an interaction between muscarinic and VIP receptors in secretory cells. However, VIP failed to induce any significant glandular contraction or relaxation, indicating a lack of VIP receptors or a difference in the subtypes of muscarinic receptors in myoepithelial cells in submucosa glands. PMID- 1706152 TI - Neuropeptides and asthma. AB - Many neuropeptides have recently been identified in human and animal airways. These peptides have potent effects on airway caliber, blood vessels, and secretions, raising the possibility that they may be involved in airway diseases such as asthma. Vasoactive intestinal peptide and peptide histidine methionine are potent bronchodilators and may be neurotransmitters of nonadrenergic bronchodilator nerves. In asthma, if these peptides are broken down more rapidly by enzymes from inflammatory cells, this might contribute to exaggerated bronchial responsiveness. Neuropeptides that are found in sensory nerves, such as substance P, neurokinin A, and calcitonin gene-related peptide, have inflammatory effects and might also contribute to the pathology of asthma if released from sensory nerve endings by an axon reflex. These findings may have important therapeutic implications for the future. PMID- 1706153 TI - A measurement of complement activity using immunoliposome. PMID- 1706154 TI - Fibrinogen-immobilized urokinase demonstrates increased thrombolytic activity in animal experiments. PMID- 1706155 TI - [Ultrastructure of human normal and pathological reassembled collagen fibers]. AB - It is possible, in vitro, to break down human skin then reassemble the collagen fibres of the dermis, essentially type I collagen. The authors have developed a biophysical process for the manufacture of type I autocollagen. They present the electron microscopic study of this collagen. These healthy collagen fibres are compared with the fibres obtained from radiation dermatitis skin and keloid scars. Major ultrastructural differences are demonstrated. PMID- 1706156 TI - ["Chinese hat" flap]. AB - A design of a new type of advancement flap is described and called "chinese hat" flap. It uses the properties of the traditional advancement flap combined with rotation at the two distal edges, that overlap the height of the defect like two claws. This allows direct suture for larger defects with a prominent dog ear that become level with time. Two cases illustrate the procedure. PMID- 1706157 TI - [Objective evaluation of the results of interventions for breast hypertrophy]. AB - It would appear to be impossible to compare completely different techniques of mammaplasty performed in very different clinical situations. However, we thought it would be useful to approach the result of mammaplasties from a more "orthopaedic" point of view. Such an approach distinguishes populations of patients with different results and in whom the postoperative assessment must take into account the result obtained, the residual scars and the final breast shape. A formula has been developed after multiple attempts: [formula: see text] This formula expresses positive factors: the quantity of glandular tissue removed (in grams), the final appearance of the scars, the appearance of the overall shape of the two reconstructed breasts and the reappearance of ptosis measured from the edges of the breast below the inframammary sulcus (in the erect position). In this way, it is possible to express these positive factors by the multiplying the scores attributed to each of the factors. For example, a resection of 150 g will be scored as 1.5 and a resection of 1,200 g will be scored as 12, i.e. the weight in grams is simply divided by 100 to give the score for weight. The scar will be scored according to an individual scale of 1 to 5. The shape will also be scored according to an individual scale of 1 to 5. The division factors include the length of the inframammary scar (segment 3); this inframammary vertical line will be included directly in the calculation. In contrast, the length of the horizontal inframammary scar will be divided by 10.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706158 TI - [Tuberous breast: clinical and therapeutic considerations. Report of 20 cases]. AB - Tuberous breast is a so-called mammary deformity which associates: deficient and contracted base, enlarged nipple-areolar complex, glandular herniation through the areola. The parenchyma is reduced, cylindrical, asymmetric, and frequently posted. Surgery reveals a basic glandular ring. The gland passes through this ring like a "glove's finger", revealing the herniation. All the cases are very different, but the surgical correction is common: concentric skin excision for areolar reduction. "V" vertical excision, allowing gland dissection. Via the skin wedge of the "V", extended dissection is carried out and half of the breast is denuded. The inferior mammary sulcus is released. The basal ring is cut and the posterior breast tissue is radially incised to expanding base, the parenchyma is stretched out. Correction of volume abnormalities: augmentation (implant), posterior reduction of symmetry. Correction of ptosis by skin redraping over the new mammary shape. PMID- 1706159 TI - [Indications and limitations of breast lifting by peri-areolar approach (round block). Report of 48 cases]. AB - The prevention of spreading of the areolar nipple complex by means of the round block technique is the origin for this review of 48 cases. After the description of the pre-operative markings and the operative technique, the author gives full details about operative indications. The latter are in order: moderate ptosis with or without the need ty for a mammary implant, replacing a mammary implant with capsulectomy and recentering of the areola. Limitations appear in some cases when 11-12 centimeters of de-epithelialization in diameter are necessary. Advantages and drawbacks are discussed. The elimination of the usual thoracic scars appears to be a major and decisive advantage in the choice of this technique which, according to the author, will play an important part in the future. PMID- 1706160 TI - [Prospective study of the specific liability of the plastic surgeon]. AB - The authors of the article first pose this question: might plastic surgery not fit into the general principles that guide any medical procedure? The general principles, especially as regards the distinction between liabilities in terms of means and liabilities in terms of results, are discussed in the first part of the survey. The authors then demonstrate that the plastic surgeon does differ from other surgeons: he has to comply with the same restraints as any surgeon. However, the survey then presents certain aspects specific to this branch of surgery, and their implications in terms of responsibilities. The article successively discusses: the patient's freedom of choice, the need for particularly thorough information and its form, the limits to the practitioner's freedom in choosing the technique, and most importantly the so-called obligation to avoid (which the authors study in particular detail in the field of analysis and responsibilities) of obviously useless operations. The authors, after recalling important recent legal precedents, come to the conclusion that it is necessary to establish an institution: "the medical ombundsman" defined by a bill in the 1988 sitting of the French Parliament. PMID- 1706161 TI - [Approach to the clivus via maxillary bipartition. Report of a case of chordoma]. AB - Large tumors of the clivus require a combination of several approaches. We review the technique previously described; they cross numerous anatomical structures and include a transcranial approach. We propose to combine transoral and transrhino septal approaches by means of maxillary bipartition which allows good exposure and an easy removal in a case of chordoma of the clivus. PMID- 1706162 TI - [The submental island skin flap. A surgical protocol. Prospects of use]. AB - The authors report their use of a previously undescribed skin flap situated in the submental region and vascularised by the submental artery derived from the facial artery. It is possible to raise flaps as large as 11 cm X 6 cm with an identical colour to that of the face. The pedicle flap can reach any part of the homolateral oral cavity or the lower 2/3 of the homolateral side of the face (with the exception of the nasal region). The free flap, using a good calibre pedicle (including facial arteries and veins, may be as long as 7 cm and appears to be extremely reliable. It is also possible to use a distal or composite pedicle taking a segment of the internal basilar margin. The donor site scar dissimulated under the mandible is perfectly acceptable. PMID- 1706163 TI - [Radical surgical techniques for pectus excavatum]. AB - Conservative techniques with insertion of a subcutaneous implant have the advantage of being simple to perform but the frequent disadvantage of a poor result. The authors present two more radical techniques used in chest surgery: modelling osteochondroplasty and resection of the deformed sternocostal plate and replacement by a prosthesis. The indications are described together with the very satisfactory results with no major complications obtained in a series of 13 cases. PMID- 1706164 TI - [Dermography: a complement to esthetic surgery]. AB - Dermography (intra-dermal dye implants) is the medical application of ancestral tattooing. This technique allows very easy correction of dyschromic cutaneous lesions, and is very helpful in surgical iatrogenic sequelae. It presents a larger field of medical or esthetic applications. PMID- 1706165 TI - [Heat-moulded plastic splint for the nose]. AB - The authors present a very simple and very comfortable splinting device for use after rhinoplasty. It consists of a splint made from malleable plastic which can be moulded by soaking in water at 60 degrees C, allowing perfect modelling to the shape of the nose. This splint is held in place by means of Velcro bands which offer remarkable flexibility of use. PMID- 1706166 TI - [Inner canthus--anatomical junction. A surgical entity]. AB - The medial canthus is a surgical entity. The anatomical features are defined from a dissection of eight orbital regions. The pathways of carcinological invasion and the anatomical barriers are considered. The conclusions are discussed in the light of the data reported in the literature. PMID- 1706167 TI - [Recurrent periprosthetic capsular retraction under the pectoralis major muscle. Treatment, 2 unusual clinical cases]. AB - Capsular contracture around breast prostheses still represents a challenge, in spite of the numerous papers published on this topic. The etiology and pathogenesis of this condition are perplexing resulting in considerable controversy concerning the rationale for surgical treatment. The authors report two cases of recurrent disease: one patient with recurrent contracture after breast augmentation, the other patient with recurrence after breast reconstruction. Both patients were operated according to an original technique which consists in a square-shaped incision of the pectoralis major muscle via an inframammary approach. This surgical technique allows the prosthesis to sink into deeper layers leading to a better shape of the breast and providing satisfactory skin cover of the prosthesis. PMID- 1706168 TI - [Esthetic surgery according to the code of deontology]. PMID- 1706169 TI - [Analysis of sequelae of the latissimus dorsi flap removal. Report of 44 cases reviewed and tested]. AB - Since Tanzini, the latissimus dorsi muscle flap has been widely used in plastic surgery. Based on the experience of two plastic surgery units, we decided to try to define the sequelae of this operation. In order to simplify our analysis we only considered free flaps. Out study is based on 42 patients (26 pure muscular flaps and 16 musculo-cutaneous flaps). The sequelae were analysed in terms of aesthetic and functional criteria. The aesthetic sequelae appeared to be minima in the case of pure muscular flaps, but more severe in the case of musculo cutaneous flaps. Functional sequelae in the shoulder were observed on muscle testing in 30% of cases, although there were no repercussions on sport or work activities. Analysis of spinal posture demonstrated a modification in the frontal plane in 40% of cases although this could not be clearly attributed to the donor site. On the basis of this study, we can conclude that the latissimus dorsi flap retains an important place in the therapeutic arsenal of plastic surgery due to its reliability and its minor cicatricial and functional sequelae at the donor site. PMID- 1706170 TI - [The examination of DNA strand breaks induced by peplomycin-using non-radioactive in situ nick translation method]. AB - In situ nick translation (ISNT) is a method to detect DNA single strand break (nick) at each cellular level. In this study, peplomycin (PEP)-induced DNA strand breaks and its repair were examined using non-radioactive ISNT method. Human fibroblasts were cultured with various concentrations of PEP for different durations, and then DNA strand breaks were evaluated by ISNT method. The signal intensity of DNA strand breaks of the PEP-treated fibroblasts was increased in a dose dependent manner. At the fixed concentration of PEP, it reached to a maximum level after 10 min. of culture and remained unchanged for at least 24 hours. In addition, DNA strand breaks induced by PEP was rapidly repaired in 10 min. after washing. Thus, the non-radioactive ISNT is thought to be a quick, sensitive and specific method not only to detect DNA strand breaks but also to observe its repair. PMID- 1706171 TI - [Timing of administration of granulocyte colony stimulating factor after cytotoxic chemotherapy in hematological malignancies]. AB - We evaluated the effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) given to 30 patients with hematological malignancies after cytotoxic chemotherapy. The first course of chemotherapy was not treated with rhG CSF (control), and in the second to fourth courses, rhG-CSF was given by one of the following three ways to the patients (not necessarily in this order): 1) 10 days of administration starting 48 hr after chemotherapy, 2) 5 days of administration starting 48 hr after chemotherapy, and 3) 5 days of administration after the leukocyte counts reached to less than 2,000/microliters. The leukocyte nadirs were significantly higher in the course with 10 days of administration compared with the control course. The time needed for recovery from the leukocyte nadir was significantly shorter in 10-day course and 5-day course after the leukocyte counts reached to less than 2,000/microliters. The therapy spans became significantly shorter with all of the three patterns of administration of rhG-CSF compared with the control course. The number of days on which the leukocyte counts became less than 2,000/microliters were significantly fewer in 10-day course and 5-day course after the leukocyte counts became less than 2,000/microliters. These findings showed that rhG-CSF prevented severe neutropenia after cytotoxic chemotherapy, and (or) assisted the rapid recovery from neutropenia. These effects depend on the timing of its administration. PMID- 1706172 TI - [Evaluation of prognostic nutritional index in chemotherapy of lung cancer]. PMID- 1706173 TI - Reversion by calcium but not by Bay K 8644 of neuromuscular blockade induced by nicardipine. AB - We compared the effects of nicardipine on indirectly and directly elicited twitches in phrenic-hemidiaphragm preparations from rats, and the effects of calcium and Bay K 8644 on nicardipine-induced neuromuscular blockade. Nicardipine (5-250 microM) decreased both directly and indirectly elicited muscular contractions in a concentration-dependent way. The inhibitory effect was greater when the preparation was indirectly stimulated. Nicardipine antagonism of indirectly elicited contractions was reversed in an apparently competitive way by calcium (1-4 mM). In contrast, the calcium channel agonist Bay K 8644 (0.1 and 1 microM) had no effects per se, nor did it reverse neuromuscular blockade induced by nicardipine. Interestingly, Bay K 8644, at 10 microM, decreased the height of indirectly elicited muscular contractions. These results suggest that the calcium antagonist nicardipine affects neuromuscular transmission to a greater degree than muscular contraction, this effect being antagonized by calcium. In contrast, the calcium agonist Bay K 8644 was unable to produce any effect as stimulator of calcium channels at the neuromuscular level, indicating that this drug may exhibit tissue specificity. PMID- 1706174 TI - Saliva composition in asthmatic patients after treatment with two dose levels of a beta 2-adrenoceptor agonist. AB - Changes are known to occur in the salivary composition of asthmatic patients treated with beta 2-adrenoceptor agonists. To evaluate the precise contribution of the agonist to the impaired saliva secretion, 15 asthmatic patients, 15-23 yr old, were given two dose levels of agonist, either terbutaline or salbutamol. The lower dose, 0.15-3.0 mg/day, represented the therapeutic level used by the patients. During a wash-out period of one month, the asthma was treated with budesonide, a corticosteroid spray. Then a daily dose of 32 mg of terbutaline or salbutamol was given for one month. Samples of whole saliva, stimulated by chewing, and parotid saliva, stimulated by citric acid, were collected on three occasions: (1) at the end of the low-dose agonist treatment; (2) at the end of the wash-out period; and (3) at the end of the high-dose agonist treatment. During the high dosing the secretion rate of parotid saliva decreased and the concentrations of its total protein, amylase, hexosamine and the ratio of hexosamine/total protein were lowered. The output per minute of total protein, amylase, hexosamine, peroxidase, lysozyme, secretory IgA and potassium decreased. There were only small differences in secretion rates or saliva composition between samples collected at the end of the low-dose and at the end of the wash out period. Thus, treatment with beta 2-adrenoceptor agonists impairs saliva secretion in asthmatics. PMID- 1706175 TI - Effects of 5-fluorouracil on protein synthesis and secretion of the rat parotid gland. AB - 100 mg/kg of FU were injected intraperitoneally once daily for three days. Animals were anaesthetized with 50 mg/kg of sodium pentobarbital before cannulation of the parotid duct. The total volume, amylase and protein content of the saliva were determined after stimulation with either 5 mg/kg pilocarpine or 5 mg/kg isoproterenol in FU-treated, pair-fed, and control animals. Saliva from FU treated animals was significantly lower (p less than 0.05) in volume, amylase and protein content than that of both control groups. SDS, anionic and cationic gel electrophoresis of parotid saliva revealed no qualitative changes in the types of proteins secreted. FU reduced the total glandular amylase per unit DNA in both unstimulated and isoproterenol-stimulated parotids (p less than 0.05). Decreased protein synthesis may be the mechanism underlying the depleted secretory protein stores because the contents of isolated secretory granules from experimental glands contained less radiolabelled protein than those of either control group, and whole-gland homogenates had marked reductions in the activities of three lysosomal enzymes and in total RNA content. The secretory granules of experimental animals contained less labelled protein than those of controls, but experimental animals secreted a greater proportion of their total glandular radiolabelled secretory protein into saliva relative to amylase, suggesting that newly synthesized secretory proteins were preferentially secreted. PMID- 1706176 TI - Orbital carcinoid tumor. PMID- 1706177 TI - A prospective study of acute central retinal artery obstruction. The incidence of secondary ocular neovascularization. AB - We conducted a prospective study to determine the incidence of ocular neo vascularization following acute central retinal artery obstruction. Only patients initially evaluated within 7 days of visual loss were eligible. Any patient with pre-existing ocular neovascularization or clinical evidence of the ocular ischemic syndrome noted at the initial evaluation was excluded. During the 18 month study, 33 consecutive patients were enrolled. Six patients subsequently developed neovascularization of the iris, an incidence of 18.2%. In these six patients, neovascularization of the iris appeared as early as 12 days to as late as 15 weeks following the artery obstructions. Five of the six patients (15.2% of the total) later developed neovascular glaucoma. Another patient in this series developed neovascularization of the optic disc without neovascularization of the iris, an incidence of 3.0%. Only two of the seven patients with ocular neovascularization had ipsilateral hemodynamically significant carotid artery disease as determined by noninvasive carotid artery testing. This study confirms results of previous retrospective studies that the incidence of ocular neovascularization after central retinal artery obstruction is higher than commonly thought. It also shows that, in the majority of cases, carotid artery disease is not responsible for the neovascularization seen after central retinal artery obstruction. PMID- 1706178 TI - Capillary neoformation in the rat heart--stereological studies on papillary muscles in hypertrophy and physiologic growth. AB - Stereological investigations on myocardial capillaries provided evidence that the common estimator of capillarity, the capillary density (i.e., number of capillary profiles per unit transverse sectional area), underestimates the true capillary supply since the capillary axes are not oriented perfectly in parallel to the myofiber axes. Recently, we studied the "true" capillarity, i.e., the length density of capillaries (LV = capillary length per capillary volume), in some experimental models of cardiac hypertrophy which have been published elsewhere. It has been shown that LV decreases in renovascular hypertension, but is maintained in physical exercise and after chronic thyroxin application. However, the growth pattern of capillaries in hypertrophic hearts has not yet been analyzed. In the present paper it is demonstrated that important information on the capillary network can be derived from the two-dimensional capillary-to-fiber ratios (2D CFR: capillary profiles per myofiber profiles in transverse sections) and from the three-dimensional capillary-to-fiber ratios (3D CFR: capillary length per unit myofiber length). Increase in both suggests neoformation of additional capillary branches in parallel connection. Retrospective analysis of the quantitative data indicates that in hypertrophy induced by physical exercise or by chronic thyroxin application capillary neoformation in parallel connection counterbalances increase of oxygen diffusion distance due to myofiber enlargement. In renovascular hypertension, capillary neoformation in parallel connection does not occur. Studies on normal growth indicated both a slight decrease of LV of capillaries, as well as a continuous neoformation of additional capillary branches. PMID- 1706179 TI - Calcium metabolism and depressed contractility in isolated human and porcine heart muscle. AB - Contractility is often depressed in isolated heart muscle. To analyze this phenomenon, we measured the derivative of left ventricular pressure (dP/dt) in intact and in isolated, blood perfused pig hearts, and peak force (F) or stress (F/mm2) in ventricular trabeculae of man and pig. When the heart was in the steady state at a priming frequency of 2 Hz an extrasystolic interval of 0.3 s was interposed, followed by four postextrasystolic intervals of 0.8 s. In the case of isolated trabeculae the priming frequency was 0.2 Hz, the extra interval 0.4 s, and the post-extrasystolic intervals were 5 s. The exponential decay of potentiation is characterized by the constant D: a low value of D indicates a rapid decay of potentiation. DP/dt was about 1000 mm Hg/s in the intact hearts, but within 1 h after isolation dP/dt decreased to about 700 mm Hg/s, and this was associated with a decrease in D from 0.63 to 0.40. Developed stress in the isolated trabeculae was about 2 mN/mm2 and D was about 0.20 under standard, in vitro conditions (a.o. 1.5 mM Ca2+. 0.2 Hz stimulus frequency). This stress is only 10% of the calculated stress in the intact heart. An increase of priming frequency, or of [Ca2+], or addition of 30 nM isoproterenol to the perfusate caused a marked increase in F and D. Properties of human and porcine trabeculae were quantitatively similar. The strong correlation between dP/dt, or F, and D suggests a causal relationship. This is consistent with the current model of e-c coupling in heart muscle, in which the activity of the Ca2+ pump of the sarcoplasmic reticulum determines the decay of potentiation and the amount of releasable Ca2+ in the reticulum determines force of contraction. Since isoproterenol stimulates the Ca2+ pump in the reticulum, the increase in D and F induced by this drug is consistent with the model. We conclude, that the decreased dP/dt, F, and D in isolated preparations was due to impaired sarcoplasmic reticulum function. The role of this phenomenon in the stunned heart syndrome, species differences and possible causes are discussed. PMID- 1706180 TI - Tumor necrosis factor-alpha increases hepatic DNA and RNA and hepatocyte mitosis. AB - Tumor necrosis factor-alpha (TNF) has been reported to increase DNA synthesis in normal rat liver. Therefore, we examined the effects of TNF on rat liver regeneration. TNF, 1.5 micrograms ip every 4 h for 5 d, significantly increased hepatic DNA and RNA contents of regenerating and sham operated livers by up to 45%. Mitotic figures in sham operated liver, usually rare, were increased substantially by TNF. ODC mRNA content and enzyme activity were increased in regenerating liver, and were further increased by TNF. These data indicate that TNF, although not specific for regenerating liver, is a potent stimulus for hepatocyte DNA synthesis and mitosis. PMID- 1706181 TI - The use of audiocassette recordings for patient education. PMID- 1706182 TI - Electroconvulsive shock produces large increases in interstitial concentrations of dopamine in the rat striatum: an in vivo microdialysis study. AB - This study examined the effects of electroconvulsive shock (ECS) on interstitial concentrations of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the serotonin metabolite 5 hydroxyindoleacetic acid (5-HIAA), and the purine metabolite uric acid, in the striatum using on-line microdialysis in freely moving rats. Interstitial striatal DA increased to 1310% of baseline when the ECS was administered 18 to 24 hours after implantation of the dialysis probe. DOPAC (+ 19%), HVA (+ 30%), 5-HIAA (+10%), and uric acid (+111%) were increased to a smaller extent. The ECS-induced increase in DA was derived from a Ca++ sensitive pool since perfusion of a modified solution in which Ca++ had been replaced with Mg++ blocked this effect. PMID- 1706183 TI - Pharmacologic significance of the structural heterogeneity of the GABAA receptor chloride ion channel complex. AB - Gamma-Aminobutyric acidA (GABAA) receptors are heterooligomeric proteins with an apparent high degree of variability in the specific assembly of their component subunits. Although the precise nucleotide and deduced amino acid sequences of many of the various GABAA receptor subunits are known, the exact quaternary structures of the native receptors are unknown. Recombinant expression of receptors with different combinations of subunits produces a variety of structurally different receptors with varying Cl- channel function and sensitivities to modulation by drugs such as benzodiazepines. Differences in the regional distribution of GABAA receptor subtypes in brain, coupled with the observed differences in the relative affinities of various anxiolytic and hypnotic drugs among these receptor subtypes, suggests a new strategy for drug development that is the targeting of drugs to specific subpopulations of GABAA receptors. This is a review of the recent striking progress in understanding the heterogeneity of the GABAA receptors and its possible significance. PMID- 1706184 TI - CD11a/CD18 (LFA-1) epitopes involved in syncytium formation among CD4+ T-cells following cell free HIV-1 infection. AB - Like particular monoclonal antibodies to CD4, monoclonal antibodies to epitopes localized at the top of the alpha-subunit (CD11a) and at the stem of the beta subunit (CD18) of the lymphocyte function associated antigen-1 (LFA-1) block syncytium formation when added to CD4+ T-cells before or shortly after (5 min) cell free HIV-1 is added. This indicates involvement of LFA-1 epitopes in the early stage of cell-free infection. Syncytium formation is still blocked by CD4 antibodies when added two hours after virus adsorption, CD11a and CD18 antibodies are ineffective at this late stage. Radio-immune precipitation experiments in acutely infected T-cells with the appropriate CD11a and CD18 antibodies suggested a link between CD18 and CD4 in the cell lines studied, possibly mediated by the CD3/TCR complex. In chronically infected cells, where little CD4 is expressed, LFA-1 antibodies still precipitate the viral envelope, pointing to a direct LFA-1 envelope interaction in that late stage of infection. Our results indicate that a limited number of LFA-1 epitopes is involved in syncytium formation among T cells, none of which is required for virus entry in the cell or virus spread in the culture. PMID- 1706185 TI - Prolactin-deficient GH3B3 cells are defective in the utilization of the endogenous prolactin promoter yet are fully competent to initiate transcription from a transfected prolactin promoter. AB - Transcription of the prolactin (PRL) gene has been analyzed in wild-type D6, PRL deficient B3, and revertant r16 GH3 cells. Levels of processed nuclear transcripts from the PRL gene were substantially reduced in the deficient line compared to wild-type cells and returned to greater than wild-type levels in the revertant line. Rare PRL transcripts in the deficient line contained the same 5' end found on transcripts in wild-type and revertant cells as judged by primer extension and S1 nuclease protection assays, implying that the cells are deficient in utilization of the normal wild-type promoter. Deficient cells also contained wild-type levels of the PRL- and growth hormone-specific transcription factor pit-1/GHF-1, and no difference was found in the ability of extracts from wild-type and deficient cells to retard various restriction fragments from both the proximal and the distal PRL promoter regions. The deficient and wild-type cells were equally competent in initiating transcription from a transfected rat PRL promoter containing both the distal and proximal promoter elements. These observations imply that PRL-deficient cells are not defective in a trans activating factor functioning on these PRL promoter fragments (trans model). Rather, inefficient use of the PRL promoter in the variant cells may reflect an increased methylation state of the PRL gene itself (cis model). PMID- 1706186 TI - The use of UV light as a cross-linking agent for cells and tissue sections in in situ hybridization. AB - UV cross-linking is introduced as a novel method to stabilize tissue on microscopic slides and to immobilize target molecules in biological material for in situ hybridization. UV illumination dramatically improves the stability of tissue sections and isolated cells on slides coated with gelatin/poly-lysine. Even during prolonged high-stringency washes, specimens remain firmly attached to the support layer. At the same time, the signal intensity is increased significantly whereas background levels remain as low as without UV illumination. These results indicate that while target RNAs and other molecules in the biological material are covalently cross-linked with their nearest neighbor molecules, tissues nonetheless remain penetrable, and target molecules remain accessible. We expect that this simple and efficient technique will find widespread applications in in situ hybridization methodology. PMID- 1706187 TI - Rat vasopressin and oxytocin genes are linked by a long interspersed repeated DNA element (LINE): sequence and transcriptional analysis of LINE. AB - Sequence analysis of the rat vasopressin and oxytocin gene family reveals that the two genes are linked by a long interspersed repeated DNA element (LINE) giving rise to seven long open reading frames encoding hypothetical proteins of 99 to 556 amino acid residues. Furthermore, although both DNA strands of LINEs serve as templates for transcription, transcripts initiated at the 3' end are more abundant than those started from the 5' end. The LINEs are transcribed preferentially in brain tissues as analyzed by Northern blot, in situ hybridization, and RNase protection experiments. The data show that most LINEs are transcribed at their entire length and that a major fraction of respective RNAs does not enter the cytoplasm but remains in the cell nucleus. PMID- 1706188 TI - Conjugates of synthetic lymphocyte-activating lipopeptides with segments from HIV proteins induce protein-specific antibody formation. AB - Lipopeptide analogues of bacterial lipoprotein activate macrophages and B lymphocytes. The products formed by coupling these lipopeptides to low molecular mass antigens can be used to induce antigen-specific antibodies in mice. In the present work, it is shown that HIV-1 gp160-derived synthetic oligopeptides coupled to the synthetic lipodipeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS) propyl]-(R)-cysteinyl-s eryl- serine (P3CSS) induce peptide-specific antibodies in mice without adding further adjuvants. Depending on the peptides applied, the conjugates exhibited different lymphocyte stimulatory activity, immunoglobulin isotype patterns, and boost reactions; lipopeptide conjugates inducing a pronounced secondary immune response are considered to possess both B- and T-cell epitopes. Antibodies induced by the lipopeptide-HIV-1-peptide conjugates were also reactive against the recombinant gp160 of HIV-1. PMID- 1706189 TI - Oxidant effects on rat and human lung proteinase inhibitors. AB - This project tested the hypothesis that inhaled oxidants could cause lung damage by inactivating the proteinase inhibitors that normally protect the lung from proteolysis. Rat alpha-1-proteinase inhibitor (alpha 1-PI)2 was purified from blood plasma, and antibodies to this inhibitor were prepared. The activity of alpha 1-PI in lung lavage fluids from rats was measured by elastase inhibition, and the immunological concentration of alpha 1-PI was quantified in an enzyme linked immunoassay. The ratio of the amount of active alpha 1-PI relative to its immunological concentration was examined as a measure of the inhibitor's functional activity. This ratio and the ratio of the immunological concentration of alpha 1-PI to the total protein concentration were determined in lung lavage fluids from rats exposed to air, 10 parts per million (ppm) nitrogen dioxide, and diesel emissions (3.5 mg/m3 particulates) for 12, 18, and 24 months. Only diesel exposures resulted in a statistically significant reduction in the functional activity of alpha 1-PI of 30 percent (p less than 0.05). Similar studies were performed on rats exposed to nitrogen dioxide (0.5 ppm background with peaks of 1.5 ppm) and ozone (0.06 ppm background with peaks of 0.25 ppm) for 12 and 18 months. No statistically significant effects were observed in the functional activity of alpha 1-PI or its immunological concentration. In other protocols, rats were acutely exposed to 0.8 ppm or 1.2 ppm ozone for two, four, or eight hours, and to 0.5 ppm or 0.8 ppm ozone in conjunction with 8 percent carbon dioxide for two or seven hours. Although these acute exposure conditions did not reduce the functional activity of alpha 1-PI, the immunological concentration of alpha 1-PI and the elastase inhibitory activity, relative to other proteins, were significantly increased in relation to the total amount of ozone inhaled. The functional activity of alpha 1-PI also was measured in the bronchoalveolar lavage fluids of human subjects exposed to nitrogen dioxide (0.05 ppm with 2 ppm peaks, or to 1.5 ppm continuously) for three hours and to ozone (0.4 ppm) for two hours during exercise. These exposures did not result in significant changes in the functional activity of alpha 1-PI or its immunological concentration.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706190 TI - Detection of bacterial mRNA using polymerase chain reaction. AB - A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern blot hybridization. Detection of mRNAs by reverse transcription-PCR Southern blot analysis is orders of magnitude more sensitive than Northern blot hybridization. PMID- 1706191 TI - [Palliative treatment of malignant neoplasms of the upper aerodigestive tract]. AB - We present a syntheses of the most important local and regional palliative measures in the treatment of the malignant tumors of the upper respiratory and digestive tract. We have considered the palliative therapeutic directed to the tumor itself, the maintenance of the air-flow and the nutrition, the treatment of the pain, of the hemorrhage, of the infection and of the alterations of the loco and regional trophism. In relation to palliative treatment directed at the tumor itself, we have analyzed the role of the chirurgie, the physiotherapy and chemotherapy. PMID- 1706192 TI - Simultaneous isolation of total cellular RNA and DNA from tissue culture cells using phenol and lithium chloride. AB - A rapid procedure for the isolation of intact total cellular RNA from cultured cells is described. This method combines the simultaneous disruption of cells and extraction of nucleic acids in a single step with the use of phenol and a buffer containing 100 mM LiCl. Total cellular RNA can be isolated in approximately 2 hours. The yield and quality of the RNA is comparable to the more widely employed methods requiring extensive preparatory steps such as extraction using guanidinium thiocyanate and subsequent CsCl gradient centrifugation. The RNA isolated using our procedure contains transcripts up to 10 kb in length and is suitable for Northern analysis. This procedure also yields high-molecular-weight DNA, which is a suitable substrate for restriction endonucleases. PMID- 1706193 TI - Acquired immunodeficiency syndrome (AIDS)--data as at 31 January 1991. PMID- 1706194 TI - Purification and characterisation of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody 83D4. AB - Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins. PMID- 1706195 TI - Endothelial leucocyte adhesion molecule-1 (ELAM-1) expression in cutaneous inflammation. AB - Endothelial leucocyte adhesion molecule-1 (ELAM-1) is a recently described endothelial surface glycoprotein which is inducible by interleukin 1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) or bacterial lipopolysaccharide (LPS). Using an immunohistochemical technique and a monoclonal antibody (1.2B6) specific for ELAM-1 we have found marked vascular endothelial expression of ELAM-1 in many cutaneous inflammatory disorders, including allergic contact dermatitis, atopic dermatitis and psoriasis, and in dermal infiltrates associated with benign, premalignant and malignant keratinocyte proliferation. In normal skin, minimal levels of ELAM-1 expression were detected. In psoriasis, double-immunoenzyme staining studies revealed a close spatial relationship between ELAM-1 expression and neutrophil margination, suggesting a functional link. Recombinant human interferon-gamma (30 micrograms) injected intradermally in normal adult human volunteers did not substantially upregulate ELAM-1 in contrast to its marked effect on intercellular adhesion molecule-1 (ICAM-1) expression, indicating that this cytokine is probably not involved in ELAM-1 induction in vivo. These results indicate that ELAM-1 is widely induced in cutaneous inflammation with a time course of expression that is longer than that observed in vitro. As ELAM-1 acts as an adhesion ligand for neutrophils, and perhaps monocytes, the expression of this molecule in cutaneous lesions is likely to be an indication of the ability of vascular endothelium to recruit these cells from the circulation. Furthermore, the cytokine inducibility of ELAM-1 is indirect evidence for functional interactions between perivascular mononuclear cells, other resident cells and the blood vessel wall. PMID- 1706196 TI - Absence of phosphatidylinositol (PI)-linked proteins in a very early human multipotential haematopoietic marrow cell. AB - A very early human haematopoietic progenitor cell population which was negative for the major histocompatibility class II antigen (HLA-DR) and positive for the CD34 (MY10) antigen was separated into two subsets according to the expression of decay-accelerating factor (DAF) on the cell surface. Using immunoadherence, cell cycle analysis, and cell culture, we determined that there is a DAF- multipotential cell and a more differentiated DAF+ lineage specific progenitor cell existing in human bone marrow. The DAF- subset was highly enriched for CFU GEMM, while the DAF+ subset contained only BFU-E and CFU-GM. The DAF- subset was approximately 0.03% and the DAF+ subset approximately 0.008% of the original bone marrow population. MIRL (membrane-inhibitory of reactive lysis), another PI linked protein, was not expressed on the DAF- population but was expressed on the DAF+ cells. These observations indicate that PI-linked proteins are absent from the multipotential stem cell but are present on an early lineage specific cell. The absence of expression of PI-linked proteins can be used to further isolate and characterize a very early multipotential haematopoietic progenitor cell population. PMID- 1706197 TI - Immunophenotypic heterogeneity of multiple myeloma: influence on the biology and clinical course of the disease. Castellano-Leones (Spain) Cooperative Group for the Study of Monoclonal Gammopathies. AB - In 112 untreated myeloma patients we have analysed the immunophenotype of plasma cells both by immunofluorescence (IF) and immunocytochemistry (APAAP). Both techniques yielded similar results pointing to an important degree of heterogeneity in antigenic expression not only between different patients but also within the same patient. The expression of CD38 and Han-PC1 antigens (Ags) was almost constant (greater than 90% positive cases), while CD9 was detected in 66% of the cases. On the other hand, less than one third of patients were positive for CD10, CD20 and HLA-DR and generally with a weak expression (less than 30% positive plasma cells). In occasional cases plasma cells were weakly positive for the myelomonocytic markers CD13 (9%), CD15 (25%) and CD14 (6%). The possibility that this heterogeneity might be the result of different stages of differentiation of the neoplastic clone is suggested both by the positive correlation in the expression of some of these antigens (CD10, CD9, CD20, HLA-DR) and by the relationship between CD10 and myeloid antigens with immature plasma cell morphology. Finally, the cALLA antigen does not seem to be of significant value in predicting survival. Moreover, none of the other markers explored showed a clear influence in the course of the disease, although the tendency towards a lower survival found for the CD20+ cases as well as the association of the expression of some antigens and advanced clinical stage, may warrant further studies in a larger series of patients. PMID- 1706198 TI - Differential effect of G-CSF and GM-CSF in acquired chronic neutropenia. PMID- 1706199 TI - Improved storage methods for epikeratoplasty. AB - A major variable in calculating the necessary curvatures and dimensions of the refractive lenticules for epikeratoplasty is the degree of swelling of the donor cornea during storage prior to lathing. The purpose of this study was to determine whether it is possible to restore donor corneas to their in vivo thickness using osmotic agents rather than physical means. The osmotic agents chosen were dextran and chondroitin sulfate. Donor porcine corneas stored in McCarey-Kaufman (MK) medium for seven days increased in thickness by almost 50%. Increasing concentrations of dextran and chondroitin sulfate, and increasing concentrations of dextran with 1.35% chondroitin sulfate added, were examined for their ability to reduce this thickness to normal levels. It was found that 15.00% dextran alone, and 10.00% dextran with 1.35% chondroitin sulfate, reduced corneal thickness to that of fresh tissue. Light and electron microscopy showed that the osmotic agents restored stromal ultrastructure to that of fresh tissue. The use of osmotic agents to reduce corneal thickness prior to lathing reduces physical deformation of the donor tissue associated with the use of a corneal press and may enhance the predictability of epikeratoplasty. PMID- 1706200 TI - [The role of gamma delta T cells in infectious disease]. AB - There have been several lines of evidence that at least significant fraction of gamma delta--T cells are specialized to recognize epitopes on mycobacterial antigens including 65 KD heat shock protein(HSP). Since HSP is known to be highly conserved in amino acid sequences from prokaryotes to eukaryotes, it is possible that the HSP--specific gamma delta--T cells may recognize the endogenous HSP expressed by autologous cells. The broad--reactive gamma delta--T cells may be responsible for protection during the period between an early stage covered mainly by phagocytes and a late stage covered by typical immunities in terms of the time sequence after microbial invasions. PMID- 1706201 TI - [NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]. AB - We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK sensitive and allospecific targets are also distinct, since killing of NK sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions. PMID- 1706202 TI - Growth inhibition of Plasmodium falciparum in in vitro cultures by selective action of tryptophan-N-formylated gramicidin incorporated in lipid vesicles. AB - We studied the differential effect of tryptophan-N-formylated gramicidin on uninfected and Plasmodium falciparum-infected erythrocytes. Trp-N-formylated gramicidin induces a much faster leakage of K+ from infected cells than from uninfected cell whereas, and at an even lower concentration, gramicidin A' causes a rapid K+ leakage from both uninfected and infected cells. We also studied the effect of Trp-N-formylated gramicidin and gramicidin A' incorporated in liposomes on the growth of Plasmodium falciparum in an in vitro culture. Incorporation of Trp-N-formylated gramicidin in the membranes of so-called 'stealth' vesicles strongly decreases the concentration needed to induce 50% inhibition of parasite growth. Moreover, no decrease in the K+ content of uninfected cells was observed when cells were exposed to liposome-incorporated Trp-N-formylated gramicidin at a concentration which causes full inhibition of parasite growth. These observations strongly suggest that Trp-N-formylated gramicidin incorporated in 'stealth' vesicles ends up specifically in the infected cell, thereby inhibiting the growth of the growth of the malaria parasite. PMID- 1706203 TI - Potassium selective ion channels in insulin-secreting cells: physiology, pharmacology and their role in stimulus-secretion coupling. PMID- 1706204 TI - Intraluminal radiation therapy per endoprosthesis: a case study. AB - Palliation of cholangeocarcinomas has been achieved by radiation delivered to the bile duct via endoprothesis. This case study supports and extends the works of others by describing the endoscopic techniques involved in the implantation of iridium seeds within a nasobiliary catheter. The case presented demonstrates endoscopic radiation treatment with no systemic radiation effects in a patient whose life extension was 19 months. PMID- 1706205 TI - Analysis of glycoprotein oligosaccharides by fast atom bombardment mass spectrometry. AB - A strategy is outlined for isolation and structural analysis of oligosaccharides derived from glycoproteins. Oligosaccharides, N- and O-linked, are released by chemical and enzymatic methods and separated using gel filtration, concanavalin A affinity chromatography and high-performance ion-exchange chromatography. Structural studies are carried out by fast atom bombardment mass spectrometry. The structural information from the latter can be extended to include determination of binding positions between monosaccharide residues. Prior to the analysis, samples are subjected to periodate oxidation, reduction and permethylation. The positions of glycosidic linkages are deduced from the spectrum by the primary and secondary sequence ions. Structural information can be obtained from mixtures of isomeric compounds. PMID- 1706206 TI - Leucocyte cellular adhesion molecules. AB - Leucocytes express adhesion promoting receptors which mediate cell-cell and cell matrix interactions. These adhesive interactions are crucial to the regulation of haemopoiesis and thymocyte maturation, the direction and control of leucocyte traffic and migration through tissues, and in the development of immune and non immune inflammatory responses. Several families of adhesion receptors have been identified (Table). The leucocyte integrin family comprises 3 alpha beta heterodimeric membrane glycoproteins which share a common beta subunit, designated CD18. The alpha subunits of each of the 3 members, lymphocyte function associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1) and p150,95 are designated CD11a, b and c respectively. These adhesion molecules play a critical part in the immune and inflammatory responses of leucocytes. The leucocyte integrin family is, in turn, part of the integrin superfamily, members of which are evolutionally, structurally and functionally related. Another Integrin subfamily found on leucocytes is the VLA group, so-called because the 'very late activation antigens' VLA-1 and VLA-2 were originally found to appear late in T cell activation. Members of this family function mainly as extracellular matrix adhesion receptors and are found both on haemopoietic and non-haemopoietic cells. They play a part in diverse cellular functions including tissue organisation, lymphocyte recirculation and T-cell immune responses. A third integrin subfamily, the cytoadhesins, are receptors on platelets and endothelial cells which bind extracellular matrix proteins. A second family of adhesion receptors is the immunoglobulin superfamily, members of which include CD2, LFA-3 and ICAM-1, which participate in T-cell adhesive interactions, and the antigen-specific receptors of T and B cells, CD4, CD8 and the MHC Class I and II molecules. A recently recognised family of adhesion receptors is the selectins, characterised by a common lectin domain. Leucocyte adhesion molecule-1 (LAM-1), which is the human homologue of the murine homing receptor, MEL-14, is expressed on leucocytes, while endothelial leucocyte adhesion molecule-1 (ELAM-1) and granule membrane protein (GMP-140) are expressed on stimulated endothelial cells and activated platelets. This review will be confined to adhesion receptors found on leucocytes, with particular emphasis on the leucocyte integrins. PMID- 1706207 TI - Treatment of hairy-cell leukemia. AB - Hairy-cell leukemia is an unusual chronic lymphoid leukemia with distinctive clinical and pathological features. The management of this disorder has been revolutionized in the last decade with the discovery of the efficacy of alpha interferon and the inhibitors of adenosine metabolism, deoxycoformycin and chlorodeoxyadenosine. The best treatment protocol for hairy-cell leukemia has not yet been defined. Patients may still die from their disease, particularly in the early phases of treatment. Conversely, some patients appear not to require treatment and others respond well to splenectomy and need no further therapy. An individualized clinical approach is recommended, with a role for splenectomy in the patient with cytopenia and a relatively low number of hairy cells in the bone marrow. The first line drug treatment remains interferon alpha given for 12-18 months, following which the patient is observed for clinical relapse. Deoxycoformycin remains a useful experimental agent but cannot be recommended for routine clinical use until issues of long term toxicity are resolved. Chlorodeoxyadenosine is a very promising experimental drug, but confirmation of the early data in larger group trials is required. Similarly the adjunctive use of granulocyte colony stimulating factor appears useful, but will need further study in larger groups of patients. There is little or no role for alkylating agents or more intensive chemotherapy in the modern management of hairy-cell leukemia. PMID- 1706208 TI - Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. AB - 1. Three analogues of L-arginine were characterized as inhibitors of endothelial nitric oxide (NO) synthase by measuring their effect on the endothelial NO synthase from porcine aortae, on the vascular tone of rings of rat aorta and on the blood pressure of the anaesthetized rat. 2. NG-monomethyl-L-arginine (L NMMA), N-iminoethyl-L-ornithine (L-NIO) and NG-nitro-L-arginine methyl ester (L NAME; all at 0.1-100 microM) caused concentration-dependent inhibition of the Ca2(+)-dependent endothelial NO synthase from porcine aortae. 3. L-NMMA, L-NIO and L-NAME caused an endothelium-dependent contraction and an inhibition of the endothelium-dependent relaxation induced by acetylcholine (ACh) in aortic rings. 4. L-NMMA, L-NIO and L-NAME (0.03-300 mg kg-1, i.v.) induced a dose-dependent increase in mean systemic arterial blood pressure accompanied by bradycardia. 5. L-NMMA, L-NIO and L-NAME (100 mg kg-1, i.v.) inhibited significantly the hypotensive responses to ACh and bradykinin. 6. The increase in blood pressure and bradycardia produced by these compounds were reversed by L-arginine (30-100 mg kg-1, i.v.) in a dose-dependent manner. 7. All of these effects were enantiomer specific. 8. These results indicate that L-NMMA, L-NIO and L-NAME are inhibitors of NO synthase in the vascular endothelium and confirm the important role of NO synthesis in the maintenance of vascular tone and blood pressure. PMID- 1706209 TI - Response of the canine bladder base during reflex micturition. AB - The response of the bladder base to filling and voiding was studied in a surgical dog model in which the bladder base was separated from the main body and closed to form a chamber. In the bladder distension phase there was a reduction in pressure in the bladder base chamber, which implies relaxation of the bladder base. In the expulsion phase the reflex bladder body activity took place with an increase in bladder base pressure, indicating contraction. Relaxation and contraction were blocked by propranolol and phentolamine respectively and both responses were abolished by trimetaphan. It was concluded that the bladder base activity in the micturition cycle is a reflex action mediated via the sympathetic nervous system. PMID- 1706210 TI - Dilatation of the prostatic urethra with 35 mm balloon. AB - A series of 54 men, 25 with acute urinary retention and 29 with cytometrically proven bladder outflow obstruction (BOO), underwent dilatation of the prostatic urethra using a 35 mm fixed diameter, low compliance balloon. In 42 patients this was performed under cystoscopic guidance and in 12 patients under fluoroscopic control. Three months following dilatation, 13/27 patients (48%) with BOO who returned for review were rendered unobstructed and 19/27 (70%) were symptomatically improved. By 6 months only 3 remained unobstructed but 15 remained symptomatically improved. Nine months after dilatation 14 patients retained symptomatic improvement but only 2 remained unobstructed. Of the 25 patients treated for acute retention only 6 voided spontaneously, 1 of these relapsing into retention at 2 months and another at 4 months. No patient was rendered unobstructed but 2 patients (who declined prostatectomy) noted an improvement in their obstructive symptoms at both 3 and 6 months. No patient developed retrograde ejaculation following dilatation. Balloon dilatation to 35 mm has no role in acute urinary retention but may have a role in younger men with BOO who wish to avoid prostatectomy and the risk of retrograde ejaculation. In these patients careful follow-up is required. PMID- 1706211 TI - Chronic relapsing experimental allergic neuritis induced by repeated transfer of P2-protein reactive T cell lines. AB - Chronic relapsing experimental allergic neuritis (crEAN) was induced by repeated transfers of P2-protein reactive T lymphocyte lines. Clinically, each intravenous transfer of P2-reactive T cells induced a relapse of the disease with weight loss and flaccid paresis of the hindlimbs followed by recovery. After multiple transfers, recovery from disease was incomplete, leading to increasing neurological deficit during the remissions. The pathology of the lesions during exacerbations was characterized by massive inflammation in the peripheral nervous system, associated with extensive endoneurial oedema, nerve fibre destruction and wallerian degeneration. Selective primary demyelination and remyelination was found in the minority of affected nerve fibres. No onion bulbs were present in chronic lesions. In the central nervous system partial degeneration of the posterior columns reflected the extent of wallerian degeneration in the peripheral nerves and spinal roots. In addition, during stages of active disease some T lymphocytes and upregulation of Ia antigen expression were found in the spinal cord. PMID- 1706212 TI - Cells secreting anti-MAG antibody occur in cerebrospinal fluid and bone marrow in patients with polyneuropathy associated with M component. AB - Occurrence and distribution of cells secreting antibodies against myelin associated glycoprotein (MAG) were studied in 9 patients with polyneuropathy associated with the monoclonal (M) component in serum. Utilizing an immunospot assay, we found that 4 of 7 patients with polyneuropathy associated with an IgM M component had cells secreting anti-MAG IgM antibody in cerebrospinal fluid (CSF) numbering between 1 per 212 and 1 per 3333 mononuclear cells. All 7 patients had cells secreting anti-MAG IgM antibody in bone marrow (median value 1 per 2000 cells). In contrast, peripheral blood from only 2 of these patients contained low numbers of such cells. One patient with polyneuropathy associated with an IgA M component had cells secreting anti-MAG IgA antibody in CSF, and 1 with an IgG M component had cells secreting anti-MAG IgG antibody in CSF; both patients also had anti-MAG IgM antibodies detectable in CSF only by ELISA. These 2 patients may thus have concurrent intrathecal production of antibodies of 2 different isotypes which are directed against the same or different epitopes of MAG. The production of antibodies directed against a component of myelin occurring in the immediate vicinity of the peripheral nervous system might be involved in the pathogenesis of the polyneuropathy. PMID- 1706213 TI - Preserved recognition of familiar personal names in global aphasia. AB - Recognition of proper and common nouns was compared in four patients diagnosed with global aphasia secondary to ischemic left-hemisphere infarction. For proper noun recognition, subjects matched the spoken or written name of a famous person to a photograph, and for common nouns, subjects were tested on standardized and special word recognition tests. As expected, common noun recognition was severely compromised in the aphasic patients. In contrast, familiar personal names, despite their greater length and complexity, were recognized equally well by aphasic and normal control subjects. The right hemisphere may mediate the ability to recognize personally familiar names, as it may be specialized for establishing personally relevant environmental stimuli. PMID- 1706214 TI - A simulation program to display specific digestion products of predicted RNA foldings. AB - A parameterizable program in Pascal was developed for VAX/VMS computers to simulate the autoradiograms of gel-separated RNA fragments generated by partial cleavage of a folded RNA molecule using five specific RNases. Each screen displays the results of cleavage by either one enzyme or all five, with the RNA molecule labeled at either of its ends (5' or 3'); each run is performed with three different lengths and against a ladder containing alkaline hydrolysis products of the same RNA molecule as size markers. The program should be useful for comparing actual results with predicted functional foldings of RNA molecules. PMID- 1706215 TI - Adenoid cystic carcinoma of the esophagus. A clinicopathologic study of three cases. AB - In a group of 245 cases of primary carcinoma of the esophagus the authors found three cases of adenoid cystic carcinoma (ACC). Clinical and pathologic data of those patients (one female and two male; age range, 49-74 years) were analyzed. Tumors were localized in the middle third of the esophagus. One patient lived 15 months after surgery. Another is a case of early ACC who has been living 4.5 years after surgery and is without specific symptoms. The third patient had not had surgery and died 13 months after the onset of dysphagia. An autopsy showed only a locally invasive tumor growing into the surroundings of the esophagus, and regional lymph node metastases without distant parenchymal metastases. These findings support pathologic and biologic similarities between ACC of the esophagus and ACC of the salivary glands. There are synchronous tumors of the esophagus and the vital localization which makes the prognosis of ACC of the esophagus worse than ACC of the salivary glands. PMID- 1706216 TI - Primary adenocarcinoma of the urinary bladder. A clinicopathologic analysis of 72 cases. AB - Adenocarcinomas account for approximately 2% of primary epithelial malignancies of the urinary bladder. The clinicopathologic features of 72 cases treated at one institution are reported; 22 cases were evaluated immunohistochemically. Twenty four tumors were urachal and 48 nonurachal. The cases were analyzed according to their stage at presentation, histologic type, and mucin staining; they were tested immunohistochemically to determine their reaction to carcinoembryonic antigen, Leu-M1, prostate-specific antigen, and prostatic acid phosphatase. Tumor stage was a highly significant predictor of outcome (P = 0.001). Nonurachal tumors tended to have a worse outcome than urachal, but the difference was not statistically significant (P = 0.07). Histologic type was not a significant predictor of outcome (P = 0.10). For adenocarcinoma of the urinary bladder, stage was the most significant predictive factor; separating urachal from nonurachal tumors was important, but mucin histochemistry and immunohistochemistry did not help in this distinction. On occasion, a few tumors may react with some polyclonal antibodies to prostate-specific antigen; thus these results must be interpreted with caution. In these instances, the possibility of using highly sensitive and specific monoclonal antibodies such as the one employed in this study should be considered. PMID- 1706217 TI - Monoclonal prostate-specific antigen in untreated prostate cancer. Relationship to clinical stage and grade. AB - The authors evaluated 440 men with clinically staged and untreated prostate cancer with a monoclonal prostate-specific antigen (PSA) assay. The serum PSA value correlated significantly with both the stage and grade of disease (P less than 0.00005). The relationships between PSA and consecutive Stages A, B, C, and D2 (alpha = 0.15) and between progressive Gleason's scores 2 to 4, 5 to 7, and 8 to 10 (alpha = 0.15) were statistically significant. Also statistically significant was the correlation between serum PSA level and intracapsular versus extracapsular disease (P less than 0.00005), although no one value can be used to differentiate reliably between patients in these two categories. The probability of clinically detectable metastasis (Stage D2) is 85% if the serum PSA level is greater than 30; however, 12% of patients without clinical evidence of metastases (Stages A, B, and C) have such a serum PSA value. Despite the statistically significant association between PSA and tumor differentiation and volume as reflected by tumor grade and clinical stage, this marker cannot be used to determine either for an individual patient. PMID- 1706218 TI - Expression of the hst-1 and c-kit protooncogenes in human testicular germ cell tumors. AB - Seventy testicular germ cell tumors were analyzed at the DNA and RNA levels for the c-kit, hst-1, and int-2 oncogenes using Northern and Southern blot analyses, respectively. There were significant differences in oncogene expression between seminomas and nonseminomas with c-kit being expressed in 24 of 30 (80%) seminomas but in only 3 of 40 (7%) nonseminomatous tumors (P = 0.0001, chi 2 test) and hst 1 being expressed in 24 of 38 (63%) nonseminomas but only 1 of 24 (4%) of seminomas (P = 0.0001, chi 2 test), demonstrating an inverse relationship in the expression pattern of these 2 oncogenes in human testicular germ cell tumors. A significant association between tumor stage and hst-1 expression in the nonseminoma group was found (P = 0.0002, chi 2 test). No gross alterations in the c-kit, hst-1, and int-2 loci were found at the DNA level and no int-2 mRNA expression was detected in any of the germ cell tumors examined. PMID- 1706219 TI - Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate. AB - The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments. PMID- 1706220 TI - Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping. AB - Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3 B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA. PMID- 1706221 TI - Catecholaminergic and peptidergic nerve components of intramural ganglia in the rat heart. An immunohistochemical study. AB - Immunohistochemical properties of the terminal nerve network in the rat heart were assessed by use of the elution-restaining method. The colocalization of the enzymes involved in catecholamine synthesis (tyrosine hydroxylase--TH. dopamine beta-hydroxylase--DBH) as well as the respective distributions of the neuropeptides associated with the adrenergic nervous system (neuropeptide tyrosine--NPY, C-terminal flanking peptide of neuropeptide Y--C-PON) were studied in series of serial sections throughout the interatrial septum and the atrioventricular junction. Our data suggest that ganglion cells of sulcus terminalis as well as the epicardial ganglia enclosed between the superior vena cava and ascending aorta are VIP- and TH-negative, but neuropeptide Y- and DBH immunoreactive. They give rise to three intraseptal nerves directed towards the specialised structures of the atrioventricular junction. These nerve fascicles contain abundant, thick TH-immunoreactive nerve fibres and scarce, thin NPY- and DBH-immunoreactive fibres. The cell bodies of the intramural ganglion cells localized between the right and left branches of the bundle of His (Moravec and Moravec 1984) are strongly TH- and DBH-immunoreactive. They are innervated by thick nerve fibres having the same immunohistochemical properties (NPY- and DBH immunoreactivities) as those of a subpopulation of the epicardial ganglion cells and seem to supply some of the TH-immunoreactive nerve fibres directed via the intraseptal nerves to the epicardial ganglia. The existence of a multicomponent nerve network, characterized by a reciprocal innervation of the sinus node and atrioventricular node areas, is suggested by our immunohistochemical data. PMID- 1706222 TI - The peptidyl-prolyl isomerase, FK506-binding protein, is most likely the 12 kd endogenous inhibitor 2 of protein kinase C. PMID- 1706223 TI - mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. AB - wee1 acts antagonistically to cdc25 in the tyrosine dephosphorylation and activation of cdc2, yet biochemical evidence suggests that wee1 is not required for tyrosine phosphorylation and its role is obscure. We show here that a related 66 kd kinase, called mik1, acts redundantly with wee1 in the negative regulation of cdc2 in S. pombe. A null allele of mik1 has no discernible phenotype, but a mik1 wee1 double mutant is hypermitotically lethal: all normal M phase checkpoints are bypassed, including the requirement for initiation of cell cycle "start," completion of S phase, and function of the cdc25+ mitotic activator. In the absence of mik1 and wee1 activity, cdc2 rapidly loses phosphate on tyrosine, both in strains undergoing mitotic lethality and in those that are viable owing to a compensating mutation within cdc2. The data suggest that mik1 and wee1 act cooperatively on cdc2, either directly as the inhibitory tyrosine kinase or as essential activators of that kinase. PMID- 1706224 TI - Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP-dependent phosphorylation. AB - Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8 bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide. PMID- 1706225 TI - Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope. AB - The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia. PMID- 1706226 TI - Ty virus-like particles in the Saccharomyces cerevisiae strain NCYC74. AB - Electron microscopic analysis of thin sections of Saccharomyces cerevisiae NCYC74 has revealed the presence of many clumped cytoplasmic particles that morphologically resemble Ty element virus-like particles (VLPs). Accumulation of Ty VLPs has only previously been observed in S. cerevisiae strains that over express a cloned Ty element. The particles in NCYC74 co-purify with Ty RNA, Ty specific antigens and a reverse transcriptase activity. Furthermore, they appear to be recognized by antibodies to Ty VLPs during indirect immunofluorescence experiments. These observations provide compelling evidence that the cytoplasmic particle in NCYC74 are indeed Ty VLPs. PMID- 1706227 TI - Macromolecule-macromolecule interaction in drug distribution: effect of alpha globulin concentration on the hepatic uptake of fractionated 3H-heparin by perfused rat liver. AB - The effect of alpha-globulin, the dominant binding protein for fractionated 3H heparin, on the hepatic uptake of 3H-heparin was studied by liver perfusion experiments in rats. Fractionated 3H-heparin concentration in the recirculated perfusate decline one-exponentially with time for each of six initial concentration levels of alpha-globulin. The hepatic uptake clearance of fractionated 3H-heparin was 0.154 ml/min/g liver in the absence of alpha globulin, and it decreased with increasing alpha-globulin concentrations. This result indicates that the hepatic uptake rate of alpha-globulin-bound fractionated 3H-heparin is lower than that of unbound fractionated 3H-heparin. On the other hand, it was indicated that almost all fractionated 3H-heparin binds to alpha-globulin at 8 mg/ml of alpha-globulin in in vitro study. However, the hepatic uptake clearance of the heparin at the concentration was of a certain value that could not be ignored. It was suggested that alpha-globulin-bound fractionated 3H-heparin also contributed to the hepatic uptake of fractionated 3H heparin. Therefore, a protein-mediated transport system, which has been reported for some low molecular weight drugs, may also exist in the hepatic uptake of such a high molecular weight compound as fractionated 3H-heparin. PMID- 1706228 TI - Mode of action of the gramicidin S analogs lacking hydrophilic amino acid residues on biomembranes. AB - The gramicidin S analog lacking basic ornithine residues, cyclo(-Val-Ala-Leu delta Phe-Pro-)2 (where delta Phe represents alpha, beta-dehydrophenylalanine), increased the K+ permeability of human erythrocytes and Staphylococcus aureus similarly to the parent gramicidin S. This analog altered the normal discoid shape of human erythrocytes to an invaginated form. The direction of the shape change was opposite to the case of gramicidin S causing crenated cells. We suppose that the analog accumulated predominantly into the inner half monolayer of membrane and destabilized the membrane structure, resulting in a break in the membrane. PMID- 1706229 TI - [Scintigraphy in breast tumors]. AB - The authors illustrate the use of nuclear medicine in tumours of the breast. The techniques can be used at various stages: 1) in the study of remote metastases (usually blood-borne)--bone and parenchymal scintigraphy; and 2) in the study of tumour spread to the lymph glands (axillary and mammary). The techniques of lymphoscintigraphy of the mammary lymphatic plexus and of axillary lymphoscintigraphy are illustrated, and scintigram readout parameters are defined. The Authors also discuss the possibility of using this examination for adequate preoperative tumour staging, as well as the possibility of obtaining even more accurate lymphoscintigraphic images, endowed with even more selective specificity, by means of the use of monoclonal antibodies labelled with radioactive substances. PMID- 1706230 TI - Serum lipase activity is increased in disease states other than acute pancreatitis: amylase revisited. PMID- 1706231 TI - Diagnostic and prognostic utility of phospholipase A activity in patients with acute pancreatitis: comparison with amylase and lipase. AB - We compared the diagnostic and prognostic utility of phospholipase A (PLA; EC 3.1.1.4) for acute pancreatitis with that of amylase and lipase by analysis of sera from 151 consecutive patients presenting with abdominal pain in whom assays of serum amylase and (or) lipase had been ordered. We determined the diagnostic accuracy for both the initial and the peak enzyme activities. Maximal diagnostic accuracy obtained for the initial activities of amylase, lipase, and PLA was 0.83, 0.83, and 0.76 at cutoff values of 650, 650, and 41 U/L, respectively. Use of peak enzyme activities showed maximal diagnostic accuracy of 0.85, 0.86, and 0.73 at cutoff values of 650, 1050, and 42 U/L, respectively. Receiver-operator characteristic curve analysis revealed the diagnostic performance of amylase and lipase to be similar, whereas that of PLA was almost random and not incremental. As with amylase and lipase, PLA activities in sera showed no relation to patients' survival; three patients who died after an attack of acute pancreatitis failed to demonstrate the dramatic increases in PLA activity previously described. We conclude that assessing the severity of acute pancreatitis by using enzyme activities still remains problematical. Measurements of amylase or lipase activities provide similar diagnostic discrimination when appropriate cutoff values are used and remain the methods of choice for diagnosis of acute pancreatitis. PMID- 1706232 TI - Lipase isoforms and amylase isoenzymes: assays and application in the diagnosis of acute pancreatitis. AB - Pancreatic juice and serum from patients with acute pancreatitis contain three enzymes that have lipolytic activity: L1 and L2, which are pancreatic isoenzymes or isoforms of lipase (EC 3.1.1.3), and L3, which is probably pancreatic carboxyl ester lipase, also known as cholesterol esterase (EC 3.1.1.13). These enzymes are readily separated electrophoretically on agarose and can be developed with an overlay of Kodak Ektachem lipase slide material. The latter acts as a dry-reagent developing substrate, with the enzymes producing blue bands in the slide material. We found L1 in about one-half of normal persons, L2 in none, and L3 in all. We assayed for amylase (EC 3.2.1.1), amylase isoenzymes, lipase, and lipase isoforms in the sera of 100 patients with suspected acute pancreatitis. L2 lipase has the greatest diagnostic efficiency for the diagnosis of pancreatitis, compared with total amylase, P3 amylase, and total lipase. Lipase and L2 could replace amylase, an inefficient test, for the diagnosis of patients with suspected acute pancreatitis. In patients receiving organ transplants, a serum amylase value of greater than 300 U/L or a lipase of greater than 1000 U/L discriminated well between patients with and without complications and (or) acute rejection. PMID- 1706233 TI - Lipase activity in serum measured with Ektachem is often increased in nonpancreatic disorders. AB - For patients with symptoms of pancreatitis, measurement of amylase in serum reportedly is more sensitive than that of lipase in acute pancreatitis, whereas lipase reportedly is more specific. However, serum lipase activities exceeding the upper reference limit (URL) have been reported for many patients who did not have pancreatitis. I reviewed the serum lipase and amylase concentrations of 493 consecutive inpatients and emergency department patients for whom both tests were ordered. Serum lipase and amylase activities, determined with an Ektachem 700 analyzer, were less than or equal to URL for 390 patients (83%) and greater than URL for 103. Medical records of 101 of these 103 were reviewed; 18 had acute or chronic relapsing pancreatitis. In this latter group, serum lipase values greater than URL had 100% sensitivity and 84% specificity; those of serum amylase greater than URL had 72% sensitivity and 88% specificity. However, the test combination of serum lipase greater than URL and serum amylase less than or equal to URL also occurred in 84% of the patients in which review of the medical records revealed nonpancreatic gastrointestinal or hepatobiliary disorders as the primary problem (n = 55). Therefore, serum lipase activity measured with the Ektachem assay is also often increased in patients with intra-abdominal disorders that appear to be nonpancreatic. PMID- 1706234 TI - Urinary fibronectin fragments (a potential tumor marker) measured by immunoenzymometric assay with domain-specific monoclonal antibodies. AB - We found that urinary fibronectin (UFN) in cancer patients was almost all fragmented and consisted mainly of the cell-binding domain. We developed a two site immunoenzymometric assay for UFN, using two monoclonal antibodies that both recognize this domain of fibronectin. The amount of UFN was expressed as arbitrary units per milligram of creatinine. Some 2% of the 623 healthy subjects had UFN above the clinical cutoff point (200 arb. units/mg creatinine), as did 14% of the 271 patients with nonmalignant diseases. In contrast, concentrations of UFN exceeded the cutoff point in 59% of the 589 patients with cancer. In 37 patients with gastrointestinal cancer tested for UFN and for one or more of three established serum tumor markers, UFN was found in 25, significantly more often than the other markers. These results indicated that UFN is a marker that may be helpful in evaluating many kinds of cancer. PMID- 1706235 TI - Expression of decay-accelerating factor is reduced on hyperplastic synovial lining cells in rheumatoid synovitis. AB - Decay-accelerating factor (DAF), a membrane inhibitor of homologous complement activation, is present in synovial cells lining joint space and detected in synovial fluid. DAF is considered to protect synovial membrane from complement mediated injury associated with articular inflammation. We studied the immunohistopathological features of DAF molecules in synovial membrane of rheumatoid synovitis using a DAF-specific monoclonal antibody, 1C6. Reacting molecules with the 1C6 antibodies in synovial tissue extracts formed a 70-kD band in Western blot analysis. DAF was strongly detected on the flat synovial lining cells, but weakly on the hyperplastic and multi-layered lining cells in rheumatoid synovitis. The latter cells reacted with anti-Leu-M3 antibodies specific for a cell surface marker of activated macrophages, sometimes accompanied by C3 and IgM deposition on the superficial synovial membrane. These results suggest that active rheumatoid synovitis characteristically with hyperplastic synovial lining cells is out of control by DAF, thereby permitting further complement-mediated injury. PMID- 1706236 TI - Human hybridomas derived from CD5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies. AB - Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B-CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT-sensitive heteromyeloma line (CB-F7). A fusion frequency of up to 10(-5) allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi-specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a 'switch-on' of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi-reactive antibody repertoire of CD5+ B cells. PMID- 1706237 TI - Expression of the neural cell adhesion molecule (CD56) by human myeloma cells. AB - Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T,B, myelomonocytic, and natural killer (NK)-cell antigens. Leu-19/NKH1 (CD56), a molecule identical to N-CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual parameter flow cytometry was used to correlate N-CAM positivity with DNA aneuploidy or cytoplasmic immunoglobulin expression as markers of myeloma cells. When N-CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N-CAM frequently exhibited pre-B cell markers, myeloid antigen, and HLA-DR, respectively (seven out of eight cases, 88%). Six out of eight N-CAM-negative myelomas were of the IgG lambda isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N-CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained. PMID- 1706238 TI - Changes in natural killer cell phenotype in patients with post-viral fatigue syndrome. AB - We analysed peripheral blood CD56+ natural killer (NK) cell subsets in 23 carefully characterized patients with post-viral fatigue syndrome (PFS), compared with 19 healthy controls, using fluorochrome-conjugated, specific monoclonal antibodies and the FACScan. We found significantly increased percentages of CD56+, and especially CD56bright+ NK cells in PFS patients. We also found significantly increased percentages of CD56+ high affinity interleukin-2 (IL-2) receptor (CD25)+ and CD56+ transferrin receptor (CD71+) subsets of cells, most of which also stained brightly for CD56. Also, we found an increased percentage of CD56+ CD3+ cells, many of which stained brightly for CD56, although there was no increase in the percentage of CD56- CD3+ T cells in these patients. These observations, in conjunction with very low percentage of CD56- CD25+ cells, suggest that there is a preferential involvement of this minor subset of CD56+ CD3+ T cells in PFS. Finally, a decreased percentage of CD56+ Fc gamma receptor (CD16)+ NK cells was identified, which suggests a reduced capacity of antibody dependent cellular cytotoxicity in PFS patients. Subsets of CD56+ NK cells co expressing CD2, CD4 or CD8 did not show any significant difference between PFS patients and healthy controls. These phenotypic changes provide laboratory evidence of immunological abnormalities in this syndrome, and, we suggest, may be consistent with persistent viral infection. PMID- 1706239 TI - Characterization and large production of human monoclonal antibodies against the HIV-1 envelope. AB - Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures. PMID- 1706240 TI - Acute-phase protein synthesis in human hepatoma cells: differential regulation of serum amyloid A (SAA) and haptoglobin by interleukin-1 and interleukin-6. AB - Interleukin-6 (IL-6, BSF-2 or IFN-beta 2) is thought to be the major regulator of the acute-phase protein response that follows tissue injury and inflammation, with interleukin-1 (IL-1), tumour necrosis factor and more recently, LIF or HSF III, slightly stimulatory on only certain acute phase proteins. The synthesis of the major acute-phase protein SAA, originally described as being synthesized in response to IL-1, has been claimed recently to be mainly under IL-6 regulation. Our results show that in the human hepatoma cell line HuH-7, IL-1 is the major stimulating cytokine increasing SAA synthesis by a factor in excess of 100-fold. We also show that under most conditions interleukin-6 and tumour necrosis factor stimulate additively in combination with IL-1. Isoelectric focusing has demonstrated that SAA1 and SAA2 alpha are expressed but not SAA2 beta. The HuH-7 cell line is IL-6 responsive since haptoglobin is stimulated mainly by IL-6. PMID- 1706241 TI - Biliary stenting for malignant jaundice. A district hospital experience. AB - Non-surgical management of malignant jaundice is becoming widespread in referral centres and the results are good. We report a retrospective analysis of 46 patients with malignant jaundice who were treated with either endoscopic or combined percutaneous endoscopic biliary stenting in a district general hospital. Out of 46 patients, 24 were stented successfully endoscopically and 19 of the remaining 22 patients were put forward for the combined procedure, and 12 had successful stenting. Eight were managed with palliative bypass surgery and two died. The procedure related mortality was 6.9% and the 30 day mortality was 4.6%. Good palliation of patients with malignant jaundice is achievable in small centres providing there is good radiological and surgical back-up. PMID- 1706242 TI - Evaluation of calcofluor white stain for detection of Pneumocystis carinii. AB - A rapid calcofluor white (CFW) stain for detecting Pneumocystis carinii was evaluated prospectively. Eighty-nine bronchoalveolar lavage (BAL) specimens, 21 open-lung biopsy (OLB) tissues, 2 induced sputums, 1 expectorated sputum, 2 tracheal secretions, and 1 bronchial secretion from 102 patients were examined for P. carinii cysts by both the CFW stain and a modified methenamine silver (MS) stain. Twenty episodes of P. carinii pneumonia were detected: 19 of these episodes were detected by CFW stain, and 16 of those episodes were detected by MS stain. Discrepancies between the two staining methods were resolved by review of the clinical histories and, in one case, by testing an OLB specimen. Neither staining procedure gave false-positive results with any specimen. More cysts were detected in CFW-stained specimens than in MS-stained specimens (p = 0.05). CFW stain is a simple, rapid, and inexpensive method for detecting P. carinii in clinical specimens and is at least as sensitive as MS stain. PMID- 1706243 TI - A comparison of calcofluor white, potassium hydroxide, and culture for the laboratory diagnosis of superficial fungal infection. AB - A total of 207 skin scrapings were prospectively studied using potassium hydroxide (KOH), calcofluor white (CW), and culture to determine the clinical usefulness of each microscopic method. For dermatophytes (prevalence 13.2%), CW had a sensitivity of 92% and a specificity 95%, giving a positive predictive value of 74% and negative predictive value of 99%. KOH had a sensitivity of 88% and a specificity of 95%, giving a positive predictive value of 73% and a negative predictive value of 98%. CW was simple, rapid, and easy to read. For dermatophyte infection, CW results are as useful as KOH results. PMID- 1706244 TI - Evaluation of endocrine parameters in clinical trials with beta-hCG vaccine. AB - This report presents data on various endocrine parameters with respect to pituitary-ovarian axis in-depth in control vs 18 subjects immunized with three beta-hCG based vaccine formulations. Hormonal parameters such as TSH, PRL, ACTH, progesterone, cortisol, T3 and T4 were measured by RIA in sera in control pre vaccine cycles as well as at 3, 6, and 12 months after primary vaccine immunization. In 6 women urinary E1G, PdG, LH and FSH were measured by ELISA/RIA tests on early morning urine (EMU) samples collected throughout the menstrual cycle in control vs post-booster vaccine cycles. The results indicated that none of the beta-hCG vaccine formulations altered the pituitary peptide or steroid hormone levels in blood at any time during the period of study. Serum P concentration was adequate and indicative of ovulatory cycle in almost all the cycles during the study. The in-depth study on urinary excretion pattern and levels of gonadotropins and E1G and PdG throughout the control vs post-vaccine booster cycles conclusively showed that pituitary-ovarian axis was not adversely affected by the vaccine. PMID- 1706245 TI - Pretranslational suppression of cytochrome P-450h (IIC11) gene expression in rat liver after administration of interferon inducers. AB - Administration of the interferon inducer polyriboinosinic acid.polyribocytidylic acid (poly rl.poly rC) (10 mg/kg, ip) to male rats suppressed the constitutive hepatic expression of the male-specific cytochrome P-450 [AH, reduced flavoprotein/oxygen oxidoreductase (RH hydroxylating), EC 1.14.14.1] isozyme P450IIC11 (P-450h) to 21% of control levels within 24 hr. The mRNA for P-450h was more rapidly suppressed by the drug, being significantly suppressed to 56% of control values within 6 hr of administration. P-450h mRNA levels were further lowered to 10% of control by 24 hr. The kinetics of suppression of P-450h apoprotein and mRNA by poly rl.poly rC indicate that the primary mechanism(s) is (are) at a pretranslational level. Tilorone analog R11-877DA (TA) (50 mg/kg, ip), also an interferon inducer, produced qualitatively similar effects to those of poly rl.poly rC, although the TA produced lesser (51% and 59%) decreases in P 450h protein and mRNA, respectively, 24 hr after injection. Again, the results indicate a pretranslational mechanism of P-450h suppression. Although both interferon inducers suppressed hepatic P-450h expression, the magnitudes of these effects were not notably greater than those on total hepatic P-450, indicating that suppression of rat liver P-450 isozymes by interferon inducers is not confined to P-450h. PMID- 1706246 TI - Gene expression in the jimpy mutant: evidence for fewer oligodendrocytes expressing myelin protein genes and impaired translocation of myelin basic protein mRNA. AB - Myelin basic protein (MBP) and proteolipid protein (PLP) gene expression was investigated in the murine dysmyelinating mutant, jimpy and age-matched normal mice. MBP and PLP mRNA expression was examined in several brain regions by in situ hybridization histochemistry between 10 and 20 days postpartum. The results showed a general reduction in both PLP and MBP mRNA expression throughout in jimpy brain due primarily to fewer numbers of jimpy oligodendrocytes expressing these genes. A less severe, but significant, reduction in the level of PLP expression per oligodendrocyte was also observed in the mutant brain. Because of the diffuse pattern of MBP mRNA distribution in the controls it was not possible to determine whether MBP expression was reduced on a per cell basis in the jimpy mutant. Silver grains representing MBP mRNAs were diffusely distributed over myelinating regions in normal mice but, in contrast, the grains were primarily clustered over oligodendrocyte cell bodies in the jimpy brains. No significant differences in jimpy oligodendrocyte morphology were evident by galactocerebroside immunohistochemistry. These results suggested that the apparent MBP mRNA clustering was not due to truncated or reduced numbers of oligodendrocyte processes. Rather, they suggest that the inability of jimpy mice to incorporate MBP into myelin may be due, in part, to a disruption in the translocation of MBP mRNAs within the oligodendrocyte. PMID- 1706247 TI - Recovery within day-time sleep after slow wave sleep suppression. AB - Six subjects had their SWS activity suppressed by acoustic stimulation during a day-time (11.00 h) recovery sleep after a 4 h night sleep (03.00-07.00 h). Sleep was disturbed for a period corresponding to 90% of the duration of a preceding undisturbed baseline sleep (also at 11.00 h and preceded by a 4 h night sleep) and thereafter allowed to continue undisturbed until spontaneous awakening. The results showed that SWS and EEG power density were significantly reduced during suppression and that full recovery occurred before spontaneous awakening. The disturbed sleep was significantly longer than the baseline sleep. The increase in duration consisted mainly of SWS, stage 2 and REM. The results suggest that the suppression of SWS activity caused a need for an extension of sleep in order to allow recovery. PMID- 1706248 TI - Contrast and stereoscopic visual stimuli yield lateralized scalp potential fields associated with different neural generators. AB - The use of dynamic random-dot stereograms (RDS) allows to investigate evoked potential components generated exclusively by cortical structures. We analyzed the scalp distribution of stereoscopically evoked or contrast evoked potential field by recording electrical brain activity in 20 channels simultaneously from an electrode array covering the occipital scalp areas. Evoked brain activity was obtained from 13 healthy adults with dynamic RDS stimuli presented as a stereoscopic checkerboard pattern in the center, or in the right or left visual half-field. Such stereoscopically evoked scalp potential distributions were compared to those elicited by a conventional 2-dimensional checkerboard reversal stimulus of the same mean luminance and retinal extent. We found that the latencies of the major evoked components were similar for contrast and stereoscopic stimuli, while significant differences were observed when we compared the strength of the evoked potential fields or the topographical pattern elicited by lateralized stereoscopic and contrast stimuli. The functional relation of evoked electrical brain activity to the retinal stimulus location was significantly different for stereoscopic and contrast stimuli. We present evidence that stereoscopic perception relies on the activation of cortical structures in the human visual system that are different from those activated by comparable contrast stimuli, supporting the conclusions derived from our earlier electrophysiological experiments on stereoscopic vision. These data on the physiological correlates of processing of stereoscopic information in humans are in line with the results obtained with single neuron recordings from the cat and monkey visual cortex. PMID- 1706249 TI - The effect of stimulus repetition rate on the diagnostic efficacy of the auditory nerve-brain-stem evoked response. AB - This study investigates the hypothesis that an increase in the click presentation rate during diagnostic testing with the auditory nerve-brain-stem response (ABR) will increase the efficiency with which lesions may be detected in the nervous system. Cats were exposed to conditions of hypoxia, hypercapnia and acidemia, and hypoglycemia was induced in rats. ABR was recorded using the standard 10/sec click rate and also a higher (55/sec) rate during both the control state and experimental state. Various parameters of the ABR were compared at the two click rates in the control and experimental states to see if the higher click rate was more effective in detecting pathology in the nervous system. It was found that in only a very few cases was the higher stimulus presentation rate more effective, and that in general ABR recordings at one stimulus rate only is quite sufficient for work in a clinical setting. PMID- 1706250 TI - BAEP amplitudes and amplitude ratios: relation to click polarity, rate, age and sex. AB - BAEP amplitudes and amplitude ratios in 47 healthy subjects aged 4-58 years have been investigated with respect to the simultaneous influence of stimulus polarity, rate, sex and age. The sex effect (larger amplitudes in women than in men) seemed to be most pronounced for wave IV-V amplitude, while the age effects (decreasing amplitudes with age) seem to be largest for the early waves. The amplitude reductions which followed an increment in the stimulus rate from 10 to 50 Hz were generally larger than the reduction following a change in the stimulus polarity from R to C clicks. The rate-induced amplitude reductions depend on click polarity for waves II and V. Thus, adaptation may be different in C click responding neurons as compared to R click responding neurons. The IV-V/I and IV V/III amplitude ratios were both independent of sex, but the IV-V/I ratio increased significantly with age. Polarity and rate effects depended on age (wave III) but not on sex. Thus, aging may involve alterations of neural function, while the sex-related amplitude differences may be explained by non-neural factors. PMID- 1706251 TI - Age effects on the P300 to novel somatosensory stimuli. AB - Event-related brain potentials (ERPs) to somatosensory task-relevant targets and task-irrelevant novel (tactile and shock) stimuli were studied in 30 subjects between the ages of 18 and 79. Target and novel P300 latencies increased linearly with age at comparable rates. P300 amplitudes and scalp topographies also changed with age. P300 amplitudes remained constant at frontal sites and decreased at central and parietal sites for both target and novel stimuli with increasing age. The current results extend the age-related novel P300 changes reported in the auditory and visual modalities to the somatosensory system. The age-related amplitude reduction at posterior scalp sites supports independent contributions of frontal and posterior association cortex to P300 generation. PMID- 1706252 TI - Relay stations and neurotransmitters between the pallidal region and the hippocampus. AB - The effects of internal pallidum and lateral habenula stimulation on epileptiform activity of cat's hippocampus were studied. A steady interictal activity was induced by locally applied sodium penicillin (PCN) solution. Both pallidal and habenular electrical stimulation caused an increase in spike frequency and amplitude. Intraperitoneally injected atropine sulphate failed to modify pallidal and habenular influences. Intraperitoneal methysergide bimaleate (5-HT antagonist) suppressed the effects of habenular stimulation. In contrast to the effects of pallidal and habenular stimulation, raphe electrical stimulation inhibited hippocampal spiking and intra-raphal muscimol (a GABA receptor agonist) enhanced hippocampal-based epilepsy. After muscimol, raphe stimulation at the same threshold parameters failed to affect hippocampal activity. In cats with habenular lesions hippocampal spike frequency and amplitude were reduced and intra-raphal muscimol did not affect the hippocampus. The results are discussed in the light of a complex interrelationship between basal ganglia and hippocampus. The role of the lateral habenula and of the medial raphe as relay stations between the two regions is emphasized. PMID- 1706253 TI - Atypical electroencephalographic pattern in a patient with subacute sclerosing panencephalitis. AB - Subacute sclerosing panencephalitis (SSPE) has become rare since the widespread use of the measles vaccine. In this patient with a 5 month history of seizures and progressive dementia due to SSPE, the atypical electroencephalographic pattern was characterized by generalized spike-and-wave discharges, maximal on the right, associated with clinical seizures. After diazepam (intravenously), more typical periodic complexes appeared. PMID- 1706254 TI - Polysomnography in locked-in syndrome. AB - Sleep patterns were evaluated in a case of 'locked-in' syndrome. This patient had an ischemic infarction involving the ventral portion of the upper half of the pons bilaterally, with a posteromedial extension into the tegmentum. Reticular structures, notably the median raphe nuclei, supposed to play a major regulatory role in sleep, were most probably involved. Unexpectedly, repeated polysomnographic studies revealed sleep patterns with only minor abnormalities. PMID- 1706255 TI - EEG artefacts caused by a Clinitron bed. AB - Clinitron beds, often used to prevent decubitus ulcers, may cause an EEG artefact by static electricity. The best way to prevent this artefact is to turn off the bed during EEG registrations. PMID- 1706256 TI - Deutsche EEG Gesellschaft, 34th annual meeting. Munster, 28-30 October 1989. Abstracts. PMID- 1706257 TI - A conceptual model of taste receptors. AB - Of all the senses, that of taste is perhaps the hardest to define and quantify and elucidation of the underlying mechanisms is unrespondingly difficult. This article describes a conceptual model--at the molecular level--for the four basic taste modalities, using apparent specific volume as a fundamental parameter. It also considers possible practical applications. PMID- 1706258 TI - The differential regulation by glucagon and growth hormone of insulin-like growth factor (IGF)-I and IGF binding proteins in cultured rat hepatocytes. AB - The liver is a major site of production of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGF-BPs). GH decisively influences IGF-I production. To study the role of GH and glucagon in the regulation of IGF-I and IGF-BP production, we examined IGF-I and IGF-BPs secreted by primary rat hepatocytes cultured in a serum-free medium. Glucagon (1 x 10(-8) M) stimulated IGF-I secretion and IGF-BP secretion. Bovine GH (bGH, 300 ng/ml) stimulated IGF-I secretion but suppressed IGF-BP secretion. Combining bGH and glucagon significantly augmented IGF-I secretion above the level seen with each individual agent. The inhibitory effect of bGH on IGF-BP secretion was reversed by glucagon. The major species of IGF-BPs secreted by hepatocytes were found, on Western ligand blotting, to be 24K and 30-34K. All species of secreted IGF-BPs appeared to be comparably affected by glucagon, bGH, and their combination. Northern analysis of IGF-I mRNA revealed three transcripts of 0.7-1.1 kilobases (kb), 1.8 kb, and 7.0 kb. Glucagon stimulated IGF-I mRNA levels 1.8- to 2.0-fold, whereas bGH stimulated IGF-I mRNA levels 2.0- to 2.5-fold. When hepatocytes were incubated with glucagon and bGH for 6 h, IGF-I mRNA levels were augmented 10 fold. Glucagon, in the presence of 50 ng/ml bGH, had a dose-dependent effect on IGF-I mRNA accumulation from a 6-fold level of stimulation at 50 ng/ml of glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon. This study has demonstrated that glucagon, as well as GH, has significant effects on the production of both IGF-I and IGF-BPs. Of particular interest was the marked augmentation of hepatic IGF-I messenger RNA levels and the reversal of the low levels of IGF-BP production seen on adding glucagon to bGH. PMID- 1706259 TI - Elevated activity of low molecular weight insulin-like growth factor-binding proteins in sera of vitamin C-deficient and fasted guinea pigs. AB - We have previously reported that scorbutic and fasted guinea pig sera contain an insulin-like growth factor-I (IGF-I)-reversible inhibitor of collagen, proteoglycan, and DNA synthesis in cultured cells. Here we report that IGF binding protein (IGFBP) activity is increased in serum containing the inhibitor [125I]IGF-I or -II bound to these sera was eluted in the 30- to 50-kDa region of an S200 gel column. [125I]IGF-I affinity cross-linking analysis revealed that a 38-kDa cross-linked species increased markedly in fasted and scorbutic sera, with a lesser increase in a 34-kDa species, while scorbutic sera also yielded a 44-kDa species. Gel filtration of unlabeled sera showed a 10-fold increase in the activity of two proteins in the 30- to 50-kDa region from the experimental sera. Their activity correlated with their ability to inhibit binding of [125I]IGF-I to its cellular receptor, suggesting that they have the potential to inhibit IGF-I dependent functions. Ligand blotting showed that 29 and 35-kDa IGFBPs were almost undetectable in normal serum, but were dramatically induced by scurvy and fasting, so that they accounted for close to 40% of the total circulating BPs. Total IGFBP-3 in the experimental sera was increased about 30%, while there was little effect of scurvy or fasting on the level of BP-3 activity isolated by acid extraction of the high mol wt region of the S200 column. An IGF-I analog with normal affinity for the 30- to 50-kDa BPs from fasted and scorbutic sera, but with reduced affinity for the cell receptor, was equivalent to IGF-I in reversing the inhibition of collagen synthesis by scorbutic guinea pig serum in human fibroblasts. Thus, reversal of inhibition appears to require initial saturation of IGFBPs. The overall results suggest that two circulating IGFBPs with unoccupied binding sites are induced in vitamin C-deficient or fasted guinea pigs and may be responsible for inhibition of IGF-I-dependent functions by sera from these animals. PMID- 1706260 TI - Determination of subunit contact-associated epitopes of the beta-subunit of human follicle-stimulating hormone. AB - Three different experimental approaches were used to assess the regions on the beta-subunit of human FSH (hFSH beta) that may be altered or masked by its association with the alpha-subunit of hFSH (hFSH alpha) in the heterodimeric hFSH molecule. In a direct approach, we tested whether synthetic peptides corresponding to hFSH beta sequences 1-20, 16-36, 33-53, 49-67, 66-85, 81-100, and 98-111 inhibited association of hFSH alpha and hFSH beta in an enzyme-linked immunosorbent assay. Synthetic peptides-(81-100), -(98-111), and -(66-85) caused greater than 50% inhibition of subunit association, whereas other peptides showed 26% or less inhibition. These data suggested that the C-terminal sequences of hFSH beta, particularly 81-100, are at a subunit interface with hFSH alpha in heterodimeric hFSH. In another approach we reasoned that antibodies with a higher affinity for free hFSH beta than for heterodimeric hFSH bind to epitopes on hFSH beta that are masked or altered by hFSH alpha subunit. To test this hypothesis, epitopes of hFSH beta were mapped using synthetic peptides of hFSH beta sequences, three monoclonal antibodies (3G3, 4D5, and 4G8), and a polyclonal antiserum (NIDDK anti-hFSH beta). Compared to 3G3 all the other antibodies exhibited minimal reactivity with hFSH, but bound strongly to hFSH beta. The epitope-mapping data with both 4D5 and NIDDK anti-hFSH beta identified peptide 81 100, which was not recognized by 3G3. The epitope map with 4G8 identified the same three peptides as with 3G3. However, in the case of 4G8 its reactivity with peptide 33-53 was the least, whereas it was ranked first for 3G3. Since both 3G3 and 4G8 had an identical affinity for hFSH beta, it was hypothesized that sequences in peptide 33-53 may be altered or masked by hFSH alpha. To test this, we determined the specificity of anti-hFSH beta-(33-53) peptide antiserum for hFSH beta and hFSH in an enzyme-linked immunosorbent assay. The antipeptide antiserum bound strongly to free hFSH beta and weakly to hFSH, suggesting that part of the sequence in peptide-(33-53) was masked or altered by association with hFSH alpha in heterodimeric hFSH. Taken together, the subunit association studies, the epitope-mapping data, and the specificity of anti-hFSH beta-(33-53) peptide antiserum have suggested that sequences in peptide-(81-100) and -(33-53) are masked or conformationally altered by hFSH alpha in heterodimeric hFSH. PMID- 1706261 TI - Insulin-like growth factor-I serum levels show a midembryogenesis peak in chicken that is absent in growth-retarded embryos cultured ex ovo. AB - Insulin-like growth factor-I (IGF-I) is the primary mediator of GH action after birth, but its role as a regulator of prenatal growth is unclear. In a previous study we showed that IGF-I mRNA was expressed in chicken embryos beginning at the blastoderm stage (day 0, newly laid egg). Here we present the ontogeny of serum IGF-I in normal and growth-retarded chicken embryos. Serum samples were pooled from multiple embryos starting on day 4 of development in ovo until hatching (day 21). Extracts of day 2 and 3 whole embryos were also studied. IGF-binding proteins were removed by filtration on Sep-Pak C-18 cartridges. IGF-I was quantitated by a heterologous RIA validated for chicken species. Embryonic IGF-I showed a HPLC profile similar to that of adult chicken serum IGF-I. Serum IGF-I was measurable on day 6 of development (approximately 0.04 ng/ml), reached a peak on day 15 (18 ng/ml), and decreased to a low concentration (0.2 ng/ml) the day before hatching. Embryos cultured ex ovo showed progressive growth retardation after day 10 of development, and by day 20 their weight was 50% of normal. The serum IGF-I concentration of ex ovo cultured embryos was normal on day 10, but remained low until day 21, without the midembryogenesis rise observed in normal embryos. These results support the concept that IGF-I may have a role in general embryonic growth in addition to any paracrine/autocrine action in individual tissues. PMID- 1706262 TI - Regulation of insulin-like growth factor-binding protein messenger ribonucleic acid levels in sheep thyroid cells. AB - The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP 2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology. PMID- 1706263 TI - Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. AB - Primary uterine cell cultures were used to study multifactor regulation of progesterone receptor (PR) and the signal transduction pathways which may serve to mediate that regulation. Increases in intracellular cAMP, brought about by treatment with cholera toxin plus isobutyl methyl xanthine or by addition of 8 bromo-cAMP, result in 6- to 7-fold increases in the intracellular content of PR as monitored by [3H]R5020 binding and by Western immunoblot using anti-PR antibodies. In these primary cultures of uterine cells isolated from 19-day-old immature rats, 8-bromo-cAMP evokes significant increases in PR by 8 h with maximal increases by 24 h. This time course and magnitude of PR stimulation are similar to those evoked by maximally effective concentrations of estradiol (3 x 10(-9) M) or IGF-I (20 ng/ml). Dose-response studies reveal that 10(-6) to 10(-4) M concentrations of 8-bromo-cAMP (8-Br-cAMP) elicit a maximal response. In contrast, 8-bromo-cGMP over a wide concentration range was unable to elevate cellular PR levels. Under these culture conditions, cell proliferation was not altered by treatment with any of these agents. Although estrogen, cAMP, and insulin-like growth factor I (IGF-I) may act via different pathways to increase PR, the effects evoked by maximally effective concentrations of these agents are not additive implying involvement of a common component. The increases in PR evoked by estradiol, cAMP, or IGF-I are markedly suppressed by treatment with antiestrogen (ICI 164,384) or the cyclic nucleotide-dependent protein kinase inhibitor H8 or the protein kinase A inhibitor PKI, indicating the involvement of the estrogen receptor and phosphorylation pathways in PR regulation by these three agents. The present studies identify cAMP, as well as estrogen and IGF-I, as important regulators of the level of PR in uterine cells and suggest that multiple factors, including those affecting intracellular cAMP levels, might influence responsiveness to progestins via regulation of the intracellular PR content. PMID- 1706264 TI - Dexamethasone inhibition of prostaglandin production in human term placental cells is protein and ribonucleic acid synthesis dependent. AB - A key enzyme in the regulation of prostaglandin (PG) synthesis is PG synthase (PGS; cyclooxygenase), which converts arachidonic acid to PGs. Since both PGs and glucocorticoids are elevated before parturition, we studied the regulation of dexamethasone (DEX; 150 nM) on PGF2 alpha synthesis and PGS expression in human placental cells in vitro. Both first trimester and term placental cells were used. DEX reduced PGF2 alpha synthesis in human term placental cells, in contrast to first trimester cells which were unaffected by the same treatment. DEX inhibition of PGF2 alpha production by term placental cells was time and dose dependent. PGS expression was analyzed by [35S]methionine metabolic labeling and immunoprecipitation using polyclonal antibodies developed in rabbits against ram seminal vesicle PGS. DEX reduced PGS expression in term placental cells, but not in first trimester cells. In contrast to the effect of DEX on PGF2 alpha, progesterone and estradiol production by cells were unaffected at any stage of gestation examined. DEX inhibition of PGF2 alpha synthesis required de novo biosynthesis of RNA and proteins. These results suggest 1) corticosteroids play a role in the regulation of placental PG synthesis during parturition; 2) the inhibition of PG synthesis and PGS expression by glucocorticoids is RNA and protein biosynthesis dependent; and 3) induction of labor by glucocorticoids is not directly related to changes in placental progesterone or estradiol biosynthesis. PMID- 1706265 TI - Growth hormone-dependent and -independent regulation of cytochrome P-450 isozyme expression in streptozotocin-diabetic rats. AB - The sexually dimorphic GH secretory pattern is thought to be the major factor regulating constitutive expression of hepatic P450IIC11 (P-450h) and P450IIC12 (P 450i). In this study we investigated whether factors other than the diabetes induced decrease in GH secretion contribute to alterations in P-450 isozyme expression in streptozotocin (STZ)-diabetic rats. In male rats, hepatic P-450h apoprotein and mRNA decreased to 13% and 24% of control male levels, respectively, within 14 days of STZ injection. STZ-diabetes had little effect on expression of P-450i in females. Treatment of diabetic male rats with GH did not reverse the suppression of P-450h. STZ treatment also suppressed P-450h expression in GH-treated hypophysectomized (Hx) male rats, but incompletely. Thus, GH can partially reverse diabetic suppression of P-450h. However, in Hx male rats without GH supplementation, STZ treatment suppressed P-450h apoprotein and mRNA expression to 16% and 6% of nondiabetic Hx male levels, respectively, demonstrating the existence of GH-independent regulation of P-450h expression. In Hx female rats, P-450h apoprotein levels were 40% of those in intact control males and were not significantly decreased by STZ. Concomitantly, STZ produced a greater decrease in serum insulin levels and a greater increase in serum glucagon in Hx male rats than in Hx females. The results provide evidence for the existence of STZ-sensitive GH-independent expression of P-450h and further document the gender differences in STZ sensitivity. PMID- 1706266 TI - Passive immunization against insulin-like growth factor-I does not inhibit growth hormone-stimulated growth of dwarf rats. AB - Passive immunization against insulin-like growth factor-I (IGF-I) was undertaken in GH-deficient rats in an attempt to elucidate the relative importance of the endocrine vs.autocrine/paracrine actions of IGF-I in stimulating growth. Antiserum against IGF-I was raised in sheep and purified by affinity chromatography. The ability of the purified antibodies to neutralize the actions of IGF-I in vitro and bind IGF-I in vivo were extensively tested using L6 myoblast and cartilage bioassays. Four groups of male rats with isolated GH deficiency were used in the study. At 49 days of age the rats received 100 microliter normal saline given sc each day for 10 days, 2 mg/kg recombinant bovine GH (bGH) given in 100 microliter, sc, each day, 2 mg/kg bGH, sc, and 300 microliter immunoglobulin G purified from normal sheep serum given daily ip, or 2 mg/kg bGH plus 300 microliter anti-IGF-I immunoglobulin G daily, ip (a dose that was able to completely inhibit IGF-I actions on sulfate uptake into cartilage). Treatment with GH significantly increased growth rates (P less than 0.001) in the rats, but there was no difference between any of the three GH-treated groups; passive immunization against IGF-I did not diminish the GH-stimulated growth in these rats. Excess antibody could be detected in the plasma of all anti-IGF-I treated rats at the conclusion of the experiment, and the antibody was capable of sequestering both free and binding protein-bound IGF-I. The absence of even a slight retardation of GH-stimulated growth in the anti-IGF-I-treated rats suggests that circulating IGF-I may not be important in mediating the growth promoting actions of GH, although the immunoneutralization probably does not affect GH stimulation of tissue IGF-I production. PMID- 1706267 TI - Testicular oxytocin gene expression in seminiferous tubules of cattle and transgenic mice. AB - We are using transgenic mice to study the regulation of the bovine vasopressin (VP) and oxytocin (OT) genes. Prompted by the observation that mice bearing a bovine OT transgene express bovine OT RNA in their testes, we investigated the expression of the VP-OT locus in normal mice and cattle. Normal wild-type mice do not have detectable levels of either VP or OT RNA in their testes. Normal cattle are also devoid of detectable VP transcripts, but have relatively high levels of testicular OT RNA. Additionally, OT, but not VP, peptide is detectable by HPLC. In situ hybridization to RNA in bovine testicular tissue sections localized OT transcripts to seminiferous tubules, with a distribution similar to that of alpha inhibin, suggesting expression in Sertoli cells. Interestingly, the bovine OT RNAs in the transgenic mouse testes were also shown by in situ hybridization to have the same distribution. These data suggest that the cis-acting regulatory sequences responsible for expression of the OT gene in bovine Sertoli testis reside within the limits of the transgene used in this study. Further, the trans acting factors present in murine testicular cells are able to recognize these elements, although they do not express the endogenous mouse OT gene in this tissue. PMID- 1706268 TI - Vasopressin and oxytocin gene expression in rat testis. AB - The vasopressin (VP) gene is expressed as three different transcripts in the rat testis. Using polymerase chain reaction (PCR) analysis we have been able to identify a VP RNA that is identical in exonic structure to that found in the hypothalamus. However, the abundance of this form is very low, and it cannot be detected by Northern blotting. Two VP RNAs with a novel structure, as shown using exon-specific probes, are present in higher abundance. By differential hybridization, sequencing of a cDNA clone, and PCR we have deduced the structure of these novel transcripts. Both of the novel testicular VP RNA species share two exons with the classical hypothalamic RNA. However, the testicular VP gene derived RNA lacks the first exon of the hypothalamic transcript, the exon that contains the sequence information for the VP nonopeptide hormone. Instead, it has novel sequence that are derived from at least two unique testis-specific exons, one of which is located 7-10 kilobase up-stream of the brain-specific start of transcription. These two unusual transcripts are probably derived by alternative splicing of at least two up-stream exons. Sequence and polysome analyses indicate that the testicular VP RNAs are probably not translated. Northern blotting revealed that the VP gene-derived RNA species are tightly regulated during postnatal development, becoming apparent by 40 days of age, although they subsequently fail to respond to a variety of physiological perturbations. Oxytocin gene transcripts are not detectable by Northern hybridization, but the authentic hypothalamic-type RNA can be detected in the rat testis using PCR analysis. PMID- 1706269 TI - Multiple abnormalities in insulin responses to nonglucose nutrients in neonatally streptozotocin diabetic rats. AB - Insulin responses to nutrient secretagogues were investigated in neonatally streptozotocin-injected (n-STZ) rats, i.e. an animal model of noninsulin dependent diabetes. In the perfused pancreas 16 mM L-glutamine induced and 10 mM octanoate tended to induce (P less than 0.2) higher responses in n-STZ than in nondiabetic rats. Addition of 3.9 mM glucose potentiated responses to glutamine and octanoate more in n-STZ (3.3- and 3.4-fold) than in nondiabetic rats (1.5- and 1.9-fold). Conversely, the succinate derivative succinate monomethylester (Succ ME) induced lesser response in n-STZ rats (57% of that in nondiabetic rats) and coperfusion with 3.9 mM glucose increased the response less in n-STZ (1.4 fold) than in nondiabetic rats (3.8-fold). Pyruvate (20 mM) mimicked the potency of 3.9 mM glucose, i.e. pyruvate potentiated the response to Succ ME only nonsignificantly (1.2-fold) in n-STZ but markedly (4.9-fold) in nondiabetic rats. Dichloroacetate (20 mM) failed to affect the response to Succ ME together with pyruvate in n-STZ rats. To investigate the role of hyperglycemia for octanoate induced secretion, nondiabetic rats were made hyperglycemic by 48-h glucose infusions. Octanoate-induced secretion from perfused pancreas was enhanced 3.8 fold after moderate hyperglycemia (13.2 +/- 0.6 mM) and 17-fold after marked hyperglycemia (22.7 +/- 0.6 mM). This positive association between response and degree of hyperglycemia was not found with a nonnutrient secretagogue, 3-isobutyl 1-methylxanthine. Results with glutamine and octanoate indicate that oxidation of nonglucose nutrients which normally do not regulate secretion is enhanced secondary to chronic hyperglycemia. Results with Succ ME and pyruvate suggest that early steps of oxidation of glucose are impaired in n-STZ rats. PMID- 1706270 TI - Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules. AB - Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once. PMID- 1706271 TI - Involvement of dihydropyridine-sensitive calcium channels in the GABAA potentiation of TRH-induced TSH release. AB - The effects of gamma-aminobutyric acid (GABA) and isoguvacine on the thyrotropin (TSH) secretion stimulated by thyrotropin releasing hormone (TRH), were investigated in vitro with perifused rat pituitaries. At nanomolar concentrations the two agonists induced potentiation of the TRH-induced TSH release. The potentiation was blocked by SR 95531 a specific GABAA antagonist. The isoguvacine potentiation of the TSH response to TRH failed to occur when cobalt (Co2+) was added to the perifused medium. Nifedipine completely blocked the GABA or isoguvacine potentiation of the TSH response while omega-conotoxin did not modify it. Pre-perifusion of the pituitaries with pertussis toxin did not change the TSH response to TRH but completely inhibited the isoguvacine potentiation of the response. Our results demonstrate that the GABA potentiation of TRH-induced TSH release occurring through the stimulation of GABAA receptor sites is a calcium (Ca2+)-dependent phenomenon, probably mediated by activation of dihydropyridine (DHP)-sensitive, omega-conotoxin-insensitive Ca2+ channels involving a pertussis toxin-sensitive G protein. PMID- 1706272 TI - Endotoxin-induced impairment of vascular smooth muscle contractions elicited by different mechanisms. AB - The current study was designed to analyse the mechanisms which are impaired in the vascular hyporeactivity to contractile agents induced by E. coli lipopolysaccharide endotoxin (LPS). Endothelium-denuded aortic rings were prepared from thoracic aorta removed from control and LPS-pretreated rats (20 mg/kg i.p., 4 h before the experiment). In order to determine whether LPS treatment altered the contractile components that depend on intracellular calcium release and extracellular calcium entry to the same extent, rings were contracted under various experimental conditions. The responses elicited by indanidine, phenylephrine (without and with nitrendipine 1 microM), (-) Bay K 8644, (+) S 202 791 and the calcium ionophore calimycin in the presence of 1.25 mM external CaCl2 were all impaired by LPS pretreatment (maximal contractions 19, 63, 44, 28, 22 and 22% of controls, respectively). Concentration-effect curves for CaCl2 made in depolarizing medium (KCl 40 and 100 mM) and in the presence of calimycin (3 microM) were shifted to the right in rings from LPS-pretreated rats. However, the LPS-induced depression of contraction was overcome by the addition of CaCl2 (up to 30 mM). Additionally, in the absence of external CaCl2, the contraction induced by caffeine (50 mM) was not significantly altered by LPS treatment. It is concluded that LPS treatment does not reduce the ability of aortic smooth muscle cells to contract. The results suggest that LPS treatment impairs mechanisms involved in calcium handling within smooth muscle cells after stimulation of calcium entry through different pathways and activation of intracellular calcium release by alpha 1-adrenoceptor agonists. PMID- 1706273 TI - Handling habituation and chlordiazepoxide have different effects on GABA and 5-HT function in the frontal cortex and hippocampus. AB - In slices of frontal cortex and hippocampus from rats that had been habituated to handling for 21 days, there was significantly less 20 mM K(+)-evoked release of [14C]GABA ([14C]gamma-aminobutyric acid) compared with rats naive to handling. Handling for 21 days also significantly increased the uptake of [14C]GABA into frontal cortex and hippocampus. The change in uptake in the hippocampus was independent of any changes in release and could account for the apparent change in evoked release; in the cortex there were no independent changes in uptake and K(+)-evoked release. When the changes in uptake were taken into account, there were no independent changes in basal release of GABA in either region. HPLC analysis showed the change in uptake was not due to differences between the groups in endogenous GABA concentrations. Acute administration of chlordiazepoxide (CDP 7.5 mg/kg i.p.) to handling naive rats also significantly reduced K(+)-evoked [14C]GABA release from the cortex and hippocampus, but basal release and GABA uptake were unchanged. Neither handling nor CDP administration significantly changed the K(+)-evoked [3H]5-HT release, however, the uptake of 5 HT and its basal release in both regions were both significantly and independently increased in animals habituated to handling, compared with handling naive animals. In the hippocampus, the endogenous 5-HT concentrations were significantly lower in the rats that had received 21 days of handling, compared with handling naive rats. In the cortex the endogenous 5-hydroxyindoleacetic acid concentrations were significantly lower in the group that had been handled for 21 days. Thus both the GABA and 5-HT systems were responsive to handling habituation. PMID- 1706274 TI - Effects of cromakalim, RP49356, diazoxide, glibenclamide and galanin in rat portal vein. AB - The study investigated the possible involvement of an ATP-sensitive potassium (K) channel in the relaxant actions of K channel openers in rat portal vein. The effects of glibenclamide on the relaxant responses and rises in 86Rb efflux evoked by cromakalim, RP49356 and diazoxide were studied. The effects of galanin and depletion of intracellular ATP concentrations [( ATP]i) were also examined. Galanin increased mechanical activity and 86Rb efflux, effects most likely mediated via galanin receptors rather than a direct action on a K channel. Glibenclamide inhibited the relaxant responses and rises in 86Rb efflux evoked by cromakalim, RP49356 and diazoxide. Reduction of [ATP]i caused relaxation and this effect was partially reversed by glibenclamide. The restored activity was abolished by cromakalim. These results suggest that an ATP-sensitive K channel is present on rat portal vein and that it may be involved in the relaxant actions of cromakalim, RP49356 and diazoxide. PMID- 1706275 TI - Ca2+ channel inhibition by a new dihydropyridine derivative, S11568, and its enantiomers S12967 and S12968. AB - Biochemical and electrophysiological techniques were used to describe the Ca2+ channel blocking properties of a new dihydropyridine derivative, S11568 (+/-)- ([(amino-2-ethoxy)-2-ethoxy]methyl)-2-(dichloro-2',3'-phenyl)-4- ethoxy-carbonyl 3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyridine and its enantiomers S12967 ((+) S11568) and S12968 ((-)-S11568). In binding studies, S11568 and S12968 displaced specifically bound [3H]PN 200-110 from cardiac and vascular smooth muscle preparations with potencies of 5.6-51 nM, respectively. S12967 was 6- to 18-fold less potent than S12968. A good correlation was found between the IC50 value for the inhibition of 45Ca2+ uptake by A7r5 aortic smooth muscle cells and binding data. Whole-cell patch clamp studies in both guinea-pig ventricular myocytes and A7r5 cells yielded similar results. At holding potential (VH) -50 mV, S12968 inhibited L-type Ca2+ current with an IC50 value near 70 nM, 2- to 3-fold more potently than S11568 and 30-fold more potently than S12967. With VH -100 mV, all three compounds were less potent, with IC50 values ranging from 500 nM to 3 microM. These results demonstrate conclusively that S12968 is the more active enantiomer. Furthermore, the pronounced voltage dependence of its actions in vitro suggests that in vivo it could exhibit good selectivity for vascular smooth muscle over cardiac muscle. PMID- 1706276 TI - Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using 1H NMR. AB - 1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis. PMID- 1706277 TI - Pyrimidine nucleotide and nucleic acid synthesis in human monocytes and macrophages. AB - In most cell types, the production of deoxynucleotides is tightly coupled to the pace of cell division, and nearly all deoxynucleotides are used for semiconservative DNA synthesis. The capacity of peripheral blood monocytes and macrophages to proliferate is controversial. However, these cells have been reported to produce and release thymidine, which can serve as a precursor or regulator of DNA synthesis by lymphocytes and other cells. To determine to what extent de novo pyrimidine nucleotide synthesis is linked to cell division in peripheral blood monocytes and macrophages, compared to human U937 promonocytes and CEM lymphoblasts, we used a precise precursor-product labeling method. The results showed that in all three cell types, the pace of pyrimidine deoxynucleotide production, and of thymidylate synthesis, was in proportion to the rate of DNA synthesis. The human blood monocytes and macrophages, in contrast to U937 cells, had extraordinarily low deoxyribonucleotide pools (less than 1 pmol/10(6) cells) and synthesized neither thymidylate nor DNA de novo during 7 days culture. Colony-stimulating factors augmented RNA synthesis in monocyte derived macrophages, and enhanced cell survival, without inducing either DNA or thymidylate synthesis. We conclude that the thymidine released by macrophages derives from dead or dying cells, and not from de novo synthesis. PMID- 1706278 TI - Cell/substratum adhesions in RSV-transformed rat fibroblasts. AB - Cell/substratum adhesions have been studied in rat fibroblasts transformed by a ts-mutant of Rous sarcoma virus (LA-29) using light and electron microscopy and a variety of preparative methods including immunolabeling. Cells were studied both during the process of transformation, i.e., shifting from 39 degrees to 35 degrees C, and in a fully transformed state (passaged at 35 degrees C continuously). The typical focal contacts observed at 39 degrees C (restrictive temperature) were replaced by "point-contacts" (100-200 per cell) which were classified by immunolabeling as podosome-like adhesions containing actin, beta 1 integrin subunit, vinculin, talin, alpha-actinin, and small membrane patches containing clathrin and integrin. Tyrosine-phosphorylated proteins and pp60src were found in association with groups of small particles on the protoplasmic surface of ventral membranes by gold immunolabeling. Both types of point-contacts were visualized by electron microscopy of ultrathin sections and shadowed replicas and characterized by gold immunolabeling wherever possible. The overall composition of podosome-like adhesions is similar to focal contacts but there are differences in the three-dimensional organization of the microfilaments and in the topography of vinculin which is associated more with actin filaments than with the plasma membrane. The presence of talin and extracellular matrix receptor in podosomes together with the adhesive properties of these actin-containing structures argues against the hypothesis that pp60src affects the interaction of actin with the plasma membrane by phosphorylating the fibronectin receptor and/or other associated proteins. PMID- 1706279 TI - Immunological localization of cystic fibrosis candidate gene products. AB - The recent identification of the cystic fibrosis (CF) gene and its putative protein product, the CF transmembrane conductance regulator (CFTR), enabled us to synthesize oligopeptides corresponding with a predicted extracellular domain (position 103-117; peptide A) and a cytoplasmic domain (position 501-515; peptide B) constituting the phenylalanine deletion (F 508) observed in the majority of CF mutations. Immunobiochemical studies with antibodies directed against these peptides revealed the presence of two CFTR candidate proteins (155 and 195 kDa) in various types of epithelial cells. Immunolocalization studies performed on slices of human duodenum showed the strongest expression in the endoplasmic reticulum (RER) of the mucus-producing Goblet cells. Labeling is also demonstrated in the RER and apical membranes of villus and crypt cells, however, to a weaker extent. PMID- 1706280 TI - [The mechanisms of the realization of the effect of glucocorticoids in aging]. AB - The interaction of glycocorticoids with serum transport proteins, plasma membranes and rat liver cytoplasmic receptors progressively declines during ontogenesis, reaching its minimum at 24 months of age. Glycocorticoid receptor complexes (GRCs) binding to the rat liver nuclei and their residual fractions as well as the glycocorticoid-induced initiation of RNA-synthesis also decrease with age. The GRCs are shown to be capable of association with nuclear envelope, nuclear matrix and RNA-containing nuclear fraction isolated from the rat liver. The intracellular glycocorticoid receptor recycling requires fresh synthesis of the RNA and protein. PMID- 1706281 TI - Signal transduction mechanisms through Fc gamma receptors on the mouse macrophage surface. AB - Mouse macrophages and macrophage cell lines such as P388D1 or J774 carry at least two distinct Fc gamma receptors (Fc gamma R): one specific for the Fc portion of IgG2a (Fc gamma aR, also classified as Fc gamma RI) and another for IgG2b (Fc gamma 2bR, also classified as Fc gamma RII beta). These Fc gamma Rs should transmit, upon binding of an appropriate ligand, a specific signal that leads to the regulation of macrophage functions, as the interaction of immune complex with cell surface receptor has been shown to lead to suppression of the humoral immune response or B cell differentiation, to the destruction of target cells by antibody-dependent cell-mediated cytotoxicity, to activation of arachidonic acid metabolic cascade, to the phagocytosis of opsonized particles, or to the generation of superoxide anion. In this review, we first describe evidence that Fc gamma 2aR and Fc gamma 2bR are associated with casein kinase II and phospholipase A2 activity, respectively. We will then discuss a potential role for these enzymatic activities in signal transduction pathways that leads to the activation of the arachidonic acid metabolic cascade and adenylate cyclase, to the regulation of phagocytosis, and to the suppression of interferon-gamma action to induce Ia antigens. PMID- 1706282 TI - The effect of long-acting somatostatin analogue on enzyme changes after endoscopic pancreatography. AB - The effect of the long-acting somatostatin analogue, octreotide acetate (Sandostatin) on enzyme elevation after endoscopic pancreatography was studied in a prospective, randomized, double-blind trial. Sixty-three consecutive patients undergoing ERCP were randomly allocated to two group. In the control group, 34 patients received isotonic sodium-chloride, and in the treated group 29 patients received 0.1 mg of octreotide acetate subcutaneously before the pancreatography. After the endoscopy, amylase levels increased to pathological range in 15 of the controls and in 3 of the treated patients, whereas lipase levels showed a pathological rise in 17 of the controls and in 5 of the treated patients. A significant difference (p less than 0.01) was observed in the amylase and lipase changes between the two groups at 90 and 180 min after pancreatography. The enzyme levels showed at 90 min, mean +/- SD amylase: controls 540 +/- 185 units/liter, treated patients 261 +/- 108 units/liter; lipase: controls 304 +/- 98 units/liter, treated patients 198 +/- 88 units/liter. These findings suggest that the use of long-acting somatostatin analogue ameliorates the enzyme increases in the serum after endoscopic pancreatography. PMID- 1706283 TI - Endoscopic four quadrant tattoo for the identification of colonic lesions at surgery. PMID- 1706284 TI - Colonic tattoo for follow-up of endoscopic sessile polypectomy. PMID- 1706285 TI - Fat necrosis and inflammatory pseudotumor due to endoscopic tattooing of the colon with india ink. PMID- 1706287 TI - Photodynamic therapy for completely obstructing esophageal carcinoma. PMID- 1706286 TI - Colonic abscess and focal peritonitis secondary to india ink tattooing of the colon. PMID- 1706288 TI - India ink colonic tattoo: blots on the record. PMID- 1706289 TI - Localization of actin and characterization of its isoforms in the hyphae of Neurospora crassa. AB - The actin of Neurospora crassa wild type Strain St. Lawrence has been purified, characterized and localized. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-Sepharose. This protein was identified as actin on immunoblots when an anti-actin monoclonal antibody raised against chicken gizzards was used as a probe. After two-dimensional gel electrophoresis three actin isoforms were detected. The distribution of actin in hyphae was examined by FITC-phalloidin staining of formaldehyde fixed hyphae. F-actin was found to be mainly concentrated in the hyphal tips in which it formed a uniform cap. Apical actin could be involved in hyphal morphogenesis, organelle motility and maintenance of polarity. PMID- 1706290 TI - Characterization of enhancer-of-white-apricot in Drosophila melanogaster. AB - The white-apricot (wa) allele differs from the wild-type white gene by the presence of the retrovirus-like transposable element copia within the transcription unit. Most RNAs derived from wa have 3' termini within this insertion, and only small amounts of structurally normal RNA are produced. The activity of wa is reduced in trans by a semidominant mutation in the gene Enhancer-of-white-apricot (E(wa). Flies that are wa and heterozygous for the enhancer have eyes which are much lighter than the orange-yellow of wa alone while E(wa) homozygotes have white eyes. This semidominant effect on pigmentation is correlated with a corresponding decrease in white RNA having wild type structure, and flies homozygous for E(wa) have increased levels of aberrant RNAs. Three reverant alleles of E(wa) generated by reversion of the dominant enhancer phenotype with gamma radiation are noncomplementing recessive lethals, with death occurring during the larval stage. The effects on wa eye pigmentation of varying doses of the original E(wa) allele, the wild type allele, and the revertant alleles suggest that the original E(wa) allele produces a product that interferes with the activity of the wild type gene and that the revertants are null alleles. We propose that the E(wa) gene product influences the activity of the downstream copia long terminal repeat in 3' end formation. PMID- 1706291 TI - Circumsporozoite protein genes of malaria parasites (Plasmodium spp.): evidence for positive selection on immunogenic regions. AB - The circumsporozoite (CS) protein is a cell surface protein of the sporozoite, the stage of the life cycle of malaria parasites (Plasmodium spp.) that infects the vertebrate host. Analysis of DNA sequences supports the hypothesis that in Plasmodium falciparum, positive Darwinian selection favors diversity in the T cell epitopes (peptides presented to T cells by host MHC molecules) of the CS protein. In gene regions encoding T cell epitopes of P. falciparum, the rate of nonsynonymous nucleotide substitution is significantly higher than that of synonymous substitution, whereas this is not true of other gene regions. Furthermore nonsynonymous nucleotide substitutions in these regions cause a change of amino acid residue charge significantly more frequently than expected by chance. By contrast, in Plasmodium cynomolgi, the same regions show no evidence of positive selection, and residue charge is conserved. The CS protein has a central repeat region, which is the target of host antibodies. In P. falciparum, the amino acid sequence of the repeat region is conserved within and between alleles. In P. cynomolgi, on the other hand, there is evidence that positive selection has favored evolution of two different repeat types within a given allele. PMID- 1706292 TI - Cloning and characterization of the scalloped region of Drosophila melanogaster. AB - Viable mutants of the scalloped gene (sd) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry+] transposon insertion at 13F associated with sd[ry+2216] served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles were characterized by genomic hybridization analysis as a means of defining the extent of the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a "hot spot" for P element activity. Four of five recessive lethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12-14 kb away from the region of viable lesions. In a sd+ genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd58, used for comparison in the transcript analysis, makes at least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones has identified three structurally related transcripts that hybridize to this genomic region. The 5' end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view that the transcripts represented by these cDNA clones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing. PMID- 1706293 TI - Identification of a sterility-inducing cytoplasm in a fertile accession line of Phaseolus vulgaris L. AB - Previous investigations into the genetic mechanism of fertility restoration in cytoplasmic male sterile Phaseolus vulgaris suggested that this is a particularly interesting system for the study of nuclear-mitochondrial interactions. This study was conducted to investigate the nature of nuclear-mitochondrial compatibility in fertile accession line G08063, the reported progenitor to the cytoplasmic male sterile line. Results from genetic analysis indicated that fertile line G08063 carried a sterility-inducing cytoplasm with a fertility restoring nuclear genotype. Mitochondrial DNA analysis indicated that the mechanism of fertility restoration by line G08063 was different from that conditioned by Fr, a previously described restorer gene. A mitochondrial DNA sequence associated with sterility and lost upon fertility restoration by nuclear gene Fr was present in the mitochondrial genome of fertile line G08063; this sequence was not carried within the mitochondrial genome of any other P. vulgaris accession line tested. PMID- 1706294 TI - Molecular mapping of the active site of an aging antigen: senescent cell antigen requires lysine(s) for antigenicity and is located on an anion-binding segment of band 3 membrane transport protein. AB - An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG binding. The active antigenic sites of the aging antigen have been localized to residues 538-554 and 778-827. Two peptides within these regions interact synergistically to generate a synthetic aging antigen that is an effective inhibitor of senescent cell IgG binding to old cells. We synthesized peptides corresponding to these residues (pep-ANION 1: SKLIKIFQDHPLQKTYN, and pep-COOH: LFKPPKYHPDVPYVKR). These are extracellular regions of band 3 containing lysines which are implicated in anion transport. The contribution of lysine to the antigenicity of the aging antigen and to anion transport was examined by chemically modifying the lysines on both synthetic peptides and whole cells, and by synthesizing peptides in which glycines or arginines were substituted for lysines. Anion transport sites were localized using 16- to 18-mer peptides followed by 6- to 8-mer peptides. Functional studies with the peptide pep-COOH indicate that it contains sulfate binding sites and inhibits sulfate transport in addition to carrying aging antigenic determinants. Substitution of arginines or glycines for lysines in pep COOH reduces the sulfate-binding properties of the peptide although significant inhibition still occurs. Residues 812-827 (pep-COOH) and 813-818 (N6, the six amino acids on the amino side of pep-COOH) and 822-839 are inhibitors of anion transport when used in equimolar amounts with sulfate suggesting that these regions may be transport regions in situ. Results of this study indicate that: (a) lysines are required for the integrity of the aging antigenic site; (b) pep COOH (residues 812-827) is part of senescent cell antigen and an anion-binding site; (c) pep-ANION 1 (538-554), which has been reported to be a transport segment of band 3, does not bind sulfate; (d) residues 588-602 are part of an anion binding/transport segment; (e) band 3 residues 822-839 are part of an anion binding/transport site, and (f) lysines contribute to anion binding but are not the only amino acid(s) required for anion binding and, thus, anion transport. PMID- 1706295 TI - Interaction of thrombin with endothelial cells in the presence of fibrinogen and alpha 2-macroglobulin. AB - Binding of thrombin to cultured endothelial cells has been studied in the presence of fibrinogen and alpha 2-macroglobulin. Both fibrinogen and alpha 2 macroglobulin inhibit the interaction of thrombin with endothelial cells. Whereas fibrinogen decreases the rate of activation by the thrombin-thrombomodulin complex of protein C, thrombomodulin inhibits the rate of inactivation by alpha 2 macroglobulin thrombin. alpha 2-macroglobulin also binds to endothelial cells; (Kd = 3 x 10(-7) M with 3 x 10(5) binding sites/cell), and the rate of binding of the alpha 2-macroglobulin to endothelial cells is faster than its complex formation with the thrombin. The data suggest that essentially the cell-bound form of fibrinogen and alpha 2-macroglobulin influences thrombin binding and functions. PMID- 1706296 TI - The effect of gamma-hexachlorocyclohexane (lindane) on blood cells, kidney and liver tissues in rabbits. AB - The effect of intragastric administration of gamma-hexachlorocyclohexane (lindane) on peripheral blood cells, kidney and liver was studied in rabbits. White blood cells were found to be most affected by lindane, as was shown by the reduced phagocytic activity of neutrophils and the increased number of lymphocytes with inactive nucleoli. The changes in the characteristics of erythrocyte cell membranes depended on the length of time lindane was administered and on its withdrawal. Histological examination of the liver and kidney showed focal degeneration of tissue and cell structure. PMID- 1706297 TI - Clear cell basal cell carcinoma. AB - We describe a case of clear cell basal cell carcinoma of the superficial type, presenting as a crusted eruption on the abdomen. Histological examination showed a solid proliferation of clear cells attached to the under-surface of an atrophied epidermis. In addition, distinct pagetoid infiltration was seen within the overlying epidermis. A focal connection between the clear cell portion and a deeper lying nodular basal cell carcinoma was demonstrated, elucidating the true nature of the lesion. Immunohistochemical studies and electronmicroscopy confirmed the epithelial derivation of the tumour. The clear cell appearance was due to multiple cytoplasmic electronlucent vacuoles which were not surrounded by membranes. PMID- 1706298 TI - Osteoclast-like giant cell tumour of the urinary bladder. AB - We report two cases of osteoclast-like giant cell tumour of urinary bladder associated with papillary transitional cell tumours. Both cases were morphologically identical to giant cell tumour of bone. The giant cells stained strongly for acid phosphatase which was resistant to tartrate digestion, a staining reaction typical of osteoclasts. In view of the ability of urinary bladder to induce metaplastic and neoplastic bone, we believe that these tumours may represent extraosseous giant cell tumours of bone. PMID- 1706299 TI - Clear cell carcinoma of minor salivary glands. AB - Two cases of carcinoma of the minor salivary glands are presented in which most cells had clear cytoplasm. Both patients had clinical histories in excess of 10 years and, in the one case with adequate follow-up, no recurrence had occurred after a further 11 years. Both tumours were locally invasive. The clear cells contained small amounts of glycogen, but no intracytoplasmic mucin. Immunohistochemical and ultrastructural studies showed epithelial features, with no evidence of myoepithelial differentiation. These tumours were very similar to the small number of previously reported cases, which were all considered to be low-grade carcinomas. Amongst the differential diagnoses, the most important is metastatic clear cell carcinoma of the kidney and this can only be confidently excluded clinically or by the use of imaging techniques. In summary, we consider intraoral clear cell carcinoma to be a distinct tumour of low malignant potential. PMID- 1706300 TI - Cerebral ganglioglioma with anaplastic oligodendroglial component. AB - We report an unusual and possibly unique example of cerebral ganglioglioma with an anaplastic oligodendroglial component. The latter was documented on both morphological and immunohistochemical grounds. Immunohistochemically, the anaplastic cells were strongly positive with the monoclonal antibody anti-Leu-7, while they lacked glial fibrillary acid protein, vimentin and neurofilaments. PMID- 1706301 TI - Cytokeratins in plasmacytomas. PMID- 1706302 TI - Malignant mixed mullerian tumors: an immunohistochemical study of 47 cases, with histogenetic considerations and clinical correlation. AB - Forty-seven cases of malignant mixed mullerian tumors were reviewed histologically and studied immunohistochemically with three major objectives: to analyze the histogenetic relationship between the carcinomatous and sarcomatous components of these neoplasms, to ascertain the practical role of immunohistochemical studies in diagnosis and classification, and to determine the prognostic significance of immunohistochemically verified rhabdomyoblastic and neuroendocrine differentiation. Epithelial differentiation (cytokeratin and/or epithelial membrane antigen expression) was confirmed in all carcinomatous components; within the sarcomatous areas, it was identified among individual cells (60% of cases) and within poorly formed clusters of cells (57% of cases). There was a statistically significant tendency for concordant expression of alpha 1-antichymotrypsin, Leu-M1, S-100, Leu-7, and neuron-specific enolase between the carcinomatous and sarcomatous components of individual cases. These two findings provide evidence of common origin for the sarcomatous and carcinomatous components of these neoplasms. Histologic review of metastases in 21 cases revealed a biphasic composition in the majority of metastatic lesions (62%), another feature that further supports a common origin for the two components. From a practical standpoint, immunohistochemistry may be helpful in accentuating the biphasic pattern of these neoplasms and in verifying the presence of rhabdomyoblastic differentiation. In most cases, however, careful morphologic examination and thorough sampling will suffice for correct diagnosis and subclassification. The presence of heterologous, rhabdomyoblastic, or neuroendocrine differentiation did not have a statistically significant influence on survival; the last of these was associated with a tendency for a more rapidly fatal course. PMID- 1706303 TI - Immunohistochemistry of endometrial stromal sarcoma. AB - Twenty-three cases (12 low grade, 11 high grade) of endometrial stromal sarcoma were studied with monoclonal antibodies to vimentin, keratin, desmin, muscle actin, epithelial membrane antigen, and collagen type IV, using the avidin-biotin immunoperoxidase method. Tumors were highly variable in the expression of these antigens. Some tumors contained both epithelial and smooth muscle-related antigens; others were immunoreactive only for the intermediate filament vimentin. Immunoreactivity patterns for metastases or recurrences were similar to the respective primary tumor and no correlation was observed between tumor grade and antigen expression. Normal myometrium, when present, was keratin-positive and variably epithelial membrane antigen-positive. We conclude that endometrial stromal sarcoma, as well as normal myometrium, may express both epithelial and/or muscle-related antigens. These findings most likely reflect a common mesodermal mullerian derivation and illustrate the intimate relationship of the endometrial stromal cell to the endometrial glands and myometrium. Knowledge of these immunoreactivity patterns is essential when evaluating poorly differentiated uterine tumors or spindle cell tumors presenting in extrauterine locations. PMID- 1706304 TI - Differential antigen preservation during tissue autolysis. AB - Immediate fixation or snap freezing of tissue is ordinarily done to maximize antigen preservation for immunocytochemistry; however, delay in tissue allocation or spontaneous lymph node infarction can render tissue suboptimal for immunostaining. To test the effects of tissue autolysis/necrosis on the preservation of various lymphoid, epithelial, and mesenchymal markers, two lymph nodes (one with reactive lymphoid hyperplasia and one with metastatic ductal breast carcinoma) were evaluated for immunocytochemically demonstrated antigen preservation at 0-, 4-, 8-, 12-, 24-, 48-, and 72-hour intervals of autolysis at 37 degrees C. All specimens were stained by frozen section and formalin-fixed paraffin section immunocytochemical reactions with antibodies against CLA (CD45), UCHL-1 (CD45RO), L-26, kappa, lambda, anti-epithelial keratins (AE-1 and AE-3), epithelial membrane antigen, and vimentin. Frozen sections were additionally stained for Leu-1 (CD5), Leu-2a (CD8), Leu-3a+b (CD4), Leu-4 (CD3), and Leu-14 (CD22). The most resilient lymphoid antigen preservation was observed with CLA and UCHL-1, both exhibiting immunoreactivity at 72 hours in both frozen and fixed preparations. L-26 showed similar reactivity in frozen sections, but detectable antigen was observed only up to 24 hours in formalin-fixed tissue. Leu-2a proved to be the most labile antigen, persisting for only 12 hours in frozen sections. The epithelial markers epithelial membrane antigen and AE-1 exhibited excellent antigenic preservation in both frozen and fixed preparations; AE-3 persisted well in frozen section but was not demonstrated in fixed tissue. Vimentin immunoreactivity was vastly superior in frozen, as compared with fixed, tissue sections. Most antigens showed remarkable preservation despite morphologic degradation; however, differential antigenic resilience was demonstrated. Knowledge of this variation in antigen decay is critical for evaluation of immunoperoxidase phenotypic studies of autolyzed or necrotic tissue. PMID- 1706305 TI - The use of immunohistochemistry in metastatic prostatic adenocarcinoma to the breast. AB - Since the introduction of hormonal therapy for the treatment of metastatic prostatic adenocarcinoma, there have been 33 reports of metastases of prostate carcinoma to the breast. We report two cases of diethylstilbestrol (DES)-treated patients with metastatic prostate adenocarcinoma who developed breast masses. The lesions had infiltrative patterns simulating primary breast carcinoma. Immunoperoxidase stains, prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP) were positive, identifying these cases as metastatic prostatic carcinoma to the breast. Differentiating primary from secondary tumors in these patients is difficult since there have been 10 reports of primary breast carcinoma occurring in DES-treated patients with prostatic adenocarcinoma. Their differentiation is important to direct appropriate therapy, and PSA and PAP immunoperoxidase stains are important in their correct classification. PMID- 1706306 TI - Characterization of a cell line derived from rhabdoid tumor of kidney. AB - Rhabdoid tumor of kidney (RTK) is a rare, highly malignant childhood neoplasm of uncertain histogenesis. Several recent studies have described considerable histochemical heterogeneity among cases of RTK, with confusing combinations of epithelial, mesenchymal, myogenous, and neuroepithelial markers in some tumors. The present study characterizes the histology, ultrastructure, histochemistry, cytogenetics, and oncogene expression in a cell line derived from RTK. The surgical specimen, nude mouse xenograft, and cell cultures demonstrated characteristic intermediate filament whorls by electron microscopy and expressed vimentin (diffusely) and cytokeratin (focally, in hyaline cytoplasmic inclusions) without detectable desmin, Thy-1, or epithelial membrane antigen. S-100 protein was absent in the surgical specimen and heterotransplant, and was seen very weakly and focally in the cell cultures. Light microscopic features of cultures were unchanged by several compounds (tissue plasminogen activator, nerve growth factor, cyclic adenosine monophosphate) which induce differentiation of some other pediatric neoplasms. The growth factor requirements of RTK cultures indicate a cell with mesenchymal features. Insulin-like growth factor-2 mRNA was detected in the RTK and in three Wilms' tumors also studied. Unlike most Wilms' tumors, RTK expresses the c-myc rather than the N-myc oncogene. PMID- 1706307 TI - Intestinal ganglioneuromatosis: mucosal and transmural types. A clinicopathologic and immunohistochemical study of six cases. AB - Six cases of intestinal ganglioneuromatosis (GN) included in this study reveal the occurrence of two morphologic patterns. Transmural GN was characterized by neural hyperplasia in all layers of the bowel wall with predominant involvement of the myenteric plexus. It was found in three patients affected by multiple endocrine neoplasia IIb. Mucosal GN, having predominant involvement of the mucosa without concomitant hyperplasia of the myenteric plexus, was associated with von Recklinghausen's disease, adenocarcinoma of the colon, and multiple adenomas with megacolon in one case each. Clinicopathologic correlations and review of the literature suggest that mucosal GN might represent a distinct entity with a lower morbidity rate than the transmural variant. Immunohistochemical stains reveal considerable heterogeneity. S-100 protein, neuron-specific enolase, and synapto physin immunostaining followed the distribution of the nervous hyperplasia in the different intestinal layers as identified morphologically and allowed precise determination of the proliferating cells. Increased reactivity for vasoactive intestinal polypeptide, opioid peptides leu-enkephalin and met-enkephalin, and substance P was present in all cases with transmural involvement; mucosal GN showed normal reactivity for opioid peptides and focal increased staining for substance P (one case) and vasoactive intestinal polypeptide (two cases) in the lamina propria. Mild increased immunoreactivity for tyrosine hydroxylase was present in the myenteric plexus of four out of four cases. Histochemical determination of acetylcholinesterase, performed in one case of transmural type, demonstrated hyperplasia of parasympathetic fibers and neurons. Electron microscopic study of another case suggested the presence of several neurotransmitters. These results indicate that the physiopathology of GN is related to a complex hyperplasia of several peptidergic, cholinergic, and probably adrenergic nerve fibers instead of a selective overgrowth of one type of nerve fiber. PMID- 1706308 TI - Identification and quantification of aberrant crypt foci and microadenomas in the human colon. AB - The objective of the present study was to determine whether aberrant crypt foci (ACF) similar to those observed in the colons of experimental animals exposed to colon carcinogens could be identified and quantified in the human colon. Twenty seven colon resections from patients affected by familial adenomatous polyposis (FAP, five cases), colorectal cancer (CRC, 12 cases), and benign diseases of the large bowel (BD, 10 cases) were collected from a pathology repository or immediately after operation. Ten or more 1-cm2 formalin-fixed, methylene-blue- stained samples of colonic mucosa from each colon were scored under light microscopy for ACF. The number of ACF per cm2 and the number of crypts per ACF for each colon were calculated. The average number of ACF per cm2 in the FAP group (20 +/- 19, mean +/- SD) was significantly higher (P less than 0.01) than those of the CRC (0.37 +/- 0.41) and BD (0.18 +/- 0.35) groups. At least one ACF was found in every colon resection from CRC patients and in six out of 10 colon resections from the BD group. The average number of crypts per ACF ranged from five to 35 with absolute values from 1 to over 100. Fifty-five histologic specimens, 43 with ACF of various size and 12 without, were prepared by sectioning the colon parallel to the mucosal surface. There was a close correlation between the number of crypts per ACF in each specimen as scored by methylene-blue and histologic examination. Twenty-six aberrant crypt foci displayed dysplasia as evident by histologic analysis. In these instances we feel the term microadenoma is appropriate and, using this unique approach of examining the human colon, they can be easily identified and quantified. These lesions may well be precursors for adenomatous polyps and colorectal cancer. PMID- 1706309 TI - A yeast artificial chromosome contig encompassing the cystic fibrosis locus. AB - The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions. PMID- 1706310 TI - Structure of the gorilla alpha-fetoprotein gene and the divergence of primates. AB - The sequence of the gorilla alpha-fetoprotein gene, including 869 base pairs of the 5' flanking region and 4892 base pairs of the 3' flanking region (24,607 in total), was determined from two overlapping lambda phage clones. The sequence extends 18,846 base pairs from the Cap site to the polyadenylation site, and it reveals that the gene is composed of 15 exons, which are symmetrically placed within three domains of alpha-fetoprotein. The deduced polypeptide chain is composed of a 19-amino-acid leader peptide, followed by 590 amino acids of the mature protein. The RNA polymerase II binding site, TATAAAA, and the promoter element, CCAAC, are positioned at -21 and -65 from the Cap site, respectively. The polyadenylation signal, AATAAA, is located in the last exon, which is untranslated. The sequence for the gorilla alpha-fetoprotein gene was compared with that of the previously published human alpha-fetoprotein gene (P. E. M. Gibbs, R. Zielinski, C. Boyd, and A. Dugaiczyk, 1987, Biochemistry 26: 1332 1343). Four types of repetitive sequence elements were found in identical positions in both species. However, one Alu and one Xba DNA repeat within introns 4 and 7, respectively, of the human gene are absent from orthologous positions in the gorilla. The Alu and the Xba DNA repeats probably emerged in the human genome after the human/gorilla divergence and became established novelties in the human lineage. There are 363/21,523 mutational changes between human and gorilla, amounting to 1.69% DNA divergence between the two primate species. The value of 1.69% is lower than the 2.27% obtained from melting temperatures of hybrids between human and gorilla genomic DNA (C. G. Sibley and J. E. Ahlquist, 1984, J. Mol. Evol. 26: 99-121). At the protein level, Homo sapiens differs from Gorilla gorilla only at 4 of 609 amino acid positions (0.66%) in the alpha-fetoprotein sequence. This difference signifies a lower rate of molecular divergence for the alpha-fetoprotein gene in primates, as compared to rodents. PMID- 1706311 TI - Immune complexes as immunizing agents to increase the number of monoclonal antibody producing hybrids and to deviate the response to poorly immunogenic epitopes. AB - The use of immune complexes (IC) in an antibody excess, as immunizing agent, led to a large increase in the mouse polyclonal response to human SIgA. This enhanced response, as compared to SIgA alone, was analysed with mouse polyclonal anti alpha chain antibodies (Ab). A kinetic study showed an early rise (between days 14 and 21) of the antibody response against the discontinuous epitopes of SIgA while the anti-IgA response increase was delayed. Induction of hybridomas with an IC consisting in SIgA containing an excess of anti-alpha chain Ab, increased 10 fold the number of positive wells. Moreover, two of these MAb were specific for weakly immunogenic epitopes. One recognized only SIgA (anti-C), i.e. the association between the alpha chain and the secretory component (SC), while the other mainly combined to IgA dimers (anti-P). Both these MAb will be useful tools for structural studies and for the dosage of secretory Ab. PMID- 1706312 TI - Monoclonal antibodies that recognize human granulocyte-macrophage colony stimulating factor and neutralize its bioactivity in vitro. AB - We have produced monoclonal antibodies to bacterially synthesized, human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and have studied in detail the characteristics of three strongly neutralizing antibodies. The antibodies reacted with GM-CSF at high dilution (EC50 = 0.1-1.7 nM) in an indirect ELISA but did not react with murine GM-CSF or other cytokines. They also recognized glycosylated hGM-CSF produced by human lymphocytes. The antibodies were able to immunoprecipitate rhGM-CSF, but only reacted weakly with rhGM-CSF on Western blots, indicating that they recognized a conformation-dependent epitope. Cross-blocking studies showed that the three antibodies recognized overlapping epitopes. The antibodies inhibited binding of 125I-labeled rhGM-CSF to HL-60 cells at nanomolar concentrations and neutralized GM-CSF activity in two different bioassays. These antibodies thus provide a useful tool for analyzing the specificity of bioassays and for further studies of the production and function of GM-CSF in vitro and in vivo. PMID- 1706313 TI - Monoclonal antibodies specific to human ETS-2 oncoprotein: recognition of epitopes clustered on the B domain. AB - Six monoclonal antibodies were prepared from mice immunized with a bacterially expressed human ets-2 protein. These antibodies specifically recognize the two human ets-2-encoded proteins p56 and p54 but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ets-2 protein. Differential reactivities of these monoclonal antibodies with the peptide fragments generated by partial proteolytic digestion of the bacterially expressed ets-2 protein indicated that the six antibodies recognize at least three distinct epitopes in the B domain of the ets 2 protein. Immunoprecipitation experiments comparing native and denaturing conditions suggested that the ets-2 domain detected by the monoclonal antibodies is masked in the native condition by either protein folding or interacting proteins. The biochemical analysis of the ets-2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ets-2 protein in heterologous expression systems. PMID- 1706314 TI - Monoclonal antibodies against cardiac myosin heavy chain. AB - Three mouse IgG1 monoclonal antibodies (MAbs), named FA1, FA2, and FA3, against cardiac myosin heavy chain (MHC) with high specificity have been obtained. The immunogen used to generate these MAbs was the high-salt- and detergent-insoluble fraction of adult rat myocardial tissue. Western blots showed that these MAbs reacted with a 200 kD protein band, which comigrated with the heavy chain of purified rat cardiac myosin in SDS-PAGE. Immunofluorescence microscopy revealed that the antigen recognized by these MAbs was localized at the A-band of isolated myofibrils. The tissue-, species-, and isoform-specificities of these MAbs were examined by Western blots on various muscle samples. FA2 recognized fish, frog, chicken, rabbit, bovine, mouse and rat cardiac MHC, as well as rabbit skeletal and rat aorta smooth muscle MHC. This antibody reacted equally well with both alpha- and beta-isoforms of MHC. FA1 did not crossreact with any MHC tested so far but with rat cardiac MHC. It appeared to react only with alpha-isoform of MHC. FA3 recognized only rat, bovine and rabbit cardiac MHC with the specificity to bovine and rabbit atrial MHC. Elisa competition assay revealed that different epitopes on the antigen molecules were recognized by these three MAbs, although there was a partial overlap between the epitopes for FA1 and FA2. These anti-MHC MAbs will be most useful in investigating the expression of MHC during myocardial development. PMID- 1706315 TI - Production of monoclonal antibodies specific for platelet activation antigens and their use in evaluating platelet function. AB - Monoclonal antibodies (MAb) were used to identify platelet membrane molecules that are expressed after platelet activation. Balb/C mice were immunized with fixed thrombin-activated human platelets and their spleen cells were fused with the murine myeloma cell line NS1-Ag4/1. The resulting hybridomas were screened for antibody production against fixed thrombin-activated platelets and fixed resting platelets by flow cytometry. Two MAbs 2C8 (an IgM) and 1E3 (an IgG2a) demonstrated significant binding to fixed thrombin-activated platelets while reacting minimally with fixed resting platelets. The reactivity of 2C8 and 1E3 were compared to MAb's S12 and AC1.2, both of which have known specificity for an alpha-granule membrane protein (GMP-140) expressed on the surface of activated platelets. In radioimmunoprecipitation studies, both 2C8 and 1E3 immunoprecipitated a protein of approximately 140 kDa similar to that precipitated by S12 and AC1.2. Immunodepletion studies, with S12, AC1.2, 1E3, and 2C8 confirm that they all react with the same antigen. 2C8 may recognize the same epitope as S12, whereas 1E3 appears to recognize a different epitope of the same molecule. The use of these MAbs to measure platelet activation in whole blood correlates well with the results of conventional platelet aggregometry. PMID- 1706316 TI - Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by recombinant P. aeruginosa lipoprotein I and lipoprotein I-specific monoclonal antibodies. AB - Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of Oprl and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20). PMID- 1706317 TI - Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae. AB - The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae. Five monoclonal antibodies to P2 of four strains of nontypeable H. influenzae were developed by immunizing mice with whole bacterial cells. All five antibodies recognized epitopes on P2 in immunoblot assays of whole organism lysates, purified outer membrane, and purified P2. Competitive enzyme-linked immunosorbent assays and immunoblot assays of cyanogen bromide digested P2 showed that two antibodies to the P2 protein of strain 1479 recognized different epitopes on the molecule. Immunofluorescence and immunoelectron microscopy demonstrated that each of the five antibodies recognized epitopes that were abundantly expressed on the bacterial surface. Analysis of 120 H. influenzae strains indicated that three of the five antibodies were reactive exclusively with the homologous strain. The remaining two antibodies were reactive with less than 3% of the strains. These studies indicate that the P2 protein expresses a highly strain-specific and immunodominant epitope on the bacterial surface. The expression of strain-specific and immunodominant epitopes on the bacterial surface may represent a mechanism by which the bacterium induces antibodies that will protect against recurrent infection by the homologous strain but will not protect against infection by heterologous strains. PMID- 1706318 TI - Cell walls of normal and lysozyme-damaged blastoconidia of Candida albicans: localization of surface factor 4 antigen and vicinal-glycol staining. AB - The fungicidal effect of lysozyme on Candida albicans involves ultrastructural modifications previously described (G. Marquis, S. Montplaisir, S. Garzon, H. Strykowski, and P. Auger, Lab. Invest. 46:627-636, 1982). To further define the action of lysozyme on the yeast cell wall, we used the following: (i) the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method to highlight vicinal-glycol-reactive sites of complex carbohydrates; (ii) a monospecific antiserum and a protein A-gold complex to study the expression of surface factor 4, a major Candida antigen; and (iii) the periodic acid-silver methenamine method to stain cell wall glycoproteins. All Candida cells were found to express surface factor 4 antigen. In normal blastoconidia, surface factor 4 was located in a glycoprotein-rich cell wall layer, underneath radially oriented bundles of filaments which form the outermost wall layer. In lysozyme-treated blastoconidia, this glycoprotein-rich layer was lost and the regular brushlike organization of the outer fibrillogranular layer was disrupted. PA-TCH-SP staining and localization of surface factor 4 antigen demonstrated an altered arrangement of bundles of filaments in the outer wall layers of blastoconidia which were morphologically intact but had abnormal cell wall appearance. Next, there was a reduction in thickness of the outer layer and the expression of surface factor 4 antigen was limited to the cytoplasmic membrane area. Later on, the cell wall was almost uniformly highlighted by PA-TCH-SP staining. These data evinced a highly plastic architecture of the cell wall in C. albicans. PMID- 1706319 TI - Isolate and epitope variability in susceptibility of Giardia lamblia to intestinal proteases. AB - The surface antigens of Giardia lamblia differ. To determine whether the unique surface antigens found in variants and isolates could differentially protect the parasite from digestion by intestinal protease, G. lamblia clones WB-2X (WB), GS/M-H7 (GS/M), and B6, each of which expresses a unique surface variant antigen, were exposed to alpha-chymotrypsin and trypsin at concentrations up to 20 mg/ml in culture medium. The number of surviving trophozoites and morphologic changes were assessed over time. After 24 h, there was a significant decrease in the number of surviving trophozoites of WB (80.5 and 94.2% for trypsin and alpha chymotrypsin treatments, respectively, compared with controls) and B6 (78.9 and 95.5% for trypsin and alpha-chymotrypsin treatments, respectively, compared with controls) at 10 mg of enzyme per ml compared with culture medium alone. Cytotoxicity was prevented by the presence of soybean trypsin inhibitor, indicating the effects were due to protease activity. In contrast, there was no significant cytotoxicity after exposure of GS/M to either enzyme at the same enzyme concentration. After exposure to alpha-chymotrypsin, susceptible G. lamblia became rounded and then lysed, but after exposure to trypsin, G. lamblia appeared plastered onto the surface of the well and was intertwined and surrounded by finely granular material. Effects were concentration and time dependent; at least 6 h of treatment was required to observe changes 12 to 18 h later. Trophozoites surviving alpha-chymotrypsin or trypsin exposure became stably resistant to protease treatment. In vitro, the variant surface antigen of GS/M, but not those of WB or B6, resisted digestion by trypsin or alpha chymotrypsin, suggesting that the variant surface antigens impart susceptibility or resistance to digestion. The initial surface variant antigens of WB and B6 were replaced in resistant cultures. Trophozoites differ in their ability to survive after exposure to intestinal proteases, which may enable certain G. lamblia isolates or isolates possessing certain surface variant antigens to survive in the small intestine. PMID- 1706320 TI - Recombinant derivatives of Pasteurella multocida toxin: candidates for a vaccine against progressive atrophic rhinitis. AB - Potential vaccine components for protection against atrophic rhinitis in pigs were developed. This was achieved by deletion mutagenesis of the gene encoding the Pasteurella multocida toxin. Four purified toxin derivatives lacking different and widely separated regions in the amino acid sequence were characterized by a lack of toxic activity. One such component was shown to induce efficient protection of vaccinated female mice and their offspring against challenge with purified P. multocida toxin. PMID- 1706321 TI - Identification of linear B-cell determinants of pertussis toxin associated with the receptor recognition site of the S3 subunit. AB - Receptor recognition of pertussis toxin is mediated by the B oligomer consisting of subunits S2, S3, 2xS4, and S5. One possible way to interfere with toxin action would be the inhibition of recognition and binding of the cellular receptor(s) by preformed toxin-directed antipeptide antibodies. A prerequisite for this approach is the localization of linear antigenic determinants followed by the identification of inhibitory epitopes. Anti-S2 peptide antibodies have been shown to inhibit binding of the holotoxin to in vitro model receptor systems. For the elucidation of linear antigenic and immunogenic determinants harbored in the S3 subunit, synthetic peptides corresponding to selected linear amino acid sequences of S3 have been prepared and used to raise peptide-specific antibodies in rabbits. All peptides elicited a strong homologous response. Four synthetic peptides reacting with anti-pertussis toxin antibodies (R36-51, R87-95, R134-150, and R147-160) have been identified. Seven synthetic peptides (R1-12, R12-23, R14 29m, R36-51, R95-107, R134-150, and R164-178) induced antibodies recognizing pertussis toxin. Thus, these segments correspond to linear antigenic determinants. Analogous to the S2 subunit, the N terminus of S3 proved to be immunorecessive in the native toxin. The highly homologous S2 subunit was only bound strongly in Western blotting (immunoblotting) by antiserum directed at peptide R164-178, which is identical in the S2 and S3 subunits. A weak recognition of S2 in Western blotting was observed with anti-R95-107 antiserum. The ability of affinity-purified anti-S3 peptide antibodies to interfere with pertussis toxin binding was investigated by hemagglutination of goose erythrocytes as a model receptor system for S3-mediated receptor recognition. Antipeptide antibodies directed at R1-12, R12-23, R14-29m, and R36-51 inhibited hemagglutination of goose erythrocytes. This indicates that the corresponding antigenic regions in the S3 subunit are associated with the formation of the receptor binding domain. Inhibition of B-oligomer-mediated pertussis toxin binding to cellular receptors by preformed antipeptide antibodies of sufficient affinity should not only block the detrimental effects of the S1 subunits, but also interfere with the mitogenic effects attributed to the B oligomer. PMID- 1706323 TI - Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide. AB - A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism. PMID- 1706322 TI - Mapping of B-cell epitopes on the outer membrane P2 porin protein of Haemophilus influenzae by using recombinant proteins and synthetic peptides. AB - The P2 protein of Haemophilus influenzae type b has a porin activity and is the most abundant protein in the outer membrane. We have employed fusion protein constructs and synthetic peptides along with monoclonal antibodies to map B-cell epitopes in this protein. A linear, surface-exposed epitope was identified between residues 158 and 174. A second surface-exposed epitope was identified near the carboxy-terminal end of the protein (residues 319 to 341). Two additional B-cell epitopes were identified. One was localized between residues 28 and 55, whereas the other was located between residues 148 and 174. These epitopes were not present on the surface of intact H. influenzae cells. Thus, four distinct immunogenic and antigenic regions on the P2 protein have been identified. PMID- 1706324 TI - Escherichia coli O128 strains from infants with diarrhea commonly show localized adhesion and positivity in the fluorescent-actin staining test but do not hybridize with an enteropathogenic E. coli adherence factor probe. AB - Twenty-nine strains of Escherichia coli O128 isolated from infants with diarrhea that did not produce heat-stable enterotoxin, heat-labile enterotoxins, or Vero cytotoxin showed localized attachment to HEp-2 cells (LA). Only four strains hybridized with the enteropathogenic E. coli adherence factor (EAF) probe. One of the 25 LA+ EAF- strains attached to 72% of cells, while a plasmid-negative variant attached to 0.5% of cells. LA+ EAF- and LA+ EAF+ strains gave a positive fluorescent-actin staining test that correlates with the ability to cause attaching and effacing lesions in the intestine. The use of the EAF probe alone to detect LA+ strains is inadequate for epidemiological studies. PMID- 1706325 TI - Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D', I', and L2'. AB - The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp. PMID- 1706326 TI - A novel prostate carcinoma-associated glycoprotein complex (PAC) recognized by monoclonal antibody TURP-27. AB - A prostate carcinoma-associated antigen recognized by MAb TURP-27 was characterized immunohistochemically and biochemically. TURP-27 antigen was found localized in the cell membrane and cytoplasm of the ductal epithelial cells of normal (10%), benign (75-100%) and malignant (20-100%) prostate cells. Fetal prostate tissues were also found to express the TURP-27 antigen, suggesting expression early in development. This antigen was not expressed by non-prostate tumors examined, but significant cross-reactivity was observed in myelinated nerves while minor cross-reactivity was seen in certain lymphocyte subsets, cells in the adrenal medulla and chief cells of stomach. Immunoblotting and biochemical data demonstrated that the TURP-27 antigen is a sialic-acid-containing glycoprotein complex with major molecular species in prostate tissues of 310-250, 180, 140, 115, 95-90, 69, and 40- to 35-kDa. Immunoblotting patterns similar to those observed for prostate tissues were also seen in CNS extracts with the exception of the 69 and 40- to 35-kDa proteins. This prostate carcinoma associated sialoglycoprotein complex (PAC) recognized by MAb TURP-27 is likely to represent a novel tumor antigen expressed by prostate tumors. PMID- 1706327 TI - The high lysability by LAK cells of colon-carcinoma cells resistant to doxorubicin is associated with a high expression of ICAM-1, LFA-3, NCA and a less differentiated phenotype. AB - A human colon-carcinoma cell subline resistant to doxorubicin (LoVo/Dx), previously shown to be more lysed than the chemosensitive subline LoVo/H by different immune effectors, is reported here to be similarly susceptible to direct, anti-proliferative effect of soluble cytokines (TNF-alpha and/or IFN gamma). More adhesion molecules ICAM-1, LFA-3 and NCA were expressed on LoVo/Dx than on LoVo/H, while no significant amounts of CEA were detectable on the cell surface or in culture supernatant of either tumor subline. Anti-ICAM-1, anti-LFA 3 and anti-NCA monoclonal antibodies (MAbs) caused a marked reduction of lysis by interleukin-2 (IL-2) activated lymphocytes (LAK) of LoVo/Dx, whereas a lower effect was evident on LoVo/H. A pool of these antibodies was able to further increase the inhibition of the LAK lysis of both sublines. LoVo/Dx displayed a less differentiated phenotype as assessed by morphology, in vitro growth and altered or increased expression of markers such as desmoplakin and vimentin respectively, and disappearance of mucin. Treatment of LoVo sublines with differentiating agents (dimethylformamide and retinoic acid) led to a decreased expression of all adhesion molecules studied, accompanied by increased resistance to LAK-mediated lysis. These data indicate that sensitivity of chemoresistant tumor cells to cytotoxic effectors depends on the level of expression of adhesion molecules, including NCA, and is related to differentiation stage. PMID- 1706328 TI - In vitro and in vivo effect of salbutamol on histamine release from human basophils. AB - The effect of salbutamol, a selective beta 2-adrenergic agonist, on immunological release of histamine from leukocytes isolated from blood of 14 allergic, asthmatic patients, was investigated after in vivo or in vitro drug administration. Simultaneously, the effect of this drug on peak expiratory flow rate (PEFR) was estimated. Histamine was assayed by the single isotope-enzymatic method. Obtained results have confirmed that salbutamol is a rather poor inhibitor of histamine release from basophils. Although both administered salbutamol doses (0.5 or 1 mg, i.v.) significantly increased PEFR (p less than 0.01), inhibitory effect on histamine release by allergen was seen only after higher dose administration in vivo (p less than 0.01 against control). Similarly, a small but significant decrease in histamine release by both concanavalin A (by 20%, p less than 0.01) and allergen (by 30%, p less than 0.025) was seen in vitro after incubation of leukocytes with 8 x 10(-6) mol/l salbutamol, whereas 4 x 10( 7) mol/l salbutamol was without effect. PMID- 1706329 TI - Tumor localization and in vivo antitumor activity of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C-dextran conjugate. AB - The tissue distribution and in vivo antitumor activity of a novel monoclonal antibody-mitomycin C conjugate (A7-MMCD) composed of anti-human MAb A7 and MMC dextran conjugate were investigated using tumor-bearing mice. A7-MMCD was prepared via an anionic dextran intermediate for the purpose of keeping the non specific uptake by the reticuloendothelial system to a minimum. 111In-labeled A7 MMCD showed about a 5-times-greater accumulation in SW1116 (targeted tumor) than in S180 (non-targeted tumor) 48 h after injection, and produced a tumor-to-blood ratio which was 3 times higher in SW1116-bearing mice than in S180-bearing mice 96 h after injection. Accumulations in the liver, spleen, and kidney were also observed to some extent. Pharmacokinetic analysis revealed that A7-MMCD had nearly the same properties in the body as MMCDan (MMCD with an anionic charge), i.e., those of a negatively charged macromolecule. Both A7-MMCD and MMCDan had relatively similar tissue uptake rate indices for the liver and spleen. The tumor uptake rate index for SW1116 was about 2.5 times greater than that for S180, and the total amount of 111In-A7-MMCD accumulated in SW1116 was calculated to be approximately 5 times greater than the amount in S180. These results indicated that A7-MMCD could achieve site-specific targeting in the body. Furthermore, in the therapeutic experiment using SW1116 implanted subcutaneously, A7-MMCD suppressed tumor growth significantly, compared to free MMC and MMCDan. These results suggest that in designing an monoclonal antibody-drug conjugate via an intermediary, the physicochemical properties of intermediate macromolecules must also be taken into consideration to obtain a high degree of efficacy in vivo. PMID- 1706330 TI - Effect of weaning at different ages on serum insulin-like growth factor I (IGF I), IGF binding proteins and serum in vitro mitogenic activity in swine. AB - We investigated the effects of weaning or fasting of 21- or 35-d-old swine by monitoring serum mitogenic activity, circulating insulin-like growth factor I (IGF-I) and its binding proteins using L6 myoblast bioassays, RIA and ligand blotting techniques. Serum samples were collected from 21- or 35-d-old animals just before and 36 h after weaning or fasting. Sera from 21- and 35-d-old weaned animals were not significantly altered in their ability to promote myoblast proliferation, whereas sera from 21- and 35-d-old fasted animals caused 29 and 21% decreases (P less than .05) compared with preweaning. The mitogenic activity of control serum was inhibited by serum from fasted animals but not by preweaned or weaned sera. Serum IGF-I levels were decreased 65 to 70% (P less than .05) with weaning or fasting at both ages. Unoccupied binding sites on circulating IGF binding proteins in the 155 kDa range decreased 18 to 19% with weaning at both ages and decreased 40% (P less than .05) with fasting at 21 d but only 17% at 35 d. Ligand blotting revealed that the 43 and 39 kDa IGF binding protein bands decreased with weaning and fasting at both ages, whereas the 29-kDa band increased with weaning and fasting. These data indicate that serum IGF-I and specific IGF binding protein bands decrease during weaning or fasting at 21 and 35 d of age. However, serum mitogenic activity did not always follow serum IGF-I levels. PMID- 1706331 TI - Central action of tachykinins on activity of expiratory pumping muscles. AB - The central effects of tachykinins (substance P, neurokinin A, and neurokinin B) on the distribution of the motor activity to rib cage and abdominal expiratory muscles were studied in anesthetized tracheotomized spontaneously breathing dogs and cats. Intracisternal application of substance P (11 dogs) in doses of 10(-5) to 10(-4) M caused diaphragm electrical activity to change insignificantly from 19.3 +/- 1.9 to 24.8 +/- 3.2 units (P greater than 0.05), produced a moderate increase of triangularis sterni activity from 12.6 +/- 2.2 to 19.2 +/- 2.2 units (P less than 0.05), and stimulated a large increase of transversus abdominis activity from 9.4 +/- 2.7 to 28.5 +/- 2.6 units (P less than 0.01). Comparable effects were seen with similar doses of neurokinin A (8 dogs) and neurokinin B (3 dogs) administered intracisternally. Local application of substance P to the ventral medullary surface (5 dogs and 4 cats) also caused expiratory muscle activity to increase more than diaphragm activity, and in addition transversus abdominis activity increased to a larger extent than triangularis sterni activity. Furthermore, administration of the substance P antagonist [D-Pro2,D Trp7,9]-SP to the ventral medullary surface decreased respiratory motor output, with expiratory muscles activity being attenuated to a greater extent than diaphragm activity. Application of neurotensin and N-methyl-D-asparate to the ventral surface of the medulla produced responses similar to those observed as a result of central administration of tachykinin peptides. The results suggest that 1) mammalian tachykinins are involved in the regulation of thoracic and abdominal expiratory muscle activity, 2) these muscles manifest substantial differences in their electrical responses to excitatory neuropeptides acting centrally, and 3) inputs from modulatory neurons located in this vicinity of the ventral medullary surface seem to be distributed unevenly to different expiratory premotor and/or motoneurons. PMID- 1706332 TI - Cystic fibrosis, vasoactive intestinal polypeptide, and active cutaneous vasodilation. AB - The transmitter substance for the active cutaneous vasodilation that accompanies sweating during hyperthermia in humans is unknown. Hokfelt et al. (Nature Lond. 284: 515-521, 180) hypothesized that it is vasoactive intestinal polypeptide (VIP) that is cotransmitted with acetylcholine. Heinz-Erian et al. (Science Wash. DC 229: 1407-1408, 1985) reported that VIP innervation is sparse in the skin of persons with cystic fibrosis (CF). A corresponding attenuation of active vasodilation in these subjects would be evidence that VIP is involved in this effector mechanism of human thermor-regulation. Immunocytochemical analysis of skin biopsies from four men with CF confirmed that VIP innervation was sparse. We also analyzed immunoreactivity for calcitonin gene-related peptide (CGRP; normal), substance P (normal), and neuropeptide Y (low). VIP-immunoreactive Merkel cells were abnormal. Despite sparse VIP-immunoreactive innervation, our CF subjects' cutaneous vascular responses to hyperthermia were normal. Because VIP was not completely absent, this evidence is insufficient to rule out VIP as the vasodilator transmitter. However, the CGRP and substance P innervation we observed could mean that release of one or both of these peptides was the mechanism of the fully developed active cutaneous vasodilation. PMID- 1706333 TI - Hyperamylasemia and salivary gland enlargements in patients with eating disorders. PMID- 1706334 TI - G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain. AB - Attachment of heterotrimeric G-proteins to the inner face of the plasma membrane is fundamental to their role as signal transducers by allowing interaction with both receptors and effectors. Certain G-protein alpha subunits are anchored to the membrane by covalent myristoylation. The beta gamma complex is required for G protein interaction with receptors and is independently membrane associated through an unknown mechanism. A series of carboxyl-terminal modifications including isoprenylation which may contribute to membrane attachment has been identified recently in G-protein gamma subunits. Expression and membrane targeting of beta and gamma subunits were examined in COS cells. The expression of either subunit was found to require cotransfection with both beta and gamma cDNAs. Mutation of the carboxyl-terminal cysteine residue of gamma shown to undergo isoprenylation and carboxymethyl-esterification preserved beta gamma expression but blocked isoprenylation and membrane attachment. These results implicate the carboxyl-terminal processing of G-protein gamma subunits and beta coexpression as necessary and sufficient for membrane targeting of the beta gamma complex. PMID- 1706335 TI - GMP-140 binding to neutrophils is inhibited by sulfated glycans. AB - GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP 140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP 140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal. PMID- 1706336 TI - Endosomes are acidified by association with discrete proton-pumping vacuoles in Dictyostelium. AB - The endocytic compartment in the amoeba Dictyostelium discoideum was labeled by feeding fluorescein 5-isothiocyanate-dextran. In homogenates containing 2 mM Mg2+, the compartments so labeled copurified with all of the vacuolar H(+)-ATPase activity in a dense peak. The fluorescence properties of the probe showed that these dense vacuoles were inherently acidic. Furthermore, after purging their residual acidity, they could be re-acidified by the addition of ATP. These data suggest that the H(+)-ATPase was structurally and functionally coupled to the endocytic space. The association of the H(+)-ATPase and endocytic compartment was reversed by the removal of either Mg2+ or traces of the cytosol. Endocytic vacuoles prepared in this way were deficient in vacuolar H(+)-ATPase activity and were not acidified upon addition of MgATP. The missing proton pumps were recovered in large buoyant vacuoles that lacked ingested fluorescein 5 isothiocyanate-dextran, acid hydrolases, and residual acidity. These vacuoles were also less susceptible than endosomes to disruption by digitonin, suggesting that their bilayers were low in sterols. These results indicate that the endocytic circuit in Dictyostelium is acidified by a discrete and separable proton-pumping organelle. PMID- 1706337 TI - The RNA component of RNase P from the archaebacterium Haloferax volcanii. AB - RNase P, an endoribonuclease responsible for generating the mature 5' termini of tRNA precursors, is composed of both RNA and protein. It has been demonstrated that the eubacterial RNase P RNA will, under the appropriate reaction conditions, exhibit catalytic activity in vitro. Evidence has not been obtained for catalytic activity by the RNAs of eukaryotic RNase P enzymes. Using a cDNA probe prepared from RNA copurifying with RNase P activity from the archaebacterium Haloferax volcanii, we have characterized the gene encoding the RNase P RNA. The proposed transcript from this gene can assume a structure resembling the eubacterial RNase P RNA and includes many of the highly conserved sequences of these RNAs. This RNA was incapable of cleaving pre-tRNA substrates in the absence of protein under a variety of in vitro conditions. Catalytic activity was observed when this RNA was combined with the protein subunit of the Bacillus subtilis RNase P complex. Similarities among the archaebacterial, eubacterial, and eukaryotic RNase P RNA sequences and structures are discussed. PMID- 1706338 TI - Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase. AB - The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled). PMID- 1706339 TI - Polyclonal antibodies against RNase L. Subcellular localization of this enzyme in mouse cells. AB - RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40 kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation. PMID- 1706340 TI - Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor. AB - Acidic fibroblast growth factor (aFGF) is a broad spectrum mitogen that is stabilized by complexation with heparin and heparan proteoglycans. The monomeric human protein contains 3 reduced cysteine residues of unknown function, the first 2 of which are conserved among all seven known fibroblast growth factors. The influence of these free sulfhydryl groups on the level, stability, and heparin dependence of the mitogenic activity at physiological temperature and pH is characterized using a complete set of site-directed mutants in which either any 1, 2, or all 3 of the cysteine residues are converted to serines. Mutants of aFGF in which either any 2 or all 3 cysteine residues are substituted by serines are more active, have longer activity half-lives, and are less heparin dependent than wild-type aFGF. In contrast, wild-type aFGF and the three mutants that each retain 2 cysteine residues inactivate more rapidly in the absence of heparin by a nonproteolytic mechanism but are markedly stabilized by heparin. This cysteine mediated destabilization of aFGF not only diminishes its activity in the absence of heparin in tissue culture but also could functionally restrict its activity in vivo to the vicinity of mast cell-derived heparins and heparan proteoglycans associated with cell surfaces and basement membranes. PMID- 1706341 TI - The kinetics of vimentin RNA and protein expression in interleukin 2-stimulated T lymphocytes. AB - In this paper, we examine the regulation of vimentin expression during interleukin 2-induced proliferation of the cloned helper T cell line, L2. We observe a 10-20-fold increase in steady-state vimentin RNA that is accompanied by a 14-fold increase in the rate of vimentin protein synthesis. This indicates that translation is occurring, and thus that vimentin expression is regulated, at least in part, at the level of transcription and/or RNA stability, rather than at the level of protein synthesis. In contrast to the increases in steady-state vimentin RNA and the rate of protein synthesis, steady-state vimentin protein levels increase maximally only 1.3-3-fold in proliferating L2 cells. The rate of vimentin protein turnover remains relatively constant throughout L2 cell activation, active proliferation, and return to quiescence, indicating that an increased rate of turnover does not account for the minimal increase in vimentin content. These observations are consistent with a transient increase in vimentin protein synthesis necessary to support L2 cell growth and/or vimentin network reorganization. Because cell division occurs before an equilibrium state is reached, the maximal increase in steady-state vimentin protein content is never attained. PMID- 1706342 TI - A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion. AB - The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C'' and FG. This structure is consistent with C'C'' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein. PMID- 1706343 TI - Expression of liver fatty acid-binding protein/human growth hormone fusion genes within the enterocyte and enteroendocrine cell populations of fetal transgenic mice. AB - The intestinal epithelium establishes and maintains a precise spatial organization despite its continuous and rapid renewal. We have used transgenic mice containing liver fatty acid-binding protein/human growth hormone (L FABP/hGH) fusion genes to begin to define the molecular mechanisms which are responsible for appropriate regional and cell-specific expression of genes in the gut. Multilabel immunocytochemical methods were employed to characterize the patterns of expression of two transgenes in the enteroendocrine and enterocytic populations of late gestation fetal mice at the time of initial cytodifferentiation of the gastrointestinal epithelium (fetal days 16-19). Surveys of the enteroendocrine cell population using a panel of antibodies directed against 11 neuroendocrine products revealed that these cells are scarce prior to fetal day 17, show a progressive increase in number through day 19, and while the relative proportion of subpopulations (defined by their principal peptide product) are somewhat different than in adults, their geographic distribution along the duodenal to colonic and intervillus(crypt) to villus axes are very similar to that encountered in adult (2-5 month old) mice. Immunoreactive L-FABP is first detectable at fetal day 17 and at this time of first appearance shows an adult pattern of regional enterocytic expression: i.e. it is present in cells overlying nascent villi but not those in the intervillus zone, it is highest in proximal small bowel, declines distally, and is absent from colonocytes. Colocalization studies indicate that L-FABP is not present in enteroendocrine cells during fetal life. Mapping studies indicate that nucleotides -596 to +21 of the rat L-FABP gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) expression in fetal transgenic mice (with the exception that a subset(s) of enteroendocrine cells, typically containing immunoreactive gastric inhibitory peptide, support transgene but not L-FABP expression). This is in marked contrast to adult transgenic mice where inappropriate hGH accumulation occurs in crypt-associated epithelial cells, in colonocytes, and in many enteroendocrine populations. These studies indicate the importance of considering developmental stage when interpreting the results of any mapping study of cis-acting elements that regulate cell-specific and regional expression of genes in the perpetually renewing intestinal epithelium. Moreover, they also raise the possibility of using transgenes to define fundamental temporal changes in the gut's epithelial cell populations. PMID- 1706344 TI - A genetic element that increases the frequency of gene amplification. AB - A repetitive mammalian genetic element, HSAG-1, has been shown to promote the amplification of the vector, pSV2-DHFR, containing the functional cDNA for dihydrofolate reductase (DHFR). LR-73 cells, a line of Chinese hamster ovary cells, were transfected with this recombinant construct or with the parent vector, then subjected to culture in selective medium containing steadily increasing concentrations of methotrexate, a drug which specifically inhibits DHFR. Cultures transfected with the HSAG-1-containing construct acquired drug resistance faster than those transfected with the parent vector. This acceleration of acquisition of drug resistance was due to an increased probability of the generation and subsequent selection of cellular variants with increased copy numbers of the vector. The effect has also been observed in CHO(DHFR-) and HeLa cell lines. Possible mechanisms for the effect of the HSAG-1 element on gene amplification are discussed. PMID- 1706345 TI - Bioinorganic chemistry. PMID- 1706347 TI - Electrolyte transport in the lungs. AB - The crucial physiologic role of such transport is illustrated by cystic fibrosis. In that lethal genetic disease, transepithelial sodium absorption and chloride secretion are respectively increased and decreased, and pulmonary mucus is dehydrated. Mechanisms that regulate normal sodium and chloride movement are discussed, as are the derangements in cystic fibrosis. PMID- 1706346 TI - Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation. AB - The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF) stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors. PMID- 1706348 TI - The nasal route for drug administration. PMID- 1706349 TI - Comparative study of reaction kinetics in membrane and agarose bead affinity systems. AB - Compared to conventional agarose bead affinity supports, a microporous nylon membrane exhibits greatly improved reaction kinetics as quantified in the reaction between gamma-globulin and immobilised protein A. The improvement is only observed when the solution of gamma-globulin is forced through the membrane pores. In the absence of flow in the pores, it is possible to relate approximately the rate of uptake onto either type of affinity support to independently determined diffusion coefficients. In the presence of flow, the reaction rate is similar for membranes having 0.45 and 3.0 microns diameter pores, and considerably smaller than predicted by the Smoluchowski formula. PMID- 1706350 TI - Insulin-like growth factor binding proteins in human endometrium: steroid dependent messenger ribonucleic acid expression and protein synthesis. AB - The insulin-like growth factor (IGF) autocrine/paracrine system is believed to play a role in endometrial differentiation and trophoblast growth. The IGFs bind with high affinity to a family of binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. IGFBP-1, one of three well characterized IGFBPs in humans, has been shown by numerous investigators to be present in secretory but not proliferative endometrium; however, no information is available with regard to two other human IGFBPs, IGFBP-2 and IGFBP-3, in normal human endometrium. We have examined expression of the messenger RNAs (mRNAs) encoding IGFBP-2 and IGFBP-3 in human proliferative endometrium [under the influence of estradiol (E2)] compared to secretory endometrium (under the influence of E2 and progesterone). Using a complementary DNA probe specific for IGFBP-2, by Northern analysis, expression of a 1.4 kilobase mRNA was found to be differentially expressed in secretory compared to proliferative phase endometrium. Using a complementary DNA probe encoding IGFBP-3, a 2.4 kilobase mRNA was found in endometrium and was also found to be differentially expressed in secretory compared to proliferative phase endometrium. Endometrial stromal cells were established in culture and revealed constitutive synthesis and secretion of IGFBP-2 and IGFBP-3 into the conditioned medium (CM), as detected by Western ligand blot analysis of the CM. Identification of the IGFBPs in the CM was made using IGFBP-2 and IGFBP-3 specific antiserum. In the presence of E2 and progesterone, there was an enhancement in the amount of IGFBP-3 and a marked enhancement of IGFBP-2 in the conditioned media, suggesting sex steroid dependence of IGFBP-2 and, to a lesser extent, IGFBP-3 protein synthesis. Western ligand blot analysis of IGFBPs in serum throughout the menstrual cycle revealed no hormonal dependence in the serum IGFBPs, suggesting local action of the IGFBPs in endometrium. These data thus show that mRNAs encoding IGFBP-2 and IGFBP-3 are expressed in human endometrium, that their expression is differentially regulated in secretory compared to proliferative phase endometrium (different steroidogenic environments), and that the synthesis of both IGFBP-2 and IGFBP-3 proteins is regulated by steroid hormones. PMID- 1706351 TI - Insulin-like growth factors (IGFs) and IGF-binding proteins in the developing rhesus monkey. AB - Rhesus monkeys follow a developmental pattern of serum insulin-like growth factor I (IGF-I) levels similar to that found in humans. In these monkeys, serum IGF-I levels peak during puberty (2.5-4.5 yr of age in males). We have examined the developmental pattern of IGF-binding protein-1 (IGFBP-1), -2, and -3 in serum by Western ligand blotting, the levels of IGFBP-3, IGF-I, and IGF-II in serum by RIA, and the IGFBP mRNA levels of IGFBP-1, -2, and -3 in the livers of rhesus monkeys from fetal life through adulthood by Northern analysis. The pattern of the serum levels of the IGFBPs reflected the liver mRNA levels of the IGFBPs. The IGFBP-1 and IGFBP-2 liver mRNA and serum levels were highest in the fetus and first year of life and were very low after 4 yr of age. Conversely, the IGFBP-3 liver mRNA and serum levels were relatively low early in life and peaked during puberty. The serum levels of IGF-I and IGF-II were strongly correlated with the level of IGFBP-3. We conclude that the developmental pattern of IGFBPs in the rhesus monkey is similar to that in the human, and that serum IGFBP levels are probably regulated by the rate of IGFBP mRNA synthesis. PMID- 1706352 TI - Acute-phase protein detection and quantification in gingival crevicular fluid by direct and indirect immunodot. AB - The development of an assay for markers of active periodontitis, obtained directly from gingival crevicular fluid (GCF) and simply quantified, would be of great importance to the dental practitioner. The purpose of this study was to evaluate direct and indirect immunodot techniques as to their potential in easily quantifying acute-phase proteins within periodontally diseased and healthy site GCF. Indirect immunodots (GCF eluates dotted onto nitrocellulose membrane) using monoclonal antibodies and a radioactive isotope label were used to identify and establish relative amounts of C-reactive protein (CRP) and alpha-2-macroglobulin (A2M) in 2 diseased and 2 healthy sites in 24 periodontitis patients. Periodontally diseased sites were found to contain significantly lower concentrations of A2M than healthy sites (p less than 0.001), but CRP levels did not vary significantly between healthy and diseased locations. Using a direct immunodot assay (GCF absorbed directly into nitrocellulose membrane strips), A2M levels quantified with radioactive isotopes at healthy and diseased sites could be correlated with A2M levels determined by enzyme-linked antibody-colormetric probes at those same sites. Such a direct sampling and quantification system shows promise for future "in-office" diagnostic methodology. PMID- 1706353 TI - Evaluation of the activity of chemically identified enteric neurons through the histochemical demonstration of cytochrome oxidase. AB - The measurement of the density of the reaction product produced by the histochemical demonstration of cytochrome oxidase activity provides a method for the visual identification of physiologically active enteric neurons. The current study utilized the cytochrome oxidase technique in order to evaluate the metabolic history of neurons in different regions of the bowel and in chemically identified types of neuron. In addition, the effect of drugs or neurotoxins commonly used in the immunocytochemical identification of enteric neuronal phenotypes was also analyzed. Cytochrome oxidase activity was visualized with a blue-black reaction product resulting from the cobalt-intensified oxidation of 3,3'-diaminobenzidine. Peptides or 5-hydroxytryptamine (5-HT) were localized with biotinylated secondary antibodies and alkaline phosphatase-labeled avidin. Bound avidin or endogenous alkaline phosphatase was visualized with a red reaction product in the presence or absence, respectively, of levamisole. Use of measured without interference from a simultaneously demonstrated histo- or immunochemical marker. A multi-peptidergic class of cholinergic submucosal secretomotor neuron containing neuropeptide Y (NPY) and calcitonin gene related peptide (CGRP) immunoreactivities was found to be less metabolically active than the average of all submucosal neurons. In contrast, a non-cholinergic submucosal secretomotor neuron containing dynorphin (which is also known to contain vasoactive intestinal peptide) immunoreactivity was more metabolically active than submucosal neurons that do not contain this peptide. On average, submucosal neurons were more metabolically active than those of the myenteric plexus, and levels of metabolic activity in the myenteric plexus were found to be higher in the duodenum and the cecum than in the jejunum-ileum or colon. Myenteric neurons characterized by CGRP or NPY immunoreactivities or by endogenous alkaline phosphatase activity, were all less metabolically active than the average of all neurons in myenteric ganglia. Colchicine, which stimulates intestinal motility, was observed to increase cytochrome oxidase activity in enteric neurons, suggesting that an effect on the enteric nervous system contributes to its action on the bowel. The neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine (5,7-DHT) were each found to stimulate neuronal metabolic activity. 5,7-DHT appeared to activate excitatory subtypes of 5-HT receptor since its effects were blocked or mimicked by compounds that act as antagonists or agonists, respectively, at these receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706354 TI - On variables that affect estimates of the true sizes and densities of radioactively labeled cell nuclei. AB - Tritiated thymidine has been widely used as a nuclear marker of cell birth. The true diameters and packing densities (nuclei/microns 3) of such radioactively labeled nuclei cannot be measured directly from tissue sections. Here we show that existing stereological corrections cannot be applied to data from radioactively labeled nuclei. We empirically measured the number of silver grains exposed by nuclei containing tritiated thymidine. The nuclei were separated from the photographic emulsion by known thicknesses of fixed, embedded avian telencephalon. The results of this experiment were used to develop an equation that estimates the number of silver grains exposed by a cell nucleus of any given diameter, containing a given amount of radioactive label, and located at any given distance from the photographic emulsion. The equation also allows one to calculate the probability that a label-containing nucleus will be correctly classified as labeled. Simulations of the equation revealed that not all label containing nuclei are correctly classified by using commonly employed identification procedures and that larger nuclei are less likely to be correctly classified than smaller nuclei, given the same amount of label. The equation can be used to modify one class of existing stereological equations so as to be applicable to measurements of radioactively labeled nuclei. Finally, we discuss the assumptions and limitations of this modification. PMID- 1706355 TI - Cholecystokinin innervation of monkey prefrontal cortex: an immunohistochemical study. AB - Knowledge of the circuitry of chemically identified systems in primate prefrontal cortex is limited. Although cholecystokinin is very abundant in prefrontal cortex (Geola et al.: Journal of Clinical Endocrinology and Metabolism 53(2):270-275, 1981; Taquet et al.: Neuroscience 27(3):871-883, 1988), the organization of cholecystokinin-containing structures in primate prefrontal cortex has not been investigated. Using immunohistochemical and retrograde transport techniques, we characterized the cholecystokinin innervation of prefrontal cortex in macaque monkeys. The use of two antibodies directed against different portions of the cholecystokinin molecule revealed that distinct forms of the molecule were differentially localized in the same cortical neurons. These small, nonpyramidal cholecystokinin-positive neurons had a variety of somal morphologies and the density of labeled cells did not differ among cytoarchitectonic regions. Labeled neurons had a distinctive laminar distribution with the greatest density of cells present in layers II-superficial III. Labeled fibers also had a distinctive laminar pattern of distribution that differed from that of the immunoreactive neurons. In granular prefrontal cortex, terminal fields were evident in layers II, IV, and VI, with the greatest density in layer VI. Agranular area 24 exhibited a bilaminar pattern of immunoreactivity with a band in layer II and a very dense terminal field in layers V-VI. A high density of cholecystokinin binding sites has been found in layers III-IV of prefrontal cortex and other association areas in the monkey; this finding has been attributed to possible cholecystokinin-containing afferents from the thalamus (Kritzer et al.: Journal of Comparative Neurology 263:418-435, 1987). The mediodorsal nucleus of the thalamus is known to be a source of afferents which terminate in layer IV of prefrontal cortex. However, combined retrograde transport and immunohistochemical techniques failed to reveal the presence of cholecystokinin-positive neurons in the mediodorsal nucleus of the thalamus that project to prefrontal cortex. These findings, and other observations, suggest that the terminal field in layer IV is formed by descending axons that arise from cholecystokinin-containing neurons in layers II and superficial III. This study demonstrates that the cholecystokinin innervation of prefrontal cortex has a laminar specific organization that is preserved across cytoarchitectonic regions. This distribution of immunoreactive structures suggests a distinctive role of cholecystokinin in cortical circuitry that is common to every region of prefrontal cortex. PMID- 1706356 TI - Development of the anterior olfactory nucleus in normal and unilaterally odor deprived rats. AB - The development of a second order structure in the olfactory pathway, the anterior olfactory nucleus, was examined in both normal rat pups and in subjects which underwent unilateral naris closure on postnatal day 1 (P1). Naris occlusion in neonatal rats produces a constellation of changes within the first relay in the pathway, the olfactory bulb, including a 25% reduction in total volume. Such large changes suggest that higher order structures might also be affected. Anterior olfactory nucleus development was quantified in several ways. Laminar volumes were computed by using serial section planimetry. In control animals differential development was observed, with regions extending most rostrally (e.g., pars externa and pars lateralis) exhibiting the least growth. The anterior olfactory nucleus on the "deprived" side of subjects with a single naris occluded was identical in size to that observed in controls, development within the pars lateralis was examined in control animals at P10, P20, P30, and adults. Developmental increases in numbers of both branches per cell and spines were noted, but mean branch length remained relatively constant. Finally, the effects of naris occlusion on histological patterns of succinate dehydrogenase (SDH) staining and 2-deoxyglucose uptake within pars lateralis were examined at P20 to test for more subtle effects of naris occlusion. SDH staining was quite similar in deprived and control rats at P20. However, 3H-2-DG uptake was decreased in rostral areas of the anterior olfactory nucleus ipsilateral to the deprived olfactory bulb, suggesting that naris closure does affect the structure. PMID- 1706357 TI - Somatodendritic morphology of on- and off-cells in the rostral ventromedial medulla. AB - The rostral ventromedial medulla (RVM) contains two classes of physiologically defined neurons, on-cells and off-cells, that are implicated in nociceptive modulation. In a continuing effort to detail the neural circuitry that underlies the activity of these two distinct neuronal types, the somatodendritic morphology of on- and off-cells was studied in the cat, rat, and ferret. In lightly anesthetized animals, on-cells increased and off-cells decreased their discharge rate during a withdrawal reflex evoked by noxious stimuli. Following their physiological characterization by using intracellular recording, on- and off cells were injected with either horseradish peroxidase or biocytin and their somatodendritic arborizations were examined. Labeled on- and off-cells included fusiform and stellate cells of all sizes as well as large multipolar neurons. Although the somatic shape of both on- and off-cells in RVM was heterogeneous, off-cells tended to be fusiform neurons whose long axis was oriented mediolaterally. The dendritic domains of both on- and off-cells extended bilaterally past the lateral edge of the trapezoid body or pyramid and ventrally to, and sometimes including, the trapezoid body or pyramid. In contrast to their extensive mediolateral spread, the dendritic domains of both cell types were limited to the ventral half of the reticular formation and were compressed along the rostrocaudal axis. The dendritic arbor of individual on- and off-cells extended well beyond the cytoarchitectonic boundaries of any single nuclear region, within the domain delineated as the RVM. The spatial domains of the dendritic arbors of on- and off-cells are further evidence that the on- and off cells throughout the RVM constitute an integrated unit in the modulation of nociceptive transmission. PMID- 1706358 TI - Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer's disease: II. Primary and secondary visual cortex. AB - In this study we investigated the primary and secondary visual areas of normal and Alzheimer's disease brains by using the SMI32 antibody. It is known that in Alzheimer's disease primary sensory areas are usually less devastated than association cortices, although visual symptomatology has been documented early in the course of the disease. In area 17, the SMI32 antibody primarily labeled the perikarya and dentritic tree of the large Meynert cells and cells in layer IVB. Smaller neurons in layers III, V, and VI were also immunoreactive (ir). In area 18, very large SMI32-ir pyramidal neurons in layers III and V were observed. In both areas, staining intensity was correlated with cell size, the largest neurons being the most intensely stained. Only a few changes were observed in the Alzheimer's disease cases. The only statistically significant differences in SMI32-ir neuron counts between control and Alzheimer's disease brains occurred in layer IVB cells and Meynert cells in area 17, and in layer III cells in area 18. In contrast with association cortices, there were no changes in staining intensity in the visual areas. There were fewer neurofibrillary tangles and neuritic plaques in these areas than in prefrontal and inferior temporal cortex, and a correlation between neurofibrillary tangle counts and SMI32-ir neuron loss was only observed in layer III of area 18. These observations show that in the primary and secondary visual cortex, SMI32 also labeled a distinct subset of pyramidal cells that are known from data obtained in the monkey brain to furnish long corticocortical as well as subcortical projections. Interestingly, although there is much less cell and/or neurofibrillary tangle formation in these occipital regions than in prefrontal and temporal association areas, there is significant loss within key subsets of pyramidal cells. The selective loss of this particular subpopulation of pyramidal neurons will disrupt association pathways linking primary visual cortex with areas involved in higher level visual processing. The partial disconnection of such pathways may be relevant to the visual symptomatology frequently observed in Alzheimer's disease patients. These data further support the hypothesis that subtypes of pyramidal neurons with specific anatomical and molecular profiles may display a differential vulnerability in Alzheimer's disease. PMID- 1706359 TI - Simultaneous labeling of vagal innervation of the gut and afferent projections from the visceral forebrain with dil injected into the dorsal vagal complex in the rat. AB - The vagal innervation of the different layers of the rat gastrointestinal wall was identified with the fluorescent carbocyanine dye Dil, injected into the dorsal motor nucleus of the vagus (dmnX). Multiple, bilateral injections were used to label all dmnX preganglionic motoneurons, and as a consequence, most of the vagal primary afferents that terminate in the adjacent nucleus of the solitary tract (nts) were also retrogradely and transganglionically labeled. With Fluorogold used to label the enteric nervous system completely and specifically, the Dil-labeled vagal profiles could be visualized and quantified in their anatomical relation to the neurons of the myenteric and submucous ganglia. In the myenteric plexus, vagal fibers and terminals were found throughout the gastrointestinal tract as far caudal as the descending colon, but there was a general decreasing proximodistal gradient in the density of vagal innervation. All parts of the gastric myenteric plexus (fundus, corpus, antrum), as well as the proximal duodenum, were extremely densely innervated, with vagal fibers and terminals in virtually every ganglion and connective. Further caudally, both the percentage of innervated myenteric ganglia and the average density of label within the ganglia rapidly decreased, with the exception of the cecum and proximal colon, where up to 65% of the ganglia were innervated. In the gastric and duodenal submucosa very few and in the mucosa no vagal fibers and terminals were found. With both normal epifluorescence and laser scanning confocal microscopy, highly varicose or beaded terminal structures of various size and geometry could be identified. The Dil injections, which impregnated the dmnX as well as the adjacent nts, resulted in retrograde and anterograde labeling of all the previously reported forebrain connections with the dorsal vagal complex. We conclude that the myenteric plexus is the primary target of vagal innervation throughout the gastrointestinal tract, and that its innervation is more complete than previously assumed. In contrast, vagal afferent (and efferent) innervation of mucosa and submucosa seems conspicuously sparse or absent. Furthermore, the use of more focal injections of Dil offers the prospect to simultaneously identify specific subsets of vagal preganglionics and their central nervous inputs. PMID- 1706360 TI - Marginal neurons in the urodele spinal cord and the associated denticulate ligaments. AB - Marginal neurons have been described in the spinal cords of a variety of vertebrates including lamprey, reptiles, birds, and mammals but not in amphibians. There has been speculation about a motor function for these neurons but recent experimental evidence in lampreys indicates that they are intraspinal mechanoreceptor neurons. Additional evidence on reptiles and birds demonstrates that the marginal neurons are closely associated with the denticulate ligaments. In the present investigation, we have examined the spinal cords of Necturus, Ambystoma tigrinum, and A. mexicanum with light and electron microscopic techniques. Marginal nuclei were found in the ventrolateral position immediately internal to the pia and to the denticulate ligament. The marginal neurons were scattered in a continuous column of neuropil without segmental accumulation. They were approximately 30 to 50 microns in diameter and fusiform with dendrites extending from the poles, parallel with the length of the spinal cord. Neuronal fingerlike processes, like those found in peripheral mechanoreceptors and in the marginal nuclei of reptiles, were also found in the three species of urodeles studied. The structure of the denticulate ligaments, similar in the three different amphibians, was composed of collagen, elastin, and fibroblasts, all of which were concentrated in the segmental lateral processes. PMID- 1706361 TI - The cells of origin of the spinohypothalamic tract in cats. AB - Various cutaneous and visceral stimuli alter the discharge rates of neurons in the hypothalamus. Changes in the activity of hypothalamic neurons are thought to play important roles in eliciting neuroendocrine, autonomic, and affective responses to somatosensory and visceral stimuli. Information from peripheral structures has been considered generally to reach the hypothalamus via multisynaptic ascending pathways. Recently, a direct projection from the spinal cord to the hypothalamus was demonstrated in rats. The goal of this study was to determine whether a similar projection exists in cats. Either wheat germ agglutinin conjugated to horseradish peroxidase, a mixture of this tracer and the B subunit of cholera toxin conjugated to horseradish peroxidase, or fast blue was injected into the hypothalamus of cats. Injections were centered in the hypothalamus in seven cats and did not spread to the thalamus, zona incerta or midbrain. After these injections, retrogradely labeled neurons were observed bilaterally in each of the 17 spinal segments that were examined. A total of approximately 400-500 labeled neurons was observed in alternate sections through these segments in the most effective cases. Roughly 70% of the labeled neurons were located contralaterally. Labeled neurons were found predominantly in the deep dorsal horn, the intermediate zone/ventral horn and in the area surrounding the central canal. A few were also noted in the superficial dorsal horn. The first and second sacral segments contained the largest numbers of retrogradely labeled neurons in the spinal cord. The number of spinohypothalamic tract neurons observed in this study in cats was roughly an order of magnitude smaller than that previously reported for rats. This finding suggested either that the spinohypothalamic tract is relatively small in cats or that our tracing techniques did not label many spinohypothalamic tract neurons in cats. To test the sensitivity of one of our tracing techniques, control injections of wheat germ agglutinin conjugated to horseradish peroxidase that filled the ventrobasal thalamus were made in two cats. In both cases, thousands of spinal cord neurons were labeled. In summary, our results indicate that a spinohypothalamic tract exists in cats. However, our findings also suggest that the total number of spinohypothalamic tract neurons in cats may be an order of magnitude smaller than it is in rats. PMID- 1706362 TI - Galanin mRNA in the nucleus basalis of Meynert complex of baboons and humans. AB - Galanin, a 29-amino acid peptide, has been shown by immunocytochemistry to occur in most large acetylcholinergic neurons of the complex that includes the nucleus basalis of Meynert and the nucleus of the diagonal band of Broca in nonhuman primates. In contrast, several studies have reported that most large neurons of the human nucleus basalis of Meynert complex appear to lack galanin immunoreactivity. We investigated this apparent species-difference by hybridization histochemistry for galanin messenger ribonucleic acid (mRNA) in humans and baboons. The results confirm previous immunocytochemical data; very few large neurons of the nucleus basalis of Meynert complex in humans contained detectable galanin messenger RNA, whereas most such cells in baboons were labeled by the oligodeoxynucleotide probe. The few labeled neurons in humans were primarily medial or ventral to the main body of the nucleus basalis of Meynert and corresponded in location to a minor population of relatively intensely labeled cells in baboons. These findings indicate that the indetectability of immunoreactive galanin in most cells of the nucleus basalis of Meynert complex in humans is due to a paucity or an absence of galanin messenger RNA and not to differences in posttranslational processing or transport of the peptide. Inasmuch as the probe labeled neurons in several other nuclei of both species, it is unlikely that differences in galanin messenger RNA sequences underlie the species related disparity in hybridization in the nucleus basalis of Meynert complex. The indetectability of galanin messenger RNA in most cells of the human nucleus basalis of Meynert complex indicates that the expression of the galanin gene is regulated by as yet unidentified influences that differ in human and nonhuman primates. The varying phenotypes of galanin in primates suggest potentially important species-differences in the function of galanin in neurons of the nucleus basalis of Meynert complex. PMID- 1706363 TI - Connections between the central nucleus of the amygdala and the midbrain periaqueductal gray: topography and reciprocity. AB - Previous reports indicate that the midbrain periaqueductal gray and the central nucleus of the amygdala are interconnected but the organization of these projections has not been characterized. We have analyzed this reciprocal circuitry using anterograde and retrograde tracing methods and image analysis. Our findings reveal that innervation of periaqueductal gray from the central nucleus of the amygdala is extensive and discretely organized along the rostrocaudal axis of periaqueductal gray. In addition, the reciprocal projection from periaqueductal gray to the central nucleus of the amygdala is more extensive and more highly organized than previously suggested. Multiple or single discrete injections of wheatgerm agglutinin-horseradish peroxidase into several rostrocaudal levels of periaqueductal gray retrogradely labeled a substantial population of neurons, predominantly located in the medial division of the central nucleus of the amygdala. Tracer injections into the central nucleus revealed a high degree of spatial organization in the projection from this nucleus to periaqueductal gray. Two discrete longitudinally directed columns in dorsomedial and lateral/ventrolateral periaqueductal gray are heavily targeted by central amygdalar inputs throughout the rostral one-half to two-thirds of periaqueductal gray. Beginning at the level of dorsal raphe and continuing caudally, inputs from the central nucleus terminate more uniformly throughout the ventral half of periaqueductal gray. In addition, a substantial population of periaqueductal gray neurons were retrogradely labeled from the central nucleus of the amygdala; these were heterogeneously distributed along the rostrocaudal axis of periaqueductal gray, and included both raphe and non-raphe neurons. Thus, the present study demonstrates that periaqueductal gray receives heavy, highly organized projections from the central nucleus of the amygdala and, in turn, has reciprocal connections with the central nucleus. Previous studies have demonstrated that longitudinally organized columns of output neurons located in dorsomedial and lateral/ventrolateral periaqueductal gray project to the ventral medulla. Thus, there may be considerable overlap between the two longitudinally organized terminal input columns from the central nucleus of the amygdala and the two longitudinal columns of descending projection neurons from periaqueductal gray to the ventral medulla. The central nucleus of the amygdala has been implicated in a variety of emotional/cognitive functions ranging from fear and orienting responses, defensive and aversive reactions, associative conditioning, cardiovascular regulation, and antinociception. Many of these same functions are strongly represented in the periaqueductal gray. It is noteworthy that the present results demonstrate that lateral periaqueductal gray, a preeminent central trigger site for behavioral and autonomic components of the defense/aversion response, is heavily targeted by inputs from the central nucleus of the amygdala at all levels of periaqueductal gray.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706364 TI - Peptide-immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Manduca sexta. AB - Antisera against a variety of vertebrate and invertebrate neuropeptides were used to map cerebral neurosecretory cells in the sphinx moth Manduca sexta. Intense immunoreactive staining of distinct populations of neurosecretory cells was obtained with antisera against locust adipokinetic hormone, bovine pancreatic polypeptide, FMRFamide, molluscan small cardioactive peptide (SCPB), leucine enkephalin, gastrin/cholecystokinin, and crustacean beta-pigment dispersing hormone (beta PDH). Other antisera revealed moderate to weak staining. Each type of neurosecretory cell is immunoreactive with at least one of the antisera tested, and most of these neurons can be identified anatomically. The staining patterns provide additional information on the organization of cerebral neurosecretory cells in M. sexta. Based upon anatomical and immunocytochemical characteristics, 11 types of neurosecretory cells have been recognized in the brain, one type in the suboesophageal ganglion, and one in the corpus cardiacum. Extensive colocalization experiments show that many neurosecretory cells are immunoreactive with several different antisera. This raises the possibility that these cells may release mixtures of neuropeptides into the hemolymph, as has been demonstrated in certain other systems. The immunocytochemical data should be helpful in efforts to identify additional peptide neurohormones released from the brain of this and other insects. PMID- 1706365 TI - Distributions of choline acetyltransferase and acetylcholinesterase activities in the retinal layers of the red-tailed hawk and road runner. AB - The activities of choline acetyltransferase and acetylcholinesterase were assayed in submicrogram samples from layers of red-tailed hawk and road runner retina. Both enzyme activities were concentrated in and near the inner plexiform layer. Within the inner plexiform layers of both species, activities of each enzyme were concentrated in two bands, one in each half of this layer. Little choline acetyltransferase activity was found superficial to the middle third of the inner nuclear layer. The distributions of acetylcholinesterase activities corresponded well to those of choline acetyltransferase, except in the outer plexiform layer and the outer margin of the inner nuclear layer of the hawk. These distributions of enzyme activities indicate that populations of amacrine cells in the retinae of these species are cholinergic. In addition to these same cells and presumably cholinoceptive amacrine and ganglion cells, acetylcholinesterase activity in the hawk was associated with a population of horizontal cells that may be unrelated to synaptic cholinergic neurotransmission. Choline acetyltransferase activities associated with amacrine somata and processes were about four times greater in the hawk than in the road runner, suggesting important differences in the density and function of cholinergic elements between species. Possible synaptic relationships in the inner plexiform layer consistent with the interspecies differences in enzyme activities are considered. PMID- 1706366 TI - Fine structure of synapses and retinal innervation of substance P and adenosin deaminase containing neurons in the superior colliculus of the rat. AB - The fine structure of substance P (SP) and adenosine deaminase (ADA) immunoreactive structures in synaptic contacts localized to the superficial layers of the superior colliculus of the rat was investigated by means of immunoelectron microscopy. We also examined the possibility of retinal innervation of SP- and ADA- containing neurons by immunohistochemistry after degeneration of retinal terminals caused by enucleation. SP-like immunoreactive presynaptic terminals of the stratum griseum superficiale (SGS) formed both asymmetric and symmetric synaptic contacts. Presynaptic dendritelike structures were also observed. SP immunoreactive postsynaptic elements made contacts with terminals showing diverse features. ADA-like immunoreactive structures were seen only as postsynaptic elements to different kinds of nonimmunoreactive terminals and were mostly localized in the ventral third of the SGS and the dorsalmost stratum opticum (SO). After enucleation, degenerating retinal terminals were found to form synaptic contacts with SP and ADA immunoreactive structures. The highest number of such degenerating terminals on ADA immunoreactive structures was observed 2 days after retinal denervation, very few being seen after 5 days. These degenerating terminals were restricted to the ventral SGS and dorsal SO. SP immunoreactive structures postsynaptic to degenerating retinal terminals were most numerous 5 days after enucleation and mainly localized in the dorsal SGS. Occasionally, SP immunoreactive dendritelike processes forming synapses with degenerating retinal terminals were simultaneously presynaptic to other nonimmunoreactive profiles, defining, therefore, serial synapses. The present results suggest that SP-I and ADA-I collicular neurons may be part of distinct channels carrying visual information to the lateral posterior and lateral geniculate nuclei of the thalamus, respectively. PMID- 1706367 TI - Stump the experts. PMID- 1706368 TI - Occupational allergy to pancreatic powder: characterization of IgE-binding antigens in pancreatic extract by immunoblotting. AB - The IgE response to inhaled dust of pancreatic powder was studied with sera from two employees of a pharmacy. IgE from both sera was directed against both porcine and bovine trypsin preparations. The major IgE-binding structures had an isoelectric point in the pH 8 to 9 range. The molecular weight of the IgE-binding structures was different for both patients. In one patient, most IgE was directed against a 28 to 30 kd structure, whereas the second patient had IgE directed against components with a molecular weight of 35 and 45 kd, respectively. PMID- 1706369 TI - Studies of desensitization and cross-desensitization to immunologic and nonimmunologic stimuli that evoke contraction and histamine release in superfused guinea pig trachea. AB - This study examined the possibility that there is cross-desensitization between immunologic and nonimmunologic stimuli that evoke contraction and histamine release (HR) in the isolated guinea pig trachea. Compound 48/80 and D tubocurarine were found to cause homologous and heterologous desensitization for both contraction and HR from superfused trachea. Specific antigen challenge of trachea obtained from animals sensitized with either IgG1 (ovalbumin [OA]) or IgE (oxazalone-human serum albumin [OX-HSA]) also resulted in homologous desensitization for both contraction and HR. However, in experiments with animals sensitized with both IgG1 and IgE antibodies, prechallenge with OA resulted in cross-desensitization to OX-HSA, whereas the reverse sequence was ineffective in eliciting this phenomenon. This may be related to the type of desensitization produced by each antigen (specific versus nonspecific) or to heterogeneity of mast cells in the tissue. Prechallenge of the trachea with compound 48/80 or D tubocurarine failed to alter subsequent effects of antigen after active sensitization with OA or passive sensitization with either IgG1 or IgE antibodies. Small but statistically significant decreases in tracheal responses to D-tubocurarine were observed after antigen prechallenge to active both IgG1 and IgE antibodies. This is the first study to demonstrate a cross desensitization between compound 48/80 and D-tubocurarine and the first to examine cross-desensitization with IgG1 and IgE antibodies in the guinea pig trachea. The overall conclusion is that there is no major overlap in the desensitization mechanisms between immunologic and nonimmunologic stimuli in the guinea pig trachea. PMID- 1706370 TI - Ultrastructural demonstration of specific IgG and IgE antibodies binding to Aspergillus fumigatus from patients with aspergillosis. AB - Aspergillus-induced diseases usually demonstrate elevated circulating antibodies belonging to different isotypes. The antigens currently used to detect antibodies are crude culture filtrate and mycelial extracts of A. fumigatus (Af). Most Af associated diseases result from the inhalation of the spores of the organisms present in the environment. However, it is not known whether specific circulating antibodies directed only against spore or mycelia of Af exist in the sera of patients with Af-induced diseases. With colloidal gold we have investigated thin sections of spores and hyphae of Af for their reactivity with Af-specific IgG and IgE antibodies. The results indicate that both spores and hyphae reacted identically with IgG and IgE antibodies from patients. None of the sera from normal control subjects reacted in this system, although low levels of antibodies were detected in the sera by ELISA. Sera from both patients with allergic bronchopulmonary aspergillosis or aspergilloma reacted with cell envelope antigens, whereas sera from patients with invasive aspergillosis also bound to cell sap. This method therefore demonstrates localization of antigens binding to different isotypes in the sera from different clinical forms of aspergillosis and may be useful in purifying specific antigens for immunodiagnosis. PMID- 1706371 TI - A coupled enzyme assay for measurement of sialidase activity. AB - A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination. PMID- 1706372 TI - Spectrophotometric determination of amylase activity in coloured solutions. AB - A simple procedure for spectrophotometric determination of amylase activity in coloured solutions is described. Starch cross-linked with a bifunctional reagent in the presence of black drawing ink is used as an insoluble chromolytic substrate. The absorbances of reaction mixture filtrates are read in the near infrared region (at 800-900 nm) where the majority of coloured substances does not absorb; on the contrary, black drawing ink released from the substrate during the action of amylase absorbs strongly even at these wavelengths. PMID- 1706373 TI - Management of slides by departments of medical illustration and medical libraries in university teaching hospitals. AB - The main results of a research study into the management of slide collections in university teaching hospitals are described. The collections of both departments of medical illustration and medical libraries are surveyed and the recommendation is made that increased and more widespread co-operation is desirable between departments of medical illustration, medical libraries and professionally interested individuals to improve the effectiveness of centralized medical slide collections. PMID- 1706374 TI - Age-dependent reversal of the lobular distribution of androgen-inducible alpha 2u globulin and androgen-repressible SMP-2 in rat liver. AB - Hepatocytes situated at pericentral and periportal zones of the liver lobule show differences in the expression of several liver-specific genes, such as androgen inducible alpha 2u globulin and androgen-repressible senescence marker protein-2 (SMP-2). A marked temporal difference in the expression of these two androgen regulated genes has also been observed. The liver of the pre-pubertal male rat is insensitive to androgen, and during this period hepatocytes synthesize only SMP 2. During young adult life (greater than 40 days), the liver becomes androgen sensitive and concomitant synthesis of alpha 2u globulin and repression of SMP-2 occur. In the senescent male rat (greater than 750 days), the liver again becomes androgen insensitive when the decline in alpha 2u globulin is accompanied by an increase in SMP-2 synthesis. In this article we present results to show a correlation between the temporal and spatial (intralobular) changes in the expression of the androgen-inducible alpha 2u globulin and the androgen repressible SMP-2 in rat hepatocytes. Results indicate that the temporal changes in hepatic androgen sensitivity are dictated by the intralobular location of the hepatocytes. Hepatocytes located around the central vein (pericentral/perivenous) may benefit from a paracrine advantage for the expression of a subset of genes, including the gene for the androgen receptor. PMID- 1706375 TI - Expression of blood group A and B antigens in nuclear heterochromatin in mucous cells of human cervical glands. AB - Using a post-embedding immunogold labeling procedure, we found that monoclonal antibody against A (MAb-A) or B antigen (MAb-B) reacted with nuclear heterochromatin regions, as well as secretory granules, in mucous cells of human cervical glands. Systematic and critical observation of specimens from 24 individuals of different blood groups revealed that the labeling pattern with MAb with strictly dependent on the blood group (A,B, or O) of the donors, i.e., MAb-A reacted with the heterochromatin from blood group A and AB but not with B and O individuals. Labeling with MAb-B was also specific for the heterochromatin from blood group B donors. On the other hand, MAb against H antigen did not react with the heterochromatin from any individuals examined, despite the fact that H antigens were detected by the MAb in secretory granules. Such specific reactions provide evidence that certain types of blood group-related antigens exist in the nuclear heterochromatin in mucous cells of human cervical glands. In contrast to the secretory granules in which ABH antigens were recognized by blood group specific lectin, heterochromatin regions had little or no affinity for these lectins. Furthermore, the secretory status of individuals affected the staining intensity with MAb in secretory granules but not in the heterochromatin. These results suggest that the blood group substances found in the heterochromatin may have different molecular properties from those in the secretory granules, although both have the same determinant structures of ABH antigens. PMID- 1706376 TI - Immunohistochemical localization and quantification of desmoplakins I & II and keratins 1 and 19 in plastic-embedded sections of human gingiva. AB - We developed immunohistochemical and image analytical techniques to localize and quantify keratins and desmoplakins in sections of plastic-embedded human gingiva. Acetone fixation followed by plastic embedding of gingiva provided excellent morphology and permitted immunohistochemical detection of keratins 1 and 19 and desmoplakins I & II after 2.5-min trypsin digestion. Quantitative image analysis demonstrated that different volume densities of staining of each marker were associated with specific epithelial strata. Keratin 1 stained most heavily in granular strata, followed by corneal and spinous strata; keratin 19 stained most strongly in the basal layer; desmoplakins I & II stained most strongly in the granular and corneal strata. These findings confirm that variations of keratin and desmoplakin expression in these epithelial are associated with regional patterns of epithelial differentiation. PMID- 1706377 TI - A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry. AB - We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB. PMID- 1706378 TI - CD40 signaling activates CD11a/CD18 (LFA-1)-mediated adhesion in B cells. AB - Cell-cell adhesion events play critical roles in the sequential migrations and multiple specific cell-cell interactions which B cells undergo during normal development and function. We have observed that mAb to several B cell-associated molecules, including mAb to CD19, CD37, and CD40, induce homotypic aggregation of freshly isolated human B cells. The aggregation of B cells induced by CD40 mAb was due to activation of a cell-cell adhesion system, and not due to agglutination by mAb, because 1) in addition to being energy dependent and cation dependent, the aggregation was blocked by inhibitors of messenger RNA and protein synthesis; and 2) a mouse B cell line transformed with intact human CD40 aggregated in response to CD40 mAb, whereas a line expressing surface CD40, but lacking the cytoplasmic tail and previously shown incapable of transmitting a signal from the cell surface, did not aggregate. The aggregation, although of slow onset, was persistent and of high avidity. In addition, CD40 mAb induced increased surface expression of intercellular adhesion molecule-1 (CD54), a ligand for CD11a/CD18 (LFA-1), and CD18 mAb blocked aggregation. CD40 mAb also augmented the ability of dense B cells to stimulate the proliferation of allogeneic T cells via a CD18-dependent process. We conclude that signaling through CD40, elicited by cross-linking the CD40 protein on the cell surface, activates the CD18/intercellular adhesion molecule adhesion system; in addition, CD40 cross-linking may activate a second adhesion system since CD40 mAb induced aggregation of the B cell line Ramos, which does not express surface CD18. B cell adhesion may be triggered by signaling through multiple surface proteins, thereby lending specificity of activation to adhesion systems which are broadly expressed. PMID- 1706379 TI - Human T cell responses to IL-1 and IL-6 are dependent on signals mediated through CD2. AB - We investigated the involvement of IL-1 and IL-6 in activation of resting human T lymphocytes via the Ti-Ag receptor/CD3-dependent and the CD2-dependent pathways, respectively. When lymphocytes were triggered through CD3-Ti, neither IL-1 nor IL 6 nor the combination of both cytokines was capable of inducing a proliferative response, whereas addition of monocytes or IL-2 to such a system mediated DNA synthesis and cellular mitosis. In contrast, in the presence of submitogenic concentrations of mAb directed at CD2, IL-1 and/or IL-6 produced marked comitogenic dose-dependent effects. Moreover, although the action of IL-1 was clearly dependent on expression of the IL-2/IL-2R system, proliferation to CD2 antibody plus IL-6 could not be blocked by mAb directed at the IL-2R and/or IL-4. T cell responsiveness to both IL-1 and IL-6 was facilitated in the presence of CD58-like signals as delivered by human rCD58, SRBC or a mAb (anti-T111A), which binds to an interaction site for CD58 on the human CD2 molecule. These findings indicate that CD2 and its ligand CD58 play an important role in T cell/monocyte interactions during primary immune responses by means of upregulating T cell susceptibility to monocyte-derived cytokines. PMID- 1706380 TI - CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation. AB - Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation. PMID- 1706381 TI - Alloreactive T cells can distinguish between the same human class II MHC products on different B cell lines. AB - Certain allele-specific alloreactive T cell clones do not recognize the products expressed by some B cell lines that, according to typing methods other than sequencing, carry the allelic molecules recognized by these clones. In order to characterize the naturally occurring sequence polymorphisms putatively responsible for the differential allorecognition of these class II molecules, we have determined the third and/or second exon nucleotide sequences of HLA-DRB1, DRB3/4/5, -DQB1, and -DQA1 genes from 35 representative lymphoblastoid cell lines. In some cases, the lack of recognition correlates with the presence of single amino acid substitutions in either the second or third hypervariable region (HVR) of the first domain of these molecules. In other cases, the differentially allorecognized class II molecules have identical second and/or first domain amino acid sequences. These findings indicate that a) class II MHC alloreactive T cell clones can distinguish between molecules with identical amino acid sequences expressed by B cell lines established from unrelated individuals; b) allorecognition of class II molecules is sensitive to naturally occurring single amino acid substitutions in either the second HVR of class II molecules, which is unavailable to interact with TCR residues, or the third HVR. Our results also suggest that 1) in different B cell lines, identical class II molecules may present different endogenous peptides, which may behave as histocompatibility Ag; 2) the peptide-binding specificity of a class II molecule may be affected by amino acid substitutions in its second HVR (Ag-binding site); and 3) human class II allorecognition may be restricted by epitopes contributed by residues of their third HVR. PMID- 1706383 TI - A Waldenstrom's macroglobulin with anti-G3m(b1) antibody activity. AB - A human monoclonal macroglobulin (IgM, K) from a patient (KI) with Waldenstrom's macroglobulinemia was shown to have antibody activity against a human IgG (Gm) allotype. In hemagglutination tests, only one anti-D serum with G3m(b0b1) reacted with macroglobulin KI. Antiglobulin specificity of macroglobulin KI was determined to be an anti-G3m(b1) antibody by hemagglutination inhibition tests. Fab fragments from macroglobulin KI could react with human IgG3 protein possessing G3m(b1), but Fc fragments could not react. Gm phenotype in IgG isolated from serum KI was determined to be Gm(a,z,g,b0,s,t,u). This is the first report of a Waldenstrom's macroglobulin with antiglobulin specificity against a Gm allotype. PMID- 1706382 TI - CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production. AB - IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease. PMID- 1706384 TI - Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice. AB - Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101 148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121 140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations. PMID- 1706385 TI - The naive repertoire of human T helper cells specific for gp120, the envelope glycoprotein of HIV. AB - The envelope glycoprotein of HIV gp120 is a T cell Ag in experimental animals and in humans infected with HIV or deliberately immunized with gp120 in various forms. Inasmuch as T cell responses result from the interaction of Ag processed and presented by APC with the unprimed T cell repertoire, we have investigated the human T cell repertoire specific for gp120 in seronegative, normal individuals. T cell lines and clones specific for HIV gp120 were generated by repeated in vitro stimulation of peripheral blood T lymphocytes with gp120-pulsed APC, followed by IL-2 expansion. We observed that the T cell response to whole gp120 involved single restricted immunodominant epitopes in gp120 that differ between responding individuals. Focusing of the response to limited regions of gp120 when the whole Ag is used for priming suggests that one or more adjacent epitopes are immunodominant and mask responses to "immunorecessive" epitopes. We have been able to generate primary in vitro responses to recessive epitopes by stimulation in vitro with synthetic peptides of gp120. The results indicate that a much broader T repertoire can be detected when individual peptides are used for priming in vitro rather than gp120. This information has important implications for the development of vaccination protocols aimed at eliciting diverse immune responses to "immunorecessive" regions of envelope glycoprotein. PMID- 1706386 TI - Characterization of the human complement receptor 2 (CR2, CD21) promoter reveals sequences shared with regulatory regions of other developmentally restricted B cell proteins. AB - Expression of human complement receptor 2 (CR2, CD21, C3d,g/EBV receptor) is developmentally restricted on human B lymphocytes to cells of the late-pre and mature stages. CR2 is also a member of the genetically linked regulators of complement activation family found on human chromosome 1q32. Regulators of complement activation proteins are variably expressed in plasma, on cell membranes, and in nonvascular extracellular fluid sites. To begin to understand the mechanisms that control both tissue specific and B cell developmental restriction of CR2 expression, we have cloned and characterized the CR2 promoter upstream of a single apparent transcriptional initiation site. Within this region are sequences with significant similarity to previously characterized TATA, SP1, AP-2, AP-1-like, and Ig enhancer E motif DNA protein binding sites, in addition to direct and inverted repeats. Significant regions of identity are also found between CR2 promoter sequences and those of the CD23 promoter, another protein whose expression is developmentally restricted on B cells. The CR2 promoter will direct transcription of the reporter gene chloramphenicol acyltransferase when transiently transfected into the human Raji B cell line. Therefore, we have identified the promoter for a human B cell protein whose expression is developmentally restricted. Further analysis of this region and the transcriptional regulation of CR2 gene expression should lead to significant insights into the molecular mechanisms by which B cells mature and are activated. PMID- 1706387 TI - Substance P acts directly upon cloned B lymphoma cells to enhance IgA and IgM production. AB - The IgA producing murine B lymphoma, CH12.LX.C4.4F10 (4F10) and the IgM producing murine lymphoma, CH12.LX.C4.5F5 (5F5) were found to express substantial numbers of substance P (SP) receptors having dissociation constants equal to 0.69 nM. Binding of SP by these B lymphoma cells was via the tachykinin-specific C terminus sequence, Phe-X-Gly-Leu-Met-NH2, because SP, SP antagonist (D-Pro2-D Phe7-D-Trp9-SP), eledoisin, and substance K could effectively inhibit radiolabeled SP binding, whereas the SP N-terminus fragment, SP (1-4), could not. The functionality of these receptors could be demonstrated by the ability of subnanomolar concentrations of SP to induce Ig secretion in a dose-dependent fashion. However, the presence of a second stimulus in these cultures was required to obtain maximal increases. IgA secretion by 4F10 cells was elevated only 25 to 37%, and IgM secretion by 5F5 cells was not significantly increased in cultures in which nanomolar concentrations of SP were present. Conversely, coculturing 5F5 cells with a suboptimal concentration of LPS (50 ng/ml) and 10( 10)M SP resulted in an approximate threefold increase in supernatant IgM when compared to control cultures stimulated with LPS alone. While not as dramatic, 10(-10) M SP also enhanced IgA secretion of LPS-stimulated 4F10 cells by approximately 45%. This enhancement of Ig secretion was SP-specific, as evidenced by the ability of 1000-fold excess of SP antagonist to block SP-induced, but not LPS-induced, Ig production. Clearly, SP could act synergistically with LPS to enhance Ig secretion; therefore, we questioned whether this augmentation was also reflected at the level of H chain mRNA expression. 10(-9)M SP induced modest increases (50 to 60%) in mu-chain mRNA expression by LPS-stimulated 5F5 cells when compared with cells stimulated with LPS alone. The 4F10 cells did not display this magnitude of difference for alpha-chain mRNA expression. Thus, although SP-induced increases of mu-chain mRNA by 5F5 cells may contribute to the increased Ig secretion observed by these LPS-activated lymphocytes, it is unlikely that increased mRNA expression can totally account for the threefold increases in secretion that were observed. PMID- 1706388 TI - Immunodominance: intramolecular competition between T cell epitopes. AB - We have used an approach of linking previously characterized T cell epitopes into immunologically complex synthetic peptides in order to investigate the mechanism of immunodominance. Our results show that first, cI12-26 is highly dominant following immunization with the lambda repressor (cI) protein, but is a minor epitope in the context of the cI:NP peptide. In contrast, the dominant epitope in response to the cI:NP peptide is a new junctional epitope, which is composed of sequences derived from both the cI and influenza nucleoprotein (NP) segments of the composite peptide. Second, T cell recognition of cI:NP is not significantly altered by Ag processing, based on results from glutaraldehyde-fixed APC. Third, the relative affinities of cI and cI:NP for MHC binding are similar, based on in vitro competition, excluding competition at the level of MHC binding as the determinant of immunodominance. Taken together, these results are consistent with the hypothesis that immunodominance of cI:NP is determined by peptide conformation, which affects the configuration of peptide binding to MHC, thus altering T cell recognition. In conclusion, immunodominance is not simply a function of the primary amino acid sequence, but is a function of the context of the epitope within the protein molecule. PMID- 1706389 TI - Development of a monoclonal antibody, anti-6C2, which is involved in the interaction of CD4 T helper cells and activated B cells. AB - We have developed a mAb anti-6C2, by immunizing mice with T cell line derived from the Callithrix jacchus (common marmoset). Anti-6C2 is reactive with approximately 50% of unfractionated T cells, 50% of CD4+ cells, and 40% of CD8+ cells. Regarding CD4+ cells, anti-6C2-reactive cells substantially overlap with the CD29+CD45RO+ Th cell population. Moreover, anti-6C2 can divide these T cells into 6C2+ and 6C2- subpopulations. The CD4+CD45RO+6C2+ cells maximally respond to soluble Ag such as tetanus toxoid and provide strong helper function for PWM driven B cell IgG synthesis. Most interestingly, anti-6C2 was also reactive against activated B cells but not resting B cells; furthermore, this epitope was inducible through activation of resting B cells or B cell line. Biochemical characterization showed that anti-6C2 precipitated two glycoproteins with the relative molecular weights of 180,000 and 95,000 from 125I-surface labeled cell lysate. Sequential immunoprecipitation studies demonstrated that these two glycoproteins were the lymphocyte function-associated antigen (LFA-1) Ag complex (CD11a/18). Significantly, although this antibody did not inhibit cytotoxic killer T cell responses and Ag-induced T cell proliferation as did conventional anti-LFA-1, it did inhibit PWM-driven B cell IgG synthesis. Because 6C2 expression was induced after B cell activation, the above results strongly suggest that the 6C2 molecule may play a role in the interaction of CD4 helper cells and activated B lymphocytes. PMID- 1706390 TI - Infection of CD8+ T lymphocytes with HIV. Requirement for interaction with infected CD4+ cells and induction of infectious virus from chronically infected CD8+ cells. AB - In this study, we have investigated the basic requirements for HIV-1 infection of CD8+ lymphocytes in vitro. Unfractionated PBL obtained from healthy HIV-1 seronegative donors were activated with PHA and infected in vitro with HIV-1LAV. Based on immunofluorescent labeling, the vast majority of cells (85 to 97%) surviving peak virus replication belonged to the CD8+ subset and only a small percentage (0.5 to 1.5%) were CD4+. Amplification of HIV-1 proviral sequences by polymerase chain reaction performed on the sorted surviving CD8+ cells demonstrated that CD8+ cells harbored HIV-1 proviral DNA. In addition, stimulation of these HIV-1-infected, CD8(+)-sorted cells either with PHA or anti CD2 mAb resulted in induction of virus replication, as measured by reverse transcriptase activity. Electron microscopic analysis of CD8+ cells chronically infected with HIV-1 and stimulated with PHA showed typical virions budding from, and associated with, the surface of cells immunolabeled with gold beads directed toward the CD8 molecule. Infection of CD8+ cells with HIV-1 occurred only when CD4+ cells were present in the PHA-activated lymphocyte population exposed to HIV 1 at the beginning of the culture or when sorted CD8+CD4- lymphocytes were cocultured with autologous sorted CD8-CD4+ cells that had been previously infected with HIV-1. Coculture of these cells with PHA-blasts and incubation of their supernatants with a CD4+ cell line showed that these chronically infected CD8+ cells could spread HIV-1 infection to uninfected CD4+ cells after stimulation with PHA or anti-CD2 mAb. Therefore, these results suggest that the minimal requirement for in vitro infection of CD3+CD8+CD4- lymphocytes is the presence of infected CD4+ cells and that infected CD8+ T lymphocytes can further spread the infection to CD4+ cells. PMID- 1706391 TI - Myasthenia gravis. T epitopes on the delta subunit of human muscle acetylcholine receptor. AB - Autoimmune T cell lines specific for muscle nicotinic acetylcholine receptor (AChR) were propagated from the blood of three myasthenia gravis patients by the use of a pool of synthetic peptides (delta-pool) corresponding to the complete sequence of the delta-subunit of human muscle AChR. Propagation of AChR-specific T cell lines was attempted unsuccessfully from four other myasthenia gravis patients and from four healthy controls. The lines had CD3+, CD4+, CD8- phenotype, strongly recognized the delta-pool, and cross-reacted vigorously with non-denatured AChR purified from mammalian muscle. They did not cross-react detectably with pools of similar overlapping synthetic peptides corresponding to the complete sequences of the alpha- and gamma-subunits of human muscle AChR. The sequence segments of the delta-subunit that contain T epitopes were identified by investigating the response of the three CD4+ T cell lines to the individual synthetic peptides forming the delta-pool. Each line had an individual pattern of peptide recognition. Although no immunodominant region, recognized in association with different DR haplotypes, could be identified, the sequence segments most strongly recognized by the CD4+ T cell lines were clustered within residues 121 290. One of the peptides more strongly recognized by the T cells corresponded to a sequence segment with high predicted propensity to form an amphipathic alpha helix, a structural motif proposed to be typical of T epitopes. PMID- 1706392 TI - Selective stimulation of thymocyte precursors mediated by specific cytokines. Different CD3+ subsets are generated by IL-1 versus IL-2. AB - The sequence of activation signals that stimulate proliferation, differentiation, and selection of mature T cell subsets from immature, dull-CD5+/CD4-, CD8- double negative (bCD5), (dCD5/DN) thymocytes are still unclear. However, it is likely that cytokines play integral roles in these events. Here we report that IL-1, in the presence of Con A, supports the proliferation and differentiation of highly purified dCD5/DN precursors into bright-CD5+ DN, CD2- lymphocytes with an apparently mature phenotype. These cells express CD3 and preferentially express the products of two TCR gene families, V beta 8 and V beta 6, whose expression is dependent on the allelic expression of the Mls-1 locus. Experiments, using DN thymocytes mixed with purified dCD5 subset of DN cells from a congenic strain of mice (i.e., expressing two different alleles of CD5) have shown that the cells that are stimulated by IL-1 and comitogen are derived from the immature dCD5 subset and not from the mature bCD5 cells contained within the DN subset. In contrast, IL-2 with the co-mitogen stimulates three- to fourfold higher levels of proliferation, from the same purified immature precursor population, and nearly a twofold increase in cell yield. However, the cells that were generated from precursor thymic cells stimulated with IL-2 represent a completely different T cell subset compared to IL-1-generated cells; these IL-2-stimulated cells express comparable levels of CD3, but also express substantial levels of CD2 and the TCR gamma/delta, and a subset expresses CD8. These data suggest that these two TCR alpha/beta and TCR-gamma/delta subsets of mature thymocytes use different cytokines and therefore possibly different stromal interactions to initiate differentiation. PMID- 1706393 TI - Single amino acid changes in DR and antigen define residues critical for peptide MHC binding and T cell recognition. AB - Single amino acid substitutions of Ag and MHC were used to analyze the fine structure of the influenza hemagglutinin (HA)-derived epitope (HA 307-319) recognized in the context of DR7 molecules by a T cell clone. Putative T cell (HA 308, 310, 311, 313, and 316) and DR (HA 309, 312, and 317) contact residues of the Ag were identified by the use of single amino acid-substituted analogs that were tested for their T cell-activating and DR-binding capacities. The peptide DR7-T cell interaction was further characterized by the use of a panel of 13 site directed DR7 mutant transfectants analyzed for their capacity to present Ag to T cells, and for their purified mutant DR7 molecules to bind HA 307-319 or its single amino acid-substituted analogs. Eight mutants lost their Ag-presenting function, whereas only one had any decrease in peptide binding. Finally, for three of the mutants it was possible to correct the deleterious effects of mutation by using a particular single amino acid-substituted analog of the peptide molecule. The observed pattern of complementation led to a model that predicts that the Ag assumes an extended conformation, with a turn, in the binding groove, such that the following residues are in close proximity: DR 86-HA 309, DR 71-HA 312, DR 30-HA 314, and 315. PMID- 1706394 TI - Autoantibodies to the centrosome (centriole) react with determinants present in the glycolytic enzyme enolase. AB - Autoantibodies to cellular Ag are found in the sera of patients with systemic rheumatic diseases. Identification and characterization of the reactive autoantigens has helped clinicians to define subsets of rheumatic diseases and has assisted biologists in defining the function within the cell of these molecules. We have studied autoantibodies from patients that react with the centrosome (centriole) region of the cell. We found by immunoblotting techniques that these antibodies react with a 48-kDa protein. Additional immunoblotting and affinity purification studies indicate that the Ag may be the glycolytic enzyme enolase. PMID- 1706395 TI - Inhibition of homologous complement by CD59 is mediated by a species-selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9. AB - The capacity of the human complement regulatory protein CD59 to interact with terminal complement proteins in a species-selective manner was examined. When incorporated into chicken E, CD59 (purified from human E membranes) inhibited the cytolytic activity of the C5b-9 complex in a manner dependent on the species of origin of C8 and C9. Inhibition of C5b-9-mediated hemolysis was maximal when C8 and C9 were derived from human (hu) or baboon serum. By contrast, CD59 showed reduced activity when C8 and C9 were derived from dog or sheep serum, and no activity when C8 and C9 were derived from either rabbit or guinea pig (gp) serum. Similar specificity on the basis of the species of origin of C8 and C9 was also observed for CD59 endogenous to the human E membrane, using functionally blocking antibody against this cell surface protein to selectively abrogate its C5b-9 inhibitory activity. When E bearing human CD59 were exposed to C5b-8hu, CD59 was found to inhibit C5b-9-mediated lysis, regardless of the species of origin of C9, suggesting that the inhibitory function of CD59 can be mediated through recognition of species-specific domains expressed by human C8. Consistent with this interpretation, CD59 was found to bind to C5b-8hu but not to C5b67hu or C5b67huC8gp. Although CD59 failed to inhibit hemolysis mediated by C5b67huC8gpC9gp, its inhibitory function was observed for C5b67huC8gpC9hu, suggesting that, in addition to its interaction with C5b-8hu, CD59 also interacts in a species-selective manner with C9hu incorporated into C5b-9. Consistent with this interpretation, CD59 was found to bind both C5b67huC8gpC9hu and C5b-8huC9gp, but not C5b67huC8gpC9gp. Taken together, these data suggest that the capacity of CD59 to restrict the hemolytic activity of human serum complement involves a species-selective interaction of CD59, which involves binding to both the C8 and C9 components of the membrane attack complex. Although CD59 expresses selectivity for C8 and C9 of human origin, this "homologous restriction" is not absolute, and this human complement regulatory protein retains functional activity toward C8 and C9 of some nonprimate species. PMID- 1706396 TI - Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules. AB - To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site. PMID- 1706397 TI - Altered surface expression of CD11 and Leu 8 during human basophil degranulation. AB - Immunofluorescence and flow cytometric techniques have been used to study changes in surface Ag expression and viability that occur during human basophil degranulation. Treatment with polyclonal anti-IgE, FMLP, or the calcium ionophore A23187 induced histamine release, along with rapid and sustained unimodal increases in basophil CD11b mean fluorescence intensity. In contrast, treatment with anti-IgE or FMLP resulted in a decrease in Leu 8 expression. Degranulation did not significantly affect basophil viability (as determined by exclusion of propidium iodide), scatter characteristics, or percentage of identifiable IgE bearing cells, and an inconsistent association was seen between percent histamine release and reduction in the percent of cells identified by light microscopy after staining with alcian blue. For anti-IgE, dose-dependent changes in CD11b, CD11c, and Leu 8 expression were seen (optimal at 0.1, 0.1, and 1 microgram/ml, respectively), although CD11a expression remained unchanged. Histamine release was optimal at 0.3 microgram/ml anti-IgE, and at superoptimal concentrations, reduced CD11b expression was observed which paralleled decreases in histamine release; reduction of the expression of Leu 8, however, occurred equally at optimal and superoptimal concentrations of anti-IgE. Kinetic analyses of these responses revealed that CD11b up-regulation proceeded more rapidly than histamine release, whereas Leu 8 down-regulation was much slower and did not plateau until 120 min of stimulation. Although changes in CD11b mean fluorescence intensity correlated with the magnitude of histamine release, exposure to stimuli in the absence of calcium (which blocked degranulation) resulted in similar alterations in CD11b and Leu 8, suggesting that degranulation was not required for changes in the surface expression of these adhesion molecules. Interestingly, pretreatment of basophils with drugs that either inhibited or enhanced histamine release (isobutylmethylxanthine and cyclosporin A vs cytochalasin B, respectively) significantly decreased the magnitude of anti-IgE-induced CD11b up-regulation; down-regulation of Leu 8 expression was also partially inhibited by treatment with isobutylmethylaxanthine. These studies demonstrate that activation of human basophils by secretagogues in vitro results in a variety of phenotypic changes including alterations in surface expression of adhesion molecules, and suggest that degranulation in vivo may be accompanied or preceded by changes in adhesion related functions. PMID- 1706398 TI - FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells. AB - FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK 506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/ 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK 506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein). PMID- 1706399 TI - Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti alpha-galactosyl antibodies. AB - Antibodies that lyse trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of trypomastigotes by antiGal. Lysis of metacyclic trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection. PMID- 1706400 TI - Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus induced demyelinating disease. IV. Identification of an immunodominant T cell determinant on the N-terminal end of the VP2 capsid protein in susceptible SJL/J mice. AB - Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease serves as a relevant animal model of human multiple sclerosis. Myelin damage induced by TMEV infection appears to be immune mediated. Disease susceptibility correlates best with the temporal development of chronic, high levels of TMEV specific, MHC class II-restricted delayed-type hypersensitivity (DTH) responses. We have proposed a model wherein these responses result in CNS demyelination via a macrophage-mediated terminal nonspecific bystander response. As virus-specific DTH responses appear to be intimately involved in the pathogenicity of CNS demyelination, it is critical to determine the specificity of these responses so that effector T cells specific for potential pathogenic epitopes can be targeted to serve as the focus of specific immunoregulatory processes. In the current study, the capsid protein specificity of the TMEV-susceptible SJL/J and TMEV resistant C57BL/6 mouse strains was examined. DTH and Tprlf responses in both infected and immunized SJL/J mice were found to be predominantly directed toward the VP2 capsid protein, specifically to an epitope(s) contained within the N terminal 150 amino acids of VP2. This same epitope was also found to be dominant in priming SJL/J mice for responses to challenge with intact virions. In contrast, the T cell-mediated responses of TMEV-resistant C57BL/6 mice did not show preferential reactivity towards VP2, because all three major capsid proteins (VP1, VP2, and VP3) elicited responses with essentially equal potency. The relationship of the restricted VP2 T cell epitope to predicted neutralizing antibody sites on the VP2 protein is discussed as is the potential use of this epitope for prevention and/or treatment of TMEV-induced demyelinating disease via the induction of epitope-specific tolerance. PMID- 1706401 TI - A granulocyte-colony-stimulating factor gene promoter element responsive to inflammatory mediators is functionally distinct from an identical sequence in the granulocyte-macrophage colony-stimulating factor gene. AB - A number of mesenchymal cells produce hemopoietic growth factors in response to inflammatory mediators in vitro and in vivo. Induced transcription from the hemopoietic growth factor genes is at least partially responsible for their increased expression. We have previously identified a sequence, cytokine (CK)-1, in the granulocyte (G)-CSF gene promoter that responds to TNF-alpha and binds a transcription factor, NF-GMa. We report here that the CK-1 sequence responds in a time- and dose-dependent manner to IL-1 beta and that the mutations which affect NF-GMa binding correlate with decreased transcriptional activity after stimulation with either TNF-alpha or IL-1 beta. The CK-1 sequence also responds to the human T lymphotrophic virus-1 transactivator, tax, so that this promoter element may contribute to the overall response of the G-CSF gene to these various agents. Although NF-GMa binding is seen in a number of cell types, the ability of the G-CSF CK-1 sequence to act as a transcriptional enhancer is specific for fibroblasts and not T cells. Furthermore, we show that an identical sequence in the granulocyte macrophage CSF gene, although apparently binding the same protein in vitro, cannot respond to any of these stimuli in either fibroblasts or T cells. Modification interference experiments, using the CK-1 region in the context of the granulocyte macrophage-CSF and G-CSF genes, indicated that the contact points for NF-GMa differ in each case and suggest that differences in sequences flanking the 10-bp CK-1 region probably leads to an altered DNA:protein conformation, which may explain the differential response of this conserved promoter element. PMID- 1706402 TI - Genetic construction and characterization of a fusion protein consisting of a chimeric F(ab') with specificity for carcinomas and human IL-2. AB - A genetic construct was created incorporating gene fragments encoding the H chain V region of the human carcinoma specific antibody L6, the CH1 domain of human IgG1, a linker region, and human IL-2. This construct was cotransfected with a chimeric L6 L chain construct into the murine myeloma cell line Ag8.653 for expression. First round clones produced the fusion protein at an estimated 5 to 10 micrograms/ml based on idiotypic reactivity. Dual binding activity was demonstrated through specific interaction with the L6 Ag on human tumor cells and the IL-2R on activated human T cells. The IL-2 portion of the molecule was shown to support the growth of the IL-2-dependent T cell line CTLL2, and the qualitative nature of the IL-2 signal was found to be the same as rIL-2 with respect to induction of tyrosine-phosphorylation of intracellular protein substrates. Tumor cells coated with the fusion protein were shown to cause T cell proliferation and the presence of the fusion protein was found to enhance cell mediated destruction of human tumor cells. PMID- 1706403 TI - [Histochemical and ultrastructural observations of limb muscle in spontaneous diabetic mice]. AB - Spontaneous non-obese diabetic (NOD) mice were used to study the morphological change of muscle fibers in diabetes mellitus. The gastrocnemius and soleus muscles were removed from the NOD mice suffering from diabetes mellitus for 1, 3, 6 or 9 weeks. Muscle fibers were observed electronmicroscopically and histochemically, by staining with Sudan black B. Single fiber atrophy was noted in white fibers at an early stage of the illness. At the age of 6 and 9 weeks, white fibers showed extensive damage, characterized by the atrophy and wide separation of myofibrils, irregularity and thinness of myofilaments and massive glycogen deposition. The damage in the myofibril itself, however, was less pronounced in red and intermediate fibers, and those fibers had more abnormal mitochondria and a decreased affinity for sudan pigment. Morphological changes were varied in the three types of fibers. It is concluded that diabetes mellitus had different effects on each three types of muscle fibers neurogenically and metabolically. PMID- 1706404 TI - Effects of exogenous porcine growth hormone on serum insulin-like growth factor binding proteins in growing pigs. AB - The insulin-like growth factor-binding proteins (IGFBPs) in sera of growing pigs were partially characterized with respect to their size, immunological relationships to other known IGFBPs and their regulation by porcine (p) GH. Castrated male pigs (14-16 weeks of age) were treated with either vehicle or pGH (up to 100 micrograms/kg body weight per day) by daily i.m. injection for 7 days. Blood samples were collected by jugular venepuncture at the time of injection. Five IGFBPs of 43, 40, 34, 30 and 26 kDa were identified on ligand blots of porcine sera. A 30 kDa IGFBP, in addition to the 43 and 40 kDa IGFBPs, was immunoprecipitated by antiserum to pIGFBP-3 and found to contain N-linked carbohydrate suggesting that it is a fragment of pIGFBP-3 as has been noted for a 29 kDa N-glycosylated IGFBP in rat sera. The 34 kDa IGFBP in pig sera was precipitated by antisera to rat IGFBP-2 and contained no N-linked carbohydrate. Administration of pGH to normal growing pigs not only increased pIGFBP-3 levels but elicited a dose-dependent suppression of levels of the 34 kDa IGFBP as well. In summary, the Mr pattern of IGFBPs in the sera of growing pigs is similar to that observed in fetal and maternal pig sera and in other species. Furthermore, we report that administration of pGH to normal pigs suppresses the expression of an IGFBP-2-like IGFBP in pig sera while increasing expression of pIGFBP-3. PMID- 1706405 TI - Secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins from bovine mammary tissue in vitro. AB - In vitro, insulin-like growth factor-I (IGF-I) promotes both growth and development of bovine mammary tissue. In vivo, the effects of IGF-I may encompass endocrine, paracrine or autocrine mediation. We addressed the possibility of paracrine/autocrine effects of IGF-I in the mammary gland by examining the in vitro secretion of IGF-I and IGF-binding proteins (IGFBPs) from bovine mammary tissue. Bovine mammary explants from pregnant non-lactating and lactating non pregnant animals were found to synthesize and secrete IGF-I and IGFBPs. Mammary acini cultures, representative of mammary secretory epithelia, secreted both IGF I and IGFBP, but synthesized only IGFBP. Concentrations of IGF-I in conditioned media from explants were 1.54 and 0.72 fmol/micrograms DNA for pregnant and lactating animals respectively. Concentrations of IGFBPs in conditioned media from explants were similar for both physiological states at 2529 pmol 125I labelled IGF-I bound/micrograms DNA. Ligand/Western blotting procedures identified four IGFBPs of 29, 33, 37 and 44 kDa for acini cultures and five IGFBPs of 28, 31, 36, 44 and 46 kDa for explant cultures. Similar affinities for IGF-I and IGF-II were shown by IGFBP, using 125I-labelled recombinant human IGF-I as the competing ligand (median effective dose (ED50) of 0.085 pmol). When 125I labelled bovine IGF-II was used as the ligand, only bovine IGF-II (ED50 of 0.25 pmol) inhibited binding. The addition of prolactin, insulin and cortisol, with or without GH, did not affect secretion of either IGF-I or IGFBP. This report describes the ability of normal mammary tissue to synthesize and secrete IGF-I and IGFBPs. PMID- 1706406 TI - Activation of the serotonergic system in chick spinal cord during hatching. AB - The objectives of the present study were to determine the levels of serotonin (5 HT), its major catabolic metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) in chick spinal cord before, during, and after hatching and also to determine if changes in the levels of these chemicals are directly related to the hatching behavior. The levels of 5-HT, 5-HIAA, and NE were measured by high performance liquid chromatography with electrochemical detection in whole spinal cords of 20-day-old "pre-hatching" embryos, 21-day-old "normal hatching" embryos, 0-day-old "post-hatching" chicks, and 0-day-old "glass egg hatching" chicks. NE was measured but no significant differences were found in NE levels among experimental groups. The concentration of 5-HT was elevated in chick embryo spinal cords during normal hatching compared to pre-hatching embryos and post-hatching chicks. The concentration of 5-HIAA was elevated during and after normal hatching compared to pre-hatching embryos. However, neither 5-HT nor 5 HIAA levels were found to be elevated in chick spinal cords during glass egg hatching compared to pre-hatching embryos or post-hatching chicks. Therefore, there appears to be an activation of the serotonergic system in chick spinal cord related to the specific event of hatching but this activation is not directly related to the movements common to both hatching and glass egg hatching. PMID- 1706407 TI - Interferon production from peripheral blood, synovial fluid, and synovial tissue lymphocytes in patients with rheumatoid arthritis and ankylosing spondylitis. AB - A previous study demonstrated that interferon was present in the serum of 30% of the patients with systemic lupus erythematosus (SLE), which was significantly higher than the 4.5% found in normal controls. We also recently reported that interferon production was deficient from SLE mononuclear cells, which has been attributed to immunodeficiency of the lymphocytes. In this study, interferon measurement included lymphocytes obtained from peripheral blood (PB), synovial fluid (SF) and synovial tissue (ST) in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). PB from normal subjects (NS) was used as a control. The results showed with PHA stimulation, that the interferon level in PBL (L = lymphocyte) in NS (70.0 +/- 67.5) was significantly higher when compared with PBL in RA (27.9 +/- 21.6). However, there was no difference between PBL in NS and AS. With ConA stimulation, the interferon level was significantly higher in the PBL of NS (130 +/- 59) and as compared with the PBL in RA (83.6 +/- 53.5). The SFL in RA (67.8 +/- 31.1) and the STL in RA (77.2 +/- 93.2) were also significantly different. It is concluded that interferon production was deficient not only in PBL in RA, but also in SF and STL in RA. The reduced interferon production from PB, SF and ST lymphocytes in RA patients may be due to previous release or immunodeficiency. Lymphocyte interferon production was normal in AS, which suggests that the lymphocyte abnormality between RA and AS may be different. PMID- 1706408 TI - Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies. AB - Monoclonal antibodies (MAbs) were produced against two African arenaviruses, Lassa virus and Mopeia virus. Competitive binding analysis of MAbs identified four antigenic sites on the nucleoprotein (NP), two on glycoprotein 1 (GP1) and six on glycoprotein 2 (GP2) of the Josiah strain of Lassa virus. 64 virus isolates from western, central and southern Africa were all consistently distinguishable by MAbs to certain epitopic sites on GP1, GP2 and NP viral proteins. Furthermore, MAbs to Lassa virus GP1 and NP uniformly distinguished viruses from the West African countries of Sierra Leone, Liberia and Guinea from those of Nigeria. GP2-directed MAbs to two African arenaviruses reacted broadly with South American arenaviruses demonstrating that an epitopic site on GP2 may be the most highly conserved antigen in the arenavirus group. PMID- 1706409 TI - Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine. AB - Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection. PMID- 1706410 TI - Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4. AB - Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G + C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4. PMID- 1706411 TI - Distribution and characterization of pedal peptide immunoreactivity in Aplysia. AB - Pedal peptide (Pep) is a very abundant neuropeptide in Aplysia. A radioimmunoassay (RIA) was developed to quantify Pep-like immunoreactivity (IR Pep) in tissue extracts. IR-Pep was present in very high concentrations in the central nervous system (CNS) and two peripheral tissues: the large hermaphroditic duct (LHD) and the foot. RIA of fractions from high-pressure liquid chromatography (HPLC) indicated that Pep itself was the predominant immunoreactive species in each of these tissues. Lower concentrations of Pep were found in a number of other peripheral tissues. Incorporation of labelled amino acid indicated that Pep was synthesized in the LHD, whereas Pep in the foot was synthesized primarily in central neurons and transported to the foot. IR-Pep was further localized by immunocytology. All peripheral IR-Pep appeared to be associated with neuronal fibers, most commonly varicose axons. Immunoreactive innervation of the LHD and foot was particularly dense but positive staining was also observed in other tissues including tegument, gill, gut, and heart, IR-Pep innervation in all tissues including the LHD appeared to be localized predominantly in muscular portions of the tissue. Spontaneous contractions of isolated LHD were accelerated by the application of Pep. Pep appears to act as a transmitter or neuromodulator at a number of different sites in Aplysia. PMID- 1706412 TI - Activity-driven sharpening of the regenerating retinotectal projection: effects of blocking or synchronizing activity on the morphology of individual regenerating arbors. AB - Both blocking activity with intraocular tetrodotoxin (TTX) and synchronizing activity with a xenon strobe light (1 Hz) prevent retinotopic sharpening of regenerating optic projection in goldfish (Meyer, 1983; Schmidt, 1985; Cook and Rankin, 1986). In this study, we tested, in both normal and regenerating projections, the effects of these two treatments on individual optic arbors. Arbors were stained via anterograde transport of HRP, drawn in camera lucida from tectal whole mounts, and analyzed for spatial extent in the plane of the retinotopic map, order of branching, number of branch endings, depth of termination, and the caliber of the parent axon. In normal tectum, fine, medium, and coarse caliber axons gave rise to small, medium, and large arbors, which averaged 127 microns, 211 microns and 275 microns in horizontal extent, and terminated at characteristic depths. All three classes averaged roughly 21 branch endings. Optic arbors that regenerated with normal patterns of activity returned to a roughly normal appearance by 6-11 weeks postcrush: the same three calibers of axons gave rise to the same three sizes of arbors at the same depths, but they were much less stratified and well on average about 16% larger in horizontal extent. At this time point, arbors regenerated under TTX or strobe were on the average 71 and 119% larger, respectively, than the control-regenerated arbors (larger in all classes), although they had approximately the same number of branch endings and were equally poorly stratified. Synapses formed under strobe were also normal in appearance. Thus the only significant effect of both strobe and TTX treatment was to enlarge the spatial extent of arbor branches. Arbors that were not regenerating were very slightly (but significantly) enlarged by TTX block of activity or strobe illumination. As previous staining showed that regenerating axons initially make widespread branches and later retract many of those branches (Schmidt, Turcotte, Buzzard, and Tieman, 1988; Stuermer, 1988), the present findings support the idea that blocking activity or synchronizing activity prevents retinotopic sharpening by interfering with the elimination of some of the errant branches. PMID- 1706413 TI - Hot knife microtomy for large area sectioning and combined light and electron microscopy in neuroanatomy and neuropathology. AB - The technical details given in this paper meet the demand in neuroanatomy and neuropathology for methods which combine broad light microscopical surveys with detailed ultrastructural studies of logically selected areas in well perfused brain material, and emerge directly from experiences in Palay's laboratory at the National Institutes of Health in 1956. Using the procedures recommended will give good sections of exceptionally large areas (up to, and above 1 cm x 1 cm) of fully hardened blocks available at all points for electron microscopy. On such large blocks fully correlative, combined light and electron microscopy may be carried out easily. The process is termed 'hot knife microtomy'. In three different laboratories, primary aldehyde fixation by perfusion and hot knife microtomy have given uniformly excellent data from normal, diseased, and virus infected brain tissues. These techniques permit full neuroanatomical control and orientation, make comprehensive correlative mapping throughout the CNS feasible, and allow study of the time course of infective processes. PMID- 1706414 TI - Distribution and morphology of Alz-50-immunoreactive cells in the developing visual cortex of kittens. AB - The population of interstitial cells of the white matter in the postlateral gyrus of the cat was studied at different postnatal ages using the antibody Alz-50. These neurons are among the first cells to develop in the cortex, and many of them are transitory, disappearing by cell death during the first postnatal days. In the present study, we found that immunoreactivity to Alz-50 is expressed during the first three postnatal weeks, and that positive neurons were not detected after postnatal day (P) 23. In addition to marking cells in the white matter, Alz-50 also recognizes many pyramidal cells in the cortical layers II-III and V of the visual cortex at postnatal day 4. The staining of cortical cells was not observed at other ages. We found that the number of positive cells in the white matter decreases by postnatal days 12 and 16, showing an apparent increase in number at postnatal day 23. In this study we also attempted to correlate the morphology of Alz-50-immunoreactive cells with the interstitial cells of the white matter, as seen in Golgi preparations. We conclude that Alz-50 immunoreactivity may be related to specific developmental changes and not particularly associated to the occurrence of cell death. PMID- 1706416 TI - Axonless horizontal cells of the rabbit retina: synaptic connections and origin of the rod aftereffect. AB - An axonless horizontal cell (AHC) of the rabbit retina was penetrated with a microelectrode and stained with horseradish peroxidase after recording its light responses. The cell was then serially sectioned and its connections examined with the electron microscope. Physiologically, the cell exhibited cone-dominated responses and a minor rod influence known as rod aftereffect. Electron microscopy showed that this AHC was only connected to cones. Therefore, the rod aftereffect could only invade the cell through the gap junctions between the synaptic endings of rod and cone photoreceptors. In the synaptic invaginations of the cone pedicles contacting the cell, only one of the lateral elements was stained. This suggests that the two lateral elements of each cone-invaginating synapse belong to two different horizontal cells. By staining intracellularly adjacent AHCs, we showed that the two lateral processes may originate from two horizontal cells belonging to the same morphological type. PMID- 1706415 TI - Ultrastructural morphometric analysis of GABA-immunoreactive terminals in the ventrocaudal periaqueductal grey: analysis of the relationship of GABA terminals and the GABAA receptor to periaqueductal grey-raphe magnus projection neurons. AB - The periaqueductal grey (PAG) plays an important role in the descending modulation of nociception. Inhibitory influences of GABAergic terminals, located within the periaqueductal grey, are thought to play a role in antinociception by influencing the activity of neurons that project to the nucleus raphe magnus and adjacent reticular nuclei. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of GABA immunoreactive terminals, and to visualize the neuronal distribution of the GABAA receptor in the ventrolateral periaqueductal grey of the rat. Of particular interest was a quantitative description of the interaction between GABA immunoreactive axon terminals and periaqueductal grey neurons that were retrogradely-labelled from the nucleus raphe magnus and adjacent medullary reticular nuclei. Most terminals were observed to be immediately apposed only to two or three dendrites, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal grey, 37.5% of all GABA immunoreactive terminals were adjacent to periaqueductal grey-nucleus raphe magnus and periaqueductal grey-reticular nucleus projection neurons. Symmetrical synapses with these retrogradely-labelled neurons were formed by 17% of GABA immunoreactive terminals in the ventrolateral periaqueductal grey. We also noted that 13.2% of the GABA-immunoreactive terminals formed symmetrical synapses with GABA-immunoreactive dendrites in the periaqueductal grey, and occasionally those dendrites were retrogradely labelled. Only 0.8% of the GABA-immunoreactive terminals formed putative symmetrical synapses with other GABA-immunoreactive terminals. Consistent with these findings, GABAA receptor immunoreactivity was only associated with dendrites and perikarya in neurons of the ventrolateral PAG. These results are consistent with an inhibitory role for GABA on PAG neurons, a configuration required by hypothetical models for opoid disinhibitory circuitry within the PAG. In addition, the data further suggest that other kinds of GABAergic connections may be important in descending antinociception, and that a population of GABAergic PAG projection neurons exists that may be inhibitory within nucleus raphe magnus and the adjacent reticular nuclei. PMID- 1706417 TI - P0 glycoprotein mRNA distribution in myelin-forming Schwann cells of the developing rat trigeminal ganglion. AB - A biotinylated P0 cDNA was hybridized in situ to aldehyde-fixed vibratome sections of trigeminal ganglia from day 2, day 7, day 15, day 30 and adult rats. Nickel-enhanced horseradish peroxidase (HRP) was used in an antibody sandwich method to detect hybridization. After postfixation in osmium tetroxide, the sections were dehydrated in ethanol and embedded in epon. At each age, some vibratome sections were used to count the HRP-positive and HRP-negative myelin forming Schwann cells. The percentage of HRP-positive myelin-forming Schwann cells in ganglia from day 2, 7, 15, 30, and adult rats were 31%, 56%, 47%, 12% and 3%. In sections of ganglia from 2-day-old rats, studied by light and electron microscopy, peroxidase reaction product localizing hybridized P0 mRNA was found on profiles of granular (rough) endoplasmic reticulum (RER) in perinuclear regions of Schwann cells which had formed two to three compact myelin lamellae. Peroxidase deposits were larger and more numerous in the cytoplasm cells with thicker myelin sheaths. At day 7, some Schwann cells had long external mesaxons; the cytoplasm between these mesaxons and the cell surface often contained abundant HRP-stained profiles of RER. In sections from day 7 and day 15 ganglia, substantially more reaction product was found. In each myelin-forming Schwann cell, the amount was generally proportional to the size of the newly formed myelin sheath. HRP deposits were observed all along the outer surfaces of myelin segments at these ages, and their distribution corresponded to that of the RER.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706418 TI - Neuronal communication breakdown in neurotransmitter systems in Alzheimer's and Parkinson's dementias. PMID- 1706419 TI - Gingival HIV detection studied. PMID- 1706420 TI - Deadly dietary deceptions. PMID- 1706421 TI - Comprehensive financial planning for dentists in the '90s. PMID- 1706422 TI - New approaches to esthetic periodontal reconstruction. PMID- 1706423 TI - Infection control, OSHA and a hazards communication program. PMID- 1706424 TI - Rightful vs. wrongful termination of dental employees. PMID- 1706425 TI - Importance of image. PMID- 1706426 TI - Characterization of the extracellular matrix of the primate temporomandibular joint. AB - The distribution of type I and II collagens, fibronectin and the fibronectin integrin receptor, tenascin, laminin, link protein, and cartilage-specific glycosaminoglycans was examined in the primate temporomandibular joint complex using an immunohistochemical approach. In general, type I collagen, fibronectin, and the fibronectin-integrin receptor were found to co-distribute throughout the joint complex. Immunostaining for these proteins was notably intense in the prechondroblastic and mineralization zones of the articular cartilages of the joint. Tenascin was identified in several structures of the joint, including the articular cartilages, where intense staining was observed in the prechondroblastic and cartilagenous zones. Laminin was detected only in the adventitia of blood vessels located in the attachment tissues of the disc and joint synovium. Cartilage-specific glycosaminoglycans and type II collagen were observed in the cartilagenous zones of the articular cartilages of the mandibular condyle and temporal bone. In addition, immunostaining for cartilage-specific glycosaminoglycans also was detected throughout the extracellular matrix surrounding "chondrocyte-like" cells located in the joint disc. Despite the localization of cartilage-specific glycosaminoglycans in the disc, type II collagen was not detected in this structure. It is suggested that a fibronectin integrin receptor mechanism may be involved in the regulation of growth of the articular cartilages of the temporomandibular joint. PMID- 1706427 TI - Staining of zonules during posterior chamber lens implantation in a cadaver eye model. AB - A cadaver eye model was used to study the effects of extracapsular lens removal and implantation on the zonules and capsule. Five staining solutions were instilled in the posterior and anterior chambers of human cadaver eyes and washed out three to five minutes later. Gomori's chrom hematoxylin-eosin most clearly revealed the zonules. Three eyes stained in this way had lens removal (including the continuous circular capsulorhexis technique for anterior capsulectomy) and intraocular lens implantation. The effects of the surgery on the zonules and capsule were clearly visualized and appeared quite satisfactory. This staining technique may prove useful in other cadaver eye models. PMID- 1706428 TI - Effects of [N-(L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (SCH32615), a neutral endopeptidase (enkephalinase) inhibitor, on levels of enkephalin, encrypted enkephalins and substance P in cerebrospinal fluid and plasma of primates. AB - In halothane-anesthetized and -ventilated cynomologus macaque monkeys, the effects of administering vehicle (n = 3) or the neutral endopeptidase inhibitor N [L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (16 mg/kg, n = 5; or 100 mg/kg, n = 3, intravenously) was examined. Cisternal CSF aliquots were examined by radioimmunoassay: 1) for Met enkephalin; 2) after trypsin and carboxypeptidase B treatment for encrypted enkephalin (X-ENK); 3) for substance P; and 4) for unmetabolized drug. Similar measures were carried out in femoral artery and femoral venous plasma, except that substance P was not assayed. In CSF, prior to drug, low, but measurable levels of enkephalin (61 pg/ml), X-ENK (285 pg/ml) and substance P (16 pg/ml) were observed. Vehicle-injected animals showed no change from baseline levels over a 4-hr sampling period in either plasma or CSF levels. In contrast, following 16 mg/kg, in CSF, there was a significant 9-fold increase in MET and 11-fold increase in X-ENK at 30 min. CSF substance P levels rose also by a factor of 2, with the peak effect observed at 60 min. All levels displayed a significant reduction by 4 hr. There was no statistical difference between the maximum effects observed with either the 16- or 100-mg/kg dose. Plasma peptide levels of enkephalin and X-ENK were not altered by drug. CSF displayed significant drug levels by 30 min, which were between 0.1 and 1% of levels observed concurrently in plasma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706429 TI - Toluene diisocyanate contracts guinea pig bronchial smooth muscle by activating capsaicin-sensitive sensory nerves. AB - This study was designed to evaluate the mechanism of action of toluene diisocyanate (TDI) and the role of endogenous neutral endopeptidase in modulating in vitro contractile responses to TDI in guinea pigs. TDI (0.01-1 mM) produced a concentration-dependent contraction of the guinea pig main bronchi. Sensory nerve desensitization with capsaicin greatly reduced and in some cases almost abolished TDI-induced contractions. The neutral endopeptidase inhibitor phosphoramidon significantly increased the contractile response to TDI. Pretreatment with the substance P antagonist (D-Arg1,D-Pro2,D-Trp7,9,Leu11)-substance P greatly reduced TDI-induced contractions. These results suggest that TDI activates the "efferent" function of capsaicin-sensitive sensory nerves and that neutral endopeptidase may play a role in modulating the response in guinea pigs. PMID- 1706430 TI - Neurohormonal regulation of ion transport in the porcine distal jejunum. Enhancement of sodium and chloride absorption by submucosal opiate receptors. AB - The actions of opiates in modulating ion transport across the porcine distal jejunal mucosa were examined in vitro. Opiate receptors were functionally characterized using [D-Pen2,D-Pen5]-enkephalin (DPDPE), [D-Ala2,N-Me-Phe4,Gly5 ol]-enkephalin (DAMGO) and U-50,488 (trans-3,4-dichloro-N-methyl-N-[2-(1 pyrrolidinyl)- cyclohexyl]-benzeneacetamide), agonists selective for delta, mu and kappa receptor types, respectively. Serosal administration of opiate agonists produced concentration-dependent decreases in basal short-circuit current (Isc. DPDPE and DAMGO also increased tissue conductance (Gt). DPDPE (EC50 = 4 nM) was 7 and 86-fold more potent in decreasing basal Isc than DAMGO and U-50,488, respectively. U-50,488 displayed the greatest efficacy in decreasing Isc. Serosal naloxone decreased DPDPE and DAMGO potencies under basal conditions with Ke values of 11 and 6 nM, respectively. DPDPE- and DAMGO-induced decreases in basal Isc were associated with increases in net Cl absorption; in addition, DAMGO produced an increase in net Na absorption. 8-Bromocyclic AMP (0.3 mM) increased Isc, decreased Gt and inhibited net Na and Cl absorptive fluxes. Selective opiate agonists decreased cyclic AMP-induced elevations in Isc with a rank order of potency (DPDPE, EC50 = 3 nM) of DPDPE greater than DAMGO greater than U-50,488. DPDPE reversed the action of cyclic AMP on Isc and Cl absorption but had no effect on net Na transport. Cyclic AMP-induced decreases in Gt were not altered by DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706431 TI - 9-methyl-7-bromoeudistomin D, a powerful radio-labelable Ca++ releaser having caffeine-like properties, acts on Ca(++)-induced Ca++ release channels of sarcoplasmic reticulum. AB - 9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706432 TI - Isovolumic hemodilution with dextran 40 in the rat: effect on the development of peripheral edema and various physiologic parameters. AB - Low molecular weight dextran 40 (D40), 40,000 daltons, is a potential therapeutic agent for cerebral ischemia because it increases local cerebral blood flow. However, the evaluation of D40 in the rat has been difficult due to systemic effects. We evaluated the effects of isovolumic hemodilution with D40 on the development of peripheral edema, mean arterial pressure, hematocrit (Hct) and total blood volume in 18 rats, during 30 min or 4 hr i.v. infusions, in animals with and without previous challenge with D40. Reduction of Hct without peripheral edema to a mean of approximately 31% was only achieved in the animals challenged with i.p. D40 24 hr before hemodilution and who received D40 over a period of 4 hr. Infusion of D40 over a period of 30 min was associated with shorter survival time, compared to the 4-hr infusion group (P less than .005). In the pretreated, rapidly infused group, total blood volume per body weight decreased significantly over time (P less than .005) and the mean arterial blood pressure dropped, but not significantly (P less than .07), whereas no change in Hct was detected and there was a trend toward increased peripheral edema, relative to the slowly infused groups. We conclude that reduction of Hct can be achieved successfully with i.p. administration of D40 24 hr before the study combined with infusion of the agent during a 4-hr period, without significant peripheral edema and early hypotension. This procedure should be used to avoid allergic reactions when evaluating hemodilution with D40 in rats. PMID- 1706433 TI - Ex vivo binding of t-[35S )butylbicyclophosphorothionate: a biochemical tool to study the pharmacology of ethanol at the gamma-aminobutyric acid-coupled chloride channel. AB - The effects of acute administration of ethanol on t [35S]Butylbiclophosphorothionate (35S-TBPS) binding measured ex vivo in unwashed membrane preparations of rat cerebral cortex were investigated. Ethanol, given i.g., decreased in a dose-related (0.5-4 g/kg) and time-dependent manner the binding of 35S-TBPS. This effect was similar to that induced by the administration of diazepam (0.5-4 mg/kg i.p.). Scatchard plot analysis of this radioligand binding revealed that ethanol, differently from diazepam, decreased the apparent affinity of 35S-TBPS recognition sites whereas it failed to change the density of these binding sites. The effect of ethanol on 35S-TBPS binding could not be reversed by the previous administration to rats of the benzodiazepine receptor antagonist, Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5 methyl-6-oxo-4H- imidazo[1,5a][1,4]benzodiazepine-3-carboxylate). Vice versa, the benzodiazepine receptor partial inverse agonist, Ro 15-4513 (ethyl-8-azido-5,6 dihydro-5-methyl-6-oxo-4H- imidazo[1,5a][4,4]benzodiazepine-3-carboxylate) (8 mg/kg i.p.), prevented completely ethanol-induced decrease of 35S-TBPS binding. The ability of Ro 15-4513 to prevent the action of ethanol was shared by the anxiogenic and proconvulsant beta-carboline derivatives, FG 7142 (N-methyl-beta carboline-3-carboxamide) (12.5 mg/kg i.p.) and ethyl-beta-carboline-3-carboxylate (0.6 mg/kg i.v.), which, per se, enhanced this parameter. Moreover, ethanol (0.5 4 g/kg) was able to reverse the increase of 35S-TBPS binding elicited by the s.c. injection of isoniazid (350 mg/kg) and to clearly attenuate the severity of tonic clonic seizures produced by this inhibitor of the GABAergic transmission.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706434 TI - Cl- channels in basolateral renal medullary membranes: III. Determinants of single-channel activity. AB - We evaluated the effects of varying aqueous Cl- concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl- channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for channel activity, and the effects of varying Cl- concentrations and/or PGO on the relation between holding voltage (VH, mV) and open-time probability (Po). Reducing cis Cl- concentrations, in the range 50 320 mM, produced a linear reduction in fractional open time (Po) with a half maximal reduction in Po at cis Cl- approximately 170 mM. Channel activity was sustained by equimolar replacement of cis Cl- with F-, but not with impermeant isethionate. For trans solutions, the relation between Cl- concentration and Po was negatively cooperative, with 50% reduction in po at 10 mM Cl-. Reducing cis Cl- had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent energy (delta G) for channel opening. Phenylglyoxal (PGO) reduced Z and altered delta G for Cl- channel activity when added to cis, but not trans solutions. Furthermore, in the presence of cis PGO, reducing the cis Cl- concentration had no effect on Z but altered delta G. Thus we propose that cis PGO and cis Cl- concentrations affect separate sites determining channel activity at the extracellular faces of these Cl- channels. PMID- 1706435 TI - Identification of the cardiac sarcolemmal Na(+)-Ca2+ exchanger using monoclonal antibodies. AB - We have previously partially purified the sarcolemmal Na(+)-Ca2+ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K.D., Longoni, S., Ward, R. 1988. Biochim. Biophys. Acta 945:298-306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50-75% of solubilized Na(+)-Ca2+ exchange activity. The MAb's appear to be reactive with native conformation-dependent epitopes on the Na(+)-Ca2+ exchanger since they do not react on immunoblots. An indirect method was used to identify Na(+)-Ca2+ exchange proteins. A column containing Na(+)-Ca2+ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteins of apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our previous association of Na(+)-Ca2+ exchange with these proteins. PMID- 1706436 TI - Co-crystals of gramicidin A and phospholipid. A system for studying the structure of a transmembrane channel. AB - Single crystals of a complex of gramicidin A, a transmembrane channel-forming polypeptide, and dipalmitoyl phosphatidylcholine, a phospholipid, have been prepared and characterized by X-ray diffraction. They belong to space group P222(1), with unit cell dimensions a = 26.8 A, b = 27.5 A, c = 32.8 A. The asymmetric unit appears to be a complex of one gramicidin monomer and two phospholipid molecules. The unit cell dimensions, space group, and chemical composition are compatible with lipids packing in a bilayer-like motif, and an end-to-end association of gramicidin monomers to form a functional dimeric unit. The crystals diffract to 2 A and are suitable for structural studies by single crystal X-ray analysis. This represents the first example of a well-ordered crystalline channel complexed with lipids, and solution of its structure may give insight into mechanisms of ion transport across membranes. PMID- 1706437 TI - Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P. AB - The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed. PMID- 1706438 TI - Modulation of N-nitrosomethylurea-induced mammary tumor promotion by dietary fiber and fat. AB - A test of the anticancer effects of dietary fiber was conducted using the N nitrosomethylurea (NMU)-induced rat mammary tumor model. Starting 3 days after NMU treatment, four different groups of F344 rats (30 rats in each group) were fed as follows: Group 1 received a high-fat diet; group 2, a high-fat plus fiber diet (soft white wheat bran, 10% wt/wt); group 3, a low-fat diet; and group 4, a low-fat plus fiber diet. The rats remained on these diets for 15 weeks. Tumor incidence in group 1 was 90% compared with 66% in group 2 (P less than .001). Tumor incidence in group 3 was 63% compared with 47% in group 4 (P greater than .4). These results show that supplemental dietary fiber exerts an inhibitory effect on the promotional phase of NMU-induced mammary carcinogenesis in rats when fed a high-fat but not a low-fat diet. To test whether fiber may exert its antipromoting effect by reducing circulating estrogens, serum 17 beta-estradiol was assayed. No changes were observed in serum 17 beta-estradiol levels among the four groups, suggesting that the protective effect of fiber in this animal model is not mediated by a fiber-induced reduction of circulating 17 beta-estradiol. PMID- 1706439 TI - Prostate specific antigen in patients with clinical stage C prostate cancer: relation to lymph node status and grade. AB - The preoperatively drawn sera from 84 previously untreated patients who had clinical stage C prostate cancer and underwent staging pelvic lymph node dissections were sent for monoclonal Hybritech analysis to assess the usefulness of prostate specific antigen (PSA) in predicting lymph node status. Of the 84 patients 47 (56%) had positive lymph nodes at surgery. The median PSA value for all patients with nodal metastases was 11.4 ng/.ml., and for those without it was 11.2 ng./ml. Relative to Gleason score, median PSA values were 11.35 for 2-4, 12.2 for 5-7 and 10.9 ng./ml. for 8-10. Within each M.D. Anderson grade median PSA values were 10.15 for grade I, 13.2 for grade II, 12.7 for grade III and 10.5 ng./ml. for grade IV. Simultaneously drawn preoperative frozen serum samples for 28 of these patients were independently analyzed by the Yang radioimmunoassay. Comparing Hybritech and Yang methods revealed strong statistical co-association (correlation coefficient R2 = 97.36, p less than 0.00001) but neither assay was statistically associated with nodal metastasis. Although no PSA level excluded the presence of nodal disease, we suggest that a Hybritech PSA of greater than 30 ng./ml. and a Yang PSA of greater than 50 ng./ml. may serve as a weak adjunctive marker predicting nodal metastasis. PMID- 1706440 TI - Prostate specific antigen in hormonally treated stage D2 prostate cancer: is it always an accurate indicator of disease status? AB - The clinical significance of serum prostate specific antigen (PSA) values in hormonally treated prostate cancer patients and the effect of hormonal therapy on the serum PSA concentration, independent of the response observed from its antitumor activity, are not well defined. To elucidate further the influence of antiandrogen therapy on serum PSA expression, 81 randomly selected patients with stage D2 prostate cancer were evaluated with respect to serum PSA concentration. These patients were divided into 2 groups on the basis of previous hormonal therapy. Group 1 consisted of 43 patients 55 to 89 years old (mean age 71 years) who had received no prior therapy for prostate cancer. Group 2 included 38 men 58 to 84 years old (mean age 72 years) who had received only androgen deprivation therapy with either bilateral orchiectomy or diethylstilbestrol. The mean interval between initiation of antiandrogen therapy and evaluation of these patients was 14 months (range 8 to 31 months). At the time of PSA determination both groups were similar in all respects, including tumor grade, disease symptoms and bone scan findings. The median serum PSA concentration was 96.0 ng./ml. in group 1 and 16.5 ng./ml. in group 2 (p less than 0.001), despite both groups having similar symptoms and widespread metastatic disease on radionuclide bone scan. In group 1 only 1 patient (2%) had a serum PSA level less than 4.0 ng./ml., whereas 13 men (34%) in group 2 had a serum PSA concentration below 4.0 ng./ml. (p less than 0.001). Of the patients in group 1, 2% and of the men in group 2, 45% had a serum PSA concentration less than 10 ng./ml. (p less than 0.001). These findings demonstrate that the serum PSA level in prostate cancer patients treated hormonally may have a significantly different meaning than the same serum PSA value in patients without hormonal therapy. In addition, these observations suggest that PSA expression may be under hormonal regulation and that androgen deprivation therapy may have a direct effect on the serum PSA concentration, independent of the response obtained from any antitumor activity. However, the exact mechanism of this androgenic influence on PSA expression awaits further investigation at the cellular level. PMID- 1706441 TI - Elevated serum tumor markers in patients with testicular cancer after induction chemotherapy due to a reservoir of markers in cystic differentiated mature teratoma. AB - Elevated serum tumor markers in patients with testicular cancer after induction chemotherapy indicate in most instances the presence of residual malignant disease. We describe 2 patients with elevated tumor markers after chemotherapy and before retroperitoneal lymph node dissection who did not prove to have residual malignant disease but cystic differentiated mature teratoma with a high content of alpha-fetoprotein and beta-human chorionic gonadotropin, respectively, in the cysts. It is postulated that leakage of the contents of these cysts to the plasma compartment was responsible for maintaining elevated serum tumor marker levels. Recognition of such entities is of consequence since unnecessary salvage chemotherapy in these patients may be avoided. PMID- 1706442 TI - Pregnancy hormone levels signal trisomy 21, improved screening, lower costs possible. PMID- 1706443 TI - [IL-2 responsiveness in antigen-specific lymphocyte proliferation test with sulfhydryl drug-sensitized murine lymphocytes--using rapid fluorochromasia assay]. AB - Response of murine lymphnode cells (LNC) sensitized with sulfhydryl drugs to recombinant interleukin 2 (rIL-2) was studied in antigen-specific lymphocyte proliferation test (LPT). Mice were primed with tiopronin (TP) and gold sodium thiomalate (GTM) and the secondary response to LNC was measured in a proliferative assay in vitro. Rapid fluorochromasia assay with propidium iodide (RFP) was used for the quantitative measurement of LPT instead of 3H-thymidine uptake. There was no difference in proliferative response to specific antigen between TP or GTM-primed LNC and control ones. In contrast, a significant proliferation was observed when LNC from sensitized mice were cultured with sensitizing antigen and rIL-2. The strength of response was dependent on the concentration of rIL-2. It was considered that adding rIL-2 to LPT of TP or GTM sensitized mice enhanced antigen-specific lymphocyte proliferative response and the measurement of IL-2 responsiveness using RFP method might be useful to detect sulfhydryl drug allergy in man. PMID- 1706444 TI - [Production of human monoclonal antibody reactive with gastrointestinal carcinoma]. AB - Lymphocytes obtained from regional lymph nodes and spleen in the patients with gastrointestinal carcinoma were fused with the human B lymphoblastoid cell line GC01 and human hybridomas producing human monoclonal antibody (MoAb) were derived. Human MoAb No. 235 (IgM) derived from spleen cell of a gastric cancer patient reacted with adenocarcinoma of stomach, colon, and pancreas in the new immunohistochemical assay, modified direct immunoperoxidase method, and reacted with KATO III cells in cultured cell lines. The antigenic determinant of this antibody was suspected to be protein moiety after enzyme treatment. The competitive binding inhibition assay indicated that its epitope was different from anti-CEA monoclonal antibodies (KM10, A10, B9, AH3, JA4) and KM01. These findings suggested the possible use of human MoAb No. 235 for clinical application of targeting cancer chemotherapy in the future. PMID- 1706445 TI - Characterization of tachykinin receptors in urinary bladder from guinea pig. AB - The contractile responses to substance P (SP), neurokinin A (NKA), Tyro neurokinin B (Tyr-NKB), senktide (NK3 receptor selective agonist) and SP methyl ester (SPOMe, NK1 receptor selective agonist) were investigated in detrusor strips from guinea pigs. Except for senktide, all drugs induced a concentration related contraction with the following rank order of potency: SPOMe greater than SP greater than NKA greater than or equal to Tyr-NKB. After desensitization of NK1 receptors with SPOMe, the rank order of potency was NKA greater than or equal to Tyr-NKB greater than SP greater than SPOMe. Both NK1 and NK2 receptors exist in the detrusor strip from guinea pigs. PMID- 1706446 TI - [The use of lithium carbonate in treating patients with diffuse toxic goiter]. PMID- 1706447 TI - Articular lymphoscintigraphy in human knees using radiolabeled dextran. AB - Whereas joint lymphatics are inaccessible to conventional (oil-contrast) lymphography, articular lymphatic dysfunction can be assessed by lymphoscintigraphy (isotope lymphography). Using 99mTc-labeled dextran (molecular weight-70,000 daltons), we performed dynamic lymphoscintigraphy in 38 patients with degenerative osteoarthropathy of the knee. Comparison with the normal (contralateral knee) in 25 patients demonstrated that tracer disappeared at 24 hours more slowly from the abnormal side (86.5 +/- 3.3% retention in the abnormal joint compared with 77.1 +/- 4.6%; p less than 0.01), but accumulated more intensely in regional lymph nodes on the pathologic side (3.8 +/- 2.4% vs 1.9 +/- 0.3%; p less than 0.01). The findings suggest deranged macromolecular transport and lymphatic dysfunction in degenerative knee joint disease. PMID- 1706448 TI - [Immunological markers for the study of sweat gland tumors]. PMID- 1706449 TI - Where to hit MS. PMID- 1706450 TI - Hyperdynamic circulation in cirrhosis: a role for nitric oxide? AB - Hypotension, low systemic vascular resistance, and a reduced sensitivity to vasoconstrictors are features of cirrhosis. These cardiovascular changes might be the result of increased synthesis of a vasodilator. Nitric oxide (NO), a potent vasodilator, is synthesised in and released from peripheral blood-vessels in man. Studies in animals indicate that bacterial endotoxin and cytokines induce NO synthase expression in vessel walls, with sustained NO release and consequent hypotension. Endotoxaemia is a common feature of cirrhosis; persistent induction of NO synthase may account for the associated haemodynamic changes. PMID- 1706451 TI - Cross-reactivity between antibodies to thyroid microsomal antigens and myeloperoxidase. PMID- 1706452 TI - Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations. AB - The molecular nature of the 'unconventional viruses' that cause slow, progressive brain deterioration is still poorly understood. As part of a reinvestigation of potential agent-specific nucleic acids, we developed a protocol for enriching agent-specific sequences. This protocol uses extensive micrococcal nuclease digestion followed by rate zonal sucrose sedimentation. Most of the infectivity in the gradient (84%) had a characteristic mean size of approximately 120S, and was resolved from 70% of a host glycoprotein (PrP) that can cosediment with infectivity. In infectious size fractions, nucleic acids were reduced approximately one million-fold with respect to starting brain homogenate, and specific purification of infectivity was approximately 100,000-fold with respect to nucleic acid. Using a novel polymerase chain reaction strategy, we were able to amplify RNA species in these fractions. Remarkably, host polyadenylated sequences of 1 to over 4 kb were detected in the nuclease-protected infectious fractions. These strategies set the stage for the identification of similar nucleic acids that may be specific for the CJD agent. PMID- 1706453 TI - Characterization of pathological dystrophin transcripts from the lymphocytes of a muscular dystrophy carrier. AB - Both normal and pathological transcripts of tissue-specific genes may be detected by polymerase chain reaction (PCR) amplification in tissues not normally considered to express the gene product. The exploitation of constitutive basal mRNA levels ("ectopic" transcription) would be a major boon to diagnostic medicine since it promises both to simplify the analysis of complex genes and to avoid the requirement for an expressing tissue that is sometimes obtainable only by biopsy. We have demonstrated the feasibility of this novel strategy by characterizing a mutation in the X-chromosomal Duchenne (or Becker) muscular dystrophy (DMD/BMD) gene encoding dystrophin. The massive size of this gene has in the past often hindered carrier detection due to the high frequency of recombination and the high proportion of new mutations. In this study a deletion was identified in both a BMD patient and a heterozygous carrier using only a minimal volume of peripheral blood. Following specifically primed reverse transcription of lymphocyte RNA, the relevant region of the pathological cDNA was PCR-amplified. Sequence analysis indicated an in-frame deletion of exons 45 to 47. PMID- 1706454 TI - HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide. AB - The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell. PMID- 1706455 TI - Distribution of msDNAs among serotypes of enteropathogenic Escherichia coli strains. AB - A genetic element, called a retron, is present in certain Escherichia coli strains. It consists of genes for the production of a covalently linked DNA-RNA compound and a reverse transcriptase. The presence of a retron can be detected by testing for a satellite DNA band by polyacrylamide gel electrophoresis. This DNA band consists of the DNA portion of the DNA-RNA compound and is called msDNA (multicopy single-stranded DNA). In a survey of intestinal E. coli isolates we detected msDNAs in classical enteropathogenic (EPEC) strains and in strains with aggregative adherence to tissue-culture cells (AA), but not in enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains. Among 76 EPEC strains belonging to 14 different serotypes, msDNA was found to be present in 7 serotypes. In total, five different types of msDNA were found, although within each serotype, the msDNAs were the same. These results suggest that different retrons are clonally inherited. PMID- 1706456 TI - The variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome. AB - The relapsing fever agent Borrelia hermsii avoids the host's immune response by the strategy of multiphasic antigenic variation. A given Borrelia cell can express one of a number of alleles for polymorphic outer-membrane proteins, known as Vmp proteins. The genes for the variant-specific Vmp proteins of serotypes 7 and 21 of B. hermsii strain HS1 were sequenced. The genes, which were designated vmp7 and vmp21, were obtained from populations of borreliae before and after a switch in serotypes from 7 to 21. The analysis showed that vmp7 and vmp21 are 77% identical in terms of their coding sequence. The deduced translation products of vmp7 and vmp21 are polypeptides of 369 (37.2 kD) and 364 amino acids (37.1 kD), respectively. Vmp7 and Vmp21 have sequence features of prokaryotic lipoproteins and are processed as such during expression in E. coli. The secondary structure predictions of the Vmp proteins reveals analogous structures to the VSG proteins of the African trypanosome. PMID- 1706457 TI - Involvement of OmpF during reception and translocation steps of colicin N entry. AB - [125I]-colicin N binds to OmpF receptor sites (70,000 per cell) with an average Kassoc of 3.2 x 10(6) M-1 at 23 degrees C. Monoclonal antibody directed against a cell-surface-exposed epitope of OmpF is able to complete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross-react with various monoclonal antibodies directed against colicin A. PMID- 1706458 TI - Analysis and manipulation of yeast mitochondrial genes. PMID- 1706459 TI - Preparation of high molecular weight RNA. PMID- 1706460 TI - Epitope tagging and protein surveillance. AB - The epitope tagging approach offers advantages of economy, universality, and precision over the use of antibodies raised directly against a protein of interest. The latter strategy promises a potentially greater diversity of reagents and obviates the need to modify the protein, but it may not yield sufficiently high-affinity, abundant, or specific antibodies. The major uncertainty in an epitope-tagging strategy, namely, the ability of the altered protein to function in vivo, is readily resolved in yeast by testing complementation of a null allele by the modified gene. Modification of the protein is easily accomplished by addition of the epitope coding sequence to the gene via oligonucleotide-mediated site-directed mutagenesis. The uniqueness of the epitope in the genome and the use of the monoclonal antibody assure a high affinity, specific, and abundant antibody. Unrelated but identically modified proteins can be immunoprecipitated and affinity purified under the same conditions. Only extraction conditions and possibly a simple initial fractionation step need vary. Moreover, otherwise identical but differentially tagged proteins can be separated. Even proteins completely defective in an essential in vivo function can be purified and studied. Finally, polypeptides coprecipitating with the protein of interest are normally difficult to distinguish from those merely cross-reactive with the antibody used. As an alternative to defining a complex of proteins using a battery of antibodies, complexes are defined as a set of immunoprecipitable polypeptides present only in extracts containing the modified protein. PMID- 1706461 TI - In vitro protein synthesis. PMID- 1706462 TI - Methods for studying the yeast vacuole. PMID- 1706463 TI - Nucleolar-specific positive stains for optical and electron microscopy. PMID- 1706464 TI - Reassessment of antigenic determinant of Saccharomyces cerevisiae serotype Ia. AB - We examined the immunochemical structure of the antigenic determinant of S. cerevisiae serotype Ia. The specific factor serum for S. cerevisiae serotype Ia was obtained either from factor 18 serum by adsorption with heat-killed cells of Candida glabrata, or from anti-S. cerevisiae Ia (M 6001) serum by adsorption with heat-killed cells of S. cerevisiae Ib (IFO 0751). We designated this adsorbed serum as factor 18a. Acetolysis of S. cerevisiae cell wall D-mannan gave five oligosaccharides. Signals of 1H-nuclear magnetic resonance spectra of mannooligosaccharides derived from S. cerevisiae mannan were assigned for their linkages by the aid of those of alpha-1,3'-linked mannooligosaccharides derived from glucuronoxylomannan of capsule of Cryptococcus neoformans serotype A-D. Agglutination-inhibition experiments revealed that the mannopentaose from S. cerevisiae mannan was the most effective inhibitor. Moreover, inhibitory activities of alpha-1,3'-linked mannotriose, mannotetraose, and mannopentaose which were derived from glucuronoxylomannan of C. neoformans were shown to be higher than those of mannotetraose with one terminal alpha-(1-3) linkage from homologous S. cerevisiae mannan. These results indicate that mannopentaose with terminal two alpha-(1-3) linkages is responsible for the specificity of S. cerevisiae Ia. PMID- 1706465 TI - Pleurectomy for mesothelioma. AB - OBJECTIVE: To assess the effectiveness and safety of parietal pleurectomy in establishing a tissue diagnosis and controlling pleural fluid accumulation in patients with pleural mesothelioma, and to assess the success of this procedure in effecting palliation. DESIGN AND SETTING: Fifty consecutive patients with pleural mesothelioma who underwent thoracotomy at the cardiothoracic units at Concord and Royal Prince Alfred Hospital were reviewed retrospectively. The male:female ratio was 4:1 and the mean age was 63 years. In only 11 of the 50 patients was a tissue diagnosis of mesothelioma made before surgery. INTERVENTIONS: At thoracotomy, subtotal parietal pleurectomy was performed in 45 of the 50 patients. In two patients biopsy alone was performed and three patients were treated by a chemical pleurodesis only, as pleurectomy was not technically possible. Pulmonary decortication was required in 28 patients to allow full expansion of the underlying lung for effective pleurodesis. RESULTS: There was one postoperative death. The morbidity rate was 16%. Excluding the patient who died in the postoperative period, the median survival was 16 months, and ranged from three to 54 months, with 21% of patients surviving for more than two years. Only one patient developed a reaccumulation of pleural fluid. CONCLUSIONS: Pleurectomy, with decortication when required, provides both a tissue diagnosis and effective control of pleural fluid accumulation and therefore excellent palliation in patients with pleural mesothelioma. We advocate early thoracotomy in these patients. PMID- 1706466 TI - Nucleo-cytoplasmic incompatibility in cybrid plants possessing an Atropa genome and a Nicotiana plastome. AB - Twenty-nine cybrids possessing an Atropa belladonna nuclear genome and a Nicotiana tabacum plastome were selected from two independent protoplast fusion experiments. In contrast to the previously described reciprocal, green and fertile cybrids with a Nicotiana nuclear genome and an Atropa plastome (Kushnir et al. 1987), the plants obtained were totally chlorophyll-deficient. An Atropa nuclear genome and a Nicotiana plastome from these chlorophyll-deficient cybrids were combined with an Atropa or a Scopolia plastome and a Nicotiana nuclear genome, respectively, in control fusion experiments. All of these nuclear genome/plastome combinations gave rise to normal, green plants. Therefore, we conclude that an N. tabacum plastome is incompatible with an A. belladonna nuclear genome. PMID- 1706467 TI - Dual transcriptional initiation sites from the pyrC promoter control expression of the gene in Salmonella typhimurium. AB - Expression of the Salmonella typhimurium pyrC gene encoding dihydroorotase is negatively regulated by CTP and stimulated by GTP. This regulation does not occur at the level of transcription initiation but appears to involve translation attenuation of the transcripts. Alterations of specific bases in a region of hyphenated dyad symmetry located in the leader established that base pairing in the 5' terminal region of the pyrC leader transcript is required for normal regulation of dihydroorotase synthesis. Primer extension experiments on RNA from mutant strains that permit manipulation of the CTP and GTP pools showed that pyrC transcription may start at either a cytosine or a guanine residue, 2 bp apart. The ratio between G-starts and C-starts appeared to be determined by the intracellular [GTP]/[CTP] pool ratio. The larger transcript, starting with a C, is able to form a stable hairpin in the 5' end, sequestering part of the ribosome binding site in the stem. The leader of the shorter transcript, however, cannot form this secondary structure. Thus, translational initiation will occur unhindered only from the shorter transcript. PMID- 1706468 TI - Noncompetitive inhibition of gamma-aminobutyric acidA channels by Zn. AB - The action of zinc on chloride currents evoked by gamma-aminobutyric acid (GABA) was examined on cultured hippocampal neurons using whole cell voltage clamp and outside-out patch recording. Zn (5-30 microM) noncompetitively blocked responses evoked by GABA (0.5-100 microM), but did not affect either the time-to-peak or desensitization of the macroscopic current. In outside-out patches, Zn had no effect on the mean conductance or lifetime of the 19 or 30 pS openings of the GABA channel; however, the frequency of channel opening was markedly decreased in a voltage-independent manner. Zn inhibition of GABA responses appeared to be independent of the benzodiazepine binding site as Zn was effective in the presence of either diazepam or Ro15-1788, a competitive antagonist of benzodiazepine agonists and inverse agonists. In contrast to prior reports, Zn also inhibited GABA currents in a similar manner on cultured superior cervical ganglion neurons. These results suggest that Zn acts at an extracellular site on the GABAA receptor complex, which is distinct from either the GABA or benzodiazepine binding sites. The structural similarity of the Cys-Cys loop of the alpha and gamma GABAA receptor subunits to some Zn-binding proteins suggests one possible region for a Zn binding site. PMID- 1706469 TI - Short chain and long chain alkanols have different sites of action on nicotinic acetylcholine receptor channels from Torpedo. AB - At nicotinic acetylcholine receptors, short chain n-alcohols (alkanols) have excitatory actions, whereas long chain alkanols inhibit channel activity. This study tests a previously proposed unitary hypothesis that suggests that these contrasting actions can be accounted for by interaction at just one hydrophobic site within the ion channel lumen. All alkanols bind to this site, but only long chain alkanols are large enough to completely block the channel. Short chain alkanols are too small to cause any channel occlusion, and in binding to the site they stabilize the open state of the receptor and enhance ion flux. In this study, we assay integrated agonist-stimulated ion efflux over 15 msec, as a measure of receptor activity. In nicotinic acetylcholine receptor-rich membrane vesicles from Torpedo, we show that, in contradiction to this elegant model, long chain and short chain alkanols appear to act at different sites. Firstly, ethanol and octanol do not compete for a single site on the receptor. Secondly, alkanol chain length dependencies for inhibition and for flux enhancement are significantly different. Thirdly, intermediate length alkanols do not partially inhibit channels, as required by the model; high concentrations of these alkanols completely inhibit the response. Fourthly, careful measurements, including determination of the free alkanol concentration, of inhibitory potencies of alkanols from propanol to decanol show no evidence for a steric contribution to the ability of an alkanol to inhibit the ion channel. Furthermore, our results suggest that the inhibitory effect of long chain alkanols may be mediated by a discrete site on nicotinic acetylcholine receptors, whereas there is no evidence that a protein site is involved in the excitatory mechanism of short chain alkanols. Indeed, it seems more likely that short chain alkanols may have a nonspecific site of action. PMID- 1706470 TI - Characterization of two novel cholecystokinin tetrapeptide (30-33) analogues, A 71623 and A-70874, that exhibit high potency and selectivity for cholecystokinin A receptors. AB - Based on their relative affinities for cholecystokinin octapeptide (26-33) (CCK 8), cholecystokinin tetrapeptide (30-33) (CCK-4), desulfated CCK-8, and gastrin, cholecystokinin (CCK) receptors have been classified as CCK-A (alimentary) and CCK-B (brain). Selective nonpeptide antagonists of CCK-A and CCK-B receptors, as well as highly selective CCK-A and CCK-B peptide agonists, have been described. We report here the characterization of two novel CCK-4-based peptides, A-71623 and A-70874. In radioligand binding assays, the IC50 values for A-71623 and A 70874 were 3.7 and 4.9 nM in guinea pig pancreas (CCK-A) and 4500 and 710 nM in cerebral cortex (CCK-B), respectively. Both were agonists in stimulating pancreatic amylase release, and their stimulatory effects were potently inhibited by the CCK-A antagonist L-364,718. A-71623 was a full agonist and A-70874 was a partial agonist (approximately 80%) in stimulating phosphoinositide breakdown in pancreas. Both peptides also were potent agonists in stimulating CCK-A receptors in the ileum. They were, however, weak and behaved as partial agonists in calcium studies in NCI-H345 cells, which possess CCK-B/gastrin receptors. In guinea pig gastric glands, the affinities of A-71623 and A-70874 for the CCK-B/gastrin receptor were 11 and 1.6 microM, respectively. These results demonstrate that A 71623 and A-70874 are potent and selective agonists at CCK-A receptors. The preferential interaction of these novel CCK-4 analogs with CCK-A receptors is in contrast to other CCK-4-based peptides, which are primarily selective for CCK-B receptors. In addition, A-71623 and A-70874 are the first two examples of potent CCK-A agonists that do not contain a tyrosine residue whose sulfation is required for potent CCK-A agonist activity of larger peptides. PMID- 1706471 TI - Carbachol activates a novel sodium current in isolated guinea pig ventricular myocytes via M2 muscarinic receptors. AB - Carbachol induces a novel tetrodotoxin-resistant Na+ current in guinea pig ventricular myocytes bathed in Tyrode's solution with 20 mM Cs+. This action of carbachol, which initiates a series of reactions that culminates in a catecholamine-independent positive inotropic effect, occurs through muscarinic rather than nicotinic cholinoceptive sites. The concentrations of muscarinic antagonists required to suppress the carbachol-induced current by 50% were 2.1 nM, 270 nM, and 1700 nM for atropine, AF-DX 116, and pirenzepine, respectively. These results indicate that an M2-selective antagonist, AF-DX 116, is more potent than an M1-selective antagonist, pirenzepine, as an inhibitor. The M1-selective agonist McN-A-343 did not induce an inward current and blocked that caused by carbachol, in a rapid and reversible manner. This finding is also consistent with the conclusion that the muscarinic receptor involved in the regulation of myocardial Na+ channels by carbachol cannot be distinguished from the M2 subtype of such receptors. Treatment with pertussis toxin did not affect the ability of carbachol to induce an inward current in ventricular myocytes and reversed the current activated by carbachol in atrial cells from outward to inward. The electrophysiological and pharmacological nature of the carbachol-induced current in ventricular myocytes is very similar to that of the acetylcholine-induced current in Xenopus oocytes transfected with porcine M2, but not M1, muscarinic receptors. In both preparations, Na+ is the dominant charge carrier, intracellular Ca2+ is not involved in opening the Na+ channel, and an M2 receptor is involved. PMID- 1706472 TI - Serum-induced sensitization of cyclic AMP accumulation in 1321N1 human astrocytoma cells. AB - Exposure of 1321N1 human astrocytoma cells to fresh medium containing fetal bovine serum induced a marked increase in the subsequent ability of isoproterenol and forskolin to stimulate cAMP accumulation in intact cells, compared with cells exposed to fresh medium without serum. This "sensitization" of cAMP accumulation by serum was dose dependent, occurred rapidly, was maintained in the continuing presence of serum, and reversed rapidly upon removal of serum. Preliminary characterization of the sensitizing factor(s) in serum has been performed, but the factor(s) remain to be identified. Sensitization appeared to result from an increase in maximal response and not from changes in the potency of isoproterenol or forskolin. The protein kinase C inhibitor staurosporine inhibited serum induced sensitization. Furthermore, down-regulation of protein kinase C almost completely eliminated the subsequent ability of serum to induce sensitization, indicating involvement of protein kinase C in the serum effect. Pretreatment of cells with pertussis toxin also markedly reduced subsequent sensitization induced by serum, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in the pathway for serum-induced sensitization. The rate of cAMP degradation was not changed in sensitized cells, but some increase in adenylyl cyclase activity was retained in broken cell preparations from sensitized cells, suggesting increased synthesis of cAMP by adenylyl cyclase as the mechanism for sensitization. PMID- 1706473 TI - A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression. AB - The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70 Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle. PMID- 1706474 TI - Control of protein phosphatase 2A by simian virus 40 small-t antigen. AB - Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity. PMID- 1706475 TI - Cyclic AMP-independent activation of transcription factor NF-kappa B in HL60 cells by tumor necrosis factors alpha and beta. AB - No correlation exists in HL60 cells between NF-kappa B activation by tumor necrosis factor (TNF alpha) and TNF beta and intracellular levels of cyclic AMP. Cyclic AMP levels did not increase upon treatment of cells with each of these cytokines, although NF-kappa B was activated. Forskolin or 1-isobutyl-3 methylxanthine drastically increased intracellular levels of cyclic AMP, but neither activated NF-kappa B nor influenced TNF-induced NF-kappa B activation. PMID- 1706476 TI - Spontaneous release of a factor with interleukin-3-like activity by human lymphocytes and monocytes. AB - The production of a factor with interleukin-3-like activity (IL-3-LA) by cultured human peripheral-blood mononuclear cells has been studied. Culture supernatants spontaneously derived from lymphocytes or monocytes stimulated the proliferative activity of 3 IL-3-dependent cell lines. Purification of this factor by gel filtration-high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing demonstrated the presence of a glycoprotein with a molecular weight of 25-30 kilodaltons and an isoelectric point of 7.6. The biochemical characteristics of the IL-3-LA derived from human monocytes and lymphocytes were very similar. The biological activity of the semipurified factor was tested on mononuclear cells from fetal cord blood. It was found that after 21 days 40% of the cells in the culture were mature basophils which released 15-18 ng histamine/10(6) cells. PMID- 1706477 TI - Genotoxicity of bleomycin. PMID- 1706478 TI - Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. AB - Nerve growth factor (NGF) is a neurotrophic factor responsible for the differentiation and survival of sympathetic and sensory neurons as well as selective populations of cholinergic neurons. NGF binds to specific cell-surface receptors but the mechanism for transduction of the neurotrophic signal is unknown. Several experiments using the NGF-responsive pheochromocytoma cell line, PC12, have implicated tyrosine phosphorylation in NGF-mediated responses, although no NGF-specific tyrosine kinases have been identified. Here we show that NGF induces tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product, a tyrosine kinase receptor whose expression is restricted in vivo to neurons of the sensory spinal and cranial ganglia of neural crest origin. Tyrosine phosphorylation of trk by NGF is rapid, specific and occurs with picomolar quantities of factor, indicating that the response is mediated by physiological amounts of NGF. Activation of the trk tyrosine kinase receptor provides a possible mechanism for signal transduction by NGF. PMID- 1706479 TI - Biotechnology patents. Amgen scores a knockout. PMID- 1706480 TI - A superantigen encoded in the open reading frame of the 3' long terminal repeat of mouse mammary tumour virus. AB - Mice express a collection of superantigens, which bind to class II major histocompatibility proteins and interact with T cells bearing particular V beta chains as part of their alpha beta receptors. These superantigens have been suggested to be encoded by exogenous or endogenous mouse mammary tumour viruses. One such superantigen is now shown to be encoded in the open reading frame of the long terminal repeat of a mammary tumour virus, a gene of previously unknown function. PMID- 1706481 TI - Determination of the subunit stoichiometry of a voltage-activated potassium channel. AB - The voltage-activated K+, Na+ and Ca2+ channels are responsible for the generation and propagation of electrical signals in cell membranes. The K+ channels are multimeric membrane proteins formed by the aggregation of an unknown number of independent subunits. By studying the interaction of a scorpion toxin with coexpressed wild-type and toxin-insensitive mutant Shaker K+ channels, the subunit stoichiometry can be determined. The Shaker K+ channel is found to have a tetrameric structure. This is consistent with the sequence relationship between a K+ channel and each of the four internally homologous repeats of Na+ and Ca2+ channels. PMID- 1706482 TI - Human gamma-globin genes silenced independently of other genes in the beta-globin locus. AB - Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes. PMID- 1706483 TI - [Bleomycin and pulmonary function changes in children with malignant lymphomas]. AB - In 25 patients under 18 years of age with Hodgkin's disease or non-Hodgkin lymphoma treated with bleomycin as part of the treatment with several cytostatics, the diffusion capacity of the lung for carbon monoxide (DLCO) was determined before, during and after this treatment to investigate the damaging effect of bleomycin on the lungs. The DLCO decreased in 18 of the 25 children; the degree of decrease depended both on the total dosage (max. 120 mg/sq.m body surface) and on the dose per administration (5 or 10 mg/sq.m). Eight of these 18 children were followed up for some time after discontinuation of bleomycin treatment. During the relatively brief follow-up period of one year on average, complete recovery of pulmonary function was seen in none of these children; in two, partial recovery occurred. It is necessary to study the changes of DLCO for a longer period after bleomycin treatment, as well as the factors that influence recovery of pulmonary function in children. PMID- 1706484 TI - Antimouse laminin antibodies in IgA nephropathy and various glomerular diseases. AB - IgG, IgA and IgM class antibodies to mouse laminin and human fibronectin in sera from patients with various glomerular diseases (50 cases of IgA nephropathy, 5 cases of minimal-change nephrotic syndrome; 6 cases of membranous nephropathy, 5 cases of systemic lupus erythematosus, 2 cases of Henoch-Schonlein purpura, 3 cases of poststreptococcal nephritis and 4 cases of preeclampsia) and from 30 normal controls were tested using a solid-phase enzyme-linked immunosorbent assay method. IgA antimouse laminin antibody titers in sera from IgA nephropathy patients were significantly higher (p less than 0.05) than in controls. There were no statistical differences in IgA antimouse laminin antibody titers between patients with other glomerular diseases and normal controls. IgM antimouse laminin antibody was significantly raised (p less than 0.01) in sera from patients with preeclampsia. The reaction of mouse laminin with the IgA nephropathy and preeclampsia sera on each of the IgA and IgM assay systems was inhibited by the antigen at up to 5 micrograms/ml. However, it was not inhibited by anti-C3d, anti-C1q, anti-J chain and antisecretory component sera or saccharides. The reaction of mouse laminin with an exceptionally high-titer IgA antimouse laminin antibody serum from a normal control on the IgA assay system was clearly inhibited by 1 mM of melibiose, which contains alpha-galactosyl residues. The same concentration of melibiose, however, did not inhibit the reaction of mouse laminin with IgA nephropathy sera on the same assay system. Treatment of mouse laminin with alpha-galactosidase did not alter any binding from IgA nephropathy sera but binding was lost from an exceptionally high-titer normal control serum. There were no correlations between serum IgA level and IgA antimouse laminin antibody titer in sera from IgA nephropathy patients. Immunoblot techniques revealed the presence of antibody in sera from IgA nephropathy patients reacting with both subunits A and B of laminin, somewhat stronger with laminin A. None of the sera tested contained antifibronectin antibodies. These results indicate that the IgA antimouse laminin antibody is a specific antibody in IgA nephropathy and might play a role in the pathogenesis of the nephritis since mouse laminin and human mesangial laminin present a common epitope. PMID- 1706485 TI - A novel substance P binding site in rat brain regions modulates TRH receptor binding. AB - Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [3H](2 Me-His3)TRH ([3H]MeTRH) on membranes from rat brain regions at 0 degrees C for 5 h. Amygdaloid membranes bound [3H]MeTRH with high-affinity (Kd = 3.1 +/- 0.5 nM (n = 4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC50 for SP = 65 microM). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP greater than nonapeptide (3-11) greater than hexapeptide (6-11) greater than heptapeptide (5-11) greater than pentapeptide (7-11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [3H]MeTRH binding in hippocampus greater than spinal cord greater than retina greater than n. accumbens greater than hypothalamus greater than amygdaloid greater than olfactory bulb greater than or equal to pituitary greater than pons/medulla in parallel assays. In amygdaloid membranes SP (50 microM) reduced the apparent maximum receptor density by 39% (p less than 0.01) without altering the binding affinity, and 100 microM SP induced a biphasic dissociation of [3H]MeTRH with kinetics faster than those induced by both TRH (10 microM) and serotonin (100 microM). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [3H]MeTRH binding to amygdaloid membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706486 TI - Differential extraction of axonally transported proteoglycans. AB - Axonally transported proteoglycans were differentially solubilized by a sequence of extractions designed to infer their relationship to nerve terminal membranes. Groups of goldfish were injected unilaterally with 35SO4 and contralateral optic tecta containing axonally transported molecules were removed 16 h later. Tecta were homogenized in isotonic buffer and centrifuged at 100,000 g for 60 min to create a "total supernatant" fraction. Subsequent homogenizations followed by recentrifugation were with hypotonic buffer (lysis extract), 1 M NaCl, Triton X 100 or alternatively Triton-1 M NaCl. Populations of proteoglycans in each extract were isolated on DEAE ion exchange columns and evaluated for content of glycosaminoglycans (GAGs). Results show the distribution of transported proteoglycans to be 26.3% total soluble, 13.7% lysis extract, 13.8% NaCl extract, 12.2% Triton extract, and 46.2% Triton-NaCl extract. Proteoglycans from all fractions contained heparan sulfate as the predominant GAG, with lesser amounts of chondroitin (4 or 6) sulfate. The possible localizations of transported proteoglycans suggested by the extraction results are discussed. PMID- 1706487 TI - Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse. AB - In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [32P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [3H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [3H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane. PMID- 1706488 TI - Cleavage of the P0 glycoprotein of the rat peripheral nerve myelin: tentative identification of cleavage site and evidence for the precursor-product relationship. AB - The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37 degrees C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24 kDa protein with a concomitant decrease of P0 protein. The conversion of P0 into 24 kDa protein was blocked by heating isolated myelin at 100 degrees C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24 kDa protein. Similarly, addition of CaCl2 to isolated myelin did not accentuate the formation of 24 kDa protein suggesting that the conversion of P0 into 24 kDa protein may not be due to Ca2+ activated protease. It is postulated that the formation of 24 kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24 kDa protein was purified and characterized. The N-terminal sequence of 1-17 amino acid residues of 24 kDa protein was identical to P0. 24 kDa protein was immunostained and immunoprecipitated with anti-P0 antiserum indicating the immunological similarities between P0 and 24 kDa protein. Labeling of 24 kDa protein with [35S]methionine provided evidence that P0 may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24 kDa protein was phosphorylated, glycosylated and acylated like P0. Phosphorylation of 24 kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706489 TI - Frontostriatal relationships on normal and pathological aging of the human brain. PMID- 1706490 TI - Receptor glomeruli and their ultrastructural organization in the arteries and pia mater of the human brain and spinal cord. PMID- 1706491 TI - Polyneuropathy syndromes associated with serum antibodies to sulfatide and myelin associated glycoprotein. AB - We studied a series of 64 patients with sensory +/- motor peripheral neuropathies by comparing clinical and physiologic features to serum antibody reactivity against compounds containing sulfated carbohydrate moieties. We determined antibody reactivity by an enzyme-linked immunosorbent assay (ELISA) using purified glycolipids and glycoproteins as antigens, and we used high-performance thin-layer chromatography and Western blotting to test the specificity of results. Twelve patients with high titers of IgM antibodies directed against the myelin-associated glycoprotein (MAG) had sensory-motor polyneuropathies with physiologic evidence of demyelination. IgM antibody reactivity to MAG was associated with an IgM serum M protein in five patients. Eight other patients, most with sensory greater than motor polyneuropathies, had high titers of antibody reactivity to sulfatide but not of IgM to MAG. Two had an associated IgM paraprotein. None of the patients with selective serum antisulfatide activity had predominantly demyelinating features on physiologic testing. We conclude that (1) high ELISA titers of antibodies to MAG may be more common than previously suspected in patients with chronic demyelinating sensory-motor neuropathies, and (2) the presence of high titers of antisulfatide antibodies in serum may provide clues to the pathogenesis of otherwise idiopathic, axonal, predominantly sensory neuropathies. PMID- 1706492 TI - HTLV-I-associated myelopathy in a Californian: diagnosis by reactivity to a viral recombinant antigen. AB - A male patient developed leg numbness and weakness, and bowel, bladder, and erectile dysfunction. Examination revealed an isolated thoracic myelopathy, with lower-extremity spasticity, decreased vibration and position sense, hyperreflexia, and Babinski's signs. Serum and CSF showed antibody reactivity to human T-cell lymphotropic virus type I or II (HTLV-I/II), suggesting HTLV-I associated myelopathy. Antibody reactivity to a unique HTLV-I recombinant protein provided definitive diagnosis of HTLV-I infection. PMID- 1706494 TI - Special certification in cardiac pacing. PMID- 1706493 TI - Study of mepartricin possible interactions with antilipemic drugs. Controlled clinical trials in patients with benign prostatic hyperplasia. AB - In order to more throughly study mepartricin pharmacodynamic characteristics, 2 groups of 15 patients with BPH and coexistent lipid metabolism disorders were studied in conformity with a sequential experimental design during which also systemic-acting (procetofen) and endoluminal-acting (cholestyramine) fat-lowering drugs were tried. The data obtained confirmed mepartricin specific effect on symptomatology and function in BPH and demonstrated the absence of positive and/or negative pharmacological interactions between the substance in question and the fat-lowering agents tried. PMID- 1706495 TI - Therapeutic irradiation over a permanent cardiac pacemaker. AB - Therapeutic irradiation of fields containing cardiac pacemakers presents a unique problem to pacemaker physicians and radiation oncologists alike. The present case involved a proposed radiation field containing both subclavian pockets. As an alternative solution, the Cordis model 334A implantable pulse generator was irradiated using a backup temporary pacemaker that was kept outside of any significant radiation exposure. A total of 60 Gy was delivered in 30 fractions, with backup temporary pacing and continuous ECG monitoring used for the first 5 fractions. Frequent re-evaluation of pacing and sensing function revealed no changes as a result of irradiation; following radiotherapy, transtelephonic monitoring showed normal pacemaker function for a 4 month follow-up. This represents a useful alternative, particularly for nonmultiprogrammable pacemakers that are made of more radiation-resistant technology. PMID- 1706496 TI - Candida albicans pacemaker site infection. AB - A 69-year-old man with a history of diabetes and episodic lymphocytopenia underwent pacemaker implantation for complete heart block. Despite prophylactic antibiotics, pocket irrigation, and strict sterile technique, a fungal (Candida albicans) pacemaker site infection developed that required pacemaker explanation and systemic amphotericin B therapy. After 3 days of temporary pacing, a second pulse generator was implanted on the opposite side. At 2-year follow-up, he has had no recurrence of pacemaker infection. This report underscores the predilection of diabetics for infections, and in particular, their susceptibility to Candida albicans. PMID- 1706497 TI - Resolution of high initial epicardial patch defibrillation thresholds following chronic implantation. AB - To determine what effect chronic implantation of automatic implantable cardioverter defibrillator epicardial patch electrodes had on initial high defibrillation thresholds at implant, six patients were studied. There were five men, one woman, mean age 61 years. Three had coronary artery disease and three had dilated cardiomyopathies. Mean ejection fraction was 20%. Two patients underwent concomitant coronary artery revascularization and one underwent mitral valve replacement. No patient was on antiarrhythmic drugs. At the time of initial implant, adequate defibrillating thresholds could not be obtained in any patch configuration despite the use of up to 40 joules. Further testing was precluded in each patient due to the development of profound hypotension (less than or equal to 70 mmHg systolic) that was poorly responsive to pressors. The patch electrodes were then implanted in an arbitrary anterior-posterior position and the leads were tunneled to an abdominal pocket. After 10-15 days (mean 11), the lead ends were exposed and defibrillation testing was performed again. In all six patients, adequate defibrillation thresholds were obtained (mean 18 joules). We conclude that if adequate defibrillation thresholds cannot be obtained at implant and if further testing cannot be performed without jeopardizing the life of the patient, the patch electrodes should be implanted and retesting performed at 10 15 days. PMID- 1706498 TI - Pacemaker infection with Mycobacterium avium complex. AB - A 21-year-old. HIV negative, malnourished, homeless woman with congenital heart block had a pacemaker implanted at 7 years of age and multiple procedures thereafter. The most recent of these procedures was replacement of a pulse generator in the right pectoral region. Four months later she had fever, pain, and swelling over the implant site resulting from infection with mixed flora and Mycobacterium avium complex. The pacemaker system was removed by thoracotomy via a median sternotomy and a new DDD pacemaker simultaneously implanted. She was treated with systemic antibiotics--isoniazid, rifampin, ethambutol--for 2 weeks. Six months later she was healthy, pacing well, and apparently free of infection, off all medications. PMID- 1706499 TI - Natural history of sinus node chronotropy in paced patients. AB - The natural history of chronotropic incompetence is not clear. To assess this, we evaluated corrected sinus node recovery time (cSNRT) and sinus node chronotropy at rest and during exercise in two groups of syncopal patients with sinus node disease. Group A comprised patients with resting bradycardia but normal cSNRT and group B had resting bradycardia and prolonged cSNRT (greater than 1000 ms). An additional two groups (C and D) were studied. Group C comprised patients with complete AV (CAVB) and no evidence of sinus node disease and group D were asymptomatic controls of similar age. At diagnosis, patients with symptomatic bradycardia but normal cSNRT and no evidence of carotid sinus syndrome (group A) had resting bradycardia and impaired peak heart rate (PHR-I) on exercise compared to controls (P less than 0.001 and P less than 0.05, respectively), but no reduction in exercise duration. At follow-up group A patients demonstrated an increase in resting rate that was significantly slower than the controls (P less than 0.01). Peak heart rate (PHR-II) also remained significantly slower (P less than 0.05). There was no difference in exercise duration between groups A and D at follow-up. Group B was further subdivided according to follow-up findings of preservation of atrial activity in seven patients (group B-1) and junctional rhythm without any atrial activity in four patients (group B-2). Retrospective analysis showed no significant difference in resting heart rate at initial examination but group B-2 showed a significantly lower peak heart rate on exercise compared with B-1 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706500 TI - Differentiation of arrhythmias by measurement of intracardiac pressures in man. AB - Antitachycardia devices currently use sustained high rate, abrupt changes in cycle length, and probability density function to determine the onset of ventricular tachycardia. Hemodynamic changes occur with the onset of tachycardia and may provide a method of discriminating supraventricular from ventricular tachycardia. In this study, patients had atrial and ventricular pressures measured during rapid atrial and ventricular pacing. Right atrial pressure increased significantly with ventricular pacing but not with atrial pacing. Right ventricular pressures did not significantly differ with atrial or ventricular pacing. The change in atrial pressure compared to baseline was greater, with ventricular pacing compared to atrial pacing. Right ventricular pressure increased compared to baseline with atrial or ventricular pacing, but there was no significant difference between pacing modalities. Measurement of right atrial pressure might prove useful in discriminating supraventricular from ventricular tachycardia. PMID- 1706501 TI - Efficacy of ticlopidine in the prevention of thromboembolic events in patients with VVI pacemakers. AB - This study was designed to evaluate whether long-term treatment with ticlopidine reduces the incidence of thromboembolic episodes in patients with a VVI pacemaker. One hundred eleven patients with a VVI pacemaker were randomly assigned to two groups: group A (52 patients) was treated with ticlopidine at the dose of 250 mg a day; and group B (59 patients) was not treated and served as a control group. The primary analysis of efficacy of ticlopidine was based on the occurrence of thromboembolic episodes and of cardiovascular and cerebrovascular deaths. The mean follow-up period was 66 months. In group A, there was a significant reduction in the incidence of thromboembolic episodes (P less than 0.05) with a smaller incidence of total cardiovascular and cerebrovascular deaths (8 in group A and 18 in group B; P = 0.05) as compared with group B. Twelve percent of patients had moderate side effects with 1 dropout (epistaxis). Our data confirm the high incidence of thromboembolic events in patients with a VVI pacemaker and demonstrate the efficacy of ticlopidine in preventing them. PMID- 1706502 TI - Benefit of single setting rate responsive ventricular pacing compared with fixed rate demand pacing in elderly patients. AB - In order to assess the value of a simple, single setting rate response option to VVI pacing, 12 patients (mean age 75.1 +/- 6.2, range 62-83 years, seven males, five females) with symptomatic complete heart block were entered into a double blind, randomized crossover trial of VVI versus VVIR (single setting rate responsive) pacing using Medtronic Activitrax pacemakers. Assessment was by time taken in seconds (sec) and Borg scale symptom score (6-20) for simple activities (standing from chair x 30; walking 800 meters; 52 steps on stairs [slow and fast pace], and incremental, noninclined maximal treadmill exercise), performed after a 4-week period with the patient in each pacing mode. Times were significantly improved in VVIR mode for standing from chair [mean +/- SD] (78.7 +/- 22.5 vs 70.7 +/- 19.5 sec; P less than 0.05), for 800 m walk (1032 +/- 80 vs 885 +/- 59 sec; P less than 0.05), fast ascent of stairs (29.5 +/- 7.7 vs 26.5 +/- 5.6 sec; P less than 0.02), and treadmill exercise (626.7 +/- 189.5 vs 741.0 +/- 170.2 sec, P less than 0.005) although no difference in time for slow stair ascent was demonstrated. Symptom scores were significantly less in VVIR for standing from chair (12.7 +/- 2.8 vs 10.3 +/- 1.8; P less than 0.01), 800 m walk (10.9 +/- 2.7 vs 9.0 +/- 2.4; P less than 0.01), slow ascent of stairs (11.6 +/- 2.1 vs 10.0 +/ 2.0; P less than 0.01), and fast ascent of stairs (13.0 +/- 2.0 vs 11.7 +/- 1.9; P less than 0.02) but unchanged for treadmill exercise.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706503 TI - Anxiety and depression in patients with life-threatening ventricular arrhythmias: impact of the implantable cardioverter-defibrillator. AB - In order to assess the psychological responses to the automatic implantable cardioverter-defibrillator (AICD), 18 patients with a history of life-threatening ventricular arrhythmias were requested to complete the Spielberger State-Trait Anxiety Inventory and the Beck Depression Inventory. The patients were divided into three groups of six and matched for age, sex, underlying cardiac disease, ejection fraction, and NYHA Functional Classification. Group I had experienced conscious discharges from the AICD, group II had the AICD but without discharges, and group III without the AICD were treated with antiarrhythmic medications alone based on electrophysiological guided testing. Patients with the AICD were also requested to complete a questionnaire directed specifically at their experiences with the AICD. All of the 18 patients completed the study responses and results were analyzed by blinded review. There were no significant differences in anxiety and depression scores in the three groups studied, nor any significant differences in responses to the questionnaire in group I versus group II. One patient in group I reported experiencing adverse psychological responses to the AICD. Although there appears to be no significant differences in psychological responses as a result of the AICD implantation in patients with life-threatening ventricular arrhythmias, further study with larger patient groups is needed to identify and support patients who may develop adverse responses to the AICD. PMID- 1706504 TI - Suppression of incessant polymorphic ventricular tachycardia by selective intracoronary ethanol infusion. AB - Two weeks after an extensive anterior myocardial infarction, a 68-year-old man developed incessant polymorphic ventricular tachycardia (PMVT), unresponsive to all conventional treatment modalities. After requiring greater than 40 direct current cardioversions in less than 3 hours, he underwent attempted intracoronary chemical ablation of his arrhythmia as a treatment of last resort. An infusion catheter was positioned selectively in the subtotally occluded left anterior descending (LAD) coronary artery, the putative "tachycardia-related vessel." Fifty percent ethanol was delivered to the anterior wall through this catheter by slow, constant infusion. Following selective intracoronary ethanol infusion, spontaneous, unprovoked episodes of PMVT ceased, despite discontinuation of all antiarrhythmic drugs. The LAD remained patent. Several days later, the patient underwent coronary artery bypass surgery and implantation of an implantable defibrillator, succumbing in the early postoperative period from recrudescent intractable ventricular fibrillation and cardiogenic shock. Slow intracoronary infusion of 50% ethanol does not cause abrupt vessel occlusion such as occurs after rapid injection of higher concentrations of ethanol. Selective intracoronary infusion of 50% ethanol may provide temporary lifesaving suppression of otherwise intractable polymorphic ventricular tachycardia. PMID- 1706505 TI - Validation of a method for choice of pacing mode in carotid sinus syndrome with or without sinus bradycardia. AB - A new method for selection of the pacing mode in 60 consecutive patients with severe cardioinhibitory or mixed carotid sinus syndrome was prospectively validated. DDD pacing was preferred for 26 patients with: (1) the cardioinhibitory form and who had symptomatic pacemaker effect; (2) mixed type I form, (cardioinhibitory and vasodepressor) with symptomatic pacemaker effect, ventriculoatrial conduction or orthostatic hypotension; (3) mixed type II; or (4) severe bradycardia. VVI pacing was selected in the remaining 34 patients without these symptoms. During a 32 +/- 10 month follow-up period syncope and severe dizziness persisted in five patients in the VVI group (15%) and in three patients in the DDD group (12%). Symptomatic relief occurred in 87% (52/60) of patients. Minor symptoms persisted in 47% of the VVI group and 42% of the DDD group. No patient developed cardiac insufficiency or intolerance to pacing. During a 2 month duration a single-blind, randomized, cross-over study compared VVI and DDD pacing, 69% of the patients programmed from DDD to VVI suffered more frequent, severe, and intolerable symptoms. (1) Thirty four of 60 patients (57% of the entire group) in whom VVI pacing was satisfactory were identified prior to pacemaker implant. In the remainder, VVI pacing was contraindicated as it produced frequent side effects. (2) The preimplant predictive value that VVI pacing would be successful was 85% for those eventually receiving VVI pacemakers and the preimplant predictive value that VVI pacing would fail was 69% for those who underwent DDD implant. PMID- 1706506 TI - Activity-sensing rate responsive versus conventional fixed-rate pacing: a comparison of rate behavior and patient well-being during routine daily exercise. AB - Rate responsive single chamber pacing (VVIR) may be the pacemaker of choice in patients who are not suitable candidates for a dual chamber system. Several studies, most of them performed in an exercise laboratory, have shown a significantly higher exercise capacity demonstrating an improvement in cardiac output and anaerobic threshold compared to conventional fixed rate pacing (VVI). Expressing our idea that stress testing in an "artificial environment" on a bicycle or motor driven treadmill has its limitations and may be difficult to extend into patient's daily life, we designed an outdoor study imitating patient's daily activity. Twenty-one patients with an activity-sensing rate responsive pacemaker performed in a double blind fashion in VVI and VVIR mode the following test circuit: walking 170 meters on flat ground, 210 meters incline, climbing a flight of stairs, and the same circuit in reverse order, and therefore "downhill". Heart rate behavior was recorded by Holter monitoring and patients subjective feelings of well-being, i.e. fatigue and dyspnea were also evaluated. VVIR pacing responded promptly to exercise, i.e., walking on a flat ground, but no further significant increase in pacing rate was observed in relationship to the strength of physical activity while walking inclined or climbing stairs. While patients became exhausted, a nonphysiological decrease in heart rate sometimes occurred. Despite these limitations 6 of 12 patients who had a paced only rhythm while exercising in both VVI and VVIR mode reported feeling significantly better in the VVIR mode, expressing less dyspnea and fatigue. In contrast, two of nine patients having only intermittently paced rhythm preferred the VVIR mode. Patients with lower ejection fraction (EF) were more likely to show subjectively a benefit while exercising in VVIR mode, compared to those with less reduced or normal EF. Despite the technical limitations of using a piezo crystal for rate adaptation, VVIR pacing is an important option in paced-only patients, but it seems less beneficial in patients with only intermittent paced rhythm. PMID- 1706507 TI - Bandwidth-induced errors in parameters used for automated activation time determination during computerized intraoperative cardiac mapping: theoretical limits. AB - Two parameters commonly used when determining the time of local activation during computerized intraoperative cardiac mapping are the time of the peak in bipolar electrograms (BP) and the time of the largest negative slope (LNS) in monopolar electrograms (MP). The dependence of these parameters upon bandwidth was studied. A database of fast MP and BP was compiled from intraoperative recordings collected from epicardial sock arrays in man. Based on 95%, 99% and 99.9% of total signal power, the bandwidths of each response were determined. After removing specified frequencies using the Fourier transform, errors in determining these parameters were computed and characterized with respect to mean square error (MSE), percent power bandwidth and frequency content. The Fourier transform was used to abruptly filter out undesired upper frequencies. Filtering BP at 200 Hz produced a 1-ms shift in the peak in 17.0% of the electrograms, 0.4% of the peaks shifted by 2 ms and none shifted by more than 2 ms. Comparable shifts in the LNS occurred when filtering MP at 400 Hz. Retention of 95% of the power in a BP rarely (0.9%) changed the time of its peak by more than 1 ms. For MP, retention of 99.9% of the power was required for similar performance (3.6%). Thus, although BP have greater signal power in the higher frequencies than MP, a greater bandwidth is required when recording monopolarly in order to preserve the small amount of power necessary to define the time of the LNS with accuracy comparable to determination of the peak in BP. PMID- 1706508 TI - Current concepts for selecting the location, size and shape of defibrillation electrodes. PMID- 1706509 TI - Transvenous placement of a pacemaker lead through an introducer despite long standing subclavian-vein occlusion. AB - Under ordinary circumstances complete chronic subclavian vein occlusion precludes using that vein for the pacemaker lead. There are circumstances, however, in which a patent vein beyond the obstruction can be reached with an introducer needle, thus permitting transvenous access similar to any other introducer assisted implantation. A case exemplifying this point is described. Patency of the right subclavian vein beyond an occlusion was demonstrated by angiography, and a new dual-chamber pacemaker lead was inserted through that site by use of the introducer method. PMID- 1706510 TI - Endless loop tachycardias in DDD pacing are a well-known complication. PMID- 1706511 TI - Electrophysiological features and the clinical follow-up of patients affected by ventricular tachycardias. PMID- 1706512 TI - AgNOR staining in benign hyperplasia and carcinoma of the prostate. AB - We have modified existing techniques for silver staining of nucleolar organizer regions of intact interphase cells by hypotonic swelling and by formic acid treatment to reduce background staining. This allowed the microscopic identification and counting of individual AgNORs in the nucleoli. The method was used on nine adenomatous prostatic samples (including one of normal prostate tissue outside a localized tumor) and on seven prostatic adenocarcinomas. In general, the adenomatous samples displayed fewer AgNORs (mean 13 dots/cell) than did the carcinomas (mean 24 dots/cell). Although no cells with very high AgNOR counts were found in specimens from nonmalignant tumors, two of the adenomatous prostates did have AgNOR profiles that to a large extent overlapped with those of carcinomas. A highly differentiated carcinoma (of which only very small amounts were present in the sample) had low AgNOR counts. The three moderately differentiated carcinomas had more silver-positive material than the nonmalignant prostates but less than the three poorly differentiated carcinomas. The latter tumors also had a substantial proportion of cells with greater than 60 AgNOR counts, something that was never seen in carcinomas with higher differentiation. The data indicate that analysis of silver staining-positive material in intact interphase cells may help distinguish between benign and malignant prostatic tumors and between highly malignant and low malignant carcinomas. PMID- 1706513 TI - DNA flow cytometric study of the hyperplastic and neoplastic canine prostate. AB - Flow cytometric DNA measurements were carried out on 45 formalin-fixed, paraffin embedded canine prostate tissues. The tissues were categorized as normal, hyperplastic, or neoplastic on the basis of light microscopic examination, and DNA ploidy was compared with histologic classification. Ten normal prostate samples showed diploid DNA histograms, but with proliferation greater than that found in the normal human prostate. Thirteen specimens of benign prostate hyperplasia showed diploid or near-diploid DNA histograms. Of 22 prostatic carcinomas, 12 were diploid and 10 were aneuploid, with the majority of the aneuploidy being near-triploid. The frequency of DNA aneuploidy recognized in canine prostatic carcinoma is similar to findings in human prostatic carcinoma if all their grades of malignancy are included. PMID- 1706514 TI - Localization of type IV collagen in the basement membranes of human prostate and lymph nodes by immunoperoxidase and immunoalkaline phosphatase. AB - The object of these studies was to examine the localization of type IV collagen (Coll-IV) in the basement membranes (BM) of epithelial and stromal elements (smooth muscle, nerves, vessels) in normal, hyperplastic, and neoplastic (primary and metastatic) prostate. We also examined the relationship of Coll-IV distribution to the degree of tumor differentiation (Gleason grading system). We compared immunoperoxidase (IP) and immunoalkaline phosphatase (AP) techniques in these studies and in selected samples we also evaluated immunofluorescence (IF) localization of Coll-IV and the effects of tissue fixation and pepsin digestion. We found that IF localization of Coll-IV was intense in unfixed sections. IP and AP reactions were absent in fixed, paraffin-embedded sections but pepsin treatment yielded intense and uniform reaction products in these same preparations. Both the IP and AP techniques showed similar localization of Coll IV in the BM of normal, hyperplastic, and well-differentiated tumor. In most of the higher-grade tumors Coll-IV localization was reduced and a similar pattern of distribution was observed after IP and AP techniques. However, in some high-grade tumors the IP technique showed good localization but AP did not, and vice versa. Such discrepancies were noted in the BM of the tumor cells, as well as in the BM of the stromal elements and in lymph nodes with metastatic tumor. Thus, our study shows decreased Coll-IV localization in higher-grade tumors and suggests that the use of a single technique (IP or AP) may exaggerate this apparent loss of Coll-IV BM components. The exact cause of these discrepancies is unknown but they must reflect variable losses in the ability of the tumor cells to form BM, degradation or decreased synthesis of BM components by high-grade tumors, or a combination of the above. PMID- 1706515 TI - Design, synthesis, and functional expression of a gene for charybdotoxin, a peptide blocker of K+ channels. AB - A gene encoding charybdotoxin (CTX), a K+ channel blocker from scorpion venom, was designed, synthesized, and expressed as a cleavable fusion protein in Escherichia coli. A sequence-specific protease, factor Xa, was used to cleave the fusion protein and thus release the toxin peptide. The recombinant toxin was purified, oxidized to form disulfide bonds, and treated to form N-terminal pyroglutamate. Recombinant CTX is identical to the native venom CTX with respect to high-performance liquid chromatography mobility, amino acid composition, and N terminal modification. With single Ca2(+)-activated K+ channels as an assay system, recombinant CTX shows blocking and dissociation kinetics identical to the native venom toxin. The synthetic gene and high-level expression of functionally active CTX make it possible to study the fundamental mechanism of the toxin-ion channel interaction. PMID- 1706516 TI - Dendritic location of neural BC1 RNA. AB - In nerve cells, a specialized protein synthetic machinery is thought to operate in local compartments of dendrites, in particular beneath synaptic junctions, and thereby to facilitate swift adjustments of the postsynaptic protein repertoire in situ. This notion has been supported by the identification of polyribosomes and selected mRNAs in those compartments. In this study, we report the discovery of a specific RNA polymerase III transcript in dendrites. This RNA, a noncoding, 152 nucleotide-long, single-gene transcript known as BC1 RNA, is expressed almost exclusively in the nervous system. In adult rats as well as in immature rats in late developmental stages, BC1 RNA has been located in the dendrites and somata of a subset of neurons in the central and peripheral nervous system. The colocalization of BC1 RNA with dendritic mRNAs and polyribosomes may indicate a role--possibly within the functional unit of a high molecular mass ribonucleoprotein particle--in specific pre- or posttranslational processes in postsynaptic compartments of neurons. PMID- 1706517 TI - Ultrastructural localization of beta-amyloid, tau, and ubiquitin epitopes in extracellular neurofibrillary tangles. AB - Neurofibrillary tangles (NFTs), a hallmark of Alzheimer disease, are commonly located in perikarya of neurons. In advanced cases of Alzheimer disease, however, NFTs are observed also in the extracellular space. As extracellular NFTs (E NFTs), and occasionally intracellular NFTs (I-NFTs), are recognized by antibodies to beta-amyloid protein (beta AP), beta AP may be present not only in amyloid deposits but also in paired helical filaments (PHFs), the primary components of NFTs. We compared the antigenic characteristics of I-NFTs and E-NFTs with light- and electron-microscopic immunocytochemistry by using several antibodies to noncontiguous epitopes of the microtubule-associated protein tau and of ubiquitin (Ub) as well as an antiserum to beta AP. At variance with I-NFTs, E-NFTs were made predominantly of straight filaments (SFs), rather than PHFs, that were often separated by astroglial processes and in close association with small beta AP deposits. Occasionally, E-NFTs were made of bundles of amorphous material, which showed no resemblance to SFs, PHFs, or amyloid fibrils. The antigenic changes in E-NFTs suggest that when NFTs become extracellular they lose the N and, possibly, the C termini of tau while maintaining the intermediate region of the molecule; they also lose the N-terminal two-thirds of Ub while the C-terminal conjugation site of Ub is preserved. A small subset of E-NFTs reacted with antibodies to both beta AP and tau. Although in most E-NFTs, the epitopes recognized by tau and Ub antibodies were located in typical PHFs and SFs, the epitopes recognized in this subset of anti-beta AP and anti-tau-positive E-NFTs were located exclusively in the bundles of amorphous material. It is suggested that either beta AP epitopes are present but inaccessible in PHFs and SFs and become exposed after conformational changes occurring in the extracellular space or PHFs and SFs become closely associated with beta AP in the extracellular space. PMID- 1706518 TI - Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott). AB - Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein. Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane alpha-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum. PMID- 1706519 TI - Straight and paired helical filaments in Alzheimer disease have a common structural unit. AB - The presence of abundant neurofibrillary tangles in certain areas of the brain constitutes one of the defining pathological characteristics of Alzheimer disease. The predominant component of the tangle is an abnormal fibrous assembly known as the paired helical filament (PHF). The PHF is formed by a twisted double helical ribbon of subunits that gives rise to an image alternating in width between 8 nm and 20 nm with a cross-over spacing of 80 nm. Also found in tangles is the straight filament (SF), a different kind of abnormal filament, about 15 nm wide, that does not exhibit the marked modulation in width shown by the PHF. It is reported herein that PHFs and SFs form hybrid filaments displaying both morphologies, that PHFs and SFs share surface epitopes, and that computed maps reveal a similar C-shaped morphological unit in PHFs and SFs, though differing in relative arrangement in the two types of filament. The observations imply that the SF is a structural variant of the PHF and establish a common unit of assembly for these two pathological filaments. PMID- 1706520 TI - Ultrasensitive fluorometric detection of carbohydrates as derivatives in mixtures separated by capillary electrophoresis. AB - Reducing monosaccharides and oligosaccharides, after reductive amination, were separated and detected as their 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) derivatives by capillary electrophoresis/laser-induced fluorescence. Under optimized conditions, the minimum detectable quantities for monosaccharide solutes were assessed at low attomole levels (0.5 amol for the CBQCA derivative of galactose). The system has shown considerable promise for high-sensitivity analysis of both neutral and amino sugars in glycoproteins. Complex oligosaccharides, isolated from bovine fetuin by hydrazinolysis, were also successfully "mapped." PMID- 1706521 TI - Transcript elongation and termination are competitive kinetic processes. AB - In this paper, we develop a kinetic approach to predict the efficiency of termination at intrinsic (factor independent) terminators of Escherichia coli and related organisms. In general, our predictions agree well with experimental results. Our analysis also suggests that termination efficiency can readily be modulated by protein factors and environmental variables that shift the kinetic competition toward either elongation or termination. A quantitative framework for the consideration of such regulatory effects is developed and the strengths and limitations of the approach are discussed. PMID- 1706522 TI - Potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) by 5-ethyl-6-phenylthiouracil derivatives through their interaction with the HIV-1 reverse transcriptase. AB - In the search for 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT) derivatives, we have found several 5-ethyl-6-(phenylthio)uracil analogues to be highly potent and selective inhibitors of human immunodeficiency virus (HIV) type 1. 1-Benzyloxymethyl-5-ethyl-6-phenylthiouracil, the most potent congener of the series, inhibits HIV-1 replication in a variety of cell systems, including peripheral blood lymphocytes, at a concentration of 1.5-7.0 nM, which is lower by a factor of 10(3) than the 50% antivirally effective concentration of the parent compound HEPT. The 5-ethyl-6-(phenylthio)uracil analogues, like HEPT itself, do not inhibit HIV-2 replication but do inhibit replication of 3'-azido-3' deoxythymidine-resistant mutants of HIV-1. 1-Benzyloxy-methyl-5-ethyl-6 phenylthiouracil and its congeners are targeted at the HIV-1 reverse transcriptase (RT). They do not inhibit HIV-2 RT. They do not need to be metabolized to exert their inhibitory effect on HIV-1 RT. Yet this inhibitory effect is competitive with the natural substrate dTTP. The HEPT derivatives represent a group of RT inhibitors with a unique mode of interaction with HIV-1 RT. PMID- 1706523 TI - Adhesion protein GMP140 inhibits superoxide anion release by human neutrophils. AB - The respiratory burst of blood neutrophils has a critical role in the destruction of microorganisms and tissue damage in inflammation. Neutrophils adhere in a dose dependent fashion to granule membrane protein 140 (GMP140), a member of the LEC CAM (lectin/epidermal growth factor/complement-binding domain cell adhesion molecule) family of adhesion proteins when it is immobilized onto plastic surfaces. Adherence to GMP140 was associated with less superoxide anion generation than adherence to other surfaces, an effect that is especially remarkable after activation of neutrophils with tumor necrosis factor alpha, an agent that on other surfaces promotes adhesion and spreading. However, on GMP140 the cells fail to spread and instead remain rounded and refractile. Neutrophils adhering to GMP140 were also deficient in superoxide anion generation to formylmethionylleucylphenylalanine. Furthermore, fluid-phase GMP140 also inhibited the superoxide generation by neutrophils stimulated by tumor necrosis factor alpha. The effect of GMP140 was reversible by washing and was inhibited by anti-GMP140 Fab antibody. GMP140 appears to be a natural antiinflammatory molecule that may prevent the inappropriate activation of neutrophils in the circulation. PMID- 1706524 TI - Restricted T-cell receptor V beta gene usage by myelin basic protein-specific T cell clones in multiple sclerosis: predominant genes vary in individuals. AB - Recent studies in experimental autoimmune encephalomyelitis as a model for multiple sclerosis (MS) have demonstrated limited heterogeneity in T-cell antigen receptors (TCR) specific for myelin basic protein (MBP). To investigate restricted beta-chain variable-region (V beta) gene usage in humans, we analyzed TCR gene rearrangements in two lines and 34 MBP-specific T-cell clones that were isolated from five MS patients and two healthy subjects. The T cells were characterized for their specificity to MBP epitopes and HLA-restricting molecules. We demonstrate here that MBP-specific T-cell clones from these different MS patients and healthy individuals, in contrast to T cells from rodents, display a more diverse V beta gene usage as evidenced by their TCR V beta gene rearrangements. However, the different MBP-specific T-cell clones isolated from each individual MS patient showed a common V beta gene usage, suggesting individual-specific TCR restriction. Out of 16 MBP-specific clones derived from a single MS patient, 12 clones (75%) utilized the V beta 15 gene for their TCR gene rearrangement. MBP-specific clones isolated from four other MS patients also showed a consistent tendency for a predominant, but different, TCR V beta gene rearrangement. These results suggest a TCR heterogeneity among MBP specific T-cell clones from different individuals but a limited TCR V beta gene usage among MBP-specific T-cell clones of the same individual. The predominant V beta gene used by the MBP-specific T-cell clones studied here was not found to correlate with the epitope specificity of T cells or with their restricting HLA molecule. These findings may support the possibility of intervention with monoclonal antibodies to specific V beta gene products as an approach to immune therapy of MS but also imply the necessity for an individual-specific immunotherapeutic approach. PMID- 1706525 TI - Regional differences in calcium-release channels from heart. AB - The heart is a heterogeneous tissue composed of several cell types tailored for specialized functions. We found that intracellular channels also exhibit regional specialization. In cardiac and skeletal muscle these channels are called the calcium-release channel and are identified by activation with either calcium or caffeine and inhibition by the hexavalent cation ruthenium red. The calcium release channel of the sarcoplasmic reticulum from the interventricular septum has a smaller conductance (31 pS vs. 100 pS) and has longer open and closed times when compared with the channel from left-ventricular free wall. An additional calcium-permeable channel with an even smaller conductance (17 pS) was found in the septum, and this channel is similar to the inositol 1,4,5-trisphosphate-gated channel from smooth muscle and different from the calcium-release channel (ryanodine receptor) from skeletal and cardiac muscle. The inositol 1,4,5 trisphosphate-activated channel may be derived from specialized conducting tissue that is relatively abundant in the septum, whereas the other calcium-release channels may be derived from regionally specialized myocardial cells in the septum and free wall. PMID- 1706526 TI - The ubiquitous glucose transporter GLUT-1 belongs to the glucose-regulated protein family of stress-inducible proteins. AB - In mammals, glucose transport is mediated by five structurally related glucose transporters that show a characteristic cell-specific expression. However, the rat brain/HepG2/erythrocyte-type glucose transporter GLUT-1 is expressed at low levels in most cells. The reason for this coexpression is not clear. GLUT-1 is negatively regulated by glucose. Another family of proteins, glucose-regulated proteins (GRPs), is also ubiquitously expressed and stimulated by glucose deprivation and other cellular stresses. We therefore hypothesized that GLUT-1 may be a glucose-regulated stress protein. This was tested by subjecting L8 myocytes and NIH 3T3 fibroblasts to glucose starvation or exposure to the calcium ionophore A23187, 2-mercaptoethanol, or tunicamycin, all known to increase GRP levels. The mRNA for GLUT-1 was augmented by 50-300% in a time-dependent manner, similarly to the changes in GRP-78 mRNA. Ex vivo incubation of rat soleus muscles induced a marked and concomitant rise in the mRNA levels of GLUT-1 and GRP-78. Finally, calcium ionophore A23187 and 2-mercaptoethanol induced a 2- to 3-fold increase in the levels of the GLUT-1 protein and hexose uptake. In all instances in which GRP-78 and GLUT-1 responded to stress, the transcription of the cell specific muscle/adipocyte-type insulin-responsive glucose transporter (GLUT-4) did not change. Thus, despite the lack of structural similarity, GLUT-1 and GRP 78 expression is regulated similarly, whereas the regulation of GLUT-4, which is structurally related to GLUT-1, is different. We propose that GLUT-1 belongs to the GRP family of stress proteins and that its ubiquitous expression may serve a specific purpose during cellular stress. PMID- 1706527 TI - Primary structure of the precursor for the sea anemone neuropeptide Antho-RFamide (less than Glu-Gly-Arg-Phe-NH2). AB - Neuropeptides containing the carboxylterminal sequence Arg-Phe-NH2 are found throughout the animal kingdom and are important substances mediating neuronal communication. Here, we have cloned the cDNA coding for the precursor protein of the sea anemone neuropeptide (Antho-RFamide) less than Glu-Gly-Arg-Phe-NH2. This precursor is 334 amino acids in length and contains 19 copies of unprocessed Antho-RFamide (Gln-Gly-Arg-Phe-Gly), which are tandemly arranged in the C terminal part of the protein. Paired basic residues (Lys-Arg) or single basic residues (Arg) occur at the C-terminal side of each Antho-RFamide sequence. These are likely signals for posttranslational cleavage. The processing signals at the N-terminal side of each Antho-RFamide sequence, however, include acidic residues. Processing at these amino acids must involve either an amino- or an endopeptidase that cleaves C-terminally of aspartic acid or glutamic acid residues. Such processing is, to our knowledge, hitherto unknown for peptidergic neurons. The Antho-RFamide precursor also contains two copies of the putative Antho-RFamide related peptide Phe-Gln-Gly-Arg-Phe-NH2 and one copy of Tyr-Val-Pro-Gly-Arg-Tyr NH2. In addition, the precursor protein harbors four other putative neuropeptides that are much less related to Antho-RFamide. This report shows that the biosynthetic machinery for neuropeptides in coelenterates, the lowest animal group having a nervous system, is already very efficient and similar to that of higher invertebrates, such as mollusks and insects, and vertebrates. PMID- 1706528 TI - Intrinsic regulation of apical sodium entry in epithelia. AB - In the past 30 years the basic features of Na+ absorption by epithelia have been unraveled and generally accepted cell models have been established. However, these cell models of transepithelial Na+ transport represent, for the most part, a static view of cell function, i.e., all transport parameters are assumed to be in a steady state. Today the focus is on the dynamic properties of epithelia, the non-steady-state condition, and the adaptation to environmental or transport changes. This review deals with mechanisms intrinsic to the epithelium that regulate apical membrane Na+ permeability in response to changes in transport load and ambient conditions. Together with parallel autoregulatory events concerning the basolateral K+ conductance, the described mechanisms controlling apical membrane Na+ permeability serve to maintain the intracellular ionic composition within the limits that are compatible with cell function and survival. Extraepithelial factors that influence epithelial Na+ transport such as mineralocorticoids and glucocorticoids, ADH, catecholamines, and other neurotransmitters are discussed elsewhere. Apical membrane Na+ permeability appears to be determined by several intrinsic or autoregulatory mechanisms. The PmNa of epithelia with channel-mediated apical Na+ entry is downregulated by increases in the Na+ concentration of the apical bathing solution (self inhibition) and by procedures that inhibit basolateral Na+ extrusion (feedback inhibition). The underlying mechanisms of both regulatory systems are unclear. With the use of current-noise (fluctuation) analysis, on the one hand, and single channel recordings, on the other hand, conflicting results were obtained concerning the saturability of single-channel conductance with increasing external Na+ concentrations. Results from Na(+)-uptake studies in apical membrane vesicles from amiloride-sensitive epithelia render it unlikely that cell Na+ itself is the mediator of feedback inhibition. Both self-inhibition and feedback inhibition of PmNa are prevented by titrating superficial sulfhydryl groups in the apical membrane. Elevations of cell Ca2+ decrease apical Na+ entry, possibly via an indirect mechanism involving protein kinase C. The PmNa is markedly dependent on cell metabolism and pHc; inhibition of ATP supply and lowering cell pH reduce PmNa. Additionally, PmNa may be altered by exocytotic expansion and endocytotic retrieval of the apical membrane area or by insertion of channel proteins into the apical membrane without increasing the apical membrane area. The diversity of regulatory systems may insure the high degree of flexibility and plasticity of epithelia in their response to environmental changes. PMID- 1706529 TI - In vitro HIV infection of primate lymphocytes. AB - Peripheral blood mononuclear cells (PBMC) obtained from four different primate species were tested for their respective ability to support the "in vitro" replication of the human immunodeficiency viruses, HIV-1, and HIV-2. PBMC of Cebus apella, patas (Erythrocebus patas), green (cercopithecus aethiops sabaeus) and rhesus monkeys (Macaca mulatta) were infected "in vitro" with either HIV-1 or HIV-2. Cultures were assayed weekly for particle-associated reverse transcriptase activity. Both viruses were found to be cytolytic for all these monkey's PBMC. Low levels of HIV-1 and HIV-2 infection were observed in Cebus cells. However, productive infection was only detected in HIV-2 infected rhesus PBMC. The capacity of HIV-2 to replicate in rhesus cells may provide a useful model for evaluating antiviral drugs and vaccines. PMID- 1706530 TI - [Theoretical mechanism of ethionine hepatocarcinogenesis]. PMID- 1706531 TI - [The effect of 6% (40/0.5) hydroxyethyl starch and Ringer's lactate on blood coagulation, laboratory parameters and circulation during peridural anesthesia]. AB - We investigated the effects of 6% hydroxyethyl starch (HES 40/0.5) and lactated Ringer solution (LRS) on blood coagulation tests and laboratory parameters during epidural anesthesia. Additionally, the efficacy of this prophylactic intravenous fluid supply in preventing sympathetic blockade induced hypotension was studied. METHODS. A single shot lumbar epidural block was given to 55 patients using 14-18 ml of bupivacaine 0.75%. The patients were randomized to receive either 1000 ml 6% HES 40/0.5 or 1000 ml LRS starting 5 min before the epidural blockade was set. The first 500 ml was infused during a 15-min period and the remaining 500 ml solution during the next 30 min. Cardiovascular parameters were recorded and blood samples were taken 30, 60, 120 and 240 min after the start of the infusion. RESULTS. No significant differences were found in the cardiovascular parameters, although in patients with a cranial spread of epidural blockade above T 10, patients who received LRS showed more episodes of severe hypotension. Serum osmolarity, potassium and sodium remained constant throughout the observation period. HES 40/0.5 caused a significantly greater hemodiluting effect than LRS, which was evident in more pronounced temporary decreases in serum protein concentration, hemoglobin concentration, hematocrit, fibrinogen and platelets. In coagulation parameters LRS caused no changes of PTT and Quick, whereas HES 40/0.5% led to a significant prolongation of PTT and a decrease in Quick. CONCLUSION. Fluid supply with either LRS or 6% HES 40/0.5 cannot prevent the epidural blockade induced hypotension entirely. In epidural anesthesia with spread of blockade above T 10, 6% HES 40/0.5 is superior to LRS in the prevention of severe hypotension. The temporary increase in plasma volume after infusion of 6% HES 40/0.5 results in a greater hemodilution with a concomitant decrease of blood viscosity and improved microcirculation flow. These might be of interest in prevention of thromboembolic complications. The specific effects of the two solutions were also determinable during epidural blockade. PMID- 1706532 TI - Comparison of endoscopic Nd:YAG laser therapy and oesophageal tube in palliation of oesophagogastric malignancy. AB - The clinical results of 96 patients with upper gastrointestinal malignancy have been evaluated retrospectively. Sixty-nine patients with a mean age of 72 years (35 men and 34 women) were treated with endoscopic laser therapy, and 27 patients with a mean age of 67 years (16 men and 11 women) with insertion of an oesophageal tube. After laser therapy the bulk of the tumour was reduced in 87%, and in 55% clear signs of relieved dysphagia were seen. The insertion of an oesophageal tube was successful in 89%. In the laser group no fatal complications occurred, and the overall complication risk was 8.7%. The 1-year survival in all laser patients was 12%, and in patients with impassable tumour stenosis the survival was 6%. The mortality related to the insertion of an oesophageal tube was 11%, and complications occurred in 48% of the patients. The 1-year survival of the tube group was nil. It is concluded that endoscopic laser therapy and insertion of oesophageal tube are both effective methods in palliation of oesophagogastric malignancy, but the mortality and risk for complications were markedly lower after laser therapy. PMID- 1706533 TI - Effect of the histamine-1 antagonist astemizole alone or with omeprazole on rat gastric mucosa. AB - The stimulation of acid secretion by gastrin may in the rat be explained solely by gastrin-induced histamine release. This study was done to examine whether histamine could mediate the general trophic effect of gastrin on the oxyntic mucosa, by using a long-acting selective histamine-1 antagonist (astemizole) alone or with omeprazole-induced hypergastrinaemia for 90 days in female Sprague Dawley rats. At day 90, isolated vascularly perfused rat stomachs were prepared to study maximal gastrin- and histamine-stimulated acid and pepsinogen outputs and maximal gastrin-stimulated histamine release. Oxyntic mucosa morphometry, mucosal histamine and pepsinogen contents, and plasma gastrin and histamine levels were also determined. For the first time, omeprazole has been found to inhibit gastric emptying and to increase plasma histamine. As compared with controls, astemizole alone did not influence plasma gastrin, increased plasma histamine in only some rats, and gave a slight increase in all other variables. Together with omeprazole, it further increased variables already stimulated by omeprazole. Thus, mucosal thickness, histamine concentration, and chief-cell density in oxyntic mucosa were significantly higher in astemizole/omeprazole treated rats than in omeprazole-treated rats. Gastrin-stimulated histamine release was increased in both astemizole- and omeprazole-treated rats. For all rats plasma histamine was significantly correlated with plasma gastrin and with numerical fundic argyrophil cell density. In conclusion, the present study confirms the trophic effect of gastrin and shows a slight trophic effect of astemizole on the oxyntic mucosa. It also shows that plasma histamine may reflect the argyrophil cell density in the oxyntic mucosa and that omeprazole inhibits gastric emptying. PMID- 1706534 TI - GABAergic pathway from zona incerta to neocortex: clarification. PMID- 1706535 TI - Failure to elicit neuronal macroscopic mechanosensitive currents anticipated by single-channel studies. AB - Mechanosensitive channels can be observed in most cell types during single channel recording and have been implicated in many cellular processes. Potassium selective single-channel currents, both stretch-activated and stretch inactivated, can be observed in growth cones and cell bodies of Lymnaea stagnalis neurons. Equivalent macroscopic mechanosensitive currents could not, however, be elicited while applying various mechanical stimuli. This discrepancy suggests that single-channel mechanosensitivity is an artifact of patch recording. PMID- 1706536 TI - Prenatal screening for open neural tube defects, Down's syndrome, and other major fetal disorders. PMID- 1706537 TI - Chemically distinct rat olivocochlear neurons. AB - We have produced a neurochemical map of the cell bodies of origin of the cochlear efferent terminals in rat by combining glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), or calcitonin gene-related peptide (CGRP) immunocytochemistry with retrograde transport of horseradish peroxidase. The locations of cochlear efferent cell bodies are in general agreement with the medial and lateral systems described by White and Warr (J. Comp. Neurol. 219:203 214, 1983) with some minor modifications. The lateral system consists of at least two pools of chemically distinct neurons located within the lateral superior olive (LSO) ipsilateral to the injected cochlea. One pool immunostains with an antibody to GAD while the other immunostains with antibodies to ChAT and to CGRP. The medial efferent system consists of periolivary neurons that are almost exclusively large and ChAT-positive but CGRP-negative. They are located both ipsilateral and contralateral to the cochlea they project to. There are a few GAD positive small neurons in the medioventral and rostral periolivary regions that project ipsilaterally, but these may prove tobe ectopic neurons. The ipsilateral lateroventral periolivary region (LVPO) contains some efferent neurons, all of which are ChAT-positive but CGRP-negative. Additional cochlear efferent neurons, some of which are ChAT-positive and others GAD-positive, are present within and immediately dorsal to the fiber capsule surrounding the medial limb, and to a lesser extent the lateral limb, of the ipsilateral LSO. Not all GAD-positive or ChAT-positive olivary cells project to the cochlea. We have complemented the results in the brainstem by demonstrating two immunocytochemically distinct populations of efferent terminals in the cochlea simultaneously, one CGRP positive and the other GAD-positive. Approximately equal numbers of boutons immunoreactive for both markers are present beneath inner hair cells throughout the entire length of the cochlea. Surprisingly high numbers of GAD-positive and CGRP-positive boutons are also present on outer hair cells, with each class having its spatially and morphologically distinct features. The lack of CGRP positive periolivary cells that are retrogradely labeled by cochlear injections of HRP suggests that the lateral olivocochlear system sends projections to outer hair cells. Our results raise questions about species differences in the organization of targets of the lateral and medial olivocochlear systems. PMID- 1706538 TI - Immunohistochemical demonstration of alpha 1-antichymotrypsin and alpha 1 antitrypsin in salivary gland pleomorphic adenomas of children. AB - Twenty-five benign pleomorphic adenomas of salivary glands in children were studied with immunohistochemical techniques in order to characterize the cell types comprising the epithelial and so-called "mesenchymal" regions of the tumors. The antisera against alpha 1-antichymotrypsin (alpha 1-ACT) and alpha 1 antitrypsin (alpha 1-AT) were used to stain in normal salivary gland tissue as well as in pleomorphic adenoma. In normal salivary glands, alpha 1-ACT was localized to the intercalated duct and serous acinar cells. On the other hand, there was positive staining for alpha 1-AT in the intercalated and striated duct cells. Twenty-five cases (100%) of pleomorphic adenomas in children displayed positivity to alpha 1-ACT staining and 22 cases (88%) showed a positive staining for alpha 1-AT. alpha 1-ACT staining was particularly intense in chondrocyte-like cells of 20 cases (80%), in inner tubular cells of 16 (64%) and cyst-lining cells of 12 (52%). The limited number of tumor cells which were called plasmatoid or hyaline cells and squamous epithelial cells, were positive for alpha 1-ACT. None of the outer tubular cells and hyalinous material was positively stained for alpha 1-ACT. A strong positive reaction for alpha 1-AT was observed in chondrocyte-like cells of 15 cases (60%). Inner tubular cells were positive for alpha 1-AT in 12 cases (48%), plasmatoid or hyaline cells in 10 (40%) and cyst lining cells in 8 (35%). Squamous epithelial cells, clear cells, secretory product and hyalinous material were positive for alpha 1-AT in some cases. Chondroid matrix and myxoid stroma had no reaction with both antibodies. The biological role of alpha 1-ACT and alpha 1-AT with a wide immunohistochemical distribution in pleomorphic adenomas of children may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases. PMID- 1706539 TI - Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: biochemical and histological studies. AB - Chlordecone (CD) pretreatment is well known to greatly potentiate CCl4 toxicity. Previous work has shown that suppression of hepatocellular regeneration permits an ordinarily limited liver injury to progress in an irreversible manner. Insufficient hepatocellular energy has been proposed as a mechanism for suppressed hepatocellular regeneration. Since cyanidanol reportedly increases cellular ATP, this compound was employed to test the above hypothesis. The present study was designed to investigate the sequential biochemical and histological changes over a time course of 120 hr after CCl4 administration. Male Sprague-Dawley rats (125-150 g) were maintained on 10 ppm CD diet for 15 days and were challenged with either a standard protocol dose (100 microliters/kg) or a low (50 microliters/kg, L) dose of CCl4. Cyanidanol pretreatment at 48, 24, and 2 hr before CCl4 administration to rats maintained on CD diet resulted in 100 or 70% animal survival, for CCl4 (L) or the standard dose of CCl4, respectively. Preliminary studies indicated that neither simultaneous nor subsequent administration of cyanidanol with CCl4 challenge affords such protection. Prior treatment with cyanidanol and a latency period were found necessary for protection. Without cyanidanol, CD + CCl4 combination caused 50 and 100% lethality after CCl4 (L) and the standard dose, respectively, while the same doses of CCl4 alone did not cause lethal effects. Plasma enzymes (alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase) in control rats showed only moderate and transient increases after CCl4 challenge. The combination of CD + standard dose of CCl4 resulted in progressive and marked elevations of all three serum enzymes at all time intervals until the death of animals. Cyanidanol pretreatment resulted in significant decline in the plasma enzyme elevations at later time points. Cyanidanol pretreatment increased hepatic ATP synthesis in control or CD rats. CCl4 administration to control rats did not alter hepatic ATP levels, while in CD-fed rats hepatic ATP levels were significantly decreased. Cyanidanol pretreatment to CD + CCl4 combination-treated rats did not significantly prevent the decline in hepatic ATP and glycogen levels. However, in the surviving rats a recovery in these parameters was observed. Light microscopic examination of livers from animals that received CCl4 alone revealed only marginal cellular injury, at early time points only. However, CCl4 challenge to rats maintained on CD resulted in progressive injury, characterized by the appearance of ballooned cells, necrotic cells, and cells with lipid droplets in the liver. Cyanidanol pretreatment to these rats caused decreased vacuolation and significantly reduced the progression of liver necrosis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706540 TI - Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: regeneration studies. AB - Previous work has shown that chlordecone (CD)-amplified CCl4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl4 challenge describe sequential changes in hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125-150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr before a single ip injection of either a standard protocol dose (100 microliters/kg) or a low dose (50 microliters/kg, L) of CCl4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol stimulated [3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl4 (100 microliters/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increased putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl4(L) combination, the [3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl4 group of rats prevented the decrease in the hepatic DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706541 TI - The effect of cyclosporine on exocrine function of the rat pancreas--an in vitro study. AB - We have studied the effect of cyclosporine on the exocrine pancreas of rats submitted to treatment with different parenteral doses (15, 30, 50 mg/kg/day) for different time periods (7 and 30 days). Serum amylase levels, pancreas weight, and total pancreatic amylase and protein content showed a dose-dependent decrease. Basal amylase secretion from isolated lobules remained unchanged. Furthermore, the pancreatic lobules from animals under cyclosporine treatment exhibited a decreased response to bethanechol stimulation. Our data suggest that cyclosporine produces a toxic effect on exocrine pancreatic function. PMID- 1706542 TI - Reduction of sensitization induced by blood transfusion in the rat by monoclonal antibodies. AB - Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three transfusions of DA blood; the initial transfusion was either untreated or pretreated with monoclonal antibody directed to class I antigens or other cell surface markers. Mean antibody activity in recipient sera against class I DA antigens was significantly decreased by pretreatment with the monoclonal antibodies. The most marked suppression was associated with pretreatment by antibodies to the four major nonoverlapping epitopes of the RT1Aa antigen. Subsequent transfusions of DA blood failed to stimulate a secondary response. Crossreactivity of the alloantibody reactivity with BDIX antigens was diminished by pretreating the transfusions with rat anti-RT1A antibodies and, to a lesser extent, with a mouse monoclonal antibody (OX-18) to a common class I determinant. Monoclonal antibody pretreatment had no effect on the humoral response to class II DA antigens. These studies indicate that blood transfusions pretreated with monoclonal antibodies induce a less-potent cytotoxic humoral immune response and that reactivity is most effectively suppressed by completely masking the class I antigen. This technique may prove of clinical value in preventing the sensitization caused by blood transfusions in potential transplant recipients. PMID- 1706543 TI - The effect of cyclosporine on the release of endothelium-derived relaxing factor from isolated human epicardial coronary arteries. PMID- 1706544 TI - Receptor-gated ion channels may be selective CNS targets for ethanol. PMID- 1706545 TI - Progress in the development of potent bombesin receptor antagonists. AB - Bombesin and the mammalian-related peptides gastrin-releasing peptide (GRP), GRP and neuromedin B have been shown to have numerous actions in the CNS, gastrointestinal tract and on growth. However, the role of the peptides in various physiological processes has remained unclear because of the lack of potent antagonists. Recent in vitro studies have described four different classes of bombesin receptor antagonist, some of which are active in the nanomolar range and in vivo. Robert Jensen and David Coy describe recent insights into peptide structural determinants of biological activity. Evidence from structure-function studies have resulted in identification of some analogues that function as potent antagonists in all systems examined. Furthermore, various subtypes of bombesin receptors can now be differentiated by these various classes of antagonist. PMID- 1706546 TI - Solid cell nests of the thyroid gland. AB - The histogenesis and clinical significance of solid cell nests (SCN) of the thyroid are not fully understood. From August 1987 to December 1989 a total of 2544 patients with thyroid and parathyroid diseases underwent surgery at Ito Hospital, and SCN were revealed within the thyroid parenchyma in 21 (0.8%). Distribution of SCN was not limited to the upper one-third of the lateral lobe, and SCN were found even in the isthmus lobe. In 5 cases microcysts were also noted within SCN, and their content was thought to be acidic proteoglycan. Immunohistochemical study revealed that SCN were negative for thyroglobulin and calcitonin but positive for carcinoembryonic antigen. Thirteen of 21 cases showed positive immunostaining with cytokeratin. Scattered calcitonin-positive cells were noted around the SCN. It is suggested from these findings that SCN of the thyroid are closely related to certain cells of ultimobranchial body vestiges which may be not of neuroectodermal origin but of endodermal origin. PMID- 1706547 TI - Expression of keratin 13 in human epithelial neoplasms. AB - The distribution of the 52 kDa keratin 13 was evaluated immunohistochemically, using the AE8 monoclonal antibody. Various squamous and transitional cell epithelial lesions and representative control tissues were studied. This antibody performed adequately in formalin-fixed and paraffin-embedded tissue, but like keratin immunohistochemistry in general, required protease pretreatment. Keratin 13 was found consistently in the suprabasal layers of squamous epithelia of oral cavity, tonsils, larynx, esophagus, lower female genital tract, and transitional urothelium, but it was absent in the epidermis. Generally, various forms of squamous metaplasia were AE8-positive. In dysplasia, AE8 reactivity was considerably decreased or even absent despite the presence of apparent suprabasal maturation. In differentiated squamous cell carcinomas, AE8 immunoreactivity was usually limited to a few cells in the center of the keratinized foci. However, in 10% of squamous cell carcinomas, a significant number of tumor cells was positive. Only well-differentiated urothelial carcinomas showed AE8 immunoreactivity, while poorly differentiated tumors were negative. Interestingly, a Brenner's tumor showed a high number of AE8-positive epithelial cells. Our results show that the expression of keratin 13, as immunohistochemically determined by AE8 antibody, is significantly down-regulated in squamous cell malignancies. Its possible value as an adjunct to diagnosis of dysplasia should be investigated further. PMID- 1706548 TI - The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses. AB - Monoclonal antibodies which reacted with four different epitopes were used to select neutralization-resistant variants of Australian bluetongue virus serotype 1 (BTV1AUS; isolate CS156). Nucleotide sequencing of the VP2 outer coat protein gene of these variants showed that two of them contained alterations within the previously defined neutralization site at amino acids 328 to 335 (Gould et al., 1988). Comparison of VP2 sequences of several BTV serotypes, in addition to nucleotide sequence changes in a number of variants, suggested that this neutralization site was larger and contained 19 amino acids, the conformation of which could be affected by other regions of the VP2 protein. Nucleotide sequencing of neutralization-resistant variants revealed a total of four other regions of VP2 affecting the ability of monoclonal antibodies to neutralize the virus and these results support the notion that the neutralization site in VP2 was conformation dependent. The complete nucleotide sequence of the VP2 gene of virulent BTV1AUS (C5156) was determined directly from viral nucleic acid isolated from the blood of a sheep suffering clinical bluetongue disease. Comparison of the VP2 sequence of this virulent virus with that previously published for an avirulent, laboratory strain (Gould, 1988), indicated that the passage of virulent virus approximately 20 times in tissue culture over the last decade, not only led to attenuation but resulted in the appearance of ten nucleotide changes in the VP2 gene. Six of these nucleotide changes were silent, two resulted in conservative amino acid substitutions and two generated radical amino acid changes. However, in a separate experiment, a single passage of the virulent virus in tissue culture while leading to attenuation did not result in a nucleotide change in the VP2 outer coat protein gene. PMID- 1706549 TI - [Use of automated microscopic image analysis for the determination of microscopic hyphomycetes (Fusarium sp.)]. AB - The method of automated microscopic image analysis was tested in intention to support the taxonomy of Fusarium. The catalogue of morphometric object features of the software system AMBA provides size and shape parameters which can be utilize to discriminate populations of macroconidia of different Fusarium species and varieties. The applied colouring of macroconidia (methylene blue) has no significant effect on these features. The results of statistical analysis show fitness of chosen technique. PMID- 1706550 TI - [Changes in rheological properties of blood in multiple sclerosis and their correction]. AB - As many as 45 patients with multiple sclerosis were examined for rheological blood properties. As compared to controls, the group under examination manifested the rise of plasma viscosity, acceleration of red blood cell aggregation. 26.2% of patients demonstrated an appreciable increase of blood viscosity. It is assumed that these changes contribute to the deterioration of microcirculation and aggravate the demyelinating process. Correction of the rheological properties of the blood by plasmapheresis coupled with other methods of pathogenetic therapy turned out effective. PMID- 1706551 TI - Neuroendocrine cells of the human lung express substance-P-like immunoreactivity. AB - The occurrence of substance P (SP) in the neuroendocrine population of human lungs was investigated by immunohistochemical methods. All individuals studied (n = 16) had SP-like immunoreactive cells, being more numerous in lungs of fetuses and newborn infants than in adults. These cells, both solitary and forming neuroepithelial bodies, were found at all levels of the respiratory mucosa. Solitary neuroendocrine cells and neuroepithelial bodies were found in the bronchial and bronchiolar mucosa, while at the alveolar level neuroepithelial bodies were also seen. A more intense SP-like immunoreactivity was found in the basal cytoplasm of these cells. Occasionally they show cytoplasmic prolongations which interdigitate with neighboring epithelial cells. These facts suggest that SP-like immunoreactive cells may have a paracrine or local secretion function, acting over surrounding epithelial cells or structures situated in the lamina propria. The evidence of great numbers of SP-like immunoreactive neuroendocrine cells in fetuses and infants might be the expression of a functional role of SP in lung development. PMID- 1706552 TI - Absence of right-left asymmetries in the rat hippocampus as demonstrated by Timm staining. AB - Although biochemical and behavioural studies have shown right-left differences in several areas of the rat limbic system, some anatomical studies reported no significant right-left differences in several morphological parameters of the hippocampus. The purpose of the present study was to determine whether there are asymmetries in the micro-anatomy of the rat hippocampus by examining the intensity of Timm staining in various hippocampal fields and the area occupied by mossy fibres by the use of combined microdensitometric and quantitative image analysis techniques. Timm staining demonstrates the distribution of intrahippocampal association pathways because it is a histochemical marker of zinc and other heavy transition metals. There were no right-left differences in the density of Timm staining at the level of the dentate gyrus, in the dendritic layer of CA1 and CA2 fields, in the mossy fibre area or in the subiculum. These findings provide further evidence of a lack of morphological asymmetry in the rat hippocampus. PMID- 1706553 TI - [Development of the subclavian artery in the loggerhead turtle (Caretta caretta) studied by the injection method]. AB - In the turtle, the left aorta and the pulmonary trunk originate from the right ventricle, while the right aorta takes its origin from the left ventricle as a functional systematic arch. The subclavian artery arises from the brachiocephalic artery on each side, and passes ventral to the vagus nerve and the jugular vein. These features are basically the same as in birds, and the subclavian artery of the adult turtle corresponds to a secondary artery from the viewpoint of comparative anatomy. Many investigators, including one of the present authors (Suzuki, 1987), have studied the development of the aortic arch and the subclavian artery in the chick embryo, but not in the turtle. The present authors examined it in Loggerhead turtle (Caretta caretta) embryos, from 14 days of incubation to completion of the aortic arch (27 days incubation). All blood vessels were injected with Berlin blue solution using a fine glass needle inserted into the aortic trunk through the ventricle of the heart. The following results were obtained. 1. In the turtle embryo the primary subclavian artery develops first, but is replaced by the secondary subclavian artery as in the chick. 2. The primary subclavian artery arises from the 12th dorsal intersegmental artery and passes dorsal to the posterior cardinal vein. In the 16 day embryo, it gives rise to capillary nets both cranially and caudally at the base of the forelimb bud along the inner surface of the thoracic wall. 3. At 19 days of incubation, a small blood vessel arises from the aortic sac at the origin of the third aortic arch and passes laterally, ventral to the anterior cardinal vein. The vessel then extends caudally, and finally, at 21 days of incubation, connects to the cranial part of the capillary net of the primary subclavian artery at about the middle of the lateral thoracic wall. After the completion of the connection, the vessel from the aortic sac is called by the name "the secondary subclavian artery." 4. The secondary subclavian artery gradually increases in size, while the proximal part of the primary one begins to atrophy and finally disappears at 27 days of incubation. After this, the forelimb bud receives its blood supply only from the newly-formed secondary subclavian artery. 5. In conclusion, in the turtle, the secondary subclavian artery is formed by connection of the primary artery with the caudally extending artery arising from the aortic sac, while in the chick it is derived from an outgrowth of the primary artery.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706554 TI - Carcinosarcoma of the submandibular gland. An autopsy case. AB - We present a rare case of submandibular gland carcinosarcoma occurring in a 45 year-old male patient. His clinical history revealed that the carcinosarcoma had developed from a carcinoma ex mixed tumor in three years. In spite of repeated resection, intensive chemotherapy and irradiation, the tumor recurred and grew rapidly, and the patient died of hemothorax caused by rupture of a pulmonary metastatic tumor. The fourth recurrent tumor and autopsy specimens showed features of carcinosarcoma consisting of three tumor components, i.e., undifferentiated carcinoma, and chondrosarcomatous and osteosarcomatous growth. The metastatic nodules in both lungs and pulmonary hilar lymph nodes showed the same pattern. Immunohistochemically, the chondrosarcomatous cells were positive for vimentin and S-100 protein, and for epithelial markers such as epithelial membrane antigen (EMA) and cytokeratin (MA-902). Undifferentiated carcinoma cells, on the other hand, were partially positive for muscle actin other than cytokeratin (KL 1). Ultrastructurally, desmosome-like structures were seen in the chondrosarcomatous cells. These findings suggest that the sarcomatous lesions might have originated from epithelial cells. PMID- 1706555 TI - Primary adenocarcinoma of the female urethra with three histologic patterns and partial AFP positivity. AB - A rare case of adenocarcinoma of the female urethra with alpha-fetoprotein (AFP) positivity in a 52-year-old woman is reported. The tumor was papillary polypoid, localized in the posterior wall of the mid-urethra and microscopically showed three histologic components. Upon immunostaining and histochemical staining, the tumor was characterized by intestinal-type cells positive for epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), EMA-negative and AFP-positive columnar vacuolated cells and mainly EMA-positive clear cells. On the basis of these features together with the known embryogenesis of the urethra, an endodermal origin of the tumor is suggested, possibly arising from the reserve or stem cells in the urethral mucosa. This case and its immunohistochemical features are quite unique and the histologic combination is meaningful when considering the oncogenesis and histogenesis of urethral tumors. PMID- 1706556 TI - Malignant mixed mullerian tumor of the ovary. Report of a case studied by immunohistochemistry. AB - A case of malignant mixed mullerian tumor of the ovary in a 57-year-old woman is reported along with the results of an immunohistochemical study. The tumor, measuring 16 x 10 x 9 cm, was composed predominantly of adenocarcinoma with a smaller amount of anaplastic carcinoma as an epithelial component and chondrosarcoma, liposarcoma, fibrosarcoma and rhabdomyoblasts as mesenchymal elements. Immunohistochemistry using paraffin sections demonstrated cytokeratin (CK) and epithelial membrane antigen (EMA), generally regarded as epithelial markers, not only in the epithelial component but also in chondrosarcoma cells. Vimentin and desmin, generally regarded as mesenchymal markers, were exhibited partly in carcinoma cells as well as in mesenchymal elements. Positive staining for S-100 protein was obtained not only in chondrosarcoma and liposarcoma cells, but also partly in adenocarcinoma cells. This intricate immunohistochemical picture reflected the histologic findings. It is noteworthy that both carcinoma cells and chondrosarcoma cells demonstrated simultaneous expression of CK, EMA, vimentin, desmin and S-100 protein. This somewhat unusual antigen expression by tumor cells may indicate a change in the nature of tumor cells due to microenvironmental factors. PMID- 1706557 TI - Acidic fibroblast growth factor (aFGF) is present in the fluid of brain tumour pseudocysts. AB - Fluid samples from brain tumour pseudocysts were examined for the presence of Fibroblast Growth Factor (FGF). Fluids were collected from 6 patients with differentiated low grade gliomas (group A), 3 anaplastic gliomas (group B) and 3 metastases (group C). For FGF assays pooled fluids from group A, B, and C were subjected to affinity chromatography on a Heparin-Sepharose column. Each pool contained endothelial cell mitogenic activity which eluted in the 1.2 M NaCl fraction and to a lesser degree in the 0.6 M fraction of Heparin-Sepharose high affinity chromatography. Mitogenic activity in the 1.2 M NaCl fraction of Heparin Sepharose chromatography suggests the presence of acidic FGF (aFGF). PMID- 1706559 TI - Improved methods for analysis and biological characterization of fiber. AB - Dietary fibers are not uniform, chemically or in their nutritive and biological properties, the only common ground being their resistance to mammalian digestive enzymes. The AOAC method for total fiber is subject to inferences from ash, protein, tannins and resistant starches. These interferences can be reduced by urea enzymatic dialysis. The measurement of soluble and insoluble fiber is nutritionally relevant, since physical properties greatly modify dietary effects of fiber. Insoluble fiber is conveniently measured as neutral-detergent fiber. This procedure has been improved by reducing the starch interference and the time of analysis. Physical and biological properties of dietary fiber can be measured by using relevant procedures for hydration capacity, metal ion exchange capacity and rate of fermentation. The lignin and tannin content modify the characteristics of dietary fiber. PMID- 1706558 TI - Epithelial cysts in the central nervous system, characteristic expression of cytokeratins in an immunohistochemical study. AB - Nineteen epithelial cysts in the central nervous system including six colloid cysts of the third ventricle, seven Rathke's cleft cysts in the sella, two enterogenous cysts in the posterior fossa, two epithelial cysts in the spinal canal and two neuroectodermal cysts in the cerebrum were examined immunohistochemically for expression of intermediate filament proteins-simple type, stratified type and skin type cytokeratins and GFAP. Colloid cysts of the third ventricle, Rathke's cleft cysts in the sella and epithelial cysts in the spinal canal expressed complex type cytokeratins while enterogenous cysts and neuro-ectodermal cysts showed only simple type cytokeratins. In addition, Rathke's cleft cysts expressed GFAP in occasional lining cells. The characteristic composition and distribution of cytokeratins in various kinds of epithelial cysts in the central nervous system are demonstrated and discussed with regard to their origins. PMID- 1706560 TI - Analysis of foodstuffs for dietary fiber by the urea enzymatic dialysis method. AB - Total dietary fiber (TDF) values for cereal grains, fruits, vegetables, processed foods, and purified or semi-purified dietary fiber products were determined by a new method using 8M urea and enzymes (urea enzymatic dialysis, UED, method). The results are compared with the official AOAC procedure. Soluble and insoluble dietary fiber were determined for several of these foodstuffs and compared with the NDF values. Crude protein and ash contamination was usually lower with the UED method compared with the AOAC method, particularly for samples that formed gels during ethanol precipitation. Urea and the heat stable amylase were effective in removing starch even at relatively low temperatures of the assay (50 degrees C). The new assay is relatively economical in use of equipment, enzymes, and reagents. Studies are currently in progress to minimize the assay time for the UED method while further improving its flexibility and robustness. The results of the studies will be discussed. PMID- 1706561 TI - Analytical electron microscopy and monoclonal antibody techniques applied to the human inner ear. PMID- 1706562 TI - [Histamine release and cardiovascular reactions to implantation of bone cement during total hip replacement]. AB - Cardiovascular reactions to acrylic bone cement in patients with total hip replacement are a common complication. Hypotension and arrhythmias are the most frequently observed symptoms. Elderly patients with fractures of the femoral neck constitute a special risk group. In some patients these reactions can be fatal. The mechanisms suggested to explain these reactions are embolism of air, polymer or fat, reaction to the heat, and toxic or vasodilating effects of the acrylic monomer. In a pilot study and in a case report a significant rise of the plasma histamine was described following cementation of the femur. We therefore performed an investigation to find whether application of bone cement to the femur caused histamine release in elective hip surgery, and, independently of this, also investigated whether premedication with H1- + H2-antagonists had any effect on the cardiovascular reactions due to bone cement implantation into the femoral shaft in elderly patients with hip fracture. METHODS. Part I. In all, 40 patients, scheduled for elective surgical hip replacement were anesthetized by general or epidural anesthesia. Patients were continuously monitored by ECG. Blood pressure was recorded noninvasively at 2-min intervals during the study. Blood samples for the determination of the plasma histamine were taken immediately before implantation of the bone cement into the femur, and 2, 5, and 10 min after. Part II. A further group of 20 patients aged greater than or equal to 70 years with fractures of the femoral neck and in whom total hip replacement was planned were included in the study. In this group, 10 patients were randomly assigned to receive 4 mg clemastine + 400 mg cimetidine i.v. about 15 min before implantation of the bone cement. All patients were operated on under general anesthesia. ECG was monitored continuously and blood pressure was monitored at 2 min intervals during the study. Changes of the blood pressure and heart rate and therapeutic interventions following the implantation of the bone cement were documented. RESULTS. Part I. In 11 of the 40 patients (27.5%) plasma histamine increased by greater than 0.5 ng/ml (9 patients greater than 1 ng/ml). In comparable groups (patients with a control systolic blood pressure less than or equal to 130 mmHg) the histamine responders showed a significantly greater reduction in systolic blood pressure (-5.7 +/- 14.7 vs -17.7 +/- 8.6 mmHg). Part II. In the control group we observed a significantly greater fall in systolic blood pressure than in premedicated patients (41.5 +/- 25.4 vs 11.0 +/- 13.4 mmHg). In the control group 7 of the 10 patients required therapeutic interventions, while in the premedicated group only one therapeutic intervention was necessary (P less than 0.05). DISCUSSION. We have demonstrated that the implantation of acrylic bone cement into the femur may increase plasma histamine by greater than 1 ng/ml. In elderly patients with preexisting cardiac diseases or/and hypovolemia even moderate histamine release can cause serious, sometimes potentially fatal, cardiovascular complications. In this special risk group with hip fractures we found a significant reduction in the frequency of cardiovascular reactions to bone cement implantation in patients premedicated with H1 + H2 antagonists. Because we also observed significant falls in systolic blood pressure in premedicated patients, we assume that the pathogenesis of cardiovascular reactions to bone cement implantation is multifactorial. It may be that potentially lethal complications only occur if two or more of the predisposing factors (hypovolemia, myocardial insufficiency, arrhythmia, embolism, histamine release) are present simultaneously. Pre- and intraoperative measures therefore have to be instituted to eliminate all possible risk factors. PMID- 1706563 TI - A multipurpose solid-phase method for protein determination with Coomassie brilliant blue G-250. AB - The multipurpose method for protein determination (MMPD) is based in the Coomassie brilliant blue G-250 binding to immobilized and washed proteins in paper filter disks, and the A600 measurement of the eluted protein-dye complexes. The analysis requires 5-microliters samples, put in 7-mm paper filter disks, which can be stored for up to 2 weeks, without sensible changes in their protein content. The A600 is a logarithmic function of the log of bovine serum albumin quantity, between 2.5 and 250 micrograms with two linear segments used as standard curves: one between 2.5 to 20 micrograms and the other between 20 and 80 micrograms. Results with the MMPD were quantitatively comparable with those obtained with the method of Lowry et al., and those reported by other workers, assaying the following material: (i) bovine serum albumin, in doubly distilled water or in presence of several substances that interfere with the current methods; and (ii) total homogenates and their respective trichloroacetic acid insoluble extracts, which were obtained from several phenol-rich vegetal specimens and complex animal products. The MMPD is proposed as an alternative for protein determination for its versatility and reliability, in both vegetal or animal products, especially when the analysis with traditional methods is made difficult by the presence of natural or added interfering substances and the sample volume is too small to discard them, or when the material must be stored for relatively long periods of time, prior to its processing. PMID- 1706564 TI - A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening. AB - We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times. PMID- 1706565 TI - DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. AB - A simple assay is described using bisbenzimidazole (Hoechst 33258) to determine cellular DNA content in 96-well tissue cultures plates. At time points of interest, the plates are emptied of media and stored frozen. When the assay is to be performed, cultures are briefly incubated in distilled water and frozen again. This process lyses the cells and allows rapid and thorough mixing of the fluorochrome and cellular DNA. Freezing permits convenient storage of cultures until the time of assay. Experiments can be batched, further reducing processing time and giving better intra- and interexperimental standardization. The assay generates a linear standard curve for DNA fluorescence versus cell number, the range of which is appropriate for microculture wells. This enables the rapid and accurate measurement of cell number involving minimal processing time, making this assay well suited for cell proliferation studies. PMID- 1706566 TI - Insulin-like growth factor binding protein measurement: sodium dodecyl sulfate stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting. AB - The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF. PMID- 1706567 TI - Perivascular innervation of the cerebral arteries in spontaneously hypertensive rats--an immunohistochemical study. AB - Perivascular innervation in cerebral arteries of spontaneously hypertensive rats and of normotensive Wistar-Kyoto rats was studied. Adrenergic nerve fibers and neuropeptide Y-containing nerve fibers, indicative of vasoconstrictor nerves, were denser in all cerebral arteries of spontaneously hypertensive rats than those of Wistar-Kyoto rats. In contrast, cholinergic nerve fibers and vasoactive intestinal polypeptide, substance P-containing nerve fibers, indicative of vasodilator nerves, remained unchanged in all cerebral arteries of spontaneously hypertensive rats, as compared with findings in the Wistar-Kyoto rats. Thus, not only adrenergic nerve fibers but also neuropeptide Y-containing nerve fibers may play an important role in preventing the disruption of the blood-brain barrier and the development of hypertensive encephalopathy in spontaneously hypertensive rats. PMID- 1706568 TI - Adverse cutaneous reactions due to macrolides. AB - Macrolides, which are widely prescribed and seldom produce hypersensitivity reactions, are considered to be safe drugs. We present five patients with generalized skin reactions due to erythromycin and/or spiramycin, proved by oral challenge tests. One patient showed reactions to both erythromycin and spiramycin. All skin prick and patch tests and histamine release tests with both macrolides were negative. PMID- 1706569 TI - Structure-function studies of HIV reverse transcriptase. AB - The retroviral RT is properly under intensive study as the major target of antiviral therapy. The enzyme exhibits a number of features that make it an attractive target: it is crucial for viral replication; its RNA-dependent DNA polymerase activity is probably unique to viral replication, or if not unique, is generally unimportant in host cell function; its activities are readily monitored; and powerful lead compounds in the form of nucleotide analogues are already in hand. Our laboratory has been involved in studies to elucidate the structure and function of the HIV-1 RT and to develop a formal genetics of the enzyme. Working with constructs expressing RT in bacteria, we been able to use in vitro mutagenesis to localize functions on the molecule; by coupling mutagenesis with high-throughput screening of colonies, we have been able to isolate mutants with specific, rare, phenotypes. We believe that extensions of these efforts will help us to understand the functions of the protein and, coupled to a detailed three-dimensional structure, should facilitate the development of new and better inhibitors. PMID- 1706570 TI - DNA polymerases versus HIV reverse transcriptase in AIDS therapy. PMID- 1706571 TI - Antiviral drug resistance. AB - Antiviral drug resistance is an area of increasing importance in acquired immunodeficiency syndrome (AIDS), not only in terms of the human immunodeficiency virus (HIV), but also opportunistic pathogens such as herpes simplex virus (HSV) and human cytomegalovirus (CMV). Studies of drug resistance in these and other viruses have proven valuable both for the molecular dissection of drug mechanisms and drug targets and for predicting the features of drug resistance in clinical settings: Drug-resistance mutations arise readily, due in part to a lack of fidelity of viral polymerase. Both biochemical and genetic analyses are generally required to understand the basis of drug resistance. Novel drug targets, such as a CMV gene product that contributes to ganciclovir phosphorylation, can be identified by analysis of such mutations. Regions of drug targets that are involved in drug recognition can be identified by sequencing of drug-resistance mutations. Analysis of drug-resistant viruses, obtained either in the laboratory or from patients, reveals a broad spectrum of alterations and points to the importance of heterogeneous populations of virus in resistance and pathogenesis. PMID- 1706572 TI - Correlation of molecular conformation and activity of reverse transcriptase inhibitors. PMID- 1706573 TI - Characterization of reverse transcriptase activity and susceptibility to other nucleosides of AZT-resistant variants of HIV-1. Results from the Canadian AZT Multicentre Study. PMID- 1706574 TI - Antiretroviral activity, biochemistry, and pharmacokinetics of 3'-azido-2',3' dideoxy-5-methylcytidine. AB - 3'-Azido-2',3'-dideoxy-5-methylcytidine (CS-92, AzddMeC) is an antiviral nucleoside analogue structurally related to 3'-azido-3'-deoxythymidine (AZT). CS 92 is a potent and selective inhibitor of HIV-1 reverse transcriptase and HIV-1 replication in human lymphocytes and macrophages. The EC50 for CS-92 in HIV-1 infected human PBM cells was 0.09 microM. In HIV-1-infected human macrophages, the EC50 was 0.006 microM. This compound was also effective against human immunodeficiency virus type 2 in lymphocytes. The replication of Friend murine virus was only weakly inhibited, and no effect was observed against herpes simplex virus type 1 and type 2 and coxsackievirus B4. CS-92 was not toxic to PBM or Vero cells when tested up to 200 microM and was, furthermore, at least 40 times less toxic to granulocyte-macrophage and erythroid precursor cells in vitro than was AZT. The interaction of the 5'-triphosphate of CS-92 with HIV-1 reverse transcriptase indicated competitive inhibition (the inhibition constant, Kis, was 0.0093 microM) with a 30-fold greater affinity for CS-92-TP than for ddCTP. CS-92 TP inhibited HIV-1 reverse transcriptase by 50% at a concentration 6,000-fold lower than that which was required for a similar inhibition of DNA polymerase alpha. Pharmacokinetic studies showed that CS-92 was not deaminated to AZT in rats, but this compound was found to have a half-life of 2.7 hours. In rhesus monkeys, however, a compound with a retention time and ultraviolet spectra characteristics similar to AZT was detected. The mean half-life in rhesus monkeys for CS-92 was 1.52 and 1.74 h after intravenous and oral administration, respectively, and the oral bioavailability was about 21 percent. Additional preclinical studies with CS-92 will determine the ultimate utility of this antiviral agent for the treatment of HIV-1 infections. PMID- 1706575 TI - Anabolism and mechanism of action of Ro24-5098, an isomer of 2',3' dideoxyadenosine (ddA) with anti-HIV activity. PMID- 1706576 TI - Rational targets for design and synthesis of anti-HIV agents. PMID- 1706577 TI - Antimyristoylation of GAG proteins in human T-cell lymphotropic and human immunodeficiency viruses by N-myristoyl glycinal diethylacetal. PMID- 1706578 TI - Antibody-targeted photolysis. Bacteriocidal effects of Sn (IV) chlorin e6-dextran monoclonal antibody conjugates. AB - Antibody-targeted photolysis is a technique for damaging or killing cells using light and an antibody-bound photosensitizer. In the present study, immunoconjugates were constructed to selectively kill Pseudomonas aeruginosa bacteria using tin (IV) chlorin e6 which was linked to dextran and then bound to the carbohydrate moiety of a monoclonal antibody specific for Pseudomonas aeruginosa Fisher type I polysaccharide antigen. Killing of Pseudomonas during mid-log phase growth was shown to be dependent upon light dose with complete bacterial cell killing observed at an irradiation dose of 80 J/cm2. Individual components of the immunoconjugates (e.g., antibody or chlorin alone) showed no bacterial cytotoxicity and immunoconjugates constructed with nonbinding antibodies were also ineffective as cytotoxic agents. These studies demonstrate that killing of gram negative bacteria using photoradiation is feasible and suggest that this methodology may be applicable in treatment of infections in man. PMID- 1706579 TI - Electronic control of iontophoretic drug delivery. PMID- 1706580 TI - Retrobulbar alcohol injection in blind painful eyes. AB - We studied 39 blind painful eyes in 39 patients who were treated with retrobulbar injection of absolute (96%) alcohol for their severe ocular pain at the King Khaled Eye Specialist Hospital from January 1984 to January 1987. There were 21 (54%) male and 18 (46%) female patients; all were followed for at least three months. The protracted ocular pain was mainly due to: end-stage (absolute) glaucoma in 31 (80%) eyes, uveitis or endophthalmitis in four (10%) eyes, or corneal ulcer in two (5%) eyes. One eye had painful phthisis bulbi, and one eye had infraorbital neuralgia. The complications encountered were transient and included blepharoptosis in eight (21%) eyes, external ophthalmoplegia, and corneal epithelial defect. The effective time of the injection to relieve pain ranged from two weeks to two years (mean, 29 weeks). The authors believe that there is still a place for retrobulbar alcohol injection for blind painful eyes when enucleation or evisceration is not possible. PMID- 1706581 TI - Expression of intermediate filament proteins in the adult human cochlea. AB - The immunohistochemical localization of intermediate filament proteins was studied in frozen sections of chemically fixed, nondecalcified adult human cochleas. Cytokeratins were found in all epithelial cells lining the cochlear duct (including most supporting cells of the organ of Corti) but were absent in the hair cells. Neurofilament proteins were present in the nerve endings at the hair cells, in the neural bundles, and in the ganglion cells. Vimentin staining occurred in most of the supporting structures and was roughly complementary to the regions showing cytokeratin staining and neurofilament staining. However, the region of the spiral prominence and outer sulcus, as well as the pillar cells and Deiters' cells in the organ of Corti, showed coexpression of vimentin and cytokeratins. No definite immunostaining was observed with antibodies to desmin and glial fibrillary acidic protein. PMID- 1706582 TI - Pathophysiology of Streptococcus pneumoniae otitis media: kinetics of the middle ear biochemical and cytologic host responses. AB - Streptococcus pneumoniae is an important bacterial pathogen in the pathophysiology of otitis media. To elucidate the inflammatory responses that occur during pneumococcal otitis media, the kinetics of the biochemical and cytologic middle ear responses to heat-killed encapsulated and nonencapsulated pneumococci were studied in the chinchilla model. Inoculation of the middle ear cavity with at least 10(6) S pneumoniae cells induced an early, brief vascular response with leakage of small (albumin) followed by larger (alpha 2 macroglobulin) proteins, followed by sustained influx of acute inflammatory cells and lysozyme. The threshold for a sustained lysozyme response was 1,000 times lower for nonencapsulated than for encapsulated pneumococci. These results indicate that nonviable S pneumoniae organisms with an intact envelope initiate the middle ear inflammatory response. Therefore, interventions that enhance the clearance of pneumococcal cells from the middle ear may reduce the inflammatory response and prevent chronic middle ear inflammation. PMID- 1706583 TI - Intimal staining for visibility enhancement during microanastomoses. AB - Absolute perfection in microvascular surgery continues to be thwarted by technical inadequacies in construction of the microanastomosis. Occasionally this is a consequence of limited visualization or misidentification of the intima proper predisposing to thrombosis. At least in rats, we have demonstrated that intimal staining with clinically inert vital dyes has enhanced gross appreciation of the exact intimal location. This maneuver has simplified suture placement without evidence of any associated increased risk of anastomotic failure. PMID- 1706584 TI - The fasciovascular pedicle for revascularization of other tissues. AB - A fasciovascular pedicle based on the epigastric vessels was developed in a rat model to determine if it could be used as a "universal carrier" to revascularize a new composite flap. The effects of time course, carrier size, and flap ischemia on the revascularization process were studied. A 2.5 x 4-cm or 1 x 4-cm fascial patch pedicled on the vessels was transferred under bipedicled 2.5 x 4-, 6-, or 8 cm abdominal panniculocutaneous flaps. At different time intervals, the flap was raised as an island flap connected only by it vascular bundle and then sutured back in place. The skin perfusion by dermofluorometry and flap survival were both markedly increased on day 5 (p less than 0.001). The wide carrier had a 93% survival area, whereas the narrow carrier had only 71%. The wide carrier induced relatively faster and better revascularization (p less than 0.05). Moderate ischemia promoted revascularization (p less than 0.01). An india ink injection study and histological examination provided visual evidence of revascularization. This fasciovascular pedicle is a promising model for prefabrication of complex new composite flaps and for studying the process of revascularization between the layers. Based on these findings and further investigations, a thin, prefabricated abdominal free flap was successfully transferred for facial resurfacing in humans. PMID- 1706585 TI - Quantitative enzyme-histochemical analysis of tryptase- and chymase-containing mast cells in psoriatic skin. AB - Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymase- and tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72-73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activity in the upper dermis may lead to an imbalance in the biochemical regulatory systems. PMID- 1706586 TI - [Transrectal echography of the prostate: current evaluation]. AB - Our results with transrectal ultrasonography of the prostate are updated herein. Since the specifically urologic ultrasound (Aloka SSD 520) equipped with a sectorial transrectal probe of 5 MHz, became available in our service in 1984, we have performed over 1,700 transrectal ultrasound procedures. From the outset, we established several lines of investigation whose partial results have been published over these past years. Our main interest has been and continues to be that of describing an unequivocal echo pattern that would define prostate cancer in any stage, particularly in its early stages. To date, no finding has proved to be pathognomonic. However, we believe that we have encountered some aspects that have been poorly described to date that allow us to determine the current role of transrectal US in the urologic setting. We support the concept of integral urologic ultrasonography, one that encompasses all of our activities. The present article is a synthesis of a major part of our investigative effort and includes our statistical data and current points of view. Our own experience has taught us that things may turn out to be different from what they initially appear to be. Thus, we cannot rule out that our views will not change in the future. We have therefore attempted not to be rigid with respect to our claims. We must add, however, that except for numerical changes and variations in the degree of significance of specific findings, no major modifications have had to be made during this period. We have divided the study into various parts. Each one reviews a specific pathology (cancer, prostatitis, adenoma...), some procedures (biopsy, puncture, sonometry), and a comparative study between classical and transrectal US (digital rectal examination and US). In all of the foregoing, we have presented our results and some considerations in the discussion. Finally, our current views regarding transrectal US are presented succinctly in the conclusions. PMID- 1706587 TI - [Comparative study of psychometric results of 2 french speaking populations with hypothyroidism detected at birth]. AB - The authors compare the data from the psychological assessment of a cohort of 191 French speaking hypothyroid children detected by neonatal screening: 99 were followed in Quebec and 92 in Lille, according to two independent programs. Three age levels were selected: 12-18 months, 5 and 7 years. Data were similar and, in both studies, results of the later ages were slightly lower than the norms of the tests or results obtained in control groups. In both populations correlations were found between some results and two factors related to hypothyroidism: delayed bone surface development and thyroxinemia at the time of diagnosis. On the other hand, etiology and time of treatment had no repercussions on any group results. PMID- 1706588 TI - [Retinitis in an infant infected with HIV]. AB - A 7 month-old HIV-infected infant of African origin was admitted to the hospital. A routinely performed fundoscopy showed the presence of an unilateral left retinitis. Urine culture was positive for cytomegalovirus (CMV). Treatment with AZT (3.5 mg/kg/6 hr) was started concomitantly with monthly 350 mg/kg gammaglobulin intravenous administration. The retinitis resolved after 3 weeks and no recurrence was observed after 4 months of follow-up. The difficulties in establishing a diagnosis and in evaluating a response to treatment are briefly discussed. PMID- 1706589 TI - Molecular and functional analysis of the virus- and interferon-inducible human MxA promoter. AB - The virus- and interferon-inducible human MxA (IFI-78k) gene is a homologue of the murine influenza resistance gene Mx1. Three overlapping human cosmid clones covering most of the gene including its promoter region were isolated. Sequencing the 5' MxA cDNA derived by RT-PCR (reverse transcriptase-polymerase chain reaction) confirmed the most 5' putative transcriptional start site. The MxA promoter does not contain a TATA or CCAAT box but has three Interferon Stimulated Response Element (ISRE) motifs. Strong induction with type I interferons was demonstrated with a fragment containing only two ISREs in human L132 cells. This induced expression was not adversely affected by 2-aminopurine. However, the promoter showed constitutive expression in transiently or stably transfected murine LM cells. PMID- 1706590 TI - Generation and characterization of the human immunodeficiency virus type 1 mutants. AB - Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene. PMID- 1706591 TI - Synthetic peptides corresponding to the F protein of RSV stimulate murine B and T cells but fail to confer protection. AB - We have previously located a major neutralization site of the fusion protein of respiratory syncytial virus (RSV) in the polypeptide region extending from amino acids Ile221 to Glu232. In this report, 8 peptides corresponding to the six major hydrophilic regions of the F1 subunit were selected to analyse their immunogenic and protective capacities as well as their ability to block the high neutralization activities of 4 monoclonal antibodies (MAbs). Only 5 of the 8 peptides tested induced specific antibodies while all induced an in vitro interleukin-2 response of splenocytes from immunized mice. Peptide 3 (Ile221 Phe237) was able to elicit neutralizing antibodies, confirming our previous hypothesis concerning the location of a neutralization site. However, immunization with the latter did not induce significant reduction of virus in lungs of BALB/c mice upon challenge, probably due to an inadequate level of circulating neutralizing antibodies. Interestingly, peptides 2 (Asn216-Glu232), 3 (Ile221-Phe237), and 5 (Ser275-Ile288) blocked in vitro neutralization by four different F1 specific MAbs. A hypothesis is proposed to explain these results. PMID- 1706592 TI - Operational overlapping of cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus serotype 3. AB - VP7-specific neutralizing monoclonal antibodies (N-MAbs) to serotype 3 human rotavirus were produced to analyze serotype 3-specific and cross-reactive neutralization epitopes on VP7. On the basis of the reactivity patterns in neutralization tests with various human and animal strains, a total of 10 N-MAbs could be classified into four groups; five antibodies specific to serotype 3 were divided into two groups, and five antibodies consisted of two groups which are cross-reactive with strain 69 M (serotype 8) or strain WI61 (serotype 9). Seven N MAbs showed the same reactivity patterns to the virus strains in both neutralization tests and enzyme-linked immunosorbent assay (ELISA), while three N MAbs specific to serotype 3 in neutralization showed a cross-reactivity with the serotype 8 strain in ELISA. Neutralization-resistant mutants of serotype 3 strains P and YO were selected by the N-MAbs. Cross-neutralization tests between the mutants and the MAbs indicated the presence of two serotype-specific (S1 and S2) and three cross-reactive (C1, C2, and C3) epitope groups. S1, S2, and C3 epitope groups overlapped operationally each other, and the S1 epitope group had an overlapping with the C1 epitope group. However, C2 epitope group identified by the MAbs which neutralized serotypes 3 and 9, had no operational overlapping with any other epitope groups. PMID- 1706593 TI - Characterization of monoclonal antibodies against feline infectious peritonitis virus type II and antigenic relationship between feline, porcine, and canine coronaviruses. AB - Seven monoclonal antibodies (MAbs) with neutralizing activity against feline infectious peritonitis virus (FIPV) strain 79-1149 (type II) were prepared. When the polypeptide specificity recognized by these monoclonal antibodies (MAbs) was investigated by Western immunoblotting, all of the MAbs reacted with peplomer glycoprotein (S) of the virus. By competitive binding assay these MAbs were found to recognize at least 3 different epitopes. The reactivity of these MAbs with 6 viruses classified as FIPV type I (UCD-1, UCD-2, UCD-3, UCD-4, NW-1, and Black), feline enteric coronavirus (FECV) type II strain 79-1683, canine coronavirus (CCV) strain 1-71, and transmissible gastroenteritis virus (TGEV) strains TO-163 and SH was examined by neutralization tests. All MAbs neutralized FECV strain 79 1683, CCV strain 1-71, and TGEV strains TO-163 and SH, while they did not neutralize the 6 FIPV type I viruses. Moreover, the MAb against TGEV strain TO 163, which has strong neutralizing activity against 7 TGEV viruses, neutralized CCV strain 1-71, FECV strain 79-1683, and FIPv strain 79-1146, but did not neutralize the 6 FIPV type I viruses. These results demonstrated that there are at least 3 epitopes involved in the neutralization of FIPV type II strain 79 1146, and that these epitopes are not present in FIPV type I viruses but are present in FECV strain 79-1683 which does not induce feline infectious peritonitis, TGEV strains TO-163 and SH, and CCV strain 1-71. These results suggest the presence of 2 serotypes of FIPV which can be clearly distinguished by the neutralization test using MAbs. PMID- 1706594 TI - Peripheral lymph node biopsies in children under 14 years at Christchurch Hospital 1982-88: an audit. AB - OBJECTIVE: To review peripheral lymph node biopsies in children under 14 years of age. METHOD: Retrospective audit of biopsy specimens. RESULTS: Four patients had had no histological diagnosis and no follow-up. Two patients had no histological diagnosis evident and limited follow-up. Thirteen patients were diagnosed at biopsy to have non-diagnostic reactive. PMID- 1706595 TI - Endogenous cleavage of the Arg-379-Ala-380 bond in vitronectin results in a distinct conformational change which 'buries' Ser-378, its site of phosphorylation by protein kinase A. AB - Activation of blood platelets by thrombin was previously shown to specifically release protein kinase A, which in human plasma singles out and phosphorylates one protein, identified as vitronectin. This protein is known to be involved in processes that follow platelet stimulation, specifically, in the binding of heparin (interfering with the heparin-mediated inhibition of thrombin and Factor Xa by antithrombin III), in the growth of endothelial cells and in fibrinolysis. This paper shows that phosphorylation of vitronectin by protein kinase A is stoichiometric (approx. 1 mol/mol), that it is targeted to one site (Ser-378) at the C-terminal edge of the heparin-binding domain, and that it distinguishes between the two physiologically occurring forms of vitronectin: the one-chain (75 kDa) form, and the nicked two-chain (65 + 10 kDa) form, held together by an interchain disulphide bridge. Protein kinase A phosphorylates the one-chain form but not the two-chain form, although Ser-378 and the complete recognition sequence of the kinase are still present in the clipped 65 kDa chain. Cleavage of the Arg-379-Ala-380 bond results therefore in a conformationally distinct form of vitronectin in which Ser-378 is 'buried'. This is demonstrated by our finding that Ser-378 is present in the 65 kDa chain of clipped vitronectin but inaccessible to phosphorylation at physiological pH. Upon binding heparin, the phosphorylation site becomes exposed and able to undergo a stoichiometric phosphorylation at physiological pH. PMID- 1706596 TI - Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites. AB - The precise localization of the epitopes for six monoclonal antibodies specific for the N-terminal region of human platelet glycoprotein IIIa (GPIIIa) was determined. The epitope for P37, a monoclonal antibody that inhibits platelet aggregation, was found at GPIIIa 101-109, flanked by the epitopes for P23-3 (GPIIIa 16-28), P23-4 (GPIIIa 83-91), P23-5 (GPIIIa 67-73), P23-7 (GPIIIa 114 122) and P40 (GPIIIa 262-302), and very close to the early chymotryptic cleavage site of GPIIIa in whole platelets (Phe-100). When the amino acid sequence of GPIIIa was searched for peptide sequences hydropathically complementary to the fibrinogen gamma-chain C-terminal (gamma 400-411) and A alpha-chain RGD containing peptides, none was found for the gamma 400-411, two (GPIIIa 128-132 and 380-384) were found complementary to fibrinogen A alpha 571-575 and two (GPIIIa 109-113 and 129-133) were found for A alpha 94-99. Two of these putative fibrinogen-binding sites overlap with each other, and a third one overlaps with the epitope for P37. These findings reinforce the earlier suggestion that the N terminal region of GPIIIa is involved in fibrinogen binding, and suggest the existence in GPIIIa of either multiple or alternative RGD-binding sites or one RGD-binding domain with several moieties. Finally, early chymotryptic cleavage of GPIIIa in whole platelets liberates to the soluble fraction the peptide stretch Ser-101-Tyr-348, which carries the epitope for P37 and the putative binding sites for fibrinogen. The rest of the molecule, together with the GPIIb-resistant moiety, remains membrane-bound. This leads us to propose that the fibrinogen binding domain of GPIIIa is not involved in the binding to GPIIb to form the Ca2(+)-dependent GPIIb-GPIIIa complex. PMID- 1706597 TI - Characterization and organization of the genes encoding the A-, B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q. AB - A partial cDNA clone for the A-chain of human complement subcomponent C1q was isolated from a monocyte library. Use of the A-chain cDNA clone, and a previously characterized B-chain cDNA clone [Reid (1985) Biochem. J. 231, 729-735] allowed the isolation of overlapping cosmid clones that were shown to contain the genes encoding the A-, B- and C-chains of human C1q. The three genes were found to be aligned, 5'----3', in the same orientation, in the order A-C-B on a 24 kb stretch of DNA on chromosome 1p. The A-, B- and C-chain genes are approx. 2.5, 2.6 and 3.2 kb long respectively, and each contains one intron, located within a codon for a glycine residue found half-way along the collagen-like region present in each chain. These glycine residues are located just before the point where the triple-helical portions of the C1q molecule appear to bend when viewed in the electron microscope. Southern-blot analyses indicated that there is only one gene per chain, and preliminary examination of genomic DNA from several C1q-deficient patients showed no evidence for major deletions or insertions within the A-, B- or C-chain genes. The DNA sequence of the coding region of the C-chain gene allows the completion of the entire derived amino acid sequence for the human C1q molecule. The globular, C-terminal, regions of the chains of C1q show a strong similarity in amino acid sequence to the non-collagen-like, C-terminal, regions of the type VIII and type X collagens, indicating structural and evolutionary relationships between these three molecules. PMID- 1706599 TI - Role of ultrasound and informed consent in the evaluation of elevated maternal serum alpha-fetoprotein. AB - Informed consent of medical procedures should include a discussion of both the risk of the procedure and the probability of the malady in question. Many centers in the United States currently recommend amniocentesis for all women with elevated levels of maternal serum alpha-fetoprotein (MSAFP) unexplained by targeted ultrasound examination. Prospective clinical data from our unit support the use of an algorithm that provides for a 90% reduction of the predicted risk of neural tube defect from MSAFP alone. The predicted risk is revised only if the ultrasound shows normal fetal cranial size and shape, normal ventricular size, normal posterior fossa anatomy, and normal spinal anatomy. Preliminary results supported this approach with no reduction in sensitivity, while substantially reducing the need for invasive testing. The additional experience reported here of 20,211 patients resulted in 451 ultrasound examinations for an elevated MSAFP, but only 54 amniocenteses. During this period, nine open neural tube defects were detected among patients with elevated MSAFP using ultrasound; none was missed. All fetuses with defects had ultrasound findings of cranial and intracranial changes first reported by Campbell. These data support the premise that, prior to amniocentesis, informed consent should include discussion of the ultrasound evaluation. PMID- 1706598 TI - Further evidence that cyclosporin A protects mitochondria from calcium overload by inhibiting a matrix peptidyl-prolyl cis-trans isomerase. Implications for the immunosuppressive and toxic effects of cyclosporin. AB - The Ki values of cyclosporins A, G and H for the peptidyl-prolyl cis-trans isomerase (PPIase) of liver and heart mitochondria are about 2, 20 and 500 nM respectively. This parallels their profile as inhibitors of non-specific pore opening of mitochondria induced by supraphysiological Ca2+ concentrations. The novel immunosuppressant FK-506 gave little inhibition of either process at 5 microM. These data support our previous hypothesis [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] that pore opening involves an interaction between matrix PPIase and the adenine nucleotide translocase. It is suggested that this model may help to clarify the mechanism of action of cyclosporin as an immunosuppressant and its toxic effects on the liver and kidney following prolonged therapy. PMID- 1706600 TI - Transplacentally-transported 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) affects the catecholamine and indoleamine levels in the fetal mouse brain. AB - The effects of a dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on the amounts of dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT) and 5 hydroxyindoleacetic acid (5-HIAA) were examined in the whole brains of fetal mice and maternal mice after its administration to pregnant mice. DA and DOPAC concentrations were decreased significantly in both the fetal and maternal brains. At 3 hr after injection, reduction of the DOPAC concentration was more marked than that of DA in both the fetal and maternal brains. Increase of 5-HT concentration was observed until 12 hr after injection in the fetal brains and 6 hr in the maternal brains. These results indicate that 1-methyl-4-phenyl pyridinium ion (MPP+) and MPTP affect the levels of catechol- and indoleamines in the brain of premature stage as well as in the mature brain. PMID- 1706601 TI - Discrimination of cell types in primary transitional cell carcinoma by monoclonal anti-cytokeratin antibodies. AB - The morphologic discrimination between low and high grade malignant tumor cells arising in the urinary bladder is a major diagnostic problem in cytopathology. Using immunochemical peroxidase staining of cytokeratins (CKs) of human bladder exfoliative cytology specimens, we have been able to discriminate between transitional cell carcinoma cells, atypical cells and normal bladder cells. Commercially available monoclonal antibodies used in this study were: anti-CK 13 (Sigma K8.12), anti-CK 5, 7 and 8 (Sigma K8.13), anti-CK 19 (Sigma K4.62), and anti-CK 18 (Transformation Res. 1091). When normal bladder cells are stained with these antibodies, CK 18 is specific for superficial cells; CK 13 is specific for the basal type cells and CKs 5, 7, 8 and 19 are expressed by all urothelial cell types. Four cases diagnosed by cytopathological criteria as 'atypical' and 7 diagnosed as 'positive' (various grades) were used in this study. Cytologic diagnosis was confirmed by histopathology in 7 cases. Tissue was not available for histopathology in 4 cases. Malignant cells with a 'basal' or 'immature' phenotype preferentially stained with CK 13 and were associated with increased metastatic or malignant potential. Patients with higher grade tumors had more cells positive for CK 13 than did patients with atypical or lower grade malignancies. Patients with well-differentiated, low grade tumors predominantly shed cells that expressed CK 18 and CK 19. High grade, invasive bladder lesions were characterized by many cells expressing CK 13, and fewer cells expressing CK 19. The combination of diagnosis by morphologic criteria on Papanicolaou-stained specimens with immunochemical characterization of the same cells for CKs facilitate an accurate diagnosis of bladder lesions. PMID- 1706602 TI - Light microscopic, ultrastructural and immunocytochemical spectrum of malignant lacrimal and salivary gland tumors, including malignant mixed tumors. AB - Ten malignant myoepithelial tumors of the salivary glands and one of lacrimal gland origin were studied by light, electron microscopy and immunocytochemistry. The light microscopic appearance of the tumors varied from primarily spindle cell neoplasms (two cases), to others with predominantly epithelial components (four cases) and mixed varieties (five cases). Therefore, they can be confused with other epithelial and mesenchymal neoplasms. The electron microscopic spectrum varied from tumors with widespread and typical myoepithelial differentiation (i.e. myofilament bundles at the cell periphery, attachment plaques and intercellular junctions) to some with diffusely distributed filaments, without associated spindle densities but with attachment plaques, and others with evidence of duct formation and containing scattered cells showing intracytoplasmic tonofilaments. Often the tumors revealed mixed ultrastructural features; the relative numbers of the different cellular components was variable. The eleven neoplasms were S-100 protein, actin and keratin positive, either focally or diffusely, with varying degrees of intensity. Ten of the eleven tumors were positive for vimentin and nine of ten tested expressed carcinoembryonic antigen. Only two of nine were focally positive for glial fibrillary acidic protein. The study emphasizes the variable light microscopic appearances of these neoplasms and their immunocytochemical and ultrastructural spectrum. Accurate determination of myoepithelial differentiation sometimes requires careful evaluation of the light, ultrastructural and immunocytochemical findings. If all three diagnostic modalities are not utilized, it is likely that some of these neoplasms will be improperly classified. PMID- 1706604 TI - [Rhythmic profiles of the late phase of acute myocardial infarction as a function of age]. AB - OBJECTIVES: To evaluate the incidence of ventricular arrhythmias in the late phase of acute myocardial infarction (AMI) and to compare it with the following clinical parameters: age, sex, AMI localization, ventricular function (Killip classes), maximal creatinokinase (CK max) and the presence of sinus tachycardia. DESIGN: Prospective study, during a period of 31 months, of a non-selected group of patients with AMI. SETTING: Coronary Care Unit (UTIC-Arsenio Cordiero). PATIENTS: Non-selected group of 153 patients with acute myocardial infarction who survived the second week of disease. MATERIAL AND METHODS: 24-hour Holter ECG performed between the 4th and the 25th day of AMI. The patients were divided into two groups according to the hourly frequency of premature ventricular beats (PVB): less than 3 per hour (PVB less than 3/h) and 3 or more per hour (PVB greater than or equal to 3/h). RESULTS: PVB greater than or equal to 3/h occurred in 36 patients (24%). There was no differences in the occurrence of ventricular arrhythmias between sex, AMI localization, AMI size evaluated by CK max, and the presence of sinus tachycardia. Patients in Killip class III had more ventricular arrhythmias (67%) than patients in Killip class I (23%) (p less than 0.005), in Killip class II (18%) (p = 0.007), and in Killip IV (0%) (p = 0.017). In patients with serious left ventricular failure (classes III + IV) the ventricular arrhythmias were not significantly higher (40%) than in patients without serious left ventricular failure (classes I + II) (22%) (chi 2 = 2.5; p less than 0.25 NS). Patients with less than 41 years old had less PVB greater than or equal to 3/h (4%) than patients between ages 41-69 (24%) (p less than 0.05), and than patients over 70 years old (47%) (p = 0.00075). CONCLUSIONS: The majority of patients (76%) showed a low risk rithmic profile (PVB less than 3/h) in the late phase of AMI. Among all parameters the age of the patients was the one best related to the occurrence of ventricular arrhythmias. Sex, AMI localization, AMI size, and the presence of sinus tachycardia were not related to the presence of PVB. A slight tendency was found in patients with heart failure to have more PVB. On the other hand the elder group carried a statistically significant risk factor for a higher occurrence of ventricular premature beats. PMID- 1706603 TI - Pathophysiological role of augmented atrial natriuretic polypeptide gene expression in DOCA-salt hypertension. Effects of atrial natriuretic polypeptide monoclonal antibody. AB - To elucidate the pathophysiological significance of endogenous atrial natriuretic polypeptide (ANP) in hypertension, we investigated the biosynthesis and secretion of ANP in deoxycorticosterone acetate (DOCA)-salt hypertensive rats and also examined the effect of passive immunization with monoclonal antibody raised against alpha-rat ANP on the development of hypertension in DOCA-salt rats. The plasma ANP level in DOCA-salt rats was significantly elevated in comparison to control rats. While the atrial ANP concentration in DOCA-salt rats was not significantly altered, the atrial level of ANPmRNA in DOCA-salt rats was two-fold higher than that in control rats. These results suggest the augmented biosynthesis of ANP in the atrium and the oversecretion of ANP from the heart in DOCA-salt rats. The levels of ANP and ANPmRNA in the ventricle of DOCA-salt rats exhibited significant increases, compared with those in control rats, suggesting the induction of ANP gene expression in the ventricle of DOCA-salt hypertensive rats under pre/after overload. Weekly intravenous administrations of monoclonal antibody with high affinity for alpha-rat ANP significantly augmented the rise in blood pressure of DOCA-salt rats. These results support the hypothesis that the augmented secretion of ANP is one of the defensive mechanisms to counteract high arterial pressure in this model of hypertension. PMID- 1706605 TI - Projections of chemically-specified neurons in the guinea-pig colon. AB - The arrangement of the enteric nerve plexuses in the colon of the guinea-pig and the distributions and projections of chemically specified neurons in this organ have been studied. Immunoreactivity for neuron specific enolase was used to examine the total population of neurons and individual subpopulations were studied using antibodies raised against calbindin, calcitonin gene-related peptide (CGRP), leu-enkephalin, gastrin releasing peptide (GRP), galanin, gamma aminobutyric acid, neurokinin A, neuropeptide Y (NPY), somatostatin, substance P, tyrosine hydroxylase and vasoactive intestinal peptide (VIP). Neuronal pathways within the colon were lesioned using myotomy and myectomy operations and extrinsic pathways running between the inferior mesenteric ganglia and the colon were also severed. Each of the antibodies revealed nerve cells and nerve fibres or only nerve fibres within the wall of the colon. VIP, galanin and GRP were in anally projecting pathways in the myenteric plexus, as they are in other species. In contrast, there are differences in the projection directions of enkephalin, substance P, NPY and somatostatin nerve fibres between regions and species. Surprisingly, somatostatin and NPY fibres have opposite projections in the small intestine and colon of the guinea-pig. The majority of nerve fibres that innervate the circular muscle, including fibres with immunoreactivity for VIP, enkephalin, substance P, NPY, galanin and GRP come from the myenteric ganglia. The mucosa is innervated by fibres from both the myenteric and submucous ganglia. The present results suggest that the guinea-pig distal colon is a suitable place in which to determine relations between structure, neurochemistry and functions of enteric neural circuits. PMID- 1706606 TI - SIV, STLV-I and type D retrovirus antibodies in captive rhesus macaques and immunoblot reactivity to SIV p27 in human and rhesus monkey sera. AB - The prevalence of simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus type 1 (STLV-I), and type D retrovirus (SRV-D) antibodies was determined for 1229 rhesus monkeys (Macaca mulatta) from two research colonies. Serum samples were tested by using enzyme-linked immunosorbent assay (ELISA), immunoblot (IB), and radioimmunoprecipitation assay (RIPA). Seropositive results for the three retroviruses tested were 0 for SIV, 270 (22%) for STLV-I, and 103 (8.4%) for type D retrovirus. Of the rhesus monkey sera, 61 (5.0%) were reactive to SIV gag p27 only, when tested by IB, but were negative when further tested by RIPA. Virus isolation was attempted from cultured peripheral blood mononuclear cells of 35 monkeys whose sera contained only p27 reactivity and none were positive by reverse transcriptase and core antigen assays to detect SIV. No overt clinical signs of immunodeficiency disease or unexplained deaths were evident in either monkey colony. Additionally, 63 of 165 (38%) human sera from various groups (primate center workers, normal donors, health care workers) had weak to moderate IB reactivity only to SIV p27, but 31 of 31 sera tested were negative by RIPA. These sera remained reactive to SIV p27 following absorption with an uninfected cell lysate, after blocking IB strips with various blocking solutions and were reactive to different SIV antigen preparations while remaining negative to human immunodeficiency virus type 1 (HIV-1) by IB and negative to HIV-2 by ELISA. These data underscore the need to adopt criteria for a positive SIV serologic test requiring reactivity against more than one viral gene product. These results also illustrate a potential problem in the testing of human sera for antibodies against simian retroviruses and demonstrate the need for caution in the interpretation of immunoblot results. PMID- 1706607 TI - Characterization of serum antibody responses to recombinant HIV-1 gp160 vaccine by enzyme immunoassay. NIAID AIDS Vaccine Clinical Trials Network. AB - An enzyme immunoassay (EIA) was developed to measure serum antibody responses of healthy adult volunteers vaccinated with 40 or 80 micrograms of human immunodeficiency virus type 1 (HIV-1) recombinant gp160 (rgp160) vaccine at 0, 1, 6, and 18 months. This assay, which used purified rgp160 as antigen, was compared with the Biotech/Du Pont HIV-1 Western blot and the Abbott HIV-1 EIA. Of 33 volunteers who received three doses of rgp160 vaccine, seroresponses were detected in 91% by rgp160 EIA, 97% by Western blot, and 30% by HIV-1 EIA. The level of IgG rgp160 EIA antibody (mainly IgG1) peaked after the third immunization; 64% of 33 vaccinees still had detectable antibody by 12 months. The fourth immunization induced anamnestic IgG EIA antibody in 23 of 24 vaccinees, with titers ranging from 1:200 to 1:25,600. Neutralizing antibody was not detected in postvaccination sera by microtiter syncytium formation inhibition assay. Additional testing of sera by EIA indicated that the immune response to the vaccine was directed toward epitopes on both gp120 and gp41. Seroresponses to the immunodominant epitopes on gp41 were infrequent and none were detected to the neutralization epitope in the V3 region of gp120. This highly sensitive EIA is useful for characterizing HIV-1-specific antibody responses induced by an HIV-1 gp160 subunit vaccine. PMID- 1706608 TI - Expression, purification, and crystallization of the HIV-1 reverse transcriptase (RT). AB - The HIV-1 pol gene proteins (protease, reverse transcriptase, and endonuclease) were expressed in Escherichia coli N4830-1 by the use of the inducible expression vector pWS60 into which the pol gene was inserted. The p66/p51 heterodimer of reverse transcriptase (RT) was isolated in a highly pure and active form. Crystals of the p66/p51 heterodimer were obtained by the vapor diffusion hanging drop technique. The present crystal quality is still not adequate for high resolution X-ray investigation. PMID- 1706609 TI - Estrogen receptors appear undetectable in the C-cells of the human thyroid gland. AB - An attempt was made to explain the bone-preserving effect of estrogen by analysis of estrogen receptors (OER) in the calcitonin-producing C-cells of the human thyroid gland. Thyroid tissue from twenty patients with benign hyperthyroidism and three patients with medullary thyroid carcinoma was used. The C-cells were identified immunohistochemically using a polyclonal antibody to calcitonin, and by a similar staining technique the adjacent sections were stained for the OER protein using a monoclonal antibody (H 222-ABBOTT, USA). In spite of an intense nuclear staining of the positive control tissue (an OER positive breast carcinoma) no specific OER-staining was identified in C-cells or any other cells of the thyroid gland. Neither a dextran-coated charcoal assay nor a solid-phase immunoenzyme assay revealed any quantitative OER activity in tissue homogenates. Various observations point to a regulating mechanism between calcitonin and estrogen. The nature of this mechanism is not known, but according to our study it is unlikely to be a direct one. PMID- 1706610 TI - Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). AB - The prognostic significance of phenotype in malignant lymphomas (ML) is a matter of controversy. Here we analyze the clinical presentation, response to treatment, and survival of the 361 phenotyped patients with ML who were treated by the LNH 84 regimen. Histologic subtypes were diffuse-small cell in 10 patients, diffuse mixed in 69, diffuse large-cell in 177, immunoblastic in 94, and anaplastic large cell in 11. One hundred and eight patients (30%) had a peripheral T-cell ML and 253 (70%) a B-cell ML. Most of B-cell MLs were of diffuse large-cell type, and the T-cell MLs were distributed among diffuse mixed and immunoblastic subtypes. T cell MLs had an aggressive presentation with more patients having advanced stage (21% versus 41% stage II, 53% versus 45% stage IV, p = .0002), and B symptoms (58% versus 42%, p less than .01). The only significant difference in clinical manifestations were the higher frequency of gastrointestinal involvement in B cell MLs (20% versus 2%, p less than .0001) and the more frequent spleen (39% versus 21%, p = .0005) and skin (19% versus 3%, p less than .0001) involvement in T-cell MLs. There was no difference in response to the LNH-84 regiment between the two subgroups, but T-cell ML patients relapsed at a higher rate (43% versus 29%, p less than .001). T-cell ML patients have a significantly shorter freedom from-relapse (FFR) survival (median: 34 months versus not reached, logrank test: p = .002) and a non-significant shorter overall survival (median: 42 versus 50 months).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706611 TI - Distribution of multi-drug resistance-associated P-glycoprotein in normal and neoplastic human tissues. Analysis with 3 monoclonal antibodies recognizing different epitopes of the P-glycoprotein molecule. AB - P-glycoprotein is a molecule strongly associated with multi-drug resistance to certain cytostatic drugs, including adriamycin, vincristine, and daunorubicin. Using 3 monoclonal antibodies directed at different epitopes of this molecule (JSB-1, MRK16, C219) we investigated the tissue distribution of P-glycoprotein in normal and malignant human tissues, employing a routine immunoperoxidase technique. P-glycoprotein was found in the gastrointestinal epithelium, epithelia of the bronchi, mammary gland, pancreatic ducts, renal tubules, prostate gland, salivary gland, sweat glands of the skin, as well as in bile canaliculi and ductules, the adrenal and in endothelium of capillaries in various organs, most notably the brain. Mostly the pattern of reactivity of the 3 antibodies was similar, but some distinctly different staining reactions were seen. Reactivity was found in a variety of human tumors, arising from tissues normally expressing P-glycoprotein. Knowledge of the distribution and function of this molecule in both normal and malignant tissues may predict resistance and thus may influence choice of therapy. The role of P-glycoprotein in normal tissues and its implications for chemotherapy is discussed. PMID- 1706612 TI - The cell membrane and cell signals: new targets for novel anticancer drugs. AB - In the concluding Discussion session, emphasis focussed on the potential for interfering selectively with cell membranes and cell signalling in tumour as against normal tissues. There could be no doubt that tremendous advances are being made in our understanding of the molecular changes associated with malignancy and that the information available for the rational design of inhibitors of particular signalling pathways is increasingly sophisticated. There was a consensus that we need more information on the qualitative and quantitative differences in the structure and function of membranes and the signalling machinery in various normal tissues as compared to their cancerous counterparts. Ideally we will develop drug against, for example, specific forms of, let us say, protein kinase C or tyrosine kinase which are found to be predominantly active in neoplastic cells. This may well prove possible, at least in some instances, in which case a safe therapeutic margin will be assured. But differences may in other situations turn out to be in the level of expression rather than purely qualitative in nature, and the scale of the disparate expression may not always be great. Even in such situations, adequate therapeutic selectivity may still be achieved. This may derive from a "damping down" of signalling in the hyperactive tumour. Although there are legitimate concerns regarding the possible toxic effects of administering signal-wrecking molecules in man, we should not be pessimistic as there are clear precedents elsewhere in medicine for drugs acting on membrane signals proving to be safe and effective against expectation informed by hindsight. There may also be concerns about new forms of drug resistance. But this will be so for any new agent or novel target. And with mechanism of action clearly to the fore we should be able to predict resistance pathways in advance and devise appropriate circumvention strategies or targeted second line therapies. There was a palpable buzz at the meeting that this is a valid, different and above all rational approach. Not only that, but the new therapeutic molecules which we discover will themselves prove to be valuable tools with which to probe further into the mechanisms of malignancy and signal transduction. We had expected to see a bewildering amount of new information from the basic sciences of molecularbiology and cell physiology, and we got it. But it was also impressive to witness the number of new compounds coming through which look like real drugs or at least exciting lead compounds. The membrane-active ether lipids are in clinical trial. Bryostatin 1 will shortly join them.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706613 TI - Carcinoma of unknown primary: identification of a treatable subset? AB - We initiated a phase II study with combination chemotherapy consisting of cisplatin, etoposide and bleomycin in a subset of patients with carcinomas of unknown primary site characterized by the presence of at least one of the following criteria: 1) age below 50 years; 2) clinical evidence of rapid tumour growth; 3) tumour located predominantly in a midline distribution; 4) good response to previous administered radiotherapy. In 34 evaluable patients an objective response rate of 53% (95% confidence limits 35%-70%) was achieved. For patients with poorly differentiated adenocarcinomas the response rate was 35%, and, in most instances, of short duration. A response rate of 79% including complete responses and long-term survivals was achieved in patients with undifferentiated carcinomas. This difference in response rate was statistically significant (p = 0.02). No supplementary prognostic factors predicting response to chemotherapy could be identified. One patient with an initial diagnosis of undifferentiated carcinoma proved to have a malignant lymphoma after additional immunohistochemical investigation. Until a better characterization of this syndrome is possible patients with undifferentiated carcinomas of unknown primary site should be challenged with cisplatin-based chemotherapy. PMID- 1706614 TI - Salvage chemotherapy in Hodgkin's disease. Results in patients relapsing more than twelve months after first complete remission. AB - Forty-nine patients with Hodgkin's disease who relapsed after a first complete remission of more than 12 months following primary chemotherapy were treated with salvage therapy regimens. A total of 41 patients (84%) achieved complete remission. In particular, complete response was documented in 17 of 19 patients re-treated with the same initial drug combination. The five-year freedom from progression, relapse-free and overall survivals were 51%, 57% and 65%, respectively. In our experience, consolidation radiotherapy following drug induced remission failed to improve the five-year relapse-free survival. Present findings indicate that about half of patients relapsing after a disease-free interval exceeding 12 months can remain alive and disease-free 5 years after starting salvage chemotherapy. PMID- 1706615 TI - Treatment of epidemic Kaposi's sarcoma with combination chemotherapy (vincristine and bleomycin) and zidovudine. AB - Advanced AIDS-associated Kaposi's sarcoma often requires systemic cytotoxic chemotherapy. Despite high response rates, the majority of the patients die of opportunistic infections (OIs). Effective anti-retroviral agents in combination with cytotoxic chemotherapy may be useful in preventing the development of OIs in addition to increasing the tumor response. Twelve patients with extensive EKS were treated with a non-myelosuppressive drug regimen consisting of bleomycin and vincristine (BV) in combination with the anti-retroviral agent, zidovudine (ZDV). The dose of ZDV was 200 mg orally every four hours (full dose) in eight patients (Group I) or 100 mg orally every four hours (half dose) in four patients (Group II). Toxicity was acceptable with only 3 patients (all from Group I) requiring blood transfusions. ZDV dose reduction due to granulocytopenia was required in 6 patients (5 in Group I and 1 in Group II). Only two patients developed OIs during 27.5 cumulative months of therapy. The overall response rate was 83% in both groups with 4 patients achieving complete remission (CR) and 6 patients acheiving a partial remission (PR). We conclude that a combination of (BV) chemotherapy and ZDV can be used safely with high response rates. Prospective studies of such combination regimens are currently in progress. PMID- 1706616 TI - Identification of autophosphorylation sites of HER2/neu. AB - HER2 or c-erbB-2 is a putative growth factor receptor with sequence homology to the epidermal growth factor receptor. It is the human homologue of the rat protooncogene neu and may have an important role in human malignancies such as breast and ovarian cancers. Like other growth factor receptors, HER2 has intrinsic protein tyrosine kinase activity and undergoes autophosphorylation. Recently, we have demonstrated that, similar to the epidermal growth factor receptor, all autophosphorylation sites of HER2 are localized in the carboxyl terminus of this protein. In the present study, immunopurified HER2 was allowed to autophosphorylate, and tryptic phosphopeptides were generated. After purification of these phosphopeptides by high performance liquid chromatography, microsequencing was performed. Utilizing this approach, two autophosphorylation sites were unequivocally identified at Y1023 and Y1248. The sequences of two other tyrosine phosphorylated tryptic peptides were determined, but the exact site of autophosphorylation could not be determined because multiple tyrosines were located on each peptide. However, each of these peptides contains tyrosines that correspond to major autophosphorylation sites of the epidermal growth factor receptor, suggesting that, in addition to Y1023 and Y1248, Y1139 and Y1222 also serve as autophosphorylation sites of HER2. PMID- 1706617 TI - Deregulated c-myc expression abrogates the interferon- and interleukin 6-mediated G0/G1 cell cycle arrest but not other inhibitory responses in M1 myeloblastic cells. AB - The proliferation of M1 myeloblastic cells can be specifically restricted at the G0/G1 phase of the cell cycle by exposure to alpha- and beta-interferons or to interleukin 6. The latter cytokine also induces the morphological and functional differentiation of these myeloblasts toward monocytes. Each of these two different cytokines suppresses the expression of the c-myc nuclear oncogene, and the selective reduction in c-myc mRNA and protein precedes the cell cycle changes. In order to investigate whether one or more of the growth-suppressive effects of interferon and interleukin 6 are mediated by c-myc reduction, M1 cells were transfected with SV40-driven c-myc plasmid, whose expression fails to be turned off by these two cytokines. A detailed analysis of the responses to interferon and to interleukin 6 revealed that all of the myc-transfected clones have lost the cytokine-mediated G0/G1 type of growth arrest. However, not all of the growth responses to these cytokines were rescued by this specific genetic manipulation, and the cytokine-treated transfected cells stopped to proliferate in a new fashion which was not cell cycle specific. In addition, the myc transfected cells developed the differentiated phenotype in response to interleukin 6, as determined by the morphological change, expression of Fc receptors, and cytochemical analysis, suggesting that these molecular events can occur in the monocyte cell lineage in spite of the abnormal constitutive expression of c-myc.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706618 TI - Regulation of pp60c-src expression in rat and mouse fibroblasts by an inducible antisense gene: effects on serum regulation of growth and polyoma virus middle T function. AB - Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum. PMID- 1706619 TI - Multiple subtypes of glycine-immunoreactive neurons in the goldfish retina: single- and double-label studies. AB - The glycinergic system in goldfish retina was studied by immunocytochemical localization of glycine antiserum at the light-microscopical level. Numerous amacrine cells, a type of interplexiform cell, interstitial cell, and displaced amacrine cell were glycine-immunoreactive (IR). Amacrine cells, accounting for 97% of the glycine-IR neurons, were of four types based solely on their level of dendritic stratification: stratified amacrine cells of the first, third, and fifth sublayers and bistratified amacrine cells of the first and fifth sublayers. Double-labeling experiments were carried out to determine possible co localization of glycine-IR with GABA-IR, serotonin-IR, substance P-IR and somatostatin-IR. No evidence for co-localization of glycine-IR with these other transmitter substances was found, despite reports of co-localization of these substances in retinas of other species. Glycinergic neurons in goldfish retina appear to consist of a heterogeneous population of at least seven morphologically distinct subtypes that are also neurochemically distinct in regard to GABA, serotonin, substance P, and somatostatin. Since dendritic stratification in the inner plexiform layer is correlated with ON-, OFF-response types, we suggest that the subtypes of glycine-IR amacrine cells play different roles in the encoding of visual information. PMID- 1706620 TI - Major population differences in T cell response to a malaria sporozoite vaccine candidate. AB - Using a complete series of overlapping peptides, we have identified the T cell epitopes of a malaria vaccine candidate, the circumsporozoite (CS) protein, that are recognized by sporozoite-exposed residents of a non-endemic country. This protein and subunits from it are being considered as malaria sporozoite vaccine candidates, as CS-specific antibodies and cytotoxic T lymphocytes have been shown to have a role in protection. The rationale for developing an antibody-based vaccine is that in Plasmodium falciparum the immunodominant B cell epitope of the protein, (Asn-Ala-Asn-Pro)n [(NANP)n], is invariant. However, the ideal vaccine must contain CS protein-derived T cell antigenic epitopes to allow natural boosting of the antibody response following sporozoite exposure. Here, we show that major differences occur between the CS-specific T cell responses of non endemic Caucasians and an endemic African population. HLA differences between the populations are, in part, responsible. Subunit malaria vaccines for one population may be ineffective in a different population. PMID- 1706621 TI - Cholera toxin- and forskolin-induced cyclic AMP accumulations of pig skin (epidermis). Modulation by chemicals which reveal the beta-adrenergic augmentation effect. AB - Effects of cholera toxin and forskolin on pig epidermal adenylate cyclase system were investigated. Both agents increased cyclic AMP levels of epidermis. Marked accumulations were observed in the presence of cyclic AMP phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX). It has been known that hormone stimulated adenylate cyclase responses are modified by various chemical treatments. Following long term incubation with hydrocortisone, Ro10-1670, and colchicine, the epidermal beta-adrenergic adenylate cyclase response was increased without the alteration of cyclic AMP phosphodiesterase activity. Adenosine-, and histamine-adenylate cyclase responses were unchanged by hydrocortisone treatment, and were decreased by Ro10-1670 and colchicine treatments. Following the long term incubation with these chemicals, effects of cholera toxin and forskolin were investigated. Colchicine-treated skin revealed the increased cholera toxin-, and forskolin-induced cyclic AMP accumulations. Neither hydrocortisone- nor Ro10-1670-treated skin revealed alterations of cholera toxin-, and forskolin-effect. The stimulatory effect of colchicine on the cholera toxin-, and forskolin-effect was observed at doses of the beta-adrenergic augmentation effect. Our results indicate that among the chemicals which reveal the beta-adrenergic augmentation effect, colchicine is unique in that it also increases cholera toxin-, and forskolin-induced cyclic AMP accumulations of epidermis. PMID- 1706622 TI - Catalytic RNA: a Nobel Prize for small village science. PMID- 1706623 TI - Mx proteins: antiviral proteins by chance or by necessity? AB - The interferon-inducible Mx1 protein is responsible for inborn resistance of mice to influenza. It is now recognized that this protein is a member of a family of interferon-inducible, putative GTP-binding proteins found in many organisms. Thus, these proteins, called the Mx proteins, are found in species that are naturally infected with influenza virus, and also in species that are not. Some Mx proteins display a broader antiviral profile than the one observed for Mx1 in mice. Others, however, may not be antiviral. Two recently discovered GTP-binding proteins, Vps1p in yeast and dynamin in rat, are also related to Mx1. These proteins are synthesized constitutively and serve basic cellular functions. PMID- 1706624 TI - Glutamate: three meetings but how many receptors? Excitatory Amino Acids 1990: a Fidia Research Foundation Symposium, Padua, Italy, May 21-26, 1990. Excitatory Amino Acid Receptors in the Brain: Functions and Disorders, Montreal, Canada, June 23-24, 1990. Excitatory Amino Acids: an Update Official Satellite of the XIth IUPHAR Congress, Flims, Switzerland, June 28-29, 1990. PMID- 1706625 TI - Interferon-dependent transcriptional activation: signal transduction without second messenger involvement? AB - Two specific macromolecular interactions are known to underlie the demonstrated transcriptional stimulation of different sets of genes incident to the binding of different polypeptide ligands to cells. The initial polypeptide-receptor interaction is widely recognized to be specific. It is also well established that the binding of specific transcription factors to well-defined DNA sites activates specific genes. In this paper, we suggest that a third, equally specific protein protein interaction links the first two specific interactions and ensures the high specificity required in these pathways. We propose that a receptor recognition protein is required to recognize the bound receptor. This receptor recognition protein might itself be part of a transcription factor or might interact directly with a transcription factor that would thereby be activated and translocated to the nucleus to participate in gene activation. According to this hypothesis, no global changes in second messenger concentrations are necessary, and the enzymatic properties of the receptor-recognition protein(s) need not be specified. Phosphorylations of, or by, receptor-bound proteins would not, of course, be excluded. But such modifications during ligand-mediated signal transduction would not depend on global second messenger changes. This model is derived from studies of the proteins involved in interferon (IFN)-stimulated gene transcription. It is now established that interferon-alpha (IFN alpha) activates a multisubunit transcription factor in the cell cytoplasm, and that this factor then moves to the nucleus to activate a set of IFN-stimulated genes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706626 TI - Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex. AB - The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M proteinase PMID- 1706627 TI - Extrachromosomal chromatin: novel target for bleomycin cleavage in cells and solid tumors. AB - The preference of bleomycin, a DNA strand scission antitumor agent, to damage extrachromosomal (episomal) DNA was investigated. These episomes contain transcriptional promoters, replication origins, and oncogenes from MMTV, BPV, and v-Ha-ras and confer a neoplastic phenotype to a mouse fibroblast cell line. We found that bleomycin induces dose-dependent single- and double-stranded cleavage of intracellular episomes as measured by topological forms conversion. Bleomycin scission of episomes occurs within 1 min, and upon drug removal, damaged episomes are as rapidly repaired. By expressing the episomal and genomic damage as breaks per nucleotide, bleomycin has a 30-50-fold cleavage preference for episomal chromatin compared to genomic DNA. The episomes have preferred regions of the bleomycin-induced damage, particularly within the MMTV LTR and BPV origin of replication. Also, it is possible to assess bleomycin action on episomes in solid tumors in mice. Single intravenous injections of BLM into tumor-bearing mice result in single- and double-stranded cleavage of episomes that are dose related and occur within 1 min. Specific double-stranded breaks occur in the same regulatory regions of episomes in solid tumors and in cultured cells. Finally, we observe that damage to the episomal drug target occurs at therapeutic doses in mice. PMID- 1706628 TI - Preservation of the lung: comparison of flush perfusion with cold modified blood and core cooling by cardiopulmonary bypass. AB - Clinically applied techniques for lung preservation of flush perfusion with cold modified blood and core cooling by means of cardiopulmonary bypass were compared in a canine model of single left lung transplantation with immediate ligation of the contralateral bronchus and pulmonary artery. In the cold modified blood group (n = 5), after pretreatment with the synthetic prostacyclin analogue iloprost (20 ng/kg/min), lung preservation was achieved by pulmonary artery perfusion with a solution of autologous donor blood, albumin, and mannitol. In the cardiopulmonary bypass group (n = 7), donors were cooled to an esophageal temperature of 10 degrees C before harvest of the heart-lung bloc. Donor organs were stored at 4 degrees C for 6 hours. After transplantation, artificial ventilation at a fixed oxygen concentration was continued until death or until animals were killed at 24 hours. Assessment of lung preservation was by recipient survival, blood gas analyses, and measurement of total lung water content. All cold modified blood recipients survived for 24 hours; four of seven bypass recipients died within 7 hours of operation. There was no change in oxygenation in the cold modified blood group at 1 hour after contralateral ligation: paO2, 212.2 mm Hg +/- 8.5 preligation; 227.2 mm Hg +/- 11.5 postligation. Oxygenation in the bypass group was reduced at 1 hour (paO2, 182.6 +/- 7.6 mm Hg preligation; 139.8 +/- 32.1 mm Hg postligation), but the difference did not reach statistical significance. The difference between groups at 1 hour was significant (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706629 TI - Inositol trisphosphate analogues induce different oscillatory patterns in Xenopus oocytes. AB - Agonists that utilize the calcium-mobilizing second messenger inositol(1,4,5)trisphosphate Ins(1,4,5)P3 usually generate oscillations in intracellular calcium. Such oscillations, based on the periodic release of calcium from the endoplasmic reticulum, can also be induced by injecting cells with Ins(1,4,5)P3. The mechanism responsible for oscillatory activity was studied in Xenopus oocytes by injecting them with different inositol trisphosphates. The plasma membrane of Xenopus oocytes has calcium-dependent chloride channels that open in response to calcium, leading to membrane depolarization. Oscillations in calcium were thus monitored by recording membrane potential. The naturally occurring Ins(1,4,5)P3 produced a large initial transient followed by a single transient or a burst of oscillations. By contrast, two analogues (Ins(2,4,5)P3 and Ins(1,4,5)P(S)3) produced a different oscillatory pattern made up of a short burst of sharp transients. Ins(1,3,4,5)P4 had no effect when injected by itself, and it also failed to modify the oscillatory responses to either Ins(2,4,5)P3 or Ins(1,4,5)P(S)3. Both analogues failed to induce a response when injected immediately after the initial Ins(1,4,5)P3-induced response, indicating that they act on the same intracellular pool of calcium. The existence of different oscillatory patterns suggests that there may be different mechanisms for setting up calcium oscillations. The Ins(2,4,5)P3 and Ins(1,4,5)P(S)3 analogues may initiate oscillations through a negative feedback mechanism whereby calcium inhibits its own release. The two-pool model is the most likely mechanism to describe the Ins(1,4,5)P3-induced oscillations. PMID- 1706630 TI - [Latrotoxin channels. Permeability for divalent cations]. AB - The dependence of Ca2+ transport in synaptosomes along the channels formed by alpha-latrotoxin on [Ca2+]in and the feasibility of transport along these channels of other bivalent cations were studied. It was found that the concentration dependence of Ca2+ influx is nonlinear and is described by the Michaelis-Menten kinetics (Km = 1.07 +/- 0.19 mM). Mg2+, Ba2+, Sr2+, Mn2+ and Co2+ competitively inhibited the Ca2+ influx via latrotoxin channels. Studies with the use of the fluorescent Ca2+ probes, Quin-2 and Fura-2, revealed that these cations can also penetrate inside synaptosomes via latrotoxin channels. The bivalent cation influx via latrotoxin channels caused a decrease of the membrane potential of synaptosomes. The similarity of properties of latrotoxin and endogenous Ca2+ channels is discussed. PMID- 1706631 TI - Elimination of electronic offset and physiological background activity in magnetocardiographic localization. AB - A method has been developed to eliminate disturbing magnetic signals in the biomagnetic localization of arrhythmogenic sources in the heart. The procedure consists of two steps: Superimposed background activity of the heart is eliminated by subtraction of a template of pure background activity. Systematic and electronic offset is subsequently eliminated by baseline-correction during periods of zero activity. The method was applied to several kinds of arrhythmias. It was demonstrated that elimination of background activity is the prerequesite for exact localization and that the proposed procedure yields correct results. PMID- 1706632 TI - Radiotherapy in the treatment of pancreatic cancer. AB - In the last two decades, we have witnessed revolutionary advances in pancreatic imaging as well as increased availability of megavoltage radiotherapy equipment and sophisticated radiotherapy planning devices. Several advances in the radiotherapy of pancreatic cancer have been made for the patient with resectable disease. Postoperative radiotherapy combined with chemotherapy confers a survival advantage after 'curative' resection. Preoperative and intraoperative intraoperative radiotherapy may do the same, but this requires further evaluation. Preoperative irradiation may improve the resectability rate, but the clinical data are still very limited. For locally unresectable disease, PHD radiotherapy with adjuvant 5-FU should now be the standard treatment in suitable cases with a median survival time of about one year. High LET radiation beams have failed to produce improved survival in two prospective randomized studies. Intraoperative radiotherapy is an effective means of pain control and enhances control of local disease, but has not been shown to improve survival rate significantly. Interstitial radiotherapy also improves local control, but it is associated with a high mortality rate and an even higher major complication rate. Wide-area radiation therapy and preoperative radiotherapy both seem to show promise in this group of patients. Patients with metastatic disease should be treated palliatively on an individual basis. PMID- 1706633 TI - Non-operative palliation of pancreatic cancer. PMID- 1706634 TI - The activation and silencing of gene transcription in the liver. PMID- 1706635 TI - In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine dextran-amine, Fluoro-Ruby, within the CNS. AB - A number of fluorescent dextrans were screened for axonal transport properties within the rat CNS. One compound, Fluoro-Ruby (FR), was found to be particularly sensitive for demonstrating retrograde and particularly anterograde axonal transport. The tracer may be either pressure or iontophoretically injected, and the fixed tissue can be examined without histochemical processing. The technique can be combined with a wide variety of other neuroanatomical methods. PMID- 1706636 TI - MPTP-treated young mice but not aging mice show partial recovery of the nigrostriatal dopaminergic system by stereotaxic injection of acidic fibroblast growth factor (aFGF). AB - Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that acts as a neurotrophic factor for certain central and peripheral neurons. Acidic FGF was injected stereotaxically into the striatum of young (2-month-old) and aging (12-month-old) C57BL/6 mice that were treated 1 week before with systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP treatment (4 x 20 mg/kg, i.p. given 12 h apart) reduced tyrosine hydroxylase (TH) immunoreactive (IR) fibers in the striatum and reduced dopamine (DA) concentration to 32% of the controls in young and 20% of the controls in aging mouse brain 5 weeks after administration. Although the DA concentration recovered to 43% of the controls in young mice following stereotaxic injection of aFGF 5 weeks after MPTP treatment, aging mice with such treatment did not show a significant recovery of DA concentration. Computerized image analysis of TH-IR fibers in the striatum also showed significant recovery in young mice treated with aFGF, while aging mice did not show a significant recovery. We conclude that treatment of MPTP-depleted young mice with aFGF results in partial recovery in the nigrostriatal DA system but such benefits decline with age. PMID- 1706637 TI - Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats. AB - Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to 29 +/- 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E 64c (P less than 0.05) to increase MAP2 levels to 55 +/- 25.7% of control levels. E-64c had no significant effect on the ischemia-induced depletion of myelin associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia. PMID- 1706638 TI - The effects of nanoliter ejections of lidocaine into the pontomedullary reticular formation on cortically induced rhythmical jaw movements in the guinea pig. AB - In the ketamine/urethane anesthetized guinea pig, electromyographic (EMG) responses of the anterior digastric muscle were studied when loci within the lower brainstem were microejected with lidocaine (2%) during rhythmical jaw movements (RJMs) evoked by repetitive electrical stimulation of the masticatory area of the cortex. The area investigated was between the trigeminal motor nucleus (Mot V) and the rostral pole of the inferior olive. Microejections of lidocaine, contralateral to the cortical stimulus site, into the ventral-medial portion of Mot V where digastric motoneurons are known to be located, resulted in reduction or complete abolishment of the digastric EMG activity ipsilateral to the ejection with no effective change in mean cycle duration (CD) or mean percent normalized integrated amplitude of the contralateral digastric EMG. Microejections of lidocaine, contralateral to the cortical stimulus site, into the ponto-medullary reticular formation in areas that included portions of the caudal nucleus pontis caudalis (PnC), nucleus gigantocellularis (GC), medial nucleus parvocellularis (PCRt), and dorsal paragigantocellularis (dPGC), in most cases produced a bilateral reduction in the mean normalized integrated amplitude and a bilateral increase in the mean cycle duration. In these sites, the bilateral increase in mean cycle duration of digastric EMG bursts was also associated with a significant increase of coefficient of variation in CD. In many cases, microejection of lidocaine completely abolished rhythmical digastric activity, bilaterally. HRP injections into Mot V were performed to determine the locations of trigeminal premotoneurons and their relationship to effective lidocaine sites for rhythmical jaw movement suppression. Retrogradely labeled cells were found mainly in the mesencephalic nucleus of V; trigeminal principal and spinal V sensory nuclei, bilaterally; and within the intermediate and lateral regions of reticular formation, bilaterally. No labeling was found in the medial reticular formation, including the nucleus gigantocellularis and dorsal paragigantocellularis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706640 TI - Diabetes affects retrograde but not anterograde transport of sciatic nerve phosphofructokinase in Sprague-Dawley rats. AB - Phosphofructokinase activity was measured in the sciatic nerve of streptozotocin induced diabetic and nondiabetic rats. Average steady-state phosphofructokinase activity was obtained from three consecutive segments of the mid-femoral region in the left sciatic nerve in both diabetic (4 and 24 weeks) and nondiabetic, age matched animals. Over time, phosphofructokinase activity significantly decreased (p less than 0.05) with diabetes, with no effect demonstrated within similar age groups. The accumulation of phosphofructokinase activity was accomplished by ligating the mid-femoral region of the right sciatic nerve for 24 h. Anterograde and retrograde axonal transport of phosphofructokinase was measured in the 3-mm segment proximal and distal to the ligature, respectively. There was a trend (p = 0.0627) towards a decline in net proximal accumulation (mean proximal minus mean background) with age. Net distal (mean distal minus mean background) activity declined by 80% (p less than 0.05) in the control group between 4 and 24 weeks of the diabetic state. However, diabetic animals did not experience the same age related decline in retrograde transport. The findings suggest that diabetes affects the age-associated evolution of retrograde transport, presumably a reflection of the neuropathy occurring in the distal axon branches, without altering anterograde transport to any appreciable extent. PMID- 1706639 TI - Osteoclast cytomorphometry demonstrates an abnormal population in B cell malignancies but not in multiple myeloma. AB - Increased bone resorption in the vicinity of myeloma cells is mediated by local stimulating factors. Other malignancies of the B cell lineage are also able to produce resorbing factors responsible for increased bone resorption. We have studied three groups of subjects: 10 patients with overt multiple myeloma, 10 patients with a B cell malignancy, and 10 healthy human subjects as controls. Patients were studied at the time of diagnosis and had a transiliac bone biopsy. Osteoclasts were evident on histological sections by their acid phosphatase activity. A software was developed on an automatic image analyzer (Leitz TAS+) for measuring the maximal Feret's diameter (Oc.Le) of each osteoclast (corresponding to the osteoclast length). The histogram of Oc.Le frequency distribution was supplied in each group. In myeloma patients, the Oc.Le frequency distribution was similar to that in normal subjects and showed the histogram to be asymetric with a positive skew (maximum peak at 20-25 microns). With a graphical analysis, this distribution was shown to follow a lognormal distribution corresponding to a homogeneous osteoclast population. In other B cell malignancies, Oc.Le displayed a bimodal distribution with a peak at 20-25 microns and a lower peak at 10-15 microns. The graphical analysis showed that small (mononucleated?) osteoclasts are present in B cell malignancies with normal osteoclasts. This might reflect the secretion of different soluble factors by malignant cells of the B lymphocyte lineage. PMID- 1706641 TI - Caring for seriously ill and dying patients: the philosophy and ethics. AB - The care of seriously ill and dying patients necessitates a philosophic and ethical basis, without which unacceptable patterns of practice may develop. Several problems are described: inadequate or unskilled communication of information, withdrawal by the physician, patient labelling and poor health care. Palliative care must be based on a philosophy that acknowledges the inherent worth and dignity of each person. In addition, it must take place within the framework of four ethical principles: autonomy, beneficience, nonmaleficience and justice. The first and most important of these is the need to regard patients as unique people with a right to compassion, gentle truth, autonomy in decision making and excellence in physicial and psychospiritual care. Beneficence obliges us not only to relieve suffering but also to enhance the patient's quality of life whenever possible. Nonmaleficence and justice require allocation of sufficient health care resources of the type necessary to provide high-quality care and prevent patients from coming to harm. PMID- 1706642 TI - Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody. AB - Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15-30 mg. After the mAb infusions the number of circulating tumour cells was temporarily reduced, but in some cases antibody coated cells remained in the circulation for several days. mAb penetrated to extravascular tumour sites; in general higher doses were required to saturate cells in the lymph nodes than to sensitize tumour cells in the bone marrow. mAb doses of up to 250 mg were given i.v. over 4 h without major toxicity. One patient twice achieved a partial remission after two periods of mAb treatment with an 8-month interval; the second remission lasted for 9 months. One patient showed a minor response. None of the patients made antibodies against the mouse immunoglobulin. Serum immunoglobulin levels were followed as a measure of the function of the normal B cell compartment; no significant changes were seen up to 6 months after mAb treatment. PMID- 1706643 TI - Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation. AB - Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1-0.01 microns) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized 3H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released. PMID- 1706644 TI - Deposition of extracellular matrix along the pathways of migrating fibroblasts. AB - Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development. PMID- 1706645 TI - Qualitative and quantitative comparison of the distribution of phosphorylated and non-phosphorylated neurofilament epitopes within central and peripheral axons of adult hamster (Mesocricetus auratus). AB - The distribution of phosphorylated and non-phosphorylated neurofilament epitopes was determined immunocytochemically in adjacent 2 microns-thick sections of sciatic nerve, ventral root and spinal cord. Staining was scored as either intense, moderate or absent and the proportion of labeled axons was calculated for each category. Nearly all sciatic nerve and ventral root axons were immunoreactive with both antibodies against phosphorylated and non-phosphorylated neurofilaments and there were no significant differences in the number of intensely- or moderately-labeled axons. Within the spinal cord however, while the majority of large caliber axons was stained with both antibodies, there was a significant number of small caliber axons which stained only with antibodies against phosphorylated neurofilaments. These results show that phosphorylated and non-phosphorylated neurofilaments are extensively codistributed in CNS and PNS axons, and that in the CNS, staining intensity for non-phosphorylated epitopes is less in the smaller axons. PMID- 1706646 TI - Distribution of the 3-fucosyl-N-acetyl-lactosamine (FAL) epitope in the adult mouse brain. AB - The distribution of the 3-fucosyl-N-acetyl-lactosamine (FAL) epitope within the adult mouse brain was studied by immunohistochemistry using the monoclonal antibody Leu-M1. Leu-M1-positive elements comprised astrocytes and neurons. FAL positive astrocytes were particularly abundant in barrier structures of the brain, but were also prominent at the periphery of most medullated fiber tracts. Their intracerebral distribution led to a distinct pattern of organization, which in some locations, including the cerebral cortex, could be used for an extended regional architectonic description. Since only some FAL-positive astrocytes were also positive for glial fibrillary acid protein (GFAP), the emerging topography of the FAL-positive astrocytes often differed from the GFAP-distribution. In the cerebellum, Bergmann glia cells expressed the FAL epitope and, in the vermis, their arrangement had a band-like appearance. Positive oligodendrocytes could not be identified. The common ependymal cells were negative, whereas tanycytes were highly immunoreactive. The Leu-M1 antibody also stained some neurons. These occurred in selected neocortical regions, within the dorsal and ventral striatum, in the globus pallidus, the nucleus basalis of Meynert, the nucleus diagonalis and some hypothalamic areas. In some instances, their morphology and location indicated an association with neurochemically specified cell groups. PMID- 1706647 TI - [Anticancer spectrum of pingyangmycin in vitro]. AB - Pingyangmycin (PYM), produced by Streptomyces pingyangensis n. sp., was found to be identical to bleomycin A5. In the present study, a comparative observation was carried out in 10 human cancer cell lines. As determined by a colony-forming assay, the dose-response curves obtained from cells exposed to PYM for 1 h were of one type only: biphasic exponential. The sensitivities of these cells derived from different types of tumors, however, varied with a broad range of ID50 values (0.03-0.82 microgram/ml). A hepatoma cell line (BEL-7402) and three lines derived from squamous carcinomas of the esophagus (Eca109 and CaEs17) or the nasopharynx (CNE) were relatively sensitive (ID50 less than 0.20 microgram/ml) to PYM which is known to have clinical activity against these diseases. Two gastric adenocarcinoma cell lines (MGc80-3 and BGC-823) and a pulmonary adenocarcinoma cell line (SPC-A-1) appeared to be less sensitive to the drug, with ID50 values of 0.21-0.47 microgram/ml. PYM was 7-fold more effective against LTEP-78 cells derived from pulmonary squamous carcinoma as opposed to SPC-A-1 cells, resulting in a low ID50 value of 0.04 microgram/ml. However, PYM as a single agent has not yet received full evaluation in relation to this type of lung cancer. In contrast with other cell lines of squamous cancer origin, HeLa and CC-801 cells derived from uterine cervix carcinomas which have been evaluated as highly responsive to PYM had the highest ID50 values (greater than 0.70 microgram/ml). PMID- 1706648 TI - Review: cytokines and malaria. AB - Malaria, which is caused by hemoprotozoan parasites of the genus Plasmodium, has once again reached epidemic proportions. The resurgence of malaria has occurred because the parasite has developed resistance to the anti-malarial drugs and the mosquito vector has developed resistance to the insecticides. Added to these impediments is the problem that, in spite of intense efforts by researchers world wide, there is yet no effective anti-malarial vaccine. Our lack of knowledge concerning the exact mechanism of the host immune response to infection with Plasmodium parasites has contributed significantly to the lack of an effective and safe vaccine. The role of an antibody-independent, cell-mediated mechanism which can result in the generation of soluble mediators or cytokines by T lymphocytes and macrophages in host defense against blood stage malaria is being actively investigated in humans and in mice with malaria. With the availability of recombinant lymphokines and monokines and neutralizing antibodies against these reagents it is now possible to determine the role of cytokines in the development of protective anti-malarial immunity. In this review, we discuss recent evidence from human studies and experimental murine models concerning the possible roles of cytokines in malaria. PMID- 1706649 TI - Screening for biotinidase deficiency in children with unexplained neurologic or developmental abnormalities. AB - To test the hypothesis that the frequency of biotinidase deficiency is greater in children with unexplained developmental delay or neurologic abnormalities than in the general population, we studied children seen at a large outpatient clinic over a four-year period who had one or more of these neurologic abnormalities and for whom no specific cause for their abnormalities could be found. The group totaled 274 children (163 boys; 111 girls) whose ages ranged from 2 weeks to 17 years. Characteristics were IQ/DQ, 30 to 70 in the 115 for whom scores were available; 41% had seizures; 15% had sensorineural hearing loss; 54% showed gross motor delay or ataxia; and 27% had decreased muscle tone. One patient with a classical clinical picture of biotinidase deficiency was diagnosed during the study period and was not included in the study. None of the patients with nonclassic findings had a deficiency of biotinidase activity. Our results suggest that biotinidase deficiency does not account for a large proportion of children with unexplained neurologic abnormalities or developmental delay. This does not negate the importance of biotinidase testing in children with clinical patterns specifically suggestive of the deficiency. PMID- 1706650 TI - Interleukin-1 and inhibition of interleukin-1 in hemodialysis patients. PMID- 1706651 TI - Cytoskeletal filament typing of human corneal endothelial cells. AB - Flat mounts of human corneal endothelial cells (HCECs) were examined immunohistochemically by using a wide assortment of monoclonal antibodies against the five classes of intermediate filaments (IFs) and actin and myosin. HCECs showed uniform immunostaining with monoclonal antibodies against the 40-kD (CK 19) and 45-kD (CK 18) cytokeratin (CK). Only part of the endothelial cells reacted with monoclonal antibodies against the 52-kD (CK 8) and 54-kD (CK 7) cytokeratin polypeptides and with monoclonal antibodies against vimentin. Monoclonal antibodies against the low- and middle-molecular-mass neurofilament proteins produced positive staining of all HCECs. No positivity was obtained with antibodies against desmin or glial fibrillary acidic protein. In addition, positive immunostaining with monoclonal antibodies against actin and slow myosin demonstrate that these proteins form part of the cytoskeleton of HCECs. The results of this study show that immunostaining of flat cell preparations is very useful for studies on HCECs. HCECs display an unusual combination of cytokeratin IFs and neurofilaments, together with vimentin, and are heterogeneous with respect to their IF makeup. These findings are discussed in relation to the presumed origin of HCECs. PMID- 1706652 TI - Interleukin-2 induces corneal neovascularization in A/J mice. AB - Mitogen-stimulated lymphocytes induce a highly reproducible form of corneal neovascularization (CNV) in inbred mice. To determine if supernatants derived from stimulated lymphocytes and their constituent mediators were also angiogenic, we injected conditioned medium (CM) from mitogen-stimulated lymphocytes, control non-conditioned medium, recombinant interleukin-2 (rIL-2), or control bovine serum albumin (BSA), a component of rIL-2, into the corneas of syngeneic A/J mice. Control, nonconditioned medium was not angiogenic. While the other injections all induced some CNV, the CM and rIL-2 both induced a significantly greater area of CNV than BSA (p less than 0.05). The area of CNV induced by the CM was greater than that induced by IL-2 (p = 0.03). These data show that IL-2, which stimulates vascular endothelial cells in vitro and is elaborated during corneal immune reactions in vivo, is one of the potential mediators of immunologically mediated CNV. PMID- 1706653 TI - Giant papillary conjunctivitis associated with elevated corneal deposits. AB - A patient presented with central corneal scarring and neovascularization associated with elevated deposits that were shown to be keratin and calcium. Giant papillary conjunctivitis (GPC) was noted at a corresponding location in the palpebral conjunctiva. The lid reaction resolved after the elevated corneal deposits were debrided. A rigid gas-permeable contact lens was then fitted for visual rehabilitation. Either foreign bodies or elevated corneal deposits may cause GPC. PMID- 1706654 TI - Colorectal adenocarcinoma in patients less than 40 years of age. AB - From 1973 to 1985, 105 patients under 40 years of age were treated for colorectal adenocarcinoma at Roswell Park Cancer Institute. There were 51 males and 54 females. The mean age was 32 years. The majority of patients were treated for left colon or rectal carcinomas. Ninety-seven of 105 patients underwent surgical resection of their primary cancer, 70 (67 percent) of which were potentially curative. Twenty-seven patients underwent palliative resections. Dukes' A or B lesions were not seen in patients less than 20 years old, whereas these early lesions were seen in 11 percent of patients 20 to 29 years old and in 26 percent of patients greater than 30 years of age. The mean survival for patients between 20 and 29 years was 39 months and 46 months for patients 30 years and older. PMID- 1706655 TI - Effects of the optical isomers of a novel 5-HT2 antagonist and (-)-Bay K 8644 on spontaneous mechanical activity in rat portal veins. AB - The effect of the (+)-enantiomer (Ir) and the (-)-enantiomer (Lu) of a novel 5 HT2 antagonist was studied on spontaneous mechanical activity in rat portal veins and compared to that of the Ca-agonist (-)-Bay K 8644. Ir and Lu (10(-8) to 10( 5) M) were found to equally increase the spontaneous mechanical activity in rat portal veins. At high concentrations (10(-4) M) Ir and Lu totally abolished spontaneous activity. The frequency of the spontaneous activity was equally decreased by the two enantiomers. The increase in spontaneous activity induced by Ir and Lu was abolished in Ca-free medium and restored by readdition of Ca. The augmenting effect of the enantiomers on mechanical activity was not influenced by atropine but was totally eliminated by the Ca-antagonist nifedipine. The Ca agonist (-)-Bay K 8644 was more potent in increasing the amplitude of the mechanical activity than the enantiomers and had a concentration-dependent increasing effect on the frequency of the spontaneous activity. The results indicate that in rat portal veins Ir and Lu have a dual non-stereoselective effect which may include a Ca-agonistic stimulating mechanism of action as well as a pacemaker blocking effect. PMID- 1706656 TI - Metabolism of thioamide antithyroid drugs. PMID- 1706657 TI - Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis. PMID- 1706658 TI - Analysis of the conformational transitions of proteins by temperature-gradient gel electrophoresis. AB - Temperature-gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins. A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field. Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins. Using alpha-amylase as an example we show the kinds of results which may be obtained from such measurements. Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature. The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves. The protein bands are detected by silver and/or activity staining. The thermal denaturation of alpha-amylase from Aspergillus oryzae showed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility. The discontinuity is due to an irreversible denaturation process under the gel conditions. The transition temperature was measured as a function of several parameters, e.g., the concentration of Ca(+)+, dithiotreithol, urea and the pH value. The structural transition of alpha-amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution. The structural transitions of two other alpha-amylases from Bacillus subtilis and Bacillus licheniformis were also studied. The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706659 TI - Intensification of peroxidase-diaminobenzidine staining using gold-sulfide silver: a rapid and highly sensitive method for visualization in immunoblotting. AB - A highly sensitive and rapid visualization method for protein detection by immunoblotting is described. Proteins blotted onto a Durapore membrane were visualized by the following procedure: after conventional peroxidase-based staining with 3,3'-diaminobenzidine (DAB), the produced DAB precipitates were intensified by treating with (i) gold trichloride (acid), (ii) sodium sulfide, and (iii) a developer containing silver nitrate. This postintensification method was employed for the detection of the genetic polymorphism of human proteins, such as deoxyribonuclease I in urine, and group specific component, transferrin and alpha 1-antitrypsin in serum after polyacrylamide gel-isoelectric focusing, followed by immunoblotting. This postintensification technique was found to be simple, giving up to 16- to 64-fold amplification of the conventional peroxidase DAB staining. PMID- 1706660 TI - Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity. AB - L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre. PMID- 1706661 TI - Multiple apomucin translation products from human respiratory mucosa mRNA. AB - Poly(A)-rich RNA was purified from a pool of five human tracheobronchial mucosa. After in vitro translation in a reticulocyte lysate and immunoprecipitation of the translated products, using either a polyclonal antiserum or a monoclonal antibody to deglycosylated respiratory mucin peptides, the products were characterized by SDS/PAGE. The respiratory mucin precursors migrated as a very large smear from almost the top of the resolving polyacrylamide gel to an area corresponding to a molecular mass of about 100 kDa. After hybridization with mucin cDNA probe TH 29 described by Crepin et al. [Crepin, M., Porchet, N., Aubert, J. P. & Degand, P. (1990) Biorheology 27, 471-484] respiratory mucin mRNAs also appeared polydisperse. Although degradation or incomplete translation of high-molecular-mass mRNA cannot be entirely ruled out, these results suggest that human respiratory apomucins consist of a family of peptides which share some common epitopes. This possibility is in agreement with (a) the diversity of mucin precursors observed previously with pulse/chase experiments performed with explants of human respiratory mucosa and (b) the polydispersity of secreted respiratory mucins observed by electron microscopy. PMID- 1706662 TI - Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes. AB - Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV. PMID- 1706663 TI - Characterization of a murine beta 1-4 galactosyltransferase expressed in COS-1 cells. AB - We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGT1-directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGT1-transfected cells was approximately eightfold higher than that in mock- or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1-transfected cells and mock- or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal beta-galactosidase, most of the linkage created by this enzyme was in the Gal beta 1-4GlcNAc group. The acceptor specificity of the pCMGT1-directed enzyme was changed from N-acetylglucosamine to glucose by adding alpha lactalbumin in the reaction mixture. Alpha-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent Km values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP Gal are 2 mM, 60 microM and 24 microM, respectively. These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a beta 1-4 linkage. PMID- 1706664 TI - Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies. Proteins L44/L45 and L44' have different functional roles. AB - In order to characterize the acidic ribosomal proteins immunologically and functionally, a battery of monoclonal antibodies specific for L44, L44' and L45, the three acidic proteins detected in Saccharomyces cerevisiae, were obtained. Eight monoclonal antibodies were obtained specific for L45, three for L44' and one for L44. In addition, two mAbs recognizing only the phosphorylated forms of the three proteins were obtained. The specific immunogenic determinants are located in the middle region of the protein structure and are differently exposed in the ribosomal surface. The common determinants are present in the carboxyl end of the three proteins. An estimation of the acidic proteins by ELISA indicated that, in contrast to L44 and L45, L44' is practically absent from the cell supernatant; this suggests that protein L44' does not intervene in the exchange that has been shown to take place between the acidic proteins in the ribosome and in the cytoplasmic pool. It has also been found that, while IgGs specific for L44 and L45 do not inhibit the ribosome activity, the anti-L44' effectively blocks the polymerizing activity of the particles. These results show for the first time that the different eukaryotic acidic ribosomal proteins play a different functional role. PMID- 1706665 TI - Reduction of peripheral blood macrophages/monocytes in Kawasaki disease by intravenous gammaglobulin. AB - The effects of intravenous gammaglobulin (IVGG) on changes in the peripheral blood mononuclear cell subsets during acute Kawasaki disease (KD) were studied by a random selection trial of IVGG plus Aspirin (group G) compared to Aspirin alone (group A). Group G received IVGG with 200 mg/kg per day x 5 dose. The absolute counts of peripheral blood mononuclear cell subsets were assayed by a fluorescence-activated cell sorter using monoclonal antibodies of Leu series. Before therapy, patients in each treatment group had increased counts of CD14 + macrophage/monocytes compared to healthy childhood controls (P less than 0.01). After IVGG treatment group G underwent a greater decrease in their CD14 + macrophage/monocyte counts (P less than 0.01) than group A. The changes of CD3+ T cells. Leu 7+ NK/K cells and CD19+ B cells in the peripheral blood mononuclear cell subsets with treatment in group G, were similar to those in group A. These results suggest the possibility that IVGG therapy is effective in KD by modulating macrophages/monocytes. PMID- 1706666 TI - Hormone-resistant metastatic prostate cancer. Comparisons between estramustine phosphate and low-dose epirubicin treatments. AB - We compared the effect and toxicity of estramustine phosphate and weekly low-dose epirubicin in a prospective randomized trial in 41 patients with metastatic prostate cancer refractory to hormonal manipulation. No significant difference between treatment modalities was seen. Palliation was reached in over 60% of patients. The median survival was 15 months in both groups. Toxicity was mild. Further, we investigated the effect of epirubicin after the failure of preceding estramustine phosphate therapy in additional 20 patients. Pain relief was achieved in 50% of these patients. The median survival was 10 months. Toxicity was acceptable. PMID- 1706667 TI - The antisecretory effects of somatostatin and analogues in rat descending colon mucosa. AB - Somatostatin-14 (SS-14) and somatostatin-28 (SS-28) produce concentration dependent reductions in short-circuit current in rat colonic mucosa. EC50 values of 15.0 and 13.3 nM were obtained for SS-14 and SS-28 respectively while the N terminal fragments of SS-28, namely somatostatin-(1-12) (SS1-12) and somatostatin (1-14) (SS1-14) were inactive. Cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) and cyclo(Pro-Tyr D-Trp-Lys-Thr-Phe) were potent antisecretory peptides, like SS-14 and SS-28; while the putative somatostatin antagonist, cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys Thr[Bzl]) exhibited neither agonist nor antagonist effects. Responses to SS-14 could be regulated by agents which affected the secretory state of the epithelium. Antisecretory effects of SS-14 were markedly attenuated by piroxicam and were restored following piroxicam plus either forskolin or vasoactive intestinal polypeptide (VIP). SS-14 also attenuated secretory responses produced by carbachol, substance P (SP), VIP and alpha- and beta-calcitonin gene related peptide (alpha-, beta-CGRP). Therefore, SS-14 exhibits broad spectrum antisecretory effects in rat descending colon mucosa. PMID- 1706668 TI - [Characteristics on staining of leukocytes in experimental animals]. AB - The staining characteristics of the peripheral blood cells from mouse, rat, guinea pig, rabbit, dog, marmoset and monkey were studied. In marmoset, it is easy to distinguish neutrophils from eosinophils by using the phosphate-buffered solution of pH 5 or 6. It was found in the special staining methods that neutrophil granules showed intense peroxidase and Sudan black B reactions in marmoset in comparison with those in the other species of experimental animals. Neutrophil granules rabbit was, however, intensely stained with esterase and acid phosphatase. PMID- 1706669 TI - Distributional change of achromatic epithelial cells of the intestine in the fetuses and young of rats. AB - The achromatic epithelial cells (AEC), whose cellular materials remain unstained by several stains, e. g., H.E, PAS, Alcian blue, Orange G Anilin blue and Mucicarmin, are detected in the intestinal mucous epithelium of the Iar: Wistar Imamichi rat from the 19th day of gestation to the 21st day after birth. The results of light microscopic and transmission electron microscopic observations suggest that the rats AEC are equivalent to the previously reported vacuolar cells (VC) in cattle and in mice. The present paper describes that rat intestinal AEC appear first in the small intestine, and spread progressively toward the anal direction, while they disappear first in the cecum, and finally disappear at the ileum and the colon 22 days after birth. PMID- 1706670 TI - Bioactivity of synthetic human cholecystokinin (CCK)-33 in vitro and in vivo. AB - The relative potencies of synthetic human cholecystokinin (h-CCK)-33, porcine CCK 33 (p-CCK-33) and CCK-8 were examined by measuring pancreatic secretion in the conscious rat (in vivo) and amylase release from rat pancreatic acini using a perifusion study (in vitro). The increments of protein output during an 1-hr infusion of 100 pmol/kg/hr of h-CCK-33, p-CCK-33 and CCK-8 were 27.0 +/- 2.9 mg/hr (M +/- SE), 19.3 +/- 2.8 and 14.0 +/- 1.8 mg/hr, respectively. H-CCK-33 and p-CCK-33 showed significantly higher responses of protein output than CCK-8 in a same molar ratio, in vivo. In vitro, the stimulation with 10(-10) M h-CCK-33, p CCK-33 and CCK-8 led to a similar biphasic amylase release in a perifusion study. Twenty-five microM CR-1409, an antagonist for CCK receptor, completely inhibited the 10(-10) M h-CCK-33-stimulated amylase release. Although it was found that h CCK-33 and p-CCK-33 were more potent than CCK-8 in vivo, 10(-10) M CCK-8, h-CCK 33 and p-CCK-33 were equipotent on rat pancreatic acini in vitro. It is suggested that the discrepancy in potencies of the large molecular form and small molecular form of CCK in vivo and in vitro may be attributed to the delay of degradation of the large molecular form of CCK in vivo. PMID- 1706671 TI - Co-expression of X-hapten-like antigen and antigen YH206 on mucin molecules. AB - The asialocarbohydrate antigen YH206 is expressed on adenocarcinoma-associated mucin molecules which lack epitopes of CA19-9 and DU-PAN-2. To further characterize this molecule, the monoclonal antibody BM2 against the affinity purified antigen YH206 was established. It was demonstrated by an inhibition test that antigen BM2 was an X-hapten-like structure, one of the representative oncodevelopmental antigens. Although the sensitivity of antigen BM2 in sera of stomach and pancreas cancer patients did not appear to be superior to that of antigen YH206, both antigens were complementary to each other resulting in the improvement of sensitivity. Interestingly, double-determinant enzyme immunoassays showed that antigen BM2 and YH206, both having a cryptic nature for neuraminidase, were co-expressed on the same mucin molecule in sera of patients with stomach cancer or liver cirrhosis. These data suggest that mucin molecules in serum might be classified into several groups based on the distribution of tumor-associated epitopes. PMID- 1706672 TI - The diagnostic value of serum pancreatic phospholipase A2 (PLA2) in pancreatic diseases. AB - The diagnostic significance of serum immunoreactive pancreatic phospholipase A2 (PLA2) was studied in 119 patients with pancreatic disease, 200 with various non pancreatic disease, and 203 healthy controls using radioimmunoassay (RIA) specific to human pancreatic PLA2. This newly developed RIA using monoclonal antibody was satisfactorily sensitive and reliable. Serum PLA2 was elevated in all six patients with acute pancreatitis. Frequency of abnormal serum PLA2 levels was 60% in chronic pancreatitis (n = 52) and 67% in pancreatic cancer (n = 61). Serum PLA2 levels were low in chronic pancreatitis with severe exocrine insufficiency and advanced pancreatic cancer. In chronic pancreatitis, patients with low serum PLA2 level showed lower enzyme output in secretin test than patients with normal or high serum PLA2 level. Frequency of abnormal PLA2 levels was 27% in non-pancreatic disease and, in particular, patients with renal failure showed high PLA2 levels. Sensitivity (62%) and efficiency (69%) of serum PLA2 assay in pancreatic disease were superior to those of amylase. In conclusion, serum PLA2 determination using RIA was useful for the diagnosis of acute pancreatitis by high serum PLA2 levels and the diagnosis of severe exocrine pancreatic insufficiency by low serum PLA2 levels. PMID- 1706673 TI - [DNA diagnosis of beta-thalassemia. Study of restriction fragment length polymorphisms in families with affected children]. AB - A kit of DNA-probes directed at the cluster of human beta-globulin genes was used to study the incidence rate of 7 polymorphic restriction sites in beta thalassemia patients and normal donors in the Azerbaijan SSR. Informative polymorphic sites Hind III were detected in GJ and AJ fetal globin genes, Hinc II in psi beta and Hinc III in 3' area of psi beta gene and Ava II in beta-globine gene differing in the incidence rate in the patients and donors. An analysis of haplotypes with respect to informative sites was made in two Azerbaijan families with an affected child. It has been found that the analysis with respect to one informative site is sufficient for prenatal diagnosis of the status of the following children. PMID- 1706674 TI - [Evaluation of the effectiveness of infusion solutions based on electron microscopic studies]. AB - Two combined hemocorrectors have been developed for the treatment of shock terminal phases. The effectiveness of these infusion solutions was evaluated in the study of the liver and myocardium ultrastructure in experimental animals during the development of ischemic shock and its treatment. It was shown that a combination of the known agents (Rheopolyglucin, lactasol, mannitol and sodium succinate) favourably influenced the structure of the microcirculation bed, but did not affect metabolic disorders typical for a "shock cell". Inclusion into the combined hemocorrector of a molecular autocomplex, a derivative of 1,4 naphthoquinone, producing a membrane-protective effect, has significantly promoted the preservation and recovery of intracellular structures of the liver and myocardium cells. PMID- 1706676 TI - Memory is a property of an ion channels pool: ion channels formed by Staphylococcus aureus alpha-toxin. AB - The short-time depolarization effects on the integral conductance induced by S. aureus alpha-toxin (ST) in planar lipid bilayer membranes has been studied. Ion channels formed by ST were found to have several potential-induced nonconductance (closed) states. The transitions of ion channels between the states are only through one conductance state. The transition of ST-channels from closed to open state is induced by membrane depolarization. The amplitude current after a series of voltage pulses is a function of pulse number, and is effectively independent of the time interval between the neighbouring pulses. Therefore, a membrane which contains a pool of ion channels "remembers" its previous existence. A simple model can be used to explain this phenomenon. PMID- 1706675 TI - Effects of ruthenium red on excitation and contraction in muscle fibres with Ca2+ electrogenesis. AB - The effect of ruthenium red (RR) on the electrical and contractile responses, membrane Ca currents, staining patterns of the external and internal membrane system were tested in intact and mechanically skinned muscle fibres of the crayfish Astacus fluviatilis. The following results were obtained: 1. Depression of the contractile responses following membrane depolarization (twitch, tetanus, potassium contractures). 2. Caffeine contractures were unaffected in intact (100 mumol/l - 1 mmol/l RR) and blocked in skinned fibres (30 mumol/l RR). 3. Mechanical threshold and mechanical latency were increased and/or prolonged. 4. The rate of depolarization of the action potentials (AP) was decreased and decremental spread of AP was recorded. 5. Both fast and slowly inactivating Ca ionic currents were decreased and the time constants of activation (tau(m] and inactivation (tau(h] were prolonged after RR (100 mumol/l) pretreatment. 6. The penetration of RR into the T-system was inversely related to its binding to the sarcolemma. The depression of depolarization-induced contractions was most pronounced in fibres with unstained sarcolemma and stained T-tubules. In intact fibres, neither terminal cisternae nor other elements of SR were stained. On the contrary, all internal membrane structures were stained in skinned fibres. There was a gradient of staining intensity from surface toward the interior. PMID- 1706677 TI - DHP-sensitive Ca2+ channels from crayfish skeletal muscle T-tubules incorporated into planar lipid bilayers. PMID- 1706678 TI - [Expression of genes controlling modification of amylase in hybrids and original representatives of a cattle subfamily (Bovinae)]. AB - Modifications of similar type were noted in the number of multiple AMY-1 forms of amylase isozyme for various species of Artiodactyla. It is supposed that these modifications are linked to periodical activation during evolution of genes responsible for modification of the molecules. Data obtained on Bovinae hybrids testify to this point of view. Inactivation of active and reinactivation of "silent" genes responsible for modification of AMY-1 molecules are observed in hybrids of bison x cow and bison x aurochs combinations. PMID- 1706679 TI - [Para-aortic lymph node excision in cervix cancer]. PMID- 1706680 TI - Autoimmunity to collagen II and myelin basic protein: comparative studies in humans and rodents. PMID- 1706681 TI - Antigen recognition and peptide-mediated immunotherapy in autoimmune disease. PMID- 1706682 TI - Speculations on mechanisms of HLA associations with autoimmune diseases and the specificity of "autoreactive" T lymphocytes. PMID- 1706683 TI - [Cutaneous angiogenesis associated with vulvar epidermoid carcinoma]. AB - A case of cutaneous angiogenesis, probably induced by tumour angiogenic factors (TAF), is described. The patient, a 39-year-old female, affected by a vulvar squamous cell carcinoma, developed some erythematoangiomatous lesions on the lower abdominal quadrants, contiguously to the tumour. Histopathologically, the lesions were characterized by vascular neoformation in the superficial dermis and a slight lymphohistiocytic lesions resolved after the polychemotherapy which preceded enlarged vulvectomy. This supports the hypothesis of a close correlation between neoangiogenesis and TAF. PMID- 1706684 TI - Electrophoretic pattern of rodent seminal vesicle proteins as revealed by silver staining. AB - Electrophoresis of seminal vesicle secretions (SVS) from several rodents showed a very simple pattern composed of 3-5 main protein bands when an anionic dye (Coomassie brilliant blue) was used. However, use of a silver staining method showed a more complex protein spectrum, and several minor components of 12-90 kD, were clearly revealed. Western blotting using antibodies to SVS demonstrated that these minor protein components were not serum contaminants. Rat, mouse and hamster SVS shared antigenic determinants which were not related to rabbit SVS. PMID- 1706685 TI - Pharmacokinetics of iloprost in patients with chronic renal failure and on maintenance haemodialysis. AB - Iloprost is a potent, chemically stable prostacyclin-mimetic for which therapeutic efficacy has been proven in patients with peripheral arterial occlusive disease (PAOD) and in those suffering from Raynaud's phenomenon. In volunteers and PAOD-patients the pharmacokinetics of iloprost after intravenous (i.v.) infusion treatment was characterized by dose-dependent steady-state plasma levels, a terminal half-life of approximately 20-30 min, and a total clearance of 15-20 ml/min/kg. Bioinactivation was mainly due to beta-oxidation. In the present study the pharmacokinetics of iloprost was investigated in 21 patients suffering from renal insufficiency, which either required haemodialysis or not. They were treated by one hour i.v. infusion with 1 ng/kg/min and blood samples were taken during and after the end of infusion. Due to technical sampling problems iloprost pharmacokinetics could only be calculated for seven dialysis and eight non dialysis patients. In the dialysis patients steady-state levels were 114 to 320 pg/ml as compared to 36 to 70 pg/ml in the non-dialysis group. Half-lives were similar in both groups: alpha-phase: 0.05 h and beta-phase: 0.5 h. The total clearance was 2.6 to 8.0 ml/min/kg (dialysis patients) and 13.2 to 25.8 ml/min/kg (non-dialysis patients). The present study demonstrated that the pharmacokinetic profile of iloprost in patients with renal failure (not subject to haemodialysis) was similar to that observed in PAOD-patients and volunteers. In patients on maintenance haemodialysis, iloprost clearance was reduced by a factor of four. The iloprost dose regimen required in general (due to interindividual variability in response) a careful dose titration. PMID- 1706686 TI - Myasthenia gravis: prototype of the antireceptor autoimmune diseases. PMID- 1706687 TI - Presynaptic effects of toxins. PMID- 1706688 TI - Mechanisms of chemosensory transduction in taste cells. AB - The application of new techniques to the study of taste cells has revealed much about both the basic physiology of these cells and also about the mechanisms of taste transduction. The taste cells are electrically excitable cells with a variety of voltage-dependent ion currents. These ionic currents have an important role in the transduction of salt taste in mammals and frogs. In mudpuppies different ion channels are involved in the transduction of acidic-sour stimuli. The role of ion currents in the transduction of sweet taste is less clear. Some proposed mechanisms suggest an important role for ion currents and others suggest that the transduction process may be a biochemical event involving cell surface receptors and intracellular second messengers, possibly cAMP. The transduction of bitter taste seems to be a biochemical event involving cell surface receptors and intracellular second messengers in the inositol trisphosphate pathway. Thus, one cannot talk about "the mechanism" of taste transduction. Different taste modalities are transduced by different mechanisms. A corollary to this is that taste cells are not a homogeneous population of cells. In order to provide animals with the ability to discriminate between different taste modalities the taste cells consist of distinct subpopulations of cells based on their primary taste modality. The primary taste modality in a given cell is determined by the receptors and transduction mechanism(s) expressed in that cell. Evidence suggests that modality-specific receptors are expressed in a segregated manner in distinct subpopulations of taste cells. Secondary responses observed in gustatory axons may arise due to a lack of absolute specificity in the transduction processes and nonspecific effects of low pH and high ionic strength and osmolarity on the taste cells. An interesting area for future work will be to elucidate the mechanism(s) by which basal cells become committed to a given taste modality and how the gustatory neurons influence this process of differentiation. The involvement of the gustatory neurons is critical as they must synapse with taste cells of the correct taste modality to preserve the integrity of the information transferred to the CNS. This process of synaptogenesis is presumably mediated by the expression of taste-modality-specific, cell surface antigens on the basolateral domain of a taste cell and receptors on the appropriate neurons, but much work will be necessary to elucidate this process.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706689 TI - A case of mistaken identity. PMID- 1706690 TI - Binding of 16 alpha-[18F]fluoro-17 beta-estradiol to alphafetoprotein in Sprague Dawley female rats affects blood levels. AB - To examine the relationship between blood levels of 16 alpha-[18F]fluoro-17 beta estradiol(18F-ES) and serum alphafetoprotein (AFP) concentration, we undertook a study in which serum from various aged (20-33 days old) Sprague-Dawley female rats injected with 18F-ES was analyzed for both blood activity levels and AFP. There is a strong positive correlation between serum AFP concentration and 18F-ES blood levels (r = 0.914, P less than 0.001), suggesting that the binding of 18F ES by AFP has a significant effect on blood activity levels. The AFP concentration and ultimately the AFP-18F-ES binding is dependent on the age and weight of the rat: younger, as well as low weight rats exhibited high AFP concentrations and consequently increased 18F-ES blood activity. The rats most suitable for comparative studying of labeled estrogens are 25-28 days of age and weigh a minimum of 50-55 g. Thus, the use of the immature rat model to compare labeled estrogens requires a careful consideration of possible interference from blood binding proteins (i.e. AFP), as well as potential receptor binding competition from endogenous estrogens produced during the estrous cycle. Comparable consideration of blood binding proteins (sex steroid binding protein, SBP) and endogenous estrogens must be made in human studies, as well. PMID- 1706691 TI - Hb F-Catalonia or alpha 2G gamma(2)15(A12)Trp----Arg. AB - Hb F-Catalonia, a G gamma chain variant with a Trp----Arg substitution at position gamma 15(A12), was observed in two Spanish newborn babies from Northeastern Spain. Analyses were performed with different reversed phase high performance liquid chromatographic procedures that allowed the identification of the amino acid replacement in only a minute quantity of the isolated G gamma X chain. PMID- 1706692 TI - A simple approach to the determination of the gamma chain composition of HB F in adult human blood samples. AB - A procedure for the determination of the gamma chain heterogeneity of adult human blood samples with low Hb F has been developed. It consists of the isoelectrofocusing of lysates at a rather high hemoglobin concentration, the isolation of the focused Hb F by elution from the gel, and the analysis of the isolated hemoglobin by reversed phase high performance liquid chromatography for the separation and quantitation of globin chains. Depending on the isoelectrofocusing apparatus, many samples with as little as 1% Hb F may be focused in a single run and the gamma chain composition of the recovered hemoglobin (enriched from 15% to 95% in Hb F) easily analyzed by high performance liquid chromatography. The same procedure might be used for the isolation of low level hemoglobin variants for structural studies. PMID- 1706693 TI - Expression patterns of mRNAs for alpha-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity. AB - In developing and normal adult rat liver the expression patterns of the mRNAs for alpha-fetoprotein (AFP) and albumin (ALB) were analysed by in situ hybridization using specific 35S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto central distance decreasing in concentration going from the portal to the central area. Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung; furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moorman et al., 1990), suggesting that common regulatory factors are operative during development. PMID- 1706694 TI - Catecholamine-synthesizing enzymes and neuropeptides in rat heart epicardial ganglia; an immunohistochemical study. AB - The subepicardial atrial ganglia of rat hearts were examined using immunohistochemical techniques and antibodies against the catecholamine-synthetic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), and the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and met-5 enkephalin (ENK). Some of the ganglion cells present in the ganglia exhibited DBH like immunoreactivity (LI) and NPY-LI, whilst these cells never exhibited TH-, VIP-, CGRP-, SP- or ENK-LI. Groups of small cells exhibiting an intense TH-LI, corresponding to cells referred to as catecholamine-containing cells and sometimes small intensely fluorescent cells in the literature, were observed in the ganglia. A subpopulation of these cells exhibited immunoreactivity to one of the neuropeptides tested, namelyu SP. Only a few of the cells showing TH-LI displayed DBH-LI. Nerve fibres showing SP-, CGRP-, DBH- and TH-LI were present in the ganglia; some of these fibres being closely associated with the ganglion cells or with the cells showing TH-LI. The observation provide new information on the catecholamine-synthetic enzyme/neuropeptide expression of the ganglion and catecholamine-containing cells and of the associated nerve fibres of rat heart subepicardial ganglia. PMID- 1706695 TI - An improved technique for hyaluronan histochemistry using microwave irradiation. AB - A hyaluronan binding protein (HABP), extracted from cartilage, was biotin labelled and used for histochemical localization of hyaluronan (HA) in tissue sections. Various tissues were fixed for a mixture of formaldehyde and glutaraldehyde during microwave irradiation. The microwave oven when set at 700 W and 45 degrees C yielded an intense and specific staining of HA. Under these conditions the relative proportion of the two aldehydes did not influence the staining intensity. Aldehyde fixation during microwave irradiation for HA histochemistry, (1) save time, (2) eliminates the use of cetylpyridinium chloride (CPC), and (3) improves the reproducibility. PMID- 1706696 TI - Isolation, characterization, and in vitro culture of larval and adult epidermal cells of the frog Xenopus laevis. AB - Methods for the isolation and in vitro culture of larval and adult Xenopus laevis epidermal cells have been developed. Epidermal cells of stage 52-54 tadpoles and adult epidermal cells were enzymatically dissociated and purified (98%) by Percoll-density centrifugation and unit-gravity sedimentation. Both cell types attached on fibronectin-coated dishes and proliferated for 1 wk when the proper medium was used. There were four significant differences between larval and adult cells: a) Adult cells had a greater buoyant density than larval cells. b) Keratin synthesis patterns were markedly different. c) A combination of medium F12 and Eagle's minimum essential medium was optimal for growth of larval cells whereas MCDB151 medium was optimal for adult cells. d) Adult cells needed fetal bovine serum (greater than 5%) whereas larval cells grew without fetal bovine serum. In contrast to these differences, larval and adult cells had two similar properties: a) Insulin had a potent effect on the growth of both cells, and b) The optimal Ca++ concentration for cell growth was quite low for both cell types; 0.1 mM for larval cells and below 0.05 mM for adult cells. These results suggest that low Ca++ levels are essential for both cornifying (adult) and uncornifying (larval) amphibian keratinocytes. The culture techniques described herein for larval and adult epidermal cells provide a new in vitro model for analyzing development of the epidermis during amphibian metamorphosis. PMID- 1706697 TI - Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins. AB - Primary rat alveolar type II cells and early passage rat lung fibroblasts were co cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to "model", in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and "panning". Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co culture system at early passage. The type II cells formed a monolayer of dedifferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha2-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured. PMID- 1706698 TI - Fibronectin, not laminin, mediates heparin-dependent heparin-binding growth factor type I binding to substrata and stimulation of endothelial cell growth. AB - Fibronectin and heparin-binding growth factors (HBGF) are essential for growth of cultured endothelial cells. The stimulation of endothelial cell growth by HBGF type one (HBGF-1) in particular requires heparin or a similar glycosaminoglycan. The requirement for fibronectin and heparin for HBGF-1-stimulated endothelial cell growth may be related. HBGF-1 absorbed to the natural subcellular matrix of endothelial cells supports cell growth. [125I]HBGF-1 specifically associates with a sequentially reconstituted matrix of collagen-fibronectin-heparin, and HBGF-1 absorbed to the reconstituted matrix supports growth of the endothelial cells. A reconstituted matrix of collagen-laminin-heparin neither supported binding of [125I]HBGF-1 nor HBGF-1-stimulated endothelial cell growth. Association kinetics of [125I]HBGF-1 to heparinlike sites and membrane receptor sites on endothelial cell monolayers suggest that fibronectin-heparinlike binding sites in the subcellular matrix may be an obligatory reservoir of active HBGF-1 that binds to specific cell membrane receptors. PMID- 1706699 TI - Human endometrial epithelial cell lines for studying steroid and cytokine actions. AB - Recent studies suggest that the proliferation and expression of HLA-DR molecules in endometrial epithelium may be regulated by systemic steroids and local cytokines. To test the interacting influences of cytokines and steroids on the expression of HLA-DR and proliferation of epithelial cells, an endometrial cell model is required that is sensitive to both signals. In this study, we characterize cells of carcinoma cell lines of endometrial lineage for their responsiveness to cytokines and steroids. Independently developed for its response to steroid hormones from a well-differentiated adenocarcinoma of human endometrium, EnCa101AE cell line is further cloned for the expression of progesterone receptor. Immunohistochemical localization using monoclonal antibodies demonstrates that both EnCa101AE cell line and cloned ECC1 cells are purely epithelial, as evidenced by the expression of cytokeratin and epithelial membrane antigen, express estrogen receptors, and concomitantly exhibit IFN-gamma receptor. Experiments using radioiodinated IL-1 reveal that these cell lines also possess high affinity receptors for IL-1. As indicated by the induction of HLA-DR molecules, and alterations in morphologic characteristics, these cell lines are sensitive to both IFN-gamma and IL-1 action. The class II molecules (HLA-DR, HLA DP, and HLA-DQ) are differentially induced by IFN-gamma treatment in carcinoma cell lines, with HLA-DR being the prevailing induced molecule. IFN-gamma inhibits and estradiol-17 beta promotes growth of ECC1 cells in a dose- and time-dependent manner. These findings indicate that the interacting effect(s) of the cytokines and steroid hormones on endometrial epithelium may be studied in these unique steroid- and cytokine-sensitive epithelial cell lines. PMID- 1706700 TI - Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation. AB - The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability. Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold. Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message. The magnitude of the latter effect was strongly dependent on the sequences at the ribosome-binding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one. The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack. Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites. PMID- 1706701 TI - Negative autoregulation of cysB in Salmonella typhimurium: in vitro interactions of CysB protein with the cysB promoter. AB - CysB protein positively regulates genes of the Salmonella typhimurium cysteine regulon and negatively autoregulates cysB. The cysB promoter was characterized by primer extension of cellular RNA, which gave products identifying a major in vivo transcription start site located 95 bp upstream of the cysB start codon and two minor sites located 9 and 10 bp downstream of the major site. Gel shift binding studies and DNase I footprinting experiments showed that CysB protein binds to the cysB promoter from position -10 to +36 relative to the major transcription start site. We have designated this binding site CBS-B. CysB protein inhibited transcription initiation at the cysB promoter in an in vitro runoff assay, indicating that cysB is negatively autoregulated by the binding of CysB protein to the cysB promoter, where it acts as a repressor. N-Acetyl-L-serine, an inducer of the cysteine regulon, inhibited the binding of CysB protein to the cysB promoter and partially reversed the ability of CysB protein to inhibit transcription initiation. These effects are in contrast to those observed in studies of positively regulated cys promoters, in which N-acetyl-L-serine stimulates binding and causes CysB protein to activate transcription initiation. PMID- 1706702 TI - First step toward a virus-derived vector for gene cloning and expression in spiroplasmas, organisms which read UGA as a tryptophan codon: synthesis of chloramphenicol acetyltransferase in Spiroplasma citri. AB - Spiroplasmas are wall-less procaryotes in which the UGA codon serves not as a stop signal but as a code for the amino acid tryptophan. Spiroplasma genes that contain UGA codons thus cannot be studied in the usual Escherichia coli cloning and expression systems. Although this problem can be circumvented by using UGA suppressor strains of E. coli, spiroplasmas themselves would provide a more efficient cloning and expression host. We have now successfully employed the replicative form (RF) of a filamentous spiroplasma virus (SpV1) to clone and express the E. coli-derived chloramphenicol acetyltransferase (CAT) gene in Spiroplasma citri. The CAT gene was inserted in one of the four intergenic regions of the SpV1 RF and introduced into cells by electroporation. Both the RF and the virion DNA produced by the transfected cells contained the CAT gene sequences. Northern blot analysis, primer extension, and S1 mapping showed that transcription of the CAT gene started from a promoter located on the SpV1 RF and was terminated downstream of the CAT gene, still within the viral RF. Expression of the CAT gene was demonstrated by acetylation of chloramphenicol by cell-free extracts from the transfected spiroplasmas. PMID- 1706703 TI - Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes. AB - The plasmid pPR20 contains the gene tgt, which encodes tRNA guanine transglycosylase (Tgt), on a 33-kbp DNA insert from a region around 9 min on the Escherichia coli linkage map. The plasmid was subcloned to determine the sequence and organization of the tgt gene. Tgt is a unique enzyme that exchanges the guanine residue with 7-aminomethyl-7-deazaguanine in tRNAs with GU(N) anticodons. After this exchange, a cyclopentendiol moiety is attached to the 7-aminomethyl group of 7-deazaguanine, resulting in the hypermodified nucleoside queuosine (Q). Here we give the complete sequence of a 3,545-bp StuI-BamHI DNA fragment where we found the tgt gene and three previously unknown genes encoding proteins with calculated molecular masses of 42.5 (Tgt), 14, 39, and 12 kDa. The gene products were characterized on sodium dodecyl sulfate gels after synthesis in a combined transcription-translation system. The mRNA start sites of the open reading frames (ORFs) were determined by primer extension analysis. Plasmids containing the ORF encoding the 39-kDa protein (ORF 39) complemented a mutation in Q biosynthesis after the Tgt step. This gene was designated queA. The genes are arranged in the following order: ORF 14 (transcribed in the counterclockwise direction), queA, tgt, and ORF 12 (all transcribed in the clockwise direction). The organization of the promoter sequences and the termination sites suggests that queA, tgt, and ORF 12 are localized on a putative operon together with the genes secD and secF. PMID- 1706704 TI - Pleiotropic effects of a relC mutation in Streptomyces antibioticus. AB - Ochi (Agric. Biol. Chem. 51:829-835, 1987) has isolated a relaxed mutant of Streptomyces antibioticus, designated relC49, relC49 accumulates significantly lower levels of ppGpp than the parent stain, IMRU3720. At its maximum, the ppGpp level in relC49 was only one-fourth that observed in strain IMRU3720. Interestingly, a burst of ppGpp synthesis between 18 and 22 h of growth in IMRU3720 coincided with the onset of actinomycin production in that strain. As shown previously, the activity in protein synthesis of ribosomes from strain IMRU3720 decreases with the age of the culture. The decrease in activity was less pronounced in cultures of relC49. relC49 mycelium contains reduced levels of phenoxazinone synthase, a key enzyme involved in actinomycin biosynthesis. The rel mutation prevents the normal increase in the activity of one of the other enzymes required for production of the antibiotic, 3-hydroxyanthanilate-4 methyltransferase, and a third enzyme, actinomycin synthetase I, appears to be completely absent from relC49 mycelium. Levels of phenoxazinone synthease mRNA were examined by RNA dot blotting with the cloned phenoxazinone synthase gene as a probe. mRNA levels for phenoxazinone synthase were dramatically reduced in relC49 compared with strain IMRU3720. These results are discussed in terms of the possible regulation of the onset of actinomycin production by ppGpp. PMID- 1706705 TI - Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K 12. AB - Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12. Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion. In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of beta-galactosidase synthesis upon adding the missing amino acid was determined. Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments). Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids. Although codons for arginine, serine, and proline are interspersed among the codons for the three branched chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs. Deletions removing the terminator stem from the leader removed all ilv-specific control, indicating that the attenuation mechanism is the sole mechanism for ilv specific control. PMID- 1706706 TI - Characterization of the Cephalosporium acremonium pcbAB gene encoding alpha aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains. AB - A 24-kb region of Cephalosporium acremonium C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of Penicillium chrysogenum. A 3.2-kb BamHI fragment of this region complemented the mutation in the structural pcbC gene of the C. acremonium N2 mutant, resulting in cephalosporin production. A functional alpha aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6-kb EcoRI BamHI DNA fragment, as shown by complementation of an ACV synthetase-deficient mutant of P. chrysogenum. Two transcripts of 1.15 and 11.4 kb were found by Northern (RNA blot) hybridization with probes internal to the pcbC and pcbAB genes, respectively. An open reading frame of 11,136 bp was located upstream of the pcbC gene that matched the 11.4-kb transcript initiation and termination regions. It encoded a protein of 3,712 amino acids with a deduced Mr of 414,791. The nucleotide sequence of the gene showed 62.9% similarity to the pcbAB gene encoding the ACV synthetase of P. chrysogenum; 54.9% of the amino acids were identical in both ACV synthetases. Three highly repetitive regions occur in the deduced amino acid sequence of C. acremonium ACV synthetase. Each is similar to the three repetitive domains in the deduced sequence of P. chrysogenum ACV synthetase and also to the amino acid sequence of gramicidin synthetase I and tyrocidine synthetase I of Bacillus brevis. These regions probably correspond to amino acid activating domains in the ACV synthetase protein. In addition, a thioesterase domain was present in the ACV synthetases of both fungi. A similarity has been found between the domains existing in multienzyme nonribosomal peptide synthetases and polyketide and fatty acid synthetases. The pcbAB gene is linked to the pcbC gene, forming a cluster of early cephalosporin biosynthetic genes. PMID- 1706707 TI - Transcription of the stability operon of IncFII plasmid NR1. AB - The stability (stb) locus of IncFII plasmid NR1 is composed of an essential cis acting DNA site located upstream from two tandem genes that encode essential stability proteins. The stb locus was found to be transcribed from a promoter site just upstream from the first gene, stbA. This promoter was active for transcription both in vivo and in vitro and was located within the region that includes the essential cis-acting site. Transcripts initiated from this site were approximately 1,500 to 1,600 nucleotides in length. Northern (RNA) blot analysis indicated that the transcripts traversed both stbA and the downstream gene, stbB. Mutants from which the promoter had been deleted failed to produce detectable transcripts from either stbA or stbB. Transcription of a third open reading frame, stbC, which is contained within the stbB gene in the opposite DNA strand, could not be detected. For a mutant in which a transposon had been inserted in stbA, no transcription of stbB was detected. After deletion of most of the transposon, which left behind a 35-bp frameshift insertion in stbA, transcription of stbB was restored, although the insertion still had a polar effect on stbB function. The rate of in vivo transcription of the stb locus was measured by pulse-labeling of RNA followed by quantitative RNA-DNA hybridization. Mutants deleted of stbB had an approximately 10-fold increase in the rate of transcription, whereas those deleted of the promoter region had at least a 10 fold reduction in transcription rate. The half-life of stb mRNA was approximately 2 min. These data suggest that stbA and stbB are cotranscribed as an operon that may be autoregulated. PMID- 1706708 TI - Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids. AB - Minireplicons from the I-complex plasmids R387 (IncK) and pIE545 (IncZ) were constructed, and the nucleotide sequences of their replication regions were compared with that of the B plasmid, pMU720. The coding sequence of the putative replication protein, RepA, of each plasmid was located. RepA of K and B plasmids were homologous, whereas RepA of Z resembled RepA1 of FII plasmid. Sequences upstream of RepA were conserved in the three I-complex plasmids. Group B and Z plasmids were incompatible. PMID- 1706709 TI - The E1 functional epitope of the human interferon gamma is a nuclear targeting signal-like element. Mapping of the E1 epitope. AB - Eight neutralizing monoclonal antibodies (mAbs) directed against the human interferon gamma (HuIFN-gamma) that were classified in the E1 epitope group were mapped by the synthetic peptide approach. A set of 136 octapeptide homologs of the 143-residue primary sequence of the HuIFN-gamma, each one with a 7-residue sequence overlap with successive peptide, was synthesized. Based on the similar reactivity patterns of all the mAbs with this set of synthetic peptides, the E1 functional epitope was localized to residues 84-94 on the HuIFN-gamma. The epitope sequence is: Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Gln-Lys. The fact that eight independently isolated mAbs binding to the same domain can neutralize the HuIFN-gamma activity suggests that the E1 domain must be at or adjacent to a functional site. Within this domain is a sequence element, Lys-Lys-Lys-Arg, that resembles the nuclear location signals known to effect the intracellular transportation of a number of nuclear proteins, such as the large tumor antigen (T antigen) of simian virus 40 (SV40) and polyoma virus and steroid hormone receptors. This observation suggests that the HuIFN-gamma molecule and/or its complex with the receptor must function in the nucleus to effect transcription regulation that results in the various biological activities. The signal for that intracellular transportation must be provided by the HuIFN-gamma molecule. PMID- 1706710 TI - The amphipathic alpha-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells. AB - Nine monoclonal antibodies (mAbs) against apoA-I reacting with distinct but overlapping epitopes covering more than 90% of the sequence have been used to block the interaction of 125I-labeled high density lipoprotein (125I-HDL) with HepG2 cells in order to delineate the cell binding domain of apolipoprotein A-I (apoA-I). While 2 mAbs reacting with epitopes exclusively localized in the N terminal region (residues 1 to 86) enhanced slightly association of 125I-HDL, all other mAbs, which react with epitopes localized in the regions of amphipathic alpha-helical repeats, inhibited that association by 9 to 15%. Although this inhibition is not significant compared to the effect of an irrelevant mAb, combination of these mAbs could significantly inhibit the association of 125I-HDL (32 to 43%) as could polyclonal antibodies (up to 95%). These results are compatible with the concept of HDL binding to these cells via the nonexclusive interaction of each of the amphipathic alpha-helical repeats of apoA-I. When the same approach was applied to block the association of 3H-cholesteryl ether (CE) labeled HDL to HepG2 cells, each anti-apoA-I could inhibit by 15 to 25% the cellular association of cholesteryl ether while mAbs in combination or polyclonal antibodies could inhibit this association up to 45% or 60%, respectively. The cholesteryl ether radioactivity that remained associated with the cells (40%) in the presence of polyclonal antibodies could be effectively blocked by addition of an antibody against the receptor binding domain of apoE (1D7). Therefore, the differential cellular association of cholesteryl ether compared to apolipoprotein can be explained by the presence of apoE secreted by HepG2 and apoE or apoB/E receptors. Thus, we conclude that the optimum uptake of both cholesteryl ether and apoA-I of HDL by cells requires the accessibility of the entire apoA-I and the cooperative binding of the amphipathic alpha-helical repeats to HepG2 cell membranes. This type of interaction would explain the competitive binding observed for apoA-I, -A-II, and -A-IV by others. PMID- 1706711 TI - An inhibitory monoclonal antibody binds in close proximity to a determinant for substrate binding in cytochrome P450IIC5. AB - We used the expression of chimeric proteins and point mutants to identify amino acids of the hepatic progesterone 21-hydroxylase P450IIC5 which are part of an epitope recognized by an inhibitory monoclonal antibody and which affect substrate binding. Three amino acids of P450IIC5 at positions 113, 115, and 118 were introduced into P450IIC4, which is 95% identical to P450IIC5. The resultant chimeric protein acquired binding of the monoclonal antibody 1F11, which is highly specific and inhibitory for P450IIC5. Point mutants in P450IIC4 showed that two of the three changes, T115S and N118K, contribute to the epitope recognized by this antibody. The T115S mutant bound the antibody weakly (Kd greater than 30 nM) whereas the N118K mutant bound the antibody as tightly as P450IIC5 (Kd less than or equal to 0.7 nM). Thus, residues 115 and 118 are located on the surface of these enzymes, and the Lys/Asn difference at amino acid 118 is largely responsible for the high degree of discrimination which this antibody exhibits between P450IIC5 and P450IIC4. The valine to alanine mutation at position 113 conferred to P450IIC4 a lower apparent Km for progesterone 21 hydroxylation. Because antibody binding was not affected by this mutation, it is tempting to speculate that this residue is buried in the protein where it exerts its effect on the catalytic activity by interaction with the substrate or alters the positions of residues of the active site. The close proximity of the epitope at positions 115 and 118 to Ala113 suggests that the inhibitory monoclonal antibody interferes with substrate binding. PMID- 1706712 TI - Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. AB - The enzyme reverse transcriptase (RT) is crucial in the early steps of the life cycle of retroviruses. We have expressed in bacteria the RTs from human immunodeficiency viruses (HIV) types 1 and 2 in order to study the structural functional relationships of these two multifunctional enzymes that share a relatively high degree of amino acid sequence homology. For comparison purposes, we have analyzed several catalytic functions of both enzymes. The two HIV RTs show a high similarity in many aspects studied but exhibit profound differences in several other properties. For instance, the specific RNase H activity of HIV-2 RT is about 10 times lower than the corresponding activity of HIV-1 RT. There are also significant dissimilarities between some of the apparent Km values calculated for the DNA polymerizing functions of both enzymes. Furthermore, the heat stability of the DNA polymerizing activity of HIV-2 RT is about 15-fold higher than that of HIV-1 RT. On the other hand, the susceptibility of the RNase H activities of the two enzymes to heat inactivation was found to be similar. Other treatments also enable discrimination between the RNase H and DNA polymerizing catalytic properties of the two enzymes (although both reverse transcriptases respond similarily). Thus, the RNase H activity was inactivated by N-ethylmaleimide, suggesting the possible involvement of cysteine residues in performing this activity, whereas the DNA polymerizing functions of the two enzymes were fully resistant to this chemical modification. The zinc chelator 1,10-phenanthroline affected the DNA polymerase activities of both enzymes to a significantly higher extent than the RNase H activity. In all, the two HIV RTs were shown to be substantially different one from the other in several of their properties and also distinct from other RTs thus far studied. PMID- 1706713 TI - N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine. AB - Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine. PMID- 1706714 TI - Cotranscription, deduced primary structure, and expression of the chloroplast encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1. AB - The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology. PMID- 1706715 TI - Ionic events induced by epidermal growth factor. Evidence that hyperpolarization and stimulated cation influx play a role in the stimulation of cell growth. AB - Charybdotoxin, a blocker of K+ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca2+ channels, were employed in mouse NIH-3T3 fibroblasts overexpressing the epidermal growth factor (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell growth. The [Ca2+]i response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (greater than 3 x 10(-8) M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na+ and Ca2+. Pretreatment with 10 microM SC38249 induced only minor changes of the intracellular Ca2+ release by EGF (the process responsible for the initial phase of the [Ca2+]i and membrane potential responses) and blocked the persistent, second phase [Ca2+]i and the hyperpolarization responses, both dependent on Ca2+ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and growth of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, growth stimulated by EGF was inhibited, moderately (-20%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca2+]i sustained by Ca2+ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis. PMID- 1706716 TI - Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5. AB - Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes. PMID- 1706717 TI - The effects of actinomycin on the structure of dAn.dTn and (dA-dT)n regions surrounding its GC binding site. A footprinting study. AB - The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding. PMID- 1706718 TI - Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity. AB - The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication. PMID- 1706719 TI - Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31. AB - A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed. PMID- 1706720 TI - Heavy and light chain variable region sequences and antibody properties of anti phosphotyrosine antibodies reveal both common and distinct features. AB - Phosphotyrosine and similar analogs have been used to elicit antibodies that have found widespread use in the study of cellular tyrosine phosphorylation. In order to better understand the anti-phosphotyrosine immune response and to elucidate the details of the specific association between a tyrosine phosphate and an antibody combining site, we have undertaken a detailed comparison of antibody stability, specificity, apparent affinity, and primary structure for eight different anti-phosphotyrosine antibodies derived from immunizations with three different antigens. Two of these, 2G8 and 1G2, were derived from an immunization using azobenzylphosphonate conjugated to carrier, and five others, Py2, Py20, Py42, Py54, and Py69, were the products of an immunization with phosphotyrosine conjugated to carrier. Each of these anti-hapten antibodies was an IgG. One antibody, 129, an IgM, was the result of an immunization with a mixture of tyrosine-phosphorylated proteins which had been purified from growth factor treated cells. We found that antibody binding was significantly inhibited by millimolar levels of divalent cations or high concentrations of monovalent salt, with the exception of the antibody 129 where binding was significantly enhanced by both. Under optimal conditions, the highest apparent affinities for phosphotyrosine were observed for antibodies Py69 and Py20 (10(-6)-10(-7) M) and the lowest for 129 and 1G2 (10(-3)-10(-4). The heavy and light chain variable regions of seven of these antibodies were cloned and sequenced and a predominant anti-phosphotyrosine response was observed. The light chains of these antibodies could be assigned to one of two major VK groups, VK10 and VK19, with sequence identity between the different light chains of each class ranging from 65 to 100% at the amino acid level. Similar sequence identity was found among the heavy chain sequences (89-98% identity at the amino acid level) with the exception of one antibody, 2G8, which was only distantly related to the others (61-64% amino acid identity). These heavy chains belong to the same heavy chain family, J558. Two of the antibodies, Py20 and Py69, were clearly derived from the same progenitor cell since both share a highly unusual apparent V-D-D-JH organization. However, a significant level of somatic mutation has occurred between the two antibodies resulting in subtle changes in their apparent affinity and specificity. PMID- 1706721 TI - Protein-DNA interactions within the rat histone H4t promoter. AB - The histone H4t gene is actively transcribed in rat testis germinal cells and in several non-testis cell types including cells from liver. This histone H4 gene is expressed most actively in testis premeiotic pachytene spermatocytes, and its expression is down-regulated in postmeiotic early spermatids. The histone H4 gene promoter is functional as demonstrated by finding significant levels of chloramphenicol acetyltransferase (CAT) mRNA in mammalian cells transiently transfected with a histone H4-promoted CAT expression vector compared to cells transfected with an expression vector lacking of the promoter. Examination of the proximal promoter of the histone H4 gene by electrophoretic mobility shift assays indicates that specific protein-DNA interactions occur when pachytene spermatocyte nuclear proteins are mixed with promoter fragments containing regions designated site I and site II, consensus sequence elements essential for regulating transcription of the histone H4 gene. These specific protein-DNA interactions are eliminated by competition with identical DNA fragments, but are not eliminated by competition with nonhomologous DNA fragments. Significant differences are found in mobility shift patterns upon comparison of nuclear proteins from germinal cell populations enriched in pachytene spermatocytes where the gene is actively expressed and early spermatids where the gene is not expressed. PMID- 1706722 TI - Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells. AB - Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates. PMID- 1706723 TI - Effects of retinoic acid on NIH3T3 cell transformation by the H-ras oncogene. AB - Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 microM retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 microM retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming gene aph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14-20) resulted in alterations in focus morphology. A transformed cell line containing H ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes. PMID- 1706726 TI - Gas chromatographic assay for N,N-dimethylglycine in urine. AB - A gas chromatographic method for the determination of N,N-dimethylglycine in urine has been developed. After clean-up by cation-exchange, N,N-dimethylglycine was derivatized with ethanol and hydrochloric acid to form the corresponding ethyl ester. After evaporation of solvent, N,N-dimethylglycine ethyl ester was extracted into methylene chloride and chromatographed on a gas chromatograph equipped with a packed column containing 10% Carbowax 20 M. The detection limit of the method is 0.01 mM N,N-dimethylglycine in urine. This method has been used to detect N,N-dimethylglycine in urine from healthy subjects as well as in urine from patients with metabolic disorders. These findings were verified by gas chromatography-mass spectrometry. PMID- 1706724 TI - The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. AB - We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth. PMID- 1706725 TI - An internally positioned signal can direct attachment of a glycophospholipid membrane anchor. AB - All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition. PMID- 1706727 TI - Characterization of noncapsulate Haemophilus influenzae by whole-cell polypeptide profiles, restriction endonuclease analysis, and rRNA gene restriction patterns. AB - Thirty-four clinical isolates of noncapsulate Haemophilus influenzae representing isolates with either related or dissimilar patterns of whole-cell polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were further characterized by restriction enzyme analysis (REA) and rRNA gene restriction patterns. Total cellular DNA was extracted by a rapid, microcentrifuge-scale method and digested with BamHI, which gave a pattern of about 18 discrete bands. This confirmed the five closely related groupings suggested by SDS-PAGE. Isolates dissimilar by SDS-PAGE were also distinguishable by REA. However, there was no correlation between the degrees of similarity estimated from whole-cell polypeptide profiles and those obtained from REA for the dissimilar isolates. Therefore, inferences of genetic relatedness made on the basis of these data should be interpreted with caution. rRNA gene restriction patterns also confirmed the groupings suggested by the other two techniques. We conclude that the three methods were highly discriminatory and that whole-cell polypeptide patterns or REA with BamHI would be appropriate techniques for epidemiological studies of noncapsulate H. influenzae. PMID- 1706728 TI - Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. AB - The purpose of the study was to examine intercenter variability in the interpretation of Gram-stained vaginal smears from pregnant women. The intercenter reliability of individual morphotypes identified on the vaginal smear was evaluated by comparing them with those obtained at a standard center. A new scoring system that uses the most reliable morphotypes from the vaginal smear was proposed for diagnosing bacterial vaginosis. This scoring system was compared with the Spiegel criteria for diagnosing bacterial vaginosis. The scoring system (0 to 10) was described as a weighted combination of the following morphotypes: lactobacilli, Gardnerella vaginalis or bacteroides (small gram-variable rods or gram-negative rods), and curved gram-variable rods. By using the Spearman rank correlation to determine intercenter variability, gram-positive cocci had poor agreement (0.23); lactobacilli (0.65), G. vaginalis (0.69), and bacteroides (0.57) had moderate agreement; and small (0.74) and curved (0.85) gram-variable rods had good agreement. The reliability of the 0 to 10 scoring system was maximized by not using gram-positive cocci, combining G. vaginalis and bacteroides morphotypes, and weighting more heavily curved gram-variable rods. For comparison with the Spiegel criteria, a score of 7 or higher was considered indicative of bacterial vaginosis. The standardized score had improved intercenter reliability (r = 0.82) compared with the Spiegel criteria (r = 0.61). The standardized score also facilitates future research concerning bacterial vaginosis because it provides gradations of the disturbance of vaginal flora which may be associated with different levels of risk for pregnancy complications. PMID- 1706729 TI - Use of monoclonal antibodies against Rickettsia tsutsugamushi Kawasaki for serodiagnosis by enzyme-linked immunosorbent assay. AB - Monoclonal antibodies (MAbs) against Rickettsia tsutsugamushi Kawasaki were prepared. The crossreactivity tests of the MAbs performed by using antigenically distinct strains of R. tsutsugamushi in immunofluorescence and immunoblotting analyses indicated that the Kawasaki strain contains a strain-specific epitope and also contains a common epitope on the 56-kDa polypeptide cross-reactive with the Gilliam strain, group- and subgroup-specific epitopes on the 46-kDa polypeptide, and a subgroup-specific epitope on the 25-kDa polypeptide. By using the strain-specific MAb for serodiagnosis of tsutsugamushi disease (or scrub typhus fever), we have established a method which was designated the inhibition enzyme-linked immunosorbent assay. The principle of the method is to measure the percentage of inhibition of antigen absorption on a MAb-coated plate by antibody positive sample sera which were mixed with the antigen suspension. The advantages of this test for practical use are that (i) crude antigen can be used, i.e., purification of the antigen is not required; (ii) the test is more sensitive than immunofluorescence; (iii) the final judgment of plus or minus is clear-cut; and (iv) rickettsial antigenic types in the patients can be distinguished by this test. PMID- 1706730 TI - Genus-specific epitope on the 60-kilodalton Legionella heat shock protein recognized by a monoclonal antibody. AB - A monoclonal antibody (MAb) immunoglobulin G2a (2125) was produced against a 60 kDa Legionella heat shock protein (HSP), recognizing a unique epitope common to all species of the genus Legionella. The antibody reacted in the immunoblot with 59 Legionella species and serogroups that were tested and showed no cross reactivity with other bacteria, including Acinetobacter spp., Bordetella spp., Pseudomonas spp., Mycobacterium spp., and Escherichia coli. Two other MAbs (2122 and 2130) reacted with the 60-kDa Legionella protein as well but showed different cross-reactivities with other gram-negative bacteria in the same molecular mass range. The genus-specific MAb 2125 as well as the cross-reacting MAbs 2122 and 2130 were shown to be reactive with the expressed protein of the cloned gene of the 60-kDa HSP of Legionella micdadei and Legionella pneumophila. These antibodies demonstrate that Legionella-specific and nonspecific epitopes are present on this protein. A sandwich enzyme-linked immunosorbent assay (ELISA) in which the genus-specific MAb is used both as a capture antibody and as a biotinylated second antibody has been established. With this test it is possible to detect Legionella whole cells, sonicated cells, and cell fractions containing the 60-kDa HSP. The main part of the 60-kDa HSP is found in the cytoplasmic fraction. The sandwich ELISA can be used to demonstrate the increased expression of the 60-kDa protein in Legionella cells following heat shock as well as marked differences in the detection of the 60-kDa HSP on whole cells of different Legionella strains. The high specificity and sensitivity of the sandwich ELISA for sonicated cells might be very useful to screen on a genus level for Legionella cells or the 60-kDa antigen in environmental isolates or body fluids of patients. PMID- 1706731 TI - Prospective study of the association between serum antibodies to lipopolysaccharide O antigen and the attack rate of shigellosis. AB - A means for determining immune status against shigellosis would significantly improve the design and evaluation of interventional and other epidemiologic studies. Previous case-control studies have indicated the potential role of humoral antilipopolysaccharide antibodies. To test this proposition, 190 soldiers serving in a field unit were monitored prospectively for 2.5 months for shigellosis. Blood samples were taken at the beginning of the follow-up period and tested for serological evidence of prior exposure to Shigella sonnei and Shigella flexneri. The risk for acquiring S. sonnei shigellosis was 3.7 times higher for individuals lacking homologous antibodies (P less than 0.02). The risk for acquiring S. flexneri shigellosis was 2.4 times higher for individuals lacking antibodies, although a low attack rate for S. flexneri resulted in numbers too small to achieve statistical significance. While the importance of the serum antilipopolysaccharide antibodies in protection against the disease remains unclear, these findings demonstrate that they are strong markers of acquired immunity. Serological markers should be incorporated in epidemiologic studies of shigellosis and in the design and evaluation of future trials of potential anti-Shigella vaccines. PMID- 1706732 TI - Identification of coagulase-negative staphylococci by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and rRNA restriction patterns. AB - A total of 1,417 staphylococcal and micrococcal strains were collected from the beards and scalps of 10 subjects over a period of 8 months. Sixteen strains identified as Staphylococcus epidermidis with an API system had distinctive yellow colonies on nutrient agar plates and sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell polypeptide profiles similar to those of Staphylococcus capitis; this identification was confirmed by analysis of rRNA gene restriction patterns. PMID- 1706733 TI - Distribution of acetylcholinesterase in the hippocampal region of the mouse: I. Entorhinal area, parasubiculum, retrosplenial area, and presubiculum. AB - The distribution of acetylcholinesterase (AChE) was examined in the multilayered posterior part of the hippocampal region of the adult mouse (Mus musculus domesticus), namely, the entorhinal area, the parasubiculum, the presubiculum, and those parts of the retrosplenial cortex that extend into the posterior hippocampal region (area retrosplenialis 29d and 29e). A modification of the Koelle copper thiocholine method was employed for the histochemical demonstration of AChE. The AChE staining resulted in a distinctly stratified pattern, which has been compared in detail with the fields and layers defined by cyto- and fibro architecture. Most of the enzyme activity was located in the neuropil, but both moderately and intensely stained nerve cell bodies were observed too. In the entorhinal area two main subfields were identified, which have been designated pars medialis and pars lateralis. In pars medialis, the superficial two thirds of layer I, the interstices between the stellate cell bodies in layer II, and layers IV and VI showed moderate to high content of AChE, whereas layer V and, especially, layer III were poor in enzyme activity. A particular feature was the occurrence of cone-shaped, darkly stained areas within layer II and, occasionally, the deep part of layer I. The staining of pars laterais differed in several respects from that of pars medialis, the most prominent feature being a less conspicuous stratification. In addition, intensely stained somata occurred more frequently than in pars medialis, although they still constituted only a very small minority of the total number of nerve cell bodies. In the parasubiculum, a clear cytoarchitectural subdivision into a posterolateral parasubiculum a and an anteromedial parasubiculum b was observed. These subfields showed, however, only minor differences in AChE staining. Thus, in both subfields, layers I and IV stained intensely, whereas layers II and III showed moderate to intense staining. Layers V and VI did not differ in appearance from the corresponding layers of the entorhinal area. The retrosplenial areas 29d and 29e appeared very light in the AChE pattern, area 29e being the better stained. The presubiculum was very rich in AChE, with layers, I, III and IV being particularly intensely stained. The small nerve cell bodies of layer II were unstained, whereas the intervening neuropil was intensely stained. The distribution of AChE in the mouse was compared with that in the rat, guinea pig, and rabbit, described previously. The staining pattern is largely similar in all four species, but striking species-specific differences do exist.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706734 TI - "Sunbursts" in the inner plexiform layer: a spectacular feature of Muller cells in the retina of the cat. AB - A previously unrecognised structure in the cat retina is described. Seen in Golgi impregnated, wholemounted retinas, each such structure comprises processes radiating across the inner plexiform layer from a dense, vellate core. The processes are numerous, and largely unbranched, and give the impression of rays radiating from a point source; the structure is therefore termed a "sunburst." Evidence is presented from Golgi-impregnated retinas, and from retinas labelled with monoclonal antibodies to Muller cells, that the core of each sunburst is the inner process of a Muller cell. The sunbursts are numerous and overlap extensively, so that when neighbouring sunbursts are impregnated, they are seen to form a dense mat of processes extending across the IPL. It is suggested that each Muller cell forms a sunburst and that sunbursts form a major glial component of the neuropil of the inner plexiform layer. PMID- 1706735 TI - Trigeminal primary afferent projections to "non-trigeminal" areas of the rat central nervous system. AB - The central projections of rat trigeminal primary afferent neurons to various "non-trigeminal" areas of the central nervous system were examined by labeling the fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from the trigeminal ganglion. This technique produced a clear and comprehensive picture of trigeminal primary afferent connectivity that was in many ways superior to that which may be obtained by using degeneration, autoradiography, cobalt labeling, or HRP transganglionic transport techniques. Strong terminal labeling was observed in all four rostrocaudal subdivisions of the trigeminal brainstem nuclear complex, as well as in the dorsal horn of the cervical spinal cord bilaterally, numerous brainstem nuclei, and in the cerebellum. Labeling in the ipsilateral dorsal horn of the cervical spinal cord was very dense at C1, moderately dense at C2 and C3, and sparse at C4-C7. Numerous fibers crossed the midline in the medulla and upper cervical spinal cord and terminated in the contralateral pars caudalis and dorsal horn of the spinal cord from C1-C5. The latter axons terminated most heavily in the mandibular and ophthalmic regions of the contralateral side. Extremely dense terminal labeling was observed in the ipsilateral paratrigeminal nucleus and the nucleus of the solitary tract, moderate labeling was seen in the supratrigeminal nucleus and in the dorsal reticular formation, and small numbers of fibers were observed in the cuneate, trigeminal motor, lateral and superior vestibular nuclei, and in the cerebellum. The latter fibers entered the cerebellum in the superior cerebellar peduncle and projected to the posterior and anterior lobes as well as to the interposed and lateral deep cerebellar nuclei. Most projections in this study originated from fibers in the dorsal part of the spinal tract of V, suggesting a predominantly mandibular origin for these fibers. Projections from the ophthalmic and maxillary divisions, in contrast, were directed mainly to the cervical spinal cord bilaterally, to contralateral pars caudalis, and to certain areas of the reticular formation. In conclusion, this study has demonstrated that somatosensory information from the head and face may be transmitted directly to widespread and functionally heterogeneous areas of the rat central nervous system, including the spinal cord dorsal horn, numerous brainstem nuclei, and the cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1706736 TI - Scabies presenting as bullous pemphigoid-like eruption. AB - Several diseases, at times, may be confused with scabies. We report the diagnosis and treatment of scabies in two patients. Their eruptions recurred and persisted and eventually developed blisters. The skin biopsy specimens submitted for light microscopy and immunofluorescence were considered to be consistent with bullous pemphigoid. Both patients were treated successfully with lindane and remained disease free for up to 6 months of follow-up. PMID- 1706737 TI - Modified Fontan procedure for hypoplastic left heart syndrome after palliation with the Norwood operation. PMID- 1706738 TI - Bleeding time prolongation and bleeding during infusion of recombinant tissue type plasminogen activator in dogs: potentiation by aspirin and reversal with aprotinin. AB - Thrombolytic therapy is associated with a bleeding tendency that may be exacerbated by adjunctive antiplatelet agents. The effect of recombinant tissue type plasminogen activator (rt-PA) alone or in combination with aspirin on serial measurements of template bleeding time, ex vivo platelet aggregation and coagulation factors and the frequency of bleeding was studied in dogs. During infusion of rt-PA (15, 30 or 60 micrograms/kg per min for 90 min), a dose-related increase in bleeding time was observed. In a randomized blinded study of 25 dogs, the baseline bleeding time (mean +/- SD) was 3.5 +/- 1 min in control animals and 4 +/- 2 min after oral aspirin (15 mg/kg body weight). Infusion of rt-PA (15 micrograms/kg per min for 90 min) prolonged the bleeding time to a maximum of 15 +/- 12 min. In contrast, combined aspirin and rt-PA therapy produced an increase to greater than 30 min during infusion, reverting to 13 +/- 10 min within 2 h after cessation of infusion. Recurrent continuous bleeding from incision sites occurred in one of six dogs given aspirin alone, two of seven given rt-PA alone and all six dogs given both aspirin and rt-PA (p = 0.02). Bleeding time greater than 9 min correlated significantly with bleeding frequency (p less than 0.0001), with a sensitivity of 100% and a specificity of 87%. Intravenous bolus injection of aprotinin (20,000 kallikrein inhibitor units/kg body weight) in six dogs given both rt-PA and aspirin produced a decrease in bleeding time from greater than 30 min to 9.5 +/- 9 min and resulted in cessation of bleeding. Thus, bleeding and bleeding time prolongation in this canine model are potentiated by a marked interactive effect of rt-PA and aspirin that is rapidly reversible. Template bleeding times may provide a useful quantitative index for monitoring the bleeding tendency associated with thrombolytic therapy. PMID- 1706739 TI - The biological basis for clinical use of interferon. AB - Interferons represent the body's most rapid defence against virus infection. Natural recovery from viral infections is correlated with interferon production; the inhibition of interferon enhances the severity of infection and interferon has been shown to protect animals from some viral infections. beta-Interferon is produced by fibroblasts in response to viral infection. Leucocyte (a) interferon may be induced by foreign or infected cells or by viruses, and it is continuously released into the bloodstream during infection, declining with time. gamma Interferon are produced later in viral infection by virus-sensitized T lymphocytes. The antiviral action of interferon is induced by interaction with specific receptors on cell surfaces leading to translation of antiviral proteins. Interferons may also enhance immune responses by increasing expression of lymphocyte surface antigens and by increasing the cytotoxic activity of natural killer cells. In chronic hepatitis B and C, it seems likely that interferon's antiviral activity is responsible for the early fall of viral products, while its immunomodulatory activity is responsible for long-term effects, including the destruction of infected hepatocytes, with persistent loss of viral antigens and seroconversion. PMID- 1706740 TI - The hepatitis B virus and the host response. AB - The clearance of hepatitis B virus infected cells from the liver is probably dependent on an interplay between the interferon system and the cellular limb of the host immune response. Although the importance of the nucleocapsid proteins as targets for sensitized cytotoxic T cells is established in chronic hepatitis B infection, further studies are needed during the early phase of acute infection. The relative importance of pre-S sequences as inducers and targets of the virus neutralizing humoral immune response is becoming established but their precise place will await the development of in vitro models of hepadna virus infection and precise definition of the mechanism of viral uptake. In adult life, deficient production of alpha-interferon and suppression of the ability of the host to respond to interferon are probably important factors underlying the development of chronic infection. In the neonate, however, specific suppression of the cell mediated immune response may be involved. The presence of a mutation in the pre core region of some virus isolates has recently been described. Hepatocytes infected with this virus cannot produce hepatitis B e antigen and the course of the liver disease is fairly rapid. Whether this mutant causes liver damage in the same way as the wild virus or is directly cytopathic needs further study. In adult-acquired chronic hepatitis B virus infection, alpha-interferon produces hepatitis B e antigen clearance in 26-88% of cases and is followed by resolution of the hepatic inflammation. Results in neonatally acquired infection are less impressive and prednisolone priming followed by interferon may be needed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706741 TI - Possible prevention of chronic hepatitis B by early interferon therapy. AB - A study is currently underway to investigate the efficacy of interferon therapy in patients with prolonged (greater than or equal to 10 weeks but less than 6 months) hepatitis B infection. To date, a total of 15 patients have been enrolled in the study and randomly assigned to receive either placebo for 24 weeks (n = 8) or interferon 5 million units subcutaneously 3 times a week for 24 weeks (n = 7), with follow up for 1 year. Thirteen patients have completed the follow-up period: seven patients in the placebo group and six in the treated group. Five of the six treated patients completely eradicated the infection during interferon therapy, with clearance of hepatitis B e and surface antigens, and seroconversion to antibody positivity in each case. Two of the eight placebo patients seroconverted during the placebo period. Clearance of hepatitis B e antigen was associated with a sudden rise in serum transaminase levels and an exacerbation of hepatitis, a phenomenon that has also been reported in chronic hepatitis B patients who have responded well to interferon therapy. Therapy was well tolerated in all cases. Our results suggest that interferon treatment of patients with prolonged hepatitis B infection may prevent progression to chronicity. If confirmed by further study, they should trigger more vigilant screening for patients with raised serum transaminase levels and viral markers of hepatitis B infection. PMID- 1706742 TI - The pattern of fibrosis in the acinar zone 3 areas in early alcoholic liver disease. AB - The degree of fibrosis and the pattern of collagen distribution in the acinar zone 3, as well as the thickness of the terminal hepatic vein walls (THV) were analyzed in 48 consecutive liver needle biopsies from 48 alcoholics with preserved liver architecture. The fibrosis occurred to more or less the same degree in the whole circumference of each THV. A positive correlation was found between the degrees of perisinusoidal/pericellular fibrosis (PSF) and the occurrence of incomplete or complete septa. The thickness of THV walls (TTHV) varied from 1.0-12.5 microns with a median of 3.9 microns. No relationship was found between TTHV and PSF. The results were compared to similar data obtained in liver biopsies from 117 non-alcoholics with normal morphology or slight non specific changes. No significant difference concerning TTHV and THV diameter was found between alcoholic and non-alcoholic patients. The results suggest that the initial liver fibrosis in alcoholics is slightly asymmetrical distributed in each acinar zone 3 area. With progression, the fibrosis tends to be more uniformly distributed and septa appear, eventually linking THV with portal tracts. Apparently, thickening of the THV walls does not occur in early alcoholic liver disease. PMID- 1706743 TI - Production and characterization of the murine monoclonal antibody 2G10 to a human T4-tyrosinase epitope. AB - Employing as immunogen a short-term passaged, highly pigmented human melanoma cell line, we have produced the murine MoAb 2G10 of the IgG1 isotype. The antibody immunoprecipitated from 35S-methionine and 3H-glucosamine metabolically labeled human melanoma cells with a single-chain glycoprotein of 75 kD molecular weight. No such molecule could be precipitated from murine melanomas. To further investigate the fine specificity of the MoAb, immunochemical and immunohistochemical studies were performed. These studies demonstrated that MoAb 2G10 binds a significant fraction of tyrosinase activity from cell lysates, completely immunodepletes soluble cell extract of T4-tyrosinase molecules, and produces immunostaining patterns superimposable on those obtained with anti-T4 tyrosinase antibodies. Thus, MoAb 2G10 appears to recognize a human-specific determinant carried by either T4-tyrosinase or a closely related molecule. The functional relevance of this epitope remains to be evaluated. PMID- 1706744 TI - Regulation of collagen gene expression by transformed human fibroblasts: decreased type I and type III collagen RNA transcription. AB - The regulation of collagen gene expression in normal diploid human fetal fibroblasts (KMS-6 cells), and fibroblasts immortally transformed by treatment of KMS-6 with Co-60 gamma rays (KMST-6 cells) was compared to that of ones tumorigenically transformed by treatment of KMST-6 cells with Harvey murine sarcoma virus (KMST-6-Ras cells). Synthesized collagenous protein decreased to approximately 30% of that of normal fetal fibroblasts in both transformed cell lines, and the relative rate of collagen synthesis to total protein synthesis decreased about sixfold in KMST-6 cells and twelvefold in KMST-6-Ras cells. The m RNA levels of type I collagen in both of these cell lines decreased to approximately 20% of that of the control fibroblasts, whereas type III collagen m RNA levels decreased to only 9% of that of the control. The copy number of the collagen gene in both transformed cell lines was unaltered. The transcriptional rates of collagen alpha 1(I) and collagen alpha 1(III) in both cell lines decreased to 20% and 7% respectively of that of control. These data indicate that collagen synthesis was reduced at the transcriptional level in these transformed human fibroblasts. PMID- 1706745 TI - The monoclonal antibodies TMH-1 and TMH-2 specifically bind to a protein encoded at the murine b-locus, not to the authentic tyrosinase encoded at the c-locus. AB - Three hybridomas, TMH-1, TMH-2, and TMH-3, were previously reported by Tomita et al to produce monoclonal antibodies against murine and human T4-tyrosinase localized in melanosome for the formation of melanin pigment. However, TMH antibodies were unable to react with K1735 cells transfected with the authentic tyrosinase-cDNA construct, but did react with those transfected with the pMT4 cDNA construct. The cDNA pMT4 was initially cloned as a putative tyrosinase cDNA by Shibahara et al, but it is now known to encode mouse brown (b) locus protein, which was named "tyrosinase-related protein" by Jackson or "b protein" by Hearing and Jimenez. Furthermore, TMH antibodies recognize hair bulbs of C57BL/6J-c2J/c2J mouse (B/B, c/c) lacking tyrosinase activity, but do not recognize hair bulbs of b-locus mutated DBA/2 mouse (b/b, C/C), which have authentic tyrosinase. Considering these observations, we conclude that TMH antibodies specifically recognize the protein encoded at b-locus. PMID- 1706746 TI - Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis. AB - The expression of three immunoregulatory surface antigens by epidermal keratinocytes was studied in irritant contact dermatitis (ICD), in order to assess whether keratinocytes have a modulatory role in the pathogenesis of this disorder. Biopsies were taken from 48-h patch test reactions to six structurally unrelated irritants, and frozen sections immunolabeled with monoclonal antibodies to the major histocompatibility complex class II antigen, HLA-DR, intercellular adhesion molecule-1 (ICAM-1), and the 88-Kd glycoprotein CD36 (OKM5), as well as to the CD3 (T cells) and CD11a (lymphocyte function associated antigen-1, LFA-1) antigens. We found that there was very limited expression of HLA-DR by keratinocytes, with no correlation between the extent of HLA-DR positivity and the degree of T cell infiltration into the epidermis and dermis, suggesting that interferon gamma may not be a significant mediator of ICD at 48 h. In contrast, keratinocytes showed extensive upregulation of ICAM-1, with an excellent spatial association between ICAM-1 expression and LFA-1 positive leucocytes in the epidermis. This indicates that keratinocyte ICAM-1 induction is not restricted to diseases in which antigen presentation is pivotal, but that it has a generalized role in cutaneous inflammatory reactions, promoting the infiltration of leucocytes into the epidermis. Immunolabeling with OKM5 revealed that CD36 is present to a variable degree on keratinocytes in normal skin. Differential changes in the pattern of keratinocyte expression occurred between irritants, in a manner that suggested that the CD36 antigen does not act as an adhesion molecule in ICD, but rather that its expression is related to the proliferative state of the epidermis. The results of this study demonstrate that immune associated antigens are selectively expressed on the surface of keratinocytes in 48-h ICD biopsies, implying that these cells play an important regulatory role in the development of the inflammatory response to irritant chemicals. PMID- 1706747 TI - Adhesion molecules on the plasma membrane of epidermal cells. III. Keratinocytes and Langerhans cells constitutively express the lymphocyte function-associated antigen 3. AB - It is now becoming clear that a collection of adhesion molecules is required on the surface of epidermal cells (EC) to establish the cell interactions that are necessary for skin immunologic reactions. In previous studies, we showed that human resting Langerhans cells (LC) express at least two members of the "integrins" family of adhesive molecules, as well as the intercellular adhesion molecule-1, which is a member of the immunoglobulin-related superfamily of molecules. This latter family includes another adhesive moiety, namely, the lymphocyte function-associated antigen-3 (LFA-3), which is the ligand for the T lymphocyte-associated CD2 molecule, and has a broad tissue and organ distribution. In the present investigation the colloidal gold immunoelectronmicroscopy immunostaining system and a quantitative analysis of the labeling provided decisive evidence for the weak but clear LFA-3 expression on virtually all keratinocytes (KC) and LC freshly isolated from normal human skin. Such constitutive expression of LFA-3 molecule on EC may be relevant for a number of functional interactions between LFA-3-positive EC and CD2-positive T lymphocytes within the cutaneous environment. PMID- 1706748 TI - [Drug therapy of bacterial infections in patients with granulocytopenia]. PMID- 1706749 TI - [A case of primary liver squamous cell carcinoma producing granulocyte colony stimulating factor]. PMID- 1706750 TI - Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. AB - Transgenic murine lines have been constructed that express a chimeric class I molecule composed of the alpha 1 and alpha 2 domains of HLA-A2.1 and the alpha 3, transmembrane, and cytoplasmic domains of H-2Kb. Upon immunization with influenza virus, transgenic mice developed a strong A2.1Kb-restricted cytotoxic T lymphocyte (CTL) response specific for the same matrix protein epitope that serves as the dominant A2.1-restricted determinant in the equivalent human response. Fine specificity analysis of CTL clones using truncated peptides revealed strong similarity between the response repertoire of transgenic mice and that previously reported using influenza-specific A2.1-restricted CTL clones from humans. This suggests that even when considering T cell responses by different species, the alpha 1 and alpha 2 domains of the restriction element play a dominant role in determining the CTL specific repertoire. Thus, substituting the alpha 3 domain of A2.1 with a murine counterpart has permitted development of a transgenic strain that should serve as an excellent model system in studies of HLA-restricted responses. PMID- 1706751 TI - T cell receptor-independent CD2 signal transduction in FcR+ cells. AB - CD2 subserves both adhesion and signal transduction functions in T cells, thymocytes, and natural killer (NK) cells. In mature T lymphocytes, CD2-mediated signaling function apparently requires surface expression of T cell receptors (TCRs). In contrast, in CD2+ CD3- NK cells and thymocytes, signal transduction through CD2 is TCR independent. To resolve this paradox and characterize TCR independent triggering mechanisms, we transfected a human CD2 cDNA into a murine mast cell line, C1.MC/57 (Fc epsilon RI+, Fc gamma RII+, Fc gamma RIII+), which is known to produce interleukin 6 (IL-6) as well as release histamine in response to crosslinking of Fc epsilon RI. In the CD2 transfectant, a combination of anti T11(2) + anti-T11(3) monoclonal antibodies (mAbs) induced a rise in intracellular free calcium [( Ca2+]i), IL-6 production, and histamine release. As expected, no activation was mediated by the same mAbs in C1.MC/57. F(ab)'s fragments of the activatory combination of anti-T11(2) + anti-T11(3) mAbs induced IL-6 in the CD2 transfected mast cells, demonstrating an Fc gamma receptor ectodomain-independent triggering mechanism. In addition, either intact anti-T11(2) or anti-T11(3) IgG alone, which failed to induce [Ca2+]i mobilization in the transfectant, was able to induce IL-6 production. A mAb directed against both Fc gamma RII (previously denoted as Fc gamma RIIb) and Fc gamma RIII (previously denoted as Fc gamma RIIa) inhibits this induction. These results indicate that: (a) Ca2+ mobilization is not essential for IL-6 production; and (b) crosslinking of CD2 and Fc gamma receptors via intact anti-CD2 IgG stimulates IL-6 production. Thus, CD2-mediated IL-6 production occurs by both Fc receptor ectodomain-independent as well as Fc receptor ectodomain-dependent mechanisms in these nonlymphoid cells. Northern blot analysis demonstrates that although the mast cells do not express CD3 zeta or CD3 eta mRNA, they express Fc epsilon RI gamma mRNA. The latter is a known component of Fc gamma RIII as well as Fc epsilon RI, has significant homology to CD3 zeta/eta, and is thought to have a signal transduction function. In these mast cells, CD2 signaling machinery does not require CD3 zeta/eta and may be linked to the Fc epsilon RI gamma subunit. We predict that this subunit or a related structure may confer a TCR-independent signal transduction pathway upon CD2 in CD3- NK cells, thymocytes, and certain B lymphocytes. PMID- 1706752 TI - Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo. AB - We have investigated the effect of granulocyte colony-stimulating factor (G-CSF) delivery at the site of tumor growth by transducing, via retroviral vector, the human (hu) G-CSF gene into the colon adenocarcinoma C-26 and assaying the ability of transduced cells to form tumors when injected into syngeneic mice. As a control, the same tumor cells were infected with retroviruses engineered to transduce an unrelated gene, the human nerve growth factor receptor, or carry the neomycin resistance gene only. Only cells transduced with the huG-CSF were unable to develop tumors, although huG-CSF was expressed and produced at low level as estimated by both RNA analysis and enzyme-linked immunosorbent assay, indicating that G-CSF can exert an antitumor effect at a physiological dose. Implication of G-CSF as mediator of tumor inhibition was proven by reversing the nontumorigenic phenotype of G-CSF-expressing cells with anti-huG-CSF monoclonal antibody injected at the tumor site. No tumors were formed by injecting C-26 infected cells into nu/nu mice, while neoplastic nodules appeared after injection into sublethally irradiated mice; such tumors, however, regressed when mice normalized their leukocyte counts after irradiation. Tumors were also formed after injection of a mixture of infected and uninfected C-26 cells, although critical delay in tumor formation occurred when infected cells were 10 times more represented in the mixture. Histological examination of tissues surrounding the site of injection showed infiltration of neutrophilic granulocytes, whose number correlated with that of G-CSF-expressing C-26 cells in the injected mixture. These results indicate that G-CSF may have a potent antitumoral activity when released, even at low doses, at the tumor site. The antitumoral effect is mediated by recruitment and targeting of neutrophilic granulocytes to G-CSF releasing cells. PMID- 1706753 TI - Structural assignment of novel and immunodominant antigenic sites in the neutralizing antibody response of CBA/Ca mice to influenza hemagglutinin. AB - Information on the antigenic structure of influenza hemagglutinin (HA) has been deduced previously from sequence analyses of laboratory mutant viruses selected, in vitro, with neutralizing monoclonal antibody (mAb) established exclusively from BALB/c (H-2d) mice; and there has been no attempt to investigate the influence of host genetic background, or natural route of infection, on the protective antibody repertoire. CBA/Ca mice are extremely sensitive to X31 virus infection, and in the present study a structural analysis was made of the antibody repertoire, by direct sequencing of the HA genes of laboratory mutant viruses selected, in ovo with mAb from CBA/Ca mice primed by natural infection with X31 virus at two different infectious doses. Single nucleotide substitutions in the HA genes of mutant viruses identified both novel and immunodominant antigenic sites on the HA1 subunit: a majority of mAbs, from different donors, were of the IgG2a isotype and were specific for HA1 158 Gly. In addition, novel laboratory mutants were obtained containing substitutions in the HA1 subunit that had not been reported previously for H3 subtype viruses, either natural variants or laboratory mutants, at residues: HA1 62 Ile----Arg; HA1 165 Asn----Ser (resulting in the loss of a N-glycosylation site); and HA1 273 Pro----Leu. Our findings suggest that host genetic background and/or a natural route of infection may be significant factors in the selection of different and distinct neutralizing antibody responses to influenza HA and therefore be of some relevance in our further understanding of the immune pressure for antigenic drift, and the immunogenic features of a protective antigen. PMID- 1706754 TI - Infection of human thymocytes by Epstein-Barr virus. AB - The Epstein-Barr Virus (EBV) causes infectious mononucleosis, and has been strongly associated with certain human cancers. The virus is thought to exclusively bind to B lymphocytes and epithelial cells via receptors (CR2/CD21) that also interact with fragments of the third component of complement (C3). Recent evidence, however, has challenged this belief. We have used two-color immunofluorescence analysis using biotin-conjugated EBV and streptavidin phycoerythrin along with fluorescein-conjugated anti-T cell antibodies and demonstrated that CD1-positive, CD3-dull (immature) human thymocytes express functional EBV receptors. In four replicate experiments, the binding of EBV to thymocytes ranged between 8 and 18%. This interaction is specific as evidenced by inhibition with nonconjugated virus, anti-CR2 antibodies, aggregated C3, and an antibody to the gp350 viral glycoprotein that the virus uses to bind to CR2. EBV can infect the thymocytes as evaluated by the presence of episomal EBV-DNA in thymocytes that had been incubated with the virus as short as 12 days or as long as 6 weeks. Episomal DNA analysis was performed by Southern blotting with a EBV DNA probe that hybridizes to the first internal reiteration of the viral DNA. The presence of the EBV genome is also supported by the detection of EBV nuclear antigen 1 in infected thymocytes, assessed by Western blotting with EBV-immune sera. The EBV infection is specific as determined by blocking experiments using anti-CR2 and anti-gp350 antibodies. Finally, virus infection of thymocytes can act synergistically along with interleukin 2 and induce a lymphokine-dependent cellular proliferation. In view of previously reported cases of EBV-positive human T cell lymphomas, the possibility is raised that EBV may be involved in cancers of T lymphocytes that have not been previously appreciated. PMID- 1706755 TI - Current recording from sensory cilia of olfactory receptor cells in situ. I. The neuronal response to cyclic nucleotides. AB - The olfactory mucosa of the frog was isolated, folded (the outer, ciliated side faced outward), and separately superfused with Ringers solution on each side. A small number of sensory cilia (one to three) were pulled into the orifice of a patch pipette and current was recorded from them. Fast bipolar current transients, indicating the generation of action potentials by the receptor cells, were transmitted to the pipette, mainly through the ciliary capacitance. Basal activity was near 1.5 spikes s-1. Exposure of apical membrane areas outside of the pipette to permeant analogues of cyclic nucleotides, to forskolin, and to phosphodiesterase inhibitors resulted in a dose-dependent acceleration of spike rate of all cells investigated. Values of 10-20 s-1 were reached. These findings lend further support to the notion that cyclic nucleotides act as second messengers, which cause graded membrane depolarization and thereby a graded increase in spike rate. The stationary spike rate induced by forskolin was very regular, while phosphodiesterase inhibitors caused (in the same cell) an irregular pattern of bursts of spikes. The response of spike rate was phasic tonic in the case of strong stimulation, even when elicited by inhibitors of phosphodiesterase or by analogues of cyclic nucleotides that are not broken down by the enzyme. Thus, one of the mechanisms contributing to desensitization appears to operate at the level of the nucleotide-induced ciliary conductance. However, desensitization at this level was slow and only partial, in contrast to results obtained with isolated, voltage-clamped receptor cells. PMID- 1706756 TI - Effect of osmotic compression on the force-velocity properties of glycerinated rabbit skeletal muscle cells. AB - The force-velocity relations of single glycerinated rabbit psoas muscle fibers at 5 degrees C were studied at maximum and half-maximum activation in the presence of 0 (control) and 39-145 g/liter dextran T-70. Resting fiber diameter decreased progressively to approximately 70% of the nondextran control as the dextran concentration was increased. Isometric force at full activation increased to a maximum of 136% of control at 111 g/liter dextran and then fell to 80% of control in 145 g/liter dextran. Maximum velocity, which fell to 49% of the control value in the highest concentration of dextran, was nearly constant at approximately 65% control over the range of 58-111 g/liter dextran. Relative maximum power, which gives an estimate of changes in intermediate velocity, was not significantly reduced by dextran concentrations up to 76 g/liter, but then fell progressively to 62% of control in the highest concentration of dextran. At half-maximum activation, maximum velocity and relative maximum power were not significantly different from the values at full activation. The results obtained at partial activation indicate that the decline of velocity seen in the presence of dextran is not due to a passive internal load and that the dextran does not cause a viscous resistance to shortening. The increased velocity in the absence of dextran can be explained by the reduced ability of cross-bridges to resist shortening, as proposed by Goldman (1987. Biophys. J. 51:57). PMID- 1706757 TI - Lymphoproliferative and cytotoxic responses of human peripheral blood mononuclear cells to mannoprotein constituents of Candida albicans. AB - Two major proteoglycan constituents (designated F1 and F2) of the cell wall of Candida albicans were separated by ion-exchange chromatography from a crude carbohydrate-rich extract (GMP), and investigated for their chemical and molecular composition, antigenicity and immunomodulatory properties in cultures of human peripheral blood mononuclear cells (PBMC). Both fractions consisted predominantly of Periodic acid-Schiff (PAS) and concanavalin A (Con A)-reactive material consisting of greater than 90% mannose, 3-5% protein and small amounts of phosphorus; each was recognized by an anti-Candida rabbit serum as well as by a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope present on the fungal cell surface. When F1 and F2 were subjected to SDS-PAGE, transblotted and stained with enzyme-conjugated mAb AF1 or Con A, most of the antibody or lectin bound to high molecular mass (greater than 200 kDa) polydisperse material, some of which was present in F2 (as in the starting GMP extract) but absent in F1. This difference was also observed in PAS-stained gels of the two fractions. The F2, but not the F1, constituent was as active as the unfractionated GMP extract in inducing lymphoproliferation, production of the cytokines interleukin-2 and interferon-gamma, and generation of cytotoxicity against a natural-killer-sensitive target cell line (K562). These immunomodulatory properties were, like those possessed by GMP, protease-sensitive and heat-stable. Treatment of PMBC cultures with a modulatory anti-T-cell receptor antibody abolished the lymphoproliferation induced by GMP and F2 but not that induced by phytohaemagglutinin, showing that the mannoprotein materials of C. albicans acted through interaction with the antigen receptor complex. PMID- 1706758 TI - Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies. AB - Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing. PMID- 1706760 TI - A physical map of the genome of Haemophilus influenzae type b. AB - Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (PFGE) was used in combination with Southern hybridization to construct a genomic restriction map for the pathogen Haemophilus influenzae serotype b, strain Eagan. Sites for four restriction endonucleases, EagI, NaeI, RsrII and SmaI, were located on the 2100 kbp circular chromosome. Twelve potential virulence loci have been placed on the map together with certain loci essential for growth of the bacteria (e.g. ribosomal RNA operons). PFGE also provided a valuable tool for characterizing ten capsulated, type b isolates (other than Eagan) known to be genetically heterogeneous and two laboratory-derived variants (transformants) derived through complex recombinational events involving random uptake of high molecular-mass donor genomic DNA. PMID- 1706759 TI - Effect of fixation on activity and cytochemistry of hydrogenosomal enzymes in Trichomonas vaginalis. AB - The effect of fixation on the activity of malate dehydrogenase (decarboxylating) and pyruvate synthase was investigated in Trichomonas vaginalis. Subsequently a cytochemical staining method was developed for the demonstration of malate dehydrogenase activity in hydrogenosomes. After fixation of cells in low concentrations of glutaraldehyde and incubation in the presence of malate and the tetrazolium compound 2-(2'-benzothiazolyl)-5-styryl-3-(4' phthalhydrazidyl)tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. During the whole procedure strictly anaerobic conditions were required. Attempts to develop an analogous procedure for pyruvate synthase failed because even low concentrations of glutaraldehyde strongly inhibited enzyme activity. When cells were fixed in low concentrations of glycolaldehyde and acetaldehyde, a high enzyme activity was retained, but no staining could be achieved. Application of both staining methods to the sapropelic ciliates Trimyema compressum and Plagiopyla nasuta gave negative results. PMID- 1706761 TI - Fetal organ response to maternal protein deprivation during pregnancy in swine. AB - To test the hypothesis that maternal protein deprivation in early pregnancy retards body and organ growth of 63-d (midterm) pig fetuses, 16 primiparous domestic four-way crossbred swine were fed a diet adequate in protein (13% protein) (A) or a protein-restricted (0.7% protein) diet (PR) from d 1 to 63 of pregnancy or to parturition. Maternal body weight, plasma protein, hematocrit, and heart and spleen weights were reduced, and indices of body fatness were increased by the PR diet. Fetal body weights were reduced and fetal placental weights were increased at d 63 by protein restriction. Length from crown to rump and weights of liver, kidney, gastrointestinal tract, and cerebrum were lower in PR than in A fetuses. Concentrations of protein, RNA and DNA in liver, cerebrum and longissimus muscle were unaffected by maternal diet, but total amounts of all three constituents in liver and cerebrum were lower in PR than in A. Relative organ weights were similar in A and PR fetuses except that kidneys and gastrointestinal tract were greater in A fetuses. Newborn body weights and absolute organ weights were generally lower in PR than in A, but relative weights were similar. The observed reduction in body and organ weights and amounts of protein, RNA and DNA in 63-d fetuses and newborn progeny of PR swine establishes that the stunting effect of maternal protein restriction can be initiated by midterm, preceding the period of most rapid accretion of body tissues during prenatal life in the pig. PMID- 1706762 TI - Effect of pyrrolidone derivatives on lipid membrane and protein conformation as transdermal penetration enhancer. AB - Effect of pyrrolidone derivatives on lipid membrane and protein conformation has been assessed to obtain fundamental information about a mechanism of transdermal penetration enhancer. Their effects on a release of 6-carboxyfluorescein from liposome were examined. The lipophilic derivatives enhanced a release of dye and the enhancing effect showed a concentration dependency. Especially 1-lauryl-2 pyrrolidone showed the highest effect at the lowest concentration. The pyrrolidone derivatives also increased a hemolysis of rat erythrocytes. The derivatives slightly liberated SH group of keratin but did not change the electrophoresis pattern of keratin. 1-Methyl-2-pyrrolidone increased and retained a hydration of rat skin although 1-hexyl- and 1-lauryl-2-pyrrolidone showed no increase. These results suggest that the high enhancing effect of II, III and IX, as shown in the previous study, may be predominantly due to their interaction with skin lipid and their penetration behavior into the lipid. PMID- 1706763 TI - Heterocycles related to nucleotides. X. Reaction of thiouridine with 2 chloromercurio-4-nitrophenol. AB - 2-Chloromercurio-4-nitrophenol reacts reversibly with thiouridine, this reaction was applied to a chemical modification of E. coli transfer ribonucleic acid. PMID- 1706764 TI - Heterocycles related to nucleotides. XI. An organomercurial column chromatography for thiouridine. AB - By the organomercurial column: chromagel or Sephadex coupled to p aminophenylmercury chloride, 4- and 2-thiouridine were selectively separated from the mixtures with major nucleotides. Oligonucleotides containing 4-thiouridilic acid were also separated from the ribonuclease-T1 digests of unfractionated transfer ribonucleic acid of E. coli. PMID- 1706765 TI - Leukotriene receptor antagonism and augmentation of beta-receptor-mediated events by LY171883. AB - LY171883, (1-[2-hydroxy-3-propyl-4-[4(1H-tetrazol-5-yl)butoxy)phenyl]etha none), a leukotriene (LT) D4/E4 receptor antagonist, was assessed in comparison with two well known phosphodiesterase inhibitors, isobutylmethyl-xanthine (IBMX) and theophylline, for its ability to augment beta-receptor-mediated responses. Relaxation of carbachol-contracted guinea-pig trachea by isoprenaline was enhanced by the three agents in a dose-dependent manner. A two-fold enhancement of isoprenaline-induced smooth muscle relaxation was produced by 2.5 microM IBMX, 28 microM LY171883, or 140 microM theophylline. Similar concentrations of IBMX or theophylline did not antagonize LTE4-induced tracheal contractions; LY171883 totally inhibited the response and had significant LTE4 receptor antagonist activity even at 10-fold lower concentrations. Antigen-induced release of histamine and LTC4 from guinea-pig lung was reduced by isoprenaline. Prior treatment with LY171883, IBMX, or theophylline did not enhance this action. Isoprenaline reduced histamine-induced bronchospasm in anaesthetized guinea-pigs. LY171883, 30 mg kg-1, or IBMX, 1 mg kg-1, did not affect the isoprenaline-induced decrease in the histamine response. IBMX, 3 mg kg-1, and theophylline, 30 mg kg 1, augmented the isoprenaline-induced bronchodilation. LTE4-induced bronchoconstriction was not affected by IBMX or theophylline whereas LY171883 antagonized this response at doses as low as 3 mg kg-1. Therefore, in both in vitro and in-vivo test systems, LY171883 functioned primarily as a leukotriene receptor antagonist with minimal pharmacological activity attributable to its ability to potentiate isoprenaline. PMID- 1706766 TI - The effect of Fluosol-DA and Hespan haemodilution on S-warfarin elimination in the rat. AB - Fluosol and Hespan haemodilution in rats did not change S-warfarin elimination within 72 h although previous studies had indicated that Fluosol haemodilution caused an increased cytochrome P450b and P450e activity at 48 h. This study showed that the increased activity at that time was specific for those isoenzymes. PMID- 1706767 TI - Improved method for morphine determination in biological fluids and tissues: rapid, sensitive and selective. AB - Morphine was assayed using a simple two step solvent extraction--acid back extraction sample preparation method, coupled with normal phase high-performance liquid chromatography (HPLC) and dual electrode coulometric detection. HPLC is performed with a 1.0 M Tris-methanol (5:95) mobile phase with subtle pH adjustments to separate morphine and internal standard from any interfering compounds. The use of normal phase HPLC (silica column) substantially reduces problems from interfering lipophilic substances sometimes encountered with reverse phase HPLC following solvent extraction and which would otherwise require more time-consuming sample preparation. Dual electrode detection further improves the selectivity for morphine and gives excellent sensitivity (0.5 ng mL-1), reproducibility and stability for automated sample injection. This method has proven suitable for pharmacokinetic studies of morphine. PMID- 1706768 TI - MHC-restricted cytotoxicity against HIV. PMID- 1706769 TI - A capture enzyme immunoassay for detection of HIV-2/SIV antigen. AB - An antigen capture enzyme immunoassay was developed for the demonstration of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV). The assay (HIV-2 CE) has a sensitivity of approximately 250 pg/ml of HIV 2/SIV antigen. The HIV-2 CE was 4-16 times more sensitive than the Abbott HIV-1 antigen assay for detection of HIV-2/SIV antigen in cell culture supernatant. The sensitivity for detection of HIV-2/SIV antigen in serum or plasma was 98.5% in the HIV-2 CE and 95.5% in the Abbott HIV-1 antigen assay. The specificity of the HIV-2 CE was 99.2% (one false positive among 119 negative sera) whereas the Abbott assay was 100% specific. The HIV-2 CE detected antigen in supernatants from cultures of peripheral blood mononuclear cells of macaque monkeys infected with HIV-2 or SIV about 1 week before a reverse transcriptase (RT) microassay was positive and remained positive for at least 1 week after the RT assay had become negative. Three cultures from persistently infected monkeys were positive only in the HIV-2 CE, reflecting a higher sensitivity compared to the RT microassay. Thus, the HIV-2 CE was more sensitive than the Abbott HIV-1 antigen assay for detection of HIV-2/SIV antigen in culture supernatants as well as in serum/plasma. Furthermore, the HIV-2 CE showed a higher sensitivity than the RT microassay for detection of HIV-2/SIV in cell culture supernatants. PMID- 1706770 TI - Human immunodeficiency virus type-1 infection of homosexual men is accompanied by a decrease in circulating B cells. AB - As part of the multidisciplinary effort to characterize the natural history of human immunodeficiency virus type 1 (HIV-1) infection, the cell-surface phenotypes of lymphocytes from a cohort of homosexual men were analyzed in detail and related to clinical and laboratory parameters associated with HIV-1 infection. The present study represents a cross-sectional analysis of coded specimens from 153 homosexual men, of whom 74 were seronegative and 79 seropositive for HIV-1. Fewer circulating B lymphocytes (CD19+) were found in HIV 1-seropositive subjects relative to a seronegative reference group. HIV seropositivity was not associated with decreased numbers of CD8+ T cells or activated T cells, which suggests that the number of circulating B cells specifically decreased. In addition to CD19, B cells were measured by CD20 and CD21 in a subset of subjects, and decreases in circulating CD20+ and CD21+ B cells were also apparent in HIV-1-seropositive subjects. The decrease in B-cell numbers was present at the earliest stages of HIV-1 infection (asymptomatic, clinically silent) and became more pronounced at more advanced stages of HIV-1 infection. The absolute B-cell numbers correlated with absolute CD4+ cell numbers (r = 0.59, p less than 0.001). These data suggest that HIV-1 infection is associated with progressive, selective decreases in the numbers of circulating CD4+ T cells and B cells. PMID- 1706771 TI - Restoration of endodontically treated anterior teeth: an evaluation of coronal microleakage of glass ionomer and composite resin materials. AB - A glass ionomer material was evaluated for coronal microleakage in permanent lingual access restorations of endodontically treated anterior teeth. The material was tested as a restoration, placed over a zinc oxide-eugenol base, and as a base with an acid-etched composite resin veneer and a dentinal bonding agent. Restored teeth were thermocycled, immersed in silver nitrate, developed, and sectioned to assess microleakage. Significant coronal leakage was observed with all materials used. PMID- 1706772 TI - Postanesthesia care of the cocaine abuser. AB - The use of cocaine is increasing in the United States. An increasing number of cocaine users will be admitted to the PACU, requiring nurses to be knowledgeable about this hazardous drug. Cocaine produces harmful effects on the central nervous, respiratory, and cardiovascular systems. Nursing process can be used to guide the patient's care. Changes in physical, mental, and emotional status must be assessed often. Multiple nursing diagnoses are appropriate for this patient in the PACU. Maintaining airway and alleviating pain are nursing interventions that challenge the PACU nurse. The goal of nursing care is to provide a safe, quiet, and comfortable recovery from anesthesia. Rehabilitation is the long-range goal for the patient who needs expert nursing care, a therapeutic environment, and understanding. PMID- 1706773 TI - Psychology of the injured athlete. AB - The current interest in physical fitness has had a profound effect on all facets of society, especially on the field of sports medicine. Although scientific needs result in rapidly changing technology, patients' psychological needs never change. PACU nurses are still the first line of support for the injured athlete. The authors review the emotional needs and guidelines for care of the patient in the PACU. PMID- 1706774 TI - Practical points in the postoperative management of a craniotomy patient. AB - Presented in this article is a brief overview of cranial anatomy and physiology, highlights of surgical indications, and the surgical procedure for craniotomy. Nursing interventions and their rationales are emphasized. PMID- 1706775 TI - Celebrations and bridges. PMID- 1706776 TI - The power of one. PMID- 1706778 TI - What makes us so special? PMID- 1706777 TI - Scope of practice--post anesthesia nursing. PMID- 1706779 TI - Human chorionic gonadotropin, its free subunits and gestational trophoblastic disease. AB - Monoclonal antibody-based immunoassays can detect specifically intact human chorionic gonadotropin (hCG), the free alpha-subunit and the free beta-subunit. The differential production of hCG and its free subunits is important in gestational trophoblastic disease. Using well-defined epitope-specific monoclonal antibodies in an immunoradiometric assay format, specific and sensitive assays have been developed. Serum levels of free beta-hCG were abnormally high in patients with gestational trophoblastic disease. In contrast, free alpha-hCG levels in serum were not increased. PMID- 1706780 TI - Modification of lysine residues of Staphylococcus aureus alpha-toxin: effects on its channel-forming properties. AB - Staphylococcus aureus alpha-toxin opens an ion channel in planar phospholipid bilayers, which is selective for anions over cations, supposedly because of the presence of positively charged groups along the ion pathway. To remove some positive charges of this protein toxin, we chemically modified part of its lysine residues either with diethylpyrocarbonate, followed by histidine regeneration with hydroxylamine, or with trinitrobenzenesulfonic acid. The extent of chemical modification can be followed accurately by native polyacrylamide gel electrophoresis and isoelectric focusing. Ethoxyformilation of two to three lysine residues per toxin monomer does not impair hemolysis of rabbit red blood cells nor formation of pores in model membranes. It reduces the conductance and the anion selectivity of the channel and changes the shape of its current-voltage characteristic. This indicates that positively charged lysine residues are actually important in determining the electrical properties of the pore. Ethoxyformilation of channels preassembled in planar bilayers produces the same changes as modification of toxin monomers before channel formation. Furthermore, it can be performed by adding diethylpyrocarbonate on either side of the bilayer. This suggests that the lysine residues relevant for the electrical properties of the pore are located inside its lumen where they can be reached by diethylpyrocarbonate diffusing from either entrance of the channel. PMID- 1706781 TI - Reconstruction and analysis of human Alu genes. AB - The existing classification of human Alu sequences is revised and expanded using a novel methodology and a larger set of sequence data. Our study confirms that there are two major Alu subfamilies, Alu-J and Alu-S. The Alu-S subfamily consists of at least five distinct subfamilies referred to as Alu-Sx, Alu-Sq, Alu Sp, Alu-Sc, and Alu-Sb. The Alu-Sp and Alu-Sq subfamilies have been revealed by this study. Alu subfamilies differ from one another in a number of positions called diagnostic. In this paper the diagnostic positions are defined in quantitative terms and are used to evaluate statistical significance of the observed subfamilies. Each Alu subfamily most likely represents pseudogenes retroposed from evolving functional source Alu genes. Evidence presented in this paper indicates that Alu-Sp and Alu-Sc pseudogenes were retroposed from different source genes, during overlapping periods of time, and at different rates. Our analysis also indicates that the previously identified Alu-type transcript BC200 comes from an active Alu gene that might have existed even before the origin of dimeric Alu sequences. The source genes for Alu pseudogene families are reconstructed. It is assumed that diagnostic differences between reconstructed source genes reflect mutations that have occurred in true source Alu genes under natural selection. Some of these mutations are compensatory and are used to reconstruct a common secondary structure of Alu RNAs transcribed from the source genes. The biological function of Alu RNA is discussed in the context of its homology to the elongation-arresting domain of 7SL RNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706782 TI - Nucleotide sequence and evolution of coding and noncoding regions of a quail mitochondrial genome. AB - Segments of the Japanese quail mitochondrial genome encompassing many tRNA and protein genes, the small and part of the large rRNA genes, and the control region have been cloned and sequenced. Analysis of the relative position of these genes confirmed that the tRNA(Glu) and ND6 genes in galliform mitochondrial DNA are located immediately adjacent to the control region of the molecule instead of between the cytochrome b and ND5 genes as in other vertebrates. Japanese quail and chicken display another distinctive characteristic, that is, they both lack an equivalent to the light-strand replication origin found between the tRNA(Cys) and tRNA(Asn) genes in all vertebrate mitochondrial genomes sequenced thus far. Comparison of the protein-encoding genes revealed that a great proportion of the substitutions are silent and involve mainly transitions. This bias toward transitions also occurs in the tRNA and rRNA genes but is not observed in the control region where transversions account for many of the substitutions. Sequence alignment indicated that the two avian control regions evolve mainly through base substitutions but are also characterized by the occurrence of a 57 bp deletion/addition event at their 5' end. The overall sequence divergence between the two gallinaceous birds suggests that avian mitochondrial genomes evolve at a similar rate to other vertebrate mitochondrial DNAs. PMID- 1706783 TI - Isolation and localization of a slow troponin (TnT) gene on chromosome 19 by subtraction hybridization of a cDNA muscle library using myotonic dystrophy muscle cDNA. AB - Subtraction hybridization techniques were used to isolate 91 cDNA clones which are overexpressed in normal control skeletal muscle relative to muscle from patients with myotonic muscular dystrophy. The gene responsible for myotonic dystrophy (DM) has been localized to the 19q13.2-13.3 region of chromosome 19. To test as a candidate gene for DM, clones which represent differences in transcription are analyzed for localization to chromosome 19. One clone, designated MSL 366, was found to be on the long arm of chromosome 19 distal to the CKMM gene at 19q13.2. Sequence analysis confirmed that MSL 366 is the cDNA for human slow skeletal muscle troponin T. A genomic clone has been isolated and linkage studies with DM are in progress. PMID- 1706784 TI - Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor. AB - Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface (Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, Sehgal A [1986]: Science 232:518-521). Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross linked to [125-I]-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added [125-I]-labeled NGF. PMID- 1706785 TI - Common and subtypic determinants of hepatitis B surface antigen particles: susceptibility to reduction and/or alkylation evaluated with monoclonal antibodies. AB - The specificity of five monoclonal antibodies, three raised against hepatitis B surface antigen (HBsAg) particles and two against envelope polypeptides, was tested for on a panel of 366 sera containing HBsAg of various subtypes (131 adw, 146 adr, 39 ayw and 50 ayr). Three monoclonals bound to HBsAg irrespective of subtypes, and therefore, were directed to the common antigenic determinants of HBsAg. Of these, two raised against particles (No. 824 and No. 7922) did not bind with reduced HBsAg particles. The other raised against peptides (No. 5124) bound to reduced HBsAg particles. It did not, however, bind to reduced and alkylated HBsAg particles, thereby indicating that it was directed to an epitope involving cysteine residues not contributing to the conformation. The remaining two monoclonals were directed to subtypic determinants not identical to any of d, y, w and r determinants. The subtypic determinant detectable by one of them (No. 4403), raised against HBsAg polypeptides, markedly increased after reduction of HBsAg particles with or without alkylation. In contrast, the subtypic determinant, detectable by the other monoclonal (No. 2155) raised against particles, substantially decreased after reduction. Non-identity of common or subtypic determinants detectable by the five monoclonals were established by blocking tests in which labeled antibody was competed by non-labeled antibody, of a homologous or heterologous specificity, for the binding with HBsAg. These monoclonals would be useful in studies for immunochemical configuration of HBsAg particles and epidemiology of novel subtypic determinants. PMID- 1706786 TI - Production of monoclonal antibody against human prostatic adenocarcinoma and its immunohistochemical properties. AB - We have generated a monoclonal antibody against prostatic adenocarcinoma, PC-Ab (IgM) was derived from a fusion using the fresh prostatic tissue of adenocarcinoma as the immunogen. Initial screening was performed by enzyme-linked immunosorbent assay (ELISA) on the soluble fraction of the immunogen. The specific analysis was performed by the avidin-biotin-complex immunoperoxidase method on paraffin-embedded sections of normal, benign and malignant neoplastic tissues from the prostate and various organs. PC-Ab reacted with well differentiated adenocarcinoma (83.3%), moderately differentiated adenocarcinoma (92.2%), and poorly differentiated adenocarcinoma (90.2%), respectively. According to classification by stage, the reaction rates with stage T0, T1, T2, T3 and T4 were 77.3%, 80.0%, 95.2%, 91.3% and 92.9%, respectively but no significant differences in the stages were seen among these groups. PC-Ab reacted with the epithelium of the normal prostate and benign prostatic adenoma, and human fetal tissues. Molecular weight and isoelectric point of the antigen recognized by this PC-Ab was estimated to be 57,000 daltons and 7.0, respectively. These results indicate that PC-Ab reacts with the antigen associated with human prostatic adenocarcinoma. PMID- 1706787 TI - Lymphocyte responses to peptide M and to retinal S-antigen in uveitis patients. AB - A synthetic amino acid, peptide M18 (18 amino acids in length which correspond to the amino acid positions 303-322 of bovine S-antigen: DTNLASSTIIKEGIDKTV), is capable of inducing experimental autoimmune uveitis in monkeys and Hartley guinea pigs as well as Lewis rats. Recently, the amino acid sequence of human S-antigen corresponding to peptide M was found to be virtually identical to that of bovine S-antigen. To investigate the role of the epitope corresponding to peptide M in human uveitis, 21 patients with uveitis were tested for proliferation of peripheral mononuclear cells against peptide M18 and bovine S-antigen. Six of the 21 patients responded to S-antigen and 4 of these 6 patients responded to peptide M18. These findings show that peptide M18 is immunogenic in man and provide additional support to the hypothesis that the amino acid sequence which corresponds to peptide M might be involved in uveitic conditions in man. PMID- 1706788 TI - Immunosuppressive effect of gramicidin S on experimental ocular neuritis and allergic encephalomyelitis. AB - Investigations were carried out on the immunosuppressive effect of gramicidin S (GrS), a cyclic peptide antibiotic produced by Bacillus brevis, on the onset of experimental ocular neuritis and allergic encephalomyelitis in Lewis rats immunized with rat brain homogenates. The criteria for evaluation of the drug effect were changes in body weight, activity of 2',3'-cyclic nucleotide 3' phosphohydrolase (CNPase), clinical manifestations such as paralysis of lower extremities and histopathological changes. Clinical symptoms and body weight reduction were effectively prevented by GrS treatment of immunized animals. The activity of the myelin marker enzyme CNPase was markedly decreased in the lumbar spinal cord of encephalitogen-immunized animals on day 16 (ie 16 days after immunization) and the decrease of enzymatic activity was partially prevented by GrS administration. On the other hand, the CNPase activity of the retrobulbar optic nerve of inoculated animals remained essentially the same as that of healthy control animals, although inflammatory changes were prominent in the optic nerve. Histopathological changes observed in the optic nerve and spinal cord of diseased animals were virtually absent in GrS-treated animals. A possible mechanism of the immunosuppressive activity of GrS is discussed. PMID- 1706789 TI - Long-term prognosis in galactosaemia: results of a survey of 350 cases. AB - An international survey of the long term results of treating galactosaemia has shown poor results. These do not seem to be related to any of the relevant variables studied, for example delayed diagnosis or poor dietary compliance. PMID- 1706790 TI - Play-language relationships in young children with developmental delays: implications for assessment. AB - The purpose of this longitudinal study was to determine whether the reported parallels between symbolic play and normal language development were evidenced in 6 children with developmental delays of varying etiologies. Subjects' play and language behavior over a 6-month period was videotaped and analyzed during free play and modeling tasks. Although results supported the correspondences previously reported between normal language development and symbolic play, the variability across observations in the present subjects was more marked than expected. Implications for clinical assessment are discussed. PMID- 1706791 TI - Amiodarone, verapamil, and quinidine do not affect equilibrium binding of digoxin. AB - The binding of [3H]digoxin to purified canine cardiac sarcolemmal vesicles was characterized. Scatchard analysis of saturation isotherms yielded linear plots with a maximal binding capacity of 174 +/- 31.9 pmol/mg, a dissociation constant of 31.7 +/- 4.59 nM, and a Hill coefficient of 0.947 +/- 0.02 (mean +/- SEM), suggesting that [3H]digoxin bound to a single class of sites. In contrast to their marked effect on steady-state serum digoxin levels when administered in combination, quinidine, verapamil, and amiodarone were without effect on equilibrium binding of [3H]digoxin. Thus, increased steady-state serum concentrations of digoxin resulting from combination therapy with these particular drugs probably will have cardiac effects that may increase the risk of digitoxicity to the patient. PMID- 1706792 TI - High-dose iron-chelator therapy during reperfusion with deferoxamine-hydroxyethyl starch conjugate fails to reduce canine infarct size. AB - Iron catalyzes reactions during ischemia and reperfusion that contribute to myocardial injury. The iron-chelator deferoxamine blocks these reactions, but undesirable side effects limit the clinical potential of deferoxamine to decrease injury. We tested whether intravenous (i.v.) administration of high doses of a well-tolerated deferoxamine-hydroxyethyl starch (DEFHES) iron-chelator during the last 10 min of ischemia and the first 10 min of reperfusion would decrease canine infarct size. Fourteen chloralose-anesthetized mongrel dogs were randomized to therapy in a blinded fashion with deferoxamine conjugate (75 mg/kg deferoxamine) or hydroxyethyl starch (HES) vehicle alone. Nine other untreated dogs served as controls. Infarct size as a percentage of area at risk (MI/RISK) was not reduced by therapy with deferoxamine conjugate. The deferoxamine dose was five times greater than the maximally tolerated dose of free deferoxamine. Arterial deferoxamine concentrations in the deferoxamine-conjugate group were 0.69 +/- 0.09 mM at onset of reperfusion and 1.37 +/- 0.05 mM at 10 min of reperfusion. Area at risk, ischemic collateral blood flow, and heart rate-blood pressure (HR/BP) product were similar in the groups. Chelation of intravascular iron at the time of reperfusion does not reduce myocardial necrosis in an in vivo model of myocardial ischemia-reperfusion injury. PMID- 1706793 TI - Chronic propranolol treatment promotes left ventricular dilation without altering systolic function after large myocardial infarction in rats. AB - In rats with chronic myocardial infarction (MI), we have examined the effects of prolonged beta-adrenergic blockade with propranolol on left ventricular (LV) performance, weight, and volumes. Sham-operated rats and rats with large MI (greater than 30%) were evaluated. Four groups of rats were studied: control, sham-operated (n = 12); control, MI (n = 12); propranolol (500 mg/L of drinking water)-treated, sham-operated (n = 10); and propranolol treated, MI (n = 10). Treatment was started 3 weeks after coronary ligation. After 5-6 weeks, LV, systemic arterial, and right atrial pressures in addition to aortic blood flow before and during volume loading were measured. LV pressure-volume relations were measured ex vivo. The rats with chronic MI demonstrated expected decreases in LV systolic performance and increased LV end-diastolic and right atrial pressures. Propranolol had no independent effect on LV systolic pressure, LV end-diastolic pressure, resting cardiac index, stressed cardiac index during volume loading, peak developed aortic pressure during aortic occlusion, or ejection fraction index in either sham-operated or infarcted rats; however, heart rate was decreased. LV weight/body weight was 2.17 +/- 0.04 mg/g in control sham-operated rats, which was not different from the propranolol-treated sham-operated rats (2.09 +/- 0.04 mg/g). The LV weight/body weight was increased (p less than 0.01) to 2.21 +/- 0.08 mg/g in the propranolol-treated MI group from 1.94 +/- 0.06 mg/g in the control MI group. The LV pressure-volume relation was not altered by propranolol in the sham-operated rats but was shifted to the right by MI.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706794 TI - Pharmacology of RG W-2938: a cardiotonic agent with vasodilator activity. AB - The cardiovascular effects of RG W-2938, 6-[6-(3,4-dihydro-3-methyl-2(1H)-2 oxoquinazolinyl)]-4,5-dihydro-3 (2H-pyridazinone, a new nonglycoside, noncatecholamine cardiotonic/vasodilator agent were examined in vivo in anesthetized and conscious dogs and in vitro in isolated guinea pig hearts; in the latter, RG W-2938 5 nmol-5 mumol increased contractility in a dose-related fashion. RG W-2938 30-300 micrograms/kg administered intravenously (i.v.) to anesthetized dogs increased contractile force while decreasing arterial pressure and total peripheral resistance (TPR) in a dose-related manner. Heart rate (HR) was only slightly increased, and aortic flow was not appreciably altered. A single oral dose of RG W-2938 0.3 mg/kg administered to conscious chronically instrumented dogs produced a marked and sustained increase in contractility 15 240 min after treatment while only slightly increasing HR. The effects of RG W 2938 30-300 micrograms/kg, i.v. were studied in a mecamylamine-propranolol induced model of heart failure. RG W-2938 effectively reversed the drug-induced heart failure by increasing myocardial contractility and decreasing arterial pressure while only slightly affecting HR. These studies show that RG W-2938 is an orally effective positive inotropic/vasodilator agent. PMID- 1706796 TI - Sotalol and mexiletine: combination of rate-dependent electrophysiological effects. AB - The rate-dependent electrophysiological effects of sotalol (30 microM), mexiletine (10 and 18 microM), and their coadministration were examined in isolated dog cardiac Purkinje fibers following abrupt changes in pacing cycle length. Combination of 30 microM sotalol with 10 microM mexiletine significantly lengthened premature action potential durations at diastolic intervals of less than 50 ms while the basic action potential duration evoked at a stimulus frequency of 2 Hz was not affected. This effect on the premature action potential duration was attenuated when the higher mexiletine concentration (18 microM) was coadministered with sotalol. The fast time constant for restitution of the action potential duration was significantly slowed by either combination. Coadministration of sotalol and mexiletine, like mexiletine alone, produced a rate-dependent depression of Vmax that displayed a second slow time component during recovery. This slow component for recovery of Vmax was not distinguished in the absence of drug or in the presence of sotalol alone. Sotalol-induced lengthening of the action potential duration observed at slow pacing frequencies was also attenuated by addition of mexiletine; and, under these conditions, Purkinje fiber early afterdepolarizations were prevented. In addition, the range of premature action potential durations was significantly decreased by mexiletine and by the combination, while sotalol alone increased this range slightly. These results indicate that coadministration of sotalol and mexiletine may provide beneficial electrophysiological effects expected to provide enhanced antiarrhythmic efficacy and fewer proarrhythmic complications in patients. PMID- 1706795 TI - Cardiac electrophysiologic effects of McN-5691, a new calcium-channel blocking antihypertensive agent. AB - The purpose of this study was to evaluate the cardiac electrophysiological effects of McN-5691, a new calcium-channel blocking antihypertensive drug. In anesthetized dogs, the primary electrophysiological effect of McN-5691 was dose related prolongation of AV-nodal conduction time and refractoriness (0.1-1.0 mg/kg i.v.), which correlated with McN-5691 plasma levels. There were no significant effects on atrial or ventricular conduction times, QTc, or ventricular monophasic action potential duration. This profile was similar to that of verapamil. McN-5691 caused concentration-related, rate-dependent reductions in Vmax and amplitude of slow-response action potentials in guinea pig papillary muscle: ED-20% for depression of Vmax was 0.72 +/- 0.32 microM. Verapamil was more potent in depressing these action potentials: ED-20% for depression of Vmax was 0.03 +/- 0.01 microM. McN-5691 also caused rate-dependent reduction in Vmax and amplitude of canine Purkinje fiber action potentials, but only at relatively high concentrations: ED-20% for depression of Vmax was 55 +/- 12 microM. McN-5691 also reduced the action potential duration (0.3-30 microM) without affecting the slope of phase 4 depolarization and the maximum diastolic potential. Verapamil also reduced Vmax in Purkinje fibers (ED-20% for depression of Vmax was 32 +/- 3 microM) and shortened the action potential duration. The results show that McN-5691 has cardiac electrophysiological effects consistent with blockade of the slow inward calcium current, and that this activity occurs at concentrations well below those having local anesthetic activity. In addition, its lower potency in comparison to verapamil in depressing slow responses suggests a lesser propensity for negative inotropic effects. PMID- 1706797 TI - Effect of nimodipine on cerebral blood flow in human volunteers. AB - The present study was undertaken to examine the effect of a clinically relevant dose of nimodipine (30 micrograms/kg/h) on the autoregulation of CBF in 12 young healthy volunteers. Mean arterial blood pressure (MABP) was measured intraarterially (i.a.), and changes in cerebral blood flow (CBF) were estimated by the arteriovenous-oxygen [(a-v)O2]-difference method. The lower limit (LL) of CBF autoregulation was calculated by a computerized program and tested for different factors for correction of the PaCO2-induced changes in CBF. MABP was increased by norepinephrine (NE) and decreased by ganglion blockade (trimethaphane camphosulfonas) in combination with lower body negative pressure. The MABP manipulations were performed 1 h after infusion of nimodipine. MABP was reduced by 13 mm Hg (8-15 mm Hg), and CBF was increased by 8% (3-12%) during nimodipine infusion. Autoregulation was preserved in 11 of the 12 volunteers. A CO2-correction factor of 1% CBF/0.1 kPa was used. The LL was 75 mm Hg (71-80 mm Hg) [SE 3 mm Hg (2-4 mm Hg)] and not significantly different from a previous control group of healthy volunteers. No side effects were observed. The present study shows a maintained autoregulation of CBF during nimodipine infusion; however, this could be obtained only by reducing the correction for changes in carbon dioxide to 1%/0.1 kPa from 3%/0.1 kPa, which was used in a similar study in healthy volunteers. PMID- 1706798 TI - Determinants of acute hemodynamic and electrophysiologic effects of verapamil in humans: role of myocardial drug uptake. AB - To examine the relationship between myocardial verapamil content (MVC) and acute effects in humans, coronary sinus catheterization was used in 22 patients to determine myocardial uptake of verapamil after bolus intravenous (i.v.) verapamil (4 mg) injection. Verapamil-induced effects on hemodynamic and electrophysiologic parameters were measured simultaneously and correlated with MVC per unit baseline coronary sinus blood flow (MVC:F). Myocardial uptake of verapamil was rapid: peak MVC (1.2 +/- 0.2% of injected dose) occurred at 5.4 +/- 0.4 min; at 30 min, residual MVC was 71.1 +/- 3.4% of maximum. Peak MVC:F in individual patients was inversely related to the extent of coronary artery disease (p less than 0.005) but not to left ventricular (LV) systolic function. Verapamil produced significant (p less than 0.001) early reductions in arterial pressure and systemic vascular resistance (SVR); cardiac index (CI) increased, left ventricular (LV) positive dP/dt was unchanged. Verapamil prolonged (p less than 0.01) PR and AH intervals (maximum at 12-18 min) and atrioventricular (AV) nodal effective and functional refractory periods (ERP, FRP) (maximum at 30 min). In individual patients, the extent of changes in AH intervals (r = 0.69; p less than 0.05) and LV dP/dt (r = 0.62; p less than 0.05) correlated with peak MVC:F. We conclude that after i.v. injection, verapamil uptake by the human myocardium is rapid and more extensive in patients with minor coronary artery disease. Despite the hysteresis between MVC and drug effects, MCV is a determinant of inotropic and electrophysiologic effects of verapamil. PMID- 1706799 TI - Modulation of adrenoceptor-mediated cardiovascular effects by short-term in vivo infusion of isoproterenol in rats. AB - After a 16-h in vivo infusion period of isoproterenol (400 micrograms/kg/h) from a minipump implanted subcutaneously (s.c.) in rats, we observed an increase in heart weight due to tissue edema. In isolated perfused heart preparations, the EC50s of isoproterenol to induce positive inotropy and increase in coronary flow were increased approximately 7 and 4 times, respectively. Isoproterenol dose response curves performed in trachea preparations, were shifted to the right by about fivefold as compared with the control group. In the presence of phentolamine, the isoproterenol induced maximal relaxation in the isolated aorta from isoproterenol-pretreated animals was reduced from 46.5 to 9.2% as compared with saline-pretreated rats. In the absence of phentolamine, the epinephrine (EPI) and norepinephrine (NE) cumulative dose-response curves in this preparation were not affected by isoproterenol pretreatment with respect to EC50 and maximal effect. However, in the presence of phentolamine, the contractile response to a supramaximal dose of NE (10 microM) amounted to 30 and 70% in the saline- and isoproterenol-pretreated rats, respectively. In the presence of both phentolamine and propranolol, a similar response of 70% was observed by this supramaximal dose of agonist in the saline-pretreated rats as well. In anesthetized rats, 120 min after removal of the minipumps, sodium nitroprusside induced increase in heart rate (HR) was reduced after isoproterenol pretreatment, whereas for salbutamol the decrease in diastolic blood pressure (DBP) was also reduced. As compared with salbutamol, a marked increase was observed in the ratio of mean arterial blood pressure (MAP) to HR for SNP after isoproterenol pretreatment. The phenylephrine induced increase in MAP was increased after isoproterenol pretreatment. We conclude that in the pathogenesis of heart failure the beta-adrenoceptor-mediated effects of catecholamines are attenuated and alpha-adrenoceptor-mediated effects become progressively important. PMID- 1706800 TI - Pharmacokinetics and antihypertensive effects of lisinopril in hypertensive patients with normal and impaired renal function. AB - The antihypertensive effects and pharmacokinetic properties of lisinopril, an angiotensin-converting enzyme (ACE) inhibitor, were investigated in hypertensive patients with normal renal function (NRF, mean serum creatinine 1.0 mg/dl, n = 9) and those with impaired renal function (IRF, mean serum creatinine 1.7 mg/dl, n = 8). Lisinopril was administered orally (10-mg dose once daily for 5 or 8 days). Measurement of blood pressure (BP) and sampling of blood specimens were made on the first and last days of treatment. During consecutive dosing of lisinopril, its antihypertensive effects were sustained for greater than or equal to 12 h with less diurnal variation of BP. Serum ACE activity was markedly suppressed for 24 h. Plasma levels of lisinopril in the IRF group were higher than those in NRF with significant differences in the peak levels and areas under the plasma concentration time curve (AUC). A significant inverse correlation was found between the creatinine clearance and the AUC for lisinopril. These results suggest that lisinopril has a long-lasting action and that it is a useful antihypertensive agent for controlling BP in patients with either NRF or mild IRF. When administered for an extended period, however, more careful consideration should be given to the dose in patients with IRF than in patients with NRF to minimize the possibility of untoward side effects. PMID- 1706801 TI - Hemodynamic, hormonal, and renal effects of the prostacyclin analogue iloprost in conscious dogs with and without heart failure. AB - To test the efficacy of exogenous prostaglandins for vasodilator therapy in heart failure, we studied the effects of the prostacyclin-derivative iloprost (1.5-150 ng/kg/min) in seven conscious dogs before and after induction of heart failure by right ventricular pacing (250/min. 10 days). In healthy dogs, iloprost (150 ng/kg/min) decreased mean arterial blood pressure (MAP) (-45%) by a decrease in total peripheral resistance (TPR) (-55%), and increased cardiac output (CO) (+24%) and heart rate (HR) (+20%) with no effect on right atrial and pulmonary arterial pressures (RAP, PAP). Plasma norepinephrine (NE) (+47%), renin (+351%), and aldosterone (+126%) were increased. Urine flow (-70%) and Na excretion (-53%) were decreased. Iloprost (15 ng/kg/min) increased renal blood flow (RBF) (+29%), but did not change glomerular filtration rate (GFR). In dogs with heart failure, iloprost decreased arterial BP (-31%), TPR (-42%) and pulmonary vascular resistance (-28%) and increased CO (+29%), with no change in RAP and PAP. Plasma NE (+34%), renin (+385%), and aldosterone (+146%) were increased. RBF was unchanged. GFR (-24%) and filtration fraction (FF) (-30%) were decreased, as was urine flow (-65%). In experimental heart failure, iloprost is a potent arteriolar dilator, increasing CO with no preload effect. These beneficial effects are limited, however, by further neurohumoral activation and deterioration of renal function. PMID- 1706802 TI - Effects of SQ 31,765, a new calcium channel blocker, on stress and myosin light chain phosphorylation in swine carotid media. AB - The vasorelaxant activity of SQ 31,765 and diltiazem was studied in intact and Triton X-100 detergent-skinned fibers of the swine carotid media. Both calcium channel blockers were used to relax or prevent contractions induced by depolarization with 110 mM KCl. The potencies of SQ 31,765 and diltiazem were similar for relaxing or preventing the tonic component of KCl-induced contractions of intact vascular smooth muscle strips. A significantly higher concentration of either compound was required to prevent the phasic component of the contraction. The possible direct effects of these calcium channel blockers on contractile function were examined in the detergent-skinned carotid medial fiber contracted with 3 microM Ca2+. Both stress and myosin light chain (MLC) phosphorylation levels were determined in detergent-skinned fibers in the presence and absence of the channel blockers. Neither compound affected stress or MLC phosphorylation in Ca2(+)-contracted detergent-skinned fibers, indicating a lack of direct effect on the contractile proteins. Thus, SQ 31,765 appears to act selectively at the level of the sarcolemma. PMID- 1706803 TI - Hemodynamic effects of dexmedetomidine, an alpha 2-adrenergic agonist, in autonomically denervated dogs. AB - The hemodynamic effects of the alpha 2-adrenergic agonist, dexmedetomidine (DM), were studied in eight anesthetized, autonomically denervated dogs. Autonomic block decreased mean arterial pressure (MAP) and cardiac index (CI) by approximately 20% to 95 +/- 8 mm Hg and 4.1 +/- 0.1 L/min/m2, respectively (mean +/- SEM), and reduced norepinephrine (NE) and epinephrine plasma levels to almost undetectable levels. DM, administered intravenously (i.v.) either by bolus injection or by slow (20 min) infusion in doses between 1 and 30 micrograms/kg, had no effect on heart rate (HR), increased MAP significantly by 98%, decreased CI by 59%, and increased calculated systemic vascular resistance index (SVRI) significantly by 376%, maximally. The effect of the lowest dose was mediated mainly by arteriolar vasoconstriction, and that of higher doses was mediated by vasoconstriction and decreased CI. Left ventricular end-diastolic pressure (LVEDP) increased significantly from 6 +/- 2 to greater than 30 mm Hg. maximally. The effects were cumulative, and the first dose caused near maximal pressor effect; the resistance increase was as great with slow infusion as with bolus injection. Prazosin (1 mg/kg) did not affect the changes, but 0.3 mg/kg atipamezole, a selective alpha 2-antagonist, completely antagonized them. These observations demonstrate potent constriction of both arteriolar resistance and venous capacitance vasculature in dogs. The combination of decreased CI and increased filling pressure implies marked decrease in cardiac function which was, however, fully reversible by atipamezole. PMID- 1706804 TI - Effect of food on oral availability of apresoline and controlled release hydralazine in hypertensive patients. AB - Hydralazine is a vasodilator antihypertensive drug that has been in use for many years. Efficacy after oral administration correlates well with the levels of the drug in blood. Factors such as food ingestion that affect blood levels of hydralazine may therefore be of importance. There is dispute regarding the effect of food intake on blood levels of hydralazine and on the antihypertensive response. This randomized cross-over study examined the effect of food (642 K calories, 25 g protein, 43 g fat, 40 g carbohydrates, 32 mEq sodium, 17 mEq potassium) ingested immediately before hydralazine (taken as Apresoline, Ciba Geigy, or as slow-release hydralazine, SRH, Pennwalt Corporation) on the blood levels of hydralazine in 16 essential hypertensive patients who were slow acetylators currently taking at least 100 mg Apresoline daily. Peak blood hydralazine levels were reduced by food after both Apresoline and SRH, by 69 and 66%, respectively. Time to peak blood hydralazine concentration was delayed significantly with SRH. We could detect a statistically significant food-related reduction of area under blood hydralazine concentration versus time curves (AUC) only with Apresoline (by 44%). The AUC for SRH was decreased only 29% by food. Hydralazine should be taken at a consistent time with respect to meals. PMID- 1706805 TI - Renal effects of ACE inhibition in ovine heart failure: a comparison of intermittent and continuous ACE inhibition. AB - Results of uncontrolled studies suggest that the duration of action of an ACE inhibitor may be an important determinant of renal impairment when using these agents to treat patients with heart failure. To determine whether there is experimental evidence for this hypothesis, we compared the effects of intermittent (captopril, 25 mg i.v. bolus twice daily) and continuous (captopril. 25 mg bolus, then 50 mg/day by constant infusion) ACE inhibition in an ovine model where heart failure was induced by rapid left ventricular pacing (LVP). Six sheep underwent three 4-day periods of LVP with intermittent, continuous, or no treatment (control) given in random order from the onset of LVP. Despite evidence that intermittent captopril administration allowed significant recovery of serum ACE activity (4.6 +/- 1.2 vs. 1.1 +/- 0.5 pmol/L before and after captopril bolus on day 4, p less than 0.001) and restitution of arterial pressure between successive boluses (48 +/- 7 vs. 41 +/- 4 mm Hg, p less than 0.01), there was no difference in the renal effects of intermittent and continuous ACE inhibition (creatinine clearance was 44 +/- 14 and 47 +/- 8 ml/min on day 4 of the intermittent and continuous phase, respectively). Nevertheless, there was a significant correlation between the decline in arterial pressure and fall in creatinine clearance induced by ACE inhibition (r = 0.65, p less than 0.05), with evidence that drug accumulation may potentiate hypotension and renal impairment should arterial pressure be reduced below the threshold for renal autoregulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706806 TI - Effects of in vivo SIN1 treatment on nitrovasodilator relaxation and on EDRF mediated responses in rat aorta. AB - SIN1 (the active metabolite of molsidomine), nitroglycerin, and endothelium derived relaxing factor (EDRF) produce vasodilation by activation of soluble guanylate cyclase. Therefore, prolonged exposure to SIN1 might affect not only the responses to SIN1 itself and to nitroglycerin but also to EDRF. In vivo treatment of rats consisted of subcutaneous injections of either SIN1 (60 mg/kg) for the treated group or placebo for the control group, twice daily for 3 days. Thoracic aortas from the treated group were threefold and sixfold less sensitive to nitroglycerin and SIN1, respectively. The endothelium-dependent relaxations to acetylcholine were, nevertheless, similar in both groups. Moreover, the concentration-response curves to phenylephrine, which are known to be modulated by the endothelium, were similar in both groups. In addition, incubation with methylene blue (10 microM for 30 min), which blocks the vasodilator action of EDRF, potentiated in the same way the contractions to this alpha-adrenergic agonist. The increase in resting tone induced by methylene blue incubation was also equivalent in the two groups. The present results show that SIN1 treatment for several days in rats is associated with slight tolerance development not only to SIN1 itself but also to nitroglycerin, while the endothelial function remains operative. We conclude that the mechanisms involved in the activation of guanylate cyclase by SIN1 and nitroglycerin are probably different than those of EDRF-mediated responses. PMID- 1706807 TI - Inhibition of coronary artery thrombosis by SIN-1, a donor of nitric oxide. AB - Molsidomine and its metabolite, SIN-1, a donor of nitric oxide, are potent coronary vasodilator and anti-ischemic agents. Recently, SIN-1 and nitric oxide have also been shown to inhibit platelet adhesion and aggregation in vitro. The present study in dogs was designed to evaluate the in vivo antithrombotic properties of SIN-1. Coronary intimal damage and stenosis are known to induce coronary cyclic flow variations that reflect platelet thrombus formation followed by disaggregation and embolization (Folts preparation). This model of coronary artery thrombosis appears to simulate the combination of some of the factors contributing to unstable angina and myocardial infarction in human. SIN-1 infusion (10 micrograms/kg/min) significantly reduced the frequency of cyclic flow variations: 4.9 +/- 6.2/h vs. 14 +/- 4.6/h (before treatment, p less than 0.03, n = 6). Results were similar to those obtained with aspirin (5 mg/kg, bolus i.v.: 1.5 +/- 0.6/h vs. 11.7 +/- 3/h, p less than 0.03, n = 5) whereas saline had no effect (17.8 +/- 2.2/h vs. 19.3 +/- 2.4/h, n = 5). As expected, blood pressure was decreased only in the SIN-1 group: 56.2 +/- 7.8 vs. 87.3 +/- 9.3 mm Hg (p less than 0.02) (mean arterial blood pressure). The present results suggest that the well-documented anti-ischemic properties of SIN-1 could be partly due to its antithrombotic activity, clearly demonstrated with the model of coronary thrombosis used here in the dog. PMID- 1706808 TI - Selective beta 1-receptor full agonists, T-0509 and T-1583, increase the force monophasically and cyclic AMP biphasically in canine ventricular muscle. AB - In canine right ventricular muscle, we investigated the mechanism of action of the positive inotropic effects of T-0509 and T-1583, derivatives of denopamine, a new selective beta 1-partial agonist. T-1583 has already been characterized as a selective beta 1 full agonist (pD2 = 7.39). T-0509 also behaved as a full agonist (pD2 = 8.27) and its positive inotropic effect was antagonized competitively by atenolol (pA2 = 7.53) and noncompetitively by carbachol, and potentiated by 3 isobutyl-1-methylxanthine. With increasing concentrations of T-0509 (10(-9) to 10(-7) M) and T-1583 (10(-8) to 10(-6) M), cyclic AMP increased and increases reached plateaus approximately 40% above the baseline levels with approximately 10(-7) M T-0509 and approximately 10(-6) M T-1583, at which their positive inotropic effects reached maxima. However, with further increasing concentrations, cyclic AMP again started to increase and increases amounted to approximately 120% above the baseline levels with 10(-5) M T-0509 and with 10(-4) M T-1583. These results suggest the following: Like denopamine, the selective beta 1 full agonists, T-0509 and T-1583, at lower concentrations produce positive inotropic effects accompanied by only a small increase in cyclic AMP via stimulation of high-affinity beta 1-receptors. In higher concentrations, unlike denopamine, the two full agonists produce large increases in cyclic AMP loosely coupled to positive inotropy via stimulation of low-affinity beta 1-receptors. PMID- 1706809 TI - Different mobilization of calcium in endothelin-1-induced contractions in human arteries and veins: effects of calcium antagonists. AB - We studied the role of extra- and intracellular Ca2+ in endothelin-1-induced contractions of the isolated human internal mammary artery and vein. Veins were more sensitive to the peptide than arteries (concentration shift:3.2-fold; n = 4 10, p less than 0.05). The Ca2+ antagonists darodipine, verapamil, and diltiazem (10(-7)-10(-6) M) did not prevent the response to endothelin-1 in both vessels. In contrast, darodipine (10(-8)-10(-6) M), added after the contraction had developed, partially reversed the response in the artery (26 +/- 7%) and particularly in the vein (67 +/- 5%; n = 4, p less than 0.005 compared to the artery). Removal of extracellular Ca2+ reduced the contractions to endothelin-1 (10(-8) M) in the artery (control: 89 +/- 4% of 100 mM KCl; Ca2(+)-free: 68 +/- 4% n = 4-6, p less than 0.01), but not in the vein except at low concentrations (10(-9) M) of the peptide. After removal of intracellular Ca2+ with caffeine in the artery, endothelin-1 still evoked a contraction (17 +/- 3%, n = 3; p less than 0.005 vs. control), while in the vein the response was abolished. Thus, mobilization of Ca2+ during endothelin-1-induced contractions differs in the human internal mammary artery and vein. In the artery, the contraction depends on extracellular Ca2+, intracellular caffeine-sensitive Ca2+ stores, and a caffeine insensitive component, while in veins, mobilization of intracellular Ca2+ is most important. Ca2+ antagonists do not prevent, but partially reverse, endothelin-1 induced contractions indicating that voltage-operated Ca2+ channels do not initiate but contribute to the maintenance of the response. PMID- 1706810 TI - Inhibitory effects of diltiazem, verapamil, nifedipine, and nicardipine on sympathetic tachycardia in decentralized hearts of anesthetized dogs. AB - Intravenous diltiazem (10-300 micrograms/kg), verapamil (10-300 micrograms/kg), nifedipine (1-100 micrograms/kg) and nicardipine (1-100 micrograms/kg) inhibited the tachycardia caused by cardiac sympathetic nerve stimulation (SNS, 0.5-4 Hz) in decentralized hearts of anesthetized dogs. The dose range of each drug required to inhibit the SNS-induced tachycardia was almost equivalent to that required to produce the increase in coronary blood flow and the decrease in blood pressure. Nifedipine and nicardipine were equi-active and about 10 times more potent than diltiazem and verapamil in inhibiting the SNS-induced tachycardia. They produced a slight but dose-dependent slowing of the resting heart rate. The negative chronotropic potency was approximately nicardipine, verapamil greater than nifedipine, diltiazem. Bay K 8644 (30 micrograms/kg) antagonized the inhibitory effects of diltiazem (100 micrograms/kg) and nifedipine (30 micrograms/kg) on the SNS-induced tachycardia. Tachycardia induced by intracoronary norepinephrine (0.03-0.3 micrograms) was suppressed by diltiazem (30-300 micrograms/kg) and nifedipine (10-100 micrograms/kg). The inhibitory effects of calcium entry blocking drugs on the sympathetic tachycardia appear to be explained by the interference of the beta-adrenoceptor-mediated increase in slow inward current in the sinoatrial (SA) node. It is also suggested that other mechanisms different from calcium entry blocking action contribute to the negative chronotropic response to these calcium entry blocking drugs. PMID- 1706811 TI - Supersensitivity to vasoconstrictor action of serotonin precedes the development of atheroma-like lesions in the rabbit. AB - We have studied the relationship between the early morphological changes and arterial responsiveness to vasoactive agents in a new animal model that is proposed to mimic the events of early human atherosclerosis. Atheroma-like lesions were produced by positioning a hollow Silastic collar (referred to as a cuff) around the common carotid arteries of rabbits. Following a period of either 48 h or 1, 2, or 4 weeks after surgery, vessels from both cuffed and sham operated animals were removed, and vascular reactivity to cumulative concentrations of agonists were studied in isolated rings in organ baths. The contralateral arteries were perfused and fixed, studied by light microscopy, and the degree of intimal thickening was quantified by computer-assisted morphometric analysis and expressed as changes in the ratios of the cross-sectional areas of the intima and media in each artery. At 48 h, rings prepared from cuffed arteries were sixfold more sensitive to the contractile effects of serotonin (5-HT) than the corresponding controls. Histologically, such vessels showed some perivascular inflammation but no other morphological abnormality. At 7 days, cuffed vessels were again sixfold more sensitive to 5-HT than controls, and showed a thickened intima with marked smooth muscle proliferation and some infiltration by monocytes. Intimal/medial cross-sectional area ratios remained elevated at 2 and 4 weeks, but the supersensitivity to 5-HT diminished by 2 weeks to threefold and was absent at 4 weeks. The augmented reactivity to 5-HT at 48 h was specific, in that it did not occur for the alpha-adrenoceptor agonist, phenylephrine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706812 TI - Omega-conotoxin GVIA, the N-type calcium channel inhibitor, is sympatholytic but not vagolytic: consequences for hemodynamics and autonomic reflexes in conscious rabbits. AB - We investigated the effects of the N-type calcium channel blocking agent, omega conotoxin GVIA, on the resting hemodynamics and on some autonomic reflexes in the conscious rabbit. omega-Conotoxin 3 and 10 micrograms/kg i.v. reduced mean arterial blood pressure by 16 +/- 2 and 25 +/- 3 mm Hg, respectively, over 30 min accompanied by a tachycardia. Renal vascular conductance (Doppler flowmeter) increased by 27.6 +/- 3.7 and 38.6 +/- 10.3% at 30 min after omega-conotoxin 3 and 10 micrograms/kg, respectively. Vasodilatation was also observed but to a lesser extent in the hindquarter and mesenteric vascular beds. The baroreceptor heart rate reflex was evoked by a drug method (bolus injection of sodium nitroprusside and phenylephrine) and by inflation of perivascular balloons implanted on the thoracic vena cava and aorta. omega-Conotoxin (3 micrograms/kg) abolished the sympathetic component of the cardiac baroreceptor reflex without affecting vagal efferent activity. In addition, marked vagal-mediated bradycardia from (a) the "Bezold-Jarisch-like" reflex evoked by serotonin (1-10 micrograms/kg i.v.) and (b) the nasopharyngeal reflex evoked by cigarette smoke were unaffected by omega-conotoxin (3-10 micrograms/kg). We conclude that omega-conotoxin-induced N-type calcium channel blockade abolishes sympathetic but not vagal cardiac efferent activity. The hypotension and peripheral vasodilatation are probably due to the prejunctional sympatholytic action of the peptide. These N-type calcium channels are thus limited to the sympathetic varicosities in the rabbit. PMID- 1706813 TI - Pertussis toxin-sensitive G-protein activation does not influence the response to Bay K 8644 in embryonic chick myocytes. AB - Pretreatment of embryonic chick cardiac myocytes with pertussis toxin (1-2.5 micrograms ml-1 for 22 h) abolished the negative chronotropic effects of carbachol but not the positive inotropic effects of Bay K 8644. Neither guanosine 5'-O-3-thiotriphosphate (GTP gamma S 500 microM intracellularly), nor pertussis toxin (0.5-1 microgram ml-1 for 22 h) modified the agonist effects of Bay K 8644 on calcium channel currents recorded under whole-cell voltage clamp. These findings indicate that pertussis-sensitive guanine nucleotide binding (G) proteins does not modulate the effects of calcium channel activators in embryonic chick cardiac myocytes. PMID- 1706814 TI - Lugol stain for intraoperative determination of the proximal surgical margin of the esophagus. AB - An adequate proximal surgical margin is difficult to determine particularly in cases of esophageal carcinoma with surrounding intraepithelial invasion. We report here readily facilitated intraoperative approaches for detection of the exact margin of carcinomatous invasion of the esophagus. The resected specimen of the esophagus is incised longitudinally and placed in a 1% Lugol bath for 2-3 minutes. The normal squamous epithelium includes glycogen that interacts with the iodine of Lugol's solution and the normal epithelium of the esophagus becomes a uniform greenish-brown. A squamous cell carcinoma does not include glycogen, hence is not stained with this solution and a clear identification is feasible. Thus, a carcinomatous infiltration not recognizable in routine examinations becomes macroscopically visible when Lugol's solution is used. PMID- 1706815 TI - Tumors in undescended testis. AB - We herein report our experience in the management of 21 patients with tumors in undescended testis. The primary tumor was in the abdominal testis in six patients and in the inguinal testis in 15 patients. Seventeen patients had unilateral involvement and four had bilateral. Prior orchiopexy was reported in six (30%) patients, unilateral in three and bilateral in the other three, although tumors occurred in one side of three bilateral orchiopexy patients. Clinical staging showed five as stage I, six at stage IIb, two as stage IIc, seven as stage III, and in one no stage was possible. Microscopy showed seminoma, non-seminoma, and mixed tumors in 12, six, and three patients, respectively. As per protocol, stages I and IIb had either radiotherapy or retroperitoneal node dissection, giving three and five year survival of 11/11 (100%) and 7/7 (100%). All nine patients with stage IIc and stage III received induction chemotherapy (VAB-6) first and showed complete response (CR) in four (45%) and partial response (PR) in five (55%). Three and 5 year survival was 45% and 33%, respectively. Overall, 3 and 5 year survival was 70% and 69% respectively in all patients. Early stage disease (stages I, IIb) had excellent survivals, showing the adequacy of treatment, while patients with advanced tumor can still be salvaged with a combination of surgery, chemotherapy, and radiotherapy. PMID- 1706816 TI - Auto-anti-D blocking D determinants on red cells in pregnancy. AB - Positive direct antiglobulin test due to IgG1 autoantibody of anti-D specificity on red cells of an apparently healthy pregnant woman was found. At the period of the highest autoantibody activity D epitope were completely blocked and even by special methods used for Rh typing in patients with AIHA the detection of D antigen was not possible. Free auto-anti-D caused benign HDN in the infant. The autoantibody production was transient and stopped after delivery. D variant included into category V on the red cells of the patient under study and her two siblings was recognized. This finding, confirmed by in vitro study, gives the explanation of the complete blocking of D epitopes by anti-D autoantibody. PMID- 1706817 TI - [FK-506. A new immunosuppressive drug]. AB - In this brief review, we outline the properties of the new macrolide immunosuppressant FK-506. This selective anti-T cell agent has a similar mode of action to cyclospirin A (CSA). It is, however, more powerful, has the capacity (unlike CSA) to reverse liver allograft rejection and appears to have a higher therapeutic index than CSA in patients receiving organ transplants. Preliminary data suggest that renal function is better in recipients of liver transplants given FK-506 as primary therapy compared with those given CSA. In addition, evidence has been presented that FK-506-treated patients have shorter hospital stay than those receiving treatment with CSA. Other factors require to be taken into account, but it may be that it will prove less expensive in the future to treat patients with FK-506 than with CSA. The potential of FK-506 for the control of autoimmune diseases has been demonstrated in rodent models of rheumatoid arthritis, type I diabetes, posterior uveitis, allergic encephalomyelitis and glomerulonephritis. In the clinical field, FK-506 has been used in two cases of nephrotic syndrome resulting from glomerulonephritis; there was improvement in proteinuria and no decline in renal function. Studies to date have been restricted to one clinical centre although many hundreds of organ graft recipients have been studied. Both longer term and multi-centre investigations are urgently required, but it appears that FK-506 may offer considerable potential as an immunotherapeutic agent. PMID- 1706818 TI - [Effect of recombinant human granulocyte-colony stimulating factor (rhG-CSF) on growth of stomach cancer cell lines: preliminary report]. PMID- 1706819 TI - [HCG producing malignant teratoma of the testis: a rare cause of precocious isosexual maturation in boys]. AB - Human chorionic gonadotropin (HCG) from a testicular tumor histologically diagnosed from its metastases to be a malignant teratoma induced elevated testosterone levels and subsequent precocious isosexual development in a 12-year old boy. The endocrinologic consequences of long term ectopic HCG production in the prepubertal male are discussed. The case report illustrates the value of HCG serum levels as a marker for tumor activity. PMID- 1706820 TI - Preferences of HIV-infected patients for aggressive versus palliative care. PMID- 1706821 TI - Cystic fibrosis. Back to the chloride channel. PMID- 1706822 TI - Two types of orientation-sensitive responses of amacrine cells in the mammalian retina. AB - Neurons sensitive to the orientation of light stimuli exist throughout the mammalian visual system, suggesting that this spatial feature is a fundamental cue used by the brain to decipher visual information. The most peripheral neurons known to show orientation sensitivity are the retinal ganglion cells. Considerable morphological and pharmacological data suggest that the orientation sensitivity of ganglion cells is formed, at least partly, by the amacrine cells, which are laterally oriented interneurons presynaptic to the ganglion cells in the inner plexiform layer. So far there have been few studies of the responses of amacrine cells to oriented visual stimuli and their role in forming orientation sensitive responses in the retina remains unclear. Here I report the novel finding of a population of amacrine cells in the rabbit retina which are orientation-sensitive. These amacrine cells can be divided into two subtypes, whose orientation sensitivity is manufactured by two distinct mechanisms. The orientation sensitivity of the first subtype of amacrine cell is formed from the interactions of excitatory, centre-receptive field synaptic inputs and inhibitory inputs of opposite polarity, whereas that for cells of the second subtype seems to be the product of a marked asymmetry in their dendritic arbors. PMID- 1706823 TI - Electron-microscopic and immunohistochemical study of beta-2-microglobulin related amyloidosis. AB - beta 2-Microglobulin (beta 2-MG)-related amyloidosis has been reported as a complication in long-term hemodialysis patients. We observed beta 2-MG amyloid deposits in synovial sheaths, bone cysts and gastric mucosa. They showed unique ultrastructural features, that is bundles or nodules consisting of curved or linear amyloid fibrils, associated with various cell reactions. The electron microscopic histochemical study showed that they strongly stained with periodic acid-silver methenamine stain. A similar phenomenon was noticed in the spicules or bundles of amyloid fibrils in primary and secondary renal amyloidosis. With the cationic reagent toluidine blue 0, proteoglycan-like structures were observed around amyloid bundles and nodules, but not on each fibrils. Based on these results, we postulate that there is a close relationship between ultrastructural features and histochemical characteristics in beta 2-MG amyloid fibrils. PMID- 1706824 TI - Intermediate filaments in the endolymphatic sac of the guinea pig. AB - The expression of the five main groups of intermediate filaments and their subgroups, especially cytokeratins, was investigated in the guinea pig endolymphatic sac at the light microscopic level using immunohistochemistry. Immunostaining for cytokeratin (PKK1, PKK2, PKK3) was found in the epithelial cell layer of the sac. Vimentin was seen to stain epithelial cell layer as well as subepithelial tissue. These findings may indicate that the cytokeratins are closely related to the inner ear fluid transport. The coexpression of cytokeratin and vimentin seemed to indicate the dual function of absorption and secretion of the endolymphatic sac epithelial cell to regulate the inner ear fluid homeostasis. PMID- 1706825 TI - Resection of endobronchial tumors using a tracheoscope and neodymium:YAG laser. AB - A modified technique for palliative resections of malignant endobronchial tumors with the Nd:YAG laser is described. A special tracheoscope was used in conjunction with the instrument guide of a laser bronchoscope. The main advantage of this technique is easy and simultaneous access to both lungs for ventilation and surgical procedures. The method helps to separate surgical from anesthetic manipulations. The experience of over 70 operations using this technique provides evidence that the use of a tracheoscope is a safe and efficient method of endoscopic resections of larger intrabronchial tumors. PMID- 1706826 TI - Comparative studies of the outer membranes of Bacteroides gingivalis, strains ATCC 33277, W50, W83, 381. AB - Outer membrane fractions from Bacteroides gingivalis strains ATCC 33277, 381, W50, and W83 were isolated by French pressure cell disruption and the distribution of major and minor proteins was determined by SDS-PAGE electrophoresis after treatment with 2% Sarkosyl and 2% Triton X-100. Heat modifiability of the outer membrane proteins (OMPs) from these B. gingivalis strains was also determined after treatment at 100 degrees C and analysis by both 1- and 2-dimensional SDS-PAGE. The distribution of the OMPs on the surface of these B. gingivalis strains was determined by 125I labelling. For the most part of the OMPs of B. gingivalis presented a complex distribution, with OMPs observed between 123 kD and 13 kD. While the distribution of the OMPs was strain specific, OMPs common to all of the strains were observed. Two percent Sarkosyl treatment of the OMs at room temperature resulted in the solubilization of approximately 60% of the OMP. The Sarkosyl-insoluble MOMPs had relative molecular weights between 110 kD and 20 kD. Many of the OMPs which were separated at room temperature were heat-modified at either 65 degrees C or 100 degrees C. Heating of the OMs at 100 degrees C resulted in the heat modification of the majority of those OMPs observed at room temperature. Sarkosyl-100 degrees C OMs displayed MOMPs at apparent molecular weights between 90 kD and 15 kD. Radioiodination of the B. gingivalis strains ATCC 33277, 381, W83 and W50 revealed between 7 and 14 MOMPs at the cell surface depending upon the strain. The complexity of the OM of these B. gingivalis strains indicated the possibility of identifying and separating those OMPs involved in a variety of biological functions, including virulence, transport, and cell interaction. PMID- 1706827 TI - [Changes in the severity of burn shock in irradiated animals under the influence of blood serum alpha- and beta-globulins]. AB - Injection of blood serum alpha-beta-globulins during burn shock normalizes the amount of middle mass molecular peptides, acid proteinase activity, acid-base balance, and hemodynamics in irradiated rats. The survival of animals in this period increases from 23% to 52%. PMID- 1706828 TI - AgNOR distribution in serous tumours of the ovary. AB - The present paper investigated the distribution of AgNOR in serous tumours of the ovary, with a particular attention to borderline lesion and carcinomas. AgNOR are classified as large AgNOR (LN) and small AgNOR (SN) and are counted separately in 100 nuclei for each tumor. Total number of AgNOR was also recorded (TN). The study shows that the mean values of LN, SN and TN increase from adenomas to borderline lesions and carcinomas, with highly significant differences (p less than 0.001). LN show the most impressive differences between borderline lesion and carcinomas, without overlap of values. The follow up of the patients is not long enough for any correlation between AgNOR counts and prognosis, but preliminary data suggest that high AgNOR counts in borderline tumors should be interpreted very cautiously, because they do not seem to have any correlation with a more aggressive behaviour. PMID- 1706829 TI - AgNOR distribution in normal and dysplastic laryngeal mucosa and in laryngeal epidermoid carcinomas. AB - Samples of normal and dysplastic laryngeal mucosa and of laryngeal epidermoid carcinomas were submitted to the AgNOR silver staining technique. AgNORs were subdivided in large (LN) and small (SN) and counted separately along their sum (TN). Overlap of values is great between normal and mildly dysplastic mucosa and between moderate and severe dysplasia. Grouping together normal with mildly dysplastic mucosa, and moderate with severe dysplasia, their mean values show minimal overlap and the differences between them are highly significant. Plotting the mean values of these two groups and of carcinomas, the linear interpolations show a clearcut increase of values. PMID- 1706831 TI - The implantable cardioverter defibrillator. PMID- 1706830 TI - Calcium currents in the A7r5 smooth muscle-derived cell line. AB - We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patch-clamped cells, as well as cytosolic calcium ([Ca2+]i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of approximately 1 microM. Isoproterenol (1 microM) or forskolin (10 microM) increased currents in approximately half of the cells tested. Forskolin (10 microM) increased single-channel activity in five of eight cell attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca2+]i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca2+]i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca2+]i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca2+]i transients were due to synchronous action potentials in electrically coupled cell monolayers. PMID- 1706832 TI - CARDIOSTIM '90. Proceedings of the implantable cardioverter defibrillator sessions. June 20-23, 1990, Nice. PMID- 1706833 TI - Sudden death mortality in implantable cardioverter defibrillator patients. AB - Implantable cardioverter defibrillator (ICD) prevention of sudden cardiac death (SCD) is not absolute and our experience was reviewed to determine the frequency and nature of SCD in this population. The incidence and cause of mortality in 56 consecutive patients, who underwent ICD implantation beginning May 1982 with follow-up through May 19, 1990 were analyzed. Twenty-one patients died, 33% of the mortality was due to SCD, and 52% of deaths may be considered arrhythmic. The cumulative 1, 3, and 5 year SCD survivals were 93%, 89%, and 75%. All seven patients dying of SCD presented initially with SCD, all received previous shocks prior to SCD, and two of the seven patients had devices that were probably inactive at the time of death. We conclude that ICDs reduce but by no means eliminate arrhythmic death, particularly in those at highest risk for SCD. Arrhythmic death remained the most common cause of death in this population. PMID- 1706834 TI - ICD clinical update: first decade, initial 10,000 patients. PMID- 1706835 TI - Long-term community hospital experience with the internal defibrillator. AB - Seventy-seven patients with drug refractory ventricular tachycardia (57) and ventricular fibrillation (20) received the implantable defibrillator. There were 55 men and 22 women with a mean age of 63 +/- 10 years. The anatomical diagnoses were coronary artery disease in 61 patients, cardiomyopathy in 15 patients, and aortic stenosis in one patient. The mean ejection fraction was 32 +/- 12%. Concurrent surgery at defibrillator implantation was coronary bypass in eight patients and aortic valve replacement in one patient. There were no intraoperative mortalities. The mean ventricular fibrillation termination threshold was 13 +/- 6 joules. During a follow-up period of 16 +/- 10 months (range 2-40 months) four patients died: electrical mechanical dissociation (two patients), respiratory failure, and sepsis. Thirty-eight patients (51%) continued receiving antiarrhythmic drug therapy, with quinidine sulfate and procainamide being the most frequently utilized agents. Fifty-four patients (72%) have received a mean of 9 +/- 10 shocks (range 1-44). Implantable defibrillators are often needed in patients seen in large community hospitals. This technology can be administered successfully in this setting with complications and results comparable to those reported from university hospitals. Implantable defibrillators are effective in preventing arrhythmic death and can be used with low risk to the patients. PMID- 1706836 TI - Programmed stimulation-guided therapy compared with implantable cardioverter defibrillator device therapy in the treatment of ventricular tachyarrhythmias. PMID- 1706837 TI - Occurrence of ICD shocks and patient survival. AB - Fifty-six consecutive patients who underwent initial implantation of an implantable cardioverter defibrillator (AICD) between May 1982 and January 1990 were analyzed. During a mean follow-up of 31.5 +/- 25 months, 32 (60%) patients experienced a spontaneous shock from their device. Their clinical characteristics and survival were compared to those of 21 patients without shocks. No statistically significant difference was found in the distribution between the two groups in age, sex, cardiac diagnosis, New York Heart Association Class, presenting arrhythmia, or mean follow-up (F/U). The group with shocks had a higher incidence of previous MI (P = 0.021) a lower mean ejection fraction (P = 0.023) and had been tried on a greater number of medical regimens (P = 0.003). The 1-, 3-, and 5-year cumulative survivals were 84%, 69%, and 37% in the group with shocks and 93%, 93%, and 93% for the group without shocks. Our data suggests that the occurrence of a shock is a negative prognostic indicator and that the excellent prognosis of patients without shocks contributes in large part to the favorable outcome of AICD patients. PMID- 1706838 TI - Clinical performance of the implantable cardioverter defibrillator: electrocardiographic documentation of 101 spontaneous discharges. AB - Records of 105 patients, who received an automatic implantable cardioverter defibrillator (AICD), were studied to investigate the causes of spontaneous AICD discharges and to correlate the symptoms with the arrhythmias triggering AICD discharges. During a follow-up period of 13 +/- 8 months, 46/105 (44%) patients had 566 spontaneous AICD discharges. A total of 101 discharges were documented with Holter monitoring in 23 patients. In this study group, there were 8 (8%) AICD discharges for 5 episodes of ventricular fibrillation, and 68 (67%) discharges for 63 episodes of sustained ventricular tachycardia. Patients lost consciousness in all episodes of ventricular fibrillation, but were symptomatic prior to only 36 (53%) discharges in ventricular tachycardia. Nonsustained ventricular tachycardia persisting for a period of 7.5 +/- 2 seconds resulted in 20 AICD discharges; patients were symptomatic prior to 13 (65%) discharges. Supraventricular tachycardias triggered three discharges. One patient had two spurious discharges during sinus rhythm. In conclusion, most of the spontaneous AICD discharges were appropriate for the detected rhythms, but only clinically appropriate for the management of arrhythmias in 75% of the cases. A significant portion of the patients with sustained or nonsustained ventricular tachycardias triggering AICD discharges were asymptomatic prior to discharge, which requires further assessment of the physiology of the arrhythmia as a component of the detection algorithm. PMID- 1706839 TI - Fate of explanted ICD patients. AB - Of 56 consecutive patients who underwent an initial AICD implantation at our center, we analyzed eight patients who subsequently had their units explanted and not replaced by other antitachycardia devices. The mean age was 57.8 years, mean ejection fraction was 28.4%; six patients had coronary disease and two had cardiomyopathy. The presenting arrhythmia was sudden death in four patients and sustained ventricular tachycardia in four others. Mean follow-up from implant to explant was 25 +/- 22 months, and 22 +/- 10 months from explant to end of follow up. Reasons for explantation were: infection in five patients, lead fracture in one patient, battery depletion in one patient, and one patient underwent cardiac transplantation. Devices were not reimplanted because of: patient refusal in three patients, physician discretion in two patients (one recurrent infection, one received no shocks over 24 months), cardiac transplantation in one patient, ablation of VT focus in one patient, and one patient died while being treated for infection. Three patients died 2, 21, and 26 months after device explantation of nonsudden cardiac, sudden cardiac and noncardiac causes, respectively. CONCLUSIONS: Preoperative clinical parameters were not indicative of a lower risk of arrhythmic events in these patients as compared to the general population of AICD implantees. Of eight patients, two received alternate nonmedical therapy, one died while receiving treatment for a device-related infection; of the five remaining patients none died of cardiac causes. Termination of AICD therapy for malignant ventricular arrhythmias does not imply imminent sudden cardiac death for most patients treated by alternate modes of therapy. PMID- 1706840 TI - Survival in patients with depressed left ventricular function treated by implantable cardioverter defibrillator. AB - Mortality in patients with cardiovascular disease is generally due to pump failure or lethal ventricular arrhythmias. In patients with ventricular tachycardia (VT) or ventricular fibrillation (VF) and poor left ventricular (LV) function the death rate is particularly high. The overall incidence of premature arrhythmic death rate in patients with poor LV function is not totally clear. Since implantable cardioverter defibrillator (ICD) could prevent arrhythmic death in any population, we proceeded to analyze mortalities in patients with poor LV function who received ICD. Among a total of 200 consecutive patients receiving ICD at our institution, 68 (34%) had LV ejection fraction (LVEF) of less than 30%. Thirty-one of these (45%) experienced appropriate ICD discharges and 17/31 (55%) had multiple shocks. Survival curves in this population revealed a 5 year projected overall survival of 11% whereas an actual survival was 60%. Even those who ultimately died from nonsudden causes, life was prolonged by ICD in a significant number of cases. Based upon these findings it is concluded that ICD has a major impact on survival in patients with poor LV function suggesting that many of these patients die prematurely from arrhythmia causes. PMID- 1706841 TI - The implantable cardioverter-defibrillator: clinical results. AB - To evaluate the effectiveness of the automatic implantable cardioverter defibrillator (AICD), a 7-year experience, from 1983-1990, was reviewed. A total of 111 patients received an AICD device. Their ages ranged between 8 and 83 years. Mean age was 63.9 years. There were 91 men and 20 women. Eighty of the patients received the AICD following an out-of-hospital cardiac arrest, while 31 were suffering from intermittent symptomatic ventricular tachycardia. The underlying etiology in 97 patients (87%) was ischemic coronary artery disease, in 11 patients (10%) dilated cardiomyopathy, and in 3 patients (3%) idiopathic ventricular fibrillation. Mean ejection fraction was 33.2%. Implantation of the AICD was performed via a left thoracotomy in 39 patients, median sternotomy in 49 patients and subxiphoid-subcostal approach in 23 patients. In-hospital mortality occurred in one patient who suffered an acute myocardial infarction 4 hours postoperatively. Out-of-hospital mortality was observed in 19 patients. There were two arrhythmic deaths. Follow-up was available for 107 patients. Mean follow up was 33.1 months. Sixty-six patients (62%) had AICD shocks. The initial appropriate shocks occurred during the first postimplantation year in 91% of the patients. In 53 of the survivors, initial AICD shocks took place within 4.4 +/- 4.7 months from implantation. Thirteen of the 20 patients who died had received appropriate AICD shocks. In these patients, the time between implantation and first shock was 2.7 +/- 3.6 months whereas the time between implantation and death was 11.3 +/- 10.3 months (NS). We conclude that the AICD is effective in converting ventricular tachyarrhythmias and prolongs survival. PMID- 1706842 TI - Device interaction--antitachycardia pacemakers and defibrillators for sustained ventricular tachycardia. AB - We evaluated the combined use of permanent automatic antitachycardia pacemakers and implanted defibrillators in ten patients with recurrent monomorphic sustained ventricular tachycardia (VT). Pacemaker programming was VVI-T automatic burst in eight patients, VVI-T magnet mode in one patient, and VVI in one patient. Device interactions occurred in four patients, requiring changes in pacemaker programming. These included defibrillator multiple counting during pacing, inappropriate pacemaker bursts initiating VT, inappropriate reset of the pacemaker antitachycardia mode by defibrillation, defibrillator discharge after pacemaker VT termination, and defibrillator VT reinitiation. Two patients required pacemaker programming out of the antitachycardia mode, and two required a change in antitachycardia pacing parameters. Seven patients remain in automatic VVI-T and three in VVI modes. Mean follow-up is 13 months and all patients are alive. Thus, although pacemaker/defibrillator combinations function well for patients with more than one VT rate, device interactions occur frequently and may require pacemaker reprogramming or elimination of the overdrive mode. Combined use of these devices should be cautiously considered when single device therapy is unsatisfactory. Devices that combine both pacing and defibrillation features may reduce adverse interaction. PMID- 1706843 TI - A prospective study utilizing a transtelephonic electrocardiographic transmission program to manage patients in the first several months post-ICD implant. AB - We prospectively enrolled 20 consecutive patients (11 men and 9 women; mean age 63 +/- 9.5 years) post-AICD implant in a transtelephonic electrocardiographic transmission (TET) program. The monitor was chosen for its retrograde (30 seconds) and antegrade memory capabilities (45 seconds). The patients were discharged from the hospital after receiving instructions to utilize the system for any cardiac symptoms. The monitor was worn 1-3 months (mean 2.5 +/- 0.7 months). During the follow-up period there were 54 TETs received. Nine were for documented AICD discharges, 19 were for symptoms associated with arrhythmias (11 of these 19 reported AICD discharges that were not documented), and 26 for symptoms not associated with arrhythmias. Eight of the 9 AICD discharges documented were appropriate for ventricular tachycardia (mean 185 +/- 40 beats/min). The arrhythmias associated with symptoms were: atrial fibrillation (12); nonsustained ventricular tachycardia (3); ventricular couplets (2); ventricular premature beats (10); and atrial premature contractions (2). Several TETs documented multiple arrhythmias. The most common symptoms not associated with arrhythmias were shortness of breath, dizziness, chest pain, and nervousness. Office interrogation of the AICDs revealed 12 of the 20 patients (60%) had received AICD discharges, with 5 of these 12 patients unaware of this occurring. We found the TET monitoring system a useful tool in the management of the AICD patient the first several months postoperatively. We were able to assess device function and avoid unnecessary office visits and/or hospitalizations. PMID- 1706844 TI - The impact of antitachycardia pacing with defibrillation. AB - Chronic recurrent ventricular tachycardia (VT) can be reproducibly terminated by programmed endocardial right ventricular stimulation. However, antitachycardia pacing can be associated with possible acceleration of VT, while frequent episodes of VT and patient discomfort can limit treatment by an implantable cardioverter defibrillator (ICD). The combined use of antitachycardia pacing and the AICD (automatic implantable cardioverter defibrillator) was evaluated in 6 out of 51 patients (age 57 +/- 11 years) in whom the AICD had been implanted because of recurrent VT. In each instance VT could be terminated by temporary overdrive pacing. The interactive mode of VT termination by a pacemaker (Tachylog) as well as by the AICD was assessed after implantation. In the automatic mode, the Tachylog functioned as a bipolar, ventricular inhibited (VVI) device with antitachycardia burst stimulation capability, allowing two to five stimuli at intervals of 260-300 ms and one or two interventions. During follow-up of 47 +/- 24 months, the Tachylog terminated VT reliably 50-505 times per patient. When burst stimulation accelerated VT, termination was achieved by AICD discharge. Thus, drug resistant VT can be terminated by antitachycardia pacing to avoid patient discomfort. In the event of tachycardia acceleration, VT was terminated by the AICD. A universal pacemaker-defibrillator should combine antibradycardia and antitachycardia pacing with back-up cardioversion defibrillation. PMID- 1706846 TI - Biosensor applications to antitachycardia devices. AB - Current arrhythmia detection algorithms are unable to adequately distinguish stable from unstable tachycardias; therefore application of a biosensor to antitachycardia devices has been proposed to improve their performance. Right heart pressures and impedance have been investigated for incorporation into these systems. Integration of other parameters (oxygen saturation, preejection period, pH, cardiac output, flow, and temperature) into these devices might also prove useful. The status of these biosensor arrhythmia detection algorithms and their application to antitachycardia devices are described below. PMID- 1706845 TI - Implantable cardioverter defibrillator: data storage and retrieval with Patientlog. AB - A pacemaker management system (Patientlog), implemented on a IBM personal computer (AT), was adapted for the control and the administrative management of patients with an implantable cardioverter defibrillator (ICD). Several parameters used for pacemakers were also suitable for ICDs, while some fields were defined within the available frame of the database, to cope with the specific technical information of ICDs. "Intervention-cards" display diagnostic (threshold) as well as complex surgical information (approach, lead systems, etc.). "Follow-up-cards" present charge time and the number of shocks in such a way that decision making is very easy. Fourteen patients were followed with a total of 21 interventions, six different types of devices, and more than 150 follow-ups. A correct control of ICD functions is easy. The system is adapted for programmable ICDs. Updated reports containing necessary information are generated immediately after each procedure or follow-up. PMID- 1706847 TI - A time-domain analysis of intracardiac electrograms for arrhythmia detection. AB - The analysis of intracardiac electrogram morphology has been proposed as a complementary method for accurate discrimination between sinus rhythm (SR), supraventricular dysrhythmias, and ventricular dysrhythmias by automatic antitachycardia and cardioverter defibrillator devices. In this study, the performance of a traditional time-domain method for surface electrocardiogram interpretation--Correlation Waveform Analysis (CWA) and a newly developed technique--Bin Area Method (BAM) were used to analyze unfiltered intraatrial and intraventricular electrograms obtained from 47 patients during routine cardiac electrophysiology studies. Nineteen patients had 31 distinct, sustained, monomorphic ventricular tachycardias (VTs) induced; 13 patients had paroxysmal bundle branch block of supraventricular origin (BBB) induced; 19 patients had retrograde atrial activation during ventricular overdrive pacing. Three patients were common to two or more groups. Using a best fit electrogram alignment, both CWA and BAM distinguished VT from SR in 28/31 cases (90%), BBB from SR in 15/15 patients (100%), and anterograde from retrograde atrial activation in 19/19 patients (100%). We conclude that the use of time-domain techniques that are independent of amplitude and baseline fluctuations appear to be reliable for discrimination of retrograde atrial activation, paroxysmal BBB, and VT from SR using intracardiac electrograms. Reduction of computational time and power constraints, without sacrificing reliable dysrhythmia discrimination, is possible. These features may make real-time morphology analysis of intracardiac electrograms feasible for automatic antitachycardia and cardioverter defibrillator devices. PMID- 1706848 TI - Recognition of multiple tachyarrhythmias by rate-independent means using a small microcomputer. AB - New implantable devices are now available that can offer different therapies for different arrhythmias but they need a method of discriminating between these rhythms. Heart rate analysis is predominantly used to discern between sinus rhythm (SR) and pathological tachycardias but this may be of limited value when the rates of the rhythms are similar. An enhanced form of Gradient Pattern Detection (GPD) has been developed using an 8-bit microcomputer that can distinguish between SR and up to three other arrhythmias in real time. This is a method based on electrogram morphology where each rhythm's specific electrogram is classified by a sequence of gradient 'zones'. The microprocessor of the computer is of similar processing power to ones used in current pacemakers. Five patients with multiple arrhythmias were studied. Four had ventricular tachycardia (VT) and one had three conduction patterns during supraventricular tachycardia (SVT). Bipolar endocardial right ventricular electrograms were recorded during SR and tachycardia in all patients. The computer would first 'learn' about each different rhythm by a semi-automatic means. Once all the rhythms were learned the program would enter the GPD analysis phase. The computer would output a series of real-time rhythm specific marker codes onto a chart recorder as it recognized each rhythm. Sixteen different arrhythmias (13 VT, 3 SVT) were examined for this study. All rhythms (including SR) were distinguished from each other except in the case of one patient with six VTs where two VTs had identical shapes and therefore could not be detected apart. The method would be a useful addition to heart rate analysis for future generations of microprocessor assisted pacemakers. PMID- 1706849 TI - Long-term testing of defibrillator batteries. AB - This article describes the life testing of lithium/silver vanadium oxide defibrillator batteries. Batteries were tested at 37 degrees C under conditions which simulate clinical conditions. A total of 788 cells are currently under test. No failures have been observed, and cells are performing normally. PMID- 1706850 TI - A packaged solution to the problems of testing arrhythmia control devices at implant. AB - A composite implantable arrhythmia control device (ACD) implements the functions of a bradycardia pacemaker, an antitachycardia pacemaker, a cardioverter, and a defibrillator in an integrated fashion. Given this broad spectrum of functionality, the implant testing for these devices can become a formidable endeavor requiring a large ensemble of expensive, complex support equipment, and a significant amount of time. However, if this procedure is not carried out correctly, the device might later fail to defibrillate. This article presents a unique packaging system for an ACD that allows the device to be used while it is still in its sterile package. The device may then be used during implant testing as the defibrillation test unit. This collapses the amount of support equipment that is required to just the ACD, its programmer, and an optional switch box. By providing additional support specifically for implant testing through the ACD programmer, implant testing may be reduced to a quick, easy-to-manage procedure. Since the device used during implant testing is the same device that will be implanted, this packaging system offers the further advantage that the physician can be confident that, once implanted, the ACD will function correctly. PMID- 1706851 TI - Can changes in transcardiac impedance appropriately detect ventricular fibrillation? AB - Transcardiac impedance, as measured between two implanted defibrillator patches, has been shown to vary in peak-to-peak magnitude in response to the contractions of the ventricles. These variations in transcardiac impedance are known as delta Z. This study was aimed at determining if there was a significant change in the magnitude of delta Z between that during normal sinus rhythm, that during ventricular fibrillation (VF), and if this change could be used as a detector of the onset of VF. This study was performed acutely on 14 anesthetized dogs. PMID- 1706852 TI - Differences in the pathological changes in dogs' hearts after defibrillation with extrapericardial paddles and implanted defibrillator electrodes. AB - A comparison was made between the pathological changes in the myocardium of eight dogs, each receiving about 90 joules of energy in a series of defibrillation discharges, delivered either between paddles placed against the pericardium (3 dogs) or between implanted Telectronics 040-105 defibrillation patch electrodes (5 dogs). The changes in the myocardium were most pronounced where the paddles had been applied to the pericardium. There was transmural damage beneath the left and right paddle positions and in the surrounding tissues. Extensive subepicardial and subendocardial myocyte damage was obvious histologically in the right ventricle of one of the patch dogs and in all of the paddle dogs. The percentage of damaged myocardial mass, both right ventricular and total involvement, was higher in the three paddle dogs compared with the five patch dogs. There was septal damage in the heart of one paddle dog. Necrosis of the right ventricular wall was observed in three of the patch dogs and in all the three paddle dogs. Scattered necrotic myocytes and some patches of mild necrosis up to 1-mm deep were observed in the left ventricle of the patch dogs (severity score 1-3). The necrosis was more extensive in the paddle dogs, ranging from mild necrosis less than 1-mm deep to marked necrosis incorporating half-to-whole ventricular wall thickness (severity score 3-5). PMID- 1706853 TI - Comparison between two versus three patches single pulse shock defibrillation in pigs. AB - The aim of the study was to test the hypothesis that defibrillation with a single pulse shock can be obtained at lower energy using three epicardial patches configuration (one cathode and two anodes) instead of the conventional two patches. The total surface area of the two- and three-patches configuration was the same (10 cm2 vs 9.9 cm2). Epicardial spatial configuration was planned by using a computerized heart model. In ten anesthetized open-chest pigs, ventricular fibrillation was induced by using AC current through the mesh plaque epicardial custom-designed electrodes, and the minimum energy requirement for defibrillation was determined 15 seconds after the onset of stable ventricular fibrillation. Results were as follows (mean +/- standard deviation): Defibrillation Threshold 16 +/- 9 J 9 +/- 5 J P less than 0.01 CONCLUSIONS: three epicardial patches configuration significantly reduces energy requirements for defibrillation compared with two patches when single pulse shock is used. PMID- 1706854 TI - Relationship between QT interval duration and electrical induction of ventricular tachycardia. AB - The relation of inducible ventricular tachycardia (VT) to QT interval duration of ventricular paced rhythm has not been evaluated. To clarify this relation we measured corrected QT interval duration (QTc) during sinus rhythm and QT interval duration during ventricular paced rhythm (QT-V) in patients with coronary artery disease without (non-VT group = group B) and with inducible VT (VT group = group A). Duration of QT-V was greater in the VT group (n = 20) compared with non-VT group (n = 20) during ventricular pacing at cycle lengths of 600 ms (424 +/- 26 vs 396 +/- 19 ms, P less than 0.01), of 500 ms (407 +/- 20 vs 383 +/- 21 ms, P less than 0.01), and of 400 ms (390 +/- 21 vs 362 +/- 17 ms, P less than 0.001). During sinus rhythm the mean values of QTc were similar in both groups (408 +/- 25 vs 413 +/- 20 ms, NS). During ventricular stimulation the percentage of patients with values of QT-V exceeding 380 ms was 35% in non-VT group and 95% in VT group (P less than 0.01) at cycle length of 500 ms and 5% versus 60%, respectively, (P less than 0.01), at cycle length of 400 ms. Thus, a trend toward longer QT values of ventricular paced rhythm exists in patients with inducible VT. PMID- 1706855 TI - Ventricular tachycardia/fibrillation: therapeutic alternatives. AB - It is now clear that no single therapy is appropriate for a consecutive series of patients with ventricular tachycardia or ventricular fibrillation (VT/VF). Drug responders by electrophysiological studies, patients who are not inducible following surgery, and patients treated with an implantable cardioverter defibrillator (ICD) all can have similarly low sudden death rates and virtually identical long-term mortality. However, many patients fail to respond to drugs, and surgical risks are excessive in others, and always higher than for an ICD implant. Nevertheless, overall survival in each of these groups (and probably for patients treated with antitachycardia pacers and ablation) is about 60% at 60 months. Major challenges now are: (1) choosing therapy to maximize risk-benefit ratio; and (2) treatment of the pump failure and progressive disease that now accounts for most cardiac mortality. PMID- 1706856 TI - Prophylactic use of implantable cardioverter defibrillators: medical, technical, economic considerations. PMID- 1706857 TI - Implantable pharmacological defibrillator (AIPhD): preliminary investigations in animals. AB - The treatment of ventricular fibrillation (VF) by means of automatic implantable cardioverter defibrillators (AICD) poses many severe problems and limitations at the present time. In order to overcome these problems, we propose a totally new way to terminate VF or ventricular sustained tachycardia (VST). Our proposal consists of replacing the electric shock, which is dangerous, delayed, and sometimes ineffective, with a "chemical" shock: i.e., a chemical bolus retroperfused in the coronary sinus (CS) immediately after VF arises. The possible device is hypothesized and preliminary investigations in animals, performed to verify the theoretical assumption, are presented. In rabbits, and in larger animals (sheep and swine). Drugs were perfused in the coronary bed: lidocaine was used in 86% and bretylium tosylate in 14% of the animals. The results were: lidocaine immediately terminated VF in 100% and sinus rhythm was restored in rabbits; lidocaine terminated VF in VST in sheep; and in swine, bretylium immediately produced sinus rhythm in one case; in another one, only delayed sinus rhythm was achieved but lasted a short time; in the last case ventricular tachycardia at 128 beats/min appeared. Because new drugs, which are really "defibrillating" drugs, are available (bretylium tosylate, bethanidine, clofilium, tricyclic antidepressants, phenotiazine derivatives), we plan to investigate these defibrillating drugs in isolated hearts, found in suitable animals like dogs (sheep and swine are difficult to defibrillate) and in humans during routine electropharmacological studies. PMID- 1706858 TI - Ichthyosis follicularis in two girls: an autosomal dominant disorder. AB - Ichthyosis follicularis (IF) is a rare disorder of keratinization that has been described primarily in males and proposed as a possible X-linked disorder. We report two black girls with nonscarring alopecia; photophobia; follicular hyperkeratoses; hyperkeratosis of the extensor aspects of the hands, knees, and elbows; fixed, erythematous, perineal plaques; and angular cheilitis who seem to fit the clinical criteria for IF. One girl also had gingival hypertrophy and a hearing deficit. One child's father had identical symptoms. We propose that these girls may have a variant of IF that is inherited as an autosomal dominant trait. PMID- 1706859 TI - [Inter-alpha-trypsin inhibitor and its derivatives in inflammatory syndromes]. AB - Modifications of inter-alpha-trypsin inhibitor (ITI) in inflammatory syndromes were determined by studying its serum components: ITI 80 (the native form) and serum derivatives (SD), as well as urinary ITI derivatives (UID) excretion in 31 controls and 128 patients with inflammatory of various origins. The patients were divided into 4 groups: Group I bacterial infections (n = 29); Group II cancers (n = 50); Group III inflammatory diseases (n = 14); Group IV inflammatory syndromes due to other causes (n = 35). Other markers of inflammation were also studied. In bacterial infections and cancers ITI 80 concentrations were significantly decreased, with values of 0.55 +/- 0.15 g/l and 0.54 +/- 0.15 g/l respectively vs 0.65 +/- 0.11 g/l in controls. SD concentrations were significantly increased in all 4 groups: Gr I: 0.31 +/- 0.12 g/l; Gr II: 0.30 +/- 0.11 g/l; Gr III: 0.25 +/- 0.08 g/l; Gr IV: 0.24 +/- 0.10 g/l, as compared with 0.16 +/- 0.09 in controls. UID excretion was increased in all cases, particularly in bacterial infections and cancers (10.8 +/- 13.4 and 6.0 +/- 8.8 mg/mmol of creatinine vs 1.5 +/- 1.7 g/mmol). A significant correlation was observed between CRP levels and SD levels. In bacterial infections and cancers, a fall in ITI associated with a rise in SD and an increase in UID excretion is suggestive of degradation of the native form. In inflammatory diseases and inflammatory syndromes of other causes, the rise in SD without significant variations in ITI 80 suggests and increase in SD synthesis. The correlation between CRP and SD seems to indicate that SD are produced in the early stage of inflammatory syndromes. PMID- 1706860 TI - [Effectiveness of intravenous immunoglobulins in polymyositis and dermatomyositis. An open trial in 15 patients]. AB - Polymyositis (PM) and dermatomyositis (DM) are dysimmune diseases usually treated with corticosteroids and immunosuppressants. Human polyvalent immunoglobulins administered intravenously (IgIV) are known to be effective in some dysimmune diseases. Between August 1987 and September 1989 we conducted an open trial of IgIV in 15 patients (mean age 44 +/- 14 years) with either PM (12 cases) or DM (3 cases) associated with a collagen disease in 2 patients. In 14 of these 15 patients the conventional treatments (corticosteroids, immunosuppressants, plasmapheresis, total body irradiation, lymphopheresis) had failed. One patient was seropositive for picornavirus and received IgIV as initial treatment. IgIV infusions were given 4 +/- 3.9 years on average after the onset of PM or DM. Twelve of the 15 patients received another treatment, starting at least 6 weeks before IgIV and pursued without dosage increase, which consisted of corticosteroids (11 cases), methotrexate (5 cases) or plasmapheresis (1 case). Human polyvalent immunoglobulins for intravenous use were prescribed in doses of 2 g/kg/monthly course. All but two patients (1 course) received 3 to 6 courses on average. The IgIV infusions were well tolerated in 12 patients; 3 patients showed allergic manifestations which regressed. Therapeutic effectiveness was evaluated by muscle testing and by repeated assays of creatine phosphokinase (CPK). Clinical improvement, usually perceptible after the first course, was observed in 13/15 patients; it was associated with a more than 30 percent decrease of the initial CPK level in 13 patients and with a reduction of associated therapies in 9 patients. In the entire patient population a statistically significant lowering of mean CPK value was observed as early as in the first course (P less than 0.001). In view of their effectiveness, rapid action and safety, intravenous Ig infusions may be regarded as an interesting treatment in PM or DM patients. PMID- 1706861 TI - Systemic sulpiride increases dopamine metabolites in the lateral hypothalamus. AB - In rats with microdialysis probes in the perifornical lateral hypothalamus (PFH) a single injection of the D2 receptor blocker 1-sulpiride (20 mg/kg IP) significantly increased extracellular dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), but not 5-hydroxyindoleacetic acid (5-HIAA). This suggests that sulpiride crosses the blood-brain barrier and blocks D2 dopamine receptors in the PFH leading to increased dopamine turnover reflected in increased extracellular DOPAC and HVA. We conclude that D2 blockade in the hypothalamus could play a role in the hyperphagia and body weight gain observed in female rats under chronic administration of the antipsychotic drug, sulpiride. PMID- 1706862 TI - Neuronal sites mediating locomotor hyperactivity following central neurokinin agonist administration. AB - The stable substance P analog, DiMeC7, increases spontaneous locomotor activity after infusion into the lateral ventricles or median raphe nucleus (MRN). The elevated locomotion observed after intracerebroventricular (ICV) infusion of DiMeC7 was attenuated, but not blocked, by bilateral 6-hydroxydopamine (6OHDA) lesions of the ventral tegmental area (VTA). In contrast, bilateral 6OHDA lesions of the nucleus accumbens (NAS) blocked the motor activity induced by ICV administration of DiMeC7. Similar lesions did not affect the increases in motor behavior observed after MRN infusions of DiMeC7. However, the hyperactivity following MRN microinjections of DiMeC7 was attenuated by intraperitoneal administration of the dopamine (DA) antagonist, haloperidol. The results suggest that ICV infusions of DiMeC7 increase locomotor activity by acting directly on neurons in the NAS and in part by influencing, directly or indirectly, the activity of DA cells in the VTA. The increased motor activity seen after MRN administration of DiMeC7 appears to depend on DA neurons but not on projections to the NAS. PMID- 1706863 TI - Effects of axotomy on the cholinesterase and carbonic anhydrase activities of axons in the proximal and distal stumps of rabbit sciatic nerves: a temporal study. AB - The temporal changes in transected sciatic nerves of rabbits were studied using recently developed histochemical techniques for discriminating between sensory and motor axons. A segment of the nerve was removed to inhibit spontaneous regeneration across the gap. Staining characteristics of the proximal and distal nerve stumps were studied at 1, 2, 3, 4, 9, and 35 days following axotomy and compared with control sciatic nerves. In the control and test nerves, subpopulations of myelinated sensory axons were identified histochemically by carbonic anhydrase (CA) staining, and a subset of alpha motor axons were visualized by cholinesterase (CE) staining. Axon staining patterns were reciprocal; i.e., sensory axons were CA-positive and CE-negative, whereas motor axons were CE-positive and CA-negative. Histochemical activities persisted at day 35 after axotomy in the proximal stump and until day 9 after axotomy in the distal stump. This implies that these techniques may aid in both immediate and delayed primary nerve repair. With time, there is buildup of scar tissue adding to the proximal and distal stumps. Therefore, sections for demonstrating sensory and motor axons must be taken progressively further back from the nerve stump ends. Histochemical axon typing indicated that sensory axons regenerated earlier and to a greater degree than motor axons in the developing neuroma. Use of both the carbonic anhydrase and cholinesterase staining methods is more accurate than either technique alone as an adjunct for examining normal and injured peripheral nerves. PMID- 1706864 TI - Vimentin and keratin are expressed in the neurogenic tissue of the rabbit embryo during primary neurulation. AB - Against the commonly held belief that differential expression of keratins is a sign of neurogenic commitment amongst ectodermal cells of the early vertebrate embryo we show here that the same keratins (8 and 18) are expressed in the epidermal ectoderm and the neurectoderm throughout primary neurulation of the early rabbit embryo, i.e. between 8.5 and 11 days post conceptionem (d.p.c.). However, keratin expression decreases during this developmental period and, by the time primary neurulation is completed, keratin expression is virtually absent in the cells of the neural tube. Vimentin expression is weak, at first, but increases in a reciprocal manner as compared to the decreasing keratin expression until it has reached a high and stable level of expression in the established neural tube of the 10 to 11 day old rabbit embryo. PMID- 1706865 TI - Immunohistologic analysis of neuron-specific enolase and neuropeptides in stromal cells of cerebellar hemangioblastomas. PMID- 1706866 TI - Concentration of hyaluronectin and anionic glycoconjugates in perineuronal glial cell processes at GABAergic synapses of rat cerebellum. AB - Hyaluronectin was detected in the rat cerebellum using monospecific polyclonal antibodies at the light and electron microscopic level. The immunoreactivity was associated in the grey matter with glial (astrocytic) cell processes of perineuronal glial sheets but could not be found in glial cell bodies nor in neurons. This molecular specialization of grey matter astrocyte expansions was distinctly expressed at known sites of GABAergic synaptic transmission. PMID- 1706867 TI - The sterical order of the sialic acid in the microvillous layer of the human duodenal mucosa. PMID- 1706868 TI - Cell adhesion molecules in brain. PMID- 1706869 TI - Histochemical and ultracytochemical studies on complex carbohydrates in the cyclic rat endometrium. PMID- 1706870 TI - Application of the chromogenic reaction to conventional silver staining, the Ag NOR staining and the silver-intensified immunogold technique. AB - The principle of the chromogenic reaction and the transformation of "black and white" histochemical staining results or immunohistochemical signals to coloured microscopic images is described. The chromogenic reaction was optimized and is, so far, possible with either cyan-blue or magenta-red reaction products. The application of the chromogenic reaction to conventional silver stain was optimal in the Lendrum staining resulting in red or blue stained reticulin fibres. The Ag NOR staining of the nucleolus organizing region (NOR) could be transformed by the same reaction to coloured reaction products as well as the silver-intensified immunogold technique in immunocytochemistry. PMID- 1706871 TI - Histophotometry of protein thiols and disulphides in tissue samples from the human uterine cervix and the skin reveals a "field effect" as well as an "extended field effect" of malignant tumours. AB - Fresh frozen and fixed serial sections were stained with 2,2'-dihydroxy-6,6' dinaphthyldisulfide (DDD) and Fast blue B for reactive protein thiols (PSHr) and total reactive protein sulfur (TRPS). The mean optical densities of PSHr and TRPS determined histophotometrically at a distinct part of a tissue were related to each other (PSHr: TRPS). If this quotient has been determined, e.g. for normal epithelium and the adjacent stroma, both quotients can be related to each other by a double quotient (Q PSHr: TRPS). With the aid of the double quotient highly significant differences could be found between tumours and normal tissue from patients without tumour of the human uterine cervix. Similar differences exist between normal skin from healthy patients and skin tumours. Q PSHr: TRPS revealed similar differences to exist between normal tissue of patients without tumour and apparently normal tissue in the neighbourhood of tumours of the uterine cervix and of skin ("field effect" of tumours). Histophotometric investigations on abdominal skin (and skin of breast) showed highly significant differences between normal skin of patients without tumour and patients with various kinds of tumours of the uterine cervix, ovaries, liver and breast ("extended field effect" of tumours). PMID- 1706872 TI - The influence of embedding on the stoichiometry of the pararosaniline-Feulgen stain in histological material. AB - In the present study the influence of the embedding technique on the dye substrate-interaction of the Feulgen reagent has been investigated. Pieces of rat liver and of human lung tumours were fixed in formaline, Carnoy's or Bouin's solution or in SuSa and embedded in paraffin (P) or in glycolmethacrylate (GMA). Sections were stained with the pararosaniline-Feulgen-reagent. Mean optical density (MOD) of cell nuclei was measured with an image analyzer. GMA required longer times for hydrolysis and staining than P. the plateau phase of acid hydrolysis was longer in GMA. In general MOD was significantly lower in GMA than in P. The ratio of MOD of lymphocyte nuclei versus diploid basal cell nuclei in lung tissue was 0.97 in GMA and 0.88 in P, respectively. We presume that cross linking of GMA monomers during polymerization stabilizes hydrolysed DNA. Steric factors are responsible for the slow diffusion of the dye-molecule. The influence of the embedding medium on the stoichiometry of the Feulgen stain has to be carefully considered for statistical evaluation of DNA-histograms. PMID- 1706873 TI - F3: a new developmentally regulated member of the HNK-1 family. PMID- 1706874 TI - Neurofilament expression in bovine chromaffin cells. AB - Neurofilament (NF) expression was examined in adult bovine adrenal chromaffin cells by immunocytochemistry using a series of monoclonal antibodies directed against either nonphosphorylated or phosphorylated epitopes of the heavy NF subunit. In situ, this NF subunit was not detected in chromaffin cells. However, chromaffin cells grown in primary culture under standard conditions contained NF proteins, but only in a nonphosphorylated state. Phosphorylation of NFs could be induced under culture conditions favouring the development of a neuronal phenotype--40% of the cells developed neurites within a week while NF phosphorylation occurred later. Phosphorylated NFs were restricted to neurites, unlike nonphosphorylated NFs which were observed in both perikarya and neurites. PMID- 1706875 TI - Neural and endocrine markers as diagnostic tools in pancreatic and gastrointestinal endocrine tumors. PMID- 1706876 TI - Immunogold labelling of membrane receptors for scanning electron microscopical investigations of genetically determined disorders of lipid metabolism. AB - Receptors binding of low-density lipoprotein at surfaces of human lymphocytes was studied by immunochemical and scanning electron microscopical methods. Using colloidal gold sol binding ability of cells from several probands could be demonstrated by immunolabelling. We investigated cell material from normolipidaemic persons and from patients with heterozygote autosomal monogenic hypercholesterolaemia. We found a distinct decrease of labelled gold particles at the cell surface of patients with this disorder of lipid metabolism. The simple extraction possibility of lymphocytes from human venous blood and the demonstrated scanning electron microscopical results recommend the described procedure for genetic application. PMID- 1706877 TI - Electron microscopic study of receptor mediated endocytosis of a monoclonal antibody (RoMo-1) against the surface marker CD 14 of human monocytes. AB - The internalization of the gold labelled monoclonal antibody RoMo-1 recognizing the CD 14 surface molecule of human monocytes was studied by electron microscopy. Monocyte enriched mononuclear cells (MNC) from the peripheral blood of healthy donors were incubated for 15, 30, 60, and 90 min with gold marked RoMo-1 in Eagle MEM at 37 degrees C. The process of internalization of RoMo-1 occurs as a receptor mediated endocytosis (RME), as described for other ligands. PMID- 1706878 TI - [Fine structure studies and identification of lectin receptors on the viral envelope of two HIV-isolates]. AB - The electron microscopic particle findings were compared with the levels of revertase in corresponding samples over a longer period of time, and a good correlation was found. Comparative investigations of the fine-structure of two HIV isolates did not reveal any morphological differences. It can be assumed, on the basis of the comparative studies on lectin receptors using Helix pomatia lectin, that the viral envelopes of the two isolates are equipped similarly with N-acetyl-d-galactosamine. The differences are not significant with mature particles. PMID- 1706879 TI - [Binding of monoclonal antibodies to an insulin-producing rat tumor cell (RIN) after partial cell synchronization]. AB - Murine monoclonal antibodies against pancreatic islet cell antigens were generated by somatic cell hybridization and their binding to the insulin producing rat insulinoma cell line (RIN-5 AH) was evaluated in an indirect immunofluorescence test. An ascites dilution of 1:50 was used for immunostaining using RIN cells harvested from monolayer cultures in RPMI 1640 supplemented with 7.5% fetal calf serum (controls) or test cultures supplemented with 10 mmol/l hydroxyurea to collect the cells in G1/S phase. The five mc-ICSA tested showed a wide variety in their individual binding from 95 up to 30% but their binding was not influenced by hydroxyurea use for synchronization. The antigenic determinant recognized by the anti-glucolipid mc-ICSA 56a F3 was not present on all RIN-5 AH cells. In average, only 35% of the RIN cells showed a striking binding of the monoclonal 56a F3. PMID- 1706880 TI - Heparin-induced thrombocytopenia. PMID- 1706881 TI - Vitronectin. PMID- 1706882 TI - Sequence and analysis of BECV F15 matrix protein. AB - Clones from the bovine enteric coronavirus (F15) cDNA library were cloned in pBR322 and sequenced by the method of Sanger and Coulson. This led to the identification of a sequence of 1,300 bases which contained a single open reading frame of 690 bases yielding a protein having properties of the matrix protein (M). It was comprised of 230 amino acids with a molecular weight of 26,376 Da. It was hydrophobic and had a net charge of +8 at neutral pH. Analysis of its secondary structure could not establish a simple transmembrane arrangement of the amino acids. Comparison of its nucleotide sequence with that of BECV Mebus strain showed only a two-base change resulting in a 100% homology between the two amino acid sequences. Furthermore, a very conserved structure of M appeared on comparison with the Dayoff optimal alignment of MHV-A59, MHV-JHM, TGEV, IBV Beaudette and IBV 6/82M amino acid sequences. As the two strains of BECV, F15 and Mebus present some antigenic differences, this led us to reconsider the role of M in viral antigen specificity. A hypothesis is that, as it seems to possess the necessary information on its transmembrane region, it is an ideal candidate for the viral budding process. PMID- 1706883 TI - Relative frequency of rotavirus serotypes in Yamagata, Japan, over four consecutive rotavirus seasons. PMID- 1706884 TI - Reflex and chemical responses of tracheal submucosal glands in piglets. AB - In adult animals, airway fluid secretion is enhanced reflexly via central nervous system pathways, and locally by mediators such as substance P. To evaluate the role of maturation on these regulatory mechanisms, we compared the effects of reflex stimulation and intravenous substance P administration on airway secretion in anesthetized, paralyzed and artificially ventilated piglets, 9 to 22 days of age, and older piglets all aged 10 weeks. Airway secretion was monitored by counting the hillocks appearing in the upper trachea in an exposed field of tracheal epithelium (1.2 cm2) coated with powdered tantalum. In younger animals, mechanical stimulation of the larynx had no discernible effect on tracheal submucosal gland secretion. Neither excitation of airway irritant receptors nor stimulation of pulmonary C-fiber receptors by capsaicin caused a significant increase of fluid secretion from tracheal submucosal glands. In addition, stimulation of peripheral chemoreceptors by ventilating animals with 12% O2 in N2, and 6% O2 in N2, failed to induce a substantial change in airway secretion, when compared with number of hillocks in the control period. Furthermore, administration of sodium cyanide had little or no effect on baseline secretion. In contrast, to the weak reflex responses in younger piglets electrical stimulation of the vagus nerve caused the number of hillocks to increase on average by 16.3 +/- 2.3 (P less than 0.01). In addition, local application of a pledget soaked in solution of methacholine caused the number of hillocks to increase by 32.1 +/- 5.2 (P less than 0.01). Intravenous administration of substance P also induced an augmentation in fluid secretion. Increase in concentration of substance P (10(-8), 10(-7), 10(-6), and 10(-5) M, 1 ml) was associated with a concomitant elevation in the number of activated submucosal glands (5.3 +/- 2.6, 10.0 +/- 4.4, 27.1 +/- 4.5, 41 +/- 5). In older piglets, stimulation of laryngeal mucosa, airway irritant receptors, as well as stimulation of pulmonary C-fiber receptors induced a significant increase in tracheal secretion, although stimulation of peripheral chemoreceptors had no effect on airway secretion. These data suggest that reflex responses of submucosal glands are weak during early postnatal development, however, tracheal submucosal glands do respond to exogenously administered cholinergic substances and tachykinin peptides. PMID- 1706885 TI - Role of vasoactive substances in the segmentary vasomotor response following spinal cord stimulation. An experimental study. AB - It is presumed today that spinal cord stimulation induces local delivery of vasoactive substances, such as prostacyclins, histamine, substance P, and vasoactive neuropeptides, in the perivascular environment and the vascular wall to mediate the segmental vasodilator response. To investigate this mechanism, 9 dogs were subjected to low thoracic spinal cord stimulation. Venous and arterial blood samples from the paraesthesic area in the lower limbs were obtained before and 120 min after stimulation to measure changes in the plasma concentration of vasoactive intestinal peptide, substance P, and histamine. The results were compared with those obtained from vessels of the upper limbs. Blood flow changes following stimulation were recorded by electromagnetic flowmeters. Local arterial vasoactive intestinal peptide showed a mean increase of 33% after 60 min of stimulation. Changes concerning substance P were inconclusive. Local arterial and venous histamine concentrations increased 26 and 29%, respectively, after 60 min of stimulation. PMID- 1706886 TI - Xenogeneic neural transplantation: role of vasculature and MHC antigen in immunological rejection. AB - The reconstruction of blood vessels in a graft is one of the important events inducing immunological rejection. First, we investigated the reconstruction of blood vessels in a graft by using a monoclonal antibody against mouse endothelial surface antigen 1. Secondly, the lymphocyte proliferative response to Ia antigen were examined in mixed lymphocyte cultures by using monoclonal antibodies against Ia antigen. The results show that the blood vessels originating in the host tissue inoculate with those originating in the donor graft tissue and that the expression of Ia antigen on the vascular endothelial cells plays an important role in the immunological rejection through sensitization of peripheral lymphocytes. PMID- 1706887 TI - Effects of interstitial edema on brain cell transplantation. AB - Fetal raphe cells were transplanted into the anterior part of the corpus callosum of serotonin denervated hydrocephalic rats using a cell suspension method. Hydrocephalus was induced by intracisternal injection of kaolin. The survival of the transplanted cells and fiber outgrowth were evaluated according to the level of serotonin and its metabolite, hydroxyindoleacetic acid, using high-performance liquid chromatography with electrochemical detection in the anterior and posterior parts of the corpus callosum 1-2, 5-6, and 7-8 weeks after transplantation. The results suggest favorable effects of interstitial edema associated with hydrocephalus on the survival of transplanted raphe cells and fiber outgrowth. PMID- 1706888 TI - Retrograde adriamycin sensory ganglionectomy: novel approach for the treatment of intractable pain. AB - Selective sensory ganglionectomy by means of retrograde suicide transport of adriamycin was performed on 3 patients with neuropathic pain in the areas of the trigeminal and intercostal nerves, producing significant pain relief, particularly from hyperalgesic pain. Adriamycin ganglionectomy is considered as a less invasive and highly selective pain treatment, which may possibly become an alternative for surgical ganglionectomy or rhizotomy. PMID- 1706889 TI - [Creativeness as a potential for survival in the abyss between fear of and longing for death]. AB - On the background of a creative psychotherapy with a young man covering the antagonism between Eros and Thanatos creativity is presented as a power not only mediating between destructive and constructive processes but integrating itself into the personal image and sense of life. Thanatophobic regression metamorphosizes into courage for life, the ability to face transitoriness and to discover the power of phenix in it which creates new creations. PMID- 1706890 TI - A rebound-like pattern of REM sleep induced by guinea pig myelin basic protein (MBP) in cats. AB - In order to investigate the effects of myelin basic protein (MBP) on sleep parameters, adult cats received 10 micrograms of guinea pig MBP into the third ventricle of the brain and were subsequently recorded for 8 hours. In comparison with both saline solution (0.1 ml) and 10 micrograms basic proteins (protamines and histones), MBP specifically produced a rebound-like pattern of the REM sleep (REMS); it decreased the REMS latency, increased the number of REMS episodes and dramatically increased the REMS percent during the 3rd and 6th hour of recording. In the light of the strong depolarisation effect and of the presence of specific binding sites for serotonin, the results are discussed as suggesting that MBP enhances the REMS triggering mechanisms. PMID- 1706891 TI - Dose- and time-dependent increase of lysosomal enzymes in embryonic cartilage in vitro after ionizing radiation. AB - Radiation doses of 20, 50 or 100 Gy caused the same time related decrease for RNA and proteoglycan (PG) synthesis in embryonic cartilage in vitro (4 days culture). In this paper, participation of lysosomes in this radiation response is investigated. Therefore, we employ a cytochemical method using beta glycerophosphate as substrate for acid phosphatase (AP) detection. Increase of AP was found 2 days after irradiation and increased during the whole culture period. The increase was more pronounced with a higher radiation dose. Stimulation of AP activity explains the observed radiation response of RNA and PG synthesis. PMID- 1706892 TI - Flow cytometric DNA analysis of hepatocellular carcinoma: preliminary report. AB - Flow cytometric DNA analysis was performed in 50 paraffin-embedded specimens of clinical hepatocellular carcinoma (HCC) after hepatic resections. The DNA distribution pattern was classified in two types, diploid and aneuploid, according to the degree of dispersion on the DNA histogram. The major DNA pattern of HCC in this report proved to be aneuploid (78%), although 22% of tumors revealed a diploid pattern. The serum alpha-fetoprotein level exceeded 40 ng/ml in 86.1% of the aneuploid tumors and in 13.9% of the diploid tumors (p less than 0.05). We found no correlation between DNA distribution and hepatitis B surface antigen positivity, the presence of liver cirrhosis or tumor size. Additionally we noted no significant correlation between the DNA pattern and survival rates in patients with HCC who underwent hepatic resection. PMID- 1706893 TI - Cytologic patterns in juice from human pancreatic transplants: correlation with histologic findings in the graft. AB - In 19 patients who had undergone pancreatic transplantation with temporary exteriorization of the pancreatic juice, graft tissue became available for histologic examination. In these patients the cytologic patterns in the pancreatic juice were compared with the histologic findings in the graft specimens. In five samples the diagnosis by cytologic studies was rejection. Acute rejection was confirmed in all the histologic specimens. In eight cytologic samples, graft pancreatitis was suspected because of the increased amounts of neutrophils, degenerating cells, epithelial cells, monocytes, and some macrophages, with or without necrotic tissue fragments. All eight histologic specimens showed findings characteristic of pancreatitis. In three cytologic samples, bacteria or fungi were observed. Histologic examination of these patients showed graft pancreatitis. In four patients the cytologic findings were normal. Graft histologic factors were normal in two instances. In one of these grafts there was graft pancreatitis, and in one graft chronic vascular rejection was seen. Our study shows that two different pathologic events occurring in the pancreatic graft (i.e., acute rejection and pancreatitis) are reflected by characteristic changes in pancreatic juice cytology. PMID- 1706894 TI - Suppressive effects of two bioresponse modifiers, krestin and levamisole, on 5 azacytidine-induced digital defects in rats. AB - The effects of two bioresponse modifiers, krestin (PSK) and levamisole hydrochloride (levamisole), on 5-azacytidine (5-AC)-induced syndactyly, brachydactyly, and ectrodactyly were investigated. Both PSK and levamisole suppressed 5-AC-induced digital malformation in the rat. The effect of PSK was significant when given 24 h before to 1 h after 5-AC treatment, and levamisole when given 12 h before to 3 h after treatment. Both agents showed an immune stimulating effect and have been used as cancer chemotherapeutic drugs. However, as the contribution of fetal or maternal immune functions or the alleviation action is unknown, further investigations are required to clarify the mechanism. PMID- 1706895 TI - Thrombocytopenia with or without thrombosis after pentosan polysulfate treatment. PMID- 1706896 TI - Inhibition of thrombin-induced tissue-type plasminogen activator release by agents which increase intracellular cyclic AMP. PMID- 1706897 TI - Antigenic relationship between the venom of the night adder Causus maculatus and venoms of other viperids. AB - Monovalent antivenoms were raised in mice against the venoms of Causus maculatus, Vipera ammodytes, Echis carinatus, Cerastes cerastes, Bitis arietans, Agkistrodon rhodostoma and Bothrops atrox. These antivenoms as well as four commercially available antivenoms were tested against the venoms of 15 viperid species by means of immunoelectrophoresis and/or ELISA. Cross-reactive protein bands were determined by immunoblot. ELISA cross-reactions of C. maculatus antivenom were low with all heterologous venoms. When investigating the other viperine antivenoms in ELISA stronger cross-reactions were observed with several heterologous venoms. In immunoblot, two heterologous antivenoms cross-reacted with one or two protein bands of C. maculatus venom whereas there were at least four heterologous antivenoms cross-reacting with each of the other venoms. The findings indicate that there is little antigenic affinity between C. maculatus venom and the other venoms investigated. Broad in vitro cross-reactions between viperine antivenoms and Causus venom which were reported in literature seem to be attributable to the use of antivenoms of commercial grade. Specificity of commercially produced, mono- or polyvalent antivenoms may not be strictly limited to those venoms, against which potency is claimed on the label of the product. PMID- 1706898 TI - [The polymorphism of alpha-amylase]. AB - Individual phenotypes, phenotypic and genetic frequencies of alpha-amylase enzyme were determined by means of population genetic study. The results of this study revealed absence of genetic linkage between alpha-amylase phenotypes, haptoglobins and serum factor G1m(1) of gammaglobulin system (Gm). PMID- 1706899 TI - Evaluation of video tape and a simulator for instruction of basic surgical skills. AB - Twenty first-year veterinary students with no prior participatory experience in surgery were randomly paired and assigned into two study groups. Ten students (group V) viewed a hemostatic technique video tape until they thought they could competently perform and assist in performing a hand-tied ligature on a blood vessel in a live animal. Ten students (group VS) wer also given a simulator for technique practice. Paired students were video recorded and blindly evaluated on their ability to perform and assist proper ligation of a bleeding vessel. Inexpensive hemostasis models were very helpful for teaching students essential surgeon and assistant skills involved in hand-tied ligature placement. Students who practiced with simulators performed significantly better as surgeon and assistant, and in total psychomotor skill evaluation, then students watching the video only. Students using simulators performed ligation with significantly more accuracy and tended to be more expeditious at this task. Further training is needed for students to acquire skills necessary for efficient bleeding vessel exposure and isolation. PMID- 1706900 TI - [The treatment of patients with acute coronary failure by ultraviolet irradiation of the blood]. PMID- 1706901 TI - [A comparative study of serum complement (C3 and C4) in inflammatory joint diseases]. AB - The third and the fourth fraction of the complement (C3 and C4), haptoglobin, fibrinogen, alpha 1-glycoprotein, alpha 2-macroglobulin and transferrin were examined in 692 patients with inflammatory joint disease--rheumatism, rheumatoid arthritis, ankylosing spondylarthritis, psoriatic arthritis, Reiter's syndrome, sacroiliitis [correction of sacroileitis], reactive arthritis, gout, osteoarthrosis and nosologically undefined arthritis in active or nonactive phase and in 60 healthy controls. The complement fractions studied show an increase of various degree and importance in almost all groups of patients in both phases studied. The relations between the complement fractions and the other acute phase indices show significant correlations between them and the other acute phase indices. C3 and to a certain degree C4 could be added to the acute phase reacting indices. Their place in the downgrade scale is as follows: fibrinogen, haptoglobin, alpha 1-glycoprotein, C3, C4, alpha 2-macroglobulin, transferrin. PMID- 1706902 TI - In vitro evaluation of bleomycin-induced cell lethality from plastic and glass containers. AB - To optimize cancer chemotherapy, a considerable amount of research has been expended to study pharmacologic, pharmacokinetic, biochemical, and pharmaceutic properties of antineoplastic agents. However, published data on the stability and compatibility of these agents in various administration fluids and containers are few in number. Evidence of a significant decrease in stability as shown by high performance liquid chromatography has been reported when bleomycin was infused in plastic containers for prolonged periods (over 24 hours) as compared with the same procedure with glass containers. Because administration of bleomycin is usually given as a continuous infusion, we undertook this study to determine whether the drug loss of stability that occurs in plastic containers results in a therapeutic loss of efficacy (cytotoxicity). By using a tumor stem-cell assay we compared the quantitative effects of bleomycin in plastic and glass containers on cell lethality. The results from our assay showed no significant difference in cell lethality by bleomycin from its aqueous solution stored in glass and plastic containers over the time periods observed. If these results had been statistically significant, the tumor stem-cell assay may have been shown to be a more sensitive means of determining the clinical significance of these stability studies. PMID- 1706903 TI - [Comparative study of parenteral and oral immunization against influenza in a large clinical trial. 2. Results of immunologic studies]. AB - In a multicentric trial 350 persons (19-24 years) were immunized with influenza vaccines containing the following virus antigens: A/Singapore/6/86, (H1N1); A/Mississippi/1/85, (H3N2); B/Ann Arbor/1/86. 174 received an i.m. injection of 0.5 ml "Influmun" vaccine from SSW Dresden/GDR. 176 persons were immunized twice within 60 days with enteric-wated capsules each containing approximately 60 micrograms hemagglutinin of all three virus strains. The volunteers were clinically observed in an interval of 6 months. The majority of orally immunized persons with low pre-immunization titers responded with fourfold or higher IgA antibody titer rises in nasal secretions, but no significant IgG antibody titer increase in sera could be observed. Secretory antibody titers remained elevated for 2 to 4 months. Parenterally immunized persons showed antibody titer rises in sera but not in nasal secretions. In both groups the highest antibody titer increases were observed after application of the A/Singapore/6/86 virus antigen. Volunteers with high pre-immunization titer did not show an antibody increase neither after parenteral nor oral immunization. PMID- 1706904 TI - The effect of drugs on lung hyperreactivity in Sephadex treated rats. AB - The injection of Sephadex G200 intravenously into rats induced a blood eosinophilia and an hyper-reactivity of lung parenchymal strips to 5 hydroxytryptamine (5HT) and carbachol. The blood eosinophilia and the hyper reactivity to 5HT reached a maximum at the same time and before that to carbachol. It was shown previously that the reduction of the blood eosinophilia by treatment with dexamethasone, dapsone, phenidone, isoprenaline and aminophylline also reduced the hyper-reactivity to 5HT. In this study we found that only dexamethasone reduced the hyper-reactivity to carbachol. PMID- 1706905 TI - Characterisation of the neurokinin receptors mediating contraction of isolated tracheal preparations from a variety of species. AB - The effects of neurokinin (NK) agonists on isolated tracheal preparations from rat (RT), pig (PT), rabbit (RbT) and guinea-pig (GPT) have been investigated. None of the NKs contracted RT, suggesting that this preparation lacks NK receptors mediating contraction, whereas NKs caused concentration- related contractions of PT, RbT and GPT. In PT, NK1-receptors mediate contraction since only substance P (SP) and the NK1-receptor selective agonists, SP methylester (SPOMe) and GR73632 were highly potent. In contrast, in RbT, only NKA and the selective NK2-receptor agonist, GR64349 were potent, indicating the presence of NK2-receptors. However, in GPT both NK1- and NK2-receptors appear to mediate contraction to NKs since NKA, GR73632 and GR64349 were highly potent and SP and SPOMe moderately potent agonists. This study demonstrates apparent species differences in the NK-receptor populations present in tracheal smooth muscle. PMID- 1706906 TI - Inhibition by calcitonin gene-related peptide of agonist-induced bronchoconstriction in various mammals including man. AB - The effect of calcitonin gene-related peptide (CGRP) on the smooth muscle of mammalian airways has been investigated in vitro. CGRP itself lacked contractile activity but reduced responses evoked by bronchoconstrictor agents via a post junctional action. The inhibitory effect of CGRP was concentration-related and greatest in distal airways. The results suggest that CGRP may contribute to regulating muscular tone in the tracheobronchial tree. PMID- 1706907 TI - Experimental short-bowel syndrome: effect of an elemental diet supplemented with short-chain triglycerides. AB - To determine whether short-chain triglycerides (SCTs, 1:1 triacetin:tributyrin, wt:wt) enhance intestinal adaptation in short-bowel syndrome (SBS), male Sprague Dawley rats underwent 60% distal small-bowel resection with cecectomy and received either a chemically defined diet (CD) or a CD containing 40% of nonprotein energy as either medium-chain triglycerides (MCTs) or SCTs. After 12 d the SCT group had significantly increased jejunal mucosal weight compared with the MCT and CD groups and had significantly increased segment weight and mucosal protein compared with the CD group. In the colon the SCT group had significantly increased segment and mucosal weights and mucosal protein and DNA compared with both the MCT and CD groups. Body-weight change and measurements of serum ketones, albumin, glucose, and triglycerides revealed no significant differences among groups. SCTs improved jejunal and colonic adaptive growth and maintained comparable nutritional status in SBS when compared with CD alone or CD with MCTs. PMID- 1706908 TI - Heffalumps, jagulars, and cheshire cats. A commentary on cytokeratins and soft tissue sarcomas. AB - Historically, antibodies to cytokeratin intermediate filaments have been models of target specificity. In most diagnostic settings, the utility of these antibodies was unquestioned; reactivity for cytokeratin was dogmatically equated with epithelial differentiation. Recently, however, the diagnostic importance of these antibodies has been challenged, prompted by the demonstration of cytokeratin reactivity in a variety of "nonepithelial" neoplasms. In this review, the evolving literature on this topic is explored, and the practical implications of these findings are discussed. PMID- 1706909 TI - Pulmonary function in workers exposed to diesel exhausts: the effect of control measures. AB - To assess the protective effect of exhausts pipe filters or respirators on pulmonary function, 15 workers in a tunnel construction site, truck and loading machine drivers, rock workers, and others were studied. The total and respirable dust, combustible matter in respirable dust, carbon monoxide, nitrogen monoxide and nitrogen dioxide were measured for each subject during entire work shifts. The effect of the exposure on the lung function variables was measured by dynamic spirometry, carbon monoxide single breath technique, and nitrogen single breath wash-out. The exhaust pipe filtering had a protective effect, directly discernible in the drivers on vital capacity and FEV1.0 and for the whole group on FEV% and TLco. The dust respirators had no effect, probably because of the difficulties in correctly using personal protection under the circumstances in the tunnel. In the absence of a true exposure assessment, control measures for diesel exhausts can be tested by medical effect studies. Catalytic particle filters of diesel exhausts are one method of rendering the emissions less irritant, although they will not remove irritant gases. An indicator of diesel exhaust exposure should include the particle fraction of the diesel exhausts, but a discrimination between different sources of organic dust must be possible. PMID- 1706910 TI - Steroid-induced reduction of histamine release does not alter the clinical nasal response to cold, dry air. AB - In some persons, cold, dry air (CDA) provokes symptoms of rhinitis that are associated with increased levels of histamine and other inflammatory mediators in nasal lavages. Because the patterns of mediators released during the early reaction to antigen and CDA-induced rhinitis are similar, we believe that mast cell activation is part of the reaction to CDA. In view of our previous finding that 1-wk pretreatment with topical steroids reduced symptoms and mediator release in the early nasal response to antigen of allergic subjects, we examined the effect of beclomethasone dipropionate on the response to CDA. Using a double blind, crossover design, 84 micrograms of beclomethasone or placebo were administered in each nostril twice a day to 13 volunteers for 7 days prior to CDA challenge. The reaction to CDA was monitored by measuring the levels of histamine, N-alpha-p-tosyl-L-arginine methyl ester (TAME)-esterase activity and albumin in nasal lavages before and after provocation. Overall symptom scores, as well as scores for rhinorrhea and congestion, were also obtained. Cold, dry air challenge resulted in elevation over baseline of all parameters after placebo pretreatment. After beclomethasone, a significant reduction in histamine levels, but not in TAME-esterase activity or albumin levels or in number of symptoms, was observed. These results indicate that 1-wk pretreatment with beclomethasone affects mast cells, reducing histamine release after CDA, as it did in antigen induced rhinitis. They also indicate that histamine may not be essential for the development of the immediate nasal reaction to CDA. PMID- 1706911 TI - Protected bronchoalveolar lavage. A new bronchoscopic technique to retrieve uncontaminated distal airway secretions. AB - We tested the effectiveness of protected bronchoalveolar lavage (PBAL), performed through a protected transbronchoscopic balloon-tipped (PBT) catheter, in collecting distal airway secretions with a minimal degree of contamination. The PBAL had less than or equal to 1% squamous epithelial cells in 91% of specimens and an absence of bacterial growth in 59% of patients without pneumonia. Using a threshold of 10(4) cfu/ml we had one false positive result in 33 patient without pneumonia and one false negative in 13 patients with pneumonia. Quantitative bacterial cultures of the PBAL specimens had a diagnostic sensitivity of 97% and a specificity of 92%, with a positive predictive value of 97% and a negative predictive value of 92%. The diagnostic efficiency was 96%. The presence of intracellular organisms in much greater than or equal to 2% of the recovered alveolar cells (Giemsa stain) was seen in all but two patients with pneumonia (on corticosteroids) and in none of the patients without pneumonia. Gram stains of the PBAL specimens were positive in all but one patient with pneumonia and negative in all but one patient without infection (patient with endobronchial narrowing secondary to neoplasm with false positive cultures). Either the Giemsa or the Gram stain was positive in all patients with pneumonia, allowing early and accurate diagnosis of lower respiratory tract infection before the results of cultures were available. The time off antibiotic therapy before bronchoscopy did not affect the result of PBAL cultures, contrary to what we observed for the protected brush specimen. PMID- 1706912 TI - Depressed DNA synthesis in vitro by early prepubertal undescended human testis. AB - To study the spermatogenic potential of the early prepubertal inguinal testis and the effect of temperature on spermatogenesis, the levels of incorporation of 3H thymidine and 14C-uridine into the testicular tissue were studied at 31 degrees C and 37 degrees C in vitro and compared with those of scrotal (descended) testis. 3H-thymidine incorporation into the inguinal testicular tissue at 31 degrees C, which is close to normal testicular temperature, was significantly lower than that of the scrotal testis. There was no significant differences in 14C-uridine incorporation between inguinal and scrotal testis at that temperature. At 37 degrees C, which is close to normal body temperature, 3H-thymidine and 14C uridine incorporation into the tissues of inguinal and scrotal testes was significantly higher than at 31 degrees C. It would appear that DNA synthesis of the inguinal testis is lower than that of the scrotal testis at 31 degrees C, and higher temperature, at least for a short while, did not contribute to the impairment of spermatogenic potential in early prepubertal inguinal testis. PMID- 1706913 TI - In vitro temperature sensitivity of DNA, RNA, and protein syntheses throughout puberty in human testis. AB - To evaluate the optimal temperature for DNA, RNA, and protein syntheses in the prepubertal, pubertal, and postpubertal human testis, the levels of incorporation of 3H-thymidine, 14C-uridine, and 14C-leucine into cultured testicular tissue were studied at 28 degrees C, 31 degrees C, 34 degrees C, 37 degrees C, 40 degrees C, and 43 degrees C. The maximum level of 3H-thymidine incorporation in prepubertal testes occurred at 37 degrees C, whereas the maximum incorporation in the pubertal and postpubertal testes occurred at 31 degrees C. Incorporations of 14C-uridine and 14C-leucine in the three groups were temperature dependent (28 degrees C to 37 degrees C). DNA synthesis by germ cells in the pubertal and postpubertal testes was maximum at 31 degrees C and was impaired at body temperature (37 degrees C), whereas in the prepubertal testis it was temperature dependent with a maximum at 37 degrees C. RNA and protein synthesis in the three groups was temperature dependent at 28 degrees C to 37 degrees C, was depressed at 40 degrees C, and remarkably depressed at 43 degrees C. PMID- 1706914 TI - Recurrent or metastatic disease in select patients with adrenocortical carcinoma. Aggressive resection vs chemotherapy. AB - In a retrospective, nonrandomized comparison of patients with first recurrence of adrenocortical cancer, 18 patients were treated with chemotherapy (primarily mitotane) and 15 patients were treated with surgical resection plus similar chemotherapy. Surgical resection of recurrent adrenocortical cancer was often extensive, with morbidity in 20% of patients and no mortality. Mitotane therapy was ineffective at controlling tumor growth. Median survival from the time of diagnosis for all patients was only 23 months and no patient was cured. Disease free interval greater than 12 months was associated with prolonged survival, but it only occurred in six patients (18%), with a similar frequency in both treatment groups. Surgical resection of recurrent disease was associated with prolonged survival from the time of first recurrence. The potential benefit of this resection was evident in the 5 patients (33%) who were able to live greater than 5 years from the time of first recurrence with improvement in symptoms and signs of hypercortisolism. Although no patient with recurrent adrenal cancer could be cured, resection of recurrent disease was associated with a slight prolongation of survival and good palliation of Cushing's syndrome. PMID- 1706915 TI - Differential effects of sodium butyrate and hexamethylene bisacetamide on growth and secretion of cultured human endocrine tumor cells. AB - Advanced gastrointestinal endocrine tumors respond poorly to conventional chemotherapy. In this study we examined the effects of two agents that promote cellular differentiation, sodium butyrate and hexamethylene bisacetamide, on the in vitro growth and secretory responses of a human pancreatic carcinoid (BON) and human gastrinoma (PT-2 and PT-SM) cell lines that have been established in our laboratory. We found that both sodium butyrate and hexamethylene bisacetamide strongly inhibited growth of BON, PT-2, and PT-SM cells. With continuous exposure of BON cells to sodium butyrate (2 mmol/L), the doubling time was prolonged, from 60 hours in controls to 156 hours, and saturation density was reduced to 28% that of controls. Hexamethylene bisacetamide (4 mmol/L) reduced saturation density to 37% that of controls in BON cells and prolonged the doubling time, from 60 hours to 103 hours. Antiproliferative effects of similar magnitudes were observed in the gastrinoma cell lines. In contrast, differential effects were produced on amine biosynthesis in BON cells; sodium butyrate stimulated levels of 5 hydroxytryptamine in the cells, whereas hexamethylene bisacetamide caused a profound dose-dependent inhibition of amine biosynthesis. The significant antiproliferative activity of sodium butyrate and hexamethylene bisacetamide and the inhibitory effects of hexamethylene bisacetamide on amine biosynthesis warrant evaluation of these agents or analogues for treatment of metastatic carcinoid and gastrinoma. PMID- 1706916 TI - Lindane induced changes in morphology and lipids profile of testes in rats. AB - Intraperitoneal treatment of adult male rats with lindane at dosages of 4 and 8 mg/kg over a period of 45 days caused retardation in body and testicular growth. Testicular degeneration was conspicuous over a period of 45 days in both dosages of lindane. After treatment with lindane, histochemical and biochemical studies revealed the accumulation of testicular lipid components, i.e. total lipids, triglycerides and cholesterol along with fatty degeneration in testicular tissues. Moreover, the loss of male accessory organ weight indicated androgen deficiency in treated rats. PMID- 1706917 TI - Preparation of dextran-bound recombinant hirudin and its pharmacokinetic behaviour. AB - Recombinant desulphatohirudin was bound via lysine residues to oxidized dextrans. The hirudin-dextran conjugates inhibit thrombin like free hirudin. The Ki-values are in the same range. In rabbits and rats the pharmacokinetic behaviour following i.v. administration of these conjugates was examined in comparison to that of hirudin. The hirudin-dextran conjugates were more slowly eliminated than hirudin, the distribution volumes in steady-state were significantly reduced, and, in comparison to hirudin, 5 to 15 times larger areas under the plasma concentration-time-curves were obtained. PMID- 1706919 TI - Role of microtubules on Ca2+ release from the endoplasmic reticulum and associated histamine release from rat peritoneal mast cells. AB - In order to study the role of cytoskeletons on histamine release from mast cells, the effects of cytoskeleton-inhibiting agents were investigated. Since neither colchicine, vinblastine nor cytochalasin D was effective in inhibiting the IP3 formation, it is possible that neither microtubules nor microfilaments of rat peritoneal mast cells participate in the initial membrane events of the histamine release. However, both colchicine and vinblastine, but not cytochalasin D, were effective in inhibiting Ca2+ release from the intracellular Ca store. It was accordingly suggested that the microtubules, rather than microfilaments, are intimately related to the Ca2+ releasing process from the endoplasmic reticulum. The fluorescence intensity of the mast cells stained with FITC-labeled anti tubulin antibody reflects the amount of tubulin polymers inside the cell, and colchicine treatment decreased the fluorescence intensity, indicating that colchicine is effective in depolymerizing the microtubules of rat mast cells. By contrast, the amount of tubulin polymer in the mast cells increased by compound 48/80, indicating that the rearrangement of microtubules took place in the mast cells, leading to histamine release. When permeabilized mast cells were exposed to potassium antimonate solution, microtubules attached themselves to the endoplasmic reticulum and many Ca antimonate dots were observed. From the present results, it was concluded that microtubules play an important role in the processes leading to Ca2+ release from the intracellular Ca store and subsequent histamine release. PMID- 1706918 TI - Production and some properties of monoclonal antibodies against human pancreatic secretory trypsin inhibitor. AB - Hybridomas that secrete monoclonal antibodies against human pancreatic secretory trypsin inhibitor (PSTI) were established by fusion of spleen cells obtained from mice immunized with PSTI with mouse NS-I-Ag 4/1 myeloma cells. One of three resulting monoclonal antibodies (KN-1) was found to recognize the N-terminal moiety of the inhibitor, while the others (KN-2 and KN-3) reacted with other as yet undefined parts of the molecule. Trypsin inhibitory activity of PSTI treated with KN-1 monoclonal antibody was the same as that of PSTI itself, thus indicating no relationship between the N-terminal moiety of the PSTI molecule and its inhibitory activity. We further examined the applicability of one of the monoclonal antibodies (KN-1) for immunohistochemical study of human pancreatic cancer tissue including the normal as a model, and found granular staining of the cytoplasm of the normal acinar and duct cells and also of that of adenocarcinoma cells in formalin-fixed, paraffin-embedded tissue sections. PMID- 1706920 TI - Enhancement by beraprost sodium, a stable analogue of prostacyclin, in thrombomodulin expression on membrane surface of cultured vascular endothelial cells via increase in cyclic AMP level. AB - Prostacyclin and beraprost sodium (beraprost), a stable analogue of prostacyclin, increased cyclic AMP (cAMP) levels of cultured human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. The elevation of cAMP by beraprost was sustained longer than that by prostacyclin. The expression of thrombomodulin (TM) on membrane surface of HUVEC was enhanced by beraprost and prostacyclin, and the persistence of the increase in TM expression by beraprost was greater than prostacyclin. Dibutyryl cAMP (db-cAMP) mimicked the effects of beraprost and 3-isobutyl-1-methylxanthine enhanced the effects. Beraprost, prostacyclin and db-cAMP also effectively blocked the interleukin-1- and tumor necrosis factor-induced depression of TM expression substantially. These results suggest that TM expression is positively regulated by cAMP in HUVEC, and that beraprost may be potentially effective for reducing thrombotic events through the mechanism which initiates the stimulation of cAMP/TM system in vascular endothelial cells. PMID- 1706921 TI - Effect of valproic acid, its unsaturated metabolites and some structurally related fatty acids on the binding of warfarin and dansylsarcosine to human albumin. AB - The sites to which valproic acid and its main unsaturated metabolites (2-en-2 propyl pentanoic acid and 4-en-2-propyl pentanoic acid) bind to on human albumin were investigated by (1) measuring their ability to displace the fluorescent probes warfarin and dansylsarcosine and (2) by assessing the extent to which they inhibited the hydrolysis of 4-nitrophenyl acetate. Valproate and its metabolites displaced both warfarin and dansylsarcosine, and they also inhibited the hydrolysis of 4-nitrophenyl acetate. The order of potency for inhibition of both binding and hydrolysis was: 2-en-2-propyl pentanoic acid greater than 4-en-2 propyl pentanoic acid greater than or equal to valproate. It is concluded that valproic acid and its unsaturated metabolites can displace ligands from the warfarin binding site (site I) and the benzodiazepine/indole binding site (site II), but the primary interaction is with site II. Furthermore, the introduction of a double bond into the carbon backbone of valproate increases affinity for albumin at both sites. PMID- 1706922 TI - Inhibition of bleomycin-induced cellular DNA strand scission by 1,10 phenanthroline. AB - Inhibition by 1,10-phenanthroline of cellular DNA strand scission induced by the antitumor antibiotic bleomycin in Ehrlich ascites tumor cells was studied. DNA alkaline elution was performed on cells after 1-hr bleomycin treatments. Pretreatment for 24 hr with initial 1,10-phenanthroline concentrations of 0.2 nmol/10(5) cells, which depletes cells of ferritin iron by 80%, had no consistent effect on bleomycin strand breakage. However, simultaneous treatment with 3.1 nmol of 1,10-phenanthroline/10(5) cells and with bleomycin concentrations from 5 to 25 microM decreased both apparent double-stranded breaks and random breakage. When cells were treated with both 3.1 nmol of 1,10-phenanthroline/10(5) cells and 25 microM bleomycin, washed free of both drugs, and incubated at 35 degrees for 1 hr, the resulting breakage was equivalent to that found in cells treated with bleomycin only. When the combination treatment was extended to 4 hr, cell washing and reincubation resulted in increased strand scission, as compared with strand scission in cells treated with bleomycin only. Growth inhibition by bleomycin was not affected appreciably by temporary suppression of DNA strand breakage activity. PMID- 1706923 TI - [A peptide, containing the universal antigenic determinant of tryptophanyl-tRNA synthetase]. AB - Among clostripain hydrolysate peptides of beef pancreas tryptophanyl-tRNA synthetase the peptide Ile-Ser-Phe-Pro-Ala-Ile-Asn-Gln-Phe-Ala-Ala-Pro-Ser-Gln Phe-Ser-Ile-Arg was revealed which contains the continuous antigenic determinant for monoclonal antibody Am1. This antibody specifically cross-reacts with tryptophanyl-tRNA synthetases of procaryotes, eucaryotes and archebacteriae. The synthetic peptide with identical amino acid sequence plus N-terminal Arg residue (S-peptide), being immobilized on enzyme immunoassay (EIA) microtitration plate, also binds with Am1. Am1 affinity constant (M-1) measured by non-competitive EIA was (3.0 +/- 0.3).10(7) for S peptide and (1.4 +/- 0.3).10(9) for the native enzyme. The sequence of immunoreactive peptide adopts with high probability the secondary structure including beta-turn(s) and antiparallel beta-sheet composed of inverted repeats. At the same time, the analysis of circular dichroism spectrum (in the far UV) of the peptide dissolved in water comes closest to 16% beta-turn and only 8% beta-sheet. The binding of Am1 with peptide was not observed in aqueous solution. PMID- 1706924 TI - [Exposition of epitopes of transmembrane protein gp41 of the human immunodeficiency virus on surface capsids of the hepatitis B virus core antigen]. PMID- 1706925 TI - [Organochlorine compounds in human milk]. AB - The impact of life style characteristics and chemical exposure conditions of about 80 nursing women on the contents of Organochlorine compounds (OCC) in their breast milk is analyzed and described by a robust statistical procedure. Information on independent variables such as age, diet, smoking, occupation and chemical exposures is obtained by a questionnaire. Concentrations of HCB alpha-, beta-, gamma-HCH, Heptachlorepoxide, Dieldrin, DDE and PCB in milk samples are measured and serve as dependent variables. By logistic regression we quantify the influence of combinations of several independent variables on the probability of being extremely contaminated with at least one OCC. We also study the pattern in which OCC are associated with each other. There are distinct pairwise correlations of the four main contaminants: HCB, beta-HCH, DDE and PCB. These correlations entail that a woman with a high PCB concentration, for example, is more likely of being also contaminated above average with the remaining contaminants. We define a simple load score and compare its different theoretical distributions under the assumption of presence or absence of the correlations. By this we can show that the proportion of heavily contaminated women is underestimated, if the OCC associations are ignored. We therefore recommend that the pattern of OCC-correlations in human milk is further studied in more representative samples. These investigations should also comprise occupationally exposed women. PMID- 1706926 TI - Psychological profile and behavioural characteristics in 12 patients with Prader Willi syndrome. AB - In the present study medical, psychological and behavioural aspects in 12 patients with Prader-Willi syndrome (PWS), aged between 13 months and 28 years are presented. In half of the patients the diagnosis of PWS was made before the age of one year. The contribution of cytogenetic investigations with the detection of a 15q11 deletion in half of the PWS patients is discussed. In the evaluation of the intellectual performances IQ levels were spread between 45 and 95 with a mean IQ of 54. A specific behavioral profile in all patients with characteristic fluctuations with age was observed. Rapid changes in behaviour increased with age and after puberty, mostly related with withholding of food. The present study reinforces the importance of early diagnosis in the PWS with adequate and intelligible information towards the parents. It may prevent the dramatic obesity which leads to severe physical problems and psychological burdens in PWS adolescents and adults. PMID- 1706927 TI - New therapeutic modalities for cancer. AB - A review is given on new biological approaches to cancer therapy based on knowledge concerning interferons, interleukins. LAK-cells, tumour-infiltrating lymphocytes, tumour necrosis factor, colony-stimulating factors, monoclonal antibodies and oncogenes. There are many potential permutations for the application of biological therapy for cancer. One of the most important developments has been the increased understanding of the molecular mechanisms of malignancy through which biological manipulation can be tailored to an individual tumour. Although current clinical studies are not demonstrating high response rates they may well be analogous to the advances seen with chemotherapy in its early days. As yet only relatively small numbers of patients have had access to, or been suitable for, treatment. With further refinements in production, administration, and an increase in the specificity of treatment, the possibility of curing metastatic solid tumours may become a reality. PMID- 1706928 TI - Benefits of developmental screening. PMID- 1706929 TI - Liposomes and biotherapeutics. AB - Application of liposomes as delivery system for biotherapeutic peptides and proteins may offer important therapeutic advantages over existing delivery methods. Several approaches towards achieving improved delivery of biotherapeutics with liposomes are outlined. Although the literature on this topic is sporadic and frequently incomplete, enough of a research foundation exists to justify the conclusion that liposomes can play an important role in the formulation and delivery of biotherapeutics. However, it will be necessary to understand more fully the mechanisms of action before optimum liposomal dosage forms can be designed. PMID- 1706930 TI - Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants. AB - Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5 phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-trisphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources. PMID- 1706931 TI - AIDS surveillance in the Americas. PMID- 1706932 TI - Eradicating indigenous transmission of wild poliovirus in the Americas. An update. PMID- 1706933 TI - Semantic relations of the word in aphasics. AB - The study of the semantic relations of the word in aphasics was performed by an associative experiment applied in two samples, one of 20 aphasic patients and the other of 40 normal subjects. Following application of this associative experiment in normal subjects, a model design was achieved based on the hierarchy of the categories within the semantic field. In this model, the associations were grouped by meaning into semantic categories, while the categories were hierarchized according to the number of words included and to their frequency of association. Comparison of the single model design performed by each aphasic with the normal model design showed a deterioration of the semantic categories, hierarchy, but on a background of a marked reduction of the number of categories within the semantic field of the stimulus. The aphasics lost 2/3 of the associations found in normals but the functionality of the 1/3 left was decreased. This may be the basic reason for the unsuccessful word-finding process in aphasics. PMID- 1706934 TI - A bioassay for the evaluation of antiproliferative potencies of progesterone antagonists. AB - A bioassay which allows quantification of the antiproliferative potency of progesterone antagonists on the mammary gland was developed. For this purpose, ovariectomized rats were substituted with oestrone and progesterone and a further group simultaneously treated with the progesterone antagonists Mifepristone (= RU 38.468), Onapristone (= ZK 98.299), or ZK 112.993 (Schering AG, Berlin). A morphometric analysis of the tubulo-alveolar buds in the inguinal mammary glands revealed a dramatic antiproliferative effect of the progesterone antagonists after as little as 3 days of treatment. Several less specific mammary gland growth parameters (weight, DNA- and RNA-content) proved to be less sensitive. This bioassay measures the potency of progesterone antagonists to competitively antagonize the specific effects of progesterone on the target organ mammary gland. Further advantages of this bioassay are the use of a hormonally standardized biological system, the quantitative results, the small amount of test compound necessary, as well as the substitution with progesterone and oestrone since the antiproliferative potency of progesterone antagonists on experimental hormone dependent mammary carcinomas is most potently displayed in ovariectomized animals substituted with both sex hormones. PMID- 1706935 TI - Comparison between the cardiac effects induced by muzolimine and furosemide in guinea-pig atria. AB - Muzolimine (10-500 microM) induced a concentration-dependent reduction of both the contractile force and frequency in spontaneously beating atria and in electrically driven left atrium from reserpine-treated guinea pigs. This negative inotropic response was unaffected by the addition of atropine to the perfusion fluid, and it was highly sensitive to changes in external Ca2+ concentration. Both in spontaneously beating and in electrically driven atrium, muzolimine (50 400 microM) antagonized, in an apparently competitive manner, the increase in contractile force induced by cumulative addition of CaCl2 (0.68-9.59 mM) to the bathing fluid. Muzolimine (50-100 microM) reduced the inotropic response to low (5-30 nM), but not high (50-100 nM) concentrations of Bay K 8644, a calcium channel agonist. The inotropic effects of 8-phenyltheophylline and of ouabain were antagonized by muzolimine (10-100 microM) in a noncompetitive manner, while the response to noradrenaline was not altered. Similar to muzolimine, verapamil at a concentration suitable to block calcium channels inhibited, in a noncompetitive way, the inotropic effect induced by 8-phenyltheophylline and by ouabain without altering the contractile response to noradrenaline. Furosemide (10 and 100 microM) did not influence the contractile force or the frequency of spontaneously beating atria, nor the inotropic effect induced by CaCl2, 8 phenyltheophylline, ouabain, or noradrenaline. These results indicate that the influence of muzolimine on guinea-pig atria originates from an inhibition of Ca2+ influx into cardiac cells and that furosemide does not mimic the effect of muzolimine at this level. PMID- 1706936 TI - The galactose-binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands prepared by chemical modification of glycopeptides containing triantennary N-linked oligosaccharides. AB - A glycopeptide containing a triantennary N-linked oligosaccharide from fetuin was modified by a series of chemical and enzymic reactions to afford a reagent that contained a terminal residue of 6-(N-methylamino)-6-deoxy-D-galactose on one branch of the triantennary structure and terminal galactose residues on the other two branches. Binding assays and gel filtration experiments showed that this modified glycopeptide could bind to the sugar-binding sites of ricin. The ligand was activated at the 6-(N-methylamino)-6-deoxy-D-galactose residue by reaction with cyanuric chloride. The resulting dichlorotriazine derivative of the ligand reacts with ricin, forming a stable covalent linkage. The reaction was confined to the B-chain and was inhibited by lactose. Bovine serum albumin and ovalbumin were not modified by the activated ligand under similar conditions, and we conclude, therefore, that the reaction of the ligand with ricin B-chain was dependent upon specific binding to sugar-binding sites. Ricin that had its galactose-binding sites blocked by the covalent reaction with the activated ligand was purified by affinity chromatography. The major species in this fraction was found to contain 2 covalently linked ligands per ricin B-chain, while a minor species contained 3 ligands per B-chain. The cytotoxicity of blocked ricin was at least 1000-fold less than that of native ricin for cultured cells in vitro, even though the activity of the A-chain in a cell-free system was equal to that from native ricin. Modified ricin that contained only 1 covalently linked ligand was also purified. This fraction retained an ability to bind to galactose affinity columns, although with a lower affinity than ricin, and was only 5- to 20-fold less cytotoxic than native ricin. PMID- 1706937 TI - Structure of an unusually stable RNA hairpin. AB - The structure of a very common RNA hairpin, 5'GGAC(UUCG)GUCC, has been determined in solution by NMR spectroscopy. The loop sequence, UUCG, occurs exceptionally often in ribosomal and other RNAs, and may serve as a nucleation site for RNA folding and as a protein recognition site. Reverse transcriptase cannot read through this loop, although it normally transcribes RNA secondary structure motifs. A hairpin with that loop displays unusually high thermodynamic stability; its stability decreases when conserved nucleotides are mutated. The three dimensional structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR using distance geometry, followed by restrained energy minimization. The structure was well-defined despite the conservative use of interproton distances, by constraining the backbone conformation by means of scalar coupling measurements. A mismatch G.U base pair, with syn-guanosine, closes the stem. This hairpin has a loop of only two nucleotides; both adopt C2'-endo sugar pucker. A sharp turn in the phosphodiester backbone is stabilized by a specific cytosine-phosphate contact, probably a hydrogen bond, and by stacking of the cytosine nucleotide on the G.U base pair. The structural features of the loop can explain the unusual thermodynamic stability of this hairpin and its sensitivity to mutations of loop nucleotides. PMID- 1706939 TI - Vitamin K-dependent carboxylation of poly-L-glutamate. AB - Poly-L-glutamate preparations of varying chain length were used as substrates for bovine liver vitamin K-dependent carboxylase. The quality of these substrates (as measured by their apparent kinetic constants) was comparable to that of the more commonly used tri- and pentapeptides, but the maximal reaction rate increased at increasing chain length. PMID- 1706938 TI - On the structure of platelet-derived growth factor AA: C-terminal processing, epitopes, and characterization of cysteine residues. AB - The complete amino acid sequence analysis of the "short" form of rPDGF-AA expressed in baby hamster kidney cells revealed the absence of posttranslationally modified amino acid. Approximately 50% of the proteins were shortened by two to three amino acid residues at the C-terminus. Trypsin treatment of BHK rPDGF-AA lead to the identification of two internal epitopes that correspond to the two previously described domains in rPDGF-BB [Vogel, S., & Hoppe, J. (1989) Biochemistry 28, 2961-2966]. Cysteine residues at positions 37, 46, 47, and 93, respectively, were converted by site-directed mutagenesis into serine residues, and the monomeric proteins were prepared through expression in Escherichia coli. None of the mutant proteins was able to dimerize, but all of them exhibited to various extents a reversible conformational change which may reflect an intermediate prefolded monomer. An intramolecular disulfide bridge between Cys-10 and Cys-91/93 was identified in these monomers. From a mixture of the mutant proteins 37 and 46, an active dimer was reconstituted, suggesting an intermolecular cysteine bridge between these two residues. PMID- 1706941 TI - Induction of nitrite production in mouse spleen cells by immunization. AB - Changes of nitrite production in mouse spleen cells of in vitro secondary antibody response were investigated. Mouse spleen cells immunized with gamma globulin fraction of rat serum produced nitrite 3 days after in vitro challenging with the same antigen. Nitrite production of rabbit IgG-challenged spleen cells was found to be about 2.9-times higher than that of spleen cells primed with the gamma globulin fraction of rat serum. Nitrite production in this system was completely suppressed by T cell depletion (99.7% inhibition). Furthermore, nitrite production in these cells significantly decreased by addition of anti interferon gamma antibody (62.9% inhibition). These data indicate that nitrite production in antigen-immunized spleen cells is affected with the immunogenicity of an antigen and regulated by T cells, especially interferon (IFN) gamma. PMID- 1706940 TI - Evidence for protein kinase C--independent pathways mediating phorbol ester induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells. AB - The effects of phorbol esters on many cell types are known to be mediated through activation of the protein kinase C (PKC) signal transduction pathway. By using the specific inhibitor of this enzyme 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine dihydrochloride (H7) we have assessed the role of PKC activation in phorbol ester (phorbol 12-myristate 13-acetate, PMA)-induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells (B-CLL) as a model of terminal differentiation of human B lymphocytes. H7 affected a dose-dependent inhibition of PMA-induced thymidine and uridine uptake with ID50 values of 41 microM and 32 microM, respectively. A comparable ID50 value (34 microM) was obtained for H7 inhibition of B-CLL PKC activity in a cell-free system. PMA induced changes in cell morphology, expression of CD20, CD37 and FMC7 surface antigens together with increased secretion of immunoglobulin were variably abrogated by H7 suggesting that PKC activation is more important in B cell activation/DNA synthesis than in the differentiative response. Consistent with this, expression of a sizable proportion of PMA-inducible genes was not significantly affected by H7. These data are consistent with the existence of a PMA-activated, PKC-independent signal transduction pathway which may be important, though by itself apparently insufficient, for eliciting full terminal differentiation in B lymphocytes. PMID- 1706943 TI - Stable RNA secondary structure in a retroviral vector insert terminates reverse transcriptase elongation in vitro but not in cultured cells. AB - We wished to test whether an RNA signal that causes termination of elongation by reverse transcriptase in vitro would affect retroviral vector function. A synthetic oligonucleotide containing a sequence capable of forming a very stable RNA secondary structure was subcloned into the retrovirus vector N2. The integration of this sequence into N2 causes termination of elongation by reverse transcriptase in vitro at the precise positions previously reported in a different sequence context. However, no premature termination of DNA synthesis was observed in the unintegrated DNA of vector transduced cells. Likewise, there was no deleterious effect of the sequence insert on vector titer. These results indicate that termination signals defined in in vitro systems cannot be used as predictors of in vivo function and suggest that viral proteins in addition to reverse transcriptase play an important role in transcript initiation and elongation. PMID- 1706944 TI - Modified Blalock-Taussig shunts in the treatment of tetralogy of Fallot. PMID- 1706942 TI - Amplification and expression of mdr genes and flanking sequences in verapamil hypersensitive hamster cell lines. AB - The properties of several multidrug resistant (MDR) Chinese hamster ovary (CHO) cell lines which are verapamil hypersensitive have been investigated, extending our earlier study of two such cell lines. It was observed that increasing levels of multidrug resistance are associated with increasing verapamil and nicardipine sensitivity, although the cell lines are not hypersensitive to cyclosporin A. Although there is appreciable amplification of the P-glycoprotein gene at higher levels of multidrug resistance/verapamil hypersensitivity, there is only very low or no amplification of five flanking genes, including the sorcin gene. Low levels of resistance (3-10 fold) appear to involve increased P-glycoprotein gene expression at the level of transcription. P-glycoprotein levels of the cell lines have been measured by flow cytometry using the monoclonal antibody C219, and there is a general correlation between P-glycoprotein overexpression, increased levels of the corresponding mRNA and the level of verapamil hypersensitivity. PMID- 1706945 TI - Inhibited morphological terminal differentiation and enhanced proliferation of cultured mouse epidermal cells at different concentrations of dimethyl sulphoxide. AB - Dimethyl sulphoxide (DMSO), at concentrations of 1-2%, induces terminal differentiation in several different cell types in vitro and enhances the growth of newborn mouse epidermal cells in primary culture under conditions that also permit terminal differentiation. We have found that DMSO concentrations approaching 4% reversibly inhibited (with little overt toxicity) terminal differentiation of normal epidermal cells from newborn SENCAR mice. Cells cultured in medium containing 4% DMSO and calcium in excess of 1 mM did not stratify extensively or slough large amounts of keratinized debris into the medium as occurred in control cultures, nor did they form large numbers of squamous cells or keratin bundles, as revealed by light and electron microscopy. The number of detergent-insoluble cornified envelopes was similarly reduced. Long term growth of epidermal colonies in secondary culture was optimum in 1% DMSO, this concentration also permitting normal terminal differentiation of these cells. Since DMSO had these effects on epidermal cells in vitro, it may also affect epidermal cell proliferation and terminal differentiation in vivo, an important consideration should DMSO ever be approved for topical use in the US. PMID- 1706946 TI - The G1 interval in the mammalian cell cycle: dual control by mass accumulation and stage-specific activities. AB - The temporal determinants of the G1 cell cycle interval were investigated using nine mammalian cell lines. In each case, cells were allowed to proliferate for many cell cycles under conditions that slowed progress through S phase without an equivalent impairment of overall mass accumulation. This disproportionate inhibition of progress through the cell cycle caused newly produced cells to be more massive than usual. Under these growth conditions, the determinants of the length of the G1 interval became evident. For two cell lines, HeLa S3 and NIH 3T3, a protracted S phase, and the resultant increase in mass, resulted in a dramatically shortened G1 interval. Thus, for these cell lines, a major portion of G1 time exists to accommodate mass accumulation needed to initiate the subsequent S phase. Nevertheless, under conditions that protracted S phase and shortened the G1 interval, cells still exhibited a measurable G1 time, reflecting the stage-specific activities within G1. One activity that may be responsible for this obligatory G1 time is the synthesis of a labile protein. For other cells studied here, protraction of S phase also caused proliferating cells to become more massive, but in these cases there was no diminution of the G1 time. For these cells, the entire G1 interval must accommodate G1-specific activities necessary to initiate a new cell cycle. A unifying view of the G1 interval recognizes the two distinct influences that determine the time spent in G1: the need to accumulate sufficient mass to initiate a new DNA-division sequence; and the stage-specific events necessary for the subsequent S phase. The length of the G1 interval is dictated by the longer of these two time-consuming activities. PMID- 1706947 TI - Different combinations of regulatory elements may explain why placenta-specific expression of the glycoprotein hormone alpha-subunit gene occurs only in primates and horses. AB - Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses. PMID- 1706948 TI - Characterization of insulin-like growth factor-binding proteins of porcine ovarian follicular fluid. AB - Using competitive ligand-binding studies, ligand blotting, and immunoprecipitation, we have characterized the insulin-like growth factor (IGF) binding proteins (BPs) of porcine follicular fluid. Competitive ligand-binding studies revealed a preference of ovarian IGFBPs for IGF-II over IGF-I. Follicular fluid from small, 1-3-mm follicles had nearly twice the binding capacity for IGFs as that from large, 6-10-mm follicles. Ligand blots of porcine follicular fluid resolved 5 major bands of IGF-binding activity having apparent molecular sizes of 44, 40, 34, 29, and 22 kDa. The 40-44-kDa bands were immunoprecipitated by an antibody to porcine IGFBP-3, the acid-stable subunit of the 150-kDa growth hormone-dependent IGF-binding protein complex of porcine serum. The 34-kDa band was immunoprecipitated by an antibody to rat IGFBP-2, the major IGF-binding protein found in fetal rat serum. To date we have been unable to immunoprecipitate the 29- and 22-kDa bands with any of the antibodies tested, including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGF-binding protein. The 40-44-kDa species (IGFBP-3) was the predominant form and was equally abundant in fluid from large and small follicles. In contrast, the smaller forms, including IGFBP-2 and the 29- and 22-kDa forms were significantly more prominent in fluid from small follicles. In view of other studies indicating a significant effect of IGFBPs on ovarian cell function, follicular IGFBPs may play an important role in the IGF autocrine/paracrine regulatory system of the ovary. PMID- 1706949 TI - General continuum theory for multiion channel. I. Theory. AB - It is assumed that the channel is completely characterized by three factors: (a) its geometric shape, (b) the potential energy interaction between an ion and the channel wall, and (c) the potential energy interaction between two ions at arbitrary positions in the channel. The total potential energy of an ion in a multiion channel can be described by a summation over factors b and c. The ion water interaction is described by a continuum diffusion coefficient which is determined by the channel geometry (c). Given this physical description, a theory is described that predicts the flux of all the ion species that are present, with no additional assumptions about, e.g., the maximum number of ions allowed in the channel, location of binding sites or shape of energy barriers. The solution is based on a combination of the Nernst-Planck and Poisson equation. The Poisson potential is corrected for the ion's self potential. A hard sphere ion-ion interaction is included that prevents ions from piling up on top of each other in regions where the channel wall has a high charge density. An exact analytical solution is derived for the region in the bulk solution, far from the channel mouth and this solution is used as a boundary condition for the numerical solution. The numerical solution is obtained by an interactive procedure that is surprisingly efficient. Application of the theory to the acetylcholine receptor channel is described in the companion paper (Levitt, D. G. 1990. Biophys. J. 59:278-288). PMID- 1706950 TI - General continuum theory for multiion channel. II. Application to acetylcholine channel. AB - The general theory (Levitt, D. G. 1990. Biophys. J. 59:271-277) is applied to a model channel that resembles the acetylcholine receptor channel (ACH). The model incorporates the known features of the ACH geometry and fixed charge locations. The channel has a wide mouth facing the outer solution, tapering to a narrow region facing the interior of the cell. Rings of fixed negative charge are placed at the two surfaces where the bilayer begins, corresponding to the known charges at the ends of the M2 segment. It is assumed that the forces acting on the ion are electrostatic: ion-channel wall, ion-ion, Born image and applied voltage. Analytical expressions for these forces are derived that take account of the low dielectric lipid region. In addition, there is a local hard sphere repulsive force that prevents ions from piling up on each other in regions of the channel with a high fixed charge density. A classical continuum theory is used to obtain an expression for the diffusion coefficient in the channel. The model can mimic the major qualitative and, in many cases, quantitative experimental features of the ACH channel: current-voltage relation, conductance versus concentration and interaction between monovalent and divalent ions. The model calculations were also compared with the site directed mutagenesis experiments of Imoto, K., C. Busch, B. Sakmann, M. Mishina, T. Konno, J. Nakai, H. Bujo, Y. Mori, K. Fukuda, and S. Numa. (1988. Nature (Lond.). 335:645-648) in which the charge at the ends of the channel was systematically varied. PMID- 1706952 TI - Monte Carlo simulation of the water in a channel with charges. AB - A Monte Carlo simulation of water in a channel with charges suggests the existence of water in immobile, high density, essentially glasslike form near the charges. The channel model has a conical section with an opening through which water molecules can pass, at the narrow end of the cone, and a cylindrical section at the other end. When the charges are placed near the narrow section of the model, the "glass" effectively blocks the channel; with the charges removed, the channel opens. The effect can be determined from the rate of passage of the water molecules through the pore, from the average orientation of the water molecule, and from distortion of the distribution of molecules. In the simulations carried out to date, no external ions have been considered. In addition to the energy, the Helmholtz free energy has been calculated. PMID- 1706951 TI - Nonstationary fluctuation analysis and direct resolution of single channel currents at postsynaptic sites. AB - In order to measure unitary properties of receptor channels at the postsynaptic site, the noise within the decay phases of inhibitory postsynaptic currents (IPSCs) and of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents (EPSCs) in rat hippocampal neurons was studied by nonstationary fluctuation analysis. Least squares scaling of the mean current was used to circumvent the wide variation in amplitude of postsynaptic currents. The variance of fluctuations around the expected current was analyzed to calculate single channel conductance, and fluctuation kinetics were studied with power spectra. The single channel conductance underlying the IPSC was measured as 14 pS, whereas that underlying the EPSC was 42 pS. Openings of the EPSC channel could also be resolved directly in low-noise whole-cell recordings, allowing verification of the accuracy of the fluctuation analysis. The results are the first measurements of the properties of single postsynaptic channels activated during synaptic currents, and suggest that the technique can be widely applicable in investigations of synaptic mechanism and plasticity. PMID- 1706953 TI - Interleukin-5 is a human basophilopoietin: induction of histamine content and basophilic differentiation of HL-60 cells and of peripheral blood basophil eosinophil progenitors. AB - Cytokine-induced differentiation of basophils may contribute to various inflammatory processes. We examined the effects of recombinant human interleukin 5 (IL-5) and other human cytokines in vitro on myeloid colony formation in methylcellulose and on alkaline passaged HL-60 basophilic cell differentiation. Myeloid colonies (CFU-C) at day 14, formed in the presence of either IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or G-CSF included peripheral blood-derived progenitors of the eosinophil/basophil lineage. IL-5 stimulated a greater proportion of basophil-containing, histamine-positive, eosinophil-type colonies compared with GM-CSF, IL-3, or G-CSF. IL-5 also stimulated dose-dependent increases in histamine content of alkaline-passaged, butyrate cotreated HL-60 cells. The concentration of IL-5 required for half maximal induction of HL-60 histamine content was similar within twofold to that needed for half-maximal stimulation of the multifactor dependent TF-1 erythroleukemic cell line. Neutralizing rat monoclonal antibodies to human IL-5 were developed and used to demonstrate that each of these IL-5 bioactivities could be specifically blocked. We conclude that in addition to its previously described eosinophil differentiation activity, IL-5 may be considered a basophilopoietin. PMID- 1706954 TI - Accessibility of epitopes on fibrin clots and fibrinogen gels. AB - Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241-476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII induced fibrinogen gel networks. A different MoAb (4-2/xl-f, anti gamma 392-406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15-21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1-reactive epitopes. An antibody that does not recognize fibrin (antibody 1-8C6, antiB beta 1-21) predictably did not bind to fibrin clots and bound to 35% of the 1-8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent. PMID- 1706955 TI - Growth factor requirements of childhood acute T-lymphoblastic leukemia: correlation between presence of chromosomal abnormalities and ability to grow permanently in vitro. AB - Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL 103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL-103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, -105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny. PMID- 1706956 TI - Serum beta-2-microglobulin (beta 2m) in myeloma: toward a simple prognostic stratification using beta 2M and acute-phase proteins? PMID- 1706957 TI - CD5+, CD11c+, CD20+ hairy cell leukemia. PMID- 1706958 TI - Bleomycin-iron complexes and DNA radiation damage. AB - Radiation in the form of high-energy electrons dose-dependently activates bleomycin-Fe3+ in oxygen-containing DNA solutions. This activation causes a DNA fragmentation and a release of oxidative degradation products from the DNA. During irradiation, bleomycin-chelated Fe3+ is reduced to Fe2+. The kinetics of DNA base propenal-formation (measured by reaction with thiobarbituric acid) and iron-reduction (measured by bathophenanthroline chelation) are similar, with a yield of 1 mol base propenal/6 mol Fe3+ reduced. The activation of bleomycin-Fe3+ by irradiation could be instrumental in the synergistic action of radiotherapy and bleomycin observed on simultaneous administration in vivo. PMID- 1706959 TI - Recurrent homogeneously staining regions in 8p1 in breast cancer and lack of amplification of POLB, LHRH, and PLAT genes. AB - In a cytogenetic study of 125 primary and untreated breast cancers, 107 were selected for the quality of their metaphases permitting detection of amplifications:homogeneously staining regions (HSRs), abnormally banded region (ABRs), and double minutes (dmins). HSRs and ABRs were detected in 62 cases (58%), but no cases of dmins were observed. The localizations of HSRs and ABRs were not random because they were observed in the 8p1 position in 14 cases. The possible amplifications of five sequences, MOS (8q1), LHRH (8p21.1), POLB (8p11.2), PLAT (8p12), and D8Z2 (8c) were investigated in three tumors with HSR on the short arm of chromosome 8. Because these sequences were not amplified, two interpretations can be proposed: 1) there is a frequent amplification of a sequence from the 8p1 region, located between the investigated sequences; and 2) the amplifications do not occur in 8p1, but HSRs or ABRs of undetermined origin have a strong tendency to be translocated onto 8p. Because cases with HSR(8p) have less complex karyotypes than with HSRs in other locations, the first interpretation is the most likely: HSRs may be formed in 8p and further translocated on other chromosomes in the course of tumor progression. PMID- 1706960 TI - Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity. AB - An analogue of human platelet factor 4 (PF4) lacking affinity for heparin was specifically designed to evaluate the importance of this property in the antitumor effects of recombinant PF4. The purified protein, recombinant PF4-241 (rPF4-241), failed to bind heparin but retained the ability to suppress the growth of tumors in mice. Daily intralesional injections of rPF4-241 significantly inhibited the growth of the B-16 melanoma in syngeneic mice without direct inhibitory effects on B-16 cell growth in vitro. Similar antitumor effects were observed with the human colon carcinoma, HCT-116, grown in nude mice, indicating that the inhibitory activity was neither tumor-type specific nor T cell dependent. rPF4-241 inhibited endothelial cell proliferation in vitro with dose dependence similar to the native sequence rPF4. Both rPF4 and rPF4-241 inhibited angiogenesis in the chicken chorioallantoic membrane. The analogue, however, was inhibitory at lower concentrations than rPF4 in the chorioallantoic membrane system and its inhibitory effects were not abrogated by the presence of heparin. The present findings support the conclusion that both rPF4 and rPF4-241 inhibit tumor growth by suppression of tumor-induced neovascularization. The finding that this activity is independent of heparin binding may allow the development of PF4-based angiostatic agents with reduced toxicity and improved bioavailability. These results also suggest that PF4 may play a more specific role in modulation of blood vessel development than previously recognized. PMID- 1706961 TI - In vitro and in vivo antitumor effects of murine monoclonal antibody NCC-ST-421 reacting with dimeric Le(a) (Le(a)/Le(a)) epitope. AB - A murine monoclonal antibody (MAb), NCC-ST-421 (IgG3), was raised by using a human gastric cancer xenograft St-4 as immunogen. Immunization was achieved by transferring immunocompetent normal BALB/c mouse spleen cells into BALB/c-nu/nu mice bearing St-4 tumors. Hybridomas were produced from spleen cells of the mice after rejection of the tumors and were screened for preferential reactivity with cancers on formalin-fixed paraffin sections, as described previously for establishment of MAb NCC-ST-439 (M. Watanabe et al., Jpn. J. Cancer Res., 76: 43 52, 1985). The immunobiological and immunochemical properties of the new MAb NCC ST-421 are described here. The MAb is essentially directed to a structure with dimeric Le(a) (V4III4Fuc2Lc6Cer) epitope (Gal beta 1----3[Fuc alpha 1----4]GlcNAc beta 1----3Gal beta 1----3[Fuc alpha 1----4]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 Cer). It cross-reacts with Le(a) but does not show any effect on Le(a)-positive RBC in vitro or on Le(a)-positive tissue loci in vivo. ST-421 strongly induced antibody-dependent cellular cytotoxicity using human peripheral blood leukocytes as effector cells with a variety of human tumor cells, using the short-term 51Cr release assay. It also showed striking complement-dependent cytotoxicity with a human complement source and was able to produce lysis of a variety of human cancer cell lines, supporting its observed ability to cause cytotoxic suppression of tumor growth in nude mice. In another series of experiments, i.p. injection of ST-421 completely inhibited growth of human tumor xenografts in nu/nu mice, and this inhibitory activity was closely dependent on expression of the dimeric Le(a) antigen on the cell surface. While Le(a) antigen was expressed in the kidneys of nu/nu mice, infusion of ST-421 in these mice did not cause histological change in kidney tissue. This finding suggests that the MAb does not damage normal cells or tissues which contain cross-reacting Le(a) antigen. These results demonstrate that ST-421 exerts a significant antitumor effect in vitro as well as in vivo, does not affect Le(a) antigen expressed on normal tissues, and therefore has potential application in therapy of certain types of human cancer which express the dimeric Le(a) antigen. PMID- 1706962 TI - A comparison of the HL-60 cell line and human granulocytes to effect the bioactivation of arylamines and related xenobiotics. The binding of 2 aminofluorene to nucleic acids as the result of the respiratory burst. AB - Studies were made on the ability of the leukemic cell line, HL-60, to substitute for normal human granulocytes in research concerned with the bioactivation of arylamines. The arylamine carcinogen, 2-aminofluorene (2-AF), was used as the model substrate in the form of 2-[9-14C]AF, and was incubated with HL-60 cell cultures, both in the presence and absence of phorbol myristate acetate (PMA) which induces the respiratory burst. The HL-60 cultures were generally employed after having been induced to undergo differentiation to neutrophils by the action of dimethyl sulfoxide (DMSO). Comparisons of the amounts of DNA and RNA binding by 2-AF between HL-60 and normal human granulocyte cultures demonstrated close similarities in the amount and nature of nucleic acid binding by this arylamine substrate. HL-60 cells that had been induced to differentiate to neutrophils to the extent of about 80% showed high levels of the respiratory burst along with extensive covalent binding of 2-[9-14C]AF to cellular nucleic acids. Although normal human granulocytes tended to metabolize 2-AF slightly faster than did highly differentiated HL-60 cells, the extent of nucleic acid binding relative to the amount of 2-AF metabolized was similar. A major difference in the metabolic fate of 2-AF in these cell cultures was the unique ability of HL-60 cultures at all stages of differentiation to effect the slow N-acetylation of 2-AF to give 2 acetylaminofluorene (2-AAF). Extensive analyses of incubation extracts showed that the major differences in apparent metabolites were quantitative. With few exceptions, both activated HL-60 and granulocyte cell cultures produced the same metabolites, most of which remain unidentified. Studies with inhibitors such as catalase, superoxide dismutase and azide ion further suggest that these two related cell cultures metabolize 2-AF in similar manner. The DMSO-differentiated HL-60 culture is proposed as a convenient model with which to investigate the metabolism and bioactivation of arylamines by human granulocytes or pure neutrophils. PMID- 1706963 TI - Ionic currents activated during hyperpolarization of single right atrial myocytes from cat heart. AB - Whole-cell recording techniques were used on single right atrial myocytes to study the ionic currents that may be responsible for the diverse diastolic voltage characteristics of atrial tissue. Ionic currents were activated by hyperpolarizing voltage pulses negative to -30 mV. In general, four different types of cells were identified based primarily on the ionic currents elicited during hyperpolarization. The first cell type exhibited an inward current that decayed with time at more negative voltages, reversed near the potassium equilibrium potential, inwardly rectified at more positive voltages, increased in elevated extracellular potassium, and was blocked by 3 mM barium or 10 mM cesium. This current was identified as the potassium current iK1. A second cell type exhibited a time-dependent inward current that increased at more negative voltages, had an activation range between -50 and -110 mV, had a reversal potential of -26 mV, and was blocked by 3 mM cesium. This current was identified as an if current. A third cell type exhibited an inward current that initially decayed and then became more inward with time. Barium (3 mM) abolished the initial inward current and revealed a time-dependent increasing inward current that was blocked by 3 mM cesium. This current was composed of both the iK1 and if currents. A fourth cell type exhibited only small time-independent leak currents in response to hyperpolarization. These results indicate that individual cells within the right atrium are electrophysiologically heterogeneous with respect to the types of ionic channels present in their sarcolemmal membranes. This specialization in ionic currents partially explains the diverse diastolic voltage characteristics and functional properties of atrial tissue. PMID- 1706964 TI - Heterotopic cardiac transplantation decreases the capacity for rat myocardial protein synthesis. AB - Heterotopic cardiac isografts are vascularly perfused hearts that maintain structural and functional integrity for prolonged periods of time. When placed in an infrarenal location, the heart is hemodynamically unloaded and undergoes negative growth, leading to cardiac atrophy. At 7 and 14 days after transplantation, the transplanted heart was decreased in size compared with the in situ heart (p less than 0.001). To assess the possible mechanism(s) to account for this reduction in size we studied in vivo rates of total left ventricular (LV) protein synthesis, total LV RNA content, and 18S ribosomal RNA content by nucleic acid hybridization. The LV protein synthetic rate was 4.7 and 5.3 mg/day in the in situ heart and was significantly decreased to 2.9 and 2.7 mg/day in the transplanted hearts at 7 and 14 days, respectively. LV RNA content of the transplant declined to 53% and 48% of the in situ value at 7 and 14 days, respectively. Hybridization studies revealed that LV 18S ribosomal subunit content was reduced proportionately to total RNA in the heterotopic hearts. As a result of these changes, there was no significant difference in the efficiency of total LV protein synthesis between the in situ and transplanted hearts. The present studies demonstrate that the hemodynamic unloading and cardiac atrophy that is characteristic of heterotopic cardiac transplantation is accompanied by a decrease in LV total RNA content and 18S RNA, resulting in a decreased capacity for myocardial protein synthesis. PMID- 1706965 TI - Comparison of dextran and hematocrit effects in the pulmonary microcirculation. AB - We have compared the effects of an increase in perfusate viscosity induced by dextran and by hematocrit (Hct) on segmental vascular resistance in the pulmonary circulation and the effect of flow on microvascular resistance in lungs perfused with dextran and red blood cells. We isolated and perfused lungs of 39 neonatal rabbits weighing 670 +/- 250 g. To determine the effect of dextran, group 1 (n = 8) lungs were perfused with both 5% and 20% dextran 70 solutions (Hct, approximately 25%); to determine the effect of Hct, group 2 (n = 5) lungs were perfused alternately with 5% and 40% Hct solutions (5% dextran 70). In group 1 and group 2 lungs, experiments were done at constant flow and pressure. Group 3 and group 4 (each n = 4) lungs were perfused with 5% and 20% dextran 70, respectively (0 Hct); group 5 (n = 5), with 10% dextran 70; group 6 (n = 7), with 5% dextran 500; and group 7 (n = 6), with 20% dextran 70 (Hct, approximately 5%). All lungs were perfused in zone 3; airway and left atrial pressures were 6 and 8 cm H2O, respectively. To partition the circulation, we measured pressures in the pulmonary artery and left atrium continuously and pressures in 20-50-microns arterioles and 20-50-microns venules by micropuncture. We found that an increase in both the concentration and molecular weight of dextran increased perfusate viscosity and the resistance in all three longitudinal vascular segments: arteries, microvessels, and veins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706966 TI - Effect of positive inotropic agents on myosin isozyme population and mechanical activity of cultured rat heart myocytes. AB - To examine whether catecholamines have a direct effect on myosin heavy chain expression of heart myocytes or whether they act via an altered work load, myocytes from neonatal rat hearts were cultured in thyroid hormone-free media containing various positive inotropic and chronotropic agents. The velocity and frequency of contraction of the myocytes were monitored using an optoelectronic system. After 3-5 days of culture, myosin isozyme populations, cellular cAMP content, and 2-deoxy-D-glucose uptake of the myocytes were determined. Compared with myocytes cultured in the absence of inotropic agents (32.6 +/- 3.5% V1), the proportion of myosin V1 was significantly (p less than 0.05) increased in the case of 1 microM isoproterenol (48.2 +/- 5.9% V1), 1 microM forskolin (57.1 +/- 11.7% V1), and 1 mM dibutyryl cAMP (79.1 +/- 2.0% V1). Dibutyryl cAMP increased V1 to a similar level as 30 nM triiodothyronine did (70.2 +/- 13.0% V1). Only a small increase was observed in myocytes cultured in the presence of 10 microM phenylephrine (40.4 +/- 8.4% V1), 10 microM ouabain (40.6 +/- 11.9% V1), or 10 microM Bay K 8644 (40.7 +/- 11.7% V1). The agents with a marked effect on myosin heavy chain expression resulted in a higher cAMP content; isoproterenol and forskolin also stimulated 2-deoxy-D-glucose uptake. All agents resulted in a higher velocity of contraction; with the exception of ouabain, frequency of contraction was also increased. A change in Ca2+ concentration in the medium from 1.3 to 2.4 mM resulted in a small increase in V1 (40.7 +/- 5.2% V1) but had the same effect on contraction velocity as dibutyryl cAMP did. Furthermore, 10 nM isoproterenol also increased V1 in myocytes that were arrested with 10 microM verapamil. The increase in V1 in the case of dibutyryl cAMP, isoproterenol, and forskolin is thus most probably not a correlate of the increased mechanical activity but of the high cellular cAMP content. PMID- 1706967 TI - Biobehavioral factors in Cardiac Arrhythmia Pilot Study (CAPS). Review and examination. AB - The behavioral studies component of the multicenter Cardiac Arrhythmia Pilot Study (CAPS) was designed to examine the relation of biobehavioral factors and frequency of ventricular premature complexes (VPCs), efficacy of antiarrhythmic therapy, and disease end points in a study population that had experienced recent myocardial infarction and significant ventricular ectopy. Biobehavioral factors included both psychosocial (depression, anxiety, social support, type A behavior, mood, defensiveness, and anger expressiveness) and psychophysiological (heart rate and blood pressure reactivity to a videogame stressor) variables. Data were collected at baseline and at 3-, 6-, 9-, and 12-month follow-ups. Of the 502 patients enrolled in CAPS, 353 participated in the behavioral studies component. At baseline, assessments of psychosocial variables revealed the CAPS study population to be generally similar to other heart disease populations, and no relation between these variables and psychophysiological reactivity or arrhythmias was found. At follow-up among patients assigned to the placebo condition, biobehavioral variables were not related to levels of VPCs or VPC suppression. Cox regression analyses revealed that type B behavior, depression, and reduced heart rate reactivity were associated with increased clinical events, even after controlling for baseline left ventricular ejection fraction, myocardial infarction before the qualifying event, use of beta-blockers, use of digitalis, Q wave of qualifying myocardial infarction, and presence of unsustained ventricular tachycardia on baseline electrocardiogram. It is hypothesized that the relation among reduced heart rate reactivity, depression, and clinical events is mediated by diminished cardiac vagal tone. PMID- 1706968 TI - Treatment of chronic viral hepatitis. PMID- 1706969 TI - CD5+ B cells in autoimmune disease and lymphoid malignancy. AB - Evidence is accumulating that CD5+ B cells belong to a developmental lineage distinct from that of conventional B cells and mainly participate in natural immunity. They have attracted attention because of their involvement in autoimmunity and lymphoid malignancy in both mice and humans. Patients with rheumatoid arthritis and Sjogren's syndrome were found to show a striking increase in the number of CD5+ B cells. B cell-chronic lymphocytic leukemia cells frequently express CD5. However, there are arguments against the role for CD5+ B cells in autoimmune disease, particularly in murine and human systemic lupus erythematosus (SLE). Whereas most IgM anti-DNA antibodies are produced by CD5+ B cells, high-affinity, pathogenic IgG antibodies are produced mainly by CD5- B cells. Either of two possibilities can explain the failure of CD5 expression of B cells responsible for producing IgG anti-DNA antibodies: either the cells are conventional B cells or the cells are CD5+ B cells that lack CD5 expression. In studies using SLE-prone NZB x NZW F1 mice and their H-2-congenic progeny, we discussed herein the possibility that CD5+ B lineage cells are also responsible for the pathogenic IgG autoantibody production by phenotypic switching from CD5+ to CD5-, probably under a particular genetic background. A line of H-2-congenic NZB x NZW F1 progeny failed to produce IgG anti-DNA antibodies, but in turn, showed a marked clonal proliferation of CD5+ B cells. Thus, it appears that genetically determined signals for either proliferation or differentiation would lead CD5+ B cells to cause distinct disease, i.e., autoimmune disease or lymphoid malignancy. Further studies using H-2-congenic New Zealand mice may provide insights into the correlation between autoimmunity and related lymphoid malignancy. PMID- 1706970 TI - Monoclonal antibodies to human neutrophil Fc gamma RIII (CD16) identify polypeptide epitopes. AB - Human neutrophils constitutively express two low-affinity Fc gamma R, Fc gamma RII (CD32) and Fc gamma RIII (CD16). Eleven monoclonal antibodies (mAb) to CD16 were used to identify antigenic differences among Fc gamma RIII-bearing cells, to define functional epitopes of Fc gamma RIII on neutrophils, and to characterize biochemically the epitopes identified by some of these mAb. Flow cytometry demonstrated that 9 of the 11 mAb reacted with neutrophils, 10 of the 11 reacted with natural killer cells, and 9 of 11 reacted with monocytes and monocyte derived macrophages. These mAb reacted with CD16 positive cells with varying fluorescence intensities. The ability of anti-CD16 mAb to block the binding of 125I-labeled immune complexes to neutrophils was examined. Four monoclonal antibodies strongly inhibited (87-96%) the binding to neutrophils of 125I-labeled immune complexes. Competitive binding assays were performed to determine whether any other anti-CD16 mAb identify the epitope identified by mAb 3G8. Two other mAb, CLBFCGRAN 1 and CLBGRAN 11, blocked binding of 125I-3G8 IgG to neutrophils. Six of the anti-CD16 mAb efficiently immunoprecipitated polypeptides of broad mobility ranging from 45 to 84 kDa from 125I-labeled neutrophils. When Fc gamma RIII, a complex sialoglycoprotein consisting of almost 50% oligosaccharides, was immunoprecipitated from neutrophils with 3G8 Fab Sepharose and subsequently digested with N-glycanase, 5 of the 6 mAb were capable of immunoprecipitating a deglycosylated polypeptide migrating at 29 kDa. These results demonstrate that these 5 mAb identify polypeptide epitopes of Fc gamma RIII, whereas 1 mAb, YFC120.5, may react with a glycosyl moiety or a determinant whose conformation is dependent on the presence of oligosaccharides. PMID- 1706972 TI - Immunoreactive neuropeptides in nerves in ligamentous tissue. An experimental neuroimmunohistochemical study. AB - A search for neuropeptide nerves in the healing of the experimentally ruptured medial collateral ligament (MCL) of the rabbit knee used specific antisera to the neuropeptides Substance P, calcitonin gene-related peptide (CGRP), and galanin. Sutured and unsutured MCLs were studied four and 14 weeks postoperatively. Both fluorescent thin nerve strands and small dotlike nerve terminals were regularly seen in the healing zone and in the adjacent normal ligamentous tissue, suggesting innervation of such structures by neuropeptide nerves. All three neuropeptides were more abundant in sutured ligaments than in unsutured ligaments, which may suggest beneficial effects of the apposition of the torn ligament ends on local nerve regeneration. Active involvement of the neural elements in the healing process was also suggested by kinetic studies showing a decrease in Substance P and CGRP staining as well as an increase in galanin staining during the study period. These changes in the periphery parallel the reactive changes earlier described in the dorsal root ganglion and dorsal horn cells occurring after a peripheral nerve injury. This may depend on the antidromic transport to the periphery of neuropeptides synthesized in the central nervous system. This experimental neuroimmunohistochemical mapping study and the known effects of neuropeptides on blood vessels, macrophages, and fibroblasts should stimulate further work on the role of innervation in ligamentous healing. PMID- 1706971 TI - Specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. AB - To determine the specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA, sera from BALB/c mice immunized with single stranded DNA from Escherichia coli (EC) were tested for binding to a panel of synthetic DNA and RNA homopolymers as well as duplexes. Results of these studies indicate that sera from EC DNA immunized mice preferentially bind certain DNA and RNA homopolymers as well as DNA duplexes. Furthermore, the specificity of the antibodies from immunized mice resembled those of sera from autoimmune MRL lpr/lpr mice in terms of the synthetic antigens recognized, although some differences were noted in the magnitude of the response to individual duplexes. These results suggest that anti-DNA antibodies induced by bacterial DNA bind to DNA structures dependent on both the base and the sugar phosphate moieties of the nucleic acid antigen and may resemble some anti-DNA antibodies expressed in spontaneous autoimmune disease in these binding properties. PMID- 1706974 TI - Perioperative considerations for patients treated with bleomycin. PMID- 1706973 TI - Traumatic aphasia in children and adults: a comparison of clinical features and evolution. AB - We compared the performance on test of language, apraxia, acalculia and intelligence of 32 children--age 5 to 16 years--and 31 adults, seen at our Aphasia Unit for the sequelae of a brain trauma. Logorrhea was never present among children who had nonfluent aphasia more frequently than adults. Apraxia and acalculia were equally present in the two groups. Twelve children and 15 adults of the first study were retested at variable distance from trauma. No group difference was found in the recovery of auditory comprehension (Token Test) or oral expression (oral confrontation naming and telling-of-an-event). On a battery of verbal and spatial memory tests 6 of the 12 children had pathological scores in one or more tests. Scholastic achievements were compared with those of the nearest kin. We conclude that while aphasia profiles are different in children and adults, the incidence of apraxia and acalculia and the recovery rate do not discriminate the two groups. PMID- 1706975 TI - Dosage compensation of a retina-specific gene in Drosophila miranda. AB - The X1R chromosome of Drosophila miranda and the 3L autosome of Drosophila melanogaster are thought to have originated from the ancestral D chromosomal element and therefore may contain the same set of genes. It is expected that these genes will be dosage compensated in D. miranda because of their X linkage. To test these possibilities and to study evolution of the dosage compensation mechanism, we used the 3L-linked autosomal head-specific gene 507 ml of D. melanogaster to isolate the homologous gene (507mr) from a D. miranda genomic library. In situ hybridization showed that gene 507 is located at the 12A region of the X1R chromosome of D. miranda, indicating that the chromosomal homology deduced by cytogenetic means is correct. Restriction analysis and cross-specific DNA and RNA blot hybridization revealed the presence of extensive restriction pattern polymorphism and lack of sequence similarity in some areas of the 507mr and 507 ml DNA, including the 3' portion of the transcribed region. However, the 5' portion of the transcribed region and the DNA sequences, located approximately 0.8 kb upstream and 3 kb downstream from the 507 ml gene showed a high degree of similarity with the DNA sequences of comparable regions of the 507mr gene. In both species gene 507 codes for a highly abundant 1.8 kb RNA which is expressed in the retina of the compound eye. Although in D. miranda the males have one and the females have two copies of the 507 gene, the steady-state levels of the 507 mRNA in both sexes were found to be similar, indicating that gene 507 is dosage compensated in D. miranda.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1706976 TI - Changes in haemostasis and proteins of the acute phase following cardiac surgery. AB - After cardiac surgery performed with extracorporeal circulation (ECC) involving heart valve prostheses (PHVS, n 12) and after a bypass of the coronary arteries by a venous graft (CABG, n 19), the authors investigated the dynamics of changes of haemostasis on the 1st, 3rd, 6th, 10th and 21st day after operation. As anti thrombotic treatment after PHVS anticoagulants were used, after CABG thrombocyte inhibitors. On the 1st day after operation in both groups thrombocytes decline, while after the 10th day their numbers increase. From the 6th day there is in both groups a rise of fibrinogen and other proteins of the acute phase (alpha-1 antitrypsin, orosomucoid and ceruloplasmin, while there was a drop of transferrin. On the 3rd and 6th day after operation fibrinolysis activators decline (euglobulin fibrinolysis). These findings suggest an increased risk of thrombophilia during the postoperative period and are probably associated with the release of interleukin-1 after Ecc and the stress of cardiac surgery. In patients with CABG on the 1st day a major drop of thrombocytes occurs, on the 6th day an elevated fibrinogen value was recorded and on the 10th day a reduced fibrinolytic activity, as compared with patients with PHVS. These changes will be, however, associated rather with a greater development of general atherosclerosis in patients with CABG, which leads to a further alteration of haemostasis, rather than with the applied antithrombotic treatment. PMID- 1706977 TI - [Gn-RH analogs and benign prostatic hypertrophy]. PMID- 1706978 TI - Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos. AB - Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish. PMID- 1706979 TI - Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig. AB - 125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst. PMID- 1706980 TI - Vimentin downregulation is an inherent feature of murine erythropoiesis and occurs independently of lineage. AB - In mammalian erythropoiesis, the mature cells of the primitive lineage remain nucleated while those of the definitive lineage are anuclear. One of the molecular and structural changes that precedes enucleation in cells of the definitive lineage is the cessation in the expression of the gene for the intermediate filament (IF) protein vimentin and the removal of all vimentin filaments from the cytoplasm. We show here that in immature primitive cells vimentin is synthesized and forms a cytoplasmic network of IFs. As differentiation proceeds in vivo, vimentin gene expression is downregulated in these cells; this is accompanied by the loss of vimentin filaments from the cytoplasm. This loss temporally coincides with the nucleus becoming freely mobile within the cytoplasm, suggesting that, while IF removal is not directly linked to the physical process of enucleation, it may be a prerequisite for the initiation of nuclear mobility in both lineages. These changes are also observed in early primitive cells cultured in vitro, suggesting that they constitute an intrinsic part of the murine erythroid differentiation program independent of lineage and hematopoietic microenvironment. PMID- 1706981 TI - Where are we in the quest for vaccines for malaria? PMID- 1706982 TI - Foscarnet. A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. AB - The pyrophosphate analogue, foscarnet, selectively inhibits the DNA polymerase of human herpes viruses, including cytomegalovirus, and the reverse transcriptase of HIV. Viral replication is therefore prevented, but resumes when the drug is cleared from infected cells. In vitro, the combination of foscarnet and zidovudine (azidothymidine) has an additive effect against cytomegalovirus and acts synergistically against HIV. An improvement in cytomegalovirus retinitis is obtained in over 85% of affected AIDS patients during foscarnet induction therapy, but relapse usually occurs within a month of ceasing treatment. There is a similar duration of remission during maintenance therapy given for 5 days each week, but this can be extended 4- to 5-fold with daily administration of higher doses. In allograft recipients, progression of retinitis can be halted by foscarnet until immune function recovers and eradicates the virus. The incidence of acute renal failure, which is common during foscarnet therapy, may be reduced by dosage adjustment and adequate prehydration. Anaemia, phlebitis, nausea and vomiting, and disturbances in serum calcium and phosphate levels, perhaps resulting from uptake of foscarnet into bone or chelation with ionised calcium, have also been associated with administration of the drug. Cytomegalovirus retinitis is difficult to treat, with few therapeutic options available. Although treatment with foscarnet produces some severe adverse effects, with care these can be minimised, and the drug produces clinical improvement in a large proportion of patients; this is a highly encouraging finding at this stage in its development. Preliminary comparative data indicate that foscarnet and ganciclovir are similarly effective, but foscarnet may have some theoretical advantages in AIDS patients since it can be used in combination with zidovudine without potentiating myelosuppression. PMID- 1706983 TI - 5-HT1A partial agonists. What is their future? PMID- 1706984 TI - Bopindolol. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy. AB - Bopindolol is a nonselective beta-adrenoceptor antagonist [corrected] with partial agonist activity which is used in the treatment of hypertension. The drug is rapidly metabolised to an active hydrolysed form. The antihypertensive effects of bopindolol 0.5 to 4 mg are sustained for more than 24 hours after once daily dosing, and the drug appears similar in efficacy to propranolol, metoprolol, atenolol, pindolol and slow release nifedipine in the treatment of mild to moderate forms of this disease. In limited trials bopindolol has also successfully reduced symptoms in patients with angina pectoris, anxiety and essential tremor. Thus, bopindolol is an effective and well-tolerated beta adrenoceptor antagonist. PMID- 1706985 TI - Antibiotic prophylaxis in hysterectomy and induced abortion. A review of the evidence. AB - By the early 1980s, perioperative prophylaxis in vaginal hysterectomy had been shown consistently to be of such value in reducing postoperative infection that some authors maintained that further placebo-controlled studies were no longer ethical. The benefit of prophylaxis in abdominal hysterectomy was less uniformly demonstrated, in studies which were prospective, placebo-controlled, double-blind and randomised. Prophylaxis may significantly reduce the incidence of febrile morbidity and/or wound/pelvic infection, the duration of hospital stay, or the total usage of antibiotics. It is therefore generally agreed that each centre should itself scientifically evaluate the efficacy of prophylaxis before a decision on its routine use in abdominal hysterectomy is made. In comparative studies, agents which were active against both anaerobic and aerobic organisms were more efficacious than those active against anaerobes only. Antibiotics with similar spectra of activity showed similar efficacy in both types of hysterectomy. Multiple- and single-dose regimens of the same antibiotics also showed equal efficacy. The new cephalosporins with a longer half-life were attractive theoretically as agents in single-dose regimens; ceftriaxone, however, has been shown to have an adverse effect on the normal gut flora. With the increased numbers of induced abortions carried out in the UK and other parts of the world in recent years, the need to reduce postabortal infection is generally appreciated. The results of early studies using tetracyclines as the prophylactic agents were difficult to evaluate because of the incomplete follow-up and different definitions of pelvic infections. No benefit was demonstrated in 2 studies using a single preoperative dose of tinidazole, whereas oral metronidazole in 3 doses and penicillin/pivampicillin for 4 days were shown to be efficacious in reducing postabortal infection. In a recent study with doxycycline, significant benefit was shown in patients with negative preoperative screening for gonococcal and chlamydial infection. These genital infections, together with a history of previous pelvic inflammatory disease (PID)/gonorrhoea, nulliparity with multiple partners, young age of the patient and gestational age have been described as significant risk factors. Some researchers hold the view that selective prophylaxis based on these risk factors should be practised instead of mass prophylaxis. All agree that an antibiotic regimen that is both efficacious and well tolerated has yet to be found. PMID- 1706986 TI - Long term treatment of duodenal ulcer. A review of management options. AB - Duodenal ulcer is a chronic disease characterised by remission and relapses. The duration of this relapsing tendency is unpredictable for the individual patient, but in most cases it lasts for many years and perhaps the entire lifetime. Various therapeutic strategies have been suggested to maintain the disease in remission: continuous, intermittent and on-demand treatment with H2-antagonists, or surgery. Continuous maintenance treatment with the currently available H2 blockers has proved to be superior to all the other strategies in terms of efficacy, and should therefore be regarded as the long term treatment of choice for duodenal ulcer patients. The duration of maintenance treatment is still uncertain, but probably it should not be less than a few years. Intermittent treatment or surgery could be proposed to patients unsuitable for continuous maintenance, depending on whether they have mild or aggressive disease, respectively. PMID- 1706987 TI - Paediatric analgesia. Which drug? Which dose? AB - The pharmacological management of paediatric pain is an area which is undergoing considerable development. Improvements in pain management are coming from appreciation of the special problems of children, increased knowledge of drug pharmacology and the development of better methods of drug delivery. Traditional methods of postoperative analgesia such as intramuscular injections are disliked by children and are being replaced by intravenous infusions, patient-controlled analgesia (PCA) and epidural opioids. Local anaesthetic blocks offer the benefit of fewer side effects and for certain procedures can provide complete pain relief in the immediate postoperative period. Inhaled analgesics such as nitrous oxide can be adapted for use in children and provide excellent analgesia for short painful procedures. The pain from needles is reduced considerably by the use of local anaesthetic creams. There has been greater appreciation of the benefits of drug combination, particularly with cancer pain management, and the importance of providing long term analgesia for these patients and patients with burns. Considerable scope exists for future developments such as transmucosal and transdermal drug delivery systems and other methods of drug delivery which are suited to the special needs of children. PMID- 1706988 TI - Guidelines for drug treatment of male infertility. AB - The prerequisite for rational therapy of male fertility disorders is an exact diagnosis. While the possibilities of influencing disturbances of spermiogenesis are limited, male adnexal diseases can be successfully treated in many cases. Drugs for the treatment of fertility disorders must be applied with this in mind, and empiric therapy is often performed in addition to causal treatment which, however, may be quite rationally determined. The therapeutic spectrum in andrology includes antibiotic and antiphlogistic agents, mast cell blockers, zinc, vitamins, and immunosuppressive drugs (corticosteroids). These agents are used for the treatment of inflammatory diseases of the testes and the accessory glands or for suppression of antispermatozoal antibodies. Hormonal disturbances are infrequently encountered by the andrologist, but they can be treated, with proven efficacy, with gonadotrophins, gonadotrophin-releasing hormone (GnRH) or androgens. In certain cases that are not hormonally related, the use of antiestrogens (clomifene, tamoxifen) as stimulating agents may be successful. Furthermore, tissue hormone releasing proteases (kallikrein) can be used both therapeutically (especially in motility disturbances that are not due to structural flagellar defects) and diagnostically (in order to distinguish between inflammatory and noninflammatory testicular damage). Anticholinergics and alpha sympathomimetics are applied to ameliorate ejaculation or emission failure. In addition to a review of these treatment forms, the development of new concepts, e.g. angiotensin converting enzyme (ACE) inhibitors, is discussed. PMID- 1706989 TI - Treatment options for the relief of pain during childbirth. AB - Despite its severity, the disposition of women towards pain during childbirth is influenced by many complex personal and cultural factors. Such influences may inspire a degree of stoicism towards labour pain which would be extraordinary in other painful circumstances. Nevertheless, the majority of women who deliver in a modern obstetric unit request some form of pharmacological pain relief. An important component of proper antenatal education, therefore, is to provide impartial information about the various analgesic alternatives which are available within each centre. Regimens of analgesia which depend on the systemic absorption of drugs (e.g., parenterally administered opioids; inhalational analgesia) are simple to administer but they have limited efficacy and are commonly associated with unpleasant central side effects. While some innovations in actual drug administration have been introduced, it is unlikely that any further major improvements will be feasible using the systemic approach to analgesia. Epidural analgesia has become established as the most effective and consistently reliable method of providing pain relief in labour. Recent advances have demonstrated that many of the adverse effects traditionally associated with epidural analgesia can be substantially reduced by administering local anaesthetics in smaller doses. It is becoming apparent that additional patient benefits are possible when epidural opioids are also used in combination with local anaesthetics. Techniques which allow the mother to exercise personal control over her epidural analgesia requirements are received more favourably and may help reduce the need for obstetric intervention. PMID- 1706990 TI - Torasemide. A review of its pharmacological properties and therapeutic potential. AB - Torasemide (torsemide) is a high-ceiling loop diuretic which acts on the thick ascending limb of the loop of Henle to promote rapid and marked excretion of water, sodium and chloride. Like furosemide (frusemide), its major site of action is from the luminal side of the cell. Torasemide is at least twice as potent as furosemide on a weight-for-weight basis, produces equivalent diuresis and natriuresis at lower urinary concentrations and has a longer duration of action, allowing once-daily administration without the paradoxical antidiuresis seen with furosemide. Torasemide also appears to promote excretion of potassium and calcium to a lesser extent than furosemide. In trials of up to 48 weeks' duration in patients with mild to moderate essential hypertension, torasemide, administered as a single daily dose, has been shown to achieve adequate blood pressure control reaching steady-state within 8 to 12 weeks. Those patients not responding initially have generally responded to a doubling of the dose. Comparative trials of up to 6 months show torasemide is as effective as indapamide, hydrochlorothiazide or a combination of triamterene/hydrochlorothiazide in maintaining control of blood pressure. Torasemide has also been used successfully to treat oedematous states associated with chronic congestive heart failure, renal disease and hepatic cirrhosis. In short term trials control of blood pressure, bodyweight and residual oedema has been sustained. Torasemide appears to be a useful alternative to furosemide in these patients, providing potent and long-lasting diuresis while being relatively potassium and calcium sparing. In clinical trials to date torasemide has been well tolerated with adverse effects of a mild, transient nature reported by only small numbers of patients. Changes in biochemical parameters have been common, including decreases in plasma sodium and potassium levels and increases in plasma creatinine and uric acid levels. These changes are typical of loop diuretics. No changes were clinically significant nor were clinically relevant changes noted in glucose metabolism, cholesterol or triglyceride levels or in haematological values. Thus, torasemide is an interesting new loop diuretic with potential use in the treatment of mild to moderate essential hypertension and of oedematous states in which diuretic therapy is warranted. Preliminary studies suggest it to be as efficacious as other diuretics in common use and to have some advantage over furosemide in duration of action and in effects on potassium and calcium. However, further long term trials in larger groups of patients are needed to delineate the place of torasemide in therapy fully, both as a single agent and in combination with other currently accepted drug regimens. PMID- 1706991 TI - Release of substance P from guinea pig trachea leukotriene D4. AB - Coordinated studies of leukotriene D4 (LTD4)-mediated contractile responses and LTD4-evoked release of the tachykinin substance P (SP) in both intact and epithelium abraded guinea pig tracheal smooth muscle preparations were performed. A partial contribution by axon reflex mechanisms to the magnitude of LTD4-induced tracheal contractions was suggested by a maximum inhibition of 21% and 28% by 5 x 10(-6) M tetrodotoxin (TTX) in abraded and intact preparations, respectively. SP induced contractions were antagonized by the SP analog [DPro4DTrp7,9]-SP 4-11 in both types of preparation. The SP analog produced 58% and 72% inhibition of contractile responses to 10(-8) M LTD4 in abraded and intact preparations, respectively. Direct measurement of SP release by radioimmunoassay of the bathing medium showed TTX-sensitive LTD4-evoked release of SP. Inhibition by 5 x 10(-6) M TTX of LTD4-evoked SP release was 77%. The SP antagonist produced greater inhibition of LTD4-evoked contractions (58% in abraded, and 72% in intact preparations) than maximum TTX inhibition of LTD4-evoked contractions (21% in abraded, and 28% in intact). However, LTD4 (10(-8) M)-evoked SP release was at least 77% blocked by maximum doses of TTX. We therefore suggest that an additional agent, released by TTX-insensitive mechanisms, but whose contractile effects are also antagonized by [DPro4DTrp7,9]-SP 4-11, may participate in the LTD4 response. PMID- 1706992 TI - 89Strontium-induced bone marrow depression suppresses the early inflammatory response and fibrosis caused by intratracheal bleomycin. AB - To investigate the effect of bone marrow depression on the development of bleomycin-induced lung injury, F-344/Crl rats were given an intraperitoneal (IP) injection of 89SrCl2 (2 mCi/kg body weight) 7 days prior to the intratracheal (IT) instillation of 7.0 U/kg body weight bleomycin (Sr-bleomycin group). A second group of rats was given an IP injection of saline followed 7 days later by IT bleomycin (bleomycin group). Additional rats were given 89Sr IP and saline IT (Sr group) or saline IP and saline IT (saline group). Rats were sacrificed at 0, 3, 10, 21, and 30 days after the intratracheal instillations. 89Sr administration resulted in significantly lower numbers of circulating blood neutrophils and monocytes in the Sr-bleomycin group compared with the bleomycin group through at least the first 21 days following the IT instillations. Lymphocyte numbers were also depressed in the Sr-bleomycin group at days 3 and 21. Analysis of bronchoalveolar lavage fluid (BALF) revealed significantly reduced protein and lymphocyte numbers in the BALF from the 89Sr-bleomycin group compared with the bleomycin group at day 3, but not at later time points. Neutrophils in BALF were also lower (though not significantly) in the 89Sr-Bleomycin group at day 3. There was no difference in the number of BALF macrophages between the Sr-bleomycin and bleomycin groups at any time point throughout the study. Histology and morphometry showed the same trends as the BALF data with much less severe lesions in the 89SR-Bleomycin group compared with the bleomycin group at day 3, but not at later time points. At day 10, hydroxyproline values were significantly higher in the bleomycin group (47% increase above saline group) than the Sr-bleomycin group (only 18% increase above Sr group), but by day 21, there was no longer a significant difference between these two groups. These results demonstrate that bone marrow depression significantly suppresses the early inflammatory response and collagen deposition caused by a single IT dose of bleomycin, but has little effect on the resolution of bleomycin-induced injury. PMID- 1706995 TI - Increased frequency of chromosomal damage in peripheral blood lymphocytes up to nine years following curative chemotherapy of patients with testicular carcinoma. AB - The presence of micronuclei (MN) in binucleated peripheral blood lymphocytes of 37 testicular carcinoma patients was studied 1.5 to 9.3 years following curative chemotherapy. All patients received cisplatinum, 35 patients also received bleomycin. In addition, most patients were treated with another cytotoxic drug, i.e., vinblastine (n = 23) and/or etoposide (n = 24). The mean time interval between cessation of chemotherapy and the micronucleus assay was 4.6 years. The median frequency of MN in binucleated cells in treated patients (0.059) was significantly higher than that in 12 untreated cancer patients (0.036; P = 0.003) or that in 26 healthy age-matched controls (0.034; P less than 0.001). Frequencies of MN in the 12 untreated cancer patients (including 7 patients with disseminated testicular carcinoma) did not differ from those in the 26 healthy controls (P = 0.890), suggesting that chromosomal damage in lymphocytes of treated testicular cancer patients must be attributed to the chemotherapy. Results indicate that lymphocytes containing chemically induced chromosomal damage persist for up to 9.3 years following cessation of chemotherapy. The implications of these findings with regard to the increased risk for secondary tumors are discussed. PMID- 1706993 TI - Changes in 7SL RNA conformation during the signal recognition particle cycle. AB - The structure of 7SL RNA has been probed by chemical modification followed by primer extension, using four substrates: (i) naked 7SL RNA; (ii) free signal recognition particle (SRP); (iii) polysome bound SRP; and (iv) membrane bound SRP. Decreasing sensitivity to chemical modification between these different substrates suggests regions on 7SL RNA that: bind proteins associated with SRP might interact with ribosomes; and are protected by binding to membranes. Other areas increase in chemical sensitivity, exemplified by a tertiary interaction present in naked 7SL RNA but not in free SRP. Such changes suggest that 7SL RNA changes its conformation during the SRP cycle. These conformational changes could be a necessary component to move through the SRP cycle from one stage to the next. PMID- 1706996 TI - Expression of alpha-fetoprotein by differentiating fetal rat hepatocytes in vitro. AB - Previous studies have established that, under appropriate conditions, fetal rat hepatocytes will differentiate in culture. This is characterized by the acquisition and loss of enzyme markers which are observed during liver development in vivo. The expression of alpha-fetoprotein (AFP), which declines during normal development, is examined in cultured hepatocytes derived from 15- and 19-day gestation rats. Secretion of AFP and relative levels of AFP mRNA and gene transcription were measured. Initially, AFP expression was greater in 15-day gestation hepatocytes, and in both instances AFP secretion and AFP mRNA decreased during culture. The decline in AFP expression by 15-day gestation fetal hepatocytes in vitro was not significantly altered by various manipulations of the culture conditions. It is proposed that cultured fetal hepatocytes continue to differentiate in vitro by repressing AFP expression while the expression of other liver-specific genes is being initiated. The fetal hepatocyte culture model therefore adequately reflects in vivo changes in developmentally regulated liver specific genes. PMID- 1706994 TI - The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation. AB - Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions. PMID- 1706997 TI - Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane. AB - The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle. PMID- 1706999 TI - De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins. AB - We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages. PMID- 1706998 TI - Keratins 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells in the human hair follicle. AB - Morphological maturation of the inner root sheath (IRS) and cuticle of the human hair follicle reveals analogies to differentiation processes in other keratinizing epithelia. Detailed biochemical analysis of respective differentiation products, however, has been largely restricted by their low solubility. Herein we provide further evidence for the existence of K1 and K10 derivatives in IRS and hair cuticle based on protein analysis of isolated fractions and immunofluorescence in situ, substantiating our earlier data (Stark, H. J., et al. Differentiation 35, 236-248 (1987)). Extracts from both compartments showed on two-dimensional (2D)-polyacrylamide gels a group of presumptive K1 and K10-turnover products in a wide pI (basic to acidic) and Mr range (56,000-65,000), named IC-I to III and IC-IV, respectively. These components (also found in nail plate) reacted with specific antibodies (to K1 and K10) on Western blots. Weak but distinctive radiolabeling of presumptive precursor spots close to authentic K1 and K10, respectively, and their presence in lower follicle fractions (distant from infundibulum) largely precluded epidermal contamination. Two-dimensional tryptic peptide maps of excised 2D spots from the IC-I to III series revealed high homology to K1, and those from IC-IV components to K10. Immunodetection in frozen sections was improved by trypsin pretreatment and showed distinguished staining for K1 and K10 in IRS ranging from the lower bulbus region up to the "keratinizing zone" of the follicle. Above, the reaction was abruptly abolished which coincides with ultrastructural "melting" of distinct filaments in the intracellular matrix. Thus, our data suggest that differentiation in these follicular compartments (IRS and cuticle) might follow common principles of keratinization. PMID- 1707000 TI - Inhibition of B cell proliferation with anti-CD19 monoclonal antibodies: anti CD19 antibodies do not interfere with early signaling events triggered by anti IgM or interleukin 4. AB - The 95-kDa antigen recognized by the anti-CD19 panel of monoclonal antibodies is found on the surface of most cells of the B cell lineage. Anti-CD19 antibodies inhibit B cell proliferation in response to anti-Ig plus interleukin 4 (IL4), but enhance the response to mitogenic concentrations of either phorbol 12-myristate 13-acetate (PMA) or Epstein-Barr virus. This dichotomy in the effect of anti-CD19 antibodies suggested that the inhibitory action may be directed at the transmembrane signaling pathways utilized by anti-IgM and IL4. To investigate this hypothesis, an attempt was made to determine the mechanism of signal transduction utilized by the CD19 antigen, and elucidate its effect on transmembrane signaling invoked by anti-immunoglobulin and IL4. Binding of anti CD19 antibody to B cells did not promote activation of either the phosphoinositide or cAMP signaling pathways. In addition, anti-CD19 antibody did not inhibit phosphatidylinositol bisphosphate (PIP2) hydrolysis induced by anti IgM or IL4, nor did it interfere with cAMP induction by IL4. We also found that anti-CD19 antibody inhibited PMA plus calcium ionophore-induced B cell proliferation. This evidence indicates that anti-CD19 mAb interrupts the signaling cascade at a point distal to receptor-mediated breakdown of PIP2 and/or activation of adenyl cyclase. This conclusion was fully consistent with experiments in which anti-CD19 antibody was shown to inhibit DNA but not RNA synthesis, and the observation that anti-CD19 antibody must be present between 6 h and 20 h after the initiation of the culture suggesting that anti-CD19 mAb exerts its inhibitory effect in late G0 or G1, after the initial signaling events. PMID- 1707001 TI - Na+/H+ antiporter has different properties in human B lymphocytes according to CD5 expression and malignant phenotype. AB - In B chronic lymphocytic leukemia (B-CLL) cells, lipopolysaccharide (LPS) and phorbol esters fail to activate the plasma membrane-associated Na+/H+ antiporter and, subsequently, to elicit a rise in cytosolic pH. Since these events are thought to be a prerequisite for LPS-induced proliferation of B normal lymphocytes, we analyzed the kinetic properties of Na+/H+ antiporter in B-CLL cells as compared to both CD5- and CD5+ normal B lymphocytes. In the present work we report that Na+/H+ exchange rate after acid loading is drastically decreased in B-CLL cells, as compared to normal CD5- B lymphocytes, although the antiporter affinity for external Na+ and internal H+ is not significantly different in both cell populations. Kinetic data account for a reduction in the number of operating antiport units in B-CLL. The Na+/H+ antiporter of CD5+ normal B lymphocytes exhibits both an exchange rate and an ion affinity significantly higher than that observed in both CD5+ B-CLL cells and CD5- B normal lymphocytes, thus suggesting a possible explanation for their activated phenotype. PMID- 1707002 TI - Chronic experimental autoimmune encephalomyelitis induced by the 89-101 myelin basic protein peptide in B10RIII (H-2r) mice. AB - Development of experimental allergic encephalomyelitis (EAE) in the SJL (H-2s) mice is associated with a T cell-dependent autoimmune response to the C-terminal part of the myelin basic protein (MBP). In this study the influence of both H-2 and non-H-2 genetic background on EAE induced with the MBP89-101 peptide is described. Analysis of different H-2q haplotype strains, B10G, B10Q, SWR and NFR/N, showed that the B10 background is relatively resistant to disease induction. Both SWR and NFR/N were susceptible to EAE showing that the H-2q haplotype is permissive for EAE development induced with MBP89-101 and that the T cell receptor (TcR) haplotype or complement C5 deficiency exert no significant influence on disease susceptibility. In a series of H-2-congenic strains on the B10 background only B10RIII (H-2r) mice were susceptible to EAE. The B10RIII mice developed a severe EAE with early onset and chronic progressive or relapsing course of disease. In addition, B10RIII mice treated with Freund's complete adjuvant and pertussis toxin alone showed an early monophasic disease. The clinical observations were confirmed by immunohistopathologic analysis of the central nervous system. In these studies, we also applied antibodies to different TcR V beta elements which showed no specific limitation of the used TcR among infiltrating T cells in the target tissue in any of the strains. It is concluded that an MBP peptide-specific disease can be induced in three different haplotypes and it is possible that shared structures between the As, Aq and Ar molecules are of importance for the trigger of encephalitogenic T cells with different TcR V elements. The presently described chronic EAE model induced in the B10RIII mice will be of value as a model for multiple sclerosis. PMID- 1707003 TI - T cell proliferation induced by monoclonal antibodies to a phosphatidylinositol linked differentiation antigen of guinea pig lymphocytes. AB - Monoclonal antibodies (mAb) to differentiation antigens frequently influence the in vitro function of antigen-bearing cells. We characterized a 32-36-kDa membrane protein expressed on guinea pig lymphocytes and Langerhans cells. A series of independently derived mAb to this protein, now called guinea pig T cell activation antigen (gpTAA), induced strong proliferation of T cells in vitro. Cross-linking of the mAb by a secondary antibody (rabbit anti-mouse Ig) and costimulation with phorbol 12-myristate 13-acetate were required for activation. Treatment of the cells with phosphatidylinositol-specific phospholipase C greatly reduced the amount of antigen expressed on the cell surface as measured by flow cytometry analysis. This finding indicates that the antigen is anchored to the cell membrane via phosphatidylinositol linkage as shown similarly for other membrane proteins with T cell activating properties, e.g. Thy-1 and Ly-6. The guinea pig protein differs, however, in its molecular weight and tissue distribution from similar proteins identified in the mouse or in the rat system. Unlike Thy-1, gpTAA is also expressed on B Lymphocytes and Langerhans cells. Considering the previously described involvement in cellular adhesion, and the functional characteristics reported here, gpTAA might represent a new species of differentiation antigen with T cell-activating capacity. PMID- 1707004 TI - Isoforms of the E2 molecule: D44 monoclonal antibody defines an epitope on E2 and reacts differentially with T cell subsets. AB - Human T cell rosetting with erythrocytes is clearly dependent on the CD2-CD58 interaction. We have previously demonstrated that other T cell molecules are involved in the rosette phenomenon, including the E2 molecule, a 32-kDa transmembrane glycoprotein. In this report we show that the D44 monoclonal antibody (mAb), previously shown to subdivide T cells into subpopulations with distinct functional repertoires and to identify 70% of lymphocytes from bronchoalveolar lavage from HIV+ patients, defined a new epitope on the E2 molecule. This was illustrated by the reactivity of the D44 mAb in Western blot experiments performed with the immunoaffinity purified E2 molecule. Moreover, double-labeling experiments showed that the E2 molecule exhibited varying epitopes when expressed in different cell types. The D44 and 12E7 epitopes were restricted to distinct subpopulations of T cells and, more importantly, the D44 expression was limited to the CD29+ population including the memory subset. The O662 and L129 epitopes are present on all T cells. Thus, the E2 molecule has both common and variable epitopes in its extracellular domain, and only the common epitopes seem to be involved in T cell adhesion processes. PMID- 1707005 TI - Suppressor cells of popliteal lymph node origin are involved in the in vivo and in vitro control of experimental allergic encephalomyelitis effector cells in the Lewis rat. AB - Lewis rats immunized in the hind footpads with total guinea pig spinal cord tissue in mycobacteria-enriched complete Freund's adjuvant develop chronic relapsing experimental allergic encephalomyelitis. It was previously shown that popliteal lymph node cells (LNC) isolated at the time of the first recovery (day 16) and transferred into naive syngeneic recipients protect from active induction of the disease. On the other hand, inguinal LNC taken at the onset of the disease (day 11) induce under similar conditions an acceleration of the appearance of the clinical symptoms. In this report, we show that the in vivo suppressive activity of popliteal LNC is associated with the absence of production of interleukin 2 in this compartment. The lack of production of this lymphokine and the suppressive activity can be detected only in the popliteal compartment and appear as early as day 11 after immunization. We show that this suppressive population displays in vitro inhibitory activity on the proliferative response of the disease effectors (inguinal LNC) to guinea pig myelin basic protein. This suppressive activity is not abrogated by addition of interleukin 2, suggesting that these suppressor cells do not inhibit the proliferation by absorption of the released lymphokine or by inhibition of its production. PMID- 1707006 TI - A lectin-binding soluble factor released by CD8+CD57+ lymphocytes from AIDS patients inhibits T cell cytotoxicity. AB - CD8+CD57+ T cells, expanded in peripheral blood lymphocytes of AIDS patients, inhibit the effector phase of HLA-specific cytotoxic T lymphocytes, natural killer and lymphocyte-activated killer cells in a 4-h chromium-release assay. This inhibitory activity present in supernatants of purified sorted CD8+CD57+ cells is mediated by a non-antigen-specific inhibitory factor which is distinct from prostaglandin E2, T cell growth factor (TGF)-beta, latent-TGF-beta, tumor necrosis factor (TNF)-alpha and TNF-beta. Partial biochemical characterization demonstrates that the CD8+CD57+ inhibitory activity (a) is heat, trypsin and acid resistant, (b) binds to concanavalin A columns, indicating its glycosylation state and (c) is mediated by a 20-30-kDa soluble molecule. PMID- 1707007 TI - Unusual phenotype of intestinal intraepithelial lymphocytes in the rat: predominance of T cell receptor alpha/beta+/CD2- cells and high expression of the RT6 alloantigen. AB - The phenotype of intraepithelial lymphocytes (IEL) from the small intestine of adult rats was studied by flow cytometry. Using appropriate monoclonal antibodies the expression patterns of the T cell receptor alpha/beta (TcR2), CD2, the alloantigen RT6 and several other T cell antigens were analyzed. The vast majority of rat IEL expressed TcR2 which is in contrast with data reported for the mouse. The comparison of IEL with lymph node cells revealed major phenotypic differences. Whereas CD2 was present on virtually all lymph node T cells it was found on only less than 5% of IEL. The T cell-specific differentiation antigen RT6 present on only a fraction of lymph node cells was found on about 99% of IEL demonstrating uniform expression with an approximately tenfold higher density. Identity of the detected molecule with RT6 was proven by using congenic controls and by the demonstration of phosphatidylinositol linkage to the IEL membrane. About 86% of IEL expressed CD8 but a substantial proportion of these cells co expressed the CD4 molecule (34%). Two-color analysis revealed that the CD4+CD8+ double-positive subset completely lacked CD45RB suggesting that they represent memory cells. In the CD4-CD8+TcR2+ subset there was a remarkable heterogeneity of CD5 expression. A substantial number of these cells did not express CD5 despite high density of TcR2. Phenotypic peculiarities found on all or most IEL such as the lack of CD2 and the increased expression of RT6 indicate that the intestinal epithelial environment exerts strong effects on the development and maturation of these cells. PMID- 1707008 TI - Evidence for a restricted idiotypic and epitopic specificity of anti thyroglobulin autoantibodies in patients with autoimmune thyroiditis. AB - Anti-thyroglobulin (TG) autoantibodies from patients with autoimmune thyroiditis express a cross-reactive alpha idiotype (Id) termed T44 which is not expressed by IgG from normal individuals. The present study demonstrates that the expression of the T44 Id is strongly associated with the recognition by anti-TG autoantibodies of a specific epitopic cluster on human TG. The epitopic reactivity of anti-TG autoantibodies was determined using a competitive inhibition assay with a panel of 15 monoclonal antibodies that define six antigenic clusters on TG. All T44+ autoantibodies from patients recognized cluster II, whereas no anti-TG IgG from healthy individuals reacted with this region. Affinity columns of Sepharose-bound intravenous therapeutic immunoglobulins which contain anti-T44 activity, retained both T44 Id-expressing antibodies and a subset of region II-specific anti-TG autoantibodies from patients with Hashimoto's disease. Restricted idiotypic and epitopic specificity may demarcate disease-associated from natural anti-TG autoantibodies, suggesting that qualitative rather than quantitative criteria should be used to identify pathological autoantibodies. PMID- 1707009 TI - Transfection of human CD59 complementary DNA into rat cells confers resistance to human complement. AB - We have examined the role of the human CD59 antigen in inhibiting complement mediated lysis by transfer and expression of a CD59 cDNA in rat cells. A cDNA encoding CD59 was subcloned into the expression vector pSFSVneo and stably transfected into the rat T cell line NB2-6TG. Indirect immunofluorescence staining using the anti-CD59 monoclonal antibody YTH53.1 demonstrated the presence of human CD59 antigen on transfected cells and its attachment to the cell surface by a rat glycolipid anchor. Transfected cells were found to contain a single 3.3-kb species of CD59 mRNA by Northern blot hybridization. Immunoblotting revealed that this encoded a protein band of the same size as that observed in human erythrocytes. To determine the biological effect of expression of human CD59 in rat cells, an assay was devised which measured the relative lysis of transfected cells compared to untransfected cells in the presence of human complement and a lytic monoclonal antibody. It was observed that CD59 transfected rat cells are less susceptible to lysis by human complement and that this effect was blocked by a F(ab')2 fragment of YTH53.1. These experiments provide a direct demonstration that CD59 can function as an homologous complement restriction factor for nucleated cells. PMID- 1707010 TI - [The influence of inhibitors of kinin biosynthesis, prostaglandins and angiotensin II on the vascular and tubular effects of strophanthin in the rat kidney]. AB - Strophanthin was shown to increase the blood flow in the external, internal zones of the cortex and the external medulla of the rat kidney without influencing the blood supply to the internal zone of the medulla. Contrikal eliminates the vasodilating effect of the drug in the mentioned zones of the renal tissue and significantly increases its natriuretic action. Indomethacin also prevents the hemodynamic shift occurring in the kidneys under the influence of strophanthin but fails to change the character of the natriuretic effect of the drug. Captopril exerts no influence on the increase of the blood flow in the external medulla but eliminates the vasodilating effect of strophanthin in the cortex and significantly potentiates its natriuretic action. PMID- 1707011 TI - [The interferon system: inhibitors of interferon action]. AB - The review of the literature data and the authors' data on interferon action inhibitors being one of the components of interferon system is presented. The methods of the production of various types of interferon action inhibitors, their characteristics and the main differences between them are described. The possible mechanisms of inhibitors' actions are considered. It was shown that the study of interferon action inhibitors is important in both the theoretical and practical aspects. PMID- 1707013 TI - Iloprost improves femoro-distal graft flow after a single bolus injection. AB - A double-blind, randomised, placebo-controlled trial was conducted to study the effect of the stable prostacyclin analogue iloprost on femoro-distal graft blood flow. After completing femoro-distal reconstruction, 3000 ng of iloprost or placebo was injected into the graft over 2 min. Graft blood flow, measured by electromagnetic flowmetry, increased by a mean (range) of 94% (12 to 192%) in patients receiving iloprost (n = 15) compared to 6% (-34 to 53%) in controls (n = 16; p less than 0.0001, t-test). Increased graft flow, measured by duplex ultrasound, was maintained in the iloprost group over a 7 day period postoperatively (F = 5.2, p = 0.03; analysis of variance) and remained higher at 7 days (p = 0.007, t-test). Iloprost produces an immediate, sustained increase in graft blood flow after femoro-distal reconstruction and may therefore be of benefit in reducing the incidence of early graft failure. PMID- 1707012 TI - Demonstration of a transcription element in vitro between the capping site and translation initiation site of the mouse myelin basic protein gene. AB - A transcription element was identified, by in vitro analyses, just downstream from the capping site of the mouse myelin basic protein (MBP) gene. Deletion of this element caused a dramatic drop of transcription efficiency in mouse brain, rat liver and HeLa cell nuclear extracts, regardless of the form of DNA being closed circular or linear form. DNase I footprint analysis demonstrated the presence of a ubiquitous trans-acting factor for this region. This element functioned even when it is located in the normal direction downstream from the adenovirus major late promoter. Mutation analysis suggested that an essential part of the downstream element was located between +25 and +45. PMID- 1707014 TI - Laser laparoscopic management of large endometriomas. AB - Forty-seven patients underwent laser laparoscopic management of endometriomas from 3 to 12 cm in diameter. Eighteen patients had infertility, 15 had pelvic pain, and 14 had both. The types of laser used were the carbon dioxide, argon, and potassium-titanyl-phosphate. There were no surgical complications. Twelve of 32 patients with infertility achieved pregnancy after the initial procedure. Subsequently, 2 patients conceived after a second-look procedure. Twenty-three of 30 patients with pelvic pain reported improvement or resolution. We confirm the efficacy of operative laparoscopy using lasers in the management of large ovarian endometriomas. PMID- 1707015 TI - Snoods: a periodic network containing cytokeratin in the cortex of starfish oocytes. AB - An extensive fibrous cytoskeletal component in the cortical cytoplasm of oocytes of the starfish Pisaster ochraceus reproducibly stains with anticytokeratin antibody and hence contains cytokeratin. The large-meshed network resembles a snood (hair net). Snood fibers form loops and branches throughout the cortex of a premeiotic oocyte, except at the animal pole where they emanate from a nonstaining zone surrounding the centrosomes. By immunofluorescence microscopy of isolated cortices and electron microscopy of isolated cortices and intact oocytes, snood fibers exhibit complex striations with a periodicity of approximately 0.75 micron. Snoods are not colocalized with the cortical arrays of microtubules and are unaffected by drugs that disrupt microtubules or microfilaments. Stimulation of oocyte maturation by 1-methyladenine causes snoods to disappear, presumably by disassembly, about halfway to the time of germinal vesicle breakdown. They do not reappear during meiosis, fertilization, or development to the two-cell stage, and their functional importance, if any, during oogenesis or development remains to be elucidated. PMID- 1707016 TI - Tissue-specific, temporal changes in cell adhesion to echinonectin in the sea urchin embryo. AB - Echinonectin is a dimeric, glycoprotein found in the hyaline layer of the developing sea urchin embryo. It was found that echinonectin supports adhesion of embryonic cells in vitro. Previous studies have shown that the protein hyalin also supports adhesion. The purpose of this study was to examine the specificity of cell-echinonectin interactions during sea urchin development. Primary mesenchyme cells (PMCs) ingress into the blastocoel during gastrulation. In the process the PMCs lose contact with the hyaline layer. It was found experimentally that differentiating PMCs decreased their adhesion to hyalin at the time of ingression. It was of interest, therefore, to determine whether there was a coordinate loss of adhesion to echinonectin at ingression as well. When cell echinonectin interactions were quantified using a centrifugal force-based adhesion assay, it was shown that micromeres adhered well to echinonectin. At the time of ingression, PMCs displayed reduced adhesion to echinonectin just as had been found when hyalin was tested as a substrate. There was no change in adhesion of presumptive ectoderm or endoderm to echinonectin over the same time period. Early in gastrulation presumptive ectoderm and endoderm adhered to echinonectin only half as strongly as to equimolar concentrations of hyalin. After gastrulation endoderm cells were observed to retain the same relative affinity to hyalin and echinonectin, while ectoderm cells became equally adhesive for both hyalin and echinonectin. Quantitatively, this represents an overall increase in the affinity of ectodermal cells for echinonectin. Adhesion to combined substrata of echinonectin and hyalin was reduced but not abolished by monoclonal antibodies specific for echinonectin. The antibodies did not cross-react with hyalin. We conclude that both echinonectin and hyalin independently act as adhesive substrata for the developing sea urchin embryo. PMCs lose an affinity for echinonectin and ectodermal cells later increase their affinity for this substrate. PMID- 1707017 TI - Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis. AB - The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum. PMID- 1707018 TI - T-lymphocyte requirement for diabetes in RT6-depleted diabetes-resistant BB rats. AB - Diabetes-prone (DP) BB rats develop spontaneous autoimmune insulin-dependent diabetes mellitus (IDDM). The cell populations involved in the expression of diabetes are not precisely known but probably include natural killer (NK) cells, macrophages, and T lymphocytes. Because the DP rat has few lymphocytes of the CD5+/CD+ phenotype, cytotoxic T lymphocytes (Tc) are not believed to be important in the process. Diabetes-resistant (DR) BB rats that are depleted of RT6+ T lymphocytes also become diabetic and provide an additional model of IDDM. We report that diabetes in DR rats depleted of RT6+ T lymphocytes is prevented by the concomitant depletion of either the CD5+ or the CD8+ population. In contrast, coadministration of anti-asialogangliosideM1 (alpha-ASGM1), an antiserum that principally recognizes NK cells, failed to prevent hyperglycemia in RT6-depleted rats. We propose that the initiation of diabetes in both DP and RT6-depleted DR rats is T-lymphocyte dependent. However, the final common pathway leading to autoimmune beta-cell destruction in IDDM may be different in these models. The RT6-depleted DR rat requires a cell that is sensitive to anti-CD8 (possibly a Tc), whereas the DP rat requires an anti-ASGM1-sensitive cell. PMID- 1707019 TI - Function follows form: generation of intracellular signals by cell deformation. AB - Cells are exposed during their lifetimes to an array of physical forces ranging from those generated by association with other cells and extracellular matrices to the constant forces placed on cells by gravity. Alterations in these forces, either with differentiation and development or changes in activity or behavior, result in modifications in the biochemistry and adaptation in structure and function of cells. Also, a variety of differentiated cells have unique shapes that relate to extremely specialized functions, with structure and function emerging concurrently. These observations lead to the concept that the forces perceived by cells may dictate their shape, and the combined effects of external physical stimuli and internal forces responsible for maintaining cell shape may stimulate alterations in cellular biochemistry. This review examines the state of our knowledge concerning the mechanisms through which physical forces are converted to biochemical signals (mechanotransduction), and speculates on the molecular structures that may be involved in mechanotransduction. PMID- 1707020 TI - Single ion channel's view of classical receptor theory. AB - The nature of drug agonism has been the central mystery of two conceptually different approaches: classical receptor theory, which does not require any knowledge of mechanism, and mechanistic theories, which do. Ligand-activated ion channel macromolecules that contain both the agonist receptor site and molecular machinery to generate a response present a unique experimental system with which to explore agonism and antagonism. Electrical recordings from one channel at a time offer phenomena and a perspective quite different from that usually encountered in studies of the drug-receptor interaction. This review describes patch-clamp recordings that illustrate the ligand-evoked behavior that gives rise to classical phenomena. A comparison of the channel currents recorded in the presence of different agonists reveals how these drugs act as full or partial agonists. At higher concentrations of agonist, the conformational and kinetic transitions that underlie desensitization can be observed. Receptor conformational changes induced by agonist and antagonist binding further expand our ideas about what these drugs do, and contribute to the growth of concepts that will further our understanding of drug agonism. PMID- 1707021 TI - Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis. AB - Hepatocytes are stimulated to produce nitric oxide (NO.) from L-arginine in response to conditioned Kupffer cell medium or a combination of cytokines. Associated with the production of NO.in hepatocytes, there is a profound decrease in total protein synthesis ([3H]leucine incorporation). This report demonstrates that authentic NO.and the NO.-generating compound S-nitroso-N-acetylpenicillamine inhibit hepatocyte total protein synthesis in a reversible and concentration dependent fashion. In parallel with the suppression of hepatocyte total protein synthesis, authentic NO.inhibits the production of two specific hepatocyte proteins, albumin and fibrinogen, without influencing the quantity of albumin mRNA. Although authentic NO.induces a rapid increase in cGMP levels in hepatocytes, the addition of the cGMP analog 8-bromoguanosine 3':5' cyclic monophosphate to unstimulated HC cultures does not reproduce the inhibition of total protein synthesis. These data show that NO.is the hepatocyte L-arginine metabolite that inhibits protein synthesis. Furthermore, these findings indicate that NO.does not inhibit hepatocyte protein synthesis solely through the activation of soluble guanylate cyclase but appears to affect a translational or posttranslational process. PMID- 1707022 TI - Chemotherapy in recurrent and advanced cervical cancer. AB - Twenty-five patients, ranging from 21 to 61 years of age (median = 45 years), with histologically proven recurrent and advanced cervical cancer were treated with chemotherapy using a combination of bleomycin, ifosfamide, and cis-platinum (BIP). Twenty-one patients were evaluable for response. Ninety percent of patients achieved a subjective response. An objective response was noted in 14 of 21 (66.6%) patients: complete in 4 (19%) and partial in 10 (47.6%). Side effects were mainly nausea/vomiting, alopecia, myelosuppression, reversible encephalopathy, and impaired renal function. One patient died from the toxic effects of chemotherapy. These results indicate that BIP is an active combination in recurrent cervical cancer with acceptable toxicity. PMID- 1707023 TI - Utility of anti-carcinoembryonic antigen monoclonal antibodies for differentiating ovarian adenocarcinomas from gastrointestinal metastasis to the ovary. AB - The distinction between primary ovarian tumors and metastatic cancers to the ovary is frequently ambiguous. Recently, we reported that the D-14 monoclonal antibody (MAb), which is directed against a specific epitope of carcinoembryonic antigen (CEA), always reacts with colorectal adenocarcinomas and only rarely with neoplasms of non-gastrointestinal origin [J. Cancer Res. Clin. Oncol. 116, 51-56 (1990)]. We report here an analysis of the reactivity of four different anti-CEA MAbs with formalin-fixed tissue sections of human primary and metastatic colorectal and ovarian carcinomas. The four monoclonal antibodies employed were D 14, CEJ065, ZCEA1, and SP-625. D-14, CEJ065, and SP-625 MAbs reacted with essentially every colorectal adenocarcinoma. In contrast, ZCEA1 was the least reactive and 10 tumor samples showed no reactivity to this MAb. All four anti-CEA MAbs demonstrated scarce immunoreactivity with ovarian carcinomas and appear to be useful for distinguishing between ovarian carcinoma and colorectal metastasis to the ovary. Adenocarcinomas of the stomach and breast were also examined to determine CEA reactivity with the D-14 MAb, since these tumors need to be considered in the differential diagnosis of an ovarian mass. The majority of stomach adenocarcinomas were immunoreactive. In contrast, only 3 of 36 breast carcinomas were weakly immunoreactive, indicating that D-14 MAb is of no assistance in identifying breast carcinoma metastasis to the ovary. PMID- 1707025 TI - Expression of ras oncogene product and EGF receptor in cervical squamous cell carcinomas and its relationship to lymph node involvement. AB - The expression of ras oncogene product p21 and epidermal growth factor (EGF) receptor was studied immunohistochemically in tissues obtained from 52 patients with squamous cell carcinoma of the uterine cervix. We examined the relationship between p21 and EGF receptor expression and lymph node metastasis in cervical cancer. The data demonstrate that the patients with positive staining for ras p21 in cervical carcinomas have a higher incidence of lymph node metastasis than the patients with negative staining for p21 (P = 0.027). Although the levels of p21 expression in the metastatic sites were reduced compared to those in the primary sites, tumor cells in metastatic lymph nodes also expressed p21. No relationship was found between EGF receptor expression and lymph node metastasis. These results suggest that expression of ras oncogene product may be associated with the biological aggressiveness of cervical carcinomas. PMID- 1707024 TI - A phase II evaluation of cisplatin, bleomycin, and mitomycin-C in patients with recurrent squamous cell carcinoma of the cervix. AB - Cisplatin, bleomycin, and mitomycin-C were used to treat 25 patients with recurrent squamous cell carcinoma of the cervix. Six patients had a partial response, yielding a total response rate of 27%. Nine patients had stable disease. The median survival for the whole group was 30 weeks. The median survival for responders was 32 weeks. The median progression free interval for the whole group was 12 weeks and the median progression-free free interval for responders was 14 weeks. The toxicities noted were primarily nausea, vomiting, and myelosuppression. The combination of cisplatin, bleomycin, and mitomycin-C has modest effectiveness in the treatment of recurrent squamous cell carcinoma of the cervix, but represents no improvement over single-agent chemotherapy. PMID- 1707026 TI - [Pharmacotherapy of voice and articulation disorders in aphasia]. AB - The phonation and articulation disorders in a group of aphasics with aphonia/dysarthrophonia could be considerably reduced by the use of medication. The group consisted of 10 apoplectic patients whose resulting aphasia could not be classified because of the vocal impairment. Extrapyramidal motion disorders were proved by laryngoscopy and stroboscopy. A definite improvement of phonation and articulation was observed after L-dopa medication. PMID- 1707027 TI - Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus. AB - The hepatitis B virus genome encodes a transcriptional transactivator protein designated HBxAg. We have investigated whether this antigen is a target structure for human T-lymphocytes. Using recombinant HBxAg protein, we found HBxAg-specific stimulation of peripheral blood mononuclear cells in patients with acute hepatitis B virus infection (6 of 6) and chronic hepatitis B virus infection (6 of 17) but not in healthy individuals. With HBxAg-specific synthetic polypeptides, several T-cell epitopes were identified. Most were located in the carboxyterminal half of the HBxAg protein. Five T-cell clones specific for a T cell epitope located at the carboxyterminal region of HBxAg were established and found to belong to the CD2/CD4-positive, CD8-negative subtype. These data establish for the first time HBxAg as an antigen in the cellular immune response. PMID- 1707028 TI - Expression of insulin-like growth factor II, alpha-fetoprotein and hepatitis B virus transcripts in human primary liver cancer. AB - Insulin-like growth factor II is a fetal growth factor structurally and functionally related to insulin and insulin-like growth factor I. Its mRNA expression is developmentally regulated in human liver, the reexpression of insulin-like growth factor II fetal transcripts being often observed in primary liver cancer. Insulin-like growth factor II and alpha-fetoprotein mRNAs were studied in 16 human primary liver cancers, most of which were highly differentiated. Hepatitis B virus transcripts were also analyzed in the tumors from hepatitis B virus chronic carriers. alpha-Fetoprotein mRNA was detected in only four tumors and in one nontumorous cirrhotic tissue; all these samples also displayed insulin-like growth factor II fetal transcripts. Furthermore, fetal insulin-like growth factor II mRNAs were observed in five tumors and six nontumorous cirrhotic areas not expressing alpha-fetoprotein mRNA. The presence of hepatitis B virus RNA was only observed in tissues not expressing alpha fetoprotein or fetal insulin-like growth factor II mRNA. In conclusion, fetal insulin-like growth factor II transcripts are more frequently observed than alpha fetoprotein mRNA in highly differentiated liver cancers and in surrounding cirrhotic areas. The reexpression of fetal insulin-like growth factor II transcripts might then be a marker of early steps of liver cell transformation. PMID- 1707029 TI - Bellini duct carcinoma: further evidence for this rare variant of renal cell carcinoma. AB - Bellini duct carcinomas have recently been identified as a new entity in the spectrum of renal cell carcinomas and 10 cases have now been reported. The present paper adds detailed clinical and morphological data on six new cases. In addition, immunohistological and electronmicroscopical results support the origin of these tumours from the renal collecting ducts, especially the papillary ducts (Bellini ducts). A set of immunohistological reactions, including reactions to cytokeratins 13 and 19, vimentin and UEA-1 was found to facilitate the differential diagnosis of Bellini duct carcinomas from other renal cell carcinomas and infiltrating urothelial carcinomas of renal pelvis. PMID- 1707030 TI - Human aminoacylase-1: cloning, regional assignment to distal chromosome 3p21.1, and identification of a cross-hybridizing sequence on chromosome 18. AB - Aminoacylase-1 (ACY1, EC 3.5.1.14) is a cytosolic enzyme with a wide range of tissue expression and has been postulated to function in the catabolism and salvage of acylated amino acids. ACY1 has been assigned to chromosome 3p21, a region reduced to homozygosity in small-cell lung cancer and renal cell carcinoma, and has been reported to exhibit reduced or absent expression in small cell lung cancer cell lines and tumors. Using monoclonal antibodies to human ACY1, we have isolated cDNA clones from a liver lambda gt11 cDNA library. As proof of identity, the fusion protein encoded by a putative ACY1 cDNA displayed ACY1 enzymatic activity. Additionally, it was determined that the putative ACY1 cDNA clones hybridize to an EcoR1 restriction fragment that has been mapped to chromosome 3p. Both ACY1 activity and this restriction fragment have been further demonstrated to be syntenic to distal 3p21.1 through the use of a panel of human rodent somatic cell hybrids containing fragments of chromosome 3. An additional EcoR1 restriction fragment to which the probe hybridizes has been assigned to chromosome 18. The major mRNA species to which the ACY1 cDNA hybridizes is 0.9 kb; faint hybridization to a 4.2-kb mRNA species is also detected. These studies further refine a region of interest in the investigation of gene inactivation in small-cell lung cancer and provide a new marker on chromosome 18. PMID- 1707031 TI - Cloning of human hepatic nuclear factor 1 (HNF1) and chromosomal localization of its gene in man and mouse. AB - HNF1 is a transcription factor that is required for hepatocyte-specific expression of several genes, including albumin and fibrinogen. Rat HNF1-encoding cDNAs have recently been cloned, revealing that this factor is a distant member of the homeoprotein family. We have now isolated HNF1 clones from a human liver cDNA library by using a rat HNF1 cDNA-derived probe. The longest clone, HCL20, contains a sequence corresponding to the intact rat HNF1-coding region followed by a 3' nontranslated region and a poly(A) tail, hence representing an almost full-length HNF1 cDNA. Alignment of the human and rat sequences shows that HNF1 is highly conserved between the two species. The HNF1 gene was mapped by in situ hybridization and by RFLP analysis of interspecific mouse backcrosses to chromosomes 12q24.3 and 5F in human and mouse, respectively, establishing a new segmental homology between these two chromosomes. PMID- 1707032 TI - Single amino acid substitutions determine mouse CD8 allotypes: epitope mapping of mouse CD8. PMID- 1707033 TI - Comparative analysis of the expression of the extracellular matrix protein tenascin in normal human fetal, adult and tumor tissues. AB - Tenascin (TN) is a high-molecular-mass oligomeric glycoprotein of the extracellular matrix (ECM) endowed with a programmed expression in embryonic life and a neo-expression in the interstitium of some malignancies. Using monoclonal antibodies (MAbs) which identify human tenascin, we have conducted an extensive immunohistochemical analysis of TN expression in normal fetal and adult human tissues as well as in a wide variety of human tumors. Results of this study demonstrate that TN (1) is detectable in embryonic and fetal tissues at least from the 10th week of gestation; (2) is present in the interstitium of a variety of adult tissues of different embryonic origin; (3) may be neo-expressed in the stroma of benign and malignant tumors; (4) has the ability to accumulate in a highly variable manner in the ECM of tumors of the same and of different histotypes. PMID- 1707034 TI - Hodgkin's disease in adults in Saudi Arabia. Clinical features, prognostic factors and an analysis of therapy. Outcome of combination chemotherapy only, for both localized and advanced disease. AB - Fifty evaluable, previously untreated, adult patients with clinically staged (CS) early and advanced Hodgkin's disease were treated with chemotherapy alone, using various regimens. Their mean age was 31.9 years. Fifteen patients (30%) had CS I or II and 35 (70%) had CS III or IV. Eighty-eight per cent of patients had one or more of the B symptoms and 64% had an unfavorable histology. Complete remission (CR) was achieved in 43 out of 50 patients (86% with 95% confidence interval of 76% to 96%), partial remission in 3 (6%) and treatment failure in 4. Adjusted analysis, using all possible subset regression, showed that unfavorable histology, bulky disease and receiving a total dose-intensity (TDI) less than or equal to 0.80 were negatively associated with the likelihood of achieving CR. At a median follow-up of 36 months (range, 6-90), 84% of patients were alive and 82% were disease-free. The overall median survival has not been reached, but the projected 5-year survival probability was 79%. Time-to-relapse was also estimated for those who achieved initial CR. The estimated 5-year relapse-free survival was 87%. The Cox proportional hazards model predicted that unfavorable histology, bulky disease and TDI less than or equal to 0.80 had an independent, adverse influence on survival. We conclude that the results of chemotherapy alone are encouraging and the rationale is practical and acceptable in those countries where the availability of radiotherapy units is limited. PMID- 1707035 TI - Characterization of a novel monoclonal antibody, 3H-1, reactive with squamoproliferative lesions and squamous-cell cancers. AB - Immunization of mice with membranes from a virus-transformed human keratinocyte cell line (KJD-I/SV40) yielded an IgM monoclonal antibody (MAb 3H-I) which reacted with the membrane and cytoplasm of KJD-I/SV40 cells and in the perinuclear region of a squamous-cell carcinoma line (Colo-16). Immunohistochemistry of formalin-fixed, paraffin-embedded sections using MAb 3H-I gave intense staining of proliferating squamous epithelium in several characteristic patterns. Acanthotic squamous epithelium and well-differentiated squamous-cell carcinomas (SCC) demonstrated a membranous staining pattern whereas psoriatic skin and undifferentiated SCC of the head and neck exhibited diffuse cytoplasmic, focal cytoplasmic or, in some tumours, no staining. Simple squamous epithelium was unreactive. Western blotting revealed an antigen of 55 kDa. The epitope recognized by MAb 3H-I may be a marker for particular stages of squamous proliferation and differentiation. PMID- 1707036 TI - The effect of fetal calf serum on growth arrest caused by activators of protein kinase C. AB - The growth of human-derived A549 lung carcinoma cells is inhibited by activators of protein kinase C (PKC) such as 12-O-tetradecanoylphorbol- 13-acetate (TPA). In this study, the effect of serum deprivation on TPA-induced growth retardation has been investigated. Cells cultured with 10% FCS and TPA (10(-8) M) stopped growing for 6 days, whereas inhibition of DNA synthesis caused by TPA in cells which were grown in medium containing the serum substitute ultraser lasted for less than 48 hr. The ability of cells to respond to the growth-inhibitory potential of TPA decreased with decreasing amounts of FCS in the cellular medium. Addition of fetuin or epidermal growth factor (EGF) to incubates with serum-deprived cells increased the ability of TPA to affect growth, but addition of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) or retinoic acid (RA) was without effect. Growth arrest caused by bryostatin I, another PKC activator, was equally transitory in serum-supplemented and serum-deprived cells. Cytosol of serum-deprived cells contained only 32% of specific phorbol ester binding sites compared to cells grown with FCS; PKC enzyme activity and immunodectable protein were similarly reduced in cells grown without FCS. There was no difference in rate of TPA-induced down-regulation of PKC activity and cytosolic phorbol ester receptor sites between cells grown with or without serum. PMID- 1707037 TI - Time course of pressure and flow in ascending aorta during ejection. AB - To analyze aortic flow and pressure relationships, 10 closed-chest anaesthetised dogs were instrumented with electromagnetic aortic flow probes and micromanometers in the left ventricle and ascending aorta. Left ventricular ejection time was divided into: time to peak flow (T1) (both pressure and flow rising), peak flow to peak pressure time (T2) (pressure rising, flow decreasing), and peak pressure to dicrotic notch time (T3) (pressure and flow both decreasing). These time intervals were expressed as percent of total ejection time. Load-active interventions rose markedly T2 (from 4.2 +/- 5.5 to 19.4 +/- 3.5 after phenylephrine (p less than 0.02); from 4.2 +/- 6.5 to 21.2 +/- 5.3 after dextran (p less than 0.02)). Conversely, dobutamine reduced T2 from 4.4 +/- 5.9 to -2.5 +/- 6.5 (p less than 0.05). Thus, during load-active interventions aortic pressure increases for a longer T2 time although forward flow is decreasing, as a result of higher aortic elastic recoil during ejection. Conversely, beta 1-adrenergic stimulation significantly shortens T2. Dynamic pressure-flow relationship is thus continuously changing during ejection. T2 seems to be inversely related to the efficiency of left ventricular ejection dynamics. PMID- 1707038 TI - Chorioretinal degenerative changes in the tilted disc syndrome. AB - Ninety patients (163 eyes) with tilted disc syndrome were examined in order to show possible chorioretinal degenerative lesions associated with the typical ectasia of the infero-nasal fundus observed in this anomaly. Eighteen out of the 163 eyes had pigmentary accumulations, either branched, linear or dotted and five had roundish areas of chorioretinal atrophy. Furthermore, in 7 eyes areas of pigmentary atrophy at the posterior pole were observed. The most serious lesions were represented by macular choroidal neovascular membranes, seen in 3 eyes, which were responsible for the loss of vision in these eyes. A relationship was found between the depth of the ectasia, the degree of tilt of the optic disc and the development of chorioretinal degeneration. Because of possible complication by macular choroidal neovascularization, the tilted disc syndrome can not be regarded in every case as a benign and not evolutive ocular anomaly. PMID- 1707040 TI - Effect of experimental autoimmune encephalomyelitis on pregnancy: studies in rabbits and rats. AB - The influence of experimental autoimmune encephalomyelitis (EAE) on the course and outcome of pregnancy, and the effect of pregnancy on EAE development, was investigated in rabbits and rats. Animals were immunized with encephalitogenic antigen in complete Freund's adjuvant (CFA) either before or during pregnancy. Abortion or fetal resorption was observed in most of the rabbits immunized before or during pregnancy, but not in pregnant rabbits injected with CFA or saline alone. Fetal loss was higher in those rabbits that developed clinical EAE. In rats, fetal loss occurred only when immunization was carried out during the first half of pregnancy. The appearance of EAE in pregnant rabbits, but not in rats, was delayed until after abortion or termination of pregnancy. The incidence of EAE in rabbits was lower, with milder severity and longer duration. Serum antibody levels to myelin basic protein, the autoantigen of EAE, was lower in pregnant rabbits, but not in rats, as compared to non pregnant animals. These results indicate that in species where pregnancy has a suppressive influence on the development of experimental autoimmune demyelinating disease, immunization with the neuroantigen induces a high rate of fetal loss. PMID- 1707039 TI - Cerebrospinal fluid markers in neurological disorders. AB - Cerebrospinal fluid (CSF) markers are a useful tool for determining disease progression or activity in some neurological disorders which need parameters both for evaluating treatments and investigating pathobiological evolution in research oriented follow-up. A number of CSF proteins are reviewed with data on biological properties, analytical methods, clinical usefulness of: myelin basic protein, S 100 protein, glial fibrillary acidic protein, neural-cell adhesion molecule, neuron-specific enolase and others. PMID- 1707041 TI - [Vitronectin in normal human skin and in skin lesions]. AB - Vitronectin (S-protein of complement, serum spreading factor) is a multifunctional glycoprotein present in human plasma and in the elastic tissue of various organs. It belongs to the group of adhesion proteins and is of importance in the terminal stages of both the coagulation and complement system and in fibrinolysis. In human skin it is localized on dermal elastic fibers and on pathologically altered elastic material (solar elastosis, pseudoxanthoma elasticum) as well as on keratin filament material such as keratin bodies in lichen planus or amyloid deposits in localized cutaneous amyloid. It is also found in the abnormally thickened cutaneous blood vessels in erythropoietic protoporphyria and porphyria cutanea tarda. Late-stage inhibition of the complement cascade in bullous disorders in which activation of the complement system is of pathogenetic significance may be an additional important function of vitronectin in skin diseases. PMID- 1707042 TI - [Condylomata acuminata--topical and systemic interferon therapy]. AB - An open study was carried out to test the effect of systemic administration of interferon (IFN) gamma and local application of IFN beta as monotherapy and adjuvant treatment. The topical application of IFN beta gel had no effect as monotherapy and when it was given as adjuvant therapy the rate of recurrence was not significantly reduced. IFN gamma was given for monotherapy in two different doses (100 and 200 micrograms per s.c. injection). The response rate to the cyclic treatment was 45% in the group (20 patients) receiving a dosage of 100 micrograms, and 57% in the group (26 patients) receiving a dosage of 200 micrograms. Patients with a duration of the disease longer than 18 months and patients with immune deficiency did not respond to the monotherapy. A group of 15 patients with resistant genital warts received adjuvant treatment with IFN gamma over 7 days after surgical treatment. In patients with inconspicuous immune status it was possible to reduce the recurrence rate. PMID- 1707043 TI - Tissue distribution of 99mTc, 111In and 123I-OV-TL 3 Fab' in ovarian carcinoma bearing nude mice. PMID- 1707044 TI - Contrasting of Lowicryl K4M thin sections. AB - A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections. PMID- 1707045 TI - A role for hyaluronan in joint development. AB - Hyaluronan, a common connective tissue component, influences cell adhesion, migration and cytodifferentiation in vitro, and because of these properties has been postulated to have a role in morphogenesis. Its role in the development of cartilage-associated structures, such as joints, has yet to be defined. Using a biotinylated hyaluronan-binding region-link protein complex, free hyaluronan binding sites have been localised in the joint region concomitant with the first signs of cavitation (Stage 37), whereafter it is localised in the joint space and is maintained here as this enlarges. The application of our results is discussed in the context of a primary role for hyaluronan in joint cavity formation. PMID- 1707046 TI - The role of trophoblastic binucleate cells in implantation in the goat: a morphological study. AB - In the goat conceptus individual intra-epithelial trophectodermal binucleate cells first appear 18 days post coitum and their incidence rapidly increases where the trophectoderm is apposed to the caruncular and intercaruncular sites of initial attachment to the uterine epithelium. Special staining techniques reveal that these cells, when mature, contain prominent Golgi bodies and numerous characteristic granules. Our evidence shows that at 19 days post coitum the binucleate cells migrate to the microvillar junction and fuse with individual uterine epithelial cells to form hybrid feto-maternal trinucleate cells. It is proposed that subsequent continued binucleate cell migration and fusion with trinucleate cells produce the syncytial plaques typical of the remainder of pregnancy. It is further suggested that the fusion is important in facilitating the delivery of the characteristic granules to the base of the uterine epithelial layer with subsequent exocytosis of their contents into maternal tissue. PMID- 1707047 TI - The antituberculous effect of bleomycin. PMID- 1707048 TI - Potent vasoactive properties of endothelin 1 in human skin. AB - The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance). The coadministration of endothelin 1 (1-100 pmol) with the neuropeptide vasodilator calcitonin gene-related peptide (CGRP) inhibited the vasodilator response to CGRP (10 pmol) by up to 82.7 +/- 9.2% (with 100 pmol endothelin, P less than 0.001). The response of the prostanoid vasodilator prostaglandin E2 (10 pmol) was inhibited by endothelin in a similar manner. In addition to the vasoconstrictor effects, endothelin 1 produced a dose-dependent flare that surrounded the area of pallor, and this was associated with a significant increase in blood flow (P less than 0.05) within the flare area. The H1 antagonist terfenadine (120 mg po) significantly reduced the flare area associated with endothelin 1: flare 5 min after intradermal endothelin (10 pmol, placebo treated), 668 +/- 405 mm2; terfenadine treated, 201 +/- 257 mm2 (P less than 0.05). The flare was also significantly attenuated when endothelin (10 pmol) was injected into local anesthetic-treated skin. Thus intradermal injection of endothelin in humans causes long-lasting vasoconstriction at the site of injection and a surrounding flare. Results suggest that the flare component is partially histamine dependent and the result of an axon reflex. This study demonstrates the potent activity of endothelin in human skin. It is possible that endothelin could be relevant to the local response of skin to injury. PMID- 1707049 TI - Pathways of substance P stimulation of canine tracheal ciliary beat frequency. AB - Substance P (SP), an inflammatory neuropeptide, may be released by intraepithelial nerves in response to an irritant or inflammatory stimulus. To investigate the neural and humoral pathways mediating the response of tracheal ciliary beat frequency (CBF) to topically applied SP, CBF was measured on the ventral midtracheal surface of anesthetized beagles by using heterodyne-mode correlation analysis laser light scattering. In the first study, aerosolized SP, delivered to the lungs of eight beagle dogs, stimulated CBF in a dose-dependent manner from a baseline of 4.9 +/- 0.4 Hz to a maximum of 14.9 +/- 1.5 Hz at dose of 10(-7) M. In the second study, the tracheal lumen was isolated from the bronchial airways by inflating the cuff of an endotracheal tube near the carina. Intravenous hexamethonium bromide (2 mg/kg), ipratropium bromide (0.5 micrograms/kg), and indomethacin (2 mg/kg) were used as blocking agents to inhibit the nicotinic, muscarinic, and cyclooxygenase pathways, respectively. Aerosolized 10(-9), 10(-8), or 10(-7) M SP was delivered sequentially to the tracheal lumen for 3 min at 30-min intervals. SP caused two distinct CBF stimulatory episodes at 4 min (mean time of the maximal response) and at 18 min (mean time of the maximal response) after onset of delivery and returned to baseline after 25 min. SP stimulated CBF from the baseline of 5.1 +/- 0.4 Hz to a maximum of 14.2 +/- 2.5 Hz during the first episode (P less than 0.01) and to 10.4 +/- 0.6 Hz during the second episode (P less than 0.01) at dose of 10(-8) M. These responses were inhibited by all the blocking agents. These data suggest that SP stimulates CBF via a cyclooxygenase-dependent parasympathetic reflex. PMID- 1707050 TI - Leakage of macromolecules in ventilated and unventilated segments of preterm lamb lungs. AB - The movement of macromolecules into and out of unventilated lung segments was evaluated in prematurely delivered and ventilated lambs. Seven lambs at 130 days gestational age had a bronchial balloon placed at birth before the first breath to obstruct the left lower lobe. Surfactant and 131I-albumin were instilled into the left lower lobe while surfactant and 125I-albumin were instilled into the remaining lung, and 70,000 molecular weight [3H]dextran was given into the vascular space at birth. Twenty-five percent of the lung by weight was not ventilated, and 24% of the total leak of dextran from the vascular space was recovered in the unventilated lungs at 3 h. An epithelial leak of protein from the two lung regions was documented by the loss of 11.4 and 18.4% of the labeled albumins in the nonventilated and ventilated lung regions, the appearance of 4.9 and 7.5% of the airway-instilled albumin in the vascular space from the nonventilated and ventilated lung regions, and the recovery of the labeled albumins in the carcasses of the lambs. The bidirectional flux of macromolecules was larger in the ventilated than in the nonventilated lung regions, indicating that ventilation can increase the leak of protein in the preterm lung. The lung areas that were never exposed to ventilation or oxygen also demonstrated a large bidirectional flux of macromolecules, a finding not present in the fetus, fullterm newborn, or adult. These findings indicate that ventilation is not solely responsible for the increased protein leak found in preterm lungs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707051 TI - Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse. AB - Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver. PMID- 1707052 TI - Cloning and sequence analysis of cDNA for Trimeresurus flavoviridis phospholipase A2, and consequent revision of the amino acid sequence. AB - A cDNA clone of Trimeresurus flavoviridis phospholipase A2 was isolated from the venom gland cDNA library using DNA amplified by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminal sequences of this enzyme. The amino acid sequence deduced from the cDNA nucleotide sequence determined by the dideoxy termination method was found to be inconsistent in the segment comprising the 69th to 81st positions from that reported previously [Tanaka, S. et al. (1986) J. Biochem. 99, 281-289]. Reinvestigation of the amino acid sequence of the peptide covering the sequence in question showed that the amino acid sequence predicted from the nucleotide sequence is correct. The sequence contains Asn-Asn-Gly (positions 69-71) and it was found that the Asn-Gly bond easily undergoes alpha-beta transpeptidation when digested with Achromobacter protease I at pH 9.0 but not seriously at pH 6.8. It is likely that the transpeptidation reaction caused a failure in the previous sequence determination. The cDNA clone obtained was 597 base pairs long and contained an open reading frame of 414 base pairs coding for 138 amino acid residues. A typical signal peptide sequence (16 amino acid residues long), located at the N terminal moiety of the deduced sequence, was immediately followed by a polypeptide which corresponds to the mature enzyme. Northern blot analysis showed a single transcript only in the poly(A)+ RNA fraction of the venom gland but not in those of many other organs tested. PMID- 1707053 TI - Effects of aprotinin on prostaglandin metabolism and platelet function in open heart surgery. AB - The effects of the protease inhibitor, aprotinin, on plasma prostaglandin levels and platelet function during and after cardiopulmonary bypass (CPB) were studied in a group of 23 patients which consisted of 11 untreated patients (control group) and 12 aprotinin-treated patients (aprotinin group). Thromboxane B2 (TXB2, a stable metabolite of thromboxane A2) and beta-thromboglobulin levels in the control group increased significantly during CPB compared with preoperative values. These increases were significantly suppressed in the aprotinin group. 6 Keto-PGF1 alpha (stable metabolite of prostacyclin) increased significantly during CPB in both groups, and there was no significant difference between the two groups. In the aprotinin group, the TXB2/6-Keto-PGF1 alpha ratio decreased significantly during CPB compared with the preoperative value, whereas no significant decrease was observed in the control group. Platelet counts decreased significantly during and after CPB in both groups. Platelet aggregability decreased significantly during CPB in the control group, whereas no significant decrease was found in the aprotinin group. In conclusion, aprotinin treatment improved prostaglandin metabolism and preserved platelet function during open heart surgery. PMID- 1707054 TI - Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development. AB - Myosin isoforms contribute to the heterogeneity and adaptability of skeletal muscle fibers. Besides the well-characterized slow and fast muscle myosins, there are those isoforms that appear transiently during the course of muscle development. At a stage of development when two different myosins are coexpressed, the possibility arises for the existence of heterodimers, molecules containing two different heavy chains, or homodimers, molecules with two identical heavy chains. The question of whether neonatal and adult myosin isoforms can associate to form a stable heterodimer was addressed by using stage specific monoclonal antibodies in conjunction with immunological and electron microscopic techniques. We find that independent of the ratio of adult to neonatal myosin, depending on the age of the animal, the myosin heavy chains form predominantly homodimeric molecules. The small amount of hybrid species present suggests that either the rod portion of the two heavy chain isoforms differs too much in sequence to form a stable alpha-helical coiled coil, or that the biosynthesis of the heavy chains precludes the formation of heterodimeric molecules. PMID- 1707055 TI - Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis. AB - Radixin is a barbed end-capping actin-modulating protein which was first identified in isolated cell-to-cell adherens junctions from rat liver (Tsukita, Sa., Y. Hieda, and Sh. Tsukita, 1989. J. Cell Biol. 108:2369-2382). In the present study, we have analyzed the distribution of radixin in dividing cells. For this purpose, an mAb specific for radixin was obtained using chicken gizzard radixin as an antigen. By immunofluorescence microscopy with this mAb and a polyclonal antibody obtained previously, it was clearly shown in rat fibroblastic cells (3Y1 cells) that radixin was highly concentrated at the cleavage furrow during cytokinesis. Radixin appeared to accumulate rapidly at the cleavage furrow at the onset of furrowing, continued to be concentrated at the furrow during anaphase and telophase, and was finally enriched at the midbody. This concentration of radixin at the cleavage furrow was detected in all other cultured cells we examined: bovine epithelial cells (MDBK cells), mouse myeloma cells (P3 cells), rat kangaroo Ptk2 cells, mouse teratocarcinoma cells, and chicken fibroblasts. Furthermore, it became clear that the epitope for the mAb was immunofluorescently masked in the cell-to-cell adherens junctions. Together, these results lead us to conclude that radixin is present in the undercoat of the cell-to-cell adherens junctions and that of the cleavage furrow, although their respective molecular architectures are distinct. The possible roles of radixin at the cleavage furrow are discussed with special reference to the molecular mechanism of the actin filament-plasma membrane interaction at the furrow. PMID- 1707056 TI - The ectopic expression of myelin basic protein isoforms in Shiverer oligodendrocytes: implications for myelinogenesis. AB - The myelin basic proteins (MBPs) are a set of peripheral membrane polypeptides that are required for the compaction of the major dense line of central nervous system myelin. We have used primary cultures of oligodendrocytes from MBP deficient shiverer mice as host cells for the expression by cDNA transfection of each of the four major MBP isoforms. The distributions of the encoded polypeptides were studied by immunofluorescence and confocal microscopy and compared with patterns of MBP expression in normal mouse oligodendrocytes in situ and in culture. The exon II-containing 21.5- or 17-kD MBPs were distributed diffusely in the cytoplasm and in the nucleus of the transfectants, closely resembling the patterns obtained in myelinating oligodendrocytes in 9-d-old normal mouse brains. By contrast, the distribution of the 14- and 18.5-kD MBPs in the transfectants was confined to the plasma membrane and mimicked the distribution of MBP in cultures of normal adult oligodendrocytes. Our results strongly suggest that the exon II-containing MBPs are expressed first and exclusively during oligodendrocyte maturation, where they may play a role in the early phase of implementation of the myelination program. In contrast, the 14- and 18.5-kD MBPs that possess strong affinity for the plasma membrane are likely to be the principle inducers of myelin compaction at the major dense line. PMID- 1707058 TI - Separation of serum proteins by high-performance gel-permeation column systems. AB - Combined TSK-Gel G4000SW-G3000SW and Zorbax GF450-GF250 columns and a Superose 6 column were evaluated for the analytical gel permeation chromatographic separation of serum proteins. Serum elution profiles showed four distinct peaks, which were attributed to immunoglobulin M (IgM), alpha 2-macroglobulin, IgG and albumin. A reproducible shoulder on the IgG peak could be attributed to IgA. These purified serum proteins and other commercially obtained proteins with relative molecular masses between 1000 kDa and 10 kDa were chromatographed on the combined systems and the separate columns. The frictional coefficient-based hydrodynamic radii of these proteins showed a linear relationship with the inverse error function complement of their partition coefficients. Using this relationship, theoretical summation plots of the data obtained from the separate columns of a system correspond to plots that were calculated by treating the combined systems as one column. The best resolution was obtained with the TSK colums, but all three column systems were suitable for the separation of clinically important immunoglobulins in serum. The retention of positively charged proteins by the TSK columns becomes noticeable after relatively short usage and is a first sign of progressive loss of resolution. PMID- 1707057 TI - Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells. AB - Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707059 TI - Efficient method of haemoglobin F enrichment in adult haemolysate for determination of the gamma-chain isoforms by high-performance liquid chromatography. AB - An alkali denaturation procedure was developed for the efficient enrichment of haemoglobin F (Hb F) in human adult haemolysate. In contrast to the previous procedures, the method is readily applicable to small amounts of blood containing less than 1% of Hb F, providing reproducibly a sufficient level of Hb F (at least 11%) to allow an accurate high-performance liquid chromatographic determination of the three gamma-chain isoforms. Further enrichment was possible by combination with CM-Sephadex chromatography. PMID- 1707060 TI - Endogenous insulin-like growth factor (IGF) binding proteins cause IGF-1 resistance in cultured fibroblasts from a patient with short stature. AB - The ED50 of insulin-like growth factor (IGF)-I-stimulated alpha-aminoisobutyric acid (AIB) uptake (mean +/- SD) in cultured fibroblasts from a child with short stature that we have reported (1.40 +/- 0.24 nM), is significantly higher than the ED50 of IGF-I-stimulated AIB uptake in fibroblasts from 11 normal subjects (0.42 +/- 0.12 nM) and from 127 short children (0.35 +/- 0.11 nM). Similarly, the ED50 of IGF-I-stimulated thymidine incorporation in fibroblasts from this child is 2.8 times higher than that in fibroblasts from four normal subjects. To minimize potential modulation of IGF-I action by endogenous IGF binding proteins in these assays, fibroblast responsiveness to [Q3,A4,Y15,L16]IGF-I, an IGF-I variant that has a 600-fold reduced affinity for serum IGF binding proteins, has been examined. The biological activity of this variant is comparable in the patient's and normal fibroblasts, suggesting that the resistance to IGF-I action cannot be attributed to a defective IGF-I receptor. To investigate directly the possibility that IGF-I sensitivity in the patient's fibroblasts is reduced by endogenous IGF binding proteins (IGFBP), binding proteins that are secreted into AIB assay buffer during a 3-h collection and that are cell-associated at the end of the collection have been analyzed. Ligand blot analysis of conditioned AIB assay buffer demonstrates that fibroblasts from the patient secrete 1.3-2.2 times more of Mr 46,400/42,900, 32,000, and 26,800 binding proteins than normal fibroblasts. The major difference between fibroblasts from the patient and from normal subjects is a striking 10-fold increase in the amount of a cell surface Mr 32,000 binding protein in the patient's fibroblasts. The Mr 32,000 binding protein is similar in size to IGFB-1 and different from IGFBP-2 and IGFBP-3, but it does not cross-react with an antibody against IGFBP-1. We conclude that the resistance to IGF-I action in the patient's fibroblasts is caused by an abnormal production and/or cell association of IGF binding proteins. PMID- 1707061 TI - Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus. AB - We have studied the effects of oral administration of vanadate, an insulinometic agent and a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) in vitro, on blood glucose and PTPase action, in two hyperinsulinemic rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Oral administration of vanadate (0.25 mg/ml in the drinking water) to ob/ob mice for 3 wk lowered blood glucose level from 236 +/- 4 to 143 +/- 2 mg/dl without effect on body weight. Administration of vanadate to db/db mice produced a similar effect. Electron microscopic examination revealed no signs of hepatotoxicity after 47 d of treatment. There was a slight reduction in insulin receptor autophosphorylation when tested by immunoblotting with antiphosphotyrosine antibody after in vivo stimulation, and the phosphorylation of the endogenous substrate of the insulin receptor, pp185, was markedly decreased in the ob/ob mice. Both cytosolic and particulate PTPase activities in liver of ob/ob mice measured by dephosphorylation of a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation were decreased by approximately 50% (P less than 0.01). In db/db diabetic mice, PTPase activity in the cytosolic fraction was decreased to 53% of control values (P less than 0.02) with no significant difference in the particulate PTPase activity. Treatment with vanadate did not alter hepatic PTPase activity as assayed in vitro, or receptor and substrate phosphorylation as assayed in vivo, in ob/ob mice despite its substantial effect on blood glucose. These data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase. PMID- 1707062 TI - Immunohistochemical demonstration of acetaldehyde-modified epitopes in human liver after alcohol consumption. AB - Acetaldehyde, the toxic product of ethanol metabolism in the liver, covalently binds to a variety of proteins. Recent studies indicate that such binding can stimulate the production of antibodies against the acetaldehyde adducts. We raised rabbit antibodies which recognized various protein-acetaldehyde conjugates but not the corresponding control proteins. Such antibodies were used in immunohistochemical studies to find out whether acetaldehyde-generated epitopes can be detected from liver specimens of 13 human subjects with different degrees of alcohol consumption. While the specimens obtained from alcohol abusers (n = 4) and alcoholics (n = 3) exhibited marked positive staining for acetaldehyde adducts inside the hepatocytes in a granular uneven pattern, the control samples (n = 6) were almost devoid of immunoreactivity. In the alcohol abusers with an early stage of alcohol-induced liver damage, staining was detected exclusively around the central veins. The data indicate that intracellular acetaldehyde adducts occur in the centrilobular region of the liver of individuals consuming excessive amounts of alcohol. Immunohistochemical detection of such adducts may prove to be of value in the early identification of alcohol abuse and in elucidating the mechanisms of alcohol-induced organ damage. PMID- 1707063 TI - CD8+ cell anti-HIV activity correlates with the clinical state of the infected individual. AB - The extent of antiviral activity exhibited in vitro by CD8+lymphocytes from individuals infected by HIV-1 correlates significantly with their clinical status. CD8+ lymphocytes from asymptomatic subjects were found to inhibit HIV-1 replication by 90% or greater at effector/target (E/T) ratios ranging from as low as 0.05 to 0.25. CD8+ cells from 17 of 19 (89%) of these subjects suppressed replication at an E/T ratio of 0.10 or less. CD8+ lymphocytes from symptomatic patients (non-AIDS) inhibited HIV-1 replication at E/T ratios ranging from 0.05 to 1.0, and CD8+ cells from 8 of 13 (62%) required ratios greater than 0.10. As a group, patients with AIDS exhibited the lowest degree of anti-HIV activity with their CD8+ lymphocytes. The effective range of E/T ratios from AIDS patients was 0.10-2.0, and 9 of 10 (90%) required E/T ratios greater than 0.25. This anti-HIV activity exhibited by CD8+ cells also correlated significantly with the subject's peripheral blood CD4+ cell count. The relative extent of CD8+ cell anti-HIV-1 activity was not found dependent on variations in the CD4+ target cells and viruses used. These findings suggest that the decreased CD8+ cell antiviral activity is related to progression to disease in HIV-infected individuals. PMID- 1707064 TI - Sex difference in the bed nucleus of the stria terminalis of the human brain. AB - A quantitative analysis of the volume of the darkly staining region of the posteromedial bed nucleus of the stria terminalis was performed on the brains of 26 age-matched male and female human subjects. We suggest the term "darkly staining posteromedial" component of the bed nucleus of the stria terminalis (BNST-dspm) to describe this sexually dimorphic region of the human brain. The volume of the BNST-dspm was 2.47 times greater in males than in females. This region in humans appears to correspond to an area of the bed nucleus of the stria terminalis in laboratory animals that exhibits volumetric and neurochemical sexual dimorphisms, concentrates gonadal steroids, and is anatomically connected to several other sexually dimorphic nuclei. Furthermore, the bed nucleus of the stria terminalis is involved in sexually dimorphic functions, including aggressive behavior, sexual behavior, and gonadotropin secretion, which are also influenced by gonadal steroids. Therefore, it is possible that in human beings as well, gonadal hormones influence the sexual dimorphism in the BNST-dspm and that this morphological difference, in part, underlies sexually dimorphic function. PMID- 1707065 TI - Ventral respiratory group projections to phrenic motoneurons: electron microscopic evidence for monosynaptic connections. AB - The hypothesis that excitatory drive is transmitted monosynaptically from bulbospinal medullary respiratory neurons to spinal respiratory motoneurons was tested by an ultrastructural analysis of the phrenic motoneuronal pool in the rat. Combined anterograde labeling of the principal inspiratory bulbospinal neuron population (ventral respiratory group) and retrograde labeling of the phrenic motoneuron pool demonstrated the presence of labeled synaptic profiles, indicating that at least some bulbospinal inspiratory neurons make monosynaptic contacts with phrenic motoneurons. The synaptic boutons of ventral respiratory group neurons that were labeled in the phrenic nucleus had asymmetrical membrane densities at sites of synaptic contact with labeled phrenic somal or dendritic profiles, supporting the notion that this bulbospinal pathway has excitatory contacts with phrenic motoneurons. The morphological types of labeled boutons included three of the eight previously identified bouton types in the phrenic nucleus (Goshgarian and Rafols: Journal of Neurocytology 13:85-109, 1984), including the "S"-terminal, the "NFs"-terminal, and the "F"-terminal. There was no conclusive evidence of labeled double synapses, indicating that this type of synaptic contact is not common in the intact bulbospinal pathway. PMID- 1707066 TI - Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. AB - Motoneurons of adult frog spinal cord have been retrogradely labeled with the carbocyanine derivative diI. Spinal cords were then dissociated and the labeled motoneurons partially purified by centrifugation over a bovine serum albumin (BSA) density gradient. The resulting cell suspension was plated on a substrate of innervated muscle extracellular matrix (ECM) to which the motoneurons attached and extended processes. Labeled adult motoneurons survived for more than 4 weeks in a defined medium in the absence of added serum or growth factors. These cultures of adult motoneurons provide a favorable preparation for studying molecular factors that influence process outgrowth and synapse formation. PMID- 1707067 TI - GABA-like immunoreactivity in a population of locust intersegmental interneurones and their inputs. AB - Intracellular labelling of locust intersegmental interneurones with lucifer yellow or horseradish peroxidase was carried out in combination with light and electron microscope immunocytochemistry by using an antibody raised against gamma amino butyric acid (GABA). Fifteen percent (four out of 27) of intracellularly stained interneurones showed GABA-like immunoreactivity. This is in agreement with previous physiological observations that 20% of the interneurones in this population make inhibitory output connections in the metathoracic ganglion. GABA like immunoreactivity was also found in processes presynaptic to the interneurones in the mesothoracic ganglion. The presence of such immunoreactive inputs onto the intersegmental interneurones correlates well with physiological evidence that their receptive fields are in part shaped by direct input from GABA ergic spiking local interneurones. PMID- 1707068 TI - Quantitative study of the tectally projecting retinal ganglion cells in the adult frog: I. The size of the contralateral and ipsilateral projections. AB - The proportion of ganglion cells connected to the several central targets of the retinal projection varies in different species. In the frog, the retinotectal projection is clearly the largest branch of the optic pathway and the relative size of the tectally projecting population can be expected to be correspondingly great. However, there have been no studies aimed at quantifying the size of this population and at partitioning its contralateral and ipsilateral components. We injected the tectum with horseradish peroxidase (HRP) dried onto fine needles to count the numbers of retinal ganglion cells labeled by retrograde transport. The retinas were prepared as flat-mounts to facilitate the cell counting. The tecta were injected either unilaterally or bilaterally in mirror-symmetric loci. Specimens included completely normal frogs and frogs which had undergone unilateral optic nerve regeneration, although only normal retinas are presented in the current study. The retrograde transport interval was varied progressively (from 3 to 5 days), and single or multiple injections of HRP were placed singly or as clusters, in order to increment the cell counts toward a level of saturation. Approximately 70.9% of the neurons in the ganglion cell layer could be labeled by this method. Correcting for the presence of displaced amacrine cells, estimated to comprise approximately 16% of the neurons in the ganglion cell layer (Scalia et al., '85, Brain Res. 344:267-280), we calculate that approximately 84.4% of the retinal ganglion cells project contralaterally to the optic tectum. Flat-mounted retinas ipsilateral to unilaterally injected tecta of completely normal frogs were also examined for labeled cells. The results of injections in the rostrolateral, caudomedial, and caudolateral tectum were studied. We found that ipsilaterally labeled cells comprised no more than 2.3% of the overall population of ganglion cells in the ganglion cell layer. The ipsilaterally projecting cells were found in loci which were approximately mirror symmetric to the regions of maximal cell labeling in the contralateral retinas from the same animals. The ipsilateral population was always displaced toward the periphery of the retina with respect to the contralateral population, regardless of whether the contralateral locus was centered in the temporal, ventronasal, or dorsonasal sector of the retina. Because the ipsilaterally projecting ganglion cells form such a minor population, and because they exist in the monocular as well as the binocular parts of the retina, it seems likely that they may not play a significant role in visual function in the frog. PMID- 1707069 TI - Descending neurons supplying the neck and flight motor of Diptera: organization and neuroanatomical relationships with visual pathways. AB - In dipterous insects, a volume of behavioral and electrophysiological studies promote the contention that three wide-field motion-sensitive tangential neurons provide a necessary and sufficient input to specific channels that drive the torque motor during flight. The present studies describe the results of neuroanatomical investigations of the relationships between motion-sensitive neuropil in the fly optic lobes and descending neurons that arise from a restricted area of the brain and supply segmental neck and flight motor neuropil. The present observations resolve at least 50 pairs of descending neurons supplying flight motor centers in the thoracic ganglia. The majority of descending neurons receive a distributed output from horizontal motion-sensitive neurons. However, the same descending neurons are also visited by numerous small field retinotopic neurons from the lobula plate as well as hitherto undescribed small tangential neurons. Neuroanatomical studies, using cobalt, Golgi, and Texas red histology, demonstrate that these smaller inputs onto descending neurons have dendrites that are organized at specific strata in retinotopic neuropil and that these correspond to horizontal and vertical motion sensitivity layers. Conclusions that only a restricted number of wide-field neurons are necessary and sufficient for visually stabilized flight may be premature. Rather, neuroanatomical evidence suggests that descending neurons to the flight motor may each be selectively tuned to specific combinations of wide- and small-field visual cues, so providing a cooperative descending network controlling the rich repertoire of visually evoked flight behavior. PMID- 1707070 TI - Descending neurons supplying the neck and flight motor of Diptera: physiological and anatomical characteristics. AB - In Diptera, dorsal neuropils of the pro-, meso-, and metathoracic ganglia supply motor neurons to neck and flight muscles. Motor circuits are supplied by more than 50 pairs of descending neurons (DNs) whose dendritic trees in the brain are restricted to dorsal neuropils of the deutocerebrum where they are grouped together into discrete clusters. Each cluster is visited by wide-field motion sensitive neurons and by morphologically small-field retinotopic elements. This organization suggests that flight descending neurons should respond to complex stimuli reflecting panoramic movement and small-field motion. Intracellular recordings, combined with dye filling, confirm this. Certain descending neurons responding to visual flow fields terminate bilaterally in superficial pterothoracic neuropils, at the level of indirect (power) flight muscle motor neurons. Other DNs terminate laterally, and provide segmental collaterals to areas containing neck and direct (steering) flight muscle motor neurons. Such DNs are activated by wide-field directional stimuli corresponding to pitch, roll, or yaw, and to small-field stimuli. Appropriate directional mechanosensory stimuli also activate dorsal descending neurons. The significance of dorsal descending neurons for the control of flight is discussed and compared with studies on course deviation neurons in other insects. It is suggested that, in Diptera, dorsal descending neurons may separately be involved in the control of velocity, stabilization, and steering manoeuvres. PMID- 1707071 TI - The effects of aging on the secretion of the common alpha-subunit of the glycoprotein hormones in men. AB - To investigate the effects of aging on the secretion of the common alpha-subunit of the glycoprotein hormones, we measured basal levels of luteinizing hormone (LH), testosterone (T) and alpha-subunit in 176 normal men aged 19 to 89 years. In addition, in two groups of young (less than 65 years; n = 25) and old (greater than 65 years; n = 15) subjects, the effects of luteinizing hormone-releasing hormone (LHRH) on LH and alpha-subunit secretion were determined. Age-related increases in serum alpha-subunit and LH were noted only in the oldest men while T levels decreased progressively with advancing age. LHRH stimulation resulted in significantly greater secretion of alpha-subunit in the old subjects while no difference in LH release between young and old men was observed. Moreover, there was a delay to peak LH and alpha-subunit levels after LHRH in the old subjects. These data suggest that the aging process in males involves deficits in both testicular and gonadotroph functions as demonstrated by (1) the relative hypogonadotropic hypogonadism seen until the ninth decade; (2) the hypergonadotropic hypogonadism apparent in men greater than 80 years; (3) the delay in the timing of peak responses of LH and alpha-subunit after LHRH administration; and (4) the disproportionate increase in the secretion of alpha subunit relative to intact LH. PMID- 1707072 TI - Seizures after lindane therapy. PMID- 1707073 TI - [Prenatal diagnosis of molar pathologies coexisting with a fetus. Review of the recent literature and a case report]. AB - A case of twin pregnancy combining a complete mole and a normal pregnancy is reported. A spontaneously aborted partial triploid mole was found in the past medical history of the patient. Prenatal investigations showed an heterogenous mass suggestive of a trophoblastic disorder coexisting with a normal placenta and a morphologically normal fetus on sonography associated with increased levels of hCG and normal levels of AFP in the maternal serum. High-resolution color Doppler imaging showed no blood flow within the suspect mass, excluding a myoma in necrobiosis or a large placental chorioangioma. The patient did not presented the severe complications classically described in classical mole and a passive conservative attitude was adopted. The pregnancy ended prematurely and the patient delivered at 27 weeks of gestation of a phenotypically normal infant. The mother and the baby had an unremarkable post-partum course. The review of the recent literature showed that partial hydatidiform mole could be separated in four categories: triploid partial moles; twin pregnancies combining a complete mole and a normal pregnancy; diploid partial mole; and pseudo-moles. Detailed sonographic examination and evaluation of maternal serum hCG and AFP should allow prenatal differential diagnosis of these pathological entities. PMID- 1707074 TI - Immunocytochemical features of the vestibular nuclei in the monkey and cat. AB - Immunocytochemical studies of the vestibular nuclei (VN) were done in the squirrel monkey and cat using polyclonal antisera. Brain stem sections were processed using the Avidin-Biotin peroxidase complex with diaminobenzidine as the chromagen. Choline acetyltransferase immunoreactivity (ChAT-IR) was most prevalent in the caudal medial (MVN), inferior (IVN) and peripheral superior (SVN) VN. Nearly all cells of groups x and z were ChAT-positive. None of the giant cells of the lateral vestibular nucleus (LVN) was ChAT-IR. Glutamate immunoreactivity (GLU-IR) was abundant in all VN and in cells of the vestibular ganglion (VG). Gamma-aminobutyric acid immunoreactivity (GABA-IR), was found in cells of rostral MVN, cell group y and in granules about giant cells in dorsal LVN. Substance P immunoreactive (SP-IR) was present in a small cells in MVN, IVN and the VG and in granules surrounding all large cells in LVN in both monkey and cat; SP-IR granules were most intense in ventral LVN in the monkey. Some cells in the dorsal parts of the fastigial nucleus (FN) were outlined by SP-IR granules in both species. Leucine-enkephalin immunoreactivity (ENK-IR) was identified only in granules surrounding cells of group x in the monkey. GLU was the only immunoreactive substance found in the giant cells of LVN. The disposition of ChAT IR in the VN suggested participation in commissural systems, as well as projections to spinal cord and/or cerebellum. Small GABA-IR neurons in MVN probably represented both commissural and projection neurons; GABA-IR granules about cells in dorsal LVN and some cells in MVN and SVN appeared to represent Purkinje cell (PC) terminals. SP-IR granules surrounding cells in ventral LVN appeared to represent terminals of small SP-positive VG cells. The source of SP IR granules around cells in dorsal LVN and some cells in FN and SVN remains unknown, but these fibers may originate from portions of the reticular formation known to contain large numbers of SP-positive neurons. PMID- 1707075 TI - [Cytoarchitecture and topography of the ventromedial hypothalamic nuclei of swine (Sus scrofa domestica)]. AB - The topography and cytoarchitecture of nc. hypothalamicus ventromedialis, nc. hypothalamicus dorsomedialis and nc. infundibularis were descripted in the pig. The nc. hypothalamicus ventromedialis is the dominant nucleus in the tuber cinereum. The rostral pole lies caudal to the nc. hypothalamicus anterior. Its caudal pole is bounded by the nc. premamillaris and the nc. hypothalamicus ventrolateralis. Scattered neurons separate the nc. hypothalamicus ventromedialis dorsal from the nc. hypothalamicus dorsomedialis. Dorsolateral it is limited by the fornix and medial from the ventricular wall by the nc. hypothalamicus periventricularis ventralis and the nc. infundibularis. The neurons are medium sized. They are compactly arranged at the periphery but in the centre the texture is much looser. The nc. hypothalamicus dorsomedialis is a poorly delimited nucleus in the rostral part of the medial hypothalamus. Ventral it is separated from the nc. hypothalamicus ventromedialis by a narrow relative cell free zone. The area hypothalamica rostralis limits it dorsolateral and the nc. paraventricularis dorsomedial. The caudal boundary is formed medial by the nc. periventricularis dorsalis and lateral by the fornix and the area hypothalamica lateralis. Small and medium-sized neurons predominate in this nucleus. The nc. infundibularis surrounds the infundibulum and reaches the recessus mamillaris. The two lateral parts are united at the floor of the third ventricle. Its small neurons are associated with the ependym. The topography and cytoarchitecture of the pig tuber cinereum was compared with the nuclear configuration of other Artiodactyla, Perissodactyla, Carnivora and Rodentia. PMID- 1707076 TI - Fine structure of regenerating caudal spinal cord in adult tuatara (Sphenodon punctatus). AB - A report on the presence of cerebrospinal fluid contacting neurons (CSFCN) inside the regenerating caudal spinal cord of adult specimens of the tuatara Sphenodon punctatus is given. The cells in question are easily distinguished from amongst ependymal tanicytes of the regenerating cord. Large granules of high electron density are particularly common in these neural cells which generally show clear cytoplasm of low electron density. The ultrastructural findings strongly support the idea of a secretory activity of these neurons directed toward the central canal. PMID- 1707077 TI - Relation of the insular claustrum to the neocortex in Insectivora. AB - The claustra of 9 species of Insectivora (Sorex araneus, Sorex minutus, Tenrec ecaudatus, Solenodon paradoxus, Neomys fodiens, Erinaceus europaeus, Talpa europaea, Desmana moschata, Potamogale velox) were investigated. In all examined animals we found two parts of the insular claustrum: the main part called by us the pars principalis and more medially situated lamina profunda claustri. In the "basal" Insectivora the main part is in close contact with the layer VIa of the neocortex. In some more developed "basal" and in all "progressive" Insectivora the area capsularis appears. Dorsolaterally it separates the main part of the insular claustrum from the neocortex and possesses, besides neurons, also numerous fibers of the extreme capsule. The above data strongly suggest that in the phylogenesis the insular claustrum originates from the cortex from which it gets separated by the extreme capsule. Lamina profunda claustri is rather a narrow band of neurons situated on the medial side of the pars principalis and mostly separated from it by a thin lamina of white substance. Lamina profunda is continuous with the layer VIb of the neocortex. PMID- 1707078 TI - Correlation between differences in the structure of dendrite bundles and cytoarchitectonic patterns in the cerebral cortex of the rabbit. AB - The bundling patterns of apical dendrites are investigated in the neocortex of adult rabbits using 10-15 microns paraffin serial sections. In order to extend observations to 12 neocortical areas, four vertical planes of sectioning differing with regard to their angle with the frontal plane are chosen. In alternate sections, stained with either Kluver-PAS or a modified Gomori-method, the shape and structure of dendrite bundles are investigated in relation to the cytoarchitectonic pattern. In all 12 areas, bundling of apical dendrites is found both in lamina IV and II/III. Differences in bundle structure are found to be more pronounced amongst bundles in lamina IV than in lamina II/III. The principal difference between the bundling patterns in the precentral (motor) and occipital (visual) areas known from previous studies is confirmed, i.e. in the precentral areas short and stout bundles with a complex arrangement of apical dendrites are encountered, whereas in the occipital areas long and slender bundles with a simpler alignment of dendrites are found. Bundling patterns in the temporal cortex resemble the ones found in the occipital areas, whereas in the postcentral areas bundles are seen which show characteristics common to bundles of both the precentral and occipital areas. PMID- 1707079 TI - Subthalamic nucleus of the monkey: connections and immunocytochemical features of afferents. AB - Retrograde and anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was studied in 7 squirrel monkeys with discrete injections of the subthalamic nucleus (STN). Injections labeled: (1) the lateral two-thirds of the nucleus (63% and 47%), (2) ventrolateral parts caudally (20%), (3) dorsomedial parts caudally (18%), (4) rostromedial parts (21%), (5) the medial third (38%) and (6) the lateral pole of the nucleus (9%). Afferents to the lateral two-thirds of the STN originated from two parallel cellular arrays in dorsal parts of the middle third of the lateral pallidal segment (LPS) and a single array in the rostral third of the LPS. Medial regions of the STN received input from cells in the rostral LPS. Small numbers of cells were retrogradely labeled in the centromedian-parafascicular (CM-PF) and the pedunculopontine (PPN) nuclei. No cells were labeled in the frontal cortex, the striatum, the substantia innominata (SI), the substantia nigra (SN) or the dorsal nucleus of the raphe. Virtually all pallidal neurons, including identified pallidosubthalamic neurons, were immunoreactive (IR) for gamma-aminobutyric acid (GABA). Pallidosubthalamic neurons were most numerous in regions of the LPS with the lowest density of leucine enkephalin-IR fibers. Substance P-IR fibers, found mainly in the medial pallidal segment, bore no relationship to pallidal afferents to the STN. Choline acetyltransferase-IR cells in the SI and the PPN were not retrogradely labeled with WGA-HRP granules. Anterograde transport in fibers and terminal fields surrounded retrogradely labeled cells in the LPS, suggesting a reciprocal relationship. The caudal third of the LPS and ventral region of the middle third of this nucleus, appeared to project few fibers to, or to receive few fibers from, the STN. A small number of STN efferents entered the medial border of the putamen, but no terminal fields were identified. STN projections to the pars reticulata of the SN appeared to represent about 10% of the projection to the LPS. No STN efferents were identified in the frontal cortex, the SI or the PPN. The hypothesis that STN afferents from the frontal cortex and CM-PF may represent collaterals of projections to other loci is discussed. PMID- 1707080 TI - Rapid and sensitive detection of Salmonella (O:6,7) by immunomagnetic monoclonal antibody-based assays. AB - An immunomagnetic technique to detect and identify Salmonella serogroup C1 has been developed. Monoclonal antibodies (MAbs) specific for the O antigen 6,7 of Salmonella lipopolysaccharide (LPS) coupled to magnetic beads were used to isolate the salmonellae. Captured bacteria were easily identified by acridine orange staining and measured by enzyme immunoassays with a conjugate anti-LPS MAb as the detector probe. The whole detection process required 2-3 h and the sensitivity was 10(3)-10(4) bacteria/ml. The presence of blood (10%, v/v) or stool (1%, w/v) components did not interfere with the immunomagnetic assay performance. PMID- 1707081 TI - Immortalization of growth factor-dependent mouse splenic macrophages derived from cloned progenitors. AB - We describe a nonviral transformation strategy for the establishment of permanent cell lines derived from the progeny of individual mouse splenic macrophage (M phi) progenitors. These colony stimulating factor-1 (CSF-1)-dependent cell lines possess many features of mature M phi s, including antibody-dependent phagocytic and cellular cytotoxic activities, ability to secrete lysozyme, and expression of the Mac-1 antigen and mRNA for the CSF-1 receptor. It was also possible to immortalize selected clones of splenic M phi s differing in their constitutive antigen-presenting activities with the retention of the antigen-presenting phenotype in the resultant cell lines. The approach described in this report should be useful in obtaining additional cell lines of M phi s expressing other phenotypes of interest. PMID- 1707082 TI - The reduction of nonspecific binding in chemiluminescent sandwich enzyme immunoassays. AB - The reduction in nonspecific binding obtained in EIA procedures based on glucose oxidase (GO)-labelled antibody is determined by the origin of the antibodies used for both the solid phase and for GO labelling. We have examined the relationship between nonspecific binding and various antibody combinations for the purpose of establishing highly sensitive chemiluminescent sandwich enzyme immunoassays. It was shown that nonspecific binding could be reduced by the following combination of solid phase and GO labelled antibodies: (i) guinea pig IgG-guinea pig IgG, (ii) goat IgG-rabbit IgG, (iii) goat IgG-rabbit Fab'. On the basis of these results, we succeeded in establishing highly sensitive chemiluminescent sandwich enzyme immunoassays for human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP). The detection limit was 2.5 mIU/ml for hCG using combination (i), 1 ng/ml for AFP using combination (ii), and 0.05 ng/ml (70 amol/assay) for AFP using combination (iii). PMID- 1707083 TI - [Cancers of the prostate and cyproterone acetate. Value of blood testosterone and PSA levels for treatment surveillance]. AB - On the basis of a retrospective series of 74 patients with adenocarcinoma of the prostate, the authors study the survival of these patients according to the clinical stage of the lesion when treated with cyproterone acetate. Biologically speaking, the authors have studied the evolution of the blood testosterone level, as well as of PSA and PAPs. Their conclusion is that the PSA is an excellent indicator of the efficacy of the cyproterone acetate treatment. Its fast decrease proves treatment efficacy, while a new rise indicates the resumption of evolution of the neoplastic process and must lead to change the therapeutic approach. PMID- 1707084 TI - Antibodies to core lipopolysaccharide determinants: absence of cross-reactivity with heterologous lipopolysaccharides. AB - Using monoclonal antibodies directed against defined epitopes of endotoxin core, this study demonstrated that the presentation of lipopolysaccharide (LPS) to antibodies is critical for measuring the specific binding of antibodies to LPS structures. False cross-reactive reactions apparently were observed when free core LPS or lipid A were used as antigens in ELISA, whereas coating with complexes of high-density lipoproteins with core LPS increased both the sensitivity and the specificity of the test compared with coating with free core LPS, so that nonspecific binding of antibodies was largely avoided. Using this technique, it was not possible to find broadly cross-reactive core LPS antibodies after immunization of rabbits and humans with rough mutants of gram-negative bacteria. These observations underscore the need for careful evaluation of the potential for cross-reactivity of antisera and of monoclonal antibodies directed against endotoxin core. PMID- 1707085 TI - Overview of pancreatic cancer, 1989. AB - Pancreatic cancer remains a leading cause of death from cancer. Its incidence is rising throughout the world, not solely because of improved diagnostic techniques. Diagnosis is followed relatively rapidly by the demise of the patient, and reasons for this rapid course have been sought. Both patient and physician delay play roles in this course. In addition, spread of the lesion outside the confines of the pancreas is usually present at the time of diagnosis. Symptoms, age and sex distribution, pathologic types, location within the pancreas, and physical findings are discussed. The various diagnostic techniques are evaluated and compared. Choices of operations are discussed, including risks associated with each, survival results, and the recent decrease in operative mortality reported from several institutions. Some of the newer approaches to therapy are discussed--combinations of radiotherapy, operation, and palliative procedures. The importance of a diagnostic method that will detect the lesion at an earlier stage is stressed. PMID- 1707086 TI - Severe congenital neutropenia: clinical effects and neutrophil function during treatment with granulocyte colony-stimulating factor. AB - We studied neutrophil function and clinical responses in seven patients with severe congenital neutropenia (SCN) after they received treatment with recombinant human granulocyte colony stimulating factor (rhG-CSF). Two subpopulations of patients with SCN were defined by their pattern of absolute neutrophil response, superoxide production, and cytochrome b559 levels. One group had an oscillating absolute neutrophil count and reduced ability to produce superoxide and cytochrome b559 (n = 4), and the second group had a relatively constant absolute neutrophil count response with normal superoxide and cytochrome levels (n = 3). Neutrophils from both groups had decreased surface expression of FcRIII and abnormal upregulation of the C3bi receptor (CR3). All patient neutrophils, however, had normal contents of the primary granule constituent, beta-glucuronidase, and the specific granule constituent, vitamin B 12 binding protein. The clinical response to rhG-CSF was evident by marked improvement in the degree of periodontitis and reduction in the number of oral ulcers in both groups of patients. Although neutrophil function is not completely normal in patients with SCN, it is likely that enough redundancy exists in neutrophil bactericidal capacity to promote normal host response to inflammation. PMID- 1707087 TI - Kinetics of antibody response to hepatitis B virus determinants and to recombinant vaccines in Italy. AB - The kinetics of antibody response to the group determinant a and subdeterminants d and y of hepatitis B virus were studied after infection and following immunisation with two recombinant DNA yeast-derived hepatitis B vaccines. The initial antibody response was to the subdeterminant epitopes, whereas anti-a antibody, which provides protection against different subtypes of the virus, was not detected for some weeks or months. The delay in the development of anti-a antibody after active immunisation raises important issues of early protection against infection. PMID- 1707088 TI - Short-term action of insulin on Aplysia neurons: generation of a possible novel modulator of ion channels. AB - In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at -35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger. PMID- 1707089 TI - Accelerated response to reinoculation in experimental allergic encephalomyelitis: histopathologic study. AB - Experimental allergic encephalomyelitis (EAE) was induced in Lewis rats by the injection of spinal cord tissue or myelin basic protein and adjuvants (Freund's or carbonyl iron or pertussis vaccine), or by adoptive immunization. After an interval of five to 12 weeks, the recovered rats were reinoculated by a different route and usually with a different adjuvant. The onset of the second attack was determined by the histologic detection of EAE lesions at intervals during the incubation period. In each of ten experiments, the second attack of EAE occurred one or two days earlier than in naive controls injected at the same time. Residual EAE lesions left over from the first attack could not explain the findings in the reinoculated rats. The accelerated response to the second inoculation may be related to the anamnestic response of classical immunology or to residual damage to the blood-brain barrier. Resistance to a second attack was not encountered in this histopathologic study. PMID- 1707090 TI - Immunocytochemical double labeling of glial fibrillary acidic protein and transferrin permits the identification of astrocytes and oligodendrocytes in the rat brain. AB - Sequential immunocytochemical double labeling of glial fibrillary acidic protein (GFAP) and transferrin (Tf) was used for the simultaneous identification of astrocytes and oligodendrocytes in paraffin-embedded sections of the rat brain. Paraformaldehyde or formalin-alcohol-acetic acid fixation followed by protease treatment of paraffin-embedded sections provided the most satisfactory double labeling. The results demonstrated GFAP-positive astrocytes and Tf-positive oligodendrocytes as two subpopulations of glia with no overlap. Our results suggest that, with some exceptions, immunocytochemical double labeling of GFAP and Tf has a potential application for the simultaneous identification of astrocytes and oligodendrocytes in the rat brain. PMID- 1707091 TI - Increased susceptibility of human fetal astrocytes to human T-lymphotropic virus type I in culture. AB - Human T-lymphotropic virus type I (HTLV-I) has been considered as an agent responsible for tropical spastic paraparesis and HTLV-I associated myelopathy. However, the pathogenesis of the diseases remains unclear. In a previous study we demonstrated that HTLV-I could infect adult human astrocytes and oligodendrocytes in vitro, although the rates of infected cells were low, at a rate of 0.1% and 0.01-0.05% respectively. Since mother-to-child transmission has been proposed as one of the major pathways for the prevalence of HTLV-I endemic, in the present study we investigated the susceptibility of human fetal astrocytes to HTLV-I in culture. After two days of co-culturing fetal brain cells with irradiated MT-2 cells (an HTLV-I-producing T-cell line), immunofluorescence staining revealed many positive astrocytes for HTLV-I p19 antigen. Multinucleated giant cells doubly immunoreactive to glial fibrillary acidic protein and HTLV-I antigen were frequently observed and showed a characteristic feature of hairy or fluffy external appearance. The percentage of infected astrocytes became as high as 19.4% at Day 21 of co-culture and then decreased. Electron microscopic examination revealed type C virus-like particles in astrocytes. These results indicate that human fetal astrocytes are more susceptible to HTLV-I infection than adult human astrocytes in tissue culture. PMID- 1707092 TI - Dopamine differentially regulates dynorphin, substance P, and enkephalin expression in striatal neurons: in situ hybridization histochemical analysis. AB - Dopamine regulation of the levels of dynorphin, enkephalin, and substance P messenger RNAs in rat striatal neurons was analyzed with in situ hybridization histochemistry (ISHH). Relative levels of peptide mRNA expression in the patch and matrix compartments of the dorsolateral striatum were compared among control rats, rats treated for 10 d with apomorphine, rats with unilateral 6 hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic system, and rats with nigrostriatal dopaminergic lesions followed 2 weeks later by 10 d of apomorphine treatment. Image analysis of ISHH labeling demonstrated that the number of neurons expressing each peptide mRNA remained constant, whereas the relative level of peptide mRNA per neuron changed significantly, depending on the experimental treatment. Dynorphin mRNA expression increased following chronic apomorphine treatment: striatal patch neurons increased to an average of 100% above control values, whereas striatal matrix neurons showed only a 25% increase. Dynorphin mRNA expression decreased following 6-OHDA lesions: patch neurons showed an average 75% reduction in expression, whereas matrix neurons showed no significant change. In animals with 6-OHDA lesions followed by apomorphine treatment, both patch and matrix neurons showed an average increase in dynorphin expression of 300% above control levels. Changes in dynorphin mRNA levels with these treatments were matched by qualitative changes in dynorphin immunoreactivity both in the striatum and in striatonigral terminals in the substantia nigra. Neither substance P nor enkephalin mRNA levels showed a significant difference between the striatal patch and matrix compartments in any experimental condition (in the dorsolateral striatum). Substance P mRNA expression was increased an average of 50% after 10 d of apomorphine treatment and showed an average decrease of 75% following 6-OHDA lesions of the mesostriatal system. There was no significant change in the expression of substance P mRNA in striatal neurons compared to control values in rats with combined 6-OHDA lesion and apomorphine treatment. Enkephalin mRNA expression was not significantly altered by chronic apomorphine treatment but showed an average increase per cell of some 130% above control levels following 6-OHDA-induced lesions of the mesostriatal system. In animals with a 6-OHDA lesion and apomorphine treatment, enkephalin mRNA was also elevated but not significantly above the levels produced by the lesions alone. These data show that the expression of dynorphin, enkephalin, and substance P is differentially regulated by the mesostriatal dopaminergic system and, further, suggests that the mechanisms by which this regulation occurs may be different for the 3 peptide families. PMID- 1707093 TI - Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin. AB - Tetramethylrhodamine-conjugated omega-conotoxin was used as a fluorescent stain (Jones et al., 1989) to determine the spatial distribution of voltage-gated Ca2+ channels along frog motor nerve terminals. Like native omega-conotoxin, the fluorescent toxin blocked neuromuscular transmission irreversibly. The fluorescent staining was confined to the neuromuscular junction and consisted of a series of narrow bands (in face views) or dots (in side views) approximately 1 micron apart. This characteristic staining pattern was prevented by pretreatment with omega-conotoxin and by prior denervation for 5-7 d. Combined fluorescence and phase-contrast optics indicated that the stain was on the synaptic rather than the nonsynaptic side of the nerve terminal. The bands and dots of stain proved to be in spatial register with the postsynaptic junctional folds, as revealed by combined staining of ACh receptors. It is concluded that the voltage gated Ca2+ channels on frog motor nerve terminals are concentrated at active zones. The findings are consistent with the suggestion (Heuser et al., 1974; Pumplin et al., 1981) that the large intramembraneous particles seen at freeze fractured active zones are voltage-gated Ca2+ channels. PMID- 1707094 TI - Substance P- and enkephalin-like immunoreactivities are colocalized in certain neurons of the substantia gelatinosa of the rat spinal cord: an ultrastructural double-labeling study. AB - The finding that certain cells of the substantia gelatinosa of the rat spinal cord contain both substance P (SP)- and enkephalin (ENK)-like immunoreactive material offers new insights into the mechanisms of action of these peptides in the processing of nociceptive sensory information. The simultaneous detection of these immunoreactivities was obtained in the superficial dorsal horn of the rat spinal cord at the ultrastructural level using monoclonal antibodies. An internally radiolabeled monoclonal antibody (against SP or ENK) was used to recognize one antigenic site, while the other antigenic site was identified by either a bispecific monoclonal antibody (for SP) or a monoclonal antibody (for ENK). The bispecific anti-SP antibody recognized HRP, whereas a secondary bispecific antibody recognized both the IgG of the anti-ENK monoclonal antibody and HRP. In laminae I-III, SP-like immunoreactivity (SP-LI) and ENK-like immunoreactivity (ENK-LI) were colocalized in a significant number of axonal varicosities, which contained round or pleomorphic synaptic vesicles. Such double labeled varicosities, however, were not found to be components of synaptic glomeruli. Most of the immunostained boutons of lamina I were SP-like immunoreactive only. In rats pretreated with colchicine, SP-LI and ENK-LI were colocalized in small perikarya of lamina II and in some lamina I cells. These findings indicate that SP and ENK occur in a significant population of interneurons of the superficial dorsal horn. It is suggested that some of these neurons may correspond to stalked cells and release one or the other substance depending on physiological conditions. PMID- 1707095 TI - Effect of cycloheximide and actinomycin D on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy. AB - Thymidylate synthase and thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, are rate determining enzymes in DNA synthesis. The increases in the activities of hepatic thymidylate synthase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats that had been administered cycloheximide. Concomitantly, other regenerative parameters such as the liver weight and contents of protein, RNA, and DNA were also significantly reduced in 24-h regenerating liver of cycloheximide-treated rats. When actinomycin D was injected, the activity of thymidine kinase, the liver weight, and contents of protein, RNA, and DNA were completely depressed in 24-h regenerating liver. However, the activity of thymidylate synthase in actinomycin D-administered rats rose to the level similar to the control (70% partially hepatectomized). The immunoblotting assay showed that thymidylate synthase is newly synthesized during liver regeneration after partial hepatectomy without being affected with actinomycin D. PMID- 1707096 TI - [Identification and characterization of the two promoters of plasmid pKYM]. AB - Plasmid pKYM is a multicopy plasmid isolated from Shigella sonnei and multiples stably in Escherichia coli. The plasmid encodes Rep protein which is essential for its multiplication and synthesizes cop ribonucleic acid (RNA) which is a short RNA complementary to the 5' region of rep m-RNA. This RNA controls the copy number and the incompatibility of the plasmid. The previous analysis located the promoters of rep m-RNA RNA (PR) and cop RNA (PL) in the inc region. This report confirmed the presence of these promoters by analyzing the RNAs isolated from the cells carrying pKYM and those synthesized in vitro. The initiation sites of these transcriptions were also determined. Analysis of in vivo RNA suggested that the quantity of cop RNA whose size was about 90 nucleotides was larger than that of rep m-RNA and these RNAs easily formed RNA-RNA hybrid. The analysis also suggested that the synthesis of rep m-RNA was repressed by cop RNA and Rep protein itself. PMID- 1707097 TI - Structure and inheritance of sense and anti-sense transcripts from a transposon in the green alga Chlamydomonas reinhardtii. AB - We have studied the transcription pattern of a 5700 base-pair transposon (TOC1) in Chlamydomonas reinhardtii. Northern blotting and nuclease S1 protection experiments define three classes of major TOC1 RNAs that accumulate to different levels in a number of strains and segregate independently in the progeny of crosses: class 1 RNAs are unstable near full-length sense transcripts whose 5' end maps to the left 217 base-pair repeat of TOC1, class 2 and class 3 RNAs are large, discrete chimaeric transcripts containing full-length sense (class 2) and anti-sense (class 3) copies of TOC1. Sequence motifs common to the 5' non transcribed regions of C. reinhardtii genes were found upstream from the putative initiation site of class 1 transcripts. A functional polyadenylation site was located in the far-right 237 base-pair repeat of TOC1. Class 1 TOC1 transcripts are initiated, and probably terminated, within the terminal repeats of TOC1 and may represent retrotransposition intermediates. Class 2 and 3 TOC1 transcripts co segregate with specific TOC1 elements identified on Southern blots. The loci that control the production of high levels of class 1 transcripts could correspond to specific TOC1 elements, i.e. only a few TOC1 elements are transcribed, or to a regulatory locus. The accumulation of an 11,500 to 12,000 base sense transcript (class 2) is reduced two- to fourfold by the presence of a 9500 to 9700 base antisense transcript (class 3). In contrast, the accumulation of the 5' ends of class 1 transcripts are unaffected by the anti-sense TOC1 transcript. PMID- 1707098 TI - An RNA replisome as the ancestor of the ribosome. AB - The ribosome is proposed to have evolved from an earlier RNA-replisome, which synthesized RNA. Ancestral tRNA molecules originally were loaded with trinucleotide sequences and donated them to growing RNA chains. The enzymatic addition of the C-C-A trinucleotide to presentday transfer RNA molecules is a carryover from this function. The strategies of reading RNA sequences by triplet codons and of housing information genetically in special repository molecules predates the origin of protein and DNA. These latter two polymers arose together at the time when the RNA replisome was converted to a ribosome. PMID- 1707099 TI - Distribution of the salmonid Hpa 1 family in the salmonid species demonstrated by in vitro runoff transcription assay of total genomic DNA: a procedure to estimate repetitive frequency and sequence divergence of a certain repetitive family with a few known sequences. AB - An in vitro runoff transcription assay of total genomic DNA was developed. As an example of use of this assay, analysis of a highly repetitive sequence in the cherry salmon (Oncorhynchus masou) is described. Total genomic DNA of the cherry salmon was completely digested with Hpa 1, whose site is known to be in the tRNA unrelated region of the cherry salmon Hpa 1 family. On transcription of the digested DNA in a HeLa cell extract, a discrete-sized RNA of about 100 nucleotides, constituting 70% of the transcripts, was produced, whereas on transcription of the undigested total DNA, only smeared RNA was obtained. In a fingerprint, the oligonucleotides of the discrete transcript from the digested total DNA were very distinct and exactly corresponded to those of a transcript from an Hpa 1 digest of a cloned DNA, but with few extra oligonucleotides. These results showed that the cherry salmon Hpa 1 family constitutes a major repetitive family in the genome of the cherry salmon. For determination of the distribution of the salmonid Hpa 1 family in other salmonid species, the same analysis was applied to DNAs from the chum salmon (Onchorhynchus keta), brown trout (Salmo trutta), Japanese common charr (Salvelinus leucomaenis pluvius), and Japanese huchen (Hucho perryi). The results showed that the salmonid Hpa 1 family is widespread in the genomes of salmonid species. A method and equations are also presented for estimating the relationship between the ratio of a given repetitive family to all the Pol III genes and its average sequence divergence by calculating the molar ratio of the runoff transcript to all the in vitro Pol III transcripts. PMID- 1707100 TI - Norepinephrine and serotonin release upon impact injury to rat spinal cord. AB - Microdialysis sampling was used to characterize the release of norepinephrine and serotonin upon impact injury to the rat spinal cord. Increases in extracellular norepinephrine concentrations in response to injury were small and of short duration. In contrast, serotonin concentrations quickly rose 35-90 times following injury and took 30-45 min to return to control levels. Bleeding caused by injury was probably the major source of the increased serotonin levels. Our results allow a role for serotonin in secondary damage upon injury to the spinal cord but suggest that norepinephrine is not a very significant contributor to such damage. PMID- 1707101 TI - Emesis-related biochemical and histopathological changes induced by cisplatin in the ferret. AB - Cisplatin is an effective antineoplastic agent that causes severe vomiting due to unknown mechanism. The ferret, an animal model useful in the determination of emetic activity, was used to clarify the emesis-related biochemical and histopathological changes that were induced by cisplatin. Cisplatin (5 to 10 mg/kg) was administered intraperitoneally and a 7-10 mg/kg i.p. dose evoked dose dependent emesis in the ferret. Almost all cisplatin-vomiting episodes occurred within a 6 hour observation period. All ferrets receiving cisplatin in this study died within 3-5 days. Significant increases in ileal mucosal levels of serotonin and norepinephrine were observed in cisplatin-treated ferrets. There were no significant changes in the levels of serotonin and dopamine in the gastric mucosa. Also, karyorrhexis was observed in the epithelial cells of the ileum and in the lymph follicles of the spleen of cisplatin-treated ferrets. These significant biochemical and pathological changes in the ileum may play an important role in cisplatin-induced emesis. PMID- 1707102 TI - [Application of C8-2Ag-Ab system for the diagnosis of hepatitis C]. PMID- 1707103 TI - [Development of hemopoietic stem cell research]. PMID- 1707104 TI - [An immunohistochemical study on cell differentiation in the outer root sheath of the normal human anagen hair follicles with antikeratin monoclonal antibodies]. AB - The expressions of several cytokeratins (CKs) in the outer root sheath (ORS) of the human anagen hair follicles were immunohistochemically studied using 11 antikeratin monoclonal antibodies (MoAbs) and 10 specimens from scalp. CKs 1, 10, 11, which are markers for differentiating keratinocytes, were exclusively found in the intermediate cells and the granular cells at the infundibulum. In cytokeratin expression, a distinct linear demarcation between the infundibulum and the isthmus was observed. Trichilemmal keratinization appeared to go in an inner-upward direction toward the hair canal. CK 19, a marker of undifferentiated stem cells, was found in outermost cells of the ORS at the isthmus and in some cells of the lower ORS. CK 16, a marker of hyperproliferative keratin, was detected in the outermost cells of the infundibulum and all the cells of the ORS below the isthmus. Therefore, the keratinocytes at the infundibulum may show a differentiation similar to that of the interfollicular epidermal keratinocytes. The ORS cells below the isthmus seem to move up to inner-upward direction along the hair axis with differentiation. PMID- 1707105 TI - [A method of hemostasis in transvesical adenomectomy]. PMID- 1707106 TI - End-organ protection and antihypertensive therapy. Introduction. PMID- 1707107 TI - End-organ protection and antihypertensive therapy. Proceedings of a symposium. Interlaken, Switzerland, January 12-13, 1990. PMID- 1707108 TI - Blood pressure variability: mechanisms and clinical significance. AB - Studies have shown that the circadian blood pressure profile is similar in normotensive and hypertensive subjects, and that the observed increase in blood pressure variability in hypertensive patients is proportional to the increase over the normotensive baseline blood pressure, suggesting that transient blood pressure oscillations on a percentage basis are not altered by hypertension. It has also been found that although antihypertensive therapy tends to reduce mean arterial blood pressure (MAP), the tendency to oscillate around a mean pressure is modified to a lesser extent. The significance of this observation is unknown; however, this effect should possibly be considered and studied in view of the incomplete protection against cardiovascular complications that is afforded by the current management of hypertension. PMID- 1707109 TI - Calcium channel blockers for kidney protection. AB - Acute postischemic renal failure (ARF) is a major complication in surgery and in particular in renal transplantation. Calcium channel blockers (CCBs) are able to prevent or ameliorate ARF in different experimental models when given before ischemic injury. Because ARF, which is observed in 20-60% of graft recipients, carries the risk of undetected rejections and nephrotoxic injury and is also expensive, we tested the hypothesis, suggested by animal experiments, that there is also a beneficial effect of CCBs when given after ischemic injury. A total of 134 recipients of first or second cadaver grafts were randomly assigned to a diltiazem pretreatment (DZ) or control (C) group. Kidney grafts were pretreated immediately prior to transplantation by reperfusion with 500 ml Euro-Collins (preservation fluid), with or without DZ. The DZ patients also received a 74-h infusion of DZ (0.12 mg/kg/h), starting 2 h prior to surgery. DZ was continued (90 mg b.i.d. p.o.) until day 30. Immunosuppression consisted of low-dose steroids and cyclosporine A (CSA) (10 mg/kg p.o.). CSA was instituted 6 h after surgery and later adjusted to achieve whole-blood trough levels of 300-600 ng/ml [by polyclonal radioimmunoassay (RIA)]. A total of 129 patients were available for efficacy analysis. There were no significant differences concerning donor demographics, human lymphocyte antigen (HLA) match, or ischemic times. ARF was defined as a need for dialysis in the first week. Our data show a significant reduction in ARF in grafts pretreated with DZ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707110 TI - Reversal of left ventricular hypertrophy: a desirable therapeutic goal? AB - It is controversial whether myocardial hypertrophy in essential hypertension represents an adaptive physiologic or pathological response to increased pressure load imposed on the heart. Cardiac structural adaptations in endurance athletes fulfill the criteria of physiologic hypertrophy: maintained cardiac performance and full reversibility of cardiac structural changes following cessation of endurance training. In contrast, there is some evidence that cardiac hypertrophy in arterial hypertension is a pathological process. Diastolic function of the left ventricle is impaired; systolic function, although maintained in the early stages of myocardial hypertrophy, becomes reduced beyond a critical mass; and cardiac structural changes are not fully reversible. An increasing number of prospective studies document that left ventricular hypertrophy in essential hypertension represents an independent risk factor for cardiovascular mortality and morbidity, in particular for sudden cardiac death. Ventricular arrhythmias were regarded as the pathophysiologic link between cardiac structural adaptation and the increased risk of sudden death due to left ventricular hypertrophy. Structural changes of the coronary circulation may represent one confounding factor in the relationship between left ventricular hypertrophy and its cardiovascular risk. As a consequence, optimal antihypertensive therapy should aim at reducing both arterial hypertension and left ventricular hypertrophy. Whether regression of myocardial hypertrophy indeed reduces cardiovascular risk cannot be definitively answered from the available data. PMID- 1707111 TI - Antihypertensive therapy and coronary prevention. AB - Antihypertensive therapy reduces cardiovascular risk in patients with malignant hypertension, but has not yet been shown to reduce risk in patients with mild to moderate hypertension. The lack of significant coronary prevention in the latter group, in large-scale trials, may have resulted from methodologic problems, statistical limitations, the adverse effects of antihypertensive medications, or failure to recognize the prognostic importance of concomitant regression of left ventricular hypertrophy. To date, primary prevention studies have focused on the use of beta-blockers and diuretics, both of which may have adverse metabolic effects and neither of which is capable of modifying left ventricular hypertrophy. Future research should be directed toward evaluating coronary prevention in patients with nonmalignant hypertension who have been treated with newer agents, such as calcium channel blockers and angiotensin-converting enzyme inhibitors, each of which has been shown to regress left ventricular hypertrophy and not to have adverse metabolic effects. PMID- 1707112 TI - A review of calcium antagonists and atherosclerosis. AB - Many detailed studies have demonstrated that calcium antagonists can suppress development of diet-induced atherosclerosis in the thoracic aorta of animals. A number of possible mechanisms have been proposed based on in vitro work, but the exact mechanism remains unclear. Alteration of serum lipid levels and blood pressure does not appear to be the common pathway. Differing effects between calcium antagonists of different classes indicate that the voltage-dependent calcium channel-blocking action common to all calcium antagonists is not the sole mechanism. Preliminary results of several major quantitative angiographic studies in human coronary artery disease have recently become available and indicate that calcium antagonists are able to retard the progression of existing lesions in humans also. There is also evidence that calcium antagonists may prevent the development of new lesions and, in some cases, induce lesion regression. Longer follow-up and further trials are required to assess the appropriateness of widespread clinical application of these agents in coronary artery disease, to determine the optimal timing for their introduction, and to define their mechanism of action in influencing the natural history of atherosclerosis. PMID- 1707113 TI - Diltiazem in ischemia-induced and reperfusion arrhythmias. AB - The beneficial effect of the calcium antagonist diltiazem on ischemia-induced and reperfusion arrhythmias has been documented in several animal studies. This effect may be due to prevention or reversal of spasms (secondary to prevention of intracellular calcium overload), due to reduction of conduction delay, suppression of after-potentials and triggered activity, or a consequence of inactivation of increased sympathetic activity. The clinical importance of these arrhythmias in humans is still a matter of debate. PMID- 1707114 TI - The effects of calcium channel blockers on blood fluidity. AB - Although vasodilation, direct cardiac actions, or both represent the main properties of calcium channel blockers, there are further pharmacologic effects that may be therapeutically relevant. For example, hemorrheological effects, which have been demonstrated for a variety of calcium antagonists, have received relatively little attention to date. Hemorrheology describes the mechanics of blood and its components. It is of particular interest in the context of cardiovascular disease, as it has been shown that under certain conditions (reduced pump function, impaired vasomotor reserve), parameters of blood fluidity may be crucial for tissue perfusion. Whole-blood viscosity is the dominating factor in large arteries. For geometrical reasons, plasma viscosity and the rheological properties of blood cells may become of paramount importance at the microcirculatory level. In ischemic states, erythrocytes may be depleted of ATP, which they need for maintenance of normal shape and for transformation. This results in rigidification of the red blood cell and hindrance of its passage in the microcirculatory bed. Hence, blood flow deteriorates with the consequence of further unfavorable changes of the "milieu interieur," leading to the induction of a vicious cycle. Although effects on several hemorrheological parameters, for example, whole-blood viscosity, plasma viscosity, and red cell aggregation, can be demonstrated for various calcium channel blockers, the main rheological effects of these compounds are believed to consist in the improvement of erythrocyte deformability. When the ATP-dependent calcium pump is impaired in ischemia, calcium channel blockers may inhibit the slow inward transmembrane calcium flux and prevent the accumulation of intracellular calcium. PMID- 1707115 TI - Arguments supporting the use of calcium antagonists in idiopathic dilated cardiomyopathy: theoretical considerations and clinical results. AB - There is increasing evidence that sarcolemmal disorders in idiopathic dilated cardiomyopathy (IDC), due either to viral infection, catecholamine excess, ethanol intoxication, microcirculatory disorders, or autoimmune responses, induce enhanced Ca2+ inflow. This may lead to mitochondrial Ca2+ accumulation and activation of phospholipase and proteases, resulting in left ventricular dysfunction. The acute beneficial hemodynamic response to diltiazem in IDC patients, in comparison to other calcium blockers, enabled us to perform an open trial investigating the effects of chronic diltiazem (60-90 mg t.i.d.) in patients with IDC. The results of this trial suggested that adjunctive diltiazem in IDC had beneficial effects on mortality, hemodynamics, and symptoms. Thus a multicenter, double-blind, placebo-controlled trial was initiated. PMID- 1707116 TI - Secondary prevention after myocardial infarction: in favor of beta-blockers. AB - A large number of pharmacological trials have been carried out, attempting to reduce the mortality (10-15%) in the year following of an acute myocardial infarction (MI) and/or the recurrence of ischemic events. Thrombolytic therapy and beta-blockade are the only interventions to be associated with a significant decrease in cardiac mortality. Early intervention with intravenous beta-blockers aims at limiting infarct size and at decreasing mortality. The Swedish study using intravenous (15 mg) followed by oral (200 mg/day) metoprolol showed a 36% reduction in mortality after the first week, a benefit persisting after 1 year. The combination of streptokinase and intravenous atenolol is safe and may be beneficial in selected patients. Large-scale controlled multicenter studies have shown that beta-blockers introduced within the first 3 days after acute MI significantly reduce total mortality and/or sudden death in the year following the acute event. Some of these studies demonstrate a reduction in recurrence of MI. The reduction (averaging 25%) in mortality may be explained by the anti ischemic action of beta-blockers and the prevention of arrhythmia-induced death. Introduced early, beta-blockers may reduce the size of the initial MI as well as subsequent infarction and/or ischemia. Furthermore the antistress action of beta blockers results in a decrease in free fatty acids, with their untoward effect in acute MI. Antiplatelet aggregation may also play a role. These properties of beta blocking agents should be utilized in every patient with acute MI in the absence of any major contraindication. Elective indications include patients with hypertension, angina pectoris, and/or ventricular arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707117 TI - Management of non-Q-wave myocardial infarction: role of diltiazem versus beta blocker therapy. AB - Several large-scale, multicenter trials have been conducted to evaluate the effects of currently available calcium channel blockers on a variety of cardiac end points in patients with myocardial infarction. Results indicate that careful subgroup stratification is necessary if morbidity and mortality are to be favorably altered. Certain groups of patients who are at high risk of recurrent myocardial infarction (MI) or death must be targeted for more aggressive diagnosis and therapy. Subset analysis of the Multicenter Diltiazem Postinfarction Trial (MDPIT) provides detailed information about diltiazem's long term benefit following non-Q-wave or inferior Q-wave MI, and its lack of efficacy in patients with extensive or prior MI. Concerning use of beta-blockers versus calcium channel blockers as secondary prevention following acute MI, the pathogenesis, clinical course, prognosis, anatomy, and histology of non-Q-wave MI differs appreciably from Q-wave MI, and hence it is logical to assume that secondary prophylaxis post-MI should differ for non-Q-wave versus Q-wave MI. It would appear that beta-blockers (particularly those agents without intrinsic sympathomimetic activity) are best suited for secondary prevention after Q-wave MI, whereas diltiazem is the only therapy of proven benefit for use after non-Q wave MI. PMID- 1707118 TI - Arterial vasodilator and antihypertensive effects of diltiazem. AB - The arterial vasodilator properties of the calcium antagonist diltiazem were investigated by measurement of changes of forearm blood flow during brachial artery infusions of eight dosages of diltiazem in eight hypertensive patients. Forearm blood flow increased and calculated forearm vascular resistance decreased dose-dependently (maximum decrease of 83.5 +/- 8.6%). When comparing the effects of calcium influx inhibition by diltiazem with stimulation of the vascular cyclic guanosine monophosphate (GMP) system by sodium nitroprusside, the vasodilation by diltiazem was approximately 1.6 times greater, attesting to its potent arterial vasodilator activity. The clinical efficacy and feasibility of diltiazem monotherapy was evaluated in 40 patients with mild to moderate essential hypertension treated with a slow-release formulation of diltiazem, 90 mg twice daily over 8 weeks, and in a subgroup of 21 patients with two tablets of 90 mg once daily in the morning over a period of 2 weeks. Blood pressure was lowered significantly and to a similar extent by either twice- or once-daily administration of diltiazem. This effect was maintained during open long-term therapy over a mean of 11 months. Heart rate and body weight did not change. Thus, the vasodilator properties of diltiazem can be utilized for effective long term treatment of hypertension. The possibility of once-daily dosing may prove useful with respect to drug compliance in the long-term treatment of a generally asymptomatic disease such as hypertension. PMID- 1707119 TI - Biliary and pancreatic metastases of breast carcinoma: is surgical palliation indicated? AB - Obstructive jaundice developed in a patient concomitantly with the diagnosis of breast carcinoma. Abdominal exploration disclosed a metastatic tumor in the head of the pancreas, the distal bile duct, and the gallbladder. A cholecystectomy and choledochojejunostomy were performed and later, because of intestinal obstruction, the patient underwent gastrojejunostomy. Pathological examination demonstrated metastatic lobular carcinoma of breast with strongly positive staining for estradiol. Additional hormonal therapy has been given to the patient since the operation. The patient is alive 16 months after the diagnosis of her disease. This case suggests that a vigorous diagnostic approach should be adopted in every jaundiced patient with metastatic breast cancer in order to exclude causes of jaundice other than diffuse metastatic involvement of the liver. Patients with extrahepatic biliary metastasis should be treated by aggressive surgical treatment, combined with systemic therapy which can offer them significant palliation and better survival. PMID- 1707120 TI - A functionalized superparamagnetic iron oxide colloid as a receptor directed MR contrast agent. AB - We have synthesized a surface functionalized superparamagnetic iron oxide colloid whose clearance from the vascular compartment was inhibited by asialofetuin but not fetuin. Unlike other particulate or colloidal magnetic resonance (MR) contrast agents, the agent of the current communication is not withdrawn from the vascular compartment by cells of the macrophage-monocyte phagocytic system, as indicated by its selective increase in hepatic relaxation rates. Because of this we refer to this colloid as a hepatic selective (HS) MR contrast agent. At 20 mumol Fe/kg the HS MR agent darkened MR images of liver. The HS MR agent exhibited no acute toxicity when injected into rats at 1800 mumol Fe/kg. Based on these observations, surface functionalized superparamagnetic iron oxide colloids may be the basis of MR contrast agents internalized by receptor mediated endocytosis generally, and by the asialoglycoprotein receptor in particular. PMID- 1707121 TI - The observation of endothelial cells in vein grafts by the en face silver staining method. AB - The introduction of en face silver staining to microsurgery has provided useful, detailed information about endothelial cells with regard to their size, shape, and number. With this staining method, endothelial cells of rat vein grafts have been morphologically investigated following division of the site of observation into three areas: the recipient artery, the proximal part of the vein graft, and the midportion of the vein graft. The results show that the arterial endothelial cells are small and spindle-shaped, whereas the cells in the proximal vein graft location are larger. Finally, large, rounded endothelial cells are seen in the midportion of the graft. These results are quantitated through a computerized graphic analysis system, which provides estimations of the cell size, Feret's diameters, and irregularities in the cell borders. The combination of en face silver staining and computerized graphic analysis has been especially useful for comparing minute changes that occur in recovering/regenerating endothelial cells. PMID- 1707122 TI - Mechanism of post-segregational killing by the hok/sok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. AB - The hok/sok system of plasmid R1, which mediates plasmid stabilization via killing of plasmid-free segregants, encodes two genes: hok and sok. The hok gene product is a potent cell-killing protein. The expression of hok is regulated post transcriptionally by the sok gene-encoded repressor, an antisense RNA complementary to the hok mRNA leader region. We show here that the hok mRNA is very stable, while the sok RNA decays rapidly. We also observe a new hok mRNA species which is 70 nucleotides shorter in the 3'-end than the full-length hok transcript. The appearance of the truncated hok mRNA was found to be regulated by the sok antisense RNA. Furthermore, the presence of the truncated hok mRNA was found to be correlated with efficient expression of the Hok protein. On the basis of these findings, we propose an extended model in order to explain the killing of plasmid-free segregants by the hok/sok system. PMID- 1707123 TI - The importance of the 5'-region in regulating the stability of sdh mRNA in Bacillus subtilis. AB - The decay of the polycistronic Bacillus subtilis sdh mRNA was analysed using probes specific for each of the component cistrons, sdhC, sdhA and sdhB. In exponentially growing cells, the entire sdh mRNA seems to decay with an 'all or nothing' mechanism and with a uniform half-life of 2-3 min for all cistrons. In stationary-phase cells, the half-life of the 5'-part had dropped to about 0.6 min whereas that of the 3'-part was about 1.2 min. Decay of sdh mRNA was also measured in exponentially growing cells containing a 'down-mutation' in the ribosomal binding site preceding sdhC which decreases the expression of sdhC by about 90%. The mutation has a moderate effect on expression of the downstream cistron sdhA. In this mutant, the half-life of the 5'-part of sdh mRNA was about 0.5 min (i.e. the same as in stationary phase wild-type cells) and the half-life of the 3'-part about 1.3 min. Also, analysis of the decay of an sdh-cat fusion transcript revealed that the sdh (5') part decayed more rapidly than the cat part and this difference was more pronounced in stationary-phase cells compared to exponentially growing cells. The results of these experiments demonstrate the importance of the 5'-segment of sdh mRNA in controlling the stability of the transcript under different growth conditions. PMID- 1707124 TI - Early infectious complications after peripheral blood stem cell autografts in children. AB - Seventeen children underwent marrow-ablative high-dose chemotherapy with peripheral blood stem cell autografts and were studied retrospectively to determine the type, frequency, and outcomes associated with infectious complications 3 months postgraft. The patients were kept in isolated rooms with a laminar air flow facility, but no decontamination procedures, such as gut sterilization with nonabsorbable antibiotics, nonmicrobial diet, and skin cleansing, were used. They were under their mothers' daily care to maintain good psychological conditions. After the completion of marrow-ablative chemotherapy and the infusion of stem cells, the absolute granulocyte count exceeded 0.5 x 10(9)/liter with a mean of 17.9 days (range 6-65 days). Fifteen patients developed a total of 16 febrile episodes during the first 4 week period, and the confirmed diagnoses were mucositis (12), enterocolitis (nine), septicemia (four), central venous catheter-associated infection (three), pneumonia (one), perianal abscess (one), and possible invasive fungal infection (one). All episodes were successfully treated with parenteral antibiotic therapy, and no patient died of infectious complications. The observations suggest that high-dose chemotherapy can be performed safely with simple and efficient patient management protocol followed by peripheral blood stem cell autografts. PMID- 1707125 TI - Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation. AB - In this study we report the preparation of a human osteosarcoma cell cDNA library and describe the isolation and sequence determination of a clone encoding the complete sequence of a novel human insulin-like growth factor (IGF)-binding protein (hIGFBP-4). Previous work indicated that hIGFBP-4 is the predominant IGFBP expressed by human osteoblast-like cells, and that IGFBP-4 binds and inhibits the mitogenic activities of IGF-I and IGF-II. Sequence determination revealed that hIGFBP-4 is a unique gene product with significant amino- and carboxy-terminal sequence similarity to three other known IGFBPs. Identical alignment of 18 cysteines in IGFBP-4 and the three other IGFBPs is a key structural feature of this protein family. In vitro studies of human osteoblast like cells suggest that PTH regulates the expression of hIGFBP-4 and that the PTH effect is mediated through a cAMP mechanism. hIGFBP-4 mRNA was also expressed in skin fibroblasts, and thus, this inhibitory IGFBP could be an important physiological regulator of IGF actions in bone cells and other cell types as well. PMID- 1707126 TI - Human prolactin gene expression: positive correlation between site-specific methylation and gene activity in a set of human lymphoid cell lines. AB - From the human B-lymphoblastoid cell line IM-9-P, we derived the IM-9-P series of clonal sublines that differ from each other in the degree of human PRL (hPRL) production. To elucidate the mechanisms underlying the different levels of hPRL gene activity in these cell lines, we investigated the methylation status of the gene, since the methylation pattern of cytosine-residues in CpG dinucleotides has been implicated with the transcriptional activity of eukaryotic genes. Restriction enzyme analysis of the hPRL gene with methylation-sensitive endonucleases disclosed no correlation between the extent of methylation and gene activity for ThaI, AvaI, and HhaI recognition sequences. Hypermethylation of a MspI site (CCGG) in the second protein-coding exon, however, was found to coincide with hPRL gene activity. Exposure of the cells to the nucleoside 1-beta D-arabinofuranosylcytosine, which has been reported to increase enzymatic DNA methylation, led to an elevation of hPRL production that persisted after removal of the drug. However, treatment of PRL-positive cells with the demethylating cytidine analog, 5-azacytidine, caused a distinct and heritable reduction of hPRL secretion and hPRL mRNA abundance, concurrent with hypomethylation of the specific MspI site that is hypomethylated in PRL-negative cell lines of the IM-9 P family. Contrasting the generally favored inverse relationship between methylation and transcriptional activity of a gene we describe a system in which site-specific methylation is positively correlated with gene expression. PMID- 1707127 TI - Synergistic enhansons located within an acute phase responsive enhancer modulate glucocorticoid induction of angiotensinogen gene transcription. AB - The hepatic transcription of the angiotensinogen gene is regulated by both glucocorticoids and cytokines generated as products of the acute phase reaction. We have identified a multimodular enhancer in the 5'-flanking region of the rat angiotensinogen gene that mediates these responses and consists of an acute phase response element (APRE) flanked on both sides by adjacent glucocorticoid response element consensus motifs (GREs). Induction of transcription by the cytokine interleukin-1 (IL-1) is glucocorticoid dependent and mediated through the APRE. The APRE binds in a mutually exclusive manner a cytokine/phorbol ester-inducible protein (BPi), indistinguishable from nuclear factor kB, and a family of constitutive liver proteins (BPcs) related to the heat-stable transcription factor C/EBP. Using mutated 5'-flanking sequences of the angiotensinogen gene fused to a firefly luciferase reporter gene transfected into hepatoblastoma (HepG2) cells, we have mapped enhanson sequences required for the transcriptional response to glucocorticoids. Two functionally distinct GREs are identified by deletion and site-directed mutagenesis, both of which mediate glucocorticoid stimulated transcription in vivo. Glucocorticoid-induced transcription mediated by the angiotensinogen gene enhancer is, furthermore, dependent on the occupancy of the APRE by either the BPi or a member of the BPc family because a mutant APRE that binds neither BPi nor BPc exhibits an attenuated glucocorticoid responsiveness. Mutant APREs that permit exclusive binding of either BPi or BPc synergistically transmit the glucocorticoid response mediated by one or the other of the adjacent GREs. Thus, the induction of angiotensinogen gene transcription involves interaction between the glucocorticoid receptor and either one of the APRE-binding proteins: either the cytokine-inducible NFkB or the constitutive family of C/EBP-like proteins, bound to adjacent enhansons in a mutually synergistic enhancer complex. PMID- 1707128 TI - Gonadotropin subunit messenger RNA concentrations after blockade of gonadotropin releasing hormone action: testosterone selectively increases follicle-stimulating hormone beta-subunit messenger RNA by posttranscriptional mechanisms. AB - Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or indirect effects of steroids mediated through hypothalamic GnRH. We aimed to define the effects of testosterone (T) on alpha, LH beta, and FSH beta mRNA expression in the male rat after blockade of GnRH action on the gonadotrope. A water-soluble GnRH antagonist was administered iv to castrate male rats (increased endogenous GnRH secretion) and to castrate T replaced rats in which gonadotropin subunit mRNAs had been increased by prior treatment with exogenous GnRH pulses. In castrate male rats, GnRH antagonist resulted in a fall in all three subunit mRNAs. Alpha and LH beta declined at slower rates (half-disappearance after 50 and 65 h, respectively), and neither fell to values present in intact rats over 84 h. In contrast, FSH beta mRNA declined more rapidly, with a half-disappearance after 20 h. In castrate T replaced rats, alpha mRNA declined at a rate similar to that in castrates (half disappearance after 50 h). LH beta declined more slowly, and the rate of FSH beta decline was markedly prolonged in the presence of T (half-disappearance time increased from 20 to 50 h). These results suggest that T exerts direct effects on FSH beta transcription or mRNA stability which are independent of GnRH action. To assess these possibilities, a long-acting GnRH antagonist (Detirelix) was administered to castrate male rats, which also received T or sham implants 4 days after castration. FSH beta mRNA levels fell during the 4 days of Detirelix alone, but the addition of T on day 4 resulted in a 2-fold rise in FSH beta mRNA, restoring FSH beta mRNA to levels present in intact rats. Serum FSH closely paralleled FSH beta mRNA concentrations. Alpha mRNA was reduced by 25%, and LH beta mRNA concentrations were unchanged in the presence of T. The rate of alpha mRNA transcription was markedly reduced and that of LH beta tended to fall in T treated rats, but T had no significant effect on the FSH beta transcription rate. Thus, the action of T to increase concentrations of cytosolic FSH beta mRNA appears to be exerted at a posttranscriptional level, possibly via effects of T on FSH beta mRNA stability. This may represent a mechanism by which T can effect differential regulation of gonadotropin subunit mRNA concentrations. PMID- 1707129 TI - Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. AB - The amphibian tetradecapeptide bombesin and its mammalian homolog gastrin releasing peptide are neurotransmitters and paracrine hormones, and are mitogenic for fibroblast and small cell lung carcinoma cell lines. cDNAs encoding the bombesin/gastrin-releasing peptide receptor (BR) expressed by murine Swiss 3T3 fibroblasts were isolated using electrophysiological and luminometric Xenopus oocyte expression assays. Oocytes microinjected with BR transcripts responded to concentrations of bombesin from 1 x 10(-10) to 1 x 10(-6) M. These responses showed homologous desensitization and could be specifically blocked by bombesin antagonists. Sequence analysis showed that the BR has seven membrane-spanning domains and five potential N-linked glycosylation sites. Data base analysis showed that the BR is most homologous to the tachykinin receptors. Although tyrosine kinase activity has been associated with BR function, no tyrosine kinase homologies occur within the BR sequence. PMID- 1707130 TI - Ductal heterogeneity of cytokeratins, gene expression, and cell death in the rat ventral prostate. AB - The rat ventral prostate is a complex gland composed of numerous ducts. The epithelial cells that line the lumen of the ducts are surrounded by stromal cells. The epithelial cells display a characteristic morphology that is dependent on their anatomical location within the ducts; the cells that line the lumen in the region of the ducts close to the urethra (the proximal region) are cuboidal, while those in the distal regions of the ducts are tall columnar cells. We have examined the regional expression of two genes that are expressed in the prostate: prostate steroid-binding protein (PSBP; a marker for androgen-dependent protein synthesis) and TRPM-2 (a marker for programmed cell death). We have demonstrated that the expression of PSBP, in the presence of androgens, and TRPM-2, in the absence of androgens, is restricted to the luminal epithelial cells in the distal regions of the prostatic ducts. Neither of the genes is expressed in the proximal regions of the ducts. In view of the probable effects of the epithelial-stromal interactions in the gland we have also characterized the cytokeratin composition of the epithelial cells lining the prostatic ducts. We have established that the basal epithelial cells of the prostate are primarily localized in the proximal region of the ducts. We propose that these cells may attenuate the influence of the stromal cells on the luminal epithelium and exert a negative influence on the cytodifferentiation of the secretory epithelial cells. The results also suggest that PSBP, which has been considered to be an androgen-dependent gene may, in fact, be a sequence that is constitutively expressed in the luminal cells that die in the absence of androgens. This has significant implications on the mechanism of androgen action in the rat ventral prostate. PMID- 1707131 TI - Cloning of the rat insulin-like growth factor-binding protein-2 gene and identification of a functional promoter lacking a TATA box. AB - We have isolated clones encoding the rat insulin-like growth factor-binding protein-2 (IGFBP-2) gene and determined its organization and nucleotide sequence. The rat IGFBP-2 gene spans at least 8 kilobases and consists of four exons, each of which contains protein-coding sequences. The amino acid sequences of exons 1, 3, and 4 are 32-50% identical to the corresponding exons of human IGFBP-1 and IGFBP-3, and 87-91% identical to those of human IGFBP-2. The 18 cysteines in the mature binding proteins are conserved. Exon 2 shows negligible homology. Primer extended reverse transcription indicated that the 5' end of IGFBP-2 mRNA is 151 nucleotides up-stream from the translation start site [designated nucleotide (nt) -151]. Consistent with this result, IGFBP-2 mRNA protected a genomic fragment terminating at approximately nt -148, as well as smaller fragments. A 1260 nt fragment containing 1144 nt of 5' flanking region had promoter activity when inserted in the correct orientation into a plasmid containing a promoterless luciferase reporter gene and transiently transfected into BRL-3A rat liver cells, which express IGFBP-2, but not when transfected into H4-II-E cells, which do not express IGFBP-2. The IGFBP-2 gene lacks a TATA box immediately up-stream from the transcription initiation site. It is GC rich (66% between nt -270 and +385) and contains GC boxes that might be recognized by transcription factors Sp1 or ETF. The promoter region contains multiple direct and indirect repeats. One direct repeat contains a variant Sp1 site (-158 to -150) near the consensus Sp1 site at nt -138 to -130. The 5' flanking region also contains motifs that might be recognized by transcription factors AP-1 (Jun/Fos), AP-2, and liver factor B1. The role of these sites in basal and regulated expression of the IGFBP-2 gene remains to be determined. PMID- 1707132 TI - Recognition of disparate HA and NS1 peptides by an H-2Kd-restricted, influenza specific CTL clone. AB - CTLs (CD8+) are known to recognize exogenous peptide in the context of class I MHC molecules. We observed that an influenza subtype H1 and H2 cross-reactive CTL clone B7, which was stimulated by a fusion protein containing a portion of HA2 subunit of A/PR/8 virus HA, recognized a synthetic peptide (residues 515-526) of the HA2 subunit of A/PR/8 virus strain. This CTL clone also recognized a structurally disparate NS1 peptide 50-68 of the same A/PR/8 virus. We examined the recognition of the NS1 peptide 50-68 and the HA peptide 515-526 by the subcloned CTL clone, B7-B7. Cold target inhibition experiments showed that the recognition of the HA peptide by the CTL clone B7-B7 could be competed by NS1 peptide-treated target cells and vice versa. The recognition of both NS1 peptide and HA peptide by the CTL clone B7-B7 was restricted by the same allele, H2Kd. In addition, this NS1 peptide requires approximately a 600-fold higher concn for optimal CTL recognition than did the HA peptide. We conclude that the TCR on clone B7-B7 recognizes the HA peptide or the NS1 peptide as comparable complexes with the same class I MHC molecules, although there is no obvious homology in the primary sequences of HA 515-526 and NS1 50-68 peptides. CTLs elicited with certain antigens appear to recognize distinctively different antigens complexed to the same presenting MHC molecule. PMID- 1707133 TI - Human B cells express membrane-bound and soluble forms of the CD14 myeloid antigen. AB - The expression of the myeloid differentiation antigen CD14 on the B lineage was analyzed. A CD14-specific monoclonal antibody was used to isolate the antigen from normal B, B-type chronic lymphocytic leukemia cells, and a representative Epstein-Barr virus-transformed B lymphoblastoid cell line (EBVLCL). A soluble form of this protein was detected in the culture supernatant of all the B cell types tested. The molecule expressed in the normal B and B-type chronic lymphocytic leukemia cells was identical in size to the 52,000 mol. wt monocyte isolated CD14 glycoprotein. A 64,000 mol. wt antigen was isolated from the lymphoblastoid cell line. Similar 2-D gel electrophoretic patterns to that of the monocyte-derived CD14 were obtained from the normal B and B-type chronic lymphocytic leukemia cell-isolated molecules. These similarities were reflected in minor isoelectric point (pI) differences between the polypeptide spots (pI 4.8), in the first dimension, and identical molecular weight (52,000) in the second dimension. The EBVLCL-isolated polypeptide, when analyzed by 2-D gel electrophoresis, showed a pI identical to that of the myeloid antigen (pI 4.6). The isolated soluble form was of smaller (47,000 mol. wt, normal B and B-type chronic lymphocytic leukemia cells) or similar size (64,000 mol. wt, lymphoblastoid cell line) compared with their corresponding membrane-bound forms. Interestingly, two-colour immunofluorescence analysis showed that only two out of four CD14-specific mAb tested bound to the B cells. We conclude that the CD14 antigen is, in fact, expressed in the B lineage. Its cell surface expression and serum level in the prognosis of B-type chronic lymphocytic leukemia patients needs to be evaluated. PMID- 1707134 TI - Structural and functional characterization of complement C8 gamma, a member of the lipocalin protein family. AB - Human complement component C8 exhibits an unusual structure in that it contains three chains, two of which, alpha and beta, display high sequence homology to other complement and CTL pore-forming proteins. The third chain, C8 gamma, is covalently linked to C8 alpha by a disulfide linkage; it is demonstrated that Cys40 of C8 gamma is linked to Cys164 of C8 alpha, a unique cysteine located in a loop located between the cysteine-rich LDL-receptor class A module and the membrane-inserting region of C8 alpha. C8 gamma was recently identified as a member of the lipocalin protein family, in which all proteins were either shown to, or are believed to bind small hydrophobic ligands. The present results now demonstrate that C8 gamma incorporates retinol and retinoic acid in the presence of 2 M NaCl. Molecular modeling of C8 gamma, based on the crystal structure of the homologous beta-lactoglobulin, reveals a structure of eight antiparallel beta strands, bearing a highly hydrophobic binding pocket. The residues participating in the pocket formation are highly conserved when compared with the structures of beta-lactoglobulin and retinol-binding protein, both of which are known to interact with retinol. It is therefore proposed that C8 gamma may act as a retinol transporting protein in plasma. PMID- 1707135 TI - Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses. AB - Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN II in Balb/c strain only, indicating differential antigen presentation. PMID- 1707136 TI - Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes. AB - The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones. PMID- 1707138 TI - Antigenic structure of human tumour necrosis factor: recognition of distinct regions of TNF alpha by different tumour cell receptors. AB - TNF alpha is a cytokine which causes cytolysis of tumour cell lines in vitro as well as haemorrhagic necrosis of many transplanted tumours in vivo. In association with these activities, the cytokine manifests a high degree of toxicity in vivo. The in vitro and in vivo effects of a panel of 13 monoclonal antibodies against human TNF alpha have been investigated. Of these MAbs, eight neutralized TNF alpha activity in the WEHI-164 cytotoxicity assay as well as in the binding of TNF alpha to receptors on these cells. The effects of this group of antibodies on TNF alpha-induced regression of WEHI tumours in vivo correlated with their in vitro neutralizing activities. One MAb which inhibited cytotoxicity, receptor interaction and tumour regression in the WEHI model (MAb 37) failed to inhibit TNF alpha-receptor binding and tumour regression in Meth A models. This observation indicates that different classes of receptor specificity may exist on different tumour cells. Together the antibodies define six non overlapping epitopic domains on TNF alpha and within these regions there are at least nine overlapping epitopes. Inhibitory MAbs, when co-injected into tumour bearing mice with radiolabelled TNF alpha, resulted in the diversion of TNF alpha away from both tumour and lung, which correspond to the sites of highest TNF alpha uptake in control MAb-TNF alpha treated mice. In contrast, uptake of TNF alpha by the liver was increased and overall, biodistribution studies showed that very little TNF alpha reached the target tumour but was rapidly and widely dispersed throughout the body. Preliminary studies with these MAbs show that segregation of TNF alpha activities and receptor binding may be possible. PMID- 1707137 TI - Antigenic structure of bovine growth hormone: location of a growth enhancing region. AB - Site-directed antisera generated by peptide immunization have been used to study the antigenicity of bovine growth hormone (bGH). Prediction of sequential antigenic sites has been performed using secondary structure information derived from the 'Protean' prediction routine. The structures predicted by this programme agree closely with the corresponding structure of GH recently derived from crystallographic studies. We have previously shown that the binding of monoclonal antibodies of particular epitope specificity to human or bovine GH results in significant enhancement of hormonal activity in vivo; however, the sites recognized by these antibodies were not known. Here we identify a sequence region, corresponding to a loop structure joining helices 3 and 4, which, is associated with the growth enhancement phenomenon. Antisera raised to either of two overlapping peptides (residues 120-140 and 134-154) significantly increase the biological activity of GH in vivo. Antisera directed to other regions on the GH molecule failed to demonstrate this property. Coincidentally, the sites recognized by the growth-enhancing anti-peptide antisera overlap with the site on GH which is highly susceptible to proteolytic cleavage; such cleavage has been shown in some cases to result in hormone enhancement. PMID- 1707139 TI - Cross-linking of a monoclonal antibody-antigen complex enables detection of parasite antigen in immunoblots and in an expression library. PMID- 1707140 TI - Measurement of prostate-specific antigen in serum as a screening test for prostate cancer. AB - BACKGROUND: Prostate-specific antigen (PSA) is secreted exclusively by prostatic epithelial cells, and its serum concentration is increased in men with prostatic disease, including cancer. We evaluated its usefulness in the detection and staging of prostate cancer. METHODS: We measured serum PSA concentrations in 1653 healthy men 50 or more years old. Those with PSA values greater than or equal to 4.0 micrograms per liter then underwent rectal examination and prostatic ultrasonography. Ultrasound-directed prostatic needle biopsies were performed in the men with abnormal findings on rectal examination, ultrasonography, or both. The results were compared with those in 300 consecutively studied men 50 or more years old who underwent ultrasound-directed biopsy because of symptoms or abnormal findings on rectal examination. RESULTS: Serum PSA levels ranged from 4.0 to 9.9 micrograms per liter in 6.5 percent of the 1653 men (107). Nineteen of the 85 men in this group (22 percent) who had prostatic biopsies had prostate cancer. Serum PSA levels were 10.0 micrograms per liter or higher in 1.8 percent of the 1653 men (30). Eighteen of the 27 men in this group (67 percent) who had prostatic biopsies had cancer. If rectal examination alone had been used to screen the men who had biopsies, 12 of the 37 cancers (32 percent) would have been missed. If ultrasonography alone had been used to screen these men, 16 of the 37 cancers (43 percent) would have been missed. Serum PSA measurement had the lowest error rate of the tests, and PSA measurement plus rectal examination had the lowest error rate of the two-test combinations. CONCLUSIONS: The combination of measurement of the serum PSA concentration and rectal examination, with ultrasonography performed in patients with abnormal findings, provides a better method of detecting prostate cancer than rectal examination alone. PMID- 1707141 TI - Association of p60c-src with polyoma virus middle-T antigen abrogating mitosis specific activation. AB - Polyoma middle-T antigen is required for tumorigenesis in animals and for viral transformation of a variety of cells in culture (reviewed in ref. 1). Middle-T associates with and thereby activates p60c-src, a cellular tyrosine kinase homologous to the oncogene product of Rous sarcoma virus. Activation of p60c-src by middle-T is accompanied both by dephosphorylation of tyrosine 527, a site which negatively regulates src kinase src kinase activity (reviewed in refs 4-6) and by autophosphorylation on tyrosine 416 (refs 7-10). Phosphoprotein p60c-src is subject to cell cycle-specific regulation. It is most active during mitosis and repressed in interphase. Here we report that mitotic p60c-src is dephosphorylated at tyrosine 527. We also show that in cells expressing middle-T, src kinase activity is high both in mitosis and during interphase. An oncogenic mutant src protein, p60c-src(527F), where tyrosine 527 is substituted by phenylalanine, is also highly active in all phases of the cell cycle. PMID- 1707142 TI - Breath mints for the dragon. Lindane toxicity. PMID- 1707143 TI - Detection of cancer metastases in regional lymph nodes: comparative histological and immunohistological investigations with the broad-range anticytokeratin monoclonal antibody A45-B/B3. AB - A total of 113 patients with carcinomas of breast, testis, stomach and colon were examined for lymph node metastases by means of an exact case-by-case comparison by conventional histology and by immunohistochemistry using the anticytokeratin monoclonal antibody A45-B/B3. Among 891 examined lymph nodes, 90% of metastases were recognized by both methods, about 2% by histology alone, and more than 10% by immunohistochemistry alone. The method can be applied for intraoperative frozen section diagnosis. PMID- 1707144 TI - Asymmetric PCR-based strategy for genetic analysis of the p53 tumor suppressor gene in cell lines and tumor tissues. AB - A novel strategy for genetic analysis of the p53 tumor suppressor gene is described, based on direct sequencing of the asymmetric polymerase chain reaction (PCR) products. A set of 10 PCR primers was designed which allows to amplify and sequence highly conserved regions of the molecule, i.e. the target areas of p53 mutations. The stepwise optimization of RNA isolation, cDNA synthesis, PCR amplifications and sequencing resulted in a procedure which is faster and more reliable than the techniques used to search for p53 mutations so far. This and similar strategies should be applicable to the study of genetic alterations in antioncogenes or other classes of genes which suffer from subtle mutations potentially scattered along large segments of the molecule. PMID- 1707145 TI - Peptidergic innervation of the pars distalis of the adenohypophysis. AB - Substance P-like immunoreactive nerve fibers have been found in the pars distalis of the adenohypophysis in the monkey, dog, and rat. Calcitonin gene-related peptide-like immunoreactive nerve fibers have also been identified in the dog and rat. The peptidergic fibers are closely related to the glandular cells with synaptic contacts demonstrated between them. It is suggested that neural factors may be directly involved in the regulation of the activities of the anterior pituitary. PMID- 1707146 TI - Selection of brain choline acetyltransferase synthetic peptides. AB - Choline acetyltransferase (ChAT) of pig brain is a 640 amino acid enzyme. The sequence has recently been published (Berrard et al., 1988). We identified potentially antigenic epitopes on porcine ChAT by primary and secondary structure analysis. We used 12 structural parameters derived from the amino acid sequence, especially surface antigenicity, internal repetitions and protease cleavage site clusters and superposed gliding averages for the complete ChAT protein. Four peptides from 13 to 21 amino acids in length were defined for antibody production. In detail, we describe a nine-step selection process and discuss costs of synthesis and purification. PMID- 1707147 TI - Participation of serotonergic mechanisms in the pathophysiology of experimental neoplastic spinal cord compression. AB - We evaluated the role of serotonin (5-HT) in neoplastic cord compression in paraplegic rats harboring a thoracolumbar epidural tumor. We measured serotonin and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), in the C-1 to C 7, T-1 to T-6, T-7 to T-12, and T-13 to L-3 spinal segments of tumor-free and tumor-bearing rats. In normal controls, a consistent rostral-to-caudal gradient for 5-HT and 5-HIAA was evident, but the 5-HIAA/5-HT ratio remained constant. In paralyzed rats, 5-HT levels were unchanged, but the 5-HIAA/5-HT ratio was elevated, especially in the compressed segments. Treatment with either dexamethasone or indomethacin delayed onset of paraplegia but had no effect on 5 HT metabolism. Blocking 5-HT receptors by cyproheptadine, evaluated 30 hours after onset of paralysis and treatment, resulted in a reduction in the high water content, vascular permeability, and prostaglandin E2 synthesis in the compressed cord. Early administration of cyproheptadine effectively delayed the onset of paraplegia. These data indicate that receptor-activated serotonergic mechanisms participate in the disruption of the blood-spinal cord barrier and that these effects can be manipulated pharmacologically. PMID- 1707148 TI - Cells secreting antibodies to myelin basic protein in cerebrospinal fluid of patients with Lyme neuroborreliosis. AB - An autoimmune response to myelin basic protein (MBP) has been proposed to participate in the development of the chronic neurologic manifestations that may accompany Borrelia burgdorferi-induced Lyme disease. Using an immunospot assay, we counted cells secreting antibodies to MBP. Anti-MBP IgG antibody-secreting cells were detected in CSF from eight of 13 consecutive patients with Lyme neuroborreliosis irrespective of stage of disease. The numbers were between 1/370 and 1/5,000 CSF cells (mean, 1/1,250 in the 13 patients). The highest numbers were encountered in two patients with severe signs of CNS involvement. The numbers decreased in parallel with clinical improvement after treatment. Anti-MBP IgG antibody-secreting cells were also observed in the CSF from patients with a variety of other inflammatory diseases of the nervous system, and their role in the development of tissue damage remains unsettled. Anti-MBP IgG antibody secreting cells were not detected in the patients' blood, reflecting accumulation of this autoantibody response to CSF. PMID- 1707149 TI - The effects of sucralfate suspension and diphenhydramine syrup plus kaolin-pectin on radiotherapy-induced mucositis. AB - A prospective, double-blind study compared the effectiveness of sucralfate suspension with diphenhydramine syrup plus kaolin-pectin in reducing severity and pain of radiation-induced oropharyngeal mucositis. Fourteen patients who received at least 4600 cGy to the oral cavity used one of the mouth rinses four times a day, beginning at 1600 cGy. Data were collected on daily perceived pain and helpfulness of mouth rinse, weekly mucositis grade, weight change, and interruption of therapy. Analysis of data revealed no statistically significant differences between the two groups in any parameter. A retrospective review of 15 patients who had received at least 4600 cGy radiation to the oropharynx but had not used a daily mouth-coating rinse, was compared with the study group. Comparison of the two groups suggested that consistent daily oral hygiene and use of a mouth-coating agent will result in less pain and may reduce weight loss and interruption of radiation because of severe mucositis. PMID- 1707150 TI - Anchorage-independent growth and the expression of cellular proto-oncogenes in normal human epidermal keratinocytes and in human squamous cell carcinoma cell lines. AB - The expression of multiple cellular proto-oncogenes and the in vitro anchorage independent growth of normal human epidermal keratinocytes and several human squamous cell carcinoma cell lines were studied and correlated. Squamous cell carcinoma cell lines KB, Si Ha, HEp-2, and Fa Du showed high anchorage independency, and MS 751 and A-253 cell lines had minimum independency. However, the normal keratinocytes and the A-431 cell line did not show anchorage independent growth. Both the normal human epidermal keratinocytes and cancer cell lines expressed multiple proto-oncogenes such as src, erb B-1, abl, fos, raf, H ras, and myc, and the amount of expression of these oncogenes was notably higher in the cancer cell lines than in the normal keratinocytes. The expression of proto-oncogenes from the monolayer cultures of the cancer cell lines is poorly correlated with the anchorage independency of the cells. These data indicate that the anchorage independency is not directly linked to the expression of specific cellular proto-oncogene(s) of the monolayer cancer cell cultures. PMID- 1707151 TI - Experimental oral foreign body reactions: vegetable materials. AB - Foreign bodies and tissue reactions to foreign materials are commonly encountered in the oral cavity. Exogenous materials most commonly causing foreign body reactions are metallic in origin (usually amalgam). Of the nonmetallic materials seen during biopsies, suture materials and vegetable matter are most often observed. Since many foodstuff foreign materials are unidentifiable histologically, common vegetables were experimentally implanted subcutaneously in rats to assess local host responses and to characterize the nature of these materials microscopically. The histologic characteristics of these vegetable foreign body reactions are detailed herein. The implanted materials correspond to reactions seen in human subjects. PMID- 1707152 TI - Conservation of structure and expression of the c-yes and fyn genes in lower vertebrates. AB - The src-gene family in mammals and birds consists of 9 closely related protein tyrosine kinases. We have cloned the c-yes and fyn homologues of the src-family from the teleost fish Xiphophorus helleri. Both genes show a high degree of sequence conservation and exhibit all structural motifs diagnostic for functional src-like protein tyrosine kinases. Sequence comparisons revealed three domains (exon 2, exons 3-6, exons 7-12) which evolve at different rates. Both genes exhibit an identical expression pattern, with preferential expression in neural tissues. No transcripts of c-yes were found in liver which is contrary to the situation in higher vertebrates. In malignant melanoma, elevated levels of c-yes and fyn were detected indicating a possible function during secondary steps of tumor progression for src-related tyrosine kinases. PMID- 1707153 TI - Decrease of c-erbB-2 and c-myc RNA levels in tamoxifen-treated breast cancer. AB - The c-myc, c-erbB-2, hst and int-2 oncogenes are frequently amplified and/or overexpressed in human breast carcinomas. We studied the effect of tamoxifen on RNA levels of these oncogenes in 19 breast cancer patients treated for 3 weeks prior to surgery as compared with 22 control patients. RNA levels were measured by in situ hybridization coupled with computer-aided quantification. c-myc and c erbB-2 expression was high in the control population (mean values: 23.4 and 29.1 grains/cell respectively) and significantly decreased in the tamoxifen-treated population (mean values: 14.6 and 7.4 grains/cell respectively) (P = 0.018, P = 0.003 respectively); hst and int-2 RNA levels were low (2-6 grains/cell) and not significantly altered by the treatment. There was a correlation between gene amplification and expression for c-erbB-2 (P = 0.0005) and hst (P = 0.02) in the control population. Elevated c-erbB-2 RNA level was correlated with the absence of estrogen (P = 0.02) or progesterone (P = 0.05) receptors. In the ER+ population, the tamoxifen-treated group had significantly lower c-myc expression levels than the control group (P = 0.04) which is in agreement with the estrogen induction of c-myc in ER+ T47D cell line and its inhibition by antiestrogens. Surprisingly, c-erbB-2 expression in the tamoxifen-treated group was significantly diminished in the ER- (P = 0.02) and PR- (P = 0.01) populations. This effect was not observed in the ER- BT474 cell line. These results suggest that in vivo tamoxifen decreases c-myc and c-erbB-2 RNA levels in breast cancer cells via two different mechanisms. To our knowledge this is the first evidence of in vivo down regulation of a gene by tamoxifen in ER- breast cancer cells. PMID- 1707154 TI - Kinetics of early HIV-1 gene expression in infected H9 cells assessed by PCR. AB - The time course of viral gene expression in H9 cells acutely infected with HIV-1 was analysed by the polymerase chain reaction (PCR). Virus-specific sequences were first detected in genomic DNA of H9 cells 1-2 h after infection. RNA for the regulatory genes such as the tat and nef appeared 2-3 h post-infection and RNA for the gag and env at 3 h. The results demonstrate that viral DNA synthesis occurs rapidly after infection of target cells followed by synthesis of viral RNA. Cell-associated reverse transcriptase activity increased after 24 h, while culture supernatant enzyme activity increased later, between 1-5 days. The delay in virus release after rapid integration and transcriptional activity suggests the involvement of additional factors, perhaps both cellular and viral, that control the formation and budding of mature virions. PMID- 1707155 TI - [Use of substance P fragments in experimental treatment of alcoholism]. AB - Experiments on unbred male mice demonstrated qualitative differences in the pharmacological effect of substance P fragments (P1-4 and P5-11) injected intraperitoneally in a dose of 250 mcg/kg. Fragments P1-4 and P1-4 dodecylamide on the one hand, had a positive effect on the level of motivation which was displayed by lesser consumption of alcohol by the animals, and, on the other, possessed immunocorrective properties manifested by restoration of macrophage phagocytic activity and immune response to sheep erythrocytes, which were impaired by chronic alcoholic intoxication. Fragment P5-11 had no effect on alcoholic motivation and produced a directly contrary action on the immune system, it inhibited the formation of antibody-forming cells. PMID- 1707156 TI - Follow-up of infants treated with extracorporeal membrane oxygenation for newborn respiratory failure. AB - Follow-up studies were conducted to assess the medical and developmental outcome of 92 infants treated with extracorporeal membrane oxygenation at the University of Michigan. Of 118 near-term (greater than 34 weeks' gestation) infants who received extracorporeal membrane oxygenation, 103 (87%) were surviving and available for follow-up at between 1 and 7 years of age. Ninety-two of these children were seen on at least one occasion. Each visit included a history and physical examination, an evaluation by a physical therapist, and developmental testing by a pediatric psychologist. Medical outcome during year 1 found 31% of the children rehospitalized, primarily with respiratory illness. Outpatient treated lower respiratory tract illness was seen in an additional 31% of the children. New or nonstatic neurologic problems were noted in 6% of the children. Abnormal growth during year 1 occurred in 26% of the children. At last clinic visit 16% of the children exhibited moderate-to-severe neurologic abnormalities, and 8% had moderate-to-severe cognitive delay. Sensorineural hearing loss occurred in 4% of children. Nine percent of the children were receiving speech and language therapy; screening tests showed that an additional 6% had speech and language delay. Overall, at last visit 16 (20%) of the children exhibited some type of handicap. A review of the literature on follow-up studies of non extracorporeal membrane oxygenation-treated infants with persistent pulmonary artery hypertension produced an impairment rate of 18.5%. Outcome post extracorporeal membrane oxygenation appears similar to that seen in less ill cohorts of infants treated with more "conventional" therapy. Long-term follow-up of all such infants remains essential. PMID- 1707157 TI - Ten years of extracorporeal membrane oxygenation: neurodevelopmental outcome. AB - Cf the 87 survivors of extracorporeal membrane oxygenation over a 10-year period, 67 participated in a follow-up study which included neurologic examination (n = 67), cognitive testing (n = 67), and audiologic assessment (n = 33). Matched control subjects for those older than 5 years were also evaluated. Outcome was defined as normal for cognitive scores greater than or equal to 85 and normal neurologic examination results, suspect for cognitive scores 70 through 84 or nonfocal neurologic findings such as hypertonia/hypotonia, and abnormal for cognitive scores less than 70 or abnormal neurologic examination results. Of the 10 school-aged children studied, 9 were normal and there were no differences in mean cognitive scores between subjects and controls (IQ subjects = 109 +/- 12 [SD], IQ controls = 107 +/- 13). For preschoolers aged 2.7 through 4.11 years, the mean cognitive score was 91 +/- 11 and 7 (70%) were normal. For infants 6 through 30 months, the mean cognitive score was 101 +/- 22 and 27 (57%) were normal. A total of 7 children (21% of those studied) had abnormal audiologic assessments. Three children demonstrated mild high-frequency and 4 moderately severe high-frequency sensorineural hearing loss which was bilateral in 3 and of undetermined laterality in 1. Abnormal neurodevelopmental outcome was significantly associated with cerebral infarction and chronic lung disease. Outcome was not related to demographic or perinatal variables, illness severity prior to extracorporeal membrane oxygenation, or underlying diagnosis. Neurodevelopmental outcome among survivors of extracorporeal membrane oxygenation in this series is consistent with previous reports of morbidity among neonates with severe respiratory failure treated conventionally. PMID- 1707158 TI - Hyperpolarization-activated cationic channels in smooth muscle cells are stretch sensitive. AB - The properties of hyperpolarization-activated channels were studied in single smooth muscle cells from the stomach of the toad, Bufo marinus, using the patch clamp technique. In cell-attached patches, inward channel currents were activated by hyperpolarizing pulses from a holding potential of -20 mV to potentials more negative than -60 mV. The activity of the channels increased and their latency of activation decreased as the hyperpolarization was increased. The slope conductance of the channels with standard high sodium concentration pipette solution was 64.2 +/- 9.1 pS (SD, n = 17). Stretching the patch, by suction applied to the back of the patch pipette, also increased the activity and shortened the latency of activation. We designate these channels as HA-SACs (hyperpolarization- and stretch-activated channels). HA-SACs were observed in 83% (175/210) of the patches studied. HA-SAC currents were carried by sodium and potassium ions, but their amplitude was increased by replacing extracellular sodium with potassium. Extracellular magnesium and calcium ions significantly reduced the single-channel conductance of HA-SACs. These permeation characteristics and the single-channel conductance of HA-SACs were indistinguishable from those of stretch-activated channels (SACs) previously described in these cells. The following observations are consistent with HA-SACs being a subset of SACs. First, SACs were at times found in cell-attached patches which lacked HA-SACs. Second, the number of channels in a cell-attached patch simultaneously activated by stretch (usually 5-10 and often more) exceeded by far the number simultaneously activated by hyperpolarization (usually one or two).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707159 TI - Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses, studied in adult vertebrate muscle. AB - Skeletal muscles of adult mice and frogs were dissociated enzymatically and prepared for patch-clamping within less than 6 h. Outside-out patches were superfused with repetitive pulses of acetylcholine (ACh) with switching times of about 0.2 ms. Peak responses were reached within 1 ms. In mouse muscle the average channel conductance was 65 pS and the average open time 1 ms (20 degrees C). Between 1 and 10 microM ACh, the peak responses increased proportional to the second to third power of the ACh concentration, and less steeply between 10 and 1000 microM ACh. The apparent Km of the dose-response curve was about 100 microM. After the peak, channel opening probability declined with time constants decreasing from about 1 s with 1 microM ACh to 15-50 ms with 1 mM ACh. After 100 ms desensitization the channel opening had decreased to less than 1/300 peak value. The rate of desensitization increased with rising temperature, with Q10 values of 1.7-2.5 between 10 and 30 degrees C. The desensitization characteristics of channels from frog muscle were similar to that from mice. With pulses of 100 microM ACh the channels opened with a probability of 0.55, the open probability declining with a time constant of about 60 ms and dropping to less than 0.001 after 300 ms. The results support the view that three binding steps of ACh are necessary for opening of the channel. Desensitization in the presence of high ACh concentrations is slower than the decay of synaptic currents. PMID- 1707160 TI - MspI polymorphism at the human complement component C6 gene (C6). PMID- 1707161 TI - Sequence polymorphism in the human alpha-2-macroglobulin (A2M) gene. PMID- 1707162 TI - The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer. AB - Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism. PMID- 1707163 TI - DNA protein interactions at the interferon-responsive promoter elements: potential for an H-DNA conformation. AB - The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two B1 and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the B1 complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OL1 were recovered in B2. For each probe, the possibility of the part of the sequence involved in B1 complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secondary structure, the formal existence of a H-DNA structure has not been demonstrated. Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations. PMID- 1707164 TI - Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies. AB - By sequencing cDNA clones which cover the complete coding region of human tenascin (TN), we have established its primary structure. This confirms that, as in the case of chicken, TN is mainly made up of three groups of sequences with a high homology to Epidermal Growth Factor (EGF) fibronectin (FN) type III repeat and fibrinogen. Furthermore, we have determined the amino-terminal sequence of the mature peptide directly on purified TN. The main differences with respect to the chicken TN molecule are that in the human there are 14 and half EGF-like repeats compared to 13 and half in the chicken and that, as previously reported, there are 15 FN-like repeats compared to 11 in the chicken. By Polymerase Chain Reaction (PCR) amplification we have also studied the different splicing patterns of the TN pre-mRNA in cultured cells. The results show the presence of at least four different isoforms containing different numbers of FN-like type III repeats. Using purified human TN as immunogen, we have obtained numerous monoclonal antibodies (Mabs) to TN. By screening a human melanoma cDNA library in the expression vector lambda gt11 with these Mabs and subsequently sequencing the insert of the positive clones, we have been able to localize, within the TN molecule, the epitopes recognized by two of these Mabs: BC-4, which recognizes an epitope within the EGF-like sequence and BC-2 which recognizes an epitope within the FN like type III repeats whose expression is regulated by alternative splicing of the TN pre-mRNA. Thus, while the Mab BC-4 may be useful in studies on TN distribution (since it recognizes all different TN isoforms) BC-2 may be useful in the study of the expression of particular TN isoforms generated by the alternative splicing of the TN primary transcript. PMID- 1707165 TI - Fidelity of reverse transcriptase of the simian immunodeficiency virus from African green monkey. AB - The in vitro fidelity of highly purified recombinant reverse transcriptase from simian immunodeficiency virus of African green monkeys (SIVagm) was determined. By using the phi X174am16 reversion assay an overall error rate of 1/19,000 was determined. This is 2.4-fold higher than the overall accuracy of purified recombinant HIV-1 reverse transcriptase, measured in parallel. The evaluation of error frequencies from nucleotide pool bias studies suggest an even higher accuracy for the SIVagm-derived reverse transcriptase. T:dGMP mismatches were formed most frequently with an error rate of 1/155,000, followed by G:dGMP (1/230,000), A:dGMP (1/315,000), G:dAMP (1/340,000), T:dCMP (1/540,000), T:dTMP (1/790,000), and A:dCMP (1/1,050,000) mispairs. Thus, according to pool bias effects and depending on the mismatch under consideration SIVagm reverse transcriptase appears to be 2 to 20-fold more accurate than the homologous enzyme from the human immunodeficiency virus type 1. This higher accuracy is not due to a co-purifying exonuclaease activity. Like the enzyme from HIV-1, the simian monkey-derived enzyme was found to be devoid of a proofreading 3' to 5' exonuclease. PMID- 1707166 TI - RNA blots: staining procedures and optimization of conditions. PMID- 1707167 TI - A new polymorphic probe on chromosome 3p: lambda LIB13-49' (D3S723). PMID- 1707169 TI - IXth World Symposium on Cardiac Pacing and Electrophysiology. May 28-31, 1991, Washington, D.C. Abstracts. PMID- 1707168 TI - Mspl RFLP at the D5S122 locus tightly linked to APC. PMID- 1707170 TI - Plasma protein synthesis and secretion in the visceral yolk sac of the fetal rat: gene expression, protein synthesis and secretion. AB - This report compares the relative levels of messenger RNA species coding for plasma proteins in rat visceral yolk sac and fetal liver from 12.5 days to 21.5 days gestation. Transthyretin, retinol-binding protein, transferrin and alpha 1 fetoprotein mRNAs were detected in both tissues, although relative levels were much higher in the yolk sac compared to fetal liver, in early gestation. Messenger RNA coding for the positive acute phase proteins thiostatin, fibrinogen, alpha 2-macroglobulin and alpha 1-antitrypsin were detected at a low but significant level in yolk sac, while the levels in fetal liver steadily increased from 16.5 days gestation and, with the exception of alpha 1 antitrypsin, reached levels higher than those found in adult liver just prior to birth. Albumin, inter-alpha 1-trypsin inhibitor, alpha 1-acid glycoprotein, haptoglobin, vitamin D-binding protein and ceruloplasmin messenger RNA levels were either very low or undetectable in yolk sac and fetal liver. Secretion of proteins by yolk sac endoderm occurred largely across the basolateral surface, i.e. towards the fetal compartment. These data support the hypothesis that one function of the yolk sac in the rat is the synthesis and secretion of a select group of plasma proteins to maintain homeostasis in the fetal compartment in the period before the fetal liver has matured sufficiently to carry out this function. PMID- 1707171 TI - Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties. AB - We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer. PMID- 1707172 TI - Differential induction of interferon gamma gene expression after activation of CD4+ T cells by conventional antigen and Mls superantigen. AB - We have analyzed cytokine gene expression by a murine CD4+ T-cell clone that expresses three forms of T-cell recognition. The clone employs a V beta 6 containing T-cell receptor to recognize (i) a self class II major histocompatibility complex and an ovalbumin-derived peptide (OVA), (ii) an I-Ab alloantigen, and (iii) Mls-1a. All three responses are accompanied by similar levels of cell proliferation. However, although interferon gamma gene expression is strongly induced during both physiological recognition of the OVA peptide and allogeneic major histocompatibility complex recognition, expression of this gene was not detected during the Mls response. These studies indicate that Mls recognition is functionally distinct from T-cell recognition of peptides and alloantigens and leads to an alternative pattern of cytokine gene expression. They also suggest the possibility that encounter with these two classes of T-cell antigen in vivo may generate subsets of T helper cells that display different patterns of cytokine gene expression. PMID- 1707173 TI - Neuronal NADPH diaphorase is a nitric oxide synthase. AB - NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide. PMID- 1707174 TI - Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA. AB - Constructs expressing the core, surface, X, or polymerase proteins of hepatitis B virus were transfected into human cells. In transient assays, only the polymerase inhibited the responses to interferons alpha and gamma (IFN-alpha and -gamma). Stable expression of the polymerase was achieved in the cell line 2fTGH, which carries an IFN-inducible marker gene, by growth under conditions that select for inhibition of the response to IFN-alpha, but the clones grew poorly. When expressed alone, the terminal protein domain of the polymerase gene inhibited the response to IFN-alpha and the reverse transcriptase plus RNase H domains appeared to be toxic. Clones of cells expressing terminal protein alone, selected for the loss of response to IFN-alpha, grew normally and had no detectable response to IFN-alpha, IFN-gamma, or double-stranded RNA. Binding of IFN-alpha to these cells was not impaired but did not lead to activation of the E alpha subunit of the IFN induced transcription factor E. These observations are of potential importance in relation to the pathogenesis of chronic hepatitis B virus infection and the resistance of such infection to IFN-alpha therapy. PMID- 1707175 TI - Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential. AB - Addition of exogenous acidic fibroblast growth factor (aFGF) to NBT-II epithelial carcinoma cells results in fibroblastic transformation and cell motility. We have generated aFGF-producing NBT-II cells by transfection with recombinant expression vectors containing human aFGF cDNA, or the human aFGF cDNA coupled to a signal peptide (SP) sequence. The effects of the nonsecreted and the secreted 16-kDa growth factor on the morphology, motility, and cell invasive potential (gelatinase activity) were compared. aFGF coupled to a SP was actively secreted out of the producing cells. The secretion of aFGF was not necessary for induction of gelatinase activity, as this was observed in NBT-II cells producing aFGF with or without SP. Production of aFGF, whether secreted or not secreted, resulted in increased in vitro motility of most isolated clones; however, there was no correlation between aFGF level and motility rate. The data suggest that expression of aFGF in NBT-II cells induces metastatic potential through an autocrine or intracrine mechanism. PMID- 1707176 TI - Photosensitization is required for inactivation of equine infectious anemia virus by hypericin. AB - Hypericin, a photoreactive polycyclic quinone, was found to dramatically reduce infectivity of cell-free stocks of equine infectious anemia virus. However, the antiviral activity of hypericin was completely dependent on the presence of light. Short periods of photosensitization resulted in a partial loss of reverse transcriptase activity and complete inhibition of viral infectivity. These results suggest that the photodynamic effect of hypericin interferes with more than one stage in the virus replication cycle. PMID- 1707177 TI - [Intra-arterial chemoembolization of non-resectable hepatocellular carcinoma]. AB - During a period of nine months, fifteen patients with extensive hepato-cellular carcinomas were subjected to palliative arterial chemo-embolisation using doxorubicin and cisplatin in an emulsion with Lipiodol and/or Spherex particles. Two patients (stage Child C) died within two weeks as the result of liver failure. The remaining thirteen patients showed improvement in their general condition. Subsequent CT showed evidence of tumour reduction and reduction of ascites. Gas production in the tumour was considered as evidence of tumour necrosis. Biochemical results confirmed these findings with a fall of AFP as tumour marker indicating tumour regression. PMID- 1707178 TI - [Treatment of Crohn disease]. AB - Crohn's disease is a chronic inflammatory disease of the bowel for which there is no curative treatment. The purpose of treatment is to reduce mortality to the absolute minimum and to give the patients a normal quality of life. Acute episodes of low severity are treated with sulfasalazine and its most recent derivatives such as 5-aminosalicylate. More severe episodes require oral corticosteroid therapy which, when prescribed in adequate doses, results in clinical remission in over 90% of the cases. To this must be added parenteral nutrition in patients with very severe symptoms or when a major nutritional deficit is present. During remissions, a maintenance treatment with 5 aminosalicylate or azathioprine is justified when the acute episodes are frequent and/or severe. These continuous chronic forms require prolonged low-dose corticosteroid therapy or azathioprine. Artificial nutrition is the best way of treating corticosteroid-resistant episodes. In children, corticosteroids must be avoided as much as possible, and low-flow rate enteral nutrition is often used as primary treatment of salicylate-resistant episodes. The surgical treatment of Crohn's disease consists of resection of the lesions with anastomosis or ileostomy in case of total proctocolectomy. Following resection-anastomosis, the cumulative relapse rate is about 50% ten years after surgery; it is lower after colostomy and proctocolectomy. Surgery is indicated in case of complications (abscesses, stenosis, fistulae, perforation) and when the disease does not respond to a well-conducted medical treatment. Specialized teams including physicians, surgeons, proctologists and specialists in nutrition are essential to a correct treatment of Crohn's disease. PMID- 1707179 TI - Evidence of intracellular activation of serine proteases in acute cerulein induced pancreatitis in rats. AB - It is believed that activation of zymogen proteases occurs in the early development of acute pancreatitis. This hypothesis was proved on subcellular fractions of rat pancreas after induction of pancreatitis by infusion of high doses of cerulein for 2 h. Secretory enzyme activities were measured spectrophotometrically in subcellular fractions obtained by differential ultracentrifugation. Additionally, trypsin and chymotrypsin activities were detected by enzyme blots after isoelectric focusing. Finally immunoblotting (Western-blot analysis) for amylase, lipase, trypsin/ogen, and chymotrypsin/ogen was carried out on fractions separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In cerulein pancreatitis, subcellular fractions of secretory granules and vacuoles showed significant amounts of free trypsin and chymotrypsin activities compared with controls. The presence of free activities of serine proteases was paralleled by the appearance of numerous low molecular weight peptides detected by 2-dimensional electrophoresis and SDS-PAGE, which in part represented proteolytically cleaved secretory proteins. It is concluded that the intracellular activation of serine proteases that occurs in cerulein pancreatitis could contribute to further acinar cell destruction. PMID- 1707180 TI - Activated CD3- CD16+ natural killer cells express a subset of the lymphokine genes induced in activated alpha beta + and gamma delta + T cells. AB - In this study we analysed the potential of highly purified polyclonal TcR alpha beta+, TcR gamma delta + and CD3- NK cells, to produce lymphokines in response to mitogenic stimulation. RNA hybridizations were performed to detect with high sensitivity the induction of multiple lymphokine genes. Upon stimulation with lectin and phorbol ester TcR gamma delta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes, which included IL-2, -3, 4, -5, GM-CSF, TNF alpha and beta, IFN gamma. In contrast, a more limited set of lymphokine genes (GM-CSF, TNF alpha and beta, IFN gamma) was induced in activated CD3- NK cells, thus indicating that this subpopulation of cells may display different regulatory functions, with respect to CD3+ T lymphocytes. PMID- 1707181 TI - Regulation and functional involvement of distinct determinants of leucocyte function-associated antigen 1 (LFA-1) in T-cell activation in vitro. AB - The expression of leucocyte function-associated antigen 1 (LFA-1) was studied by immunofluorescence method on human peripheral blood mononuclear cells (PBMC) stimulated by the phytohaemagglutinin lectin (PHA). Monoclonal antibodies (MoAb) Mas 191c, IOT18 (directed against the beta-chain, 95 kDa, CD18) and IOT16, SPVL7, MHM24 (identificating the alpha-chain, 180 kDa, CD11a) were used, defining the 'CD11a/CD18' antibody panel. By means of cross-linking or competitive experiments, we showed that these antibodies recognized at least four distinct and spatially distant domains on the LFA-1 molecule. Immunofluorescence analysis revealed that the up-regulation of LFA-1 expression was a late event, similar to the expression kinetics of the HLA DR and CD38 molecules, and followed the appearance of CD25 and CD71 molecules. Moreover, it was established that the LFA 1 up-regulation required mRNA and protein synthesis. Functional activity comparison of the different anti LFA-1 MoAb showed that the CD11a MoAb significantly inhibited the proliferation of lymphocytes stimulated by the phytohaemagglutinin to various extents, as the LFA-1 alpha determinant identified. By contrast, the CD18 MoAb did not influence strongly this cell process. We observed only a dim inhibitory effect with the CD18 MoAb recognizing an epitope common or very close to an LFA-1 alpha determinant. These results suggested that the LFA-1 antigen was important, at a molecular level, in the regulation of T-cell activation. PMID- 1707182 TI - The influence of T cells on the immunoglobulin repertoire and the affinity maturation of the immune response against dextran B512 in C57BL/6 mice. AB - A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency and antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of kappa light chain. A major anti dextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response. PMID- 1707183 TI - Auto- and polyreactivity of IgM from CD5+ and CD5- cord blood B cells. AB - The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population. PMID- 1707184 TI - [Variability of RNA viruses as exemplified by Togaviruses]. PMID- 1707185 TI - A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis. AB - Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication. PMID- 1707186 TI - Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. AB - The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded. PMID- 1707187 TI - Anterior and combined anteroposterior fusion for lumbar disc pain. A preliminary study. AB - This article reports on a study of 51 consecutive patients (83 lumbar discs) with back pain who underwent anterior interbody lumbar fusion or combined anterior and posterior fusion at the same operation during a 2-year period. All patients met the criteria for diagnosis of a painful internal disc disruption and/or failed back syndrome and have had a lengthy trial of conservative treatment consisting of rest, physical therapy, back support, nonsteroidal anti-inflammatory drug therapy, and guarded activity; this treatment was often supplemented by epidural cortisone injections, pain management, and functional rehabilitation. Patients with prolonged back pain who failed with conservative care after a minimum of 12 months of severely disabling symptoms were selected for surgery on the basis of a positive dynamic discogram reproducing their exact pain and demonstrating a morphologically degenerative disc (internal disc disruption). For the purpose of this study, patients were categorized into three groups and followed up for 15-36 months after the operation. There were no deaths or major complications, and the overall success in achieving measurable diminution of preoperative pain was 80%. This article discusses preliminary conclusions on the efficacy and safety on anterior and anteroposterior fusion for lumbar disc pain. PMID- 1707188 TI - Stem cell factor (SCF), a novel hematopoietic growth factor and ligand for c-kit tyrosine kinase receptor, maps on human chromosome 12 between 12q14.3 and 12qter. AB - Recently a novel hematopoietic growth factor, stem cell factor (SCF), was cloned and demonstrated to be the ligand for the c-kit tyrosine kinase receptor. In the mouse, SCF is encoded by Sl (steel), a gene critical to the development of several distinct cell lineages during embryonic life and which has important effects on hematopoiesis in the adult animal. The Sl/SCF locus maps to the distal region of mouse chromosome 10, in the vicinity of genes that have been mapped to human chromosome 12. Here we report the use of somatic cell hybrid lines to localize SCF to the long arm of human chromosome 12, between 12q14.3 and 12qter. In addition to localizing the Sl homolog in man, these data provide further evidence for the conservation of synteny between the long arm of human chromosome 12 and the distal end of mouse chromosome 10. PMID- 1707190 TI - Senile dementia and presenile dementia. AB - Neurotransmitters including acetylcholine, dopamine, norepinephrine, serotonin, GABA and vasopressin were examined in control subjects and patients with Alzheimer-type dementia, involving presenile and senile dementia. Neurotransmitters exhibited various mode of changes with aging. Abnormalities found in senile or presenile dementia were not always parallel to the age-related changes. These results suggest that Alzheimer-type dementia cannot be understood as an accelerated senescence, but other etiological factors might be introduced for the manifestation of the dementia. Moreover, the disturbance in neurotransmitters revealed a difference between presenile Alzheimer's disease and senile dementia, indicating that further studies should be carried out taking the age of onset into consideration. PMID- 1707189 TI - Immunoreactive calcium-binding protein (calbindin-D28k) in interneurons and trigeminothalamic neurons of the rat nucleus caudalis localized with peroxidase and immunogold methods. AB - Calbindin-D28k is a highly abundant protein found in neurons in selected brain regions, including cells in sensory systems of the brainstem. Because of its capacity to bind cytosolic Ca++, calbindin-D28k is thought to contribute to the regulation of compartmental Ca++ concentrations in neurons. In this study of the rat spinal trigeminal nucleus, calbindin-D28k was localized with immunoperoxidase and immunogold methods. Results showed that immunoreactive calbindin-D28k neurons were widely distributed to all regions of the nucleus, but were particularly numerous in the substantia gelatinosa. Some trigemino-thalamic neurons that were identified by retrograde labeling of a conjugated wheat-germ agglutinin with horseradish peroxidase also contained calbindin-D28k immunoreactivity. Most of the calbindin-D28k labeling was found in cell bodies and dendrites. Axon terminals were rarely stained. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also heavily associated with euchromatin within nuclei. These findings show that immunoreactive calbindin-D28k is localized to both interneurons and projecting neurons of the spinal trigeminal nucleus. Many of these cells are likely to receive glutamatergic afferent inputs, which may act in part by increasing Ca++ flux into the neurons. Calbindin-D28k has a high capacity for buffering Ca++ and under some conditions may protect neurons against glutamate-induced excitotoxicity. We speculate that calbindin-D28k may function to regulate calcium concentrations in spinal trigeminal neurons. PMID- 1707191 TI - [The effect of the high-molecular components of the blood serum--gamma-globulin and albumin--on the sodium channel activity of neuroblastoma cells]. AB - Blood serum gamma-globulin and albumin were tested for their effect on the sodium currents of voltage clamped internally perfused serum deprived neuroblastoma cells. Albumin in the concentration corresponding to its content in 5% blood serum (22 microM) produced an increase in sodium channel currents and shifts in activation and inactivation curves along the voltage axis towards more negative or positive potentials, respectively. These results both qualitatively and quantitatively reproduce the effects of 5% blood serum observed previously (Zubov, Sal'nikov, 1984, 1986). The addition of gamma-globulin failed to change the parameters registered. The data obtained led us to an assumption of the role of albumin as an active substance responsible for the effect of blood serum on the potential-dependent sodium-transporting system of neuroblastoma cell membrane. PMID- 1707192 TI - [Thymic cysts apropos of 2 cases]. PMID- 1707193 TI - Malignant myoepithelioma of the larynx with massive metastatic spread to the liver: an ultrastructural and immunocytochemical study. AB - A malignant myoepithelioma arising in the submucosal glands of the larynx of a 71 year-old man is reported. The patient presented with a neck mass and massive metastatic involvement of the liver. Light microscopy of a liver biopsy specimen and fine-needle aspiration sample of the neck mass revealed a poorly differentiated carcinoma. Electron microscopic study of a second liver biopsy specimen demonstrated unequivocal features of myoepithelial differentiation; this was further confirmed by the strong cytokeratin and S-100 protein positivity and carcinoembryonic antigen negativity of the tumor cells. Myoepitheliomas are rare tumors, and most reported cases have been benign or of low-grade malignancy. The present case is unique because of its mode of presentation and fulminant course. It also underscores the usefulness of electron microscopy as a diagnostic modality in the work-up of metastatic lesions. PMID- 1707194 TI - Phenotypic cytoskeletal heterogeneity in melanoma, a challenge to the surgical pathologist evaluating a poorly differentiated neoplasm: an illustrative case. AB - Characterization of poorly differentiated neoplasms can be a challenging task for the surgical pathologist. It is essential that the entire spectrum of immunomorphologic findings of various tumors be recognized to avoid improper characterization of a given neoplasm, which may in turn adversely affect patient management. Tumor characterization is complicated by the immunomorphologic transformations that malignant cells may undergo by virtue of which they may depart from expression of expected features and acquire new, unexpected characteristics. Traditionally, amelanotic melanomas have been difficult to characterize because of the diversity of their light microscopic morphology (epithelioid, spindle, and combined varieties). As a result, several other neoplasms are usually considered in the differential diagnosis. This report describes a primarily spindle-cell amelanotic melanoma that created a diagnostic dilemma, which could only be resolved by combining the information obtained from extensive evaluation by means of several diagnostic techniques. This case also stresses the phenotypic heterogeneity of the cytoskeleton of malignant melanomas and therefore their varied immunomorphologic characteristics. PMID- 1707195 TI - [The interferon status in different diseases]. PMID- 1707196 TI - [Selection of antigenic variants of the influenza virus on the cells of different hosts]. AB - Antigenic differences were found in influenza B virus variants isolated and propagated in different systems: chick embryos (E variants) and MDCK cell culture (M variants). The antigenic differences in M and E variants were detected in HI tests with polyclonal mouse sera and monoclonal antibodies as well as in biological neutralization tests in chick embryos and MDCK cell culture, and confirmed when M and E variants were used as antigens for antibody detection in human sera. By protein mobility in PAGE, M and E variants did not differ from each other and were also identical with the reference B/Victoria/87 strain. PMID- 1707197 TI - [Production of virus-induced and mitogen-induced interferons by canine cells]. AB - Cells of monocytic-phagocytic series and immunocompetent cells derived from dogs: bone marrow, leukocytes, spleen, thymus, lymph nodes (with the exception of the liver) were found to be capable of producing interferon (IFn) in vitro in response to inoculation of viral and non-viral inducers. This capacity is manifested in different ways: thymus and lymph node cells were less active interferon producers. Studies of physicochemical properties of the resulting interferons confirmed the belonging of the virus-induced IFn to alpha interferons, PHA-induced IFn to immune alpha-IFn. The above-mentioned refers both to bone marrow and leukocyte interferons. PMID- 1707198 TI - [The antigenic specificity of the hemagglutinin and neuraminidase of influenza B virus reproducing in different cell systems]. PMID- 1707199 TI - Local recurrences following colorectal operations. AB - In about 15-20% of patients operated for colorectal tumours local recurrences develop mainly in the first postoperative year. This large number can only be reduced by adequately radical operations taking into account the patient's age, tumour site and the tumour-biological factors. Indispensable factors are the organized care and regular control of the operated patients with an emphasis, beside CEA test on US and CT studies. In local recurrences attempt should be made at removal of the tumour by an additional operation which is implementable in 20 30% of cases. For palliative treatment first of all radiotherapy can be applied for pain relief. PMID- 1707200 TI - Clinical significance of age-related changes of the palpebral fissures between age 2 and 18 years in healthy Caucasians. AB - The development of surface measurements of the palpebral fissures and age-related changes in the quality of the relationships between the individual measurements were followed in 1552 healthy Caucasians between ages 2 and 18. At age 2, the height of the palpebral fissure and the biocular width (ex-ex) were the most developed features (93.3% and 86%) and the least developed was the intercanthal width (77.6% to 82.9%). The measurements reached adult size between ages 8 (intercanthal width in girls) and 16 (palpebral fissure inclination in boys). The rate of growth in the orbital measurements was usually moderate, seldom above average and fast only in intercanthal width between ages 3 and 4. The study determined the periods with minimal growth (approximately ages 5 to 7 and 9 to 10) for each of the measurements. After maturation, the changes in measurements were minimal. A knowledge of the developmental levels of the measurements at an early age, their changes with age and their maturation times are of great importance in timing early or final corrective surgical procedures. PMID- 1707201 TI - Management of extensive burns in children. AB - 2723 children were treated in the Burn Department of the Pediatric Clinical Hospital No. 9, Moscow, during 3 years (1986-1988), among them 1465 children being under 3 years of age. Since 1985, the Department has been using the beds of "Clinitron" type, 79 children (3 months-14 years of age) with extensive burns having been treated on the Clinitron beds with air-fluidized pillows. During the treatment, the children received the usual therapy accepted in the Department. The use of the air-pillows beds promoted rapid drying of the wounds, accelerated epithelialization of the superficial burns, reduced the periods of the wound preparation for autodermoplasty for deep burns, prevented rejection and lysis of the replanted grafts. PMID- 1707202 TI - Applications of islet musculocutaneous flap from the trapezius muscle for primary plastic operations of oral cavity defects. AB - 13 patients after major resection of oral cavity malignancies had primary plastic operation performed using trapezius muscle musculocutaneous flaps to cover the defect. The flaps were mobilized on a vascular pedicle supplied by the superficial cervical artery and drained by the concomitant vein. Partial flap necrosis was seen in two patients--mainly due to thrombosis of the vein of the pedicle. The vein should have an inside diameter of at least 0.4 cm. In the remaining cases, satisfactory functional and aesthetic results were obtained. In the flap mobilization region there is, as a rule, no growth of hair, which is important as the skin layer of the flap provides the inner lining of the oral cavity. PMID- 1707203 TI - Assessment of disinfectant topical agents in keratinocyte cell culture grafting technique in severely burned patients. AB - On the basis of previous experiences and of international literature data the authors emphasize the importance of making a "targeted" choice of the topical disinfectant in the therapy of burn wound infections. The objective of the investigation is to reach the highest rate of take of the autologous keratinocyte cultures in burn wounds. PMID- 1707204 TI - Thumb reconstruction using free flap transfer. AB - The authors present a choice of thumb reconstruction method using free transfer of sensitive radial artery flap together with autologous bone graft implantation in a single-time operation. They regard this approach as more advantageous than the standard tubed flap techniques. The advantages are the following: 1. single time operation, 2. flap tissue sensitivity, and 3. satisfactory shape of the reconstructed thumb, requiring no subsequent modellation. Photographs show the surgical procedure and three other casuistics. PMID- 1707205 TI - Arthroplasty for major defects of articular areas of fingers. AB - The costal perichondrium is a suitable material for the reconstruction of major damage to articular areas, even bone tissue in small joints of the hand. A modelled silicone implant conditions and simultaneously defines the shape of the reconstructed areas. Together with the perichondrium reconstruction, it helped to fill the defect and for a suitable shape of the articular areas. Morphological findings of the perichondrium transformation showed a steady growth and a rising frequency of hyalinization of larger foci containing chondrocyte-like cells. After 8 years the cartilage surface showed a thick layer of hyaline cartilage. PMID- 1707206 TI - Immunoreactivity of canine and feline polyglucosan bodies for monoclonal antibody against human polyglucosan. AB - With the use of monoclonal antibodies, raised against the human polyglucosan, positive staining of polyglucosan bodies (PGB) was detected in the brain, spinal cord and cecum of aged dogs. PGB in feline brain were also positively stained with these antibodies. These findings indicate that animal PGB share common antigenicity with human PGB. PMID- 1707207 TI - Polar spongioblastoma: an immunohistochemical and electron microscopical study. AB - A case is reported of a 9-year-old boy with a cerebral polar spongioblastoma. This neoplasm, first described by Russell and Cairns in 1947, is morphologically a distinct entity characterized by bipolar tumor cells with palisading nuclei. In the case under study immunoreactivity for neuron-specific enolase was found and ultrastructural features of developing neuronal elements were present. A neuro endocrine nature was suggested by de Chadarevian et al. (1984) in a morphologically similar case. These findings are in contrast with the long-held view that the polar spongioblastoma is cytogenetically related to the embryonal radial glial cells. PMID- 1707208 TI - A case of I-cell disease. AB - A case of I-cell disease is reported. The patient suffered from several episodes of pneumonia, and died of pneumonia at 12 months of age. Tissue specimens obtained at autopsy were stained with colloidal iron to demonstrate acid mucopolysaccharides. Characteristic foamy changes were observed in organs such as the heart, kidneys, liver, spleen and brain. An interesting finding in this case was that not only the interstitial cells but the alveolar epithelium in the lung showed the same foamy changes. The major causes of death of patients with I-cell disease are congestive heart failure and recurrent respiratory infections. However, there have been few reports on the histological changes in the lungs, and none have described the changes in the alveolar epithelium. Further cases must be investigated to examine the pathological relation between the histological changes in the lungs and the cause of death, because recurrent respiratory infections are the major contributor to death in patients with I-cell disease. PMID- 1707209 TI - [Combined pharmacokinetic--pharmacodynamic model analysis of N-acetyl procainamids following intravenous infusion in rabbits]. AB - The pharmacokinetic and pharmacodynamic profiles of N-acetyl procainamide were analyzed by integrated PK-PD model following intravenous infusion to rabbits. No significant differences between the PK parameters estimated from iv administration and intravenous infusion were found. However, two of the PD parameters were shown to be significantly different. The values of Emax, Keo, S, EC50 were found to be 120 +/- 13.2 ms, 0.0182 +/- 0.007 min-1, 2.26 +/- 0.93, 6.31 +/- 0.71 microgram/ml respectively following intravenous infusion; the corresponding values following iv administration were 53.6 +/- 2.5 ms, 0.061 +/- 0.017 min-1, 2.19 +/- 0.39, 6.21 +/- 1.74 micrograms/ml respectively. PMID- 1707210 TI - Endogenous nitric oxide as a probable modulator of pulmonary circulation and hypoxic pressor response in vivo. AB - The objective of this study was to investigate the role of endogenous nitric oxide, formed from L-arginine, in the regulation of pulmonary circulation in vivo, with special reference to the hypoxic pressor response. In artificially ventilated open-chest rabbits, pulmonary vascular resistance at normoxic ventilation (FIO2 = 21%) was 78 +/- 16 cmH2O ml-1 min 1000-1 (mRUL). Hypoxic ventilation (FIO2 = 10%) increased pulmonary vascular resistance to 117 +/- 17 mRUL. N omega-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase, increased pulmonary vascular resistance at normoxic ventilation to 192 +/- 28 mRUL and during hypoxic ventilation to 462 +/- 80 mRUL. During N omega nitro-L-arginine methylester infusion there was also an increase in mean arterial blood pressure as well as a decrease in cardiac output that was even more pronounced during hypoxic ventilation. L-arginine reversed the effect of N omega nitro-L-arginine methylester on pulmonary vascular resistance at normoxic ventilation to 140 +/- 26 mRUL and at hypoxic ventilation to 239 +/- 42 mRUL. In spontaneously breathing closed-chest rabbits, N omega-nitro-L-arginine methylester evoked a marked decrease in arterial PO2 and increases in respiration frequency and central venous pressure, while blood pH, PCO2 and base excess remained unchanged. Taken together these findings indicate that endogenous nitric oxide, formed from L-arginine, might be a regulator of ventilation-perfusion matching at normoxic ventilation, and that nitric oxide acts as an endogenous modulator of the hypoxic pressor response. PMID- 1707211 TI - Striatal dopamine and glutamate release: effects of intranigral injections of substance P. AB - The levels of extracellular striatal dopamine and glutamate were measured simultaneously in halothane-anaesthetized rats using microdialysis. Unilateral injections of substance P (0.07 nmol) into the substantia nigra, pars reticulata enhanced the levels of dopamine and glutamate in the ipsilateral striatum. Intranigral injections of neurokinin A (0.09 nmol) enhanced the levels of striatal dopamine, and intranigral injections of gamma-aminobutyric acid (300 nmol) or dynorphin A (0.5 nmol) decreased the levels of striatal dopamine, but none of these had any effect on the levels of striatal glutamate. Local perfusion with the dopamine agonists apomorphine (D1/D2), SKF 38393 (D1) or pergolide (D2) (each at 10(5) M) decreased the levels of striatal dopamine and enhanced the levels of striatal glutamate. In unilateral 6-hydroxydopamine-lesioned rats, basal striatal glutamate levels were decreased bilaterally. Furthermore, on the denervated side intranigral substance P stimulation of striatal glutamate levels was enhanced, while on the intact side intranigral substance P stimulation of striatal dopamine and glutamate levels was similar to that seen in normal rats. These findings suggest that striatonigral substance P provides a stimulatory regulation of ipsilateral striatal glutamate release. Furthermore, it is indicated that striatal glutamate release can also be regulated by dopamine terminals. PMID- 1707212 TI - Effects of intrathecal substance P and a substance P antagonist on a reflex to noxious heat are independent of changes in tail skin temperature. AB - Substance P or the substance P receptor antagonist (D-Arg1,D-Trp7.9,Leu11) substance P (Spantide) was injected into the lumbar subarachnoid space in mice, and the ability to change the tail-flick reflex and the tail skin temperature was investigated. Tail-flick latency (the time needed to evoke the tail-flick reflex by noxious radiant heat) was reduced for 1-4 min after intrathecal administration of substance P (5 micrograms), but the tail skin temperature was not significantly changed. Nor was the tail skin temperature significantly changed after intrathecal injection of Spantide (5 micrograms), but this compound significantly increased tail-flick latencies 5-30 min after injection. Analysis of co-variance showed that the effects of substance P or Spantide on tail-flick latency were significant, whereas the influence of tail skin temperature on tail flick latency was non-significant. Thus, intrathecal substance P induces a short lasting increase in nociceptive sensitivity, and intrathecal Spantide produces an antinociceptive effect of longer duration. The results seem not to be the result of changes in tail skin temperature. PMID- 1707213 TI - Increased negativity of interstitial fluid pressure contributes to development of oedema in rat skin following application of xylene. AB - Intradermal interstitial fluid pressure (Pi) has been studied in rat skin during formation of inflammatory oedema caused by application of xylene. Pi was measured with sharpened micropipettes connected to a servocontrolled counter-pressure system. Control Pi averaged -1.3 +/- 0.6 (SD) mmHg. Following xylene application Pi decreased to -5.0 mmHg after 5 min and then increased to stabilize at about 0 mmHg at 45-60 min and later. When the transvascular fluid shifts accompanying the inflammatory reaction were prevented by inducing circulatory arrest prior to xylene application, Pi fell to -7.5 mmHg within 5 min and remained at this level throughout the observation period of 90 min. Aprotinin in large doses (80,000 KIE kg-1) before xylene application reduced the fall in Pi, whereas indomethacin had no effect. The increased negativity in Pi will add directly to a normal transcapillary net filtration pressure of about 0.5 mmHg, resulting in a 10- to 20-fold increase in this pressure. The present experiments therefore suggest that the interstitium plays an active role in oedema formation in the initial phase of xylene-induced inflammation in rat skin through the development of an increased negativity of Pi. PMID- 1707214 TI - [Pathophysiology of intraocular neovascularization]. AB - The pathology of intraocular neovascularization was studied in human and animal eyes by means of electron microscopy and histochemistry, and also by tissue culture of bovine retinal small vessels. The newly formed vessels in the vitreous obtained at the time of vitrectomy from the eyes with proliferative diabetic retinopathy and retinal vein occlusion lacked tight junction and formed fenestration in the endothelial cells. When 1 microgram of the basic fibroblast growth factor (b-FGF) enveloped in ethylene vinyl acetate copolymer was implanted into the vitreous of monkey eyes, new vessels were formed in the iris in all eyes and in the vitreous in 12 eyes out of 14 experimental eyes. The origins of new vessels were the iris vessels in the iris, and both stromal vessels of the ciliary body and retinal vessels in the vitreous. These vessels showed fenestration in the endothelial cells. The activity of the lysosomal enzyme detected by acid phosphatase increased in the epithelial layer of the ciliary body. The new vessels in the vitreous of rabbits were seen in 9 out of 10 eyes when b-FGF was implanted in the vitreous with an intravitreal injection of amino adipic acid solution (1 mg in 0.2 ml of physical saline), although none of the 8 eyes formed new vessels when sodium iodate was injected intravenously after implantation of b-FGF with aminoadipic acid. Corneal neovascularization was formed by implantation of 250 ng of b-FGF into the corneal micropockets of the guinea pigs, and regressed after the b-FGF was removed. The endothelial budding and protrusion were frequently seen during the course of neovascularization. Immunohistochemical detections showed positive stainings for fibronectin in most front-end lesions, for laminin and type 4 collagen associated with the endothelial cells, and for factor VIII only on the endothelial cells which formed the vascular lumen. The acid phosphatase activity was detected on the leucocytes infiltrating in the corneal stroma. In the course of regression of corneal neovascularization, initial pathological change was thrombus formation followed by disappearance of endothelial cells, although the basement membrane of the endothelial cells remained. Fibronectin reduced its activity in the early stage of regression, laminin and type 4 collagen remained even after the vascular lumen had subsided, and factor VIII was stained in a geographically irregular manner. Migrating activity of the cultured-bovine retinal small vessels was accelerated by fibronectin and fetal bovine serum. PMID- 1707215 TI - [Clinical effect on tumor regression and tissue concentration of peplomycin treated with peplomycin emulsion in hydroxypropylcellulosum]. AB - Peplomycin emulsified in hydroxypropyl cellulosum (HPC-PEP) was prepared for intravesical chemotherapy. Clinical efficacy of HPC-PEP and tissue concentration of peplomycin (PEP) were studied in 12 patients with bladder tumor. Histopathology showed transitional cell carcinoma; 2 in grade 1,8 in grade 2, and 2 in grade 3. The total volume of 30 ml HPC-PEP was prepared from a mixture of 2% HPC and 90 mg PEP in 15 ml saline, and was intravesically administered through a urethral catheter and retained for two hours. Clinical evaluation 7 days after the initial instillation demonstrated good tumor regression in 2, good response in 5, and no change in 5. The mean PEP level in tumor tissue was 0.36 microgram/gr after 7 days and 0.19 microgram/gr even after 14 days. These clinical observations and tissue levels of PEP suggest that HPC-PEP might be useful as an intravesical instillation agent for bladder tumor. PMID- 1707216 TI - [Response to chemotherapy in endocrine therapy-relapsed and -resistant prostate cancer]. AB - Effects of chemotherapy to endocrine therapy (castration with estrogen/antiandrogen)-relapsed (24 cases) or endocrine therapy-resistant (14 cases) prostate cancer were compared. Pretreatment clinical stages in these groups were stage D1 (3 cases) and D2 (35 cases). Regimens of chemotherapy in this study were as follows: cis-platinum (CDDP) (1 case), phosphamide (3 cases), combination of vincristine, phosphamide and peplomycin (5 cases), combination of cyclophosphamide, adriamycin (ADM) and CDDP (8 cases) and combination of phosphamide, ADM, and CDDP (21 cases). Response to chemotherapy and subsequent survival in these two groups were examined. When evaluated at 3 months after the start of the chemotherapy, partial response and stable cases were 50% and 36% in endocrine therapy-relapsed and -resistant groups, respectively. Because the worse performance status contained more cases in the endocrine therapy-resistant group, the response was compared at the same base of performance status, and the response was almost equal in the two groups. Survival in the endocrine therapy relapsed group was better than that in the therapy-resistant group. When compared at the same base of performance status, the difference in survival time between the two groups was not evident. In conclusion, the response of chemotherapy was similar between endocrine therapy-relapsed and -resistant patients, and performance status was a main factor influencing the prognosis of endocrine therapy-refractory prostate cancer. PMID- 1707217 TI - [Relationship between reactivation and tumor markers in prostatic cancer]. AB - Twenty-seven cases of reactivated prostatic cancer between 1979 and 1990 were investigated. Reactivation took place in the form of local aggravation in 3 cases, occurrence or aggravation of metastasis to bones in 8 cases, and in both forms in 16 cases. The elevation of tumor markers preceded the clinical findings in 11 cases (41%). In 75% of the cases with occurrence or aggravation of metastasis, the elevation of tumor markers preceded the clinical findings. This showed that tumor markers were useful in most cases for early detection of reactivation. However, in 3 cases with local aggravation, the clinical findings preceded the elevation of tumor marks. Therefore, it is also important to check the clinical findings at the follow-up. At the time of reactivation, positive rates of prostatic acid phosphatase (PAP), gamma-seminoprotein (gamma-Sm) and prostatic specific antigen (PA) were 78%, 83% and 80%, respectively. Thus gamma Sm and PA appeared to be more reliable than PAP for monitoring of prostatic cancer. PMID- 1707218 TI - [Study of the long-term administration of ofloxacin to the patients following transurethral resection of the prostate]. AB - The present study was undertaken to evaluate the clinical efficacy of long-term administration of ofloxacin (OFLX) to the patients following transurethral resection of the prostate. The patients were randomly divided into two groups: A and B. All the patients were administered flomoxef (FMOX) intravenously for 3 days following transurethral resection of the prostate (TUR-P). In group A, 100 mg of OFLX twice daily was thereafter administered for 4 to 15 weeks to 22 patients until they showed an improvement in pyuria. In group B, which served as a control, neither OFLX nor any other antibiotics were administered to 26 patients until they showed an improvement in pyuria. No patients complained of urination trouble due to infection. At the same time, cultures of bacillus in the urine were also examined 4 days, 7 days and 2 weeks after TUR-P with these two groups. The mean days necessary for the improvement of pyuria were 64.9 +/- 20.5 in group A, 66.3 +/- 18.4 in group B. At 2 weeks after TUR-P, bacillus in the urine were negative in 19/22 patients in group A, and 14/26 in group B. Chi square test showed significance for these two groups. Accordingly, OFLX was useful for bacillus in the urine, but OFLX was not so useful for shortening the continuance of pyuria of post TUR-P. No patients complained of nausea or any other complications during the study. PMID- 1707219 TI - A new method for estimating preexcitation index without extrastimulus technique and its usefulness in determining the mechanism of supraventricular tachycardia. AB - The preexcitation index has been shown to be useful in determining the mechanism of paroxysmal supraventricular tachycardia (SVT) and the site of the accessory pathway in atrioventricular (AV) reentrant tachycardia. To test whether a preexcitation index could be computed analytically instead of by scanning the whole SVT cycle with extrastimuli, 19 patients with SVT were studied. The new index was computed using the following formula: (AV conduction time during SVT) + (ventriculoatrial conduction time during ventricular pacing at the SVT cycle length) - (SVT cycle length). There was a strong correlation between the preexcitation index determined by the extrastimulus technique and the new index in 15 patients in whom the preexcitation index could be determined (r = 0.99, p less than 0.01). The value on the new index was greater than 90 ms only in patients with dual AV nodal pathways. In the 4 patients in whom the preexcitation index could not be determined by the extrastimulus technique, the new index could differentiate AV reentrant tachycardia (index for 2 patients, 60 and 60 ms, respectively) from AV nodal reentrant tachycardia (index for 2 patients, 100 and 105 ms, respectively). In conclusion, the new index provided help in determining the mechanism of SVT, even when retrograde atrial preexcitation by a ventricular extrastimulus did not occur. PMID- 1707220 TI - Usefulness of the electrophysiology laboratory for evaluation of proarrhythmic drug response in coronary artery disease. AB - Two potential manifestations of proarrhythmic responses to type IA antiarrhythmic agents in the electrophysiology laboratory were evaluated in 122 patients with chronic coronary artery disease and previous myocardial infarction: (1) conversion of uniform nonsustained ventricular tachycardia (VT) into sustained VT after drug administration, and (2) induction of sustained VT by fewer extrastimuli after drug administration. Forty-two patients were evaluated for nonsustained VT. Eighty patients were evaluated for sustained VT: 30 of these had spontaneous sustained VT only while receiving empiric therapy with quinidine or procainamide, whereas the remaining 50 developed spontaneous VT in the absence of antiarrhythmic drugs. All patients underwent programmed stimulation in the baseline state and after procainamide. Four patients had conversion of induced uniform nonsustained VT into the same morphology, but sustained VT after procainamide administration. These responses only occurred in patients evaluated for nonsustained VT. Over 90% of patients presenting with sustained VT had uniform sustained VT induced at the baseline study and after procainamide, regardless of whether the spontaneous arrhythmia occurred only in the presence or absence of antiarrhythmic drugs. There was no significant difference in the change in mode of induction from baseline to procainamide study, regardless of whether patients had developed spontaneous VT only in the presence or absence of antiarrhythmic drugs. One patient with no inducible VT at the baseline study had inducible uniform sustained VT after procainamide administration, and 1 patient with inducible VT at baseline developed spontaneous sustained uniform VT after procainamide administration. Both patients had developed spontaneous sustained VT only while receiving therapy with type IA agents.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707221 TI - Effectiveness of glibenclamide on myocardial ischemic ventricular arrhythmias in non-insulin-dependent diabetes mellitus. AB - Glibenclamide, a hypoglycemic sulfonylurea, is a blocker of the adenosine triphosphatase-modulated potassium ion channels. The opening of these channels in the myocardial cells, induced by acute myocardial hypoxia, can be responsible for ischemic ventricular arrhythmias. To evaluate the antiarrhythmic effects of this drug 19 non-insulin-dependent diabetic patients were selected. They had coronary artery disease and evidence on Holter monitoring of ventricular premature complexes or nonsustained ventricular tachycardia, or both, induced by transient myocardial ischemia. In all patients, 24-hour electrocardiographic monitoring was performed to evaluate the number and duration of myocardial ischemic events, the frequency of ventricular premature complexes and nonsustained ventricular tachycardia per minute of ischemia and the percentage of ventricular premature complexes versus total ischemic beats. Selected patients were classified in 2 groups: group A (9 patients) received metformin (placebo) and group B (10 patients) was treated with glibenclamide. On the fourteenth day patients underwent 24-hour control monitoring. Then a crossover between the 2 groups was made and a new Holter monitoring sequence was performed at the end of the second phase. Results indicate that glibenclamide significantly (p less than 0.001) reduced both the frequency of ventricular premature complexes and the episodes of nonsustained ventricular tachycardia during transient myocardial ischemia, but did not change the number and duration of acute myocardial ischemic attacks and did not reduce the spontaneous ventricular arrhythmias. Thus, glibenclamide appears to have an antiarrhythmic effect in preventing ventricular arrhythmias induced by transient myocardial ischemia. PMID- 1707222 TI - Ventricular late potentials and induced ventricular arrhythmias after surgical repair of tetralogy of Fallot. AB - Ventricular tachycardia (VT) and sudden death are rare but recognized complications after surgical repair of tetralogy of Fallot. We prospectively studied 31 patients (19 boys and 12 girls, mean age +/- standard deviation 7 +/- 4 years) with postoperative tetralogy of Fallot, by means of right-sided cardiac catheterization, 24-hour Holter monitoring, body-surface and intracavitary signal averaging (gain 10(5) to 10(6), filters of 100 and 300 Hz) and programmed ventricular stimulation (1 and 2 extrastimuli, 3 basic cycle lengths, right ventricular apex and outflow tract). All patients were asymptomatic and none had documented or suspected ventricular arrhythmias. Ventricular late potentials were detected in 10 of 31 patients (32%) and spontaneous ventricular arrhythmias in 12 of 31 patients (39%). No sustained VT was induced by programmed ventricular stimulation but nonsustained VT was induced in 3 patients (10%). Patients with inducible VT more often had late potentials (3 of 3 vs 7 of 28, p less than 0.01), and spontaneous ventricular premature complexes (VPCs) during Holter monitoring (3 of 3 vs 9 of 28, p less than 0.05). To predict VT inducibility, late potentials had a sensitivity of 100%, a specificity of 75%, a positive predictive value of 30% and a negative predictive value of 100%. For spontaneous VPCs, the figures were 100, 68, 25 and 100%, respectively. It is concluded that shortly after repair of tetralogy of Fallot, the presence of both spontaneous VPCs and ventricular late potentials are associated with an increased incidence of inducible VT. Conversely, the absence of VPCs and ventricular late potentials may identify patients at low risk of subsequent ventricular arrhythmias. PMID- 1707223 TI - Boronate affinity chromatography of gamma-glutamyltransferase in patients with hepatocellular carcinoma. AB - We analyzed the serum gamma-glutamyltransferase (gamma-GT) by boronate affinity chromatography to ascertain the presence or absence of any changes in the binding properties of gamma-GT toward boronate gels in patients with hepatocellular carcinoma and liver cirrhosis, and in normal controls. The mean gamma-GT activity ratio of the bound (peak 2) and nonbound (peak 1) fraction in patients with hepatocellular carcinoma was significantly higher than that in patients with liver cirrhosis or in normal controls. Thus, the gamma-GT, which has adjacent cis hydroxyl groups in its carbohydrate moieties, was found to increase in the serum of patients with hepatocellular carcinoma. The positivity rate was examined in patients with hepatocellular carcinoma and liver cirrhosis, using a cut-off level for the peak 2:peak 1 ratio of 1.05 (mean + 2 SD of liver cirrhosis). Nineteen (42.2%) patients with hepatocellular carcinoma had a ratio of peak 2:peak 1 higher than 1.05. Nine of the 19 patients who had serum alpha-fetoprotein levels below 100 ng/ml had an elevated peak 2:peak 1 ratio. In total, 77.8% of the occurrence of hepatocellular carcinoma could be detected by a combination of these two markers. Three patients who had developed hepatocellular carcinoma during the course of cirrhosis but remained negative for alpha-fetoprotein throughout the course developed higher levels of peak 2:peak 1 ratio when hepatocellular carcinoma occurred. These results indicate that the two markers, the peak 2:peak 1 ratio of serum gamma-GT activity and serum alpha-fetoprotein level, may be considered to serve as complementary markers for the diagnosis of hepatocellular carcinoma. PMID- 1707224 TI - Modulation of tissue plasminogen activator biosynthesis by phosphatidylinositol liposomes in human fetal lung fibroblasts. AB - Phosphatidylinositol (PI) liposomes at 40 microM increased tissue plasminogen activator (t-PA) biosynthesis by human fetal lung fibroblasts IMR-90 (FLF), after 5 days of incubation by 7.4 +/- 1.4 times of the control level. Other phospholipid liposomes, such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylglycerol (PG), had no effect on t-PA biosynthesis by FLF. The induction of t-PA biosynthesis by PI liposomes was inhibited by specific inhibitors of phosphoinositide pathway: gentamycin and lithium chloride. Thus, gentamycin inhibited the effect of PI liposomes on t-PA biosynthesis by 76% (P less than 0.001), while it had no effect on control FLF. Likewise, lithium chloride inhibited t-PA biosynthesis of both PI-treated and control FLF by greater than 84%. The induction of t-PA biosynthesis by PI liposomes was dependent on RNA transcription and independent of DNA biosynthesis. PMID- 1707225 TI - Sickle cell anemia with few painful crises is characterized by decreased red cell deformability and increased number of dense cells. AB - The frequency and severity of the painful sickle cell crisis vary greatly among affected patients. Aside from a high level of Hb F(greater than 20%) there is no established parameter which may modulate the clinical severity of the disease. In this paper we describe two groups of adult patients with homozygous SS and present their characteristics. The division into these two groups was on the basis of relatively low RBC deformability (less than or equal to 37% of control) and high RBC deformability (greater than 65% of control) in the steady state. None of the patients had alpha-gene deletion and all had Hb F level less than 6.0%. Each patient was followed for a minimum of 3 years. The number of dense cells was quantitated by centrifugation on discontinuous Stractan gradient. RBC deformability index in isotonic medium (DI 290) was determined by ektacytometry and expressed as % of control. The patients with low RBC deformability had significantly less painful crises and more leg ulcers than those patients with high RBC deformability. The average number of dense cells was 22.2% and 9.8% of total circulating cells in the first and second group respectively. Moreover, the group with high red cell deformability had 33% mortality during the study period whereas no deaths occurred in the group with low RBC deformability. The data indicate that there is a subset of patients with SS who have relatively few painful crises despite low Hb F level. We wish to designate these by the acronym MIDDD syndrome: Mild disease as far as painful crises are concerned, increased number of Dense cells, and Decreased red cell Deformability. In addition these patients have high incidence of leg ulcers, have low incidence of urinary tract infection, and less mortality. Cellular factors seem to contribute to the incidence of painful crises. PMID- 1707226 TI - Blood zinc protoporphyrin is elevated only in sickle cell patients with low fetal hemoglobin. AB - Increased levels of various porphyrin species have been reported in sickle cell anemia (SS) patients in the absence of lead poisoning and iron deficiency anemia, but conflicting data remain. Suspecting that SS patients may be heterogenous for this abnormality, we have studied zinc protoporphyrin (ZPP) and protoporphyrin IX (PPIX) blood levels and find abnormally elevated levels of ZPP in those with low peripheral fetal hemoglobin (%HbF) levels. Two groups exist: one with less than 9% HbF and elevated ZPP, and one with greater than or equal to 9% HbF and normal ZPP levels (P less than 8.1 x 10(-4). There is a strong negative correlation of ZPP levels with %Hb F (r = -0.83, P less than 8.0 x 10(-5], and a moderate one with total hemoglobin levels (r = -0.55, P less than 0.05). These results suggest that ZPP may indeed contribute to the pathophysiology of the disease and/or serve as a marker of the severity of the disease. PMID- 1707227 TI - Growth and development of children of mothers treated with chemotherapy during pregnancy: current status of 43 children. AB - To evaluate the potential teratogenicity of modern cancer treatment, 43 children born to mothers with hematological malignancies (18 with non-Hodgkin lymphoma, 14 Hodgkin disease, seven acute leukemia, and four with chronic granulocytic leukemia) who received chemotherapy during some portion of their pregnancy, including 19 of these 43 who received chemotherapy during the first trimester, were examined for physical health, growth, and development. Immunological, hematological, and cytogenetic status also were evaluated. The children's ages ranged from 3 to 19 years. The children had a careful history and physical examination to detect any abnormal symptoms or signs and the mother's previous chemotherapy was carefully documented. In all of the children studied, physical, neurological, psychological, hematological, immune function, and cytogenetics were normal. These results suggest that chemotherapy can be administered during pregnancy, even during the first trimester, because it is not hazardous to the fetus; nevertheless, this study is inadequate in size to exclude the possibility of teratogenesis, and more reports are necessary to define the best treatment from cancer during pregnancy. PMID- 1707228 TI - Role of leukocytes in the activation of intravascular coagulation in patients with septicemia. AB - To elucidate the role of leukocytes in intravascular coagulation in patients with septicemia, plasma levels of thrombin-antithrombin III complex (TAT), soluble fibrin monomer complex (SFMC) and fibrinogen (Fbg) were determined in 33 patients with septicemia. Twenty of 33 patients revealed marked leukopenia caused by suppression of hematopoiesis by the administration of chemotherapeutic agents for the treatment of hematological malignancies; the total leukocyte count of these patients was less than 1,000/microliters. Thirteen of 33 patients showed normal or increased leukocyte counts. Plasma levels of TAT and SFMC in septicemic patients without leukopenia were significantly higher than in patients with leukopenia. Although plasma TAT and SFMC levels correlated well with the number of leukocytes, a more significant positive correlation was found between the number of monocytes and the levels of TAT and SFMC. Plasma levels of Fbg were significantly lower in patients without leukopenia than in patients with leukopenia. No significant correlation was found between the number of leukocytes and the levels of Fbg. However, a significant negative correlation was found between the number of monocytes and the levels of Fbg. TAT levels did not correlate with the number of platelets. The fibrinolytic system was activated only in septicemic patients without leukopenia, which may be explained by secondary fibrinolysis following leukocyte-activated coagulation. These findings suggest that leukocytes, in particular monocytes, may play a critical role in the pathogenesis of intravascular coagulation in septicemia. PMID- 1707229 TI - Acute lymphoblastic leukemia with azurophilic granules that contain ultrastructural myeloperoxidase activity. AB - We present a case of acute lymphoblastic leukemia (ALL) in which the leukemic cells had cytoplasmic azurophilic granules. Surface marker studies revealed that the leukemic cells expressed CD10 (CALLA), CD19, CD20, and HLA-DR antigens. Cytochemical studies by light microscopy revealed that the blasts were negative for myeloperoxidase, PAS staining, and double esterase staining, supporting the diagnosis of ALL. However, an ultrastructural study demonstrated that some of the cytoplasmic granules were myeloperoxidase (MPO) positive. Our findings suggested that leukemic transformation in this case may have taken place at a stage ontogenetically close to the pluripotent stem cell. Furthermore, the present case indicates the existence of a new form of ALL is characterized by MPO-positive granules detectable by ultracytochemistry and lymphoid-associated surface markers. PMID- 1707230 TI - Systemic treatment of AIDS-related Kaposi's sarcoma: results of a randomized trial. AB - PURPOSE: Patients with acquired immunodeficiency syndrome (AIDS)-related epidemic Kaposi's sarcoma generally respond well to cytotoxic chemotherapy. However, due to the associated myelosuppression, these patients are at risk for developing complicating infections that may affect survival. We therefore conducted a multi center randomized clinical trial comparing single-agent against combination chemotherapy in advanced AIDS-related Kaposi's sarcoma. Low-dose chemotherapy was employed to evaluate its role in combination therapy for this disease and the toxicities associated with the lower intensity. PATIENTS AND METHODS: Sixty-one patients with extensive mucocutaneous Kaposi's sarcoma or visceral involvement were randomized for treatment with low-dose Adriamycin (doxorubicin, 20 mg/m2) alone (31 cases) or in combination with bleomycin and vincristine (ABV) (30 cases). Patients were randomized within strata based on prognostic features associated with shorter survival in prior studies. Both treatment arms were evenly matched at study entry. RESULTS: Complete and partial tumor remissions were significantly higher with ABV (88%) than with Adriamycin alone (48%) (p = 0.004). The median survival was 9 months in both groups. Study entry criteria significantly associated with shorter survival included CD4 lymphocyte counts less than 100/mm3, hemoglobin level less than 10 g/dL, a history of constitutional symptoms, and a prior history of opportunistic infection(s). Toxicities were similar in both arms, and the regimens were well tolerated. Neutropenia (granulocyte count less than 1,000/mm3) occurred in 34% of patients receiving Adriamycin alone and in 52% of patients receiving ABV and was progressive in successive courses of chemotherapy in both treatment arms. The development of AIDS-defined opportunistic infections was relatively infrequent during therapy (14%). CONCLUSIONS: Low-dose ABV is an effective chemotherapy regimen for the treatment of extensive Kaposi's sarcoma. ABV chemotherapy is associated with significantly higher responses than Adriamycin alone and with acceptable toxicity. PMID- 1707231 TI - A new mutation in the proteolipid protein (PLP) gene in a German family with Pelizaeus-Merzbacher disease. AB - A C-to-T transition in exon 4 of the PLP gene was found in 2 affected males and two obligate carriers in a German family with Pelizaeus-Merzbacher disease. The mutation, which causes loss of an HphI site and changes amino acid 155 from threonine to isoleucine, was absent from 108 normal chromosomes. There are 5 concordances and 1 discrepancy between these results and those obtained by magnetic resonance imaging in this family. PMID- 1707232 TI - Immunohistochemical detection of P-glycoprotein in endometrial adenocarcinoma. AB - P-glycoprotein (Pgp) has emerged as the central mediator in classic multidrug resistance in model systems in vitro. High levels of Pgp also have been detected in many normal human tissues and tumors; and its role in clinical drug resistance is currently under investigation. Recently significant levels of Pgp were localized to gravid and secretory endometrium; and it was demonstrated that the combination of estrogen and progesterone is sufficient to induce high levels of both Pgp mRNA and Pgp in uterine secretory epithelium. These findings suggest that increased Pgp expression also may be present in hormone-responsive malignancies such as endometrial adenocarcinoma. To determine whether Pgp is expressed in endometrial adenocarcinoma, 36 endometrial adenocarcinomas (grade I [n = 17]; grade II [n = 6]; grade III [n = 13]) were investigated retrospectively by the avidin-biotin-complex immunohistochemical procedure using three murine monoclonal antibodies (MAb) MAb C219, MAb C494, and MAb JSB-1, which recognize spatially distinct cytoplasmic epitopes of Pgp. Seventy-two percent of the tumors showed positive immunostaining with at least one MAb; 67% showed immunostaining with MAb C219, 50% with MAb C494, and 62% with MAb JSB-1. Forty-six percent of tumors were immunoreactive to two and 29% to all three antibodies. Membranous and Golgi/paranuclear type staining patterns were observed. Overall the intensity of immunostaining varied from one sample to another for a given tumor type, and considerable heterogeneity of expression was commonly seen within a given tumor. Strong to moderate immunoreactivity was seen in diffusely infiltrating, adenosquamous, and serous papillary carcinomas. In general, immunoreactivity to MAb C494 was weaker than MAb C219 or MAb JSB-1. Adenomatous and non-neoplastic endometrium adjacent to the tumors displayed strong membranous immunostaining with MAb JSB-1. Endometrial capillaries showed weak-to-moderate immunostaining to all three antibodies. It is concluded that Pgp is commonly expressed in endometrial adenocarcinoma and may be a significant factor responsible for their drug-resistant nature subject to modulation by progesterone. PMID- 1707233 TI - p53 expression in colorectal tumors. AB - The expression of the nuclear phosphoprotein p53 was studied immunohistochemically in a series of 150 benign and malignant colorectal tumors. Using monoclonal antibody PAb1801, tumors divided unequivocally into two groups on the basis of immunohistochemistry. Forty of the carcinomas (46.5%) showed positive staining but only 4 of the adenomas (8.7%) were positive (P less than 0.001). The few positive adenomas always showed moderate or severe dysplasia. Metaplastic polyps (n = 9) and small familial adenomatous polyposis-related adenomas (n = 9) were uniformly negative. Carcinomas with p53 expression did not differ from those without in terms of site, differentiation or the prognostic indicators of Dukes' stage, DNA ploidy, or tumor histology. The improved morphologic resolution available in periodate lysine paraformaldehyde dichromate (PLPD)-fixed, paraffin-embedded tissue permitted several conclusions to be made: p53 is confined to neoplastic nuclei; staining in positive tumors is heterogeneous and often more marked at the infiltrative margins; and staining intensity is dramatically reduced in mitotic cells. It is concluded that expression of immunohistochemically detectable p53 (probably representing mutated forms of the protein) occurs in some adenomas around the time of transition to carcinoma. Therefore there is an association with the appearance of infiltrative behavior but not with degree of tumor progression (including metastasis) at the time of resection. PMID- 1707234 TI - Alternative splicing of human VCAM-1 in activated vascular endothelium. AB - Vascular cell adhesion molecule 1 (VCAM-1)/inducible cell adhesion molecule 110 is a mononuclear leukocyte-selective adhesion molecule, expressed on vascular endothelium following activation by certain cytokines or endotoxin. This inducible transmembrane protein and member of the immunoglobulin gene superfamily was previously reported to contain six immunoglobulinlike domains. Using the polymerase chain reaction, a VCAM-1 cDNA was obtained from mRNA of interleukin-1 (IL-1)-treated cultured human umbilical vein endothelial cells (HUVEC). The cDNA clone contained an additional 276 base-pair (bp) domain, located between domains 3 and 4. This new domain is most homologous to the existing N-terminal domain (domain 1). The internal 276-bp region is encoded by a single exon of the human VCAM-1 gene, indicating that the two forms of mRNA arise by alternative splicing. Both forms of VCAM-1 mRNA were detected by polymerase chain reaction in IL-1 stimulated HUVEC, although the seven-domain form appeared predominant. On the surface of HUVEC only a 110-kd polypeptide, consistent with the seven immunoglobulinlike domain form of VCAM-1, was detectable by immunoprecipitation. Alternative splicing of the VCAM-1 gene in cytokine-activated endothelium may generate functionally distinct cell-surface adhesion molecules. PMID- 1707235 TI - Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide. AB - This study was designed to evaluate the effect of the synthetic peptide Gly-Arg Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis. PMID- 1707236 TI - Rosenthal fibers share epitopes with alpha B-crystallin, glial fibrillary acidic protein, and ubiquitin, but not with vimentin. Immunoelectron microscopy with colloidal gold. AB - Ultrastructural immunoreactivities of alpha B-crystallin, glial fibrillary acidic protein (GFAP), ubiquitin, and vimentin in Rosenthal fibers (RFs) isolated from an Alexander's disease brain were investigated using nonosmium and low temperature embedding technique. The morphology of RFs embedded in Lowicryl K4M resin was well preserved after treatment with 0.5% Triton X-100. alpha B crystallin immunoreactivity was present in RFs of various sizes and was the strongest in loosely scattered deposits, which were considered to be the initial stage of RFs. Glial fibrillary acidic protein immunoreactivity in RFs was heavy, homogeneous throughout RFs, and equivalent to that in networks of glial filaments. Immunoreactivities of both alpha B-crystallin and GFAP were mainly restricted to the high electron-dense areas within RFs and were proved to exist close to each other by double immunolabeling. Rosenthal fibers were negative for vimentin. Ubiquitin immunoreactivity was relatively homogeneous in RFs with small diameters, but in RFs with large diameters, the immunoreactivity diminished in the center. Based on these observations, combined with the tendency of self aggregation of alpha B-crystallin, it is conceivable that RFs are huge aggregation products of alpha B-crystallin involving GFAP, and that ubiquitination may be a consequent phenomenon, as it may be in other intracytoplasmic inclusions, such as neurofibrillary tangles and Lewy bodies. PMID- 1707237 TI - Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas. AB - Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis. PMID- 1707238 TI - A novel chain of basement membrane-associated collagen as revealed by biochemical and immunohistochemical characterizations of the epitope recognized by a monoclonal antibody against human placenta basement membrane collagen. AB - Biochemical and immunohistochemical characterizations of the epitope recognized by a monoclonal antibody, JK-132, originally produced against human type IV collagen showed that it was distinct from the previously reported monoclonal antibody, JK-199 (Kino et al, J Biochem 1988, 103:829-835). The bound fraction of a crude pepsin extract of human placenta on JK-132 antibody-coupled resin showed close similarity to type IV collagen in a triple-helical conformation in terms of the amino acid composition and circular dichroism spectrum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the fraction showed six peptide bands with molecular weights of 50,000 or below, both before and after reduction. Four of the peptides reacted with JK-132 on immunoelectroblotting, but none reacted with JK-199. JK-132 reacted with two additional bands with molecular weights of 100,000 and 120,000, which were not visible on direct staining with Coomassie Brilliant Blue R-250. Two peptides (molecular weights 40,000 and 15,000) bound on a JK-199 antibody affinity column were sequenced, and both contained the same amino-terminal sequences as alpha 1(IV) chain. Conversely the sequences of three of the peptides (molecular weights 50,000, 32,000, and 23,000) eluted from a JK-132 antibody affinity column did not match either the alpha 1(IV) or the alpha 2(IV) sequence reported. Immunohistochemically, JK-132 reacted strongly with basement membranes of blood capillaries in skeletal muscle tissues but not with the basement membranes of muscle fibers in frozen sections of periodate-lysine-paraformaldehyde-fixed tissue, suggesting heterogeneity or tissue specificity of basement membrane collagen. By immunoelectron microscopy, the reaction products were found on the basal laminae of endothelium and of smooth muscle cells around blood vessels. These findings suggest the presence of a new collagen chain associated with basal laminae. PMID- 1707239 TI - Cellular events associated with inflammatory angiogenesis in the mouse cornea. AB - The aim of this study was to establish an angiogenesis model in the mouse and to define immunohistochemically the cellular events that precede angiogenesis. After chemical cauterization of the murine cornea, neovascularization was observed within 36 hours. The cellular infiltrate was analyzed by using antibodies on cryostat and paraffin sections and by histochemical staining for mast cells. It was found that neither T lymphocytes nor mast cells nor macrophages in a more mature stage of development were part of the infiltrate that preceded the ingrowth of new blood vessels. Instead, the infiltrating cells appearing from 3 hours on were granulocytes and inflammatory monocytes, as detected by an antibody against the calcium-binding protein MRP14. The authors conclude that the induction of angiogenesis during nonspecific inflammation is associated with the early influx of myelomonocytic cells, but not with the infiltration of mature macrophages, T lymphocytes, or mast cells. This study shows that immunohistochemical analysis of cauterized murine corneas presents a useful tool for further studies on cells and cell products involved in the angiogenic process. PMID- 1707240 TI - Age-related decline in prostacyclin synthesis by human aortic endothelial cells. Qualitative and quantitative analysis. AB - To investigate the functional alteration of human aortic endothelial cells with aging, prostacyclin synthesis was qualitatively and quantitatively examined. The endothelial cells of human aortas and umbilical veins or inferior vena cavae were immunohistochemically examined and found positive for prostacyclin, but the intensity of aortic endothelial cells from older subjects was low. In addition to the endothelial cells, smooth muscle cells in the thickened intima, not the media, of the aorta were also immunoreactive. Endothelial cells were successfully cultured from human aortas obtained from infants through aged subjects and were subdivided into three groups: young, middle, and old. Prostacyclin synthesis by endothelial cells from all types of blood vessels was extremely great at the primary culture, but decreased abruptly in the following subcultures. Among the aortic endothelial cells, the young group synthesized the largest amount of prostacyclin in a conventional culture condition, with synthesis progressively decreasing in the older groups. The in vitro prostacyclin biosynthesis was supported by the qualitative analysis on the tissue sections. These results indicate that prostacyclin synthesis of the aortic endothelial cells decreases with age, but intimal smooth muscle cells potentially have a back-up mechanism and substitute this synthesis to some extent. The decreased synthesis of prostacyclin with age may play an important role in the development and advancement of thrombosis and atherosclerosis. PMID- 1707241 TI - Charge selectivity in kidney ultrafiltration is associated with glomerular uptake of transport probes. AB - The isolated perfused kidney exhibits substantial charge selectivity, as in vivo, in relation to fractional clearance of [3H]dextran sulfate and [3H]dextran. When cycloheximide is present in perfusate, fractional clearance of dextran sulfate is increased and proteinuria becomes significant, but glomerular filtration rate remains essentially unchanged compared with control. The possible role of cells in affecting transglomerular transport was demonstrated when isolated glomeruli from control perfused kidneys showed a very significant resident concentration of [3H]dextran sulfate and [3H]albumin, whereas there was no corresponding accumulation of [3H]dextran or [3H]inulin. Glomerular concentration of dextran sulfate and albumin was significantly reduced by cycloheximide. Kinetics of uptake and release of glomerular dextran sulfate indicated that it had a half life of glomerular residence of approximately 2-3 min and that this half-life was considerably extended in the presence of cycloheximide. The half-life for glomerular residence of albumin was in the range of 30-40 min. The conclusion from this work is that glomerular charge selectivity for dextran sulfate could be quantitatively rationalized on the basis of transient uptake and release by glomerular cells. PMID- 1707242 TI - Endothelium reduces DNA synthesis in isolated arteries. AB - We evaluated effects of endothelium removal and of endothelium-derived vasoactive agents on DNA synthesis and contractility in the isolated arterial wall. The experiments were performed on renal artery segments that had been 1) isolated from adult rats, 2) suspended in tissue culture for 3 days in the continuous presence of fetal calf serum, and 3) exposed for the last 24 h to 5-bromo-2' deoxyuridine. Nuclear incorporation of this thymidine analogue was visualized by immunohistochemistry and used as an index of DNA synthesis. Tissue culture did not alter relaxing responses to guanosine 3',5'-cyclic monophosphate (cGMP) generating agents but promoted relaxing responses to adenosine 3',5'-cyclic monophosphate (cAMP)-generating agents. It stimulated DNA synthesis in the endothelium, media, and adventitia. Mechanical removal of endothelium increased intra-arterial DNA synthesis. This was most prominent in the media. In preparations with endothelium, indomethacin and methylene blue did not enhance DNA synthesis. In segments that had been denuded of endothelium, atrial natriuretic factor, forskolin, iloprost, and prostaglandin E2, but not isobutylmethylxanthine or sodium nitroprusside, significantly reduced intra arterial DNA synthesis. These data indicate that endothelium removal promotes the mitogenic response of the arterial wall to exogenous growth factors. This cannot be attributed to inhibitory influences of endothelium-derived relaxing factor or prostaglandins released by the endothelium under basal conditions. PMID- 1707243 TI - Heterogeneity in conducted arteriolar vasomotor response is agonist dependent. AB - Microiontophoresis of acetylcholine onto cheek pouch arterioles of the pentobarbital-anesthetized hamster results in both a local response at the pipette tip and a conducted dilator response. The conducted response is not dependent on blood flow, and its magnitude decays with distance from the site of stimulation. In an attempt to define the mechanism responsible for activation of arteriolar conduction, vasoactive agonists directed toward different vascular wall cell types, receptor types, and second messengers were applied to arterioles by pressure-pulse microejection. As expected, microapplication caused a consistent arteriolar response at the site of application with each of the agonists tested (local response). However, a high degree of variability was observed among agonists in their ability to produce conducted responses. Acetylcholine, muscarine, and phenylephrine, invariably induced both local and conducted responses. In contrast, bradykinin, substance P, papaverine, isoproterenol, and adenosine, though consistently inducing local responses, displayed a highly variable ability to induce the conducted responses. When conduction was observed, the arteriolar response was similar regardless of the agonist used to induce the response. Microejection of sodium nitroprusside or arginine vasopressin produced local arteriolar responses with no evidence of a conducted response regardless of the dose. These studies reveal previously undetected heterogeneity among microvessel responses and may reflect variations in the coupling mechanisms linking the local vasomotor response to the conducted response. PMID- 1707244 TI - Hyperoxemic reperfusion does not increase myocardial infarct size. AB - We tested the hypothesis that arterial hyperoxia during myocardial reperfusion increases reperfusion injury and infarct size. The anterolateral marginal coronary artery of 35 anesthetized rabbits was occluded for 45 min, then reperfused for 3 h with either normoxic [arterial PO2 (PaO2) = 96.7 +/- 22.9 mmHg)] or hyperoxic (PaO2 = 554.8 +/- 61.7 mmHg) blood. In the hyperoxic group only, PaO2 was adjusted 10 s before the onset of reperfusion by raising inspired oxygen concentration to 100%. The area of infarction (AI) was defined by triphenyltetrazolium staining, and the area at risk (AR) by fluorescent microspheres. These areas were measured by planimetry. Heart rates and blood pressures did not differ between the two groups during occlusion or reperfusion. Infarct size (AI/AR) was 49.1 +/- 16.5% in the normoxic group (n = 17) and 40.8 +/- 16.1% in the hyperoxic group (n = 18). From these data, 90% confidence limits establish that the maximal true increase in AI/AR caused by hyperoxia would be 0% 1%. Hyperoxic reperfusion of ischemic myocardium compared with normoxic reperfusion does not significantly increase myocardial infarct size. PMID- 1707245 TI - Pre- and postjunctional effects of some prostanoids in human isolated vas deferens. AB - The effects of prostaglandin (PG) E1, PGE2, the thromboxane A2 analogue U-44069, and the prostacyclin derivative iloprost were studied on isometric contractions induced by norepinephrine (NE) and by electrical field stimulation of nerves in isolated preparations of the human vas deferens. The effects of these agents on the electrically induced release of 3H from preparations preincubated with [3H]NE were also investigated. PGE1 and PGE2 inhibited the electrically induced contractions concentration dependently. U-44069 augmented the contractions without affecting baseline tension, and in preparations where the contractions had been inhibited by PGE1 or PGE2, U-44069 restored the contractions almost to starting levels. The thromboxane A2-receptor antagonist BM 13505, having no effect or inhibitory effects on electrically induced contractions, abolished the stimulatory effect of U-44069. Contractions induced by exogenous NE were augmented by U-44069, whereas PGE1 and BM 13505 were without effects. The electrically induced release of 3H was inhibited by PGE1 and PGE2 in a concentration-dependent manner, whereas U-44069 and BM 13505 increased the release of 3H. Furthermore, the inhibitory effect of PGE1 on 3H release was partly counteracted by U-44069. Iloprost had no significant effect on electrically induced contractions or on 3H release. These results suggest that, in the human vas deferens, thromboxane A2 augments contractions predominantly through a postjunctional site of action, whereas PGs of the E type have a prejunctional inhibitory effect. In addition, the pre- and post-junctional effect profiles of U-44069 and BM 13505 suggest that there may be more than one thromboxane receptor. PMID- 1707246 TI - Temporal relationships of viremia, interferon activity, and antibody responses of sheep infected with several bluetongue virus strains. AB - Sheep had viremias that were first detected on day 3 (+/- 1) after infection with several strains of bluetongue virus (BTV) representing United States serotypes 10, 11, 13, and 17. Diphasic peaks of infectivity were attained on days 6 and 10 (+/- 2). Interferon (IFN) was first detected in serum samples on day 5 (+/- 1), and reached greatest concentrations on day 6 (+/- 2), which coincided with the first viremic peak; IFN concentrations then decreased toward zero by day 10 (+/- 2). Interferon peak concentrations induced approximately a 90% decrease in virus titer. The decrease in IFN concentrations by day 9 (+/- 2) corresponded with the second viremic peak on day 10 (+/- 2). Onset of the decrease in detectable concentrations of virus after the second peak of viremia corresponded to the initial detection of serum antibody to BTV by day 10 (+/- 2). Virus titer decreased and antibody production increased until approximately days 21 to 28, when the titers plateaued and virus was not detected. Febrile responses peaked on day 7 (+/- 1) during the peak viremic period. The WBC count was depressed at the time the virus titer increased, but returned to normal values while the sheep were still viremic. Diphasic viremias in BTV-infected sheep were attributed to induction of high concentrations of IFN concurrent with the first virus titer peak, followed by production of antibody to specific BTV strains and a subsequent reduction in viremia at the second virus titer peak. PMID- 1707247 TI - Spontaneous dural carotid-cavernous fistula with central retinal vein occlusion and iris neovascularization. AB - Spontaneous dural carotid-cavernous fistulas are dural vascular malformations that usually run a benign course. We present a case of a spontaneously occurring dural carotid-cavernous fistula complicated by central retinal vein occlusion and iris neovascularization that led to progressive visual failure. PMID- 1707248 TI - The anatomic basis of maternal serum screening. AB - Fetal serum markers, such as alpha fetoprotein (AFP), must traverse one of two very different pathways to reach maternal serum, either from fetus to amnion fluid, membranes and decidua or from fetal to maternal circulation through the placental villi. Alpha fetoprotein usually enters the amnion fluid through body wall defects uncovered by skin or through urine. Placental AFP leakage may be from villous hemorrhage or injury. These observations from anatomic pathology suggest that biochemical markers may exist to identify the source of elevated maternal serum AFP. PMID- 1707249 TI - Human microglial cells: characterization in cerebral tissue and in primary culture, and study of their susceptibility to HIV-1 infection. AB - Neuropathological studies have shown that human immunodeficiency virus type 1 infected cells within the brain express several markers characteristic of macrophages and could either be microglial cells, or monocytes invading the CNS, or both. To better define the target cells of human immunodeficiency virus type 1 within the brain, we have studied human microglial cells, both in vivo and in vitro, and compared them to monocytes for their antigenic markers and their susceptibility to human immunodeficiency virus type 1 infection. Brain-derived macrophages were isolated from primary cortical and spinal cord cultures obtained from 8 to 12-week-old human embryos. The isolated cells presented esterase activity, phagocyted zymosan particles, expressed several (Fc receptors, and CD68/Ki-M7 and CD11b/CR3 receptors) of the macrophagic antigenic markers, and appeared to be resident microglial cells from human embryonic brain. Conversely, brain-derived macrophages did not express antigens CD4, CD14, or CD68/Ki-M6, which are easily detected on freshly isolated monocytes. Using these antigenic differences between isolated microglial cells and monocytes, we have observed that two populations of macrophages could be individualized. In the normal adult brain, microglial cells were numerous in both the gray and the white matter. The infrequent cells sharing antigens with monocytes were found almost exclusively around vessels. In 8 to 12-week-old human embryos, microglial cells were found in both the parenchyma and the germinative layer. Cells sharing antigens with monocytes were only found at the top of and inside the germinative layer. In brain tissue from patients with human immunodeficiency virus type 1 encephalitis, cells sharing antigens with monocytes are abundant not only around the vessels but also in the parenchyma. In double-labeling experiments, human immunodeficiency virus type 1-infected cells showed monocyte antigens. Finally, microglial cells also differ from monocytes in their in vitro susceptibility to human immunodeficiency virus type 1 infection; after stimulation by r-TNF alpha or GmCSF, monocytes but not microglial cells can replicate human immunodeficiency virus type 1. This in vitro difference in human immunodeficiency virus type 1 susceptibility between monocytes and microglial cells together with the presence of monocytic antigens within the brain tissue of human immunodeficiency virus type 1-infected patients suggest that human immunodeficiency virus type 1 infected cells within the brain are either monocytes that have crossed the blood brain barrier and spread through the tissue or perivascular microglial cells that, after phagocyting infected blood lymphocytes, subsequently contain viral antigen and migrate to brain tissue. PMID- 1707250 TI - Feminism, eating, and mental health. AB - Eating disorders are prevalent health problems for women today. The traditional biomedical or psychiatric approaches offer a narrow perspective of the problem, its courses, and its treatment. Analyzing disordered eating from a feminist perspective, this article discusses cultural, political, and social phenomena that have had a significant impact on the development of these disorders. Parallels of eating disorders and other women's mental illnesses and the medicalization of their symptoms is explored. A "new view" of disordered eating in women is proposed that can be advanced only through feminist research. PMID- 1707251 TI - Identification of mutans streptococci with monoclonal antibodies. AB - Mutans streptococci have been correlated with dental caries. The identification of the species within this group is still a problem. The characterization of a monoclonal antibody (Mab) OMVU10 against S. sobrinus as well as the isolation and characterization of Mabs against S. mutans (OMVU30 and OMVU31), S. cricetus (OMVU40) and mutans streptococci (OMVU2) is described. The epitope specificity for OMVU10 and OMVU31 was cell-wall antigen B in both cases although both Mabs recognized different species-specific epitopes. OMVU40 was cross reactive with Streptococcus sanguis taxon 3. All other Mabs were specific for one species. Using these Mabs, a key to the identification of mutans streptococci is developed. This key was tested for 85 wild type isolates of mutans streptococci and proved to be highly reliable and easy to perform. PMID- 1707252 TI - Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria. AB - Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium. PMID- 1707253 TI - [Basic and clinical aspects of IL-6]. AB - Interleukin-6 (IL-6) is a pleiotropic cytokine regulating immune response, production of acute phase reactants in hepatocytes, growth of hematopoietic stem cells and other cellular functions in many cell lineages. The increased production of IL-6 is often seen in infections diseases, chronic inflammatory diseases, and certain tumors which accompany polyclonal B cell activation and increased level of CRP. Recent progress in the study of the basic aspects on IL-6 will be discussed, which includes the regulation mechanisms of IL-6 gene, the structure of IL-6 receptor complex (IL-6, 80 KDa IL-6 receptor and signal transducing gp130) and IL-6 signal transduction pathways. PMID- 1707254 TI - Basement membrane zone as a target for human neutrophil elastase in psoriasis. AB - Human neutrophil elastase was found, by indirect immunofluorescence using rabbit anti-elastase anti-serum, to be bound to basement membrane of psoriatic plaques in vivo. The enzyme was also identified inside the migrating neutrophils in the reticular dermis and dermal papillae, as well as outside the cells in micro abscesses in psoriatic skin. In vitro incubation of normal skin with human neutrophil elastase resulted in the destruction of hemidesmosomes and separation of the epidermis from the dermis above localizations of bullous pemphigoid antigen. These findings are direct evidence that human neutrophil elastase could play a role in psoriasis in in vivo destruction of the epidermal-dermal junction. PMID- 1707255 TI - Prospective, randomized trial of palliative treatment for unresectable cancer of the esophagus. AB - To evaluate the best method of palliation for obstructing nonresectable squamous cell carcinoma of the mid or distal esophagus, 27 patients were prospectively randomized to one of three treatment arms: (1) esophageal intubation with an Atkinson tube (AT, 10 patients), (2) esophageal intubation followed by radiation therapy (AT/RT, 8 patients), and (3) endoscopic laser therapy followed by irradiation (L/RT, 9 patients). Pretreatment characteristics were similar in the three groups. There was no procedure-related mortality. There were eight total complications related to the tube and none related to laser treatment (p = 0.02). Mean survival was 119 days in the AT group, 72 days in the AT/RT group, and 169 days in the L/RT arm (p = not significant). Quality of survival was most dependent on swallowing ability, and the swallowing score increased by 2.3 units in the AT group, 1.8 units in the AT/RT group, and 1.4 units in the L/RT group (p = not significant). Adding RT to laser therapy significantly increased time in treatment (mean, 38.7 days) when compared with the AT group (mean, 12.5 days) (p less than 0.001). However, only 1 patient required repeat laser ablation. It is concluded that AT and L/RT result in good palliation as measured by relief of dysphagia and survival time. However, morbidity of AT is significantly greater than that of L/RT. Laser and radiation therapy with a reduced total dosage of RT or with a change in fractionation schedule to limit treatment time is the preferred method of palliation. PMID- 1707256 TI - Pleuroperitoneal shunting for intractable pleural effusions. AB - Pleuroperitoneal shunts were implanted in 17 patients with intractable pleural effusions, 15 of which were malignant and 2 benign. Complicating factors included 13 instances of severe trapped lung and 3 cases of synchronous ascites. There was one hospital death. Palliation of dyspnea at rest was achieved in all patients, although 3 required oxygen with exertion. Four shunts became occluded between 1 and 10 months after placement. Two of these were replaced. The remaining conduits continued to function to the present or until the patients' deaths between 1 and 28 months. Shunting allowed hospital discharge and provided symptomatic relief in a group of patients in whom other approaches had failed or were not applicable. PMID- 1707257 TI - Palliative repair of aortic atresia associated with tricuspid atresia and transposition of the great arteries. AB - Successful palliative repair of aortic atresia and hypoplastic aortic arch associated with tricuspid atresia in a neonate is described. The repair consisted of reconstruction of the hypoplastic aortic arch with an equine pericardial patch, division of the patient ductus arteriosus, connection of the pulmonary artery to the aorta, implantation of the proximal part of the ascending aorta into the main pulmonary artery, and anastomosis of a polytetrafluoroethylene graft 5 mm in diameter between the right ventricular outflow tract and the central pulmonary artery, which was transferred anteriorly to the main pulmonary artery. PMID- 1707258 TI - Afferent connections to the abducent nucleus in the cat. AB - The afferent connections to the abducent nucleus in the cat were studied by means of retrograde transport of WGA-HRP after implantations of the tracer in crystalline form. Retrogradely labelled cells were found bilaterally in the medial and descending vestibular nuclei, mainly in their ventral and medial portions, in the rostral part of the ipsilateral gigantocellular reticular nucleus, in the medial part of the contralateral caudal pontine reticular nucleus and bilaterally in the oculomotor nucleus, mainly in its dorsolateral division. Some labelled cells were also found bilaterally in the mesencephalic reticular formation, the periaqueductal grey and the nucleus of the trapezoid body. PMID- 1707259 TI - [Prognostic value and development of late potentials after aortocoronary bypass. A prospective study of 100 patients]. AB - Ventricular late potentials are post-infarction markers of the risk of ventricular tachycardia and sudden death. In order to assess their prognostic value and evolution after coronary bypass surgery, 100 patients underwent signal averaged electrocardiographic recordings 24 hours before and 9 days after surgery, and were then prospectively followed up for 40 +/- 8 months. Patients who displayed late ventricular potentials underwent an additional recording at 5 months with 24 hour Holter monitoring. The average age of the patients was 57.0 +/- 8.4 years; 55 had previous myocardial infarction; 32 had triple vessel disease; the mean left ventricular ejection fraction was 59.7 +/- 12.4%. Ventricular late potentials were recorded in 17 patients before surgery and their left ventricular ejection fraction was significantly lower (51.4 +/- 11.5% vs 61.4 +/- 11.9%: p less than 0.05). There was one operative death in a patient with late ventricular potentials. After surgery, late ventricular potentials were only recorded in 6 patients: at the 9th postoperative day in 3 cases and at the 5th postoperative day in 3 cases. Ventricular late potentials appeared postoperatively in 5 patients, 4 of whom had suffered perioperative myocardial infarction. The recordings became normal at the 5th month in 2 of these 5 patients. Holter monitoring at the 5th month compared with a control group, showed a significant correlation between left ventricular potentials and frequent repetitive or polymorphic ventricular extrasystoles. The 40 month survival rate was excellent: 2 patients were lost to follow-up; there were 3 cardiac deaths, one of which was sudden and 4 non-cardiac deaths. All patients with late ventricular potentials were still alive. These results show that late ventricular potentials persist after coronary bypass surgery in 2/3 of patients; their prognostic significance is not obvious. The low incidence of postoperative sudden death could be attributed to the favourable overall effects of revascularisation rather than on the arrhythmogenic substrate. PMID- 1707260 TI - The value of clinical laboratory studies in acute pancreatitis. PMID- 1707261 TI - Acute leukemia and related entities. Impact of new technology. AB - Twenty-seven cases of acute leukemia and related entities were evaluated by morphologic examination, cytochemical study, terminal deoxynucleotidyltransferase study, immunophenotyping, cytogenetic analysis, ultrastructural cytochemical study, and gene rearrangement analysis to determine the impact on the determination of the French-American-British (FAB) classification and the definitive diagnosis. The definitive diagnosis contained prognostic, diagnostic, and treatment information beyond the FAB classification that affected the disease course and patient management. All diagnostic variables were evaluated in each case and were labeled essential, ambivalent, supportive, or noncontributory. Except for gene rearrangement analysis, all variables we studied contributed essential data to establish the definitive diagnosis. Ambivalent findings were rare but could be explained with the knowledge of the total data. All variables, except cytochemical study, whose results were almost always essential, contributed supportive data. Noncontributory data only occurred with cytogenetic analysis in cases that demonstrated normal karyotypes. The FAB classification was established in 20 (74%) of the cases by use of morphologic examination, cytochemical study, and terminal deoxynucleotidyltransferase study. With use of the same variables, however, the definitive diagnosis, whose determination required all data, was established in only 15 (55.5%) of the cases. The addition of immunophenotyping increased the definitive diagnosis to 25 (92.5%) of the cases. The use of ultrastructural myeloperoxidase and platelet peroxidase analysis enabled us to definitively diagnose the remaining two cases (27 cases [100%]). Cytogenetic analysis revealed four cases in which essential information was added to the diagnosis. However, because the cytogenetic information usually was not immediately available, the result did not affect the immediate diagnosis or treatment. Surprisingly, the gene rearrangement studies did not yield essential data in any case and in a few cases contributed ambivalent data. This finding should not exclude gene rearrangement analysis in selected cases; however, the data should always be interpreted in light of all clinical and laboratory findings. This study clearly demonstrates the importance of a multifaceted approach to the understanding of the acute leukemias and related entities and shows the impact of newer technologies on reaching a definitive diagnosis. PMID- 1707262 TI - Reevaluation of the periodic acid-Schiff stain in acute leukemia with immunophenotypic analyses. AB - To determine the sensitivity and specificity of the periodic acid-Schiff (PAS) stain in the diagnosis of acute leukemia in light of the finer characterization of this disorder now available through immunophenotyping, we examined the blasts from 51 patients with newly diagnosed acute leukemia by morphological, cytochemical, and immunophenotypic analyses. The 51 patients represented every new case of acute leukemia subjected to cytochemical stains and flow cytometry between July 1987 and February 1989. By cell-surface marker analysis, 29 exhibited lymphocytic lineage, while 21 were myelocytic. One was mixed lineage. The PAS positivity, defined by the presence of blocks or coarse granules in 5% or more of the blasts, was found in 15 of 29 lymphoblastic leukemias and in four of the myeloblastic leukemias. However, PAS-positive lymphoblastic leukemias were negative with the other cytochemical stains: myeloperoxidase, Sudan black B, and alpha-naphthyl butyrate esterase. The PAS-positive myeloblastic leukemias were positive with at least one other stain. Three cases of myeloblastic leukemia exhibited greater than 10% PAS-positive blasts, with all three being acute monoblastic leukemia. Thus, the sensitivity and specificity of the PAS stain alone for lymphoblastic leukemia was 52% (15 true positives of 29) and 81% (four false positives), respectively. The sensitivity of a cytochemical-staining combination of PAS positivity and myeloperoxidase, Sudan black B, and alpha naphthyl butyrate esterase negativity in defining cases of lymphoblastic leukemia remained at 52%; however, the specificity of this combination for lymphoblastic leukemia was 100% (no false positives). Thus, a positive PAS stain, in combination with negative myeloperoxidase, Sudan black B, and alpha-naphthyl butyrate esterase stains, continues to have a diagnostic role in the distinction between lymphoblastic and myeloblastic leukemia, and greater immunologic sophistication serves to support this position. PMID- 1707263 TI - Clinical evaluation of an algorithm for the interpretation of hyperamylasemia. AB - Total amylase concentration in serum continues to be widely determined in the diagnosis of acute pancreatic disease. Accumulated experience has made clear, however, that this determination has distinct limitations. Consequently, the knowledge of the origin of hyperamylasemia may have an important influence on treatment, hospitalization, and extent of clinical investigations. We undertook a logical and systematic approach to the interpretation of hyperamylasemia through the use of an algorithm that can be applied in clinical situations without the need for the integration of radiologic procedures or clinical data. The proposed algorithm was tested for effectiveness in 97 consecutive hospitalized patients with hyperamylasemia (amylase level greater than twice the upper reference limit) for a 2-year period. The majority (52.5%) of these patients had acute pancreatitis. The algorithm assigned the correct diagnostic categories in 95.8% of cases, with a disagreement between patient diagnosis and algorithm-generated diagnosis in only four cases. These four patients (two with acute biliary disease, one with bacterial peritonitis, and one with chronic renal failure) had pancreatic lipase values greater than five times the upper reference limit, so that the algorithm classified their condition as acute pancreatitis. The clinical trial indicated that the proposed decision tree, which requires only knowledge of biochemical data that are readily available, is useful in the evaluation of elevated amylase activity and facilitates arrival at a definitive diagnosis. PMID- 1707264 TI - Mechanism of fibrous capsule formation surrounding hepatocellular carcinoma. Immunohistochemical study. AB - In 14 cases of hepatocellular carcinoma with capsule, we studied the mechanism of capsule formation by the immunoperoxidase technique using antibodies to types I, III, and IV collagen, antilaminin antibody, and anti-prolyl hydroxylase antibody. Marked round cell infiltration was observed in the noncancerous side of the capsule and around compressed hepatocytes near the capsule. Thin capsules were composed primarily of type III collagen produced by an increased number of fibroblasts, transitional Ito cells, and hepatocytes near the capsule. In thickened capsules, the noncancerous side consisted primarily of type III collagen and the cancerous side of types I and III collagen. Type I as well as type III collagen was produced by fibroblasts, transitional Ito cells, and hepatocytes. The capsule thus formed is suggested to be part of the defense mechanisms against the growth of hepatocellular carcinoma. PMID- 1707265 TI - Squamous metaplasia of the peritoneum. AB - The capacity of the peritoneal serosa to undergo metaplasia to mullerian-type epithelium is well recognized. We report a case of squamous metaplasia of the peritoneum that was studied by light microscopy. Immunohistochemical techniques, and electron microscopy. The pathogenesis of peritoneal squamous metaplasia is obscure, but may be a response to chronic irritation. PMID- 1707266 TI - [Ultrastructure of breast cancer vessels]. AB - Vascular bed of the infiltrating mammary gland carcinomas characterized by the incomplete maturity, atypia and polymorphism is studied electron microscopically. Two groups of vascular structures were investigated: avascular part represented by pseudovessels and vascular part with protocapillaries, capillaries, sinusoids and venulae that are distinguished by their morphofunctional properties and the degree of maturity. The vascular bed is characterized by a structural and functional heterogeneity. Ultrastructural properties characterizing the stages of the vascular bed morphogenesis are presented: angiogenesis activation, relative vascular differentiation and regression of some parts which occur asynchronically and are due to the properties of the stromal parenchymal relations in different microareas, and to the heterogeneity of the tumour on the whole. PMID- 1707267 TI - [Method of bronchoalveolar lavage for performing cytochemical examination in chronic nonspecific pulmonary inflammation]. AB - The method suggested includes the use of homogenizer terrilytin for removing excessive mucus from broncho-alveolar lavage (BAL). Cytochemical investigation of BAL neutrophils is reasonable to be performed if the viability of the cell suspension is not lower than 60%. Cytospectrophotometric examination is recommended for an unbiased evaluation of the neutrophil functional state. PMID- 1707268 TI - Distribution of elastic fibres in the developing rabbit craniomandibular joint. AB - The biomechanical properties of the CMJ disc depend upon the composition and organization of the extracellular matrix. Elastic fibres are important elements of the matrix and may be in part responsible for the resilience of the disc during jaw movements. Elastic fibres first appeared after the establishment of a miniature CMJ at 23 days of prenatal development. The first elastic fibres appeared in the antero-inferior and postero-inferior attachment regions of the disc. In the newborn rabbit there were elastic fibres in the articulating surfaces of the joint and by one week fibres could be seen in the intermediate zone portion of the disc. At two weeks, when the animals were beginning to experiment with solid food, the disc band areas showed accumulations of elastic fibres and proteoglycans. The findings suggest that the elastic elements of the disc, squamosal and condylar articulations may have a resilience function which develops in response to functional loads placed upon the joint as the rabbit grows and changes diet. PMID- 1707269 TI - Effect of smokeless tobacco use in humans on mucosal immune factors. AB - To assess the effects of smokeless tobacco on the secretory immune system and dental caries, we examined users of smokeless tobacco and non-tobacco users. There were no significant differences in the prevalence of DMFS between users and non-users. There was significantly more salivary IgA, IgA2 and J-chain in users. Levels of salivary lysozyme and lactoferrin were significantly lower in users than controls. Because there was no difference in levels of secretory component in relation to the increased IgA levels of smokeless tobacco users, this suggests an effect of smokeless tobacco on secretory epithelial cells responsible for synthesis of secretory component, lysozyme and lactoferrin, and for the packaging of secretory component on IgA. There were only slight differences in salivary or serum antibody levels to Streptococcus mutans. These findings indicate that although smokeless tobacco has a significant influence on the synthesis of secretory IgA, the numbers of DMFS were similar between smokeless tobacco users and controls. PMID- 1707270 TI - Down syndrome and low maternal serum alpha fetoprotein. AB - The early pregnancy maternal serum alpha-fetoprotein (MSAFP) results for 35 patients who delivered a baby with Down syndrome (DS) were analysed. These results were collected from 1981 to mid-1989. Eight of the 35 showed an MSAFP result less than 0.5 multiples of the median (MOM). These MSAFP results were corrected for maternal weight. The results support other workers' conclusions that mid-trimester hormonal analyses may be helpful in diagnosing the presence of DS in women otherwise not considered at risk. PMID- 1707271 TI - CO2 laser laparoscopic salpingotomy for treatment of tubal ectopic pregnancies: potential limitations. AB - Linear salpingotomy was performed on 16 patients using the CO2 laser laparoscopically directed. Median operating time was 60 minutes (range 40-100) and all patients were discharged on the first postoperative day. There were 4 patients in whom persistence of trophoblast activity was detected, 2 of whom were treated surgically and 2 by oral methotrexate therapy. PMID- 1707272 TI - Multiple antigenic determinants on type III collagen. AB - Eight monoclonal antibodies have been produced against human pepsin-soluble type III collagen. All antibodies were shown to be highly specific for type III collagen and did not cross-react with a range of other collagen types or connective-tissue proteins. Examination of type III collagen from other species showed that these antibodies had a wide range of species specificities, indicating that several distinct epitopes were being recognized. The location of the epitopes was investigated by using reactivity of the antibodies to CNBr fragments and to sequential fragments formed by tryptic digestion of renaturing type III collagen. These data also indicated that several distinct epitopes were recognized and that they were located over the length of the type III collagen. PMID- 1707273 TI - Rat tyrosine kinase inhibitor shows sequence similarity to human alpha 2-HS glycoprotein and bovine fetuin. AB - Human alpha 2-HS glycoprotein and bovine fetuin, abundant proteins of fetal plasma, are structural members of the fetuin family within the cystatin superfamily. They are characterized by the presence of two N-terminally located cystatin-like units and a unique C-terminal sequence segment not present in the other members of the cystatin superfamily. Search for related sequences revealed that the natural inhibitor of the insulin receptor tyrosine kinase [Auberger, Falquerho, Contreres, Pages, Le Cam, Rossi & Le Cam (1989) Cell (Cambridge, Mass.) 58, 631-640] shows sequence similarity to the mammalian fetuins. The sequence identity between rat tyrosine kinase inhibitor, human alpha 2-HS glycoprotein and bovine fetuin is 56 and 60% respectively (percentage of residues in identical positions). The sequence similarity extends over the entire protein structures, except the extreme C-terminal portions. In particular, the number and relative positions of the cysteine residues are invariant among the proteins, suggesting that the characteristic array of linearly arranged and tandemly repeated disulphide loops of the cystatin superfamily is also present in rat tyrosine kinase inhibitor. We conclude that rat tyrosine kinase inhibitor may be classified as a novel member of the mammalian fetuin family. PMID- 1707274 TI - The effect of microwave radiation on the stability and formation of gramicidin-A channels in lipid bilayer membranes. AB - The effects of microwaves on the single-channel kinetics of gramicidin-A channels in lipid bilayer membranes were examined. Attempts were made to separate thermal and athermal effects by accurate measurements of temperature at the site of the membrane and by relating the measured parameters to their previously characterized temperature dependence. It was found that microwave radiation does not affect single-channel conductance or channel life time to a degree that is significantly different from that expected of a purely thermal effect. On the other hand, the rate of channel formation is decreased during exposure, which is opposite to that expected of a purely thermal effect. The mechanism of this effect is discussed in terms of the dimerization process of channel formation. PMID- 1707275 TI - Activation of CD16+ effector cells by rheumatoid factor complex. Role of natural killer cells in rheumatoid arthritis. AB - The role of natural killer (NK) cells in rheumatoid arthritis (RA) remains unclear. A pathogenetic function of rheumatoid factors (RF) also has not been defined. In the present studies, natural killer (NK) cells were examined as a model for FC gamma receptor type III-positive (FC gamma RIII+) cells, with regard to their interaction with RF. NK cell antigen CD16 (FC gamma RIII) and CD56 expression and functional NK and antibody-dependent cell-mediated cytotoxicity (ADCC) activity were compared in peripheral blood lymphocytes and autologous synovial fluid lymphocytes (SFL) of RA patients. Peripheral blood lymphocytes and SFL showed normal CD56 expression. In contrast, both the frequency and the density of CD16 antigen were decreased in SFL. Furthermore, diminished NK cytotoxicity and a significant decrease in ADCC were observed in SF NK cells. In subsequent in vitro studies with normal fresh NK cells, it was demonstrated that IgG-containing RF complexes from RA patients induced a modulation of FC gamma RIII structure from the NK cell surface, a decrease in NK activity, and a complete loss of ADCC. When purified RF was incubated with NK-enriched cell lines from RA patients, increased transcription and subsequent production of interferon gamma and tumor necrosis factor alpha were observed. These data suggest a direct involvement of RF complexes in the pathogenetic process of chronic inflammation in RA. PMID- 1707276 TI - Antagonists of bombesin/gastrin releasing peptide based on [D-Ala24]GRP(20-26) heptapeptide. Modifications leading to potent analogues with prolonged duration of action. AB - Analogues of gastrin releasing peptide (GRP) and bombesin based on His-Trp-Ala Val-D-Ala-His-Leu, the 20-26 heptapeptide sequence of [D-Ala24]GRP, have been synthesized and tested in vitro for their ability to inhibit GRP (18-27)-induced mitogenesis in Swiss 3T3 cells. Compounds identified as potent antagonists in this test system were also tested in vivo for their ability to inhibit bombesin induced amylase secretion in rats. The Trp-Ala-Val sequence was found to be a very important feature of the antagonist activity; most substitutions in this region led either to much less potent or inactive analogues. In contrast, amino acid replacements in other parts of the molecule were more tolerated and sometimes led to marked increases in the in vitro and in vivo activity. The most potent analogues were obtained by replacing Leu26 by MeLeu and His25 by Lys(X) where X = Z, PhCO, PhCH2CO or Ph(CH2)2CO. Thus 4-pyridylcarbonyl-His-Trp-Ala-Val D-Ala-Lys(CO-CH2-CH2-Ph)-Leu- NHMe (86) and 4-pyridylcarbonyl-His-Trp-Ala-Val-D Ala-Lys(Z)-MeLeu-OMe (87) had IC50 values of less than 20 micrograms/kg s.c. in vivo, and their effects lasted for more than 3 hr. PMID- 1707277 TI - Antitumor characteristics of the conjugate of N4-(4-carboxybutyryl)-ara-C with ethylenediamine-introduced dextran and its resistance to cytidine deaminase. AB - By oxidation of dextran, and reduction of the Schiff bases formed by reaction of the oxidised dextran with diaminoalkanes, several diaminoalkane-introduced dextrans were prepared and evaluated as drug carriers. Conjugates between N4-(4 carboxyburyryl)-1-beta-D-arabinofuranosylcytosine (glu-ara-C) and such drug carriers were prepared, and selected conjugates were tested in vivo, and investigated for inhibitory effects on cytidine deaminase. Ethylenediamine introduced dextran prepared under 10% oxidation conditions was found to be most useful as a drug carrier from its chemical characteristics and toxicity evaluation in BDF1 mice. The conjugate obtained from glu-ara-C and ethylenediamine-introduced dextran 2000 showed high antitumor activity, significant at the relatively low dose of 100 mg equivalent ara-C/kg, in BDF1 mice bearing L1210 leukemia cells. Glu-ara-C and the conjugate were unaffected by cytidine deaminase under conditions in which 1-beta-D-arabinofuranosylcytosine was degraded rapidly to 1-beta-D-arabinofuranosyluracil. PMID- 1707278 TI - Cloning and characterization of human placental catechol-O-methyltransferase cDNA. AB - Catechol-O-methyltransferase (COMT) cDNA clones were isolated from a human placental cDNA library using synthetic oligonucleotides as probes. All four positive clones isolated contained an open reading frame, which potentially coded for a 24.4-kD polypeptide, presumably corresponding to the cytoplasmic form of the COMT (S-COMT). In addition to the S-COMT sequences, two of the clones carried extensions in the 5' end, which potentially coded for a 50-amino-acid peptide extending the S-COMT reading frame. This sequence contained a stretch of signal sequence-like hydrophobic amino acids in its amino terminus. The deduced human COMT polypeptide had 80% similarity with the previously characterized rat COMT. Expression of one of the cDNA clones in human K-562 cells resulted in cell clones with 3- to 10-fold increased COMT activity. Cell-free translation of transcripts synthesized in vitro from one of the short cDNAs yielded a 26-kD product, similar in size to human S-COMT. Translation of transcripts from one of the long cDNAs gave 30-kD and 26-kD polypeptides, suggesting translation initiation from two different AUG initiation codons. The 30-kD protein, but not the 25-kD protein, associated with microsomal membranes in translation lysates. A potential polyadenylation signal AATTAA was detected in the 3' ends of two of the clones 265 nucleotides downstream from the COMT translation termination codon. RNA blotting on human placental RNA revealed a 1.5-kb-long COMT-specific transcript. DNA analysis suggested that human, as well as rat, canine and monkey cells have one gene for COMT. PMID- 1707279 TI - Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae. AB - The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids. We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter. P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21. The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17 hydroxyprogesterone. These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells. The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature. The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein. Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20 100 lower Vmax values; yet it retained normal affinity toward both substrates. This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm. PMID- 1707280 TI - Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor. AB - We have isolated and characterized a cDNA encoding a chicken beta homolog of c erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene. PMID- 1707281 TI - Distribution and origins of substance P-immunoreactive projections to the paraventricular and supraoptic nuclei: partial overlap with ascending catecholaminergic projections. AB - Anatomical and pharmacological evidence suggests a role for substance P (SP) in the control of vasopressin secretion, but the origins of SP-immunoreactive (IR) projections to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus have not yet been identified. Combined axonal transport, immunohistochemical, and ablation approaches were used to characterize the organization of SP-IR projections to the PVH. The results may be summarized as follows: (1) SP-IR projections are broadly and prominently distributed throughout the SO and both the magnocellular and parvicellular divisions of the PVH. The distribution within the PVH is quite uniform. (2) Combined retrograde transport immunohistochemical analyses identified multiple potential sources of SP-IR inputs to the PVH. These included a number of hypothalamic cell groups, the laterodorsal and peduculopontine tegmental nuclei, and the rostral and caudal aspects of the ventrolateral medulla. Portions of the tegmental and medullary SP IR neurons that were retrogradely labelled following tracer deposits in the PVH also stained positively for choline acetyltransferase or tyrosine hydroxylase, respectively. (3) To evaluate the distribution and prominence of medullary SP-IR projections to the PVH and SO, staining for SP and catecholamine-synthesizing enzymes was carried out in animals that had previously received knife cuts at the level of the pontomedullary border. Pronounced, and roughly parallel decrements in staining for peptide and amines were seen in the magnocellular division of the PVH and in the SO; less marked reductions in SP-IR varicosities are in a position to influence multiple visceral regulatory cell types in the PVH and SO. Inputs to the magnocellular neurosecretory system arise in large measure from medullary neurons in which SP coexists with catecholamines. SP-IR projections to the parvicellular division of the PVH appear to originate from a number of sources. PMID- 1707282 TI - Ciliary ultrastructure in a child with Kartagener's syndrome. A transmission electron microscopic study using tannic acid staining. AB - Kartagener's syndrome has been characterized by a primary ultrastructural abnormality of the cilia which consequently impairs their movements. We used transmission electron microscopy with tannic acid staining to investigate the fine structure of the cilia from the nasal mucosa of a 7-year-old girl with Kartagener's syndrome. The staining technique employed was useful for visualizing the dynein arms and protofilaments of the microtubules of the cilia. Although 15% of the cilia examined demonstrated microtubular disarrangements, these findings were considered to be acquired changes due to chronic sinusitis. No abnormal ciliary ultrastructures specific to Kartagener's syndrome, such as absence of dynein arms, were detected in this study. In such cases without any abnormal ciliary ultrastructures, it is conceivable that some other unknown factor may be involved in the impaired ciliary movement. PMID- 1707283 TI - The expression of vimentin in epithelial cells from human nasal mucosa. AB - The results of an immunohistological study of the normal human nasal mucosa show that there are frequently vimentin-positive cells detectable in addition to cytokeratins in the respiratory epithelium. The vimentin cells are probably ciliated and/or goblet type in origin. Furthermore, some co-expressing cells were found in basal parts of the submucous glands. PMID- 1707284 TI - Effect of glycyrrhizin on pain and HLA-DR antigen expression on CD8-positive cells in peripheral blood of herpes zoster patients in comparison with other antiviral agents. AB - Glycyrrhizin (GL) is a saponin widely used as an anti-inflammatory agent. Pain intensity and HLA-DR antigen expression on CD8+ cells were assessed during and after treatment with GL. Other agents such as acyclovir, gamma-globulin and interferon beta were also administered for comparison. Pain resolved most rapidly among those treated with acyclovir followed by those treated with GL. Pain resolution correlated with the regression of HLA-DR+ in CD8+ subpopulations in peripheral blood. GL is suggested to be an alternative or additive antiviral agent to herpes zoster. PMID- 1707285 TI - Hyaluronan and its binding proteins, the hyaladherins. PMID- 1707286 TI - Artiodactylan phylogeny: an immunogenetic study based on comparative determinant analysis. AB - The phylogenetic relationships of major artiodactylan taxa were investigated by means of comparative determinant analysis (CDA). Monospecific antisera against taurine cattle albumin, transferrin, C3 and IgM were used to derive determinant formulas of their homologues in 21 species (plus 12 other mammals for outgroup comparison). Fifteen accepted mutations could be demonstrated in Artiodactyla, permitting recognition of nine immunologically defined species groups. Results with phylogenetically relevant implications include the clear immunogenetic separation of the vicugna from true ruminants, a complex pattern of accepted mutations rendering a genealogical analysis of the principal pecoran radiation difficult, one synapomorphic mutation combining the goitred gazelle with bovines but excluding Caprinae, and the immunological recognition of the three grades of wild cattle evolution. This study demonstrates the suitability of CDA as a tool of phylogenetic systematics above the level of genera. PMID- 1707287 TI - Alpha-fetoprotein in vaginal fluids. The early diagnosis of premature rupture of membranes. AB - Alpha-fetoprotein, one of the most characteristic proteins of pregnancy, is present in the amniotic fluid during the whole period of pregnancy. In the case of premature rupture of the membranes it is appearing in the vaginal fluid. The level of AFP was measured by the ELISA technique in the vaginal fluid of pregnant women. In 70 cases of the rupture of membranes and in 33 cases of splitting of the membranes the AFP was measurable in the vaginal fluid and it was not detectable in 31 cases of urine and in 16 cases of vaginal fluids of pregnant women without any sign of premature rupture of the membranes. PMID- 1707288 TI - Radiotherapy in the elderly. PMID- 1707289 TI - Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. AB - In cell culture the cytotoxicity of some anticancer drugs, especially bleomycin, can be greatly enhanced by exposing cells to non-cytotoxic electric pulses. Nude or conventional mice bearing subcutaneous transplanted tumours were treated with intramuscular doses of bleomycin followed by local delivery of electric pulses similar to those used in vitro. Tumors were reduced and even eradicated after this electrochemotherapy. Thus the antitumour effects of bleomycin in mice can be considerably potentiated by local electric pulses. PMID- 1707290 TI - Electrochemotherapy of spontaneous mammary tumours in mice. AB - Electrochemotherapy delivers external electric pulses to the tumour site to induce local potentiation of the antitumour activity of intramuscular injections of bleomycin. C3H/Bi mice with spontaneous mammary carcinomas received weekly injections of 50 micrograms bleomycin followed by electric pulses 30 min later. All the 38 tumours treated exhibited at least a partial regression. 23 complete remissions were observed, 3 of which were cures. One difficulty in assessing the cure rate in this model is that frequent parallel or sequential tumours cause early death. Electrochemotherapy appears similarly efficient in spontaneous tumours as in previously studied transplanted tumours. PMID- 1707291 TI - The fibrinolytic response to venous occlusion and the natural anticoagulants in patients with antiphospholipid antibodies both with and without systemic lupus erythematosus. AB - Patients with systemic lupus erythematosus (SLE) have an increased risk of thrombosis and this is increased in the presence of antiphospholipid antibodies (APA). These APA are also associated with thrombosis in patients who do not have SLE. We compared haemostatic parameters in SLE patients with and without APA, and also compared patients who had APA but not SLE with healthy normal controls. No relationships between the natural anticoagulants, antithrombin III, heparin cofactor II, protein C and protein S, and the presence of APA were found. In the patients with SLE both tissue plasminogen activator antigen and plasminogen activator inhibitor (PAI) were increased, but these changes were not due to APA which had no effect on fibrinolysis in these patients. In the patients with APA who did not have SLE the fibrinolytic response to venous occlusion was reduced due to raised levels of PAI; similar changes have, however, been reported in some patients with idiopathic thrombosis. PMID- 1707292 TI - The haematology of homozygous sickle cell disease after the age of 40 years. AB - Haematological indices have been studied in 181 patients with homozygous sickle cell (SS) disease aged 40-73 years. Cross-sectional analyses in 5-year age bands indicated age-related decreases in HbF (males only), total haemoglobin and platelet counts. Longitudinal studies within individuals confirmed the downward age-related trend in haemoglobin and platelets and also revealed a falling reticulocyte count, most significant when expressed as absolute values. Total nucleated cells also fell although the decline was significant only in females. These observations are consistent with a progressive bone marrow failure which is not explained by the commonly occurring renal impairment in older SS patients since the changes persisted in analyses confined to patients with normal creatinine levels. The mechanism of this bone marrow failure is currently unknown. PMID- 1707293 TI - AIDS-associated Kaposi's sarcoma. PMID- 1707294 TI - Advances in the diagnosis of Pneumocystis carinii pneumonia. AB - There has been a dramatic improvement in the ability to diagnose P. carinii pneumonia since the beginning of the AIDS epidemic. Currently at the NIH over 90% of patients with P. carinii pneumonia can be diagnosed within a few hours of presentation by examination of an induced sputum specimen. Improved diagnosis has led to earlier initiation of therapy and an improvement in survival. However, as clinical management of AIDS patients improves, it is possible that there will be a change in the clinical presentation of P. carinii pneumonia, with an associated increased difficulty in making the correct diagnosis. PMID- 1707295 TI - New developments in antiretroviral drug therapy for human immunodeficiency virus infections. PMID- 1707296 TI - Palliative care in oncology: making quality the endpoint. PMID- 1707297 TI - Quality-of-life assessment during a palliative care programme. AB - By means of a cross-sectional study, 115 terminal cancer patients (53 males, 62 females) who were no longer responsive to anticancer treatment, were investigated. The sample included all patients who had been undergoing palliative care (PC) during a single week (at the out-patient clinic, in hospital or at home). From the start of PC, the quality of the patients' lives was assessed by a weekly self-descriptive record comprising 32 items at four levels of intensity. The responses given on the questionnaire during the sample week were compared to those given by the same patients at the beginning of PC (T0). From T0 to time during treatment, figures show a significant increase in the percentage of patients who reported drowsiness, and a significant decrease in pain, weakness, functional impairment and psychological distress. The global judgment of not feeling well was reduced from 49% of the patients at TO to 31% during the period of treatment (p less than 0.01). This result shows that, although the disease progressively develops, PC can enhance the quality of the lives of patients during the terminal stages of illness. The subjective judgment of not feeling well was much more closely correlated with physical, functional and psychological symptoms. Of the physical symptoms, pain has the closest correlation with feeling bad. However, pain has a low number of statistically significant correlations with respect to the other items, in marked contrast to the high number of correlations regarding psychological and functional items.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707298 TI - Placebo controlled phase I/II study of subcutaneous GM-CSF in patients with germ cell tumors undergoing chemotherapy. AB - Patients with metastatic germ cell tumors undergoing five-day chemotherapy with etoposide, vinblastin, bleomycin and cisplatinum were given recombinant GM-CSF (mammalian glycosylated, Sandoz/Schering-Plough) at increasing dose levels of 75, 150, 300 or 600 micrograms protein/day in a double blind placebo controlled study. The drug was administered SC twice a day for 5 days starting 24 hours after completion of chemotherapy. Fourteen treatment courses, 10 with GM-CSF and 4 with placebo in 11 patients were evaluable for assessment of toxicity and hematological recovery, and 2 were not evaluable due to complications of progressive germ cell tumor. One patient receiving the highest dose level developed a delayed skin reaction at the site of injection. Fever under 38.5 degrees C and a flu-like syndrome were observed in 4/5 patients receiving the higher two dose levels, but not with lower dose levels or placebo. Two patients experienced mild bone pain. The neutrophil nadir was similar in the two groups, but the duration of neutropenia was significantly shorter in the GM-CSF group. At day 21 of chemotherapy the neutrophil count was 2.57 +/- 1.37 10(9)/l with GM CSF, and 1.01 +/- 0.56 10(9)/l with placebo (p less than 0.05). Patients receiving GM-CSF could be retreated on day 21, whereas in patients given placebo, retreatment was delayed for an average of 7 days (p less than 0.05). Thus, a 5 day treatment with GM-CSF given subcutaneously resulted in a significant shortening of neutropenia and allowed for the timely administration of the subsequent cycle of chemotherapy. PMID- 1707299 TI - Differential keratin gene expression in developing, differentiating, preneoplastic, and neoplastic mouse mammary epithelium. AB - Two keratins whose expression has been associated with proliferation (K14) and hyperproliferation (K6) in mouse epithelia were detected in normal, preneoplastic, and neoplastic mouse mammary tissues. K6 and K14 keratins were independently expressed in distinct epithelial cell populations in developing mammary anlage. K6 was confined to a small number of mammary epithelial cells associated with the growing end buds and among the proximal luminal epithelium, whereas K14 expression appeared in basally located fusiform cells that correspond to the location of mammary myoepithelial cells. This pattern was maintained in mature glands and through full functional differentiation with the exception that K6-positive cells were only rarely detectable. During lobuloalveolar growth in early pregnancy, K6 and K6/K14 coexpressing cells were observed among the luminal and suprabasal cells in the expanding lobular epithelium. This K6/K14 coexpressing epithelial subset persisted throughout pregnancy, lactation, and involution, albeit in much smaller numbers than observed in early pregnancy. Two patterns of K6 and K14 expression in preneoplastic and neoplastic lesions of mouse mammary glands were induced by various carcinogenic stimuli. In one, increased numbers of K6- or K14-positive cells were present in distinct cellular populations; in the other, coexpression of K6/K14 was found in a large subpopulation of both preneoplastic and neoplastic mammary epithelium. These observations suggest that expression of K6 and K14 keratins in the mouse mammary gland is associated with growth and expansion of specific mammary epithelial cell populations, and as such these keratins may be useful probes with which to identify mammary epithelium-specific primordial cells. In agreement with this possibility, K6/K14 expression was demonstrated within a distinct subset of morphologically distinct luminal mammary epithelial cells that have been reported to possess kinetic properties in vitro consistent with those expected of latent mammogenic stem cells. PMID- 1707300 TI - Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2 aminopurine and 6-dimethylaminopurine. AB - The protein kinase inhibitors 2-aminopurine (2-AP) and 6-dimethylaminopurine (6 DMAP) were used to examine the effects of protein dephosphorylation on the control of mitosis in mammalian cells. Both 2-AP and 6-DMAP induced premature mitosis in hamster fibroblasts that were arrested in S phase. This response was characterized by changes in cell morphology, breakdown of the nuclear envelope, and premature chromosome condensation. Premature mitosis was followed by a return to interphase morphology and reformation of the nuclear envelope around decondensed and fragmented chromatin to form numerous micronuclei. The activity of both compounds was dependent upon new protein synthesis but not new RNA synthesis. 2-AP and 6-DMAP acted cooperatively with each other and with caffeine, suggesting a common mechanism of action. In exponentially growing cells, 2-AP and 6-DMAP did not induce premature mitosis but did increase the frequency of binucleated cells by blocking cytokinesis. These findings support a role for protein dephosphorylation in the control of mitosis and indicate that cell cycle perturbations can modify this regulation. PMID- 1707301 TI - [Recent advances in gastrointestinal hormones]. AB - Recent advances in the field of peptide chemistry and gene technology have resulted in an explosive accumulation of information on the chemical structures of gastrointestinal hormones. Based on the information, chemical syntheses of these peptides or their shorter fragments and analogs have been performed. Synthetic peptides related to the hormones have now become important tools in searching for functions of peptides and in producing region-specific antisera to the respective peptides. Using these antisera, hormone-producing cells were clearly identified and the post-translational biosynthetic processings in the cells were demonstrated. Recent immunohistochemical studies have also revealed that cells contain and can release a variety of peptides or amines that are capable of influencing target cells and acting as hormone, neurotransmitter or neuromodulator. In addition, recent studies on galanin and glucagon-like peptide 1 (GLP-1) are described. PMID- 1707302 TI - Experimental membranous glomerulonephritis in rats induced with gp 700, a glomerular protein. AB - To investigate the antigenicity of glomerular cell products for the induction of Heymann nephritis (HN), eight Lewis rats were immunized with an isolated glomerular protein (GLP) with only limited tubular cell contamination. Four out of four rats immunized with 240 micrograms GLP showed proteinuria at week 12. Their kidney specimens taken at week 16 showed contiguous granular deposits of IgG along the glomerular basement membrane by direct immunofluorescence and corresponding electron-dense deposits; the findings were identical to those in classical HN induced by the brush border protein. By immunoprecipitation, IgG eluted from isolated glomeruli of rats that received 240 micrograms GLP precipitated only a 700 kd glycoprotein (gp 700) in GLP, whereas 330, 440, and 700 kd glycoproteins were precipitated in the crude preparation (Fx1A), which is derived mainly from renal tubules. Two different monoclonal antibodies (anti-gp 330 and 14C1) against gp 330 precipitated the antigens in the same fashion as the glomerular eluate. These data reveal that gp 700, gp 440, and gp 330 share epitopes and that the gp 700 is demonstrable both in the glomerulus and in tubular brush border but that gp 440 and gp 330 are present only in the brush border. In addition, glomerular gp 700 was shown to induce Heymann-type glomerulonephritis just as gp 330 did. PMID- 1707303 TI - Characterization of a 70Z/3 pre-B cell derived macrophage clone. Differential expression of Hox family genes. AB - An adherent cell line was established from the much studied pre-B cell line 70Z/3. The adherent cell line had the morphological features of a macrophage and stained positive for non-specific esterase. In addition, this line expressed high levels of RNA for the macrophage specific enzyme, lysozyme. Although the 70Z/3 macrophage variant has lost the ability to respond to lipopolysaccharide and interferon-gamma by expressing RNA transcripts for immunoglobulin light chain, it exhibited a new response characterized by increased levels of I-A RNA transcripts. Both the pre-B and macrophage variants also expressed RNA transcripts which hybridize to a Hox 2.3 probe. In contrast, transcripts which hybridize to Hox 1.1, 2.1, and 6.1 probes are expressed in the pre-B line but could not be detected in the macrophage. PMID- 1707304 TI - Cell-mediated immune response to copolymer I in multiple sclerosis measured by the macrophage procoagulant activity assay. AB - The macrophage/monocyte procoagulant activity (MPCA) assay, a sensitive and specific in vitro test for cell-mediated immunity, has been used to ascertain the reactivity of MS peripheral blood mononuclear cells (PBM) to copolymer I (Copl), a synthetic peptide analogue of myelin basic protein (MBP) currently being tested as a possible therapeutic agent in multiple sclerosis (MS). Because the suppressive effect of Copl is believed to lie in its possible cross-reactivity with MBP, the reactivity of PBM of MS patients to MBP was also tested. MS patients either at the stable phase of the relapsing/remitting form or with chronic progressive disease were investigated and compared with patients with other diseases and with healthy subjects. The reactivity to Copl was significantly increased in patients with chronic progressive disease but not in stable MS patients or in control subjects. No difference in reactivity to MBP between MS patients and healthy subjects was found regardless of disease status. However, in the control group comprising patients with other diseases, MBP reactivity was significantly elevated. In chronic progressive MS patients, a relationship was found between the response to Copl and that to MBP, supporting the possibility of an immunological cross-reactivity between these two antigens. There was no significant difference in reactivity to the non-specific stimulant, lipopolysaccharide, between the MS and control groups. PMID- 1707305 TI - Cell division-associated expression of an epitope, KH17, on early developing thymocytes. AB - A newly established monoclonal antibody, KH17, detects a unique epitope temporarily expressed on early developing CD3-thymocytes confined to a cycling stage. KH17 is detectable on a part of CD4-CD8-,CD4-CD8+, and CD4+CD8+ cells, but not on CD4+CD8- thymocytes. By four-color flow cytometry analysis using KH17, we were able to define the heterogeneity of immature CD4-CD8- thymocytes by the expression of KH17 and IL-2R. In Thy-1-congeneic bone marrow chimeras, the appearance of KH17-IL-2R+ thymocytes preceded the increase of KH17+IL-2R- cells. The antibody could also divide CD3-CD4-CD8+ cells into two subpopulations, KH17+ and KH17-, which showed a continuum. In the fetal thymus there was a rapid and dramatic increase of KH17-CD4+CD8+ thymocytes concomitant with a decrease of KH17+CD4-CD8+ thymocytes in later gestation days. KH17 is not expressed on resting peripheral T cells, but is expressed on a large proportion of Con A activated blastic spleen cells. The KH17 molecules precipitated from Con A activated spleen cells were 55 and 75 kd polypeptides, but different from IL-2R subunits. PMID- 1707306 TI - Inhibition of T cell proliferation specific for acetylcholine receptor epitopes related to myasthenia gravis with antibody to T cell receptor or with competitive synthetic polymers. AB - Long-term T cell lines and clones of C3H.SW origin specific to synthetic peptides representing immunogenic epitopes of the human aetylcholine receptor alpha subunit were established. Using these lines and clones, it was possible to characterize the T cell recognition process of myasthenic epitopes. Testing a panel of N-and/or C-terminal truncated peptides it could be demonstrated that the deletion of the two C-terminal amino acids of peptides p195-212 and p259-271 resulted in a loss or reduction of the stimulatory capacity of the peptides towards the specific T cell lines. In contrast, no substantial effect on the stimulation of the line could be observed by shortening peptide p195-212 by up to five amino acids at the N-terminal end. The proliferation of T cell lines and clones specific to peptide p195-212 was inhibited by a mAb directed against the V beta 8 region of the T cell receptor. Furthermore, it was possible to block the peptide-specific proliferative responses of the lines and clones by the I-Ab restricted synthetic polypeptide (T, G)-A--L but not by the I-Ak restricted polypeptide antigen (H,G)-A--L. Similarly, p195-212 inhibited the proliferative response of the TCSW259-271 T cell line and p259-271 inhibited the specific proliferative response of the TCSW195-212 line. Moreover, the C-terminal shortened peptides inhibited significantly the in vitro stimulation of the T cell lines by the immunogenic peptides p195-212 and p259-271. The inhibition by the synthetic peptides or by (T,G)-A--L may be due to competitive blockade of the MHC binding site for the T cell line stimulating AChR peptides. PMID- 1707307 TI - Translational diffusion and intrinsic viscosity of globular proteins. Theoretical predictions using hydrated hydrodynamic models. Application to BPTI. AB - In this paper, we present a way to make hydrodynamic models of globular proteins, including the hydration shell associated with them in aqueous solutions. Theoretical calculations using these models are made in order to determine the hydrodynamic properties of these proteins, employing rigorous and approximate methods of calculation. These will be applied to the bovine pancreatic trypsin inhibitor, BPTI. Several hydrodynamic models are constructed: the A-model for the unhydrated protein BPTI and a set of H-models for hydrated protein with different hydration degrees. Theoretical results for the translational diffusion coefficient Dt and the intrinsic viscosity [eta] are obtained from different models. From the analysis of the A-model and hydrodynamic properties, there is not a clear assignation of an ellipsoidal shape to this protein molecule. An amount of approximately 0.5 g H2O/g protein could be assigned to the BPTI. PMID- 1707308 TI - Symmetry of disciform scars in bilateral age-related macular degeneration. AB - The size of the final macular scar in subretinal neovascularisation (SRNV) is one of the most important determinants of final visual function in patients with subfoveal disease. We studied patients with bilateral macular scars from age related subretinal neovascular membranes retrospectively in order to determine whether or not fellow eyes behave similarly. We found a significant correlation between eyes in terms of final scar size (r = 0.50, p less than 0.01). We found that 50% of fellow eyes with large macular scars (greater than 3 x 10(6) microns2) had similar sized lesions, while only 16% of fellow eyes with small macular scars (less than 0.5 x 10(6) microns2) had large scars (p less than 0.01). We discuss the significance of these findings in relation to the pathogenesis of subretinal neovascular membranes, and their implications for treatment. PMID- 1707309 TI - Activation of interferon-inducible genes in mice by poly rI:rC or alloantigens. AB - We have examined the effects of synthetic dsRNA (poly rI:rC) treatment or of immunization with irradiated allogeneic cells on the expression in vivo of several interferon (IFN)-inducible genes. For this purpose, DBA/2 mice were injected i.p. once with poly rI:rC, or once and then again 3 weeks later, with irradiated C3H/He spleen cells and the effect of these treatments on the levels of the following mRNAs was determined: 202, 2',5'-oligoadenylate synthetase (2-5A synthetase), class I and class II major histocompatibility antigens, and beta actin. After poly rl:rC treatment, the levels of the 202 and 2-5A synthetase mRNAs in the spleen and in the bone marrow peaked between 12 and 24 h and decreased thereafter. The class I mRNA levels started to increase at 12 h, peaked at 24 h, and declined thereafter. No increase in class II mRNA expression was observed after poly rl:rC injection, whereas beta-actin levels remained unchanged. Pretreatment of DBA/2 mice with sheep anti-murine IFN-alpha/beta antibodies before poly rI:rC injection strongly diminished the induction of 202 mRNA, indicating that IFN-alpha/beta mediated this induction. When irradiated C3H/He spleen cells were injected into DBA/2 mice, the class I and class II mRNAs in the spleen, but not in the bone marrow, started to increase at 12 h, peaked between 48 and 96 h, and decreased thereafter. No increase in the levels of 202 and 2-5A synthetase mRNAs was detected, whereas beta-actin levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707310 TI - Proteolysis of the cytochrome d complex with trypsin and chymotrypsin localizes a quinol oxidase domain. AB - The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the two electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I. This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex. The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E. coli as the substrate for the proteolysis. Hence, the protease-sensitive locus is periplasmic in the cell. Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I. The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop. This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies. Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme. It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane. PMID- 1707311 TI - Rotational diffusion of band 3 in erythrocyte membranes. 1. Comparison of ghosts and intact cells. AB - The rotational diffusion of eosin-labeled 3 in human erythrocyte cells and hemoglobin-free ghosts at 37 degrees C has been studied in detail by polarized delayed luminescence. The time-resolved anisotropy with both cells and freshly prepared ghosts is similar, decaying with well-resolved rotational correlation times of 0.03, 0.2, and greater than or equal to 1 ms. Mild proteolytic removal of the water-soluble 41-kDa cytoplasmic domain of band 3 in ghosts results in a drastic increase in the fractional contributions of the two fastest depolarizing components. Our results, taken together with other data in the literature, imply that several classes of band 3 that differ greatly in mobility exist in ghosts and intact cells. The mobility of one class is hindered due to complexation with other membrane or cytoplasmic proteins mediated via the 41-kDa cytoplasmic domain. However, another class of band 3 molecules exists as homo-or heterooligomeric complexes larger than a dimer that are stabilized by hydrophobic interactions involving the intramembranal domain. Finally, the presence of the (previously undetected) 0.03-ms anisotropy component strongly suggests that a significant fraction of band 3 in both ghosts and intact cells is highly mobile and diffuses at the rate expected for a freely rotating dimer in the erythrocyte membrane. PMID- 1707312 TI - Ion channels formed by a highly charged peptide. AB - A peptide (MA-beta) corresponding to a segment of the nicotinic acetylcholine receptor (AChR) that has amphipathic alpha-helical periodicity forms ion channels in artificial phospholipid bilayers. The MA-beta ion channels are very stable, comprise two discrete conductance states, and undergo rapid, flickering-type closings. The discrete-conductance ion channels formed by MA-beta contrast with the continuous-conductance ion channels formed by a peptide (M2-delta) identical in sequence with M2 [Oiki, S., Danho, W., Madison, V., & Montal, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8703-8707], a putative transmembrane segment of the AChR. Neither MA-beta nor M2-delta sufficiently mimics the electrophysiological properties of the native AChR. We suggest that peptide ion channels can be classified into at least three general groups: discrete-conductance channels, such as MA-beta; continuous-conductance channels, such as M2-delta; and membrane disruptors, such as those formed by short, amphipathic alpha-helical peptides. PMID- 1707313 TI - Phosphorylation sites of the nicotinic acetylcholine receptor. A novel site detected in position delta S362. AB - The delta-subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue isolated form receptor purified in the absence of protein phosphatase inhibitors contains a total of four phosphate groups. Three of these are shown to represent phosphoserine groups. The fourth possible represents phosphotyrosine. The phosphate groups are localized within the primary structure: We found phosphoserine in positions delta S361 and delta S377, the predicted sites phosphorylated by PKA and PKC, respectively. In addition, we found that position delta S362 is also phosphorylated. Phosphorylation experiments with the synthetic peptide delta L357-delta K368 show that phosphorylation of this novel site can be catalyzed by PKA and by PKC. It is concluded that the delat-subunit of the acetylcholine receptor is stably and not transiently phosphorylated. Implications for the physiological functions of receptor phosphorylation are discussed. PMID- 1707314 TI - [Selectivity of latrotoxin channel in lipid bilayers]. AB - It is established that high initial K-TEA and Ca2(+)-K+ selectivity of channel form by latrotoxin in lipid bilayers (LT-channel) may be reduced by lowering the pH value and by increasing electrolytes concentration of solution. It is suggested that LT-channel is water-filled pore cation selectivity which is defined by the electrostatic potential on the mouth of the channel, which is induced by the ionogenic group of toxin. PMID- 1707315 TI - [Microfluorometric study of pH in frog nerves at rest and during rhythmic stimulation]. AB - With the use of microspectrofluorimetry the pH-dynamics in the frog nerve at rest and during rhythmic excitation was investigated. Rhythmic excitation results in an increase of hydrogen ion concentration in the nerve fibres. Metabolic and ion channel inhibitors affect intracellular pH changes. The results obtained suggest different role of various transport systems (sodium pump and ion channels) in the regulation of pH at rest and during rhythmic excitation. PMID- 1707316 TI - Clinical applications of the polymerase chain reaction. PMID- 1707317 TI - [Stimulation of cardiac lymphatic drainage by obzidan, heparin and rheogluman in acute myocardial ischemia]. AB - The state of the lymphatic heart perfusion has been studied experimentally on dogs with intact myocardium (control 1), with acute myocardial ischemia (AMI) model (control 2) and during injection of heparin, rheogluman and obsidan in AMI. It has been stated that AMI induces acute distress of the lymphatic heart perfusion system occurring at the first minutes after the onset of the focal myocardial ischemia. Heparin, rheogluman and obsidan demonstrated selective stimulating effect on the function of the lymphatic myocardial system. PMID- 1707318 TI - [The immunochemical identification of beta-globulin in human seminal plasma]. AB - Using affinity chromatography there was isolated a protein from human seminal plasma, which was named beta-globulin of seminal plasma (beta-GSP). It was demonstrated, that beta-GSP possesses heterogeneity, consists of two subunits with the M. W. about 19 and 15 kD, it has electrophoretic mobility of b1-globulin and possesses affinity to immobilized 17-beta-estradiol. Relatively high concentration of beta-GSP was found in seminal plasma. Besides, this protein was determined in small amounts in saliva. One can suppose, that beta-GSP is a secretory protein. PMID- 1707320 TI - [Seasonal changes in lysosomal pH of peripheral blood cells of healthy subjects]. AB - We studied pH values in lysosomes of peripheral blood white cells of 49 blood donors using the two-wavelengths (470 and 450 nm) microspectrophotometry of neutral red after supravital in vitro staining. In polymorphonuclear leucocytes lysosomal pH reaches the highest values in August-September (6.98 +/- 0.04) and the lowest--in February-March (6.23 +/- 0.09), p less than 0.05. PMID- 1707319 TI - [Endogenous intoxication of the body in acute myocardial infarct during lymph stimulation]. AB - It was stated experimentally in dogs that the elevation of the lymph toxicity was more expressed than that of the blood in acute myocardial infarction. The shortening of the half-life period of paramecia evidenced the above mentioned fact. The injection of the lymphogogue preparations (obsidan, heparin, rheogluman) after coronary artery occlusion resulted in distinct rise of blood and lymph toxicity in early periods because of the "washing out" of toxic products from the ischemic myocardium, followed by normalization that had been more quicker than in controls. PMID- 1707321 TI - Neuropeptide-immunoreactive cells in human thymus. AB - The outer cortex of the human thymus contains a one- to two-cell-thick layer that is immunoreactive with antisera against beta-endorphin, (Leu)- and (Met) enkephalin, bombesin, and substance P. The epithelial nature of these immunostained cells is revealed by immunoelectron microscopic studies showing the presence of desmosomal junctions. The presence of peptide-containing cells in the outer cortex, where the most immature and recently immigrated thymocytes are found, emphasizes the role of neuropeptides in regulating the microenvironment for T cell development. PMID- 1707322 TI - Teaching junior doctors practical procedures. PMID- 1707323 TI - Lithium maintenance: 1. A standard education programme for patients. AB - A videotape lecture and written hand-out containing factual information about lithium were given to 30 attenders at a lithium clinic. A further 30 patients acted as a control group and were not given the programme until later in the study. The educational programme resulted in substantial and significant increases in patient knowledge about lithium, such that knowledge increased from a baseline comparable with that of social workers to a level similar to that of community psychiatric nurses. Patients' attitudes to lithium also became more favourable after education. PMID- 1707324 TI - Urinary chromatographic profiles in psychiatric diseases. PMID- 1707325 TI - Acute effects of intranigral application of MPP+ on nigral and bilateral striatal release of dopamine simultaneously recorded by microdialysis. AB - Intracerebral microdialysis in freely moving rats was used to investigate the effects of perfusions with the 1-methyl-4-phenylpyridinium ion (MPP+) in the substantia nigra (SN) on the extracellular levels of dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5 hydroxyindoleacetic acid (5-HIAA) in the perfused SN and in the ipsi- and contralateral striata. Following MPP+ perfusion, the release of DA in the SN increased markedly from nondetectable basal levels to about 105 fmoles/min, whereas the output of DOPAC, HVA and 5-HIAA decreased below 25% of basal levels. The intranigral MPP+ application induced, at the same time, an almost immediate, long-lasting decrease in the release of DA in the ipsilateral striatum to less than 20% of basal levels and a moderate increase in the DOPAC and HVA levels, without affecting 5-HIAA output. In the contralateral striatum, the extracellular levels of DA, DOPAC, HVA and 5-HIAA remained unchanged during the entire perfusion experiment. These results suggest that infusion of 10 mM MPP+ into the SN produces an almost immediate blockade of neuronal impulse flow, as shown by the rapid decline in DA release from the ipsilateral striatal nerve terminals. The simultaneously occurring massive increase of the extracellular DA in the SN is, therefore, probably the result of destruction of the nigral cell bodies and/or dendrites following locally applied MPP+. This study clearly illustrates the possibilities of simultaneous microdialysis in various brain areas, allowing pharmacological manipulations on the levels of the cell bodies, while monitoring events in the terminal areas. PMID- 1707326 TI - Chronic nerve growth factor treatment of normotensive rats. AB - The objectives of this study were to examine the effects of chronic nerve growth factor (NGF) administration on vascular innervation and blood pressure in neonatal rats. Newborn Wistar-Kyoto (WKY) rats bred from normotensive parents were chronically treated with NGF for 8 weeks. Littermate controls received saline. Sympathetic ganglia of treated animals were greatly enlarged and in the superior cervical ganglion neuronal numbers were increased 200% and nuclear areas by 46%. The catecholamine contents of several sympathetically innervated tissues were determined biochemically and found to be significantly elevated in mesenteric arteries, aorta, ileum, adrenal and salivary glands from treated compared to control animals. The catecholamine concentrations were similar to, or exceeded those of the spontaneously hypertensive rat. Histochemically, an aberrant nerve supply was evident occupying a greater volume of the adventitia of the caudal artery and mesenteric arteries. In addition, nerve fibres could be seen penetrating the vessel wall to emerge within the lumen of mesenteric blood vessels. Analysis of the smooth muscle wall of the caudal artery revealed that a small but significantly hyperplastic response had been induced. Systolic blood pressures of NGF-treated and control animals were taken at one week intervals from 5 to 8 weeks of age utilizing the tail cuff method. The blood pressure of treated animals were within the normotensive range. It is concluded that chronic NGF treatment leads to changes in vascular innervation and muscle thickness that are similar to those seen in hypertensive animals. Furthermore, the results suggest the elevated levels of NGF seen in peripheral tissues of the spontaneously hypertensive rat are likely to be responsible for the hyperinnervation and resulting hyperplastic responses within vascular tissues, but not exclusively responsible for the elevated blood pressure. PMID- 1707327 TI - Substance P in the habenulo-interpeduncular system of the goldfish. AB - The distribution of substance P (SP)-like immunoreactivity has been studied in the habenulo-interpeduncular system of the goldfish in normal conditions and after habenular ablation. In normal conditions intense SP-like immunoreactivity was observed in the neuropilar structure of the interpeduncular nucleus (IPN). No SP-like immunoreactive cell bodies were observed in the habenular nuclei (HBN), but some SP-like immunoreactive fibres were localized in the central core of the nucleus. Following surgical habenular ablation SP-like immunoreactivity was reduced in the IPN. The image analysis performed on the IPN showed clear-cut transmittance changes in the area examined. The results suggest that SP is involved in connecting HBN and IPN in goldfish, and are consistent with the data of mammals and other vertebrates. PMID- 1707328 TI - Substance P injections into the ventral tegmentum affect unit activity in mesolimbic terminal regions. AB - Microinjections of substance P (SP) into the ventral tegmental area (VTA) increase locomotor activity in rats, and this effect is thought to be produced by activation of the mesolimbic dopamine system. In the present study, firing rates of neurons in areas receiving projections from the mesolimbic dopamine system were recorded during injections of SP (3 microgram in 0.5 microliters saline) into the VTA of rats anesthetized with chloral hydrate. Significant changes in firing rates were observed in 84% of the units recorded in nucleus accumbens and olfactory tubercle. There were mostly decreases in nucleus accumbens (NAC, 21 of 25 units affected by SP) and mostly increases in olfactory tubercle (OT, 13 of 18 units affected by SP). In contrast, neither saline injections into VTA nor SP injections 2 mm dorsal to VTA had any effect on NAC or OT neurons. Haloperidol (0.5 mg/kg IV) blocked the effects of SP, suggesting that effects were mediated, at least in part, by the mesolimbic dopamine system. Results indicated that activation of dopaminergic neurons by SP injections into VTA can produce changes in the activity of neurons in NAC and OT, areas which receive mesolimbic dopaminergic projections. PMID- 1707330 TI - Expression of low molecular mass cytokeratins in oocytes of Schistosoma mansoni. AB - We studied the distribution of acidic 45 kDa keratin 18 and 40/42 kDa keratin 19 in Schistosoma mansoni, a trematode of medical importance in many tropical regions. The monoclonal antibodies which were produced against the cytoskeleton of mammary carcinoma cell line BT-20 recognized cytokeratins preferentially in parasite oocytes. As has been described in mammalian oocytes, the acidic cytokeratins were present in a nonfibrillar form. The two monoclonal antibodies also recognized testicular cells. No keratin immunoreactivity could be demonstrated by immunofluorescence microscopy at the larval stage, the miracidium. In immunoblotting, the molecular mass as determined by SDS polyacrylamide gel electrophoresis of schistosome cytokeratins was about 15 kDa higher than that of the corresponding cytokeratins recognized by the monoclonal antibodies in BT-20 cells. The results suggest that acidic low molecular mass cytokeratins in trematodes have a phylogenetically conserved major function in oocytes which is unrelated to the documented cytoskeletal role in differentiated mammalian epithelial cells. PMID- 1707329 TI - Partial attenuation of chronic fluphenazine-induced changes in regional monoamine metabolism by D-alpha-tocopherol in rat brain. AB - Recent evidence has suggested a role for free radicals in tardive dyskinesia. We, therefore, investigated the effects of chronic administration of fluphenazine decanoate (FLU) and/or vitamin E (VIT E) on regional monoamine metabolism in rat brain. Chronic FLU caused significant increases in dopamine (DA) in nucleus accumbens and brainstem, significant decreases in dihydroxyphenylacetic acid (DOPAC) in frontal cortex, nucleus accumbens and hippocampus and significant decreases in homovanillic acid (HVA) in nucleus accumbens, caudate-putamen and brainstem. Coadministration of FLU and VIT E normalized HVA in caudate-putamen, nucleus accumbens and brainstem as well as DOPAC in nucleus accumbens and hippocampus. Chronic FLU caused significant increases in norepinephrine (NE) levels in all regions studied. VIT E attenuated FLU-induced increases in NE levels in nucleus accumbens and hippocampus. Significant increases in serotonin (5-HT) levels occurred in nucleus accumbens and hippocampus whereas significant decreases in 5-hydroxyindole-acetic acid (5-HIAA) occurred in all brain regions after chronic FLU. Coadministration of VIT E attenuated the changes observed in hippocampal 5-HIAA but potentiated the FLU-induced increases in 5-HT in this region. Our data suggest that VIT E can attenuate some of the FLU-induced changes in monoamine metabolism. Results are discussed in relation to possible involvement of free radicals in monoamine metabolism during chronic neuroleptic use. PMID- 1707331 TI - Appearance of the keratin pair K3/K12 during embryonic and adult corneal epithelial differentiation in the chick and in the rabbit. AB - Sequential expression of the K3/K12 keratin pair was studied during epithelial corneal differentiation, using monoclonal antibodies coupled with electrophoretic analysis. In the chick embryo, K12 appears on day 12, while K3 is present at least from day 11. By contrast, in the rabbit embryo, the expression of K12 (at 17 days) precedes that of K3 (at 21 days). In the adult rabbit, K3 is expressed without K12 in part of the limbus. Thus, within the corneal-type keratin pair, either the acidic or the basic partner appears first, according to the developmental stage or the species. PMID- 1707332 TI - Production and characterization of monoclonal antibodies against major allergens of American cockroach. AB - From several fusion experiments between spleen cells obtained from BALB/c mice immunized with partially purified Cr-PI of American cockroach and NS-1 cells, growth was observed in many wells. Seven stable subclones secreting monoclonal antibodies (mAbs) against Cr-PI, as determined by enzyme-linked immunosorbent assay (ELISA) with high absorbance values and immunoblot analysis, were obtained. All seven mAbs were characterized as IgG1 subclass by immunodiffusion, and reacted strongly with 72 kilodaltons (kD) of Cr-PI which have been identified as a major allergen of American cockroach. Six mAbs were found to have similar epitope specificities against Cr-PI by ELISA. The remaining mAb was found to have different epitope specificities with others. Interestingly, all mAbs did not react with any components of crude extracts of Oriental and German cockroaches as determined by immunoblot analysis and ELISA. A mAb-based double-antibody sandwich ELISA was developed, and the ELISA was dose-dependent and capable of detecting as little as 140 ng of Cr-PI allergen. PMID- 1707333 TI - Ketotifen reduces sneezing but not histamine release following nasal challenge with antigen. AB - We evaluated the effect of pre-treatment with 1 and 2 mg b.i.d. of ketotifen on the early response to nasal challenge in a double-blind cross-over trial. Weekly nasal challenges were performed in 10 allergic subjects after 1 hr and 1, 2, 3 and 4 weeks of ketotifen administration. The response to nasal challenge was monitored by counting the number of sneezes, the assessment of subjective symptoms, and by measuring the levels of histamine and TAME-esterase activity in recovered nasal lavages. The number of sneezes diminished significantly after a single dose of drug with both the 1 and 2 mg doses. Prolonged pre-treatment did not improve the results. The levels of histamine and TAME-esterase activity in recovered nasal lavages were not changed significantly by either pre-treatment at either dose. Although the number of subjects was small, our data suggest that ketotifen diminishes allergic symptomatology by its antihistaminic properties rather than by inhibiting histamine release from mast cells. As we did not look into the effects of ketotifen on other products generated by mast cells (prostanoids, leukotrienes and tryptase), we cannot fully rule out an effect on mast cells. PMID- 1707334 TI - Serial immunological investigations in a patient who had a life-threatening reaction to intravenous protamine. AB - Reactions to intravenous protamine include rash, urticaria, bronchospasm, hypotension, and/or pulmonary artery pressure elevation. We have previously shown that in diabetic patients receiving daily protamine-insulin injections, the presence of anti-protamine IgE or IgG antibodies are significant risk factors for acute, life-threatening reactions when protamine is given intravenously. To study protamine reactions further, we measured serum anti-protamine IgE and IgG antibody levels, in-vitro basophil histamine release and intracutaneous skin testing to protamine serially in an NPH-insulin dependent diabetic who had a severe, protracted anaphylactic reaction to protamine. At the time of his protamine reaction, his serum contained 8.5 ng/ml of anti-protamine IgE and 1.3 micrograms/ml of anti-protamine IgG antibody. One month following the reaction both anti-protamine IgE and IgG increased to 16 ng/ml (twofold rise) and 90.5 micrograms/ml (70-fold rise), respectively. With time, both anti-protamine IgE and IgG antibody declined. Serial intradermal skin tests using protamine sulphate did not discriminate between the protamine reactor and nine normal control subjects who had no prior exposure nor any demonstrable serum IgE antibody to protamine. In-vitro basophil histamine release to protamine sulphate was inconclusive in discriminating between the protamine reactor and normal control subjects. We postulate that protamine may be an incomplete or univalent antigen that must first combine with a tissue macromolecule or possibly heparin to become a complete multivalent antigen capable of eliciting IgE antibody-dependent mediator release. PMID- 1707335 TI - Epitope mapping of two monoclonal antibodies to the central portion of human osteonectin. AB - In this study preliminary characterization of two monoclonal antibodies against osteonectin was undertaken. One monoclonal originally raised against bovine bone osteonectin cross reacts with human bone and platelet osteonectin. The other monoclonal antibody has been reported to react with osteonectin derived from human bone and bovine bone but not to the same extent with that from platelets. Initial mapping of the antigenic determinants for both monoclonals was done by testing their ability to bind to the expressed forms of osteonectin in two overlapping SaOS-2 lambda gt11 osteonectin cDNA clones. One clone contains a 0.54 kb insert and is comprised of 50 nucleotides of 5' noncoding and a coding segment for a 17 amino acid signal peptide and 146 amino acids of the N-terminal region of the mature protein. The other clone has a 1.9 kb insert, and includes amino acid no. 18 to the C-terminus of the molecule (amino acid no. 286), a single termination codon, and 1115 nucleotides of 3' noncoding sequence. Both monoclonals recognized expressed osteonectin from the two lambda gt11 SaOS-2 cDNA clones. These results localize the epitope to a region between amino acids 18-146 of osteonectin. PMID- 1707336 TI - Successful reinduction therapy with amsacrine and cyclocytidine in acute nonlymphoblastic leukemia in children. A report from the Childrens Cancer Study Group. AB - Amsacrine (AMSA) and cyclocytidine were studied as retrieval therapy in 122 pediatric patients with acute nonlymphoblastic leukemia (ANLL). Patients either failed to achieve sustained initial remissions or were in relapse. Induction therapy consisted of intravenous (IV) AMSA (75 mg/m2) from days 1 to 5 and subcutaneous cyclocytidine (600 mg/m2) from days 1 to 7. Maintenance therapy consisted of IV etoposide (VP-16) (100 mg/m2) for 5 days and IV AMSA (100 mg/m2) on day 1. Of 122 patients, 109 were evaluable. There were 13 early deaths. Ninety six patients received adequate therapy defined as completion of two courses of therapy. Of these 96 patients, 52 achieved complete remission. Fifteen of 33 patients who failed initial induction achieved complete remission. Eighteen of 39 patients who were resistant to anthracyclines had complete responses. There was no direct evidence of AMSA-induced cardiotoxicity. Remission duration was 28 days to 3 or more years (median, 98 days). AMSA and cyclocytidine were effective retrieval therapy for patients who were in relapse or unresponsive to frontline therapy. Duration of remission was short (median, 98 days). PMID- 1707337 TI - Evolution of neuropathy and myopathy during intensive vincristine/corticosteroid chemotherapy for non-Hodgkin's lymphoma. AB - Neuropathy and myopathy are common sequelae of intensive chemotherapy protocols that contain vincristine and corticosteroids. The authors prospectively monitored the evolution of neuropathy and myopathy during an intensive 12-week chemotherapy program for patients with intermediate and high-grade non-Hodgkin's lymphoma. In this study, vincristine was administered by bolus injection followed by a 3-day continuous intravenous (IV) infusion (total dose of 2.0 mg/m2 every other week); the maximum dose of vincristine was not arbitrarily limited. Cronassial, a mixture of four naturally occurring gangliosides, was administered in a randomized double-blind test to evaluate whether this agent could prevent vincristine-induced neuropathy. High doses of dexamethasone (50 mg/d for 3 days weekly or every other week) were also prescribed. Patients were monitored every 4 weeks with comprehensive physical and neurologic examinations and electrophysiologic studies of peripheral nerve function. Twenty-seven patients were fully evaluable. Weakness was a prominent adverse reaction in this study, and all patients had moderate to severe signs and symptoms of neuropathy and myopathy. Cronassial (100 mg) administered by intramuscular (IM) injection daily provided no protection against the development of neuropathic symptoms. Vincristine typically impaired fine-motor coordination initially, whereas corticosteroids were associated with delayed development of proximal muscle weakness. Results of electrodiagnostic studies did not add to the clinical examination results. The authors conclude that symptomatic weakness due to neuropathy or myopathy appears in a predictable manner during intensive vincristine/corticosteroid-based treatment protocols. Simple clinical tests can be used to rapidly distinguish between toxic effects due either to vincristine or corticosteroids, and routine implementation of these tests can prevent inappropriate dose attenuation of these agents. PMID- 1707338 TI - A randomized trial comparing radiation therapy versus concomitant radiation therapy and chemotherapy in carcinoma of the thoracic esophagus. AB - From September 1982 to December 1985, 59 previously untreated patients with Stage II squamous cell carcinoma of the thoracic esophagus were randomly assigned to receive radiation therapy (RT) alone versus the concomitant use of RT and chemotherapy (CT) with 5-fluorouracil (5-FU), mitomycin C, and bleomycin (RT + CT). Thirty-one patients were randomized to the RT regimen and 28 to the RT + CT regimen. The complete local response rate was 58% for the RT group and 75% for the RT + CT group (P = 0.77). The median duration of response was 8 months for both groups. The overall 5-year survival rates were 6% and 16% (P = 0.16) for the RT and RT + CT groups, respectively. Acute toxicities were more pronounced in the RT + CT group. This clinical trial did not detect a difference in outcome with combined-technique therapy. This result must be interpreted with caution because of the small number of patients entered in this trial. Confirmation of the value or lack of value for combined therapy will require additional larger clinical trials. PMID- 1707339 TI - Spindle cell carcinoma of the lung. A clinicopathologic study of three cases. AB - Two cases of monophasic spindle cell carcinomas and one case of adenosquamous carcinoma with the spindle cell component located in the lower respiratory tract are presented. In the biphasic tumor, areas of transition from carcinoma to sarcomatous spindle cells were clearly found. The two monophasic tumors and the spindle cell component of the biphasic tumor were histologically characterized by sheets of spindle cells. However, by electron microscopic and immunohistochemical study, several features of squamous epithelial differentiation were found in the spindle cell areas of all cases. Keratin and vimentin were, in various degrees, coexpressed in all the cases. Therefore, it is supposed that the spindle cell component displays a spectrum of phenotypes originating from squamous cell carcinoma, and monophasic spindle cell carcinoma is considered as a kind of the extreme phenotype of squamous cell carcinoma pretending mesenchymal differentiation. PMID- 1707340 TI - Depressed adenoma of the stomach, revisited. Histologic, histochemical, and immunohistochemical profiles. AB - For 52 patients with depressed adenomas of the stomach, histopathologic studies were done on 56 tumors and for 43 of them, histochemical and immunohistochemical features were examined. In addition, nondepressed adenomas (n = 57) and the depressed type of early gastric adenocarcinomas of the well-differentiated variety (n = 44) were studied as the controls. Depressed adenomas in the majority (73%) involved the entire thickness of the mucous membrane of the stomach with tubules of atypical epithelium, presenting a severe grade in many of the cases (41%). Paneth's cells were found in cases of a depressed adenoma, in significantly higher percentages (61%) than in those with a nondepressed adenoma (P less than 0.01). The frequency of cases with argyrophil cells was also higher in depressed adenoma (63%) than in nondepressed adenoma (36%) or in cases of early gastric carcinoma (32%). Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 were noticed in tumor cells, immunohistochemically in 28% of the cases with depressed adenoma, the frequency being intermediate between cases of a benign nondepressed adenoma (6% for both CEA and CA 19-9) and cases of early gastric carcinoma (71% for CEA and 66% for CA 19-9). No difference was noticed in lectin reactivity and mucin content between depressed and nondepressed adenomas, whereas tumor cells in the early depressed carcinoma had a higher lectin reactivity and less mucin content than those seen in the adenomas. It would thus appear that depressed adenoma is a benign neoplastic lesion; however, the malignant potential of this lesion is somewhat higher than the nondepressed counterpart, as indicated by the immunoreactivity to tumor markers and follow-up results reported by colleagues previously. PMID- 1707341 TI - The usefulness of simultaneous determinations of glucosaminylation and fucosylation indices of alpha-fetoprotein in the differential diagnosis of neoplastic diseases of the liver. AB - The degrees of glucosaminylation (glucosaminylation index) and fucosylation (fucosylation index) of alpha-fetoprotein (AFP) were determined in serum samples of 351 patients with hepatocellular carcinoma (HCC), 47 with carcinoma metastatic to the liver from digestive organs, five with mixed cholangiocellular and HCC, and 176 with benign liver diseases. The glucosaminylation index of AFP in patients with carcinoma metastatic to the liver (42 +/- 23%, mean +/- SD) was significantly higher than that in patients with HCC (5 +/- 7%, P less than 0.001) or that in patients with benign liver diseases (2 +/- 4%, P less than 0.001). The fucosylation indices of AFP in patients with carcinoma metastatic to the liver, with HCC, and with benign liver diseases were 76 +/- 25%, 42 +/- 30%, and 4 +/- 6%, respectively. Thus, the fucosylation indices of AFP were high in two neoplastic liver diseases (carcinoma metastatic to the liver and HCC) and low in benign liver diseases, whereas the glucosaminylation indices were high in carcinoma metastatic to the liver but low in HCC and benign liver diseases. When the values of 30% and 80% were used as the level of the glucosaminylation and fucosylation indices, respectively, to discriminate carcinoma metastatic to the liver from HCC, 40 of 47 patients with carcinoma metastatic to the liver (85%) were able to be discriminated from HCC (sensitivity). The specificity, the positive predictive value, and the overall accuracy were 86% (302/351), 45% (40/40 + 47 + 3 - 2) and 86% (40 + 302/47 + 351), respectively. These data suggest that the combined information in these two indices provides a potent criterion for the diagnosis of neoplastic diseases of the liver. PMID- 1707342 TI - A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. AB - Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior. PMID- 1707343 TI - A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii. AB - In C. reinhardtii, the mature psaA mRNA is assembled by a process involving trans splicing of three separate transcripts encoded at three widely scattered loci of the chloroplast genome. At least one additional chloroplast locus (tscA) is required for trans-splicing of exons 1 and 2. We have mapped this gene by transformation of a deletion mutant with a particle gun. The 0.7 kb region of the chloroplast genome that is sufficient to rescue tscA function has been subjected to insertion mutagenesis, showing that it does not contain significant open reading frames. We suggest from these experiments that the product of the tscA gene may be a small chloroplast RNA that acts in trans in the first trans splicing reaction of psaA. A model for the mode of action of this RNA is presented, in which the characteristic structure of group II introns is assembled from three separate transcripts. PMID- 1707344 TI - Genetic and structural homology of stem cell factor and macrophage colony stimulating factor. PMID- 1707345 TI - Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. AB - Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases. PMID- 1707346 TI - Influence of a muramyl dipeptide on human blood leukocyte functions and their membrane antigens. AB - Murabutide, which belongs to the immunomodulator family of muramyl peptides, was applied directly to fresh human blood to evaluate changes in leukocyte properties. After blood incubation with murabutide, lymphocytes presented a higher responsiveness to T-mitogens, and monocytes and polymorphonuclear cells exhibited an increase in their capacity to produce hydrogen peroxide. In addition, murabutide treatment enhanced phagocytic activity of neutrophils, whereas monocytes presented a decrease in this activity. Some surface markers were also investigated in the distinct leukocyte populations. After incubation with murabutide, a larger number of lymphocytes expressed Ta1 antigen (CD W26) and transferrin receptor (CD 71). In contrast, expression of interleukin-2 receptor (CD 25) was slightly decreased. Monocytes from treated blood displayed a larger number of receptors for C3bi (CD 11b), whereas the surface marker CD 14 and the class I receptor for the Fc portion of IgG were down-regulated. Activation of polymorphonuclear cells by murabutide was confirmed by the up regulation of the C3bi receptor, Fc receptor, and CD 14 surface antigen. The effects of murabutide on leukocytes described in this paper may contribute to understanding mechanisms of the modulating activity of muramyl peptides on specific and nonspecific immunity. PMID- 1707347 TI - Characterization of a CD4-positive T-cell line derived from an athymic (nu/nu) mouse. AB - We have isolated a Thy-1+, CD3+, CD4+ T-cell line from the spleen of a 12-week old nu/nu (nude) BALB/c mouse. The cell line is clonal, and it expresses an alpha beta T-cell antigen receptor. Upon activation, these cells secrete IL-2 but not IL-4, putting them in the Th1 category. The cells can be triggered to proliferate and secrete lymphokines in the presence of irradiated syngeneic or allogeneic splenic feeder cells that express a variety of MHC haplotypes. This response is MHC class II-specific, because it can be blocked by either anti-Ia or anti-CD4 antibodies. From the response pattern of this T-cell line, we conclude that it recognizes a common determinant on class II MHC antigens. This nude mouse T lymphocyte presumably has not undergone thymic selection. Therefore its unique specificity may reflect both the bias of T-cell antigen receptor genes for encoding receptors that recognize MHC molecules and the requirement for functional thymic epithelial cells for the efficient education of a self-MHC restricted repertoire. PMID- 1707348 TI - Cytokeratin expression in human cell lines derived from liver tumors. AB - Immunohistochemical staining of cell lines derived from human liver tumours showed that five cell lines derived from hepatocellular carcinoma (HCC) and hepatoblastoma were stained positively with monoclonal keratin antibodies, CK-5 (Ker-18-specific) and KL-1 (broad specificity), but not with CK-7 (Ker-7 specific). On the other hand, four carcinoma cell lines derived from the biliary system were stained positively with not only CK-5 and KL-1, but also CK-7. PMID- 1707349 TI - [Initial results of monitoring hepatitis C virus antibodies in selected population groups in Slovakia. Preliminary results]. PMID- 1707350 TI - Carboplatin-based combination chemotherapy for advanced carcinoma of the cervix. AB - A combination of carboplatin, vincristine, methotrexate and bleomycin (COMB) was given to 29 patients with locally advanced, metastatic or recurrent cervical carcinoma. A total of 85 cycles of chemotherapy were given, with half of the patients receiving greater than 3 cycles. Both the response rate (32.1%) and the median duration of response (30 weeks) were relatively disappointing. Renal toxicity was minimal, but one-third of our patients experienced severe (WHO grades 3 and 4) nausea and vomiting. This combination showed little advantage over carboplatin used as a single agent. PMID- 1707351 TI - Alterations in keratin expression in hamster tracheal epithelial cells exposed to benzo[a]pyrene. AB - Changes in keratin expression were documented in cultures of hamster tracheal epithelial (HTE) cells exposed to vitamin A (retinol 10(-6) M) and benzo[a]pyrene (B[a]P), two agents known to affect the differentiation of tracheal epithelial cells in vivo. Keratin protein patterns were determined after 10 days in culture in the presence and absence of B[a]P in order to determine whether the expression of these proteins was altered by this carcinogen. HTE cells maintained in the presence of vitamin A expressed four simple epithelial keratins (7,8,18 and 19) while vitamin A deficient HTE cells expressed four additional cytokeratins (5,6,14 and 17). No effect of B[a]P on keratin expression was observed in vitamin A treated cells. However, vitamin A deficient HTE cells exposed to B[a]P (0.05-10 micrograms/ml) demonstrated a decrease in the expression of the four differentiation-related keratins while the simple epithelial keratins appeared to be unaffected. These observations were verified at the RNA level employing Northern blot analysis using cDNA probes for human keratins 5,6 and 14. Results demonstrate that B[a]P alters the expression of differentiation-related genes, the cytokeratins, in cell types known to develop into tumors of the respiratory tract. PMID- 1707353 TI - Linking in accessory pathways. Functional loss of antegrade preexcitation. AB - BACKGROUND: Concealed retrograde activation has been proposed as a mechanism for antegrade conduction block in the bundle branches and atrioventricular accessory pathways. We studied this hypothesis (linking) in 10 patients with the Wolff Parkinson-White syndrome in whom antegrade preexcitation could be persistently blocked by overdrive atrial pacing. METHODS AND RESULTS: An atrial pacing protocol, with a decremental ramp followed by an incremental ramp, defined a range of atrial paced cycle lengths (linking window) associated with both persistent conduction and block in the accessory pathway. Within the limits of the linking window, the ability of an atrial impulse to conduct over the accessory pathway was dependent on the preceding state (i.e., conduction or block). The observed linking window ranged from 70 to 290 msec (mean, 185 +/- 68 msec) and closely approximated the measured delay in retrograde activation of the accessory pathway during persistent antegrade block. The mean antegrade effective refractory period of the accessory pathways was long (486 +/- 156 msec), and in each case, it exceeded the antegrade refractory period of the normal atrioventricular pathway. Critically timed premature ventricular extrastimuli, delivered while linking was maintained in the accessory pathway, were able to interrupt the linking and restore antegrade accessory pathway conduction. CONCLUSIONS: These observations suggest that accessory pathway linking is associated with bidirectional block in the accessory pathway. The ability to initiate linking (and the stability of the phenomenon) depends on a critical relation between antegrade accessory pathway refractoriness and the magnitude of retrograde accessory pathway activation delay. PMID- 1707352 TI - Induction of 8-hydroxydeoxyguanosine but not initiation of carcinogenesis by redox enzyme modulations with or without menadione in rat liver. AB - Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione. PMID- 1707354 TI - Release of nitrogen oxides from cultured bovine aortic endothelial cells is not impaired by calcium channel antagonists. AB - BACKGROUND: The endothelium-derived relaxing factor has been shown to be nitric oxide or a related nitroso compound, synthesized by the enzyme nitric oxide synthetase, which oxidizes the guanidono nitrogens of arginine. This enzyme is activated by increases in cytosolic calcium. The effect of the clinically used calcium channel antagonists on this process is controversial. The present study was performed to determine whether calcium channel blockade with these pharmacologic agents would alter the activity of nitric acid synthetase in intact endothelial cells. METHODS AND RESULTS: A specific and sensitive chemiluminescence assay was used to measure the release of nitrogen oxides (nitric oxide and one-electron oxidation products of nitric oxide) from bovine aortic endothelial cells grown in culture. Under basal conditions, the release of nitrogen oxides was about 0.2 nmol/100 micrograms protein/hr. Bradykinin doubled this response. Removal of extracellular calcium abolished basal and bradykinin stimulated release of nitrogen oxides. Neither diltiazem, verapamil, nor nifedipine in concentrations that are encountered clinically altered the release of nitrogen oxides. CONCLUSIONS: These experiments show that although the production of nitrogen oxides is dependent on extracellular calcium, the clinically used calcium channel antagonists do not inhibit the release of the endothelium-derived relaxing factor. PMID- 1707355 TI - Electrocardiography and postextrasystolic potentiation: utilization of the two methods to predict postrevascularization segmental myocardial function. AB - We previously reported that postextrasystolic potentiation (PESP) is a useful predictor of changes in systolic wall function (SWF) following coronary revascularization. In the current study we analyzed ECG changes related to corresponding myocardial segments to determine their correlation with PESP and SWF. We found: (1) The PESP response in a jeopardized segment was a valid predictor of improved SWF even when Q waves, ST-segment changes, or T-wave changes were present. (2) However, when Q waves were present in two or more of the corresponding leads, positive PESP was less likely to be observed. (3) Thus Q waves in two leads predicted the least postrevascularization improvement. (4) Segments with no corresponding Q-wave postrevascularization usually improved SWF. (5) Furthermore, a continuum of responsiveness to PESP was found, ranging from T wave changes, ST-segment changes to Q-wave changes, indicating dissociation between electrical and mechanical events. In conclusion, the ECG together with PESP provide good predictive information relative to the efficacy of revascularization. PESP is a more valuable predictive indicator. ECG alone may be of value in that the occurrence of Q waves in two or more corresponding leads predicts a low probability of improved SWF. Further studies are indicated to investigate the dissociation between electrical and mechanical events. PMID- 1707356 TI - Pacing-induced myocardial ischemia does not affect the endothelial release of coagulant and fibrinolytic factors into the coronary circulation. AB - The present study addresses the potential effects of pacing-induced myocardial ischemia on the secretion of coagulant and fibrinolytic factors within the coronary circulation. In 6 patients undergoing programmed ventricular stimulation with repeated induction of clinical ventricular tachycardia, the coronary release of tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PAI) capacity, von Willebrand factor antigen (WF:Ag), and prostacyclin (6-keto-PGF 1a) was measured. Blood samples were collected simultaneously from the ascending aorta and the coronary sinus at baseline and immediately after the induction of ventricular tachycardia. The occurrence of pacing-induced myocardial ischemia was established by myocardial net lactate production. Myocardial ischemia was induced in every patient by repeated pacing trials. Pacing-induced ischemia did not affect the coronary release of any of the above factors. Consequently, there was no alteration of transcardiac gradients of thrombin antithrombin complexes and D-dimer. The present results indicate that pacing induced myocardial ischemia does not affect the release of coagulant and fibrinolytic endothelial factors or prostacyclin into the coronary circulation. PMID- 1707357 TI - Increased in vitro sensitivity of Candida albicans to fluconazole. AB - Sodium dioctyl sulfosuccinate (SDSS), an anionic surfactant used at a non antimicrobial concentration, increased the sensitivity of Candida albicans to fluconazole in complex media (such as Sabouraud). The conditions were assessed to determine the in vitro sensitivity to fluconazole. In this connection, the use of a liquid medium at a non-alkaline pH is important. The presence of SDSS in complex media does not seem to affect the neutralization of a particular substances but favours the activity of fluconazole. PMID- 1707358 TI - Saving the bathwater. AB - This article presents an account of the development and use of the Neonatal Behavioral Assessment Scale (NBAS) over the past 15 years. It deals with both the formal and informal ways in which administering the NBAS has increased our understanding of the forces for development in the newborn, of states of consciousness in the infant, of the potential for predicting from one point in development to the next, and of a clinician's opportunities to share information with parents and to participate in the enterprise of parenting. It draws attention to the value of the qualitative insights that are gained by investigators, alongside the more quantitative fruits of a research endeavor. PMID- 1707359 TI - [Pancreatitis associated with 5-aminosalicylic acid]. AB - Acute pancreatitis with severe belt-like upper abdominal pain developed within 1 4 weeks of starting medication in three patients (29-year-old man with ulcerative colitis; 43-year-old woman and 22-year-old woman with Crohn's disease) treated, for the first time, with 5-aminosalicylic acid (mesalazine), 500 mg three times daily. Concentrations of lipase initially were 545, 1182 and 3000 U/l, and of amylase 243, 449 and 129 U/l, respectively. Symptoms receded within a few hours after the drug had been discontinued, enzyme levels returning to normal in the course of the next 2-3 weeks. On repeating the drug in two of the patients, in lower dosage, the pancreatitis recurred within a few days. These observations support the view that 5-aminosalicylic acid can cause acute pancreatitis, perhaps as an allergic reaction. PMID- 1707360 TI - Differential effects of 3 beta blockers on lipid peroxidation in hyperthyroid muscle. AB - To determine whether beta blockade protects against the acceleration of lipid peroxidation in hyperthyroid rat soleus (slow-oxidative) muscle, in vivo chronic (3 weeks) effects of 3 beta blockers with different ancillary properties on mitochondrial oxidative enzymes, antioxidant enzymes, and thiobarbituric acid reactive substances were investigated. The rats were rendered hyperthyroid by the administration of thyroxine and treated simultaneously with either carteolol (a nonselective blocker with partial agonist activity; 30 mg/kg/day), atenolol (a beta 1-selective blocker; 50 mg/kg/day), or arotinolol (a nonselective blocker with weak alpha-blocking action; 50 mg/kg/day) over a 3 week period. Hyperthyroidism induced tachycardia, an increase in the mitochondrial oxidative enzymes, manganese (mitochondrial) superoxide dismutase and thiobarbituric acid reactive substances, and a decrease in the other antioxidant enzymes. The tachycardia was alleviated completely by either atenolol or arotinolol, but partially by carteolol. Arotinolol, but neither carteolol nor atenolol, inhibited the increase in oxidative enzymes and thiobarbituric acid-reactive substances. The levels of antioxidant enzymes were minimally affected by the beta-blocker treatment. Beta 2-, and possibly alpha- as well, but not beta 1-, blockade suppressed mitochondrial hypermetabolism and protected against peroxidative injury in the hyperthyroid soleus muscle. Partial agonist activity was not beneficial. PMID- 1707361 TI - An in vitro evaluation of adrenergic action on rat pancreatic amylase secretion: lack of stimulatory effect. AB - To assess direct evidence of adrenergic stimulation in pancreatic amylase secretion, effects of catecholamines on amylase release and intracellular cyclic AMP accumulation were examined with rat dispersed pancreatic acini. We first carried out control studies with CCK-8 and carbamylcholine to evaluate the usefulness of the material for the examination of amylase secretion, and examined VIP-induced cyclic AMP accumulation to assess the agonist evoked intracellular response. As a result, significant effects of CCK-8, carbamylcholine and VIP were observed, which confirmed that dispersed pancreatic acini used in this study were useful in examining exocrine pancreatic secretion. However, catecholamines failed to stimulate amylase release from pancreatic acini, although a significant increase in intracellular cyclic AMP accumulation was observed. Thus the present study strongly suggests that direct involvement of catecholamine is unlikely in pancreatic amylase secretion, in contrast to results reported previously. PMID- 1707362 TI - The utility of postmortem lung for RNA studies; variability and correlation of the expression of surfactant proteins in human lung. AB - Postmortem human lung tissue was evaluated for its utility in studies of the mRNAs for the surfactant proteins. Data obtained from different analytical procedures indicated that surfactant protein mRNAs are quite stable in these tissues with a half-life of 10 to 12 h. These analyses revealed no major regional differences in the mRNA levels for the surfactant protein A (SP-A) and surfactant protein B (SP-B) although small differences were present in the levels for the surfactant protein C (SP-C). Analysis of adult surgical lung specimens indicated that there is greater individual variation in the mRNA levels for SP-A and SP-B compared to SP-C among individuals. Furthermore, in a given individual the level of SP-A mRNA correlated well with that of SP-B, whereas the level of SP-C mRNA did not correlate with either that of SP-A or SP-B. PMID- 1707363 TI - Monoclonal antibodies that inhibit binding of propolypeptide of von Willebrand factor to collagen. Localization of epitopes. AB - We reported previously that bovine propolypeptide of von Willebrand factor (pp vWF) binds to type I collagen. To determine the collagen-binding sites of pp-vWF we generated monoclonal antibodies (mAbs) against bovine pp-vWF. One mAb, designated TC8, very strongly inhibited the binding of pp-vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp-vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp-vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while TC6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N-terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp-vWF, the locations of these fragments in the primary structure were estimated to be Phe570--Lys682 for the 21-kDa fragment and Glu281--Lys375 for the 18-kDa fragment. These data suggest that pp-vWF contains at least two collagen binding sites which lie within or close to the regions between Phe570--Lys682 and Glu281--Lys375. PMID- 1707364 TI - Leucocyte sequestration in endotoxemia and the effect of low-molecular-weight dextran. AB - Leucocyte sequestration in various organs during endotoxin-induced shock in sheep was studied using leucocytes labelled with indium 111 oxine. A moderate dose of Escherichia coli endotoxin (10 micrograms/kg body weight) was slowly infused intravenously in 16 sheep, 9 of which subsequently received a continuous i.v. infusion of low-molecular-weight dextran (LMWD) given at an infusion rate of 15 ml/h over 4 h, starting 30 min after administration of the endotoxin. By that time, signs of acute lung injury had developed, thus mimicking a clinical situation. The remaining animals were untreated and served as controls. A marked increase in lung, liver and kidney leucocyte sequestration, together with a sharp, corresponding drop in splenic activity and leucocyte count in peripheral blood, occurred shortly after the endotoxin infusion in both groups. However, after 90 min there was a significantly lower leucocyte activity in the lungs, liver and kidneys of LMWD-treated animals as compared with controls. Less marked hemodynamic and respiratory alterations were also observed in animals treated with LMWD. The present study confirms previous reports that significant leucocyte sequestration in the lungs occurs early during endotoxemia. Furthermore, we found that leucocyte sequestration also occurs in the liver and kidneys, which could explain the development of multi-organ failure, frequently described in clinical sepsis. Even after injury to organs, LMWD infusion seems to be beneficial by significantly lowering leucocyte sequestration and could therefore be justified as an addition to the arsenal of interventions used in the treatment of endotoxemia. PMID- 1707365 TI - Hypothalamic 5-hydroxytryptamine metabolism is not influenced by streptozotocin diabetes induced at neonatal age. AB - Rats were rendered diabetic at neonatal age by an intraperitoneal injection of streptozotocin (90 mg/kg). At 8-10 weeks of age, their 5-HT and 5-HIAA levels in different hypothalamic nuclei (ventromedial, paraventricular, periventricular, supraoptical and suprachiasmatic nuclei) were similar to control animals which received vehicle only. It is concluded that the combination of mild hyperglycemia and hypoinsulinemia does not affect hypothalamic 5-HT metabolism in rats. PMID- 1707366 TI - Histological studies on eyelid opening in normal male mice and hemizygotes for the mutant gene Tabby (Ta) with and without epidermal growth factor treatment. AB - Previous work has shown that mice hemizygous or homozygous for the mutant gene Tabby have delayed eyelid opening, as compared to unaffected, wildtype littermate controls; exogenous treatment with epidermal growth factor reverses this delay. We performed histological studies to explore the mechanisms of action of the Tabby gene and of epidermal growth factor in these processes. These show that eyelid opening is associated with keratinization of the fusion junction and conjunctival sac formation. Both these processes occur earlier in normal male mice (days 4 and 7 respectively) than in Tabby hemizygotes (days 7 and 10, respectively). After epidermal growth factor injection, keratinization and conjunctival sac formation are both observed on postnatal day 1 in all control and mutant pups. Thus epidermal growth factor appears to accelerate eyelid opening by stimulating these morphological processes and the Tabby gene appears to delay eyelid opening by impairing them. It is possible that deficiency of epidermal growth factor at the tissue level may be involved in the development of some of the traits seen in Tabby mutants. In addition to analysing the effects of the Tabby gene and of epidermal growth factor on eyelid opening in the mouse, this study appears to be the first detailed histological description of normal eyelid opening. The findings have potential clinical significance; firstly, because the Tabby gene shows genetic homology to the human gene for hypohidrotic ectodermal dysplasia, and disturbed eyelid opening is a trait of some forms of human ectodermal dysplasia, and secondly, because the gene for epidermal growth factor receptor is an oncogene. PMID- 1707367 TI - Increase in ocular blood flow induced by isobutylmethylxanthine and epinephrine. AB - Alterations in regional blood flow of the eye were studied in rabbits using the radioactively labelled microsphere technique. The animals were topically treated with 1% IBMX, 0.1% epinephrine in combination with 1% isobutylmethyl-xanthine (IBMX), or with 0.1% epinephrine alone. After IBMX there was a tendency towards an increase in blood flow in the iris, ciliary processes and sclera, whereas the choroidal blood flow tended to decrease. After epinephrine the iridial blood flow increased about 50% at 6 hr. After IBMX combined with epinephrine a biphasic response was obtained: an initial decrease in blood flow of the iris, the ciliary processes and to some extent the choroid was followed by a marked and long lasting increase in blood flow from 2.5 to 7.5 hr in the iris (up to 220%), ciliary processes (123%) and sclera (115%). The choroidal blood flow was not increased. The marked increase in blood flow of the iris and the ciliary processes indicates that the reduction of intraocular pressure seen after topical treatment with a combination of epinephrine and IBMX, is not based on a vascular mechanism. The increase of ocular blood flow after a combination of IBMX and epinephrine is probably based on increased cAMP secondary to inhibition of phosphodiesterases. This may indicate the presence of beta-adrenergic receptors in the ocular vasculature. PMID- 1707368 TI - Further studies on galanin-, substance P-, and CGRP-like immunoreactivities in primary sensory neurons and spinal cord: effects of dorsal rhizotomies and sciatic nerve lesions. AB - The peptides galanin (GAL), substance P (SP), and calcitonin gene-related peptide (CGRP) were analyzed with immunohistochemistry and radioimmunoassay in the spinal cord, dorsal root ganglia, dorsal roots, and sciatic nerve of normal rats and rats subjected to several experimental procedures, including ligation, crush, and/or sectioning of nerves. The results show that peripheral nerve transection induces a dramatic increase in GAL content both in dorsal roots and sciatic nerve, demonstrating that this lesion causes an increased out-transport of the newly synthesized peptide both into the central and peripheral branches of the primary sensory neurons. In contrast evidence was obtained for decreased out transport of SP and CGRP. The functional significance of these findings remains to be analyzed. PMID- 1707369 TI - Both regenerating and late-developing pathways contribute to transplant-induced anatomical plasticity after spinal cord lesions at birth. AB - Fetal spinal cord transplants prevent the retrograde cell death of immature axotomized central nervous system (CNS) neurons and provide a terrain which supports axonal elongation in the injured immature spinal cord. The current experiments were designed to determine whether the axons which grow across the site of the neonatal lesion and transplant are derived from axotomized neurons and are therefore regenerating or whether the axons which grow across the transplant are late-growing axons that have not been axotomized directly. We have used an experimental paradigm of midthoracic spinal cord lesion plus transplant at birth and temporally spaced retrograde tracing with the fluorescent tracers fast blue (FB) and diamidino yellow (DY) to address this issue. Fast blue was placed into the site of a spinal cord hemisection in rat pups less than 48 h old. After 3-6 h to allow uptake and transport of the tracer, the source of fast blue was removed by aspiration and the lesion was enlarged to an "over-hemisection." A transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. The animals survived 3-6 weeks prior to the injection of the second tracer (DY) bilaterally into the host spinal cord caudal to the lesion plus transplant. Neurons with late-developing axons would not be exposed to the first dye (FB), but could only be exposed to the second tracer, diamidino yellow. Thus, neurons with a diamidino yellow-labeled nucleus are interpreted as "late developing" neurons. Neurons axotomized by midthoracic spinal cord lesion at birth could be exposed to the first tracer, fast blue. If after axotomy they regrew caudal to the transplant, they could be labeled by the second tracer as well. We interpret these double-labeled neurons as regenerating neurons. If neurons labeled with fast blue and axotomized by the spinal cord hemisection either failed to regenerate or grew into the transplant but not caudal to it, they would be labeled only by the first dye. We have examined the pattern and distribution of single (FB or DY)- and double (FB + DY)-labeled neurons in the sensorimotor cortex, red nucleus, locus coeruleus, and raphe nuclei. The sensorimotor cortex contains only DY-labeled neurons. The red nucleus contains both FB- and FB + DY-labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707370 TI - The need for cellular elements during axonal regeneration in the sea lamprey spinal cord. AB - Spinal axons in the larval sea lamprey regenerate following a complete spinal transection. It is not known whether regenerating growth cones require contact with cellular elements or whether the basement membrane and collagenous meninx primitiva which surround the spinal cord are sufficient for neurite out-growth. To determine this, a freeze lesion was made which severed axons, destroyed neuronal perikarya, and greatly reduced the number of glial cells. After at least 10 weeks of recovery, 50 neurites from 31 Muller and Mauthner axons were labeled by intracellular injection of HRP. Eighty-six percent of these neurites did not regenerate into the lesion site. No neurites grew through the lesion. No animals recovered coordinated swimming. These results suggest that glial and/or neuronal surfaces are required for axonal regeneration. Moreover, a monolayer of glial cells appears to be suboptimal and a three-dimensional matrix of cells may be necessary to promote regeneration in the lamprey spinal cord. PMID- 1707371 TI - Chloroplast phosphoribulokinase associates with yeast phosphoriboisomerase in the presence of substrate. AB - Pea chloroplastic phosphoribulokinase and yeast phosphoriboisomerase partition independently of one another in a two-phase polyethyleneglycol, dextran system, but apparent interaction is seen when ribose-5-phosphate is added to the two phase system. It appears that the pea leaf kinase recognizes yeast isomerase when it is carrying metabolite. PMID- 1707372 TI - Direct cleavage of a DNA fragment by a bleomycin-oligonucleotide derivative. AB - Bleomycin A5 oligonucleotide derivative was used for direct cleavage of a DNA target. In the presence of Fe2+ ions and 2-mercaptoethanol, Blm-R-pd(CCAAACA) (I) damaged the target. pd(TGTTTGGCGAAGGA), with the yield of 80% , without affecting its own oligonucleotide tail. The sites of cleavage were T3-T5 and G6-G7. Unbound bleomycin A5 damaged the G6-G7-C8 site. Reagent I formed more stable complementary complexes with the target than parent oligonucleotide (ATm = 11 C). PMID- 1707373 TI - The structure of porin from Rhodobacter capsulatus at 1.8 A resolution. AB - The structure of the porin from Rhodobacter capsulatus was determined at a resolution of 1.8 A. The analysis started from a closely related crystal structure that had been solved at a medium resolution of 3 A using multiple isomorphous replacement and solvent flattening. The new structure contains the complete sequence of 301 amino acid residues. Refinement of the model is under way; the present R-factor is 22% with good geometry. Except for the lengths of several loops, the resulting chain fold corresponds to the medium resolution model. The membrane channel is lined by a large number of ionogenic side chains with characteristic segregation of differently charged groups. PMID- 1707374 TI - Short-term effect of kainic acid administration and its dependence on the circadian rhythm on cytoplasmic RNA content in neurons of the hypothalamus of mice. AB - Kainic acid in a dose of 12 micrograms/g given 1 hour before decapitation strongly modifies the content of cytoplasmic RNA, as determined cytophotometrically, in neurons of the supraoptic, paraventricular, and arcuate nuclei of the hypothalamus of adult mice. This stimulatory action of kainic acid was strongest during the night hours when the control animals exhibited the lowest amounts. PMID- 1707375 TI - Who me? Present a CE program? PMID- 1707376 TI - Essential fatty acids and their metabolites in signal transduction. AB - The above examples argue that the diversity of eicosanoid structures is reflected in distinct actions, that the membrane-permeable nature of these molecules provides a system for interactive cellular signalling that may be particularly important in the nervous system, and that convincing evidence exists for direct actions of eicosanoids on both ion channels and kinases, as well as G-protein coupled receptors. PMID- 1707377 TI - Monoclonal antibodies against chondroitin sulphate isomers: their use as probes for investigating proteoglycan metabolism. PMID- 1707378 TI - Molecular modelling of integral membrane proteins. PMID- 1707379 TI - Mapping of B-cell epitopes on the Leishmania donovani 70 kDa heat-shock protein. PMID- 1707380 TI - Structure of rodent urinary proteins. PMID- 1707381 TI - A comparison of proteoglycan arrangement in normal and keratoconus human corneas. PMID- 1707382 TI - Gamma-interferon production in response to hepatitis B core protein and its synthetic peptides in patients with chronic hepatitis B virus infection. AB - The nucleocapsid of hepatitis B virus (HBV) is an efficient immunogen in activating T cells to produce interferon-gamma (IFN-gamma) in patients with chronic HBV infection. We investigated hepatitis B core antigen (HBcAg)-specific T cell recognition, which seems to be implicated in the pathogenesis of chronic liver disease. IFN-gamma production by peripheral-blood mononuclear cells of patients with chronic HBV infection [25 patients with chronic active hepatitis (CAH) and 14 asymptomatic carriers of HBV (ASCs)] was measured by an enzyme linked immunosorbent assay. P19 polypeptide, which is derived from recombinant HBcAg particle (rHBcAg), increased IFN-gamma production in patients with CAH, but its effect was weaker than that of rHBcAg. P19 had no stimulating effect on T cells from ASCs. The fine specificity of T cell recognition of HBcAg was examined using 8 kinds of synthetic peptides. T cells from the patients who responded against P19 polypeptide recognized the sites within the common sequences of HBcAg and HBeAg (p72-90, P90-99, P108-122 and P126-146). These results suggest that HBcAg and P19 are cross-reactive at the T cell level, and that these T cells recognize the sites within the common sequences of HBcAg and HBeAg in HBV infected patients. PMID- 1707383 TI - Gastric mucosal damage by ethanol is mediated by substance P and prevented by ketotifen, a mast cell stabilizer. AB - To elucidate the possible role of substance P in the pathogenesis of acute gastric mucosal damage, rats were treated intragastrically with 1.0 mL 96% ethanol, 0.6N HCl, or 25% NaCl, with or without IP coadministration of substance P, senktide, or septide (1 mumol/L per 100 g). All three peptides were found to double the mean lesion area when compared with that induced by ethanol, whereas substance P antagonist (1 mumol/L per 100 g) prevented the expansion of damage extent. The increased damage was associated with increased gastric mucosal levels of platelet activating factor, leukotriene B4, and leukotriene C4. Substance P antagonists also reduced by half the extent of the gastric damage induced by ethanol when administered by itself. WEB 2086 (platelet-activating factor antagonist; Boehringer Ingelheim KG, Germany), hydroxyzine (H1 blocker), and cimetidine (H2 blocker) reduced lesion area by 50%, but only in rats treated with both substance P and ethanol. Ketotifen (mast-cell stabilizer) (100 micrograms/100 g), administered orally 30 minutes before damage induction, totally abolished the extent of the damage induced by either ethanol or the coadministration of ethanol and peptides in the surface epithelium of the entire mucosa. The protective effect of ketotifen was accompanied by significant reduction in mucosal generation of platelet-activating factor, leukotriene C4, and leukotriene B4. Similar mucosal protection was afforded by ketotifen against damage induced by 0.6N HCl, 25% NaCl, or indomethacin. Therefore, it is suggested that substance P is involved in the pathogenesis of acute ethanol-induced gastric mucosal damage. The effective mucosal protection provided by ketotifen indicates the important role of mast cells and their mediators in the pathogenesis of acute gastric mucosal damage and may have therapeutic implications. PMID- 1707384 TI - Synthesis and prostaglandin E2-induced secretion of surfactant phospholipid by isolated gastric mucous cells. AB - Lipids, particularly surface-active phospholipids, have been proposed to provide an important protective barrier in the gastric mucosa. The predominant surface active phospholipid in the pulmonary surfactant complex is dipalmitoylphosphatidylcholine. To determine whether the gastric epithelium synthesizes and secretes this phospholipid, primary cultures of canine gastric mucous cells isolated by counterflow elutriation were studied. During the 24-hour period of culture, the gastric mucous cells incorporated 3H-choline into phosphatidylcholine, with dipalmitoylphosphatidylcholine representing 13.8% +/- 0.6% of the phosphatidylcholine synthesized. When mucous cell preparations with greater chief cell contamination were studied, they incorporated significantly less precursor into dipalmitoylphosphatidylcholine. Administration of prostaglandin E2, a cytoprotective agent, to the cultured mucous cells for 1 hour led to a significant increase in phosphatidylcholine release, reaching a maximum of 120.4% +/- 4.2% (P less than 0.001) at 10(-6) mol/L. No significant stimulation of phospholipid release by prostaglandin E2 was seen in the fractions containing a greater proportion of chief cells. To further establish the relationship between mucin and phospholipid secretion, two gastric cancer cell lines, Hs746T and KATO III, were studied. Using immunocytochemical and biochemical techniques, mucin synthesis and secretion were confirmed by these cell lines. The Hs746T cells were significantly more active in the secretion of both mucin and phospholipid than the KATO III cells. The Hs746T line secreted 5.7 fold more mucin and 7.3-fold more phospholipid than KATO III cells during a 24 hour period of culture. The association between mucin and phospholipids in an aqueous solution was also studied. Purified mucin in the concentration of 0.5-2 mg/mL of glycoprotein led to a significant dose-dependent increase in phospholipid solubility, suggesting the formation of a glycoprotein-phospholipid complex. The current studies indicate that the gastric mucous cell is the source of surfactant phospholipids as well as mucin. The synthesis and release of mucin and phospholipid are functions of the mucous cell that play a critical role in the primary defense of gastric epithelium. PMID- 1707385 TI - Expression of enzymatically active sucrase-isomaltase is a ubiquitous property of colon adenocarcinomas. AB - Adenocarcinoma of the colon is one of the most prevalent and lethal of all human malignancies. The early diagnosis and management of this disease could be improved if biological markers, whose expression was restricted to malignant colon cells, were identified. Sucrase-isomaltase is a glycoprotein hydrolase expressed throughout the small intestine and fetal colon but not in the normal adult colon. This study shows that the expression of enzymatically active sucrase isomaltase is a ubiquitous property of primary and metastatic colon adenocarcinoma. Significant sucrase enzyme activity (i.e., greater than 5 mU/mg protein) was observed in 16 colon carcinomas but not in adjacent normal colon mucosa. Sucrase-isomaltase messenger RNA was identified in all tumors using reverse transcriptase polymerase chain reaction. Using a quantitative polymerase chain reaction analysis, this study shows that the amount of sucrase-isomaltase messenger RNA in tumors examined (3.4 x 10(-8) to 3.19 x 10(-7) micrograms/micrograms total RNA) was greater than in adjacent mucosa (0 to 3.4 x 10(-8) micrograms/micrograms total RNA). This induction of sucrase-isomaltase messenger RNA and enzyme activity was corroborated by immunostaining. Of 30 colon adenocarcinomas examined, 80% were positive for sucrase-isomaltase. In addition, all colon carcinoma metastases examined were positive for sucrase-isomaltase. The staining pattern was distinct and demarcated tumor cells from the surrounding histologically normal tissue. No sucrase-isomaltase staining was seen in normal mucosa from the same patients. With the exception of lung, no sucrase-isomaltase immunostaining was observed in a variety of examined noncolonic adenocarcinomas. Thus, the specificity and ubiquity of sucrase-isomaltase expression in adenocarcinomas of the colon can be exploited to improve the clinical management of this disease. In addition, studies on the structure of the sucrase-isomaltase gene and its regulatory elements should contribute toward understanding the alteration of gene expression by oncogenic transformation of the colonic mucosa. PMID- 1707386 TI - A prospective comparison of laser therapy and intubation in endoscopic palliation for malignant dysphagia. AB - There is little objective long-term follow-up comparing laser therapy with intubation for palliation of malignant dysphagia. In a prospective, nonrandomized two-center trial 43 patients treated with the neodymium:yttrium-aluminum-garnet laser were compared with 30 patients treated by endoscopic intubation; the two groups were comparable for mean age and tumor position, length, and histology. Dysphagia was graded from 0 to 4 (0, normal swallowing; 4, dysphagia for liquids). Pretreatment mean dysphagia grades were similar: laser-treated group, 2.9 (SD, 0.6); intubated group, 3.2 (SD, 0.55). For thoracic esophageal tumors, the percentage of patients achieving an improvement in dysphagia grade by greater than or equal to 1 grade initially and over the long term was similar (laser, 95% and 77%; intubation, 100% and 86%). For tumors crossing the cardia, intubation was significantly better (laser, 59% and 50%; intubation, 100% and 92%, respectively; P less than 0.001). In patients palliated over a long period, however, the mean dysphagia grade over the remainder of their mean dysphagia grade over the remainder of their lives (mean survival: laser, 6.1 months; intubation, 5.1 months) was better in the laser group (1.6 vs. 2.0; P less than 0.01); 33% of laser-treated and 11% of intubated patients could eat most or all solids (P less than 0.05). For long-term palliation, laser-treated patients required on average more procedures (4.6 vs. 1.4; P less than 0.05) and days in the hospital (14 vs. 9; P less than 0.05). The perforation rate was lower in the laser-treated group (2% vs. 13%; P less than 0.02); no treatment-related deaths occurred in either group. For individual patients, the best results are likely to be achieved when the two techniques are used in a complementary fashion in specialist centers. PMID- 1707387 TI - Hydrophobia, lectin binding, and molecular order in the stratum corneum of adult and neonatal rats and in human breast cells in culture. AB - A search for differences due to ANS staining (hydrophobia), Con A and PNA binding capacity, and birefringence was carried out on stratified epithelia of rat skin and human breast cells (HBC) in culture. Microfluorimetric measurements confirm that the ANS fluorescence of the stratum corneum from adults is higher than that of newborns. HBC exhibited an unexpected deep ANS-fluorescence. Differences in the binding capacity of the epithelial layers to Con A and PNA were detected with advancing age. Retardation measurements revealed that the form birefringence of the stratum corneum is higher in adult animals specially as revealed by the fact that its form birefringence curve branch from n = 1.414 to n = 1.479 is steeper, i.e. depict higher values. The strong birefringence of the cytoplasmic tonofilaments presented by cultured human breast cells was considered an unexpected finding and attributed to changes that the cells underwent following the in vitro conditions. PMID- 1707388 TI - Cloning and characterization of the Mycobacterium leprae putative ribosomal RNA promoter in Escherichia coli. AB - The putative promoter region of the 16S ribosomal RNA-encoding gene (rRNA) of Mycobacterium leprae was cloned and characterized in Escherichia coli. A 932-bp HaeIII restriction fragment, containing the 5' end of the 16S rRNA gene and flanking upstream region, was cloned in front of a promoterless reporter gene in the shuttle vector, pMH109, to generate the plasmid, pYA1101. This clone exhibits promoter activity both in Gram-(E. coli) and Gram+ (Bacillus subtilis) bacteria. Sequence analysis and primer extension experiments with mRNA derived from the M. leprae clone were used to determine the structure and the location of the promoter, as well as the transcription start point in E. coli. The promoter region contains sequences that resemble the -35 and -10 consensus sequences found in many bacteria. A region located 34 bp distal to the promoter is a putative rRNA processing signal, based on sequence homology with processing signals involved in the maturation of the rRNA precursor in B. subtilis and several Mycoplasma species. PMID- 1707390 TI - Palliation of malignant dysphagia. PMID- 1707389 TI - Oxygen free radicals in acute pancreatitis of the rat. AB - This study aimed to assess the role of oxygen free radicals in acute pancreatitis. Acute pancreatitis was induced in rats by infusion of the CCK analogue cerulein (5 micrograms/kg per hour) for 30 minutes, 3.5 hours, and 12 hours. After the infusion, serum enzymes and conjugated tissue dienes and malondialdehyde were measured and tissue samples were subjected to electron and light microscopy. Electron microscopy after 30 minutes showed moderate intracellular alterations. After 3.5 hours of cerulein infusion interstitial oedema and intravascular margination of granulocytes in the pancreatic gland were seen. After 12 hours histological evaluation showed pronounced zymogen degranulation, extensive tissue necrosis, and migration of granulocytes into the tissue. Amylase and lipase activities increased 15 and 35-fold respectively during this time. After 30 minutes of cerulein infusion conjugated dienes and malondialdehyde increased, they reached their peak after 3.5 hours and decreased to normal values after 12 hours. Treatment with superoxide dismutase (100,000 U/kg/hour) and catalase (400,000 U/kg/hour) either before or after the start of the cerulein infusion prevented lipid peroxidation and reduced zymogen degranulation and tissue necrosis. Tissue oedema and inflammatory response, however, were not affected in any of the treated rats. Oxygen free radicals are instrumental in the development of acute pancreatitis. Even after its onset, scavenger treatment reduced the tissue damage normally observed. PMID- 1707391 TI - Comparative studies of ovarian histopathology and the mating behaviour of Periplaneta americana, Linn. (Orthoptera) treated with sublethal concentrations of BHC and DDT. AB - Cockroaches are familiar pests of grain stores. They are prolific breeders and can also transmit various diseases. Adult females were therefore treated with sublethal BHC and DDT concentrations to control their fecundity and viability. Ovulation was arrested as a result of damage to and resorption of the terminal oocytes. Histopathological changes were clearly expressed in the follicular epithelium of pathological oocytes and also in immature oocytes. Vitellogenesis was arrested and protein yolk precursors were broken down and destroyed by the pycnotic follicular epithelium. Fibrosis and thickening of the tunica propria gradually diminished. Prolonged treatment resulted in atrophy of the ovaries, together with a large number of immature and pathological oocytes. Since the effect of DDT is cumulative, the histopathological changes it caused were severer than those induced by BHC. Treated adults kept in pairs showed prolonged duration of mating, followed by a 2-3 days' delay in ootheca formation; no first instar nymphs emerged from these oothecae, however, owing to the failure of fertilized eggs to develop any further in the oothecal lumen. PMID- 1707392 TI - Changes in gill structure induced by BHC, lindane and endosulfan in the fresh water teleost Puntius ticto (Ham.). AB - The effect of three organochlorinated pesticides on the gill structure of Puntius ticto, a freshwater teleost, was investigated. Fish exposed to sublethal concentrations of BHC (0.17 ppm), lindane (0.19 ppm) and endosulfan (0.20 ppm) were studied. The pesticides were detected qualitatively in the gill tissue by thin-layer chromatography (TLC). The results showed that they could be detected after 15 days' exposure, but not after 96 h exposure. Histopathological examination revealed several structural and functional changes in the gills. Exposure to BHC was followed by an inflammatory reaction and complete dystrophy of the lamellar structure of the gills. Lindane-treated fish showed disruption of the epithelial covering of the gills and excessive haemorrhage in the blood vessels. In exposure to endosulfan the gill lamellae shrank and became thinner. PMID- 1707393 TI - Malignant lymphoma with myxoid stroma: a new pattern in need of recognition. AB - We report a case of malignant lymphoma in the soft tissues exhibiting prominent myxoid stromal changes and cord-like cellular arrangement, mimicking the architectural as well as cytological features of myxoid chondrosarcoma, except for the absence of tumour lobulation. The only clue to the possible lymphomatous nature of the lesion was the past history of lymphoma. Immunohistochemical studies showed that this represented a B-cell lymphoma, staining positively for leucocyte common antigen and five B-lineage markers L26, MB2, B1 (CD20), B4 (CD19) and To15 (CD22). We conclude that malignant lymphoma should not be excluded from consideration when one encounters a myxoid tumour. PMID- 1707394 TI - Intra-abdominal neuroectodermal tumour of childhood with divergent differentiation. AB - Two cases are reported of intra-abdominal small cell tumours expressing concomitant neural and epithelial differentiation. These features were discernible on conventional microscopy and supported immunocytochemically. Immunoreactive vimentin was also revealed in both tumours, and, in addition, one showed focal desmin positivity. Epithelial differentiation in both tumours was confirmed ultrastructurally. The tumours were interpreted to represent a variant of peripheral primitive neuroectodermal tumour, and the report serves to emphasize a potential among such tumours for complex differentiation. The neoplasms are compared with other similar tumours reported recently in children. PMID- 1707395 TI - Pseudosarcomatous lesions of the urinary bladder. AB - The clinical, microscopical, immunocytochemical and ultrastructural features of five cases of benign mesenchymal proliferative lesions of the urinary bladder, mimicking sarcoma, are presented. Four of the five patients are alive and disease free following diagnosis, an interval ranging from 9 months to 9 years, mean 4 years. A fifth patient, who had a pseudosarcomatous stromal response adjacent to a urinary transitional cell carcinoma, now has invasive transitional cell carcinoma. The lesions revealed a striking microscopical, immunocytochemical and ultrastructural similarity to nodular fasciitis, suggesting the lesions represented a bizarre mesenchymal proliferative response to inflammation. PMID- 1707396 TI - Endodermal sinus tumour of the liver. PMID- 1707397 TI - Immunoreactivity to desmin in secretory epithelium of eccrine sweat glands. PMID- 1707398 TI - Identification of antigens which stimulate T lymphocytes of Salmonella enteritidis 11RX immunized mice. AB - The technique of using sodium dodecylsulfate-polyacrylamide gel electrophoresis fractionated antigens (Ag) transferred to nitrocellulose filters was adopted to analyse T cell responses to Salmonella enteritidis 11RX Ag. Employing in vitro proliferation assays with T cells from S. enteritidis 11RX-primed (BALB/c x C57BL/6J)F1 mice as the measure of T cell stimulation, we have identified Ag able to stimulate T cells in the regions containing 16, 24, 34 and 50-60 kDa proteins, with dominant Ag activity at about 16 kDa. These results were confirmed with long term, Ag-specific L3T4+ T cell lines which responded to molecules in the same four Mr regions, suggesting that no selection by a single antigenic determinant had occurred during more than 3 months of in vitro culture, or that all the molecules which were stimulatory shared at least one antigenic determinant. Because the seven clones we examined responded only to 16 kDa molecules, the former alternative is the more likely. Standard immunoblot analysis indicated that these Ag also act as major B cell stimulating determinants. T cells of BALB/c mice, which are 5-10 times more resistant to S. enteritidis 11RX than C57BL/6J mice, showed the same pattern of reactivity as F1 mice whereas the major antigenic region for T cells of C57BL/6J mice was located between 50 and 60 kDa. PMID- 1707399 TI - Concentrations of cefpodoxime in plasma, ejaculate and in prostatic fluid and adenoma tissue. AB - Twenty-four healthy volunteers and 24 patients undergoing transurethral resection of the prostate received an oral dose of 200 mg of cefpodoxime as proxetil ester in a fasting state. At the same time 3.235 g of iohexol, a renal contrast medium, was injected intravenously to indicate possible urinary contamination of the prostatic fluid. The subjects were divided into three groups each. After 3, 6 and 12 h the cefpodoxime concentrations were measured in plasma, urine, prostatic fluid and ejaculate in volunteers and in plasma, prostatic fluid and prostatic adenoma tissue in patients by a bioassay as well as by an HPLC method. In general, the concentrations measured by bioassay were higher than those by HPLC. The median plasma concentrations (bioassay) in volunteers (patients) after 3, 6 and 12 h were 2.28 (2.34) mg/l, 0.95 (1.17) mg/l and 0.12 (0.28) mg/l, respectively. The median ejaculate concentrations after 6 and 12 h were 0.95 mg/l and 0.19 mg/l, respectively. Only in three volunteers and in one patient prostatic fluid concentration without urinary contamination could be measured after 3 h with a median fluid to plasma ratio of 0.10. The prostatic adenoma tissue concentrations (bioassay) after 3 and 6 h were 0.50 mg/kg and 0.24 mg/kg with tissue to plasma ratios of 0.30 and 0.26, respectively. After 3 h about half of the volunteers and after 12 h about half of the patients showed no detectable concentration in ejaculate (volunteers) and prostatic tissue (patients), respectively. It was concluded that the cefpodoxime should be administered 3 to 6 h prior to surgery if used for perioperative prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707400 TI - Substance P-induced histamine release from human basophils, skin and lung fragments: effect of nedocromil sodium and theophylline. AB - We compared histamine release induced by substance P with those obtained with classical secretagogues on human basophils, lung and skin fragments. We also tested the capacity of nedocromil sodium and theophylline to inhibit histamine release in these 3 experimental models. Substance P (10(-4) M) caused a noncytotoxic histamine release (about 10% of total) from basophils, lung and skin fragments. Substance P-induced histamine release was always smaller than that obtained with optimal doses of anti-IgE, formyl-methionine phenylalanine or compound 48/80. Nedocromil sodium did not prevent secretagogue-induced histamine release from basophils or sliced skin. In contrast, it significantly inhibited anti-IgE- or substance P-induced histamine release from human lung. Theophylline caused a dose-related inhibition on these 3 models. We conclude that substance P is a modest secretagogue for human basophils and mast cells, and that skin and lung mast cells are heterogeneous with respect to their response to nedocromil sodium. PMID- 1707401 TI - Rat mast cell granules reacting with a mouse monoclonal antibody M6764 which recognizes the same epitope of a mouse monoclonal antibody HNK-1. AB - Rat mast cell granules and plasma membrane fractions were obtained by homogenization of highly purified rat mast cells and isolation in a Percoll gradient and a sucrose gradient, respectively. Immunostaining of rat mast cells, granules and plasma membrane fractions was performed with mouse monoclonal antibody M6764 which was produced against the crude membrane fractions of the neural tubes. Rat mast cells and granules were immunostained with the monoclonal antibody, but not the plasma membrane fractions. The granules fixed with glutaraldehyde-paraformaldehyde showed ring-like forms. Chloroform-methanol treatment did not effect the staining of rat mast cells and granules with the monoclonal antibody. Western blotting analysis of rat mast cells and granules with the monoclonal antibody showed broad protein bands ranging from 100 to 250 kD. PMID- 1707402 TI - Histamine release from basophils after in vivo application of recombinant human interleukin-3 in man. AB - Interleukin 3 (IL3) is known to stimulate progenitor cell proliferation and maturation as well as differentiated cell functions, e.g. direct or anti-IgE mediated histamine release (HR). We investigated 14 patients with malignant diseases being treated with recombinant human IL3 (rhuIL3) as a daily subcutaneous bolus injection for 15 days. For analysis, patients were combined in a 'low-dose' [30 (n = 1), 60 (n = 3) and 125 (n = 2) micrograms/m2/day] and a 'high-dose' [250 (n = 6) and 500 (n = 2) micrograms/m2/day] therapy group. In the high-dose group there was a 2-fold increase in total leukocytes, and 8-fold increase in basophils, and a 23-fold increase in eosinophils. Histamine content per basophil decreased rapidly after rhuIL3 administration. Anti-IgE-induced HR increased in a dose-dependent manner after rhuIL3 therapy (low-dose group: HRmax 47.5 vs. 53.3%; high-dose group: HRmax 57.8 vs. 75.4%, p less than 0.05). In contrast to anti-IgE-induced HR, HR with ionophore (78.0 vs. 50.3%, p less than 0.05), FMLP (32.3 vs. 11.4%, p less than 0.05), sodium chloride (31.8 vs. 22.6%, n.s.) and mannitol (56.3 vs. 47.8%, n.s.) decreased. There was no histamine release from basophils upon in vitro stimulation with rhuIL3 alone. The kinetics of the increase in anti-IgE-induced histamine release did not parallel the rapid histamine depletion of cells. We conclude that rhuIL3 therapy may cause a rapid HR from basophils which cannot be observed after stimulation of cells in vitro. The clinical importance of this HR remains unclear as side effects could not be correlated with HR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707403 TI - Epitope-specific regulation of the antibody response against alpha-lactalbumins in the mouse. AB - The immune responsiveness to human and bovine alpha-lactalbumin (HuALA and BoALA) was found to be under the control of immune response (Ir) gene(s) linked to the major histocompatibility complex. H-2k mice responded to both HuALA and BoALA, whereas H-2d,s,and f mice respond only to HuALA; H-2b mice were nonresponders to both HuALA and BoALA. A survey with B10.A recombinant mouse strains enabled us to map the Ir gene in the I-A subregion. The responsiveness was shown to be dominant in F1 mice. The coimmunization of BoALA and HuALA resulted in the suppressed secondary antibody response to HuALA in B10.S (H-2s) and BALB/c (H-2d) but not in C3H (H-2k) suggesting that the low responsiveness against HuALA in these strains is due to an active suppression. The transfer of splenic T cells of B10.S mice primed with BoALA into syngeneic animals suppressed the response to HuALA. T cells specific for a particular epitope present on BoALA appeared to suppress the immune response to other epitopes on HuALA. Thus, the presence of epitope specific suppressor T cells seems to account for this Ir-gene-controlled low responsiveness to ALA in H-2s mice. PMID- 1707404 TI - Susceptibility to proteolipid apoprotein and its encephalitogenic determinants in mice. AB - We investigated in mice strain differences in induction of experimental allergic encephalomyelitis by proteolipid apoprotein and studied encephalitogenic determinants. SJL/J, C3H/He, CBA/J and A/J mice were high responders, BALB/c and AKR/J mice were moderately susceptible, and DBA/2, B6 and congenic strains of B10 background were low responders. Synthetic peptide 136-150 was encephalitogenic for SJL/J mice, and 215-232 was encephalitogenic for C3H/He mice. These encephalitogenic derterminants are present in the extracellular portion of proteolipid apoprotein in myelin. PMID- 1707405 TI - High temperature enhances cytotoxicity of mercury (HgCl2) on HeLa S3 cells. AB - The combined effect of mercury (HgCl2) and high temperature on the growth and synthesis of nucleic acid and protein, and on the cell cycle of HeLa S3 cells was investigated. The subsequent growth of the cells was dose-dependently inhibited by mercury at 37.2 degrees and 41.2 degrees C. The inhibitory effect of mercury on subsequent growth was enhanced at the higher temperature. IC50 values for DNA and RNA synthesis but not protein synthesis, at 41.2 degrees C, were significantly lower than those at 37.2 degrees C (P less than 0.05, P less than 0.01, respectively). Flow cytometric analysis using synchronous cells indicated the possibility of blocking of cell cycle progression in the early part of S phase by the combined treatment. These results suggest that the cytotoxicity of mercury to cell growth was enhanced at the higher temperature and that this enhancement is related to the increased inhibitory effect of mercury on DNA and RNA synthesis and on the cell cycle at high temperatures. PMID- 1707406 TI - A seral epidemiological study of HIV transmitted through human seral gamma globulin preparations. AB - In order to study the potential risk of transferring HIV through human seral gamma-globulin preparations (immunoglobulin), indirect immunofluorescent antibody test (IFA) and Western Blot (WB) assay were applied to 343 random samples (sera) with previous injection of imported human seral gamma-globulins (Ig) positive for Human Immunodeficiency Virus (HIV) antibodies between 1981-1987 for the detection of HIV antibodies. All results were negative and tests on all 23 controls who had previously received Ig made in China also gave negative results. However all 12 batches of imported Ig collected from the above-mentioned users, were positive for HIV antibodies when tested by WB and IFA. This study shows that under normal conditions, human seral gamma-globulin does not transmit HIV. PMID- 1707407 TI - Characterization of epithelial cell cultures derived from human tracheal glands. AB - Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 micrograms/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 micrograms/ml), and bovine pituitary extract (25 micrograms/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide beta-D-galactose-(1----3)N acetyl D-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl- channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. PMID- 1707408 TI - Establishment of a human fetal cardiac myocyte cell line. AB - Human cardiac myocytes undergo degeneration, cytolysis, and necrosis in a number of clinical disease conditions such as myocarditis, dilated cardiomyopathy, and during episodes of cardiac allograft rejection. The precise cellular, biochemical, and molecular mechanisms that lead to such abnormalities in myocytes have been difficult to investigate because at present it is not possible to obtain and maintain viable cell cultures of human adult cardiac myocytes in vitro. However, human fetal cardiac myocytes are relatively easy to maintain and culture in vitro, but their limited availability and growth, variability from one preparation to another, and varying degrees of contamination with endothelial and epithelial cell types have made it difficult to obtain reliable data on the effect of cardiotropic viruses and cardiotoxic drugs on such myocytes. These thoughts prompted us to attempt to derive a cell line of human cardiac origin. Highly enriched human fetal cardiac myocytes were transfected with the plasmids pSV2Neo and pRSVTAg and gave rise to a cell line (W1) which has been maintained in culture for 1 yr. Morphologic and phenotypic analyses of W1 cells by flow microfluorometry and immunoperoxidase techniques indicate that the W1 cell line shares many properties of human fetal cardiac myocytes, but appears not to react with specific antibodies known to react with markers unique to human endothelial, epithelial, skeletal muscle, and dendritic cells. These preliminary data suggest that the W1 cells may provide a unique source of an established cell line that shares many properties ascribed to human cardiac myocytes. PMID- 1707409 TI - Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin binding growth factor 1/acidic fibroblast growth factor. AB - Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF 1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. PMID- 1707410 TI - Influence of antithyroidal therapy on asthma symptoms in the patients with both bronchial asthma and hyperthyroidism. AB - To investigate the possible influences of hyperthyroidism on the pathophysiology of bronchial asthma, we clinically observed the effect of antihyperthyroidal therapy on asthmatic symptoms in 7 patients who shared both disorders. In five cases, asthma symptoms were affected by the therapy. Asthma symptoms improved after normalization of thyroid function in two cases, but worsened after therapy for hyperthyroidism in three cases. There was no apparent correlation between the pathophysiology of the two diseases after therapy in the two other cases. These results showed that thyrotoxicosis does not uniformly affect the pathophysiology of asthma and emphasizes the need to follow asthmatic patients closely following treatment for hyperthyroidism. PMID- 1707411 TI - Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism. AB - Extracellular amylase in Streptomyces lividans was undetectable in starch supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57 kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly. PMID- 1707412 TI - Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium. AB - The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIII fragment revealed an open reading frame which could encode a protein of 407 amino acids with an Mr of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane beta-sheet structures, implying a possible role for the protein in translocation of hydrophilic O-antigen-containing materials. Salmonella strains of groups A, B, and D1 contained rfc-homologous DNA, but strains of groups C1, C2, C3, D2, and E2 did not. PMID- 1707413 TI - Low-molecular-weight heparinoid compared with warfarin for prophylaxis of deep vein thrombosis in patients who are operated on for fracture of the hip. A prospective, randomized trial. AB - In a randomized, prospective trial, a low-molecular-weight heparinoid (Org 10172 [Lomoparan]) was compared with warfarin for efficacy and safety in preventing deep-vein thrombosis in 263 patients who had an operatively treated fracture of the hip. One group of patients received Org 10172 in a dose of 750 units subcutaneously every twelve hours until the ninth postoperative day; on the seventh postoperative day, warfarin was added to the regimen. The other group received only warfarin. Both drugs were begun preoperatively, immediately after the admission evaluation. In the patients who received warfarin, the desired prothrombin time was one and one-half times the control level. Deep-vein thrombosis was detected by 125I-fibrinogen scanning and impedance plethysmography and was confirmed by phlebography and compression ultrasonography. Deep-vein thrombosis was found in nine (7 per cent) of the 132 patients who received Org 10172 and in twenty-eight (21 per cent) of the 131 patients who received warfarin (p less than 0.001). Adverse reactions were not significantly different in the two groups. Major bleeding complications occurred in eight patients in the Org 10172 group, only four of whom were receiving the drug at the time of bleeding, and in five patients who were receiving warfarin (not significant). There was no difference in intraoperative loss of blood or in requirements for transfusion. We concluded that the low-molecular-weight heparinoid Org 10172 is a safe, convenient, effective antithrombotic agent for the prevention of venous thrombosis after an operation for fracture of the hip. PMID- 1707414 TI - Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells. AB - The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2) activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56+ or CD57+ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56+ and CD57+ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients. PMID- 1707415 TI - Antigenic properties of myelin and the role of myelin in pathology. Naples, Italy, 22-23 October 1990. PMID- 1707416 TI - Measurement of acute phase proteins for assessing severity of Plasmodium falciparum malaria. AB - Seventeen adult patients with acute Plasmodium falciparum malaria, admitted to the Hospital for Tropical Diseases, were studied. Serial measurements of the serum concentration of C-reactive protein, serum amyloid A protein, and percentage parasitaemia were determined, together with initial measurement of serum electrolytes, liver function, haemoglobin, white cell and platelet counts. Initial C-reactive protein and serum amyloid A concentrations were increased (C reactive protein mean 49.0 mg/l serum amyloid A 28 mg/l) falling towards the normal range by the seventh day of treatment. There was a significant correlation between the pretreatment parasite count and clinical and laboratory markers of inflammation. C-reactive protein and serum amyloid A concentrations correlated inversely with the serum sodium. These results indicate that measurement of acute phase reactants such as C-reactive protein and serum amyloid A may prove valuable in assessing the severity of P falciparum malaria, and in following the response to antimalarial treatment. PMID- 1707417 TI - Cells containing factor XIIIa and pulmonary fibrosis induced by bleomycin. AB - To show the clinical importance of cells containing FXIIIa in pulmonary fibrosis induced by bleomycin, the distributions of FXIIIa and collagenous components were investigated immunohistochemically in both normal lung tissues and those affected by bleomycin. In the normal tissues FXIIIa-containing cells were sparse, but they were numerous in the pulmonary fibrotic tissues, especially in the subpleural area and around the blood vessels of alveolar septa, where slight to moderate fibrosis was seen, and in the intra-alveolar fibrinous exudate. In the collagenous scar-like areas, however, these cells were fewer in number and their FXIIIa expression was depleted. These findings suggest that cells containing FXIIIa have an important role in the development of pulmonary fibrosis induced by bleomycin. PMID- 1707418 TI - Recovery of CD3+ and CD5- lymphocyte subpopulation after autologous bone marrow transplantation and chemotherapy. AB - Although most circulating T cells in normal subjects express both CD3 and CD5 antigens on the cell surface, a small number lack the CD5 antigen. Recipients of allogeneic bone marrow transplants develop increased numbers of CD3+ CD5- cells, particularly those who develop graft versus host disease (GVHD). This CD3+ CD5- population may rise transiently in patients who have received an autologous bone marrow transplant (BMT) and in patients following completion of intensive chemotherapy for acute myeloid leukaemia (AML). These findings suggest that these CD3+ CD5- cells are a normal component of the regenerating lymphoid system after BMT or chemotherapy. PMID- 1707419 TI - Marking resection margins in surgical biopsy specimens. PMID- 1707420 TI - Thalamo-cortical processing of vibrissal information in the rat. I. Intracortical origins of surround but not centre-receptive fields of layer IV neurones in the rat S1 barrel field cortex. AB - The receptive fields of cells restricted to the D1 cortical barrel territory in the S1 cortex of the rat were examined before and after substantial lesions of the D2 barrel. We tested 131 cells (N = 62, unlesioned controls; N = 69, lesioned animals) for modal latency and response magnitude to standard vibrissal deflections of 1.14 degrees. Lesions ranged in size to encompass 22-95% of the volume of the D2 barrel hollow and 5-75% of its neighbouring septal region, as calculated from cytochrome oxidase and Nissl staining of alternate sections. Negligible loss (mean 1.1%) of other barrel hollows and their septal regions (6.3%) occurred. A mean loss of 58% of the D2 barrel hollow and 27% of its accompanying septa was paralleled by a highly significant deficit in response magnitude (57.3%; p less than 0.005) of D1 barrel cells to D2 vibrissal stimulation, when compared with controls. The best-fit relationship between deficit and volumetric loss of the D2 barrel hollow was linear (regression coefficient -0.91). In the extreme case where 95% loss of D2 barrel hollow occurred, there was a 92% deficit in response of D1 barrel cells to the D2 input. No significant loss in response magnitude to other vibrissae, including the principal D1 input, occurred. Postlesioned animals exhibited some increase in excitability to the D1 vibrissa, and to vibrissae whose principal barrel territories were undamaged (delta, gamma, C1). Lesioning of the D2 barrel caused a highly significant mean increase (60%) in latency of residual responses to stimulation of the D2 vibrissal input (15.2 ms controls; 24.3 ms experimentals). No significant changes in response latency to other vibrissae compared to controls occurred. These results suggest that an intact D2 barrel is essential for the generation of responses of D1 barrel cells by the D2 vibrissa, and further imply that surround receptive fields of layer IV barrel cells are largely generated intracortically by barrel-to-barrel relay. The implications of these findings to cortical processing of tactile information and plasticity in the somatosensory system are discussed. PMID- 1707421 TI - Morphological relationships among extensor digitorum longus, tibialis anterior, and semitendinosus motor nuclei of the cat: an investigation employing the retrograde transport of multiple fluorescent tracers. AB - To determine the morphological relationships among extensor digitorum longus (EDL), tibialis anterior (TA), and semitendinosus (St) motor nuclei in the spinal cord of the cat, these nuclei were retrogradely labeled with three different fluorescent tracers. The fluorochromes--bisbenzimide, nuclear yellow, and propidium iodide--were applied by intramuscular injection or soaking the muscle nerve. The positions of the labeled motor nuclei were bilaterally symmetrical. The EDL and TA motoneurons were located in close proximity to one another, in the lateral regions of lamina IX in spinal segments L6 and L7. Although the boundaries of each nucleus were tightly opposed, the EDL and TA motor nuclei overlapped minimally, with the somata of EDL motoneurons positioned dorsal to those of TA. The St motor nucleus was located ventromedial to that of EDL and extended from the caudal portion of L6 through S1. Supplemental studies of the reflex effects evoked in EDL, TA, and St muscles by cutaneous nerve stimulation provided physiological observations that may be related to these anatomical results. PMID- 1707422 TI - Synaptic organization of projections from basal forebrain structures to the mediodorsal thalamic nucleus of the rat. AB - The synaptic organization of the mediodorsal thalamic nucleus (MD) in the rat was studied with the electron microscope, and correlated with the termination of afferent fibers labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Presynaptic axon terminals were classified into four categories in MD on the basis of the size, synaptic vesicle morphology, and synaptic membrane specializations: 1) small axon terminals with round synaptic vesicles (SR), which made asymmetrical synaptic contacts predominantly with small dendritic shafts; 2) large axon terminals with round vesicles (LR), which established asymmetrical synaptic junctions mainly with large dendritic shafts; 3) small to medium axon terminals with pleomorphic vesicles (SMP), which formed symmetrical synaptic contacts with somata and small-diameter dendrites; 4) large axon terminals with pleomorphic vesicles (LP), which made symmetrical synaptic contacts with large dendritic shafts. Synaptic glomeruli were also identified in MD that contained either LR or LP terminals as the central presynaptic components. No presynaptic dendrites were identified. In order to identify terminals arising from different sources, injections of WGA-HRP were made into cortical and subcortical structures known to project to MD, including the prefrontal cortex, piriform cortex, amygdala, ventral pallidum and thalamic reticular nucleus. Axons from the amygdala formed LR terminals, while those from the prefrontal and insular cortex ended exclusively in SR terminals. Fibers labeled from the piriform cortex formed both LR and SR endings. Based on their morphology, all of these are presumed to be excitatory. In contrast, the axons from the ventral pallidum ended as LP terminals, and those from the thalamic reticular nucleus formed SMP terminals. Both are presumed to be inhibitory. At least some terminals from these sources have also been identified as GABAergic, based on double labeling with anterogradely transported WGA-HRP and glutamic acid decarboxylase (GAD) immunocytochemistry. PMID- 1707423 TI - Midget ganglion cells of the parafovea of the human retina: a study by electron microscopy and serial section reconstructions. AB - In this study we used serial section electron microscopy and three-dimensional reconstructions to examine four midget ganglion cells of the human retina. The four cells were located in the parafoveal retina 2.5 mm or 8 degrees from the foveal center. Both type a (with dendritic trees in distal inner plexiform layer) and type b (with dendritic trees in proximal inner plexiform layer) midget ganglion cells have been studied. These cells have dendritic trees of 7-9 microns diameter, and their complete dendritic trees in the neuropil of the inner plexiform layer can be analyzed, as well as the bipolar cell axon terminals having synaptic input, by a study of 100-150 serial ultrathin sections. Type a midget ganglion cells appear to be in a one-to-one relationship with flat midget bipolar cell axon terminals ending in distal inner plexiform layer. Type b midget ganglion cells are in a one-to-one synaptic relationship with invaginating midget bipolar cell axon terminals in proximal inner plexiform layer. The midget bipolar cells primarily involved with the midget ganglion cells do not contact other ganglion cell dendrites. In other words, midget bipolar cells appear to be in exclusive contact with single midget ganglion cells in the human retina. The midget ganglion cells receive most of their input from their associated midget bipolar cells in the form of ribbon synapses at dyads or monads (55-81 ribbons total), although ribbonless synapses are seen occasionally. In all four midget ganglion cells reconstructed, one or two other bipolar cell axon terminals, presumed to be from wide-field bipolar types, provide 1-3 ribbon synapses each. The number of amacrine synapses upon a midget ganglion cell's dendritic tree is approximately equal to the number of bipolar ribbon inputs (43%-56% bipolar ribbons: 44%-57% amacrine synapses). We assume from our knowledge of response characteristics of ganglion cells in other mammalian retinas (Nelson et al., '78: J. Neurophysiol. 41:427-483), that the type a midget ganglion cell and its exclusive connectivity with a flat midget bipolar cell forms a single cone connected OFF-center pathway, whereas the type b midget ganglion cell with its exclusive connectivity to an invaginating midget bipolar cell forms a single cone connected ON-center pathway, through the retina to the brain. PMID- 1707424 TI - Striatonigral projection neurons: a retrograde labeling study of the percentages that contain substance P or enkephalin in pigeons. AB - Two largely separate populations of neuropeptide-containing striatonigral projection neurons have been distinguished in pigeons, one population whose neurons contain substance P (SP) and dynorphin (DYN) and a second population whose neurons contain enkephalin (ENK) (Reiner, '86a; Anderson and Reiner, '90a). In the present study, we investigated the abundance of these two types of neurons relative to all striatonigral projection neurons by combining retrograde labeling by the fluorescent dye fluorogold with immunofluorescence labeling for SP and ENK. Pigeons received large intranigral injections of fluorogold to retrogradely label the striatonigral projection neurons, and several days later they were treated with colchicine (32 hours before transcardial perfusion). Adjacent series of sections through the basal ganglia were labeled for SP and ENK using immunofluorescence techniques. The tissue was examined using fluorescence microscopy and the percentages of retrogradely labeled neurons containing either SP or ENK were quantified. We found that 85-95% of the fluorogold-labeled striatonigral neurons were SP+, whereas only 1-4% were ENK+. Thus the majority of striatonigral projection neurons in pigeons appear to contain SP, whereas a small percentage contain ENK. Only a small percentage of striatonigral neurons did not contain either. Since striatal projection neurons also contain GABA (Reiner, '86b), the present results suggest that a high percentage of striatonigral projection neurons coexpress SP, DYN and GABA, whereas a small fraction coexpress ENK and GABA. The available data are consistent with the conclusion that this is true in reptilian and mammalian species as well. PMID- 1707425 TI - Plasticity of spinal systems after unilateral lumbosacral dorsal rhizotomy in the adult rat. AB - Plasticity of spinal systems in response to lumbosacral deafferentation has previously been described for the cat, by using immunocytochemistry to demonstrate plasticity of tachykinin systems and degeneration methods to demonstrate plasticity of descending systems. In this study, we describe the response to lumbosacral deafferentation in the adult rat. Application of immunocytochemical methods to visualize tachykinins (predominantly substance P magnitude of SP), serotonin (5-HT), and dopamine B-hydroxylase (DBH), the synthesizing enzyme for norepinephrine, permits us to compare the response of SP systems in rat and cat spinal cord and to examine the response of two descending systems, serotoninergic and noradrenergic, to deafferentation. We used image analysis of light microscopic preparations to quantify the immunoreaction product in the spinal cord in order to estimate the magnitude, time course and localization of changes induced by the lesion. The distribution of SP, serotoninergic (5-HT), and noradrenergic staining in the spinal cord of rat is very similar to that of the cat. Unilateral lumbosacral rhizotomy elicits a partial depletion, followed by a partial replacement of tachykinin immunoreactivity in laminae I and II. This response was similar to that described for the cat, although characterized by a longer time course, and, as in the cat, is likely due to plasticity of tachykinin containing interneurons. The same lesion elicits no depletion but a marked and permanent increase in 5-HT immunoreactivity in laminae I and II, which develops more rapidly than the response by the SP system. These results indicate sprouting or increased production of SP and 5-HT in response to deafferentation. No change was seen in DBH immunoreactivity, indicating that the noradrenergic system does not show plasticity in response to deafferentation. Our results demonstrate that dorsal rhizotomy evokes different effects in different systems in the adult spinal cord of the rat and thus suggests that the response of undamaged pathways to partial denervation of their target is regulated rather than random. PMID- 1707426 TI - Sexually dimorphic distribution of a galanin-like peptide in the central nervous system of the teleost fish Poecilia latipinna. AB - Immunohistochemical techniques were used to visualize areas of the brain and spinal cord containing a galanin-like peptide in the teleost fish, the sailfin molly. Galanin-like immunoreactivity (GAL-LI) in both males and females was identified in neurons in the nucleus preopticus periventricularis, nucleus lateralis tuberis, and nucleus commissuralis. GAL-LI fibers had a comparable distribution in the forebrain, preoptic, hypothalamic, and visceral sensory areas of both sexes. In striking contrast to these areas, the optic tectum, torus semicircularis, brainstem tegmentum, and spinal cord of the male contained much higher levels of GAL-LI than the female. GAL-LI in these dimorphic areas in the female was limited to single fiber bundles in the ventromedial tegmentum and in the trigeminal system. Additionally, a population of neurons in the preoptic nucleus was found to contain GAL-LI in the male only. Sexual dimorphism was especially prominent in the spinal cord, where extensive GAL-LI fibers were found in the male only. These fibers were oriented in the longitudinal plane and confined largely to the gray matter. Comparative studies were performed on the goldfish spinal cord, in which GAL-LI was localized solely in the dorsal horn and exhibited no sexual dimorphism. Further, examination of spinal cord material from neonatal mollies revealed a lack of spinal GAL-LI at this developmental stage. As the extent of GAL-LI in the male molly spinal cord differs from both the goldfish and from that reported for the mammalian spinal cord, and a prominent sexual dimorphism in GAL-LI extends from the diencephalon to the caudal spinal cord, it is suggested that a galanin-like peptide may play a unique, sex-specific role in this species. PMID- 1707427 TI - Source of sexually dimorphic galanin-like immunoreactive projections in the teleost fish Poecilia latipinna. AB - A galanin-like peptide has a sexually dimorphic distribution in the teleost fish, the sailfin molly. An extensive system of galanin-like immunoreactive (GAL-LI) fibers has been described in the brainstem and spinal cord of the male molly, which is absent in the female (Cornbrooks and Parsons, companion paper). As GAL LI in the mammalian spinal cord has been localized to neurons of origin in the dorsal root ganglia and dorsal and ventral horns, the present study was undertaken to determine whether the sexually dimorphic GAL-LI in the male molly may originate in part from corresponding sources in this species. Colchicine treatments of the spinal cord and dorsal root ganglia did not result in GAL-LI staining in neuronal somata in these regions. Following complete transection of the spinal cord and at any level of the spinal cord, there was a complete absence of GAL-LI caudal to the lesion site. In fish that received unilateral spinal transection, there was a loss of GAL-LI ipsilateral and caudal to the lesion. Finally, in fish that received lesions in the rostral hypothalamus, there was a complete loss of GAL-LI in the sexually dimorphic fiber system in the brainstem and spinal cord, but not in non-dimorphic GAL-LI regions of the brainstem. Thus the sexually dimorphic fiber system in the male molly may originate in neurons of the preoptic nucleus that are sexually dimorphic for a GAL-LI peptide. This preoptico-spinal pathway may mediate sex-specific behaviors in this species. PMID- 1707428 TI - Neutrophils and mast cells: nedocromil sodium inhibits the generation of neutrophil-derived histamine-releasing activity (HRA-N). AB - The effect of nedocromil sodium on the generation of neutrophil-derived histamine releasing activity (HRA-N) from human peripheral blood neutrophils and on the secretory effects of HRA-N and anti-IgE on rat basophil leukemia (RBL) cells was studied. Nedocromil sodium caused dose-related inhibition of HRA-N generation by human neutrophils (10(-7) to 10(-4) mol/L; inhibitory concentration of 50%, 1.5 x 10(-8) mol/L). In contrast, the presence of nedocromil sodium had no effect on HRA-N-mediated serotonin release from RBL cells. In five experiments, with one crude and four partially purified HRA-N preparations, serotonin release from RBL cells exposed to native HRA-N or HRA-N in the presence of nedocromil sodium (10( 4) mol/L) was 28% +/- 6% and 26% +/- 5%, respectively. Similar results were found with all concentrations of nedocromil sodium studied. Preincubation of RBL cells with nedocromil sodium (10(-4) mol/L) for 0 to 60 minutes also did not affect HRA N-mediated serotonin release. Likewise, nedocromil sodium (10(-7) to 10(-4) mol/L) had no effect on anti-IgE-induced serotonin release from RBL cells. In 10 experiments, anti-IgE alone or in the presence of nedocromil sodium (10(-4) mol/L) induced 32% +/- 6% and 32% +/- 5% serotonin release, respectively. Preincubation of RBL cells with nedocromil sodium before anti-IgE exposure had no further effect. This is the first study of pharmacologic manipulation of the generation of a histamine-releasing factor. It is hypothesized that one possible mechanism whereby nedocromil sodium protects against asthma and allergic rhinitis is through inhibition of HRA-N generation. PMID- 1707429 TI - Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity. AB - The technique of Fmoc chemistry has been applied successfully to the synthesis of branched peptides. The immunogenicity of branched peptides has been compared quantitatively with those of protein-conjugated and resin-linked peptides. Six different peptide sequences were used to immunise rabbits and both antipeptide and anti-protein titres were determined for each serum. The data show that the titres of sera from rabbits immunised with branched peptides were higher than those of rabbits immunised with protein-conjugated peptides which in turn were higher than those immunised with resin-linked peptides. The effect was demonstrated with two strains of rabbits. PMID- 1707430 TI - A simple method for coating native polysaccharides onto nitrocellulose. AB - A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disks. These were analyzed by blotting with size matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS. PMID- 1707431 TI - [Changes in serum levels of gynecological tumor markers throughout the period from early gestation to puerperium]. AB - The aim of this study is to elucidate the change in serum levels of gynecological tumor markers throughout the period from the early gestational stage to puerperium. We measured eight tumor markers of--CA 125, TPA, SCC, AFP, haptoglobin, ferritin, CA19-9 and CEA--in 17 healthy women with a normal course of pregnancy, delivery and puerperium, and obtained the following results: 1) Profiles of change in serum levels of CA125, SCC, haptoglobin and ferritin were similar during pregnancy, with those levels being the highest at 4-15 weeks of gestation and declining gradually from 16 to 27 weeks. Serum levels of these four markers decreased significantly (p less than 0.01) at 16-27 and 28-40 weeks of gestation, respectively. 2) A significant (p less than 0.01) increase in CA125 and SCC was observed 2 hours after delivery compared with the levels in the first stage of delivery. However, these two markers decreased to the normal range after the fifth day postpartum. 3) Serum TPA decreased significantly (p less than 0.05) in 16-27 weeks of gestation, comparing with those of 4-15 weeks. Serum CA19-9 and CEA remained almost unchanged within the normal range throughout the period from pregnancy to puerperium. 4) Tumor markers of CA125, TPA, SCC, haptoglobin, ferritin and CEA of which serum levels decreased during the course of pregnancy and puerperium might be a clue to judge whether gynecological tumors in pregnant women are malignant or benign. PMID- 1707432 TI - Chemical abortion in patients with recurrent fetal loss. PMID- 1707433 TI - Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls. AB - This report describes essential requirements for the validation of a radioimmunoassay (RIA) for insulin-like growth factor-I (IGF-I) and presents solutions to some problems and pitfalls commonly observed. The preparation of IGF I to be used as radioligand or standard has to be selected carefully since some IGF-I preparations are contaminated with variants which demonstrate different potencies for different antisera used in the RIA. Accurate assessment of IGF-I levels in blood plasma requires an efficient extraction method for the IGF binding proteins (IGFBPs). Extraction methods to remove the influence of IGFBPs in the RIA were compared using blood plasma of considerable differences in IGF I/IGFBP ratios. Acidification of plasma before column chromatography on Sephadex G-75 (G75) is generally considered to be the most reliable extraction method, but it is very time-consuming. The acid-ethanol extraction (AE) of plasma is not valid in many situations. Non-parallel displacement to the IGF-I standard was observed with AE-extracted plasma samples in the RIA. In addition, a comparison of IGF-I values obtained in the RIA after AE or G75 extraction of fetal ovine plasma has shown no significant correlation. We report an extraction technique based on a modified AE extraction followed by cryo-precipitation (AEC). AEC extraction on blood plasma reduced residual IGFBPs to a level that did not interfere in the assay. Furthermore, AEC-extracted plasma samples showed parallel displacement in the RIA to highly purified preparations of authentic IGF-I. We observed high correlations, with a slope close to unity, of IGF-I values obtained in the RIA using the AEC or G75 extraction for plasma from different species including adult and fetal sheep, rat, mouse and man. The AEC extraction provides a rapid and simple alternative to G75 extraction for blood plasma from a variety of species provided that high-affinity antisera are used for the RIA. PMID- 1707434 TI - Differential effects of epidermal growth factor on the differentiation of type A spermatogonia in adult mouse cryptorchid testes in vitro. AB - The effect of epidermal growth factor (EGF) on testicular germ cell differentiation was investigated. Testicular fragments from surgically prepared cryptorchid testes of adult mice were cultured for 9 days in serum-free media containing various concentrations of EGF. Histological sections of testis were examined under a light microscope and each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. EGF at concentrations ranging from 100 to 200 ng/ml induced differentiation of type A spermatogonia. The observed maximal stimulatory activity of EGF at a concentration of 100 ng/ml was 30% of the positive control cultures treated with calf serum. EGF at concentrations ranging from 1 to 100 ng/ml significantly inhibited the mitotic activity of FSH, FSH plus retinol, or FSH plus fetuin on type A spermatogonia and their differentiation. The number of type A spermatogonia in testes cultured with FSH, FSH plus retinol, or FSH plus fetuin decreased when EGF was added. On the other hand, EGF stimulated the differentiation of type A spermatogonia induced with fetuin but did not influence retinol-induced differentiation. It is proposed that EGF inhibits testicular germ cell differentiation by blocking the proliferation of type A spermatogonia stimulated by FSH. PMID- 1707435 TI - Developmental changes in growth hormone, insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in plasma of young growing pigs. AB - The relationship between plasma concentrations of normally secreted GH and insulin-like growth factor-I (IGF-I) was investigated in pigs after weaning. Frequent blood sampling for between 12 and 24 h showed that plasma GH was pulsatile in pigs of 10, 20 and 35 kg liveweight. Pulses were brief in duration, low in amplitude and variable in frequency. Basal and average daily plasma concentrations of GH changed significantly with development, increasing by about 50% between 10 and 20 kg liveweight. Concentrations of IGF-I in plasma showed little or no evidence of diurnal periodicity and were not increased by GH pulses. Average daily concentrations of both IGF-I and IGF-II in plasma progressively increased between 10 and 35 kg liveweight, as did the total desaturated IGF binding protein (IGFBP) activity of plasma. A strong positive correlation was observed between the total concentration of IGFs (IGF-I plus IGF-II) in the circulation and plasma IGFBP activity. The developmental rise in IGFBP activity of plasma was associated with increased labelling with 125I-labelled human IGF-II in ligand blots of binding proteins of apparent molecular masses greater than 200, 50, 43 and 29 kDa. One class of binding proteins of 34.5 kDa decreased with development. This study of young growing pigs shows that normally secreted endogenous GH exerts no significant immediate control over plasma IGF-I concentrations, and that plasma levels of IGF-I and IGF-II increase with maturation in this species.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707436 TI - Dopamine-dependent postnatal development of enkephalin and tachykinin neurons of rat basal ganglia. AB - The influence of deprivation of the neurotransmitter dopamine (DA) on the development of [Met5]-enkephalin (ME) and substance P (SP) neuropeptide systems of the striatum was investigated in Sprague-Dawley rats. The neurotoxin 6 hydroxydopamine (6-OHDA) was used to induce DA deficiency on postnatal day 3 in rats, and the animals were killed at different postnatal time points until 35 days of age. The levels of ME and SP were determined by radioimmunoassay, and the abundance of preproenkephalin (PPE) and preprotachykinin (PPT) mRNA in the striatum was assessed by Northern blot hybridization analysis. The concentrations of DA, 5-hydroxytryptamine (5-HT), and their acid metabolites were determined by HPLC with electrochemical detection. The postnatal development of the PPE-derived peptide ME and the PPT-derived peptide SP closely paralleled the appearance of the respective mRNAs coding for these peptides. The dopaminergic lesion with 6 OHDA led to a marked depletion of DA and its metabolites but produced an increase in content of 5-HT and its metabolite in the striatum. The lesion did not affect the ME and PPE mRNA levels in the striatum up to 25 days but increased the levels at 35 days. In contrast, a decreased developmental expression in SP and PPT mRNA was observed throughout the observation period. The lesion failed to influence the development of the mRNA coding for the structural protein beta-actin. The results indicate that the normal development of enkephalin, tachykinin, and 5-HT systems of the striatum is dependent on the availability of DA, the integrity of dopaminergic neurons, or both. The studies provide evidence for an interrelationship and interdependence between the development of neurotransmitter and neuropeptide systems. It is suggested that an early developmental abnormality in the DA system could permanently alter the neuropeptide systems, which in turn could influence the progression and expression of the DA-deficiency state parkinsonism, Lesch-Nyhan disease, or both. PMID- 1707437 TI - Mechanism of inhibition of N-methyl-D-aspartate-stimulated increases in free intracellular Ca2+ concentration by ethanol. AB - Dissociated brain cells were isolated from newborn rat pups and loaded with fura 2. These cells were sensitive to low N-methyl-D-aspartate (NMDA) concentrations with EC50 values for NMDA-induced intracellular Ca2+ concentration ([Ca2+]i) increases of approximately 7-16 microM measured in the absence of Mg2+. NMDA stimulated [Ca2+]i increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mM KCl prior to NMDA addition. Under these predepolarized conditions, 100 mM ethanol inhibited 25 microM NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA-induced increases in [Ca2+]i in a concentration dependent manner at 4, 16, 64, and 256 microM NMDA. Whereas NMDA-induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 microM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 microM) enhanced 25 microM NMDA-induced increases in [Ca2+]i by approximately 50%. Glycine (1-100 microM) prevented the 100 mM ethanol inhibition of NMDA stimulated [Ca2+]i observed in the absence of exogenous glycine. MK-801 (25-400 nM) inhibited 25 microM NMDA-stimulated rises in [Ca2+]i in a concentration dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707438 TI - Effect of tryptophan on extracellular concentrations of tryptophan and 5 hydroxyindoleacetic acid in the striatum and cerebellum. AB - The effects of L-tryptophan (50 mg/kg i.p.) on extracellular concentrations of tryptophan and the 5-hydroxytryptamine (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the rat striatum and cerebellum, regions with rich and poor 5-HT innervation, respectively. Determinations were on perfusates from dialysis probes in the brains of conscious, freely moving rats. The pharmacokinetic profiles of dialysate tryptophan after tryptophan load (peak concentration, time to peak concentration, area under curve, and half-life) in the two regions did not differ significantly. The dialysate 5-HIAA concentration in the striatum rose two- to threefold after the administration of tryptophan. Therefore, as 5-HIAA was undetectable in the cerebellum either before or after the administration of tryptophan, the increase of 5-HIAA in the striatum is unlikely to depend appreciably on its production within the cerebral vasculature or outside the brain or on its entering the striatum through a blood-brain barrier damaged by placement of the dialysis probe. Overall, the findings strengthen previous evidence that extracellular 5-HIAA concentrations determined by cerebral dialysis are a valid measure of the metabolism of 5-HT of brain neuronal origin. PMID- 1707439 TI - Immunocytochemical localization of chondroitin and chondroitin 4- and 6-sulfates in developing rat cerebellum. AB - Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707440 TI - Inhibition of the chick pineal rhythm in rate of thymidine incorporation by cyclic AMP. AB - Chick pineal glands in organ culture showed a circadian rhythm in the rate of thymidine incorporation. Thymidine incorporation was very markedly inhibited when 3-isobutyl-l-methylxanthine (IBMX) was continuously present. When IBMX was added to cultures in control medium during the photoperiod of the second day in culture, the extent of inhibition of incorporation during that photoperiod increased with the increase in length of the photoperiod remaining. Incorporation did not resume at the start of a second photoperiod if IBMX was added within the first 10 h of the first photoperiod. Corresponding results were obtained with glands continuously cultured in constant darkness. Similar results were also obtained using glands treated with 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), 7 beta-acetoxy-8,13-epoxy-1 alpha, 6 beta, 9 alpha-trihydroxy-1abd 14-ene-11-one (forskolin), or 8-bromo-cyclic AMP, but not with 8-bromo-cyclic GMP. When glands cultured with IBMX were transferred to control medium, incorporation remained inhibited until the start of the next photoperiod. We conclude that the increase in the rate of thymidine incorporation at the start of each new photoperiod is dependent on a "switch" process that is inhibited by elevated concentrations of cyclic AMP. PMID- 1707441 TI - Levels of proteolipid protein mRNAs in peripheral nerve are not under stringent axonal control. AB - The proteolipid protein (PLP) is the major protein in the myelin sheath of the CNS. It was recently reported that PLP coding transcripts are also found in the PNS, although the protein was not detectable in peripheral nerve myelin. In the present investigation, levels of mRNA for PLP in sciatic nerve were studied during development and following transection and crush injury. Results were compared to those for P0, the major PNS myelin protein, and the myelin-associated glycoprotein (MAG). PLP transcript levels were very low at 21 days in sciatic nerve and remained unchanged in the adult sciatic nerve. This contrasts markedly with P0 and MAG mRNAs, which are expressed at high levels during development and decrease in content significantly by adulthood. The level of PLP messages was reduced approximately 40% in the quiescent Schwann cells in the distal segment of the sciatic nerve at 21 days after permanent transection, yet P0 mRNA levels were very low, and MAG mRNAs were undetectable in this tissue. The distal segment of the crush-injured sciatic nerve is characterized by transient demyelination followed by rapid myelination. PLP mRNA levels remained comparatively unaffected in the 3-week period following crush injury. RNase protection experiments using two antisense riboprobes confirmed that levels of PLP-derived protected fragments, corresponding to PLP and DM-20 messages, remained unchanged in the developing and adult sciatic nerve. These results indicate that myelin-specific P0 and MAG genes are tightly controlled at the level of transcription through Schwann cell-axonal interactions, whereas PLP transcription in the peripheral nerve remains nearly dissociated from axonal influences. PMID- 1707442 TI - Characterization of extracellular histamine in the striatum and bed nucleus of the stria terminalis of the rat: an in vivo microdialysis study. AB - The intracerebral microdialysis technique, coupled with a sensitive radioenzymatic assay, was employed to study histamine release in the striatum and in the bed nucleus of the stria terminalis (BNST) in conscious, freely moving rats. In these brain regions, extracellular histamine concentrations decreased by 20% when calcium was omitted from the perfusion solution. Extracellular histamine was insensitive to the addition of tetrodotoxin to the perfusion medium. In striatum, extracellular histamine concentrations declined in an apparent biexponential manner after the administration of alpha-fluoromethylhistidine, an inhibitor of histamine synthesis. The half-lives for the disappearance of histamine were 32 min and 7.7 h, indicating the presence of at least two histamine pools. Histidine loading resulted in a nearly twofold increase in histamine outflow in striatum. In the BNST, yohimbine increased the extracellular histamine content by 50%, suggesting that histamine release is subject to alpha 2 adrenergic regulation in vivo. The extent to which histamine detected in cerebral microdialysis samples is of neurogenic origin remains to be established. PMID- 1707444 TI - Practice transitions in the 1990s. PMID- 1707443 TI - From recent grad to working professional. PMID- 1707445 TI - Financing a startup. PMID- 1707446 TI - Slow passage to power. PMID- 1707447 TI - Keep your image polished. PMID- 1707448 TI - Oral pathology: erythema multiforme. PMID- 1707449 TI - Binding of ubiquitin to experimentally induced murine AA amyloid. AB - Amyloid enhancing factor (AEF) activity has recently been demonstrated in ubiquitin purified from amyloidotic murine tissues and Alzheimer brain extract. Since AEF is known to bind to amyloid fibrils and 'fibril-AEF' on passive transfer induces accelerated amyloidogenesis in the recipient animals, it was of interest to investigate whether ubiquitin binds to amyloid. Immunohistological studies were carried out on liver sections from amyloidotic mice. Biotin strepavidin-peroxidase methods using monospecific rabbit anti-mouse AA amyloid IgG (RAAG) and rabbit anti-bovine ubiquitin IgG (RABU) antibodies were employed to immunostain the amyloid and ubiquitin deposits, respectively. RABU-treated liver sections were counterstained with thioflavine S. RAAG reacted strongly with the amyloid, indicating that it is AA type, and RABU-positive immunodeposits were found bound to the thioflavine-S-positive AA deposits. Treatment of the liver sections with 0.1 M sodium acetate containing 0.5 M NaCl, pH 4, for 2-3 h at 37 degrees C nearly completely desorbed the AA amyloid-bound ubiquitin. Since ubiquitin demonstrates AEF activity in vivo and binds non-covalently to AA amyloid, we suggest that ubiquitin may indeed be 'fibril-AEF' and may play a crucial role in the pathogenesis of amyloidosis. To our knowledge, this is the first time that ubiquitin bound to extracellularly deposited amyloid has been demonstrated. PMID- 1707450 TI - True histiocytic neoplasm of Langerhans' cell type. AB - Malignant histiocytic neoplasms of dendritic cell lineage are rare and most are derived from the dendritic cells of the T-cell and B-cell areas of the lymph node, respectively. Only one case of a malignant histiocytic neoplasm of Langerhans' cells, the dendritic cell of the skin, has been reported. We report the occurrence of a true histiocytic neoplasm of Langerhans' cell type in a 23 year-old female patient. The diagnosis of this highly aggressive neoplasm is supported by histochemical, immunohistochemical, ultrastructural, and genotypic analysis--techniques which allow differentiation from malignant melanoma and large cell anaplastic lymphoma. PMID- 1707451 TI - Eosinophilic granuloma of the bone in Hand-Schuller-Christian disease: extensive in vivo eosinophil degranulation and subsequent binding of released eosinophil peroxidase (EPO) to other inflammatory cells. AB - The eosinophils from bone granuloma, bone marrow, and peripheral blood of a patient with Hand-Schuller-Christian disease (HSCD) were studied by electron microscopy and cytochemistry. Impressive eosinophil degranulation was observed. Extracellular release of eosinophil peroxidase (EPO) and EPO binding to surrounding cells were seen in the granuloma and bone marrow. Cells with peroxidase-positive plasma membrane were also observed in peripheral blood. The pattern of eosinophil degranulation showed quite different features from those described so far. In the granuloma, the process begins with intracytoplasmic release of the granule matrix content, as revealed by both extensive extragranular accumulation of EPO and progressive decrease of the matrix electron density. Core dissolution follows thereafter, leading to complete disappearance of the granules. At the end of the process, the cells show rupture of the plasma membrane and release of their content into the surrounding environment. This pattern of secretion was also observed in blood and marrow eosinophils of the patient. In view of the previously reported findings that EPO binding to inflammatory cells influences their functions, EPO release and binding to surrounding cells in HSCD may play a role in the evolution of the inflammatory lesion in the disease. PMID- 1707452 TI - A critical evaluation of AgNOR counting in benign naevi and malignant melanoma. AB - There is considerable variation in the quoted mean numbers of AgNORS per nucleus for benign melanonaevi and malignant melanomas. This is partly attributable to different approaches to AgNOR counting. This study summarizes our experience in devising an optimal technique for counting AgNORs. We show that it is essential to count intra-nucleolar AgNORs in addition to those lying outside the nucleolus to obtain clear separation of naevi from melanoma. Although this seems an onerous task, we further demonstrate that a maximum of only 30 nuclei need to be counted to obtain a mean AgNOR count per nucleus which is representative of the whole lesion. This compares with the arbitrary figure of 100 nuclei chosen by most workers. Only by optimizing and standardizing all aspects of the AgNOR technique including fixation, staining, and counting will mean AgNOR counts per nucleus become a useful quick, reproducible method which can be applied to lesions which pose diagnostic problems such as borderline melanocytic lesions. PMID- 1707453 TI - Monoclonal antibodies to cultured human glomerular mesangial cells. I. Reactivity with haematopoietic cells and normal kidney sections. AB - The aim of this study was to produce monoclonal antibodies to cultured human glomerular mesangial cells in order to obtain specific markers for these cells and to aid the study of their function. Using standard monoclonal antibody techniques, 29 hybridomas producing antibodies directed to cultured mesangial cells were obtained. Most of these antibodies were not reactive with normal or neoplastic haematopoietic cell lines by flow cytometry. Fourteen of the 29 culture supernatants bound to various components of normal human kidney sections stained by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) method. Ten of these supernatants reacted with components within the glomerulus, with six binding to the mesangium. These studies suggest that (1) mesangial cells in culture may show significant de-differentiation, because most supernatants which reacted with mesangial cells in culture did not do so in tissue sections; (2) antibodies reactive with haematopoietic cells may not detect the majority of immunogenic surface antigens on cells in tissues; and (3) some of the antibodies which we have produced may prove to be useful markers for mesangial cells in glomerular disease. PMID- 1707454 TI - Effects of dihydroergotamine and etilefrine on experimentally-induced postural hypotension in dogs. AB - The effects of dihydroergotamine and etilefrine on experimentally-induced postural hypotension were examined. Although dihydroergotamine at 3 and 10 micrograms/kg (i.v.) increased blood pressure (BP), it did not affect cardiac output (CO). However, dihydroergotamine at 10 micrograms/kg reduced the decrease in CO induced by the tilt. Therefore, it is suggested that the increase in BP is induced by the contraction of resistance vessels, and that the inhibition of the decrease in CO due to tilt is induced by the contraction of capacitance vessels. Etilefrine at 0.1 mg/kg (i.v.) increased BP and heart rate (HR), however, it did not attenuate the decrease in BP induced by the tilt. Although it tended to increase CO, it did not attenuate the decrease in CO. It is suggested that the increase in BP is due to the contraction of resistance vessels, and to the increase in cardiac contractile force and HR. In this study, dihydroergotamine and etilefrine did not attenuate the decrease in BP due to tilt, though dihydroergotamine inhibited the decrease in CO due to tilt. As an explanation, it is suggested that dihydroergotamine induces contraction of resistance vessels as well as capacitance vessels, however the effects of the drug on resistance vessels is weak, and that etilefrine has little or no effect on capacitance vessels. In our previous study, midodrine, an alpha-1 agonist, attenuated the decreases in BP and CO due to tilt, and it has been suggested that the inhibition was induced by the contraction of capacitance vessels. Therefore, dihydroergotamine, etilefrine and midodrine show different pahrmacological profiles in experimentally-induced postural hypotension. PMID- 1707456 TI - Cytotoxic drugs and the aquatic environment: estimation of bleomycin in river and water samples. AB - A radioimmunoassay has been used to determine levels of the anticancer drug bleomycin in sewage treatment works effluent, river and potable water samples. Samples were concentrated 100-fold by lyophilisation and a final limit of detection of 5 ng L-1 was achieved. Concentrations of immunoreactive bleomycin of between 11 and 19 ng L-1 were found in the effluents but a lower concentration range less than 5-17 ng L-1 was found in river and potable water samples. The risk to human health of ingesting water (in SE England) with such low levels of this cytotoxic drug appears to be minimal in relation to the normal chemotherapeutic doses administered (20-30 mg m-2). PMID- 1707455 TI - Potentiating effect of daunorubicin on vasocontractile responses to KCl and BAY K 8644 in rat aorta. AB - The effects of daunorubicin on vasoconstraction by several agonists have been investigated on isolated aortic strips from rats. Pretreatment of the strips with daunorubicin (17.7 microM) potentiated the contractile response to low concentrations of KCl or to BAY K 8644 but not to phenylephrine or clonidine. The maximal contractile response to KCl was not affected by the pretreatment while that to BAY K 8644 was increased. The potentiated response to KCl could be eliminated by addition of nifedipine (1 microM) or use of a calcium-free solution. The maximal contractile response to BAY K 8644 was greatly increased by partial depolarization with KCl (10 mM, final concn) in the control solution but only slightly increased by the partial depolarization in the solution with daunorubicin. These results suggest that daunorubicin facilitates activation of the voltage-dependent calcium channel and increases the contractile responses to KCl and BAY K 8644 in rat aorta. PMID- 1707457 TI - Effect of an anti-SRS-A agent, NZ-107, on airway responses induced by ovalbumin and A23187 in the guinea-pig. AB - The effects of the anti-SRS-A agent NZ-107 on antigen-(ovalbumin) and calcium ionophore A23187-induced airway responses in the guinea-pig have been investigated. In the presence of 5 microM indomethacin, NZ-107 (3 microM) did not affect the peak response in ovalbumin-induced contraction but did inhibit the prolonged response following the peak response in the tracheal strip. A higher concentration of NZ-107 (10 microM) completely blocked both peak and prolonged responses. Inhibitory effects of NZ-107 on ovalbumin responses were less in the lung parenchymal strip. The potency of NZ-107 in inhibiting ovalbumin-induced tracheal contraction was not changed in the absence of indomethacin but was reduced in the presence of 45 mM serine-borate, an inhibitor of the conversion of LTC4 to LTD4. NZ-107 inhibited A23187-induced contractions in both tracheal and parenchymal strips but in both cases the inhibitory potency was less than that on ovalbumin response. NZ-107 was a more potent inhibitor of ovalbumin-induced SRS-A release than histamine release in lung fragments but was ineffective in inhibiting A23187-induced SRS-A and histamine release. NZ-107 at a concentration of 10 microM more effectively inhibited LTC4- and histamine-induced tracheal contractions than it did LTC4 in the presence of 45 mM serine-borate. These results suggest that NZ-107 selectively inhibits antigen-induced SRS-A responses in airway tissues of the guinea-pig. PMID- 1707458 TI - Production of two species of interferon by Large White and Meishan pig conceptuses during the peri-attachment period. AB - Antiviral activities present in uterine flushings from pregnant Large White, Large White 'hyperprolific', and prolific Meishan gilts, between Days 8 and 20 of gestation were compared. Flushings (20 ml) from all gilts between Days 14 and 20 were positive in an in-vitro interferon (IFN) assay using vesicular stomatitis virus as a challenge infection. Highest antiviral activities (of up to 400,000 1,200,000 total Units/flushing) were obtained at Day 16 of gestation, i.e. clearly after the beginning of attachment. There was no major difference between breeds although, at Day 14, flushings from Meishan gilts yielded significantly higher titres than those from the other two, suggesting a correlation with the previously described earlier trophoblast elongation in Meishan gilts. Conceptus cultures contained antiviral activity, with values very close to those obtained in vivo, but the difference between breeds was not significant. Cultures from Day 20 on contained very little antiviral activity. The antiviral activity was associated with a mixture of at least two IFNs, one of which was IFN-alpha like, and the other was serologically identified as an IFN-gamma, that is an 'immune IFN', previously found to be secreted only by T lymphocytes. This finding may have implications for our understanding of the immunology of early pregnancy. PMID- 1707459 TI - Nerves in inflammatory synovium: immunohistochemical observations on the adjuvant arthritis rat model. AB - Previous evidence has been presented that neurogenic input may influence adjuvant induced arthritis (AA) in rats. We now present evidence of alterations in synovial nerves in AA. Nerves were studied in well perfused and fixed rats, using immunohistochemistry with the sensitive avidin-biotin peroxidase complex (ABC) method and heterologous antisera to cytoskeletal protein gene product 9.5 (PGP) and the neuropeptides substance P and calcitonin gene related peptide (CGRP). The innervation of synovium was compared in normal rats and rats with AA. Observations concordant with what has been reported for neuropeptide nerves in the synovium of patients with rheumatoid arthritis (RA) are presented. It has been suggested that neural peptide substances are reduced in nerves of synovium from patients with RA. In the AA rat a specific reduction of lining zone and sublining nerves in the synovium was noted. The AA rat model is very suitable for studying the involvement of synovial nerves in arthritis, permitting optimal preservation of immunoreactive neural epitopes. PMID- 1707460 TI - Peptide containing nerves in human synovium: immunohistochemical evidence for decreased innervation in rheumatoid arthritis. AB - The innervation of normal and rheumatoid human synovium was studied by immunofluorescence microscopy. Antibodies against the general neuronal marker protein gene product (PGP) 9.5 and specific neuropeptides were used. We observed sensory nerves containing substance P (SP) and calcitonin gene related peptide (CGRP) as well as autonomic sympathetic fibers immunoreactive for neuropeptide tyrosine (NPY), its C terminal peptide (C-PON) and the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). Three subpopulations of nerve fibers labelled with SP and CGRP were identified: some stained for SP or CGRP only and others contained both peptides. NPY/C-PON and TH labelled predominantly perivascular nerves. Quantification of immunostained nerves revealed a significantly decreased innervation of rheumatoid synovia. The densities of both PGP 9.5 and neuropeptide containing nerves were lower in all rheumatoid samples. Our results are compatible with a local release of neuropeptides into joint fluid and point to a disturbed neuronal control of rheumatoid synovial tissue. PMID- 1707461 TI - Expression of the interleukin 6 gene in rheumatoid synovial fibroblasts. AB - A number of fibroblastoid synovial cell lines have been established from rheumatoid joints. These cell lines were shown to express the interleukin 6 (IL 6) gene constitutively, and exposure of these cells to 5 ng/ml of recombinant human interleukin 1 beta (IL-1 beta) increased IL-6 gene expression. Other recombinant human lymphokines, namely interferon-gamma, tumor necrosis factor alpha, and granulocyte-macrophage colony stimulating factor had no enhancing effect on IL-6 gene expression. Dexamethasone added to the cultures at 10(-7) M concentration suppressed the constitutive expression of the IL-6 gene. At a concentration of 10(-5) M, dexamethasone partially suppressed the IL-1 enhanced expression of IL-6. The IL-6 gene probe also hybridized to RNA from unfractionated synovial fluid cells, peripheral blood T cells and non-T cells but not Epstein-Barr virus transformed peripheral blood B cells of patients with rheumatoid arthritis. Our results suggest that in rheumatoid arthritis, synovial fibroblasts actively participate in joint inflammation by lymphokine production. The coexpression of both IL-1 and IL-6 by one synovial fibroblast line suggests a mechanism for the perpetuation of synovitis. PMID- 1707462 TI - Staining sections of water-miscible resins. 1. Effects of the molecular size of stain, and of resin cross-linking, on the staining of glycol methacrylate embedded tissues. AB - Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. 'Large' dyes (greater than 1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. 'Small' dyes (less than 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dye of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted. PMID- 1707463 TI - Recent approaches to the treatment of sickle cell anemia. PMID- 1707464 TI - A new experimental trial using repeated heating every 24 hours for local hyperthermic therapy with bleomycin in vivo. AB - This report presents the effect of repeated heating every 24 hrs using bleomycin (BLM) which, although seemingly contrary to the usual agreement that hyperthermia should be carried out with a long interval due to thermotolerance, holds many possibilities. FM3A cells on the foot pad of C3H mouse were immersed in a heated water bath at 43 and 44 degrees C for 30 min. The effect of repeated heating was appreciated by an improved growth curve and 50 day survival compared to mice which received heating twice with a 96-hr interval. Repeated heating every 24 hrs 5 times with BLM suppressed tumor growth significantly as compared to heating twice with a 96-hr interval without BLM. The longest survival time was obtained by the repeated heating with BLM among all protocols. There is therefore a good possibility that more effective results could be obtained clinically by repeated heating over a short period. PMID- 1707465 TI - Thyrotoxic crisis in Graves' disease: indication for immediate surgery. AB - Thyrotoxic crisis (thyroid storm) is a rare complication of hyperthyroidism. It can be observed not only in thyroid autonomy with latent hyperfunction after exposure to iodine, but also in Graves' disease with overt hyperfunction. Adequate management of thyrotoxic crisis is still controversial. We report about four patients (four women, mean age 75 years) with Graves' disease who developed thyrotoxic crisis during therapy with antithyroid drugs so that surgical intervention became necessary. The patients had been admitted to the hospital for nonspecific symptoms such as headache, cachexy, and psychosis. Thyroid hormone levels had reached twice the normal range prior to surgery. All patients showed severe neurological deficits leading to coma. In three cases euthyroidism was achieved within two days after surgery. The neurological symptoms disappeared after an average of four days. The postoperative course did not show severe complications and all patients recovered completely. Especially in the elderly a monosymptomatic or nonspecific course of thyroid storm with neurological symptoms may represent a severe and life-threatening situation. In these cases surgery can become necessary even if euthyroidism has not been achieved preoperatively. PMID- 1707466 TI - Acute phase response after myocardial infarction: correlation between serum levels of cytokines and C-reactive protein. PMID- 1707467 TI - An argyrophil-III method for the selective demonstration of mitochondria in aldehyde-fixed rat brain. AB - Based on recent achievements in physicochemical aspects of histological silver staining, a selective and reliable method was elaborated for the demonstration of mitochondria in frozen sections of aldehyde-fixed rat brain. Areas of similar architecture were stained with comparable intensity both within one section or in serial sections. The distribution patterns of mitochondria can be examined at low magnification or even with the unaided eye. At high magnifications, rounded and elongated profiles with the size and shape of mitochondria clearly stand out against a pale background. Electron microscopy revealed a high degree of selectivity and completeness of the mitochondrial staining. PMID- 1707468 TI - Effects of rGM-CSF and rG-CSF on the cisplatin sensitivity of the blast cells of acute myeloblastic leukemia. AB - Recombinant growth factors have been shown to alter the sensitivity of acute myeloblastic leukemia (AML) blast cells to cytosine arabinoside (ara-C) in culture. The mechanism is controversial and suggestions for it include changes in ara-C metabolism, changes in cell cycle parameters, and changes in the balance between self-renewal and determination in blast stem cells. We addressed this issue by measuring the cisplatin sensitivity of freshly obtained AML blasts in rG CSF, rGM-CSF, or the two together. For comparison, simultaneous measurements of ara-C sensitivity were made. We found that exposure to different factors in suspension altered the cisplatin sensitivity of AML blasts in the same direction as the change observed in ara-C sensitivity. Similar changes in cisplatin sensitivity were seen when cells were briefly exposed to the drug, washed, and then grown in suspension in the presence of different growth factors. Control experiments showed that the conditions in suspension, not in the clonogenic assay in methylcellulose, were responsible for the changes in cisplatin sensitivity. The capacity of high specific activity to inactivate clonogenicity was tested at several times under growth conditions which altered the sensitivity of cells to cisplatin. Whereas changes in survival after 3HTdR and cisplatin both were seen with time, growth conditions that altered cisplatin sensitivity were not associated with changes in 3HTdR toxicity. The data do not support explanations of the effects of growth conditions on drug toxicity which depend either on drug metabolism or cell cycle effects. Instead, the findings are consistent with a model that postulates an association between drug sensitivity and the balance between self-renewal and differentiation in the blast population. PMID- 1707469 TI - Heterogeneity in response to interleukin 6 (IL-6), expression of IL-6 and IL-6 receptor mRNA in a panel of established human multiple myeloma cell lines. AB - The controversial role of interleukin-6 (IL-6) as an auto- or paracrine growth factor for human multiple myeloma (MM) cells was studied using a panel of six well characterized feeder-cell dependent and independent MM cell lines as models. With respect to the effect of IL-6 on growth and survival, three types of lines were found: (1) U-1958, dependent on IL-6 both for growth and survival; (2) U 1996, dependent on IL-6 for growth but not survival; and (3) U-266-1984, Fravel, L363, and Karpas 707, independent of IL-6. Feeder-cell supernatants were as efficient as feeder-cell monolayers in stimulating growth and contained IL-6 as the only growth promoting activity. IL-6 was growth stimulatory and sustained the growth of U-1958 only when the medium contained fetal calf serum. The nature of the serum factor(s) is unknown, but it was excluded to be the IL-6 carrier protein a2-macroglobulin. IL-1, IL-2, IL-3, TNF-alpha, GM-CSF, IGF-1, and insulin were neither co-stimulatory with IL-6 nor stimulated growth on their own. Only U 266-1984 expressed IL-6 mRNA. IL-6 receptor mRNA was expressed in all lines except the L363 and Fravel. We conclude that the response to IL-6 is heterogeneous among the MM lines and that IL-6 acts as a paracrine growth factor for two of six lines. In a third line, U-266-1984, the IL-6 mRNA expression suggests the possibility of an autocrine growth stimulation. PMID- 1707471 TI - Isolation of retrovirus from patients with multiple sclerosis. PMID- 1707470 TI - Replacement of donor lymphoid tissue in small-bowel transplants. AB - The presence of recipient lymphocytes in grafts is thought to equate with rejection. Thus, we wished to follow the fate of lymphocytes after transplant of the small bowel. Three complete small-bowel transplants, two with the liver from the same donor also transplanted, were done successfully. Patients were immunosuppressed with FK 506. 5 to 11% of lymphocytes in the recipients' peripheral blood were of donor origin during the early postoperative period when there were no clinical signs of graft-versus-host disease. However, donor cells were no longer detectable after 12 to 54 days. Serial biopsy specimens of the grafted small bowel showed progressive replacement of lymphocytes in the lamina propria by those of the recipient's HLA phenotype. Lymphoid repopulation was complete after 10 to 12 weeks but the epithelial cells of the intestine remained those of the donor. The patients are on enteral alimentation after 5, 6, and 8 months with histopathologically normal or nearly normal intestines. Re examination of assumptions about the rejection of intestinal grafts and strategies for its prevention are required following these observations. PMID- 1707472 TI - Exploiting angiogenesis. PMID- 1707473 TI - Maternal serum alpha-fetoprotein in congenital hypothyroidism. PMID- 1707474 TI - HLA-B27 subtypes. PMID- 1707475 TI - Complementation of DNA repair-deficient Escherichia coli by the yeast Apn1 apurinic/apyrimidinic endonuclease gene. AB - The Saccharomyces cerevisiae APN1 gene encoding an AP endonuclease/3'-diesterase was engineered in vitro for expression in Escherichia coli. The expression vector directs the synthesis in E. coli of a Mr 40,500 protein that reacts with anti Apn1 antibodies and has the DNA-repair activities characteristic of Apn1 isolated from yeast. A band corresponding to Apn1 was observed in DNA repair activity gels only with extracts of E. coli harbouring the APN1 expression plasmid. Expression of Apn1 conferred resistance to oxidants and alkylating agents in E. coli lacking exonuclease III and endonuclease IV. For H2O2 damage, this rescue effect was correlated with the repair of oxidative lesions in the bacterial chromosome by the Apn1 protein. Thus, Apn1 can function in bacteria in a manner similar to its proposed multiple functions in yeast. PMID- 1707476 TI - Repression and derepression of conjugation of plasmid R1 by wild-type and mutated finP antisense RNA. AB - The finP gene of plasmid R1 is located between the genes traM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5' non-translated sequences, the ribosome-binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1-19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R1 19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1-19 in trans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II. PMID- 1707477 TI - Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA. AB - The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication. PMID- 1707478 TI - DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. PMID- 1707479 TI - High-resolution DNA measurements using the nuclear isolation medium, DAPI, with the RATCOM flow cytometer. PMID- 1707480 TI - Rapid DNA content analysis by the propidium iodide-hypotonic citrate method. PMID- 1707481 TI - Cell division analysis using bromodeoxyuridine-induced suppression of Hoechst 33258 fluorescence. PMID- 1707482 TI - Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258 ethidium bromide bivariate flow cytometry. PMID- 1707483 TI - Detection of bromodeoxyuridine-labeled cells by differential fluorescence analysis of DNA fluorochromes. PMID- 1707484 TI - Using monoclonal antibodies in bromodeoxyuridine-DNA analysis. PMID- 1707485 TI - Washless double staining of a nuclear antigen (Ki-67 or bromodeoxyuridine) and DNA in unfixed nuclei. PMID- 1707486 TI - Detection of intracellular virus and viral products. PMID- 1707487 TI - Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. PMID- 1707488 TI - Flow cytometric analysis of double-stranded RNA content distributions. PMID- 1707489 TI - Simultaneous fluorescent labeling of DNA, RNA, and protein. PMID- 1707490 TI - Flow cytometric cell-kinetic analysis by simultaneously staining nuclei with propidium iodide and fluorescein isothiocyanate. PMID- 1707491 TI - Flow cytometric methods for studying isolated nuclei: DNA accessibility to DNase I and protein-DNA content. AB - Two FCM methods utilizing isolated nuclei were described. A DNase I sensitivity assay, employing changes in binding and digestion kinetics of the enzyme as well as the binding of intercalating fluorochrome was used to observe structural changes of chromatin rendered by physical and chemical agents. A nuclear DNA protein staining method was used to study changes in nuclear protein content, redistribution of populations in the cell cycle, and unbalanced growth, manifested as an extraordinary accumulation of nuclear protein brought about by physical and chemical perturbation. PMID- 1707492 TI - Flow cytometric analysis of male germ cell quality. PMID- 1707493 TI - Cell sorting with Hoechst or carbocyanine dyes as perfusion probes in spheroids and tumors. PMID- 1707494 TI - Plant cell cycle analysis with isolated nuclei. PMID- 1707495 TI - Supravital cell staining with Hoechst 33342 and DiOC5(3). PMID- 1707496 TI - Specific staining of DNA with the fluorescent antibiotics, mithramycin, chromomycin, and olivomycin. PMID- 1707497 TI - Mechanisms of hyperbaric-oxygen inhibition of growth and net biosynthesis of RNA, DNA, protein and lipids in Escherichia coli. AB - The effects of hyperbaric oxygen at 4.2 atmospheres on growth, induction of stringency and net synthesis of RNA, DNA, protein and lipid by Escherichia coli were evaluated by measuring culture absorbance and incorporation of specific 14C labelled substrates. Combinations of different bacterial strains, various nutritional conditions and chloramphenicol were used to suppress or allow induction of stringency and to mitigate or enhance other consequences of reported sites of oxygen toxicity. In minimal medium with glucose as the sole organic nutrient, there was almost instantaneous inhibition of net biosynthesis of four major macromolecular constituents: lipid, RNA, DNA and protein. Net lipid biosynthesis primarily failed indirectly due to induction of stringency. Protein net synthesis declined due to induction of stringency and lack of amino acids. RNA net biosynthesis stopped from a direct effect and indirectly via induction of stringency. Net DNA biosynthesis was indirectly primarily impaired. PMID- 1707498 TI - [Intratumoral injection of OK-432 in conjunction with fibrinogen greatly enhances antitumor effect on colorectal carcinomas]. AB - We found that antitumor effect of OK-432, a lyophylized preparation of an attenuated strain of streptococcus pyogenes, on colorectal carcinoma was greatly augmented when it was injected intratumorally in conjunction with fibrinogen. Twenty cases of colorectal cancer received intratumoral injection of 5 KE of OK 432 mixed with 80mg of fibrinogen including factor-XIII and 1ml of aprotinin at the time of endoscopic examination. Changes in the shape of the tumors were observed endoscopically within a few days after injection, and in most cases, decrease in the height of tumor margin was noted. Histopathological findings on surgically resected specimens revealed that the fine meshwork of fibrin was formed at the injected site soon after the injection, and a marked infiltration of inflammatory cells including neutrophils, plasma cells, macrophages, eosinophils and lymphocytes. Such granulomatous changes developed over 7 days after injection, and the degradation of tumors were observed. By 14 days after the injection, tumor tissues were largely replaced with granulomas, and shrinkage of tissues were observed. These findings indicated that fibrinogen including factor-XIII and aprotinin has a potential ability to augment the immunoreaction induced by biological response modifiers, and intratumoral injection of OK-432 in conjunction with fibrinogen solution was superior to intratumoral injection of OK 432 alone as the local immunotherapy of colorectal cancer. PMID- 1707499 TI - [Modern variations of human influenza group A viruses at the molecular level]. AB - The authors own results on the variety of the genomic primary structures in human influenza A viruses participating in the epidemic process, including the atypical viruses. The comparative studies revealed new trends in the HA gene antigenic drift on the late stages and the PB1 gene shift. Modifications occurring in the primary structure of the influenza A viruses native genomes during laboratory treatment (adaptation to new hosts, vaccine preparation, egg passaging) have been analyzed. Sequencing of several types of "antigenic anachronisms" revealed the direct links between some of such viruses and the anthropogenic pollution of the biosphere by vaccine strains. Modifications in the HA genes of influenza A viruses during the persistent infection have also been studied. PMID- 1707500 TI - Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. AB - An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue. Using the previously assigned structural alerts to DNA reactivity (electrophilicity), the chemicals were split into 154 alerting chemicals and 147 non-alerting chemicals. The alerting chemicals were split into three chemical groups; aromatic amino/nitro-types, alkylating agents and miscellaneous structurally-alerting groups. The non-alerting chemicals were subjectively split into three broad categories; non-alerting, non-alerting containing a non-reactive halogen group, and non-alerting chemical with minor concerns about a possible structural alert. The tumor data for all 301 chemicals are re-presented according to these six chemical groupings. The most significant findings to emerge from comparisons among these six groups of chemicals were as follows: (a) Most of the rodent carcinogens, including most of the 2-species and/or multiple site carcinogens, were among the structurally alerting chemicals. (b) Most of the structurally alerting chemicals were mutagenic; 84% of the carcinogens and 66% of the non-carcinogens. 100% of the 33 aromatic amino/nitro-type 2-species carcinogens were mutagenic. Thus, for structurally alerting chemicals, the Salmonella assay showed high sensitivity and low specificity (0.84 and 0.33, respectively). (c) Among the 147 non-alerting chemicals less than 5% were mutagenic, whether they were carcinogens or non-carcinogens (sensitivity 0.04). PMID- 1707501 TI - Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. AB - Many hormones, neurotransmitters and growth factors, on binding to G protein coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits. PMID- 1707502 TI - Effects of neuropeptide-Y and substance-P on the secretory activity of dispersed zona-glomerulosa cells of rat adrenal gland. AB - Neuropeptide-Y (NPY) and substance-P (SP), two peptides contained in the chromaffin granules of adrenal medullary cells, were found to partially inhibit both basal ACTH-stimulated release of aldosterone and 18-hydroxy-corticosterone by isolated rat zone-glomerulosa cells, without affecting the overall post pregnenolone yield or basal progesterone output. Conversely, the exposure to both peptides increased 11-deoxy-corticosterone and corticosterone secretion. These data indicate that NPY and SP are able to exert a direct suppression of 18 hydroxylase activity in rat zona-glomerulosa cells, without conceivably altering the earlier steps of aldosterone synthesis. The possible physiological implications of these findings are discussed in light of previous studies suggesting a net adrenoglomerulotrophic effect of NPY and SP in vivo. PMID- 1707503 TI - Substance P and substance K in the median eminence and paraventricular nucleus of the rat hypothalamus. AB - We have used specific radioimmunoassays coupled with reversed-phase high performance liquid chromatography (HPLC) to measure and characterise substance P (SP) and substance K (SK) in subdivisions of the rat hypothalamus. SP and SK levels in the paraventricular nucleus (PVN) were 968 +/- 61 and 381 +/- 22 pg respectively; in the supraoptic nucleus (SON) were 210 +/- 21 and 79 +/- 8 pg; and in the median eminence/arcuate nucleus (ME) were 1044 +/- 66 and 451 +/- 20 pg. Reversed-phase HPLC revealed that immunoreactive (ir) SP was present solely in the non-oxidised form in all tissue extracts. The principal form of ir-SK in the PVN and SON coeluted with synthetic SK on HPLC, but some immunoreactivity eluted in a later position. This material represented less then 5% of the total ir-SK in extracts of the PVN and SON, but increased to 35-40% of the total in the ME. Gel chromatography and HPLC characterised this compound as being slightly smaller and more hydrophobic than SK. These results establish that ir-SK is present within the hypothalamus in varying amounts and molecular forms. The location of significant amounts of both SP and SK in the PVN and ME, the principal regions of CRF-41 synthesis and release, is compatible with a role for neurokinins in the modulation of CRF-41 and consequently ACTH release. PMID- 1707504 TI - Effects of experimental spinal cord transection on substance P receptors: a quantitative autoradiography study. AB - The autoradiographic localisation of Substance P (SP) receptors was investigated in the rat spinal cord following cordotomy at the thoracic 8-9 level. The binding of [125I]-BH-SP was measured in discrete gray matter structures above (C6-C7 level) and below (L2-L3 level) the injury site, and expressed in fmol/mg protein. There was a statistically significant increase in SP receptor density 1 week after cordotomy in the dorsal horn and in the central canal of lumbar region, in laminae 3-4-5 and also in the ventral horn of cervical spinal cord. Elevated SP receptor density was also noted in cervical ventral horn at 3 weeks after thoracic cordotomy, whereas the increase seen at 1 week was normalized at 3 weeks everywhere else. Since SP concentration has been reported to show a similar localized increase below a spinal transection, the present results are consistent with an up-regulation of SP receptors. A similar direct relationship between SP receptors and SP content appears to occur also in the ventral horn above the injury site. PMID- 1707505 TI - High-dose intravenous gamma globulin: does it have a role in the treatment of severe erythroblastosis fetalis? PMID- 1707506 TI - Properties of epitopes of Pfs 48/45, a target of transmission blocking monoclonal antibodies, on gametes of different isolates of Plasmodium falciparum. AB - We have studied the properties of epitopes on Plasmodium falciparum gamete surface protein Pfs 48/45, a target antigen of malaria transmission blocking antibodies. Using a two site immunoradiometric assay we have defined three spacially separate, non-repeated, epitope regions on the peptides representing this antigen. Epitope region I is a target of monoclonal antibodies (MoAbs) which strongly suppress infectivity of gametocytes of P. falciparum to mosquitoes; the effect is complement independent and is mediated as effectively by the monovalent Fab fragments as by intact MoAb. Epitope region II consists of two spacially close subregions, IIa and IIb; variant forms of epitopes IIa and IIb occurred in different isolates of P. falciparum. Epitope region III also showed slight structural modification between isolates. MoAbs against regions II or III were relatively ineffective in suppressing gametocyte infectivity compared to MoAbs against region I. However, certain combinations of MoAbs against regions II and III together acted synergistically to suppress infectivity to mosquitoes. All these epitopes failed to react with MoAb when the antigen was presented in reduced form. A fourth epitope, however, was identified which reacted strongly with MoAb when the antigen was presented in reduced form. The MoAb against this epitope had no effect on the infectivity of gametocytes of P. falciparum to mosquitoes. PMID- 1707507 TI - Clonal repertoire analysis of murine B cells specific for repeat sequence antigens of Plasmodium falciparum. AB - Clonal analysis of the murine B-cell repertoire has been used to investigate the possible role of tandem repeat sequence epitopes of Plasmodium falciparum in immune evasion. A limiting dilution culture system was used whereby murine spleen cells were stimulated with the B-cell mitogen lipopolysaccharide (LPS) in the presence of 3T3 fibroblast filler cells. One in three B cells were shown to produce clones secreting immunoglobulin measurable by an ELISA. The frequency of antibody forming cell precursors (AFCp) specific for the 3' repeat epitopes of the ring injected erythrocyte surface antigen (RESA) was estimated in non-primed mice and found to be low. However, an accurate frequency determination was not possible using this method since the detection of the few positive cultures was found to depend on the presence of more than one AFCp or its products. Limiting dilution analysis was used to assess the frequency and repertoire of splenic AFCp at various times after immunization with a synthetic peptide of the RESA 3' repeat epitope (8 x 4-mer), presented in various ways. There was no marked increase in LPS-responsive AFCp specific for this antigen at the level of either IgM or IgG secretion. This was in marked contrast to the antibody response in vivo, where moderate IgG antibody titres, normally indicative of a secondary response, were seen in the serum of the same mice used for AFCp assay. This discrepancy between serum titre and AFCp frequency following immunization was not apparent with a non-malarial antigen, keyhole limpet haemocyanin (KLH). It was concluded that the LPS-stimulated limiting dilution culture system was not registering RESA-specific memory AFCp. These results raise the possibility that the malarial antigens are deficient in memory B-cell generation, or that secondary responses to these determinants may arise from a distinct B-cell progenitor which is non-responsive to LPS in vitro. PMID- 1707508 TI - Antigenic variation in Giardia lamblia: cellular and humoral immune response in a mouse model. AB - Neonatal mice (CR:NIH:S) were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7) and the surface antigens of the intestinal trophozoites, as well as the cellular and humoral immune responses, were analysed during the course of infection. Infections in mice peaked 2-3 weeks after inoculation and were self-cured by day 42 post-infection (p.i.). The proportion of trophozoites expressing the Mr 72,000 surface antigen of the initial inoculum had decreased by day 12 and approached zero by day 22 p.i., similar to infections in humans. The predominant parasite-specific humoral response was an IgM- and IgG-isotype directed to the original Mr 72,000 surface antigen as well as other antigens. T lymphocytes (predominantly LY4(CD4)+) isolated from Peyer's patches 12 days p.i. and later showed a significant proliferative response to Giardia lamblia antigens. Spleen and lymph node cells showed no lymphoproliferative response. T cell blot analysis revealed the presence of dominant T-cell epitopes in the areas of Mr 200,000-75,000 and less than 50,000 polypeptides. No response was demonstrated in the Mr 72,000 region (migration site of the major surface antigen), suggesting T-cell dependent mechanisms are most likely not responsible for the surface antigen switch which occurred during the course of infection. This model infection can be used to study the role of immunological mechanisms in Giardia lamblia variant antigen switching and in the control of infections. PMID- 1707509 TI - Trichuris muris: antigen recognition and transfer of immunity in mice by IgA monoclonal antibodies. AB - Mesenteric node lymphocytes from mice that had been infected with the nematode Trichuris muris, and then boosted with adult worm excretory-secretory antigens were fused with myeloma cells to produce a panel of 9 monoclonal antibodies (MoAbs). Five of the MoAbs were of the IgA isotype. The antigen recognition profiles of these MoAbs were studied using SDS-PAGE and immunoblotting; three major profile patterns were identified. Five MoAbs recognized a major band in the MW range 43-48 kD; all recognized a range of antigens. Three MoAbs were used to localize antigens in the bodies of adult worms. Granules within the anterior stichocytes were recognized strongly, as was material within the eggs and pseudocoelom. Two MoAbs stained the cuticle. Although the phosphorylcholine (PC) determinant was widely distributed within worm tissues none of the MoAbs tested recognized PC. Passive transfer of immunity was achieved using two of the IgA monoclonals; no immunity was transferred by the IgM and IgG MoAbs used. The limited recognition profiles of these IgA MoAbs, and the ability to stain stichocyte granules, suggest that their protective activity results from an interaction with ES antigens. PMID- 1707510 TI - Fasciola hepatica: immunoprecipitation analysis of biosynthetically labelled antigens using sera from infected sheep. AB - The antigenicity of the biosynthetically labelled somatic and excretory/secretory proteins of adult Fasciola hepatica was investigated over 20 weeks of an infection with F. hepatica in sheep. The antibody response was initially detected by ELISA 2 weeks after infection, and was sustained at this level for the remainder of the infection. Immunoprecipitation analysis indicated that a large number of proteins were recognized by the sheep, with several dominant antigens occurring in the 29-31 kilodalton molecular weight range. No differences were found between the antigens recognized by sheep in the early or late stages of infection suggesting that there are similarities between the antigens of the immature and mature forms of the parasite. PMID- 1707511 TI - Antigenic components of Paragonimus heterotremus recognized by infected human serum. AB - The antigenic components of a Paragonimus heterotremus saline extract were revealed by SDS-PAGE and Western blot analysis using sera from 32 patients with P. heterotremus infection, 60 with other helminthiasis, and 15 normal human sera. It was found that the worm extract was comprised of more than 13 polypeptides, among which 5 components were strongly recognized by paragonimiasis sera. These components had approximately showed molecular weight of less than 12.3, 12.3, 18.5, 31,.5 and 38 kD. Only the 31.5 kD component was recognized by all 32 paragonimiasis sera. Sera from other helminth infections and from uninfected did not produce detectable bands with the worm extract. The present findings suggested that the 31.5 kD component may be sensitive and specific for the diagnosis of human paragonimiasis heterotremus. PMID- 1707512 TI - Evolving concepts of infection by Pneumocystis carinii. PMID- 1707513 TI - Effect of pseudomonas elastase on human mononuclear phagocyte alpha 1-antitrypsin expression. AB - The net balance of neutrophil elastase and its inhibitor, alpha 1-antitrypsin (alpha 1-AT), is a critical determinant of connective tissue turnover during homeostasis and in disease states. In addition to liver-derived alpha 1-AT, which translocates from blood to tissues, this elastase-alpha 1-AT balance is maintained by expression of alpha 1-AT at the local tissue level in resident mononuclear phagocytes. Our previous studies have shown that this elastase-alpha 1-AT balance is also tightly controlled at a cellular level in that addition of exogenous neutrophil elastase (serpine-type elastase) to cultured mononuclear phagocytes is associated with an increase in expression of the alpha 1-AT gene. Subsequent studies have demonstrated that this novel regulatory loop involves interaction between exogenous neutrophil elastase and endogenous alpha 1-AT inducing a structural rearrangement in the alpha 1-AT molecule and exposing highly conserved conformation-specific domain of alpha 1-AT, which can then be recognized by a specific cell surface receptor, the serpine-enzyme complex receptor. In the following study, we examined the effect of a bacterial metalloelastase, Pseudomonas aeruginosa elastase, on expression of alpha 1-AT in human mononuclear phagocytes. We show that pseudomonas elastase inactivates monocyte-derived alpha 1-AT by limited proteolysis but, in so doing, alpha 1-AT becomes recognized by the serpine-enzyme complex receptor and mediates an increase in de novo synthesis of alpha 1-AT in these cells. However, the concentrations of pseudomonas elastase needed to proteolytically inactivate alpha 1-AT in monocyte culture fluid are higher than those required for inactivation of purified plasma alpha 1-AT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707514 TI - Spontaneous and agonist-induced openings of an acetylcholine receptor channel composed of bovine muscle alpha-, beta- and delta-subunits. AB - During the development of mammalian muscle the gamma-subunit of the nicotinic acetylcholine receptor (AChR) is replaced by the epsilon-subunit to produce well defined alterations in the conductance and gating of the channel. To gain a better understanding of the functional role of the gamma- and epsilon-subunits, we have studied the properties of an AChR channel lacking these subunits. The AChR expressed in Xenopus oocytes injected with the bovine alpha-, beta- and delta-subunit-specific mRNAs (referred to as alpha beta delta-AChR) is unusual in that its channel opens spontaneously at a high frequency in the absence of agonist. From a comparison of the alpha beta delta-AChR with complete receptors containing either the gamma- or epsilon-subunit, we conclude that the gamma- and epsilon-subunits influence most channel properties, including agonist binding, and are especially important for stabilizing the closed state of the unliganded receptor channel. The alpha beta delta-AChR can form when a complete set of four subunit-specific mRNAs is injected. The ease with which it is assembled raises the possibility that the alpha beta delta-AChR contributes to some of the variations in receptor properties that occur during development. PMID- 1707515 TI - Effects of arachidonic acid on the gap junctions of neonatal rat heart cells. AB - Myocytes were isolated from neonatal rat hearts and grown in tissue-culture dishes for 1-2 days. Spontaneously formed cell pairs were used to study the conductance of gap junctions. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Exposure to arachidonic acid (AA) produced a quasi dose-dependent decrease in junctional conductance, gi (binding constant, Kd = 4 microM; Hill coefficient, n = 0.75). AA-dependent uncoupling was reversible. Addition of 1 mg/ml albumin to the bath solution accelerated the recovery. During control, cell pairs exhibited a gradual decrease in gi (16.4% in 6 min). Exposure to 20 microM 4-bromophenacyl bromide, a phospholipase inhibitor, suppressed the decay in gi (1.8% in 6 min), suggesting that endogenous AA may be involved in spontaneous uncoupling. The effect of AA on gi was specific. Arachidic acid (100 microM) and arachidonamide (10 microM), structural analogues of AA, had no effect on gi. Currents recorded shortly before complete uncoupling caused by AA, or early during recovery from uncoupling, revealed random opening and closing of single channels. The single channel conductance, gamma i, was not affected by the concentration of AA (1 microM - 100 microM). The mean gamma i turned out to be 33.5 pS. The results suggest that AA-dependent uncoupling was caused via decrease in open channel probability, presumably mediated by a direct action on channel proteins. PMID- 1707516 TI - The effects of quinidine on sodium-dependent calcium efflux in isolated rod photoreceptors of the salamander retina. AB - The effect of quinidine on the membrane current generated by the Na:Ca, K exchange has been investigated in the outer segment of isolated rod photoreceptors from the retina of the larval tiger salamander. The inward exchange current associated with the efflux of Ca2+ was selectively recorded by introducing a Ca2+ load through the light-sensitive channels, and then shutting these channels with a bright light. Quinidine (20-1000 microM) reduced the magnitude of the exchange current and slowed its decay during the removal of a Ca2+ load. Quinidine did not alter the form of the relation between the exchange current and the total concentration of exchangeable calcium remaining within the outer segment. [Ca]T, showing that it does not change the affinity of the exchange mechanism for internal Ca2+. The relation between exchange current inhibition and the quinidine concentration could be described by a simple Michaelis relation with a Ki of 287 microM and a maximum inhibition of 50%. The incomplete block of the Na:Ca, K exchange current by quinidine shows that it does not act by simple competition with external Na+, and suggests that the inhibition of the exchange by quinidine may be non-specific. PMID- 1707517 TI - Possible involvement of GTP-binding proteins in the deactivation of an inwardly rectifying K+ current in enterocytes isolated from guinea-pig small intestine. AB - The possible regulation of guinea pig enterocyte ion channels by GTP-binding proteins has been investigated by using the whole-cell recording mode of the patch-clamp technique and non-hydrolysable analogues of GTP. The main K+ currents in these cells are mediated by inwardly-rectifying K+ channels. Intracellular dialysis with hydrolysis-resistant GTP analogues leads to the deactivation of the inward K+ currents. Non-hydrolysable analogues of ATP or GDP were without effect. The effect occurs after a lag phase of 2 to 7 min, suggesting a multistep mechanism. Cl currents were not affected by any of the nucleotide analogues. It is suggested that inwardly-rectifying K+ currents are deactivated by a G-protein dependent process. PMID- 1707518 TI - Atrial natriuretic factor increases cyclic GMP and cyclic AMP levels in a directly photosensitive pineal organ. AB - Atrial natriuretic factor (ANF) stimulates accumulation of cyclic GMP in a photosensitive organ, as evidenced for the first time in cultured trout pineals. Stimulation was rapid (within a few min), dose-dependent, and stronger in organs cultured in darkness than in those cultured under light. After 30 min in the dark, (i) cyclic AMP levels were slightly increased at 10(-7) mole/l of ANF, (ii) cyclic GMP and cyclic AMP increased dramatically after inhibition of the phosphodiesterases by isobutylmethylxanthine (IBMX), (iii) ANF and IBMX effects were more than additive on cyclic GMP, (iv) pertussis toxin decreased the cyclic GMP response to ANF. These responses were affected by light. The possibility that cyclic GMP might be a second messenger of both light and chemical (ANF) inputs, in pineal photoreceptor cells, is hypothetized. PMID- 1707519 TI - SRP-RNA sequence alignment and secondary structure. AB - The secondary structures of the RNAs from the signal recognition particle, termed SRP-RNA, were derived buy comparative analyses of an alignment of 39 sequences. The models are minimal in that only base pairs are included for which there is comparative evidence. The structures represent refinements of earlier versions and include a new short helix. PMID- 1707521 TI - A novel 40S multi-snRNP complex isolated from rat liver nuclei. AB - Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx. 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions. MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e. the 32-45KD core proteins and polypeptides of 60-80 and 110 130KD). MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD. Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit. The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component. Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi snRNP entity. PMID- 1707520 TI - Retrotransposon-like nature of Tp1 elements: implications for the organisation of highly repetitive, hypermethylated DNA in the genome of Physarum polycephalum. AB - The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by the Tp1 family of highly repetitive retrotransposon like sequences. Tp1 elements consist of two terminal direct repeats of 277bp which flank an internal domain of 8.3kb. They are the major sequence component in the hypermethylated (M+) fraction of the genome where they have been found exclusively in scrambled clusters of up to 50kb long. Scrambling is thought to have arisen by insertion of Tp1 into further copies of the same sequence. In the present study, sequence analysis of cloned Tp1 elements has revealed striking homologies of the predicted amino acid sequence to several highly conserved domains characteristic of retrotransposons. The relative order of the predicted coding regions indicates that Tp1 elements are more closely related to copia and Ty than to retroviruses. Self-integration and methylation of Tp1 elements may function to limit transposition frequency. Such mechanisms provide a possible explanation for the origin and organisation of M + DNA in the Physarum genome. PMID- 1707522 TI - The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. AB - Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification. PMID- 1707523 TI - A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences. AB - We describe a new computer program that identifies conserved secondary structures in aligned nucleotide sequences of related single-stranded RNAs. The program employs a series of hash tables to identify and sort common base paired helices that are located in identical positions in more than one sequence. The program gives information on the total number of base paired helices that are conserved between related sequences and provides detailed information about common helices that have a minimum of one or more compensating base changes. The program is useful in the analysis of large biological sequences. We have used it to examine the number and type of complementary segments (potential base paired helices) that can be found in common among related random sequences similar in base composition to 16S rRNA from Escherichia coli. Two types of random sequences were analyzed. One set consisted of sequences that were independent but they had the same mononucleotide composition as the 16S rRNA. The second set contained sequences that were 80% similar to one another. Different results were obtained in the analysis of these two types of random sequences. When 5 sequences that were 80% similar to one another were analyzed, significant numbers of potential helices with two or more independent base changes were observed. When 5 independent sequences were analyzed, no potential helices were found in common. The results of the analyses with random sequences were compared with the number and type of helices found in the phylogenetic model of the secondary structure of 16S ribosomal RNA. Many more helices are conserved among the ribosomal sequences than are found in common among similar random sequences. In addition, conserved helices in the 16S rRNAs are, on the average, longer than the complementary segments that are found in comparable random sequences. The significance of these results and their application in the analysis of long non-ribosomal nucleotide sequences is discussed. PMID- 1707524 TI - Modulatory effects of snuff, retinoic acid, and beta-carotene on DMBA-induced hamster cheek pouch carcinogenesis in relation to keratin expression. AB - The hamster cheek pouch (HCP) serves as an excellent model system not only for the studies on initiation and promotion but also for the modulation of experimental oral carcinogenesis. In our studies, HCPs treated with 7,12 dimethylbenz[a]anthracene (DMBA) showed both cheek pouch and stomach papillomas. Utilizing this model system, we tested and compared the modulatory effects of snuff, retinoic acid, and beta-carotene on the incidence of tumors and the keratin expression pattern. HCPs treated with snuff, either alone or in combination with DMBA, resulted in stomach papillomas. HCPs treated with snuff showed no cheek pouch tumors, and those treated with snuff and DMBA showed only 10-15% tumor incidence. Both beta-carotene and retinoic acid showed a total inhibition of DMBA-induced carcinogenesis in the HCP as well as in the stomach. The keratin expression pattern showed alterations depending on the experimental conditions. PMID- 1707525 TI - Automated estimation of epithelial volume in breast cancer sections. A comparison with the image processing steps applied to gynecologic tumors. AB - The paper describes an image analysis technique for automated estimation of the epithelial percentage in standard paraffin tissue sections of invasive ductal breast cancers. Two staining procedures are evaluated: Feulgen (pararosanilin) and CAM 5.2-demonstrating the presence of cytokeratin 8 and 18-, both counterstained with naphthol yellow. In the technique, one image is recorded with a filter to visualize where the epithelium lies. This filter is chosen corresponding to the type of staining: it is yellow for Feulgen and blue for anti cytokeratin CAM 5.2. To visualize where the stroma lies, the same image can be used for anti-cytokeratin CAM 5.2, whereas for Feulgen, a second image has to be recorded from the same microscope field with a blue filter. The image processing steps to determine the total tissue area comprise correction for shading, segmentation of the tissue area, and restoration of the segmented image by removal of small artefacts and closure of small tears in the tissue. The method for determination of the epithelial area consists of the following steps: correction for shading, gaussian blurring, segmentation of nuclei or epithelial cells, and editing of the segmented image by removal of small objects and closure of small spaces between the epithelial nuclei or cells. These image processing steps are compared to those for quantification of the epithelial percentage in gynecologic tumors of epithelial origin. For the Feulgen stain, the method is evaluated on 30 breast cancers of the ductal type (4 grade I, 12 grade II, and 14 grade III).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707526 TI - Juvenile xanthogranuloma of the paravertebral soft tissue in infancy: a report of two cases. AB - We report 2 cases of paravertebral soft tissue lesions with the histologic features of juvenile xanthogranuloma, both of which occurred in infants. Juvenile xanthogranuloma situated in the soft tissue is rare. We describe 2 cases with similar clinical and pathologic features; there has been no recurrence at 1 and 2 years after excision, respectively. PMID- 1707527 TI - Infective arthritis secondary to bladder outflow obstruction. AB - We describe two cases of septic arthritis occurring in association with lower urinary tract infection in elderly men. In both cases the organism isolated from both the joint and the urine was Staphylococcus aureus. Further investigation of the urinary tract in both individuals identified bladder outflow obstruction secondary to benign prostatic hyperplasia predisposing them to infection. The urinary tract should be suspected as a focus of infection in septic arthritis in elderly men and further investigation of the urinary system may disclose a surgically correctable lesion. PMID- 1707528 TI - Irreversible renal failure in men with outflow obstruction: is it a preventable disease? PMID- 1707529 TI - [Complex evaluation of the defense systems in patients with infiltrative pulmonary tuberculosis]. AB - Patients with infiltrative pulmonary tuberculosis at its degenerative stage were examined for parameters of hemostasis, serum proteolytic inhibitors, immunity status and complement. In doing so, the following changes were detected: simultaneous intensification of plasma coagulability and fibrinolytic properties, sputum-induced increase in fibrinolysis; disproportion in the content of the main protease blockers (higher alpha 1-protease inhibitor and lower alpha 2 macroglobulin); T-lymphocyte deficiency; stimulation of the immunity humoral link; rise in the count of O-lymphocytes and higher concentration of the complement component C3. The correlation between the indices of the examined systems was observed, thus indicating the interdependence of the body defensive factors. The findings can be used in the clinical practice when pathogenetic drugs are chosen. PMID- 1707530 TI - Many peptide fragments of alien antigens are homologous with host proteins, thus canalizing T-cell responses. AB - All proteins of this world are constructed in compliance with the same rule. Accordingly, two totally unrelated proteins, on the average, share 30 identical tripeptides, two tetrapeptides, and one pentapeptide per 500 residues. With this in mind, the 221-residue-long influenza virus hemagglutinin II (IVHA-II), as a representative of alien antigens, was compared with three diverse proteins representing the host: 533-residue-long chicken c-src protein kinase (c-src product of the cellular oncogene of Rous sarcoma virus), 595-residue-long human estrogen receptor, and 585-residue-long human serum albumin. Forty-three tripeptides, two tetrapeptides, and one pentapeptide of IVHA-II were also found in one or the other of the three host proteins. Six regions of IVHA-II (9-22 residues long) in which oligopeptides were clustered that were identical to their host oligopeptides were defined as "host-homologous" regions, and the remaining regions were called "nonself" or "pathogen-specific" regions. Because the total number of host proteins is vastly more than three, host-homologous regions were no doubt underestimated, while only one or two regions of IVHA-II must remain as truly pathogen-specific. Nevertheless, oligopeptide analysis of two known T-cell response-eliciting peptide fragments and one known inert peptide fragment of a virus and a malarial protozoan readily revealed the latter to be a host homologous region. Of the two known T-cell response-eliciting peptide fragments, one was more nonself than the other. Not surprisingly, the more nonself fragment elicited helper T-cell response from individuals of diverse major histocompatibility complex haplotypes, whereas the less nonself fragment elicited cytotoxic T-cell response only from HLA-A2 human individuals. PMID- 1707531 TI - Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. AB - Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells resident in the pancreatic islets. We recently discovered that the pathogenesis of diabetes in NOD strain mice was associated with T-cell reactivity to an antigen cross-reactive with a mycobacterial 65-kDa heat shock protein. To identify peptide epitopes critical to the insulin dependent diabetes mellitus of NOD mice, we studied the specificities of helper T cell clones capable of causing hyperglycemia and diabetes. We now report the identification of a functionally important peptide within the sequence of the human variant of the 65-kDa heat shock protein molecule. T-cell clones recognizing this peptide mediate insulitis and hyperglycemia. Alternatively, the T cells can be attenuated and used as therapeutic T-cell vaccines to abort the diabetogenic process. Moreover, administration of the peptide itself to NOD mice can also down-regulate immunity to the 65-kDa heat shock protein and prevent the development of diabetes. Thus, T-cell vaccination and specific peptide therapy are feasible in spontaneous autoimmune diabetes. PMID- 1707532 TI - Detection of human immunodeficiency virus type 1 clinical isolates with reduced sensitivity to zidovudine and dideoxyinosine by RNA.RNA hybridization. AB - A quantitative rapid assay to detect resistant clinical human immunodeficiency virus type 1 (HIV-1) strains remains an important medical goal. A system incorporating a quantitative RNA.RNA hybridization assay that measures the amount of intracellular HIV-1-specific RNA has been employed to detect the level of inhibition by nucleoside analogues in sensitive and resistant HIV-1 strains. The RNA.RNA hybridization assay readily distinguished previously published zidovudine (ZDV; 3'-azido-3'-deoxythymidine)-resistant isolates from ZDV-sensitive isolates of HIV-1. The 50% inhibitory concentration (IC50) of ZDV for HTLV-IIIB and sensitive clinical HIV-1 isolates is between 0.01 and 0.04 microM. HIV-1 strains from three patients on long-term ZDV therapy displayed a greater than 20-fold increase in the ZDV IC50 compared to sensitive strains. The drug sensitivity system was confirmed by showing that mutations in the HIV reverse transcriptase gene from a ZDV-resistant isolate resulted in four amino acid changes (Leu-125--- Trp, Ile-142----Val, Thr-215----Tyr, and Pro-294----Thr) including one change (Thr-215----Tyr) that has been previously reported to be associated with resistance. One clinical HIV strain with high-level ZDV resistance displayed a 5 fold increase in 2',3'-dideoxyinosine IC50 compared to that of HTLV-IIIB. A drug sensitivity assay employing RNA.RNA hybridization may be useful for extensive screening of HIV isolates from patients enrolled in clinical trials and permit the correlation of in vitro resistance with clinical outcome. PMID- 1707533 TI - Localization of the estrogen-binding site of alpha-fetoprotein in the chimeric human-rat proteins. AB - Rat alpha-fetoprotein (AFP) has been demonstrated to bind estrogen, whereas human AFP lacks the activity. We constructed four chimeric molecules from cDNAs encoding these AFPs with the use of two restriction sites common to them and expressed them as well as rat and human AFP cDNA in yeast. The recombinant molecules were purified, characterized, and found to have the predicted structures. Analyses of estrogen binding indicated that a rat AFP sequence composed of residues 423-506 that contains 31 rat-specific amino acids is essential for the activity. PMID- 1707534 TI - Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by anti-idiotype antibodies. AB - The CD36 molecule expressed by human endothelial cells is a receptor for the adhesion of erythrocytes infected with the human malaria parasite Plasmodium falciparum. A CD36-specific monoclonal antibody, OKM8, inhibits the adhesion of malaria-infected erythrocytes (IRBC) to purified CD36 and cells expressing CD36. Monospecific polyclonal anti-idiotype (anti-Id) antibodies, raised against monoclonal antibody OKM8, expressed determinants molecularly mimicking the CD36 binding domain for the adhesion of IRBC. Purified rabbit anti-Id antibodies reacted with the surface of IRBC by immunofluorescence, directly supported the adhesion of wild-type P. falciparum malaria isolates, and inhibited IRBC cytoadherence to melanoma cells. An approximately 270-kDa protein was immunoprecipitated by the anti-Id antibodies from surface-labeled and metabolically labeled IRBC and was competitively inhibited by soluble CD36. These results support the hypothesis that CD36 is a receptor and the approximately 270 kDa protein, sequestrin, is a complementary ligand involved in the adhesion of IRBC to host-cell endothelium. Sequestrin is a candidate malaria vaccine antigen, and anti-Id antibodies that recognize this molecule may be useful for passive immunotherapy of cerebral and severe P. falciparum malaria. PMID- 1707535 TI - Cytokine regulation of substance P expression in sympathetic neurons. AB - The nervous and immune systems interact in a bidirectional fashion. For example, the neuropeptide substance P (SP) has been implicated in a variety of immune responses. Conversely, cytokines, a class of immunoregulatory glycoproteins, affect the synthesis of neurotransmitters and neurotrophic factors. This paper examines the role of cytokines in regulating neuropeptide expression in sympathetic neurons. Exposure of cultured explants of the rat superior cervical ganglion to the cytokine interleukin 1 beta (IL-1 beta) increased levels of SP. IL-1 beta increased neuronal SP expression in dissociated cultures of ganglion neuronal and nonneuronal cells but had no effect on peptide content in pure neuronal cultures. By contrast, treatment with a differentiation-promoting protein, leukemia inhibitory factor, increased SP in both pure neuronal and mixed cultures, indicating a different mechanism of action for the two molecules. The specificity of the IL-1 beta effect was further demonstrated by the lack of response to treatment with other cytokines, including interleukin 2, interleukin 6, and tumor necrosis factor alpha. The cell type necessary for the IL-1 beta activity is probably the ganglion Schwann cell. Treatment with a synthetic immunosuppressant glucocorticoid, dexamethasone, blocked the increase in SP after treatment with IL-1 beta. These observations support the hypothesis that neuropeptide expression is regulated, in part, by interactions with specific immunoregulators. In addition, the data suggest a role for SP in mediating the response of the superior cervical ganglion to injury of the ganglion itself or to the fibers innervating it. PMID- 1707536 TI - Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis. AB - The rapid synthesis and breakdown of mRNA in prokaryotes can impose a significant energy drain on these cells. Previous in vivo studies [Duffy, J. J., Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 565-579; Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 581-591] indicated that while RNA turnover in Escherichia coli was hydrolytic, it was nonhydrolytic in Bacillus subtilis. Here we provide an explanation for these observations based on enzymatic analysis of extracts of these two organisms. RNA degradation to the mononucleotide level in E. coli extracts is due solely to two active ribonucleases, RNase II and polynucleotide phosphorylase, which act hydrolytically and phosphorolytically, respectively. RNase II activity represents close to 90% of the total activity of the extract, as expected for predominantly hydrolytic degradation in this organism. In contrast, RNase II is absent from B. subtilis extracts, and the primary mode of RNA degradation is phosphorolytic, employing the Bacillus equivalent of polynucleotide phosphorylase and releases nucleoside diphosphates as products. A low level of a Mn2(+)-stimulated, hydrolytic ribonuclease is also detectable in B. subtilis extracts. Overall, E. coli and B. subtilis extracts differ by about 20- to 100-fold, depending on the substrate, in their relative use of hydrolytic and phosphorolytic routes of RNA degradation. The relation of the mode of mRNA degradation to the environment of the cell is discussed. PMID- 1707537 TI - Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme. AB - An in vitro model was used to investigate the potential for different structural forms of endogenous antigen to be processed and presented by major histocompatibility complex class II molecules. For this purpose the class II restricted presentation of an immunodominant epitope of hen egg lysozyme [HEL-(46 61)] was studied in class II-positive B-lymphoma cells (M12.C3) transfected with genes encoding HEL molecules either (i) secreted in high (hi) or low (lo) amounts as soluble antigen [sHEL(hi/lo)], (ii) localized within the endoplasmic reticulum (ER)/salvage compartment (ER-HEL), or (iii) anchored on the cell surface as an integral membrane protein (mHEL). The corresponding sHEL, ER-HEL, and mHEL gene products were expressed as predicted except that HEL determinants accumulated in the culture supernatant as well as on the cell membrane of mHEL-transfected cells. Class II-positive cells endogenously expressing all three forms of HEL antigen constitutively presented the immunodominant HEL-(46-61) determinant with differential efficiency (mHEL, sHEL greater than ERHEL) to a class II-restricted T hybridoma. A second T hybridoma recognized endogenous HEL-(46-61) determinants constitutively presented on sHEL(hi) and mHEL transfectants but not on sHEL(lo) or ERHEL transfectants. The formation of HEL-(46-61)/I-Ak complexes in the ERHEL and sHEL(lo) transfectants was therefore limiting. Mixing experiments with different antigen-presenting cells indicated that the HEL-(46-61) determinant was derived from endogenous antigen rather than by reuptake of shed or secreted HEL determinants. We conclude that MHC class II molecules can present some antigenic determinants derived from endogenous proteins that are sequestered in the ER/salvage compartment as well as distally transported in the form of secretory or membrane antigens. PMID- 1707538 TI - Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. AB - Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368 390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria. PMID- 1707539 TI - Acidic and basic fibroblast growth factors are survival factors with distinctive activity in quiescent BALB/c 3T3 murine fibroblasts. AB - Platelet-derived growth factor (PDGF), epidermal growth factor, and insulin-like growth factor have previously been identified as survival factors with distinctive activities for the density-inhibited quiescent BALB/c 3T3 murine fibroblasts. Fibroblast growth factor (FGF), like PDGF, renders quiescent BALB/c 3T3 cells competent to respond to epidermal growth factor and insulin-like growth factor, which mediate cell-cycle traverse through G1 into S phase [Stiles, C. D., Pledger, W. J., VanWyk, J. J., Antoniades, H. N. & Scher, C. D. (1979) Proc. Natl. Acad. Sci. USA 76, 1279-1283]. We now show that FGF possess marked cell survival-enhancing activity distinctive from that of PDGF. Both acidic FGF (aFGF) and basic FGF (bFGF) markedly enhance short-term (3-hr) survival of quiescent cells. bFGF is the more active of the two factors and shows marked long-term (20 hr) survival-promoting activity alone, whereas aFGF requires heparin for long term activity. Protection by bFGF or aFGF plus heparin is not associated with cell-cycle traverse into S phase. Both the short-term (3-hr) and long-term (20 hr) protective actions of aFGF and bFGF critically depend on protein synthesis, whereas those of PDGF do not. The accumulated evidence shows that several growth factors can contribute to maintenance of the integrity of quiescent murine fibroblasts and that their action can involve protein kinase A- and C-mediated processes as well as protein synthesis. Different growth factors display distinctive modes of action. PMID- 1707540 TI - Human response against NP-4, a mouse antibody to carcinoembryonic antigen: human anti-idiotype antibodies mimic an epitope on the tumor antigen. AB - Anti-idiotype antibodies (Ab2) were purified from a cancer patient treated with NP-4, a murine monoclonal antibody to carcinoembryonic antigen (CEA). These Ab2 were specific for NP-4 and inhibited the binding between NP-4 and CEA. BALB/c mice immunized with these human Ab2 produced anti-Ab2 antibodies that were also reactive with the CEA epitope recognized by NP-4. These results indicate that human Ab2 to NP-4 can antigenically mimic the CEA epitope recognized by NP-4. PMID- 1707541 TI - Tyrosine phosphorylation of components of the B-cell antigen receptors following receptor crosslinking. AB - Crosslinking membrane immunoglobulin (mIg), the B-cell antigen receptor, stimulates tyrosine phosphorylation of a number of proteins. Since many receptors are phosphorylated after ligand binding, we asked if components of the mIg receptor complexes were tyrosine-phosphorylated after mIg crosslinking. Both mIgM and mIgD are noncovalently associated with at least two other proteins. mIgM is associated with the MB-1 protein, which is disulfide-linked to a protein designated Ig-beta. mIgD is not associated with MB-1 but is with IgD-alpha, which is also disulfide-linked to Ig-beta. Using immunoprecipitation with a specific anti-MB-1 antiserum followed by anti-phosphotyrosine immunoblotting, we found that crosslinking mIgM stimulated tyrosine phosphorylation of MB-1, Ig-beta, and a previously unidentified 54-kDa polypeptide associated with MB-1. In mature splenic B cells that express both mIgM and mIgD, mIgM crosslinking stimulated tyrosine phosphorylation of the 32-kDa MB-1 protein, whereas mIgD crosslinking stimulated tyrosine phosphorylation of MB-1-related proteins of 33 and 34 kDa. The 32-kDa MB-1 protein was only associated with mIgM, whereas the 33- and 34-kDa MB-1-related proteins were specifically associated with mIgD and are most likely IgD-alpha. Thus, crosslinking either mIgM or mIgD stimulated tyrosine phosphorylation only of the MB-1-related proteins associated with that receptor. PMID- 1707542 TI - Three-dimensional structure of human basic fibroblast growth factor. AB - The three-dimensional structure of human basic fibroblast growth factor (bFGF) has been determined by x-ray crystallography and refined to a crystallographic residual of 17.4% at 2.2-A resolution. The structure was initially solved at a nominal resolution of 2.8 A by multiple isomorphous replacement using three heavy atom derivatives. Although the map clearly showed the overall fold of the molecule, electron density was not observed for the first 19 amino-terminal and the last 3 carboxyl-terminal amino acids, suggesting that they are disordered. The bFGF crystals were grown from 2.0 M ammonium sulfate at pH 8.1 in space group P1 with cell dimensions a = 30.9 A, b = 33.4 A, c = 35.9 A, alpha = 59.5 degrees, beta = 72.0 degrees, and gamma = 75.6 degrees. There is one molecule per unit cell and the crystals diffract to spacings beyond 1.9 A. The overall structure of bFGF can be described as a trigonal pyramid with a fold very similar to that reported for interleukin 1 beta, interleukin 1 alpha, and soybean trypsin inhibitor. An apparent sulfate ion is bound within a basic region on the surface of the molecule and has a ligands the main-chain amide of Arg-120 and the side chains of Asn-27, Arg-120, and Lys-125. This is suggested as the presumed binding site for heparin. Residues 106-115, which are presumed to bind to the bFGF receptor [Baird, A., Schubert, D., Ling, N. & Guillemin, R. (1988) Proc. Natl. Acad. Sci. USA 85, 2324-2328], include an irregular loop that extends somewhat from the surface of the protein and is about 25 A from the presumed heparin binding site. The backbone structure of this putative receptor-binding loop is very similar, although not identical, to the corresponding region of interleukin 1 beta. PMID- 1707544 TI - Metabolism and mechanism of action of 5-fluorouracil. AB - This is a review on the mechanism of action of FUra. Three main areas are addressed: metabolism, RNA-directed actions of FUra, and DNA-directed actions of FUra. Key words for bibliographic purposes: metabolism, RNA, rRNA, mRNA, tRNA, DNA primase, DNA, thymidylate synthetase, uracil N-glycosylase, FUra, FUrd, FdUrd, FdUMP, RNA splicing, 5,10-methylene tetrahydrofolate, FUTP. PMID- 1707543 TI - BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light. AB - Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification. PMID- 1707545 TI - Uterine junctional zone: correlation between histologic findings and MR imaging. AB - Uterine zonal anatomy as visualized on T2-weighted (repetition time, 2,500 msec; echo time, 80 msec) magnetic resonance (MR) images consists of a high-intensity central (endometrial) zone, a subjacent low-intensity junctional zone of myometrium, a moderately intense zone of myometrium, and a thin, low-intensity subserosal zone of myometrium. To better define the histologic correlates of these diagnostically significant zones, T2-weighted MR images of 17 in vivo and 13 extirpated human uteri were compared with histologic sections of 17 uteri stained with hematoxylin-eosin, Mallory trichrome, and immunofluorescence staining for actin. Morphometric and electron microscopic observations of uterine surgical specimens were also made. The observations indicate that both the junctional zone and the subserosal zone consist of compact smooth muscle fibers with little extracellular matrix compared with the myometrium proper. Also, the junctional zone is divided into a compact region and a transitional region. The compact region correlates well with the hypointense MR appearance of the junctional zone. PMID- 1707546 TI - Mechanisms of the early and late response of the kidney to contralateral nephrectomy. AB - The immediate (1 day, D1) and late (90 days, D90) effects of unilateral nephrectomy on contralateral renal hemodynamics, and the renal handling of electrolytes and water were investigated in the whole animal. The immediate and late ability of the remnant kidney to autoregulate perfusate flow and glomerular filtration rate (GFR) was studied in the isolated perfused kidney of the rat. In the whole animal, in D1 rats as compared to controls, GFR calculated for a single kidney increased from 0.85 +/- 0.3 to 1.1 +/- 0.2 ml/min (p less than 0.05). In D90 rats GFR increased further and was similar to prenephrectomy GFR (1.4 +/- 0.5 vs. 1.7 +/- 0.5 ml/min, p NS). Urinary prostanoid excretion in 24 h, calculated for one kidney, increased by 50-500% in D1 rats, but returned to prenephrectomy values in D90 rats. In the isolated perfused kidney, decreasing perfusion pressure (PP) from 100 to 70 mmHg did not change the renal vascular resistance (RVR) in control and D90 kidneys, but in D1 kidneys RVR decreased from 8.6 +/- 1.3 to 7 +/- 1.3 mm Hg/ml/min (p less than 0.05). In D90 kidneys RVR was significantly lower as compared to control and D1 kidneys at all perfusion pressures. Decreasing PP from 100 to 70 mm Hg resulted in a significant decrease in perfusion flow in control, D1 and D90 kidneys, while with the increase in PP from 100 to 130 mm Hg the perfusion flow increased significantly in all three kidney groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707547 TI - Effects of endothelin on the renal microcirculation of the split hydronephrotic rat kidney. AB - We have examined the effects of endothelin (ET) on the renal microcirculation by in vivo microscopy using the model of the split hydronephrotic rat kidney. ET, a potent vasoconstrictor peptide synthesized by vascular endothelial cells, showed marked and long-lasting effects on glomerular blood flow and vessel diameters in various segments of the renal vascular bed. Intravenously applied ET (100 ng/min/kg) increased systemic blood pressure from 123 +/- 7 to 156 +/- 4 mm Hg, decreased glomerular blood flow by 70%, and preferentially constricted larger preglomerular vessels, e.g. the arcuate artery. The competitive leukotriene antagonist FPL55712 significantly attenuated the vasoconstrictor response of the larger vessels. Local ET administration decreased glomerular blood flow in a dose dependent manner (50% reduction at a concentration of 2.6 +/- 0.7 x 10(-9) M) and constricted smaller vessel segments, e.g. the afferent and efferent arterioles near the glomerulus. The constriction induced by ET was not significantly affected by the Ca2+ channel blocker nitrendipine (2.8 x 10(-6) to 1.1 x 10(-5) M). We conclude that intravenous ET effects are probably mediated by leukotrienes, inducing constriction of larger renal vessels. Locally administered ET acts directly on the renal vasculature, especially on smaller vessels. PMID- 1707548 TI - Establishment of renal cell lines derived from S2 segments of the proximal tubule. AB - The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology. These cell lines were maintained in culture for at least 24 passages, then cells were frozen. One of the cell lines, the RKPC-2, was selected and further characterized. RKPC-2 cells formed domes on impermeable supports, indicating fluid and solute transport. RKPC-2 cells formed continuous monolayers of low transepithelial resistance on collagen-coated filters. They were able to accumulate tetraethylammonium, an organic cation; however, no significant transcellular transport could be measured. We conclude that this cell line which shows characteristics of epithelial cells has maintained certain properties of intact proximal tubules, in particular the capacity to accumulate organic cations. PMID- 1707549 TI - Kinetics of glucose decarboxylation in the substrate-limited isolated perfused kidney. AB - Rat kidneys were perfused with a cell-free perfusate containing substrate-free albumin, different glucose concentrations (0.20-5.0 mmol/l), and uniformly labeled 14C-glucose. The rate of glucose decarboxylation (Qox), as a function of [glucose]p, displayed saturation kinetics [Vmax = 0.35 mumol/(g.min); Km = 0.87 mmol/l]; saturation occurred at [glucose]p = 1.0-2.0 mmol/l. Although the presence of as low as 0.2 mmol/l of glucose significantly increased fractional sodium reabsorption (%TNa), there was no correlation between [glucose]p or Qox and % TNa. However, free water clearance (CH2O or CH2O/GFR) was directly proportional to [glucose]p and independent of Qox. We conclude that (1) in the absence of other substrates, renal glucose Qox saturates at hypoglycemic levels of glucose and (2) glucose plays an important role in the generation of solute free water, a role that is unrelated to glucose Qox. PMID- 1707550 TI - Differences in rat kidney morphology between males, females and testosterone treated females. AB - Kidneys of normal female and male Wistar-Kyoto rats were studied by standard morphological techniques and morphometry in order to evaluate possible differences in the overall kidney morphology between both sexes. Furthermore, we investigated the role of testosterone (DHT) on kidney morphology by treating females with daily DHT injections. Kidney weight and volume in relation to body weight were not significantly different between males and females and were not affected by DHT. Differences were found in the volume distribution among the kidney zones. The cortex was larger in males than in females, whereas the medulla was conspicuously larger in females than in males. The greater volume of the cortex in males was mainly due to a more extensive development of proximal tubules. DHT treatment in females increased the volume of their proximal tubules. Glomerular volume was similar among the three groups. Within the medulla, the difference was most prominent in the inner stripe (14.9% of the total kidney volume in females vs. 8.9% in males) and was also important in the inner medulla (7.0 vs. 4.8%). The absolute epithelial volume of thick ascending limbs in this zone was larger in females than in males. This difference was more pronounced in short loops (approximately 20%) than in long loops (approximately 10%). The values of the DHT-treated females ranged in between. In spite of the greater development of medulla and thick ascending limbs in females, urine concentration was higher in males than in females and maximum urinary concentrating ability after 48 h dehydration was not different between both sexes. PMID- 1707551 TI - [Interdisciplinary follow-up in patients with tumors]. AB - There are two main reasons for routine follow-up examinations after treatment of cancer patients: the assessment of treatment efficacy and detection of relapse and the rating of drug side-effects. Routine controls can only efficiently be performed with a profound knowledge of the biology of the tumor and of the therapeutic efficacy of the available treatments. The frequency and the type of the follow-up examinations depend mainly on the curative or the palliative treatment possibilities. Interdisciplinarity is important again only in case of new therapeutic considerations. Examples of useless controls are mentioned. Through the prevention of unnecessary examinations the primary-care physician could play an important role in the cut-down of healthcare costs. PMID- 1707552 TI - Structural limits for evolutive capacities in complex molecular systems. AB - The possibilities of evolution for a system with and without a code of translation from nucleic acids into proteins are evaluated. Our interest is mainly centred on the enzymatic RNA case since this molecule has, at the same time, reproductive and functional properties. After scanning the evolutive capacities of the enzymatic RNAs, including the possibility to play the role of "synthetase" which would match nucleic acids with amino acids as a transition step towards a code, we will try to show that due to their own functional limitative factors, the matching system (code) is necessary. This would be the only way to transform the formal complexity--complexity which has not entered into action before the translation process--into functional information to drive the instructive self-reproductive process. Once this stage is reached, the system could evolve without a limit. PMID- 1707553 TI - Patient attitudes toward testing for maternal serum alpha-fetoprotein values when results are false-positive or true-negative. AB - We investigated the attitudes toward the maternal serum alpha-fetoprotein (MSAFP) test of patients whose results from MSAFP testing were either false-positive or true-negative. Forty-six women who had previous false-positive results from MSAFP testing and 46 who had previous true-negative values participated. Patients were matched for age and socioeconomic status, and all were delivered of normal neonates. A significantly larger number of patients in the group with false positive results believed they had not understood the MSAFP test than in the group with true-negative results (19.6% vs 0%). Significantly more women in the "false-positive" group felt that the test caused anxiety than in the "true negative" group (65.2% vs 17.4%). Fewer women in the false-positive group than in the true-negative group said they would have MSAFP testing in a subsequent pregnancy (58.7% vs 91.3%). Further, the women in the false-positive group were less likely than those in the true-negative group to recommend MSAFP testing to a friend (52.1% vs 80.4%). In patients whose results were false-positive, there was a significant relationship between the patient's perception of understanding the MSAFP test and her attitude toward MSAFP testing. PMID- 1707554 TI - Improving palliation in pancreatic cancer: intraoperative celiac plexus block for pain relief. AB - Most patients with pancreatic carcinoma are not curable. Surgical palliation of obstructive jaundice and gastric outlet obstruction leaves many patients with severe pain from pancreatic carcinoma. Anesthesiologists have drawn increasing attention to the successful use of postoperative percutaneous celiac plexus block for the treatment of pancreatic pain. Ironically, little attention has been paid to celiac plexus block during laparotomy. We reviewed the cases of 12 patients with pancreatic carcinoma and severe abdominal pain who were treated surgically. All patients had operative celiac plexus block with absolute alcohol at the time of exploratory laparotomy for biliary bypass, gastroenterostomy, or tumor biopsy. Complete postoperative pain relief was obtained in 10 of the 12 patients; two had only partial relief. No operative complications were related to celiac plexus block; one patient died postoperatively of pneumonia. Average postoperative hospital stay was 13 days and average postoperative survival was 3 1/2 months. Most patients had excellent pain relief for at least 2 months or until death. Because most patients treated surgically for pancreatic carcinoma are receiving only palliation with biliary bypass or gastroenterostomy, surgeons should pay increased attention to pain relief. Operative celiac plexus block is easy, safe, and highly effective in relieving the agonizing pain of pancreatic carcinoma. PMID- 1707555 TI - Endoscopic palliative management of rectal cancer. AB - Laser ablation and bipolar coagulation have been used to palliate rectal cancer and avoid surgery. Indications are distal metastatic disease, extensive local invasion, obstruction and bleeding from nonresectable rectal tumor, or refusal of surgery. From Jan 1, 1986, to Jan 1, 1989, I saw 26 patients who met those criteria; 19 already had metastatic disease and three repeatedly refused abdominoperineal resection. A two-laser approach using both CO2 and Nd:YAG lasers was used in patients with low-lying lesions; others were treated by the Nd:YAG laser only. For rectal tumors, bipolar esophageal tumor probes were used via the rigid sigmoidoscope. The number of laser sessions averaged three per patient, and the number of bipolar coagulation sessions averaged five per patient. Bleeding followed bipolar coagulation in one patient. There were no perforations in either treatment group, and no patient has required colostomy. Of the 19 patients who already had metastatic disease, 12 are still alive, the longest survival being 20 months. Of those medically unfit for surgery, three have died of coincidental disease, and one is alive with controlled rectal cancer after 16 months. All three patients who refused surgery are alive; the longest survival is 13 months. PMID- 1707556 TI - Pregnancy after Fontan repair of tricuspid atresia. AB - We have described a patient who had a cyanotic congenital heart disease (type Ib tricuspid atresia), with initial palliation accomplished in childhood via a Glenn procedure. In 1985, she had a Fontan repair with the Bjork modification; 3 years later she achieved her first pregnancy at age 27. Maternal Doppler echocardiography in early pregnancy showed good flow through the constructed conduit, with normal left ventricular size and function. Fetal echocardiography at 22 weeks' gestation via two-dimensional, M-mode, pulsed Doppler, and color flow mapping revealed no evidence of fetal cardiac disease. At 25 weeks' gestation recalcitrant preterm labor developed, and the infant was delivered spontaneously. Labor, delivery, and puerperium were uncomplicated, and the newborn (though too premature to survive) was of appropriate weight for gestational age, without evidence of congenital heart disease or other anomalies. We believe this is the first report of pregnancy and spontaneous delivery in a patient who has had Fontan repair of a congenital heart defect. PMID- 1707557 TI - [Deontological problems in the study of arrhythmias (as an example of the need for a differentiated approach in individual deontology)]. PMID- 1707558 TI - [Characteristics and clinical use of immunostimulants]. PMID- 1707559 TI - Cystic fibrosis. 4. Abnormalities of airway epithelial function and the implications of the discovery of the cystic fibrosis gene. AB - Details of ion transporting abnormalities in cystic fibrosis airway epithelium are now known. The central hypothesis, that excessive drying of the airway surfaces is a primary event that leads to all the manifestations of the respiratory insufficiency in cystic fibrosis, is not proved. The hypothesis is, however, consistent with the known transporting abnormalities and is strengthened by the modest clinical improvement produced by a strategy designed to correct the transporting abnormalities. The discovery of the cystic fibrosis gene, together with the presumed structure of the protein product, provides a focal point that must eventually connect the functional abnormalities with the genetic defect. The cellular function of the cystic fibrosis transmembrane regulator must now be the major target in research on cystic fibrosis. Strategies for treatment based on known cellular and molecular abnormalities are beginning to emerge but will be undoubtedly more focused once the responsibility of the cystic fibrosis transmembrane regulator is known. PMID- 1707560 TI - Iloprost and tissue-type plasminogen activator differentially affect platelet aggregation in platelet-rich plasma and in whole blood. AB - Platelet aggregation in platelet-rich plasma (PRP) is more sensitive to agonists and more resistant to antagonists than that in whole blood. In this study, we show that the presence of neutrophils in whole blood accounts at least in part for diminished platelet aggregation in whole blood. The platelet-inhibitory effect of neutrophils relates to the release of nitric oxide and not to elastase or adenosine. Furthermore, two unrelated platelet inhibitors, iloprost and tissue type plasminogen inhibitor, decrease platelet aggregation to a greater extent in whole blood than in PRP. However, these agents exert cumulative or synergistic inhibitory effects with neutrophils on platelet aggregation in PRP. Thus, the inhibition of platelet aggregation in vivo may relate to the interaction between neutrophils and platelet-inhibitory agents. PMID- 1707561 TI - Characterization of bolesatine, a toxic protein from the mushroom Boletus satanas Lenz and it's effects on kidney cells. AB - Protein, DNA, and RNA syntheses were assayed in Madin Darby canine kidney cells (MDCK) treated by bolestaine, a toxic glycoprotein from the mushroom Boletus satanas (single chain, Mr 63,000 +/- 3000, pI 8.3 +/- 0.1, disulphide intrachain bridge) previously shown to be an inhibitor of in vitro protein synthesis. Cellular protein and DNA syntheses are inhibited in a dose-dependent manner after 24 h of incubation, whereas the uptake of the labelled precursors of proteins, DNA and RNA biosyntheses into the cells is not affected. The IC50 of bolesatine for protein synthesis is 0.14 microM in the cell culture medium. RNA synthesis is not inhibited at this concentration. The IC50 for DNA synthesis is 0.32 microM. When galactose is added to the culture medium, it decreases or even abolishes the toxic effects, indicating that it prevents the toxin from binding on the membrane and penetrating inside the cells. The profiles of polysomes in MDCK cells treated with bolesatine, compared to the untreated ones, show an increasing pool of polysomes indicating that the toxin acts on the peptidyl elongation step in the protein synthesis. PMID- 1707562 TI - Effects of in vivo treatment with FK506 on natural killer cells in rats. PMID- 1707563 TI - [Chemotherapy of penile carcinomas]. AB - The value of chemotherapy in treatment of squamous epithelial carcinoma of the penis is still uncertain, and its clinical relevance is speculative. In locally delimited carcinoma (T1, T2), partial penectomy is still the first-line therapy, and the alternatives of bleomycin monotherapy or combination therapy with bleomycin and radiation should be applied only when patients rigorously reject surgery. The prospects of success are rather uncertain with these forms of treatment. Palliative chemotherapy for metastasized carcinoma has so far yielded data indicating that methotrexate administration is of therapeutic benefit, even if only for a short time. Elaborate multiple combinations must be considered critically, especially because of the appreciable burden on the patient, a rate of response that in the final analysis is still uncertain, and the small number of cases treated so far. PMID- 1707564 TI - Prostate-specific antigen is not excreted by human kidney or eliminated by routine hemodialysis. AB - Serum levels of prostate-specific antigen have assumed a prominent clinical role in the management of prostate cancer. Little is known about the production of prostate-specific antigen, its mechanism of entry into the serum in normal and pathologic states, and mechanism(s) of removal from the serum. This study examined nephrostomy urine specimens, finding no significant excretion of prostate-specific antigen into the renal pelvic urine. Similarly, no significant lowering of serum prostate-specific antigen levels occurred during routine hemodialysis in men with chronic renal failure, nor were ambulatory, pre-dialysis serum prostate-specific antigen levels in these same men with chronic renal failure elevated. It appears unlikely that the kidney plays a significant role in the clearance of prostate-specific antigen from human serum. PMID- 1707565 TI - Radionuclide assessment of bladder-emptying function in normal male population and in patients before and after prostatectomy. AB - Radionuclide assessment of the bladder-emptying function was evaluated in 82 normal individuals and in 16 patients before and after prostatectomy. The parameters evaluated were: average flow rate (AFR), peak flow rate (PFR), corrected peak flow rate (CPFR = PFR/[bladder volume] 0.5), ejection fraction (EF) of the bladder, and post-voiding residual urine (RU) volume. A good interobserver reproducibility was found in 19 measurements. Urinary flow rates, EF, and RU showed a highly significant statistical difference between normal individuals and patients before surgery: AFR, 9.2 +/- 5.1 vs. 2.9 +/- 1.5 mL/sec; PFR, 19.5 +/- 9.2 vs 7.4 +/- 3.2 mL/sec; CPFR, 1.17 +/- 0.34 vs 0.54 +/- 0.22; EF, 95.6 +/- 4.6 vs 68.2 +/- 23.2 percent; and RU, 11.8 +/- 15.8 vs 93.4 +/- 115 mL; respectively. After prostatectomy the urinary flow rates showed a highly significant improvement and did not differ from the normal individuals: AFR, 7.9 +/- 2.7 mL/sec; PFR, 19.0 +/- 6.4 mL/sec; and CPFR, 1.32 +/- 0.57. The EF after surgery (91.7 +/- 10.9%) was lower than in normal individuals, but showed a significant improvement compared with EF before surgery. The RU after surgery (27.4 +/- 48.0 mL) although lower than before surgery did not differ significantly and was greater than in the normal individuals. No relationship between age and flow was found in this study. Both average and peak flow rates were related to the bladder volume. This method involves a single, noninvasive procedure which enables determination of bladder-emptying function. PMID- 1707566 TI - The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity. AB - Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been generated in vitro using infectious cDNA clones and used to demonstrate that strong determinants of the attenuation and temperature-sensitive phenotypes of P2/Sabin reside in the 5' 492 nucleotides. In this region of the genome the viruses differ only at nucleotides 437 and 481. The ts phenotype associated with the 5' noncoding region is expressed at different temperatures in different cell lines, suggesting an involvement of cellular factors which may be species specific. Suppression of both the ts and attenuation phenotypes correlates with an A-G mutation at nucleotide 481, although other changes are also involved. PMID- 1707567 TI - The open reading frame L2 of cottontail rabbit papillomavirus contains antibody inducing neutralizing epitopes. AB - Polyclonal antisera were generated against bacterially derived fusion proteins of the open reading frames (ORFs) of the capsid proteins of cottontail rabbit papillomavirus (CRPV). The carboxy-terminal two-thirds of CRPV L1 and the carboxy terminal half of CRPV L2 were cloned into a bacterial expression vector and induced proteins were used as antigen and immunogen. The polyclonal antisera were tested in a series of immunological assays, including ELISA, Western blot, and neutralization of CRPV. ELISA demonstrated that the polyclonal antisera raised against expressed L1 proteins reacted strongly to disrupted CRPV virion antigen and weakly both to intact CRPV virion and disrupted BPV-1 virion. Anti-CRPV L2 antisera reacted strongly only to intact and disrupted CRPV virion antigen. Viral capsid proteins of CRPV were detected in Western blots of HPV-11, BPV-1, and CRPV virus particles by these polyclonal antisera. The anti-L1 sera recognized the major capsid protein (60 kDa) and the anti-L2 sera identified a 76-kDa viral protein of CRPV. Only the antisera generated against expressed L2 neutralized CRPV. The neutralizing titer of the anti-L2 sera, however, was several orders of magnitude lower than the titer of a neutralizing polyclonal antiserum that was generated by immunizations with intact CRPV virions. PMID- 1707568 TI - A prominent antigenic surface polypeptide involved in the biogenesis and function of the vaccinia virus envelope. AB - Polypeptides of the vaccinia virus envelope exposed on the surface were identified by means of sulfo-N-hydroxysuccinimidobiotin as a surface tag. Among surface expressed polypeptides is the 35-kDa antigen, previously designated Ag35. Both monoclonal (mAb) and monospecific affinity pure antibodies directed against Ag35 neutralized vaccinia infectiousness, indicating that this prominent surface antigen has a function during early virus-host cell interactions. The binding of several monoclonal antibodies to various regions of Ag35 was tested by reacting CNBr fragments, derived from the polypeptide, employing Western blotting. All mAbs tested reacted with the same region of Ag35. Estimation of the molecular weights (MW), based on migration of the CNBr peptides in sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealed that those partial digestion products which contained a proline-rich 99 amino acid limit digest fragment were present at a position approximately 12.5 kDa larger than that predicted from the DNA sequence. By contrast, partial and limit digest products lacking the proline rich fragment migrated to the MW position expected from the length of the DNA sequence. This observation demonstrates that departure from a predicted 22.3 kDa to an anomalous MW of Ag35 is conferred by the proline-rich peptide. The surface location of Ag35 was confirmed by immune electron microscopy. In a competition test the binding specificity of mAb and affinity-purified antibodies at the surface of virions could be demonstrated. Evidence for an association of Ag35 with the virus envelope at various stages during biogenesis of vaccinia was obtained by immune electron microscopy of whole mounts and thin sections. Presence of Ag35 as an early component of immature and mature virions, probably residing in the bilayer membrane structure was detected. A distinction can, therefore, be made between Ag35 and several other vaccinia envelope polypeptides which are synthesized as late functions and added during late stages of envelope assembly. PMID- 1707569 TI - [Natural killer cells in the interferon therapy of cancer patients]. PMID- 1707570 TI - [Chemotherapy in disseminated malignant skin melanoma with a combination of nitrosomethylurea, vincristine, peplomycin or bleomycetin]. AB - A study of combination chemotherapy with nitrosomethylurea, vincristine and peplomycin or bleomycetin given an 5-6-day cycles showed the cytotoxic regimens to be more effective than single-agent treatment with dacarbazine for disseminated melanoma of the skin with metastases to the subcutaneous fat, lymph nodes, lungs and other viscera. In a group of 129 such patients, complete (12%) and partial remission (38%) was observed with the former combination whereas with nitrosomethylurea, vincristine and bleomycetin, complete and partial response rates were 7.6 and 26.9%, respectively. PMID- 1707571 TI - [The morphological differentiation of bronchogenic cancer]. AB - Ontogenetic differentiation of the lung was shown to be subject to determination, divergence and cell differentiation. Delay in determination was followed by proliferation of stem and precursor cells resulting in various types of dysplasia. Disorders of tissue divergence were observed during differentiation whereas various degree of cell differentiation disturbances was registered in moderately and poorly differentiated tumors. PMID- 1707572 TI - The use of three different therapeutic protocols according to the immunologic pathology present in three women with recurrent spontaneous abortion and fetal death for immunologic reasons: flucortolone and salicylates; high-dose intravenous gammaglobulins; subcutaneous heparin. AB - The authors report three cases of immunologic spontaneous abortions and fetal death. In the first patient there was lupus-like anticoagulant activity with a diagnosis of sub-clinical autoimmune disease; the second showed inadequate blocking factor activity, while the third subject presented excessive lymphocytotoxicity. In these cases three different therapeutic protocols were successfully used: flucortolone and salicylates, high-dose intravenous gammaglobulins and subcutaneous heparin. PMID- 1707573 TI - In situ hybridization of muscle mitochondrial mRNA in mitochondrial myopathies. AB - To determine whether a mitochondrial mRNA deficiency exists in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were studied using in situ hybridization. Histochemistry and immunohistochemistry were performed along with hybridization. Hybridization reactions were widely distributed over the sarcoplasm of all muscle fibers in the patient with MELAS. In the patient with CPEO, 80% of the fibers showed a marked decrease in density of autoradiographic grains. This marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochrome c oxidase (CCO). The isotope-labeled cDNA probe used in in situ hybridization in this study complements a part of subunit I of CCO and a part of subunit II of complex I in the mitochondrial gene. Our results suggest a defect in the mRNA in this CPEO patient. PMID- 1707575 TI - The cytoarchitectonic distribution of senile plaques in three aged monkeys. AB - The density of senile plaques (SP) was determined in 55 cytoarchitectonic areas of the cerebral cortex in three aged (27+ years) macaque monkeys. In silver stained sections the SP distributions pattern was variable, with a predilection for frontal areas and the primary somatosensory cortex. In one monkey, SP density in motor and premotor areas reached a level comparable to that found in Alzheimer's disease (AD). Lower SP densities were found in the amygdala and insula, and in cingulate, limbic temporal, and temporal, occipital, and parietal association cortices. Then lowest densities were in the hippocampus and in the primary auditory and primary visual cortices. SP stained with Congo red, to identify the older amyloid-containing plaques, showed a similar distribution but were fewer in number. There was at times a marked shift in SP density between adjacent cytoarchitectonic fields, suggesting that cytoarchitectonics or connectivity may play a role in determining SP distribution. The distribution of the SP in the normal aged human brain according to cytoarchitectonic areas is not known. Their pattern of distribution in these three primates appears to differ from that found in AD, which emphasizes the hippocampus, amygdala, entorhinal cortex, and temporal and parietal lobe. PMID- 1707574 TI - Induction of epitopes associated with neurofibrillary tangles in clonal mouse neuroblastoma (S20Y) cells. AB - Accumulation of paired helical filaments (PHF) in neurofibrillary tangles is a key neuropathological hallmark in Alzheimer's disease (AD). To date, PHF have been found primarily in humans. Cultured murine cholinergic neuroblastoma (S20Y) cells, following exposure to a serum-free medium or a differentiation medium, developed immunoreactivity to anti-PHF antibodies, and to the Alz-50 by immunocytochemical and immunoblot analyses. Electron microscopic examination revealed abundant fascicles of 10-nm filaments coursing tortuously amongst organelles, such as mitochondria, endoplasmic reticulum and dense-core vesicles, in perikarya and in neuritic extensions. However, subcellular structures identical or similar to PHF could not be found in these non-human cells. This convenient cell culture model may prove to be useful for studying certain aspects of the mechanisms underlying the abnormal cytoskeletal alterations which are characteristic of AD and related neurodegenerative disorders. PMID- 1707576 TI - Proteoglycan epitope in synovial fluid in gonarthrosis. 28 cases of tibial osteotomy studied prospectively for 2 years. AB - High tibial osteotomy was performed for medial gonarthrosis in 28 patients. Preoperatively, and at 3, 12, and 24 months after surgery, clinical and radiographic examinations were made, and joint-fluid samples were aspirated. Arthroscopy was performed preoperatively and at 24 months. Immunoassay of proteoglycan epitope in joint fluid showed an increase in concentration at all times as compared with a reference population with normal knee joints. An increase in both the concentration and the total amount of proteoglycan epitope in joint fluid was noted at 3 months postoperatively with a return to preoperative values at later times. Regrowth of fibrocartilage did not correlate with proteoglycan epitope data. PMID- 1707577 TI - Response of the endolymphatic sac d.c. potential to asphyxia. AB - The effect of asphyxia on the endolymphatic sac d.c. potential (ESP) was examined in the guinea pig. Asphyxia was caused for 1.5 min by stopping the respirator. The ESP decreased in amplitude during asphyxia. After the termination of asphyxia the ESP showed a diphasic recovery pattern. When respiration was resumed, the ESP decreased again following a transient recovery. Thereafter, the ESP showed a gradual recovery. beta-blocker (propranolol) inhibited a temporary decrease in the ESP after the resumption of respiration, but not alpha-blocker (phentolamine). The result indicates that the ESP decrease after the resumption of respiration is induced by beta-adrenergic action. PMID- 1707578 TI - Plasma levels of beta-endorphin and substance P in the first year of life in full term and preterm infants. PMID- 1707579 TI - A comparison of substance P peptide and preprotachykinin mRNA levels during development of rat medullary raphe and neostriatum. AB - Substance P (SP) and the mRNA coding for its precursor peptide, preprotachykinin (PPT), were measured in medullary raphe nuclei (MRN) and neostriatum (NS) over development in order to determine (1) whether PPT mRNA levels correlate with peptide development, and (2) whether changes in PPT mRNA might help to account for the apparent decline in SP seen immunohistochemically in certain brain areas postnatally. Total RNA was quantified in dissected tissue pieces using the sensitive orcinol reaction. When MRN PPT mRNA levels measured by Northern blot analysis were adjusted to total RNA levels, PPT mRNA per MRN increased over development with a profile similar to that seen for SP peptide. Moreover, both peptide and mRNA levels exhibited a similar decline after postnatal day 15. Therefore, developmental regulation of SP biosynthesis in the MRN may, in part, explain previous evidence documenting a postnatal decline in SP there. In the NS, SP peptide and PPT mRNA increased with a similar profile from E18 through the first postnatal week. Thereafter, SP increased less rapidly than its mRNA, indicating incongruities in prohormone message and processed peptide in the NS. PMID- 1707580 TI - Fetal alcohol delays the developmental expression of myelin basic protein and transferrin in rat primary oligodendrocyte cultures. AB - This study has examined the development of immunoreactive myelin basic protein and transferrin in primary glial cell cultures. Cultures were initiated from control and experimental Sprague-Dawley rats 1-2 days postnatally. Experimental treatment involved exposure to 5% (w/v) ethanol in a liquid diet during the last two weeks of gestation. Prenatal alcohol administration delayed the expression of myelin basic protein and transferrin during the first three weeks postnatally. Other oligodendroglial and astroglial markers were little affected, if at all, by fetal alcohol exposure. PMID- 1707581 TI - Sphingosine inhibition and promotion of histamine release from isolated rat mast cells. AB - Sphingosine inhibited the histamine release induced by antigen, compound 48/80 with and without calcium, and the combination of TPA and the ionophore A23187. The inhibition occurred in the concentration range 1-3 microM, where no sign of cytotoxicity was noted. Preincubation for 5-10 min was needed for inhibition, and the effect persisted after washing of the cells. No inhibition was found with optimal concentrations of the ionophore or with TPA present during the preincubation. Sphingosine in combination with suboptimal concentrations of the ionophore could induce a considerable histamine release. This response was dependent on energy and was potently inhibited by the flavonoid phloretin. After preincubation with TPA, sphingosine exerted a pronounced potentiation of the response to very low concentrations of the ionophore. The findings regarding inhibitory effects of sphingosine do not seem to be compatible with a selective action on protein kinase C. The ability to synergize with the ionophore and to potentiate the effect of preincubation with TPA resembles previous findings with palmitoylcarnitine and suggests that sphingosine can stimulate mast cells by activation of protein kinase C. PMID- 1707582 TI - The determination of histamine in challenged human leukocyte preparations by high performance liquid chromatography. AB - A highly sensitive and rapid method was developed for the determination of histamine in challenged human leukocyte preparations by high-performance liquid chromatography. The assay is based on the Shore's OPT-reaction of the unpurified sample and on a specific separation of the derivate with analytical reversed phase phenyl column combined with spectrofluorometric detection. The detection limit of histamine by this method was 0.07 pmol (signal to noise ratio 2:1) and the within-day variation for peak height was 3.6% and for retention time 0.8%. A good linear standard curve ranging from 12.5 pg to 500 pg (0.07 pmol-2.7 pmol) was obtained with correlation coefficient of 0.998. The histamine release from human basophils in mixed leukocyte preparation was induced by the calcium ionophore A 23187. A concentration of 0.4 micrograms/ml ionophore was required for 50% histamine release with a Ca2(+)-concentration of 1.8 mmol/l. The measured total histamine content was 1.5 pg/basophil. PMID- 1707583 TI - Effect of inhibitors on histamine release from mast cells recovered by bronchoalveolar lavage in basenji-greyhound and mongrel dogs. AB - Studies of rodent mast cells have demonstrated that subpopulations differ in regard to their response to inhibitors of histamine release. To determine whether such compounds have different effects on mast cells from Basenji-Greyhound (BG) dogs with airway hyperreactivity and from mongrel dogs, we investigated the effect of cromolyn sodium, nedocromil sodium, theophylline, and quercetin on calcium ionophore A23187-induced histamine release from mast cells recovered by bronchoalveolar lavage. Mast cells recovered from BG and mongrel dogs were similar in respect to morphology and spontaneous and calcium ionophore-induce histamine release. Histamine release from mast cells from both BG and mongrel dogs was inhibited by quercetin (10(-4) M) and nedocromil sodium (5 x 10(-5) M). In contrast, only the histamine release from mast cells recovered from mongrel dogs was inhibited by cromolyn sodium (10(-4) M) and theophylline (5 x 10(-3) M). Thus, mast cells that are similar in regard to morphology and response to histamine liberators may differ in their response to inhibitors of histamine release. PMID- 1707584 TI - Effect of interleukin 3 on the differentiation and histamine content of cultured bone marrow mast cells. AB - Mouse bone marrow hematopoietic stem cells were isolated from mouse femur bone and cultured in RPMI 1640 supplemented medium with 20 units/ml of the purified T cell lymphokine, interleukin 3 (IL-3), IL-3 was uniquely able to induce the proliferation and differentiation of mature mast cells in vitro. The sparse granulation of the bone marrow-derived mast cells (BMMC) can be seen by day 5, progressing to definable mast cells by day 7, the mast cells appear morphologically mature and comprise a 96% pure population after 14 days of the culture. The monocytes macrophages, eosinophils and neutrophils disappeared by day 9. After 4 weeks of tissue culture, mast cells are fully mature and completely granulated at 98% cell purity. The BMMC are mononuclear, oval or round in shape and appear smaller than rat peritoneal mast cells. BMMC are stable over 3-5 months in conditioned medium. The homogeneous mast cell population possesses membrane receptors and mediators, such as histamine in their metachromatic granules. The histamine content of BMMC in culture between 2 to 4 weeks rose from 1.43 to 1.82 pg/cell. Moreover, the percentage of histamine release caused by 0.1 microM and 1.0 microM ionophore A23187 was 15% and 35%, respectively. By contrast, the histamine releasing activity of 0.01% and 0.001% compound 48/80 were 12 +/- 2% and 59 +/- 7% respectively. The granular density, histamine content and histamine release activity of BMMC are different from that of peritoneal mast cells. PMID- 1707585 TI - Mediators of substance P-induced inflammation in the rat knee joint. AB - Substance P (SP) injected into the synovial cavity of the rat knee resulted in an inflammatory response as measured by plasma protein extravasation into the joint capsule. This response was dose dependent over the range of approximately 4 microM to approximately 200 microM. Part of this inflammatory response was mediated via mast cells as pre-treatment of the animals with a mast cell degranulator (compound 48/80) resulted in a 66% reduction of the response. A direct effect of SP on the vascular receptors may also contribute to the inflammatory response as pre-treatment with the substance P antagonist (SPA) D Pro4 D-Trp7,9,10 SP4-11 also reduces the inflammatory response. Intra-articular injections of the H1 blocker diphenhydramine or the H2 blocker cimetidine significantly blocked the SP-induced inflammatory response. The 5 hydroxytryptamine (5-HT) antagonist methysergide proved to be even more potent in blocking the SP-induced inflammatory response. No synergistic inhibition was observed with combinations of the different antagonists. Intra-articular injections of 5-HT elicited a much more pronounced inflammatory response than that produced by a 10-fold higher concentration of histamine. The results suggest that SP produces increased vascular permeability partly via direct actions on the blood vessels and partly via mast cells. The inflammatory response occurring via mast cells appears to be mediated by histamine and to a greater extent by 5-HT. PMID- 1707586 TI - Effect of substance P on the membrane potential of coronary arterial endothelial cells in situ. AB - The membrane potential of the endothelial cells on a strip of pig coronary artery was recorded. The fluorescent dye lucifer yellow was injected into the recorded cell in order to prove its identity. We show that substance P transiently hyperpolarized these cells. The function of the endothelial cell hyperpolarization could be important for the role played by the endothelium and the kinins in inflammation. PMID- 1707587 TI - Clear cell cribriform hyperplasia of the prostate. Immunohistochemical and DNA flow cytometric study. AB - Clear cell cribriform hyperplasia (CCCH) of the prostate is an unusual form of benign prostatic hyperplasia characterized by a nodular proliferation of clear cells with small, uniform nuclei. The authors studied 15 cases of CCCH by immunohistochemistry and 13 of them by DNA flow cytometry to establish the immunohistochemical and DNA profile of this lesion. Patients ranged in age from 58 to 88 years (mean, 68 years). Follow-up of a mean of 22 months showed all patients alive with no evidence of malignant prostatic disease. All 13 CCCHs showed diploid DNA content; in contrast, among 4 papillary/cribriform carcinomas of the prostate used for comparison, 3 were aneuploid and 1 was diploid. A basal cell layer was demonstrated in all 15 CCCHs by the use of the 34 beta E12 anti high-molecular-weight keratin antibody (EAB-903) that reacts with the basal cells but not with the acinar cells of the prostate. A continuous basal cell layer was not evident in the carcinomas. The blandness of the epithelium, the well-defined nodular configuration, the presence of a basal cell layer demonstrable by immunocytochemistry, and the lack of aneuploidy as determined by DNA flow cytometry together lend support to the concept that CCCH is a benign lesion. PMID- 1707588 TI - Malignant peritoneal mesothelioma in childhood with long-term survival. AB - A diffuse, well-differentiated, malignant peritoneal mesothelioma (MPM) developed in a nine-year-old girl. She received limited chemotherapy and radiation therapy and is alive and well without clinical evidence of disease 109 months after diagnosis. The neoplastic cells stained immunohistochemically for cytokeratin and epithelial membrane antigen but were unreactive with B72.3, anti-carcinoembryonic antigen, and anti-Leu-M1. Ultrastructurally, the tumor cells had abundant desmosomes, numerous tonofilament bundles, and variable-length microvilli. These findings confirm the mesothelial nature of the cells. Features consistent with malignancy included DNA aneuploidy by flow cytometric analysis and diffuse peritoneal involvement. The three previously described survivors with MPM were also premenarchal girls. Some MPMs in premenarchal girls have an indolent biologic behavior similar to that of low-grade peritoneal serous neoplasia or well-differentiated papillary mesothelioma in adult women. PMID- 1707589 TI - Vascular proliferation as an unusual cause of hemorrhagic diathesis in myelofibrosis. AB - One year after splenectomy, a patient with myelofibrosis developed spontaneously large hematomas that were not due to coagulation abnormalities or functionally defective platelets. At autopsy, the liver, muscle, and skin showed extramedullary hematopoiesis associated with capillary proliferation and extravasation of blood. These findings indicate that neovascularization can be an additional cause of bleeding in myeloproliferative disorders and might be induced by neoplastic hematopoietic cells. PMID- 1707590 TI - Comparison of Papanicolaou's stain with the Gomori methenamine silver (GMS) stain for the cytodiagnosis of Pneumocystis carinii in bronchoalveolar lavage (BAL) fluid. AB - The cytodiagnosis of Pneumocystis carinii (PC) in bronchoalveolar lavage (BAL) fluids has traditionally required a special stain such as Gomori's methenamine silver (GMS) stain. Recent reports indicate that identification of foamy alveolar casts (FACs) with Papanicolaou's (Pap) stain may provide a sensitive and less complicated way of making the diagnosis. To confirm these observations, results on a series of 318 BALs were reviewed. PC was identified on 65 (20%) specimens from 54 patients. Pap stains and GMS stains were positive on 56 (86%) of these BALs. Pap stains were positive on seven (11%) specimens that had negative GMS stains. PC was later confirmed on these specimens by other methods. Only two (3%) BALs had positive GMS stains and negative Pap stains. The results of this study confirm other reports that show that PC can be sensitively diagnosed with the Pap stain. The authors suggest that routine special stains for PC are unnecessary on BALs. PMID- 1707591 TI - The identification of prostate-specific antigen. PMID- 1707592 TI - Human CYP1A1 gene: cosegregation of the enzyme inducibility phenotype and an RFLP. AB - The human CYP1A1 (cytochrome P1450) gene encodes an enzyme involved in the activation of procarcinogens, such as benzo[a]pyrene, to the ultimate reactive intermediate. Approximately 10% of the human population exhibit high CYP1A1 inducibility, and Kouri et al. reported that the high-inducibility phenotype might be at greater risk than low-inducibility individuals for cigarette smoke induced bronchogenic carcinoma. In one 3-generation family of 15 individuals, we show here that the high-CYP1A1-inducibility phenotype segregates concordantly with an infrequent polymorphic site located 450 bases downstream from the CYP1A1 gene. Our findings are consistent with the study of Kawajiri et al., who demonstrated an association between this polymorphism and an increased incidence of squamous-cell lung cancer. Our data suggest that the CYP1A1 structural gene, or a region near this gene, might be correlated with the inducibility phenotype. PMID- 1707594 TI - c-myc protooncogene polypeptide expression in endometriosis. AB - The c-myc protooncogene and its polypeptide product are important regulators of cell proliferation and differentiation, and ovarian steroids are believed to stimulate growth of various uterine cell types through altered expression of the c-myc gene. To determine whether c-myc expression may also be involved in the growth and development of endometriosis, we assessed c-myc expression in eutopic and ectopic endometrial tissue obtained from women undergoing surgery for endometriosis. Immunocytochemistry using a monoclonal antibody to the c-myc protein demonstrated positive staining of glandular and stromal cell nuclei, and cytoplasmic staining of glandular but not stromal cells in both eutopic and ectopic endometrium. These findings suggest that c-myc expression may be an important regulator of cell proliferation in endometriotic tissue. PMID- 1707593 TI - Exonic point mutations in NADH-cytochrome B5 reductase genes of homozygotes for hereditary methemoglobinemia, types I and III: putative mechanisms of tissue dependent enzyme deficiency. AB - We analyzed the NADH-cytochrome b5 reductase gene of hereditary methemoglobinemia type I and type III, by using PCR-related techniques. The mutation in type I is a guanine-to-adenine substitution in codon 57 of exon 3 of the NADH-cytochrome b5 reductase gene, and the sense of this codon is changed from arginine to glutamine. In type III the mutation is a thymine-to-cytosine transition in codon 148 of exon 5, causing leucine-to-proline replacement in type III. The former mutation abolishes the MspI recognition site. Homozygosity for the former mutation in a patient with type I was confirmed by restriction analysis of PCR amplified fragments and by dot blot hybridization of amplified products with allele-specific oligonucleotide probes. The latter mutation generates a recognition site for MspI. Amplification of exon 5 by PCR followed by digestion with MspI revealed homozygosity for this mutation in patients with type-III. Putative mechanisms of tissue-dependent enzyme defects in hereditary methemoglobinemia are discussed. PMID- 1707595 TI - Immunohistochemical localization of gonadotropin-releasing hormone during implantation in the New Zealand white rabbit. AB - Gonadotropin-releasing hormone was localized immunohistochemically during implantation (gestational days 6 to 14) in the New Zealand White rabbit. During early implantation (days 7 to 9), intense gonadotropin-releasing hormone immunostaining was localized predominantly to the cytoplasm of the nonknob cytotrophoblast with light to moderate staining in the cytoplasm of the syncytiotrophoblast (knob). In later gestation, light to moderate staining of the cytoplasm of the trophoblast at the true placental site was detected. No appreciable change in staining was noted after day 10. Fetal membranes, identified after day 10, showed intense and unchanging immunostaining for gonadotropin-releasing hormone. Obplacental giant cells showed light to moderate nuclear and cytoplasmic gonadotropin-releasing hormone immunostaining. Light to moderate gonadotropin-releasing hormone immunostaining was also noted in the cytoplasm of uterine epithelium and glands. We conclude that immunoreactive gonadotropin-releasing hormone is present in the cytotrophoblast at the time of the earliest embryo-uterine interactions and may play a significant role in implantation and embryo survival. PMID- 1707596 TI - The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers. AB - Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C 8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9 fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1. PMID- 1707597 TI - Production and separation of peptides from proteins stained with Coomassie brilliant blue R-250 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AB - Proteins stained with Coomassie brilliant blue on polyacrylamide gels were digested with lysylendopeptidase in the presence of sodium dodecyl sulfate. Peptide production was similar to that under ordinary conditions of digestion. Peptides were recovered easily and efficiently from the gel pieces and separated by HPLC. The present method for preparation of peptides from proteins separated by sodium dodecyl sulfate gel electrophoresis is quite simple and can be used for sequence analysis of proteins in general at the subnanomolar level. PMID- 1707598 TI - Biotinylated antibodies bound to streptavidin beads: a versatile solid matrix for immunoassays. AB - Streptavidin was covalently bound to commercially available polyacrylamide beads (3-10 microns diameter) by peptide bond formation between the carboxyl groups on the solid matrix and the amino groups of the soluble protein. Biotinylated antibody or lectin was linked to the polyacrylamide beads via the streptavidin molecules. Immunoassays for human IgA1, IgA2, IgE, and vitronectin were developed utilizing the antibody or lectin as a capture ligand. The protein being assayed was quantitated colorimetrically at 492 nm via horseradish peroxidase-conjugated antibodies. PMID- 1707599 TI - Proteins of the complement system and acute phase reactants in sera of patients with spinal cord injury. AB - Complement activity was studied in patients with spinal cord transection. In some sera acute phase reactants: haptoglobin, C-reactive proteins, ceruloplasmin, as well as fibrinogen and fibrin degradation products, and immune complexes were monitored. Complement and acute phase reactants are increased in a majority of patients. Continuing inflammation and release of inflammatory mediators could be responsible for poor healing that commonly occurs in spinal cord injury. Urinary tract and other infections are associated with some but not all of the protein abnormalities. These proinflammatory proteins may contribute to the lack of healing of spinal transection. PMID- 1707600 TI - Effects of sample handling on adrenocorticotropin concentration measured in canine plasma, using a commercially available radioimmunoassay kit. AB - A commercially available radioimmunoassay (RIA) kit for measurement of human adrenocorticotropin (hACTH) was validated for use in dogs. Assay sensitivity was 3 pg/ml. Intra-assay coefficient of variation (x 100; CV) for 3 canine plasma pools was 3.0 (mean +/- SD, 33 +/- 0.99 pg/ml), 4.2 (71 +/- 2.4 pg/ml) and 3.7 (145 +/- 3.7 pg/ml) %. Interassay CV for 2 plasma pools measured in 6 assays was 9.8 (37 +/- 3.6 pg/ml) and 4.4 (76 +/- 3.4 pg/ml) %, respectively. Dilutional parallelism was documented by assaying 2 pools of canine plasma at 3 dilutions and correcting the measured result for dilution. Corrected mean concentrations for the first pool were 33 (+/- 0.99), 36 (+/- 4.3), and 33 (+/- 6.8) pg/ml; corrected mean concentrations for the second pool were 145 (+/- 5.4), 141 (+/- 10.8) and 125 (+/- 3.4) pg/ml. Recovery of 1-39hACTH added to canine plasma (6.25, 12.5, 25.0, 50.0, and 100.0 pg/ml) was linear and quantitative (slope = 0.890, R2 = 0.961). To test whether anticoagulant or the protease inhibitor, aprotinin, influences ACTH concentration in canine plasma, ACTH was measured in canine blood collected in 4 tubes containing anticoagulant: heparin (H), heparin + 500 kallikrein inhibitor units (KIU) of aprotinin/ml (HA), EDTA (E), and EDTA + aprotinin (EA). Plasma ACTH concentration was the same when samples containing H and HA, or HA and E were compared, and was significantly (P less than 0.01) lower in samples containing EA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707601 TI - Deconvolution study of the absorption rate and disposition kinetic values of lindane in sheep. AB - Absorption rate and plasma and fat disposition of lindane after various lindane percutaneous treatments in shorn and unshorn sheep were investigated. To analyze data with a deconvolution method, IV administration was performed to determine the basic pharmacokinetic values of lindane in sheep. After IV administration, the steady state volume of distribution was very high (8.07 +/- 3.60 L/kg of body weight), and the mean residence time was long (28.1 +/- 11.7 hours). Deconvolution analysis indicated that lindane absorption was continuous until 33 to 41 days after spraying with a 0.025% lindane solution. Total amount of absorbed lindane in shorn (15,171 +/- 4,463 micrograms/kg) sheep was about twice that in unshorn (7,615 +/- 3,128 micrograms/kg) sheep; from deconvolution analysis, it was calculated that the time required for 50% of the available dose to be absorbed was between 115 and 179 hours. After percutaneous lindane administration, the fat concentration was compared with the available lindane dose. The apparent half-life of lindane elimination in fat was 225 +/- 47.4 hours, which is similar to the value calculated for the absorption rate constant. By comparing fat and plasma concentrations, it was calculated that for a mean plasma concentration of 5 ng/ml, the fat lindane concentration was 1.65 +/- 0.87 micrograms/g (ie, lower than the generally accepted tolerance level of 2 micrograms/g). PMID- 1707602 TI - [Choice of vascular-loading solutions for treating hypovolemia in adults]. PMID- 1707603 TI - Immunoenhancing properties and antiviral activity of 7-deazaguanosine in mice. AB - The nucleotide analog 7-deazaguanosine has not previously been reported to possess biological (antiviral or antitumor) properties in cell culture or in vivo. Up to 10(5) U of interferon per ml was detected in mouse sera 1 to 4 h following oral (200-mg/kg of body weight) and intraperitoneal (50-mg/kg) doses of the compound. 7-Deazaguanosine also caused significant activation of natural killer and phagocytic cells but did not augment T- and B-cell blastogenesis. Intraperitoneal treatments of 50, 100, and 200 mg/kg/day administered 24 and 18 h before virus inoculation were highly protective in mice inoculated with lethal doses of Semliki Forest or San Angelo viruses. Less but still significant survivor increases were evident in treated mice infected with banzi or encephalomyocarditis viruses. In most cases, the degree of antiviral activity was similar to that exhibited by the biological response modifier 7-thia-8 oxoguanosine. 7-Thia-8-oxoguanosine was more potent than 7-deazaguanosine against encephalomyocarditis virus in mice, however. Oral efficacy was achieved with 7 deazaguanosine treatments of greater than or equal to 100 mg/kg against all virus infections, whereas 7-thia-8-oxoguanosine is reported to be devoid of oral activity in rodents. Thus, 7-deazaguanosine represents the first reported orally active nucleoside biological response modifier exhibiting broad-spectrum antiviral activity against particular types of RNA viruses. PMID- 1707604 TI - Inhibition of human immunodeficiency virus type 1 morphogenesis in T cells by alpha interferon. AB - Some murine retroviruses exhibit altered release of virus when cells are treated with alpha interferon (IFN-alpha), resulting in the accumulation of intracellular virions in cytoplasmic vacuoles. In studies of the inhibitory effect of IFN-alpha (Wellferon) on acute human immunodeficiency virus type 1 infection of human T cell lines, we found that in C3 cells, the 50% effective concentration was 9 U/ml and the 90% effective concentration was 310 U/ml. There was no apparent accumulation of intracellular particles detected by p24 antigen levels or by processing the cells for electron microscopy. Extracellular reverse transcriptase activity and p24 levels decreased in parallel with increasing IFN, whereas the intracellular viral proteins decreased only slightly. By electron microscopy, cells treated with higher concentrations of IFN (512 U/ml) disclosed very few particles budding into extracellular spaces; no intracellular particles could be seen, despite nearly normal levels of intracellular viral protein detected by the p24 antigen assay and correct processing detected by Western blot (immunoblot) analysis. Thus in human immunodeficiency virus-infected cells, the major block produced by IFN-alpha appeared to be late in the viral cycle at the morphogenesis stage of virion production. Chronically infected Jurkat cells treated with IFN appeared to be inhibited in growth rate, as virus production decreased proportionally with cell number. PMID- 1707605 TI - Isolation and characterization of Lactococcus lactis subsp. lactis promoters. AB - DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained. PMID- 1707606 TI - Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum. AB - The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains. PMID- 1707607 TI - [XIVth Congress of the Society of Biomechanics. Marseille, 6-7 September 1989]. PMID- 1707608 TI - Tissue electrical admittance (electrolyte concentration) in rat renal medulla: effects of furosemide and acetazolamide. AB - Fluctuations of total electrolyte concentration in the renal medulla were estimated from continuous measurement of tissue electrical admittance (reciprocal impedance) by means of needle electrodes placed in the kidney of anaesthetized rats. To compare effects of two diuretic agents with different sites of action, rats received either furosemide, 0.3 mg/kg i.v. followed by an infusion at 0.3 mg/kg.h, or acetazolamide, a single injection of 10 mg/kg. At this dosage similar increases in renal excretion were obtained with either drug. After furosemide (a loop diuretic) admittance fell sharp within first 10 min, then partly recovered and reached a plateau 35 min after injection. Acetazolamide (inhibitor of proximal reabsorption) caused no changes in admittance compared to the pattern observed in untreated control animals. We conclude that dissipation of tissue electrolytes from the renal medulla is not simply a consequence of diuresis and natriuresis but depends critically on the site of transport inhibition in the nephron. PMID- 1707609 TI - Intestinal calcium transfer and alkaline phosphatase activity in relation with vitamin D and glucide diet. AB - For four weeks after weaning, rats were fed either on a diet without any calcium utilization factors (-D) or on the same diet with cholecalciferol (+D) or sorbitol (S). In the -D group, blood calcium levels decreased whilst alkaline phosphatase activities in blood and bone were increased. For +D and S groups, these parameters were normal. Using everted or in situ ligatured loops, calcium transfer from a CaCl2 + 45Ca solution was measured in the duodenum, the jejunum and in the ileum. Alkaline phosphatase activity from these regions was also measured. For the three diets and for all regions of the intestine, there was a good correlation between calcium transfer and phosphatase activity. These values were higher in the duodenum than in the ileum or jejunum, and also higher in the ileum in the +D group than in the -D and S groups although this was not significant. These low levels in the S group which were, sometimes, even lower than those seen in the -D group contrasted with blood and bone levels of alkaline phosphatase, which were normal for the S and +D groups. There was also a discrepancy between the low values found for both phosphatase activity and calcium transfer in rats S in the experiments where the calcium transfer assay was carried out in calcium solution and those found in experiments were both calcium and carbohydrate were present. In the latter, enhanced levels of intestinal phosphatase activity were observed, as well as a marked, albeit delayed, increase in intestinal calcium transfer. Onset latency and rapid offset are reminiscent of induction of bacterial enzymes by carbohydrates. PMID- 1707610 TI - Evidence that adenosine is not involved in the non-adrenergic non-cholinergic relaxation in the rat duodenum. AB - In rat isolated duodenal segments, adenosine induced, in the presence of atropine and guanethidine, a dose-dependent, long-lasting (about 20 s), tetrodotoxin (TTX) resistant relaxation both in endoluminal pressure and in isometric tension. Electrical field stimulation (EFS) induced, in the presence of atropine and guanethidine, a TTX-sensitive short-lasting (about 6 s) relaxation followed by a sustained rebound contraction. Theophylline, a P1 receptor antagonist, at the concentration of 100 microM caused a marked inhibition of the adenosine-induced relaxation, while the EFS-induced relaxation was not modified. Our results suggest that adenosine induces relaxation of the rat duodenal smooth muscle acting on P1 receptors localized at muscular level. However, differences in the morphology and in the sensitivity to theophylline between adenosine- and EFS induced relaxation ruled out adenosine as neurotransmitter of the non-adrenergic, non-cholinergic inhibitory system. PMID- 1707611 TI - The effects of cafeteria diet induced obesity on rat blood amino acid compartmentation. AB - In female virgin Wistar rats, the effects of a cafeteria-diet induced obesity on blood amino acid levels and their distribution between plasma and blood cells have been studied in fed and 24-hour starved states. Cafeteria diet induced obesity provoked a decrease in total blood cell amino acid content, both in fed and starved situations when compared with controls. Whether is a causal factor for developing obesity due to imbalance in tissue amino acid supply for protein biosynthesis processes, or represents some signal related to hypothalamic control of feeding, or is a consequence of the obesity remains to be established. PMID- 1707612 TI - Hexadecanoic and neuraminic acid incorporations in two rat colon carcinoma cell lipids: selective influence of 1-O-octadecyl 2-O-methyl-3-phosphocholine on glycerolipid and ganglioside biosynthesis. AB - [3H] hexadecanoic and N-acetyl [14C] neuraminic acids were incorporated in glycerolipids or gangliosides of 2 rat colon carcinoma cell lines, having (PRO cells), or not (REG cells) invasive capacities when inoculated in syngeneic BD IX rats. The cells were cultured (48 h) in presence of 1-0-octadecyl-2-0-methyl-3 phosphocholine (ET 18-0-CH3) 20 or 40 microM, which, on transformed cells, inhibits the cell growth, modifies the glycerolipid biosynthesis, and activates the sialyltransferases. ET 18-0-CH3 20 microM activated, in PRO and in REG cells the incorporation of [3H] hexadecanoate in monosialogangliosides (1.45 fold compared to controls), but not in disialogangliosides and the distribution of this fatty acid between monosialo- (82%) and disialogangliosides (18%) was unchanged with controls. After [14C] neuraminic acid labelings, and for control experiments, the total radioactivities in gangliosides, in PRO cells, were twice higher than in REG cells, a difference which, probably, reflects the ganglioside content. ET 18-0-CH3 20 microM did not increase the incorporation of the [14C] neuraminic acid in PRO and in REG cells, and did not change its distribution between monosialo (70-80% for controls and experiments with ET 18-0-CH3) and disialogangliosides (20-30%). Similar results were obtained with ET 18-0-CH3 40 microM for the distribution of [14C] neuraminic acid in monosialo- and disialogangliosides. Whatever the precursor, the trisialogangliosides were never radiolabeled. Analysis of the [3H] glycerolipids (the main radiolabeled lipid classes in controls were: phosphatidylcholines, triglycerides, sphingomyelins and phosphatidyl-inositols) revealed that ET 18-0-CH3, compared to controls, did not activate the incorporation of [3H] hexadecanoate in total glycerolipids (PRO or REG cells). It activated (3 fold) its incorporation in triglyerides, inhibited it (0.5-0.6 fold) in phosphatidylcholines, sphingomyelins and phosphatidyl-inoditols and all these most noticeable differences were observed in PRO and in REG cells. These findings reflect the impossibility of ET 18-0-CH3 to activate the sialyltransferases during the ganglioside biosynthesis in colon carcinoma cells, while it modified ceramide, glycerophospholipid and neutral glycerolipid biosynthesis. PMID- 1707614 TI - [Changes in the blood flow of the primary carotid and its branches during modifications of the O2 and CO2 composition of alveolar gas]. AB - We measured common carotid blood flow using a range gated Doppler velocimeter, and internal and external blood velocities using a continuous Doppler in 20 lowlanders at sea level, under normal barometric pressure, in 10 subjects in an altitude chamber under a barometric pressure of 462 Torr (61.6 KPa) and then in 5 of them over a 3-weeks period at 3850 m of elevation (475 Torr = 63.3 KPa). The same measurements were also performed in 20 permanent residents at 3850 m. Common carotid blood flow was 15% higher in all subjects exposed to high altitude, due to a lowering in downstream resistances since systemic blood pressure did not change at high altitude. The increase in common carotid blood flow was the result of an immediate increase in internal carotid blood velocities observed in the altitude chamber as well as after the arrival at high altitude, but a few days later those velocities in the internal carotid artery declined to values similar to those observed at sea level. In the same time velocities in external carotid artery rose at high altitude, remained steadily elevated and the result is a permanent increase in common carotid blood flow at altitude. In all subjects we performed the same measurements, during an acute inhalation of gas mixtures to try to quantify the mechanisms controlling the changes in common carotid blood flow while changing gas inhalation. In the limits of the variations in PO2 (60 to 400 Torr) and in PCO2 (30 to 50 Torr) the stimulation by CO2 is twice more efficient than the O2 stimulation on vasomotion. PMID- 1707613 TI - Interaction of fatty acid binding protein with microsomes: removal of palmitic acid and retinyl esters. AB - [14C] palmitic acid or [3H] retinyl esters incorporated in microsomal membranes were removed by a cytosolic fraction enriched in fatty acid binding protein. When mouse liver cytosol was fractionated by 70% ammonium sulphate, a precipitate and a soluble fraction were obtained. The soluble fraction containing the fatty acid binding protein was able to remove from microsomal membranes, [14C] palmitic acid or [3H] retinyl esters, whereas the precipitate fraction had no removal capacity. Retinoid analysis indicated that 70% ammonium sulphate soluble fraction was enriched in endogenous retinyl esters with regard to cytosol or 70% ammonium sulphate precipitate fraction. PMID- 1707615 TI - Non-shivering thermogenesis and brown adipose tissue activity in essential fatty acid deficient rats. AB - The effects of essential fatty acid (EFA) deficiency on energetic metabolism and interscapular brown adipose tissue (BAT) activity were examined in the cold acclimated rat. Weanling male Long-Evans rats were fed on a low fat semipurified diet (control diet, 2% sunflower oil; EFA deficient diet, 2% hydrogenated coconut oil) for 9 weeks. They were exposed at 5 degrees C for the last 5 weeks. In EFA deficient rats, compared to controls, growth retardation reached 22% at sacrifice. Caloric intake being the same in the two groups, it follows that food efficiency was decreased by 40%. Resting metabolism in relation to body surface area was 25% increased. Calorigenic effect of norepinephrine (NE) in vivo (test of non-shivering thermogenesis) underwent a marked decrease of 34%. BAT weight was 21% decreased but total and mitochondrial protein content showed no variation. A 26% increase in purine nucleotide binding per BAT (taken as an index of thermogenic activity) was observed, suggesting that the enhancement in resting metabolism observed was mainly due to increased BAT thermogenesis. However, BAT mitochondria respiratory studies which are more direct functional tests showed a marked impairment of maximal O2 consumption of about 30% with palmitoyl-carnitine or acetyl-carnitine (both in presence of malate) or with alpha-glycerophosphate as substrate. It is likely that this impaired maximal BAT oxidative capacity may explain the impaired NE calorigenic effect in vivo. A possible increase in mitochondrial basal permeability is also discussed. PMID- 1707616 TI - Neonatal isoerythrolysis in Himalayan kittens. PMID- 1707617 TI - The amylase gene-enzyme system of chickens. II. Biochemical characterization of allozymes. AB - The chicken amylase allozymes, AmyF and AmyS, were extracted from pancreatic tissues of AmyF/F and AmyS/S individuals and purified. Activities were measured under various reaction conditions (= treatments) to assess whether the allozymes were functionally different. The amylases had properties typical of alpha amylases, i.e., both were inhibited by ethylenediaminetetraacetate and alpha amylase inhibitor from wheat, had pH optima between 7.0 and 8.0, and could utilize a variety of substrates containing alpha 1,4 linkages. The amylases were also found to be inhibited by potassium phosphate buffer and p chloromercuribenzoate. In terms of substrate specificity, both amylases could utilize all of the substrates tested with activity observed in the following order: amylopectin greater than potato starch greater than dextrin greater than glycogen greater than amylose. Statistical analysis indicated significant functional differences between the two allozymes in terms of specific activities, substrate specificities, and inhibitor sensitivities. AmyF had a significantly lower specific activity than did AmyS. The amylases responded differently to the substrate amylose, with AmyF better able to digest this substrate. AmyS was less sensitive than AmyF to alpha-amylase inhibitor from wheat. PMID- 1707618 TI - Changes in alpha-globin gene expression in mice of two alpha-globin haplotypes during development. AB - Adult alpha-globin in mice is synthesized in large amounts during development, first in the primitive, nucleated erythrocytes of yolk sac origin and later in the definitive, nonnucleated erythrocytes that differentiate in the fetal liver, spleen, and bone marrow. Isoelectric focusing analysis of hemoglobins of mice with the Hbag2 and Hbac haplotypes shows that the ratios of alpha chain 1 to chain 5m and alpha chain 1 to chain 4 in adult hemoglobins from Hbag2 and Hbac mice, respectively, change between day 11.5 and day 16.5 of gestation in nucleated red cells, while no change occurs in nonnucleated red cells. The percentage ratios of the two different alpha-globin chains are different in Hbag2 and Hbac mice for EII, EIII, and adult hemoglobin. In nucleated red cells of yolk sac origin, differences and changes in alpha-globin ratios are a composite of changing globin gene transcription and posttranslational competitive affinities among globins to form embryonic and adult hemoglobin tetramers. PMID- 1707619 TI - [Partial purification of glycosyltransferases participating in the biosynthesis of Salmonella anatum and S. kentucky O-antigens using high performance gel chromatography]. AB - The solubilized glycosyltransferases which catalyse the biosynthesis of Salmonella anatum and S. kentucky O-specific polysaccharides were partially purified by HPLC on Superose 12. Two mannosyl transferases from S. kentucky were separated by gel chromatography; these transferases were found useful for chemical-enzymic synthesis of polyprenylpyrophosphate derivatives of trisaccharides Tal-Man-Gal and Man-Tal-Gal. PMID- 1707620 TI - [Uncommon acidic monosaccharides--components of O-specific polysaccharides of Vibrio bacteria]. PMID- 1707621 TI - Pharmacodynamic profile of isbufylline, a new antibronchospastic xanthine devoid of central excitatory actions. AB - 1,3-Dimethyl-7-isobutylxanthine (isbufylline, TE/06; CAS 90162-60-0) is a newly synthetized xanthine derivative. This compound exhibits remarkable antibronchospastic properties both in in vitro and in vivo (after oral or intravenous administration) experimental models. Isbufylline is significantly more effective than theophylline in antagonizing bronchospasms elicited by spasmogens (capsaicin, arachidonic acid, PAF and antigen) which mainly act by a local release of biologically active substances proposed to be involved in the pathogenesis of asthma. Isbufylline, unlike theophylline, possesses little or no CNS excitatory properties. This reduced neuroexcitatory action is probably related to the poor affinity of isbufylline, as compared to theophylline, to A1 purinoceptor. Indeed, isbufylline is ineffective in antagonizing 2Cl-adenosine induced EEG synchronization. In normotensive and hypertensive rats oral administration of isbufylline resulted in small and transient positive chronotropic and hypotensive response, markedly lower than those elicited by theophylline or enprofylline. Finally isbufylline exhibits phosphodiesterase inhibitory properties, although at concentration 50-100 times higher than those exerting spasmolytic effects in isolated bronchial tissues. As a whole the pharmacodynamic profile of isbufylline is promising and, in the clinical setting, this compound might exert enhanced antiasthma effects coupled to a low incidence of central and cardiovascular adverse effects. PMID- 1707622 TI - Cloning and expression of the complete SIVagm pol region in E. coli. Purification and partial characterization of the reverse transcriptase. AB - The complete pol region of the simian immunodeficiency virus from African green monkeys was cloned and expressed in E. coli. The reverse transcriptase was purified to high specific activity and could be shown to contain both reverse transcriptase activity as well as an associated RNase H activity. As is observed with other reverse transcriptases the enzyme is composed of two subunits which cannot be separated by conventional techniques. When comparing the recombinant enzyme with the authentic enzyme isolated from virus no differences were found by biochemical, enzymological, or immunological criteria. Moreover, the action of inhibitors against this enzyme did not show significant differences when compared to reverse transcriptases from HIV-1 and HIV-2. PMID- 1707623 TI - Liver histology, blood biochemistry and RNA, DNA and subcellular protein composition of various skeletal muscles of rats with experimental cirrhosis: implications for alcoholic muscle disease. AB - (1) Liver cirrhosis was induced in male rats by treatment with carbon tetrachloride and phenobarbitone for 130-142 days. Detailed histological examination showed all livers from rats treated with carbon tetrachloride had annular fibrosis, necrosis, loss of normal hepatic architecture and other features that were consistent with an established micronodular cirrhosis. (2) Plasma biochemical analysis showed a significant reduction in total protein concentration (13%), which was due entirely to a reduction in plasma albumin (29%). There were also large increases in the plasma activities of alkaline phosphatase (110%) and aspartate aminotransferase (159%), when compared to phenobarbitone-treated controls. Plasma cholesterol was also increased (67%), but other plasma analytes were not significantly altered. (3) The soleus (Type I), plantaris (Type II) and gastrocnemius (Types I and II) muscles were dissected and examined for possible differential effects. There were minor reductions in all three muscle weights, but these changes did not reach statistical significance. The protein, RNA and DNA concentrations, total muscle content and content relative to body weight in cirrhotic rats were also not significantly altered in any of the muscles. Cirrhosis did not cause any perturbations in derived parameters, i.e. amount of synthetic apparatus per cell, RNA/DNA ratio, apparent cell size, protein/DNA ratio and the capacity for protein synthesis or RNA/protein ratio. (4) The gastrocnemius was fractionated into soluble, stromal and myofibrillar proteins. The concentrations and contents of all three proteins were unaltered in cirrhotic animals, compared to controls. (5) It is concluded that in this experimental model of cirrhosis there were no effects on those skeletal muscle variables which are strikingly altered by chronic alcohol feeding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707624 TI - [How to say it with slides]. PMID- 1707625 TI - Isolation and characterization of monoclonal antibodies directed against different epitopes of type-1-like fimbriae from a multifimbriated E. coli strain. AB - Monoclonal antibodies (MAbs) were raised against purified fimbriae isolated from the uropathogenic E. coli strain WF96 (O7:K1:H6:F11rel,F10). This strain expresses at least four different types of fimbriae. 11 MAbs were selected for further characterization. They are directed against epitopes of a fimbrial type which is composed of 19.5 kDa subunits. It resembles type 1 fimbriae with regard to its high resistance to disruption by SDS. The MAbs were tested for crossreactivity to type 1 fimbriae and other fimbriae with known F-serotypes by ELISA. Two of these MAbs, Pili III 2F7 and Pili III 68C5, were directed against an epitope which was also found on MS fimbriae (type 1). Thus type 1 like fimbriae of E. coli WF96 share at least one epitope with MS fimbriae. Nevertheless, the antigenic properties of these two fimbrial types were found not to be completely identical, since all the other 9 MAbs were not crossreactive. The MAbs were not able to inhibit haemagglutination of erythrocytes of different species and thus not directed against adhesive sites of the fimbriae. All the epitopes detected by MAbs were accessible on native fimbriae; some of them were also detectable on denatured fimbrial subunits. Electron micrographs revealed that these epitopes were evenly distributed on the fimbrial organelle. PMID- 1707627 TI - A prototype bioreductive DNA groove binding ligand. AB - Molecular mechanics calculations have been used to evaluate the potential bioreductive behaviour of several DNA minor groove binding ligands containing quinone/hydroquinone redox systems. The proposed structures are analogues of the Hoechst 33258 molecule with modifications of the benzimidazole rings. Binding energies of simple analogues indicate the reduced forms bind more strongly to the DNA minor groove. N-methylation of the imidazole ring(s) produces structures which can form extended quinone methides. These also show stronger binding in the reduced form and it is speculated that such structures might provide a basis for the design of groove binding ligands which will act as bioreductive alkylating agents. PMID- 1707626 TI - Intermediate filaments vimentin and desmin share epitopes with M 1 protein of group A streptococci. AB - The cross reactivity of the murine monoclonal antibody PM II 40 against human glomeruli with streptococcal type 1 M protein was investigated. The antibody PM II 40 recognized a protective epitope on a glomerulonephritis-associated M type 1 strain. A 23 kD streptococcal surface protein extracted by limited pepsin digestion reacted with PM II 40 in the Western immunoblot. The aminoterminal sequence of this peptide was identical to the known aminoterminal amino acid sequence of type 1 M protein. Human renal podocytes carry the cross-reactive antigen of the antibody PM II 40 as could be shown by electron microscopy. The podocytes cultured from isolated human glomeruli showed a fibrillar pattern with the antibody in the immunofluorescence test. An anti-vimentin antibody and the antibody PM II 40 recognized the same proteins with molecular weights of 54, 52 and 43 kD of SDS-extracted isolated human glomeruli suggesting that vimentin is the glomerular antigen. The antibody PM II 40 not only react with vimentin but also with desmin suggesting that the recognized epitopes are distinct from that described by Kraus et al. for vimentin and type 1 M protein. PMID- 1707628 TI - Modulation of inositol lipid hydrolysis in human breast cancer cells by two classes of bombesin antagonist. AB - Inositol lipid hydrolysis was monitored in the human breast cancer cell line MCF 7 in response to various bombesin (BN) and substance P (SP) analogues. Both members of the BN family of peptides, i.e. BN and gastrin-releasing peptide (GRP), stimulated a dose-related increase in total inositol phosphate production, with a similar half-maximal effective dose (ED50) around 1 nM. The BN receptor antagonist [Leu13-psi-CH2NH-Leu14]-BN (LLBN) at 1 microM was devoid of agonist activity and displaced the BN dose-response to the right, resulting in a tenfold increase in the ED50 for BN. BN also stimulated a dose-related increase in 45Ca2+ efflux which was also inhibited by LLBN. Two SP analogues [DArg1,D-Pro2,D Trp7,9,Leu11]-SP and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-SP ([APheTL]-SP), both antagonized BN-stimulated inositol lipid hydrolysis. [APheTL]-SP (60 and 80 microM) alone also exhibited considerable agonist activity which was not antagonized by LLBN. Indeed, a sub-threshold dose of [APheTL]-SP (40 microM) in the presence of LLBN (10 microM) potentiated the inositol lipid hydrolysis response. BN, GRP, LLBN and [APheTL]-SP all inhibited binding of 125I-labelled GRP to MCF-7 cells, to 50% of that occurring in the absence of the peptides, at concentrations of 150 pM, 150 pM, 150 nM and 600 nM respectively. These data are consistent with the presence of separate but interacting receptors or binding sites for BN and SP analogues, which are coupled to a common signal transduction pathway in human breast cancer cells. PMID- 1707629 TI - A marsupial beta-lactoglobulin gene: characterization and prolactin-dependent expression. AB - Analysis of the tammar wallaby beta-lactoglobulin cDNA and inferred amino acid sequences reveal extensive sequence divergence from the eutherian beta lactoglobulins. Conserved residues include the cysteines and a number of individual amino acids involved in structure and ligand-binding. The only region of extended similarity is a heptapeptide sequence which may impart specificity to its interaction with a receptor protein. Northern analysis of total mammary RNA revealed two transcripts which result from differential utilization of polyadenylation signals. The concentration of beta-lactoglobulin mRNA increased in late lactation and correlates with increases in milk production and levels of milk fat. Quantification of beta-lactoglobulin mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the gene is dependent on prolactin alone and that expression is not modulated by other hormones known to play a role in the initiation of lactation in eutherians. PMID- 1707630 TI - [False asthmas of the adult]. AB - Many conditions may simulate asthma, particularly tracheobronchial lesions that are dominated by tumoral stenoses, laryngeal anomalies and cardio-vascular diseases. At the time of the initial assessment of all sibilant dyspneas, certain other complementary examinations should systematically be made: pulmonary radiography, ORL examination and exploration of respiratory function. PMID- 1707631 TI - Gene expression of macrophage colony-stimulating factor and its receptor in human placenta and decidua. AB - Macrophage colony-stimulating factor (M-CSF) induces proliferation of monocyte/macrophage progenitor cells and can also activate some functions of mature cells including fetally derived placental cells. To study the role of M CSF in the pregnant female reproductive tract, the expression of M-CSF mRNA and its receptor, c-fms proto-oncogene, in human placenta and decidua was identified. M-CSF and c-fms mRNAs, 4.7Kb and 3.9Kb respectively, were detected by Northern blotting in the early stage placenta and subsequently increased during pregnancy. These mRNAs were not detected in the nonpregnant endometrium but were strongly induced in maternal decidua with the same mRNA size as in the placenta. Northern blot hybridization on the endometrium of a pseudopregnant uterus revealed that the expression of endometrial M-CSF and c-fms mRNAs is regulated by synergistic action of female sex steroid hormones. These findings indicate that, in an autocrine and/or paracrine manner, M-CSF is deeply involved in the local proliferation and differentiation of cells at the materno-fetal interface, and support the placental immunotrophism hypothesis. PMID- 1707632 TI - Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis? AB - "Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253 1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis. PMID- 1707633 TI - Signal transduction by P2-purinergic receptors for extracellular ATP. AB - Extracellular adenosine triphosphate (ATP), at micromolar/nanomolar concentrations, has been shown to induce significant functional changes in a wide variety of normal and transformed cell types. While ATP can be nonspecifically released from the cytosol of damaged cells, it is also co-packaged in certain exocytotic vesicles/granules containing conventional neurotransmitters and hormones. The diverse biologic responses to ATP appear to be mediated by a variety of so-called P2-purinergic, cell surface receptors that are activated upon binding ATP and other nucleotides. Recent physiologic, biochemical, and pharmacologic studies suggest that there are multiple ATP receptor subtypes. These include: (1) G-protein-coupled ATP receptors, which stimulate inositol phospholipid hydrolysis, Ca2+ mobilization, and activation of protein kinase C; (2) ATP receptors that directly activate nonselective cation channels in the plasma membranes of a variety of excitable cell types; and (3) ATP receptors that, via the rapid induction of surface membranes pores permeable to both ions and endogenous metabolites, can produce cytotoxic or activation responses in T lymphocytes and other immune effector cells. In addition to these functional criteria, these putative ATP receptor subtypes can be distinguished by characteristic selectivities for a variety of structurally modified ATP analogs. Current research is directed towards the identification, isolation, and structural characterization of these receptors by both biochemical and molecular biologic approaches. PMID- 1707634 TI - In situ expression of transforming growth factor beta in streptococcal cell wall induced granulomatous inflammation and hepatic fibrosis. AB - The expression of transforming growth factor beta (TGF-beta) was examined during the evolution of streptococcal cell wall (SCW)-induced hepatic granulomas in rats to evaluate the role of TGF-beta in chronic inflammation progressing to fibrosis. As determined by immunocytochemistry, Kupffer cells rapidly expressed TGF-beta 1 following intraperitoneal (i.p.) injection of SCW, and TGF-beta was expressed by mononuclear phagocytes in the earliest cell aggregates as well as by mononuclear phagocytes within the capsule of mature lesions. Interestingly, apparent extracellular TGF-beta was observed in mature lesions at the interface of the capsule and the cellular core, a region of active fibrogenesis. Granulomas isolated 3, 6, and 12 weeks post-SCW injection elaborated nanogram (ng) quantities of latent and active TGF-beta into culture supernatants, and expressed high levels of 2.4 and 1.9 kb TGF-beta 1 transcripts. Expression of procollagen type I and III mRNAs were observed in parallel with the expression of the TGF beta 1 transcripts. Thus, TGF-beta is expressed throughout SCW-granuloma development, and, based on known bioactivities, it appears that TGF-beta mediates, in part, the recruitment and activation of monocytes and fibroblasts and deposition of collagen in SCW-granulomas and likely other chronic inflammatory lesions progressing to fibrosis. PMID- 1707635 TI - TGF-beta regulates production of growth factors and TGF-beta by human peripheral blood monocytes. AB - Transforming growth factor beta 1 (TGF-beta 1) and its closely related homologue, TGF-beta 2, rapidly induce growth factor gene expression by freshly isolated human peripheral blood monocytes. Within 3 h of exposure to TGF-beta, mRNA species specific for interleukin-1 (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) were observed. By 14-18 h, cytokine bioactivity and protein were detected in the culture supernatants. Furthermore, not only TGF-beta 1, but also TGF-beta 2 mRNA are expressed constitutively in unstimulated monocytes. However, in response to exogenous TGF-beta (beta 1 or beta 2), only TGF-beta 1 gene expression is upregulated, and the expression of TGF-beta 2 mRNA is unchanged. This selective autoinduction of TGF-beta 1 appears to be controlled at both transcriptional and post-transcriptional levels. These paracrine and autocrine activities of TGF-beta suggest potential mechanisms through which an inflammatory response can be initiated and amplified. In addition, the TGF-beta enhancement of growth factor generation may promote fibrosis and angiogenesis relevant to physiological tissue repair as well as pathological fibrotic sequelae. PMID- 1707636 TI - Characterization of two preparations of antibodies to basic fibroblast growth factor which exhibit distinct patterns of immunolocalization. AB - Immunoglobulins reactive against basic fibroblast growth factor (bFGF) were obtained from the serum of a single rabbit immunized against residues [1-24] of bFGF conjugated to keyhole limpet hemocyanin (KLH). Pure immunoglobulin preparations no. 1 and no. 2 were prepared using different affinity chromatography columns and preabsorption to KLH-coupled Sepharose for preparation no. 1. Both preparations no. 1 and no. 2 were specific for bFGF in in vitro assays. Competition with synthetic peptides suggests that preparations no. 1 and no. 2 recognize predominantly epitope(s) within residues [16-24]bFGF or residues [1-10]bFGF, respectively, in situ. Furthermore, no. 2 (but not no. 1) antibodies can react with tissue-(heparin-)-bound antigen. When used in indirect immunofluorescence for bFGF in frozen heart sections, preparation no. 1 stained predominantly muscle intercalated discs (IcDs); muscle nuclei were also stained, in an overall punctate fashion. Preparation no. 2 stained muscle nuclei strongly, in association with the nuclear envelope; it also stained basement-membrane associated bFGF. Differences in immunostaining were also observed in uterine smooth muscle and kidney sections but not in skeletal muscle. It is plausible that accessibility of various epitopes within the amino-terminal region depends strongly on the local interactions of bFGF. Our data illustrate the importance of using several different antibodies to localize bFGF in a tissue. PMID- 1707637 TI - Increased oxidative metabolism in phytohemagglutinin-stimulated lymphocytes from patients with systemic lupus erythematosus is associated with serum SSA antibody. AB - We have examined oxidative metabolism in phytohemagglutinin (PHA)-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE) because increased oxygen free radicals would explain the DNA abnormality previously observed in these cells. Almost no oxidative activity was found in freshly isolated control or lupus lymphocytes or control lymphocytes stimulated with PHA. However, increased oxidative metabolism, measured by nitroblue tetrazolium (NBT) conversion to formazan, was found in PHA-stimulated lymphocytes from 14 of 21 lupus patients. A time course study showed that NBT activity appeared in positive lupus lymphocytes at 1-2 days of PHA stimulation, increased to a maximum at 2-4 days, and diminished thereafter. NBT activity was not related to specific disease symptoms, drug therapy, or serum dsDNA, Sm, RNP, or SSB (La) antibodies. The selected population of lupus patients studied precluded conclusions about NBT activity and disease severity. However, the intensity of NBT response in stimulated lupus lymphocytes was positively correlated with the presence of serum SSA (Ro) antibody. We suggest that increased oxidative activity of SLE lymphocytes generates a chemical change in endogenous DNA in vivo and may be a primary event in the pathogenesis of autoimmunity. Absence of detectable oxidative activity in stimulated lymphocytes in a subgroup of lupus patients suggests that at least two different mechanisms are associated with the altered DNA profiles observed in this disorder. PMID- 1707638 TI - Is there a neutralization epitope in the second conserved domain of HIV-1 envelope protein? PMID- 1707639 TI - Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines. AB - A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24 h. HIV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HIV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines. PMID- 1707640 TI - Epitopes of the HIV-1-negative factor (nef) reactive with murine monoclonal antibodies and human HIV-1-positive sera. AB - Murine monoclonal antibodies (MAbs) raised against a recombinant nef protein fragment of human immunodeficiency virus type 1 (HIV-1) strain BH10 were characterized by an epitope mapping system using overlapping decapeptides. Four different immunogenic regions were identified. Ten human HIV-1-positive sera were tested in the same epitope mapping system, seven of these were reactive with four immunogenic regions. Two of the nef-specific epitopes recognized by human sera overlapped with the epitopes defined by the murine monoclonal antibodies. The reactivity of the monoclonal antibodies with the recombinant nef protein and with infected and uninfected cells were investigated in a variety of test systems. The results are discussed with respect to homologous regions of nef and cellular proteins. PMID- 1707641 TI - Re-evaluation of the involvement of the adhesion molecules ICAM-1/LFA-1 in syncytia formation of HIV-1-infected subclones of a CEM T-cell leukemic line. AB - The role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and LFA-1 in human immunodeficiency virus type 1 (HIV-1)-induced cell fusion was investigated in subclones of a T-cell leukemic cell line (CEM) with differing abilities to form syncytia. Addition of monoclonal antibodies 84H10 directed against ICAM-1 and MHM23 directed against the common beta subunit of LFA-1 (CD18) resulted in greater than 50% suppression of syncytia formation in cultures of these clones infected with cell-free virus. Two subclones, 2G5-144-84 and 2G5-1, were deficient in their ability to form syncytia and expressed reduced amounts of LFA-1 compared with the parental line. The expression of ICAM-1 but not LFA-1 was upregulated on the clones following treatment with interferon-gamma (IFN gamma); however, this did not overcome the delay in syncytia formation observed in these cells. The syncytia-positive subclones 1B11-39 and 17D-9 expressed high levels of LFA-1. Basal expression of ICAM-1 was upregulated on these cells by treatment with tumor necrosis factor-alpha (TNF alpha), which also accelerated and enhanced syncytia formation. However, anti-ICAM-1 and anti-LFA-1 (CD18) antibodies did not reverse the TNF alpha-induced enhancement of syncytia formation of HIV-1-infected clones 1B11-39 and 17D-9. Under conditions of low viral expression, adhesion molecules may contribute to syncytia formation if adequate levels of both receptor and ligand in the ICAM-1/LFA-1 complex are expressed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707642 TI - Inhibition of human immunodeficiency virus (HIV-1) replication in vitro by noncytotoxic doses of camptothecin, a topoisomerase I inhibitor. AB - We examined the effects of topoisomerase inhibitors on human immunodeficiency virus type 1 (HIV-1) infection of H9 cells in cell culture. Infection is blocked or substantially reduced by the topoisomerase I inhibitor camptothecin (CPT), but not by two topoisomerase II inhibitors. Significant reduction (greater than or equal to 90%) in the amount of virus released, as measured by reverse transcriptase, is obtained if the cells are treated for 1 h with 0.01-0.02 microM CPT at the time of virus infection, and expression of viral proteins is also blocked. CPT is also shown to reduce the level of infection when chronically infected cells are cocultivated with uninfected cells. These results with CPT suggest that this compound may represent a new class of drugs with antiretroviral potential. PMID- 1707643 TI - A monocyte-derived factor interferes with detection of reverse transcriptase in HIV-1 infection. AB - Culture supernatants from the rabbit macrophage cell line 6083 infected with a retrovirus, human immunodeficiency virus type 1 (HIV-1), were negative for reverse transcriptase (RT) expression although the line was shown to be productively infected by all other criteria tested. Supernatants from uninfected cultures of 6083, the human monocyte line U937, and from freshly isolated peripheral human monocytes, were found to contain a monocyte-derived inhibitory factor (MDIF) which interferes with a standard assay for RT. MDIF is a heat labile activity of approximately of 40 kD. Both substrates and products of the reverse transcriptase assay are degraded by MDIF which is not affected by reduction and alkylation of disulfide bonds. MDIF is inhibited by the addition of a particular thioated oligonucleotide (S-dG30) to the reaction mixture but this addition also inhibits RT. The optimum method to minimize MDIF interference in the RT assay is by addition of ethylene glycol bis-(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA); MDIF requires divalent cations for activity and has a strong preference for calcium which is preferentially chelated by EGTA. The potential presence of this inhibitory activity should be considered when using RT levels as a measure of retroviral infection. PMID- 1707644 TI - Susceptibility of primary human glial fibrillary acidic protein-positive brain cells to human immunodeficiency virus infection in vitro: anti-HIV activity of memantine. AB - Primary human glial fibrillary acidic protein-positive (GFAP+) brain cells (enriched population) have successfully been infected with human immunodeficiency virus type 1 (HIV-1) in vitro, when cocultivated with HIV-1-producing H9 cells. Direct incubation of brain cells with HIV-1 resulted only in a limited infection. The percentage of HIV+ cells increased from 5% in passage 1 to 40% in passage 8. Simultaneously with the increase of infected cells, the reverse transcriptase activity in the culture medium increased and reached maximal values in passage 8. The infected cells also produced intact viral particles. In the early phase of cultivation the HIV-infected cells displayed a significantly higher proliferation rate than the uninfected controls. At passage number 8 the HIV-infected GFAP+ cells had almost totally lost the ability to grow, while the controls proliferated at a rate almost unimpaired from the beginning of the cultivation. Up to 10 to 15% of the HIV-infected GFAP+ cells contained at passage number 5 more than 3 nuclei. Memantine (1-amino-3,5-dimethyladamantane), a blocker of the N-methyl-D-aspartate receptor channels, was found to display a significant anti HIV effect (at a concentration of 1 microgram/ml) on enriched cultures of GFAP+ cells in vitro. PMID- 1707645 TI - Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2. AB - Five mouse monoclonal antibodies were raised against a recombinant protein comprising the complete sequence of gag24 protein from HTLV-IIIB. All monoclonal antibodies recognized the native protein in enzyme-linked immunosorbant assay (ELISA) and Western blots. All monoclonal antibodies also cross-reacted with an human immunodeficiency virus type 2 (HIV-2) strain in western blots, whereas only one antibody detected HIV-2 p25 in ELISA. By using synthetic peptides, cross reacting epitopes were mapped and three regions were defined. The conserved immunogenic sites were located in the carboxyterminal region of the protein. Inhibition experiments with human sera showed that this region is also immunogenic in humans. PMID- 1707646 TI - The effects of enalapril and sulindac on the dermal response to substance P and neurokinin A. AB - The effects of pretreatment with enalapril, and sulindac, on the weal response to intradermal injections of substance P and neurokinin A were assessed in a randomised, double-blind, placebo-controlled study. Weal responses to both substance P and neurokinin A depended significantly on dose. Neither enalapril nor sulindac, nor the combination of these agents influenced the responses to either tachykinin. These results do not suggest any role for substance P or neurokinin A in the clinical effects of angiotensin converting enzyme inhibitors. PMID- 1707647 TI - Sodium hyaluronate increases vascular ingrowth in the rabbit ear chamber. AB - The rabbit ear-chamber model was used to study the effect of sodium hyaluronate (NaHe, Healon) on the rate of ingrowth of vascular structures of healing granulation tissue. The chamber area covered by granulation tissue was determined by in-vivo microscopy at regular intervals during a period of 32 days. Daily injections of 1% NaHe into the ear chamber, 50 microliters from day 0 to day 7 and 25 microliters from day 8 to day 21, significantly inhibited ingrowth as observed between days 20 and 26, compared with buffer-injected controls. There was no difference between the latter and non-injected chambers. Intermittent injections of 1% NaHe, 50 microliters on days 1 and 5 and 25 microliters on days 9, 13 and 22 significantly increased the ingrowth as observed between days 6 and 18. It was noted that wound macrophages internalized fluorescein-labelled NaHe. The inhibitory effect of daily injections on angiogenesis was probably due to physical hindrance caused by the NaHe. The stimulatory effect of intermittent administration of NaHe on angiogenesis may have several explanations, including activation of macrophages and their release of angiogenetic factors. PMID- 1707648 TI - Detection of blood group A and H-related antigens in normal and neoplastic bladder epithelium: a comparative study using monoclonal antibodies with defined fine specificities. AB - Three monoclonal anti-blood group H and four monoclonal anti-blood group A antibodies directed at the blood group antigens on Type 1 or Type 2 backbone structures were evaluated as immunohistochemical reagents by indirect immunofluorescence of normal and neoplastic bladder epithelia. The results were compared with fluorescence using polyclonal rabbit anti-A or H sera or Ulex europaeus lectin. The monoclonal antibodies gave less intense or more restricted immunofluorescence than the conventional reagents but showed considerable variation in the extent of their reactivities with urothelial samples from different individuals. In some cases they failed to give immunofluorescence with tissue samples known to contain the immunodominant blood group structures they recognize. In addition, hitherto unsuspected heterogeneity was revealed in the expression of the Type 2-based blood group H and A-structures in the endothelia of neighbouring small blood vessels. PMID- 1707649 TI - Evidence for an alpha 2-macroglobulin with complement-inhibiting activity in rat serum. AB - alpha 2-Macroglobulin (alpha 2M) was purified both from the serum of male rats developing an acute turpentine-induced inflammatory reaction where its concentration is greatly increased (3-4 mg/ml) and from the serum of healthy males where it is present at low levels (15-30 micrograms/ml). A three-step purification procedure involving gel filtration, anion exchange chromatography on DEAE cellulose and negative immunoaffinity was used. A pure native alpha 2M, as assessed by biochemical and immunological tests, was obtained. This alpha 2M differed from other subforms in terms of its electric charge and its complement inhibiting activity in a complement-dependent immune haemolysis test. Moreover, this inhibitory activity was not affected by complexing with trypsin or modification by interaction with methylamine showing that this newly described property is not linked to the well known antiproteinase function of alpha 2M. PMID- 1707650 TI - Degenerative and regenerative changes in murine skeletal muscle after injection of venom from the snake Bothrops asper: a histochemical and immunocytochemical study. AB - The degenerative and regenerative changes in murine skeletal muscle after injection of Bothrops asper venom were studied by histological, lectin histochemical and immunocytochemical techniques. According to our observations, the process was divided into four main stages: (a) During the first 3 days prominent degenerative events took place in skeletal muscle fibres, capillaries, arteries, veins and intramuscular nerves. An inflammatory infiltrate was abundant after the first day and removal of necrotic material was well advanced by the third day. (b) Muscle regeneration was evident by the fourth day. From 4 to 6 days there were two populations of regenerating muscle fibres, one of apparently normal fibres located in areas where capillary vessels were abundant, and another population of groups of regenerative fibres showing signs of degeneration. This second type of fibre was predominant in areas where the number of capillaries was greatly reduced. (c) One and 2 weeks after envenomation areas of small regenerative fibres of normal morphology and areas of degenerating regenerative fibres were observed. The latter were abundant in regions of dense fibrotic tissue and scarce capillaries. (d) Finally, at 4 and 8 weeks after envenomation there were both areas of fibrosis and areas where regenerating muscle fibres predominated. However, the diameter of these fibres was abnormally small, an indication that they may have been atrophic fibres. It is suggested that muscle regeneration is partially impaired after myonecrosis induced by Bothrops asper venom, probably due to the damage induced by this venom on muscle microvasculature and nerves. PMID- 1707651 TI - Presence of acute phase response in coal workers' pneumoconiosis. AB - To evaluate the role of personal factors in pneumoconiosis, several acute phase proteins were studied in 62 coal miners without acute illnesses and classified as having no pneumoconiosis (n = 19), simple pneumoconiosis (n = 23), or complicated pneumoconiosis with progressive massive fibrosis (n = 20). Groups were similar for age, years of work at high risk jobs, chronic bronchitis, and forced expiratory volume in one second (FEV1). C-reactive protein concentration was significantly higher in the simple and complicated pneumoconiosis groups in comparison with the no pneumoconiosis group. The C-reactive protein concentration was above the upper normal value in 12 (27.9%) out of 43 cases with simple and complicated pneumoconiosis. On the other hand only one case of no pneumoconiosis was above the upper normal range (5.3%), a significant difference taking into account a stratified analysis for chronic bronchitis. Fibrinogen concentration was significantly increased in the simple pneumoconiosis group compared with the no pneumoconiosis group. The value of fibrinogen was above the upper normal limit in 17 out of the 43 cases with pneumoconiosis (39.5%) by contrast with two cases in the no pneumoconiosis group (10.5%). No significant differences in alpha 1 antitrypsin and ceruloplasmin concentrations were found between groups. In conclusion, an alteration in some acute phase proteins related to pneumoconiosis was found in miners. This could be used as a marker of disease activity and personal response against the pathogenic agent. PMID- 1707652 TI - Convergence of processing channels in the extrastriate cortex of monkeys. AB - The first (V-I) and second (V-II) visual areas of primates contain three types of anatomical segregations of neurons as parts of hypothesized "P-B" or "color", "P I" or "form," and "M" or "motion" processing channels. These channels remain distinct in relays of P-B and P-I information to the inferior temporal lobe via V II and dorsolateral visual cortex for object recognition, and "M" information to posterior parietal cortex via the middle temporal visual area (MT) for visual tracking and attention. The present anatomical experiments demonstrate another channel where "P-B" modules in V-I and "P-B" and "M" modules in V-II merge in the projections to the dorsomedial visual area (DM), which relays to MT and posterior parietal cortex. This integrative area may function in unifying our perception of the visual world, and may allow "color" as well as "motion" to play a role in visual tracking and attention. PMID- 1707653 TI - [Tumor-degenerating factor (TDF)]. AB - A new human fibroblast-producing factor which degenerates human tumor cells in vitro was found and designated as tumor-degenerating factor (TDF). The characteristics of the TDF were as follows: 1) The molecular weight of the TDF was estimated to be 30k by SDS-PAGE. 2) It may be basic and glycosylated. 3) Its activity was inhibited by fibronectin and, vice versa, by binding TDF molecule with fibronectin molecule. 4) Its activity was enhanced by interferons. It is possible that the TDF is an important substance concerning the interaction between tumor cells and interstitial cells. PMID- 1707654 TI - [Establishment and characterization of a human hepatocellular carcinoma cell line JHH-7 producing alpha -fetoprotein and carcinoembryonic antigen--changes in secretion of AFP and CEA from JHH-7 cells after heat treatment]. AB - A human hepatocellular carcinoma cell line, JHH-7, was established from resected liver tumor of a 53 year old male with hepatitis B virus infection. JHH-7 was composed of polygonal epithelial cells and functionally synthesized and secreted human albumin, AFP, CEA and ferritin. No HBsAg was detected in the culture supernatant of JHH-7 cells. Changes of secretion of AFP and CEA from JHH-7 cells after heat treatment was studied using a temperature gradient incubator. Secretion of AFP decreased along with the inhibition of cell proliferation by heat treatment. Secretion of CEA, however, did not decrease even though the cells were damaged. PMID- 1707655 TI - Degenerate binding of immunogenic peptides to HLA-DR proteins on B cell surfaces. AB - Binding of linear fragments of protein antigens to class I or class II molecules of the MHC is necessary for the stimulation of a cellular immune response. This report describes the binding of a biotinylated T cell determinant from influenza hemagglutinin to class II proteins on the surface of Epstein-Barr virus transformed B lymphocytes. The rapid, simple, and quantitative binding assay involves flow cytometric analysis of transformed B cells stained with fluoresceinated streptavidin following incubation with the biotinylated peptide. Binding of the biotinylated peptide required cell surface expression of human class II molecules, and was inhibited by an anti-HLA-DR monoclonal antibody as well as the unbiotinylated natural determinant. Rates of association and dissociation of the peptide were similar to those reported for purified MHC class II proteins, and the peptide bound only approximately 1% of the DR molecules expressed on the cell surface. When assayed on many different DR-homozygous B cell lines, the biotinylated hemagglutinin T cell determinant bound to HLA-DR on each cell line. The degeneracy of peptide binding to B cell lines was not unique to the hemagglutinin peptide because three other biotinylated T cell determinants failed to bind to class II deficient B-lymphoblastoid cells but bound to varying degrees to multiple DR-homozygous lines. PMID- 1707656 TI - Different types of antigen-presenting cells affect the induction of experimental autoimmune arthritis. AB - We obtained a type II collagen-specific murine T cell line containing at least two T cell clones, one reacting with only native collagen II and the other with both denatured and native molecules. Only the former could induce arthritis. The arthritogenic T cell clone(s) was preferentially stimulated to grow when epidermal cells were used as antigen-presenting cells. Conversely, the non arthritogenic T cell clone(s) was mainly stimulated when spleen cells were used. Thus, it is speculated that different types of antigen-presenting cells preferentially present different epitopes on the same antigen, affecting the resulting in vivo immune phenomena. PMID- 1707657 TI - Differential expression of the interleukin 2 receptor beta (p75) chain on human peripheral blood natural killer subsets. AB - The interleukin-2 receptor (IL-2R) is composed of at least two subunits, the IL 2R alpha (P55, CD25/Tac) and IL-2R beta (p70-75) chains. In the present study we have identified the quantitative expression of IL-2R beta on subsets of NK cells in peripheral blood using flow cytometry and a recently established mAb against IL-2R beta (TU27). We demonstrate that NK cells are not a homogeneous population but that phenotypic subsets exist, and that the levels of IL-2R beta expression correlate with these subsets. The levels of IL-2R beta expression on NK subsets are CD3- CD16- CD56bright NK cells (immature NK) greater than CD16+CD4- NK cells (mature NK) greater than CD16+CD57+ NK cells (more mature NK) in adult peripheral blood lymphocytes. The level of IL-2R beta expression on CD16+ NK cells in cord blood is significantly higher than that in adult PBL. These findings suggest that there may be a inverse relationship between IL-2R beta expression and the differentiation of NK cells. IL-2R beta, preferentially expressed on the NK cells, may play an important role in differentiation and maturation of NK cells. PMID- 1707658 TI - All T15 Id-positive antibodies (but not the majority of VHT15+ antibodies) are produced by peritoneal CD5+ B lymphocytes. AB - After adult irradiation and reconstitution with autologous bone marrow, BALB/c and C.B20 mice no longer utilize the T15 Id in response to phosphorylcholine. T15 Id expression can be restored by transfers of peritoneal B cells or by FACS purified CD5+ IgM+ lymphocytes (but not by T lymphocytes) from syngeneic donors. Using bone marrow and peritoneal cell donors that are congeneic for heavy and light chain allotypes, the exclusive origin of the T15 Id in peritoneal B cells was ascertained. These conclusions have been essentially confirmed by immunization with either anti-T15 Id or anti-VHT15 antibodies conjugated to lipopolysaccharide. Thus, the production of VHT15-positive antibodies continues at control levels in bone marrow-reconstituted animals while no T15 Id production can be stimulated even in this protocol of direct B cell stimulation. These results constitute the first formal demonstration of the exclusive production of and Id by CD5+ B-cells. PMID- 1707659 TI - The human class I MHC gene HLA-F is expressed in lymphocytes. AB - Genomic and cDNA clones encoding a human, non-classical class I gene, Dew3, have been isolated. The complete coding sequence has been determined. The sequence is capable of directing expression of a protein with high sequence homology to HLA A,B,C molecules but with a shortened cytoplasmic tail. Sequence comparisons demonstrate that this gene is from a separate locus to the 'classical' HLA-A,B,C and 'non-classical' HLA-E and HLA-G loci. Dew3 is equally distantly related to all of these previously described, functional class I genes. It is, however, extremely homologous to a third 'non-classical' gene, HLA-5.4, and to the chimpanzee gene, Ch28. The RNA species it transcribes is shorter than that of the classical genes, due to an altered acceptor splice site which results in the loss of exon 7. The transcription of Dew3 RNA shows a unique pattern of tissue distribution, being expressed in B cell lines and peripheral blood lymphocytes and absent from T cell lines, fibroblasts and a myelomonocytic leukaemia. A Dew3 protein product was detected after transfection into a human EBV-transformed B cell line but was located intracellularly. The HLA-5.4 gene has been recently designated HLA-F. The Dew3 and X5.1 clones thus represent two new alleles of the HLA-F locus in man. Sequence comparison with its chimpanzee homologue suggests that selective pressure for conservation of amino acid sequence is still maintained at this locus. PMID- 1707660 TI - Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein specific T cell clones correlates with encephalitogenicity. AB - Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the context of I-Au. A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-alpha) mRNA and biological activity on L929 cells and their capacity to induce paralysis, the clinical hallmark of experimental allergic encephalomyelitis (EAE). No correlation was found between interleukin-2 or gamma interferon production and encephalitogenicity. LT and/or TNF-alpha may play a central role in the pathogenesis of EAE. PMID- 1707661 TI - Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface. AB - It was previously reported that rat keratinocytes grown at the air-liquid interface on collagen gels or on nylon membranes produce multilayered cultures of uniformly stratified cells, comparable to the epidermis in situ by morphological and biochemical criteria. A protocol has now been developed by which primary human keratinocytes grown for two weeks submerged on microporous nylon membranes and raised to the air-liquid interface for an additional three weeks, exhibit most of the comparable characteristics of the epidermal cells in vivo. Staining with fluorescein isothiocyanate-conjugated monoclonal antibodies indicated the presence of 56,5 and 65-67 kDa keratins as well as filaggrin-type proteins in the upper cellular layers. Desmosomes, lamellar granules and keratohyalin-like granules were observed. Cultures were covered with layers of cornified cells. This study differs from the majority of other investigations on human keratinocytes in that no feeder layers or other biological substrata were used. This system should be useful in toxicological studies of chemicals which are to be applied topically to the skin. PMID- 1707662 TI - Fluctuation analysis of amiloride-blockable currents in membrane patches excised from salt-taste receptor cells. PMID- 1707663 TI - The chloride-stimulated K(+)-secretion by insect midgut and its modification in the presence of osmotic gradients: a short-circuit current and noise-analysis study. AB - K(+)-secretion in the midgut of the larval moth, Manduca sexta, was studied by measuring the kinetics of the lumen-directed short-circuit current (Isc) and the conduction noise from basolateral K+ channel block by Ba2+. Hemolymph chloride as well as hypotonicity both stimulate this K+ current (IK). The kinetic nature of the stimulation is, however, different in each case. Analysis of blocker noise supports, to a large degree, the interpretation obtained from kinetics, namely: chloride ions do not act via changes in cell volume but influence ion turnover and channel number. PMID- 1707664 TI - Epileptogenicity and cellular currents in rat hippocampus during ontogenesis. PMID- 1707665 TI - Ionic channels and proteins in synaptic vesicles: facts and speculations. PMID- 1707666 TI - Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex. AB - The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2 nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension. PMID- 1707667 TI - Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase. AB - Succinylfluorescein-labeled dideoxyTTP has been used as a substrate for reverse transcriptase from HIV-1. On addition to the 3'-end of a primer molecule, there is a reduction of fluorescence yield of a factor of ca. 4. Release of a fluorescent DNA/DNA primer/template duplex from its complex with reverse transcriptase results in a reduction of fluorescence by a further factor of 2. The fluorescent nucleotide is incorporated somewhat less efficiently than 3' azidoTMP and TMP, which show similar incorporation kinetics. Fluorescent chain terminated primers have been used to investigate the interaction of normal and chain-terminated primer/template complexes with reverse transcriptase. The dissociation constant of a 36/18-mer was 0.65 nM, whereas that of the same complex after the addition of the fluorescent chain-terminating nucleotide to the primer was 3 nM at 25 degrees C. The rate of dissociation of the latter complex from the enzyme was 0.04 s-1. This was decreased by a factor of ca. 10 at high concentrations (greater than 200 microM) of the nucleotide triphosphate complementary to the next position of the template. The results obtained suggest that potent inhibition of reverse transcriptase activity in in vitro assays results from formation of a slowly dissociating complex between the enzyme and chain-terminated primer/template complexes. However, arguments are presented that lead to the conclusion that this is not the mode of inhibition in cells invaded by HIV. At the prevailing relative concentrations in this situation, chain termination resulting in incomplete transcription is likely to be the major factor. PMID- 1707668 TI - Thyrotropin-releasing hormone regulation of thyrotropin beta-subunit gene expression involves intracellular calcium and protein kinase C. AB - Our previous studies demonstrated TRH stimulation of TSH beta gene expression in rat pituitary cell cultures and GH3 tumor cells in a transient expression assay. To begin to characterize the gene-proximal elements of the pathways involved in TRH stimulation of TSH beta gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca2+]i, or activate protein kinase C on TSH beta promoter activity in transfected GH3 cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSH beta promoter activity at 8 h similar to TRH (2-3-fold). TPA did stimulate protein kinase C activation without [Ca2+] mobilization. The calcium ionophore ionomycin increased cytoplasmic free [Ca2+] by stimulating both calcium influx and release from internal stores without affecting protein kinase C. Ionomycin also stimulated a dose-dependent increase (2-fold) in TSH beta promoter activity at 8 h. However, the voltage-dependent Ca2+ channel agonist Bay K 8644, which increased influx of extracellular calcium, had little or no effect on TSH beta gene expression until 48 h (5-fold). Similar effects on prolactin/mRNA levels were observed in these cells. Effects of these factors were not additive, suggesting a common pathway(s) to stimulate gene expression. Inhibition of intracellular calcium mobilization by treatment with 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) inhibited ionomycin effects on gene expression without affecting phorbol ester activity, and, conversely, inhibition of protein kinase C activity by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707669 TI - Induction of several acute-phase protein genes by heavy metals: a new class of metal-responsive genes. AB - Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals, whereas very little is known about the effect of heavy metals on acute phase-reactant genes. We have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants alpha 1-acid glycoprotein, C reactive protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. alpha 1-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd greater than Pb greater than Cu greater than Ni greater than Zn greater than Mg. None of the metals affected the mRNA levels of albumin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced alpha 1-acid glycoprotein and C reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the alpha 1-acid glycoprotein and C-reactive protein promoters. Additionally, an alpha 1-acid glycoprotein expression vector, pAGP(-595)CAT, responded to Hg and Cd when transfected into human HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707670 TI - N-ethylmaleimide and mercurials modulate inhibition of the mitochondrial inner membrane anion channel by H+, Mg2+ and cationic amphiphiles. AB - Previously it has been shown that the mitochondrial inner membrane anion channel is reversibly inhibited by matrix Mg2+, matrix H+ and cationic amphiphiles such as propranolol. Furthermore, the IC50 values for both Mg2+ and cationic amphiphiles are dependent on matrix pH. It is now shown that pretreatment of mitochondria with N-ethylmaleimide, mersalyl and p-chloromercuribenzenesulfonate increases the IC50 values of these inhibitors. The effect of the mercurials is most evident when cysteine or thioglycolate is added to the assay medium to reverse their previously reported inhibitory effect (Beavis, A.D. (1989) Eur. J. Biochem. 185, 511-519). Although the IC50 values for Mg2+ and propranolol are shifted they remain pH dependent. Mersalyl is shown to inhibit transport even in N-ethylmaleimide-treated mitochondria indicating that N-ethylmaleimide does not react at the inhibitory mercurial site. However, the effects of N-ethylmaleimide and mersalyl on the IC50 for H+ are not additive which suggests that mercurials and N-ethylmaleimide react at the same 'regulatory' site. It is suggested that modification of this latter site exerts an effect on the binding of Mg2+, H+ and propranolol by inducing a conformational change. It is also suggested that a physiological regulator may exist which has a similar effect in vivo. PMID- 1707671 TI - Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor--a model study. AB - The inhibitory glycine receptor (GlyR) of rat spinal cord contains an intrinsic transmembrane channel mediating agonist-gated anion flux. Here, synthetic peptides modelled after the predicted transmembrane domains M2 and M4 of its ligand-binding subunit were incorporated into lipid vesicle membranes and black lipid bilayers to analyze their channel forming capabilities. Both types of peptides prohibited the establishment of, or dissipated, preexisting transmembrane potentials in the vesicle system. Incorporation of peptide M2 into the black lipid bilayer elicited randomly gated single channel events with various conductance states and life-times. Peptide M4 increased the conductance of the bilayer without producing single channels. Exchange of the terminal arginine residues of peptide M2 by glutamate resulted in a significant shift towards cation selectivity of the respective channels as compared to peptide M2. In conclusion, the peptide channels observed differed significantly from native GlyR in both conductivity and ion-selectivity indicating that individual synthetic transmembrane segments are not sufficient to mimic a channel protein composed of subunits with multiple transmembrane segments. PMID- 1707672 TI - Reverse enzyme synthesis in microemulsion-based organo-gels. AB - Lipase from three different sources has been immobilised in microemulsion-based gels (MBGs) with retention of catalytic activity. Such lipase-containing MBGs prove to be novel solid-phase catalysts for use in apolar organic solvents such as n-heptane. Using these systems, preparative-scale synthesis of a wide variety of esters under mild conditions was possible with products easily isolated and obtained in high yield. Stereoselective esterification of octan-2-ol was observed for all three lipases with Chromobacterium viscosum (CV) lipase yielding product with an enantiomeric excess of 92%. Repeated usage of a CV lipase-containing MBG resulted in a visually unchanged gel whose activity was 75% of the initial value after 30 days. The sectioned MBGs were well suited for use in column flow reactors and were also found to be effective esterification catalysts at temperatures as low as -20 degrees C. PMID- 1707673 TI - Mapping epitopes on the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum using fusion proteins. AB - Epitopes for a number of monoclonal antibodies (mAbs) binding (Ca(2+)-Mg2+) ATPase purified from skeletal muscle sarcoplasmic reticulum have been defined by studying binding to fusion proteins generated from cDNA fragment libraries. Comparison of these results with those of previous studies of binding of mAbs to proteolytic fragments of the ATPase have allowed the definition of the epitopes to within approx. 100 residues and for one (mAb 1/2H7) to within 45 residues. The experiments suggest considerable exposure of the nucleotide binding domain of the ATPase on the top surface of the protein. Those mAbs that were found to inhibit steady-state ATPase activity were found to bind to epitopes in the nucleotide binding domain of the ATPase. PMID- 1707674 TI - Cellular mechanisms of bradykinin-induced hyperpolarization in renal epitheloid MDCK-cells. AB - Previous studies have demonstrated that bradykinin hyperpolarizes the cell membrane of subconfluent MDCK cells by increase of the potassium conductance. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of bradykinin on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate diester (TPA). In untreated cells, bradykinin leads to a transient increase of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, increase of Cai, activation of potassium channels and hyperpolarization of the cell membrane. The effects of bradykinin on PD and Cai are still present in the absence of extracellular calcium. In cells pretreated with pertussis toxin the effect of bradykinin on inositol trisphosphate formation is almost abolished but bradykinin still leads to a transient increase of Cai and PD in the presence and absence of extracellular calcium. In cells pretreated with TPA the bradykinin-induced increase of inositol trisphosphate formation is blunted, the bradykinin-induced increase of Cai abolished, but the bradykinin-induced hyperpolarization still present. The observations indicate that bradykinin increases Cai in part by phorbol ester and pertussis toxin sensitive activation of phospholipase C. In addition, bradykinin is capable of enhancing Cai by utilizing pertussis toxin insensitive mechanisms. Furthermore, bradykinin is able to transiently enhance the potassium conductance without a general increase of intracellular calcium. PMID- 1707675 TI - 5-Azacytidine increases the total cellular copper content and basal level metallothionein mRNA accumulation of human Hep G2 cells. AB - In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed. PMID- 1707676 TI - Constitutive expression of exogenous c-myb gene causes maturation block in monocyte-macrophage differentiation. AB - A nuclear protooncogene c-myb has been hypothesized to play an important role in hematopoiesis, but little is known about the physiological function of the c-myb gene products. To study the role of c-myb gene expression in monocyte-macrophage differentiation and proliferation, we introduced exogenous c-myb gene into murine myelomonocytic leukemia WEHI-3B(D+) cells which can be induced to differentiate into mature monocytes with granulocyte-colony stimulation factor (G-CSF) and actinomycin D. Expression of the transfected gene was found to result in elevated levels of c-myb transcripts, which were not subject to normal down-regulation by differentiation induction. This constitutive expression of c-myb gene allowed the c-myb transfectants to differentiate into promonocytes with G-CSF and actinomycin D, but blocked further maturation from promonocytes to mature monocytes. It is concluded that normal down-regulation of c-myb gene expression during monocyte macrophage differentiation is required for the maturation of promonocytes to mature monocytes. PMID- 1707677 TI - Inter-species complementation of a rosy deficiency in Drosophila melanogaster. AB - A chimeric Xdh gene was constructed in vitro, by recombining DNA sequences from the Dipterans Drosophila melanogaster and Calliphora vicina. The ry506 strain, an eye-colour mutant of Drosophila that is deficient for Xdh, was genetically transformed with the recombinant gene. Transformed flies with ry+ eye phenotype and increased resistance to purine were obtained, showing that the chimeric XDH is physiologically active in Drosophila. XDH activity was detected in crude extracts from transformed flies, yet at lower levels than in wild-type controls. The amounts of Xdh transcripts in the transformants were found to be 8 to 16% of the amount of wild-type ry mRNA, suggesting that Calliphora Xdh sequences may be relatively inefficient for mRNA production in Drosophila, or may produce unstable mRNA. PMID- 1707678 TI - In vitro transcription analysis of the Drosophila tropomyosin and other muscle genes. AB - Nuclear transcription extracts were prepared from embryos of Drosophila melanogaster to study the in vitro transcription of the tropomyosin genes. Several non-muscle gene promoters, including the non-muscle promoter of the Tropomyosin II gene, were shown to be efficiently transcribed in vitro. The Tropomyosin I gene and the muscle promoter of the Tropomyosin II gene, as well as two other contractile protein muscle genes, were not transcribed in vitro. The embryonic extract did, however, contain developmental-specific proteins that bound to the muscle enhancer regulatory region of the Tropomyosin I gene. PMID- 1707679 TI - Isolation of the human cytochrome P-450 IIC8 gene: multiple glucocorticoid responsive elements in the 5' region. AB - The 5' portion of the cytochrome P-450 IIC8 gene has been isolated from a human genomic library. A 3.5 kb genomic fragment including 2477 bp of 5' flanking region and exon 1 of the gene was sequenced. S1 nuclease mapping and specific primer extension experiments localized the transcription start site 23 bp upstream from the ATG. Two and three putative TATA and CAAT boxes were identified together with seven core motifs for glucocorticoid responsive elements, an interesting feature in this constitutive P-450 subfamily. PMID- 1707680 TI - Ribonucleases. Introduction. PMID- 1707681 TI - Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors. AB - Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions. PMID- 1707682 TI - Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+. AB - A precursor to 10Sa RNA accumulates in an rne mutant. However, the present studies indicate that RNase III is the enzyme that processes this RNA. Cell extracts prepared from an rne mutant failed to cleave p10Sa RNA, whereas E coli wild type, rne and rnp cell extracts processed p10Sa RNA under specific assay conditions that require the presence of Mn2+ but not under the customary conditions used for assaying RNase III. That the p10Sa cleaving activity is solely RNase III was confirmed by comparing the increase in p10Sa and poly(A).poly(U) cleaving activities in a strain harboring a plasmid carrying an RNase III gene as compared to a normal E coli strain. It is of interest that these 2 substrates are cleaved by RNase III efficiently, but under 2 different assay conditions. In all strains tested, with normal or elevated levels of RNase III, RNase III fractionates predominantly with the membrane. Further characterization of the maturation of 10Sa RNA revealed that the processing of 10Sa RNA is a 2 step reaction involving 2 separate activities, both sensitive to heat and proteinase K treatment. The first step is catalyzed by RNase III, and results in the formation of a molecule, p10Sa', which is larger than the mature 10Sa RNA. The second activity catalyzes the conversion of p10S' to 10Sa RNA, and this step does not require a divalent cation. The second activity is not any of the known processing endoribonucleases, RNase III, E or P, but could be a new enzyme having no obligate requirement for a divalent cation. PMID- 1707683 TI - RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA. AB - Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors. It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products. Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules. The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme. The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi. Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function. RNA chains as long as 40 residues can be synthesized in this system. As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction. PMID- 1707684 TI - The disappearance of macromolecules from the peritoneal cavity during continuous ambulatory peritoneal dialysis (CAPD) is not dependent on molecular size. AB - The transport of macromolecules from the circulation to the peritoneal cavity is a size-selective restricted process, while the transport of these solutes from the peritoneal cavity is probably mainly by lymphatic absorption. If so, it should be independent of molecular size. Therefore, we studied with a clearance technique the disappearance of intraperitoneally administered inulin and polydisperse dextran 70 in nine continuous ambulatory peritoneal dialysis (CAPD) patients and compared the results with the simultaneously measured appearance clearance of serum proteins. Using gel permeation chromatography 18 dextran fractions with different molecular radii could be analyzed. Inulin clearance (2.94 mL/min) was higher than total dextran clearance (1.30 mL/min). The maximal dextran concentration in all dialysate samples was found in the 50.4 A fraction. The clearances of the dextran fractions were the same of different molecular sizes. All disappearance clearances were higher than the appearance clearances: the protein/dextran clearance ratio ranged from 0.15 for albumin/36 A to 0.04 for alpha 2-macroglobulin/91 A. This confirms that the appearance of a macromolecule, but not its disappearance is dependent on molecular size. It is concluded that the disappearance of macromolecules from the peritoneal cavity is mainly a size independent convective process, possibly by lymphatic uptake. This implies that total dextran 70 clearance can be used for measurement of lymphatic absorption in CAPD patients. PMID- 1707685 TI - Modulation of neonatal rat myeloid kinetics resulting in peripheral neutrophilia by single pulse administration of Rh granulocyte-macrophage colony-stimulating factor and Rh granulocyte colony-stimulating factor. AB - Rh granulocyte-macrophage (GM) colony-stimulating factor (CSF) and rh granulocyte (G) CSF have been demonstrated to induce proliferation and maturation of myeloid stem cells and release of mature polymorphonucleocytes (PMNs) from human and animal adult bone marrows. Unfortunately, reduced bone marrow progenitor cells, neutrophil storage pool (NSP) depletion and peripheral neutropenia are characteristic of human and animal newborn bone marrows. We investigated the effect of administering intraperitoneal rhGM-CSF and rhG-CSF to Sprague-Dawley newborn rats (less than 36 h). Newborn rats treated with intraperitoneal CSF (3.0 micrograms/kg) demonstrated significant leukocytosis at 6 and 24 h: rhGM-CSF vs. control, WBC (10(3)/mm3), at 6 h, 8.0 +/- 0.5 vs 4.3 +/- 0.9 (p less than or equal to 0.003), and at 24 h, 7.7 +/- 1.7 vs. 3.8 +/- 0.2 (p less than or equal to 0.008); rhG-CSF vs. control WBC (10(3)/mm3) at 6 h, 6.6 +/- 1.2 vs 4.3 +/- 0.1 (p less than or equal to 0.03), and at 24 h, 8.1 +/- 0.2 vs. 3.75 +/- 0.2 (p less than or equal to 0.003). The absolute neutrophil count was also significantly elevated at 6 h following intraperitoneal CSF (3.0 micrograms/kg): RhGM-CSF vs. control 1,827 +/- 25 vs. 379 +/- 10 (p less than or equal to 0.001); rhG-CSF vs. control, 1,698 +/- 40 vs. 371 +/- 10.1 (p less than or equal to 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707686 TI - Hypothalamic--pituitary--adrenal axis and monoamine transmitter activity in depression: a pilot study of central and peripheral effects of electroconvulsive therapy. AB - Central and peripheral measures of hypothalamic--pituitary--adrenal (HPA) axis and monoamine neurotransmitter activity were assessed in 8 depressed patients during a medication-free period and again after completion of a course of electroconvulsive therapy (ECT). Seven patients responded fully to ECT. At baseline there was corresponding activation of the HPA and noradrenergic systems, with apparent elevation in cerebrospinal fluid (CSF) concentrations of corticotropin-releasing hormone (CRH) in some patients. Neither CRH nor adrenocorticotropin (ACTH) in CSF changed significantly after ECT, with a mean 10% decline in CSF CRH. Urinary free cortisol (UFC) excretion was high both before and after treatment. Although peripheral noradrenergic hyperreactivity at baseline appeared to normalize with ECT, CSF concentrations of the principal norepinephrine metabolite, 3-methoxy-4-hydroxyphenyl-glycol (MHPG) were unaffected and remained correlated with CSF CRH. In contrast, there were increases in the CSF levels of the main metabolite of serotonin in half the patients. PMID- 1707687 TI - CSF spinal gradients of 5-HIAA. PMID- 1707688 TI - The presence of pregnancy-associated plasma protein-A in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state. AB - Pregnancy-associated plasma protein-A (PAPP-A) is a high-molecular-weight glycoprotein primarily secreted by syncytiotrophoblasts of human placenta. It is not known, however, whether human CL of menstrual cycle or pregnancy also contain this protein. Therefore, light and electron microscope immunocytochemical studies were undertaken to investigate the presence, cellular and subcellular distribution, and dependence of luteal PAPP-A content on reproductive state. Human CL from early, mid, and late luteal phases and from term pregnancies immunostained specifically for PAPP-A. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states. Immunostaining was not found in negative control tissues, i.e. human liver or bovine CL of pregnancy. As expected however, term-pregnancy human placenta used for a positive control tissue immunostained intensely for PAPP-A. The luteal immunostaining was highest in early luteal phase, decreased progressively from early to mid and from mid to late luteal phases, and then disappeared in corpora albicantia. The relative intensity of immunostaining in early luteal phase human CL was similar to that in term-pregnancy human placenta and higher than in term-pregnancy human CL. The immunogold particles due to PAPP-A were primarily associated with secretory granules of large luteal cells. A small number of gold particles were also found in rough endoplasmic reticulum and cytoplasm. In conclusion, human CL contain immunoreactive PAPP-A. The luteal content varies with reproductive state, with the highest amount found in early luteal phase CL.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707689 TI - Denaturation behaviour of DNA-protein-complexes detected in situ in metaphase chromosomes in suspension by Hoechst 33258 fluorescence. AB - The denaturation behaviour of DNA-protein complexes in metaphase chromosomes in suspension was analysed in situ by Hoechst 33258 fluorescence. The results indicate that due to the stability of the dye molecule and the product of the molecular extinction coefficient and the quantum yield at different temperatures, Hoechst 33258 is a suitable probe for the detection of double-stranded DNA. Thus, it is possible to monitor the concentration of double-stranded DNA in a suspension by measuring the total fluorescence intensity. The fluorescence denaturation profiles of DNA (calf thymus) were found to be comparable to absorption measurements. The decrease in fluorescence of metaphase chromosomes in suspension with increasing temperature may therefore be used to detect conformational changes of DNA in situ. PMID- 1707690 TI - Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide absorption bands of polypeptides and fibrous proteins in alpha-, beta-, and random coil conformations. AB - Infrared spectra of poly(D,L-alanine), poly(L-glutamic acid), poly(L-lysine), silk fibroin, and tropomyosin have been registered for various conformations of the polypeptide chain. Assuming additivity of the main- and side-chain absorption, spectral parameters of amide I and II absorption bands corresponding to alpha-, beta-, and random coil conformations have been derived. The amide I band parameters for H2O and D2O have been compared. PMID- 1707691 TI - Transglutaminase involvement in the secretion of insulin from electropermeabilised rat islets of Langerhans. AB - Ca(2+)-Induced insulin release from electropermeabilised islets is inhibited by the transglutaminase inhibitors monodansylcadaverine, glycine methylester, methylamine and cystamine but not by the control compounds dimethyl monodansylcadaverine and sarcosine methylester which lack the primary amine group. Neither monodansylcadaverine nor glycine methylester inhibited insulin secretion induced by either cAMP or the phorbol ester PMA at basal levels (10 nM) of Ca2+. These data provide further evidence for the involvement of transglutaminase in Ca2+ induced insulin secretion, they also suggest that insulin secretion induced by either cAMP or PMA may act in part by a mechanism independent of that induced by Ca2+. PMID- 1707692 TI - Pentoxifylline (Trental) decreases the replication of the human immunodeficiency virus type 1 in human peripheral blood mononuclear cells and in cultured T cells. AB - Pentoxifylline (Trental), used routinely for the treatment of intermittent claudication, has been shown previously to decrease the levels of tumor necrosis factors-alpha (TNF-alpha) RNA in cancer patients and to lead to a general improvement of well being. Increased TNF-alpha levels have been observed not only in cancer patients but also in cachectic patients with the acquired immunodeficiency syndrome (AIDS), and TNF-alpha is known to increase the expression of the human immunodeficiency virus type 1 (HIV-1) via activating its long terminal repeat (LTR). Moreover, TNF-alpha decreases the therapeutic efficacy of zidovudine (AZT). Here we show a significant decrease in HIV-1 replication by pentoxifylline in infected human peripheral blood mononuclear cells. The reduction was proportional to the downregulation of expression of a reporter gene, the bacterial gene for chloramphenicol acetyl transferase, linked to the HIV-1 LTR in human monocytoid cells. We conclude that patients with AIDS may benefit from pentoxifylline treatment because of its blockage of TNF-alpha mediated HIV-1 upregulation, from increased efficacy of AZT, and also from improvement in TNF-alpha-induced cachexia. PMID- 1707693 TI - Lack of effect of granulocyte-macrophage and granulocyte colony-stimulating factors on cultured human endothelial cells. AB - The hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% +/- 45% (mean +/- SEM, n = 5, P less than .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 beta (IL-1 beta) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% +/- 376% (n = 7, P less than .005) and 247% +/- 71% (n = 4) of control levels, respectively. gamma-Interferon (gamma-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1 beta. There was no direct or priming effect of GM CSF (1 ng to 1 microgram/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 microgram/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI-1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% +/- 72% of control levels, n = 5, P less than .025). The production of PAI-1 was increased by TNF 200 U/mL (241% +/- 44% of control, n = 3, P less than .005), thrombin 4 U/mL (180% +/- 12% of control, n = 5, P less than .0005) and IL-1 beta 10 U/mL (275% +/- 44% of controls, n = 5, P less than .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 +/- 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG CSF on the proliferation activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC. PMID- 1707694 TI - Macrophage-active colony-stimulating factors enhance human immunodeficiency virus type 1 infection in bone marrow stem cells. AB - To define the relationship between human immunodeficiency virus type 1 (HIV-1) infection in hematopoietic stem cells and virus production by their progeny, we performed kinetic studies infecting bone marrow (BM) stem cells and culturing them in the presence of hematopoietic growth factors. CD34-positive (CD34+), CD4 negative (CD4-) BM cells were isolated and infected in vitro with the monocytotropic HIV-1JR-FL strain or the laboratory-maintained HTLV-IIIB strain at a high multiplicity of infection. The cells were susceptible to productive infection only with HIV-1JR-FL, and virus production as measured by p24 protein release was markedly increased (more than fivefold) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL 3). Macrophage CSF (M-CSF) was less stimulatory and granulocyte CSF (G-CSF) had no effect on virus production. Virus production coincided with proliferation of mononuclear phagocytes but was not related to granulocytic proliferation in G-CSF treated BM cultures. Although peak virus production from GM-CSF-treated macrophages occurred 2 to 3 weeks after infection, peak virus production in infected stem cells was observed 5 to 6 weeks after. Enhancement in virus production had a more rapid onset when CD34+/CD4- cells were cultured in the presence of both GM-CSF and IL-3 for 7 or 14 days. Under these conditions there was a 10-fold enhancement in virus production after 7 days of preincubation and a 50-fold enhancement after 14 days. These data indicate that while the stem cell compartment may be susceptible to infection with a monocytotropic HIV-1 strain, productive and sustained infection is realized only after macrophage differentiation. The lack of effect of G-CSF on virus production is likely because of the limited effect of this hematopoietin on mononuclear phagocyte generation and function. PMID- 1707695 TI - Transforming growth factor-beta trans-modulates the expression of colony stimulating factor receptors on murine hematopoietic progenitor cell lines. AB - Transforming growth factor beta (TGF-beta) is a potent and selective growth inhibitor of early hematopoietic progenitors and leukemic cells. The cellular mechanism(s) underlying this antiproliferative effect is, however, currently unknown. In the present study, we demonstrate that TGF-beta inhibits the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and granulocyte-CSF (G-CSF) receptors on murine factor dependent and independent hematopoietic progenitor cell lines without a significant change in receptor affinity. A maximum reduction in GM-CSF receptor numbers of 65% to 77% was observed by 96-hour incubation with TGF-beta. The TGF beta induced trans-down-modulation of GM-CSF receptors was prolonged, noncytotoxic but reversible, and not due to endogenous production of GM-CSF. The TGF-beta induced reduction in CSF receptor numbers preceded TGF-beta's growth inhibitory action. In addition, the ED50 (1 to 10 pmol/L) for TGF-beta's CSF receptor modulatory and antiproliferative effect was similar. The effect of TGF beta on cell surface CSF receptor expression was specific, because the expression of other cell surface proteins (Ly 5 and Ly 17) was not affected by TGF-beta treatment, and because other growth inhibitors (tumor necrosis factor and interferon) did not affect CSF receptor expression. These data suggest that the downregulation of the growth of hematopoietic progenitor cells by TGF-beta involves reducing the cell surface expression on growth factor receptors. PMID- 1707696 TI - Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. AB - The CD34 antigen is expressed by 1% to 4% of human and baboon marrow cells, including virtually all hematopoietic progenitors detectable by in vitro assays. Previous work from our laboratory has shown that CD34+ marrow cells can engraft lethally irradiated baboons. Because the CD34 antigen has not been detected on most solid tumors, positive selection of CD34+ cells may be used to provide marrow cells capable of engraftment, but depleted of tumor cells. In seven patients with stage IV breast cancer and two patients with stage IV neuroblastoma, 2.5 to 17.5 x 10(9) marrow cells were separated by immunoadsorption with the anti-CD34 antibody 12-8 and 50 to 260 x 10(6) positively selected cells were recovered that were 64 +/- 16% (range 35% to 92%) CD34+. The patients received 1.0 to 5.2 x 10(6) CD34-enriched cells/kg after marrow ablative therapy. Six patients engrafted, achieving granulocyte counts of greater than 500/mm3 at 34 +/- 10 (range 21 to 47) days and platelets counts of greater than 20,000/mm3 at 46 +/- 14 (range 28 to 66) days posttransplant. Five of these patients showed durable engraftment until the time of death 82 to 386 days posttransplant. One patient failed to sustain engraftment associated with metastatic marrow disease. Three patients died at days 14, 14, and 17 posttransplant, two of whom had evidence of early engraftment. These studies suggest that CD34+ marrow cells are capable of reconstituting hematopoiesis in humans. PMID- 1707697 TI - The majority of peripheral blood monoclonal IgM secreting cells are CD5 negative in three patients with mixed cryoglobulinemia. AB - The mixed cryoglobulinemia is considered to be a nonmalignant human B-cell proliferation that frequently produces a monoclonal IgM with anti-IgG activity (rheumatoid factor). Using murine monoclonal anti-idiotypic antibodies specific for private or minor idiotopes on monoclonal IgM from three patients suffering from nonmalignant mixed cryoglobulinemia, we investigated the presence of the CD5 antigen on the monoclonal IgM producing cells in these patients. It is shown by two-color cytofluorometric analysis that the majority of the peripheral blood monoclonal IgM rheumatoid factor secreting cells is CD5 negative in these three patients. One of the monoclonal rheumatoid factor K variable regions was sequenced at the protein level and belongs to the human VK III group, as a high proportion of monoclonal rheumatoid factors and some B-cell chronic lymphocytic leukemia (CLL) membrane bound Igs. Thus, despite the preferential use of similar VK genes and the absence of somatic mutation affecting these variable regions in both malignant B-cell CLL and nonmalignant mixed cryoglobulinemia, these proliferating B cells differ in the CD5 membrane expression. PMID- 1707698 TI - Differentiation of a human eosinophilic leukemia cell line (EoL-1) by a human T cell leukemia cell line (HIL-3)-derived factor. AB - Differentiation of a human eosinophilic leukemia cell line, EoL-1, induced by the culture supernatant of a human ATL cell line, HIL-3 (HIL-3 sup) was compared with differentiation induced by defined cytokines. HIL-3 sup induced EoL-1 cells to express eosinophilic granules and segmented nuclei after 6 to 9 days of incubation. HIL-3 sup also induced the expression of Fc epsilon receptor II (Fc epsilon RII/CD23) and an eosinophil differentiation antigen EO-1 mainly on eosinophilic granule (+) cells. Furthermore, HIL-3 sup induced EoL-1 cells to respond to an eosinophil chemotactic factor, platelet activating factor. HIL-3 cells express messenger RNA (mRNA) of interleukin-5 (IL-5), macrophage colony stimulating factor (M-CSF), and IL-3 but not granulocyte CSF (G-CSF). Granulocyte macrophage CSF (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) were detected in the HIL-3 sup. Recombinant IL-2 (rIL-2), rIL-3, rIL-4, rIL-5, rM-CSF, and rGM CSF did not induce eosinophilic granules. rG-CSF induced a few eosinophilic granule (+) cells, and TNF-alpha, which did not induce eosinophilic granules by itself, enhanced the ability of G-CSF to induce them. However, G-CSF and TNF alpha did not induce the expression of Fc epsilon RII and EO-1 antigen. Moreover, anti-G-CSF, anti-TNF-alpha, anti-GM-CSF, anti-IL-3, and anti-IL-5 antibodies did not suppress the effect of HIL-3 sup on the differentiation of EoL-1 cells. All the data suggest that HIL-3 sup contains an unidentified factor that induces differentiation of EoL-1 cells, and that EoL-1 cells and HIL-3 sup provide an important model for the examination of differentiation mechanisms and functions of eosinophils. PMID- 1707699 TI - Production of endothelial cell stimulating angiogenesis factor (ESAF) by chondrocytes during in vitro cartilage calcification. AB - The aim of this study was to identify the stimulus for production of the latent collagenase and angiogenic activator ESAF by growth plate chondrocytes. Stimulation correlated most closely with matrix calcification. Alkaline phosphatase was necessary for calcification (and so stimulation of ESAF production) but we could find no evidence for a direct link with ESAF production. ESAF production was also stimulated by addition of preformed mineral to non calcified cultures but was inhibited by dexamethasone. Protein synthesis was necessary for the stimulation of ESAF production by calcification, though ESAF is not itself a protein. Based on these findings we suggest that chondrocytes, at a suitable stage of maturation in the growth plate, are stimulated to produce ESAF by the proximity of crystals in the matrix. Stimulation, which may consist of the induction of an enzyme or transport protein, leads to the release of this potent activator of collagenolysis as part of the angiogenic cascade. PMID- 1707700 TI - Golgi complex and lysosomes in rabbit derived Pneumocystis carinii. AB - The ultrastructural morphology of Pneumocystis carinii obtained from nonimmunosuppressed rabbit is described in details. Golgi complex and primary lysosomes of P carinii are described here for the first time. They are easily revealed by the zinc iodide-osmium tetroxide cytochemical reagent. Thiamine pyrophosphatase and beta-glycerophosphatase activities are found in the parasite but cytidine 5' monophosphatase activity is not observed. A weak thiamine pyrophosphatase activity is detected in Golgi vesicles. An endomembranous saccular structure, present from the intracystic body stage to the precystic stage, apparently plays the role of secondary lysosome. A second type of endomembranous saccular structure, only present in the well developed trophozoitic and precystic forms is also described. The presence of carbohydrates in the cell wall of the parasite was demonstrated by periodic acid thiosemicarbazide-silver proteinate staining and lectin concanavalin A labeling. The development of Golgi vesicles preceded the transition from double-layered to three-layered parasite stages. PMID- 1707701 TI - Acute transient parotitis after high dose etoposide and autologous bone marrow transplantation. AB - Etoposide is an important component of several intensive therapy regimens in allogeneic and autologous bone marrow transplantation for advanced hematologic malignancies. We observed the occurrence of transient acute parotid and submandibular sialoadenitis in nine of 19 patients receiving high dose etoposide and melphalan followed by autologous bone marrow rescue. Manifestations included pain, tenderness and swelling of the parotid and submandibular glands. Symptoms arose 4-16 h after completion of etoposide infusion and resolved within 72 h. Elevation of serum amylase accompanied the symptoms, and was also observed in some patients who were asymptomatic. Discomfort was controlled with analgesics and the clinical course was uncomplicated in all cases. Transient parotitis is a relatively frequent and benign complication of high dose etoposide therapy. PMID- 1707702 TI - Pharmacological characterization of tachykinin-stimulated inositol phospholipid hydrolysis in peripheral tissues. AB - 1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of rat parotid gland, hamster urinary bladder and guinea-pig ileum longitudinal muscle. 2. In the presence of lithium, substance P and other naturally-occurring and synthetic tachykinins induced large, dose-dependent increases in [3H] inositol monophosphate accumulation. 3. In slices of rat parotid gland, [pGlu6,L Pro9]SP(6-11) was considerably more potent in stimulating inositol phospholipid hydrolysis than [pGlu6,D-Pro9]SP(6-11). 4. In contrast, in slices of hamster urinary bladder, [pGlu6,D-Pro9]SP(6-11) exhibited greater potency in evoking inositol phospholipid breakdown than [pGlu6,L-Pro9]SP(6-11). 5. The differential selectivity of these C-terminal fragments of substance P suggests that they may be useful tools for distinguishing between NK1 and NK2 receptors. 6. L-659,837 and L-659,874 antagonized eledoisin-stimulated inositol phospholipid hydrolysis in slices of hamster urinary bladder. Neither compound significantly reduced substance-P evoked inositol phospholipid breakdown in slices of rat parotid gland, or senktide-induced inositol phospholipid hydrolysis in slices of guinea pig ileum. 7. L-659,837 and L-659,874 had no effect on the atropine-sensitive, carbachol-stimulated inositol phospholipid hydrolysis in slices of rat parotid gland. 8. These data further support the notion that L-659,837 and L-659,874 are potent and selective NK2 receptor antagonists. PMID- 1707703 TI - The effects of drugs on Sephadex-induced eosinophilia and lung hyper responsiveness in the rat. AB - 1. Rats given an intravenous injection of Sephadex particles (0.5 mg of G200 in 1 ml of saline) on days 0, 2 and 5 had a blood eosinophilia which was maximal on day 7. 2. On day 7, broncho-alveolar lavage (BAL) fluids taken from the rats contained an increased number of eosinophils and fewer mononuclear cells but there was no change in the small number of neutrophils. In addition the rats were hyper-sensitive to the increase in resistance to artificial respiration produced by 5-hydroxytryptamine (5-HT), given intravenously, with a shift to the left of the log dose-response curve. Lung parenchymal strips, taken from the rats on days 6, 7 and 8, were hyper-reactive to 5-HT with an increase in slope of the log dose response curve. 3. Compounds with a wide variety of activities were evaluated for their effects on the blood eosinophilia on day 7 when given before each injection of Sephadex. The eosinophilia was reduced by glucocorticosteroids, beta adrenoceptor agonists, aminophylline, dapsone and phenidone. 4. Dexamethasone, isoprenaline, dapsone and phenidone at doses that reduced the blood eosinophilia also reduced the changes in number of leucocytes in the BAL fluids and the hyper responsiveness to 5-HT in vivo and in vitro, except that the effects of dapsone on the hyper-sensitivity to 5-HT in vivo did not reach significance. Aminophylline was the least effective of the drugs at reducing the blood eosinophilia and its effects on the other changes did not reach significance. Sodium cromoglycate reduced the BAL eosinophilia but had no effect on the other changes produced by Sephadex. 5. The correlation coefficients between blood eosinophil numbers and reactivity to 5-HT in vitro and sensitivity in vivo were r = 0.76, (n = 88; P < 0.001) and r = 0.53, (n = 61; P < 0.001) respectively. 6. Doses of dexamethasone, isoprenaline, dapsone and phenidone that reduced the blood eosinophilia when given before each injection of Sephadex were inactive when given up to 8 h after the Sephadex. 7. These data show an association between blood eosinophilia and hyper-responsiveness of the lung. The blood eosinophilia in the rats was triggered within the first few hours of injecting the Sephadex and drugs have been identified which inhibit this trigger. PMID- 1707704 TI - Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats. AB - 1. When rats were fed with clenbuterol for 7 days skeletal muscle mass increased by 21% in the tonic soleus and phasic plantaris muscles and a 16% hypertrophy of the heart was also induced. Fenbufen, fed to rats for the same period, blocked the hypertrophy of the heart but not that of the skeletal muscles. 2. When feeding of fenbufen commenced 3 days before the administration of clenbuterol, plasma prosta-glandin F2 alpha (PGF2 alpha) was reduced by 79%; there was again no effect of fenbufen on clenbuterol-induced increases in the RNA or protein content of plantaris, nor in the increased area of fast or slow twitch fibres in the soleus. In the heart the clenbuterol-induced increases in the RNA (+21%) and protein content (+20%) were totally inhibited. 3. The effects of clenbuterol on heart muscle appear to be mediated by a cyclo-oxygenase metabolite of arachidonic acid whilst the effects on skeletal muscle are not. PMID- 1707705 TI - Effects of repeated infusions of substance P and vasoactive intestinal peptide on the weights of salivary glands subjected to atrophying influences in rats. AB - 1. The long-term influence of substance P (SP) and vasoactive intestinal peptide (VIP) on rat salivary gland weight was investigated after parasympathetic denervation or on feeding soft food. 2. The parotid gland lost about one-third of its weight within 4-5 days following parasympathetic post-ganglionic denervation or change in dietary regimen, from pellets to liquid diet, thought to reduce nerve reflex activity. 3. Daily i.v. infusions with SP or VIP diminished or largely prevented the fall in parotid gland weight, whereas infusions with pentagastrin, bethanechol and saline had no effect. The infusions were preceded by administration of alpha- and beta-adrenoceptor antagonists; these antagonists were also given to the control animals. 4. The effect of SP and VIP on the parotid gland weight appeared to be related to cell size rather than to cell number, as judged by measurements of RNA and DNA. 5. Observations on the two other major salivary glands underlined the fact that different gland types in the same animal behave differently. Parasympathetic preganglionic denervation (decentralization) lowered the weights of the sublingual and submandibular glands, whereas liquid diet only reduced the weight of the sublingual gland. SP and VIP did not affect the weights of the submandibular glands, but VIP prevented the slight fall in sublingual gland weight induced by liquid diet. 6. The present results suggest a trophic role in rats for SP and VIP on parotid glands and for VIP on sublingual glands. Such an influence may be exerted naturally as a result of their release from nerves containing these peptides around acini. PMID- 1707706 TI - Effects of cyclic AMP-affecting agents on contractile reactivity of isolated mesenteric and renal resistance arteries of the rat. AB - 1. Effects of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-affecting agents were compared in mesenteric and renal resistance arteries that had been isolated from 20 week old Wistar-Kyoto rats, chemically sympathectomized, stretched to their optimal diameter for mechanical performance and made to contract in response to 30 mM potassium. 2. In mesenteric resistance arteries, isoprenaline, dopamine, NaF, forskolin, isobutyl-methylxanthine, milrinone and dibutyryl-cyclic AMP induced relaxation. Clonidine induced further increases in tension that could be reduced by pertussis toxin and prazosin but not by yohimbine. Clonidine also reduced relaxant responses to isoprenaline. 3. In renal resistance arteries, isoprenaline and dopamine failed to induce relaxation. Compared to mesenteric resistance arteries, renal vessels were less sensitive to the relaxant effect of NaF, forskolin and isobutyl-methylxanthine. Relaxant responses to dibutyryl cyclic AMP did not differ between the two resistance arteries. 4. Indirect evidence thus suggests that in mesenteric resistance arteries, adenylate cyclase is susceptible to pharmacological activation and inhibition and is functionally coupled to relaxation. The refractory nature of renal resistance arteries to the relaxant effects of isoprenaline and dopamine could be due primarily to absence of appropriate receptors and to a relatively low activity of adenylate cyclase. PMID- 1707707 TI - The antinociceptive activity of paracetamol in zymosan-induced peritonitis in mice: the role of prostacyclin and reactive oxygen species. AB - 1. Oral administration of high doses of paracetamol (600 mg kg-1 or more) resulted in inhibition of the writhing and reduced the levels of prostacyclin (PGI2, measured as 6-keto-PGF1 alpha) induced by intraperitoneal administration of zymosan in mice. The high oral doses of paracetamol required were accompanied by behavioural toxicity which may have contributed to the inhibition of writhing. 2. The number of writhes per mouse and the proportion of mice writhing at least once correlated significantly with the levels of 6-keto-PGF1 alpha. However, inhibition of writhing by paracetamol occurred at higher levels of 6-keto-PGF1 alpha than was previously observed with acidic non-steroidal anti-inflammatory agents. 3. When injected i.p., PGI2, carbacyclin and iloprost (agonists at the PGI2 receptor) induced writhing. Intraperitoneal injection of PGI2 reversed the inhibition of writhing induced by indomethacin (1 mg kg-1, p.o.) but not that induced by oral administration of paracetamol. 4. Paracetamol at 800 mg kg-1, p.o., inhibited carbacyclin-induced writhing but indomethacin at 1 mg kg-1 p.o. did not. Paracetamol administered i.p. at 100 mg kg-1 reduced the peritoneal levels of 6-keto-PGF1 alpha and inhibited zymosan-induced but not carbacyclin induced writhing and did not produce behavioural toxicity. 5. The in vitro potency of paracetamol as a prostaglandin synthesis inhibitor is known to be reduced by the presence of lipid peroxides. However, no lipid peroxides, measured as thiobarbituric acid reactive material, were detected in the peritoneal lavage fluid of zymosan-injected mice. 6. Intraperitoneal administration of a mixture of superoxide dismutase and catalase reduced detectable superoxide anion by 98% without inhibiting the writhing response to zymosan or the antinociceptive potency of paracetamol. 7. The data are consistent with the suggestion that inhibition of PGI2 synthesis in the peritoneal cavity by paracetamol is responsible for only a part of its antinociceptive activity in this test. However, extremely high oral doses of paracetamol were required which produced behavioural toxicity which clearly contributed to the inhibition of writhing. The low potency of paracetamol in this model cannot be attributed to the generation of lipid peroxides via the oxidative burst. PMID- 1707708 TI - Differential effects of calcium antagonists and Bay K 8644 on contractile responses to exogenous noradrenaline and adrenergic nerve stimulation in the rabbit ear artery. AB - 1. The effects of three calcium antagonists (nifedipine, verapamil, diltiazem) and the calcium agonist Bay K 8644 were compared on contractile responses of similar amplitude elicited by noradrenaline (NA) and electrical nerve stimulation (ENS) in the rabbit isolated ear artery. 2. Contractions induced by both NA (3 x 10(-7) M) and ENS (10 Hz, 10s) were almost exclusively mediated by alpha 1 adrenoceptors, since 10(-7) M prazosin abolished (NA) or almost abolished (ENS) the responses, and prazosin was more than three orders of magnitude more potent than rauwolscine on both types of response. 3. ENS-induced contractions were considerably less inhibited by nifedipine, verapamil and diltiazem than were those elicited by NA. Bay K 8644 enhanced responses to NA more than those to ENS. 4. The inhibitory effect of nifedipine and Ca2+ deprivation on NA-induced contractions decreased with increasing NA concentration. Reduction of the NA response by prazosin or phenoxybenzamine increased the nifedipine inhibition. 5. Reduction of the ENS-induced contractions by prazosin or phenoxybenzamine, or by use of a lower stimulation frequency did not increase the inhibitory effect of nifedipine. 6. In conclusion, the differential effects of the calcium antagonists on NA- and ENS-induced contractions were not related to differences in alpha adrenoceptor subtype (alpha 1/alpha 2), receptor reserve or response amplitude, but may rather reflect temporal and spatial differences in alpha-adrenoceptor activation between the responses. PMID- 1707709 TI - The use of oxyhaemoglobin to explore the events underlying inhibition of platelet aggregation induced by NO or NO-donors. AB - 1. Full inhibition of thrombin-induced platelet aggregation was elicited by the least maximal platelet inhibitory concentrations of nitric oxide (NO; 7 +/- 1 microM) or NO-donors which included sodium nitroprusside (NaNp; 80 +/- 13 microM) 3-morpholinosydnonimine (SIN-1; 3 +/- 0.1 microM) or endothelial cells (EC; 2.36 +/- 0.12 x 10(5) added 1 min before thrombin. Oxyhaemoglobin (oxyHb; 10 microM) administered 30s to 10 min after stimulation with thrombin caused a time dependent reversal of the inhibition induced by these agents. OxyHb was ineffective when these agents were co-incubated with the non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 0.05 mM). 2. OxyHb did not reverse the platelet inhibition with IBMX (0.2 mM) or that caused by a selective guanosine 3'; 5'-cyclic monophosphate (cyclic GMP) phosphodiesterase inhibitor 2-O-propoxyphenyl-8-azapurin-6-one, (M & B 22948; 20 microM). In addition, oxyHb did not reverse the inhibition with iloprost (1 nM) which inhibits platelet aggregation through stimulation of adenylate cyclase. 3. The inhibition of platelet aggregation by NO (7 +/- 1 microM) or NaNp (80 +/- 13 microM) was accompanied by a 13 fold increase in cyclic GMP levels occurring within 15 s of addition of these agents. In the continued presence of NO or NaNp, the reversing effect of oxyHb given 1 min after thrombin was associated with a pronounced decrease in cyclic GMP levels. 4. We conclude that the inhibition of platelet aggregation by activators of guanylate cyclase depends in the first few minutes on continuous stimulation of the enzyme in order to maintain intracellular concentrations of cyclic GMP, except when its breakdown is inhibited. 5. The addition of agents such as oxyHb after the inhibition of platelet aggregation offers another way of investigating the biochemical changes involved in maintaining platelets in an inactive state. PMID- 1707710 TI - Tachykinin receptors in the circular muscle of the guinea-pig ileum. AB - 1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7] neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1 agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4 10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family. The response to activation of NK3-receptors is totally neurogenic and partially mediated by endogenous acetylcholine, the response to activation of NK1-receptors is partly neurogenic and largely myogenic and the response to activation of NK2-receptors is totally myogenic. PMID- 1707711 TI - (+)-isradipine but not (-)-Bay-K-8644 exhibits voltage-dependent effects on cat adrenal catecholamine release. AB - 1. Catecholamine release from cat adrenal glands perfused at a high rate (4 ml min-1) at 37 degrees C with polarizing (1.2 or 5.9 mM K+) or depolarizing (17.7, 35, 59 or 118 mM K+) solutions, was triggered by 5 or 10 s pulses of Ca2+ (0.5 or 2.5 mM) in the presence of various concentrations of K+. 2. In polarized glands, secretion was greater the higher the K+ concentration present during the secretory K+/Ca2+ test pulse. Thus, in 17.7 mM K+, catecholamine released was 162 +/- 27 ng per pulse, while in 118 mM K+ secretion rose to 1839 +/- 98 ng per pulse. In depolarized glands, secretion reached a peak of around 1000 ng per pulse in 35-59 mM K+; in 118 mM K+, secretion did not increase further, suggesting that voltage changes are implicated in the control of the secretory process. 3. Blockade of secretion by increased concentrations of (+)-isradipine was much more manifest in polarized glands. The higher the degree of depolarization was (35, 59 or 118 mM K+), the lower the IC50 s were. So, the ratios between the IC50 s in polarized and depolarized glands rose from 3.92 in 35 mM K+ to 26.7 in 118 mM K+. 4. In contrast, the Ca2+ channel activator (-)-Bay K 8644 potentiated catecholamine release evoked by K+/Ca2+ pulses equally well in polarized or depolarized glands. The ratios between EC50 s in polarized or depolarized glands were, respectively, 0.30, 0.59 and 0.69 for 17.7, 35 and 118 mM K+. 5. In simultaneous experiments, the two enantiomers of Bay K 8644 exhibited opposite effects on secretion. (+)-Bay K 8644 (a Ca21 channel blocker) inhibited secretion better in depolarized than in polarized glands, whilst (-) Bay K 8644 potentiated secretion in a voltage-independent manner. 6. Potentiation of secretion by (-)-Bay K 8644 was concentration-dependent from 10-8 to 10-6M. At 10- 5M, such potentiation largely disappeared in both polarized and depolarized glands. However, this dual effect of (-)Bay K 8644 was better seen in depolarizing conditions, suggesting that using the same enantiomer, the voltage dependence is only seen when blockade of secretion dominates. 7. In the presence of increasing concentrations of (-)Bay K 8644 (3 x 10-9, 3 x 10-8 and 3 x 10-7M), the concentration-response curves for (+)isradipine to inhibit secretion were displaced to the right. However, a Schild plot of (dose ratio - 1) against (-) Bay K 8644 concentrations gave a slope of 0.6, suggesting that the interactions between (+)-isradipine and (-)Bay K 8644 were non-competitive in nature. The pA2 for (-)-Bay K 8644 was 9.13. 8. Overall, the results suggest that potentiation of secretion by (-)Bay K 8644 (a voltage-independent phenomenon), and blockade by (+)-isradipine or (+-Bay K 8644 (a voltage-dependent phenomenon) might be exerted through binding of the dihydropyridines activators and blockers to separate sites on chromaffin cell L-type Ca2 + channels. PMID- 1707712 TI - The differential contribution of endogenous prostaglandins to the release of acetylcholine from the myenteric plexus of the guinea-pig ileum. AB - 1. Prostaglandin E (PGE) may be essential for maintaining the sensitivity of the myenteric plexus of guinea-pig ileum to nicotine. The contributions of prostaglandins to nervous activity evoked by different stimuli have now been investigated by measuring the amount of acetylcholine (ACh) released from the myenteric plexus of the guinea-pig ileum. 2. The amount of ACh released in response to dimethylphenylpiperazinium (DMPP) or substance P was depressed to about 40% of control by 2.8 microM indomethacin (Ind), whereas the release of ACh induced by 5-hydroxytryptamine (5-HT) was not affected. The inhibitory effects of Ind were overcome by 14.3 nM PGE2. 3. Mepacrine 5 microM, an inhibitor of phospholipase A2, depressed the release of ACh in response to DMPP and substance P to the same extent as Ind. These inhibitory effects of mepacrine were overcome by arachidonic acid (10 microM), but not by arachidonic acid plus Ind. The release of ACh evoked by 5-HT or electrical field stimulation (EFS) was also inhibited to about 60% of control by mepacrine but these inhibitions were overcome by arachidonic acid (10 microM) either in the absence or the presence of Ind. 4. The results suggest that endogenous prostaglandins and arachidonic acid contribute to the maintenance of the excitability of the myenteric plexus by DMPP and substance P. By contrast, the release of ACh induced by 5-HT and EFS may be regulated by arachidonic acid and not by prostaglandins. PMID- 1707713 TI - Calcitonin gene-related peptide and human epicardial coronary arteries: presence, release and vasodilator effects. AB - 1. In the present study, the levels of calcitonin gene-related peptide (CGRP) like immunoreactivity (-LI) in human cardiopulmonary tissue were determined in combination with studies on CGRP-LI release from the left anterior descending coronary artery (LAD) and functional effects of CGRP on coronary arterial tone. 2. The highest levels of CGRP-LI were found in the LAD followed in declining order by the bronchus, right atrium, pulmonary artery, lung and left ventricle. 3. Exposure to capsaicin evoked a clear-cut increase in CGRP-LI outflow, suggesting release from isolated large specimen of the LAD. This release was Ca2(+)-dependent and was markedly attenuated by incubation with the mitochondrial Ca2(+)-inhibitor, ruthenium red. Exposure to potassium also released CGRP-LI in a Ca2(+)-dependent fashion from the LAD. 4. In functional experiments on human epicardial coronary arteries with an inner diameter of 0.4 to 0.8 mm, human CGRP alpha and beta relaxed the potassium-precontracted arteries equipotently. Substance P (SP) also relaxed these precontracted arteries but the relaxation could be prevented by incubation with methylene blue, an inhibitor of endothelium derived relaxing factor (EDRF)-mechanisms, which did not influence the effect of CGRP. 5. Capsaicin evoked a ruthenium red-sensitive relaxation of the potassium precontracted arteries. However, ruthenium red did not affect the relaxations induced by CGRP or SP. Furthermore, the capsaicin effect was not influenced by methylene blue. 6. It is concluded that CGRP-LI is present in human cardiopulmonary tissue and can be released upon exposure to high concentrations of capsaicin as well as potassium. CGRP causes relaxation of arteries independently of EDRF activation and closely resembles the vasodilator effects of capsaicin. This supports the view that the coronary vasodilatation observed upon sensory nerve activation is mediated by CGRP. Ruthenium red inhibits capsaicin induced CGRP-LI release and functional effects and may thus serve as an experimental tool in evaluating the function of capsaicin-evoked stimulation of peripheral nerve terminals. PMID- 1707714 TI - Novel selective agonists and antagonists confirm neurokinin NK1 receptors in guinea-pig vas deferens. AB - 1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl [L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B). This estimate is similar to that we obtained for NK1-mediated (substance P methyl ester) contraction in the guinea-pig ileum preparation (pKB= 7.86+ 0.05). 5. Tachykinin action appears not to depend on release of a number of intermediary mediators including acetylcholine, histamine or cyclo-oxygenase products, nor to involve interaction with neuronal mechanisms including alpha 2-adrenoceptor feedback, noradrenergic Uptake-I or opioid-release, since antagonism or inhibition of these mechanisms did not modify responses to tachykinins. 6. We conclude that tachykinin action in the field-stimulated guinea-pig vas deferens preparation is mediated through interaction with a predominant neurokinin NK, receptor population and this preparation can therefore be used to study NK, modulation of sympathetic neurotransmission. PMID- 1707715 TI - Cytoreductive surgery in disseminated non-seminomatous germ cell tumours of testis. AB - Between 1977 and 1988, 67 patients underwent surgical removal of residual metastatic deposits following an aggressive chemotherapy regimen (cisplatin, vincristine, methotrexate and bleomycin alternating with etoposide, actinomycin D and cyclophosphamide) for disseminated germ cell tumours of the testis (stage IIB or above). Ninety-one surgical procedures were performed. There were 63 (69 per cent) retroperitoneal lymph node dissections, 16 (18 per cent) thoracotomies, three (3 per cent) hepatic resections, three (3 per cent) craniotomies, five (5 per cent) delayed orchidectomies and one anterolateral decompression of the vertebral column. Nine (13 per cent) patients required a repeat retroperitoneal node dissection and one patient needed a repeat thoracotomy to remove recurrent metastatic deposits during the period of follow-up. Multivisceral resections and vascular reconstruction procedures were required in 20 (30 per cent) patients undergoing retroperitoneal node dissection. Fifty-five (82 per cent) patients remain in complete remission with a mean follow-up period of 49.6 months (range 2 121 months). Nine (13 per cent) patients died with metastatic disease between 2 months to 4 years after operation. There were three deaths in the perioperative period (4 per cent). The histology of the resected metastases revealed undifferentiated active tumour in 20 (30 per cent) patients, differentiated mature teratoma in 29 (43 per cent) patients and fibrosis/necrosis in 18 (27 per cent) patients. Twelve (60 per cent) patients with undifferentiated elements and 15 patients (60 per cent) with raised preoperative tumour markers (poor prognostic categories) are in complete remission. Cytoreductive surgery in patients with metastatic germ cell tumours offers the best chance of remission following chemotherapy even in poor prognostic group categories. PMID- 1707716 TI - Pyramidal neurons are immunopositive for peptides, but not GABA, in the temporal cortex of the macaque monkey (Macaca fascicularis). AB - Areas 20, 21 and 22 of the temporal neocortex of the macaque monkey (Macaca fascicularis) were studied with immunocytochemical and electron-microscopic techniques to localise neurons immunoreactive to the neuropeptides vasoactive intestinal polypeptide, substance P and somatostatin, and to gamma-aminobutyric acid (GABA). GABAergic neurons were found in all cortical layers, but especially in layers II, IV and VI. They were all of non-pyramidal morphology, comprising small round cells, and bipolar or multipolar forms. Presumed GABAergic axon terminals were also common. Peptidergic neurons were also found in all layers, but they consisted of cells of many morphological types, including pyramidal cells. Compared with previous descriptions in other cortical areas and in other animals, we find a greater proportion of peptidergic temporal cortical neurons compared to the GABAergic population. The immunopositive neurons were easily recognisable ultrastructurally from non-reactive neurons by the dense labelling of the cytoplasm and nucleus. Immunopositive and negative neuronal somata were often contiguous, providing evidence for the specificity of the immune reaction. Stem dendrites were often labelled for a short distance from the soma, and other strongly reacting dendritic segments were found in the neuropil, as were labelled axons. Neurons labelled for GABA had features typical of non-pyramidal cells, but neuropeptides were also found in cells with pyramidal characteristics. PMID- 1707717 TI - The selectivity of the channel coupled to the 5-HT3 receptor. AB - The 5-HT3 receptor is unusual among receptors for biogenic amines in that it is directly coupled to an ion channel that is highly permeable to Na+ and K+. We have studied the permeation properties of this channel in order to achieve a more detailed understanding of its physiological function and to extend the comparison with other ligand gated channels. The 5-HT3 receptor channel is significantly permeable to the organic cations Tris, choline, and N-methyl-glucamine, with permeabilities decreasing with size. The permeability ratios for Tris and choline are similar to those determined for the nicotinic receptor; the permeability ratio for Tris is also similar to that of a non-N-methyl-D-aspartate (non-NMDA) excitatory amino acid receptor. This suggests that the diameters at the narrowest parts of these 3 channels are similar. The Ca2+ permeability of the 5-HT3 receptor channel is relatively low, with an upper bound to PCa/PNa estimated as 0.076. The single channel conductance, as determined by noise analysis, was also relatively low, with a value of 4.4 +/- 0.5 pS. Thus, both the Ca2+ permeability and single channel conductance are lower than those of the nicotinic receptor. In these respects, the 5-HT3 receptor is closer to non-NMDA excitatory amino acid receptors. These results are interpreted in terms of a model of the 5-HT3 receptor channel in which the interior has a lower polarizability, and possibly a greater length, in comparison with the nicotinic acetylcholine receptor channel. PMID- 1707718 TI - The adaptation of enkephalin, tachykinin and monoamine neurons of the basal ganglia following neonatal dopaminergic denervation is dependent on the extent of dopamine depletion. AB - This study examined whether dopamine (DA) is necessary for the normal development of striatal enkephalin and striatonigral tachykinin peptide systems. The neurotoxin, 6-hydroxydopamine (6-OHDA) was used to induce DA deficiency on the third day of the postnatal period in Sprague-Dawley rat pups. The animals were sacrificed at 60 days of age. The levels of Met5-enkephalin (ME) and substance P (SP) were determined by radioimmunoassay and preproenkephalin (PPE) and preprotachykinin (PPT) mRNA abundance in the striatum were assessed by hybridization analysis. The concentrations of DA, 5-hydroxytryptamine (5-HT) and their acid metabolites were determined by high-pressure liquid chromatography with electrochemical detection. The lesioned animals were grouped on the basis of the degree of loss of DA, and changes in ME, SP and 5-HT systems were correlated with respect to the degree of DA loss. The nature and extent of the changes in these systems were dependent on the degree of DA depletion. A loss of more than 90% DA was necessary to result in increased levels of ME and its PPE mRNA and reduced levels of SP and its PPT mRNAs; however, increased levels of 5-HT could be observed at a lower degree of DA loss. The results indicate that the normal development of enkephalin and tachykinin and 5-HT systems of basal ganglia are dependent on the availability of DA and/or the integrity of the nigrostriatal dopaminergic neurons. The results are relevant to our further understanding of the neurobiology of DA deficiency disorders. PMID- 1707719 TI - Effects of combined serotonin depletion and lesions of the nucleus basalis magnocellularis on acquisition of a complex spatial discrimination task in the rat. AB - The purpose of the present experiment was to determine the effects of lesions of cholinergic neurons originating from the nucleus basalis magnocellularis (NBM), alone or in combination with central serotonin depletion, on learning and memory in rats trained in the Stone 14-unit T-maze--a complex, positively-reinforced spatial discrimination task. Lesion of cholinergic neurons within the NBM was accomplished by bilateral infusion of ibotenic acid. Serotonin depletion was accomplished by the systemic administration of p-chloroamphetamine (PCA). The results show that PCA-induced serotonin depletion enhanced learning. This effect was completely prevented by NBM lesions, despite the fact that NBM lesions alone did not affect the performance of rats in this task. The results of this study support the view that the cholinergic and serotonergic systems may functionally interact in learning and memory processes. The significance of this interaction in the etiology and treatment of dementia should be further investigated. PMID- 1707720 TI - Biochemical and anatomical consequences of adult infraorbital nerve transection for serotonergic afferents within rat trigeminal subnuclei interpolaris and caudalis. AB - Immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used, more than 76 days after infraorbital nerve (ION) transection, to examine the distribution and density of serotonin-immunoreactive (5-HTIR) axons, as well as serotonin (5-HT) and 5 hydroxyindoleacetic acid (5-HIAA) content, within the infraorbital (IO) regions of subnuclei caudalis (SpVc) and interpolaris (SpVi). In SpVi, increases in 5-HT concentration and in density of 5-HTIR axonal varicosities were observed on the lesioned side. No changes were seen in SpVc. PMID- 1707721 TI - Distribution of galanin-immunoreactive nerve fibers in the rat nasal mucosa. AB - Galanin-like immunoreactivity was found in nerve fibers beneath and within the epithelium of the rat mucosa by the use of immunohistochemical techniques. Immunoreactive fibers were also noted close to blood vessels and seromucous glands in the lamina propria. Fast blue applied to the nasal mucosa underwent retrograde transport to some immunoreactive neurons of the trigeminal ganglion. Thus, the rat nasal mucosa was shown to be innervated by galanin-containing sensory nerves. PMID- 1707723 TI - Galanin inhibits insulin and corticosterone release after injection into the PVN. AB - The neuropeptide galanin (GAL, 1 microgram/0.3 microliters) was injected into the paraventricular nucleus (PVN) at two different times of the 12:12 h light/dark cycle, namely 2 h before the dark ('pre-dark') and before the light ('pre-light') periods. Blood samples were collected 15 min after injection and examined for serum levels of insulin (INS), corticosterone (CORT) and glucose (GLUC). Results indicate that PVN GAL injection in the pre-dark period strongly inhibits both CORT and INS release but has no effect on GLUC levels. These hormone changes, however, were not detected in the blood samples collected in the pre-light period. At this time, baseline levels of CORT were significantly lower than in the pre-dark period, while INS and GLUC levels were generally similar at both time periods. These results indicate that the effects of PVN GAL on CORT and INS secretion are inhibitory in nature and are temporally linked to the diurnal cycle. PMID- 1707722 TI - Systemic administration of thyrotropin-releasing hormone enhances striatal dopamine release in vivo. AB - We examined the effects of systemically administered thyrotropin-releasing hormone (TRH) on the release of dopamine (DA), as assessed by brain microdialysis within the corpus striatum of anesthetized rats. A single dose (10 micrograms i.v.) elevated DA levels in brain extracellular fluid (ECF) by 240% above baseline levels after 150 min. Systemic tyrosine ([TME] 20 mg/kg i.v.) also increased DA release (by 190% after 150 min), while combined treatment with both agents was associated with significant potentiation of the DA response (to 640% after 150 min). None of the treatments significantly altered striatal tissue levels of DA or its metabolites. A large dose of TRH (50 micrograms i.v.) significantly increased DA release (by 1150%) whether or not animals had received an active or denatured prolactin (PRL) antiserum prior to the experiment, suggesting that the TRH effect is not mediated by PRL. Although TRH is rapidly metabolized in plasma and penetrates the blood-brain barrier only poorly, our results suggest that even relatively small doses of the hormone can affect striatal dopaminergic neurotransmission. PMID- 1707724 TI - Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior. AB - The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior. PMID- 1707725 TI - Anatomical interconnections of the pedunculopontine tegmental nucleus and the nucleus prepositus hypoglossi in the cat. AB - The Pedunculopontine tegmental nucleus (PPT) is a mesopontine structure containing predominantly cholinergic neurons, and physiological data indicate its neurons transfer eye-movement gated ponto-geniculo-occipital (PGO) waves to thalamus during the rapid eye movement phase of sleep. The present study, using anterograde and retrograde tracing of wheat germ agglutinin-conjugated horseradish peroxidase, found that the medullary nucleus Prepositus hypoglossi (PH), whose neurons are known to have eye-movement-related information, projects densely to PPT, and PPT has reciprocal projections to PH. The PH-PPT projection has some topographic organization, with rostral PH to rostral PPT and caudal PH to caudal PPT projections dominating. The PH-PPT projection may furnish the anatomical substrate for input of eye movement-related information into the rostral PGO wave system. PMID- 1707726 TI - Antigenic profile of plaques and neurofibrillary tangles in the amygdala in Down's syndrome: a comparison with Alzheimer's disease. AB - Most patients with Down's syndrome (DS) undergo a premature cognitive decline with aging, and eventually develop the neuropathologic changes of Alzheimer's disease (AD), including amyloid-containing neuritic plaques, and the formation of neurofibrillary tangles. The amygdala is a focus of marked neuropathologic change in older patients with DS and in AD. We examined the amygdala with immunocytochemical and histochemical methods in 6 cases with DS, ages 19, 20, 27, 29, 56 and 64 years and compared them to 4 cases with AD, ages 54, 76, 77 and 80 years. An antiserum to the A4 amyloid peptide demonstrated amyloid deposition in plaques in all 10 cases. Plaques were also revealed in all cases by the Alcian blue stain for glycosaminoglycans and by the Bielschowsky and Bodian silver stains. An antiserum to alpha-1-antichymotrypsin (ACT) showed plaques in the AD cases and in the 19, 56 and 64 year old DS cases. Neurofibrillary tangles were observed with silver stains only in the older DS and in the AD cases, and not in the 19, 20, 27 and 29 year old DS cases. Likewise, antisera to paired helical filament, to microtubule associated proteins tau and microtubule associated protein-2 (MAP-2), and to ubiquitin, all of which are components of neurofibrillary tangles, reacted with tangles and abnormal neurites only in the older DS and the AD cases. An antiserum to neurofilament epitopes labeled NFTs in the older DS cases and the AD cases, but not in the younger DS cases, except for two intraneuronal NFTs in the 27 year old case.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707727 TI - Glial and neuronal markers in bass retinal horizontal and Muller cells. AB - Retinal horizontal cells (HCs) are second-order neurons that integrate information from photoreceptors over large retinal areas, mediating the lateral spread of visual signals in the distal retina. The 'glial' vs. 'neuronal' nature of the HC has been widely debated. For example, carbonic anhydrase (CA), glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP) are considered 'glial' markers, yet both CA and GFAP have been previously reported in HCs of the teleost retina in species-specific patterns. In contrast, the neurofilament triplet (NFT) proteins are considered 'neuronal' markers; these proteins have been immunolocalized to a mammalian HC, but are absent from teleost HCs. We have studied these cytochemical characteristics in HCs from the white bass, by immunolabeling both cryosections of intact retina and freshly isolated, identified cells attached to coverslips. We found that both HCs (neurons) and Muller cells (MCs; glia) immunolabeled with antisera to CA. Both type 1 (external) HCs and MCs immunolabeled with an antibody to vimentin. Only MCs immunolabeled with antisera to GS and GFAP. Neither HC perikarya (and their major dendrites) nor MCs immunolabeled with an antibody to the 160-kDa subunit of NFT protein. Thus, bass HCs and MCs share the presence of CA and vimentin epitopes and absence of the NFT 160-kDa epitope. Moreover, retinal cell isolation, by itself, does not affect cell-type specific immunolabeling patterns in identified cells, except for what may be lost with the finer processes of the various cells. Isolated cell studies can aid in interpreting immunolabeling patterns observed in the intact retina, especially in retinal layers where several cell types may be present. PMID- 1707728 TI - Peptidergic innervation of the cremaster nucleus. I. A sexually dimorphic population of substance P-containing intraspinal neurons exists in the substance P pathway to the rat cremaster nucleus. AB - The cremaster nucleus (CN) lies in the lumbar spinal cord and is sexually dimorphic: the male CN contains three times as many motoneurons as the female. The substance P (SP) innervation of the CN is also sexually dimorphic with males receiving a very prominent innervation which is greatly diminished in females. These investigations examined SP-containing neurons located in the ventral half of lamina IV and the lateral aspects of laminae V, VII, and IX, in lumbar spinal levels 1,2. SP-containing intraspinal neurons in these laminae are at least three times as numerous in males than females. This provides the first demonstration of a sexually dimorphic population of spinal neurons which is not motor or preganglionic in nature. These SP-containing interneurons are found within, or adjacent to, the SP-containing fibers which constitute the massive SP pathway to the male CN. Processes of these SP-containing neurons were observed to contribute to the formation of the SP pathway to the male CN. The immunohistochemically demonstrable presence of these lumbar 1,2, laminae IV-IX, SP-containing neurons validates former studies which suggested their existence (Gibson et al., Brain Research, 301 (1984) 243-251; Uda et al., Neurosci. Lett., 57 (1985) 185-190). PMID- 1707729 TI - Anatomical and neurochemical evidence for suicide transport of a toxic lectin, volkensin, injected in the rat dorsal hippocampus. AB - Volkensin, a ribosome-inactivating toxic lectin which has been proposed as a 'suicide transport' agent in the CNS, was unilaterally injected in the rat dorsal hippocampus at a dose of 1.2 ng. Three to 5 days after the injection, degenerating neurons were observed at the electron microscope in the medial septum-diagonal band area ipsilateral to the injection. Ten days after the injection, the number of pyramidal neurons in the CA3 region of the contralateral hippocampus, which are the major source of hippocampal commissural fibers, was obviously decreased. At the same survival time, the number of choline acetyltransferase (ChAT) immunoreactive neurons in the ipsilateral medial septum diagonal band area was moderately but significantly decreased. These neurons are known to be the major source of the septohippocampal cholinergic projection. Concomitantly, microchemical assays of ChAT levels revealed a 25% decrease of enzyme activity in the medial septum-diagonal band area ipsilateral to the injection. This was accompanied by a 33% decrease of ChAT in the ipsilateral ventral hippocampus which was interpreted to be due, at least in part, to the degeneration of cholinergic septal neurons projecting to both the dorsal and the ventral hippocampus. Taken together, these results provide clear evidence that volkensin is taken up by nerve terminals in the injected area of the brain and retrogradely transported to the cell bodies originating the projection, which are killed by the toxin. The usefulness of the strategy of 'suicide transport' in the CNS is, therefore, confirmed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707730 TI - Changes of substance P-like immunoreactivity in the dorsal horn are associated with the 'phasic' behavioral response to a formalin stimulus. AB - Substance P (SP) has been proposed as a nociceptive transmitter/modulator in the dorsal horn of the spinal cord. Formalin used as a nociceptive stimulus has been shown to increase, in a biphasic manner, the amount of immunoreactive SP in the dorsal horn. The time course of the changes in substance P-like immunoreactivity (SPLI) caused by formalin is similar to both the electrical activity of dorsal horn neurons and licking behaviors. The administration of morphine reduces stereotypic behaviors caused by a formalin injection but actually increases the amount of SPLI in the dorsal horn. Therefore, the extent to which SP in the dorsal horn is involved with nociception as a result of formalin remains uncertain. To test the involvement of SP with chemogenic nociception, we utilized lidocaine to block afferent activity prior to an injection of formalin and studied the time course of behaviors and SPLI changes in the dorsal horn. Our results showed that formalin produced two distinct phases of nociceptive behaviors as measured by stereotypic licking of the injected paw: an acute 'phasic' response followed by a longer-lasting 'subacute' or 'tonic' response. Lidocaine reduced both phases of stereotypic behaviors, but only reduced the first increase of SPLI in the dorsal horn. These results suggested a direct involvement of SPLI in the dorsal horn with only 'phasic' behavioral responses to a formalin stimulus. PMID- 1707731 TI - Reduction of neurotensin immunoreactivity in the amygdala in Alzheimer's disease. AB - The density of neurotensin immunoreactivity (NT-IR) was dramatically decreased in 6 of 12 amygdaloid nuclear subregions in patients with Alzheimer's disease (AD) compared to age-matched normals. Diminution of NT-IR was most pronounced in amygdaloid regions containing the greatest number of senile plaques. This contrasts to our previous findings of little, if any, loss of substance P or somatostatin immunoreactivity within these same regions. The present findings corroborate biochemical reports of a decrease in NT-IR in the AD amygdala and suggest that this peptide may be selectively affected relative to other neuropeptides. PMID- 1707732 TI - The subparafascicular thalamic nucleus of the rat receives projection fibers from the inferior colliculus and auditory cortex. AB - The sources of afferent fibers to the subparafascicular thalamic nucleus (SPF) of the rat were investigated by the retrograde WGA-HRP and anterograde PHA-L methods. Layer V of the areas 3, 1 and 2 of the temporal cortex as well as the dorsal and external cortices of the inferior colliculus were found to send projection fibers to the whole rostrocaudal extent of the SPF bilaterally with a clear-cut ipsilateral dominance. The results indicate that the SPF of the rat may constitute a relay nucleus in the central auditory pathways. PMID- 1707733 TI - Inhibition of N-methyl-D-aspartate activated ion current by desmethylimipramine. AB - The tricyclic antidepressant desmethylimipramine (DMI) interacts with the NMDA receptor/ionophore complex; however, the site of the interaction has not been clearly established. Although evidence from receptor binding assays suggests that DMI interacts with the Zn2+ binding site, other binding studies and electrophysiological studies suggest otherwise. Using the whole-cell patch clamp technique to record from cultured hippocampal neurons, we report that recovery of NMDA-activated current from block by DMI is time-dependent and this time dependent component was not observed following preexposure of neurons to Zn2+. These observations favor the hypothesis that DMI interacts at a binding site within the NMDA receptor/complex channel pore and not at the Zn2+ binding site. PMID- 1707734 TI - Topographical projections from the thalamus, subthalamic nucleus and pedunculopontine tegmental nucleus to the striatum in the Japanese monkey, Macaca fuscata. AB - Topographical projections from the thalamus, subthalamic nucleus (STN) and pedunculopontine tegmental nucleus (PPN) to the striatum were examined in the Japanese monkey (Macaca fuscata) by using the retrograde axonal transport technique of WGA-HRP (wheat germ agglutinin-conjugated horseradish peroxidase). After WGA-HRP injection in the head of the caudate nucleus (CN) or putamen (Put), labeled neuronal cell bodies in the thalamus were distributed mainly in the nucleus ventralis anterior (VA)-nucleus ventralis lateralis (VL) complex and the nucleus centrum medianum (CM)-nucleus parafascicularis (Pf) complex, and additionally in the paraventricular, parataenial, rhomboid, reuniens, centrodorsal, centrolateral, paracentral, and centromedial nuclei. The data indicated that the pars principalis of VA (VApc) projected mainly to CN and additionally to Put, and that the pars magnocellularis of VA (VAmc) or pars oralis of VL (VLo) projected selectively to CN or Put, respectively. It was also indicated that CM projected to the middle and caudal parts of Put, while Pf projected to CN and the rostral part of the Put. The data further indicated that the dorsomedial, ventromedial, or lateral part of CM projected respectively to the dorsolateral, ventromedial, or intermediate part of Put, and that the medial or lateral part of Pf projected respectively to the medial or lateral part of the head of CN. Direct projections from STN and PPN to the striatum were confirmed. The subthalamostriatal projections showed a mediolateral topography. The PPN was shown to project bilaterally to the striatum with an ipsilateral predominance. PMID- 1707735 TI - Evidence for decreased transport of tryptophan hydroxylase in Alzheimer's disease. AB - Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of serotonin and a specific marker for serotonergic neurons. These neurons are affected in Alzheimer's disease (AD) in several ways: serotonin is decreased in axon terminals, serotonin neurons accumulate neurofibrillary protein, and these neurons are lost in AD brains. One subcellular mechanism which may underlie degeneration of neurons in AD is decreased axonal transport with accumulation of enzymes and their potentially toxic metabolites in the cell body. To determine whether there is a defect in axonal transport in serotonin neurons in AD we measured TPH activity, serotonin and its oxidative metabolite 5 hydroxyindoleacetic acid (5-HIAA) in dorsal raphe cell bodies from Alzheimer and control cases. TPH activity is increased 4.7-fold in raphe neuron cell bodies in Alzheimer brains. Serotonin and 5-HIAA are increased by 4.0- and 2.0-fold, respectively in Alzheimer compared to control raphe cell bodies. In contrast, in synaptic terminals of the amygdala 5-HT and 5-HIAA were decreased by 41% and 50%, respectively in the same AD cases. We propose that the accumulation of TPH and its products in the raphe perikarya in AD results from a diminished transport of TPH to axon terminals. The accumulation of oxidative metabolites of serotonin may contribute to the degeneration of serotonergic neurons in AD. PMID- 1707736 TI - Phosphorylation-related immunoreactivity and the rate of transport of neurofilaments in chronic 2,5-hexanedione intoxication. AB - Axonal transport of neurofilaments and the phosphorylation of epitopes on neurofilament proteins was studied in rats chronically intoxicated with 2,5 hexanedione. Sensory axons arising from the L5 dorsal root ganglion exhibited accelerated transport and a reduced abundance of neurofilament proteins. The binding of an antibody to phosphorylated neurofilament epitopes was compared to the binding of an antibody to non-phosphorylated epitopes by quantitative ELISA. This immunochemical index of neurofilament phosphorylation was reduced in dorsal roots, proximal peripheral sensory axons and ventral roots, but not in a distal nerve (the nerve to the soleus). Axotomy produced a reduction in neurofilament protein abundance comparable to hexanedione without any change in the immunochemical phosphorylation index. These results are consistent with the hypothesis that the state of phosphorylation of neurofilaments in proximal axons is correlated with the rate of neurofilament transport. PMID- 1707737 TI - A subpopulation of primate corticocortical neurons is distinguished by somatodendritic distribution of neurofilament protein. AB - In recent immunohistochemical studies of human and monkey neocortex we observed that the somatodendritic distribution of neurofilament protein appears to be restricted to a subpopulation of pyramidal neurons. To further characterize this apparent specificity in cytoskeletal organization, combined retrograde tract tracing and immunohistochemical methods were used to examine the extent to which neurons from different cortical areas providing a projection to prefrontal cortex have a somatodendritic distribution of neurofilament proteins. These studies revealed that the proportion of neurons providing a projection from different cortical areas to prefrontal cortex varied from nearly 30% to 90%, and appeared to be related to the functional nature of the projection. PMID- 1707738 TI - K+ efflux pathways and neurotransmitter release associated to hippocampal ischemia: effects of glucose and of K+ channel blockers. AB - Ischemia of hippocampal slices leads to 86Rb+ efflux and to amino acid neurotransmitter release. This 86Rb+ efflux which corresponds to the massive K+ efflux from neuronal cells observed in ischemic animals is inhibited by glucose (IC50 = 1.7 mM). Glucose also inhibits the ischemia induced liberation of GABA and aspartate. 86Rb+ efflux is insensitive to any type of known blockers for ATP sensitive, Ca2(+)-sensitive and voltage-sensitive K+ channels. PMID- 1707739 TI - Afferent projections of the superior and recurrent laryngeal nerves. AB - The central afferent projections of the superior and recurrent laryngeal nerves were investigated in the rat, utilizing the transganglionic transport of WGA-HRP. Labelled superior laryngeal nerve terminal fields were found bilaterally in the interstitial and medial subnuclei of the nucleus tractus solitarii with the ipsilateral being more dense. The distribution of the recurrent laryngeal nerve terminals were similar to that of the SLN with two major differences: the projections were ipsilateral, and there was a marked decrease in the terminal field density. The terminal field density differences were confirmed by quantitatively identifying the labelled ganglion cells of the vagus nerve. These findings accurately delineate the first integrative components in the mediation of the complex laryngeal reflexes. PMID- 1707740 TI - Subregions of the caudate nucleus and their in- and output channels in oro-facial dyskinesia: a behavioural and retrograde tracing study in the cat. AB - Previous studies have shown that the feline caudate nucleus contains DPI sensitive (caput nuclei caudati, anterodorsal part; r-CRM) and DPI-insensitive (caput nuclei caudati, rostromedial part; CRM) regions. Stimulation of dopamine receptors within the r-CRM by dopamine or DPI are known to elicit oro-facial dyskinesia (OFD), i.e. a syndrome of tic-like contractions of the facial muscles in combination with tongue protrusions. OFD is also elicited from the sub commissural part of the globus pallidus (scGP), a first order output station of the r-CRM, but not from the CRM. On the basis of these data it has been hypothesized that (1) OFD is a specific feature of the r-CRM, but not the CRM; (2) effects elicited from the r-CRM are funneled via the scGP, and that (3) r-CRM and CRM are differentially innervated. Cats were bilaterally equipped with cannulas directed at the CRM or r-CRM and scGP. Following recovery from the operation the cats received bilateral injections of DPI into CRM (5 micrograms/5 microliter) or r-CRM (5 and 10 micrograms/5 microliter), the latter in combination with muscimol (50 and 100 ng/1 microliter) into the scGP or its solvent. Subsequently, behaviour was analyzed. OFD, quantified in number of tongue protrusions, was only elicited from the r-CRM, but not from the CRM confirming previously reported data in this respect. Furthermore the effect varied according to the dose used. The OFD elicited from the r-CRM was found to be blocked at the level of the scGP by local injections of muscimol, a GABA agonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707741 TI - [Monoclonal antibodies obtained against G-protein epitopes: comparison of immunoreactions seen in the brain, retina and striated muscular tissue]. AB - Monoclonal antibodies have been obtained against a purified fraction of brain G proteins containing the Gi alpha, G0 alpha, G beta, and G gamma subunits. After characterization, two monoclonal antibodies have been used to detect the cellular distribution of the two epitopes in neural, retinal and muscular tissues: ELISA, cross-dot and Western blot demonstrated that F.IV.5 is an anti-G beta antibody specific for the 36 kDa beta-subunit. ELISA, cross-dot and immunocytochemical distribution of the epitopes recognized by F.VII.9 suggested that this antibody recognizes epitopes which are also detected with polyclonal anti-G0 alpha antibodies. With both monoclonal antibodies, we confirmed that G proteins demonstrated a sub-membranous distribution as well as extensively cytoplasmic, axoplasmic or sarcoplasmic distributions in different cell types. PMID- 1707742 TI - Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes. AB - Isolated rat hepatocytes were used as an in vitro model to investigate a possible interaction between oxytetracycline (OXT) and aflatoxin B1 (AFB1). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB1 inhibited RNA and protein synthesis at a concentration of 10(-7) M and 10(-6) M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10(-3) M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB1 inhibited the accumulation of glycogen from 10(-6) M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB1 on glycogen accumulation. If the "in vivo" protection, provided by OXT against AFB1 induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB1-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB1. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism. PMID- 1707743 TI - Deuterium oxide reduces agonist and depolarization-induced contraction of rat aortic rings. AB - The influence of deuterium oxide (D2O) on calcium-dependent vascular smooth muscle contraction was investigated. The effect of D2O on receptor-operated calcium channels was investigated with phenylephrine-induced contraction in the rat aortic ring preparation. D2O depressed the contraction response in a dose dependent manner with 50% inhibition of maximum contraction observed with 60% D2O. The effect of 60% D2O on phenylephrine-induced contraction was reversible and not dependent on an intact endothelium. Sixty percent D2O also reduced potassium chloride induced contractions by 50%, indicating an effect on voltage operated calcium channels. Studies with Bay K 8644, and L-type calcium channel activator, confirm an effect on utilization of extracellular calcium sources and on the voltage-operated calcium channel. Sixty percent D2O also depressed a calcium contraction dose-response curve by approximately 25%. Likewise, a change in the pD2' for nifedipine in the presence of D2O may indicate an effect on the nifedipine binding site and (or) the voltage-dependent calcium channel. Further studies were performed to determine whether the D2O effects were nonspecific or selective effects on the receptor- and voltage-operated calcium channels. Sucrose induced contaction in the presence of 60% D2O was found to be inhibited by approximately 50%. D2O similarly affected isoprenaline relaxation, which would suggest a nonspecific D2O effect on the vascular smooth muscle contractile process. PMID- 1707744 TI - Mechanisms underlying anoxic hyperpolarization of hippocampal neurons. AB - The outward current evoked in CA1 neurons by brief anoxia is strongly voltage dependent and is abolished by an atropine-sensitive action of carbachol (and also when recording with a GTP gamma S-containing microelectrode). In this respect, it closely resembles the M-type K current, but the involvement of other, voltage independent, carbachol-sensitive K channels has not been excluded. When the anoxic outward current is eliminated, an anoxic inward current is revealed, which may be Cl- mediated. It is suggested that an early release of Ca2+ from a dantrolene (and perhaps GTP)-sensitive internal store activates Ca-sensitive Cl channels, as well as carbachol-sensitive (mainly M-type?) K channels. The opposing Cl- and K currents would account for the variable depolarizing and hyperpolarizing effects of anoxia. PMID- 1707745 TI - A phase II study of days 1 and 8 cisplatin and recombinant alpha-2B interferon in advanced non-small cell lung cancer. AB - Preclinical data from studies of human lung cancer xenografts suggest that the cytotoxic effects of cisplatin are enhanced by alpha-interferon. To verify the above observations, the authors initiated a Phase II trial in advanced non-small cell lung cancer (NSCLC). Cisplatin was given at 100 mg/m2 during a 28-day cycle in a divided day 1 and day 8 schedule. Starting on day 1, alpha-2B interferon was administered intramuscularly at a dose of 5 million units three times a week continuously for a minimum of 2 months. Between January 1989 and September 1989, 30 patients were evaluated for response and toxicity. According to the staging system proposed by Mountain, 20 patients had Stage IV disease, 7 had Stage IIIB disease, and 3 had Stage IIIA disease. Expression of neuron-specific enolase (NSE) and Leu-7 was immunohistochemically investigated to evaluate possible relationship to treatment response. The response rate was 13.3% (95% confidence interval [CI]: 1.2% to 25%). The four responders showed positivity for NSE, and two of them were positive for Leu-7. An average of three cycles was given. The mean dose intensity administered was 83% of the projected dose for cisplatin and 92% of the projected dose for alpha-2B interferon. A standard scale was used to assess interferon toxicity. Hematologic, renal, and systemic side effects were not significant. In advanced NSCLC the addition of alpha-2B interferon did not increase the cisplatin-induced response rate. Further studies should be performed to determine the real value of chemotherapy response in tumors showing positive immunoreactivity for neural markers such as NSE and Leu-7. PMID- 1707746 TI - The effect of palliative radiation therapy on epidural compression due to metastatic malignant melanoma. AB - The efficacy of palliative radiation therapy in the treatment of spinal cord and cauda equina compression due to metastatic malignant melanoma was evaluated in 38 sites in 35 patients treated between 1970 and 1990. All patients had radiographic documentation of epidural compression. The median dose of radiation therapy was 2850 cGy (range, 500 to 4000 cGy), with daily fractions ranging from 200 to 800 cGy. Twenty-eight sites in 26 patients were evaluable 1 month after completion of radiation therapy, and symptoms responded completely in 11 of 28 (39%) sites. Fourteen sites (46%) showed a partial response of symptoms. Response lasting until death was documented in 21 of 26 patients (81%). Patients receiving a total dose of 3000 cGy or greater were more likely to achieve a complete response than those receiving less than 3000 cGy (62% versus 20%) by univariate (P = 0.025) and multivariate (P = 0.048) analyses. A treatment program of radiation therapy and corticosteroids is effective in palliating the symptoms of epidural compression due to metastatic malignant melanoma. It is recommended to deliver an accelerated course of radiation therapy to a dose of 3000 cGy or greater without exceeding spinal cord tolerance (e.g., 3000 cGy in ten fractions at 300 cGy per fraction). PMID- 1707748 TI - Influence of hepatitis B virus infection and age on mode of growth of hepatocellular carcinoma. AB - According to the extent of hepatic involvement of the tumor and that of portal vein invasion at the time of initial diagnosis, patients with hepatocellular carcinoma (HCC) were grouped into three or four groups. Correlations among the extent of hepatic involvement, extent of portal vein invasion, and prevalence of hepatitis B surface antigen (HBsAg) and age distribution were examined. The extent of hepatic involvement of the tumor and that of portal vein invasion were significantly greater in patients with positive HBsAg compared with findings in the negative patients (P less than 0.001). For cases of both positive and negative HBsAg, patients with a more extensive HCC were significantly younger. Results of the multivariate logistic regression analysis showed that hepatitis B antigenemia and younger age were statistically significant and independent positive predictors of extensive HCC. These results strongly suggest that hepatitis B surface antigenemia and age play an important role in the growth mode and the kinetics of HCC in Japanese patients. PMID- 1707747 TI - In vivo measurements of the fraction of dose of bleomycin labeled with cobalt 57 delivered to human tumors. AB - Concentrations of bleomycin labeled with cobalt 57 (Co-bleo) over time were measured in vivo in 17 patients with 32 sites of lymphoma and 18 patients with lung tumors after administration of the same dose of bleomycin. There were marked variations in individual tumor drug concentrations even among tumors with the same histologic type, indicating that the tumor concentration of this drug in individuals cannot be predicted from the administered dose. Also, tumor concentration could not be predicted from the area under the concentration over time curve (AUC) of Co-bleo in the blood; there was no correlation (r = 0.53) between the AUC and the concentration in the tumor at any point in time between 30 minutes and 8 hours after injection. There was no significant difference in the percent of the injected dose per milliliter (%ID/ml) which was delivered to the tumor when low and high amounts of bleomycin were administered to the same patient. Also, a good correlation (r = 0.88) between the %ID/ml over time was found when injection of low and high doses of bleomycin were compared. The results indicate that using quantitative single photon emission computed tomography (SPECT) and a labeled tracer dose it is possible to predict what fraction of the dose of a chemotherapeutic drug will concentrate in an individual patient's tumor in vivo. They also show that, for bleomycin, escalation of dose will result in a proportional increase of tumor concentration. This increase depends on individual properties of tumors which can be measured quantitatively in vivo by SPECT and are expressed as percent of %ID/ml of tumor tissue. PMID- 1707749 TI - (6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid effects on nucleotide metabolism in CCRF-CEM human T-lymphoblast leukemia cells. AB - (6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid [(6R)DDATHF] is a folate antimetabolite with activity specifically directed against de novo purine synthesis, primarily through inhibition of glycinamide ribonucleotide transformylase. This inhibition resulted in major changes in the size of the nucleotide pools in CCRF-CEM cells. After a 4-h incubation with 1 microM (6R)DDATHF, dramatic reductions in the ATP and GTP pools were observed, with almost no effect on CTP, UTP, and deoxyribonucleotide pools. When the incubation was continued in drug-free medium, recovery of ATP and GTP pools was protracted. ATP did not return to normal until 24-36 h, and GTP pools were only partially repleted by 48 h. The ATP and GTP pools were not affected when the initial 4-h incubation with (6R)DDATHF was conducted in the presence of 100 microM hypoxanthine. Addition of hypoxanthine to the medium after a 4-h incubation with (6R)DDATHF caused rapid recovery of the ATP and GTP pools. Similar effects were seen when the purine precursor aminoimidazole carboxamide was used in place of hypoxanthine. The effect of (6R)DDATHF on nucleotide pools and the capability of hypoxanthine or aminoimidazole carboxamide to prevent or reverse this phenomenon correlated directly with the inhibition of cell growth. Presumably as a consequence of the decrease in purine nucleotide triphosphate levels, the conversion of exogenously added uridine, thymidine, and deoxyuridine to nucleotides was markedly decreased. These effects were protracted for almost 48 h and were also reversed by hypoxanthine. Differential repletion of ATP and GTP pools after (6R)DDATHF pre-treatment demonstrated that diminished precursor phosphorylation is primarily a consequence of GTP rather than ATP starvation. PMID- 1707750 TI - Comparative study of monoclonal antibodies TURP-27 and HNK-1: their relationship to neural cell adhesion molecules and prostate tumor-associated antigens. AB - The murine monoclonal antibodies TURP-27 and HNK-1 have been shown to detect antigens which are heavily expressed by benign prostatic hyperplasia and carcinoma of the prostate. Western blot analysis of prostate extracts showed that monoclonal antibodies TURP-27 and HNK-1 bound glycoproteins with molecular weights of 180,000, 140,000, 120,000, 100,000, 90,000, and 69,000. Studies have shown that the HNK-1 carbohydrate epitope may be involved in cell adhesion and that it is a component of several characterized adhesion proteins. TURP-27 was found to bind at least three of these adhesion proteins: neural cell adhesion molecules; myelin-associated glycoprotein; and a second myelin glycoprotein, P0. Western blot analysis of prostate extracts showed that an antineural cell adhesion molecule serum bound the Mr 180,000 and 140,000 proteins. Based on reciprocal blocking and chemical tests, it was determined that the TURP-27 and HNK-1 epitopes are not identical. These data imply that the TURP-27 epitope may be a variant of the HNK-1 epitope or that the two epitopes are closely linked and that the TURP-27 and HNK-1 epitopes on prostate cells are positioned on neural cell adhesion molecule-like proteins. PMID- 1707751 TI - N,N',N''-triethylenethiophosphoramide (thio-TEPA) oxygenation by constitutive hepatic P450 enzymes and modulation of drug metabolism and clearance in vivo by P450-inducing agents. AB - The cancer chemotherapeutic drug N,N',N''-triethylenephosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N'' triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio TEPA (100 microM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was approximately 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15-20% inhibitable by these antibodies but was 80-85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min-1 P450-1, respectively, at 100 microM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, less than or equal to 0.15 min-1 P450-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707753 TI - Preferential expression of c-kit protooncogene transcripts in small cell lung cancer. AB - As an initial step to understand rapid growth of small cell lung cancer (SCLC), a complementary DNA library prepared from a SCLC cell line was screened with viral oncogene probes encoding protein-tyrosine kinases, which are known to play an important role in regulation of cell growth. Fifteen clones hybridizing with v fms probe were isolated, and, by partial sequence analysis, four of them were identified to be c-kit protooncogenes. Northern blot study demonstrated that most of the SCLC tumors and cell lines expressed c-kit transcripts, while non-SCLC tumors and cell lines did not. Neither amplification nor rearrangement of the c kit gene was demonstrated in SCLC cell lines by Southern blot analysis, however. Our results suggested that c-kit expression in SCLC reflects the unique biological nature of the tumor cells different from non-SCLC and further suggested that the c-kit product may participate in autocrine or paracrine stimulation of SCLC growth. PMID- 1707752 TI - Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate. AB - 2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5' triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2 fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707754 TI - B16 melanoma metastasis to an "artificial organ" implant. AB - The mouse B16 melanoma metastasizes in two stages, first to the lungs and then from lung metastases to systemic organs. Despite widespread dissemination, visible metastases generally occur only in the brain, adrenals, kidneys, ovaries, pancreas, and mesentery. As a novel approach to investigate the basis of metastatic patterning in this system, the possibility was explored that an implantable "artificial organ" could serve as a site for the occurrence and experimental modulation of secondary-stage metastasis. Each implant consisted of a cellulose disc 4 mm in diameter, with a central 1-mm polymer pellet to effect local sustained release of angiogenic or growth factors in a s.c. environment. During the secondary spread of tumors initiated with the B16 melanoma clone G3.12 and with the more metastatic variant G3.12/BM2, metastatic involvement of implants containing angiogenic factors was mainly as invisible micrometastases demonstrable by bioassay; visible metastases were rare and were located in implant blood vessels. Metastasis occurred in about 30% (G3.12) and 50% (G3.12/BM2) of implants with vasculature induced by ethylene-vinyl acetate copolymer alone. Endothelial cell growth factor and heparin promoted greater vascularization but did not significantly alter metastatic involvement of implants. Release of tumor cell mitogenic activity from pellets containing a crude extract of mouse lungs increased the incidence of G3.12/BM2 metastasis in implants to over 70% and stimulated growth of visible metastases within the cellulose matrix. In contrast, liver extract inhibited metastasis growth. Colonization of implants following intracardiac injection of G3.12/BM2 cells was generally similar to metastasis, but visible colonies formed more readily and were less dependent on the influence of lung extract. These results indicate that metastasis and colonization can occur regularly in implants and that the relative favorability of the implant environment for secondary tumor growth can be altered by incorporation of tumor cell growth modulators. PMID- 1707755 TI - Structural studies of the O-specific polysaccharide from Salmonella kentucky strain 98/39 (O:8, H:i, Z6). PMID- 1707756 TI - Modified malto-oligosaccharides as inhibitors of human alpha-amylases. PMID- 1707757 TI - Synthesis of a tetrasaccharide of the genus-specific lipopolysaccharide epitope of Chlamydia. AB - Allyl 2-acetamido-2-deoxy-3,4-O-(1,1,3,3-tetraisopropyldisiloxan-1,3- diyl)-beta D- glucopyranoside was coupled with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha D-manno-2-octulopyranosyl bromide)onate (1) to give a good yield of the alpha-(2- --6)-linked disaccharide, isolated after deacetylation and regioselective conversion into the corresponding 7',8'-O-carbonyl or 7',8'-O-(1,1,3,3 tetraisopropyldisiloxane-1,3-diyl) derivatives, respectively. Subsequent glycosylation with 1 gave a high yield of the alpha- and beta-(2"----4')-linked trisaccharide derivatives 16 and 18, whereas block synthesis using the alpha-(2-- -8)-linked Kdo-disaccharide bromide derivative 19 afforded a low yield of the corresponding alpha- and beta-(2"----4')-linked tetrasaccharide derivatives 20 and 22. Removal of the protecting groups furnished the disaccharide allyl O (sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----6)-2-acetamido -2 deoxy- beta-D-glucopyranoside, the trisaccharide allyl O-(sodium 3-deoxy-alpha-D manno-2-octulopyranosylonate)-(2----4)-(sodium 3-deoxy-alpha-D-manno-2 octulopyranosylonate)-(2----6)-2-acetamido -2-deoxy- beta-D-glucopyranoside, and the tetrasaccharide allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate) (2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-(sodium 3 deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----6)-2-acetamido -2-deoxy- beta-D glucopyranoside in high yield. Copolymerization of the allyl glycosides with acrylamide gave artificial polyvalent haptens suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydia lipopolysaccharides. PMID- 1707758 TI - Electrophysiological basis of ventricular tachyarrhythmias. PMID- 1707760 TI - FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes. AB - This report compares the ability of cyclosporin A and FK-506 to inhibit human T cell activation triggered via cell surface molecules that utilize different intracellular processes. We stimulated highly purified peripheral blood T lymphocytes with mitogens (Con A and PHA), ionomycin + PMA, or monoclonal antibodies specific for cell surface antigens involved in activation (CD2, CD3, CD28) either in combination with each other or in conjunction with PMA. Using measurements of the proliferative response, IL-2 production, and changes in intracellular Ca2+ ([Ca2+]i), we demonstrate that FK-506 exerts its inhibitory effect on early events of T-cell activation in a manner indistinguishable from that of CsA. An important finding in this study is the strict correlation between those activation pathways that are inhibited by FK-506 and CsA and the requirement that the sensitive pathways induce a measurable rise in [Ca2+]i. This correlation held even for the CD28/CD2 pathway which was previously shown to be calcium-independent; however by employing FACS analysis of [Ca2+]i within individual cells, a subset of cells activated via CD28/CD2 was found to respond with a measurable rise in [Ca2+]i. We also noted that the proliferative response induced by certain stimuli, such as ionomycin + PMA and PHA + PMA, was partially resistant to FK-506 and CsA, while IL-2 production was completely suppressed. The partial FK-506/CsA-resistance of these responses was shown to be determined by the amount of PMA added to the cultures. We conclude from our investigations that FK-506 and CsA inhibit highly similar signal transduction pathways in human T lymphocytes. PMID- 1707759 TI - The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins. AB - Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins. PMID- 1707761 TI - Augmentation of interleukin 2-activated cytotoxicity after treatment of cells with inhibitors of interferon production. AB - Low-density human lymphocytes cultured with recombinant interleukin 2 (rIL-2) generated a high level of interferon(s) (IFN). Consistently more IFN including IFN-tau was produced during the first 3 days of culture with rIL-2 than during the subsequent 4 days. That ability was mainly associated with mepacrine+ cells and was decreased by low concentrations of leucine methyl ester (Leu-O-Me) or ammonium chloride. Leu-O-Me was employed either for the pretreatment of cells before the culture or as the additive to culture medium. The decrease in IFN production after pretreatment was associated with enhanced rIL-2-activated cytotoxicity. Similarly, 1 mM of ammonium chloride or 1 mM of Leu-O-Me added to rIL-2 supplemented cultures for 3 days showed an association between inhibition of IFN-tau generation and increased activation of cytotoxic activity. Thus NK cells appear to regulate their own response to rIL-2 activation and the control mechanism seems to be associated with the ability of the cells to produce IFN(s) and possibly other cytokines. PMID- 1707762 TI - Differential responses of CD4+CD45RA+ and CD4+CD29+ subsets to activated CD8+ cells: enhanced stimulation of the CD4+CD45RA+ subset by cells from patients with multiple sclerosis. AB - To examine whether functionally different CD4+ cells respond uniformly to the immunoregulatory influences of allogeneic activated CD8+ cells (*CD8+), we subfractionated the CD4+ population into two subsets, based on the high expression of either CD45RA or CD29. We confirmed that the CD45RA+ cells proliferated poorly in response to soluble anti-CD3 mAb, compared to the vigorous response obtained with the CD29+ subset; the CD45RA+ cells were more responsive to stimulation with Con A. Using normal healthy controls, we found that whereas *CD8+ had a significant suppressive effect on the proliferation of the CD29+ subset, they augmented the mitogen-induced proliferative response of the CD45RA+ cells. We further demonstrated that *CD8+ derived from MS patients augmented the response of the CD45RA+ subset to a significantly higher degree compared to healthy age- and sex-matched controls. There were no significant differences between the degree of suppression exerted by the *CD8+ of either the MS or the control group on the CD29+ cells. These results demonstrate that helper/memory CD4+CD29+ cells are more sensitive to the suppressive influences of *CD8+ compared to the CD4+CD45RA+ subset. In addition, in MS, *CD8+ may contribute to a more pronounced "on" signal for virgin CD4+CD45RA+ cells, which might serve as a means to perpetuate the autoimmune disease process. PMID- 1707763 TI - Immunospecific suppression of encephalitogenic-activated T lymphocytes by chimeric cytotoxin IL-2-PE40. AB - We examined the action of a chimeric protein, IL-2-PE40, on the development of a T cell-mediated disease of the central nervous system with numerous similarities to multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). EAE is caused by IL-2 receptor-bearing T cells specific for myelin basic protein (BP). We report here that the treatment of Lewis rats with IL-2-PE40 delayed and shortened the course of EAE induced by BP in adjuvant and dramatically prevented EAE mediated by anti-myelin basic protein T line cells. The absence of paralytic signs, the absence of cell infiltration in the central nervous system, and the abatement of cellular immunity to myelin basic protein in the treated rats are direct consequences of the specific mechanism of action of IL-2-PE40. Our data support the notion that IL-2-PE40 may be efficient as an immunosuppressive agent for those disorders in which activated T cells play a crucial role. PMID- 1707764 TI - Differential effects of poly (Glu60, Phe40) (GPhe) on murine TH1 and TH2 cells. AB - The random amino acid copolymer (Glu60, Phe40)n (GPhe) was previously shown to augment antigen-dependent proliferation of the murine TH2 cell lines DCL-2 and D10.G4.1. In the present study, the addition of GPhe to (Glu36, Lys24, Ala40)n (GLA)-primed BALB/c primary lymph node (1 degree LN) T cell cultures, the source of DCL-2, resulted in significant suppression of both the proliferative and lymphokine response to GLA. Suppression by GPhe of the 1 degree LN response was subsequently shown to be neither antigen- nor haplotype restricted, and was inhibitable by polyclonal anti-GPhe antibodies. Studies were extended to a GLA reactive T cell hybridoma clone (DL.4G6.1). where significant suppression by GPhe of GLA-stimulated lymphokine production was observed as measured by markedly decreased HT-2 stimulatory activity of the collected supernatants. Subsequent antibody blocking experiments employing the monoclonal anti-murine IL-4 antibody 11B11 revealed that BALB/c GLA-reactive 1 degree LN T cells and DL.4G6.1 did not produce detectable levels of IL-4 in their culture fluids when stimulated by GLA, which suggested that these cells, unlike DCL-2, were TH1-like in nature. The addition of GPhe to the TH1 clones 5.2 and 5.9 resulted in significant suppression of proliferation to homologous antigen (ovalbumin), in contrast to the augmentation observed with the TH2 cell lines DCL-2 and D10.G4.1. It was concluded from these data, that the addition of GPhe to various T cell cultures lead to unusual suppressive and augmenting activities specific for TH1 and TH2 cells, respectively. Although the mechanism for these dichotomous effects of GPhe is as yet undetermined, several possibilities are considered. PMID- 1707765 TI - Further characterization of the autologous mixed lymphocyte reaction: induction of double negative gamma delta T lymphocytes. AB - To investigate the specific nature of the autologous mixed lymphocyte reaction (AMLR), we applied a method in which mixtures of NY-nonadherent responder cells and NY-adherent stimulator cells were treated with neuraminidase before culture and then cultured to assay the AMLR. This method produced a marked enhancement of DNA replication in the responder cells and the results were reproducible, regardless of the individuals tested. Using this method, we were able to make the following observations regarding the specific nature of the AMLR. (i) The AMLR is an IL-2-independent reaction, as revealed by bioassay to detect the presence of IL-2 by a blocking test using anti-IL-2R sera and as shown by the absence of mRNA for IL-2 in Northern hybridization. (ii) It is also HLA-DR dependent as proven by the fact that anti-DR sera almost completely inhibited the reaction. (iii) The AMLR was also found to induce the generation of activated CD4+ helper T cells in direct response to stimulation by NY-adherent cells, in which HLA-DR antigens were involved. (iv) Also, it induced the generation of CD4-CD8- double-negative (DN) lymphocytes, including gamma delta T cells with a cytotoxic activity against NK-resistant target cells and with a variety of lymphocyte activation markers (CD56, HLA-DR, CD25, transferrin receptors, CD38, and LFA-1). However, the AMLR did not induce the generation of NK cell markers CD16 and CD57. (v) The DN lymphocytes and gamma delta T cells appeared to be generated from the precursors of CD4-CD8- DN cells, in direct response to the stimulator cells. These results strongly suggest that the AMLR may be a phenomenon which induces the proliferative response of gamma delta T cells and their precursors, in addition to that of alpha beta T cells, particularly of CD4+ helper T cells. PMID- 1707766 TI - Human alpha-fetoprotein (AFP) causes a selective down regulation of monocyte MHC class II molecules without altering other induced or noninduced monocyte markers or functions in monocytoid cell lines. AB - Human alpha-fetoprotein (AFP) purified from human amniotic fluid was investigated for its effect on human monocytoid cell lines, including U 937 cells with established subclones. The impact of AFP on the expression of surface markers (MHC class I and II, CD4, CD18, CD45, Fc receptors for IgG) was analyzed using known inducers of monocyte-macrophage differentiation such as phorbol esters and IFN-gamma. Furthermore we investigated the effect of AFP on the induction of macrophage antibody-dependent cell-mediated cytolytic activity (ADCC). AFP did selectively induce a rapid down regulation of surface MHC class II expression. No evidence of alterations was found in the endogenous or differentiation-induced expression of other markers on the surface on monocytes, nor did AFP affect the functional maturation of surface Fc receptors or the ability to express ADCC. PMID- 1707767 TI - The effect of antibody isotype and antigenic epitope density on the complement fixing activity of immune complexes: a systematic study using chimaeric anti-NIP antibodies with human Fc regions. AB - A systematic study has been carried out to investigate the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement and for the complexes subsequently to bind to erythrocyte C3b-C4b receptors (CRI). For this purpose, a series of chimaeric monoclonal anti-NIP antibodies was used, which all shared the same combining site but had different human constant domains. Antigen epitope density was varied by coupling different numbers of NIP hapten molecules to bovine serum albumin. All three parameters affect complement fixation. In general, complement activation is better in antibody excess and at equivalence than it is in antigen excess, and better at high epitope density than at low epitope density, although the effects are variable for different immunoglobulin isotypes and for the two pathways. It has been confirmed that IgG1 and IgG3 are good activators of the classical pathway and are tolerant to variations in both epitope density and antigen/antibody ratio. IgG4 and IgA do not activate the classical pathway in any circumstances. IgG2 activates the classical pathway only at high epitope density and at equivalence or antibody excess. IgM activates the classical pathway well only at the higher epitope densities and at equivalence or antibody excess but, in addition, shows an interesting and unexpected prozone phenomenon where immune complex in antibody excess inhibits complement activation by the classical pathway. The results of the alternative pathway activation are strikingly different. IgA is by far the best activator of the alternative pathway and is relatively tolerant to epitope density and to antigen/antibody ratio. IgM, IgG1 and IgG3 do not significantly activate the alternative pathway in any circumstances. IgG2 is the best IgG subclass for alternative pathway activation but requires high epitope density and equivalence or antibody excess. Binding to CR1 in general parallels the amount of complement fixed independent to the pathway by which it is fixed. However, IgG1 and IgG3 complexes in antigen excess activate complement well but bind poorly to CR1. Nascently formed complexes seem to bind complement in a way that is similar to that bound by preformed complexes, but are then less able to bind to red cell CR1. These observations help to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others. PMID- 1707768 TI - Recent advances in diagnosis and classification of leukemias and lymphomas. PMID- 1707769 TI - Analysis of CD1 molecules on haematological malignancies of myeloid and lymphoid origin. I. Cell surface antigen expression. AB - One hundred and ninety well-characterized acute and chronic leukaemias were studied for the expression of CD1a antigen by indirect immunofluorescence (IIF). CD1a was detected on 28 per cent of mature B cell lymphoproliferative disorders, 26 per cent of acute non-lymphoblastic leukaemias (ANLL), 21 per cent of chronic granulocytic leukaemias in blast crisis (CML-BC), 53 percent of T acute lymphocytic leukaemias (T-ALL) and in only one out of 35 common acute lymphoblastic leukaemias (c-ALL). In some cases the expression of the CD1a antigen on the surface of leukaemic cells showed a spontaneous fluctuation after a short period of incubation in vitro. CD1b and CD1c molecules were also detected on B cells and acute non-lymphoblastic leukaemias. The presence of CD1 antigens was confirmed using a dot blot assay (DBA) on the lysate of leukaemic cells. PMID- 1707770 TI - Analysis of CD1 molecules on haematological malignancies of myeloid and lymphoid origin. II. Intracellular detection of CD1 antigens. AB - The surface and cytoplasmic expression of CD1a molecules was analysed by indirect immunofluorescence (IIF) and dot blot assay (DBA) in a panel of 40 acute and chronic leukaemias. Thirty-two per cent of the samples were positive by IIF but, surprisingly, 72 per cent of the patients were positive by DBA, suggesting the intracellular presence of these molecules, CD1b and CD1c were also detected by DBA at similar percentages. Immunocytochemical staining of cytocentrifuge preparations confirmed the intracellular presence of CD1a, CD1b, and CD1c in leukaemic cells of pre-B, B, T, and non-lymphoid lineages. PMID- 1707771 TI - Clinical significance of noninvasive recordings of micropotentials of the heart- experience over a two years' period. AB - To assess the risk of malignant ventricular tachycardia (VT) and sudden cardiac death, clinical data including Holter monitoring, programmed ventricular stimulation and highly amplified signal averaged ECG were employed. Among 394 patients, 175 had late potentials. Close correlations were demonstrated between the presence, duration and voltage of late potentials and left ventricular function disturbances, arrhythmia profile, presence and frequency of VT. Signal averaging contributes to better identification of patients at risk. During a mean follow-up of two years 32 patients died, 20 suddenly. 17 of the latter had late potentials of long duration and 12 previous ventricular tachycardia. The predictive value of LP is superior to the other methods used. PMID- 1707772 TI - Holter monitoring in the long QT syndrome of children and adolescents. AB - Results of Holter monitoring (HM) of ECG in 45 patients with long QT syndrome (LOTS) and 26 healthy control children are presented. 15 patients had a history of syncopal attacks (group I), other patients were symptomless (group II). Heart rate-corrected QT interval (QTc) on standard resting ECG exceeded 460 ms in 73.3% of patients in group I and in 44.8% of group II patients. On Holter monitoring, it was present in 100% of group I and 83.9% of group II patients. T wave alternation was registered in 66.6% of group I and 56.7% of group II patients. Ventricular arrhythmia was not found on standard resting ECG in any of the examined children, whereas Holter monitoring revealed ventricular extrasystoles in 53.3% of group I and 6.7% of group II patients (p less than 0.05). In one case early ventricular extrasystoles accompanied an attack of ventricular tachycardia. Mathematical analysis of 24-h extracardial heart rhythm regulation revealed its disturbance in LQTS patients compared to healthy children, as well as asthenic signs in the sympathetic nervous system. These changes were more pronounced in patients with a more severe course of the disease (group I). PMID- 1707773 TI - Neurophysiological effects of substance P in primate hypertension models. Preliminary report. AB - Peripheral administration of the neuropeptide Substance P (SP) in rhesus monkeys and baboons caused a long lasting decrease in blood pressure, accompanied by a gradual normalization function of stress-induced bioelectrical activity changes in brain structures, relevant for cardiovascular regulation (frontal cortex, ncl. raphe, locus coeruleus). In addition, SP was found to normalize the disturbed interaction of brain structures of both hemispheres. Use of SP as a putative agent to treat essential hypertension in humans needs further studies. PMID- 1707774 TI - Competency-based orientation program for a surgical intensive therapy unit--Part 1. PMID- 1707775 TI - [Effects of calcium hydroxide on DNA and RNA synthesis of rat pulp fibroblasts in vitro]. PMID- 1707776 TI - [Distribution of intermediate filaments in the salivary glands and salivary gland tumors]. AB - 13 cases of salivary glands and 30 of salivary gland tumors were studied by ABC method with 6 monoclonal antibodies to intermediate filaments and one to microfilament. The results showed that the distribution of intermediate filaments in salivary glands had their regularity. According to the reaction to the antibodies, these tumors could be divided into 3 groups and 3 subgroups. The findings also suggested that in the salivary gland tissue the epithelial cells which mainly contained the 54 Kd keratin and the epithelial cells which mainly contained 57/66 Kd keratin were the origin of the salivary gland tumors. The actin-positive myoepithelial cells might play a role in some tumor formation. PMID- 1707777 TI - [The laboratory examination of Candida albicans]. AB - This study compares the laboratory methods in detecting candidas causing oral candidiasis and suggests that smear method is the main one. PAS stain is the best for diagnosis, and the potassium hydroxide method is the easiest and quickest in handling. As to those uncertained by smear method, culture should be done. For those positive on Sabouraud's medium, the germ tube test should be performed and positive results can be regarded as candida albicans. If negative the chlamydospore production as well as the carbohydrate fermentation and assimilation should be done. PMID- 1707778 TI - [Use of signal averaging system in the study of ventricular late potentials]. AB - Signal-averaged electrocardiograms (SAECGs) were performed in 93 patients with various heart diseases and 20 dogs with experimental acute myocardial infarction. SA-ECGs were recorded with a high-pass bidirectional digital filter Mode FDS which was made by Shanghai Fudan University and Suzhou Medical College. All the patients and dogs were also examined with ART 1200 EPX recorder. Ventricular late potentials (VLPs) were directly recorded from the epicardium with experimental infarction by using composite electrodes and signal averaging system in late stage of coronary ligation. The results showed: VLPs were detected by body surface SA-ECG only in half of the dogs with VLPs detected by the real time mapping on infarction region, but the abnormal SAECGs did reveal the delayed ventricular activity; the data obtained with FDS or ART SA-ECG were similar. PMID- 1707779 TI - [Granulocyte colony-stimulating factors]. PMID- 1707780 TI - Opportunities for bioactive compounds in transgenic plants. AB - A variety of bioactive compounds have now been introduced into plants through recombinant DNA techniques. Early examples included genes encoding proteins conferring herbicide tolerance and insect or virus resistance. More recently, pharmacologically useful compounds such as enkephalin and immunoglobulin have been produced in transgenic plants. Modification of existing compounds to provide better nutritional value or improved functional properties is exemplified in the case of seed storage proteins. The value of RNAs as bioactive compounds for suppression of undesirable products and viral infection has now been demonstrated in plants. The developmentally regulated expression of novel bioactive compounds in defined tissues, and their targeting to specific subcellular locations, is becoming of ever increasing economic and sociological importance as knowledge of the molecular mechanisms involved accumulates. PMID- 1707781 TI - [Palliative pulmonary resection for primary lung cancer]. AB - From January 1961 through December 1984, 253 of 2048 patients who have undergone surgical treatment for primary lung cancer were retreated by palliative pulmonary resection. The indications of palliative resection were: there was partial carcinoma or metastatic lymph node left in the thorax; microscopically, residual tumor was found on bronchial stump margin. Operation modes: partial pulmonary resection 135, total pneumonectomy 118. Postoperative complications occurred in 25 cases and 17 died in the hospital with in 30 days. 236 cases were followed-up for 1 to 21 years. The 1-year, 3-year and 5-year survival rates after operation were 51.3%, 13.1% and 8.1% respectively. The survival rates after palliative pulmonary resection for squamous and adenocarcinoma were higher than thoracotomy but the survival rates of large undifferentiated, small cell and mixed cancer were similar to those of thoracotomy. Besides, patients who had both subcarinal lymph node involvement and incomplete excision in resection had the worst prognosis. The authors consider that squamous and adeno carcinoma of the lung are the main indication for palliative resection. Subcarinal lymph nodes must be excised as much as possible while operation, otherwise local radiation and/or chemotherapy should be performed after operation. PMID- 1707782 TI - [Ventricular tachyarrhythmias treated with berberine]. AB - The effects of berberine on 100 cases with ventricular tachyarrhythmias observed with 24 to 48 hour ambulatory monitoring were reported. The results showed that 62% of patients had 50% or greater, and 38% of patients had 90% or greater VPC suppression. The mean value of VPCs in whole group was significantly decreased by berberine from 452 +/- 421.8 beats per hour to 271 +/- 352.7 beats per hour (P less than 0.001). These results revealed that berberine is effective for ventricular tachyarrhythmias. There were no severe side effects, only mild gastroenterologic symptoms were observed in some patients. PMID- 1707783 TI - [Evaluation of risks and treatment of asymptomatic ventricular arrhythmias]. PMID- 1707784 TI - Embryonic gene expression patterns of TGF beta 1, beta 2 and beta 3 suggest different developmental functions in vivo. AB - We have compared the expression of the genes encoding transforming growth factors beta 1, beta 2 and beta 3 during mouse embryogenesis from 9.5 to 16.5 days p.c. using in situ hybridisation to cellular RNAs. Each gene has a different expression pattern, which gives some indication of possible biological function in vivo. All three genes appear to be involved in chondroossification, though each is expressed in a different cell type. Transcripts of each gene are also present in embryonic epithelia. Epithelial expression of TGF beta 1, beta 2 and beta 3 RNA is associated with regions of active morphogenesis involving epithelial-mesenchymal interactions. In addition, widespread epithelial expression of TGF beta 2 RNA can be correlated with epithelial differentiation per se. The localisation of TGF beta 2 RNA in neuronal tissue might also be correlated with differentiation. Finally both TGF beta 1 and beta 2 transcripts are seen in regions actively undergoing cardiac septation and valve formation, suggesting some interaction of these growth factors in this developmental process. PMID- 1707785 TI - T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo. AB - Trunk neural crest cells and motor axons move in a segmental fashion through the rostral (anterior) half of each somitic sclerotome, avoiding the caudal (posterior) half. This metameric migration pattern is thought to be caused by molecular differences between the rostral and caudal portions of the somite. Here, we describe the distribution of T-cadherin (truncated-cadherin) during trunk neural crest cell migration. T-cadherin, a novel member of the cadherin family of cell adhesion molecules was selectively expressed in the caudal half of each sclerotome at all times examined. T-cadherin immunostaining appeared graded along the rostrocaudal axis, with increasing levels of reactivity in the caudal halves of progressively more mature (rostral) somites. The earliest T-cadherin expression was detected in a small population of cells in the caudal portion of the somite three segments rostral to last-formed somite. This initial T-cadherin expression was observed concomitant with the invasion of the first neural crest cells into the rostral portion of the same somite in stage 16 embryos. When neural crest cells were ablated surgically prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that the metameric T-cadherin distribution occurs independent of neural crest cell signals. This expression pattern is consistent with the possibility that T-cadherin plays a role in influencing the pattern of neural crest cell migration and in maintaining somite polarity. PMID- 1707786 TI - Retinoic acid-binding protein, rhombomeres and the neural crest. AB - We have investigated by immunocytochemistry the spatial and temporal distribution of cellular retinoic acid-binding protein (CRABP) in the developing nervous system of the chick embryo in order to answer two specific questions: do neural crest cells contain CRABP and where and when do CRABP-positive neuroblasts first arise in the neural tube? With regard to the neural crest, we have compared CRABP staining with HNK-1 staining (a marker of migrating neural crest) and found that they do indeed co-localise, but cephalic and trunk crest behave slightly differently. In the cephalic region in tissues such as the frontonasal mass and branchial arches, HNK-1 immunoreactivity is intense at early stages, but it disappears as CRABP immunoreactivity appears. Thus the two staining patterns do not overlap, but are complementary. In the trunk, HNK-1 and CRABP stain the same cell populations at the same time, such as those migrating through the anterior halves of the somites. In the neural tube, CRABP-positive neuroblasts first appear in the rhombencephalon just after the neural folds close and then a particular pattern of immunoreactivity appears within the rhombomeres of the hindbrain. Labelled cells are present in the future spinal cord, the posterior rhombencephalon up to rhombomere 6 and in rhombomere 4 thus producing a single stripe pattern. This pattern is dynamic and gradually changes as anterior rhombomeres begin to label. The similarity of this initial pattern to the arrangement of certain homeobox genes in the mouse stimulated us to examine the expression of the chicken Hox-2.9 gene. We show that at stage 15 the pattern of expression of this gene is closely related to that of CRABP. The relationship between retinoic acid, CRABP and homeobox genes is discussed. PMID- 1707787 TI - The utility of proximal nerve conduction in radiculopathies: the pros. PMID- 1707788 TI - The utility of proximal nerve conduction in radiculopathies: the cons. PMID- 1707789 TI - Acoustic trauma in extracranial magnetic brain stimulation. AB - The effects of the magnetic coil acoustic artifact (MCAA) associated with extracranial magnetic field stimulation (EMFS) of the brain were studied in normal hearing rabbits. Spectral and intensity analyses showed that the MCAA is a high intensity transient signal with peak energy between 2 and 5 kHz, and peak amplitudes in the first 100-200 mu sec. At EMFS levels of 50-100% of maximum output (2.0 Tesla), the corresponding MCAA levels were 131-142 dB sound pressure level (peak hold) at the outer ear and amplified by the external meatus to reach 145-157 dB sound pressure level (SPL) at the position of the tympanic membrane in rabbits. Measurements of the acoustic middle ear muscle reflex (AMR) in non anesthetized rabbits indicated that exposure to EMFS levels of 50-100% resulted in correspondingly increasing compound threshold shifts (CTS) and permanent threshold shifts (PTS) in the unprotected ears of the experimental animals. Auditory brain-stem responses (ABR) measures on the same and additional animals corroborated these findings. Morphological studies showed evidence of substantial cochlear trauma at EMFS levels as low as 50%, with increasing severity up to 100% EMFS. Morphological examination of inner ear structures following exposure to the MCAA in the acute preparation (fixed within hours after exposure) showed ruptures between pillar cells and a detached organ of Corti. Preparations examined 3 or more weeks after exposure showed damaged pillar cells, a widespread loss of outer hair cells, fused and fractured inner hair cell stereocilia, and kinocilium outgrowth on inner hair cells. Although this extremely short impulse contains approximately 2 orders of magnitude less acoustic energy than a continuous noise exposure of 131 dB for 15 min, it is substantially more injurious to the cochlea. The present findings suggest that the acoustic artifact produced by the EMFS coil in some clinical instruments may pose a potential risk for temporary and permanent hearing loss in patients and clinicians when held in close proximity to the unprotected ear. Initial studies suggest that the magnetic field alone did not appear to cause permanent hearing impairment. We recommend the use of ear protectors for the patient and clinician during EMFS as a precautionary measure to prevent possible hearing loss from the MCAA. PMID- 1707790 TI - Anatomical localization revealed by MEG recordings of the human somatosensory system. AB - A 14-channel cryogenic magnetometer system (BTi) was used to record the magnetic fields over the left hemisphere of 3 human subjects in order to locate the sources of responses to tactile stimulation of the index, the thumb and the little finger of the right hand. The locations of the active dipole sources determined using the spherical model were then projected onto the magnetic resonance image (MRI) of the individual subjects providing an anatomical localization. The MRI slices were also used to construct a 3-dimensional image to enhance visualization of the area of the calculated sources. The locations of the dipole sources from the 3 fingers were distinct from one another in all subjects. An analysis of variance ('ANOVA') showed the most significant (P less than 0.05) difference in source location between the little finger and the thumb with the former being superior to the sources of the other 2 fingers in all of the subjects. In all cases, the sources were found to be located on the postcentral gyrus. The strength of the equivalent dipole sources and the amplitudes of the responses to stimulation for all 3 fingers showed a consistent trend among all of the 3 subjects, with the thumb having the largest response. In general, no signs of habituation were found. PMID- 1707791 TI - Differences between morning-types and evening-types in the dynamics of EEG slow wave activity during night sleep. AB - During baseline nights in a sleep laboratory electrophysiological sleep records were made for 8 morning-type subjects (M-types) and 8 evening-type subjects (E types). As compared with the E-types, the M-types were relatively advanced with respect to the times of maximum and minimum rectal temperature and sleep times. Also, the M-types showed a larger initial temperature drop after sleep onset. Comparisons of the outcomes of visual sleep scoring revealed for the M-types a shorter sleep latency and a longer sleep duration. In addition, the M-types reported a higher subjective sleep quality than the E-types. For the M-types sleep stages 3 + 4 and EEG delta (0.5-3.5 Hz) energy declined monotonically across the first 4 NREM/REM cycles. For the E-types, however, no decrement was observed over the first 2 cycles. Analysis of the wave forms of delta energy, employing a pattern recognition technique independent of visual sleep scoring, substantiated this finding. These results are discussed in relation to the differences in circadian characteristics between M-types and E-types. PMID- 1707792 TI - EEG power density during recovery sleep in the morning. AB - Sleep was recorded under baseline conditions (waking prior to sleep 16 h; lights off 23.00 h) and during recovery sleep in the morning (waking prior to sleep 24 h; lights off 07.00 h). Slow-wave activity (SWA; EEG power density in the range of 0.75-4.5 Hz) declined progressively over consecutive nonREM-REM cycles in both conditions despite the different circadian phase at which sleep occurred. SWA in nonREM sleep in the first 5 h of sleep was significantly higher in recovery than in baseline. Also SWA within the first 20 min of nonREM-episodes 2 and 3 was significantly higher in recovery sleep, and a tendency in the same direction was seen for nonREM-episode 1. These data show that homeostatic processes are expressed in the EEG also when sleep is initiated at a circadian phase where REM sleep propensity is high. However, comparison of the power spectrum in the first cycle of day-time recovery sleep with published data on recovery sleep at various circadian phases suggests that circadian factors influence the EEG spectra. PMID- 1707793 TI - Pyridoxine-dependent epilepsy: EEG investigations and long-term follow-up. AB - The EEG features and clinical correlates were investigated before, directly after, and on long-term follow-up after initiation of pyridoxine therapy in 6 patients with B6-dependent epilepsy. At each phase, the EEG provided important diagnostic and prognostic information. Pre-B6 3 neonates manifested a unique EEG pattern of generalized bursts of 1-4 Hz sharp and slow activity. This pattern has not been previously described in neonates with B6 dependency and in this age group appears to be highly suggestive of the diagnosis. Five patients experienced an apparent initial response to traditional antiepileptics. The parenteral pyridoxine test, performed in all 5, and repeated in 3, proved to be a highly reliable and reproducible diagnostic test. After 50-100 mg of B6 there was cessation of clinical seizures within minutes and of paroxysmal discharges within hours. On long-term follow-up (3-28 years) all 6 patients were seizure free on B6 (10-100 mg/day) monotherapy. Recurrences of seizures and of specific sequential EEG changes (background slowing, photoparoxysmal response, spontaneous discharges, stimulus-induced myoclonus, generalized seizures) occurred upon B6 withdrawal. Long-term prognosis correlated with the EEG. Two patients had persistently abnormal EEG backgrounds and were moderately to severely retarded, while 4 had normal EEGs with normal or near normal development. PMID- 1707794 TI - EEG changes during recovery from acute severe neonatal citrullinemia. AB - We report our observations of serial clinical and EEG examinations in 3 neonates during recovery from acute severe encephalopathy due to citrullinemia. Their electroclinical picture closely resembles the clinical stages of experimental models of hyperammonemia in monkeys. The length of the EEG interburst interval, a quantitative measure of EEG background abnormality, correlated with elevated serum levels of ammonia and suggests that hyperammonemia itself is a key figure in the genesis of encephalopathy in this condition. Finally, the manner in which the EEG normalizes during recovery from hyperammonemia in this setting suggests that burst-suppression resembles an exaggerated regression to the discontinuity of the very premature infant. PMID- 1707795 TI - A comparison between electroencephalography and somatosensory evoked potentials for outcome prediction following severe head injury. AB - The value of somatosensory evoked potentials (SEPs) for the prediction of outcome following severe head injury (HI) is established. The role of the electroencephalogram (EEG) in this setting is uncertain. In this comparative study, SEPs and EEGs were recorded within 3 days of severe HI in 90 patients, and the results related to outcome at 6 months. Patients with an isoelectric EEG or an EEG with repeated isoelectric intervals died. Reactivity of the EEG to external stimulation tended to be associated with favorable outcome. Grading of the EEGs on the basis of frequency composition otherwise provided no prognostic information. The presence of SEP scalp potentials bilaterally predicted favorable outcome, particularly if the central conduction times were normal. Conversely, the absence of one of both scalp potentials was associated with unfavorable outcome. EEGs thus provided useful prognostic information in only a minority of patients. By comparison, SEPs allowed prediction of both favorable and unfavorable outcomes in a much larger number of patients, and were therefore prognostically superior. PMID- 1707796 TI - Polarity reversal of N20 and P23 somatosensory evoked potentials between scalp and depth recordings. AB - From depth and scalp electrodes, we recorded MN-SSEPs of a 33-year-old man with right parietal dysfunction and refractory right temporal seizures. A depth lead with 8 electrodes was implanted deep in each parietal-temporal region. Stimulation and recording parameters followed American EEG Society guidelines. Scalp recordings had well-defined P9, P13-14, N18, N20, and P23 potentials with normal conduction times bilaterally. Depth recordings showed potentials of greater number, voltage, and coherence. P13-14 and N18 were recorded at all depth sites with latencies similar to those at the scalp. N18 had markedly greater voltage and duration near the thalamus, with multiple fast components on its ascending phase. In the deep parietal region there was a positivity corresponding to the scalp N20 and a negative potential equal in latency to scalp P23. These findings support an origin of P13-14 caudal to the thalamus, multiple thalamic and possibly rostral brain-stem generators for N18, and generation of N20 and P23 in sensory cortex of subjacent white matter. PMID- 1707797 TI - The instruction to refrain from blinking affects auditory P3 and N1 amplitudes. AB - Often subjects have been instructed to refrain from blinking lest their evoked EEG potentials should be distorted. We studied whether these very instructions have any impact on P3 amplitude. Two tones were presented in random order, and subjects had to count the high-pitched tones. Half the subjects were instructed not to blink, whereas this instruction was omitted for the other subjects. Target tones evoked larger P3s than non-targets in the latter group but not in the former, in particular not in those subjects that actually blinked rarely. The groups also differed in their N1 amplitudes. These findings might be relevant to P3 studies working with patients and controls: the harder some frequently blinking subjects try to refrain from blinking, the smaller might become their P3 amplitudes. Omitting the instruction and using off-line blink subtraction procedures seems a viable alternative. This study was actually motivated by discrepant findings on the effects of the preceding tone sequence on P3. These discrepancies could be largely resolved by the instructional variable, in conjunction with different tone intensities. It is suggested that subjects who are discouraged from blinking try to protect themselves against the arousing effects of stimuli. PMID- 1707798 TI - An EEG-based brain-computer interface for cursor control. AB - This study began development of a new communication and control modality for individuals with severe motor deficits. We trained normal subjects to use the 8 12 Hz mu rhythm recorded from the scalp over the central sulcus of one hemisphere to move a cursor from the center of a video screen to a target located at the top or bottom edge. Mu rhythm amplitude was assessed by on-line frequency analysis and translated into cursor movement: larger amplitudes moved the cursor up and smaller amplitudes moved it down. Over several weeks, subjects learned to change mu rhythm amplitude quickly and accurately, so that the cursor typically reached the target in 3 sec. The parameters that translated mu rhythm amplitudes into cursor movements were derived from evaluation of the distributions of amplitudes in response to top and bottom targets. The use of these distributions was a distinctive feature of this study and the key factor in its success. Refinements in training procedures and in the distribution-based method used to translate mu rhythm amplitudes into cursor movements should further improve this 1-dimensional control. Achievement of 2-dimensional control is under study. The mu rhythm may provide a significant new communication and control option for disabled individuals. PMID- 1707799 TI - Magnetic transcranial brain stimulation and multimodality evoked potentials in an adrenoleukodystrophy patient and members of his family. AB - In a patient with adrenoleukodystrophy (ALD), prolonged conduction times along the corticospinal tract and afferent sensory pathways were demonstrated using magnetic transcranial brain stimulation and multimodality evoked potentials, respectively. On investigation of two healthy family members (one an obligate carrier), slight latency increases in evoked potentials suggested impaired conduction along the respective pathways. Thus, the combined use of these methods (1) is helpful in assessing the location and extension of demyelinating lesions and (2) may raise the sensitivity in detecting subclinical involvement in patients with ALD and in carriers. PMID- 1707800 TI - Acute effects of diphenylhydantoin on peripheral and central somatosensory conduction. AB - We studied the acute effects of an intravenous loading dose of DPH (16 mg/kg body weight) on peripheral and central somatosensory conduction in 10 normal volunteers. Somatosensory evoked potentials were recorded before and at regular intervals after DPH infusion. There was no effect on peripheral conduction. DPH significantly delayed N13 peak latency without changing conduction in the posterior spinal columns. Although the N13-N20 interpeak interval remained stable because of the parallel shift of the 2 peaks, the central conduction time measured from onset latencies of N11 and N20 significantly increased. We conclude that acute administration of DPH at serum levels below 30 micrograms/ml induces a reversible delay of synaptic transmission in spinal and central somatosensory structures. PMID- 1707801 TI - Evoked potentials in a man with a complete large myelinated fibre sensory neuropathy below the neck. AB - Cortical somatosensory evoked potentials (SEPs) were recorded from a man with a severe neuropathy without touch and proprioception below the neck. Peripheral neurophysiological tests showed a complete large myelinated fibre sensory neuropathy. Sensory threshold to electrical stimulation of the median nerve was 15 mA (normal 2-4 mA). With a stimulus of 39 mA, duration 400 microsecons, applied at the wrist a cortical SEP was recorded with a latency of 84 msec, giving a propagation velocity of 11.9 m/sec. At stimulation rates of above 3.3 Hz the SEP was absent. It is concluded that the SEPs recorded were conducted along A delta peripheral fibres. PMID- 1707802 TI - Adaptive Fourier series modeling of time-varying evoked potentials: study of human somatosensory evoked response to etomidate anesthetic. AB - Evoked potentials (EPs) have traditionally been analyzed in time domain, with amplitude and latency of various signal components used in clinical interpretation. A new approach, called adaptive Fourier series modeling (FSM), is presented here. Dynamic changes in magnitudes of Fourier coefficients are analyzed for diagnostic purposes. In order to estimate the time-varying changes in the Fourier coefficients of noisy signals, a least mean-square filtering algorithm is applied. Results of computer simulations as well as experimental data are presented. Time-varying trends are presented in a new compressed evoked spectrum format. These techniques are applied to the study of alterations in human somatosensory EPs caused by the intravenous administration of etomidate during neurosurgical procedures. Amplitude increases of the order of 200-500% occurring within a time span of about 100 sec were captured. Due to its superior convergence properties, the adaptive FSM technique estimates more rapid changes in amplitude and latency than exponentially weighted averaging or moving window averaging schemes. PMID- 1707803 TI - Short latency trigeminal evoked potentials: normative data and clinical correlations. AB - A very short latency trigeminal evoked potential (STEP) to electrical stimulation of the upper lip has been recorded from over the scalp. This potential consists of 5 distinct peaks within the 12 msec range. Normative data were obtained from 25 healthy volunteers. The impact of the stimulus rate and intensity on the response was studied in each subject. These results were compared to those of 19 patients suffering from lesions involving the trigeminal system in its peripheral aspect or the brain-stem. The STEP was consistently abnormal whenever the involved side was stimulated. Changes in peak latencies and in interpeak latency differences (IPLD) correlated well with clinical and radiological findings and improved with the removal of the offending lesion. The STEP proved to be a reliable method for evaluating the trigeminal system in its peripheral and central pathways; it may thus serve as an additional parameter for studying brain stem functions. PMID- 1707804 TI - Distribution of lumbar spinal evoked potentials and their correlation with stimulation-induced paresthesiae. AB - In 7 awake patients with neuropathic lower extremity pain, spinal somatosensory evoked potentials (SEP) were elicited from the non-painful leg by electrical stimulation of the peroneal nerve and mechanical stimulation of the hallux ball. Recording was made epidurally in the thoraco-lumbar region by means of an electrode temporarily inserted for trial of pain-suppressing stimulation. In response to peroneal nerve stimulation, two major SEP complexes were found. The first complex consisted, as has been described earlier, of an initial positivity (P12), a spike-like negativity (N14), a slow negativity (N16) and a slow positivity (P23). The second complex consisted of a slow biphasic wave, conceivably mediated by a supraspinal loop. Both complexes had a similar longitudinal distribution with amplitude maxima at the T12 vertebral body. The SEP evoked by mechanical hallux ball stimulation had a relatively small amplitude, and there was no significant second complex. The relationship between stimulus intensity and SEP amplitude was negatively accelerating. The longitudinal distribution of spinal SEP was compared with the somatotopic distribution of paresthesiae induced by stimulation through the epidural electrode. It was found that stimulation applied at the level of maximal SEP generally induced paresthesiae in the corresponding peripheral region. Therefore, spinal SEP may be used as a guide for optimal positioning of a spinal electrode for therapeutic stimulation when implanted under general anesthesia. An attempt was made to record the antidromic potential in the peroneal nerve elicited from the dorsal columns by epidural stimulation. The antidromic response was, however, very sensitive to minimal changes of stimulus strength and body position of the patient, and was also contaminated by simultaneously evoked muscular reflex potentials. Thus, peripheral responses evoked by epidural stimulation appeared too unreliable to be useful for the permanent implantation of a spinal electrode for therapeutic stimulation. PMID- 1707805 TI - Short and middle latency vestibular evoked responses to acceleration in man. AB - We have succeeded in recording short and middle latency vestibular evoked responses in human subjects. The head was held rigidly in a special, patented head holder, constructed individually for each subject, which gripped the teeth of the upper jaw. The stimulus consisted of 2/sec steps of angular acceleration impulses produced by a special motor with intensities of about 10,000 degrees/sec 2 and with a rise time of 1-2 msec. The electrical activity was recorded as the potential difference between special forehead and mastoid electrodes having a large, secure contact area with the skin. The activity was digitally filtered and averaged in 2 separate channels by means of a Microshev 2000 evoked response system. The short latency responses, with peaks at about 3.5 msec (forehead positive), 6.0 msec (forehead negative) and 8.4 msec (forehead positive; bandpass: 200-2000 Hz; average of 1024 trials), had amplitudes of about 0.5 microV. The middle latency responses had peaks at about 8.8 msec (forehead positive), 18.8 msec (forehead negative) and 26.8 msec (forehead positive; 30-300 Hz; N = 128 trials), with larger amplitudes (about 15 microV). These responses were consistently recorded in the same subject at different times and were similar in different normal subjects. Strenuous control experiments were conducted in order to ensure that these responses are not artefacts due to the movement of conducting media (head, electrodes and leads) in the electromagnetic field of the motor and are elicited by activation of normal labyrinths.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1707806 TI - Scalp potentials following sudden coherence and discoherence of binaural noise and change in the inter-aural time difference: a specific binaural evoked potential or a "mismatch" response? AB - When uncorrelated random noise signals presented to the two ears suddenly become identical (coherent), a centrally located sound image is abruptly perceived and long latency scalp potentials are evoked. When the same signals are presented monaurally there is no perceived change and no potentials are evoked: hence the response must be purely a function of the binaural interaction. P70, N130 and P220 components were consistently recorded to both coherence and discoherence. N130 was usually largest at Fz and P220 at Cz. No potentials of shorter latency were identified, even after averaging 5000 or more sweeps. When the noise became coherent with an inter-aural time difference (delta T) of +/- 0.5 msec (giving rise to an off-centre sound image), the responses were of slightly longer latency and showed no significant asymmetries between C3 and C4. In binaurally coherent noise, delta T changes of +/- 0.5 or +/- 1.0 msec evoked similar responses which showed no significant asymmetries on the scalp. N130 was of longer latency when delta T was changed from +/- 0.5 msec to zero, as compared with the converse change. In view of the similarity of all these responses it is considered unlikely that they were due to specific populations of binaurally responsive cortical neurones. The N130 and P220 components are thought to be non-specific potentials which are elicited by any perceptible change in steady auditory stimulus conditions, due to a "mismatch" between the stimulus and the contents of a short-term auditory memory. PMID- 1707807 TI - Subpially recorded cervical spinal cord evoked potentials in syringomyelia. AB - Intraoperative spinal cord evoked potentials (SCEPs) to median nerve stimulation were detected subpially from the dorsal surface of the cervical spinal cord in 5 patients with cervical syringomyelia and were compared to normal SCEPs obtained from the unaffected side in 6 patients during intraoperative monitoring of dorsal root entry zone lesion. Normal SCEP began with a positive deflection P9 and a complex N11/N13 with several low amplitude short potentials superimposed on the N11/N13. The complex was followed by a second negative potential N2 and a late prolonged positivity, P. In the 4 patients in whom median nerve somatosensory evoked potentials (SEPs) were present preoperatively, SCEP consisted of the N11 potential and the following low amplitude short (LAS) potentials, while the N13 wave was missing. In the fifth patient, in whom the preoperative median nerve SEP was missing, SCEPs were of much lower amplitude and shorter duration than normal. The potentials N2 and P were not recorded in any of our 5 patients. Changes in N13 wave, N2 and P potentials noted in syringomyelia were presumed to be the result of destruction of the spinal cord dorsal horn neurons caused by spinal cord central cavitation. PMID- 1707808 TI - Effect of spatial frequency on simultaneous recorded steady-state pattern electroretinograms and visual evoked potentials. AB - Pattern electroretinograms (P-ERGs) and visual evoked potentials (VEPs) to 4 Hz alternating square-wave gratings were simultaneously recorded in 23 subjects. Responses were Fourier analyzed and amplitude and phase of the 2nd and 4th temporal harmonics were measured. The spatial frequency-amplitude function of the P-ERG 2nd harmonic component displayed either a bandpass tuning behavior, or a low-pass behavior. The peak amplitude for subjects with bandpass tuning was at 1.5 c/deg. The phase of the P-ERG 2nd harmonic decreased monotonically as spatial frequency increased. The VEP 2nd harmonic had a bimodal spatial frequency function with a peak at 3 c/deg and a second increase at spatial frequencies below 1 c/deg, regardless of the P-ERG characteristics. The phase of VEP 2nd and 4th harmonic had an inverted U-shaped function with peak at 3 c/deg and 1.5 c/deg respectively. Comparison of simultaneously recorded P-ERG and VEP spatial frequency functions demonstrated different tuning behavior for cortical and retinal responses. It is concluded that the proposed technique permits the separate analysis of retinal and cortical processing of visual information. The 2nd and 4th harmonic components of bEP behave independently of each other suggesting they may be generated by different subsystems. PMID- 1707809 TI - Clinical relevance of phase of steady-state VEPs to P100 latency of transient VEPs. AB - Pattern visual evoked potentials (VEPs) to transient and steady-state stimulation were recorded in 10 normal subjects at 4 levels of luminance (180, 57, 22 and 11 cd/m2). VEPs were also recorded in 5 patients with optic neuropathy at a fixed luminance (180 cd/m2). The relationship between P100 latency of transient VEPs (T VEPs) and the phase of steady-state VEPs (S-VEPs) was analyzed. As luminance decreased in normal subjects, P100 latency was prolonged and the phase lag increased. A significant linear relationship between the P100 latency and phase was found. Patients showed both the prolonged P100 latency and the delayed phase. The simple linear regression line of the phase-P100 latency function of normal subjects closely matched the patients' values. These results suggest that changes in the phase may be equivalent to changes in the P100 latency. S-VEPs, therefore, may be clinically useful in assessing visual function. PMID- 1707810 TI - Somatosensory evoked potentials in neonates and infants: developmental and normative data. AB - Fifty-two sets of cortical somatosensory evoked potentials (SEPs) were recorded from 23 normal children between the ages of 1 day and 13 weeks with median nerve stimulation. Two bandpass settings 5-1500 Hz and 30-3000 Hz were used; rate of stimulation was 1.1 Hz and sweep-time was 200 msec. The state of wakefulness was documented, but SEPs were obtained and evaluated independently of the child being awake or asleep during the recording. SEPs were present in every recording. The bandpass 30-3000 Hz best differentiated positive and negative early potentials. The bandpass 5-1500 Hz was helpful in some cases, as late slow waves were recorded with this setting. Normative data were established. Mean values were calculated for 3 age groups: 0-2 weeks, 2-6 weeks and 7-13 weeks. P15 and N20 were the first components seen in the newborn, with the P22 becoming the major component by 2-3 weeks of age. The study indicates that maturation of the somatosensory system is fastest during the first 3 weeks of life. PMID- 1707811 TI - Cell/cell channel formation involves disulfide exchange. AB - The oocyte cell/cell-channel assay was used to identify amino acids involved in the process of cell/cell-channel formation. The expression of the rat liver gap junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol-specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin-32 mutants were generated by site-directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process. PMID- 1707812 TI - Sensitive titration microcalorimetric study of the binding of Salmonella O antigenic oligosaccharides by a monoclonal antibody. AB - The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4. PMID- 1707813 TI - Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia. AB - The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60 90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump. PMID- 1707814 TI - Primary carcinoma of the vagina. Case report. AB - An unusual case of Primary Carcinoma of the vagina was recently observed. The patient was treated surgically in 1981 for squamous cervical carcinoma in situ (CIN 3); eight years later the same patient was treated for squamous carcinoma of the vagina that involved the upper, median and lower tract. She was treated with radical colpectomy and VBP chemotherapy for three courses. The pathology, natural history and treatment are discussed. PMID- 1707815 TI - Release and vascular activity of endothelium-derived relaxing factor in atherosclerotic rabbit aorta. AB - A diet containing 0.3% cholesterol was given to male New Zealand rabbits for 16 weeks; this produced atherosclerotic lesions (fatty streaks) on 80% of the intimal surface of the thoracic aorta and on 45% of the intimal surface of the abdominal aorta. The endothelium-dependent relaxations induced by acetylcholine, substance P and ionophore A23187 were inhibited in the atherosclerotic aortas. Besides the endothelium-independent relaxations induced by nitroglycerine, the relaxations induced by atrial natriuretic peptide (ANF) were also significantly reduced in the more atherosclerotic thoracic aorta. In bioassay experiments it was found that acetylcholine and substance P caused a smaller release of endothelium-derived relaxing factor (EDRF) from atherosclerotic thoracic aortas than from control thoracic aortas: the EDRF released by the vasodilators evoked less relaxation in atherosclerotic detector abdominal aortas than in control detector abdominal aortas. Nitric oxide evoked significantly less transient relaxation in the atherosclerotic thoracic and abdominal aortas than in the respective control tissues. The data indicate that as experimental atherosclerosis in the rabbit progresses, both vascular activity and EDRF release become affected; this leads to a complete loss of endothelium-dependent relaxation in the more atherosclerotic blood vessels. PMID- 1707816 TI - Cardiovascular responses to milrinone in pertussis toxin-pretreated pithed rats. AB - The modulating effects of pertussis toxin on angiotensin II and B-HT 920-evoked hemodynamic changes were compared with those of milrinone to evaluate the possible role of guanine nucleotide regulatory proteins (G proteins) in the mechanism of action of milrinone. Both milrinone and pertussis toxin shifted the blood pressure dose-response curves of B-HT 920 to the right, but the responses to angiotensin II were decreased after milrinone pretreatment only. The increase in cardiac frequency evoked by milrinone and isobutylmethylxanthine (IBMX) was not sensitive to pertussis toxin. In contrast, the decrease in systolic blood pressure elicited by milrinone could be prevented by pertussis toxin pretreatment, suggesting the involvement of a regulatory protein. Milrinone and IBMX did not influence the effects of arecoline on blood pressure or heart rate in either normal or pertussis toxin-pretreated rats. It is concluded that milrinone may affect a G protein, but not the adenylate cyclase-associated inhibitory protein, Gi. PMID- 1707817 TI - Galanin inhibits somatostatin release by the rat islet cell tumor in culture, Rin m. AB - Using a rat islet cell tumor in culture, Rin-m, we studied the effects of the neuropeptide, galanin, on somatostatin release. Galanin applied to the incubation medium inhibited pancreatic hormone release rapidly and dose dependently with an IC50 at 4 nM and the maximal effect (40% inhibition) was elicited by 100 nM peptide. Pretreatment of Rin-m cells with pertussis toxin abolished the inhibitory effect of galanin on somatostatin release. The results suggest that galanin probably controls the function of the pancreatic delta cell through a pertussis toxin-sensitive pathway. PMID- 1707818 TI - Relaxation response of isolated canine veins to agents that act on the adenylate cyclase-cyclic AMP system: further investigation. AB - The present study was undertaken to clarify whether there is a deficiency in the relaxation mediated by the adenylate cyclase-cyclic AMP (cAMP) system in the portal vein as compared to the saphenous vein. Longitudinal strips of the portal vein and helical preparations of the saphenous vein were used. The relaxation response to various agents was examined under conditions such that the venous preparations were previously contracted by methoxamine in equipotent concentrations (EC80), i.e., 10(-6) M for portal vein and 10(-5) M for saphenous vein. The saphenous vein relaxed fully in response to isoproterenol but the portal vein relaxed only to 29% of the maximum relaxation induced by papaverine 10(-4) M. However, dibutyryl cAMP and 8-bromo-cAMP, membrane permeable derivatives of cAMP, 3-isobutyl-1-methylxanthine and papaverine, phosphodiesterase inhibitors, and forskolin, a direct stimulator of adenylate cyclase, relaxed portal and saphenous veins similarly though with quantitative differences. The results suggest that there is no profound deficiency in the adenylate cyclase-cAMP system but there may be a deficiency in the coupling between surface beta-adrenoceptors and adenylate cyclase or there may be a low density of beta-adrenoceptors in the portal vein. PMID- 1707819 TI - Evidence that endogenous nitric oxide modulates oedema formation induced by substance P. AB - The possibility that nitric oxide (NO) could have a role in the modulation of inflammatory oedema formation was investigated in rat skin using selective inhibitors of NO synthesis. Intradermally injected substance P (0.03-1 nmol) induced oedema which was inhibited by concurrent administration of the inhibitor of NO synthesis L-NG-nitro arginine methyl ester (L-NAME), but not by the enantiomer D-NAME. L-Arginine reversed the inhibitory effect of L-NAME. A second inhibitor of NO formation, L-NG-monomethyl arginine (L-NMMA), had a similar inhibitory effect on substance P-induced oedema. The results suggest that endogenous NO has a modulatory role in oedema formation induced by mediators of increased microvascular permeability. PMID- 1707820 TI - Impaired cyclic AMP generation in outer medullary tubules of gentamicin-treated rats. AB - We have examined the effects of chronic gentamicin treatment on arginine8 vasopressin (AVP)-dependent cyclic AMP (cAMP) metabolism in rat medullary collecting tubules (oMCT) and medullary thick ascending limbs of Henle's loop (mTALH). Gentamicin attenuated AVP-stimulated cAMP accumulation to a greater extent in the mTALH (delta -51%) than in the oMCT (delta -25%). The mechanism of attenuation differed between segments, and could not be attributed to either direct inhibition of adenylate cyclase activity nor direct potentiation of cAMP phosphodiesterase activity. These data suggest that the gentamicin-induced decrease in renal concentrating ability may be due at least in part to reduced AVP-dependent cAMP accumulation in the oMCT and mTALH. PMID- 1707821 TI - ATP-producing and consuming processes of Ehrlich mouse ascites tumor cells in proliferating and resting phases. AB - The extents of ATP-yielding and consuming processes in Ehrlich mouse ascites tumor cells during the proliferating and resting growth phase were compared. In the resting phase the total ATP production was decreased by one-third. The ATP supply by oxidative phosphorylation was drastically reduced, whereas the rate of glycolysis stayed nearly constant. All ATP-consuming processes investigated, i.e., protein turnover, Na+/K(+)-ATPase, Ca2(+)-ATPase, and RNA synthesis, were decreased proportionally with the total ATP consumption. PMID- 1707822 TI - Vitronectin-induced haptotaxis of vascular smooth muscle cells in vitro. AB - Vitronectin, a multifunctional glycoprotein present in the plasma and interstitial tissues, has recently been found to be localized in atherosclerotic lesions. In this study we examined the effects of vitronectin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to fluid-phase vitronectin in a concentration-dependent fashion. The cells also migrated to membrane filter surfaces precoated with vitronectin for a few minutes in the absence of additional vitronectin in the fluid phase, suggesting that this substance binds easily to the filters and stimulates cell migration by haptotaxis under the conditions described. These observations suggest that vitronectin deposited in the intima may be involved in the pathogenesis of atherosclerosis by recruiting smooth muscle cells from the media into the intima. PMID- 1707823 TI - Characterization of p51/52, a cell-growth regulated protein of WI-38 cells. AB - A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52 kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53. PMID- 1707824 TI - The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin. AB - Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces. PMID- 1707825 TI - Identification of epitopes within the circumsporozoite protein of Plasmodium vivax recognized by murine T lymphocytes. AB - The murine cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax was characterized using five synthetic peptides, some of which we identified as corresponding to T cell epitopes. The peptides P308-320, P344 355 and P353-364 were immunogenic, inducing a genetically restricted proliferative response, due to the activation of CD4+ T cells. The peptide P308 320 was recognized only by the lymphocytes of B10 (H-2b) mice. The other two peptides were recognized by primed lymphocytes of H-2a and H-2k mice. Of interest was the finding that one of these peptides, P353-364, induced a proliferative response of a large percentage of immune outbred Swiss mice. Our data provide evidence that, at least in mice, there is recognition of multiple T cell epitopes within the major surface antigen of P. vivax sporozoites. PMID- 1707826 TI - Gliotoxin treatment selectively spares M-CSF- plus IL-3-responsive multipotent haemopoietic progenitor cells in bone marrow. AB - Gliotoxin, an epipolythiodioxopiperazine, is a fungal metabolite that causes genomic DNA degradation preferentially in certain blood cell types including T lymphocytes and macrophages. Gliotoxin has previously been used to treat murine allogeneic bone marrow prior to transplantation into irradiated recipients, and in this situation the drug prevents development of graft-versus-host disease, and permits the establishment of allogeneic bone marrow chimeras. We have examined the nature of the cells that survive gliotoxin treatment and report here that gliotoxin selectively spares a unique class of haemopoietic stem cell that forms large (HPP) colonies in the presence of mixtures of M-CSF and IL-3. We confirm that the cells which survive gliotoxin treatment are capable of reconstituting the haemopoietic system in allogeneic lethally irradiated mice. PMID- 1707827 TI - Human granulocyte colony-stimulating factor receptors in acute myelogenous leukemia. AB - Human granulocyte colony-stimulating factor (G-CSF) receptors on human acute leukemia cells were investigated using human G-CSF iodolabeled by the lactoperoxidase method. Among various human leukemic cell lines, only cells of myelogenous lineage including HL-60, THP-1 and U937 had one type of high-affinity receptor for G-CSF, as shown by Scatchard analysis. Fresh leukemia cells from 19 patients with acute myelogenous leukemia (AML) were then studied. Specific receptors for G-CSF were demonstrated on blast cells in all 19 cases, the mean number of G-CSF receptors per AML cell ranging from 95 to 1436. G-CSF receptors on AML cells appeared to be a single affinity type, although some variations were observed. The mean number of G-CSF receptors on leukemic cells from patients with either FAB M3 or FAB M2 was greater than that of cells from patients with M1 (p less than 0.01, p less than 0.10, respectively). Moreover, the mean number of receptors for G-CSF on CD13- and CD34-positive AML cells was higher than that on CD13-negative and CD34-positive AML cells (p less than 0.01), and the mean number of G-CSF receptors on CD7-positive AML cells was lower than that for CD7-negative AML cells (p less than 0.10). Since the FAB classification and surface phenotypes reflect maturation stages, our findings indicate that the distribution of G-CSF receptors, even on AML cells, may be related to the maturation process. PMID- 1707828 TI - 5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1. AB - The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed. PMID- 1707829 TI - Penetration enhancement across a model membrane by liposomally entrapped drugs using N,N'-diacylcystine as a bilayer lipid. AB - A liposomal system for percutaneous absorption and transdermal drug delivery was developed and evaluated using a model apparatus connecting two chambers by a membrane. When N,N'-long chain diacyl L-cystine was incorporated into the liposomal bilayer as well as lecithin and cholesterol, liposomally entrapped calcein as the model drug transferred well across the keratin membrane functioning as the skin model. PMID- 1707830 TI - Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development. AB - Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in lambda ZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The 'neonatal (N) GlyR' has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR). Injection of a single RNA transcribed from the NGlyr-cDNA into Xenopus oocyte induced functional formation of glycine-gated Cl- channels, however, its pharmacological property differed from that of AGlyR. PMID- 1707831 TI - cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells. AB - The relationship between activation of the cAMP-dependent protein kinase (cAMP PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane. PMID- 1707832 TI - AMPA and kainate-operated channels reconstituted in artificial bilayers. AB - Cationic channels which can be activated by alpha-amino-3-hydroxy-5 methylisoxazole-4-propionate (AMPA) and kainate play a key role in the generation of excitatory postsynaptic potentials in the brain of all vertebrates. On a protein carrying binding sites for both these ligands, affinity-purified from the Xenopus nervous system, electrical measurements have been performed to investigate its functional properties after the pure complex had been incorporated into planar lipid bilayers. Domoate, AMPA or kainate added to the reconstituted protein activated cationic channels, which were blocked by typical antagonists for this neurotransmitter system. These data suggest that the reconstituted protein is an ionotropic receptor of the unitary non-NMDA subtype. PMID- 1707833 TI - The chloroplast gene for ribosomal protein CL23 is functional in tobacco. AB - Chloroplast rpl23 loci potentially coding for a polypeptide homologous to the E. coli L23 ribosomal protein are frame-shifted in spinach and several other plants, indicating that these loci are pseudogenes. In tobacco, rpl23 constitutes a continuous open reading frame of 93 codons and its transcript initiates at least 66 bp upstream from the initiation codon. The N-terminal amino acid sequence of a 13 kDa protein from the 50 S subunit of tobacco chloroplast ribosomes matches that derived from the tobacco rpl23 locus. This shows that rpl23 is a functional gene in tobacco. PMID- 1707834 TI - Chondrosarcoma of the pelvis: the role of palliative debulking surgery. AB - Curative resection of pelvic chondrosarcoma is sometimes technically impossible. In such cases, surgical debulking on one or more occasions may provide symptomatic relief. In a series of 12 patients with pelvic chondrosarcoma, three have undergone a single debulking procedure and three two or more such procedures. All six patients obtained symptomatic relief and five remain alive and well at a median of 12 months from surgery. Two patients have no clinical evidence of recurrent disease 21 and 25 months after the last debulking procedure. PMID- 1707835 TI - Perspectives in chemotherapy of pancreatic cancer. PMID- 1707836 TI - Future prospects of radiotherapy in pancreatic cancer. AB - To clarify the role of radiation therapy in the treatment of carcinoma of the pancreas studies with radiotherapy alone, combined interventional or concurrent radio-chemotherapy are reviewed. It is shown that in the case of inoperable tumour radiotherapy alone is inadequate, but by the means of a combination of chemotherapy, external beam or interventional (intra-operative, interstitial) irradiation, improvements in local control rates and median survival can be achieved. Following so-called curative resection, a number of studies have shown that adjuvant radio-chemotherapy or interventional treatments possibly prolong survival. PMID- 1707837 TI - Amount and speed of fast axonal transport in diabetes. AB - Abnormalities in axonal transport have been observed in human and experimental diabetes and may be related to the pathogenesis of diabetic neuropathy. Axonal transport has previously been evaluated by indirect methods. In this study, direct-measurement techniques were applied (with computer-enhanced video-recorded images) for the first time to evaluate intra-axonal organelle speed and frequency (the amount of organelle traffic) in both the anterograde fast component (AFC) and retrograde fast component (RFC) of axonal transport in diabetic nerve. Sciatic nerve and dorsal and ventral nerve roots were studied in the animal model of insulin-dependent diabetes (BB/Wistar rat) and sciatic nerve in the non insulin-dependent (streptozocin-induced) model of diabetes (STZ-D rat). STZ-D rats were studied at 1 mo, and BB/Wistar rats were studied at 1 and 2 mo of diabetes duration. Statistically significant decreases in peripheral axon organelle speed were found only for RFC at 1 mo of diabetes in both the BB/Wistar (8.1%) and STZ-D (5.4%) rats. The difference was no longer significant in BB/Wistar rats at 2 mo of diabetes. This recovery suggests that the underlying abnormality is reversible. No differences were seen in AFC of any axons, and the only other difference seen was a 5.1% decrement in RFC at 2 mo in the ventral roots. No significant difference was observed in any group for organelle frequencies. Other factors should be considered to explain the decrease in materials transported in accumulation studies. The transient deficits in RFC speed observed remain of undetermined significance in the pathogenesis of diabetic neuropathy. PMID- 1707838 TI - Alpha-fetoprotein in Angelman syndrome. PMID- 1707839 TI - Increased hyaline droplet formation in male rats exposed to decalin is dependent on the presence of alpha 2u-globulin. AB - A peculiar decalin-induced male rat nephropathy associated with the altered renal handling of filtered protein appears limited to the accumulation of the protein, alpha 2u-globulin. Several strains of male rats that produce alpha 2u-globulin (Fischer-344, Sprague-Dawley, Buffalo, and Norway Brown) demonstrate spontaneous renal cortical hyaline droplets which are exacerbated after exposure to decalin. In all cases, a close correlation exists between hyaline droplet formation observed histologically and alpha 2u-globulin accumulation measured biochemically. In stark contrast, the NCI-Black-Reiter strain, which does not produce measurable quantities of alpha 2u-globulin, neither forms hyaline droplets nor accumulates any filtered protein in its kidney cortex either spontaneously or after exposure to decalin. Also, female rats injected ip with male rat alpha 2u-globulin exhibit increased hyaline droplet formation and alpha 2u-globulin accumulation when treated with decalin. These data provide evidence that the presence of alpha 2u-globulin is key in understanding why this nephropathy appears unique to the male rat. PMID- 1707840 TI - [The prognostic significance of tumor cell detection in bone marrow of patients with breast cancer]. AB - In 95% of patients with primary breast cancer, the extent of metastases cannot be proven by conventional methods. Nevertheless, more than 50% of these patients have a relapse within five years. To improve the predictive value for recurrency, we examined bone marrow aspirates of 128 patients with primary breast cancer. Bone marrow aspirates from 2-6 sites of the skeleton (iliac crest and sternum) were taken as well as biopsies for histological examination. The immunohistochemical studies were carried out on interphase smears and stained with cytoceratin antibodies (PKK 1) and antibodies against tumor-specific antigen TAG 12 (12 H 12). All patients were screened for distant metastases (X-ray, ultrasound, bone scan). Tumor cells and micrometastases in bone marrow were detected in 41 patients (32%). Their presence was correlated to other prognostic factors (tumor size, lymph node status, oestrogen/progesterone receptors). The median duration of follow-up was 39.5 months. 14 patients (45%) in the tumor cell positive group relapsed, compared to only 4 out of 36 patients in the tumor cell negative group. In 29% we found bone metastases. The relapse free interval was shorter for patients with micrometastases (8 vs. 15.8 months). The presence of tumor cells in bone marrow aspirates detected at the time of primary surgery, is a useful prognostic factor and a good predictor of metastases and may help in selecting patients for systemic adjuvant treatment. PMID- 1707841 TI - [Change in tubal pregnancy 1983-1989. 7-years experiences with surgical laparoscopy]. AB - The authors report on the changing aspects in diagnostics and surgical treatment of tubal pregnancy during a period of 7 years (1983-89). In that period, 432 cases of tubal pregnancy were diagnosed and treated, 311 of which were treated by endoscopic abdominal surgery. Diagnosis of tubal pregnancy now depends on radioimmunology for identifying the presence of beta-HCG in the serum as well as on sonographic diagnosis. This may supply proof of tubal pregnancy in many cases as early as 3-4 weeks after conception. Surgery should not be initiated too early, but should also not be too late. We found that the 4th to 5th week after conception yields the best results. During 1986-88, 156 patients were surgically treated by laparoscopy because of tubal pregnancy. These operations and the subsequent fate of the patients are analysed. 66 patients continued to actively wish for a child. In 15% of these there was a recurrence of tubal pregnancy. 62% of these patients became again pregnant in utero. PMID- 1707842 TI - [Early amniocentesis for cytogenetic diagnosis]. AB - This study was designed to assess the feasibility of amniocentesis and amnion cell culture for prenatal diagnosis in early weeks of gestation (less than 15 weeks). Within a period of 18 months (1/88-6/89) 135 diagnostic amniocenteses were performed between 10 and 14 weeks and amniotic fluid was obtained in all instances. In all cases but one, sufficient cell cultures and chromosomal analyses were achieved. In the follow up, one miscarriage (0.7%) occurred, in two cases transient amniotic fluid leakage ceased spontaneously. All of the aforementioned complications were related to amniocenteses in weeks 11 and 12. The course of the continued pregnancies as well as fetal outcome were without any further complications. Comparing specimens of early and regular (16-18 weeks) amniocenteses in terms of time required for cell culture and karyotyping as well as the quality of chromosomal banding no significant differences were found. In contrast to maternal serum AFP which increased continuously, amniotic AFP levels could be seen to increase to a peak at 13 weeks' gestation but afterwards gradually declined. Based on these first experiences and results of early amniocenteses the specific problems of this method are discussed. In summary, it is concluded, that early amniocentesis between 12 and 14 weeks of gestation is feasible with regard to both the technique of amniotic fluid retrieval and the technique of chromosomal analysis. Early amniocentesis, therefore, could fill up the diagnostic gap between chorionic villi sampling (CVS) between 9 and 11 weeks of gestation and "classical" amniocentesis during weeks 16-18. PMID- 1707843 TI - Isolation of a second S-locus-related cDNA from Brassica oleracea: genetic relationships between the S locus and two related loci. AB - Self-incompatibility in Brassica oleracea is controlled by the highly polymorphic S locus. Isolation and subsequent characterization of the S-locus-glycoprotein (SLG) gene, which encodes the S-locus-specific glycoprotein (SLSG), has revealed the presence of a self-incompatibility multigene family. One of these S-locus related genes, SLR1, has been shown to be expressed. In this study we present the isolation and preliminary characterization of a second expressed S-locus-related sequence, SLR2. Through restriction fragment length polymorphism (RFLP) linkage analysis we demonstrate that the SLR1 and SLR2 loci reside approximately 18.5 map units apart in one linkage group that segregates independently of the S-locus. The identification of a second SLR gene expressed in stigmas suggests that loci unlinked to the S-locus may play a role in the self-incompatibility response, or in pollination in general. PMID- 1707844 TI - Cytogenetic analysis of the second chromosome heterochromatin of Drosophila melanogaster. AB - This paper reports the cytogenetic characterization of the second chromosome heterochromatin of Drosophila melanogaster. High resolution cytological analysis of a sample of translocations, inversions, deficiencies and free duplications involving the pericentric regions of the second chromosome was achieved by applying sequential Hoechst 33258 and N-chromosome banding techniques to larval neuroblast prometaphase chromosomes. Heterochromatic rearrangements were employed in a series of complementation assays and the genetic elements previously reported to be within or near the second chromosome heterochromatin were thus precisely assigned to specific heterochromatic bands. The results of this analysis reveal a nonhomogeneous distribution of loci along the second chromosome heterochromatin. The l(2)41Aa, l(2)41Ab, rolled (l(2)41Ac) and l(2)41Ad loci are located within the proximal heterochromatin of 2R, while the nine remaining loci in the left arm and two (l(2)41Ae and l(2)41Ah) in the right arm map to h35 and to h46, respectively, the most distal heterochromatic regions. In addition, a common feature of these loci revealed by the cytogenetic analysis is that they map to specific heterochromatic blocks but do not correspond to the blocks themselves, suggesting that they are not as large as the Y fertility factors or the Rsp locus. Mutations of the proximal most heterochromatic loci, l(2)41Aa and rolled, were also examined for their phenotypic effects. Extensive cell death during imaginal disc development was observed in individuals hemizygous for either the EMS 31 and rolled mutations, leading to a pattern of phenotypic defects of adult structures. PMID- 1707845 TI - Transcriptional analyses of the uncoordinated region of Drosophila melanogaster. AB - RNA blotting experiments reveal that a genomic region encompassing the uncoordinated complementation group of Drosophila melanogaster produces two classes of transcripts. The first class contains five polyadenylated RNAs, of 4.35, 8.7, 9.0, 11.5, and 13.0 kb, and the second class contains a transcript of 7.3 kb that can be detected in both the total cellular and the poly(A)+ fractions. Transcripts of the first class are found predominantly in the early larval, pupal, and adult life stages, whereas the 7.3-kb species is present throughout the life cycle and is also found in the heads of adult flies. The two classes of RNAs derive from different combinations of exons. Analyses of RNAs derived from genotypes that are deficient for the uncoordinated complementation group and from seven uncoordinated mutant alleles support the suggestion that the uncoordinated gene (unc) has been isolated and encodes the multiple transcripts. PMID- 1707846 TI - Synthesis and characterization of the Kunitz protease-inhibitor domain of the beta-amyloid precursor protein. AB - To understand the pathological process by which amyloid is deposited in Alzheimer's disease, it is important to characterize the proteolytic processing events of the beta-amyloid precursor protein (beta-APP) from which the amyloid forming fragment is excised. A potentially important component in beta-APP processing is the 57-amino acid (aa) Kunitz serine protease inhibitor (KPI) located within the extracellular domain of both the 751- and 770-aa isoforms of beta-APP. We have synthesized DNA encoding the 57-aa KPI domain as a necessary step in identifying the role of the protease inhibitor in beta-APP processing and amyloid formation. A bacterial secretion system directed by the alkaline phosphatase signal peptide of Escherichia coli linked to a synthetic gene encoding KPI was used to produce soluble, extracellular recombinant KPI (reKPI) protein. The reKPI protein was purified to homogeneity from bacterial supernatants and was biochemically and biologically characterized. Complete aa sequence analysis confirmed the fidelity of the reKPI, and fast-atom bombardment mass-spectral analysis was used to document that reKPI was of the predicted Mr. The reKPI is as active on a molar basis as the inhibitor-containing beta-APP when assayed for inhibition of trypsin activity. Together these data suggest that reKPI protein is properly folded and lacking in modified aa. Hence, this reKPI will be an important reagent in gaining a better understanding of the role of the KPI domain in beta-APP function and metabolism, as well as in the proteolytic events involved in beta-amyloid formation. PMID- 1707847 TI - Spin-trapping studies of the oxidation-reduction reactions of iron bleomycin in the presence of thiols and buffer. AB - The reaction of ferrous bleomycin with dioxygen is reexamined to clarify whether radical species derived from molecular oxygen are generated. Detection of low levels of spin-trapped oxyradicals confirm the production of OH during this reaction when bleomycin is present in excess, but not when iron and drug concentrations are equal. In phosphate buffer, hydroxyl radicals continue to be spin trapped for at least 15 min after Fe(II)bleomycin has been oxidized to Fe(III)bleomycin. In HEPES buffer, detection of a HEPES radical in the absence of spin trap over the same period independently supports the conclusion that reactive radicals are present after the initial oxidation of Fe(II)bleomycin is complete. When glutathione is included in the aerobic reaction mixture, thiyl radical species are spin trapped. The reaction of Fe(III)bleomycin with cysteine produces thiyl radical without spin-trapped hydroxyl radical. PMID- 1707848 TI - Deletion and non deletion hereditary persistence of fetal hemoglobin in Italy. PMID- 1707849 TI - Islet transplantation in experimental diabetes of the rat. XIII. Cryopreservation reduces MHC class II but not class I antigens of rat pancreatic islets. AB - Pretreatment of islet allografts prior to transplantation may reduce islet immunogenicity and prolong graft acceptance. We have studied the MHC antigen reducing effect of cryopreservation onto rat pancreatic islets performing indirect immunofluorescence tests and peroxidase-anti-peroxidase staining (PAP). Three different freezing programs were used. Program A: 0.5 degrees C/min to -35 degrees C and 1 degree C/min from -35 to -100 degrees C. Program B: 2 degrees C/min to -35 degrees C and 6 degrees C/min from -35 to -100 degrees C. Program C: 0.25 degrees C/min to -40 degrees C. Cryopreservation clearly reduced the number of class II antigen positive cells per islet in all cases. Program A was most effective with 45.5% of class II antigen negative islets compared to 6.4% of class II antigen negative fresh islets as shown by indirect immunofluorescence. The class II antigen reducing effect of cryopreservation proved to be permanent and not only temporary. Reduced class II antigen expression of cryopreserved islets could not be reestablished by incubation of the islets with rat IFN. A combination of cryopreservation followed by a 10 day culture period proved to be most effective with 85.6% of class II antigen negative islets. In contrast, we could not show any effect of cryopreservation on class I antigen expression. Viability of the cryopreserved rat islets was shown in-vitro by glucose stimulated insulin secretion. PMID- 1707850 TI - Expression of an EL4 tumour-associated determinant on subpopulations of murine T cells in normal and lympho-proliferative autoimmune mice. AB - It has been demonstrated that the single autosomal recessive lpr and gld genes are responsible for the accumulation of unusual T-cell subsets. Although these subsets have been assigned to the T-cell lineage, they share certain antigenic cell surface markers with mature B lymphocytes. Consequently the maturational pathway(s) of these cells has been difficult to fit in the currently accepted models of T-cell differentiation. Previous work has determined that the YE1/19.1 monoclonal antibody (mAb), developed against the EL4 tumour line, reacts with the accumulating T cells in lpr-expressing mice. In this study we report that YE1/19.1 could also be used as a marker for the accumulating T cells in gld expressing mice and that the hyporesponsiveness seen in gld mice correlated with these accumulating cells. We then demonstrated that the YE1/19.1 antibody also reacts with a subpopulation of neonatal thymocytes as well as a mitogen non responsive subpopulation of 'double negative' T cells from the spleens and thymuses of non-autoimmune mice. Our findings indicate that the YE1/19.1 mAb will be a useful probe for helping in the eludication of the intra-thymic maturational pathways of T lymphocytes. PMID- 1707851 TI - Detection of Leu-19 (CD56) antigen on human thyroid epithelial cells by an immunohistochemical method. AB - The Leu-19 (CD56) antigen, which is recognized by anti-Leu-19 and NKH-1 monoclonal antibody, is a 200,000-220,000 molecular weight (MW) glycoprotein that is expressed predominantly on human natural killer (NK) cells that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity. However, cross reactivity of this antibody has been observed in lung cancer, and in muscle and neural tissues. In the present study, we used the immunoperoxidase technique to examine the expression of Leu-19 antigen in human thyroid epithelial cells. In normal thyroid tissues (n = 4), thyroid tissues from Graves' patients (n = 7) and benign thyroid tumours (n = 7), thyroid epithelial cells expressed Leu-19 antigen in all cases. In thyroid papillary carcinoma (n = 6) there was no expression in four cases. This staining pattern of anti-Leu-19 antibody is similar to that of anti-thyroid peroxidase antibody. These findings implicate that the expression of Leu-19 antigen is closely related to the differentiation of thyroid epithelial cells. PMID- 1707852 TI - Effect of variations in peptide sequence on anti-human milk fat globule membrane antibody reactions. AB - Monoclonal anti-mucine antibodies BC1, BC2 and BC3 produced using human milk fat globule membrane react with a synthetic peptide p1-24 (PDTRPAPGSTAPPAHGVTSAPDTR) representing the repeating amino acid sequence of the mucin core protein. The minimum epitope recognized by these three monoclonal antibodies (mAb) in p1-24 was contained in the five amino acids APDTR. To analyse the variation of position of the epitope, various modifications of the APDTR sequence were made by synthesizing peptides and testing by direct binding and inhibition enzyme-linked immunosorbent assays. Firstly, peptides p13-32 and C-p13-32, in which the epitope APDTR was placed in the middle instead of the C-terminal as in p1-24, were examined. These peptides had a greater reaction with mAb BC1, BC2 and BC3 compared with the reaction with p1-24. Secondly, A-p1-24 and TSA-p1-24 were made wherein two APDTR epitopes were present--these peptides were shown to bind two IgG antibody molecules. Finally, the contribution of each amino acid in the APDTR epitope was studied using the pepscan polyethylene rods, making all 20 of the amino acid substitutions in each position for SAPDTR (the minimum epitope APDTR with an adjacent amino acid S). In the 120 peptides examined there were some 'permissible' substitutions in A, D and T but not in P or R for BC1 and BC2; there were more 'permissible' substitutions for BC3; different substitution patterns were found with each antibody and some substitutions gave an increased reaction compared with the native peptide SAPDTR. The studies are of value in analysing the reaction of antibodies with epitopes expressed in breast cancer and in determining the antigenicity of synthetic peptides. PMID- 1707853 TI - Fluoride: an adjuvant for mucosal and systemic immunity. AB - Fluoride, the agent responsible for reduction of dental caries worldwide, and a recognized proliferative agent, is a potent adjuvant when given intragastrically to rats. Intragastric fluoride causes increases in the size and cellularity of the Peyer's patches and mesenteric lymph nodes as well as the number of plasma cells secreting IgG and IgA antibodies to ovalbumin given in their drinking water. Rats ingesting NaF and fed OA showed a significant increase in surface immunoglobulin expression on lymphocytes from the Peyer's patches and mesenteric lymph nodes. The frequency of CD4+ T cells in these lymphoid tissues was elevated while that of CD8+ T cells was significantly decreased. In separate experiments, rats parenterally immunized with myelin basic protein (MBP) and fed NaF twice weekly, had significantly elevated serum IgG antibody activity to MBP compared to similarly immunized rats not receiving NaF. The supplemental fluoride prescribed for infants and especially that which is inadvertently ingested by children and adults given fluoride gels, is within the concentration range of that which produced the effects we observed in rats. The adjuvant effect we describe thus has relevance for fluoride therapy worldwide. PMID- 1707854 TI - Monoclonal antibodies to monomeric rat liver metallothionein-I: the immunoreactivity of lysine residues in metallothionein. AB - Regardless of the weak immunological response against the low-Mr metallothioneins (MTs) the production of murine monoclonal antibodies (mAbs) to monomeric rat liver MT-I was successful. ELISA revealed two groups of mAbs which exhibited different specificities as examined on the native and the lysine-residue modified antigen (Ag). One "lysine-directed mAb" group, consisting of three mAbs, exhibited a specific immunoreactivity with the lysine-containing epitopes of MTs. Their role in the antigenicity of MTs was examined by modifying these residues using glutaraldehyde (GA). Titration with GA resulted in a progressive decline in Ag recognition in the immunoblot; this was completely leveled off when equimolar concentrations were reached. A similar response employing the GA-modified protein in the ELISA was noticed. The second group of mAb cross-reacted with various MTs of different origin, indicating that the common, lysine-free NH2-terminus is exclusively recognized. In direct ELISA of cross-linked MTs, the observed reactivities were much more pronounced. Iodoacetamide (IA) modification of the lysines confirmed the above observations of the GA-derived Ag. Notably, the immunoreactivity was not affected when the cysteine residues were IA carboxymethylated, nor did the subsequent loss of metals diminish the immunological response in the immunoblot. PMID- 1707855 TI - Neutralizing antibodies and antigens in AIDS. AB - Until recently, much of the effort put into development of an AIDS vaccine has focussed on the elicitation of a neutralizing antibody response. The viral target of neutralization, HIV envelope glycoprotein, has been produced in bulk through recombinant techniques, but has had little success as a vaccine. The specific epitopes to which neutralizing antibodies bind have been mapped, and although the major epitope is hypervariable, others are conserved. This allows the design of second generation vaccines. Meanwhile, vaccine studies in the SIV animal model simply using inactivated virus as immunogen have demonstrated that an effective vaccine is at least possible. A variety of HIV vaccine preparations are now under investigation and the outlook for the future is promising. PMID- 1707856 TI - Comparison of the anaphylactoid response induced in rats by castanospermine and dextran. AB - The plant alkaloid castanospermine (10 mg/kg or higher, i.p.; 400 mg/kg, p.o.) induced in some rat strains an anaphylactoid reaction similar to that induced by dextran, i.e. erythema and edema of the snout, ears and paws, for several hours after administration. 86% of the rats from a responsive strain responded to castanospermine while 79% responded to dextran. Rats which responded to castanospermine showed marked, but transient, tachyphylaxis to a second dose of castanospermine or dextran. Rats maintained on a complex-carbohydrate-free diet also responded to castanospermine, excluding the possibility that the effect was due to absorption of dextran-like, dietary, complex carbohydrates. These data raise the possibility that some apparent food allergies in man could be due to the presence in the diet of plant alkaloids with properties similar to those of castanospermine. PMID- 1707857 TI - Characterization and quantification of cellular infiltrates in nasal mucosa of patients with grass pollen allergy, non-allergic patients with nasal polyps and controls. AB - Little is known about cellular infiltrates in nasal mucosa and the differences between these infiltrates in allergic and non-allergic patients. A reproducible and objective method making use of monoclonal antibodies for the quantification and characterization of cellular infiltrates in biopsy specimens of nasal mucosa is described. This method was used to study quantitative differences in cellular infiltrates in the epithelium and lamina propria of the nasal mucosa of patients with isolated grass pollen allergy, non-allergic patients with nasal polyps, and controls. A surprisingly wide variation was found in all groups. In all groups the T lymphocytes were much more numerous than the B lymphocytes. The number of CD8+ cells exceeded the number of CD4+ cells in the epithelium but in the lamina propria the numbers were approximately equal. Significant differences between the three groups were found with respect to the number of CD1+, IgE+, neutrophils and cytoplasmic IgG4+ cells. No significant differences were found in the numbers of CD4+, CD8+, CD14+, CD22+, HLA-DR+, IgG1-3+ cells or eosinophils. The use of biopsy in combination with monoclonal antibodies is an easy and well-tolerated method to study immunological reactions in the nasal mucosa. The results of this study indicate a possible role for a T-cell-mediated response in allergic rhinitis. PMID- 1707858 TI - Experimental cedar pollinosis in rhesus monkeys. AB - In order to develop an experimental model for human cedar pollinosis, 4 rhesus monkeys (Macaca mulatta) were subcutaneously immunized with crude cedar pollen antigen, which contained 1.0 microgram or 10 micrograms protein with aluminium hydroxide gel, six times every month. All 4 animals showed immediate-type cutaneous reactivity. Serum IgE antibody responses against the pollen antigen, as quantitated by the Prausnitz-Kustner reaction, were seen in 3 of 4 monkeys. Radioallergosorbent test scores increased in 2 monkeys and remained at higher levels throughout the study. Histamine release from leukocytes was positive in all animals. In conjunctival provocation tests, they showed clinical signs such as eye-scratching after the antigen was dropped into their eyes. This reaction is similar to that observed in humans and suggests this animal model might be useful in studying human cedar pollinosis. PMID- 1707859 TI - Measurement of gene expression in human retinal microvessels by solution hybridization. AB - Changes in gene expression could play a central role in the phenotypic abnormalities of the retinal vascular cells observed in diabetic retinopathy and other retinal diseases. To measure gene expression in human retinal microvessels, a RNA-probe excess solution hybridization assay was used. Retinal microvessels were isolated from eyes obtained within 36 hr of death, and intact RNA was extracted by the guanidine method. Hybridization of poly(A)+ RNA northern blots revealed only the cytoskeletal beta-actin message; by using the more sensitive solution hybridization assay, the plasminogen activator-inhibitor 1 (PAI-1) and von Willebrand factor (vWF) mRNAs were quantified. The prevalence of these transcripts in the retinal microvessels was 0.04 x 10(6) copies/ng RNA for PAI-1 and 0.14 x 10(6) copies/ng RNA for vWF, much less than the prevalence in human umbilical vein endothelial cells (1.93 x 10(6) and 3.90 x 10(6), respectively). The PAI-1 mRNA levels in retinal microvessels isolated from five type II diabetic patients were significantly higher than those in vessels isolated from ten age matched controls (0.06 x 10(6) versus 0.04 x 10(6) copies/ng RNA, P less than 0.05). The solution hybridization assay accurately measured low-abundance mRNAs in human retinal microvessels; determination of gene expression in these cells could aid in understanding the pathogenesis of important ophthalmologic diseases such as diabetic retinopathy. PMID- 1707860 TI - Anion channels in the apical membrane of mammalian corneal epithelium primary cultures. AB - Chloride ion (Cl-) secretion by the rabbit corneal epithelium involves a catecholamine-stimulated Cl- conductance at the apical membrane, but the characteristics of the ion channel(s) responsible are unknown. A primary cell culture system was developed that induces stratified epithelial differentiation of rat and rabbit corneal epithelial cells, as detected by differential interference contrast light and transmission electron microscopy. In patch clamp studies, gigaOhm seal, were obtained easily during the first 1-4 days in vitro, and patches contained a voltage-dependent outwardly rectifying channel. Single channel conductance at 24 degrees C was about 10, 29, and 72 pS in symmetric 150 mM NaCl at membrane potentials of -60, 0, and +60 mV, respectively. The current voltage relationship measured with 75 mM NaCl and sucrose in the bath indicated anion selectivity. Increasing the temperature to 37 degrees C and replacing HEPES buffer with tricine, increased channel conductance and decreased rectification. Characteristics of this channel and a low conductance Cl- channel are compared with those of anion channels of other vertebrate epithelia. PMID- 1707861 TI - Selective effects of experimental glaucoma on axonal transport by retinal ganglion cells to the dorsal lateral geniculate nucleus. AB - Rapid-phase axonal transport to the dorsal lateral geniculate nucleus (dLGN) was determined autoradiographically in seven macaque monkey eyes with chronic intraocular pressure (IOP) elevation, in four eyes with an acute IOP elevation, and in three eyes with normal IOP. The monkeys with chronic IOP elevation showed a greater decrease in radioactive labeling of the magnocellular layers of the dLGN than the parvocellular layers by qualitative examination. Grain counts in selected specimens confirmed that transport to the magnocellular layers was less than to the parvocellular layers in monkeys with chronic IOP elevation. This selectivity was present in mildly damaged specimens and increased with greater ganglion cell loss. In monkeys with acute IOP elevation, qualitative evaluation suggested no consistent difference in transport among the dLGN layers; one animal in this group had less transport to the parvocellular than to the magnocellular layers by grain counts. Starting in early stages of the disease, chronic experimental glaucoma causes preferential damage to the ganglion cells that project to the magnocellular layers of the dLGN. PMID- 1707862 TI - Nonpigmented cells of the rabbit ciliary body epithelium. Tissue culture and voltage-gated currents. AB - The aqueous humor of the eye is thought to be secreted by the epithelium of the ciliary body. This epithelium has been difficult to study, in part because of its complicated morphology. The authors attempted to circumvent this difficulty by growing the epithelial cells in tissue culture. A procedure is described for producing pure primary cultures of rabbit nonpigmented ciliary body epithelial cells. This procedure was used with whole-cell patch-clamp recording to characterize voltage-activated currents in the nonpigmented cells. These experiments show that most nonpigmented cells contain two kinds of currents: a rapidly activating and inactivating inward current, carried by Na+ and blocked by tetrodotoxin (TTX), and a more slowly activating and inactivating outward current, blocked by tetraethylammonium (TEA+), Ba2+, and 4-aminopyridine (4-AP) and presumably carried by K+. Both of these currents have been observed in freshly dissociated cells and in cultures up to 7 days old. The voltage-gated currents in ciliary body epithelial cells are remarkably similar to those of neurons and raise the possibility that these epithelial cells are capable of spike propagation. PMID- 1707863 TI - Crystallin mRNA concentrations and distribution in lens of normal and galactosemic rats. Implications in development of sugar cataracts. AB - It is well established that high concentrations of sugar in the lens of the eye eventually lead to fiber cell destruction and cataracts. In these studies the decrease in crystallin mRNAs was quantified as a result of influx of high concentrations of galactose into the lens of rats. The alpha A-, alpha B1-, and gamma-crystallin mRNA concentrations were assessed in normal lens and in lens undergoing development of sugar cataracts by northern blot and in situ hybridization methods. In a normal, 28-day-old lens, alpha A-crystallin mRNA accumulated to high levels throughout the fiberplasm, and alpha B-crystallin mRNA was present at low levels in epithelial cells, with increased expression in elongating epithelial and fiber cells. The beta B1-crystallin mRNA was distributed to about the same grain density throughout the fiberplasm but at significantly lower levels than alpha A-crystallin mRNA. The gamma-crystallin mRNA first emerged in the terminally differentiated fiber cell, with insignificant amounts detected in the elongating epithelial and fiber cells at the bow. Measurements of hybridization levels on the same RNA population isolated from a single lens showed that in the controls, alpha A-crystallin mRNA comprised about ten times the level of alpha B-crystallin mRNA and twice the level of beta B1- and gamma-crystallin mRNAs. In the cataractous lens the rate of decrease in the concentrations of alpha A-, alpha B- and beta B1-crystallin mRNAs was the same; the decrease in gamma-crystallin mRNA was far more severe. By 20 days of feeding of galactose, at the age of 48 days, gamma-crystallin mRNA diminished to about 9% of the control levels, alpha A-crystallin mRNA to 49%, alpha B crystallin mRNA to 55%, and beta B1-crystallin mRNA to 65%. In the normal lens, at 48 days of age, the levels of alpha A-, alpha B-, and beta B1-crystallin mRNAs showed no significant changes; the gamma-crystallin mRNA level decreased significantly, to about 70% of the day-28 level, the time at which galactose feeding began. Overall, these data suggest that the loss in crystallin mRNAs in response to the development of galactose cataracts follows this order of decline: gamma greater than alpha B greater than alpha A greater than beta B1. PMID- 1707864 TI - Effects of oxidants on lens transport. AB - Hydrogen peroxide is associated with the development of cataracts. As an oxidant, it can act on the sulfhydryl groups of proteins and alter the transport properties of membranes. A nearly impermeant sulfhydryl binding agent is p chloromercuriphenylsulfonate (p-CMPS). The changes in the current-voltage relationship of the equatorial potassium current produced by hydrogen peroxide and p-CMPS are similar. The authors studied the effects of p-CMPS to determine the possible effects of binding extracellular sulfhydryl groups. With a vibrating probe and microelectrodes, the authors saw three sequential effects of 0.5-5.0 microM p-CMPS. The first phase was a shift of the reversal potential, which is equivalent to the potassium equilibrium potential, to more negative values. The current-voltage relationship (J vs PD) shifted in a manner opposite to that produced by ouabain. The 86Rb uptake was stimulated. Ouabain blocked this initial phase. The second phase was a decrease in the resistance. The effects seen were similar to those described in other tissues after the intracellular injection of small amounts of Ca++. This second phase was inhibited by the removal of Ca++ from the medium and also by the addition of quinine to the medium. The third phase consisted of a depolarization of the lens. This effect has been described by others with larger concentrations of p-CMPS and is accompanied by an influx of Na+ and Ca++. The results suggested that micromolar quantities of extracellular p CMPS sequentially stimulate the Na, K-pump; activate Ca(++)-dependent K+ channels; and open nonspecific channels. It is suggested that the second phase may play a role in cateractogenesis. PMID- 1707865 TI - Immune inhibition of virus release from human and nonhuman cells by antibody to viral and host cell determinants. AB - Immune inhibition of release of the DNA viruses, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti viral sera (not anti-host cell sera). Simian virus 40 and surprisingly two herpes viruses, bovine mamillitis and equine abortion, were not inhibited by either anti viral or anti-host sera. Using the herpes simplex virus model, inhibition of virus release was detected in different cells of human and nonhuman origin with cross-inhibition between cell lines of different origin; thus, this form of immunotherapy may not require antibody to be tissue or organ specific. Evidence of inhibition of virus release from neoplastic and leukemic cell lines suggests possible application of this approach to control of virus-mediated leukoproliferative pathology (e.g. Burkitt's lymphoma or adult T cell leukemia). PMID- 1707866 TI - Dimethyl sulfoxide inhibits human immunodeficiency virus production in vitro. AB - LDV/7, H9, and MOLT-4, three cell lines infectible by human immunodeficiency virus were incubated with dimethyl sulfoxide, an inducer of cell differentiation. It was shown that this is a powerful inhibitor of viral production, but its effect is transient: viral production resumes when the compound is removed from the culture medium. It does not inactivate the virus, and it fails to prevent viral infection or to inhibit expression of p24 on the surface of the infected cells. PMID- 1707867 TI - [Current pathophysiologic aspects of allergic rhinitis. III]. PMID- 1707868 TI - Antiserum to an inhibin alpha-chain peptide neutralizes inhibin bioactivity and increases ovulation rate in sheep. AB - A synthetic fragment representing the N-terminal 25 amino acid residues of the alpha-subunit of ovine inhibin (alpha-IF) was coupled to human alpha-globulin (h alpha-G) and used as an antigen. In Exp. 1, ovine antiserum generated against alpha-IF-h alpha-G was shown in vitro to neutralize inhibin bioactivity contained in ovine follicular fluid. In Exp. 2, 18 lambs were immunized with .3, .6 and 1.2 mg alpha-IF-h alpha-G or equivalent doses of h alpha-G. Antibody titer to alpha IF was detected only in serum from lambs immunized against alpha-IF-h alpha-G and was first detected 27 +/- 2 d after primary immunization. Thereafter, antibody titers increased steadily. The degree of antibody responses was unrelated to antigen dose and differed among lambs. Plasma FSH concentrations were unchanged, whereas LH concentrations were lower (P less than .001) in sheep immunized against alpha-IF-h alpha-G. Ovulation rate was increased (3.5 +/- .5 vs 1.5 +/- .1; P less than .01) in lambs immunized against alpha-IF-h alpha-G. Ovulation rate was similar among animals receiving different antigen doses and increased with time after primary immunization (P less than .01). At estrous periods occurring approximately 34, 50, 74 and 107 d after primary immunization, respective ovulation rates were 157, 169, 207 and 450% of control values. Ovulation rate and antibody titer were correlated positively (pooled r = .95; P less than .01) within lambs. In Exp. 3, three lambs were immunized with .25 mg unconjugated alpha-IF; this was nonantigenic. In conclusion, the use of a synthetic fragment of the alpha-subunit of ovine inhibin as a hapten elicits an antibody capable of neutralizing inhibin bioactivity in vitro and increasing ovulation rate in vivo. PMID- 1707869 TI - Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer. AB - Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure. PMID- 1707870 TI - DNA-DNA hybridization and analysis of restriction endonuclease and rRNA gene patterns of atypical (catalase-weak/negative) Campylobacter jejuni from paediatric blood and faecal cultures. AB - Sixteen strains of atypical (catalase-weak or negative), hippurate-hydrolysing campylobacters from paediatric blood and faecal cultures were identified by DNA DNA slot hybridization. All were closely related (greater than or equal to 67%) to Campylobacter jejuni and representative strains had G + C contents of 30 +/- 1 mol%. Numerical analysis of chromosomal DNA HaeIII digest patterns revealed two clusters of strains at the 55%S level corresponding to C. jejuni subsp. jejuni and C. jejuni subsp. doylei; most strains belonged to the latter subspecies. No two strains had identical patterns but within each subspecies two subgroups were identifiable, corresponding to Lior biotypes I and II. Southern blot hybridization analysis with a 16 + 23S rRNA cistron probe from Escherichia coli also showed differences between the various strains, and in a numerical analysis three groupings were formed at 70%S corresponding to C. jejuni subsp. jejuni Lior biotypes I and II, and C. jejuni subsp. doylei. Four of the subspecies doylei strains contained a 3.4-MDa plasmid. These analyses showed that catalase-negative C. jejuni subsp. jejuni were genomically distinguishable from C. jejuni subsp. doylei as were Lior biotypes within subsp. jejuni. Ability to produce catalase is not a feature common to all C. jejuni strains, and our results confirm that some strains of subspecies jejuni may be negative in that character although typical in other respects. DNA pattern heterogeneity was consistent with serological differences between strains. PMID- 1707871 TI - Deadenylation and turnover of interferon-beta mRNA. AB - The pathway of degradation of human interferon-beta (IFN-beta) mRNA was examined in murine C127 cells that carry an expression vector for this mRNA. The IFN-beta mRNA decayed with a half-life of approximately 45 min in actinomycin D-treated cells and became gradually shorter. This mRNA was superinduced in cycloheximide treated cells, but it also became gradually shorter. However, apparently full length species of IFN-beta mRNA accumulated after prolonged incubation with cycloheximide. The shortened IFN-beta mRNA species were partially deadenylated and less stable than full-length species. These findings suggest that at least two nuclease activities are involved in degrading IFN-beta mRNA; one deadenylates this mRNA and decays in cycloheximide-treated cells, while the other apparently breaks down deadenylated mRNA. PMID- 1707872 TI - Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats. AB - Annexins are structurally related proteins that bind phospholipids in a Ca2(+) dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes. PMID- 1707873 TI - Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1). AB - Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4 expressing cell lines. PMID- 1707874 TI - Deletion mutation in an eye lens beta-crystallin. An animal model for inherited cataracts. AB - The most prevalent proteins in the lens of the eye are called crystallins, and it is thought that aberrant crystallins may cause opacification of lens tissue. The Philly mouse, a strain with an inherited cataract, has an abnormal beta B2 crystallin, the principal beta-crystallin in the mouse. The cDNA that codes for the beta B2-crystallin protein has been cloned and sequenced from both the normal and the cataractous Philly mouse. The normal mouse beta B2 cDNA is 756 nucleotides in length with 618 nucleotides of open reading frame. An in-frame deletion of 12 nucleotides has occurred in the Philly mouse cDNA, which results in the loss of 4 amino acids. The sequence of the mutant beta B2 was analyzed against the reported structure of the normal bovine beta B2-crystallin determined by x-ray crystallography. The region, in which the deletion of the amino acids occurs near the COOH terminus, is essential for the formation of the tertiary structure of the beta B2-crystallin. The loss of these residues could explain the alterations that are seen with the Philly beta B2 protein and may account for the instability of the Philly beta B2 protein. This abnormal beta B2-crystallin may be the cause of the cataract in this animal. PMID- 1707875 TI - Interaction of C-terminal sequences of human immunodeficiency virus reverse transcriptase with template primer. AB - We have raised a rabbit monospecific antibody (designated C2003) against a synthetic peptide (CTP66) derived from a conserved sequence in the C-terminal portion of the p66 component of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (DeVico, A.L., Copeland, T.D., Veronese, F.D., Oroszlan, S., Gallo, R. C., and Sarngadharan, M. G. (1989) AIDS Res. Hum. Retroviruses 5, 51-60). This antibody directly inhibits the polymerase activity of HIV-1 RT and of RTs from a variety of retroviruses. HIV-1 RT is protected from this inhibition by preincubation of the enzyme with template primer prior to treatment with the antibody. Such protection is abrogated when the pretreatment is conducted under conditions of high ionic strength. Kinetic studies showed that the antibody-mediated inhibition is competitive with respect to template primer concentration. These results indicate that C2003 antibody acts to interfere with the template binding function of the enzyme and further indicates that conserved residues recognized by the antibody may be directly involved in this function. PMID- 1707876 TI - Tyrosine phosphorylation of a common 57-kDa protein in growth factor-stimulated and -transformed cells. AB - Protein tyrosine phosphorylation was studied in macrophages and fibroblasts to identify putative components of post-receptor mitogenic pathways that might be functionally conserved in different cell types. Nondenaturing conditions were established for the approximately quantitative recovery of anti-phosphotyrosine antibody (alpha PY)-reactive proteins from cells. A common, 57-kDa alpha PY reactive protein was identified by V8 protease peptide mapping in colony stimulating factor-1 (CSF-1)- or granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated BAC1.2F5 macrophages, in platelet-derived growth factor stimulated NIH-3T3 cells, and in CSF-1-stimulated NIH-3T3 cells expressing the c fms/CSF-1 receptor. The 57-kDa protein was phosphorylated on serine and tyrosine and was the only alpha PY-reactive protein band whose phosphorylation was reproducibly increased in GM-CSF-stimulated cells. The effect of the growth factors on the tyrosine phosphorylation of the 57-kDa protein could be mimicked by treatment of the cells with orthovanadate, a phosphotyrosine protein phosphatase inhibitor. In the absence of growth factors, tyrosine phosphorylation of the 57-kDa protein was higher in v-fms or c-fms (F969, S301)-transformed NIH 3T3 cells than in untransformed NIH-3T3 (c-fms) and NIH-3T3 (c-fms, F969) cells. These data indicate that the 57-kDa protein is a common target for growth factor stimulated tyrosine phosphorylation and potentially important for growth factor mitogenic signaling. PMID- 1707877 TI - Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity. AB - The choriogonadotropin beta subunit is unique in the human glycoprotein hormone family in containing a carboxyl-terminal extension, with four sites of O glycosylation, that is not present in the other beta subunits. We have used site directed mutagenesis to define boundaries on the extent to which truncations can be made at the COOH terminus without abolishing subunit assembly and biological activity. Two COOH-terminal deletion mutant chains of human choriogonadotropin beta, des(93-145) and des(101-145), were prepared and expressed in Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The heterologous gonadotropin, bovine alpha-human choriogonadotropin des(101-145) beta, formed a heterodimer and, when assayed with transformed murine Leydig cells in vitro, competed with the binding of standard human choriogonadotropin and stimulated both cAMP and progesterone production, albeit with a reduced potency relative to bovine alpha-human choriogonadotropin beta wild type. In contrast, human choriogonadotropin des(93-145) beta, expressed under identical conditions in the presence of bovine alpha, failed to form heterodimer and thus exhibited no competitive binding and was without effect on cAMP and progesterone levels. Consequently, removal of the putative determinant loop region of the beta subunit (residues 93-100), which is believed to be important in determining receptor specificity, abolishes association with alpha. Hence, in addition to its possible role as a receptor determinant, this region of the molecule appears to be critical for proper folding or subunit interaction. The truncated form of human choriogonadotropin beta lacking residues 101-145 is the shortest form of the subunit yet described that retains biological activity. Moreover, these results demonstrate that the proposed disulfide between Cys-26 and Cys-110 is not required for subunit assembly or for receptor binding and subsequent intracellular signaling. PMID- 1707878 TI - Molecular cloning and characterization of the acidic 80-kDa protein kinase C substrate from rat brain. Identification as a glycoprotein. AB - The complete amino acid sequence of 80 K, the major acidic protein kinase C (PKC) substrate of rat brain, was deduced from a cDNA nucleotide sequence. An open reading frame of 927 bases predicted a protein of 309 amino acid residues (Mr = 29,796, pI = 4.06). 58% of the deduced protein sequence was confirmed by Edman degradation of peptides generated by proteolysis of purified 80 K. The absence of internal methionine residues in the deduced amino acid sequence was confirmed by the inability to cleave 80 K with CNBr. Antiserum raised against a synthetic peptide corresponding to residues 298-309 of the predicted amino acid sequence recognizes the 80-kDa polypeptide in Western blots. The protein shows 65% sequence identity with a closely related PKC substrate from bovine brain. Genomic Southern blot analysis using a probe corresponding to a segment of the 80 K gene devoid of introns showed one major band. Northern blot analysis of rat brain RNA reveals a prominent transcript of 2.2 kilobases which hybridizes to 80 K cDNA. The amino acid composition and hydropathicity plot suggest an extended structure with no hydrophobic domains. The amino acid sequence showed many short repeats as well as several potential phosphorylation sites, five of which were for PKC, one was for both PKC and cyclic AMP-dependent protein kinase, and one for casein kinase II, and potential glycosylation sites. Indeed, carbohydrate moieties were detected on electroblots of purified 80 K using both a specific glycan stain and Galanthus nivalis plant lectin which binds to terminal D-mannose in the glycan moiety. This is the first time that this major PKC substrate has been identified as a glycoprotein. PMID- 1707879 TI - Differential regulation of lymphotoxin and tumor necrosis factor genes in human T lymphocytes. AB - Lymphotoxin (LT) and tumor necrosis factor (TNF) are related cytokines that share many biological effects. The genes for LT and TNF are adjacent to each other on chromosome 6 in man, but previous data indicate that the kinetics of their production differ markedly. To explain the mechanisms for this difference, we compared the regulation of these two genes in human T lymphocytes, isolated from peripheral blood, after stimulation with the mitogens concanavalin A and phorbol myristate acetate. Differences in the kinetics of protein secretion were paralleled by differences in cognate mRNA accumulation. TNF mRNA accumulated rapidly after stimulation, peaked by 6 h, and returned to unstimulated (base line) levels by 24 h. In contrast, LT mRNA accumulated slowly after stimulation, usually peaked at approximately 18 h, and remained increased above base-line levels at 48-72 h. By nuclear transcription run-on assays, increased transcription of TNF mRNA and LT mRNA was demonstrated after stimulation. However, TNF transcription peaked earlier and appeared to be 4-10 times greater than that of the LT gene. In contrast, the half-life of LT mRNA was 8-10-fold longer than that of TNF mRNA as demonstrated by actinomycin D pulse-chase experiments. Cycloheximide did not block LT or TNF mRNA accumulation, indicating that new protein synthesis was not required for induction of either gene. These results suggest strongly that the LT and TNF genes are regulated differently in human T lymphocytes after mitogen stimulation. TNF mRNA accumulates rapidly primarily because of increased transcription and decreases rapidly related to its brief half-life. In contrast, LT mRNA accumulates more slowly but persists much longer; the accumulation of this mRNA appears to be controlled largely by post transcriptional mechanisms. PMID- 1707880 TI - Demonstration of alternative splicing of a pre-mRNA expressed in the blood stage form of Plasmodium falciparum. AB - By screening of a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum raised against a 41-kDa protein band, which was shown to confer protective immunity to monkeys, the phage clone 41-3 was identified. The entire 41-3 gene was isolated, and its coding regions were determined by amplification and sequencing of 41-3 specific mRNA fragments. The 41-3 gene has a complex structure consisting of nine exons, encoding 375 amino acids in total with a calculated molecular weight of 43,400. Provided that the N-terminal hydrophobic residues function as signal sequence which is cleaved off, the molecular weight of the 41-3 protein decreases to 41,200 and could therefore be considered to be a component of the protective Mr = 41,000 protein band. Indeed, a 41-kDa protein could be detected by Western blot analysis using antisera raised against different recombinant expression products of the 41-3 gene. We furthermore demonstrate an alternative splice process for the mRNA precursor transcribed from the 41-3 gene to yield at least three distinct mRNAs. The major splice product carries all exons E1 to E9, whereas at least two minor 41-3 mRNA species can be identified which show deletions in the region between exons E5 and E7. The possible role of this differential splice process for the parasite is discussed. PMID- 1707881 TI - The genomic structure of the human glucocorticoid receptor. AB - We have determined the structure of the human glucocorticoid receptor (hGR) gene after the isolation and characterization of cosmid clones mapping to discrete regions of the cDNA. The gene contains a total of 10 exons and has a minimum size of 80 kilobases. Exon 1 consists solely of 5'-untranslated sequence, and exon 2 encodes the amino-terminal portion of the receptor. The two putative zinc fingers are separately encoded by two exons, and a total of five exons combine to form the cortisol-binding domain. By restriction mapping and sequence analysis of cosmids located on the 3'-end of the gene, we have established that the two receptor isoforms, hGR alpha and hGR beta, originate from the same gene by alternative splicing. Each hGR isoform is encoded by nine exons, of which the first eight are identical, whereas the ninth exons are heterologous. Multiple GC boxes and no obvious TATA or CAAT elements have been found in the 5'-flanking region. S1 nuclease analysis yielded one major band, and the transcription start site is localized to the *C residue within TAC*CCTC. Alignment of sequences around the splice junctions of hGR with those of other members of the steroid receptor superfamily revealed three different splice positions within the DNA binding domain. This comparison also permitted the prediction of the positions of the splice sites and the sizes of the putative exons in the human mineralocorticoid receptor. PMID- 1707882 TI - Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein. AB - A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein. PMID- 1707883 TI - Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line. AB - We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites. PMID- 1707885 TI - [Biological glue and obstruction of bronchial stumps. Experimental study. Sutureless bronchial closure of lobectomies in dogs]. AB - From April, 1989 to January, 1990, we have been "inducing bronchial fistulae" in dogs by performing 10 lobectomies, 5 middle and 5 caudal, in both the right and the left lung. The bronchial stump was not sutured but closed by a "Tissucol vicryl mesk" implant, which can be adapted to the size of the stump and is fully absorbed in the long term and replaced by a natural fibrous plug. The results seem to be fairly satisfactory: no dog died; all stumps--followed up a D10, D12... up to the 11th month--were obturated without complications, at least in these animals whose bronchi were normal and not infected. Bronchial fistulae still are a serious complication of lung resections, and while the smaller fistulae (2-3 mm) can be managed with biological glue delivered by simple endoscopy, severe fistulae--especially after pneumonectomy, above all in the right lung--often demand difficult, high-risk surgery. This points out to the interest of this small experimental series. Two conditions are essential to successfully implement this alternative procedure: 1) perfect sterilization of the excision pocket; a window is often required, which in addition has the advantage of facilitating the insertion and observation of the implant; 2) a high concentration of aprotinin (10,000 units) delaying the dissolution of the glue plug, thus allowing it to be integrated by the natural healing process. PMID- 1707884 TI - Regulation of desmosome assembly in MDCK epithelial cells: coordination of membrane core and cytoplasmic plaque domain assembly at the plasma membrane. AB - Desmosomes are major components of the intercellular junctional complex in epithelia. They consist of at least eight different cytoplasmic and integral membrane proteins that are organized into two biochemically and structurally distinct domains: the cytoplasmic plaque and membrane core. We showed previously that in MDCK epithelial cells major components of the cytoplasmic plaque (desmoplakin I and II; DPI/II) and membrane core domains (desmoglein I; DGI) initially enter a pool of proteins that is soluble in buffers containing Triton X 100, and then titrate into an insoluble pool before their arrival at the plasma membrane (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685; Pasdar. M., and W. J. Nelson. 1989. J. Cell Biol. 109:163-177). We have now examined whether either the soluble or insoluble pool of these proteins represents an intracellular site for assembly and interactions between the domains before their assembly into desmosomes at the plasma membrane. Interactions between the Triton X-100-soluble pools of DPI/II and DGI were analyzed by sedimentation of extracted proteins in sucrose gradients. Results show distinct differences in the sedimentation profiles of these proteins, suggesting that they are not associated in the Triton X-100-soluble pool of proteins; this was also supported by the observation that DGI and DPI/II could not be coimmunoprecipitated in a complex with each other from sucrose gradient fractions. Immunofluorescence analysis of the insoluble pools of DPI/II and DGI, in cells in which desmosome assembly had been synchronized, showed distinct differences in the spatial distributions of these proteins. Furthermore, DPI/II and DGI were found to be associated with different elements of cytoskeleton; DPI/II were located along cytokeratin intermediate filaments, whereas DGI appeared to be associated with microtubules. The regulatory role of cytoskeletal elements in the intracellular organization and assembly of the cytoplasmic plaque and membrane core domains, and their integration into desmosomes on the plasma membrane is discussed. PMID- 1707886 TI - Gas chromatography of tryptophan together with other amino acids in hydrochloric acid hydrolysates. AB - The classical hydrolysis of proteins with hydrochloric acid using tryptamine [3 (2-aminoethyl)indole] as additive revealed that tryptophan can be measured without destruction together with other amino acids by gas chromatography. An extensive study was made to establish the optimum conditions for protein hydrolysis (time and temperature of hydrolysis, amount of tryptamine) and for the derivatization of amino acids. The amino acid contents (including tryptophan) of standard proteins such as lysozyme, bovine and human albumin, human gamma globulin, casein and alpha-chymotrypsin and protein matrices (meat and fish meals, sunflower) were determined, after hydrochloric acid hydrolysis (4 h, 145 degree C) in the presence of tryptamine. as N, O, (S)-trifluoroacetyl isobutyl esters with SE-30 as the stationary phase. The reproducibility of the measurements was 4.6% (relative standard deviation) or less. PMID- 1707887 TI - Feather keratin as a ligand in an affinity chromatographic technique for isolation of protease from Trichophyton verrucosum. AB - A technique of affinity chromatography was developed and optimized for proteases from the postculture fluid of Trichophyton verrucosum. The technique employs porous glass with adsorbed feather keratin or keratin covalently bound to the glass. Modifications of the amounts of proteases introduced into the columns and the manner of elution (pH gradient, buffer concentration, EDTA) made it possible to achieve yields of the isolated enzymes of the order of 80%. The degree of purification of four fractions isolated by the proposed technique was about 6 fold and allowed electrophoretically almost homogeneous enzymatic forms to be isolated. Substrate and inhibition tests on the four chromatographically purified proteolytic enzymes indicated a specifically keratinolytic nature of the enzymes studied. PMID- 1707888 TI - Use of chromatography for the preparation of homologous tetanus antitoxin. AB - To obtain immunoglobulin of selected action with a higher titre, techniques for isolating antibodies from a donor's normal immunoglobulin by means of affinity chromatography were used. To obtain an affinity sorbent, tetanus toxoid was used as a ligand. Using an immunosorbent based on the purified ligand, tetanus antibodies were prepared with a specific activity of 65 IU/mg. According to gel permeation chromatography, the tetanus antibodies obtained were represented mainly by IgG monomer (greater than 90%). The anticomplementary activity of the preparations obtained, being at least 11 mg of protein per 2CH50, does not rule out the possibility of intravenous administration of tetanus antibodies. PMID- 1707889 TI - Use of monoclonal antibodies in an ELISA for the diagnosis of bovine leukaemia virus infection. AB - An ELISA diagnostic test for detection of bovine leukaemia virus (BLV) infected animals was developed. The test is based on the use of a mixture of monoclonal antibodies (MAbs) against envelope glycoprotein and against viral structural protein p24. The sensitivity and specificity of the test were found to be dependent on the relative proportions of MAbs of the appropriate epitope specificity. Polystyrene microtitre plates, wells or sticks were firstly coated with a mixture of purified MAbs and then non-purified viral antigens were adsorbed from tissue culture fluid obtained from BLV-producing cells. The optimal conditions for adsorption of MAbs and viral antigens as well as for the ELISA procedure were established. The test is more sensitive and cheaper (no need for virus antigen purification) than the routinely used ELISA using purified virus antigens. The assay is highly specific, rapid, practical and could be easily automated. It is suitable for the detection of BLV-antibodies in blood serum or milk in the large-scale screening programs for BLV-infected animals. PMID- 1707890 TI - Method for improving accuracy of virus titration: standardization of plaque assay for Junin virus. AB - Titrating infective virus is one of the most important and common techniques in virology. However, after many years of widespread use, the parameters governing the accuracy of titration values are still not well understood. It was found that under conditions currently used for virus titration, only a small percentage of virus in the inoculum is adsorbed onto the cells and thereby detected in the titration assay. The objective of our work was to establish the conditions for a plaque assay which could estimate more accurately the titer of Junin virus. Two different stain methods were compared and several parameters governing plaque formation were studied. The volume of the inoculum appeared as the most important factor affecting observed titer. A linear relationship between the volume of inoculum and the reciprocal apparent titer allowed us to estimate an absolute titer by extrapolation. The approach described here is likely to be applicable to the more accurate estimation of the titer of a wide range of virus. PMID- 1707891 TI - Analysis of RNA stability and (-) strand content in viral infections using biotinylated RNA probes. AB - Non-radioactive biotinylated RNA probes specific for plus (+) and minus (-) sense RNAs of brome mosaic virus (BMV) were synthesized in vitro from a plasmid bearing a 200 base pair fragment complementary to the 3' terminus of each of the three genomic RNAs of the virus. Using virion RNAs isolated from BMV infected barley plants, the sensitivity of biotinylated RNAs as hybridization probes was compared with that of 32P-labeled probes in Northern hybridization assays. Although the sensitivity of biotinylated and 32P-labeled probes is similar (approximately 5 pg), the time required to detect the RNA bands was much less than for autoradiography; (-) sense RNAs could be detected in 30 min whereas 48 h or more were required by autoradiography. The value of biotinylated probes for following RNA stability was exemplified by the detection of supplied inocula in protoplasts 24 h post-inoculation. Quantitation of relative accumulation of progeny (+) and ( ) sense RNAs by densitometry of the Northern blots probed with biotinylated RNAs paralleled that of radiolabeled probes. The application of these probes was extended to the detection of RNAs in barley protoplasts and BMV infected plant sap by dot hybridization. In these tests, viral RNAs were detected in as few as 250 protoplasts and sap dilutions up to 1:2000. The merits of these non radioactive probes in monitoring the replication events by the detection and quantitation of mutant progeny RNAs of BMV are discussed. PMID- 1707892 TI - Detection of pome fruit viroids by enzymatic cDNA amplification. AB - We have studied the detection of apple scar skin, dapple apple, and pear rusty skin viroids in nucleic acid extracts of infected pome fruit tissues by reverse transcription-polymerase chain reaction (RT-PCR) with viroid cDNA-specific primers. Analysis of RT-PCR-amplified products by gel electrophoresis or by Southern blot hybridization indicated that the new procedure is more sensitive than existing detection methods and provides information about viroid detection without requiring large samples or using molecular hybridization. Our results also suggest the potential utility of the PCR technology in detecting other viroids and possibly other plant pathogens. PMID- 1707893 TI - Detection of an arbovirus in an invertebrate and a vertebrate host using the polymerase chain reaction. AB - The ability of the polymerase chain reaction (PCR) to diagnose an arboviral infection in an arthropod vector or a mammalian host was examined. Dugbe (DUG) viral RNA was detected in RNA extracts from infected tissue samples by reverse transcription and enzymatic amplification of the resulting cDNA using Taq DNA polymerase, followed by characterisation of the amplified product by agarose gel electrophoresis or dot-blot hybridisation. Viral RNA was detected in the organs and haemolymph of infected Amblyomma variegatum ticks, and in the brain and blood of infected mice. The PCR technique was found to be as sensitive as a plaque assay for detecting DUG virus, but not as sensitive as intracerebral inoculation of mice. The sensitivity of the technique was greatest using crude RNA extracts combined with dot-blot analysis of the resulting PCR products using a DUG specific cDNA probe. A result was obtained within 48 h using PCR whereas biological assays took at least 8 days to diagnose the virus infection. PMID- 1707894 TI - Rapid serotyping of infectious pancreatic necrosis virus by one-step enzyme linked immunosorbent assay using monoclonal antibodies. AB - A one-step ELISA has been developed for detection and serotyping of infectious pancreatic necrosis virus (IPNV) in infected cell cultures using monoclonal antibodies (mAb) raised against strains representing the Sp, Ab and VR 299 serotypes of IPNV. This assay uses a serotype-specific mAb as capture and a mAb directed against a common epitope as detector antibody. Avidin-peroxidase was employed for amplification. The assay was performed in 90 min by simultaneous incubation of the samples, the biotin labelled mAb and the avidin-peroxidase, and detected 37 ng/ml of purified virus. Serotyping of 34 isolates from different areas of Europe, Asia and America showed a total concordance with the results obtained by the neutralization assay. PMID- 1707895 TI - Ventral temporal cortex in the rat: connections of secondary auditory areas Te2 and Te3. AB - The present study in the rat deals with the hodological organization of two cytoarchitectonically distinct areas lying caudoventrally (Te2) or ventrally (Te3) to the primary auditory area (Te1). The afferent and efferent systems of connections were identified by using the properties of retrograde and anterograde transport of wheat germ agglutinin conjugated with horseradish peroxidase (WGA HRP). Large tracer deposits in the ventral temporal cortex involving Te2, Te3, and the dorsal bank of the perirhinal cortex induced a dense retrograde and anterograde pattern of labeling in the following nuclei of the medial geniculate (MG) complex: caudodorsal (MGCD), dorsal (MGD), medial (MGM), suprageniculate (SG), and peripeduncular area (PPA). The ventral nucleus (MGV) was only slightly labeled in its caudal division. Several extrageniculate structures were also labeled. Retrograde cell labeling occurred in centers giving rise to ascending systems of diffuse projections: locus coeruleus (LC), dorsal raphe nucleus (DR), and basal magnocellular nucleus (B). Slight anterograde labeling was present in the dorsal and external cortices of the inferior colliculus (IC), central gray, deep layers of the superior colliculus (SC), reticular thalamic nucleus (RT), and caudate putamen (CPU). Callosal connections were also noted with the contralateral homotopic cortex. In the cases in which there was a notable extension of the zone of diffusion of the tracer into the dorsal bank of the perirhinal cortex, a characteristic pattern of labeling in the subparafascicular, reuniens and paraventricular thalamic nuclei, mammillary complex, lateral and dorsal hypothalamic nuclei, amygdaloid complex, laterodorsal tegmental nucleus, subiculum, and retrosplenial cortex was displayed. Tracer deposits restricted to Te2 induced a dense labeling of the caudal, ventrolateral MGD, lateral PPA and, to a lesser extent, MGCD. The MGM and SG were only slightly labeled. Extrageniculate afferents essentially consist of sparse projections from LC, DR, and B, whereas efferent fibers are directed to the dorsal cortex of the IC, central gray, deep SC layers, and CPU. Callosal connections were also identified. Following tracer deposits restricted to Te3, dense labeling occurred in the MGD, mostly in its medial division, in the caudal MGM, and in the PPA. The MGCD, SG, and MGV were only sparsely labeled. Extrageniculate afferents arise from LC, DR, and B, and efferents are directed to the RT and dorsal cortex of the IC. Contralateral connections with the homotopic cortical area were also noted. Te2 and Te3 share some degree of similitude in their pattern of connections with the MG complex.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707896 TI - Light and electron microscopic evidence for a GABAergic projection from the caudal basal forebrain to the thalamic reticular nucleus in rats. AB - Neurons in the magnocellular nucleus of the caudal basal forebrain extend an axonal projection which arborizes within the reticular nucleus of the thalamus. The present study addresses the ultrastructure and neurochemistry of this projection in rats. Many labeled terminals are apparent within the thalamic reticular nucleus following Phaseolus vulgaris leucoagglutinin injections into the caudal basal nucleus; anterogradely labeled axon terminals most commonly contact both somata and dendrites of reticular nucleus neurons with symmetric membrane specializations. Thus, the majority of the labeled terminals examined contrast with choline acetyltransferase positive terminals which have been previously identified as contacting dendrites and forming asymmetric synapses within this nucleus. Many of the neurons within the caudal basal nucleus which are retrogradely labeled following tracer injections into the thalamic reticular nucleus are gamma-aminobutyric acid (GABA) immunoreactive. In addition, following injections of Phaseolus vulgaris leucoagglutinin or fluoro-ruby into the caudal basal forebrain, some of the labeled axonal swellings and boutons within the thalamic reticular nucleus also contain glutamic acid decarboxylase. These results indicate that a significant component of the projection is GABAergic. These anatomical observations suggest that the projection from the caudal basal nucleus onto the thalamic reticular nucleus could facilitate the relay of information through the dorsal thalamus by inhibiting reticular nucleus neurons, and thus, in turn, disinhibiting thalamic relay neurons. PMID- 1707897 TI - Quantitative Nissl study of the neuronal types, and recognition of cytoarchitectural subdivisions, within the rabbit periaqueductal gray. AB - A quantitative analysis of 4,621 Nissl-stained neurons within the periaqueductal gray of the rabbit found that there were four main cell types (stellate/round, ovoid, spindle, and triangular) distributed throughout this complex. Further statistical analysis on these neurons confirmed that there were morphological grounds to subdivide the periaqueductal gray into four cytologically distinct regions: ventral, lateral, dorsal, and medial. Neurons in the narrow medial zone, which completely surrounds the aqueduct, were orientated essentially parallel to the aqueduct. The majority of these neurons were small, ovoid, or spindle in shape, and highly basophilic. The cells in this region had the lowest packing density of those in any periaqueductal gray subdivision. The dorsal subdivision, a small midline region, contains the largest cells of any division and the highest packing density of glial cells. The neurons in this region show no preference for orientation, tend to be round, and are moderately basophilic. Cells in the lateral zone are also quite large and demonstrate a preferred orientation either parallel or perpendicular to the aqueduct. The average cell density within lateral PAG is considerably higher than in other regions. Most of these neurons are round or ovoid, and moderately basophilic. Neurons in the ventral zone are mainly ovoid, of medium size, highly basophilic, and lie fairly sparsely arrayed and are orientated essentially parallel to the aqueductal surface. PMID- 1707898 TI - Representation of the cecum in the lateral dorsal motor nucleus of the vagus nerve and commissural subnucleus of the nucleus tractus solitarii in rat. AB - Motor fibers of the accessory celiac and celiac vagal branches are derived from the lateral columns of the dorsal motor nucleus of the vagus nerve. These branches also contain sensory fibers that terminate within the nucleus of the tractus solitarii. This study traces the innervation of the intestines by using the tracer cholera toxin-horseradish peroxidase. In 53 rats, the tracer was injected into either the stomach, duodenum, jejunum, terminal ileum, cecum, or ascending colon. With all cecal injections, prominent retrograde labeling of cell bodies occurred bilaterally in the lateral columns of the dorsal motor nucleus of the vagus nerve above, at, and below the level of the area postrema. Dendrites of laterally positioned neurons projected medially and rostrocaudally within the dorsal motor nucleus of the vagus nerve and dorsomedially into both the medial subnucleus and parts of the commissural subnucleus of the nucleus of the tractus solitarii. Sensory terminal labeling occurred in the dorsolateral commissural subnucleus at the level of the rostral area postrema and the medial commissural subnucleus caudal to the area postrema. Additionally, there was sensory terminal labeling within a small confined area of the dorsomedial zone of the nucleus of the tractus solitarii immediately adjacent to the fourth ventricle at a level just anterior to the area postrema. Stomach injections labeled motoneurons of the medial column of the entire rostrocaudal extent of the dorsal motor nucleus of the vagus nerve and a sensory terminal field primarily in the subnucleus gelatinosus, with less intense labeling extending caudally into the medial and ventral commissural subnuclei. Dendrites of gastric motoneurons project rostrocaudally and mediolaterally within the dorsal motor nucleus of the vagus nerve and dorsolaterally within the nucleus of the tractus solitarii. They are most pronounced at the level of the rostral area postrema where many dendrites course dorsolaterally terminating primarily within the subnucleus gelatinosus. Injections of the duodenum labeled a small number of the cells within the medial aspects of the dorsal motor nucleus of the vagus nerve. Jejunal, ileal, and ascending colon injections labeled cells sparsely within the lateral aspects of the dorsal motor nucleus of the vagus nerve bilaterally. No afferent terminal labeling was evident after injection of these areas of the bowel.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707899 TI - Projection of the mammalian superior colliculus upon the dorsal lateral geniculate nucleus: organization of tectogeniculate pathways in nineteen species. AB - Anterograde and retrograde transport methods have been used to analyze the projection of the superior colliculus upon the dorsal lateral geniculate nucleus in 19 mammalian species. Our retrograde findings reveal that tectogeniculate neurons are relatively small, and lie dorsally within the superficial gray. These small tectogeniculate neurons are spatially related to a dense tier of W-cell retinal input. Our anterograde tracing results show that tectogeniculate axons are visuotopically distributed to small-celled regions of the lateral geniculate in all nineteen species. In the majority of these species, the small-celled, tectally innervated regions of the lateral geniculate lie adjacent to the optic tract and contain W-cell-like neurons. Our findings suggest that neuroanatomical demonstration of the tectogeniculate projection is a relatively simple and straightforward way of revealing regions of the lateral geniculate which contain W-cells. This is true even in species in which the lateral geniculate lacks obvious cellular laminae, and in regions of the lateral geniculate where W-cells are few in number. The present data are especially interesting in light of the cortical projections of tectally innervated, small-celled regions of the lateral geniculate to the patches or puffs within layer III of area 17. Since these regions of small-celled geniculocortical axons are co-extensive with zones ("blobs") rich in cytochrome oxidase, it might be that information carried over the tectogeniculate circuitry plays an important role in the functions of the blob system. PMID- 1707900 TI - Distribution of vasoactive intestinal peptide- and substance P-containing nerves originating from neurons of airway ganglia in cat bronchi. AB - This study examined the possibility that vasoactive intestinal peptide (VIP)- and substance P (SP)-containing nerve fibers in bronchial smooth muscle, glands, epithelium, and blood vessels originate from neurons of airway ganglia. Explants of airway walls were maintained in culture with the expectation that nerve fibers from neurons of airway ganglia would remain viable, whereas fibers originating from neurons not present in the airway walls would degenerate. Airways were dissected and placed into culture dishes containing CMRL 1066 medium for 3, 5, and 7 days. In controls (noncultured), VIP- and SP-like immunoreactivity was observed in nerve fibers associated with bronchial smooth muscle, glands, and blood vessel walls and in nerve cell bodies of airway ganglia. Nerve fibers containing SP were also observed within the bronchial epithelium. After 3, 5, and 7 days in culture, VIP- and SP-containing fibers were identified in all of the same locations except in the airway epithelium where SP-containing fibers could not be demonstrated. VIP and SP were frequently colocalized in the same nerve fibers of bronchial smooth muscle and glands in controls and cultured airways. There were no statistically significant differences in nerve fiber density for either VIP- or SP-containing fibers in bronchial smooth muscle between controlled and cultured airways. VIP concentrations in cultured airways were significantly less than in controls. The results suggest that a large proportion of VIP- and SP containing nerve fibers supplying bronchial smooth muscle, glands, and blood vessels in the airways originate from neurons of airway ganglia. PMID- 1707901 TI - Use of the perforated balloon catheter to infuse marker substances into diseased coronary artery walls after experimental postmortem angioplasty. AB - A perforated balloon catheter was used in human coronary arteries after postmortem angioplasty had been performed. The catheter used has a standard angioplasty balloon with a pattern of laser-produced holes, 25 microns in size, which generate streams of fluid under pressure. Studies of the routes by which marker substances enter diseased arterial tissue when infused by the perforated balloon after experimental angioplasty are described. A colored marker dye entered the new crevices and dissection planes created by the angioplasty, but did not extend greater than 2 cm either proximal or distal to the perfused segment. Horseradish peroxidase entered tissue not only from the lumen and adventitia as occurs with its infusion into normal tissue with the perforated balloon, but also extended from new crevices and dissection planes created by the angioplasty. Platelet aggregation, coagulation and cell proliferation, the likely causes of restenosis after angioplasty, originate in the sites of greatest tissue disruption and blood stasis. These postmortem studies suggest that active drugs are delivered to the arterial wall in a manner likely to be effective in preventing these events. PMID- 1707902 TI - Nutritional rehabilitation of developmentally disabled residents in a long-term care facility. PMID- 1707903 TI - A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures. AB - We developed a double staining technique for simultaneous demonstration of astrocytes and microglial cells in histological brain sections and cell cultures. The procedure included a histochemical stain specific for microglial cells and an immunocytochemical stain specific for astroglial cells, with postponement of the final visualization of the staining products until both reactions had been performed. First, microglial cells were specifically but invisibly labeled by histochemical reaction for nucleoside diphosphatase (NDPase). Then the astroglial cells were labeled by performing the first parts of the immunocytochemical reaction for glial fibrillary acidic protein (GFAP). Finally, in a series of intervening steps, the NDPase reaction product was visualized and stabilized by treatment with ammonium sulfide and silver nitrate, while the 1-naphthol basic dye method was used to visualize the GFAP immunoreactive product. As an end product, the NDPase-positive microglial cells were brown and the GFAP-reactive astroglial cells blue. The two types of glial cells were clearly distinguishable in vibratome sections of rat brain tissue and in primary astroglial cell cultures, and we never observed cells that stained for both NDPase and GFAP. When the GFAP antibody was replaced by the OX-42 antibody, which recognizes microglial cells and macrophages, double staining of microglial cells was observed. The staining protocol has wide applications in studies of the functional interactions between microglial and astroglial cells in the normal brain and in different pathological states with neuronal or axonal degeneration, just as it can be used for experimental studies in cell cultures. PMID- 1707904 TI - Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone. AB - Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix. PMID- 1707905 TI - Simultaneous detection of highly phosphorylated proteins and viral major DNA binding protein distribution in nuclei of adenovirus type 5-infected HeLa cells. AB - Highly phosphorylated proteins in situ in sections of Lowicryl-embedded cells are preferentially stained by bismuth, provided that the reactivity of the amino groups is blocked by glutaraldehyde fixation. This study showed that bismuth staining can be preceded by indirect immunocytochemistry using gold particles as markers. As a result, both immunostained and bismuth-stained proteins can be detected concomitantly on the same section. This was also carried out on sections of formaldehyde-fixed cells which were immunolabeled, then post-fixed with glutaraldehyde, and finally exposed to bismuth stain. These procedures were applied to sections of adenovirus Type 5-infected HeLa cells. Bismuth ions and viral anti-72 KD antibody bound concomitantly to intranuclear virus-induced single-stranded DNA (ssDNA) accumulation sites, structures in which viral replicative activity is intermittent, and also to the fibrillogranular peripheral replicative zones which surround the ssDNA accumulation sites and in which replication of viral genomes is continuous. The delicate fibrillar network enclosed within virus-induced compact rings of unknown function is slightly bismuth stained and binds few antibodies to viral 72 KD protein. Three intranuclear structures were stained exclusively with bismuth: the fibrillar component of the nucleolus, which is involved in ribosome formation; the interchromatin granules; and the virus-induced "fibrillar spots" of unknown significance. Thus, not all highly phosphorylated proteins in adenovirus-infected cells are viral 72 KD protein. In glutaraldehyde-fixed Miller spreads of nucleic acid molecules from adenovirus-infected cells, bismuth deposits occurred over unique thick filaments, the only portion of the viral deoxyribonucleoprotein molecules shown to be associated with viral 72 KD protein. In vitro studies revealed that the latter protein, known to be multiply phosphorylated, concomitantly binds anti-72 KD antibody and bismuth ions. These data have broadened the scope of the use of bismuth staining. Taken together, they indicate that in adenovirus infection highly phosphorylated proteins accumulate over intranuclear structures related to both replication of viral genomes and alteration of ribosomal metabolism. PMID- 1707906 TI - Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes. AB - To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5' cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture. PMID- 1707907 TI - Regulation of expression of CD27, a T cell-specific member of a novel family of membrane receptors. AB - CD27 belongs to a newly defined family of transmembrane R, including the nerve growth factor R, two distinct TNF R and CD40. The function of CD27 is unknown, but on the basis of structural and functional properties, we postulate that it plays a role in the events subsequent to T cell activation, possibly as a cytokine R. We have analyzed the mechanisms underlying the regulation of CD27 protein expression. Membrane expression of CD27 strongly increases after T cell activation via the TCR/CD3 complex or the CD2 molecule. In contrast, direct stimulation of protein kinase C by phorbol esters markedly down-regulates CD27 surface expression. This down-regulation most likely does not result from CD27 phosphorylation, because both anti-CD3 mAb and PMA induce hyperphosphorylation of CD27 on serine residues. Rather, membrane expression seems to be regulated primarily at the RNA level. Stimulation of T cells with anti-CD3 mAb strongly increases steady state CD27 mRNA levels, whereas PMA treatment greatly reduces these transcript levels. Dissection of the TCR/CD3-induced signaling pathways showed that cytoplasmic cAMP as well as Ca2+ concentrations contribute to the increase of CD27 expression. These data indicate that upon Ag-specific T cell stimulation, membrane expression of CD27 is regulated at the RNA level through the joint action of distinct TCR/CD3-associated signaling pathways. PMID- 1707908 TI - Specific triggering of gamma, delta T cells by K562 activates the gamma, delta T cell receptor and may regulate natural killer-like function. AB - Freshly isolated and resting gamma/delta T cell lines, although capable of lysing a variety of MHC-unrestricted targets, fail to lyse K562. Yet, the killing of K562 can be specifically induced by antibodies to CD3 or delta-chains. Although this phenomenon may be caused by redirected lysis, it also raised the possibility that K562 may possess ligands capable of specifically interacting with the gamma/delta receptor. We found that K562 specifically induced both CD3 and delta modulation as well as IL-2R expression and IL-2 production by gamma/delta cells, supporting the idea that the TCR-gamma/delta is specifically triggered by K562 cells. Moreover, although the gamma/delta cell clones lysed other target cells (e.g., Molt 4, U937, Jurkat etc.), these latter targets did not induce delta modulation or IL-2R expression. In addition, F(ab)2 anti-CD3 antibodies inhibited activated gamma/delta T cells from killing K562 but did not inhibit the lysis of the other targets. Taken together, these results suggest that gamma/delta cells lyse some targets by utilizing receptors (perhaps NK-like) distinct from the gamma/delta receptor. We also found that triggering of the gamma/delta receptor by K562 inhibited the capacity of resting gamma/delta to lyse Molt 4 cells under conditions in which the K562 cells were not lysed. These findings suggest that the gamma/delta receptor maybe directly involved in the lysis of certain targets (i.e., K562) and, importantly, may potentially regulate the function of NK-like receptors that are involved in the lysis of other targets. PMID- 1707909 TI - Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes. AB - The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist. PMID- 1707910 TI - Reconstitution of an active surface CD2 by DNA transfer in CD2-CD3+ Jurkat cells facilitates CD3-T cell receptor-mediated IL-2 production. AB - To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL 2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex. PMID- 1707911 TI - Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells. AB - Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL 2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines. PMID- 1707912 TI - Studies on the induction of anterior chamber-associated immune deviation (ACAID). 1. Evidence that an antigen-specific, ACAID-inducing, cell-associated signal exists in the peripheral blood. AB - The anterior chamber of the eye is an immunologically privileged site. Recent evidence indicates that this privilege is an actively acquired immune state in which a unique form of systemic immune deviation exists, anterior chamber associated immune deviation (ACAID). ACAID is characterized in part by the generation of Ag-specific splenic T lymphocytes that mediate suppression of induction and expression of delayed hypersensitivity and that suppress production of C-fixing antibodies. Delayed hypersensitivity and C fixation are usually associated with extensive nonspecific inflammation and innocent bystander tissue injury. It is, therefore, believed that ACAID represents physiologic adaptations of the immune system that mitigate wanton destruction of the anatomically delicate visual axis, thereby preserving sight, while at the same time providing selective immune protection. As a means of examining the potential contribution of the eye to ACAID induction, we have studied the immune properties of blood harvested from mice that had received BSA into the anterior chamber 48 h earlier. We found that an Ag-specific "signal" is present in the blood of these mice; when blood was transfused into naive syngeneic mice, regulatory cell populations were induced and the recipients were unable to display BSA-specific delayed hypersensitivity. Further analysis of this signal revealed it to be associated with the leukocyte fraction, but not with either the plasma or RBC components of whole blood. Among leukocytes, the suppression-inducing activity correlated positively with cells bearing the mature macrophage marker F4/80 and negatively with cells bearing Thy-1, surface Ig, and class II MHC molecules. These findings are discussed with regard to the potential intraocular sources of the ACAID inducing signal and the possible mode of action of this factor. PMID- 1707913 TI - Effects of transforming growth factor-beta and IL-1 alpha on prostaglandin synthesis in serum-deprived osteoblastic cells. AB - We investigated the effects that the combination of IL-1 alpha and transforming growth factor-beta (TGF-beta) had on PGE2 production in a murine clonal osteoblastic cell line MC3T3-E1 and primary rat calvarial osteoblast-like cells. In serum-supplemented medium, IL-1 alpha was a potent stimulator of PGE2 production in MC3T3-E1 cells (50-fold increase with 0.1 ng/ml). TGF-beta (10 ng/ml) had only a small effect alone and no additional effect on IL-1 alpha induced responses. In serum-deprived MC3T3-E1 cells, PGE2 responses to IL-1 alpha were either absent or markedly reduced. TGF-beta alone had small effects. However, simultaneous addition of TGF-beta with IL-1 alpha to MC3T3-E1 cells partially restored the ability of IL-1 alpha to generate a PGE2 response (10-fold increase in PGE2 with 0.1 ng/ml of both IL-1 alpha and TGF-beta). As with MC3T3 E1 cells, serum-deprived primary fetal rat calvarial osteoblastic cells also did not respond to IL-1 alpha, unless TGF-beta was present in the medium (sixfold increase in PGE2 with 0.1 ng/ml IL-1 alpha and 10 ng/ml TGF-beta). The synergistic effect of TGF-beta and IL-1 alpha was specific for PGE2 responses, because these factors did not synergistically affect cell proliferation, collagen and noncollagen protein synthesis, or alkaline phosphatase activity. The observed synergy was not associated with changes in the steady state cyclooxygenase (PGH synthase) mRNA levels. However, it did correlate with increased release of [3H]arachidonic acid from prelabeled serum-depleted MC3T3-E1 cells. Hence, the synergistic interactions of IL-1 alpha and TGF-beta on PGE2 appear to occur through an increase in the release of arachidonic acid substrate from phospholipid pools. These effects may be important for both normal bone turnover and the responses of bone to inflammatory and immune stimuli. PMID- 1707914 TI - Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system. AB - The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR. PMID- 1707915 TI - Murine IgE and IgG responses to melittin and its analogs. AB - Melittin, a bee-venom peptide of 26 amino acids, induces IgE and IgG responses in man and animals. The antibody response was shown previously to be specific primarily for the C-terminal 6 residues and its T cell epitope in H-2d restricted mice was shown to be in residue 11-19 of melittin. To study the relationship of peptide structure and immunogenicity in mice, we have prepared a series of melittin analogs varied in length and composition at the C-terminus. Immunogenicity of the analogs for IgG and IgE responses was found to correlate with two factors: a peptide length of more than 24 residues and the presence of a hydrophilic C-terminal region preferably with two to four cationic groups. These factors result in the ability of peptide to bind to cell membranes. Analogs that possess these features are good immunogens whereas those lacking any of these features are weak immunogens. PMID- 1707916 TI - Epitopes of the p70 and p80 (Ku) lupus autoantigens. AB - High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601 609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes. PMID- 1707917 TI - Regulation of Ig-induced eosinophil degranulation by adenosine 3',5'-cyclic monophosphate. AB - We have investigated the effects of cAMP on Ig-induced human eosinophil activation. Stimulation of human normodense eosinophils with IgG- or secretory IgA (sIgA)-coated Sepharose beads induced cellular degranulation, as measured by the release of the granule protein, eosinophil-derived neurotoxin (EDN). Pretreatment with cAMP analogs (N6,O2,-dibutyryl adenosine-3,':5' cyclic monophosphate; 8-bromoadenosine 3':5' cyclic monophosphate; or N6 benzoyladenosine 3':5' cyclic monophosphate) or cAMP phosphodiesterase-inhibitors (theophylline or isobutylmethyl xanthine (IBMX] strongly inhibited Ig-induced human eosinophil degranulation. The beta-adrenoceptor agonists, isoproterenol and salbutamol, induced relatively low level increases in intracellular cAMP, and weakly suppressed EDN release induced by IgG-coated beads. However, cellular pretreatment with IBMX synergistically enhanced the inhibitory effects of isoproterenol or salbutamol on both IgG and sIgA-induced eosinophil degranulation. Similarly, PGE2 treatment increased intracellular cAMP concentrations in eosinophils and correspondingly inhibited the Ig-dependent cellular degranulation response: co-incubation with IBMX further enhanced both effects of PGE2. Finally, cholera toxin, which irreversibly activates the stimulatory guanine nucleotide-binding protein linked to adenylyl cyclase, strongly inhibited the release of EDN from IgG- or sIgA-stimulated eosinophils. The time-dependent accumulation of cAMP in cholera toxin-treated cells closely paralleled the time courses of inhibition of IgG- and sIgA-induced EDN release after toxin exposure. These data indicate that the cAMP-dependent signal transduction mechanism in eosinophils exerts a negative modulatory effect on the cellular degranulation responses induced by sIgA or IgG. The inhibitory effects of cAMP on eosinophil activation may provide an important physiologic and a clinically relevant therapeutic mechanism for limiting the release of eosinophil derived cytotoxic proteins during certain allergic or inflammatory responses in vivo. PMID- 1707918 TI - Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway. AB - A molecular explanation for "suppressor" macrophage inhibition of lymphocyte proliferation is described. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of the nitric oxide synthetase pathway, markedly augments Con A-induced proliferation of rat splenic leukocytes. Macrophages are necessary and sufficient for NGMMA-releasable-suppression, as indicated by a loss of suppression after either pretreatment of isolated splenic macrophages with NGMMA or their depletion by plastic adherence or L-leucine methyl ester. L- (but not D-) arginine overrides NGMMA-releasable suppression, and suppression is blocked by RBC as would be expected if nitric oxide were the effector molecule. Unlike rats, NGMMA did not augment Con A-induced proliferation of normal mouse splenic leukocytes. However, NGMMA did augment Con A-induced proliferation of mouse splenic leukocytes induced to contain suppressor macrophages by intravenous injection of Corynebacterium parvum, which suggests a quantitative, not qualitative, difference in suppressor macrophages between rats and mice. Nitrite production, as an indicator of nitric oxide synthesis, correlated with suppressor macrophage activity in rats and mice and was inhibited by NGMMA. Finally, NGMMA also markedly enhanced proliferation with every other mitogen examined (PHA, protein A, PWM, and LPS). It is concluded that immunoregulation of lymphocyte proliferation by suppressor macrophages is mediated, in part, directly or indirectly by products of the nitric oxide synthetase pathway. PMID- 1707919 TI - Dexamethasone selectively modulates basal and lipopolysaccharide-induced metalloproteinase and tissue inhibitor of metalloproteinase production by human alveolar macrophages. AB - To define the capacity of glucocorticoids to regulate tissue damage associated with inflammation more clearly, we have studied the effects of dexamethasone on human alveolar macrophage secretion of both a variety of metalloproteinases and also the counter-regulatory tissue inhibitor of metalloproteinases (TIMP). We found that dexamethasone selectively and coordinately inhibited expression of the following human metalloproteinases: interstitial collagenase, stromelysin, and the 92-kDa type IV collagenase, as well as TIMP. Both basal and LPS-stimulated cells exhibited similar degrees of inhibition, with greater than 50% decrease in secretion of all enzymes and TIMP observed at dexamethasone concentrations of greater than or equal to 10(-8) M in serum-containing medium. The effects of dexamethasone were mediated at a pretranslational level. In summary, our results indicate that glucocorticoids suppress the matrix-degrading phenotype that is characteristic of mature human mononuclear phagocytes, and block the effects of the most potent known signal for upregulation of metalloproteinase secretion. Similar actions in vivo would serve to limit tissue damage associated with the inflammatory response. PMID- 1707920 TI - Leishmania donovani lipophosphoglycan selectively inhibits signal transduction in macrophages. AB - The effect of purified lipophosphoglycan (LPG) of Leishmania donovani on signal transduction and gene expression in murine bone marrow-derived macrophages was investigated. LPG stimulated the rapid expression of both c-fos and TNF genes within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. Macrophages pretreated with LPG for 3 h became unresponsive to subsequent stimulation with LPS and the activators of protein kinase C, 1-oleoyl-2-acetyl-glycerol, and calcium ionophore A23187. Moreover, LPG induced a rapid down-modulation of TNF receptors. In contrast, the ability of macrophages to express the c-fos gene in response to the cAMP analogue, dibutyryl cAMP, was not impaired by LPG. Fragmentation of LPG revealed that the inhibitory activity of LPG required both the repeating phosphorylated disaccharides and the phosphosaccharide core. Collectively, these data demonstrate that LPG selectively impaired signal transduction in macrophages and suggest a role for this molecule in the survival of the parasite within the macrophage. PMID- 1707921 TI - Early appearing gamma/delta-bearing T cells during infection with Calmette Guerin bacillus. AB - To search for a potential role of TCR gamma/delta T cells in host-defense against mycobacterial infection, we analyzed the kinetics, repertoire, specificity, and cytokine production of gamma/delta T cells in the peritoneal exudate cells (PEC), lymph node (LN) cells and spleen cells during an i.p. infection with a sublethal dose (5 x 10(5) of viable Bacillus Calmette-Guerin (BCG) in mice. In the PEC on day 7 after infection, approximately 26% of the CD3+ cells were CD4-CD8-, most of which expressed TCR gamma/delta on their surface. However, the PEC on day 28 contained an increased number of alpha/beta T cells that were CD4+8- or CD4-8+ and the proportion of gamma/delta T cells in the PEC reciprocally decreased to 18% of the CD3+ cells. The kinetics of gamma/delta and alpha/beta T cells in the LN during BCG infection showed in much the same pattern as that seen in the PEC. When purified CD4-CD8- cells in the LN on day 7 after BCG infection were cultured with sonicated BCG lysate, PPD derived from Mycobacterium tuberculosis or recombinant 65 kDa heat shock protein derived from Mycobacterium bovis, the gamma/delta T cells on this stage significantly proliferated and secreted IL-2 in response to sonicated BCG lysate and PPD but not to 65 kDa heat shock protein. V gene segment usage analysis with PCR method revealed that purified protein derivative-reactive gamma/delta T cells preferentially used V gamma 1/2/V delta 6, whereas gamma/delta T cells polyclonally expanded in response to the BCG lysate. These results suggest that gamma/delta T cells specific for mycobacterial antigens preceding alpha/beta T cells in appearance during infection may serve as a first line of defense against mycobacterial infection. PMID- 1707922 TI - Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. AB - The human pathogen CMV, is a major cause of morbidity and mortality in immunocompromised hosts. The CD8+ class I-restricted CTL response to CMV assists in preventing progression of CMV infection to life-threatening disease; however, the viral Ag recognized by CD8+ CTL are not well characterized. In general, virus specific CTL recognize endogenously synthesized viral proteins processed and presented associated with class I MHC molecules. Although proteins or inactivated virions have been experimentally delivered to the cytoplasm to result in class I MHC presentation, this mode of Ag delivery to the class I processing pathway after natural viral entry has not been documented in humans. Our data demonstrate that the CMV-specific class I-restricted CTL response in individuals latently infected with CMV is predominantly specific for selected structural virion proteins introduced into the cell after viral penetration and efficient recognition occurs in the absence of de novo viral gene expression. This CTL response may provide a biological advantage for limiting the spread of infection after CMV reactivation because infected cells are lysed before viral assembly. PMID- 1707923 TI - Phorbol esters down-regulate transcription and translation of the CD4 gene. AB - This study investigated the effects of PMA on biosynthesis and transcription of the CD4 molecule and gene in order to define mechanisms resulting in reduced cell surface expression of the CD4 molecule after treatment with PMA. Cells treated with PMA showed reduced biosynthesis of the CD4 molecule but not of class I HLA molecules. Furthermore, PMA treatment resulted in reduced steady-state levels of CD4 mRNA and inhibition of the relative rate of transcription of the CD4 gene. Cells expressing transfected CD4 cDNA gene products modulated in response to PMA, however, re-expressed CD4 earlier than cells expressing the product of the wild type CD4 gene. These data suggest that the cell surface expression of the CD4 molecule is probably down-regulated at the level of the protein, as well as the gene, and that inhibition of transcription may affect the kinetics of CD4 expression. These observations provide further insight into the mechanisms by which HIV affects expression of CD4. PMID- 1707924 TI - Signal transduction through the T cell antigen receptor. Activation of phospholipase C through a G protein-independent coupling mechanism. AB - The T cell Ag (Ti-CD3) receptor complex has been proposed to regulate phosphoinositide-specific phospholipase C (PLC) through a cholera toxin (CTX) sensitive guanine nucleotide-binding (G) protein. In this study, we have used CTX and staurosporine as pharmacologic probes to further define the linkage between the Ti-CD3 receptor and PLC activity in the human T cell line, Jurkat. CTX pretreatment inhibited Ti-CD3 receptor-dependent phosphoinositide hydrolysis and, concomitantly, protein tyrosine kinase activation in intact cells. Studies with electrically permeabilized Jurkat cells revealed that guanosine 5'-(3-O-thio) triphosphate stimulated an increase in PLC activity, that unlike the response to Ti-CD3 receptor ligation, was not affected by cellular pretreatment with CTX. In contrast, the phosphotyrosine phosphatase inhibitors, orthovanadate and molybdate anions, stimulated phosphoinositide hydrolysis in permeabilized cells through a CTX-sensitive mechanism of PLC activation. Additional studies with a known PTK inhibitor, staurosporine, supported the results obtained with CTX. Staurosporine pretreatment inhibited the phosphoinositide hydrolysis induced by anti-CD3 antibodies or phosphotyrosine phosphatase inhibitors, but failed to alter the G protein-dependent PLC activation response to guanosine 5'-(3-O-thio) triphosphate. The results of this study indicate that PLC activity(s) in Jurkat cells are regulated by both G protein- and PTK-dependent coupling mechanisms. However, the differential inhibitory effects of CTX and staurosporine on these PLC activation pathways strongly suggest that a protein tyrosine kinase activation event, rather than a G protein, mediates the functional linkage between the Ti-CD3 receptor and PLC activity in Jurkat cells. PMID- 1707925 TI - A T helper cell hybridoma produces an antigen-specific regulatory activity. Relationship to the T cell receptor by serology and antigenic fine specificity. AB - We have previously shown that a T cell hybridoma, A1.1, constitutively produces an Ag-specific regulatory factor with specificity for poly-18, a synthetic polypeptide. This cell also responds to poly-18 plus I-Ad by producing lymphokines. The antigenic specificity of the factor and the T cell appeared to be the same. This suggested the possibility that some part of the TCR, responsible for antigenic specificity of the cell, also imparts specificity to the A1.1-derived factor. This was supported by the observation that the factor was bound and eluted from a monospecific anti-TCR antiserum. Further, we demonstrated that antisense oligodeoxynucleotides corresponding to the TCR V alpha of A1.1 (but not TCR V beta) block production of the Ag-specific factor. Herein, we report recent findings that strengthen the proposed relationship between the TCR and the A1.1-derived factor. The factor was bound and eluted from a monoclonal anti-TCR C alpha antibody, but not from anti-TCR beta, anti-V beta 6, nor anti-CD3 epsilon. The anti-TCR C alpha antibody bound a Mr 46-kDa protein from A1.1 supernatants, which is the same apparent size at which activity could be eluted from an SDS-PAGE gel separation of concentrated factor. Antigenic fine specificity analysis revealed that two amino acids in poly-18 are critical for the recognition of the antigen by the Ag-specific factor. These two amino acids appear to be those recognized by the TCR. The factor that was bound and eluted from the monoclonal anti-TCR C alpha showed this fine-specificity as well. This, combined with our earlier studies, supports the view that the A1.1-derived factor is encoded, at least in part, by TCR-alpha. PMID- 1707926 TI - Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I. AB - Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation. PMID- 1707927 TI - Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes. AB - C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146 162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility. PMID- 1707928 TI - Analysis of the pivotal residues of the immunodominant and highly uveitogenic determinant of interphotoreceptor retinoid-binding protein. AB - Interphotoreceptor retinoid-binding protein (IRBP), a retinal-specific Ag, induces experimental autoimmune uveitis in a variety of animals. We have previously shown that sequence 1169-1191 of bovine IRBP is the immunodominant epitope of this protein in Lewis rats and is highly immunogenic and uveitogenic in these rats. The active site of peptide 1169-1191 was determined by testing its truncated forms. The shortest peptide to be immunologically active was found to be 1182-1190 (WEGVGVVPD). To determine the role of individual residues of this sequence, we have tested the immunologic activities of nine analogs of peptide 1181-1191, in which each of residues 1182-1190 was substituted with alanine (A). The tested activities included the capacity to induce experimental autoimmune uveitis and cellular responses in immunized rats, as well as the capability to stimulate lymphocytes sensitized against IRBP or the parent peptide 1181-1191. Analogs that did not stimulate these lymphocytes were also tested for their capacity to competitively inhibit the proliferative response to 1181-1191. Analogs A(1184), A(1186), and A(1187) resembled 1181-1191 in their activities, whereas the other analogs exhibited remarkably reduced activities, with several patterns being noticed. Analog A(1182) was inactive in all tests. Analog A(1190) was very weakly uveitogenic and non-immunogenic, but it stimulated lymphocytes sensitized against IRBP or 1181-1191 when added at exceedingly high concentrations. Analogs A(1183) and A(1185) resembled A(1190) in being weakly uveitogenic and A(1185) was also found to be poorly immunogenic. In addition, relatively high concentrations of A(1183) and A(1185) were needed to stimulate lymphocytes sensitized against IRBP or 1181-1191. However, a different pattern of activities was exhibited by analogs A(1188) and A(1189). These peptides were uveitogenic and immunogenic, but failed to stimulate lymphocytes sensitized to IRBP or 1181-1191. Furthermore, A(1188) and A(1189), but not A(1182), also inhibited the response to 1181-1191 of a cell line specific toward this parent peptide. The data are interpreted to show that residues 1188 and 1189 are involved in the interaction of the peptide with the TCR, whereas residues 1182 and 1190 and, perhaps, 1183 and 1185, are pivotal for the binding of peptide 1181 1190 to the MHC molecules on APC. PMID- 1707929 TI - Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1. AB - Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system. The effect of the immunosuppressive molecule transforming growth factor-beta, (TGF-beta 1) on chronic relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was studied. TGF-beta 1 markedly inhibited the activation and proliferation of myelin-basic protein-specific lymph node cells in vitro. This reduced the capacity of these cells to transfer EAE. In addition, administration of TGF-beta 1 in vivo consistently resulted in an improved clinical course, even when given during ongoing disease. Immunopathologic study demonstrated a marked reduction in central nervous system damage and expression of cell-surface lymphocyte function-associated Ag-1 and class II MHC molecules in TGF-beta 1-treated mice. These findings have identified TGF-beta 1 as a possible therapeutic agent for the human demyelinating disease multiple sclerosis. PMID- 1707930 TI - Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines. AB - We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL 6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression. PMID- 1707931 TI - Involvement of the 55- and 75-kDa tumor necrosis factor receptors in the generation of lymphokine-activated killer cell activity and proliferation of natural killer cells. AB - In this study we investigated the expression of the 55 kDa (p55) and the 75 kDa (p75) TNF receptors in CD56+ NK cells and their role in NK and lymphokine activated killer cells cell functions. By using mAb against the p55 and p75 TNF R, NK cells were found to express both p55 and p75 upon activation, and both receptors were involved in the generation of lymphokine-activated killer cells activity. Proliferative activity of IL-2 stimulated NK cells was inhibited by anti-TNF-alpha mAb, indicating that endogenously produced TNF-alpha is important for optimal proliferation of NK cells. Furthermore, addition of rTNF-alpha increased the IL-2-induced proliferation of NK cells. mAb to p55 and p75 inhibited the IL-2-induced proliferation indicating that both TNF-R are involved in mediating this effect. PMID- 1707932 TI - Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation. Studies with phorbol myristate acetate and staurosporine. AB - We have investigated whether TNF-induced changes in human endothelial cell (EC) surface Ag expression are mediated by protein kinase C (PKC). This suggestion arose from the observations that PMA, a potent PKC activator, can mimic TNF by inducing expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), and class I MHC molecules on human EC. However, in contrast to the actions of PMA, TNF neither causes membrane translocation of PKC nor induces the phosphorylation of the myristoylated alanine-rich C kinase substrate, two measures of PKC activation. Moreover, the PKC inhibitor staurosporine can block PMA-induced endothelial leukocyte adhesion molecule 1 expression at 4 h, but does not inhibit the actions of TNF. At 24 h, staurosporine itself induces intercellular adhesion molecule 1 and class I MHC, and acts additively with TNF. Twenty four hour treatment with PMA causes loss of PKC. We propose that at 24 h, staurosporine and PMA share a mechanism of action, namely diminution of PKC activity. However, 24 h treatment with TNF does not reduce the amount of PKC nor does it prevent activation of PKC by PMA. We conclude that TNF effects in EC are not mediated by PKC activation or inactivation. PMID- 1707933 TI - Conformational structure of a monoclonal anti-idiotypic antibody to the monoclonal anti-adenocarcinoma-associated carbohydrate antibody YH206. AB - The mAb AI206 (IgG1) is an anti-Id antibody of mAb YH206 (IgM) to adenocarcinoma associated carbohydrate Ag and inhibits the reaction of mAb YH206 to YH206 Ag at low concentrations. By Western blot analysis, mAb AI206 only reacted with unreduced mAb YH206, whereas it did not react with reduced mAb YH206. Furthermore, mAb AI206 reacted with IgM subunit (180 kDa), F(ab')2 (110 kDa), and F(ab) (50 kDa) of pepsin-treated unreduced mAb YH206. Thus, mAb AI206 recognized the structure of F(ab) of mAb YH206. The mAb YH206 reacted with unreduced mAb AI206, F(ab')2 (110 kDa), and F(ab) (50 kDa) of pepsin-treated unreduced mAb AI206. It is presumed that mAb YH206 and mAb AI206 recognize each other in an unreduced condition but not a reduced condition. The recognition of such a conformational Id on F(ab) is important. Because mAb YH206 recognized the carbohydrate on YH206 Ag as well as the peptide on mAb AI206, the conformation on F(ab) of mAb AI206 may mimic the carbohydrate structure on YH206 Ag. In fact, YH206 antibody activity was induced in syngeneic mouse serum immunized with mAb AI206. These observations suggest that the internal image of YH206 carbohydrate Ag is preserved within the conformational Id on F(ab) of mAb AI206. PMID- 1707934 TI - CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen. AB - p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97 specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response. PMID- 1707935 TI - [Prostatic adenocarcinoma: pathological stage C. I: Diagnosis, technical aspects and significance]. PMID- 1707936 TI - The interferon-gamma receptor: a comparison with other cytokine receptors. AB - During the past few years, identification of cytokine receptors has become a major goal in cytokine research. A great deal has been learned from the plethora of receptor structures that have been elucidated recently. Although, evidently, the complexity of the cytokine network extends to the signaling pathways involved, some of these pathways have now become more accessible. The challenge in the coming years will be to fill the gap between the receptors and the gene regulatory events induced by the various cytokines. Another challenge lies in the potential use of cytokine receptors as targets for modulating cytokine action. Since the topic of interferon (IFN) receptors has been reviewed recently, this minireview will focus on the current knowledge on the IFN-gamma receptor in the context of recent advances on other cytokine receptors. Some new receptor models that may be of consequence for characterizing IFN receptors will be presented briefly. PMID- 1707937 TI - The binding of the 69- and 100-kD forms of 2',5'-oligoadenylate synthetase to different polynucleotides. AB - Three major forms of 2',5' oligoadenylate (2-5A) synthetase are induced in interferon (IFN)-treated human cells: 40-46, 69, and 100 kD. Here we studied the binding and activation of the 69- and 100-kD forms to single-stranded (ss) or double-stranded (ds) RNAs. The 69- and 100-kD form enzymes purified by immunoaffinity chromatography, were shown to be activated by synthetic dsRNAs poly(I).poly(C) or poly(A).poly(U) whereas ssRNAs poly(I), poly(C), poly(A), poly(U), and poly(G) had no effect. Both enzymes were also partially purified by binding to dsRNA or ssRNA-Sepharose. The synthetases bound to dsRNA Sepharose were partially activated but required the addition of soluble poly(I).poly(C) for maximal activity. The synthetases bound to ssRNA-Sepharose manifested no activity but became activated in the presence of soluble dsRNA, poly(I).poly(C). However, activation of such ssRNA-bound enzymes by dsRNA did not result in their dissociation from the ssRNA-Sepharose. These results indicate the presence of different polynucleotide binding sites on the 69- and 100-kD forms of 2-5A synthetase: a specific dsRNA binding site essential for activation and another polynucleotide binding site or sites which, although not specific, might be important for the optimal conformation of these proteins in cells. PMID- 1707938 TI - The effect of interferon treatment on 14 human colorectal cancer cell lines: growth and carcinoembryonic antigen secretion in vitro. AB - The effect of interferons (IFNs) on growth inhibition and carcinoembryonic antigen (CEA) secretion by 14 established human colorectal carcinoma cell lines was studied in vitro. The cell lines were characterized by morphologic differentiation, level of CEA production, and rate of growth. All cell lines were treated in vitro with recombinant human IFN (alpha, beta, and gamma) and the effect of treatment on growth rate and CEA secretion determined. Each cell line exhibited an individual pattern of growth inhibition that was independent of degree of differentiation, level of CEA production, and rate of growth. IFN-beta treatment did not increase CEA secretion in any of the cell lines studied. IFN alpha and IFN-gamma resulted in increased CEA production (2- to 81-fold increase) primarily in the moderately to well-differentiated cell lines. IFN-gamma was a more potent inducer of enhanced CEA secretion than IFN-alpha. The more poorly differentiated cell lines did not produce CEA and could not be induced to do so by any of the IFNs. PMID- 1707939 TI - The effects of various cytokines on interleukin-6 and interferon-alpha synthesis in human peripheral blood mononuclear cells. AB - The effect of various recombinant cytokines on the induction of interleukin-6 (IL 6) synthesis induced in adherent and nonadherent cells of human peripheral blood mononuclear cells (PBMNC) by bacterial lipopolysaccharide (LPS) or concanavalin A (CA) was studied. The results showed that human interferon-(HuIFN)-alpha, -beta, and gamma at a concentration of 100-10,000 IU/ml enhanced the LPS-induced IL-6 production in the adherent cell fraction of PBMNC. However, in nonadherent cells, treatment with HuIFN-alpha or -beta inhibited the CA-stimulated IL-6 production in a dose-dependent manner. Recombinant (r) IL-2 enhanced the IL-6 production of the adherent cells, while rIL-1 alone in the absence of other inducer induced IL 6 production in the nonadherent cell fraction. Other cytokines such as the recombinant tumor necrosis factor-alpha (rTNF-alpha) or rIL-6 itself did not modulate IL-6 production in human PBMNC. TNF and the interleukins studied did not affect the Sendai virus-induced IFN production in the adherent cells. In contrast, the different IFNs exerted a significant priming effect. PMID- 1707940 TI - Effect of demineralized bone powder on osteoblast-like cells in culture. A potential rapid quality control assay. AB - Demineralized bone powder (DBP) has been shown to induce osteogenesis in a variety of bone defects and extra-osseous sites. Previous investigations have been carried out in animal models which are time-consuming and expensive. We studied the effect of DBP on well-established populations of osteoblast and non osteoblast-like cells in culture to establish an inexpensive, efficient and reliable assay for bone induction. DBP and BP (non-demineralized powder), of particle size 38-53 microns, were prepared from rat long bones. ROS (rat osteosarcoma) 17/2.8 and ROS 24/1 cell lines were subcultured weekly. For both 17/2.8 (well differentiated) and 24/1 (poorly differentiated) cells, proliferation, i.e. cell count, was significantly greater in DBP enriched medium when compared with control or medium with BP. Cell counts for wells with BP were no different from controls. The increased cell count in DBP-enriched medium was significant on days 2-5 (peak effect 2-3 days). Alkaline phosphatase production reached peak levels after day 3 when proliferation was beginning to taper off. In this study a consistent increase in osteoblast proliferation and alkaline phosphatase production under the influence of DBP was demonstrated. The tissue culture assay for proliferation must now be correlated with bone induction in vivo. In future, the method may be useful for investigating the mechanism of bone induction. PMID- 1707941 TI - [Molecular structure and function of Clostridium botulinum neurotoxin]. PMID- 1707942 TI - [Pathogenesis induced by bacterial proteinases--versatile pathological mechanisms at molecular level]. PMID- 1707943 TI - [DNA topology and regulation of transcription in eukaryotes]. PMID- 1707944 TI - Electrolyte transport through a cation-selective ion channel in large intestinal enterocytes of Xenopus laevis. AB - Electrogenic ion transport through the colon epithelium of the African clawed toad (Xenopus laevis) was investigated with electrophysiological methods in vitro. Interest was focused on a previously described phenomenon, that removal of Ca2+ from the mucosal Ringer's solution increases electrogenic sodium absorption. Our results clearly show that Ca2+ removal reveals an apical ion channel that is not a specific Na+ channel, but a non-selective cation channel with an 'apparent' ion selectivity of the order K+ greater than Na+ = Rb+ greater than Cs+ greater than Li+. This Ca2(+)-sensitive current increased linearly with the mucosal pH, and could be inhibited by other divalent cations (Mg2+, Ba2+) and the organic ion channel blockers quinidine and verapamil. The mucosal Ca2+ concentration that induced a half-maximal inhibition of the Ca2(+)-sensitive current was about 1 mumol l-1 and was independent of the mucosal pH. Owing to the high Ca2+ sensitivity, a regulation of the channel conductivity by extracellular Ca2+ is ruled out. It is concluded that this channel, which is almost identical to similar channels found in amphibian skin and bladder, acts as a pathway for cation absorbing or secreting processes. Possibly the binding of extracellular Ca2+ is related to selectivity changes of the Ca2(+)-sensitive ion channel. PMID- 1707945 TI - The development of specific rRNA-derived oligonucleotide probes for Haemophilus ducreyi, the causative agent of chancroid. AB - Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family. PMID- 1707946 TI - Detection of phocid distemper virus RNA in seal tissues using slot hybridization and the polymerase chain reaction amplification assay: genetic evidence that the virus is distinct from canine distemper virus. AB - Slot hybridization and the polymerase chain reaction (PCR) after reverse transcription (RT) were used to detect RNA extracted from tissues of seals after naturally occurring disease and experimental infection with phocid distemper virus (PDV). A phosphoprotein (P) gene-specific cDNA served as a probe for both slot hybridization and the identification of PCR-generated fragments by Southern blotting. As primers for the PCR assay PDV P gene-derived oligonucleotides were used. Hybridization, PCR and partial nucleic acid sequence analysis clearly demonstrated that PDV is a distinct virus (most closely related to canine distemper virus) within the morbillivirus group. PCR, when combined with Southern blot hybridization, was clearly superior to slot hybridization and more sensitive than cell culture isolation and immunofluorescence assays for the detection of virus in tissues. Considerable amounts of viral RNA could be demonstrated in the lungs and spleens. In experimentally infected animals a large quantity of virus specific RNA was additionally found in colon samples. Using RT-PCR in combination with Southern blotting. PDV could be demonstrated in buffy coat cells using a simple and fast cell lysis procedure. PMID- 1707947 TI - Breakdown of the blood-brain barrier during dengue virus infection of mice. AB - A breakdown of the blood-brain barrier occurred in mice inoculated intracerebrally (i.c.) or intraperitoneally (i.p.) with dengue virus type 2 (DEN2). This resulted in leakage of protein-bound Evans blue dye and 51Cr labelled erythrocytes into the brain tissue. The leakage increased with time after infection and coincided with an increase of a DEN2-induced cytokine, the cytotoxic factor (CF), in the spleens of such mice. The titres of virus in the brain increased exponentially in i.c. inoculated mice but the virus was not detected in brains of mice given DEN2 by the i.p. route. Similar breakdown of the blood-brain barrier also occurred in mice inoculated intravenously with CF; the damage was dose-dependent and the vascular integrity was restored during the 3 h period after inoculation. Treatment of mice with antihistamine drugs, blocking H1 or H2 receptors, decreased the DEN2-induced protein leakage by up to 50% in i.c. inoculated mice and up to 92% in those inoculated i.p. Indomethacin, a prostaglandin synthetase inhibitor, had no effect. In i.c. inoculated mice protein leakage was inhibited by about 60% by treatment with CF-specific (CFA) or DEN2-specific antisera (DEN2A) whereas protection was complete with the combined treatment with both antisera. On the other hand, in i.p. inoculated mice the inhibition of protein leakage was 80 to 89% with CFA. These findings show a breakdown of the blood-brain barrier leading to cerebral oedema during DEN2 infection which is mediated via the release of histamine by a virus-induced cytokine. PMID- 1707948 TI - Studies on glycoprotein 13 (gp13) of equid herpesvirus 1 using affinity-purified gp13, glycoprotein-specific monoclonal antibodies and synthetic peptides in a hamster model. AB - Hamsters were immunized with either an affinity-purified preparation of equid herpesvirus 1 (EHV-1) glycoprotein 13 (gp13) or synthetic peptides representing three sequences within the homologous glycoprotein of EHV-4, resulting in the production of anti-peptide (in the case of peptide-immunized animals) or antivirus antibodies. The sera from gp13-immunized hamsters contained antibodies which showed virus-neutralizing activity and complement-mediated antibody lysis of EHV-1-infected target cells. These hamsters were protected from EHV-1 challenge. The characteristics of a panel of anti-gp13 monoclonal antibodies (P28, P17, 14H7, 16E4 and 16H9) were assessed both in vivo and in vitro. 16E4 and P28 showed high levels of complement-mediated neutralization of virus, complement mediated lysis of virus-infected target cells and passive protection of hamsters. Furthermore, epitope mapping studies demonstrated that this glycoprotein contains a neutralizing epitope recognized by EHV-1-immune horse serum. The data imply that gp13 has potential as a candidate antigen for a molecular vaccine. PMID- 1707949 TI - Analysis of neutralization-escape mutants selected from a mouse virulent type 1/type 2 chimeric poliovirus: identification of a type 1 poliovirus with antigenic site 1 deleted. AB - A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neurovirulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice. PMID- 1707950 TI - Protection of mice by a protease inhibitor, aprotinin, against lethal Sendai virus pneumonia. AB - Proteolytic activation of Sendai virus in the lungs of mice is necessary to cause pneumopathogenicity. Using Sendai virus-infected lung block cultures, protease inhibitors were tested for their antiviral effect by examining inhibition of proteolytic activation. Among the inhibitors tested, a serine protease, aprotinin, was shown to be most effective. In vivo protection experiments demonstrated that aprotinin, when administered intranasally, could confer protection on mice against lethal Sendai virus pneumonia through the same mechanism as observed in the in vitro system. The present study provides an experimental basis for the use of protease inhibitors as antiviral drugs. PMID- 1707951 TI - The nucleoproteins of human parainfluenza virus type 1 and Sendai virus share amino acid sequences and antigenic and structural determinants. AB - The complete nucleotide sequence of the nucleoprotein (NP) gene of human parainfluenza virus type 1 (hPIV-1) was determined from a cDNA clone of mRNA. The mRNA is 1683 nucleotides long (excluding polyadenylic acid) and encodes a protein of 524 amino acids with a predicted Mr of 57,548. An amino acid identity of 83% was predicted between the NPs of the human pathogen hPIV-1 and the murine paramyxovirus, Sendai virus, compared to 72% similarity at the level of the nucleotide sequence. In contrast, the amino acid sequence identity between the NPs of hPIV-1 and hPIV-3 was 59%, suggesting a more distant evolutionary relationship. The NP amino acid sequences of hPIV-1 and Sendai virus were highly conserved in the amino-terminal half of the molecule, in which 395 of the first 420 amino acids were identical. Of 11 monoclonal antibodies (MAbs) targeted against the Sendai virus NP, five cross-reacted with the hPIV-1 NP. The MAbs that cross-reacted recognize epitopes within regions of high amino acid similarity between the NPs of the two viruses. Also, five of the eight MAbs raised against hPIV-1 NP cross-reacted with Sendai virus NP. Taken together, our observations suggest that the essential amino acid sequence determinants of the NP structures of hPIV-1 and Sendai virus are conserved despite changes in their nucleotide sequences during evolution. This implies that there was a selective pressure to maintain the important functional domains of the protein. PMID- 1707952 TI - Hepatitis C virus seroprevalence in Italian haemophiliacs injected with virus inactivated concentrates: five year follow-up and correlation with antibodies to other viruses. AB - The overall prevalence of anti-HCV antibody in a group of 125 haemophiliacs was 62%. Four patients who had never received replacement therapy were anti-HCV negative. Of the 121 patients injected regularly with commercial concentrates, 76 were already anti-HCV seropositive in 1985 and remained so throughout the follow up. Two patients seroconverted in 1987 without obvious signs or symptoms of hepatitis. Our patients were treated with dry heat-treated concentrates since 1985 and with wet heat- or solvent/detergent-treated concentrates since 1988. The absence of further seroconversions and of symptoms of acute post-transfusion non A, non-B hepatitis since 1988 suggest that present virucidal treatments of concentrates are effective in preventing HCV transmission. Anti-HCV positivity appeared to be unrelated to the type and degree of haemophilia as well as to the presence of antibodies to hepatitis B virus, human immunodeficiency virus type 1, and human herpesvirus type 6. PMID- 1707953 TI - Detection of myelin basic protein-like material in cerebrospinal fluid of multiple sclerosis patients by immunoblot assay. AB - Myelin basic protein (MBP)-like material in 15 cerebrospinal fluid (CSF) samples from patients with multiple sclerosis (MS) was analyzed by isoelectric focusing (IEF) followed by immunoblot assay using rabbit antiserum against human MBP- peptide 69-89, which contains the dominant epitope for MBP-like material. Samples from seven of 10 MS patients with disease in the exacerbation stage showed one band and in three other samples, a number of faint bands also appeared in the alkaline pH region in addition to the one band. CSF from five MS patients whose disease was in remission showed no detectable bands. Our results are consistent with those obtained by quantitative assay, reported in the literature. PMID- 1707954 TI - Low-dose ACOP-B and VABE: weekly chemotherapy for elderly patients with advanced stage diffuse large-cell lymphoma. AB - Elderly patients with advanced-stage diffuse large-cell lymphomas (DLCLs) are either excluded from or under-represented in most clinical trials of combination chemotherapy regimens because they tolerate treatment poorly and usually have a worse outcome. We report two brief weekly chemotherapy regimens designed specifically for elderly patients. Eligible patients were aged 65 to 85 years, had advanced-stage DLCL (diffuse mixed, diffuse large-cleaved or noncleaved, immunoblastic, or diffuse large-cell not otherwise specified). Advanced stage was defined as Ann Arbor stage III or IV or stage I or II with a mass greater than 10 cm or B symptoms. Low-dose doxorubicin, cyclophosphamide, vincristine, bleomycin, and prednisone (LD-ACOP-B) accrued 40 patients between March 1983 and September 1985; 65% achieved a complete response (CR), there were two toxic deaths, the actuarial failure-free survival (FFS) is 19%, disease-specific survival (DSS) 30%, and overall survival (OS) 28%, with a maximum follow-up of 6 years. The regimen of etoposide, doxorubicin, vincristine, bleomycin, and prednisone (VABE) accrued 32 patients between July 1985 and June 1987; 63% achieved a CR, there were two toxic deaths, and the actuarial FFS is 34%, DSS 45%, and OS 36%, with a maximum follow-up of 4 years. There is no difference in FFS, DSS, or OS between these two regimens. VABE caused more myelosuppression and infectious complications, although the toxic death rates were similar. We prefer LD-ACOP-B because follow-up is longer and toxicity is less. PMID- 1707955 TI - Combination chemotherapy of intermediate-grade and high-grade non-Hodgkin's lymphoma with MACOP-B: a Southwest Oncology Group study. AB - One hundred nine assessable patients with measurable stage II, III, or IV intermediate- or high-grade lymphoma were treated with methotrexate with leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) by members of the Southwest Oncology Group (SWOG) between November 1985 and June 1986 to confirm the activity of the program as initially described by Klimo and Connors and to test the safety of using third-generation regimens in a cooperative group. The median age was 53.5 years, and stage II was seen in 30% of patients and diffuse large-cell histology in 63%. Complete remission (CR) was achieved in 50% of all patients and partial remission (PR) in 33%. Response rates did not differ by histology. Median follow-up is 46 months with 51% of patients alive at 3 years and 63% of CR patients free of disease at 3 years. Severe (grade 3) or worse hematologic toxicity was seen in 51% of all treated individuals, and 29% had severe mucositis. We failed to confirm the high response rates as originally reported. Whether MACOP-B is superior to other treatment regimens requires the prospective trial currently being conducted by the SWOG. PMID- 1707956 TI - Multivariate analysis of prognostic factors in stage IV follicular low-grade lymphoma: a risk model. AB - We analyzed the records of 96 previously untreated patients with stage IV follicular low-grade lymphoma (FLGL) uniformly treated with cyclophosphamide, doxorubicin, vincristine, prednisone, and bleomycin (CHOP-Bleo) chemotherapy from 1972 to 1982. The overall complete remission (CR) rate was 77%. At a median follow-up of 138 months, the 10-year cause-specific survival rate was 42% with a median survival of 100 months. Failure-free survival (FFS) was 15% at 10 years with a median FFS of 30 months. Multivariate analysis showed peripheral lymph node size (LN), degree of marrow involvement, and sex, in that order, to be important for FFS, while the number of extranodal sites (#ENS), LN, sex, and degree of marrow involvement were important for cause-specific survival. We devised a tumor burden (TB) model, incorporating #ENS, LN, and degree of marrow involvement. Three groups were identified with statistically significant differences in cause-specific survival and FFS. Those with low TB (one ENS exclusive of extensive marrow and nodal disease less than 5 cm) had a 10-year cause-specific survival of 73% compared with 24% for patients with high TB (greater than or equal to two ENS and nodal disease greater than or equal to 5 cm) (P less than .001) and 40% for those with intermediate TB (either greater than or equal to 2 ENS, or extensive marrow only, or nodal disease greater than 5 cm) (P = .050). Patients with low TB had a 10-year FFS rate of 32%, while the intermediate and high TB groups had 10% and 9% FFS, respectively (P = .003). Because sex was a very strong prognostic variable, we created a risk model for survival and FFS based on TB and sex. Females with low TB had the best prognosis (92% survival and 50% FFS at 10 years) and males with high TB had the worst outlook (median survival and FFS, 43 and 12 months, respectively). Other TB-sex combinations defined two groups with statistically significant differences in survival but comparable FFS. This model should aid in the design and analysis of future trials. PMID- 1707957 TI - Prognostic factors in unselected patients with nonseminomatous metastatic testicular cancer: a multicenter experience. AB - Between 1981 and 1986, 200 consecutive patients with metastatic nonseminomatous testicular cancer were entered into the Swedish Norwegian Testicular Cancer (SWENOTECA) project from 14 hospitals. The treatment plan was four chemotherapy cycles (cisplatin, vinblastine, and bleomycin) followed by surgical resection of residual tumor masses. After a median observation time of 75 months, the overall 5-year survival rate was 82%. In a univariate analysis, the following parameters influenced the prognosis significantly: the extent of the disease (Medical Research Council [MRC] grouping); the prechemotherapy levels of serum alpha fetoprotein (AFP), human chorionic gonadotropin (HCG), and lactate dehydrogenase (LDH); the patients' age; the presence of extrapulmonary hematogeneous metastases; and/or particularly large lymph node metastases. Patients fared better when more than 3 weeks elapsed between orchiectomy and start of chemotherapy as compared with those who were treated within this interval. The place of treatment (a large oncology unit v smaller units) also represented a significant prognostic factor for patients with large-volume (LV) and very-large volume (VLV) disease combined. Multivariate analysis (Cox regression proportional hazards model) performed in all 193 assessable patients showed the following adverse prognostic factors: high-volume metastatic burden, age older than 35 years, prechemotherapy AFP greater than 500 micrograms/L and/or HCG greater than 1,000 U/L, and an interval between orchiectomy and start of chemotherapy of less than 3 weeks. The place of treatment also significantly influenced the final outcome. If patients with LV and VLV disease were combined, the presence of two of the following risk factors represented an additional prognostic factor: AFP greater than 1,000 micrograms/L, HCG greater than 10,000 U/L, liver metastases, brain metastases, bone metastases, retroperitoneal tumor greater than or equal to 10 cm, and mediastinal tumor greater than or equal to 5 cm. PMID- 1707958 TI - A clinical phase II study of ProMACE-CytaBOM. PMID- 1707959 TI - Transforming growth factor alpha and epidermal growth factor in human pancreatic cancer. AB - Overexpression of the epidermal growth factor receptor (EGFR) has been reported as an important molecular abnormality in human pancreatic cancer. There is in vitro evidence that simultaneous overproduction of one of its ligands, transforming growth factor alpha (TGF-alpha), might result in an autocrine loop with an increased proliferation signal. We analysed by immunocytochemical staining a retrospective series of human pancreatic cancers, chronic pancreatitis, and normal fetal and adult pancreatic tissues for the presence of TGF-alpha and epidermal growth factor (EGF). Ductal epithelial cells showed TGF alpha immunoreactivity in both normal tissue and chronic pancreatitis, and 95 per cent of tumours showed strong immunoreactivity. In contrast, EGF immunoreactivity was not found in normal pancreas, but was expressed in 12 per cent of pancreatic carcinomas. Well-defined areas of EGF immunoreactivity in exocrine ducts showing reactive changes in pancreatitis might represent a benign response to tissue damage similar to that previously described in the gastric mucosa. PMID- 1707960 TI - Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry. AB - Five peptides, corresponding to regions of the predicted protein sequence of the oestrogen-regulated pNR-2 protein which is expressed in oestrogen-responsive human breast cancer cells, were synthesized. Two peptides were immunogenic in rabbits and antisera against one peptide reacted with the pNR-2 protein in sections of formalin-fixed, paraffin-embedded breast tumour. There was a significant correlation between the extent of pNR-2 protein expression detected by immunohistochemistry and pNR-2 mRNA levels determined by hybridization with a cDNA probe in a series of primary breast tumours. pNR-2 expression was assessed immunohistochemically in a panel of normal tissues. Expression was detected in normal breast, small intestine, and stomach (body and antrum). PMID- 1707961 TI - Anti-allergic effect of N-acetylneuraminic acid in guinea-pigs. AB - The in-vivo anti-allergic effect of N-acetylneuraminic acid (NANA) in guinea-pigs passively sensitized with anti-ovalbumin rabbit serum has been studied. NANA (20 mg kg-1 i.v.) inhibited bronchial anaphylaxis and the release of histamine into bronchoalveolar lavage fluid. NANA dose-dependently inhibited heterologous passive cutaneous anaphylaxis and haemorrhaging in the passive Arthus reaction. However, it did not inhibit the release of histamine from sensitized minced lung tissue. PMID- 1707962 TI - In vitro assays show a dissociation of reverse transcriptase activity and core antigen (p24) production in two HIV-1 isolates from a patient receiving long-term treatment with zidovudine (ZDV). AB - Two HIV-1 isolates were obtained from a patient receiving long-term treatment with zidovudine (ZDV). The in vitro sensitivity to ZDV triphosphate of the reverse transcriptase (RT) from both isolates appeared to be unchanged compared to that of the LAV-Bru HIV-1 reference strain. When isolates were grown in CEM cells (a T-lymphoblastoid tumor cell line) and their RT activity and core antigen (p24) production were determined, the level of p24 production compared to RT activity was high; in infected CEM cells treated with ZDV, RT activity was at background level while the p24 production was still significant, thus indicating a dissociation of RT activity and core antigen production. PMID- 1707963 TI - Blockage of synaptic release by brief hyperpolarizing pulses in the neuromuscular junction of the crayfish. AB - 1. Synaptic currents were evoked at the neuromuscular junction of the deep extensor abdominal muscle of the crayfish by direct depolarization of motor nerve endings. 2. Quantal content and time course of neurotransmitter release were determined from delay histograms of unitary release events recorded with a macropatch clamp technique. 3. Synaptic facilitation was elicited by pairing depolarizing pulses at intervals ranging from 10 to 200 ms. At 14 degrees C the duration of facilitation was about 50 ms. Reducing activity of the Nao(+)-Cai2+ exchange by lowering [Na+]o by 50% resulted in prolonged facilitation, which lasted approximately 150 ms. 4. Normalized synaptic delay histograms at normal [Na+]o and 50% [Na+]o were the same for the first and the facilitated second response, indicating that activity of the Na(+)-Ca2+ exchange does not determine the time course of release. 5. The application of a hyperpolarizing post-pulse after the first depolarizing stimulus reduced release and altered its time course to a similar extent both in normal and in 50% [Na+]o. However, it did not affect the level and the time course of release of the facilitated response. 6. A hyperpolarizing post-pulse given after the first and second pulses of a pair reduced release to the same extent for the two depolarizing pulses. 7. These results indicate that whereas manipulations thought to increase [Ca2+]i (i.e. reducing activity of the Nao(+)-Cai2+ exchange or facilitation) affect the quantal content, they do not influence the time course of release. However, changes of membrane potential do affect the quantal content, and more importantly the time course of release, thus suggesting a contributory role of membrane potential in the control of synaptic release. PMID- 1707964 TI - Stretch-activated channels in single early distal tubule cells of the frog. AB - 1. Single stretch-activated channels have been studied in cell-attached and excised patches from single early distal tubule (diluting segment) cells of Rana temporaria. 2. The channels can be reversibly activated, in both cell-attached and excised patches, by the application of negative pressure to the pipette causing mechanical stretching of the cell membrane. In cell-attached patches, application of 14.8 cmH2O negative pressure to the patch pipette increased reversibly the open probably from 0.11 to 0.87. 3. The channel conductance in the cell-attached configuration with standard Ringer solution in the pipette is 21.3 pS. 4. The channel is non-specific. In excised inside-out patches ion substitution experiments show that the channel does not discriminate between sodium and potassium ions, nor does it appear to select for cations over anions. 5. The channel is voltage sensitive such that depolarizing the cell opens the channel. The open probability at the resting membrane potential, 0.89, was reduced to 0.26 at a hyperpolarizing potential of 100 mV (holding pressure of 20.1 cmH2O or -206 Pa). 6. The sensitivity of the channel to mechanical stretching suggests that the channel may be involved in cell volume regulation. PMID- 1707965 TI - Competitive antagonists and partial agonists at the glycine modulatory site of the mouse N-methyl-D-aspartate receptor. AB - 1. Kynurenate (Kyn), 7-chlorokynurenate (7-Cl-Kyn), 3-amino-1-hydroxypyrrolid-2 one (HA-966) and D-cycloserine are known to bind to the glycine site that modulates the N-methyl-D-aspartate (NMDA) response of vertebrate central neurones. The effects of these compounds were investigated with patch-clamp and fast-perfusion techniques on mouse cortical neurones in primary culture in an effort to establish whether they act as antagonists, partial agonists and/or inverse agonists of glycine. A fast drug application method allowed the study of both steady-state and transient responses. 2. The analysis of steady-state responses indicates that the main effects of Kyn and 7-Cl-Kyn are those expected from competitive antagonists of glycine, with a dissociation constant of 15 microM for Kyn, and of 0.3 microM for 7-Cl-Kyn. Concentration jumps indicate that at all concentrations of glycine, and in particular in the absence of added glycine, the blockade by Kyn and 7-Cl-Kyn develops at a rate which is close to the rate of dissociation of glycine from its binding site and is independent of antagonist concentration. 3. The main effects of D-cycloserine and of HA-966 are those of partial agonists of high and low efficacy, respectively. In the absence of added glycine, D-cycloserine always produced a potentiation, while HA-966 produced either a potentiation or an inhibition. This can be explained by assuming the presence of a variable level of contaminating glycine. With both D cycloserine and HA-966, concentration jumps produced biphasic relaxations in which the onset rate of the slow component was, here again, close to the rate of dissociation of glycine from its binding site. 4. These results can be interpreted by assuming that (1) Kyn and 7-Cl-Kyn are competitive antagonists of glycine, (2) HA-966 and D-cycloserine are partial agonists, (3) in the absence of added glycine some glycine is present in the extracellular solution and (4) the response in the total absence of glycine is very small or negligible. PMID- 1707966 TI - Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. AB - 1. Synaptically connected neurones were identified in the granule cell layer of slices of 17- to 21-day-old rat hippocampus. Whole-cell current recording using the patch-clamp technique revealed synaptic currents ranging from less than 10 to 200 pA in symmetrical Cl- conditions, at a holding potential of -50 mV. These currents were blocked by 2 microM-bicuculline, indicating that they result from the activation of postsynaptic gamma-aminobutyric acid receptor (GABAA-receptor) channels. 2. Addition of tetrodotoxin (TTX, 1 microM) resulted in the loss of most currents of more than 40 pA in amplitude. Currents which disappeared after TTX treatment were assumed to be the result of spontaneous presynaptic action potentials. The currents seen in the absence of TTX are referred to as spontaneously occurring inhibitory postsynaptic currents (IPSCs); those remaining in the presence of TTX were defined as miniature IPSCs. 3. Similar currents were observed when recording in the whole-cell configuration while extracellular stimulation was applied to a nearby neurone. These currents were also completely blocked by 2 microM-bicuculline and by 0.5 microM-TTX. They were thus defined as stimulus-evoked IPSCs. 4. The half rise time of both miniature and stimulus evoked IPSCs was fast (less than 1 ms). The time course of decay of both miniature IPSCs and stimulus-evoked IPSCs could be well fitted with the sum of two exponentials. At a membrane potential of -50 mV, the mean decay time constants of the two components were 2.0 +/- 0.38 and 54.4 +/- 18 ms (mean +/- S.D.) for miniature IPSCs (six cells) and 2.2 +/- 1.3 and 66 +/- 20 ms (three cells) for stimulus-evoked IPSCs. 5. Stimulus-evoked IPSCs varied in amplitude from less than ten to hundreds of picoamperes. In eight of eleven cells histograms of IPSC amplitudes showed several clear peaks which, when fitted with the sum of Gaussian curves, were found to be equidistant. This is consistent with the view that stimulus-evoked IPSC amplitudes vary in a quantal fashion. The quantal size varied between 7 and 20 pA, at a membrane potential of -50 mV. 6. Decreasing the Ca2+ and increasing the Mg2+ concentration in the extracellular solution decreased the number of peaks in the IPSC amplitude histogram but did not affect the size of the quantal event.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707967 TI - The response induced by intracellular cyclic AMP in isolated olfactory receptor cells of the newt. AB - 1. Responses induced by intracellular cyclic nucleotides were analysed in isolated olfactory receptor cells of the newt under a voltage-clamp condition by using the patch pipette in a whole-cell recording configuration. Cyclic nucleotides were applied by diffusion from the patch pipette. 2. Introduction of either cyclic AMP or cyclic GMP caused a transient inward current in cells held at -50 mV. The response amplitude was dose-dependent with the Hill coefficient of 3 and half-saturating concentration of 300 microM (concentration in the pipette) for both cyclic AMP and cyclic GMP. Cyclic CMP was less effective than those two nucleotides. 3. The response to intracellular cyclic AMP was seen in all cilia bearing cells, but not in cells which lost the cilia during dissociation. The response latency was shorter when cyclic AMP was introduced into the ciliated terminal swelling (ca 0.2 s) rather than into the cell body (ca 1.4 s). These results suggest that the sensitivity to intracellular cyclic AMP is confined to the cilia. 4. The cyclic AMP-induced current was transient (half decay time, ca 2.3s) despite the fact that cyclic AMP was continuously loaded from the patch pipette. The response time course was controlled by Ca2+; the reduction of external Ca2+ concentration (replaced with Mg2+) or loading the cell with 50 mM EGTA prolonged the cyclic AMP-induced responses. The Ca2(+)-induced suppression was reversible. 5. The reversal potential of the cyclic AMP-induced transient current was -4.8 +/- 3.8 mV, and that of the current re-induced by Ca2+ removal was 1.5 +/- 2.1 mV, suggesting that both currents flowed through the same ionic channel. The channel permeates all alkali metal ions with the permeability ratios of PLi:PNa:PK:PRb:PCs = 0.93:1:0.93:0.91:0.72, but not Cl- or choline ions. 6. These results demonstrate that the cyclic AMP-induced response and the odorant induced response of the isolated olfactory cell have nearly identical characteristics. The present study supports the notion that cyclic AMP is the internal messenger mediating olfactory transduction. PMID- 1707968 TI - Irreversible desensitization of ATP responses in developing chick skeletal muscle. AB - 1. In developing chick skeletal muscle, extracellular adenosine 5'-triphosphate (ATP) elicits an early excitatory conductance increase followed by a late potassium conductance increase. Both of these responses desensitize profoundly. Intracellular recordings and whole-cell voltage-clamp recordings were made in order to examine the time course and mechanism of desensitization and the recovery from desensitization. 2. Most of the loss of responsiveness to ATP occurred during the first minute of exposure to ATP. For the excitatory conductance, the loss of responsiveness to ATP resulted in part from long-lasting activation of the ATP-sensitive channels and in part from entrance into an inactive (non-conducting) state. In contrast, desensitization of the potassium conductance was entirely the result of a relatively fast transition to an inactive state. 3. Recovery from desensitization took many hours for both responses and was quite sensitive to temperature. 4. Recovery from desensitization for both responses was prevented by preincubation with the glycosylation inhibitor, tunicamycin. Several lines of evidence suggest that tunicamycin treatment blocked the delivery of new ATP receptors to the cell surface. 5. The recovery of the early response to ATP following exposure to two non-competitive inhibitors of the ATP response was also examined. These two compounds are thought to covalently modify the receptor. After exposure to either of these inhibitors, responsiveness to ATP returned over a time course that was similar to the time course of recovery from desensitization. 6. These results indicate that, following activation, ATP receptors do not become available for reactivation, and that recovery from desensitization is due to the insertion of newly synthesized receptors into the plasma membrane. PMID- 1707969 TI - Spontaneous activity at long-term silenced synapses in rat muscle. AB - 1. The impulse activity in the sciatic nerve of rats was blocked for 30-59 days by a chronic infusion of tetrodotoxin (TTX) into a cuff around the nerve from an external mechanical pump. After this treatment the extensor digitorum longus muscle was isolated and the electrical activity at the endplates was recorded by intracellular electrodes. Endplates in the paralysed muscles were still functional as the muscle contracted briskly upon stimulation of the nerve distal to the block. 2. The spontaneous miniature endplate potentials (MEPPs) differed from those in normal muscles in having a more variable amplitude; 24% of the events were more than twice as large as the modal amplitude. In 30% of the fibres the largest of these 'giant' MEPPs (GMEPPs) triggered muscle action potentials. The amplitude distributions often had suggestive peaks indicating that the GMEPPs might consist of multiple quanta of the same size as those constituting the nerve impulse-evoked endplate potentials (EPPs). 3. The GMEPPs were more prolonged, but were similar in shape to MEPPs from normal muscles, with a smooth, relatively fast rising phase and a more prolonged decay. The mean time-to-peak was higher than for impulse-evoked EPPs of the same size, suggesting that the spontaneous release was more distant or less synchronized than after a nerve impulse. The half-decay time of the GMEPPs showed no large increase with increasing amplitude suggesting that the release of transmitter was not focal. The half-decay times were, however, longer than for impulse-evoked EPPs of the same size, suggesting that the spontaneous release might be less distributed than impulse-evoked release. 4. GMEPPs were not influenced by TTX, they were larger than the impulse evoked EPPs in solutions containing high Mg2+ and low Ca2+, and they were not increased by high extracellular Ca2+ concentrations. Thus, the GMEPPs were not caused by spontaneous action potentials and probably not by Ca2+ influx. 5. In most cases the frequency of large and small MEPPs in paralysed muscles was influenced in the same way as those in normal muscles. It was increased by an increase in the extracellular K+ concentration or osmolarity, and reduced by a decrease in Ca2+ or an increase in Mg2+. The frequency was increased by Ruthenium Red. Also, like MEPPs in normal muscles, the frequency of small MEPPs in paralysed muscles was increased by increasing the extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707970 TI - The effect of external pH changes on responses to excitatory amino acids in mouse hippocampal neurones. AB - 1. The whole-cell and outside-out configurations of the patch-clamp technique were used to record responses to excitatory amino acids in mouse hippocampal neurones in cell culture at different pH. The amino acids kainate, quisqualate, N methyl-D-aspartate (NMDA) and L-glutamate were applied by a rapid perfusion system. 2. In the whole-cell recording mode the responses to NMDA or to low concentrations of glutamate, recorded in the absence of Mg2+ and with glycine in the extracellular superfusion solution, were antagonized by acidic pH and potentiated by an alkaline extracellular solution. Decrease in pH from 7.3 to 6.0 reduced NMDA responses to 33 +/- 2% and an increase in pH from 7.3 to 8.0 potentiated it to 141 +/- 6%. The responses to quisqualate and kainate were only slightly changed by altering the pH from 7.3 to 6.3 or 8.3. 3. The equilibrium dissociation constant (Kd) for H+ antagonism of responses to NMDA, estimated from the fit of a single-binding-site adsorption isotherm, was calculated to be 0.25 +/- 0.06 microM, corresponding to pH 6.6 +/- 0.1. The H+ attenuation of NMDA current was voltage independent at membrane potentials -60 to +30 mV. 4. H+ antagonism of responses to NMDA was reduced when the NMDA concentration was lowered. In the pH range 6.3-8.3 the H(+)-induced reduction did not vary with the concentration of glycine or Mg2+. The sensitivity of NMDA current to Zn2+ was unchanged in the pH range 6.3 +/- 8.0. These results suggest that H+ ions do not directly interfere with the binding of NMDA to its agonist recognition site or with the binding of glycine, Mg2+ and Zn2+ to the specific allosteric sites on the NMDA receptor-channel complex. 5. In outside-out patches held at -60 mV, unitary NMDA-activated currents were recorded at pH 7.3 and 6.3. The mean NMDA single-channel conductance (gamma) obtained for the largest and most frequent openings were: gamma 7.3 = 52.5 +/- 0.8 pS and gamma 6.3 = 51.8 +/- 0.9 pS. The duration of the mean channel open time, tau o, decreased from 4.75 +/- 0.25 ms in the control at pH 7.3 to 3.59 +/- 0.21 ms at pH 6.3. The mean burst duration, tau b, was reduced from 8.51 +/- 0.78 ms at control pH 7.3 to 5.1 +/- 0.34 ms at pH 6.3. The frequency of NMDA channel bursts was reduced by 31%.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707971 TI - Alterations in cat knee joint blood flow induced by electrical stimulation of articular afferents and efferents. AB - 1. Experiments were performed in cats anaesthetized with pentobarbitone. Laser Doppler flowmetry was used to assess the responses of knee joint blood vessels to nerve stimulation under control conditions and in the presence of different adrenoceptor antagonists in order to establish the nature of neurotransmitters released from articular nerve fibres. 2. The posterior articular nerve (PAN) supplying the knee was stimulated at different intensities, and frequency response curves were obtained. In fourteen animals electrical stimulation of PAN produced an initial vasoconstriction during stimulation which in eight of these was followed by a prolonged dilatation on cessation of stimulation. The constrictor response was increased as a function of frequency but was little altered with increasing intensity beyond a threshold level. 3. The constrictor response to electrical stimulation of PAN was markedly reduced by the alpha adrenergic antagonist phentolamine (10(-5) M, the alpha 1-blocker prazosin (10( 5) M), and guanethidine (10(-5) M) which inhibits the release of noradrenaline, ATP, and neuropeptide Y from sympathetic nerve endings. 4. The constrictor response to PAN stimulation was unaffected by the alpha 2-blocker rauwolscine and the P2-purinoceptor desensitizer alpha,beta-methylene ATP. 5. The dilator response was due to activation of afferent fibres as it could also be produced by direct electrical stimulation of the L7 dorsal roots. 6. The dilator response to stimulation of PAN or the L7 dorsal root was reduced by prior intra-articular injection of 100 micrograms of the substance P antagonist D-Pro4-D-Trp7,9,10-SP4 11. 7. These results suggest that the vasoconstrictor response to electrical stimulation of PAN is most likely to be mediated via noradrenaline acting mainly upon alpha 1-adrenoceptors. As the dilator response to articular nerve stimulation is reduced by a substance P antagonist, the mediator inducing this response may be substance P or a related neurokinin. PMID- 1707972 TI - Prognostic implication of silver-binding nucleolar organizer regions (AgNORs) in oral squamous cell carcinoma. AB - Silver-binding nucleolar organizer regions (AgNORs) were counted in sections from formalin-fixed, paraffin-embedded tissue blocks of oral squamous cell carcinoma. Thirty-nine cases, that comprised poor prognostic group (n = 19) and good prognostic group (n = 20), were examined with respect to the relation between AgNOR counts and histologic grading, and correlation between AgNOR counts and prognosis. The pooled mean AgNOR counts were: Grade 1 carcinomas, 6.39 +/- 1.67 (mean +/- SD; n = 35); Grade 2, 9.74 +/- 1.72 (n = 3). Mean AgNOR count of Grade 3 was 6.19 +/- 2.37 (n = 1). The pooled mean AgNOR count in poor prognostic group was higher than that in good prognostic group. Five-year survival rate of the cases with high AgNOR counts (greater than or equal to 6.5) was significantly lower than that with low AgNOR counts (less than 6.5). High AgNOR counts are highly suggestive of poor prognosis in oral squamous cell carcinoma. PMID- 1707973 TI - Plasma protein changes in chronic osteomyelitis of the jaws. AB - In the present study, quantification of different serum proteins known as inflammatory reactans was performed during different stages of chronic osteomyelitis of the jaws, to find a suitable tool for evaluation of treatment. In all 46 sera from 17 osteomyelitis patients and 6 healthy subjects were analyzed. Repeated measurements of alfa-1-antitrypsin, orosomucoid and haptoglobin could be recommended for following-up the effect of treatment, although too extensive conclusions should not be drawn from single measurements. The serum levels of these proteins seemed to co-vary with the clinical activity of the disease. Since the synthesis of the immunoglobulins only indirectly reflects the inflammatory activity, they are not considered to be suitable markers of inflammation, although their concentration in serum varied with the clinical activity. A certain "mass of inflammation" seemed to be necessary before raised values of C-reactive protein were detected. No information was gained from albumin concentration. PMID- 1707974 TI - Are the polarization colors of picrosirius red-stained collagen determined only by the diameter of the fibers? PMID- 1707975 TI - Prostatectomy in a district hospital. AB - In order to monitor the safety and efficacy of a new service for transurethral prostatectomy, an audit was performed, prospectively, over a period of 7.25 years. Of 304 prostatectomies performed, 91% were by transurethral prostatectomy. The proportion of patients with retention was 52%, 16% were uraemic and the incidence of carcinoma of the prostate was 21%. The operative mortality rate was 1.0%. An outline of the treatment policy and the data on complications and revision operations are presented. Comparisons are made with the experience of teaching centres and other district hospitals. Transurethral prostatectomy can be performed safely in the district general hospital and is a service which is essential to the smooth running of the surgical department. PMID- 1707976 TI - Synthesis and properties of some peptide analogues of actinomycin D. AB - Analogues of actinomycin D (AMD) were synthesized in which amino acid replacements were made at various sites in the peptide moieties. These include (i) replacement of both N-methylvalines by N-methylleucine, (ii) replacement of both sarcosines by N-[2-(methoxycarbonyl)ethyl]glycine, and (iii) replacement of one or both D-valines by D-threonine. The purpose of replacements ii and iii was to ascertain the effect upon biological activity of introducing a new side chain which could be functionalized to allow the attachment of carrier molecules such as antibodies. NMR data indicated that none of the analogues had solution conformations significantly different from that of AMD. Difference spectra with DNA revealed that replacement i enhanced binding while the other analogues bound less strongly to DNA. All the analogues had lower antimicrobial activities than AMD. In contrast, 5,5'-(MeLeu)2AMD displayed in vitro antitumor activity comparable with that of AMD at approximately 100-fold lower concentrations. PMID- 1707977 TI - Crystallization and preliminary characterization of mitogillin, a ribosomal ribonuclease from Aspergillus restrictus. AB - Mitogillin is a ribonuclease secreted by the fungus Aspergillus restrictus. The substrate for mitogillin is a short, universally conserved, sequence in ribosomal RNA. Cleavage of this sequence inactivates protein synthesis. Mitogillin was crystallized by a two-chamber vapor/liquid diffusion method using ethanol as the precipitant. This method has wider potential in the use of volatile organic solvents as precipitants. Crystals of mitogillin diffract X-rays to lattice d spacings of at least 1.6 A, and belong to the monoclinic space group P2(1), with a = 50.4 A, b = 82.4 A, c = 38.2 A and beta = 99.8 degrees. PMID- 1707978 TI - Experimental determination of torsion angles in the polypeptide backbone of the gramicidin A channel by solid state nuclear magnetic resonance. AB - An analytical method for the determination of torsion angles from solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopic data is demonstrated. Advantage is taken of the 15N-1H and 15N-13C dipolar interactions as well as the 15N chemical shift interaction in oriented samples. The membrane-bound channel conformation of gramicidin A has eluded an atomic resolution structure determination by more traditional approaches. Here, the torsion angles for the Ala3 site are determined by obtaining the n.m.r. data for both the Gly2-Ala3 and Ala3-Leu4 peptide linkages. Complete utilization of the orientational constraints derived from these orientation-dependent nuclear spin interactions in restricting the conformational space is most effectively achieved by utilizing spherical trigonometry. Two possible sets of torsion angles for the Ala3 site are obtained (phi, psi = -129 degrees, 153 degrees and -129 degrees, 122 degrees), both of which are consistent with a right-handed beta-helix. Other functional and computational evidence strongly supports the set for which the carbonyl oxygen atom of the Ala3-Leu4 linkage is rotated into the channel lumen. PMID- 1707979 TI - Solid-state nuclear magnetic resonance derived model for dynamics in the polypeptide backbone of the gramicidin A channel. AB - The dynamics of the backbone of the gramicidin A transmembrane cation channel in dimyristoylphosphatidylcholine bilayers have been investigated using solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopy. With the temperature dependent fluidity of the bilayer, the rates of motions in the helical gramicidin channel can be modulated. It is shown that in the gel phase, all substantial motions of the channel are slow on the timescale of the n.m.r. experiment (3.5 kHz). The use of oriented samples in which the axis of global channel rotation is aligned parallel to the magnetic field enables separation of global and local dynamics. Spectra obtained from oriented bilayer samples containing single-site 15N-labeled gramicidin at 8 degrees C are analyzed to yield a spatial model for local backbone motion. This model includes the axis of motion, the mean orientation, and the maximum amplitude of displacement for individual peptide planes. Specific sites in the first turn of the amino terminus were investigated, with emphasis on the Ala3 and Leu4 linkages, for which the orientation of the 15N chemical shift tensor with respect to the molecular frame has been determined. The effect of two well-characterized bilayer defect structures, parabolic focal conics and oily streaks, is included in the spectral simulations. It is found that only relatively small amplitude motions are possible at the two sites, with amplitudes of not more than +/- 8 degrees and +/- 15 degrees for the Ala3 and Leu4 sites, respectively. Detailed characterization of the bilayer surface geometry in the oriented samples is presently the major limiting factor in the use of this technique for probing the spatial extent of local motions in integral membrane proteins. PMID- 1707980 TI - Evidence that less-than-full-length pol gene products are functional in hepadnavirus DNA synthesis. AB - Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides. PMID- 1707981 TI - Homooligomerization of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 occurs before the acquisition of correct intramolecular disulfide bonds and mature immunoreactivity. AB - The posttranslational maturation of the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza type 3 virus (PIV3) was investigated in pulse chase experiments in which folding was monitored by immunoprecipitation with conformation-dependent antibodies and gel electrophoresis under nonreducing conditions and oligomerization was monitored by chemical cross-linking and sedimentation in sucrose gradients. The acquisition of mature immunoreactivity and the formation of correct intramolecular disulfide bonds were concurrent events, with half-times of approximately 10 to 15 min. The finding that newly synthesized HN had little reactivity with postinfection cotton rat serum or with most of the members of a panel of HN-specific monoclonal antibodies indicated that the major epitopes of the PIV3 HN protein are highly conformational in nature. Chemical cross-linking studies indicated that the mature HN protein is present in homoligomers, which are probably tetramers. These findings are consistent with recent observations for the HN protein of Sendai virus (S.D. Thompson, W.G. Laver, K.G. Murti, and A. Portner, J. Virol. 62:4653--4660, 1988; S. Vidal, G. Mottet, D. Kolakofsky, and L. Roux, J. Virol. 63:892--900, 1989). Surprisingly, analysis of pulse-labeled HN protein by sedimentation on sucrose gradients after labeling periods of as little as 2 min indicated that it was present intracellularly only in oligomeric form. The same results were obtained when the labeling period was preceded by a 1.5-h cycloheximide treatment to clear the endoplasmic reticulum of presynthesized HN protein, which indicated that the oligomerization did not involve the incorporation of newly synthesized monomers into partially assembled oligomers. Subsequent chase incubations did not significantly alter the sedimentation profile or stability of the oligomeric forms, suggesting that oligomers detected after short labeling periods were tetramers. Association with cellular proteins did not appear to be responsible for the sedimentation of newly synthesized HN protein as an oligomer. The absence of a detectable monomeric form of intracellular HN protein raised the possibility that oligomerization is cotranslational, and it is possible that the type II membrane orientation of the HN protein might be an important factor in its mode of oligomerization. PMID- 1707982 TI - Characterization and sequence analyses of antibody-selected antigenic variants of herpes simplex virus show a conformationally complex epitope on glycoprotein H. AB - Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu 86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707983 TI - Identification of neutralizing antigenic sites on VP1 and VP2 of type A5 foot-and mouth disease virus, defined by neutralization-resistant variants. AB - Five neutralizing monoclonal antibodies (nMAbs) obtained against type A5 Spain-86 foot-and-mouth disease virus were used to generate a series of neutralization resistant variants. In vitro and in vivo assays showed that the variants were fully refractory to neutralization by the selecting nMAb. On the basis of cross neutralization and binding assays, two neutralizing antigenic sites have been located on the virus surface; one, located near the C-terminus of VP1, displayed a linear epitope, and the second, located on VP2, displayed two conformational epitopes. Nucleotide sequencing of RNA of the parental and variant capsid protein coding region P1 has placed the amino acid changes at position 198 of VP1 for the first site and at positions 72 and 79 of VP2 for the related epitopes in the second site. The relative importance of these two sites in the biological properties of foot-and-mouth disease virus is discussed. PMID- 1707984 TI - Immunomodulation of encephalomyocarditis virus-induced disease in A/J mice. AB - The E variant of encephalomyocarditis (EMC) virus causes an encephalomyelitis and coagulative necrosis of the pancreas and parotid glands in some but not all strains of inbred and outbred mice. In other models of disease caused by picornaviruses, depletion of specific lymphocyte subsets abrogates the development of tissue lesions. In this study, severe encephalomyelitis and acinar pancreatitis and parotitis developed in adult male A/J mice infected with 100 PFU of EMC virus. Depletion of the CD4+ subset of T lymphocytes in vivo with a monoclonal antibody (MAb) prior to EMC virus inoculation protects mice from developing encephalomyelitis, pancreatitis, and parotitis. This effect is also seen when animals are treated with anti-CD4 and anti-CD8 in combination, but the anti-CD8 MAb alone does not ameliorate the disease. Overall, concentrations of virus in tissues from anti-CD4-treated animals are lower than in immunologically intact control mice. Small-plaque variants of virus were also recovered from the tissues in some animals in this group. CD4+ lymphocytes are involved in the expression of EMC virus-induced pancreatitis and parotitis in A/J mice. This specific subset of T cells would appear to influence EMC viral tropism or replication in various organs. PMID- 1707985 TI - Continuous epitopes of the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein and reactivity of human sera to synthetic peptides representing various HIV-1 isolates. AB - Immunoreactive regions of human immunodeficiency virus type 1 (HIV-1) gp41 were mapped by reacting HIV-1 antibody-positive human sera with overlapping synthetic peptides which covered the transmembrane protein. Three immunoreactive domains were identified, and five different and partially overlapping epitopes recognized by HIV-1-positive human sera were found within one immunodominant region. The effect on antibody recognition after single amino acid substitutions within one defined epitope was also studied. The reactivity of various HIV-1-positive sera to synthetic peptides with amino acid substitutions representing known isolates suggests an important substitution in the major epitope of African HIV-1 strains. PMID- 1707986 TI - Identification and characterization of human immunodeficiency virus type 1 gag pol fusion protein in transfected mammalian cells. AB - Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27 pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity. PMID- 1707987 TI - Prostate specific antigen levels after definitive irradiation for carcinoma of the prostate. AB - Prostate specific antigen (PSA) levels were determined in 78 patients judged clinically to be free of disease at intervals of 36 or more months (range 38 to 186 months, median 87 months) after completion of irradiation therapy by 125iodine implantation or external beam radiation. Of this select group of patients 38% had undetectable serum PSA levels (0.5 ng./ml. or less) and 38% had PSA levels that were within normal limits (4.0 ng./ml. or less). All stages and grades were represented. Undetectable PSA levels were only rarely found (3%) in patients with carcinoma of the prostate before treatment. In 24 of these 78 patients a negative biopsy of the irradiated prostate had been obtained 18 to 42 months after treatment. When the PSA level was drawn, which ranged from 7 to 16 years after treatment, an equal percentage of these biopsied patients had either an undetectable, normal or elevated level. Irradiation is able to decrease PSA to undetectable levels in some patients with prostatic carcinoma. Whether this reflects suppression of marker production alone or, more importantly, ablation of prostate cancer producing that marker remains to be determined. PMID- 1707988 TI - Urophonographic studies of benign prostatic hypertrophy. AB - The sonic detection and recording systems of urethral sounds generated during micturition were developed. This procedure was tentatively postulated as "urophonography" and its recording diagram as a "urophonogram". Classification of urophonograms was done on the basis of analyzing normal healthy male volunteers and patients with benign prostatic hyperplasia. Four types of urophonograms were demonstrated according to the shape and characteristics. Types 1, 2, 3 and 4 were characterized by a diamond shape, irregular occurrences of sound spikes, the mixture of Types 1 and 2 and no remarkable sound spikes respectively. Types 1, 2, and 3 were found in BPH, while Type 4 was demonstrated in normal healthy male volunteers. After prostatectomy a high percentage of Type 4 was demonstrated. The frequency (Hz) of these sounds was around 650. Diamond shape sound showed higher value of power gain (dB) than irregular type sound. The wave length was around 0.50 (m). Comparison of urophonographic studies with conventional uroflowmetric investigation was undertaken. Urophonography was useful for investigations of dysfunctional voiding and lower urinary tract obstruction. PMID- 1707989 TI - Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. AB - PSA is a kallikrein-like, serine protease that is produced exclusively by the epithelial cells of all types of prostatic tissue, benign and malignant. Physiologically, it is present in the seminal fluid at high concentration and functions to cleave the high molecular weight protein responsible for the seminal coagulum into smaller polypeptides. This action results in liquefaction of the coagulum. PSA is also present in the serum and can be measured reliably by either a monoclonal immunoradiometric assay or a polyclonal radioimmunoassay. The calculated half-life of serum PSA ranges from 2.2 to 3.2 days and the metabolic clearance rate of this tumor marker follows first-order kinetics. Digital rectal examination, cystoscopic examination and prostate biopsy all can cause spurious elevations of the serum PSA concentration. Conditions such as bacterial prostatitis and acute urinary retention also can falsely elevate the serum PSA level. Because approximately 25% of the patients with BPH only will have an elevated serum PSA concentration and BPH tissue contributes to this PSA value in a variable manner from patient to patient, it is unlikely that PSA by itself will become an effective screening tool for the early diagnosis of prostate cancer. However, if combined with digital rectal examination and/or transrectal ultrasound it may become a vital part of any early detection program. Prostatic intraepithelial neoplasia also may be associated with moderately elevated serum PSA levels. Although there is a direct correlation between the serum PSA concentration and clinical stage, PSA is not sufficiently reliable to determine the clinical stage on an individual basis. This finding also applies to pathological stage. As a result, the preoperative serum PSA concentration cannot be used to decide whether to recommend radical prostatectomy for potential cure. Low preoperative serum PSA concentrations in patients with previously untreated prostate cancers are predictive of a negative bone scan. Thus, in these select patients a staging bone scintigram may not be necessary. With respect to monitoring patients after definitive therapy, PSA is an exquisitely sensitive tumor marker. Irrespective of the treatment modality (radical prostatectomy, radiation therapy or antiandrogen treatment), PSA reflects accurately the tumor status of the patient and is prognostic of eventual outcome; this tumor marker is capable of predicting tumor recurrence months before its detection by any other method. PSA is also a most useful immunocytochemical marker. Its sensitivity and specificity to identify tissue of prostatic origin approach 100%. When compared to PAP, PSA is a more precise and meaningful marker in all clinical situations.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1707990 TI - From the Food and Drug Administration. PMID- 1707991 TI - [Anti-insulin receptor antibody--its measurement and significance]. PMID- 1707992 TI - [Clinical significance of analysis of alpha 2-macroglobulin in the diagnosis of diabetic diseases]. PMID- 1707993 TI - [Clinical significance of somatomedin-C measurement in the diagnosis of diabetic diseases]. PMID- 1707994 TI - [Clinical significance of urinary alpha 1-microglobulin and beta2-microglobulin in the diagnosis of diabetes mellitus]. PMID- 1707995 TI - [Clinical significance of urinary alpha 2-macroglobulin excretion rate in non insulin-dependent diabetes mellitus]. PMID- 1707996 TI - [Immunological abnormalities in diabetes mellitus]. PMID- 1707997 TI - [Comparison of the anti-arrhythmic effectiveness of ethacizine, ethmozine, cordarone, mexitil and ritmilen in elderly patients with ventricular extrasystole]. PMID- 1707998 TI - [Surgical treatment of ventricular allorhythmia and non-ischemic tachycardia]. AB - The paper deals with the problems in the diagnosis and treatment of patients with the most malignant rhythm disturbances, i. e. ventricular arrhythmias. A great emphasis is laid on the so-called "primary" arrhythmias appearing without a manifest cardiac abnormality. The arrhythmias proceed unfavorably and are followed by profound morphofunctional myocardial changes occurring chiefly in the upper portions of the ventricular septum, more frequently on the right. Cryodestruction of arrhythmogenic areas is an effective method for surgical correction of arrhythmias having an unfavorable course. PMID- 1707999 TI - [Epidemiology, invasion and growth of non-iridic choroid melanomas and associated intraocular changes: histologic study of 223 eyes]. AB - 223 complete eyes enucleated because of a malignant uveal melanoma were studied histologically to evaluate patterns of growth and invasion and secondary intraocular changes. Eyes with primary iris melanomas or with a previous operation were excluded. The most important secondary changes are the retinal detachment usually responsible for the loss of visual function as well as the direct tumor invasion of the chamber angle and the rubeosis iridis, both able to cause a secondary glaucoma, which is often difficult to treat. The rubeosis iridis increased in frequency with increasing tumor prominence and was in no case combined with preretinal neovascularisations. The growth of malignant uveal melanomas may occur in three directions, i.e. outwards (through the outer envelope of the eye), inwards (towards the vitreous) or in the uveal plane. In any case intraocular borders manifesting different resistance against tumor invasion and thereby influencing prognosis of the disease have to be penetrated. Changes at the front of tumor invasion like loosening of the cellular union and transformation to a higher malignant cell type seem plausible but could not be demonstrated light-microscopically. PMID- 1708000 TI - Histopathological studies on senile plaques in brains of patients with Down's syndrome. AB - Senile plaques (SPs) as a hallmark of Alzheimer's pathology were studied in the brains of 5 cases of Down's syndrome (DS), using silver stains including modified Bielschosky and new methenamine silver method which had been reported to show almost the same staining pattern as that of beta-protein immunostaining. SPs were observed in the CA1, the subiculum, the molecular layer of the gyrus dentatus, and the cerebral neocortices of all cases examined. In the striatum and the cerebellum, SPs were found in 2 out of 4 cases, both over the age of 30. In the cerebral cortex and the hippocampal formation, SPs were almost exclusively composed of the diffuse type below the age of 30, while primitive and classical plaques first appeared over the age of 30. In case 1 (aged 16), the cerebral cortex contained a few perivascular plaques associated with amyloid angiopathy known as dyshoric angiopathy. Our results suggest that diffuse plaques and dyshoric angiopathy are possibly earliest stages of SP formation in DS brains. PMID- 1708001 TI - Hypertension: the facts and the future. A satellite meeting of the XI World Congress of Cardiology. Manila, Philippines, February 11-16, 1990. PMID- 1708002 TI - Genealogy of the spontaneously hypertensive rat and Wistar-Kyoto rat strains: implications for studies of inherited hypertension. AB - The spontaneously hypertensive rat (SHR) is the most commonly used animal model of hypertension. For many years, it has been widely accepted that the most appropriate control strain is the Wistar-Kyoto (WKY) rat to which SHR rats are genetically related. However, recent concerns have been raised about genetic differences between the various colonies of SHRs and, in particular, genetic differences between colonies of WKY rats. It has been further emphasized that the only way to establish that a genetic trait is an etiological factor in the development of hypertension is through studies of F2 backcrosses between SHR and WKY rats. The present article details the history of the SHR and WKY strains and demonstrates why there is high likelihood of genetic variability between rats of both strains from different colonies around the world. It suggests that the WKY strain is not the most suitable for backcross studies because of the incidence of spontaneous hypertension and the somewhat higher levels of blood pressure in these rats. A central reference strain is proposed using SHRs inbred at Kyoto University, where brother/sister inbreeding has continued for more than 80 generations. PMID- 1708003 TI - Blood pressures in a random sample of the New Zealand population: preliminary data from the life in New Zealand survey. AB - Blood pressures (BPs) were measured as part of a health check in a randomly selected sample of the New Zealand population in the Life in New Zealand survey. A total of 1,410 men and 1,605 women over 15 years of age were studied. Measurements were made by trained observers using the Hawksley random zero instrument. Systolic BP (SBP) and diastolic BP (DBP) increased with age in men and women. There were 29% of men and 24% of women over the age of 45 years with BPs over 160/95 mm Hg, of whom 24% of men and 33% of women indicated they were on treatment for hypertension. No regional or urban/rural differences were seen in either SBPs or DBPs. A history of hypertension in the fathers of respondents related to BPs in the highest tertile of SBPs in males, and SBPs in females. The association was not seen between mothers of respondents, except for DBP in women respondents. PMID- 1708004 TI - Antihypertensive therapy: achievements, failures, and prospects. AB - Several studies have shown that treatment of hypertension does not return risk to normal. Reasons include imperfect clinic organization, poor patient compliance, adverse drug effects, inadequate blood pressure control, and, particularly, failure to affect the atheromatous complications of hypertension. Future therapeutic efforts, and especially drug design, should be directed to these needs. PMID- 1708005 TI - Optimum drug treatment in hypertension. AB - The ultimate objective in treating hypertension is to select an agent directed at the specific mechanism causing blood pressure elevation in the individual case. Such a choice is not possible in most cases at present; it is usually more important to match the profile of drug activity to the characteristics of the patient. In combination therapy, it is important also to match drug with drug, so as to obtain synergistic actions and to avoid adverse interactions. Finally, drugs that allow once-daily dosing are advantageous. These principles are used in deriving an optimum approach for practical application of the antihypertensive drug armamentarium available in 1990. PMID- 1708006 TI - Treatment of hypertension: the view from general practice. AB - All patients aged 35-64 years on a computerized age/sex register at Mornington Health Centre, Dunedin, were invited to meet with a Research Nurse for the assessment of their blood pressure. Results are presented from the 1,335 people attending (57% response). These show that 33 people (2%) had hitherto undiagnosed hypertension and that 157 (12%) had their hypertension diagnosed and treated. Evidence will also be presented of the level of control that indicates a marked improvement in published figures on the level of identification, treatment, and control in a community-based group. PMID- 1708007 TI - Treatment of hypertension in pregnancy. AB - A better understanding of the hemodynamic abnormalities in gestational hypertension together with the use of effective antihypertensive agents have resulted in more rational therapeutic approaches and a substantial improvement in maternal and fetal welfare. In normal pregnancy, there is reduced vascular reactivity with peripheral pooling and decreased circulatory responses to pressor agents. These are prostacyclin-dependent processes. In gestational hypertension, the normal increase in plasma volume and cardiac output with pregnancy is attenuated and prostacyclin-dependent processes are impaired, resulting in persistent vasoconstriction, enhanced responses to pressor agonists, and failure to develop adequate uteroplacental interchange. Among the modern antihypertensive agents, alpha- and beta-adrenergic antagonists and calcium ion entry blockers have permitted safe and effective long-term blood pressure control with sustained fetal growth. The development of proteinuria that can occur in chronic hypertension or in previously normotensive women (toxemia of pregnancy) can be prevented by the use of beta-adrenergic blocking agents and possibly by low-dose aspirin (75 mg/day). Maternal prostacyclin-thromboxane imbalance, important in the pathogenesis of gestational hypertension, is corrected by low-dose aspirin treatment. With the prevention of pre-eclampsia, the adverse maternal and fetal prognosis in gestational hypertension has been improved. PMID- 1708008 TI - Ten-year follow-up of worksite hypertension control programs. AB - We have analyzed the control of hypertension and the demographics of 898 actively treated (AT) (visits within past 12 months) and inactive patients (IAT) (no visit for at least 12 months) in six worksite hypertension programs established in 1977. Results were analyzed using Student's t test and correlation coefficients. Patients are treated at the worksites by a nurse-physician team using a stepped care protocol. In the AT group (n = 436), the mean age was 52 +/- 0.5 years, initial blood pressure (BP) was 152 +/- 1/97 +/- 0.5 mm Hg, and current BP was 136 +/- 0.7/86 +/- 0.04 mm Hg (p less than 0.001). Of the 436 patients, 194 were men and 242 were women (217 Caucasian and 156 black). In the IAT group (n = 462) mean age was 51 +/- 0.5 years, initial BP was 154 +/- 1/99 +/- 0.5 mm Hg, and last BP was 138/88 +/- 0.4 mm Hg (p less than 0.001). Of these, 171 were men and 291 were women (238 Caucasian and 171 black). No differences in initial and current BP were noted when comparing the AT with the IAT group. The percent of Caucasian (43%) and blacks (36%) men in the AT group was similar to those in the IAT group (37%). Control of hypertension at worksites where blacks predominate was similar to sites where Caucasians predominate. We conclude that worksite control of hypertension over a 10-year period maintains approximately 50% of the initial population under treatment. Control rates greater than 80% to less than 90 mm Hg diastolic are present despite heterogeneity in demographics. PMID- 1708009 TI - Anemia and angiotensin-converting enzyme inhibition in renal transplant recipients. AB - The renin-angiotensin system has been shown to have an effect on erythropoietin synthesis and hemoglobin concentration. We present a retrospective study of stable renal transplant recipients who received an angiotensin-converting enzyme (ACE) inhibitor as treatment of hypertension. Fifteen patients were eligible, with a mean hemoglobin concentration of 130.7 +/- 22.7 g/L (SD). Within 6 months of ACE-inhibitor therapy, the mean hemoglobin had fallen significantly to 110.5 +/- 23.2 g/L (p less than 0.001). No parallel change in leukocyte or platelet counts was evident. Prospective studies are needed to clarify the effect of inhibition of the renin-angiotensin system on erythropoietin synthesis and release. PMID- 1708010 TI - Sensitivity to Ca2+ and the effects of a calcium channel antagonist in resistance vessels from two strains of genetically hypertensive rat. AB - Ca sensitivity and sensitivity to diltiazem were studied in two strains of genetically hypertensive rat [spontaneously hypertensive rats (SHRs) and genetically hypertensive (GH) rats] and their normotensive control strains [Wistar-Kyoto (WKY) and normal Wistar (N) rats] at two ages before and after establishment of significant hypertension. Ca sensitivity was equal in both hypertensive/normotensive strain pairs from young rats but significant relative increases in Ca sensitivity were present in older rats. Sensitivity to the vasodilating action of diltiazem in vessels precontracted using K+ was also present in adult rats. These results suggest that the abnormality in Ca sensitivity seen in genetic hypertension in rats is an acquired characteristic not involved in the etiology of hypertension. PMID- 1708011 TI - First-choice antihypertensive drug use in the Glasgow Blood Pressure Clinic. AB - The evolution of antihypertensive drug use in the Glasgow Blood Pressure Clinic between 1969 and 1986 was determined, from computerized data, by extracting percentages of new patients prescribed different drugs at their first clinic visit. Prescribing of adrenergic neuron blockers and centrally acting drugs (methyldopa and clonidine) was common in the early years but declined rapidly until, after 1975 and 1980, respectively, it remained at 10% or less. Diuretics, mainly thiazides, were prescribed for 20% of patients in 1969 to a peak of 55% in 1980. beta-Blockers were first used during the early 1970s and their use peaked in 1980, when they were prescribed for over 60% of new patients. They remained a first-choice treatment in more than 40% of patients. Calcium channel blockers were first used in 1980 and by 1986 were prescribed for 15% of new patients. Angiotensin-converting enzyme inhibitors were used from 1982 and in 1986 were prescribed for 7% of new patients. These two classes of drugs are more expensive than older drugs. However, because of their low usage, this did not greatly influence treatment costs up to 1986, as beta-blockers and thiazides remained widely used. PMID- 1708012 TI - Changes in clinical characteristics and drug treatment of hypertension over 40 years at the Dunedin Hypertension Clinic. AB - The clinical characteristics of the 4,170 hypertensive patients referred to the Dunedin Clinic from 1950 to 1989 have been compared for eight successive 5-year periods. A gradual decrease in the severity of referred hypertension and an increase in the proportion of patients already on treatment at the time of referral (currently 50%) were noted. For male patients, mean +/- SD initial lying blood pressure was 179 +/- 27/116 +/- 19 mm Hg in 1950-1954 and 158 +/- 25/91 +/- 14 mm Hg in 1985-1989. Corresponding prevalence data for target organ damage among male patients were retinal grade 3 or 4, 49% and 3%; cardiomegaly on chest radiograph, 60% and 26%; electrocardiogram left ventricle strain pattern, 28% and 3%; and serum urea levels greater than 10 mmol/L, 16% and 5%, respectively. For women there was a similar trend. The number of patients on drugs in each of nine categories and the percent use of each drug category for each year during 1950 1989 was recovered from computerized data files. The percentage peak usage of ganglion blockers was in 1950-1958, adrenergic neuron blockers in 1963-1970, centrally acting drugs in 1965-1968, diuretics in 1960-1982, beta-blockers in 1974-1987, alpha-blockers in 1980-1987, and angiotensin converting enzyme inhibitors and calcium antagonists in 1989. The diuretics have been the most enduring drugs, followed by the beta-blockers. PMID- 1708013 TI - Treating mild to moderate hypertension: cost-effectiveness and policy implications. AB - Results from major clinical trials reported during the 1980s have led to renewed debate about the costs and benefits of treating mild to moderate hypertension. There is general agreement that existing cost-effectiveness analyses of antihypertensive therapy are outdated, and in need of reappraisal. Based on the pooled results of clinical trials, the benefits of treating mild to moderate hypertension [diastolic blood pressure (DBP) of 90-114 mm Hg] were re-examined. Using actuarial methods and estimates of health state utilities, the benefits of treatment were expressed in "quality-adjusted life years" (QALYs). After lifelong treatment for hypertension, the gain in QALYs ranged from 1.8 to 11.5 months in men and from 2.5 to 11.3 months in women. The cost-effectiveness ratios ranged from $30,200 per QALY gained (for 50-year-old men with DBP of 110 mm Hg) to $547,700 per QALY gained (for 30-year-old women with DBP of 110 mm Hg), in 1988 New Zealand dollars, discounted at 5%. In several categories of patients, the analysis suggested a net negative impact on QALYs, i.e., the adverse effects of drug treatment outweighed the benefits. These results have policy implications for both resource allocation and clinical practice. PMID- 1708014 TI - Mitogenesis in cultured vascular smooth muscle cells from two rat models of hypertension in response to fetal calf serum and angiotensin II. AB - Hypertension may result from vascular hypertrophy or hyperplasia due to enhanced growth of vascular smooth muscle cells (VSMCs), which has been demonstrated in VSMCs from spontaneously hypertensive rats (SHRs) compared to Wistar-Kyoto (WKY) rats. To determine whether this enhanced mitogenesis is peculiar to SHRs or a general phenomenon in genetic models of hypertension, we have measured indices of cell growth [3H]-thymidine uptake in VSMCs from SHRs and New Zealand genetically hypertensive (GH) rats and controls [WKY and normal Wistar (N) rats] cultured in fetal calf serum (FCS) or angiotensin II (Ang II, 0.1 microM) in either 3% heat treated FCS or serum-free medium. SHR cell numbers increased faster in response to both mitogens compared to WKY rats. However, GH and N rat responses to FCS were the same. Ang II caused a significant but similar increase in cell numbers in both GH and N rat cells (i.e., Ang II caused hyperplasia in all four strains) but [3H]thymidine uptake was significantly greater in GH rat cells. Ang II increased the total well protein content but not protein normalized on cell number, i.e., no hypertrophic effect of Ang II was seen in these actively dividing cells. We conclude that (a) growth properties of VSMCs from rats with genetic hypertension vary between strains; the differences in growth may reflect strain-specific variation in the activity of intracellular signalling systems subserving mitogenesis; and (b) Ang II causes VSMC hyperplasia. PMID- 1708015 TI - Induction of thrombospondin expression in vascular smooth muscle cells by angiotensin II. AB - Vascular smooth muscle cells (VSMCs), unlike cardiac or skeletal myocytes, are capable of undergoing reversible phenotypic modulation from "contractile" to "proliferative/synthetic" cells in vivo. We have investigated the ability of angiotensin II (Ang II) to influence this process via modulation of extracellular matrix synthesis. Ang II induced a rapid (within 2 h), dose (10(-6)-10(-9) M) dependent stimulation (14-fold) of thrombospondin (TSP) gene expression in rat VSMCs in the absence of additional factors. This was followed by an enhanced platelet-derived growth factor (PDGF) A chain and transforming growth factor-beta (TGF beta) gene expression. These effects of Ang II could be negated by the simultaneous addition of saralasin (IC50 approximately 10(-9) M for Ang II at 10( 7) M) to cells. Transcription levels for TSP were further enhanced (to 28 X control values) by 6 h, at which time the synthesis of PDGF A chain mRNA was maximal. Although exposure of cells to TSP (5 X 10(-8) M) stimulated signal transduction pathways, it did not enhance levels of either PDGFA or TGF beta transcripts. The glycoconjugate content of extracellular matrices elaborated by cells chronically exposed to Ang II was elevated compared to control cultures and there was a small increase in cell number. PMID- 1708016 TI - Regulation of vasoactive intestinal peptide release and metabolism by sodium. AB - To determine whether vasoactive intestinal peptide (VIP) might act as a humoral mediator for a proposed portal or hepatic sodium monitor, we measured plasma VIP levels and urinary sodium excretion after portal and intravenous sodium loading in rabbits equilibrated on normal and low sodium diets. Sodium excretion was significantly less after intravenous (2 h: p less than 0.005; 4 h; p less than 0.005; 8 h; p less than 0.025) than after portal sodium administration in rabbit on a low-sodium diet. Plasma VIP levels fell after intravenous (p less than 0.005) but not after intraportal sodium in this group. To determine whether this fall in VIP levels reflected increased metabolism or decreased secretion, metabolic clearance studies were performed in rabbits on a low sodium diet. The metabolic clearance rate of VIP and its theoretical secretion rate increased after intravenous sodium loading in rabbits on a low salt diet (MCR, p less than 0.025; SR, p less than 0.05). We conclude, therefore, that in rabbits on a low salt diet, intravenous sodium increases VIP metabolism, causing a decrease in plasma levels that may explain the difference in sodium excretion. PMID- 1708017 TI - The effects of a hyperosmotic challenge in vivo on tissue composition of rat cardiac and skeletal muscle. AB - The effects of hyperosmotic stress under both acute and chronic conditions were investigated in two types of rat muscle tissue: cardiac and skeletal. Under acute conditions of a 4-h infusion, both types of muscle behaved as osmometers. However, under chronic hyperosmotic stress (several days), skeletal muscle behaved as predicted as an osmometer, but cardiac muscle did not. The levels of free amino acids in this tissue increased markedly--in particular, taurine. This phenomenon is discussed with respect to the capacity of this tissue for volume regulation. PMID- 1708018 TI - The control of body sodium in relation to hypertension: exploring the Strauss concept. AB - The overall control of body sodium relies on mechanisms that have close links to blood pressure control and hypertension. The Strauss concept of a basal level of body sodium, below which any available sodium is retained and above which any extra sodium is excreted (at a rate exponentially related to the amount present in the body), has been confirmed in rat studies. Total body sodium, on an average sodium intake, thus consists of basal plus extra: the proportions of these can vary and this makes interpretation of total body sodium difficult. Basal body sodium is, in theory, the level at which delivery of sodium to the distal nephron equals distal reabsorption of sodium. The latter is largely determined by aldosterone and, indeed, basal body sodium is high in primary aldosteronism and low in Addison's disease. The half-life of extra sodium is short in primary aldosteronism and long in Addison's disease: it is also short in some hypertensive subjects but in general lengthens with age. The exact mechanisms involved are still uncertain. Most of this work is based on step reductions in sodium intake. Step increases in sodium intake appear to lead to more complicated adjustment processes, with a delay in commencing excretion followed by some under damping of the system before a new higher level of body sodium and a new equilibrium of intake and excretion is reached. PMID- 1708019 TI - Hypertension after Intersalt: prospects for prevention. AB - Special methods of statistical analysis of the Intersalt data permitted a better comparison between populations and between age groups than was previously possible, but these methods led to an exclusive focus on the distribution of blood pressure (BP) rather than on the prevalence of hypertension. Big changes in prevalence that accompany small changes in mean BP were implied but not illustrated. A plot of prevalence against log Na excretion from the tabulated data suggests that a Na excretion rate greater than 100 mmol/day could not be recommended under any circumstances. However, a rate less than 40 mmol/day would be expected to prevent hypertension and to prevent the BP from rising with age. From the intersalt data, an intermediate range of Na excretion of 40-100 or 50 100 mmol/day, which are public health targets in Australia and the United States, respectively, would be expected to be protective in the presence of other aspects of a healthy lifestyle, especially if the K intake preserves the natural Na/K ratio (less than or equal to 1.0). PMID- 1708020 TI - A salt-hypertension hypothesis. AB - In urban Australia, the risk of retiring with hypertension is greater than 40%, and the basic abnormality--a rise in blood pressure (BP) with age--is almost universal. A hypothesis linking this with salt, therefore, concerns everyone. The diet of early humans was unsalted, and the Na content of breast milk (6 mmol/kg) shows how little NaCl is needed even during the most rapid period of growth. The hypothesis that the hypertonic concentration needed to preserve food causes the BP to rise with age is based partly on the normotensive status of contemporary "salt-free" societies and partly on experimental evidence. "Salt-free" populations seldom use alcohol and happen to be lean and active, with a low fat intake and largely vegetarian diet, but Westerners with similar virtues do not escape hypertension. Ideally, the prophylactic effect of avoiding salt would be ascertained in large-scale, prospective trials, but practical, ethical and, economic factors impose serious design problems. Nevertheless, a public health intervention based on this hypothesis would be incomplete without a serious attempt to measure the outcome. PMID- 1708021 TI - Is atrial natriuretic factor a physiological regulator of sodium excretion? A review of the evidence. AB - Recent debate has centered on the possible role of atrial natriuretic factor (ANF) as a physiological regulator of natriuresis. Plasma ANF rises with increased dietary sodium, during intravenous infusion of a saline load, and with change from upright to recumbent posture. Peptide secretion is proportional to atrial distension such that plasma concentrations rise 10-15 pM for each mm Hg increment in either right or left atrial pressures. ANF administered to humans in a low dose (0.75 pmol/kg/min) produces an increase in plasma concentrations within the normal range and induces natriuresis, excretion of cGMP, and suppression of renin-angiotensin-aldosterone activity. These results are consistent with a role for ANF in the physiological regulation of sodium excretion. Factors that clearly modify the natriuretic effect of ANF include volume/sodium status and renal perfusion pressure. Variations in these modifying factors may account for some of the reported inconsistencies in natriuresis observed in clinical high-ANF states such as congestive heart failure and tachycardia and in experimental circumstances including balloon-induced atrial distension in the cardiac-denervated dog. Overall, current data favor a role for ANF in the physiological regulation of sodium excretion in humans but more definitive evidence must await the advent of a specific ANF antagonist. PMID- 1708022 TI - Modulation of the renin-angiotensin system by atrial natriuretic peptide. AB - Many hormonal systems are involved in salt homeostasis and blood pressure control, including the renin-angiotensin-aldosterone system, the kallikrein-kinin system, and atrial and brain natriuretic peptides. Many of these hormonal peptides have actions both in the central nervous system and the periphery that are complementary. There is also increasing evidence that many of them may act as both circulating endocrine systems as well as local paracrine autocrine systems. The atrial natriuretic peptides may be viewed as endogenous inhibitors of the renin-angiotensin system. Angiotensin-converting enzyme (ACE) and renin inhibitors have provided important information on the role of the renin angiotensin system in physiological and pathophysiologic states. The development of specific atriopeptidase inhibitors, kinin antagonists, and kallikrein inhibitors offer the same promise for these systems. PMID- 1708023 TI - Fasting and 24-h urinary sodium/creatinine values in young and elderly women on low-salt and salt-supplemented regimens. AB - A high sodium intake is a risk factor for both hypertension and osteoporosis. High-salt diets cause calciuresis and may influence blood pressure (BP) by stimulating calcium-regulating hormones and augmenting intracellular calcium concentrations. Urinary calcium rises in parallel with urinary sodium. However, aging may influence the rate of excretion of a dietary sodium load. This study compares sodium/creatinine (Na/Cr) values in 2-h urines collected after an overnight fast (12 h) with values obtained in 24-h urines from normotensive women aged (a) 19-23 years (n = 6) and (b) 65-70 years (n = 9) who were consuming either 70 mmol of Na/day [low-sodium regimen (LSR)] or 170 mmol of Na/day [supplemental sodium regimen (SSR)]. Young and elderly women excreted similar amounts of sodium per 24-h period. The 24-h urinary sodium excretion matched dietary sodium input appropriately on LSR and SSR, and SSR raised calcium excretion by 30%. However, whereas in the young women Na/Cr values were similar in fasting and 24-h collections on both LSR and SSR, these values were higher in 24-h than in fasting urines on SSR in the elderly women. We attribute this response to temporal differences in urinary sodium excretion associated with aging. We conclude that assessing sodium excretion from fasting urinary sodium measurements may underestimate total sodium loss (and hence calcium loss) in subjects on a stable sodium intake, particularly in the elderly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708024 TI - Sodium-lithium countertransport is not a marker of proximal tubular sodium clearance. AB - The reported inverse relationship between fractional urinary clearance of lithium (FCLi) and erythrocyte sodium-lithium countertransport (Na-Li CT) in normotensive and hypertensive subjects suggests that Na-Li CT may be a marker of proximal tubular sodium reabsorption. We have refuted this hypothesis in a multiracial study of 57 Caucasian and 48 Maori normotensive and hypertensive males aged 20-40 years. Na-Li CT was measured in vitro by standard Li efflux methodology, and in vivo by the Li cell:plasma (Li C:P) ratio 24 h after a 1 g oral dose of lithium carbonate. The Na-Li CT and Li C:P ratio were not significantly different in the two races and were strongly correlated within race, confirming the validity of the Li C:P ratio as an in vivo index of in vitro Na-Li CT. There was no correlation in either race between in vitro or in vivo erythrocyte membrane sodium transport indices and FCLi. PMID- 1708025 TI - Body sodium in normal subjects predisposed to hypertension. AB - Exchangeable sodium is lower than normal in young male patients with essential hypertension. This may reflect a primary or secondary abnormality. To investigate this question, exchangeable body sodium was measured in 31 normotensive men with positive and 31 normotensive men with negative family history of essential hypertension on a normal sodium intake (150 mmol/day). Blood pressure (BP) tended to be higher in the former group (p less than 0.005) but age, urinary sodium excretion, plasma renin activity, and aldosterone levels or creatinine clearance were comparable. Exchangeable sodium averaged 100.8 +/- 7.1% in subjects with positive and 100.2 +/- 6% in those with negative family history. In both groups, exchangeable sodium was unrelated to arterial pressure. The response of exchangeable sodium to variations in dietary sodium intake was further investigated in 13 subjects with and 10 subjects without family history. The change from a low (17 mmol/day) to a high (270 mmol/day) sodium diet elevated exchangeable body sodium to a comparable extent, despite a greater increase in BP in subjects with positive family history. Sodium-dependent suppression of renin, angiotension II, aldosterone, and plasma catecholamines was comparable between the two groups. These findings suggest that body sodium is normal and adapts normally to variations in dietary sodium intake in normotensive subjects with familial predisposition to hypertension. Body sodium depletion in early hypertension appears to be a secondary rather than a primary event. PMID- 1708026 TI - Dopaminergic activity and water-sodium handling in the kidneys of essential hypertensive subjects: is renal dopaminergic activity suppressed at the prehypertensive stage? AB - To investigate whether the suppression of the renal dopaminergic system in hypertension is primary or secondary, renal dopaminergic activity was compared between young healthy normotensive subjects without a family history of hypertension [FH(-)] and those with a family history of hypertension [FH(+)]. A significant decrease in urinary dopamine excretion was recognized, and the responses of urine volume, urinary sodium excretion, fractional excretion of sodium, and urinary kallikrein and kinin activity to infused dopamine were significantly augmented in FH(+) subjects. In addition, a normal level of L-dopa delivery into the kidney and at the renal proximal tubules and a significant reduction of the conversion from L-dopa to dopamine in the kidney were found in FH(+) subjects. These findings suggest that renal dopaminergic activity is already suppressed at the prehypertensive stage, and a reduction in the conversion from L-dopa to dopamine in the proximal tubules may contribute to the attenuation of renal dopaminergic activity in FH(+) subjects. PMID- 1708027 TI - Kinetics of sodium-lithium countertransport in normotensive and hypertensive subjects. AB - The kinetic parameters Vmax and Km of erythrocyte sodium-lithium countertransport (Na-Li CT), and the lithium cell to plasma ratio (Li C:P) measured 24 h after a 1 g oral dose of Li2CO3, were determined in 14 normotensive (NT) and 14 untreated mild hypertensive (HT) males matched for age and weight. Li C:P and Na-Li CT were strongly correlated (r = -0.73), p less than 0.001), confirming the validity of Li C:P as an in vivo index of Na-Li CT. No differences in Li C:P (NT: 0.27 +/- 0.07; HT: 0.23 +/- 0.06), Na-Li CT (NT: 0.39 +/- 0.10 mmol/L/h; HT: 0.43 +/- 0.10 mmol/L/h), Vmax (NT: 0.58 +/- 0.16 mmol/L/h; HT: 0.68 +/- 0.20 mmol/L/h), or Km (NT: 1.5 +/- 0.4 mmol/L; HT: 1.6 +/- 0.9 mmol/L) were found between NT and HT. Our data do not support the marker concept of erythrocyte Na-Li CT in human hypertension. PMID- 1708028 TI - Salt appetite, body sodium, handling of a NaCl load, renin, and aldosterone in genetically and spontaneously hypertensive rats. AB - Salt appetite, body sodium, handling of a NaCl load, plasma renin activity (PRA), and plasma aldosterone concentration (PAC) were compared in New Zealand genetically hypertensive (GH) and Japanese spontaneously hypertensive rats (SHRs) and their respective normotensive controls [normal Wistar (N) and Wistar-Kyoto (WKY) rats]. Salt appetite was increased in SHRs compared with GH, N, and WKY rats when rats were on salt-free chow and given a choice of distilled water and NaCl solution. Body sodium, measured by whole body counting, was higher in SHRs than in the other strains but did not differ among GH, N, and WKY rats. The rate of excretion of a NaCl load was not increased in GH rats and was slightly increased in SHRs only when on a very low NaCl intake. PRA and PAC (radioimmunoassay) were lower in SHRs than in GH, N, and WKY rats. PAC had a significant negative correlation with body sodium across the four strains. There is no evidence of any abnormality in sodium regulation in GH rats. However, the SHRs have an increased salt appetite and an increased body sodium even when sodium intake is limited; PRA and PAC appear to have responded appropriately to the increased body sodium. PMID- 1708029 TI - Diet and lifestyle in hypertension: changing perspectives. AB - It is now recognized that dietary and other lifestyle or environmental factors are critical for the phenotypic expression of a genetic predisposition to blood pressure (BP) elevation. These environmental factors influence the entire frequency distribution of BPs in any given population, and hence affect the prevalence of hypertension using whatever arbitrary cutoff point is chosen to categorize patients in this way. "Dose-response" relationships with BP have been demonstrated with body fat, alcohol consumption, sodium intake, vegetarian vs. meat-related diets, and physical activity. Possible relationships between these and other "environmental" factors have not yet been fully clarified, although this is of considerable importance for primary prevention of hypertension as well as for specifying advice for individual patients. Knowledge of the extent to which varying dietary or other lifestyle factors operate in different patients is also likely to be necessary for those trying to resolve the nature of the pathophysiological and genetic mechanisms underlying high BP. Finally, several of the factors causing hypertension independently predispose to atherosclerosis, and have compounded the risk of cardiovascular disease in hypertensive patients. PMID- 1708030 TI - Perspectives of hypertension in black patients: black vs. white differences. AB - Hypertension is a major disease in the black population of sub-Saharan Africa and the U.S. The prevalence of hypertension varies from 1-30% of the adult population. Differences in blood pressure (BP) between black and white patients have been documented. In this review, genetic, endocrine, and environmental characteristics, renal physiology, and cardiac function are reviewed. Racial differences in renal physiology and socioeconomic status seem to account for BP differences. Black hypertensive patients in sub-Saharan Africa are prone to cerebral hemorrhage, malignant hypertension leading to uremia, and congestive heart failure, whereas coronary artery disease is uncommon. Responses to hypotensive agents like beta-blockers and angiotensin-converting enzyme inhibitors are poor unless these agents are combined with a thiazide diuretic. Black hypertensive patients respond best to diuretics, vasodilators, or calcium channel blockers. A profiled approach to the treatment of hypertension is suggested. PMID- 1708031 TI - Determinants of blood pressure in the first decades of life. AB - The blood pressure (BP) in children has been studied since the beginning of this century, and in the past decade the potential association between childhood BP levels and adult hypertension has gained increasing interest. From several longitudinal studies, many of them comprising large numbers of children and youngsters, it appears that the BP of children is significantly associated with BP on follow-up measurements and that childhood BP is related to adult levels. Whether the objective is to predict future BP, or the aim is to shed light on the early pathogenesis of primary hypertension, it is of major importance to find out why BP rises in some and stays the same in others. To achieve this, characteristics need to be detected that are related to changes in BP in the first decades of life. Although not many reports on these dynamic relations are presently available, age, height, body weight, initial BP level, and a family history of hypertension have been put forward as determinants of children's BP change over time. Moreover, there are data to support the effect of dietary factors, most notably certain electrolytes, on BP regulation early in life. Also, certain hemodynamic and neural characteristics, such as changes in cardiac output and left ventricular mass, renal blood flow, and sympathetic nervous system activity, may be related to a subsequent rise in BP and future hypertension. Findings from nonexperimental and experimental studies on determinants of BP in children and youngsters will be reviewed. PMID- 1708032 TI - Coronary artery disease can be prevented by antihypertensive therapy: experiences from the MAPHY Study. AB - The present randomized primary prevention study in hypertensive men aged 40-64 years (n = 3,234) was aimed at investigating whether metoprolol given as initial treatment would prevent coronary artery disease (CAD) better than thiazide diuretics. Two hundred fifty-five patients had a definite CAD event during the 15,730 patient-years of follow-up; 25% of these events were fatal, and 38% were definite acute myocardial infarctions. The incidence of CAD was significantly lower during follow-up in patients randomized to metoprolol than in patients randomized to diuretics: 111 vs. 144 cases (p = 0.001). Stroke mortality was significantly lower in the metoprolol group than in the diuretic group, but the overall stroke incidence was similar in the two treatment groups. A majority of events occurred among smokers in both treatment groups although only one-third of patients were smokers at baseline. Blood pressure (BP) control was similar in the two treatment groups; therefore, the difference between the groups in CAD events is mediated via mechanisms other than the BP-reducing effect of metoprolol. PMID- 1708033 TI - Smoking, antihypertensive treatment benefit, and comprehensive antihypertensive treatment approach: some thoughts on the results of the International Prospective Primary Prevention Study in Hypertension. AB - Several major studies investigated the possibility of a primary preventive effect of beta-blockers. The International Prospective Primary Prevention Study in Hypertension (IPPPSH) compared a beta-blocker-containing vs. a non-beta-blocker containing antihypertensive regimen in 6,357 moderate-severe hypertensive men and women treated over 3-5 years. Blood pressure (BP) control was comparable with either regimen. beta-Blocker treatment was associated with less hypokalemia, earlier electrocardiogram normalization, and fewer withdrawals for uncontrolled hypertension. In agreement with the Medical Research Council (MRC) trial on mild hypertension and the Heart Attack Primary Prevention in Hypertension (HAPPHY) trial, but at variance with the Primary Prevention Metoprolol in Patients with Hypertension (MAPHY) study, cardiac event rates were similar in beta-blocker- and non-beta-blocker-treated patients. With either regimen, in-study BP reduction was associated with a lower rate of stroke as well as of cardiac events. In a subgroup analysis, nonsmokers appeared to derive beta-blocker benefit, the results being similar to those of the MRC. Smokers required higher doses of drugs to achieve diastolic target pressure, had a higher heart rate and hematocrit, and a higher cardiac event rate than nonsmokers at any given level of diastolic pressure. Except for the MAPHY trial, these primary prevention studies do not support the concept of a cardiac primary preventive effect of antihypertensive beta-blockade but stress the importance of good BP control and a comprehensive risk factor prevention approach in the management of hypertensive patients. PMID- 1708034 TI - Changing pattern of cardiovascular disease in the Japanese population in relation to hypertension control programs. AB - The changing pattern of cardiovascular disease during the last 30 years in Japan is described. The most striking change was a reduction in deaths caused by hypertensive cerebral hemorrhage. There was no increase in ischemic heart disease in the rural area studied but such a trend was shown in the urban area. A change in risk factors for cardiovascular disease, reduction in blood pressure, and increase in serum cholesterol were more marked in the rural than the urban area. Institution of a hypertension control program and marked changes in lifestyle, including diet, could explain the relatively rapid change in the frequency and the type of cardiovascular disease, particularly cerebral stroke. The prevention of atherosclerotic complications such as cerebral infarction and ischemic heart disease is becoming a more important task in preventive cardiology. PMID- 1708035 TI - Hypertension and cardiovascular diseases in an epidemiological study in Hokkaido, Japan. AB - The present study describes the results from the 10-year follow-up data of a prospective epidemiological study for hypertension and cardiovascular diseases in two communities of rural agricultural districts in Hokkaido, Japan. The number of incidences of cerebrovascular accidents (CVAs) in persons who were normotensive, borderline hypertensive (BHT), untreated hypertensive (HT), well-controlled HT [blood pressure (BP) less than 150/90 mm Hg], and poorly controlled HT (BP greater than or equal to 150/90 mm Hg) were 0.46, 3.24, 4.17, 3.49, and 12.76 per 1,000 person-years. respectively: CVAs were markedly high in poorly controlled HT persons. The winter-summer mean BP differences in the first year were significantly and positively correlated with the differences in mean BP between the tenth and the first year, and were significantly higher in the progression to hypertension group than in the nonprogression group in both towns. Multivariate analysis indicated that the winter-summer mean BP difference was a significant variable for indication of progression to hypertension. From these results, we concluded that (a) good control of hypertension could considerably prevent CVA, (b) cold environment may contribute to the progression to hypertension, and (c) winter-summer variation in BP may predict the future course of BP. PMID- 1708036 TI - Risk predictors in treated hypertension. AB - The data for patients referred to the Dunedin Hypertension Clinic (975 men, 1,348 women) between 1953 and 1977 have been examined by the Cox proportional hazards method for significant age-corrected predictors of 8-year cardiovascular and total mortality. For men, some significant predictors were systolic and diastolic blood pressure (BP), indices of target organ damage (heart and eyes) and smoking at presentation, and achieved BP and serum cholesterol at follow-up. For women, serum cholesterol, diabetes, target organ damage (heart and eyes) and smoking at presentation, and achieved BP level at follow-up were predictors. Relative risk for total or cardiovascular mortality was increased in both sexes most by smoking and by increased levels of achieved BP. PMID- 1708037 TI - Blood pressure, change in blood pressure, and cardiovascular event rates in placebo-treated patients in the Medical Research Council Trial. AB - The relationship between cardiovascular event rate (stroke + coronary events), entry blood pressure (BP), change in BP over 6 months, and other variables was examined in the 8,654 placebo-treated patients in the Medical Research Council (MRC) trial. Entry BP was greater than screening pressures and fell significantly after randomization to placebo. The entry pressure was significantly greater but the fall after entry was less in those who had a cardiovascular event during 5.5 years of follow-up. Thus, patients with high entry pressure that remained elevated during follow-up had the highest event rates. This pattern was marked for stroke but was also found for coronary events. Other variables strongly related to event occurrence were sex, age, and smoking habit. Discriminant analysis based on these variables correctly identified 67% of those who had an event but incorrectly classified 40% of the much larger group who did not. These results suggest that failure of BP to fall after screening examinations is a cardiovascular risk factor. Thus, high-risk patients may be identified by repeated measurements of BP before treatment is started. PMID- 1708039 TI - Fallacies in the interpretation of the large-scale trials of treatment of mild to moderate hypertension. AB - Data from large-scale trials of treatment of mild to moderate hypertension are being misinterpreted and unjustifiably extrapolated to general populations. The main errors include acceptance of "entry" pressures as typical in spite of evidence that true blood pressure was much lower, extrapolation of results in low risk volunteers to the whole population, neglect of treatment given to the most endangered control subjects, and the assumption that the burden of side effects seen in a rigid high-dose trial is typical of that seen when the same or better drugs are used in clinical practice. The health risks of hypertension and the benefits conferred by antihypertensive therapy are being played down unjustifiably on the basis of inappropriate data. PMID- 1708038 TI - Flow cytometric analysis of cell cycle of cultured aortic smooth muscle cells from two strains of genetically hypertensive rats. AB - The growth curves of aortic smooth muscle cells (SMCs) from spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar-Kyoto (WKY) rats, and genetically hypertensive (GH) rats and their normotensive outbred controls (N) were compared. The proportion of cells in the various phases of the cell cycle was measured by flow cytometry. SHR cells showed significantly shorter doubling time than WKY rat cells, but doubling times for GH and N rats did not differ and were nearly the same as for SHRs. SHRs showed significantly greater proportion of S-phase (DNA-synthesizing) cells compared to WKY rat cells in the exponential growth phase. No significant difference was observed between GH and N rats in the proportion of cells in other phases of the cell cycle. GH and N rat cells stopped dividing in fetal calf serum (FCS)-free Dulbecco's Modified Eagle Medium (DMEM), whereas SHR and WKY rat cells continued to progress through the cell cycle in serum-free DMEM. The responses of FCS-deprived-arrested cells to the addition of FCS were similar in GH and N rats. SHRs showed a prolonged increase in S-phase cells in response to FCS compared to WKY rat cells, but maximum effects were similar. Enhanced cell proliferation in SHRs is not present in the other hypertensive strain of rats and is not explained by a simple hyperresponse to FCS. PMID- 1708040 TI - Future directions for randomized trials of cardiovascular disease prevention in hypertensive patients. AB - Coronary artery disease (CAD) is the major cause of death in hypertensive patients in Western populations and further efforts are required to determine how best to reduce its incidence. Large-scale placebo-controlled trials of blood pressure reduction could resolve the uncertainty about the effects of antihypertensive treatment on CAD incidence, but at present, such trials could probably only be conducted in patients with mild hypertension (or normotension). If such patients were selected on the basis of clinically manifest vascular disease, the sample size necessary to detect plausible treatment effects may be practicable (i.e., several thousand patients). In moderately or severely hypertensive patients, "active-controlled" trials could be conducted to determine the relative effects of different anti-hypertensive drugs on CAD morbidity and mortality. However, because any difference between such regimens is likely to be modest in magnitude (and smaller than the difference between treatment and no treatment), the sample size required to detect such differences would be very large (i.e., tens of thousands). With somewhat smaller numbers of patients, it would be possible to conduct placebo-controlled trials to study the effects on CAD of other potentially cardioprotective interventions, such as cholesterol reduction and aspirin, both of which may offer effective means for CAD prevention in hypertensive patients. PMID- 1708041 TI - Interactions between enalaprilat and doxazosin at rat tail artery alpha 1 adrenoceptors. AB - Using paired isolated perfused rat tail artery segments, it was found that enalaprilat, an ACE inhibitor, augmented 1.6-fold the contractile responses to phenylephrine (PE), an alpha 1-adrenoceptor agonist. Similarly, enalaprilat potentiated 1.9-fold the alpha 1-adrenoceptor antagonist activity of doxazosin in paired rat tail artery segments. In rats treated with deoxycorticosterone acetate (DOCA) 20 mg/kg i.m. twice weekly for 5 weeks, plasma renin activity fell from a control value of 5.73 +/- 0.93 to 0.04 +/- 0.01 ng of AI/ml/h. The inhibition of circulating renin activity in these animals was associated with a loss of the potentiating effects of enalaprilat upon the alpha 1-adrenoceptor antagonist action of doxazosin. The results are interpreted as indicating that angiotensin II (AII) can modulate the functional activity of alpha 1-adrenoceptors in vascular smooth muscle. PMID- 1708042 TI - SD-3211, a novel benzothiazine calcium antagonist, alone and in combination with a beta-adrenoceptor antagonist, produces antihypertensive effects without affecting heart rate and atrioventricular conduction in conscious renal hypertensive dogs. AB - We compared the effects of SD-3211, a novel calcium antagonist, on blood pressure, heart rate, and atrioventricular conduction with those of diltiazem using conscious renal hypertensive dogs (one-kidney, one-clip type). We also examined the combined effects of these calcium antagonists with a beta adrenoceptor antagonist, propranolol, on these variables. Oral administration of SD-3211 (1.25, 2.5, and 5 mg/kg) resulted in a dose-dependent decrease in blood pressure without affecting heart rate. SD-3211 at all three doses significantly decreased systolic blood pressure. At 2.5 and 5 mg/kg the compound elicited significant decreases in mean blood pressure and diastolic blood pressure. Hypotension obtained with the highest dose of SD-3211 lasted for at least 9 h. No significant alteration in PR interval was observed in electrocardiograms after administration of SD-3211. Diltiazem, given orally at doses of 2.5 and 5 mg/kg but not 1.25 mg/kg, produced significant hypotension with little change in heart rate. The duration of hypotension induced by the highest dose of diltiazem was only 3 h. Diltiazem prolonged PR interval in a dose-dependent manner, causing second-degree atrioventricular block in some dogs. Combined administration of SD 3211 or diltiazem (2.5 mg/kg) with propranolol (30 mg/kg) resulted in enhanced hypotension with no alteration in heart rate. SD-3211 plus propranolol had little effect on the PR interval, whereas diltiazem plus propranolol caused a markedly enhanced prolongation. These results indicate that SD-3211 is an antihypertensive agent with long-lasting action and little effect on heart rate and atrioventricular conduction and, when administered alone or in combination with propranolol, may be useful in the treatment of hypertension. PMID- 1708043 TI - Stimulation of prostacyclin synthesis by defibrotide: improved contractile recovery from myocardial "stunning". AB - Prostacyclin (PGI2) improves regional contractility of postischemically dysfunctional ("stunned") myocardium. We determined whether defibrotide, a fraction of mammalian DNA known to stimulate endogenous formation of PGI2, also improves contractile recovery of stunned myocardium. Anesthetized, open-chest minipigs were subjected to coronary occlusion of 5 min (left anterior descending branch, LAD) followed by 120 min of reperfusion. The animals were treated with defibrotide (32 mg x kg-1 x h-1, intravenously, i.v.) or vehicle throughout the experimental period. Defibrotide improved regional contractility in the ischemic reperfused area from 30 (vehicle) to 78% of the preischemic control without altering the contractility of nonischemic myocardium. Transcardiac PGI2 formation, determined from the difference between coronary venous and arterial plasma concentrations, was elevated from 437 (preischemic control) to 869 pmol x l-1 in defibrotide-treated animals, but was unchanged in the vehicle-treated and a sham-operated group. Thromboxane A2 (TXA2) release was not modified. Defibrotide reduced ischemia-induced formation of platelet aggregates but did not affect the activity of polymorphonuclear neutrophil granulocytes. The data demonstrate an improvement of contractile recovery from stunning by defibrotide that may be related to an inhibition of ischemia-induced platelet activation and (or) membrane protection owing to enhanced transcardiac formation of PGI2. PMID- 1708044 TI - Mode of action of a new class IC drug (ORG 7797) against atrial fibrillation in conscious dogs. AB - In five chronically instrumented conscious dogs, we studied the antifibrillatory action of the experimental class IC drug Org 7797 ((16 alpha,17 beta)-17 methylamino-oestra-1,3,5(10)-triene-3,16-diol- (Z)-2-butenedioate). Under control conditions, paroxysms of atrial fibrillation were induced by burst pacing (50 Hz; inducibility 100%) and persisted on the average for greater than 3 min. Org 7797 (1, 2, and 3 mg/kg/h) significantly reduced both inducibility and duration of atrial fibrillation to 25 and 10%, respectively. To elucidate the electrophysiologic mechanisms of this potent antifibrillatory action, we measured the effects of Org 7797 on conduction velocity, effective refractory period (ERP), and wavelength of the atrial impulse. Org 7797 decreased atrial conduction velocity significantly by 18-25% and lengthened ERP by 18-29% (pacing 2-5 Hz). The maximal pacing frequency (Fmax) was decreased from 8.3 to 6.2 Hz. During Fmax, Org 7797 decreased the conduction velocity by 23% and lengthened ERP by 75%, resulting in a prolongation of the wave-length from 9.8 +/- 2.3 cm (control) to 13.7 +/- 2.6 cm (3 mg/kg/h; p less than 0.01). These results indicate that the antifibrillatory action of Org 7797 is based on diminution of the physiologic rate-dependent shortening of refractoriness, resulting in a prolongation of the wavelength during maximal heart rates (HRs). This electrophysiologic effect of the drug will decrease the number of multiple wavelets during fibrillation, thus increasing the statistical chance of spontaneous termination of the fibrillatory process. PMID- 1708045 TI - Ontogeny of the adenylate cyclase system in the pulmonary vascular smooth muscle of the fetal lamb. AB - The enzyme adenylate cyclase (AC) plays a critical role in regulation of vasodilatation in the developing pulmonary circulation. We characterized the ontogeny of the function of the AC system in the pulmonary vascular smooth muscle (VSM) of fetal lambs during the third trimester. Basal (nonstimulated) AC activity and activity with targeted stimulation of the components of the AC system were determined in pulmonary VSM plasma membranes from fetal lambs at 110 115 days (F-1) and 125-135 days of gestation (F-2) (with term being 144 +/- 3 days) and from pregnant ewes (adult) for comparison. We assessed beta-adrenergic receptor-mediated activity so that we could perform parallel studies of the VSM receptor binding characteristics. Basal AC activity declined 48% from F-1 to F-2 and an additional 13% from F-2 to adult. beta-Adrenergic receptor-stimulated activity was demonstrable only in adult pulmonary VSM membranes even though receptor density and affinity for the agonist were similar in fetal and adult pulmonary VSM. AC activity with NaF stimulation at the level of the G proteins declined 65% from F-1 to F-2 and was similar in F-2 as compared with adult. This indicates that the function of the G proteins or the catalytic subunit of the enzyme decreases from F-1 to F-2. The latter is suggested by the observation that AC activity with direct stimulation of the catalytic subunit with forskolin decreased 45% over this time period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708046 TI - Platelet function during antihypertensive treatment with quinapril, a novel angiotensin converting enzyme inhibitor. AB - The effect of various antihypertensive medications on platelet function is of increasing interest. Conflicting effects of captopril on platelet function are reported and the impact of angiotensin converting enzyme (ACE) inhibitors not containing a sulfhydryl group such as enalapril, lisinopril, and quinapril on platelet function remains unstudied. Therefore, the aim of the present study was to examine the effect of antihypertensive treatment with quinapril, a novel ACE inhibitor not containing a sulfhydryl group, on platelet function. Ten white men (age range of 32-61 years) with untreated mild-to-moderate essential hypertension (supine diastolic blood pressure greater than 95 mm Hg) were treated with 4 weeks each of placebo and quinapril in a double-blind, randomized, crossover design. Quinapril (20 mg twice a day) significantly lowered systolic (p less than 0.01) and diastolic blood pressure (p less than 0.01) without any significant effect on heart rate or plasma catecholamines. No significant change was noted for in vitro platelet aggregation induced by epinephrine, ADP, or collagen. Plasma concentrations of the platelet release factors beta-thromboglobulin and platelet factor 4 did not change, nor did the platelet content of norepinephrine, platelet weight (mg/10 ml of blood), circulating platelet count, or platelet size. Thus, as assessed by a broad spectrum of platelet parameters, we found that antihypertensive treatment with quinapril has no significant effect on platelet function in patients with mild-to-moderate essential hypertension. These "platelet-neutral" properties of quinapril suggest that quinapril, both from a thromboembolic and a hemostatic point of view, may be a rather safe agent for treatment of hypertension. PMID- 1708047 TI - Cardiovascular and renal actions of AHR-16303B, an antagonist of 5-HT2 receptors and calcium channels, in hypertensive and normotensive rats. AB - The cardiovascular and renal responses to AHR-16303B, a novel antagonist of 5 hydroxytryptamine (5-HT2) receptors and calcium channels, were examined in spontaneously hypertensive (SHR) and normotensive rats (NTR) and compared with verapamil and ritanserin. In SHR, AHR-16303B (10-300 mg/kg orally, p.o.) produced dose-related reductions in mean arterial blood pressure (MABP), accompanied by modest isokaliuretic diuresis and unchanged heart rate (HR). In NTR, 10-30 mg/kg p.o. AHR-16303B had no effect on MABP or renal excretory function; 100 and 300 mg/kg reduced MABP but had only transient effects on HR; 100 mg/kg produced antidiuresis in NTR. Both strains of rats tolerated doses of AHR-16303B as high as 300 mg/kg. In both SHR and NTR, verapamil (10-100 mg/kg p.o.) produced dose related reductions in MABP, antinatriuresis at 60 and 100 mg/kg, and variable effects on HR. Oral ritanserin had no effect on MABP of SHR or NTR at 3 or 10 mg/kg. AHR-16303B is unique in that it simultaneously antagonizes 5-HT2 receptors and produces safe and effective reduction of elevated BP without altering HR or triggering renal compensatory antidiuresis. At effective 5-HT2/calcium antagonistic doses, AHR-16303B has no effect on cardiorenal homeostasis in normotensive animals. PMID- 1708048 TI - Hemodynamic and myocardial energetic effects of CK-3197, a selective positive inotropic agent. AB - CK-3197 was developed as a selective positive inotropic agent for the treatment of congestive heart failure. We compared the hemodynamic and myocardial energetic effects of CK-3197 to ouabain in the pentobarbital-anesthetized dog. Fifteen minutes after intravenous (i.v.) administration of CK-3197 (0.1, 0.3, and 1.0 mg/kg) to five dogs, mean left ventricular (LV) dP/dt increased by 24, 68, and 109% and mean arterial pressure (MAP) decreased by 4, 9, and 18%, respectively, from basal values. CK-3197 was 11 times more potent as a positive inotropic agent than as a vasodilator. Heart rate (HR) increased by 5, 14, and 24% after these doses of CK-3197, whereas LV end diastolic pressure (LVEDP) decreased by 4 mm Hg after the highest dose of compound. LV oxygen consumption (MVO2) and stroke MVO2 increased by 9, 25, and 102% and 1, 8, and 58%, respectively, at the peak of the increases in LV dP/dt. Ouabain (0.02 and 0.03 mg/kg, i.v.) increased MAP (12 and 22%), HR (2 and 20%), and LV dP/dt (19 and 36%), with a 14 and 16% increase in LV MVO2 and a 12 and -6% change in stroke MVO2. Thus, CK-3197 is a selective, positive inotropic agent with preload reducing activity in the dog. CK-3197, similar to ouabain, produced energy-efficient positive inotropic responses with either no increase in MVO2 or increases in myocardial oxygen consumption that were less than the expected 1:1 ratio with LV dP/dt. Therefore, CK-3197 may have significant utility in the clinical treatment of congestive heart failure. PMID- 1708049 TI - Altered responsiveness of saphenous vein grafts to norepinephrine and tyramine: relation to tissue catecholamine stores. AB - This study was designed to investigate early changes in reactivity in relation to the adrenergic innervation of venous grafts. Saphenous vein grafts were implanted into the carotid artery by the end-to-end technique in mongrel dogs. After 1 week, the grafts were harvested and dose-response curves to norepinephrine and tyramine were determined and compared with those of nongrafted saphenous veins and carotid arteries. Tissue norepinephrine levels of the blood vessels were measured by HPLC with electrochemical detection. Grafted vessels demonstrated an enhanced sensitivity to norepinephrine and a significant attenuation to tyramine. Additionally, grafted saphenous veins exhibited a significant depletion of norepinephrine content when compared to nongrafted veins. These differences suggest that denervation of the saphenous vein produces a supersensitivity to catecholamines that could account for enhanced vascular reactivity observed following implantation of saphenous vein grafts. PMID- 1708050 TI - Probucol reduces myocardial dysfunction during reperfusion after short-term ischemia in rabbit heart. AB - The effects of probucol, a lipophilic antioxidant, on the myocardial dysfunction (stunning) observed during reperfusion after 15-min ischemia in rabbit heart were studied. Rabbits received food with or without 1% probucol for 3 weeks. They were then anesthetized and prepared for recording of myocardial segment shortening, arterial blood pressure (BP), left ventricular pressure (LVP), rate of development of LVP (dP/dt), and a lead II ECG. Regional myocardial ischemia was produced by acute occlusion of the first marginal branch of the left coronary artery. Myocardial segment shortening was depressed after reperfusion in control rabbits. In comparison, myocardial segment shortening was significantly greater in probucol-treated rabbits than in control rabbits during reperfusion, indicating a beneficial effect. No hemodynamic or ECG changes measured could explain this difference. The number of premature ventricular contractions was reduced in the probucol-treated group, although the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF) were not. Concentrations of probucol in serum and heart of five rabbits were 15.0 +/- 1.2 micrograms/ml and 17.5 +/- 2.5 micrograms/g (mean +/- SEM), respectively. Only probucol concentrations in the serum were positively correlated with the improvement in myocardial segment shortening (r = 0.91, p = 0.03). We conclude that a clinically relevant serum concentration of probucol reduces ischemia-induced myocardial stunning in the rabbit. PMID- 1708051 TI - Effects of hydroxyethyl starch conjugated deferoxamine on myocardial functional recovery following coronary occlusion and reperfusion in dogs. AB - The effect of hydroxyethyl starch-conjugated deferoxamine (HES-DFO) on the recovery of regional myocardial function after 15 min of coronary artery occlusion followed by 3 h of reperfusion of the left anterior descending coronary artery (stunned myocardium) was investigated in anesthetized dogs. Regional myocardial blood flow was measured by radioactive microspheres and regional myocardial segment shortening (%SS) by sonomicrometry. HES-DFO (equivalent of 50 mg/kg DFO), iron saturated HES-DFO (HES-FO), deferoxamine (DFO, 50 mg/kg), or saline were administered by intravenous infusion starting 30 min before occlusion and throughout occlusion. Ischemic bed size and collateral blood flow were similar in all four groups. HES-DFO significantly improved %SS in the ischemic reperfused region during reperfusion; however, HES-FO and DFO had no effect on %SS as compared to the saline-treated group. HES-DFO and HES-FO had no effect on hemodynamics; however, DFO produced a marked reduction in systemic blood pressure. Since HES-FO had no effect on the recovery of %SS, the beneficial effect of HES-DFO is thought to be due to its iron chelating characteristics. Plasma concentrations of HES-DFO not only reached a higher peak level but also had a longer half life (3 h) than that of regular DFO (20 min). Thus, high molecular-weight HES-DFO is effective in enhancing the recovery of regional wall motion in stunned myocardium. The reason for the lack of efficacy of DFO compared to HES-DFO at this high dose may be related to the formation of a toxic deferoxamine free radical species. PMID- 1708052 TI - Endotoxin tolerance differentially alters hemodynamic responses to a thromboxane A2 mimetic and phenylephrine. AB - Repeated sublethal doses of endotoxin render rats tolerant to lethal doses of endotoxin and reduce thromboxane (Tx) A2 synthesis. Endotoxin-tolerant rats are also more resistant to lethal doses of U46619, a stable TxA2 mimetic. These observations raised the possibility that tolerance may alter hemodynamic responses to TxA2 via one or more mechanisms. Mean arterial pressure (MAP) responses to i.v. injections of the stable TxA2 mimetic U46619 at doses ranging from 0.17 to 8.4 micrograms/kg were determined. Despite an initial lower systemic vascular resistance in tolerant rats compared to control rats (2.4 +/- 0.3 vs 3.1 +/- 0.2 mm Hg/ml/min/100 g of body weight, respectively, p less than 0.05), the maximum pressor response to U46619 was significantly greater (p less than 0.05) at the higher doses of U46619 in tolerant rats compared to control rats. Tolerant and control rats also exhibited qualitatively different changes in MAP in response to U46619. Control rats manifested an initial hypotensive response (15 s) not observed in tolerant rats. In contrast, tolerant rats exhibited no depressor response, but a higher peak pressor response to U46619 than that seen in controls. Since prostaglandins may modulate vascular responses to U46619, subsequent studies were conducted in indomethacin-pretreated or essential fatty acid (EFA) deficient rats that were depleted of arachidonic acid substrate. Either indomethacin or EFA deficiency significantly prevented the initial hypotensive response in control rats, suggesting that prostaglandins may mediate this response to U46619. In additional studies, the MAP response in tolerant and control rats to the alpha 1-adrenergic agonist phenylephrine was determined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708053 TI - Vascular interactions of calcium and reactive oxygen intermediates produced following photoradiation. AB - This study was designed to examine vascular smooth muscle contractile properties following enhanced production of reactive oxygen intermediates (ROIs), which were produced by pretreating rat caudal arteries and aortas with a photosensitizer, hematoporphyrin derivative, and then illuminating them with red laser light. This treatment produced a long-lasting contraction that was dependent on the presence of extracellular calcium. Reduction in extracellular calcium relaxed the smooth muscle and replacement of calcium 30 min later increased the tension. Oxygen radical scavengers did not block the contractile effect postillumination when calcium was returned to the bathing solution; however, verapamil (5.5 microM) and nifedipine (10 microM) attenuated this contraction. The contractions were dependent on oxygen in the aerating gas mixture. Production of ROIs by isolated blood vessels was supported biochemically by a significant increase in both bath and tissue levels of oxidation products, reactive with thiobarbituric acid, and by a reduction in the tissue stain, nitroblue tetrazolium. These ROI-induced contractions were observed in vitro on large conduit arteries and also in vivo on small ear arteries. The vascular response following this acute production of ROIs may be similar to vascular abnormalities in certain pathological conditions where ROI production is reported to be elevated. Therefore, these results could contribute to a further understanding of mechanisms involved in these ROI dependent vascular changes. PMID- 1708054 TI - Effects of cilazapril, a novel angiotensin converting enzyme inhibitor, on the structure of pulmonary arteries of rats exposed to chronic hypoxia. AB - Chronic hypoxia is known to be associated with a thickening of the media of pulmonary arteries. The goal of the present study was to assess if cilazapril, a novel long-acting angiotensin converting enzyme (ACE) inhibitor, could prevent this thickening. For this purpose, three groups of rats were studied. One group was kept in normal room air. Two other groups were exposed to chronic hypoxia (inspired fraction of oxygen equal to 8% during 4 weeks). One group of hypoxic rats was treated with placebo and the other group received cilazapril (as food admixture of approximately 3 mg/kg/day). After 4 weeks, rats were anesthetized and pulmonary artery pressure and hematocrit measured. Then, the lungs were perfused and fixed and morphometry of the pulmonary arteries was performed. Hypoxia induced an increase in pulmonary artery pressure and hematocrit associated with a dramatic increase in the thickness of the media of the pulmonary arteries. Cilazapril completely prevented the thickening of the media of the pulmonary arteries but did not significantly decrease the pulmonary artery pressure or right ventricular weight. PMID- 1708055 TI - AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels. AB - In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) [3H]ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADP (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) [3H]nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing. PMID- 1708056 TI - Effects of pirmenol on electrical induction of sustained ventricular tachycardia in a seven-day-old canine myocardial infarction. AB - Effects of pirmenol on electrical induction of sustained ventricular tachycardia (VT) were examined in 14 dogs with 7-day-old myocardial infarctions. Before administration of the drug, sustained VT was induced in 8 of 14 dogs. After administration of 3 mg/kg pirmenol, induction of VT was suppressed in 2 dogs but remained inducible in 6 dogs. After cumulative administration of 5 mg/kg pirmenol, VT was no longer inducible in 3 dogs but in the other 3 dogs VTs were still inducible at increased cycle lengths. After 7 mg/kg pirmenol, VT was not inducible in the remaining three dogs. Arrhythmias could not be provoked in any postinfarction dogs after pirmenol administration. Plasma concentrations after sequential and cumulative administration of 3, 5, and 7 mg/kg pirmenol averaged 0.43, 0.65, and 1.15 micrograms/ml, respectively. Administration of pirmenol increased the effective refractory period (ERP) and paced QRS duration in both the normal and infarcted ventricular myocardium. In the infarcted myocardium, prolongation of the ERP for the second and third extrastimuli was greater than for the first one (p less than 0.05). Results indicate that pirmenol is effective for prevention of sustained VT owing to prolongation of both the ERP and conduction time in recent myocardial infarction. PMID- 1708057 TI - Opioids potentiate contractile response of rabbit myocardium to the beta adrenergic agonist isoproterenol. AB - Opioid agonists and antagonists have both been reported to augment myocardial contractile force in vitro. We reported that the strong opioid agonists morphine and levorphanol, the weak agonist dextrorphan (an optical isomer of levorphanol), and the opioid antagonist naloxone all potentiate the stimulatory effects of the beta-adrenergic agonist isoproterenol on isometric tension generated by isolated rabbit right ventricular myocardium. The EC50 of isoproterenol was found to be shifted leftward 2.7-, 5.4-, 5.3-, and 3.4-fold respectively (p less than 0.05 when compared with controls), when the opioids were added at a final concentration of 1 x 10(5) M. Lower concentrations of opioid or antagonist did not potentiate the effects of isoproterenol. The rank order potency for potentiation thus differs markedly from that of opioid analgesia. The observed potentiation is therefore not agonist specific and not stereospecific. Furthermore, the drugs alone at a range of concentration from 10(-8) to 10(-5) M had no effect on isometric tension generated. We conclude that opioid agonists and antagonists potentiate the response of ventricular myocardium to the effects of beta-adrenergic stimulation by a novel mechanism unrelated to the binding of these drugs to opioid receptors. The paradoxical augmentation of myocardial contractility by either class of agent under a variety of clinical and experimental conditions is thus explained by these findings. Either agent may interact with myocardial tissue to cause increased sensitivity to stimulation by circulating catecholamines. PMID- 1708058 TI - Constrictive effect of serotonin on visible renal arteries: a pharmacoangiographic study in anesthetized dogs. AB - Serotonin (5HT) has been implicated in the pathogenesis of several arterial disorders. Experimental intrarenal infusion of 5HT has produced initial decreases, and later sustained increases in total renal blood flow in the dog, and flow limiting renal artery spasm in other species. We assessed renal angiographic responses to 5HT infused into one renal artery of nine dogs before and after treatment with indomethacin (I). On blinded assessment of arteriograms, 5HT-induced narrowing was detected (p less than 0.001), and the effect was accentuated by I (p less than 0.001). Ketanserin (K), a 5HT-2 receptor antagonist, reversed the arteriographic vasoconstrictor effect of 5HT in five I treated dogs (p less than 0.05). Linear cortical defects and decreased contrast intensity were seen in the nephrogram phase during 5HT infusion after I. This appearance resembled the arteriographic findings in hemorrhagic shock, suggesting profound impairment of renal cortical perfusion. This study suggests that 5HT constricts visible renal arteries via interaction with the 5HT-2 receptor, and that this in vivo effect is modified by arterial synthesis of vasodilator prostaglandins in the anesthetized dog. PMID- 1708059 TI - Effects of verapamil and nifedipine on mechanisms of arrhythmia in an in vitro model of ischemia and reperfusion. AB - The effects of verapamil and nifedipine on cellular mechanisms of arrhythmia were examined in isolated canine Purkinje fiber-papillary muscles. Microelectrode recordings were made simultaneously from both tissues. Preparations were superfused with Tyrode's solution modified to mimic specific conditions of ischemia for 40 min with or without calcium channel blockers. Verapamil or nifedipine resulted in significantly greater depolarization of Purkinje tissue in response to ischemic conditions and increased the incidence of inexcitability or conduction block in Purkinje and muscle tissues. These calcium channel blockers caused only minor changes in ischemia-induced depolarization of muscle. In Purkinje tissue, return to nonischemic conditions in the absence of drugs caused, in sequence, oscillatory afterpotentials, temporary depolarization to inexcitability, and a phase of automaticity at low membrane potential. These events did not occur in muscle. Verapamil or nifedipine abolished oscillatory afterpotentials and low membrane potential automaticity in Purkinje tissue. However, reperfusion-induced depolarization and inexcitability of Purkinje tissue was delayed but not attenuated. This study demonstrates that verapamil or nifedipine exacerbate depolarization and depression of conduction in Purkinje tissue exposed to ischemic conditions. However, verapamil and nifedipine suppress some but not all potential mechanisms of arrhythmia induced by reperfusion. PMID- 1708060 TI - The effect of cilazapril, a new angiotensin converting enzyme inhibitor, on peak and trough blood pressure measurements in hypertensive patients. AB - Cilazapril (CLZ) is a new, long-acting nonsulfhydril converting enzyme inhibitor (ACE-I). Its effect on peak and trough sitting diastolic blood pressure (SDBP) was studied in a total of 85 patients with uncomplicated, essential hypertension at three centers. After 4 weeks of a single-blind placebo (PLA) run-in period, patients whose SDBP was between 100 and 115 mm Hg, were randomized into active treatment with either PLA (n = 27), CLZ 2.5 mg (n = 29), or CLZ 5 mg (n = 29) once daily in a double-blind fashion for another 8 weeks. At the end of the PLA run-in and after 4 and 8 weeks active therapy, an hourly blood pressure (BP) profile during 1-10 and 21-24 h postdose was performed. The drop in SDBP at the end of the active treatment period at peak and trough was statistically and clinically significant for both CLZ doses in comparison with the PLA group. The peak/trough ratio after subtraction of the PLA effect was 62% for CLZ 2.5 mg and 59% for CLZ 5 mg. These results indicate that the dose regimen of CLZ 2.5-5 mg once daily is adequate and effective for 24 h. PMID- 1708061 TI - Myocardial contractile behavior of a new sotalol derivative. AB - The effects of E4031, a new class III antiarrhythmic agent similar to sotalol, were tested in isometrically contracting rabbit papillary muscles and in anesthetized, open-chest dogs. In papillary muscles, E4031 caused a modest dose dependent increase of 26 +/- 8% in developed tension and 38 +/- 8% in its maximal rate of rise. Since there was no significant change in the maximal rate of relaxation, the ratio between both maximal velocities increased from 0.92 +/- 0.03 to 1.19 +/- 0.10. Time to peak tension did not change significantly, whereas time to half relaxation increased from 72 +/- 3 to 85 +/- 4 ms. The effective refractory period in the rabbit papillary muscles increased from 179 +/- 10 to 414 +/- 45 ms. In the open-chest dog, the i.v. administration of E4031 did not induce significant changes in heart rate, mean arterial pressure, or left ventricular end diastolic pressure. +dP/dt increased from 1,839 +/- 162 to 2,470 +/- 247 mm Hg/s with no significant change in -dP/dt after 100 micrograms/kg of E4031. Consequently, (+dP/dt)/(-dP/dt) increased from 0.97 +/- 0.07 to 1.18 +/- 0.08. To further evaluate the effects of E4031 on myocardial relaxation, the time constant of isovolumic left ventricular pressure decay was measured by two different methods (tau 1 and tau 2) before and after administering 10 micrograms/kg E4031. Tau 1 increased from 27 +/- 1.8 to 33 +/- 1.6 ms and tau 2 increased from 30 +/- 2.3 to 41 +/- 3.3 ms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708062 TI - Influence of the endothelium on histamine-induced relaxation of rat middle cerebral arteries in vitro. AB - Histamine relaxed PGF2 alpha-precontracted rat isolated middle cerebral arteries (ID approximately 230 microns) concentration dependently with a pD2 of 5.31 (EC50: 5 x 10(-6) M). Cimetidine induced a concentration-dependent rightward shift of the histamine concentration-response curve of endothelium-intact arteries. The slope of the Schild plot was indistinguishable from unity, and the estimated pA2 for cimetidine was 6.14. The selective H2-receptor agonist dimaprit induced a concentration dependent relaxation of the cerebral arteries similar to that induced by histamine. This indicates that the histamine receptor mediating the relaxation in rat middle cerebral arteries belongs to the H2-receptor subtype. 2-Pyridylethylamine, a selective H1-receptor agonist, induced a small concentration-dependent contraction of the arteries with a pD2 of 4.16. Mepyramine, a selective H1-receptor antagonist had no potentiating effect on the relaxation induced by histamine, suggesting either that the contractile effect of 2-pyridylethylamine is nonselective or that H1 receptors mediating contraction are of minor importance for the overall histamine response. The selective H3 receptor agonist, (R)-alpha-methylhistamine, was without effect in a specific concentration range (10(-7)-10(-5) M) excluding participation of H3 receptors in the histamine-induced relaxation of these vessels. Indomethacin did not affect the vessel response to histamine, but removal of the endothelium and treatment of endothelium-intact arteries with 3 x 10(-6) M methylene blue induced a similar 0.5 log rightward shift of the histamine concentration-response curve.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708063 TI - Electropharmacodynamics of N-acetylprocainamide in the immature mammalian heart- evidence for a prominent class III effect in the newborn. AB - The electrophysiologic effects of N-acetylprocainamide (NAPA), a metabolite of procainamide reported to exert significant Class III antiarrhythmic effects in the adult heart, were evaluated in 12 neonatal mongrel canines (ages 7-14 days). Following transvascular placement of quadripolar electrical catheters in the right atrium and ventricle, and a tripolar catheter in the region of the His bundle, electrophysiologic assessments were made of sinus and AV nodal function and atrial and ventricular refractoriness utilizing intracardiac stimulation and recording techniques following cumulative intravenous doses of 20 and 40 mg/kg of NAPA (serum concentrations 13.1 +/- 1.9 and 25.6 +/- 3.4 micrograms/ml). NAPA resulted in an overall increase in sinus cycle length of 18%. No changes were observed in the paced cycle length resulting in AV nodal Wenckebach. Consistent with NAPA's reported Class III action, NAPA increased the effective and functional refractory periods of the ventricle by 35 and 34%, respectively (p less than 0.001). Even larger increases were observed in atrial refractoriness (effective 75%, functional 57%, p less than 0.001). In separate experiments (n = 6), the effects of intravenous NAPA (40 mg/kg) on the duration of the monophasic action potential recorded from the neonatal atrium and ventricle were determined. NAPA increased APD90 of the atrium by approximately 50% and that of the ventricle by 60%, thus confirming a significant effect on myocardial repolarization in the neonate. PMID- 1708064 TI - A model of ion channel kinetics using deterministic chaotic rather than stochastic processes. AB - Models of ion channel kinetics have previously assumed that the switching between the open and closed states is an intrinsically random process. Here, we present an alternative model based on a deterministic process. This model is a piecewise linear iterated map. We calculate the dwell time distributions, autocorrelation function, and power spectrum of this map. We also explore non-linear generalizations of this map. The chaotic nature of our model implies that its long-term behavior mimics the stochastic properties of a random process. In particular, the linear map produces an exponential probability distribution of dwell times in the open and closed states, the same as that produced by the two state, closed in equilibrium open, Markov model. We show how deterministic and random models can be distinguished by their different phase space portraits. A test of some experimental data seems to favor the deterministic model, but further experimental evidence is needed for an unequivocal decision. PMID- 1708065 TI - Myelin basic protein (MBP) displays significant homologies with gag core proteins of HTLV retroviruses. PMID- 1708066 TI - [Usefulness of the latex test for rapid detection of Salmonella O antigen in bacterial culture fluids and clinical specimens. III. Diagnostic field studies]. AB - The aim of this study was to evaluate a latex reagent prepared in our laboratory for a routine diagnosis of Salmonellosis in humans. Liquid cultures in selenite broth (SF) (18-24 hr), previously inoculated with faeces samples of individuals suspected of being infected with Salmonella were subjected to the study. In these cultures, after 15 min. of heating at boiling temperatures, group antigens of Salmonella with an aid of polyvalent latex reagent A-E and monovalent reagents B, C1, C2, D, and E were searched. The results of latex test were compared to the results obtained by routine bacteriological examination. Studies performed in 13 laboratories of Sanitary Epidemiological Stations included 5246 faeces samples. Out of these samples 1835 (35%) reacted with monovalent latex reagent and 1897 (36.2%) samples were positive, for Salmonella by culture technique and belonged to 14 genera of group B, C1, D, and E. S. enteritidis was the most frequently isolated and encountered for 98.6% of all isolated strains. Latex test with A-E reagent was positive in 2246 (42.8%) of culture samples in SF medium, of which 1736 were positive by culture and 510 samples were negative for Salmonella in routine bacteriological examination. The samples positive in culture and with A-E latex reagent reacted in 97.2% with one monovalent reagent. Out of bacteriologically negative samples and reacting with A-E latex reagent 28.8% were positive with monovalent latex reagents. In summary, we can conclude that latex test used in a survey studies can be an usefull test in addition to routine bacteriological examination, since after 18-24 hr it allows with high credibility of 95% to confirm or exclude Salmonella in a tested sample. Such a procedure due to a shortening of routine diagnostic course brings significant savings. Moreover, latex test makes possible rapid detection of mixed infections with Salmonella of different serological groups. The use of extremely carefully, properly prepared selenite broth constitutes a basic condition for agreement between results of latex test and routine bacteriological investigation. PMID- 1708067 TI - [Sedation gives relief to seriously ill patients with cancer]. PMID- 1708068 TI - Cutaneous sensitivity to thiacetazone. PMID- 1708069 TI - Hypersensitivity to dextran in BCG vaccine. PMID- 1708070 TI - Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat. AB - This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis. PMID- 1708071 TI - Tachykinin receptors mediating airway macromolecular secretion. AB - Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with 3H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). All the tachykinins tested stimulated HMWG release to an approximately equal degree. Stimulation was concentration-related, with log concentrations giving half-maximal effects (EC50) as follows: SP -9.47, NKA 7.37, NKB -5.98, PHY -8.08, and ELE -7.68. This rank order of potency (SP greater than PHY greater than or equal to ELE greater than or equal to NKA greater than NKB) is most consistent with NK1 receptors. To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers. The potency of SP was not diminished by pretreatment with atropine, propranolol, or chlorpheniramine, and atropine actually increased the magnitude of the secretory response. The SP receptor antagonist [D-Arg1,D Phe5, D-Trp7,9, Leu11]-SP blocked SP-induced secretion. These findings indicate that SP is a potent stimulus of airway macromolecular secretion. This effect occurs through the action of NK1 receptors, and is not dependent upon cholinergic, beta-adrenergic, or H-1 histamine receptors. The facilitation by atropine of SP stimulation suggests the existence of a mechanism of cholinergic inhibition of SP-induced stimulation. PMID- 1708072 TI - Characterization of the stimulatory effect of galanin on growth hormone release from the rat anterior pituitary. AB - The present study was undertaken to investigate the direct actions of rat galanin (R-GAL) on growth hormone (GH) release from the rat anterior pituitary in vitro. R-GAL modestly but significantly stimulated GH release without an increase in intra- and extracellular cyclic AMP levels in monolayer cultures of rat anterior pituitary cells. This stimulatory effect of R-GAL was dose-dependent but not additive with that of GH-releasing factor (GRF). R-GAL-stimulated GH release was less sensitive to the inhibitory effect of somatostatin than was GRF-stimulated GH release. In perfusions of rat anterior pituitary fragments, R-GAL induced a gradual and sustained increase of GH release. Incremental GH release derived in part from preformed stored GH. These data confirm that R-GAL acts at the pituitary level to stimulate GH release by a mechanism distinct from that of GRF. PMID- 1708073 TI - [The combined radionuclide diagnosis of rectal cancer recurrences]. AB - Of 153 patients with confirmed rectal cancer CEA was recorded in 139 (90.8%); erroneous results were noted in 14 (9.2%) patients; in scintigraphy with 67Ga citrate and 111In-bleomycin diagnoses coincided in 147 (96.1%) patients, and false-negative results were noted in 6 (3.9%) patients. During the investigation 16 patients who had undergone radical operation with any signs of recurrence and 36 patients with suspected tumor recurrence, scintigraphic false-positive results were observed in 12.5 and 30.6% patients, and a false-positive increase in the CEA titer was observed in 12.5 and 11.1%. Of 11 patients with false-positive scintigraphic results a rise of the CEA level was noted in 2 cases making it possible to rule out with a great degree of assurance the presence of a recurrent tumor. The results obtained suggest the appropriateness of the combination of these methods to improve the diagnosis of rectal cancer recurrences. PMID- 1708074 TI - Kainic acid-induced seizures stimulate increased expression of nerve growth factor mRNA in rat hippocampus. AB - The influence of kainic acid (KA)-induced limbic seizure activity on the expression of mRNA for nerve growth factor (NGF) in adult rat brain was studied using in situ hybridization and S1 nuclease protection techniques with RNA probes complementary to murine and rat NGF mRNA. Within hippocampus, intracerebroventricular injection of 0.5 microgram KA caused a dramatic bilateral increase in hybridization of the 35S-labeled cRNA within stratum granulosum. This increase was first evident 1 h post-KA, appeared maximal at approximately 20-fold control levels at 2-3 h post-injection, and declined to control levels by 48 h post-injection. During the period of maximal hybridization, all but the deepest cells within stratum granulosum appeared to be autoradiographically labeled. Hybridization of the NGF cRNA probe was also increased within superficial layers of piriform and entorhinal cortex and, to much lesser extent, within scattered neurons of layers II and III of neocortex in KA-treated rats. In olfactory cortical areas, hybridization was maximally elevated 15.5-24.5 h after KA injection. In contrast to these effects, KA treatment did not consistently influence the density of hybridization, or number of neurons labeled, within the dentate gyrus hilus or the hippocampus proper (CA1-CA3). In agreement with the in situ hybridization results, S1 nuclease protection assay detected KA-induced increases in hybridization within pooled dentate gyrus/CA1 samples, but not hippocampal CA3 samples. These data support the conclusion that seizure activity stimulates a transient increase in NGF expression by select populations of forebrain neurons and indicates that experimental seizure paradigms might be further exploited for analyses of the mechanisms of NGF regulation and processing in the adult brain. PMID- 1708075 TI - Antibiotics cause changes in the desensitization of ACh receptors expressed in Xenopus oocytes. AB - ACh receptors (AChRs) synthesized in Xenopus oocytes which were cultured in medium containing gentamicin desensitized much more rapidly than those expressed in the absence of gentamicin. The effect caused by 24 h incubation in gentamicin could not be reversed by leaving oocytes in culture medium without gentamicin for 24 h. In addition, gentamicin exhibited a direct reversible blocking action on the function of AChRs. Our experiments indicate that antibiotics should be used cautiously in culturing oocytes when studying the function of induced neurotransmitter receptors and ion channels. PMID- 1708076 TI - The genes encoding nerve growth factor and its receptor are expressed in the developing female rat hypothalamus. AB - Nerve growth factor (NGF), its messenger RNA (mRNA) and its receptor protein and mRNA are found in several brain regions. Little attention has been given to the possibility that the hypothalamus, which controls the endocrine system, may produce NGF and/or express NGF receptors. This would indicate the existence in this brain area of neuronal populations which are either target for NGF responsive cells or sensitive to NGF, respectively. Blot hybridization of polyadenylated (poly(A)+) RNA to a mouse NGF cRNA probe revealed the presence of NGF mRNA in both the suprachiasmatic region (henceforth, called preoptic area [POA]) and medial basal hypothalamus (MBH) of developing female rats. The mRNA was already detectable on fetal day 18 and reached maximal levels around postnatal day 3 (MBH) and 12 (POA), declining thereafter. This pattern was temporally different than that of areas known to produce NGF, i.e., the neocortex (Cc) and hippocampus (Hc). In agreement with previous reports, NGF mRNA levels in these areas were negligible before birth, became maximal between the second and third week of postnatal life and decreased moderately thereafter. The concentration of NGF protein, measured by a two-site enzyme immunoassay, was 3 times higher in the POA than in the MBH at an infantile age (day 12), increasing 2-fold in the POA of juvenile animals (day 28) but not in the MBH. This developmental pattern was similar to that seen in the Hc, though the NGF concentrations were significantly lower in the POA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708077 TI - Co-expression of cholecystokinin mRNA and tyrosine hydroxylase mRNA in populations of rat substantia nigra cells; a study using a combined radioactive and non-radioactive in situ hybridization procedure. AB - Improvements in the sensitivity of non-radioactive in situ hybridization histochemistry methods for detection of mRNA now make it feasible to combine the use of non-radioactive and radioactive in situ methods to visualize two mRNAs on the same tissue section. The method reported here allows the simultaneous detection of two mRNAs in one cell and therefore is ideally suited to the studies of co-expression. Here we demonstrate the co-expression of tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA in the ventral mesencephalic dopaminergic neurones of the rat. The distribution of dopaminergic neurones containing both TH and CCK transcripts suggests, on the basis of earlier anatomical studies that these CCK/TH-containing doubled-labelled cells project mainly to the striatal matrix. Dopamine neurones believed to project to the patch compartment did not contain CCK mRNA. PMID- 1708078 TI - Comparison of Streptococcus mutans and Streptococcus sanguis receptors for human salivary agglutinin. AB - Oral streptococci vary in their susceptibility to salivary agglutinin-mediated aggregation. To understand the molecular basis of this specificity, the structure and function of receptors for agglutinin from Streptococcus mutans KPSK2 (MSL-1) and Streptococcus sanguis M5 (SSP-5) were compared. Immunological screening of an S. mutans KPSK2 genomic DNA library yielded two identical clones expressing a streptococcal protein that co-migrated with a 220 kDa peptide in SDS extracts from this organism. This protein inhibited agglutinin-mediated aggregation of S. mutans KPSK2 in a dose-dependent manner. The MSL-1 gene is homologous to the S. mutans SpaP and pac genes although single base substitutions alter several amino acids. MSL-1 is also similar to the agglutinin receptor (SSP-5) cloned from S. sanguis M5. All three proteins, MSL-1, P1, and SSP-5 share at least one epitope since monoclonal and polyclonal anti-SSP-5 antibodies react with both MSL-1 and P1. However, other monoclonal antibodies are specific for SSP-5 and appear to react with a peptide domain exhibiting little homology to MSL-1 or P1. Sugar inhibition studies showed that agglutinin-mediated aggregation of S. mutans KPSK2 was most potently inhibited by fucose and lactose. Sialic acid, a potent inhibitor of S. sanguis aggregation, had no effect on the interaction of agglutinin with S. mutans KPSK2. These results suggest that while the MSL-1 and SSP-5 proteins are genetically and immunologically related, their specificity for binding sites on agglutinin differs. PMID- 1708079 TI - Retinoid-binding proteins in embryonal carcinoma cells. PMID- 1708080 TI - Vitamin A-mediated regulation of keratinocyte differentiation. PMID- 1708081 TI - Retinoid effects on photodamaged skin. PMID- 1708082 TI - Retinoids and control of epithelial differentiation and keratin biosynthesis in hamster trachea. PMID- 1708084 TI - A patient's right to a good death. PMID- 1708083 TI - The role of interferons in the resistance to murine cytomegalovirus. AB - The protective effect of IFN alpha/beta or IFN-gamma against MCMV infection in mouse embryo fibroblasts (MEF) of genetically resistant and susceptible strains have been examined. For this purpose, MEF derived from Balb/c, C3H, DBA2, C57BL6 mouse strains were used. Cells were pretreated for 24 hrs with either IFN alpha/beta or IFN-gamma and subsequently infected with MCMV at different M.O.I. No difference in susceptibility to MCMV was observed between untreated Balb/c, C3H, C57 and DBA2-MEF when infected at a M.O.I. of 5 whereas at a M.O.I. of 1 or 0.5 untreated DBA2-MEF displayed the highest susceptibility to viral infection. Pretreatment of MEF with IFN alpha/beta showed that the degree of protection to MCMV was dependent on the M.O.I. used. At a M.O.I. of 5, IFN alpha/beta did not inhibit viral replication in any MEF tested, whereas at a M.O.I. of 1 or 0.5 was protective in C3H-MEF and to a lesser extent in Balb/c-MEF. No protection was observed in DBA2 and C57-MEF. Different results were observed when MEF were pretreated with IFN-gamma. C57-MEF were protected when infected at a M.O.I. of 5 or 1; no protection was observed at a M.O.I. of 0.5. Balb/c-MEF were protected only at a M.O.I. of 1. No protection was observed in C3H and DBA2-MEF. PMID- 1708085 TI - [Palliative surgical treatment of cancer of the esophagus]. AB - Palliative surgery, including cleaning resection, colon or stomach bypass, intubation or fistula is all one can offer to the majority of patients suffering from oesophageal carcinoma due to an usually advanced tumor stage at the time of diagnosis. The primary goal is to prevent or resolve dysphagia. A prolongation of survival time can - with the exception of cleaning resection - not be expected by these procedures. The average survival time was 9.4 months after cleaning resection and 4.7 months after other palliative procedures in our series. If a cleaning resection is not possible, the endoscopic implantation of an endotube is the treatment of choice because of its low mortality and morbidity. PMID- 1708088 TI - Action of 9-beta-D-arabinofuranosyl-2-fluoroadenine on RNA metabolism. AB - The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on RNA metabolism was evaluated both in whole cells and in cell-free systems. F-ara-A was converted to its 5'-triphosphate, F-ara-ATP, in cells and then incorporated into RNA as well as DNA. F-ara-A inhibited RNA synthesis in cultured cells in a concentration-dependent manner. This inhibition was mediated mainly by F-ara-ATP. Experiments using isolated nuclei demonstrated that RNA polymerases I, II, and III accounted for 24, 73, and 3% of the total RNA synthesis activity, respectively. About 88% of the total inhibition was attributed to the suppression of RNA polymerase II activity. In cultured cells, F-ara-A was preferentially incorporated into the poly(A)+ RNA fraction. Approximately 78% of the incorporated F-ara-A monophosphate residues were located at the terminal position of the RNA chain. The incorporation of F-ara-A monophosphate into mRNA resulted in premature termination of the RNA transcript and impaired its functioning as a template for protein synthesis. The inhibitory action of F-ara-A on RNA metabolism is a unique property of this compound, differing from the action of arabinosylcytosine and arabinosyladenine. PMID- 1708087 TI - Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli. AB - The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA+ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA+ gene was expressed in the presence of the inducer isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30 degrees C, or after inactivation of DnaA by a shift to 42 degrees C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom- plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708089 TI - Regulation of dopamine D2 receptors in a novel cell line (SUP1). AB - A prolactin-secreting cell line, SUP1, has been established from rat pituitary tumor 7315a. In radioligand binding experiments, the D2 receptor antagonist (S)-( )-3-[125I]iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamide ([125I]IBZM) labeled a single class of sites in homogenates of SUP1 cells (Kd = 0.6 nM; Bmax = 45 fmol/mg of protein). The sites displayed a pharmacological profile consistent with that of D2 receptors. Inhibition of the binding of [125I]IBZM by dopamine was sensitive to GTP, suggesting that D2 receptors in SUP1 cells are coupled to guanine nucleotide-binding protein(s). In the presence of isobutylmethylxanthine, dopamine decreased the level of cAMP accumulation in SUP1 cells. Dopamine also inhibited prolactin secretion from SUP1 cells. Both the inhibition of cAMP accumulation and the inhibition of prolactin secretion were blocked by D2 receptor antagonists, suggesting that these effects of dopamine were mediated by an interaction with D2 receptors. The regulation of D2 receptors in SUP1 cells by D2 receptor agonists was investigated. Exposure of SUP1 cells to dopamine or to the D2 receptor agonist N-propylnorapomorphine led to increased expression of D2 receptors, with no change in the affinity of the receptors for [125I]IBZM. An increase in the density of D2 receptors in SUP1 cells was evident within 7 hr of exposure to dopamine. Spiroperidol, a D2 receptor antagonist, blocked the effect of dopamine on receptor density. These results suggest that exposure of D2 receptors in SUP1 cells to agonists leads to an up-regulation of D2 receptors. Dopamine retained the ability to inhibit cAMP accumulation in SUP1 cells exposed to dopamine for 24 hr, suggesting that D2 receptors in SUP1 cells are not desensitized by prolonged exposure to agonist. SUP1 cells should be a useful model system for future studies of the regulation of the expression and function of D2 receptors in cultured cells. PMID- 1708086 TI - Social and developmental biology of the myxobacteria. AB - Myxobacteria are soil bacteria whose unusually social behavior distinguishes them from other groups of procaryotes. Perhaps the most remarkable aspect of their social behavior occurs during development, when tens of thousands of cells aggregate and form a colorful fruiting body. Inside the fruiting body the vegetative cells convert into dormant, resistant myxospores. However, myxobacterial social behavior is not restricted to the developmental cycle, and three other social behaviors have been described. Vegetative cells have a multigene social motility system in which cell-cell contact is essential for gliding in multicellular swarms. Cell growth on protein is cooperative in that the growth rate increases with the cell density. Rippling is a periodic behavior in which the cells align themselves in ridges and move in waves. These social behaviors indicate that myxobacterial colonies are not merely collections of individual cells but are societies in which cell behavior is synchronized by cell cell interactions. The molecular basis of these social behaviors is becoming clear through the use of a combination of behavioral, biochemical, and genetic experimental approaches. PMID- 1708090 TI - Characterization of VSG gene expression site promoters and promoter-associated DNA rearrangement events. AB - The expressed variant cell surface glycoprotein (VSG) gene of Trypanosoma brucei is located at the 3' end of a large, telomeric, polycistronic transcription unit or expression site. We show that the region 45 kb upstream of the VSG gene, in the expression site on a 1.5-Mb chromosome, contains at least two promoters that are arranged in tandem, directing the transcription of the expression site. DNA rearrangement events occur specifically, at inactivation of the expression site, and these events delete the most upstream transcribed region and replace it with a large array of simple-sequence DNA, leaving the downstream promoter intact. Because of the placement of simple-sequence DNA, the remaining downstream promoter now becomes structurally identical to previously described VSG promoters. The downstream promoter is repetitive in the genome, since it is present at several different expression sites. Restriction fragment length polymorphism mapping allows grouping of the expression sites into two families, those with and those without an upstream transcription unit, and the DNA rearrangement events convert the expression sites from one type to the other. Deletion of the upstream transcription unit also leads to the loss of several steady-state RNAs. The findings may indicate a role for promoter-associated DNA rearrangement events, and/or interactions between tandemly arranged promoters, in expression site transcriptional control. PMID- 1708091 TI - Peptide antisera to human colony-stimulating factor 1 receptor detect ligand induced conformational changes and a binding site for phosphatidylinositol 3 kinase. AB - A peptide antiserum (anti-A) directed to the intracellular, juxtamembrane region (residues 552 to 574) of the human colony-stimulating factor 1 receptor (CSF-1R) precipitated only ligand-activated, native receptors from solution but bound to unstimulated forms after their denaturation. Two peptide antisera (anti-KI1 and KI2), directed to residues 679 to 700 and 701 to 721, respectively, in the CSF-1R kinase insert (KI) domain and including mapped sites of ligand-induced phosphorylation at Tyr-699 and Tyr-708, bound at least 80% of the receptor molecules expressed in either CSF-1-stimulated or unstimulated cells. Immune complexes formed with anti-KI1, anti-A, or a peptide antiserum to the CSF-1R carboxyl terminus (anti-C-ter) coprecipitated CSF-1R complexed to a phosphatidylinositol 3-kinase (PtdIns 3-K) from CSF-1-stimulated cells, whereas anti-KI2 serum did not. In an in vitro assay, binding of CSF-1R to PtdIns 3-K required receptor tyrosine phosphorylation but not CSF-1R-mediated phosphorylation of the lipid kinase, and the association was specifically blocked by anti-KI2 or antibodies to phosphotyrosine. Neither anti-KI1, anti-A, nor anti C-ter serum inhibited binding. We conclude that (i) only a minority of ligand activated receptors form a stable complex with PtdIns 3-K in vivo, (ii) efficient binding of the lipid kinase requires receptor tyrosine phosphorylation within the CSF-1R KI domain, and (iii) a region within the KI domain defined by residues 701 to 721 at least partially overlaps the PtdIns 3-K binding site. PMID- 1708092 TI - Retinoic acid increases zif268 early gene expression in rat preosteoblastic cells. AB - In this study we demonstrate that retinoic acid (RA) increases the expression of transcription factor zif268 mRNA in primary cultures of fetal rat calvarial cells and in simian virus 40-immortalized clonal rat calvarial preosteoblastic cells (RCT-1), which differentiate in response to RA, but not in the more differentiated RCT-3 and ROS 17/2.8 cells. The increased expression of zif268 mRNA is rapid (maximal within 1 h), transient (returns to basal levels by 3 h), detectable at RA doses of 10(-12)M, and independent of protein synthesis. The relative stimulation of zif268 mRNA by RA was much larger than that of other early genes, including c-fos, c-jun, and junB. The rate of transcription of RA stimulated RCT-1 cells, estimated by nuclear run-on assays, was elevated, suggesting that RA regulation of zif268 gene transcription was at least in part transcriptional. Moreover, RA stimulated the transcriptional activity of a Zif268CAT (chloramphenicol acetyltransferase) plasmid containing 632 bp of zif268 5' regulatory sequences in RCT-1 cells but not in the more differentiated RCT-3 cells. These in vitro data support the in vivo observations which localize zif268 and RA receptor-gamma transcripts to bone and cartilage during development, suggesting that both RA and zif268 may play a role in osteoblast differentiation. PMID- 1708093 TI - Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts. AB - Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle. PMID- 1708095 TI - Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis. AB - The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20 fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme. PMID- 1708094 TI - Two regions of the mouse mammary tumor virus long terminal repeat regulate the activity of its promoter in mammary cell lines. AB - In vivo expression of the mouse mammary tumor virus (MMTV) is restricted to a few organs, with the highest rate of transcription found in the mammary gland. Using a series of mammary and nonmammary murine cell lines, we have identified two regulatory elements, located upstream of the hormone responsive element, that specifically regulate the MMTV promoter. The first element displays an enhancerlike activity and is coincident with the binding of a nuclear factor (designated MP4; position -1078 to -1052 in the long terminal repeat) whose presence is apparently restricted to mammary cell lines. The second regulatory region mediates a repressive activity and is mapped to the long terminal repeat segment from -415 to -483. This repression is specific for a particular subtype of mammary cells (RAC cells) able to grow under two differentiation states (A. Sonnenberg, H. Daams, J. Calafat, and J. Hilgers, Cancer Res. 46:5913-5922, 1986). The MMTV promoter in mammary cell lines thus appears to be modulated by two cis-acting elements that are likely to be involved in tissue-specific expression in vivo. PMID- 1708096 TI - Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells. AB - To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR) CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication. PMID- 1708098 TI - Repression of the human glycoprotein hormone alpha-subunit gene by glucocorticoids: evidence for receptor interactions with limiting transcriptional activators. AB - Expression of the glycoprotein hormone alpha gene is regulated divergently by glucocorticoids in different cell types. Coexpression of the glucocorticoid receptor (GR) with an alpha-CAT reporter gene caused activation of alpha promoter activity in fibroblasts, but repression in JEG-3 choriocarcinoma cells, indicating that cell-specific factors dictate positive vs. negative regulation of this promoter by GR. Cell-specific sequences and other enhancer elements in the the alpha gene have been relatively well characterized in JEG-3 cells, and this model was used to further examine the mechanism of transcriptional repression by glucocorticoids. Promoter mutagenesis indicated that the degree of GR-mediated repression was impaired by a variety of deletional and site-directed mutations between -171 and -111 bp, a region that includes both cell-specific and cAMP response elements (CREs). In an attempt to further localize a negative glucocorticoid response element (GRE) sequence, binding studies were used to assess GR interactions with alpha promoter DNA sequences. Using avidin-biotin complex DNA binding assays, a series of overlapping alpha promoter DNA sequences between -170 to 29 basepairs were tested, but each failed to bind GR, whereas a control GRE avidly bound receptor. Similarly, in competition assays in transfected CV-1 cells, the alpha gene 5'-flanking sequence did not compete for GR stimulation of a glucocorticoid responsive reporter gene, whereas a sequence that contains known GR-binding sites (murine mammary tumor virus) effectively inhibited GR-mediated expression. The absence of high affinity GR-binding sites in the alpha promoter suggested that mutations that affected GR inhibition may have eliminated recognition sites for transactivators, which are themselves targets for the GR, rather than altering specific negative GRE sites in the DNA sequence. To examine this possibility, GR repression was studied using chimeric transcription factors. The transcription-activating domains of several different proteins (CREB, thyroid hormone receptor, or VP16) were linked to the DNA-binding domain of Gal-4, and transcription was driven by the Gal-4 recognition site (UAS). GR markedly repressed transactivation by Gal-4-CREB and, to a lesser degree, the Gal-4-thyroid hormone receptor and Gal-4-VP16 chimeric proteins. Repression occurred when UAS was linked to either the alpha promoter or to the E1B promoter. Thus, inhibition occurs in the absence of either the CRE or the proximal alpha promoter. These results support a mechanism in which GR-mediated repression in JEG-3 cells occurs by receptor interference with the transactivating potential of enhancer-binding proteins or associated transcription factors. PMID- 1708097 TI - Modulation of c-kit mRNA and protein by hemopoietic growth factors. AB - We examined the effects of various hemopoietins on c-kit mRNA and protein expression. Interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and erythropoietin, but not IL-4, down-regulated levels of c-kit mRNA expressed by mast cells and stem cell progenitors. The effect of IL-3 was dominant and independent of cell growth or viability and was paralleled by reduced expression in c-kit protein. These observations indicate that regulation of c-kit expression is closely interlinked with the molecular mechanisms triggered by erythropoietin, IL-3, and granulocyte-macrophage colony-stimulating factor. PMID- 1708099 TI - Insulin-like growth factor-mediated phosphorylation and protooncogene induction in Madin-Darby canine kidney cells. AB - We have characterized the role of tyrosine phosphorylation in protooncogene induction mediated by insulin-like growth factors I and II (IGF-I and IGF-II) in the Madin-Darby canine kidney (MDCK) cell line. These cells possess few, if any, insulin receptors, thus allowing determination of the effects of these growth factors in the absence of any secondary signal mediated through the insulin receptor. We found that IGF-I produced a specific stimulation of tyrosine kinase activity of the 97-kDa beta-subunit of the IGF-I receptor, resulting in autophosphorylation of the receptor and an increase in kinase activity toward a synthetic peptide substrate. This was associated with a gradual decrease in the level of phosphorylation of pp120, the major constitutive phosphotyrosine containing protein of MDCK cells, and an increase in the ratio of serine to tyrosine phosphorylation. This was followed by a rapid, but transient, induction of c-fos gene expression, with no change in the levels of c-myc mRNA. Cycloheximide treatment resulted in a superinduction of both c-fos and c-myc and prevented any further stimulation by IGF-I. IGF-II did not stimulate tyrosine phosphorylation of its own receptor, but was 25% as active as IGF-I in stimulating phosphorylation of the IGF-I receptor. Despite this, IGF-II did not significantly enhance the expression of either nuclear protooncogene. Insulin also produced a delayed stimulation of IGF-I receptor phosphorylation, but was unable to stimulate biological effects in these cells. Under these conditions neither of the IGFs nor insulin produced any significant stimulation of thymidine incorporation into DNA. These data indicate that the IGF-I receptor can be activated upon binding of IGF-I, and to a lesser extent IGF-II, in intact cells to mediate cellular events. The nature of the signal generated by the IGF-I receptor appears to vary depending on the ligand that occupies it. PMID- 1708100 TI - 3,5,3'-triiodothyronine receptor auxiliary protein (TRAP) enhances receptor binding by interactions within the thyroid hormone response element. AB - We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell. PMID- 1708101 TI - Protein antigenicity: a thermodynamic approach. AB - This article summarizes computer-aided analyses of X-ray crystallographic data aimed at understanding the immunologically important aspects of the structure of antibody combining sites and protein antigens. In these calculations we use an empirical free energy potential function to estimate the atomic origin of binding specificity. By evaluating contributions of individual amino acid residues towards the Gibbs free energy of antibody-antigen complex formation, we arrive at a better understanding of the essential antigenic features of protein surfaces, as well as the inherent "binding" properties of the antibody combining sites. Such an "energetic" understanding of antigenicity may well be of practical importance in vaccine design. This article both reviews published data and discusses new results, i.e. delta G calculations on the HyHEL-10 complex with lysozyme, and an alternative treatment of the McPC 603 complex with phosphoryl choline. PMID- 1708102 TI - Mechanisms of T cell recognition with application to vaccine design. AB - Both helper and cytotoxic T lymphocytes generally recognize protein antigens not in their intact form, as antibodies do, but on the surface of another cell, after "processing" by that cell to unfold or cleave the protein into fragments and after association of the processed antigen with major histocompatibility complex (MHC) molecules on that cell. This complex process leads to immunodominance of certain segments from the protein, which depends not only on structural features intrinsic to the antigenic segment itself, but also on antigen processing and on the structure of the MHC molecules of the responding individual. We have explored all three of these factors, including the enzymes involved in processing, the way peptides bind to MHC molecules, and structural features such as helical amphipathicity that seem to favour T cell recognition. We have used this information to locate and characterize antigenic sites of proteins of interest for vaccine development, including proteins from the malaria parasite and the AIDS virus, HIV. For HIV, we have identified both helper and cytotoxic T cell sites, coupled a helper site to a B cell site to produce a synthetic immunogen that elicits neutralizing antibodies, and studied the effect of viral sequence variation on cytotoxic T cell recognition and binding of the immunodominant peptide to MHC molecules. This information suggests strategies for the rational design of synthetic or recombinant vaccines. PMID- 1708103 TI - Strategies for exploiting the immune system in the design of vaccines. AB - There are three types of vaccines in medical use today--live, attenuated agents, mainly viruses; inactivated whole organisms; and subunit preparations. Though there are examples in each category of successful preparations, live attenuated viruses have almost invariably given long-lasting immunity after one or two administrations. Contributing reasons for this are gleaned, not from human vaccine studies, but from model systems and it is concluded that a vaccine needs to achieve four goals: activation of antigen-presenting cells; overcoming genetic variability in T cell responses; generation of high levels of T and B memory cells; and persistence of antigen for recruitment of B memory cells. Of the newer approaches to vaccine development, synthetic peptides have substantial limitations but should be successful in some cases. Subunit preparations may also be limited as a general method. Some live viral or bacterial vaccines, used as vectors of nucleic acid coding for other antigens, hold considerable promise as is illustrated by recent examples. PMID- 1708104 TI - Detoxification of pertussis toxin by site-directed mutagenesis: a review of connaught strategy to develop a recombinant pertussis vaccine. PMID- 1708105 TI - Epitope specificity of three anti-pertussis toxin monoclonal antibodies with dissimilar effects in assays of toxin neutralizing activity. AB - The epitope specificity of two monoclonal antibodies against the S1 subunit (A4, A12) and one MAb against the S3 subunit (B9) of pertussis toxin, all protective in the mouse aerosol model of B. pertussis infection, but with different effects in assays of toxin-neutralizing activity, was examined in competitive binding enzyme immunoassays using biotinylated anti-pertussis toxin monoclonal antibodies or biotinylated goat anti-pertussis toxin polyclonal antibody after preincubation with unlabelled antibody. Biotinylated A4 was blocked by A4, A12, and B9; A12 was blocked by A4, A12, and B9. In contrast, biotinylated B9 was blocked by B9 and A4, but not by A12. All three monoclonal antibodies successfully blocked the anti pertussis toxin polyclonal antibody; a mixture of the three anti-pertussis toxin monoclonal antibodies was more effective than any monoclonal antibody alone P less than or equal to 0.01). These data suggest that these three anti-pertussis toxin monoclonal antibodies recognize separate, but closely linked epitopes on pertussis toxin, and that epitopes on the S1 subunit and B-oligomer may induce protective immunity. PMID- 1708106 TI - Adjuvant-independent immunization by immunotargeting antigens to MHC and non-MHC determinants in vivo. AB - Using avidin as a model protein antigen, and biotinylated monoclonal antibodies as a convenient means of forming stable complexes with avidin, we have investigated the adjuvant-independent immunization of three mouse strains, C57BL/6, C3H and (C57BL/6 x C3H)F1, with immunoconjugates targeted to different class II MHC and non-MHC sites. The results confirm the effectiveness of anti-I Ak and anti-I-Ab immunoconjugates with respect to priming for secondary IgG responses in (H-2b x H-2k)F1 mice, while indicating a lack of response in strains which are homozygous for the targeted allele. In terms of non-MHC targets in the monocyte-macrophage lineage, neither anti-MAC-1 nor anti-MAC-2 immunoconjugates were effective in any of the three strains. However, the 33D1 anti-dendritic cell antibody gave significant responses in all three strains, with the F1 response being more than 10-fold greater than the anti-class II immunoconjugates in either strain. These findings indicate that immunotargeting a protein antigen to a non MHC determinant on dendritic cells in vivo can be an effective means of inducing an adjuvant-independent serological response, and that this approach can have significant advantages over anti-class II MHC immunotargeting. PMID- 1708107 TI - Detection of Mycoplasma bovis and Mycoplasma agalactiae by oligonucleotide probes complementary to 16S rRNA. AB - The partial sequences of 16S rRNA from Mycoplasma bovis and M. agalactiae were determined by dideoxynucleotide sequencing using reverse transcriptase. Two oligonucleotides complementary to different evolutionary variable regions of 16S rRNA from these two species were synthesized. The oligonucleotides were end labelled with 32P and used as probes in filter hybridization experiments with different bovine, caprine and ovine mycoplasmas as samples. One of the probes, complementary to a sequence of the V8-region of both M. bovis and M. agalactiae, did not cross-hybridize to any bovine, caprine or ovine mycoplasmas except M. bovigenitalium and M. californicum. This probe is thus not useful for analysis of bovine samples, but can be used for detection of M. agalactiae in samples from goats and sheep, since M. bovigenitalium and M. californicum have never been isolated from these hosts and M. bovis only occasionally. The other probe, complementary to a sequence of the V6-region of M. bovis, gave some cross hybridization with M. agalactiae but not with bovine mycoplasmas. M. agalactiae has never been isolated from cattle and this probe is therefore useful for rapid screening of bovine samples for M. bovis. PMID- 1708108 TI - The possible role of probiotics as dietary antimutagen. AB - Possible antimutagenic actions of probiotics--mainly lactic acid bacteria--were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli) and its cell-free culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animals were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli. PMID- 1708109 TI - Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes. AB - A 3-h exposure of V79 Chinese hamster cells with the sensory irritant 2 chlorobenzylidene malonitrile (CS) caused apolar mitoses in a dose-dependent manner. With a preparation and staining technique that allows for the visualization of the spindle apparatus and the chromosomes it was found that unlike in Colcemid-induced c-metaphases residual spindle fibers or microtubule material were present in the majority of CS-induced c-metaphases. The observation suggests different mechanisms for the induction of the c-mitotic effect by the two spindle poisons. PMID- 1708110 TI - Structure and function of telomeres. AB - The DNA of telomeres--the terminal DNA-protein complexes of chromosomes--differs notably from other DNA sequences in both structure and function. Recent work has highlighted its remarkable mode of synthesis by the ribonucleoprotein reverse transcriptase, telomerase, as well as its ability to form unusual structures in vitro. Moreover, telomere synthesis by telomerase has been shown to be essential for telomere maintenance and long-term viability. PMID- 1708111 TI - Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups. AB - Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules. PMID- 1708113 TI - Ventricular fluid neuropeptides in Parkinson's disease. II. Levels of substance P like immunoreactivity. AB - Substance P-like immunoreactivity (SPLI) was determined in cerebrovascular fluid of patients with extrapyramidal motor diseases. Patients with Parkinson's disease (PD) showed a SPLI concentration decreased by 30% compared with patients without extrapyramidal disease. No differences were apparent for patients with dystonia. Fluid obtained from the foramen Monro showed higher SPLI concentrations than fluid from a lateral ventricle, indicating that hypothalamic sources are important for ventricular substance P. Lateral ventricular SPLI was particularly low in parkinsonian patients which raises the possibility of a decreased SPergic activity in basal ganglia occurring in PD. PMID- 1708112 TI - Neurochemical mediators of the behavioural effects of receptor-selective substance P agonists administered intrathecally in the rat. AB - Recently, two compounds have been developed, designated septide and senktide, which are highly selective agonists for the substance P receptor, types NK-1 and NK-3, respectively. Each of these, when injected intrathecally in awake rats, produced a distinct and non-overlapping constellation of sensory and behavioural effects which were subsets of the symptoms evoked by intrathecal administration of substance P. Prior systemic administration of 5-hydroxytryptamine (5-HT), alpha-adrenergic and opiate receptor antagonists, at doses sufficient to block the behavioural effects of the corresponding receptor agonists, did not alter responses to intrathecally injected septide or senktide. This was so, even for symptoms which suggested inhibitory mediation, hypoalgesia and (transient) motor flaccidity. Septide and senktide, administered by lumbar puncture and by indwelling catheter, produced identical results. Finally, in contrast to some other peptides, flaccid paralysis induced by senktide was not accompanied by spinal necrosis. PMID- 1708114 TI - The relationship between ventral striatal efferent fibers and the distribution of peptide-positive woolly fibers in the forebrain of the rhesus monkey. AB - Peptidergic fibers in the globus pallidus of the monkey appear in the morphological form referred to as woolly fibers. These fibers are composed of a dense plexus of thin beaded axons which ensheath an unstained central core. Such structures are not confined to the globus pallidus, but are also present in the bed nucleus of the stria terminalis, the hypothalamus, the dorsal part of the amygdala, and ventrally in the basal forebrain. The present study describes the relationship between projections from the rostral and ventral striatum and the enkephalin- and substance P-positive woolly fibers. Following injections of either tritiated amino acids or the lectin Phaseolus vulgaris-leucoagglutinin in the ventral striatum, anterogradely labeled fibers and terminals in the forebrain were visualized simultaneously with enkephalin- or substance P immunoreactivity in the same tissue section in order to determine: (i) the extent to which the woolly fiber distribution represents striatal output systems; (ii) whether woolly fibers can be considered as a marker for the entire striatal forebrain projection; and (iii) whether enkephalin and substance P are involved differentially in distinct ventral striatopallidal pathways. Phaseolus vulgaris leucoagglutinin labeling is seen in the globus pallidus and adjacent structures either as single, beaded fibers or in a profile strikingly similar to that of woolly fibers. In tissue sections treated for a double immunohistochemical protocol, following which the Phaseolus vulgaris-leucoagglutinin-immunoreactive fibers turn black and the peptidergic woolly fibers brown; many of the lectin positive fibers are seen to enter the peptide-positive woolly fiber plexus. Likewise, following the injections with tritiated amino acids in the ventral striatum, coarse structures that have dimensions resembling those of the woolly fibers are identified. In sections immunohistochemically stained and subsequently treated for autoradiography, peptide-positive woolly fibers can be identified underlying the silver grains. In sections stained for both peptide immunoreactivity and tracer substances, enkephalin or substance P-positive woolly fibers are present in all pallidal regions that receive ventral striatal input. However, the ventral striatum also sends fibers to the hypothalamus, bed nucleus of the stria terminalis, the dorsal part of the amygdala, the septum, the preoptic area, and other areas of the basal forebrain. In these nuclei the peptide-positive woolly fiber distribution is less extensive than the terminal labeling. The distribution of substance P-positive fibers in the subcommissural pallidal region is more limited than the distribution of enkephalinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1708115 TI - Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex: comparison to activity staining. AB - Distribution patterns of activity and immunohistochemical staining for glutamate dehydrogenase were compared during the postnatal development of rat hippocampal formation and cerebellar cortex. On postnatal day 5, dendritic layers of the hippocampal formation showed only a very weak enzyme activity. Similarly, when studied at the same age, the external granule cell layer and Purkinje cells of the cerebellar cortex exhibited a very faint and moderate staining, respectively. With advancing age, in both brain regions a marked postnatal increase in glutamate dehydrogenase activity occurred in neuropil area as glutamatergic structures matured. However, compared to activity staining, both brain regions of early postnatal stages showed a relatively high level of glutamate dehydrogenase like immunoreactivity. In this case, the immunohistochemical staining of hippocampal dendritic layers and of the molecular layer of the cerebellar cortex was rather diffuse, being not very similar to parameters of the maturation of the respective glutamatergic structures. In contrast to the activity staining for the enzyme, the immunohistochemical labelling in adult rats revealed a selective predominance of immunoreactivity in astroglial cells from postnatal day 5 onwards. The Bergmann glia in the cerebellar cortex exhibited the strongest intensity of immunoreactivity. Generally, the patterns of immunoreactivity were found to depend on the fixation procedure adopted. Concluding from our results, glutamate dehydrogenase is demonstrable in glial and in neuronal cell elements as well. Therefore, it is recommended that activity staining and the immunohistochemical procedure be combined to study qualitative and quantitative aspects of glutamate dehydrogenase in nervous tissues. PMID- 1708116 TI - [Use of BICAP by endoscopic route in the palliative treatment of neoplastic stenosis of the esophagus]. AB - Over the years the palliative treatment of neoplastic stenosis of the esophagus in patients who cannot be operated has seen a variation of endoscopic methods which aimed to reopen the alimentary canal either using simple dilatation, or the insertion of endoprostheses, or sclerosing injection or antiblastic therapy, or lastly using disobstructive laser therapy. In particular, the use of Neodymium YAG laser in endoscopic therapy for the deobstruction of neoplastic esophageal stenosis is currently widely used. More recently deobstruction of the stenosis may also be achieved using bipolar diathermocoagulation with BICAP following esophageal dilatation. Recent comparative studies of the use of BICAP and laser therapy in the treatment of neoplastic esophageal stenosis have tended to reveal the complementary characteristics of the two techniques. The present paper reports the Authors' experience in this respect which has been satisfactory with regard to both methods, in line with the findings of other studies. In the study of two groups of 8 patients treated with BICAP and laser therapy respectively, recanalisation was obtained in 100% of cases with good functional results in 75% of patients treated with BICAP and 87.5% of those receiving laser therapy. The time interval between one treatment session and the next in relation to the efficacy of the therapy was similar in both methods and ranged from a minimum of 4 weeks to a maximum of 12 weeks. Complications were scarce in both groups. PMID- 1708117 TI - Corneal endothelial permeability after anterior chamber silicone oil. AB - One of six silicone oils, differing in both viscosity and manufacture, was infused into the anterior chambers of rabbit eyes. Polydimethylsiloxane oil, 5000 cps, caused an increased corneal endothelial permeability to inulin and dextran at 24, 96, and 168 hours after placement into the eye. Intraocular pressures were slightly elevated in the experimental eyes, compared with contralateral controls, at 24 and 144 hours after infusion. The effects of five other oils on corneal endothelial permeability were examined 168 hours after infusion. All oils increased permeability and caused thinning of endothelial cells, together with the appearance of a retrocorneal membrane, except Dow Corning Medical Fluid 360. The results indicated that contact of most silicone oils with corneal endothelium rapidly induces physiologic and morphologic changes. PMID- 1708118 TI - Low power laser biostimulation of chronic oro-facial pain. A double-blind placebo controlled cross-over study in 40 patients. AB - The efficacy of low power laser stimulation in the treatment of chronic oro facial pain conditions was investigated in a double-blind placebo controlled modified cross-over study in 40 patients. The laser was an invisible infrared (IR) diode laser with an emission at 904 nanometer (nm). Treatment effect was evaluated by means of VAS-scales and global assessment of pain. Outcome of treatment was correlated to changes in urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA). The clinical impression was that placebo was superior to laser stimulation. No statistically significant difference between the analgesic effect of the laser and placebo irradiation was found on VAS-scales. A significant (P = 0.05) increase in 5-HIAA excretion was found in the placebo group. It is concluded that the possibility of a substantial placebo response should be taken into consideration using 904 nm (IR) lasers for pain treatment in patients with this type of chronic oro-facial pain. PMID- 1708119 TI - SDS-PAGE analysis of protein and lipopolysaccharide of extracellular vesicules and Sarkosyl-insoluble membranes from Bacteroides gingivalis. AB - We have compared outer membranes (OM) of Bacteroides gingivalis ATCC 33277 isolated by the following 3 techniques: 1) high speed centrifugation after mechanical cell shearing; 2) sonication of the bacteria, followed by solubilization of the cytoplasmic membrane with N-Laurylsarconsinate (Sarkosyl), after which the Sarkosyl-insoluble membranes were recovered by centrifugation; 3) ammonium sulfate precipitation of extracellular vesicules from culture supernatant, followed by centrifugation and dialysis. Electron microscopy showed that the 3 preparations consisted of closed vesicules. Analysis by SDS-PAGE revealed that all 3 contained up to 28 polypeptides, most of which were common to each extract. The extracellular vesicules and Sarkosyl-insoluble preparation yielded similar protein patterns, although quantitative differences were observed. The sheared-cell preparation contained 8 additional proteins. The level of contamination of OM material by peptidoglycan and cytosol components was 1.8% in the sheared-cell preparation, and was null or lower than 0.8% in the other preparations. All 3 preparations showed the presence of LPS with a multiple banding pattern typical of smooth LPS. The sheared-cell preparation had a slightly lower LPS content than the other 2 preparations. Since extracellular vesicules are naturally released during bacterial growth, and are relatively simple to obtain, such native entities seem an appropriate source of OM components for use in studying the immunobiology of B. gingivalis surface antigens. PMID- 1708120 TI - Heterogeneity and co-expression of intermediate filament proteins in adenoid cystic carcinoma of salivary glands. AB - Among a series of 76 adenoid cystic carcinomas (ACC), 51 cases with cibriform or trabecular patterns were selected for an immunohistochemical study. These tumors were composed of three types of cells: basaloid, myoepithelial and ductal luminal acinous or tubular cells with a variable amount of each cell type from one case to another. Monoclonal antibodies (MoAb) against keratins (PKK1, KL1, K8.12, K8.13, K4.62 anti-cytokeratins antibodies) and against vimentin were used. Tubular cells were characteristically marked by anti-cytokeratins MoAb, but with a great heterogeneity of keratin distribution. In myoepithelial cells, keratin was absent or slightly positive and vimentin was present, with co-expression of the two types of filaments in some cells. Like myoepithelial cells, basaloid cells were positive to anti-vimentin antibody and negative or slightly positive to anti-keratin antibodies, with sometimes a co-expression of vimentin and keratin filaments in the same cell. Histogenesis of adenoid cystic carcinomas was discussed. Progenitor intercalated duct reserve cells may change into ductal luminal and myoepithelial tumor cells. Otherwise, basaloid cells, arising also from intercalated duct reserve cells, are able to acquire some secretory organites. PMID- 1708121 TI - New synthetic routes to synthons suitable for 2'-O-allyloligoribonucleotide assembly. AB - New synthetic routes have been devised for the high yield preparation of protected 2'-O-allylribonucleoside-3'-O-phosphoramidites, exemplified by the ribonucleosides guanosine and 2,6-diaminopurine riboside (2-aminoadenosine). Key features are the use of versatile intermediates and an easy allylation step. The development of a novel synthon based on 2'-O-allyl-2,6-diaminopurine riboside enables short 2'-O-allyl-oligoribonucleotide probes to be synthesized with adenine replaced by 2-aminoadenine. Thus very stable hybrids with complementary RNA target sequences can be formed due to the formation of the three hydrogen bond 2-amino A.U base pairs. PMID- 1708122 TI - Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3). AB - Using synthetic oligonucleotides, a gene encoding the HIV-1 replication primer, tRNA(Lys,3), was constructed and placed downstream from a bacteriophage T7 promoter. In vitro transcription of this gene yielded a form of tRNA(Lys,3) which lacks the modified bases characteristic of the natural species and the 3' -C-A dinucleotide. Synthetic tRNA(Lys,3) annealed to a pbs-HIV1 RNA template can prime cDNA synthesis catalysed by recombinant HIV-1 reverse transcriptase. Trans-DDP crosslinking indicates that this synthetic tRNA is still capable of interacting with HIV-1 RT via a 12-nucleotide portion encompassing the anticodon domain. Gel mobility shift and competition analyses imply that the affinity of synthetic tRNA for RT is reduced. In contrast to earlier observations, synthetic tRNA is readily competed from RT by natural tRNA(Pro). The reduced affinity of synthetic tRNA(Lys,3) for RT is not appreciably affected by mutations in positions within the loop of the anticodon domain. These results would imply that the overall structure of the anticodon domain of tRNA(Lys,3) is an important factor in its recognition by HIV-1 RT. In addition, modified bases within this, although not absolutely required, would appear to make a significant contribution to the enhanced stability of the ribonucleoprotein complex. PMID- 1708123 TI - Activation of junB by PKC and PKA signal transduction through a novel cis-acting element. AB - The product of the junB gene, a gene homologous to the proto-oncogene c-jun, is a component of transcription factor AP-1. JunB expression is modulated by a wide variety of extracellular stimuli, such as serum, growth factors, phorbol esters (TPA) and activators of protein kinase A (PKA). In order to study the molecular basis of this complex regulation, we have cloned the mouse junB gene from a genomic testis library, and characterized the junB promoter. Here we show that the junB promoter is activated by serum, TPA, and activated PKA. Sequences located between -91 and -44 are necessary for induction. These sequences contain a CAAT box, a G-C rich region and a previously undescribed inverted repeat (IR). The IR element can mediate induction by TPA and PKA when coupled to a heterologous promoter, and specifically binds a protein of 110 kD. PMID- 1708124 TI - A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. AB - Modifiers of position-effect-variegation in Drosophila encode proteins that are thought to modify chromatin, rendering it heritably changed in its expressibility. In an attempt to identify similar modifier genes in other species we have utilized a known sequence homology, termed chromo box, between a suppressor of position-effect-variegation, Heterochromatin protein 1 (HP1), and a repressor of homeotic genes, Polycomb (Pc). A PCR generated probe encompassing the HP1 chromo box was used to clone full-length murine cDNAs that contain conserved chromo box motifs. Sequence comparisons, in situ hybridization experiments, and RNA Northern blot analysis suggest that the murine and human sequences presented in this report are homologues of the Drosophila HP1 gene. Chromo box sequences can also be detected in other animal species, and in plants, predicting a strongly conserved structural role for the peptide encoded by this sequence. We propose that epigenetic (yet heritable) changes in gene expressibility, characteristic of chromosomal imprinting phenomena, can largely be explained by the action of such modifier genes. The evolutionary conservation of the chromo box motif now enables the isolation and study of putative modifier genes in those animal and plant species where chromosomal imprinting has been described. PMID- 1708125 TI - Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop. AB - The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl) uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA. PMID- 1708126 TI - DNA binding properties of a new class of linked anthramycin analogs. AB - We have investigated the DNA binding properties of the anthramycin analogues 4, 5, and 6 using fluorescence spectroscopy. A considerable fluorescence enhancement occurs when pyrrolo [1,4] benzodiazepines (P[1,4]Bs) are covalently attached to duplex DNA, which was used to show that neither the presence of RNA, single stranded DNA, or protein had any effect on the degree of fluorescence enhancement resulting from the incubation of 5 and 6 with DNA. The enhancement was found to be dependent on the presence of the imine functionality in each of the compounds. A wavelength of 320 nm was used to excite the chromophore and its emission wavelength maximum was 420 nm. Additionally, we have discovered that the P[1,4]B ring system exhibits exceptionally favorable fluorescence polarization anisotropy (FPA) decay characteristics. For these more detailed fluorescence measurements, we used the structurally simpler analogue 4,. The time resolved maximum FPA for 4 in glycerol at 25 degrees C is 0.28. This result indicates that the P[1,4]B family of antibiotics could serve as sensitive probes of DNA dynamics in the 0.1 to 35 ns time scale. PMID- 1708127 TI - Cloning and analysis of the mobile element gypsy from D. virilis. AB - The homologue of the Drosophila melanogaster mobile element gypsy was cloned from the distantly related species D. virilis. It has three ORFs highly homologous to those of the element from D. melanogaster. gypsy from D. virilis appears to be actively transcribed and is capable of transposition. Comparison of the untranslated regions of both elements revealed conserved sequences including those which had previously been demonstrated to be important in transcription regulation. Distribution of gypsy among the different strains of D. virilis and different species within the D. virilis group was analyzed. Possible involvement of horizontal transmission in the process of spreading and evolution of gypsy is discussed. PMID- 1708128 TI - Antiproliferative effects of antisense oligonucleotides directed to the RNA of c myc oncogene. AB - Several groups have reported the use of antisense oligonucleotides to inhibit c myc gene expression and study its biological role. However high concentrations of free oligonucleotides were generally needed. To lower their concentration and stabilize the antisense effect against c-myc, oligonucleotides were covalently linked to poly(L-lysine) and administered in ternary complexes formed with heparin (100 micrograms/ml). A sequence specific growth inhibition was observed at concentrations lower than 1 microM, while oligonucleotide-poly(L-lysine) conjugates alone were inefficient. Similar results occurred with other polyanionic compounds. Inhibition of proliferation was correlated to a reduction of c-myc protein and to a transient decrease in c-myc mRNA level. However, implication of RNase H in this process could not be demonstrated. PMID- 1708129 TI - MspI RFLP for microtubule associated protein-2 (MAP2). PMID- 1708130 TI - A new polymorphic probe on chromosome 3p: lambda LIB37-97'' (D3S724). PMID- 1708131 TI - A new polymorphic probe on chromosome 3p: lambda LIB31-38 (D3S726). PMID- 1708132 TI - A new polymorphic probe on chromosome 3p: lambda LIB43-55' (D3S728). PMID- 1708133 TI - A new polymorphic probe on chromosome 3p: lambda LIB03-23 (D3S573). PMID- 1708134 TI - Substance P affects the GABAergic system in the hypothalamo-pituitary axis. AB - In the present work we examined the effect of the neutralization of endogenous substance P by the administration of an anti-substance P serum (ASPS) on GABA concentration in the anterior pituitary in hyperprolactinemic conditions induced by 5-hydroxytryptophan or by grafting anterior pituitaries. ASPS reduced the increase in the anterior pituitary GABA concentration induced by hyperprolactinemia. In vitro experiments showed that substance P inhibited K(+) evoked GABA efflux from hypothalamic fragments and decreased GABA concentration in the anterior pituitary but ASPS increased it. Our results demonstrate that substance P modifies hypothalamic GABA release and anterior pituitary GABA concentration and suggest that an interaction exists between substance P and GABA. PMID- 1708135 TI - Characterization of the bombesin receptor on mouse pancreatic acini by chemical cross-linking. AB - Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18-27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of 125I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used 125I-GRP and chemical cross-linking techniques to characterize the mouse pancreatic BN receptor. After binding of 125I-GRP to membranes, and incubation with various chemical cross-linking agents, cross linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18-27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of 125I-GRP to membranes or cross linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure. PMID- 1708136 TI - C-terminal peptides of calcitonin gene-related peptide act as agonists at the cholecystokinin receptor. AB - We have previously described that [Tyr0]CGRP(28-37) acts as a receptor antagonist of rat CGRP in guinea pig pancreatic acini. We therefore examined other C terminal peptides of CGRP for such activity. CGRP-acetyl(28-37) acetate did act as a rat CGRP antagonist. However, C-terminal CGRP peptides of 4 to 8 amino acid residues did not antagonize the actions of rat CGRP but stimulated amylase secretion. In pancreatic acini, a maximally effective concentration of rat CGRP (100 nM) caused a 2.1-fold increase in amylase secretion. When the C-terminal peptides of CGRP were tested in at 100 microM, CGRP(34-37) caused a 1.8-fold increase in amylase secretion, CGRP(33-37) a 2.8-fold increase. CGRP(32-37) a 9.2 fold increase, CGRP(31-37) a 4.1-fold increase, and CGRP(30-37) a 5.1-fold increase. Further studies with the most effective peptide, CGRP(32-37), demonstrated that it did not cause release of lactate dehydrogenase, and thus did not cause amylase release by cell damage. Unlike rat CGRP, CGRP(32-37) did not increase cellular cyclic AMP, but did stimulate outflux of 45Ca. CGRP(32-37) stimulated amylase release was not inhibited by the substance P receptor antagonist, spantide, by the bombesin receptor antagonist, [D-Phe6]bombesin(6-13) propylamide, or by the muscarinic receptor antagonist, atropine, but was inhibited by the CCK receptor antagonist L364,718. C-terminal peptides of CGRP inhibited binding of 125I-BH-CCK-8, with the relative potencies of the peptides being the same as their relative potencies for stimulating amylase secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708137 TI - Microwave irradiation increases recovery of neuropeptides from brain tissues. AB - The effect of focused high energy microwave treatment (MW) on brain concentrations and molecular forms of substance P, neurokinin A, neuropeptide Y, neurotensin, galanin and calcitonin gene-related peptide was investigated. Groups of rats were treated as follows: 1) MW, storage for 60 min at 22 degrees C, 2) Decapitation, storage for 60 min at 22 degrees C. 3) Decapitation, storage for 60 min at 22 degrees C, MW treatment, 4) MW, decapitation, storage for 2 min at 22 degrees C and 5) Decapitation, storage for 2 min at 22 degrees C. Peptide concentrations were in all instances highest in the MW sacrificed groups. MW increased the concentration of intact peptides by rapid inhibition of peptidase activity and increase in peptide solubility/extractability. PMID- 1708138 TI - Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system. AB - The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin. PMID- 1708139 TI - Substance P innervation of the rat thymus. AB - Immunohistochemistry for substance P (SP) in the rat thymus revealed fine varicose neural profiles in specific regions of the thymus. Thymic SP innervation was abundant within the capsule and interlobular septa. The majority of SP+ nerve fibers within the septa were free of vascular association, although some fibers were associated with the vasculature deep within the septa. SP+ nerve fibers entered the thymic cortex from the septa and distributed among cortical thymocytes and mast cells. Along the corticomedullary junction, SP+ nerve fibers were found in association with the vasculature. The medullary region of the thymus received only a sparse innervation of SP+ fibers. In addition, SP+ nerve fibers coursed adjacent to OX-8+ cells and mast cells in the extrathymic connective tissue surrounding the thymus. The present study provides evidence that SP is present in nerve fibers in the thymus, and may be available to interact with thymocytes, mast cells, and other cells in the thymus, and affect their development and function. PMID- 1708140 TI - The pediatrician and early intervention for the developmentally disabled or handicapped child. AB - New federal legislation involving infants at risk for handicaps and their families, in the form of Public Law 99-457, will rely on interaction between pediatricians and other professionals to maximize health and social benefits. Involvement in early identification and remediation of infants at risk is a role well suited to the primary care pediatrician. Early Intervention Programs offer remediation and enhancement of development for children at biologic or environmental risk. Pediatricians should be alert to screen, identify, and assess children who may be helped by Early Intervention Programs. The primary care pediatrician should work with children who have these problems, help coordinate care, and serve as an advocate for the child and family. PMID- 1708141 TI - [Pericardial C-reactive protein. A marker of agonal cardiac disease ?]. AB - We studied the influence of the agonal period on the concentrations of acute phase proteins in biological fluids obtained from 26 autopsy cases. We found significant differences for C-reactive protein concentrations in serum and in pericardial fluid, between short and long agonies. The other acute phase proteins studied (alpha-1 antitrypsin, alpha-2 macroglobulin, haptoglobin) failed to show any significant difference in serum and pericardial fluid levels between the two types of agony. The increase in C-reactive protein level in the pericardial fluid is attributed to an "agonal pericarditis" which may result from an agonal myocardial necrosis. Our results could be of interest in forensic medicine. PMID- 1708142 TI - Glutamate activation of a single NMDA receptor-channel produces a cluster of channel openings. AB - Activations of the N-methyl-D-aspartate (NMDA) receptor by glutamate were studied in outside-out patches from CA1 cells in rat hippocampal slices. Very low glutamate concentrations (20-100 nM) were used so that individual receptor activations would be well separated. The shut-time distribution contained at least five components, only the longest component being obviously concentration dependent. The three briefest shut-time components had time constants of 56 microseconds, 0.68 ms and 10.1 ms; all of these were independent of glutamate concentration. An individual activation of the receptor therefore produces a long cluster of channel openings that contains longer gaps than have been reported for receptor activations by other fast neurotransmitters. In addition, (i) some activations may contain still longer (mean 78 ms) shut periods generating 'super clusters', and (ii) a significant amount of NMDA current may be carried by prolonged ('high P(open)') periods during which the channel is open for most of the time. Such periods occur intermittently even at these very low glutamate concentrations. It is suggested that the slow time course of the NMDA receptor mediated synaptic currents may be determined mainly by the channel activation kinetics. PMID- 1708143 TI - Location of a threonine residue in the alpha-subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel. AB - By the combination of cDNA manipulation and functional analysis of normal and mutant acetylcholine receptor (AChR) channels of Torpedo expressed in Xenopus laevis oocytes determinants of ion flow were localized in the bends bordering the putative M2 transmembrane segment (Imoto et al. 1988). We now report that in the rat muscle AChR, substitution of a threonine residue in the alpha-subunit localized in the M2 transmembrane segment increases or decreases the channel conductance, depending on the size of the amino acid side chain located at this position. This threonine residue (alpha T264) is located adjacent to the cluster of charged amino acids that form the intermediate anionic ring (Imoto et al. 1988). This effect is pronounced for the large alkali cations Cs+, Rb+, K+ whereas for Na+ the effect is much smaller. Taken together the results suggest that the threonine residues at position 264 in the two alpha-subunits together with the amino acids of the intermediate anionic ring form part of a narrow region close to the cytoplasmic mouth of the AChR channel. PMID- 1708144 TI - Behavioural changes on diet selection and serotonin (5-HT) turnover in rats under pizotifen treatment. AB - The effects of pizotifen on protein and carbohydrate self-selection in rats over a seven-day period, and on 5-HT turnover was studied. Four groups of male Wistar rats were individually caged and ad lib fed with a standard (SD) and (50%) carbohydrate-enriched diet (CED), sweet (diet group I) or not (diet group II). Food intake was measured daily 4 hr after IP injection of pizotifen (2.5 mg/kg) or vehicle. 5-HT and 5-HIAA in the hypothalamus (Hy), striatum (St) and hippocampus (Hi) were assayed on the 8th day of the experiment. Pizotifen increased the consumption of SD. The absolute intake of CED remained totally and daily unchanged, while the percentage proportion was reduced. Total food intake was increased by the drug which seemed to affect the proportion rather than the absolute amounts of carbohydrate and protein consumed. This effect was independent of the carbohydrate taste. There was a decrease of 5-HT levels in the Hi, while 5-HIAA/5-HT ratio was increased in the Hy and in the Hi of animals that consumed sweet carbohydrate. The above data suggest a role of pizotifen on 5-HT central metabolism and diet selection and support the view that changes of 5-HT metabolism in the Hy and Hi are responsible for protein selection and the regulation of SD/CED ratio, but they cannot explain drug's effect on total food intake. PMID- 1708145 TI - Discrimination of butorphanol and nalbuphine in opioid-dependent humans. AB - The purpose of the study was to evaluate the agonist and antagonist stimulus properties of the mixed opioid agonist antagonists butorphanol and nalbuphine in opioid-dependent subjects. Opioid-dependent volunteers (methadone 30 mg/day, PO) were trained in a three-choice drug discrimination procedure to discriminate between the effects of saline (2 ml), hydromorphone (10 mg/70 kg) and naloxone (0.15 mg/70 kg) administered IM. Subjects earned monetary reinforcement for correctly identifying the training drugs by letter code. Other subjective, behavioral and physiological measures were also collected. Hydromorphone and naloxone increased drug-appropriate responses and other characteristic subjective effects measures. Butorphanol and nalbuphine produced increases in naloxone appropriate discrimination responding and in those subjective effect measures increased by naloxone. Butorphanol produced greater than 80% naloxone-appropriate responding at 1.05 mg/70 kg; nalbuphine produced 100% naloxone-appropriate responding at 2.1 mg/70 kg. Neither butorphanol nor nalbuphine showed opioid agonist-like effects in these subjects maintained at moderate levels of physical dependence. In opioid-dependent subjects, the stimulus effects of butorphanol and nalbuphine are antagonist-like. PMID- 1708146 TI - Sex differences in the effects of inescapable footshock on central catecholaminergic and serotonergic activity. AB - In two experiments sex differences in changes in central noradrenergic, dopaminergic and serotonergic activity were measured immediately after a 30-min session of inescapable footshocks. In Experiment 1 concentrations of noradrenaline, dopamine, serotonin and their major metabolites were determined in the frontal cortex, hypothalamus, amygdala, striatum, mesencephalon and the medulla-pons area. Inescapable shock increased the activity of all 3 transmitter systems, as evidence by increased metabolite concentrations in specific brain areas. Shock-induced increments in metabolite levels were larger in females than in males, especially for the serotonergic system. In addition, shock presentation resulted in a decrement in the noradrenaline content in most areas studied. In the frontal cortex, noradrenaline was reduced by inescapable shock in males but not in females. In Experiment 2, sex-dependent neurochemical consequences of predictable versus unpredictable shocks were studied in the frontal cortex and the medulla-pons area. Similar to Experiment 1, both brain parts showed large shock-induced increments in the activity of the catecholaminergic systems. Differential effects of predictable and unpredictable shock were not found (frontal cortex) or were rather small (medulla-pons) and appeared sex-dependent for serotonin in this area. The sex differences in neurochemical change found in the first experiment were largely replicated in the second experiment. The relevance of the observed sex differences in central neurotransmitter reactivity for sex differences in behavior is discussed. PMID- 1708147 TI - Muscarinic receptor subtypes and sexual behavior in female rats. AB - Cholinergic muscarinic systems are involved in the regulation of female sexual behavior in rats and hamsters. This series of experiments was designed to determine whether sexual behavior in female rats is controlled preferentially by one of the traditional muscarinic receptor subtypes. Intraventricular infusion of the muscarinic antagonist scopolamine (10 micrograms bilaterally) which binds with high affinity to both M1 and M2 subtypes inhibited sexual behavior, as indicated by the incidence of lordosis, in ovariectomized rats treated with estrogen and progesterone. In contrast, the M1-selective antagonist pirenzepine failed to reduce the incidence of lordosis following intraventricular infusion (10 to 80 micrograms bilaterally). Biochemical analyses revealed that intraventricular infusion of scopolamine (10 micrograms bilaterally) inhibited both M1 and M2 binding in brain tissues while intraventricular infusion of pirenzepine (10 micrograms bilaterally) completely inhibited M1 binding without affecting M2 binding. Intraventricular infusions of the acetylcholinesterase inhibitor physostigmine (10 micrograms bilaterally), the cholinergic agonist carbachol (1 microgram bilaterally), and the muscarinic agonist oxotremorine-M (0.1 micrograms bilaterally) activated lordosis in ovariectomized females primed with low doses of estrogen. In contrast, the putative M1 agonist McN-A-343 failed to significantly increase lordosis following intraventricular infusions (1, 10, 20 micrograms bilaterally). According to biochemical results, the ability of these agents to activate lordosis in female rats was related to their affinities for M2 binding sites not M1 binding sites. In a final experiment, estrogen treatment of ovariectomized rats did not alter muscarinic subtype binding in several brain areas as measured by the M1-selective ligand [3H] pirenzepine and the M2-selective ligand [3H] oxotremorine-M. The results of these experiments confirm that muscarinic systems contribute to the regulation of lordosis in female rats and indicate that M2 binding sites rather than M1 binding sites may be a critical component of this regulation. PMID- 1708149 TI - Metapsychology, symbol formation, and the work of Susan Deri. PMID- 1708148 TI - Intra-midbrain raphe injections of the neurokinin-3 agonist senktide inhibit food and water intake in the rat. AB - Microinjection and lesion studies have implicated the midbrain dorsal (DR) and median raphe (MR) nuclei in behavioral arousal. This behavioral state is manifested as locomotor hyperactivity, hyperphagia, hyperdipsia and increases in plasma corticosteroid release. Intra-midbrain raphe injections of the GABAA agonist muscimol elicit this behavioral activation. We have demonstrated that similar infusions of tachykinins produce locomotor hyperactivity through activation of neurokinin-3 (NK-3) receptors located on serotonin cell bodies. The purpose of the present study was to determine the effects of intra-MR and DR infusions of senktide, an NK-3 agonist, on food and water consumption in nondeprived rats. Male Sprague-Dawley rats were implanted with indwelling intra MR or intra-DR cannula. Infusions of muscimol (25 ng/0.5 microliters) into the MR increased water intake, while MR and DR infusions increased food consumption. In contrast, intra-MR injections of senktide decreased water intake and intra-MR and DR injections decreased food intake. The results suggest that the behavioral states induced by muscimol and neurokinin infusions into the raphe are distinct and that raphe/neurokinin pathways are involved in consummatory mechanisms. PMID- 1708150 TI - The homeostatic and the representational function of the symbolic process; with reference to the "Rat Man's" obsessive ideation. PMID- 1708151 TI - Case presentation. Pathologic use of word symbols. PMID- 1708152 TI - The preconscious and potential space. AB - Greenberg and Mitchell (1983) have suggested that the drive/structure model and the relational/structure model are mutually exclusive models of psychic life. We regard their contribution as an invaluable one, which makes explicit the fundamental divergences in psychoanalytic theory. We have examined a derivative tendency in the field, for drive and relational theorists alike, to present psychic life as a dichotomy between inner experience and outer experience. We see a tendency to equate the drive model with unconscious motivation, and to the primacy of internal experience. There seems to be an equivalent tendency to equate the relational model with conscious perception and motivation, and to the primacy of external experience. We are advocating, for drive and relational theorists alike, greater focus on the process of intermediation between internal and external experience in the psychic life of the individual. Within the context of the drive model, precedent for such a focus is found in Freud's conception of the preconscious, an essential third dimension whose function was to mediate between the conscious and the unconscious. Within the context of the relational model, Winnicott's notion of potential space serves as a bridge between interior experience and external reality in the life of the individual. Finally, we have argued that by constructing three-part models of psychic life, these theorists have laid the groundwork for a synthetic theory. Though for Freud the drive state is primary, and for Winnicott the relationship between the infant and its environment (mother) is primary, each theorist posits an intermediating zone that fulfills a similar function in the psychic life of the individual. Whether we choose to call that zone the preconscious or potential space, its function is to translate bidirectionally between the infinitely dimensioned realm of interior, or unconscious, experience and the time-and space-bound realm of external, or conscious, experience. By highlighting the parallel constructs, we are not claiming to have created a synthesis between the theories. Our claim is that the eventual road to synthesis appears to reside in the direction of a movement away from the dichotomy between the primacy of inner or outer experience, and toward the common meeting ground of the primacy of an intermediating function. PMID- 1708153 TI - Receptors, phosphoinositol hydrolysis and plasticity of nerve cells. AB - Excitatory amino acid neurotransmission has been shown to be necessary but may not be sufficient, for the production of LTP and other prolonged changes in synaptic transmission. Excitatory neurotransmission may produce depolarization induced increases in intracellular calcium that cause PI hydrolysis and synergistically potentiate receptor-G protein induced PI hydrolysis. This synergistic potentiation of phosphoinositide hydrolysis, and increased [Ca]i due to positive cross stimulation, may lead to depolarization block, a persistent increase in protein kinase activation, altered morphology, oncogene activity and other plasticity changes important in memory. PMID- 1708154 TI - Genetic conservation of cyclo-oxygenase. AB - Homology between sheep and mouse cyclo-oxygenase cDNA and a range of other species was investigated using "zoo blots". Both the sheep and mouse cDNAs showed a high degree of homology to other species. Human cyclooxygenase appears to have greater homology to sheep and other ruminants than to mouse. Both sheep and mouse cDNAs detected identically sized transcripts in human placental total RNA. PMID- 1708155 TI - Modification of postischemic increase of leukocyte adhesion and vascular permeability in the hamster by Iloprost. AB - This study of the postischemic events in the hamster cheek pouch showed that there is an increase in number of leukocytes adhering to the venular endothelium after reperfusion. It also showed that the stable prostacyclin analogue Iloprost could counteract both the postischemic increase in leukocyte adhesion and the postischemic increase in vascular permeability to macromolecules. The hamsters were anesthetized and the cheek pouch was everted and prepared for intravital microscopy. Temporary ischemia (30 min) was obtained using an expandable cuff placed around the proximal part of the cheek pouch. Fluorescein labelled dextran (FITC-dextran, Mw 150,000) was used as a tracer of macromolecular leakage from the postcapillary venules. Iloprost, given either topically (0.1 nM) or as an intravenous infusion (40 ng/kg/min), reduced the postischemic permeability increase (P less than 0.05) but did not affect the hemodynamics or the permeability response induced by histamine. It is proposed that these effects could be due to inhibition of leukocyte activation by Iloprost, indicating that these cells could play a role in the permeability increase during reperfusion after ischemia. PMID- 1708156 TI - U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells. AB - The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3 isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC. PMID- 1708157 TI - Families of children with developmental disabilities: an examination of family hardiness. AB - Using the Typology Model of Adjustment and Adaptation, a family stress model, relationships among family hardiness, family stressors, family appraisal, coping, social support, and satisfaction with family functioning were examined in a sample of 57 families of children with developmental disabilities. Family hardiness was associated with family appraisal, social support, parental coping related to maintaining family integration, and satisfaction with family functioning. Higher satisfaction with family functioning was correlated with coping-integration, network support, functional support, and hardiness. Lower satisfaction with family functioning was associated with higher family stressor scores, social support loss, and increased parental age. Over 42% of the variance in family functioning was accounted for by family hardiness, functional support, family stressors, and parental age. The results highlight the value of continued investigations of hardiness in families. PMID- 1708158 TI - Use of monoclonal antibodies for the detection and quantitation of HIV1 core protein p25: comparative evaluation of in vitro HIV1 infection by immunofluorescence, antigen capture ELISA and reverse transcriptase assays. AB - A two-site enzyme linked immunosorbent assay (ELISA) was developed to detect and quantify the HIV1 core protein p25 in the cell-free supernatant from virus infected CEM cell culture, and compared with other assays. The assay, based on a sandwich method, employs two monoclonal antibodies (mAb) directed against different epitopes on the p25 core protein of HIV1, one used for p25 antigen capture and the other as a biotinylated probe. This immunoassay is sensitive enough to detect as little as 30 pg/ml of recombinant p25, the range of sensitivity of commercial kits, and therefore compares favourably with the conventional reverse transcriptase assay. Moreover, several hundred assays can be monitored quite conveniently by this simple ELISA procedure, which represents a useful tool for detection of HIV1 replication in microculture systems and rapid screening of antiretroviral agents using the reference strain HIV1-BRU as a model system. PMID- 1708159 TI - Cytomegalovirus-antibody-negative lymphocytes have increased permissiveness for human immunodeficiency virus. AB - Synergistic activity between cytomegalovirus (CMV) and human immunodeficiency virus (HIV) was observed. In vitro, lymphocytes which were CMV-antibody-negative showed greater cytopathic effects due to HIV than those which were antibody positive. These cytopathic effects included increased cell death and greater virus production. In vitro stimulation of host lymphocytes with CMV was found to be analogous to that of interleukin-2 stimulation in terms of HIV production and cytopathic effects. PMID- 1708160 TI - Discrete association of CD3 and CD4 molecules in T-cell stimulation acting through the autologous mixed lymphocyte reaction and the CD3/T-cell receptor complex in human autoreactive T-cell clones. AB - To investigate autologous antigen recognition, we developed two autoreactive CD4+ T-cell clones, A2 and A10, maintained with non-T cells and IL-2. Autologous non-T cell stimulation of the T-cell clones resulted in a decrease in cell surface expression of CD4, whereas the expression of CD2, CD3, and WT31 was unchanged. Activation of the autoreactive T-cell clones by cell surface binding anti-CD3 MoAb, as a specific antigen stimulator of the T-cell receptor complex, induced cell surface antigen comodulation of CD3, CD4, and WT31. These data suggest a discrete association of CD3 and CD4 molecules in T-cell stimulation, acting through the autologous mixed lymphocyte reaction and the CD3/T-cell receptor complex. PMA treatment resulted in concomitant down-regulation of CD3 and CD4 but calcium ionophore treatment did not. Thus, it appears that phosphorylation of CD3 leads to the down-regulation of surface antigens of CD4. PMID- 1708161 TI - Selective enhancement of Leu-CAM expression by interleukin 6 during differentiation of human promonocytic U937 cells. AB - Interleukin 6 (IL-6) is a cytokine with multiple biological activities on various tissues and cells. We have investigated the effect of recombinant human IL-6 (rhIL-6) on growth and differentiation of U937. Recombinant human IL-6 induced a dose-dependent growth inhibition, apparent around day 4, of up to 50% by day 8 of culture. Concomitant with this, changes in cytochemical activities were observed, indicative of differentiation. A panel of U937 antigens was analysed after culture with rhIL-6. Expression of the majority of these membrane antigens was unaffected, with the exception of two classes. First, rhIL-6 had a profound effect on members of the leucocytic cell adhesion molecules (Leu-CAM) family. Expression of the alpha-chain of CR3 (complement receptor 3; CD11b) was enhanced in a dose-dependent fashion, with maximal expression around day 7. Parallel to this a simultaneous increase in beta-chain (CD18) expression was found. Furthermore, enhanced expression of CR3 was, accompanied by increased rosetting with sheep erythrocytes sensitized with C3bi. A much lower, but significant, enhancement was observed for the alpha-chain of the p150,95 antigen (CD11c). Expression of leucocyte function-associated antigen-1, (LFA-1), (CD11a/CD18) remained unchanged. Remarkably, however, expression of a ligand of LFA-1, intercellular adhesion molecule-1 (ICAM-1; CD54), was enhanced with similar kinetics as CR3 and p150,95. A specific anti-rhIL-6 antiserum completely inhibited the IL-6 effect. These observations provide further support for an important role of IL-6 in the regulation of myeloid cell development in man. PMID- 1708162 TI - Expression of CD40 and CD43 during activation of human B lymphocytes. AB - CD40 and CD43 are two cell-surface glycoproteins that appear to be functionally involved in the growth stimulation of human B cells. Whereas CD40 is structurally similar to the NGF receptor and is present on all resting B cells, CD43 displays no homology to other known proteins and is expressed only on a subpopulation of these cells. To further understand the extra- and intracellular signals regulating these molecules and in which stage of activation they may play a role, we used various activation strategies and studied their expression on tonsillar B cells. As expected, activation of protein kinase C by TPA increased both CD40 and CD43. In contrast, a rise in intracellular Ca2+, e.g. by ionomycin, did not influence the expression of these antigens. However, in the presence of TPA, ionomycin further up-regulated CD43 but not CD40. Anti-IgM behaved similarly to ionomycin suggesting that the effect of this reagent was due primarily to its ability to increase intracellular Ca2+. Of three interleukins (IL-2, IL-4 and IL 6) only IL-4 had a significant effect when used alone in that it up-regulated CD40 but not CD43. However, in the presence of anti-IgM, both IL-2 and IL-4 synergistically up-regulated the two antigens. Complementation of antigen receptor stimulation with TPA or IL-4 increased CD40 during the first 24 h, whereas up-regulation of CD43 did not occur until 24 to 48 h after stimulation. With regard both to up-regulation in response to different stimuli and to kinetics, CD40 expression paralleled that of the early activation antigen CD23, whereas CD43 was induced in parallel with the transferrin receptor (CD71). Taken together, our results suggests that the expression of CD40 and CD43 is regulated by different intracellular signals and that CD40 may be important during early activation, whereas CD43 may have its major function during later stages of B cell differentiation. These assumptions are in line with the observations that CD40 antibodies can directly activate resting B cells and that CD43 are retained on plasma cells. PMID- 1708163 TI - A monoclonal IgM antibody to a methylcholanthrene-induced tumour. I. Specificity for alpha-N-acetylgalactosamine but with no cross-reactivity to the human blood group A determinant. AB - A monoclonal IgM antibody, H17, has been obtained from rats immunized with mouse fibrosarcoma cells from an in vitro established methylcholanthrene (MCA)-induced tumour. H17 shows specific and very selective binding to alpha-N acetylgalactosamine (GalNAc alpha) when tested for reactivity to a panel of glycolipids. It cross-reacts with GalNAc alpha on the Forssman antigen extracted from dog small intestine, but not from the human blood group A determinant, a finding not commonly observed among antibodies with this specificity. Despite its specificity, H17 does not react with TA3-Ha, a mouse mammary adenocarcinoma, known to express the Tn antigen (GalNAc alpha-O-Ser/Thr). The uniqueness of H17 probably relates to the fact that it has been generated against an MCA-induced tumour rather than against the pure saccharide itself. Minimum energy conformation structures of different GalNAc alpha containing saccharide molecules were computer modelled to allow a plausible interpretation of the accessible site of GalNAc alpha for successful interaction with the H17 paratope as compared to other GalNAc alpha binding antibodies. PMID- 1708164 TI - Non-immune VH-binding specificity of human protein Fv. AB - The specificity of human F(ab)-binding Protein Fv (previously called Protein F), a sialoprotein released into the digestive tract mainly during hepatitis, was investigated with fragments or chains of monoclonal immunoglobulins. Protein Fv bound an unreduced H-chain dimer of a monoclonal human IgA2m(1) molecule but neither the corresponding L-chain dimer, nor several Bence-Jones molecules. Using enzymatic subfragments of F(ab)mu, or F(ab')2 gamma, a significant binding was observed with Fv fragments (VH + VL), while Fb fragments (CH1 + CL) were inactive. Taken altogether, these results prove that the structure recognized by Protein Fv is located in the VH domain. This structure probably involves discontinuous epitopes linked by a disulphide bond, which are destroyed by combined reduction and dissociation. Protein Fv does not interfere with the antigen-binding site, since there was no reciprocal inhibition with the antigen antibody reaction. PMID- 1708165 TI - CD5+ B-lymphocytes are as dependent on T-helper cells and as responsive to T suppressor cells in pokeweed mitogen-stimulated activation as conventional CD5- B lymphocytes. AB - Pokeweed mitogen (PWM)-stimulated cultures were established with varying numbers of CD4+ or CD8+ cells and CD5+ immunoglobulin-secreting cells were subsequently identified using a combination of haemolytic plaque formation and rosetting with immunobeads coated with anti-Leu-1. It was found that CD5+ and CD5- B-cells required similar numbers of CD4+ cells for half-maximum plaque-forming cell (PFC) induction and that 50% suppression of both CD5+ and CD5- PFC occurred with similar numbers of CD8+ cells. Suppression of both CD5+ and CD5- PFC could occur when T-helper signals were supplied by a PWM-stimulated culture supernatant, indicating that both subsets could be directly suppressed by CD8+ cells rather than by indirect suppression of T-helper cells. It is concluded that CD5+ B-cells are as responsive to T-regulatory influences as conventional CD5- B-cells. PMID- 1708166 TI - Oligopeptide induction of a secondary cytotoxic T-cell response to Epstein-Barr virus in vitro. AB - Three Epstein-Barr virus (EBV) nuclear antigen (EBNA)-encoded oligopeptide epitopes have been mapped, each capable of acting as a recognition determinant for class I-restricted lysis by CD8+ cytotoxic T lymphocytes (CTL). This report shows that each peptide, when presented on an appropriate autologous antigen presenting cell (APC), also stimulates EBV-specific memory T cells present in peripheral blood mononuclear cell (PBMC) populations to develop in vitro into peptide-specific CTL. These CTL specifically lysed autologous EBV-infected lymphoblastoid cell lines (LCL) and peptide-sensitized uninfected targets. Identical viral oligopeptides could therefore function as recognition determinants for both the induction and commission of class I-restricted specific cytotoxicity. A model system is described in which autologous phytohaemagglutinin (PHA) blasts present exogenous peptide during the stimulation phase. The magnitude of the peptide-specific CTL response was dependent on the concentration of peptide added to the APC and specific lysis was inhibited by anti-class I monoclonal antibody (MoAb) but not anti-class II MoAb. Cultures depleted of CD8+ T cells by cell separation with immunomagnetic beads prior to stimulation invariably failed to generate a peptide-specific CTL response. However, the effect of CD4 depletion on CTL activity was equivocal and indicated that a need for CD4+ T cells as accessory helper cells may depend on the efficiency of the APC to elaborate their own help. This model has advantages in the analysis of events involved in the development of CTL activity in vitro. PMID- 1708167 TI - Human natural killer cells express different integrins and spread on fibronectin. AB - Human natural killer (NK) cells adhered and most of them also actively spread on cellular fibronectin (cFn), plasma Fn (pFn) and its Mr 120,000-140,000 or Mr 105,000 cell-binding proteolytic Fn-fragments as well as on heparin-binding Fn fragments containing the alternative cell binding site. The cells did not spread on vitronectin, laminin or collagens. Adhesion on Mr 105,000 Fn fragment containing the cell binding site, could be prevented by the synthetic peptide GRGDS but not by an inactive peptide, whereas adhesion on heparin-binding Fn fragments was unaffected by the peptide. Spreading of the NK cells led to a distinct reorganization of F-actin. Immunoprecipitation with monoclonal antibodies (MoAb) against the beta 1 integrin subunit of radioactively surface labelled cells revealed a broad polypeptide band of Mr 140,000 under reducing conditions and a polypeptide doublet of Mr 160,000 and Mr 110,000 under non reducing conditions. Identical polypeptides, corresponding to the alpha- and beta subunits of the Fn-receptor complex, were bound to the Mr 105,000 chymotryptic Fn fragment coupled to Sepharose. Similar experiments with small lymphocytes did not reveal any polypeptides. Immunofluorescence results with McAbs suggested that among the alpha-subunits of integrins, the alpha 3, alpha 4, and alpha 5 subunits are expressed in NK cells. The present results suggest that non-activated NK cells, but not small lymphocytes, express beta 1-integrins, and that at least the Fn-receptors alpha 4 beta 1 and alpha 5 beta 1 may function in the adhesion and migration of NK cells. PMID- 1708168 TI - Recombinant virus vaccine-induced SIV-specific CD8+ cytotoxic T lymphocytes. AB - Evidence indicates that cytotoxic T lymphocytes (CTLs) may be important in containing the spread of the human immunodeficiency virus (HIV) in the infected host. Although the use of recombinant viruses has been proposed as an approach to elicit protective immunity against HIV, the ability of recombinant viral constructs to elicit CD8+ CTL responses in higher primates has never been demonstrated. A live recombinant virus, vaccinia-simian immunodeficiency virus of macaques (SIVmac), was used to determine whether such a genetically restricted, T lymphocyte-mediated antiviral response could be generated in a primate. Vaccinia SIVmac vaccination elicited an SIVmac Gag-specific, CD8+ CTL response in rhesus monkeys. These CTLs recognized a peptide fragment that spans residues 171 to 195 of the Gag protein. The rhesus monkey major histocompatibility complex (MHC) class I gene product restricting this CTL response was defined. Both the vaccinated and SIVmac-infected monkeys that shared this MHC class I gene product developed CTLs with the same Gag epitope specificity. These findings support the use of recombinant virus vaccines for the prevention of HIV infections in humans. PMID- 1708169 TI - Immunopathologic and clinical features of hemolytic anemia due to cold agglutinins. PMID- 1708170 TI - A comparative assessment of commonly employed staining procedures for the diagnosis of cryptosporidiosis. AB - Following an increase in the number of reports of Cryptosporidium infections and the problems encountered in detecting these organisms in faecal smears, a comparative assessment of a modification of the Sheather's flotation technique and other commonly employed staining procedures proved the modified Sheather's technique to be most useful in identifying Cryptosporidium oocysts in diarrhoeal stools. This technique not only detected the parasite in the highest number of stools but also proved to be cost-effective and the least time-consuming. Other staining techniques assessed were the modified Ziehl-Neelsen, safranin-methylene blue and auramine-phenol fluorescence. Both the modified Ziehl-Neelsen and the auramine-phenol fluorescence procedures produced nonspecific staining, while the safranin-methylene blue method was found to be the least sensitive technique. PMID- 1708171 TI - Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro. AB - AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10. PMID- 1708172 TI - The amino acid sequence Gly-Ala-Pro-Leu appears to be a fibrinogen binding site in the platelet integrin, glycoprotein IIb. AB - The amino acid sequence Gly-Ala-Pro-Leu-Arg-Val is predicted by the anticomplementarity hypothesis to be a fibrinogen binding site on human platelet fibrinogen receptors. The peptide Ala-Pro-Leu-Arg-Val binds fibrinogen and inhibits platelet aggregation and clot retraction. The peptide Gly-Ala-Pro-Leu is the shortest sequence within the predicted sequence which potently inhibits the adhesion of platelets to fibrinogen and platelet aggregation. The sequence Gly Ala-Pro-Leu is present as residues 309-312 in glycoprotein IIb, the alpha-subunit of the glycoprotein IIb/IIIa complex, the fibrinogen receptor. The sequence Gly Ala-Pro-Leu is present in 4 of 8 integrin alpha-subunits and Gly-Ala-Pro is present in 8 of 8 integrin alpha-subunits. PMID- 1708173 TI - Orellanine inhibits protein synthesis in Madin-Darby canine kidney cells, in rat liver mitochondria, and in vitro: indication for its activation prior to in vitro inhibition. AB - Pure orellanine, a nephrotoxic compound extracted from the mushroom Cortinarius orellanus, which is known to induce severe kidney damage several days or weeks after ingestion, is found to inhibit strongly the synthesis of macromolecules (proteins, RNA and DNA) in Madin-Darby canine kidney (MDCK) cells and in rat liver mitochondria, although the uptake of labelled precursors of the above macromolecules is not significantly altered. Direct addition of orellanine to a cell-free system of rabbit reticulocyte lysate does not produce any inhibition of protein synthesis. However, when orellanine is pre-incubated with activating rat liver microsomal systems, this inhibition occurs. Thus, the in vivo inhibition of protein synthesis is most likely due to a metabolite of orellanine. PMID- 1708174 TI - Liver lipid peroxidation-related parameters after short-term administration of hexachlorocyclohexane isomers to rats. AB - Rats treated with diets containing 20 ppm of alpha- or gamma hexachlorocyclohexane (HCH) for 15 or 30 days showed increased levels of liver cytochrome P-450 followed by increased production of both thiobarbituric acid reactants by liver homogenates and microsomes and superoxide anion production by liver microsomes. In these animals superoxide dismutase (SOD) activity was also increased. In consequence, the ratio between SOD activity and microsomal superoxide radical (O2-.) production showed a slight increase after 15 days of treatment. However, after 30 days, there was a tendency for this ratio to decrease. Other parameters studied were liver glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase and catalase (CAT) activities. Among them, only CAT activity showed a 26% and 38% increase after 15 or 30 days of treatment with the alpha-isomer. It is suggested that when lipid peroxidation is involved in the mechanism of toxicity of a xenobiotic, this parameter can be used to determine the no-observed-effect level. PMID- 1708175 TI - [The clinical and diagnostic characteristics of Sjogren's syndrome (disease) and of nonspecific chronic sialoadenitis]. AB - Analysis of clinical and laboratory parameters of nonspecific chronic sialadenitis, among which essential symptoms of Sjogren's syndrome (disease) have been distinguished, has lead the author to a conclusion that Sjogren's syndrome and disease develop as a symptom complex in the presence of all chronic sialadenitis forms, i.e., parenchymatous, interstitial, and sialodochitis. PMID- 1708176 TI - Gene isolation is 25 years old this month. PMID- 1708178 TI - Radiation therapy combined with chemotherapy for inoperable pancreatic carcinoma. AB - From March 1985 to July 1989, 22 patients with unresectable pancreatic adenocarcinoma entered the study to receive external beam irradiation with chemotherapy. Radiation therapy consisted of 60 Gy in 3 courses (20 Gy each course) delivered over a period of 2 weeks, with a 2-week rest between the courses. Chemotherapy consisted of 5 fluorouracil, 500 mg/m2, plus cisplatinum, 20 mg/m2, administered on days 1, 2 and 3 of each radiation therapy course. Of the 22 evaluable patients, 10 were males and 12 females; their median age was 63 years (range, 32-77), and their median performance status was 80 (range, 60-90). After treatment, 12 partial remissions and 6 no changes were reported. In 4 cases, abdominal progression of disease during treatment required interruption of the therapy program. At the start of treatment, abdominal pain was the most important symptom in 17 patients; improvement of abdominal pain was observed in 10 cases (76%) after treatment and lasted for a median of 5 months. Median survival time was 7.5 months, and time to progression was 6.2 months. Median follow-up was 7 months (range, 14 days -38). In 2 cases, persistent hematologic toxicity did not permit completion of therapy, and in another 3 cases grade II hematologic toxicity required a 2-week rest period over the normal split-course program. In another 4 cases, grade I hematologic toxicity did not require any delay in the therapy program. Our results are comparable with those achieved in other major studies and are acceptable in terms of survival time, palliation of symptoms and toxicity. In our experience, the combination of radiotherapy plus 5 fluorouracil and cisplatinum does not seem to offer any advantage over the combination of radiation therapy and 5-fluorouracil. PMID- 1708177 TI - Frequent occurrence of Ha-rasl allelic deletion in human ovarian adenocarcinomas. AB - Fourteen human adenocarcinoma specimens were analyzed for somatic abnormalities affecting genes of the ras family. No amplification of the 3 ras genes was detected. Allelic deletion of the Ha-rasl gene (11p15.5) was found to be a very common abnormality in human ovarian adenocarcinomas (4 out of 7 informative cases). However, in these neoplasm deletion of a presumed normal Ha-rasl allele is not a contributory factor in strengthening the tumorigenic effect of a mutated allele. More probably, Ha-rasl allelic losses are markers of larger chromosomal deletions. Analyses at gamma globin loci (11p15.5) and int-2 locus (11q13) provided evidence that the deletions may extend from Ha-rasl locus towards the centromere but never involve loss of the entire chromosome 11. These findings may suggest that a putative tumor suppressor gene closely linked to Ha-rasl in 11p15.5 is involved in ovarian cancerogenesis. PMID- 1708179 TI - Lectin histochemistry on squamous metaplasia in different epithelial tumors of dogs. AB - Biotinylated lectins and avidin-biotin-peroxidase complex were used to study the correlation between cellular glycoconjugates' expression and squamous maturation in normal canine skin and in various epithelial neoplasms. Normal skin tissue was obtained from five, male, random-source dogs, 5 to 7 years old. The tumors tested, selected from the files of our Department, were fifteen squamous cell carcinomas from different tissue origin, five hepatoid perianal gland adenocarcinomas with squamous metaplasia, and fourteen solid mammary carcinomas with and without histologic evidence of squamous metaplasia. Except for mammary gland carcinomas, all tumors had been surgically excised from male dogs. Intermediate filament aggregation of twelve solid mammary gland carcinomas were studied electron microscopically. The basal and the lower spinous cells in normal skin and the less differentiated cells in squamous cell carcinomas stained moderately with Griffonia simplicifolia agglutinin-I. Spinous and granular cell layers stained strongly with Phytolacca americana mitogen and Arachis hypogaea agglutinin. Both lectins stained well-differentiated cells in squamous cell carcinomas. The electron microscopic study carried out in solid carcinomas of mammary glands revealed some relationship between the presence of intracytoplasmic tonofibrils and the binding of Griffonia simplicifolia agglutinin-I and Phytolacca americana mitogen to the tumors tested. Our results suggest that the glycosylation pattern occurring during normal keratinocyte differentiation is conserved in squamous cell carcinomas and that Griffonia simplicifolia agglutinin-I and Phytolacca americana mitogen may represent useful tools in distinguishing poorly differentiated squamous cell carcinomas from other poorly differentiated mammary epithelial tumors. PMID- 1708181 TI - The effect of "end to side" portacaval anastomosis on regeneration ability of white rat liver. AB - The study evaluated the effect of portacaval anastomosis on the regeneration ability of the liver after partial hepatectomy in rat. The regeneration of liver was evaluated using the following tests: deoxyribonucleic acid synthesis, the activity of tyrosine aminotransferase (TAT) and the concentration of DNA and RNA in subcellular fractions of liver obtained by differential centrifugation. The results indicate the harmful effect of the portacaval shunting on the liver proliferation, which was near 50% decreased and the peak of the regeneration was delayed for about 12 hours. The most significant changes were observed in the nuclear and cytoplasmic fraction. In addition, high mortality ratio was found in the animals with portacaval anastomosis followed by partial hepatectomy. PMID- 1708180 TI - [Diagnosis of rejection after pancreatic transplantation in the minipig model]. AB - An allogenic transplantation of the total-pancreas with duodenum was carried out in the minipig model. The oral duodenum was closed blindly and the aboral part was led out as a stoma. The portal vein of the transplant was anastomosed end-to side in the inferior vena cava and the celiac artery of the transplant in the distal aorta. Investigations for early diagnosis of rejection were carried out in this model parallelly respectively one by one. CT/MRT, selective angiography, laboratory chemical parameters were unsuitable for early detection. The pancreatic succus cytology and nuclear medical investigations show reproducible changes caused by rejection before the blood-sugar level was increased. PMID- 1708182 TI - [Sudeck disease]. AB - In 1900, the Hamburg surgeon P.H.M. Sudeck was the first to describe the clinical and radiological symptomatology of the disorder to which he later lent his name. He devoted his scientific career to this disorder, "acute inflammatory atrophy" of bone and soft-tissue as he called it. He elaborated the histology of the disorder with his students and advocated the pathogenic peripheral humoral theory. In later years seven further attempts to explain the pathogenesis were published. The present paper notes that this disorder, which can be divided into three distinct stages, is diagnosed mainly on the basis of clinical factors, above all in the early stage, and that x-rays in the early stages are difficult to differentiate from those of immobilisation osteoporosis since both disorders can manifest as patchy rare fraction and fibrous osteolysis in subchondral bone and old growth plates. Moreover, the author also notes that 10% of all Sudeck cases are not preceded by trauma; these must be understood as spontaneous Sudeck or as Sudeck after distant disturbances. A few sources also refer to Sudeck's dystrophy in the presence of lymphatic congestion. Another, special form of Sudeck's atrophy, is the shoulder-hand syndrome which, however, cannot always be differentiated precisely from rheumatic disorders or dystrophic contractures. PMID- 1708183 TI - Recognition of a hepatitis B virus nucleocapsid T-cell epitope expressed as a fusion protein with the subunit B of Escherichia coli heat labile enterotoxin in attenuated salmonellae. AB - Two overlapping T-cell sites of the nucleocapsid antigen (HBc) of Hepatitis B Virus (HBV) (amino acids (aa) 120-140) and a B-cell epitope of the pre-S(2) region of the HBV surface antigen (aa 133-140) were expressed as a fusion protein with the subunit B of Escherichia coli heat labile enterotoxin (LT-B) in attenuated salmonellae (aroA Salmonella dublin SL1438). When Balb/c (haplotype H 2d) mice were fed salmonellae expressing LT-B or the LT-B/HBV fusion protein they developed serum IgG anti-LT-B antibodies and splenic cells reactive to LT-B. C57BL/10 (H-2b), in contrast, showed anti-LT-B antibody titres, but no splenic cell priming to LT-B. Neither in Balb/c nor in C57BL/10 mice could an antibody response to the fused HBV antibody binding site be demonstrated. In C57BL/10, however, an HBc T-cell epitope fused to LT-B primed a splenic cell response to an analogous synthetic peptide (HBc aa 121-145) in four out of five mice after three oral immunizations. This is the first description of the priming of a cellular immune response to a defined heterologous epitope expressed in attenuated salmonellae and delivered by the oral route. PMID- 1708184 TI - Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus. AB - Under laboratory conditions coronaviruses were shown to have a high frequency of recombination. In The Netherlands, vaccination against infectious bronchitis virus (IBV) is performed with vaccines that contain several life-attenuated virus strains. These highly effective vaccines may create ideal conditions for recombination, and could therefore be dangerous in the long term. This paper addresses the question of the frequency of recombination of avian coronavirus IBV in the field. A method was sought to detect and quantify recombination from sequence data. Nucleotide sequences of eight IBV isolates in a region of the genome suspected to contain recombination, were aligned and compared. Phylogenetic trees were constructed for different sections of this region. Differences in topology between these trees were observed, suggesting that in three out of eight strains in vivo RNA recombinant had occurred. PMID- 1708185 TI - Concentration of some proteinase inhibitors: alpha 1-antitrypsin and alpha 2 macroglobulin in rabbit blood serum in two models of experimental atherosclerosis. AB - In rabbits with experimental atherosclerosis induced by a cholesterol-rich diet, alpha 1-antitrypsin concentration was decreased as compared with control, by 34%, whereas alpha 2-macroglobulin concentration was increased by 86%. In animals fed a methionine-rich diet changes in concentration of both inhibitors involved in elastase metabolism were but slight, if any. PMID- 1708186 TI - Quantitation of nanogram amounts of nucleic acids in the presence of proteins by the ethidium bromide staining technique. AB - Modification of the method for determining low amounts of RNA and DNA is proposed. It consists in nucleic acid staining in solution with EtBr (1 microgram/ml) followed by photography of 10 microliters drops on a UV-transparent plate under UV illumination. Densitometric measurements of the Polaroid negatives were used to construct standard concentration curves in the range of 1-16 micrograms/ml of DNA or RNA. This permitted to determine nucleic acid in amounts as little as 10 micrograms. The measurements were not influenced by the presence of proteins such as bovine serum albumin, DNase, RNase or proteinase K, thus the method proposed may be useful in determining the nucleic acid content of very small samples or of scarce biological material. PMID- 1708187 TI - Analysis of suppressor activity in experimental allergic encephalomyelitis inhibited by graft-versus-host reaction. AB - The induction of local graft-versus-host reaction (GvH) prior to the encephalitogenic challenge resulted in the conversion of acute experimental allergic encephalomyelitis (EAE) to the chronic-like EAE. This inhibitory effect of GvH on EAE development was cyclophosphamide (CY) sensitive. Cell-free supernatants of peripheral blood lymphocytes (PBL) isolated from guinea pigs with chronic-like EAE and during recovery from EAE showed suppressor activity on the in vitro proliferative response of myelin basic protein (MBP) sensitized PBL. The appearance of anaphylactic anti-MBP antibodies and a change in the ratio complement fixing: haemagglutinating (CF/HA) antibodies was also registered. PMID- 1708188 TI - [Effects of neutrophils on histamine release from mast cells]. AB - To determine whether neutrophils (NP) contribute to immediate anaphylaxis, the effects of NP on histamine release from mast cells (MC) were studied and changes in the NP morphology were investigated. When rat peritoneal MC and pleural NP collected from actively sensitized rats were mixed and challenged with antigen (trichosanthin), histamine release from MC was significantly increased. This promotive effect of NP on antigen-induced histamine release from MC was dosage dependent. The observation of scanning and transmission electron microscopy showed that NP were activated and secretion was resulted. It is suggested that NP may be activated in immediate anaphylaxis and secrete some mediators which could promote histamine release from MC. Quercetin may inhibit the promotive effect of the NP on histamine release and the secretion of the NP while ketotifen and isoproterenol have no influence on this promotive effect. PMID- 1708189 TI - The role of histamine release in shock. AB - More than 80 years after its discovery and nearly 70 years after its first being implicated in the mechanism of shock, the precise role of histamine (H) in the pathophysiology of the condition remains to be determined. The prevailing view over the decades has been that H is a noxious mediator contributing to the fatal outcome of shock. An adequate assessment of its role has long been hampered by the lack of a sufficiently sensitive and specific method for the measurement of H and by deficiencies of design in many studies. We now know that H is released in all types of shock. In low-output, high-resistance states, as in hypovolemic and cardiogenic shock, its effect (contrary to previous notions) appears to be beneficial, probably through the inhibition of excessive vasoconstriction and through a positive inotropic effect. In endotoxin shock the results are conflicting, but seem to indicate that H is not a lethal factor. In human septic shock, patients who died had higher H levels. The role of H release in the various forms of shock, both experimental and human, clearly needs an adequate, critical reevaluation, in carefully designed and executed studies using satisfactory methodology. PMID- 1708191 TI - Use of local hyperthermia for the treatment of benign prostatic hyperplasia. PMID- 1708190 TI - Effects of clomipramine treatment on cerebrospinal fluid monoamine metabolites and platelet 3H-imipramine binding and serotonin uptake and concentration in major depressive disorder. AB - In an open study of 12 inpatients who met the DSM-III criteria for a major depressive episode, the effects of clomipramine (CI) on the monoamine metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 4-hydroxy-3 methoxyphenyl glycol (HMPG) in cerebrospinal fluid (CSF) were measured simultaneously with the effects on 3H-imipramine binding, serotonin (5-HT) uptake and 5-HT concentration in platelets after 3 and 6 weeks of treatment. Drug (CI and desmethylclomipramine) plasma concentrations were determined. The concentrations of 5-HIAA and HMPG decreased substantially, and the concentration of HVA remained unchanged. There was also a large and significant reduction of the number of imipramine binding sites (Bmax) and of the platelet 5-HT concentration. The 5-HT uptake was not measurable after 3 weeks of treatment. None of the parameters changed significantly between weeks 3 and 6. There were no significant correlations between antidepressant effect (measured by the Montgomery-Asberg Depression Rating Scale) and plasma drug concentrations, although a tendency to a significant correlation between antidepressant effect and CI was observed at 3 weeks. There were no significant intercorrelations between the different 5-HT parameters and no other significant correlations between the biochemical measures and clinical outcome. PMID- 1708192 TI - Local microwave hyperthermia and benign prostatic hyperplasia induced bladder outlet obstruction. PMID- 1708193 TI - Verbal communication impairment in dementia research frontiers in language and cognition. PMID- 1708194 TI - [Age and development related changes in counseling priorities for pediatric home visits]. AB - More home visits should be made in the context of prophylactic care for children up to three years of age, in order to come to grips with problems of child development likely to results from the inadequate knowledge and experience of parents and to promote age-adjusted motivation for the undisturbed development of their children. In this sense home visits can be an effective approach to primary prevention and a contribution to harmonious development in childhood. The priorities of counselling and their content are subject to change, depending on the age and development of the children involved. Examples are given of age adjusted stimulation of development for one-year-old children. They are based on results of empirical investigations conducted between 1984 and 1987. PMID- 1708195 TI - [The role of school admission examination in a preventive health program for children 3-7 years old]. AB - This paper reports on new aspects of the prophylactic care programme, with particular emphasis being laid on prophylactic examination for children of pre school age. An account is given of the interrelations between the various check ups. Prophylactic examination in the fifth year of age--mass screening No. 10 as the first school entrance examination--is the main priority during this period. The major issues relating to case history, physical, locomotor, and psychosocial development, behaviour, and classical status are described in detail and are offered as a graduated programme. PMID- 1708196 TI - [Preventive health care for children in Sweden]. AB - A review is given of structures, organization, function, and contemporary problems of preventive health care for children in Sweden. The importance of health education for their parents is emphasized. PMID- 1708197 TI - Food allergy in children: diagnosis and treatment with sodium cromoglycate. AB - Food allergy (FA) is a very important problem affecting numbers of infants and children with protean manifestations which are frequent challenges to the pediatrician and other specialists working with children. Adverse reactions to food are very complex, frequently mediated bu IgE mechanisms, and often by other mechanisms. To make the correct diagnosis and to arrive at a proper therapeutic approach requires all the skill a physician can gather. Only an extensive knowledge of the various mechanisms and pharmacologic agents that can be used to prevent or treat these adverse reactions will allow the physician to approach the problem scientifically and come to a reasonable solution for the patient. The role of dietary factors in atopic dermatitis (AD) has long been a subject of controversies. However, it has been shown that FA plays a role in some children with AD. Therefore, the management of this multifaceted disorder is a challenge for pediatricians, dermatologists, and allergists. SCG, which is the salt of a bis-chromone carboxylic acid, has been shown to be of proven efficacy in the prophylaxis of bronchial asthma, allergic rhinitis, and of other disorders associated with mast cell degranulation. The drug has different modes of action, such as inhibition of rat passive cutaneous anaphylaxis, and the antigen-induced histamine release from passively sensitized peritoneal cells. Recently, clinical studies indicated that SCG has a direct effect on inflammatory cells, inhibiting either various leukocyte functions (membrane receptor expression, cytotoxic capacity), or "in vitro" activation of human neutrophils, eosinophils and monocytes. Although SCG has been widely used for the management of respiratory allergy, conflicting results of FA treatment have been reported by several authors. We have reviewed 18 papers on the use of SCG in the management of children with FA, which included 341 children aged 0.5-15 years. In this paper we discuss 12 studies reporting 281 children affected with AD. PMID- 1708198 TI - Antiarrhythmic actions of tocainide in canine models of previous myocardial infarction. AB - The antiarrhythmic and antifibrillatory efficacies of the class IB antiarrhythmic agent tocainide were characterized in conscious dogs in the early subacute phase of anterior myocardial infarction (48 hours after infarction) and in anesthetized dogs with ventricular tachyarrhythmias that were inducible by programmed stimulation more chronically (10.8 +/- 1.0 days) after anterior myocardial infarction. The frequency of spontaneous premature ventricular complexes in the early subacute postinfarction phase was reduced significantly from 48.4% +/- 10.5% to 8.2% +/- 5.0% of total complexes (p less than 0.01) by the cumulative intravenous administration of 10 mg/kg tocainide. In the late postinfarction setting, the intravenous administration of tocainide (6 mg/kg intravenous loading dose + 100 micrograms/kg/min intravenous maintenance infusion) suppressed the initiation of ventricular tachyarrhythmias by programmed stimulation in 5 of 12 animals that were tested and slowed the rate of tachycardia in 3 additional animals. However, the incidence of ventricular fibrillation and of the total number of arrhythmia-related deaths that resulted from the occurrence of a secondary ischemic insult in the presence of previous infarction did not differ significantly between tocainide (75% [9 of 12] incidence of both ventricular fibrillation and of total number of arrhythmia-related deaths) and saline-vehicle control groups (80% [12 of 15] incidence of ventricular fibrillation; 93.3% [14 of 15] incidence of total number of arrhythmia-related deaths). These findings suggest that although class I agents such as tocainide may be efficacious in the suppression of spontaneous premature ventricular complexes and ventricular arrhythmias immediately after myocardial infarction, they may be of limited value in the prevention of malignant ischemic arrhythmias that occur later after myocardial infarction. PMID- 1708199 TI - Sinus parasystole. AB - Sinus parasystole is the expression of a protected nondominant sinus pacemaker, which is totally independent of the dominant rhythm. Two forms of sinus parasystole are described: (1) an active form, where both the dominant and the parasystolic pacemakers are located within the sinus node and (2) a passive form, where the basic rhythm is ectopic and the sinus pacemaker is protected as a result of complete retrograde SA block. Three cases of sinus parasystole are analyzed. In the active form of the arrhythmia the parasystolic sinus P waves are identical to those of the basic sinus rhythm. The diagnosis is suggested by variably coupled premature sinus P waves occurring with mathematically related intervals. This relationship between the parasystolic intervals can not be precise whenever complicating factors such as modulation occur. The recognition of active sinus parasystole is difficult, since the parasystolic P waves do not differ from basic P waves, so that the pattern resembles that of sinus arrhythmia or sinus extrasystoles. The passive form of sinus parasystole is more easily recognized due to the clear-cut difference between the dominant ectopic atrial waves and the "parasystolic" sinus P waves, which manifest with variable coupling intervals and reflect mathematically related intervals in between. PMID- 1708200 TI - Balloon valvuloplasty in pulmonary valve atresia. PMID- 1708201 TI - Physical and genetic mapping of the CDR gene with particular reference to its position with respect to the FRAXA site. AB - This study narrows down the localization of the gene coding for the cerebellar degeneration-related protein (CDR 34) to the upper boundary of the FRAXA and reports the finding of two common RFLPs respectively identified at an RsaI site flanking the 3' end of the gene and at a Hincll site flanking its 5' end. Segregation analysis carried out in the CEPH-pedigrees for the new CDR/RsaI-RFLP versus other polymorphic loci of the region has established a tight linkage with the markers DXS105/DX98 and absence of measurable linkage with two clusters of markers respectively located proximally to the FRAXA (F9, DXS102, DXS51, and DXS369) or distally to it (DXS52, DXS304). In addition, two recombinants were found among 23 scorable sibs identified in the Sardinian pedigrees segregating for the Martin-Bell Syndrome (MBS) and the CDR/RsaI variants. The overall evaluation of the in situ and genetic data reported suggest that the CDR locus 1) is located at the upper boundary of the FRAXA site; 2) is distal to DXS51 and proximal to DXS 389; and 3) segregates in a close linkage association with the loci DXS98 and DXS105 and, to a lesser extent, with the locus for MBS. PMID- 1708202 TI - Thy-1 expresses two signals for apical localization in epithelial cells. AB - Recent work has shown that anchoring via glycosyl phosphatidylinositol (GPI) results in apical targeting for a variety of endogenous and transfected plasma membrane proteins expressed in epithelial cells. To further determine the correlation between GPI anchoring and apical localization, we expressed GPI anchored and secretory forms of Thy-1 in Madin-Darby canine kidney cells by transfection. Native GPI-anchored Thy-1, normally expressed in thymocytes and neurons, was localized to the apical surface. A truncated form of Thy-1, lacking 22 out of 31 hydrophobic amino acids at the COOH-terminus, was also constructed; this deletion blocked attachment of the GPI anchor and resulted in apical secretion of Thy-1. In combination with previous results, our observations indicate that Thy-1 contains apical targeting information in its protein sequence as well as in the GPI anchor. PMID- 1708203 TI - Primary action of endothelin on Ca release in bovine coronary artery smooth muscle cells. AB - Intracellular free Ca concentrations (Cai) were determined by fura-2 microfluorometry in single freshly dispersed cells to differentiate endothelin (ET)-induced Ca release from Ca influx through voltage-gated Ca channels (VGCC). In physiological solution ET (10(-8) M) significantly (P less than 0.05) increased Cai 23 +/- 3% (+/- SE) above baseline; this increase was not significantly attenuated by 2 x 10(-4) M lanthanum, a blocker of VGCC, or Ca-free solution. When the sarcoplasmic reticulum was depleted of Ca by prolonged treatment with 5 x 10(-3) M caffeine, depolarization with 80 mM K (80K; or 30K) plus ET did not increase Cai above that induced by 80K (or 30K) in caffeine alone. In contrast, 10(-6) M BAY K 8644, instead of ET in the protocol, significantly (P less than 0.05) increased Cai above that induced by 80K (or 30K). ET released Ca from the caffeine-sensitive internal store but was not rapid and transient like caffeine-induced release, which elicited a peak Cai increase in less than 1 min; instead, release was more gradual and prolonged with Cai peaking in greater than 2 min, thus resembling the response to 10(-5) M ryanodine. With two ET exposures, either a transient nonrepeatable increase in Cai or a delayed, but sustained, increase in Cai resulted, similar to the response to ryanodine. These data indicate that in freshly dispersed bovine cells the predominant mechanism by which ET increases Cai is release of Ca from the sarcoplasmic reticulum; if any increase in L-type voltage-gated Ca influx occurred, it was minimal and matched by efflux. PMID- 1708204 TI - CaMKII mediates stimulation of chloride conductance by calcium in T84 cells. AB - We used the secretory colonic cell line T84 to study the regulatory pathways controlling the Ca-stimulated Cl conductance [GCl(Ca)]. Under whole cell patch clamp, basal (unstimulated) current levels averaged 73 +/- 9 pA/20 pF (n = 93) and increased to 600 +/- 100 pA/20 pF (n = 53; at +100 mV) on exposure to 1-2 microM ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50 microM) reversibly inhibited GCl(Ca), whereas the protein kinase C antagonists H7 and phloretin (50 microM) were without effect. This suggests that increases in intracellular Ca stimulate GCl(Ca) via a CaM-dependent process rather than activating Cl channels directly. To assess the involvement of protein kinases in the Ca-dependent stimulation of Cl conductance, we employed pseudosubstrate peptide inhibitors of protein kinase C (PKC) and the Ca/CaM-dependent protein kinase II (CaMKII). Cellular concentrations of inhibitors during whole cell recording were estimated to be 4 20 times the inhibitory constant values for kinase inhibition observed in vitro. Pipette solutions containing the PKC peptide inhibitor PKC-(19-36) (7.5 microM) had no effect on GCl(Ca). In contrast, stimulation of GCl(Ca) by ionomycin was abolished when pipette solutions contained 10 microM CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20 microM) lacks the CaMKII inhibitory domain and did not affect GCl(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells. PMID- 1708205 TI - Chloride channel blockers inhibit ACTH secretion from mouse pituitary tumor cells. AB - The effect of several chemically related chloride channel blocking drugs was investigated on the adrenocorticotropic hormone (ACTH) secretory process in mouse clonal AtT-20 corticotrophs. When cells were simultaneously exposed to diphenylamine-2-carboxylate (DPC) or related substances (Hoechst compounds 131, 143, and 144) and the adenylate cyclase activator forskolin, ACTH secretion was inhibited by 76-95% [half-maximal inhibitory concentration (IC50) 450, 15, 84, and 32 microM, respectively]. All four compounds also blocked forskolin stimulated adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in AtT-20 cells by 51-87% (IC50 190, 29, 100, and 130 microM for DPC and compounds 131, 143, and 144, respectively). Pertussis toxin pretreatment of cells caused a partial reversal of DPC-inhibited forskolin-stimulated cAMP formation. The toxin had no effect on inhibition of forskolin-stimulated ACTH secretion by DPC. Secretion of ACTH in response to cAMP-independent stimulants such as the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate or the calcium channel agonist BAY K 8644 were blocked by compound 131 as was the secretory response to 8 bromoadenosine 3',5'-cyclic monophosphate. These results suggest that phenylanthranilic acids have adenylate cyclase inhibiting action but that the postcyclase activity is more relevant to the ability of these compounds to block ACTH secretion. DPC also blocked 125I efflux (an index of Cl- secretion) from AtT 20 cells. Because an increase in osmotic strength of the culture media reduced forskolin-stimulated ACTH secretion, these data suggest that DPC and related compounds may negatively modulate chloride-dependent osmotically driven ACTH secretion from AtT-20 cells. PMID- 1708206 TI - Sarcosine kinetics in pigs by infusion of [1-14C]sarcosine: use for refining estimates of glycine and threonine kinetics. AB - To investigate in vivo the interconversion between glycine (Gly) and its N-methyl product sarcosine (Sar), [1-13C]Gly and [1-14C]Sar were infused into hourly fed pigs receiving diets with low- and high-threonine levels. An open two-pool model was developed to calculate Sar demethylation (DM) and Gly methylation (GM). During [1-14C]Sar infusion, intracellular Gly specific radioactivities (SA) in the liver and kidney were higher than plasma Gly SA, suggesting that demethylation of Sar occurred in those tissues. DM estimated by using hippuric acid (HA) as the production pool had a mean value of 1.55 mumol.kg-1.h-1, similar to the Sar production rate (mean 1.85 mumol.kg-1.h-1). GM was undetectable (less than 0.5 mumol.kg-1.h-1). These results suggest that, in fed pigs, Sar is produced mainly from choline catabolism and is degraded only to Gly in liver and kidney. On the assumption that Sar degradation gave rise only to Gly, the production rate of Gly (Gly PR) was calculated from [1-13C]Gly and [1-14C]Sar infusions using either the primary pools (plasma Gly and HA, respectively) or the secondary pools (HA and plasma Gly, respectively). The results were explained by a liver-plasma Gly exchange model. The whole body Gly irreversible loss, i.e., direct loss from plasma and liver, was calculated from this model to be 832 +/- 58 mumol.kg-1.h-1, showing that the estimation of Gly PR with [1-13C]Gly infusion and plasma Gly enrichment (599 +/- 56 mumol.kg-1.h-1) was a significant underestimate of the true value.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708207 TI - Gastrin-releasing peptide is a transmitter mediating porcine gallbladder contraction. AB - We studied the role of gastrin-releasing peptide (GRP) for porcine gallbladder motility. Immunohistochemistry visualized nerve fibers containing GRP-like immunoreactivity in muscularis. GRP concentration dependently stimulated contractions of muscularis strips (ED50, 2.9 nM). Neuromedin B was less potent (ED50, 0.1 microM), suggesting existence of GRP-preferring receptors. GRP-induced contractions were unaffected by muscarinic antagonism (1 microM atropine), axonal blockade (1 microM tetrodotoxin), cholecystokinin (CCK) receptor antagonism (10 microM MK-329), or substance P desensitization (1 microM), supporting the existence of myogenic GRP receptors. The bombesin (BN) analogue D-Phe6-BN-(6 13)propylamide (PA) stimulated contractions (ED50, 3.3 nM) with low efficacy (29% of that of GRP). D-Phe6-BN-(6-13)PA (1 microM) shifted GRP concentration-response curves one log to the right. D-Phe6-BN-(6-13)PA interacted specifically with GRP receptors; while abolishing responses to GRP (1 nM), responses to substance P (0.1 microM) and CCK-8 (1 nM) were unchanged. Electrical stimulation (10 Hz, 0.5 ms, 10 V) caused a rapid onset-slow offset, tetrodotoxin-sensitive excitation. Atropine reduced the amplitude to 58% and caused a delayed, slow onset-slow decline response. D-Phe6-BN-(6-13)PA reduced the amplitude to 59% and caused a very rapid onset-rapid decline response. Atropine plus D-Phe6-BN-(6-13)PA abolished responses to nerve stimulation. Nerve stimulation caused significant release of GRP-like immunoreactivity. Thus two neural inputs were defined: a cholinergic rapid onset-rapid offset excitation and a delayed, slow onset-slow offset excitation caused by release and subsequent binding of GRP to GRP preferring receptors. PMID- 1708208 TI - Slow-wave activity in colon: role of network of submucosal interstitial cells of Cajal. AB - The present study compares the electrophysiological properties of two preparations dissected from the canine colon circular muscle layer: first, containing the submucosal network of interstitial cells of Cajal (ICC) with two to four associated smooth muscle cell layers, and second, a circular muscle preparation devoid of the submucosal ICC network. In the ICC-rich preparations, consistent slow-wave activity was observed with prolonged plateau potentials of approximately 10-s duration. The plateau potentials were sensitive to D 600. In approximately 45% of circular muscle preparations devoid of the submucosal ICC network (confirmed using electron microscopy) slow waves, of different waveshape, were recorded at frequencies identical to those in whole circular muscle preparations. These slow waves did not show a plateau potential. Compared with ICC-rich preparations with a resting membrane potential of about -80 mV, circular muscle preparations had lower membrane potentials, about -70 mV when active, and about -60 mV when quiescent. Heptanol (1 mM) electrically uncoupled cells, since it abolished electrotonic current spread and allowed measurement of the input resistance by intracellular current injection. Heptanol also affected ionic conductances. Heptanol abolished slow waves; the underlying mechanism needs further investigation. In the presence of heptanol, cells in the isolated ICC network and in circular smooth muscle preparations showed spontaneous hyperpolarizing potential fluctuations at a frequency of four to six per second. These oscillations were abolished by current-induced hyperpolarization and TEA (30 mM) and are therefore likely due to spontaneously active K+ conductance. PMID- 1708209 TI - cAMP increases synthesis of surfactant-associated protein A by perfused rat lung. AB - Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8 BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27-36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A. PMID- 1708211 TI - Cutaneous and basophilic sensitivity to substance P and gastrin in non-atopic versus atopic subjects. AB - We compared the cutaneous reaction to intradermal injection of substance P, gastrin and histamine in asymptomatic atopic subjects with a history of hay fever and/or asthma versus non-atopic healthy volunteers. We also studied in these two groups the basophilic histamine release induced by substance P and gastrin with that obtained with anti-human IgE and Con A. Intradermal injection of substance P (3-300 pM) and gastrin (3-30 pM) caused a wheal and flare reaction which was comparable in both groups of subjects. Substance P 10(-4)M caused a mean basophilic histamine release of about 15% in atopic and non-atopic subjects. Gastrin was not effective in this model. Anti-IgE and Con A-induced histamine release was significantly higher in atopic than in non-atopic volunteers. PMID- 1708210 TI - Primary translation products of pulmonary surfactant protein D. AB - Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate binding protein that is synthesized by alveolar type II epithelial cells. To further characterize SP-D, we isolated RNA from adult rat lungs and rat type II cells and translated mRNAs in vitro. [35S]methionine-labeled translation products were precipitated with antibodies to rat SP-D, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and visualized by fluorography. Immune precipitates of translation reactions for rat lung or rat type II cells demonstrated a single collagenous polypeptide (39.3 kDa) that was smaller than surfactant-associated SP-D (43 kDa, reduced) but larger than the mature secreted form of rat SP-A. This component was not identified in translation reactions of rat liver, gut, brain, mammary gland, or rat L2 cell RNA. There was a fivefold enrichment of SP-D mRNA in freshly isolated type II cells relative to lung; however, the levels of translatable SP-D mRNA decreased rapidly during the first 24 h of cell culture. The SP-D translation product migrated faster than the major cellular form of SP-D but approximately 1 kDa slower than cellular SP-D synthesized in the presence of 2,2'-dipyridyl plus tunicamycin. Translation in the presence of canine pancreatic microsomes gave a single glycosylated, endoglycosidase F-sensitive form (40.6 kDa) and demonstrated cleavage of a small signal peptide. These results indicate that SP-D is a secretory product of differentiated type II epithelial cells and that SP-D is secreted in a mature form that does not undergo further proteolytic processing in vivo. PMID- 1708212 TI - Bacteria-induced histamine release from human bronchoalveolar cells and blood leukocytes. AB - Histamine release induced by Staphylococcus aureus was examined in cells obtained by bronchoalveolar lavage (BAL) in non-atopic individuals. Approximately half of the individuals responded with mediator release to the bacterium, and the release was found to be time- and concentration dependent. No difference was found between the patients who responded and those who did not respond in regard to age, sex, smoker/non-smoker, % recovery of BAL-fluid, total cell count, differential cell counts, histamine content per mast cell, or diagnoses. Also stimulation of the BAL-cells with the calcium-ionophore A23187 resulted in histamine release. S. aureus-induced histamine release from basophils was examined in leukocyte suspensions obtained from the same individuals, and in all experiments release was found. The dose-response curves were similar to those obtained with BAL cells. The bacteria-induced mediator release from superficially lying cells in the airways epithelium might be of importance for the precipitation or exacerbation of bronchial asthma in respiratory tract infections. PMID- 1708213 TI - [Chronobiology of the benign growth of the prostate gland]. PMID- 1708214 TI - Pentamorphone for management of postoperative pain. AB - The efficacy, duration, and safety of the synthetic opioid pentamorphone in the treatment of acute postoperative pain were evaluated in a randomized, double blind study of 72 patients given 0.08, 0.16, or 0.24 micrograms/kg of pentamorphone or a placebo intravenously in the recovery room after major abdominal or orthopedic surgery. Only patients given 0.24 micrograms/kg of pentamorphone experienced decreased pain intensity and increased sedation, both transient in duration. Although the two higher doses of pentamorphone delayed the patient's request for supplemental morphine, the total amount of morphine required within the first hour was not different between treatments. No acute cardiorespiratory changes were observed. Pentamorphone (0.08-0.24 micrograms/kg) was ineffective for treating acute postoperative pain after major surgery. PMID- 1708215 TI - [Evaluation and treatment of aphthous ulcerations of the mouth]. PMID- 1708216 TI - [Cutaneous vascularization. Angiogenesis. Structure and function of endothelial cells]. PMID- 1708217 TI - The activity of locally applied cytotoxics to breast cancer cells in vitro. AB - The ability of commonly used operative lavage solutions to destroy breast cancer cells was investigated. The cytotoxicity of solutions of Savlon, noxythiolin, povidone iodine, hydrogen peroxide, bleomycin and water on two human breast cancer cell lines was measured in vitro. Viable cells were determined by ability to exclude trypan blue. Results have been analysed with standard non-parametric tests and demonstrate that all solutions tested significantly (P less than 0.01) reduced the number of viable cells recovered when compared with a control solution of phosphate buffered saline. Solutions of Savlon, 2.5% noxythiolin and povidone iodine were more effective than the other agents in reducing the number of recovered viable cells. PMID- 1708218 TI - Different malignant phenotypes induced in a stable, subdiploid, benign epithelial clone by DNA transfection. AB - Progression to malignancy in carcinomas has been studied in a stable, benign, subdiploid, cloned epithelial cell line (A5P/B10) sensitive to Geneticin at 100 micrograms/ml. A total of 28 cell lines were selected for Geneticin - resistance and inoculated into the footpads of syngeneic animals following co-transfection with pSV2neo and genomic DNA, or transfection with plasmid constructs containing neo and the activated Ha-ras oncogene. The behavior of 12 cell lines cotransfected with normal genomic DNA and inoculated into 146 footpads was the same as the A5P/B10 cells. Low grade primary tumors were produced in 122 footpads by 13 cell lines transfected with Ha-ras, and a proportion (61/122) produced well differentiated lymph node metastases. One of 3 cell lines cotransfected with genomic DNA from a malignant cell line (BC1) produced 8 anaplastic primary tumors with anaplastic metastases. Cell lines from lymph nodes involved by these anaplastic tumors were sensitive to Geneticin, and genomic DNA from 2 clones of these cells failed to produce a malignant phenotype when co-transfected into the A5P/B10 cells. These results indicated that the progression to a malignant phenotype induced in benign cells from a spontaneous epithelial tumor by co transfection with genomic DNA from malignant cells was different from that induced by the ras oncogene. PMID- 1708219 TI - Evidence for distinct isoenzymic forms of a cell surface protease on normal colonic cells and on carcinoma cells from the same colon. AB - Both normal and carcinoma cells of the colon possess the cell surface protease guanidinobenzoatase (GB). GB is similar to plasminogen activator. In fixed sections, the insolubilised GB can be located by the fluorescent probe 9-amino acridine (9-AA) which binds to the active centre of GB, causing cells possessing active GB to fluoresce yellow. Both cell types possess cytoplasmic proteins which can be extracted in isotonic saline and were shown to inhibit their respective cell surface GB. The formation of an enzyme-inhibitor complex was demonstrated by the failure of 9-AA to react with the cell surface GB inhibitor complex. The data presented indicate that the colonic carcinoma cells possess an isoenzymic form of GB not recognised by the inhibitors of GB extracted from normal colonic cells. PMID- 1708220 TI - Expression of polymorphic HLA-A,B epitopes in human urothelial cell lines. AB - Malignant human urothelial cell lines propagated in vitro have previously been demonstrated to express low amounts of monomorphic HLA-A,B,C as compared to premalignant urothelial cells. In this study the expression of polymorphic HLA A,B epitopes in human urothelial cell lines have been investigated in greater detail. The expression of HLA-B locus coded epitopes in malignant TGrIII cells was demonstrated to be low or absent as compared to pre-malignant TGrII or slightly transformed TGrI cells, suggesting a mechanism by which malignant cells could escape from the host immune response. The extreme polymorphism of HLA-A,B,C antigens suggests that HLA typing could be used as a method to identify the origin of cell lines which is essential in the study of the process of malignant transformation in vitro, or when correlating in vitro data with clinical observations of the patient. Two urothelial cell lines classified as slightly transformed (TGrI) and two as pre-malignant (TGrII) could, according to their expression of polymorphic HLA-A,B epitopes, be identified as genuine independent cell lines. One TGrII cell line previously designated Hu1734 shared the same HLA A,B phenotype as the genuine HCV29 (TGrII) cell line and is therefore suspected to be a subline of the latter. Out of 18 cell lines and sublines classified as TGrIII the fidelity of four (Hu1922 and three sublines of T24) was proved by their HLA-A,B phenotype. Mistaken identity either by contamination or false labelling of the cultures was suspected in samples of six TGrIII cell lines and sublines. In four of these the HLA-A,B type characteristic for the T24 cell line could be demonstrated, but in general HLA typing of TGrIII cell lines as a method to identify the origin of the individual cell lines failed, primarily due to the decreased expression of HLA-A,B,C antigens and the apparent selective loss of HLA B locus coded antigens. PMID- 1708221 TI - Hematopoietic growth factors. AB - Hematopoiesis is a complex process that underlines the production of multiple highly specialized cells. The intricate mechanisms involved in this process include both positive and negative feedback by humoral activities, pluripotent stem cell selfrenewal and differentiation, and local interactions between stromal components of the hematopoietic microenvironment and various stem and progenitor cells. A group of hematopoietic growth factors, as well as their genes and chromosomal locations, have been identified. Advances in biochemistry and molecular biology led to the purification, genetic sequencing and molecular cloning of these glycoproteins. They include interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and erythropoietin (EPO). The biologic specificity of these substances is defined by their ability to support proliferation and differentiation of hematopoietic cells in a semisolid clonal assay system. These factors share certain characteristics, including their ability to stimulate the function of mature cells, their overlapping activity affecting progenitor cells of several lineages, and their direct and indirect actions on nonhematopoietic cells. Trials using hematopoietic growth factors demonstrated their remarkable efficacy in a variety of clinical settings. PMID- 1708222 TI - Generation of diversity in retroviruses. AB - Retroviruses are unique in that their propagation includes transfer of genetic information from RNA to DNA. Two enzymes (RT and RNA polymerase II) that participate in their replication process do not encode editing functions and are thus "error prone." Current estimates indicate that up to one nucleotide substitution per genome occurs per retrovirus replication cycle. In addition, rearrangements can occur during reverse transcription. These mutations result in viral populations in which the wild-type sequence can only be defined by consensus. Retroviruses are also unusual among viruses in their high recombination frequency, which is the result of the copackaging, and then reverse transcribing of two different RNA genomes in the same particle. Recombinants are formed during copying of RNA templates into DNA. With the ability to mutate and recombine genetic information at a higher rate, retroviral populations are poised to respond to selective forces, which may increase or decrease replication of particular genotypes. Interspecies transmission of retroviruses or retroviral genes also likely plays a role in generating diversity. Endogenous proviruses exist in the germline of many vertebrates; pedigrees indicate that recent infections of the germline have also occurred. Thus, the selective forces that mold the retroviral genome may be opposing: selection for efficient replication as exogenous viruses versus selection for passive replication as endogenous proviruses. The latter may be more advantageous over the course of evolution since the survival of the retrovirus is ensured by survival of the host organism. Segments of endogenous viruses may reappear in exogenous viruses through recombination and thus these endogenous sequences are perpetuated. PMID- 1708223 TI - Activities of pefloxacin and ciprofloxacin against experimental malaria in mice. AB - We investigated the in vivo antimalarial activities of pefloxacin and ciprofloxacin in Swiss albino mice infected intravenously with 5 x 10(6) Plasmodium yoelii N67 parasites 1 h before treatment. Groups of 20 mice received a subcutaneous injection of 40, 80, or 160 mg of ciprofloxacin or pefloxacin per kg of body weight every 8 h for 3 days. Parasitologic activity was assessed on day 4, and survival was assessed on day 21. Control mice had a fulminant course with a parasitemia of 61.3% +/- 12.1% on day 4, and 90% of the mice were dead on day 21. The lower dosages of pefloxacin and ciprofloxacin (40 and 80 mg/kg) were not efficient. With 160 mg/kg, ciprofloxacin achieved an 85.8% reduction in parasitemia and 17 of 20 mice survived. Pefloxacin achieved a 92.8% reduction in parasitemia, and all mice survived. All treated, noninfected control mice survived. With ciprofloxacin, the antimalarial activity was similar with injections of 240 mg/kg every 12 h but was strongly diminished with injections of 160 mg/kg every 12 h. With pefloxacin, similar activities were observed with injections of 160 mg/kg every 8 h or injections of 160 or 240 mg/kg every 12 h. With both drugs, this activity was highly reduced when the treatment was delayed by 24 h. This underlines the need to provide treatment within the first hours after infection to achieve an optimal effect in this rapidly lethal experimental model of malaria. Pefloxacin and, to a lesser extent, ciprofloxacin are potent antimalarial drugs which might prove useful in the treatment of less rapidly aggressive human malaria. PMID- 1708224 TI - Correlation of quinolone MIC and inhibition of DNA, RNA, and protein synthesis and induction of the SOS response in Escherichia coli. AB - The effects of nalidixic acid and four fluoroquinolones on DNA, RNA, and protein synthesis in the presence and absence of 20 mg of chloramphenicol per liter were examined by comparing the killing kinetics, MIC, morphological response, and maximum concentration to induce recA in Escherichia coli. All agents demonstrated paradoxical killing kinetics, in that above an optimum concentration the rate of bactericidal action was slower. Filamentation of E. coli AB1157 was observed with all quinolones up to the optimum bactericidal concentration. Addition of chloramphenicol reduced the bactericidal activity, inhibited filamentation, and abolished recA induction, but it had no effect on DNA synthesis inhibition by any of the agents. Excellent correlation was obtained between the concentration required to inhibit DNA synthesis by 50%, the MIC, the maximum concentration to induce recA, and the optimum bactericidal concentration. Evidence from this study and previously published data suggest that the primary mechanism of action of quinolones is independent of the SOS response and does not require active protein synthesis; however, induction of recA and SOS responses is consequential and enhances cell death. PMID- 1708225 TI - Inhibition of HIV replication in lymphocyte cultures of virus-positive subjects in the presence of sho-saiko-to, an oriental plant extract. AB - An oriental remedy, Sho-saiko-to (SST) consisting of a mixture of aqueous extracts from seven different plants and whose most active component is the chemically defined compound baicalein was tested for its ability to inhibit the production of the human immunodeficiency virus (HIV). The testing was done with cultures of human lymphocytes obtained from HIV-positive asymptomatic subjects and patients with ARC or AIDS. The replication of the virus was monitored by quantitative assay of the reverse transcriptase (RT) activity and of the synthesis of antigen p24. The lymphocyte cultures (LC) were maintained in the absence and in the presence of 25, 50 or 100 micrograms/ml of SST, and monitored for up to 5 weeks. The results showed that in LC from asymptomatic subjects RT activity and synthesis of p24 was completely inhibited by low concentrations of SST. High concentrations of SST inhibited virus replication in 80% of LC from ARC patients, but were completely ineffective in LC from AIDS patients. It was observed that the RT activity was more sensitive to inhibition by SST than the synthesis of p24, and that the antiviral effect was dependent on the virus load of the LC. PMID- 1708226 TI - Inhibition of HIV replication by Hyssop officinalis extracts. AB - Crude extracts of dried leaves of Hyssop officinalis showed strong anti-HIV activity as measured by inhibition of syncytia formation, HIV reverse transcriptase (RT), and p17 and p24 antigen expression, but were non-toxic to the uninfected Molt-3 cells. Ether extracts from direct extraction (Procedure I), after removal of tannins (Procedure II), or from the residue after dialysis of the crude extract (Procedure III), showed good antiviral activity. Methanol extracts, subsequent to ether, chloroform and chloroform ethanol extractions, derived from procedure I or II, but not III, also showed very strong anti-HIV activity. In addition, the residual material after methanol extractions still showed strong activity. Caffeic acid was identified in the ether extract of procedure I by HPLC and UV spectroscopy. Commercial caffeic acid showed good antiviral activity in the RT assay and high to moderate activity in the syncytia assay and the p17 and p24 antigen expression. Tannic acid and gallic acid, common to other teas, could not be identified in our extracts. When commercial products of these two acids were tested in our assay systems, they showed high to moderate activity against HIV-1. Hyssop officinalis extracts contain caffeic acid, unidentified tannins, and possibly a third class of unidentified higher molecular weight compounds that exhibit strong anti-HIV activity, and may be useful in the treatment of patients with AIDS. PMID- 1708227 TI - Cardiac tamponade due to ovarian carcinoma. AB - We describe a patient with ovarian carcinoma who presented with cardiac tamponade with subsequently development of cardiac arrest as the initial symptom. After successful resuscitation and pericardiocentesis our patient was given an intrapericardial infusion of bleomycin and there was no recurrence of pericardial effusion at 14 months follow-up. PMID- 1708228 TI - Neutralizing epitopes of feline calicivirus. AB - A new collection of eighteen neutralizing monoclonal antibodies (N-MoAbs), raised against feline calicivirus (FCV), was used to analyze neutralizing epitopes of the F4 strain of FCV, the prototype strain of FCV in Japan. By cross neutralization tests with the 20 FCV strains including Japanese. American, Swiss, and New Zealand isolates, the 18 N-MoAbs were categorized into six groups. One N MoAb (1 D 7) neutralized all the strains tested: eight N-MoAbs neutralized only FCV-F 4; while the others neutralized the FCV strains in various degrees. In order to confirm this grouping, eight N-MoAbs were used to select neutralization resistant variants of FCV F4. Although no variant against 1 D 7 was obtained, antigenic variants against other N-MoAbs were obtained. Neutralization tests using these variants revealed that there are six neutralizing epitopes on FCV F4 and that several epitopes are functionally related. One of these epitopes was the same epitope as one of the two epitopes identified by another panel of N-MoAbs we produced previously. Therefore, a total of seven neutralizing epitopes on FCV F4 were identified. Immunoblot analysis indicated that four of the seven epitopes existed on the 67 kDa capsid protein of the virus. PMID- 1708229 TI - Cytokeratins as molecular markers in the evaluation of the precise differentiation stage of human gingival epithelium reconstituted in vitro. AB - Cytokeratins are considered to be molecular markers for different types of epithelial differentiation. They were used to investigate the precise differentiation stage of gingival epithelium, reconstituted in vitro, following two different culture procedures. Human trypsin-dissociated gingival keratinocytes were seeded either on a feeder layer of irradiated mouse 3T3 fibroblasts or on a connective tissue equivalent (lattice) made up of human fibroblasts in a collagen gel. The cytokeratins were extracted and analysed by two-dimensional gel electrophoresis. Although both methods showed on histological sections that cultured gingival keratinocytes formed a multilayered non keratinizing epithelium, the cytokeratins patterns showed great differences. The gingival epithelium-like structure reconstituted on 3T3 feeder layer expressed some cytokeratins characteristic of the in situ gingival epithelium (K 5, 6, 14, 16, 17) and some which do not exist in the normal tissue (K 8, 18, 19, traces of K 13 and K 15) and are specific for embryonic, simple and tumour epithelia. However, the gingival epithelium reconstituted on connective tissue equivalent expressed all the cytokeratins present in the normal tissue (K 5, 6, 14, 16, 17), except those specific for terminal differentiation (K 1, 2, and 10/11). These findings suggest that the culture of gingival keratinocytes on connective tissue equivalents allows them to reproduce physiological stages of differentiation. PMID- 1708230 TI - Identification of T- and B-cell epitopes in synthetic peptides derived from a Streptococcus mutans protein and characterization of their antigenicity and immunogenicity. AB - Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 Mr Streptococcus mutans antigen was found in human subjects. Significant serum IgG antibodies were detected both to the native streptococcal antigen and to the SP17, containing essentially residues 1-15. A series of short peptides with deletions at the amino- and carboxy-termini were then tested to identify the B cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies but only the former consistently inhibited human antibodies, suggesting that residues 8-13 constitute a major B-cell epitope. The human CD4 subset of T-cells was then examined and this showed a significant uptake of [3H]-thymidine when stimulated with both the native streptococcal antigen and the SP17. The series of short peptides was then used to stimulate CD4 cells, in order to determine the T-cell epitope. The synthetic peptide with residues 6-15 was the shortest peptide that stimulated significant [3H]-thymidine uptake and this peptide was designated as a T-cell epitope. The immunogenicity and antigenicity of SP17 was also investigated in macaques. Immunization of monkeys with the free SP17 failed to elicit serum antibodies or T-cell responses. However, immunization with SP17 linked to tetanus toxoid as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the synthetic peptide but also to the native streptococcal antigen. As in the human studies a B-cell epitope was found in residues 8-13, whereas an overlapping T-cell epitope was located in residues 7 15.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708231 TI - Molecular approaches to leucotoxin as a virulence component in Actinobacillus actinomycetemcomitans. AB - A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56. PMID- 1708232 TI - Massive fetomaternal hemorrhage: case report. AB - Massive fetomaternal hemorrhage (FMH) is rare. Early diagnosis is important because FMH has a poor prognosis in the antepartum or early postpartum period. A case of massive FMH in which 400 ml blood was lost is presented here. Fetal anemia was suspected from the sinusoidal fetal heart rate pattern. The hemorrhage in the maternal circulation was diagnosed before delivery by using the Kleihauer Betke test. An emergency cesarean section was performed. The cord hemoglobin level was 3.7 g/dl. The baby's severe anemia was treated with a neonatal blood transfusion. In this case, the baby had a good clinical course. Early diagnosis and treatment of massive FMH are necessary for a good prognosis. PMID- 1708233 TI - Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin. AB - 1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations. PMID- 1708234 TI - Soluble fibrin preparations inhibit the reaction of plasmin with alpha 2 macroglobulin. Comparison with alpha 2-antiplasmin and leupeptin. AB - The kinetics of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP), alpha 2 macroglobulin (alpha 2M) and leupeptin were studied in the presence of fibrin monomer (Fn) and CNBr fragments of fibrinogen (Fg-CNBr). Active plasmin was detected in continuous and discontinuous assays using the chromogenic substrate D Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251). The two 'fibrin-like' preparations functioned as hyperbolic mixed-type inhibitors of S-2251 hydrolysis. The dissociation constants (KF) for the binding of plasmin to Fn and Fg-CNBr were 22 nM and 17 nM respectively. Fn and Fg-CNBr inhibited the reaction of plasmin with alpha 2AP: the extent of inhibition depended on the fibrin concentration. In the presence of 800 nM-Fn or 800 nM-Fg-CNBr, the experimental second-order rate constant (K"app.) was decreased from 2.4 x 10(7) M-1.s-1 to 1.2 x 10(6) and 5.3 x 10(5) M-1.s-1 respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by alpha 2M was even greater. The k"app. value was decreased from 4.0 x 10(5) M-1.s-1 to 8.0 x 10(2) and 1.3 x 10(3) M-1.s-1 in the presence of 800 nM Fn and -Fg-CNBr respectively. By contrast, the fibrin preparations caused only a small change in the rate of plasmin inhibition by leupeptin. The maximum change in k"app. was 3-fold. All plasmin inhibition curves were linear, suggesting that free and fibrin-bound forms of plasmin remained in equilibrium during the course of reaction with proteinase inhibitors. Fn and Fg-CNBr had no effect on the reaction of miniplasmin with S-2251, alpha 2AP or alpha 2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of alpha 2AP and alpha 2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to alpha 2AP. These experiments demonstrate that the reaction of plasmin with alpha 2M is inhibited by the non-covalent binding of plasmin to fibrin. We propose that plasmin bound to the surface of a clot is protected from inhibition by alpha 2M as well as by alpha 2AP. PMID- 1708235 TI - Effect of protonmotive force on the relative proton stoichiometries of the mitochondrial proton pumps. AB - The rate of phosphorylation of ADP by isolated mitochondria respiring on succinate was set by addition of ATP, ADP or ADP plus malonate. We measured the rates of phosphorylation and respiration and the protonmotive force under each of these conditions. We measured the oxygen consumption required to drive the proton leak at the protonmotive force reached under each condition and subtracted it from the respiration rate during phosphorylation to determine the oxygen consumption driving phosphorylation. By dividing the rate of phosphorylation by the rate of respiration driving phosphorylation we calculated the mechanistic P/O ratio (number of molecules of ADP phosphorylated per oxygen atom reduced). This ratio was the same at high, intermediate and low values of protonmotive force, indicating that the relative stoichiometries of the mitochondrial protonmotive force-producing and protonmotive-force-consuming pumps (i.e. H+/O:H+/ATP) are independent of the protonmotive force. This greatly weakens the case for a decrease in stoichiometry, or 'slip', in the mitochondrial proton pumps at high protonmotive force. PMID- 1708236 TI - Transforming growth factor beta 2 inhibits interleukin 1 beta- and tumour necrosis factor alpha-induction of nitric oxide synthase in rat renal mesangial cells. AB - Treatment of mesangial cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) has been shown to induce nitric oxide (NO) synthase with subsequent autocrine stimulation of soluble guanylate cyclase (Pfeilschifter and Schwarzenbach, 1990, FEBS Lett. 273, 185-187). Here we report that transforming growth factor beta 2 (TGF beta 2) dose-dependently inhibits IL 1 beta- and TNF alpha-stimulated cGMP formation in mesangial cells. Half-maximal inhibition is observed at concentrations of 0.4 and 0.06 ng/ml of TGF beta 2, respectively. Maximum inhibition of cGMP formation over a 24 h period requires the presence of TGF beta 2 during the first 4 h of induction. In addition, the inhibitory effect of TGF beta 2 on cytokine-induced cGMP formation is not affected by the potent cyclo-oxygenase inhibitor indomethacin, thus excluding prostaglandins as mediators. PMID- 1708238 TI - Vascular cell responses to a hybrid transforming growth factor-beta molecule. AB - Functional biological assays were performed using a hybrid molecule of Transforming Growth Factor-Beta (TGF-5 beta) where nine amino acids near the cleavage site of TGF-beta 1 were substituted with nine amino acids located in the identical position of TGF-beta 2. Bovine aortic endothelial and smooth muscle cells as well as rat epididymal fat pad microvascular endothelia were studied in three distinct bioassays examining proliferation, migration and angiogenesis. The data suggested TGF-5 beta elicited results that do not differ significantly from the TGF-beta 1 isoform, while TGF-beta 2 expressed unique characteristics. We have also shown that these amino acid substitutions to TGF-beta 1 do not, in fact, alter the biological functions of the growth factor. PMID- 1708237 TI - Elongation factor-2 in chick embryo is phosphorylated on tyrosine as well as serine and threonine. AB - An endogenous 95 kDa chick embryo cytosolic protein (p95) was phosphorylated in the presence of [gamma-32P]ATP and the kinase activity for p95 was mostly associated with particulate fraction. Phosphorylation of p95 was prominent in embryos of early developmental stage. Hydrolysis of p95 phosphoprotein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native p95 was also tyrosine-phosphorylated. p95 phosphoprotein was purified by DEAE-Sephacel chromatography and immunoprecipitation with anti-phosphotyrosine antibody and the amino acid sequence was determined. The N-terminal sequence, Val-Asn-Phe-Thr-Val Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp- Lys-Lys-Ala-Asn-Ile-Arg-Asn-Met-, was found to be identical to those of elongation factor-2 (EF-2) of both rat and hamster. Our results suggest the presence of other EF-2 kinase in chick embryo cell than the previously reported Ca2+/calmodulin-dependent protein kinase III. PMID- 1708240 TI - Analysis of integrin mRNA in human and rodent tumor cells. AB - Several human and rodent tumor cell lines were examined for the presence of integrin mRNAs by dot- and Northern-blot analysis. All tumor cells tested expressed mRNAs for alpha 5, alpha IIb, beta 1 and beta 3. The mRNA of beta 2 integrin was not detectable and that of alpha V integrin was found only in certain cells. Northern blotting was carried out in three selected tumor cell lines: Clone A, HEL and B16a. An apparent difference in the mRNA species coding for the alpha IIb beta 3 integrin, but not for alpha 5 and beta 1, was found. Our result suggests that alternative splicing of integrin genes may be one of the important mechanisms in regulating alpha IIb beta 3 expression and function in different tumor cells. PMID- 1708239 TI - Differential regulation of thrombin- or ATP-induced mobilization of intracellular Ca2+ by prostacyclin receptor in mouse mastocytoma cells. AB - Thrombin induced an increase in [Ca2+]i in mouse mastocytoma P-815 cells. This increase was markedly reduced by prior exposure to pertussis toxin (PT) but not by removal of extracellular Ca2+, suggesting that thrombin stimulates phospholipase C via a PT-sensitive GTP-binding protein. ATP also induced an increase in [Ca2+]i. This increase was insensitive to PT but completely suppressed on removal of extracellular Ca2+, suggesting that ATP stimulates Ca2+ influx in a PT-insensitive manner. Iloprost, a stable prostacyclin analogue, increased the cellular cAMP level and dose-dependently inhibited the thrombin induced increase in [Ca2+]i, whereas the ATP-induced increase in [Ca2+]i was markedly enhanced by iloprost. Cyclic AMP analogues, dibutyryl cAMP and 8-bromo cAMP, also inhibited the increase in [Ca2+]i induced by thrombin and promoted that by ATP, indicating that the inhibitory and stimulatory effects of iloprost are mediated by cAMP. These results suggest that the prostacyclin receptor differentially regulates two distinct Ca2+ mobilizing systems via cAMP in mastocytoma cells. PMID- 1708241 TI - Interleukin-1 beta induces the production of an L-arginine-derived relaxing factor from cultured smooth muscle cells from rat aorta. AB - The effect of interleukin-1 beta on the production of non-prostanoid vasoactive factors by cultured rat aortic smooth muscle cells was investigated. Under bioassay conditions, the perfusate from a column of confluent cells grown on beads and treated with interleukin-1 beta (1 ng/ml for 18 to 24 hr) abolished the contraction of a canine coronary ring without endothelium contracted by phenylephrine (1 microM), while the perfusate from control cells had no effect. The relaxing activity of the perfusate was observed when transit times were increased from 1 sec to 5 min. Nitro L-arginine (100 microM) reversed the relaxations and L-arginine stereoselectively restored the relaxations. Interleukin-1 beta (1 ng/ml) evoked a time-dependent accumulation of cyclic GMP but not cyclic AMP in cultured smooth muscle cells. The transfer of fresh or stored (-70 degrees C) conditioned culture medium from interleukin-1 beta-treated cells but not from control cells, to cultured smooth muscle cells stimulated the production of cyclic GMP. These observations demonstrate that interleukin-1 beta induces the production of transferable factor which relaxes vascular smooth muscle and stimulates the production of cyclic GMP. PMID- 1708242 TI - Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI beta of cAMP-dependent protein kinases. AB - Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RI beta protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others. PMID- 1708243 TI - Location of the C-terminus of titin at the Z-line region in the sarcomere. AB - Limited proteolysis of titin with trypsin yielded a number of polypeptides which were electrophoresed and transferred to a nitrocellulose membrane. Proteolytic removal of the C-terminal residues on the nitrocellulose-bound polypeptides was achieved by using carboxypeptidase Y. The species of the polypeptides left after the digestion was quantified by immunoblotting with two distinct monoclonal anti titin antibodies A2 and A12 of which the epitopes were located at 0.74 micron and 0.69 micron away from the center of an A-band, respectively. Two polypeptides (266 kd and 84 kd) reactive to both antibodies were identified in the control group. Fifteen minutes after the digestion, the immunoreactivities of A2 on 266 kd and 84 kd polypeptides were disappeared, while those of A12 on these polypeptides were not affected. The results indicate that the C-terminal end of titin is located near the Z-line region and the N-terminal end at the M-line region in the sarcomere. PMID- 1708244 TI - Calcium chelation induced glutathione efflux from tumor cells and prevention by ruthenium red or neomycin. AB - Cultured human lung carcinoma cells (A549) were incubated in a calcium-free medium containing calcium chelators (EGTA, 1-10 mM or BAPTA, 5 mM) for 1 hour at 37 degrees C. With limited toxicity, the presence of calcium chelators resulted in a decrease of cellular GSH and detachment of the cells from the tissue culture flask. The permeable EGTA tetraacetoxymethyl ester (0.5mM-5 mM) caused a decrease in the cellular GSH content without cell detachment. GSH was not oxidized to GSSG nor formed mixed disulfides with protein thiols. AT-125, a gamma-glutamyl transpeptidase inhibitor, prevented detachment, but not the efflux of cellular GSH. Pretreatment with two impermeable compounds (ruthenium red, 100 microM and neomycin, 0.5-10 mM) protected the cells from detachment and prevented the decrease in intracellular GSH. The presence of calcium in the medium during the EGTA and BAPTA treatments also protected the cells. Calcium associated with the cytoplasmic membrane phospholipids or proteins appears important to limit membrane permeability for GSH efflux and to maintain cell attachment. PMID- 1708245 TI - Induction of cytochrome P-450IA1 in fetal rat liver by a single dose of 3 methylcholanthrene. AB - Pregnant Sprague-Dawley rats were treated with a single ip dose of either olive oil or 40 mg/kg of 3-methylcholanthrene on gestation day 20 and sacrificed at various times after injection. Determination of aryl hydrocarbon hydroxylase activity 24 hr after injection revealed that treatment with 3-methylcholanthrene resulted in a 10.5-fold stimulation of enzymatic activity in liver 800 x g supernatants. Western blot analysis with monoclonal antibody 1-7-1 confirmed these results and demonstrated the presence of a 3-methylcholanthrene-inducible P 450 isozyme. Using Northern and slot blot techniques, the induction of steady state levels of CYPIA1 RNA was shown to occur as early as 4 hr following 3 methylcholanthrene injection. CYPIA1 RNA levels were induced 31.6-fold over values obtained from oil-treated tissues at this time. This appears to be the optimal time to study changes in the levels of CYPIA1 RNA gene expression in the fetus following transplacental exposure to polycyclic aromatic hydrocarbons. By 12 to 24 hr postinjection, the induction of CYPIA1 RNA levels declined to 3.5- to 8.5-fold above control values. These results demonstrate that the kinetics of induction of the CYPIA1 gene during the fetal period differed from that seen in adults. PMID- 1708246 TI - Inhibition of protein kinase C by calphostin C is light-dependent. AB - Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, inhibits protein kinase C by competing at the binding site for diacylglycerol and phorbol esters. Calphostin C is a polycyclic hydrocarbon with strong absorbance in the visible and ultraviolet ranges. In characterizing the activity of this compound, we unexpectedly found that the inhibition of [3H]phorbol dibutyrate binding was dependent on exposure to light. Ordinary fluorescent light was sufficient for full activation. The inhibition of protein kinase C activity in cell-free systems and intact cells also required light. Light-dependent cytotoxicity was seen at concentrations about 5-fold higher than those inhibiting protein kinase C. PMID- 1708247 TI - Expression cDNA cloning of fibroblast growth factor (FGF) receptor in mouse breast cancer cells: a variant form in FGF-responsive transformed cells. AB - Expression cDNA library of fibroblast growth factor (FGF)-responsive mouse breast cancer cells (SC-3) was prepared and screened using chick FGF receptor (FGF-R) cDNA as a probe. Two positive clones were isolated. Sequence analysis revealed that two clones have an identical full open reading frame. Compared with sequence data on mouse brain FGF-R, SC-3 cells were found to contain FGF-R with 12 amino acids insertion near N-terminal region. This insertion was mainly composed of hydrophobic amino acids. Additionally, the deletion of two amino acids in extracellular domain and the substitution of one amino acid in C-terminal region were identified in SC-3 cell FGF-R. Transfection of this clone into CHO cells resulted in a significant increase in basic FGF (bFGF) binding. PMID- 1708248 TI - Avian thymic hormone and chicken (muscle) parvalbumin are distinct proteins: isolation of a muscle parvalbumin cDNA fragment by PCR. AB - Access to the nucleotide sequence of parvalbumin from chicken muscle was gained via the polymerase chain reaction. In the absence of specific amino acid sequence data, the PCR primers were based on consensus data for the two parvalbumin Ca2(+) binding sites. The 137 bp fragment obtained by amplification clearly codes for a parvalbumin, as judged by the presence of 10 invariant codons within the sequence flanked by the primers. When used to probe poly(A)+ RNA from chicken muscle, the fragment recognizes an 800 nucleotide transcript. The translated nucleotide sequence of the muscle protein is unmistakably distinct from that of the thymus specific parvalbumin known as avian thymic hormone. Of the 31 amplified residues, the two proteins differ at 14. The presence of a distinct parvalbumin in chicken thymus is consistent with the potent effector role proposed for the protein. PMID- 1708249 TI - Hydrogen peroxide: an endogenous smooth muscle cell hyperpolarizing factor. AB - Hydrogen peroxide can be released by different cells such as the nerves, the endothelial or phagocytotic white blood cells which can all interact with vascular smooth muscles. We show that hydrogen peroxide hyperpolarizes and relaxes pig coronary artery smooth muscle cells. The possibility that the endothelium derived hyperpolarizing factor released by the endothelium in response to bradykinin and substance P being hydrogen peroxide was tested using catalase, an enzyme which hydrolyses hydrogen peroxide. We find that this particular endothelial hyperpolarizing factor and hydrogen peroxide are two distinct molecules. PMID- 1708250 TI - Purification of a Cl-(-)channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system. AB - A Cl- channel protein of sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in CHAPS and fractioned by anion exchange, gel filtration, and affinity chromatography with concanavalin A. All fractions in each step were reconstituted into vesicles with asolectin by dialysis and their channel activities were assayed after the vesicles had been fused into a planar lipid bilayer. A 100-kDa protein, different from Ca2(+)-ATPase, was found to form anion channels. PMID- 1708251 TI - Demonstration of cross-linked cytokeratin polypeptides in transplantable rat hepatoma cells. AB - Covalently cross-linked multimers of cytokeratins were shown to be present in transplantable Morris hepatoma 7777 cells. These high molecular weight antigens were not detectable in normal rat liver cells. However, identical high molecular weight antigens were also demonstrated in rat liver cells when the cells were homogenized in solutions containing Ca2+. The cross-linking reaction was suggested to be mediated by the action of tissue transglutaminases. PMID- 1708252 TI - Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth. AB - Hepatocyte growth factor (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates growth and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0 fold occurred with 10 ng/ml HGF. This stimulatory effect was additive with both acidic and basic fibroblast growth factors, while it was inhibited by transforming growth factor-beta 1. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of 22 pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes. PMID- 1708253 TI - Increases in cytosolic Ca++ down regulate thyrotropin receptor gene expression by a mechanism different from the cAMP signal. AB - Thyrotropin (TSH) receptor mRNA levels in rat FRTL-5 thyroid cells are decreased by treatment with the calcium ionophores, A23187 or ionomycin, as well as with TSH, cholera toxin, forskolin, and 8-bromo-cAMP. Down regulation is, in each case, associated with a decrease in [125I]TSH binding and a decreased ability of TSH to increase cAMP levels. The ionophore does not alter cAMP levels and ethylene glycol-bis-(beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) in the medium prevents down regulation of TSH receptor mRNA levels by the ionophore, but not by TSH; the EGTA action is reversed by the simultaneous addition of Ca++. Whereas down regulation by TSH and its cAMP signal requires the presence of insulin and/or serum in the medium; down regulation by a calcium ionophore is still evident in their absence. Down regulation of TSH receptor mRNA levels and receptor desensitization by TSH/cAMP or an ionophore is lost in cells transfected with a full length TSH receptor cDNA devoid of regulatory elements, but able to reconstitute TSH receptor signal generation. PMID- 1708254 TI - Effects of the immunosuppressant FK-506 and its analog FK-520 on hepatic and renal cytochrome P450 mixed-function oxidase. AB - The novel immunosuppressant FK-506 and its analog FK-520 were found to inhibit the hepatic microsomal mixed-function oxidase system in male Sprague-Dawley rats. At 5 and 10 mg/kg/day, s.c., for 6 days they caused 30-80% decreases in cytochrome P450 levels, NADPH-cytochrome P450 reductase, and benzphetamine N demethylase activities. The metabolism of FK-506 itself was inhibited by 50%. FK 506 and FK-520 had a minimal effect on the renal cytochrome P450 levels unlike cyclosporin A which produced a 67% increase after six daily 25 mg/kg doses. A single dose of FK-506 (25 mg/kg, s.c.) had a minimal effect on the hepatic or renal metabolizing enzyme system. In vitro, addition of FK-506 and FK-520 to human and control rat liver microsomes resulted in a concentration-dependent inhibition of benzphetamine N-demethylation (10-20% at 50 microM, 60-75% at 250 microM). We suggest that in view of its potential to inhibit hepatic cytochrome P450-dependent mixed-function oxidase, resulting in the inhibition of its own metabolism, FK-506 should be administered with caution to transplant patients. PMID- 1708255 TI - Metabolism of substance P and bradykinin by human neutrophils. AB - The catabolism of substance P and bradykinin, two peptides involved in inflammation, by human neutrophils was investigated. Substance P was cleaved by unstimulated neutrophils, but the rate of hydrolysis increased greatly (about 4 fold) when the cells were lysed by freezing and thawing or stimulated to release with fMet-Leu-Phe and cytochalasin B. The enzyme responsible for cleaving substance P was cathepsin G, hydrolyzing the Phe7-Phe8 bond. Neutral endopeptidase 24.11 (enkephalinase) became the main inactivating enzyme only when neutrophil cytoplasts (containing plasma membrane but no subcellular particles) or washed plasma membrane enriched high speed sediments were tested. Subcellular fractionation showed the highest substance P degrading activity to be in the granules. Purified cathepsin G readily cleaved substance P with a Km of 1.13 MK, a kcat of 6.35 sec-1 and a kcat/Km of 5639 M-1 sec-1, similar to kinetic constants previously reported for the best peptide substrates of cathepsin G. Despite the high Km, purified cathepsin G did hydrolyze SP at a much lower substrate concentration (down to 1 nM) as determined by radioimmunoassay. Bradykinin was also hydrolyzed by intact neutrophils but, in contrast, was not inactivated by cathepsin G, but by neutral endopeptidase at the Pro7-Phe8 bond. The inactivation of bradykinin by intact neutrophils was decreased by phorbol 12 myristate 13-acetate, probably due to down-regulation by endocytosis of the neutral endopeptidase on the plasma membrane. Thus, both bradykinin and substance P are inactivated by human neutrophils, although by different enzymes. In spite of the less favorable kinetics in vitro than with neutral endopeptidase, cathepsin G is the main inactivator of substance P in neutrophils. This may be due to the estimated 300 to 3600-fold higher concentration of cathepsin G in neutrophils than that of the neutral endopeptidase. PMID- 1708256 TI - Changes in protein, RNA and DNA and rates of protein synthesis in muscle containing tissues of the mature rat in response to ethanol feeding: a comparative study of heart, small intestine and gastrocnemius muscle. AB - 1. The relative sensitivity of heart, small intestine and skeletal muscle to chronic ethanol feeding was investigated in mature Wistar rats fed ethanol as 36% of total energy intake; controls were fed the same diet in which ethanol was substituted by isoenergetic glucose. 2. Chronic ethanol feeding had no apparent effect on the protein, RNA and DNA contents of heart homogenates (atria and ventricles). The ratios of RNA/protein (synthetic capacity), RNA/DNA (synthetic material per nucleus) and protein/DNA (DNA-unit or apparent cell size) were also unaltered in the hearts of alcohol-fed rats. Fractional rates of cardiac protein synthesis (ks), and synthesis relative to RNA (kRNA) and DNA (kDNA) and absolute rates of protein synthesis (Vs) were unaffected by ethanol feeding. The total content of cardiac soluble proteins was unaltered by chronic ethanol feeding, but there were small and statistically significant decreases in the contents of the myofibrillar and stromal protein fractions. There were no differences in ks in any of the cardiac subcellular protein fractions. 3. In the small intestine, ethanol feeding had no statistically significant effect on either protein or RNA contents, but there was an apparent increase in RNA when expressed relative to either protein or DNA, though the DNA-unit was unaltered. There were also substantial decreases in ks, kRNA, kDNA and Vs of approximately 15-35%. 4. In the gastrocnemius, RNA contents were significantly reduced by ethanol feeding but protein and DNA contents were unaffected. Indices of the synthetic capacity and synthetic material per nucleus were also reduced, but the DNA-unit was unaltered. These observations were accompanied by approx. 15-30% reductions in ks, kRNA, kDNA and Vs in response to ethanol feeding. 5. It is concluded that various aspects of protein metabolism in the heart, small intestine and skeletal muscle are adversely affected by chronic ethanol toxicity. The characteristics and magnitude of the responses in each tissue differ. Effects in the heart may be subtle, though haemodynamic indices may ensue. The ethanol-induced alterations in the small intestine and skeletal muscle may be responsible for gastrointestinal disturbances in motility and skeletal muscle weakness, respectively. PMID- 1708257 TI - Antibodies against acetaldehyde-modified epitopes: presence in alcoholic, non alcoholic liver disease and control subjects. AB - Several studies have recently shown the presence of antibodies against acetaldehyde-modifications in the sera of alcoholic patients. To assess the specificity of this immune response, plasma immunoreactivity with proteins modified in vitro by acetaldehyde was measured using an enzyme-linked immunosorbent assay in 97 alcoholic patients with varying degrees of alcoholic liver disease, in 35 patients with non-alcoholic liver diseases and in 33 control subjects who were social drinkers. All three groups showed some response against acetaldehyde-modified epitopes. The highest plasma reactivities were found in alcoholics, especially those with steatosis and alcoholic hepatitis. Plasma from patients with non-alcoholic liver disease and control subjects also reacted with the acetaldehyde conjugates, but to a lesser extent than plasma from alcoholics. The reliability of these antibodies as markers of either alcohol abuse or alcoholic liver disease therefore appears to be low. However, further studies using more precisely modified proteins or elucidation of the classes of immunoglobulin involved in the immune response against the modified proteins may give clearer differences between the three groups. PMID- 1708258 TI - Functional and structural adaptation of the parotid gland to medium-term chronic ethanol exposure in the rat. AB - Male rats were maintained on a regimen of twice daily intragastric administration of ethanol or a calorifically equivalent sucrose solution for thirty days. A second control group received no intragastric solution and all groups received chow and water ad libitum. Parotid saliva elicited by pilocarpine was collected by unilateral duct cannulation. The parotid flow rate over the initial post stimulatory five minute period was raised by 44% in ethanol-dosed rats and the salivary sodium concentration was also raised, in line with higher flow rate. There were no histopathological changes related to ethanol or sucrose dosing, but stereological analysis showed a 64% increase in the proportional volume of intralobular vascular tissue in ethanol-dosed rats. These quantified histological findings suggest that parotid intralobular haemodynamics may be altered after chronic ethanol-dosing and this may contribute to the hypersecretory response exhibited by the ethanol-dosed rats. PMID- 1708259 TI - [Models and manikins in dental education]. AB - Different available types of dental models and manikin used for preclinical odontological education are analyzed. On the basis of their composition are classified in biological, artificial, and mixed models. According to their usage, those ones are classified in demonstration, exploration, and working models. Each of them are studied, remarkable aspects in odontological education are emphasized. PMID- 1708260 TI - Monoclonal anti-La antibody derived from a mouse with experimental SLE is similar to human anti-La antibodies. AB - The La antigen is a highly conserved protein, originally defined by sera of patients with Sjogren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity. PMID- 1708261 TI - A repeated proline-rich sequence in Sm B/B' and N is a dominant epitope recognized by human and murine autoantibodies. AB - Reactivity of a murine monoclonal anti-Sm B antibody was localized to a cDNA clone T3/2 encompassing codons 177-221 of Sm N and to a series of 10-residue synthetic peptides containing a proline-rich motif. Human sera containing anti-B antibodies also bound to this proline-rich motif in the form of a heptamer, PPGMRPP, and could distinguish the N/B sequence from similar sequences present in the RNP A and C polypeptides in a number of cases. Monoclonal antibodies binding to either this peptide or to a determinant present on both B and D polypeptides inhibited the majority of anti-B binding in 50% of anti-B sera. The region of N/B containing PPGMRPP also bears a second epitope, sterically blocked by monoclonal antibody KSm 5. This hotspot binds 40-60% of anti-B activity in 30% of anti-B sera. These data suggest that the anti-Sm B epitope recognition repertoire is restricted in SLE patients. PMID- 1708262 TI - Expression of intercellular adhesion molecule-1 and lymphocyte function associated antigen-3 on human thyroid epithelial cells in Graves' and Hashimoto's diseases. AB - Human endocrine thyroid epithelial cells (TEC) from autoimmune thyroiditis which express HLA Class II antigens have been shown to present autoantigens to T cells for a TEC-specific immune response. Since the initiation of a specific immune response also involves antigen-receptor independent interactions between accessory molecules, such as lymphocyte function-associated antigen-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-3 (LFA-3) with CD2, it was of interest to determine whether TEC can express the adhesion molecules (ICAM-1 and LFA-3) which augment the efficiency of antigen presentation. Cultured TEC were studied for their expression of ICAM-1 and LFA-3 by immunofluorescence. Those derived from Graves' disease expressed these molecules after stimulation with recombinant human interferon-gamma (IFN gamma) or with recombinant human tumour necrosis factor alpha (TNF alpha). However, using the same stimuli, TEC from non-toxic goitre were induced to express ICAM-1, but not LFA-3. To establish whether ICAM-1 and LFA-3 on TEC were expressed in vivo during the disease process, antibodies against these molecules were incubated with frozen sections of autoimmune thyroiditis, including Graves' and Hashimoto's diseases, and non-toxic goitre. Both ICAM-1 and LFA-3 were highly expressed in the autoimmune diseases, but not in non-toxic goitre. These findings establish that TEC are able to express adhesion molecules and suggest the possible involvement of these adhesion molecules in the TEC-specific immune response in autoimmune thyroiditis. PMID- 1708263 TI - T-cell epitopes on the 70-kDa protein of the (U1)RNP complex in autoimmune rheumatologic disorders. AB - High-titre IgG antibodies against the immunodominant 70-kDa protein of the (U1)ribonucleoprotein (RNP) complex are present in virtually 100% of patients with mixed connective tissue disease (MCTD), and less commonly in a variety of other autoimmune rheumatic diseases. As T-cell 'help' is assumed to be required for this potentially pathogenic form of immune response, investigations to define T-cell epitopes on the 70-kDa protein were undertaken. In prior studies we expressed the 70-kDa protein and a number of its fragments, spanning most of the molecule, as recombinant fusion proteins using the pGEX expression-vector system. These fusion proteins were used as antigens in the epitope mapping studies reported here. PBMC were isolated from patients with (U1)RNP-positive rheumatic diseases and from both normal controls and rheumatologic patients with other autoantibody reactivities, including those to Ro, La and dsDNA. Reactivity to the purified 70-kDa protein was assayed by thymidine incorporation and was evident only in anti-(U1)RNP positive patients but was not restricted to MCTD patients, being present also in patients with SLE and rheumatoid arthritis. The stimulation indices (SIs) observed were in the two- to five-fold range. Using the 70-kDa protein fragments, a T-cell stimulatory epitope was localized to the C-terminal 63 amino acids of the autoantigen. A T-cell line, derived from PBMC of a (U1)RNP positive patient with MCTD, also reacted predominantly with this C-terminal fragment but with an SI of approximately 15-fold. Thus, we have demonstrated the presence and specificity of autoreactive T lymphocytes to a defined peptide epitope in systemic rheumatic disease. PMID- 1708264 TI - Isolation of HIV-1 from seropositive people living in Cotonou, Benin. AB - Benin is located in West Africa and is situated between HIV-2 and HIV-1-endemic zones. The first cases of HIV-1 infection in Benin were reported in 1987. Since then, AIDS cases have been diagnosed there and the number of known HIV seropositive people has rapidly increased. Blood samples were collected from 14 seropositive and 11 seronegative patients living in the main city, Cotonou, and their peripheral blood mononuclear cells were cultured. In seven of the seropositive cases, a retrovirus was detected by measurement of Mg2(+)-dependent reverse transcriptase activity and electron microscopy. HIV-1 antigen assay and genomic analysis indicated that the isolated viruses belong to the first serotype. In each positive case, an HIV-1 DNA probe hybridized to the RNA extracted from the virus and six isolates were found positive by the polymerase chain reaction using HIV-1-specific primers. PMID- 1708265 TI - Efficacy of recombinant human granulocyte colony-stimulating factor on neutropenia in patients with AIDS. AB - The efficacy of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutropenia was evaluated in 14 patients with AIDS and AIDS-related complex (ARC). In all patients, including 11 neutropenic patients, 100 or 200 micrograms/m2 of rhG-CSF significantly increased the neutrophil counts. The response was greater in patients with higher neutrophil counts before the treatment, and was also dose-dependent. Although the effect seemed to be less potent, the agent also increased the neutrophil counts even when zidovudine (azidothymidine, AZT) and other myelosuppressive antiviral agents were administered simultaneously. These observations indicate that rhG-CSF may be beneficial in preventing and treating some secondary infections, and will make it easier to continue therapy with antiviral agents in patients with AIDS or ARC. PMID- 1708266 TI - Cellular localization of PNA binding in colorectal adenomas: comparison with differentiation, nuclear:cell height ratio and effect of desialylation. AB - The lectin Arachis Hypogaea (Peanut Agglutinin, PNA) was used to study the cellular localization of the Thomsen-Friedenreich (T) disaccharide Gal-beta (1-3) GalNAc alpha 1-R in 22 formalin-fixed paraplast-embedded colorectal adenomas of varying cellular dysplasia. An indirect immunoperoxidase method was used prior to and after neuraminidase treatment. Detailed information on the cellular localization of PNA binding was obtained. In addition, morphometric measurements of the nuclear: cell height ratios were performed on staining-filtered micrographs of crypts from all adenomas. We found 1) a statistically significant increase in the nuclear:cell height ratio with increasing grade of dedifferentiation (p less than 0.003), 2) a statistically significant smaller nuclear:cell height ratio in crypts that were PNA-positive in the Golgi region when these were compared to crypts that were PNA-positive on luminal cell membranes, 3) a decreasing number of crypts expressing PNA binding sites in the Golgi region with increasing dedifferentiation, leading to complete absence of PNA binding sites in Grade IV adenomas, 4) neuraminidase pretreatment increased the number of crypts expressing PNA binding sites in cytoplasm and on luminal membranes, whereas no changes were detected in crypts expressing PNA binding sites in the Golgi region. Our results confirm the general concept of accumulation of precursors of carbohydrate antigens in dedifferentiated cells. On the basis of the results presented, we conclude that the nuclear:cell height ratio shows a good correlation with the cellular localization of PNA binding, cellular differentiation and classic pathologic grading. PMID- 1708267 TI - Immunohistologic separation of B-cell-positive granulomas from B-cell-negative granulomas in paraffin-embedded tissues with special reference to tumor-related sarcoid reactions. AB - Frozen and formalin-fixed paraffin-embedded tissue sections were studied concurrently in 17 cases of granulomatous lesions of different etiologies using antibodies recognizing either fixation-sensitive or fixation-resistant antigens. In fixed tissues, the antibodies 4KB5 and L26 for B cells and UCHL1 and MT1 for T cells gave results similar to those obtained in frozen tissues with anti-leu 12/leu-14 for B cells and T-3 for T cells. Paraffin-embedded sections from 35 additional cases of granulomatous lesions were studied retrospectively using the same markers. The combined results from all 52 cases show that granulomas can be divided into two main "families" according to the presence or absence of B cells within the granulomas: one is a B-cell-negative family of lesions to which sarcoidosis and mycobacterial infection belong; the other is a B-cell-positive family of lesions to which toxoplasmosis, granulomatous lesions of unknown significance and tumor-related sarcoid reactions belong. PMID- 1708268 TI - Pulmonary atresia with intact ventricular septum: is neonatal repair advisable? AB - The optimal management of pulmonary atresia with an intact ventricular septum in the neonate remains controversial. The introduction of balloon septostomy and prostaglandin has significantly reduced early mortality but early surgical intervention is necessary to obtain a more adequate pulmonary blood flow. Fourteen neonates with pulmonary atresia and an intact ventricular septum were admitted to the Wessex Cardiothoracic Unit, Southampton from 1979 to 1986. Thirteen patients underwent cardiac catheterization. Cardiac catheterization data and right ventricular angiograms were reviewed retrospectively. Four patients with tripartite ventricles underwent total repair. The others received various palliative operations (valvotomy + modified Blalock-Taussig shunt or modified Blalock-Taussig shunt alone). Retrospective analysis of the angiograms indicated that right ventricular morphology alone is not a satisfactory criterion for surgical management. We have been able to demonstrate that there is a good correlation between the diameter of the tricuspid valve and the diameter of the infundibulum and that successful neonatal repair is possible when the tricuspid valve diameter is above 80% of the normal value for weight and when the tricuspid valve diameter to infundibular diameter ratio (TV/Inf ratio) is 2.2 or less. In patients with a tripartite ventricle but inadequate TV diameter and TV/Inf ratio, a closed pulmonary valvotomy with a modified Blalock-Taussig shunt remains the treatment of choice. PMID- 1708269 TI - Reversible low-molecular-weight proteinuria in patients with distal renal tubular acidosis. AB - Four patients with untreated renal tubular acidosis had a urinary excretion of low-molecular-weight (LMW) proteins which was restored to normal by alkali therapy. Hypokalaemic proximal tubular damage in untreated patients with distal renal tubular acidosis is believed to be the cause of LMW proteinuria. An examination of urinary excretion of LMW proteins is useful for determining hypokalaemic proximal tubular dysfunction, as well as the efficiency of alkali therapy. PMID- 1708270 TI - Human monoclonal anti-basement membrane zone antibodies derived from virally transformed lymphocytes of a patient with bullous pemphigoid recognize epitopes associated with hemidesmosomes. AB - The purpose of this study was to determine the ultrastructural localization of the epitopes recognized by two human monoclonal antibodies designated 5E and 10D, that were derived from virally transformed lymphocytes from a patient with bullous pemphigoid (BP). Previous immunoblotting showed that 5E but not 10D reacted with a 230-kDa protein in extracts of normal human skin. In the present study, indirect immunofluorescence showed that both 5E and 10D bound to the epidermal side of 1 M NaCl-separated normal human skin. Immunoperoxidase electron microscopy showed that 5E and 10D formed discontinuous focal deposits along the basal region of the epidermal basal cells in normal human skin in a similar localization to that seen with whole BP sera. Post-embedding immunogold electron microscopy demonstrated binding of both monoclonal antibodies to the intracellular components of hemidesmosomes. As far as we are aware, 5E and 10D are the first monoclonal antibodies to be described that recognize epitopes of BP antigen associated with hemidesmosomes and should serve as useful probes for future studies. PMID- 1708271 TI - Tissue kallikrein and kininogen in human sweat glands and psoriatic skin. AB - The cellular localization of immunoreactive tissue kallikrein and kininogen was studied in normal and psoriatic human skin. Immunoreactivity to both enzyme and substrate was observed in secretory granules of the dark cells in the secretory fundus (acinus) of the sweat glands. Double immunostaining revealed a segmental distribution of the two antigens. Each acinar section contained either tissue kallikrein or kininogen. However, there appeared to be a junctional zone in which both were present, but in separate dark cells. Immunoreactivity for both antigens was also observed in close apposition to the luminal microvilli of the duct cells. No specific immunostaining was seen in sebaceous glands, hair follicles, keratinocytes and other cells of the secretory unit such as myoepithelial or clear cells. In psoriatic skin there were in addition many neutrophils immunoreactive for tissue kallikrein in the epidermis and psoriatic scales. Mitogenic action of kinins may account to some extent for the characteristic accelerated turnover of epidermal cells in psoriasis and locally applied kinin antagonists may prove of value in the treatment of this disease. PMID- 1708272 TI - Innervation zone of orbicularis oculi muscle and implications for botulinum A toxin therapy. AB - Motor points (areas of maximal sensitivity to electrical stimulation) were found in constant locations over orbicularis oculi when measured in both eyes of six normal subjects. All subjects had a motor point at the lateral terminus of the upper lid crease and the medial extent of the lower lid crease. A study of the innervation zone [distribution of neuromuscular junctions (NMJ)] was conducted on strips of pretarsal and preseptal portions of the upper eyelid orbicularis that had been removed routinely during involutional ptosis surgery. There was no significant difference in NMJ concentration between the medial and lateral sections, as determined by cholinesterase staining. Therefore, we concluded that the innervation zone is diffuse for the orbicularis muscle within this portion of the upper eyelid. Single-point injections of botulinum toxin were then compared to the conventional multiple injection sites on separate eyes in 10 patients with benign essential blepharospasm. Eight of the 10 patients reported greater relief on the side given injections into multiple points; the other two patients experienced no difference between the two methods. Both histologic data and clinical observation of response to botulinum toxin injection suggest the innervation zone for the upper orbicularis is diffuse. Thus, we conclude that multiple injections are superior to the injection of a single motor point. PMID- 1708273 TI - Stereoscopic external photography and the free-stereo viewing technique. AB - Stereoscopic external photography is a simple but helpful aid in teaching oculoplastic surgical anatomy. Binocular slides or photographs may be taken freehand and may be viewed stereoscopically without special equipment (the free stereo viewing technique). PMID- 1708274 TI - Are primary alloresponses truly primary? AB - Proliferative T cell responses against major histocompatibility complex (MHC) incompatible stimulator cells in the mixed lymphocyte reaction are conventionally regarded as primary. However, it is generally accepted that the recognition of allogeneic MHC products results from a cross-reaction by self-MHC-restricted cells. These two assumptions were tested by examining the contribution of previously primed and naive T cells to 'primary' alloresponses. Peripheral blood T cells were separated into LFA-3+, memory, and LFA-3-, naive, populations by fluorescence-activated cell sorting. In contrast, to recall antigen responses to Candida albicans which were almost entirely confined to the LFA-3+, memory, population, the proliferative response to MHC incompatible stimulator cells, including HLA-DR-expressing mouse L cell transfectants, was equally distributed between the two T cell subsets in 5 day assays. Furthermore, limiting dilution analysis showed that the frequency of alloreactive T cells did not differ significantly between the two populations. The kinetics of proliferation in the two populations differed but were consistent with their naive and memory phenotype, in that after 3 days of culture the LFA-3+ cells proliferated more strongly to MHC alloantigens. These results show that a substantial proportion of 'primary' alloresponses are contributed by previously primed cells. In addition, the evidence for the cross-reactive hypothesis is supported and extended from the clonal to the population level. PMID- 1708275 TI - Cytotoxic T lymphocyte recognition of the H-2-erbB hybrid gene product lacking the complete H-2 domain structure. AB - The chimeric mouse MHC class I gene derived from a recombinant H-2Kb gene, in which the coding region for a large part of alpha 1 and alpha 2 extracellular domains was replaced with a partial avian erythroblastosis virus erbB gene segment encoding the kinase domain, was successfully introduced into a mouse mastocytoma line P1.HTR (H-2d) and transcribed to mRNA. The transfectant cells expressed the chimeric gene product, which was reactive to a phosphotyrosine specific antibody. When the chimeric gene transfectant was inoculated into CDF1(H 2d) or BDF1(H-2d/b) mice, it grew at an early time but regressed thereafter. Transfectant-specific as well as parental P1.HTR-specific antibody activities were demonstrated in the sera of these mice. Transfectant-specific cytotoxic T lymphocytes (CTL) were generated in the antigen-sensitized culture of spleen cells from the transfectant-immune mice. The CTL-mediated lysis of target chimeric gene transfectant cells was poorly inhibited by anti-H-2d antiserum, which blocked the lysis of parental P1.HTR cells by anti-tumor CTL developed in parallel. The former was, however, inhibited by either anti-transfectant antiserum or anti-phosphotyrosine antibody, which was ineffective for blocking the latter. Target cell lysis by either anti-transfectant or anti-tumor CTL was blocked by anti-CD8 monoclonal antibody but not by anti-CD4 antibody. It was suggested from these results that the H-2K-erbB hybrid gene product, which lacks complete three-domain class I structure, was recognized by CTL in a manner that was endogenous H-2 class I-independent but CD8-dependent. PMID- 1708276 TI - Bypass by an alternate 'carrier' of acquired unresponsiveness to hCG upon repeated immunization with tetanus-conjugated vaccine. AB - We report here the use of an alternative carrier diphtheria toxoid (DT) in human subjects to overcome antigen-specific unresponsiveness upon immunization with a hapten/ligand-carrier conjugate. In the phase I clinical trial of a birth control vaccine using gonadotrophin subunits linked to tetanus toxoid, some of the subjects failed to evoke a booster antibody response to human chorionic gonadotrophin (hCG). Presentation of the ligand on DT instead in subsequent immunizations restored anti-hCG response. PMID- 1708277 TI - Modification of the T cell responsiveness to synthetic peptides by substituting amino acids on agretopes. AB - T cell receptors, major histocompatibility complex molecules, and antigens constitute tri-molecular complexes which induce T cell activation. T cells in I Ab mice generate proliferative responses to a synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58) and its analogs with substitution at position 50 (50A, 50V, 50L, 50N, 50Q, 50K, and 50M). However, none of these peptides stimulate T cells in I-Ak mice. We substituted two residues at positions 46 and 54 of p43-58(50D), 50V, 50L, 50E, and 50K with two amino acids on agretopes of the I-Ak binding HEL52-61 peptide and immunized I-Ak mice with these newly synthesized peptides: 46D50D54R, 46D50V54R, 46D50L54R, 46D50E54R, and 46D50K54R. Apart from 46D50D54R, these peptides elicited T cell responses in I-Ak mice in an immunogen-specific manner, but did not stimulate those in I-Ab mice. Further, 46D50V54R inhibited competitively the responses of I-Ak restricted T cell hybridomas specific for 46D50E54R. These results demonstrate that the residues at positions 46 and 54 on the peptides act as an agretope and the residue at position 50 acts as an epitope in I-Ak mice, as in I-Ab mice, and provide the possibility of opening up a new method to prepare peptide antigens which induce T cell responses in each murine strain by introducing appropriate amino acids on agretopes. PMID- 1708278 TI - Blood leukocytes bind platelet glycoprotein (IIb-IIIa)' but do not express the vitronectin receptor. AB - Within the integrin family of Arg-Gly-Asp(RGD)-binding adhesion receptors, the subfamily defined by the beta chain known as beta-3 or glycoprotein (GP)IIIa is known to contain two individual receptors. These are the GPIIb-IIIa complex of platelets, where the alpha chain of the heterodimer is GPIIb, and the vitronectin receptor (VnR) containing the alpha V subunit. The presence of either GPIIb-IIIa and/or the VnR on blood leukocytes has been controversial. We have investigated this problem by performing immunoprecipitation and immunoblotting studies with rabbit and monoclonal antibodies (mAb) to each of the subunits of GPIIb-IIIa and the VnR. On cultured cells of different origin, it was established that almost all expressed the VnR but none had GPIIb-IIIa, and the only polypeptide associated with beta 3 was alpha V. Platelets expressed predominantly GPIIb-IIIa, and weakly, the VnR. Monocytes and neutrophils freshly isolated from blood did not express the VnR but bore on their surface a modified form of GPIIb-IIIa. This molecule appeared identical to GPIIb-IIIa but an epitope on GPIIb was masked on the intact cell and was only revealed after immunoblotting. We have termed this modified form of GPIIb-IIIa, GP(IIb-IIIa)'. With differentiation in culture, monocytes rapidly lost surface GP(IIb-IIIa)' and concurrently began to express the VnR. Evidence is presented that GP(IIb-IIIa)' is derived from particles released by activated platelets and is bound firmly to the leukocyte membrane. Its primary function does not seem to be to mediate attachment to matrix proteins; thus, although U937 cells bearing platelet-derived GP(IIb-IIIa)' bound fibrinogen in an RGD-dependent manner, isolated blood monocytes did not. It is suggested that this transfer of membrane proteins from platelets to monocytes and neutrophils may regulate the expression of the leukocyte VnR and also serve as a means of facilitating leukocyte procoagulant activity. PMID- 1708279 TI - Lysis of interferon-gamma activated Schwann cell by cross-reactive CD8+ alpha/beta T cells with specificity for the mycobacterial 65 kd heat shock protein. AB - Heat shock protein (hsp) 65 is a major T cell antigen of Mycobacterium leprae. The hsp 65 of M. leprae is nearly identical in M. bovis/M. tuberculosis (greater than 95% protein sequence homology) and surprisingly similar in man (65% protein sequence homology). Recently, we had provided evidence in a murine model that CD8+ T cells recognize and lyse Schwann cells presenting M. leprae antigen in the context of major histocompatibility (MHC) class I gene products. Because murine Schwann cells are class I negative, antigen presentation requires prior stimulation with interferon-gamma (IFN-gamma). CD8+ T cells were activated against tryptic fragments of mycobacterial hsp 65. These T cells recognized epitopes of hsp 65 which had been generated through the cytoplasmic class I processing pathway. They were also capable of lysing Schwann cells which had been activated by IFN-gamma and not primed with nominal hsp 65 peptides. In contrast, T cells activated against tryptic ova peptides only lysed Schwann cells which had been both stimulated with IFN-gamma and primed with ova peptides. Evidence is presented that class I (H-2D) restricted, CD8+ alpha/beta T lymphocytes with specificity for the mycobacterial hsp 65 recognize IFN-gamma-stimulated Schwann cells probably because they are specific for a(n) epitope(s) shared by the bacterial hsp and a host cognate. Activation of autoreactive T cells with specificity to shared epitopes could contribute to nerve damage in tuberculoid leprosy which is characterized by low to absent M. leprae in Schwann cells. PMID- 1708280 TI - PC-based molecular modeling in the classroom: applications to medicinal chemistry and biochemistry. AB - Among the most difficult aspects of medicinal chemistry and biochemistry for the student to master are the three-dimensional (3D) nature of drugs and bio-organic substances and the interaction of these substances with 3D targets. Compounding this problem is the fact that such relationships are very difficult to illustrate in a lecture or discussion format. While skeletal molecular models serve a useful role in the learning process, the techniques of PC-based desktop molecular visualization provide a more powerful and effective alternative to the lecture format. These techniques can be implemented on standard MS-DOS PC hardware using one of the commonly available data projection systems. The approach has found considerable use in several areas, including the generation of computer-based lecture aids, the illustration of the molecular shapes of drugs and biochemical structures, the superposition and comparison of drug substances with common pharmacophores, and the illustration of enzyme-substrate interactions. Another related technique, molecular animation, has proven to be quite successful at illustrating the essentials of enzyme mechanisms in the classroom. The "film clips" resulting from this technique may have use beyond the classroom, and further work in this area is underway. PMID- 1708281 TI - Rapid nuclear accumulation of injected oligodeoxyribonucleotides. AB - The intracellular transport and fate of nucleic acids is poorly understood. To study this process, we injected fluorescent oligodeoxyribonucleotides (oligos) into the cytoplasm of CV-1 epithelial cells and primary human fibroblasts. Rapid nuclear accumulation was found with the phosphodiester (PD), phosphorothioate (PT), and methylphosphonate (MP) forms of a 28-mer oligo complimentary to the rev mRNA of the human immunodeficiency virus type 1. Migration of the oligos in the cytoplasm was slower than diffusion of a coinjected dextran, but the oligos freely diffused into the nucleus. Nuclear incorporation was temperature but not energy dependent. The intranuclear distribution of the oligos was influenced by the chemistry of internucleoside linkages. The PD oligos and, to a lesser extent, the PT oligos colocalized with small nuclear ribonucleoproteins (snRNPs), whereas the MP oligos colocalized with concentrated regions of genomic DNA. These data have important implications for our understanding of the transport and accumulation of exogenous nucleic acids in mammalian nuclei, and the assay described could potentially be used for testing the efficacy of oligos designed as therapeutic agents. PMID- 1708282 TI - A developmentally regulated, nervous system-specific gene in Xenopus encodes a putative RNA-binding protein. AB - A gene from Xenopus laevis that is expressed specifically in the nervous system beginning at the stage of neural plate formation has been isolated and several cDNAs have been sequenced. The sequence of the predicted protein contains two copies of a presumed RNA-binding domain, each of which includes two short conserved motifs characteristic for ribonucleoproteins (RNPs), called the RNP-1 and RNP-2 consensus sequences. We name this gene Xenopus nrp-1, for nervous system-specific RNP protein-1. Sequence comparisons suggest that the nrp-1 protein is a heterogeneous nuclear RNP protein, but it is clearly distinct from previously reported hnRNP proteins such as the A1, A2/B1, and C1 proteins. nrp-1 RNA undergoes an alternative splicing event giving rise to two predicted protein isoforms that differ from each other by seven amino acids. In situ hybridization to tadpole brain shows that the nrp-1 gene is expressed in the ventricular zone where cell proliferation takes place. The occurrence of an RNP protein with nervous system-limited expression suggests that it may be involved in the tissue specific control of RNA processing. PMID- 1708283 TI - Noncovalent drug-DNA binding interactions that inhibit and stimulate (A)BC excinuclease. AB - (A)BC excinuclease from Escherichia coli catalyzes the initial step of nucleotide excision repair. It recognizes and binds to many types of covalent modifications in DNA and incises the damaged strand on both sides of the lesion. We employed a variety of noncovalent DNA binding drugs to examine in vitro the mechanisms and the nature of the DNA-drug interactions responsible for two phenomena: inhibition of excision repair by caffeine and other noncovalent DNA binding compounds; incision of undamaged DNA produced by (A)BC excinuclease in the presence of the bisintercalating drug ditercalinium. All of the chemicals examined (e.g., actinomycin D, caffeine, ethidium bromide, and Hoechst 33258) inhibited incision of a covalent adduct by (A)BC excinuclease, and direct evidence is given for a common mechanism in which UvrA is depleted by binding to drug-undamaged DNA complexes. In the absence of significant amounts of undamaged DNA, another mechanism of inhibition was observed, in which enzyme bound to noncovalent drug DNA complexes in the vicinity of the lesion prevents formation of preincision complexes at the lesion. Ditercalinium and unexpectedly all of the other drugs examined promoted the incision of undamaged DNA when the enzyme was present at high concentration. Thus, this activity contrary to previous assumptions is not unique to bisintercalators. Another unexpected finding was stimulation of incision at certain sites of photodamage in DNA produced by low concentrations of noncovalent DNA binding chemicals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708284 TI - Deuterium quadrupole coupling in N-acetylglycine and librational dynamics in solid poly(gamma-benzyl-L-glutamate). AB - To study the dynamics of peptide groups in solid proteins, we have accurately determined the principal components and molecular orientation of the electric field gradient tensor for the exchangeable deuterons in monoclinic N acetylglycine by single-crystal deuterium nuclear magnetic resonance. These results are compared with the principal components of the amide deuterons in solid poly(gamma-benzyl-L-glutamate) measured in powder samples over a wide temperature range (140-400 K). The comparison indicates that in the solid polypeptide the N-D bonds undergo a small-amplitude torsional reorientation (libration) perpendicular to the peptide plane. To estimate dynamic rates, longitudinal relaxation times (T1 values) are reported for N-acetylglycine and poly(gamma-benzyl-L-glutamate). T1 values for the carboxyl and amide deuterons in N-acetylglycine are approximately 100 s, whereas for the amide deuterons in the polypeptide T1 approximately 1 s, also indicating that the N-D bonds are not stationary in the polypeptide. We determine from the reduced quadrupole coupling tensor the mean-square amplitude for the libration and show that it increases linearly with temperature. A simple qualitative theory for the relaxation times is presented on the basis of the assumption that the N-D reorientation is described either as a diffusion process in a square well or as a damped Langevin oscillator with a harmonic restoring force. The conclusion is that the short relaxation times of the polypeptide amide deuterons result from substantial frictional effects on reorientation that increase with temperature. PMID- 1708285 TI - Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis. AB - The use of deoxyribonucleotide substitution in RNA (mixed RNA/DNA polymers) permits an evaluation of the role of 2'-hydroxyl groups in ribozyme catalysis. Specific deoxyribonucleotide substitution at G9 and A13 of the ribozyme decreases the catalytic activity (kcat) of the ribozyme by factors of 14 and 20, respectively. The reduction of the reaction rate concomitant with the absence of these 2'-OHs or the 2'-OH of the substrate U7 position can be partially compensated by increasing the Mg2+ concentration above 10 mM. The KMg of the all RNA ribozyme is 5.3 mM, and the lack of either of the three influential 2'-OHs increases this value by a factor of approximately 3. These and other reaction constants for the ribozyme and the deoxy-substituted analogues have been determined by assuming a three-step mechanism. The data presented here provide the basis for the formulation of a molecular model of ribozyme activity. PMID- 1708288 TI - The video deposition--"you are the witness". PMID- 1708286 TI - Intracellular mechanisms involved in short-term regulation of net protein synthesis in pancreatic acini. AB - The mechanisms regulating the net synthesis of digestive enzymes during short term stimulation by agonists were examined in pancreatic acini isolated from the rat. Dispersed pancreatic acini were stimulated for up to 60 min with various concentrations of cholecystokinin octapeptide (CCK-OP), carbachol, A23187, 4 beta phorbol 12-myristate 13-acetate (PMA). The effects of these agonists on net protein synthesis was determined by measuring the incorporation of [3H]leucine or [35S]methionine into protein. Carbachol, PMA, A23187 and concentrations of CCK-OP of 100 pM and greater caused inhibition of protein synthesis. Fluorography of [35S]methionine labeled acinar cell proteins separated by one-dimensional SDS polyacrylamide gel electrophoresis demonstrated that the agonists inhibited the synthesis of the digestive enzymes. Northern blot analysis using cDNA probes revealed that CCK-OP, carbachol and PMA did not alter the cellular content of amylase, lipase and elastase mRNA. The protein kinase C inhibitors 1-(5 isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine failed to reverse the inhibitory effects of CCK-OP, carbachol and PMA on protein synthesis. CCK-OP and PMA activated phospholipase A (PLA) which liberated lysophosphatidylcholine (LPC) and free fatty acids from membrane phosphatidylcholine. Exogenously added PLA2 (Naja naja venom) inhibited protein synthesis and increased LPC to a similar extent as CCK and PMA. The results suggest that the inhibitory effects of CCK and carbachol on net protein synthesis are due to their effects on intracellular calcium and PLA-mediated breakdown of phosphatidylcholine rather than protein kinase C activation. PMID- 1708287 TI - TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells. AB - In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species. PMID- 1708289 TI - Increased alpha-1-acid glycoprotein in depression lowers free fraction of imipramine. PMID- 1708290 TI - Platelet 3H-imipramine binding, serotonin uptake, and plasma alpha 1 acid glycoprotein in disruptive behavior disorders. PMID- 1708291 TI - Monoclonal antibody YB5.B8 identifies the human c-kit protein product. AB - The c-kit proto-oncogene encodes a 145- to 160-Kd transmembrane tyrosine kinase, which is a member of the platelet-derived growth factor receptor family and is allelic with the murine white spotting locus (W). W mutations affect several aspects of hematopoiesis, most notably erythroid progenitors and mast cells. A monoclonal antibody, YB5.B8, had been raised against the leukemic blasts of a patient with M1-type acute myelocytic leukemia (AML) and it precipitates a 150-Kd cell surface glycoprotein from leukemic cells. The YB5.B8 epitope is expressed on mast cells, on up to 3% of normal mononuclear bone marrow cells, and it identifies a sub-group of AML patients with a poor prognosis. In view of similarities noted between the cell surface antigen identified by YB5.B8 and the c-kit protein product, we performed experiments to determine whether they are identical. c-kit RNA expression in the cell lines HEL (human erythroleukemia) and A172 (glioblastoma) was shown to parallel the expression of the YB5.B8 epitope in these lines as measured by flow cytometry. Immunoprecipitation analysis with anti kit serum and YB5.B8 antibody indicated that the two antibodies identified proteins of identical size in HEL (155 Kd) and A172 (145 Kd) cells, and sequential immunoprecipitations with the kit and the YB5.B8 antibodies demonstrated that the two antibodies recognize the same molecule. The proteins identified by both the anti-kit and YB5.B8 antibodies displayed in vitro autophosphorylation activity in immune complex kinase assays. In addition, YB5.B8 was able to inhibit the binding of the kit ligand to HEL cells. These studies provide evidence that the YB5.B8 antigen and the c-kit protein product are identical and raise certain hypotheses regarding the role of c-kit in AML. PMID- 1708292 TI - Dexamethasone and 1,25-dihydroxyvitamin D3, but not cyclosporine A, inhibit production of granulocyte-macrophage colony-stimulating factor in human fibroblasts. AB - Tumor necrosis factor alpha (TNF alpha) stimulates granulocyte-macrophage colony stimulating factor (GM-CSF) production in human fibroblasts and other mesenchymal cells. However, relatively little is known about agents that downregulate cytokine production in these cells. In the present report we show that dexamethasone (Dexa), a synthetic glucocorticoid, markedly reduced GM-CSF production in TNF alpha-stimulated fibroblasts at both the protein and the RNA levels. CSF activity, GM-CSF protein, and RNA levels, determined by an in vitro colony-forming assay in normal human bone marrow cells, by an enzyme immunoassay, and by Northern blotting assay, were reduced to greater than 90% of control values by Dexa (1 mumol/L). Similarly, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a hormone with possible physiologic immunoregulatory significance, reduced GM-CSF expression in a concentration- and time-dependent manner. However, this repression was less pronounced than that of Dexa, and in part due to a decreased proliferative activity. In contrast, cyclosporine A (CsA), another immunosuppressive agent, did not alter GM-CSF expression in TNF alpha-stimulated fibroblasts. Our in vitro studies suggest that by inhibiting GM-CSF production in fibroblasts, glucocorticoids and possibly 1,25(OH)2D3, but not CsA, may attenuate TNF alpha-mediated inflammatory processes and influence the regulation of hematopoiesis. PMID- 1708293 TI - Increased serum levels of granulocyte colony-stimulating factor in patients with severe congenital neutropenia. AB - Severe congenital neutropenia (SCN), also known as Kostmann Syndrome, is characterized by a maturation arrest of myelopoiesis at the level of promyelocytes with absence of neutrophils in bone marrow (BM) and blood. Hypotheses of the pathophysiology of SCN include (1) defective production of granulocyte colony-stimulating factor (G-CSF), and/or (2) defective response to G CSF. To exclude defective G-CSF production we tested sera from patients with SCN for the presence of G-CSF using Western blot analysis and NFS-60 proliferation assay. Using these assays we were able to detect increased G-CSF serum levels in SCN patients (150 to 910 pg/mL) as compared with normal controls (between undetectable and 100 pg/mL). These results suggest that patients with SCN have no defect in G-CSF production but a defective response of neutrophil precursors to endogenous G-CSF. PMID- 1708294 TI - The specific activity of plasminogen activator inhibitor-1 in disseminated intravascular coagulation with acute promyelocytic leukemia. AB - In disseminated intravascular coagulation (DIC) with acute promyelocytic leukemia (APL) in the absence of severe infection, marked fibrinolysis was noted in comparison with normal levels of antithrombin III, which is a major inhibitor of the coagulation system. Increased plasminogen activator inhibitor-1 (PAI-1) antigen levels in plasma from patients with septicemia decreased the ratio of the plasma clot lysis rate induced by an anti-alpha 2-plasmin inhibitor monoclonal antibody to the tissue-type plasminogen activator (t-PA) concentration. This decrease was not as prominent in plasma from patients with DIC, especially those with APL. To explore the character of PAI-1 in these plasmas, we measured the specific activity of PAI-1 by determining the ratio of active PAI-1 antigen to t PA-unbound PAI-1 antigen. To calculate the amount of active PAI-1 antigen, the amount of t-PA/PAI-1 complex before and after the addition of a fixed amount of t PA to the sample was measured by a sandwich solid-phase enzyme-linked immunosorbent assay using anti-PAI-1 and anti-t-PA monoclonal antibodies. The assay to measure total PAI-1 antigen used three monoclonal anti-PAI-1 antibodies and had similar sensitivities to free active, latent, vitronectin-bound and t-PA bound PAI-1. The specific activity of PAI-1 decreased in patients with DIC (43.7% +/- 30.6%) and in DIC cases with APL (10.3% +/- 6.0%) in comparison to patients with septicemia (83.7% +/- 20.2%) or normal controls (85.8% +/- 27.3%). In DIC associated with APL, degraded forms of PAI-1 were detected in plasma by immunoblotting. These results suggest that a decrease in the specific activity of PAI-1 and an increase in secondary fibrinolysis result in a hyperfibrinolytic state in DIC patients with APL. PMID- 1708295 TI - A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope. AB - We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK 2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation. PMID- 1708296 TI - Possible mechanism of selective killing of myeloid leukemic blast cells by lymphokine-activated killer cells. AB - Major histocompatibility complex-unrestricted lymphokine-activated killer (LAK) cells have been proposed as therapy for a variety of hematologic malignancies. Because these cells recognize and kill their targets independently of their antigen specific CD3 receptor, it is unclear how they might discriminate between normal and malignant cells. We now propose one such mechanism for the selective killing of myeloid leukemia blasts. While both CD2+ and CD2- activated killer cells may inhibit the clonogenic growth of myeloid leukemia cells, only the CD2+ subset effectively inhibits the growth of normal myeloid (granulocyte-macrophage and granulocyte) progenitors. This difference appears to reflect differential requirements for cell adhesion molecule recognition between normal and malignant progenitor cells. Inhibition of the growth of normal granulocyte-macrophage colonies by CD2+ LAK cells is blocked by antibodies to the CD2-lymphocyte function-associated antigen 3 (LFA-3) (CD58) cell adhesion system. In contrast, these antibodies have no effect on CD2+ LAK-mediated inhibition of malignant cell clonogenic growth. Instead, antibodies to the LFA-1 (CD11a/CD18)-intercellular adhesion molecule 1 (ICAM-1) (CD54) adhesion system reduce inhibition. These differences correspond to differential expression of the CD54 cell adhesion molecule by normal and malignant myeloid progenitor cells because less than 15% of normal CD34 positive cells are CD54+ while greater than 85% of CD34+ acute myeloid leukemia blasts express the CD54 antigen. LFA-3, the ligand for CD2, is strongly expressed by erythrocytes, and these cells competitively inhibit killing of normal but not malignant clonogenic cells in an analogous way to the effects of monoclonal antibody to the CD2-LFA-3 adhesion system. The operation of this effect in vivo may be a basis for selective cytotoxicity by CD2+ LAK against clonogenic myeloid blast cells, and could be exploited further with infusion of appropriate monoclonal antibodies. PMID- 1708297 TI - DDT in Mytilus edulis: spatio-temporal variations in the Punta Banda Estuary, Baja California, Mexico. PMID- 1708298 TI - Effects of lindane and acetone on the development of larvae of the southern king crab (Lithodes antarcticus Jaquinot). PMID- 1708299 TI - Changes in the toxicity of three pesticides as a function of environmental pH and temperature. PMID- 1708300 TI - Persistent organochlorine residues in game-ranched bison in Saskatchewan, Canada. PMID- 1708301 TI - Determination of chlorpyrifos and its major breakdown products in technical formulations. PMID- 1708302 TI - Structure determination of O-linked glycopeptides by tandem mass spectrometry. AB - A new method for characterizing O-linked glycopeptides without chemical degradation is presented. Collision-induced dissociation (CID) analysis of intact O-linked glycopeptides containing mono- and disaccharides was performed. For glycopeptides containing one hexose unit, both the peptide sequence and the site of attachment of the sugar moiety were obtained from a single high-energy CID spectrum. However, in a glycopeptide bearing multiple sugar residues per site, the CID spectrum was dominated by fragments resulting from cleavages of the carbohydrate substituents and the gas-phase deglycosylated peptide, thus obviating the concomitant observation of peptide sequence ions. Hence, information on the structures of the carbohydrate substituents was obtained, but not on the sites of attachment of these residues to the peptide. Subsequent CID analysis of the gas-phase deglycosylated peptide ion can be used to obtain the sequence of the peptide backbone from the same sample. This method holds promise for simultaneously determining the carbohydrate structure and the peptide sequence of intact O-linked glycopeptides without chemical degradation. PMID- 1708303 TI - Fibroblast growth factors stimulate nerve growth factor synthesis and secretion by astrocytes. AB - Nerve growth factor (NGF) is produced and secreted by astrocytes and fibroblasts, but not by microglia, in a primary non-neuronal cell culture derived from newborn rat brains under a standard culture condition. NGF secretion by astrocytes was highest just after passage and then gradually decreased. There is no significant difference in NGF secretion by astrocytes from five sites of origin tested: cerebral cortex, striatum, hippocampus, septum, and cerebellum. Acidic and basic fibroblast growth factors (aFGF and bFGF) significantly increased NGF secretion by astrocytes. The effect of aFGF was greater than that of bFGF, and the effect of both FGFs was not additive at the maximum concentration. The peak of NGF secretion stimulated by aFGF occurred 3-12 h after the addition of aFGF. On the other hand, the dramatic increase in cell numbers was observed 12-48 h after stimulation, and the morphological change became significant 24 h after aFGF stimulation. NGF synthesized by astrocytes is rapidly secreted into the culture medium and aFGF enhances NGF secretion from the transcription level, because cycloheximide and actinomycin-D completely inhibited NGF secretion by astrocytes in the presence or absence of aFGF. Epidermal growth factor (EGF), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) also increased NGF secretion by astrocytes to a certain extent. NGF secretion by astrocytes in the presence of a maximum dose of aFGF was enhanced by the addition of IL-1 beta or TNF-alpha, but not EGF. However, platelet-derived growth factor, interleukin-3, and interleukin-6 had no significant effects. FGFs also enhanced NGF secretion by fibroblasts derived from meninges, but not by microglia. PMID- 1708304 TI - Retarded Wallerian degeneration following peripheral nerve transection in C57BL/6/Ola mice is associated with delayed down-regulation of the P0 gene. AB - Wallerian degeneration is markedly retarded in C57BL/6/Ola mice, the majority of axons remaining intact for up to 10 days after sciatic transections. By 5 days after axotomy in normal mice P0 mRNA is markedly down-regulated, whereas high expression is present in the mutant mice at 7 days and is not reduced by a second distal axotomy. However, dissociated Schwann cells cultured without neurons down regulate P0 in the normal manner. The data suggest that the continued expression of P0 mRNA is probably dependent on a relatively stable axonal signal with a low turnover that does not require continuous fast anterograde or retrograde transport. This may indicate an intrinsic axolemmal molecule(s) is involved in the neuronal signal. PMID- 1708305 TI - Neurofilament M and calbindin D28k are present in mutually exclusive subpopulations of enteric neurons in the rat submucous plexus. AB - Immunocytochemical methods have been used to examine the localisation of 3 neurofilament proteins and the calcium binding protein, calbindin D28k, in whole mount preparations of the submucous plexus in the Wistar rat. Neurofilament-M (160 kDA protein) was present in 40% of the submucosal neurons, staining fine filaments in the soma and the axonal processes. Calbindin D28k was present in 40% of the submucosal neurons staining both the soma and nerves within the plexus. The neurofilament proteins and calbindin D28k were never observed within the same neurons. Neurofilament-M was co-localised with substance P and calcitonin gene related peptide but not somatostatin or the other neuropeptides investigated. Calbindin D28k was co-localised with vasoactive intestinal polypeptide and neuropeptide Y. Galanin- and somatostatin-immunoreactive neurons did not contain either the neurofilament proteins or calbindin D28k. The results demonstrate the presence of subsets of submucosal neurons that can be distinguished by the presence of neurofilament-M or calbindin D28k. PMID- 1708306 TI - Inhibition of CRH-41 release by substance P, but not substance K, from the rat hypothalamus in vitro. AB - Substance P is one of a series of tachykinins which is present throughout the central nervous system, and has potent effects on neuroendocrine function. Recent studies have suggested that it inhibits pituitary-adrenal activity at a site above the level of the pituitary. We have therefore used a well-validated rat hypothalamic incubation system to investigate the effects of substance P on the release of the principal corticotrophin-releasing hormone, CRH-41. Substance P caused a dose-dependent inhibition of the 28-mM KCl-stimulated release of CRH-41, with a maximum effect at 100 nM (P less than 0.01). This effect was attenuated by 10 microM of the substance P antagonist (D-Pro2,-D-Trp7,9)-substance P. No statistically significant effect of substance K was seen at 1 or 100 nM. Substance P, at a dose of 100 nM, did not alter the 28-mM KCl-stimulated release of CRH-41 from isolated median eminences in vitro. It is concluded that substance P is a potent inhibitor of the stimulated release of CRH-41, probably acting at a site within the hypothalamic paraventricular nucleus. PMID- 1708307 TI - PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. AB - PDGF binding to its receptor promotes the association with and stimulates the phosphorylation of PLC-gamma 1 at tyrosine and serine residues. Also, PDGF induces an increase in the hydrolysis of inositol phospholipids by PLC. How PDGF activates PLC was investigated by substituting phenylalanine for tyrosine at PLC gamma 1 phosphorylation sites 771, 783, and 1254 and expressing the mutant enzymes in NIH 3T3 cells. Phenylalanine substitution at Tyr-783 completely blocked the activation of PLC by PDGF, whereas mutation at Try-1254 inhibited and mutation at Tyr-771 enhanced the response. Like the wild type, PLC-gamma 1 substituted with phenylalanine at Tyr-783 became associated with the PDGF receptor and underwent phosphorylation at serine residues in response to PDGF. These results suggest that PLC-gamma 1 is the PLC isozyme that mediates PDGF induced inositol phospholipid hydrolysis, that phosphorylation on Tyr-783 is essential for PLC-gamma 1 activation. These results provide direct evidence that growth factor receptors activate the function of intracellular protein by tyrosine phosphorylation. PMID- 1708308 TI - Differential abilities of Th1 and Th2 to induce polyclonal B cell proliferation. AB - Human gamma globulin-specific T helper cell (Th) clones, activated by HGG in the presence of antigen (Ag)-presenting cells, stimulated polyclonal B cell proliferation. Both Th1 and Th2 clones induced B cell proliferation, but Th1 clones were generally 5- to 10-fold less efficient than Th2 in this capacity. Th1 and Th2 each induced proliferation of both small and large B cells, although Th1 induced less B cell proliferation than Th2, regardless of B cell size. Th1 induced B cell proliferation was increased significantly by stimulating the Th1 clones with immobilized anti-CD3 mAb. The B cell response to Ag-activated Th1 clones was also increased by the addition of rIL-4 or culture supernatants from activated Th2 clones, and this enhancement was abolished by addition of anti-IL-4 mAb. The differential capacity of the Th subsets to stimulate B cells could not be attributed to differences in the degree of Ag-induced activation of the Th clones as reflected by Th proliferation or Th expression of activation markers, RL388 Ag, IL-2R, or TfR. Taken together the results suggest that even though Th1 and Th2 are similarly activated by Ag-presenting cells, Ag-activated Th2 interact more effectively with B cells than Ag-activated Th1. It is possible that inefficient interaction and subsequent intercellular signaling between Th1 and B cells results in inefficient Th1-induced B cell proliferation, and that this deficiency may be circumvented by signals (e.g., lymphokines) provided by Th2, or by the stimulation of Th1 with plate-bound anti-CD3 Ab rather than Ag. PMID- 1708309 TI - Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice. AB - A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE. PMID- 1708310 TI - Activation of macrophages for ADCC in vitro: effects of IL-4, TNF, interferons alpha/beta, interferon-gamma, and GM-CSF. AB - Macrophages in varying states of activation differ in their ability to perform antibody-dependent cellular cytotoxicity (ADCC) and antibody-independent macrophage-mediated tumor cytotoxicity (MTC). To define further the activation requirements for macrophages to perform various cytolytic functions, we stimulated peptone-elicited peritoneal macrophages, which are only poorly cytolytic, with one of a panel of cytokines and then quantified three distinct cytolytic capacities. The peptone-elicited macrophages, after stimulation with IFN-alpha/beta, IL-4, or TNF, had increased ability to perform both the rapid and slow variants of ADCC but not to perform MTC. Stimulation with high doses of IFN gamma, however, increased the macrophages' ability to perform all three cytolytic functions. GM-CSF had no effects on any cytolytic capacity. The effects of IL-4, TNF, IFN-gamma, and IFN-alpha/beta on the macrophages' capacity for both forms of ADCC were dose-dependent. IFN-gamma and IFN-alpha/beta increased the macrophages' capacity for both variants of ADCC within 4 hr of treatment, whereas IL-4 and TNF did so only after prolonged treatment. These results suggest that three forms of macrophage cytolytic capacity can be enhanced by cytokine treatment but that the requirements for enhancing each of the three forms of macrophage cytolytic capacity differ. PMID- 1708311 TI - Role of protein kinase C in bradykinin-induced increases in microvascular permeability. AB - The goal of this study was to determine whether protein kinase C mediates bradykinin-induced increases in microvascular permeability. Permeability of the hamster cheek pouch was evaluated using intravital fluorescent microscopy and fluorescein isothiocyanate (FITC)-dextran (MW 70,000). We examined effects of sphingosine, a protein kinase C inhibitor, on bradykinin-induced increases in permeability. Increases in permeability were quantitated by counting the number of leaky sites and calculating the clearance of FITC-dextran. During bradykinin (10(-6) M), leaky sites increased from 0 to 40 +/- 4 (mean +/- SEM) sites/0.11 cm2, and clearance increased from 1.7 +/- 1.0 to 22 +/- 9 ml/sec x 10(-6). The bradykinin type-2 receptor antagonist D-Arg,[Hyp3,Thi5,8,D-Phe7]-bradykinin virtually abolished formation of leaky sites in response to bradykinin. To determine whether changes in microvascular pressure contribute to the increase in leaky sites, venular pressure was measured using a micropipette and survo-null device. Increases in cheek pouch venular pressure were similar during application of bradykinin and adenosine, which increased permeability, and isoproterenol, which did not increase permeability in the cheek pouch. Thus, increases in permeability were not linked to changes in microvascular pressure. The protein kinase C inhibitor, sphingosine (10(-6) M), markedly attenuated responses to bradykinin. Leaky sites increased from 0 to only 2 +/- 1 sites/0.11 cm2, and clearance increased from 3.9 +/- 1.4 to only 6.7 +/- 2.2 ml/sec x 10(-6). To test the specificity of sphingosine, we examined effects of adenosine (10(-6) M). Sphingosine did not significantly alter increases in microvascular permeability in responses to adenosine. We also examined effects of 1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H-7), another protein kinase C inhibitor, on responses to bradykinin and adenosine. H-7 greatly attenuated formation of leaky sites during stimulation with bradykinin and did not alter the number of leaky sites produced during adenosine. The findings suggest that protein kinase C may mediate increases in vascular permeability in response to bradykinin. PMID- 1708312 TI - Myocardial angiogenesis and coronary perfusion in left ventricular pressure overload hypertrophy in the young lamb. Evidence for inhibition with chronic protamine administration. AB - In contrast to young growing animals, pressure-overload hypertrophy in adults is frequently associated with diminished myocardial capillary density and maximal coronary flow per gram. To determine the role of angiogenesis in maintaining perfusion capacity in the hypertrophying heart, the angiogenesis inhibitor protamine sulfate was administered to young lambs during the development of left ventricular (LV) pressure-overload hypertrophy. Baseline and maximum (adenosine) myocardial perfusion was measured in four groups of chronically instrumented 10 week-old lambs subjected to 1) ascending aortic bands since the age of 4 weeks (LVH group, n = 10), 2) sham operation at the age of 4 weeks (SHAM group, n = 8), 3) aortic bands and twice daily injections of protamine since the age of 4 weeks (LVH + P group, n = 9), 4) sham operation and injection of protamine (SHAM + P group, n = 8). Capillary density was measured postmortem. Peak LV pressure and the LV/body weight ratio were similarly increased in LVH and LVH + P compared with sham-operated lambs (p less than 0.001). In LVH lambs, LV capillary number increased by 32% compared with sham-operated lambs (p less than 0.05), and capillary density, coronary flow reserve, and minimal coronary resistance remained normal. In contrast, LVH + P lambs had no significant increase over SHAM lambs in LV capillaries and total maximum coronary flow. The LVH + P lambs had lower LV subendomyocardial capillary density and higher minimal coronary resistance per gram (p less than 0.05 versus LVH lambs). Right ventricular capillary density and minimal resistance were similar in all groups. These findings support the hypotheses that myocardial angiogenesis with pressure overload hypertrophy is important in maintaining maximal LV coronary flow in the young and that impairment of angiogenesis results in diminished coronary flow capacity. PMID- 1708313 TI - Healthy individuals and patients with systemic lupus erythematosus have unique, person-specific spectra of antibodies detectable on immunoblots. AB - Through the technique of immunoblotting, the spectrum and organ specificity of antibodies in healthy individuals and patients with systemic lupus erythematosus were examined. It was demonstrated that healthy individuals and patients with autoimmune disease have antibodies, some tissue specific and some not tissue specific, which are present in a pattern that is unique to each individual. PMID- 1708314 TI - Monoclonal antibodies against human anti-F(ab')2 antibodies react with light chain epitopes. AB - Anti-F(ab')2 antibodies affinity isolated from sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or normal SLE relatives were used to produce monoclonal antibodies (mAbs) in Balb/c and NZB mice. Four of five mAbs showed only primary light chain specificity. Only one mAb produced in an NZB mouse against anti-F(ab')2 from a single SLE patient showed anti-mu-chain specificity. Parallel identical control immunizations with IgG or a single human IgG kappa myeloma produced mAbs with a predominant gamma-chain/Fc fragment specificity. Anti-light chain specificity of mAbs was demonstrated to involve epitopes requiring tertiary structure of the entire light chain instead of antigens confined to Ckappa/lambda or Vkappa/lambda fragments. Anti-kappa specificity of three mAbs was extremely similar but not identical to that defined by anti-Km1 allotyping systems. No evidence was obtained with any of the mAbs produced for antigens unique to SLE or RA anti-F(ab')2 antibodies. The light chain antigenic prominence of many anti-F(ab')2 antibodies may reflect structural features shared by this group of immunoglobulins somehow important for their biologic function. PMID- 1708315 TI - Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL2 receptor p55 chain (TAC, CD25). AB - Using a high-sensitivity immunofluorescence procedure, a proportion of blood lymphocytes can be shown to express the p55 chain of the IL-2 receptor (CD25, TAC) without in vitro stimulation. In this study, we measured the proportion of peripheral blood lymphocytes expressing CD25 in patients with a diagnosis of HIV infection and compared the results with cells from controls. The mean value for HIV-positive samples was 15%, while controls gave a mean value of 31%. The difference between the groups was highly significant (P less than 0.001, Mann Whitney U test). When expression of CD25 was examined separately in CD4-positive and CD8-positive T cells and in B cells, there was a wider spread of values in the HIV group compared with controls, but no systematic difference. In the patient group studied (110 samples from 53 patients) there was a weak correlation between CD25 and CD4 expression (correlation coefficient r = 0.21, P less than 0.02), but there were patients with low CD25 and high CD4 as well as patients with high CD25 and low CD4, suggesting that CD25 can vary independently of changes in CD4 lymphocytes. Although the majority of very low values of CD25 (less than 10%) were found in patients with stage IV disease, such low values were also common in Stage II disease. PMID- 1708316 TI - Epitopes of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins recognized by antibodies in the sera of HIV-1-infected individuals. AB - Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENVgp) that were recognized by antibodies. Two epitopes of HIV ENVgp, ENVP466 (amino acids 466-481) and ENVP497 (amino acids 497-509), were recognized by antibodies in the sera of most HIV infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIV's ENVgp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections. PMID- 1708317 TI - CAR-3 and CA 19-9 serum levels in pancreatic cancer: any differences between two epitopes of the same mucin-like glycoprotein? AB - The aim of this study was to compare the utility of two recently identified tumour markers of pancreatic cancer, CA 19-9 and CAR-3, and to ascertain the roles of some factors influencing both antigens. CA 19-9 and CAR-3 were measured in sera of 18 control subjects, 27 patients with pancreatic cancer, 25 with chronic pancreatitis, and 29 with extra-pancreatic diseases. CA 19-9 and CAR-3 were, respectively, found to be increased in 85 per cent and 44 per cent of patients with pancreatic cancer, 28 per cent and 0 per cent with chronic pancreatitis and 72 per cent and 28 per cent with extra-pancreatic diseases. The ROC curves showed that, for any serum value considered, CA 19-9 is more effective than CAR-3 in discriminating between pancreatic cancer and control subjects and chronic pancreatitis. With the combined use of both antigens the results were no better than those given by CA 19-9 alone. Correlations were found between liver function tests and CA 19-9 levels and between cholestasis indices only and CAR-3 values. Our findings show that CAR-3 is not a sufficiently reliable marker of pancreatic cancer, due to its low sensitivity. Nor does it offer any more information than CA 19-9. Both assays are influenced, at least in part, by the extent of the neoplasia. Cholestasis which can greatly influence a serum glycoproteic marker such as CA 19-9, was found also to affect, to a lesser extent, CAR-3, an epitope on the same mucin molecule. PMID- 1708318 TI - Modulation of class II histocompatibility antigen expression by maternal serum. AB - Human maternal serum has been shown to down-regulate the expression of MHC class II antigen in three distinct circumstances. Cord blood mononuclear cells, incubated in the mother's own serum, showed significant modulation of class II antigen expression. This was also the case for unrelated donor lymphocytes, incubated in pooled maternal serum. One neoplastic line (Daudi) was further shown to down-regulate class II antigen expression. In this last case, the down regulating effect persisted over a 10-day period during which maternal serum was renewed regularly. Retroplacental serum was more MHC class II-inhibiting than peripheral serum. This down-regulating effect does not apply to maternal lymphocytes. The inhibitory effect is thought to be due to a factor, yet to be defined, included in the maternal IgG fraction. Regular assays made throughout pregnancy showed that the class II inhibiting component appears early (5th week), reaches its peak value at the 12th week, and disappears 2 or 3 weeks after delivery. PMID- 1708319 TI - Antibodies to recombinant HIV-1 nef protein detected in HIV-1 infection as well as in nonrisk individuals. PMID- 1708320 TI - Scaling and attainment of goals in family-focused early intervention. AB - Evaluating the impact of early intervention as a means to prevent or ameliorate developmental disabilities has been a long standing problem and the issue of effectiveness continues to be debated. This study explored the utility of Goal Attainment Scaling as a planning and evaluation tool whereby intervention outcomes for infants and families could be documented. The 23 families in this study were participants in a larger research effort evaluating the implementation of community based, family-focused intervention. An average of 5.9 goals were written for each family, with approximately 60% of goals written for infants and 40% for families. Attainment of goals was evident in a mean T-score of 51.9 for post-test values and in documentation that approximately two-thirds of all goals were attained at least at the expected level. The practical features of Goal Attainment Scaling and the correspondence of goal attainment scores with other measures of change suggest that it may be a valuable approach to complement traditional evaluation strategies. PMID- 1708321 TI - Factor B reference typing report. AB - In a factor B (BF) Reference Typing of the VIth Complement Genetics Workshop and Conference, Mainz, FRG, 1989, 99 samples from 13 laboratories, including 18 families, were investigated with the majority of presently known typing procedures. Among the major ('standard') allotypes BF SO4 was found to be new. For the group of common BF F subtypes samples from 11 laboratories including complete family data from 5 laboratories were compared. The subtypes BF FA and FB were recognized and confirmed to be identical in the samples from all groups. Within a third group rare subtype variants of F and S were compared and characterized. In samples submitted from individuals with assumed non-expressed (BF*QO) alleles unexpected and hypomorphic gene products were seen. The investigation of DNA samples for restriction fragment length polymorphisms from the same set of individuals revealed a correlation of the Msp I 0.7-kb fragment with BF F, and confirmed the correlation of a Taq I 6.6-kb fragment with BF FA. PMID- 1708322 TI - BF DNA reference typing. AB - A study of the RFLP patterns of the samples submitted, using TaqI and MspI restriction enzymes has allowed a confirmation of the correlation previously reported on the BF protein types and subtypes. The 6.6-kb band obtained with TaqI is closely correlated to the BF FA type and the MspI 0.7-kb band with BF F. PMID- 1708323 TI - C4 reference typing report. AB - Human C4 is most polymorphic at the protein level, distinction between allotypes of the C4A and C4B proteins resting on electrophoretic migration patterns and difference in hemolytic activity. The aim of the C4 reference typing has been the definition of reference variants, the assignment of rare variants, and the investigation of duplicated, deleted, or non-expressed and hybrid genes. Samples from 136 individuals, predominantly with known segregation, from 16 laboratories were investigated by standard electrophoretic techniques, for their relative hemolytic activity, reactivity with monoclonal antibodies and Rg/Ch reagents, alpha-, and beta-chain types, relative electrophoretic migration distance, as well as the C4/21-OH-TaqI RFLPs. The results were evaluated in three groups; they consisted in the definition of the eight most common C4 alleles, and the ten Rg/Ch standard phenotypes in group I. In group II twelve C4A and fourteen C4B duplications among 96 complotypes, as well as eighteen deleted/non-expressed C4A and twenty-two C4B alleles, and hybrid alleles were seen by correlation of lytic activity, electrophoretic mobility, and monoclonal and/or Rg/Ch reactivity. Group III consisted of the newly defined allotypes A 8, A 7, A 58, A 55, A 45, B 45, B 35, and B 22, furthermore of alleles subdividing the A 1/A 91, and the B 13/B 12/B 11 regions. The reference typing has allowed reclassification of the majority of described C4 allotypes and resulted in a revision of the C4 nomenclature. PMID- 1708324 TI - C4: Rodgers and Chido typing. AB - Rodgers (Rg) and Chido (Ch)-typing, by means of haemagglutination inhibition, has been performed on 136 plasma/serum samples selected by or submitted to the 1989 Workshop. All were tested for two Rg and six Ch antigenic determinants and a proportion were also tested for WH. The established interrelationships for the antigenic determinants have been fully supported and previously reported associations with C4 allotypes have been maintained. A second example of the Ch phenotype Ch:-1, 2,-3,4,5-6 was detected in an Italian family with the B3 allotype. PMID- 1708325 TI - Aprotinin treatment of pseudomonal corneal infection. AB - Pseudomonas aeruginosa produces proteolytic enzymes capable of causing severe corneal degradation. In this study we tested aprotinin, a broad-spectrum protease inhibitor, on rabbit corneas infected with P. aeruginosa to determine whether any benefit could be derived. Corneas treated with aprotinin and tobramycin topically and subconjunctivally were not clinically better than corneas treated with tobramycin alone. PMID- 1708326 TI - Idiopathic lipid corneal degeneration. AB - Two patients with no history of eye disease or systemic disorder exhibited bilateral corneal lipid annular infiltrates, together with deep stromal vascularization. Penetrating keratoplasty was performed on one eye of each patient, and one patient presented with an acute graft rejection. PMID- 1708327 TI - Familial pulmonary hypertension in association with an abnormal hemoglobin. Insights into the pathogenesis of primary pulmonary hypertension. AB - A kindred with a familial hemoglobinopathy and familial primary pulmonary hypertension with autosomal dominant transmission has been identified. Affected family members were obvious from their cyanosis due to a reduced affinity for oxygen by the hemoglobin variant. The mother and one child had clinical pulmonary hypertension, whereas two siblings had cyanosis and preclinical pulmonary vascular disease as evidenced by abnormal perfusion lung scans and elevated levels of fibrinopeptide A in the face of normal pulmonary hemodynamics. In one, pulmonary hypertension could be induced with exercise. The studies on this family support the hypothesis that primary pulmonary hypertension may be initiated by abnormalities of the pulmonary vascular bed that predispose to in situ thrombosis. The possible common genetic transmission of the two diseases offers the speculation that the gene that confers predisposition to pulmonary hypertension may be located near the gene responsible for beta globulin. PMID- 1708329 TI - Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in the rat. Single and multiple dosing studies. AB - The pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were investigated in male Sprague-Dawley rats at a dose of 5 micrograms/kg. The serum concentrations of rhG-CSF were monitored using a specific sandwich enzyme immunoassay. In single-dose studies, the influence of routes of administration were evaluated. For iv administration, the serum concentration-time data showed the rapid disappearance of rhG-CSF from the systemic blood and a mean residence time (MRT) of 1.341 hr. For sc, im, and ip administration, lower peak serum levels were observed, but after 2 to 3 hr, rhG CSF levels were higher than those for iv administration. The MRTs after sc, im, and ip injections were 3.918, 2.894, and 3.538 hr, respectively. The serum concentration profiles after extravascular injections showed that an im injection gave slightly faster absorption kinetics of rhG-CSF from the injection site into systemic blood than did sc and ip injections. In multiple-dose studies, rhG-CSF was injected into animals iv and sc at 5 micrograms/kg/day for 7 days. On the day 7 the serum concentration-time profiles after rhG-CSF administration were compared between single and multiple dosing. The AUC after iv multiple dosing decreased by 17.4%, although half-lives and the volume of distribution were not significantly different between single and multiple dosing groups. The AUC after sc multiple dosing decreased by 25.6%; however, the bioavailability and observed maximum serum concentration of rhG-CSF were not significantly different. These results showed that the clearance of rhG-CSF increased after multiple dosing, although the mechanism of increased clearance was not apparent. PMID- 1708328 TI - Immunohistochemical localization of cytochrome P-450IA1 induced by 3,3',4,4' tetrachlorobiphenyl and by 2,3,7,8-tetrachlorodibenzoafuran in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup). AB - The regulation of different cytochrome P-450 forms and their functions in different organs and cell types could determine the susceptibility of those cells and organs to toxic effects of xenobiotics, including chemical carcinogenesis. Here we describe the cellular localization of cytochrome P-450E (P-450IA1) induced in 10 major organs or organ systems of a marine vertebrate species, the fish, Stenotomus chrysops (scup). Scup were injected ip with 3,3',4,4' tetrachlorobiphenyl (TCB) at 1 mg/kg, or with 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 micrograms/kg. Induction was verified by Western blot analysis of microsomes from selected organs (liver, kidney, and gill) using monoclonal antibody (MAb) 1-12-3 to scup P-450IA1. The localization of P-450IA1 was subsequently determined in sections prepared by standard histological methods (10% buffered formalin fixation, paraffin embedding), and stained with MAb 1-12-3 and peroxidase-labeled second antibody. P-450IA1 was induced in epithelial and endothelial cells in liver (including pancreatic tissue), kidney, gill, gut, spleen, testis, and ovary. Induction also was detected in endothelial cells, but not other types, in heart, brain, and red muscle. In heart, the staining was present in the endocardium as well as in the endothelium of the coronary vasculature and great vessels. Although TCDF and TCB both induced P-450IA1 in various cells of all organs examined, the effect of TCB was in most cases greater than that of TCDF. This may be due to a relatively higher TCB dosage. A wider staining distribution was seen in gut, gill, kidney, and gonad of TCB-treated fish, which might be explained by a greater penetration, or by excretion of parent TCB, as opposed to TCDF. In any case, the results show that these important environmental agents induce P-450IA1 in generally similar patterns in all organs examined. The common finding of a strong induction of P-450IA1 in endothelial cells in all organs examined supports the suggestion that the endothelium may be a primary site of P-450IA1 induction. PMID- 1708330 TI - Expression of a surface epitope on cells that link branches in the tracheal network of Manduca sexta. AB - A monoclonal antibody (MAb 2F5) to a cell surface epitope labels a small subpopulation of tracheal epithelial cells in each thoracic and abdominal segment of Manduca. These cells (nodes) represent the sites within the tracheal network at which invaginating tracheal tubes join during embryonic establishment of the tracheal network. Tracheal nodes are also the sites at which tracheal cuticle fractures during each molt. Since tracheal cuticle is shed through each spiracle, a tracheal node lies between each pair of contralateral spiracles within a segment (commissural node) and between each pair of adjacent, ipsilateral spiracles (lateral longitudinal node). MAb 2F5 first labels presumptive nodal cells of tracheal epithelium immediately prior to the linking of epithelial tubes from successive and opposite spiracles. One cell at the tip of each invaginating tracheal branch labels with MAb 2F5. The highly localized expression of the cell surface epitope recognized by MAb 2F5 may be instrumental in the orderly coupling of tracheal branches during embryonic development. On the basis of immunolabeling of Western blots and tissues, MAb 2F5 is believed to recognize Manduca fasciclin II, a member of a class of molecules involved in cell adhesion/recognition. PMID- 1708331 TI - Hypertrophy and increased gene expression of neurons containing neurokinin-B and substance-P messenger ribonucleic acids in the hypothalami of postmenopausal women. AB - We have previously described hypertrophy of neurons containing estrogen receptor mRNA in the infundibular nucleus of postmenopausal women. In the present investigation we identified peptide mRNAs in the hypertrophied neurons and determined whether postmenopausal neuronal hypertrophy was accompanied by changes in gene expression. In the first study in situ hybridization was performed on sections from hypothalami of postmenopausal women (n = 3) using synthetic 35S labeled cDNA probes complementary to mRNAs encoding estrogen receptor, substance P (SP), neurokinin-B (NKB), POMC, cholecystokinin, dynorphin, CRF, enkephalin, galanin, neuropeptide-Y, GH-releasing hormone, and tyrosine hydroxylase. Neuronal cross-sectional areas and cell densities were measured with the aid of a computer microscope system. Neurons labeled with the NKB and SP probes were comparable in size, morphology, and distribution to the hypertrophied neurons containing estrogen receptor mRNA. In contrast, neurons labeled with other cDNA probes were sparsely distributed (CRF and dynorphin), smaller in size (neuropeptide-Y, galanin, GH-releasing hormone, enkephalin, cholecystokinin, and POMC), or located anterior to the hypertrophied population (tyrosine hydroxylase). In the second study sections from hypothalami of premenopausal (n = 3) and postmenopausal (n = 3) women were incubated with cDNA probes complementary to SP or NKB mRNAs. The mean cross-sectional areas of postmenopausal infundibular neurons containing NKB and SP mRNAs increased to 194% and 176% of premenopausal values, respectively. The autoradiographic grain densities of infundibular neurons labeled with either probe were also significantly increased in the postmenopausal group. Finally, the numbers of labeled neurons/tissue increased 6-fold (SP) and 15-fold (NKB) in the postmenopausal infundibular nucleus. These data demonstrate that human menopause is associated with marked increases in hypothalamic NKB and SP gene expression. We propose that neurons containing estrogen receptor, SP, and NKB mRNAs participate in the hypothalamic circuitry regulating estrogen negative feedback in the human. PMID- 1708332 TI - Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells. AB - Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 microM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8-bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum. PMID- 1708333 TI - Ontogeny of insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in porcine serum during fetal and postnatal development. AB - The insulin-like growth factors (IGF-I and IGF-II) circulate in plasma in association with IGF-binding proteins (IGFBPs). As a first step to understanding the regulation of expression of these proteins in pigs, we characterized the ontogeny of circulating IGFs and IGFBPs during fetal and early postnatal development. Serum IGFs were separated from IGFBPs, before IGF RIA, by acidification and chromatography on C18 Sep-Pak cartridges. The IGF-I levels increased during the latter half of fetal life from 11 +/- 1 ng/ml on day 60 to 37 +/- 3 ng/ml on day 112 (2-3 days before term) and further increased postnatally to 227 +/- 21 to 265 +/- 26 ng/ml) and also increased higher than IGF I levels, with no obvious developmental pattern, during fetal life (170 +/- 21 to 265 +/- 26 ng/ml) and also increased postnatally by 2-fold (463 +/- 29 ng/ml on day 42). These results support the view that IGF-II is a fetal and postnatal growth factor, whereas IGF-I is primarily a postnatal growth mediator in pigs. Serum IGF-binding proteins were identified by Western ligand blotting. Five IGFBPs with apparent mol wt of 43K, 39K, 34K, 31K, and 26K were detected in fetal and postnatal sera. The two largest proteins were shown to be glycoproteins and immunologically related to porcine (p) IGFBP-3, suggesting that they are glycosylation variants of pIGFBP-3. The abundance of these two IGFBPs increased coincidently with increasing serum IGF-I levels. The 34K IGFBP was immunologically related to rIGFBP-2 and was 2- to 3-fold more abundant in fetal serum than in postnatal serum. The 31K IGFBP was resolved into a triplet and also was a component of pIGFBP-3 immunoprecipitates. Similarly, the 26K IGFBP was present in pIGFBP-3 immunoprecipitates. The 31K and 26K IGFBPs represented a minor portion of serum IGF-binding activity in fetal and postnatal pigs and exhibited no obvious developmental patterns. It is hypothesized that the postnatal increases in serum IGF-I and 43K and 39K IGFBPs as well as the decrease in the 34K IGFBP are driven by GH action. PMID- 1708334 TI - Tissue-specific expression and thyroid hormone regulation of the endogenous placental growth hormone variant and chorionic somatomammotropin genes in a human choriocarcinoma cell line. AB - Human (h) placenta-derived choriocarcinoma cell lines (BeWo, JAR, and JEG-3) were examined for expression of pituitary GH (hGH-N) as well as placental GH variant (hGH-V) and chorionic somatomammotropin (hCS, encoded by the hCS-A or hCS-B gene). RNA was isolated and assessed using hGH-N complementary DNA since hGH and hCS genes share more than 90% sequences similarity. The relative expression is BeWo greater than JAR greater than JEG-3. In BeWo cells expression of placental hCS-A, hCS-B, and hGH-V genes, but not pituitary hGH-N, is observed using polyadenylated RNA and oligonucleotide probes specific for the different family members. The absence of hGH-N expression in BeWo cells is not due to deletion or gross rearrangement of the gene. No difference was seen between the hGH/hCS genes in genomic DNA from these cells and the DNA from placenta and pituitary when analyzed by restriction digestion and blotting. Treatment of BeWo cells with 10 nM T3 results in a 6-fold increase in messenger RNA from placental members of the hGH gene family. Levels of hCS-A, hCS-B, and hGH-V transcripts are all elevated. Cellular and secreted proteins from BeWo cells were analyzed by Western blotting, and a band of about 22 kilodaltons was detected using a polyclonal antibody which cross-reacts with hGH-V and hCS. The level of 22 kilodalton band in samples of cellular as well as released protein was increased by T3 treatment. BeWo cells provide a model system for studying hGH-V and hCS regulation as well as tissue specific expression. PMID- 1708335 TI - Ovarian glycosaminoglycans potentiate angiogenic activity of epidermal growth factor in mice. AB - Epidermal growth factor (EGF) has been shown to induce capillary proliferation. In the course of our investigation on the interaction of ovarian components with EGF, we observed that partially purified glycosaminoglycans (GAGs) isolated from mouse ovaries enhanced the angiogenic activity of EGF when applied simultaneously to the lateral wall of the sheath of musculus rectus abdominis. Mouse EGF from submandibular glands embedded in Elvax 40 implanting on the musculus rectus abdominis induced neovascularization in a dose-dependent manner. When 0.5 micrograms ovarian GAGs was embedded in the implant with a low amount of EGF that induced only slight neovascularization (0.5 or 1 microgram/implant), the angiogenic activity of the growth factor was markedly enhanced. The active GAG component was isolated by chromatography on Dowex 1-x2. The fraction eluted with 0.5 M NaCl possessed the greatest activity to potentiate the angiogenic activity of EGF. When the reaction mixture of GAGs and EGF was treated with 1% cetylpyridinium chloride, the angiogenic activity was identified with the supernatant. On the other hand, after incubating EGF with 0.5 M NaCl fraction, the angiogenic activity of EGF was identified with the precipitate (GAG fraction) of the cetylpyridinium chloride-treated reaction mixtures. These findings show that ovarian GAGs potentiate the angiogenic activity of EGF by interacting or complexing with EGF. PMID- 1708336 TI - Tachykinin (substance-P) gene expression in Leydig cells of the human and mouse testis. AB - Specific substance-P immunoreactivity can be detected in the Leydig cells, particularly of human testes, and to a lesser degree in mouse Leydig cells, but not in the rat. Using a modified polymerase chain reaction (PCR) assay, preprotachykinin-A (substance-P) mRNA could be detected in extracts of human, mouse, and bovine testes, but not in rat or boar testes or in bovine thyroid or corpus luteum used as negative controls. This assay is able to discriminate among the alpha, beta, and gamma transcripts of the gene and shows that only the beta and gamma transcripts are present in the testes. Sequencing analysis of the PCR products from bovine hypothalamus, mouse brain, and human testis confirmed the structure of these transcripts, which encode both substance-P and neurokinin-A (substance-K) neuropeptide hormones. Using a variant of this assay it was possible to identify tachykinin transcripts in as few as 500 freshly prepared purified mouse Leydig cells. In parallel studies PCR analysis was also able to confirm the presence of mRNA for both substance-P and neurokinin-A receptors in human testes. Thus, the tachykinins substance-P and neurokinin-A must now be added to the list of potentially paracrine substances regulating intratesticular function. PMID- 1708337 TI - Further characterization of insulin-like-growth factor binding proteins in rat osteoblast-like cell cultures: modulation by 17 beta-estradiol and human growth hormone. AB - Insulin-like growth factors (IGF-I and IGF-II) are endocrine and autocrine factors affecting bone growth and metabolism. Binding proteins for IGFs (IGFBPs) are synthesized by the target tissues of IGF actions. Thus, IGFBPs may act as modulators for the biological functions of IGFs. We have characterized the rat IGFBPs (rIGFBPs) and studied their regulation by 17 beta-estradiol (beta E2) and human GH (hGH) in rat osteoblast-like (ROB) cell cultures. ROB cells were prepared from 19- to 20-day fetal rat calvariae by sequential collagenase digestion and studies were performed on the serum and phenol red-free conditioned medium of confluent cultures. [125I]IGF-I ligand blots showed that the major rIGFBP in the ROB is a nonglycosylated protein of 31 kilodaltons. This protein was immunoprecipitated by a specific antibody to rIGFBP-2 and messenger RNA for rIGFBP-2 was detected by RNA hybridization indicating that the rIGFBP-2 is the major rIGFBP of ROB. A minor band at 24 kilodaltons is likely to be the rat homologue of the newly isolated inhibitory IGFBP-4. The predominant glycosylated adult form of rIGFBPs of rat serum, rIGFBP-3, was undetectable. When cultures were treated with beta E2 for 2 days, there was a dose-dependent biphasic response which showed an inhibition of the rIGFBP-2 at low doses of 10(-11) to 10(-9) M and a stimulation at 10(-6) M. These changes in rIGFBP-2 parallel the changes in the endogenous IGF-I level. rIGFBP-2 level was not affected by 17 alpha-estradiol at the same concentration range. hGH, on the other hand stimulated the levels of rIGFBP-2 and rIGFBP-4 at doses ranging from 10(-11) to 10(-9) M without changing the IGF-I secretion. The alteration of the rIGFBPs by beta E2 and hGH suggests a role for these hormones in bone by modulating the biological functions of IGFs via their binding proteins. PMID- 1708338 TI - Intracellular distribution of the endogenous and transfected beta form of thyroid hormone nuclear receptor visualized by the use of domain-specific monoclonal antibodies. AB - To study the regulation, tissue distribution, and subcellular localization of nuclear receptor for thyroid hormone, monoclonal antibodies (mAbs) against the human placental c-erbA (hTR beta 1) protein were prepared. hTR beta 1 was expressed in Escherichia coli and purified to apparent homogeneity. The purified hTR beta 1 was used to produce monoclonal antibodies. Three hybridomas, secreting mAb J51, J52, and J53, were isolated. All of these mAbs recognized hTR beta 1. J51 and J52 belong to the immunoglobulin G1-k subclass; J53 is an IgM. To evaluate cross-reactivity with other classes of c-erbAs, the three mAbs were used to immunoprecipitate the in vitro translation products of human (h) TR alpha 1, TR alpha 2, rat (r) TR beta 1, TR alpha 1, and TR alpha 2. None of these three mAbs reacted with h- or rTR alpha 1 and TR alpha 2. J51 did not react with rTR beta 1, but J52 and J53 cross-reacted with rTR beta 1 with the same activity as hTR beta 1. To localize the epitopes in the hTR beta 1 molecule, [35S]methionine labeled and truncated hTR beta 1 containing the hormone-binding domain E (Lys235 Asp456; Lys201-Pro414), domain D (Met169-Asp456), or the DNA-binding domain C (Glu100-Asp456) were expressed in E. coli and purified. Immunoprecipitation of the above truncated hTR beta 1 with mAbs indicated that the epitopes for J51 and J52 were located in two different sites in the A/B domain. The epitope for J53 was located in the E domain. Using immunocytochemistry and mAb J52, the endogenous TR beta 1 in rat pituitary GH3 cells was visualized to be exclusively present in nuclei. The transfected hTR beta 1 in monkey COS-1 and human choriocarcinoma JEG-3 cells was recognized by both J51 and J52. Interestingly, the intracellular localization of the transfected hTR beta 1 or rTR beta 1 in the above two cell lines depended on the level of expression. TR beta 1 expressed at low levels was found exclusively in nuclei. However, for high level expression of TR beta 1, cytoplasmic localization was also detected. J53, however, failed to detect nuclear fluorescence of the endogenous and transfected TR beta 1 in fixed cells, suggesting that its antigenic site might be occluded. Localization of the endogenous and transfected TR beta 1 in nuclei indicated that these two receptor proteins are structurally indistinguishable.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1708339 TI - A human monoclonal IgG reactive with a private idiotope of a monoclonal IgM with autoantibody activity against myelin-associated glycoprotein. AB - One hundred and thirteen sera from patients with monoclonal IgG were tested for reactivity against a panel of 13 human monoclonal IgM having various autoantibody activities: 6 to myelin-associated glycoprotein (MAG), 2 to vimentin intermediate filament protein and 5 to red blood cell antigens [cold agglutinins with specificity directed to I antigen (3 cases), i antigen (1 case) or Pr antigen (1 case)]. One IgG was found to react with a monoclonal IgM with anti-MAG activity. This reactivity was characterized as idiotypic and directed against a private idiotope of the monoclonal IgM. This work provides further evidence for the existence of anti-idiotypic antibody activity of monoclonal Ig occurring in human B cell neoplasias. PMID- 1708340 TI - Surface expression by mononuclear phagocytes of an epitope shared with mycobacterial heat shock protein 60. AB - Bone marrow-derived macrophages were stained with a variety of monoclonal antibodies (mAb) with specificity for the mycobacterial heat shock protein (hsp) 60 or with specific rabbit antiserum raised against mycobacterial hsp 60. The mAb ML30 as well as the specific antiserum brightly stained bone marrow-derived macrophages whereas all the other mAb as well as normal rabbit antiserum gave no or negligible reactivity. Surface expression of the shared epitope is observed by day 3 of in vitro cultivation and markedly increased by interferon-gamma stimulation on day 9. Although hsp 60 is thought to be restricted to the mitochondrial compartment, antibody responses to this molecule have been implicated in autoimmunity. Our finding that bone marrow-derived macrophages express a cross-reactive epitope recognized by an mAb with specificity for amino acids 275 to 295 of the mycobacterial hsp 60 is consistent with such a role. PMID- 1708341 TI - Differential activation of human basophils by anti-IgE and formyl-methionyl leucyl-phenylalanine. Indications for protein kinase C-dependent and -independent activation pathways. AB - Upon activation, basophilic granulocytes release inflammatory mediators such as histamine. We studied histamine release of human basophils (64.1 +/- 9.6% pure) after cross-linking of membrane-bound IgE via anti-IgE, or after binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). A variability in the extent of histamine release upon stimulation by either anti-IgE or fMLP was found between donors. Kinetic studies revealed that the histamine release induced by anti-IgE (t1/2 greater than 240 s) was more than 20-fold slower than the almost instantaneous release upon stimulation with fMLP (t1/2 less than 10 s). Differences in the cell activation pathways triggered by these stimuli were further analyzed with staurosporine, an inhibitor of protein kinase C (PKC) and with wortmannin, an inhibitor of a PKC-independent pathway. Inhibition of PKC resulted in a partial inhibition of the anti-IgE-induced release, whereas the fMLP-induced release was slightly potentiated. The anti-IgE-induced release was completely inhibited in the presence of wortmannin. This inhibitor too, had no effect on the fMLP-induced release. We conclude that major differences exist in the signal-response coupling between the anti-IgE and fMLP-induced histamine release in human basophils. The so-called releasability of human basophils may be due to the availability of different cell activation pathways. PMID- 1708342 TI - The structural basis of serological specificity in Shigella flexneri O-antigens. PMID- 1708343 TI - A two-site immunometric assay for arginine vasopressin. PMID- 1708344 TI - Design and synthesis of bombesin/gastrin-releasing peptide antagonists. PMID- 1708345 TI - Reprogramming of myosin light chain 1/3 expression in muscle heterokaryons. AB - Fast myosin light chain (MLC) 1/3 is one of the few genes which regulates transcript production at both transcriptional and post-transcriptional levels, utilizing two functionally distinct promoters coupled with alternatively spliced exons. The transcriptional process controlling expression from this single gene locus is developmentally regulated, such that MLC 1 precedes MLC 3 during myogenesis. Results from our RNA analyses demonstrate that in differentiated rat L6E9 muscle, MLC 3 is the sole isoform expressed from the MLC 1/3 locus. However, we also show that by generating rat L6E9:mouse C2 muscle heterokaryons, MLC 1 expression from the L6E9 MLC locus can be induced. In addition to novel rat MLC 1 expression in the C2:L6E9 heterokaryons, we show that the synthesis profile of rat MLC 3 mRNA is also altered relative to L6E9 muscle cultured alone. Additional experiments demonstrate that the reprogramming of rat MLC 1 and 3 expression in the muscle heterokaryons requires that C2 and L6E9 nuclei be contained within a common cytoplasm. These results demonstrate that expression from the MLC 1/3 gene is "plastic," and is not under the control of a strict developmental program but, rather, can be modified by the environmental milieu. PMID- 1708346 TI - Identification of cell-type-specific genes of Volvox carteri and characterization of their expression during the asexual life cycle. AB - Volvox carteri possesses two morphologically and functionally distinct cell types: somatic cells and gonidia (asexual reproductive cells). To define the developmental programs involved in the differentiation of these two cell types, we have isolated 31 nonhomologous cDNA clones that hybridized to RNAs that were significantly more abundant in one cell type than the other. Details of the cell type- and stage-specificity of expression of the transcripts detected by these cDNAs (plus five genes previously characterized by others) were examined by Northern-blot analysis. Accumulation patterns for the 19 gonidial transcripts fell into two distinct classes: transcripts of one gene were maximally abundant in very early cleavage, whereas transcripts of the other 18 did not reach maximal abundance until quite late in gonidial development. Similarly, the 12 somatic cell-specific transcripts fell into two categories: transcripts of 5 "early" somatic genes became abundant soon after the completion of embryogenesis, whereas transcripts of 7 "late" somatic genes were not detected until later developmental stages. Expression of 3 other genes (two involved in flagellar development and one that encodes an extracellular matrix component) was also found to be restricted largely to somatic cells. These studies indicate that phenotypic differences between somatic cells and gonidia can be at least partially explained by differential regulation of RNA accumulation, and that there appear to be multiple patterns of accumulation of cell-type-specific transcripts within each cell type. PMID- 1708347 TI - Distribution and density of substance P receptors in the feline gastrointestinal tract using autoradiography. AB - Autoradiography was used to localize and quantify substance P receptors in the feline gastrointestinal tract. The specific binding of 125I-Bolton Hunter substance P was determined in the esophagus, lower esophageal sphincter, antrum, pylorus, duodenum, jejunum, ileum, ileocecal sphincter, and colon. Competitive binding studies indicated that substance P binding sites or NK-1 receptor sites were demonstrated. The concentration of NK-1 receptors was greatest in the distal half of the gastrointestinal tract, with the highest concentrations in the proximal colon. The circular muscle layer contained the greatest amount of substance P binding. The location and density of binding sites for substance P may be important in understanding the relative importance of both the pharmacological responses to this neuropeptide and the immunohistochemical evidence of the peptide at different sites in the intestine. PMID- 1708348 TI - Role of galanin in the gut. PMID- 1708349 TI - Perspectives on rapid paracentesis: positional and pressure effects. PMID- 1708350 TI - [Analogies between the thymus and epidermis]. AB - In this paper, similarities between epidermis and thymus are reviewed. Both epidermis and thymus deal with an epithelial stroma harbouring dendritic cells, which are bone-marrow derived. Both epithelia are keratinized, and a map can be constructed illustrating histo-topographic and antigenic similarities between thymic epithelial cells distributed in various thymic zones (i.e. subcapsular cortex, outer cortex, inner cortex, medulla, outer layers of Hassall's bodies, inner layers of Hassall's bodies) and keratinocytes of different epidermal layers. By contrast, a possible similarity between thymocytes and Langerhans cells is not so easy to demonstrate, although both cell types are CD1 positive. Rather, in our opinion a comparison is preferable of thymocytes to Thy-1 positive dendritic epidermal cells, due to morphological, antigenic, functional and especially lineage similarities. Similarities between thymus and epidermis are clearly important dealing with analogous molecular interactions, namely, thymic epithelial cells/thymocytes versus keratinocytes/T-lymphocytes. Indeed, our recent investigations demonstrated that a subset of keratinocytes is ICAM-1 positive, and the whole keratinocyte population is LFA-3 positive. Since the interaction thymic epithelial cells (ICAM-1 and LFA-3 positive)/thymocytes (LFA-1 and CD2 positive) has been shown to be necessary for promotion of activation and maturation of thymocytes, the interaction keratinocytes (ICAM-1 and LFA-3 positive, as we demonstrated)/T-lymphocytes (LFA-1 and CD2 positive as well) might be, by analogy, important not only for the "homing" of T-lymphocytes within the epidermis, but also for the epidermis being considered a peripheral inductive site for T-cell activation and maturation. PMID- 1708351 TI - [Solar keratosis: a histochemical study on the distribution of SH groups and SS bonds]. AB - The distribution of free SH groups and SS covalent linkages in hypertrophic, atrophic, acantholytic, bowenoid, pigmented solar keratoses (SK) and squamous cell carcinoma was evaluated. The sulphydryl groups were present in cytoplasms with a granular pattern and nucleoli mainly in atrophic, hypertrophic and bowenoid SK; the distribution of SS linkages appeared as a brilliant ovoid fluorescence localized in living layers, due to individually keratinized cells in SK. Similar results were found in squamous cell carcinoma. Our results agree with the opinion which considers SK as in situ carcinomas. PMID- 1708352 TI - Immunogenicity of anti-idiotypic antibodies and of their F(ab')2 fragments. AB - Within the idiotype/anti-idiotype network, immunoglobulins act alternatively as reactive molecules and as antigens. To investigate the antigenic properties of immunoglobulins, we evaluated the immunogenicity in rabbits of three murine monoclonal anti-idiotypic antibodies and of their F(ab')2 fragments. These antibodies, bearing the internal image of a human melanoma antigen, may be useful in view of a human therapeutic application. The effect was evaluated as specific anti-anti-idiotypic response, related to the immunogenicity of the idiotypic epitopes in the combining sites of the immunoglobulins, and as total anti-murine immunoglobulin response, which represents the recognition of all the immunological determinants of the molecule. The results showed that the administration of the F(ab')2 fragments results in either higher or similar degrees of anti-anti-idiotypic immunization, compared to those induced by the whole immunoglobulins. Nevertheless, when anti-anti-idiotypic immunogenicity was increased, the anti-murine response did not increase proportionally. This suggests that the use for in vivo administration of F(ab')2 fragments is more convenient than the use of their original molecules, since this results, at least, in a similar or eventually in an increased specific immunogenicity, while the possibility of aspecific recognition is reduced. PMID- 1708353 TI - Function of idiotypic networks in vivo: immunisation with idiotype-bearing (Id1) antibody induces further production of Id1 antibody. AB - Injection of BALB/c mice with an anti-foot-and-mouth disease virus (FMDV) monoclonal antibody (mAb) apparently induced the idiotype network to produce more anti-FMDV (idiotype-bearing) antibody, as determined by hybridoma production. Anti-idiotype antibodies were also induced, detected by binding directly to the mAb used for the immunizations (the "immunising" antibody). Many of the anti idiotype antibodies were directed against regions in or near the paratope of the immunising mAb, since they competed for the binding of the latter mAb to 146S antigen. The induced idiotype-bearing (anti-FMDV) antibodies also competed for the binding of the immunizing mAb to 146S antigen, demonstrating that both antibodies were of similar epitope specificity. Consequently, it would appear that an idiotype-bearing (Id1) antibody can induce the idiotypic networks to produce more Id1 antibody of the same specificity as that used for the initial stimulation, demonstrating the in vivo functioning of the idiotype network. PMID- 1708354 TI - Comparison of properties of virulent, avirulent, and interferon-resistant Rickettsia prowazekii strains. AB - Several properties of virulent, avirulent, and interferon-resistant Rickettsia prowazekii strains were compared. All of the interferon-resistant rickettsial strains (which were derived from the avirulent Madrid E strain) resembled the virulent Breinl strain in that they grew well in untreated mouse macrophagelike RAW264.7 cells. In contrast, the avirulent Madrid E strain grew poorly in untreated RAW264.7 cells. Pretreatment of interferon-resistant rickettsiae or R. prowazekii Breinl with antirickettsial serum or immunoglobulin G suppressed the ability of the rickettsiae to grow in untreated RAW264.7 cells. Interferon resistant R. prowazekii strains, like the Madrid E and Breinl strains, rapidly killed a substantial proportion of RAW264.7 cells that had been treated with gamma interferon or very high concentrations of alpha/beta interferon. Untreated infected RAW264.7 cells and interferon-treated mock-infected RAW264.7 cells were not killed during the same period. In cultures of RAW264.7 cells treated with either alpha/beta interferon (120 to 1,200 U/ml) or a subsaturating concentration of gamma interferon (0.5 U/ml), R. prowazekii Breinl organisms killed a higher percentage of the cells than did comparable numbers of R. prowazekii Madrid E organisms or interferon-resistant rickettsiae. Although R. prowazekii Breinl (like R. prowazekii Madrid E) was quite sensitive to gamma interferon in mouse L929 cells, the Breinl strain was resistant to murine alpha/beta interferon compared with the Madrid E strain and the two strains selected for resistance to murine gamma interferon. One of the interferon-resistant strains (strain 60P, which was selected for resistance to murine alpha/beta interferon) differed from the other R. prowazekii strains in that it induced little or no detectable interferon in L929 cell cultures. PMID- 1708355 TI - Monoclonal antibodies to surface antigens of a pathogenic Mycoplasma hominis strain. AB - Three monoclonal antibodies (MAbs) were prepared against an arthritogenic strain of Mycoplasma hominis isolated from the joint aspirates of a patient with chronic septic arthritis. Immunoblots of polyacrylamide gel-electrophoresed proteins before and after surface proteolysis showed that the predominant antigenic determinants were on surface-exposed polypeptides. These polypeptides have extensive hydrophobic characteristics, as demonstrated by Triton X-114 phase partitioning. The electrophoresed proteins from cells grown in medium containing [14C]palmitate were blotted onto nitrocellulose which was both reacted with the MAbs and exposed to X-ray film. Superimposable bands on both the immunoblots and the exposed film suggested that the proteins might be acylated. The MAbs were further tested for reactivity with 16 other strains of M. hominis isolated from patients with septic arthritis (1 strain), septicemia (10 strains), or nongonococcal urethritis (1 strain); from the cervix (1 strain), rectum (1 strain), or surgical wound (1 strain) of patients; and from a contaminated cell culture. No single protein was consistently recognized from strain to strain, although a 94-kDa protein from 16 of the 17 strains tested was bound by at least one of the MAbs. The apparent antigenic heterogeneity among strains of M. hominis, including those isolated from the same tissue source and/or from patients with the same type of clinical disease, might be misleading in that all strains express epitopes associated with a discrete number of proteins to which one, two, or all three MAbs bind. The expression of the epitopes on multiple proteins from the same or different strains may reflect a mechanism for generating antigenic diversity. PMID- 1708356 TI - The alpha-L-(1----2)-trirhamnopyranoside epitope on the group-specific polysaccharide of group B streptococci. AB - A number of epitope specificities associated with the group antigen (group B polysaccharide) of group B streptococci have been identified in a polyclonal antiserum induced in rabbits by a nonencapsulated variant strain of group B streptococci. This was achieved by using a series of oligosaccharide inhibitors, obtained by both synthetic and degradative procedures, to inhibit the binding of the group B polysaccharide to the polyclonal antiserum. While the dominant epitope expressed in the antiserum was alpha-L-Rhap(1----2)alpha-L-Rhap(1--- 2)alpha-L-Rhap, specificities associated with alpha-L-Rhap and alpha-L-Rhap(1--- 3)alpha-D-Galp(1----3)beta-D-Glcp-NAc(1----4)alp ha-L-Rhap were also identified. The dominant expression of the former epitope is consistent with its terminal location on the group antigen and also with highly branched multiantennary structure of this antigen. Antibodies specific for the alpha-L trirhamnopyranoside epitope were purified by affinity chromatography, using the synthetic trisaccharide glucitol as the hapten. Oligosaccharide inhibition studies indicate that the specificity of these antibodies is identical to that of a murine monoclonal antibody induced by the same nonencapsulated strain of group B streptococci. PMID- 1708357 TI - Characterization of microneme antigens of Cryptosporidium parvum (Protozoa, Apicomplexa). AB - Two monoclonal antibodies (MAbs) raised against purified excysted oocysts and sporozoites of cryptosporidium parvum reacted in an immunofluorescence assay with antigens located at the anterior pole of the zoites. On Western blots of purified oocysts, these MAbs reacted with a series of bands between 210 and 40 kDa; several of these bands were recognized by both MAbs; others were specific. One MAb (TOU) did not react after periodic acid treatment and was therefore considered to recognize a carbohydrate epitope; as determined by immunoelectron microscopy, this MAb reacted on micronemes of sporozoites and merozoites and also with the peripheral cytoplasm and the parasitophorous vacuole of trophozoites and macrogametes. The other MAb (HAD) reacted with an epitope that was insensitive to periodate treatment but did not react in the immunoelectron microscopy assay. However, the similar labeling pattern obtained with the immunofluorescence assay with both MAbs and the fact that the two antibodies share common bands on Western immunoblots suggest that both MAbs react with molecules located in Cryptosporidium micronemes, one reacting with a glycannic epitope and the other reacting with a peptidic epitope. PMID- 1708358 TI - Production of recombinant Bordetella pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica: utility of Escherichia coli gene expression signals. AB - Serotype-specific fimbriae of Bordetella pertussis are considered potential components of new-generation vaccines against whooping cough. Attempts to characterize fimbriae, and indeed other virulence determinants, produced by B. pertussis have been frustrated on one hand by low yields from B. pertussis itself and on the other by an inability to produce native recombinant products in Escherichia coli. In order to try to circumvent this problem, we have examined the expression of B. pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica from native as well as E. coli expression signals. These studies revealed that the fimbrial gene product was expressed from the original B. pertussis promoter and Shine-Dalgarno sequence in both B. parapertussis and B. bronchiseptica. The transcriptional start site of the gene was located 146 nucleotides upstream of its ATG start codon. A recombinant fimbrial subunit gene containing PLAC and the atpE translation initiation region of E. coli was also expressed in B. bronchiseptica. In all cases in which gene expression was detected the gene product was expressed as serotype 2-specific fimbriae as determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopic investigation of the bacterial cell surface. PMID- 1708359 TI - Murine natural killer cells are fungicidal to Cryptococcus neoformans. AB - Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus neoformans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans. PMID- 1708360 TI - Characterization of monoclonal antibodies against alpha-hemolysin of Escherichia coli. AB - Monoclonal antibodies (MAbs) were raised against native and denatured alpha hemolysin (HlyA) of Escherichia coli. Binding of the MAbs to native, denatured, and erythrocyte-complexed active wild-type hemolysin and mutant derivatives was tested. All 15 MAbs analyzed bound to native hemolysin, even when the toxin was complexed with human erythrocytes. While some MAbs were unable to bind to a specific native mutant hemolysin, others could not even bind to mutant hemolysin carrying deletions remote from their actual binding sites. A rough determination of the binding sites of 15 MAbs on HlyA was performed by Western immunoblot analysis using CNBr fragments of HlyA and mutant hemolysin proteins. Interestingly, the binding sites of the MAbs against native hemolysin seem to be more randomly distributed on HlyA than are those of MAbs against denatured hemolysin. Three MAbs inhibited the hemolytic activity significantly. Two of these MAbs bound to the hydrophobic region, and the other one bound to the repeat domain of HlyA. The use of synthetic peptides from these regions allowed determination of the linear epitopes for two of these MAbs. PMID- 1708361 TI - Vascularity, perfusion rate and local tissue oxygenation of tumors derived from ras-transformed fibroblasts. AB - Tumors derived from ras-transformed rat fibroblasts were investigated in order to gain insight into possible interrelationships between oncogenic transformations and therapeutically relevant parameters of the metabolic micromilieu of solid tumors in vivo. Tumors grew in nude mice after injection of in vitro-passaged cells. Growth rates, early stages of angiogenesis, perfusion and tissue oxygenation were assessed. Compared with the parental cell line, both ras transformants grew very rapidly and exhibited an early onset of angiogenesis. Perfusion rates of one ras-transformed tumor line were similar to those of the parental tumors whereas reduced flow values were detected in tumors of the other ras line. The better perfused tumors exhibited more adequate tissue oxygen levels whereas reduced oxygen levels were obvious in the poorly perfused ras line. These results indicate that ras transformation alters therapeutically relevant parameters of the tumor micromilieu. However, the tumor phenotype cannot be predicted even if the transforming oncogene is known. PMID- 1708362 TI - Indirect immunotargeting of cis-Pt to human epidermoid carcinoma KB using the avidin-biotin system. AB - Cis-diamminedichloroplatinum (II) (cis-Pt) complexed to a carboxymethyl dextran avidin conjugate was targeted to biotin-monoclonal antibody 108 (b-MAb 108). This MAb recognizes the extracellular domain of the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma (KB) cells over-expressing EGF-R. Cis-Pt carboxymethyl-dextran-avidin (Pt-dex-Av) containing 60-90 M cis-Pt/M avidin was administered 24 hr following b-MAb108 containing 3-5 M biotin/M MAb. This treatment was potentially more effective in suppressing the growth of established KB tumor xenografts, or in inhibiting the development of lung metastases in nude mice, than free MAb 108, free drug or MAb 108 followed by drug. Replacing b-MAb 108 by unbiotinylated antibody or by b-MAb of a different specificity also yielded lower suppressive effects. The sequential administration of Pt-dex-Av following b-MAb was more effective than introduction of the Pt-dex-Av when already complexed to b-MAb 108. The results presented in this preliminary investigation suggest that Pt-dex-Av is specifically removed from the circulation by b-MAb 108 concentrated at the tumor site. PMID- 1708363 TI - Androgen receptors in endocrine-therapy-resistant human prostate cancer. AB - Despite the initial androgen-dependent growth of most human prostate cancers, eventually all prostate cancers become androgen-independent at varying intervals after androgen ablation or anti-androgen therapy. In order to gain more insight into the role of the androgen receptor (AR) in this process, AR and prostate specific antigen (PA) expression was evaluated immunohistochemically in prostatic tumour tissues from patients who developed urinary flow obstruction between 4 and 107 months after onset of treatment. AR expression was evaluated with a monoclonal antibody (MAb) specific for the N-terminal domain of the human AR. To substantiate the progressive tumour growth, proliferative activity was assessed immunohistochemically by staining with MAb Ki-67. Ki-67-defined tumour-growth fractions varied from 0.8-64.7%. In 13 of the 17 examined tumours over 80% of the tumour cells were AR-positive, 3 tumours showed a considerable heterogeneity in AR expression and in 1 tumour almost all tumour cells seemed to be AR-negative. Two-thirds of the examined tumours contained variable proportions of PA-positive tumour areas. These observations contrast with the view that androgen ablation induces a preferential outgrowth of receptor-negative tumour cells. PMID- 1708364 TI - Prognostic significance of argyrophilic nucleolar organizer staining in soft tissue sarcomas. AB - The utility of argyrophilic stain for nucleolar organizer region (AgNOR) for estimating proliferative activity and prognosis of soft-tissue sarcomas (STS) was examined. Formalin-fixed and paraffin-embedded sections of 38 cases with STS were used; the reaction product of AgNOR stain was observed as dots mainly in the nucleoli. The mean number of AgNOR dots per nucleus of tumor cells was calculated in 200 cells (AgNOR count). The AgNOR count, ranging from 1.4 to 16.1 (mean, 7.5), showed a good correlation with cellularity (r = 0.483, p less than 0.003) and histologic grade (r = 0.626, p less than 0.00005), but less shown with mitotic counts (r = 0.350, p less than 0.04). The prognosis of cases with AgNOR low-count group (5-year survival rate was 74.6%) was much better than those in high count group (33.3%) (p less than 0.0005). The AgNOR count correlated well with reactivity of tumor cells for Ki-67 staining, which was available only in freshly prepared sections. These findings suggested that the AgNOR staining is a simple and useful method for estimating tumor-cell proliferation and prognosis of patients with STS. PMID- 1708365 TI - Retinoic acid suppression of squamous differentiation in human head-and-neck squamous carcinoma cells. AB - Retinoids (vitamin A analogues) inhibit the squamous differentiation of normal and malignant epithelial cells. This study investigated the ability of the head and-neck squamous-cell carcinoma (HNSCC) cell line 1483 to undergo squamous differentiation in the absence and presence of beta-all-trans retinoic acid (RA). The growth of these cells in culture is accompanied by an increase in keratinocyte transglutaminase, involucrin and keratin KI, 3 established markers of squamous cell differentiation. Higher levels of these differentiation markers were detected in cells cultured in delipidized serum (DLS), from which endogenous retinoids have been extracted, than in cells cultured in fetal bovine serum (FBS), which contains retinoids. Treatment with I microM RA decreased the levels of the various differentiation markers in cells cultured in either FBS or DLS as revealed by immunofluorescent labelling of permeabilized cells and by immunoblotting of cell extracts using specific monoclonal or polyclonal antibodies. The cells' ability to cross-link proteins to form envelopes under the plasma membrane was stimulated in the presence of calcium ionophore but inhibited by RA. These results indicate that the malignant 1,483 HNSCC cells recapitulate the main characteristics of normal squamous-cell differentiation in culture and that RA suppresses this differentiation as it does in normal keratinizing epithelial cells. PMID- 1708366 TI - Use of combination of monoclonal antibodies directed against three distinct epitopes of a tumor-associated antigen: analysis of cell binding and internalization. AB - Three monoclonal antibodies (MAbs), MOv17, MOv18 and MOv19 with tumor-restricted specificity for human ovarian carcinoma, were tested alone or in double combination with the aim of analyzing their binding and internalization behavior on different in vitro cell lines. Biochemical studies indicated that the 3 MAbs were directed against 3 epitopes of the same 38 kDa surface molecule. By immuno electron-microscopy they exhibited a different internalization behavior since MOv17 induced evident endocytosis through coated vesicles, whereas MOv18 gave rise to occasional uncoated vesicles and MOv19 was completely unable to promote internalization of the relevant molecule. When tested 2 by 2 there was a binding synergy in one of the 9 possible combinations (125I-labelled MOv18 and unlabelled MOv19), but no change in the internalization behavior. The binding synergy, which was highly reproducible, was temperature-dependent and was also evident on glutaraldehyde-fixed cells. A metabolism involvement is therefore unlikely. This could be attributed to an easier accessibility of the CaMOv18 due to a conformational change of the molecule after MOv19 MAb binding. PMID- 1708367 TI - Differentiating effect of sodium butyrate on human hepatoma cell lines PLC/PRF/5, HCC-M and HCC-T. AB - The in vitro effect of sodium butyrate (SB) on human hepatoma cell lines PLC/PRF/5, HCC-M and HCC-T was investigated. SB was added at the non-toxic but cytostatic concentration of 1 mM. In all these cell lines, SB reduced cell proliferation and changed the morphology of the cells into a fibroblast-like shape. In PLC/PRF/5, alpha-fetoprotein production and c-myc expression were inhibited. In contrast, gene expression of albumin, one of the normal liver-cell products, and that of integrated hepatitis B virus genome, was increased. In HCC M and HCC-T, c-myc expression, which was enhanced in the naive state, was reduced. In HCC-M, fos expression was inhibited but the expression of N- and K ras genes did not change. SB seemed to induce normal or mature properties of hepatocytes in human hepatoma cell lines. PMID- 1708368 TI - Tumour-growth suppression in nude mice by a murine monoclonal antibody: factors hampering successful therapy. AB - The murine MAb C215 has been shown to mediate ADMMC in vitro and to have a tumour growth-suppressive effect on xenografted COLO 205 human colocarcinoma cells in nude mice. To overcome the limitations of MAb therapy, it is necessary to understand the underlying mechanisms of tumour-growth suppression. In the present work, we have used C215 to define the importance of different parameters involved in tumour therapy with murine IgG2a antibodies. The results show that there exists a period of roughly 2 days after inoculation into animals during which the tumour cells are sensitive to an inhibitory antibody-mediated effect. After this initial period, the in-vivo sensitivity of tumour cells to antibody-mediated inhibition is much reduced. Tumour cells can remain "dormant" and, despite ongoing antibody treatment, develop into tumours with a reduced growth rate, which is not caused by outgrowth of antigen-deficient tumour cells. Finally, a pronounced dependence of antibody-mediated tumour suppression on antibody dose was observed. PMID- 1708369 TI - Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties. AB - As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1 14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225 232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations. PMID- 1708370 TI - Morphogenetic behavior of simian virus 40-transformed human mammary epithelial stem cell lines on collagen gels. AB - Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned, epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The myoepithelial related cell lines give rise only to the branching rays. Epidermal growth factor stimulates the production of the thick protrusions, whereas cholera toxin stimulates the production of the degenerating areas. Immunocytochemical staining of these cultures using reagents directed against the cell surface-extracellular matrix or the cellular cytoskeleton confirms the epithelial and myoepithelial nature of the cells, and demonstrates that the degenerating areas are undergoing squamous metaplasia. The fingerlike protrusions consist of cords of cells composed of inner, epithelial and outer, myoepithelial-related cells sometimes surrounding a central lumen reminiscent of ducts. The saclike structures resemble alveoli. Ultrastructural analysis confirms the identification of the basic cell types and also identifies indeterminate cells possessing features of both epithelial and myoepithelial cells. It is suggested that the epithelial cell lines represent human mammary stem cells that can undergo processes of morphogenesis and differentiation in vitro to form many of the three-dimensional structures found within the breast. PMID- 1708371 TI - Three cell lines from hamster buccal pouch tumors induced by topical 7,12 dimethylbenz(a)anthracene, alone or in conjunction with herpes simplex virus inoculation. AB - Three squamous carcinoma cell lines HBPC-1, HBPC-2, and HBPC-3 were established from hamster buccal pouch tumors induced by topical 7,12 dimethylbenz(a)anthracene (DMBA) treatment alone, topical DMBA treatment in conjunction with type 1 herpes simplex virus (HSV-1) inoculation, and topical DMBA application in combination with type 2 HSV (HSV-2) inoculation, respectively. The cells were epithelial in morphology, had a doubling time of approximately 18 h, and required bovine serum for optimal growth. They demonstrated an in vitro anchorage-independent growth and produced squamous cell carcinomas when transplanted into normal hamster pouch submucosa. The carcinoma cell lines equally expressed cellular hst, src, abl, and raf proto-oncogenes that were not expressed in the normal hamster pouch epithelial cells. An equal amount of fos gene expression was noticed in the normal pouch epithelial cells, HBPC-1 and HBPC-3, but the HBPC-2 expressed less fos poly(A+)RNA than the other cell lines. The myc proto-oncogene was also expressed both in the normal pouch epithelial cells and in the cancer cell lines. However, the size and number of expressed myc poly(A+)RNA in the normal cells and cancer cell lines differed. Although the normal cells and HBPC-1 expressed a single myc transcript, 1.7 kilobase (kb) and 2.3-kb, respectively, both HBPC-2 and HBPC-3 expressed two myc poly(A+)RNAs, 1.7-kb and 2.3-kb. PMID- 1708372 TI - Retinoic acid regulates, in vitro, the two normal pathways of differentiation of human laryngeal keratinocytes. AB - We have investigated the regulation of the two normal differentiation pathways followed by laryngeal epithelium. Using a tissue culture system that permits growth of cells at the air-liquid interface in serum-free medium, we found that modulating the concentration of retinoic acid is sufficient to determine which pathway is used. At 10(-8) M retinoic acid, the cells form a stratified squamous epithelium which expresses the differentiation-specific keratin K13. At 10(-7) M retinoic acid, the cells form a ciliated pseudostratified epithelium, with no expression of K13. These results are distinct from those seen with foreskin keratinocytes, which have only a single pathway of normal differentiation. PMID- 1708373 TI - Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells. AB - F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco's modified Eagle's medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells. PMID- 1708374 TI - Mitosis and angiogenesis in microwell endothelial cell culture. PMID- 1708375 TI - Characterization and mutagenesis of sulfur-regulated genes in a cyanobacterium: evidence for function in sulfate transport. AB - A sulfur-regulated gene (cysA) that encodes the membrane-associated ATP-binding protein of the sulfate transport system of the cyanobacterium Synechococcus sp. strain PCC 7942 was recently isolated and sequenced. Adjacent to cysA and transcribed in the opposite direction is a gene encoding the sulfate-binding protein (sbpA). Two other genes, cysT and cysW, encode proteins that may form a channel for the transport of sulfate across the cytoplasmic membrane. A fourth gene, cysR, located between cysT, and cysW, encodes a polypeptide that has some homology to a family of prokaryotic regulatory proteins. Mutant strains in which cysA, cysT, or cysW was interrupted by a drug resistance marker were not viable when grown with sulfate as the sole sulfur source and exhibited essentially no sulfate uptake. In contrast, sbpA and cysR mutants grew on sulfate, although they did not exhibit the 20-fold increase in the Vmax (concentration of sulfate at half-maximal transport rate) for sulfate transport characteristic of wild-type cells grown under sulfur-limiting conditions. Three of the sulfur-regulated genes in Synechococcus sp. strain PCC 7942 are similar to genes encoded by the chloroplast genome of the primitive plant Marchantia polymorpha. These data suggest that a sulfate transport system similar to that of Synechococcus sp. strain PCC 7942 may exist in the chloroplast envelope of photosynthetic eukaryotes. PMID- 1708376 TI - Isolation and characterization of a sulfur-regulated gene encoding a periplasmically localized protein with sequence similarity to rhodanese. AB - During sulfur-limited growth, the cyanobacterium Synechococcus sp. strain PCC 7942 loses most of its photosynthetic pigments and develops an increased capacity to acquire sulfate. Sulfur deprivation also triggers the synthesis of several soluble polypeptides. We have isolated a prominent polypeptide of 33 kDa that accumulates specifically under sulfur-limiting conditions. This polypeptide was localized to the periplasmic space. The gene for this protein (designated rhdA) was isolated and discovered to lie within a region of the Synechococcus sp. strain PCC 7942 genome that encodes components of the sulfate permease system. The mRNA for the 33-kDa protein accumulates to high levels within an hour after the cells are deprived of sulfur and drops rapidly when sulfur is added back to the cultures. The amino acid sequence of the protein has similarity to bovine liver rhodanese, an enzyme that transfers the thiol group of thiosulfate to a thiophilic acceptor molecule, and a rhodaneselike protein of Saccharopolyspora erythraea. A strain in which rhdA was interrupted by a drug resistance marker exhibited marginally lower levels of rhodanese activity but was still capable of efficiently utilizing a variety of inorganic sulfur sources. The possible role of this protein in the transport of specific sulfur compounds is discussed. PMID- 1708377 TI - Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons. AB - A physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 was constructed by using the contour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one- and two-dimensional separations to analyze restriction digests of high-molecular-weight genomic DNA. The map, which shows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, is circular and yields a total size of 2.58 megabase pairs for the L. lactis subsp. lactis DL11 chromosome. By using rDNA from Mycoplasma capricolum to probe Southern blots of pulsed-and fixed-field digestion patterns, six putative rRNA operons were identified in L. lactis subsp. lactis DL11 and placed on the map of the chromosome. Five of these loci are clustered in a region representing only 20% of the chromosome. The presence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon to be deduced. PMID- 1708378 TI - Identification and nucleotide sequence of a developmentally regulated gene encoding a eukaryotic histone H1-like protein from Chlamydia trachomatis. AB - A lambda gt11 recombinant library of Chlamydia trachomatis serovar L2 chromosomal DNA was screened with a 29-mer synthetic oligonucleotide specific to the N terminal amino acids of a predominant 18-kDa chlamydial protein. One recombinant clone, designated lambda gt11/L2/RKA10, was selected on the basis of its strong hybridization signal. Restriction endonuclease analysis and complete nucleotide sequencing of the recombinant revealed a 2,633-bp insert containing one complete open reading frame (ORF2) and two partial ORFs (ORF1 and ORF3). The deduced amino acid sequence of ORF2 matched perfectly at its N-terminal end with the derived amino acid sequence. The 375-bp ORF is capable of encoding a protein comprising 125 amino acids with a molecular mass of 13,689. A sequence compatible with a Shine-Dalgarno ribosome-binding site was located 9 bp upstream from the initiation codon, while the sequence distal to ORF2 revealed a rho-independent terminator. The protein, designated CTH1, possesses an estimated pI of 10.71 due to its high lysine content. This highly basic protein contains no tryptophan or phenylalanine. A protein data base search identified significant homology between CTH1 and painted sea urchin histone H1. Northern (RNA) blot analysis of Chlamydia infected host cells demonstrated transcripts at 12 h postinfection. The recombinant plasmid encoding ORF2 expressed a gene product of approximately 18 kDa, similar to the native chlamydial protein as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein appears to represent one of the few eukaryotic histonelike proteins described to date in prokaryotes. PMID- 1708379 TI - Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis. AB - Monoclonal antibodies (MAb) 3F11 and 06B4 recognize epitopes that are conserved on gonococcal lipooligosaccharides (LOS), present on some meningococcal LOS, and conserved on human erythrocytes. LOS of some group B and C prototype meningococcal LOS strains (LOS serotypes L1 to L8) treated with neuraminidase showed increased expression of the 3F11 and 06B4 MAb-defined epitopes. Neuraminidase-treated LOS separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stained showed a shift in migration from a component with a mass of approximately 4.8 kDa to a component with a mass of between 4.5 and 4.6 kDa. The same strains grown in medium with excess CMP-N-acetylneuraminic acid had LOS that shifted in migration to a slightly higher component (mass, approximately 4.8 kDa). Chemical analysis of the neuraminidase-digested products from one LOS indicated it contained approximately 1.5% sialic acid. Covalent linkage between sialic acid and the LOS was confirmed by analysis of de-O acylated and dephosphorylated LOS by liquid secondary ion mass spectrometry. Three studies show that some meningococci contain sialic acid in their LOS, that the sialic acid is cleaved and lost in conventional acetic acid hydrolysis, and that the sialic acid alters the expression of MAb-defined epitopes. PMID- 1708380 TI - Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli. AB - soxR governs a superoxide response regulon that contains the genes for endonuclease IV, Mn2(+)-superoxide dismutase, and glucose 6-phosphate dehydrogenase. The soxR gene encodes a 17-kDa protein; some mutations of this gene cause constitutive overexpression of the regulon. Induction by paraquat (methyl viologen) requires both soxR and a new gene, soxS. soxS is adjacent to soxR, it encodes a 13-kDa protein, and it is required for paraquat resistance. These functions were revealed by studies in which the sequence of the 1.1-kb soxR soxS region was determined, the 5' ends of the mRNAs were mapped, and complementation tests were performed with soxRS plasmids containing deletions of known sequence. The two genes are divergently transcribed, and the transcripts overlap. The soxS promoter is within the 85-nucleotide intergenic region, whereas the soxR promoter is within soxS. soxS mRNA increases after induction. Both protein products have possible DNA-binding (helix-turn-helix) domains. SoxR contains four cysteines (CX2CXCX5C) that might be part of a sensor region. SoxS shows 17 to 31% homology to the C-terminal portions of members of the AraC family of positive regulators. PMID- 1708381 TI - Gene encoding two alkali-soluble components of the spore coat from Bacillus subtilis. AB - We report the cloning and characterization of a gene called cotF from Bacillus subtilis that encodes alkali-soluble polypeptides of 5 and 8 kDa that are components of the spore coat. The 5- and 8-kDa polypeptides are generated by proteolytic cleavage of the primary product of the cotF gene, which is 160 codons in length and is capable of encoding a polypeptide of 19 kDa. Amino acid sequence analysis indicates that the 5-kDa species is derived from the NH2-terminal portion of the primary gene product and that the 8-kDa species is derived from the COOH-terminal portion. A mutant bearing an in vitro-constructed cotF null mutation produced normal-looking spores that contained an apparently complete set of coat proteins except for the absence of the 5- and 8-kDa polypeptides. The map position of cotF is 349 degrees. Transcription of cotF commenced coincidently (during h 6 of sporulation) with genes known to be under the control of sporulation transcription factor sigma kappa. PMID- 1708382 TI - Purification of a Flavobacterium pentachlorophenol-induced periplasmic protein (PcpA) and nucleotide sequence of the corresponding gene (pcpA). AB - A pentachlorophenol (PCP)-induced periplasmic protein (PcpA) with a molecular weight of 30,000 has been identified and purified from Flavobacterium sp. strain ATCC 39723. Results suggest that PcpA may be involved in PCP mineralization. The induction of PcpA correlated with the induction of PCP-degrading activity. When PcpA was released from the periplasmic space by EDTA or osmotic shock treatment, PCP-degrading activity was arrested. Two PCP- mutants of ATCC 39723, which do not degrade PCP, did not produce PcpA following induction with PCP. The N terminus of PcpA was sequenced, and two degenerate primers were synthesized for use in DNA amplification of a 68-bp probe which hybridized to a genomic library clone containing pcpA. The nucleotide sequence of pcpA was determined and found to encode an open reading frame of 816 bp. pcpA was found to be part of a 1.35-kb transcript with a transcriptional start site 67 bp upstream of the translational start site. Data base search comparisons yielded no sequences of high similarity to pcpA or its protein product. PMID- 1708383 TI - Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin. AB - The paucity of crystallographic data on the structure of intrinsic membrane proteins necessitates the development of additional techniques to probe their structures. The colicin E1 ion channel domain contains one prominent hydrophobic region near its COOH terminus that has been proposed to be an anchor for the assembly of the channel. Saturation site-directed mutagenesis of the hydrophobic anchor region of the colicin E1 ion channel was used to probe whether it spanned the bilayer once or twice. A nonpolar amino acid was replaced by a charged residue in 29 mutations made at 26 positions in the channel domain. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic region and the periphery of the hydrophobic region caused a large decrease in the cytotoxicity of the purified mutant colicin E1 protein. This result implies that the hydrophobic domain spans the membrane bilayer twice in a helical hairpin loop, with the center of this domain residing in an aqueous or polar phase. The lengths of the trans-membrane helices appear to be approximately 18 and 16 residues. The absence of significant changes in ion selectivity in five of nine mutants indicated that these mutations did not cause a large change in the channel structure. The ion selectivity changes in four mutants and those previously documented for the flanking Lys residues imply that the hydrophobic hairpin is part of the channel lumen. Water may "abhor" the hydrophobic side of the channel, explaining the small effects of residue charge changes on ion selectivity. PMID- 1708384 TI - Membrane topography of ColE1 gene products: the immunity protein. AB - The topography of the colicin E1 immunity (Imm) protein was determined from the positions of TnphoA and complementary lacZ fusions relative to the three long hydrophobic segments of the protein and site-directed substitution of charged for nonpolar residues in the proposed membrane-spanning segments. Inactivation of the Imm protein function required substitution and insertion of two such charges. It was concluded that the 113-residue colicin E1 Imm protein folds in the membrane as three trans-membrane alpha-helices, with the NH2 and COOH termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The approximate spans of the three helices are Asn-9 to Ser-28, Ile-43 to Phe-62, and Leu-84 to Leu-104. An extrinsic highly charged segment, Lys-66 to Lys-74, containing seven charges in nine residues, extends into the cytoplasmic domain. The specificity of the colicin E1 Imm protein for interaction with the translocation apparatus and the colicin E1 ion channel is proposed to reside in its peripheral segments exposed on the surface of the inner membrane. These regions include the highly charged segment Lys-66 to Lys-83 (loop 2) and the short (approximately eight residue) NH2 terminus on the cytoplasmic side, and Glu-29 to Val-44 (loop 1) and the COOH-terminal segment Gly-105 to Asn-113 on the periplasmic side. PMID- 1708385 TI - Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1. AB - Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1 ---4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4) D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1-- -6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose. PMID- 1708386 TI - Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. AB - The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled. PMID- 1708387 TI - The inhibition of tissue type plasminogen activator by plasminogen activator inhibitor-1. The effects of fibrinogen, heparin, vitronectin, and lipoprotein(a). AB - Plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis by inhibiting tissue type plasminogen activator (t-PA). Fibrinogen, heparin, and vitronectin enhance the rate of inhibition of t-PA by PAI-1. Kinetic studies indicate that both fibrinogen and heparin increase the second-order inhibition constant by a maximum of approximately 4-fold, whereas vitronectin increases the rate constant by a maximum of approximately 6-fold. The dissociation constants of fibrinogen, heparin, and vitronectin for the inhibition reaction were 200 nM, 20 nM, and 600 pM, respectively. In addition, PAI-1 inhibition of t-PA may be regulated by the presence of lipoprotein(a) (Lp(a)). Previous studies demonstrated that Lp(a) competes with plasminogen for the active site of fibrinogen- and heparin-bound t PA. Kinetic studies described here demonstrate that Lp(a) prevents the inhibition of t-PA by PAI-1 in the presence of fibrinogen and heparin, but has no effect on the reaction in the presence of vitronectin or in the absence of either fibrinogen or heparin. The data suggest that fibrinogen and heparin may enhance the rate of inhibition through an interaction with t-PA, and that vitronectin may enhance the inhibition through an interaction with PAI-1. In addition, these experiments indicate that Lp(a) may regulate fibrinolysis by competing with PAI-1 and plasminogen for fibrinogen- and heparin-bound t-PA. These data suggest that PAI-1 inhibition of t-PA in vivo is primarily mediated via interaction with fibrinogen, heparin, vitronectin, and Lp(a), and therefore, the functional levels of PAI-1 activity in the vasculature may be regulated by the presence of these components. PMID- 1708388 TI - Effects of sodium butyrate on proliferation-dependent insulin gene expression and insulin release in glucose-sensitive RIN-5AH cells. AB - A rat islet tumor subclone, RIN-5AH-T2-B, was cultured with 2 mmol/liter of the proliferation-arresting compound sodium butyrate (NaB). Insulin gene expression and glucose-stimulated insulin release were analyzed and compared with logarithmically proliferating and confluent control cells cultured without NaB. Logarithmically proliferating control cells revealed high insulin gene expression. In the presence of amino acids, these cells showed a dose-dependent insulin response to glucose with a half-maximal and maximal 6.5-fold stimulation by 0.8 and 5.6 mmol/liter D-glucose, respectively. However, as the control cells approached growth arrest, insulin gene expression subsided to below detectability, an occurrence that is associated with decreased insulin release and accumulation of cells in the G1 phase of the cell cycle. In contrast, NaB arrested cells showed continuous insulin gene expression throughout the experiment. Despite this, insulin release in response to glucose was lost. NaB revealed a biphasic effect on the cell-cycle: after an initial leaky G1 arrest during the first 24 h, the 5AH-B cells were arrested in G2 during the following 3 days. These data suggest that insulin gene expression and glucose-stimulated insulin release are affected by the cell cycle. These glucose-sensitive RIN-5AH T2-B cells may be useful in studies of insulin secretion and gene regulation. PMID- 1708389 TI - Tumor necrosis factor alpha and interferon gamma modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells. AB - In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes. PMID- 1708390 TI - Synthesis and secretion of apolipoprotein A-I by chick skin. AB - Chick skin slices were incubated with [35S]methionine and labeled apoA-I was immunoprecipitated from incubation medium and tissue homogenate. ApoA-I accounted for approximately 13 and 2.5% of radioactive medium and cell proteins, respectively. After ultracentrifugation of the medium, 55% of labeled apoA-I was found as a constituent of lipoproteins (d less than 1.210 g/ml) and 45% in a lipid-poor form (1.210-1.260 g/ml). To ascertain whether this large proportion of lipid-poor apoA-I was due to a dissociation of this peptide from medium lipoproteins during ultracentrifugation, labeled incubation medium was applied to an anti-chick apoA-I immunoaffinity column. The material bound to the column was analyzed by nondenaturing polyacrylamide gradient gel electrophoresis and found to contain three subpopulations of lipoproteins with a particle size of 12, 11, and 9 nm, respectively. The radioactivity of these subpopulations accounted for 82% of total radioactive medium apoA-I. ApoA-I was localized by immunohistochemistry in the viable cells of the epidermis and in the stratum corneum. Rat skin slices were found to synthesize and secrete apoE but no apoA-I. ApoA-I and apoE secreted by chick and rat skin, respectively, may play a role in the secretion of lipids from the differentiating keratinocytes and thus contribute to the formation of the hydrophobic barrier of the skin. PMID- 1708391 TI - Syndecan from embryonic tooth mesenchyme binds tenascin. AB - Syndecan is a cell surface heparan sulfate-rich proteoglycan found on various epithelial cells but also in some embryonic mesenchymal tissues. We have immunoisolated syndecan from embryonic tooth mesenchyme that appeared as a 250 300-kDa molecule (Kav = 0.3 in Sepharose 4B), containing only heparan sulfate side chains (Mr = 35,000). Northern analysis of whole tooth germs and tooth mesenchymes also revealed high expression of syndecan mRNAs (2.6 and 3.4 kilobases). In the binding assay utilizing nitrocellulose as a solid phase to immobilize matrix molecules, syndecan immunoisolated from tooth mesenchyme revealed binding to tenascin, and this interaction was shown to be mediated via heparan sulfate side chains. In contrast, syndecan from mouse mammary epithelial cells showed only weak interaction with tenascin. We propose that syndecan and tenascin may represent interactions of a cell surface receptor and a matrix ligand involved in mesenchymal cell condensation and differentiation during early organogenesis. PMID- 1708392 TI - Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis. AB - The enzyme sterol 27-hydroxylase catalyzes the first step in the oxidation of the side chain of sterol intermediates in the bile acid synthesis pathway. Human sterol 27-hydroxylase cDNAs were isolated from a liver cDNA library by cross hybridization with a previously cloned rabbit cDNA probe. DNA sequence analysis of hybridization-positive clones predicted a human sterol 27-hydroxylase consisting of a 33-amino-acid mitochondrial signal sequence followed by a mature protein of 498 amino acids. RNA blotting experiments demonstrated sterol 27 hydroxylase mRNAs of approximately 1.8 to 2.2 kilobases in liver and fibroblast cells. The steady state levels of the mRNA did not change when cultured cells were grown in the presence or absence of sterols. Introduction of the sterol 27 hydroxylase cDNA into Simian COS cells resulted in the expression of active enzyme capable of catalyzing multiple oxidation reactions (R-CH3----R-CH2OH----R COOH) at carbon 27 of sterol intermediates of the bile acid synthesis pathway. PMID- 1708393 TI - The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I. AB - The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3' untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene. PMID- 1708394 TI - The long-term clinical outcome of patients with tricuspid atresia. II. Influence of surgical procedures. AB - One-hundred and eleven patients with tricuspid atresia were seen during 1972-1982 and thirteen of these were not operated upon. The remaining 98 patients have undergone at least one operation. Those with low pulmonary blood flow had 1-4 shunt procedures; in 15 patients with high pulmonary blood flow the pulmonary artery was banded. The mortality rate after the first shunt was 10% (8/82), 9% after the second shunt (3/32) and 12% after the third and fourth shunts. Three out of 15 (20%) patients died after pulmonary artery banding. Twenty three patients underwent a Fontan operation and five died (22%); eighteen survivors are clinically well (after 1-7 years). One patient required a pacemaker implantation and revision of his Fontan procedure. The survivors of the palliative operations and the unoperated patients were more restricted, the older ones having developed pulmonary vascular obstructive disease, chronic heart failure or arrhythmias. PMID- 1708395 TI - Palliative care. Physical symptoms II. PMID- 1708396 TI - Biopsy resection of the external gland of the prostate by transurethral enucleation of the prostate for early detection of prostate cancer. PMID- 1708397 TI - Cultured gingival epithelium. A possible suitable material for pre-prosthetic surgery. AB - In cultivating gingival epithelium from people up to the age of 66 years it is possible to gain a differentiated mucosal tissue sheet that is more than 100 times the size of the original biopsy surface. The morphological characteristics are a prickle cell layer with polygonal cells and a basal cell layer with flattened cells. Desmosomes, tonofibrils and microplicae at the superficial side of the epithelium are typical features. There is a cell biological differentiation as the distinct binding of lectins and of a cytokeratin antibody proves. The multiplication in surface area, the possibility of using cells of senior patients and the morphological and cell biological differentiation of cultured gingival epithelium are advantages in its application for the autologous bridging of intraoral defects. PMID- 1708399 TI - Bone marrow involvement by Whipple bacillus. PMID- 1708398 TI - Safety and immunogenicity of Escherichia coli O18 O-specific polysaccharide (O PS)-toxin A and O-PS-cholera toxin conjugate vaccines in humans. AB - O-specific polysaccharide (O-PS) isolated from serotype 18 Escherichia coli lipopolysaccharide (LPS) was covalently coupled to either Pseudomonas aeruginosa toxin A (TA) or or cholera toxin (CT). The conjugates were nontoxic and nonpyrogenic. The conjugates were well tolerated on parenteral administration to human volunteers, with only mild, transient local reactions reported. Immunization engendered an IgG antibody response to both the O-PS and carrier protein. Anti-LPS antibody promoted the uptake and killing of an E. coli O18 strain bearing the K1 capsule by human polymorphonuclear leukocytes, which was complement dependent. Antibody to carrier protein neutralized the activity of native TA or CT in cell culture assays. Passively transferred IgG isolated from the serum of immunized donors provided a significant (P less than .01) degree of protection against fatal experimental E. coli O18 sepsis in mice. This study illustrates the potential use of such conjugates as vaccines against E. coli extraintestinal infections. PMID- 1708400 TI - Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by the dipyridodiazepinone BI-RG-587. AB - The dipyridodiazepinone human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitor BI-RG-587 was tested for its ability to inhibit HIV-1 replication in both acutely and chronically infected cell lines. The ability of BI-RG-587 to inhibit steps in the virus replicative cycle other than reverse transcription was also assessed. BI-RG-587 was found to be a potent inhibitor of HIV-1 replication in acutely infected cells (50% inhibitory concentration [IC50] = 37.2 nM), and the sensitivity and kinetics of that inhibition was similar to the known RT inhibitor zidovudine (AZT). Even at 100x IC50, BI-RG-587 had no effect on gp120/CD4 interaction, syncytia formation, or envelope glycoprotein processing. In addition, no inhibition of viral replication or protein production was noted in a chronically infected cell line that produces viral products in an RT-independent manner. Finally, no inhibition of acute HIV-2 replication was noted, even with very high (2500x IC50 for HIV-1) concentrations of BI-RG-587. These results demonstrate that BI-RG-587 is a potent inhibitor of HIV-1 replication and that this inhibition occurs at the point of reverse transcription. PMID- 1708401 TI - Detection of polyadenylated RNA in hepatitis B virus-infected peripheral blood mononuclear cells by polymerase chain reaction. AB - Polymerase chain reaction (PCR) with a reverse transcriptase step characterized a specific transcription activity in hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) in two patients (1 and 2) with chronic hepatitis positive for antibody to hepatitis B core antigen (HBc). Patient 1 was also coinfected with human hepatitis delta virus. A patient who cleared HBV replication after antiviral treatment with vidarabine served as negative control. HBV-specific RNA poly A sequences were detected by PCR in PBMC of patients 1 and 2 even without detectable HBV DNA (patient 2) as shown by dot blot and PCR assays. RNA sequences were found in both the nucleus and cytoplasm. The demonstration of HBV mRNA sequences within PBMC suggests the transcription of viral DNA, in agreement with the findings of HBV surface antigen in PBMC. The results in patient 1 demonstrated HBV mRNA sequences in leukocytes even without PCR-detectable HBV DNA sequences, likely due to ongoing hepatitis delta virus replication. PMID- 1708402 TI - Submandibular endodermal sinus (yolk sac) tumor in a female infant. A case report. AB - A submandibular endodermal sinus (yolk sac) tumor (EST-YST) is reported. The patient was a 1.5-year-old girl exhibiting a rapidly growing tumor in the submandibular region. The lesion showed the typical microscopic features including Schiller-Duval bodies and colloid bodies. Alpha-fetoprotein immunoreactivity was expressed by most tumor cells. There was no clinical or radiological evidence of the presence of this tumor elsewhere in the body. PMID- 1708403 TI - Serotonin and 5-hydroxyindoleacetic acid in plasma. Potential use as peripheral measures of MAO-A activity. AB - We have explored the possibility of using measures of 5-HT (extracellular 5-HT and 5-HIAA in blood) as peripheral indexes of MAO activity. Depressed patients treated with phenelzine for 6 weeks display a dramatic increase of plasma 5-HT (270% of basal values) together with a decrease of plasma 5-HIAA. After treatment with irreversible MAO inhibitors, rat plasma 5-HIAA decreased in a similar fashion than MAO-A activity in liver, lungs and brain, measured using 5-HT as substrate. These results support the use of plasma 5-HT and 5-HIAA in humans to evaluate the action of MAOI on the serotonergic system. PMID- 1708404 TI - Effects of L-deprenyl and amantadine in an MPTP-model of parkinsonism. AB - Mongolian gerbils of both sexes received a single daily dose of 40 mg/kg of 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over 4 consecutive days. On the fifth day the animals were treated with 15 mg/kg i.p. of L-deprenyl or amantadine or the combination of both drugs. At different time intervals (1, 2, 5 hours) the animals were sacrificed. In the caudate nuclei dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) were measured by an HPLC technique. MPTP affected the dopaminergic (HVA -25%) as well as the serotoninergic system (5-HT 54%, 5-HIAA -31%). L-deprenyl and amantadine accumulated DA and 5-HT in the MPTP affected caudates. Synergistic effects of the drug combination could be proven. PMID- 1708405 TI - New proazulene guaianolides from Thapsia villosa. AB - Four new guaianolides 2-5 possessing the terpenoid skeleton of archangelolide [6] were isolated from a specimen of Thapsia villosa [chromosome number 2n = 66 (= 6x)]. Attempts to hydrolyze the natural products 2-6 yielded azulenes, mainly 7 acetyl-1,4-dimethylazulene [9]. PMID- 1708406 TI - Alterations of somatostatin and its modulation by levodopa in MPTP-treated mouse brain. AB - We examined the changes in the concentrations of neuropeptides in various regions of the mouse brain 1, 2 or 6 weeks after 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) treatment (30 mg/kg i.p. twice/day for 5 days) and further examined the effects of levodopa injections (200 mg/kg i.p.) for 14 days starting 4 weeks after MPTP treatment on regional somatostatin (SRIF) concentrations. Substance P, cholecystokinin-octapeptide and thyrotropin releasing hormone did not show any significant changes up to 6 weeks after MPTP treatment, whereas the SRIF concentration increased 1 week after MPTP treatment but decreased thereafter, showing a marked decrease in the striatum and hippocampus after 6 weeks. In the striatum, the decreased concentration of SRIF recovered to the normal level with levodopa injections. This SRIF depletion could be a change secondary to dopamine depletion. On the other hand, in the cerebral cortex, while showing no change in the SRIF concentration after MPTP treatment, the concentration decreased significantly with levodopa injections. In the hippocampus, the decreased SRIF levels were still low after levodopa treatment. Since it has been reported that SRIF concentrations are significantly reduced in the frontal cortex and hippocampus of demented parkinsonians and patients with senile dementia of the Alzheimer type and that levodopa treatment induced various psychiatric side effects, the results of the present study suggest some relationship among levodopa treatment, SRIF depletion and the demented state. PMID- 1708407 TI - Acute reduction and long-term improvement of chorea with ceruletide (cholecystokinin analogue). AB - We studied the effects of ceruletide, a cholecystokinin analogue, on choreic involuntary movement in several neurological diseases by clinical scoring and electromyography in 11 patients. Ceruletide brought about a brief reduction of choreic movement reaching its maximum within 60 min and another long-lasting improvement over several weeks by single administration. The levels of homovanillic acid in cerebrospinal fluid before treatment were significantly higher in cases with long-lasting improvement than those in cases without improvement. We suggest that ceruletide may reduce choreic movement for a long period through effects on the central dopamine system and speculate that such a long-term effect may be accounted for by the change in transmission after the second messengers in neurons. PMID- 1708409 TI - Internalization of plasma proteins by cerebellar Purkinje cells. AB - Animal studies suggest that Purkinje cells internalize proteins from the blood and CSF. This process may relate to the pathogenesis of paraneoplastic cerebellar degeneration in patients with anti-Purkinje cell antibodies. To determine if human Purkinje cells may also internalize plasma proteins, cerebellar tissue was taken from routine autopsies of eight patients without neurologic or neoplastic disease. Several plasma proteins including IgG, IgA, IgM, transferrin, albumin and alpha-2-macroglobulin were detected by immunohistochemistry within the cytoplasm of Purkinje cells. Internalized proteins frequently filled the entire soma and major dendrites, sparing the nucleus. Vascular structures were also immunolabeled, while glia internalized plasma proteins differentially, with oligodendrocytes selectively internalizing transferrin. Purkinje cells were the most numerous and heavily labeled neuronal cell type in spite of their small numerical representation in the cerebellar neuronal population. Our results are compatible with previous animal studies, and suggest that internalization of specific antibodies could contribute to the pathogenesis of Purkinje cell loss in paraneoplastic cerebellar degeneration. PMID- 1708408 TI - Immunoglobulins within the central nervous system in primary Sjogren's syndrome. AB - Cerebrospinal fluid (CSF) and sera from 17 patients with primary Sjogren's syndrome (PSS) with or without clinical evidence of nervous system involvement were studied. Intrathecal IgG synthesis as measured by oligoclonal IgG bands on agarose isoelectric focusing or elevated IgG index in CSF was found in 6 of 8 patients with clinical nervous system involvement but also in 5 of 9 patients without clinical nervous system involvement. Elevated IgM-index in CSF was found in 7 of 8 patients with clinical nervous system involvement and in 6 of 9 patients without clinical nervous system involvement. By immunoblotting, CSF IgG antibodies against myelin basic protein (MBP) were found in 3 of 12 patients with multiple sclerosis (MS), but in none of the patients with PSS or in the 12 controls. Intrathecal anti-viral IgG-antibodies, as measured by immunoblotting against measles, mumps, varicella or herpes simplex, were found in 8 of 17 patients with PSS, and in 7 of 12 patients with MS, but were not detected in the controls. Our observations support the concept that the central nervous system (CNS) is included in the multiple immunological phenomena of PSS. Interestingly, in some PSS patients intrathecal IgG synthesis occurred without overt clinical nervous system involvement and thus the clinical significance of intrathecal IgG synthesis in PSS is uncertain. The similarities with MS regarding intrathecal antiviral antibody production may be interpreted as the result of polyclonal B cell activation. PMID- 1708410 TI - Keeping up. PMID- 1708411 TI - First lobbyist. PMID- 1708412 TI - For whom the PAC toils. PMID- 1708413 TI - Dentists shaping their own future. PMID- 1708414 TI - Unexpected effects of legislation. PMID- 1708415 TI - Issues on the horizon. PMID- 1708416 TI - Proper or premature? PMID- 1708417 TI - 1990 USC esthetic dentistry symposium. PMID- 1708418 TI - Achieving esthetic ceramic restorations. PMID- 1708419 TI - What do you say before you say hello? PMID- 1708420 TI - Hygiene maintenance of dental implants. PMID- 1708421 TI - Jean Savage. PMID- 1708422 TI - Cleft lip and palate: developmental effects. AB - Children with cleft lip and/or palate are at risk for societal disapproval and/or discrimination and emotional adjustment problems due to facial disfigurement, speech impairment, and learning difficulties. This article reviews the potential psychosocial problems from a developmental perspective and offers suggestions as to the role of nurses at each developmental stage. Nurses may be prone to the same negative attitudes and biases as the general population and may engage in subtle discrimination against children with cleft lip and/or palate. PMID- 1708423 TI - Effect of atrial natriuretic factor and 8-bromo cyclic guanosine 3':5' monophosphate on [3H]acetylcholine outflow from myenteric-plexus longitudinal muscle of the guinea pig. AB - We report that atrial natriuretic factor (ANF) inhibits electrically induced cholinergic twitches of longitudinal muscle in whole intestinal segments and myenteric-plexus longitudinal muscle (MPLM) strips from the guinea pig ileum. To elucidate the possible presynaptic mechanism of ANF's action, we studied spontaneous and stimulation-evoked radiolabeled acetylcholine (ACh) outflow from MPLM after incubation with [3H]choline. We developed a method of mounting and treating MPLM preparations, which allowed us, at the same time, to record isometric contractions and to determine [3H]ACh outflow upon electrical stimulation by a train of three pulses. ANF (5 x 10(-8) M), norepinephrine (2 x 10(-7) M) and 8-bromoguanosine 3':5'-cyclic monophosphate (10(-3) M) in nearly equieffective concentrations caused a similar inhibition of cholinergic twitches. However, ANF had no effect on [3H]ACh outflow, whereas norepinephrine was found to suppress [3H]ACh outflow and 8-bromoguanosine 3':5'-cGMP to enhanced [3H]ACh outflow. ANF (5 x 10(-8) M) caused a 7.0-fold increase of cGMP over control values, predominantly in muscle layers, whereas Escherichia coli heat-stable toxin (12.5 U/ml) elicited a 35-fold increment of cGMP in the extramuscular layer. Thus, ANF is able to elevate cGMP in intestinal smooth muscle and to inhibit cholinergic contractions of MPLM. This inhibition is mimicked by exogenous cGMP and by endogenously generated cyclic nucleotides. We suggest that the depressive action of ANF on cholinergic contractions of MPLM is mediated via its postsynaptic impact implicating elevation of cGMP in smooth muscle. PMID- 1708424 TI - Release and metabolism of 5-hydroxytryptamine in the cat spinal cord examined with microdialysis. AB - The primary objective of this work was to investigate how in vivo administration of depolarizing agents increases extracellular concentrations of 5 hydroxytryptamine (5-HT) and simultaneously decreases levels of its chief metabolite 5-hydroxyindole-3-acetic acid (5-HIAA). To this end the effects of a number of agents on extracellular levels of 5-HT and 5-HIAA in cat spinal cord were determined by microdialysis and high-pressure liquid chromatography with electrochemical detection. The semipermeable portion of the dialysis probe was centered in the dorsal horn. Artificial cerebrospinal fluid containing elevated K+ or drugs was introduced into the dorsal horn via the dialysis probe. Increases in recovered 5-HT were assumed to reflect increased levels in the extracellular fluid due to release, reduced reuptake and/or reduced metabolism. Depolarizing agents (K+, veratridine and aconitine) increased 5-HT, but dramatically decreased 5-HIAA levels. Extracellular 5-HT returned to near base-line levels with removal of the depolarizing agents. The reduction in 5-HIAA was transient after potassium infusion, but outlasted the infusion of veratridine or aconitine by several hours. Reduction of 5-HIAA could be blocked or reversed with the Na+ channel blockers, lidocaine and tetrodotoxin. Evidently, the decrease in 5-HT metabolism followed activation of sodium channels. Parachloroamphetamine caused an increase in 5-HT levels, presumably reflecting increased 5-HT release, but did not change 5-HIAA levels. Although fluoxetine increased 5-HT, it reduced 5-HIAA, presumably through blockade of 5-HT reuptake leading to a secondary reduction in metabolism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708425 TI - A prostacyclin analog impairs the response to tissue-type plasminogen activator during coronary thrombolysis: evidence for a pharmacokinetic interaction. AB - Inhibition of thromboxane (TX) A2 with aspirin enhances the response to coronary thrombolysis. However, experimental evidence suggests that platelet activation during coronary thrombolysis is mediated by a number of agonists, in addition to TXA2. As a consequence, greater benefit would be expected with antiplatelet agents that have a broader spectrum of activity. However, a recent clinical trial, combining tissue plasminogen activator (t-PA) with the prostacyclin analog, iloprost, did not detect such a benefit. To address the mechanism of this response, we compared the effect of iloprost, a stable analog of prostacyclin, with GR32191, a TXA2/prostaglandin endoperoxide receptor antagonist, on the response to i.v. t-PA in a closed chest, canine model of coronary thrombosis. GR32191 reduced the time to reperfusion by 47% (n = 6, P less than .05), consistent with a role for TXA2-mediated platelet activation in impairing thrombolysis. In contrast, iloprost increased the time to reperfusion by 50% (n = 5, P = NS) and in four of nine animals reperfusion failed to occur despite inhibition of platelet aggregation. In a separate series of experiments, steady state plasma t-PA clearance increased by 38% (407 +/- 49 vs. 294 +/- 42 ml/min; n = 8, P less than .02) during infusion of iloprost and recovered after its withdrawal. This appeared to be a specific effect, as infusion of nitroglycerin at a dose which induced a similar fall in blood pressure altered neither the time to reperfusion nor plasma t-PA. Iloprost impairs the thrombolytic response to t PA via an increase in the clearance of this agent. PMID- 1708426 TI - Altered inotropic responses in diabetic cardiomyopathy and hypertensive-diabetic cardiomyopathy. AB - To understand the mechanisms of diabetic cardiomyopathy and the consequences of combined hypertension and diabetes, cardiac tissue responses to various inotropic agents were measured in experimental diabetes. Streptozotocin was injected into Wistar rats, spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs). Six weeks after the injection diabetic rats showed a subsensitivity to beta adrenergic stimulation in ventricular tissue and a supersensitivity and hyper responsiveness to Ca++ and alpha adrenergic stimulation (except in WKYs) in ventricular tissues and left atria. A supersensitivity to BAY K 8644 in SHR left atria and a hyper-responsiveness to verapamil in ventricular strips were also noted. These alterations may be due to a change in receptor number or to postreceptor alterations. Diabetic SHRs exhibited greater changes in several of the drug responses (responses to isoproterenol, phenylephrine and BAK 8644) were more hyperlipidemic and had a high mortality as compared with Wistar rats and WKY diabetics. These findings confirm that the combination of hypertension and diabetes results in greater cardiac pathology than is seen with either disease alone. PMID- 1708427 TI - Enhancement of glucose transport in clone 9 cells by exposure to alkaline pH: studies on potential mechanisms. AB - Incubation of a nontransformed rat liver cell line, Clone 9, at pH 8.5 resulted in an approximately 16-fold stimulation of cytochalasin B-inhibitable 3-O methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells "depleted" of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K1 approximately 10(-7) M). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated an approximately 2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane. PMID- 1708428 TI - Evidence for permanent water channels in the basolateral membrane of an ADH sensitive epithelium. AB - The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (Pd) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by "perforating" the apical membrane with amphotericin B. The addition of this antibiotic increased Pd from 1.12 +/- 0.10 x 10(-4) cm/sec (n = 7) to 4.08 +/- 0.33 x 10(-4) cm/sec (n = 7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl2 (10(-3) M) decreased Pd by 52% and parachloromercuribenzoic acid (pCMB) (1.4 x 10(-4) M) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52 +/- 0.23 kcal/mol (n = 3), in the control situation, to 9.99 +/- 0.91 kcal/mol (n = 4) in the presence of 1.4 x 10(-4) M pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders. pCMB (0.5 or 1.4 x 10(-4) M) was found to inhibit water exit by 91 +/- 2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present. PMID- 1708430 TI - [Characterization of the factor VIII inhibitor in a patient with chronic renal failure]. AB - A 29-year-old women, who had been treated by hemodialysis for 5 years because of chronic renal failure, developed bleeding tendency in March 1989. Laboratory data showed prolonged activated partial thromboplastin time which was not corrected by addition of normal plasma; factor VIII activity was less than 1% and factor VIII inhibitor 70 Bethesda units/ml. The inhibitor was eluted in the second peak which corresponded to IgG when the plasma was subjected to Sephacryl S 200 column. The further purified IgG fraction by passing through protein A column showed a factor VIII inhibitor activity of 52 Bethesda units/ml. The factor VIII inhibitor epitopes were examined by western blotting technique using factor VIII purified by monoclonal antibody as the antigen. The factor VIII preparation used was composed of a doublet of light chain (Mr 80,000) and three heavy chains (Mr 160,000-200,000) when examined by immunoblotting using anti-factor VIII light and heavy chains monoclonal antibodies after SDS-PAGE. Factor VIII inhibitor that arose in a hemophilia A patient recognized the light chain, and the inhibitor in this case reacted to the heavy chain of factor VIII. PMID- 1708429 TI - Fluid resuscitation with deferoxamine prevents systemic burn-induced oxidant injury. AB - We studied the effect of deferoxamine (DFO) infused after burns on hemodynamic stability as well as local and systemic inflammation and oxidant-induced lipid peroxidation. Eighteen anesthetized sheep were given a 40% of total body surface burn and fluid resuscitated to restore oxygen delivery (DO2) and filling pressures to baseline values. Animals were resuscitated with lactated Ringer's (LR) alone or LR plus 1,500 ml of a 5% hetastarch complexed with DFO (8 mg/ml). Animals were killed 6 hours postburn. The sheep resuscitated with LR and LR plus hetastarch demonstrated significant lung inflammation and significant increases in lung and liver malondialdehyde (MDA) from controls of 47 +/- 6 and 110 +/- 7 nMol/gm to 63 +/- 13 and 202 +/- 59 for LR and 67 +/- 4 and 211 +/- 9 for LR + hetastarch, respectively. The group resuscitated with hetastarch alone required 15% less fluid. VO2 returned to baseline values in both groups by 2 hours. Resuscitation with the 5% hetastarch-DFO decreased total fluids by 30% over LR and prevented the increase in lung and liver MDA. In addition, postburn VO2 increased by 25% above baseline values. Burn tissue edema, measured as protein rich lymph flow, was significantly increased with the administration of DFO compared with the other groups. We conclude that DFO used for burn resuscitation prevents systemic lipid peroxidation and decreases the vascular leak in nonburn tissues while also increasing O2 utilization. Resuscitation with hetastarch-DFO may accentuate burn tissue edema, possibly by increased perfusion of burn tissue. PMID- 1708431 TI - [Effect of administration with chlorpyrifos on electroretinogram in rats]. AB - Changes in electroretinograms (ERG) due to administration of chlorpyrifos were examined in rats. Male Wistar rats were intraperitoneally injected with chlorpyrifos (0.2 mmol/kg body weight), and their cholinesterase (ChE) activities and ERG were measured at various times after the injection. Amplitudes of A and B waves in ERG were more than 50% decreased and latencies of the waves were prolonged 10-20% from 5 h to 2 d after the injection. At the same time, activities of ChE in plasma, erythrocytes, brain and retinochoroid were remarkably decreased. However, the amplitudes and latencies recovered to the control level more rapidly than the ChE activities of the erythrocytes, brain and retinochoroid. Injection into rats of 0, 0.002, 0.01, 0.05 or 0.25 mmol/kg of chlorpyrifos showed that the chlorpyrifos-induced changes of ERG were dose dependent, and that a level of 0.05 mmol/kg caused a 50% decrease in the A or B wave 5 h after the injection. These results indicate that chlorpyrifos caused abnormal ERG characterized by decreased amplitudes and prolonged latencies of A and B waves and that the abnormal ERG did not always correspond to the decreased retinochoroid or brain ChE activities. PMID- 1708432 TI - Comparative studies on fine-needle aspiration cytology with ultrasound scanning in the assessment of thyroid nodule. AB - The clinical usefulness of ultrasound scanning in the assessment of thyroid nodule was examined. The total diagnostic accuracy for aspiration biopsy cytology (ABC) in the differentiation of benign from malignant thyroid nodules was 92.4% in 26 patients, which is similar to that of previous reports when compared with the findings of pathological examination by surgery. Additionally, the diagnostic efficacy of high resolution ultrasonography was evaluated based on the results of ABC. When the echogram of 90 patients with a thyroid nodule was divided into eight patterns according to Obara's classification, only 64.3% of the carcinoma exhibited the typical malignant pattern of ultrasonography and 21.4% of the carcinoma exhibited the ultrasonographically homogenous nodule with a clear margin, which was often observed in the benign nodules. Therefore, it is necessary to be very careful when differentiating malignant from benign thyroid nodules by ultrasonography. In conclusion, although high resolution ultrasonography provides useful information for the assessment of most thyroid nodules, all ultrasonography (including high resolution ultrasonography) should be combined with ABC for the final diagnosis. PMID- 1708433 TI - Interleukin-4 as a growth regulator of clonogenic cells in acute myelogenous leukemia in suspension culture. AB - Using two complementary culture systems, suspension and clonal cultures, and with a method of graphic display (star diagram), we studied the effects of recombinant human interleukin-4 (IL-4) on leukemic stem cell renewal and differentiation in acute myelogenous leukemia (AML). The interactions between IL-4 and other recombinant human cytokines, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF) and interleukins-1 alpha, -2, -3, -5, and -6 were also studied. IL-4 alone had significant effects on both self-renewal and differentiation of blast progenitors in some cases; in clonogenic assay, IL-4 stimulated blast colony formation and in one case IL-4 was the most powerful stimulator among the nine growth factors tested. Star diagrams, constructed using the data from both suspension and clonal cultures, showed that IL-4 could influence the balance between self-renewal and differentiation of clonogenic cells. Negative and positive interactions were detected between IL-4 and other cytokines in suspension culture. These results indicate that IL-4 is a cytokine with a potential role in regulating the growth of myeloid leukemic stem cells, and that IL-4 may be useful in treating selected AML patients. PMID- 1708434 TI - Childhood undifferentiated leukemia with early erythroid markers and c-myb duplication. AB - Ultrastructural, flow cytometric, and molecular studies were performed on leukemia cells from bone marrow and pleural effusion of a 6-year-old boy diagnosed with undifferentiated (MO) leukemia, using routine histology and immunostains at diagnosis and relapse. Ultrastructurally, surface and/or intracellular ferritin particles were present on or in some blasts and the majority of blasts contained identifiable acid ferrocyanide reactive inorganic iron comparable to that seen in normal early erythroblasts. The cells lacked other evidence of differentiation, including diaminobenzidine-reactive or immunoreactive hemoglobin. Flow cytometric analysis of malignant cells showed a lack of lymphoid or myeloid markers. Anti-transferrin receptor antibody was positive on 93% of cells and antibody to glycophorin A reacted with 23% of cells. RNA blot analysis of leukemia cells with myeloperoxidase (MPO) showed an absence of appreciable levels of MPO mRNA. Chromosome analysis showed 51,XY, t(1;16)(p31;q24), +6, +10, +15, +19, +21. The oncogene c-myb, which is specifically expressed and regulated in hematopoietic cells and produces a DNA binding protein responsible for myeloid differentiation, was found to be duplicated in the patient's tumor cells. Expression of c-jun, N-ras, c-myc, and p53 was normal. The data indicate that the malignant cells in this patient are of early erythroid lineage at diagnosis and relapse and that classification of cell lineage can be enhanced by ultrastructural Prussian blue staining. The failure of this otherwise undifferentiated leukemia to express or evolve into a myeloid phenotype is biologically and clinically distinct from previously described cases of erythroid and myeloid leukemia and may represent a previously unidentified phenotype which should be included in the spectrum of 'undifferentiated' childhood leukemia. PMID- 1708435 TI - Therapy of myelodysplastic syndromes: advances and perspectives. Innsbruck, Austria, October 7-10, 1990. PMID- 1708436 TI - Stimulation of fatty acid oxidation in myocytes by phosphodiesterase inhibitors and adenosine analogues. AB - The effect of various phosphodiesterase inhibitors, and adenosine analogues on palmitate oxidation, were studied in isolated rat myocytes. Enoximone, milrinone, and dipyridamole, at a concentration of 250 microM, stimulated palmitate oxidation by 78%, 40%, and 43%, respectively. The specific A1-agonist, N6 cyclopentyladenosine, increased palmitate oxidation by 56%, at a concentration of 250 microM. Moreover, the nucleoside transport inhibitor, S-(P-Nitrobenzyl-)6 thioinosine, increased palmitate oxidation by 40%, at a concentration of 100 microM. These data suggest that the stimulation of palmitate oxidation by enoximone and adenosine analogues may be mediated via the inhibition of the uptake and/or the oxidation of glucose in myocytes. PMID- 1708437 TI - Uncharged tRNA, protein synthesis, and the bacterial stringent response. AB - Uncharged tRNA has been shown in vivo to have an active role both in the stringent response, and in modulating the rate of translational elongation. Both of these effects appear to be mediated by codon-anticodon interactions on the ribosome. Although the involvement of uncharged tRNA in the stringent response was expected from in vitro experiments, it has only recently been confirmed in vivo. Inhibition of translation by cognate uncharged tRNA was not expected, and a model is proposed in which excess uncharged tRNA competes with charged tRNA (in ternary complex) for the 30S component of the ribosomal A site. When uncharged tRNA is in sufficient excess over charged tRNA, interaction of uncharged tRNA with the 50S component of the A site occurs as well, leading to a stringent response. The cell has a continuum of responses to decreasing aminoacyl-tRNA levels: in moderately limited conditions, the proportion of uncharged tRNA increases, and the translation rate is slowed; under more severe limitations, uncharged tRNA provokes a stringent response, with pleiotropic consequences for the cell. PMID- 1708438 TI - RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus. AB - Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7 kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease identified as having a general role in the chemical decay of E. coli mRNA. PMID- 1708439 TI - Induction of a phi C31 prophage inhibits rRNA transcription in Streptomyces coelicolor A3(2). AB - A lysogen of Streptomyces coelicolor A3(2) containing a thermoinducible mutant of the temperate phage phi C31 (phi C31 cts1) was used to obtain synchronous phage development. Filter hybridization experiments indicated a marked reduction in rRNA synthesis after prophage induction. S1 nuclease mapping showed that transcription from each of the four promoters of one rRNA gene set (rrnD) was reduced to approximately the same extent, and that inhibition required protein synthesis. Crude preparations of RNA polymerase from induced lysogens had enhanced transcribing activity for phi C31 DNA which was lost upon further purification. The purified preparations were unimpaired in their ability to transcribe from the rrnD promoters in vitro and apparently unchanged in polypeptide composition. The factor(s) responsible for stimulating phage transcription, and possibly for inhibiting rRNA synthesis, may have been separated from the enzyme during purification. PMID- 1708440 TI - Mapping of the msDNA operon in the chromosome of Escherichia coli B. AB - An msDNA operon, consisting of genes for msDNA and a reverse transcriptase, is present in Escherichia coliB and absent from E. coliK12. We have found that the msDNA operon is located on a DNA fragment, longer than 15kb, that is absent from E. coliK12. Using conjugation, P1 transduction, and nucleic acid hybridization between E. coliB and E. coliK12 strains, we have located the position of the msDNA operon on the E. coliB chromosome at a site that corresponds to minute 19 on the genetic map and to position 900 on the physical map of the E. coliK12 chromosome. PMID- 1708441 TI - [Application of microcarriers to Bacillus subtilis culture]. AB - Microcarriers, first applied to cell cultures. It was reported that microcarriers affected cell growth positively. In this study Cytodex I microcarriers were used. Because of their surface characteristics mammalian cells can easily attach to Cytodex I microcarriers and grow well. B. subtilis cells also attached to microcarriers and grew well. In this study Cytodex I microcarriers were used for productive bacterial growth. PMID- 1708442 TI - [Hematopoietic growth factors in the limelight. New actors on the pharmacotherapeutic stage]. PMID- 1708443 TI - CD16+ and CD56+ cells as markers of necrotizing and crescentic glomerulonephritis and active IgA glomerulonephropathy. PMID- 1708444 TI - Mechanism of the re-buildup phenomenon in moyamoya disease--analysis of local cerebral hemodynamics with intra-arterial digital subtraction angiography. AB - The authors investigated the mechanism of the re-buildup phenomenon on electroencephalogram in 14 patients of moyamoya disease with superficial temporal artery-middle cerebral artery anastomosis. Visualization of the lateral view of the common carotid angiography was performed with intra-arterial digital subtraction angiography (IA-DSA), using a 4/sec x 3 sec + 2/sec x 5 sec + 1/sec x 5 sec film sequence. The catheter tip was inserted into C5/6 level and 250 mgI/ml of iopamidol was used as the contrast agent; 6 ml in total was injected over 1.5 seconds. Circulation times of the common carotid artery (C3 portion)-ascending parietal vein (delta TTPs) and common carotid artery-internal cerebral vein (delta TTPD) were measured before hyperventilation (HV), immediately after HV, and 3 minutes after HV during pre- and postoperative periods. delta TTPD in the preoperative period was prolonged by HV and was normalized at 3 minutes after HV but delta TTPs were prolonged immediately after and 3 minutes after HV. In the postoperative period, however, these values did not change significantly immediately after and 3 minutes after HV. These findings indicate that delayed cerebral blood flow response to HV is a pathogenetic factor of the re-buildup phenomenon in moyamoya disease. PMID- 1708445 TI - Reconstruction for the vertebral artery stenosis at its origin--bypass surgery using a saphenous vein graft with external shunting. AB - Eight patients with vertebral artery (VA) stenosis at its origin were surgically treated by a new reconstructive technique. Two patients suffered vertebrobasilar transient ischemic attacks, and five had brainstem and/or cerebellar infarction. All patients had multiple stenotic lesions, including bilateral VA stenoses in one and unilateral VA stenosis plus contralateral VA occlusion in seven. They underwent a subclavian artery to VA bypass using a saphenous short vein graft with external shunting. Postoperatively, no patients had aggravated neurological symptoms, and angiography showed all bypasses to be patent. One had an episode of transient loss of consciousness 8 months postoperatively, but the others showed no episodes of ischemia during an average follow-up period of 2 years and 10 months. The results indicate that this procedure is safer and more useful than other surgical procedures. PMID- 1708446 TI - Follow-up study on ruptured aneurysms treated by wrapping. AB - The authors experienced 1903 cases of ruptured aneurysm between 1961 and 1986. Most cases were treated by clipping or ligating the aneurysmal neck. However, these procedures were impossible in 52 cases (2.7%), for which wrapping alone (21 cases), aneurysmal body clipping and wrapping (11 cases), or body ligation and wrapping (20 cases) was performed. We studied the long-term results in 44 of these 52 cases. Rerupture occurred in only two cases with incomplete wrapping. We concluded that complete wrapping, and wrapping of residual portions following aneurysmal body clipping or body ligation produce favorable results. PMID- 1708448 TI - Microsurgical thromboendarterectomy of the cavernous carotid artery--case report and surgical technique. AB - A 53-year-old male suffered a transient right hemiparesis and left monocular blindness. Angiography revealed 80% stenosis of the cavernous carotid artery. Microsurgical thromboendarterectomy was performed by a direct approach through Parkinson's triangle. During surgery, the carotid circulation was transiently trapped between the cervical and the supraclinoid segment and the trapped arterial lumen was irrigated with heparinized saline. Soft elastic lesion was easily removed. Cavernous carotid thromboendarterectomy through a direct approach is considered as a suitable operation for the solitary and localized stenotic lesions of the cavernous carotid artery, although this operation has not yet been reported to date. PMID- 1708447 TI - Cerebellar hemorrhage after supratentorial craniotomy--report of three cases. AB - We report on three cases of remote cerebellar hemorrhage after supratentorial craniotomy, which had much in common in their computed tomographic, operative, and clinical findings. We speculate that, when the patient is in the supine position, displacement of the cerebellum causes stretching of the superior vermian veins and their tributaries, resulting in tearing of these vessels. Postoperative cerebrospinal fluid overdrainage or massive air reflux into the cranial cavity through the drainage tube may accelerate this process. Meticulous management of the drainage system is necessary to prevent this postoperative complication. PMID- 1708449 TI - Intra-abdominal cyst following revision of ventriculoperitoneal shunt--case report. AB - An intra-abdominal cyst is a rare complication of ventriculoperitoneal (VP) shunt. A 19-year-old male was admitted complaining of abdominal pain and distension, dysuria, constipation, headache, and fever. He had undergone a VP shunt for obstructive hydrocephalus caused by a cerebellar astrocytoma 16 years earlier, and had received shunt revision twice, 5 years and 3 months earlier, respectively. Examination on admission revealed neck stiffness, early papilledema, a mass in the lower abdomen, and abdominal muscular guarding with rebound tenderness. Laboratory studies showed leukocytosis of the peripheral blood and pleocytosis of the cerebrospinal fluid (CSF). Abdominal ultrasonograms and computed tomographic scans demonstrated a cystic lesion. Under the diagnosis of meningitis and local peritonitis with an intra-abdominal cyst, we sistemically administered antibiotics and externalized the shunt. However, since the cyst fluid could not be aspirated through the abdominal catheter, it was exchanged with a flexible catheter under fluoroscopic control, according to Seldinger's method. A total of 400 ml of cyst fluid was drained. Staphylococcus epidermidis was detected in both the cyst fluid and the CSF. After meningitis subsided, repositioning of the abdominal catheter into the other side of the abdomen was performed but resulted in shunt malfunction and meningitis due to the same organisms. After meningitis again subsided, the VP shunt was converted to a ventriculoatrial shunt. The clinical course was uneventful thereafter. PMID- 1708450 TI - Unusual intra- and extracranial arteriovenous communication--case report. AB - The authors report an unusual case of arteriovenous communication between extracranial and intracranial vessels, accompanied by incidentally detected bilateral arachnoid cysts of the middle cranial fossa. A 52-year-old male was admitted with a sudden onset of headache, vomiting, and conjunctival hyperemia of the right eye followed by progressive chemosis and proptosis. He had undergone a craniotomy for hypertensive right putaminal hemorrhage 4 months previously. Angiography showed the main feeding artery to be the superficial temporal artery and the draining veins to be the superficial Sylvian veins and the basal vein of Rosenthal. Partial obstruction of the right cavernous sinus was also shown. At surgery, granulation tissue continued to the dura mater through the skull aperture of previous craniotomy and adhered to the underlying damaged cerebrum. The extremely unusual nature of the communication, the operative findings, and the atypical fistulous figures suggested that communication had occurred postoperatively via newly generated vessels in granulation tissue. PMID- 1708451 TI - Multicentric intracranial fibrous xanthoma--case report. AB - Intracranial fibrous xanthoma is extremely rare; only 11 cases have been reported so far. The authors report a case of multicentric intracranial fibrous xanthoma. Precontrast computed tomographic (CT) scans revealed a left frontal subdural mass, which had previously been diagnosed as a chronic subdural hematoma at another hospital. However, operation disclosed no hematoma but a granulomatous tumor. The biopsied specimen was histologically diagnosed as fibrous xanthoma. Postoperative postcontrast CT scans showed intense homogeneous enhancement at the clival and the left frontal regions, both of which appeared as iso- to low intensity areas on T1-weighted magnetic resonance (MR) images and as low intensity areas on T2-weighted MR images. She gradually recovered by conservative treatment but suddenly died of cerebral infarction. Autopsy revealed fibrous xanthomas in the left frontal and the clival regions. This is the first report of the use of MR imaging for intracranial fibrous xanthoma, and its features differ from those of common intracranial parenchymal tumors, MR imaging can be helpful in the diagnosis of this tumor. PMID- 1708452 TI - Primary pituitary carcinoma with spinal cord metastasis--case report. AB - The authors report a rare case of non-functioning pituitary carcinoma with spinal cord metastasis. A 37-year-old female presented with a 2-month history of right retro-orbital ache and vomiting. She had a pituitary adenoma removed 3 years prior to admission. Neuroradiologically, a mass lesion was demonstrated in the right middle cranial fossa. The tumor was removed through craniotomy and was histologically diagnosed as pituitary carcinoma. One week after the operation, tetraplegia developed and CT scans demonstrated a spinal canal lesion. Although the tumor was removed through C3-C7 laminectomies, she gradually deteriorated and died. At autopsy, a tumor was disclosed in the right temporal lobe and basal ganglia. Moreover, the tumor invaded into the middle cranial fossa and the parasellar region. PMID- 1708453 TI - Intracranial hemorrhage due to brain metastasis from hepatocellular carcinoma- case report. AB - The authors report a case of repeated brain metastases from hepatocellular carcinoma (HCC) in a 70-year-old male, who had underwent liver segmentectomy for HCC 5 years earlier. He developed intracerebral hemorrhage in the right parietal region, which was considered to be intratumoral because the metastatic tumor was detected in the same region. Total removal of the tumor and hepatic artery embolization followed by ethanol injection for recurrent HCC were performed. One month later, a metastatic tumor was discovered in the upper vermis and was totally removed. Both metastatic brain tumors were histologically verified as Edmondson grade 2 HCC. Four months later, multiple metastases to the left frontal region and the upper vermis occurred, and he died of pneumonia. Brain metastasis from HCC is rare; nine such cases have been reported in the literature, of which eight cases developed intracranial hemorrhage as in the present case. PMID- 1708454 TI - Immunohistochemical detection and correlation between MHC antigen and cell mediated immune system in recurrent glioma by APAAP method. AB - As part of an on-going clinical trial of immunotherapy for recurrent malignant gliomas, using alkaline phosphatase-anti-alkaline phosphatase method with monoclonal antibodies, we investigated the correlation between expression of the major histocompatibility complex (MHC) and the subpopulation of tumor infiltrating lymphocytes (TILs) in 38 glioma specimens (20 grade IV, 11 grade III, and 7 grade II) from 33 patients. Thirty specimens (78.9%) were positive to class I MHC antigen and 20 (52.6%) were positive to class II MHC antigen. The correlations between class I MHC antigen expression and the number of infiltrating T8 (p less than 0.01), and also between class II MHC antigen expression and the number of infiltrating T4 (p less than 0.05) were significant. We conclude that TILs are the result of immunoreaction (host-defense mechanism). 31.6% of specimens had perivascular infiltration of T cells. The main infiltrating lymphocyte subset in moderate to marked perivascular cuffing was T4. Our results may indicate that lack of MHC antigen on the glioma cell surface has a share in the poor immunogenicity in glioma-bearing patients. In addition, considering the effector/target ratio, the number of infiltrating lymphocytes against glioma cells was too small, so the immunological intervention seems to be essential in glioma therapy. Previous radiation therapy and chemotherapy, including steroid therapy, did not influence lymphocyte and macrophage infiltration. PMID- 1708455 TI - Addition computed tomography with stable xenon--special reference to ischemic cerebrovascular diseases. AB - Stable xenon (Xes) is used as a contrast agent because it freely diffuses to cerebral tissues through the blood-brain barrier. In this study, 2 axial levels for Xes enhancement analysis were selected from a baseline series of computed tomographic (CT) scans and 6 serial CT scans were obtained every 20 seconds for each scan level during the 240 seconds inhalation period of 30% Xes in 10 volunteer controls and in 52 patients with ischemic cerebrovascular diseases (ICVD). The serial CT scans were added and averaged in each pixel. This was used to make a new CT picture (addition CT scan). The CT scans before the Xes inhalation, the scan at the end of the Xes inhalation, and the addition CT scan were compared to see whether gray matter and ischemic areas could be differentiated from white matter. The addition CT scans could differentiate the three structures very well in both the acute and chronic stages of ICVD. This technique is thought to be a very simple and useful method to detect the small infarcted areas and low perfusion areas that cannot be visualized on precontrast CT scans. PMID- 1708456 TI - Hemodynamic evaluation before and after the STA-MCA anastomosis--with special reference to measurement of regional transit time with intra-arterial digital subtraction angiography. AB - Twenty-seven patients with minor completed and major stroke in the chronic stage underwent superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis. The regional cerebral blood flow (rCBF), using inhalation of stable xenon and computed tomographic scanning (Xes CT-CBF study), and the mode of transit time (MTT) in the MCA territory using intra-arterial digital aortography were measured. Activated rCBF and MTT was measured 20 minutes after the administration of acetazolamide (10 mg/kg) in 14 patients. Nineteen of the 23 patients with minor stroke (Group 1) showed immediate improvement in their neurological state within a few days of the operation, while four patients with minor stroke (Group 2) and four patients with major stroke (Group 3) showed no improvement. Based on the rCBF obtained with the Xes CT-CBF study, affected side rCBF/unaffected side rCBF and %f [(peak DSA number/affected side MTT)/(peak DSA number/unaffected side MTT)] were compared. There was a significant positive correlation. Affected side MTT in Group 1 was 6.41 +/- 1.16 sec, preoperatively, and significantly decreased to 5.13 +/- 0.91 sec after the operation. On the other hand, preoperative MTT in Group 2 was 4.40 +/- 0.81 sec and 4.76 +/- 0.89 sec, postoperatively. Preoperative %f in Group 1 was 0.514 +/- 0.143 and significantly increased to 0.739 +/- 0.154, postoperatively. Group 2 showed no change. Vasodilatory capacity with acetazolamide showed a marked improvement in Group 1, postoperatively. Our study indicated that if MTT is moderately lengthened, %f is moderately decreased, and vasodilatory capacity is impaired, in patients with minor ischemic stroke will benefit from STA-MCA anastomosis. PMID- 1708457 TI - Changes in coagulation and fibrinolysis and effects of ticlopidine and cisternal drainage in the acute phase following aneurysm rupture. AB - In this study, changes in blood coagulation and fibrinolysis in the acute stage of subarachnoid hemorrhage (SAH) from ruptured intracranial aneurysms were investigated, and the effects of the platelet aggregation inhibitor ticlopidine and cisternal drainage were evaluated. All of the 53 patients underwent early surgery, and 27 patients received ticlopidine postoperatively. Cisternal drainage was implemented in 15 cases of severe SAH treated after late 1986. Of the hematologic factors studied, the activated partial thromboplastin time, fibrin and fibrinogen degradation products, fibrinogen, and platelet aggregation rate were found to be abnormal in the acute phase of SAH, and the "intravascular factor" scale obtained was also noted to be significantly (p less than 0.01) related to the outcome. Ticlopidine was consistently effective in reducing platelet aggregation but had little effect on other blood parameters. Cisternal drainage resulted in a significantly (p less than 0.01) lower incidence of symptomatic vasospasm and higher rate of good outcome. These results suggest that ticlopidine therapy plus cisternal drainage is highly beneficial in SAH, and that coagulation and fibrinolysis data are useful in determining the prognosis. PMID- 1708458 TI - Tumors at the trigone of the lateral ventricle--clinical analysis of eight cases. AB - Tumors at the trigone of the lateral ventricles are relatively rare. The authors have operated on eight cases with trigonal tumors during a 10-year period. Four cases were true intraventricular tumors arising from the ventricular walls, consisting of two meningiomas, one cavernous angioma, and one choroid plexus papilloma. On the other hand, the remaining four cases were paraventricular tumors originating in the adjacent brain and consisted of three astrocytomas and one glioblastoma multiforme. Although these trigonal tumors were readily detected with computed tomographic (CT) scanning, differential diagnosis was difficult because of their similar appearances on CT scans. The initial symptoms were headache in seven, and the neurological examination revealed personality changes, choked disc, visual field defects, hemiparesis, etc., in four, and no deficits in the remaining four cases. All cases were operated on via superior or middle temporal gyrus incision, and the surgical results were good except for one case who died of postoperative brain edema. In the four cases with tumors located in the dominant hemisphere, two were left with sensory aphasia, dyslexia, dyscalculia, and hemianopsia which improved within 6 months. In these two cases, postoperative CT scans revealed cerebrospinal fluid retention with severe edema along the surgical route which disappeared spontaneously within 3 months. We consider that the temporal gyrus incision was the safest approach, even though the tumor was located in the dominant side. PMID- 1708459 TI - Diffuse low-density areas in white matter on CT scans after intracarotid ACNU infusion--report of three cases. AB - Since 1984, we have treated 11 malignant glioma patients with intracarotid infusion of ACNU [1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)- 3 nitrosourea hydrochloride] in addition to surgical removal and irradiation. We experienced three patients, who showed clinical manifestation of leukoencephalopathy and computed tomographic (CT) findings of diffuse low-density areas in the white matter on the side of ACNU infusion. Two of the three patients showed an additional CT finding of ring enhancement in the temporo-occipital region. The histological diagnosis of the first case was radiation necrosis, while that of the others was recurrent tumor with coagulation necrosis in the surrounding brain. Our experience suggests that intracarotid ACNU infusion increases the hazard of radiation necrosis, and the optimum dose and effective mode of administration should be evaluated. PMID- 1708460 TI - Dolichoectatic basilar artery treated by reducing hemodynamic stress--report of two cases. AB - Two cases of dolichoectatic basilar artery with a mass effect to the brainstem structure were treated by reducing hemodynamic stress. In one case, angiograms showed the dolichoectatic basilar artery creating a turbulent flow in the vertebrobasilar junction, and unilateral vertebral artery clipping in addition to posterior fossa decompression was performed. The other case, a combination with bilateral internal carotid artery occlusion, underwent bilateral superficial temporal artery-middle cerebral artery anastomoses. The possible surgical treatments of dolichoectasia are discussed. PMID- 1708461 TI - Subependymal giant cell astrocytoma associated with tuberous sclerosis: with special reference to cell kinetic studies--case report. AB - The authors report a case of subependymal giant cell astrocytoma associated with tuberous sclerosis in a 15-year-old boy. Computed tomographic scans showed a large intraventricular mass with peritumoral calcification and a cyst in the left lateral ventricle. Left dominant unilateral hydrocephalus was also revealed. Magnetic resonance images clearly demonstrated the lesion. The tumor was subtotally removed and a ventriculoperitoneal shunt was performed because of the hydrocephalus. The proliferation potential was assessed by measuring the bromodeoxyuridine (BUdR) labeling index employing the in vitro labeling method, and determining the deoxyribonucleic acid (DNA) content by flowcytometry. BUdR positive cells were found to be rare, and the DNA histogram demonstrated no evidence of high proliferative activity or aneuploidy. PMID- 1708462 TI - Primary intracranial melanoma--case report. AB - We present a rare case of primary intracranial melanoma in the right occipital region of a 76-year-old male. Magnetic resonance imaging showed an isointensity mass with shorter T1 and T2 relaxation times than those of the opposite hemisphere. A well-defined, dark black tumor was totally removed and histologically diagnosed as malignant melanoma. Eight months postoperatively, however, the tumor recurred and was excised again. He was doing well 1 year after the second operation without additional treatment. In our case, 1) no systemic melanomas were found in close clinical examinations; 2) there was a single nodular tumor attached to the leptomeninges; and 3) a favorable outcome was obtained by surgical treatment alone. These results are consistent with the diagnosis of primary intracranial melanoma. PMID- 1708463 TI - Choroid plexus hemangioma with port-wine nevus of the face: relationship to Sturge-Weber syndrome--case report. AB - The authors describe a case of choroid plexus hemangioma in a 49-year-old male. Computed tomographic scan showed an isodense mass at the trigone of the right lateral ventricle with homogeneous enhancement. He also displayed a port-wine nevus on the ipsilateral side of the face. At operation, the tumor was found not to adhere to the lateral ventricular wall but to be connected to the choroid plexus, and was colored similarly to the facial nevus. Histological examination showed a capillary hemangioma with many crowded capillaries. This case was not included in the category of Sturge-Weber syndrome but is thought to be closely related, considering the syndrome from the viewpoint of generalized neurocutaneous hemangiomatosis. PMID- 1708464 TI - The influences of hyperosmolality and synaptic inputs on galanin and vasopressin expression in the hypothalamus. AB - Galanin is a neuropeptide that is widely distributed throughout the rat central nervous system. It is co-localized with vasopressin in magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. Vasopressin biosynthesis is increased there by various hyperosmolar stimuli, including drinking 2% saline. We previously demonstrated that the chronically hyperosmolar Brattleboro rat has increased biosynthesis of galanin in the paraventricular and supraoptic nuclei. In this report we show using hybridization histochemistry that drinking 2% saline also increased galanin transcripts in these nuclei. We also demonstrate using hybridization histochemistry and immunohistochemistry that knife cuts that sever hypothalamo-hypophysial fibers transiently elevated galanin expression in the supraoptic nucleus ipsilateral to the lesion and depressed vasopressin expression ipsilaterally. Pituitary stalk transections also elevated galanin and decreased vasopressin transcripts. In addition, various knife cuts in the caudal hypothalamus were able to dissociate the expression of vasopressin and galanin, although co-localized and similarly affected by hyperosmolality in the supraoptic nucleus. Unilateral sagittal knife cuts that divided the posterior hypothalamus but avoided the hypothalamo-hypophysial pathway, as well as hemisections at the level of the premammillary area, resulted in ipsilateral elevations of galanin transcripts without significantly affecting vasopressin expression. These results indicate that independent intracellular signal transduction pathways exist for regulating expression of the two genes. PMID- 1708465 TI - Serotonin release from rat brain mast cells in vitro. AB - Mast cells are primarily localized in connective tissues, where they secrete numerous mediators. They have also been identified in the mammalian central nervous system on the basis of their histochemical and morphological properties, but their role there remains unknown. A perfusion system was used to investigate in vitro mediator release from rat brain mast cells. Compound 48/80, the classic mast cell secretagogue of connective tissue mast cells, induced dose-dependent and non-cytotoxic release of serotonin, histamine and beta-hexosaminidase from mast cells in the rat thalamus and hypothalamus, but not in the cerebellum which was used as a negative control. Detailed studies were performed on thalamic mast cells, which were identified on the basis of metachromasia with Toluidine Blue and Safranin-positive staining with the Alcian Blue/Safranin technique. Their secretion was characterized by: (a) parallel release of serotonin, histamine and beta-hexosaminidase; (b) lack of dependence on extracellular calcium; (c) susceptibility to inhibition by disodium cromoglycate; and (d) lack of lactate dehydrogenase release. These results indicate that the morphology and secretory characteristics of thalamic mast cells resemble those of connective tissue mast cells. The ability of brain mast cells to secrete their mediators is discussed in the context of their possible involvement in brain pathophysiology. PMID- 1708466 TI - Histochemical and ultrastructural characteristics of rat brain perivascular mast cells stimulated with compound 48/80 and carbachol. AB - Mast cells, known for their involvement in allergic reactions where they secrete numerous chemicals in response to immunoglobulin E and specific antigens, have recently been localized in the central nervous system. The function of these brain mast cells has remained speculative as they have not been the subject of any combined functional or detailed morphological studies. Here it is shown that these cells are primarily perivascular and stain metachromatically with Toluidine Blue, but red with Alcian Blue counterstained with Safranin, indicating that they contain proteoglycans quite similar to those of peritoneal, but not mucosal mast cells. Intracardiac administration of the classic mast cell secretagogue, compound 48/80, or the acetylcholine analog, carbachol, caused ultrastructural changes in brain mast cells consistent with secretion, but without exocytosis. However, it is known that the same concentration of carbachol has no effect on homogeneic peritoneal mast cells. These results indicate that brain mast cells share histochemical characteristics with serosal mast cells, but differ in their reactivity to secretagogues, and apparently in the mechanism of secretion. Their ability to respond to neurotransmitters and their distinct mechanism of secretion, which may be selective, expands their possible role in brain pathophysiology. PMID- 1708467 TI - Co-expression of neuropeptides in the cat's striatum: an immunohistochemical study of substance P, dynorphin B and enkephalin. AB - The expression of tachykinin-like and opioid-like peptides was studied in medium sized neurons of the caudate nucleus in tissue from adult cats pretreated with colchicine. Two methods, a serial thin-section peroxidase-antiperoxidase technique and a two-fluorochrome single-section technique, were applied. Quantitative estimates were made mainly with the peroxidase-antiperoxidase method. The numbers of neurons expressing substance P-like, dynorphin B-like, and enkephalin-like immunoreactivity were recorded in regions identified, respectively, as striosomes and extrastriosomal matrix. Striosomes were defined by the presence of clustered substance P-positive and dynorphin B-positive neurons and neuropil. Tests for the co-existence of enkephalin-like peptide and glutamate decarboxylase-like immunoreactivity were also made with the peroxidase antiperoxidase method. Co-expression of substance P-like and dynorphin B-like immunoreactivities was the rule both in striosomes and in the matrix. In striosomes, substance P-like immunoreactivity was found in 96% of dynorphin B immunoreactive neurons, and in the matrix 89% of dynorphin B-positive cells contained substance P-like immunoreactivity. Substance P/dynorphin B-positive neurons corresponded to over half (57%) of the neurons in striosomes but only 39% of the neurons in the matrix. Both in the matrix and in striosomes, about two thirds of all neurons (63% and 65%, respectively) were identified as enkephalin positive. Among all substance P/dynorphin B-positive medium-sized neurons, 76% also contained enkephalin-like antigen. The enkephalin-positive neurons characterized by triple peptide co-existence (enkephalin/substance P/dynorphin B) represented a mean of 63% of striosomal enkephalin-positive neurons (41% of all striosomal neurons) and 35% of matrical enkephalin-positive neurons (26% of all matrical neurons). Finally, nearly all enkephalin-positive neurons were immunoreactive for glutamate decarboxylase, and therefore probably GABAergic, but only about half the glutamate decarboxylase-positive population was enkephalin immunoreactive. These findings suggest that neuropeptides from three distinct precursors may be co-localized in single medium-sized neurons in the striatum, and that the differential patterns of co-expression of substance P-like, dynorphin B-like, and enkephalin-like peptides may confer functional specializations upon subpopulations of GABAergic neurons giving rise to the efferent projections of the striatum. The linked expression of substance P-like and dynorphin B-like peptides in single neurons both in striosomes and matrix suggests that some regulatory mechanisms controlling peptide expression apply regardless of compartment.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1708468 TI - Substance P-containing neurons in the pontomesencephalic tegmentum of the human brain. AB - We have employed immunohistochemical and computerized morphometric procedures to study substance P-containing neurons in the tegmentum of adult humans. An estimated 192,500 +/- 40,500 substance P-containing neurons were found in three main cytoarchitectural regions: the mesencephalic reticular formation, the central gray, and the pontine reticular formation. The morphology of the immunoreactive neurons varied according to the region in which they were found. On the basis of size alone two types of substance P-containing neurons, large and small, were readily distinguishable by eye and measurement. Within each of the three main regions it was possible to distinguish distinct subgroups using cell size, morphology and position. Large neurons were concentrated in the caudal midbrain (pedunculopontine tegmental nuclei), in the oral pontine reticular nucleus and in the lateral dorsal tegmental nucleus. In contrast, small neurons were concentrated in the rostral mesencephalic reticular formation (cuniform nuclei). Both small and large neurons were found in the midbrain and pontine raphe nuclei. In addition, small neurons were concentrated in discrete midline regions (the periaqueductal gray, the tegmental nuclei of the pontine central gray, and the interpeduncular nucleus). The findings suggest that the majority of neurons in the brainstem tegmental nuclei previously identified as cholinergic also contain substance P in humans. PMID- 1708469 TI - Localization of acetylcholinesterase positive neurons and substance P and enkephalin positive fibers by histochemistry and immunohistochemistry in the sympathetic intermediate zone of the developing human spinal cord. AB - Localization of acetylcholinesterase positive neurons and substance P and enkephalin fibers were studied by histochemistry and immunohistochemistry in the intermediate sympathetic zone of the spinal cords of 39 human embryos/fetuses from gestation ages five to 40 weeks. Acetylcholinesterase positive neurons were observed in the nucleus intermediolateralis pars principalis as early as the fifth week of gestation. By the ninth to 13th weeks of gestation, positive neurons were also seen in the nuclei intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis. Increase in amount of these positive acetylcholinesterase neurons was demonstrated till term. Substance P and enkephalin fibers were initially observed by the eighth gestation week in the intermediolaterlis pars principalis nucleus and positive fibers were then detected in the nucleus intermedialis pars funicularis as well as the nucleus intercalatus spinalis by the 14th week of gestation. By the 26th week of gestation, all the major nuclei intermediolateralis par principalis, intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis has substance P and enkephalin fibers. Initial demonstration of acetylcholinesterase positive neurons appeared to be at an earlier stage than that of our substance P and enkephalin positive fibers. PMID- 1708470 TI - Effect of insulin on serotonin, 5-hydroxyindole-acetic acid, norepinephrine, epinephrine and corticosterone contents in chick. AB - Pineal serotonin (5-HT), 5-hydroxyindole-acetic acid (5-HIAA), norepinephrine (NE), epinephrine (E) and adrenal corticosterone, NE and E contents were measured spectrofluorometrically 5, 15, 30 and 60 min after insulin treatment to induce hypoglycemic stress. In the pineal gland, hypoglycemic stress caused dramatic decrease of 5-HT, resulted in initial decrease followed by an increase of 5-HIAA and brought about changes of NE and E. Corticosterone content in the adrenal gland increased significantly following treatment with insulin. Adrenal NE and E contents plummetted to control value followed by quick resynthesis. The findings indicate that insulin-induced hypoglycemia greatly modulates the activities of pineal and adrenal glands. PMID- 1708471 TI - Tyrosine phosphorylation systems in Alzheimer's disease pathology. AB - Immunohistochemical techniques have been used to assess the distribution of phosphotyrosine-containing compartments in Alzheimer's disease (AD) pathology. Elevated levels of phosphotyrosine are apparent in the somatodendritic compartment of tangle-bearing neurons, in the neuritic plaque (NP) and in dystrophic neurites coursing through the neuropil. The only neuronal staining observed in non-AD tissue is in developing neurites. This suggests that some neuronal elements involved in AD pathology may be recapitulating a developmental profile or, alternately, that elevated phosphotyrosine levels may reflect a role for tyrosine kinase/phosphatase systems in the degeneration process directly. Cells in the neuritic plaque which strongly resemble microglia also contain elevated levels of phosphotyrosine compared to non-activated ramified microglia in the same tissue section. Thus, tyrosine phosphorylation systems may be involved in the response of microglia to degeneration in AD pathology. Implications of these results are discussed. PMID- 1708472 TI - Contralateral degeneration in the cat spinal trigeminal nucleus following unilateral retrogasserian trigeminal rhizotomy. AB - Retrogasserian trigeminal rhizotomy was used to study the central projections and patterns of degeneration in the spinal trigeminal nucleus (STN). At survival times of 3-20 days, reduced silver stains show extensive degeneration throughout the ipsilateral STN and in addition, well delineated degeneration was identified in the periobex region of the contralateral STN that varied with survival time. The results suggest that primary afferents may contribute to this contralateral projection. PMID- 1708473 TI - Acidic fibroblast growth factor-like immunoreactivity in brain of Alzheimer patients. AB - Localization of acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined in postmortem human brain tissue of Alzheimer and age-matched control cases using a rabbit polyclonal antibody specific for aFGF. In control cases, a small number of glial cells were stained very weakly in white but not in gray matter. In Alzheimer cases, some astrocytes were strongly stained for aFGF in both gray and white matter. These intensely staining cells were frequently observed surrounding senile plaques, but represented a small proportion of the total astrocytic population. The present study suggests that aFGF may be upregulated in areas of Alzheimer pathology. PMID- 1708474 TI - Human neuroblastoma cells treated with aluminium express an epitope associated with Alzheimer's disease neurofibrillary tangles. AB - A number of studies have implicated aluminium as a possible factor in the pathogenesis of Alzheimer's disease (AD). Following an examination of the uptake of aluminium by human neuroblastoma cells in culture, treated with a range of concentrations of aluminium complexed with ethylene-diaminetetra-acetic acid (EDTA), we have now carried out an immunocytochemical study. Using an antibody to phosphorylated tau protein, which reacts specifically with AD neurofibrillary tangles (NFT), we have found that after treatment periods of 16 days to 8 weeks with aluminium-EDTA, the cells show positive staining with this antibody. No such reaction was detected in cells grown in medium alone, nor in aluminium-EDTA treated cells subjected to the same immunocytochemical procedure but without added primary antibody. Cells grown in medium plus EDTA, which contains a low level of aluminium contamination, showed a slight reaction. Our system may provide a suitable model for studying the early changes which lead to NFT formation. PMID- 1708475 TI - Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections. AB - For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling. PMID- 1708476 TI - Distribution of nerve growth factor receptor immunoreactivity in the human hippocampus. AB - Nerve growth factor (NGF) receptor-like immunoreactivity has been demonstrated in the normal adult human hippocampus, using minimally fixed cryostat sections obtained from snap-frozen tissue and incubated with the mouse monoclonal antibody, ME 20.4. The majority of the reactivity was associated with nerve fibre processes and their terminals. Numerous fibres were apparent in the alveus, originating from the fornix, and extending into the stratum oriens and pyramidal layer of the hippocampal formation. A more diffuse particulate reactivity, presumed to be nerve terminal, was observed around the pyramidal neurons and in the stratum radiatum, stratum lacunosum moleculare and also in the dentate fascia. The pattern of hippocampal NGF receptor immunoreactivity was broadly similar to acetylcholinesterase histochemical localization, indicating a principal localization on cholinergic axons innervating this area. Preliminary observations indicate an overall reduction in NGF receptor-immunoreactive axons and terminals in old age and Alzheimer's disease. PMID- 1708477 TI - Craniovascular application of capsaicin activates nociceptive thalamic neurones in the cat. AB - In cats anaesthetized with alpha-chloralose and urethane, extracellular recordings were made in ventrobasal thalamus from cells responding to electrical stimulation of the superior sagittal sinus and middle meningeal artery. Capsaicin, but not vehicle, evoked an increase in the firing rate of nociceptive cells (5 of 6 wide dynamic range and the only nociceptive specific cell). Non nociceptive cells did not respond to either capsaicin or vehicle. Cells with long latencies to electrical stimulation were excited by capsaicin but cells with short latencies were not. Capsaicin-responsive cells were found in the ventroposteromedial nucleus and the medial nucleus of the posterior complex and mostly had receptive fields involving the first trigeminal division. PMID- 1708478 TI - Sensory innervation of the mystacial pad fur of the ferret. AB - The guard and vellus hairs on the muzzles of many mammalian species possess a complex sensory innervation. The observation in the primate that the sources of the endings may be segregated by afferent type to separate terminal nerve branches prompted us to examine the innervation of the mystacial fur on the ferret muzzle. Sections were prepared with reduced silver techniques and serially reconstructed for analysis. The types and orientations of sensory endings in the ferret are similar to those described for the primate. However, we found that any terminal nerve branch could be composed of the full set of afferent types and therefore, there was no consistent segregation. The implications of these results on the afferent to target specificity during development and on multiple cortical representations are discussed. PMID- 1708479 TI - Reduced NMDA receptor-ion channel function in the vitamin B-6 restricted neonatal rat brain. AB - We have measured the binding of [3H]-MK-801 to cortical and hippocampal membrane preparations from the vitamin B-6 restricted neonatal rat. The results indicate a reduced potency and efficacy of glutamate and glycine in enhancing [3H]-MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor-ion channel. Scatchard analysis of [3H]-MK-801 binding isotherms showed a significantly lower number of [3H]-MK-801 binding sites in the cortex and hippocampus of vitamin B-6 restricted rats. These results indicate a reduced function of the NMDA receptor-ion channel in the brain of these animals. PMID- 1708480 TI - Somatotopic distribution of corneal afferent neurons in the guinea pig trigeminal ganglion. AB - The size and somatotopic distribution of corneal afferent neurons in the guinea pig trigeminal ganglion were determined using a retrograde axonal tracing technique. Wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was applied to the central cornea of the guinea pig and the animals were perfusion-fixed 48 h later. In addition, a preliminary study examined corneal afferent neurons in two animals latently infected with the herpes simplex virus by corneal inoculation. The majority of WGA-HRP-labelled neurons were located in the ophthalmic division of the ipsilateral ganglion. A clear dorsoventral somatotopic arrangement of labelled corneal afferent neurons was noted. The size of the neurons averaged 23 microns and the number of cells per ganglion averaged 205. By contrast, the number of labelled neurons in latently infected ganglia averaged less than 50. No size or morphological distinctions could be made between neurons from uninfected or latently infected ganglia. The results of this study have provided for the first time the precise location and somata diameter of primary afferent corneal neurons within the guinea pig trigeminal ganglion. PMID- 1708481 TI - Immunohistochemical localization of aspartate in corticofugal pathways. AB - In the present study we used immunohistochemistry, with an antibody directed against aspartate (Asp), in conjunction with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) histochemistry to examine the Asp-containing neurons in the motor and somatosensory cortices of the rat that project to the caudate/putamen or the pons. Injections of WGA-HRP in the caudate/putamen labelled cells in layer V of both cortical areas. In the motor cortex, retrogradely labelled neurons were distributed throughout layer V; 59% of these cells were also Asp-immunoreactive. In the somatosensory cortex labelled neurons were found mainly in layer Vb; 57% of these cells were double labelled. A similar percentage of cells were double labelled in this cortical area following WGA-HRP injections into the pontine nuclei. These observations taken together with previous findings (Exp. Neurol., 105 (1989) 36-44) suggest that nearly equal proportions of neurons that give rise to the corticostriatal and corticopontine pathways may use either Asp or glutamate (Glu) as putative neurotransmitters. PMID- 1708482 TI - The possible involvement of tetrodotoxin-sensitive ion channels in ischemic neuronal damage in the rat hippocampus. AB - To determine the role of tetrodotoxin-sensitive ion channels in post-ischemic selective neuronal death, the effect of tetrodotoxin on ischemia-induced brain cell injury was studied in rats. The animals were subjected to 20 min of cerebral ischemia in a four vessels occlusion model. Thirty min before ischemia, tetrodotoxin at a dose of 10(-7) or 10(-6) M was topically applied into the hippocampal CA1 subfield. Morphological changes in the CA1 subfield were evaluated 7 days after ischemia and compared with those of a vehicle-injected group. The average cell density of CA1 pyramidal neurons ipsilateral to the injection (cells/mm, mean +/- S.E.M.) was 27 +/- 7 (n = 6) in the vehicle-treated group, and 56 +/- 13 (n = 6) and 83 +/- 17 (n = 6) in the group treated with tetrodotoxin at doses of 10(-7) and 10(-6) M, respectively. Tetrodotoxin mitigated the ischemic hippocampal neuronal damage in a limited but dose dependent manner. This suggests that activation of tetrodotoxin-sensitive ion channels might contribute to the process of the ischemic neuronal damage. PMID- 1708483 TI - A monoclonal antibody to common acute lymphoblastic leukemia antigen (neutral endopeptidase) immunostains senile plaques in the brains of patients with Alzheimer's disease. AB - We immunostained brain tissues of patients with Alzheimer's disease (AD) together with non-demented aged and younger controls with a battery of anti-human hemopoietic cell monoclonal antibodies (OK series, Ortho Diagnostics Co., Ltd. and some others) by the avidin-biotin-peroxidase complex (ABC) method to see if any epitopes are shared with the nervous system or might contribute to the neurodegenerative changes in this disease. One out of 29 monoclonal antibodies, OKBcALLa, which recognizes common acute lymphocytic leukemia antigen (CALLA, CD10), immunostained senile plaques in the brains of patients with AD. The pattern and intensity of this staining, using cryopreserved samples, was almost identical to that obtained with anti beta-protein. Thus, senile plaques in the Alzheimer's brain share an epitope with CALLA. PMID- 1708484 TI - [125I]SCH 23982, a new tool for rapid visualization of dopaminergic neurons in lower vertebrate retinas. AB - Incubation of turtle or Xenopus retinas in 0.1 nM [125I]SCH 23982, a dopamine D1 receptor antagonist for 30-45 min and subsequent fixation in paraformaldehyde/glutaraldehyde resulted in strong blue formaldehyde-induced fluorescence of inner retinal neurons. On the basis of their morphological features, the labeled cells were classified as dopaminergic cells, an identification which was confirmed by double-labeling experiments using an antiserum against tyrosine hydroxylase. The whole experiment can be conducted in less than 2 h (whole mount), the label is very stable and allows the use of high magnification objectives for detailed morphological investigation of dopaminergic retinal neurons. PMID- 1708485 TI - Effects of glucose on calcium channels in neural cells. AB - Hyperglycemia has been reported to alter outcome following experimental and clinical cerebral ischemia, but the mechanisms involved are incompletely understood. Since glucose influences the function of dihydropyridine-sensitive, voltage-gated Ca2+ channels in some non-neural cells, and since cellular Ca2+ overload has been implicated in the pathogenesis of ischemic neuronal injury, we examined whether glucose regulates Ca2+ channel function in a cultured neural cell line. Physiologic concentrations of glucose had no effect on free intracellular Ca2+ levels in PC12 cells, but 4-fold elevation of glucose above physiologic levels reduced the dihydropyridine-sensitive, depolarization-induced increase in Ca2+. This effect would not account for exacerbation of ischemic brain injury by hyperglycemia, but may contribute to attenuation of ischemic injury by glucose in certain settings. PMID- 1708486 TI - Dystrophic neurites around amyloid plaques of human patients with Gerstmann Straussler-Scheinker disease contain ubiquitinated inclusions. AB - Dystrophic neurites have been previously observed around prionic protein-derived amyloid plaques of Gerstmann-Straussler-Scheinker (GSS) disease. Ubiquitin (Ubq) immunohistochemistry reveals the presence of dot-like stainings around many of these plaques. In order to determine the nature of ubiquitinated deposits, we performed an immunogold electron microscope study on autoptic samples from the cerebellum of a GSS patient. Both pre- and post-embedding staining methods showed Ubq-positive dense bodies and filamentous structures, belonging to dystrophic neurites. They are analogous to ubiquitinated neuritic processes described around cerebellar amyloid plaques of Alzheimer's disease (AD). These results suggest that amyloid deposition is responsible for the degeneration of adjacent axon terminals in both AD and GSS. PMID- 1708487 TI - Light-dependent development and asymmetry of visual projections. AB - The projections from thalamus to visual Wulst in chicks are asymmetrical and their development is determined by exposure to light just before and after hatching. The asymmetry results from the orientation of the embryo in the egg, the left eye being occluded and not the right. We have shown that this asymmetry can be eliminated by incubating the embryos in darkness or light so that both eyes receive the same stimulation. PMID- 1708488 TI - Low affinity nerve growth factor receptor binding in normal and Alzheimer's disease basal forebrain. AB - The binding characteristics of radiolabelled beta-nerve growth factor ([125I]NGF) have been determined on membrane preparations of basal forebrain from Alzheimer's disease (AD) brain and age-matched normal brains. [125I]NGF binds in a specific fashion indicative of a single receptor and is not displaced with microM concentrations of cytochrome c, insulin or epidermal growth factor (EGF). The mean dissociation constant (Kd) and the mean capacity (Bmax) of the NGF receptor were not significantly different between the 5 AD and 5 normal basal forebrain samples examined. Choline acetyltransferase (ChAT) activity was significantly reduced (P less than or equal to 0.001) in AD cerebral cortical samples compared with normal tissue. PMID- 1708489 TI - Labelling of restricted numbers of axons by solid rhodamine implantation into nerve trunks. AB - Solid rhodamine B isothiocyanate microsurgically implanted into mammalian peripheral nerve labels a small number of axons within that nerve. These axons can be observed in whole mounts or slices following fixation of the tissue approximately 16 h after the rhodamine is applied. The labelling appears to involve axoplasmic transport in both directions, for axons can be followed over long distances in the whole mounts. Since only some axons are labelled, their structure can be observed within large populations of cells in a whole nerve. This is valuable in interpreting axon counts in developmental and regenerative studies, and in observing the pattern of sprouting at regenerating axon terminals. PMID- 1708490 TI - [Laser-induced occlusion of corneal blood vessels with variable emission and absorption]. AB - Laser light has repeatedly been proposed for the occlusion of corneal neovascularizations. Priority has to be given to minimize laser energy in order to limit side effects such as corneal burns. We investigate the influence of variable emission wavelength using a dye laser or dye-enhanced absorption with intravenous sodium fluorescein. Laser energy can be reduced using intravenous dyes at vessel diameters less than 50 microns. Permanent occlusion is achieved in 43% instead of 28% without dye-enhanced absorption. Vessels measuring greater than 50 microns in diameter can be occluded best at lowest laser energy levels when 575 nm laser light is used, which approximates the absorption maximum of hemoglobin. PMID- 1708491 TI - Autistic children: diagnosis and clinical features. AB - Autism is one of the behaviorally defined developmental disorders of brain function. It has a variety of genetic and nongenetic etiologies, with etiology being unknown in the majority of children. Boys are more frequently affected than girls. Manifest in the preschool years, autism always affects sociability, communication, and the child's repertoire of activities and interests. Autism encompasses children with a broad range of severities and a variety of other signs of brain dysfunction. These include motor signs, notably stereotypies; abnormal responses to a variety of sensory stimuli; and disorders of affect and attention. A significant proportion of autistic children experience epileptic seizures and have abnormal EEGs. Neuroimaging, preferably magnetic resonance imaging, discloses abnormalities of brain development in a minority of autistic persons. The level of intelligence may range from profound mental deficiency to giftedness. The pattern of cognitive skills is likely to be uneven, typically with better nonverbal than verbal skills. In the preschool years, all autistic children have a developmental language disorder. Verbal expression may range from total lack of language to verbosity with echolalia; comprehension and language use are invariably impaired. While there is no specific pharmacologic agent to mitigate the fundamental disorder, children may benefit from drugs to treat specific symptoms such as attention disorder and seizures. Although autistic behaviors are the consequence of a static disorder of brain function, their character changes with maturation and appropriate intervention. Communication skills and sociability remain deficient but improve in all but the most severely affected children. Outcome is a function of both innate cognitive competence and the effectiveness of early intervention focused on the development of appropriate social skills and meaningful communication. Intelligent autistic adults may be educable, employable, and able to live independently, while more severely handicapped ones require a lifelong protected environment. PMID- 1708492 TI - Etiology of autism: genetic influences. AB - In summary, there are a few specific genetic conditions that can be associated with autism. Among cases of unknown etiology, there is ample evidence for a higher genetic liability to autism in siblings of autistic probands than expected from the population prevalence. It appears likely that both parents and siblings have a higher liability for social and cognitive deficits that are milder but conceptually similar to those found in autism. Others factors may alter this underlying genetic liability such as sex, IQ, and prenatal and perinatal injury. In the future, genetic analyses and genetic linkage studies will need to consider using a broader definition of the autism phenotype to include not only autism but severe cognitive and social deficits. The exact genetic mechanisms and genes involved are the subject of current investigations by several research groups. Investigations in this area are likely to continue to provide important information about the causes of autism. PMID- 1708493 TI - Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity. AB - The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase. PMID- 1708494 TI - Cloning, expression and characterization of the human transcription elongation factor, TFIIS. AB - The cDNA for the human elongation factor, TFIIS, has been cloned and expressed in E. coli with the T7 expression system. This 280-amino acid TFIIS protein is shorter by 21 residues than that of the mouse. The missing 21 residues are located in the amino-terminal region, which is not thought to be required for transcriptional stimulation. Apart from this gap, human and mouse proteins reveal 96% overall identity and 98.5% sequence similarity if conservative substitutions are taken into account. The bacterially expressed human protein and the purified calf thymus proteins are indistinguishable in their ability to stimulate transcript elongation by purified RNA polymerase II. Estimation of the native molecular size of the human protein in solution indicates that it exists as a dimer. PMID- 1708496 TI - Complete nucleotide sequence of a 16S ribosomal RNA gene from Streptomyces griseus subsp. griseus. PMID- 1708495 TI - Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damage in vitro: homology with bacterial repair enzymes. AB - Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli. PMID- 1708497 TI - A new polymorphic probe on chromosome 3p: lambda LIB41-51 (D3S632). PMID- 1708498 TI - Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes. AB - The entire nucleotide sequences of the rice, tobacco and liverwort chloroplast genomes have been determined. We compared all the chloroplast genes, open reading frames and spacer regions in the plastid genomes of these three species in order to elucidate general structural features of the chloroplast genome. Analyses of homology, GC content and codon usage of the genes enabled us to classify them into two groups: photosynthesis genes and genetic system genes. Based on comparisons of homology, GC content and codon usage, unidentified ORFs can also be assigned to each of these groups such that it is possible to speculate about the functions of products which may be produced by these ORFs. The spacer regions and intron sequences were compared and found to have no obvious homology between rice and liverwort or between tobacco and liverwort. PMID- 1708499 TI - Prolonged nausea and vomiting associated with buprenorphine. AB - We conducted a trial to evaluate the histamine-releasing characteristics of morphine, meperidine, and buprenorphine when administered intravenously to 20 healthy adults without pain. Substantially more nausea and vomiting occurred with buprenorphine than with the other compounds, and was more intense on ambulation. High-affinity receptor binding may play a role in the long duration of nausea and vomiting after a single dose of this agent. PMID- 1708500 TI - Renal oncocytoma: diagnostic utility of cytokeratin-containing globular filamentous bodies. AB - Sixty-six renal cortical epithelial tumors were classified by light and electron microscopy into 18 oncocytomas and 48 renal carcinomas, and their pattern of cytokeratin and vimentin reactivity was evaluated by immunoperoxidase using paraffin-embedded tissue. We found by electron microscopy that most oncocytomas (11 of 15) contain globular filamentous bodies that consist of a complex of intermediate filaments and organelles. These structures were found to correlate on immunohistochemistry with a discrete punctate cytoplasmic pattern of cytokeratin reactivity, provided the antibody preparation contained specificity for cytokeratins 8 and 18. A similar punctate finding was not observed in four oncocytomas nor in the 48 renal carcinomas. Although 11 oncocytomas failed to express vimentin, seven tumors showed focal reactivity restricted to rare individual cells in areas of sclerosis (five tumors) or in cell clusters bordering central scars (two tumors). We conclude that many oncocytomas contain a potentially diagnostically useful punctate pattern of cytokeratin reactivity and that focal vimentin reactively may be observed in otherwise typical oncocytomas, restricted to tumor cells appearing to be undergoing atrophy. PMID- 1708501 TI - The immunophenotype of hemangiopericytomas and glomus tumors, with special reference to muscle protein expression: an immunohistochemical study and review of the literature. AB - Glomus tumors and hemangiopericytomas have traditionally been described as neoplasms of pericytes. Ultrastructurally, smooth muscle features have been identified in the cells of the glomus tumor, while the cells of the hemangiopericytoma have been described as more closely resembling normal pericytes. Immunocytochemical studies were performed to demonstrate the immunophenotype of these two tumors and to particularly evaluate expression of muscle-specific actin and desmin. Using the avidin-biotin immunoperoxidase method, formalin-fixed, paraffin-embedded tissue from 16 glomus tumors and 11 hemangiopericytomas was evaluated for the presence of vimentin, low-molecular weight cytokeratins (35 beta H11), muscle actins (HHF35), desmin (clone 33), S100 protein, nerve growth factor receptor (NGFR5), myelin-associated glycoprotein (CD57), Factor VIII-related antigen, and Ulex lectin. Muscle actins were found in 14 of 16 tumors, and desmin was found in three of 16 of the glomus tumors. None of the 11 hemangiopericytomas expressed either desmin or muscle actins. Variable numbers of both tumors were positive with antibodies to CD57, with the nerve growth factor receptor, and with antibodies to S100 protein. In conclusion, these studies provide immunocytochemical evidence of smooth muscle differentiation in glomus tumors. Although muscle differentiation has been identified in the normal pericyte by expression of muscle-specific actin (HHF35), we find no evidence for analogous differentiation in the population of cells comprising hemangiopericytomas. PMID- 1708502 TI - Image analysis for quantitation of estrogen receptor in formalin-fixed paraffin embedded sections of breast carcinoma. AB - Monoclonal antibody to human estrogen receptor (ER) provides a useful immunohistochemical tool for the evaluation of ER content in breast carcinoma, but visual interpretation is subjective. Computer-assisted image analysis has proved effective in immunohistochemical quantitation of ER in fresh tumor imprints and cryostat sections. We examined the usefulness of this technique in 5 microns-thick formalin-fixed paraffin-embedded tissue sections of 66 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis. Immunohistochemistry was automated and performed on a Code-on slide stainer (Instrumentation Laboratories, Lexington, MA) using Pronase predigestion, a monoclonal antibody (ER-ICA; Abbott, Chicago, IL), and a biotin-labeled secondary antibody. Detection was achieved with an avidin-alkaline phosphatase conjugate and nitroblue tetrazolium (NBT) bromochloroindoyl phosphate (BCIP) substrate. The immunohistochemical ER staining was analyzed visually and with the CAS/200 image analyzer (Elmhurst, IL). The visual semiquantitative histologic scores (HSCORE), the automated quantitative assays including the percentage of positive nuclear areas (PNA), and the quantitative immunocytochemical scores (QIC SCORE = PNA x % of positive stain/10) were compared with the corresponding DCC results. Linear correlations were demonstrated between all immunohistochemical assays and the logarithm of DCC, the strongest correlation seen with PNA (r = 0.91). Threshold points for positive HSCORE, QIC SCORE, and PNA assays were extrapolated using DCC as the reference. ER immunodetection by PNA as compared with visual examination alone was enhanced by 18% (up to 88%) in sensitivity and 34% (up to 94%) in specificity, and the DCC concordance rate increased by 26% (up to 91%). A comparative chart extrapolating DCC from PNA was thus established.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708503 TI - Dehydroandrographolide succinic acid monoester as an inhibitor against the human immunodeficiency virus. AB - Dehydroandrographolide succinic acid monoester (DASM) is the dehydroandrographolyl ester of succinic acid; and andrographolide, from which DASM is made, is the major diterpenoid lactone found in the Chinese medicinal herb, Andrographis paniculata. DASM has been found to be an inhibitor against the human immunodeficiency virus (HIV) in vitro. It was nontoxic to the H9 cell at the concentrations of 50-200 (average, 108) micrograms/ml and was inhibitory to the HIV-1 (IIIB) at the minimal concentration of 1.6-3.1 (average 2.0) micrograms/ml. It was also inhibitory to two other strains of HIV-1 and a strain of HIV-2. This inhibitory effect could also be demonstrated in cultures of activated human blood mononuclear cells; the 50% toxic dose and the 50% HIV inhibitory dose were about 200-greater than or equal to 400 and 0.8-2 micrograms/ml, respectively. At the subtoxic concentration, DASM partially interfered with HIV-induced cell fusion and with the binding of HIV to the H9 cell. Presumably, it also interfered with HIV replication at another unidentified step(s). PMID- 1708504 TI - Immunological characterization of hepatitis delta antigen. PMID- 1708505 TI - Viroids and viroid-like satellite RNAs: a phylogenetic analysis. AB - With the discovery that certain RNAs possess catalytic properties (Kruger et al., 1982; Guerrier-Takada et al., 1983), the possible significance of certain small plant pathogenic RNAs, namely viroids and viroid-like satellite RNAs, as relics of precellular evolution deserves consideration. As a first step in this endeavor, I report here the results of a phylogenetic study of the computer aligned nucleotide sequences of these RNAs that is based on the parsimony principle (Felsenstein and Sober, 1986). PMID- 1708506 TI - Characterization of a subgenomic HDV-specific poly(A)+ RNA species isolated from human hepatoma cells supporting HDV RNA replication. PMID- 1708507 TI - Disruption of keratin intermediate filaments by ultraviolet radiation in cultured human keratinocytes. AB - Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect. PMID- 1708508 TI - Evaluation of a prostacyclin analog, iloprost, and a thromboxane A2 receptor antagonist, daltroban, in experimental intimal hyperplasia. AB - This study evaluated the efficacy of a prostacyclin analog, iloprost, and a thromboxane A2 receptor antagonist, daltroban, as inhibitors of experimental intimal hyperplasia. The vascular injury model used is based on an endothelial injury induced by a brief infusion of air into an isolated segment of the common carotid artery in the rat. Iloprost and daltroban were administered by continuous IV infusion for two weeks. The infusion rates were 0.1 micrograms/kg/min for iloprost and 0.1 mg/kg/hr for daltroban; these dosing rates are associated with significant alterations in eicosanoid-related pharmacologic effects. The animals were sacrificed at two weeks and the carotid arteries fixed in situ for light microscopy. The myointimal thickening was measured as the intima to media area (I/M) ratio. The control animals developed marked intimal thickening, with an I/M ratio of 0.76 +/- 0.12 (mean +/- SEM; N = 7). There was no inhibition of intimal hyperplasia (P greater than 0.05) after either iloprost (I/M ratio: 1.04 +/- 0.13; N = 8) or daltroban (I/M ratio: 0.70 +/- 0.04; N = 6). It is concluded that neither of these two modulators of eicosanoid activity, iloprost and daltroban, inhibit intimal hyperplasia following experimental endothelial injury. PMID- 1708509 TI - Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy. AB - Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of Iloprost at a dose of 10 micrograms, at a rate of 0.1 micrograms/kg/h, twice daily for two weeks. The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen in vitro. In the one patient tested, thermography revealed an increase in skin temperature by more than 2 degrees C. Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy. PMID- 1708510 TI - [Serologic study of patients with leptospirosis using TR antigen]. AB - This paper deals with 254 monosera of patients with leptospirosis verified by macroagglutination test with thermoresistant antigen (TR antigen) and passive hemaglutination (HA), as reference technique. The TR antigen was elaborated according to the method described by Mailloux and coworkers, 1974, with modifications, and it showed a wide generic reactivity. It was specific when serologic test were performed with hyperimmune sera obtained from febrile disease producing bacteria and the causal agent of syphilis. The results obtained by both serologic tests were compared and it was observed that 106 sera (41.73%) were reactive to HA, which showed a highly significant statistical difference between the two techniques; so a lower sensibility of TR macroagglutination test was determined. PMID- 1708511 TI - Inside the thymus, Mls antigen is exclusively presented by B lymphocytes. AB - The ability to stimulate an Mls-1 mixed lymphocyte reaction (MLR) is predominantly expressed by low density B lymphocytes in the spleen and peritoneal cavity of normal adult mice, and is absent in splenic B cells 1 month after lethal irradiation and reconstitution from autologous bone marrow. Coreconstitution of these mice with normal syngeneic peritoneal cells restores the stimulatory potential of splenic B cells, but sorted CD5+ or CD5- IgM+ lymphocytes from peritoneum are equally good stimulators, suggesting that functional Mls-1 expression may require long life spans and selection. Bone marrow-reconstituted DBA/2 mice that fail to express Mls-1 antigens in the periphery nevertheless maintain T-cell receptor V beta 6 and 8.1 deletions among the newly formed T cells. These findings led us to directly investigate the Mls stimulatory ability of purified antigen-presenting cell populations inside the thymus. We report here that thymic B lymphocytes seem to represent the only intrathymic cell population able to stimulate Mls-1 MLR. PMID- 1708512 TI - Glycolipid anchorage of Plasmodium falciparum surface antigens. AB - Human red blood cells (RBC) were infected with the malarial parasite Plasmodium falciparum, the anchoring of schizont proteins to RBC membranes by glycoinositol phospholipids was demonstrated by three criteria: (1) metabolic incorporation of 3H-ethanolamine and 3H-myristate into the protein; (2) release of 35S-methionine labelled protein into the supernatant after incubation with phosphatidylinositol specific phospholipase C; and (3) the exposure of a glycoinositol phosphate epitope on the methionine-labelled protein following phospholipase C cleavage. Labelled proteins were analysed by immunoprecipitation, polyacrylamide gel electrophoresis in sodium dodecylsulphate and gel fluorography. Several candidate proteins were observed when each criteria was investigated. Among these, 3 proteins which met all three criteria were identified by immunoprecipitation with monospecific sera or monoclonal antibodies. These included 3 possible vaccine candidates, the p190 major surface antigen, the p76 serine protease and the p71 protein which is thought to be a member of the family of heat-shock Hsp70 proteins. PMID- 1708513 TI - Photocoagulation scar expansion after laser therapy for choroidal neovascularization in degenerative myopia. AB - The authors performed a prospective study of 36 eyes affected by pathologic myopia with macular subretinal neovascularization (SRNV) successfully treated with either argon green, dye orange (590 nm), or krypton red lasers. Re-treated eyes were excluded. Evolution of the laser scar was carefully monitored for 12 months. Scar expansion was noted in 35 cases (97%), without a significant loss of visual acuity during the first year after treatment. The scar enlarged more conspicuously during the first 3 months after photocoagulation, with a mean increase of 103% in the first year. Expansion was not related to laser wavelength, patient age, SRNV size, or degree of myopia. Scar enlargement was greatest in the same direction as that of maximal extension of the myopic peripapillary crescent; inspection of the morphologic configuration of the peripapillary crescent before laser treatment may be helpful in evaluating the potential risk of post-laser scar expansion toward the fovea. PMID- 1708514 TI - Thermal papillitis after dye red photocoagulation of a peripapillary choroidal neovascular membrane. AB - Thermal papillitis is a previously unrecognized complication of photocoagulation for peripapillary choroidal neovascularization. This report describes a patient in whom transient thermal papillitis, choroidal ischemia, and two small branch retinal arteriolar occlusions developed after dye red photocoagulation of an idiopathic peripapillary choroidal neovascular membrane. PMID- 1708515 TI - Profound central visual loss and ocular neovascularization in idiopathic recurrent branch retinal arterial occlusion. AB - The authors report a 65-year-old healthy, white man who experienced a dramatic loss of central vision. Iris neovascularization, rubeotic glaucoma, disc neovascularization and subhyaloid hemorrhage developed after multiple, recurrent, idiopathic branch retinal arterial occlusions. Vitreous and perivascular inflammation were prominent associated clinical features. Systemic steroids were useful in suppressing intraocular and perivascular inflammation, yet neither steroid nor anticoagulant therapy effectively prevented recurrent occlusive episodes. Retinal neovascularization and rubeotic glaucoma were successfully managed with scatter panretinal photocoagulation. Episodic intraocular inflammation and ocular neovascularization have been noted in one-third of patients sustaining recurrent idiopathic branch retinal arterial occlusions. PMID- 1708516 TI - Neovascularization of the disc in pars planitis. AB - Neovascularization of the disc (NVD) was present in 9 of 163 patients with pars planitis. In all cases, NVD was unilateral and observed in eyes with recent exacerbation of the inflammatory process. To reduce intraocular inflammation, all nine eyes were treated with varying combinations of topical, periocular, and systemic corticosteroids. In addition to corticosteroids, one eye received Argon laser photocoagulation, two eyes underwent peripheral cryotherapy, and one eye was treated with both Argon laser photocoagulation and peripheral cryotherapy. With decrease or disappearance of intraocular inflammation, NVD resolved in all cases without recurrence during follow-up study, which ranged from 6 to 189 (mean, 81) months. Rhegmatogenous retinal detachments developed in two eyes treated with peripheral cryotherapy. Both detachments were successfully repaired with surgery. Control of intraocular inflammation appears to be the key factor for regression of NVD in pars planitis. If NVD does not regress or vitreous hemorrhage occur, photocoagulation and peripheral cryotherapy may be beneficial. PMID- 1708517 TI - Retinal hemangioma-like lesions in eyes with retinitis pigmentosa. AB - The authors report two patients with bilateral vascular masses of the peripheral retina associated with primary pigmentary dystrophy of the retina (retinitis pigmentosa). Although they are most similar to the retinal capillary hemangiomas of von Hippel, the affected patients had no clinical history or clinical findings suggestive of that syndrome. They differ from the calcified retinal hamartomas that have been associated with retinitis pigmentosa because they do not show the extensive telangiectasia and exudation seen with the exudative retinopathy that has been described with retinitis pigmentosa. They do not show the fluorescein angiographic pattern that characterizes peripheral choroidal neovascularization. Their main complication seems to be vitreous hemorrhage rather than exudative retinopathy. The authors discuss the possible relationship of these acquired retinal vascular masses to the retinitis pigmentosa. PMID- 1708518 TI - [Care of scars: what kind of information does the patient understand?]. PMID- 1708519 TI - [Artificial sphincter: surgical results]. PMID- 1708521 TI - [Long live the media library in the hospital!]. PMID- 1708520 TI - [Palliative care in adults]. PMID- 1708522 TI - Release of immunoreactive human neutrophil proteinase 4, normally and in peritonitis. AB - A specific enzyme-linked immunoassay (ELISA) has been developed for the determination of neutrophil proteinase 4 (NP4) in human plasma/serum and tissue fluids. Comparison of the sequence for the first 20 N-terminal amino acids of NP4, neutrophil elastase and cathepsin G shows that NP4 is distinct from the other two proteases. However, all three show considerable homology. Neither elastase nor cathepsin G show any immunoreactivity when tested in the present ELISA. Normal human plasma contains about 38 micrograms/l of NP4, identified as alpha 1-proteinase inhibitor complexes. This represents about 50% of the total amount of NP4 released in plasma. The remaining 50% is bound by alpha 2 macroglobulin. Blood coagulation leads to a rapid release of NP4 from the leukocytes. Peritonitis is accompanied by a pronounced release of NP4, as shown by a three-to 10-fold increase of NP4 plasma levels and by the NP4 level in peritoneal exudates, which reaches about 40 mg/l in severe cases. PMID- 1708523 TI - The influence of pH, pCO2 and concentrations of dyshemoglobins on the oxygen dissociation curve (ODC) of human blood determined by non-linear least squares regression analysis. AB - In the context of the ODC algorithm of Siggaard-Andersen the effects of pH, carbon dioxide tension (pCO2) and concentrations of dyshemoglobins: Carboxyhemoglobin (x COHb), methemoglobin (x MetHb) and fetal hemoglobin (x HbF) in terms of five constants, a1(0) to a5(0), were determined by a non-linear least squares analysis of 11700 records of data containing measured values of oxygen tension (pO2), oxygen saturation (sO2), as well as pH, pCO2, x COHb, x MetHb, x HbF. The standard values of a1(0) to a3(0) used in the ODC were found to be significantly different from those giving the best fit to the present data whereas the presently used coefficient for methemoglobin was found to be acceptable. The coefficient for fetal hemoglobin, a5(0), could not be determined due to insufficient data. pH and pCO2 effects were found to be correlated in the data and this fact accounts for some of the deviations of a1(0) and a2(0) from the standard values. The analysis evaluated the effect of pH as being stronger and the effect of pCO2 as being weaker than assumed in the standard model. Finally the effects of the concentrations of met- and carboxyhemoglobin were found to be correlated but no explanation of this fact is known at present. PMID- 1708524 TI - Accurate measurements of hemoglobin oxygen saturation, and fractions of carboxyhemoglobin and methemoglobin in fetal blood using Radiometer OSM3: corrections for fetal hemoglobin fraction and pH. AB - The differences in the visible absorption spectra between fetal and adult oxyhemoglobin and carboxyhemoglobin result in errors in the measurements og hemoglobin oxygen saturation (SO2) and carboxyhemoglobin fraction (FCOHb) in fetal blood, if not corrected for the actual fetal hemoglobin fraction (FHbF) in the sample. In 11 fully oxygenated umbilical cord blood samples (mean FHbF = 77%), we found a mean positive bias in SO2 of 4.7%, and in FCOHb of 2.7%, when measured with a dedicated spectrophotometer (OSM3, Radiometer A/S, Denmark), and using the matrix of absorption coefficients for adult hemoglobin. Accurate measurements were obtained by using OSM3's correction for FHbF in the blood specimen after measurement of FHbF by OSM3. The effects of plasma pH on the measurements of SO2 and FCOHb in fully oxygenated fetal blood were found to be similar to those found for adult blood. From plasma pH 7.05 to 8.02, measured SO2 increased 1.3% and FCOHb 0.6%. Correction for the pH of fetal blood samples should be considered when calibrating OSM3 and in connection with research studies. The effects of FHbF and pH on the measurement of methemoglobin fraction (FMetHb) were less than 0.2%, and can be ignored. FHbF measured by OSM3 at pH 7.4 is about 14% too high compared to alkali denaturation rate method. However, the presence of a metabolic acidemia, which is common in fetal blood specimens, decreases this bias, so that for example in our study, FHbF, measured by OSM3 and uncorrected for pH changes was on average only 6% too high. We recommend that OSM3's factor of 18.6 is reduced to 16.4, and that correction is made for pH. PMID- 1708525 TI - An RNA first: it's part of the gene-copying machinery. PMID- 1708526 TI - A class III transcription factor composed of RNA. AB - It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease. PMID- 1708527 TI - Determination of primary motoneuron identity in developing zebrafish embryos. AB - The developmental determination of primary motoneurons was investigated by transplanting identified motoneurons in embryonic zebrafish to new spinal cord positions. Some cells moved from the new positions in which they were placed back to their original positions, thus it was difficult to evaluate whether they were determined. Among cells that remained in their new positions, those transplanted about 1 hour before axogenesis developed axonal trajectories that were appropriate for their original soma positions, whereas those transplanted 2 to 3 hours before axogenesis developed morphologies appropriate for their new soma positions. These results suggest that motoneuronal identity is determined before axogenesis. PMID- 1708528 TI - Thromboprophylaxis in patients with hip fractures: a prospective, randomized, comparative study between Org 10172 and dextran 70. AB - A prospective, randomized, assessor-blind trial has been undertaken to compare the thromboprophylactic effect and safety of the heparinoid Org 10172 (a mixture of low molecular-weight sulfated glycosaminoglycuronides) and dextran 70 in patients operated on for hip fracture. Prestudy biostatistical calculations led to the need for 260 patients. Three hundred eight patients were randomized and 19 were excluded after randomization, the majority because of postponed surgery. Analyses were made on the 289 patients on an intention-to-treat basis, as well as on the 247 patients given correct prophylaxis. Diagnosis of deep vein thrombosis was based on bilateral ascending phlebography on postoperative days 10 through 12. The frequency of deep vein thrombosis on an intention-to-treat basis was 10% in the Org 10172 group and 30% in the dextran 70 group and, on the basis of correct prophylaxis, 12% and 31%, respectively, both differences being significant (p less than 0.001). Two-month mortality rates were equal in the groups. Three fatal pulmonary emboli were seen in the dextran group. Significantly more patients in the dextran group received postoperative transfusions; no other differences in various hemorrhagic parameters were seen. Thus it can be concluded that Org 10172 has a significantly better thromboprophylactic effect than does dextran in patients with hip fractures without significant side effects. PMID- 1708529 TI - [Nursing. Lumbar disk prolapse]. PMID- 1708530 TI - Studies for revealing a possible sensitization to hirudin after repeated intravenous injections in baboons. AB - The immunogenic potential of natural and recombinant hirudin was investigated using baboons. Animals received 4 intravenous hirudin injections of 1,000 antithrombin units/kg at certain times (day 1, 3, 8 and 42) and were checked for an immune response. None of the tests performed (in vitro histamine-release, ELISA for detection of humoral hirudin-specific IgG and IgM antibodies by indirect ELISA, direct skin test) revealed any kind of sensitization to hirudin. Urinary excretion of hirudin was not diminished by successive injections, if diuresis was within normal ranges. In summary, these results confirm previous observations indicating the weak antigenic potential of hirudin. Therefore, immune reactions to hirudin in man during therapeutic measures requiring several exposures to the inhibitor should not to be expected under normal circumstances. PMID- 1708531 TI - Differential effects of aprotinin and tranexamic acid on cerebral bleeding and cutaneous bleeding time during rt-PA infusion. PMID- 1708532 TI - Myelin basic protein-mRNA used to monitor trimethyltin neurotoxicity in rats. AB - Trimethyltin (TMT) is an alkyltin that targets neurons of the limbic system. A gene probe (i.e., mRNA) for myelin basic protein (MBP), a major component of central nervous system myelin, was used to monitor this toxic neuropathy in Sprague-Dawley rats. Animals were administered a single intraperitoneal injection of TMT-hydroxide at a neuropathic (8.0 mg/kg/body wt) or nonneuropathic (0.8 mg/kg/body wt) dose and sampled at 1, 3, or 7 days postexposure to correlate the progression of hippocampal neuropathology with probe (i.e., MBP-mRNA) levels. Microscopic examination of the brain showed only moderate but progressive damage over the 7-day postexposure period in animals treated with the neuropathic dose. Neuronal loss was first observed in the dendate gyrus and CA4 at 1 day postexposure, and progressed to the CA3c sector at 3 and 7 days postexposure. Elsewhere in the brain, minimal involvement of the entorhinal cortex neurons occurred 3 days postexposure and intensified by 7 days. No histological damage was seen at the nonneuropathic (0.8 mg/kg) dose. For gene probe analysis, the brain was divided into anterior and posterior halves. In rats treated with the neuropathic dose of TMT, the anterior brain showed progressive depressions of MBP mRNA levels over the 1-, 3-, and 7-day postexposure period that correlated with increasing hippocampal neuropathology. The posterior brain showed no significant changes in MBP-mRNA levels with respect to that of controls over the same time period. At the nonneuropathic dose (0.8 mg/kg) a significant depression of MBP mRNA levels occurred in the anterior brain at 7 days postexposure in the absence of overt histological damage. PMID- 1708533 TI - Chlorpyrifos pharmacokinetics and metabolism following intravascular and dietary administration in channel catfish. AB - The pharmacokinetics and metabolism of a model organophosphorothioate, chlorpyrifos, was investigated in channel catfish (Ictalurus punctatus). Chlorpyrifos exhibited multicompartmental disposition after oral and intravascular administration, indicating slower distribution to peripheral storage tissues. The oral bioavailability of chlorpyrifos was 41%, substantially higher than in mammals. Muscle contained less than 5% of the oral dose, with an elimination half-life of about 3.3 days. Residues in the whole fish were greater than 95% chlorpyrifos, while excreta (bile and urine) primarily contained metabolites. The dephosphorylated metabolite, trichloropyridinol (TCP), was the major metabolite in the blood, while the glucuronide conjugate of TCP was the major metabolite in urine and bile. The toxic metabolite, chlorpyrifos oxon, was not detected in any samples of blood, tissues, or excreta. The metabolism of chlorpyrifos in catfish appeared similar to other species of fish and mammals. Extensive metabolism resulted in a low potential for chlorpyrifos to accumulate in catfish from dietary exposure. PMID- 1708534 TI - Interesting times for dopamine receptors. PMID- 1708535 TI - Do calcium channel classifications account for neuronal calcium channel diversity? AB - Calcium (Ca2+) ions are involved in the development and control of a variety of neuronal properties and functions such as channel expression, synaptic transmission and neurosecretion. The main pathway by which Ca2+ enters the intracellular space is through voltage-activated Ca2+ channels that can be classified according to their different biophysical and pharmacological properties. Identification and characterization of these channel types are prerequisites for understanding the mechanisms that underlie Ca2(+)-controlled processes. In this article we summarize the efforts made to identify neuronal Ca2+ channel types, and we attempt to evaluate how useful existing classifications are in assigning specific properties and functions to distinct channel types in neurons. PMID- 1708536 TI - Neuroscience in Poland. AB - Earlier this year I tried to locate and count the scientists who may possibly want to join the Polish Neuroscience Society. A first rough estimate indicated about 200-250 scientists, including graduate students, who do research on the nervous system. It seemed worthwhile to attempt to highlight their work so that Polish neuroscience might share in the impetus coming from the historical changes sweeping across Europe. PMID- 1708537 TI - Parallel processing in the basal ganglia: up to a point. PMID- 1708538 TI - Glutamate, nitric oxide and cell-cell signalling in the nervous system. AB - Nitric oxide (NO) is a recently discovered and highly unorthodox messenger molecule. Current evidence indicates that, in the CNS, NO is produced enzymatically in postsynaptic structures in response to activation of excitatory amino acid receptors. It then diffuses out to act on neighbouring cellular elements, probably presynaptic nerve endings and astrocyte processes. In several peripheral nerves, and quite possibly in parts of the CNS as well, NO might be formed presynaptically and thus act as a neurotransmitter. In both cases, a major action of NO is to activate soluble guanylate cyclase and so raise cGMP levels in target cells. PMID- 1708539 TI - Plateau potentials and active integration in the 'final common pathway' for motor behaviour. AB - Most studies of vertebrate spinal motoneurones have suggested that they possess relatively simple membrane properties, causing them to behave merely as passively driven output neurones in motor behaviour. According to this concept, motoneurones passively transform the net synaptic drive from pre-motoneuronal levels into spike trains. Recent research has demonstrated a more complex picture by showing that motoneurones can express nonlinear intrinsic response properties, such as plateau potentials and endogenous oscillatory properties. This work suggests that the 'final common pathway' is actively involved in shaping motor behaviour. PMID- 1708540 TI - Common patterns of plasticity contributing to nociceptive sensitization in mammals and Aplysia. AB - In contrast to innocuous stimuli, which only have transient effects when applied to the body surface, noxious stimuli generate persistent changes in the nervous system. This nociceptive memory manifests itself most prominently as a post injury sensitization where, after tissue damage, the avoidance reaction and pain that result from subsequent stimuli are exaggerated and prolonged and can be initiated by low intensity stimuli. Similarities between nociceptive sensitization in mammals (including humans) and the mollusc Aplysia californica suggest that fundamental mechanisms contributing to injury-induced behavioral modifications might be widespread in the animal kingdom. PMID- 1708541 TI - Contributions of topography and parallel processing to odor coding in the vertebrate olfactory pathway. AB - Odor information appears to be encoded by activity distributed across many neurons at each level in the olfactory pathway. Thus olfactory circuits function as parallel distributed processors. New methods for observing distributed activity in such systems permit computer simulations to be constructed that are constrained by patterns of activity observed in the real system. Analysis of the system using a combination of physiological measurements and computational approaches might elucidate the principles by which odors are discriminated. PMID- 1708542 TI - [Cancer of the exocrine pancreas]. PMID- 1708543 TI - Workshop findings on the ovine homologue of CD5. PMID- 1708544 TI - Workshop findings on the ovine homologue of CD8. PMID- 1708545 TI - Competitive binding with putative Bo5 (CD5) cluster of monoclonal antibodies. AB - The relationship of seven monoclonal antibodies, putatively to the Bo5 (CD5) antigen, was tested. Five of the mAbs were confirmed to be directed against the Bo5 antigen. Three mAbs, CC29, BLT-1 and 8C11, effectively blocked binding to bovine PBM of mAb CC17, previously reported to be directed against this antigen. MAb 8-3F4 also blocked binding of mAb CC17, but less effectively than the others. MAbs IL-A67 and 79-5 did not inhibit binding of mAb CC17 because of antibody allelic specificity or technical reasons. PMID- 1708546 TI - Epitopes of the T19 lymphocyte surface antigen are extensively conserved in ruminants. AB - The reactivity of five monoclonal antibodies (mAbs SBU-T19, 197, IL-A29, CC-15 and CC-39) specific for the T19 molecule on sheep and cattle CD4-CD8- T cells was compared. MAbs SBU-T19 and 197 were shown to recognise separate epitopes on T19. All mAbs reacted with lymphocytes from several different ruminant species and the tissue distribution and frequency of positive cells was similar in each case. None of the mAbs reacted with horse, pig or camel lymphocytes. The extensive conservation of T19 epitopes in ruminants during the mammalian radiation could indicate an important role for this molecule in the ruminant immune system. PMID- 1708547 TI - Expression of the "T19" and "null cell" markers on gamma delta T cells of the sheep. AB - A 215 kDa molecule termed T19 marks CD4-CD8- T cells in sheep and cattle. In this report, we analysed the T19 or "null cell" panel of mAbs against gamma delta T cells of sheep, using a mAb specific for the gamma delta TCR. By two-colour immunofluorescence, all of the mAbs in the T19 panel reacted with gamma delta T cells or subsets thereof, although staining intensities and percentages of cells stained by the different mAbs indicated considerable heterogeneity for the T19 molecule. This probably results from differential expression of certain epitopes on T19. The reactivity of most of the mAbs for the 215 kDa T19 molecule was also confirmed by immunoprecipitation and SDS-PAGE. PMID- 1708549 TI - Immunohistology of workshop monoclonal antibodies to the bovine homologue of CD1. AB - Six monoclonal antibodies putatively to the BoCD1 antigen were compared by immunohistology on cryostat sections from a range of tissues. The different staining patterns observed allowed the mAbs to be placed in three groups (a) 20 27, (b) CC13, CC14, TH97A and (c) CC20, CC40. An ovine mAb VPM5 did not stain bovine tissues sufficiently strongly to enable a comparison with the other CD1 mAbs. PMID- 1708548 TI - Analysis of the CD1 cluster in sheep. AB - The anti-CD1 monoclonal antibodies submitted to the 1st International Workshop on Leucocyte Differentiation Antigens of Cattle, Sheep and Goats were tested for their reactivity on sheep skin, thymus and lymph node and for their reactivity with sheep efferent and afferent lymph and peripheral blood. With the exception of 20-27 they all stained that same cell populations. The antibodies precipitated molecules with a heavy chain of 46,000 apparent molecular weight and a light chain of 14,000 apparent molecular weight. VPM5 and CC14 antigens were purified by affinity chromatography. All the antibodies cross-reacted with these molecules. The results show that 20-27 recognises the same molecules as the other antibodies and suggest that 20-27 is a pan CD1 monoclonal antibody and the other monoclonal antibodies are homologues of the human CD1b molecules. PMID- 1708550 TI - Polymorphism of the CD4 and CD5 differentiation antigens in cattle. AB - Monoclonal antibodies (mAbs) specific for bovine CD4 and CD5 antigens have been found to identify polymorphic determinants on these molecules. In the case of CD5, mAb IL-A67 recognises one allotypic form of the antigen while four other CD5 specific mAbs in the workshop (CC17, CC29, BLT-1 and 8C11) recognise a second allotype. The CD4-specific mAbs submitted to the workshop reacted with the cells of all animals tested. However, a further two mAbs (CC26 and IL-A18) specific for CD4 were found to react with cells only from about 85% of animals tested. Sequential immuno-precipitation experiments together with family studies showed that the allotypes of CD4 and CD5 are both inherited in a simple Mendelian manner and are co-dominantly expressed. One of the CD5 allotypes was not detected in Bos taurus animals while the gene frequency of the second allotype was only about 10% in the B. indicus animals tested. The gene frequency of the CD4 allotype detected by CC26 and IL-A18 was similar in the two sub-species. PMID- 1708551 TI - Reactivity of the workshop CD5 panel antibodies on western blot of cell lysate. PMID- 1708552 TI - Reactivity of the workshop CD5 panel antibodies with the purified CD5 antigen. PMID- 1708553 TI - Individual antigens of cattle. Bovine CD1 (BoCD1). AB - The results support the view that these mAbs can be regarded as equivalent to CD1 mAbs in man, as has been concluded for certain of them previously. Although mAb 20-27 did not cluster with the other mAbs, the finding that it precipitated the same molecule from thymocytes indicates that it should be included in BoCD1. Studies in man indicate that more than one CD1 molecule exists (Boumsell and Knowles, 1988; Cattoretti et al., 1988) and a similar situation is apparent in cattle with 20-27 probably being equivalent to CD1c and the others to CD1b. PMID- 1708554 TI - Individual antigens of cattle. Bovine CD2 (BoCD2). AB - The data obtained in the workshop provide further evidence that CH128A and IL-A26 and the 12 new mAbs that form a cluster recognise the bovine orthologue of CD2. The mAbs inhibit rosetting with SRBC, stain cells in primary and secondary lymphoid organs in patterns consistent with those obtained in humans with anti CD2 mAbs, and the 11 IgG mAbs all immunoprecipitate a peptide with a Mr of 58-62 kDa. It is not clear from the studies whether the epitopes defined by the mAbs correspond with the region I and II epitopes present on CD2. None of the data suggest that any of the mAbs recognise the region III (CDD2R) epitope (Peterson and Seed, 1987; Knapp et al., 1989). Further studies are now needed to define the physical and functional relation of the epitopes and establish whether antibody mediated activation corresponds with that noted in humans. Data reported in one study (Baldwin et al., 1988) with IL-A26 suggest possible differences in the requirements for activation. In addition, further studies are needed to demonstrate how many cell types express BoCD2. In mice, evidence has been presented which shows the mouse orthologue is expressed on some B cells (Yagitta et al., 1989). Studies in cattle have clearly shown CD2 is present on the majority of CD4+ and CD8+ T-cells and a small population of CD4-/CD8- cells (Baldwin et al., 1988; Davis, unpublished observations). Evidence presented in this workshop has shown that some CD2+ cells express a WC2 molecule (Sopp et al., 1991).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708555 TI - Individual antigens of cattle. Bovine CD5 (BoCD5). PMID- 1708556 TI - Individual antigens of cattle. Bovine CD6 (BoCD6). PMID- 1708557 TI - Individual antigens of cattle. Bovine CD8 (BoCD8). PMID- 1708558 TI - Individual antigens of cattle. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2). AB - The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric molecule expressed in a subpopulation of the WC1+ cells and on an additional small population of T lymphocytes. The cell populations recognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor molecules. PMID- 1708559 TI - Individual antigens of cattle. Antigens expressed predominantly on monocytes and granulocytes: identification of bovine CD11b and CD11c. PMID- 1708560 TI - Workshop studies on the ovine CD1 homologue. PMID- 1708561 TI - [Control over treatment efficacy in pulmonary tuberculosis patients by determining the amount of nucleic acids in the blood]. AB - The authors formulated for the first time a conception of the possibility of controlling the course of pulmonary tuberculosis treatment efficacy by means of determination of the amount of nucleic acids in the blood. A dependence has been established of changes of the amount of nucleic acids of the blood on the degree of clinical improvement of patients with pulmonary tuberculosis. PMID- 1708562 TI - [Pathology in Aachen]. PMID- 1708563 TI - [Proceedings of the German Society for Pathology. 74th meeting. Aachen 5-9 June 1990]. PMID- 1708564 TI - [The stem cell system of hematopoiesis: physiological and pathophysiological concepts]. AB - It is the purpose of this review to describe the physiological as well as the pathophysiological principles of the hematopoietic stem cell system. The concept of hemopoietic stem cells has a long history which is now understood on the basis of its embryogenesis and after collecting extensive experimental and clinical experience using stem-cell transplantations as a means to restore hematopoietic function of the bone marrow after appropriate conditioning. The hemopoietic stem cells cannot be distinguished by light microscopy from "lymphocytes" considered to be a heterogeneous group of mononuclear cells. These stem cells can respond to specific regulatory factors with specific differentiation and proliferation, and are very radiosensitive and resistant to cryopreservation. The system responds to perturbations in a manner characteristic for feed back regulation and is bound in its physiology to an intact stromal matrix. PMID- 1708565 TI - [Reticular dysgenesis: primary disorder in differentiation of hematopoietic stem cells?]. AB - Reticular Dysgenesis (RD) basically represents a lymphopenic severe combined immunodeficiency (SCID) in association with congenital agranulocytosis. It is presumed that RD results from a primary defect of pluripotent hematopoietic stem cells (HSC). Alternatively RD might be due to an alloreaction induced by T-cells derived from intrauterine transfusion of maternal cells into an immunoincompetent host. In the past 15 years, among 49 newborns with SCID taken care of in the University Hospital of Ulm, 5 children (4 boys, 1 girl) exhibited the characteristics of RD. In 3 of 4 cases studied, maternal T-cells were detected by HLA-typing. All 3 children showed signs of graft-versus-host-disease (GvHD), confirmed histologically. However, 9 of 45 newborns with SCID without congenital agranulocytosis also disclosed maternal T-cell-engraftment; 5 of the 9 had signs of GvHD. Therefore, it is unlikely that RD is caused by GvHD secondary to maternofetal transfusion. The fact that erythropoiesis, thrombopoiesis and the monocyte-macrophage-system basically are intact argues against a global maturation defect of HSC in RD. PMID- 1708566 TI - [Effects on bone marrow during stimulation of hematopoietic stem cells with recombinant human interleukin-3]. AB - Bone marrow aspirates and biopsies of 20 patients with malignant tumors (n = 6), bone marrow failure due to chemotherapy (n = 4), myelodysplastic syndromes (n = 5) and aplastic anemia (n = 5) were analysed before and after treatment with recombinant human interleukin-3. rhIL-3 led to increased overall bone marrow cellularity with trilinear stimulation of hematopoietic cells. In all patients marked eosinophilia and, in some, fibrosis was observed. These results show that rhIL-3 stimulates the proliferation and differentiation of pluripotent hematopoietic progenitor cells and may contribute to the treatment of bone marrow failure. PMID- 1708567 TI - [In vitro and in vivo effects of recombinant human interleukin-3 on hematopoietic progenitor cells]. AB - As part of a phase I/II clinical trial with recombinant human Interleukin-3 (rhIL 3), bone marrow and peripheral blood cells from patients treated with rhIL-3 were assessed in vitro for effects on the cycling status and frequencies of erythroid, myelomonocytic and multilineage progenitor cells. Treatment with rhIL-3 induced a pronounced increase in S-phase rate of BFU-E, CFU-GEMM and day 14 CFU-GM. Circulating CFU-GEMM and day 14 CFU-GM were increased on day 7 of therapy whereas circulating BFU-E were reduced in the majority of patients. After i.v. bolus injection of IL-3 an acute effect on the peripheral blood count with neutrophilia and, after an initial nadir, monocytosis was observed. IL-6 serum-levels increased in 5 out of 14 patients after IL-3 administration with concomitant side effects in 2 patients in whom IL-6 levels increased above 75 pg/ml. In the majority of the patients a stimulation of peripheral leukocytes and platelets was observed during the 15 day treatment course. In conclusion rhIL-3 induces a multilineage response in vivo by stimulating proliferation of hemopoietic progenitor cells with a resulting elevation in peripheral blood counts. PMID- 1708568 TI - [Hematopoietic stem cells (CFU-S) in retrovirus-induced murine malignant histiocytosis]. AB - The murine malignant histiocytosis sarcoma virus (MHSV) is a replication defective murine retrovirus which contains a ras-oncogen. Upon intravenous infection of normal adult NIH-mice a rapidly fatal systemic proliferation of transformed mononuclear phagocytes develops. We have investigated the involvement of other mature hematopoietic lineages and of hematopoietic stem cells during the course of the disease. The erythroid lineage is affected by a profound anemia which is temporarily accompanied by an increase of erythroid precursors in bone marrow and spleen and a final ablation of erythroid stem cells. A granulopenia occurs with a concurrent decrease of granulocytic precursors and stem cells. The platelets fall to very low levels early during the disease despite of an increase of megakaryocytes and megakaryocytic stem cells. Multipotential stem cells show temporarily a slight increase followed by an almost total terminal ablation. The results show that MHSV has distinct effects on other hematopoietic lineages apart from its transforming properties. PMID- 1708569 TI - [Importance of DNA-cytometric parameters in the prediction of blast crisis in the course of chronic myelogenous leukemia]. AB - In a retrospective study the DNA-cytometric parameters stemline ploidy, stemline shoulder fraction and proliferative fraction were followed during the course of the disease in 20 patients with chronic myelogenous leukemia. Stemline ploidy and stemline shoulder fraction significantly increased whereas the proliferative fraction steadily decreased during the disease most of these changes taking place during chronic phase before the clinical onset of blast crisis. Prognostically relevant cutpoints indicating disease transformation during the next 12 months could be defined. PMID- 1708570 TI - [Fine structure of megakaryocyte precursor cells (promegakaryoblasts) derived from smears and sections of bone marrow tissue in patients with chronic myeloid leukemia and so-called primary osteomyelofibrosis with accompanying thrombocythemia]. AB - An immunomorphometric study (antiglycoprotein III a - Y2/51) was performed on routinely processed trephine biopsies in 24 patients with chronic myeloid leukemia (CML) as well as so-called primary osteomyelofibrosis (OMF) to determine the frequency and fine structure of promegakaryoblasts (PMB). With respect to controls, there was a significant increase in megakaryocytes in both entities with an orderly expansion of the precursor subpopulation in CML, but a relative decrease in OMF. In comparison with smears of aspirates, sizes of PMB were reduced by about 25% (factor 1.4) in the corresponding bone marrow sections. PMID- 1708571 TI - [Pattern analytic investigations of blast cells in myelodysplastic syndrome and secondary acute myeloid leukemia]. AB - Blast-cells in Romanowsky-Giemsa stained bone marrow smears from 14 cases of primary myelodysplastic syndrome which consequently developed an acute myeloid leukemia and 28 cases of primary acute myeloid leukemia were analysed by a computer aided high resolution pattern recognition system. As control we used blast-cells from reactive affected bone marrow. Whereas blast-cell types in MDS and secondary AML showed overlapping features and a heterogenous distribution we could distinguish blasts in primary AML compared to "reactive" blasts. Opposite to this blasts in secondary AML showed no different pattern compared to "reactive" blast. PMID- 1708572 TI - [Cytogenetic examination of bone marrow cells in stem cell diseases of myelodysplastic syndromes]. AB - Cytogenetic examination of bone marrow cells was performed in 43 patients with myelodysplastic syndrome (MDS). MDS was diagnosed from bone marrow biopsies and smears according to the FAB classification. Of all 43 patients 24 (56%) had clonal karyotype changes including frequently monosomies of chromosomes #5 and #7 as well as interstitial deletions of the long arm of #5, 5 q-. Chromosome aberrations were observed in patients belonging to all FAB-subgroups, e.g. 11/15 patients with RA, 6/10 with RAEB, 5/9 with RAEB/T, and 1/9 with CMMoL. Complex chromosome aberrations involving more than 2 chromosomes occurred predominantly in patients with RAEB/T (4/9) but also in patients with RA (2/15) and RAEB (2/10), and correlated significantly (p less than 0.002) with a shorter survival. No correlation was found between chromosome aberrations and the development of ANLL in 9/43 patients (21%). PMID- 1708573 TI - [Myelosclerosis in myelodysplastic syndromes (MDS). Retrospective analysis of 232 patients with MDS]. AB - In a retrospective study, 45 (19.4%) out of 232 patients with MDS revealed myelosclerosis (MS) in bone marrow biopsy (BMB). Histological classification according to FAB criteria showed the following distribution: RA 21 (47%), RARS 1 (2%), RAEB 9 (20%), RAEB-T 3 (7%), and CMMol 11 (24%). Sclerosis occurred in all subtypes of MDS, but with a higher incidence in CMMol. Clinical data showed lower values of hemoglobin and lower platelet counts in MDS.MS. Life expectancy was reduced to 7.8 months, compared with 15.0 months in MDS without MS (p = 0.0026). In RA, the survival times were 9.7 months in MDS.MS, compared to 27.9 months in MDS without MS (p = 0.0035). 21 (47%) of the patients with MDS.MS experienced a transformation into ANLL. Myelosclerosis therefore seems to herald a bad prognosis. PMID- 1708574 TI - [Comparison of HIV-associated dyshemopoiesis in myelodysplastic HIV-negative patients]. AB - Bone marrow biopsies of 23 HIV-1 infected patients and 29 patients with myelodysplastic disorders were examined histologically by immuno- and enzyme histochemical techniques and morphometrical methods. The nucleolar organizer regions (AgNOR) were demonstrated and visually counted. Hypercellularity in marrows of AIDS patients is substantially caused by an increase of erythropoiesis and CD68+ macrophages/reticular cells. However, hypercellularity in preleukemia results from hyperplasia of all hematological cell lines and CD68+ bone marrow macrophages/reticular cells. AgNOR count is positively correlated with granulo- and erythropoiesis in preleukemia. In AIDS there is such a positive correlation between AgNOR count and granulopoiesis whereas an inverse relationship appears in erythropoiesis. The results suggest that HIV-associated bone marrow alterations differ from merely reactive changes and are related to myelodysplastic disorders. PMID- 1708575 TI - CD5-positive B-cells of the fetal and adult spleen lymphoid tissue: an immunophenotypical study. AB - The lymphoid tissue of the human fetal spleen at various stages of gestation was studied on frozen and paraffin sections and with two-colour flow cytometry. On the sections scattered lymphoid cells and perivascular lymphoid aggregates were found starting from the 15th week of gestation. CD3, CD5, CD19, CD20, CD21, CD22, CD24, CD35, CD38-positive cells were observed. No CD10- and CD23-positive cells were detected. Flow cytometry showed a prevalence of B-lymphocytes. The majority of them expressed CD5 antigen. These cells were also IgM/D, CD19, CD20, CD21, CD22, CD24 and CD35-positive. Only few of them expressed CD10 and CD23. A similar phenotype was found in human cord blood. By contrast, in adult spleens CD5 B cells never exceeded 8% of the B-cells. The comparison between CD5 B-cells of fetal spleen and CD5 B neoplastic cells of 72 cases of small B-cell lymphomas showed that CD23, which was usually expressed by a high percentage of the neoplastic cells, particularly in B-CLL, was not displayed by the majority of the CD5 non neoplastic cells. The reverse was shown by CD35. These findings suggest that different states of activation distinguish the normal CD5 B-cells from their malignant counterpart. PMID- 1708576 TI - [A new pan-macrophage antibody Ki-M1P stains plasmacytoid cells in paraffin sections of lymph nodes]. AB - Plasmacytoid, T cells (PTC) occurring in cases of chronic non-specific lymphadenitis were investigated using a panel of monoclonal antibodies and cryostat sections. From the typical T cell antigens only CD4 was detectable on PTC. Antibodies directed against myelo-monocytic antigens such as Ki-M2, Ki-M6 (CD68), and Ki-M7 revealed positive reaction with these cells. The recently established monoclonal antibody Ki-M1P reactive with monocyte/macrophages shows a surface and granular cytoplasmic reactivity with PTC. This observation may indicate the myelo-monocytic origin. Ki-M1P detects a formalin resistant antigen and is thus applicable to conventionally processed paraffin sections as demonstrated in twenty cases of hystiocytic necrotizing lymphadenitis (KIKUCHI). PMID- 1708577 TI - [Quantitative methods in diagnostic pathology]. AB - The classical pathological diagnosis is of decisive importance for adequate therapy choice. Unfortunately, inconsistency in judgement of the same case and disagreement in diagnosis between different pathologists can be pronounced. Nevertheless, techniques of quantitative pathology largely improve the reliability of diagnosis because of their, in principle, high objectivity and reproducibility. Besides stereology, morphometry and flow cytometry recently also digital image processing, three dimensional laser scan microscopy and multimedial integrated image-database expert systems are becoming increasingly applicated. However, to gain maximum profit of these quantitative methods, sufficient measurement error handling is indispensable. In addition, concepts of standardisation and ongoing quality control should be available to assure confident measurements as a valuable aid in the diagnostic process. PMID- 1708578 TI - Automated image cytometry in cytopathology. AB - Image cytometry is used more and more for the study of clinical cytology, notably for the determination of morphometrical and densitometrical values, the quantification of monoclonal antibody labelling and the detection of DNA probes after in situ hybridisation. Aspects of automated and interactive image cytometry are discussed, including a brief evaluation of limitations and advantages of the image technique in connection to flow cytometry. Some new technologies such as a sampling technique for paraffin embedded tissue and a new automated microscope, which are of special interest to the pathologist, are described in more detail. Applications in image cytometry include diagnostic and prognostic studies. Examples of diagnostic studies are the automated screening for cervical cancer and the detection of rare remaining cancer cells (minimal residual disease) in the peripheral blood. The use of archival material in image cytometry allows interesting retrospective studies with regard to the relation of the course of the disease with the ploidy characteristics of the tumor. PMID- 1708579 TI - [DNA cytometry and automation in clinical diagnostics]. AB - The biological basis for DNA-cytometric assistance in the diagnostic evaluation of dysplasias and borderline lesions is "chromosomal aneuploidy" as a marker for neoplasia. The detection of its equivalent in DNA-cytometry ("DNA-aneuploidy") may be taken as a marker for neoplastic transformation. Examples for the DNA cytometric detection of prospective malignancy are reported for cervical dysplasias, myelodysplasias and borderline cystadenomas of the ovary. Further examples are given for diagnostic assistance in the cytological detection of malignant cells in touch preparations from soft tissue and bone tumours and in urine samples. The basis for "DNA-grading" of malignancy is the fact that the modal chromosomal aneuploidy and its variability in many tumours correlate with the patients prognosis. The prognostic validity of their DNA-cytometric equivalents (modal DNA-ploidy = stemline-ploidy and 2c Deviation Index resp.) are demonstrated for chronic myelogenous leukemias and urinary bladder cancers. Other DNA-cytometric parameters may also be prognostically valid. Data, concerning the prognostic relevance of DNA-cytometry are known for at least 18 different tumour sites. The reproducibility of "DNA-grading" of malignancy is high as compared with subjective morphological grading systems. Modern technological equipment for Feulgen-staining and interactive DNA-measurements for diagnostic purposes is described. PMID- 1708580 TI - [Properties of the cell surface of normal and malignant cells: investigations on polysialic acid and terminal sialic acid residues in specific linkages]. AB - Data are reported to demonstrate the usefulness of a monoclonal anti-polysialic acid antibody for (i) the visualization of immature and mature neural elements in human teratomas, and (ii) the distinction of small cell lung carcinoma from bronchial carcinoids as well as squamous cell and adenocarcinomas of the lung. Lectins which discriminate between the various types of sialylated sequences, such as the Sambucus nigra L. I lectin specific for Neu5Ac alpha 2,6 Gal/GalNAc and the leukoagglutinin from Maackia amurensis (MAL) specific for Neu5Ac alpha 2,3 Gal beta 1,4 GlcNAc have been applied to the study of human colonic mucosa. The Neu5Ac alpha 2,3 Gal beta 1,4 GlcNAc sequence was detectable in normal and transitional mucosa and carcinomas, whereas the Neu5Ac alpha 2,6 Gal/GalNAc sequence was found in carcinomas. The lectin Amaranthin reacts with Gal beta 1,3 GalNAc-alpha (the T antigen) and NeuAc alpha 2,3 Gal beta 1,3 GalNAc-alpha (the cryptic T antigen). It stained normal and transitional colonic mucosa as well as carcinoma. The reactivity was solely due to the cryptic T antigen and indicates that the T antigen may not represent a general carcinoma autoantigen. PMID- 1708581 TI - The pluripotent hemopoietic stem cell: its identification and applications. AB - The experiments leading to the concept of a pluripotent hemopoietic stem cell (PHSC) are reviewed. The identification and morphological characteristics of the PHSC are presented and a number of current and prospective applications of grafts of PHSC are described. PMID- 1708582 TI - [The modular image analysis computer for assessment of prognosis in testicular tumors]. AB - Since 1981 a total of 107 patients with non-seminomatous germ cell tumors of the testis (NSGCT) in clinical stage I were assigned to a "wait and see" protocol. At a median follow-up of 40 months (4-100) thirty seven pts. (35%) relapsed with 84% of these within the first year. Employing the Hedley-technique out of 50 primary tumor-tissues available nuclear suspensions were obtained, staining according to the DNA-Feulgen-procedure and evaluated by the modular image analysis computer (MIAC, Leitz, FRG). In 67% hyperpentaploidy was found in the cases with progression while only 23% exhibited a greater than 5c-rate in the NED-group. With logistic regression achieving a p-value of 0.0296 hyperpentaploidy has to be considered a significant prognosticator in NSGCT/CS I. PMID- 1708583 TI - [Methodological requirements for precise measurements using DNA single cell cytometry]. AB - Methodological aspects on DNA single cell cytometry were investigated. Specimens can be homogeneously Feulgen-stained if a high constancy of temperature is realized in the staining cuvette during acid hydrolysis. For the TV image analysis system under investigation (TAS-plus, Leitz) a linear correlation (r greater than 0.99) between physically defined optical transmission values and resulting electronic signals was found. Shading effects can be controlled by using a narrow mask width (VQ less than 1.5%). As an internal standard blood cells or epithelial cells can be used if a correction factor, that was found to be mainly constant, is calculated. PMID- 1708584 TI - [DNA cytometry in gastric cancer--a comparison between static cytometry on cytologic and histologic preparations and flow cytometry]. AB - Static cytometry on cytologic and histologic preparations and flow cytometry are the basic techniques of DNA cytometry with certain advantages and drawbacks. An exact and reliable assessment of DNA ploidy and stem line heterogeneity of paraffin-embedded specimens is best performed with static cytometry on smears. The high resolution of DNA histograms in flow cytometry is reduced by the often strong portion of detritus and therefore the internal reference peak or small tumor stem lines might not be identified. The use of histologic preparations is advantageous for the analysis of correlations between histomorphologic tumor heterogeneity and stem line heterogeneity. PMID- 1708585 TI - [Histomorphometric study of normal, atypical and neoplastic pancreatic duct epithelium]. AB - Morphometric and densitometric measurements were applied to nuclei of normal, atypical (incl. hyperplastic) and neoplastic pancreatic duct epithelium in cases of inconspicuous pancreas, chronic pancreatitis and carcinomatous pancreas. These datas and their central moments and medians were used in linear discriminant analysis. A gradual shift of nuclear size and deviation from spherical form results in normal epithelium from normal pancreas to chronic pancreatitis and to carcinomatous pancreas. The atypical or hyperplastic epithelium has denser caryoplasma in chronic pancreatitis than in carcinomatous pancreas. PMID- 1708586 TI - [Centroblast morphology: a morphometric evaluation by means of computer-aided image analysis]. AB - In cytologic preparations, cell images from 30 cases of high grade malignant Non Hodgkin's lymphoma (NHL) and 10 cases of tonsillitis have been analysed using a colour TV microscope system, high resolution scanning and image processing. Multivariate analysis of 30 cell features allowed satisfying recognition and discrimination of centrocytes, centrocyte-like centroblasts, ordinary centroblasts and multilobated centroblasts. In addition, according to their image analytical data, the ordinary centroblasts from NHL formed 5, and those from tonsillitis 4 well defined, homogeneous clusters. Even at simultaneous analysis, there was an essential overlap only between centroblast subtype 3 from NHL and subtype 9 from tonsillitis, but reliable discrimination of all the rest. PMID- 1708587 TI - [Granulopoiesis in chronic myeloproliferative disorders (CMPD): electron microscopy/morphometric investigations]. AB - This pilot study comprises quantitative ultrastructural investigations of the normal, non-leukemic granulopoiesis in the bone marrow of four patients suffering from heart diseases, compared with the granulopoiesis of four patients with different types of CMPD. By evaluation of the morphometric parameters it could be demonstrated that normal granulopoiesis is subject to a strong functional order of organelles in the course of maturation, aiming at the preparation of mitoses of immature cells and the production of functionally capable specific granules. Contrary to this, the studied cases of CMPD exhibited striking differences of morphometric parameters, which may be interpreted as functional deficiencies or defects of organelles involved in the production of specific granules, with the phenomenon of dissociation of nuclear and cytoplasmic maturation. PMID- 1708588 TI - [New methods for the morphometric analysis of anisotropic tissues]. AB - Some unbiased, design-based stereological methods that have recently been developed for the study of anisotropic tissues like muscle, myocardium, brain, cartilage, and skin, are briefly reviewed. Vertical sections permit the unbiased estimation of surface density and mean volume-weighted particle volume from microscopic sections. The available experience includes various studies on malignant melanomas. In addition, the surface area of total organs (e.g., the pleural surface area) can be determined with vertical sections, which was hitherto not feasible. The orientator is a simple method to generate isotropic sections in biological material. With the orientator method it is possible to determine not only the surface density, but also the length density of the objects. Thus the method is suitable for the study of fascicular systems (tubules etc.), and for the study of vascularisation in particular. PMID- 1708589 TI - [Prerequisites for the application of statical morphometry in diagnostics]. AB - The aims of the present paper were: 1. to test a set of parameters suitable for quantifying morphological features; 2. to create a generally valuable strategy for data storage and for a user friendly data management; 3. to examine the parameter set and the conception for an optimal data management by the quantification of cytological specimens of pleural effusions. The significance of a distinction between "primary parameters" (data directly captured during the measurement) and "secondary data" (data calculated from the primary data) is explained. In the first step of classification of the cytological specimens 81.8% of the patients with reactive alterations of mesothelial cells (n = 11) and 100% of the patients with tumours (mesotheliomas and metastasis of adenocarcinomas) (n = 19) were correctly classified with the aid of a little "expert system". In the second step (discrimination between mesotheliomas (n = 6) and metastasis of adenocarcinomas (n = 13] all patients were correctly classified. PMID- 1708590 TI - [Comparison of flow cytometric, static cytometry and tumor cytogenetic investigation results human renal cell carcinomas]. AB - Published data about the frequency of DNA-aneuploidy of renal cell tumours show a great variation. The own study was performed to clarify whether this fact is due to methodological discrepancies or to intratumoural heterogeneity of DNA ploidy. The comparison of DNA ploidy assessed by flow cytometry (fresh and paraffin embedded tissue) and static cytometry (paraffin-embedded tissue) in equivalent tumour regions showed identical results. 71% of clear cell renal carcinomas showed intratumoural heterogeneity of DNA ploidy. In 55% there was either a combination of diploid and aneuploid stem lines and/or a combination of different aneuploid stem lines ("heterogenous aneuploidy"). The rate of aneuploidy was found to be higher with increasing cytoplasmic eosinophilia of the clear cell renal carcinoma cells. Studies which address the prognostic significance of DNA ploidy of renal carcinomas should specifically investigate on the significance of heterogenous aneuploidy for prognosis. PMID- 1708591 TI - [Retrospective flow-cytometric analysis of the DNA content in colorectal carcinomas and the test of the prognostic significance of DNA ploidy]. AB - The nuclear DNA content of 163 colorectal carcinomas was determined by flow cytometry (FCM) on formalin-fixed and paraffin-embedded tissue. DNA-aneuploidy was found in 97 cases (59.5%), without correlation to sex, mean age, tumor-stage (DUKES and pTNM) and grading. The frequency of aneuploidy was statistically significantly higher in patients younger than 70 years of age (p less than 0.01) and in tumors localized in the left colon and rectum (p less than 0.002). The tumors in which different areas could be analyzed (n = 80) showed a heterogeneous DNA-ploidy pattern in 18%. The comparison of DNA-content in primaries and in lymph-node metastases (n = 49) resulted in a difference of DNA-ploidy in 38% of the DNA-aneuploid tumors, but only in 6% of the DNA-diploid carcinomas (p less than 0.02). Carcinomas with DNA-aneuploidy showed a trend to a higher rate of loco-regional recurrences, a higher S-phase fraction (13.5% +/- 5.9 vs. 8.1 +/- 7.0), and proved to be associated with a poorer prognosis (p = 0.04). The statistically significantly higher mortality of patients with DNA-aneuploid carcinomas in DUKES A and B stages indicates that DNA-aneuploidy could perhaps be regarded as a stage-independent additional risk factor. PMID- 1708592 TI - [Comparative structural analyses of the spongy bone of lumbar and cervical vertebral bodies. Radiological-morphometric and statistical studies]. AB - To investigate the question as to a possible insufficiency of the osteoblasts with increasing age, autopsy material obtained from 105 deceased of both sexes at an age of 16 to 91 in whom clinically manifest bone diseases had been excluded, was studied. The structure of the spongy bone of the 3rd to 5th lumbar vertebral bodies (LVB) and of the 5th to 7th cervical vertebral bodies (CVB) was examined quantitatively-morphometrically, and the results were submitted to a statistical evaluation. In the three LVB, Vv, Sv and S/V behave in a similar manner. Here, Vv and Sv decrease after the age of 50 by more than 1/3, while S/V remains constant throughout live. In all the structural parameters, the three lower CVB have higher values than do the LVB. The age-dependent changes, in contrast, are only very slight. This differing behaviour of the spongy bone in the two regions of the spinal column is an expression of the characteristic loading forces in the respective regions: LVB loading is predominantly static, CVB loading mainly dynamic. Thus, from this functional point of view, physiological osteoporosis due to aging, so-called, is merely an expression of an aging bone adapting - in the same way as the bone of a young adult - to the current loading forces acting upon it. PMID- 1708593 TI - [Vertebral trabecular bone in various age groups and in osteoporosis-- morphometry and bone matrix biochemistry]. AB - Vertebral trabecular bone was analysed by morphometry and bone matrix biochemistry. Trabecular bone volume (TBV) and mean trabecular plate thickness (MTPT) decreased with age. TBV was significantly correlated with MTPT and mean trabecular plate density (MTPD). The individual structure of trabecular bone could be described by both MTPT and MTPD together, but changes of these parameters, that were pathognomonic for osteopenia, were not found. By measuring TBV 3 cases of severe osteopenia were identified (TBV less than 2s of controls); 2 of them showed matrix abnormalities so far not described. In one case (a 67 year old woman without risk factors for osteoporosis) an abnormal high content of type III collagen was found, in the other case (a 44 year old woman with acromegaly) bone matrix analysis atypically revealed a significant fraction of type II collagen. Further studies will be needed to assess the pathogenetic or diagnostic importance of these new findings. PMID- 1708594 TI - [AgNOR cytometry by means of automatic image analysis--a contribution to standardization]. AB - The silver staining of nucleolar organizer regions (AgNORs) was evaluated in a total of 697 tumours from ten different tissues. By means of qualitative light microscopy, type, duration and delay of fixation proved to be influential in determining the AgNOR result. For standardization of the AgNOR technique, staining time series in 30 tissue blocks of the ten types of tissue were evaluated using digital image analysis. The silver incubation time which rendered the most distinct diagnostic difference in the AgNOR content of benign and malignant tissue varied considerably. Accordingly, staining time has to be adjusted to the individual argyrophilia of each tissue block or tissue section, for which the use of internal staining standards such as lymphocytes or connective tissue was found to be mandatory. For routine purposes, the appropriate silver incubation time is achieved if AgNORs are visible as black dots mainly within the nucleoli of proliferating cells. PMID- 1708595 TI - [The role of hematopoietic growth factors in the regulation of normal and pathological hematopoiesis]. AB - Normal hematopoiesis is regulated by a family of hematopoietic growth factors that mediate the proliferation and maturation of blood cell precursors and pluripotential stem cells. Several of these factors have been isolated, biochemically characterized and are now being produced by recombinant DNA techniques in quantities sufficient for preclinical studies and clinical use. Three factors in particular have recently received considerable attention for their potential clinical use: granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and Multi-CSF or Interleukin 3 (IL-3). G-CSF is apparently lineage-specific for the proliferation of and differentiation of neutrophil granulocyte precursors. GM-CSF and IL-3 are less restricted in their action, affecting various lineages, such as eosinophil granulocytes, neutrophil granulocytes, monocytes, erythrocytes and megakaryocytes. The CSFs have many potential clinical applications including enhancing granulocyte production and function in neutropenic patients and rendering myeloid leukemic cells more susceptible to killing by cycle-specific agents. PMID- 1708596 TI - [Single cell fluorescence photometry for quantification of antigens on alveolar macrophages]. AB - The quantitation of cell surface antigens on alveolar macrophages (AM) using flow cytometry is complicated by strong autofluorescence and wide variation of cell- size and autofluorescence. Therefore a microscope-fluorometric method was developed which allows simultaneous assessment of fluorescence of fluorochrome labelled monoclonal antibodies and size in individual cells. Autofluorescence of smokers AM was found to be variable and considerably stronger than autofluorescence of nonsmokers AM. By means of a multi step determination of autofluorescence and specific fluorescence using different filter combinations of the microscope fluorometer density of membrane bound HLADR-antigens could be efficiently determined also in strongly autofluorescent smoker AM. PMID- 1708597 TI - [Microscopic DNA cytophotometry of axillary lymph node metastasis in mammary carcinomas]. AB - 65 primary mammary carcinomas and their corresponding axillary lymphnode metastases were studied. DNA-histograms were measured in imprints using microscopical Feulgen-cytophotometry (CAS 100). Comparison between DNA histograms according to AUER-typing showed a close consent in all except five cases; with a shifting from euploidy to aneuploidy (one case), and from aneuploidy to euploidy (four cases). The clinical value of these findings should be proved. PMID- 1708598 TI - [Histochemical and histomorphometric studies on growth fractions in human breast carcinoma]. AB - Nucleolar organizer regions (NOR) can easily be recognized on paraffin sections of bioptic material with an argyrophilic stain. NORs are meant to to correlate with mitotic activity of proliferating cells. This study examine wether NOR activity corresponds with the proliferations marker KI-67. 10 fibroadenomas and 40 invasive ductal breast carcinomas were studied. For objective tumor grading the size of the nuclei was determined morphometrically. NORs, KI-67 and nuclei sizes correlate closely in fibroadenomas. In carcinomas, however, a correlation between NORs, KI-67, and the size of the nuclei could not be found. NORs and KI 67 evidently mark different cell activities. For prognostic evaluation of malignant tumors they should therefore not be used alternatively but complementarily. PMID- 1708599 TI - [Capillary length in the human heart during physiological growth and under pathological conditions]. AB - Knowledge about the capillary length in mammalian and human heart is still scanty. Because it is very difficult to examine this parameter directly we used an indirect method. The distance between the arterioles can be used as an approximately measurement for the double capillary length. We tried to measure the distances between arterioles in about 30 postmortem injected hearts using India ink to produce a kind of reflected imitated tabby (tiger) heart pattern. In addition we measured the distances between arterioles with orcein staining in histological preparation of 130 human hearts including 24 hearts with congenital vitia. Our results indicate that from birth there is a determined continuous growth of capillary length with increasing heart weight during physiological development and under pathological conditions. The capillary length in hypertrophic hearts may be an additional functional impairment of the pathological hearts. PMID- 1708600 TI - [Differential diagnosis of papillary carcinomas of the thyroid, using image analysis and three dimensional reconstruction from serial sections]. AB - Papillae with fibrovascular cores are characteristic of papillary carcinoma of the thyroid. Papillae may be found in diffuse hyperplasia, nodular hyperplasia, Hashimoto's disease and follicular adenoma. Tissues from ten benign hyperplasias and ten papillary carcinomas were reconstructed from serial sections with three dimensional reconstruction programs. Significant qualitative and quantitative differences were found between the hyperplasia and the carcinoma. The principal differences between papillae of papillary carcinoma and hyperplasia were more clearly seen in the three dimensional reconstruction, than by means of morphometric methods. Certain criteria, e.g. the volume of papillae, were useful only with regard to the third dimension. Nevertheless, three dimensional reconstruction of biological tissue is a time consuming procedure which is not yet suitable for routine examination. PMID- 1708601 TI - [Proliferation-associated antigens PCNA and Ki-67 in two- and three-dimensional experimental systems of human squamous epithelial carcinomas]. AB - Multiparameter-flow-cytometry was used to compare the levels of two proliferation associated antigens, proliferating cell nuclear antigen (PCNA) and the Ki-67 related antigen, in squamous carcinoma cells grown as monolayers, multicellular spheroids (MCTS), and xenograft tumors. While the level of Ki-67-positive cells decreased with time of culture, the percentage of PCNA-positive cells stayed high in all experimental states investigated (up to 5 weeks of growth). The reduction of the mean fluorescence of PCNA/cell indicated a different way of regulation of this antigen in squamous carcinoma cells in comparison to other cell types. PMID- 1708602 TI - [Morphological demonstration of function-dependent cellular calcium redistributions in secretory cells by x-ray microanalysis, LAMMA and fluorescence cytometry]. AB - Cytosolic calcium and its parathyroid hormone induced increase was evaluated in rat osteosarcoma cells by quin 2 fluorometry. Electron microscopic calcium detection in depot organelles (i.e. the recently defined calciosomes) is improved by a new precipitation method with hydroxylamine naphthoic acid and fluoride combined with X-ray microanalysis. As shown in submandibulary gland and pancreatic B cells, a new combination of GBHA staining with laser microprobe mass analysis (LAMMA) for the first time enables direct kinetical studies of calcium at the microscopical level using stable calcium isotopes (i.e. Ca44) as tracers. PMID- 1708603 TI - [Proliferation and differentiation in megakaryopoiesis]. AB - Megakaryocytic progenitor cells only complete a limited number of mitotic events; early in megakaryopoiesis, these cells loose their capacity for cell division and acquire the ability for endoreduplication--a phenomenon that is unique to the megakaryocytic lineage. There is growing evidence, that at least two humoral activities affect proliferation of progenitor cells and maturation of its progeny. However, the cytokines that mediate these functionally defined, overlapping activities are not yet precisely known. This review summarizes current understanding of these processes and will discuss the interactions of megakaryocytic cells with the bone marrow microenvironment as well as their role in disorders of hematopoiesis. PMID- 1708604 TI - [Histology, immunocytochemistry and DNA cytophotometry of adrenocortical tumors- a clinicomorphological study of 72 tumors]. AB - Surgical specimens of 72 adrenocortical tumours were investigated by conventional histology, immunocytochemistry and DNA-cytophotometry. Histologically, 57 tumours were classified as adenomas and 15 as carcinomas. Nine adenomas weighed more, 2 carcinomas less than 50g. Only in 9 of the latter cases were distant metastases and/or lethal outcome of disease recorded, while the clinical course of the remaining patients was uneventful. No significant differences in DNA content were found between adenomas and carcinomas or between carcinomas with aggressive and indolent behaviour. Neither could immunocytochemistry discriminate between these conditions. Immunostaining with the monoclonal antibody D 11 proved to be the only effective means to definitely type adrenocortical neoplasia. Thirty-one cases exhibited positivity upon immunostaining with a polyclonal antiserum against synaptophysin. This phenomenon has so far not been encountered in non neuroendocrine neoplasia. PMID- 1708605 TI - [Histology, immunocytochemistry and DNA cytophotometry of adrenal glandpheochromocytoma (PCC)--a morphologic clinal study of 64 tumors]. AB - Surgical specimens of 64 adrenal PCCs were investigated by conventional histology, immunocytochemistry and DNA-cytophotometry. Tumour weights of more than 200 g were recorded for each of the 6 malignant, but for only one of the 58 clinically benign neoplasms. Increased mitotic activity (greater than 5 mitoses/10 HPF) was, apart from one benign lesion, only seen among malignomas. Immunocytochemically, all malignomas were entirely devoid of S100-positive sustentacular cells and, as compared to benign PCCs, showed reduced expression rates of different neuropeptides. Upon cytophotometry, only 5/43 tumours exhibited euploid DNA histograms, all these cases belonging to the group of benign PCC. According to these findings, morphology does not enable a definite prediction of the clinical course of individual PCC cases, but renders the definition of risk groups possible. A benign diagnosis can be made in multihormonal euploid tumours weighing less than 200 g. In larger neoplasms and in cases lacking sustentacular cells and showing increased mitotic activity, an unfavourable prognosis is to be suspected and the same therapeutic procedures should be applied as for tumours in which malignancy is evident in metastatic growth. PMID- 1708606 TI - Detection of infectious agents by molecular methods at the cellular level. AB - The role of molecular methods for the detection of infectious agents at the cellular level continues to grow. For the future PCR and in situ PCR methods appear to hold great potential as adjuncts to current methods, but not without overcoming some obstacles. PMID- 1708607 TI - [PCR: DNA amplification from histological sections]. AB - Specific DNA sequences can be amplified from tissue material by means of the polymerase chain reaction (PCR) using oligonucleotides homologous to upstream and downstream flanking regions as primers for repeated cycles of Taq polymerase mediated DNA synthesis (primer extension) in vitro. The amplification product provides the unique possibility to analyze genomic alterations (mutations, deletions, translocations) which may play a role during pathogenetic processes, or to detect heterologous (viral, bacterial) nucleic acids with maximum sensitivity. PCR with morphologically defined material from histologic sections gives the chance to bridge the gap between morphological description of a disease and the underlying molecular alteration. PCR from sections can be performed even from paraffin-embedded material of archival specimens. As an example a ras gene mutation analysis of human colorectal cancers and their metastasis and of human seminomas is presented. Only minute amounts of biological material are required for PCR, as exemplified with material punched from defined preneoplastic areas in rat liver cryostat sections. Using this material, not only a thorough mutational analysis of DNA of preneoplastic foci is possible after a simultaneous PCR amplification of various genomic sequences, but also an investigation of transcription activity after reverse transcription of mRNA into cDNA, as shown for c-myc expression during preneoplasia. The extremely high sensitivity of the method requires severe precaution with respect to contamination, and product control by Southern blots or sequencing. PCR from histological sections will become a valuable tool for analyzing molecular mechanisms of disease based on the classical morphological parameters of pathology. PMID- 1708608 TI - [Oncogenes and oncogene products--possibilities and significance of their detection]. AB - Diagnosis- and/or prognosis-related alterations of (proto) oncogenes may be detected in neuroblastoma (N-myc), carcinoma of breast and ovary (HER2/neu), NHL (c-myc, bcl-2), CML (c-abl/bcr), and some other neoplasias. A wide variety of methods for the detection of gene alterations can be applied. The methods of detection have to be chosen according to the expected mechanisms of oncogene activation, the availability of adequately prepared tissue, and the technical standard of the laboratory. The sensitivity, specificity, and quantitation of morphological techniques (immunohistochemistry and in situ hybridization) is restricted and their results have to be interpreted most carefully. Whenever possible, at least two different techniques should be used, preferably on two different levels, i.e. RNA/DNA and protein. Furthermore, the combination of morphological and non morphological methods should be aspired. PMID- 1708609 TI - [Tumor suppressor genes: identification and role in malignant transformation]. AB - Two lines of evidence have suggested that the loss of tumor suppressor gene function contributes to the multi-step process of tumorigenesis. Tumorigenicity and transformed phenotypes are frequently suppressed in somatic cell hybrids of tumor and normal cells. Consistent chromosomal aberrations indicating gene losses and mutations have been found in hereditary and sporadic human tumors. Tumor suppressor genes can be molecularly cloned, even if the encoded protein products have not been characterized biochemically. Here we describe a functional assay based on DNA transfection permitting the molecular identification of candidate human tumor suppressor genes (R. SCHAFER et al., Proc. Natl. Acad. Sci. USA 85, 1590-1594, 1988). A molecular clone has been identified capable of suppressing the neoplastic phenotype induced by an oncogene belonging to the family of ras genes, whose activation is frequently found in tumors. PMID- 1708610 TI - [Cytoskeleton--function and pathology: studies on the pathology of the intermediate filament cytoskeleton of liver cells]. AB - Microfilaments, microtubules and intermediate filaments (IF) are major filamentous components of the cytoskeleton and play a role in the modulation of cell shape, in cellular movements, cellular stability, intracellular organisation as well as cell-to-cell and cell-to-stroma interactions. Particular interest was concentrated in the last few years on IF because of their cell type-specificity and, consequently, their suitability as cell markers in diagnostic pathology. Despite their apparent stability, IF are dynamic structures which may be modified under pathologic conditions. In recent years, pathologic alterations related to the IF cytoskeleton have been described in a diversity of chronic and degenerative disorders, including alcoholic hepatitis and neurologic diseases (e.g. M. Alzheimer, M. Parkinson). Our studies were particularly devoted to the elucidation of the pathogenesis of severe alcoholic liver injury (alcoholic hepatitis), which is associated with inflammation, liver cell degeneration and necrosis and morphologically characterized by the appearance of cytoplasmic hyaline inclusions (i.e., Mallory bodies). In the present review morphologic, immunologic and biochemical studies on nature and pathogenesis of Mallory bodies are summarized. Moreover, similarities between Mallory bodies and other cytoskeleton-related inclusion bodies suggest common routes of pathogenesis. Consequently, studies along these lines may not only lead to the understanding of mechanisms involved in alcoholic injury but may also provide information on general principles of cell damage as well as on regulation and function of the IF cytoskeleton. PMID- 1708611 TI - [Immunophenotypic and genotypic characterization of T-cell populations in thymomas]. AB - The T cell component of eight thymomas has been studied by immunohistochemistry and Southern blot analysis. In the cortical areas of thymomas the T cells expressed an immature cortical phenotype. In cortical and medullary areas T cell Receptor (TcR) alpha-beta lymphocytes heavily predominated over TcR gamma-delta lymphocytes. No genomic rearrangement of TcR and Immunoglobulin genes could be detected, thus supporting the non neoplastic nature of the lymphocyte component in thymomas. PMID- 1708612 TI - [Significance of neuronal acetylcholine receptors as differentiation antigens in neuroblastomas and paragangliomas]. AB - Using a panel of monoclonal antibodies to various epitopes of the alpha-, beta- and gamma-subunit of the muscle acetylcholine receptor (AchR), two different immunohistochemical reactivity patterns--corresponding to different neuronal AchRs--were identified in ganglia of the peripheral and central nervous system. The immunoreactivity pattern of neuroblastomas and paragangliomas was identical to the pattern found in the peripheral nervous system and has not been encountered in any other tumor type. Both the intensity of neuronal AchR immunoreactivity and the transcription of the neuronal AchR alpha-gene seem to correlated with a higher degree of neuroblastoma differentiation. PMID- 1708613 TI - [Phenotypic and genotypic characterization of splenic infiltrates in hairy cell leukemia]. AB - Cells of splenic infiltrates of five patients with hairy cell leukemia have been analyzed for the expression of immunoglobulin by immunohistochemistry and for rearrangement of T cell receptor and immunoglobulin genes by the Southern blot technique. In each case surface immunoglobulins were found with monoclonal expression of light chains. Concomitantly, heavy and light chain gene rearrangements were present. These results confirm the B cell nature of hairy cells. PMID- 1708614 TI - [Cytogenetic and clinical features of Philadelphia chromosome positive leukemias]. AB - The Philadelphia chromosome defines chronic myeloid leukemia, and is mostly based on a translocation t(9;22) with a typical BCR-ABL rearrangement which also occurs in so called atypical translocations. The transformation of chronic myeloid leukemia is associated with clonal evolution in 80% of cases. The appearance of an isochromosome 17q unequivocally heralds the onset of a myeloid type of blast crisis. Treatment of Ph-positive CML has still to be considered palliative except for allogeneic bone marrow transplantation. The Philadelphia chromosome is also found in about 20% of patients with acute lymphoblastic leukemia and in about 2% of patients with nonlymphoblastic leukemia. It is associated with a poor prognosis. Molecular and cytogenetic findings help differentiating between de novo acute leukemia and blast crisis of chronic myeloid leukemia. PMID- 1708615 TI - Characterisation of the osteoblastic phenotype by the use of differential hybridization. AB - To identify genes active in cells of the osteoblast lineage we have begun to characterise the phenotype of the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. We have used the method of differential hybridization to identify cDNA clones encoding mRNA species which are expressed in ROS 17/2.8 cells but not in a non-osteoblast-like osteosarcoma cell line (ROS 25/1). We have identified a number of gene products exhibiting this pattern of expression. PMID- 1708616 TI - [Comparative studies on collagen expression of chondrocytes in monolayer and spheroid culture]. AB - Dedifferentiation of cells is well known in cell culture biology. This phenomenon is examined in comparative studies on collagen expression of chondrocytes in monolayer and spheroid culture. Immunohistochemical studies were carried out by the indirect peroxidase technique. In the differentiated state a positive reaction for collagen type II was found. This was lost as dedifferentiation took place, in which case positivity for collagen types I, III, and V increased. PMID- 1708617 TI - [Application of silver acetate autometallography in histopathology: a new detection method for use in immunogold silver staining, lectin histochemistry and in situ hybridization]. AB - Modern histochemical techniques allow the specific detection of tissue constituents in situ. Routinely formalin fixed, paraffin embedded tissues may present problems to the pathologist since destruction of substances can lead to false negative results. Immunogold-silver staining (IGSS) can be a way to overcome some of these problems. A highly efficient method allows silver enhancement to be carried out without the necessity of a dark box (silver acetate autometallography). To test the new modification, antisera to a variety of antigens were used on paraffin and semithin resin sections. The system was also tested for its applicability in multiple immunostaining techniques and for the in situ hybridisation of viral DNA and for demonstration of carbohydrates by lectin histochemistry. It showed a very high detection efficiency with low background staining. PMID- 1708618 TI - [Immunohistologic photometric quantification of pyruvate kinase content of rat tumors]. AB - 86 benign and malignant tumours were investigated histologically and immunohistologically. Staining intensity was measured by scanning-photometry. The computed optical density was digitalized and evaluated with an image analysis system. The results indicate a significantly higher amount of M2-PK in malignant tumours. In the adenocarcinomas of the mammary gland, the content of the M2-PK increased parallel with malignancy (p less than 0.01; r = 0.743). The M2-PK can serve as an indicator of the grade of malignancy in mammary tumours of the rat. PMID- 1708619 TI - [Demonstration of polysialic acid and N-CAM in neuroendocrine tumors]. AB - The neural cell adhesion molecule (N-CAM) is a general calcium-independent cell adhesion molecule. A common structural feature of N-CAM is the presence of homopolymers of alpha-2,8-linked sialic acid residues, which regulates the homophilic adhesive properties of N-CAM. N-CAM undergoes a change from a highly sialyated to a less sialylated form from embryonic to adult life. We have investigated the presence of N-CAM, using a monoclonal antibody against polysialic acid, and polyclonal antibodies against N-CAM. We found the presence of highly sialylated N-CAM in pheochromocytomas, medullary carcinomas of the thyroid, neuroblastomas, small cell cancer of lung and pituitary adenomas. We failed to visualize N-CAM in neuroendocrine tumors of various organs (carcinoids), melanomas and pancreatic endocrine tumors. The visualization of polysialic acid and N-CAM could serve as an additional criterium for the differential diagnosis of neuroendocrine tumors. PMID- 1708620 TI - [DNA extraction and Southern blot analysis in paraffin embedded material]. AB - DNA was extracted from formaldehyde fixed and paraffin embedded tissue by the use of a modified extraction protocol. In all cases the recovered DNA was more degraded than DNA from fresh or frozen tissue. Fixation times of more than 4 days made it impossible to use the extracted DNA for Southern Blotting; DNA from paraffinized tissues not older than 1 or 2 years could be used for Southern Blotting after digestion with restriction enzymes, but we did not succeed in showing bands with restriction fragments more than 10 kb in length. We conclude that SBA analysis for malignant lymphomas with DNA extracted from routinely paraffin-embedded tissues isn't possible, because the restriction fragments looked for are in many cases longer than 10 kb. PMID- 1708621 TI - [Human lymphocyte transformation after immortalization by transfection with cytoplasmic DNA from mouse L cells of the cytokines IL-1alpha, IL-6 and TNF]. AB - Human lymphocytes from peripheral blood were induced to proliferate indefinitely in vitro by transfection with cytoplasmic DNA isolated from transformed mouse L929 cells. Two cell lines analyzed proliferate in chemically defined, serum-free media without addition of cytokines. Using RIA and bioassays, the cells were found to secrete IL-1 alpha, IL-6, and TNF into the culture medium whereas no IL 2, IL-4, BCGF, CSF, or IFN-gamma were detected. Northern blot hybridizations revealed TNF alpha mRNA (1.5 kb) as well as TNF beta mRNA (1.4 kb) transcripts in RNA of the immortalized cells. Since the immortalized lymphoid cell lines neither form colonies in soft agar medium nor induce tumors after injection into immunodeficient nude mice, we suggest that these cytokines may be involved in autocrine growth stimulation of human lymphoid cell lines immortalized by transfection with cytoplasmic DNA from mouse L-cells. PMID- 1708622 TI - [Epstein-Barr virus and malignant lymphomas--studies on incidence and localization of viral DNA]. AB - Tissue specimens from 198 cases of Hodgkin's disease and 151 non-Hodgkin lymphomas as well as 34 non-malignant lymph node biopsies were examined for the presence of Epstein-Barr virus (EBV) DNA by polymerase chain reaction. EBV specific DNA sequences were detected in DNA extracts of 58% of Hodgkin's disease biopsies. Non-Hodgkin lymphomas and benign lymph node lesions were associated with EBV in far smaller proportions. In Hodgkin's disease biopsies, subsequent in situ hybridization revealed an exclusive localization of EBV DNA in the tumour cells, suggesting an involvement of EBV in the pathogenesis of Hodgkin's disease in a substantial proportion of cases. PMID- 1708623 TI - [Demonstration of DNA tumor viruses in carcinomas of the upper respiratory tract]. AB - In situ hybridization (ISH) using 35S-labeled probes was applied to the detection of viral DNAs in 115 epithelial tumours from various sites. Epstein-Barr virus (EBV) DNA was detected in 83% of undifferentiated nasopharyngeal carcinomas (NPC) while none of seven squamous cell NPC displayed an EBV-specific autoradiographic signal. Also, all other tumours investigated, including thymomas, thymic carcinomas, tonsillar carcinomas and medullary breast carcinomas were negative upon ISH to EBV-specific probes. These results suggest a unique association of undifferentiated NPC with EBV. 28 tonsillar carcinomas were further analyzed for the presence of human papillomavirus (HPV) DNA. Six carcinomas showed an HPV16 specific signal while hybridization to HPV6- and HPV11-specific probes yielded negative results. No HPV-DNA was detected in any of 30 tonsils with chronic inflammation, thus excluding that the finding of HPV16 in a proportion of tonsillar carcinomas is merely circumstantial. These results point to a possible etiologic role of HPV16 in the pathogenesis of some tonsillar carcinomas. PMID- 1708624 TI - [Molecular biological demonstration of parvovirus infection in fetal tissue]. AB - We report on the morphological findings in 16 fetuses with serologically confirmed maternal parvovirus B19 infection. Typical routine morphological findings of the hydropic fetuses were the presence of abnormal erythroblasts with typical nuclear inclusions. These infected cells positively stained immunohistochemically with antibodies against a recombinant virus protein, as well as they ultrastructurally contained virus particles. Using in-situ hybridization techniques, we were able to demonstrate the presence of parvovirus B19 genome in the infected cells, while no other cell type was shown to contain virus genome. According to our results the differential diagnosis of fetal parvovirus B19 infection should be considered in each case with hydrops fetalis of unknown origin. The careful routine microscopic examination of fetal tissue may provide evidence for parvovirus infection which should be confirmed by in situ hybridization analysis. PMID- 1708625 TI - [Virus demonstration and pathologic changes in different phases of coxsackievirus B myocarditis in mice]. AB - A/J mice between 15 days and 10 weeks of age were infected intraperitoneally with Coxsackievirus B3 (CVB3). To search for virus in the myocardium various methods were applied: virus isolation from the myocardium, RNA extraction for dot blot hybridization and in situ hybridization. Two different RNA probes, one specific for CVB3 the other cross-reacting with other enteroviruses, were radioactively labeled with 35S or 32P by in vitro transcription. In paraffin sections histological alterations were assessed semiquantitatively. The animals developed acute myocarditis with myolysis and virus in the myocardium until 14 days after infection. The second stage of the disease was characterized by a persistent inflammatory infiltrate. At this stage no virus could be shown in the myocardium. Antibodies against cardiac myosin appeared 16 days after infection. Autoimmune mechanisms thus seem to be a most relevant factor for persistent inflammation after the acute viral phase of the disease. PMID- 1708626 TI - [Patterns of acute and persistent infections in enteroviral heart diseases]. AB - Enteroviruses are considered as the major etiologic agents of myocarditis in humans. Recent in situ hybridization studies on endomyocardial biopsies indicate that not only in acute myocarditis (pattern of acute infection), but also in chronic dilated cardiomyopathy enterovirus RNA can be detected (pattern of persistent infection). Our experimental studies on murine coxsackievirus B3 myocarditis provided evidence that persistent infection occurs also in mice. Quantitative in-situ hybridization and immunohistochemistry as well as electron microscopic in situ hybridization experiments were performed on ACA/SnJ mice three to thirty days after infection. The pattern of acute infection (days 3-9 p.i.) is characterized by rapid progression of myocardial lesions, an increasing number of inflammatory cells and a high number of infected myocytes. Hallmarks of the persistent pattern (day 15-30 p.i.) are reduced inflammation, reduced numbers of persistently infected cells and a slow progression of myocardial lesions. Infection is primarily restricted to degenerated, atrophic myocytes and to fibroblasts. PMID- 1708627 TI - [Nicotinic acetylcholine receptors in tumors with rhabdomyomatous differentiation. Immunohistochemical amd molecular genetic demonstration]. AB - The nicotinic acetylcholine receptors (AChRs) of the skeletal muscle consist of pentameric ion channels, each of which is composed of 4 kinds of subunits. During the development of the muscle a change from a fetal type (alpha beta alpha gamma delta) to an adult type (alpha beta alpha epsilon delta) of AChR is due to the replacement of the gamma-subunit with the epsilon-subunit. We investigated the expression and transcription of the AChRs in rhabdomyosarcomas (5 cases) and in nephroblastomas with rhabdomyomatous differentiation (2 cases) by immunohistochemistry using monoclonal antibodies to the alpha-, beta- and gamma AChR-subunit, and by mRNA dot blot and in situ hybridization applying cDNA-probes of the alpha-, beta-, gamma-, delta- and epsilon-AChR-subunit. The results indicate an intratumorous coexpression of fetal and adult types of AChRs. The presence of gamma-subunits of fetal AChRs may serve as a novel selective marker of tumors with rhabdomyomatous differentiation. PMID- 1708628 TI - [Demonstration of activated oncogenes of the ras family in human thyroid tumors using the polymerase chain reaction]. AB - Activation of ras-oncogenes in 180 different thyroid tumours, was examined by using polymerase chain reaction (PCR) amplification and subsequent analysis of the amplification products by oligonucleotide probing, direct sequence analysis of single stranded DNA and primer-mismatch analysis. In contrast to recently published results we found a very low frequency of activated ras-genes occurring mainly in undifferentiated carcinomas and follicle cell carcinomas which show a transition to undifferentiation. In none of thirty benign tumors an activated ras gene was found. The predominant ras-oncogene activated in thyroid neoplasias was the Harvey-ras gene. Our results suggest Harvey-ras activation at a later evolutionary step in tumour genesis. PMID- 1708629 TI - [Demonstration of the differential expression of IGF-II in various embryonal kidney tumors using slot-blot hybridization and in-situ hybridization]. AB - IGF-II may play a decisive role in the development of Wilms tumors since an elevated expression of this important embryonal growth factor has been reported in the majority of nephroblastomas. In our series of 15 typical triphasic or blastemal predominant nephroblastomas slot-blot-hybridization revealed a marked increase of IFG-II-mRNA in the tumor tissue of 11 patients. Compared to normal kidney tissue IGF-II-expression was elevated up to 64 times. Apart from nephroblastoma a number of other embryonal renal tumors with either a much better or much worse prognosis was investigated. A moderately increased expression of IGF-II was noted in 2 congenital mesoblastic nephromas. IGF-II-mRNA was only slightly increased in a clear cell sarcoma of the kidney but was not elevated in 2 malignant rhabdoid tumors of the kidney. In-situ-hybridization allowed precise localization of the markedly increased IGF-II mRNA to blastemal cells. Differentiation to epithelial structures such as tubules or glomeruli or to stromal cells was associated with a marked loss in IGF-II expression. PMID- 1708630 TI - [Pattern of intermediate filaments in thymomas]. AB - The cytokeratin pattern of medullary, mixed and cortical thymomas and thymic carcinomas were analyzed by two-dimensional equilibrium electrophoresis. Extracts from a medullary thymoma and normal, total thymi showed a similar pattern. On the other hand in cortical thymomas and very similar in thymic carcinomas there were marked changes in the cytokeratin pattern. These findings support the classification of thymomas as described by MULLER-HERMELINK. PMID- 1708631 TI - [Diagnosis of multiple endocrine neoplasms type IIa using DNA analysis]. AB - The gene causing MEN IIa has recently being assigned to the pericentromeric region of chromosome 10. We performed linkage analysis using DNA-markers closely related to the chromosomal locus at chromosome 10: MCK II, retinol binding protein cDNA and cTBIRBP-9. Available for the study were EDTA blood from two families. The analysis was positive in two asymptomatic offsprings in one family (B), whereas the markers were not informative in the other family (A). Genetic distance of the informative marker of family A to MEN IIa gene is 2 cM, i.e. a likelihood of 98% (95% up a confidence limit with 5%) for the gene carrier status of two children aged 11 and 7 y old. The following clinical investigation including pentagastrin test, plasma catecholamines and 24 hour urine catecholamines and parathormone was negative until now. We recommend early linkage analysis for establishing the genetic status in offspring of MEN IIa families to focus further screening to those, who are predicted to be gene carrier. PMID- 1708632 TI - Stem cell origin of human myeloid blood cell neoplasms. AB - Studies with G6PD and molecular probes indicate that the myeloid leukemias and the chronic myeloproliferative disorders are clonal diseases. The G6PD data indicate that chronic myelogenous leukemia, polycythemia vera and essential thrombocythemia involve stem cells pluripotent for granulocytes, erythrocytes, megakaryocytes and lymphocytes. Agnogenic myeloid metaplasia is also a clonal disease that involves multipotent hematopoietic stem cells. However, myelofibrosis, the predominant clinical manifestation, occurs secondarily and is not a component of the abnormal clonal proliferation. Acute nonlymphocytic leukemia is a clonal disease, but G6PD studies suggest that there are at least two forms of this leukemia. In one type of ANL, the involved stem cells exhibit pluripotent differentiative expression. In another type of ANL, differentiative expression is largely restricted to the granulocytic pathway. The heterogeneity of ANL has both clinical and pathogenetic implications. PMID- 1708633 TI - [Induction of primitive neuroectodermal tumors by oncogene complementation]. AB - Primitive neuroectodermal tumors (PNET) represent a family of undifferentiated neural neoplasms which predominantly occur in children. PNETs are likely to originate from precursor cells and exhibit a marked potential for neuronal, glial and ependymal differentiation. A prominent example is the medulloblastoma of the cerebellum. In a model system using a novel transgenic CNS transplantation model, we have introduced a combination of ras and myc oncogenes into cell suspensions from fetal forebrain (E14) and postnatal cerebellum (P2) of the rat. Oncogene transfer into fetal forebrain grafts resulted in a high incidence of anaplastic neural tumors predominantly derived from glial precursors. Cell lines established from these neoplasms expressed high levels of both oncogenes. In a second experiment, the ras/myc vector was introduced into cell suspensions from neonatal cerebellum. The transformed cells were cultured for 3 weeks. Following stereotaxic transplantation, tumors of a similar morphology were observed. However, one animal developed a neoplasm with features of a cerebellar medulloblastoma. A cell line which exhibits a marked capacity for neurite extension and synaptogenesis was established from this tumor. Since these cells neither express ras nor myc, an insertion mutagenesis event appears to be responsible. Experiments to characterize this mutation are in progress. Our results indicate a potent transforming effect of ras and myc on neural precursor cells in vivo. PMID- 1708634 TI - [Evaluation of the quality of remission in leukemias using molecular genetic methods]. PMID- 1708635 TI - [Clonality and stem cell defects in the molecular pathology of chronic myeloproliferative disorders]. AB - The chronic myeloproliferative disorders (CMD) are characterized by sustained or progressive proliferations of bone marrow cells, affecting one or more lineages in varying combinations. They are classified in 4 subgroups but transitional forms and transformations among the different entities are common. Analysing RFLPs and methylation patterns of X-chromosomal genes we could show, that in each entity granulocytes as well as bone marrow cells are of monoclonal origin. These findings support the view that all forms of CMD have in common that they arise from a multipotent hematopoietic stem cell. CML can be differentiated from all other forms of CMD by the Philadelphia-chromosome which on the molecular level has been revealed as bcr-abl gene junction. We investigated 258 cases of CMD for rearrangement of the bcr gene and found that it selectively occurred in CML. The methods applied can be of diagnostic value in differentiating reactive from neoplastic proliferations by analysis of clonality, and in differentiation of CML from other types of CMD by detection of the bcr-rearrangement. PMID- 1708636 TI - [Pathology of hematopoietic stem cells. 21st Fall Meeting. Mainz, 19-21 October 1990]. PMID- 1708637 TI - [Role of hemopoietic stem cells and progenitor cells in preleukemia]. AB - Cases of myelodysplastic syndrome (MDS) that have evolved into overt leukemia may be retrospectively termed preleukemia. Studies with X-chromosomal markers in females strongly suggest that preleukemia originates from transformation of a very early hemopoietic stem cell. Growth characteristics of myeloblasts in preleukemia indicate that some of the cases already represent early stages of acute leukemia. In other preleukemias clonal evolution towards overt leukemia can be observed. This apparently involves neoplastic transformation of a granulocytic determined progenitor cell. PMID- 1708638 TI - [List of members]. PMID- 1708639 TI - [Stem cell proliferation and differentiation of lymphopoiesis]. AB - During differentiation and maturation well defined genetic rearrangements of B- and T-cell receptors are followed by a stepwise expression of defined phenotypic properties. Transformational events on such defined steps of cell differentiation result in different types of malignant lymphomas, some of which have these events during early steps of cell differentiation but show further maturation or activation. This is shown for the plasmacytoma but may also be true for Hodgkins disease or large cell anaplastic lymphoma. Inducing signals for maturation, differentiation, and activation are given by cytokines. The loss of a regular function of these cytokines may result in para- or autocrine growth signals in malignant lymphomas. PMID- 1708640 TI - [Function and pathology of bone marrow stromal cells]. AB - For their lineage commitment and differentiation haematopoietic tem cells need a specific microenvironment. This milieu is formed by bone marrow stroma, which includes lymphocytes, macrophages, fibroblasts, reticular cells, adipocytes, osteogenic and endothelial cells. These cells play different regulatory roles in supporting haemopoiesis. The stroma-stem cell interaction take place by immediate cellular contact. Growth factors and extracellular matrix molecules produced by stromal cells influence the regulation of haemopoiesis as well. This cellular context could be established in different animal models. Impairment of stromal cell function with increased fibroblast proliferation and increased collagen synthesis is an impressive morphological feature of myelofibrosis. PMID- 1708641 TI - Mapping of the ARS-like activity and transcription initiation sites in the non canonical yeast mitochondrial ori 6 region. AB - The insert-containing, non-canonical ori 6 region of yeast mitochondrial DNA of Saccharomyces cerevisiae was dissected into 15 different segments that were ligated to the integrative yeast vector YIp5. Six recombinant plasmids exhibited replicative ability in yeast and carried consensus sequences similar to the previously described 11 bp motifs active as autonomous replication sequences (ARS). In addition, all active constructions carry one or more of the characteristic GC-rich domains A, B or C present in the ori 6 region, thus confirming and expanding the study of Blanc (Gene 30 (1984) 47-61) with the canonical ori 5. Also a new transcriptional origin is activated in the ori 6 region, apparently circumventing a disruption by insertion of a GC-rich sequence that, in this ori, removes the mitochondrial promoter usually present next to the C element. The ARS-positive constructions correspond to the retained segments of spontaneous well-characterized suppressive or neutral petite genomes that contain segments of the ori sequence. PMID- 1708642 TI - Routine immunohistochemical characterization of short term in vitro explants from human intracranial tumours. AB - 35 intracranial tumours, 18 gliomas, 12 meningiomas, one neurilemmoma (neurinoma), one malignant melanoma and two metastases were successfully grown in vitro and were submitted to immunocytochemical reactions, including cytokeratin, glial fibrillary acid protein (GFAP), vimentin, fibronectin, S-100 protein, neurofilament proteins, neuron-specific enolase (NSE) and basic myelin protein (MBP). Cytokeratin in metastases, GFAP and vimentin in gliomas, vimentin in meningiomas were consistently positive. S-100 protein was weakly and partially positive in gliomas, meningiomas, the neurilemmoma and malignant melanoma. Positive demonstration of fibronectin within cells was interpreted as a consequence of phagocytosis, except in meningiomas where fibronectin expression next to cell membranes seemed genuine. All other tested markers proved negative. The most important result seems to be that cells expressed markers irrespective of cellular shape and cytological morphology. It can be concluded that the cellular population as a whole consisted of tumour cells during the short time under observation and that supportive cell contamination during this early growth period was negligible. PMID- 1708643 TI - Histological and immunocytochemical findings in a case of fetal choroid plexus papilloma. AB - Reported in this paper is a case of a fetus delivered in the 24th week of pregnancy whose intracranial space was found to be almost totally filled up by a choroid plexus papilloma. Co-expression of vimentin and cytokeratin 8, 18 of the epithelium was immunocytochemically observed, as had been also described in normal fetal choroid plexus. PMID- 1708644 TI - Adult Wilms' tumour. A case report with immunohistochemical studies. AB - The case of a 27-year-old female patient presenting with a Wilms' tumour is reported. The tumour was dominantly of blastemal type showing minor epithelial (tubular) areas and anaplastic portions. Thorough immunohistochemical examinations were performed to solve differential diagnostic problems. Keratin positivity was found to be of differential diagnostic value in the distinction of this tumour from the so called small round cell tumours. Prognostic aspects of Wilms' tumour are also discussed. PMID- 1708645 TI - Diffusion-weighted MR imaging and T2-weighted MR imaging in acute cerebral ischaemia: comparison and correlation with histopathology. AB - Diffusion-weighted MR imaging is a new technique which measures the microscopic motion of water protons. Signal hyperintensity on diffusion-weighted images correlates closely with evidence of ischaemic damage on histopathologic sections. Following occlusion of the middle cerebral artery (MCA), diffusion-weighted images indicate the presence of early pathophysiologic changes occurring first in the basal ganglia and, subsequently, in cortical gray matter within the MCA vascular territory. Diffusion-weighted images also better define the anatomic locus of ischaemic tissue injury than T2-weighted images. Diffusion-weighted imaging thus appears to facilitate early detection and thereby possible therapeutic intervention in patients with acute stroke. PMID- 1708646 TI - Survival and fibre outgrowth of neuronal cells transplanted into brain areas associated with interstitial oedema. AB - The influence of interstitial oedema on the survival of fetal raphe cells transplanted into serotonin (5-HT)-denervated rats and the fibre outgrowth from these cells was investigated. Fetal raphe cells were transplanted into the corpus callosum in which long-lasting interstitial oedema had been induced by intracisternal kaolin injection. The 5-HT and 5HIAA levels in the corpus callosum were restored to their maximum within 5-6 weeks post-transplantation regardless of whether interstitial oedema was induced or not. Furthermore, it was appeared that the presence of interstitial oedema even facilitated fibre growth as demonstrated by the 5-HT immunohistochemistry and the restoration of the 5-HT and 5-HIAA levels in brain areas distant from the transplantation sites. These results imply favourable effects of interstitial oedema on the survival of transplanted raphe cells and their fibre outgrowth. PMID- 1708647 TI - Autoradiographic patterns of brain interstitial fluid flow after collagenase induced haemorrhage in rat. AB - Cerebral oedema accompanies intracerebral haemorrhage. We induced intracranial bleeding by the intracerebral injection of bacterial collagenase. There was oedema observed both at the haematoma site in the caudate/putamen and bilaterally in the hippocampal regions. To determine the role of vasogenic oedema spread from the site of injury, we studied by autoradiography the distribution of extracellular markers injected along with the collagenase. Both 14C-dextran (m.w. 70,000) and 14C-sucrose (m.w. 341) spread away from the injection site into both hippocampal regions in a similar pattern, suggesting bulk flow. Vasogenic oedema secondary to a haemorrhagic lesion in the caudate/putamen is an important cause of the oedema observed in both hippocampal regions in our model. PMID- 1708648 TI - Blood-brain barrier damage in traumatic brain contusions. AB - Plasma proteins were used as an endogenous marker of blood-brain barrier damage in 19 patients dying with traumatic cortical contusions. Patients survived for a few hours to 31 days after head injury. Eight proteins (M WT 61-2,500 x 10(3) were demonstrated with standard immunohistochemical techniques. Proteins were not found in "control" brains or in macroscopically normal parasagittal cortex in the head injury patients. Proteins were found in all of the macroscopic contusion in all brains. Protein leakage appeared to be from the contusion itself. Protein staining around histologically normal vessels was unusual. There was a gradient of staining from the macroscopic contusion into the surrounding brain. There was a trend for staining to be most marked between 3 and 8 days survival after head injury. There was no gradient of leakage by molecular size of the protein. PMID- 1708649 TI - Passage of DMP across a disrupted BBB in the context of antibody-mediated MR imaging of brain metastases. AB - To study the possible application of monoclonal antibody/dextran-magnetite conjugates in specific MR imaging of brain metastases, both components of these conjugates were tested for their ability to penetrate the endothelium during conditions of local blood-brain barrier (BBB) impairment. The passage of dextran magnetite particles (DMP) across a disrupted blood-brain barrier was studied in a freezing lesion model using electron microscopy (EM) and MR imaging. One hour after i.v. injection, focal accumulation of DMP in capillary endothelial cells within the freezing lesion was shown by EM. In parallel with this, MR imaging indicated a strong contrast enhancement in the lesion. EM observations showed that the particles were still present in the endothelial cells four and eight hours after injection. The passage of an anti-small cell lung cancer (SCLC) monoclonal antibody across the endothelium of intracerebrally xenografted human SCLC was studied using immunohistological techniques. It was found that passage across endothelial cell occurred in the tumor within four hours after injection. PMID- 1708650 TI - Human gammaglobulin use in the treatment of severe thrombocytopenia associated with sarcoidosis. AB - Thrombocytopenia associated with sarcoidosis is an uncommon, yet potentially lethal, complication. The traditional treatment for the thrombocytopenia has been steroid therapy followed by splenectomy if steroid therapy fails. The use of human immunoglobulin as a potential therapy in a patient afflicted with thrombocytopenia and sarcoidosis is reviewed. PMID- 1708651 TI - [Evaluation of fetal antigenic stimulation in physiological and complicated pregnancy]. AB - Fetal hemoglobin and trophoblastic beta 1-globulin levels have been determined in women with normal and complicated pregnancy in order to evaluate the rates of fetal antigenic stimulation. It was demonstrated that trophoblastic beta 1 globulin may not exactly represent fetal antigenic structures, but it is a marker of functional feto-placental activity. An elevated percentage of HbF in blood in threatened+ abortion and early toxemia of pregnancy confirmed feasibility of using it as an indirect marker of fetal antigenic stimulation and permeability of the fetoplacental barrier. PMID- 1708652 TI - [Effect of sideropenia in mothers on hematologic indicators and iron stores in newborn infants]. AB - The concentrations of total hemoglobin, transport iron, serum ferritin, serum total iron binding, the transferrin saturation rate, unbound erythrocyte porphyrins have been examined in 633 mothers and their newborns. A direct correlation was found between the neonatal and maternal iron status. The formation of the iron store in newborns was significantly influenced by the duration and severity of maternal iron deficiency. Deficient iron storage in antenatal life due to maternal iron deficiency was a major cause of sideropenia and anemia in the infants. Preventive iron therapy during pregnancy prevented iron-deficient anemia in mothers and provided a better neonatal iron status to meet the requirements of the first year of life. PMID- 1708653 TI - Effects of acute normovolemic hemodilution on splanchnic oxygenation and on hepatic histology and metabolism in anesthetized pigs. AB - Perioperative hemodilution (HD) has become an accepted means of reducing transfusion requirements. Therefore, the effects of limited (decrease in hematocrit [Hct] from 30 to 20%, "HD1") and severe (decrease in Hct from 20 to 14%, "HD2") acute normovolemic HD with 6% hydroxyethyl starch on splanchnic blood flows (electromagnetic flow probes), O2 uptakes and deliveries, surface O2 tensions (PO2) (Clark-type electrode), hepatic metabolism (organic acids), and hepatic histology (liver biopsies) were studied in nine pigs anesthetized and paralyzed with ketamine/flunitrazepam and pancuronium. HD1 caused significant (P less than 0.05) increases in cardiac output and all splanchnic flows. Only hepatic arterial blood flow increased twice as much as did cardiac output. Except for hepatic arterial O2 delivery, all splanchnic O2 deliveries decreased. Splanchnic O2 extractions increased, and O2 uptakes remained unchanged. There were no changes in mean surface PO2 values or in surface PO2 histograms of liver and small intestine; in portal or hepatic venous pH; and in hepatic uptake of pyruvate and lactate. In contrast, during HD2 (despite further increases in flows and O2 extractions) portal and hepatic venous pH decreased; mean surface PO2 of liver and small intestine decreased; and the liver surface PO2 histogram showed broadening and a shift to the left. However, hepatic uptake of lactate and pyruvate, and splanchnic O2 uptake remained unchanged, and histologic examination did not reveal significant cell injury. These data indicate that in this experimental model limited acute normovolemic HD was well tolerated by the splanchnic organs. After severe HD, gross liver function remained intact, but there was evidence that compensatory mechanisms (increases in flow and O2 extractions) were no longer fully able to counteract the decrease in splanchnic O2 delivery. PMID- 1708654 TI - Hypertonic saline/dextran resuscitation of dogs with experimentally induced gastric dilatation-volvulus shock. AB - We investigated small-volume (5 ml/kg) 7% NaCl in 6% dextran 70 (HS/D70) as an alternative to large-volume (60 ml/kg) 0.9% NaCl for treatment of experimentally induced canine gastric dilatation-volvulus (GDV) shock. The stomach was surgically displaced and then distended with an intragastric balloon in 11 dogs anesthetized with pentobarbital. All dogs were subjected to GDV for 180 minutes before partial decompression and resuscitation. Hemodynamic values, blood gas values, and plasma volume were measured during control, shock, and resuscitation periods. Resuscitation started with 1 group (n = 6) receiving 5 ml of HS/D70/kg, iv, over 5 minutes, and the other group (n = 5) receiving 60 ml of 0.9% NaCl/kg, IV, over 60 minutes. Both groups received a surgical maintenance dosage (20 ml/kg/h) of 0.9% NaCl after initial resuscitation. Resuscitative effects of small volume HS/D70 were similar to large-volume 0.9% NaCl during the first hour of treatment; however, cardiac output was significantly higher in the HS/D70 group for the last 2 hours of resuscitation. Changes in heart rate, left ventricular pressure change, and systemic vascular resistance appeared to be responsible for improved perfusion. Mixed venous oxygen partial pressure data supported improved perfusion in the HS/D70 group. Packed cell volume remained higher in the HS/D70 group, indicating less hemodilution and improved oxygen delivery. Resuscitation of this GDV-induced shock model was better sustained with small-volume HS/D70, compared with conventional large-volume 0.9% NaCl. PMID- 1708656 TI - Personality and cerebrospinal fluid monoamine metabolites in alcoholics and controls. AB - Alcoholics as a group have been consistently reported to show differences from controls on various personality inventories. Moreover, neurobiologic substrates have been postulated to underlie personality dimensions. Therefore, we compared alcoholics with controls on measures of personality and investigated relationships between measures of personality and cerebrospinal fluid monoamine metabolite concentrations. The alcoholics were significantly different from controls on many personality measurements. There were significant, negative correlations between interview-derived lifetime aggression scores and cerebrospinal fluid concentrations of both the serotonin metabolite 5 hydroxyindoleacetic acid and the dopamine metabolite homovanillic acid. However, there were no significant correlations between any cerebrospinal fluid monoamine metabolite concentrations and scores on personality inventories. PMID- 1708655 TI - Early afterdepolarizations and arrhythmogenesis. Experimental and clinical aspects. AB - There is growing evidence that early afterdepolarizations (EADs) and EAD-induced triggered activity play a significant role in the clinical syndrome of long QTU and polymorphic ventricular tachyarrhythmias better known as Torsade de Pointes (TdP). This evidence is briefly examined in this report. The three steps required for the manifestation of EAD-induced triggered activity are: 1) critical prolongation of the repolarization phase, 2) a net depolarizing current carrying the charge for EAD, and 3) propagation of EADs which are locally generated to capture the entire heart resulting in one or more extrasystoles. The majority of pharmaceutical interventions associated with EADs could be grouped as acting predominantly through one of three different mechanisms 1) a delay of one or both potassium currents IK and Ikl, 2) an increase of transsarcolemmal calcium current (ICa), and 3) a delay of sodium current (INa) inactivation. Two experimental models in the dog utilized cesium and anthopleurin-A to produce bradycardia dependent long QTU and polymorphic ventricular tachyarrhythmias that may resemble the clinical syndrome of long QTU and TdP. In both in vivo models, monophasic action potential (MAP) recordings demonstrated EADs-like deflections more prominent in endocardial than in epicardial recordings. The clinical syndrome of long QTU and TdP can be either congenital, idiopathic or acquired. Several observations suggest a common underlying mechanism with a greater predominance of adrenergic influence in the congenital or idiopathic long QTU syndrome. Adrenergic influence can act by enhancing the depolarizing current of EAD as well as EAD transmission in the intact heart.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708657 TI - Clinicopathologic study of changes in prolapsed intervertebral disks. AB - To study the various factors that might influence the detection of edge neovascularization that is seen in prolapsed intervertebral disks, clinical features of 112 patients were reviewed. Edge neovascularization, which was seen in nearly one half of surgical specimens, was identified more frequently in lumbar disks (61.2%) than in cervical disks (3.8%). Although the characteristic change was more likely to be found in entirely than partially submitted specimens, the difference was not statistically significant. There was a direct relationship between neovascularization and the duration of symptoms. The frequency of finding the specific change increased from 12.5% in patients with disease for less than a month to 82% of patients who had symptoms for 6 months or more. Specific changes in prolapsed disks probably reflect a reparative phenomenon that is influenced by the degree and duration of mechanical forces. PMID- 1708658 TI - Differential diagnosis of adult hemoglobin A, F, and S conditions. A case of G gamma-beta(+)-hereditary persistence of fetal hemoglobin. AB - Hemoglobin (Hb) S/beta(+)-thalassemia is a hemoglobinopathy of variable but potentially severe clinical course. The condition is usually confirmed by the presence of a microcytic anemia and elevated levels of Hbs S, F, and A2 by electrophoresis. However, other less common disorders of Hb structure and synthesis may exhibit laboratory findings that mimic Hb S/beta(+)-thalassemia but have a more favorable prognosis. We present a case occurring in a man with clinical and laboratory features that were suggestive of Hb S/beta(+)-thalassemia but with normocythemia. Although nonmicrocytic variants of beta(+)-thalassemia, including concomitant nutritional deficiencies, were considered, high-pressure liquid chromatography revealed nearly all of the patient's fetal Hb to contain only G gamma chains. This pattern is most consistent with the rate but clinically benign condition of Hb S/G gamma-beta(+)-hereditary persistence of fetal Hb, a nondeletional type of hereditary persistence of fetal Hb. We discuss a diagnostic approach to adult Hb A, F, and S conditions, including thalassemias and thalassemia-like syndromes. PMID- 1708659 TI - [Anlage of the initial part of the thoracic duct in human embryogenesis]. AB - Canalization of the jugular lymphatic sac is accompanied with disengagement from the blood stream of the thoracic subcardinal and the thoracic parts of the superior mesocardinal veins, forming the paired thoracic anlage of the thoracic duct (ThD). Canalization of the retroperitoneal lymphatic sac (RLS) is combined with differentiation of the antevertebral plexus of the abdominal veins, formation of the ThD cistern. Lateroaortal, retrocaval and retroaortal lumbar trunks (LT) become its roots. Together with RLS and the ThD cistern they form the lymphatic "muff" around the abdominal aorta and inferior vena cava. The degree of its parts separation and its substitution by collaterals of LT and by lymph nodes determines the variants of the definitive organization of the ThD initial part. Already at the stage of anlage the intestinal trunk is not included in the ThD root system, but serves as the RLS anterior tributary, or its lumbar, preaortic tributary. PMID- 1708660 TI - [Rapid histochemical staining of histological specimens]. PMID- 1708661 TI - [A method of detecting the nucleoli in the nuclei of the cells of various tissues]. PMID- 1708662 TI - [Neuro-vascular relations in the intramural neural plexus of the gastrointestinal tract]. PMID- 1708663 TI - The biocompatibility testing of some dental amalgams in vivo. AB - The biological responses to some dental amalgams were determined in vivo and compared with those of dental porcelain. The technique of implantation employed in the study addressed some of the vagaries of the Recommended Standard Practices for Biological Evaluation of Dental Materials (RSP) and considered both cellular responses (inflammation, infiltration and fibrogenic cell activity) and the organizational status of the resultant encapsulation. The implantation sites for both the experimental and control were biogeometrically similar, unlike those currently recommended in RSP. At the end of the test period, all the dental amalgams tested caused minor responses reflected by the formation of thin capsules with an acceptable matrix organization. The Australian manufactured dental amalgams--Permite C, Lojic, F400, New Ultrafine and GS80 all produced even capsules with quiescent cells. By one hundred days, the capsule around Dispersalloy, although generally well formed, showed some areas of cellular activity and matrix variability. The biological responses to all the dental amalgams examined were mild and considered to be acceptable for clinical usage. The matrix organization of enveloping capsules must be considered in the determination of the biocompatibility of a dental restorative material. PMID- 1708664 TI - Phosphorylation of hormone sensitive phosphodiesterase in isolated adipocytes. AB - Cyclic AMP phosphodiesterase in rat adipocytes is stimulated by insulin and also by agents that increase cyclic AMP levels. When the enzyme is immunoprecipitated from a solubilised microsomal preparation from adipocytes prelabelled with radioactive phosphate and separated on SDS polyacrylamide gels, label is found in a protein band at the expected Mr for adipose tissue phosphodiesterase. Treatment of the adipocytes with isoproterenol or methyl isobutylxanthine increased the labelling of this band. Insulin alone had no effect on its labelling but did decrease the incorporation of label caused by isoproterenol. PMID- 1708665 TI - Human milk fat globule membrane glycoproteins express blood group-related determinants primarily on mucin-like epithelial membrane antigens and gp70. AB - Milk fat globule membranes (MFGM) were prepared from 21 human milks and dot blotted. MFGM samples were compared with reference to 8 blood group-related antigens reactive with monoclonal antibodies and lectins. All preparations contained epithelial membrane antigen (EMA) and the majority was positive for type 1 Lewis a and b antigens, whereas only trace amounts of sialyl Lewis a were found. For type 2 Lewis antigens, most MFGM reacted intensely for X, but only weakly and infrequently for the Y antigen. Reactivities for H were also infrequent and antigens related to A and/or B (types 1 and 2) were not found. Western blot analyses established that these antigenic determinants were borne mainly by mucin-like components and gp70. PMID- 1708666 TI - Increased plasma level of endothelin with vasoconstriction after dextran infusion. AB - Our previous study demonstrated that volume expansion with dextran produced blood pressure elevation due to vasoconstriction 3 hours after the cessation of infusion. To examine whether endogenous endothelin contributes to this vasoconstriction, we measured plasma level of endothelin before, immediately after, and 3 hours after the administration of dextran. Plasma level of endothelin decreased immediately after the administration (from 1.5 +/- 0.3 pg/ml to 1.1 +/- 0.2 pg/ml, P less than 0.05), and increased 3 hours after the administration (2.1 +/- 0.3 pg/ml, P less than 0.05). However, the changes in the plasma level of endothelin did not significantly correlated with those in blood pressure or total peripheral resistance. Thus, vasoconstriction after dextran infusion was accompanied by an increase in the plasma level of endothelin, but further evaluation is needed for the direct role of this peptide in the vasoconstrictive blood pressure elevation. PMID- 1708668 TI - [Nursing care of surgical patients with AIDS]. AB - In this work the nursing intervention of the surgical patient with AIDS preoperatively, operatively and postoperatively is briefly described. Emphasis is given to the precaution procedures of the patient's care in the operating department. PMID- 1708667 TI - Aldrin and lindane impact on acid and alkaline phosphatase activities of prawn, Metapenaeus monoceros: in vitro study. AB - Activity levels of acid and alkaline phosphatases in hepatopancreas, stomach, muscle, gill and brain tissues of penaeid prawn, Metapenaeus monoceros were studied after in vitro addition of different concentrations of aldrin and lindane, the organochlorine insecticides. The activity levels of both these enzymes were inhibited significantly in all the tissues of prawn and the degree of inhibition is increased with increase in the concentration of insecticide. The significant inhibition creates disturbances in the normal functioning such as disturbances in the normal functioning such as disturbances in protein synthesis and different biochemical lesions in selected tissues of prawn, M. monoceros. PMID- 1708669 TI - Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg). AB - The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3' dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated. PMID- 1708670 TI - Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract. AB - Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges. PMID- 1708671 TI - Inter-alpha-trypsin inhibitor and pre-alpha-trypsin inhibitor in health and disease. Determination by immunoelectrophoresis and immunoblotting. AB - Inter-alpha-trypsin inhibitor (ITI) consists of 3 polypeptides cross-linked by chondroitin sulphate, which is o-glycosidically linked to the smallest of the polypeptides, designated bikunin. Pre-alpha-trypsin inhibitor (p alpha I) consists of bikunin and a fourth polypeptide, also associated by chondroitin sulphate. Crossed immunoelectrophoresis (CIE) of plasma, using immunoglobulins to ITI, revealed 3 precipitation-lines, two of which increased in size during disease. Molecular mass determination by polyacrylamide gel electrophoresis showed that the immunoprecipitates contained mixtures of proteins. Therefore CIE is unfit for quantitation of the individual proteins related to ITI. Immunoblotting suggested that the plasma concentrations of p alpha I and of bikunin was increased in uraemia, rheumatoid arthritis and after trauma. The plasma concentrations of ITI and of p alpha I were decreased in a patient with endocarditis. PMID- 1708672 TI - A point mutation at the X-chromosomal proteolipid protein locus in Pelizaeus Merzbacher disease leads to disruption of myelinogenesis. AB - A group of inherited neurological disorders are the X-chromosome linked dysmyelinoses, in which myelin membranes of the CNS are missing or perturbed due to a strongly reduced number of differentiated oligodendrocytes. In animal dysmyelinoses (jimpy mouse, msd-mouse, md rat, shaking pup) mutations of the main integral myelin membrane protein, proteolipid protein, have been identified. Pelizaeus-Merzbacher disease (PMD) or sudanophilic leucodystrophy is an X-linked dysmyelinosis in humans. We report here on the molecular basis of the defect of affected males of a PMD kindred. Rearrangements of the PLP gene were excluded by Southern blot hybridisation analysis and PCR amplification of overlapping domains of the PLP gene. Sequence analysis revealed one single C----T transition in exon IV, which leads to a threonine----isoleucine substitution within a hydrophobic intramembrane domain. The impact of this amino-acid exchange on the structure of PLP in the affected cis membrane domain is discussed. A space filling model of this domain suggests a tight packing of the alpha-helices of the loop which is perturbed by the amino-acid substitution in this PMD exon IV mutant. The C----T transition in exon IV abolishes a Hph I restriction site. This mutation at the recognition site for Hph I (RFLP) and allele-specific primers have been used for mutation screening the PMD kindred. PMID- 1708673 TI - Structure of the human alpha 1-microglobulin-bikunin gene. AB - Alpha 1-Microglobulin (protein HC) and bikunin (formerly HI-30, urinary trypsin inhibitor, inhibitor subunit of inter-alpha-(trypsin) inhibitor) are abundant serum glycoproteins. They belong to two distinct protein families, the lipocalin family, a family of transport proteins for small hydrophobic molecules and the Kunitz-family of proteinase inhibitors. Mature alpha 1-microglobulin and bikunin result from a common precursor. Now we have isolated and sequenced the human gene coding for this precursor protein. The gene consists of 10 exons which span 1.3 kb and 9 introns with an aggregate length of about 16.5 kb. The largest intron (6.5 kb) separates exon 6 (coding for the C-terminal sequence of alpha 1 microglobulin) from exon 7 (coding for a linker peptide and the N-terminal peptide of bikunin). Repetitive DNA sequences of the Alu-type occur downstream of the polyadenylation site, within introns 4 and 6, and upstream of the putative promoter region which has been defined by sequence comparison and transcription start site determination. The gene also contains several sequence motifs reminiscent to known enhancer sequences. PMID- 1708674 TI - A histochemical study of changes observed in the mouse diaphragm after organophosphate poisoning. AB - A sublethal dose of sarin (GB, isopropyl methylphosphonofluoridate) was administered to mice. The animals were killed up to 28 d after dosing and frozen sections were made of the excised diaphragms which were stained using haematoxylin and eosin and a modified Gomori trichrome method. Muscle fibre degeneration and mononuclear infiltration were seen, notably at 24 h and 3 d. A number of histochemical procedures were carried out, including the GBHA procedure for ionized calcium. Calcium accumulation, seen at 4 h, was the earliest abnormality observed. All changes were rapidly regressing by 5 d and histological appearances were normal by 14 d. It was concluded that sarin produced myopathic changes preceded by calcium accumulation. PMID- 1708675 TI - An evaluation of the putative human mammary tumour retrovirus associated with peripheral blood monocytes. AB - The primary aims of this study were purification and molecular cloning of a putative retrovirus designated human mammary tumour virus (HMTV). However, our preliminary unpublished data of negative reverse transcriptase (RT) activity in ostensibly 'infected' cells led us to re-examine the evidence for this virus; namely multinucleate giant cell (MNGC) formation and RT activity in cultured blood monocytes from breast cancer patients versus benign breast tumour and normal control subjects. MNGCs from by fusion of monocytes and we estimated the total number of cell fusions which had occurred after 10 days of culture in vitro by counting cells with two, three, four and five or more nuclei (n) and by measuring the density of adherent mononuclear cells for each subject studied. We found no clear-cut difference in MNGC formation between the three subject groups. Moreover, a substantial number of cultures, encompassing the three groups, showed far more MNGCs per 10(5) monocytes than previously reported. Various parametric and nonparametric statistical analyses were performed on the multinucleate cell data and only one parametric test, which utilised the density of monolayers as a co-variate, showed a statistically significant difference at the 5% level between the breast cancer and the normal subject groups. We observed marked subject-to subject variation in multinucleate cell formation and we suggest that the evidence for a difference between the breast cancer and the normal groups is marginal. Further, MNGC formation by breast cancer monocytes may not be attributed to the presence of a retrovirus since 5'-Azacytidine (AZA), an agent known to stimulate replication of latent retroviruses showed no effect on the MNGC formation. In addition, culture supernatants from the three groups were assayed for RT activity and no test sample gave a significant signal above background. Preliminary transmission electron microscopy analysis failed to identify viral particles in MNGCs. PMID- 1708676 TI - Epitope-specific binding of CD8 regulates activation of T cells and induction of cytotoxicity. AB - Functions of the CD8 molecule were examined to determine whether CD8 is merely a ligand-binding molecule and/or is involved in signal transduction. Using KT112 (anti-CD8 beta), CD8 was demonstrated to transduce an activation signal leading to cytotoxicity. Conformational changes of the CD8 molecule might be responsible for the activation, because (i) KT15 (anti-monomorphic CD8 alpha), but not antibodies specific for polymorphic CD8 alpha determinants, abrogated KT112 (anti CD8 beta)-induced cytotoxicity without blocking the binding of KT112, whilst (ii) KT112 (anti-CD8 beta) inhibited KT15 (anti-monomorphic CD8 alpha)-mediated augmentation of proliferation triggered by a V beta 11-specific antibody without blocking the binding of KT15. PMID- 1708677 TI - Functional degeneracy of residues in a T cell peptide epitope contributes to its recognition by different T cell hybridomas. AB - Synthetic antigen Poly EYK(EYA)5 induces T cells of narrowly defined fine specificity as represented by the two I-Ad-restricted T cell hybridomas, A.1.1 and B.1.1. Both these hybridomas recognize the minimum 15-amino-acid peptide sequence EYK(EYA)4. We have characterized the residues involved in the recognition of EYK(EYA)4 peptide by these hybridomas with synthetic peptides and discovered a distinct functional hierarchy for the residues in the sequence. Even with the repeating tripeptide (EYA)5, which is recognized by B.1.1 cells, the residues that are essential cluster near the middle of the sequence but not near the N- or C-terminal region. Different MHC binding and TCR contacting residues were found for each of the hybridomas. The results suggest that different T cells either recognize different parts of the peptide MHC complex or that the peptide binds to MHC in multiple conformations. This was supported by the fact that Poly EYK(EYA)5 is alpha-helical but the peptides used here showed only a slight propensity to adopt this structure and it did not correlate with their functional activity. We also found that (EYA)5 does not compete with EYK(EYA)4 in the stimulation of A.1.1 cells despite its obvious capacity to interact with I-Ad when it stimulates B.1.1 cells. This may be because these peptides have a low affinity for Ia and therefore only appropriate TCR interactions would stabilize the antigen-Ia complex. In conclusion, antigen-MHC-TCR interaction appears to be a dynamic process which allows recognition of different residues of a T cell determinant by different T cells. PMID- 1708678 TI - Lactic acid neovascularisation. PMID- 1708679 TI - Solubilization, partial purification, and reconstitution of glutamate- and N methyl-D-aspartate-activated cation channels from brain synaptic membranes. AB - L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. The affinity batch chromatography procedure described previously [Chen et al. (1988) J. Biol. Chem. 263, 417-427] was used to obtain a fraction enriched in glutamate-binding proteins. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. [14C]Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L Glutamate caused an increase in the rate of [14C]methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. The increase in methylamine influx was dependent on the concentration of L-glutamic acid with an estimated Kact for L glutamate equal to 0.2 microM for synaptic membrane proteins and 0.32 microM for purified proteins. Of the major glutamate receptor agonists, only N-methyl-D aspartate activated cation fluxes in liposomes reconstituted with glutamate binding proteins. Glutamate-activated methylamine flux was completely inhibited by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D aspartate- or glutamate-induced influx of Na+ led to a transient increase in the influx of the lipid-permeable anion probe S14CN-. Electrophoretic analysis of partially purified proteins reconstituted in liposomes indicated enrichment of several bands, the most prominent being those of molecular size equal to approximately 69, 60, 35, and 25 kDa. Antibodies raised against the purified 71- and 63-kDa glutamate-binding proteins reacted strongly with the approximately 69 kDa band of reconstituted proteins and markedly decreased the initial rate of glutamate-activated cation flux. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the approximately 69-kDa protein in the function of these ion channels. PMID- 1708680 TI - AIDS in the Nineties: from science to policy. Pregnancy and paediatrics. PMID- 1708681 TI - [Haemophilus parainfluenzae as a cause of urinary tract infection]. PMID- 1708682 TI - Granulocyte colony-stimulating factor. AB - Granulocyte colony-stimulating factor was discovered during attempts to define the normal regulators present in cell supernatants that could induce terminal differentiation of the murine myeloid leukemic cell line WEHI-3B D+. The purification and subsequent cloning of both murine and human G-CSF allowed the normal functions of this molecule to be elucidated and indicated that it was a relatively lineage-specific stimulator of the survival, proliferation, and differentiation of precursor cells of the neutrophilic granulocyte cell lineage as well as an activator of mature neutrophil function. Murine and human G-CSFs as well as the ligand-binding domains of G-CSF receptors have been strongly conserved so that the biological activities and receptor-binding characteristics of G-CSFs are completely species cross-reactive. The evolutionary conservation of G-CSFs is suggestive of an important role for the molecule in maintaining neutrophil levels and activity in vivo, a role amply supported by a series of animal experiments and clinical trials that have been performed recently. These experiments and trials have suggested that G-CSF will be clinically useful in augmenting patients' resistance to certain infections, in enhancing the neutrophil responses of some patients with reduced granulocyte counts or defective granulocytes, and in reducing the decline in granulocytes that occurs during chemotherapy or irradiation therapy and bone marrow transplantation. The special capacity of G-CSF among the CSFs to induce terminal differentiation in some myeloid leukemias is not understood in molecular terms. However, the capacity of G-CSF to stimulate proliferation in some myeloid leukemias as well argues that caution needs to be exercised in the timing of G-CSF administration and patient selection before considering this form of therapeutic approach. PMID- 1708683 TI - How the field of controlled-release technology began, and its central role in the development of angiogenesis research. AB - The first controlled-release methodology was developed in 1962 and was based upon diffusion of small molecules (molecular weight less than 500) through the wall of silicone rubber tubing. The problem of controlled-release of large molecules such as proteins was not solved until more than a decade later, when it was discovered that microchannels formed around lyophilized proteins embedded in polymers such as ethylene vinyl acetate copolymer (EVA) and poly(hydroxyethylmethacrylate) (pHEMA), as water permeated through the polymer. These studies led to the development of a 5-year contraceptive implant, e.g., Norplant; rapid progress in the field of angiogenesis research; and a method for regulating cell shape and growth control in vitro. PMID- 1708684 TI - Sex chromosome anomalies: prenatal diagnosis and the need for continued prospective studies. PMID- 1708685 TI - Sex chromosome aneuploidy: the Denver Prospective Study. PMID- 1708686 TI - [Molecular biology: a lexicon]. PMID- 1708687 TI - "State of the art" transrectal ultrasound imaging in the assessment of prostatic disease. PMID- 1708688 TI - [In situ transcription and determination of mRNA in human thymic antigen 1a positive cells within epidermis]. AB - This paper introduces a method of study on the characteristics of cell surface protein. We used human thymic antigen 1a anti-sense oligonucleotide primer, radiolabeled deoxynucleotide and in situ transcription with cells. The results of in situ transcription and determination of mRNA in CD1a positive cells isolated from the epidermis of the prepuce of normal neonate showed that most of the positive cells contained specific CD1a mRNA, and the specificity and sensitivity of this method were higher and the procedure was relatively simple. PMID- 1708689 TI - Asymmetrical effects of increases in hydrostatic pressure on macromolecular movement across the airway mucosa. A study in guinea-pig tracheal tube preparations. AB - This study employed isolated guinea-pig tracheal tube preparations in order to examine effects of increases in hydrostatic pressure on the movement of macromolecular solutes (fluorescein isothiocyanate-conjugated dextran; FITC-D, MW 70 kD; kept either in serosal or mucosal bathing fluids) across the mucosa. An asymmetry of the mucosal barrier was demonstrated by the finding that under baseline zero-pressure difference conditions luminal entry of serosal FITC-D was greater than serosal entry of luminal FITC-D. Furthermore, an increased serosal pressure (5 cm H2O) moved significant amounts of serosal FITC-D into the lumen, whereas a corresponding pressure applied on the luminal side only marginally increased mucosal crossing of luminal FITC-D. By raising the luminal pressure to 10 and 20 cm H2O (which may be used as positive end-expiratory pressures (PEEP) in vivo in patients) mucosal penetration of luminal FITC-D was as marked as that induced in the opposite direction by the low (5 cm H2O) serosal pressure increase. Another aspect of the asymmetry of the airway mucosal barrier was evident from experiments examining the effect of a serosal pressure increase on mucosal penetration of luminal FITC-D. Neither during nor after the period of sustained serosal pressure increase was luminal FITC-D crossing the mucosa to a greater extent than under baseline zero-pressure conditions. This finding agrees with in-vivo data demonstrating that plasma exudation into the airway lumen may not be associated with an increased absorption of luminal solutes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708690 TI - The heterogeneity of human IgE exemplified by the passive transfer of D2O sensitivity. AB - Basophil responsiveness to histamine-releasing factors (HRF) is limited to cells from atopic donors; this response can be transferred passively to non-reactive basophils by IgE molecules from the sera of donors who are intrinsically responsive to HRF. Deuterium oxide (D2O) also causes mediator release from the basophils of atopic asthmatic subjects. To assess whether basophil responsiveness is IgE dependent, and, if so, whether this release revealed IgE heterogeneity, we tested the ability of sera from HRF responders (IgE+) and non-responders (IgE-) to sensitize basophils to D2O. Both purified IgE+ and unpurified sera from an HRF responder were passively used to sensitize basophils whose IgE had been removed by lactic acid treatment. As a control, an IgE- myeloma-containing serum was used for passive sensitization. In five experiments, histamine release in the presence of 44% D2O was 9 +/- 2% and 46 +/- 4% using control IgE- and IgE+ sensitized cells, respectively. The non-responder serum, even at higher IgE levels, did not sensitize the cells for D2O release. If the IgE receptors on lactic-acid treated cells were first exposed to serum from an IgE- donor, sensitization to D2O by IgE+ was blocked. The percentage histamine release to D2O was directly related to both the amount of IgE+ used for passive sensitization and the concentration of D2O used for release. These experiments further support the concept of IgE heterogeneity and suggest that the occupancy of IgE receptors on the basophil surface 'activate' the cell to make it more responsive to various stimuli. PMID- 1708691 TI - A correlation of phylogenetic diversity in the Proteobacteria with the influences of ecological forces. AB - The Proteobacteria are physiologically and morphologically diverse, although they form a coherent set of four main lineages on phylogenetic analysis of ribosomal RNA. A rational and consistent taxonomic arrangement bringing today's phenotypic and phylogenetic conclusions about them into register is not yet possible. It is also difficult to understand the selective forces involved in their evolution that fostered such diversity. This latter problem is addressed in this essay and is based on the assumption that bacterial evolution could only have occurred in ecological consortia whose products of metabolism modified the environment, provided nutrition, and have a basis for selection of new capabilities. PMID- 1708692 TI - Fusion protein based epitope mapping of the MPB57 protein from Mycobacterium bovis BCG and its epitope insertion into the native protein. AB - The gene coding for the 12-kDa protein (MPB57) of Mycobacterium bovis BCG has recently been cloned and sequenced (R. Yamaguchi, K. Matsuo, A. Yamazaki, S. Nagai, K. Terasaka, and T. Yamada. 1988. FEBS Lett. 240: 115-117). To map linear B-cell epitopes by beta-galactosidase fusion proteins, we have constructed convenient vectors (pUR278S, pUR288S, and pUR289S) with the SmaI site. Based on recognition by polyclonal antibodies, two epitope regions on the MPB57 protein were identified, both of which corresponded to the amino acid sequences Glu20 to Val45 (26 residues, epitope I region) and Ile78 to Leu86 (9 residues, epitope II). Complementary oligonucleotides encoding epitope II were synthesized, polymerized by a ligase reaction, inserted into the native MPB57 protein gene, and expressed in Escherichia coli, giving rise to epitope-inserted proteins. Their stability and potential uses are described. PMID- 1708693 TI - Antileukemic activity of Viva-Natural, a dietary seaweed extract, on Rauscher murine leukemia in comparison with anti-HIV agents, azidothymidine, dextran sulfate and pentosan polysulfate. AB - An antileukemic activity of partially purified polysaccharide of an edible seaweed. Viva-Natural, against Rauscher murine retrovirus-induced erythroleukemia has been demonstrated. This antileukemic effect is compared with standard anti human immunodeficiency virus (HIV) agents, azidothymidine (AZT), dextran sulfate and pentosan polysulfate. Pretreatment with Viva-Natural, as an immunomodulator, on day 3 prior to the virus inoculation demonstrated definite prophylactic activity, while pretreatment with the other three anti-HIV agents showed no prophylactic activity. The replication of Rauscher virus in BALB/3T3 cell cultures accompanied by direct cytopathic effect (syncytia formation) was suppressed in the presence of Viva-Natural or the other anti-HIV agents in the culture medium. In spite of the antiviral potentials of the four agents in vitro, only Viva-Natural and AZT demonstrated therapeutic efficacy against Rauscher leukemia in mice. PMID- 1708694 TI - Reversal by 5-azacytidine of the S-adenosyl-L-methionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis. AB - The development of gamma-glutamyltranspeptidase (GGT)-positive foci, in Wistar rats, initiated with diethylnitrosamine and subjected to selection according to 'resistant hepatocyte' protocol, was coupled, 7 weeks after initiation, with liver DNA hypomethylation and with a fall in S-adenosylmethionine/S adenosylhomocysteine (SAM/SAH) ratio, and in 5-methylthio-adenosine (MTA) content. A 15-day treatment with SAM, started 1 week after selection, caused a dose-dependent decrease in the development of GGT-positive foci, recovery of liver SAM/SAH ratio and MTA level, and liver DNA methylation. A 12-day treatment with 20 mumol/kg per day of 5-azacytidine (AzaC), starting 1 week after selection, enhanced growth of GGT-positive foci, caused strong DNA hypomethylation, and partially counteracted the inhibition of GGT-positive foci growth, without affecting recovery of SAM/SAH ratio and MTA level, induced by SAM. These results suggest a role of DNA methylation in the antipromoting effect of SAM. PMID- 1708695 TI - Coexistence of autologous antibodies and decay-accelerating factor, an inhibitor of complement, on human renal tumor cells. AB - Autologous immunoglobulin was detected on the cell surface of tumor cells freshly isolated from cancerous kidneys of patients with renal cell carcinoma by flow cytometry after staining with murine anti-human IgG monoclonal antibodies. Cells isolated in parallel from macroscopically normal regions of the tumorous kidneys were not specifically stained with the anti-human IgG reagents. In further studies, tumor cells were stained with an antibody to decay-accelerating factor (DAF), a known inhibitor of complement. Flow cytometry of these cells revealed that nearly all tumor cells expressed DAF, and that the intensity of staining with the anti-DAF monoclonal antibody correlated with the staining of cells with anti-IgG. The results suggest that tumor cells coated with autologous antibody may be resistant to complement-mediated cytotoxicity in vivo through the expression of high levels of DAF. PMID- 1708696 TI - Cell lineages and oval cell progenitors in rat liver development. AB - We determined whether the formation of the hepatic primordium in the rat is associated with the expression of liver-specific markers. Further, we examined the origin of intra- and extrahepatic bile ducts and tried to establish whether there are cell types in the developing liver that might correspond to "stem-like" cells ("oval cells") that proliferate during carcinogenesis and toxic injury in adult livers. Using in situ hybridization and immunohistochemical methods, we show that alpha-fetoprotein (AFP) mRNA is detected in cells of the ventral foregut at 10.5 days of development and that the protein is first detected 1 day later. Thus, AFP transcription occurs before liver morphogenesis, and translation of the protein is first detected when liver cords are being formed, indicating that AFP expression in endodermal cells signals their commitment toward the liver lineage. Although albumin is considered a trait of differentiated hepatocytes, its mRNA was first detected just 1 day later than the AFP message. An analysis of the expression of lineage-specific cytokeratins (cytokeratins 7, 9, 18, and 19), surface markers, and histochemical determination of gamma-glutamyl transferase activity and glycogen revealed that (a) hepatoblasts undergo gradual maturation throughout liver development, (b) AFP- and albumin-containing hepatoblasts gave rise to intra- and extrahepatic bile ducts, and (c) hepatoblasts forming primitive intrahepatic bile ducts during liver development have markers similar to those expressed by stem-like cells that proliferate during liver carcinogenesis. PMID- 1708697 TI - Thrombospondin binds to the surface of human osteosarcoma cells and mediates platelet-osteosarcoma cell interaction. AB - We have previously shown that thrombospondin (TSP) is synthesized and secreted by human MG-63 osteosarcoma cells. In this study, the secretion and cell surface expression of TSP by two different human osteosarcoma cell lines (MG-63 and TE 85) as well as the involvement of TSP in the platelet-aggregating activity of these tumor cells were studied. Using a sandwich enzyme-linked immunosorbent assay, MG-63 cells secreted 3-fold as much TSP as TE-85 cells at 48 h (0.17 +/- 0.01 (SD) versus 0.06 +/- 0.006 micrograms/10(6) cells, P = 0.007). Binding of exogenous 125I-TSP to MG-63 and TE-85 cells in monolayer indicated that binding was time and concentration dependent, saturable, and inhibited by excess cold TSP. However, despite a similar affinity, MG-63 cells had 10-fold more TSP binding sites than TE-85 cells (402,394 +/- 130,346 versus 36,748 +/- 7,708 TSP binding sites/cell; P = 0.002). Similar binding differences of 125I-TSP were observed with both osteosarcoma cell lines in suspension. A fluorescence activated cell-sorting analysis was used in conjunction with an anti-TSP polyclonal antibody, and binding of endogenous TSP to MG-63 and TE-85 cells in suspension was investigated. Addition of an anti-TSP antibody to MG-63 and TE-85 cells in suspension increased the mean fluorescence intensity 50-fold when compared to an irrelevant antibody. Moreover, the fluorescence intensity of MG-63 cells with an anti-TSP polyclonal antibody was increased by 40% when compared to TE-85 cells. Since TSP was expressed on the surface of osteosarcoma cells, the involvement of this glycoprotein in the platelet-aggregating activity of MG-63 and TE-85 cells was therefore investigated using an anti-TSP polyclonal antibody and two monoclonal antibodies (P10 and MA-II), the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment and the Mr 25,000 heparin-binding fragment of TSP, respectively. Preincubation of MG-63 cells (1 x 10(6) cells/ml) with either an anti-TSP polyclonal antibody (100 micrograms/ml) or monoclonal antibody P10 (15 micrograms/ml) inhibited by 80% other platelet-aggregating activity of these tumor cells, while anti-TSP monoclonal antibody MA-II (15 micrograms/ml) had no effect. In sharp contrast, the anti-TSP polyclonal antibody (100 micrograms/ml) only exhibited a slight inhibitory effect on platelet aggregation induced by TE-85 cells when using a low concentration of tumor cells (0.6 x 10(6) cells/ml).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1708698 TI - Vacuole formation and cytokeratin rearrangement of hepatoma cells induced by teleocidin are not associated with down-regulation of protein kinase C. AB - PLC/PRF/5 human hepatoma cells cultured with teleocidin reduced the rate of cell proliferation and were transformed into large cells with many vacuole-like subcellular structures. In these vacuolated cells, the protein content per cell increased without changing the total cellular protein synthesis. Cytokeratin was one of the proteins which increased quantitatively. This intermediate filament formed fibrous network structures throughout the enlarged cytoplasm. The assembly of other cytoskeletal proteins such as actin, tubulin, and vimentin was not altered remarkably, suggesting that teleocidin morphologically transformed the hepatoma cells by changing the assembly of cytokeratin protein selectively. On the other hand, the alterations of cell proliferation, cell morphology, and cytokeratin assembly induced by teleocidin were not associated with either down regulation of protein kinase C or reduced number of epidermal growth factor receptors. In addition, these teleocidin effects were not mimicked by the protein kinase C agonist 1-oleoyl-2-acetylglycerol or inhibited by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. From these results it can be speculated that the morphological transformation and reduced cell proliferation induced by teleocidin may be mediated by still unknown mechanisms unrelated to protein kinase C. PMID- 1708699 TI - Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E. AB - A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 micrograms. Parallel control groups received analogous inoculations of an isotype matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer sensitive rat T cell lymphoma G1-Tc1. The cytotoxic cell population was more than 90% CD4+. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals. PMID- 1708700 TI - Immunoregulatory cytokine release in rat spleen cell cultures after treatment with bleomycin and its analogues in vivo. AB - We have studied the immunological effects that accompany a change in the chemical structure of a group of antineoplastic antibiotics by comparing the immunoregulatory cytokine release during mitogen-stimulated spleen cell culture after in vivo drug treatment. Whereas bleomycin and peplomycin increased cytokine levels in culture supernatants when compared with supernatants from untreated control rat spleen cell cultures, liblomycin generally reduced cytokine levels under the same culture conditions. We then compared these results with the antitumor effects of equivalent doses of the three drugs against a highly antigenic rat fibrosarcoma, KMT-17, both in vivo and in vitro. The results suggest that the immunoaugmenting effects of these antitumor antibiotics are essential for an optimal antitumor effect in vivo, and that these effects can be drastically altered by modification of the chemical structure of the drugs employed. PMID- 1708701 TI - HIV-related alterations in CD8 cell subsets defined by in vitro survival characteristics. AB - Previously we showed that over 50% of CD8 cells from HIV-infected persons do not survive in 3-day cultures of mononuclear cells; this loss occurred preferentially in subsets with phenotypes indicative of in vivo activation. In the studies reported here, we asked if cytokines enhanced CD8 cell survival. Of IL1, IL2, IL4, IL6, tumor necrosis factor, and interferon-gamma only IL2 specifically enhanced CD8 survival in the HIV group, compared to the control group. Further studies thus focused on characterizing CD8 cell survival in the presence of IL2. In both study groups, three subsets of CD8 cells were identified based on in vitro survival: (a) those surviving in culture medium alone (survivors), (b) those surviving only when IL2 was included in the culture medium (IL2-dependent survivors), and (c) those failing to survive even in the presence of IL2 (nonsurvivors). By dual-color cytofluorometry, the CD8 survivor subset was similar in the two study groups, and expressed nonactivated phenotypes (Leu8+, CD45RA+, HLA-DR-). The IL2-dependent survivor subset was also similar in the two study groups and expressed the phenotypes Leu8-, CD45RA+, CD57+, HLA-DR+, and CD38+, suggesting prior activation. The CD8 nonsurvivor subset, in contrast, was markedly different in the study groups: compared to the control group, the HIV group contained significantly higher proportions of CD8 cells expressing the phenotypes Leu8-, CD57+, and HLA-DR+, also suggesting activation. These findings indicate that, in HIV infection, the activated CD8 cell subsets that do not survive in medium alone consist of a "normal" component that requires IL2 for survival and an "abnormal" component that does not survive even in IL2. PMID- 1708702 TI - Suppressor cells and T cell synergy in the primary MLR: interleukin-2 is required for the maintenance of rat CD8+ suppressor cells. AB - The requirements for maintenance of allospecific CD8+ Ts cells generated in the rat primary MLR were investigated. Allospecific CD8+ Ts cells rapidly lose their activity over 72 hr in secondary culture with media alone, whereas low concentrations of rIL-2 (less than 1 U/ml) are able to maintain potent CD8+ Ts cell activity. This Ts cell activity is maintained at rIL-2 concentrations which do not result in significant cell proliferation. Therefore, cell proliferation per se is not a requirement to maintain Ts cell activity, although the CD8+ Ts cells can proliferate to rIL2 in a concentration-dependent manner. An anti-IL-2 receptor monoclonal antibody significantly inhibited the maintenance of Ts cell activity. Two-color flow cytometric analysis demonstrated that Ts cells cultured in rIL-2 maintain upregulation of their high-affinity IL-2 receptor. Although allospecific Ts cells maintained in secondary culture with rIL-2 for 48 hr maintained antigen specificity, there is also the induction of an antigen nonspecific population. After 168 hr in secondary culture the Ts cells have lost allospecificity, although Ts activity can be maintained with rIL2 in continuous culture for up to 4 weeks. PMID- 1708703 TI - In vivo modulation of the distribution of thymocyte subsets: effects of estrogen on the expression of different T cell receptor V beta gene families in CD4-, CD8- thymocytes. AB - Estrogen treatment of mice has been shown to deplete CD4+, CD8+ double-positive (DP) thymocytes and to alter the relative proportion of CD4+ and CD8+ single positive (SP) thymocytes. In this work, we have studied the effect of the steroid hormone 17 beta-estradiol (E2) on the different subsets of CD4-/CD8- double negative (DN) thymocytes by analyzing the expression of CD5, CD3-epsilon and of several V beta gene family products of the T cell antigen receptor (TCR). After in vivo administration of E2 a significant decrease in the number and proportion of dull CD5+, CD3-, beta-TCR- DN thymocytes was observed. In contrast E2 treatment significantly increased the proportion of bright CD5+, CD3+, beta-TCR+ DN cells. The E2-induced increase in DN/TCR+ cells was observed for subsets expressing V beta 6, V beta 8, and V beta 11, but not V beta 3 gene products of the TCR. Thus, estrogen administration results in a selective inbalance of the DN thymocyte subsets by depleting an immature, dull CD5+, CD3-, TCR beta- DN subset, while enriching a mature, bright CD5+, CD3+, TCR beta+ DN subset of cells. In addition to TCR beta+ DN thymocytes, an increased proportion of CD4+ and CD8+ SP thymocytes expressing V beta 8, V beta 6, and V beta 11, but not V beta 3, TCR proteins was also observed after E2 administration. An involvement of intrathymic cytokine production in mediating the hormone action is suggested by the ability of estrogen to increase the levels of IL-1 alpha mRNA of intact thymus. Our data suggest that estrogen exerts its effects on a broad range of immature cells, including dull CD5+, CD3-, beta-TCR- DN and DP thymocytes. PMID- 1708704 TI - Thymic stromal cells in culture. I. Establishment and characterization of a line which is cytotoxic for normal thymocytes and produces hematopoietic growth factor(s). AB - Lines of thymic stromal cells have been established. One of these, designated TS 9, has been cloned and studied extensively. This line expresses both acid and alkaline phosphatases. Despite repeated cloning, TS-9 cells remain morphologically heterogeneous. The origin of these cells is not clear. They express low levels of immunologically identifiable cytokeratins, produce laminin, a basement membrane protein, but express antigens typically found on bone marrow stromal cells. The TS-9 cells are MHC Class I+ but Class II-. They express the Thy-1, Pgp-1, and Mac-2 antigens but not other lineage markers of T cells or macrophages. Coculturing TSC with normal thymocytes or with the CTLL-1 cell line leads to a profound inhibition of lectin-induced and/or IL-2 induced T cell proliferation. This requires direct cell-cell contact and ultimately results in the death of the bound lymphocytes. It cannot be reproduced by culturing the thymocytes with TSC culture supernatants. These supernatants do contain hematopoietic growth factor(s) which augment the growth of some T lineage cells and support the growth of monocytic colonies in semi-solid culture medium. Both normal thymocytes and a variety of T cell tumors bind to TSC but only the normal cells are killed as a consequence of this interaction. Neither the binding nor the killing appear to be MHC restricted. We suggest that this killing may provide a model for the effector mechanism of the negative selection imposed by the thymus on developing T cells. PMID- 1708705 TI - Outcome of 100 randomly positioned children of very low birthweight at 2 years. AB - Prematurely born children often show a tendency to adopt extensor motor patterns during the first years of life. These children and especially those children of very low birthweight are considered to be at high risk for abnormal development. Positioning in an ordinary manner or in a more flexed position imposed at random during the neonatal period until discharge from hospital did not have a statistically significant influence on the development of these children at 24 months after term. Analysis of the optimality score of the 100 randomly selected children, born consecutively at the University Women's Hospital, Bern, showed a significant influence of prenatal optimality and congenital malformations on their later outcome. PMID- 1708706 TI - Models of parent partnership and child development centres. AB - Four models (and an emergent fifth model) of parent partnership are described and their relevance to UK child development centres is explored. Clinical, management and service development issues are drawn out. PMID- 1708707 TI - Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase. AB - This paper deals with the postnatal development of afferent and efferent connections of the rat striatum as revealed by the transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). Tracer was injected weekly from birth to the end of the first postnatal month in the head of the caudate nucleus. To control for transport from cortical areas contaminated by the micropipette, injections in newborn rats were made by either vertical or lateral penetrations. In addition some newborn and 14-day-old animals were injected only in the cortex. The results showed that at birth there was retrograde transport to the thalamus, substantia nigra and raphe nuclei. Labelling in the cortex was seen at birth but was probably due to cortical contamination. Transport from the striatum was clearly established on day 7, when a few labelled neurons were observed on both the ipsi and contralateral sides. These neurons increased in number and were distributed through layers III to VI by day 14. At this time labelled cell bodies were observed in the claustrum and lateral amygdaloid nucleus as well as in the globus pallidus and entopeduncular nucleus. On day 21 the contralateral labelling of the lateral amygdaloid nucleus was apparent. The anterograde transport from the striatum to globus pallidus, entopeduncular nucleus and substantia nigra was already visible at birth although its intensity increased during the first postnatal month. PMID- 1708708 TI - A lead-associated nuclear protein which increases in maturing brain and in differentiating neuroblastoma 2A cells exposed to cyclic AMP-elevating agents. AB - The lead-associated nuclear protein, p32/6.3, increases significantly in the postnatally developing rat cerebral cortex (Egle and Shelton, J. Biol. Chem., 261 (1986) 2294-2298). In the present study, this increase has been identified with late development of the cerebral cortex or forebrain because p32/6.3 reached adult levels 10 to 14 days after birth in guinea pig (a precocial animal) and after hatching in chicken. Comparison with other developmental processes indicates that p32/6.3 reaches adult levels just before or during the period of synapse maturation. Thus p32/6.3 may prove useful as a biochemical indicator of nuclear maturation in this period. The developmental regulation of p32/6.3 was further studied in mouse neuroblastoma 2a (Nb2a) cells. In vitro induction of differentiation of Nb2a cells by serum withdrawal from the culture medium increased p32/6.3, implicating p32/6.3 with differentiating neurons. This association was further strengthened when treatment of the Nb2a cells for 24 h with dibutyryl cAMP (1-5 mM), papaverine (5-12.5 micrograms/ml) or 3-isobutyl-1 methylxanthine (IBMX; 50-250 microM) increased the abundance of p32/6.3 1.5- to 3 fold more than serum withdrawal alone. 8-Bromo-cAMP (2-4 mM), N6-benzoyl cAMP (4 mM) and forskolin (10 microM) also increased the abundance of p32/6.3 in Nb2a cells, arguing that cAMP is involved in p32/6.3 regulation. These results, in conjunction with the postnatal increase of p32/6.3 in cerebral cortex, suggest a relationship between p32/6.3 levels and neuronal maturation. PMID- 1708709 TI - Influx of urea during bronchoalveolar lavage depends on the permeability of the respiratory membrane. AB - In 6 healthy controls and 23 patients with pulmonary diseases the influx of urea during bronchoalveolar lavage was measured by comparing the concentrations of albumin and urea in the sequential samples recovered. It varied between -28 and 151 mumol/l. In the bronchoalveolar lavage fluid and serum we measured alpha-2 macroglobulin (A2M) and ceruloplasmin (CP). The bronchoalveolar lavage fluid to serum ratios were calculated (QCP and QA2M). QA2M/QCP was taken as a measure of the respiratory membrane permeability; it varied between 0.05 and 0.53. Influx of urea during lavage was higher according as the QA2M/QCP ratio was higher. We conclude that concentrations of substances in the epithelial lining fluid calculated with the urea correction method have to be corrected for the influx of urea. PMID- 1708710 TI - Toxicity of lindane, atrazine, and deltamethrin to early life stages of zebrafish (Brachydanio rerio). AB - Fertilized eggs of zebrafish were exposed under flow-through conditions to several concentrations of the following pesticides: lindane 40, 80, 110, 130, and 150 micrograms/liter; atrazine 300, 1300, and 9100 micrograms/liter; deltamethrin 0.5, 0.8, and 1.2 micrograms/liter. Hatching, abnormalities in development (external deformations, edema, etc.), and mortality were recorded over a period of 35 days. At the end of the experiment, the body lengths of the fish were measured. Survival of juvenile fish after 35 days was reduced by increasing concentrations of all xenobiotics tested: lindane enhanced the mortality from 110 micrograms/liter and atrazine from 1300 micrograms/liter, and deltamethrin showed an effect even at the lowest test concentration (0.5 micrograms/liter). Other parameters were affected differently: hatching rate was reduced only by deltamethrin (from 0.8 micrograms/liter): lindane caused a decrease in growth (40 micrograms/liter) but had no effect on the other parameters. Atrazine increased the number of deformations and edema (1300 micrograms/liter) but did not influence hatching rate and growth. The sensitivity of the early life stages to the pesticides was compared with acute toxicity data (LC50) of adult zebrafish. The early life stages were less sensitive to lindane (118 versus 75 micrograms/liter), whereas in the case of atrazine (1300 versus 37,000 micrograms/liter) and deltamethrin (0.5 versus 2 micrograms/liter; 0.5 micrograms/liter was the lowest concentration tested) larvae were more sensitive. PMID- 1708711 TI - Problems and prospects for pharmaceutical education in the Americas. PMID- 1708712 TI - Conduction velocity and temporal dispersion of the nerve volleys evoked by air puff stimulation of the index finger and palm. AB - Air-puff stimuli were delivered to 5 successive sites (3 cm increments) over the index finger and palm to record propagating sensory nerve action potentials (SNAPs) from surface electrodes over the median nerve at the wrist. SNAPs consisted of a series of individual peaks (N1, P1, P2, N2 and P3) and the corresponding peaks in the records with stimulation at the various locations could be identified. The apparent conduction velocity of the first peak determined from the stimulation point at the finger tip to the wrist was 38 m/sec. With stimulation of the more proximal locations it became even slower. This paradoxical slowing is due to an increasing effect in the calculation of conduction velocity of the utilization time at the receptors for air-puff stimulation at progressively proximal sites. Segmental conduction velocity estimated between adjacent stimulus sites was 10-20 m/sec faster than the distal conduction velocity between the finger tip and the wrist. Within each segment, the conduction velocities of the individual peaks were not significantly different. These findings, together with invariant durations of the negative components (N1 and N2) in the propagating SNAPs along the ascending digital nerves, lead to the conclusion that the separate peaks are not the result of temporal dispersion due to differences in conduction velocity of skin afferents, but are primarily due to a more peripheral receptor mechanism involving variable delays in activation of different classes of mechanoreceptors. PMID- 1708713 TI - Randomized double pulse stimulation for assessing stimulus frequency-dependent conduction in injured spinal and peripheral axons. AB - Injury compromises the ability of axons to conduct action potentials at high frequencies. To study stimulus frequency-dependent conduction in injured spinal and peripheral axons, we developed a new stimulation paradigm which applies trains of double pulses at 5 Hz and randomly varied interpulse intervals of 3, 4, 5, 8, 10, 30, 50, and 80 msec. In each double pulse, the first pulse was used to condition the response activated by the second test pulse. Responses elicited by double pulses with 80 msec intervals served as controls. The L5 dorsal root was stimulated to activate dorsal column and dorsal root compound action potentials in pentobarbital anesthetized rats. To injure the spinal cord, we compressed the cord stepwise (0.25 mm every 5 min) until action potential conduction across the compression site was abolished and then decompressed the spinal cord 10 min later. Before injury, conditioning pulses applied 3-80 msec before the test pulses did not alter dorsal column responses except for a slight amplitude augmentation at 20 msec interpulse intervals (mean +/- S. E., + 4.2 +/- 0.8%, P less than 0.02) compared to controls. Injury had 3 effects on the responses. First, it significantly reduced response amplitudes and increased response latencies at 3-5 msec interpulse intervals, i.e., responses activated with 3 msec intervals were 26.0 +/- 7.4% (P less than 0.002, paired t test, n = 6) smaller and had 108 +/- 45 microseconds (P less than 0.04) longer latency than control responses. Second, response amplitude increases at 20 msec interpulse intervals (9.0 +/- 0.7%, P less than 0.0001) significantly exceeded those observed before injury (P less than 0.02, paired t test). Third, injury accentuated response amplitude declines during the stimulus train, most prominently at 80 msec intervals. Spinal cord injury did not affect the dorsal root responses. L5 root compression injury depressed dorsal root action potentials at 3-5 msec interpulse intervals (36.9 +/- 8.4%, n = 4, P less than 0.0001) but had no other effect on the responses. Our data indicate that randomized double pulse evoked potentials are sensitive detectors of acute axonal dysfunction and can be used to quantify stimulus frequency-dependent conduction deficits in injured central and peripheral axons. PMID- 1708715 TI - Bereitschaftspotential in a simple movement or in a motor sequence starting with the same simple movement. AB - The Bereitschaftspotential (BP) recorded from 3 derivations (vertex, left and right precentral areas) in 20 right-handed, normal young subjects was compared in 2 kinds of motor task: a simple movement (task A) and a motor sequence (task B) starting with the simple movement (A). Differences in the onset time and amplitude of the BP were observed: the onset was earlier and the amplitude was larger in the sequential motor task (B) than in the simple one (A). These differences were more important at the vertex (Cz) and in the right precentral area (C4) than in the left contralateral precentral area (C3). These results suggest that the preparatory processes involved in a motor sequence do not exclusively concern the initial movement but also the remainder of the motor task and that the BP is dependent upon the duration or the complexity of the motor task to be executed. The BP seems on temporal grounds to be a global and not a partial expression of a motor task. The changes in the onset time and amplitude of the BP are maximal at the vertex and this could be related to a greater and perhaps earlier activation of SMA in complex sequential motor tasks. PMID- 1708714 TI - Developmental and age-related changes in reflexes of the human jaw-closing system. AB - Reflex responses of the jaw-closing system to innocuous mechanical stimulation of the tongue and palate were examined in a group of 25 girls aged 7-8 years and in a group of 25 women aged 70-80 years. Responses were measured both as changes in background biting force and from bilateral recordings of masseter EMGs. For comparative purposes, results from an earlier study of 35 young adult women (aged 18-25 years) were available. Compared to younger groups of subjects, reflex responses of the elderly were reduced in numbers and amplitude, were characterized by fewer initial excitatory component responses, and had longer latency to onset. Analyses of responses of the children indicated that age 7-8 years is a transitional period. Some children show adult-like responses, while others display responses that appear to represent earlier forms or transitional responses. These results suggest that oral-motor reflexes are not fixed response patterns upon which more complex motor skills, such as speech, are built. Rather, oral reflex development appears to occur in concert with the acquisition of complex motor skills. Systematic changes in reflex responses also occur in the period from young adulthood to seventh decade of life. This result indicates a continuous evolution of oral sensorimotor systems throughout the human life span. PMID- 1708716 TI - Changes in electromyographic responses to muscle stretch, related to the programming of movement parameters. AB - Three experiments are reported that used the advance information paradigm which consists of providing subjects with either no or partial information about an upcoming movement. Subjects moved handles to control the vertical displacements of CRT beams, to point to eight targets. The illumination of different combinations of these targets prior to movement execution provided advance information about which hand, movement direction, or movement extent would be required. Reaction time (RT), integrated EMG activity in the forearm extensor and flexor muscles, and M1, M2, and M3 components of the stretch reflex responses triggered in these muscles were analysed as a function of the precued movement parameter. Compared to the no-information condition, RT decreased in all precue conditions; however, the reduction was greater when direction than when hand was precued, and greater when hand than extent was precued. The EMG activity of forearm muscles increased during the preparatory period in all precue conditions, but generally did not differ among them. An overall facilitation of the stretch reflex components was observed in all precue conditions. This facilitation: (1) was greater for flexor than extensor muscles, (2) was similar regardless of the degree of extent precued, (3) differed for the M2 and M3 components depending on whether the responding hand precued was ipsilateral or contralateral. When the precued movement direction was considered, similar changes in the M3 component were found in extensor and flexor muscles. M3 was facilitated when the muscle was precued as an agonist and was inhibited when it was precued as an antagonist. Collectively these data provide support for a motor programming conception of movement organization. PMID- 1708717 TI - Finite limb dimensions and finite muscle length in a model for the generation of electromyographic signals. AB - A volume conductor model is presented in which the aspects of finite volume conductor dimensions and finite muscle fiber length are combined. The effects of these aspects on single muscle fiber action potentials (SFAPs) and on motor unit action potentials (MUAPs) are shown and verified with surface recorded motor unit action potentials. It is demonstrated that the influence of the fiber length being finite is enhanced significantly by the finite limb dimensions of the volume conductor model, for single fiber action potentials as well as for motor unit action potentials. The model described is found to be capable of generating surface MUAPs which show a very good resemblance with measured surface MUAPs. Recorded MUAPs illustrate clearly the effects caused by finite muscle fiber length. The effect of finite limb dimensions in simulated intramuscular MUAPs was evidently less dominant than in simulated surface MUAPs. PMID- 1708718 TI - Latency of motor evoked potentials to focal transcranial stimulation varies as a function of scalp positions stimulated. AB - We recorded motor evoked potentials (MEP) to transcranial magnetic stimulation from abductor pollicis brevis (APB), flexor carpi radialis (FCR), biceps brachii and deltoid muscles at rest and during slight voluntary activation. An 8-shaped coil connected to a Cadwell MES-10 magnetic stimulator was positioned over different scalp positions 1 cm apart. At least 24 stimuli were delivered at each location. Latencies of MEPs were compared with those obtained by electrical and magnetic stimulation during muscle activation. Progressively longer MEP latencies were obtained in 5 groups depending on the type and position of stimulation. The shortest latencies were obtained with (1) electrical stimulation during muscle contraction and (2) non-focal magnetic stimulation during muscle contraction; magnetic stimulation at rest produced longer latencies with stimulation of (3) an optimal position, (4) a suboptimal position, and (5) a non-optimal position. Mean latency differences between successive groups were 1.9, 2.0, 1.6, and 2.6 msec for APB. Similar latency differences were found for the other arm muscles. The results are compatible with the hypothesis that the different latencies evoked by stimulation at different scalp locations are determined by the summation at spinal motoneurons of excitatory postsynaptic potentials generated by successive numbers of I waves. PMID- 1708719 TI - Non-invasive evaluation of central motor tract excitability changes following peripheral nerve stimulation in healthy humans. AB - The interval between muscle stretch and the onset of the long latency electromyographic responses (LLRs) has been theoretically fragmented into an afferent time (AT), taken at the peak of wave N20 of somatosensory evoked potentials and an efferent time (ET), calculated by means of magnetic transcranial stimulation (TCS), the two being separated by a cortical interval (CI). If this were the case, the afferent input should progressively 'energize' the sensorimotor cortex during the CI and change the excitability of cortico spinal tracts. To investigate this, motor evoked potentials (MEPs) from thumb flexor muscles were recorded, whilst a conditioning stimulation of median or ulnar nerve randomly preceded (10-48 msec intervals) magnetic brain TCS. Nerve stimulation was adjusted to motor threshold and amplitudes of conditioned and test MEPs at different nerve-TCS interstimulus intervals were evaluated. Conditioned MEPs were significantly attenuated with nerve-TCS intervals between 16 and 20 msec for elbow and 20 and 22 msec for wrist stimulation. This was followed by MEP potentiation with nerve-TCS intervals corresponding to the sum of AT + CI (mean 23.2 msec, range 21.7-24.8). The onset latency of facilitated conditioned MEPs was about 1 msec briefer than that of test MEPs, but invariably longer than the latency of MEPs facilitated by a voluntary contraction. This protocol did not demonstrate amplitude facilitation of the segmental H reflex, corroborating the idea that the facilitated part of the conditioning nerve-TCS curve receives a transcortical loop contribution. PMID- 1708720 TI - The NCS3 mutation: genetic evidence for the expression of ribosomal protein genes in Zea mays mitochondria. AB - A deletion eliminating part of a transcribed region of mitochondrial DNA (mtDNA) has been found in the maize nonchromosomal stripe 3 (NCS3) mutant. This results in the specific loss of a set of three mitochondrial RNAs consisting, in normal plants, of a 4.9 kb transcript, its 1.8 kb intron and the resulting processed mRNA of approximately 2.9 kb. In the NCS3 mitochondrial genome the DNA encoding the putative promoter and 5' end of the affected RNAs is missing. This transcribed region of normal maize mtDNA has been sequenced and the intron splice junction has been determined. The 2.9 kb processed mRNA carries two overlapping open reading frames (ORFs) with predicted amino acid sequences that show similarity to two Escherichia coli ribosomal proteins, S3 (rps3) and L16 (rpl16). The presence of severe stunting and striping in NCS3 plants correlates absolutely with the molecular changes described here. This fact and the impaired ability for mitochondrial protein synthesis by NCS3 plants indicate that one or both of these reading frames are translated to functional ribosomal proteins in normal maize mitochondria. PMID- 1708721 TI - Mammalian genes expressed in Drosophila: a transgenic model for the study of mechanisms of chemical mutagenesis and metabolism. AB - Mammalian cytochrome P450s provide our first line of defence against the toxic effects of environmental chemicals. Ironically these enzymes also convert some compounds to their ultimate toxic or mutagenic species. Our knowledge of these mammalian enzymes and the role they play in chemical toxicity and mutagenesis has stemmed mostly from in vitro studies. In order to establish the role of specific enzymes in the toxicological response in vivo we have generated transgenic Drosophila which express mammalian cytochrome CYP2B1, which is a member of a large gene family encoding several important drug metabolising enzymes. The gene was fused to a Drosophila promoter which confers expression in the larval fat body. Using the Somatic Mutation And Recombination Test (SMART) we have demonstrated that transgenic larvae expressing the P450 are hypersensitive to the anticancer drug cyclophosphamide, a procarcinogenic substrate which is activated by the enzyme. This work demonstrates the potential of such transgenic Drosophila strains as an in vivo model for studying the role of specific mammalian drug metabolising enzymes in the pathways and metabolic cascades associated with the action of cytotoxic and carcinogenic chemicals, and also the chemical properties of specific classes of mutagen to be determined. PMID- 1708722 TI - Properties of promoter regions of mdg1 Drosophila retrotransposon indicate that it belongs to a specific class of promoters. AB - A sequence 30 bp downstream from the start site of the Drosophila melanogaster retrotransposon mdg1 is shown to be responsible for correct and precise initiation of mdg1 RNA synthesis in combination with the RNA start-site sequence TCAGTT. A sequence-specific DNA binding protein is demonstrated to interact with the +30 sequence, and the efficient binding of this factor is necessary for in vivo transcriptional activity of the plasmid constructs containing mdg1 promoter fragments. The nucleotides -8/+34 of mdg1 represent a minimal promoter which is able to provide correct initiation of transcription by RNA polymerase II at basal levels. A comparison with properties of some other retrotransposable elements and several developmentally regulated cellular genes allows us to conclude that together they form a specific class of RNA polymerase II promoter. This promoter class characteristically lacks upstream sequences necessary for transcription initiation, such as TATA boxes, but requires a specific downstream promoter element within 40 bp downstream of the RNA start site. The level of transcription can, however, be modulated by upstream regulatory elements. The identified sequence-specific downstream initiation factor may be responsible for transcription initiation on promoters of some genes which belong to this class. PMID- 1708723 TI - Conserved genes encode guide RNAs in mitochondria of Crithidia fasciculata. AB - RNA editing is the post-transcriptional alteration of the nucleotide sequence of RNA, which in trypanosome mitochondria is characterized by the insertion and deletion of uridine residues. It has recently been proposed that the information for the sequence alteration in Leishmania tarentolae is provided by small guide (g) RNAs encoded in the mitochondrial DNA [Blum et al. (1990) Cell, 60, 189-198]. We are studying the mechanism of RNA editing in the insect trypanosome Crithidia fasciculata and report that: (i) a full length, conventional DNA gene or an independently replicating RNA gene that could encode the edited MURF3 transcript is absent when probed for in sensitive, calibrated assay systems; (ii) in all cases (seven) investigated in C. fasciculata so far, putative gRNA genes are found in a position in the mitochondrial DNA virtually identical to that in L. tarentolae and (iii) also in C. fasciculata, the putative gRNA genes are transcribed into small RNAs with discrete 5' ends. These results provide strong evolutionary evidence in support of the participation of gRNAs in RNA editing. Remarkably, in C. fasciculata the basepaired region of some putative gRNA:mRNA hybrids contains a C:A non-Watson-Crick basepair. PMID- 1708724 TI - Assessment of 1,2-dimethylhydrazine in bone marrow micronucleus assay: variations in protocol and response. AB - The effect of variations in experimental protocol on the assessment of the genotoxicity of 1,2-dimethylhydrazine (DMH) in the bone marrow micronucleus assay was determined. The incidence of micronuclei (MN) in the bone marrow of CBA mice treated with DMH (either intraperitoneally (i.p.) or orally) was found to be significantly greater than that observed in C57B1/6J mice using the same dose and dosing regimen. With i.p. injection, DMH, at doses of 20 and 50 mg/kg, was found to be positive in the bone marrow MN test in CBA mice only. In C57B1/6J mice, DMH (i.p.) was found to be positive at only the 50 mg/kg dose. With oral administration, DMH was positive in the MN test only at the 50 mg/kg dose and only in CBA mice. No significant difference in the percentage of MN was observed when 300, 500, or 1,000 polychromatic erythrocytes (PCEs) were scored following a single treatment of DMH. Cyclophosphamide (CY) was found to induce a dose dependent increase in the percentage of MN observed in the bone marrow of C57B1/6J mice. DMH tested positive in the colon nuclear aberration (NA) assay in both strains of mice using both i.p. and oral routes of administration, although C57B1/6J mice were found to be more sensitive than CBA mice. No significant difference was observed regarding the percentage of NAs observed in the colon between mice injected i.p. or orally gavaged. PMID- 1708725 TI - Sensory neurons and motoneurons of the jaw-closing reflex pathway in rats: a combined morphological and physiological study using the intracellular horseradish peroxidase technique. AB - Motoneurons and muscle spindle afferents of the rat masseter muscle were physiologically and morphologically characterized. Their soma-dendritic morphology and axonal course were investigated using the intracellular horseradish peroxidase method. Following electrical stimulation of the masseter nerve, individual motoneurons were identified by antidromic all-or-none action potentials and individual sensory neurons by orthodromic action potentials. Using threshold separation an excitatory input from muscle spindles to a masseter motoneuron was demonstrated. The short latency difference of 0.34 ms between the mean orthodromic response in the sensory neurons and the beginning of the synaptic potential in the masseter motoneuron suggests a monosynaptic connection between the spindle afferents and the motoneurons. Following intrasomatic horseradish peroxidase injection large multipolar cell bodies of masseter motoneurons were found within the motor nucleus. Their positions corresponded to the topographic organization of the motor trigeminal nucleus as described in retrograde tracing studies. Dendrites of masseter motoneurons were complex and could be found far beyond the nuclear borders. Distal dendrites extended to the mesencephalic trigeminal nucleus, the supratrigeminal nucleus, the lateral lemniscus and the reticular formation. Within the reticular formation dendrites were seen in the intertrigeminal nucleus and the peritrigeminal zone. Unipolar cell bodies of muscle spindle afferents were found in the mesencephalic trigeminal nucleus after intra-axonal injection of horseradish peroxidase. For all reconstructed sensory neurons a similar axonal course was found. Axonal terminals were found ipsilateral in the motor trigeminal nucleus, indicating a direct connection between sensory neurons and motoneurons. Further collaterals were found ipsilateral in the supratrigeminal nucleus and caudal to the motor trigeminal nucleus in the parvocellular reticular nucleus alpha. Since the latter termination areas are important for bilateral control of jaw-movements, the muscle spindle afferents are likely to participate not only in a monosynaptic motor reflex, but also in more complex neuronal circuits involved in jaw movements. PMID- 1708727 TI - Free calcium transients and oscillations in nerve cells. AB - Changes in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xanthine induced release. IP3 and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated. PMID- 1708726 TI - Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat. AB - In situ hybridization was used to determine whether genes for neuropeptides [substance P/neurokinin A (SP/NKA), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide tyrosine (NPY) and cholecystokinin (CCK)] are expressed in inferior ganglia of the vagus (nodose) and glossopharyngeal (petrosal) nerves. Synthetic oligodeoxyribonucleotides, complementary to the cognate, mRNAs were labeled with [32P] or [35S], and hybridized to 10 microns thick sections of unperfused tissue which were then processed for film and emulsion autoradiography. We found numerous, clustered neuronal perikarya throughout the nodose and petrosal ganglia that expressed preprotachykinin A (SP/NKA) and CGRP mRNAs to varying degrees. Neurons expressing preproSOM mRNA were less abundant and more scattered throughout both ganglia. Notably, we found mRNA for NPY in cells (usually 5-10 per section) in both ganglia. To our knowledge, this is first evidence for NPY in these sensory ganglia. In contrast to previous immunohistochemical findings, we found no evidence for expression of preproCCK in either the nodose or petrosal ganglia. The present findings demonstrate that cells of the nodose and petrosal ganglia express the genes for a number of neuropeptides that are presumably involved with transmission of visceral sensory afferent information to higher order neurons of the central nervous system. PMID- 1708728 TI - Renal lesions in hypocalcemic conditions. Phenotypic manifestations of genetical background and renal expressions. PMID- 1708729 TI - High amylase activity in pleural fluid and primary bronchogenic adenocarcinoma. AB - We report a case of primary bronchogenic adenocarcinoma, complicated by pleural effusion, in which very high pleural amylase activity was found, whilst serum amylase was normal. Isoamylase determination showed a salivary-type amylase. Concerning the origin of this enzyme, ultrastructural study of the malignant cells obtained from the pleural fluid suggested a local amylase synthesis. The pathophysiological significance of electron-dense granules found in these cells is also discussed. PMID- 1708730 TI - Anaphylactic reaction because of intrauterine 32% dextran-70 instillation. AB - Anaphylactic reaction associated with hysteroscopy using 32% dextran-70 as a distending medium is a rare phenomenon. We report anaphylactic reaction associated with hysteroscopy using dextran that was delivered manually in three otherwise healthy women occurring within a 6-month period. The rapidity and pressure of dextran administered might facilitate intravascular absorption and predispose patients to this phenomenon. PMID- 1708731 TI - Oral contraceptives increase insulin-like growth factor binding protein-1 concentration in women with polycystic ovarian disease. AB - Insulin-like growth factor-I (IGF-I) stimulates ovarian androgen production. Insulin-like growth factor binding protein-1 (IGFBP-1) inhibits IGF actions in vitro. OBJECTIVE: To investigate the effect of oral contraceptive (OC) pills, given for 3 months, on serum gonadotropin, androgen, IGF-I, and IGFBP-1 concentrations, and glucose tolerance in seven women with polycystic ovarian disease (PCOD) and in five healthy control subjects. PATIENTS: Seven women with PCOD and five healthy control subjects. INTERVENTIONS: An oral glucose tolerance test (OGTT) was performed before and after treatment with OC. RESULTS: After treatment with OC, serum luteinizing hormone, androstenedione, and free testosterone levels decreased, and sex hormone-binding globulin concentration increased in the women with PCOD as well as in the control subjects. The cumulative response of serum insulin to OGTT was larger in the women with PCOD than in the control subjects both before and after treatment. Serum IGF-I concentration, which was unchanged during OGTT, decreased from basal level of 326 +/- 70 micrograms/L to 199 +/- 28 micrograms/L after treatment with OC in the women with PCOD, whereas no change was found in the control subjects (from 235 +/ 11 micrograms/L to 226 +/- 11 micrograms/L). Treatment with OC caused an increase of the mean basal IGFBP-1 concentration from 24 +/- 7 micrograms/L to 73 +/- 14 micrograms/L in the women with PCOD. This increase was constant during the OGTT. In the control subjects, treatment with OC did not result in any significant change in IGFBP-1 concentrations (from 44 +/- 11 micrograms/L to 61 +/- 9 micrograms/L). CONCLUSION: The combination of decreased total IGF-I concentration and increased IGFBP-1 concentration induced by OC may decrease ovarian androgen production in PCOD. PMID- 1708732 TI - Cellular and tissue distribution of a single-strand-specific nuclease. AB - 1. Specific antibodies which were raised against a single-strand-specific nuclease isolated from rat liver microsomes were used for characterizing this enzyme and determining its cellular and tissue distribution. 2. The single-strand specific nuclease does not show any homology with other known nucleolytic enzymes. 3. It is mainly localized in microsomes and cytosol; traces of it are also found in nuclei, but it could not be detected in mitochondria. 4. Using the same specific antibodies we attempted to detect this nuclease in some other tissues which exhibit high metabolic rates, namely kidneys, heart and spleen. 5. Thus, with the aid of immunological techniques we were able to determine that at least part of the total poly(U) nucleolytic activity observed in kidney and heart is due to a nuclease immunologically identical to the enzyme under study. Kidneys were the best source for this enzyme. PMID- 1708733 TI - Evidence that angiotensin II decreases mitochondrial calcium in the glomerulosa cell. AB - The present studies were performed using primary monolayer cultures of bovine glomerulosa cells to determine whether the elevation in cytosolic calcium concentration produced by angiotensin II was accompanied by an elevation in mitochondrial calcium. Exchangeable mitochondria calcium content was assessed indirectly by measuring the changes in cytosolic calcium concentration and calcium efflux produced by the mitochondrial uncoupler, carbonyl cyanide m chlorophenylhydrazone (CCCP). Total mitochondrial calcium content was also assessed directly by atomic absorption spectroscopy. CCCP had a direct effect to promote calcium release from an oligomycin/antimycin-sensitive (mitochondrial) calcium pool in permeabilized cells. In intact cells, CCCP caused rapid reductions in cellular ATP content and the ratio of ATP to ADP. Still, its effects on calcium dynamics were exerted primarily at the mitochondrial level as evidenced by inhibition with ruthenium red, but not dantrolene. As expected, angiotensin II produced a rapid increase in calcium efflux and an equally rapid and sustained increase in cytosolic calcium concentration. Nonetheless, CCCP stimulated elevations in cytosolic calcium concentration and calcium efflux were reduced by angiotensin II in a concentration-dependent manner. Total mitochondrial calcium content was also lower in angiotensin-treated than in control cells. These results indicate that angiotensin II causes a net decrease in mitochondrial calcium stores. On the basis of these data, it is proposed that alterations in calcium metabolism initiated by angiotensin II are exerted not only at the membrane and cytosolic levels but also at the level of the mitochondria. Changes in mitochondrial calcium dynamics may directly contribute to the regulation of mitochondrial steroidogenic enzymes by angiotensin II. PMID- 1708734 TI - Effects of nonenzymatic glycosylation of mesangial matrix on proliferation of mesangial cells. AB - Cross-linking of cell matrix components by nonenzymatic glycosylation may contribute to diabetic glomerulopathy. We examined the effects of modification of matrix by nonenzymatic glycosylation on mesangial cell function. Matrix was generated by growing mesangial cells in tissue culture for 2 wk and removing the cells with a detergent cell-lysis solution. By indirect immunofluorescence and Northern-blot analysis, the remaining matrix contained laminin, fibronectin, and collagens type I and IV. The matrix was modified by incubation for 24 h with 50 mM glycolaldehyde, a highly reactive cross-linking nonenzymatic glycosylation product, or for 2 wk with 200 mM glucose-6-phosphate (G6P). Modification was carried out with or without equimolar aminoguanidine, an inhibitor of cross-link formation. Nonenzymatic glycosylation of the matrix by glycolaldehyde or G6P was confirmed by fluorometry and [14C]G6P incorporation and was prevented by aminoguanidine. [3H]thymidine incorporation for 24 h by mesangial cells plated onto unmodified or modified matrix was then performed. Modification of matrix had no effect on attachment of mesangial cells, determined 4 h after plating. Nonenzymatic glycosylation of matrix by glycolaldehyde or G6P significantly inhibited thymidine incorporation by mesangial cells. This effect was partially reversible by aminoguanidine. Aminoguanidine-modified matrix had no effect on thymidine incorporation. Thymidine-incorporation results were confirmed by direct cell counting. We conclude that modification of matrix by nonenzymatic glycosylation influences growth of mesangial cells, which could contribute to the mesangial abnormalities of diabetic glomerulopathy. PMID- 1708735 TI - Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts. AB - We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16,K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: "keratinizing" (gingiva, hard palate), "typical nonkeratinizing" (inner cheek, floor of mouth, ventral tongue) and "special non keratinizing" (soft palate), all of which differed from the epidermal keratinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult "special nonkeratinizing" subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation. PMID- 1708736 TI - Heterogeneity of intermediate filament expression in human testicular seminomas. AB - Testicular seminoma has in the past been considered to represent a germ cell tumor incapable of further differentiation. In recent years this view has been challenged on the basis of morphologic and chromosomal studies. Moreover, studies of intermediate filaments (IF) of seminoma cells have provided evidence of the capability of seminoma cells to differentiate in different directions. In the present study of the IF protein profile of 26 human testicular seminomas, using frozen as well as formalin-fixed, paraffin-embedded tissues, we report evidence of a heterogeneous differentiation potential inherent in these neoplasms. Thus, in 4 of the seminomas neither cytokeratins nor vimentin were detected; 3 showed vimentin positive cells but no cytokeratins; in 4 seminomas only cytokeratins were detected. In the remaining 15 cases both cytokeratins and vimentin were present, with occasional cells demonstrating coexpression of cytokeratin and vimentin. While the cytokeratins present were mostly of the "simple epithelial type", in 2 instances seminoma cells also contained cytokeratins 4 and 17, normally found in stratified and/or complex glandular epithelia. Furthermore, in 3 cases scattered tumor cells stained for desmin and in 2 other seminomas neurofilaments were identified. All of the cases showed variable positive staining for desmoplakins and desmoglein, indicative of the presence of desmosomes. It can therefore be concluded that, while some seminomas seem to be devoid of IFs, most of them show varied differentiation patterns usually with epithelial features but occasionally also with components commonly regarded as characteristic of myogenic or neurogenic differentiation. These observations may help to elucidate the relationship of seminomas to other germ cell tumors, and also contribute to our understanding of the histogenesis of these neoplasms. PMID- 1708737 TI - A cellular logic for G protein-coupled ion channel pathways. AB - A vast array of cellular signal transduction processes arise from combinations of many different types of agonists, receptors, effectors, and coupling molecules such as heterotrimeric G proteins or protein kinases that connect receptors to effectors. Receptors, effectors, G proteins, and kinases are being newly identified at bewildering speeds and in the process it seems that our understanding of how cells respond to specific stimuli may have diminished just as we lose sight of the forest when we are buried in the trees. Evolution would suggest that there may be a logic to the response provoked by a given stimulus and, using our recently acquired knowledge of G protein pathways between receptors and ion channel effectors, I will attempt to decipher what the underlying logic might be. PMID- 1708738 TI - Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus. AB - The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed. PMID- 1708739 TI - Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene. AB - The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein. PMID- 1708740 TI - Molecular characterization of the murine argininosuccinate synthetase locus. AB - The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2. PMID- 1708741 TI - [Genetic techniques in drug production. Current status and future perspectives. 2: Approved recombinant substances]. PMID- 1708742 TI - [Changes in plasma vitronectin, fibronectin, and serum laminin P1 levels and immunohistochemical study of vitronectin in the liver of patients with chronic liver diseases]. AB - Vitronectin (VN), fibronectin (FN) and laminin (LM), which are known to be important glycoproteins in cell attachment, are produced by such liver cells as hepatocytes, Kupffer cells endothelial cells and Ito cells. In this study, the levels of plasma VN, FN and serum LM P1 in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma accompanied with cirrhosis were examined and compared with those in normal subjects. Plasma VN levels in patients with chronic hepatitis, compensated cirrhosis and decompensated cirrhosis were less than that in normal subjects. As hepatic dysfunction deteriorated, plasma VN level decreased in chronic liver diseases. Plasma FN levels in patients with compensated and decompensated cirrhosis were also less than that of patients with chronic hepatitis, which was not significantly different from that of normal subjects. Plasma VN and FN levels in patients with hepatocellular carcinoma were similar to those in patients with compensated cirrhosis. Plasma VN and FN levels in patients with chronic liver diseases including hepatocellular carcinoma showed positive correlations with serum albumin content, cholinesterase activity, and normalized normo test value. On the other hand, serum LM P1 levels in patients with chronic hepatitis, compensated cirrhosis and decompensated cirrhosis were higher than that of normal subjects. As hepatic dysfunction deteriorated, serum LM P1 level increased in chronic liver diseases. Level of serum type IV collagen 7S, which is related to hepatic fibrosis, was similar to that of serum LM P1; serum LM P1 concentration in patients with chronic liver diseases showed a significant positive correlation with that of serum type IV collagen 7S. Immunolocalization of VN in liver tissue from patients with chronic hepatitis and cirrhosis was examined by the method of avidin-biotin-complex staining, and positive reaction was observed in enlarged portal tracts, central veins and fibrous septa. These results suggest that decreased levels of plasma VN and FN and increased level of serum LM P1 in patients with chronic liver diseases are related to hepatic dysfunction, and that changes in the levels of these glycoproteins involved in cell attachment are important in the development of hepatic fibrosis in patients with chronic liver diseases. PMID- 1708743 TI - G protein-mediated ion channel activation. AB - Guanine nucleotide binding proteins couple a wide variety of receptors to ion channels via both "direct" or membrane-delimited and "indirect" second messenger mediated pathways. This tutorial summarizes current approaches to defining the mechanisms of guanine nucleotide binding protein-mediated ion channel activation. Two well-characterized ion channels in the heart, namely, the beta-adrenergic receptor-activated calcium channel and the muscarinic receptor-activated potassium channel, are used to illustrate the criteria that can distinguish between direct and indirect guanine nucleotide binding protein-transduced pathways. PMID- 1708744 TI - Insulin-like growth factor I gene expression in vascular cells. AB - Insulin-like growth factor I (IGF I), a potent growth factor in vitro, is present in blood and in multiple tissues and is a major mediator of the effects of growth hormone on postnatal growth. IGF I is internalized and retained largely intact in cultured vascular endothelial cells. Neovasculature transiently expresses IGF I immunoreactivity, but it is not known whether this represents internalization of the circulating growth factor or vascular cell synthesis of IGF I. As an initial approach to defining the role of endogenous production of IGF I in the growth program of the vessel wall, Northern hybridizations were performed with RNA from cultured rat aortic smooth muscle cells and bovine aortic endothelial cells. Rat aortic smooth muscle cells expressed three primary IGF I messenger RNA transcripts sized 8.2, 1.7, and 0.9-1.2 kb. Bovine aortic endothelial cells expressed one major and one minor IGF I transcript of 2.1 and 1.6 kb, respectively. IGF I gene expression in smooth muscle cells was also demonstrated by ribonuclease protection assays using a rat exon 3 riboprobe. Both endothelial and vascular smooth muscle cells secreted IGF I, as detected by radioimmunoassay of conditioned medium after separation of IGF I from its binding proteins by gel filtration chromatography. Because IGF I stimulates growth of vascular cells, characterization of IGF I gene expression in blood vessels may be key to understanding developmental as well as abnormal growth in the cardiovascular system. PMID- 1708745 TI - Solid phase synthesis of the retroviral nucleocapsid protein NCp10 of Moloney murine leukaemia virus and related "zinc-fingers" in free SH forms. Influence of zinc chelation on structural and biochemical properties. AB - The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX2 CysX4 HisX4 Cys zinc finger-like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn2+ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn2(+)-dependent, allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 10(6) M-1. The synthetic protein, in the presence of Zn2+ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNA(Pro) onto MoMuLV RNA. PMID- 1708746 TI - Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes. AB - Several cell types participate in the formation of vitreoretinal traction membranes in proliferative intraocular disorders. The communication between these cells involves hormones, growth factors, and the interaction with extracellular matrix molecules. We have previously demonstrated a partial colocalisation of two potent mediators of cell attachment, fibronectin and vitronectin, in periretinal membranes from patients with proliferative vitreoretinopathy (PVR). We found a similar pattern of vitronectin and fibronectin deposition in proliferative diabetic retinopathy (PDR) (n = 6). Now we show the expression of the corresponding cell surface receptors, integrins, for fibronectin and vitronectin by proliferating cells in 22 periretinal membranes, including traumatic (n = 8) and idiopathic (n = 8) PVR as well as PDR membranes (n = 6). Integrins are membrane receptors for extracellular matrix macromolecules which are involved in such basic biological phenomena as embryogenesis and metastasis. Future studies on the pathogenesis of vitreoretinal proliferation will have to focus on the initiation, maintenance, and regulation of this intercellular communication network involving attachment proteins and integrins. PMID- 1708747 TI - Suppurative keratitis in rural Bangladesh: the value of gram stain in planning management. AB - External eye disease which result in corneal scarring are an important cause of blindness in Bangladesh and at the Chittagong Eye Infirmary and Training Complex (EITC) over 200 cases of suppurative keratitis are managed each year. We reviewed the records of 127 cases of microbial keratitis to determine the relative contributions of Gram stain and culture to diagnosis of the causative organism. There were 107 culture-proven cases of microbial keratitis amongst the 127 patients in this study. Gram stain was positive in 89 cases which represents 70% of the total and 83% of all culture-proven cases. Streptococcus pneumoniae and Pseudomonas sp were the commonest bacteria isolated and Aspergillus sp and Fusarium sp the commonest fungi. In 20 cases (16%) no organism was isolated on Gram stain or culture. Our results support the use of both Gram stain and culture in isolation of the causative organism in cases of suppurative keratitis in Bangladesh. However the low cost of Gram stain and its useful recovery rates for both bacteria and fungi support its use as an initial investigation for microbial keratitis at the secondary level of eye care in rural Bangladesh. PMID- 1708748 TI - Vitronectin and proliferative intraocular disorders. I. A colocalisation study of the serum spreading factor, vitronectin, and fibronectin in traction membranes from patients with proliferative vitreoretinopathy. AB - The presence of a scaffold for cellular spreading and proliferation is a precondition for the development of traction membranes in proliferative vitreoretinopathy (PVR). This study shows the presence of the serum spreading factor, vitronectin, in the extracellular matrix of periretinal membranes removed during vitreoretinal surgery. By means of a double label immunofluorescence protocol, a partial colocalisation of vitronectin with fibronectin at the magnification of light microscopy can be detected. Fibronectin is a high molecular glycoprotein with multiple biological functions including the mediation of cell attachment and migration. Both proteins share a special cell recognition site which could be a target for experimental pharmacological approaches to PVR. Preliminary studies of vitreous aspirates using electrophoresis and Western blotting indicate that vitronectin may play a more important role in post traumatic PVR as compared to PVR following rhegmatogenous retinal detachment. PMID- 1708749 TI - Standardization of biological dyes and stains: pitfalls and possibilities. AB - The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review. PMID- 1708751 TI - Immunohistochemical identification of tubular segments in percutaneous renal biopsies. AB - To identify the renal cortical tubular segments involved in tubulo-interstitial disease in formalin-fixed, paraffin-embedded percutaneous kidney biopsies, we developed multiple immunolabeling protocols using segment-specific tubular markers. The present study of biopsies from patients with minimal change or thin basement membrane nephropathy provides a baseline for interpretation of histopathology. Proximal tubules were stained either by the PAS reaction or by the biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E)-streptavidin-gold silver system (brush borders black). The anti-Tamm-Horsfall (THP) antibody immunoperoxidase (aminoethylcarbazole, AEC-IPO), and anti-epidermal cytokeratins (ECK) antibodies-immunoalkaline-Fast Blue BB methods marked the distal straight tubules and the cortical collecting system red-brown and blue, respectively. When these immunolabelings were combined, the coapplication of AEC-PO-labeled peanut agglutinin (PNA) or anti-epithelial membrane antigen antibody-AEC-IPO technique (both are markers for distal nephron) visualized the apical membranes of distal convoluted tubules. In the protocol PHA-E + PNA + THP + ECK, the tubular basement membranes were outlined by the anti-laminin antibody-AEC-IPO staining, carried out simultaneously. The protocol PNA + THP + ECK + PAS was found to be quite appropriate multiple immunolabeling method for the tubules, and is recommended for use as a tool in the study of tubulo-interstitial diseases. PMID- 1708750 TI - Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins. AB - We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins. In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to alpha-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal alpha-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal beta 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to beta-Gal and Limax flavus (LFA) which is specific to alpha-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules. The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells. PMID- 1708752 TI - Antigenicity of HLA-A2 and HLA-B7. Loss and gain of serologic determinants induced by site-specific mutagenesis at residues 62 to 80. AB - The contribution of the hypervariable region spanning amino acid residues 62 to 80 to the serologic determinants of HLA-A2 and HLA-B7 has been examined by site directed mutagenesis. Three HLA-A2 mutants, having changes as in HLA-B7 at positions 62, 76, and at the complete 65-to-80 segment, respectively, were obtained and expressed on class I HLA-deficient human cells upon transfection. The reactivity of 19 monoclonal antibodies (mAbs) against both broad public and allospecific determinants on HLA-A2 and HLA-B7 was analyzed. The results indicate that: (1) the change at residue 62 abrogated recognition of the corresponding HLA A2 mutant by mAb MA2.1 (anti-A2 + B17); (2) the change at residue 76 did not effect any of the determinants analyzed, although its side chain is easily accessible at the surface of the molecule; (3) the replacement of the whole 65-to 80 segment in HLA-A2 by that from HLA-B7 abrogated recognition by MA2.1 and by 108-2C5, a mAb recognizing a public determinant from the HLA-A locus. Such replacement led to gaining the determinants recognized by mAbs GS145.2 (anti-B7 + B27) and SFR8-B6 (anti-Bw6); and (4) the HLA-A2-reactive mAbs whose reactivity was known to be abrogated by changes in alpha 2 were unaffected by the changes introduced in alpha 1, underlining the frequent segregation of serologic determinants on class I antigens to single domains. PMID- 1708753 TI - Humans with OKT4-epitope deficiency have a single nucleotide base change in the CD4 gene, resulting in substitution of TRP240 for ARG240. AB - The OKT4 epitope of the CD4 cell-surface protein has been shown to be polymorphic in white, black, and Japanese populations. The variable phenotypic expression is due to an alteration of the OKT4 epitope, since those persons lacking reactivity with OKT4 monoclonal antibody (mAb) are reactive with OKT4A-F mAb as well as other mAb specific for CD4. To determine the nature of this polymorphism at the gene level, we sequenced polymerase chain reaction-amplified genomic DNA containing the CD4-V3 and -V4 exons from American black subjects who are OKT4 normal, OKT4-negative heterozygous, or OKT4-negative homozygous. Comparison of the sequences revealed that the two CD4 exons are identical except for a cytosine to-thymidine transition occurring at nucleotide position 868. This alters the first codon position of mino acid 240 and results in a tryptophan residue replacing an arginine residue. The change was also found in white and Japanese persons who are OKT4-negative. PMID- 1708754 TI - Higher frequency of point mutations in the c-K-ras 2 gene in human colorectal adenomas with severe atypia than in carcinomas. AB - Human colorectal carcinomas may be induced from adenomas or they may occur de novo. To examine which is the main pathway, we analyzed point mutations at codon 12 in the c-K-ras 2 gene in 73 colorectal carcinomas, 13 metastatic tumors, 72 adenomas and 30 normal tissues. The c-K-ras 2 codon 12 mutation frequency was 0/30 in normal tissues, 0/17 in adenomas with mild atypia, 3/37 (8.1%) in adenomas with moderate atypia, 15/18 (83.3%) in adenomas with severe atypia, 19/73 (26.0%) in primary carcinomas and 3/13 (23.1%) in metastatic tumors. The mutation frequency in adenomas with severe atypia was much higher than that in carcinomas. These results indicate that many colorectal carcinomas may not be induced through adenomas with severe atypia. PMID- 1708755 TI - Trial to induce prostatic cancer in ACI/Seg rats treated with a combination of 3,2'-dimethyl-4-aminobiphenyl and ethinyl estradiol. AB - In an attempt to induce prostatic adenocarcinoma at higher incidence in a shorter period, we administered diet containing 0.75 ppm of ethinyl estradiol (EE) for three weeks to ACI/Seg rats, which are predisposed to develop a high incidence of microscopic adenocarcinoma of the prostate at higher age. Then, feeding was changed to basal diet and a single subcutaneous injection of 50 mg/kg body weight of 3,2'-dimethyl-4-aminobiphenyl (DMAB) was given two days after the change. We repeated this schedule 10 times. The rats were killed in week 60 of the experiment and subjected to routine autopsy. The average body weight of rats in group 1 given EE and DMAB was lower than that of control rats in group 2. The incidence of adenocarcinoma was not significantly different in the two groups, i.e., 6/74 (8.1%) in group 1 and 2/54 (3.7%) in group 2. The lesions were all microscopic. The incidence of atypical hyperplasia was significantly higher in group 1 at 17 of 74 rats (23.0%) whereas in group 2, it was only 2 of 54 rats (3.7%). Simple hyperplasia was also observed in 25 of 74 rats (33.8%) in group 1, which was significantly higher than that in group 2, where six of 54 rats (11.1%) had this lesion. The reduced growth of animals due to treatments with EE and DMAB probably suppressed the development of prostate cancer in this experiment. Further studies are needed to develop an appropriate model to induce prostate carcinoma at higher incidence in a shorter period. PMID- 1708756 TI - Purification of ornithine decarboxylase-inducing factor from cell-free ascites fluid of Ehrlich ascites tumor and its characteristics. AB - The ornithine decarboxylase-inducing factor (ODC factor) was purified about 1,000 fold in 42% yield from the ascites fluids of an Ehrlich ascites tumor by a combination of centrifugation and concanavalin A (ConA) treatment. A single ip injection of 0.5 micrograms of the purified factor per mouse resulted in half maximum induction of liver ODC. The factor was found to be a trypsin- and chymotrypsin-resistant, acidic glycoprotein (pI about 4.43) with a minimum molecular weight of about 70 kilodaltons, containing a disulfide bond(s) in its functional domain. It did not react with ConA. This factor induced retrodifferentiation of liver function, causing a marked increase of prototype M2 isozyme of pyruvate kinase. It reduced liver catalase activity, and also modified thyroid hormone metabolism, reducing the serum levels of T4 and T3. These results suggest that the ODC factor is multifunctional and induces many of the changes observed in a tumor-bearing host. PMID- 1708758 TI - Substance P-induced contraction of rabbit airways: mechanism of action. AB - The mechanism by which substance P induces contraction of airway smooth muscle has been the subject of numerous reports. It has been suggested that in rabbit airways the action of substance P is indirect, via the release of endogenous acetylcholine, whereas this is not so in other species. The present detailed study investigated whether substance P-induced contraction in rabbit isolated bronchus and trachea is due to the release of endogenous acetylcholine or in bronchus is due to histamine release and whether substance P is metabolized by the enzymes enkephalinase and acetylcholinesterase. Isometric contraction to cumulative addition of substance P was measured in the presence of 10(-6) and 10( 4) M atropine, 10(-6) M pyrilamine, 10(-5) M phosphoramidon, or 3 x 10(-7) M neostigmine. Neither atropine nor pyrilamine had any effect on the substance P responses. Phosphoramidon, however, produced a 12-fold shift to the left in the response curve with a decrease in the 50% effective concentration from 7.0 x 10( 8) to 6.1 x 10(-9) M (n = 4 control and 5 treated; P less than 0.05). In contrast, neostigmine at a concentration that produced a sixfold shift to the left in the acetylcholine response curve had no effect on substance P responses. We conclude that, in rabbit airways in vitro, substance P-induced contraction is not mediated by release of endogenous acetylcholine or histamine. In addition, endogenous enkephalinase but not acetylcholinesterase may be involved in the degradation of substance P. Our results show that, in contrast to previous studies in rabbits, the mechanism of action of substance P may resemble that described in humans. PMID- 1708757 TI - Effect of co-administration of granulocyte colony-stimulating factor on interferon therapy. AB - The effect of co-administration of granulocyte colony-stimulating factor (G-CSF), as an antineutropenia agent, on interferon therapy was examined in a mouse model, in anticipation of an enhancement of interferon efficacy, because neutrophils induced by G-CSF are thought to act as antitumor effectors. G-CSF was intraperitoneally co-administered with human interferon alpha A/D (IFN) on Day 6 to Day 10 after intradermal inoculation of Meth A fibrosarcoma. Although the co administration of G-CSF could protect against neutropenia and leukopenia induced by IFN, it did not enhance the regression of tumor, and rather reduced the prolongation of survival time and the long-term survival incidence of IFN therapy. The subsequent in vitro study showed that the antiproliferative activity of peripheral blood leukocytes from Meth A-bearing mice given both IFN and G-CSF was much weaker than that of mice given IFN alone. Whether the observed nullifying effect of G-CSF on IFN therapy is also the case with tumors other than Meth A is open to further study. PMID- 1708759 TI - Hematologic effects of AIDS therapies. AB - Hematologic abnormalities remain an important feature of HIV infection, and they often limit the therapy of this disorder. This is currently a field of intense research, and some progress has already been made. The current development of newer, less myelosuppressive therapies for HIV infection is encouraging. In addition, as more is learned about the biochemistry, molecular biology, and pathophysiology of HIV and its effects upon its host, more effective, HIV specific, and less toxic therapies may be developed. Finally, the use of various cytokines to ameliorate toxicities and possibly stimulate the functioning of myeloid cells is a promising area of research and should be pursued further. However, in spite of these efforts, much more needs to be done. In particular, therapy of HIV-associated lymphoma, which is likely to be increasingly observed as AIDS patients live longer, is often entirely unsatisfactory, in part because patients cannot tolerate the hematologic toxicity of the regimens employed. As in the general field of oncology, hematologic toxicities in AIDS remains a major limitation of therapy, and advances in this area have the potential to markedly improve both survival and quality of life. PMID- 1708760 TI - Interferon and other biologic agents for the treatment of Kaposi's sarcoma. AB - Interferon-alpha is an effective treatment for a subset of patients with AIDS associated Kaposi's sarcoma. When given at high doses to patients who lack systemic signs, symptoms, and opportunistic infections associated with advanced HIV infection and who maintain some degree of cell-mediated immune function, tumor regression may be observed in a high proportion of patients. Although the addition of chemotherapy to IFN-alpha appears to confer no added benefits, the combination of IFN-alpha with zidovudine has induced high tumor response rates in preliminary studies, including responses in some patients considered unlikely to respond to IFN-alpha alone. IFN-alpha-induced tumor regression has also been associated with suppression of HIV, as measured by serum p24 antigen concentrations and peripheral blood virus cultures. Other biologic agents, including interferons beta and gamma, tumor necrosis factor, and IL-2, have also been tested, to a lesser extent, in patients with Kaposi's sarcoma. Although systemically administered IFN-beta and intralesional TNF injections have led to tumor regression in some cases, the role of these biologics has been incompletely defined. Additional studies of these agents in combination with nucleoside reverse transcriptase inhibitors such as zidovudine will be required to fully assess their role in the treatment of Kaposi's sarcoma and HIV infection. It can also be anticipated that newer biologic agents, which specifically inhibit the production or action of angiogenic factors believed to be involved in the genesis of Kaposi's sarcoma, will be studied in the near future. PMID- 1708761 TI - Energy dependence of O-antigen synthesis in Salmonella typhimurium. AB - The uncoupler 2,4-dinitrophenol prevents in vivo synthesis of O antigen in Salmonella typhimurium by inhibiting the first reaction of the pathway, formation of galactosyl-pyrophosphoryl-undecaprenol. Inhibition was observed only in intact cells; dinitrophenol had no effect on activity of the synthase enzyme in isolated membrane fractions. In vivo inhibition could not be explained by changes in intracellular nucleotide pools or a shift in the equilibrium of the reaction and appeared to be specific for the first step in the pathway. Neither the subsequent mannosyl transferase, which catalyzes formation of the trisaccharide-lipid intermediate, mannosyl-rhamnosyl-galactosyl-pyrophosphoryl-undecaprenol, nor O antigen polymerase was inhibited. In addition, incorporation of galactose into core lipopolysaccharide was only modestly inhibited under conditions in which O antigen synthesis was abolished. The results suggest that maintenance of proton motive force is required for access of substrate, UDP-galactose and/or undecaprenyl phosphate, to the active site of the galactosyl-pyrophosphoryl undecaprenol synthase enzyme. PMID- 1708762 TI - Evidence for energy-dependent transposition of core lipopolysaccharide across the inner membrane of Salmonella typhimurium. AB - The uncoupler 2,4-dinitrophenol blocks the final step of lipopolysaccharide assembly--transfer of O antigen from undecaprenyl pyrophosphate to core lipopolysaccharide--in intact Salmonella typhimurium but not in isolated membrane fractions. The O-antigen ligase enzyme is not inhibited by dinitrophenol in vitro, and core lipopolysaccharide synthesized in the presence of uncoupler in vivo is functional as acceptor of O antigen in vitro. The evidence strongly suggests that maintenance of proton motive force is required for transmembrane transposition of core lipopolysaccharide to the active site of O-antigen ligase at the periplasmic face of the inner membrane. PMID- 1708764 TI - Antigenic changes in lipopolysaccharide I of Rhizobium leguminosarum bv. viciae in root nodules of Vicia sativa subsp. nigra occur during release from infection threads. AB - Three different monoclonal antibodies raised against the O antigen-containing lipopolysaccharide (LPS I) of free-living cells were used in an immunocytochemical study to follow the fate of LPS I on the outer membrane of Rhizobium leguminosarum bv. viciae 248 during the nodulation of Vicia sativa subsp. nigra. After immunogold labeling, the LPS I epitopes were detected on the outer membrane of bacteria present in infection threads throughout the nodule. Epitopes were not detectable on bacteria released from the infection thread. The data show that the LPS I epitopes present on rhizobia in infection droplets disappear shortly before or during endocytosis of the bacteria into the host plant cell cytoplasm. The abruptness of the change suggests an active degradation or modification of LPS I epitopes rather than only a repression of their synthesis. PMID- 1708763 TI - Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria). AB - Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria. PMID- 1708765 TI - Cloning, characterization, and nucleotide sequence analysis of a Zymomonas mobilis phosphoglucose isomerase gene that is subject to carbon source-dependent regulation. AB - The Zymomonas mobilis gene encoding phosphoglucose isomerase (pgi) was cloned by genetic complementation of an Escherichia coli pgi mutant. An enzyme assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the presence of excess amounts of phosphoglucose isomerase in E. coli clones carrying the Z. mobilis pgi gene. The pgi gene is present in only one copy on the Z. mobilis genome. Nucleotide sequence analysis of the pgi region revealed an open reading frame of 1,524 bp preceded by a strong Shine-Dalgarno sequence. The pgi gene encodes a 507-amino-acid protein with a predicted molecular weight of 55,398. Z. mobilis phosphoglucose isomerase is between 38 and 43% identical to the enzyme from other species. Northern (RNA) blot analysis showed that the pgi transcript is 1.8 kb in length. The level of the pgi transcript was found to be influenced by the phase of growth and by the carbon and energy sources. Transcript levels increased with respect to total RNA during logarithmic growth and were threefold higher when grown on fructose than on glucose. These changes in transcript levels paralleled phosphoglucose isomerase activities in the cultures. Differential mRNA stability was not a factor, since the half-life of the pgi transcript was 6.3 min in glucose-grown cells and 6.0 min in fructose-grown cells. Thus, an increase in the rate of transcription appears to be at least partially responsible for the increased levels of phosphoglucose isomerase observed for Z. mobilis grown on fructose. PMID- 1708766 TI - Cotranscription of the electron transport protein genes nifJ and nifF in Enterobacter agglomerans 333. AB - A nucleotide sequence showing extensive homology to the nifF gene, which codes for a flavodoxin involved in nitrogen fixation in Klebsiella pneumoniae, was localized on the plasmid pEA3 of Enterobacter agglomerans and determined. The analysis of transcriptional fusions, as well as transcript protection assays, indicated a novel nif gene organization, that is, the cotranscription of nifJ and nifF. PMID- 1708767 TI - endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1. AB - The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a 1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2. PMID- 1708768 TI - Preparing a generic (multipurpose) videodisc. PMID- 1708769 TI - Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. AB - Extracellular ATP4- opens pores in the plasma membrane of mouse macrophages and the J774 macrophage-like cell line that allow molecules as large as fura-2 (831 daltons) to enter the cytoplasmic matrix of the cells. The functional similarity of the ATP-induced pores to gap junctions led us to examine whether these pores were related to members of the connexin family of gap junction proteins. Under conditions of high stringency, RNA isolated from J774 cells hybridized with cDNA for connexin-43 but not with cDNA for connexin-32, -26, or -46. RNA isolated from several variant J774 cell lines that do not permeabilize in response to extracellular ATP (ATPR cells) did not hybridize with connexin-43 cDNA. Immunoblots demonstrated that J774 cells, but not the variant ATPR B2 cell line, expressed connexin-43 protein. These studies demonstrate that mouse macrophages express the connexin-43 gap junction mRNA and protein and strongly suggest that in these cells connexin-43 forms "half-gap junctions" in response to extracellular ATP4-. PMID- 1708770 TI - Promoter-dependent phosphorylation of RNA polymerase II by a template-bound kinase. Association with transcriptional initiation. AB - The largest subunit of eukaryotic RNA polymerase II (RNAP II) has a serine- and threonine-rich C-terminal domain (CTD) that may interact both with DNA and with the activating region of transcription factors. It has been proposed, in one model, that a protein kinase phosphorylates the promoter-associated CTD, facilitating the transition between promoter-binding and RNA-elongating forms of RNAP II. An immobilized template transcription system was used to test the predictions of this model directly. A protein kinase that phosphorylated the CTD at multiple sites was detected. This activity was tightly bound to the template, as evidenced by continued association after multiple rounds of washing. Phosphorylation was promoter sequence-dependent and exhibited the same nucleotide substrate specificity as the previously characterized ATP-requiring step in initiation. It was necessary for [gamma-32P]ATP and initiating rNTPs to be present simultaneously in the reaction in order to efficiently chase-radiolabel into elongating RNAP II-containing complexes, consistent with the idea that initiation and phosphorylation are temporally associated reactions. PMID- 1708771 TI - Amino acid sequence and post-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium. AB - Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. C., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138. PMID- 1708772 TI - Localization of distinct functional domains on prekallikrein for interaction with both high molecular weight kininogen and activated factor XII in a 28-kDa fragment (amino acids 141-371). AB - The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain. PMID- 1708773 TI - Protein engineering of novel plasminogen activators with increased thrombolytic potency in rabbits relative to activase. AB - Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy for patients with acute myocardial infarction. Due to its rapid rate of clearance from the circulation, continuous intravenous administration of approximately 100 mg over 3 h is recommended. We have previously characterized novel thrombolytic variant forms of t-PA which offer the potential of administration by bolus injection and reduced dosage due to their slower rates of clearance, relative to t-PA. This study was undertaken to quantitatively compare the pharmacokinetics, thrombolytic activity, and hemostatic effects of two of these variant forms, called delta FE1X and delta FE3X plasminogen activator (PA), with commercially available recombinant t-PA (Activase). These evaluations were performed in rabbits after bolus intravenous injection of the proteins. Following injection of 0.25 mg of protein/kg of body weight, the rates of clearance for delta FE3X and delta FE1X PA antigen were decreased approximately 9- and 18-fold, respectively, relative to Activase. Plasma plasminogen activator activity was also measured and the rates of clearance of delta FE3X and delta FE1X PA activity were similarly decreased by approximately 9- and 22-fold, respectively, relative to Activase. To quantitate thrombolytic activity we used the rabbit jugular vein thrombosis model and demonstrated that approximately 50% thrombolysis was achieved with delta FE1X and delta FE3X PA at approximately an 8.6- and 3-fold lower dose than Activase, respectively. No major differences in fibrinogen and alpha 2-antiplasmin depletion were observed among the agents at doses required to produce 50% thrombolysis, indicating similarities in fibrin specificities among these agents. These results demonstrate a reciprocal relationship between thrombolysis and rate of clearance for these thrombolytic proteins. The 8.6-fold increase in potency of delta FE1X PA relative to Activase supports the future clinical testing of this novel engineered protein as a thrombolytic agent. PMID- 1708774 TI - Regulation of alpha-fetoprotein gene expression by antagonism between AP-1 and the glucocorticoid receptor at their overlapping binding site. AB - We show here that the alpha-fetoprotein gene (AFP) promoter can be regulated by AP-1 activity using transient transfection assays. AFP promoter activity induced by c-jun/c-fos can be repressed by cotransfected glucocorticoid receptor. The DNA sequence conferring AP-1 activity was located in the proximal promoter region. Gel retardation assays using the AFP proximal promoter identified an AP-1-like sequence which can bind to bacterially expressed c-jun protein. This AP-1-like element, when cloned into the tk promoter, responds to the AP-1 activity of c jun/c-fos products in both CV-1 and F-9 cells. The element overlaps with a consensus glucocorticoid-responsive element which was shown to confer negative modulation of AFP promoter activity. A 23-base pair DNA element containing the overlapping glucocorticoid-responsive element and AP-1 sites can be positively regulated by glucocorticoid receptor in the absence of c-jun/c-fos products. When plasmids expressing glucocorticoid receptor, c-jun and c-fos are cotransfected together, they repress each other. Thus, these data demonstrate that negative regulation of the AFP gene by glucocorticoid may be due to the interference of AP 1 activity by glucocorticoid receptor either by direct competition for DNA binding or via protein-protein interaction. They provide another example of transcriptional regulation of developing-associated genes between two major signal transduction pathways in response to extracellular stimuli. This supports the model that expression of alpha-fetoprotein is regulated during development by the effect on transcription of antagonism between glucocorticoid receptor and fos/jun. PMID- 1708775 TI - The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA. AB - To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes. PMID- 1708776 TI - Synthesis of albumin and acute-phase proteins in perfused liver after burn injury in rats. AB - The acute-phase response that follows injury and sepsis is characterized by increased hepatic synthesis of specific secreted proteins while production of albumin is decreased. The effect of burn injury on specific synthesis rates of secreted hepatic proteins has not been reported. In this study, Sprague-Dawley rats received either a 30% flame burn (n = 12) or a sham burn (n = 12) and were allowed to recover for 11 days. Burned animals showed slower weight gains and a 25% to 30% higher resting energy expenditures compared with controls. On postburn day 11, synthesis of secreted hepatic proteins was measured by incorporation of leucine during a 2-hour isolated liver perfusion. Synthesis of total secreted proteins, the seromucoid fraction, and complement component C3 was significantly increased in burned animals, whereas synthesis of albumin was unaltered. In spite of unchanged albumin synthesis, plasma albumin concentrations were 50% lower in burned animals than in control animals throughout the postburn period. These findings suggest that decreased albumin synthesis is not the only mechanism responsible for persistent hypoalbuminemia that follows burn injury. PMID- 1708777 TI - Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells. AB - Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation. PMID- 1708778 TI - Induction of cell cycle-dependent genes during cell cycle progression of arterial smooth muscle cells in culture. AB - Serum stimulation of arterial smooth muscle cells in culture induces a progression through the cell cycle and cell proliferation. Most genes previously described as cell cycle-dependent in various cell types also demonstrate a cell cycle-dependent expression in arterial smooth muscle cells. As in other cell types, these genes can be classified into three groups according to their mode of expression: "immediate early" genes (c-fos, c-myc, ...), "delayed early" genes (2F1, ...), and "late-G1" genes (proliferating cell nuclear antigen, thymidine kinase, . . .). In addition to these previously described genes, three genes isolated from a cDNA library of stimulated smooth muscle cells have been demonstrated to be cell cycle-dependent: A21, the rat JE gene, and L51 can be classified as "immediate early" genes, while M11 represents a new member of the "delayed early" gene family. PMID- 1708779 TI - Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells. AB - The decidualized endometrium during the first trimester of pregnancy synthesizes and secretes a 32-kDa insulin-like growth factor-binding protein (termed hIGFBP 1) at high levels. IGFBP-1 is the major soluble protein product of this tissue and is principally localized to the differentiated endometrial stromal cell, the decidual cell. In the present study long term culture of stromal cells from the nonpregnant endometrium have been employed to elucidate the hormonal requirements for IGFBP-1 production. Immunoreactive IGFBP-1 was undetectable in control cultures. However, inclusion of medroxyprogesterone acetate (MPA) induced rates of 0.35 +/- 0.09 microgram/0.1 mg cell DNA.day (mean +/- SEM; n = 5) after 20-30 days. In these cultures cells exhibited morphological changes consistent with decidual cell differentiation. In all cultures removal of MPA after exposure for 10-16 days, with or without subsequent inclusion of relaxin (RLX), increased production of IGFBP-1 450- to 4600-fold to rates of 150-710 micrograms/0.1 mg cell DNA.day or 26-131 micrograms/10(6) cells.day on days 24-26. The rates tended to be higher with the inclusion of RLX and were sustained in contrast to cultures without RLX, where rates fell by day 30. Individual cultures responded differently to RLX when added from the initiation of culture, with either a response similar to MPA alone or a cyclical change in production, achieving maximal rates of 190-290 micrograms/0.1 mg cell DNA.day. Cultures in which RLX alone induced high IGFBP-1 high production were obtained from endometrium during the progesterone-dominated luteal phase. In cultures exhibiting high rates of immunoreactive IGFBP-1 production, the protein represented their major secretory protein product. This was confirmed by [35S]methionine incorporation and the presence of IGFBP-1 as the predominant protein in serum-free culture medium. The immunoreactive IGFBP-1 isolated from culture medium was found to be identical, by a number of criteria, with IGFBP-1 derived from decidual tissue. These results were consistent with a primary role of progestin exposure, whether in vivo or in vitro, in converting endometrial stromal cells to cells potentially able to exhibit the high rates of IGFBP-1 production typical of the decidualized endometrium of pregnancy. PMID- 1708780 TI - Patterns of antibody reactivity to selected human immunodeficiency virus type 1 (HIV-1) gp160 epitopes infected individuals grouped according to CD4+ cell levels. AB - We examined sera from 160 HIV-infected individuals for antibodies reactive to HIV 1 gp160 epitopes defined by seven synthetic peptides. Seropositive individuals were placed into three groups based upon levels of circulating CD4+ cells. These groups consisted of individuals with (1) more than 400 CD4+ cells, (2) 200-400 CD4+ cells, and (3) fewer than 200 CD4+ cells/mm3. The percentage of sera containing antibodies reactive with two immunodominant gp160 epitopes (a.a. 304 321 and 600-611) was unchanged between groups, regardless of CD4 cell numbers. The percentage of sera containing antibodies reactive with weakly immunogenic gp160 epitopes, such as those defined by peptides 425-448 and 846-860, declined in the groups as CD4 values decreased. Our results suggest that the patterns of antibody reactivity to gp160 epitopes change as CD4 levels decline. A narrowing of the humoral immune response to epitopes on the envelope of HIV-1 appears to occur with disease progression. PMID- 1708781 TI - Generation and analysis of clonal IgM- and IgG-producing human B cell lines expressing an anti-DNA-associated idiotype. AB - This study describes a methodology for generating stable, cloned, EBV-transformed IgG- and IgM-producing human B cell lines. Using these lines we have characterized immunoglobulin V gene utilization in an anti-DNA-associated idiotypic system. The 31 anti-DNA-associated idiotype is encoded preferentially by the VK1 gene family, and, in all probability, reflects a germ line gene encoded framework determinant. Analysis of these lines indicates that the DNA binding antibodies produced by B cell lines from SLE patients may differ from DNA binding myeloma proteins and from natural autoantibodies. PMID- 1708782 TI - Mechanisms of disruption of the articular cartilage surface in inflammation. Neutrophil elastase increases availability of collagen type II epitopes for binding with antibody on the surface of articular cartilage. AB - We recently observed that specific antibodies to type II collagen do not bind in appreciable amounts to the intact surface of articular cartilage, whereas antibodies to the minor collagen types V, VI, and IX do. These results suggest that the outermost cartilage surface layer prevented interaction of the antibodies with the major collagen type in articular cartilage. The present studies were designed to investigate the pathogenic mechanisms involved in the disruption of the cartilage surface layer in inflammatory arthritis. Articular cartilage obtained from rabbits undergoing acute antigen-induced arthritis of 72 h duration showed a significant increase in binding of anti-type II antibody to cartilage surfaces compared with normal control cartilage (P less than 0.01). Augmentation of anti-type II binding was also observed upon in vitro incubation of bovine articular slices or intact rabbit patellar cartilage for 1 h with human polymorphonuclear neutrophils (PMN), PMN lysates, or purified human PMN elastase. This increase was not inhibited by sodium azide, nor was it enhanced by incubation of cartilage with the strong oxidant hypochlorous acid. Chondrocyte mediated matrix proteoglycan degradation in cartilage explants cultured in the presence of cytokines failed to increase antibody binding appreciably. The augmentation in antibody binding seen with PMN lysates was inhibited by the nonspecific serine-esterase inhibitor PMSF, but not by the divalent metal chelator EDTA. The elastase-specific inhibitor AAPVCMK also inhibited most of the PMN-induced increase in antibody binding, whereas the cathepsin G-specific inhibitor GLPCMK was much less effective. Incubation of intact cartilage with purified human PMN elastase indicated that this serine esterase could account for the increase in anti-type II collagen antibody binding to intact cartilage surfaces. These studies suggest that in an inflammatory response, PMN-derived elastase degrades the outer layer of articular cartilage, exposing epitopes on type II collagen. They also help clarify the pathogenic mechanisms involved in early articular cartilage damage in inflammatory joint diseases. PMID- 1708783 TI - Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor alpha and insulinlike growth factor I. AB - We report methods allowing the culture of rapidly dividing gastric epithelial cells to investigate the regulation of mucosal cell replication. Cells from canine fundic mucosa were dispersed by enzyme treatment, enriched by filtration and elutriation, and cultured on collagen gel in DMEM/F12 medium. After 48 h, greater than 95% of the cells displayed immunoreactivity with antibody to cytokeratin, an epithelial marker. The cells formed confluent monolayers by 72 h with a transmembrane resistance of 1,600 ohm.cm2 when mounted in a Ussing chamber indicating retention of epithelial cell characteristics. Calf serum (0.1-2%) produced a dose-dependent mitogenic effect evident by increases in [3H]-thymidine incorporation into acid-precipitated material and in cell number. After an 18-24 h incubation with [3H]-thymidine, approximately 55% of the cells cultured in 2% serum showed evidence of DNA synthesis by autoradiography and all of the replicating cells were cytokeratin positive. Using comparable culture conditions, a similar proportion of cells incubated for 18-24 h with bromodeoxyuridine displayed nuclear anti-bromodeoxyuridine immunoreactivity, thus indicating that over half of the cells in these cultures synthesized DNA during this period. As with serum, epidermal growth factor and transforming growth factor alpha (TGF alpha) (10 pM to 1 nM), insulin (10 nM to 1 microM) and insulinlike growth factor I (IGF-I, 1-100 nM) increased [3H]-thymidine uptake. The greater potency of IGF I, compared to insulin, suggests the presence of IGF-I receptors. We conclude that this culture preparation is composed of fundic mucosal epithelial cells and contains a predominance of dividing epithelial cells. EGF/TGF alpha and IGF-I are potential factors directly regulating proliferation of fundic mucosal cells. PMID- 1708784 TI - Association of circulating receptor Fc gamma RIII-positive monocytes in AIDS patients with elevated levels of transforming growth factor-beta. AB - Monocytes in the circulation of normal individuals express two receptors for the constant region of immunoglobulin, Fc gamma RI and Fc gamma RII. In contrast, we have observed that AIDS monocytes express significant levels of a third Fc gamma R, Fc gamma RIII (CD16), which is normally associated with activation or maturation of the monocyte population. By dual-fluorescence analysis using a monoclonal antibody specific for Fc gamma RIII (MAb 3G8), 38.5 +/- 3.2% of the LeuM3 (CD14)-positive monocytes in AIDS patients were CD16 positive as compared to 10.4 +/- 1.0% for healthy individuals (n = 29; P less than 0.005). Furthermore, AIDS monocytes expressed Fc gamma RIII-specific mRNA which is expressed minimally or not at all in control monocytes. As a recently identified inducer of Fc gamma RIII expression on blood monocytes, transforming growth factor-beta (TGF-beta) was found to be elevated in the serum and/or plasma of AIDS patients. Moreover, incubation of normal monocytes with AIDS serum or plasma induced CD16 expression which correlated with serum TGF-beta levels (r = 0.74, P less than 0.001) and was inhibited with a neutralizing antibody to TGF-beta. Thus, the increased CD16 expression on peripheral blood monocytes in AIDS patients may be the consequence of elevated circulating levels of the polypeptide hormone TGF-beta. PMID- 1708785 TI - Expression of endothelial-leukocyte adhesion molecule-1 in elicited late phase allergic reactions. AB - To better understand the events involved in the local migration of inflammatory cells into sites of allergic reactions, we studied expression of the cytokine inducible endothelial cell (EC) neutrophil adhesion molecule, endothelial leukocyte adhesion molecule (ELAM-1), in sequential skin biopsies from patients with respiratory allergy during the late phase reaction (LPR) between 20 min and until 24 h after intradermal allergen (ragweed or dust mites) injection. In 7 of 7 atopic patients but in only 1 of 4 apparently normal controls, allergen induced appearance of ELAM-1 on EC. ELAM-1 expression occurred concurrently with the development of inflammatory cell infiltrates by 3-4 h after intradermal injection. Saline injected sites in all subjects were negative. Skin organ cultures demonstrated that allergen could produce the same EC changes in vitro whether allergen was injected in vivo 20 min before culture or added during skin culture. These EC changes in organ culture were inhibited by the presence of combined anti-sera to both TNF-alpha and IL-1, but not by antisera to either cytokine alone. We conclude that EC activation occurs in elicited LPR and suggest that cytokine-induced EC activation may play a role in the migration of inflammatory cells into allergic skin reactions. Furthermore, resident cells in the skin rather than infiltrating leukocytes appear to be the source of the cytokines that mediate endothelial activation. PMID- 1708786 TI - Targeted enzyme therapy of experimental glomerulonephritis in rats. AB - We sought to determine whether systemic administration of proteases ameliorates membranous nephritis induced in rats by immunization and challenge with cationic bovine gamma globulin, and whether targeting of protease to glomerular capillaries increases efficacy. Proteases substituted with biotin were targeted via the cationic protein avidin A, which by virtue of its charge has affinity for the glomerular basement membrane. Despite identical pretreatment proteinuria, rats given untargeted protease (biotin-conjugated without avidin, or unconjugated plus avidin) had significantly less proteinuria than saline-treated controls and nephrotic rats given avidin plus biotin-conjugated (targeted) protease had even less proteinuria and reduced glomerular rat IgG and C3. Among more severely nephrotic rats, targeted protease was again more effective than untargeted protease at reducing proteinuria, and also decreased the size of electron-dense glomerular deposits, hypercholesterolemia, and creatininemia. Inactivated targeted proteases had no effect on proteinuria, hypercholesterolemia, or azotemia. Finally, active targeted protease did not affect proteinuria in the nonimmune mediated nephrosis induced by puromycin aminonucleoside. We conclude that systemic protease can specifically diminish glomerular immune deposits, proteinuria, hyperlipidemia, and creatininemia associated with experimental immune complex glomerulonephritis but not toxic nephrosis, and that targeted protease is more effective than untargeted protease. PMID- 1708787 TI - Distribution of motoneurons supplying cat sartorius and tensor fasciae latae, demonstrated by retrograde multiple-labelling methods. AB - Sartorius (SART) and tensor fasciae latae (TFL) in the cat hindlimb are functionally heterogeneous muscles with regions that differ in their skeletal actions and electromyographic recruitment during normal activity. The topographical organization of motoneurons supplying different regions of SART or TFL has been investigated by exposing cut nerve branches supplying different peripheral territories to a combination of retrograde tracers, including Fast Blue (FB), Fluorogold (FG), and horseradish peroxidase (HRP). Motoneurons supplying medial, central, and anterior regions of SART were intermixed extensively throughout a single columnar nucleus located in the ventrolateral part of segments L4 and L5. With this column, motoneurons supplying medial SART tended to lie more rostrally than those supplying anterior regions, but the gradient was modest and showed some cat-to-cat variation. Two major branches entered anterior SART at different proximodistal levels. When these two branches were exposed to different tracers, most motoneurons contained a single tracer; only a few double-labelled cells were apparent. The labelling suggests that anterior SART may contain two separate, in-series divisions of motor units. In TFL, motoneurons supplying nerve branches to posterior, central, and anterior parts of the muscle were intermingled indiscriminately in a single ventrolateral cell column in L6 and rostral L7. These results suggest that topographical organization in lumbar motor nuclei does not always reflect the highly ordered biomechanical and functional specialization evident in the peripheral organization of the muscles themselves. PMID- 1708788 TI - Immunocytochemical localization and development of multiple kinds of neuropeptides and neuroendocrine proteins in the chick ultimobranchial gland. AB - The ultimobranchial gland is an endocrine organ consisting of C cell groups. In chickens, the glands are richly supplied by nerve fibers immunoreactive for neurofilaments. It was found by immunocytochemical staining that C cells of chick ultimobranchial glands showed immunoreactivities for multiple kinds of neuropeptides and neuroendocrine proteins in addition to calcitonin, i.e., calcitonin gene-related peptide (CGRP), somatostatin, neurotensin, chromogranin A, and tyrosine hydroxylase. Furthermore, enkephalin-immunoreactive cells that showed long cytoplasmic processes and large cell bodies, being distinct from the C cell feature, were detected. The densities of these cells per unit area of ultimobranchial gland were assessed using computer-assisted image analysis system; calcitonin cells were 42.9 +/- 10.0%; CGRP cells 26.9 +/- 5.6%; neurotensin cells 8.6 +/- 6.9%; somatostatin cells 3.1 +/- 1.4%; chromogranin A cells 11.8 +/- 1.8%; tyrosine hydroxylase cells 10.0 +/- 5.2%; enkephalin cells 2.9 +/- 1.3%. Dense distributions of peptidergic nerve fibers were also detected in chick ultimobranchial glands. Numerous varicose fibers immunoreactive for substance P were distributed in the close vicinity to C cell clusters and blood vessels. Enkephalin-immunoreactive fibers were also prominent around C cell clusters. Galanin-, vasoactive intestinal peptide (VIP)-, and tyrosine hydroxylase-immunoreactive fibers were distributed around blood vessels only. Subsequently, the ontogeny of these neuropeptides, neuroendocrine proteins, and peptidergic innervations was examined in chickens at various developmental stages. In 10-day-old embryos, weak to moderately intense immunoreactivity for calcitonin was already present in almost all C cells. Immunoreactivities for somatostatin, CGRP, and tyrosine hydroxylase began to appear at this age. At 12 days of incubation, substance P-immunoreactive fibers were first detected in the parenchyma of ultimobranchial glands. Considerable numbers of enkephalin immunoreactive fibers and cells were also observed. At 14 days of incubation, the largest populations of somatostatin- and enkephalin-immunoreactive cells were attained; the densities of somatostatin- and enkephalin-immunoreactive cells per unit area were 21.2 +/- 3.2% and 12.9 +/- 3.1%, respectively. Substance P immunoreactive fibers became numerous throughout the gland at this age. Thereafter, calcitonin-, CGRP-, tyrosine hydroxylase-immunoreactive cells progressively increased in number with embryonic age, whereas somatostatin- and enkephalin-immunoreactive cells started to decrease. Chromogranin A- and neurotensin-immunoreactive cells began to appear at 16 days and 18 days of incubation, respectively. Galanin-, VIP-, and tyrosine hydroxylase-immunoreactive fibers were inconspicuous during embryonic life. PMID- 1708790 TI - Pigmented neuroectodermal tumor of infancy. A light microscopic and immunohistochemical study. AB - We studied two cases of pigmented neuroectodermal tumor of infancy (PNTI) by routine light microscopy and immunohistochemistry on formalin fixed, paraffin embedded tissues using antibodies to HMB-45 "melanoma associated" antigen, S-100 protein, neuron specific enolase (NSE), Leu-7 antigen, chromogranin, epithelial membrane antigen, collagen Type IV, alpha-fetoprotein and muscle-specific actin and to the intermediate filaments cytokeratin (CK), vimentin, desmin and neural filaments. We found that the large epithelioid cells, many of which contained melanin pigment, were strongly positive for CK and HMB-45, and less intensively positive for vimentin and NSE. The small neuroblast-like cells revealed only focal, weak NSE positivity. Both cell types were negative for S-100 protein and for the other antigens examined. Our results suggest that: (1) the large and small cell populations in PNTI have different immunophenotypes; (2) the expression of CK and HMB-45, together with the S-100 negativity, appears unique for the pigmented cells; and (3) this profile may be helpful in the exclusion of melanoma and peripheral neuroblastoma from the differential diagnosis. PMID- 1708789 TI - The reticular thalamic nucleus (RTN) of the rat: cytoarchitectural, Golgi, immunocytochemical, and horseradish peroxidase study. AB - Experiments have been performed on adult albino rats in order to study the cellular organization of the thalamic reticular nucleus. For this purpose four approaches have been used: Nissl stain, Golgi impregnation, retrograde transport of horseradish peroxidase after injection in different thalamic nuclei, and immunocytochemistry with antibodies against GABA and glutamic acid decarboxylase. In sections through the horizontal plane, three morphologically different neurons have been observed. Cells with round perikarya and with multipolar dendrites were found predominantly in the rostral pole of the nucleus. Neurons with large fusiform cell body and with dendrites arborizing mainly on the horizontal plane were detected through the whole extent of the nucleus. Small fusiform neurons were observed almost exclusively in the medial third of the dorso-ventral extent of the nucleus. The Golgi impregnation method demonstrated that dendrites of small fusiform neurons develop in the vertical plane perpendicular to the dendritic arborization of large fusiform neurons. In coronal sections neurons with round perikarya and with large fusiform cell bodies are detectable while small fusiform neurons are only rarely visible. These data have been confirmed by statistical form factor analysis. Moreover, by means of the horseradish peroxidase and the immunocytochemical study, it has been confirmed that all three groups of neurons project within the thalamus and that they are GABAergic. The data concerning the distribution within the nucleus of the three morphologically different neurons are discussed in relation to the topographic distribution of cortical sensory afferents and to the topographic maps within different sectors of the reticular nucleus. PMID- 1708791 TI - Quantitative immunohistochemical analysis of keratins and desmoplakins in human gingiva and peri-implant mucosa. AB - Quantitative immunohistochemistry was used to compare the distributions of keratins and desmoplakins in human gingiva and peri-implant mucosa (three specimens each). In gingiva, keratin 1 (a marker of cornification) and desmoplakins I & II (markers of desmosomes) stained most heavily in granular strata followed by corneal strata; keratin 13, a marker of non-cornifying stratified squamous cells, stained most heavily in suprabasal strata of oral sulcular epithelium. Keratin 19, a marker for junctional epithelium, stained the basal stratum of oral sulcular epithelium most heavily. In peri-implant mucosa, the patterns of staining were similar, except that staining for desmoplakins I & II was generally significantly reduced compared with gingiva, and junctional epithelium co-expressed keratins 13 and 19. Peri-implant junctional epithelial cells attached to titanium implant abutments were removed by trypsin/EDTA digestion, and also exhibited co-expression of keratins 13 and 19. Inflammatory cell infiltration was associated with reduction of keratin 1 staining in gingiva. The data indicate that the epithelia of gingiva and peri-implant mucosa are not composed of identical cell populations. PMID- 1708793 TI - Role of supravital staining in detection of malignancies in otorhinolaryngology. PMID- 1708792 TI - Cardiovascular responses to combined microinjection of substance P and acetylcholine in the intermediolateral nucleus of the rat. AB - As microinjection of either substance P (SP) or acetylcholine (ACh) into the right intermediolateral cell nucleus (IML) at the T2 level elicits increases in heart rate (HR) in the anesthetized rat, we investigated the possibility of a synergistic effect on HR and arterial pressure (AP) of ACh and SP microinjected in this nucleus. Moreover, we studied the effect on HR and AP of microinjection of either ACh or SP into the IML combined with activation of cardiovascular neurons in the ipsilateral rostral ventrolateral medulla (RVLM) by microinjection of glutamate (Glu). Male Wistar rats (n = 16) were anesthetized with urethane (1.4 g/kg i.p.), artificially ventilated, and the dorsal medulla and spinal cord (T1-T3) were exposed. Micropipettes containing SP and ACh were positioned in the right IML at the T2 level. Microinjection of threshold amounts of ACh (5 x 10(-2) M, 2-10 nl) and SP (3 x 10(-6) M, 2-10 nl) that caused small or no changes in HR or AP (less than 10 bpm or mmHg) elicited statistically significant synergistic increases in HR (22.9 +/- 3.3 bpm) but no changes in AP. Threshold microinjections of Glu (0.18 M, 2-10 nl) into the right RVLM combined with microinjections of threshold amounts of SP or ACh into the ipsilateral IML elicited significant synergistic increases in HR of 13.1 +/- 1.9 bpm and 10.6 +/- 1.9 bpm and in AP of 9.7 +/- 1.9 mmHg and 10.8 +/- 1.7 mmHg, respectively. These results indicate that SP and ACh interact to influence cardioacceleratory spinal preganglionic neurons (SPN) and interact with the transmitter released in the IML by RVLM stimulation to elicit increases in HR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708794 TI - Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis. AB - Trichohyalin, a protein contained in granules in the cells of the hair-follicle inner root sheath and in the medulla of the hair shaft, has been purified previously from sheep hair bulbs and is also a major protein of filiform papillae of tongue epithelium. Polyclonal affinity-purified antibodies and a monoclonal antibody raised to purified pig tongue trichohyalin both stained the inner root sheath of hair follicles and the medulla of hair fibers and identified human trichohyalin as a single 220-kDa band on immunoblots of human hair bulb proteins. These antibodies were used to examine human epidermis by immunofluorescence and immunoblotting. The antibodies decorate granules in cells in the granular layer and stratum corneum of non-hair-bearing human skin, and immunoblots identify a protein in epidermis comigrating with trichohyalin from human hair and human tongue epithelium. Absorption of antibody to trichohyalin on a trichohyalin affinity column abrogated staining of the epidermis and the bands on the immunoblots. Trypsin-separated epidermis contained 220 and 160 kDa bands identified as trichohyalin, but epidermis shaved from skin and quickly frozen showed only a single 220-kDa band, indicating that the 160-kDa protein was generated by proteolysis. Double immunofluorescence for trichohyalin and filaggrin showed that some cells containing filaggrin also contain trichohyalin. These studies show that trichohyalin is not limited to hair and tongue but is present in isolated cells in the granular layer and stratum corneum of normal epidermis. PMID- 1708795 TI - Neuropeptides enhance irritant and allergic contact dermatitis. AB - It is supposed that neuropeptides participate in the regulation of delayed-type hypersensitivity (DTH) reactions. However, their function in this kind of immune response is not known presently. Therefore, in vivo studies were initiated to test the effect on allergic (ACD) and irritant contact dermatitis (ICD) of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin (SOM), which are released from afferent neurons in the skin. Each neuropeptide was applied topically at the site of contact with the allergen (oxazolone) or irritant (croton oil) during the challenge and sensitization phase of contact dermatitis. The intensity of the inflammation was measured as an increase of ear-swelling response, which represents the degree of plasma extravasation in the early phase of inflammation. Neuropeptides alone led only to a distinct vasodilation. All three neuropeptides were equally able to increase allergic and irritant inflammation. Even minor irritant stimuli were enhanced. Beyond that, CGRP was able to boost sensitization, whereas SOM and SP did not show any effects on the sensitization process. The results presented demonstrate that neuropeptides increase plasma extravasation independent of the pathogenesis of inflammation and may act as priming substances for other mediators of increased vascular permeability. In addition, CGRP enhances the sensitization process. PMID- 1708796 TI - Human junctional epithelium: demonstration of a new marker, its growth in vitro and characterization by lectin reactivity and keratin expression. AB - We have studied lectin reactivity in normal human junctional, sulcular, and attached gingival epithelia with 15 lectins and identified the epithelia by parallel staining with monoclonal anti-keratin antibodies. Dolichos biflorus agglutinin reacted uniquely with junctional epithelium, not staining other gingival cells of non-blood group A1 donors. We have demonstrated that the moiety recognized in junctional epithelium is not blood group A1 antigen or Tn antigen. Using a panning technique with this lectin to isolate the cells, we have grown keratinocytes from human junctional epithelium, and compared their phenotype in vitro to that of cells grown from the sulcular and attached gingival epithelium. Colonies established from each epithelial type were examined in frozen section with the anti-keratin antibodies. All expressed keratin 14 (keratinocyte marker), keratins 4 and 13 (suprabasal non-cornification markers), and keratins 7, 18, and 19 (simple epithelia keratins). Keratins 1, 10, and 8 were not expressed. Vimentin, the intermediate filament of mesenchymal cells, was also expressed by all types of cells in culture. Thus we have shown that when cells from the three areas of the gingiva were grown in culture they revert to one phenotype, at least with respect to their keratin expression. These results support the hypothesis that the epithelial phenotype is influenced by the sub-epithelial mesenchyme, and it is this that is responsible for the unique phenotype of the junctional epithelium. PMID- 1708797 TI - Isoprinosine treatment of alopecia areata. PMID- 1708798 TI - Vitronectin: effects on keratinocyte motility and inhibition of collagen-induced motility. AB - Epibolin, a plasma protein, was initially purified on the basis of its ability to enhance spreading of keratinocytes. It is now known that epibolin is identical to serum spreading factor, S protein, and vitronectin, and the current name for the protein is vitronectin. Studies of vitronectin on cultured keratinocytes showed that it caused spreading and epiboly but not cellular adhesion to the substratum. In studies with other types of cells, vitronectin increased migration of several types of cells in a Boyden chamber. Because some agents that enhance spreading and adhesion, such as collagen and fibronectin, also increase motility, we tested whether vitronectin increased motility of keratinocytes. By photographing and quantitating motility of keratinocytes plated on a bed of colloidal gold particles, we determined that vitronectin increased local movement of keratinocytes in a concentration-dependent fashion, resulting in clearing of gold particles in a circular pattern around the cells, but did not cause the production of tracks found in cultures plated on collagen or fibronectin. The small increases in clearing of the gold particles that occurred in the presence of vitronectin were abolished by antibody to vitronectin. Furthermore, the marked increase in motility produced by type I collagen was significantly reduced when the keratinocytes were treated with vitronectin. Antibody to vitronectin also abrogated the vitronectin-induced reduction in collagen-stimulated motility, confirming that this action was specific for vitronectin. Serum, which contains vitronectin, stimulated motility in a fashion identical to purified vitronectin, but serum lacking vitronectin was inactive. These studies show that vitronectin causes a localized increase in movement associated with spreading resulting in a halo around individual cells, that vitronectin does not enhance directional motility of keratinocytes in this assay but in contrast antagonizes such motility produced by collagen, and that vitronectin is the factor in serum responsible for this effect. The findings with vitronectin and collagen show that these agents stimulate different types of motility. The roles in wound healing of agents stimulating different types of motility are unclear and require further study. PMID- 1708799 TI - Tissue vitronectin in normal adult human dermis is non-covalently bound to elastic tissue. AB - Vitronectin is a multifunctional human plasma glycoprotein that is also found in constant association with elastic tissue fibers in normal adults. We have investigated the nature of the association of vitronectin with elastic tissue, and compared it to that of other elastic fiber-associated proteins, namely fibrillin and amyloid P component. Samples of normal human dermis were incubated with a variety of extraction agents, including high molar salt solution, non ionic detergent (Nonidet P-40), the reducing agents dithiothreitol or 2 mercaptoethanol, and the chaotropic agents sodium dodecyl sulfate or guanidine hydrochloride. Vitronectin purified from serum typically migrates as two bands of 75 and 65 kD. By contrast, immunoblotting studies of residual dermal material after extraction with the various agents revealed only lower molecular weight (58, 50, 42, 35, and 27 kD) anti-vitronectin reactive bands. Although these bands may represent degradation products of vitronectin generated as a result of the extraction procedure, we cannot exclude the possibility that tissue vitronectin is distinct from plasma vitronectin. Anti-vitronectin reactive polypeptides co migrating with the 58-, 50-, and 42-kD bands were solubilized following extraction with sodium dodecyl sulfate or guanidine hydrochloride, but not with the other extraction agents. Immunofluorescence studies using residual dermal material after extraction with guanidine hydrochloride demonstrated a marked reduction in elastic fiber staining intensity with anti-vitronectin and anti amyloid P component, but not with anti-fibrillin. Thus the majority, if not all of dermal vitronectin, is, like amyloid P component, non-covalently associated with, and not an integral constituent of, elastic fibers. PMID- 1708800 TI - The expression of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in experimental cutaneous inflammation: a comparison of ultraviolet B erythema and delayed hypersensitivity. AB - Endothelial cell adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are cytokine-regulated cell surface molecules involved in leukocyte adhesion. We have studied two forms of cutaneous inflammation to investigate in vivo the kinetics of adhesion molecule expression in relation to tissue accumulation of leukocytes. Immunohistology was performed on skin biopsies taken from human volunteers at 1, 6, 24, 72 h, and 1 week after two minimal erythema doses (MED) of ultraviolet B (UV-B) or intra-cutaneous tuberculin-purified protein derivative (PPD) (10-100 U). ELAM-1 expression on vascular endothelium and polymorphonuclear leukocyte infiltration were first observed at 6 h and maximal at 24 h after both UV-B and PPD. At 72 h and 1 week, however, endothelial ELAM-1 was more strongly expressed in PPD biopsies. VCAM-1 was minimally expressed in control skin, and was induced above background levels on endothelium, on some perivascular cells, and on stellate-shaped cells in the upper dermis at 24 h after injection of PPD; it was maintained up to 1 week. In contrast, no induction of VCAM-1 was seen following challenge with either 2 or 8 MED UV-B. Following PPD, but not UV-B, there was marked induction of ICAM-1 expression on basal keratinocytes. In these biopsies, the inflammation induced in response to PPD therefore differed from UV-B-induced inflammation in showing prolonged expression of endothelial ELAM-1, induction of VCAM-1 on endothelium and other cells, and induction of keratinocyte ICAM-1. These differences may result from differences in the cytokines released and may in turn be responsible for the differences in the nature of the leukocytic infiltration during the two types of inflammatory response. PMID- 1708801 TI - Effects of 13-cis-retinoic acid, all-trans-retinoic acid, and acitretin on the proliferation, lipid synthesis and keratin expression of cultured human sebocytes in vitro. AB - The aim of this study was to determine the effects of 13-cis-RA, all-trans-RA, and acitretin on the proliferation, lipid synthesis, and keratin expression of human sebocytes in vitro and to elucidate possible mechanisms of retinoid action on sebaceous glands at the cellular level. It was found that 13-cis-RA and all trans-RA decreased sebocyte proliferation in a dose- and time-dependent manner, with a 13-cis-RA-IC50 of 10(-5) M (after 7 d) and 10(-6) M (after 14 d) and an all-trans-RA-IC50 of 10(-7) M (after 14 d; no IC50 after 7 d). Acitretin inhibited sebocyte proliferation only at 10(-5) M. Furthermore, 13-cis-RA was the most potent inhibitor of acetate incorporation into lipids, which indicated lipid synthesis (48.2% reduction), followed by all-trans-RA (-38.6%), and by acitretin (-27.5%). All retinoids tested markedly decreased the synthesis of triglycerides, wax/stearyl esters, and free fatty acids in cultured sebocytes, whereas squalene synthesis remained uninfluenced and cholesterol synthesis slightly increased. On the other hand, keratin 5 was down-regulated, keratin 17 was up-regulated, and the expression of keratin 13 was virtually unaffected by all retinoids tested. Keratins 6 and 16 were down-regulated by 13-cis-RA and by all-trans-RA, keratin 14 was down-regulated by 13-cis-RA only, and keratin 19 was up-regulated by all trans-RA. These investigations indicate that 13-cis-RA and, to a lesser extent, all-trans-RA are potent inhibitors of both cell proliferation and lipid synthesis in human sebocytes in vitro, whereas acitretin only decreases lipogenesis in this model. In addition, retinoids may modify the differentiation of sebocytes in vitro by modulating keratin expression. Models of cultured human sebocytes are useful tools for further investigations on the sebaceous gland and its activity at the cellular level. PMID- 1708802 TI - Hair growth regulation: a molecular biologic approach. PMID- 1708803 TI - Aberrant intercellular adhesion molecule-1 (ICAM-1) expression by hair-follicle epithelial cells and endothelial leukocyte adhesion molecule-1 (ELAM-1) by vascular cells are important adhesion-molecule alterations in alopecia areata. PMID- 1708804 TI - Techniques and results of therapeutic catheter embolization of congenital vascular defects. AB - Transcatheter embolization in congenital vascular malformations (CVM) is an alternative or additive method in the curative and palliative treatment of hyperdynamic arteriovenous malformations (AVM). Its indications and its mostly interdisciplinary treatment ought to be managed by teamwork. The safe and adequate transcatheter vasoocclusion depends furthermore on an optimal choice of applicating devices (catheters, guide wires etc.) and suitable embolic materials. Semi-fluid Ethibloc is in our eyes actually the most effective embolizing agent for getting a safe and permanent central occlusion of congenital av-fistulae. PMID- 1708805 TI - Regulation of bile acid synthesis. V. Inhibition of conversion of 7 dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis. AB - In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7 dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis. PMID- 1708806 TI - Finding QRS complexes in severe noise by intelligent use of context and experience. PMID- 1708807 TI - Application of fractal geometry to the analysis of ventricular premature contractions. PMID- 1708808 TI - Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex. AB - The complement system augments the humoral immune response to low concentrations of antigen. This effect may be partly mediated by complement receptors on the surface of B lymphocytes that bind immunogenic complexes bearing fragments of C3 and C4. We have shown by immunoprecipitation analysis that the two complement receptors expressed by B lymphocytes, complement receptor 1 (CR1) and CR2, form a detergent-sensitive complex on the surface of tonsillar B lymphocytes and on K562 erythroleukemia cells that were co-transfected with cDNAs encoding CR1 and CR2. The CR1/CR2 complex is distinct from the CR2/CD19 complex and may assist B cell activation by efficiently capturing C3b-containing immunogens and maintaining such immunogens on the B cell after CR1 and factor I-mediated cleavage to iC3b and C3dg. The complement activating immunogen may then trigger signal transduction by the CR1/CR2 complex, the CR2/CD19 complex, or membrane immunoglobulin. PMID- 1708809 TI - The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-langley, and York blood group antisera. AB - Erythrocytes (E) lacking high incidence blood group antigens were screened by an antiglobulin test with a monoclonal antibody to human complement receptor type 1 (CR1; C3b/C4b receptor; CD35). Some examples of E lacking Knops, McCoy, Swain Langley, and York antigens, a serologically related group, were not agglutinated. Moreover, E of the null phenotype for these same antigens were nonreactive. To further explore this relationship, E expressing these antigens were surface labeled, solubilized, and incubated with the corresponding blood group-specific antisera. CR1 was immunoprecipitated, indicating that the epitopes recognized by each of these antisera are expressed on CR1. E of two individuals, putative null phenotypes for the Knops, McCoy, and Swain-Langley blood group antigens, expressed a very low number of CR1 (less than 30/E; approximately 10% of the normal mean). This observation accounts for their lack of reactivity in the antiglobulin test and their prior designation as null phenotypes. Also, the previously reported low as well as variable expression of CR1 on E explains prior difficulties in the serologic analyses of these blood group antigens. PMID- 1708810 TI - The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells. AB - Pluripotent hematopoietic stem cells (PHSC) are very rare cells whose functional capabilities can only be analyzed indirectly. For a better understanding and possible manipulation of mechanisms that regulate self-renewal and commitment to differentiation of PHSC, it is necessary to purify these cells and to develop assays for their growth in vitro. In the present study, a rapid and simple, widely applicable procedure to highly purify day 14 spleen colony-forming cells (day 14 CFU-S) is described. Low density bone marrow cells (rho less than or equal to 1.078 g/cm3) were enriched by two successive light-activated cell sorting procedures. In the first sort, cells within a predetermined light scatter (blast cell) window that are wheat germ agglutinin/Texas Red (WGA/TxR) positive and mAb 15-1.4.1/fluorescein isothiocyanate negative (granulocyte-monocyte marker) were selected. In the second sort, cells were selected on the basis of retention of the supravital dye rhodamine 123 (Rh123). Cells that take up little Rh123 (Rh123 dull cells) and those that take up more Rh123 (Rh123 bright cells) were 237-fold and 132-fold enriched, respectively, for day 14 CFU-S. Both Rh123 fractions were cultured for various time periods in vitro in the presence of mast cell growth factor (MGF), with or without interleukin 3 (IL-3) or IL-1 alpha. Both Rh123 fractions proliferated in response to MGF alone as determined by a [3H]TdR assay or by counting nucleated cells present in the cultures over time. MGF also acted synergistically with both IL-3 and IL-1 alpha to promote stem cell proliferation. Stimulation of both Rh123 fractions with MGF alone did not result in a net increase of day 14 CFU-S. Stimulation with MGF + IL-3 or MGF + IL-alpha resulted in a 4.4- or 2.6-fold increase of day 14 CFU-S in the Rh123 dull fraction, and an 11.6-fold or 2.6-fold increase of day 14 CFU-S in the Rh123 bright fraction, respectively. The data presented in this paper indicate that in vitro MGF acts on primitive hematopoietic stem cells by itself and also is a potent synergistic factor in combination with IL-3 or IL-1 alpha. PMID- 1708811 TI - Different stromal cell lines support lineage-selective differentiation of the multipotential bone marrow stem cell clone LyD9. AB - An interleukin 3-dependent multipotential stem cell clone, LyD9, has been shown to generate mature B lymphocytes, macrophages, and neutrophils by coculture with primary bone marrow stromal cells. We report here that coculture with the cloned stromal cell lines PA6 and ST2 can support differentiation of LyD9 cells predominantly into granulocyte/macrophage colony-stimulating factor (GM-CSF)- and granulocyte (G)-CSF-responsive cells, respectively. However, these stromal cell lines were unable to support lymphopoiesis of LyD9 cells. The GM-CSF-dependent line, L-GM, which was derived from LyD9 cells cocultured with PA6 stromal cells, could differentiate into macrophages and granulocytes in the presence of GM-CSF. The L-GM line can further differentiate predominantly into neutrophils by coculture with ST2 stromal cells. The G-CSF-dependent line, L-G, which was derived from LyD9 cells cocultured with ST2 stromal cells, differentiated into neutrophils in response to G-CSF. Although the stromal cell-supported differentiation of LyD9 cells required the direct contact between LyD9 and stromal cells, a small fraction of LyD9 cells that were pretreated with 5 azacytidine could differentiate into neutrophils and macrophages without direct contact with stromal cells. These results indicate that different stromal cell lines support lineage-selective differentiation of the LyD9 stem cell and that 5 azacytidine treatment can bypass the requirement of direct contact with stromal cells, albeit with a lower frequency. PMID- 1708812 TI - Premature expression of the macrophage colony-stimulating factor receptor on a multipotential stem cell line does not alter differentiation lineages controlled by stromal cells used for coculture. AB - We are interested to know whether expression of a lineage-specific growth factor receptor is deterministic to lineage commitment during hematopoiesis. For this purpose, we introduced the human c-fms gene into the multipotential stem cell clone LyD9 and two myeloid progenitor clones, L-GM3 and L-G3, cells that differentiate in response to granulocyte/macrophage colony-stimulating factor (GM CSF) and granulocyte (G)-CSF, respectively. Although LyD9 cells have differentiation potential to become macrophages, c-fms transfectants of LyD9 and L-GM3 cells did not differentiate in response to human macrophage (M)-CSF. However, c-fms transfectants of L-G3 cells differentiated to neutrophils in response to human M-CSF. These results indicate that the M-CSF receptor requires a specific signal transduction pathway to exert its differentiational and proliferative effects. Furthermore, the M-CSF receptor can convey a granulocyte type differentiation signal possibly by cooperating with the G-CSF receptor signal transduction pathway. The c-fms-transfected LyD9 cells as well as the original LyD9 cells differentiated predominantly into GM-CSF- and G-CSF responsive cells by coculturing with PA6 and ST2 stromal cells, respectively. The results indicate that differentiation lineage is not affected by premature expression of the M-CSF receptor. Instead, the stromal cell used for coculture apparently controls lineage-selective differentiation of the multi-potential stem cell line. PMID- 1708814 TI - Anterograde axonal transport of horseradish peroxidase in the rat postganglionic sympathetic nerves. AB - Seven minutes after initiating the experiment, horseradish peroxidase (HRP) labeled pinocytotic vesicles were observed on the plasma membranes of the postganglionic sympathetic neurons in the left superior cervical ganglia (SCG). HRP reaction product was also demonstrated in the vesicles of the smooth endoplasmic reticulum (sER) and multivesicular bodies in the neuronal perikarya. The HRP-positive vesicles of sER and multivesicular bodies were also detected in the adrenergic sympathetic nerve endings in the pineal gland 7 minutes after initiation of the experiment. This suggests that the HRP is rapidly transported anterogradely from the postganglionic sympathetic neurons in the SCG into the axon terminals in the pineal glands through the vesicles of the sER and multivesicular bodies. The present findings reveal that the rate of anterograde transport of HRP is 85 mm/h and that the majority of the adrenergic sympathetic nerves in either the left or right half of the pineal gland are derived from the postganglionic sympathetic neurons in both the left and right superior cervical ganglia. PMID- 1708813 TI - Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14. AB - Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin. PMID- 1708815 TI - Preoperative intra-arterial chemotherapy in patients with osteosarcoma: case report of seven patients below the age of 18 years. AB - In the period between 1987 and 1989, seven patients under 18 years of age with osteosarcoma of a lower limb were treated by preoperative intra-arterial chemotherapy. The age of the patients ranged from 12 to 17 years, the median age being 14.5 years. The polyethylene catheter was placed surgically into the lower epigastric artery. Two combinations of cytostatics were administered: adriamycin bleomycin-cisplatin and high-dose methotrexate-vincristine-cisplatin. All patients, after two or three courses, underwent surgical resection of limb tumor. Tumor destruction ranged from 20 to 100%. Four patients with necrosis from 80 100% remained free of disease from 18 to 30 months: two, having necrosis of 40% and 95% respectively, died. The youngest patient whose necrosis was as low as 20%, after completion of the systemic chemotherapy, developed local recurrence and pulmonary metastases. PMID- 1708816 TI - Interferon action on membrane-associated viruses: a minireview. PMID- 1708817 TI - Interactions between interferon and the human immunodeficiency virus. PMID- 1708818 TI - Translational regulation by HIV leader RNA, TAT, and interferon-inducible enzymes. AB - Every step in the replication cycle of HIV provides unique opportunities for controlling the progression of AIDS. In this regard, virus protein synthesis should be an important target for limiting HIV multiplication in cells. Molecular mechanisms in the regulation of HIV protein synthesis were therefore investigated in the context of interferon action. The interferon-inducible enzymes, 2-5A synthetase and dsRNA-dependent protein kinase, which can inhibit translation were activated by HIV-1 leader RNA. In cell-free systems, leader RNA and Tat protein of HIV inhibited and enhanced translation, respectively. An intriguing interplay of these viral and host factors were shown to influence the rate of translation in vitro. A model describing opposing actions of HIV Tat protein and interferon in HIV replication is represented. PMID- 1708819 TI - Acetylcholine-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia. AB - 1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation. 2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions. 3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 approximately 1 microM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 microM). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction. 4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (PX/PNa) followed the ionic selectivity sequence Cs+ (1.06) greater than Na+ (1.0) greater than Ca2+ (0.93). Anion substitution experiments showed a relative anion permeability, PCl/PNa less than or equal to 0.05. 5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation. PMID- 1708820 TI - Adenosine triphosphate-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia. AB - 1. The excitatory response of cultured neurones of rat parasympathetic cardiac ganglia to extracellular adenosine 5'-triphosphate (ATP) was examined using the whole-cell isolated membrane patch recording configurations of the patch clamp technique. The short latency between ATP application and activation of the membrane current (less than 20 ms) suggests a direct coupling between purinergic receptor and ion channel. The response was maintained during exposure to ATP suggesting that receptor desensitization is not a factor in current decay. 2. The current-voltage (I-V) relationship for macroscopic ATP-evoked currents showed strong inward rectification in the presence and absence of external divalent cations and a reversal potential of +10 mV (NaCl outside, CsCl inside). Unitary ATP-activated currents in cell-attached membrane patches exhibited a linear (ohmic) I-V relationship with a slope conductance of approximately 60 pS. 3. The order of agonist potency for the purinergic receptor-mediated response was 2 methylthioATP = ATP greater than ADP greater than AMP greater than adenosine = alpha,beta-methylene ATP greater than beta,gamma-methylene ATP, a sequence consistent with a P2y receptor subtype. ATP-evoked currents were attenuated by alpha,beta-methylene ATP (IC50 approximately 10 microM) and reversibly inhibited in a dose-dependent manner by Reactive Blue 2 (Kd = 1 microM). 4. The amplitude of the ATP-evoked current was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential when NaCl was replaced with mannitol indicated that the purinergic receptor channel is cation selective. The cation permeability relative to Na+ followed the ionic selectivity sequence Ca2+ (1.48) greater than Na+ (1.0) greater than Cs+ (0.67). Anions were not measurably permeant. 5. ATP and ACh-evoked responses in rat intracardiac neurones are mediated by distinct receptor channels. The ATP-activated channels in cardiac neurones may contribute to non-cholinergic, non-adrenergic neurotransmission and mediate, in part, the vagal innervation of the mammalian heart. PMID- 1708821 TI - K+ and Cl- currents in enterocytes isolated from guinea-pig small intestinal villi. AB - 1. The whole-cell configuration of the patch-clamp technique has been used to investigate the conductance properties of villus enterocytes isolated from guinea pig small intestinal epithelium. 2. With near physiological ionic gradients inward and outward rectification was observed in the hyperpolarizing and depolarizing voltage domains respectively. 3. Replacement of intra- and extracellular K+ with N-methyl-D-glucamine (NMG) eliminated inward rectification but did not alter outward currents. In symmetrical low Cl- solutions outward currents were reduced but inward rectification was not affected. Under these conditions increases in extracellular K+ shifted both the current-voltage relation and the extrapolated reversal potential as expected for a K(+)-selective current. 4. The inwardly rectifying nature of the K+ current observed here remained unaltered after chelation of internal Mg2+ with ATP or EDTA. 5. Extracellular application of 5 mM-Ba2+ or 50 micrograms ml-1 of the venom of the scorpion Leiurus quinquestriatus abolished the inward K+ current, while 5 mM extracellular tetraethylammonium (TEA) had little effect. 6. The current remaining in the presence of symmetrical Cl- solutions and in the complete absence of K+ rectified outwardly and reversed at 0 mV. The anionic nature of this current was confirmed by replacing Cl- with different anions. SCN- and Br- carried more current than Cl-, while F- and gluconate were less permeant. 7. Anionic currents of villus guinea-pig enterocytes were not stimulated by cyclic AMP and were strongly and reversibly inhibited by the Cl- channel blocker 5-nitro 2-(3-phenylpropylamino) benzoic acid (NPPB, 10(-5) M). 8. The inwardly rectifying K+ current described here shares some, but not all, characteristics with others previously described. It is postulated that this conductance might function to couple K+ permeability and the Na(+)-K+ pump rate in enterocytes. Absorption of chloride may be mediated by the Cl- channels. PMID- 1708822 TI - The actions of calcium on the mechano-electrical transducer current of turtle hair cells. AB - 1. Mechano-electrical transducer currents evoked by deflections of the hair bundle were recorded in turtle isolated hair cells under whole-cell voltage clamp. The outcome of perfusing with solutions of reduced Ca2+ concentration was investigated. 2. The transducer current was roughly doubled by lowering the concentration of divalent cations from normal (2.2 mM-Mg2+, 2.8 mM-Ca2+) to 0 Mg2+, 0.5 mM-Ca2+. No significant effects on the current's kinetics or reversal potential, or on the current-displacement relationship, were noted. 3. If the Ca2+ concentration was lowered to 50 microM (with no Mg2+), there was about a threefold increase in the maximum current but other changes, including loss of adaptation and a decreased slope and negative shift in the current-displacement relationship, were also observed. As a result, more than half the peak transducer current became activated at the resting position of the hair bundle compared to about a tenth in the control solution. 4. The extra changes manifest during perfusion with 50 microM-Ca2+ had also been seen when the cell was held at positive potentials near the Ca2+ equilibrium potential. This supports the view that some consequences of reduced external Ca2+ stem from a decline in its intracellular concentration. 5. With 20 microM-Ca2+, a standing inward current developed and the cell became unresponsive to mechanical stimuli, which may be explained by the transducer channels being fully activated at the resting position of the bundle. 6. The results are interpreted in terms of a dual action of Ca2+: an external block of the transducer channel which reduces the maximum current, and an intracellular effect on the position and slope of the current displacement relationship; the latter effect can be modelled by internal Ca2+ stabilizing one of the closed states of the channel. 7. During perfusion with 1 microM-Ca2+, the holding current transiently increased but then returned to near its control level. There was a concomitant irreversible loss of sensitivity to hair bundle displacements which we suggest is due to rupture of the mechanical linkages to the transducer channel. 8. Following treatment with 1 microM-Ca2+, single-channel currents with an amplitude of -9 pA at -85 mV were sometimes visible in the whole-cell recording. The probability of such channels being open could be modulated by small deflections of the hair bundle which indicates that they may be the mechano-electrical transducer channels or conductance about 100 pS. 9. Open- and closed-time distributions for the channel were fitted by single exponentials, the mean open time at rest being approximately 1 ms. The mean open time was increased and the mean closed time decreased for movements of the hair bundle towards the kinocilium.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1708823 TI - Effect of Mg2+ on the control of Ca2+ release in skeletal muscle fibres of the toad. AB - 1. The effect of myoplasmic Mg2+ on Ca2+ release was examined in mechanically skinned skeletal muscle fibres, in which the normal voltage-sensor control of Ca2+ release is preserved. The voltage sensors could be activated by depolarizing the transverse tubular (T-) system by lowering the [K+] in the bathing solution. 2. Fibres spontaneously contracted when the free [Mg2+] was decreased from 1 to 0.05 mM, with no depolarization or change of total ATP, [Ca2+] or pH (pCa 6.7, 50 microM-EGTA). After such a 'low-Mg2+ response' the sarcoplasmic reticulum (SR) was depleted of Ca2+ and neither depolarization nor caffeine (2 mM) could induce a response, unless the [Mg2+] was raised and the SR reloaded with Ca2+. Exposure to 0.05 mM-Mg2+ at low [Ca2+] (2 mM-free EGTA, pCa greater than 8.7) also induced Ca2+ release and depleted the SR. 3. The response to low [Mg2+] was unaffected by inactivation of the voltage sensors, but was completely blocked by 2 microM Ruthenium Red indicating that it involved Ca2+ efflux through the normal Ca2+ release channels. 4. In the absence of ATP (and creatine phosphate), complete removal of Mg2+ (i.e. no added Mg2+ with 1 mM-EDTA) did not induce Ca2+ release. Depolarization in the absence of Mg2+ and ATP also did not induce Ca2+ release. 5. Depolarization in 10 mM-Mg2+ (pCa 6.7, 50 microM-EGTA, 8 mM-total ATP) did not produce any response. In the presence of 1 mM-EGTA to chelate most of the released Ca2+, depolarizations in 10 mM-Mg2+ did not noticeably deplete the SR of Ca2+, whereas a single depolarization in 1 mM-Mg2+ (and 1 mM-EGTA) resulted in marked depletion. Depolarization in the presence of D600 and 10 mM-Mg2+ produced use-dependent 'paralysis', indicating that depolarization in 10 mM-Mg2+ did indeed activate the voltage sensors. 6. Depolarization in the presence of 10 mM Mg2+ and 25 microM-ryanodine neither interfered with the normal voltage control of Ca2+ release nor caused depletion of the Ca2+ in the SR even after returning to 1 mM-Mg2+ for 1 min, indicating that few if any of the release channels had been opened by the depolarization.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1708825 TI - Cellular basis of diarrhoea. The Croonian lecture 1989. AB - A wide range of different stimuli is perceived by the intestinal epithelium. They include luminal factors, especially bacterial toxins, and agonists such as inflammatory mediators and neuro peptides, acting from the interstitial fluid surrounding the epithelial cells. It is likely that in any individual patient with diarrhoea there is a range of stimuli acting upon the epithelium. Specific receptors on the apical and basolateral membrane, activated by these stimuli, transduce the perceived signals to stimulate a series of membrane-bound enzyme systems. They in turn generate second messengers which are liberated into the cytoplasm. These include cyclic adenosine monophosphate, cyclic guanosine monophosphate, inositol triphosphate (which goes on to liberate free calcium), and diacyl glycerol. Each of these second messengers activates a different protein kinase, each of which then induces the phosphorylation of a series of cytoplasmic and membrane-bound proteins. Each of the protein kinases is likely to influence the activity of the others so that their effects are closely integrated. The final common pathways through which intestinal secretory stimuli pass involve the opening of an anion channel in the apical membrane, together with the stimulated uptake of chloride at the basolateral membrane. Anions, especially chloride and possibly bicarbonate, are then secreted into the lumen, and sodium and water passing between the cells accompany them. The net result is secretion of salt and water, which lies at the centre of a number of diarrhoeal diseases. PMID- 1708824 TI - Voltage clamp measurements of the hyperpolarization-activated inward current I(f) in single cells from rabbit sino-atrial node. AB - 1. The kinetics and ion transfer characteristics of the hyperpolarization activated inward current, I(f), have been studied in single cells obtained by enzymatic dispersion from the rabbit sino-atrial (S-A) node. These experiments were done to assess the role of I(f) in the generation of the pacemaker depolarization in the S-A node. 2. The activation and the deactivation of I(f) in these single cells are accompanied by significant conductance increases and decreases respectively, confirming earlier findings from multicellular man-made strips of rabbit S-A node, and from mammalian Purkinje fibres. 3. The steady state activation of I(f) lies between -40 and -120 mV, and its voltage dependence can be described by a Boltzmann relation with the half-activation point at approximately -70 mV. 4. The delay or sigmoidicity in both the onset of I(f) and the deactivation of the tail currents can be accounted for semi-quantitatively by using a second-order Hodgkin-Huxley kinetic scheme. 5. The reversal potential for I(f) is -24 +/- 2 mV (mean +/- S.E.M., n = 6). It does not change significantly as a function of the amount of I(f) which is activated, indicating that ion accumulation or depletion phenomena are not important variables controlling the time course of I(f), or its selectivity. 6. The fully-activated current-voltage relationship for I(f) is approximately linear with a slope conductance of 12.0 +/ 0.88 nS per cell (mean +/- S.E.M., n = 6). 7. A simple mathematical model based on the measured values of maximum conductance, reversal potential, and kinetics of I(f) has been developed to simulate the size and time course of I(f) during typical spontaneous pacemaker activity in rabbit sino-atrial node cells. The calculations show that I(f) can change significantly during pacing and suggest that this current change is, at least in part, responsible for the pacemaker depolarization. PMID- 1708826 TI - The fat on a joint: OA and obesity. PMID- 1708827 TI - Production of platelet derived growth factor B chain (PDGF-B/c-sis) mRNA and immunoreactive PDGF B-like polypeptide by rheumatoid synovium: coexpression with heparin binding acidic fibroblast growth factor-1. AB - We present evidence supporting the hypothesis that locally produced platelet derived growth factor (PDGF) B-like polypeptides, as well as heparin binding growth factor-1 (HBGF-1), are involved in stimulating the pronounced hyperplasia of rheumatoid synovial stromal fibroblastlike cells. Explanted rheumatoid synovial tissues in vitro spontaneously secreted, in a time dependent manner, mitogenic activity for rheumatoid synoviocytes that was neutralizable by anti PDGF antibody. PDGF B/c-sis mRNA transcripts were detected in synovium from patients with rheumatoid arthritis (RA) (n = 5). Spontaneous PDGF B-like synthesis was detected by immunoprecipitation of radiolabeled PDGF B-like polypeptides secreted by explanted tissues. Furthermore, rheumatoid synovial tissues, particularly macrophage-like cells, immunostained specifically with anti PDGF B chain. The extent and intensity of staining and mononuclear cell infiltration were highly correlated. Immunostaining of osteoarthritic and normal synovial tissues was significantly less than RA synovium. PDGF-B immunostaining of synovial specimens previously characterized for expression of HBGF-1, the precursor of acidic fibroblast growth factor (aFGF), revealed that the extent and intensity of expression of HBGF-1 and PDGF-B were highly correlated. PMID- 1708828 TI - Regional distribution of mast cells and peptide containing nerves in normal and adjuvant arthritic rat synovium. AB - Simultaneous visualization of nerves and mast cells in the rat synovium was possible with double staining. Thus, a direct comparison could be made of nerves and mast cells in the ankle joints of healthy rats and in those with severe adjuvant induced polyarthritis. Nerves were studied with avidin-biotin-peroxidase complex (ABC) immunostaining, using heterologous antisera to protein gene product 9.5 (PGP 9.5), a recently discovered neural protein, and the neuropeptides substance P and calcitonin gene related peptide (CGRP). Mast cells were visualized by metachromatic staining of granule heparin. With double staining of sections, a parallel distribution of mast cells and nerves in all parts of the normal synovium was noted. In rats with adjuvant induced arthritis, a near total parallel disappearance of mast cells and nerves in the synovium occurred. In the arthritic rat such mast cell/nerve "units" were only present in the region where synovium attaches to bone. The observed regional depletion of both nerves and mast cells in arthritis may be of importance in the pathophysiology of arthritis. PMID- 1708829 TI - Is the beneficial effect of sulfasalazine due to inhibition of synovial neovascularization? AB - Angiogenesis is fundamental to support the continued synovial proliferation observed in rheumatoid arthritis. We investigated the effects of sulfasalazine and its metabolites on endothelial cell proliferation, a prerequisite to angiogenesis. At concentrations achieved in vivo sulfapyridine inhibited basal and endothelial cell growth factor stimulated endothelial cell proliferation. Sulfasalazine and 5 amino salicylic acid had no effect. PMID- 1708830 TI - Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin. AB - Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution. PMID- 1708831 TI - Incipient mitochondrial evolution in yeasts. II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution. AB - We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY ... YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution. PMID- 1708832 TI - Location of ion-binding sites in the gramicidin channel by X-ray diffraction. AB - We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (T1+, K+, Ba2+, Mg2+ or without ions). From the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for T1- at 9.6 (+/- 0.3) A and for Ba2+ at 13.0 (+/- 0.2) A from the midpoint of the gramicidin channel. The location of Ba(2+)-binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of T1(+)-binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded beta 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the T1(+)-binding site is either near the bottom of or below the first helix turn. PMID- 1708833 TI - RNA-protein interactions in a Nus A-containing Escherichia coli transcription complex paused at an RNA hairpin. AB - We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog. The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A. We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures. Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA. Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem loop. Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected. Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ration to RNA polymerase (250:1). Both the beta and beta' subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits. Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact. Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed. We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop. PMID- 1708834 TI - Tumor growth dependent on Kaposi's sarcoma-derived fibroblast growth factor inhibited by pentosan polysulfate. AB - A neoangiogenic response is critical for the unrestricted growth of solid tumors beyond a few millimeters in diameter. Release of adequate growth-stimulating activity from tumor cells is obviously required for the stimulation of blood vessel growth, and blockade of such stimulatory activity should repress tumor growth at the microscopic level. To test this hypothesis and to study appropriate inhibitors, we used a human adrenal cancer cell line (SW-13/K-fgf) engineered to secrete Kaposi's sarcoma-derived fibroblast growth factor (K-FGF), which we previously showed to induce growth of highly vascularized subcutaneous tumors in animals by autocrine and paracrine stimuli. In the present study, we tested different polysulfates for their selective inhibition of proliferation induced by K-FGF versus proliferation independent of K-FGF. Suramin and dextran sulfate showed slight selective inhibition of K-FGF-induced proliferation, ie, inhibition three- and five-fold greater, respectively, than the inhibition of proliferation independent of K-FGF. In contrast, heparin was inactive. The heparin analogue pentosan polysulfate (PPS), however, showed selective inhibition that was more than 2000-fold greater. The inhibitory effects of PPS on growth of SW-13/K-fgf cells, as well as endothelial cells, were fully reversible by an excess of added FGF. Daily intraperitoneal injections of PPS were tolerated well by athymic nude mice and prevented growth of subcutaneous SW-13/K-fgf tumor xenografts. PPS will be a useful tool to elucidate the effects of FGFs in vitro and in vivo and appears to be a prototype for the development of tumoricidal therapy based on targeting of growth factors. PMID- 1708835 TI - Successful radioimmunotherapy for lung metastasis of human colonic cancer in nude mice. AB - The potential for radioimmunotherapy as an adjuvant treatment for early disseminated colonic cancer was investigated in an experimental lung metastasis model. Nude mice receiving intravenous injection with a suspension of human colonic cancer cells (GW-39) developed multiple (10-100) tumor nodules throughout the lungs, and more than 50% of the animals died of extensive tumor involvement within 5-10 weeks. Groups of eight or nine animals bearing 7-day-old tumor transplants were treated with a single intravenous injection of radioiodinated agents: either 0.15 or 0.30 mCi of whole IgG of the NP-4 murine monoclonal antibody (MAb) against carcinoembryonic antigen (CEA) or 0.15 or 0.30 mCi of whole IgG of Immu-31, an anti-alpha-fetoprotein (anti-AFP) MAb. Treatment of animals with 0.15 or 0.30 mCi of 131I-labeled NP-4 IgG 7 days after injection of tumor cells resulted in survival for 23 weeks after tumor implantation in four of eight and seven of nine animals, respectively. Microscopic examination revealed that over 90% of the lung tumor colonies had no evidence of surviving cells. Animals treated with 0.30 mCi of anti-AFP, an irrelevant MAb, survived 4 weeks longer than controls. Toxicity was evident in four of the 17 animals given 0.30 mCi of NP-4 IgG (specific) or anti-AFP IgG (irrelevant) MAb. These animals died within 1-3 weeks after radioantibody injection, suggesting that death was related to the radiation dose. None of the animals given 0.15 mCi of 131I-MAb died within this period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708836 TI - Studies on antinephritic effect of TJ-8014, a new Japanese herbal medicine (4): Effects on accelerated passive Heymann nephritis in rats. AB - We investigated the antinephritic effect of TJ-8014, in comparison to dipyridamole, on accelerated passive Heymann nephritis in rats. TJ-8014 (4.0 g/kg/day, p.o.) given from the heterologous phase (from the day of injection of the antiserum against Fx1A) markedly inhibited the urinary protein excretion and the elevation of plasma cholesterol levels as well as glomerular histopathological changes. When the treatment was started from the autologous phase (from the 22nd day) after proteinuria was fully developed, TJ-8014 also showed a beneficial effect. Dipyridamole (0.4 g/kg/day, p.o.) had no effect when the treatment was started either from the heterologous or autologous phase. TJ 8014 decreased glomerular rat IgG and rat C3 deposits, although it affected neither the plasma antibody titer against rabbit gamma-globulin nor the plasma complement level. TJ-8014 markedly prevented the reduction of plasma and adrenal corticosterone level as well as the reduction of renal blood flow of rats with nephritis. These results suggest that TJ-8014 may be a useful drug against idiopathic membranous nephropathy and the beneficial effect of this drug may be caused by the elimination of glomerular immune deposits and C3 through the increase in renal blood flow related to the enhanced release of adrenal corticosterone. PMID- 1708837 TI - Gastric mucosal protection induced by restraint and water-immersion stress in rats. AB - We found that the gastric mucosal lesion induced by 60% ethanol containing 150 mM HCl was reduced by pre-loading the rat with restraint and water-immersion stress (22 degrees C). The resistance to ethanol injury was maximal in 1-hr stress loaded rats and gradually decreased with increasing time of stress (2-6 hr). The protective effect of stress was markedly decreased by subdiaphragmatic truncal vagotomy, and electrical stimulation of vagal nerves afforded similar protection as observed after stress. In contrast, pretreatment with atropine markedly reduced ethanol injury in both the control and 1 hr stress-loaded rats. One-hour stress produced surface epithelial cell damage in sham vagotomized rats, but the surface cells were almost intact in vagotomized rats. Pretreatment with indomethacin significantly mitigated the protective effect of stress against ethanol injury. However, the level of mucosal prostaglandin E2 was not changed 1 hr after stress, and it tended to decrease 6 hr after stress. Pretreatments with 1-hr stress and vagal stimulation weakly reduced localized staining of gastric mucosa with gentian violet. We conclude that adaptive protection can be achieved by restraint and water-immersion stress. PMID- 1708838 TI - [The fibrinolytic system and use of aprotinin for the eye]. PMID- 1708839 TI - [Arteriosclerosis, cholesterinosis: new diagnostic and therapeutic approaches]. PMID- 1708840 TI - [Pathogenetic characteristics of the development of arrhythmia in patients with ischemic heart disease]. AB - ACTH, hydrocortisone and cyclic nucleotides levels, beta-receptors sensitivity, lipid peroxidation (LPO), antioxidant system, hemodynamics were investigated in 93 coronary patients without myocardial infarction in the presence of cardiac arrhythmia and after its correction. The clinical and laboratory findings indicate that it is primarily LPO activation and LPO products excessive accumulation in the blood that are responsible for arrhythmia emergence. Experimental data support these results by showing possibility of cardiac arrhythmia onset due to LPO products which raise the density of Na-Ca channels and inhibit the activity of Ca-dependent ATPase. PMID- 1708841 TI - Carcinoid tumour of the gastrointestinal tract: prognostic factors and disease outcome. AB - This study represents retrospective analysis of 87 patients with a carcinoid tumour of the gastrointestinal tract seen and followed in the British Columbia Cancer Agency (BCCA) from 1960 to 1986. In 49 cases, the primary site was the small bowel. The rest of the cases were distributed as follows: 11 appendix, 10 rectum, 5 stomach, and 7 undetermined. We extrapolated the Dukes' and modified Astler-Coller surgicopathological classifications used for colorectal cancer for use in our cases of carcinoid tumour of the gastrointestinal tract. A strong correlation was found, using this staging, with disease-specific survival. Other prognostic factors included histologic differentiation, the presence of macroscopic residual disease after initial surgery, and level of 5 hydroxyindoleacetic acid (5-HIAA) in urine. Among 51 patients with surgically grossly removed disease, there was a tendency for the development of distant and distant/locoregional recurrence more often than locoregional recurrence alone. The liver was the commonest site of distant recurrence. Analysis of the effect of radiotherapy or chemotherapy on carcinoid tumour of the gastrointestinal tract proved unsuccessful because only a small portion of the patients had this treatment, and it was used mainly for palliation. PMID- 1708842 TI - Double-inlet ventricle presenting in infancy. II. Results of palliative operations. AB - The influence of palliation on survival was studied in 191 consecutive infants, presenting at under 1 year of age, with double-inlet ventricle (1973 to 1988, median follow-up 8.5 years). Palliative operations were performed on 154 occasions in 121 patients (63%). Survival after a systemic-pulmonary arterial shunt (n = 57) and banding of the pulmonary trunk (n = 35) was comparable (84% and 77% at 1 year, 62% and 45% at 5 years), but those who underwent repair of aortic arch obstruction fared worse (n = 18, 44% and 22% at 1 and 5 years, p less than 0.001). The remainder did not undergo an operation because of balanced physiology (n = 17, 9% of entire group), complex anatomy (n = 32, 15%), or irreversible low output (n = 19, 12%). Palliative surgery, overall, had a deleterious effect on immediate survival (greater than 1 month relative risk 6.6, p less than 0.001), but, in the survivors, medium-term outcome was improved (greater than 6 months, 0.68, p less than 0.05). This effect was most marked for those undergoing a systemic-pulmonary artery shunt (less than 1 month, 2.52; greater than 6 months, 0.43); by contrast, after banding of the pulmonary trunk, with or without additional repair of the aortic arch repair, medium-term risk was not altered (greater than 6 months, 1.13 and 0.91, respectively). These data will assist the clinician in making decisions concerning the management of infants with double-inlet ventricle and in the judicious use of palliative surgery. PMID- 1708843 TI - TNF-beta (lymphotoxin) strongly upregulates TNF-alpha gene expression in human peripheral blood lymphocytes. AB - We investigated whether TNF-beta could induce or modulate TNF-alpha gene expression in human peripheral blood lymphocytes. For these experiments, lymphocytes were cultured in serum-free medium for 48 hours with human recombinant TNF-beta in the presence or absence of human recombinant IL-2. At the end of the culture period, total cellular RNA was isolated and probed for TNF alpha mRNA using an S1 nuclease protection assay. TNF-beta alone was unable to induce lymphocyte TNF-alpha mRNA. However, when TNF-beta was used in combination with IL-2 an 8- to 12-fold increase in TNF-alpha mRNA compared to lymphocytes activated in IL-2 alone was observed. Secreted TNF-alpha was measured in the culture supernatants by TNF-alpha specific ELISA. Consistent with the results of mRNA analysis, TNF-beta alone did stimulate TNF-alpha secretion, but lymphocytes activated with TNF-beta/IL-2 demonstrated a 3- to 10-fold increase in secreted TNF-alpha over that induced by IL-2 alone. These experiments demonstrate that TNF beta can participate in the upregulation of TNF-alpha gene expression in lymphocytes and suggest that cellular dysregulations involving autocrine TNF production may be exacerbated by this positive feedback pathway. PMID- 1708844 TI - Frozen bone marrow trephine biopsy--a technical evaluation. AB - The routine study of bone marrow trephine biopsies involves fixation, decalcification, paraffin-embedment, sectioning and staining. However, this process creates artifacts, produces shrinkage of tissue, consumes time and can result in sections of unsatisfactory cytological quality. It also renders the tissue unsuitable for enzyme-histochemical and immunohistochemical analyses. Frozen section of bone marrow without decalcification was evaluated as an alternative method for the study of bone marrow. This method was found to give sections with comparable cytological quality to that of paraffin-embedment, yielded sections for interpretation within 24 hours, and allowed enzyme histochemical and immunohistochemical analyses to be applied successfully. PMID- 1708845 TI - [Changed technique improves the treatment with prostatic coil]. PMID- 1708846 TI - Lymphoproliferative disorders after FK 506. PMID- 1708847 TI - Enkephalin, dynorphin and substance P in postmortem substantia nigra from normals and schizophrenic patients. AB - Three peptide neuromodulators that are found in high concentration in the substantia nigra: dynorphin A 1-8, met5-enkephalin-arg6-gly7-leu8 and substance P, were measured by specific radioimmunoassays in nigral tissue from normals and schizophrenics postmortem. Substance P and dynorphin were unchanged between the two groups. However, the proenkephalin-derived peptide was significantly elevated in the schizophrenic group. The immunoreactivity was identified as authentic met5 enkephalin-arg6-gly7-leu8 by high pressure liquid chromatography. The data suggest that a different set of regulatory controls exists for nigral enkephalin peptides as compared to dynorphin and substance P, and that the former system may be disordered in schizophrenia. PMID- 1708848 TI - Titer and specificity of autoantibody to beta 2-microglobulin in sera from patients with rheumatic disease. AB - Sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD) possessed higher titer of antibody to human beta 2-microglobulin (beta 2m) than those from healthy controls and patients with Behcet's disease in the enzyme-linked immunosorbent assay. It was also confirmed by the immunoprecipitation method. Anti-beta 2m antibody in sera from those patients immunoprecipitated free beta 2m but not beta 2m in association with major histocompatibility complex class I antigen heavy chain. It was suggested that anti-beta 2m antibody in sera from either patients or healthy controls might be directed mainly against free beta 2m. The relationship between the anti-beta 2m antibody and anti-lymphocytotoxic antibody found in those patients is discussed. PMID- 1708849 TI - Potentiation of halomethane hepatotoxicity by chlordecone: a hypothesis for the mechanism. AB - A major toxicological issue today is the possibility of unusual toxicity due to interaction of toxic chemicals upon environmental or occupational exposures to two or more chemicals, at ordinarily harmless levels individually. While some laboratory models exist for such interactions for the simplest case of only two chemicals, progress in this area has suffered for want of a model where the two interactants are individually nontoxic. One such model is available, where prior exposure to nontoxic levels of the pesticide Kepone (chlordecone) results in a 67 fold amplication of CCl4 lethality in rats. Extensive hepatotoxicity observed in this interaction is characterized by histopathological alterations, perturbation of related biochemical parameters and is followed by complete hepatic failure. This propensity for chlordecone to potentiate hepatotoxicity of halomethanes such as CCl4, CHCl3, and BrCCl3 has been a subject of intense study to unravel the underlying mechanism. Mechanisms such as induction of microsomal cytochrome P-450 by chlordecone and greater lipid peroxidation are inadequate to explain the remarkably powerful potentiation of halomethane toxicity. Compelling experimental evidence supports the hypothesis that hepatocellular division during early time points after the administration of CCl4 is an important determinant of the progression (or repair of it) of the liver injury and consequent destruction (or restoration) of the hepatolobular architecture and function. This paper advances a hypothesis for the mechanism of hepatotoxic and lethal effect of CCl4 as being primarily related to the accelerated progression of liver injury due to suppressed hepatocellular regeneration and hepatolobular restoration. This is in contrast to the widely accepted putative mechanism, one which invokes only bioactivation followed by runaway lipid peroxidation as the events determining the course of the progressive phase of liver injury. The concept being advanced in this paper accepts bioactivation (and perhaps lipid peroxidation) as the primary initiating events of cell injury, but maintains that they are not the determinants of the progressive phase of liver injury. The biological issue of whether the cells are incapacitated from regenerating is the determinant of the progression of liver injury, and therefore, the ultimate outcome of hepatotoxicity and lethality. PMID- 1708850 TI - Pancreatoblastoma: response to chemotherapy. AB - Two cases of pancreatoblastoma in children are reported here. Only biopsies were made at laparotomy as surgical resection by duodeno-pancreatectomy was not possible. In both children a dramatic response was observed with chemotherapy: doxorubicin plus cisplatin for one, cyclophosphamide, actinomycin D, bleomycin, vinblastine sulfate, and cisplatin for the other. After completion of the chemotherapy the first patient had a local resection; then he had radiotherapy. He is alive in first remission 40 months after the end of the treatment. In the second patient, regional recurrence occurred 8 months after chemotherapy was ended. A transient second remission was obtained with ifosfamide plus etoposide alternating with epirubicin plus vincristine. The patient died 36 months after the diagnosis. Therefore, these two cases suggest that chemotherapy may be proposed before any attempt at surgical excision. Nevertheless, early consolidation by radical resection or irradiation must be considered. PMID- 1708851 TI - Biopolymers. Light on molecular recognition. PMID- 1708852 TI - Peptide selection by MHC class I molecules. AB - Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides. PMID- 1708853 TI - Social stress in tree shrews increases the whole-body RNA degradation rates. PMID- 1708854 TI - Effects of Bay K 8644 on the spontaneous electrical and mechanical activities of the rat portal vein. AB - Effects of Bay K 8644 on the spontaneous electrical and mechanical activities of the rat portal vein were studied. Bay K 8644 enhanced contractions in amplitude and duration. Bay K 8644 prolonged the duration of the spontaneous spike potential bursts and increased the number of spike potentials in a burst. These results indicate that the increase in the amplitude and duration of spontaneous contractions of the rat portal vein induced by Bay K 8644 are mediated mainly by the facilitation of the membrane electrical activity. PMID- 1708855 TI - Calcium channel modulation by dihydropyridines modifies sufentanil-induced antinociception in acute and tolerant conditions. AB - The study was aimed at elucidating the possible participation of L-type Ca2+ channel in the acute analgesic effect of an opiate and the development of tolerance to this action. Sufentanil, a selective mu agonist, and two dihydropyridines, the Ca2+ antagonist nimodipine and the Ca2+ agonist Bay K 8644, were selected. The tailflick test was used to assess the nociceptive threshold. In naive rats, nimodipine (200 micrograms/kg) potentiated the analgesic effect of sufentanil reducing the ED50 from 0.26 to 0.08 microgram/kg. Similar results were observed with its (-)-enantiomer Bay N 5248, while the (+) enantiomer Bay N 5247 was ineffective. Tolerance to the opiate was induced by chronic subcutaneous administration of sufentanil with minipumps (2 micrograms/h, 7 days). In these conditions the dose-response curve to sufentanil was displaced to the right and the ED50 was increased to 1.49 micrograms/kg. In tolerant rats, nimodipine preserved its potentiating ability and prevented the displacement to the right of the sufentanil dose response-curve (ED50 = 0.48 microgram/kg). When nimodipine was pumped (1 microgram/h, 7 days) concurrently with sufentanil, the development of tolerance to the opioid was not disturbed. However, the expression of tolerance was abolished and even the effect of acutely administered sufentanil was markedly potentiated (ED50 = 0.03 microgram/kg). Similar experiments were performed with Bay K 8644. In naive rats, Bay K 8644 at a low dose (20 micrograms/kg) that behaves as a calcium agonist, antagonized the analgesic effect of sufentanil (ED50 = 0.58 microgram/kg), whereas at a high dose (200 micrograms/kg) it potentiated this action (ED50 = 0.15 microgram/kg).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708856 TI - [Carcinoid of the appendix; appendectomy or hemicolectomy?]. PMID- 1708857 TI - [Multidisciplinary therapy using interferon and immunological evaluation for glioma patients: two-color analysis of T cell subsets]. AB - We have employed IAR therapy [combination of postirradiation, chemotherapy and interferon (IFN)] for malignant glioma patients. Changes of lymphocyte fractions in patients were evaluated before and after IAR therapy, using a recently developed two-color analysis. Eight malignant glioma patients received irradiation, chemotherapy (ACNU) and immunotherapy (OK-432 and IFN-beta). Peripheral blood lymphocytes taken during hospitalization with IAR therapy (first half and latter half), and every 3 to 6 months for 2 years at the longest after IAR therapy were double-stained with FITC- and PI-labelled antibodies and two color analysis was conducted by a FACS Analyzer. Six patients out of 8 survived for 6 months to 2 years, 2 died after 3 and 6 months, respectively. Leu-2a (suppressor/cytotoxic T), especially Leu-2a+ 15- (cytotoxic T) showed a high value. Leu-2a level decreased during treatment, and both Leu-2a+ 15- and Leu-2a+ 15+ (suppressor T) values decreased. Two thirds of the patients showing an increased Leu-2a+ 15+ level died. Leu 3a (helper/inducer T), especially Leu-3a+ 8+ (inducer T) level decreased, but Leu-3a+ 8- (helper T) level increased during treatment. The level decreased in the worse patients. Leu-3a/Leu-2a ratio was low, but it increased during treatment as compared with the results of conventional therapy. Leu-7, Leu-11a, NK activity, and gamma-IFN productivity were further studied. Treatment combined with IFN revealed an influence on the T cells resulting in an increase of helper T level and suppression of suppressor T level. PMID- 1708858 TI - Eccrine acrospiroma (clear cell hidradenoma) of the eyelid. Immunohistochemical and ultrastructural features. AB - A 46-year-old man underwent excision of a left lower eyelid mass that had enlarged over a 2-month period. Pathologic examination showed the mass to be an eccrine acrospiroma, a benign adnexal tumor that rarely arises in the eyelid. Light microscopic and ultrastructural examination showed two types of cells to comprise the tumor: eosinophilic cells with intracytoplasmic tonofilaments, and clear cells with intracytoplasmic glycogen granules. Immunohistochemical stains were positive for cytokeratins AE 1,3, epithelial membrane antigen, carcinoembryonic antigen, and muscle specific actin in tumor cells. PMID- 1708859 TI - Diagnostic spinal anaesthesia in chronic spinal cord injury pain. AB - In a double blind study, 21 patients with chronic spinal cord injury (SCI) pain underwent placement of a lumbar subarachnoid catheter and injection of placebo and lidocaine. The effects on pain intensity, distribution, altered sensations and sensory level of anaesthesia were monitored. Four patients responded briefly to placebo, while 13 demonstrated a mean reduction of pain intensity of 37.8 +/- 37% for a mean duration of 123.1 +/- 95.3 minutes in response to lidocaine. The pain response to subarachnoid lidocaine differed significantly (p less than 0.01) from placebo. Spinal anaesthesia was also associated with changes in pain distribution and altered sensation. A spinal anaesthetic-induced sensory level could not be achieved cephalad to the sensory level of neurological injury in 5 patients who presented with spinal canal obstruction. This study has demonstrated that response to diagnostic spinal anaesthesia in chronic SCI pain is complex, requiring individual interpretation in each patient and consideration of the following factors; symptomatology, etiology, pain perception, spinal canal anatomy, CSF chemistry and local anaesthetic pharmacology. PMID- 1708860 TI - Benign nerve sheath tumors: a light microscopic, electron microscopic and immunohistochemical study of 102 cases. AB - One hundred and two cases of benign nerve sheath tumors (NSTs) were studied with a combined approach using routine light microscopy (LM), immunohistochemistry (IH) for myelin basic protein (MBP) and S-100 protein as well as transmission electron microscopy (TEM) with the aim of obtaining greater insight into the true nature of these neoplasms, and also to establish the importance of IH and TEM in their diagnosis. Myelin basic protein was not identified in any of these tumors, whereas S-100 protein was positive to a variable degree in both schwannomas and neurofibromas. TEM revealed that Schwann cells predominated in tumors which were strongly positive for S-100 protein and appeared as schwannomas by LM. However, neurofibromas showing a variable patchy positivity for S-100 were composed of an admixture of Schwann cells, fibroblast-like cells and intermediate cells considered to be modified Schwann cells. Perineurial cells in typical form were not seen. It is concluded that all NSTs are basically of Schwann cell origin and that the intermediate cells and fibroblast-like cells are variants of Schwann cells. The different morphological appearances and biological behaviour of schwannomas and neurofibromas may be related to some other factors like micro environment or genetic predisposition. Further, both IH, especially for S-100 protein, and TEM play an important role in establishing their diagnosis. PMID- 1708861 TI - Solitary fibrous tumor of pleura: expression of cytokeratins. AB - A case of solitary fibrous tumor of the pleura (SFT) is presented. The histogenesis of this uncommon tumor is debated with most investigators favouring origin in submesothelial fibroblasts. Part of the evidence supporting this has been the persistent negativity of the tumor cells for cytokeratin--a feature militating against origin in mesothelial-lining cells. Our case shows unequivocal focal cytokeratin positivity in tumor cells; we feel that although this indicates mesothelial differentiation it does not militate against origin in submesothelial fibroblasts since, in reactive conditions, these are capable of mesothelial differentiation including expression of cytokeratin. Indeed, it reinforces the hypothesis that SFT is of submesothelial origin. Solitary fibrous tumors can be cellular and atypical. The reactivity of the tumor with cytokeratin, albeit rarely, should be considered in differentiating SFT from sarcomatoid mesothelioma. PMID- 1708862 TI - In spite of its validity, has Dale's principle served its purpose? A scientific paradox. PMID- 1708863 TI - Possible reasons for Neolithic skull trephining. PMID- 1708864 TI - Safe Medical Devices Act of 1990. PMID- 1708865 TI - Persistent left superior vena cava: a problem in the transvenous pacing of the heart. AB - Transvenous pacemaker implantation was impossible from the right side in a patient with persistent left superior vena cava (PLSVC) as the two cavae communicated via the coronary sinus and entry into the right heart could not be achieved. Epicardial pacing was instituted. PMID- 1708866 TI - Bradycardia and syncope in children not controlled by pacing: beta-adrenergic hypersensitivity. AB - Cardiac pacing is frequently employed in the therapy of children with syncope and documented bradycardia. This report describes two children, ages 7 and 9 years, who underwent placement of demand ventricular pacing systems for documented bradycardia and syncope. Cardiac catheterization and intracardiac electrophysiological studies failed to show evidence of structural abnormalities, sinus node or conduction system disease, inducible arrhythmias, or VA conduction in each patient. Both patients had persistent symptoms after pacemaker implantation. Autonomic function testing with continuous heart rate and blood pressure monitoring revealed exaggerated beta-adrenergic responses to simple standing and small doses of isoproterenol. Symptoms were completely eliminated with atenolol. In these two children, cardiac pacing alone was not adequate for relief of symptoms. Autonomic mechanisms of bradycardia and hypotension should be considered prior to implantation of permanent pacing systems in children. PMID- 1708867 TI - Sudden bradyarrhythmic death in patients with the implantable cardioverter defibrillator: report of two cases. AB - Two cases of sudden bradyarrhythmic death necessitating cardiopulmonary resuscitation in patients with the automatic implantable cardioverter defibrillator are described. One patient had sudden bradyarrhythmic death while being monitored in the hospital. This patient could not be resuscitated and represents the first reported sudden cardiac death secondary to bradyarrhythmia in a patient with the automatic implantable cardioverter-defibrillator. The second patient had an out-of-hospital sudden death followed by probable cardioverter-defibrillator discharge, leading to complete heart block with escape ventricular rhythm necessitating immediate temporary pacemaker insertion. These cases highlight the need for bradycardia back-up pacing and event monitoring in the newer tachyarrhythmia management devices. PMID- 1708868 TI - Intraoperative myocardial infarction during open-heart ablation of an atrioventricular accessory pathway. AB - We report a patient who suffered an intraoperative AMI from injury to the distal portion of a dominant left circumflex coronary artery in the atrioventricular groove. Depending upon coronary anatomy and location of accessory pathways, compromise of coronary circulation by nonintrinsic pathology can be a major cause of morbidity. PMID- 1708869 TI - Effect of atrial pacing on the frequency of tachycardia in patients with recurrent junctional tachycardia. AB - In order to assess whether atrial pacing reduced the frequency of tachycardia in patients with recurrent junctional tachycardias, ten patients with recurrent junctional tachycardias with atrial Intertach antitachycardia pacemakers in situ were paced in a random order in atrial demand mode at 50 ppm (AAI 50), 80 ppm (AAI 80), and 100 ppm (AAI 100) for a period of up to 1 month. The numbers of tachycardias detected by the pacemaker over this period were recorded and compared with the number seen when unpaced (000). Correct arrhythmia detection by the pacemaker was confirmed by Holter monitoring. The number of tachycardias in 000 was 44.7 +/- 19.8 (mean +/- SEM). No significant reduction in tachycardia frequency was seen in any pacing mode. Back-up atrial pacing at 50 ppm tended to reduce the frequency of tachycardias (32.3 +/- 12.8 tachycardias; P = 0.06). The higher pacing rates increased the number of tachycardias (AAI 80; 57.1 +/- 24.6 tachycardias, P = 0.20: AAI 100; 81.8 +/- 30.2 tachycardias; P = 0.31). Symptoms increased with each pacing mode and palpitations were statistically more severe in AAI 100 mode. Four patients had disabling symptoms at this rate and had to drop out. Atrial back-up pacing may be of use in some patients with junctional tachycardia, but overdrive pacing is not helpful. PMID- 1708870 TI - Maintenance of atrioventricular sequence after His-bundle ablation for paroxysmal supraventricular rhythm disorders: a unique use of the fallback mode in dual chamber pacemakers. AB - The usual mode of controlling ventricular rate after His-bundle ablation is placement of a ventricular demand rate responsive pacemaker. Atrioventricular synchrony is therefore lost in individuals whose atrial rhythm disturbances are paroxysmal. Thirty-seven His ablations were performed at this institution, since January of 1983, for atrial rhythm disturbances that were not suppressible with medical therapy alone. Of those, ten patients were identified whose atrial rhythm disturbances were intermittent and who would otherwise benefit by having proper atrioventricular sequence during sinus rhythm. These patients underwent placement of a dual chamber pacemaker system having a fallback mode. At a mean follow-up period of 17 +/- 11 months, eight patients continued to maintain proper atrioventricular sequence with ventricular pacing tracking atrial activation during sinus rhythm. Supraventricular tachyarrhythmic attacks were associated with attainment of the programmed upper rate limit at which time the fallback mode was activated and the pacemaker automatically converted to a ventricular demand (VVI) mode. Restoration of normal sinus rhythm was associated with restoration of proper atrioventricular sequence. Two patients have developed chronic atrial fibrillation and their pacemakers continue to function in the fallback mode as VVI devices. In conclusion, intermittent supraventricular tachyarrhythmias which are resistant to drug therapy can be treated with His ablation and dual chamber pacing utilizing special pacemaker features such as the fallback mode. PMID- 1708871 TI - Spontaneous occurrence of the induced cardioinhibitory vasovagal reflex. AB - ECG recording of spontaneous, neurally-mediated syncope is rare. We have observed ten patients who sustained 70 syncopal episodes in whom: (1) ECG monitoring recorded syncope caused by ventricular asystole (AV block, three patients; sinus arrest; seven patients); (2) syncope and the spontaneously observed arrhythmias were reproducible by carotid sinus massage, upright tilt test, or eyeball pressure; and (3) no discernable cause of precipitating factors were detected. Two patients had a history of cardiac disease and four patients had only mild nonclinical ECG or echocardiographic abnormalities. Syncopal episodes recorded during Holter monitoring were of sudden onset in four patients and preceded by prodromal symptoms in six patients. The maximum RR pause was 9.4 +/- 3.7 seconds (range 4.5-15). Electrophysiological evaluation was normal in seven patients. Slight sinus node dysfunction or atrioventricular conduction abnormalities were noted in three others. The clinical characteristics of spontaneous and induced episodes strongly suggest that increased vagal tone played a role in causing the spontaneous events. Vagal stimulation tests are useful for the diagnosis of syncope of unknown origin. PMID- 1708872 TI - The role of combination therapy with mexiletine and procainamide in patients with inducible sustained ventricular tachycardia refractory to intravenous procainamide. AB - This study evaluated the role of serial electropharmacological testing on combination therapy with mexiletine and procainamide in 20 patients with inducible sustained ventricular tachycardia (VT) refractory to intravenous procainamide. The clinical arrhythmias were cardiac arrest in five patients, sustained VT in 11 patients, and recurrent syncope of presumably arrhythmic origin in four patients. The mean left ventricular ejection fraction (LVEF) was 0.40 +/- 0.12 (mean +/- SD). All patients had inducible sustained VT at baseline and after administration of intravenous procainamide. All 20 patients underwent electropharmacological testing on combination therapy with mexiletine and procainamide. The mean cycle length of inducible sustained VT was 251 +/- 48 ms at baseline, 324 +/- 81 ms on intravenous procainamide (P less than 0.014 vs baseline), and 365 +/- 82 ms on combination therapy (P less than 0.0001 vs baseline, P = NS vs intravenous procainamide). Combination therapy did not suppress VT inducibility, nor did it make VT more difficult to induce in 19 of 20 patients. The remaining one patient had a partial response (runs of nonsustained VT, longest 10 seconds). Furthermore, combination therapy did not significantly prolong the VT cycle length over and above that observed during testing with intravenous procainamide. Therefore, in patients with inducible sustained VT refractory to procainamide during initial electropharmacological testing, mexiletine in combination with procainamide appears to be of little or no value and serial electropharmacological testing on these drugs is of limited usefulness. Early initiation of alternative therapy may be the preferred clinical option. PMID- 1708873 TI - Identification of ventricular tachycardia using intracardiac electrograms: a comparison of unipolar versus bipolar waveform analysis. AB - Implantable antitachycardia devices suffer a high false-positive rate of delivery of therapy because current detection schemes based upon ventricular rate and rate variations are excessively sensitive at the cost of specificity. Several methods have been proposed for providing complementary information derived from morphologic analysis of intraventricular electrograms in order to increase specificity. The majority of these techniques have utilized bipolar electrogram analysis to detect changes in ventricular activation indicative of ventricular tachycardia. Whether bipolar or unipolar intracardiac electrogram analysis might be preferred for discriminating ventricular tachycardia from sinus rhythm has not been determined. In this study, a previously demonstrated method for identification of ventricular tachycardia using intracardiac electrograms, correlation waveform analysis, was used to analyze both unipolar and bipolar signals during sinus rhythm and ventricular tachycardia recorded during electrophysiology studies of 15 patients with inducible sustained monomorphic ventricular tachycardia. Correlation waveform analysis consistently discriminated between all depolarizations during ventricular tachycardia in 14/15 patients (93%) using either electrogram configuration; 13 of the 14 patients were common to both groups. Of these patients, 8/15 (53%) had greater separation between sinus rhythm and ventricular waveforms with bipolar electrogram analysis while 7/15 (47%) had greater separation with unipolar electrogram analysis. We conclude that morphologic analysis of unipolar and bipolar electrograms may be equally effective in distinguishing ventricular tachycardia from sinus rhythm. For individual patients, either a unipolar or bipolar ventricular configuration may be preferable, and should be chosen on a patient-specific basis during electrophysiology study prior to antitachycardia device implantation. PMID- 1708874 TI - A multicenter evaluation of a single-pass lead VDD pacing system. The Multicenter Study Group. AB - Since June 1985 until April 1989, 237 patients (130 males, 107 females, aged 22 to 95 years, mean 71) with symptomatic AV conduction disturbances and competent sinus node, were implanted with a single-pass lead VDD pacing system in 30 centers and followed-up for at least 6 months. The ventricular pacing lead incorporated two atrial ring 3-cm apart electrodes, positioned within the right atrial cavity without contact with the heart wall, in order to detect the atrial activity, which is differentially processed by the pacemaker. At implant, mean atrial electrogram amplitude, derived from a custom pacemaker system analyzer (PSA) with the same input filter of the pacemaker was 1.7 +/- 0.8 mV (n = 93). In all cases, atrial sensitivity at implant was the default value +/- 0.15 mV. The atrial tracking capability of the pacing systems was assessed within the month and every 6 months after implantation by means of clinical evaluation, resting ECG, 24-hour Holter monitoring and the following tests: exercise stress testing, mental stress, isometric exercise, and nifedipine test. These tests evoke an increase of atrial rate in consequence of metabolic needs or as a reflex response. The criterion used to evaluate the correct operation of the system was the percentage of atrial synchronization. This was defined as the ratio between atrial triggered ventricular paced complexes and all ventricular paced complexes. All monitorings showed a ratio higher than or equal to 98% in a percentage of patients not lower than 95%. Mean follow-up was 385 days (range 183-1,370 days).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708875 TI - Defibrillation with low voltage using a left ventricular catheter and four cutaneous patch electrodes in dogs. AB - The purpose of this study was to determine a lower limit of defibrillation thresholds (DFTs) that could be used to evaluate nonthoracotomy lead configurations for implantable defibrillators. A lead configuration that consisted of a left ventricular catheter and four circumferential cutaneous patches was tested because it was hypothesized to create a relatively uniform electric field for defibrillation. In eight anesthetized dogs, three 8F defibrillating catheters with 6 cm platinum clad titanium tips were inserted into the right ventricle (R), right ventricular outflow tract (O), and left ventricle (L). Four cutaneous patch electrodes (4P), each with a surface area of 42 cm2, were placed on the left lateral, right lateral, anterior and posterior thorax. DFTs for ten lead configurations, consisting of different combinations of these electrodes, were evaluated. DFTs were determined by using a modified Purdue technique and applying a single capacitor biphasic shock with both phases 6 ms in duration after 15 sec of electrically induced fibrillation. The L(-)----4P+ configuration produced a lower DFT than R(-)----4P+ (3.2 +/- 1.6 J vs 8.0 +/- 4.2 J, P less than 0.001) with reduced current (2.6 +/- 0.7 A vs 4.1 +/- 1.2 A, P less than 0.001). Lowering the impedance by a mean of 40%, configurations that used four patches produced lower DFTs than those that used a single left lateral patch. The use of an O catheter produced lower DFTs only when used in conjunction with an R catheter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708876 TI - Observations on the initiation of sustained ventricular tachycardia by programmed stimulation. AB - We analyzed the initiation of sustained monomorphic ventricular tachycardia (VT) by programmed ventricular stimulation (PVS) in 50 consecutive patients who had clinical VT or aborted sudden cardiac death with remote myocardial infarction. In 25 of 50 patients, the first induced QRS complex of VT was morphologically identical to the succeeding QRS complexes of VT (type I). In 25 other patients, the first VT beat had a different morphology (type II). Type I had a significantly longer VT cycle length than type II (333 +/- 65 msec and 293 +/- 66 msec, P = 0.036). Type II VT initiation required more aggressive stimulation protocol than type I (type I: type II; number of extrastimulus required for induction 2.5 +/- 0.9 : 3.0 +/- 0.6, P = 0.026; shortest extrastimuli coupling interval 244 +/- 28 msec : 220 +/- 23 msec, P = 0.002). The interval between the last extrastimulus and the onset of the first VT beat was 408 +/- 88 msec in type I and 336 +/- 75 msec in type II (P = 0.004). Furthermore, there was good correlation between the VT cycle length and the interval from last extrastimulus to the onset of nonpaced beat in type I but not in type II.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708877 TI - Effects of stress and beta 1 blockade on the ventricular depolarization gradient of the rate modulating pacemaker. AB - Prism-CLR is a closed loop, rate modulating pacemaker that uses ventricular depolarization gradient (Gd) to continuously adjust heart rate. Heart rate response to a formal mental stress protocol, esmolol (500 mcg/kg bolus, 75-125 mcg/kg/min infusion), and mental stress during esmolol infusion were studied in six patients to investigate if Gd and paced heart rate response are under direct beta-adrenergic control. Paced heart rates increased in response to mental stress in a physiological manner (P less than 0.001). Response to esmolol infusion was paradoxical, with increased paced heart rates during esmolol bolus and infusion (P less than 0.05). There was no significant alteration in either systolic or diastolic blood pressure during mental stress or esmolol infusion (P greater than 0.05). Paradoxical increase in paced heart rates during esmolol administration suggests a primary or secondary effect of esmolol to decrease the ventricular depolarization gradient. This hypothesis was supported in four dog studies in which direct Gd measurements were made during esmolol infusion. Mental stress during esmolol infusion resulted in significantly increased paced heart rates (esmolol effect) with blunted changes in heart rate in response to the mental stress. The results of this study suggest that the physiological rate response during mental stress is attributable to sympathetic autonomic response. PMID- 1708878 TI - The construction of endocardial balloon arrays for cardiac mapping. AB - The advent of multichannel recording systems has enabled clinical mapping to be performed on a beat-by-beat basis using multi-electrode arrays. Surgical ablation of ventricular arrhythmias generally requires endocardial mapping. Clinical usage has indicated that an inflatable balloon array is the most practical design and can obviate the need for ventriculotomy by a transatrial introduction in the deflated state. Successful experience with the left ventricular balloon led to the development of a right ventricular balloon array suitably configured to extend into the outflow tract. Custom moulds are used to create an appropriate balloon from liquid latex. Nylon cloth is cut from a cardboard pattern to fashion a stretchable sock to envelope the balloon. Electrodes are formed by stitching 2 mm silver beads to the balloon sock in a preconfigured pattern. Teflon-coated 31 G multi-strand stainless-steel wires 130 mm in length connect the electrode beads by solder to the multipin connectors for easy hookup to the amplifier inputs. Tygon tubing 0.53 cm in diameter fitted to the balloon allows inflation and pressure monitoring. This basic design has been successfully implemented for the last 6 years. PMID- 1708879 TI - Autologous skeletal muscle, an alternative for cardiac assistance. AB - Original attempts to use skeletal muscle for cardiac assistance were soon abandoned when the problem of muscle fatigue could not be solved. In the last 2 decades, better understanding of muscle physiology and the development of successful protocols of electrical muscle conditioning have given new impetus to researchers around the world to proceed in the effort to identify useful applications of skeletal muscle to support the heart. More than 100 patients around the world have undergone cardiomyoplasty, mostly for cardiac failure. While subjective improvement in symptoms was noticed in the majority of the patients, only recently favorable hemodynamic changes have been documented. The other alternative that has been pursued in the laboratory is the construction of skeletal muscle ventricles that, after conditioning and vascular delay, have been shown to provide significant cardiac support when used for diastolic counterpulsation or for right heart bypass in animal models. PMID- 1708880 TI - Sudden cardiac deaths are related to malignant ventricular tachyarrhythmias. PMID- 1708881 TI - [Epithelial markers of 2 cytokeratin-negative small cell bronchial cancers]. PMID- 1708882 TI - Hormone phage: an enrichment method for variant proteins with altered binding properties. AB - Human growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl-terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13. The gene fusion was cloned into a plasmid containing origins of replication for Escherichia coli and filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage. Transcription of the hGH-gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild-type gene III protein. The hGH-gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH. Moreover, the hGH-gene III phagemid particles were enriched over 5000-fold from non-hGH phage, and 8-fold from a mutant hGH phagemid following a single hGH specific elution step from hGH receptor-coated beads. The hGH phagemid should be useful for isolating new receptor binding mutants of hGH. More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions on filamentous phage. PMID- 1708883 TI - A helix-turn-strand structural motif common in alpha-beta proteins. AB - By exhaustive structural comparisons, we have found that about one-third of the alpha-helix-turn-beta-strand polypeptides in alpha-beta barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the alpha-helix and the beta-strand is somewhat constrained, and second, the beta-strand contains a hydrophobic patch that fits into a hydrophobic pocket on the alpha-helix. The geometry of the turn does not seem to be a major determinant of the alpha-beta unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn-for instance, when the turns between the alpha-helix and the beta-strands are very long. It also occurs much less frequently in flat-sheet alpha-beta proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which alpha-beta barrels are constructed. PMID- 1708884 TI - Efficient immortalization of luminal epithelial cells from human mammary gland by introduction of simian virus 40 large tumor antigen with a recombinant retrovirus. AB - When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line. PMID- 1708885 TI - Interleukin 7 receptor ligation stimulates tyrosine phosphorylation, inositol phospholipid turnover, and clonal proliferation of human B-cell precursors. AB - Functional interleukin 7 (IL-7) receptors are expressed on the surface of multiphenotypic, biphenotypic, and immature B-lineage human lymphoid precursor cells with germ-line immunoglobulin heavy-chain genes but not on more mature B lineage lymphoid cells with rearranged and/or expressed immunoglobulin heavy chain genes. Thus, IL-7 may have an important regulatory role during the earliest stages of human B-cell ontogeny. The engagement of the surface IL-7 receptors on immature B-cell precursor cells with recombinant human IL-7 (rhIL-7) results in enhanced tyrosine phosphorylation of multiple phosphoproteins, stimulates inositol phospholipid turnover and DNA synthesis, and promotes their clonal proliferation. These effects are (i) specific for rhIL-7, since rhIL-3, rhIL-4, rhIL-5, rhIL-6, and recombinant human granulocyte colony-stimulating factor do not elicit similar activities on IL-7 receptor-positive human pro-B cells; and (ii) mediated by IL-7 receptors, since they are not observed in IL-7 receptor negative B-lineage lymphoid cell populations. rhIL-7-induced tyrosine phosphorylation on the 35-, 53-, 55-, 62-, 69-, 76-, 94-, 150-, 170-, and 190-kDa substrates as well as rhIL-7-induced stimulation of inositol phospholipid turnover are abrogated by the tyrosine kinase inhibitor genistein. These results demonstrate that the IL-7 receptor on immature human B-cell precursor populations is intimately linked to a functional tyrosine kinase pathway and tyrosine phosphorylation is an important and perhaps mandatory step in the generation of the IL-7 receptor-linked transmembrane signal. PMID- 1708886 TI - Assembly and deacetylation of N-acetylglucosaminyl-plasmanylinositol in normal and affected paroxysmal nocturnal hemoglobinuria cells. AB - Decay-accelerating factor (DAF) is anchored in cell membranes by a glycosyl plasmanylinositol (GPI) moiety that is transferred to it en bloc in the rough endoplasmic reticulum. To analyze the biochemical reactions involved in preassembly of this structure, a human hematopoietic cell-free system was employed. Incubation of cell extracts with UDP-[3H]GlcNAc and butanol partitioning of reaction mixtures yielded two products similar in TLC mobility to intermediates described in Trypanosoma brucei. Both species were sensitive to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C, indicative of association of [3H]GlcNAc label with a plasmanylinositol-containing acceptor. In contrast to trypanosome intermediates, which contain phosphatidylinositol (1,2 diacylglycerophosphoinositol), however, alkali treatment and phospholipase A2 digestion generated butanol-phase products characteristic of glycosylated plasmanylinositol (1-alkyl-2-acylglycerophosphoinositol). Kinetic and pulse-chase experiments indicated that the slower-migrating species was a product of the faster and that it, but not the faster, was sensitive to both GPI-specific phospholipase D and nitrous acid deamination, consistent with conversion of GlcNAc- to GlcN-plasmanylinositol. Accordingly, acetic anhydride acetylation retransformed the slower species back to the faster. Further incubation with cell extracts converted the slower species into more polar products. Lysates of normal and of affected blood leukocytes from two paroxysmal nocturnal hemoglobinuria (PNH) patients supported assembly of the two intermediates within 1 min. Thus, the initial enzymes mediating human GPI-anchor assembly are GlcNAc plasmanylinositol transferase and GlcNAc-plasmanylinositol deacetylase, their substrates contain plasmanylinositols, and the products of their activities are normal in affected PNH cells. PMID- 1708887 TI - Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation. AB - Magainins are an ionophoric class of vertebrate peptides with antibiotic activity against various microorganisms. Here we show that magainin 2 and synthetic analogues can rapidly and irreversibly lyse hematopoietic tumor and solid tumor target cells with a relative cytotoxic potency that parallels their antibacterial efficacy and at concentrations that are relatively nontoxic to well differentiated cells. The cytotoxicity is prevented by cell depolarization. Magainins represent a natural cytolytic agent in vertebrates and may provide another therapeutic strategy for certain tumors. PMID- 1708888 TI - Constitutive and trophoblast-specific expression of a class of bovine interferon genes. AB - The early conceptus in sheep and cattle secretes a low molecular weight protein called ovine and bovine trophoblast protein 1 (TP-1) that is critical for establishment of pregnancy. TP-1 is a type I interferon (IFN) and is most related to IFN-omega. Here we have determined if TP-1 genes are regulated similarly to other type I IFNs. Single day 18 bovine conceptuses secrete approximately 10(5) units of IFN antiviral activity per hour in culture, amounts approximately 300 times higher than those produced by Sendai virus-induced leukocytes. Although conceptuses express mRNA for IFN-alpha, IFN-omega, and TP-1, TP-1 constitutes greater than 99% of the IFN produced. In contrast, leukocytes produced predominantly IFN-alpha, although TP-1 mRNA is inducible by Sendai virus to very low levels. TP-1 mRNA is detectable by Northern analysis in conceptuses from early pregnancy but is absent in late gestation placenta and several adult tissues. Transfected bovine TP-1 genes are expressed in human choriocarcinoma (JAR) cells in the absence of any specific stimulus, whereas these cells do not secrete antiviral activity constitutively or after transfection with a bovine IFN omega gene. The transfected TP-1 gene is not expressed in nontrophoblast cells (mouse L929 and hamster Chinese hamster ovary), however. The 5' promoter region of the TP-1 gene is sufficient to direct trophoblast-specific expression onto a human growth hormone reporter gene in JAR cells. Deletion of the promoter from 450 to -126 results in a 4- to 5-fold decrease in expression. Together these data demonstrate that the genes for TP-1 are inducible by virus but are expressed preferentially in trophoblast cells and are functionally distinct from IFN-omega genes. PMID- 1708889 TI - Differential signal transduction via T-cell receptor CD3 zeta 2, CD3 zeta-eta, and CD3 eta 2 isoforms. AB - The T-cell antigen receptor (TCR) consists of an antigen-binding heterodimer, termed Ti, which is noncovalently associated with the invariant CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3 zeta and -eta subunits form either homodimeric or heterodimeric structures in turn associated with the other components of the TCR complex. This feature increases the structural complexity of TCRs by creating "isoforms." Both CD3 zeta and -eta are thought to play an important role in signal transduction triggered by antigen/major histocompatibility complex. To compare signaling functions of TCR isoforms, MA5.8, a CD3 zeta-eta- variant of the cytochrome c-specific, I-Ek-restricted T cell hybridoma 2B4.11, was stably transfected with cDNAs encoding CD3 zeta and/or CD3 eta, and resulting clones were characterized. The findings indicate that signals inducing Ca2+ mobilization, phosphatidylinositol turnover, and interleukin 2 production are each transmitted by the above TCR isoforms. In contrast, tyrosine phosphorylation of the CD3 zeta subunit but not the CD3 eta subunit follows TCR stimulation. Given the general importance of tyrosine phosphorylation for receptor signaling, it is likely that this difference between TCR isoforms plays a regulatory role in T-lineage function by qualitatively or quantitatively altering signaling events. PMID- 1708890 TI - Thymic tumorigenesis induced by overexpression of p56lck. AB - The lck gene encodes a membrane-associated protein tyrosine kinase (p56lck) that is believed to participate in lymphocyte-specific signal transduction pathways. To investigate the function of this molecule, transgenic mice were generated carrying the wild-type lck gene or a mutated lck gene encoding a constitutively activated form of p56lck (p56lckF505). Transgene expression in thymocytes was achieved in each case using the lck proximal promoter element. Mice expressing high levels of either p56lckF505 or p56lckY505 reproducibly developed thymic tumors. The sensitivity of thymocytes to p56lck-induced transformation suggests that disturbances in lck expression may contribute to the pathogenesis of some human neoplastic diseases. PMID- 1708892 TI - An intradermal assay for quantification and kinetics studies of tumor angiogenesis in mice. AB - A method is reported for the study of early phases of neovascularization in syngeneic murine tumors and human tumor xenografts in nude mice. Using this method, the effect of irradiation of tumor cells or tumor bed on tumor angiogenesis was studied. Tumor cells were injected intradermally in the abdominal skin flap, which was reopened at 2-day intervals to quantify newly formed blood vessels at the site of tumor cell injection. Both tumor cell injection and blood vessel counting were performed under a dissecting microscope. Using three syngeneic murine tumors and two clones of a human colonic adenocarcinoma, it was observed that new blood vessels started appearing within a few days after tumor cell injection and that this event preceded measurable tumor growth. The number of blood vessels increased exponentially for several days but then their further growth slowed. The extent of angiogenesis depended on the tumor type and the number of tumor cells injected. The exposure of the skin flap to ionizing radiation prior to tumor cell injection reduced neovascularization. We further observed that heavily irradiated tumor cells retained their ability to induce angiogenic response and that lymphoid cells (peritoneal exudate and spleen cells) could also elicit an angiogenic response, although it is weaker than the response elicited by tumor cells. Thus this method is suitable for quantification and kinetics of early phases of tumor angiogenesis in individual mice bearing transplants of syngeneic tumors or human tumor xenografts, and it can be useful for investigating various regulators of tumor angiogenesis. PMID- 1708893 TI - Prostatic urethroplasty with balloon catheter as a nonsurgical alternative for benign prostatic hyperplasia. AB - Prostatic urethroplasty with a balloon catheter is an easy procedure to perform, but certain guidelines must be followed to avoid complications. This procedure will reduce the overall treatment cost of benign prostatic hyperplasia significantly. Preliminary results range from 70% to 85% symptomatic improvement or resolution of the prostatism symptoms. Its recurrence rate is still not known, but a small one is expected. Nevertheless, because of the noninvasiveness, low cost, and simplicity of the procedure, it potentially could be repeated, if necessary. This procedure is one that when it is done in combination with another specialist, in this case a urologist, the patient will benefit by the use of the expertise of both the urologist's knowledge of the disease of the urinary system and the interventional radiologist's skills with catheters and guide wires. PMID- 1708891 TI - Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping. AB - The location of biologically relevant epitopes on recombinant human beta interferon in which Ser-17 replaces Cys-17 (rh[Ser17]IFN-beta) was evaluated by testing the immunoreactivity of antibodies against 159 sequential, overlapping octamer peptides. Three monoclonal antibodies (mAbs) that neutralize rh[Ser17]IFN beta biologic activity, designated A1, A5, and A7, bound to peptides spanning only residues 39-48, whereas nonneutralizing mAb bound less specifically at multiple sites near the amino terminus. The immunoreactivity of peptides spanning residues 40-47 that contained a series of single amino acid substitutions suggested that residues 41-43 (Pro-Glu-Glu) and 46 (Gln) are important for the binding of neutralizing mAbs. The reactivity of mAbs to larger synthetic peptides containing rh[Ser17]IFN-beta sequences from residue 32 through residue 56 was evaluated. All mAbs except A7 reacted with synthetic peptides representing rh[Ser17]IFN-beta residues 32-47, 40-56, and 32-56, but only mAbs A1 and A5 bound to the core peptide composed of residues 40-47. Peptide 32-56 effectively blocked the binding of mAbs A1 and A5 to rh[Ser17]IFN-beta and markedly inhibited their neutralizing activity. Biologic activity of the peptides was undetectable. Rabbit antisera raised against peptides 32-47 and 40-56 recognized rh[Ser17]IFN-beta but did not neutralize its antiviral activity. Thus, structure-function analysis by peptide mapping has permitted the identification of a linear epitope recognized by neutralizing antibody on a biologically active cytokine. We conclude that the region spanning residues 32-56 is of major importance in the expression of the biologic activity of human IFN-beta. PMID- 1708894 TI - Local supravital staining of invertebrate nervous tissue with methylene blue crystals: a reliable and simple preparation technique for wholemounts. PMID- 1708895 TI - Ultrastructural localization of glycosaminoglycans in the human mammary gland. AB - The distribution of glycosaminoglycans (GAGs) in the human breast tissue has been studied at the EM-level by means of the dye Cupromeronic Blue and Scott's critical electrolyte concentration technique. There have been several reproducable localizations: 1. Orthogonally at the main bands of collagen fibrils (probably chondroitin/dermatan sulphate). 2. Large GAGs in areas, almost free of collagen and other fibrils (probably chondroitin sulphate). 3. In the basement membranes of capillaries and glandular epithelium (probably heparan sulphate). 4. Fine GAGs at the surface of the adipocytes (probably heparan sulphate). 5. In the heterogenous granules of mast cells (probably heparin). PMID- 1708896 TI - Evaluation of proliferating cell types in human and mouse mammary gland by a double immunostaining procedure. PMID- 1708897 TI - [Effect of lysophosphatidylserine on the activation of mast cells]. PMID- 1708898 TI - [Phosphoinositide-dependent ion channel and secretion in neuron-like cells]. PMID- 1708899 TI - Evidence that calcitonin gene-related peptide contributes to the capsaicin induced relaxation of guinea pig cerebral arteries. AB - Pretreatment with capsaicin caused a depletion of substance P (SP)-, neurokinin A (NKA)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) from the trigeminal ganglion, dura mater and cerebral arteries. The effect of capsaicin on isolated basilar arteries of guinea pig resulted in a biphasic relaxant response of histamine precontracted vessels. The first phase of the capsaicin-induced relaxation was absent in capsaicin-treated guinea pigs. Furthermore, repeated administration of capsaicin decreased the first but not the second phase of relaxation, supporting the view that a stored agent was released, while the second phase probably was due to a direct effect of capsaicin per se. The biphasic effect was not modified by the SP antagonist Spantide in a concentration that blocks tachykinin response (3.10(-6) M), nor by removal of the endothelium. There was no significant difference in pD2 values (-log concentration eliciting half maximum relaxation) between relaxations induced by SP, NKA, neurokinin B, neuropeptide K or CGRP in capsaicin pretreated as compared to vehicle-treated animals. These results are in support of the assumption that CGRP is involved in the capsaicin-induced relaxation caused by release of vasoactive agents from sensory afferent nerves. PMID- 1708900 TI - Risk assessment of chemicals causing alpha 2u-globulin nephropathy. PMID- 1708901 TI - Evolving concepts of the intrarenal renin-angiotensin system in health and disease: contributions of molecular biology. AB - Previous physiological and biochemical studies suggest the existence of an endogenous renin-angiotensin system (RAS) in the kidney. However, these data cannot exclude the contribution of the circulating RAS. Proof of the local synthesis of RAS components in the kidney has been obtained recently through the use of molecular biological techniques. Using Northern blot analysis, we have demonstrated the intrarenal expression of renin, angiotensinogen, and angiotensin converting enzyme messenger RNAs. Employing in situ hybridization histochemistry, we have localized the intrarenal tissue sites of renin and angiotensinogen messenger RNA synthesis. Renin gene expression was found in cells of the juxtaglomerular apparatus. Angiotensinogen mRNA was primarily produced in the proximal convoluted tubule with lesser amounts in glomerular tufts and vasculature. These findings led us to hypothesize that the proximal tubule is a major site of renal Ang II synthesis and that locally synthesized Ang II might directly modulate tubular function. Both genes are subject to feedback regulation. Our studies showed that Ang II exerted a stimulatory effect on angiotensinogen but a negative feedback effect on renin gene expression. Dietary NaCl restriction stimulated the expression of both genes, although the onset of renin gene activation required more prolonged sodium chloride restriction. Furthermore, our data indicated that the sodium cation, irrespective of the anion, was primarily important in regulating renal angiotensinogen mRNA levels. Our studies also showed altered intrarenal renin or angiotensinogen expressions in pathophysiological states, e.g. in experimental heart failure and the spontaneously hypertensive rat. Taken together, these data support the existence of a intrarenal RAS and suggest its potential roles in the regulation of renal function in health and disease. PMID- 1708902 TI - Influence of intrarenally generated angiotensin II on renal hemodynamics and tubular reabsorption. AB - There is growing recognition that angiotensin II (ANG II) formed intrarenally exerts direct effects on renal hemodynamics and tubular reabsorption. In vivo micropuncture experiments performed in anesthetized rats have shown that peritubular capillary infusion of either ANG II or angiotensin I (ANG I), at rates that do not markedly influence baseline vascular resistance, can increase proximal tubular reabsorption rate and enhance the responsiveness of the tubuloglomerular feedback mechanism. With higher ANG II or ANG I infusion rates, pronounced preglomerular vasoconstriction occurs, resulting in reduced glomerular capillary pressure and single nephron glomerular filtration rate. The effects of peritubular capillary infusion of ANG I on glomerular function have been shown to be inhibited by the ANG II receptor antagonist, saralasin, indicating that the observed effects of ANG I on proximal tubular reabsorption and glomerular function are not due to direct effects of the decapeptide but are mediated by increases in the interstitial ANG II concentrations resulting from intrarenally generated ANG II. Interestingly, neither peritubular capillary infusion nor systemic administration of large doses of the angiotensin-converting enzyme (ACE) inhibitor, enalaprilat, elicited significant blockade of the single nephron hemodynamic responses to peritubular infusion of ANG I. These findings indicate that intrarenal conversion of ANG I to ANG II occurs, at least in part, at a site which is inaccessible to acutely administered ACE inhibitors, or that there is an alternative pathway for the intrarenal conversion of ANG I to ANG II that is not blocked by ACE inhibitors. PMID- 1708903 TI - Macula densa control of renin secretion. AB - The technique of perfusing isolated tubular segments from rabbit kidneys was used to study macula densa (MD) control of renin secretion. Renin secretion was found to be markedly and reversibly increased when perfusate NaCl concentration was reduced. This response of renin secretion occurred within the physiological concentration range, i.e. between 80 and 20 mM. The stimulatory effect of reduced NaCl was seen even when total solute concentration was kept constant. In this preparation a reduction of NaCl concentration was a stronger stimulus than a fall in NaCl load. Loop diuretics were found to stimulate renin secretion. Suppression of renin secretion was seen when most of the Na was replaced by Rb or choline, but not when Cl was replaced with isethionate or acetate. Activation of adenosine 1-receptors inhibited MD-stimulated renin secretion, an effect that was blocked by an A1-antagonist. This agent partially blocked NaCl-induced inhibition of renin release, suggesting a possible role of adenosine in MD control of renin secretion. PMID- 1708904 TI - The role of angiotensin II in renal growth. AB - Angiotensin II (AII) has many of the features of the archetypical growth factors and appears to be a growth regulator in the kidney. AII binds to specific cell surface receptors present on a number of different renal cell types including mesangial, vascular smooth muscle, tubular and interstitial cells, and activates many of the intracellular signalling pathways associated with cell growth. In vitro AII can potentiate the mitogenic effect of other growth factors such as EGF. AII induces hypertrophy of vascular smooth muscle cells but the role of AII in the growth of other renal cell types has not been systematically studied. PMID- 1708905 TI - Role of ionic currents in the physiological response to angiotensin II. AB - Studies using conventional and patch-clamp microelectrode techniques demonstrate that in a number of cell types angiotensin II (AII) causes reversible changes in transmembrane ionic currents, and that these effects can be mimicked by various membrane-associated and cytosolic messengers. AII modulates the current amplitude of ion channels, as well as their activation threshold and their open/closed time probability. Stimulatory and inhibitory effects on ion channel activity are a fundamental feature of the development of AII actions on target organs. PMID- 1708906 TI - Angiotensin II actions in the rabbit proximal tubule. Angiotensin II mediated signaling mechanisms and electrolyte transport in the rabbit proximal tubule. AB - Angiotensin II (AngII) is a potent regulator of electrolyte transport with biphasic effects on salt and HCO3-resorption in proximal tubule epithelia (PCT). In cultured PCT cells, pM to nM AngII activates a GTP-binding protein to inhibit cAMP formation and thus releases inhibition of apical Na/H exchange. Phospholipase A2 is activated by nM to microM AngII releasing arachidonate which is metabolized by a novel P450 epoxygenase to form 5,6-epoxy-eicosatrienoic acid (5,6-EET). 5,6-EET and nM apical AngII cause dihydropyridine-sensitive Ca2+ influx from the extracellular space, inhibition of apical-to-basolateral Na flux, and decrease in epithelial monolayer short circuit current. 5,6-EET also inhibits Na/K-ATPase by 50%. This P450 epoxygenase is physiologically important in the AngII-signaling system because the P450 inhibitor ketoconazole blocks AngII effects while potentiating exogenous 5,6-EET effects. Finally, these AngII mediated signaling systems are polarized in the PCT with pM basolateral AngII inhibiting adenylate cyclase and nM apical AngII activating PLA2 and subsequent generation of 5,6-EET. PMID- 1708907 TI - Angiotensin II and proximal tubule sodium transport. AB - As a target site for angiotensin II (A-II), renal proximal tubule is unique in that it may be equipped with a local A-II generating system and that both basolateral and apical membranes may be accessible for A-II's action. We have recently conducted studies to examine these possibilities. With in vitro cultured proximal tubular cells, we have demonstrated de novo synthesis of angiotensinogen and renin. With isolated renal brush border membrane (BBM), we have confirmed the presence of A-II receptors and found that A-II directly stimulated BBM Na(+)-H+ exchange. In search of the signal transduction mechanism, we have found that A-II also activated BBM phospholipase A2 (PLA) and that BBM contained a pertussis toxin-sensitive guanine nucleotide binding protein (G-protein) which mediates the effects of A-II. Further studies showed that prevention of PLA activation abolished A-II's effect on Na(+)-H+ exchange, and that activation of PLA by mellitin and addition of arachidonic acid similarly enhanced Na(+)-H+ exchange activity, suggesting that PLA activation may mediate the stimulatory effect of A II on Na(+)-H+ exchange. These results thus indicate that a local signal transduction mechanism involving G-protein mediated PLA activation exists in renal BBM which mediates A-II's effect on Na(+)-H+ exchange. Taken together, we propose that, independent of A-II in the circulation, local luminal A-II may serve as an important regulatory system on sodium transport in renal proximal tubule. PMID- 1708908 TI - Hypertonic saline/dextran improves renal function after hemorrhage in conscious swine. AB - This study was performed to determine whether resuscitation with a single bolus of 7.5% NaCl/6% Dextran 70 (hypertonic saline/Dextran, HSD) could restore renal function following hemorrhage. Chronically instrumented, conscious pigs were hemorrhaged 28 ml/kg. This level of hemorrhage reduced mean arterial pressure (MAP) and cardiac output (CO) to nearly half, renal blood flow (RBF) to approximately 25%, and glomerular filtration rate (GFR) and urine flow (V) to less than 10% of their initial values. A single, 4 ml/kg bolus injection of HSD increased MAP and RBF to approximately 80% of baseline values and restored CO and GFR to levels which were significantly different from control values. These improvements were sustained for 2 h with no further treatment. Urine flow transiently increased although not to pre-hemorrhage values, and then subsided. Plasma osmolality increased from 275 to 282 mOsm/kg H2O, and plasma sodium increased from 141 to 149 mEq/l. Recovery following administration of an equal volume of normal saline was significantly less for all variables. Euvolemic animals showed no response in MAP, CO, RBF, or GFR when treated with HSD although V, osmotic and sodium excretion increased. These results demonstrate that resuscitation with HSD following hemorrhage not only restores MAP and CO, but maintains renal function as well. PMID- 1708909 TI - [Enhancement of susceptibility to ouabain in ischemic rat heart]. AB - During 10 mins of reperfusion after 25 mins global ischemia, subtoxic doses of ouabain (50, 100 microM) were used and followed by 20 mins reperfusion with standard buffer. At these doses ouabain had no harmful effects with 29% and 45% increase in developed pressure in aerobic hearts. Intracellular Na+ (Nai), 45Ca2+ uptake and recovery of ventricular function were measured. Nai increased from 15 to 64 mumol/g dw with no increase in 45Ca2+ uptake during ischemia. Upon reperfusion with standard buffer, additional gain in Nai at 2 mins (73 mumol/g dw) was followed by a rapid decline (at 10 mins: 48 mumol/g dw). 45Ca2+ uptake increased from 0.8 to 7.5 mumol/g dw after 30 mins reperfusion with decreased recovery of function (45%) and increased LVEDP (29 mmHg). Reperfusion with ouabain accelerated initial rise in Nai (2 mins: 79 and 83 mumol/g dw) and decline of Nai was retarded (10 mins: 65 and 83 mumol/g dw). Consequently, 45Ca2+ uptake and depression of function were augmented (Ca: 10.0, 11.5 mumol/g dw; function: 27%, 18%; LVEDP: 47, 48 mmHg) even when hearts were switched back to standard buffer. Combination of high K+ (20mM) reversed the effect of ouabain. The results suggested increased susceptibility to ouabain was caused by inhibited outward Na+ transport resulting in enhanced Ca2+ influx through Na+/Ca2+ exchange. PMID- 1708910 TI - [CHOP-bleo versus m-BACOD in the treatment of intermediate and high-grade malignant lymphomas]. AB - Eighty-three patients with malignant lymphoma of intermediate and high histologic subtypes were randomly assigned to receive induction chemotherapy with either of two intensive regimens: CHOP-Bleo (cyclophosphamide, adriamycin, vincristine, prednisone and bleomycin) or m-BACOD (low-dose methotrexate, bleomycin adriamycin, cyclophosphamide, vincristine and dexamethasone). The median follow up study is 5 years. The two therapies were not significantly different in regard to response rates (52 to 59%), duration of complete remission (27 months vs 24 months) and survival, but m-BACOD was significantly more toxic than CHOP-Bleo. The relative small proportion of long-term disease free-survivors with both regimens underscores the need for other therapeutic approaches. PMID- 1708911 TI - [Serum macroamylase in a subject with non-Hodgkin's lymphoma]. AB - Macroamylasemia (MA) is a rare condition characterized by an active macromolecular complex formed by normal amylase with abnormal proteins; to our knowledge, it has not been previously described in Mexico. The size of the macromolecular complex precludes its renal excretion; thus MA is characterized by high levels of amylase in serum with normal amylasuria. We report a 53-year-old male with abdominal pain and hyperamylasemia who was erroneously diagnosed as pancreatitis. Amylase in urine was normal and a protein electrophoresis demonstrated hyperglobulinemia. Several months after the initial work-up, the diagnosis of non-Hodgkin's lymphoma was established. Serum pancreatic amylase was again found elevated with normal urinary amylase. Precipitation of amylase with polyethylene-glycol was of 81% (normal: less than 70%). This established the diagnosis of MA associated to non-Hodgkin's lymphoma. After chemotherapy, the abnormal macroamylasemia and hyperglobulinemia disappeared. PMID- 1708912 TI - [Prostatic adenomas and their treatment in 1990]. PMID- 1708913 TI - [Whipple's disease. A case report and review of the literature]. AB - A 19 year-old man had a clinical history of volvulus, ascites and abdominal pain; later, malabsorption syndrome was recognized. Jejunal biopsies obtained by endoscopic technique show changes consistent in Whipple's disease with minimal intestinal involvement and atrophy. This case report is the first in which volvulus, minimal intestinal involvement and malabsorption syndrome were recognized together. Treatment was successful with trimethoprim-sulfamethoxazole. A literature review with emphasis in recent topics, was made. PMID- 1708914 TI - [Current status of treatment of chronic hepatitis B]. PMID- 1708915 TI - [Follow the guide!]. PMID- 1708916 TI - SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. AB - Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation. PMID- 1708917 TI - Presence of an SH2 domain in the actin-binding protein tensin. AB - The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton. PMID- 1708918 TI - [Synthetic antithyroid drugs]. PMID- 1708919 TI - [76...80...89...pedagogic aids]. PMID- 1708920 TI - Quantification of functional and inactivated alpha 2-macroglobulin in sepsis. AB - Alpha 2-macroglobulin (alpha 2 M) in vitro inhibits numerous proteinases that are generated during inflammatory reactions and therefore, probably plays an important role in diseases such as sepsis. To monitor the state of alpha 2 M in sepsis, we developed novel assays for functional and inactive alpha 2M. Functional alpha 2M in plasma was measured by quantitating the binding of alpha 2M to solid-phase trypsin. Inactive alpha 2M (i alpha 2M) was assessed with a monoclonal antibody, mcAb M1, that specifically reacts with a neodeterminant exposed on i alpha 2M. This mcAb in combination with chromogenic substrates was used to detect alpha 2M-proteinase complexes. Functional alpha 2M was reduced in plasma from 48 patients with clinical sepsis compared to healthy controls (p less than 0.0001). Levels of functional alpha 2M on admission and the lowest levels encountered in 23 patients with shock were lower than in 25 normotensive patients (p = 0.023 and p = 0.009, respectively). Increased levels of i alpha 2M (greater than 30 nM) at least on one occasion were found in only 4 of the 48 patients, being not different in hypotensive compared with normotensive patients, and not in patients who died compared with those who survived. Levels of functional alpha 2M correlated significantly with levels of factor XII and prekallikrein suggesting that decreases in alpha 2M at least in part were due to contact activation. Indeed, in two patients with increased i alpha 2M, complexes between alpha 2M and kallikrein were demonstrated in addition to plasmin- and thrombin alpha 2M complexes. PMID- 1708921 TI - Lymphoscintigraphic evaluation of primary congenital lymphedema. AB - A-four-year-old girl with primary nonfamilial congenital lymphedema was evaluated by lymphoscintigraphy using 99mTc-Dextran. It was demonstrated that there was no accumulation of radioactivity in the left inguinal and femoral lymph nodes and faint accumulation in the contralateral lymph node. It was concluded that lymphoscintigraphy is a safe, reliable, noninvasive technique which is helpful in the evaluation of a patient with lymphedema, especially small children in whom conventional contrast lymphangiography offers some challenges. PMID- 1708923 TI - Prostate-specific antigen in the follow-up of prostatic adenocarcinoma treated with external beam radiation. AB - Prostate-specific antigen (PSA) has been shown to be a more sensitive tumor marker than prostatic acid phosphatase (PAP) in prostatic adenocarcinoma: PSA was positive in 54 of our 117 patients (46%) and PAP was positive in 24 (21%). In order to compare the usefulness of these markers during and after radiotherapy serum samples from 24 patients treated with external beam irradiation were analyzed. PAP was only slightly positive in 1 patient (4%) after radiotherapy. His PSA level was highly elevated and he died of progressive disease. In the other 23 patients the cancer was in local control. However, the serum PSA level remained positive in 5 of these patients indicating vital cancer cells may still have been present. An alternative possibility is that metaplastic prostatic cells which secrete PSA were left after radiotherapy, as has been shown to be the case in prostatic hyperplasia. Before radiotherapy increased PSA levels were measured in 3 patients. In 2 of them the level declined to normal within 6 months after radiotherapy. The PAP levels were normal. It is concluded that PSA (positive in 25% of patients after radiotherapy) might be more sensitive than PAP (positive in 4%) in monitoring the effect of radiotherapy in prostatic cancer patients. PMID- 1708922 TI - VAB-6 chemotherapy causes spurious elevation of alpha-fetoprotein associated with liver dysfunctions. AB - In order to elucidate the mechanism of elevation of alpha-fetoprotein (AFP) which we often observed during VAB-6 chemotherapy, we analyzed sequential changes of AFP, liver enzymes and bilirubin in 10 patients with evaluable disseminated testicular cancer who were treated with VAB-6 chemotherapy. None of the patients had previous liver disease or hepatic involvement. During the early phase of each course of chemotherapy, AFP showed a temporary elevation associated with reversible increase in liver enzymes and bilirubin. These changes returned to normal before the next course of chemotherapy. In each patient, marked tumor regression occurred as a result of VAB-6 chemotherapy. Nine of the 10 patients remain free of disease after treatment. We conclude that during VAB-6 chemotherapy, a temporary elevation of AFP is common, associated with reversible liver dysfunctions, and that this spurious elevation of the tumor marker, most likely caused by a heavy dose of cisplatin, should not be interpreted as related treatment failure. PMID- 1708924 TI - Silver-stained structures in prostatic carcinoma: evaluation of diagnostic and prognostic relevance by automated image analysis. AB - The comparison of the diagnostic and prognostic significance of histology, immunohistochemical parameters (PSA, PSP), and silver-stained nucleolar organizer regions (AgNORs) was estimated in paraffin sections taken of 63 prostatic carcinomas prior to therapy. AgNORs were visualized with a one-step silver staining technique with the appropiate staining time determined by preliminary staining-time series. The mean AgNOR number per cell (n) and the mean AgNOR area per silver-stained dot (A) were determined by means of an automatic image analysis system. Thereby prostatic carcinomas exhibited multiple small AgNORs within their nuclei (n = 4.7, A = 0.09 micron 2), whereas benign prostatic epithelium showed few but large silver-stained particles (n = 1.8, A = 0.27 micron 2; p less than 0.001). This relationship was then calculated as a quotient of AgNOR number and area (NQ = n/A) which provided additional information for the diagnosis of malignancy as well as survival. Univariate survival analysis disclosed a set of four variables predicting death from prostatic cancer; cribriform growth pattern, AgNOR quotient, histological grade, and PSA immunoreactivity. Of these parameters, immunoreactivity of PSA failed to prove its prognostic significance in multivariate survival analysis (Cox model). No relation to prognosis was found for the number as well as the area of AgNORs alone. Therefore, image analysis proved to be a prerequisit for the feasibility of this promising technique by providing objective and reproducible results. PMID- 1708925 TI - Intracavernous pharmacotherapy in psychogenic impotence. AB - Twenty-five men with psychogenic impotence but without serious psychopathology were considered for intracavernosal therapy with papaverine hydrochloride and phentolamine mesylate. A total of 20 proved suitable and began self-injection in conjunction with sex therapy; 8 patients had return of spontaneous erections without pharmacotherapy, although one of them needs to keep the medication in his refrigerator. The other 12 patients are continuing self-injection therapy. Psychotherapy with self-injection may be helpful in the management of psychogenic erectile impotence. PMID- 1708926 TI - Morphological, immunocytochemical and growth characteristics of three human glioblastomas established in vitro. AB - The human glioblastoma-derived cell lines 86HG-39, 87HG-28 and 87HG-31, used for the production of monoclonal antibodies (mAbs) against glioma-associated antigens (GAA), were characterized in terms of morphology, growth behaviour, chromosomes and antigen expression. In the primary tumours, differential expression of glial fibrillary acidic protein, S100 protein, Leu-7 and GAA as defined by mAbs MUC 2 39, MUC 2-63 and MUC 8-22 was demonstrated. Receptors for epidermal growth factor (EGFr) and nerve growth factor (NGFr) were found in many cells in short-term cultures, but the transferrin receptor (Tr) was found in only a few cells of 87HG 28. In permanent cell lines, differentiation antigens and EGFr decreased and Tr increased markedly. NGFr and GAA remained stable. Transplantation tumours of 86HG 39 were partly positive for Tr and GAA. Chromosomal analysis revealed that the 86HG-39 and 87HG-28 cell lines had a hypodiploid or diploid stem line with lines in the hypotetraploid to tetraploid region for 50 in vitro passages. The 87HG-31 cell line had chromosomal patterns in the hypotriploid to triploid region. A gain of chromosomes was seen in the groups C7, C8, C10, D14, F19, F20, G21, G22. The variability of antigens in these tumours and especially during long-term cultivation probably reveals an ability to influence the growth of malignant glioma cells via the respective effector molecules. PMID- 1708928 TI - Histiocytosis X arising in Hodgkin's disease: immunophenotypic characterization with a panel of monoclonal antibodies. AB - This report describes the antigenic profile of the proliferating cells of pulmonary histiocytosis X (HX) in a patient treated with chemotherapy for Hodgkin's lymphoma; the association of pulmonary HX and Hodgkin's disease has rarely been described in the literature. The histopathological diagnosis of HX was confirmed with the aid of monoclonal antibodies (mAbs) to CD4, CD1a, and polyclonal serum anti S-100 protein. The phenotype of HX cells has been analysed using a panel of mAbs against HLA class I A, B, C monomorphic determinants, locus A and B, beta 2-microglobulin, HLA class II distinct monomorphic determinants, DP, DQ, DR, intercellular adhesion molecule-1 (ICAM-1) and vitronectin receptors. Our results indicate that HX cells express HLA class I and II, including locus A, locus B and DP, DQ, DR, like their normal counterpart (represented by Langerhans cells) and detectable levels of ICAM-1 but not vitronectin receptors. We would like to stress the possibility of the association of HX and Hodgkin's lymphoma extending the immunophenotypic profile of HX cells. PMID- 1708927 TI - Glial fibrillary acidic protein immunoreactivity in normal and diseased human breast. AB - Immunostaining for glial fibrillary acidic protein (GFAP) identifies a minor subpopulation of immunoreactive myoepithelial cells in the normal resting human breast. The GFAP-immunoreactive cells also express a panel of myoepithelial cell markers, including cytokeratin 14 (CK 14), vimentin, smooth-muscle-specific actin isoforms, nerve growth factor receptor (NGFR) and common acute lymphoblastic leukaemia antigen (CALLA). The percentage of GFAP-immunoreactive myoepithelial cells is greatly increased in various neoplastic and non-neoplastic diseases of the breast, being highest in adenomyoepitheliomas. Furthermore, in all the instances of fibroadenoma, phyllodes tumour, epitheliosis and gynaecomastia, a variable number of epithelial cells also acquires immunoreactivity for GFAP, vimentin, CK 14, NGFR and, to a lesser extent, for CALLA. Conversely, GFAP immunoreactivity has never been encountered in the malignant cells of the different types of breast carcinoma. These findings suggest that the expression of GFAP might be a (possibly transient) feature of proliferating epithelial and myoepithelial cells in breast diseases other than carcinomas. PMID- 1708929 TI - Altered HIV expression and EBV-induced transformation in coinfected PBLs and PBL subpopulations. AB - Human immunodeficiency virus (HIV) IIIB expression and Epstein-Barr virus (EBV) B95.8-induced transformation were studied during coinfection. Coinfection of peripheral blood lymphocyte (PBL) cultures with HIV and EBV resulted in down regulation of HIV expression. EBV-induced and spontaneous transformation were markedly reduced in PBL cultures exposed to HIV before EBV. On the other hand, transformation was enhanced when PBL cultures were infected with HIV either simultaneous to or after EBV. Reconstitution of EBV-infected B cell cultures with autochthonous T cells demonstrated that HIV-infected T cells had a reduced ability to inhibit EBV-induced transformation. PHA stimulation of HIV-infected T cells eliminated their ability to inhibit EBV-induced transformation. Lymphoblastoid cell lines (LCLs) established from coinfected PBLs expressed B cell markers and were EBV positive, while a large proportion of the LCLs expressed HIV antigens, released reverse transcriptase activity into the supernatant, and produced syncytia when cocultivated with indicator cell line SupT1. HIV provirus could be detected in LCLs established from coinfected cultures by PCR amplification using specific sets of amplimers for gag and env genes of HIV. To more closely examine the role of various cell types in lymphocyte transformation and HIV replication during coinfection, experiments were carried out using subpopulations enriched for either B or T cells. Simultaneous coinfection of purified B cells with EBV and HIV resulted in a marked reduction of HIV expression, whereas EBV-induced transformation was enhanced. In contrast, spontaneous B cell transformation was inhibited by HIV. A proportion of LCLs established from purified B cells coinfected with EBV and HIV expressed HIV antigens, released reverse transcriptase activity, and produced syncytia on SupT1 cells. These results demonstrate that the IIIB strain of HIV and B95.8 strain of EBV can interact during coinfection of B cells to alter the course of virus expression. PMID- 1708930 TI - Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus. AB - Previously, an epitope recognized by a set of neutralizing monoclonal antibodies directed against the S protein of transmissible gastroenteritis has been identified. This neutralization epitope can be simulated by a single peptide combining residues 380 to 387 and 1176 to 1184 of the S protein; this combination peptide (SFFSYGEI-QLAKDKVNE) was more antigenic than it single constituents. Here we describe the immunogenicity of this combination peptide, in comparison with monomer and tandem peptides of both constituents, and with a cyclic peptide consisting of residues 373 to 398. All antisera, raised in rabbits, bound to the peptide used as immunogen. Only sera that recognized the residues 380 to 387 bound to whole virus. Three of the four antisera with the highest binding titers to whole virus also had neutralization activity. Analysis of the fine-specificity of the antisera with PEPSCAN peptides indicated that the spectrum of antibodies induced by the 380 to 387 sequence depended on the presentation of this sequence in a peptide to the immune system. The nonbinding and nonneutralizing anti-(380 to 387)-sera appeared to contain a limited spectrum of antipeptide antibodies. Furthermore, the lack of neutralization of the antiserum against the combination peptide could be explained by the immunodominance in rabbits of the 1176 to 1184 sequence over the 380 to 387 sequence. These findings demonstrate a few fundamental problems of simulating discontinuous epitopes by single synthetic peptides. PMID- 1708931 TI - Identification of a binding site in the hepatitis B virus RNA pregenome for the viral Pol gene product. AB - The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with beta galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome. PMID- 1708932 TI - Evolutionary implications of primate endogenous retroviruses. AB - Endogenous DNA sequences related to retroviruses are probably present in all primates. By using approaches based on the polymerase chain reaction, two separate studies have revealed the evolutionary history of some of these sequences. In the first study, a retrovirus-like reverse transcriptase (RT) sequence homologous to that of Baboon endogenous virus (BaEV) has been identified in both Old World monkeys and African apes, but not in humans or Asian apes. This RT sequence is highly conserved at the amino acid level, but not the nucleotide level, in the baboon, African green monkey, Java macaque, chimpanzee, and gorilla. The patterns of nucleotide substitution indicate functional conservation and suggest that this RT sequence was present in the primate germline before apes and Old World monkeys diverged about 30 million years ago. In the second study, a comparison of endogenous proviral DNAs and their adjacent sequences has been used to analyze the evolutionary history of three previously reported human endogenous retroviruses, HERV-E(4.14), HERV-R(3), and HERV-Ia. It is shown that these retroviruses have also been resident in the primate line since before the ape-Old World monkey divergence. The implications of the presence of functionally conserved RT genes in the germlines of primates, and the potential for using integration sites as tools for analyzing phylogenetic relationships among primates and their retroviruses, are discussed. PMID- 1708934 TI - Two immunodominant regions of the human papillomavirus type 16 E7 protein are masked in the nuclei of monkey COS-1 cells. AB - The human papillomavirus type 16 E7 protein is a 98-amino acid (AA)-long nuclear oncoprotein. We established eight independent mouse hybridoma cell lines producing monoclonal antibodies (MAbs) specific for the E7 protein and characterized the MAbs by competitive binding analysis with the bacterially expressed, nonfusion E7 protein and synthesized oligopeptides. The MAbs were classified into two groups; three and five MAbs recognizing epitopes in probable immunodominant regions AAs 8 to 22 (region I) and AAs 39 to 54 (region II), respectively. These MAbs were capable of immunoprecipitating the E7 protein transiently expressed in monkey COS-1 cells. By immunofluorescence staining of the acetone-fixed E7-producing COS-1 cells, the MAbs for regions I and II detected no E7 protein and only cytoplasmic E7 protein, respectively, whereas a rabbit polyclonal antiserum (anti-lac-E7) detects both nuclear and cytoplasmic E7 proteins. Like anti-lac-E7, the MAbs of the two groups stained both nuclear and cytoplasmic E7 proteins in the COS-1 cells that were denatured with formaldehyde. The results show that two immunodominant regions of the HPV 16 E7 protein are masked in the COS-1 nuclei for binding with the corresponding MAbs. The masking of the intranuclear E7 protein may result from complex formation with cellular proteins (or structures), polymerization, or self-folding. PMID- 1708933 TI - Homotypic antibody responses to fresh clinical isolates of human immunodeficiency virus. AB - Human immunodeficiency virus type 1 (HIV-1) exhibits extensive genomic and antigenic diversity, which is thought to contribute to the failure of the host's immune response to control infection and prevent clinical progression. Part of this failure may be due to utilization by the virus of antigenic variation as a means to escape protective immune responses. Antibody-escape variants of HIV-1 were studied here using fresh clinical isolates and autologous plasmas. HIV-1 was isolated from the plasma of seven people who were all seropositive for at least 2 years, and symptomatic sometime during that period. Isolated viruses were confirmed as HIV-1 by the presence of reverse transcriptase activity in infected culture supernatants, and by positive immunofluorescence using human monoclonal antibody to HIV-1 core protein. Plasma from these people were positive by Western immunoblot (DuPont) for most major HIV-1 (strain IIIB) antigens. These plasmas neutralized three laboratory strains of HIV-1 (i.e., IIIB, RF, and MN) but did not neutralize the homotypic strain in five cases, and had greatly reduced neutralizing titers against the homotypic strain in two cases. Homotypic neutralizing antibodies were absent in autologous plasma obtained 3 months later. When antibody titers were measured by fixed-cell indirect immunofluorescence assays (IFAs), high titers of IgG (1:6400 to 1:25,600) were detected against HIV 1 IIIB, while low titers of only 1:20 to 1:160 were detected against homotypic viral antigens at the time of virus isolation, and remained low 12 and 16 weeks later. No class IgA, IgD, IgE, or IgM antibodies to homotypic viral antigens, as possible IgG-blocking antibodies, were detected by fixed-cell IFAs. Cross reactions with heterologous donor's plasmas were observed in some cases, and in these cases the cross-reactions were unidirectional. Live-cell IFAs detected IgG in patient's plasma to HIV-1 IIIB-infected cells but not to cells infected with homotypic isolates. These results suggest that it is common for neutralization resistant HIV-1 variants to appear during the course of infection, and that all or most antigens of these variants are capable of escaping antibody recognition. PMID- 1708935 TI - Recognition by human A and B influenza viruses of 8- and 7-carbon analogues of sialic acid modified in the polyhydroxyl side chain. AB - To evaluate the recognition by influenza viruses of the C9-C7 polyhydroxylated moiety of sialic acid (SA) receptor determinant a novel assay has been developed based on the assessment of binding by the solid-phase immobilized virus of the enzyme-labeled sialyglycoprotein fetuin treated by periodate or periodate/borohydride to contain an 8-carbon aldehyde, 7-carbon aldehyde, or corresponding hydroxyl analogues of SA. Some features of recognition by human influenza viruses of these SA analogues were type and subtype specific, especially marked differences being found between type A and type B viruses. At the same time a significant diversity was observed among virus strains belonging to the same subtype. The assay described provides a new tool for the differentiation of influenza viruses according to receptor binding properties and for an investigation of molecular interactions in the receptor binding site of the virus. PMID- 1708936 TI - A ruthenium red-toluidine blue procedure for staining epoxy sections in the light microscopy. AB - Ruthenium red (RR) has been widely used as a fixation additive for electron microscopy on the basis of its capacity to retain acid mucopolysaccharide residues of the cell surface coat. Little is known about the properties of this compound as a direct staining agent for epoxy-resin embedded material. In this study, semithin sections of Epon-infiltrated muscle tissue samples have been treated with 1% RR followed by counterstaining with 1% toluidine blue. This 2 step staining procedure has proven to be simple, rapid, and reliable and to give a dramatic improvement in image resolution and contrast. Thus, we believe that histochemical procedures employing RR may find in the future interesting applications for the direct staining of epoxy-resin embedded tissues. PMID- 1708937 TI - [The effect of cyclic nucleotides on the rhythmogenicity of the lymphatic center in the frog Rana temporaria]. AB - Studies have been made of the effect of cAMP on the spontaneous impulse activity of lymphatic pacemaker neurons in the frog. It was shown that inhibition of phosphodiesterases by papaverine and 3-isobutyl-1-methylxanthin resulted in the increase in rhythm of bursting impulse activity of the lymphatic centre turning it into a continuous one. The same effect was produced by dibutyryl-cAMP, and to a lower extent--by cAMP. CGMP blocked rhythmic activity of the centre. Possible role of cyclic nucleotides in rhythmic activity of pacemaker cells in the spinal centre of the lymphatic hearts in the frog is discussed. PMID- 1708938 TI - [TRBC receptor on T lymphocytes of human and macaque and its ligand are different from E2 molecule and ligands for CD2]. AB - CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708939 TI - The school-to-community transition of hearing-impaired persons with developmental disabilities. A review of the empirical literature. AB - This article summarizes empirical studies of the school-to-community transition of hearing-impaired persons with developmental disabilities that have been published since 1975. Forty-three sources were located. In addition to summarizing each source, this article discusses the issues related to data collection and interpretation and makes recommendations for future research, development, and evaluation. Analysis of the sources revealed gaps in both the type and focus of the research. Recommendations include conducting more research on this population and publishing more of the results in deafness-related journals, attending to the different needs of mildly versus severely impaired persons, identifying the adult population in need of services, clarifying the terms used to describe the population, scrutinizing assessment data, and developing curriculum materials on transition specifically for this population. PMID- 1708940 TI - Coronary neovascularization as a specific sign for left atrial appendage thrombus in mitral stenosis. PMID- 1708941 TI - Placental site trophoblastic tumor in a postmenopausal woman. AB - Placental site trophoblastic tumor is a rare neoplasm that arises in the trophoblastic tissue of the placental bed. This case report is unusual because of the patient's advanced age at the time of diagnosis and the favorable response of the disease to chemotherapy. Although the clinical course is benign for most patients with placental site trophoblastic tumor, the malignant variant of the disease is characterized by recurrence, relative insensitivity to radiation and chemotherapy, and death. To the authors' knowledge, the 53-year-old woman reported is the oldest patient with histologically confirmed placental site trophoblastic tumor. Initially, surgery, radiation, and multiagent chemotherapy failed to control vaginal and pulmonary metastatic disease. After administration of four treatment cycles of a "second-line" chemotherapeutic regimen consisting of cyclophosphamide and cisplatin, complete clinical and radiologic remission was achieved. The patient's serum level of human chorionic gonadotropin has remained undetectable, and she has been without measurable evidence of disease for 16 months. PMID- 1708942 TI - Immunoglobulin expression in B-cell lymphoma. Immunohistochemical study of 345 cases. AB - Immunoglobulin expression was studied in a series of 345 cases of B-cell lymphoma by immunohistochemical studies and correlated with the histopathologic classification with the use of the Working Formulation. Immunoglobulin expression was present in 254 cases (74%) of B-cell lymphomas (IgM kappa, 122; IgM lambda, 82; light chain only, 40; mu heavy chain only, 10). Immunoglobulin expression occurred with the greatest frequency in lymphomas of small lymphocytic, small cleaved cell, and small noncleaved cell histologic types (93%, 100%, 100%, respectively) and occurred with the least frequency in lymphomas of large cell (cleaved and noncleaved) and immunoblastic histologic types (59% each). Other lymphomas demonstrated intermediate frequencies of immunoglobulin expression. An excess of cases expressing lambda light chain was noted overall (kappa-lambda ratio of 1.3; expected, 2.0) and was particularly evident for intermediate lymphocytic and follicular mixed histologic types (kappa-lambda ratios of 0.8 and 0.9, respectively). Immunoglobulin expression in B-cell lymphomas varies as a function of cellular differentiation as reflected in the histologic type and grade of the Working Formulation. An excess of cases expressing lambda light chain in specific histologic categories suggests the possibility that lymphocytes bearing the lambda light chain rearrangement may be more susceptible to certain types of lymphomatous transformation. PMID- 1708943 TI - Novel effect of cyclicization of the Arg-Gly-Asp-containing peptide on vitronectin binding to platelets. AB - Vitronectin is one of the glycoproteins that mediate cell adhesion and spreading of a variety of cells through the RGD(S) sequence. Vitronectin is demonstrated to bind to glycoprotein IIb-IIIa and play a role in platelet aggregation. Synthetic peptides containing the RGD(S) sequence can inhibit vitronectin binding to platelets, but the affinity of these peptides is less than 1/100th that of native vitronectin. The present study thus examined the ability of modified RGD(S) containing peptide to inhibit vitronectin binding to thrombin-stimulated platelets. The cyclicization of GRGDSPA peptide was done by the linkage of NH2 terminal glycin and the COOH-terminal alanin. The circular dichroism spectrum of cyclic GRGDSPA peptide only showed negative minimum at approximately 220 nm, but those of other linear peptides such as GRGDSPA and GRGESPA had no effect. This result indicated that only the cyclic GRGDSPA peptide retained some conformational structure to restrict its flexibility. Inhibition experiments revealed that the affinities of the ligands for the receptor decreased in the order of vitronectin = fibronectin = fibrinogen = von Willebrand factor (vWF) greater than cyclic GRGDSPA peptide greater than GRGDSPA peptide. GRGESPA peptide had no effect. These results demonstrate that the conformational structure of the RGD(S) sequence plays the important role for the affinity of vitronectin binding to activated platelets and the increased affinity of the modified peptide is a prerequisite for the potential antithrombotic use. PMID- 1708944 TI - Transitions in CD45 isoform expression indicate continuous differentiation of a monoclonal CD5+ CD11b+ B lineage in Waldenstrom's macroglobulinemia. AB - Waldenstrom's macroglobulinemia (WM) has been hypothesized to be a pleomorphic B cell malignancy with persistent maturation towards plasma cells in all lymphoid tissue. This proposal is based on detection of a heterogeneous density of monoclonal Ig on peripheral blood B-cells in patients with WM. We now present data derived from 2- and 3-color immunofluorescence and flow cytometric analysis that strongly supports this hypothesis. Abnormally high numbers of B lineage cells, defined by expression of CD19, CD20, and CD24, were found among peripheral blood mononuclear cells (PBMC). These B-cells are monoclonal as defined by light chain expression and by the existence of rearranged Ig genes (Southern blot analysis), although they exhibit heterogeneity in the density of surface light chain. Unlike normal PBMC B-cells, the monoclonal B-cells bear CD5 and CD10 (CALLA), express adhesion and adhesion-related molecules (CD11b, CD9), and appear to be actively differentiating during the course of the disease, based on the pattern of CD45 isoform expression. At any given point in time, the population of monoclonal B-cells is heterogeneous in differentiation stage based on transitions in the expression of CD45 isoforms from expression of CD45RA, the high molecular mass isoforms of CD45, to the low molecular mass isoform CD45R0 which appears only on very late stage B-cells and early plasma cells. For one patient, analysis of CD45 isoform expression over 2 years showed that the monoclonal B-cell population as a whole progressed towards terminal differentiation as defined by loss of CD45RA and acquisition of CD45R0. This indicates a continuously differentiating lineage of an unusual B-cell phenotype, and/or malignant transformation of a distinct lineage of B-cells in WM. PMID- 1708945 TI - Fludarabine phosphate in refractory hairy cell leukemia. AB - Our experience suggests a potential role of fludarabine phosphate in the management of hairy cell leukemia. On the basis of this experience, the activity of F-AMP needs to be further evaluated in this disease. PMID- 1708946 TI - Neuronal and glial properties of a murine transgenic retinoblastoma model. AB - Antigenic properties of a murine transgenic model for hereditary retinoblastoma, induced by a chimeric gene coding for Simian virus 40 large T antigen, an oncogene that inactivates the retinoblastoma susceptibility gene product, were studied by immunohistochemistry. All transgenic mice develop bilateral intraocular retinal tumors in the inner nuclear layer with Homer Wright-like rosettes, and one quarter develop midbrain tumors resembling trilateral retinoblastoma. Cell lines TE-1 and TM-1 were established from intraocular and metastatic tumors, respectively. Intraocular tumors reacted with antibodies to neuron-specific enolase and synaptophysin, while vimentin, glial fibrillary acidic, and S-100 proteins were detected only in reactive glia derived from adjacent retina. The midbrain tumors showed weak reactivity to synaptophysin, and they blended with reactive astrocytes positive for glial markers. The tumors were negative for cytokeratins. Finally both derived cell lines expressed synaptophysin and individual neurofilament triplet proteins in immunofluorescence and Western blotting, supporting their essentially neuronal nature. The antigenic profile resembles human retinoblastoma, but differences in morphology and antigen distribution suggest a more close relationship to neurons of the inner nuclear layer than to photoreceptor cells. PMID- 1708947 TI - Lineage-restricted clonality in biphasic solid tumors. AB - Cytogenetic analysis of two pulmonary chondroid hamartomas and nine breast adenofibromas revealed clonal chromosome aberrations in both hamartomas and in four breast tumors. To determine lineage of the cells with chromosome aberrations, a combined immunohistochemical/cytogenetic approach was developed that enabled simultaneous ascertainment of cytogenetic aberrations and immunohistochemical features in individual cells. Immunohistochemical/cytogenetic evaluation of one hamartoma and two adenofibromas demonstrated that neoplastic proliferation, in each case, was confined to the mesenchymal (stromal) component, whereas epithelial cells appeared to be reactive. Cytogenetically abnormal short term cultures of the remaining hamartoma and another of the breast adenofibromas were composed entirely of mesenchymal elements, indicating mesenchymal clonality in those tumors as well. Our findings support redesignation of pulmonary chondroid hamartomas as 'pulmonary chondromas' and suggest that carcinomas developing within fibroadenomas arise from reactive epithelial proliferation. Combined immunohistochemical/cytogenetic analysis might be useful in the development of novel therapeutic approaches that selectively target neoplastic populations within solid tumors. PMID- 1708949 TI - The proximal border of the human respiratory unit, as shown by scanning and transmission electron microscopy and light microscopical cytochemistry. AB - The epithelial cell types present in respiratory (= distal alveolarized) and terminal (= distal nonalveolarized) bronchioles in adult human lung were characterized with scanning and transmission electron microscopy (SEM, TEM) and light microscopic cytochemistry, using specific antibodies against surfactant protein SP-A and mucins, and Alcian blue/periodic acid-Schiff (AB/PAS) staining. In the respiratory bronchiole, two epithelial cell populations share the same basal lamina: one pseudostratified columnar with ciliated, secretory, and basal cells and the other predominantly simple cuboid with some interspersed flat (type I) cells. The columnar secretory cells show the ultrastructure of mucous cells. Light microscopically, they react with mucin antibodies and contain primarily periodate-reactive acid mucins. The mucous cells are the distal secretory cells described by Clara (1937). The cuboid cells are identified as type II (precursor) cells based on ultrastructural criteria for embryonic type II cells (Ten Have Opbroek et al., 1988a, 1990a), including a cuboid cell shape, a large and roundish nucleus, rough and smooth endoplasmic reticulum (ER), osmiophilic multivesicular bodies, and dense bodies. These dense bodies in turn frequently exhibit--like those in embryonic type II cells--internal vesicles or lamellae, variability in size and shape, a specific relationship to ER and a widespread cytoplasmic distribution. Finally, the cuboid cells show a cytoplasmic staining pattern for SP-A. The terminal bronchiole is lined by the columnar cell population. In the respiratory bronchiole, the columnar (bronchial) and cuboid (alveolar) cell populations occupy distinctly different zones (pulmonary artery zone versus remaining wall). The alveolar part of the respiratory bronchiole (called alveolar tubule) defines the proximal border of a true respiratory unit. PMID- 1708948 TI - Loss of antigens associated with the apical endocytotic pathway in proximal tubules from rats with heymann nephritis. AB - In addition to the glomerular lesions associated with Heymann nephritis, a rat model of human membranous nephritis, proximal tubule damage, and a perturbation of proximal tubule function also have been reported to occur in this disease. The aim of the present study was to examine in more detail the nature of the apical plasma membrane damage in proximal tubules using specific antibodies directed against clathrin, gp330, and a proton-pumping adenosine triphosphatase, all of which are components of the apical endocytotic apparatus of these epithelial cells. Immunocytochemical studies revealed a marked reduction in staining for all three antigens in proximal tubules from rats with active Heymann nephritis. Furthermore endocytotic uptake of intravenously injected FITC-dextran was considerably lower in diseased animals than in normal rats. Gp330 and rat IgG were identified as components of the luminal debris that accumulated during the course of Heymann nephritis. These results show that perturbation of proximal tubule endocytosis occurs in Heymann nephritis together with a loss of three apical antigens that are normally localized on membrane domains associated with the apical endocytotic pathway in these cells. The results also suggest that antibody-antigen complexes may be shed from the plasma membrane in both the glomerulus and the proximal tubule in this disease. PMID- 1708950 TI - Follow-up bronchoalveolar lavage in AIDS patients with Pneumocystis carinii pneumonia. Pneumocystis carinii burden predicts early relapse. AB - We performed an analysis of the value of repeat bronchoalveolar lavage (BAL) at 21 days to identify patients at risk for early relapse with Pneumocystis carinii pneumonia. Patients with P. carinii pneumonia and the acquired immunodeficiency syndrome (AIDS) were asked to participate in this study. All patients had P. carinii identified on methenamine silver stain of BAL fluid. BAL fluid was also stained with a modified Wright-Giemsa technique. The Wright-Giemsa stain was done to determine the cell differential count, and the number of P. carinii clusters associated with 500 nucleated cells was used as an estimate of P. carinii burden in the BAL. Initial and follow-up lavage was performed in 56 patients. Patients were classified based on their clinical response to anti-P. carinii therapy at 21 days. Nonresponders were patients with persistent or worsening symptoms. Responders were patients who improved and had therapy discontinued. Responders were further classified as responders with relapse if P. carinii pneumonia recurred within 6 months of the initial episode or responders without relapse if they remained disease free during the follow-up period. Responders without relapse reduced P. carinii cluster counts more than 50% in 24 of 25 cases. In responders with relapse P. carinii cluster counts were unchanged. The responders as a group had a significant decrease in the percentage of neutrophils in the BAL, with only 2 of 32 still having increased neutrophils in the follow-up lavage compared to 17 of 24 nonresponders (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708951 TI - Lymphokines and interferons: from molecular biology to clinical trials. PMID- 1708952 TI - Spectrum of biological activity of interferons. AB - The interferons comprise a group of proteins first identified by their ability to protect cells against virus infections but also capable of influencing cellular physiology. They are synthesized and secreted by a variety of cell types in response to various inducers. Their effects include antiviral action, inhibition of cell proliferation, modulation of cell differentiation and activation of various cell types in immune system. This review aims to summarize the current state of biology of interferon action with special emphasis on those aspects related to the use of these molecules in antitumoral therapy. The antitumor effects of IFNs results from pleiotropic IFN activity exerted either directly on tumor cells (i.e. antiproliferative effects, effects on oncogene expression, on cell differentiation and enhanced expression of cell surface antigens), or via indirect effects (i.e. activation of effector mechanisms of the host as modulation of the expression of the major histocompatibility antigens, effects on macrophages, NK, T and B cells). PMID- 1708953 TI - Role of biological response modifiers in immunochemotherapy of solid tumors and retroviral-induced leukemia. AB - Modulation of the immune response by the use of biological response modifiers (BRM) is aimed at amplifying the host resistance against cancer. Studies on inhibition of tumor growth on an in vitro model, in which human breast carcinoma (HBL-100) and human lung carcinoma (H125) cells were used as target tumor cells, confirmed that interferons (IFNs) alpha and beta can amplify the antineoplastic effects of immunochemotherapy by enhancing the cytotoxic activity of effector cells and by antagonizing the immunodepressive effects of radiation or anticancer drugs. Moreover, data obtained from a pilot clinical trial, designed to test the effect of low concentrations of beta-IFN on natural cell-mediated cytotoxicity, pointed out a good correlation between the in vitro and in vivo responsiveness to beta-IFN in cancer patients. The immunomodulating and antiproliferative effects of BRM were also evaluated in a model of viral leukemogenesis in vitro, after infection of cord blood derived mononuclear cells (CB-MNC) with the human leukemic retrovirus HTLV-I. Alpha-and beta-IFN were previously shown to regulate differentially the antiviral competence of recipient CB-MNC, by interfering with viral replication and delaying the emergence of the transformed clone(s). One of the mechanisms of IFN action that contributes to control HTLV-I infection in vitro can be ascribed to their property of partially counteracting the depression of cell-mediated cytotoxicity that follows exposure to HTLV-I. In the light of data previously and herein described, it seems that alpha- and beta-IFN can be considered potential candidates to define combined therapy with antiviral drugs, to control the early stages of retrovirus-associated disease in human pathology. PMID- 1708954 TI - Keratins as molecular markers of epithelial differentiation: differential expression in crypt epithelium of human palatine tonsils. AB - The expression of keratins in the stratified squamous nonkeratinizing epithelium lining the surface and the crypts of human palatine tonsils was analyzed by high resolution gel electrophoresis, immunoblotting, and immunohistochemical techniques. In contrast to the superficial epithelium, which showed a fairly constant keratin composition consisting of the neutral-to-basic keratins K4, K5, K6, and K8 and the acidic keratins K13, K14, K16, and K19, the keratin profiles of tonsillar crypt epithelial cells were found to be more variable, particularly with respect to the expression levels of K4 and K13. These were identical to those of surface epithelium, reduced, or abolished. Since K4 and K13 characterize the mature stage of differentiation in squamous nonkeratinizing epithelia, their decrease is indicative of an incomplete epithelial differentiation. Immunohistochemical analyses confirmed this hypothesis and allowed us, furthermore, to correlate the expression of K13 with the morphologic alterations of tonsillar crypt epithelium in the course of reticulation. PMID- 1708955 TI - Ultrastructural and cytochemical evidence for single impulse initiation zones in vestibular macular nerve fibers of rat. AB - Cupric ion-ferricyanide labeling methods and related ferrocyanide-stained tissues were used to locate and characterize, at the ultrastructural level, presumptive impulse initiation zones in the three types of vestibular macular nerve fibers. Large-diameter, M-type vestibular nerve fibers terminate in a calyx at the heminode, and labeling is coextensive with the base of the calyx. Intermediate, M/U-type nerve fibers have short, unmyelinated preterminal segments that sometimes bifurcate intramacularly, and small-diameter, U-type nerve fibers have long, unmyelinated preterminal axons and up to three branches. Preterminals of these nerve fibers display ultrastructural heterogeneity that is correlated with labeling patterns for sodium channels and/or associated polyanionic sites. They have a nodelike ultrastructure and label heavily from near the heminode to the base of the macula. Their intramacular branches, less organized ultrastructurally, label only slightly. Results indicate that vestibular nerve fibers have one impulse initiation zone, located near the heminode, that varies in length according to nerve fiber type. Structural heterogeneity may favor impulse conduction in the central direction, and length of the impulse initiation zone could influence nerve discharge patterns. PMID- 1708956 TI - Endoscopic laser therapy for obstructing tracheobronchial lesions. AB - The Lahey Clinic experience using laser bronchoscopy for relief of obstructive tracheobronchial lesions during a 7-year period from 1982 to 1989 involves 269 patients treated with 400 procedures. The carbon dioxide (CO2) laser was used for tracheal stenosis and granulation tissue. The neodymium:yttrium-aluminum-garnet (Nd:YAG) laser was used for all obstructing endobronchial neoplasms. Indications for therapy included severe dyspnea, hemoptysis, and postobstructive pneumonitis. All patients had relatively central lesions. A rigid bronchoscope was used to treat 88% of patients, and 12% of patients were treated with a flexible bronchoscope. One death occurred during the intraoperative period. Eleven deaths occurred within 1 week of therapy and were related to the presence of extensive malignant lesions or to coronary artery disease. Our experience indicates that bronchoscopic application of the CO2 or Nd:YAG laser affords effective palliation for patients with obstructive tracheobronchial lesions. The Nd:YAG laser is recommended for patients with bulky vascular endobronchial neoplasms, and the CO2 laser is best reserved for patients with benign tracheal stenosis and granulation tissue. PMID- 1708957 TI - [Post-extrasystolic long QT: evaluation and significance]. AB - The dynamic behaviour of the QT interval was studied in 13 patients with a prolongation of greater than 80 ms of the QT interval of the QRS complex following a post-extrasystolic pause. Normal repolarisation under basal conditions (QTc 410 +/- 30 ms) was significantly (p less than 0.001) prolonged after pauses (QTc 512 +/- 90 ms). The study protocol included the measurement of QT during a Holter recording, an exercise test, Valsalva's manoeuvre and the isoprenalin test. Hydroquidine 600-900 mg/d was given to evaluate its action on ventricular repolarisation. The longest sinus cycles seen on the night Holter tracing (mean: 1,395 +/- 666 ms) were accompanied by normal repolarisation (QTc 380 +/- 30 ms). In contrast, exercise or Valsalva's manoeuvre caused a prolongation of QTc (QTc greater than 460 ms) in 11 patients out of 13: exercise QTc 471 +/- 54 ms; Valsalva QTc 480 +/- 50 ms. No arrhythmia occurred during these dynamic manoeuvres nor during the isoprenalin test. Hydroquinidine (mean: 729 mg) induced a QTc of greater than 500 ms in 6 patients out of 13 (46%). A triplet suggestive of a "torsade de pointes" was seen in one patient only. In conclusion, patients with a post-extrasystolic T wave abnormality under basal conditions were shown to fail to appropriately adapt their QT interval during autonomic stimulation manoeuvres and the prescription of hydroquinidine at the usual dose induced a mean QTc of greater than 500 ms in 46% of them. PMID- 1708958 TI - An epidemic of dengue haemorrhagic fever and dengue shock syndrome in Delhi: a clinical study. AB - Twenty-four cases of dengue haemorrhagic fever/dengue shock syndrome were studied in Delhi in the months of September and October, 1988. The majority of these cases were boys aged 6-10 years. Classical symptoms of dengue (fever, headache, aesthesia, myalgia) occurred in all the patients. Digestive symptoms (nausea, vomiting, anorexia, abdominal pain and hepatomegaly) were also common. Haemorrhagic manifestations were present in 41.7% of the cases. Of these, 90% had gastrointestinal haemorrhages. Shock occurred in 17 cases (70.8%). Thrombocytopenia and prolongation of coagulation profile were found in 62.5% of cases. Three patients (12.5%) who presented with encephalopathy died. The other 21 patients recovered after an average period of 2-8 days. PMID- 1708959 TI - Drug-induced haemolysis and renal failure in children with glucose-6-phosphate dehydrogenase deficiency in Afghanistan. AB - Twenty children (18 boys and 2 girls) with a proven or presumptive diagnosis of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency developed intravascular haemolysis following administration of antimalarials in 9, chloroquine and chloramphenicol in 1, chloroquine, chloramphenicol and aspirin in 1, chloramphenicol and aspirin in 3, and aspirin alone in 4. Eleven of these children developed acute renal insufficiency. All were managed with supportive care, including blood transfusion, forced diuresis and peritoneal dialysis wherever indicated. Only 16 children recovered completely. The occurrence of G-6 PD deficiency is being reported for the first time in Afghanistan. PMID- 1708960 TI - Physicochemical characteristics and flora of diarrhoeal and recovery faeces in children with acute gastro-enteritis in Kenya. AB - Physiochemical characteristics and flora of diarrhoeal and recovery faeces were investigated in 14 Kenyan children with acute gastro-enteritis. Causative micro organisms were Shigella, Campylobacter, enterotoxigenic Escherichia coli, rotavirus and unknown in 6, 2, 1, 2 and 3 patients, respectively. The mean values of the pH of the diarrhoeal specimens were significantly higher than those of the recovery specimens. Large amounts of acetic acid and many other kinds of fatty acids were detected in the recovery specimens, but small amounts and few kinds of fatty acids were detected in the diarrhoeal specimens. Bacterial counts of anaerobic organisms, such as Bacteroides, Bifidobacterium, Lactobacillus and Eubacterium, were lower in the diarrhoeal specimens than in the recovery ones. The normal anaerobic intestinal flora is remarkably disturbed in patients with acute gastro-enteritis. This may result in changes in fatty acid contents and in the pH of diarrhoeal faeces. PMID- 1708961 TI - Effect of early suckling on term neonates' core body temperature. AB - The purpose of this study was to determine whether the practice of early suckling, through an effect on maternal behaviour, would improve neonatal temperature control. One hundred and sixty mothers having daytime spontaneous deliveries of healthy babies at term were randomized into two groups. The treatment group were encouraged to put the baby to the breast immediately after delivery. In the control group, the baby was placed in a cot immediately after birth and breastfeeding occurred some time later at a time of the mother's choice. Observations of the mother's behaviour towards her baby and the baby's core body temperature were recorded at 2 and 4 hours after birth and at 8 a.m. the next day. The early suckling group mothers were observed breastfeeding their babies more often than those of the control group. Significantly more of the control babies had temperatures below 36.5 degrees C at 8 a.m. the next day. Women of either group who were breastfeeding immediately prior to temperature recording were significantly less likely to have a baby with a low body temperature. It is concluded that a policy of early suckling, when compared with one of delayed contact, appears to reduce the incidence of low body temperature in the neonate. PMID- 1708962 TI - Term infant asphyxia in Kuwait. AB - In the developing nation of Kuwait, we undertook a case-control study of 43 consecutively born, asphyxiated, term infants. The asphyxia incidence of 9.4/1000 was only slightly higher than that in more developed countries. Severe morbidity occurred in 1.1/1000, and mortality in 1.1/1000. We found significant associations between asphyxia and primiparity, maternal hypertension, consanguinity, increased length of labour, and instrumental deliveries. Maternal age, socio-economic class, maternal illnesses other than diabetes, and breech delivery did not seem to play a role. The fact that the chosen method of delivery failed for a number of the asphyxiated patients, necessitating emergency Caesarian section, suggests that obstetric factors may need closer analysis. PMID- 1708963 TI - Congenital miliary tuberculosis. AB - A case of a premature baby who had the classical problems associated with congenital tuberculosis and presented a difficult diagnostic problem is described. Diagnosis was ultimately confirmed by liver biopsy. Treatment was initially with isoniazid alone, followed 2 weeks later by isoniazid and rifampicin. Isoniazid was replaced by ethambutol when an isoniazid-resistant culture was obtained. The baby responded extremely well to therapy and, at 2-year follow-up, was growing satisfactorily without any sequelae. To my knowledge, this is the first case of congenital tuberculosis with a miliary spread to be seen, treated and followed up in the developing world, where tuberculosis is prevalent. PMID- 1708964 TI - Congenital goitre and juvenile hypothyroidism in a Nigerian child. AB - A 7-year-old girl from an area where endemic goitre is not known presented with a collar-like goitre which on palpation was diffusely nodular. Investigations showed high TSH and 'low' thyroxine serum levels. She was neither mentally retarded nor spastic. The goitre was reduced in size with supplementary l thyroxine sodium but on withdrawal of therapy the gland size increased. An inborn error of thyroxine synthesis is the most likely cause of the condition. In our environment, the first line of treatment should not be surgery but rather a thoughtful approach to the causes of hypothyroidism. PMID- 1708965 TI - Cord and maternal glycosylated haemoglobin levels: a study in Sudanese pregnant diabetic women. AB - Blood glucose and glycosylated haemoglobin levels measured at delivery in 27 normal and 19 diabetic women were compared with those of the corresponding fetuses and their birthweights. In both groups, neonatal blood glucose levels were significantly higher (p much less than 0.001) than the maternal levels despite significantly lower (p much less than 0.001) glycosylated haemoglobin values in the neonates. In the control group, there was a significant correlation of maternal glycosylated haemoglobin with both cord glycosylated haemoglobin and birthweight ratio (p less than 0.01). The same was not obtained in the diabetic group, suggesting a dissociation in glucose homeostasis between the diabetic pregnant mother and her fetus. PMID- 1708966 TI - Familial hypophosphataemic rickets: experience with 24 children from Kuwait. AB - Between 1982 and 1988, familial hypophosphataemic rickets (FHR) was diagnosed in 24 children, in nine during screening of the families of index patients. The average annual incidence was 0.2/1000 live births. There were 16 boys and 8 girls in 10 families, of which nine had more than one affected child. Their ages at the onset of the disease ranged between 10 months and 14 years (mean 6.9 yrs). Growth retardation and bowing of the legs were the most prominent features, observed in all index patients and in four of the patients diagnosed by screening. Treatment with 1 alpha-hydroxyvitamin D3 and phosphates was associated with acceleration of growth in all children, healing of rickets in 21, and normalization of the serum phosphate in 22. Two children with late diagnosis are now older than 16 years with a final height below the 3rd centile. Three more pubertal children are also shorter than the 3rd centile. In areas where nutritional rickets is common, FHR is likely to be missed and the treatment delayed with grave consequences; in particular, growth retardation and bone deformity. PMID- 1708967 TI - Bilateral traumatic rupture of the diaphragm. AB - A 3.5-year-old girl sustained compression injury leading to isolated bilateral diaphragmatic rupture with extensive herniation of abdominal viscera into the pleuropericardial cavities. Reduction of the hernia and repair of the defects were readily accomplished through a thoraco-abdominal approach with excellent outcome. PMID- 1708968 TI - Conservation of the ruptured spleen in children: an African series. AB - In the Zaria region of northern Nigeria, which is endemic for malaria and schistosomiasis, laparotomy was performed for traumatic rupture of the spleen in 27 children, 10 of whom had splenomegaly. Eleven of the children were pedestrians knocked down by motor vehicles while crossing the road and six were boys who fell off mango or guava trees. Using suture techniques, 17 ruptured spleens were repaired and one was partially resected: eight of them were enlarged. Total splenectomy was performed in nine cases. Five of the children in the splenic conservation group died within 4 days of surgery owing to severe associated injuries. It is concluded that splenorrhaphy is quite feasible in both normal sized and enlarged spleens and should be encouraged in similar tropical countries where splenomegaly is a common response to endemic malaria and schistosoma. PMID- 1708969 TI - A double-blind placebo-controlled trial of prednisone in active rheumatic carditis. AB - In view of the controversy surrounding the use of corticosteroids in the management of active rheumatic carditis, a prospective double-blind controlled clinical trial comparing prednisone (2 mg/kg/24 h in three divided doses) versus placebo was carried out in 35 children with this disease, using strict clinical criteria to define carditis and excluding cases where the diagnosis was doubtful. The duration of the study, which included long-term follow-up, was 7 years. The results failed to show that prednisone at this dosage and frequency was of any benefit either in the short-term clinical response or in the long-term follow-up with regard to later requirement of valvular surgery (using the 5% level of significance). Other findings were that one-third of cases improved with time, irrespective of treatment, most of them having mild to moderate carditis initially. PMID- 1708970 TI - Clinical presentation and management of acute gastro-enteritis in in-patient children at King Khalid University Hospital, Riyadh, Saudi Arabia. AB - In a retrospective survey, case notes of all children with acute gastro-enteritis (AGE) admitted to our hospital between 1984 and 1988 were reviewed. The total number of cases was 300. The mean age was 14 months (range 1-60 mths): 67% of cases were boys and 33% girls. Eleven per cent were exclusively breastfed. The clinical presentation was diarrhoea and vomiting in 81%, diarrhoea alone in 15%, and vomiting primarily in 4%. All children had good nutritional status, i.e. both their height and weight were between the 5th and 90th percentile for their age and none showed signs of marasmus or kwashiorkor. Forty-six per cent of the children had AGE without dehydration. Mild, moderate and severe dehydration was present in 41%, 10% and 3% of cases, respectively. Isotonic, hypotonic and hypernatraemic dehydration was present in 95%, 3% and 2% of cases of dehydration, respectively. Sixty-five per cent of cases were given intravenous (IV) fluids. The mean duration of IV administration was 1 day, with a range of 1-7 days. Twenty-two per cent of the children were given oral rehydration solution (ORS) initially, and 13% were given IV plus ORS. None of the children died of gastro enteritis. It is concluded that there was excessive use of IV fluids, and that there is an urgent need to encourage the use of ORS. PMID- 1708971 TI - Necrotizing enterocolitis: an overlooked life-threatening complication of acute diarrhoeal illness in infants and children. AB - A 7-month-old girl developed necrotizing enterocolitis with extensive pneumatosis intestinalis following an acute diarrhoeal illness. This was preceded by the use of an anti-motility drug and repeated attacks of paralytic ileus. It is noteworthy that all the published cases of necrotizing enterocolitis following acute diarrhoeal illness in infants and children are from tropical countries. This might be related to the use of anti-diarrhoeal drugs and associated malnutrition resulting in hypokalaemia and hypoalbuminaemia. PMID- 1708972 TI - The effects on weight and attendance of a supplementary feeding programme operating in two under-5 clinics in Lesotho. AB - The routinely collected weight data of 2202 pre-school children who visited two under-5 clinics in Lesotho between 1978 and 1983 were used to calculate growth curves and to assess the effects of supplementary feeding. Up to the age of 5 months growth curves were above the NCHS reference. Growth started to decrease at the age of 3 months and stabilized at 11-13 months at the 10th percentile of the reference. The decrease of growth might have been due to environmental factors such as under-nutrition and disease. If this was the case, supplementary feeding might have had a positive impact on weight, but such an effect was not observed in this study. Supplementary feeding had, however, a positive effect on attendance rates and hence contributed to higher coverage of other under-5 clinic activities, such as immunization, health and nutrition education, and growth monitoring. Whether this positive effect warrants its extra cost remains open to question. PMID- 1708973 TI - Plasma lipids and lipoproteins in Nigerian children with sickle cell anemia. AB - Plasma lipids and lipoproteins were estimated in 67 Nigerian children (mean age 10.19 years) with sickle cell anaemia (HbS) and 67 age- and sex-matched non anaemic controls with normal haemoglobin (HbA). Plasma cholesterol and HDL cholesterol levels were significantly lower while the plasma triglyceride level was significantly higher in the subjects with sickle cell anaemia. LDL cholesterol levels were higher in sickle cell anaemia patients, but were not of statistical significance. These results do not support suggestions that sickle cell anaemia patients are at low risk of developing coronary artery disease. PMID- 1708974 TI - Anthropometric determinants of resting blood pressure and heart rate of Nigerian school children. AB - Blood pressure, heart rate and anthropometric parameters were measured in 807 Nigerian school-age children. There was no significant difference between the blood pressure and heart rate of boys and girls after adjusting for differences in age and anthropometric parameters. The stepwise regression analysis revealed that the strong determinants of blood pressure levels were weight, Quetelet index and triceps skinfold thickness. Based on our findings, we recommend that body weight norms rather than age should be used in evaluating abnormal blood pressure levels in Nigerian children. PMID- 1708975 TI - In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum. AB - Cryptosporidium parvum is a protozoan parasite that causes mild to severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recent studies have described small, naturally occurring antimicrobial lytic peptides with antiprotozoal activities. In the present study, the anticryptosporidial activities of three synthetic lytic peptides were determined in an in vitro sporozoite susceptibility assay. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate and propidium iodide. Sporozoite viability was reduced by 93.5% following a 60-min exposure to 10 microM Hecate-1 at 37 degrees C. Shiva-10 reduced sporozoite viability by approximately 74.0% after a 60-min exposure at 100 microM and 37 degrees C. The cecropin-b analog SB-37 reduced sporozoite viability by 6.0% following a 60-min exposure at 100 microM and 37 degrees C. A control peptide showed no anticryptosporidial activity. PMID- 1708976 TI - BI-RG-587 is active against zidovudine-resistant human immunodeficiency virus type 1 and synergistic with zidovudine. AB - A series of dipyridodiazepinones have been shown to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. The lead compound, BI-RG-587, had a 50% inhibitory concentration of 84 nM against HIV-1 reverse transcriptase activity. This compound reduced plaque formation of HIV-1 in HeLa cells expressing the CD4 receptor by 50% at 15 nM. BI-RG-587 at comparable concentrations inhibited the production of p24 antigen following the acute infection of CEM T-lymphoblastoid cells or primary human monocyte-derived macrophages with HIV-1. No inhibitory effects against HIV-2 or against three picornaviruses were detected. Zidovudine (3'-azido-3'-deoxythymidine [AZT]) susceptible and AZT-resistant isolates of HIV-1 were equally susceptible to BI-RG 587. AZT and BI-RG-587 exhibited synergistic inhibition of HIV-1BRU at all concentrations examined. PMID- 1708977 TI - Anti-human immunodeficiency virus synergism by zidovudine (3'-azidothymidine) and didanosine (dideoxyinosine) contrasts with their additive inhibition of normal human marrow progenitor cells. AB - The anti-human immunodeficiency virus (HIV) activity and hemopoietic toxicity of zidovudine (AZT) and didanosine (dideoxyinosine;ddI), alone and in combination, were assessed in a variety of cell types. AZT was more potent than ddI as an inhibitor of HIV in vitro. Synergistic inhibition of HIV by the combination of these agents was observed in MT4 cells, peripheral blood lymphocytes, and macrophages. Toxicity assessment in vitro by using progenitor (erythroid and granulocyte-macrophage) colony-forming assays with normal human bone marrow showed ddI to be less toxic than AZT. Addition of inhibitory concentrations of ddI to AZT resulted in additive inhibition of progenitor CFUs. These in vitro findings suggest that combinations of ddI and AZT at appropriately modified doses may provide an enhanced degree of selectivity in anti-HIV chemotherapy. PMID- 1708978 TI - Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine using artificial cells containing an immobilized multienzyme system and dextran-NAD. L-lactic dehydrogenase for coenzyme recycling. AB - A multienzyme system consisting of leucine dehydrogenase (EC 1.4.1.9), L-lactic dehydrogenase (EC 1.1.1.27), urease (EC 3.5.1.5), and dextran-NAD+ was microencapsulated within artificial cells. This system could convert ammonia and urea into essential amino acids, L-leucine, L-valine, and L-isoleucine. L-lactate acted as a cosubstrate for the regeneration of dextran-NADH. Greater concentrations of L-lactate favored the higher conversion ratios. The effects of ammonium salts and urea on reaction rate were also studied. The relative reaction rates in ammonium salts solutions were 44.6-78.8% of those in urea solutions. More than 90% of the original activity was retained when artificial cells were kept at 4 degrees C for 6 wk. PMID- 1708979 TI - [Low-stage prostatic adenocarcinoma and atypical lesions of the prostate]. AB - Prostate lesions whose morphological features raise doubt to the pathologist may be classed generically as being atypical hyperplasia or pseudoneoplastic lesions and are referred to in the literature using different terms. However, these have histopathological features such as dysplasia that appear to be frequently associated with small, low stage prostatic adenocarcinoma. Small atypical lesions found in the biopsy specimens of patients with stage A1 adenocarcinoma warrant a more radical therapeutic approach particularly in the younger patients. PMID- 1708980 TI - [Epidermoid carcinoma of the urinary tract. Immunohistochemical study]. AB - Six patients diagnosed as having urothelial carcinoma with varying degrees of squamous features are described. Four had pure squamous carcinoma. Two had transitional cell carcinoma (1 with squamous areas and the other had carcinoma in situ). The histological analyses of specimens were performed using immunohistochemical techniques to detect epithelial markers with low molecular weight monoclonal antibodies to cytokeratin. The tests were positive for pure squamous carcinoma and transitional cell carcinoma with squamous areas, and were negative for all areas of transitional cell carcinoma. We underscore the importance of the squamous component in the prognosis of urothelial carcinomas as well as the usefulness of this type of markers, particularly low molecular weight antibodies to cytokeratin, in differentiating squamous areas. PMID- 1708981 TI - [The Fabian prosthesis: our experience in 13 cases]. AB - The spiral intraurethral prosthesis is a device that maintains the urinary tract expedite and overcomes the prostatic obstacle. It is used as an alternative in patients requiring permanent bladder catheters due to prostatic obstruction syndrome that are at high risk for surgery and in those patients that are on the waiting list for surgery. We report the results of a one-year follow-up of 13 patients in whom a prosthesis had been implanted. The spiral prosthesis had to be removed in 6 cases (one due to calcification two months after placement); the remaining 7 patients are still being followed. We briefly analyze the micturition hydrodynamics and the changes of the continence mechanisms and attempt to explain the poor flowmetry results and incontinence observed in these patients with the urological spiral prosthesis. We have recently abandoned the metal prosthesis and now use the so-called "intraureteral catheter" which has become available. However, further experience is warranted in order to determine its results. PMID- 1708982 TI - Medication review: FK 506. AB - Overall, the absolute rate of clinical rejection in FK 506 patients is only slightly lower than with current standard therapies, but fewer steroid boluses, steroid recycles, and OKT 3 courses were needed as compared to CyA patients. FK 506 has not significantly affected allograft or patient survival or the incidence of infection as compared to CyA. It does have the benefit of allowing for the potential elimination or reduction in dose of azathioprine and prednisone. The majority of information available on FK 506 is generated by the sole human clinical trial site in Pittsburgh. Far more data from other clinical trial sites will be essential to finalize information on dosing, side effects, and drug monitoring for renal transplant recipients. The role of FK 506 in renal transplantation has yet to be well defined. The frequency and severity of side effects produced by this drug will be major determinants in its long-term usefulness for renal transplant recipients. PMID- 1708983 TI - Novel therapy for the treatment of human carcinoid. AB - Development of effective treatment for patients with carcinoid tumors has been hampered by lack of an experimental model. The authors have established the only long-term cell line of a functioning human pancreatic carcinoid tumor (BON) that produces tumors in nude mice. In this study the authors examined the effect of three agents, alpha-interferon (IFN), a somatostatin analog, SMS 201-995 (SMS), and an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), on the growth of BON tumors. BON was implanted bilaterally as 3-mm2 pieces (subcutaneously [sc]) into male BALB/c nude mice. In the first study, 23 mice were randomized to four groups: control, IFN (1 x 10(6) units, sc, four times a day), IFN + SMS (300 micrograms/kg, intraperitoneally, three times a day), and IFN + 3% DFMO in drinking water. Treatments were initiated on day of tumor implantation. In the second study, mice were randomized to six groups: control, IFN, SMS, DFMO, IFN + SMS, IFN + DFMO, and IFN + SMS + DFMO. Treatments were started on day 15 after tumor implantation. Tumor area and body weights were measured weekly. In both studies mice were killed on day 28 after BON implantation and tumors removed, weighed, and analyzed for DNA and RNA content. In the first study, IFN either alone or in combination with SMS or DFMO suppressed BON tumor growth. When treatment was initiated after established tumor growth (study 2), however, the only effective treatments for suppression of growth of BON were IFN + DFMO and IFN + DFMO + SMS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1708984 TI - Prehospital hypertonic saline/dextran infusion for post-traumatic hypotension. The U.S.A. Multicenter Trial. AB - The safety and efficacy of 7.5% sodium chloride in 6% dextran 70 (HSD) in posttraumatic hypotension was evaluated in Houston, Denver, and Milwaukee. Multicentered, blinded, prospective randomized studies were developed comparing 250 mL of HSD versus 250 mL of normal crystalloid solution administered before routine prehospital and emergency center resuscitation. During a 13-month period, 422 patients were enrolled, 211 of whom subsequently underwent operative procedures. Three hundred fifty-nine patients met criteria for efficacy analysis, 51% of whom were in the HSD group. Seventy-two per cent of all patients were victims of penetrating trauma. The mean injury severity score (19), Trauma Score plus Injury Severity Score (TRISS) probability of survival, revised trauma scores (5.9), age, ambulance times, preinfusion blood pressure, and etiology distribution were identical between groups. The total amount of fluid administered, white blood cell count, arterial blood gases, potassium, or bicarbonate also were identical between groups. The HSD group had an improved blood pressure (p = 0.024). Hematocrit, sodium chloride, and osmolality levels were significantly elevated in the Emergency Center. Although no difference in overall survival was demonstrated, the HSD group requiring surgery did have a better survival (p = 0.02), with some variance among centers. The HSD group had fewer complications that the standard treatment group (7 versus 24). A greater incidence of adult respiratory distress syndrome, renal failure, and coagulopathy occurred in the standard treatment group. No anaphylactoid nor Dextran-related coagulopathies occurred in the HSD group. Although this trial demonstrated trends supportive of HSD in hypotensive hemorrhagic shock patients requiring surgery, a larger sample size will be required to establish which subgroups of trauma patients might maximally benefit from the prehospital use of a small volume of hyperosmolar solution. This study demonstrates the safety of administering 250 mL 7.5% HDS to this group of patients. PMID- 1708985 TI - [New antineoplastic antibiotics, new analogs, modifiers of biological reactions and oncogene inhibitors]. PMID- 1708986 TI - Effect of partial starvation on in vitro spontaneous activity and glycogen levels of uterine smooth muscle from pregnant and non pregnant rats. AB - Effects of a restricted-diet (50% of the normal feeding) given during 14 days, on the isometric developed tension (IDT) of uterine horns isolated from pregnant and non pregnant (diestrous) rats, incubated in a KRB-medium without glucose, were explored. In 14 days-pregnant rats, dietary restriction did not alter the contractile activity with respect to normal-fed controls. Besides, levels of uterine glycogen, immediately after killing the animals or after 60 min incubation, remained unaltered. In advanced pregnancy partial starvation led to decay of spontaneous contractile activity after 60 min incubation. However, the considerable increment in the levels of tissue glycogen at 0 time was not modified, nor its decrease at the end of the in vivo experimental period. In non pregnant rats, a reduced feeding did not alter the development of contractile tension, but exerted a pronounced effect on the glycogen levels: these were significantly lower than controls at 0 time but suffered no changes after 60 min on in vitro activity. Indomethacin appeared to have no effect on the spontaneous contractile activity of 14 days-pregnant rats. It significantly depressed contractility in 21 days-pregnant rats. Indomethacin did not modify the levels of glycogen in any of the experimental groups. PMID- 1708987 TI - Changes in lipid parameters after intestinal resection or bypass in the rat. AB - Intestinal resection, bypass and adaptative postoperative mechanisms developed as a consequence of that surgery, are considered good methods for improving knowledge of gastrointestinal physiology as well as possible effects that the intestine could have on the general metabolism. 50% jejunoileal bypass (BP), 50% proximal (PR) and distal (DR) intestinal resections were performed on rats to compare the influence of resected intestinal segments or bypassed loop localization could exert on different serum lipid parameters. One month after surgery significant increases in total serum cholesterol and cholesterol esters were found. There was no change in free cholesterol. A decrease in triglyceride was observed after distal and proximal resection but no changes after bypass. The cholesterol/phospholipid ratio was increased after resection and after bypass. It has been suggested that the changes in lipid metabolism produced after resections and bypass depend mainly on the loss of absorptive surface rather than on the position of the resected segment. The bypass loop may itself still exert some influence on lipoprotein metabolism, mainly on high density lipoprotein cholesterol. PMID- 1708988 TI - In vivo lipid and amino acid synthesis from 3-hydroxybutyrate in 15-day-old chick. AB - The in vivo utilization of 3-hydroxybutyrate for lipid and amino acid synthesis in liver, kidney and duodenal mucosa of overnight-starved 15-day-old chicks has been investigated. Lipid synthesis was higher in liver and duodenal mucosa than in kidney. Triglycerides were the main lipids synthesized from 3-hydroxybutyrate in liver and kidney, while in duodenal mucosa a higher amount of phospholipids was observed. This tissue utilized a high percentage of 3-hydroxybutyrate for the synthesis of free cholesterol, in agreement with the major role of intestine in body cholesterogenesis. All of the assayed tissues synthesized amino acids from 3 hydroxybutyrate at a similar rate, glutamate being always the main amino acid formed. PMID- 1708989 TI - Morphometric and biochemical evaluation of rat prostate and seminal vesicle following chemical sympathectomy with guanethidine. AB - Selective chemical sympathectomy of the internal genital organs of adult male rats was undertaken by chronic treatment with low doses of guanethidine. Biochemical and morphometric methods revealed that removal of sympathetic innervation prevents fructose secretion in the prostate and seminal vesicle, in addition to promoting reduced efficiency of delivery by the latter. PMID- 1708990 TI - Stimulation of carbohydrate metabolising enzymes by synthetic hypertrehalosemic peptides in thoracic musculature of the American cockroach, Periplaneta americana. AB - The ability of the synthetic hypertrehalosemic peptides, HT-I and HT-II, to influence the activities of glycogen phosphorylase, trehalase and hexokinase via elevation of Ca++ and cAMP levels was examined in thoracic musculature of the American cockroach, Periplaneta americana. The peptides effect dose- and time dependent activation of phosphorylase, trehalase and hexokinase activities that occur concomitantly with elevated levels of intracellular calcium. In addition, HT-I increases the accumulation of cyclic AMP in muscle cells. PMID- 1708991 TI - Caerulein-induced acute pancreatitis in the rat. Pancreatic secretory response to cholecystokinin. AB - The response of pancreatic exocrine secretion to cholecystokinin (CCK), has been studied in experimental acute pancreatitis induced in rats by supramaximal doses of caerulein. Several doses of caerulein were used (4, 20 and 40 micrograms/Kg) and each one was administered by four subcutaneous injections over 3 h at hourly intervals. Pancreatic juice was collected 9 h after the first injection. The caerulein-treated animals showed a statistically significant increase in serum amylase levels. Secretory activity of ductular cells remained unchanged in all the caerulein-treated animals, but total protein and amylase secretion decreased significantly at all the caerulein doses used, both in resting conditions and under stimulation with CCK (1.25 micrograms/Kg/h). Despite this the acinar cells of rats treated with the lowest dose of caerulein retained a certain degree of secretory function since amylase activity in pancreatic juice was greater than in other groups of rats treated with higher doses of caerulein. Moreover, the percentage of increase observed in total protein and amylase in response to CCK respect to basal secretion is similar to that of the untreated animals. At higher doses (20 and 40 micrograms/Kg) the secretory capacity in response to CCK was inhibited. Therefore CCK administration in slight acute pancreatitis could be used as a therapy since it favours the secretion of pancreatic enzymes at percentual levels similar to those of the controls. PMID- 1708992 TI - Stressors and pain sensitivity in CFW mice. Role of opioid peptides. AB - Effects of several environmental situations on pain threshold were studied in CFW male mice. Immobilization induced significant and naloxone reversible analgesia. Isolation produced analgesia which was partially reversed by naloxone. One minute swimming in + 4 degrees C or + 42 degrees C water increased naloxone reversible analgesia. Isolation produced analgesia which was partially reversed by naloxone. One minute swimming in 4 degrees C or + 42 degrees C water increased naloxone irreversible pain threshold. Other situations: drinking 2% NaCl solution, disturbance of light-dark cycle or social aggregation did not produce analgesia. The role of these situations as stress-inducers, as well as the role of endogenous opioid peptides in stress-induced analgesia, were discussed. PMID- 1708993 TI - [Inhibitory activity of allene cholesteryl derivatives on the biosynthesis of ecdysone]. AB - Prothoracic glands of the migratory locust Locusta migratoria during the postembryonnic development, are the biosynthetic source of ecdysone. The production of ecdysone by these glands in vitro has been used to evaluate the inhibitory activity of four cholesteryl derivatives with an allenic function on the side chain at C-22. These molecules were devised as potential inhibitors of hydroxylation at C-22 which is an obligate step in the biosynthesis of ecdysone. Three of the four molecules tested induce a marked depressory effect of the production of ecdysone. The effect of the compound with the higher activity was dose dependent and irreversible. PMID- 1708994 TI - Effects in vitro of mercury on rat brain Mg(++)-ATPase. AB - Mercuric chloride (Hg) in micromolar concentrations inhibited Mg(++)-dependent ATPase activity in rat brain microsomes. Inhibition was higher in oligomycin sensitive (O.S.) than oligomycin-insensitive (O.I.) Mg(++)-ATPase. Hydrolysis of ATP with 15 and 50 micrograms of microsomal protein for 45 min without and with (2.10(-7M) Hg showed linear rates for 15-20 min. Altered pH vs activity demonstrated comparable inhibitions by Hg in buffered (neutral greater than acidic greater than basic) pH ranges. Inhibition of enzyme activity by Hg was found to be greater at 37 degrees C than at lower temperatures suggesting positive correlation trend. An uncompetitive inhibition with respect to the activation of Mg(++)-ATPase, O.S. Mg(++)-ATPase and O.I. Mg++ ATPase by ATP was indicated by a decrease in apparent Vmax and Km. Mg(++)-activation kinetic studies indicated that Hg causes uncompetitive inhibition of Mg(++)-ATPase and O.I. Mg(++)-ATPase and mixed inhibition of O.S. Mg(++)-ATPase. Inhibition was partially restored by repeated washings. These results indicate that the inhibition of microsomal Mg(++)-ATPase by Hg was pH, temperature, enzyme and Mg++ concentration dependent. Additionally, the data also suggest that O.S. compared to O.I. Mg(++)-ATPase is more sensitive to Hg toxicity. PMID- 1708995 TI - Effect of short-term fasting on [123I] iodohexadecenoic acid metabolism in the isolated perfused rat heart. Validation of mathematical model by comparison with experimental measurements. AB - In order to study metabolic modifications induced by short term fasting and their consequences on the uptake and intracellular fate of fatty acids iodine labelled in omega position, rats undergo a 36h fasting. Hearts are perfused in a Langendorff system with a glucose (11 mM) perfusion medium; [123I] hexadecenoic acid (IHA) is injected as a bolus. A comparison between time-activity curves p.i. demonstrates a much faster activity decrease for the hearts fasted animals. The intracellular analysis shows that short fasting did not significantly increase the myocardial uptake of fatty acids, but decreased the storage and increased the degradation of the fatty acids taken up. Mathematical analysis of the myocardial time-activity curves obtained by external detection provided results comparable to those of intracellular analysis. The coefficients of correlation between the values of the aqueous phases, organic phases and free fatty acids measured by intracellular analysis and calculated with the compartmental model are consistently higher than 0.97. Consequently, this experimental model combined with mathematical analysis of the time-course of myocardial radioactivity after 123IHA administration appears to be very promising method for studying the effects of drugs or variations of energy substrate availability on myocardial fatty-acid metabolism. PMID- 1708996 TI - The influence of lipogenic and lipolytic conditions on the pentose phosphate pathway dehydrogenases in rat-kidney-cortex. AB - The effects of various lipogenic and antilipogenic states on the activities of rat-kidney cortex glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been studied. These conditions are related to the long-term administration of different diets, such as high-carbohydrate (80%) and high-fat (23%), and also to a state of fast. Contrary to what happens in liver cells and kidney cortex during a high protein diet administration, none of these nutritional conditions produced significant changes in the kinetics of either kidney hexose monophosphate dehydrogenases. PMID- 1708997 TI - Effects of cadmium and mercury on Na(+)-K+, ATPase and uptake of 3H-dopamine in rat brain synaptosomes. AB - Effects in vivo of cadmium (Cd), mercury (Hg) and methylmercury (CH3Hg) on Na(+) K+ ATPase and uptake of 3H-dopamine (DA) in rat brain synaptosomes were studied. These heavy metals significantly inhibited the Na(+)-K+ ATPase activity in a dose dependent manner. Similarly, inhibition of DA uptake by synaptosomes was also observed in rats treated with these metals. Intraperitoneal route of metal administration was found to be more effective than per os treatment. Mercuric compounds compared to Cd elicited a higher inhibition of Na(+)-K+ ATPase and DA uptake in rat brain synaptosomes. PMID- 1708998 TI - Tropomyosin from the striated muscles of carp (Cyprinus carpio) and of icefish (Channichthys rhinoceratus). AB - Tropomyosin of fast-twitch, slow-twitch and cardiac muscles of carp and icefish has been isolated by hydroxyapatite chromatography. The subunit distribution has been investigated by polyacrylamide gel electrophoresis and by peptide mapping. The purified skeletal muscle tropomyosins all belong to the alpha family and differ from higher vertebrate tropomyosin by the lack of beta subunits. Specific alpha isotypes are however encountered in fast-twitch fibres (alpha w subunit) and slow-twitch or intermediate (pink) fibres (alpha and alpha w subunits). The amino acid compositions and the paracrystals formed by the carp alpha w alpha w and alpha alpha w tropomyosins do not differ markedly from that of rabbit alpha alpha chains. They differ however by their capability to inhibit the ATPase activity of rabbit skeletal muscle acto-HMM system. A beta-like subunit is found in carp cardiac tropomyosin, in the proportion of 25% of the native protein, but not in icefish heart. PMID- 1708999 TI - [A study of senile plaques with a combined method in brains of patients suffering from Alzheimer's disease]. AB - Combined immunocytochemistry to phosphorylated neurofilament epitopes and periodic-acid methenamine silver (PAM) were used in the study of senile plaques (SP) in 6 patients with Alzheimer disease. SP are categorized as diffuse, primitive, mature and burned-out. Amyloid deposits are found in all of them, which is loose in diffuse and primitive plaques, and condensed in mature and burned-out types. Dystrophic neurites are only found in primitive and mature plaques. Combined methods have also shown that apparently isolated dystrophic neurites in the neuropil are always associated to amyloid deposits. These features suggest that amyloid deposition is a primary event in SP formation. PMID- 1709000 TI - [Features of the histogenesis of testicular yolk sac tumors in children]. AB - 32 cases of a yolk sac tumour of the testis in children aged up to 5 years underwent retrospective morphological study. Foci of proliferation of activated gonocytes identical to the type A spermatogonia with clear nuclei and the type B spermatogonia characterized by a negative PAS-reaction and reaction to alpha fetoprotein are observed in the seminiferous tubules surrounding a tumour. Besides this areas of the primordial germinogen cell generation are found among the cells lining the system of labyrinths and channels of the yolk sac tumour. A new hypothesis of the yolk sac tumour histogenesis in the child testis is put forward--namely, through the transformation of the activated gonocytes in the seminiferous tubules into their "somatic" phase, i.e. into the yolk sac elements from which they originate. Likewise, the scheme is suggested reflecting a cyclic character of the germinogenic cells in ontogenesis. This scheme allows visual representation of the process of the parthenogenetic development of germinogenic tumours. PMID- 1709001 TI - Krypton Laser photocoagulation for neovascular lesions of age-related macular degeneration. PMID- 1709002 TI - Stimulation of tear secretion and treatment of dry-eye disease with 3-isobutyl-1 methylxanthine. AB - We examined the effect of topically applied 3-isobutyl-1-methylxanthine (IBMX), a known secretagogue, on tear secretion and dry-eye disease in a clinical study. We found that IBMX produced a dose-dependent decrease in tear film osmolarity that was significant at 3.0 mmol/L (P less than .0005) in patients with dry-eye disease. This effect was not blocked by prior administration of proparacaine hydrochloride (P less than .05). Throughout a 4-week, open-label, vehicle controlled study, IBMX decreased tear film osmolarity significantly, whereas vehicle alone did not. After 4 weeks, mean (+/- SEM) osmolarity in IBMX-treated eyes decreased from 325 +/- 3.2 mOsm/L to 312 +/- 1.8 mOsm/L but remained unchanged in vehicle-treated eyes (323 +/- 4.4 mOsm/L vs 320 +/- 4.2 mOsm/L). In our study, IBMX was significantly more effective than vehicle alone in decreasing rose bengal staining (P less than .02). Hence, topical IBMX stimulated tear secretion and decreased ocular surface disease in patients with dry-eye disease. PMID- 1709003 TI - Histamine acts directly on calcitonin gene-related peptide- and substance P containing trigeminal ganglion neurons as assessed by calcium influx and immunocytochemistry. AB - Primary cultures of rat trigeminal ganglion cells were exposed to histamine, and the intracellular free-calcium concentrations, [Ca2+]i, were measured by the calcium-sensitive dye fura-2. Histamine (10(-6)-10(-2) M) increased the [Ca2+]i of the neurons. Pretreatment of the cells with histamine H1-receptor blocker pyrilamine (10(-4) M), or chelation of extracellular calcium, abolished the response; however, the response was not altered by pretreatment with H2-blocker cimetidine (10(-2) M). Thus, the increase in [Ca2+]i was due to the influx of extracellular calcium mediated by H1-receptor. Immunocytochemical analysis showed that these cultured cells that respond to histamine were identically calcitonin gene-related peptide (CGRP)- or substance P (SP)-like immunoreactive. The findings suggested that histamine released from mast cells directly affected CGRP and SP-containing sensory neurons via H1-receptor, which convey nociceptive information. PMID- 1709004 TI - Enzymic methylation of myelin basic protein in myelin. AB - Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition. PMID- 1709005 TI - Inhibition of luteinizing-hormone exocytosis by guanosine 5'-[gamma thio]triphosphate reveals involvement of a GTP-binding protein distal to second messenger generation. AB - Dual inhibitory and stimulatory actions of guanine nucleotides on luteinizing hormone (LH) exocytosis were observed in primary sheep gonadotropes permeabilized with staphylococcal alpha-toxin. At resting cytosolic [Ca2+]free (pCa 7), 5' [gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated rapid LH exocytosis, which was maximal between 5 and 10 min. GTP[S] and p[NH]ppG had similar potencies (50% of maximum effect at 20-50 microM), but the effect of p[NH]ppG was more prolonged. Experiments carried out in the presence of saturating concentrations of phorbol 12-myristate 13-acetate (PMA), or in PMA-desensitized cells, suggested that stimulation by p[NH]ppG is mediated by a mechanism additional to protein kinase C (PKC) activation. Furthermore, p[NH]ppG stimulated LH exocytosis in the presence of saturating cyclic AMP (cAMP) concentrations, although its effect was less than additive. However, when both PMA and cAMP were present, p[NH]ppG did not stimulate a further increase in the rate of LH exocytosis. In contrast, pretreatment of cells with GTP[S] at low [Ca2+]free markedly inhibited subsequent responses to Ca2+, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTP[S] concentrations than the stimulatory effect (50% inhibition at 1-10 microM), and was not observed with p[NH]ppG. A similar inhibition was observed with adenosine 5'-[gamma-thio]triphosphate, probably by its conversion into GTP[S]. These results suggest that the stimulatory actions of guanine nucleotides can be accounted for by the combined activation of PKC and generation of cAMP, resulting from activation of conventional signal-transducing GTP-binding proteins. The inhibitory effect of GTP[S] can be clearly distinguished and indicates the involvement of a distinct GTP-binding protein in exocytosis at a site distal to second-messenger generation. PMID- 1709006 TI - Synthesis of the acid-labile subunit of the growth-hormone-dependent insulin-like growth-factor-binding protein complex by rat hepatocytes in culture. AB - Insulin-like growth factors (IGFs) circulate predominantly in a growth-hormone dependent ternary complex of 125-150 kDa. This study investigates the production of the alpha-subunit of this complex, an acid-labile glycoprotein without intrinsic IGF-binding activity, which binds to the IGF-binding protein IGFBP-3 in the presence of IGFs. Medium conditioned by primary cultures of rat hepatocytes produced alpha-subunit with similar complex-forming activity to purified rat serum alpha-subunit. Bovine growth hormone stimulated hepatocyte production of both IGF-I and alpha-subunit. IGF-I tracer bound to pure rat IGFBP-3 was converted from approx. 60 kDa to 150 kDa by serum alpha-subunit, whole rat serum or rat hepatocyte culture medium; this converting activity was destroyed by transient acidification. In contrast, IGF-I bound to hepatocyte-medium IGF binding proteins could not be converted into a high-molecular-mass from by purified rat serum alpha-subunit. Rat serum and hepatocyte-medium alpha-subunit appeared identical by electrophoretic analysis, since reaction of either with cross-linked IGF-I.IGFBP-3 tracer resulted in bands of molecular mass 130 kDa and 160 kDa, probably representing intact and partially deglycosylated complexes. However, IGF-binding proteins in rat serum and hepatocyte medium were different, in that affinity labelling of medium binding proteins, depleted of endogenous IGFs, showed no evidence of the 50-60 kDa cluster of bands characteristic of rat serum IGFBP-3. We conclude that rat hepatocytes in primary culture produce alpha subunit similar to that in rat serum; however, alpha-subunit is unable to form ternary complexes with hepatocyte IGF-binding proteins, since cultured hepatocytes do not secrete IGFBP-3. PMID- 1709007 TI - Transgenic mice carrying the guinea-pig alpha-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation. AB - We have generated transgenic mice carrying the entire guinea-pig alpha lactalbumin gene. Lactating transgenic mice expressed high levels of correctly initiated and processed guinea-pig alpha-lactalbumin mRNA in the secretory epithelium of their mammary glands, and secreted guinea-pig alpha-lactalbumin in their milk. Transcripts were detectable after 7 days of pregnancy, indicating that the transgene was under correct hormonal control. Whereas no or negligible transcription was detectable in all other tissues tested, high levels of transcripts were found in the skin of lactating transgenic mice. Guinea-pig alpha lactalbumin protein was undetectable in the skin, however. In situ hybridization analysis showed that expression was localized to the undifferentiated cells in the basal layer of the sebaceous glands. Further studies revealed high levels of endogenous beta-casein mRNA in normal lactating mouse skin, demonstrating that the transcription of milk protein genes in lactating mouse skin is a normal event, and is not peculiar to the transgene. This surprising finding highlights the developmental relationship of the mammary gland to other specialized structures of the skin, supports a role for epithelial-extracellular matrix interactions in the regulation of milk protein gene expression in vivo, and identifies the skin as a particularly accessible model system in which to study the regulation of milk protein gene expression. In addition, the guinea-pig alpha lactalbumin gene will be a source of regulatory sequences with which to direct heterologous gene expression to the sebaceous glands of transgenic mice. PMID- 1709008 TI - Effects of amyl ester of unsubstituted rhodamine on respiration and Ca2+ transport in rat liver mitochondria. AB - In rat liver mitochondria the amylrhodamine is responsible for uncoupling (respiratory stimulation in state 4) by two distinct processes. Immediately after amylrhodamine addition (2-12 microM) stimulation of respiration takes place. Respiration rate for this phase is constant in time, it is independent of the potassium or inorganic phosphate content in the medium, not inhibited by oligomycin, ruthenium red, cyclosporine A, N-ethyl-maleimide and EGTA. The second phase of the respiratory stimulation is not linear in time. Respiration rate within this phase increases with rising of potassium and phosphate content in the medium. This effect is abolished by oligomycin, ruthenium red, cyclosporine A, N ethylmaleimide and EGTA. The beginning of respiratory increment coincides with the second phase of Ca2+ release from mitochondria. PMID- 1709009 TI - Age and gender-related gene expression of hydroxysteroid sulfotransferase-a in rat liver. AB - Hepatic hydroxysteroid sulfotransferase-a (HST-a) gene expression was examined in young male (age 22-26 days) and female rats (age 22-30 days), and in older male (age 42-45 days) and female (age 49-55 days) rats. Northern and slot blot analyses of poly(A)+RNA revealed that HST-a was differentially expressed with respect to both age and gender with female rats expressing higher levels of HST-a in both age groups. Hepatic HST-a mRNA levels were approximately 4 to 6-fold higher in females compared to males in both age groups examined. HST-a expression increased with age in both male and female rats. HST-a expression was approximately 8 to 10-fold higher in 42-45 day old males relative to 22-26 day old males. HST-a mRNA levels were approximately 3 to 7-fold higher in 49-55 day old females relative to females in the 22-30 day age group. These data suggest that HST-a gene expression is transcriptionally controlled and that HST-a regulation is subject to hormonal and developmental modulation. PMID- 1709010 TI - Cloning and sequencing of human FSH receptor cDNA. AB - We have isolated and sequenced a cDNA encoding the follicle stimulating hormone (FSH) receptor. The deduced amino acid sequence (678 residues) containing seven putative transmembrane segments which displays sequence similarity to G protein coupled receptors. The receptor consists of 359 residue extracellular domain which contains four N-linked glycosylation sites. While the protein is 89% identical overall with the previously cloned rat FSH receptor, the most highly conserved regions are the putative transmembrane segments (95% similarity). PMID- 1709011 TI - Conserved positioning of proline residues in membrane-spanning helices of ion channel proteins. AB - Proline residues are a common feature of known and putative transmembrane helices of transport proteins. We find considerable consistency in the positioning of these residues within the structures. The proline residues are usually found on the hydrophilic (interior) faces of the pore-forming helices. This general observation adds considerable support to hypotheses concerning the structure of the ion-channels formed by alamethicin and melittin. As proline kinks helices, our observation suggests that the pores formed in ion-channel proteins tend to be funnel-shaped having a constriction near their center. Such a structure can aid in the capture of ions by the channel (an entropic effect) and should help in the gating mechanism of the channel. The observation will aid identification of putative transmembrane helices of ion-channels. PMID- 1709012 TI - Phosphorylation of nuclear protein is an early event in TGF beta 1 action. AB - Transforming growth factor beta (TGF beta) is a family of polypeptides that modulate growth and differentiation. TGF beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are as yet largely unresolved. In this study we report that the growth inhibitory action of TGF beta on mink lung CCl 64 cells is associated with a rapid and transient phosphorylation of a number of nuclear proteins. In parallel, a transient expression of the immediate early gene jun B is observed. The expression of jun B can be inhibited by the protein kinase inhibitor H7 and can be augmented by the phosphatase inhibitor okadaic acid. Thus, protein phosphorylation can be a possible mechanism through which TGF beta 1 initiates early genomic responses. PMID- 1709013 TI - The 56 kDa androgen binding protein is an aldehyde dehydrogenase. AB - We have described a 56 kDa protein from genital skin fibroblasts that specifically binds androgen and that is generally not expressed in genital skin fibroblasts from patients with androgen insensitivity due to genetic defects of the androgen receptor. We have isolated a partial cDNA clone for the 56 kDa protein from an expression library of genital skin fibroblasts. In vitro translation of message selected with this clone faithfully produces the 56 kDa protein which can be immuneprecipitated with an anti-56 kDa antiserum. Northern blots probed with this clone show a 2.2 kb message, which parallels the expression of the 56 kDa protein. The sequence of this 998bp clone is identical to human liver aldehyde dehydrogenase 1, the cytoplasmic isoenzyme. On activity gels of genital skin fibroblast cytosol covalently labelled with androgen, aldehyde dehydrogenase activity comigrates with the single band labelled specifically with androgen. Thus, the 56 kDa androgen binding protein is an aldehyde dehydrogenase, which is prominently expressed in normal genital skin fibroblasts, but not in non-genital skin fibroblasts. PMID- 1709014 TI - Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes. AB - Here we report the structure and bioactivity of 25 kDa glycoprotein (chondromodulin-I) as a tissue-specific functional matrix component identified and cloned for the first time. Chondromodulin-I purified from fetal bovine cartilage markedly stimulated DNA synthesis of cultured growth-plate chondrocytes in the presence of basic fibroblast growth factor (FGF). Bovine chondromodulin-I cDNA revealed that the mature protein consists of 121 amino acids with three possible glycosylation sites and is coded as the C-terminal part of a larger precursor. On northern blot analysis, expression of chondromodulin-I mRNA was observed only in cartilage. PMID- 1709015 TI - Effects of kainic acid on messenger RNA levels of IL-1 beta, IL-6, TNF alpha and LIF in the rat brain. AB - We examined the kainic acid-induced changes of mRNA levels of several cytokines such as IL-1 beta, IL-6, TNF alpha and LIF in the rat brain regions using semiquantitative RT-PCR method. IL-1 beta mRNA was markedly increased in the cerebral cortex (CC), thalamus (THL) and hypothalamus (HT) 2 h after the injection of kainic acid in a convulsive dose (12 mg/kg i.p.), and tended to decrease 4 h after the injection. IL-6 mRNA was weakly induced in the hippocampus (HPP) 2 h after the injection of kainic acid and was markedly increased in the CC, HPP, THL, and HT at 4 h. The level of TNF alpha mRNA was highly elevated in the CC, HPP, striatum (STR), THL and HT at 2 and 4 h after the injection. LIF mRNA apparently expressed in the CC and HPP of control rats and was increased in the CC, HPP and HT by the treatment with kainic acid. These results indicate that mRNAs of several cytokines are increased in various brain regions with different time-courses by kainic acid. PMID- 1709016 TI - Reduced synthesis of mtRNA in isolated mitochondria of senescent rat brain. AB - A system for studying RNA synthesis in isolated mitochondria from rat brain was set up to investigate the mechanisms responsible for the age-dependent reduction of mtRNA content. In the presence of an appropriate incubation buffer both synaptic and non-synaptic mitochondria from cerebral hemispheres were able to synthesize and process mtRNA in a way quantitatively and qualitatively similar to the in vivo transcription. The comparison of the electrophoretic pattern of mtRNAs synthesized by adult and senescent rat showed, in the senescent rat, a 50% reduction in the mtRNA synthesis rate relative to the adult value. This indicates that the age-dependent decrease of the mtRNA content is linked to a lower efficiency of the mt transcription. PMID- 1709017 TI - Isolation of a novel insulin-like growth factor (IGF) binding protein from human bone: a potential candidate for fixing IGF-II in human bone. AB - Insulin-like growth factor-II (IGF-II) is the most abundant growth factor stored in human bone. Upon release from this storage depot, IGF-II could act in bone repair and in the coupling of bone formation to bone resorption, a process inherent to bone which is a key regulatory process for maintenance of bone tissue. In this study, we report the isolation and characterization of a novel IGF binding protein (IGFBP) from human bone and describe how this IGFBP may be involved in the fixation of IGF-II in human bone. This new IGFBP has an apparent molecular weight of 29 kDa and has several fold higher affinity for IGF-II than IGF-I which could explain the much greater abundance of IGF-II than IGF-I in human bone. In terms of biological activity, this IGFBP was found to potentiate the proliferative actions of IGF-II on bone cells. This work raises the possibility that this IGFBP may participate in mediating some of the actions of IGF-II. PMID- 1709018 TI - Selectivity and specificity of new, non-peptide, quinuclidine antagonists of substance P. AB - Two members of a new class of non-peptide antagonists of substance P, (+-)-cis-3 (2-methoxybenzylamino)-2-benzhydrylquinuclidine [(+/-)-CP-96,345; I] and (+-)-cis 3-(2-chlorobenzylamino)-2-benzhydrylquinuclidine [II], were tested for their ability to antagonize neurokinin-induced contractions of the rabbit cava and jugular veins (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3 system). Compound 1 is the most potent NK-1 receptor antagonist identified until now; its apparent affinity (pA2 = 9.52) is at least two log units higher than those of other NK-1 antagonists. Compound II is less active. Both compounds have been found to be almost inactive as NK-2 and NK-3 antagonists and should, therefore, be considered as selective for the NK-1 receptor. The new compounds have no direct myotropic effects and are specific for neurokinin (NK-1) receptors since they do not affect the myotropic effects of angiotensin, noradrenaline and bradykinin in the rabbit cava and jugular veins. PMID- 1709019 TI - Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells. AB - The expression of the mRNA for SLF (the c-kit ligand), a product of the "steel" locus, has been investigated in postnatal mouse testis and homogeneous populations of testicular cells. The message was found expressed in postnatal mouse testis but not in germ cells. Studies on primary mouse Sertoli cell cultures from 18 day old mice show that Sertoli cells are the site of SLF mRNA expression in the seminiferous tubules. Treatment of Sertoli cell cultures with cAMP analogs led to a significant increase in the SLF mRNA levels. PMID- 1709020 TI - A clinical comparison of budesonide nasal aerosol, terfenadine and a combined therapy of budesonide and oxymetazoline in adult patients with perennial rhinitis. AB - The efficacy of budesonide, terfenadine and a combination of budesonide and oxymetazoline in the treatment of perennial rhinitis was evaluated by a double blind, parallel group study. Adult patients with perennial rhinitis were randomized into three groups. Group 1 patients received budesonide nasal aerosol 400 micrograms/day for 21 days and oxymetazoline nasal drops for the first three days. Group 2 and 3 patients received budesonide 400 micrograms/day and terfenadne tablet 60 mg twice/day respectively. Nasal symptoms were assessed by the patients before and daily during the treatment period using a simple scoring system. One hundred and forty-two patients were recruited and 130 completed the study. Budesonide, but not terfenadine, significantly reduced all nasal symptoms from baseline (p less than 0.05). Terfenadine could significantly relieve the nasal blockage (p less than 0.05) more than other nasal symptoms. Budesonide with or without oxymetazoline nasal drops provided a better control of nasal symptoms than terfenadine (p less than 0.05). Budesonide with oxymetazoline for the first three days showed a faster relief of nasal blockage than budesonide alone (p less than 0.05). Mild and transient adverse effects were encountered in all three groups. It is concluded that nasal symptoms of perennial rhinitis are more adequately controlled by budesonide than by terfenadine. PMID- 1709021 TI - Cooperative studies on the immunology of tuberculosis at Airlangga University, Surabaya, Indonesia. PMID- 1709022 TI - Nucleolar organizer regions in pigmented skin lesions. Value in the differential diagnosis of Spitz nevi. AB - Forty-one cases of typical melanocytic skin lesions (15 intradermal nevi, 14 Spitz nevi and 12 malignant melanomas) were used to investigate the value of staining of nucleolar organizer regions (NORs) in the differential diagnosis of such pigmented lesions. Histologic sections were stained by the silver colloid (Ag) method, with and without the prior use of a melanin blocking agent. There were statistically significant differences in the mean numbers of AgNORs per nucleus between the groups of lesions studied (1.658 for intradermal nevi, 3.0042 for Spitz nevi and 6.669 for malignant melanomas). Sections treated with potassium permanganate (melanin blocking agent) prior to staining showed an obvious increase in the AgNOR scores in all groups; this increase was highest for Spitz nevi. Although AgNOR staining allows a distinction to be made between intradermal nevi and malignant melanomas, the striking overlap between the counts for Spitz nevi and malignant melanomas precludes the use of this technique as the sole method for establishing the diagnosis of malignancy. Other clinical and morphologic data are especially required to make the diagnosis of Spitz nevi. PMID- 1709023 TI - Structural stability of paired helical filaments requires microtubule-binding domains of tau: a model for self-association. AB - Highly purified and SDS-soluble paired helical filaments (PHFs) were immunogold labeled and immunoblotted with antibodies to tau: Tau 14 (N-terminal half), AH-1 (microtubule-binding domain), and Tau 46 (C-terminal end). The main component of PHFs was modified tau of 68, 64, and 60 kd, also called A68 or PHF-tau. Trypsin digestion reduced the maximum width of PHFs by 10%-20%, increased aggregation of filaments, and abolished the binding of Tau 14, but had no effect on the binding of AH-1. The smallest tau-reactive tryptic fragments were 13 and 7-8 kd, positive with AH-1, and negative with Tau 46. Our results and the model of Crowther and Wischik suggest that by self-association and anti-parallel arrangement of the microtubule-binding domains, PHF-tau forms the backbone of PHFs. PMID- 1709024 TI - Block of kainate receptor channels by Ca2+ in isolated spinal trigeminal neurons of rat. AB - Compared with N-methyl-D-aspartate-activated channels, the interaction of Ca2+ with kainate-activated or with quisqualate-activated channels is not well understood. We have studied the effect of Ca2+ on kainate-activated currents in isolated trigeminal neurons and found that Ca2+ inhibits kainate responses. This inhibition occurs not because Ca2+ changes the affinity of kainate to its receptor, but because Ca2+ blocks monovalent cation permeation through kainate activated channels. This Ca2+ block gives rise to the outward rectification of the kainate responses. PMID- 1709025 TI - Activation of the sensory current in salamander olfactory receptor neurons depends on a G protein-mediated cAMP second messenger system. AB - Olfactory receptor neurons respond to odor stimulation with an inward cationic current. Under whole-cell patch clamp, individual, isolated olfactory receptors were exposed to pharmacological agents known to interact with distinct enzymes in a putative second messenger cascade, and their response to odors was measured. IBMX prolonged the odor-evoked current and also reduced its amplitude. cAMP and cGMP induced a current electrically identical to the odor current, but the current showed desensitization only with cAMP. GTP-gamma-s prolonged and GDP-beta s interfered with the odor-evoked current. The long latency seen in the odor response appears to be mainly due to the loading of the G protein and secondarily to the requirement for cAMP accumulation. The main source of the response decay appears to be cyclic nucleotide hydrolysis. PMID- 1709026 TI - 5-aza-2'-deoxycytidine in advanced or recurrent cancer of the uterine cervix. PMID- 1709027 TI - Changes in element concentration and distribution in breast-milk fractions of a healthy lactating mother. AB - Daily changes in components of breast milk with number of days of lactation after delivery were demonstrated by determining concentrations and distributions of several elements simultaneously. Concentrations of calcium, copper, magnesium, phosphorus, sulfur, and zinc were determined simultaneously by inductively coupled argon plasma-atomic-emission spectrometry (ICP) for whole milk and milk fractions (skimmed milk and whey) collected from 2 to 196 d postpartum from a healthy lactating mother. Calcium and phosphorus concentrations increased in transitional milk. With days postpartum, the other elements decreased from the highest concentrations in colostrum milk, the modes of decrease being characteristic for each element. Distributions of copper, iron, phosphorus, sulfur, and zinc in whey were determined on a gel-filtration column by HPLC with ICP detection (HPLC-ICP method). Distributions of the five elements and absorbance peaks at 254 and 280 nm changed dramatically day by day at the beginning (colostrum milk), resulting in constant distributions after 30 d (mature milk). These results suggest the important roles of daily changing constituents in breast milk, especially in colostrum milk, in the nutrition of the newborn. Several element peaks on a gel-filtration column were identified by comparison with standard samples. PMID- 1709028 TI - Interaction among zinc, glucose, and insulin in normal individuals during glucose and tolbutamid perfusion. AB - Reports in the literature have shown that acute or chronic zinc administration may cause hyperglycemia, with a fall in serum or insular insulin occurring in experimental animals. On the other hand, under conditions of both acute and chronic hyperglycemia, an increase, a decrease, or a normal level of blood zinc has been observed in studies conducted on humans. Thus, the objective of the investigation described here was to determine the relationship existing among zinc, glucose, and insulin under acute conditions. Thirty-six subjects of both sexes (mean age, 23 yr) were tested at 7:00 A.M. after a 12-h fast. Two antecubital veins of both forearms were punctured and maintained with physiological saline. Three experiments were performed in which zinc was administered orally, and hypertonic glucose and tolbutamid were administered intravenously. Blood samples were then collected over a period ranging from 93 to 240 min after the basal times of -30 and 0 min. Hyperzincemia did not cause changes in plasma glucose or insulin either in the absence of or during perfusion of glucose. Hyperglycemia, hypoglycemia, and hyperinsulinemia did not modify serum zinc levels. These results demonstrate that acute zinc administration did not change carbohydrate metabolism and that sudden variations in glucose and insulin levels did not modify the serum profile of zinc. PMID- 1709029 TI - Effect of an acute zinc depletion on rat lipoprotein distribution and peroxidation. AB - The aim of this study was to determine the extent to which zinc depletion leads to lipoprotein modifications by measuring both lipoprotein-fraction distribution and peroxidation in zinc-depleted rats. The animals were divided into three groups and fed for 8 wk a zinc-adequate diet (100 ppm) ad libitum (AL), a zinc deficient diet (0.2 ppm) ad libitum (ZD), or a zinc-adequate diet according to the pair feeding method (PF). Trace-element status, tissular lipids, and lipoprotein-fraction study were performed. The MDA production by the lipoprotein fraction was measured before and after induced peroxidation. Cholesterol and phospholipids were increased in ZD rats. An important increase of VLDL and IDL was observed and a significant enhanced production of MDA by the LDL was related to zinc deficiency. From this observation, we may conclude that LDL fractions of ZD rats are more susceptible to induced oxidative damage. These results suggest that in zinc deficiency, the lipoprotein fragility is an aggravating factor of peroxidation and the dyslipoproteinemia may lead to an atherogenic risk. PMID- 1709030 TI - Cadmium in hair of school children living in Tarragona Province, Spain. Relationship to age, sex, and environmental factors. AB - Cadmium concentrations were determined in the hair of 226 school children in an industrial and in a rural area of Tarragona Province (NE Spain). The influence of sex, age, hair color, smoking habits of the household members, and parents' occupation on the children's hair cadmium levels was also evaluated. Children living in the industrial area had much more cadmium in their hair than those living in the rural area (median: 0.327 vs 0.002; arithmetic mean: 0.401 vs 0.119 micrograms/g). Girls had more cadmium in their hair than boys, and cadmium levels decreased with the age independently of the sex. Smoking habits and parents' occupation also influenced the hair cadmium content in the children examined. In contrast, hair color has no influence on hair cadmium values. PMID- 1709031 TI - Combined exposure to lead and ethanol on tissue concentration of essential metals and some biochemical indices in rat. AB - The effect of daily oral administration of ethanol (2.5, 5, or 10% in drinking water for 8 wk), lead (10 mg/kg, po, once daily for 8 wk), or their combination on tissue trace-metal concentration and hematopoietic and hepatic biochemical indices was investigated in male rats. Ethanol (10%) ingestion enhanced the hepatic lipid peroxidation and decreased the calcium and magnesium content of blood and liver. Coexposure to lead and ethanol (5 and 10%) produced a more pronounced elevation of blood zinc protoporphyrin (ZPP) and hepatic lipid peroxidation. Combined lead-ethanol exposure also lowered the concentration of blood and hepatic magnesium and calcium and increased the amount of lead in the blood, liver, and brain compared to a group treated with lead alone. The results suggest that chronic alcohol ingestion results in calcium and magnesium loss. However, coexposure to lead and ethanol could result in more serious depletion of calcium and magnesium, and this could be the cause of suspected synergism between alcohol consumption and lead poisoning. PMID- 1709032 TI - Tin compounds inhibit the plasma cell response to metallic tin. Transfer of inhibition by parabiosis. AB - Injection of metallic tin powder causes intense proliferation of plasma cells in draining lymph nodes of Lewis rats. Pretreatment orally with soluble tin salts prevents this response to subsequently injected metallic tin. In the present work, pretreatment with tin salts by parenteral injection was just as effective as addition to the drinking water. This new approach made the following experiments possible. Poorly soluble tin compounds were found to be inhibitory when injected parenterally. Tin salts injected parenterally into one of two rats joined in parabiotic union prevented the plasma cell response to metallic tin in both parabionts. The transfer of the inhibitory effect via the cross-circulating blood represents significant progress toward understanding the mechanisms involved. The evidence suggests the possibility that tin salts elicit an intermediary substance or process that is responsible for inhibition of the plasma cell response to metallic tin. PMID- 1709033 TI - Influence of zinc on copper binding in tissue proteins of steers. AB - Two experiments were conducted with steers fed diets containing 270 ppm copper either with or without 2050 ppm zinc. Liver biopsies were taken from steers biweekly for 10 wk for analysis. The steers were then killed; tissues were removed, homogenized, and centrifuged, and the pellets were extracted with mercaptoethanol (BME), and selected cytosols and extracts were subjected to gel filtration (Sephadex G-75). Copper and zinc were determined on the BME extracts, pellets after extraction, cytosols, and gel-filtration fractions. Copper accumulated at about the same rate in BME extract and in the extracted pellet, with the smallest amount in the cytosol. In contrast, over 70% of the zinc was present in the hepatic cytosols. Gel filtration of BME extracts revealed the greatest amount of copper in a low-mol-wt (MW) peak in addition to three minor peaks of copper. Within the hepatic cytosols, the greatest amount of copper accumulated in proteins of MW greater than 75,000, the next greatest amount in 30,000-MW proteins, and the least amount with metallothionein (MT) of steers fed the diet with only copper added. In contrast, the greatest amount of copper was present with MT in hepatic cytosols of the steer fed a diet that included copper plus zinc. Hence the zinc status of steers influences the deposition of copper in the cytosolic proteins (as demonstrated by liver, kidney, and pancreas), but not in the intracellular fractions. PMID- 1709034 TI - Lead-induced tissue fatty acid alterations and lipid peroxidation. AB - Previous work showed that dietary lead (Pb) increases the relative concentration of arachidonic acid (20:4) as a percentage of total fatty acids, and decreases the relative proportion of linoleic acid (18:2) to arachidonic acid (18:2/20:4) in chick liver, serum, and erythrocyte membranes. The present investigation was undertaken to examine the time-course and magnitude of the fatty acid alterations with increasing dietary Pb levels. We also examined the effects of Pb on the fatty acid composition and lipid peroxide content of hepatic subcellular organelles. In Exp. 1, chicks were fed diets containing 0, 62.5, 125, 250, 500, or 1000 ppm added Pb (as Pb acetate trihydrate) from 1 to 21 d of age. After 21 d, no growth effects were observed; however, Pb lowered the 18:2/20:4 ratio and increased 20:4 concentration in total liver and serum lipids, and in total hepatic phospholipids in a dose-dependent manner. Hepatic mitochondrial membrane fatty acids were not altered, nor was there any increase in hepatic lipid peroxidation. In Exp.2, chicks were fed diets containing 0, 500, 1000, or 2000 ppm added Pb from 1 to 21 or 22 d of age. Pb depressed growth in a dose-dependent manner. In addition, Pb lowered the 18:2/20:4 ratio and increased 20:4 concentration in total liver lipids and in hepatic mitochondrial and microsomal membranes in a dose-dependent manner. Total hepatic lipid peroxidation was increased over control values by 1000 ppm Pb, and hepatic microsomal lipid peroxidation was increased by dietary Pb levels of 1000 and 2000 ppm. In Exp. 3, body weight, hepatic microsomal lipid peroxidation, and fatty acid composition were determined in 4-, 9-, 14-, 18-, and 23-d-old chicks fed 0 or 1500 ppm added Pb. Body weights of Pb-treated chicks were significantly lower than those of control chicks by day 18. Microsomal 20:4 concentration and peroxidation increased, and the 18:2/20:4 ratio decreased with age in both groups, but the changes were of greater magnitude in the Pb-treated chicks. The results suggest that some of the manifestations of Pb toxicity may be a reflection of increased concentration of 20:4 in specific membranes. Further, since the Pb-induced alterations in fatty acid composition were noted in the absence of any growth depression, we propose that fatty acid composition is more sensitive than growth rate to the presence of lead in the diet. PMID- 1709035 TI - Absorption of cadmium after a long-term oral administration of cadmium to dogs. AB - A long-term experiment using beagle dogs to investigate the absorption of cadmium was conducted. The dogs in the experimental groups were given a commercial diet and pelleted food containing 1, 3, 10, 50, and 100 mg of cadmium per day. The cadmium concentration in the blood increased continuously, gradually reaching a steady state following the administration of cadmium. The cadmium excreted daily in urine increased continuously. The cumulative excreted amount of cadmium in urine was calculated by using the trapezoidal rule based on the data of excretion of cadmium in urine. Then the absorbed fraction of administered cadmium was estimated on the basis of the relationship between the cumulative excreted amount of cadmium in urine and the cumulative administered dose of cadmium after the cadmium concentration in blood reached a steady state. The absorbed fraction of cadmium decreased with an increase in the administered dose of cadmium. A dose dependent increase between the absorbed amount and the administered dose was observed. PMID- 1709036 TI - Alteration of cellular oncogene expression in L1210 cells by a nitrosourea analog of thymidine. AB - 3'[3-(2-Chloroethyl)-3'nitrosoureido]-3'-deoxythymidine (3'-CTNU), a chloroethylnitrosourea analog of thymidine, is a potent antineoplastic agent against murine leukemia L1210. In this study, we have examined the effects of 3' CTNU on cellular oncogene (proto-oncogene) expression. We found that the expression of the c-myb proto-oncogene was dramatically enhanced in a concentration- and time-dependent manner by 3'-CTNU in murine leukemia L1210 cells, whereas the expression of the c-myc proto-oncogene was suppressed. The enhancement of c-myb gene expression was found to be cell type-specific and to involve an increase of the c-myb transcription rate rather than an alteration of c-myb gene structure or increased stability of c-myb mRNA. Further analysis demonstrated that the altered c-myb gene expression was largely due to the presence of 3'-amino-3'-deoxythymidine, a decomposition product of 3'-CNTU. The expression of five other proto-oncogenes was unaffected by 3'-CTNU treatment. Our study showed that an antineoplastic agent can increase or decrease the expression of proto-oncogenes. PMID- 1709037 TI - Modulation of melanoma antigens by interferons. PMID- 1709038 TI - Systemic suppression of contact hypersensitivity associated with suppressor lymphocytes: is a lesion in DNA an essential step in the pathway? AB - Exposure of mice to ultraviolet B (UVB) radiation and treatment with methoxsalen and UVA radiation produces a systemic suppression of contact hypersensitivity (CHS) that is associated with suppressor lymphocytes. Both UVB and methoxsalen and UVA radiation produce lesions in DNA, and this suggests that alterations in that molecule might be an essential step in the pathway. This possibility has been explored by testing the effect of other modalities that do and do not interact with DNA. Treatment of mice with superficial X radiation, which mainly produces single-strand breaks in DNA, or with 5-methylisopsoralen and UVA radiation, which produces monofunctional adducts in DNA, results in systemic suppression of CHS. In both instances, the suppression can be transferred to untreated mice by injection of lymphoid cells obtained from suppressed mice. Treatment of mice with rose bengal and visible (greater than 400 nm) radiation, a photochemical interaction that does not produce lesions in DNA, results in systemic suppression of CHS; however, in contrast to the other treatments, the suppression cannot be transferred with lymphoid cells. In addition, treatment of mice with eosin and visible radiation, which also does not interact with DNA, did not produce suppression of CHS. These findings suggest that a molecular alteration in DNA may be either an initiating or an essential step in the development of the systemic suppression of CHS that is mediated by suppressor lymphocytes. PMID- 1709039 TI - Substance P antagonist inhibits immediate and delayed type cutaneous hypersensitivity reactions. AB - The role of substance P (SP) in allergic reactions of the skin was investigated. Spantide, a competitive inhibitor of SP, was injected intracutaneously into the volar aspect of the forearm prior to the following challenges: benzalkonium chloride (irritant delayed reaction), tuberculin (immunological delayed reaction), UVB irradiation, benzoic acid (non-immunological contact urticaria), different food allergens and latex (in patients with immunological contact urticaria). Only the immunological reactions of contact urticaria and the reaction to tuberculin were suppressed by the SP antagonist, indicating that SP is involved in their pathogenesis. PMID- 1709040 TI - Multiparameter flow-cytometrical quantitation of circulating CD34(+)-cells: correlation to the quantitation of circulating haemopoietic progenitor cells by in vitro colony-assay. AB - A multiparameter flow-cytometrical method for the quantitation of CD34(+)-cells present in adult human peripheral blood cells (PBMC) has been developed. PBMC from 13 healthy adult subjects were analysed for CD34(+)-cells by flow-cytometry, with only the lymphoid population of cells negative for anti-CD3-moAb included in the analysis. At the same time mononuclear cells from the same individuals were depleted of CD3(+)- and CD14(+)-cells by immunomagnetic purging. These cells were cultured for haemopoietic colonies. A correlation coefficient of 0.92 was calculated by regression analysis of CD34(+)-cells number versus the numbers of colonies (CFU-GM, BFU-E). Purging with anti-CD34-moAb abrogated colony-growth. The flow-cytometrical method for the determination of circulating CD34(+)-cells was used for the determination of circulating haemopoietic progenitor cells in one patient, in order to time leukapheresis for subsequent autologous reinfusion following high-dose chemotherapy. Determination of CD34(+)-cells from human adult peripheral blood by multiparameter flow-cytometry promises to be a useful tool for monitoring circulating haemopoietic progenitor cells as identified by anti CD34-moAb. PMID- 1709041 TI - Complete remission in acute myeloid leukaemia after treatment with recombinant human granulocyte colony-stimulating factor and high dose intravenous methylprednisolone. PMID- 1709042 TI - Influence of optical density on the automated segmentation of rat kidney lysosomes. AB - After staining for acid phosphatase, video-images were acquired from 0.5-micron sections of rat kidney. Lysosomes in proximal tubules were automatically segmented, using a VICOM digital image processor and measured for area, number and optical density (OD). The purpose of this study is to objectively evaluate the performance of the automated segmentation algorithm at different staining intensities (a) by measuring area after staining with different incubation times, reduced substrate concentration or by adding an inhibitor and (b) by 'simulating' a decrease in OD (reducing grey-values at each point of a digitized image). The results of the experiments showed that: (1) the algorithm will underestimate the size of lysosomes (a) when the OD in close to the local background and (b) when an area is larger than or close to the area of the lowpass square filter; (2) accuracy of the segmentation can be improved by comparing the results of feature extraction after segmentation of the same image at different relative OD levels; (3) lysosomes with very low OD, compared to background are delineated with a large error or not delineated at all and this cannot be corrected. Incorrectly delineated lysosomes can be identified and excluded from further calculations, or their measured area replaced by an estimate of the true area. PMID- 1709043 TI - An unusual voltage-gated anion channel found in the cone cells of the chicken retina. AB - Using the whole-cell patch clamp technique, we have examined the voltage-gated currents present in adult chicken cone cells. When calcium and calcium-gated currents are blocked, hyperpolarizing voltage steps elicit slowly increasing inward currents as has been shown for photoreceptors in other species. Unlike the case for other species, chicken cones appear to lack the inward-rectifying cationic current Ih that contributes to the shaping of the photovoltage. Instead of Ih, these cones possess an anionic inward-rectifying current that in kinetics, activation range and probably function is remarkably similar to Ih. This anion channel is unusual in that both nitrate and acetate are more permanent than chloride ions. PMID- 1709044 TI - Depth perception and cortical physiology in normal and innate microstrabismic cats. AB - Evidence is presented that innate microstrabismus and abnormal cortical visual receptive-field properties can occur also in cats without any apparent involvement of the Siamese or albino genetic abnormalities in their visual system. A possible cause for microstrabismus in these cats may be sought in an abnormally large horizontal distance between blind spot and area centralis indicated by a temporal displacement of the most central receptive fields on both retinae. Depth perception was found to be impaired in cats with innate microstrabismus. Behavioral measurements using a Y-maze revealed in four such cats that the performance in recognizing the nearer of two random-dot patterns did not improve when they were allowed to use both eyes instead of only one. The ability of microstrabismic cats to perceive depth under binocular viewing conditions only corresponded to the monocular performance of five normal cats. Electrophysiological recordings were performed in the visual cortex (areas 17 and 18) of four awake cats, two normal, and two innate microstrabismic animals. Ocular dominance and orientation tuning of single neurons in area 17 and 18 were analyzed quantitatively. The percentage of neurons in area 17 and 18 which could be activated through either eye was significantly reduced to 49.7% in the microstrabismic animals when compared to the normal cats (74.8%). "True binocular cells," which can only be activated by simultaneous stimulation of both eyes, were significantly less frequent (1.6%) in microstrabismic cats than in normal animals (10.4%). However, subthreshold binocular interactions were identical in both groups of animals. In the strabismic animals, long-term binocular stimulation of monocular neurons did not give a clear indication of alternating use of one or the other eye. The range of stimulus orientations leading to discharge rates above 50% of the maximal response, i.e. the half-width of the orientation tuning curves, was the same in the two groups of cats. However, orientation sensitivity, i.e. the alternation in discharge rate per degree change in stimulus orientation, was higher in cortical cells of normal cats than in those of microstrabismic cats. In normal and microstrabismic cats, no clear sign of an "oblique effect," i.e. the preference of cortical neurons for vertical and horizontal orientations compared to oblique orientations, could be found neither in the incidence of cells with horizontal or vertical preferred orientation nor in the sharpness of orientation tuning and sensitivity of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709045 TI - Injection of RNA from carp retina induces the formation of a membrane potassium channel in Xenopus oocytes. AB - Total RNA was purified from freshly isolated retinas of adult carp and injected into oocytes of Xenopus laevis (stages 5-6). Two to six days after injection, depolarizing voltage-clamp steps evoked a slowly activated outward current as large as 3 microA. This current inactivated slowly with a single time constant (tau = 3.1 +/- 0.24 S.E.M., for Vm = +30 mV). The current was inhibited by tetraethylammonium (3.8 mM for half-maximal inhibition). In the presence of Co2+ (1 mM) or barium methanesulfonate (40 mM), the current-voltage relationship shifted to slightly more depolarized values (5-10 mV); the maximal value of the current that was sensitive to Co2+ or Ba2+ treatments was only a small fraction (about 10%) of the TEA-sensitive current, and its current-voltage relationship was similar to that for uninjected oocytes. The reversal potential of the membrane current was studied with [K+]0 of 1-77 mM. For [K+]0 greater than 20 mM, the reversal potential changed with a slope of 63 mV (+/- 2 mV S.E.M.) per 10 fold change in [K+]0. The conductance was induced half-maximally at 17 mV (+/- 0.9 mV S.E.M.). The depolarization required for an e-fold increase in conductance was 13 mV (+/- 0.6 mV S.E.M.). From these results, we conclude that the injection of total RNA from carp retinas induces the formation of a membrane K+ channel in Xenopus oocytes. The channel formed has many of the properties reported for the maintained outward current of goldfish horizontal and bipolar cells. PMID- 1709046 TI - Signal transmission through the B cell-specific MB-1 molecule at the pre-B cell stage. AB - Specific binding of antigens to the surface immunoglobulin M (sIgM) triggers B cells with several biochemical events involved in receptor-mediated signal transmission for proliferation and differentiation into antibody-producing cells. Recent studies with the Digitonin lysis method identified the sIgM-associated component, IgM-alpha (B34)/Ig-beta, as the possible candidate for the transducer molecule(s) in the immunoglobulin receptor-mediated signal transmission. The 34 kd protein (B34 or IgM-alpha) of this component is suggested to be encoded by the B cell-specific mb-1 gene. We prepared monoclonal antibodies which recognize the mb-1 gene product (MB-1) and studied the functional role of MB-1 in the signal transmission in B lineage cells. Using murine pre-B lymphoma cells (18-81 and 70Z/3), we demonstrated the early phase increase of the intracellular [Ca2+]i concentration and the subsequent inhibition of the proliferation by the monoclonal anti-MB-1 antibody (11-18-5). These results clearly demonstrate signal transmission through the surface MB-1 molecule on B lineage lymphomas. This MB-1 mediated signal transmission in pre-B cell lines would suggest an alternative function of MB-1 acting at the pre-B cell stage. PMID- 1709047 TI - Neonatal tolerance to MIs-1a determinants: deletion or anergy of V beta 6+ T lymphocytes depending upon MHC compatibility of neonatally injected cells. AB - Recent investigations in mice revealed that natural immunological tolerance to endogenous minor lymphocyte-stimulating locus 1a (MIs-1a) antigen correlates primarily with deletion of MIs-1a-specific V beta 6+ T lymphocytes in the thymus. Similar mechanisms account for acquired tolerance in some instances since the neonatal injection of MIs-1a-expressing MHC compatible cells in neonatal mice within the first 24 h of life causes clonal deletion of V beta 6+ T cells. Here we demonstrate that V beta 6+ T cells are not deleted in mice neonatally treated with MIs-1a spleen cells expressing allogeneic H-2 molecules. However, when such non-deleted V beta 6+ T cells were tested in vitro, no interleukin 2 (IL-2) secretion or proliferation was observed after MIs-1a stimulation. This non responsive state could be overcome by addition of exogenous IL-2, consistent with the fact that V beta 6+ cells enlarged and expressed IL-2 receptors upon MIs-1a stimulation. Furthermore, the same neonatally treated mice showed in vitro functional unresponsiveness of cytotoxic T cells but not of IL-2-secreting cells specific for the tolerated allogeneic MHC antigens. Taken together, our data indicate that neonatal tolerance to MIs-1a can be accomplished by either clonal deletion or clonal anergy, and that it does not necessarily correlate with tolerance to MHC determinants. PMID- 1709048 TI - The gene encoding the stem cell antigen, CD34, is conserved in mouse and expressed in haemopoietic progenitor cell lines, brain, and embryonic fibroblasts. AB - The human haemopoietic cell surface antigen, CD34, is a 105 - 120 kd cell surface glycoprotein whose stage-specific expression by stem cells and lineage-specific progenitor cells suggests a role in regulating early events in blood cell differentiation. A murine gene and cDNA encoding a closely homologous protein have been isolated. The gene is organized in eight exons in 22 kb of DNA. The first exon lies in a GC- and CpG-rich island. The sequence of the gene and the cDNA predict a 382 amino acid-long protein containing an N-terminal signal peptide and one transmembrane region 73 amino acids from the C-terminus. The extracellular part of the protein contains: a 140 amino acid-long-N-terminal region, 40% of whose residues are serine or threonine potential attachment sites for O-linked carbohydrate, as well as five potential attachment sites for N linked carbohydrate. Proximal to the extracellular membrane there is a 79 amino acid-long cysteine-rich region. The homology with the human sequence is highest in the intracellular domain (90% amino acid identity) and lowest in the N terminal region (43% amino acid identity). The protein is not homologous with any other proteins currently in the databases. The expression of the murine gene by a number of haemopoietic progenitor cell lines suggests that the CD34 function in haemopoiesis may be conserved between man and mouse. The high level of expression in a number of embryonic fibroblast cell lines and in brain imply a function outside of haemopoiesis. PMID- 1709049 TI - Independent regulation of interleukin 4 (IL-4)-induced expression of human B cell surface CD23 and IgM: functional evidence for two IL-4 receptors. AB - Activation of human B cells with interleukin 4 (IL-4) is known to result in increased expression of CD23 (the low-affinity receptor for IgE) and sIgM. However, whereas CD23 expression is increased by several B cell mitogens, including phorbol 12-myristate 13-acetate, Epstein-Barr virus, anti immunoglobulin (Ig), and IL-4, surface IgM (sIgM) expression is increased only with IL-4, suggesting that expression of each surface antigen is regulated independently. This was confirmed in three different ways. First, in dose response experiments, it was shown that 10 times the concentration of IL-4 was required for CD23 than for sIgM expression. Similar or even higher concentrations of IL-4 were required for proliferation. In fact, optimal sIgM expression was obtained in some experiments with concentrations of IL-4 (1-5 units/ml) which had little or no effect on either CD23 expression or B cell proliferation. Secondly, IL-4 is known to activate the phosphatidyl inositol pathway in human B cells followed 8-10 min later by an increase in cAMP. Pharmacologically mimicking this pathway by brief exposure of resting B cells to phorbol dibutyrate plus ionomycin followed 10 min later with dibutyryl cAMP resulted in an increase in expression of CD23 but not sIgM. Thirdly, CD19 monoclonal antibody, which inhibits B cell proliferation in response to IL-4 plus anti-Ig, was found to inhibit IL-4-induced CD23 but not sIgM expression. These results show that CD23 and sIgM expression are regulated independently and are consistent with the existence of two separate signal transduction pathways stimulated by IL-4, which may be coupled to distinct IL-4 receptors. PMID- 1709050 TI - Molecular and physiological properties of murine CD14. AB - We have previously cloned the murine homolog of cDNA for the human myelomonocytic differentiation antigen, CD14. We synthesized three hydrophilic peptides derived from the predicted amino acid sequence of murine CD14 (mCD14), designated MS7.1, MS7.2, and MS7.3, respectively, and raised antisera against them. Each antiserum showed specific reactivity to the same peptide used for immunization. One of the anti-mCD14 antisera directed against MS7.3 peptide (AMS7.3) demonstrated the highest titer and definitively reacted with monocytic cell lines, inflammatory polymorphonuclear cells, and macrophages. Significant cross-reactivity of AMS7.3 was observed in the human monocytic cell line, THP-1. COS-1 cells transfected with MS7 cDNA expressed an antigen recognized by AMS7.3. Resident peritoneal and alveolar macrophages both expressed mCD14. mCD14 expression in peritoneal but not alveolar macrophages increased after treatment with lipopolysaccharide. Expression of mCD14 varied among monocytic cell lines and roughly paralleled the mRNA levels except in MI cells. SDS-PAGE and isoelectric focusing analysis of immunoprecipitated mCD14 showed that mCD14 was a 53 kd disulfide-linked protein with a pI of 4.5-5.1. Reduction of molecular weight by endo F treatment demonstrated that mCD14 was an N-linked glycoprotein. Since mCD14 is shed from the cell surface membrane by phosphatidylinositol-specific phospholipase C treatment, the indication is that mCD14 is a phosphatidylinositol-linked protein. The soluble form of mCD14 was detectable. Treatment with anti-mCD14 before interferon gamma (IFN gamma) stimulation significantly enhanced IFN gamma-induced H-2 antigen expression in the macrophage cell line. PMID- 1709051 TI - Autoreactive T cells from normal mice recognize mycobacterial 65 kd heat-shock protein from Mycobacterium bovis. AB - We established a TCR V beta 6+ CD4+ autoreactive T cell line from lymph node cells of unprimed BALB/c mice bred in a specific pathogen-free condition. The T cell line proliferated in the presence of irradiated spleen cells expressing MHC class II I-Ad and/or I-Ed molecules. The proliferative response of the autoreactive T cell line was significantly enhanced in response to 65 kd heat shock protein (hsp) from Mycobacterium bovis, a member of the hsp60 family, which is highly conserved in both prokaryotes and eukaryotes. Our results suggest that the autoreactive T cells in normal mice may recognize endogenous peptides of self hsp60 bound to MHC class II gene products and are cross-reactive to mycobacterial 65 kd hsp. The autoreactive hsp-specific T cells may therefore play an important role in the immune surveillance against invasion of various pathogens and tumors by recognizing infected and transformed autologous cells that express high levels of endogenous hsp60. PMID- 1709052 TI - Ionic channels induced by surfactin in planar lipid bilayer membranes. AB - Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process. PMID- 1709053 TI - Oligomycin interaction with Na,K-ATPase: oligomycin binding and dissociation are slow processes. AB - Oligomycin interacts with the Na,K-ATPase by increasing the apparent Na+ affinity in the non-phosphorylated state of the enzyme. This property is used to estimate rate constants attributed to oligomycin binding and dissociation reactions with Na,K-ATPase. The rate constants are determined indirectly, employing stop-flow fluorimetry of eosin, the fluorescence of which is a marker for the E1 state of the enzyme, i.e. for Na+ binding. The second-order rate constants derived for oligomycin binding are in the range (6-12).10(4) M-1 s-1 at 6 degrees C for both shark rectal gland and pig kidney enzyme. Rate constants for dissociation of the enzyme-oligomycin complex are about 0.05 s-1 at 6 degrees C. The slow rates of binding and dissociation suggest that oligomycin acts from within the membrane lipid phase rather than from the aqueous phase. The dissociation constant at 6 degrees C for the enzyme-oligomycin complex can be calculated to be about 1 microM for shark enzyme and about 2 microM for kidney enzyme, at pH 7.0 in 2 mM NaCl. PMID- 1709054 TI - 5' flanking sequences of human MRP/7-2 RNA gene are required and sufficient for the transcription by RNA polymerase III. AB - Human mitochondrial RNA processing (MRP) RNA is a 270 nucleotide-long small RNA found as ribonucleoprotein particles. In this study, we isolated four human genomic clones with homology to human MRP RNA. Two of these clones contained one copy each of the real gene coding for human MRP RNA; the other two clones represented a processed psuedogene. The Southern blot with the genomic DNA showed that the haploid human genome contains one copy of real gene and a few pseudogenes for MRP/7-2 RNA. The human MRP RNA is synthesized by RNA polymerase III and the 5' flanking sequences -84 to 1 of MRP RNA gene, containing TATA and PSE-like elements, are required and sufficient for transcription in vitro. PMID- 1709055 TI - Genetics of immunoglobulins. PMID- 1709056 TI - Shared thyroglobulin epitopes in autoimmune thyroiditis. PMID- 1709057 TI - Postoperative urinary retention in men. PMID- 1709058 TI - Postoperative retention of urine: a prospective urodynamic study. AB - OBJECTIVE: To investigate the cause of post-operative retention of urine in elderly men. DESIGN: Prospective study. SETTING: Northern General Hospital, Sheffield. PATIENTS: 32 consecutive men (median age 73, range 55-85) referred to the urology department who were unable to pass urine either within 48 hours after operation and required catheterisation (23) or after removal of a catheter inserted at the initial operation (nine). INTERVENTION: Intermittent self catheterisation. MAIN OUTCOME MEASURES: Urological investigation by medium fill and voiding cystometry within four weeks after operation, and minimum follow up three months thereafter. RESULTS: 6 patients resumed normal voiding before urodynamic assessment, three proceeded straight to prostatectomy, and one was unfit for self catheterisation. Of 22 men who underwent urodynamic investigation, only five had bladder outflow obstruction, who subsequently had successful prostatectomy; 15 showed either a low pressure-low flow system (seven) or complete detrusor failure (eight) and two showed pelvic parasympathetic nerve damage. With intermittent self catheterisation spontaneous voiding returned in all but one man within a median of 8 weeks (range 6-32 weeks). Recovery of bladder function took significantly longer in men with detrusor failure than in those with an underactive bladder (median 10 weeks (range 6-32 weeks) v median 8 weeks (range 6-8 weeks); p = 0.05). Three months later all patients had re established their own normal voiding pattern with minimal residual urine on ultrasonography and satisfactory flow rates. CONCLUSIONS: Postoperative urinary retention in elderly men is not an indication for prostatectomy; a normal pattern of micturition can be re-established by intermittent self catheterisation in most men. PMID- 1709059 TI - Surgical palliation of spinal oncologic disease: a review and analysis of current approaches. AB - The clinical problem of primary and secondary vertebral tumours is encountered with increasing frequency throughout North America as oncologic management protocols evolve and the population continues to age. These lesions can present problems of pain, instability and paralysis. Optimal surgical palliation is often of benefit when more conservative methods have been unsuccessful. Vertebral tumours can be approached either anteriorly or posteriorly. These alternatives have led to the evolution in the clinical community of two "camps" championing their respective approaches. The authors have reviewed the accumulated literature up to December 1988 and have found that anterior approaches offer improved clinical outcome without substantially increased morbidity. PMID- 1709060 TI - Acute pancreatitis--30 years' experience at a teaching hospital. AB - Advances in medical technology and knowledge have influenced morbidity and mortality in surgically treated diseases. The authors have compiled four consecutive retrospective studies of demography, morbidity and mortality of patients with acute pancreatitis to summarize the experience from 1956 to 1985 at the Montreal General Hospital with 629 patients. The death rate has remained unchanged. Hypotension, gastrointestinal bleeding and respiratory failure have assumed lesser roles as major complicating factors. Renal failure and gram negative aerobic pancreatic sepsis are the common causes of death. The last two reviews revealed that surgical debridement and drainage combined with appropriate biliary procedures salvaged two-thirds of the patients with sepsis. Deteriorating nutritional status, heralded by a fall of serum albumin level below 30 g/L, is associated with a poor prognosis. Interval cholecystectomy in patients with mild biliary tract pancreatitis is associated with a low death rate (0.01%). PMID- 1709061 TI - Solitary minute hepatocellular carcinoma. A study of 14 patients. AB - Fourteen patients with clinical Stage I hepatocellular carcinoma (T1N0M0) were studied. All patients were asymptomatic, and their conditions were detected by alpha-fetoprotein (AFP) serosurvey and/or ultrasonography (US) either in the natural population in the early years of the study or in the high-risk population in the later years of the study. Cirrhosis was present in all patients. Radical resection was performed in all patients. There were no operative deaths or hospital deaths in this series. The 5-year survival rate after resection was 100%. There were seven long-term survivors in this series (14.2 years (alive), 11.3 years (alive), 8.8 years (alive), 8.8 years, 7.9 years, 7.6 years (alive), and 7.2 years after resection). The authors discuss aspects concerning early diagnosis, treatment, and prognosis of hepatocellular carcinoma (HCC). PMID- 1709062 TI - Immunohistochemical evidence of immune responses to tumor-associated antigens in lymph nodes of colon carcinoma patients. AB - The authors investigated by immunohistochemical study the drainage of three tumor associated antigens in unaffected regional lymph nodes of colon cancer patients. The study was conducted using monoclonal antibodies (MoAb) directed against different epitopes of the tumor-associated glycoprotein, TAG-72 (CC-49, CC-83, B72.3), of the carcinoembryonic antigen (CEA) (COL-4, COL-12), and of the colon associated antigen, CAA (anti-CAA). The authors detected immunohistochemical reactions of MoAb CC-49 and anti-CAA with antigen-presenting cells (APC), such as peritumoral and sinus macrophages and lymphatic endothelial cells and with specific areas of germinal centers in lymph nodes draining 11 of 24 colorectal carcinomas studied. The corresponding primary tumors expressed the TAG-72 and CAA antigens. No immunostaining was detectable in lymph nodes using the anti-CEA MoAb, even when the primary tumors strongly expressed the specific epitopes. In germinal centers of regional lymph nodes, the immunostaining was often distributed at the periphery with a characteristic crescentic or circular pattern, which strongly suggested the exposure of the specific epitopes defined by MoAb CC-49 and anti-CAA on follicular dendritic cells. This would indicate that these epitopes are selectively recognized and presented to germinal center B cells. This phenomenon may have clinical and diagnostic implications. PMID- 1709063 TI - Responsiveness of hepatocytes to epidermal growth factor, transforming growth factor-beta and norepinephrine during treatment with xenobiotic hepatic tumor promoters. AB - Previous studies with xenobiotic hepatic tumor promoters have shown that prolonged treatment with these substances decreases the level of epidermal growth factor (EGF) receptors and the response of hepatocytes to EGF. In this study we show that, in contrast to prolonged exposure, the early stage of exposure to tumor promoters is associated with enhanced responsiveness to EGF. Differences in the patterns of responsiveness to EGF are noticed between two tumor promoters, phenobarbital (PB) and alpha-hexachlorocyclohexane (alpha-HCH). Norepinephrine is one of the strongest comitogens (mitogenic amplifiers) for hepatocytes. In addition to altering responsiveness to EGF, treatment with the tumor promoters eliminated the response to norepinephrine during the early stage of treatment. Transforming growth factor-beta (TGF-beta), a well known inhibitor of EGF mitogenesis in hepatocytes, inhibited the EGF-induced DNA synthesis but did not affect the DNA synthesis stimulated directly by the tumor promoters. Long term treatment with the tumor promoters inhibited responsiveness to both EGF and norepinephrine. The implications of these findings for mechanisms of tumor promotion in the liver are discussed. PMID- 1709064 TI - Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus. AB - 1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus. PMID- 1709065 TI - [Serum alpha-1-fetoprotein in pregnancy in relation to length of gestation]. AB - In a group of 310 immunoenzymatic examinations of MS AFP by the Sevatest ELISA AFP Micro I kit in 310 women with normal pregnancies the authors detected the exponential character of the rise of serum AFP up to the 35th week of pregnancy (or foetal length of 88 mm resp.): in relation to the foetal length according to equation y(mean) = 10.544 X 1.0369x (for x = 28 to 88 mm) and in relation to the gestation period according to formula y(mean) = 7.720 X 1.1097x (for x = 15 to 35 weeks). During the subsequent course of gestation a steady decline of levels occurs and in the 41st week the mean MS AFP values decline to the level of the 22nd week of gestation (in foetal length of 99 mm the levels correspond to that during foetal length of 55 mm). PMID- 1709066 TI - [Signal transmission in plasma membranes of animal cells]. PMID- 1709067 TI - [G-proteins and their function]. PMID- 1709068 TI - [A program for evaluating ion channel conductivity using the PMD-85 microcomputer and a laboratory unit for communicating with the environment]. PMID- 1709069 TI - Diagnosis of Wiskott-Aldrich syndrome by analysis of the X chromosome inactivation patterns in maternal leucocyte populations using the hypervariable DXS255 locus. AB - The Wiskott-Aldrich syndrome (WAS) is characterized by severe recurrent infections, petachiae and chronic eczema. The syndrome involves differentiation disorders in several haematopoietic cell lineages usually manifested as T lymphocyte deficiency, dysgammaglobulinaemia and thrombocytopenia. The defect is inherited in an X-linked recessive mode. A 1-year-old boy presented with otitis, upper respiratory infections, eczema, a persistent granulocytopenia and a dysgammaglobulinaemia. In his family five males in two generations had been shown to have WAS, which entailed a significant risk for the patient to have WAS. As the WAS gene or gene product is not delineated, the symptoms of the patient presented a diagnostic dilemma. If the boy had inherited the disease, his mother should be a WAS carrier. Segregation analysis in the family using the closely linked restriction fragment length polymorphisms (RFLP) DXS7, DXS255 and DXS14 did not exclude her carriership, although the probability was low. As a result of the differentiation arrest, obligate female WAS carriers manifest a unilateral X chromosome inactivation pattern in several haematopoietic cell lineages. Methylation analysis of the X chromosomal DXS255 loci exposed random X chromosome inactivation patterns in the peripheral blood granulocytes, T lymphocytes and B lymphocytes of the patient's mother. These findings excluded her WAS carriership and therefore excluded the diagnosis of WAS in the patient. This was further substantiated in a 1-year follow up with recovery from the haematological and immunological symptoms. These results demonstrated that X inactivation analysis in maternal leucocytes is decisive in the exclusion of the diagnosis of WAS. PMID- 1709070 TI - Localization of 20-kD homologous restriction factor (HRF20) in diseased human glomeruli. An immunofluorescence study. AB - The 20-kD homologous restriction factor (HRF20), which is identical to CD59, is a membrane-associated protein which inhibits the reaction of C9 to form membrane attack complex (MAC) of homologous complements. In various human glomerular diseases deposition of complement components is frequently seen and MAC is reported to associate with immune deposits. Using a specific monoclonal antibody, 1F5, against HRF20, we attempted to study the localization of HRF20 in human glomerulonephritides and to compare the localization of HRF20 with those of immune deposits and MAC. The frozen sections of kidney specimens were fixed in acetone at room temperature before staining. In normal kidneys and kidney specimens from the patients with minimal change nephrotic syndrome, membranous nephropathy, and IgA nephropathy, HRF20 was strongly localized in the peritubular capillaries and along Bowman's capsules. A weaker but well-defined staining was obtained in the mesangial area and faint staining was seen along the glomerular capillary walls. In contrast, glomerular capillary walls were rather strongly stained in the cases with diffuse lupus nephritis which had subendothelial dense deposits. These data suggest that HRF20 (CD59) is present in the human glomeruli and its expression is enhanced under certain conditions such as lupus nephritis. PMID- 1709071 TI - Expression of the fibronectin receptor VLA-5 is regulated during human B cell differentiation and activation. AB - We examined the expression of VLA-5, a fibronectin receptor, during human B cell development and activation. VLA-5 is a member of the integrin supergene family; VLAs are heterodimers of at least six unique alpha chains sharing a common beta chain; most are involved in cell attachment to extracellular matrix (ECM). A hypothesis of haematopoietic development is that maturing cells leave the bone marrow because of the loss of VLA-5 during differentiation. However, mature B cells are not primarily circulating cells, and the role of ECM receptors in homing to peripheral lymphoid tissue and inflammatory sites is unknown. To examine the expression of VLA-5 during B cell development, cell lines blocked at specific stages of differentiation were evaluated for their synthesis and surface expression of VLA-5 using VLA-5-specific antibody and cDNA probes. VLA-5 mRNA and surface expression were found in the pre-B cell lines, REH and Nall 1, but not in more differentiated Raji cells or in several EBV-transformed peripheral B cell lines. Circulating peripheral B lymphocytes and resting tonsillar and splenic B lymphocytes expressed no VLA-5 by FACS analysis. Interestingly, mRNA and surface expression of VLA-5 were found in SKW, a highly differentiated, IgM-secreting line. In addition, low levels of staining for VLA-5 expression could be demonstrated when tonsillar or peripheral blood B lymphocytes were stimulated by Staphylococcus aureus Cowan (SAC). All cell lines expressed VLA-3 and VLA-4, two other receptors reported to mediate fibronectin binding in some cell types. Thus, our studies provided no evidence for developmental or inflammatory regulation of these receptors. Binding studies, however, demonstrated that adherence of both pre-B REH cells and SKW cells to fibronectin was almost completely inhibited by a monoclonal antibody to VLA-5 alpha. In addition, Raji cells, which lack VLA-5 but express VLA-3 and VLA-4, showed very low level binding to fibronectin. This demonstrates that for some B lymphocytes VLA-5, rather than other possible fibronectin receptors, primarily mediates attachment to fibronectin. These data also suggest that human VLA-5 expression is regulated during B cell development, with expression at a very early stage and then again after activation. This pattern of loss and reacquisition of an ECM receptor may be relevant to normal B cell maturation and to function during immunologic injury. PMID- 1709072 TI - Cord blood contains cells secreting antibodies to nervous system components. AB - Umbilical cord blood of newborns and peripheral blood of healthy adults were investigated by an immunospot assay for cells secreting IgG, IgA and IgM antibodies against myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) which represent putative antigens for an autoimmune attack in multiple sclerosis (MS) and against acetylcholine receptor (AChR) which is considered an important autoantigen in myasthenia gravis. Cells secreting antibodies against one or more of these autoantigens were detected in 18 out of 24 newborns, and in eight out of 20 adults. Eight of the cord blood samples contained cells secreting antibodies of IgG, IgA and/or IgM isotypes to one antigen, five to two antigens, two to three antigens, two to four antigens, and one to five antigens. Most prominent were anti-MBP IgG antibody secreting cells which were detected in 13 newborns at a mean number of 1/20,000 cord blood cells, and in six adults at a mean number of 1/10(5) peripheral blood cells. Anti-AChR IgG antibody secreting cells were detected in four out of 12 newborns versus four out of 14 peripheral blood specimens, at mean values of 1/10(5) cells in both instances. Cells secreting autoantibodies of IgA and IgM isotypes were less frequent both in cord blood and peripheral blood. The occurrence of nervous tissue autoantibody secreting cells in newborns must be related to a possible primary role of such autoantibodies in MS and myasthenia gravis. PMID- 1709073 TI - Other mimics of lung metastases--a reminder. PMID- 1709074 TI - Lowering of pancreatic amylase activity induced by cold exposure, fasting and adrenalectomy in rats. AB - 1. Alteration of amylase activity in the pancreas was studied in rats under different stressful situations. 2. Cold-exposed rats had reduced amylase activity, hypertrophy of adrenal glands, and lowered plasma insulin and glucose. 3. In fasted rats, amylase activity decreased and plasma insulin and glucose lowered. 4. Immobilized animals failed to produce the reduced amylase activity, despite existence of higher plasma corticosterone. 5. Adrenalectomy caused reduction of amylase activity accompanied by decreases in plasma insulin and glucose. 6. Glucocorticoid hormone is not correlated with suppression of amylase synthesis. The lowered amylase activity might involve changes in glycolytic flux and/or suppression of amylase synthesis by insulin deficiency. PMID- 1709075 TI - Isolation and characterization of fatty acid binding protein in the liver of the nurse shark, Ginglymostoma cirratum. AB - 1. A 14.5 kDa fatty acid binding protein was isolated from the liver of the nurse shark, Ginglymostoma cirratum. 2. Purified shark liver FABP (pI = 5.4) bound oleic acid at a single site with an affinity similar to that of mammalian FABP. 3. The apparent size, pI and amino acid composition of shark liver FABP indicate a close structural relationship between this protein and mammalian heart FABP. PMID- 1709076 TI - Substance P receptors in mammalian central nervous system. AB - 1. Multiple distinct affinity states or sites of substance P (SP) receptors exist in freshly-prepared rat brain membranes. 2. Substance P receptors may couple with islet-activating protein (pertussis toxin) sensitive GTP-binding protein(s). 3. Substance P receptors may be regulated Mg2+ and Na+ in an opposite manner. 4. Some important factor(s), in addition to GTP-binding protein, appear to be involved in SP binding activity. 5. An apparent molecular weight of the SP binding site is approximately 46,000 Da. PMID- 1709077 TI - Neurokinin A and B. AB - The chemical features, pharmacological and biochemical characteristics of novel mammalian tachykinin peptides, neurokinin A and neurokinin B are described and are compared with those of substance P, a representative of tachykinin family. PMID- 1709078 TI - Properties of sympathetic neuromuscular transmission and smooth muscle cell membranes in vascular beds. AB - In vascular smooth muscle tissues, the cycle of contraction-relaxation is mainly regulated by the cytosolic Ca, and many other factors, such as substances released from endothelial cells and perivascular nerve terminals (mainly sympathetic nerves). In this article, we introduce regional differences in specific features of ionic channels in vascular smooth muscle membranes (mainly on features of Ca, Na and K channels) in relation to mobilization of the cytosolic Ca. In many vascular tissues, neurotransmitters released from sympathetic nerve terminals activate post-junctional receptors, and subsequently modify ion channels (receptor-activated cation channel and voltage-dependent Ca channel), whereas in some tissues, ionic channels are not modified by receptor activations (pharmaco-mechanical coupling). However, activation of receptors, with or without modulation of ionic channels, regulates the cytosolic Ca through synthesis of second messengers. In addition, receptors distributed on prejunctional nerve terminals positively or negatively regulate the release of transmitters. Roles of neurotransmitters (mainly ATP and noradrenaline) are also discussed in relation to the generation of excitatory junction potentials. PMID- 1709079 TI - Molecular biology of myelin basic protein: gene rearrangement and expression of anti-sense RNA in myelin-deficient mutants. AB - 1. Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system (PNS). 2. Myelin basic protein (MBP) is a major protein in CNS myelin. 3. MBP is expressed specifically in the nervous system. 4. The MBP gene has been cloned and characterized. 5. Two mutant mice, Shiverer (shi) and myelin-deficient (mld. shimid), are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the central nervous system. 6. It was found that rearrangement within the MBP gene results in low expression of the gene. 7. In Shiverer, the MBP gene is partially deleted (from exons 3 to 7), and in mld, the gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. 8. In mld, anti-sense RNA complementary to exons 3 7, which correspond to the inverted segment, was detected by RNase protection studies, and presumed to be responsible for the reduced expressions of MBP. 9. The mechanism of gene rearrangement in MBP was also characterized. 10. This article reviews the recent progress in the study of the MBP gene, especially the rearrangement of the gene and its expression in mutant mice. PMID- 1709080 TI - Comparative studies on myelin proteins in mammalian peripheral nerve. AB - Myelin proteins in mammalian peripheral nerve were studied comparatively. 1. While each content of P1 and P2 in the myelin varied among species, additional content of P1 and P2 are relatively constant. 2. The antigenic determinants of P2 for induction of experimental allergic neuritis were reported. 3. Amino acid sequence analysis of P0 revealed that P0 is conserved across species and belongs to the immunoglobulin superfamily. 4. The characteristic carbohydrate chain of P0 containing sulfate and sialic acid showed a positive reaction to the molecule related immunity and adhesion. 5. Molecular architecture of the myelin is discussed. PMID- 1709081 TI - Competency-based orientation program for a surgical intensive therapy unit--Part 2. PMID- 1709082 TI - Investigation of inter-alpha-trypsin inhibitor and slow migrating proteinase inhibitors in serum and urine. PMID- 1709083 TI - Filamentous organisms in gastric brushings and gastric cancer diagnosis. AB - An association between filamentous organisms and gastric carcinoma has been reported in gastric biopsies. Review of 45 Papanicolaou-stained gastric brushing specimens revealed filamentous organisms in two cases. In both cases, gastric carcinoma was confirmed on follow-up. Cytopathologists should be aware that the identification of filamentous organisms in gastric brushings is a specific, although insensitive marker of gastric cancer. PMID- 1709084 TI - Diagnosis of Pneumocystis carinii by cytologic examination of Papanicolaou stained sputum specimens. PMID- 1709085 TI - Cytological detection of atypical cells by routine urinalysis in a cardiovascular center. AB - During the past 9 yr, 187,529 Sternheimer-Malbin-stained urinary sediments were examined as routine urinalysis specimens from patients attending the National Cardiovascular Center in Osaka, Japan. Abnormal cells were found in 20 patients who did not have clinical diagnoses of malignancy. Malignant cytological changes in 18 patients resulted in a rate of 1 case in 6,751 patients; the two remaining specimens with abnormal cells showed polyomavirus infection. This article describes our experience in the diagnosis of malignant cells of the urinary tract through the cooperation of the clinical and cytological laboratories. Since in Japan, the rate of death for bladder cancer is similar to 1 in 6,751, this method seems to be of great use in the diagnosis of urinary tract malignancies. PMID- 1709087 TI - Fine-needle aspiration cytology of papillary Hurthle-cell tumors of thyroid: a report of three cases. AB - Papillary Hurthle-cell tumors of the thyroid are composed of oxyphilic cells with a prominent papillary component. Their biologic behavior appears to be comparable with that of conventional papillary thyroid carcinomas. The literature on the fine-needle aspiration cytology of these tumors is scanty. The cytologic findings of three such cases are reported here. The findings suggest that most papillary Hurthle-cell tumors cannot be differentiated from other types of Hurthle-cell tumors cytologically. PMID- 1709086 TI - Cytology of treated and minimal Pneumocystis carinii pneumonia and a pitfall of the Grocott methenamine silver stain. AB - To clarify the role of foamy alveolar casts (FACs) in the diagnosis of Pneumocystis carinii pneumonia (PCP), we retrospectively reviewed Papanicolaou (Pap)- and Grocott methenamine silver (GMS)-stained slides from 205 bronchial specimens submitted for suspected opportunistic lung infection. FACs containing sporozoites were seen in 86 cases, all with positive GMS. FACs were absent in 119 cases with negative GMS. In patients previously treated for PCP, Pap showed FAC like material which lacked sporozoites. GMS demonstrated clumped degenerated Pneumocystis carinii (PC) cysts. In 17 GMS-positive cases there were no FACs on Pap due to sampling error and low burden of organisms. Morphologic features of macrophages in these cases can suggest the presence of PC. When FACs are lacking, a special stain is required. When GMS is used, the control must contain PC to avoid a false negative GMS. The inconsistent uptake of GMS by PC is a pitfall which is distinct from sampling error and which to our knowledge has not been previously reported. PMID- 1709088 TI - Carcinoma of the breast in a fibroadenoma: diagnosis by fine-needle aspiration cytology. AB - A case of carcinoma of the breast concurrent with a fibroadenoma in a 49-yr-old female is described in which the diagnosis was made on fine-needle aspiration cytology. Reports of such an occurrence are few, and to our knowledge none has previously documented the cytologic findings in detail. PMID- 1709089 TI - Diagnosis of pulmonary blastoma by fine-needle aspiration biopsy: cytologic and immunocytochemical findings. AB - A preoperative diagnosis of pulmonary blastoma was established by fine-needle aspiration (FNA) cytology in a patient who presented with a pulmonary mass discovered incidentally on chest x-ray. At the time of computed tomography-guided FNA, an on-site preliminary diagnosis of poorly differentiated carcinoma was made after examination of a rapid hematoxylineosin (H&E)-stained smear. Malignant epithelial and stromal elements could be demonstrated on smears subsequently stained with the H&E, Papanicolaou, and Diff-Quik methods. The cell block preparation showed a distinctly biphasic malignant tumor with classic morphologic features of pulmonary blastoma. We present the clinical, cytologic, immunocytochemical, and histologic findings of this case and re-emphasize the valuable contribution of adequate cell block preparations to accurate diagnosis of fine-needle aspiration material. PMID- 1709090 TI - Fine-needle aspiration cytology of adrenal cryptococcosis: a case report. AB - A case of adrenal cryptococcosis diagnosed by fine-needle aspiration biopsy cytology in a 58-yr-old man is presented. The organisms were easily seen with routine modified Wright stain (Diff-Quik) as variably sized yeasts, some with a brightly eosinophilic capsule. The diagnosis was confirmed with mucicarmine and silver stains. The identification of fungi with routine cytologic stains allows a rapid presumptive diagnosis of the infectious agent, collection of material for confirmatory special stains, and prompt initiation of therapy. PMID- 1709091 TI - Diagnosis of hepatic actinomycosis by fine-needle aspiration. AB - A 48-yr-old white female with a 20-yr history of intrauterine device use presented with signs and symptoms suggestive of a disseminated neoplasm with a pelvic primary. A definitive diagnosis of disseminated actinomycosis was made on the basis of computed tomography-guided fine-needle aspiration of a liver lesion. Subsequent laparotomy revealed involvement of the endometrium and of the ovaries and uterine tubes bilaterally. This article indicates the usefulness of fine needle aspiration in the diagnosis of actinomycosis in this case. PMID- 1709092 TI - [Extrasystole during extracorporeal biliary shockwave lithotripsy]. PMID- 1709093 TI - Extinction of the HPV18 upstream regulatory region in cervical carcinoma cells after fusion with non-tumorigenic human keratinocytes under non-selective conditions. AB - 'Universal fuser' clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non-selective conditions. The CAT expressing cells were fused with both non-tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non-modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non-selectable, HPV18-driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa x HaCaT cells, but still present in SiHa x SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non-tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion-amplification technique are accompanied by the reactivation of the marker gene in previously CAT- SiHa x HaCaT hybrids. These data strongly suggest that trans-acting negative regulatory factors derived from the non malignant human keratinocytes are responsible for the extinction phenomenon. PMID- 1709094 TI - FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern. AB - We have previously identified two novel members of the fibroblast growth factor receptor (FGFR) gene family expressed in K562 erythroleukemia cells. Here we report cDNA cloning and analysis of one of these genes, named FGFR-4. The deduced amino acid sequence of FGFR-4 is 55% identical with both previously characterized FGFRs, flg and bek, and has the structural characteristics of a FGFR family member including three immunoglobulin-like domains in its extracellular part. Antibodies raised against the carboxy terminus of FGFR-4 detected 95 and 110 kd glycoproteins with a protein backbone of 88 kd in COS cells transfected with a FGFR-4 cDNA expression vector. The FGFR-4 protein expressed in COS cells could also be affinity-labeled with radioiodinated acidic FGF. Furthermore, ligand binding experiments demonstrated that FGFR-4 binds acidic FGF with high affinity but does not bind basic FGF. FGFR-4 is expressed as a 3.0 kb mRNA in the adrenal, lung, kidney, liver, pancreas, intestine, striated muscle and spleen tissues of human fetuses. The expression pattern of FGFR-4 is distinct from that of flg and bek and the yet additional member of the same gene family, FGFR-3, which we have also cloned from the K562 leukemia cells. Our results suggest that FGFR-4 along with other fibroblast growth factor receptors performs cell lineage and tissue specific functions. PMID- 1709095 TI - Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium. AB - The predicted protein domains coded by exons 9-12 and 19-23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide-binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9- mRNA transcripts). In most (9 of 12), the exon 9- transcripts represented less than or equal to 25% of the total CFTR transcripts. However, in three individuals, the exon 9- transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re-evaluation of the same individuals 2-4 months later showed the same proportions of exon 9- transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon-intron junctions relevant to exon 9 deletion (exon 8-intron 8, intron 8-exon 9, exon 9-intron 9, and intron 9-exon 10) were identical except for the intron 8-exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9- transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype. PMID- 1709097 TI - Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase. AB - Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments. PMID- 1709096 TI - Cyclin B targets p34cdc2 for tyrosine phosphorylation. AB - A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts. PMID- 1709098 TI - Pumpkin malate synthase. Cloning and sequencing of the cDNA and northern blot analysis. AB - A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979 nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed. PMID- 1709099 TI - Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor beta. Comparison of transcriptional and post-transcriptional control with fibronectin and type I collagen. AB - Transforming growth factor beta (TGF-beta) and secreted protein acidic rich cysteine (SPARC) have been associated with the rapid remodeling of connective tissues that occurs in wound healing and developmental processes. To study the temporal and mechanistic aspects of TGF-beta-regulated extracellular-protein gene expression in human fibroblasts, confluent cells were pulse labeled for 30 min with [35S]methionine at various times following the single addition of 1.0 ng/ml TGF-beta. After a 4-h chase period, specific radiolabeled media proteins were isolated by either immunoprecipitation or affinity chromatography and quantitated. Stimulation of SPARC synthesis was first apparent 5 h after addition of TGF-beta, reached a maximum (3.5-fold increase) at 24 h and persisted for at least 96 h. A similar temporal response to TGF-beta was observed for the extracellular matrix proteins collagen and fibronectin. In contrast, TGF-beta induced a strong (greater than sixfold increase at 9 h after addition of TGF beta), but transient stimulation of the synthesis of endothelial-type plasminogen activator inhibitor. Northern blot analysis showed that SPARC mRNA levels were increased by TGF-beta in parallel with increase in SPARC synthesis; a maximum 3.9 fold increase in SPARC mRNA being reached at 24 h. Similarly, the levels of both collagen and fibronectin mRNA were increased by TGF-beta treatment. In each case the stimulation of mRNA was blocked by the presence of the translation inhibitor, cycloheximide. Stability of SPARC mRNA (half-life of approximately 50 h) was not significantly altered by TGF-beta. In contrast, the stability of collagen and fibronectin mRNA were both increased in the presence of TGF-beta; the increased stability being pronounced in less dense cells. In addition to effects on stability, transcription of the collagen and fibronectin genes was increased 7 h after TGF-beta addition, but returned to control levels by 24 h. However, transcription of the SPARC gene was unaffected by TGF-beta at both time points and, together with the stability data, indicates that TGF-beta regulates SPARC expression via a nuclear post-transcriptional mechanism. Differential regulation of gene expression by TGF-beta in a precise temporal pattern via transcriptional and post-transcriptional pathways may be an important aspect of the response of fibroblast cells in a wound environment. PMID- 1709100 TI - Insulin binds to type V collagen with retention of mitogenic activity. AB - The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity. PMID- 1709101 TI - Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts. AB - In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time. PMID- 1709102 TI - Localization of the 230-kilodalton bullous pemphigoid antigen in cultured keratinocytes: formation of a prehemidesmosome. AB - The hemidesmosome is the major attachment structure of the epidermal basal cell visible ultrastructurally in skin. The importance of its components to cultured cell attachment to substratum is not understood, however. A component of the hemidesmosome, the 230-kDa bullous pemphigoid antigen (p230), has been shown to be present in an insoluble or particulate fraction of cultured cells. In order to more fully characterize its potential importance for cell-matrix adhesion in cultured keratinocytes, specific antibodies were raised to the C-terminal region of p230 expressed as a bacterial fusion protein. Such antibodies recognize the hemidesmosome of epidermis, binding on the cytoplasmic region of its plaque. In addition, keratinocytes cultured in a 0.15 mM Ca(2+)-defined medium contain a detergent-resistant pool of p230 which appears to lie in the same focal plane as the culture substrate and has a patchy or irregular distribution by indirect immunofluorescence. Treatment of cultured cells at 4 degrees C with trypsin or pronase sufficient to release keratinocytes from the culture dish does not affect the electrophoretic migration of p230 on SDS-gels, suggesting that p230 is not exposed to the extracellular space. In cells cultured in 0.15 mM Ca2+, 230-kDa BP antigen is localized to discrete clusters resting near the basal plasma membrane of the cell by immunogold staining following brief detergent treatment and fixation. These clusters are approximately 0.1 micron in diameter, which is similar in size to the in vivo hemidesmosome. Fully formed electron dense hemidesmosomal plaques are not observed under the same culture conditions, however. It appears that these clusters are early precursors of the hemidesmosome. PMID- 1709103 TI - Morphological differentiation of hybrids of human mammary epithelial cell lines is dominant and correlates with the pattern of expression of intermediate filaments. AB - Hybrids have been developed from two cell lines originally derived from human mammary epithelial cells. MTSV1-7 (hygro), developed from cultured milk epithelial cells, shows a cuboidal morphology, expresses high levels of keratins (but no vimentin), and forms ball-like three-dimensional structures in collagen gels. 5.3.1E (neo), derived from a cell cultured from tissue taken from a primary breast cancer, shows an elongated morphology, expresses high levels of vimentin and low levels of keratins, and does not form structures on collagen gels. An examination of the hybrids formed by fusion of MTSV1-7 cells and 5.3.1E cells showed that while all could form three-dimensional structures in collagen gels, the type of structures formed resembled either ductal or alveolar-ball-like structures depending on the individual hybrid. Nine hybrids were examined and a clear correlation was observed between cell shape as seen on plastic, intermediate filament expression, and the form of the structures as seen in collagen gels. All the hybrids resembling the parent MTSV1-7 cells by showing a cuboidal morphology and high level of keratin expression formed ball-like structures; on the other hand, hybrids resembling 5.3.1E by showing an elongated morphology and a high level of vimentin expression formed duct-like structures in collagen gels. The results show that the ability to form structures in collagen gels was inherited in a dominant fashion from the MTSV1-7 parent. They also suggest that the profile of intermediate filament expression in the hybrids may influence both cell shape on plastic and the morphology of the three-dimensional structures formed in collagen gels. PMID- 1709104 TI - Epithelial synthesis of tenascin at tips of growing bronchi and graded accumulation in basement membrane and mesenchyme. AB - The extracellular matrix protein, tenascin, has been proposed as mediator in epithelial-mesenchymal interactions because of its characteristic distribution during embryogenesis. Here we compared the accumulation of tenascin and laminin in the early chicken lung bud. Laminin is deposited in the basement membrane, starting at the tips and increasing along the shafts of growing primary and secondary bronchi. In contrast, tenascin accumulation is highest in basement membranes and mesenchyme at sites where new bronchial branches are formed. By in situ hybridization, tenascin mRNA was found to be produced exclusively by the epithelium at sites of active growth of bronchial tubes. PMID- 1709105 TI - Microfilament distribution in cold-treated Drosophila embryos. AB - Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle. PMID- 1709106 TI - Effects of G-CSF, GM-CSF, and IL-5 on nuclear segmentation of neutrophils and eosinophils in congenital or acquired Pelger-Huet anomaly. AB - We studied the in vitro effects of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 5 (IL 5) on nuclear segmentation of neutrophils and eosinophils from three patients with congenital or acquired Pelger-Huet anomaly. After a 24-h incubation with G CSF, the majority of neutrophils showed nuclear development characterized by a bilobed appearance. In contrast to neutrophils, the nuclear segmentation of eosinophils was not induced after incubation with G-CSF, GM-CSF, or IL-5. These results suggest that G-CSF plays some role in the nuclear development of neutrophils, whereas IL-5 may not have such an effect on eosinophil maturation in the individual cases studied. PMID- 1709107 TI - Separation of functionally distinct subpopulations of primitive human hematopoietic cells using rhodamine-123. AB - Normal human bone marrow (BM) contains a small population of cells that can give rise to clonogenic progenitors after 5 weeks in long-term culture (LTC). We have previously shown that these LTC-initiating cells (LTC-IC) differ from the majority of directly clonogenic cells with respect to both light-scattering properties and surface antigen expression. In this paper we show that virtually all LTC-IC (94%) are among the 3%-5% of light-density marrow cells that take up relatively low amounts of rhodamine-123 (Rh-123). In contrast, only 70% of erythroid burst-forming units (BFU-E) and 40% of granulocyte-macrophage colony forming units (CFU-GM) are recovered in the Rh-123-dull fraction. In addition, we have found that double staining of marrow with Rh-123 and phycoerythrin-labeled anti-CD34 antibodies allows the CD34+ cells to be divided into two subpopulations, of which, on average, 35% are Rh-123-dull. Isolation of these CD34+ Rh-123-dull cells thus provides a single-step enrichment of approximately 240-fold in LTC-IC by comparison to the light-density (less than 1.077 g/cm3) fraction of normal BM. This represents an overall enrichment in LTC-IC of approximately 1000-fold. As expected from the results of staining with Rh-123 only, the majority of directly clonogenic cells are present in the CD34+ Rh-123 bright fraction, where they are enriched approximately 40-fold over their concentration in the light-density fraction. These results indicate marked differences in Rh-123 uptake between subsets of primitive human hematopoietic cells currently defined by different functional assays and suggest that RH-123 staining will be useful for the further purification and analysis of these cells. PMID- 1709108 TI - Constitutive expression of leukemia inhibitory factor RNA by human bone marrow stromal cells and modulation by IL-1, TNF-alpha, and TGF-beta. AB - Recent in vitro studies indicate that bone marrow mesenchymal elements, residing in close proximity to hematopoietic cell populations, elaborate a network of cytokines that are, at least partially, responsible for modulating the growth and maturation of the latter compartment. Leukemia inhibitory factor (LIF), a molecule with both positive and negative regulatory activities, has been implicated in murine embryogenesis and hematopoiesis. We demonstrate that cultured normal human bone marrow stromal cells constitutively express LIF message. Further, exposure of these cells to other hematopoietic modulators including interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF alpha) (but not interferon-alpha [IFN alpha]) increases the level of LIF RNA. Interestingly, cultured stromal cells derived from three of four patients with chronic myelogenous leukemia showed enhanced LIF expression. These observations suggest that LIF may participate, either alone or through interaction with other cytokines, in the bone marrow microenvironment-mediated influence on both normal and malignant hematopoietic processes. PMID- 1709109 TI - Effect of recombinant human granulocyte colony-stimulating factor administration in normal and experimentally infected newborn rats. AB - We investigated the effects of repetitive recombinant human granulocyte colony stimulating factor (rhG-CSF) administration at three different doses (every 12 h times six doses, starting at 12-24 h of age) on the kinetics of neutrophil production in Sprague-Dawley rats. We determined WBC counts, differentials, the number of total nucleated cells, the myeloid mitotic pool cells (promyelocytes and myelocytes), the storage pool cells (metamyelocytes, bands, and polymorphonuclear cells [PMNs]) and the granulocyte-macrophage (granulocyte macrophage colony-forming units, CFU-GM) and macrophage (macrophage colony forming units, CFU-M) progenitor cells of the bone marrow, spleen, and the liver before the first dose of rhG-CSF administration and 12 h after the second, fourth, and sixth dose. Control animals were given the diluent by the same schedule. Recombinant human G-CSF-treated rats showed a significant dose dependent increase in the number of total WBC and neutrophil counts at all time points compared to control rats. The total number of CFU-GM and myeloid mitotic pool cells (marrow plus spleen plus liver) progressively increased with age in both control and G-CSF groups, but the G-CSF treated groups showed a significantly larger number of mitotic pool cells at hour 24, continuing up to hour 72, compared to the control group. However, there was no significant difference at any time point in the number of CFU-G/GM as detected by the granulocyte-macrophage colony-stimulating factor (GM-CSF)-supported culture system. Priming of newborn rats with injections every 12 h of rhG-CSF times two doses, or six doses followed by inoculation of group B streptococci (GBS) did not significantly change the sepsis death rate of animals, although the neutrophil counts in infected rhG-CSF-primed animals were significantly larger than the infected control animals. Injection of human i.v. gammaglobulin 3 h following inoculation with GBS significantly improved the survival of animals compared to G CSF administration or administration of the diluent alone (control). Thus G-CSF alone may not be beneficial for the treatment of neonates with sepsis. Additional work is needed to determine whether combination of G-CSF with antibiotics or other cytokines, such as GM-CSF or interleukin 6 (IL-6) may be of benefit. PMID- 1709110 TI - Effects of recombinant interleukin 11 on human megakaryocyte progenitor cells. PMID- 1709111 TI - Demonstration of projections from the lateral nucleus to the basal nucleus of the amygdala: a PHA-L study in the monkey. AB - Previous studies of the intrinsic connections of the amygdaloid complex in the rat, cat and monkey demonstrated that the lateral nucleus projects prominently to the accessory basal nucleus and periamygdaloid cortex and lightly to several other nuclei of the amygdala. Most previous studies have emphasized the lack of a connection between the lateral and basal nuclei. As part of ongoing studies of the intrinsic circuitry of the monkey amygdala, we have placed discrete, iontophoretic injections of the lectin anterograde tracer Phaseolus vulgaris leucoagglutinin into the lateral nucleus of the Macaca fascicularis monkey. In addition to confirming the well established intrinsic connections of the lateral nucleus, heavy fiber and terminal labeling was also observed in the adjacent basal nucleus. The projection innervated all divisions of the basal nucleus and tended to be somewhat denser rostral to the level of the injection site. This hitherto unreported intrinsic connection provides a means by which the lateral nucleus, which is the principal recipient of afferents from sensory neocortex, can influence cells of the basal nucleus, which originate the major amygdalofugal projection to the neocortex. PMID- 1709112 TI - Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes. AB - As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum. PMID- 1709113 TI - Trichinella spiralis: influence of an immunodominant, carbohydrate-associated determinant on the host antibody response repertoire. AB - Host antibody responses to the G2.1 epitope, a carbohydrate-associated determinant shared by several Trichinella spiralis glycoproteins, were examined by competitive inhibition enzyme-linked immunosorbent assay (ELISA). The G2.1 epitope dominated the AKR/J mouse antibody response whether the antigens were injected or introduced through infection, as determined by the serum blocking ability of a G2.1 epitope-specific monoclonal antibody (mAb). Serum T. spiralis binding activity from several other infected mouse strains was blocked 22 to 86% by the G2.1 epitope-specific mAb. In addition to mice, the G2.1 epitope evoked powerful antibody responses in four other species. The binding activity of Trichinella-reactive antibodies from infected rats and pigs was inhibited 56 and 34%, respectively, by the mAb. Greater than 48% of the T. spiralis serum-binding activity from 4/5 infected humans was G2.1-specific. Most of the rabbit antibody response induced by injection of a previously characterized 43-kDa antigen was also directed to the G2.1 determinant. The specificities of 10 T. spiralis reactive mAb were examined, and 7 reacted with the immunodominant epitope. Finally, of nine helminth species examined, only T. spiralis and T. pseudospiralis extracts efficiently blocked G2.1-specific antibody binding to solid-phase antigens. These results suggest that the responses to G2.1 epitope may play an important role during infection. PMID- 1709114 TI - Lung cancer histology in major ethnic groups among the Jews. Israel, 1962-1982. AB - Lung cancer rates in Israel are lower than in other Western countries, not explainable by smoking habits. Due to the different relation of Squamous cell carcinoma (SqCC) and Adenocarcinoma (AC) with smoking it was of interest to study the histologic distribution in Israel. A total of 7508 histologically confirmed lung cancer cases among Jews were studied in the period 1962-82. SqCC was the leading tumor-type in Jewish men and AC in Jewish women. European-American born males in the last study period showed a decrease in SqCC rate while Asian-African born males showed a steep increase in SqCC rate, most prominent among the younger age-groups. Rates of AC increased in both, European-American and Asian-African males, but more steeply in the latter in most age-groups. Only for Large cell carcinoma were the overall rates higher in Asian-African than in European American born males. SqCC increased in European-American born females and also steeply increased in the over 55 years old Asian-African born females. AC increased in European-American born females (both young and old), but only in the young Asian-African born females (decreasing in the older). European-American born Jews still have higher rates of both, more and less smoking related lung cancer histological types, than Asian-African born Jews. The steep increase in rates of some of the histological types in the latter with the pronounced increased in the younger age-groups is expected to cause a change in the ethnic rate-ratio which has already been demonstrated for the overall lung cancer rates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709115 TI - Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima. AB - The hyperthermophilic eubacterium Thermotoga maritima uses starch as a substrate, without releasing amylase activity into the culture medium. The enzyme is associated with the 'toga'. Its expression level is too low to allow the isolation of the pure enzyme. Using cycloheptaamylose and acarbose affinity chromatography and common chromatographic procedures, two enzyme fractions are obtained. They differ in specificity, pH-optimum, temperature dependence and stability. Substrate specificity and Ca2+ dependence indicate alpha-, beta- and gluco-amylase activity. Compared with alpha-amylase from Bacillus licheniformis (Tmax = 75 degrees C), the amylases from Thermotoga maritima show exceedingly high thermal stability with an upper temperature limit at 95 degrees C. Significant turnover occurs only between 70 and 100 degrees C, i.e. in the range of viability of the microorganism. PMID- 1709116 TI - Alpha 2-macroglobulin synthesis in interleukin-6-stimulated human neuronal (SH SY5Y neuroblastoma) cells. Potential significance for the processing of Alzheimer beta-amyloid precursor protein. AB - Cultured human neuronal (SH-SY5Y neuroblastoma) cells synthesize and secrete the potent protease inhibitor alpha 2-macroglobulin (a2M) upon stimulation with interleukin-6 (IL-6) indicating that alpha 2-macroglobulin behaves as an acute phase protein in the human central nervous system. Exogenous addition of a2M to the cultured neuronal cells resulted in only a slight inhibition of Alzheimer beta A4-amyloid precursor protein (APP) synthesis, but markedly inhibited its secretion pointing to the possibility that a2M may affect the proteolytic APP processing. Evidence is provided that IL-6 and a2M are involved in Alzheimer's disease pathogenesis. PMID- 1709117 TI - A homologue of dystrophin is expressed at the neuromuscular junctions of normal individuals and DMD patients, and of normal and mdx mice. Immunological evidence. AB - Polyclonal and monoclonal antibodies, which recognize different regions and epitopes of the dystrophin molecule, bind to a protein of Mr 400,000 which is present in extracts of mdx muscle from regions which contain neuromuscular junctions (NMJ) and is absent from those which do not. This NMJ-associated homologue of dystrophin has at least 2 epitopes which are different to usual Xp21 form of dystrophin expressed along the sarcolemma of muscle fibres in normal muscles. This protein is also expressed at the NMJ of a DMD patient who lacks the first 52 exons of the Xp21 dystrophin gene and it must therefore be translated from a different gene transcript. PMID- 1709118 TI - ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins. AB - Three fifteen-amino-acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM-2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2). Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide 1 had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature- and cation-dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti-VLA-4 alpha(CD49d) or beta chain (CD29) antibodies (KD4-13 and 4B4) and anti-CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM-1; 84H10 or LB2), MHC class 1 (W6/32) and control mouse IgG had no effect. When anti-CD29 and anti-CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM-2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2-stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2 stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins. PMID- 1709119 TI - Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells. AB - The phosphorylation of epithelial-specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2-fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8-Br-cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [gamma-32P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8-Br-cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase. PMID- 1709120 TI - Structural symmetry of the extracellular domain of the cytokine/growth hormone/prolactin receptor family and interferon receptors revealed by hydrophobic cluster analysis. AB - Sequence comparison based on Hydrophobic Cluster Analysis procedures shows that the extracellular approximately 200 amino acids domains of cytokines receptors belonging to the Cytokine/Growth hormone/Prolactin receptor family and to the Interferon one are organized in two homologous subdomains. Further, comparison of the subdomains of 32 independent sequences and of a lot of already recognized homologous domains with data bases could lead to the hypothesis that these approximately 100 amino acids subdomains could possess the overall fold of the constant immunoglobulin domains and so could belong to the immunoglobulin superfamily. PMID- 1709121 TI - Molecular cloning of cDNA and a chromosomal gene encoding GPE1-BP, a nuclear protein which binds to granulocyte colony-stimulating factor promoter element 1. AB - Granulocyte colony-stimulating factor (G-CSF) is produced from macrophages in response to lipopolysaccharide (LPS). GPE1, a cis-acting element of the G-CSF gene promoter, functioned as an LPS-inducible element. We isolated cDNA and a chromosomal gene encoding the mouse GPE1-binding protein (GPE1-BP). A 150-amino acid protein deduced from the cDNA has a basic domain and a leucine zipper motif, and seems to be identical to that of recently isolated Ig/EBP. The nuclear extract from COS cells transfected with the cDNA showed GPE1-binding activity. Transcripts were ubiquitously detected, and may be spliced from two exons of a single gene. PMID- 1709122 TI - Effect of pertussis toxin on islet insulin secretion in obese (fa/fa) Zucker rats. AB - We used isolated islets of lean and obese Zucker rats to determine whether inhibitory pathways mediated by pertussis toxin-sensitive guanyl nucleotide binding (Gi) proteins contribute to hyperinsulinemia in obese rats. Epinephrine (10(-4) M) and somatostatin (10(-7) M) inhibited insulin secretion by +/- 75% in lean and fa/fa rats. Overnight culture of islets with pertussis toxin (300 ng/ml) enhanced insulin release more in lean (+/- 120%) than obese (+/- 60%) rats. In lean rats incubation of pertussis toxin-treated islets with epinephrine resulted in lower immunoreactive insulin release (p = 0.0005) than pertussis toxin-treated islets without epinephrine. However, in obese rats pertussis toxin treatment reversed this inhibition. Pertussis toxin completely reversed inhibition by somatostatin in both phenotypes. Galanin had no effect on insulin secretion. Cellular cAMP content was similar in lean and obese rats. Inhibitory hormones had no effect on cAMP production. We conclude that islets of obese rats respond normally to inhibitors of insulin release. Reversal of somatostatin-induced inhibition by pertussis toxin indicates normal function of Gi in obese rats. A subtle difference in sensitivity to pertussis toxin between lean and obese islets was noted. PMID- 1709124 TI - [Proportion of the golden section in extrasystole]. PMID- 1709123 TI - Effect of early corticosteroid therapy for Landau-Kleffner syndrome. AB - Four children treated for seizures between 1980 and 1986 were diagnosed as having Landau-Kleffner syndrome (acquired aphasia with convulsive disorder), following the onset of aphasia. They received early and prolonged ACTH or corticosteroid therapy, with high initial doses. In all four cases the EEG promptly became normal, with subsequent long-lasting remission of the aphasia and improvement of seizure control. Three to six years after discontinuation of hormone therapy the children are off medication and free from seizures and language disability. PMID- 1709125 TI - [Beta HCG in serum of women with normal pregnancy is not an artefact]. PMID- 1709127 TI - Synaptonemal complex behavior in asynaptic maize. AB - Synatonemal complexes were studied in silver-stained spread preparations of microsporocyte complements of asynaptic maize. Complexes were found predominantly in terminal regions of chromosome pairs. These tend to be aggregated in a common portion of the nucleus and to have polar orientation. As many as 19 of the 20 ends were found to be involved in relatively short paired segments. Intercalary regions of cores were not strongly organized and aligned, but some contained completed synaptonemal complex segments. The defect in asynaptic appears to represent stalling of the synaptic process at an early stage of synaptic progression. PMID- 1709126 TI - Possible involvement of adenylyl cyclase-cAMP-protein kinase a pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells. AB - Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release. PMID- 1709128 TI - Meiotic chromosome structure. Kinetochores and chromatid cores in standard and B chromosomes of Arcyptera fusca (Orthoptera) revealed by silver staining. AB - The behaviour of two chromosome structures in silver-stained chromosomes was analyzed through the first meiotic division in spermatocytes of the acridoid species Arcyptera fusca. Results showed that at diakinesis kinetochores and chromatid cores are individualized while they associate in bivalents of metaphase I; only kinetochores and distal core spots associate in the sex chromosome. Metaphase I is characterized by morphological and localization changes of both kinetochores and cores which define the onset of anaphase I. These changes analyzed in both autosomes and in the sex chromosome allow us to distinguish among three different substages in metaphase I spermatocytes. B chromosomes may be present as univalents, bivalents, or trivalents. Metaphase I B univalents are characterized by separated cores except at their distal ends and individualized and flat sister kinetochores. At anaphase I sister kinetochores of lagging B chromatids remain connected through a silver-stained strand. The behaviour of cores and kinetochores of B bivalents is identical with that found in the autosomal bivalents. The differences in the morphology of kinetochores of every chromosome shown by B trivalents at metaphase I may be related to the balanced forces acting on the multivalent. The results show dramatic changes in chromosome organization of bivalents during metaphase I. These changes suggest that chromatid cores are not involved in the maintenance of bivalents. Moreover, the changes in morphology of kinetochores are independent of the stage of meiosis but correlate with the kind of division (amphitelic-syntelic) that chromosomes undergo. PMID- 1709129 TI - Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation. AB - Transforming growth factor-alpha (TGF-alpha) is thought to be the major autocrine factor controlling growth in epidermal cells. To explore further the role of TGF alpha in epidermal growth and differentiation, we used a human keratin K14 promoter to target expression of rat TGF-alpha cDNA to the stratified squamous epithelia of transgenic mice. Unexpectedly, the only regions of epidermis especially responsive to TGF-alpha overexpression were those that were normally thick and where hair follicle density was typically low. This included most, if not all, body skin from 2-day- to 2-week-old mice, and ear, footpad, tail, and scrotum skin in adult mice. In these regions, excess TGF-alpha resulted in thicker epidermis and more stunted hair growth. Epidermal thickening was attributed both to cell hypertrophy and to a proportional increase in the number of basal, spinous, granular, and stratum corneum cells. During both postnatal development and epidermal differentiation, responsiveness to elevated TGF-alpha seemed to correlate with existing epidermal growth factor (EGF) receptor levels, and we saw no evidence for TGF-alpha-mediated control of EGF receptor (EGFR) expression. In adults, no squamous cell carcinomas were detected, but benign papillomas were common, developing primarily in regions of mechanical irritation or wounding. In addition, adult transgenic skin that was still both sensitive to TGF-alpha and subject to mild irritation displayed localized regions of leukocytic infiltration and granular layer loss, characteristics frequently seen in psoriasis in humans. These unusual regional and developmental effects of TGF alpha suggest a natural role for the growth factor in (1) controlling epidermal thickness during development and differentiation, (2) involvement in papilloma formation, presumably in conjunction with TGF-beta, and (3) involvement in psoriasis, in conjunction with some as yet unidentified secondary stimulus stemming from mild mechanical irritation/bacterial infection. PMID- 1709130 TI - Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. AB - A novel system to study early hematopoietic development is described. This report documents the in vitro capacity of murine embryonic stem (ES) cells to differentiate into hematopoietic precursors of most, if not all, of the colony forming cells found in normal bone marrow. This system is used to correlate the genetic expression of cytokines, their receptors, the beta-globins, and the hematopoietic cell surface markers throughout the time course of ES cell differentiation with the hematopoietic development that occurs in these cultures. Our results indicate that there is a strong transcriptional activation, in a well defined temporal order, of most of these genes including erythropoietin (Epo), CSF-1, IL-4, beta-globins, as well as the receptors for Epo, CSF-1, and IL-4. IL 3 and GM-CSF were not expressed during the first 24 days of ES cell differentiation. In contrast, the Steel (Sl) factor (SLF) was expressed early and underwent substantial up-regulation during this differentiation, and its receptor, c-kit, was expressed relatively constantly throughout the culture period. Our results are consistent with the conclusion that SLF, Epo, IL-4, and IL-6 are important during the early stages of ES cell differentiation and hematopoietic development. Furthermore, these results argue strongly that IL-3 and GM-CSF are not critical to early hematopoiesis. This system offers a unique in vitro model for studying hematopoietic development at the earliest possible stages. PMID- 1709131 TI - Alpha 2 macroglobulin state in acute pancreatitis. Raised values of alpha 2 macroglobulin-protease complexes in severe and mild attacks. AB - Plasma values of C reactive protein, alpha 1 proteinase inhibitor, alpha 2 macroglobulin, and complexed alpha 2 macroglobulin have been determined in serial samples from 27 patients with acute pancreatitis. Complexed alpha 2 macroglobulin was measured by a novel enzyme linked immunosorbent assay with a monoclonal antibody specific for the complexed form. Patients with severe illness had lower concentrations of total alpha 2 macroglobulin and higher concentrations of complexed alpha 2 macroglobulin than those with mild illness, and in the majority of severe attacks the abnormal amounts of complexed alpha 2 macroglobulin were present throughout the eight days of the study. The proportion of total alpha 2 macroglobulin in the uncomplexed form, however, was generally greater than 90%, and in 26% of the mild cases completely normal concentrations of uncomplexed alpha 2 macroglobulin (greater than 99% of total) were found throughout the eight days of the study. This suggests that exhaustion of alpha 2 macroglobulin in plasma is unlikely to be a major factor in the pathogenesis of acute pancreatitis. PMID- 1709132 TI - Giant colonic mucocele following palliative surgery for recurrent squamous cell carcinoma of the cervix. AB - We report an unusual complication following palliative surgery in a patient with carcinomatosis peritonei from recurrent squamous cell carcinoma of the cervix. At surgery the ascending colon was bypassed and isolated. Eight months later the patient presented with a giant mucocele. We believe this to be the first reported case in the gynecologic literature. This complication could have been prevented by adhering to the standard surgical management of fashioning a mucous fistula. PMID- 1709133 TI - Interactions of hepatitis B virus with hepatocytes: mechanism and clinical relevance. AB - A receptor for hepatitis B virus (HBV) on hepatocytes has been postulated. There is increasing evidence that the binding of the virus to the target cell surface is mediated by epitopes of the pre-S proteins of the HBV envelope. In contrast to vaccination with HBsAg, passive immunization for the prevention of HBV infection is less effective. Using antibodies against the pre-S proteins, representing the putative attachment site for HBV binding to hepatocytes, a more efficient means of protection might be achieved. PMID- 1709134 TI - The linkage of Hb Valletta [alpha 2 beta 287(f3)Thr----Pro] and Hb F-Malta-I [alpha 2G gamma 2117(G19)His----Arg] in the Maltese population. AB - We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a Thr----Pro substitution at position 87 of the beta chain. This mutation was found to be linked to that of the gamma chain variant Hb F-Malta-I with a His----Arg mutation at position 117 of the G gamma chain. Both variants were detected in the blood samples of 34 Maltese and two Italian newborn babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 beta-thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the G gamma-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the G gamma- and beta-globin genes, 27-28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency. PMID- 1709135 TI - Correction of the published sequence for the human proteolipid protein gene. AB - In the human proteolipid protein gene, the base sequence of the intronic region 5' to exon 6 was found to be 5'-ctctttcattttcctgcag-3' and not 5'-ctctttt cattttcctgcag-3' as previously reported. PMID- 1709136 TI - Assignment of the gene for human tenascin to the region q32-q34 of chromosome 9. AB - Tenascin (TN) is a hexameric extracellular matrix glycoprotein that is highly expressed in solid tumors but has a restricted distribution in normal adult tissues. Each TN subunit is composed of segments with high homology to the sequences of epidermal growth factor, fibronectin and fibrinogen. Furthermore, it has been suggested that TN could modulate epithelial-mesenchymal and neuronal glial interactions. Here, using a cDNA probe to human TN, we have carried out Southern blot analysis of the genomic DNAs from a panel of human-hamster somatic cell hybrids carrying different complements of human chromosomes. The results demonstrate that the human TN gene is located on chromosome 9. Furthermore, in situ hybridization studies demonstrate that human TN is located at 9q32-q34. PMID- 1709137 TI - A tetranucleotide repeat polymorphism in the cystic fibrosis gene. PMID- 1709138 TI - Mucosal priming of T-lymphocyte responses to fed protein antigens using cholera toxin as an adjuvant. AB - Cholera toxin is widely recognized as a potent stimulator of mucosal IgA responses after oral feeding. However, comparatively little is known of its ability to stimulate cellular responses. This is due in part to the direct inhibitory effects of cholera toxin on T lymphocytes, which can confound attempts to study primed T-cell responses in vitro. We have avoided this problem by using cholera toxin as an adjuvant to enhance the response to an unrelated protein fed simultaneously. Thus the simultaneous feeding of 10 micrograms of cholera toxin and 5 mg of keyhole limpet haemocyanin (KLH) results in the priming of T cells in the spleen, mesenteric lymph nodes, Peyer's patch and lamina propria that proliferate when restimulated with KLH in vitro. The feeding of KLH alone does not result in such responses. Both CD4- and CD8-positive antigen-specific T cells were involved in the response and the cells produced both interleukins 2 and 4 on antigen restimulation in vitro. PMID- 1709139 TI - Cytomegalovirus induced PMN adherence in relation to an ELAM-1 antigen present on infected endothelial cell monolayers. AB - In human umbilical vein endothelial cells infected with cytomegalovirus (CMV), an activation antigen recognized by monoclonal antibody (mAb) ENA1 appeared. mAb ENA1 reacts with an inducible endothelial surface antigen which has characteristics similar to those of ELAM-1. Incubation with anti-IL-1 partly inhibited this appearance and, parallel to this, the virus-induced polymorphonuclear cell (PMN) adhesion was decreased. In addition, the adhesion of PMN to virus-infected endothelial cells could be reduced by F(ab)2 fragments of mAb ENA1 to almost control level. The results obtained after incubation of PMN with mAb IB4 (against CD18) suggest that the adhesion of PMN to uninfected endothelial cells is CD18 glycoprotein dependent, and virus infection up regulates this glycoprotein-dependent mechanism. These results indicate that the virus-induced PMN adhesion is regulated by the following mechanism: virus infection of endothelial cells induces IL-1 production, and the autocrine IL-1 causes the expression of ELAM-1 on the surface of endothelial cells. In turn this activation antigen ELAM-1 binds with its putative ligand present on the PMN membrane. The virus-induced PMN adhesion occurs also through a CD18 glycoprotein dependent mechanism. PMID- 1709140 TI - Morphology and phenotype of dendritic cells from peripheral blood and their productive and non-productive infection with human immunodeficiency virus type 1. AB - Immununoelectron microscopy of human peripheral blood mononuclear cells enriched for the presence of antigen-presenting dendritic cells (DC) has revealed two morphologically distinct cell types both expressing DR and DQ major histocompatibility complex (MHC) class II antigens but lacking T, B, natural killer (NK) and monocyte/macrophage markers. The first (type 1) has an irregular surface with numerous projections and shows cytoplasmic vacuoles. The second (type 2) has a paler nucleus showing only a thin rim of dense heterochromatin, large expanses of cytoplasm devoid of organelles, fewer vacuoles and a smooth cell boundary with few processes. In addition a few cells with a morphology similar to veiled cells of the afferent lymphatics (type 3 DC) were observed. Cells with a morphology intermediate between these three types were observed, suggesting that they may represent stages of the veiled cell differentiation pathway. Type 2 and 3 DC were shown by electron microscopy to be susceptible to productive infection with human immunodeficiency virus (HIV), whilst type 1 DC did not support virus growth. Examination of infected DC preparations by in situ hybridization revealed a higher number of DC positive for viral DNA and RNA than for RNA alone. Thus, in addition to productively infected DC, there may be some that are latently infected, contain defective virus genome or replicate virus at a very low level. PMID- 1709141 TI - Bovine T cells recognize antigen in association with MHC class II haplotypes defined by one-dimensional isoelectric focusing. AB - A recently established, one-dimensional isoelectric focusing (IEF) method for distinguishing major histocompatibility complex (MHC) class II polymorphisms in an outbred species, cattle, has allowed us to analyse the involvement of the MHC in the recognition of antigen by bovine T cells. Bovine T-cell lines of Th cell phenotype (BoCD4+) specific for ovalbumin were generated from six individual high responder animals. These animals were bovine MHC (BoLA) class II typed using the IEF technique which detects bovine DR-like products. Four of the animals were shown to be heterozygous and two were homozygous for the IEF specificities. Six out of the 13 IEF specificities (EDF types) detected so far were represented by this group of animals. The cell lines were tested against a panel of IEF-typed antigen-presenting cells (APC) from unrelated donors. The lines only responded to antigen in proliferation assays when the APC shared at least one MHC class II EDF specificity with the BoCD4+ cell line. The responses did not correlate with BoLA class I specificities. However, lines from one of the animals were consistently generated to one of the two haplotypes only. This suggests that there are non responder alleles to a multi-epitope antigen, present in the cattle population. The results demonstrate that IEF of bovine MHC class II products defines haplotypes of functional relevance, and may indeed be identifying the actual restriction elements involved in presentation of ovalbumin. These results have important implications for future vaccine design in an outbred species, particularly in terms of immune response gene effects and disease associations. PMID- 1709142 TI - Differential exposure of epitopes on the human red cell antigen D (Rh). AB - The D polypeptide of the human Rh blood group system has a number of different epitopes on its surface. It is also known that there is considerable variation in the number of D antigen sites available to different human monoclonal anti-D antibodies. For instance, certain monoclonal antibodies recognise only a small number of sites on the red cell surface (about 9,000 sites/red cell on R1R2 cells) whereas other antibodies recognise a high number (about 20,000-30,000 sites/red cell). It has been found that cholesterol enrichment of the red cell membrane increases the number of sites available to those antibodies which recognize only a few sites but has no effect on those recognising many sites. The results are consistent with the view that access to some of the D epitopes is partially hindered by neighbouring molecules in the membrane and that alteration of the lipid content of the membrane changes it in such a way as to allow increased access to these obstructed epitopes. PMID- 1709143 TI - Affinity-purified soluble Fc epsilon RII/CD23 derived from RPMI-8866 cells induces histamine release from human nasal polyp mast cells through a non-IgE mediated mechanism. AB - Soluble Fc epsilon RII/CD23 (IgE-binding factor) is released spontaneously from activated B cells and most EBV-immortalised B cell lines. We have purified soluble Fc epsilon RII/CD23 from culture supernatants of RPMI-8866 cells on an IgE Sepharose column, and studied its ability to release histamine from human nasal polyp mast cells. Soluble Fc epsilon RII/CD23 induces release of a significant amount of histamine from nasal polyp mast cells in a dose-dependent manner. IgE, and a monoclonal antibody specific for the soluble form of this receptor, were shown to neutralise this effect. It was found that soluble Fc epsilon RII/CD23 was still capable of triggering histamine release from nasal polyp mast cells from which IgE had been eluted by incubation in a low pH buffer, suggesting that a non-IgE mediated mechanism was responsible for this effect. PMID- 1709144 TI - Two amino acid substitutions at residues 63 and 67 between HLA-B51 and HLA-Bw52 form multiple epitopes recognized by allogeneic T cells. PMID- 1709145 TI - [Enteral feeding in oncology]. AB - Nutrition therapy is accepted to represent an important part of therapeutic strategy in cancer patients. In correlation to general clinical state, nutritional condition and anti-cancer therapy, nutrition has to be planned according to a stepwise concept. Under certain circumstances, enteral tube feeding is indicated. This procedure is affected by tumor lesions, digestion, resorption and tumor related metabolic disorders, and side effects of particulate anti-cancer therapy. In tumor patients, elemental (chemically defined) diets are predominant. Polymeric diets, however, occasionally represent another possible nutritional tool. PMID- 1709146 TI - [Effect of a new 10% hydroxyethyl starch solution HES/270/0.5 on blood coagulation, blood loss and hemodynamics in comparison with 3.5% PPL]. AB - 20 patients scheduled for total hip replacement were given 1,000 ml of a new preparation of 10% hydroxyethylstarch (HES) (MW 270,000: 0.5) preoperatively. They were compared to a group of 20 patients who received 1,000 ml of 3.5% plasma protein solution (PPS). HES caused a more pronounced hemodilution than PPS. With HES, central venous pressure (CVP) rose significantly higher than with PPS. PTT was significantly prolonged in the HES but not in the PPS group. TT was significantly reduced by HES in comparison to PPS. PT (Quick-value %) and fibrinogen levels showed no difference in both groups. Blood loss and transfusion volume were comparable to HES and PPS until 24 h after the operation. One patient showed generalised flush after HES. This HES preparation is a colloid with volume expanding properties and appears to be without clinically apparent effects on coagulation (up to a volume of 11). PMID- 1709147 TI - Norspermidine inhibits LPS-induced immunoglobulin production in an FCS independent mechanism different from spermidine and spermine. AB - The immunosuppressive effects of several polyamines were compared. The triamines (norspermidine (NSPD) and spermidine (SPD] and the tetramines (norspermine (NSPM) and spermine (SPM] but not the diamines (1,3-diaminopropane and putrescine) inhibited IgM production from murine splenocytes stimulated with LPS. The estimated IC50 of NSPD was 2.29 x 10(-7) M. The inhibitory effect of NSPD on IgM production was associated with the inhibition of cell growth, because DNA and RNA syntheses measured by 3H-TdR and 3H-UR incorporation were also similarly reduced. Interestingly, the inhibitory effects of NSPD were over ten times greater than those of SPD in spite of the fact that their difference in chemical structure is only one carbon chain. In a FCS-free medium NSPD retained its suppressive activities on LPS-induced IgM production, but the other polyamines were remarkably weakened in their activities. These immunosuppressive effects of NSPD were prevented by adding substances of the intracellular polyamine metabolism, putrescine, SPD or SPM. These findings suggest that NSPD inhibits B cell growth and differentiation by interfering with the polyamine metabolism pathway. PMID- 1709148 TI - The role of glutathione in lymphocyte activation--II. Effects of buthionine sulfoximine and 2-cyclohexene-1-one on early and late activation events. AB - Depletion of intracellular glutathione (GSH) inhibits the lectin-induced activation response of human T lymphocytes. GSH-depleted lymphocytes undergo a partial activation response to lectins but fail to undergo blast transformation. Several lines of evidence indicate that the inhibition of lymphocyte activation in GSH-depleted lymphocytes involves relatively late activation events. Firstly, lectin stimulation induces significant 14C-AIB uptake, IL-2 production and expression of IL-2 receptor but a near complete inhibition of 3H-uridine and 3H thymidine incorporation. Comparable levels of IL-2 production and IL-2 receptor expression are seen in GSH-depleted lymphocytes allowed to recover from GSH depletion during lectin stimulation. However, in the latter case, 3H-uridine and 3H-thymidine incorporation are normal, and activation is completely restored. Exogenous IL-2 cannot restore activation in GSH-depleted lymphocytes. Furthermore, lymphocytes remain highly susceptible to inhibition by GSH depletion even after 48 h of lectin stimulation which is sufficient to induce early activation events in the Go----G1 transition, such as IL-2 receptor expression and IL-2 production. Exogenous GSH partially restores intracellular GSH levels and completely restores lymphocyte activation in GSH-depleted lymphocytes. Despite comparable degrees of GSH depletion, DL-buthionine-SR-sulfoximine and 2 cyclohexene-1-one inhibit lymphocyte activation to different degrees. The inhibition by 2-cyclohexene-1-one is consistently greater than would be predicted based on glutathione depletion per se. We conclude that GSH-dependent processes are important in relatively late steps of the activation sequence characterized by nuclear events with relative sparing of essential early steps in activation, such as IL-2 receptor expression and IL-2 production. The approximate minimal intracellular GSH concentration necessary to sustain a normal activation response is 2 nmol per 10(7) lymphocytes. PMID- 1709149 TI - The effect of clenbuterol administration in utero and throughout lactation on pre and post-natal muscle development in the rat. AB - The beta-adrenergic agonist, clenbuterol was fed to pregnant rats during gestation and throughout lactation. Changes in muscle morphology and composition were studied in foetal and weanling rats. Drug treatment did not affect mean foetal number, but mean foetal body weight was significantly reduced. Heart weights were increased and both muscle weight and secondary to primary fibre ratios were decreased in foetuses exposed to clenbuterol in utero. In animals exposed to clenbuterol throughout gestation and lactation, muscle weights, and protein, RNA and DNA content and total fibre numbers were reduced. In addition a drug induced anabolism was observed in the muscles of the dams. The data are discussed in terms of a direct drug effect on immature and differentiated muscle together with a possible indirect action through the repartitioning action of clenbuterol. PMID- 1709150 TI - Melatonin increases serum growth hormone and insulin-like growth factor I (IGF-I) levels in male Syrian hamsters via hypothalamic neurotransmitters. AB - In male Syrian hamsters daily evening melatonin injections resulted in increased circulating levels of growth hormone (GH), as well as a modest increase in body weight. A substantial increase in serum levels of insulin-like growth factor I (IGF-I) was observed in all hamsters receiving evening injections of melatonin for 10 weeks. The melatonin-induced increase in serum IGF-I levels was interpreted as a result of increased release of GH during the 10 week period of melatonin administration. The increase in serum GH and IGF-I was associated with significantly decreased hypothalamic turnover of norepinephrine (NE). Since blocking NE synthesis with alpha methyl-p-tyrosine reduced serum GH, the melatonin-induced increase in GH could not readily be attributed to decreased NE turnover. Highly significant increases in 5-hydroxyindole acetic acid (5HIAA) concentrations and in ratios of 5HIAA to serotonin (5HT) were noted in extracts of hypothalamus and in extracts of brain stem, suggesting a serotonergic component to melatonin-induced increase in GH-induced IGF secretion and subsequent growth. PMID- 1709151 TI - Evaluation of fludarabine phosphate in malignant melanoma. A Southwest Oncology Group study. AB - Twenty-seven evaluable patients with advanced malignant melanoma received fludarabine phosphate in a daily x 5 injection. Initial dosing was based on the presence of previous radiation therapy. There was no response seen in these patients despite appropriate dose escalation. Myelosuppression occurred without significant sequelae. PMID- 1709152 TI - Phase I-II study of pibenzimol hydrochloride (NSC 322921) in advanced pancreatic carcinoma. AB - Pibenzimol is a fluorescent molecule known to bind to double stranded DNA. It also induces prolongation of the G2 phase of the cell cycle, inhibition of DNA replication and cessation of the growth of some cells in late S phase after DNA content has been doubled. It has been shown to increase the life span of mice bearing intraperitoneally implanted L1210 and P388 leukemia. These factors coupled with the affinity of pibenzimol for pancreatic tissue led us to conduct a phase I-II trial of pibenzimol hydrochloride in patients with advanced pancreatic cancer. Twenty-six patients were treated with a five day continuous infusion of pibenzimol at a dose ranging from 6-28 mg/m2/d. There were no treatment related deaths. Major toxicity was hyperglycemia which was self-limited. No objective responses were noted. PMID- 1709153 TI - Phase II trial of 5,6-dihydro-5-azacytidine in pleural malignant mesothelioma. PMID- 1709154 TI - Phase II study of pibenzimol in pancreatic cancer. A Southwest Oncology Group study. AB - Twenty-three patients with advanced pancreatic adenocarcinoma were treated with Pibenzimol utilizing a daily intravenous schedule for five days. There were no objective responses seen. The major toxicity was pancreatic with grade 3 hyperglycemia in eleven patients. Pibenzimol is inactive in patients with advanced pancreatic adenocarcinoma. PMID- 1709155 TI - The tyrosine kinase lck is critically involved in the growth transformation of human B lymphocytes. AB - Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+) dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process. PMID- 1709156 TI - Alu RNA secondary structure consists of two independent 7 SL RNA-like folding units. AB - The amplification of genomic Alu elements by retroposition, i.e. by reintegration of reverse-transcribed RNA, suggests that Alu RNA plays an important role in this process. We report enzymatic studies of the secondary structure of Alu RNAs transcribed in vitro from two recently retroposed Alu elements. These experiments show that the dimeric organization of an Alu sequence is reflected in its RNA folding. Alu subunits fold independently, conserving secondary structure motifs of their progenitor 7 SL RNA molecule. Energy minimization analysis indicates that this folding pattern is also characteristic of different Alu and Alu-like sequences and has been conserved since primate divergence. By analogy to 7 SL RNA, the Alu RNA folding may be important for specific interactions with proteins. This could indicate a physiological function for Alu transcripts. However, this can be also seen as a structural adaptation leading to efficient retroposition of these sequence elements. PMID- 1709157 TI - Partial cleavage mapping of the cytoskeletal protein vinculin. Antibody and talin binding sites. AB - Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there. The use of [14C]cyanide in conjunction with DTNB showed which peptides contained the original amino terminus. Secondary cleavage with N chlorosuccinimide, a tryptophan-specific reagent, helped locate fragments, although it led to apparent increases in molecular weight of the products. These experiments revealed the location of 10 of the major DTNB fragments on the sequence. This map was used to locate binding sites. The site of interaction between vinculin and the focal contact protein talin was mapped by binding labeled talin to the separated fragments. The binding site was found to be in the amino-terminal 325 amino acids. The binding site of a commercially obtained monoclonal antivinculin antibody was mapped using Western blotting of cleaved vinculin. It proved to bind in the central area of the molecule between amino acid residues 545 and 737. Thus the cysteine cleavage reaction products provide a map of general utility for locating features on the vinculin molecule. PMID- 1709158 TI - Uptake and degradation of cytoplasmic RNA by hepatic lysosomes. Quantitative relationship to RNA turnover. AB - Past evidence has suggested that the lysosomal pathway is an important site of cytoplasmic RNA degradation in the hepatic parenchymal cell (Lardeux, B. R., Heydrick, S. J., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14507-14519). We now provide additional support for this notion by quantitating degradable RNA in lysosomes and correlating its pool size with hepatic RNA degradation. Rat livers, previously labeled with [6-14C]orotic acid, were perfused with graded levels of amino acids over the full range of induced autophagy; RNA degradation was determined from [14C]cytidine release. Close correspondence between the marker beta-acetylglucosaminidase and the breakdown of RNA to cytidine in subcellular fractions indicated that the lysosome was the main site of catabolism, a conclusion supported by the fact that degradation was enhanced when external pH was lowered from 7 to 6. Although [14C]cytidine was also released in homogenates by the action of natural ribonucleases on cytosolic RNA, this source was eliminated by unlabeled exogenous RNA. The size of the degradable RNA pool in lysosomes, determined from the total release of cytidine in homogenates, correlated directly with rates of hepatic RNA degradation over the full range of basal and induced degradation. A direct correlation was also seen between RNA degradation and cytidine pools within lysosomal particles. Because cytosolic cytidine was not taken up by lysosomes under these conditions, the pool could only have arisen from the breakdown of intralysosomal RNA. As determined by cytidine production, these findings support the view that the lysosomal-vacuolar system is the main, if not sole, site of induced and basal RNA degradation in liver. PMID- 1709159 TI - Platelet-derived growth factor (PDGF) stimulates PDGF receptor subunit dimerization and intersubunit trans-phosphorylation. AB - High affinity binding of platelet-derived growth factor (PDGF) has been proposed to involve the interaction of the dimeric PDGF ligand with two receptor subunits, designated alpha and beta. We have cloned and expressed a human PDGF receptor cDNA which differs in sequence from the beta-subunit and which has the PDGF binding properties and monoclonal antibody recognition, predicted for the alpha subunit. Scatchard analysis indicated that PDGF-AA and PDGF-AB bound to transfected alpha-subunits with affinities of Kd = 0.06 and 0.05 nM, respectively. PDGF-BB bound with a significantly lower affinity (Kd = 0.4 nM). Nevertheless, this affinity is still great enough to mediate substantial PDGF-BB binding at physiological concentrations and would be considered to be "high affinity." We have used wild-type and kinase-inactive human beta-subunits to show that PDGF binding promotes receptor subunit dimerization in intact cells. In addition, we found that PDGF stimulates tyrosine phosphorylation of the kinase inactive beta-subunit when it is expressed with alpha-subunits. The kinase inactive beta-subunits were phosphorylated at tyrosine 857 and 751, the major phosphorylation sites of the wild-type beta-subunit, indicating either that intra and intermolecular phosphorylation occurs on the same sites, or that a significant fraction of receptor tyrosine phosphorylation is intermolecular. PMID- 1709160 TI - Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake. AB - Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here. PMID- 1709161 TI - Identification and molecular cloning of two new 30-kDa insulin-like growth factor binding proteins isolated from adult human serum. AB - Three insulin-like growth factor binding proteins (IGFBP) with apparent molecular masses of 24, 28-30, and 30 kDa, nonreduced, have been isolated from human serum. The 15 NH2-terminal amino acids of the 24-kDa binding protein are identical with those of the 30-kDa BP. The apparent molecular mass of the latter is reduced to 24 kDa by N-glycanase, suggesting that the 30-kDa BP is the glycosylated form of the isolated 24-kDa BP. The complete amino acid sequences derived from the cloned cDNAs represent two new IGFBPs. They are tentatively termed IGFBP-4 and -5. The prepeptide sequences of BP-4 and -5 contain 27 and 21, the mature proteins 213 and 237 amino acids, respectively (Mr = 22,610 and 25,980). The NH2- and COOH terminal thirds of BP-4 and -5 display pronounced homology to the other three human BPs. 16 of the 16-20 cysteines and 37 of the 213-289 amino acids (12.8 17.1%) are conserved in all five mature BPs. 10 amino acid positions located in the NH2-terminal region and shared by BP-1, -2, -3, and -5 are different in BP-4. These differences may account for the preferential affinity of BP-4 for IGF II. A most intriguing homology exists between the COOH-terminal quarter of the five IGFBPs, 10 repetitive domains of human thyroglobulin, a gastrointestinal tumor associated antigen, and the invariant chain of the class II histocompatibility antigen. The cDNAs of five human IGFBPs are now available. They will allow their expression and production in sufficient quantities for in vivo studies to unravel their role in growth and metabolism. PMID- 1709162 TI - Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. AB - A 12.5-kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced. The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function. Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE. The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B. subtilis and comparisons with the corresponding sequences from numerous other species are presented. The 3' ends of the reading frames overlap the 5' ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145-base pair intercistronic region. The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1. This site is preceded by a typical B. subtilis sigma A-dependent promoter. A promoter indicator plasmid was used to show that this region carried a promoter from which transcription was regulated by pyrimidines. Transcription from this promoter was not detected in primer extension experiments using mRNA prepared from B. subtilis cells grown in the presence of excess uracil. No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough. This terminator was not regulated by pyrimidines in the growth medium under the conditions tested. The region immediately following the promoter and 5' to ORF1 has a potential transcription terminator sequence. The roles, if any, of these transcription terminators in the regulation of pyr operon expression are yet to be determined. However, deletion of the terminator immediately following the promoter abolished pyrimidine regulation of transcription in the indicator plasmid. PMID- 1709164 TI - Differential effects of lecithin and cholesterol on the immunoreactivity and conformation of apolipoprotein A-I in high density lipoproteins. AB - Recently identified epitopes in apoA-I define a distinct N-terminal region with a complex tertiary structure, characterized by multiple discontinuous epitopes. Other epitopes are constituted of short domains centered either on beta-turns or random coils or on the 22-mer amphipathic alpha-helices (Marcel, Y. L., Provost, P. R., Koa, H., Raffai, E., Vu Dac, N., Fruchart, J.-C., and Rassart, E. (1991) J. Biol. Chem. 266, 3644-3653). The compared immunoreactivity of seven epitopes studies here in response first to delipidation of high density lipoprotein (HDL) apoA-I by detergents, and second to modifications of HDL lipid composition by phospholipase A2 or by enrichment in surface lipids demonstrates that apoA-I has a flexible conformation which is readily responsive to the nature and concentration of bound lipids and that the structure of lipid-free apoA-I is significantly different from that of HDL-bound apoA-I, possibly representing a condensed molecule with several masked domains. In HDL apoA-I, these epitopes define five distinct domains which are characterized by particular responses to lipid modifications. However, two domains, each starting at the N-terminal beta turn of an amphipathic alpha-helical repeat (residues 99-121 and 186-209, respectively) have almost identical immunoreactivity whether after detergent treatment or after changes in cholesterol and phospholipid levels, a property which probably reflects the known periodicity of apoA-I structural 22-mers. The immunoreactivity of a discontinuous epitope, representative of the N-terminal domain, is inversely related to the concentration of phospholipids, a unique characteristic among the epitopes tested here which indicates that the complex N terminal region interacts with phospholipids, either directly or indirectly. These studies demonstrate that the conformation of multiple domains of HDL apoA-I is dependent on lipid phase composition and differentially affected by cholesterol and phospholipids. PMID- 1709163 TI - The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin. AB - Evaluation of the expression of the cystic fibrosis (CF) gene in human epithelial cell lines demonstrated active, but low level, transcription of the gene. Analysis of 3.8 kilobases of genomic sequences 5' to exon 1 of the CF gene demonstrated no TATA promoter element, but a high G + C content, multiple transcription start sites, and several potential Sp1 binding sites. Fragments of 5'-flanking sequences from 2.2 kilobases to as small as 102 base pairs 5' to the major transcription start site supported constitutive reporter gene expression in epithelial cells, but at low levels, and independent of the length of the 5' fragment. CF gene transcription was down-regulated by phorbol myristate acetate. Importantly, evaluation of freshly isolated normal human bronchial cells also demonstrated CF gene transcription at a relatively low rate. Together, these observations suggest that although the normal CF gene promoter has characteristics of a "housekeeping"-type gene, and the gene is expressed at low levels in cells of organs that manifest the clinical disorder "cystic fibrosis," its expression can be modulated transcriptionally, implying a possible therapeutic approach for the disease. PMID- 1709165 TI - Contact residues and predicted structure of the reovirus type 3-receptor interaction. AB - Sequence similarity between the reovirus type 3 hemagglutinin (HA3) and a anti idiotypic monoclonal antibody (87.92.6) has been shown to define the site of interaction with a neutralizing (idiotypic) monoclonal antibody (9B.G5) and the cellular receptor for the virus. A synthetic peptide (VL peptide) derived from the anti-idiotypic sequence inhibits viral binding to the receptor. In this study, variants of the VL peptide were utilized to probe specific amino acid residues involved in binding the neutralizing antibody and the receptor. These studies indicate that the--OH groups of several residues are involved in contacting the reovirus type 3 receptor, including Tyr49, Ser50, Ser52, and Thr53 in the anti-idiotypic sequence, corresponding to Tyr326, Ser327, Ser329, and Ser325 in HA3, respectively. In contrast, only Ser50 of the anti-idiotypic sequence, corresponding to Ser327 of HA3, significantly altered neutralizing antibody binding. Additional studies implicate sialic acid as a potential reovirus type 3 receptor on some cells. This includes inhibition of binding of reovirus type 3 and 87.92.6 to L cells by heavily sialylated glycoproteins. Sialic acid was therefore utilized as a candidate receptor to analyze potential interaction schemes with HA3/87.92.6. Sequence similarity to other immunoglobulin structures with similar sequences allowed modeling of the three-dimensional structure of these epitopes. These structures, in combination with peptide studies, allow the development of a model of the interaction of these epitopes with sialic acid, which serves as a reovirus type 3 receptor. These models reveal that similar amino acid residues and side-chain geometries may be utilized by the reovirus type 3 and influenza hemagglutinins in their interactions with cell surface receptors. PMID- 1709166 TI - Acute inhibition of insulin-stimulated glucose transport by the phosphatase inhibitor, okadaic acid. AB - Insulin is thought to exert its effects on cellular function through the phosphorylation or dephosphorylation of specific regulatory substrates. We have analyzed the effects of okadaic acid, a potent inhibitor of type 1 and 2A protein phosphatases, on the ability of insulin to stimulate glucose transport in rat adipocytes. Insulin and okadaic acid caused a 20-25- and a 3-6-fold increase, respectively, in the rate of 2-deoxyglucose accumulation by adipose cells. When added to cells previously treated with okadaic acid, insulin failed to stimulate 2-deoxyglucose accumulation beyond the levels observed with okadaic acid alone. Treatment of cells with okadaic acid did not inhibit the effect of insulin to stimulate tyrosine autophosphorylation of its receptor. These results indicate that okadaic acid potently inhibits the effects of insulin to stimulate glucose uptake and/or utilization at a step after receptor activation. To clarify the mechanism of inhibition by okadaic acid, the intrinsic activity of the plasma membrane glucose transporters was analyzed by measuring the rate of uptake of 3-O methylglucose by adipose cells, and the concentration of adipocyte/skeletal muscle isoform of the glucose transporter (GLUT-4) in plasma membranes isolated from these cells. Insulin caused a 15-20-fold stimulation of 3-O-methylglucose uptake and a 2-3-fold increase in the levels of GLUT-4 detected by immunoblotting of isolated plasma membranes; okadaic acid caused a 2-fold increase in 3-O methylglucose uptake, and a 1.5-fold increase in plasma membrane GLUT-4. Pretreatment of cells with okadaic acid blocked the effect of insulin to stimulate 3-O-methylglucose uptake and to increase the plasma membrane concentration of GLUT-4 beyond the levels observed with okadaic acid alone. These results indicate that the effect of okadaic acid to inhibit the effect of insulin on glucose uptake is exerted at a step prior to the recruitment of glucose transporters to the cell surface, and suggest that a phosphatase activity may be critical for this process. PMID- 1709167 TI - Keratin incorporation into intermediate filament networks is a rapid process. AB - The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements. PMID- 1709168 TI - The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers. AB - Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin. PMID- 1709169 TI - Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to cell adherens junctions where the level of tyrosine phosphorylation is elevated. AB - To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated. PMID- 1709170 TI - Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. AB - We investigated the role of the integrins alpha v beta 3 and alpha v beta 5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both alpha v-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with alpha v beta 5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to alpha v beta 5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only alpha v beta 3 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while alpha v beta 5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface. PMID- 1709171 TI - Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin sensitive arachidonic acid release without inositol phosphate accumulation. AB - Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis. PMID- 1709173 TI - [Obstruction of pancreatic ducts with a fibrin glue. Study after segmentary autotransplantation in dogs]. AB - The effects of the main pancreatic duct with a fibrin sealant have been investigated on an experimental model of segmental pancreatic transplantation in the dog. Fourteen segmental pancreatic transplantations were performed. A cephalic pancreactectomy was performed during the same operating time. The main duct was obstructed with a fibrin sealant (Tissucol modified by addition of a solution of aprotinine concentrated at 10,000 KUI per mL). Biological follow-up consisted in: 1) Intravenous Glucose Tolerance Testing at Day 0 and Day 28 with glycaemia's integral calculus and K V Alues. 2) Measurements of glycaemia and serum amylase every three days from day 0 to day 28. Histological examination of the pancreatic tissue before and after transplantation involved a microscopy analysis reporting the degree of fibrosis and necrosis. The areas of the Langherans islets and of the fibrosis were calculated with informatic area analysis. The study was carried on non diabetic dogs at Day 28. The glycaemia's calculus of IVGTT were not significantly different before and after transplantation (p = 0.291). On the other hand, there was a significant difference of the K Values before and after transplantation (p = 0.006). Histology after transplantation revealed important lesions of fibrosis and normal or hypertropic Langherans islets in most cases. Pancreatic ducts presented with linings thickened with fibrosis. There was no fibrin sealant in the lumen. Obstruction of pancreatic ducts with a fibrin sealant induces an important fibrosis of the pancreatic exocrin tissue allowing the preservation of a satisfactory endocrine function. This technic may be used in clinical practice during the segmental pancreatic transplantations or after cephalic pancreatico duodenectomy. PMID- 1709172 TI - Activation of the simian virus 40 (SV40) genome abrogates sensitivity to AVP in a rabbit collecting tubule cell line by repressing membrane expression of AVP receptors. AB - To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33 degrees C) and restrictive (39.5 degrees C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5 degrees C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33 degrees C respond only to isoproterenol (ISO, 10( 6) M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5 degrees C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10(-7) M dDarginine vasopressin (dDAVP/basal: 18.2, apparent Ka: 5 X 10(-9) M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2 type AVP receptors (approximately 50 fmol/10(6) cells) which are undetectable when SV40 genome is activated (33 degrees C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5 degrees C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells. PMID- 1709174 TI - Analysis of rat brain microdialysate by gas chromatography-high-resolution selected-ion monitoring mass spectrometry. AB - Gas chromatography-high-resolution selected-ion monitoring mass spectrometry was used to analyze catecholamine metabolites in rat brain microdialysate. Dialysate samples were collected in vials containing stable isotope analogues of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5 hydroxyindoleacetic acid (5HIAA) and analyzed as their trimethylsilyl derivatives. The metabolite levels were monitored at 20-min intervals throughout the time course of the experiment, beginning immediately after surgery and implantation of the dialysis probe and ending 4 h after amphetamine treatment. The levels of HVA were observed to decrease after amphetamine treatment, while those of MHPG and 5HIAA did not change significantly. PMID- 1709175 TI - Detection of ribose-methylated nucleotides in enzymatic hydrolysates of RNA by thermospray liquid chromatography-mass spectrometry. AB - A procedure has been developed for the detection and characterization of ribose methylated dinucleotides of the type NmpN' in enzymatic digests of RNA. Differences in reaction products from hydrolysis using RNase T2, which will not cleave NmpN', and hydrolysis by nuclease P1 are analyzed by thermospray liquid chromatography-mass spectrometry. The method is applicable to dinucleotides present in nanogram range quantities and is suited for the characterization of new or unexpected ribose-methylated nucleotides for which chromatographic mobilities are not known. PMID- 1709176 TI - Somatostatin reduces 3H-thymidine incorporation and c-myc, but not thyroglobulin ribonucleic acid levels in human thyroid follicular cells in vitro. AB - The action of somatostatin (SRIH) on 3H-thymidine (thy) incorporation and on c myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10(-7) M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent. PMID- 1709177 TI - A simple assay for proteolysis of IGFBP-3. AB - An assay for assessing the proteolysis of IGFBP-3 by a biological fluid or protein has been developed using 125IhIGFBP-3 recombinantly derived from Escherichia coli as a substrate indicator. The labelled substrate is incubated at 37 degrees in the presence of the agent being tested and proteolysis is visualized by SDS-PAGE and autoradiography. The assay showed that the pregnancy associated protease is Ca++ dependent. The substrate is also proteolyzed by rat and mouse pregnancy sera, making the assay applicable to studies in those animal systems. PMID- 1709178 TI - The computerized pupil. AB - An IBM-compatible computer graphics software program has been developed which simulates pupillary function. A light pen, electronically interfaced with the computer, simulates a pen light and, when it is placed on the computer image of the pupil, causes pupillary constriction; when it is withdrawn, the pupils redilate. A variety of abnormal pupillary responses are depicted. Anatomical diagrams and questions concerning various simulated pupillary conditions further enhances the educational experience. Medical students, residents in ophthalmology and the neurosciences, optometrists, nurses, ophthalmic assistants, and other health care professionals can utilize this program to learn a wide range of pupillary abnormalities in an interactive environment. PMID- 1709179 TI - Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography. AB - The distribution of galanin (GAL) binding sites in the visual cortex of cat and monkey was determined by autoradiographic visualization of [125I]-GAL binding to tissue sections. Binding conditions were optimized and, as a result, the binding was saturable and specific. In cat visual cortex, GAL binding sites were concentrated in layers I, IVc, V, and VI. Areas 17, 18, and 19 exhibited a similar distribution pattern. In monkey primary visual cortex, the highest density of GAL binding sites was observed in layers II/III, lower IVc, and upper V. Layers IVA and VI contained moderate numbers of GAL binding sites, while layer I and the remaining parts of layer IV displayed the lowest density. In monkey secondary visual cortex, GAL binding sites were mainly concentrated in layers V VI. Layer IV exhibited a moderate density, while the supragranular layers contained the lowest proportion of GAL binding sites. In both cat and monkey, we found little difference between regions subserving central and those subserving peripheral vision. Similarities in the distribution of GAL and acetylcholine binding sites are discussed. PMID- 1709180 TI - Origin of spinal projections to the anterior and posterior lobes of the rat cerebellum. AB - The present study was carried out to analyze the topography of spinal projections to the anterior and posterior lobes of the cerebellum and to investigate whether projections to the two lobes come from different spinocerebellar neurons or from branching axons of the same cells. We used orthograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) to identify the cerebellar areas where spinocerebellar axons terminate and retrograde double labeling techniques to estimate the incidence of spinocerebellar neurons projecting to both anterior and posterior lobes via axon collaterals. Orthograde labeling confirmed that the rat, like other mammalian species, has spinocerebellar projections to two different regions of cerebellar cortex, i.e., lobules I-V of the anterior lobe and lobule VIII of the posterior lobe, with the highest incidence in lobules II, III, and VIII. We did not observe a clear difference in the distribution of afferents coming from different spinal segments to either of the two lobes. The double-labeled cells were located primarily in the lower thoracic and upper lumbar segments, almost exclusively in Clarke's column and in the dorso-lateral part of lamina 7 (in the region of the spinal border cells). It is likely that most or all of the spinocerebellar neurons in these structures project to both anterior and posterior lobes. Therefore, the two lobes of the cerebellum are likely to receive common information from these cells, but different information from the separate populations of spinocerebellar neurons that project only to one lobe or the other. PMID- 1709181 TI - Evaluation of the influence of optic stalk melanin on the chiasmatic pathways in the developing rodent visual system. AB - In a number of mammalian species, fibre outgrowth in the developing retinofugal pathway is coincident with the presence of melanin in the retinal part of the optic stalk. The presence of melanin is transient in this developing system and has been proposed to play a role in the guidance of retinofugal fibres. Further, it has been suggested that this stalk melanin accounts for the differences between the size of the uncrossed retinal component in pigmented and nonpigmented strains. However, a recent study showed that there is no melanin in the optic stalk of Manchester rats during fibre outgrowth. Since such rats supposedly have a normal pigment distribution and a normal pattern of decussation at the optic chiasm, this finding appears to undermine the suggested role played by stalk melanin in establishing the laterality of retinal fibre projections in other mammalian species. The aim of this study was to re-evaluate the relationship between melanin in the stalk and the development of the retinofugal pathway in three strains of rat: the Wild type, Long Evans Hooded, and the Albino. The Albino rat, which lacks melanin-bearing cells entirely, was shown to have the smallest uncrossed projection, approximately 1,340 ipsilaterally projecting cells (ipc), whereas the Long Evans (2,760 ipc) and the Wild-type strain (2425 ipc) were found to have a larger uncrossed retinal component. In both pigmented strains, melanin was restricted to the eye cup and absent from the optic stalk throughout all stages of development.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709182 TI - Afferent and efferent connections of the olfactory bulbs in the lizard Podarcis hispanica. AB - The connections of the olfactory bulbs of Podarcis hispanica were studied by tract-tracing of injected horseradish peroxidase. Restricted injections into the main olfactory bulb (MOB) resulted in bilateral terminallike labeling in the medial part of the anterior olfactory nucleus (AON) and in the rostral septum, lateral cortex, nucleus of the lateral olfactory tract, and ventrolateral amygdaloid nucleus. Bilateral retrograde labeling was found in the rostral lateral cortex and in the medial and dorsolateral AON. Ipsilaterally the dorsal cortex, nucleus of the diagonal band, lateral preoptic area, and dorsolateral amygdala showed labeled cell bodies. Retrogradely labeled cells were also found in the midbrain raphe nucleus. Results from injections into the rostral lateral cortex and lateral olfactory tract indicate that the mitral cells are the origin of the centripetal projections of the MOB. Injections in the accessory olfactory bulb (AOB) produced ipsilateral terminallike labeling of the ventral AON, bed nucleus of the accessory olfactory tract, central and ventromedial amygdaloid nuclei, medial part of the bed nucleus of the stria terminalis, and nucleus sphericus. Retrograde labeling of neurons was observed ipsilaterally in the bed nucleus of the accessory olfactory tract and stria terminalis, in the central amygdaloid nucleus, dorsal cortex, and nucleus of the diagonal band. Bilateral labeling of somata was found in the ventral AON, the nucleus sphericus (hilus), and in the mesencephalic raphe nucleus and locus coeruleus. Injections into the dorsal amygdala showed that the mitral neurons are the cells of origin of the AOB centripetal projections. Reciprocal connections are present between AOB and MOB. To our knowledge, this is the first study to address the afferent connections of the olfactory bulbs in a reptile. On the basis of the available data, a discussion is provided of the similarities and differences between the reptilian and mammalian olfactory systems, as well as of the possible functional role of the main olfactory connections in reptiles. PMID- 1709183 TI - Inventory and distribution of synapses of identified uniglomerular projection neurons in the antennal lobe of Periplaneta americana. AB - Uniglomerular projection neurons in the antennal lobe of Periplaneta americana, the axons of which connect the lobe to the protocerebrum, were labeled by intracellular injection of Lucifer Yellow or biocytin. The fine structure of individual neurons within the antennal lobe was examined after the injected substances had been converted (by immunohistochemical or histochemical treatment) to electron microscopically visible reaction products. Seven projection neurons were investigated, including attractant neurons, with dendritic arbors in the macroglomerulus, and projection neurons of normal-sized glomeruli. From reconstructions of thin serial sections and examination of additional processes present at various places in the arborization regions, the distribution of synapses within the glomeruli was inferred. Although the projection neurons differ from one another in their glomerular arborization patterns, they are very similar in the spatial segregation of their input and output synapses within the arborization. Output synapses are found on the thick part of the fiber near its site of entry into the glomerulus, as well as in regions within the glomerulus where the neuron has begun to ramify into thinner fibers. In the latter regions, the many output synapses are accompanied by occasional input synapses; hence these are regarded as transitional regions. At the terminal arbors only input synapses were found. This suggests that neurons with dense terminal arborizations receive particularly numerous inputs in these regions. The large number of input synapses reflects the high degree of convergence of afferents onto projection neurons previously demonstrated physiologically. However, the presence of numerous output synapses indicates that projection neurons not only transport sensory information into the protocerebrum but are also a major component of the neuronal circuitry within the antennal lobe. PMID- 1709184 TI - Regulation of human basophil activation. I. Dissociation of cationic dye binding from histamine release in activated human basophils. AB - Human basophil activation was demonstrated by histamine release (HR) and by the decrease of the toluidine blue-positive basophils (TB+). In four experimental systems, TB+ number decreased in the absence of HR (1) in basophils from atopic subjects stimulated by allergen concentrations below the threshold for HR, (2) in basophils sensitized by anti-2,4-dinitrophenyl IgE stimulated by noncovalently linked 2,4-dinitrobenzene sulfonic acid-human serum albumin (also, the threshold for decrease of TB+ required lower concentrations of sensitizing anti-2,4 dinitrophenyl IgE than for HR), (3) in low Ca++ medium, and (4) in the presence of the Na+/H+ exchanger, monensin. These results suggest that (1) there is a lower threshold for TB+ decrease than for HR in allergen concentration, number of membrane IgE molecules, and number of IgE cross-linkings; moreover, external Ca++ requirement is lower for decrease of TB+ than for HR and (2) TB+ decrease reflects either granule exocytosis or, in the absence of HR, biochemical changes (most probably cation exchanges) altering the interaction of the basic dye with the granules. Thus, monitoring decrease in TB+ allows detection of basophil activation in the absence of HR. PMID- 1709185 TI - Release of eosinophil granule proteins during IgE-mediated allergic skin reactions. AB - To determine whether the eosinophil (EOS), a prominent component of human allergic skin reactions, releases its potentially pathogenic components in vivo, we appended collection chambers to the bases of unroofed skin blisters and challenged the sites for varying time periods with either pollen antigen (Ag) or buffer (B)-control solutions. In seven sensitive subjects, continuous challenge with pollen Ag consistently induced release of more major basic protein (MBP) and eosinophil-derived neutrophil (EDN) than did B solution. Low levels of both MBP and EDN were observed during the first hour with increased accumulation during the second to fifth hour. Comparison of Ag- versus B-challenged site responses in individual subjects demonstrated significantly higher levels of both MBP and EDN at Ag than at B sites during the second to fifth hour. Levels of both MBP and EDN in the second to fifth hour correlated significantly with histamine release in the same sites in the first hour (r = 0.66 and 0.83, respectively). Imprints of the skin bases of the chambers after 5 hours demonstrated variable numbers of EOS at the Ag-challenged sites and only occasional EOS at the B-challenged sites; most cells on the skin bases were neutrophils. However, immunofluorescence localization of MBP in biopsy specimens of the blister bases revealed striking extra cellular MBP deposition. These findings indicate that EOS components accumulate in vivo in IgE-mediated human skin reactions, even when prominent EOS accumulation is not visualized, possibly because the EOS are degranulated in the allergic-reaction site. Release of EOS components in these reactions may be linked to earlier mast cell activation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709186 TI - [Gabaergic NADPH-diaphorase-positive Martinotti cells in the visual cortex in rats]. AB - NADPH-diaphorase positive cells were described in the visual cortex of the rat in layers II-VI and in the white matter. Their somata were large or medium-sized, oval or elongated, and their cytoplasm was accumulated at the poles. Some proximal thickened coarse dendrites formed a bitufted dendritic field. These features showed a cell type impregnated with the Golgi-Kopsch-method and a Golgi deimpregnation-method described as Martinotti cell (sparsely spined polarized neurons with ascending axons). The immunocytochemical evidence of GABA in NADPH diaphorase positive neurons (double labeling) showed the GABA ergic nature of these cells, but an attempt for a double labelling of NADPH-diaphorase and Parvalbumin was negative. PMID- 1709187 TI - Distribution of hypothalamic neurons projecting to the thoracic and sacral spinal segments in the cat. AB - The distribution of hypothalamic neurons participating in hypothalamo-spinal projections (hypothalamo-spinal HSP neurons) to the thoracic and sacral segments was studied using the technique of retrograde axonal transport. Horseradish peroxidase (HRP) and nuclear yellow (NY) were injected into various thoracic and/or sacral spinal cord segments. The retrogradely labeled cells were distributed in a continuous crescent-shaped field in the posterior, dorsolateral and lateral regions of the hypothalamus: starting from the ventral tegmental area (VTA), through the posterior hypothalamic nucleus (PH), the supramammillary nucleus (SM), the dorsal- and lateral hypothalamic area (AH1) and LH), the dorsomedial nucleus (DM) as well as in the paraventricular nucleus (PV). Neurons in all of the above hypothalamic nuclei project as caudally as to the sacral segments. The projection is bilateral and the contralaterally projecting fibres cross the midline at or near their termination site. Projection to thoracic segments is mainly ipsilateral. HSP neurons projecting to upper thoracic and sacral segments showed different patterns of distribution. A sacral injection resulted in most labeled neurons in the SM and LH and less labeled neurons in the PV, than a thoracic injection of comparable size and locations. Experiments with two tracer substances suggested that some of HSP neurons had divergent axon collaterals terminating both in thoracic and sacral spinal segments. PMID- 1709188 TI - Localisation of GABA-immunopositive cells in the river lamprey spinal cord. AB - Using the immunohistochemical PAP method GABA-immunopositive cells were revealed in horizontal and transverse sections of lamprey spinal cord. The most numerous intensively stained small oval cells were located in the dorsal part of the spinal cord. The larger cells with 2-3 long nonbranching processes were located at the border of lateral column, the main part of which is composed of GABA immunonegative cells. Some of the largest, but less intensively stained cells were observed in the lateral part of the spinal cord beyond the cell column and in the ventrolateral corner of transverse sections. The latter are identified as previously described edge cells. Functional identification of the remaining GABA immunopositive cells is rather difficult. Some of them may be glial elements. PMID- 1709189 TI - Subcortical afferents to the interstitial nucleus of Cajal: an anatomical retrograde tracing study in the rabbit. AB - Organization of brainstem projections to the interstitial nucleus of Cajal (INC) in the rabbit has been studied using the method of retrograde transport of horseradish peroxidase or wheat germ agglutinin-horseradish peroxidase conjugate. Injections of tracers into INC resulted in bilateral labelling in the medial terminal nucleus of the accessory optic tract, medial region of the zona incerta, vestibular nuclei (superior, medial, inferior), rostral portion of the prepositus nucleus and several nuclei of the pontine and medullary reticular formation. Retrograde labelling on the contralateral side was noted in all 4 deep cerebellar nuclei, the lateral vestibular nucleus, group Y, the rostral interstitial nucleus of the medial longitudinal fascicle, INC and mesencephalic reticular formation dorsal and lateral to the red nucleus. Cells of origin for the ipsilateral afferents of INC were found only in the nucleus of the posterior commissure. These data are discussed in relation to other morphological and physiological studies of afferent connectivity of INC in other species. PMID- 1709190 TI - Effect of prenatal exposure to ethanol on brains of kittens: I. Changes of neurons in lateral geniculate nucleus. AB - Our investigations try to reveal the morphological changes found postnatally at different levels and centres of central nervous system of kittens of cats chronically treated with ethanol during their pregnancy and lactation. The changes found in neurons of lateral geniculate nucleus (LGN) were analyzed in Golgi-stained preparations from brains removed on the first or on the ninth postnatal days. The data were computerized. The changes measured in neurons refer to their maturation process: a significant delay of differentiation of the neurons occurs in LGN. Also a delayed gyrification of brains was observed. PMID- 1709191 TI - Pulmonary surfactant-like multilamellar bodies in human arachnoid villi. AB - Human arachnoid villi adequately treated with tannic acid before osmication ultrastructurally disclosed highly ordered multilamellar bodies in all of the 6 subjects studied. The multilamellar bodies were varied considerably in size and shape, but often showed a fingerprint-like appearance. They were found in the intercellular space, extracellular cisterns or microcores and within the cytoplasm of arachnoid cells. The number of lamellae in a single lamellar body ranged from 3 to 20, and the periodical width of the lamellae was approximately 5.0 nm. The outermost lamella sometimes showed a direct continuity with the adjacent plasma membranes of the cytoplasm or mitochondria. As multilamellar bodies were ultrastructurally quite similar to pulmonary surfactant, they were assumed to be related to the lubricated flow or absorption of the cerebrospinal fluid. PMID- 1709192 TI - Diminished lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 on neonatal neutrophils underlies their impaired CD18 independent adhesion to endothelial cells in vitro. AB - To define further the molecular basis for abnormal interactions of cord blood or neonatal neutrophils with endothelial cells in vitro, we studied neutrophil adhesion and migration under experimental conditions specifically designed to evaluate CD18-independent mechanisms. Unstimulated cord blood neutrophils of healthy term neonates demonstrated significantly diminished adhesion to IL-1 stimulated endothelial cell monolayers under conditions of shear stress (congruent to 1.85 dynes/cm2); overall levels of migration by neonatal cells were also significantly diminished, although the adherent subpopulation of these cells migrated relatively normally. A mAb (DREG-56) against the human homologue of the murine MEL-14 antigen (termed lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 (LECAM-1), a member of the LEC-CAM family of adhesion molecules) markedly inhibited adhesion of healthy adult but not cord blood neutrophils. In additional assessments of endothelial cell adhesion or migration in the absence of shear forces, cord blood neutrophils demonstrated significantly diminished values compared to adult controls. Moreover, mAb DREG-56 significantly diminished adhesion of healthy adult but not cord blood suspensions in the presence or absence of the anti-CD18 mAb R15.7. Immunofluorescence assessments of unstimulated cord blood neutrophils or neutrophils of neonates 12 to 48 h of age showed dramatically diminished levels of surface LECAM-1 compared to adult neutrophils. Chemotactic stimuli (FMLP, 10 nM, 15 min) consistently "down-regulated" surface LECAM-1 on adult neutrophils to levels approximately 10% of unstimulated suspensions and comparable to those of most unstimulated neonatal suspensions. Moreover, FMLP stimuli elicited little or no down-regulation of LECAM-1 on neonatal cells. In comparative studies, endothelial cell adhesion of unstimulated cord blood or adult control neutrophils (assessed under conditions of flow) was directly related to levels of neutrophil surface LECAM-1. Although FMLP stimulation significantly diminished both adhesion and LECAM-1 surface levels of adult control cells, the adhesion and LECAM-1 expression observed with cord blood cells were not significantly influenced by this stimulus. The mechanisms underlying diminished LECAM-1 expression and LECAM-1-dependent adhesion of neonatal neutrophils and the physiologic significance of these abnormalities deserve investigation. PMID- 1709193 TI - Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients. AB - The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels. PMID- 1709194 TI - Endocrine, neural, and immune control of secretory component output by lacrimal gland acinar cells. AB - Androgens regulate the synthesis and secretion of secretory component (SC), the IgA antibody receptor, by acinar cells from the lacrimal gland. However, this hormone action may be susceptible to significant modification by other agents from the endocrine, nervous, or immune systems. To investigate the nature of this neuroimmunoendocrine interaction, the present study examined the impact of hormones, neurotransmitters, and lymphokines on basal and androgen-induced SC production by lacrimal gland acinar cells in vitro. Our results demonstrated that vasoactive intestinal peptide, the beta-adrenergic agonist, isoproterenol, PGE2, IL-1 alpha, IL-1 beta, and TNF-alpha significantly increased media SC levels in control or androgen-containing cell cultures. In contrast, the cholinergic agonist, carbachol, significantly decreased cellular SC output. These effects may be mediated through the agents' known capacity to alter intracellular cAMP levels. In support of this hypothesis, exposure of acinar cells to stimulators or analogues of cAMP resulted in a significant enhancement of SC production. Thus, these findings indicate that SC output in lacrimal tissue may be modulated by interactions between the endocrine, nervous and immune systems. PMID- 1709195 TI - Human eosinophil, but not neutrophil, adherence to IL-1-stimulated human umbilical vascular endothelial cells is alpha 4 beta 1 (very late antigen-4) dependent. AB - Eosinophils, through their ability to generate an array of potent mediators, are thought to be the major effector cells in a number of conditions, including parasitic infection, asthma, and other allergic diseases. The mechanism(s) by which eosinophils, as opposed to neutrophils, accumulate at inflammatory sites is unknown. One possible mechanism would be an eosinophil-specific pathway of adhesion to vascular endothelium. In this study we have demonstrated that human eosinophils, but not neutrophils, constitutively express alpha 4 beta 1 (CD49d/CD29). Expression was not increased on low density eosinophils or normal density cells stimulated with platelet-activating factor. Eosinophils, but not neutrophils, specifically adhered to COS cells transfected with vascular adhesion molecule-1 in a alpha 4 beta 1-dependent manner. Eosinophil, but not neutrophil, adhesion to IL-1 stimulated human umbilical vascular endothelial cells was significantly inhibited by alpha 4 beta 1 mAb at both 5 h (p less than 0.05) and 20 h (p less than 0.001). Inhibition of both resting and platelet-activating factor-(10(-7) M) stimulated eosinophil adhesion was observed. We conclude that the alpha 4 beta 1/vascular adhesion molecule-1 adhesion pathway may be involved in specific eosinophil, as opposed to neutrophil, migration into sites of eosinophilic inflammation. PMID- 1709196 TI - Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB). AB - Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus 1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response. PMID- 1709197 TI - Role of listeriolysin-O (LLO) in the T lymphocyte response to infection with Listeria monocytogenes. Identification of T cell epitopes of LLO. AB - Using a murine model, we investigated the role of the bacterial exotoxin listeriolysin O (LLO) in cellular immunity to Listeria monocytogenes. A correlation between LLO production by infecting bacteria and generation of protective immunity to virulent LLO-producing bacteria was noted. Using isogeneic hemolysin (Hly+ or Hly-) strains of L. monocytogenes, we demonstrated that LLO production by infecting bacteria is required to elicit T cells reactive both to bacteria-associated Ag and to the secreted LLO molecule as measured by IL-2 production in vitro. Distinct sets of T cells specific for largely nonoverlapping pools of antigenic determinants represented by LLO and cell-associated Ag (heat killed L. monocytogenes) are generated after infection. We have used models for prediction of T cell epitopes based on primary structure of LLO, and synthetic amphipathic LLO peptides were evaluated as Ag in vitro or as immunogenes in vivo. Infection of several strains of mice (H-2k and H-2d) with LLO-producing L. monocytogenes resulted in the generation of T cells that could respond consistently to two peptides, LLO 215-234 and LLO 354-371. Mouse strains lacking expression of I-E molecules (e.g., B10.A(4R) and C57BL/6) responded to LLO but not to the peptides tested. With C3HeB/FeJ mice, antibodies to I-Ek blocked the presentation of LLO 215-234. The importance of the N-terminal portion of LLO 215 234 was evidenced by the drastic reduction in antigenic activity of truncated peptides (e.g., LLO 221-234 and LLO 224-234). LLO 215-234, the strongest and most consistent activator of T cells from L. monocytogenes-immune mice, fit well some models for antigenic peptides in several ways. It could be predicted to form an amphipathic alpha-helix, it contained multiple "Rothbard motifs" (charged residue or glycine, two or three hydrophobic amino acids and then a glycine or polar residue), it had a net charge of +2, and it contained the correct spacing of amino acids (five to six residues between a hydrophobic and basic amino acid) that is characteristic of I-Ek-binding peptides. Immunization with 8 of 10 synthetic LLO peptides generated T cells that recognized the immunizing peptide in vitro, but such T cells were only poorly reactive with LLO. Our results indicate that LLO is an important target Ag for stimulation of CD4+ L. monocytogenes-specific T cells, and that LLO 215-234 is antigenically dominant in C3HeB/FeJ mice. PMID- 1709198 TI - Isolation, expression, and sequence of the TAP/Ly-6A.2 chromosomal gene. AB - The murine Ly-6 locus controls the expression of a number of genes. One of the products of the Ly-6 locus, Ly-6A.2, has been implicated in the process of T cell activation. We have identified the chromosomal sequences encoding the Ly-6A.2 molecule using very stringent hybridization and washing conditions. We confirmed that this gene encoded the Ly-6A.2 molecule by transfection studies using a cell line genetically negative for the Ly-6A.2 gene as a DNA recipient. Sequence analysis showed that the Ly-6A.2 gene is made up of four exons. The start site of transcription was determined by primer extension analysis. The first exon does not contain protein coding sequences. The structure of the Ly-6A.2 gene supports previous speculation that various Ly-6 RNA can be generated by alternate splicing events. The Ly-6A.2 chromosomal gene is closely related to the previously characterized Ly-6C.1 chromosomal gene in the intron, exon, and 5' flanking regions. This analysis indicates that these genes have arisen as a consequence of gene duplication. Although endogenous Ly-6A.2 and Ly-6C genes are IFN responsive, only the latter contains a clearly identifiable IFN responsive element. A transfected Ly-6A.2 chromosomal gene that contains 3.9 kb of 5'-untranslated sequence is IFN responsive. However, a shorter chromosomal clone containing only 940 bp of 5' sequence is constitutively expressed in transfectants but does not respond to IFN. PMID- 1709199 TI - CD5+ peritoneal B cells express high levels of membrane, but not secretory, C mu mRNA. AB - We used in situ hybridization to study Ig mRNA levels in murine peritoneal and splenic B cells. Ig mRNA production fell into three distinct groups: low, intermediate, and high. Splenic B cells primarily exhibited low levels characteristic of resting B cells or high Ig mRNA levels characteristic of plasma cells. In contrast, a significant fraction of peritoneal B cells exhibited intermediate Ig mRNA levels. Intermediate Ig mRNA was T cell dependent in that congenic nu/nu mice had far fewer peritoneal cells expressing the intermediate Ig message than their wild type counterparts. CD5+ CD11b+ IgMbright+ peritoneal B cells were found to be mainly responsible for the production of intermediate Ig mRNA levels. The peritoneal CD5- CD11b+ IgMbright+ "sister" B cell subpopulation contained a lower percentage of intermediate Ig mRNA-producing B cells. CD5-CD11b IgMdull+ "conventional" B cells produced negligible levels of Ig mRNA, comparable to those of unfractionated splenic B cells. Northern analysis showed that the majority of Ig mRNA expressed in the peritoneum is of the membrane rather than the secreted form. Consistent with that result, in short-term culture, peritoneal cells showed markedly less Ig secretion than did spleen cells. These studies describe novel Ig mRNA expression by peritoneal B cells and emphasize that within the peritoneal cavity, B cells do not tend to become antibody-secreting cells. PMID- 1709200 TI - The human monocyte-like cell line THP-1 expresses Fc gamma RI and Fc gamma RII. AB - THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 53 kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.3 (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. Interferon-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U937 cells was increased six- to eight fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNF alpha), and vitamin D3 had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF alpha, PMA, or vitamin D3 had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype. PMID- 1709201 TI - Turnover of dopamine and serotonin and their metabolites in the striatum of aged rats. AB - Turnover of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], and their metabolites has been measured in adult and aged rats. Turnover rates of 3,4 dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxy-3 indoleacetic acid (5-HIAA) have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase (MAO) and from the accumulation rates by probenecid inhibition of the probenecid-sensitive transport system. DA and 5-HT turnover rates have been measured as accumulation rates of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively, after central decarboxylase inhibition by 3-hydroxybenzylhydrazine (NSD-1015) and as accumulation rates of DA and 5-HT after pargyline inhibition of MAO. The DA turnover rate after NSD-1015 was 23.9% lower in aged rats than in adults, whereas after pargyline there was no significant difference between the two age groups. The HVA fractional rate constant and turnover after pargyline were lower in aged rats than in adults, and HVA turnover after probenecid was higher in aged rats than in adults. The DOPAC-HVA pathway seems to be reinforced at the expense of DOPAC conjugation. In aged and adult rats whose 5-HT steady-state levels were not statistically different, the 5-HT turnover rate after pargyline and NSD-1015 treatment was lower in aged rats than in adults. An increase of 5-HIAA levels after pargyline and probenecid treatment in aged rats could be due to the handling stress. PMID- 1709202 TI - Pallido-nigro-luysian atrophy, progressive supranuclear palsy and adult onset Hallervorden-Spatz disease: a case of akinesia as a predominant feature of parkinsonism. AB - Few parkinsonian patients present with 'pure akinesia' or with severe akinesia accompanied by only mild rigidity, tremor and other manifestations such as ophthalmoplegia. Pathological examinations of such cases have rarely been conducted and have revealed findings compatible with progressive supranuclear palsy (PSP), pallido-nigro-luysian atrophy (PNLA) or Parkinson's disease. We report a parkinsonian patient whose main clinical feature was akinesia. A postmortem study of this patient showed findings corresponding to PNLA and PSP. Histochemical properties of the pallidal pigment granules were equivalent to those of Hallervorden-Spatz disease (HSD) and striatonigral degeneration. In addition to iron-positive pigment granules, spheroids, severe neuronal loss and gliosis in the globus pallidus and substantia nigra, formation of Alzheimer's neurofibrillary tangle (NFT) in the brainstem shares characteristics with PSP, adult onset HSD and PNLA. We suggest that the underlying pathology of 'pure' akinesia is most often situated in the globus pallidus substantia nigra and subthalamus (Luys), and that PSP, PNLA and adult onset HSD may constitute a spectrum of one disease. PMID- 1709203 TI - GABA-like and glycine-like immunoreactivities of the cochlear root nucleus in rat. AB - The cochlear root nucleus is part of the cochlear nuclear complex in small rodents. Its cells, the large root neurons, have a superficial resemblance to the globular neurons of the ventral cochlear nucleus. It has been a matter of debate, therefore, whether the root neurons and globular neurons represent the same or different types of cell. In the present study the two cell types with adjacent neuropil structures were compared by light microscopic, postembedding immunocytochemistry. Pairs of 0.5 microns sections of resin-embedded, glutaraldehyde-fixed material were treated with purified antisera raised against GABA- and glycine-glutaraldehyde-protein conjugates, respectively. Both types of cell were found to be immunonegative. Striking differences, however, occurred in what was interpreted as afferent nerve terminals. The globular cells appeared to receive numerous afferents with GABA- or glycine-like immunoreactivity on their somata. Immunoreactive terminals on the root neurons, on the contrary, were mostly GABA-positive and located on the dendrites. Although of unknown origin, the immunoreactive afferents were clearly different from the primary fibres as demonstrated both by the immunonegativity of the latter and by the different size and distribution of the terminals labelled anterogradely after horseradish peroxidase injections into the spiral ganglion. PMID- 1709204 TI - Stable clathrin: uncoating protein (hsc70) complexes in intact neurons and their axonal transport. AB - We have studied the organization of clathrin during its transport in axons. Using immunoprecipitation techniques we have confirmed earlier findings that clathrin is transported as part of slow component b, but we also detect small amounts of clathrin in fast component. As fast component is known to correspond to the transport of membraneous material, including coated vesicle membrane components, our findings suggest that some clathrin in axons undergoes transport in the form of coated membranes and that a portion of the clathrin delivered to axons and axon terminals arrives by way of fast component. The organizational form of clathrin in slow component b (SCb) was examined in more detail, as it is thought to represent a non-membrane-associated species, is relatively long-lived, and at any instant represents the major transport species in axons. We used nondenaturing immunoprecipitation methods with stringent wash procedures to identify other SCb proteins that interact with clathrin. The immunoprecipitates contained major labeled bands that corresponded to clathrin heavy and light chains, along with a prominent 70-kDa band and several minor bands that ranged in apparent Mr from 70,000 to 150,000; the 70-kDa band was shown to be the ATP dependent uncoating protein by two-dimensional gel electrophoresis. A very similar profile of polypeptides was also immunoprecipitated from extracts of cultured neurons. The results from a variety of control immunoprecipitations, including the use of antisera preadsorbed with purified clathrin trimers or clathrin light chains, indicate that coprecipitation of clathrin and uncoating protein with the other 70,000-150,000-Da polypeptides from SCb reflects specific interactions. Including exogenous uncoating protein in the lysis buffer had no detectable effect on the levels of endogenous uncoating protein recovered in the immunoprecipitates, indicating that complexes of clathrin, uncoating protein, and the other coimmunoprecipitating SCb protein existed in the intact neurons prior to lysis. Finally, a specific and functional association is further supported by the release of uncoating protein, but not the other 70,000-150,000-Da polypeptides, from the immunoprecipitated complexes on the addition of ATP. Collectively, these observations provide the first direct evidence of interaction between clathrin and uncoating protein in intact cells, lend strong support to the concept that uncoating protein plays an intimate role in clathrin dynamics within cells, and reveal a family of 70,000-150,000-Da polypeptides that form a stable nonmembranous association with clathrin in intact cells. PMID- 1709205 TI - Distribution of dihydropyridine and omega-conotoxin-sensitive calcium currents in acutely isolated rat and frog sensory neuron somata: diameter-dependent L channel expression in frog. AB - Calcium channel subtypes in adult rat and frog sensory neuron somata, acutely isolated from dorsal root ganglia (DRG neurons), were studied using Bay K 8644, nimodipine, and omega-conotoxin GVIA (omega-CgTx) as specific probes. The DRG neurons varied in diameter 15-60 microns (rat) and 20-80 microns (frog). Bay K 8644 produced a large increase in calcium currents of small-diameter rat DRG neurons and shifted channel activation and the peak of the I-V curve in the hyperpolarizing direction. At a physiological holding potential (HP) of -60 mV, nimodipine blocked 50% of the peak calcium current in small-diameter frog and rat DRG neurons, indicating a large L channel component. At HP -80 mV, nimodipine blocked a lower percentage of peak current in small-diameter rat and frog DRG neurons than expected (based on experiments at HP -60 mV) probably due to nimodipine's voltage dependence. At HP -60 mV, omega-CgTx blocked 25% and 50% of peak current in small-diameter rat and frog DRG neurons, respectively. Omega-CgTx blocked a larger percentage of current at HP -80 mV than at -60 mV, probably because of the repriming of N channels. Observation of nimodipine- and Bay K 8644 sensitive calcium current in small-diameter rat and frog DRG neurons after omega CgTx treatment, suggests that omega-CgTx is not a potent L channel blocker. The combination of omega-CgTx and nimodipine blocked all current in small-diameter frog DRG neurons but left a small portion of current unblocked in small-diameter rat DRG neurons at HP -60 mV, suggesting the possibility of omega-CgTx- and nimodipine-insensitive calcium channels in rat DRG neurons. Calcium current in most large-diameter frog DRG neurons was insensitive to nimodipine, but was completely blocked by omega-CgTx. This indicates significant variation in the expression of calcium channel subtypes in small- and large-diameter frog DRG neurons, which may subserve different sensory modalities. PMID- 1709206 TI - Dependence of Ca2+ and K+ current development on RNA and protein synthesis in muscle-lineage cells of the ascidian Boltenia villosa. AB - The early development of excitability of muscle-lineage cells of the ascidian Boltenia villosa is characterized by the appearance, just after gastrulation, of a Ca2+ current and a delayed outward K+ current, while an inwardly rectifying K+ current, present since fertilization, disappears. The muscle-lineage cells are the first cells in which we detect tissue-specific electrical properties after gastrulation. Here, we show that the development of electrical properties in these cells involves RNA and protein synthesis. If transcription or translation is blocked, the Ca2+ and outward K+ currents fail to appear, whereas the inward K+ current disappears normally. For the Ca2+ current, the sensitive period for transcription extends until just before gastrulation, while the sensitive period for translation extends until after gastrulation. The oocyte has a Ca2+ current present at about 5-10% the density of that in the muscle-lineage cells; this current disappears by gastrulation. A comparison of the oocyte and muscle Ca2+ currents indicates that they are similar in voltage dependence and inactivation mechanism. A small difference in permeability sequence can be attributed to different surface charge properties at the two stages of development. PMID- 1709207 TI - MRI pattern of progressive multifocal leukoencephalopathy (PML) in AIDS. Pathological correlations. AB - Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease which occurs in immunodepressed subjects and is particularly frequent in AIDS. Some authors having drawn attention to the protean aspect of the disease and claimed that AIDS may lose its basic characteristics and affect the grey matter as well as the white matter, we reviewed a series of 8 patients who had been biopsied and/or autopsied and had been examined at least once by MRI. In this series, contrary to what is regularly observed in toxoplasmic abscesses we did not find any lesion of the grey matter or any mass effect. On the other hand, we confirmed that PLM is not multifocal in all cases and that it course may be interrupted by prolonged remissions. The MRI criteria for PML therefore are reliable, provided multiple T2-weighted slices in coronal plane are performed, clearly showing the anatomy of the white fibres affected. However, it must be borne in mind that HIV infected patients often have other associated brain pathologies, especially when the immune deficiency increases. PMID- 1709208 TI - [Tyrosine-phosphorylated proteins in human breast carcinoma]. AB - Proteins phosphorylated on tyrosine are detectable by antibodies against phosphotyrosine (P-Tyr antibodies) in cells transformed by oncogene-encoded tyrosine kinases. We used P-Tyr antibodies to investigate the existence of abnormal levels of phosphoproteins in human breast cancer. Three human breast cancer cell lines (SK-BR-3, MCF-7 and CG-5) and 37 human breast cancer specimens were examined by Western blot analysis and "in vitro" kinase assay. In the SK-BR 3 cell line three major phosphoproteins of the approximate Mr of 185,000 (p185), 135,000 (p135) and 110,000 (p110) were detected. The former was identified as the HER-2 gene product by specific antibodies against HER-2 encoded protein. In the other cell lines, a product of the approximate Mr of 170,000 (p170), together with a p135 and a p110, were phosphorylated on tyrosine. P185 and p170 were shown to have an associated tyrosine kinase activity. Two proteins, comigrating with p135 and p110, were found to be highly phosphorylated on tyrosine in 50% of the breast cancer samples, but not in samples harvested from 12 human tumors of the gastro-intestinal tract. These data show that 50% of human breast cancer samples display an abnormal level of tyrosine phosphorylated proteins. PMID- 1709209 TI - Characterization of in vitro expressed human alpha-fetoprotein as highly reproducible reference material for clinical immunoassays. PMID- 1709210 TI - [TPA and TPS in the follow-up of cancer of the breast: preliminary evaluations]. PMID- 1709211 TI - Diagnostic value of prostatic acid phosphatase and prostate-specific antigen in patients with prostatic cancer. AB - To compare the clinical usefulness of the measurement of prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in serum of patients with prostatic carcinoma, we studied 128 patients with prostatic pathology, sixty (46.9%) of whom had prostatic cancer. Receiver-operating characteristics (ROC) curves were constructed and the underlying areas were calculated and compared to study clinical efficiency of the two markers regardless of the cutoff level selected. The area for PSA (0.90 +/- 0.30) was significantly higher (p less than 0.001) than that of PAP (0.71 +/- 0.05) showing that PSA was a better discriminator of the patients with or without prostatic cancer. The maximal clinical efficiency of the two tests at selected cutoff levels (0.8 U/L for PAP and 10 micrograms/L for PSA) was 0.787 and 0.883, respectively, confirming the superiority of PSA. However, the associated determination of the two markers improved the clinical specificity with no false-positive cases. PMID- 1709212 TI - [Value of tumor marker positivity in germinal non-seminomatous tumors of the testis]. PMID- 1709213 TI - [Prostatic diseases: tumor markers (PAP and PSA) and age at risk]. PMID- 1709214 TI - Immunological aspects of markers of joint disease. AB - There is considerable potential for using immunological methods to detect and quantify markers of joint disease in man and animal models. These markers usually result from increased matrix turnover as the disease develops, and fall into several categories. Anabolic markers are those that specifically recognize epitopes in macromolecules that are newly synthesized in the cells' initial response to repair and remodel the tissue in the early stages of the disease. Catabolic markers are those that result from degradation of preexisting matrix as the disease progresses. At present, this laboratory has available a large panel of well characterized monoclonal antibodies directed against proteoglycan epitopes that can be used to detect both anabolic and catabolic markers of joint disease. Development of immunoassay procedures to both monitor the progression of joint disease and the effect of therapeutic intervention will occur in the near future. PMID- 1709215 TI - Graft endothelialization: the role of angiogenic mechanisms. PMID- 1709216 TI - [CD5+, CD7+, and CD19+ non-Hodgkin's lymphoma in a child]. AB - The 9-year-old boy was admitted to Shizuoka Children's Hospital because of cervical lymphoadenopathy. Complete blood count showed normal RBC and platelet counts. WBC was 2700/microliters with no tumor cells. Bone marrow aspirate showed normocellularity with 34% tumor cells. Lymph node biopsy from his right neck was performed and the patient was diagnosed as non-Hodgkin's lymphoma (lymphoblastic type). Surface marker analysis disclosed that the tumor cells were positive for CD5, CD7, CD19, CD38, CD71, and Ia antigen. Chromosomal analysis of the cervical lymph node revealed 46, XY, t(7;14) (p15;q32). Molecular investigation with appropriate probe showed germ-line configurations of IgH gene, TcR beta gene, and TcR gamma gene, and one rearranged band of TcR delta gene. Monoclonality of tumor cells was demonstrated from chromosomal analysis and molecular study. CD7 and CD19 are not lineage specific antigens because CD7 is expressed on immature AML cells and CD19 is expressed on T ALL cells or AML cells. Moreover, TcR delta rearrangement is considered to occur at early phase of hematolymphoid cells. Based on these data, tumor cells of this patient is considered to originate from immature lymphoid cell, so-called lymphoid stem cell. PMID- 1709217 TI - Ultrastructure and blood-iris barrier in experimental rubeosis iridis in rabbit. AB - Iris neovascularization was produced in rabbits by hypotony following repeated aspiration of the vitreous. The hypotony was produced after 0.3 ml of vitreous fluid was aspirated using a 25-gauge needle through the pars plana of 10 rabbits. For the histochemical study, horseradish peroxidase(HRP) was injected through the ear lobe vein. After fixation of the iris tissue, the tissue was treated with diaminobenzidine and examined with both light microscopy and transmission electron microscopy. The newly-formed vessel was abundant, particularly on the upper stroma of the iris. The new vessel formation was evident due to the proliferation of endothelial cells, which may have been derived from preexisting iris vessels. The endothelial cells of the newly-formed vessels revealed prominent villous processes into the vascular lumen, formation of the marginal flap, numerous fenestrations in the endothelial junction, and reaction product onto extravascular space by the cytochemical electron microscopy. These results suggest that hypotony in the rabbit produces the disruption of the blood-iris barrier and the balance between angiogenesis-antiangiogenesis modulation. PMID- 1709218 TI - [Salicylazosulfapyridine-induced agranulocytosis in a patient with ulcerative colitis, successfully treated with granulocyte colony-stimulating factor]. PMID- 1709219 TI - Differentiation of hematuria by quantitative determination of urinary marker proteins. AB - Hematuria caused by prerenal, glomerular, postglomerular, and postrenal causes is usually differentiated by a number of noninvasive and invasive diagnostic procedures. In the present study we have applied a new analytical strategy based on observations that the various forms of hematuria can be classified by their typical protein pattern. When analyzed by quantitative turbidimetric assays, urines from postrenal hematurias contained high-molecular-weight proteins (alpha 2-macroglobulin and IgG) in proportions found in plasma. Relating excretion rates (mg/mg) of these proteins to those of albumin, ratios for alpha 2 macroglobulin/albumin and IgG/albumin were 2.0-31 x 10(-2) and 20.0-180 x 10(-2), respectively. In contrast, glomerular hematurias exhibited ratios of 0.01-2.0 x 10(-2) (alpha 2-macroglobulin/albumin) and 2.0-20 x 10(-2) (IgG/albumin). Additional determination of alpha 1-microglobulin allowed us to differentiate postglomerular hematurias caused by interstitial nephropathies from glomerular and postrenal diseases. Critical evaluation of 93 cases diagnosed by independent clinical examination including histology, sonography, and cystoscopy revealed that the criteria derived from protein measurements resulted in correct classification when urine albumin exceeds 100 mg/l. This noninvasive procedure is expected to be of considerable help in the primary care of patients with unexplained hematuria. PMID- 1709220 TI - Characterization of distinct angiotensin II binding sites in rat adrenal gland and bovine cerebellum using selective nonpeptide antagonists. AB - We investigated the characteristics of 125I-AII binding to rat adrenal and bovine cerebellar membranes in the presence and absence of new nonpeptide angiotensin II (AII) receptor ligands. The imidazole AII ligands, DUP753 and WL19, both produced biphasic competition curves to 125I-AII binding in rat adrenal glomerulosa and adrenal medulla particles, suggesting the existence of two distinct AII binding sites. Antagonist affinity (Ki) and binding capacity (Bmax) for each binding site was determined using nonlinear analysis of competition data fit to a two-site model. The high capacity site (68% of total specific 125I-AII bound) in glomerulosa had high affinity for DUP753 (4.6 +/- 0.8 nM) and low affinity for WL19 (29 +/- 3 microM), and the low capacity site had high affinity for WL19 (3.3 +/- 1.4 nM) and low affinity for DUP753 (51 +/- 9 microM). Conversely, in medulla, the high capacity site (77% total binding) had high affinity for WL19 (19 +/- 6 nM) and low affinity for DUP753 (29 +/- 8 microM), and the low capacity site had low affinity for WL19 (25 +/- 7 microM) but a high affinity for DUP753 (2.8 +/- 2.0 nM). In glomerulosa, binding parameters for the nonpeptide ligands at each site derived from monophasic competition curves obtained in the presence of either 0.3 microM DUP753 or WL19 to selectively block the high or low capacity binding site, respectively, were similar to values determined from the biphasic competition curves. Unlike the nonpeptide inhibitors, unlabeled AII yielded monophasic inhibition curves.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709221 TI - Protection against postischemic myocardial dysfunction in anesthetized rabbits with scavengers of oxygen-derived free radicals: superoxide dismutase plus catalase, N-2-mercaptopropionyl glycine and captopril. AB - Postischemic myocardial dysfunction in canine myocardium has been reported to be reduced by scavengers of oxygen-derived free radicals. One potential source of oxygen-derived free radicals in canine myocardium is xanthine oxidase, but human and rabbit myocardium either lack or possess very low levels of this enzyme. Therefore, the effects of scavengers of oxygen-derived free radicals on postischemic myocardial dysfunction produced by 15 min of ischemia and 3 h of reperfusion were evaluated in vivo in the rabbit. Superoxide dismutase (SOD) (45,000 U/kg) and catalase (55,000 U/kg) were given into the left atrium 10 min before ischemia, and followed by an additional 45,000 U/kg of SOD and 55,000 U/kg of catalase given over 85 min. This treatment reduced postischemic myocardial dysfunction, as did sulfhydryl-containing free radical scavengers N-2 mercaptopropionyl glycine (4 mg/kg, i.v.) and captopril (3 mg/kg, i.v.) given 5 min before and 60 min after reperfusion. SOD given alone at the same dose was ineffective, as was enalaprilat (0.3 mg/kg, i.v.), an angiotensin-converting enzyme inhibitor that does not scavenge oxygen-derived free radicals. Thus, postischemic myocardial dysfunction was reduced by scavengers of oxygen-derived free radicals in vivo in a species that is deficient in myocardial xanthine oxidase. This suggests that oxygen-derived free radicals derived from a source other than xanthine oxidase play a role in postischemic myocardial dysfunction. PMID- 1709222 TI - Effects of nitrendipine on sodium balance during changes in sodium intake and low dosage ANP. AB - We found previously that calcium entry blockade with nitrendipine enhanced the natriuretic effect of high-dose atrial natriuretic peptide (ANP). It is unknown whether nitrendipine also influences the effect of physiological changes in ANP. We therefore studied the effect of nitrendipine on cumulative sodium balance during changes in sodium intake as well as on natriuresis after low-dosage ANP infusion during low and high sodium (LS and HS, respectively) intake. In eight healthy volunteers, sodium balance was recorded after the switch from LS (20 mmol/day) to HS (300 mmol/day) diet. Cumulative sodium balance was equal with (441 +/- 45 mmol) or without (458 +/- 45 mmol) nitrendipine treatment. The body weight curves were also fully congruent. ANP (0.005 micrograms/kg/min for 3 h) was administered during maximal water diuresis on both sodium intake levels with and without nitrendipine. Infusion of ANP increased sodium excretion (mumol/min) from 30 +/- 7 to 81 +/- 12 (LS) and from 316 +/- 27 to 469 +/- 46 (HS). During nitrendipine, similar increments (from 36 +/- 7 to 98 +/- 24 mumol/min, HS) were found. ANP had no effect on inulin clearance and fractional excretion of lithium, but consistently depressed diluting segment reabsorption. This pattern was also similar during nitrendipine. Apparently, under the conditions of this study in normal subjects, nitrendipine has no effect on sodium balance. ANP, in physiological concentrations, increases natriuresis mainly by depressing sodium reabsorption in the distal nephron, an effect not enhanced by nitrendipine. PMID- 1709223 TI - Influence of pentobarbital and chloralose anesthesia on quinidine-induced effects on atrial refractoriness and heart rate in the dog. AB - The effects of pentobarbital and chloralose on the atrial effective refractory period (AERP), atrial and ventricular rates, and mean blood pressure and also on the effects of quinidine on the same parameters were investigated in dogs with chronic atrioventricular block and implanted atrial pacing electrodes. Pentobarbital (30 mg/kg) increased the AERP by up to 12%, atrial and ventricular rates by 39 and 40%, respectively, and after initial lowering (48%) it increased the mean blood pressure (46%). Chloralose (100 mg/kg) increased the AERP (less than 30 min) by up to 7%, the atrial rate by 49%, the ventricular rate (less than 5 min) by 18%, and the mean blood pressure by 47%. In conscious dogs, quinidine at cumulative doses of 2, 4, and 8 mg/kg, i.e., at plasma levels between 2.7 +/- 0.6 and 6.3 +/- 1.3 micrograms/ml, increased the AERP by up to 21, 28, and 46%, the atrial rate by 49, 65, and 72%, and the ventricular rate (less than or equal to 5 min) by 17, 14, and 14%, and lowered the mean blood pressure by 19, 33, and 43%, respectively. Pentobarbital increased the quinidine-induced lengthening of the AERP by up to 10, 21, and 25 ms, respectively, and reduced the corresponding atrial (38, 53, and 67 beats/min) and ventricular (4, 4, and 5 beats/min) chronotropic effects. In contrast, chloralose reduced the quinidine-induced lengthening of the AERP (5, 12, and 22 ms, respectively), but did not modify the corresponding atrial and ventricular chronotropic effects. Neither pentobarbital nor chloralose altered quinidine plasma levels or the hypotensive effects of this drug.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709224 TI - Pharmacodynamic profile of verapamil in relation to absolute bioavailability: investigations with a conventional and a controlled-release formulation. AB - The absolute bioavailability F and response (prolongation of the PR interval) of verapamil after single doses of the same oral formulation administered on two different days were investigated in 16 male subjects with an 80 mg fast dissolving and a 240 mg controlled-release preparation and compared with a bolus injection of 5 mg of verapamil. The absolute bioavailability was 23% in both investigations for the 80 mg preparation and 32% in both investigations for the 240 mg dosage form. The individual values obtained for tmax, cmax, F, and AUC0 alpha showed a wide intersubject variability; therefore, no significant differences could be observed between the two trials for each dosage, but significant differences existed between the investigations of the two preparations. After intravenous administration, concentration-effect curves were about twofold left shifted when compared with the 80 mg tablet and about threefold left shifted when compared with the 240 mg tablet. Estimation of the drug input rate showed significantly (p less than 0.05) smaller values when the controlled-release tablet was given (80 mg tablet: 95.1 and 107.7 mg/h; 240 mg tablet; 55.8 and 46.3 mg/h). Thus, the effect and bioavailability of verapamil show sufficient intersubject reproducibility if the same formulation is given. The differences between the responses and the bioavailability after administration of different preparations may be related as well to the drug absorption rate and the stereoselective first pass of verapamil as to saturation of first-pass metabolism. PMID- 1709225 TI - Naftopidil, a new alpha-adrenoceptor blocking agent with calcium antagonistic properties: characterization of Ca2+ antagonistic effects. AB - The newly developed antihypertensive agent naftopidil blocks alpha 1 adrenoceptors and inhibits Ca2+ entry via potential-dependent channels in vascular muscle. The aim of our study was to detect possible Ca2+ channel blocking activity in various isolated preparations of the guinea pig heart. Prazosin and verapamil were used for reference. In papillary muscles, 10 microM of all drugs reduced the force of contraction Fc. The action potential duration and the refractory period were hardly affected by naftopidil, decreased by verapamil, and slightly increased by prazosin. In constant-flow Langendorff hearts, the drugs reduced the perfusion pressure, decreased the Fc, and slowed the spontaneous heart rate (order of potency: verapamil much greater than naftopidil greater than prazosin). In voltage-clamped ventricular cardiomyocytes, the calcium current ICa was completely inhibited by verapamil (pD2 value of 6.9) and to 53.5% by naftopidil (pD2 value of 6.4). Prazosin (10 microM) decreased ICa by little more than 10%. There were no differences in the steady-state inhibition of ICa by the two enantiomers of naftopidil. The block of ICa was clearly use dependent. Radioligand binding studies with (+)-[3H]PN 200-110. (-) [3H]desmethoxy-verapamil, and (+)-cis-[3H]diltiazem in guinea pig skeletal muscle T-tubulus membranes demonstrated that racemic naftopidil exhibited some affinity for the three distinct drug receptor domains of the L-type Ca2+ channel. In conclusion, the present data are consistent with the hypothesis that naftopidil is a weak ligand for L-type calcium channels. It partially blocks ICa and shows no stereoselectivity. PMID- 1709227 TI - Ventricular tachycardia in an isolated guinea pig ventricular free wall model of ischemia and reperfusion. AB - Electrical activity from endo- and epicardium was recorded from isolated segments of right ventricular free walls of guinea pigs using standard differential microelectrode techniques and a high-gain electrocardiogram (ECG). Stimulation was applied to the endocardium. Tissues were exposed to ischemic conditions for 10 min and then were reperfused with "normal" Tyrode's solution. Early premature beats or rapid ventricular tachycardia (VT) occurred in 36% of hearts during "ischemia" and 79% of hearts on reperfusion. Endocardial activation was not significantly slowed by ischemic conditions or reperfusion. However, transmural conduction times increased, and muscle action potential durations decreased during ischemic conditions and early reperfusion. Rapid VT began with alternating activation of endo- and epicardium, and continuous low-voltage ECG activity bridging diastole. Activation of epicardium was essential for occurrence of early premature beats and rapid VT. Hyperkalemia during ischemia promoted arrhythmias during "ischemia," but not during reperfusion. Oscillatory afterpotentials (OAP) also occurred during reperfusion (36%), but not during "ischemia". Our study provides an isolated tissue model that reproducibly generates tachycardias, and that permits study of ischemia and reperfusion-induced transmembrane activity and defects in transmural conduction. PMID- 1709226 TI - Effect of cilazapril in hypertensive patients with renal impairment. AB - The aim of the study was to evaluate the clinical and renal hemodynamic effects of cilazapril in 10 hypertensive patients with moderate-to-severe chronic renal failure (creatinine clearance 14-50 ml/min). After 2 weeks of placebo, cilazapril 0.5 mg/day was given, and the dose was increased up to 5 mg/day if sitting diastolic blood pressure (SDBP) was not normalized (less than or equal to 90 mm Hg). Once a normal SDBP value was achieved, the patients remained on the given dose regimen for 6 months. After this period SDBP decreased from 107 +/- 2 to 95 +/- 2 mm Hg (p less than 0.001). At the end of treatment, glomerular filtration rate (GFR) remained unchanged in five patients, improved in four patients, and slightly decreased in one patient, the slope from baseline being 0.137 and the variation of GFR per unit of GFR at baseline being between -0.20 and 0.47. Likewise, effective renal plasma flow increased not significantly, showing considerable variability. Urinary protein excretion was reduced significantly from 2.51 +/- 0.75 to 0.51 +/- 0.10 g/L (p less than 0.05), suggesting that converting enzyme inhibition may exert a renal protective effect. In conclusion, it appears that cilazapril does not induce functional damage in the kidney of predialysis hypertensives. PMID- 1709228 TI - Effects of quinidine on arrhythmias and conduction in an isolated tissue model of ischemia and reperfusion. AB - Transmembrane electrical activity from endo- and epicardium and a high-gain ECG were recorded from isolated segments of guinea pig right ventricles. Endocardium was stimulated. Tissues were exposed to ischemic conditions for 15 min and then reperfused with "normal" Tyrode's solution. Ventricular tachycardia, bigeminy, or trigeminy with characteristics of transmural reentry occurred in early reperfusion in 68% of control hearts. Arrhythmias were associated with prolongation of the transmural conduction time (CT) and abbreviation of the endocardial effective refractory period (EP). Quinidine significantly suppressed reperfusion arrhythmias at 1 and 5 microM, slightly increased the incidence of arrhythmias at 10 microM, and again suppressed arrhythmias at 50 and 100 microM. At 1 and 5 microM, quinidine prevented or attenuated prolongation of the transmural CT by ischemic conditions and reperfusion. The transmural CT was not significantly changed at 10 microM, and was further prolonged at 50 and 100 microM quinidine. The endocardial ERP was prolonged by 50 and 100 microM quinidine during ischemic conditions and reperfusion. In epicardial slices, 5 microM quinidine shortened the CT transverse to the fiber orientation during reperfusion but had no effect on the longitudinal CT. Thus, antiarrhythmic efficacy of low concentrations of quinidine may occur through differential effects dependent on tissue anisotropy. PMID- 1709229 TI - Influence of hypoxia on the myocardial uptake and pharmacodynamics of quinidine in isolated perfused rabbit hearts. AB - The effects of hypoxia on the myocardial uptake and pharmacodynamics of quinidine were studied in isolated perfused rabbit hearts. Hearts were perfused with a modified Krebs-Henseleit buffer that was equilibrated with either 95% O2-5% CO2 (normoxia) or 95% N2-5% CO2 (hypoxia). The myocardial quinidine accumulation was determined from concentration differences in aortic perfusate and coronary sinus effluent. Under hypoxic conditions, the myocardial concentration of quinidine (12.0 +/- 3.6 micrograms/g) was significantly reduced compared to normoxic conditions (24.8 +/- 8.5 micrograms/g, p less than 0.01). Greater increases in QRS complex duration were observed during hypoxia (10.0 +/- 1.0 ms) compared to normoxia (7.5 +/- 1.3 ms; p less than 0.05). Greater increases in MAP duration were also observed during hypoxia (64 +/- 14 ms) compared to normoxia (37 +/- 14 ms; p less than 0.01). The myocardial concentration-effect relationships describing changes in QRS complex duration, QT interval, MAP duration, and ventricular effective refractory periods were linear in both groups. The curves of the concentration-effect relationships observed during hypoxia were shifted to the left compared to those observed during normoxia and the slopes of these relationships were also significantly greater (p less than 0.05). These pharmacokinetic and pharmacodynamic interactions may be explained by the development of acidosis during hypoxia since the pH of the coronary sinus effluent decreased significantly during hypoxia (7.10 +/- 0.04) compared to the normoxic group (7.25 +/- 0.04, p less than 0.001). Thus, although hypoxia reduces the myocardial accumulation of quinidine, greater electrophysiologic effects are observed compared to normoxic conditions. These observations likely relate to a change in responsiveness of acidotic tissue to quinidine. PMID- 1709230 TI - Presynaptic beta-adrenoceptors in guinea pig papillary muscle: evidence for adrenaline-mediated positive feedback on noradrenergic transmission. AB - Guinea pig papillary muscles were preincubated in the presence of 5 x 10(-9) mol/L unlabeled noradrenaline or adrenaline then incubated with (3H) noradrenaline and superfused. Electrical field stimulation with 180 pulses delivered at 1 or 3 Hz was used to induce overflow of radioactivity. Comparison of the effects of preexposure of the tissue to adrenaline or noradrenaline revealed that adrenaline incubation caused an enhancement of stimulation-evoked overflow of (3H)noradrenaline and a reduction of the effect of exogenously added isoprenaline. Furthermore, the selective beta 2-adrenoceptor antagonist ICI 118,551 (10(-7) mol/L), but not the selective beta 1-adrenoceptor antagonist ICI 89,406 (10(-7) mol/L), reduced electrically evoked overflow of (3H)noradrenaline in tissue preincubated with adrenaline but not in tissue preincubated with noradrenaline. The overflow-reducing effect of ICI 118.551 occurred at stimulation with 3 Hz but not at stimulation with 1 Hz. The present results support the hypothesis that noradrenergic transmission in guinea pig papillary muscle is facilitated via beta 2-adrenoceptors, and that adrenaline may serve as transmitter in this positive feedback mechanism after its incorporation into sympathetic nerves. PMID- 1709231 TI - Protective effect of cloricromene, a coumarine derivative, in hypovolemic hemorrhagic shock in the rat. AB - Hypovolemic hemorrhagic shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min until mean arterial pressure (MAP) fell to 30 mm Hg. Survival rate, MAP, plasma myocardial depressant factor (MDF) activity and plasma levels of both TxB2 and 6 keto PGF1 alpha were then evaluated. Cloricromene (0.5, 1, and 2 mg/kg) or an equal volume of vehicle (0.9% NaCl solution) were injected intravenously 5 min after the end of the bleeding. Hemorrhagic shocked rats showed enhanced plasma levels of MDF, TxB2 and 6-keto PGF1 alpha. All vehicle-treated rats died within 25 min. Cloricromene (1 and 2 mg/kg) given curatively significantly increased survival rate and blunted the rise in plasma MDF and TxB2. Moreover, cloricromene reversed the severe hypotension and the ST-segment elevation occurring during hemorrhagic shock. The data suggest that cloricromene exerts beneficial effects in experimental hypovolemic shock, probably reversing myocardial failure. PMID- 1709232 TI - Do renal tubular dopamine receptors mediate dopamine-induced diuresis in the anesthetized cat? AB - The aim of this study was to investigate whether the pentobarbitone anesthetized cat is a suitable preparation in which to characterize renal tubular dopamine receptors. Intravenous infusion of dopamine (10-100 micrograms/kg/min) resulted in a dose-related increase in mean blood pressure (MBP), urine output, sodium excretion (UNaV), and fractional sodium excretion (FENa). This diuretic effect occurred despite little change in glomerular filtration rate, suggesting that it is a consequence of decreased tubular reabsorption. Dopamine (10-100 micrograms/kg/min, i.v.) also induced marked dose-related falls in renal vascular conductance; however, renal blood flow was not significantly reduced. The increases in MBP, urine output, UNaV, and FENa induced by dopamine (10-100 micrograms/kg/min, i.v.), were unaffected by pretreatment of cats with either the selective dopamine DA1 or DA2 receptor antagonists, SCH 23390 (30 micrograms/kg, i.v.), or domperidone (100 micrograms/kg, i.v.) respectively. In contrast, pretreatment of cats with the nonselective alpha-adrenoceptor antagonist, phentolamine (1 mg/kg, i.v.) prevented dopamine-induced increases in urine output and MBP. Infusion of the selective dopamine DA1 receptor agonist fenoldopam (0.01 10 micrograms/kg/min) into the left renal artery failed to increase left renal vascular conductance, or left kidney urine output, UNaV, or FENa. In conclusion, this study provides no evidence for the involvement of renal tubular dopamine receptors in dopamine-induced diuresis in anesthetized cats. Rather, the diuretic effect appears to be linked to stimulation of alpha-adrenoceptors. PMID- 1709233 TI - Ro 40-5967, in contrast to diltiazem, does not reduce left ventricular contractility in rats with chronic myocardial infarction. AB - Ro 40-5967 is a new calcium antagonist that binds to the same binding site as verapamil but that has been shown to have a much lesser negative inotropic effect than verapamil. The goal of the present study was to compare the effects of Ro 40 5967 and diltiazem on left ventricular contractility in vitro and in vivo in normal rats and in rats with chronic myocardial infarction induced by ligating the left coronary artery. Left ventricular contractility was assessed in vitro in isolated perfused hearts and in vivo in conscious rats by measuring left ventricular dP/dtmax + and dP/dt at P 40. In vitro, both Ro 40-5967 and diltiazem did not decrease cardiac contractility up to a dose producing complete atrioventricular block. In vivo, diltiazem decreased dP/dtmax + and dP/dt at P 40. Ro 40-5967 was less negative inotropic than diltiazem. We conclude that if these results were confirmed in clinical trials. Ro 40-5967 might be a safer drug than diltiazem, especially in patients with left ventricular dysfunction. PMID- 1709234 TI - Electrophysiology and antiarrhythmic actions of E-4031 in the experimental animal model of sudden coronary death. AB - The Class III agent E-4031 was evaluated for its antiarrhythmic and antifibrillatory actions in conscious dogs 3-5 days after anterior myocardial infarction that were responsive to the induction of tachyarrhythmia by programmed electrical stimulation. The administration of E-4031 as an intravenous loading dose (100 micrograms/kg) followed by an infusion for 90 min (10 micrograms/kg/min) suppressed the induction of ventricular tachycardia by programmed electrical stimulation in 6 of 12 dogs and prolonged the cycle length of the induced arrhythmia in 5 of the 6 remaining animals. Continued administration of E-4031 in a dose regimen of 1,000 microgram/kg every 2 h provided significant protection (8 of 10 dogs) against the development of ventricular fibrillation (sudden coronary death) within the first hour after the onset of myocardial ischemia in a region of the ventricle remote from the infarct related vessel. The incidence of sudden coronary death was 80% in a comparable control group of electrically inducible postinfarcted dogs. Increases in ventricular myocardial refractoriness in the paced QT and QTc intervals suggest that Class III electrophysiologic actions contribute to the antiarrhythmic and antifibrillatory actions of E-4031. The findings suggest that E-4031 may be of clinical utility in the prevention of life-threatening arrhythmias in the setting of myocardial ischemia in the postinfarcted heart. PMID- 1709235 TI - Antihypertensive and renal activity of SQ 28,603, an inhibitor of neutral endopeptidase. AB - SQ 28,603 is a potent and selective inhibitor of neutral endopeptidase 3.4.24.11 (NEP), an enzyme that degrades atrial natriuretic peptide (ANP). In conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rats, 300 mumol/kg, i.v., of SQ 28,603 significantly lowered mean arterial pressure (MAP) from 177 +/- 12 to 154 +/- 8 mm Hg and increased urinary cyclic guanosine monophosphate (GMP) excretion from 204 +/- 70 to 1,068 +/- 326 pmol/kg/min within 2 h. Urinary sodium excretion increased within 20 min from a control 51.2 +/- 17.3 to 102.1 +/- 26.7 muEq/kg/min. Infusion of SQ 28,603 (3.7 mumol/kg/min) for 6 h in a separate group of conscious DOCA/salt hypertensive rats gradually reduced MAP from 180 +/- 7 to 142 +/- 7 mm Hg and increased urinary cyclic GMP excretion from 182 +/- 36 pmol/kg/min to 1,009 +/- 394 pmol/kg/min. Despite the continuous infusion of the inhibitor, the natriuretic response peaked during the first hour of treatment at 128 +/- 18 muEq/kg/min (vehicle = 54 +/- 10 muEq/min). Plasma ANP was significantly greater in the rats infused with SQ 28,603 than in those receiving vehicle (333 +/- 108 and 98 +/- 14 fmol/ml, respectively). SQ 28,603 also significantly reduced NEP activity by 95% in the kidneys (1.28 +/- 0.08 vs. 18.35 +/- 0.61 mumol/min after SQ 28,603 and vehicle respectively) and by 77% in the lungs (0.29 +/- 0.03 vs. 0.92 +/- 0.14 mumol/kg after SQ 28,603 and vehicle, respectively). In conclusion, inhibition of NEP activity by SQ 28,603 significantly decreased MAP and increased plasma ANP concentrations and urinary excretion of cyclic GMP in conscious DOCA/salt hypertensive rats. PMID- 1709236 TI - Effects of cromakalim on renal hemodynamics and function in dogs: comparison with nicardipine. AB - The effects of cromakalin, a potassium channel opener, on renal hemodynamics and renal function were investigated in pentobarbital-anesthetized dogs. Intrarenal infusion of cromakalim at 0.5 micrograms/kg/min resulted in significant increases in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow, and renin release. The urinary excretion rate of sodium increased by the same proportion as that of calcium. Free water reabsorption rate/osmolar clearance did not significantly change throughout the experiments. These data suggest that cromakalim did not inhibit sodium transport at the medullary portion of the ascending limb of Henle's loop and may have increased the delivery of sodium to Henle's loop. Intrarenal infusion of nicardipine increased RBF, GFR, urine flow, and the urinary excretion of electrolytes (Na, K, and Ca). The renal effects of cromakalim were very similar to those of nicardipine. Cromakalim was superimposed on a nicardipine infusion of a maximal effective dose. Superimposition of cromakalim to the nicardipine infusion did not cause any additional changes in renal hemodynamics and renal function. These data suggest that cromakalim and nicardipine exert their effects on renal hemodynamics and function via the same pathway. PMID- 1709237 TI - Electrophysiologic effects and antiarrhythmic efficacy of recainam in patients with supraventricular tachycardia. AB - Recainam is a new antiarrhythmic agent with class Ic properties. To evaluate its electrophysiologic effects and antiarrhythmic efficacy in patients with recurrent supraventricular tachycardia (SVT), programmed electrical stimulation was performed in 10 patients before and after intravenous recainam (loading dose 0.8 mg/kg, infusion 1 mg/kg/h), and in four patients on oral recainam 1,200 mg/day. Five patients had atrioventricular (AV) node reentrant tachycardia; five had AV reciprocating tachycardia. There were no significant changes in electrocardiographic and intracardiac intervals after either intravenous or oral recainam. After intravenous recainam, the ventricular effective refractory period (ERP) shortened (231 +/- 14-219 +/- 9 ms, p less than 0.05). The antegrade ERP of all three bidirectional accessory pathway markedly prolonged, but the effect on retrograde accessory pathway and AV node ERPs was unremarkable. SVT induction was prevented in three of 10 patients and SVT cycle length increased modestly in seven (357 +/- 44-374 +/- 42 ms, p = 0.07). On oral recainam, an increase in the frequency of spontaneous SVT occurred in two patients. At the doses given, recainam caused less electrophysiologic change than expected, had modest antiarrhythmic efficacy, and might have significant arrhythmogenic potential. PMID- 1709238 TI - Effects of 5-hydroxytryptamine on capillary and arteriovenous anastomotic blood flow in the human hand and forearm and in the pig hind leg. AB - The effects of intraarterially infused serotonin (5-HT) on capillary and arteriovenous anastomotic (AVA) blood flow were investigated in the hand and forearm of 19 healthy volunteers, and in the hind leg of six anesthetized pigs using radioactive microspheres with a diameter of 15 microns. The 5-HT2-receptor antagonist ketan-serin was used in an attempt to identify the receptors involved. None of the drugs in the doses used induced systemic hemodynamic effects. Low doses of 5-HT significantly increased forearm blood flow with a maximum response at the dose of 1 ng/kg/min (68 +/- 14%, p less than 0.05), whereas only at the highest dose of 80 ng/kg/min was a net decrease in forearm blood flow measured ( 28 +/- 6%, p less than 0.05). Conversely, finger blood flow was not influenced by the lower doses of 5-HT, whereas a major reduction was observed at the highest dose (-90 +/- 3%). Ketanserin increased both total forearm blood flow and AVA blood flow. The drug blunted the constrictor response to 5-HT in the forearm but only slightly attenuated this response in the finger. The percentage AVA blood flow in the human hand and forearm was not influenced by an infusion of 5-HT at 80 ng/kg/min alone. However, after pretreatment with ketanserin, which itself increased the AVA component, this dose of 5-HT significantly reduced AVA flow. In the pig, total femoral blood flow was not influenced by 5-HT, but AVA blood flow was significantly reduced and capillary skin blood flow increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709239 TI - Effects of adrenaline and mental stress on serum digoxin concentration. AB - Physical activity and pharmacological stimulation of beta 2-adrenoceptors by salbutamol increase skeletal muscle digoxin binding with a secondary decrease in serum digoxin, possibly due to increased Na-K-ATPase activity. The present study was undertaken to examine if adrenaline (ADR) infusion and sympathoadrenal stimulation by mental stress affect the serum concentrations of digoxin and potassium. After 10 days on 0.50 mg digoxin orally, 35 healthy volunteers were investigated following 2 h of supine rest. They were divided into four groups: intravenous saline (placebo, n = 10). ADR infusion at the rates of 0.1 nmol kg-1 min-1 (ADR-L, n = 8), 0.4 nmol kg-1 min-1 (ADR-H, n = 7), or subjected to a mental stress [a color-word conflict test (CWT), n = 10]. Arterial blood samples were taken before and during the active period (50 min) and during the following 60 min (at rest) to analyze serum digoxin and potassium and plasma ADR and noradrenaline (NA). All variables were stable during placebo infusion. ADR infusions caused significant and dose-dependent decreases in serum digoxin (p less than 0.05 during ADR-L and p less than 0.001 during ADR-H) and serum potassium (p less than 0.05 and p less than 0.001, respectively). CWT, on the other hand, did not reduce serum digoxin and caused a slight decrease in serum potassium only in the poststress period. Thus, ADR caused dose-dependent shifts of digoxin and potassium, whereas mental stress failed to do so, possibly due to a modest ADR response and small increases in sympathetic nerve activity in skeletal muscle. PMID- 1709240 TI - The combination of chlorthalidone with nifedipine does not exert an additive antihypertensive effect in essential hypertensives: a crossover multicenter study. AB - To evaluate whether the combination of nifedipine with chlorthalidone exerts an additive antihypertensive effect when compared to single-drug treatment, 66 uncomplicated essential hypertensives, whose diastolic blood pressure was greater than 100 and less than 115 mm Hg at the end of a 1-month washout placebo period, received, according to a randomized, double-blind, crossover design, nifedipine (20 mg b.i.d.), chlorthalidone (25 mg o.d.), the two drugs combined at the same doses, and the corresponding placebo. When compared to the randomized placebo the three active treatments significantly (p less than 0.001) reduced blood pressure without changing heart rate and body weight. However, blood pressure values were similarly reduced under nifedipine and the combination and were significantly lower (p less than 0.05) than those under chlorthalidone. Moreover, the percentage of responders and normalized patients under nifedipine and the two drugs combined were similar and significantly (normalized, p less than 0.0001; responders, p less than 0.02) greater than those under chlorthalidone. Under chlorthalidone and its combination with nifedipine, plasma potassium tended to decrease and blood glucose and serum uric acid were significantly (p less than 0.05) increased. These data show that the combination of nifedipine with chlorthalidone does not exert any additive antihypertensive effect when compared to nifedipine alone and that this combination increases both blood glucose and serum uric acid. Taken together these findings indicate that the combination of a dihydropyridine calcium antagonist with a thiazide diuretic is devoid of any clinical significance in the treatment of uncomplicated essential hypertensives. PMID- 1709241 TI - Effects of a new antiarrhythmic drug TYB-3823 on canine ventricular arrhythmia models. AB - The antiarrhythmic and direct cardiovascular effects of a new antiarrhythmic agent, TYB-3823 [1-(2,6-dimethylphenyl)-4,4-dimethyl-aminoguanidine hydrochloride], were investigated. To determine the antiarrhythmic effects, spontaneously occurring adrenaline-, digitalis-, and two-stage coronary ligation induced arrhythmias were used. TYB-3823 suppressed these three arrhythmia models. The antiarrhythmic plasma concentrations, IC50, of TYB-3823 for digitalis-, coronary ligation-, and adrenaline-induced arrhythmias were 7.8, 16.8, and 5.0 micrograms/ml, respectively. In the blood perfused sinoatrial (SA) node and papillary muscle (PM) preparations, TYB-3823 decreased the sinoatrial rate (SAR) and contractile force following an initial small positive inotropic effect and increased the intraventricular conduction time and coronary blood flow. These results indicate that TYB-3823 is similar to other class I antiarrhythmic agents, such as aprindine and nicainoprol, in the antiarrhythmic profile and is expected to become a clinically useful antiarrhythmic drug. PMID- 1709242 TI - Rapid serum carotene loading with high-dose beta-carotene: clinical implications. AB - The kinetics of serum beta-carotene loading were evaluated to help determine the minimum time required to load fatty tissues, including atherosclerotic plaque and skin, with beta-carotene. Loading atherosclerotic plaque with yellow beta carotene pigment increases absorption of blue laser radiation, potentially enhancing the selectivity and quality of laser angioplasty. Five healthy volunteers received 300 mg beta-carotene daily (the maximum FDA recommended dose) for 21 days in three divided doses with meals. Serum total carotenoid levels increased exponentially with a 10-day time constant from an average of 1.7-6.5 micrograms/ml (p less than 10(-4]. The skin began to yellow visibly at 10 days and became increasingly yellow for several weeks thereafter, suggesting that augmenting the yellow color of fatty tissues and atherosclerotic plaque with oral beta-carotene may require a minimum of several weeks to attain the maximum effect. PMID- 1709243 TI - Inhibition of L-type but not T-type calcium channel current by a new dihydropyridine derivative, S11568. AB - S11568, (+-)[((amino-2-ethoxy)-2-ethoxy]-methyl)-2-(dichloro-2', 3'-phenyl)-4 ethoxycarbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyr idine HCl, is a new dihydropyridine derivative that is water soluble and relatively insensitive to light. The Ca2+ channel inhibitory activity of S11568 was tested in whole-cell patch clamp recordings from cultured embryonic chick cardiomyocytes in 40 mM Ba2(+)-containing medium that revealed T-type and L-type components of inward current through calcium channels. S11568 inhibited L-type Ca2+ current with an IC50 value near 1 microM but was without effect on the T-type barium current. PMID- 1709244 TI - Regulation of leukocyte adhesion molecule-1 (TQ1, Leu-8) expression and shedding by normal and malignant cells. AB - The human leukocyte adhesion molecule-1 (LAM-1, TQ1, Leu-8) is involved in the binding of human leukocytes to high endothelial venules (HEV) of peripheral lymph nodes (LN). The regulation of LAM-1 expression is unique in that leukocyte stimulation induces a rapid down-modulation of LAM-1 from the cell surface. In this study, the regulation and function of LAM-1 was studied in detail in normal lymphocytes and compared with the LAM-1 of malignant leukocytes. Modulation of LAM-1 from the cell surface occurred concomitantly with the appearance of LAM-1 in the culture medium indicating that LAM-1 is cleaved from the cell surface. Shedding of LAM-1 was decreased in the presence of protein kinase C (PKC) inhibitors. As with normal lymphocytes, cells transfected with the LAM-1 cDNA and chronic lymphocytic leukemia (CLL) cells also shed LAM-1 following phorbol myristate acetate (PMA) exposure. CLL cells expressed the same Mr LAM-1 protein as normal lymphocytes and LAM-1+ CLL cells were able to specifically bind to HEV. In addition, normal lymphocytes and LAM-1+ CLL cells were capable of binding polyphosphomonester core polysaccharide (PPME) derived from yeast cell wall, a carbohydrate which mimics an essential component of the natural ligand for LAM-1, and PPME and HEV binding was specifically blocked by a new monoclonal antibody (mAb) reactive with LAM-1. The expression of LAM-1 and other adhesion molecules was examined on cells of 118 hematopoietic malignancies. LAM-1 was most frequently expressed on CLL and follicular or diffuse small cleaved cell lymphomas, whereas most other malignancies were LAM-1-. Thus, most CLL cells and some non-Hodgkin's lymphoma cells express a functionally active LAM-1 molecule which may correlate with their capacity to migrate through the circulation and disseminate into peripheral LN. PMID- 1709245 TI - Impaired response of myelodysplastic marrow progenitors to stimulation with recombinant haemopoietic growth factors. AB - The regulation of haemopoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring and comparing the in vitro response of marrow progenitors from 18 MDS patients to stimulation with recombinant haemopoietic growth factors (HGFs), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G CSF) and interleukin-3 (IL-3). A similar pattern of colony growth was detected with all three HGFs in most MDS patients, exhibiting subnormal growth of GM-CFU and markedly poor to absent growth of BFU-E and CFU-GEMM. A common severe impairment in the growth of all colony types with all three HGFs was observed in five patients, four of whom presented with pancytopenia. The stimulation of MDS marrow progenitors with a five-fold higher than control saturating dose of HGFs induced a significant increase in the frequency of one, two, or all three colony types in cultures of 14 patients, whereas colony numbers in control (n = 8) marrow cell cultures were not significantly changed. All four of the non responders were pancytopenic and three exhibited markedly impaired colony growth. Supersaturating GM-CSF, G-CSF and IL-3 increased GM-CFU numbers in six, three, and three patients, respectively. The values for BFU-E were three, six, and seven and for CFU-GEMM two, one, and five. The enhancement of MDS marrow colony numbers by supersaturating HGFs which exert their effects directly or via the action of marrow accessory cells, suggests that the progenitor cell growth abnormalities in these disorders may involve a defect in the capacity of accessory and/or progenitor cells to respond to stimulation with specific haemopoietic growth regulators. PMID- 1709246 TI - Granulocyte colony-stimulating factor corrects the neutropenia associated with glycogen storage disease type Ib. AB - A young woman with glycogen storage disease, type Ib, and chronic neutropenia had severe recurrent infections. In a life-threatening situation, treatment with granulocyte colony-stimulating factor (G-CSF) resulted in the prompt correction of neutropenia. Subsequently, daily G-CSF therapy has allowed the maintenance of a normal neutrophil count and marked clinical improvement over a period of 18 months. The spectrum of neutropenic conditions which are responsive to G-CSF should include this inherited metabolic disorder. PMID- 1709247 TI - Hyaline globules reacting positively with zidovudine antibody in brain and spinal cord of AIDS patients. AB - Histology of the central nervous system in nine AIDS showed extracellular hyaline globules in the white matter of the brain and the spinal cord. In immunohistochemical studies with a battery of antibodies, the only positive reaction of these globules was with an antibody to zidovudine. High-performance liquid chromatography showed the presence of a zidovudine isomer in eluates of brain tissue from these patients. PMID- 1709248 TI - Low-dose aprotinin for reduction of blood loss after cardiopulmonary bypass. PMID- 1709249 TI - rhG-CSF and aztreonam as prophylaxis against infection in neutropenic children. PMID- 1709250 TI - Drug regimen for tuberculosis without thiacetazone and streptomycin. PMID- 1709251 TI - [Hyperamylasemia, hyperlipasemia and acute pancreatitis in chronic inflammatory bowel diseases]. AB - 145 clinical observations of 114 patients with Crohn's disease and 65 observations of 47 patients with ulcerative colitis were analyzed prospectively concerning the prevalence of pathologically elevated levels of serumamylase or lipase and acute pancreatitis. Painless hyperamylasemia or hyperlipasemia were found in 18 of 114 patients with Crohn's disease (15.8%) and in 10 of 47 patients with ulcerative colitis (21.3%) without morphological abnormalities on ultrasound. Range of elevated serumamylase levels differs from 35 to 68 U/L (Ref. value less than 34 U/L), range of serumlipase levels varies from 199 to 858 U/L (Ref.-value less than 190 U/L). Pathologically elevated levels of serumamylase and -lipase persisted for 17.7 +/- 9.0 (5-28) days in Crohn's disease and 22.8 +/ 9.8 (7-33) days in ulcerative colitis. No relation to the activity or the duration of the disease, drug treatment or the weight loss of the patients could be shown. Acute pancreatitis was found in 4 of 114 patients (3.5%) with Crohn's disease, whereas in ulcerative colitis acute pancreatitis was diagnosed in 1 of 47 patients (2.1%). Regarding the promoting factors, drugs (azathioprine and salazosulfapyridine) and mechanical alterations of the bile duct (primary sclerosing cholangitis) or the pancreas (pancreas divisum) were found in 4 of the 5 patients. However the case of a 23 year old woman suffering from Crohn's ileocolitis who died of an idiopathic pancreatitis remains obscure. PMID- 1709252 TI - Structure of cDNAs encoding the triple-helical domain of murine alpha 2 (VI) collagen chain and comparison to human and chick homologues. Use of polymerase chain reaction and partially degenerate oligonucleotide for generation of novel cDNA clones. AB - Type VI collagen cDNAs of human and avian origin were recently obtained and characterized by screening cDNA libraries in lambda phage. Based on the published sequences of these cDNAs, we constructed partially degenerate oligonucleotide primers that we used in polymerase chain reactions for the generation of alpha 2(VI) collagen clones of murine origin. As template, we used cDNA derived from murine total RNA. We amplified, cloned and sequenced a 1043-bp fragment that contains the coding sequence for the entire triple-helical domain except for the first two amino acids that are Gly-Pro in both man and chicken. Comparison of the nucleotide and derived amino acid sequences revealed 84.6% and 92.5% identity between mouse and man at the DNA and protein levels, respectively. Comparison with chicken sequences showed 72% and 79.1% identity. The third base usage showed a distinct preference for A in the glycine codons for the three species; whereas, U is preferred in all human fibrillar collagen genes previously defined. The preference for third base codon in Y position prolines is U for the alpha 2(VI) collagen as it is for the human fibrillar collagen genes. A single cysteine at position 89, two Arg-Gly-Asp sequences, and one triple helix-interruption are conserved in mouse, man and chicken. Comparison of hydropathy plots showed great similarity between those of murine and human alpha 2(VI) collagen chains and to a lesser extent between murine and chick. Northern blot hybridization of murine poly A+ RNA with a nick-translated radiolabeled alpha 2(VI) collagen probe detected one major transcript of 3.7 kb.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709253 TI - Proliferating oral epithelial cells in culture are capable of both extracellular and intracellular degradation of interstitial collagen. AB - The potential of epithelial cells to degrade interstitial collagen was studied by culturing human masticatory mucosa on decalcified dentin matrix. Morphological changes were observed in the underlying collagen substratum and in the connective tissue of the explant. Degradation of the substratum was initiated two days after the first contact with epithelial cells exhibiting basal cell markers. Electron microscopic studies confirmed extensive collagen degradation in the vicinity of these cells. No collagen degradation was observed underneath the connective tissue portion of the explant. Experiments in which the explant was partially separated from the underlying substratum by a filter further showed that connective tissue was apparently not involved in the collagen degradation by the epithelial cells. Lysis of connective tissue of the explant was observed in association with epithelial cells that showed a disrupted basal lamina and release of vesicular material from the exposed cell membrane. Collagen fibers were visible inside some epithelial cells suggesting intracellular collagenolysis. Primary cultures of human gingival epithelial cells and porcine periodontal ligament epithelial cells (epithelial cell rests of Malassez) that expressed similar basal cell cytokeratins as the active cells of the mucosal explants secreted collagenase, gelatinase and TIMP to the culture medium. They also contained acid collagenolytic proteinases. When cultured on a porous polycarbonate membrane the epithelial cells secreted collagenolytic enzymes from the pores at cell membrane sites lacking basal lamina. These results provide evidence that proliferating basal epithelial cells have a strong capacity for collagen degradation. It seems that the absence of basement membrane is the signal for these cells to secrete matrix degrading enzymes. PMID- 1709254 TI - [Clinical exploration of nociception with the use of reflexologic techniques]. AB - In the first part of this report a methodology is described which allows an objective and specific exploration of experimental pain in man by using some electrophysiological features of cutaneous reflexes. This method can be summarized as follows: in normal and trained volunteers, we studied simultaneously the recruitment curves of the nociceptive flexion reflex of a knee flexor muscle (biceps femoris muscle) and that of pain sensation elicited by electrical stimulation of the ipsilateral sural nerve at the ankle. In this procedure, we found that the reflex threshold (Tr) was closely related to that of pain threshold (Tp) around a similar value (10 mA). In the same way, both the threshold of the maximal recruitment of the reflex (Tmr) and that of tolerance to pain (Tpt) were found to be close to 33 mA. These values are reliably reproducible in one subject from one session to the other, and in all subjects with minimal inter-individual variations and without any significant inter-sex difference. These close relationships between Tr and Tp and between Tmr and Tpt respectively constituted the basic ground for the elaboration of the methodology for investigating objectively the human nociceptive reactions. In the second part of the paper, this methodology is applied for studying the spinal mechanisms of morphine analgesia when the drug is given either by intravenous route in normal subjects and in paraplegic patients or administered epidurally in patients with acute postoperative pain. On the one hand, the resulting data strongly validate the model since they show that pain and nociceptive reflex are similarly depressed by morphine in a dose-response fashion. On the other hand, data also show that the spinal level is one of the main important sites of the mechanisms of morphine-induced analgesia since this drug is found to strongly depress selectively the nociceptive transmission directly at the spinal level. Finally, this method is applied for investigating the nociceptive reactions in patients affected either with a pathological lack of pain sensation or, by contrast, in patients complaining of acute or chronic pain from various origins. Since the nociceptive flexion reflex can be considered as a specific and objective physiological correlate of a pain sensation, it can be successfully employed as a useful tool for investigating some aspects of the human nociceptive reactions in both experimental and pathological situations. PMID- 1709255 TI - Are frequent spike-waves during non-REM sleep in relation with an acquired neuro psychological deficit in epileptic children? AB - In a population of 11 children with frequent spike waves during non REM sleep who had no neurological symptoms between birth and their first symptom, 3 groups were compared according to their neuropsychological performances. In the first group, the children had no intellectual deficit, in the second group, they had an acquired aphasia as in the Landau-Kleffner syndrome and in the third they had severe behavioural disorder and mental deterioration. The non REM sleep paroxysmic activity density tended to be highest in the third group, variable in the second group and moderate in the first group, and their topography was always generalized in the acute phase in groups II and III but asymmetrical in group I. The EEG anomalies disappeared during adolescence but in group II and III children a moderate to severe delay in school work persisted. PMID- 1709256 TI - Muscle regeneration following segmental necrosis in tenotomized muscle fibers. AB - The aim of this study was to determine how the new myotendinous junctions were re established at the proximal and distal ends of the soleus muscle after tenotomy. Both proximal and distal tendons of the soleus muscle in mature female rats were severed. The animals were killed and the soleus muscles were removed and prepared for light and electron microscopic examination 1, 3, 5, 7, 14, 21, 28, and 42 days after the operation. It was found that segmental fiber destruction followed by removal by macrophages occurred at the ends of the soleus muscle fibers. This resulted in the liberation and myogenic activation of satellite cells. By 3 days after tenotomy the fusion of myoblasts to form myotubes could be seen. The myotubes developed within the original basal lamina and reattached to the surviving non-necrotic segments and grew in both length and width so that by 6 weeks postoperation, normal myotendinous junctions had been reformed. This study is the first to show that re-establishment of the myotendinous junction following tenotomy is accomplished by regeneration of the necrotic ends of the tenotomized fibers. PMID- 1709257 TI - Clinical trials--are they ethical? PMID- 1709258 TI - Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. AB - The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase. PMID- 1709259 TI - [Ambulatory continuous intravenous infusion of fluorouracil: a feasible palliative form of chemotherapy]. AB - A study to evaluate the feasibility and toxicity of outpatient continuous intravenous infusion of fluorouracil (5-FU) was initiated at the department of Medical Oncology of the University Hospital of Utrecht. To this purpose a subcutaneous drug delivery system (Port-a-Cath) was implanted in 36 patients with various advanced cancers. Of these patients 83% had received prior chemotherapy (including 5-FU in 62%). Ambulatory continuous-infusion pumps were used to administer 5-FU in a dosage of 300 mg/m2/24 h. The treatment was continued until tumour progression was seen, and it was interrupted in case of toxicity grade 2 or more (WHO criteria). A Port-a-Cath was implanted 37 times in the 36 patients. The main complications of this infusion system were pneumothorax (2/37), arrhythmia (1/37), catheter sepsis (2/37) and thrombosis (2/37); they were easily managed. The toxicity and feasibility of this treatment were evaluable in 30 patients. They received a median of 44 g 5-FU (range 11-136, 5 g, mean 281 mg/m2/24 h) during a median infusion time of 12 weeks (range 4-32 w). Side effects were encountered in 70% of the patients and consisted of the hand-foot syndrome (14/30), nausea and vomiting (8/30), diarrhoea (8/30) and stomatitis (7/30). The toxicity was completely reversible after a short interruption of the chemotherapy. The treatment was tolerated well, and good palliation was attained in 22 of 30 patients. The best response was seen in patients with colon and breast cancer. We conclude that continuous infusion of 5-FU is a reliable outpatient chemotherapy even in this category of patients. PMID- 1709260 TI - The origin of the vestibulo-cochlear projection in the guinea pig. AB - Previous tracer studies have revealed the sacculus to be connected to the vestibular and cochlear nuclei in the guinea pig. Due to its own innervation pattern, an anterior and posterior part of the sacculus can be distinguished. The present study investigated whether the two parts differ concerning their fiber contribution to the vestibulo-cochlear projection. After tracing the nerve of the posterior sacculus with horseradish peroxidase (HRP), the vestibulo-cochlear fibers were clearly recognizeable and showed distinct terminal labeling within the cochlear nuclei. In contrast, no terminals were identified in the cochlear nuclei after tracing the anterior sacculus. These results may further substantiate the guinea pig vestibulo-cochlear projection as a central pathway for saccular acoustic sensation. PMID- 1709261 TI - Absence of recurrent axon collaterals in motoneurones to the extrinsic digit extensor muscles of the cat forelimb. AB - Forelimb alpha-motoneurones were intracellularly recorded in anaesthetized cats and iontophoretically filled with horseradish peroxidase (HRP). All motoneurones to the elbow flexors, elbow extensor and to the extensor carpi radialis muscles displayed in parallel homonymous recurrent inhibitory postsynaptic potentials (RIPSPs) and axon collaterals. Homonymous RIPSPs and axon collaterals were missing in the nuclei to the long digit extensor muscles. Two populations of motoneurones, with and without recurrent axon collaterals, seem to be present in the extensor carpi ulnaris motor nucleus. These results are consistent with the hypothesis that the motoneurones to the extrinsic digit extensors lack a recurrent axonal system. This indicates that the contribution of the recurrent Renshaw systems to motor control may be more complex than hitherto assumed. PMID- 1709262 TI - Changes of ionic channel distribution in myelinated nerve fibres from rats with experimental allergic neuritis. AB - Voltage clamp experiments were performed in single myelinated nerve fibres of rats with experimental allergic neuritis (EAN). Nerve fibres from diseased animals showed a varying degree of demyelination. In fibres with extensive demyelination large K+ currents were measured. Analysis of the contribution of slow and fast K+ channels revealed that in these fibres the relative contribution of slow K+ channels was about 50% compared to about 25% in fibres acutely demyelinated as previously reported. This indicates that chronic demyelination changes the distinct distribution of ionic channels in the rat node of Ranvier. PMID- 1709263 TI - GABA inhibits the rise in cytosolic free calcium concentration in depolarized immature cerebellar Purkinje cells. AB - gamma-Aminobutyric acid (GABA) reduced the peak rise or slowed the rate of rise in cytosolic free calcium concentration ([Ca]in) induced by quisqualate, kainate, or high KCl in immature cerebellar Purkinje cell bodies. The sustained rise or repeated transient of [Ca]in induced by tetraethylammonium, veratridine, or Bay-K 8644 was lowered to the basal level by adding GABA, although the inhibition by GABA of Bay-K-8644-induced rise in [Ca]in was only slight and transient in some cells. These findings indicate that GABA inhibits the rise in [Ca]in by hyperpolarizing the membrane potentials at Purkinje cell bodies. PMID- 1709264 TI - Efferent projections from the external parabrachial area to the forebrain: a Phaseolus vulgaris leucoagglutinin study in the rat. AB - Small iontophoretic applications of Phaseolus vulgaris leucoagglutinin (PHA-L) were used to study the ascending efferent projections in the rat from the external parabrachial (PBe) area (i.e. external lateral (PBel) and external medial (PBem) subnuclei). It was found that fibers of the caudal two third of PBe project mainly to nucleus centralis of the amygdala (Ce) with a precise pattern: the PBel subnuclei mainly project to the caudomedial subdivision of the Ce and the PBem subnuclei mainly project to the rostrolateral subdivision of the Ce. Another dense and common projection of both subnuclei was found in the ventral pallidal area adjacent to the Ce. This study delineates and extends the terminal area of the spino(trigemino)-ponto-amygdaloid nociceptive pathway demonstrated by our previous studies. PMID- 1709265 TI - Alzheimer's disease: the relationship between the density of senile plaques, neurofibrillary tangles and A4 protein in human patients. AB - The numerical density of senile plaques (SP) and neurofibrillary tangles (NFT) as revealed by the Glees silver method was compared with SP and NFT revealed by the Gallyas method and with amyloid (A4) deposits in immunostained sections in 6 elderly cases of Alzheimer's disease. The density of NFT was generally greater and A4 lower in tissue from hippocampus compared with the neocortex suggesting that A4 deposition was less important than the degree of paired helical filament (PHF) related damage in the hippocampus. The density of Glees SP was positively correlated Gallyas SP weakly correlated with A4 deposit number. A stepwise multiple regression analysis which included A4 deposit and Gallyas SP density and accounted for 54% of the variation in Glees SP density. Hence, different populations of SP were revealed by the different staining methods. The results suggested that the Glees method may stain a population of SP in a region of cortex where both amyloid deposition and neurofibrillary changes have occurred. PMID- 1709266 TI - Cholinergic neuronal loss in the globus pallidus of Alzheimer disease patients. AB - Cholinergic neurons of the ventral pallidum and the dorsal pallidum (globus pallidus) were immunohistochemically investigated in patients suffering from Alzheimer disease (AD). Measurement of cholinergic neurons, stained with an antiserum against choline acetyltransferase (ChAT), revealed that their number was significantly reduced in both the dorsal pallidum (37.5%) and the ventral pallidum (65%) of AD patients (n = 4) when compared to control subjects (n = 3). No shrinkage of these cells was observed. The number of immunostained neuropeptide Y-containing neurons in the same structures was not different in controls and AD patients, indicating that the loss of cholinergic neurons was selective. These results combined with previous reports give further information upon which specific subsets of cholinergic neurons degenerate in AD. PMID- 1709267 TI - Interaction of Mg2+ and phencyclidine in use-dependent block of NMDA channels. AB - The interaction between Mg2+ and phencyclidine (PCP) in blocking open N-methyl-D aspartate (NMDA) channels was investigated in Xenopus oocytes injected with rat brain mRNA. These receptors exhibit the pharmacological and physiological properties of the neuronal receptors, and the oocyte is readily amenable to electrical recording and application of well-controlled chemical stimuli. We found that Mg2+ at physiological concentrations greatly impeded the ability of PCP to block the NMDA channel. The interaction between Mg2+ and PCP was competitive; 0.5 mM Mg2+ caused a four-fold decrease in the potency of PCP in blocking open NMDA channels. Moreover, Mg2+ speeded the recovery from PCP block in the presence of agonist, suggesting that Mg2+ reduced reblock of NMDA channels by PCP that had escaped from open channels. Our observations suggest that the presence of Mg2+ in the channel tends to prevent PCP entry and block. Since depolarization is likely to reduce channel occupancy by Mg2+ more than that by PCP, neural activity may have an important influence on the actions of PCP and related drugs. PMID- 1709268 TI - Voltage-dependent inhibition and facilitation of Ca channel activation by GTP gamma-S and Ca-agonists in adult rat sensory neurons. AB - Intracellular application of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S, 100 microM) causes a slow down of high-threshold Ca channel activation in adult rat sensory neurons that is relieved by strong depolarizations or by the Ca agonist Bay K 8644 (5 microM). Recovery from GTP-gamma-S inhibition is usually accompanied by an increase in Ca current amplitude (facilitation) and is insensitive to holding potential (-60 to -90 mV). Inhibition and facilitation of Ca currents are also little affected by nifedipine (5 microM) or by cell incubation with omega-conotoxin (omega-CgTx, 3.2 microM). We conclude that both slowdown of Ca channel activation by GTP-gamma-S and facilitation by strong depolarizations or by Ca agonists derive from a common process in which G-protein activation, membrane voltage and Ca agonist receptors interact to modulate neuronal Ca channel gatings. PMID- 1709269 TI - Is substance P released from slices of the rat spinal cord inactivated by peptidase(s) distinct from both 'enkephalinase' and 'angiotensin-converting enzyme'? AB - Studies on the effects of peptidase inhibitors on substance P-like immunoreactive material (SPLI) released by K(+)-induced depolarization from slices of the rat spinal cord showed that bacitracin was the most potent agent to protect SPLI from degradation. Captopril and thiorphan which inhibit, respectively, angiotensin I converting enzyme and endopeptidase-24.11 also protected SPLI from degradation. However other inhibitors of these two enzymes, kelatorphan for endopeptidase 24.11 and enalaprilat for angiotensin I converting enzyme were essentially inactive, indicating that both enzymes are probably not involved in the degradation of endogenous substance P. Instead, the non-additive protecting effect of bacitracin, captopril and thiorphan might be due to the blockade of some 'bacitracin-sensitive enzyme' playing a key role in the catabolism of SP within the rat spinal cord. PMID- 1709270 TI - Quantitative histochemistry of cytochrome oxidase in rat brain. AB - A quantitative analysis of cytochrome oxidase (CO) activity in histochemically stained sections of rat brain was developed using tissue standards and computerized image processing. Standards of brain paste containing known amounts of CO were cryosectioned and stained under the same conditions as brain sections. The gray levels of the stain were converted to units of CO activity using a calibration curve derived from densitometric analysis of the standards. The technique yields reproducible quantitative values, has the superior anatomical resolution of histochemistry, and is compatible with autoradiography. PMID- 1709271 TI - Aspartate-like immunoreactivity in vestibulospinal neurons in gerbils. AB - In an effort to further characterize vestibulospinal pathways in the gerbil, immunocytochemistry was combined with retrograde identification of neurons. Vestibulospinal neurons were retrogradely labeled following injections of horseradish peroxidase into the cervical cord of anesthetized gerbils. Sections were reacted with nickel acetate-diaminobenzidine for horseradish peroxidase, giving a black reaction product. Sections were incubated in polyclonal antisera to aspartate, incubated in an avidin-biotin-peroxidase procedure, and reacted to give a brown reaction product. Alternatively, fluoro-gold was used as a retrograde tracer and aspartate-like immunoreactivity was demonstrated with avidin conjugated to Texas red. Cells stained with aspartate-like immunoreactivity, were located in all vestibular nuclei. Double-labeled cells were located in the medial nucleus and in the lateral vestibular nucleus where many of the large cells were double labeled. PMID- 1709272 TI - Target-specific projections of intrinsic ganglionic neurons with different chemical codes in the canine larynx. AB - The distribution and origin of peptide-containing intrinsic nerve fibers within the larynx were examined by immunohistochemistry and denervation experiments in the dog. In the normal larynx, a dense network of vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) fibers was seen around the acini of submucosal glands. VIP-, substance P (SP)-, or calcitonin gene-related peptide (CGRP)-IR fibers were seen in the walls of submucosal arteries, and VIP-, neuropeptide Y (NPY)-, or enkephalin (ENK)-IR fibers were seen around the arteries in the muscle tissue. Most of these peptide-IR fibers remained after bilateral denervation of the superior and inferior laryngeal nerves. Several small intrinsic ganglia were found along the peripheral branches of the laryngeal nerves. About 97% of the ganglionic neurons were VIP-IR; of these, 44% were immunoreactive to VIP alone, 22% to VIP and NPY, 13% to VIP and SP, 7% to VIP and ENK, and 14% to VIP, NPY and SP. These results reveal that the exocrine glands and blood vessels are innervated by the intrinsic ganglionic neurons and that subpopulations of ganglionic neurons with different chemical codes innervate specific target organs in the canine larynx. PMID- 1709273 TI - Hyskon complications in hysteroscopic surgery. AB - Hyskon (32 per cent dextran 70 in 10 per cent dextrose in water), a useful distension medium for hysteroscopic surgery, has significant side effects. A case of pulmonary edema following transcervical myoma resection is presented. Additional known side effects of Hyskon discussed include coagulation defects, spurious laboratory results, and anaphylaxis. Prevention and management of complications are described. PMID- 1709274 TI - Antigenic variation in Trypanosoma brucei: a telomeric expression site for variant-specific surface glycoprotein genes with novel features. AB - African trypanosomes evade the immune response of their host by periodically changing their variant surface glycoprotein (VSG) coat. Each coat is encoded by a separate VSG gene. Expressed genes are in a telomeric expression site (ES) and there are several sites in each trypanosome. To study the transcription control of VSG genes in Trypanosoma brucei we have analyzed an ES, called the dominant ES (DES), that readily switches off and on. The promoter area of the DES is very similar to that of the 221 ES (Zomerdijk et al., 1990). It can be switched off and on in vivo without detectable DNA alterations in the vicinity of the transcription start and it can drive high transient expression of a reporter gene in transfection experiments. However, there are also two major differences between the DES and the 221 ES. First, one version of the DES contains an additional upstream transcription unit overlapping the VSG gene ES promoter. The presence of this upstram transcription is dispensable, however, for the VSG gene ES promoter is active, even if transcription through this start from the upstream promoter is blocked using UV light. Moreover, a second version of the DES present in another trypanosome variant does not produce these upstream transcripts. Secondly, we find that the inactivation of DES transcription in one trypanosome variant is accompanied by DNA alterations in the DES upstream (greater than 2 kb) of the transcription start; reactivation of DES transcription is accompanied by another alteration far upstream. Although we cannot exclude that these DNA rearrangements are incidental, our results raise the possibility that the activity of ES promoters is negatively controlled in cis by far upstream sequences not included in transfection constructs and that alterations in these sequences may lead to (in)activation of the promoter. PMID- 1709275 TI - Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases. AB - We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases. PMID- 1709276 TI - Nucleotide sequence and expression of a maize H1 histone cDNA. AB - The first complete amino acid sequence of a H1 histone of a monocotyledonous plant was deduced from a cDNA isolated from a maize library. The encoded H1 protein is 245 amino acid-long and shows the classical tripartite organization of this class of histones. The central globular region of 76 residues shows 60% sequence homology with H1 proteins from dicots but only 20% with the animal H1 proteins. However, several of the amino acids considered as being important in the structure of the nucleosome are conserved between this protein and its animal counterparts. The N-terminal region contains an equal number of acidic and basic residues which appears as a general feature of plant H1 proteins. The 124 residue long and highly basic C-terminal region contains a 7-fold repeated element KA/PKXA/PAKA/PK. Southern-blot hybridization showed that the H1 protein is encoded by a small multigene family. Highly homologous H1 gene families were also detected in the genomes of several more or less closely related plant species. The general expression pattern of these genes was not significantly different from that of these genes encoding the core-histones neither during germination nor in the different tissues of adult maize. PMID- 1709277 TI - Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants. AB - The transgenic tobacco plants have been generated that express the E. coli beta glucuronidase (GUS) gene under control of the promoter from the rice proliferating cell nuclear antigen (PCNA, DNA polymerase auxiliary protein) gene. GUS expression detected in situ by staining with the chromogenic substrate, 5 bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), was restricted to meristems in the organs of the transgenic tobacco plants. This expression responded to the phytohormones which promote callus formation. Furthermore, in situ thymidine uptake showed that the GUS expression pattern corresponded well to the active sites of DNA synthesis. Deletion analysis of the 5' upstream sequence confined the GUS expression pattern to a fragment extending 263 bp upstream of the transcription start site of the rice PCNA gene. Thus, we have identified this fragment as a main regulatory element of the rice PCNA gene promoter. PMID- 1709278 TI - Ribonuclease T1 generates circular RNA molecules from viroid-specific RNA transcripts by cleavage and intramolecular ligation. AB - A 406 nucleotide long potato spindle tuber viroid (PSTVd)-specific linear RNA transcript was synthesized in vitro and subjected to limited digestion with ribonuclease (RNase) T1. Under certain conditions this guanosine-specific endoribonuclease proved to be capable of processing the longer-than-unit-length, precursor-like viroid RNA transcript by cleaving out a linear 358 nucleotide long product and ligating that to a circular RNA molecule. The new finding that RNase T1 acts as an RNA processing enzyme and, in particular, as an RNA 'circulase' can be explained by the unique structural preconditions inherent in the viroid specific substrate and by the well characterized two-step cleavage mechanism of the enzyme. These in vitro potentials of RNase T1 suggest that also in vivo procaryotic and eucaryotic RNases with a similar reaction mechanism might not only be involved in RNA degradation and trimming, but also in processing, ligation and recombination of RNA. PMID- 1709279 TI - Isolation and footprint analysis of the Escherichia coli thr leader paused transcription complex. AB - The E. coli thr operon leader region contains a cluster of transcription pause sites upstream of the attenuator. In this report, we determine the exact sites of pausing and analyze the structure of the ternary complex by footprint techniques. Under synchronized transcription initiation conditions in vitro, three closely spaced transcription pause sites were identified. These pause sites appeared downstream of the first region of dyad symmetry, which encodes an RNA hairpin in the transcript, and occurred at positions G112, G114 and A116 of the thr leader RNA. The results showed that the half-life of the thr paused complexes at G112 and G114 could be enhanced by limiting the concentration of the nucleoside triphosphate GTP in the transcription reactions. In addition, the half-life of the paused complexes was shown to increase in the presence of NusA protein. The thr leader complex that paused immediately before residues G112 and G114 of the nascent transcript was isolated and its structure was analyzed with enzymatic and chemical cleavage reagents. The footprinting studies using DNase I showed that there were approximately 35 nucleotides on both strands of the DNA that were protected by RNA polymerase from DNase I cleavage. The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites. The results from hydroxyl radical footprints also showed a similar pattern of protection at the transcription pause sites. However, no significant differences in the footprinting patterns were observed in the presence or absence of NusA protein. PMID- 1709280 TI - An MspI polymorphism for the HOX2F gene. PMID- 1709281 TI - MspI restriction fragment length polymorphism near exon 10 of cystic fibrosis (CFTR) gene. PMID- 1709282 TI - D12S17 and D12S25 identify the same MspI RFLP. PMID- 1709283 TI - MspI RFLP at 19q13.3 identified by the anonymous DNA sequence pX75B (D19S112). PMID- 1709284 TI - Immunohistochemical studies on uterine tumors. I. Invasive squamous cell carcinomas of the cervix and their precursors. AB - 40 invasive carcinomas and 80 preinvasive lesions of the uterine cervix were studied immunohistochemically; 40 benign lesions served as controls. On histological and immunohistochemical examination, invasive and preinvasive carcinomas were subdivided in the squamous (large cell, ectocervical) type and the reserve cell (small, large or clear cell, endocervical) type. Immunohistochemically, 100% of the invasive and preinvasive squamous cell carcinomas were positive with anticytokeratins 13, 14, 16 and negative with anticytokeratin 8 and anti-CEA. Most of the invasive and preinvasive reserve cell carcinomas showed a coexpression of cytokeratins 13, 14, 16, 8 and CEA. The subdivision of invasive carcinomas of the ecto- and endocervix into squamous cell and reserve cell types made by means of their structural differences is substantiated and re-evaluated by their immunohistochemical reactions. Both types of carcinomas retain the complex pattern of cytokeratins shown by their cells of origin. The reserve cell carcinomas, in addition, acquire a coexpression for CEA that indicates malignant transformation. The subdivision is of clinical importance because both types of carcinomas vary in their mode and speed of invasion and spread and in their association with HPV infection. PMID- 1709285 TI - Distribution and estimation of nucleolar organizer regions in various human lung tumours. AB - A quantitative study of nucleolar organizer regions in human lung carcinomas was carried out on routinely processed paraffin embedded tissue sections. We examined 104 lung carcinomas including 38 squamous cell carcinomas, 36 adenocarcinomas, 18 large cell anaplastic carcinomas, 6 small cell carcinomas and 6 carcinoids. No significant differences were found in mean number of NORs between squamous, adenocarcinoma and undifferentiated carcinomas including large cell and small cell carcinomas. Carcinoids had comparatively lower means except for one typical carcinoid. Considering the high incidence of overlap between ranges of NOR counts in these groups of tumours and in agreement with the only other study of lung tumours (which comprised only carcinoids and small cell carcinomas), we conclude that this technique cannot be reliably used to discriminate between various histologic types of lung cancers. However, long term follow up of these patients is needed to establish the value of the AgNOR technique for prognostic guidance. PMID- 1709286 TI - Ratio of digestive enzymes in the chick pancreas. AB - The ratio of exocrine enzymes was determined in extracts from pancreata of broiler chickens. The proteins were separated by ion-exchange chromatography to determine the elution pattern and the relative concentration of the various enzymes. Amylase was about 28.9%, the three chymotrypsinogens about 20%, and a single, anionic form of trypsinogen about 10% of the total protein. Procarboxypeptidases A and B, proelastase, lipase, and a secretory trypsin inhibitor were also present. PMID- 1709288 TI - Metabolism of 5-hydroxytryptophan in the isolated perfused rat lung. AB - The metabolism of 5-hydroxytryptophan (5-HTP) and tryptophan (TRP) in a single pass across the pulmonary circulation was studied in the isolated ventilated perfused rat lung and by high pressure liquid chromatography. The metabolism of 5 HTP was dependent on the rate of lung perfusion and the duration of infusion of 5 HTP, and was a saturable process with an apparent Km of 1.8 mM and Vmax of 0.14 mumol/g/3 min. The indoles found in the lung were 5-HTP, 5-hydroxytryptamine (5 HT) and 5-hydroxyindole acetic acid (5-HIAA); only 5-HIAA was detected in the lung effluent. The efflux of 5-HTP from the lung had two exponential components with half-lives of 0.15 and 3.65 min. After an infusion of 3H-5-HTP, the radiolabel that accumulated in lung was located mainly in the soluble fraction. An infusion of TRP resulted in the synthesis of 5-HTP, 5-HT and 5-HIAA in the lung, and 5-HTP was detected in the lung effluent. The results suggest that 5-HT can be synthesized in the intact lung from circulating TRP and 5-HTP. Since the rate of lung metabolism is low and no 5-HT is released into the lung effluent, the contribution of the lung to circulating levels of 5-HT is likely to be insignificant. Synthesis of 5-HT in intact lung suggests an intrapulmonary role for 5-HT. PMID- 1709287 TI - Identification of Duchenne muscular dystrophy genomic probe P20 constant Taql fragment corresponding to the EcoRV and Mspl polymorphisms. AB - The majority of Duchenne and Becker muscular dystrophy cases are caused by deletions observable in Southern blots with cDNA probes for the gene. When the deletion includes polymorphic probes, they may be used to determine carrier status by deletion segregation analysis: non-inheritance of parental alleles, or heterozygosity. The polymorphic genomic probe P20 is deleted in a large percentage of probands. P20 hybridizes with two constant fragments of 6.7 and 0.8 kb in Taql digests. In a number of probands, only the larger P20 Taql fragment is deleted. This study demonstrates that this fragment corresponds with the polymorphic EcoRV and Mspl fragments of P20. Families in which the upper Taql fragment is deleted may be screened for carrier status using non-inheritance of parental alleles or heterozygosity of P20 in EcoRV or Mspl digests. PMID- 1709290 TI - [Normal gamma globulin levels in liver cirrhosis: a prognostic indicator and improved survival]. AB - Serum levels of gammaglobulins were evaluated in a population of 1334 patients with liver cirrhosis (mean age +/- SD 54.8 +/- 10.9; 898 males). Hypergammaglobulinemia was not as frequently observed as described in the literature 278 patients (20.8%) had gammaglobulin levels lower than 1.5 g/dl (first group) and the other 1056 had more than 1.5 g/dl of gammaglobulins (second group). At analysis of survival curves the first group had a median survival time of over 60 months, significantly higher than the 37.6 months of the other patients, when compared with the Lee-Desu statistic (p less than 0.01). Moreover, the patients of the first group had a lower incidence of ascites as compared to the second group (28% versus 45%; p less than 0.001), hepatic encephalopathy (7% versus 14%; p less than 0.01) and hepatocellular carcinoma (5.7% versus 8.4%; n.s.). The presence and the degree of oesophageal varices were significantly lower in the patients of the first group than in the others (p less than 0.001); a number of laboratory tests for the evaluation of liver cell function and the Child class distribution were more favourable in the first group than in that of hypergammaglobulinemic patients. IN CONCLUSION: normal blood gamma globulin level is not uncommon in liver cirrhosis and it may be considered a positive prognostic factor, which identifies a group of patients with a better quality of life, a lower incidence of decompensation symptoms and a longer survival time. PMID- 1709289 TI - Dopamine and conditioned reinforcement. II. Contrasting effects of amphetamine microinjection into the nucleus accumbens with peptide microinjection into the ventral tegmental area. AB - It has been shown that infusion of certain neuropeptides into the ventral tegmental area (VTA) results in increased motor activity and enhanced dopamine turnover in the nucleus accumbens. In the present experiments, substance P (SP), neurotensin (NT), d-ala-metenkephalin (DALA) and morphine sulfate (MS) were injected bilaterally into the VTA and their effects on conditioned reinforcement were assessed. These effects were compared with infusion of amphetamine into the nucleus accumbens, which has previously been shown to strongly enhance responding for conditioned reinforcers. For these experiments, hungry rats were trained to associate a compound stimulus (light and click) with the presentation of food. In the test phase, responding on one lever (CR lever) resulted in the presentation of the stimulus but no food. Responding on the other (NCR lever) had no consequences. Different groups of animals received microinjections (0.5 microliter, bilaterally) of SP (0, 0.03, 0.3, 3.0 micrograms), NT (0, 0.025, 0.25, 0.5 microgram), DALA (0, 0.01, 0.1, 1.0 microgram) or morphine (0, 0.025, 0.25, 2.5 micrograms) into the VTA. SP infusion into the VTA resulted in a small increase in responding which was not selective for the CR lever. NT, DALA and morphine had no effect on responding for conditioned reward. In contrast, amphetamine (0, 0.2, 2.0, 20 micrograms) injected into the nucleus accumbens markedly enhanced responding for conditioned reward. These findings suggest that stimulation of the mesolimbic system at the level of the DA cell bodies, which induces a small increase in DA turnover, is not sufficient to potentiate responding for conditioned reward. On the other hand, an important requirement for potentiation may be excessive release of dopamine in the nucleus accumbens. PMID- 1709291 TI - Characterization of a radioimmunoassay to determine plasma total renin. AB - The determination of plasma total renin is useful not only as a tool to investigate the physiology of hypertension but also as a marker for Wilms' tumor. A radioimmunoassay (RIA) system to determine plasma total renin was newly developed using monoclonal antibodies specific for both inactive and active renin (inactive + active = total); in this study, an effort was made to confirm that this RIA system truly determines plasma total renin concentrations. First, it was found that this monoclonal antibody stains only the juxtaglomerular apparatus of the kidney. Second, the data determined by the RIA were compared with those obtained by the conventional enzymatic method: samples were activated, and renin activity was assayed by measuring angiotensin I. The coefficient of the data obtained by this RIA system and by the conventional method was 0.921 (p less than 0.01) based on all 89 samples, and 0.809 (p less than 0.01) based on 86 of the 89 samples whose values were less than 600 pg/ml by RIA. As a result of these studies, it was concluded that the newly developed RIA system does determine total renin levels in patients' plasma. PMID- 1709292 TI - Interferon therapy in the chronic aggressive viral hepatitis. AB - The effects of interferon (INT) therapy in the chronic aggressive hepatitis of viral origin (CAVH) are critically reviewed. The main factor which favours the progress of acute viral hepatitis to chronic forms is the persistence of viral replication in conditions of immune deficit of the host. INT has antiviral, immunomodulating and antitumoral action. The INT alpha class proved to be the most adequate for the CAVH treatment. Many recent clinical trials have demonstrated the INT alpha effectiveness in CAVH. The results of such studies are separately analysed for each hepatitis virus. The importance of various factors, i.e., dose, treatment duration, race, sex, age, histologic lesions, ALAT level, viral DNA titer, associations with adenine-arabinoside, acyclovir, corticosteroids, a.o., is discussed in relation with the rate of therapeutic response and the risk of viral infection recurrences. PMID- 1709293 TI - Reduced passage of alpha 2-macroglobulin into malignant serous effusions and diminished antifibrinolytic potential of the fluid. AB - The ratio of the relative concentrations of large and small proteins between effusions and serum was measured in patients with benign and malignant effusions. The ratio of values for a large protein, alpha 2 macroglobulin (alpha 2 M) was significantly (p less than 0.0005) lower in the 22 patients with tumoral effusion (0.436 +/- 0.016) and in the 16 patients with transudates (0.388 +/- 0.017) than in the 21 cases with inflammatory exudates (0.838 +/- 0.036) while the ratios between relative concentrations of the lower molecular weight alpha 1 antitrypsin (alpha 1AT) in serous fluids and sera were similar (close to 1.0) in all the above mentioned conditions. Most inflammatory exudates inhibited fibrinolysis in a urokinase activated system, while the activity of the inhibitors of fibrinolysis was very weak in tumoral effusions and in transudates. These findings suggest a rather selective passage of plasma proteins into a tumoral effusion, a fact that might be relevant for a pathogenic diagnosis. PMID- 1709294 TI - [GM-CSF and G-CSF: cytokines in clinical application]. AB - Leukopenia or pancytopenia as a result of bone marrow dysfunction are manifestations of various diseases or complications of therapeutic regimens. The spectrum of diseases associated with leukopenia is wide and includes congenital as well as acquired neutropenias secondary to conditions such as myelodysplastic syndromes, AIDS, malignant tumors with or without chemotherapy-enhanced neutropenia, bone marrow transplantation or therapeutic or accidental radiation. The morbidity and mortality of infectious diseases is greatly enhanced during neutropenic phases. Over the last few years attempts have been made to shorten the duration and lessen the severity of neutropenia in patients with the above conditions by administration of Granulocyte Macrophage Colony Stimulating Factor (G-CSF). Both cytokines were successfully tested in phase I and II trials. Treatment with GM-CSF or G-CSF results in a dose-dependent increase of the neutrophil count. GM-CSF also increases the number of eosinophils and monocytes in peripheral blood. The effect of both cytokines on the neutrophil count is transient as long as the underlying disease persists. This prompted the institution of maintenance therapy, which has been successfully used with either cytokine. Long-term treatment is usually well tolerated and results in a reduction in the frequency of infections as well as in the duration of antibiotic treatments. Side effects of GM-CSF or G-CSF are usually mild and include fever, myalgia, bone pain, and erythema. A number of patients developed dyspnea, hypotension, sweating, flushing and erythema after the first dose of GM-CSF in each treatment cycle. This first-dose reaction occurs more frequently after intravenous than reactions were reported with G-CSF. Some patients with myelodysplastic syndrome progressed to acute myeloic leukemia during or after treatment with GM-CSF or G-CSF. Most of these patients presented with an increased fraction of blasts in the bone marrow, which preceded the treatment with the colony stimulating factors. Since GM-CSF and possibly G-CSF may increase the risk of developing acute leukemia in patients with myelodysplastic syndrome, it appears prudent to limit the use of these cytokines in patients with this disease. The subcutaneous route of administration appears to be preferable to intravenous administration, since the incidence and severity of side effects are reduced. While many questions concerning dosage, long-term therapy and combination therapy still remain unanswered, the information presented in this review concerning the clinical use of these cytokines warrants an optimistic outlook. PMID- 1709295 TI - Duffy locus linkage and HLA antigens in hereditary motor-sensory neuropathy. AB - The Duffy system, Fya, Fyb, and HLA antigens were studied in 86 members, 39 affected, 47 healthy subjects, of 17 families with HMSN types I, II and III. The incidence of autosomal dominant as well as recessive, or sporadic forms was registered. In the autosomal dominant form of HMSN type I a close linkage was found between the genes coding the disease under study and the determinants Fya and Fyb. Recombination was found in only 9.1% of the cases. A study of HLA antigens revealed a major decrease in antigen B27 in the patients under scrutiny. An identical haplotype of most of the parents was a conspicuous feature in families with the autosomal dominant form of HMSN type I. PMID- 1709296 TI - [Psychometric studies of performance deficits in acute schizophrenic patients with special reference to gender]. AB - 19 male and 18 female schizophrenic inpatients were examined on cognitive, motor and sensorimotor performance and visual and speech related intellectual ability at the beginning and end of a therapeutic period of 4 weeks on the average. Speed of sensorimotor reaction time revealed a higher performance level with regard to male as compared to female patients. Reverse effects could be demonstrated with respect to precise motor reactions (pegboard). Starting at a higher level of performance female had an important decrease. Considering speed of unprecise movements (tapping) and tremor-parameter (steadiness) impairment of performance occurred in time course for both male and female. Especially simple tasks demanding speed to solve the problem and left-handed requirement are solved in a lower degree indicating impaired reception of information and hemispheric hyperactivation. PMID- 1709297 TI - [Assessment of patients with narcissistic neuroses using the Deneke Narcissism Inventory]. AB - In the following paper Deneke's Narzissmusinventar (1989) was used for examining 32 (17 men, 15 women) patients who, after a first interview were diagnosed having a narcissistic personality disorder respectively neurosis. We compared our results with the results Deneke found in patients with borderline personality disorders, narcissistic personality disorders and healthy persons. Our results correspond with the results Deneke found in patients with narcissistic personality disorders. Deneke's Narzissmusinventar is qualified to confirm the diagnosis of a narcissistic personality disorder respectively neurosis. PMID- 1709298 TI - Infarcts in the territory of lenticulostriate branches from the middle cerebral artery. Etiological factors and clinical features in 65 cases. AB - We studied 65 consecutive patients with a first stroke who had an appropriate CT proven small infarct in the territory of the lateral (61 patients), medial (3 patients) or both lateral and medial lenticulostriate arteries (1 patient) from the middle cerebral artery. While more than 75% of these patients were either hypertensive or diabetic (having at least one cause for small-artery disease), embolic sources were encountered in 35%, either from large vessels (28%), and/or from the heart (15%). Other causes (angiitis, migraine) were found in only 9%. The neurologic deficit was purely motor in more than 50% of the patients (in half of them with neuropsychological dysfunctions), a sensori-motor deficit was present in 30% (in half of them with neuropsychological dysfunctions), and only 20% had ataxic hemiparesis. No one had pure sensory stroke. None of the classical lacunar syndrome or the modality of sensory, motor or ataxic deficits were specific for any topographic subdivision of LS territory, but there was a tendency for clinical features to be linked with the involved basal ganglia and the topography of pathways in the internal capsule as delineated by anatomical studies. Pure motor deficits were associated with infarcts in the medial and posterior part of LS territory, visual field deficits and hemineglect always corresponded to posteriorly situated infarcts. Neuropsychological deficits were common in infarcts in the anterior and posterior subdivisions of LS territory, with a major effect of the size of infarct. Sensory deficits were not correlated with any location in LS territory, probably because thalamo-efferent fibres have a more diffuse course through the internal capsule. PMID- 1709299 TI - [Disorders of pain perception in schizophrenia]. AB - Starting from a case of marked pain insensitivity in a patient suffering from catatonic schizophrenia we state in this paper that analgesia seems to be an ubiquitous phenomenon which is not only caused by physical disorders of the central nervous system. Different models of interpretation as to be found in scientific literature are reviewed. On the basis of today's physiological knowledge, five hypotheses on causal explanation of pain insensitivity in schizophrenics are discussed: Hypalgesia and analgesia are an expression of motorial inability to react; a consequence of a disorder of consciousness; an analgetic effect of neuroleptic drugs; a basic deficit in schizophrenia and; a result of a disturbed psycho-physiological development. PMID- 1709300 TI - [Attitude of ambulatory psychiatric patients to psychopharmacologic treatment]. AB - By means of a semistructured interview 51 nonpsychotic new outpatients of the Psychiatric University Outpatient Clinic, Basle, Switzerland, were asked about their expectation of being treated with a psychotropic medication during the ensuing therapy. Their conceptualizations about the modes of action of psychopharmacological drugs were also studied. In addition, their emotionally connoted attitudes towards these modes of action were elucidated. 2/3 of those patients who have been treated with a psychopharmacological medication by their family doctor expected that also their psychiatrist at the outpatient clinic prescribed a psychotropic medication, whereas only 1/6 of those patients without a corresponding experience stated to expect a psychopharmacological treatment. Patients who have previously been treated psychopharmacologically stated significantly more often that psychotropic drugs had an effect on emotional processes and compared them significantly less often to alcohol or addictive drugs of the drug subculture than patients without the experience of previous psychotropic medication. Patients expecting to receive psychotropic drug gave significantly more often positive emotional connotations about the presumed modes of action of these drugs than patients without such an expectation. It can be concluded that previous experiences with psychotropic medication evokes the expectancy of also receiving a psychopharmacological treatment during an ensuing therapy at the Psychiatric Outpatient Clinic. Furthermore the experience of a psychopharmacological treatment induces mainly emotionally positive connotated conceptualizations about the modes of action of psychotropic medication. PMID- 1709301 TI - Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin. AB - Immunophilins, when complexed to immunosuppressive ligands, appear to inhibit signal transduction pathways that result in exocytosis and transcription. The solution structure of one of these, the human FK506 and rapamycin binding protein (FKBP), has been determined by nuclear magnetic resonance (NMR). FKBP has a previously unobserved antiparallel beta-sheet folding topology that results in a novel loop crossing and produces a large cavity lined by a conserved array of aromatic residues; this cavity serves as the rotamase active site and drug binding pocket. There are other significant structural features (such as a protruding positively charged loop and an apparently flexible loop) that may be involved in the biological activity of FKBP. PMID- 1709302 TI - Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex. AB - The structure of the human FK506 binding protein (FKBP), complexed with the immunosuppressant FK506, has been determined to 1.7 angstroms resolution by x-ray crystallography. The conformation of the protein changes little upon complexation, but the conformation of FK506 is markedly different in the bound and unbound forms. The drug's association with the protein involves five hydrogen bonds, a hydrophobic binding pocket lined with conserved aromatic residues, and an unusual carbonyl binding pocket. The nature of this complex has implications for the mechanism of rotamase catalysis and for the biological actions of FK506 and rapamycin. PMID- 1709303 TI - FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu. AB - The Drosophila homeobox segmentation gene fushi tarazu (ftz) is expressed in a seven-stripe pattern during early embryogenesis. This characteristic pattern is largely specified by the zebra element located immediately upstream of the ftz transcriptional start site. The FTZ-F1 protein, one of multiple DNA binding factors that interacts with the zebra element, is implicated in the activation of ftz transcription, especially in stripes 1, 2, 3, and 6. An FTZ-F1 complementary DNA has been cloned by recognition site screening of a Drosophila expression library. The identity of the FTZ-F1 complementary DNA clone was confirmed by immunological cross-reaction with antibodies to FTZ-F1 and by sequence analysis of peptides from purified FTZ-F1 protein. The predicted amino acid sequence of FTZ-F1 revealed that the protein is a member of the nuclear hormone receptor superfamily. This finding raises the possibility that a hormonal ligand affects the expression of a homeobox segmentation gene early in embryonic development. PMID- 1709304 TI - Ca2+ permeability of KA-AMPA--gated glutamate receptor channels depends on subunit composition. AB - NMDA (N-methyl-D-aspartate) receptors and non-NMDA receptors represent the two major classes of ion channel-linked glutamate receptors. Unlike the NMDA receptor channels, non-NMDA receptor channels have usually been thought to conduct monovalent cations only. Non-NMDA receptor ion channels that can be gated by kainic acid (KA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) are formed by the glutamate receptor subunits GluR1, GluR2, and GluR3. These subunits were expressed in various combinations in Xenopus oocytes so that their permeability to divalent cations could be studied. At physiological resting potentials, KA and AMPA elicited inward calcium currents in oocytes expressing GluR1, GluR3, and GluR1 plus GluR3. In contrast, oocytes expressing GluR1 plus GluR2 or GluR3 plus GluR2 showed no such permeability. Thus, in neurons expressing certain KA-AMPA receptor subunits, glutamate may trigger calcium dependent intracellular events by activating non-NMDA receptors. PMID- 1709305 TI - Surgical management of metastatic renal carcinoma of the spine. AB - A total of 33 patients with renal cell carcinoma metastatic to the spine underwent spinal decompression over a 5-year period; 20 were operated on for neurologic dysfunction, and the remainder for pain alone. Surgery was performed anteriorly in 21, posteriorly in 9, and combined in 3 cases. The surgical approach was determined by the preoperative anatomic localization of the tumor. Of these patients 88% had fusions with instrumentation and polymethylmethacrylate; 88% of patients had partial or complete relief of pain; and 64% of bedridden patients subsequently were able to walk. Neurologic function improved in 60% of patients with a neurologic deficit; however, only 36% of incontinent patients regained bladder control. Survival averaged 8.0 +/- 1.5 months. Survival correlated with the degree of neurologic dysfunction and the presence of other known metastases. Recurrent cord compression developed in 49% of patients, usually at the same level; 9 of these 16 patients had repeat decompression, with similar operative results as the primary procedure in terms of pain and neurologic function. Blood loss was variable but often significant. Preoperative embolization appeared to be beneficial. Precise tumor localization preoperatively directing the surgical approach and better patient selection would likely improve results and decrease morbidity. Good palliation appeared to be achieved in regards to both pain relief and improved neurologic function. PMID- 1709306 TI - Unsuspected Chlamydia trachomatis infection in heterosexual men attending a sexually transmitted diseases clinic: evaluation of risk factors and screening methods. AB - To characterize the problem of unsuspected Chlamydia trachomatis infection in heterosexual men attending a sexually transmitted diseases (STD) clinic, the authors assessed risk factors for infection and the value of screening for infection by gram-stained smears and urinalysis in 438 men who did not have conventional clinical indications for chlamydial treatment at their initial visit. Evaluations included urethral swabs for gram-stained smears and Neisseria gonorrhoeae and C. trachomatis cultures and microscopy of first-catch urine sediment. C. trachomatis was isolated from 29 subjects (6.6%) and N. gonorrhoeae from 6 subjects (1.3%), (P less than .05). The only demographic or clinical factors that were associated with C. trachomatis were age younger than 21 years and five or more lifetime sexual partners. Screening for C. trachomatis with urethral gram stain and urine sediment examination had sensitivities of only 23% and 35%, respectively. Risk factor assessment and screening with standard microscopic procedures do not adequately predict infection in this group, which accounts for almost 25% of the C. trachomatis burden in heterosexual men who visit an STD clinic. More specific chlamydia detection methods are needed for effective control programs. PMID- 1709307 TI - Evaluation of conservative therapy for exomphalos. AB - Two management patterns were identified in 36 patients with exomphalos--primary surgical closure and initial topical therapy with delayed surgical closure. Primary surgical closure of minor exomphalos was well tolerated in 15 patients, but was associated with a high local and systemic morbidity rate in 14 patients with major defects. In contrast, initial topical therapy with silver sulphadiazine and delayed closure in seven matched patients with a major defect were well tolerated and did not prolong duration of hospitalization. Enteral feeding was more readily established and subsequent fascial closure facilitated in the conservatively treated group. It was suggested that this method should be more often considered in the management of all instances of major exomphalos. PMID- 1709308 TI - Palliation of malignant tracheal strictures using silicone T tubes. AB - The use of silicone T tubes for intubation of malignant tracheobronchial strictures may provide some degree of palliation of this distressing condition. It was used in seven patients with malignant lesions and two with benign strictures (resulting from tracheal trauma and lung transplantation). Four patients (two with cancer) are still alive and well with the tube in position. All patients noted improvement in dyspnoea and stridor. The main problems were tube migration (one patient), tracheo-oesophageal fistula (one patient), and blockage of the tube by tumour (two patients) or encrusted secretions (three patients). Airway patency was restored when the tube was blocked by cleaning or by laser resection of the tumour. With careful supervision and education of the patient intubation can give useful palliation to patients with distressing upper airways obstruction. PMID- 1709309 TI - Circulating activated platelets in myeloproliferative disorders. AB - Platelet activation in patients with myeloproliferative disorders is often suggested by increased platelet alpha-granule secretion and an acquired storage pool defect of dense granules. To determine whether activated platelets circulate in patients with chronic myeloproliferative disorders, we evaluated the binding of monoclonal antibodies against activation-dependent epitopes on resting platelets (P 12, CD 63, and CD 62) in 12 patients with prominent megakaryocytic proliferation (8 patients with essential thrombocythemia, 2 with chronic myeloid leukemia, and 2 patients with polycythemia rubra vera). In addition, platelet aggregation in response to collagen, adenosine diphosphate, platelet activating factor, and agglutination with ristocetin was investigated. In 3 patients there was an increased percentage of platelets binding at least 1 activation marker. In 2 other patients, a trend towards increased antibody binding was observed. Binding of the antibody to thrombospondin (P 12) was related to expression of the GMP 140 protein (CD 62, r = 0.76, p = 0.004). There was no correlation of platelet aggregation defects in vitro to increased expression of platelet activation markers or to thrombohaemorrhagic complications. However, circulating activated platelets were detected in three out of five patients with a history of bleeding or thrombotic complications. The results of this preliminary study suggest that some but not all patients with myeloproliferative disorders showed increased amounts of circulating activated platelets. The relation of bleeding and thrombotic complications to the expression of activation-dependent epitopes on platelets in myeloproliferative disorders requires further investigation. PMID- 1709310 TI - Increased expression of thrombomodulin on the cultured human umbilical vein endothelial cells and mouse hemangioma cells by cyclic AMP. AB - We previously reported that the expression of thrombomodulin on the MEG-01, a cell line from human megakaryoblastic leukemia, was increased by agents that increase intracellular cAMP. In this paper we examine the effect of these agents on cultured human umbilical vein endothelial cells (HUVEC) and mouse hemangioma cells. Incubation of the cells with 3 mM dibutyryl cAMP (dbcAMP) increased functionally active thrombomodulin by about 2-fold on HUVEC and 4-fold on hemangioma cells. This effect was observed from 1 hour after the incubation and continued up to 24 hours. Dot hybridization of mRNA demonstrated a dose dependent increase in thrombomodulin mRNA in response to dbcAMP. Treatment of HUVEC with 20 microM forskolin or 100 microM isobutylmethylxanthine (IBMX) also increased cell surface thrombomodulin on HUVEC. These agents prevented the interleukin I (IL-I) or tumor necrosis factor (TNF)-induced decrease in thrombomodulin on HUVEC. These data suggest that the expression of thrombomodulin on HUVEC and mouse hemangioma cells may be regulated by intracellular cAMP level. PMID- 1709311 TI - Neurological deterioration under isovolemic hemodilution with hydroxyethyl starch in acute cerebral ischemia. AB - In a prospective study, we randomly allocated 70 patients with acute ischemic stroke to two therapy groups. Up to interim analysis, 33 patients underwent bloodletting, with simultaneous infusion of an identical volume of hydroxyethyl starch (10%, 200/0.5). The target hematocrit was 35%. A control group of 37 patients did not receive hemodilution. Apart from an unequal sex distribution, the two groups were comparable with regard to age, cardiovascular risk factors, and medical history. In the hemodilution group, the mean hematocrit fell from 44.4% to 37.7%. After 14 days, improvement on the neurological score scale was 3.3 points in the hemodilution group compared with 6.5 points in the control group (p = 0.12). Subgroups with early inclusion (less than 12 hours) or pronounced lowering of hematocrit (greater than 15% of initial hematocrit) also did not profit from hemodilution. Clinical deterioration observed in eight hemodilution group patients (p less than 0.01) led to discontinuation of the study for ethical reasons. PMID- 1709312 TI - Immunological comparison between prostate-specific antigen and gamma seminoprotein. AB - Prostate-specific antigen (PA) and gamma-seminoprotein (gamma-Sm) were compared by immunocytochemical, immunodiffusion and immunoblotting methods using rabbit anti-PA antibody and rabbit anti-gamma-Sm antibody. Enzyme immunoassays (EIAs) were developed for measurements of PA and gamma-Sm to determine a correlation between serum PA and gamma-Sm levels in patients with prostate cancer. The patterns of localization and distribution of PA and gamma-Sm were identical in prostate tissue sections, including benign and cancerous human prostates. The immunodiffusion study showed that the antigens with which anti-PA antibody and anti-gamma-Sm antibody reacted in seminal plasma and prostate tissue homogenates were identical to each other. In the immunoblotting study, anti-PA antibody and anti-gamma-Sm antibody recognized a single antigen corresponding to a molecular weight of approximately 33,000 both in seminal plasma and prostate tissue homogenates. The EIAs developed in this study were sensitive, specific, and reproducible, and the correlation between serum PA and gamma-Sm values determined by these EIAs was highly significant (r = 0.99, P less than 0.001). These results indicated that PA and gamma-Sm were immunologically identical and that serum PA and gamma-Sm determined by immunoassays using anti-PA antibody and anti-gamma-Sm antibody should be evaluated as identical tumor markers for serodiagnosis of prostate cancer. PMID- 1709313 TI - Additional mycotoxins of potential importance to human and animal health. PMID- 1709314 TI - [Analysis of polycyclic aromatic hydrocarbons in lipid-containing biological matrices--study of contamination of breast milk by benzo(a)pyrene along transit routes through Tyrol]. AB - 41 samples of human milk from the region of Tyrol were investigated with regard to benzo(a)pyrene content. A complicated analytical preparatory method had to be worked out. The percentage recovery of benzo(a)pyrene was 85-90. Analysis was carried out by means of capillary gas chromatography and flame ionization detection or mass spectrometry. The limit of benzo(a)pyrene detection is 0.1-1 microgram/kg (single ion monitoring). None of the investigated samples of milk showed the presence of benzo(a)pyrene. PMID- 1709315 TI - A discussion of Ambystoma kidney tubule ion channels, transporters, and pH regulation. AB - This paper explores the role and biophysical expression of the equivalent electrical circuit model as it applies to ionic conductances across the paracellular shunt, apical membrane, and basolateral membrane of the Ambystoma renal proximal tubule. Information about such conductances may be experimentally determined through transepithelial voltage and intracellular voltage measurements. The equivalent electrical circuit model has been applied extensively by investigators to define ion channels and transport mechanisms in the salamander proximal tubule. A comprehensive discussion of all known ionic conductance and transport pathways as well as pH-regulatory functions of contributory symports/antiports is examined in the Ambystoma proximal tubule. This paper explores renal physiological principles and serves as a companion to: Bock JF, Boulpaep EL: Bicarbonate transport mechanisms in the Ambystoma kidney proximal tubule: Transepithelial potential measurements. PMID- 1709316 TI - [Assessment of diagnostic lung function and diagnostic laboratory parameters in workers exposed to diesel exhaust below ground in comparison with non-exposed workers]. AB - Toxic gases caused by the use of large Diesel-driven devices in underground mines may lead to impairment of health, especially in the respiratory system. By means of occupational hygiene supervision it may be checked if the effectiveness of accomplished occupational hygiene measures are sufficient. By an enlarged examination programme of the respiratory function and laboratory diagnostic investigation miners exposed to Diesel-exhaust-gas were compared within the above mentioned supervision with a non-exposed population. In this study was not found any difference between the respiratory results attributed to influence of Diesel exhaust-gas. Among the laboratory parameters the exposition to Diesel-exhaust-gas will only affect the mercapturic acid, however, which also may be influenced by other factors. Under the conditions of the measured low degree of exposition was not found any correlation between the level of mercapturic acids and the duration of exposure as well as to the actual degree of exhaust-gas exposition. PMID- 1709317 TI - [The immune system and allergy]. PMID- 1709318 TI - [Virus infections and obstructive respiratory tract diseases]. PMID- 1709319 TI - [Bronchitis and obstructive bronchitis]. PMID- 1709320 TI - [Pathophysiologic principles of obstructive respiratory tract diseases]. PMID- 1709321 TI - Immunocytochemical characterization of large-cell carcinomas of the lung. Role, limitations and technical considerations. AB - To clarify the use of cytologic preparations, particularly those previously stained by the Papanicolaou method, for the immunocytochemical evaluation of large-cell carcinomas (LCCs), 37 cytologic preparations from cases diagnosed as LCC were examined using a battery of immunocytochemical stains for keratin, chromogranin, common leukocyte antigen (CLA) and B72.3. Thirty-two specimens were from the thoracopulmonary region (12 fine needle aspirates of the lung, 7 bronchial brushings, 5 bronchial washings, 2 sputa and 6 pleural fluids); the remaining specimens were fine needle aspirates of 3 lymph nodes, 1 vertebral body and 1 liver. Of the specimens analyzed, 30 of 37 were positive for keratin and 7 of 35 were positive for B72.3 (6 were positive for both). Only 1 of 37 was positive for CLA while none of 37 was positive for chromogranin. Six specimens showed no reaction with either keratin, B72.3 or chromogranin. These results confirm that the majority of LCCs consist of epithelial cells of either a squamous or an adenocarcinomatous type. They also show that immunocytochemistry is a useful diagnostic adjunct that can be applied to cytologic preparations previously stained by the Papanicolaou method; this is important since immunostaining is often considered after undifferentiated malignant cells are encountered in a previously stained preparation. However, a thorough understanding of some technical limitations is critical in the evaluation of the results of this technique when it is applied to cytologic specimens. PMID- 1709322 TI - Cytodiagnosis of Campylobacter pylori in Papanicolaou-stained imprints of gastric biopsy specimens. AB - The use of Papanicolaou-stained touch preparations of gastric antral biopsies for the identification of Campylobacter pylori was examined using specimens obtained from 63 consecutive patients with endoscopic evidence of antral gastritis, with the results compared to routine histologic examination and Warthin-Starry silver staining. Organisms were readily identifiable in the Papanicolaou-stained imprints of the gastric mucus. The sensitivity in detecting organisms was 92.5% for the Warthin-Starry-stained sections, 71.4% for the Papanicolaou-stained imprints and 100% for both techniques combined. False-negative imprints were attributed to poor smears and/or the submission of duodenal tissue rather than antral biopsies. Properly performed touch preparations stained by the Papanicolaou method are a cost-effective adjunct to Warthin-Starry-stained section for improving the sensitivity of gastric biopsies for the diagnosis of C pylori. PMID- 1709323 TI - Polyclonal carcinoembryonic antigen staining in the cytologic differential diagnosis of primary and metastatic hepatic malignancies. AB - In order to assess the utility of immunocytochemical staining of bile canaliculi with a polyclonal antiserum to carcinoembryonic antigen (pCEA) in the differentiation of primary hepatocellular carcinomas from metastatic malignancies, pCEA staining was performed on fine needle aspiration specimens from hepatic lesions in 60 patients. The original cytologic diagnoses were hepatocellular carcinoma in 22 patients, metastatic neoplasm or cholangiocarcinoma in 27 patients and benign hepatocytes in 11 cases. The cytologic diagnoses of malignancy were confirmed by surgical excision, autopsy or clinical investigations in 82% of the patients. Follow-up data, supported by pCEA staining, reversed the original cytologic diagnosis in three cases. Bile canalicular pCEA staining was identified in 18 of 22 cases of hepatocellular carcinoma and in all 11 benign hepatocellular aspirates. All 27 cases of metastatic malignancy or cholangiocarcinoma were negative for canalicular pCEA staining, although 11 cases exhibited cytoplasmic staining. Interpretation of pCEA staining was not affected by the intermingling of malignant cells and benign hepatocytes. Predictive values were 100% for a positive test and 87% for a negative test. These findings indicate that staining with pCEA antiserum is a useful adjunct in the differential cytologic diagnosis of malignant hepatic lesions. PMID- 1709324 TI - Epithelial atypia in gynecomastia induced by chemotherapeutic drugs. A possible pitfall in fine needle aspiration biopsy. AB - The clinical, cytopathologic and histopathologic features of a case of gynecomastia induced by chemotherapeutic drugs are described. Fine needle aspiration (FNA) smears showed epithelial atypia, and an erroneous cytologic diagnosis of carcinoma was made. Histopathologic study showed gynecomastia with epitheliosis, papillomatosis and atypical ductal hyperplasia. Review of the FNA smears showed the findings to be more typical of a reparative or regenerative process; these findings had been cytologically overinterpreted, partly due to the lack of adequate clinical information submitted with the aspirate. The possible causes of gynecomastia, the induction of epithelial atypia by cytotoxic chemotherapy and the cytologic features whose recognition may prevent false positive diagnoses in such cases are discussed. PMID- 1709325 TI - Choriocarcinoma metastatic to the breast diagnosed by fine needle aspiration. AB - Fine needle aspiration (FNA) was used to study nodules in the left breast of a patient with a previous history of uterine choriocarcinoma. The FNA smears contained numerous malignant mononucleated cells and multinucleated giant cells. The cytologic diagnosis was metastatic choriocarcinoma, which was confirmed by histologic study of excised tissue. That diagnosis would have been difficult to make cytologically if the previous history had not been known; the differential diagnosis of multinucleated giant cells in an aspirate from a breast mass is discussed. PMID- 1709326 TI - Assessing the adequacy of induced sputum for the evaluation for Pneumocystis carinii pneumonia. PMID- 1709327 TI - Phyllodes tumor with keratin cysts: a diagnostic problem in fine needle aspiration of the breast. PMID- 1709329 TI - The brain development and behaviour of autistic children. Notes on the research situation. AB - The relationships between brain functions and behaviour characteristics during various phases of the development of autistic children were looked into. In doing so, the concept of stability on the one hand as well as change on the other hand is the main topic and is illustrated with examples. The state of neuroscientific ideas and methodical possibilities on the theme is not only broached but also discussed in connection with the treatment (in the sense of an optimal coordination between brain and environment). It thus results that the connections between genetical disposition, brain development, environmental influences and behaviour in autistic children become more and more clear. Utilizing this, though, better treatment possibilities can only be expected on the basis of integrating bio-psychosocial research which emphasizes the development aspect. PMID- 1709328 TI - The relation between perinatal conditions and developmental outcome in low birthweight infants. Comparison of two cohorts. AB - We have compared the relations between perinatal conditions and developmental outcomes at age four years for two cohorts of children with birthweights 2,300 g or less, who did not develop cerebral palsy--one from Southeastern Wisconsin (children born 1975-76) and the other from Copenhagen (children born 1980-82). We examined the general effects of parental education and socioeconomic status, the use of Cesarean section, the degree of prematurity and neonatal complications on outcome. The methods of latent path structural analysis were used to form two models among 15 latent variables: one for children from Copenhagen and a similar model for children from Wisconsin. The impact of parental education and socioeconomic status was somewhat greater in Wisconsin. Several neonatal complications were related to outcome in Wisconsin: the early condition of the infant, use of a respirator, pneumothorax, and anemia/apnea. The only neonatal complication with a significant relation to outcome in Copenhagen was pneumothorax and to a much lesser degree major germinal layer haemorrhage. The degree of prematurity per se had a greater impact in Copenhagen. The use of Cesarean section and mechanical ventilation in the smallest infants was much more frequent in Denmark, but no association could be shown between this increased use and improved developmental outcome. PMID- 1709330 TI - [Effect of polyamines isolated from pilose antler (PASPA) on RNA polymerase activities in mouse liver]. AB - The incorporations of [3H] leucine into protein and [3H] uridine into RNA in mouse liver were increased when PASPA was given to mice at a dose of 30 mg/kg for 4 successive days. The RNA polymerase activity, especially the RNA polymerase II activity in the solubilized liver nuclear fraction of PASPA-treated mice was also increased. In vitro experiment demonstrated that PASPA increased the RNA polymerase activity significantly in mouse liver nuclei at a concentration of 1 microgram/ml. These results suggest that the enhancement of RNA polymerase activities, particularly RNA polymerase II activity, induced by PASPA treatment is responsible for the increase in synthesis of protein and RNA in mouse liver tissue. PMID- 1709331 TI - Reduced concentrations of galanin, arginine vasopressin, neuropeptide Y and peptide YY in the temporal cortex but not in the hypothalamus of brains from schizophrenics. AB - Postmortem investigations were performed in brains from 14 schizophrenic patients and 21 controls matched for age and autopsy latency. Concentrations of galanin, delta-sleep-inducing peptide (DSIP), corticotropin-releasing factor (CRF), arginine vasopressin (AVP), neuropeptide Y (NPY) and peptide YY (PYY) were determined in the hypothalamus and grey matter from the temporal cortex. A significant positive correlation between age and the concentrations of galanin and CRF was found in the controls. No sex differences were found except a higher mean of CRF in the hypothalamus of the women. In the temporal cortex of the schizophrenic brains, galanin, AVP, NPY and PYY were significantly reduced. DSIP reduction only bordered on significance. CRF was not reduced. Comparing neuroleptic-treated vs non-treated schizophrenics, the treatment factor could not explain the reduced concentrations of neuropeptides in the temporal lobe. A comparison of controls with schizophrenics showed no significant differences in hypothalamic neuropeptide concentrations. PMID- 1709332 TI - Human osteoblastlike cells do not respond to interleukin-6. AB - Interleukin 6 (IL-6) exerts well-established effects on cells of the immune system as well as on various other cell types. It has been implicated in the control of connective tissue cells in such conditions as rheumatoid arthritis and osteoporosis. We have investigated the effects of recombinant human interleukin-6 (rhIL-6) on human osteoblastlike cells derived from explants of trabecular bone. ROS 17/2.8 cells were used as an additional osteoblastlike cell model system. We were unable to identify any effects of rhIL-6 (5-5000 pg/ml) on the proliferation, alkaline phosphatase activity. osteocalcin production, or release of cytokines or prostaglandins by either osteoblastlike cell model system. Since we have shown previously that these cells release IL-6 in culture, we used a sheep anti-human IL-6 antibody to investigate the possibility that (1) action of added exogenous IL-6 could be masking endogenous production, and (2) endogenous IL-6 may regulate the effects of osteotropic agents on the osteoblastlike cells. Presence of the antibody exerted no detectable effects on 1,25-(OH)2D3-stimulated alkaline phosphatase or on proliferation or TNF production enhanced by IL-1. Thus IL-6 does not appear to be involved in the regulation of osteoblast activity. PMID- 1709333 TI - Chicken parathyroid hormone-related protein and its expression during embryologic development. AB - A parathyroid hormone-related protein (PTHrP) is the probable cause of humoral hypercalcemia in malignancy, but its normal physiologic role remains unknown. Since current evidence suggests that PTHrP may have a role in embryonic development, we cloned a genomic fragment that encodes chicken PTHrP (cPTHrP) and studied the expression of PTHrP in developing chick embryos. Blot hybridization of chicken genomic DNA with a cPTHrP genomic DNA probe showed a single band, suggesting that a single-copy gene encodes cPTHrP. By screening a chicken genomic library with the human probe an open reading frame was identified that corresponds to the human PTHrP (hPTHrP) exon IV. Compared to the human sequence the 5' splice junction is highly conserved and the two predicted propeptide residues are identical. The sequence predicts a mature peptide of 139 amino acids; all of the first 21 and 94 of the first 112, but only 8 of the final 27 residues of cPTHrP are identical to the human sequence. The structural features required for PTH receptor binding and activation are highly conserved between chicken and hPTHrP. Poly(A)-enriched RNA from 3-15 day chicken embryos was surveyed by hybridization to the chicken probe. A hybridizing band of 1.45 kb was found in tissues derived from all three germ layers, including brain, heart, lung, liver, gizzard, intestine, chorioallantoic membrane, yolk sac, and skeletal muscle. An additional 1.2 kb hybridizing band was found in some tissues. The conservation of the PTHrP sequence between chicken and mammals supports the view that PTHrP has an important physiologic role.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709334 TI - Chemistry and biology of alpha-fetoprotein. AB - Alpha-Fetoprotein (AFP) is a product of specific fetal tissues and of neoplastic cells of hepatocyte or germ cell origin in adults. This protein belongs to a gene family that is phylogenetically most closely related to serum albumin. Its primary, secondary, and tertiary structural aspects appear similar to the three domain concept proposed for the latter protein. The primary sequence of AFP departs most widely from serum albumin in the first 135 amino acid residues, with about 42% of the remaining 590 residues of the human proteins being identical. Some evidence exists that there are limited sequence differences in the AFP of a given animal species. AFP shows considerable charge heterogeneity that appears to relate mostly to its glycoid moiety. The proteins of some species such as the rat show more pronounced heterogeneities than that of humans. The variations in extent and type of glycosylations are evidenced by differences in the binding to various lectins. These interactions are being extensively explored in attempts to differentiate the sources of the protein produced by various normal and neoplastic cells and may provide valuable diagnostic methods. AFP, like serum albumin, shows relatively strong binding affinities for a variety of ligands. The most notable difference is the strong preferential binding of polyunsaturated fatty acids by AFP. This protein may play a role in transporting these substances to developing and to malignant cells. Various agents affect the synthesis of this protein both by specific fetal tissues and by neoplastic cells. Marked differences in the responses of cells, particularly those of neoplastic types, are indicative of variations in the genetic factors responsible for control of its synthesis. The subject of the genomic repression of the synthesis of AFP seen in fetal life upon maturation of the liver and the reoccurrence of synthesis upon malignant conversion of hepatocytes and of certain germ cells are of particular interest. The regulation of the closely related AFP and albumin genes is providing a powerful and attractive model to examine molecular events in the activation and inactivation of specific genes during development and in oncogenic processes. Extensive measurements of AFP during pregnancy and in the course of neoplasias, notably hepatoma, are being made to aid in following changes in such developments. Various specific physiological roles for this protein are also being proposed. One of these is its possible action in the regulation of immune processes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709336 TI - Radiation therapy for adjunctive treatment of adrenal cortical carcinoma. AB - Adrenocortical carcinoma is a rare disease which is primarily approached surgically. There have been few reports of the efficacy of radiation therapy and, for the most part, these have been anecdotal. This paper reports on the potential adjuvant role of radiation therapy after surgical excision of primary adrenal cortical carcinoma and also comments about the efficacy of palliative radiation therapy for metastases. We have identified eight patients treated for adrenal cortical carcinomas at Hahnemann University Hospital (HUH) from 1962 until the present and have also identified five patients with the same diagnosis at Philadelphia General Hospital (PGH) from 1962 until its close in 1975. These two groups are examined separately. In the PGH group, in which two patients were diagnosed at autopsy and only one patient was treated by radiation therapy, the median survival was between 0 and 1 month for Stage IV disease with the only patient surviving to 6 months being that patient receiving radiation therapy. In the HUH group, five of eight patients were treated adjunctively after diagnosis, one was not and two received palliative therapy. The median survival for treated Stage III patients was between 34 months and 7 years. The suggestion, based on a limited patient series, is that patients treated postoperatively to the tumor bed and nodal areas in Stage III disease may have improved survival over historic series and improved local control. PMID- 1709335 TI - [Soft tissue swelling in sports traumatology. Comparative study of the effectiveness of 2 ointments]. AB - An open randomized study design was used to compare the soft-tissue swelling in patients with fibular capsule injuries under therapy with Chomelanum (preparation A), Lasonil (preparation B) and ointment base of Chomelanum (preparation C). 90 patients were selected for participation in the study, and the results of 85 patients were available for evaluation. Concerning the initial condition there was no significant difference between the three therapy groups of the study. Assessment criteria were the difference of circumference in plane of ankle joint (injured/uninjured side) and the difference of soft-tissue above lateral malleoulus in sonography. The study showed that preparation A had a significantly larger influence on soft tissue detumescence than preparation B- and B larger than C. In observation period all patients of group B and C still showed soft tissue swelling. Between third and fourth day of treatment half of patients in group A did not need any treatment because of complete detumescence. In all assessment criteria significant differences in favor of preparation A were observed. PMID- 1709337 TI - The value of pancreatic pseudocyst amylase concentration in the detection of pseudocyst communication with the pancreatic duct. AB - The aims of the study were to compare the results of endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous cystopancreatography (PCP) in the detection of the communication between the pancreatic pseudocyst and the pancreatic duct, and to assess the reliability of the increased amylase concentration in the pseudocyst content as an indicator of the existence of communication between the pancreatic pseudocyst and the pancreatic duct system. Forty-three patients were included in the study. Pseudocystic fluid content was obtained by percutaneous aspiration. Twenty-four patients had pseudocyst amylase concentrations above 64 Wolgemuth units (WU), and 19 patients had less than 64 WU. The communication between pseudocyst and the pancreatic duct was determined in 22 patients by ERCP and in 13 patients by PCP, all in the group with increased pseudocyst amylase concentration. Due to high sensitivity and specificity of pseudocyst amylase concentration for the existence of pseudocyst communication with the pancreatic duct, we conclude that guided percutaneous aspiration of the pancreatic pseudocyst with the determination of amylase concentration in the fluid can replace ERCP as a method of choice for the detection of pseudocyst communication with the pancreatic duct. PMID- 1709339 TI - Collagenous spherulosis in chondroid syringomas. AB - We report a case of collagenous spherulosis (CS) incidentally found in a chondroid syringoma of the facial skin. The lesion was studied by routine light microscopy, special stains, immunohistochemical methods, x-ray spectrophotometry, and electron microscopy. Light microscopy revealed solitary and confluent eosinophilic globules with radiating fibrillary structures within and around the lumina of the tubuloglandular components of chondroid syringoma. The fibrillary structures stained strongly for collagen and reticulin and less intensely for acidic mucopolysaccharides. Immunohistochemically, the globules were focally positive only for collagen type IV. Electron microscopy revealed radiating collagen fibers surrounded by basal lamina-like material. No inorganic crystals were identified by x-ray spectrophotometry. We conclude that (a) CS is not specific to breast but also occurs in chondroid syringomas; (b) the term collagenous spherulosis is appropriate because collagen fibers were demonstrated histochemically and ultrastructurally in the spherules; (c) CS appears to be associated with tubular epithelial structures; (d) there was no immunohistochemical evidence of myoepithelial differentiation. The etiology and significance of CS remain obscure. PMID- 1709338 TI - Failure of vasoldilator infusion to alter pulmonary diffusing capacity in systemic sclerosis. AB - PURPOSE: Patients with systemic sclerosis (SSc) do not exhibit a normal increase in the diffusing capacity for carbon monoxide (DLCO) on assuming the supine position. We sought to determine whether a potent prostacyclin derivative and vasodilator, iloprost, would reverse this defect. PATIENTS AND METHODS: Fourteen patients with SSc were enrolled in a randomized, double-blind, placebo-controlled study of iloprost. Patients were tested before and during 3 days of iloprost or placebo infusion with both upright and supine pulmonary function studies. RESULTS: The results of baseline pulmonary function studies including DLCO were not significantly altered by iloprost. Furthermore, iloprost did not alter the abnormal postural DLCO response. CONCLUSION: These results suggest that the pulmonary vascular defects seen in this group of patients are not a consequence of reversible pulmonary vasospasm. PMID- 1709340 TI - Dysplastic melanocytic nevus. Immunohistochemical heterogeneity by melanosomal markers and S-100. AB - This study evaluates the expression of human melanosome specific antigen-1 and -2 (HMSA-1, -2) by dysplastic melanocytic nevus (DMN) and its significance by (a) immunostaining pattern, (b) other immunohistochemical markers (e.g., S-100), and (c) mesenchymal responses (e.g., lymphocytic infiltration). From 31 patients with DMN syndrome, 55 clinically characteristic and histologically (H&E) proven dysplastic nevi were assessed for HMSA expression. Approximately 70% of DMN lesions expressed the HMSA antigens; similar gross staining patterns were seen with both monoclonal antibodies (MoAbs). The expression of HMSA-2 was invariably associated with that of HMSA-1, but the converse did not apply. Two general staining patterns with MoAbs HMSA were appreciated: (a) uniform staining of the epidermal melanocytes in the DMN lesion (type I) and (b) nonuniform staining, with the epidermal melanocytes at the periphery of the DMN lesion reacting most intensely (type II). Although anti-S-100 antibody demonstrated a similar sensitivity (67%), it lacked specificity and did not distinguish different types of DMN, as compared with MoAbs HMSA. There was no correlation between these observed staining patterns and common histological features of DMN. There was a poor correlation between lymphocytic infiltrate (as judged by immunostaining for pan-T, Ti/h, Tc/s, and pan-B markers) and heterogeneity of immunostaining. We conclude that HMSA-1 and -2 are able to delineate two types of DMN and possibly bind different epitopes of the same antigen. Their potential usefulness in predicting malignant transformation of DMN has yet to be established. PMID- 1709341 TI - A comparative immunohistochemical study of adenoid cystic carcinoma of the skin and salivary glands. AB - We performed an immunohistochemical study that compared a primary adenoid cystic carcinoma (ACC) of the skin with two salivary gland ACC. All three tumors stained positively and in identical fashion for epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), broad-spectrum keratins, and low-molecular-weight keratins. Both EMA and CEA were localized to the luminal surfaces and the secreted contents of the tubular structures and the ductlike structures of the cribriform formations. The staining reactions for both types of keratin were more intense in the cells lining the tubular structures and the ductlike structures of the cribriform formations. One of the two salivary ACCs stained positively for S 100 protein; the other was positive for vimentin. The cutaneous ACC was negative for both antigens. Leu-7 antigen was not detected in either type of ACC. These results show that primary cutaneous ACC and salivary ACC have similar immunohistochemical staining patterns for a number of antigens. We believe this similarity is due to the fact that these antigens are shared by the sweat glands and salivary glands, which are considered to be the respective sites of origin for these two types of tumors. PMID- 1709342 TI - [A prognostic study of cryptogenic infantile spasms]. AB - The prognosis of infantile spasms is grim when a detectable brain lesion is present. In contrast, cryptogenic infantile spasms, in which there is no identifiable brain lesion, usually run a favorable course under treatment. Few studies have focused on the outcome in children with cryptogenic infantile spasms. Among 111 pediatric patients with a history of infantile spasms hospitalized over 18 consecutive years, 23 (21%) were given a diagnosis of cryptogenic infantile spasms. Follow-up ranged from 4 to 21 years. Outcome was as follows: the IQ was above 80 in 39% of cases and above 100 in 13% of cases; 42% of patients of school age were attending school but half of these had learning disabilities; 30% of patients had severe psychiatric disorders, and 22% had developmental delay and severe epilepsia. Early factors apparently associated with a good prognosis included the mild nature of psychomotor regression, persistence of spindles on EEGs recorded during NREM sleep, and prompt improvement of clinical status and EEG recordings under treatment. Conversely, severe regression, focalized EEG anomalies, failure of development to resume promptly after initiation of therapy, recurrence of spasms and hypsarrhythmia at discontinuation of treatment, and onset before 5 months or after 11 years of age were associated with a poor prognosis. PMID- 1709343 TI - Alteration of drug biotransformation by interferon and host defence mechanism. AB - It has been demonstrated that the liver loses its capacity to metabolise and eliminate drugs during viral infection or during the operation of host defence mechanisms. This loss in drug metabolism is due to the loss of the cytochrome P 450 component of the mixed function oxidase (the enzyme system primarily responsible for the oxidation of drugs, carcinogens and certain classes of endogenous substances such as steroids, fatty acids and prostaglandins). At present we have identified interferon and factors such as interleukin-1, interleukin-6 and tumour necrosis factor, released from Kupffer cells, as major mediators of the loss. The depression that occurs during viral infection is mediated via the production of interferon. This action of interferon requires the synthesis of an intermediate/s yet to be identified. The molecular mechanism for the decrease in cytochrome P-450 mediated drug metabolism during episodes of viral infections is caused by an interferon-mediated loss in mRNA and subsequent cytochrome P-450 synthesis in the liver. PMID- 1709344 TI - The pharmacology of immunosuppression. AB - In this paper, we review the pharmacological techniques by which graft rejection can be prevented from the early development of azathioprine to the current controversies surrounding FK 506. The basic chemistry of each drug is outlined, its source, mode of action, current uses and the clinical management are briefly documented. In addition to the more usual immunosuppressive agents, the current state of art of using monoclonal antibodies as immunosuppressives is addressed. PMID- 1709345 TI - Cyclosporine inhibition of the chloride/bicarbonate exchanger of proximal renal tubules. AB - It has previously been reported by us that the immunosuppressant agent cyclosporine inhibits the ability of renal proximal tubule cells to regulate volume. Proximal renal tubule cells exposed to hypotonic solutions rapidly swell and subsequently shrink. Their regulatory cell volume decrease (RVD) is due to Ca2(+)-calmodulin regulated KCl efflux followed by osmotically obligated water. In proximal renal tubule cells, potassium leaves the cell through a Ca2(+) activated potassium channel. While it is unknown how the volume regulatory chloride efflux occurs in humans, in teleosts its efflux is through a Cl-/HCO3- anion antiporter. Prior to this work it was known that cyclosporine (CsA) inhibits volume regulation in mammals and teleosts. It was not clear, however, if CsA inhibited the potassium channel, the chloride efflux, or both. In vivo, CsA is a potent calmodulin inhibitor whose effects on cell volume regulation are not mediated by decreased cell water permeability. The effects of the Cl-/OH- antiporter tributyltin (TBT) and the K+ channel gramicidin on CsA-associated RVD inhibition were studied in isolated proximal renal tubules of the teleost Carassius auratus (goldfish). It was found that the inhibitory effect of CsA (50 microM) could be overridden by the administration of TBT (1 microM) but could not be modified by the potassium ionophore gramicidin (0.5 microM). The inhibitory effect of CsA could not be altered by increased Ca2+ influx through the Ca2+ ionophore A23817 (10 microM), and, therefore, an altered calcium activation of KCl efflux does not appear to be involved. In conclusion, the CsA effect on RVD is due to a selective inhibition of chloride efflux most likely mediated by inhibition of the cyclophilin-calmodulin system and not by a decreased intracellular calcium signal. PMID- 1709346 TI - [Non-surgical treatment of cysts and pseudocysts of the pancreas. A study of a series of 33 cases]. AB - Pancreatic cysts and pseudocysts had to be treated by surgery until 15 years ago. Nowadays they can be aspirated (or drained) either endoscopically or by ultrasonic and computed tomographic guided punctures. The aim of this study was to see if these non-surgical treatments were efficient enough among the actual treatments. From 1984 to 1988, 33 patients were admitted in one single institution with a pancreatic cyst or pseudocyst, 22 of which were a consequence on an acute pancreatitis and 11 complicated a chronic pancreatitis. Ten cysts were connected with the Wirsung channel while 11 others were not, but the ERCP failed to give any accurate information on this point in 12 cases. As a first treatment, we abstained of any invasive procedure in 9 cases, 18 were treated by percutaneous aspiration guided by ultrasound and 6 patients underwent surgery. The mean follow-up was 30 months. All the patients who had no treatment remained symptom free; their cysts improved in 5 cases and disappeared in 4 cases. Among the cysts treated by percutaneous aspiration, 4 had to be operated, 5 disappeared, 3 improved and 6 recurred; the percutaneous aspiration obtained a lasting symptoms' relief for these patients, whatever the connection the cyst had with the Wirsung channel. This study suggests that percutaneous aspiration or drainage guided by ultrasound may be a treatment of the highest quality for acute and chronic pancreatitis cystic formations. However, it seems to act upon the symptoms rather than upon the cystic formations themselves. PMID- 1709347 TI - [Tumor markers--personal experience. The use of tumor markers for cancer of digestive organs]. AB - This review is concerned mainly with our experience in the use of tumor markers for cancer of digestive organs from study of tumor markers by the author over the past 20 years. Development of a radioimmunoassay for highly sensitive detection of alpha-fetoprotein (AFP) by Ishii et al. in 1971 enhanced the usefulness of screening for early hepatocellular carcinoma (HCC) occurring in the course of liver cirrhosis. PIVKA-II, reported as a highly specific tumor marker for HCC, was thought to be less available for detection of early HCC occurring in the course of liver cirrhosis in comparison with AFP. Carcinoembryonic antigen (CEA), a most popular and useful tumor marker for cancer of digestive organs, was frequently positive in sera of colorectal cancer patients who had no subjective complaint or physical sign. This experience supported employment of CEA as a routine screening test for colorectal cancer. A survey of routine examinations of serum CA 19-9 for a period of one month in the clinical laboratory of our hospital proved that 92% of the positive cases of low-level CA 19-9 from 37 U/ml to 75 U/ml were noncancerous. This result indicated that the cut-off value of 37 U/ml employed for serum CA 19-9, which had been evaluated as a specific and highly sensitive tumor marker for pancreatic cancer and bile duct cancer, was too low. Accordingly, it was thought necessary to investigate a change of cut off value and reevaluate CA 19-9 for pancreatic cancer and bile duct cancer in comparison with other tumor markers of carbohydrate antigen such as CA 50, sialyl SSEA-1. From our experience in the use of tumor markers, the combination assays of fetal protein such as AFP, CEA, basic fetoprotein (BFP) and carbohydrate antigen, such as CA 19-9 and CA 50, for routine examination of tumor marker, are recommended for effective screening of cancer of digestive organs. PMID- 1709348 TI - [New interesting chemotherapeutic regimens in advanced gastric cancer]. AB - We have made over view of new chemotherapeutic regimen for treatment of advanced gastric cancer 5-FU + MMC, FT + MMC and UFT + MMC therapy have been used widely for treatment of advanced gastric cancer as chemotherapeutic regimens in Japan. These regimens did not shown made than 25% in response rate as antitumor effect. Since development of CDDP, FP (5-FU + CDDP), FAP (5-FU + ADM + CDDP) and EAP (Etoposide + ADM + CDDP) is becoming gradually very important regimen for treatment of advanced stomach cancer patients. Recently, we have studied EAP therapy on 50 cases of advanced gastric cancer from January 1988 to September 1989. ADM 20 mg/m2, CDDP 40 mg/m2 and Etoposide 100 mg/m2 were administered on day 1 and 7, 2 and 8, and 4, 5 and 6, respectively, with not less than 2 courses every 3 to 4 weeks. The rate of effectiveness were obtained 43.8% with a confidence interval 95% of 30-58%. Median survival time was only 5.1 months for EAP therapy, which was highly effective but led to no prolonged survival period. Thus it is thought that good control of leukopenia, a dose-limiting factor remains to be examined. Biochemical modulation of 5-FU using such as MTX + LV and CDDP + LV (leucovorin) now under studying in the nation wide in Japan, so far it is getting better results. PMID- 1709349 TI - [Morphological changes in the pleura caused by intracavitary administration of bleomycin]. AB - We administered bleomycin to the intrapleural cavity of hamsters and observed acute and chronic histological changes of visceral pleura using light and electron microscopies. At 7 days after bleomycin challenge, we could find out the pleural thickening with infiltration of inflammatory cells, and then formation of pleural fibrosis at 28 days. However, in our study there was no abnormal findings of the lung parenchyma in morphology and intraalveolar cells, and lung distensibility. The dose (10-20 units/kg body weight) used in the study did not cause the abnormal changes in the parenchyma, although pleural thickening occurred. We suggest that intracavitary bleomycin administration is safely used for the treatment of malignant pleural effusion. PMID- 1709350 TI - [Partial parietal cystectomy and cystoplasty with a lyophilized human dura mater patch as and alternative in palliative oncological bladder surgery]. AB - Fifteen patients with infiltrating bladder carcinoma underwent partial cystectomy and cystoplasty with lyophilized human dura at our hospital from 1983 to 1988. The 5-year survival rate was 75% for stage B1, 60% for B2, 40% for C and 0% for D1. There were no intra- or post-operative deaths and the post-operative complications were few. This procedure may be useful in selected patients with bladder tumors who cannot be submitted to more radical procedures due to a coexisting pathological condition representing a high surgical risk or in elderly patients. PMID- 1709351 TI - [Role of macrophages in interferon production]. AB - The role of macrophages in production of interferons (IF) is manysided. They are able to synthesize IFs after any induction. However, the function of macrophages as producers of IFs is not, probably, basic. The levels of IFs produced by them are mainly low. When they are stimulated by inducers of "early" IF the synthesis is performed by intact macrophages whereas with the use of inducers of "late" IF there is always observed joint activity of macrophages and other immunocompetent cells. The main role of macrophages in production of IFs is in regulation of synthesis of these proteins in the host. In addition, they are able to serve as stimulating cells in inducing IF production by the majority of drugs by transmitting information on IF synthesis to lymphocytes. This function of macrophages is not species-specific. PMID- 1709352 TI - Half body irradiation for the palliation of bone metastases. AB - Half body irradiation using single large doses of photons has been reported as an effective modality for the palliation of symptoms due to widespread metastatic bone malignancy. Over a 7 year period (1982-1988) sixteen patients with disseminated malignancy were given half body irradiation at Auckland Hospital. Treatment consisted of a single dose of radiation of between 5 and 8 Gray. Either 6 or 8 MV photon beams were used. Twelve patients received treatment to the lower half body, three patients to the upper half body and one patient to both upper and lower half body. Significant pain relief occurred in fifteen patients and two patients experienced improvement with hypercalcaemia. All patients tolerated the treatment well and toxicity was minimal. PMID- 1709353 TI - Object recognition memory in the rat: the role of the hippocampus. AB - Object recognition memory of rats with fimbria-fornix or ventral temporal lesions was evaluated with a behavioral protocol (delayed non-matching-to-sample task with trial-unique stimuli) similar to that used to test recognition functions in primates. Animals with damage to the hippocampal system showed no evidence of lasting impairment on the object recognition task with retention intervals up to 30 s. In contrast, rats with fimbria-fornix lesions displayed severe and enduring deficits on a test of spatial memory, i.e. rewarded alternation, with but 5 s delays. These results provide further evidence that a dissociation exists between the types of memory that are and are not lost following damage to the hippocampus. Whereas the hippocampus is necessary for some types of mnemonic processes, other types of recognition functions (e.g. perceptual recognition) may be fully mediated in regions of sensory and/or association neocortex without the involvement of the hippocampus. PMID- 1709354 TI - Conservative treatment of Bell's palsy with steroids and dextran-pentoxiphylline combined therapy. AB - In 1980, Stennert proposed for the treatment of Bell's palsy an infusion therapy consisting of initially high dosages of steroids in combination with low molecular dextran and pentoxiphylline. Excellent results were reported as a consequence of administering this treatment scheme. This "steroid-dextran" medication was modified (SD therapy) and administered in 172 cases of Bell's palsy. The results were compared with those of a group of 59 patients who had been treated with orally administered low-dose steroids in combination with vasodilators, adenosine triphosphate and vitamins. All patients with incomplete palsies recovered completely, regardless of the mode of treatment. In cases of complete palsy, 87% of patients recovered completely when treated with SD therapy. In contrast, 68% of the patients treated with orally administered steroids recovered completely. PMID- 1709355 TI - Prostate specific antigen. A review. AB - Despite its identification in the 1970s, prostate specific antigen (PSA) has only recently gained widespread utility. In this review, we will describe significant aspects of the biochemistry of PSA, immunohistochemical applications, the effect of prostatic manipulation on the serum level of this analyte, and other considerations in the determination of the level of PSA. In addition, the effect of benign prostatic hyperplasia (BPH) on serum PSA will be described. Finally, the application of PSA in the detection, staging and monitoring of patients with prostatic carcinoma will be discussed. PMID- 1709356 TI - Is acid phosphatase (PAP) still justified in the management of prostatic cancer? AB - The usefulness of acid phosphatase (PAP) and prostate specific antigen (PSA) is compared. The author concludes that PSA is more sensitive, has better organ specificity, does less diurnal variations and correlates better with tumor which makes PSA superior for staging and monitoring of therapy in prostate cancer. PMID- 1709357 TI - Digital rectal examination versus transrectal ultrasound in detection of prostate cancer. Preliminary results from a study of a randomly selected population. AB - In a study of 2,400 randomly selected men (age 55-70 years) for early detection of prostate cancer the authors have compared the diagnostic value of digital rectal examination (DRE), transrectal ultrasound (TRUS) and prostate specific antigen (PSA). Altogether 62 prostate cancers were detected, corresponding to a detection rate of 3.5% but by use of DRE the detection rate was only 2.3%. The study showed that TRUS added significantly to the detection rate. If radical surgery is restricted to stages T1 and T2A, the combined use of DRE and TRUS detected twice as many cases fit for this treatment than DRE alone. The authors advocate randomized studies for evaluation of early radical treatment of prostate cancer. Before results of such studies have appeared they recommend methodological studies aimed at development and enhancement of the accuracy of the diagnostic tools. PMID- 1709358 TI - [Clinical care of surgically ill children in the German Federal Republic. Status quo and requirements for the future]. PMID- 1709359 TI - A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals. AB - We have identified a genetic polymorphism of factor VII that is strongly associated with plasma factor VII coagulant activity (factor VIIc) in healthy individuals from the United Kingdom. This polymorphism was detected after Msp I digestion of polymerase chain reaction-amplified genomic DNA. In a sample of 284 men, the frequency of the M2 allele (loss of cutting site) is 0.1, and individuals with the M1M2 genotype have factor VIIc levels 22% below the sample mean (p less than 0.0001). Msp I genotype was found to be the strongest predictor of factor VIIc, accounting for 20.2% of the variance, with cholesterol accounting for an additional 3.5%. The base change that gives rise to the Msp I polymorphism is a G-to-A substitution in the codon for amino acid 353, leading to replacement of arginine (Arg) with glutamine (Gln) in the protein product of the M2 allele (designated Gln 353). Three individuals homozygous for the M2 allele have both low factor VIIc and low factor VII protein concentrations. The conformation of the Gln 353 molecule may be different from that of the Arg 353 protein, affecting its intracellular processing, secretion, turnover in plasma, or activity. In view of its association with lower factor VIIc levels, possession of the M2 allele may confer protection against thrombosis and myocardial infarction. PMID- 1709360 TI - Inhibition of platelet retention on artificial microvascular grafts with monoclonal antibodies and a high-affinity peptide directed against platelet membrane glycoproteins. AB - Rapid occlusion of artificial microvascular grafts (AMGs; less than or equal to 2 mm diameter) by platelet-rich thrombi prevents the clinical use of AMGs in cardiac, vascular, and plastic surgery. Since present antiplatelet agents are unable to assure AMG patency, we have studied the role of specific platelet membrane-glycoprotein blockade on platelet retention by AMGs. In a customized in vitro perfusion chamber, retention on polytetrafluoroethylene (PTFE) AMGs (1.0-mm i.d.) of indium-111-labeled platelets in human whole blood was measured in the presence and absence of inhibitors. Specific blockade of platelet membrane glycoprotein (Gp) IIb/IIIa was achieved using monoclonal antibody 10E5 (10 micrograms/ml) and the peptide GRGDS (Gly-Arg-Gly-Asp-Ser, 0.75 mM). These inhibited 98% and 35%, respectively, of platelet retention under circumstances in which aspirin (7 mM) and dextran (4 mg/ml) inhibited 19% and 18%, respectively, of platelet retention. Nonspecific immunoglobulin G F(ab')2 (10 micrograms/ml) and nonspecific peptide (GGDA; Gly-Gly-Asp-Ala, 0.75 mM), used as control reagents, were ineffective in this setting. Monoclonal antibody 6D1 (10 micrograms/ml), which blocks platelet membrane GpIb, prevented 82% of platelet retention on PTFE. These doses of 10E5 and GRGDS completely inhibited platelet aggregation in response to 20 microM ADP, and the dose of 6D1 completely inhibited ristocetin-induced platelet agglutination. The aspirin dose prevented the second phase of ADP-induced aggregation. These data indicate that not only does initial platelet adhesion to PTFE require GpIIb/IIIa but also that GpIb plays a significant role in the early stages of platelet retention on PTFE AMGs. PMID- 1709361 TI - 2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer. AB - Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3 phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there. PMID- 1709362 TI - Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1. AB - We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E. coli F1F0 ATPase is increased significantly by interactions with F1 subunits [Pati, S., & Brusilow, W.S.A. (1989) J. Biol. Chem 264, 2640-2644]. To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes. The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations. Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits. PMID- 1709363 TI - Proton and nitrogen sequential assignments and secondary structure determination of the human FK506 and rapamycin binding protein. AB - Sequential 1H and 15N assignments of human FKBP, a cytosolic binding protein for the immunosuppressive agents FK506 and rapamycin, are reported. A combination of homonuclear and relayed heteronuclear experiments has enabled assignment of 98 of 99 backbone amide NHs, 119 of 120 C alpha Hs, 97 of 99 non-proline amide 15Ns, and 375 of 412 side-chain resonances of this 107-residue protein. Long-range NOEs are used to demonstrate that FKBP has a novel folding topology consisting of a five-stranded antiparallel beta sheet with +3, +1, -3, +1 loop connectivity. PMID- 1709364 TI - [Molecular epidemiology of Legionella pneumophila]. PMID- 1709365 TI - Low MSAFP and new biochemical markers for Down syndrome: implications for genetic counselors. PMID- 1709366 TI - Assessment of routine amniocentesis for unexplained maternal serum alpha fetoprotein elevations. PMID- 1709367 TI - Development of effective salvage treatment programs for Hodgkin's disease: an ongoing clinical challenge. PMID- 1709368 TI - Combined therapy with recombinant granulocyte colony-stimulating factor and erythropoietin decreases hematologic toxicity from zidovudine. AB - Twenty-two patients with acquired immunodeficiency syndrome (AIDS) or severe AIDS related complex and multilineage hematopoietic defects were treated with recombinant granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) in a phase I/II trial. All patients were neutropenic and anemic after withdrawal of all bone marrow-suppressive drugs. Daily, G-CSF was subcutaneously self-administered until an absolute neutrophil count (ANC) greater than 6,000/microL was achieved and maintained for 2 weeks. Subcutaneous EPO was added to the regimen and the dose increased until an increase of 15 g/L of hemoglobin was observed. Groups of patients were administered increasing doses of zidovudine to determine their tolerance. G-CSF and EPO therapy was continued with dose modification to maintain an ANC greater than 1,500/microL and hemoglobin greater than 100 g/L. The dose of zidovudine was not altered. All 22 patients responded to G-CSF with a mean 10-fold increase in neutrophils occurring in less than 2 weeks. Significant increases in CD4 and CD8 cell number, lymphocyte proliferative response, and bone marrow cellularity were seen. EPO therapy increased hemoglobin in all 20 evaluable patients within 8 weeks. Sixteen patients received 1,000 mg and four patients received 1,500 mg of zidovudine per day. The reinstitution of zidovudine resulted in a decline in reticulocytes and hemoglobin and the reappearance of transfusion requirements in eight of the 20 patients, six of whom had the study medications stopped. No patient had the study medications stopped because of neutropenia or thrombocytopenia. Toxicities were mild and did not require dose modifications. Limiting dilution plasma and lymphocyte co-cultures for HIV as well as serum p24 antigen levels did not change significantly during G CSF or combined G-CSF and EPO therapy. HIV p24 antigen decreased significantly with zidovudine therapy. Opportunistic infections occurred in 14 patients but were successfully treated with myelosuppressive antimicrobial agents, including ganciclovir, without the development of neutropenia. These results suggest that combined therapy with G-CSF and EPO may improve the neutropenia and anemia of AIDS. Combined therapy may allow the resumption of full-dose zidovudine in most patients intolerant of the hematologic effects of zidovudine without apparent alteration of HIV expression or the efficacy of zidovudine. PMID- 1709369 TI - Purification and partial characterization of a human hematopoietic precursor population. AB - This study reports the development of an assay, the Pre-colony-forming unit (CFU) assay, which detects human hematopoietic precursors. The Pre-CFU assay is based on the observation that precursors to CFU-granulocyte-macrophage (CFU-GM) that are undetectable in clonogenic assays differentiate into CFU-GM preferentially following treatment in suspension culture with recombinant human interleukin-1 alpha (rhIL-1 alpha) combined with rhIL-3. Using the Pre-CFU assay, hematopoietic precursors were detected in human bone marrow depleted of CFU-GM progenitors and differentiated hematopoietic elements via 4-hydroperoxycyclophosphamide treatment coupled with selection for CD34+ cells (4-HCresistant/CD34+ marrow). Additionally, the Pre-CFU assay detected recovery of hematopoiesis substantially earlier than the CFU-GM assay in primates following myeloablation with 5 fluorouracil. The Pre-CFU assay was used to asses purification of a phenotypically defined hematopoietic precursor population, the lin-CD34+ population. The lin-CD34+ population lacks detectable surface markers for T-cell, B-cell, natural killer cell, and myeloid lineage, possesses the CD34 antigen, is devoid of CFU-GM progenitors, and yields Pre-CFU assay values comparable with 4 HCresistant/CD34+ marrow. Using a combination of phenotypic analysis and Pre-CFU assay analysis, the action of rhIL-1 alpha plus rhIL-3 treatment on lin-CD34+ cells was further characterized. The data indicate that rhIL-1 alpha plus rhIL-3 treatment induces proliferation and differentiation of early hematopoietic precursors into progenitors and terminally differentiated cells, without inducing a significant expansion of the precursor population itself. PMID- 1709370 TI - Bone modulation in sustained hematopoietic stimulation in mice. AB - To understand the etiology of bone modulation and hypercalcemia observed in granulocytosis of a tumor-bearing animal model and to gain insight into the implication of sustained hematopoietic stimulation on the bone tissue, in vivo responses of normal mouse hematopoietic and bone tissues to long-term injections of recombinant human and murine granulocyte colony-stimulating factor (G-CSF), murine granulocyte-macrophage CSF (GM-CSF), and human erythropoietin were quantitatively analyzed. Osteoclast activation was estimated by the osteoclast endosteal ratio, determined by morphometric analyses of femoral sections. Medullary and bone areas were measured on transverse ground bone sections of the tibia. Recombinant murine G-CSF provoked marked granulocytosis associated with significant increases in the number of marrow granulocytes and their progenitors, and caused expansion of granulopoietic marrow into fatty marrow. The bone of G CSF-treated mice showed a significant increase in endosteal osteoclast numbers with medullary area enlargement and a reduction in the bone thickness; indicative of endosteal bone resorption. Although GM-CSF had little effect on granulopoiesis, it caused peritoneal macrophages to increase and induced similar bone changes as those observed in G-CSF treatment. Enhanced erythropoiesis stimulated by erythropoietin was also associated with evidence of endosteal bone resorption. Bone changes induced by these growth factors were not associated with hypercalcemia. These animal studies document association of bone modulation in sustained stimulation of hematopoiesis, and implicate important physiologic effects of hematopoietic growth factors on skeletal tissue in vivo. PMID- 1709371 TI - Effect of murine mast cell growth factor (c-kit proto-oncogene ligand) on colony formation by human marrow hematopoietic progenitor cells. AB - Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte-macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells. PMID- 1709372 TI - Myeloid cell kinetics in mice treated with recombinant interleukin-3, granulocyte colony-stimulating factor (CSF), or granulocyte-macrophage CSF in vivo. AB - Myeloid cell kinetics in mice treated with pure hematopoietic growth factors have been investigated using tritiated thymidine labeling and autoradiography. Mice were injected subcutaneously with 125 micrograms/kg granulocyte colony stimulating factor (G-CSF) (in some cases 5 micrograms/kg), or 10 micrograms/kg of granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3) every 12 hours for 84 hours. 3HTdR labeling was performed in vivo after 3 days of treatment. G CSF increased the peripheral neutrophil count 14-fold and increased the proportion and proliferation rate of neutrophilic cells in the marrow, suppressing erythropoiesis at the same time. Newly produced mature cells were released into the circulation within 24 hours of labeling, compared with a normal appearance time of about 96 hours. By contrast, GM-CSF and IL-3 had little effect on either marrow cell kinetics or on the rate of release of mature cells, although GM-CSF did stimulate a 50% increase in peripheral neutrophils. Monocyte production was also increased about eightfold by G-CSF and 1.5-fold by GM-CSF, but their peak release was only slightly accelerated. While the peripheral half lives of the neutrophilic granulocytes were normal, those of the monocytes were dramatically reduced, perhaps due to sequestration in the tissues for functional purposes. The stimulated monocyte production in the case of G-CSF required an additional five cell cycles, a level that might have repercussions on the progenitor compartments. PMID- 1709373 TI - The sequence gamma-(312-324) is a fibrin-specific epitope. AB - Fibrin accelerates the activation of plasminogen catalyzed by tissue-type plasminogen activator much stronger than fibrinogen. Detailed studies showed that (part of) this rate-enhancing effect of fibrin is brought about by two sites in the fibrin molecule: one in A alpha-(148-160) and one in the gamma-chain stretch 311-379 (also known as FCB-5). During the fibrinogen-to-fibrin conversion, A alpha-(148-160) appears to become accessible, because a monoclonal antibody against synthetic A alpha-(148-160) reacts with fibrin, but not with fibrinogen. Because a similar situation may exist for (at least parts of) FCB-5, we have prepared a monoclonal antibody against a part (ie, gamma-[312-324]) of FCB-5, and found that this is fibrin-specific and does not bind fibrinogen. We conclude that gamma-(312-324) is hidden in fibrinogen and is exposed by the formation of fibrin. PMID- 1709374 TI - Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies. AB - Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22. PMID- 1709375 TI - Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix. AB - Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to "cell adhesion sites" of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400-411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). "Hybrid" peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys-Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive. PMID- 1709376 TI - Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura. AB - Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to autoantibodies against platelets that result in their destruction. In some patients, these autoantibodies bind to platelet glycoprotein (GP) IIIa. With the aim of better defining the antigenic epitopes, plasma from 13 selected patients with chronic ITP known to have anti-GPIIb/IIIa autoantibodies was tested for reactivity with nine synthetic peptides corresponding to different regions of the GP IIIa molecule. Of these plasmas, five bound significantly (P less than .001) with either peptide 8 (amino acids 721-744) or peptide 9 (amino acids 742-762), which together form most of the carboxyterminal region presumed to be the cytoplasmic domain. Three of these positive plasmas, were tested further. In two of these positive plasmas, the anti-peptide antibodies represented greater than 80% of the detectable circulating autoantibody. To further evaluate the importance of the carboxyterminal region as an antigenic site, the chronic ITP plasmas were tested against Chinese hamster ovary cells transfected with GPIIb and either whole GPIIIa or GPIIIa lacking amino acids 728 to 762. Ten of the 13 plasmas required the presence of this region for significant autoantibody binding. We conclude that the carboxyterminal region is an important area for stimulating antiplatelet autoantibody formation in some patients with chronic ITP. It is not known whether these autoantibodies to the presumed cytoplasmic domain play an important role in the pathogenesis of the disease or occur as a secondary phenomenon during the course of platelet destruction. PMID- 1709377 TI - Granulocyte colony-stimulating factor in cerebrospinal fluid from patients with meningitis. AB - Granulocyte colony-stimulating factor (G-CSF) in the cerebrospinal fluid from patients with meningitis was measured by our modified enzyme-linked immunosorbent assay for G-CSF. The minimal detection level was 20 pg/mL G-CSF. In patients with bacterial meningitis, the G-CSF levels in the cerebrospinal fluid were extremely elevated, showing a mean value of approximately 1,500 pg/mL. On the other hand, G CSF levels in the cerebrospinal fluid from 67% patients with aseptic meningitis were moderately increased, showing a mean value of about 80 pg/mL, whereas G-CSF levels in 33% samples remained undetectable. The G-CSF levels and neutrophil counts in the cerebrospinal fluid were proven to be related by Spearman's rank correlation coefficient analysis (r = .724). These elevations of G-CSF levels at inflammation sites associated with bacterial meningitis may indicate that G-CSF plays an important role in the combat of bacterial infections. PMID- 1709378 TI - A soluble factor released by CD8+CD57+ lymphocytes from bone marrow transplanted patients inhibits cell-mediated cytolysis. AB - We report a new inhibitory activity of CD8+CD57+ peripheral blood lymphocytes from allo-bone marrow transplant patients. Positively selected CD8+CD57+ lymphocytes act as potent inhibitors of allospecific cytolytic T lymphocyte and lymphokine activated killer cell cytolytic activities. These suppressor cells are mature T cells expressing the CD2, 5, 7, CD3-TcR alpha beta complex, and lack natural killer cell markers as well as cytolytic function. Their inhibitory activity on both cytolytic processes and T-cell proliferation is mediated by a non-antigen-specific soluble factor released by CD8+CD57+ cells in culture supernatants. Preliminary characterization suggests that the CD8+CD57+ cells' inhibitory activity is mediated by a low molecular weight, glycosylated factor as indicated by its less than 1S coefficient of sedimentation and its binding to concanavalin A lectin. PMID- 1709379 TI - Prognostic value of lymphocyte surface markers in acute myeloid leukemia. AB - We studied the expression of cell surface antigens associated with myeloid and lymphoid leukemias on bone marrow-derived blast cells from 339 patients with newly diagnosed de novo acute myeloid leukemia (AML) enrolled on Cancer and Leukemia Group B (CALGB) chemotherapy protocols. Surprisingly, of 211 cases studied for the expression of CD2 (T-cell marker, sheep erythrocyte binding receptor for T lymphocytes) 45 were positive (21%). In addition, of 298 patients studied for CD19 (B-lymphocyte marker), 41 were positive (14%). Overall, of 170 patients studied for both CD2 and CD19, 56 (33%) were positive. Interestingly, central review of the French-American-British (FAB) morphology of the CD2- and CD19-positive cases showed that FAB M3 was twice as frequent, and M4E eight times as frequent compared with the CD2- and CD19-negative cases. Of 22 lymphocyte antigen-positive cases in which cells were available for studies of Ig or T-cell antigen receptor (TCR) gene rearrangement, 20 were germline, one had a rearranged Ig heavy chain gene, and one had rearranged TCR beta and Ig heavy chain genes. The presence of messenger RNA for CD2 was demonstrated in four CD2 surface antigen-positive cases, thus validating the cell surface data. Lymphocyte antigen positive cases had karyotypes commonly seen in AML; 71% of cases with an abnormal clone had t(8;21)(q22;q22), inversion 16(p13q22), t(15;17)(q22;q12), or t(9;11)(p22;q23). The patients with lymphocyte markers had a significantly higher incidence of these karyotypic abnormalities compared with patients with lymphocyte antigen-negative AML (34% v 15%, P less than .02). When the outcome to therapy of the lymphocyte antigen-positive cases was compared with that for the CD2, CD19-negative cases, we found that the CD2, CD19-positive cases actually had higher complete remission rates (75% v 59%, P = .04), and significantly longer time to failure (P = .02; 32.4% +/- 6.0% v 18.0% +/- 4.1% at 2 years) and overall survival (P = .02; 43.5% +/- 6.3% v 26.0% +/- 4.5% at 2 years). CD2 antigen positive cases also had a significantly superior survival (P = .02; 43.8% +/- 7.9% v 29.8% +/- 3.8% at 2 years). There were no significant differences (P less than or equal to .05) between the two groups in age, leukocyte count at diagnosis, incidence of extramedullary disease, or FAB classification.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709381 TI - Functional properties of the beta-globin locus control region in K562 erythroleukemia cells. AB - In this report, we compare the function of the human beta-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for "classical" enhancer activity, (2) a colony assay that detects "productive integration events," and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had approximately 10% as much colony assay activity as the entire mu LAR. However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested. PMID- 1709380 TI - Human monocytes bind to two cytokine-induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1. AB - Vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) are adhesive proteins induced on endothelium by cytokines. We examined the contribution of these adhesive proteins to human peripheral blood monocyte adherence to endothelium using transfected Chinese hamster ovary (CHO) cells stably expressing these proteins and monoclonal antibodies (MoAbs) to ELAM 1, VCAM-1, or CD49d/CD29 (VLA-4), the leukocyte receptor for VCAM-1. Monocytes bound to CHO cells transfected with cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was inhibited by MoAb to VCAM-1 or the alpha-chain of very late activation antigen-4 (VLA-4) (CD49d). Additive inhibition of adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocytes were pretreated with both MoAb to CD49d and MoAb to CD18, the common beta-chain of the leukocyte beta 2 integrin receptors. Adherence of monocytes to HUVE stimulated by recombinant human tumor necrosis factor-alpha was not reduced by MoAbs to CD18, CD49d, or ELAM-1 when used singly, but combinations of these MoAbs produced significant inhibition. We conclude that multiple receptor-ligand systems are involved in monocyte adherence to endothelium. PMID- 1709382 TI - Outcome of treatment of first relapse of Hodgkin's disease after primary chemotherapy: identification of risk factors from the British Columbia experience 1970 to 1988. AB - The outcome of treatment for a first relapse of Hodgkin's disease after primary chemotherapy was analyzed in 80 patients. They were divided into four groups: group 1 (n = 24) had initially been treated with three cycles of (mechlorethamine, vincristine, prednisone, and procarbazine [MOPP]) and wide field irradiation therapy; group 2 (n = 25) had six cycles of MOPP; group 3 (n = 15) and group 4 (n = 16) both initially received MOPP/ABVD (MOPP plus doxorubicin, bleomycin, vinblastine, and dacarbazine) or MOPP/ABV hybrid, but group 3 received conventional salvage regimens whereas group 4 was treated with high-dose chemotherapy and autologous bone marrow transplantation as salvage therapy (n = 16). Freedom from second failure (FF2F) was used as the major endpoint. Actuarial FF2F for all patients was 38% after a median follow-up of 75 months for patients who were alive. Risk factor analysis was performed on the 71 patients who had been treated with curative intent. The presence or absence of any one of three risk factors had a strong negative impact on outcome: stage IV disease at primary diagnosis, B symptoms at relapse, or a time from primary treatment to relapse of less than 1 year. Actuarial FF2F at 5 years was 17% in the group of patients with one or more of these three factors present (n = 49). If none of these factors was present, FF2F was 82% (n = 22) (P less than .001). Even high-dose chemotherapy and autologous bone marrow transplantation were not able to overcome the negative impact of one or more risk factors (FF2F = 19%, n = 12). The outcome of salvage treatments depends most on the presence or absence of these three risk factors and less on the type of salvage treatment. Patients with none of these risk factors present have an excellent outcome if they are treated with non-cross-resistant chemotherapy, or radiotherapy, or both. Novel approaches are needed for patients with one or more of these factors present. Reports on salvage treatments for Hodgkin's disease in first relapse after primary chemotherapy should include data on the proportion of patients having stage IV disease at diagnosis, B symptoms at relapse, and a time from primary treatment to relapse of less than 1 year. PMID- 1709383 TI - Mechanism of transfusion-related acute lung injury. PMID- 1709384 TI - The delta TCS1 determinant is expressed on both disulfide- and non-disulfide linked gamma delta T-cell antigen receptors. PMID- 1709385 TI - Activation of neurokinin receptors modulates K+ and Cl- channel activity in cultured astrocytes from rat cortex. AB - Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential. PMID- 1709386 TI - Ion channel involvement in hypoxia-induced spreading depression in hippocampal slices. AB - Rat hippocampal tissue slices were made hypoxic in control medium and in medium containing the ion channel blockers tetraethylammonium (TEA), 4-aminopyridine (4 AP), or tetrodotoxin (TTX). Postsynaptic evoked potentials, extracellular DC potential Vec, and in some experiments extracellular potassium concentration [K+]o were monitored in stratum pyramidale of the CA1 region. TEA (10 mM) decreased the latency of hypoxia-induced spreading depression (SD), and reduced the amplitudes of the changes in Vec and [K+]o. 4-AP (50 microM) also decreased the latency of SD but had no effect on the Vec shift. In most slices, TTX (1 microM) increased SD latency but had no effect on the Vec shift. In some slices, TTX blocked the occurrence of SD. PMID- 1709387 TI - Neonatal capsaicin treatment abolishes the nociceptive responses to intravenous 5 HT in the rat. AB - The intravenous (i.v.) administration of serotonin (5-HT) to lightly pentobarbital-anesthetized rats is known to produce a triad of reflex cardiovascular responses, distinct afferent-mediated pseudaffective reactions, and a vagally mediated inhibition of the nociceptive tail-flick (TF) reflex consistent with 5-HT acting as a noxious stimulus. In the present experiments we examined the involvement of capsaicin-sensitive afferents in mediating these responses. Lightly pentobarbital-anesthetized 16-week-old male Sprague-Dawley rats which had been treated as neonates (in the first 48 h of life) with capsaicin (50 micrograms/kg, s.c.) were compared to age-matched neonatal vehicle treated controls. Whereas the i.v. administration of 5-HT produced a dose dependent (6-96 micrograms/kg, i.v.) inhibition of the nociceptive TF reflex (ED50 = 48.1 +/- 11.3 micrograms/kg; n = 7) and distinct pseudaffective responses (usually by 24-48 micrograms/kg) in vehicle-treated rats, 5-HT (6-192 micrograms/kg, i.v.) failed to significantly alter TF latency or produce pseudaffective behaviors in the capsaicin-treated rats (n = 10). There was no difference in baseline TF latencies between the two groups. There were essentially no differences between vehicle- and capsaicin-treated rats with respect to the initial cardiopulmonary vagal afferent-mediated (Bezold-Jarisch reflex) decreases in heart rate and arterial blood pressure or the subsequent pressor phase. However, the magnitude of the late hypotensive phase was significantly greater in capsaicin-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709388 TI - Cerebral atrophy and neuropsychiatric disorders. PMID- 1709389 TI - Development and mechanisms of interferon resistance. PMID- 1709390 TI - Bronchial hyperresponsiveness and bacterial respiratory infections. AB - Some studies suggest a potential role for bacterial respiratory tract infections in the development of bronchospasm and the progression of chronic obstructive pulmonary disease (COPD). Patients with bronchiectasis or cystic fibrosis have exaggerated airway reactivity; croup in children can also cause exaggerated upper and lower airway responsiveness. Bronchial obstruction after inhalation of Haemophilus influenzae and other bacteria has been reported. Between January 1989 and June 1990 we and two other centers studied 193 patients suffering from acute exacerbation of asthma. Fifty-two (27%) of these patients had bacteria in their sputum. Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Moraxella catarrhalis, and H influenzae were the most commonly isolated bacterial species. Antibiotics may be of value in the treatment of infective lung disease, not only by killing bacteria but also by preventing increases in bacterial histamine levels within the lung airways. Moreover, an antibiotic of proven efficacy can reduce airway reactivity in patients with bacterial exacerbations of COPD or bronchial asthma. In 12 patients with acute bacterial exacerbation of asthma and high airway reactivity to methacholine, a ten-day course of treatment with cefaclor and existing bronchodilators induced microbiologic cure and a slight but nonsignificant change in airway reactivity in nine patients. Antibiotic therapy has a minor but clear role in the control of acquired bronchial hyperreactivity during bacterial respiratory infections in asthmatic patients; however, because of airway inflammation, an antibiotic's efficacy is evident only when the inflammatory process subsides. Approaches designed to minimize airway reactivity may contribute to the prevention or reversal of respiratory failure during exacerbation of COPD and bronchial asthma. PMID- 1709392 TI - [Non-ketotic hyperglycinemia]. PMID- 1709391 TI - Distribution of single-channel conductances in cultured rat hippocampal neurons. AB - 1. The nonhomogeneous spatial distribution of ionic channels in neurons has been implied from intracellular recordings at somatic and dendritic locations. These reports indicate that Na- and Ca-dependent regenerative currents are distributed differently throughout the neuron. Although a variety of K conductances and a noninactivating Na conductance have been described in intracellular studies, little is known about the spatial distribution of inward and outward currents throughout different regions of the neuron. 2. We recorded from cell-attached patches from cultured hippocampal cells from 1-day-old rats. The cells were cultured for 3-21 days. The spatial distribution of a variety of ionic channels was determined by comparing the conductances from somatic and dendritic membranes. Single-channel currents obtained from cell-attached patches were identified by the time course of ensemble (averaged) responses, voltage dependence, and the effect of channel blocking agents. 3. We consistently observed that only the rapidly inactivating inward current was localized to the soma. The other channel types that we studied, including an inward noninactivating, delayed rectifier and transient A-type currents, were observed in both the somatic and dendritic regions. 4. We suggest that the distribution of ionic conductances that we have observed may be functional in limiting excitability during development of neurons. PMID- 1709393 TI - Saponins from leaves of Acanthopanax hypoleucus Makino. AB - From the leaves of Acanthopanax hypoleucus Makino (Araliaceae), five triterpenoidal saponins, having oleanolic acid and hederagenin as sapogenins, were isolated. On the basis of chemical and spectral data, the structures of two new saponins, named hypoleucosides A (1), and B (5) were elucidated as follows: 1; 3-O-beta-D-glucopyranosyl 11 alpha-methoxy-oleanolic acid 28-O-beta-D glucopyranosyl ester, 5; 3-O-beta-D-glucopyranosyl-(1----2)-alpha-L arabinopyranosyl-(1---- 4)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D glucopyranosyl-(1----6)-beta-D-glucopyranosyl ester. PMID- 1709394 TI - Enzyme immunoassay of a substance P-like immunoreactive substance in human plasma and saliva. AB - A sensitive and specific double-antibody enzyme immunoassay (EIA) for a substance P (SP)-like immunoreactive substance (SP-IS) was developed. For competitive reactions, the SP-antibody was incubated with SP standard (or sample) and beta-D galactosidase labeled Tyr8-SP (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 10 fmol/well of SP. Using the present EIA, SP-ISs in human saliva and plasma were determined. The level of SP IS in human saliva was about 7 pmol/l, which was almost three times higher than that in human plasma. PMID- 1709395 TI - Unilateral hypoxic-ischemic injury in neonatal rat results in a persistent increase in the density of striatal tyrosine hydroxylase immunoperoxidase staining. AB - Little is known of the alterations in the neurochemical anatomy of the striatum following neonatal hypoxic-ischemic injury. In an experimental model in rodent, it has been shown that striatal biochemical markers of dopaminergic systems appear to be relatively spared. We sought to define the morphologic correlates of this finding by staining striatal tyrosine hydroxylase (TH)-positive fibers with the immunoperoxidase technique. Since this model is unilateral, it is possible to examine relative changes in the density of TH-positive staining of the striatum at the population level within the same section. We find that in immature animals (3-4 weeks) there is an increase in the relative density of striatal TH-positive staining on the injured side. There is also an increase in the relative density of staining in the nucleus accumbens, in the absence of shrinkage of that nucleus. There is no relationship between the degree of density increase in the striatum, and its degree of shrinkage. This increase in relative density is a persistent change, as it is also observed in adult rats (9-12 weeks). While the functional significance of these findings are unknown, they are compatible with a relative increase in the dopaminergic innervation of the hypoxia-ischemia injured striatum. PMID- 1709396 TI - Cell attachment and neurite stability in NG108-15 cells: what is the role of microtubules? AB - Undifferentiated NG108-15 cells forming rapid-onset neurites were acutely exposed to nocodazole or trypsin. Resorption, cell rounding and detachment were delayed or prevented by 5'-deoxy,5'-methylthioadenosine (MTA), which selectively enhanced the strength of attachment responses. However, taxol, which stabilized microtubules, did not protect cell shape appreciably when trypsin or mechanical stimuli were used to decrease the strength of attachment. Together with numerous control experiments, this evidence suggests that the mechanical properties of microtubules do not contribute acutely to maintaining cell shape, though microtubules may play an indirect regulatory role (e.g. through their interactions with actin and substratum attachment sites). Patterns of trypsin induced resorption resembled those seen 'spontaneously' in NG108-15 cells growing on laminin, and in fibroblastic CHO cells, suggesting that these results may be both physiologically relevant and applicable widely to many cell types. PMID- 1709397 TI - NZB serum factor (NZB-SF)-B precursor cell maturation factor. II. In vivo effects of NZB-SF or mAb against NZB-SF on B lineage cell populations. AB - In vivo effects of NZB serum factor (NZB-SF), which enhances the maturation of B precursor cells in vitro, were examined. Immunoaffinity-purified NZB-SF from young NZB mice was injected into B6 mice intraperitoneally twice weekly, five times total (5 micrograms/dose/mouse). Control mice were given 0.01% albumin. Then the B lineage cell populations defined phenotypically (sIg+ cells, B220+ cells, and AA4.1+ cells) or the numbers of colony-forming B lineage cells were examined. NZB-SF-treated B6 mice exhibited a decrease in the percentage of B precursor cells in marrow, even though the percentage of sIg+ cells in marrow or spleen did not differ from controls. In contrast, the frequency of colony-forming B cells in marrow and spleen, especially sIg- colony-forming B cells in marrow, increased significantly in NZB-SF-treated mice as compared to controls. In addition, monoclonal antibody (mAb) against NZB-SF was injected weekly for 9 weeks into NZB mice beginning at 7 weeks of age. mAb vs NZB-SF at a dose of 5 micrograms per mouse per injection, as stated above, prevented the decline of sIg colony-forming B lineage cells which usually occurred in the adult NZB mice (greater than 16 weeks). This treatment also prevented, in part, the decline of the B220+ cell population which normally occurs in the marrow with increasing age. Thus NZB-SF impressively influences the composition of B lineage cell populations in normal B6 mice and may account for abnormal changes of B lineage cell populations observed in NZB mice. PMID- 1709399 TI - Prolonged effect of a new platelet-activating factor antagonist on ocular vascular permeability in an endotoxin model of uveitis. AB - Platelet-activating factor (PAF) is a membrane-derived lipid mediator involved in inflammatory responses. In the present study, the effect of a new, synthetic PAF antagonist, BN 50726, on ocular-blood barrier breakdown was investigated in a model of anterior uveitis produced by injection of 5 microL 0.1% endotoxin into the midstroma of rabbit corneas. Severe keratitis and anterior uveitis were induced in 3-4 days. BN 50726 was applied once subconjunctivally and then topically four times daily for 5 days in a blind-designed experiment. Vascular permeability was measured each day with an automated fluorophotometer after injection of fluorescein-conjugated dextran. BN 50726 significantly decreased ocular vascular permeability up to the fifth day of treatment. In another series of animals, slit-lamp observation showed significant reduction in iris erythema and epithelial damage with BN 50726 treatment. These results show that the PAF antagonist reduces early and late responses in uveitis. The possibility that PAF interacts with other inflammatory mediators to affect breakdown of the blood aqueous barrier is discussed. PMID- 1709400 TI - Amino acid sequence analysis of proteins in the human corneal stromal cell membrane. AB - Plasma membrane proteins from human corneal stromal fibroblasts were isolated and separated by two-dimensional polyacrylamide gel electrophoresis. Separated polypeptides were electroeluted onto polyvinylidene difluoride (PVDF) membranes and individual polypeptides were subjected to NH2-terminal amino acid sequence analysis. Of a total of 33 polypeptides sequenced, 26 were found to be blocked at their NH2-terminus. Seven major membrane polypeptides were sequenced and further analyzed. One polypeptide, designated #18, was determined to be homologous to the beta subunit of prolyl hydroxylase/protein disulfide isomerase/thyroid hormone binding protein. The other six polypeptides were found to have no significant sequence homology with any known polypeptides, as revealed by a protein data base homology search. Polypeptide Bands #90, #102, and #103 were found to have the same NH2-terminal amino acid sequence and the same overall molecular weight, yet separated from one another according to pI. These three polypeptides probably arose from differential posttranslational modification of the same original protein. Synthetic peptides were prepared from the #18 and #19 sequence and antibodies were produced. Immunostaining of cultured human corneal stromal cells and frozen sections of corneas demonstrated that these membrane polypeptides were present in corneal keratocytes, both in vitro and in vivo. Antibody against #18 stained fixed cultured corneal fibroblasts in a very fibrous pattern, with more intense staining in the perinuclear region of the cell, while antibody against #19 stained the cell surface in a much more uniform pattern. In sections of human cornea, both antibodies stained only the keratocytes in the stroma, but they also appeared to stain epithelial cells. PMID- 1709401 TI - Rotary shadowing of elastic system microfibrils in the ocular zonule, vitreous, and ligamentum nuchae. AB - Rotary shadowing of zonular fibrils in human and bovine eyes revealed a "string of beads" configuration with multiple interconnecting filaments, identical to that recently reported in fibrils of unknown type within the vitreous. These 29 nm beaded fibrils were the only macrostructures present in zonular samples, showing ultrastructural features correlating with both the macro and microperiodicity of zonular fibrils in tissues. Interbead periodicity varied from 30-57 nm and interbead filaments appeared capable of stretching even further, possibly explaining the inherent elasticity of zonular fibrils. The junctions between outer filaments and beads were fibrillin-positive. Similar beaded fibrils were found in the human and bovine anterior vitreous along with type II and IX collagen fibrils, proteoglycan filaments and other unidentified fibrils. After collagenase and elastase digestion, bovine ligamentum nuchae showed type VI collagen fibrils and clumps of beaded fibrils like those in zonule and vitreous. This distribution indicates that the beaded fibril is the microfibril which constitutes the basic unit of the elastic system. PMID- 1709402 TI - [Relation between cardiomyopathy and ventricular arrhythmias]. AB - Ventricular arrhythmias (VA) with its relevant factors were studied in patients with cardiomyopathy and the relation between late potential (LP) and ventricular tachycardia (VT) was also evaluated. It is shown that the prevalence of VA was high in cardiomyopathy. However, its severity was not related to clinical symptoms, cardiac enlargement, cardiac function and level of plasma electrolytes. The relative risk and relative odds of LP positive were 7.33 and 20 times as high in patients with VT as in those without VT, respectively. The sensitivity, specificity, and accuracy of prediction to VT in LP positive patients were 66.2%, 90.9% and 80%, respectively. The patients with sustained VT and positive LP were more susceptible to sudden death. LP was useful to understand the mechanism and prognosis of VT. PMID- 1709403 TI - Lack of neurochemical evidence for neurotoxic effects of repeated cocaine administration in rats on brain monoamine neurons. AB - Rats were injected with cocaine (20 mg/kg, s.c. or i.p. twice daily for 8 days) or saline and killed at 1, 8, 15 or 48 days after the last injection. The concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their metabolites, assayed by HPLC-EC, in frontal cortex, hippocampus, striatum, hypothalamus, midbrain, pons-medulla and spinal cord were not significantly different from those in the saline-injected controls at any of the time points examined. These data suggest that the repeated cocaine administration in rats does not produce any long-term depletion in brain catecholamine and 5-HT content suggesting no neurotoxic effects of the drug. PMID- 1709398 TI - Pharmacokinetic interactions between theophylline and other medication (Part II). AB - Part I of this article, which appeared in the previous issue of the Journal, covered the effects or lack of effects on theophylline clearance of sympathomimetics, corticosteroids, antihistamines and other antiallergy drugs, antimicrobial agents, phenytoin, carbamazepine, barbiturates, antacids and activated charcoal. In Part II, this discussion is extended to the effects of other agents. Overall summaries, both textual and tabular, appear in Part I. PMID- 1709404 TI - Lateral inhibition and cell fate during neurogenesis in Drosophila: the interactions between scute, Notch and Delta. AB - A comparison of the patterns of expression of AS-C (T3) RNA and protein suggests that an important level of regulation occurs post-transcriptionally. First, when the RNA is abundant in the early embryo the protein is barely detectable. Later, the protein starts to accumulate in only a subset of the nuclei of those cells expressing the RNA. Only the cells in the subsets become the neuroblasts. This post-transcriptional regulation is suppressed in embryos mutant for the genes Notch and Delta; where all cells expressing RNA accumulate protein. These findings suggest that deployment of T3 protein expression is one of the causal factors that assigns specific fates to the neuroblasts and, in consequence, a basis for the mechanism of lateral inhibition is proposed. PMID- 1709405 TI - Monte-Carlo-simulations of voltage fluctuations in biological membranes in the case of small numbers of transport units. AB - Some years ago a theory of non-equilibrium voltage fluctuations in biological membranes was developed (Frehland and Solleder 1985, 1986) under a linearisation condition which is valid for a great number of transport units. In order to get an insight into the stochastic behaviour of such systems, consisting of small numbers of transport units, we carried out Monte-Carlo-simulations and compared the mean voltage course and the spectral density with the results of the previous theory. Under parameter conditions of biological relevance no significant differences from the behaviour of systems with large numbers, as predicted from the earlier theory, could be found in the case of rigid pores and ion carriers. However, in the case of small numbers, channels with open-closed-kinetics showed great deviation. With increasing number of transport units agreement with the previous theory was obtained. PMID- 1709406 TI - Effect of iloprost on reperfusion-induced arrhythmias and myocardial ion shifts in isolated rat hearts. AB - Isolated hearts excised from normotensive (NT) and spontaneously hypertensive (SH) rats subjected to transient normothermic global ischemia were used to study the effect of chronic treatment with iloprost on reperfusion-induced arrhythmias and myocardial ion shifts. After 30 min of ischemia, iloprost given s.c. in doses of 10, 50, 100 and 200 micrograms/kg per day for 14 days reduced the incidence of reperfusion-induced ventricular fibrillation (VF) in isolated hearts from the control value of 91 to 83, 75, 50 (P less than 0.05) and 25% (P less than 0.01) respectively, in NT rats. In the SH groups, the incidence of VF was also reduced from 100 to 75, 58, 33 (P less than 0.01) and 17% (P less than 0.001), respectively, with 10, 50, 100 and 200 micrograms/kg per day of iloprost. A similar reduction was observed in the incidence of reperfusion-induced ventricular tachycardia (VT). Ischemia and reperfusion caused significant changes in myocardial ion contents, i.e. an increase in Na+ and Ca2+ and a decrease in K+ and Mg2+ concentrations. The myocardial water content was also increased in parallel to the Na+ gain. The effect of iloprost given s.c. in doses of 50 and 200 micrograms/kg per day for 14 days was also measured on myocardial ion contents after 15- or 30-min ischemia and 30-min ischemia plus 10-min reperfusion. The higher iloprost dose significantly reduced the myocardial Na+, Ca2+ and water gains and the loss of K+ induced by ischemia and reperfusion in the NT and SH groups, while the decrease in Mg2+ content was alleviated only in SH rats. The results suggest that long-term iloprost treatment reduces the incidence of reperfusion-induced VF and VT by preventing Na+, Ca2+ and water accumulation as well as K+ and Mg2+ loss from myocardial tissue. PMID- 1709407 TI - Perfusion of rat colon with sennosides, rhein and rheinanthrone. Concentration related histamine release. AB - Rat colon perfused intraluminally in vitro and in vivo released histamine into the perfusates. Histamine release was increased by rhein 0.1-10 micrograms/ml and much more by rheinanthrone 0.1-10 micrograms/ml but not by sennosides A or B 1-10 micrograms/ml. The effect of rhein and rheinanthrone was reduced by tritoqualine 20 mg/kg. This raises the possibility that laxation by senna and its derivatives involves histamine formation. PMID- 1709408 TI - Selective alterations in rat cardiac mRNA induced by doxorubicin: possible subcellular mechanisms. AB - Doxorubicin (Adriamycin, ADR) is an effective antineoplastic agent with a major side effect of dilated cardiomyopathy. Previously we showed ADR selectively decreased alpha cardiac (alpha c) actin mRNA in the rat heart when compared to other mRNAs examined in heart and skeletal muscle. The present study determined if this effect was selective for mRNAs within the thin filament, related to inhibitory effects on mitochondrial transcription, and modified by pretreatment with the cardioprotective chelating agent ICRF-187. Adult Sprague-Dawley rats received ADR at 8 mg/kg intraperitoneally (ip) with or without pretreatment with ICRF-187 given at 80 mg/kg ip. After 3 days, rats were killed and myocardial RNA was extracted, electrophoresed, transferred to nitrocellulose, and hybridized with the [32]cDNA probes alpha c actin, troponin C (TnC), BamHI fragment of mouse mitochondria (MM), and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Results showed a major depressive effect of ADR on rat myocardial alpha c actin mRNA. No depression of the other mRNAs examined (TnC, MM, or G3PD) was seen. ICRF-187 did not modify the effect. We conclude that the ADR-induced decrease in alpha c actin mRNA was: (1) selective within the thin filament; (2) not related to inhibitory effects on mitochondrial transcription; and (3) not related to free radical formation. Possible subcellular mechanisms are discussed. PMID- 1709409 TI - A superfamily of Arabidopsis thaliana retrotransposons. AB - We describe a superfamily of Arabidopsis thaliana retrotransposable elements that consists of at least ten related families designated Ta1-Ta10. The Ta1 family has been described previously. Two genomic clones representing the Ta2 and Ta3 elements were isolated from an A. thaliana (race Landsberg erecta) lambda library using sequences derived from the reverse transcriptase region of Ta1 as hybridization probes. Nucleotide sequence analysis showed that the Ta1, Ta2 and Ta3 families share greater than 75% amino acid identity in pairwise comparisons of their reverse transcriptase and RNase H genes. In addition to Ta1, Ta2 and Ta3, we identified seven other related retrotransposon families in Landsberg erecta, Ta4-Ta10, using degenerate primers and the polymerase chain reaction to amplify a highly conserved region of retrotransposon-encoded reverse transcriptase. One to two copies of elements Ta2-Ta10 are present in the genomes of the A. thaliana races Landsberg erecta and Columbia indicating that the superfamily comprises at least 0.1% of the A. thaliana genome. The nucleotide sequences of the reverse transcriptase regions of the ten element families place them in the category of copia-like retrotransposons and phylogenetic analysis of the amino acid sequences suggests that horizontal transfer may have played a role in their evolution. PMID- 1709410 TI - Morbidity and mortality from chronic hepatitis B virus infection in family members of patients with malignant and nonmalignant hepatitis B virus-related chronic liver diseases. AB - Three-hundred forty-one HBsAg-positive family members of 152 patients with chronic hepatitis B virus infection (47 asymptomatic carriers, 59 with chronic hepatitis, 17 with cirrhosis and 29 with hepatocellular carcinoma) were prospectively studied to determine the morbidity and mortality from chronic hepatitis B virus infection in the family members of patients with malignant and nonmalignant hepatitis B virus-related chronic liver diseases. Most of the family members had no history of acute hepatitis, were asymptomatic and were unaware of their carrier status. However, 5.3% had stigmata of chronic liver disease, 6% had serum ALT levels that exceeded two times the upper limit of normal and 78% of those who had biopsies had chronic hepatitis with or without cirrhosis. During a follow-up period of 12 to 90 mo (median = 39 mo), 3% had symptoms of chronic liver disease; 24% had transient, recurrent or persistent elevation in serum ALT levels, 1.4% had cirrhosis and 1% had hepatocellular carcinoma. Neither hepatocellular carcinoma in the index patient nor a previous history of hepatocellular carcinoma in the family was associated with an increase in the morbidity and mortality from chronic hepatitis B virus infection in the HBsAg positive family members. PMID- 1709411 TI - Direct evidence for cytotoxicity associated with expression of hepatitis delta virus antigen. AB - It has been postulated that hepatocyte injury resulting from infection with hepatitis D virus may be caused by a direct virus cytotoxicity in contrast to immune-mediated injury associated with hepatitis B virus. We have transfected HeLa and HepG2 continuous cell lines with a recombinant plasmid containing the hepatitis D antigen gene under the inducible control of the human metallothionein promoter. The addition of zinc to the cell culture medium then led to the expression of hepatitis D antigen associated with, in the short term, a significant reduction in the rate of RNA but not DNA synthesis and, in the longer term, cell death. The necrotic cells had pyknotic nuclei and shrunken eosinophilic cytoplasm; these necrotic cells resembled the apoptotic bodies seen in hepatitis D virus-related hepatitis. The level of hepatitis D antigen in individual cells that produced these changes was similar to the level of hepatitis D antigen in hepatocytes from a chimpanzee with acute hepatitis D virus infection. We conclude that the expression of hepatitis D antigen resulted in significant cytotoxic changes in these cells, providing strong support for the view that hepatitis D antigen may be specifically cytotoxic to infected hepatocytes in vivo. PMID- 1709413 TI - [Study on the production, identification of serogroup-specific monoclonal antibodies against leptospires of Australis serogroup and detection of its antigen]. AB - Spleen cells of BALB/c mice immunized with whole Leptospira interrogans serogroup Australis serovar australis strain 620 were fused with myeloma cells line SP2/0. Specificities of four McAbs determined by MAT. 2E1 McAb (IgG3) reacted with 11 serovars, of the Australis serogroup, but did not react with 22 representative serovars of L. interrogans in 20 serogroups, L. biflexa strain patoc I and Leptonema illini strain 3055. 2E1 McAb showed serogroup specificity for Australis by agglutination and the other 3 McAbs showed partial serogroup specificity. We compared the outer envelope (OE) protein profiles of serovar australis strain 620 with those of two pathogenic L. interrogans serovar lai strain 601 and serovar hebdomadis strain 156 by SDS-PAGE. 63kd protein profile was only found in the OE of strain 620, and the quantity of 42kd protein of strain 620 was greater than that of strain 601 and 156. The immunoblotting revealed that 2E1 McAb reacted with a 34kd band in the OE preparation of serovar australis strain 620, but did not react with that of other two L. interrogans. 2E1 McAb also did not react with OE of non-pathogenic leptospires. It was suggested that 34kd protein might contain the antigenic determinants which were shared by leptospires of Australis serogroup. PMID- 1709412 TI - FK 506 ameliorates the hepatic injury associated with ischemia and reperfusion in rats. AB - The effect of FK 506 on regeneration of the liver was studied in rats after a two thirds partial hepatectomy after 60 min of ischemia of the unresected liver. The animals were divided into three distinct groups of 10 rats each. Group 1 (controls) received 0.5 ml saline solution intravenously 30 min after the induction of ischemia. Groups 2 and 3 were injected with FK 506 (0.3 mg/kg) intravenously 30 min after and 24 min before the induction of hepatic ischemia, respectively. The hepatic content of ATP and serum levels of ALT and lactate dehydrogenase were determined on each animal. In addition, the histological appearance and mitotic activity of the remnant liver was determined at regular 24 hr intervals after hepatic ischemia. All 10 control animals died within 72 hr. Treatment with FK 506 resulted in improved survival in groups 2 and 3 (30% and 80%, respectively). The improved survival seen in the FK 506-treated animals was reflected by a restoration of hepatic ATP content, a reduction in the serum levels of ALT and lactate dehydrogenase, an amelioration of hepatic necrosis and neutrophilic infiltration and an increase in the mitotic activity of the liver. These results suggest that FK 506 ameliorates the hepatic injury associated with ischemia/reperfusion and has a potent stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective agent when administered to organ donors before graft harvesting. PMID- 1709414 TI - Cytokines: applications in domestic food animals. AB - Cytokines such as human, bovine, and porcine interferons and human and bovine interleukin-1 and interleukin-2 have been used in vivo in cattle and pigs. Colony stimulating factors and tumor necrosis factor alpha have been evaluated in vitro in food animals. Studies to evaluate cytokines in domestic food animals have shown that specific and nonspecific immunomodulation is possible in immunosuppressed or pathogen-exposed animals. Cytokine prophylaxis or therapy in food animals may have the greatest potential for control of respiratory disease and mastitis. PMID- 1709415 TI - Circulatory and respiratory responses to glutamate stimulation of the lateral parabrachial nucleus of the cat. AB - Electrical stimulation of the dorsal part of the lateral parabrachial nucleus (Pbl) induced a pressor response associated with a brief hyperpneic response, whereas glutamate stimulation induced no response, suggesting that the response to electrical stimulation may be due to excitation of passing fibers. Electrical stimulation of the lateral part of the Pbl induced a pressor and hyperpneic response, while glutamate stimulation induced either a pressor-depressor or single depressor response associated with a hyperpneic and polypneic response, suggesting that the response to electrical stimulation of the lateral part may be due to excitation of cell bodies. WGA-HRP study clarified that the cells in the region around the ventrolateral edge of the brachium conjunctivum project to the cells in the C1 area and subfacial cell group of the rostral ventrolateral medulla (RVLM), suggesting that the cells in this region may send a message to the sympathetic premotor and respiratory premotor cells in the RVLM. Inversely, the cells in the C1 area and the subfacial cell group project to the lateral part of the Pbl, suggesting mutual innervation between the Pbl and RVLM. PMID- 1709416 TI - Virus-induced interferon production in human leukocytes: a low responder to one virus can be a high responder to another virus. AB - We measured the amounts of interferon (IFN) induced by four wild-type strains and two attenuated vaccine strains of poliovirus, testing each virus in 12-58 separate batches of human peripheral blood leukocytes. There were big and consistent differences in the mean amounts of IFN formed by leukocytes from different donors in response to all these polioviruses. Almost invariably, the wild-type polioviruses gave rise to more IFN than the corresponding attenuated strains, but overall, the amounts induced by one strain of poliovirus were proportional to those induced by another. There were indications of similar correlations with six different influenza viruses and with three strains of herpes simplex virus. In contrast, there was no correlation between the IFN yields induced with a poliovirus, three other enteroviruses, and various other viruses. These results suggest that the leukocytes from a given individual have a capacity to form IFN in vitro that is determined both by the genetic make-up of that individual and the particular virus concerned. If the same phenomenon applies to IFN production by cells in vivo, it may have a role in pathogenesis of viral infections. PMID- 1709417 TI - Fractionation and characterization of 2',5'-oligoadenylates by polyacrylamide gel electrophoresis: an alternative method for assaying 2',5'-oligoadenylate synthetase. AB - 2',5'-Linked oligoadenylates of varying chain lengths (2,5As) are formed from ATP by an interferon (IFN)-induced enzyme, 2',5'-oligoadenylate synthetase (2-5A synthetase). To identify these multiple forms, a method was devised utilizing electrophoretic separation of 32P-labeled 2,5As in a thin 20% polyacrylamide gel containing 7 M urea. A mixture of 2,5As synthesized from rat liver nuclear suspension was fractionated by this method. Each species was eluted from the gel for characterization by specific nucleotidylic enzymes. All major species in the gel were identified, including dimeric, trimeric, and tetrameric forms with either 5' tri- or diphosphates, as well as dephosphorylated species. Thus, in a single step, this method produced a more complete assessment of newly synthesized 2,5As and their degradative products than conventional, multistep DEAE-cellulose chromatography. It also allowed more rapid screening of multiple samples than high-pressure liquid chromatography (HPLC). This method, used to assay 2-5A synthetase induction by IFN-alpha in human T-cell H9 and CEM-CM3 lines, should be applicable for routine analysis of clinical specimens. PMID- 1709418 TI - Universal antibodies to human interferon-alpha subtypes--the production of antipeptide antibodies to conserved regions of interferon-alpha. AB - Antibodies to three conserved regions of all human interferon (IFN)-alpha 1 subtypes were raised by immunizing rabbits with short synthetic peptides coupled to a carrier. These peptides correspond to amino acid residues 29-36, 31-36, 126 131, 139-151, and 142-151 of the consensus sequence of IFN-alpha 1. The antibodies were tested for reactivity with IFN-alpha 1, -alpha 2a, -alpha 2b, alpha 4a, and -alpha 14 and IFN-beta. Most antipeptide antibodies react only weakly with IFN-alpha subtypes; only the antibodies raised against the peptide corresponding to residues 142-151 conjugated to keyhole limpet hemocyanin using glutaraldehyde react appreciably with all IFN-alpha subtypes tested. These antipeptide antibodies are potentially universal antibodies to the human IFN alpha 1 subtypes, since they exhibit similar binding affinity to all the IFN alpha subtypes tested and do not react with IFN-beta. PMID- 1709419 TI - A new approach combining interferon and bromocriptine LA to prevent spontaneous mouse mammary carcinoma. AB - The development of spontaneous mammary carcinoma in C3H mice is mainly attributed to an oncogenic virus (MMTV) and the high prolactin levels of this inbred strain. Interferon (IFN) reduces the incidence of mammary carcinoma in virgin C3H mice when administered during lactation. Bromocriptine LA, an antiprolactin drug, also decreases the incidence of this tumor in C3H mice. The combined action of mouse IFN-alpha + beta and bromocriptine LA had a significant anticarcinogenic effect more significant than either of the above agents administered independently. This antitumor action was effective in virgin C3H mice only. PMID- 1709420 TI - Insulin-like growth factor-binding proteins in tissue fluids from the lamb. AB - Heparinized samples of plasma, cerebrospinal fluid (CSF) and lymph from intestinal, prescapular and popliteal lymph nodes were collected from young lambs in order to characterize and compare the insulin-like growth factor-binding proteins (IGFBPs) in extracellular fluids with those from the circulation. Prior to collection and analysis, the superiority of heparin for plasma preparation was established over EDTA and citrate or the use of serum, according to the extent of IGF-I and IGF-II binding achieved in the high molecular mass IGFBP region in vitro. The IGFBPs were characterized by ligand blotting and competitive binding techniques using radiolabelled IGF-I, IGF-II and the truncated IGF analogue, des(1-3)IGF-I, as well as by ligand blotting of fractions after Superose 6 chromatography of incubations of fluids with labelled factors. This combined analysis demonstrated an IGF-II-specific binding protein at approximately 250 kDa that was present in plasma and each lymph type and presumably represented the soluble type-2 IGF receptor; a complex of 130 kDa containing 52 kDa and 46 kDa binding proteins that was labelled by all three IGF peptides was particularly evident in plasma and intestinal lymph and was probably a complex between IGFBP-3 and the acid-labile subunit; and a group of binding proteins that chromatographed as IGF complexes at approximately 50 kDa. This last group contained IGFBP bands of 52, 46, 32, 28 and 23.5 kDa in plasma and all lymphs as well as an IGF-II specific band of 22 kDa in prescapsular and popliteal lymphs. CSF differed qualitatively from plasma and lymph in that the 52/46 kDa IGFBP bands were undetectable in this fluid; the 35 kDa band was the predominant binding protein, and neither this nor the 28, 23.5 and 22 kDa proteins bound des(1-3)IGF-I to any significant extent. The 52, 46 and 28 kDa bands in plasma and lymph did bind this ligand. Immunoblots using antisera against bovine IGFBP-2 showed binding at 35 kDa in all fluids as well as several bands at lower molecular masses. Taken together these results show not only marked differences in the binding protein profiles of sheep plasma, lymph and CSF, but both qualitative and quantitative differences between intestinal, prescapular and popliteal lymphs. We speculate that the differences between lymphs may result from tissue-specific release of binding proteins into extracellular fluid. PMID- 1709422 TI - Microcirculatory flow changes after initial resuscitation of hemorrhagic shock with 7.5% hypertonic saline/6% dextran 70. AB - In rabbits, laser Doppler flow probes were placed in the jejunum and on the renal cortex. Pulsed Doppler probes were implanted on the abdominal aorta and superior mesenteric and femoral arteries for measuring blood flow velocity. Cardiac output was measured by thermal dilution. Either 30% or 40% of the calculated blood volume was withdrawn through a carotid catheter. After 30 or 60 minutes, an initial bolus of either lactated Ringer's (LR, 16 ml/kg) or 7.5% hypertonic saline/6% dextran 70 (HSD; 4 ml/kg) IV was followed by unlimited IV LR (administered as rapidly as possible) to restore systemic arterial blood pressure to the prehemorrhage levels. With HSD, arterial pressure corrected more rapidly (p less than 0.05), and the initial hemodilution was greater (p less than 0.05), but there were no differences by two hours. With HSD, cardiac output (90%-100% vs. 130%-160% of control; p less than 0.05), plasma Na+ (139-140 mM vs. 146-148 mM; p less than 0.05) and plasma osmolarity (292-295 mOsm vs. 308-310 mOsm; p less than 0.05) were all significantly higher than the values with LR, but there was no effect on blood flow velocities through the infrarenal aorta, femoral artery, or superior mesenteric artery. Renal cortical perfusion (56% vs. 97% of control; p less than 0.05) and jejunal mucosal perfusion (83% vs. 162% of control; p less than 0.05) were significantly higher with HSD. HSD had no detectable effect on bacterial translocation at 24 hours. Thus: 1) HSD restores blood flow more rapidly to the gut mucosal and kidney microcirculations than initial resuscitation with LR; 2) the mechanism could be associated with a transient hemodilution and persistent increases in plasma Na and osmolarity, which reduce hemorrhage-induced cell swelling and blood viscosity changes; and 3) laser Doppler analysis could aid in the diagnosis of reperfusion injury after shock. PMID- 1709421 TI - CSF monoamine metabolites, cholinesterases and lactate in the adult hydrocephalus syndrome (normal pressure hydrocephalus) related to CSF hydrodynamic parameters. AB - Monoamine metabolites, cholinesterases and lactic acid in lumbar cerebrospinal fluid (CSF) were investigated on patients with the adult hydrocephalus syndrome (idiopathic normal pressure syndrome; AHS, n = 15), Alzheimer's disease (AD, n = 14), multi-infarct dementia (MID, n = 13) and controls (n = 21). Patients had clinical and CSF hydrodynamic investigations. Monoamine concentrations were determined by reversed-phase liquid chromatography, cholinesterases and lactate were determined photometrically. In the AHS patients, CSF monoamine concentrations were not significantly different compared with controls, AD or MID patients. AHS and AD patients showed a similar reduction of CSF acetylcholinesterase activity compared with controls. Positive correlations were found in concentrations of CSF homovanillic acid, CSF 5-hydroxyindoleacetic acid and CSF lactic acid versus CSF outflow conductance (that is, resistance against CSF outflow) in the AHS patients. A similar pattern was observed in a subgroup of MID patients characterised by dilated ventricles and disturbed CSF hydrodynamics. These data suggest that a low CSF outflow conductance may facilitate the clearance of acidic substances from the arachnoid space at the probenecid sensitive active transport site. Alternative explanations would be that a pathologically low CSF outflow conductance is accompanied by an inverse caudorostral flow of CSF or a compromised trans-ependymal diffusion. PMID- 1709423 TI - Clinical features and management of distant metastases of nasopharyngeal carcinoma. AB - This is a retrospective study of 90 patients who developed distant metastases after radical radiotherapy for nasopharyngeal carcinoma. The skeleton was the commonest site of distant metastases. Clubbing, hypercalcemia and malignant fever occurred in about 10% of patients with pulmonary, skeletal and hepatic metastases respectively. An effective chemotherapeutic regimen for palliation of pulmonary and hepatic metastases was cisplatinum/carboplatin-5FU which gave a complete response rate of 29% and partial response rate of 21%. This was considered superior to some non-cisplatinum-containing regimens. One patient with hepatic metastases had good palliation by hepatic irradiation. The median survival of all patients with distant metastases was eight months. Five (6%) patients survived more than two years with one surviving free of disease at 31 months. Hepatic metastases and spinal cord compression were associated with short survivals. PMID- 1709424 TI - Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose. AB - In vivo antitumor activity of a deoxyribonucleic acid fraction obtained from Mycobacterium bovis BCG (named MY-1) increased when it was complexed with poly-L lysine (poly LL) solubilized by addition of carboxymethylcellulose (CMC). The complex of MY-1 and poly LL/CMC induced interferon in vivo at a low dose of MY-1 which alone exerted no IFN induction. With Line 10 hepatoma (L10) which is syngeneic with strain 2 guinea pigs, it was demonstrated that repeated intralesional injections of the complex resulted in delay of tumor growth and complete cure of animals from L10 tumor inoculated. Similar treatment of the animals with the same amount of MY-1 or poly LL/CMC alone had little therapeutic effect on the tumor growth. PMID- 1709425 TI - Biochemical basis of a novel T lymphocyte receptor immunodeficiency by immunohistochemistry. A possible CD3 gamma abnormality. AB - Necropsic lymphoid tissues obtained from an infant with a novel type of immunodeficiency consisting of a peripheral blood T lymphocyte antigen receptor (TCR) surface expression defect, were analyzed by immunohistochemistry for the expression of various TCR-associated epitopes. The work was aimed to characterize the biochemical basis of this kind of disorder and confirm the defect in different lymphoid tissues. Within an assessed lymphoid depletion, the patient's tissues showed a normal expression of several TCR epitopes (those associated to CD3 epsilon, CD3 delta and the clonotypic -Ti- alpha and beta chains). In contrast, the expression of the epitopes recognized by the monoclonals OKT3, WT31, and BMA031 was severely diminished. Our results therefore support that CD3 epsilon, CD3 delta, Ti alpha and Ti beta are probably not involved in this type of immunodeficiency, and strongly suggest that CD3 gamma (forming part of the epitope recognized by OKT3) may rather be the affected chain giving rise to the defective surface T cell phenotype; however, alternative interpretations are not ruled out. The disrupted TCR thus formed, containing Ti alpha beta heterodimers and CD3 epsilon and CD3 delta subunits, but lacking normal CD3 gamma, would in this scheme lack the conformational framework determinants recognized by WT31 and BMA031. PMID- 1709426 TI - Long-term culture and partial characterization of dog gallbladder epithelial cells. AB - We describe the successful isolation and maintenance of primary cultures of dog gallbladder epithelial cells. The surgically removed gallbladder was treated with trypsin/EDTA for 45 minutes and epithelial cells were collected and resuspended in Eagle's minimum essential medium with 10% fetal calf serum, and plated on Vitrogen-coated culture dishes. Each gallbladder yielded approximately 12 to 15 x 10(6) columnar epithelial cells, greater than 95% of which were viable by trypan blue exclusion. In culture, cells maintained their polarity. They were arranged and grew in small and tight clusters that coalesced at confluency. When examined using transmission electron microscopy, prominent and numerous microville were identified on the apical portion of the plasma membrane. Cells were connected by well-formed desmosomes. Scanning electron microscopy revealed clusters of polyhedral cells with numerous papillary projections. Immunohistochemical studies demonstrated uniform staining of cells to keratin 35BH11 and AE1. Histochemical studies were positive for gamma-glutamyl transpeptidase and negative for glucose 6-phosphatase and albumin. Cells incorporated [3H]uridine into intracellular proteins and [14C]glucosamine into tissue and secreted mucous glycoproteins linearly over 2 to 24 hours. Flow cytometry studies demonstrated a consistent and reproducible number of cells (10 to 12%) at S-phase. However, the number of cells at S-phase was dramatically reduced to almost negligible as cells reached confluency. This method of culturing primary dog gallbladder epithelial cells is highly reproducible and reliable. These cells preserve their state of differentiation, polarity, histochemical and immunohistochemical profile, morphologic, and metabolic integrity with repeated passaging or after being frozen. [3H]Thymidine uptake is well maintained, although doubling time shows a trend of decreased cell duplication with time. This technique offers the opportunity to study the electrophysiologic, metabolic, and immunologic properties of epithelial cells. PMID- 1709427 TI - Development of a biotinylated analog of substance P for use as a receptor probe. AB - The development of a biotinylated substance P (SP) analog for use as a receptor probe is reported. The lysine in position 3 of SP was substituted by arginine and an amino terminal extension (NTE-SP) was added consisting of Lys-Tyr-Gly-Gly-Gly Gly-Gly-Gly. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by high performance liquid chromatography and characterized by mass spectral analysis. Binding studies using human IM-9 lymphoblasts with the biotinylated SP analog (biotin-NTE[Arg3]SP) and native SP yielded dissociation curves which were identical. In addition, the biotinylated SP analog retained functional activity similar to that of native SP in altering intracellular calcium concentration of Fura-2 loaded isolated rabbit colonic myocytes. Applicability of the SP receptor probe was demonstrated by using the streptavidin-peroxidase detection system to identify SP receptors on human IM-9 lymphoblasts. In conclusion, a biotinylated SP analog has been developed which retains the functional characteristics of the native peptide and is a useful and versatile probe for receptor studies. PMID- 1709428 TI - Transcription of cRNA for in situ hybridization from polymerase chain reaction amplified DNA. AB - The synthesis of cRNA probes for in situ hybridization is usually accomplished with transcription systems using cloning vectors that contain bacteriophage RNA polymerase promoters. In this report we describe an alternative technique for generating cRNA probes that obviates the need for cDNA cloning by using the polymerase chain reaction. The segment of DNA corresponding to the desired RNA sequence was amplified from genomic DNA by the polymerase chain reaction. The bacteriophage SP6 and T7 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5' termini of the oligonucleotides used to prime the polymerase chain reaction. The promoters permitted the subsequent transcription of sense and antisense cRNA from the amplified DNA. The antisense cRNA was radiolabeled to high specific activity for use as a probe in Northern and mRNA in situ hybridization analyses. This technique offers the advantages of speed and simplicity over conventional transcription systems, which require cDNA cloning. Bypassing the need for cloning in the synthesis of cRNA probes may facilitate the use of these probes in research and diagnostic applications of mRNA in situ hybridization. PMID- 1709429 TI - Silver stained nucleolar organizer regions in cancer cells. PMID- 1709430 TI - Technical considerations in evaluating the endothelial integrity of rat aortic preparations with silver staining. AB - Assessment of endothelial integrity is an obligatory step in many pharmacological studies. Integrity of endothelium is affected by manipulations performed during the removal and cleaning of the vessel and by some of the silver-staining techniques utilized for demonstrating interendothelial junctions. When aortas were cleaned of periadventitial tissue in cold Tris-saline (once separated from the animal) by untrained personnel, only 45% of the endothelium was preserved. When cleaning was performed in situ by trained personnel while flushing with cold Krebs-Ringer-6% albumin, over 95% was left intact. AgNO3-staining performed before fixation produced a 50% loss of endothelium when using NH4Br and (NH4)2S as developers. AgNO3-staining performed after fixation produced over 95% recuperation of endothelium when 2% glutaraldehyde, 150 mM NaCl, 40 mM phosphate buffer, pH 7.4, were utilized as initial fixative, NH4Br and (NH4)2S being equally effective as developers. Chloride ions were necessary to intensify silver lines. Several patterns of deendothelization were produced by mechanical and chemical injury with saponin, NH4Br and (NH4)2S. In all cases, hematoxylin staining was employed as an auxiliary technique to interpret images of injured endothelium. Presence of albumin protected the endothelium from mechanical damage. PMID- 1709431 TI - CD7 (GP40) false-positive acute myelogenous leukemia and promyelocytic leukemia cell line HL-60. PMID- 1709432 TI - Histopathologic features of the L-tryptophan-related eosinophilia-myalgia (fasciitis) syndrome. AB - Study of 18 biopsy specimens in 11 patients with L-tryptophan-related eosinophiliamyalgia (fasciitis) syndrome showed hyaline sclerodermoid changes. Dermal scleroderma was found in eight of nine punch biopsy specimens and eight of nine excisional biopsy specimens. Fascial scleroderma was found in eight excisional biopsy specimens. One specimen obtained by excision had no fascia present. Eleven biopsy specimens showed edema of the dermis, and 13 showed dilated lymphatic structures; thus, the clinical picture of edematous sclerosis was confirmed. Mucinous fasciitis was present in five excisional biopsy specimens, in conjunction with a large number of macrophages in four. Dermal mucinosis was present in 11 biopsy specimens. Lymphocytic and macrophage inflammation was minimal in 14 biopsy specimens and pronounced in only 4. Plasma cells were present in eight cases. Eosinophils were present in substantial numbers in three biopsy specimens and only occasionally in four. Eosinophilic spongiosis was observed in one patient. Lymphocytic inflammation was noted around a single muscle spindle and around large nerve trunks in three patients. No relationship was established between these pathologic features and the duration or dose of tryptophan, prednisone treatment, or duration of symptoms. Pathologic features of the L-tryptophan syndrome consist of hyaline sclerodermoid collagen in the dermis, the septa, and the fascia. Edema, focal mucinosis, and macrophage inflammation may be features that identify this event. PMID- 1709433 TI - Anesthetic management of bleomycin-treated patients. PMID- 1709434 TI - Antiallergic effects of astemizole on immediate type hypersensitivity reactions. AB - Astemizole (0.5-5 mg/kg, p.o.) dose-dependently inhibited heterologous and homologous PCA reactions in rats at ID50 values of 1.48 mg/kg and 2.37 mg/kg, respectively. The inhibitory effect of astemizole on heterologous PCA was most remarkable when this compound was given p.o. 2 h prior to antigen challenge. Astemizole (0.1-5 mg/kg, p.o.) dose-dependently inhibited experimentally-induced asthma in guinea pigs at an ID50 of 0.86 mg/kg. Ex vivo, astemizole (0.5-5 mg/kg, p.o.) inhibited antigen-induced histamine release from lung pieces of sensitized guinea pigs. In in vitro experiments, the drug dose-dependently inhibited antigen induced histamine and SRS-A releases from guinea pig lung pieces at concentrations of 0.05-10 microM. Furthermore, astemizole (0.1-10 microM) inhibited the histamine release induced by compound 48/80 and antigen-antibody reaction from rat peritoneal mast cells, and at 0.1-500 nM inhibited both leukotriene C4- and platelet-activating factor (PAF)-induced contraction of isolated guinea pig trachea at submicromolar concentrations. Astemizole not only inhibited 45Ca uptake into rat mast cells but also prevented the Ca2+ release from the intracellular Ca store induced by compound 48/80, although this compound did not affect the histamine release from permeabilized mast cells induced by Ca2+. Our results suggest that one of the antiallergic mechanisms of astemizole may be an inhibition of signal transduction from the mast cell membrane to the intracellular systems. PMID- 1709435 TI - HCV transmission after receiving anti-c100-negative blood units. PMID- 1709436 TI - Autistic and developmental disorders after general anaesthetic delivery. PMID- 1709437 TI - Recurrent and/or metastatic head and neck squamous cell carcinoma: a clinical, univariate and multivariate analysis of response and survival with cisplatin based chemotherapy. AB - One hundred two patients with recurrent and/or metastatic head and neck squamous cell cancer were entered into four consecutive phase II trials, all cisplatinum (C-DDP, 100 mg/m2/cycle)-based. The two combinations tried were C-DDP, bleomycin, and fluorouracil (CFB) on 54 patients, and cisplatinum and vindesin in 36 patients (CV). The CFB combination was given with C-DDP by continuous infusion over 96 hours (23 patients) or on day 1 (31 patients). The CV regimen was also given in two different schedules, with VDS at 3 mg/m2/g weekly (12 patients) or by a 96-hour continuous infusion (0.6 to 1.0 mg/m2/d) in 24 patients. The following variables: sex, age, performance status, previous therapy, local recurrence, length of disease-free interval (DFI), distant metastases, weight loss, primary site, histological differentiation, type of chemotherapy, previous chemotherapy, evaluable/measurable disease, erythrosedimentation rate, and their relation with response to chemotherapy (WHO) and survival were submitted to both univariate and multivariate analysis (Cox). Overall response rate (RR:CR + PR) was 25 (28%) of 90. In the CFB protocols, RR was 12 (22%) of 54 vs. 13 (38%) of 36 (P = 0.15, NS) in the CV combination group. For the four different combinations the RR was CFB C-DDPci 7 (30%) of 23, CFB C-DDP 1 hour 5 (16%) of 31, CV VDS weekly 2 (17%) of 12, CV VDSci 11 (45%) of 24. The patient populations were very different, with the latest combination consisting of metastatic patients exclusively. Univariate analysis of multiple variables showed age less than 60 years, PS:0 or 1, no previous therapy, absence of local relapse, metastatic disease, long DFI, and that measurable disease was significant for the probability of response. Median survival was 7 months for the 90 evaluated patients, 5 months for nonresponders, and 9 months for responders (P = 0.01). In the univariate analysis, significant factors for survival were PS:0 or 1, a weight loss below 10%, long DFI, response to chemotherapy, erythrosedimentation rate (ESR) of less than 30 mm/1st hr, presence of bone metastasis, and the number of metastases. Multivariate analysis shows PS, the absence of local relapse, and disease-free interval as significant prognostic factors for response. Multivariate analysis factors of significance for survival were PS, weight loss, and response to chemotherapy. The analysis of the clinical pattern showed an evolution in RR from 3 (8%) of 36 on previously irradiated local recurrent disease to 8 (73%) of 11 in previously untreated patients with metastatic disease at presentation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709438 TI - The effect of bleomycin on the uptake and incorporation of [14C]choline into phospholipids in hamster lung tissue slices. AB - Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [14C]choline into phospholipids of lung slices in vitro. The stimulatory effect is opposite to the results obtained previously using [14C]acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis. PMID- 1709439 TI - Calcium binding protein (calbindin-D28k) and glutamate decarboxylase gene expression after kindling induced seizures. AB - In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity. PMID- 1709440 TI - Chronic treatment with SCH-23390, a selective dopamine D1 receptor blocker decreases preprotachykinin-A mRNA levels in nucleus tractus solitarii of the rabbit: role in respiratory control. AB - Acute intravenous administration of the selective D1 receptor blocker SCH-23390 resulted in an enhanced respiratory motor output as evidenced by the phrenic nerve activity, whereas local perfusion into the region of nucleus tractus solitarii had no effect. The increase in phrenic nerve activity was accompanied by a concomitant increase in the release of substance P in the region of nucleus tractus solitarii as measured by in vivo microdialysis technique. Chronic administration of SCH-23390 via subcutaneously implanted Alzet mini osmotic pumps, significantly decreased the level of preprotachykinin-A mRNA in the region of respiratory relay neurons in nucleus tractus solitarii but was without effect in the ventral medullary surface structure, wherein the central chemoreceptors are thought to be located. A smaller, but significant decrease was also seen in the striatum. The results suggest that chronic treatment with SCH-23390 leads to a disinhibition of an inhibitory dopaminergic input to the neurons in nucleus tractus solitarii from a suprapontine level, which may account for a subsequent inhibition of tachykinin-containing neurons in the nucleus tractus solitarii, the relay station for respiratory reflexes. PMID- 1709442 TI - Teaching the peasant midwife in rural Honduras. PMID- 1709441 TI - Dextranase activity of streptococcal isolates from human dental plaques. AB - Streptococci were isolated from sixty human dental plaques. Of those isolates which exhibited dextranolytic activity on culture media containing dextran, three were selected for further investigation. Two of these isolates were identified as Streptococcus mitior and the other was a strain of Streptococcus mutans. The organisms were cultivated in a dialysed medium and enzyme activities isolated from culture supernatants by precipitation with ammonium sulphate, dialysis and lyophilization. Decolourized annuli surrounding wells in agar plates containing Blue Dextran 2000 provided evidence of dextranase activity present in culture supernatants, precipitated and redissolved proteins, dialysis retentates and lyophilized materials. The pH optima for the crude enzyme preparations were determined using the modified assay of Koh and Khouw (1970). Dextranases from the Streptococcus mitior isolates had optima at pH 6, and the optimal activity for the enzyme from the Streptococcus mutans isolate occurred at pH 5.5. Additional studies of the purified enzymes are necessary to increase understanding of the possible effects of these enzymes in human dental plaques. PMID- 1709443 TI - [Promoting the development of handicapped children--possibilities and limits and responsibilities of the pediatrician]. AB - The assessment of strategies to improve the development of handicapped children is difficult. Results of empirical investigations show marked differences with regard to the underlying handicap or developmental risk. The major impact of social conditions is beyond doubt, and within these conditions the coping process of the parents plays an important role. A survey of the literature is given. From this the pediatrician's tasks are derived: Early diagnosis of severe developmental disturbances; early and sympathetic information of the parents including a realistic estimate of given possibilities of help with special regard to social conditions, and an optimal support for the parents' coping process. PMID- 1709444 TI - 5-Azacytidine inhibits the induction of transient TK-deficient cells by 5 bromodeoxyuridine. A novel hypothesis for the facilitation of hypermethylation by 5-bromodeoxyuridine. AB - This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6 thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5 methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression. PMID- 1709445 TI - Structural chromosomal rearrangements in HpaII-treated human lymphocytes. AB - Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located. PMID- 1709446 TI - A single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute Kawasaki syndrome. AB - BACKGROUND: Treatment of acute Kawasaki syndrome with a four-day course of intravenous gamma globulin, together with aspirin, has been demonstrated to be safe and effective in preventing coronary-artery lesions and reducing systemic inflammation. We hypothesized that therapy with a single, very high dose of gamma globulin would be at least as effective as the standard regimen. METHODS: We conducted a multicenter, randomized, controlled trial involving 549 children with acute Kawasaki syndrome. The children were assigned to receive gamma globulin either as a single infusion of 2 g per kilogram of body weight over 10 hours or as daily infusions of 400 mg per kilogram for four consecutive days. Both treatment groups received aspirin (100 mg per kilogram per day through the 14th day of illness, then 3 to 5 mg per kilogram per day). RESULTS: The relative prevalence of coronary abnormalities, adjusted for age and sex, among patients treated with the four-day regimen, as compared with those treated with the single infusion regimen, was 1.94 (95 percent confidence limits, 1.01 and 3.71) two weeks after enrollment and 1.84 (95 percent confidence limits, 0.89 and 3.82) seven weeks after enrollment. Children treated with the single-infusion regimen had lower mean temperatures while hospitalized (day 2, P less than 0.001; day 3, P = 0.004), as well as a shorter mean duration of fever (P = 0.028). Furthermore, in the single-infusion group the laboratory indexes of acute inflammation moved more rapidly toward normal, including the adjusted serum albumin level (P = 0.004), alpha 1-antitrypsin level (P = 0.007), and C-reactive protein level (P = 0.017). Lower IgG levels on day 4 were associated with a higher prevalence of coronary lesions (P = 0.005) and with a greater degree of systemic inflammation. The two groups had a similar incidence of adverse effects (including new or worsening congestive heart failure in nine children), which occurred in 2.7 percent of the children overall. All the adverse effects were transient. CONCLUSIONS: In children with acute Kawasaki disease, a single large dose of intravenous gamma globulin is more effective than the conventional regimen of four smaller daily doses and is equally safe. PMID- 1709447 TI - Kawasaki syndrome. PMID- 1709448 TI - Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. AB - Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding. PMID- 1709449 TI - Immunomodulation of experimental allergic encephalomyelitis by antibodies to the antigen-Ia complex. AB - Autoimmune diseases occur when T lymphocytes become activated on recognizing self antigen linked to the autologous class II molecule of the major histocompatibility complex (MHC). The resulting complex of antigen MHC T-cell receptor could be a target for treatment of autoimmune diseases. Studies in which each component is blocked separately might be limited by interference in non relevant immune responses that either use the same set of T-cell-receptor V gene segments or are linked to the same MHC. We report here an attack by a specific antibody on the unique antigenic site formed by the binding of two components of the trimolecular complex, the autoantigen bound to the self MHC. We tested its effect in experimental allergic encephalomyelitis, an acute neurological autoimmune disease which is widely regarded as a model for autoimmune disorders and which is mediated by CD4+ T cells recognizing myelin basic protein (BP), or its peptides, in association with self Ia. We made monoclonal antibodies which bound only the complex of BP and I-As. These antibodies blocked the proliferative response in vitro to the encephalitogenic determinant of BP and reduced the response to intact BP, without affecting the response to a nonrelevant antigen purified protein derivative of tuberculin presented on syngeneic macrophages. They also inhibited experimental allergic encephalomyelitis in H-2s mice. Hence, antibodies directed specifically to the autoantigen-Ia complex, may offer a highly selective and effective treatment in autoimmune diseases. PMID- 1709450 TI - Parental imprinting of the mouse H19 gene. AB - THE mouse H19 gene encodes one of the most abundant RNAs in the developing mouse embryo. It is expressed at the blastocyst stage of development, and accumulates to high levels in tissues of endodermal and mesodermal origin (H. Kim, unpublished result). After birth the gene is expressed in all tissues except skeletal muscle. It lacks a common open reading frame in the 2.5-kilobase RNA, but has considerable nucleotide sequence similarity between the genes of rodents and humans. Expression of the gene in transgenic mice results in late prenatal lethality, suggesting that the dosage of its gene product is strictly controlled. The H19 gene maps to the distal segment of mouse chromosome 7, in a region that is parentally imprinted, a process by which genes are differentially expressed on the maternal and paternal chromosomes. We have now used an RNase protection assay that can distinguish between H19 alleles in four subspecies of Mus, to demonstrate that the H19 gene is parentally imprinted, with the active copy derived from the mother. This assay will be of general use in assaying allele specific gene expression. PMID- 1709451 TI - Identification in situ and phylogeny of uncultured bacterial endosymbionts. AB - The use of Koch's technique to isolate bacteria in pure cultures has enabled thousands of bacterial species to be characterized. But for the many microorganisms that have never been cultivated, DNA amplification in vitro using the polymerase chain reaction is now making their genes accessible. Here we use this technique to study bacteria of the genus Holospora, which live in ciliates and whose phylogenetic relationship has remained unknown because they are impossible to cultivate. Species of Holospora are highly infectious and live in the nuclei of their specific host cells: H. elegans and H. undulata infect micronuclei of Paramecium caudatum, whereas H. obtusa infects the macronucleus in other strains of the same host species; Holospora species have a common developmental cycle. We have amplified, cloned and sequenced gene fragments encoding ribosomal RNA of H. obtusa. The phylogenetic position of H. obtusa in the alpha group of Proteobacteria was determined by 16S rRNA sequence analysis. The sequences were then used to design species- as well as genus-specific rRNA hybridization probes, which enabled us to detect and differentiate individual cells of the endosymbionts in situ. The large amount of rRNA in the cells indicates a high physiological activity of the endosymbionts in the host nuclei. PMID- 1709453 TI - [Molecular analysis of relationship between oncogene (N-myc and c-src) expression and major histocompatibility complex antigen gene expression in mouse neuroblastoma lines]. AB - The authors have investigated the relationship between oncogene (N-myc and c-src) expression and major histocompatibility complex (H-2 in the mouse) antigen gene expression at the molecular levels, by using mouse neuroblastoma sublines (NB-1 and NB-V). Fluorescence-activated cell sorter analysis showed that NB-1 cells exhibited positive expression to H-2 Kk, H-2 Dd, and beta-2-microglobulin, while NB-V cells were negative to all three antigens. It was found that dimethyl sulfoxide (DMSO) had a capacity to increase an H-2 class I antigen expression on NB-1 cells, whereas no change was observed on NB-V cells after DMSO treatment. Molecular analysis with deoxyribonucleic and ribonucleic acid (RNA) blot hybridization and immunoprecipitation revealed that the enhancement of H-2 antigen expression on NB-1 cells was modulated at the transcriptional control of the H-2 gene. In contrast, negative H-2 antigen expression on NB-V cells was caused by block at the level of glycosylation of the H-2 heavy chain, although an increase in messenger RNA of the H-2 gene was induced after DMSO treatment. There was neither amplification nor rearrangement of N-myc and c-src oncogenes in either neuroblastoma subline. Nuclear run-on transcription assay revealed that the N-myc gene was post-transcriptionally down-modulated by DMSO, whereas the c src gene was transcriptionally up-regulated. It was thus suspected that N-myc and c-src might be directly associated with cellular proliferation and differentiation in neuronal tumors and that in vivo tumorigenicity could be regulated by the control mechanism of oncogene expression in relation to H-2 gene expression on tumor cells. PMID- 1709452 TI - Dual effect of 1.4-dihydropyridines on Ca2+ inflow into rat pancreatic islet cells. AB - The present study aimed at comparing the effects of nifedipine, a dihydropyridine "Ca2+ antagonist", and of BAY K 8644, a dihydropyridine "Ca2+ agonist", on the short term (5 min) 45Ca uptake and the cytosolic Ca2+ concentration of rat pancreatic islet cells incubated in the presence of physiological concentrations of glucose. Nanomolar concentrations of nifedipine increased the short term 45Ca uptake while micromolar concentrations decreased it. Cd2+, an inorganic Ca2+ channel blocker, reduced the stimulatory effect of nifedipine. Low concentrations of BAY K 8644 stimulated 45Ca uptake whereas high concentrations decreased it. In contrast, verapamil, a phenylalkylamine type calcium antagonist, only provoked a dose-dependent reduction in 45Ca uptake. Lastly, low concentrations of both nifedipine and BAY K 8644 raised the fluorescence intensity of fura 2 loaded islet cells. These findings indicate that nifedipine and BAY K 8644 may exhibit agonistic and antagonistic actions on B-cell voltage-dependent Ca2+ channels. This dualistic behaviour is markedly concentration-dependent and appears to be inherent to the 1,4-dihydropyridine compounds. PMID- 1709454 TI - [Expression of major histocompatibility complex on human medulloblastoma cells]. AB - Medulloblastoma is one of the most common malignant brain tumors in childhood. These cells are immature bipotential cells that could differentiate into both neuronal and glial cells. The authors established two human medulloblastoma cell lines. One was derived from a 2-year-old girl with cerebellar tumor (designated as ONS-76) and another was from a 9-year-old girl with metastatic tumor in the right frontal lobe (ONS-81). Immunohistochemical studies showed that both cell lines possessed 145 and 200 kDa neurofilament proteins and neuron-specific enolase, without glial fibrillary acidic protein and S-100 protein. It was shown that interferon gamma could enhance or induce the expression of the major histocompatibility complex (MHC) antigens which play a major role in immune response. Also shown for the first time was the expression of MHC class II antigens on human medulloblastoma (ONS-76 and 81) with neuronal differentiation. PMID- 1709455 TI - [Cytotoxic effector mechanism and genetic control of non-immunized hybrid resistance to mouse T cell lymphoma transplanted outside and inside the brain]. AB - A Moloney virus-induced T cell lymphoma, YAC-1, derived from A/Sn (H-2a) inbred mouse origin, was tested for hybrid resistance (HyR) after subcutaneous (s.c.) or intracerebral (i.c.) tumor cell inoculation into syngeneic and semi-syngeneic mice. The F1 hybrids (H-2a/b) between A/Sn and C57BL/6 mice more strongly resisted the s.c. inoculation of 10(6) and 5 x 10(5) cells than did syngeneic recipients. In contrast, no HyR to the i.c. inoculation of 10(4) and 10(3) cells was seen in the F1 hybrid mice. Natural killer (NK) cell activity was much higher in F1 hybrids than in syngeneic mice. 125I-iododeoxyuridine-labeled YAC-1 cells were more efficiently eliminated from the highly resistant F1 hybrids than from the parental strain in both 4 and 18 hour in vivo rejection assays via intravenous (i.v.) and s.c. injection, respectively. The remaining radioactivity of the brain, however, did not differ between these mice. Thus, there was a correlation between the in vivo resistance of F1 hybrid mice to challenge s.c. inoculation of parental tumors and their expression of lymphocyte-mediated natural cytotoxicity in vitro against those tumors. T cell depletion by thymectomy followed by irradiation and fetal liver reconstitution did not abrogate the s.c. HyR against YAC-1, whereas NK cell depletion by i.v. administration of anti-asialo-GM1 antibodies resulted in the disappearance of the resistance. Furthermore, genotypic study segregating (A/Sn x C57BL/6) F1 x A/Sn backcross mice indicated that the s.c. HyR might be attributable primarily to heterozygosity within the H-2 complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709456 TI - [Experimental head injury of rabbits using pendulum impactor. An electron microscopic study]. AB - Brain damage in the early stage of experimental head injury was studied under electron microscope, especially with respect to the permeability of the blood brain barrier and damage to nerve fibers. Lightly anesthetized rabbits were treated by intravenous injection of horseradish peroxidase (150-250 mg/kg) and were subjected to occipital impacts with a pendulum impactor. The animals were then perfused with a 2.5% glutaraldehyde mixture. Small blocks of the brain were sampled, incubated, postfixed, and embedded in Epon. These sections were observed under an electron microscope without staining. The authors' criteria of concussion have been previously published. There were three nonconcussion, 11 nonlethal concussion and seven lethal concussion animals. All cases of lethal concussion showed subarachnoid hemorrhage (SAH) in the brainstem and 57% of them showed microscopic parenchymal perivascular hemorrhages in the brainstem. Fifty five percent of the nonlethal concussion cases showed SAH in the brainstem with a little parenchymal hemorrhage. The nonconcussion cases showed no hemorrhagic change except contusions and/or SAH of the cerebellum. Hemorrhagic changes were frequently seen in the cerebrum and cerebellum of nonlethal and lethal concussion cases without correlation to the severity of concussion. Under electron microscope, nonconcussion cases showed mild splitting of myelins. In nonlethal concussion cases, swelling of perivascular astrocytic feet was seen to a moderate degree in the cerebrum, pons, and medulla oblongata and damage to axons and myelins were mainly seen in the pons and medulla oblongata. Many cored dense bodies containing peroxidase were diffusely recognized in vascular endothelial cells. In lethal concussion cases, beside findings seen in nonlethal concussion, intravascular peroxidase was seen to leak from the intravascular space through an opened tight junction to the extracellular space, axons and neurophils.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709457 TI - [Experience of carotid endarterectomy]. AB - The clinical course of 45 patients treated by carotid endarterectomy over the past 5 years is described with emphasis on the following three points: 1) Diagnostic methods, namely digital subtraction angiography (DSA) and B-mode Doppler imaging technique; 2) surgical procedure using an improved shunt tube and surgical instruments; and 3) monitoring before and during surgery. All operations were conducted using a shunt. Morbidity and mortality rates were both 0%. Postoperative transient hemiparesis lasting for 6 hours was recognized in only four cases. The total percentage of correct diagnoses using intravenous DSA compared with conventional angiography was approximately 80%. The accuracy of the non-invasive B-mode Doppler technique in measuring the degree of constriction compared with conventional angiography was 84%. The shunt was made of silicone tubing and was based on a tube 30 cm in length and 3.5 mm in diameter which was a T-shaped loop. Different sized bulbs were fixed to each end of the tube to prevent extravascular deviation. Modified bulldog clamps and Sugita clips were used for fixation in the vessel. Regional cerebral blood flow (rCBF) measurement and electroencephalography (EEG) under contralateral Matas procedure were conducted before surgery, and cross circulation during short-term occlusion of the common carotid artery was evaluated. The emergence or increase of delta waves in EEG during occlusion was observed in six cases. The rCBF of the affected middle cerebral artery territory in these patients was lower than that in patients with no increase of delta waves. Furthermore, the mean stump pressure during surgery in cases with preoperative EEG changes was 40 mmHg and that in cases without changes was 63 mmHg; these values were significantly different.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709458 TI - [Clinicopathological study on low grade glioma. In relation to malignant transformation]. AB - The clinical status of patients with glioma is influenced by 1) the histological malignancy of the tumor, 2) the tumor volume, 3) secondary status such as brain edema or intracranial hypertension due to the tumor, and 4) the host immunity. Due to some improvement in at least 2) and 3) by the initial treatment, most low grade glioma cases pass through a clinically silent postsurgical period. However, at a certain point, transition to a high grade tumor malignant transformation may occur with exacerbation of the symptoms. Twenty-two cases of histologically established low grade glioma experienced over the past 7 years, in which immunological status was evaluated, were analyzed. Nine cases (41%) showed malignant transformation. Characteristic pictures of the clinical symptoms, computed tomography (CT) scan findings, immunological status, and morphological findings (mainly immunohistochemical examination) in nine cases were delineated. The findings at the time of exacerbation of the symptoms were as follows. In all cases CT scan demonstrated the change in the main lesion from low density to mixed density and were compatible with a high grade glioma. Reduction in host immunity was verified. Morphological increase in the tumor volume, increase in histological malignancy and deterioration in the secondary status due to the tumor were confirmed. Necrosis of the tumor cells as well as increase in giant cells and gemistocytes were observed. Immunohistochemical analysis revealed a decrease and irregularity in glial fibrillary acidic protein positive cells and positive processes as well as increase in vimentin intensity. These findings demonstrate change in the biological characteristics of the tumor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709459 TI - [Rapid resolution of acute subdural hematoma. Report of two cases]. AB - The authors report two cases of rapid resolution of acute subdural hematoma. Case 1, a 78-year-old male, sustained head trauma and became unconscious for a few minutes, but on arrival he was alert and neurological examination was normal. A computed tomography (CT) scan taken 5 hours after the injury showed a high-dense subdural hematoma partly containing low-density area over the right cerebral hemisphere. He was conservatively treated. A CT scan taken 33 hours after the injury disclosed diminution of the hematoma and appearance of a thin high-density layer in the subdural space adjacent to the tentorium. The high-density layer disappeared on a CT scan taken 7 days after the injury. Case 2, an 84-year-old female, struck her head in the occipital region. On arrival she was drowsy but otherwise had no neurological abnormality. A CT scan taken 1 hour after the injury demonstrated a high-dense subdural hematoma partly containing low-density area over the right cerebral hemisphere. She was conservatively managed and became asymptomatic. A CT scan taken 14 hours after the injury disclosed complete resolution of the hematoma and appearance of a thin high-density layer in the subdural space adjacent to the falx and the tentorium. This high-density layer also completely disappeared on a CT scan taken 14 days after the injury. It was suggested that a mechanism of rapid resolution of the acute subdural hematoma was attributable to washing-out by cerebrospinal fluid. PMID- 1709460 TI - [Diffuse hemispheric low density on CT scan following acute interhemispheric subdural hematoma in an infant. Case report]. AB - A 17-month-old boy was admitted to our hospital in a semicomatose state after a minor head injury. Computed tomography (CT) scans revealed a posterior interhemispheric and a thin convexity subdural hematoma. Two and a half hours later, repeated CT scans disclosed a development of the right hemispheric diffuse low density and an enlargement of the interhemispheric subdural hematoma. Decompressive craniectomy and removal of the hematoma was performed immediately. On the second postoperative day, the diffuse low density areas developed in the contralateral frontal and temporal lobes. On the 14th postoperative day, the gyri which had been low in density were markedly enhanced with contrast enhancement. On the 16th postoperative day, CT scans showed a diffuse hemispheric gyral high density with bleeding in the right parieto-occipital lobe. A right occipital lobectomy and removal of the hematoma was performed. Histological examination suggested cerebral infarction probably due to venous congestion. It is also suggested that the diffuse hemispheric gyral high density observed on the 16th postoperative day was the hemorrhagic cerebral infarction. PMID- 1709461 TI - [Chronic subdural hematoma with a markedly fibrous hypertrophic membrane. Case report]. AB - A 40-year-old female, who had taken low-dose oral contraceptives for 2 months before onset, developed transient dysarthria, left hemiparesis, and left hemihypesthesia. One month later, a computed tomography (CT) scan revealed a uniformly enhanced, convex-shaped, hypertrophic membrane with a lobulated lumen in the subdural space of the right parietal region. A right parietal craniotomy was performed. The membrane, consisting of elastic-hard, hypertrophic granulation tissue and yellowish, sticky fluid in the lumen, was readily freed and totally extirpated. Subsequently, the patient recovered without persistent symptoms. Light microscopic examination detected the sinusoidal channel layer and the fibrous layer in an alternating configuration, along with intramembranous hemorrhagic foci. Such hypertrophy must have been caused by repeated intramembranous hemorrhages and reactive granulation. Such findings of hematoma membrane have never previously been reported. Thus, this is an interesting case, clearly distinguished from typical chronic subdural hematoma. PMID- 1709462 TI - [Wide-spread spontaneous spinal subarachnoid hematoma. Case report]. AB - A 56-year-old female experienced sudden excruciating pain extending from the upper neck to the lower back. She had mild disturbance of consciousness, and a lumbar puncture revealed bloody cerebrospinal fluid. The positive neurological findings were meningitis, spastic paraparesis, hyperesthesia of the left L3 dermatome, bilateral Babinski, disappearance of anal reflex, and urinary retention. Computed tomography scans, myelography, and magnetic resonance images revealed diffuse subarachnoid hematoma and hematomyelia from Th12 to L3. Spinal angiography was tried twice before surgery but no origin of this diffuse hematoma could be found. Laminectomy was performed from Th12 to L1 and organized hematoma was found in the subarachnoid space. After the hematoma removal, non-pulsating tortuous vessels were observed on the surface of the spinal cord at the L1 level which ran into the intramedullary region. However, there was no further abnormality to define spinal arteriovenous malformation or fistula within the limits of exposure. The postoperative course was uneventful and about 2 months later she was able to walk by herself. PMID- 1709463 TI - [Arachnoid cyst of the fourth ventricle. Case report]. AB - A 65-year-old female was admitted because of progressive vertigo, truncal ataxia, and unsteadiness of gait for the past 6 years. Computed tomography (CT) and magnetic resonance imaging revealed a non-enhanced, large midline cyst in the posterior fossa and slightly dilated lateral and third ventricles. Metrizamide CT cisternography showed no communication between the cyst and the subarachnoid space. Suboccipital craniectomy, laminectomy of the atlas, and membranectomy of the cyst were performed. On light microscopic examination, the cyst wall was composed of arachnoid cells and connective tissues. Thus, this lesion was not an epithelial cyst but an arachnoid cyst occupying the fourth ventricle. An arachnoid cyst of the fourth ventricle is extremely rare, and only two cases were previously reported. PMID- 1709464 TI - [Lymphocytic adenohypophysitis. Case report]. AB - The patients was a 27-year-old female, gravida 0, para 0, with complaint of headache and visual disturbance for about 1 month. Ophthalmological examination showed impaired visual acuity and enlargement of Mariotte's spot on the left. A computed tomography scan disclosed an enhanced mass in the sellar and suprasellar region. Endocrinological examination revealed slight elevation of thyroxin, growth hormone, and prolactin. A trans-sphenoidal hypophysectomy was carried out with a preoperative diagnosis of nonfunctioning pituitary adenoma. Histological examination showed a marked infiltration of lymphocytes and interstitial fibrosis, diagnosed as lymphocytic adenohypophysitis. She received hormone replacement therapy owing to postoperative hypopituitarism. Twenty-eight cases of lymphocytic adenohypophysitis including the author's are classified clinically into two types; fulminant type and chronic type. The former type occurs frequently with disturbance of consciousness because of the end organ insufficiency and proceeds to death in a fulminant course. The latter type occurs frequently with headache and visual disturbances, closely related to pregnancy and the postpartum period and survival occurs with hormone replacement therapy. This disease should be kept in mind in the diagnosis of sellar and suprasellar mass lesions. PMID- 1709465 TI - [Rupture of intracranial aneurysm due to intravascular metastasis of choriocarcinoma. Case report]. AB - A 55-year-old female was admitted to the Juntendo Izunagaoka Hospital, because of sudden onset of motor aphasia and right hemiparesis. Neurological examination on admission revealed drowsy state, bilateral papilledema, motor aphasia, and right hemiparesis. Computed tomography showed a subcortical hematoma in the left frontoparietal region associated with perifocal edema. Left carotid angiography revealed a fusiform aneurysm at a peripheral branch of the central artery. There was no tumor stain on cerebral angiograms. A combination procedure was carried out during emergency surgery, including evacuation of the intracerebral hematoma and removal of the thrombus-like material from the aneurysmal cavity followed by wrapping the aneurysm with fascia. Histological examination of the thrombus-like material demonstrated that it was a neoplastic embolus of choriocarcinoma. Serum (2600 mIU/ml) and urinary (7200 mIU/ml) human chorionic gonadotropin levels were elevated. The features of neoplastic aneurysm due to choriocarcinoma are reviewed and discussed. PMID- 1709466 TI - [Olfactory neuroblastoma complicated by postirradiation pneumocephalus. Case report]. AB - A 56-year-old male was admitted with the complaints of nasal bleeding, gait disturbance, and disturbance of consciousness. Neurological examination revealed drowsiness, right hemiparesis, and choked discs. Computed tomography scan showed an enhanced mass at the frontal base, which extended to the left nasal and paranasal cavities. Angiography showed a tumor stain with a mass sign. The intracranial part of the tumor was removed completely and he was discharged ambulatorily. Two months after surgery, however, he was admitted again for the regrowth of the tumor. Ventriculoperitoneal shunting was placed and radiation therapy was given to the brain and nasal cavity. After 3000 rad irradiation the clinical condition suddenly became worse because of pneumocephalus. The cranial tumor disappeared after irradiation but he died of metastases and general prostration. Clinically this case was diagnosed as an olfactory groove meningioma at first, but immunohistochemical diagnosis was olfactory neuroblastoma. PMID- 1709467 TI - [Choroid plexus involvement in malignant lymphoma. Case report]. AB - Involvement of the choroid plexus by malignant lymphoma is rare. This case showed mass lesions at the choroid plexus in the first stage of systemic non-Hodgkin's malignant lymphoma. A 53-year-old female was admitted with headache, diplopia, and gait disturbance. The patient was alert but had left supraclavicular and axially lymph node swelling. Computed tomography scan revealed enhanced masses at the lateral and fourth ventricles. The lymph node biopsy disclosed non-Hodgkin's malignant lymphoma, diffuse large cell type. The immunological marker was the B cell type. At surgery, there was highly vascular white tumor at the choroid plexus of the right lateral ventricle. The tumor also involved adjacent brain tissue. Pathological examination revealed infiltration of lymphoma cells to the choroid plexus. The subependymal and perivascular spaces of the choroid plexus were filled with lymphoma cells. Virchow-Robin space and white matter of adjacent brain tissue were also involved by lymphoma. After surgery, whole brain irradiation of 35 Gy and chemotherapy were performed. However, 14 months after initial diagnosis, the patient expired because of systemic involvement by the lymphoma. PMID- 1709468 TI - [Subependymoma of the septum pellucidum. MRI features and its usefulness in planning surgery]. AB - A 61-year-old female was referred because of headache and gait disturbance. A computed tomography (CT) scan revealed a midline isodense mass lesion. The limit of the tumor was equivocal even after administration of contrast medium. Magnetic resonance imaging (MRI) study, however, clearly showed the relationship between the tumor and the surrounding structures, such as the corpus callosum, the ventricular cavities. The tumor had originated from the region of the septum pellucidum and positioned just beneath the corpus callosum from the genu to the splenium with the roof of the third ventricle pushed downward. The tumor was totally extirpated via the interhemispheric paratranscallosal route. The tumor was typical subependymoma. The post-operative course was uneventful. Transient psychiatric disturbance and inability to retain recent memory were observed and the former subsided in several weeks. CT and MRI characteristics of subependymoma are summarized and the usefulness of MRI to locate tumors and to plan proper surgical approaches are emphasized. PMID- 1709469 TI - [Ossifying fibroma of the cranial vault. Case report]. AB - A 68-year-old female was admitted with mild headache in the right frontal region. Physical and neurological findings were normal. Plain X-rays revealed a poorly circumscribed, osteoblastic lesion in the right frontal bone. Computed tomography scan showed that the diploic space was destroyed but that the inner and outer tables were intact. On 99mTc bone scan, a hot lesion was visible in the same region. The lesion was expressed as a low-signal intensity area on T1-weighted magnetic resonance (MR) image and as a high-signal intensity area on T2-weighted MR image. The tumor was broadly resected together with peripheral normal bone, and cranioplasty using a resin plate was performed. The tumor was mainly composed of mature, regularly aligned bone (lamellar bone) and intermingled fibrous tissue. Ossifying fibroma is a rare, benign fibro-osseous tumor that mainly involves the craniofacial bone. A few cases involving the cranial vault alone have been reported. The relevant literature is reviewed, and discussion focuses on the differential diagnosis between ossifying fibroma and monostotic fibrous dysplasia. PMID- 1709470 TI - [Brain metastasis from primary pericardial mesothelioma. Case report]. AB - A 50-year-old female was admitted because of nausea, vomiting, and cerebellar ataxia. Computed tomography scan revealed an enhanced mass accompanied with a cyst in the right cerebellar hemisphere. The mass situated in the subcortical region was removed. Histologically, highly vascular tumor cells lined the cavities. Postoperative radio- and chemotherapy were administered and the clinical symptoms improved gradually. Two months later, the patient complained of dyspnea. Chest X-ray on second admission demonstrated cardiomegaly. Hemorrhagic pericardial effusion amounting to 1000 ml was aspirated by pericardial puncture. Papillary clusters of tumor cells were demonstrated in the pericardial effusion. The patient died of cardiac failure. At necropsy solid tumors were located in the heart, lung, left inguinal region, and cerebellum. Histological diagnosis was mesothelioma arising from the heart. Primary pericardial mesotheliomas are rare; approximately 106 cases have been reported. Pericardial mesothelioma frequently spreads to the adjacent pleura and mediastinum, but distant metastases are extremely rare because patients with pericardial mesothelioma tend to die early due to cardiac failure or cardiac tamponade. PMID- 1709471 TI - [Clinical and histological study of pituitary fibrosarcoma following radiotherapy for pituitary adenoma. Case report]. AB - A 49-year-old male was admitted with a history of radiotherapy for a pituitary adenoma 9 years earlier. Three weeks prior to admission, he noticed visual loss in the left eye. Computed tomography (CT) scan revealed a sellar tumor. The patient underwent craniotomy and the tumor was partially resected. The histological diagnosis was benign pituitary adenoma. Two months after surgery, he began to complain of headache and left hemiparesis. CT scan at that time showed a large parasellar tumor extending into the right temporal lobe. A second craniotomy was performed and a firm tumor was partially removed. Under light microscopy, the tumor was composed of anaplastic spindle cells showing a fascicular pattern. Ultrastructurally, the tumor cells were spindle-shaped with elongated nuclei. The cytoplasm contained numerous distended rough endoplasmic reticula and free ribosomes, Golgi apparatus as well as glycogen granules. Some desmosome-like intercellular adherent were observed. Collagen fibers were scattered in the extracellular space. There was no apparent formation of a basement membrane. These findings suggested a close morphological similarity between tumor cells and fibroblasts, conforming to ultrastructural diagnostic criteria for fibrosarcoma. In spite of intensive treatment, such as a second radiotherapy and subsequent craniotomy, the patient died 9 months after admission. The clinical course and pathological findings of the post-irradiation pituitary fibrosarcoma are discussed. PMID- 1709472 TI - [Angiographic findings of vertebral dissecting aneurysm. Report of two cases and review of literature]. AB - The authors report two cases of vertebral dissecting aneurysm. The first case, a 49-year-old female, developed severe headache and computed tomography scan showed subarachnoid hemorrhage (SAH), but 4-vessel cerebral angiography failed to show an aneurysm. The second angiograms obtained 2 weeks later showed possible aneurysmal dilatation on the right vertebral artery. The third angiograms, 2.5 months after SAH, disclosed a right vertebral fusiform aneurysm on the arterial phase and it was diagnosed as a dissecting aneurysm since the contrast medium remained until the very late venous phase. The previous angiograms were reviewed using the subtraction technique, which revealed retention of the contrast medium. The second case, a 42-year-old female, suffered from SAH. Left vertebral angiography revealed a fusiform aneurysmal tapered narrowing just distal to the aneurysm, which was a typical "pearl and string sign." The subtraction film of the venous phase also showed retention of the contrast medium in the aneurysmal portion. These findings accurately diagnosed dissecting aneurysm of the vertebral artery. Since the classical true diagnostic "double lumen sign" was rarely observed in the angiograms, it was not easy to diagnose dissecting aneurysm of the vertebral artery. The authors emphasize the angiographic findings of retention of the contrast medium in the venous phase as a "true diagnostic sign" for correct diagnosis of dissecting aneurysm. PMID- 1709473 TI - [Fibromuscular dysplasia of the cervical arteries associated with a distal vertebral trunk aneurysm. Case report]. AB - A 64-year-old, hypertensive female suddenly experienced severe headache. On admission, the patient had almost clear consciousness but was slightly restless and complained of severe headache and nausea. Neurological examination revealed only neck stiffness. A computed tomographic scan revealed subarachnoid hemorrhage. Angiographically, bilateral internal carotid and vertebral arteries had the "string of beads sign" at their cervical portion, and the left internal carotid artery also had the same sign at its cavernous portion. The left vertebral artery had low-origin posterior inferior cerebellar artery and a berry shaped aneurysm at its distal trunk. A diagnosis of cervical and intracranial fibromuscular dysplasia (FMD) with a ruptured berry-shaped aneurysm of the distal vertebral trunk was made. The berry-shaped aneurysm was successfully treated with proximal clipping. Angiographically, right renal and axillary arteries also had the "string of beads sign," and the patient's hypertension seemed to be renovascular in etiology. The co-existence of intracranial FMD and cerebral aneurysm of unusual location suggests a possible relationship between the FMD and the development of cerebral aneurysm. PMID- 1709474 TI - [Solitary cerebral varix. Case report]. AB - A 69-year-old male presented with a 4-month episode of tonic seizures of the right arm and leg. Neurological examination revealed no abnormal findings. In the electroencephalogram, sharp wave trains were seen dominantly in the left frontal region. Computed tomography scans showed a spotty hyperdense lesion in the left insular area with minimal enhancement. Left carotid angiogram demonstrated no abnormalities in the arterial and capillary phases. In the venous phase, however, a varicose dilatation of the posterior insular vein was found, which was also noticed in the delayed phase. Magnetic resonance T1-weighted images failed to detect the lesion, but T2-weighted images showed a slight signal focus in the same area. After administration of 300 mg of phenytoin daily, the frequency of attacks markedly decreased and the patient was kept under close outpatient observation. PMID- 1709475 TI - [Dural arteriovenous malformation in the posterior fossa presenting with multiple intracerebral hematomas. Case report]. AB - A 59-year-old female was hospitalized because of disturbance of consciousness and convulsive seizures. She had taken a hormonal drug for 15 months after breast cancer surgery. A computed tomography scan revealed multiple high-density areas in the left temporal and frontal and the right parietal lobes. Angiography showed a dural arteriovenous malformation (AVM) in the posterior fossa fed by the occipital and the middle meningeal arteries and draining into the transverse sinus. It also demonstrated occlusion of the left sigmoid sinus in the venous phase. She complained of headache in the occipital region and dizziness. On day 13, the left occipital artery was ligated and cut, and then abnormal arterial anastomoses around the lesion were coagulated. After surgery, clinical symptoms disappeared. The etiology of dural AVM is controversial, but in this case it is suspected that sinus thrombosis due to the drug caused the dural AVM. The authors discuss the etiology and treatment of dural AVM in the posterior fossa. PMID- 1709476 TI - [Ruptured arteriovenous malformation during pregnancy with special reference to diagnostic X-ray exposure. Case report]. AB - A 32-year-old female (gravida 2, para 2) was admitted at 15 weeks of gestation with a chief complaint of left homonymous hemianopsia accompanied by frontal headache. Computed tomography scan and cerebral angiography were performed with the lead shield and probe for X-ray exposure placed on the patient's body. Angiography revealed an arteriovenous malformation (AVM) located in the posterior part of the right temporal lobe. Total dose of the diagnostic X-ray radiated to the fetus was measured 2.5 mrem at the most. The AVM and the hematoma were totally removed successfully at 19 weeks of gestation. A male infant weighing 3180 g was delivered at full term by cesarean section without congenital anomalies and he has been growing up without mental deficits. These results suggest that diagnostic X-ray examination during pregnancy has little risk and that surgical treatment should be given to the mother immediately after the rupture of an AVM if she is in good condition and the gestational date is past 12 weeks. PMID- 1709477 TI - [Trigeminal neuralgia associated with posterior fossa arteriovenous malformation and aneurysm fed by the same artery. Case report]. AB - A 45-year-old male was admitted for evaluation of left facial pain of 11-years' duration. Fifteen years prior to admission, he had had an episode of subarachnoid hemorrhage. Neurological examination revealed no definite abnormalities except for trigeminal neuralgia involving the left second division of the nerve. Computed tomography scan showed an irregular enhanced lesion involving the lateral aspect of the left cerebellar hemisphere. Vertebral angiograms revealed an arteriovenous malformation (AVM) fed by both the dilated left anterior inferior cerebellar (AICA) and superior cerebellar arteries (SCA) and draining into the superior petrosal and transverse sinuses via three enlarged draining veins. A saccular aneurysm was incidentally visualized at the proximal portion of the dilated AICA. The AVM was successfully extirpated through the left retromastoid craniectomy. The trigeminal nerve was directly compressed both rostrally and caudally at its entry zone by two dilated feeding arteries of the SCA and the AICA. The aneurysm was simultaneously obliterated. The postoperative course was uneventful, and the excruciating facial pain completely subsided. Postoperative angiograms confirmed disappearance of both the AVM and the aneurysm. Posterior fossa AVMs causing trigeminal neuralgia in the literature are reviewed, and the association of the AVM and the aneurysm in the posterior fossa is also discussed. PMID- 1709478 TI - Adenosine deaminase increases release of excitatory amino acids through a mechanism independent of adenosine depletion. AB - Addition of adenosine deaminase to cultured cerebellar neurones, led to large increases in the influx of 45Ca2+ and hydrolysis of polyphosphoinositide. These effects were inhibited or attenuated by glutamate receptor antagonists (AP5 or MK 801) and were not observed in cells stimulated by maximum concentrations of glutamate or quisqualate. Stimulation of the influx of 45Ca2+ and hydrolysis of phosphoinositide by adenosine deaminase may be secondary to an enhanced release of endogenous glutamate that in turn activates specific excitatory amino acid receptors. Accordingly, adenosine deaminase potently increased release of D [3H]aspartate, an effect that requires the presence of extracellular Na+ and is insensitive to inhibition by MK-801. None of the effects of adenosine deaminase may be simply related to a fall in endogenous adenosine. In fact, the action of adenosine deaminase was neither reversed by agonists (L-PIA or NECA), nor mimicked by antagonists (IBMX or theophylline) of adenosine receptors. It is speculated that adenosine deaminase stimulates release of neurotransmitter through a mechanism independent of depletion of adenosine. A possible direct action of adenosine deaminase should be taken into account when the enzyme is used to unmask the effects of endogenous adenosine. PMID- 1709479 TI - Neuropeptide Y modulates non-adrenergic, non-cholinergic neural bronchoconstriction in vivo and in vitro. AB - Non-adrenergic, non-cholinergic (NANC) neural mechanisms regulate airway tone in guinea-pigs, and it is possible that they may be regulated by other nerves. We have investigated effects of neuropeptide Y (NPY), a co-transmitter of adrenergic nerves, on the excitatory (bronchoconstrictor) NANC (e-NANC) response elicited both in vivo (via bilateral vagal nerve stimulation) and in vitro (via electrical field stimulation [EFS]), and on inhibitory (bronchodilator) NANC (i-NANC) responses in upper trachea elicited by EFS. NPY inhibited the e-NANC to vagal stimulation in vivo in a dose-dependent manner (90.0 +/- 3.4% inhibition at 500 micrograms/kg), but failed to alter the bronchoconstrictor response to exogenous substance P (SP). NPY also inhibited the e-NANC response to EFS in main and hilar bronchi (57.2 +/- 8.6% in main and 46.34 +/- 7.5% in hilar bronchi at 10(-6) M), whilst having no effect on the contractile response to SP. In contrast NPY had no effect on the i-NANC response in vitro. Thus, NPY exerts a powerful inhibitory action on tachykinin release from peripheral endings of capsaicin-sensitive airway sensory nerves, but has no effect on the i-NANC neural mechanisms. This suggests that adrenergic nerves may modulate e-NANC responses in the airways. PMID- 1709480 TI - Evidence for dose-dependent positively and negatively reinforcing effects of the substance P C-terminal analog DIME-C7. AB - Possible positive reinforcing and aversive effects of intraperitoneally (ip) administered substance P (SP) and its C-terminal heptapeptide analog [pGlu5, MePhe8, Sar9]-SP5-11 (DIME-C7) were investigated in rats. The 'conditioned corral preference and avoidance procedure' was used for assessing positive and negative reinforcement. Behavioural testing was conducted in a circular open field consisting of four uniform quadrants equally preferred by the rats prior to drug treatment. On 3 consecutive days, rats received an ip injection of either SP (37 nmol/kg), DIME-C7 (3.7, 7.4, 37, 185 nmol/kg) or vehicle (0.01 M acetic acid in saline) and were placed immediately into their assigned treatment corral. During the test for conditioned corral preference and avoidance, when provided a choice between the four quadrants, rats treated with SP and with the equimolar dose of DIME-C7 (37 nmol/kg) spent significant more time in the drug-paired corral, indicative of positive reinforcing effects. The doses of 3.7 and 7.4 nmol/kg DIME C7 did not influence the preference behaviour. The high dose of 185 nmol/kg DIME C7 led to a significant decrease in time spent in the previously drug-paired corral, suggestive of aversive effects of the treatment. Gross locomotor activity was not influenced by either treatment. These results are discussed in the framework of a structure/activity relationship for the reinforcing properties of peripheraly applied SP. PMID- 1709481 TI - Ultrasound detection of fetal aneuploidy in patients with elevated maternal serum alpha-fetoprotein. AB - Increasing confidence in the ability of high-resolution ultrasound to detect neural tube and ventral wall defects has enabled us to offer a revised risk estimate to the patient with an elevated maternal serum alpha-fetoprotein (MSAFP) level, such that amniocentesis may not be necessary. Recent authors have suggested that a reduced emphasis on follow-up amniocentesis fails to consider an increased risk for chromosomal anomalies in pregnancies with an elevated MSAFP, and that amniocentesis should still be performed. We reviewed our ultrasound findings from patients who underwent amniocentesis for evaluation of an elevated MSAFP and who had a karyotype prepared from the amniotic fluid sample. Four abnormal karyotypes were detected among 313 amniocenteses, and three of these were correctly predicted based on an abnormal ultrasound. The risk of an unexpected fetal aneuploidy after a normal consultative ultrasound in our series was one in 310. This is comparable to the risk of detecting abnormal chromosomes in the fetus of a 32-year-old woman, an age at which amniocentesis is not routinely offered. PMID- 1709482 TI - Sensitivity and specificity of screening for Down syndrome with alpha fetoprotein, hCG, unconjugated estriol, and maternal age. PMID- 1709483 TI - Constitutive expression of transforming growth factor alpha does not transform rat thyroid epithelial cells. AB - To evaluate the role of transforming growth factor alpha (TGF alpha) in transformed rat thyroid epithelial cells, expression and production of TGF alpha were measured in a normal rat thyroid epithelial cell line, FRTL-5, and in the same cells transformed by the Ki-ras oncogene. FRTL-5 cells transformed with Ki ras exhibit a 5- to 7-fold increase in the levels of TGF alpha-specific mRNA and a 3- to 4-fold enhancement of the amounts of TGF alpha protein in the conditioned medium, as compared with the normal thyroid cells. Although conditioned medium from the Ki-ras transformed FRTL-5 cells or authentic epidermal growth factor or TGF alpha are able to stimulate the anchorage-dependent growth of nontransformed FRTL-5 cells, neither conditioned medium nor the growth factors are able to induce the anchorage-independent growth of these cells in soft agar. FRTL-5 cells were then transfected with an expression vector plasmid containing the human TGF alpha cDNA to directly ascertain if over-expression of this growth factor is able to induce transformation in these cells. The TGF alpha-transfected FRTL-5 clones constitutively produced high amounts of TGF alpha at a level equivalent to or greater than the level found in the conditioned medium from the Ki-ras transformed FRTL-5 clones. Moreover, in contrast to the Ki-ras transformed cells, the TGF alpha transfectants were unable to grow in soft agar, did not form tumors in nude mice, and showed no reduction in the secretion of thyroglobulin. These data demonstrate that unlike Ki-ras, the constitutive expression of biologically active TGF alpha is not entirely sufficient to elicit a transformed phenotype in these cells. PMID- 1709484 TI - Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. AB - The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead. PMID- 1709486 TI - Exon skipping by mutation of an authentic splice site of c-kit gene in W/W mouse. AB - The murine mutation dominant white spotting (W) is in the proto-oncogene, c-kit. The receptor tyrosine kinase encoded by this gene has pleiotropic effects on murine development including hemopoietic cells, pigment cells, and germ cells. In this study, mutation in W homozygous mouse was identified as a single base substitution (GT----AT) at the 5'-splice donor site of the exon which encodes the transmembrane domain. Two types of aberrant exon skipping resulted from this mutation, occurred in a tissue specific manner. Either transcript lost the exon coding for transmembrane region and therefore the product might not be functional for signal transduction. Any unusual cryptic splice sites were not activated by this mutation as beta-globin gene in beta-thalassaemia. In addition, twelve base pair sequence of the 3'-end of the exon prior to the exon coding for transmembrane domain was found to be alternatively spliced. These findings should provide the genetic base for not only the receptor function but the splicing mechanism. PMID- 1709487 TI - Antigenomic Hepatitis delta virus ribozymes self-cleave in 18 M formamide. AB - The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requirements for self-cleavage. While truncating the 1679 nucleotide antigenomic HDV RNA, we have characterized the cleavage requirements of a number of ribozymes of intermediate length. Two of these, containing 186 and 106 HDV nucleotides respectively, cleaved to completion in the presence of 18 M formamide. The 186 nucleotide ribozyme also cleaved to completion in 10 M urea. Removal of an additional 10 nts from the 3' terminus of the 106 nt ribozyme resulted in a loss of the ability to cleave in high concentrations of the denaturants. The interaction of nucleotides near the cleavage site with a sequence within this 10 base region may confer unusual stability on these ribozymes. PMID- 1709485 TI - The promoter and enhancer of the inactive chicken beta-globin gene contains precisely positioned nucleosomes. AB - Core histone octamers reconstituted in vitro onto DNA fragments containing the chicken beta-globin gene promoter are precisely positioned with respect to the underlying DNA sequence [1]. Here we show that this is also true of the chicken beta-globin gene enhancer. These nucleosome binding sites are also employed within transfected COS cell nuclei, where the chicken beta-globin gene is transcriptionally inactive. Similar results were found in vivo, where positioned nucleosomes were detected over the inactive beta-globin promoter in chicken brain cells and 5-day red blood cells, and over the inactive beta-globin enhancer in brain cells. In contrast, the promoter and enhancer regions were found to be nucleosome-free in 15-day erythrocytes where the beta-globin gene is active. We argue that these results suggest a role for positioned nucleosomes in the regulation of the transcription of the chicken beta-globin gene. PMID- 1709488 TI - Ribosomal protein L27 is identical in chick and rat. PMID- 1709489 TI - An SstI RFLP at the C-Kit oncogene locus (KIT). PMID- 1709490 TI - A conserved sequence element in ribonuclease III processing signals is not required for accurate in vitro enzymatic cleavage. AB - Ribonuclease III of Escherichia coli is prominently involved in the endoribonucleolytic processing of cell and viral-encoded RNAs. Towards the goal of defining the RNA sequence and structural elements that establish specific catalytic cleavage of RNase III processing signals, this report demonstrates that a 60 nucleotide RNA (R1.1 RNA) containing the bacteriophage T7 R1.1 RNase III processing signal, can be generated by in vitro enzymatic transcription of a synthetic deoxyoligonucleotide and accurately cleaved in vitro by RNase III. Several R1.1 RNA sequence variants were prepared to contain point mutations in the internal loop which, on the basis of a hypothetical 'dsRNA mimicry' structural model of RNase III processing signals, would be predicted to inhibit cleavage by disrupting essential tertiary RNA-RNA interactions. These R1.1 sequence variants are accurately and efficiently cleaved in vitro by RNase III, indicating that the dsRNA mimicry structure, if it does exist, is not important for substrate reactivity. Also, we tested the functional importance of the strongly conserved CUU/GAA base-pair sequence by constructing R1.1 sequence variants containing base-pair changes within this element. These R1.1 variants are accurately cleaved at rates comparable to wild-type R1.1 RNA, indicating the nonessentiality of this conserved sequence element in establishing in vitro processing reactivity and selectivity. PMID- 1709491 TI - Elements involved in an in vitro block to transcription elongation at the end of the L1 mRNA family of adenovirus 2. AB - Using the 3' end of the L1 mRNA family of adenovirus 2 (Ad2) as a model system, we investigated transcription elongation following a poly(A) signal in a cell free system. The results show that RNA polymerase II can halt transcription elongation at a T-rich stretch in the non-coding DNA strand 20 nucleotides downstream of the poly(A) signal. The block to transcription elongation is enhanced when Sarkosyl is included in the elongation reaction. Deletion studies narrowed the region which directs the elongation block at the T-rich stretch, to an upstream fragment of 53 nucleotides that is very dA-rich and also contains a functional poly(A) signal. The deletion studies and analysis by site-directed mutagenesis indicate that in the present system, RNA secondary structure, the stretch of T's and the poly(A) signal are not the dominant elements responsible for the elongation block. The block to transcription elongation at the T-rich stretch was also shown to be 5 times more effective in an uninfected extract than in an Ad2 infected extract, which is reminiscent of the in vivo situation and is consistent with the suggestion that a trans-acting factor is involved in modulating the elongation block at the T-rich stretch. PMID- 1709492 TI - Structural models of ribonuclease H domains in reverse transcriptases from retroviruses. AB - Tertiary models of ribonuclease H (RNase H) domains in reverse transcriptases (RTs) from Moloney murine leukemia virus (MuLV) and human immunodeficiency virus (HIV-1) were built based upon the X-ray structure of RNase H from Escherichia coli (E. coli RNase H). In two models of RT-RNase H domains, not only active site residues but also residues, which construct a hydrophobic core and hydrogen bonds, are located in the same positions as those of E. coli RNase H. The whole backbone structure and the electrostatic molecular surface of MuLV RT-RNase H model are similar to those of E. coli RNase H. On the contrary, HIV-1 RT-RNase H model lacks the third helix and the following loop, resulting no positive charge clusters around the hybrid recognition site. Referring the complex models of RTs with their substrate hybrid, the interaction between DNA-polymerase and RNase H domains in RTs was discussed. PMID- 1709493 TI - Transcriptional termination sequence at the end of the Escherichia coli ribosomal RNA G operon: complex terminators and antitermination. AB - We have examined the termination region sequence of the rrnG operon and have observed its properties in vivo using a fusion plasmid test system. Transcription of rrnG terminator fragments was also studied in vitro. We found that termination of rrnG transcription is a complex process controlled by a tandem Rho-independent and Rho-dependent terminator arrangement which we designate rrnG-tt'. Together, these two elements were 98% efficient at terminating transcription initiated at the rrnG-P2 promoter. When the two elements were separated, however, we found that the Rho-independent structure was only 59% efficient while the Rho-dependent fragment alone could account for total transcriptional termination of the tandem arrangement. The rrnG termination region was resistant to rrn antitermination and, therefore, possesses some means of stopping antiterminated transcription. The distal rrnG sequence contains several additional noteworthy features; the rrnGt' fragment contains a REP (repetitive extragenic palindromic) sequence and homology with a small unidentified reading frame following rrnE. This sequence is followed by witA, which is homologous to a citrate transport gene, citB. Finally, our sequence, obtained from plasmid pLC23-30, contains a Tn1000 insertion that is absent from the E. coli chromosome. This insertion lies 975 bp beyond the 5S gene and is not involved in the termination events examined in this study. PMID- 1709494 TI - Direct tRNA-protein interactions in ribosomal complexes. AB - Nucleotide residues in E. coli tRNA(Phe) interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by UV-induced cross-linking. In the tRNA(Phe) molecule located in the Ab-site (pretranslocated complex) residues A9, G18, A26 and U59 are cross-linked with proteins S10, L27, S7 and L2, respectively. In tRNA(Phe) located in the Pt-site (posttranslocated complex) residues C17, G44, C56 and U60 are cross-linked with proteins L2, L5, L27 and S9, respectively. The same cross-links (except for G44-L5) have been found for tRNA in the Pb-site of the pretranslocated ribosomal complex. None of the tRNA(Phe) residues cross-linked with proteins in the complexes examined by us are involved in the stabilization of the secondary structure, but residues A9, G18, A26, G44 and C56 participate in stabilization of tRNA tertiary structure. Since translocation of tRNA(Phe) from Ab- to P-site is accompanied by changes of tRNA contacts with proteins L2 and L27, we postulate that this translocation is coupled with tRNA turn around the axis joining the anticodon loop with the CCA end of the molecule. This is in agreement with the idea about the presence of a kink in mRNA between codons located in the ribosomal A- and P-sites. In all E. coli tRNAs with known primary structure positions 18 and 56, interacting with L27 protein, when tRNA is located either in A- or P-site, are invariant, whereas positions 17 and 60, interacting with proteins only when tRNA is in the P-site, are strongly conserved. In positions 9, 26 and 59 purines are the preferred residues. In most E. coli tRNAs deviations from the consensus in these three positions is strongly correlated. PMID- 1709495 TI - The murine 2-5A synthetase locus: three distinct transcripts from two linked genes. AB - The cloning of cDNAs encoding murine 2-5A synthetase has identified three related transcripts, represented by a previously described cDNA clone, L3 and two novel cDNAs, L1 and L2. L1 contains an open reading frame coding for a protein that shows 70% conservation at the amino acid level when compared to the protein predicted to be encoded by L3. L2 recognizes an IFN-induced transcript 600-bp larger than the L3 transcript. These three cDNAs map to a cosmid, cII, containing two murine 2-5A synthetase genes, ME12 and ME5/ME8, situated in a head-to-tail orientation separated by approximately 8 kb. Southern analyses of ME12 and ME5/ME8 using L3, L1-specific and L2-specific probes indicate that these genes have a similar organization. cII was transiently and stably transfected into CV-1 cells. When treated with interferon, the transfected cells produced mature, murine 2-5A synthetase transcripts identified using L3 and L2-specific probes. Thus all transcripts present in IFN-treated mouse cells which are recognized by the available murine 2-5A synthetase cDNA probes map to an approximately 40 kb region of the mouse genome containing two 2-5A synthetase genes. PMID- 1709496 TI - A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells. AB - A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin. PMID- 1709497 TI - Nucleotide sequence and functional characterization of a mitochondrial tRNA(Trp) from Tetrahymena thermophila. PMID- 1709498 TI - Sequence and structure of Tetrahymena SRP RNA. PMID- 1709499 TI - PCR detection of the MspI (Aa) RFLP at the human phenylalanine hydroxylase (PAH) locus. PMID- 1709500 TI - Syringe drivers in the community. PMID- 1709501 TI - [Endobronchial brachytherapy: endoluminal small-volume irradiation of bronchial tumors using the high-dose-rate afterloading method]. AB - Endobronchial irradiation has been in use for over 70 years. This method of treatment has become much less of a burden to the patient thanks to developments in isotope technology, bronchoscopy and radiation therapy. It is effective in providing good palliation and in central bronchial tumours with involvement of the large airways, endobronchial irradiation has proved to be a valuable addition to our armamentarium. In the context of curative irradiation it may be combined with external beam irradiation. In tumours confined to the bronchial wall, endobronchial irradiation alone has been successfully used to achieve a cure. This method should however only be used in large thoracic centres as there are relatively few indications for its use. PMID- 1709502 TI - Ionic conductances in dissociated smooth muscle cells. PMID- 1709503 TI - In vivo administration of tumor necrosis factor-alpha is associated with antiviral activity in human peripheral mononuclear cells. AB - Tumor necrosis factor-alpha (TNF-alpha) has a spectrum of biologic effects and has been shown to exert antiviral effects in fibroblasts in vitro. The in vivo administration of TNF-alpha (40-160 micrograms/m2 intravenously over 2 hr) and its effects on vesicular stomatitis virus (VSV) replication in peripheral blood mononuclear cells (PBMC) from patients with malignancy was investigated. Blood was obtained before, during, and after infusion. The PBMC were separated and infected with VSV at a multiplicity of infection of 0.005 plaque-forming units/cell and virus yields were determined 72 h later. The TNF-alpha inhibited VSV yields by as much as 99% in a dose-dependent manner with the inhibition initially observed during the first hour of infusion. Despite a rapid reduction in TNF-alpha serum levels, the higher doses still produced antiviral effects 4 hr after the infusion. Sera obtained at identical times had no interferon activity. Human gamma-interferon (25 micrograms/ml) added in vitro augmented the TNF-alpha induced inhibitory activity in both magnitude and duration. Percentages of lymphocytes and monocytes in peripheral blood were reduced at 4 hr after TNF alpha administration and the monocyte to lymphocyte ratio was diminished and temporally coincided with the loss of TNF-induced antiviral state. These data suggest that the in vivo administration of TNF has a direct inhibitory activity on VSV replication in human peripheral blood mononuclear cells that was enhanceable by gamma-interferon and possibly monocyte mediated. PMID- 1709504 TI - Absence of luminal bile increases duodenal content of cholecystokinin in rats. AB - The effects of the removal of bile from the proximal intestine on pancreas, plasma cholecystokinin (CCK) concentration, and duodenal content of CCK were examined in rats. Bile was excluded from the duodenum and introduced into the distal ileum through a silastic cannula for 7 days. Pancreatic juice was maintained to be normally secreted into the duodenum. After 7-day bile diversion, plasma CCK concentration and duodenal CCK content were significantly increased in bile-diverted rats. Trypsin content in the proximal intestine in bile-diverted rats was one-half that in control. Pancreatic wet weight, protein content, and DNA content in the pancreas were slightly increased, and lipase content was slightly decreased, by bile diversion, but none of these changes was statistically significant. Amylase content significantly decreased and chymotrypsin content significantly increased in bile-diverted rats. Intragastric administration of camostate (trypsin inhibitor) significantly increased plasma CCK concentration in both bile-diverted and control rats, and the net increase was much greater in bile-diverted rats than in control rats. In conclusion, bile diversion increased duodenal CCK content and increased the CCK response to luminal stimulant. PMID- 1709505 TI - Phosphoramidon potentiates mammalian tachykinin-induced biting, licking and scratching behaviour in mice. AB - The effects of peptidase inhibitors were examined upon behavioural responses including scratch, bite and lick produced by intrathecal (IT) injection of substance P (SP) and neurokinin A (NK A) in mice. Phosphoramidon (0.002-2.0 nmol), an endopeptidase-24.11 inhibitor, simultaneously injected with SP or NK A, remarkably enhanced and prolonged SP- or NK A-induced behavioural response in a dose-dependent manner. The behavioural response to SP was significantly increased by 2.0 nmol of bestatin, an aminopeptidase inhibitor, but not by 1.0 nmol. Captopril, an angiotensin-converting enzyme inhibitor, was without effect on both tachykinin-induced responses. When phosphoramidon was injected together with bestatin and captopril which have no significant effect alone, SP- or NK A induced behavioral response was significantly increased. These data suggest that endopeptidase-24.11 may be an important enzyme responsible for terminating of SP- or NK A-induced behavioral response at the spinal cord level. PMID- 1709506 TI - Neurochemical consequences following injection of the substance P analogue, DiMe C7, into the rat ventral tegmental area. AB - The effect on forebrain catecholamine- and indoleamine-related neurochemical levels was investigated following stimulation of the rat ventral tegmental area with the substance P analogue, DiMe-C7. DiMe-C7 (6.0 micrograms) induced a marked hyperactivity in rats with maximal response between 15 and 30 min following the injection. Fifteen min following the DiMe-C7 injection levels of dopamine and/or its metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid) were significantly increased in the nucleus accumbens, amygdala, entorhinal cortex and striatum relative to vehicle-injected animals. Although the increase in dopamine metabolism in the nucleus accumbens is consistent with the behavioural hyperactivity, it is concluded that other forebrain nuclei may also be involved in the mediation of the hyperactivity response. PMID- 1709507 TI - msDNA of bacteria. AB - The msDNA-retron element represents the first prokaryotic member of the large and diverse retroelement family found in many eukaryotic genomes (Table II). This prokaryotic retroelement exists as a single copy element in the chromosome of two different bacterial groups: the common soil microbe M. xanthus and the enteric bacterium E. coli. It encodes an RT similar to the polymerases found in retroviruses, containing most of the strictly conserved amino acids found in all RTs. The RT is responsible for the production of an unusual extrachromosomal RNA DNA molecule known as msDNA. Each composed of a short single strand of RNA and a short single strand of DNA, msDNAs vary considerably in their primary nucleotide sequences, but all share certain secondary structural features, including the unique 2',5' branch linkage that joins the 5' end of the DNA chain to the 2' position of an internal guanosine residue of the RNA strand. It is proposed that msDNA is synthesized by reverse transcription of a precursor RNA transcribed from a region of the retron containing the genes msr (encoding the RNA portion) and msd (encoding the DNA portion) and the ORF (encoding the RT). The precursor RNA transcript folds into a stable secondary structure that serves as both the primer and the template for the synthesis of msDNA. The msDNA-retron elements of E. coli are found in less than 10% of all strains observed, are heterogeneous in nature, and have an atypical aminoacid codon usage for this species, suggesting that this element was transmitted to E. coli by some other source. The presence of directly repeated 26-base-pair sequences flanking the junctions of the Ec67-retron of E. coli also suggests that it may be a mobile element. However, the msDNA-retrons of M. xanthus appear to be as old as other genes native to this species, based on codon-usage data for the RT genes and the fact that every strain of M. xanthus appears to have the same type of msDNA. If the msDNA-retron element originated with the myxobacteria, it would place the existence of retrons before the appearance of eukaryotic cells, suggesting that the bacterial element is perhaps the ancestral gene from which eukaryotic retroviruses and other retroelements evolved.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709508 TI - Palliative radiotherapy in plasma cell myeloma. AB - Pain symptoms caused by bone lesions of multiple myeloma can be relieved by a local irradiation treatment. To estimate the influence of systemic treatment on the palliative effect of local radiotherapy the records of 70 myeloma patients treated with chemotherapy combined with or followed by local irradiation were reviewed. The local response rate, defined as complete pain relief at the irradiated site, was 80% in patients receiving irradiation during chemotherapy (melphalan and prednisone) and this palliative effect endured 31.8 +/- 3.6 months. If irradiation was started in the period without systemic treatment the local response rate was 39.6% and lasted 24.8 +/- 17.9 months. In sites treated with more than one radiotherapy course 94% response rate after the first treatment, 56% after the second treatment and no response after the third course was achieved. The duration of local pain control was positively related to the applied radiation dose. It is concluded that irradiation during concomitant chemotherapy is superior to radiotherapy performed in a period without systemic treatment. Local long-term palliation can only be achieved by a sufficient high radiation dose. PMID- 1709509 TI - Diacylglycerol accumulation is involved in the potentiating effect of A23187 on carbachol-stimulated amylase secretion from parotid gland. AB - We studied the role of sn-1, 2-diacylglycerol (DAG) accumulation in the potentiating effect of A23187 on carbachol (CCh)-stimulated amylase secretion from rat parotid acinar cells. A23187 (0.1 microM) linearly increased DAG accumulation with time for at least 30 min. At concentrations higher or lower than 0.1 microM, DAG levels increased for up to 20 min, but declined at 30 min. Dose-response curve for DAG accumulation induced by A23187 was similar to that for amylase secretion. A23187 augmented CCh-stimulated DAG accumulation and amylase secretion. These results suggest that DAG accumulation is involved in the potentiating effect of A23187 on CCh-stimulated amylase secretion. PMID- 1709510 TI - [Production, purification and action mechanism of interferon]. AB - Interferon-alpha is in clinical use for approximately 10 years. Large-scale cell culture and genetic technology had to be developed in the seventies in order to provide sufficient amounts of pure substance for meaningful clinical studies and analysis of modes of action. For its specific effects interferon-alpha has first to bind to specific receptors on the cell membrane. The resulting trans cytoplasmic signals induce a series of biochemical and cellular events, directly or indirectly responsible for anti-tumor or anti-viral effects. Essential initial events are the induction of synthesis of several new proteins such as 2',5'-oligo A-synthetase, protein-kinase P1 and major histocompatibility-complex antigens (MHC). Further effects comprise a modulation of the cell cycle, cytostatic effects, induction of differentiation as well as modulation of oncogene expression. Finally immunomodulating effects with effects on the monocyte/macrophage system, natural killer cells and indirectly cytotoxic T-cells have been noted. These effects are illustrated by clinical examples such as chronic viral hepatitis, hairy-cell leukemia and chronic myelogenous leukemia. PMID- 1709511 TI - [The efficacy of drug therapy in structural lesions of the hair and in diffuse effluvium--comparative double blind study]. AB - Growth and quality of hair was studied after treatment with Pantogar, another prescription (Verum-2) and placebo for four months in 60 patients with diffuse effluvium capillorum and agnogenic structural alternations of hair. Efficacy was assessed by measurements of swelling, dye-binding and thickness for hair-quality and evaluation of hair-density and trichograms for hair-growth. Statistical analysis of swelling properties and trichogram data indicated that Pantogar was effective, the second preparation improved quality of hair and retarded hair loss. Placebo was ineffective judged by the used parameters. Tolerance of the treatment was good and adverse effects could not be substantiated. PMID- 1709512 TI - [Chronic viral hepatitis--hope of solving a worldwide problem]. AB - Our knowledge on chronic hepatitis B and C infection has dramatically improved. For the first time it is possible, to eliminate these hepatitis-viruses at least in some patients. In addition an excellent immuno-prophylaxis is possible in hepatitis B today. However many problems are still unsolved: we still do not have a sensitive marker for hepatitis C infection and the possibility to transfer hepatitis C by blood transfusion will remain. The recurrence-rate of chronic hepatitis C after stopping treatment with Interferon is very high and many patients with hepatitis B do not respond to Interferon-treatment. These problems are especially great in third world countries. New hope comes from the possibility to successfully immunize babies immediately after birth and from the successful production of vaccine with gene technology. PMID- 1709513 TI - [Anti-allergy treatments: various observed errors]. PMID- 1709514 TI - [ Non-A, non-B hepatitis: current developments--HCV, HEV and others]. PMID- 1709515 TI - Bromotrichloromethane hepatotoxicity. The role of stimulated hepatocellular regeneration in recovery: biochemical and histopathological studies in control and chlordecone pretreated male rats. AB - It has been shown that BrCCl3 is a more potent hepatotoxin than CCl4. Pretreatment with nontoxic dietary levels of chlordecone (CD) results in amplification of BrCCl3 hepatotoxicity. The objective of this research was to investigate and compare the histopathological alterations during a time course after a low dose of BrCCl3 alone and in combination with dietary CD. Male Sprague Dawley rats were maintained on 10 ppm dietary CD or normal diet for 15 days. On day 16, they received a single ip dose (30 microliters/kg) of BrCCl3 in corn oil (CO) vehicle or corn oil alone. Blood and liver samples were collected at 0, 3, 6, 12, 24, 36, 48, 72, 96, and 120 hr for serum enzymes and histopathological examination, respectively. Serum enzymes (SDH, ALT, AST) were significantly (p less than 0.05) elevated in rats receiving the CD + BrCCl3 combination in comparison to BrCCl3 alone. For 48 hr, a continuous increase in serum enzyme activities was detected in rats treated with CD + BrCCl3 combination, but not in the rats receiving other treatments (ND + BrCCl3, ND + CO, or CD + CO). The most extensive hepatolobular necrosis was observed in rats treated with the CD + BrCCl3 combination. Thirty-six hr after the administration of BrCCl3 to rats maintained on normal diet, high mitotic activity was observed, which continued through 72 hr resulting in complete restoration of hepatolobular structure. In contrast, rats receiving the combination of CD + BrCCl3 exhibited minimal and belated hepatomitotic activity for a short period of time, resulting in progressive hepatic failure, culminating in animal death. In conclusion, hepatotoxicity of a low dose of BrCCl3 alone appeared to be overcome via stimulated hepatocellular regeneration and hepatolobular restoration. CD appears to amplify BrCCl3 hepatotoxicity via interference with this hormetic mechanism, permitting a progressive and continued hepatic injury leading to complete hepatic failure, culminating in animal death. PMID- 1709516 TI - Increased frequencies of the CD29 and CD57 markers and decreased frequency of CD45RA within CD4+ and CD8+ subsets after allogeneic bone marrow transplantation in man. AB - Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4+ and CD8+ lymphocytes into CD4+ CD45RA+, CD4+ CD29+, CD8+ CD45RA+ and CD8+ CD29+ subsets. The CD4+ CD45RA+, CD4+ CD29+ and CD8+ CD45RA+ possess suppressor inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogenic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45-227 days after the procedure. The fraction of CD4+ lymphocytes was significantly (P = 0.0005) decreased to 20 +/- 9% versus 43 +/- 3% in controls. Within the CD4+ compartment, we found an increase in the fraction of CD4+ cells that co-expressed CD29 (CD29+/CD4+) to 92 +/- 10% versus 48 +/- 15% (P = 0.008) in controls and a concomitant decrease in CD45RA+/CD4+ to 16 +/- 12% versus 56 +/- 25% (P = 0.008). Patients were also noted to have an increase in the percentage of CD8+ lymphocytes to 41 +/- 5% compared to 23 +/- 4% in controls (P = 0.0004). Examination of the CD8+ subsets revealed a significant increase in the CD29+/CD8+ fraction to 97 +/- 3% versus 64 +/- 2% in controls (P = 0.008) and a decrease in the CD45RA+/CD8+ fraction to 36 +/- 11% versus 70 +/- 21% (P = 0.008). The number of cells co-expressing CD57 were also determined within the CD4+ and CD8+ subsets. In patients CD57+/CD4+ were increased to 29 +/- 7% versus 1 +/- 1% in controls (P = 0.04), and CD57+/CD8+ to 49 +/- 12% versus 23 +/- 9% (P = 0.02). Since CD29+ and CD57+ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation. PMID- 1709517 TI - Analysis of type II collagen reactive T cells in the mouse. II. Different localization of immunodominant T cell epitopes on heterologous and autologous type II collagen. AB - The specificity of the recognition of type II collagen (CII) by T cells in the DBA/l mouse was analysed using fragments of chick and rat CII obtained by cyanogen bromide (CB) cleavage. Firstly, DBA/l mice were immunized with chick CB fragments 5, 8, 9, 10, 11 and 12. Ten days later the draining lymph node cells were cultured with rat and mouse CII and the proliferative response was determined by incorporation of [3H]thymidine. All peptides were capable of triggering T cells recognizing rat CII but only CB9 immunized mice responded well to mouse CII. Secondly, lymph node cells from DBA/l mice immunized with rat and mouse CII were cultured with the CB fragments, including rat CB10 and CB11, and the proliferative response was determined. After immunization with rat CII, the response was strongly dominated by T cells recognizing CB11 with equal responses against chick and rat CB11. After immunization with mouse CII only rat CB10 gave a strong response. It is concluded that several epitopes on the CII molecule can be recognized by T cells in the DBA/l mouse and that most of these epitopes are shared by rat and chick CII but not mouse CII. These epitopes exhibit strong immunodominance. In mice immunized with intact heterologous CII, the immunodominant response is directed against one or more epitopes on the CB11 fragment present on several heterologous CII but apparently not on mouse CII. In mice immunized with autologous CII the immunodominant response is directed against one or more epitopes on the CB10 fragment, present on rat and mouse CII. They are either absent in chick CII or located in the carboxyterminal end of the CB10 fragment where a cyanogen bromide cleavage site is present in chick CII but not in rat CII. These results suggest that the proposed importance of CB11 in collagen-induced arthritis is due to activation of T cells reactive with heterologous CII only. These cells may be important for the induction of the strong auto-antibody-response after immunization with heterologous CII. Structures of importance for direct T cell involvement in the arthritic process and recognized by autoreactive T cells are suggested to be found on CB10. PMID- 1709518 TI - Natural killer (NK) cell activity in mice with acute graft-versus-host reactions: characterization of a Thy-1+ NK-like cell with a broadened spectrum of lytic activity in the spleen and lymph nodes. AB - We have shown that acute (GVH) reactions produced in the parental-F1 hybrid combination, A/J----(C57BL/6 x A/J)F1 result in the activation of two cytotoxic cell populations: a host-derived Thy-1+/- natural killer (NK) cell with a lytic spectrum confined to YAC-1 targets, and a donor-derived Thy-1+ NK-like cell that has the ability to lyse target cells that are normally insensitive to lysis by NK cells. Cold-target inhibition (CTI) experiments have shown that the greater range of target cell killing seen in the NK-like population is mediated by a single effector cell with a broadened lytic repertoire. Percoll density fractionation studies have revealed that NK and NK-like cells co-fractionate, suggesting that both are large granular lymphocytes. We we have also shown that NK-like cells do not express either Lyt-2 or L3T4 markers. We have also observed that there is a close temporal relationship between elevated levels of interleukin-2 (IL-2) secretion by spleen cell cultures from mice with GVH disease and the subsequent emergence of splenic NK activity in both acute [A/J----(C57BL/6 x A/J)F1] and chronic (A/J----CBA x A/J)F1 GVH reactions. We have also noted that, despite high levels of IL-2 secretion, mice with chronic GVH reactions do not generate NK-like activity. Interferon (IFN) measurements have shown that, although increased IFN activity can be detected in both acute and chronic models, a preponderance of IFN alpha/beta and some IFN-gamma is produced in the acute reaction, whereas only IFN gamma can be detected in the chronic model. These results suggest that, although IL-2 may participate in augmenting conventional NK activity, IL-2 by itself does not generate NK-like activity. We suggest that IFN-alpha/beta may be the cytokine that, either alone or in concert with IL-2, triggers the NK-like cell response. The NK-like cell described in our study resembles a phenotypically identical, donor-derived large granular lymphocyte, identified by others, in close proximity to dead or dying epithelial cells in mice with GVH disease [14]. It has been suggested that these cells may mediate tissue injury. If in fact these graft derived NK-like cells are involved in the pathogenesis of acute GVH disease, our present findings suggest that they must first be activated by an appropriate complement of cytokines that includes IFN-alpha/beta.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709519 TI - The leukocyte protein L1 in plasma and synovial fluid from patients with rheumatoid arthritis and osteoarthritis. AB - L1 is a major granulocyte and monocyte protein, released during activation and turnover of such cells. Blood and synovial fluid (SF) from 41 patients with rheumatoid arthritis (RA) and 6 patients with osteoarthritis (OA), were analyzed for L1 and the acute phase proteins C-reactive protein, orosomucoid, haptoglobin, alpha 1-antitrypsin and albumin as well as for differential leukocyte count. L1 levels in plasma and SF showed highly significant differences (p less than 0.0001), between the RA and OA patients. All the OA patients had normal plasma concentrations of L1 and low concentrations of L1 in SF. All the RA patients had elevated plasma levels of L1 and high L1 concentrations in SF. In the RA patients, the ratios between the protein concentrations in SF and blood were 3.29 for L1 and less than or equal to 0.64 for the acute phase proteins. In the SF, the L1 levels did not correlate with the monocyte count, while a low, positive correlation was found between L1 and the granulocyte count. The high L1 concentrations observed in SF from RA patients probably reflected an increased turnover of leukocytes in the inflamed joints. In SF from RA patients, high L1 concentrations were found in joints with a high amount of swelling. The present study suggests that L1 may represent a marker of both local and systemic inflammation. PMID- 1709520 TI - Collagenase synthesis of rheumatoid arthritis synoviocytes: dose-dependent stimulation by substance P and capsaicin. AB - The synthesis and release of collagenase in the presence of the neuropeptide substance P (SP) and capsaicin, were investigated in vitro using identical synoviocyte cultures from patients with rheumatoid arthritis (RA). On average 10( 12) M SP augmented statistically significantly the collagenase production by approximately a factor of five. An increase in the concentrations up to 10(-6) M SP resulted in a decreased collagenase synthesis, which, however, was still above the level of that of the untreated synoviocytes. Capsaicin, a homovanillic acid derivative that acts as a releaser of SP from primary afferent neurons, caused a strong stimulation of collagenase production and release at 10(-8) and 10(-6) M (about 7 times the amount of the control). With increasing concentrations up to 10(-3) M capsaicin this effect diminished continuously. The experiments clearly show that in RA synoviocytes in vitro SP and capsaicin in low concentrations act as potent inducers of the synthesis and release of collagenase. PMID- 1709521 TI - [Human interferons: biological and clinical promises and premises]. AB - Interferons have been demonstrated by animal experimentation to have antiviral, antitumoral and immune effects. Clinical trials of human recombinant alpha interferon have shown a substantial therapeutic effect in various chronic leukemias and lymphomas. Studies concerning the mechanisms of the anti-leukemia effect of the various interferons have demonstrated that they play an important role in regulating the proliferation of normal and leukemic cells. These lymphokines interact with other important biological mediators such as interleukins, growth factors, factors associated with angiogenesis, etc. New therapeutic developments in oncology and in immunopathology may be expected in the near future. PMID- 1709522 TI - Arginine-mediated RNA recognition: the arginine fork. AB - Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R9) also bound specifically to TAR, and a mutant Tat protein that contained R9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K9) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine eta nitrogens and the epsilon nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures. PMID- 1709523 TI - Improved liver preservation for transplantation due to calcium channel blockade. AB - Intracellular calcium is an important determinant for cell death in organ hypothermic preservation for transplantation. In this study, we show that prevention of calcium entry improves the result of liver cold storage in UW solution. The isolated perfused rabbit liver was used. After 48 hr of cold storage in UW solution, bile production was reduced by 70% (P less than 0.005). However, by adding the calcium channel blockers verapamil or nifedipine (40 microM) to the UW solution, this reduction was abolished, and the livers produced the same amount of bile as unpreserved livers. Furthermore, addition of the calcium channel activator, BAY K8644 (40 microM), to the UW solution, reduced bile production by 50% (P less than 0.01) already after preservation for 24 hr. We conclude that calcium entry is of importance for liver function after preservation and cold storage, and that including a calcium channel blocker to the preservation solution makes long-term liver preservation safer. PMID- 1709524 TI - Immortalized neurons derived from tumors in transgenic mice. PMID- 1709525 TI - Neuroscience in Czechoslovakia. AB - The collapse of the communist regime in Czechoslovakia revealed not only the disastrous state of the Czechoslovak economy but also the serious problems that Czechoslovak science faces. Understanding the present situation and determining the best way to improve it requires careful analysis of the development of science during the past 40 years. PMID- 1709526 TI - The importance of the blood-brain barrier in fetuses and embryos. PMID- 1709527 TI - A role for GABAB receptors in excitation and inhibition of thalamocortical cells. AB - Gamma-aminobutyric acid (GABA) in the thalamus has mainly been associated with the inhibitory modulation of the sensory and cortical flow of information via a 'classical', chloride-dependent, GABAA receptor-mediated action. However, the discovery of a late, long-lasting potassium-dependent inhibitory postsynaptic potential (IPSP) mediated by GABAB receptors present on thalamocortical cells, has allowed new insights into our understanding of the physiological role of this neurotransmitter. In particular, work on the dorsal lateral geniculate nucleus indicates that together with a relatively weak inhibition, GABAB receptor mediated IPSPs 'prepare' thalamocortical cells for burst firing by activating low threshold calcium potentials. Thus, GABA in the thalamus can no longer be viewed only as a 'classical' inhibitory transmitter but also as a neuromodulator with a 'priming' role for burst firing excitation. This dual role of GABAB receptors in inhibition and excitation of thalamocortical cells might allow different interpretations of earlier findings in animals and humans, both in healthy and pathological conditions. It will also help to identify new functions for postsynaptic GABAB receptors in other parts of the central nervous system. PMID- 1709528 TI - Dopaminergic innervation of the cerebral cortex: unexpected differences between rodents and primates. AB - Until recently, views on the organization and role of the mesotelencephalic dopaminergic (DA) systems were mostly based on studies of rodents, and it was assumed that homology existed across mammalian species. However, recent studies of both human and non-human primates indicate that this might not be so. The mesocortical DA system in primates, which is directly involved in the pathophysiology of severe illnesses such as Parkinson's disease and psychoses, shows substantial differences from that of rodents. These differences include much larger, re-organized terminal fields, a different phenotype for the co localization of neuropeptides and a very early prenatal development. PMID- 1709529 TI - Activity-dependent modulation of nerve terminal excitation in a mammalian peptidergic system. AB - Transmitter release at nerve terminals is triggered by voltage-gated calcium influx upon arrival of an action potential. Changes in coupling between axonal impulse discharge and depolarization of the nerve ending might therefore regulate exocytotic secretion. In the magnocellular neurosecretory system of the rat, the pattern of electrical activity can modify the duration of action potentials within individual nerve endings, and alter the spatial spread of excitation into the branching terminal field of neurohypophysial axons. The observed effects are consistent with the effects of firing rate and pattern on peptide release, indicating that activity-dependent changes in axon-terminal coupling may modulate secretion in this neuroendocrine system. PMID- 1709530 TI - Migraine: a research field matured for the basic neurosciences. AB - Progress in migraine research has been rapid in recent years, from both the basic science and the clinical perspectives. A new internationally accepted headache classification with operational diagnostic criteria was published in 1988, eliminating much diagnostic uncertainty. More than a decade of study of regional cerebral blood flow (rCBF) has gradually shown a pathognomonic pattern of abnormalities, probably reflecting spreading cortical depression. Recently it has been shown that pain probably arises from excitation of perivascular pial arterial nociceptors. The innervation and receptor mechanisms of pial and extracranial arteries have been worked out in detail both in animal and humans. Involvement of calcitonin gene-related peptide (CGRP) and 5-hydroxytryptamine (5 HT) during migraine attacks has been demonstrated. A new and specific 5-HT1D receptor agonist has proved to be highly effective in treating migraine. Therefore, major research efforts recently have been concentrated on discovering the location and function of 5-HT1D receptors, extra- and intracranially. Thus, it is now possible to formulate useful neuroscientific research strategies aimed at clarifying migraine mechanisms. PMID- 1709531 TI - Plasticity of auditory maps in the brain. AB - The central auditory pathway contains maps of sound frequency that reflect the functional organization of the cochlea, as well as topographic representations of other stimulus features, such as sound location, that are synthesized within the brain. Both types of map undergo changes during development and are shaped by experience. This is particularly true of the representation of auditory space in the superior colliculus, which can be modified by alteration of auditory and visual inputs early in life. Although experience-induced plasticity in this map is restricted primarily to the developmental period, the frequency representation in the cortex of adult animals can re-organize following partial deafness. PMID- 1709532 TI - Does anesthesia cause loss of consciousness? AB - Although about 30 million operations carried out under general anesthetic are routinely performed each year in the USA alone, it is not possible to determine reliably whether or not a given anesthetized patient is conscious during surgery. As a result, some patients may be either partially aware during the operation or may be able to recall some aspects of it afterwards. It is therefore crucial to develop some experimental means to evaluate the state of consciousness of the anesthetized patient. Recent developments suggest that 40 Hz neuronal oscillations might help to solve this problem. PMID- 1709533 TI - Benzodiazepines, putative anxiolytics and animal models of anxiety. AB - Anxiety is a complex state that includes a broad range of classified symptoms. Anxiolytic treatment has been dominated by the use of benzodiazepine drugs (BZs), which is often prolonged. However, the multiplicity of anxiety states and the lack of BZ specificity preclude the potent targeting of the drugs to different states, and point to the problem of drug dependence. Furthermore, animal models of anxiety give different results when challenged with apparently similar drugs. The search for alternative drugs with a higher specificity has led to 5-HT receptor subtypes. In this review, Simon Green describes how recent research might have dual benefits: in improving drug therapy of anxiety in humans, and in analysing and categorizing more clearly the concept of anxiety itself. PMID- 1709534 TI - Activity-dependent plasticity in the visual systems of frogs and fish. AB - The retinotectal system of lower vertebrates has provided considerable insight into the cellular mechanisms underlying the development and maintenance of orderly visual projections to the brain. This review will briefly summarize some of the data on the activity-dependent components of these mechanisms and incorporate the data into a model for selective synapse stabilization of coactive synapses. The model, based on the Hebbian synapse, is similar to models of long term potentiation (LTP) of synaptic transmission, which are thought to account for the increased synaptic efficacy observed after associative conditioning paradigms. However, more recent data from two studies, one using confocal microscope analysis of migrating retinal arbors in vivo and the other investigating the requirement for protein kinase activity in map formation, point to a possible divergence in the cellular events underlying synapse stabilization in the developing visual system of the frog and LTP in the mammalian hippocampus. PMID- 1709535 TI - Subjective contours--bridging the gap between psychophysics and physiology. AB - Much is known about the initial stages of visual processing up to the striate cortex, but how is visual information represented and handled at subsequent stages? Phenomena of contour, color and movement perception have been used to identify functions of neurons and to reveal functional differences between cortical areas that application of classical receptive-field concepts has not suggested. These differences can be related to theoretical stages of visual processing that provide stability of perception under changing conditions of stimulation. PMID- 1709536 TI - The human stretch reflex and the motor cortex. AB - The spinal stretch reflex, exemplified by the tendon jerk, appears to be less important in humans than a delayed 'long-latency' response. This is easily observed when muscles of the hand are stretched while they are already contracting voluntarily. On limited evidence, many have long held that the delayed response is a transcortical reflex and have tended to neglect alternative possibilities, particularly that it might be a spinal reflex dependent upon slow afferents. New experiments have now eliminated the alternatives, leaving the transcortical hypothesis in command of the field. PMID- 1709537 TI - Neuroscience in Hungary. PMID- 1709538 TI - Basal ganglia research and Tourette's syndromes. PMID- 1709539 TI - 'Silent' nociceptors in the skin. PMID- 1709540 TI - Direct regulation of ion channels by fatty acids. AB - A variety of fatty acids regulate the activity of specific ion channels by mechanisms not involving the enzymatic pathways that convert arachidonic acid to oxygenated metabolites. Furthermore, these actions of fatty acids occur in patches of membrane excised from the cell and are not mediated by cellular signal transduction pathways that require soluble factors such as nucleotides and calcium. Thus, fatty acids themselves appear to regulate the action of channels directly, much as they regulate the action of several purified enzymes, and might constitute a new class of first or second messengers acting on ion channels. PMID- 1709541 TI - [A cryoultratomy method and its use in immunocytochemistry]. AB - A method of frozen ultrathin sectioning for tissues and suspension material is presented. Practical aspects and results in the field of cryoultratomy are observed in detail. An example of a good preservation of tissue ultrastructure and application of cryosections to immunocytochemical investigations is given. PMID- 1709542 TI - [The detection and nature of the labelling of F-actin using a phalloidin colloidal gold marker]. AB - On the basis of a conjugate of phalloidin-colloidal gold a marker has been obtained for electronmicroscopic identification and study of marking character of filament actins. Using this marker the filamentous form of actins from rabbit skeletal muscles and chicken gizzard was revealed. The marking characters of the two actins appeared to differ at the level of electron microscopy. A periodicity of the marker setting with a step of 105.4 +/- 1.7 nm on the rabbit F-actin was revealed. A possibility of differential evaluation of the filamentous and globular forms of the actins was shown when using the same marker in dot-blot analysis procedure with the sensitivity from 15 to 50 ng. PMID- 1709543 TI - Identification of epitope(s) on the internal virion proteins of rinderpest virus which are absent from peste des petits ruminants virus. AB - Monoclonal antibodies (MAb) raised against the RBOK vaccine strain of rinderpest virus were characterized by radio-immunoprecipitation (RIPA) and in the indirect ELISA using measles (MV), distemper (CDV), rinderpest (RPV) and peste des petits ruminants viruses (PPRV). Those found to be specific for the matrix (M) protein and the nucleocapsid (N) protein could be classified into different groups on the basis of the anti-morbillivirus MAb classification scheme; a number of these MAb showed a selective recognition of RPV, measles virus and distemper virus, or of different isolates of rinderpest virus, demonstrating that greater inter-isolate variation occurs than was apparent from analyses using polyclonal antisera. One group of anti-F protein MAb (group F1) reacted with all isolates of both RPV and PPRV. A second group of anti-N protein MAb (group N1/A) reacted with all RPV isolates, but not with the PPRV isolates. Furthermore, these group N1/A antibodies reacted strongly with RPV isolates which were upon original isolation of high pathogenicity, but had a weaker reaction against the isolates of this virus which were of low pathogenicity. Thus, MAb against RPV, in particular those against the N protein offered a potential superior to that of molecular analyses for "isolate fingerprinting", the differentiation of RPV from PPRV and the discrimination between rinderpest viruses which had been, upon isolation, of either high or low pathogenicity. PMID- 1709544 TI - Opsonic monoclonal antibodies against lipopolysaccharide (LPS) antigens of Pasteurella multocida and the role of LPS in immunity. AB - A panel of six monoclonal antibodies (MAbs) produced from mice immunized with Pasteurella multocida (M1404) (Heddleston serotype 2) reacted with homologous lipopolysaccharide, as indicated by enzyme immunoassay and immunoblotting. All six MAbs reacted with serotypes 2 and 5 of the 16 Heddleston serotypes. The reactive epitopes were localized on the bacterial cell surface by immunogold labelling. The antibodies could agglutinate P. multocida only if cells were first treated with 1 N HCl. All six MAbs opsonized P. multocida for phagocytosis by mouse macrophages but were not bactericidal in the presence of complement. They afforded only partial protection against infection in mice. The results, together with those of active immunization experiments with LPS, suggest a subordinate role for LPS in protection from experimental infection in mice. PMID- 1709545 TI - Prospective study on the hepatitis safety of intravenous immunoglobulin, pH 4.25. AB - Recent reports of transmission by intravenous gamma-globulin preparations of non A non-B hepatitis (NANBH), including several cases that progressed to severe liver damage and death, have raised concerns about the safety of intravenous gamma-globulin. However, the problem does not seem to be widespread. To assess this issue, we previously reported the results of liver function tests monitored in 41 patients with primary immunodeficiency treated with intravenous immunoglobulin (IGIV), pH 4.25 over periods ranging from 6 to 15 months. Eighteen of these patients at two of the three centers have now had serial serum glutamic pyruvic transaminase (SGPT) levels performed regularly at intervals of 1-5 weeks while continuing monthly intravenous infusions of nonmodified IGIV, pH 4.25 for an additional 14-26 months. The standard dosage was 400 mg per kg body weight IGIV, pH 4.25. Six lots of IGIV, pH 4.25 were used. Transient minor SGPT elevations were observed in 5 of the patients on a total of 8 occasions. None of the elevations was considered indicative of NANBH or of any chronic hepatic disease. All patients remained negative for hepatitis B surface antigen throughout the study. PMID- 1709546 TI - [Computer-assisted optimal regulation of combustion engines at work sites]. AB - This is a report about the fundamentals of computerised optimation of duration of operation and air exchange for the use of working machines and cars with internal combustion engines in workrooms. The computer programme 'EMI', which bases on the corresponding national standard TGL 33,358/01 to /03, is suitable for the dialog work with the operator. On the foundation of the ensuring compliance with the maximum working place concentration values MAKK and MAKD it is possible to optimize the contrary requirements like air exchange and duration of operation of the internal combustion engines. The programme can be used for GDR-usual computers. It is possible to order the computer programme 'EMI' including user hints at Mansfeld Industrieanlagen Dresden. PMID- 1709547 TI - Inhibitory effects of ionophore A23187 on the release of thyroid hormone and colloid reabsorption in mouse thyroid glands. AB - The effect of the ionophore A23187 on a. the release of thyroid hormone from perifused mouse thyroid glands and b. the morphological changes in follicular epithelial cells was evaluated. A23187 at a concentration of 5 mumol/l significantly inhibited both the TSH- and the forskolin-stimulated release of T3 and T4. In the presence of 3-isobutyl-1-methylxanthine or (4-(3-butoxy-4 methoxybenzyl)-2-imidazolidinone) RO 20-1724, A23187 did not affect the forskolin stimulated release of cAMP, but did inhibit the release of T3 and T4 stimulated by forskolin. Light and electron microscopic evaluation of the follicular epithelial cells of mouse thyroid tissues following 1-h stimulation with forskolin showed numerous pseudopods engulfing luminal colloid of various size and the presence of reabsorbed colloid droplets in the apical cytoplasm. Quantitative electron microscopic analysis revealed that the addition of the ionophore A23187 reduced the number of reabsorbed colloid droplets to one eighth in follicular epithelial cells. These observations suggest that the increase in intracellular Ca2+ induced by the ionophore A23187 inhibits the TSH-stimulated thyroid hormone release independently of the cAMP level, and that the suppression of thyroid hormone release may be due to an inhibition of colloid reabsorption. PMID- 1709548 TI - Histo- and immunohistochemical changes in acetylcholinesterase and choline acetyltransferase activities in the amygdaloid complex in aged rats. AB - Histo- and immunohistochemical distribution of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the amygdaloid nuclei of young adult (3 month old) and aged (26 month old) Wistar rats was compared. AChE staining and ChAT immunoreactivity showed the same regional variations in the amygdaloid nuclei of young adult rats. The density and staining of AChE- and ChAT-positive fibres, terminals, and nerve cells were reduced in aged rat amygdala. Moreover, heavily stained aberrant fibres and coarse terminals were located around the nerve cells, blood vessels, and occasionally in patches. In aged rats, atrophic AChE positive and ChAT immunoreactive nerve cells exhibited serpentine-like, thicker, and less extensively branched dendrites than those in young adult rats. These changes are similar to the age-related changes in the cholinergic enzymes in other brain regions which are targets to the basal forebrain. PMID- 1709549 TI - Immunohistochemical localization of lysozyme, lactoferrin, alpha 1 antichymotrypsin, and alpha 1-antitrypsin in salivary gland of human fetuses. AB - 26 human fetuses were examined to elucidate the immunohistochemical distributions of lysozyme, lactoferrin, alpha 1-antichymotrypsin, and alpha 1-antitrypsin in prenatal salivary glands. Development of fetal salivary glands was divided into 4 stages: The early developmental stage (EDS), the early intermediate developmental stage (EIDS), the late intermediate developmental stage (LIDS), and the late developmental stage (LDS) and were used to compare antigen localization during salivary gland development. Lysozyme (LY) staining was prominent in serous or demilune cells of the mucous acinar compartment. Lactoferrin (LF) was rarely seen in the fetal glands; only trace amounts were seen in serous cells, alpha 1 antichymotrypsin (alpha 1-ACT) was diffusely positive particularly in glandular ducts, alpha 1-antitrypsin (alpha 1-AT) was also diffusely distributed in all salivary gland elements and was more abundant in ductal cells than acinar cells. During the EDS, immunohistochemical staining of LY, LF, alpha 1-ACT, and alpha 1 AT could be observed with glandular intensity increases corresponding to the advance of cytodifferentiation of granular epithelium occurring in the subsequent EIDS and LIDS. Staining intensities were continuous during the LDS even though the amount of those materials in the fetal salivary glands was not of the extent seen in the adult salivary gland. These results suggest that production of LY, LF, alpha 1-ACT, and alpha 1-AT was positive during prenatal development of human salivary glands. The present study discusses the protective roles and defense mechanisms of LY, LF, alpha 1-ACT, and alpha 1-AT in developing human salivary glands. PMID- 1709550 TI - Prenatal development of human major salivary glands and immunohistochemical detection of keratins using monoclonal antibodies. AB - The major salivary glands were examined from 69 human fetuses ranging from 10 to 40 weeks of gestation. Prenatal growth curves of developing salivary glands could be established by histological scoring, and development was divided into the early developmental stage (EDS) from 10 to 18 weeks, early intermediate developmental stage (EIDS) from 19 to 24 weeks, late intermediate developmental stage (LIDS) from 15 to 32 weeks, late developmental stage (LDS) from 33 to 40 weeks. Characteristic morphogenesis and cytodifferentiation occurred in glandular duct cells during the period of EIDS and LIDS. In the LDS, acini and ducts of the salivary glands histologically developed into a mature state similar to adult glands. Immunohistochemical staining with monoclonal antibodies (MoAbs) PKK1, KL1, K8.12, K8.13, K4.62, RPN 1160, 1162, 1163, 1164, and 1165 was performed. During the fetal period, keratin expression as revealed by MoAbs PKK1, KL1, K8.12 was well established, and the staining pattern for each of these antibodies was comparable. Other antibodies showed rare or negative staining except K8.13 which had a diffuse, non-specific staining pattern. Accordingly, the proliferation and cytodifferentiation of fetal stage keratin staining in ductal cells as revealed by MoAbs PKK1, KL1, and K8.12 showed a heterogenic distribution in both luminal and basal cells. It is a characteristic finding that the cytodifferentiation of ductal luminal cells precedes ductal basal cells. Ductal basal cells stained with MoAb K8.12 and show heterogeneity of keratin distribution continuously until the full term of gestation. The keratin staining of oral epithelium was also examined to compare with distribution of salivary gland ductal cells and oral epithelial cells. In the present study, the developmental sequence of salivary gland cells and the immunohistochemical properties of keratin proteins in these cells were described in relation to the histogenesis of salivary gland tumours. PMID- 1709551 TI - Reproducibility of monoamine metabolites in human CSF. PMID- 1709552 TI - [Acoustic neuroma: a histopathological study]. AB - In view of recent controversy concerning the preservation of hearing in acoustic neuroma surgery, a histologic study of the nerve-tumour interface was undertaken in order to investigate cochlear nerve involvement. Twelve intact acoustic neuromas were studied by means of Masson's trichrome stain, the Luxol fast blue technique, Verhoeff's stain and an immunohistochemical technique using monoclonal antibodies to human neurofilaments. In nine out of twelve specimens, macroscopically visible adherences were present between the cochlear nerve and the tumour. The microscopical correlate was found to be the absence of a clear cleavage plane between the cochlear nerve and the tumour on the one hand, and the presence of cochlear nerve fibers, surrounded by tumoural cells, past the assumed nerve-tumour interface on the other hand. PMID- 1709553 TI - Tubular adenoma of the gallbladder with squamoid spindle cell metaplasia. Report of three cases with immunohistochemical study. AB - Three cases of tubular adenoma of the gallbladder with squamoid spindle cell metaplasia are reported. Two of the three patients, who were middle-aged Japanese, had a solitary polyp in the gallbladder, and the other had three polyps. All the lesions were detected incidentally by ultrasonography. The polyps were pedunculated with a fine or frail stalk, and ranged from 0.5 to 3.9 cm in diameter. Histologically, they were tubular adenomas accompanied by scattered foci composed of a compact collection of short-spindle or oval cells with mild atypia. These cells did not retain intercellular bridges, and lacked intracellular keratinization. Immunohistochemically, the spindle cells stained positively for high-molecular-weight cytokeratin (EAB 903, a marker of squamous cell differentiation), whereas adenoma cells lining the tubules were negative for this antigen. Therefore, the spindle cell component is considered to represent squamoid metaplasia of adenoma cells. PMID- 1709554 TI - Immunohistochemical localization of five classes of intermediate filament in a benign pelvic soft tissue tumor of rhabdoid appearance. AB - A benign pelvic soft tissue tumor from a 50-year-old woman was examined by immunohistochemistry and electron microscopy. The tumor cells had abundant eosinophilic cytoplasm with a hyaline appearance, which was filled with large aggregates of intermediate-sized filaments (IF). The cells were positively immunostained by antibodies against cytokeratin, vimentin, desmin, glial fibrillary acidic protein, and neurofilament proteins. This case represents an extreme example of the simultaneous expression of IF by neoplastic cells, and exemplifies the limited applicability of immunohistochemical detection of IF antigens for pathological diagnosis of neoplasms. PMID- 1709555 TI - Hydroxyethyl-starch in transient experimental focal cerebral ischemia. AB - In a model of focal cerebral ischaemia in the cat (transorbital occlusion of the middle cerebral artery for 60 minutes, thereafter 6 hours reperfusion by clip removal), hydroxyethyl-starch (HAES) (ELOHES; Leopold Pharma GmbH, Graz, Austria) was administered intravenously before and during the ischaemic episode as a 6% or as a 10% solution in a randomised manner (6 animals each group). The size of the developing cerebral infarct was not significantly different when comparing the 6% and the 10% group with the controls (SALINE). Collateral circulation to the infarct border (pial arteries on the suprasylvian gyrus) was also not significantly different between the two groups, except for the first hour of reperfusion, where vessels of the 6% group were wider than vessels of the 10% group. At the infarct border (ectosylvian gyrus) small resistance vessels were significantly more dilated in the 6% than in the 10% group both during the occlusion period and during the reperfusion episode after removal of the clip. Pial arteries dilated less in both HAES-groups than in the controls. It can be assumed, that HAES-incuded decrease of plasma viscosity led to an elevation of blood flow velocity and blood flow quantity (CBF). But the latter might be counteracted by autoregulation of CBF, i.e. vasoconstriction. Thus, a possible positive effect of HAES might in part be counteracted by autoregulation, which explains that no significant therapeutic effect could be achieved. PMID- 1709556 TI - Phase II study of fludarabine phosphate (NSC-312887) in patients with advanced endometrial cancer. A Southwest Oncology Group Study. AB - Nineteen evaluable patients with advanced endometrial cancer refractory to one prior chemotherapeutic regimen were treated with a 5-day schedule of fludarabine phosphate. No responses were noted. The major toxicity was grade 2 or greater leukopenia in 45% of patients. Fludarabine phosphate at this dose and schedule does not appear to be an active agent for patients with refractory endometrial cancer. PMID- 1709557 TI - Intra-abdominal desmoplastic small round-cell tumor. Report of 19 cases of a distinctive type of high-grade polyphenotypic malignancy affecting young individuals. AB - Nineteen cases of a distinctive type of malignant small-cell tumor are presented. The main features of the entity are as follows: a predilection for adolescent males (mean age: 18.6 years); predominant or exclusive intra-abdominal location, with only inconstant and secondary organ involvement; nesting pattern of growth; focal rhabdoid features; intense desmoplastic reaction; immunohistochemical reactivity for epithelial [keratin, epithelial membrane antigen (EMA)], neural [neuron-specific enolase (NSE)], and muscle (desmin) markers; and highly aggressive behavior. It is proposed that this represents yet another member of the continuously enlarging and evolving family of small round (blue) cell tumors of infancy and childhood that features, more than any other member of this group, the capacity for simultaneous multidirectional phenotypical expression. PMID- 1709558 TI - Neuroendocrine tumors of the lung with proposed criteria for large-cell neuroendocrine carcinoma. An ultrastructural, immunohistochemical, and flow cytometric study of 35 cases. AB - Based on our review of 35 cases and the literature, we found the spectrum of pulmonary neuroendocrine (NE) tumors to be too broad to fit into the traditional three-category classification scheme of typical carcinoid (TC), atypical carcinoid (AC), and small-cell lung carcinoma (SCLC). We found that a spectrum of high- and low-grade tumors exist between TC and SCLC and that in the past many of these tumors have been called AC. We chose to adhere to Arrigoni's definition of AC, as his original criteria characterized a low-grade tumor. For the higher grade non-small-cell tumors (NSCLC), we propose a fourth category of large-cell neuroendocrine carcinoma (LCNEC), which is characterized by: (a) light microscopic NE appearance; (b) cells of large size, polygonal shape, low nuclear cytoplasmic ratio (N:C), coarse nuclear chromatin, and frequent nucleoli; (c) high mitotic rate [greater than 10/10 high-power fields (HPF)] and frequent necrosis; and (d) NE features by immunohistochemistry (IHC) or electron microscopy (EM). Thus, after deciding that a pulmonary NE tumor is high grade, the major diagnostic issue is separation of LCNEC from SCLC. This distinction is based not only on cell size, but on a variety of morphologic features. We studied 20 TC, six AC, five LCNEC, and four SCLC and characterized the clinical, light microscopic, EM, IHC, and flow cytometric features of each type of tumor. We did not find any advantage to IHC, EM, or flow cytometry over light microscopy in the subclassification or prediction of prognosis; however, these methods were useful in characterizing these four types of pulmonary NE tumors and in demonstrating their NE properties. LCNEC must be distinguished from a fifth category pulmonary NE tumor: NSCLC with NE features in which NE differentiation is not evident by light microscopy and must be demonstrated by EM or IHC. Although the prognosis of LCNEC appears to be intermediate between AC and SCLC, larger numbers of patients will be needed to demonstrate significant differences in survival. PMID- 1709559 TI - Myoepithelial lesions of the breast. Myoepitheliosis, adenomyoepithelioma, and myoepithelial carcinoma. AB - The clinical and pathologic features of 31 breast lesions composed of a prominent proliferation of myoepithelial cells either admixed with epithelial cells or in pure form were studied. The lesions were divided into three categories: myoepitheliosis, adenomyoepithelioma, and malignant myoepithelioma (myoepithelial carcinoma); the latter is the only lesion composed purely of myoepithelial cells. Three multifocal, microscopic lesions located in the peripheral duct system were designated as myoepitheliosis. Twenty-seven solitary, grossly palpable, predominantly centrally located lesions qualified as adenomyoepithelioma. These were further subdivided into spindle-cell, tubular, and lobulated variants. Two lesions in the latter group had a carcinoma arising within them. Only one case, which was characterized by a solitary mass composed of an infiltrative spindle cell proliferation, qualified as malignant myoepithelioma (myoepithelial carcinoma). Two patients with adenomyoepithelioma developed recurrences; one tumor was of the tubular type, the other of the lobulated type. Both of these tumors had irregular margins. One of these patients had two recurrences and is currently well 8.5 years after the initial excision. The second patient developed a recurrence 8 months after initial excision; the recurrence presented as multiple nodules. One of the patients with myoepithelial carcinoma arising in an adenomyoepithelioma also developed a recurrence within 2.3 years. Her initial tumor was located in the axillary tail of the breast, and she had axillary node metastasis at the time of presentation. All remaining patients with follow-up are well without evidence of recurrence up to 17.3 years after the initial diagnosis (average follow-up, 6.1 years); one patient died of unrelated causes. PMID- 1709560 TI - Structure and function of myelin protein genes. PMID- 1709561 TI - Mechanisms of fast and slow axonal transport. PMID- 1709562 TI - Shifts of anti-Sm-specific antibodies in patients with systemic lupus erythematosus: analysis by counter-immunoelectrophoresis, immunoblotting and RNA immunoprecipitation. AB - We investigated serial changes in anti-Sm-specific antibodies by counter immunoelectrophoresis (CIE), immunoblotting (IB) and RNA-immunoprecipitation (RNA IP) in three patients with SLE. To assess the immuno-regulatory processes underlying anti-Sm antibody production, we compared changes in anti-Sm-specific antibodies with changes in titres of antinuclear and anti-ds DNA antibodies. IB and RNA-IP detected anti-Sm antibodies earlier than did CIE. The induction of the immune responses against the Sm-specific BB' and D polypeptides occurred separately in two of the three patients. Anti-Sm developed in all three cases after a flare-up of disease, whereas anti-ds DNA antibody levels fluctuated in parallel with disease activity. Thus, anti-Sm and anti-ds DNA antibody production seems to be independently regulated. PMID- 1709563 TI - Zidovudine resistance predicted by direct detection of mutations in DNA from HIV infected lymphocytes. AB - Zidovudine-resistant strains of HIV have recently been isolated from individuals during prolonged treatment. Analysis of the HIV reverse transcriptase (RT) gene from clinical isolates revealed that resistance was due to multiple nucleotide changes conferring specific amino acid substitutions in this enzyme. In order to correlate the degree of resistance with these amino acid changes, we constructed a series of infectious HIV variants with specific combinations of mutations in the RT gene and assessed their sensitivity to zidovudine. The reproducible nature of the mutations seen in clinical isolates has enabled the polymerase chain reaction to be used to identify lesions associated with resistance. This procedure was validated by analysis of sensitive and resistant clinical isolates with RT genes of known DNA sequence. Using a 'double' amplification procedure, zidovudine sensitivity was assessed by direct detection of specific mutations in DNA from peripheral-blood lymphocyte samples. This should make it possible to test large numbers of individuals receiving zidovudine therapy, with the aim of establishing the clinical significance of the resistant isolates. PMID- 1709564 TI - Characterization of protein-tyrosine kinase activity in the canine prostate. AB - Following the measurement of the phosphorylation of the substrate poly(Glu80Na,Tyr20) and the analysis of the alkali-resistant phosphorylation of endogenous proteins, the protein-tyrosine kinase of the canine prostate was partially characterized with regard to its subcellular localization, as well as certain kinetic and molecular properties. This kinase was mainly found in the cytosolic fraction (75%); however, its specific activity was similar to that of the residual enzyme present in the particulate fraction. Conditions for optimal activity of both fractions were determined. Under these conditions, several endogenous phosphoproteins (44-63 kilodaltons upon electrophoresis) were alkali resistant and phosphotyrosine was present in all of the major ones (pp63, pp57, pp52, and pp44). The particulate protein-tyrosine kinase activity was partially solubilized (58%) with 0.5% Triton X-100; this percentage was increased to 85% in the presence of 0.25 M KCl. Upon gel filtration, both cytosolic and particulate kinases showed an apparent molecular mass of 44 kilodaltons; these enzymes also phosphorylated similar major alkali-resistant phosphoproteins. The soluble protein-tyrosine kinase, with a sedimentation coefficient of 4.0S and an isoelectric point of 5.5, could be separated from arginine esterase and prostatic acid phosphatase. PMID- 1709565 TI - [Vasoconstriction of dog nasal mucosa caused by histamine]. AB - Nasal mucosa was obtained from 12 dogs. The vasoconstrictive effect of histamine was measured with physiological recorder. The results showed that vasoconstriction of the nasal mucosa caused by histamine could be blocked by H1 antihistamines or verapamil and not by H2 or alpha antagonists. It suggests that the effect of histamine on nasal mucosa might be mediated by H1 receptor through activation of calcium channel and not by releasing noradrenaline. PMID- 1709566 TI - Biochemical assessment of preoperative stress: a study with diazepam and measurement of monoamine metabolites and catecholamines in cerebrospinal fluid and plasma. AB - Diazepam 5 mg or an inert placebo tablet was given as preoperative hypnotic on the night before operation to two groups (n = 18 in each) of healthy women having elective Caesarean section under spinal analgesia. A third group (n = 18) received no hypnotic. The quality of the preoperative night's sleep assessed subjectively was significantly better in the diazepam-treated patients compared with those receiving no drug. The diazepam-treated patients had also smaller CSF concentrations of noradrenaline (NA) and of the dopamine metabolite, 3,4 dihydroxyphenylacetic acid (DOPAC). In comparison with the two other patient groups, in the diazepam group there was no correlation between demographic, physiological or subjectively estimated variables and CSF or plasma measurements of monoamine transmitters and their metabolites. Preoperative fear and apprehension correlated most strongly with preoperative heart rate and with the increase in heart rate from the previous day. The monoamine neurotransmitters or their metabolites were of limited use in monitoring the intensity of preoperative fear and anxiety. PMID- 1709567 TI - Anaesthesia and congenital agranulocytosis: influence of anaesthetic agent on neutrophil numbers in a patient with Kostmann's syndrome. AB - We describe a 14-month-old male patient with congenital agranulocytosis who received general anaesthesia on three separate occasions during a 6-week period for minor surgery. Granulocyte colony stimulating factor was commenced after the second anaesthetic. Each anaesthetic was followed by profound reductions in neutrophil numbers, irrespective of the agent used. Even the third anaesthetic, which avoided all the common agents thought to have a marrow suppressant effect and given during granulocyte colony stimulating factor therapy, was associated with a marked decrease in neutrophil numbers. PMID- 1709568 TI - Dextran preloading before extradural block. PMID- 1709569 TI - Interaction between cationic dyes and erythrocyte membranes in minimal change nephropathy: an electrophoretic approach. AB - A study was undertaken to clarify the usefulness of two cationic dyes, alcian blue (AB) and ruthenium red (RR) in demonstrating the defect in cellular membranes noted in minimal change nephropathy (MCN). The binding of both dyes to RBC membranes purified from normal and nephrotic children was evaluated by electrophoretic titration curves. When examined separately, AB was found to precipitate spontaneously, producing macro-aggregates with no electrophoretic mobility at pH 5. This was presumed to be the result of hydrophobic interaction of the dye with itself. The same phenomenon was observed when this dye was incubated at 37 degrees C with RBC ghost's from normal children, when AB presented a sigmoidal curve with a net positive charge for pHs higher than 5.5 and lower than 5 and no electrophoretic mobility at pH 5. However, incubation of AB with RBC ghosts from children with MCN resulted in an improvement of the solubility of the dye which then migrated with a net positive charge along the whole gradient of pH from 3.5 to 9. The presence of zwitterionic neutral detergents such as CHAPS, but not of a charged substance such as protamine sulphate, inhibited precipitation at pH 5 when incubated with membranes from normal children, supporting the hydrophobic nature of the phenomenon. When RR was used instead of AB, it was fully protonated (i.e. did not precipitate) when analysed alone, but when incubated with normal RBC ghosts, it also revealed no electrophoretic mobility at pH 5.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709570 TI - Storage, ultrastructural targeting and function of toposomes and hyalin in sea urchin embryogenesis. AB - This study compares by immunogold labeling the ultrastructural localization of a hexameric 22S glycoprotein, called toposome, with that of hyalin in unfertilized eggs and cells of hatched sea urchin blastulae. Nearly all hyalin is present in the electron translucent compartment of the cortical granules and in the translucent non-cortical pigment granules. In the blastula both of these intracellular stores have vanished and hyalin now forms a broad band below the apical lamina. By contrast, in the egg toposomes are present on the surface, as well as stored in yolk granules and in the electron dense lamellar compartment of the cortical granules. In the hatched blastula, toposomes that have been modified by limited proteolysis in the yolk granules, are associated with the plasma membranes of all newly formed cells, while the toposomes originating from the cortical granules have been incorporated as unmodified 160 kDa polypeptides into an extracellular double layer enveloping the embryo on the outside of the hyaline layer. From evidence discussed in detail, we conclude that the extracellular toposomes rivet the apical lamina to the surface and underlying cytoskeleton of the microvilli, while the modified toposomes from the yolk granules are responsible for position specific intercellular adhesion as they are released to the surface of newly formed cells. We propose that all the material stored in yolk granules is utilized for the assembly of new membranes. PMID- 1709571 TI - Regulation of glucocorticoid receptor expression. AB - A limiting factor determining the sensitivity of a cell to glucocorticoid hormones is the intracellular concentration of the glucocorticoid receptor (GR) protein. By regulating the expression of GR the cell is able to adapt to both changes in its hormone environment and to the varying requirements for biological response. Studies on the regulation of GR expression have shown this to be a complex process which involves both transcriptional and posttranscriptional mechanisms. Although GR is more or less constitutively expressed in most tissues its concentration varies under different physiological conditions. GR expression is regulated by a number of different agents including factors which act through a second messenger pathway. This allows the cell to control glucocorticoid regulated gene expression through a complex but integrated hormonal network. Here we summarize our studies on GR regulation with emphasis on: i), GR autoregulation; ii), the effect of cAMP on GR expression, and iii), GR expression during fetal rat lung development. PMID- 1709572 TI - Successful treatment of chronic idiopathic neutropenia using recombinant granulocyte colony-stimulating factor. AB - A patient with chronic idiopathic neutropenia, who had been suffering from repeated infections, was successfully treated with recombinant granulocyte stimulating factor (rhG-CSF). Subcutaneous injection of 30 micrograms/m2 rhG-CSF every two days was sufficient to maintain the neutrophil count at approximately 1,000/microliter. The patient has lived without any evidence of infection for the last 10 months using that treatment. There were no side effect caused by rhG-CSF and antibodies against G-CSF were not detected in the patient's plasma. PMID- 1709573 TI - Analysis of CD34-positive hemopoietic progenitor cells from normal human adult peripheral blood: flow-cytometrical studies and in-vitro colony (CFU-GM, BFU-E) assays. AB - Hemopoietic progenitor cells are present in minute numbers in the peripheral blood of healthy adults. By in vitro colony-assays evaluating BFU-E- and CFU-GM growth, their numbers have been estimated to be about 1,000 per 1.0 ml of whole blood. Employing a CD34-moAb, detecting an antigen present on virtually all hemopoietic progenitor cells, and by using multiparameter flow-cytometry, we have designed a flow-cytometric method for the quantitation of CD34(+)-cells in blood. Comparative studies on bone marrow and blood have shown that CD34(+)-cells from both sources display almost identical light-scatter characteristics. They differ, however, with regard to the coexpression of the CD33- and the CD19-antigens. In vitro colony-assays for BFU-E and CFU-GM in single cultures have shown that about 25% of the CD34(+)-cells from blood were clonogenic in vitro. Our data indicate that the CD34(+)-cells from peripheral blood differ substantially from bone marrow CD34(+)-cells. PMID- 1709574 TI - Chlorpyrifos exposure of workers entering sweet corn after chemigation. PMID- 1709575 TI - Liquid chromatographic analysis of mexiletine in serum, with alternate application to tocainide, procainamide, and N-acetylprocainamide. AB - A simple and precise high performance liquid chromatographic method for the determination of mexiletine in human serum or plasma is described. Following addition of N-propionylprocainamide as internal standard the specimens are extracted, under basic conditions, into methylene chloride. After removal of the aqueous layer the drug is back-extracted into dilute acid, which is then injected directly for analysis. The extraction efficiency is 79% for both mexiletine and internal standard, and the assay is linear to 4 mg/L (twice upper therapeutic concentration). Inter-run coefficients of variation are 3.0% or less. The relative retention time of mexiletine to internal standard averages 1.3. An adaptation of this method is described for an alternate application to the analysis of tocainide, procainamide and N-acetylprocainamide. PMID- 1709576 TI - Treatment of myelodysplastic syndromes. AB - Therapeutic options have been rapidly evolving for management of patients with the indolent myeloid clonal hemopathies termed myelodysplastic syndromes (MDS). Heterogeneity of MDS has been demonstrated on the basis of marrow morphology and biologic features and has been useful for prognostication into high and low risk groups for transformation to acute leukemia. Such stratification has been important for evaluating responses to various treatments. These therapeutic options include the differentiation-inducing vitamins retinoic acid and vitamin D, and cytokines such as alpha and gamma interferon, to which there has been a generally low response. The use of intensive or low dose chemotherapy has been associated with relatively low response rates, few durable responses and a high degree of hemopoietic toxicity. Allogeneic bone marrow transplantation (BMT) has shown durable responses for a subset of MDS patients, particularly those who are young and who are in the low risk subgroups. however, due to the elderly nature of the majority of MDS patients, and the toxicity associated with BMT, this option has limited utility for most of these patients. Major focus has been on the recent therapeutic use of recombinant human hemopoietic growth factors, particularly G-CSF, GM-CSF and IL3. These agents have been well-tolerated and generally produce a high incidence of sustained improvements in neutrophil counts and marrow morphology, although hemoglobin and platelet counts have generally not been altered. More extensive clinical trials evaluating the impact of these hemopoietic growth factors on the natural history of MDS are ongoing. PMID- 1709577 TI - Management of acute retention of urine: a reappraisal. AB - Our standard policy for the management of retention of urine due to prostatic hypertrophy is that the patient is catheterised and sent home, later to be seen and assessed in the Out-patient Department where he is given an admission date for operation. A detailed audit of 166 patients cared for in this way is presented and the results compared with those in 25 patients who remained in hospital in the interval between catheterisation and operation and in 402 patients not previously catheterised. Although the mortality rate was significantly higher in the retention group (3.3 vs 0.25%), we feel that this is a reflection of the fitness of the patients with retention rather than a consequence of the management policy. The advantages of the "catheterise and send home" policy are discussed. PMID- 1709578 TI - Haemodynamic evidence for per-operative cardiac stress during transurethral prostatectomy. Preliminary communication. AB - Haemodynamic changes were measured during routine transurethral prostatectomy (TURP). The heart rate and stroke volume fell progressively over the first 30 min of surgery, resulting in a steady reduction in cardiac output. There was a significant increase in left ventricular afterload from commencement of the procedure. These findings demonstrate that haemodynamic responses, which are not detectable using conventional methods of monitoring, occur during TURP. Increased left ventricular afterload indicates increased myocardial work and oxygen demand which could result in myocardial ischaemia. This may contribute to the increased cardiovascular morbidity and mortality which have been reported to occur after TURP. The possible underlying mechanisms are discussed. PMID- 1709579 TI - Peritoneal adhesion formation after lysis: inhibition by polyethylene glycol 4000. AB - Peritoneal adhesions cause much long-term postoperative morbidity. This study evaluates the efficacy of polyethylene glycol (PEG) 4000 in reducing adhesion reformation after lysis. Adhesions were induced, by abrasion, in 111 Sprague Dawley rats at a first laparotomy. At a second operation, 10 days later, these adhesions were graded and lysed, after which the animals received one of the following solutions intraperitoneally: 5 per cent PEG 4000 (n = 21), 25 per cent PEG 4000 (n = 23), 32 per cent dextran 70 (n = 22) or isotonic saline (n = 25), or were left as an untreated control group (n = 20). When the reformed adhesions were graded after a further 10 days 5 per cent PEG 4000 was found to be the only solution that inhibited adhesion reformation. The adhesions that reformed in the other four test groups were significantly worse than when they were first graded (P less than or equal to 0.033 for all groups). Therefore 5 per cent PEG 4000 may be useful in clinical practice for the reduction of adhesion formation after lysis. PMID- 1709580 TI - New and improved vaccines for the 1990s. PMID- 1709581 TI - Mediators and allergic rhinitis. PMID- 1709582 TI - The physical urticarias. PMID- 1709583 TI - Food, food additives and urticaria. PMID- 1709584 TI - Histamine releasing factors. PMID- 1709585 TI - Purification of a colon cancer cell growth inhibitor and its identification as an insulin-like growth factor binding protein. AB - We have purified a protein from serum-free conditioned medium of the HT29 human colon adenocarcinoma cell line based on its ability to inhibit the proliferation of the same cell line. The purification procedure consisted of acid gel permeation, semipreparative, and analytical reversed-phase chromatographies. The high-pressure liquid chromatography-purified colon cancer cell growth inhibitor migrates as a single band of 27 and 34 kDa on sodium dodecyl sulfate/polyacrylamide gels under nonreducing and reducing conditions, respectively. NH2-terminal amino acid sequence analysis of the first 32 residues has demonstrated that this protein belongs to the insulin-like growth factor binding protein (IGFBP) family. More precisely, this growth inhibitor appeared to be identical to the recently cloned human IGFBP-4. This IGFBP (HT29-IGFBP) has been characterized by performing ligand blotting and competitive binding experiments. The affinity of HT29-IGFBP for insulin-like growth factor (IGF) II (approximately 3.4 x 10(10) M-1) is slightly greater than its affinity for IGF-I (approximately 1.4 x 10(10) M-1). HT29 cells also produce two other isoforms (28 and 31 kDa, nonreduced) of the HT29-IGFBP having the same partial NH2-terminal amino acid sequence as the 27-kDa protein. The monoclonal antibody alpha IR-3 is known to block the mitogenic actions of IGFs. alpha IR-3 inhibited the growth of HT29 cells, thus suggesting that IGFs are required for the growth of these colon cancer cells. PMID- 1709586 TI - Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells. AB - A population of tumor-reactive cytotoxic T-cells can be propagated from tumor draining lymph nodes of patients with breast adenocarcinoma. These T-cells specifically recognize breast and pancreatic tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion but not other tumors of epithelial origin or the natural killer target K562. The tumor-specific but MHC unrestricted lytic activity of these cytotoxic T-lymphocytes (CTLs) is mediated through the alpha/beta T-cell receptor. The molecule recognized by these CTLs is ductal epithelial mucin produced by breast and pancreatic adenocarcinomas. The protein core of the mucin consists of multiple tandem repeats of a 20-amino acid sequence. Antibody SM3, directed against a determinant on the mucin protein core preferentially expressed on malignant cells is able to significantly inhibit lysis of tumor cells by the CTL, while other antibodies binding to different core epitopes are not. Normal breast epithelial lines, which also express mucin but not the SM3 epitope, are not lysed by these tumor-reactive CTLs or act as cold target inhibitors of lysis of tumor lines. The data suggest that the highly repetitive nature of the mucin allows cross-linking of the T-cell receptor on mucin-specific T-cells and therefore accounts for the lack of MHC restriction seen in this system. They further suggest that the mucin core epitope recognized on tumor cells is not expressed on normal epithelial cells in a manner that can be recognized by tumor-reactive CTLs. These findings support the role of mucins as important tumor-associated antigens mediating the cellular response to certain human cancers and suggest that epithelial mucin core sequences might form the basis for an effective vaccine to augment the antitumor immune response. PMID- 1709587 TI - Monoclonal antibody SN10 which shows a highly selective reactivity with human B leukemia-lymphoma and is effectively internalized into cells. AB - The monoclonal antibody termed SN10 (IgG1-k) which was generated and characterized in the present study shows a highly selective reactivity with fresh (uncultured) human leukemia-lymphoma cells. The antigen defined by SN10 is a cell surface glycoprotein composed of a single polypeptide chain of Mr 36,000 and designated as gp36. The primary reactivity of SN10 is against mature B-lineage leukemia-lymphoma cells. For instance, SN10 reacted with all of the 17 B non Hodgkin's lymphoma specimens, all of the 15 B chronic lymphocytic leukemia specimens, both of the 2 B prolymphocytic leukemia specimens, all of the 3 B hairy cell leukemia specimens, and 2 of the 3 B acute lymphoblastic leukemia specimens tested. Of normal peripheral blood cells, only a marginal reactivity of SN10 was detected with a minor subpopulation (less than 1-4% among different specimens) of isolated B-cells from healthy donors. No significant reactivity of SN10 was detected against any other isolated normal peripheral blood cells which include T-cells, granulocytes, monocytes, erythrocytes, and platelets. Furthermore, no significant reactivity of SN10 was detected against normal bone marrow specimens. In immunohistological studies using frozen tissue sections, SN10 reacted well with malignant lymphomas and showed varying patterns of reaction with hyperplastic reactive lymph nodes. Various normal human tissues tested were unreactive with SN10. In general, glycoprotein 36 was more abundantly expressed on fresh (uncultured) leukemia-lymphoma cells than on cultured leukemia lymphoma cell lines. No significant amount of circulating SN10 antigen was detected in the plasma of leukemia-lymphoma patients or normal healthy donors. Scatchard plot analysis of direct binding of radiolabeled SN10 to a fresh (uncultured) B non-Hodgkin's lymphoma cell specimen, a fresh B chronic lymphocytic leukemia cell specimen, and DND-39 (an American Burkitt's lymphoma cell line) showed equilibrium constants of 5.2, 5.8, and 6.8 x 10(8) liters/mol, respectively. Thus, SN10 shows a high binding avidity to each of the 3 B leukemia lymphoma cell specimens tested. Ricin A chain conjugate of SN10 killed leukemia lymphoma cells effectively, whereas the same conjugate showed no cytotoxicity against control cells. Thus, SN10 bound to target antigen on the cell surface was effectively internalized into the cell. The present results suggest the potential of SN10 for therapy as well as for diagnosis of various forms of leukemia lymphoma, particularly mature B-lineage leukemia-lymphoma. PMID- 1709588 TI - Production of insulin-like growth factor-binding proteins by human central nervous system tumors. AB - Central nervous system (CNS) tumor cells possess specific receptors for insulin like growth factors (IGFs) and respond to the growth-promoting effects of IGFs in cell culture. In the present study, we asked whether CNS tumors also produce IGF binding proteins (BPs) which may modulate the effects of IGFs on CNS tumor cells. Primary cell cultures were established from 20 CNS tumors. Dot blot analysis with 125I-labeled recombinant human IGF-I revealed IGF-binding activity in serum-free conditioned medium from 5 of 7 meningiomas, 7 of 8 malignant gliomas, and 3 of 5 other CNS tumors. Specific IGF BPs in conditioned medium were characterized further by Western ligand and immunoblotting, affinity labeling, and precipitation with specific antibodies against human IGFBP-1, -2, and -3. All conditioned media tested contained an Mr 35,000 BP which was recognized by antiserum against IGFBP-2 and an Mr 24,000 BP that was not recognized by available antisera. Medium conditioned by meningiomas (and one glioma) also contained Mr 45,000 and 50,000 IGF BPs, similar in size and/or immunological properties to growth hormone-dependent BPs present in normal human serum (IGFBP 3). Ligand blotting also showed that meningiomas produce an Mr 29,000 BP; immunoblotting and immunoprecipitation of affinity-labeled IGF-BP complexes confirmed that this BP is recognized by antiserum against IGFBP-1. Immunohistochemistry with specific monoclonal antibodies demonstrated that IGFBP 1 is abundant in pathological specimens of meningiomas and that lower amounts also are detected in malignant gliomas. We conclude that human CNS tumor cells produce a variety of IGF BPs in cell culture, including several that are similar in size and immunological properties to previously characterized human IGF BPs. Immunohistochemistry with specific monoclonal antibodies against IGFBP-1 confirms that this BP is present in vivo, further supporting the concept that IGF BPs may contribute to the regulation of growth in human CNS tumors. PMID- 1709589 TI - More than just a channel: provocative new features of protein traffic across the ER membrane. PMID- 1709590 TI - Origin of life--facing up to the physical setting. PMID- 1709591 TI - Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification. AB - Chimeric RNA molecules were detected by polymerase chain reaction amplification of kinetoplast RNA using a 3' primer specific to mRNA and a 5' primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3' oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications result in the transfer of uridine residues from the gRNA 3' oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine "guide" nucleotides in the gRNA. PMID- 1709592 TI - A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. AB - The c-myb proto-oncogene encodes a sequence-specific DNA-binding protein. To better understand its normal biological function, we have altered the c-myb gene by homologous recombination in mouse embryonic stem cells. Resulting homozygous c myb mutant mice displayed an interesting phenotype. At day 13 of gestation these mice appeared normal, suggesting that c-myb is not essential for early development. By day 15, however, the mutant mice were severely anemic. Analysis indicated that embryonic erythropoiesis, which occurs in the yolk sac, was not impaired by the c-myb alteration. Adult-type erythropoiesis, which first takes place in the fetal liver, was greatly diminished in c-myb mutants, however. Additional hematopoietic lineages were similarly affected. These results are compatible with a role for c-myb in maintaining the proliferative state of hematopoietic progenitor cells. PMID- 1709593 TI - Synergistic interactions between recombinant human interleukin-3, GM-CSF and G CSF in normal human marrow granulocyte-macrophage colony formation. AB - The network of interactions between human myelopoietic growth factors can lead to signal amplification that all regulate normal myelopoiesis. The present study identified synergistic interactions between recombinant human interleukin-3, GM CSF and G-CSF on normal human marrow day 14 CFU-GM suggesting that the interactions between these human myelopoietic growth factors are mainly confined to the early stages of normal human myelopoiesis. The synergistic combinations of recombinant human interleukin-3 plus G-CSF and GM-CSF plus G-CSF warrant clinical trial for the recovery from cancer chemotherapy or radiotherapy-induced myelosuppression and for augmenting human defence against infections. PMID- 1709594 TI - On the three-dimensional structure of quick-frozen hepatic Mallory bodies with special reference to the appearance of cytoplasmic vesicles. AB - Livers containing Mallory bodies (MBs, hyalin degenerative cytoplasmic inclusions) were examined using Heuser's and Van Harreveld's cryo-techniques. The tissues were collected from 1) a patient suffering from alcoholic hepatitis and 2) mice treated with griseofulvin (GF, an anti-mitotic drug). Normal mouse liver and isolated MBs from GF-treated mice were also analyzed by the same methods. Our results suggest that under the toxic influence of alcohol or GF on microtubular elements, MBs are generated by entanglement of elements of 10 nm filaments with microtubule elements. This in turn inhibits cellular transport processes. The reticular net of the ER-element which is usually observable in the normal tissue is changed into numerous small vesicles in the pathological and experimental tissues. The diameters of hepatocytes containing these vesicles were 1.5 to 2 times larger than control diameters. MBs have previously been described in thin sections as filamentous tangles. On replicas we found that they appear to be composed of pairs of filaments twisted in a roughly helical manner, each having a diameter less than 10 nm. The paired helical nature of the MB-filaments is reminiscent of other inclusion bodies, which are also composed of elements of 10 nm filaments, observable in various neurological diseases. PMID- 1709595 TI - Influence of assay method differences on multiple of the median distributions: maternal serum alpha-fetoprotein as an example. AB - A straightforward statistical explanation is provided to show how differences between assay methods can affect the distribution of the multiples of the median (MoM). Evaluation of the impact of assay method differences reveals that the upper tails of the MoM distribution are not affected to the same degree as the lower tails of the distribution. The disparities in MoM distributions due to assay method differences result in various sensitivity/specificity combinations for different assays having the same fixed MoM cutoffs. Disparities do not exist if risks are calculated with use of the distributions for affected and unaffected populations that are based on a center's own assay method. Applying published risk tables, however, can affect the accuracy of the risk estimates. We used maternal serum alpha-fetoprotein as an example of an assay with an established history of reporting results in MoM values; however, the concepts presented apply equally well to any assay for which results are reported in MoMs. PMID- 1709596 TI - Simple enzyme immunoassay for the simultaneous measurement of whole choriogonadotropin molecules and free beta-subunits in sera of women with abnormal pregnancies or tumors of the reproductive system. AB - A multiple enzyme immunoassay (multi-EIA) was developed to quantify whole molecule human choriogonadotropin (w-hCG) and free hCG beta-subunits (hCG-beta) simultaneously. A clone of a specific monoclonal antibody was coupled to solid phase; two other clones of different monoclonal antibodies were conjugated to horseradish peroxidase (HRP; EC 1.11.1.7) and alkaline phosphatase (AP; EC 3.1.3.1), respectively. These two enzyme conjugates were mixed together to measure w-hCG or hCG-beta, depending on the selection of the enzyme substrate. To measure w-hCG and hCG-beta simultaneously, both enzyme substrates were used with the blended enzyme conjugates. The assay is simple and reproducible, and can be completed within 2 h with high specificity and sensitivity. We measured w-hCG and hCG-beta in the sera of women with abnormal pregnancies and in patients with tumors of the reproductive system, and observed different hCG-beta/w-hCG ratios in patients with various types of trophoblastic tumors. PMID- 1709597 TI - "High-dose hook effect" in IRMA-Count PSA assay of prostate-specific antigen. PMID- 1709599 TI - Immunoelectrophoretic pattern of alpha 2-macroglobulin and alpha 2-macroglobulin protease complexes. PMID- 1709598 TI - Creatine metabolism during metabolic perturbations in patients with organic acidurias. AB - Creatine excretion was measured in two patients with methylmalonic aciduria and two patients with 3-hydroxy-3-methylglutaric aciduria. During periods of metabolic decompensation the creatine/creatinine ratio increased and fell during recovery. Prolonged periods of metabolic decompensation may result in the loss of a large proportion of the creatine pool. In one study, measurements of total daily urinary output of metabolites demonstrated that the absolute creatine excretion followed a similar qualitative pattern to the creatine/creatinine ratio. However, apparent fluctuations in methylmalonate excretion when expressed as methylmalonate/creatinine ratio were absent when absolute methylmalonate excretion was calculated. The increased creatine excretion during metabolic perturbations may result from loss from creatine containing tissues such as muscle and may represent an underlying defect in energy metabolism. Alternatively creatine transport may be disrupted by accompanying acidosis. The use of metabolite/creatinine ratios as a measure of metabolite excretion rates during metabolic decompensation whilst qualitatively sound may need a re-appraisal. PMID- 1709600 TI - Comparative efficacy of drug regimens in skin tuberculosis. AB - Three antituberculous drug regimens have been employed to study the therapeutic response in 90 patients with any one of the commonly encountered paucibacillary forms of skin tuberculosis, namely lupus vulgaris, tuberculosis verrucosa cutis and scrofuloderma. The first two regimens contained rifampicin, isoniazid and either pyrazinamide or thiacetazone, and the third regimen had rifampicin and isoniazid only. The disease was clinically defined as localized when confined to one area and widespread when the lesions were disseminated. The observations revealed that the response of lupus vulgaris and tuberculosis verrucosa cutis was alike in all the three regimens, with the localized lesions subsiding completely after 4 months of therapy and the more extensive forms taking 5 months. Patients with scrofuloderma responded similarly to both the triple drug regimens. The discharge, sinuses and ulcers cleared in 6 months but the lymph nodes took longer to regress, up to 7 months in localized and 9 months in more widespread scrofuloderma. To obtain the same results with rifampicin and isoniazid, all patients with widespread scrofuloderma and one-third of those with localized forms had to be treated for 10 and 9 months, respectively. No serious drug side effects, apart from giddiness with rifampicin and acneiform eruptions with thiacetazone, were encountered. No instances of relapse were noted in the 50% of patients who were followed-up for 3 1/2 years after therapy. Single-drug therapy with isoniazid for lupus vulgaris, as given in the past, is to be discouraged as it may promote the emergence of drug-resistant bacilli in those with an undetected focus of infection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709601 TI - Hypothalamic regulatory peptides in obese and lean Zucker rats. AB - 1. Hypothalamic concentrations of nine peptides with experimental effects on energy balance were compared in obese (fa/fa) and lean (Fa/?) male Zucker rats. To determine whether any peptide differences between obese and lean rats might be due to the overweight condition per se, separate groups of obese rats were food restricted to reduce their body weight to lean values. 2. Concentrations of neuromedin B, a bombesin-like peptide, in the central hypothalamus were significantly higher in obese than in lean rats. This difference was not affected in food-restricted obese rats. 3. Hypothalamic levels of neuropeptide Y, an extremely potent central appetite stimulant, were similar in lean and freely fed obese rats but central hypothalamic levels of neuropeptide Y rose significantly in food-restricted obese rats. 4. These findings suggest that disturbances in hypothalamic neuromedin B concentrations may be involved in the obesity syndrome of the fa/fa Zucker rat. Increased central hypothalamic levels of neuropeptide Y in food-restricted rats suggest that this peptide may help to defend body weight by stimulating eating after weight loss. PMID- 1709602 TI - The effects of age and chronic restricted feeding on protein synthesis and growth of the large intestine of the rat. AB - 1. In vivo rates of protein synthesis and growth of the large intestine were studied in ad libitum fed control and chronic diet restricted rats between 3 and 149 weeks post partum. 2. Restricted feeding (50% reduced intake) when imposed from weaning significantly extends the life span of rodents through an unknown biochemical mechanism. 3. The change in nutritional status slows the accumulation of RNA, DNA and protein in the large intestine but does not modify the fractional rate of protein synthesis. 4. It was therefore deduced, that intracellular protein degradation, or the rate of mucosal cell extrusion into the gut lumen, is accelerated by chronic restricted feeding. PMID- 1709603 TI - Recombinant human erythropoietin stimulates synthesis of fetal haemoglobin in haemodialysed patients with anaemia due to end-stage kidney. PMID- 1709604 TI - Insulin and glucagon levels in fulminant hepatic failure in man. AB - The behavior of insulin and glucagon and related metabolic substrates was assayed in plasma of patients with fulminant hepatic failure. All 12 subjects were provided the same nutritional support. High levels of insulin and glucagon were present at all times and no difference was observed between surviving patients (four) and those who died (8). Elevated values for branched-chain and aromatic amino acids as well as alanine were present. Statistically significant lower levels of aromatic amino acids and consequently a greater branched chain-aromatic amino acid ratio was found in surviving vs nonsurviving patients. A significantly greater level of alpha-fetoprotein was found in patients who survived as compared to those who died. PMID- 1709605 TI - Variation during the menstrual cycle of immune cell populations in human endometrium. AB - Morphometric analysis and immunohistology of tissue sections have been used to assess variation, during the normal menstrual cycle, of the bone marrow-derived cell populations in human endometrium. Levels of T cells and macrophages were found to be relatively constant throughout the cycle. In contrast, numbers of large granular lymphocytes, identified as being CD56-positive, were generally low between days 10 and 19, but increased sharply in the latter part of the luteal phase, decreasing again after menstruation. This LGL population is known to be abundant in first trimester pregnancy decidua, and is presumed to play a role in early pregnancy success. PMID- 1709606 TI - Propionigenium modestum: a separate line of descent within the eubacteria. AB - The complete nucleotide sequence of 16S rRNA from Propionigenium modestum was determined and compared with 380 16S rRNA sequences from representatives of all eu- and archaebacterial phyla known so far. The phylogenetic analysis of this data set indicated P. modestum to represent a new separated line of descent within the radiation of eubacterial phyla moderately related to cyanobacteria and Gram-positive bacteria with low DNA GC content. PMID- 1709607 TI - [Complex evaluation of the degree of pesticide cumulation after their simultaneous and successive administration]. PMID- 1709608 TI - Multilocular thymic cysts with pseudoepitheliomatous hyperplasia. AB - Six cases are described of benign thymic cysts of the anterior mediastinum showing focal pseudoepitheliomatous hyperplasia of the lining epithelium. The patients' ages ranged from 11 to 54 years; five cysts occurred in males and one in a female. Histologically, the lesions were characterized by exuberant proliferation of the cyst lining epithelium that grew as sheets and tongues of atypical squamous cells with large, hyperchromatic nuclei, prominent nucleoli, and scattered mitotic figures. The walls of the cyst adjacent to the areas of epithelial proliferation showed abundant hemorrhage, necrosis, and severe inflammatory changes. All cases were treated by local surgical excision. There was no evidence of recurrence or metastases over a follow-up period of up to 8 years (average follow-up, 4 years). It is proposed that pseudoepitheliomatous hyperplasia may develop in thymic cysts as an expression of regeneration of the lining epithelium in response to the inflammatory, hemorrhagic, and necrotizing changes which often accompany these lesions. This should not be mistaken for malignancy, and should be distinguished from the exceptional cases of true thymic neoplasms seen in association with thymic cysts. PMID- 1709609 TI - Pulmonary meningiomas: a report of two cases. AB - Two intraparenchymal lung tumors exhibiting the histopathologic and immunophenotypic characteristics of an intracranial meningioma are presented. The meningiomas presented as solitary asymptomatic nodules in elderly individuals. Both patients survived longer than 3 years following resection, and neither displayed clinical or radiographic evidence of a central nervous system tumor, suggesting that these are primary lung tumors. Review of the literature and discussion of other lesions in the differential diagnosis of this rare intrapulmonary neoplasm are presented. PMID- 1709610 TI - Complications of treatment for cryptosporidial diarrhoea. PMID- 1709611 TI - Palliation in AIDS--where do we draw the line? PMID- 1709612 TI - A mouse monoclonal antibody specific for an allotypic determinant of the Igha allele of murine IgM: genetic and functional analysis of Igh-6a epitopes using anti-IgM monoclonal antibodies. AB - An IgG1 mouse monoclonal antibody (MAb) specific for a mouse IgM allotypic determinant in the a, c, f, g, h, and j haplotypes was derived from a fusion of SP2/O-Ag14 mouse myeloma cells with C57BL/6 mouse spleen cells (Igh-Cb) immune to TC31, a MAb of the IgMa allotype. MAb from one hybridoma derived from this fusion (designated DS1) was demonstrated to bind in an ELISA to immunoglobulin bearing the IgMa allotype (TC31, MOPC104E), but not to immunoglobulin bearing the IgMb allotype (C.BPC112). Fluorescein-conjugated DS1 was shown to bind to the surface of BALB/cByJ splenic B cells, but was shown to have negligible binding on C57BL/6J cells. Similarly, DS1-conjugated Sepharose beads were able to stimulate in vitro proliferation of BALB/c, but not C57BL/6 splenic B cells. DS1 was unable to bind to spleen cells from BALB/c allotype congenic strains, BAB/14 (Igh-Cb) and C.AL-20 (Igh-Co), demonstrating that DS1 recognizes a determinant under the control of a gene linked to the Igh-C gene complex. Using sera from recombinant inbred lines, the determinant defined by DS1 was shown to be linked to the Igh-1 locus. Furthermore, the determinant was localized to the CH1 domain of the mu heavy chain. Sera from BALB/cByJ, NMRI, CBA/J, SEA/GnJ, RIIIs/J, and CE/J mouse strains were shown to bind to DS1 in an ELISA, while sera from A/J, SJL/J, NZB/B1NJ, AKR/J, C57BL/6J, and C57BL/10SnJ mouse strains did not bind to DS1. From these data we propose that DS1 is reactive with specificity Igh-6.1, which was originally defined by an allotypic antiserum developed by Black et al. (Immunogenetics 7:213, 1978). PMID- 1709613 TI - Immunochemical characterization of the formyl peptide receptor moieties on human neutrophils. AB - The neutrophil (PMN) receptor for formylated peptides such as N-formyl-l methionyl-l-leucyl-l-phenylalanine (FMLP) is involved in binding and subsequent response to certain chemotactic stimuli. The receptor on human PMN has been reported to consist of several glycoprotein components, ranging in size from 43 94 kDa. Furthermore, FMLP receptors on human PMN have been shown to contain both high and low affinity states. In this study, the receptor was purified by subjecting solubilized PMN plasma membrane components to FMLP-affinity chromatography, and was found to be comprised of four components, one of 68 kDa, and the others of 94, 48, and approximately 40 kDa. Only the 68, the 94, and the approximately 40 kDa components specifically bound a radioiodinated FMLP analogue. To further characterize these components, a battery of monoclonal antibodies reactive against the FMLP receptor was prepared. Seven monoclonal antibodies were selected on the basis of their reactivity with the 68 kDa receptor component. Some of these antibodies also cross-react with the 48 kDa component, suggesting that the 68 and the 48 kDa receptor moieties are immunologically related. These antibodies reacted with normal human neutrophils, but not with lymphocytes, or unstimulated HL-60 cells. Furthermore, the presence of 20 nmol of FMLP inhibited the binding of five of the anti-receptor antibodies to whole PMN. These results suggest that the epitopes recognized by these five antibodies may possibly be involved in FMLP binding. PMID- 1709614 TI - Monoclonal antibodies against human placental glutathione transferase (class pi). AB - Five monoclonal antibodies (MAbs) were produced in a mouse hybridoma system against human placental glutathione transferase (GST pi). Four of these monoclonal antibodies, named 461 to 464, were of immunoglobulin G class, whereas the monoclonal antibody 465 was of IgA class. All these MAbs specifically recognized the glutathione transferase from human placenta (class pi) showing no cross reactivity against the basic and the neutral forms of GST from human liver. When each MAb was incubated with the GST pi, no inhibition of enzymatic activity towards 1-chloro-2,4-dinitrobenzene was observed except for MAb 465 which showed a slight inhibition to a serial dilution of 1:128. PMID- 1709615 TI - Ontogeny of radial and other astroglial cells in murine cerebral cortex. AB - Three cell forms of astroglial lineage populate the prenatal and early postnatal murine cerebral wall. In the present review we consider the ontogeny of these cell forms with respect to histogenetic events of the perinatal period. Classic bipolar radial glial cells predominate prior to E17. The bipolar coexist with monopolar radial forms in the perinatal period. Both bipolar and monopolar radial forms coexist with multipolar astrocytes in the course of the first postnatal week and are ultimately succeeded by the multipolar cells. The shift from bipolar to monopolar radial forms is initially coincident with translocation of somata of bipolar cells from the ventricular zone to the upper intermediate zone and cortical strata. Arborization appears to occur both at the growing tips and along the shaft of the processes of both bipolar and monopolar radial cell types. As arborization continues, the processes of the monopolar radial cells come to resemble those of the multipolar astrocytes. Eventually the radial cells are fully transformed into the multipolar astrocytic forms. During this period of transition, radial processes in the cortex appear to be degenerating, suggesting that regressive processes contribute to the cytologic transformation. This sequence of transformations begins late in the period of neuronal migration and continues through the early stages of growth and differentiation in the murine cerebral cortex. The signals that induce these changes may arise from differentiating neurons within the cortex. These transformations occur at a time when radial glial fibers are no longer required as guides for neuronal migration, and the glial population assumes new roles related to the development and operation of cortical neuronal circuits. PMID- 1709616 TI - Tracing glial cell lineages in the mammalian forebrain. AB - Astrocytes and oligodendrocytes emerge in late gestational and early post-natal development in the mammalian CNS. The nature, and number, of progenitors for each glial type is a central question. This review will focus upon several unresolved issues relating to glial cell lineages and describe new methods to try to illuminate these issues further: 1) How can developmental patterns by which immature neuroectodermal cells give rise to classes of neurons and glia be understood in the context of lineage? 2) What are the lineage relationships among the various cell classes, how many glial lineages are there in the developing CNS, and how can recent methods of clonal analysis using stable markers be used to clarify lineage patterns? 3) Do patterns of gliogenesis vary in different regions of the CNS? 4) How do patterns of gliogenesis observed in vitro relate to those in vivo? PMID- 1709617 TI - Cell origin and identity in limb regeneration and development. AB - The occurrence of limb regeneration in adult urodele amphibians raises fundamental questions about the relationship between development and regeneration. The use of monoclonal antibodies as cell markers has provided clear evidence that blastemal cells, the progenitor cells of the regenerate, are not the same as limb bud cells in the embryo. For one of these antibodies the distinction has been traced to the relationship between limb regeneration and the nerve supply. Innervation of the limb bud during development appears to establish nerve-dependent growth control for regeneration. The cell markers have also contributed to the problem of how the blastemal cells arise after amputation, although several important questions remain to be answered. PMID- 1709618 TI - Profiles of periodontal conditions in adolescents measured by CPITN. AB - Results of more than 100 CPITN surveys from over 60 countries for the age group 15-19 years, stored in the WHO Global Oral Data Bank as of 1 August 1990, are assembled. They are presented in the form of graphs showing the mean number of sextants affected per person and arranged by country according to WHO regions. It is hoped that these overviews provide a frame of reference for the evaluation of periodontal conditions in populations and population subgroups. The most frequently observed condition in adolescents was score 2 (calculus with or without bleeding). Calculus seems to be much more prevalent in non-industrialized than in industrialized countries. Although some shallow pocketing of 4 or 5 mm was present in two-thirds of all populations observed, it affected mostly only a minority of the sample and then only in one or two sextants. However, a few surveys showed a relatively high prevalence of pocketing. As the surveys were carried out in adolescents, such high figures indicate serious problems ahead. PMID- 1709619 TI - Profiles of periodontal conditions in adults measured by CPITN. AB - Results of almost 100 CPITN surveys in more than 50 countries for the age group 35-44 years, stored in the WHO Global Oral Data Bank as of 1 August 1990, are assembled. They are presented in the form of graphs showing the percentages of persons according to the highest score per person and arranged by country according to WHO regions. It is hoped that these overviews provide a frame of reference for the evaluation of periodontal conditions in populations and population subgroups. Calculus and shallow pocketing were the most frequently observed conditions. With a few exceptions, the percentages of persons and the mean number of sextants per person with deep pockets were small to very small. The assumed differences between industrialized and non-industrialized countries with regard to periodontal health were not reflected in the survey data examined. Severe periodontal destruction seems to be a limited problem, seldom leading to tooth loss before the age of 50. For the large majority in most of the populations observed, the progress of periodontal diseases seems to be compatible with the retention of a natural dentition into older age. Nevertheless, the periodontal problem is of considerable magnitude and importance, as 5-20 per cent of populations are affected by a serious, irreversible condition at the age of 40, which is a high percentage compared with almost every other disease that afflicts mankind. PMID- 1709620 TI - Hemoglobin-based artificial blood: new polymeric derivatives of hemoglobin with low oxygen affinity. AB - To make stroma-free hemoglobin (SFHb) capable of carrying oxygen satisfactorily in vivo it is necessary both to improve its intravascular persistence and to reduce its affinity for oxygen. The method used up to now has consisted of chemical modification of SFHb to lower its oxygen affinity (fixation of permanent effector inside the phosphate binding site or intramolecular cross-linking of the deoxy form of SFHb), then polymerization or substitution with polymers. We have designed new functionalized polymers (from dextran and polyoxyethylene), capable of mimicking the effect of the natural intraerythrocyte effector, 2,3 disphosphoglycerate, i.e. of decreasing its affinity for oxygen, and we have linked these polymers chemically to oxyHb. All the resulting conjugates have lower oxygen affinity than SFHb and, when injected into rats, do not lead to hemoglobinuria. Preliminary in vivo tests also showed that these conjugates possess no acute toxicity. Further experiments with rats are now under way (60% and 80% hemorrhagic shocks) with the aim of evaluating whether these new products can be regarded as potential candidates for blood substitution. PMID- 1709621 TI - Ovarian and extraovarian mucinous tumors with solid mural nodules. AB - Three cases (two ovarian and one retroperitoneal) of mucinous tumors with solid nodules are reported. The predominant picture in the three cases was that of a mucinous cystic tumor, but small nodules of solid anaplastic carcinoma were found in all three cases. In addition, one case showed microscopic foci of microcyst rupture with histiocytic response reminiscent of sarcoma-like mural nodules, one case showed several sarcoma-like nodules, and one case showed apparent transition from anaplastic carcinoma to spindle cell sarcoma. Histologic and immunohistochemical characteristics of the lesions are given, as differentiation of these nodules is important. PMID- 1709622 TI - Lectin histochemistry of ovarian mucinous cystadenomas. AB - The lectin histochemistry of ovarian mucinous cystadenoma was studied using a panel of lectins comprising Triticum vulgaris, Lotus tetragonolobus, Arachis hypogaea, Griffonia simplicifolia I, concanavalin A, and Dolichus biflorus. All 23 cases examined in this study stained extensively with T. vulgaris. Of all the lectins, D. biflorus was the least reactive. Concanavalin A stained mainly the perinuclear zone, whereas the other lectins frequently stained the cytoplasm, glycocalyx, and extracellular luminal mucin. This pattern of lectin reactivity resembles endocervical more than intestinal epithelium and suggests that ovarian mucinous cystadenomas are of mullerian nature. The lack of difference in lectin reactivity between cystadenomas with and without goblet/Paneth cells supports a common histogenetic origin of both groups. PMID- 1709623 TI - Argyrophilic cells and ectocervical epithelium. AB - The purpose of this study was to determine the nature of the argyrophilic cells in the ectocervix and to analyze the different morphologic varieties of argyrophil cell-containing ectocervical epithelia, notably to reappraise the degree of analogy with transitional epithelium. A systematic study of 39 ectocervices was carried out using histochemical and immunohistochemical techniques. Immunodetection of cytokeratins was used to specify the differentiation of the ectocervical linings. Argyrophilic cells were detected in 43% of our specimens. They have been found in several varieties of ectocervical lining: normal-appearing or hyperkeratotic squamous epithelium, "transitional like" epithelium extending onto the portio, and immature squamous metaplasia from the transformation zone. The term "transitional-like" refers to a stratified nonsquamous epithelium that differs from true urothelium by the absence of superficial differentiation leading to the layer of "umbrella" cells. Two main types of argyrophilic cells have been delineated: serotonin cells and Merkel-type cells. The nature of the argyrophilic cells was dependent on the type of epithelium: Serotonin cells were essentially associated with "transitional-like" epithelium and Merkel-type cells with squamous epithelium. The latter cells were particularly frequent in hyperkeratotic squamous epithelium from uterine prolapse, reinforcing the homology with epidermis. PMID- 1709624 TI - Histologic and immunohistochemical analysis of nine endometrial stromal tumors: an unexpected high frequency of keratin protein positivity. AB - Nine cases of endometrial stromal tumors were examined by light microscopy and a battery of immunostains for diagnostic purposes. Eight of the cases were originally classified as endometrial stromal tumors and one as an epithelioid leiomyoma. Three of six (50%) endometrial stromal sarcomas stained positively for keratin/cytokeratin. All of these same tumors stained for vimentin. One case originally considered an epithelioid leiomyoma on light microscopy was later diagnosed as a uterine tumor with sex cord features following strong positive immunostaining with keratin/cytokeratin antibodies and negative staining with antidesmin. These results show that immunoreactivity for epithelial differentiation may be present in endometrial stromal tumors. The diverse immunostaining patterns of the stromal tumors reflect the probable histogenesis of these neoplasms from primitive totipotential stem cells which may differentiate along epithelial and various mesenchymal cell lines. PMID- 1709625 TI - Paraganglioma of the vulva. AB - A case of vulvar paraganglioma is reported. Following presentation with vulvar pain, a 1-cm nodule was excised from the labium minus of a 58-year-old woman. Histologically, the tumor was composed of nests of round eosinophilic cells with moderately pleomorphic nuclei, beneath an intact squamous epithelium. Ultrastructural studies indicated two cell types within the neoplasm: chief cells with numerous small neurosecretory granules and peripheral slender sustentacular cells. A reticulin stain confirmed the "zellballen" nature of the neoplasm, and the neoplastic cells showed moderate argyrophilia on a Grimelius stain. The immunoperoxidase stains for chromogranin and neuron-specific enolase were strongly positive in the neoplastic chief cells. Immunostaining using anti-S-100 antibody confirmed the finding of sustentacular cells by identifying many slender cellular processes among the chief cells. These light and electron microscopic findings are diagnostic of paraganglioma, an entity not previously reported in the vulva to our knowledge. PMID- 1709626 TI - Post-transcriptional and transcriptional control of collagen gene expression in normal and modulated rabbit corneal endothelial cells. AB - In a previous report, collagen synthesis did not correlate with steady-state collagen RNA levels; substantial amounts of type I collagen RNAs in endothelial cells were not translated into the respective protein. The current investigation was extended to study the level of the control mechanism in collagen gene expression in normal corneal endothelial cells or those modulated by corneal endothelium modulation factor released by polymorphonuclear leukocytes. Northern blot analysis using cloned rabbit types I and IV cDNA probes (same species as RNA sources) demonstrated specific mRNA transcripts for collagen types I and IV in the endothelial cells, although the steady-state level of these mRNAs in modulated endothelial cells was low. The turnover rate of collagen RNAs was determined; normal cells contain very stable alpha 2(I) and alpha 2(IV) mRNAs whose half-lives exceed 24 hr. The same messages decayed rapidly in the modulated cells, where they had an apparent half-life of approximately 8 hr. Using nuclear run-off transcription, the rate of transcription in normal cells was found to be slightly lower than that in modulated cells. When the relative rate of collagen gene transcription was compared, that of alpha 2(I) was the lowest and of alpha 2(IV), the highest in both cells. The relative transcriptional rates of individual collagen chains did not account for the steady-state levels, suggesting that transcriptional regulation in corneal endothelial cells was less than was translational regulation. On the other hand, during early stages of corneal endothelial cell modulation induced by factors released by polymorphonuclear leukocytes there was a differential effect on both transcriptional rate and the steady-state level of collagen RNAs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709627 TI - Localization and characterization of substance P binding sites in rat and rabbit eyes. AB - Specific and high-affinity binding sites for Substance P (SP) were found in eyes from albino rabbits and rats using an in vitro autoradiographic method with 125I Bolton Hunter SP (BHSP). autoradiograms were generated by apposing 10-20 microns thick cryostat eye sections to 3H-Hyperfilm or liquid emulsion and quantified by means of image-analysis procedures. Kinetic studies showed that equilibrium was reached after a 75-min incubation at room temperature. In rat retina, specific binding corresponding to approximately 90% of total binding, was reversible, of high affinity (dissociation constant [Kd], 0.13 +/- 0.02 nM). Half-time for dissociation of 125I-BHSP was about 15 min. Unlabeled SP and the two neurokinins (NK) A and B competed in a concentration-dependent manner for retinal sites labeled by 125I-BHSP with the following order of potencies: SP greater than NKA greater than NKB, in agreement with a pharmacologic profile of a SP receptor site. In both species, specific binding was found in the iris sphincter muscle, choroid, and retina. In rats, detectable amounts of SP-binding sites were also expressed in the corneal epithelium and iridial stroma. Quantitative analysis of the autoradiograms revealed that the highest densities of 125I-BHSP binding sites were localized in the iris sphincter muscle in rabbits and the inner retina in rats. PMID- 1709628 TI - A comparison of hypertensive and nonhypertensive coronary care patients' cardiovascular responses to visitors. AB - Patients with and without hypertension in a coronary care unit (n = 24) were compared with respect to cardiovascular responses to both a family visit and an interview by an investigator. Variables for each of the four cardiovascular indicators (systolic blood pressure, diastolic blood pressure, heart rate, and premature ventricular contractions) included the value before, the highest value during, the lowest value during, and the value after each social interaction condition. The highest group means for systolic blood pressure and heart rate were significantly higher for patients with hypertension than for patients without hypertension under both the interview and visit conditions. Differences in cardiovascular responses were not significantly greater for family visits than for interviews for patients with hypertension compared with those without hypertension. Thus, although hypertensive patients had greater cardiovascular reactivity to both social interaction conditions than nonhypertensive patients in the coronary care unit, family visits were no more physiologically stressful than a comparative interaction condition. PMID- 1709629 TI - [Urticaria following infusion of hydroxyethyl starch]. PMID- 1709630 TI - Cell killing mode of liblomycin (NK313), a novel dose-survival relationship different from bleomycins. AB - Liblomycin (NK313) is a novel derivative of bleomycin (BLM) and peplomycin (PEP). The cell kill kinetics of NK313 on rat ascites hepatoma AH66 were compared with those of PEP. NK313 induced intracellular DNA cleavage and arrested cell cycle progression at the G2 phase similarly to PEP. The cytocidal effect of NK313, however, was found to be different from that of PEP as described below: 1) The dose-survival curve for cells exposed to PEP for 1 hour was upward concave, whereas in case of NK313, the survival curve was linear. PEP was more effective to AH66 than NK313 at lower concentration, but at higher concentration, NK313 was much more effective. 2) The time-survival curve for cells treated with either NK313 or PEP was biphasic. NK313, however, did not induce temporary resistance of AH66 cells to NK313, while PEP induced resistance to PEP. 3) NK313 was effective against the cells which became temporarily resistant to PEP by the treatment of PEP. These differences suggest that NK313 might be of value to treat PEP insensitive tumor cells. PMID- 1709631 TI - Scar formation after drug-induced cochlear insult. AB - Structural and molecular changes in the guinea pig organ of Corti were studied using histochemistry and electron microscopy in the course of drug-induced hair cell degeneration. Actin filaments disappear from the cuticular plate and the stereocilia. An actin-rich bridge appears in the apical region of dying hair cells. Two supporting cells form a scar for a given hair cell. The supporting cells expand and invade the spaces of Nuel and then the region previously occupied by the hair cell. The scar region becomes cytokeratin-labeled. In this study, the apical domain of the hair cell is the last part of the cell to degenerate. Hair cell degeneration coincides temporally with scar formation. We define the resulting scar as a 'type I' scar. The results provide preliminary information about the molecular composition of the type I scar and suggest a structural basis for the dynamics of scar formation. PMID- 1709632 TI - Ventilatory responses to hypoxia and hypercapnia in awake rats pretreated with capsaicin. AB - Ventilatory responses to hypoxia and hypercapnia were measured by indirect plethysmography in unanesthetized unrestrained adult rats injected neonatally with capsaicin (50 mg/kg) or vehicle. Such capsaicin treatment ablates a subpopulation of primary afferent fibers containing substance P and various other neuropeptides. Ventilation was measured while the rats breathed air, 12% O2 in N2, 8% O2 in N2, 5% CO2 in O2, or 8% CO2 in O2. Neonatal treatment with capsaicin caused marked alterations in both the magnitude and composition of the hypoxic but not hypercapnic ventilatory response. The increase in minute ventilation evoked by hypoxia in the vehicle-treated rats resulted entirely from an increase in respiratory frequency. In the capsaicin-treated rats the hypoxic ventilatory response was significantly reduced owing to an attenuation of the frequency response. Although both groups responded to hypoxia with a shortening in inspiratory and expiratory times, rats treated with capsaicin displayed less shortening of both respiratory phases. By contrast, hypercapnia induced a brisk ventilatory response in the capsaicin-treated group that was similar in magnitude and pattern to that observed in the vehicle-treated group. Analysis of the components of the hypercapnic ventilatory responses revealed no significant differences between the two groups. We, therefore, conclude that neuropeptide containing C-fibers are essential for the tachypnic component of the ventilatory response to hypoxia but not hypercapnia. PMID- 1709633 TI - Tachykinin recovery during postmortem bronchoconstriction in guinea pig lungs. AB - We examined the role of substance P (SP) and neurokinin A (NKA) in the postmortem bronchoconstriction in guinea pig lungs using isolated lungs superfused via the trachea. Airway opening pressure (Pao) during superfusion was monitored and the superfusate collected for analysis of SP- and NKA-like immunoreactivities (SP-LI and NKA-LI, respectively). Peak Pao (39.0 +/- 3.9 cmH2O) was reached 10 min after starting superfusion; Pao decreased slowly thereafter, reaching only 9.9 +/- 2.2% of the peak value 2 h after starting superfusion (P less than 0.005); 12.6 +/- 2.6 and 34.0 +/- 9.7 fmol of SP-LI and NKA-LI, respectively, were found in the fraction corresponding to 10-20 min of superfusion. Recovered immunoreactivities decreased to 5.2 +/- 0.3 and 9.3 +/- 1.8 fmol of SP-LI and NKA-LI, respectively, in the fraction corresponding to 110-120 min of superfusion (P less than 0.05). Inhibition of neutral endopeptidase with thiorphan resulted in significantly greater increases in Pao (P less than 0.005) and augmentation of the recovery of SP-LI and NKA-LI (P less than 0.05 and P less than 0.001, respectively). Capsaicin treatment of animals 7-10 days before the removal of their lungs abolished the increase in Pao during superfusion and resulted in a significant decrease in the amount of SP-LI and NKA-LI recovered. Our data confirm that tachykinin release occurs during postmortem bronchoconstriction in guinea pig lungs and, furthermore, that tachykinin degradation by NEP modulates the intensity of this response. PMID- 1709634 TI - Changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis. AB - We investigated whether alveolar surface force increased and participated in the lung pressure-volume relationship in bleomycin-induced pulmonary fibrosis in hamsters and, if so, whether lung surfactant was hampered in the lungs. On the air-filled pressure-volume curve, decreases of lung volume from control level were significantly higher at 3-8 cmH2O pressure on day 10 than on day 30. Because the change of lung tissue elasticity evaluated from the saline-filled pressure volume curve was equal for the 2 days, the higher decrease of air volume on day 10 was due primarily to contribution of alveolar surface force. Pressure differences between deflation limbs of air-filled and saline-filled pressure volume curves, which represented net alveolar surface force, were significantly higher at any lung volume between 50 and 90% total lung capacity on day 10, but almost no significance was observed on day 30. Phospholipid concentration in bronchoalveolar lavage fluid significantly decreased on day 10 but had improved by day 30. Analysis of phospholipid species in purified lung surfactant showed decreased fractions of disaturated phosphatidylcholine and phosphatidylglycerol on day 10. Surface-active properties of the surfactant, measured by a modified Wilhelmy balance, were remarkably hampered on day 10, but most of them had improved by day 30. We consider that the quantitative and functional abnormalities of lung surfactant have a part in the aggravation of lung mechanics in the acute phase of pulmonary fibrosis. PMID- 1709635 TI - Plasma protein and apolipoprotein synthesis by human yolk sac carcinoma cells in vitro. AB - Three human yolk sac carcinoma cell lines were characterized for the expression of several markers. Each of the cell lines expressed alpha-fetoprotein, without detectable levels of chorionic gonadotropin, and the level of alpha-fetoprotein expression increased dramatically when the cultures were held without passage for extended periods. The secretion of a number of plasma proteins was documented by metabolic labeling, immunoprecipitation, and gel analysis. The major plasma proteins detected were alpha-1-antitrypsin, alpha-fetoprotein, transthyretin, beta-2 microglobulin, and plasminogen, with lower levels of transferrin and complement C4 released. Apolipoproteins B, E, and A1 were secreted in high levels as well and were found in the form of lipoprotein particles. Time course experiments on the synthesis of apolipoproteins E and A1 indicated that, as with alpha-fetoprotein, the level of synthesis increased substantially when the cultures were held without passage. The results indicate that these yolk sac carcinoma cells display a protein expression profile similar to that observed for the human yolk sac, and the possibility that the cells may have the potential to differentiate is discussed. PMID- 1709636 TI - A 3-base pair in-frame deletion of the phenylalanine hydroxylase gene results in a kinetic variant of phenylketonuria. AB - Phenylketonuria (PKU) is an autosomal recessive disease due to deficiency of a hepatic enzyme, phenylalanine hydroxylase (PAH). The absence of PAH activity results in typical PKU while persistence of a residual enzyme activity gives rise to variant forms of the disease. We report here a 3-base pair in-frame deletion of the PAH gene (delta 194) in a mild variant, with markedly reduced affinity of the enzyme for phenylalanine (Km = 160 nM), and we provide functional evidence for responsibility of the deletion in the mutant phenotype. Since the deletion was located in the third exon of the gene, which presents no homology with other hydroxylases, we suggest that exon 3 is involved in the specificity of the enzyme for phenylalanine. Finally, since none of the 98 PKU patients tested were found to carry this particular deletion, our study suggests that this molecular event probably occurred recently on the background of a haplotype 2 gene in Portugal. PMID- 1709637 TI - Effect of acute alterations in acid-base balance on rat renal glutaminase and phosphoenolpyruvate carboxykinase gene expression. AB - During chronic acidosis, the levels of the rat renal mRNAs that encode the mitochondrial glutaminase (GA) and cytosolic phosphoenolpyruvate carboxykinase (PCK) are increased 6-fold. Following acute recovery of chronic acidosis, the levels of the two mRNAs are rapidly and coordinately decreased, returning to normal within 13-17 h. In contrast, the increases in GA and PCK mRNAs during acute onset of acidosis occur with very different kinetics. The increase in PCK mRNA occurs rapidly and reaches a maximum within 7 h, whereas the GA mRNA is increased after a 4-7-h lag and then plateaus at 14-17 h. Treatment with dexamethasone or with cAMP analogs significantly increases the level of renal PCK mRNA but has no effect on the level of GA mRNA. Nuclear run-on experiments indicate that the acute induction of PCK mRNA is primarily due to an increased rate of transcription. However, transcription of GA mRNA is unaffected by acute acidosis. Therefore, the changes in the two mRNAs are temporally coordinated but occur through different mechanisms. Furthermore, the inductive effects of acidosis are not mediated solely through glucocorticoid or cAMP regulatory elements. PMID- 1709638 TI - Expression of Madin-Darby canine kidney cell Na(+)-and Cl(-)-dependent taurine transporter in Xenopus laevis oocytes. AB - Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases. PMID- 1709639 TI - Heat-shock-induced denaturation of proteins. Characterization of the insolubilization of the interferon-induced p68 kinase. AB - Heat-shock stress causes inactivation and aggregation of various cellular proteins which become further insoluble. Previous studies have shown that the interferon-induced p68 kinase activity was greatly reduced in extracts of heat shocked HeLa cells, and that the loss of activity was due to a decreased solubility of the enzyme. Here we show that the p68 kinase which is normally evenly distributed in the cytoplasm, aggregates as a thick ring around the nucleus in heat-shocked cells. The 70-kDa constitutive heat-shock proteins are major insolubilized proteins during stress and we find them to colocalize with the p68 kinase after stress. Treatments of cells with drugs which disrupt the cytoskeleton, such as colcemid and cytochalasin E, do not hinder the enzyme insolubilization during heat-shock. On the contrary, heat-protectors such as glycerol and deuterium oxide (D2O) keep the p68 kinase under a soluble and active form during heat-shock stress. Similarly, an attenuation of the insolubilization of this enzyme is observed in cells rendered thermo-tolerant by a previous heat shock, suggesting that heat-shock proteins may also contribute to the protection. During the recovery period at normal temperature after heat-shock, resolubilization occurs and most of the enzyme is again recovered under an active soluble form. PMID- 1709640 TI - cDNA sequence and bacterial expression of mouse liver sterol carrier protein-2. AB - Sterol carrier protein-2 (SCP-2) is an intracellular protein of Mr 13,096. In vitro studies have shown that it is involved in the transport and metabolism of cholesterol. This protein is believed to participate in these activities by forming a stoichiometric complex with the sterol. Because these activities occur in different intracellular locations, i.e. mitochondria, peroxisomes, and cytosol, it can be predicted that SCP-2 targets to these sites. In this report we show that a mouse cDNA (785 base pairs) encodes a precursor form of SCP-2 containing a N-terminal presequence and an additional C-terminal residue. These additional amino acid residues are found in proteins targeted to the mitochondria and peroxisomes, respectively. These signals are not found in SCP-2 purified from rat liver cytosol which is believed to be a cytosolic form. Northern analysis shows that there are four species of mRNA which hybridize to a SCP-2-specific probe at 1.0, 1.7, 2.2, and 2.9 kilobases. Southern analysis shows that the gene is distributed over a large amount of DNA or that there are multiple genes. We have cloned the cytosolic/peroxisomal form of mouse SCP-2 into the Escherichia coli expression vector pKK233-2 and have expressed and purified recombinant mouse SCP-2, Mr 13,034. The purified recombinant SCP-2 is immunoreactive to rabbit anti rat SCP-2 antibody. It also has biological activity equivalent to homogeneous rat liver SCP-2 in stimulating the microsomal conversion of 7-dehydrocholesterol to cholesterol and in the esterification of cholesterol by acyl-CoA cholesterol acyltransferase by rat liver microsomes. PMID- 1709641 TI - Epidemiological approaches to primary and secondary prevention of cancer. AB - Primary prevention of cancer requires control of both involuntary and voluntary exposures. Involuntary exposures include carcinogens in air and water, and various forms of radiation. Often these exposures are difficult to characterise individually and difficult to study epidemiologically. Although it is unlikely that they account for more than a small proportion of cancers, it is important that we refine our techniques of study to facilitate their control. Voluntary (lifestyle) exposures are responsible for the majority of cancers. In many developed countries, tobacco accounts for approximately 30% of cancer deaths, and major public health endeavours are justified to reduce this toll. Dietary factors may be as important, with dietary fat the most important risk factor, vegetables and fruits being protective. In several studies, including a cohort study in Canada, dietary fat increases breast cancer risk, though other studies have been negative. The evidence for fat increasing the risk of colorectal is more consistent. Epidemiology has shown that secondary prevention of cancer is applicable by screening for breast cancer with mammography with or without physical examination in women age 50-69, and screening for cervix cancer in women age 25-60 with cervical cytology. Organised screening programmes are essential to ensure that a high proportion of women are screened, and that the tests are high quality with adequate quality control. Under these circumstances screening every 2 years for breast cancer and every 3 years for cervix cancer is cost-effective. Screening for other cancers cannot be recommended currently. There is a time to effect that must be recognised in planning primary or secondary prevention. Full effect of most primary activities will not be achieved for decades, screening may require a decade. Available knowledge must be applied now, however, to ensure the effect will eventually be seen, as is now occurring in some countries with the downturn in lung cancer mortality following smoking reduction in men. PMID- 1709642 TI - Synergistic interactions between differentiation-inducing agents in inhibiting the proliferation of HL-60 human myeloid leukaemia cells in clonogenic micro assays. AB - All-trans-retinoic acid, hexamethylene bisacetamide and 5-azacytidine are inducers of granulocytic differentiation of HL-60 human myeloid leukaemic cells, which eventually leads to inhibition of cell proliferation. The effect of graded concentrations of all-trans-retinoic acid (RA) (1 nM-1 microM), hexamethylene bisacetamide (HMBA) (0.5-4 mM) and/or 5-azacytidine (5azaC) (1 nM-1 mM), alone and in combination with each other on colony formation and growth of HL-60 cells was studied in agar capillary clonogenic micro assays in order to identify new potential therapeutic regimens for elderly patients with acute myeloid leukaemia. ED90 concentrations, inducing 90% inhibition of colony formation for RA, HMBA and 5azaC, were 128 nM, 2.7 mM and 40 microM, respectively. The drug interactions between these differentiating agents were analysed by Berenbaum's general algebraic solution. The combinations: RA + HMBA, 5azaC + HMBA and RA + 5azaC were significantly synergistic in inhibiting HL-60 colony formation. Their interaction indices were 0.62, 0.83, and 0.97, respectively, at a specific effect level of 15%. The addition of 1 mM HMBA to 100 nM 5azaC- and 1 nM RA-treated cultures significantly increased the colony-formation inhibition from only 2.6% and 7.0% to 46.4%, and 43.1%, respectively. Also, HMBA showed marked synergism with RA and 5azaC in inhibiting colony growth. The interaction indices (I) of HMBA + RA and HMBA + 5azaC were 0.013 and 0.009, respectively, at the same specific level of 15%. Moreover, the triple combination of RA + HMBA + 5azaC showed synergism in inhibiting both the colony formation (I = 0.7) and colony growth (I = 0.4) at the same specific level of 15%. Since RA, HMBA and 5azaC were effective when administered alone in phase I clinical trials of myeloid leukaemic patients, their synergistic combinations could provide shorter and less toxic courses of treatment in elderly myeloid leukaemic patients. I is less than 1, = 1 or greater than 1 in synergistic, additive or antagonistic interactions, respectively. PMID- 1709643 TI - Combination chemotherapy with mitomycin C, vindesine and melphalan for refractory metastatic breast cancer. AB - A combination of mitomycin C, vindesine and melphalan was administered to 33 patients with heavily pretreated refractory breast cancer. The overall response rate was 27% with a mean duration of more than 10.2 months. A stabilization with a mean duration of 5.1 months was seen in 56% of cases, while 20% of patients progressed. Gastrointestinal toxicity, mostly grade 1 or 2 nausea/vomiting was seen in 85% of cases, grade 1 or 2 leukopenia in 60% of patients, and grade 1 or 2 thrombocytopenia in 42%. Considering the good compliance of this regimen and the poor prognosis of patients with refractory advanced breast cancer, this combination can be useful as a palliative treatment of breast carcinoma. PMID- 1709644 TI - Chimeric cytotoxin IL2-PE40 inhibits relapsing experimental allergic encephalomyelitis. AB - IL2-PE40 is a chimeric protein composed of human interleukin-2 (IL2) genetically fused to a modified form of Pseudomonas exotoxin lacking the cell recognition domain. IL2-PE40 is cytotoxic for IL2 receptor-bearing lymphocytes in culture and can inhibit activation of T cells in vivo. IL2-PE40 can significantly diminish antigen-stimulated proliferation of lymphocytes sensitized to myelin basic protein. Intraperitoneal administration of IL2-PE40 not only markedly inhibits the clinical manifestations of adoptively transferred relapsing experimental allergic encephalomyelitis but also dramatically reduces both inflammation and demyelination characteristic of the disease. PMID- 1709645 TI - Modulation by tumor necrosis factor-alpha of human astroglial cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF). AB - Phagocyte survival and function are enhanced by GM-CSF and G-CSF. The production of both CSFs can be induced in mesenchymal cells by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). We have recently demonstrated that IL-1 alpha and beta induced the production of GM-CSF and G-CSF by two human astroglial cell lines. In the present study, we examined the effects of TNF-alpha on the production of GM-CSF and G-CSF by U87MG, a human astroglial cell line that constitutively expresses GM-CSF and G-CSF, and U373MG, a second human astroglial cell line that does not produce CSF. We demonstrate that U87MG can be induced to increase its production of GM-CSF and G-CSF by exposure to TNF-alpha while U373MG is induced to produce GM-CSF but not G-CSF. These responses, measured by accumulation of elevated levels of CSF protein and mRNA, are rapid and sensitive. The implications of these findings to the immunopathogenesis of central nervous system infections are discussed. PMID- 1709646 TI - Conditioning nerve crush accelerates cytoskeletal protein transport in sprouts that form after a subsequent crush. AB - To examine the relationship between axonal outgrowth and the delivery of cytoskeletal proteins to the growing axon tip, outgrowth was accelerated by using a conditioning nerve crush. Because slow component b (SCb) of axonal transport is the most rapid vehicle for carrying cytoskeletal proteins to the axon tip, the rate of SCb was measured in conditioned vs. sham-conditioned sprouts. In young Sprague-Dawley rats, the conditioning crush was made to sciatic nerve branches at the knee; 14 days later, the test crush was made where the L4 and L5 spinal nerves join to form the sciatic nerve in the flank. Newly synthesized proteins were labeled in motor neurons by injecting 35S-methionine into the lumbar spinal cord 7 days before the test crush. The wave of pulse-labeled SCb proteins reached the crush by the time it was made and subsequently entered sprouts. The nerve was removed and sectioned for SDS-PAGE and fluorography 4-12 days after the crush. Tubulins, neurofilament proteins, and representative "cytomatrix" proteins (actin, calmodulin, and putative microtubule-associated proteins) were removed from gels for liquid scintillation counting. Labeled SCb proteins entered sprouts without first accumulating in parent axon stumps, presumably because sprouts begin to grow within hours after axotomy. The peak of SCb moved 11% faster in conditioned than in sham-conditioned sprouts: 3.0 vs. 2.7 mm/d (p less than 0.05). To confirm that sprouts elongate more rapidly when a test crush is preceded by a conditioning crush, outgrowth distances were measured in a separate group of rats by labeling fast axonal transport with 3H-proline 24 hours before nerve retrieval.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709648 TI - Topography of the projection from the central complex of the thalamus to the sensorimotor striatal territory in monkeys. AB - The distribution of axons arising from the central complex (or centre median parafascicular complex) and terminating in the striatum was studied in seven macaques and one squirrel monkey. Deposits of anterograde tracers were made in the two lateral-most subdivisions of the central complex, i.e., the middle part (or pars media) and the lateral part (or pars paralateralis). All injections avoided the pars parafascicularis. The intrastriatal distribution of labeled axonal endings was mapped in relation to the standard ventricular (CA-CP) system of coordinates. Labeled endings were observed in the major posterior and dorsal parts of the putamen (excluding its anteromedial and ventral parts) and also in a restricted ventrolateral part of the caudate nucleus. The topography of the central territory of the striatum, defined as the striatal space receiving axons from the central complex, was found to correspond exactly to that of the cortical sensorimotor territory delineated after cortical injections. The termination pattern of the central axons within the striatum was patchy. Viewed as a whole, the irregular and hazy patches formed oblique streaks, parallel one with the other. The three-dimensional reconstructions of data from transverse sections revealed that the streaks were bi-dimensional pictures of three-dimensional parasagittal layers covering the whole anteroposterior extent of the cortical sensorimotor territory of the striatum. Our work shows that the pars media of the central complex, which receives selectively pallidal afferent axons (Francois et al., '88: Brain Res. 473:181-186), is the main source of the centroputaminal projection. The probable implication of this in a closed sensorimotor loop of the basal ganglia is discussed. PMID- 1709647 TI - Distribution of cholecystokinin-like immunoreactivity within the stomatogastric nervous systems of four species of decapod crustacea. AB - The distribution of cholecystokinin-like immunoreactivity was studied in the stomatogastric nervous systems, pericardial organs, and haemolymph of four species of decapod crustacea, by using immunocytochemical and radioimmunoassay techniques. Whereas cholecystokinin-like immunoreactivity was found within the stomatogastric nervous systems of all four species, its distribution in each is unique. Two species (Panulirus interruptus and Homarus americanus) have cholecystokinin-like immunoreactivity within fibers and neuropil of the stomatogastric ganglion (STG); two other species (Cancer antenarius and Procambarus clarkii) do not. Further, the cholecystokinin-like immunoreactivity within the STGs of Panulirus and Homarus arise from distinct structures; from a projection of anterior ganglia in Panulirus, and from somata within the posterior motor nerves in Homarus. The staining in the other ganglia of the stomatogastric nervous system also shows some interspecies variability, although it appears to be more highly conserved than staining within the STG. These differences in staining were confirmed by measuring the amount of CCK-like peptide present in tissue extracts of ganglia by radioimmunoassay. In contrast to the variable staining within the STG, all four species have cholecystokinin-like immunoreactivity within the neurosecretory pericardial organs and thoracic segmental nerves. This cholecystokinin-like immunoreactivity is contained within fibers and within varicosities that coat the surface of these structures. The location of this staining and the presence of detectible levels of CCK-like peptide in the haemolymph suggests that CCK-like peptides in decapod crustacea may be utilized as neurohormones. PMID- 1709649 TI - Retinal afferents to the tectum opticum and the nucleus opticus principalis thalami in the pigeon. AB - The retinal afferents of the tectum opticum and the n. opticus principalis thalami (OPT) were studied with fluorescent tracers in pigeons. Injections into the tectum opticum revealed topographically related areas of high density labelling in the contralateral retina. In these areas up to 15,000 cells/mm2 were labelled. After tectal injections the soma sizes of labelled retinal ganglion cells in the area centralis ranged from 5 to 23 microns with a mean of 7.5 microns. Afferents from the ipsilateral retina could not be demonstrated. Injections into the OPT labelled neurons throughout the retina without a clear topographical relation to the locus of injection. The density never exceeded 150 cells per mm2. The soma size range was 8 to 35 microns with a mean of 14.6 microns. Independently of the injection area within the OPT, the red field in the dorsotemporal retina was always extremely sparsely labelled. The number of labelled ganglion cells in this area never exceeded 25 neurons/mm2. After OPT injections the average density of labelling per unit area was six times higher in the yellow than in the red field. The results confirm previous reports of a massive and topographically organized retinal projection onto the optic tectum. The projection onto the OPT was clearly smaller and with the retrograde tracing techniques in use, an orderly topography has not been demonstrated. The paucity of red field projections onto the OPT suggests that the role of the thalamofugal pathway in binocular integration is very limited. PMID- 1709650 TI - Histochemical demonstration of zinc in the hippocampal region of the domestic pig: II. Subiculum and hippocampus. AB - The distribution of zinc has been described in two areas of the hippocampal region of the domestic pig, viz., the subiculum and the hippocampus. Zinc was demonstrated histochemically according to the Neo-Timm method, a modification of the sulphide-silver procedure. In each of the examined areas the staining displayed a distinctly stratified pattern which has been compared in detail to fields and layers defined on the basis of cyto- and fibroarchitecture, resulting in a combined chemo- and cytoarchitectonic map. Most of the staining was confined to the neuropil, but a considerable number of stained nerve cell bodies were seen in both the subiculum and the hippocampus. In the subiculum, the plexiform layer was divided into a superficial, weakly stained subzone and a deep, better stained subzone. The cell layer was generally well stained, but displayed a complex staining pattern with differences in staining intensity of both the cell bodies and neuropil. In regio superior of the hippocampus, the stratum moleculare appeared weakly stained, with the exception of a tapering process of more darkly stained tissue projecting from the plexiform layer of the subiculum into the deepest part of the layer. Stratum radiatum and the superficial subzone of stratum oriens showed a weak staining intensity, contrasting to the relatively darkly stained pyramidal cell layer and the intensely stained deep subzone of stratum oriens. In regio inferior, the stratum moleculare was divided into a moderately stained superficial part and an unstained deep part. Stratum radiatum and stratum oriens both appeared weakly stained. The layer of mossy fibers was very intensely stained and appeared almost homogeneously black in its main suprapyramidal part, whereas the infrapyramidal part was looser in character. The pyramidal cell layer was darker than in regio superior. The distribution of zinc in the pig was compared with that in the guinea pig and rat, described previously. The staining pattern is fundamentally similar in all three species, though notable species-specific traits do exist. PMID- 1709651 TI - Axonal projections and synaptogenesis by supraspinal descending neurons in the spinal cord of the chick embryo. AB - Following the injection of horeseradish peroxidase (HRP) into the brachial spinal cord of the chick on embryonic day (E)4.5, retrogradely labeled neurons can be found in the brainstem (Okado and Oppenheim: Journal of Comparative Neurology 232: 143-161, 1985). By contrast, following high cervical spinal transection, functional (behavioral) deficits are not observed until E10 (Oppenheim: Journal of Comparative Neurology 160: 37-50, 1975). To determine whether this temporal difference between projections and function reflects a delay in synaptogenesis, we looked for the presence of anterogradely HRP-labeled pre-synaptic terminals in brachial cord following injection of HRP into the boundary between brainstem and spinal cord at ages between E3.5 and E7. HRP-labeled fibers were observed in the branchial cord by E4.5 and were diffusely distributed in the ventral and lateral marginal zones (presumptive ventral and lateral funiculi, respectively). Although some axo-dendritic and axo-somatic synapses were observed in the brachial cord prior to E6, the presynaptic profiles were always unlabeled by HRP and thus must originate from propriospinal sources. The first HRP-labeled supraspinal synapses were found in the ventral and lateral funiculi on E6. They contained several clear spherical synaptic vesicles and were axo-dendritic in nature. The cells of origin of the postsynaptic dendrites were determined by injecting HRP into the wing-bud to label the brachial motoneurons retrogradely and the presynaptic component was identified as supraspinal by HRP injections into the brainstem/spinal cord boundary to orthogradely label the descending fibers. Several double-labeled axo-dendritic synapses were found in the ventral and lateral funiculi of E6 brachial cord. Therefore, at least some descending supraspinal fibers make synapses directly onto motoneuron dendrites. We conclude that 1) there is a delay of about 1.5 days between the arrival of supraspinal fibers and synapse formation in the brachial cord, 2) the earliest synapses are axo-dendritic in nature, 3) at least some supraspinal fibers make direct contact with motoneuron dendrites as early as E6, and 4) synaptogenesis from propriospinal sources precedes that from supraspinal descending axons. These observations provide evidence indicating that the temporal difference between the onset of projections of supraspinal descending fibers and the onset of their function may be partly owing to delayed synaptogenesis. PMID- 1709652 TI - Palmoplantar keratoderma with tonotubular keratin. AB - A 61-year-old man with palmoplantar keratoderma with an unusual tonotubular keratin is reported. The histologic findings, genetic transmission, and clinical course were similar to epidermolytic palmoplantar keratoderma (Voerner type), but keratinocytes ultrastructurally displayed a tonotubular cytoskeleton, which has not been previously described, instead of a tonofilamentous one. Electrophoretically, we found no difference in the keratin pattern of normal plantar skin, skin in palmoplantar keratoderma of Voerner, and that of our patient's skin. Therefore the tubular keratin most likely formed as a result of a posttranslational change of keratin polymerization. PMID- 1709653 TI - Substance P for evaluation of coronary endothelial function after cardiac transplantation. AB - The endothelium-dependent vasodilator substance P dilates normal and diseased coronary vessels in humans in vivo and produces a maximal response similar to that seen with intracoronary isosorbide dinitrate. Twelve cardiac transplant recipients underwent intracoronary infusion of substance P after routine annual investigations. All patients were well, with no evidence of rejection and with angiographically normal coronary arteries. Substance P was infused at 2 ml/min for 2 min into the coronary artery, starting at a dose of 1.4 pmol/min and increasing by doubling increments, and followed by isosorbide dinitrate (1 mg/min) infused over 2 min. Coronary artery diameter was measured in 23 vessel segments from 12 transplant recipients. The following doses were infused: saline solution (1 ml/min), substance P (0.7 [three patients], 1.4, 2.8, 5.6, 11.2, 22.4 pmol/min) and isosorbide dinitrate (1 mg/min). The mean percent increase in diameter (+/- SEM) in response to increasing doses of substance P was as follows: 0, 6.5 +/- 2.9%, 10.9 +/- 2.9%, 12.1 +/- 2.9%, 16.5 +/- 2.6%, 19.2 +/- 3.1% and 25.8 +/- 2.2%, respectively. Half maximal dilation was produced with 1.4 to 2.8 pmol/min of substance P; the maximal response (mean percent diameter change) was 22 +/- 2.5%. This was not significantly different from that achieved with isosorbide dinitrate. It is concluded that coronary endothelial function as assessed by response to substance P is preserved in cardiac transplant recipients with angiographically normal coronary arteries. Substance P may be a suitable agent for testing endothelial function in these patients. PMID- 1709654 TI - Time course of moricizine's effect on signal-averaged and 12 lead electrocardiograms: insights into mechanism of action. AB - The mechanism of action of moricizine, a new antiarrhythmic agent used in the Cardiac Arrhythmia Suppression Trial, is incompletely characterized. In addition, because moricizine is extensively metabolized, plasma moricizine concentration has an unknown relation to myocardial drug effect. Signal-averaged and standard electrocardiograms (ECGs) were used to monitor moricizine's myocardial effects in 16 patients with frequent ventricular premature complexes taking 600 to 900 mg daily. Three signal-averaged ECG variables were measured: total filtered QRS duration (fQRS), root-mean-square voltage in the terminal 40 ms of the QRS complex (V40) and the terminal low amplitude duration less than 40 microV (LAS). At steady state, plasma samples were collected and serial recordings of signal averaged and standard ECGs were taken at 0, 1, 2, 4, 6 and 8 h after moricizine administration. A 24 h ambulatory ECG was recorded throughout the test period. Moricizine prolonged the fQRS (p less than 0.05) and decreased the V40 (p less than 0.05) of the signal-averaged ECG and prolonged the QRS (p less than 0.05) and corrected JT (JTc) intervals (p less than 0.05) of the standard ECG. The time course of the signal-averaged and standard ECG variables paralleled plasma moricizine concentration; that is, the maximal changes occurred at 1 to 2 h and declined to time 0 values at 8 h. The maximal changes were: fQRS (+8%), V40 ( 33%), QRS (+8%) and JTc (+4%). Thus, dynamic changes were observed for intraventricular conduction (fQRS, QRS) and ventricular repolarization (JTc) over the dosing interval.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709655 TI - The effect of endoscopic laser therapy on survival in patients with squamous-cell carcinoma of the esophagus. Further experience. AB - Although endoscopic laser therapy is effective for symptom palliation in esophageal cancer, few studies have investigated its effect on survival. We previously reported a 300% improvement in survival in 10 patients with squamous cell carcinoma of the esophagus after endoscopic Nd:YAG laser energy. We now report a study to determine if the survival advantage persisted after treating an additional 26 patients. Thirty-six patients with squamous-cell carcinoma of the esophagus treated with endoscopic laser therapy were compared to 20 controls identified by our hospital Tumor Registry. There was no difference between the groups with respect to age, sex, race, location of tumor, or clinical stage. More control patients (25%) had previously undergone surgery than laser patients (0%) (p less than 0.05). Survival analysis demonstrated a significant improvement in overall survival (p less than 0.05), with an improvement in median survival from 5.7 to 9.7 months (p less than 0.05). One-year survival was 38% in laser patients, compared to 20% in control patients. Our experience continues to demonstrate that endoscopic laser therapy is effective in prolonging life as well as palliating the symptoms of patients with squamous-cell esophageal carcinoma. PMID- 1709656 TI - Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. AB - We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining. PMID- 1709657 TI - Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique. AB - In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed. PMID- 1709658 TI - Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections. AB - Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression. PMID- 1709659 TI - Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique. AB - We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy. PMID- 1709660 TI - Cytochalasins enhance the proliferation of CD4 cells through the CD3-Ti antigen receptor complex or the CD2 molecule through an effect on early events of activation. AB - Cytochalasins are known to inhibit or enhance the proliferation of T cells induced by mitogens in a concentration-dependent fashion. To clarify the mechanism by which cytochalasins enhance T cell proliferation, we examined which activation pathways and events in signal transduction were affected by cytochalasins. We also examined subsets of CD4 cells for a preferential response to cytochalasins. Cytochalasins enhanced the proliferation of CD4 cells induced by optimal doses of anti-CD3 antibody or suboptimal doses of anti-CD2 antibodies. Cytochalasins, at low concentrations, enhanced the rise in intracellular Ca2+ and production of IP3 in CD4 cells activated by anti-CD2 or CD3 antibodies. Cytochalasins also enhanced the modulation of CD3 induced by anti-CD3 antibody. These results suggest that cytochalasins enhance the proliferation of CD4 cells by affecting early events in signal transduction after activation through the CD3 Ti Ag-receptor complex or CD2 molecule. At the doses used, cytochalasins appear to interact with cytochalasin-binding sites in the cell membrane. Cytochalasins predominantly enhanced CD3-mediated proliferation in the CD29-subset of CD4 cells. PMID- 1709661 TI - Recognition of peptides that are immunopathogenic but cryptic. Mechanisms that allow lymphocytes sensitized against cryptic peptides to initiate pathogenic autoimmune processes. AB - Interphotoreceptor retinoid binding protein (IRBP) is a glycoprotein that localizes in the retina and induces inflammatory changes in this tissue in immunized animals. Certain IRBP-derived peptide determinants are also immunopathogenic, and we have previously shown that these determinants could be either immunodominant or cryptic. Lymphocytes sensitized against the cryptic peptides do not recognize whole IRBP in vitro, and yet these lymphocytes must recognize the protein in vivo to initiate the autoimmune pathogenic process. We have examined here two hypothetical explanations for this dissociation: 1) It is possible that when IRBP is processed in vitro, immunodominant peptide determinants compete with the cryptic ones and inhibit their interaction with the MHC molecules on the APC. This explanation was ruled out here by the finding that the immunodominant peptide 1179-1191 ("W10") did not inhibit the response to a cryptic one, 1158-1180 ("R4"), when added at equivalent and even moderately higher concentrations. 2) The second hypothesis proposes that the cryptic antigenic sites are not generated from IRBP by the APC in vitro, whereas enzymes in the retina digest the protein to yield fragments that generate these antigenic sites upon processing by the APC. In line with this hypothesis, we have found that cleavage of IRBP by certain endoproteinases (Asp-N, Glu-C, or V-8) produced molecules that were recognized in culture by lymphocytes sensitized to the immunopathogenic but cryptic peptide R4. This study, therefore, describes a putative Ag processing mechanism that results in IRBP recognition and, consequently, the initiation of an autoimmune process by lymphocytes sensitized against a cryptic peptide. Furthermore, experiments with R4 and other cryptic peptides have shown that cleavage fragments of up to 38 residues in length can be presented by APC, to stimulate lymphocytes sensitized against these peptides. No responses were stimulated, however, by fragments of 75 or more residues. The data thus provide new insights into the processing and presentation of cryptic peptide determinants by APC. PMID- 1709662 TI - The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7. AB - The cDNA for stem cell factor was recently isolated from Buffalo rat liver cells (BRL-3A) and recombinant rat stem cell factor produced from Escherichia coli (rrSCF164). rrSCF164 synergizes with rhIL-7 to stimulate pre-B clonal growth in agar culture of mouse bone marrow cells, and in this study we have characterized the role of rrSCF164 in B cell development. The combination of rrSCF164 plus rhIL 7 stimulated increased colony numbers compared with the sum of colonies stimulated by rrSCF164 and rhIL-7 alone. Also, increased cell proliferation per colony was stimulated by the combination of rrSCF164 plus rhIL-7 compared with rhIL-7 or rrSCF164 alone. The colonies formed with rrSCF164 plus rhIL-7 and rhIL 7 alone contained exclusively pre-B cells, which expressed B220 Ag and cytoplasmic mu-chain, but were negative for surface Ig expression. Morphological examination of the cells in the colonies showed blast-like characteristics. rrSCF164 alone and in combination with rhIL-7 stimulated generation of B220+ cells in liquid culture of B220- cells, whereas rhIL-7 alone had no stimulatory effect on B220- cells. Both stem cell factor mRNA and bioactivity were detected in a mouse bone marrow-derived stromal cell line, termed OZ-11. We propose that stem cell factor is a stromal-derived factor that synergizes with IL-7 to stimulate the proliferation and differentiation of pro-B cells to pre-B cells, which become responsive to IL-7 alone. PMID- 1709663 TI - Regulation of phospholipase D-induced hydrolysis of choline-containing phosphoglycerides by cyclic AMP in human neutrophils. AB - In human neutrophils, the chemotactic tripeptide FMLP and the protein kinase C activator PMA stimulate the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3 phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) and 1-O-[3H]alkyl-2-acylglycerol ([3H]EAG) via mechanism(s) that may involve the activation of phospholipase D. We have investigated the regulation of phospholipase D by determining the effects of elevation of intracellular levels of cAMP on receptor-mediated and PMA-induced breakdown of [3H]-EAPC and formation of products. Pretreatment of neutrophils with either 10( 3) M dibutyryl-cAMP or 10(-5) M PGE2, in the presence of 4 x 10(-4) M isobutylmethylxanthine (IBMX), inhibited FMLP- and leukotriene B4-induced breakdown of [3H]EAPC and formation of [3H]EAPA and [3H]EAG. Inhibition was apparent at all time points of stimulation examined (15-120 s). In addition, the mass of diradyl-phosphatidic acid was decreased in FMLP-stimulated neutrophils pretreated with PGE2 and IBMX. In contrast, pretreatment of cells with PGE2 and IBMX did not inhibit PMA-induced breakdown of [3H]EAPC and the formation of products at 3 and 10 min after stimulation. Furthermore, formation of 1-O [3H]alkyl-2-acyl-phosphatidylethanol, produced by phospholipase D in the presence of ethanol by a transphosphatidylation reaction, was significantly inhibited by pretreatment of cells with PGE2 and IBMX in FMLP- but not PMA-stimulated neutrophils. This differential modulation by cAMP of receptor-mediated and PMA induced activation of phospholipase D suggests agonist-dependent activation of separate pathways and/or activation of separate phospholipase D enzymes. Thus, cAMP elevation may exert inhibitory effects directly on the phospholipase D activated by FMLP and/or on sites proximal to the enzyme that are involved in signal transmission. PMID- 1709664 TI - T cell and antibody reactivity with the Borrelia burgdorferi 60-kDa heat shock protein in Lyme arthritis. AB - The reactivity of cloned T cells and serum antibodies, obtained from patients with chronic Lyme arthritis, with expressed recombinant B. burgdorferi 60-kDa heat shock protein homologue (HSP60) was analyzed. The expressed recombinant Borrelia burgdorferi HSP60 was bound by antibodies in the sera of patients with Lyme arthritis, but not by control sera. A T cell clone (CR253), isolated from one of four patients examined, exhibited an HLA-DR2 restricted proliferative response to the expressed recombinant B. burgdorferi HSP60. This T cell clone specifically recognized the HSP60 of B. burgdorferi and did not proliferate in response to the human, mycobacterial, or Escherichia coli HSP60 homologues. The epitope recognized by this cloned T cell, located between amino acids 260 and 274, is in a region of the spirochetal HSP60 that is not conserved between bacteria and eukaryotes. PMID- 1709665 TI - Junctional diversity of H and L chains allows the coexpression of two mutually exclusive idiotopes (IdI104 and IdI558). AB - The molecular basis for the unexpected coexpression of the individual Id (IdI)558 and IdI104 Id by anti-alpha(1-3) DEX antibody (Ab) (126.33 and 414.2) derived from the MPW wild mouse strain has been investigated by the comparison of the structures of their VH and V lambda 1 chain regions with those of two other MPW derived Ab (262.9 and 16.3) expressing either IdI558 or IdI104 Id. Our data show that 262.9 and 16.3 Ab display identical V lambda 1 and very similar VH regions when compared with BALB/c anti-alpha (1-3) dextran Ab expressing IdI104 or IdI558, respectively. The two Ab (414.2 and 126.33) that express both IdI104 and IdI558 Id display two main features. First, their VH CDR3 are different from those found in IdI104 or IdI558 expressing anti-alpha(1-3) dextran Ab. Second, their V lambda 1 are identical to those from BALB/c origin except for the presence of an additional residue, a phenylalanine at position 95A of CDR3. This additional residue is encoded by the V lambda 1 gene segment and results from a hitherto undescribed V lambda 1-J lambda 1 junction. The alteration of the length of the V lambda 1 CDR3 loop, in conjunction with particular residues within VH CDR3, allows the coexpression of two Id that were found to be mutually exclusive in laboratory mice. PMID- 1709666 TI - The use of adenovirus-infected HeLa cells for the detection of low titer autoantibodies. AB - Following infection of HeLa cells with adenovirus type 5 the cellular La protein becomes predominantly associated with the virally encoded RNA polymerase III products VAI, and VAII, while most of the host RNA polymerase II (e.g. U1, U2, U4, U5 and mRNA) and RNA polymerase III transcription (e.g. U6 and pre-tRNAs) ceases. Other RNA polymerase III products such as the cellular Ro RNAs continue to be transcribed and assembled into ribonucleoprotein complexes containing the Ro (SS-A) antigens. Using a 32P-pulse chase-labeled, adenovirus-infected HeLa cellular extract as a source of antigen, anti-La (SS-B) and anti-Ro (SS-A) antibodies can be detected simultaneously using an immunoprecipitation assay. In the present study this method was found to be more sensitive in detecting anti-La antibodies then counter immunoelectrophoresis and immunoblotting. In studies of sera from patients suffering from rheumatic diseases the percentage positive for anti-La antibody was significantly elevated using this method, especially in patients with systemic lupus erythematosus. PMID- 1709667 TI - The influence of epitope density on the estimation of the affinity of antibody for complex antigens. AB - The influence of the epitope density of the antigen on antibody affinity values determined by fluid- and solid-phase immunoassays was assessed. The affinity of the interaction of a panel of monoclonal anti-DNP antibodies of different affinities (as determined by equilibrium dialysis) for DNP-protein conjugates of various hapten substitution ratios was used as the test system. The results obtained showed that the epitope density of the antigen markedly influences the observed affinity values obtained by both experimental approaches. However, the monoclonal antibodies were ranked in affinity terms by both assays in a similar order to that given by equilibrium dialysis. It is concluded that provided due care is exercised in choosing an appropriate epitope density for the test antigen, these methods can be used to provide rapid estimations of average antibody affinities. PMID- 1709668 TI - Serologically defined Rh determinants. AB - Forty-six numbered Rh antigens which are detected in serological tests are extant; antigens which were most recently numbered are of high or of low incidence. The identification of high incidence Rh antigens through studies involving cells with rare Rh phenotypes, including Rhnull cells, is described briefly. The serological investigations and family studies required to show that a low incidence antigen is an Rh antigen are illustrated by a description of the JAL (Rh48) antigen. Heterogeneity of polymorphic Rh antigens revealed by using monoclonal antibodies (MABS) is demonstrated by consideration of the Rh antigen D. A summary of results of serological tests shows that monoclonal anti-D antibodies define eight D epitopes. Partial D antigens of the D categories are reassessed in terms of expression of D epitopes. PMID- 1709669 TI - Monoclonal antibodies as tools in genetic studies on carbohydrate blood group antigens. PMID- 1709670 TI - Monoclonal antibodies against Rh and Rh related antigens. AB - MAB 114 and MAB 120 appeared to have the same or a very similar specificity to BRIC 125 (Avent et al., 1988). MAB 116, and possibly MAB 115, had a similar specificity to R6A (Anstee & Edwards, 1982; Avent et al., 1988). MAB 117, 118 and 119 demonstrated the MB-2D10 specificity and were, in fact, the antibodies reported by von dem Borne et al. (1990). MAB 121 was not anti-Rh34-like since it reacted with RH: 34 red cells. MAB 122 was not anti-HR since it reacted with RH: 18 cells. The specificities of MAB 121 and MAB 122 were not identified and although they probably detect different epitopes insufficient MAB 122 was available to confirm this suspicion. None of the MABS tested appeared to have the same specificity as 1D8 (Miller et al., 1987) or BS58 (Sonneborn et al., 1990). Competitive binding studies with MAB 121 provided evidence of a close association of BRIC 125 polypeptide with Rh polypeptides. PMID- 1709671 TI - Report of studies on monoclonal anti-IgG antibodies. AB - Eleven monoclonal anti-IgG antibodies were evaluated by ten laboratories for possible AHG reagent use by the ISBT/ICSH protocol. Three of the anti-IgGs were selected by tests with weak Fya sensitized cells as this represents the best screening test. One of the IgM class anti-IgGs (205) equalled ISBT/ICSH reference reagents (R3P and RIIIM) against weak anti-D, -K and Fya sensitized cells and it reacted with all four subclasses of IgG. Thus, subject to satisfactory stability and extended trials with a wide range of weak antibodies, monoclonal anti-IgGs for AHG reagent use are now a possible alternative to conventional rabbit anti IgG. PMID- 1709672 TI - Carboxylic ester hydrolase. A sensitive serum marker and indicator of severity of acute pancreatitis. AB - When using clinical criteria, both falsely positive and falsely negative diagnoses of acute pancreatitis (AP) are often made. Based on a clinical study, elevated serum levels of the pancreatic lipolytic enzyme carboxylic ester hydrolase (CEH) was recently suggested to be a highly specific marker of acute pancreatitis. To determine the sensitivity of the test for AP, a study on patients with the diagnosis set objectively was necessary. In the present study, AP was diagnosed by contrast-enhanced computed tomography in 64 patients, and histopathological examination of tissue removed at laparotomy in 18 of them. By these criteria, 42 patients suffered from acute interstitial pancreatitis (AIP), and 22 patients from necrotizing pancreatitis (NP). Based on the CEH concentrations in the first serum sample obtained in each patient, the sensitivity of CEH for pancreatitis was 98%. From the second day after admission, CEH levels in patients with NP were significantly higher than in patients with AIP. Furthermore, in patients with NP, CEH values remained at a raised level for the following 10 d, whereas a significant decrease of CEH values was noted in patients with AIP. In contrast, total serum amylase activities were higher in patients suffering of AIP than in patients suffering of NP during the observation period. We conclude, that the sensitivity of the CEH test is very high for AP. CEH concentrations remaining at a high level are suggestive of NP, whereas diminishing CEH levels are suggestive of AIP. PMID- 1709673 TI - Changes in pancreatic trophism and gene expression during a prolonged fasting period in rats. AB - To examine the effects of fasting on trophism and gene expression in pancreas, adult male rats were deprived of food from 0-6 d. Total DNA, RNA, and proteins, and specific mRNAs for rat amylase, chymotrypsinogen B, trypsinogen I, proinsulin I, and actin (assessed by employing cloned cDNAs and dot-blot hybridization) were quantitated in pancreas. Body and pancreatic wt diminished progressively to reach 65 and 75% of initial values at the 6th d of fasting. Protein/DNA and total RNA/DNA ratios decreased 2.04 and 2.31-fold, respectively, during 6 d of fasting. The concentration of amylase, chymotrypsinogen B, trypsinogen I, and actin mRNA, expressed as cpm/microgram RNA, decreased significantly throughout the study period, whereas the decrease observed in Proinsulin I mRNA concentration was not significantly different. When mRNA concentrations were refereed to the total content of DNA, however, the decrease was significant for all messengers tested. It is concluded that the prolonged absence of nutrients in the digestive tract exerts negative trophic influence on pancreas and triggers differential changes in pancreatic gene expression. These changes are gradual, asynchronic, and nonparallel. PMID- 1709674 TI - Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways. AB - Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells. PMID- 1709675 TI - Delayed thymocyte development induced by augmented expression of p56lck. AB - Accumulating evidence supports the contention that CD4 and CD8 receptor molecules play a critical signaling role during thymocyte development. The lymphocyte specific protein tyrosine kinase (p56lck), by virtue of its physical association with these surface components, provides a likely candidate for the biochemical signal transducing element required for these effects. To investigate the function of p56lck in T lymphocytes, transgenic mice were produced that carry either the wild-type lck gene or a mutated lck gene encoding a constitutively activated form of p56lck (p56lckF505). Both transgenes were expressed in thymocytes under the control of the lck proximal promoter element. A large set of founder animals was obtained in which steady-state accumulation of lck transgene mRNA directly correlated with transgene copy number, suggesting that this transgene contains a dominant control region. Progeny of these founders exhibited a transgene-dependent dose-related decrease in the production of thymocytes bearing functional antigen receptors. This effect was strictly dependent on p56lck activity, in that both wild-type and mutated versions of the genes induced similar effects with differing efficiencies. Remarkably, even a twofold increase in p56lck abundance was sufficient to substantially disrupt the appearance of functional thymocytes. These results indicate that thymocyte maturation is regulated in part by signals derived from p56lck. PMID- 1709676 TI - Evidence for the involvement of CD56 molecules in alloantigen-specific recognition by human natural killer cells. AB - In recent reports we have described the generation of natural killer (NK) lines devoid of CD3/TCR structures but with apparent specificity for allogeneic target cells. Using one such NK line as an immunogen, we now report the generation of two monoclonal antibodies (mAbs), designated 2-13 and 5-38, which bind selectively to the majority of CD3-, CD16+, CD56+ lymphocytes and inhibit the lysis of specific allogeneic target cells by a panel of alloreactive NK lines. By contrast, these mAbs had no effect on classical NK cell mediated lysis of K562 cells or major histocompatibility-restricted T cell-mediated cytolysis. Immunoprecipitation of radiolabeled NK lines followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the target molecules of both mAbs have a molecular mass of approximately 180 kD. Leu 19, a well-described anti-CD56 mAb, precipitated a 180 kD protein from NK cells, and the binding of Leu 19 to NK cells was blocked by pretreatment with both 2-13 and 5-38. However, in contrast to these mAbs, Leu 19 had no effect on the cytolytic activity of allospecific NK cells. Sequential immunoprecipitation analysis revealed that all three mAbs recognized distinct molecular species of CD56. We interpret these findings as indicating that multiple isoforms of CD56 are differentially expressed on NK lines and play critical roles in the recognition/interaction of these cells with their specific allogeneic targets. PMID- 1709677 TI - Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils. AB - Two classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium. Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with ELAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18). Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3. The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractant." This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals. PMID- 1709678 TI - Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1 activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules. AB - Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM 1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo. PMID- 1709679 TI - A cell surface/plasma membrane antigen of Candida albicans. AB - Antibody from BALB/cByJ mice immunized against a membranous fraction of Candida albicans agglutinated whole cells as well as the membranous fraction. Hybridoma techniques were used to isolate an IgM monoclonal antibody (mAb) designated 10G which agglutinated whole cells and reacted with the subcellular fraction. Yeast cells of 15 additional C. albicans strains and isolates of C. stellatoidea, C. tropicalis, C. intermedia and C. lusitaniae were also agglutinated by mAb 10G. The antigen was not detected on other fungi, including Candida krusei, C. utilis, Cryptococcus neoformans, Cr. albidus, Torulopsis glabrata, Rhodotorula spp. and Saccharomyces cerevisiae. To determine the cellular location of the epitope to which mAb 10G is specific, freeze-substitution was compared with traditional chemical fixation methods in preparation of samples for immunocolloidal gold electron microscopy (IEM). With both fixation procedures, the antigen recognized by mAb 10G was found randomly and densely concentrated on the plasma membrane on exponential-phase yeast-form cells and had a patchy distribution on the cell wall surface. Association of the antigen with the plasma membrane was confirmed by IEM of isolated membranes. On developing hyphal cells, antigen appeared first on the plasma membrane and later on the cell wall surface. Treatment of yeast cells with beta-mercaptoethanol and Zymolyase before fixation removed the antigen from the surface but left the cytoplasmic antigen undisturbed. Treatment of yeast cells or solubilized antigen with heat or proteolytic enzyme (trypsin, Pronase B, proteinase K) did not remove or destroy the antigen, suggesting a non-protein nature of the epitope. PMID- 1709680 TI - Role of ribosome release in the basal level of expression of the Escherichia coli gene pheA. AB - In Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism. In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level. To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA. The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2. However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression. These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene. PMID- 1709681 TI - Changes in macromolecular synthesis of gypsy moth cell line IPLB-Ld652Y induced by Autographa californica nuclear polyhedrosis virus infection. AB - The aberrant replication of the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) in the Lymantria dispar cell line IPLB-Ld652Y was used as a model system for the investigation of factors regulating baculovirus host specificity. A previous study of this system indicates that viral gene expression in infected cells is extremely attenuated and subsequently all cellular and viral protein synthesis is inhibited. In the present study, infection of IPLB-Ld652Y cells with AcMNPV photochemically inactivated in situ resulted in a rapid reduction in cell mitotic indices and cell growth, as well as inducing a series of distinct morphological changes in these cells. At the molecular level, infection with inactivated virus, followed by pulse labelling with [3H]thymidine, resulted in a rapid [0 to 2 h post-infection (p.i.)] and permanent inhibition of host cellular DNA synthesis. Assays of cellular DNA polymerases in isolated IPLB-Ld652Y nuclei confirmed the reduction in cellular DNA synthesis observed in intact cells and indicated an initial (0 to 2 h p.i.) reduction in the activity of aphidicolin-sensitive DNA polymerases. Activity of all cellular DNA polymerases was inhibited at later times p.i. Host cell protein synthesis was completely inhibited after 48 h p.i. Treatment of inactivated virus and virus-infected cells with various chemical and physical factors (i.e. pH and temperature) or lysosomotropic agents revealed that virus entry into cells and fusion of endocytic vesicles (containing virus) with lysosomes were essential for suppression of cellular macromolecular synthesis. The possible involvement of structural components of the AcMNPV virion in these effects is discussed. PMID- 1709682 TI - Neutralizing antibody to Mengo virus, induced by synthetic peptides. AB - A peptide from the carboxyl-terminal region of the Mengo virus capsid protein VP1, representing residues 259 to 277, can induce serum neutralizing (SN) antibodies in both the mouse and guinea-pig. This peptide, termed F164, also induces high levels of protective neutralizing antibodies in mice subsequent to immunization; 87 to 100% of mice are refractory to the effects of an intraperitoneal challenge of 100 LD50 of Mengo virus. The mouse model discussed herein will prove useful for studying the immune response to Mengo virus and evaluating the immunogenicity of individual viral components. PMID- 1709683 TI - Immunofluorescence dystrophin study in Duchenne dystrophy through the concomitant use of two antibodies directed against the carboxy-terminal and the amino terminal region of the protein. AB - Dystrophin immunohistochemical studies in muscle from Duchenne patients (DMD) have shown a population of fibers with partial labelling. In order to determine whether this is related to a cross reaction or to the presence of dystrophin. 22 DMD patients were studied immunohistochemically, through the concomitant use of antibodies from the N-terminal and the C-terminal regions of the protein. In 2, the reaction was negative while in 2 others 17 and 25% of fibers were positive with both antibodies. In the remainder, a population of partially stained fibers was seen: 11 were positive with both antibodies and in 7 only with the N-terminal one. Apparently, there is no correlation between the proportion of positive fibers and clinical progression, or the presence and pattern of DNA deletions in the central part of the gene. These observations led us to suggest that some truncated protein, intermediate synthesis or degradation products of dystrophin are present in muscle from Duchenne patients. PMID- 1709684 TI - Cisplatin, vinblastine, and bleomycin versus vinblastine, cisplatin, and etoposide in the treatment of advanced germ cell tumors of the testis. PMID- 1709685 TI - Effective treatment of small-noncleaved-cell lymphoma with high-intensity, brief duration chemotherapy. AB - Small-noncleaved-cell (SNC) lymphoma is a high-grade, biologically aggressive neoplasm notable for poor response to therapy, high relapse rate, and less than a 20% long-term survival. We treated 20 patients with SNC lymphoma with a novel chemotherapeutic regimen using intensive doses of chemotherapy at frequent intervals in the inpatient setting. All patients were previously untreated. Sixteen patients (80%) had stage IV disease. Most patients (95%) had at least one other characteristic associated with poor prognosis (bulky [greater than 10 cm] disease, multiple extranodal sites, poor performance status), and 85% had two or more characteristics associated with poor prognosis. Seventeen patients (85%) achieved a complete response (CR) to therapy, including all three patients with human immunodeficiency virus (HIV)-associated disease. There have been three relapses, all occurring less than 18 months after treatment, and two of three relapses occurred in patients who were unable to complete therapy. At a median follow-up of 29 months, 13 patients (65%) remain disease-free; the calculated 5 year actuarial disease-free survival is 60%. Toxicity, chiefly myelosuppression, was severe but manageable. There were two treatment-related deaths, both in elderly patients with poor performance status and advanced-stage disease. These data suggest that such a dose-intensive approach improves the response and survival of patients with SNC lymphoma. PMID- 1709686 TI - A randomized trial of chemotherapy followed by pelvic radiation therapy in stage IIIB carcinoma of the cervix. AB - Because of the poor results in stage III B carcinoma of the cervix with standard treatment using radiotherapy alone, we designed a randomized trial to determine whether administration of chemotherapy before pelvic irradiation would improve survival. Between May 1984 and August 1986, 107 patients with previously untreated squamous cell carcinoma were randomly assigned, after stratification by age (less than 50 v greater than 50 years), extent of parametrial involvement (unilateral v bilateral), and lymphangiographic findings (negative v positive) to pelvic radiotherapy (RT; arm A) or three cycles of chemotherapy (CT; bleomycin, vincristine, mitomycin, and cisplatin [BOMP]), followed by the same radiotherapy regimen (CT + RT; arm B). The groups were balanced by age, performance status, extent of parametrial involvement, bulkiness of cervical disease, nodal involvement, and presence of hydronephrosis. Minimal follow-up is 34 months. A complete local response was observed in 32.5% of the patients in arm A and in 47% of the patients in arm B (P = .19). Overall 5-year survival rates were 39% for the RT arm and 23% for the CT + RT approach (P = .02). Toxicity was severe in arm B and included fatal pulmonary toxicity in four patients. Locoregional and distant failures were similar in both groups. We conclude that, despite a satisfactory response rate, neoadjuvant BOMP chemotherapy adversely affects survival in stage III B cervical cancer and is associated with unacceptable toxicity. PMID- 1709687 TI - Estramustine binding protein in human brain-tumor tissue. AB - Estramustine, an estradiol-17 beta and nornitrogen mustard complex, is used in the treatment of advanced prostatic carcinoma. A specific estramustine binding protein (EMBP) is important for its cytotoxic action, and the presence of EMBP has previously been demonstrated in rat and human prostatic cancer tissue. Significant levels of EMBP were detected by radioimmunoassay in human brain-tumor tissue. The EMBP concentrations (expressed as ng/mg protein) in 16 astrocytomas (mean 2.6 ng/mg, range 0.5 to 6.2 ng/mg) and seven meningiomas (mean 5.1 ng/mg, range 0.3 to 9.3 ng/mg) were significantly higher than that found in four samples of epileptic brain (mean 0.7 ng/mg, range 0.5 to 1 ng/mg) and 18 samples of normal brain (mean 0.5 ng/mg, range 0.2 to 1.0 ng/mg). The uptake, metabolism, and antiproliferative effects of the prostatic anticancer agent estramustine have been previously demonstrated in cultured glioma cells. The presence of EMBP may suggest a selective binding and effectiveness in human brain-tumor tissue. PMID- 1709688 TI - Mutagenicity and metabolism of dimethyl phthalate and its binding to epidermal and hepatic macromolecules. AB - As an active ingredient in insect repellents, dimethyl phthalate (DMP) had previously been shown to produce chromosomal aberrations in the livers of rats following subchronic application of the phthalate to skin. When we tested DMP in the Ames mutagenesis assay, it produced in bacterial tester strain TA100 (but not TA98) a dose-related mutagenic response that was abolished by NAD- and NADP independent metabolism associated with rat liver microsomal preparations (S9). In a host-mediated mutagenesis assay, rats were injected ip with DMP (2 g/kg body weight); urine was collected for 24 h, extracted, and analyzed for mutagenic activity and phthalic acid-containing derivatives. The extracted urine was not mutagenic to TA100 and contained an equivalent of 1.96 mg phthalate/ml urine. More than 97% of the phthalic acid-containing derivatives present in the extracted urine consisted of the nonmutagenic metabolite of DMP, monomethyl phthalate (MMP). In vitro experiments showed that rat liver homogenates hydrolyzed 93% of carbonyl-labeled 14C-DMP (7.7 mM) to MMP in 2 h and bound 0.07 nmol of [14C]phthalate/mg liver macromolecules. By contrast, rat epidermal homogenates metabolized only 5% and bound 38-fold higher levels of carbonyl labeled 14C-DMP (2.66 nmol/mg of macromolecules), with no detectable binding to nucleic acids. Compared to epidermis and plasma, liver had a fivefold higher rate of DMP monoesterase activity (1240 nmol/h/mg protein), which, when inhibited by 67%, resulted in a 4.4-fold increase in phthalate-bound hepatic macromolecules (0.31 vs. 0.07 nmol of carbonyl-labeled 14C-DMP/mg macromolecules). In addition to MMP, formaldehyde was produced during the metabolism of DMP by liver. When ethanol was used to inhibit the oxidation of DMP-derived methanol by hepatic homogenates, there resulted a 74% reduction in the accumulation of formaldehyde and similar reductions of 71 and 73% in the binding of methyl-labeled 14C-DMP to nucleic acids and macromolecules. (Methyl-labeled, unlike carbonyl-labeled, 14C DMP yields a 14C-labeled methanol when hydrolyzed.) These results indicate that the DMP diester is a weak bacterial mutagen, which binds to epidermal and hepatic macromolecules other than nucleic acids, and that although the rapid hepatic metabolism of DMP to its monoester (MMP) and methanol affords protection against higher levels of phthalate binding as well as against DMP-induced bacterial mutagenesis, it also oxidizes DMP-derived methanol to formaldehyde, a metabolite that binds macromolecules, including nucleic acids. PMID- 1709689 TI - Complex expression pattern of tenascin during innervation of the posterior limb buds of the developing chicken. AB - The histological localization of the extracellular matrix glycoprotein tenascin was studied during the formation of peripheral nerves in the developing chick hindlimb (embryonic stages 21.5 to 30) by light and electron microscopic immunological methods to obtain insights into the molecule's functional role in the pathway formation by motor and sensory nerves. At stages 21.5 and 23, nerve roots and plexus were surrounded by high tenascin-immunoreactivity, whereas the not yet innervated limb bud was not immunoreactive. During innervation of the limb bud at stages 24.5 and 25, tenascin was detectable at the limb bud base and restricted in its expression to the proximal nerve regions. The nerve tips did not contact areas of elevated tenascin-immunoreactivity. At stages 26 to 28 the dorsal and ventral trunks of the crural and sciatic nerves were surrounded by tenascin-immunoreactivity, which was localized between Schwann and mesenchymal cells. The tips of the growing nerve had now reached the tenascin-positive interface between bone and muscle anlagen. This interface was contacted tangentially rather than penetrated by the nerve tips. The medial and lateral femoral cutaneous nerves were surrounded by high and weak tenascin immunoreactivity, respectively. In both nerves, tenascin-immunoreactivity was absent where the nerves branched extensively to innervate the skin. The cutaneous nerves diverging from the sciatic nerve were of very low tenascin immunoreactivity or tenascin-negative at all developmental stages tested. At stages 29 and 30, muscle nerves, having just entered the tenascin-negative muscles, exhibited strong immunoreactivity, whereas the more proximally situated trunks of the sciatic nerve were weakly and discontinuously labeled, particularly at sites where smaller nerves were branching off. Since the cutaneous branches of the sciatic nerve were always of low tenascin-immunoreactivity, the question was raised whether tenascin expression in the sciatic nerve depended on the presence of motor axons. Spinal cords of stage 19 or 20 embryos were therefore removed and tenascin expression was investigated at stages 26 and 27. Some of the residual nerves were weakly tenascin-immunoreactive, whereas others were tenascin negative. Our observations suggest that tenascin is not involved in the initial guidance of peripheral nerves to their targets. Rather, neuron-induced tenascin appears to stabilize the proximal nerve trunks during a transient time period, possibly by preventing axons and Schwann cells from intermingling with the surrounding mesenchyme, thus contributing to nerve fiber compaction. Conversely, nerve branching may be elicited by reduced levels of tenascin. Furthermore, tenascin may divert growth cones from the developing bone tissue and direct muscle afferents to their appropriate targets. PMID- 1709690 TI - Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein. AB - Several recently discovered lines of evidence support the involvement of myelin basic protein (BP)-specific T cells in multiple sclerosis (MS). To identify potentially relevant immunodominant T cell epitopes, human BP (Hu-BP)-reactive T cell lines were selected from MS and normal donors and tested for reactivity to cleavage fragments and synthetic peptides of Hu-BP. The MS T cell lines responded to more Hu-BP epitopes than did normal lines, showing biased recognition of the N terminal half of the molecule, and one region in the C terminal half, suggesting increased sensitization to BP. The MS lines also differed from normal lines in their decreased percentage of CD8+ T cells. One hundred nine T cell clones isolated from these lines confirmed the reactivity pattern of the lines but did not reflect the mixed phenotype, since all but three clones tested were CD4+. T cell clones from HLA-DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule was permissive in its ability to restrict T cell responses. Other epitopes, including the immunodominant 149-170 sequence, were restricted by several different major histocompatibility complex (MHC) molecules from both MS and normal donors. T cell receptor (TCR) V gene products could be identified on six of 38 clones tested using monoclonal antibodies. From one HLA DR2 homozygous donor, four of eight clones utilized V beta 5.2 in response to different BP epitopes, providing initial support for the preferential use of a limited set of V region genes in the human response to BP. Preferential TCR V gene use in MS patients would provide the rationale to regulate selectively BP reactive T cells through immunity directed at the TCR and thus test for the first time the hypothesis that BP-reactive T cells play a critical role in the pathogenesis of MS. PMID- 1709691 TI - Increased expression of pp60c-src protein-tyrosine kinase during peripheral nerve regeneration. AB - Since little is known about the intracellular changes that take place in response to Schwann cell-neuron interactions that occur during neurite outgrowth and myelination, we investigated the expression of a protein-tyrosine kinase, pp60c src, during peripheral nerve regeneration through a silicone tube. Segments of regenerated nerve, extracted at various times following nerve-transection, showed an induction of in vitro c-src kinase activity as measured by autophosphorylation of immunoprecipitated pp60c-src. This activity occurred at 7 days following nerve transection coincident with the onset of neurite outgrowth in vivo. This kinase activity, which peaked out between 21 and 35 days and decreased thereafter, appeared to be associated with axonal growth and myelination, but not mitogenesis in the tube. Analysis of c-src proteins levels by Western blot showed a similar expression profile as that of the kinase activity. Qualitatively, the expression of an immunoreactive c-src band, migrating slightly slower than pp60, was detected in extracts of regenerating nerve segments as well as in the corresponding L4 and L5 dorsal root ganglia. This protein may be the CNS neuronal specific form (pp60+) of the c-src protein. In situ hybridization revealed that Schwann cells and sensory and motor neurons associated with the regenerated sciatic nerve were positive for c-src mRNA during regeneration possibly accounting for the increased src protein expression during regeneration. Since the increased expression of pp60c-src in regenerated nerve segments coincides with both axonal sprouting and myelination, our findings suggest that the c-src protein may play a role in Schwann cell-neuron interactions which facilitate the occurrence of these events during regeneration. In addition, although pp60+ is generally not detectable in the mature PNS, our findings show that this protein may be induced during conditions of PNS differentiation which promote neurite outgrowth. PMID- 1709692 TI - Antiviral antibodies attenuate T-cell-mediated immunopathology following acute lymphocytic choriomeningitis virus infection. AB - The role of antiviral antibody in resistance to acute lymphocytic choriomeningitis virus infection has been examined by passive transfer of monoclonal antibodies and intracerebral challenge infection. Protection of mice from lethal T-cell-mediated acute disease was observed following passive administration of antibodies either 1 day before or up to 2 days after infection. Viral replication was suppressed in protected mice, and the cytotoxic T-cell response to virus was also diminished. Virus was cleared from the brain and other tissues of protected mice without development of lethal immunopathology, suggesting that preexisting antibody may play a significant role in modulating potentially destructive effects of T-cell-mediated immune responses to pathogens. PMID- 1709693 TI - Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles. AB - To generate nonpathogenic viral particles which express active human immunodeficiency virus type 1 (HIV-1) protease (PR), plasmids containing sequences from the genomes of HIV-1 and Moloney murine leukemia virus (M-MuLV) were constructed. Either the PR coding region alone; the gag, PR, and reverse transcriptase protein-coding regions; or the complete gag and pol protein-coding regions from HIV-1 were substituted for the corresponding regions of a full length M-MuLV clone to yield the chimeric plasmids pMoHIV-I, pMoHIV-III, and pMoHIV-IV, respectively. Cell lines which express the viral gag polyprotein were isolated for hybrids pMoHIV-I and pMoHIV-III. These cells produced viral particles which contained processed core proteins. Cleavage of the gag polyprotein in the viral particles was inhibited by the HIV-1 PR inhibitor L 687908, indicating that the viral PR is responsible for the observed processing. The hybrid virions were not infectious; analyses indicated that the viral particles contained little or no reverse transcriptase activity. In addition, particles produced by pMoHIV-III transfectants failed to package the viral genomic RNA. The cell line which expresses and processes the HIV-1 gag polyprotein is a safe and effective reagent for the in vivo evaluation of potential inhibitors of the HIV-1 PR. PMID- 1709694 TI - Expression of processed core protein of hepatitis C virus in mammalian cells. AB - A structural protein of hepatitis C virus (HCV) was expressed in monkey COS cells under the control of an exogenous promoter, and a protein of 22 kDa was identified by immunoblot analysis. This protein (p22), which was produced by processing in COS cells, reacted specifically to sera of chronic hepatitis C patients, and its coding region was mapped at the most amino-terminal part of the HCV polyprotein. These results suggested that the p22 protein is the nucleocapsid (core) protein of HCV. Moreover, the assay detecting antibody to p22 was found to be useful for early diagnosis of HCV infection. PMID- 1709695 TI - VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection. AB - We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract. PMID- 1709696 TI - Chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies against FMDV in guinea pigs. AB - Five poliovirus recombinants containing sequences corresponding to foot-and-mouth disease virus (FMDV) antigenic sites were constructed. Viable virus was recovered from four of these plasmids, in which the VP1 beta B-beta C loop (antigenic site 1) of poliovirus type 1 Sabin had been replaced with sequences derived from the VP1 beta G-beta H loop (antigenic site 1) of FMDV O1 Kaufbeuren (O1K), chimera O1.1 (residues 141 to 154), chimera O1.2 (residues 147 to 156), and chimera O1.3 (residues 140 to 160) or from the beta B-beta C loop of VP1 (antigenic site 3) in chimera O3.1 (residues 40 to 49). One chimera (O1.3) was neutralized by FMDV specific polyclonal serum and monoclonal antibodies directed against antigenic site 1 of FMDV. Chimeras O1.3 and O3.1 induced site-specific FMDV-neutralizing antibodies in guinea pigs. Chimera O1.3 was capable of inducing a protective response against FMDV challenge in some guinea pigs. PMID- 1709697 TI - Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts. AB - Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets. PMID- 1709698 TI - Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate early protein ICP27. AB - The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H 2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity. PMID- 1709699 TI - Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein. AB - cDNA clones representing the VP8 and VP5 subunits of VP4 of symptomatic human rotavirus strain KU (VP7 serotype 1 and VP4 serotype 1A) or DS-1 (VP7 serotype 2 and VP4 serotype 1B) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2 and VP4 serotype 2) were constructed and inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunization of guinea pigs with the VP8 or VP5 protein of each strain induced antibodies that neutralized the rotavirus from which the VP4 subunits were derived. In a previous study (M. Gorziglia, G. Larralde, A.Z. Kapikian, and R. M. Chanock, Proc. Natl. Acad. Sci. USA 87:7155 7159, 1990), three distinct serotypes and one subtype of VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. The results obtained by cross immunoprecipitation and by neutralization assay with antisera to the VP8- and VP5 expressed proteins suggest that the VP8 subunit of VP4 contains the major antigenic site(s) responsible for serotype-specific neutralization of rotavirus via VP4, whereas the VP5 subunit of VP4 is responsible for much of the cross reactivity observed among strains that belong to different VP4 serotypes. PMID- 1709700 TI - Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. AB - We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation. PMID- 1709701 TI - Human immunodeficiency virus infection and syncytium formation in HeLa cells expressing glycophospholipid-anchored CD4. AB - The CD4 molecule, a glycoprotein expressed primarily on the cell surface of specific T lymphocytes, is thought to function in T-cell antigen recognition and activation. In addition, CD4 serves as a receptor for human immunodeficiency virus type 1 (HIV-1) by a direct interaction with the HIV-1 surface glycoprotein (gp120). To further characterize the HIV-1-cell interaction, a HeLa cell line was established that expressed a chimeric molecule of CD4 and decay-accelerating factor (DAF). In the chimeric CD4-DAF molecule the transmembrane and cytoplasmic domains of CD4 were deleted and replaced with the carboxy-terminal 37 amino acids of DAF. This resulted in the anchoring of the extracellular domain of CD4 to the cell membrane via a glycophospholipid linkage. The glycophospholipid-anchored CD4 had a molecular size of approximately 56 to 62 kDa and was released following treatment of the cells with phosphatidylinositol-specific phospholipase C. HeLa cells expressing the CD4-DAF hybrid could be infected with HIV-1, as evidenced by reverse transcriptase activity, p24 core antigen content, and infectious virus production. In addition, transfection of the HeLa CD4-DAF cells with a plasmid that directs the synthesis of HIV-1 envelope glycoproteins or cocultivation with HeLa cells expressing the virus glycoproteins resulted in syncytium formation. These results indicate that the transmembrane and cytoplasmic domains of the CD4 molecule are dispensable for both HIV infection and syncytium formation. PMID- 1709704 TI - Cancer control following anatomical radical prostatectomy: an interim report. AB - Cancer control following anatomical radical prostatectomy was evaluated in 586 men who were followed for 1 1/2 to 8 years (median followup 4 years, 166 men followed 5 years or longer). The 5-year actuarial rate was 4% for local recurrence alone, 5% for distant metastases alone, 2% for distant metastases in association with local recurrence and 3% for death of or with disease, while 10% of the men had elevated levels of prostate specific antigen without local recurrence or distant metastases. When the actuarial status at 5 years was evaluated by clinical stage there was local recurrence alone in 0% of men with a clinical stage A1 or B1 nodule, and 4% with stage B1, 7% with stage A2 and 8% with stage B2 disease. When evaluated by pathological stage at 5 years local recurrence alone was noted in 2% of men with organ-confined disease, 8% with specimen-confined disease and 8% in whom the disease involved the surgical margin, seminal vesicles or pelvic lymph nodes. Recognizing that two-thirds to three-quarters of all local recurrences occur within the first 5 years, these data suggest that the anatomical approach to radical prostatectomy is associated with local control rates that are equal to or greater than other series reported in the literature. However, without a randomized study it is impossible to compare one clinical series to another, and followup evaluations at 10 and 15 years will be necessary to confirm these findings. PMID- 1709702 TI - Identification and characterization of the hamster polyomavirus middle T antigen. AB - Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes. PMID- 1709703 TI - Feline immunodeficiency virus infects both CD4+ and CD8+ T lymphocytes. AB - Monoclonal populations of feline T cells, derived from a specific-pathogen-free cat and expressing either the CD4 or CD8 surface antigen, were infected in vitro with two geographically distinct isolates of feline immunodeficiency virus (FIV). Both infected T-cell subsets exhibited decreased cell viability, expressed FIV encoded proteins, and generated reverse transcriptase activity. All clones examined retained their original surface phenotype after infection. It appears, therefore, that both CD4+ and CD8+ T cells may be productively infected by FIV in vivo. PMID- 1709705 TI - [Ocular complications of diabetes mellitus--classification and diagnosis]. PMID- 1709706 TI - [Ocular complications of diabetes mellitus--pathogenesis and risk factors of development]. PMID- 1709707 TI - [Photocoagulation and vitrectomy of diabetic retinopathy]. PMID- 1709708 TI - [Patho-physiological changes in the lung parenchyma caused by exposure of tobacco smoke in the hamster]. AB - Patho-physiological changes in the lung parenchyma caused by exposure of tobacco smoke was estimated in the hamster which an antioxidant enzyme system of lung tissue most resembled the human lung. In only a smoke exposure, the morphological and functional alteration was scarce. The alveolar epithelial cell composition also did not influenced by tobacco smoke exposure. But the distribution of neutrophil in the alveolar capillary space markedly influenced increasing in smoke exposure. In the latter stage of tobacco exposure, the alveolar macrophage became a large in the size and had a lot of vacuoles and lysosomal granules in the cytoplasm. When a lung injurious agent, Bleomycin, was added to the tobacco smoke exposure, the lung tissue injury became unexpectedly severe comparing only treatment of Bleomycin. These results suggest that only tobacco smoke exposure does not induce a morphological and functional alteration in the lung but could promote an induction of a synergistically severe lung injury adding another lung tissue injurious agent. Consequently it seems a tobacco smoking is an objectionable addiction. PMID- 1709709 TI - [Cigarette smoke and bronchoepithelium]. AB - Cigarette smoke is known to generate active oxygen species such as O2- and H2O2, and contains a variety of irritants capable of reducing the ciliary beat frequency (CBF). While acute cilio-inhibitory action of cigarette smoke no longer needs to be proved, little is known about the contribution of active oxygen compounds in cigarette smoke on the cilio-inhibitory action. Using cultured rabbit tracheal epithelium in vitro and CBF and the time to ciliostasis were measured by a microphoto-oscillation technique. An extract of cigarette smoke was made by bubbling smoke through 5 ml phosphate buffer saline (PBS) for 5 min. Smoke extract alone (a concentration of 10% and 20%) gave ciliostasis after 37 +/ 6 min and 17 +/- 2 min, respectively. Significantly less toxic was smoke extract (a concentration of 10% and 20%) containing 200 micrograms/ml superoxide dismutase (SOD) and 200 micrograms/ml catalase, and the time to ciliostasis were 62 +/- 5 min and 24 +/- 1 min, respectively. There was no significant difference in terms of the time to ciliostasis between 20% smoke extract alone and 20% smoke extract containing inactivated 200 micrograms/ml SOD and 200 micrograms/ml catalase (22 +/- 2 min vs. 23 +/- 3 min). The effect of smoke extract on transcellular transport of fluorescein Dextran through cultured canine bronchial epithelium was also discussed. PMID- 1709710 TI - [The role of thrombin on lung fibroblast growth and fibrosis in bleomycin-induced lung disorder]. AB - In order to clarify the role of thrombin on the development of pulmonary fibrosis in diffuse interstitial lung diseases, we examined the relationship between fibroblast growth-stimulating activity (FGA) and thrombin activity in bronchoalveolar lavage fluid (BALF) from rats with bleomycin-induced interstitial lung disorders. Male Wistar strain rats (body weight about 200 g) were given a single intratracheal injection of 0.9 mg bleomycin, and bronchoalveolar lavage was performed on days 2, 6 and 15. The BALF was centrifuged at 250 X g for 10 min to remove cells, and then the supernatant was recentrifugation at 27,000 X g for 40 min to remove pulmonary surfactants. The supernatant (10 ml) was dialyzed overnight against distilled water, frozen at -70 degrees C, freeze-dried, and resuspended in 2 ml of Dulbecco's modified Eagle medium (concentrated 5-fold). The 5-fold concentrated BALF was added to rat lung fibroblast culture media, and assayed for cytotoxic activity and FGA. Thrombin activity in 250 X g supernatant was measured by using fluorescence assay with the synthetic peptide substrate, Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. Histological examination showed a prominent increase in fibroblast number in the pulmonary interstitium on day 6, and transformation of fibroblasts into mature forms, fibrocytes, on day 15. On day 2 after bleomycin administration, no FGA was seen but cytotoxic activity was detected in the BALF. On day 6, the cytotoxic activity was not found, whereas FGA showed a significantly higher level than the control value. On day 15, the FGA decreased to the control value.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709711 TI - Proliferative response of human acute myeloid leukemia cells and normal marrow enriched progenitor cells to human recombinant growth factors IL-3, GM-CSF and G CSF alone and in combination. AB - The effects of human recombinant colony-stimulating factors (r-CSFs), interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) on inducing the growth of colonies derived from patients with acute myeloid leukemia (AML) (CFU-L) were investigated and compared to the proliferative response of CFU-GM derived from highly enriched normal blast cell populations. The effects of GM-CSF and IL-3 alone were similar. Both only minimally stimulated normal colonies derived from CFU-GM when compared to stimulation with MoCM (a mean of 28% of the total colonies and 17% of the colonies greater than 100 cells obtained with MoCM). Similarly, the number of leukemic colonies was substantially less than with MoCM (less than 30% of MoCM) in all but 3/10 AML patients and both were only able to significantly stimulate CFU-L derived colonies greater than 50 cells from 2/10 patients. G-CSF alone stimulated some CFU-L derived colony growth in 9/10 patients but the number stimulated was minimal relative to MoCM in five of the patients and significant stimulation of colonies greater than 50 cells occurred in only one patient. The mean number of normal CFU-GM derived colonies stimulated by G-CSF was 41% of the total colonies and 34% of the colonies greater than 100 cells generated by MoCM. The combination of G-CSF with GM-CSF and G-CSF with IL-3 resulted in a synergistic or additive increase in the number of CFU-L in 5/10 and 7/10 patients, respectively, and a synergistic increase in the size of CFU-L in 5/10. The same combinations resulted in a significant synergistic effect on size of normal CFU-GM derived colonies. There was no evidence of a synergistic increase in the number or size of CFU-L and CFU-GM derived colonies stimulated with GM-CSF in combination with IL-3. In addition, a combination of all three (G-CSF + GM-CSF + IL-3) did not enhance the effect of G-CSF + GM-CSF or G-CSF + IL-3. These results suggest that there is significant heterogeneity among AML patients in the pattern of responsiveness of the leukemic cells to the recombinant growth factors. In addition, their responsiveness does not significantly differ from that of normal progenitors. In view of the current clinical trials with r-CSFs and cytotoxic drugs in AML patients, this issue is important and worthy of further investigation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709712 TI - [Neoangiogenesis in aneurysmatic autologous venous transplants]. AB - In two patients with femoro-popliteal bypass using autogenous saphenous vein, aneurysmatic dilatation of the transplants occurred after a follow-up of 4 and 20 years. In the inner thrombus layer of the aneurysms side-branches could be shown by transfemoral angiography. A special microcorrosion cast technique for SEM analysis was able to make a vascular network visible, which spread within the inner thrombus layer of the aneurysms. Venules were found predominantly. According to clinical and experimental results the potential of the phylogenetic older venous system is greater than the ability of the arterial system. PMID- 1709713 TI - Comparison of different treatment strategies for diffuse large-cell lymphomas: a decision analysis. AB - There are more than 20 drug combinations in use for the treatment of diffuse large-cell non-Hodgkin's lymphoma (NHL). New treatments have been developed without prior comparison in clinical trials. The authors devised a Markov-process model to compare the efficacy of a first-generation combination chemotherapy regimen (CHOP) with that of a third-generation regimen (MACOP-B) using currently available data. For a typical 57-year-old male patient, life expectancy with MACOP-B was 12.1 years, compared with 8.3 years with CHOP. The remission rate after initial treatment and the functional status after the first remission is achieved were the most important determinants of life expectancy. It was still possible for CHOP to become better strategy provided that the initial remission rate with MACOP-B were lower than 50% or that functional status after remission was achieved were less than 66%. The analysis suggests that the most effective therapeutic regimen for diffuse large-cell lymphoma must not only result in a high remission rate but also cause minimal late sequelae. These two parameters were incorporated into the nomogram, which can serve as a basis for comparison of the efficacy of any chemotherapeutic regimen in non-Hodgkin's lymphoma. PMID- 1709714 TI - Evaluating atrial natriuretic peptide-induced cGMP production by particulate guanylyl cyclase stimulation in vitro and in vivo. PMID- 1709715 TI - Analyzing dynamic properties of intermediate filaments. PMID- 1709716 TI - A patient's right to a good death. PMID- 1709717 TI - [Interferons. Endogenous virostatic drugs, cytostatic drugs and immunomodulators]. PMID- 1709718 TI - Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplasts. AB - Deletions were made in the cauliflower mosaic virus polyadenylation sequence which was cloned downstream of the beta-glucuronidase gene (gus). The populations of mRNAs generated in tobacco mesophyll protoplasts by transient expression with the various constructs were analysed using a polymerase chain reaction procedure. When no deletion was present in the sequence, the mRNA appeared to be polyadenylated at two major polyadenylation sites. A deletion upstream from the AATAAA sequence made the population of polyadenylated mRNAs very heterogenous at their 3' ends. A deletion downstream of the AATAAA sequence had no effect on the choice of the site. Alternative polyadenylation sites were used when the native polyadenylation site was deleted. These results are discussed in relation to data obtained with other polyadenylation sequences from both plants and animals. PMID- 1709719 TI - Reduction of desensitization of a glutamate ionotropic receptor by antagonists. AB - The glutamate receptor channel subtype that responds to both quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) was expressed in Xenopus oocytes injected with rat cerebral cortex mRNA. Voltage-clamp current responses to QA, AMPA, and glutamate (GLU) exhibited a rapid increase followed by a decrease to a desensitized steady state (DS). Perfusion with high agonist concentrations produced smaller DS responses than perfusion with low concentrations. During the DS, the current was increased by lowering of the concentration of agonist or by application of low concentrations of a competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX). This paradoxical increase of the agonist-induced currents during the DS was also observed in cultured Purkinje cells with another competitive antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Dose-response curves obtained in oocytes were bell shaped, with a negative slope for high concentrations of QA. DNQX shifted these bell-shaped curves to the right. Together, these results indicate that the agonists are able to reversibly inhibit the AMPA receptor. The classical desensitization model of Katz and Thesleff [J. Physiol. (Lond.) 138:63-80 (1957)] cannot account for our observations. PMID- 1709720 TI - Tetrahydroaminoacridine block of N-methyl-D-aspartate-activated cation channels in cultured hippocampal neurons. AB - The action of tetrahydroaminoacridine (THA), a centrally active cholinesterase inhibitor that may provide symptomatic benefit in Alzheimer's disease, was studied on responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons, using whole-cell voltage-clamp and single-channel recording techniques. THA produced a concentration-dependent block of NMDA-evoked inward current responses (IC50, 190 microM at -60 mV), without affecting responses to quisqualate or kainate. THA block of NMDA responses was voltage dependent and was nearly completely relieved at positive holding potentials. Analysis of the voltage dependency indicated that the THA binding site senses 56% of the transmembrane electrostatic field. In single-channel recordings from outside-out membrane patches, THA appeared to reduce the frequency and duration of NMDA-evoked single-channel currents, without affecting the single-channel amplitude. The effects of THA on NMDA responses occur at concentrations 1-2 orders of magnitude greater than the therapeutic serum concentrations and, therefore, blockade of NMDA receptor-mediated responses is unlikely to contribute to the putative therapeutic action of THA. However, because NMDA receptors may play a critical role in cognitive and memory function, THA has the potential to produce undesirable central nervous system side effects at high doses. PMID- 1709721 TI - Sternal-wound infections after cardiac surgery. PMID- 1709722 TI - Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. AB - The crystal structures of major histocompatibility complex (MHC) molecules contain a groove occupied by heterogeneous material thought to represent peptides central to immune recognition, although until now relatively little characterization of the peptides has been possible. Exact information about the contents of MHC grooves is now provided. Moreover, each MHC class I allele has its individual rules to which peptides presented in the groove adhere. PMID- 1709723 TI - Tetramerization of an RNA oligonucleotide containing a GGGG sequence. AB - Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5' GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature. PMID- 1709724 TI - [A test for the detection of speech and language disorders in the acute phase after stroke. Development and clinical application]. AB - No standardized assessment of aphasic symptoms for stroke patients in the acute phase has as yet been published in the German language. Current test batteries such as the AAT are too time-consuming and cannot be performed on severely impaired patients. A short test for the examination of aphasic and dysarthric symptoms has been developed which contains 7 subtests in three main areas: spontaneous speech, comprehension and planning of movements, speech and language abilities. Since abilities in acute cases may only be functionally impaired and often only detectable after stimulation, standardized stimulation is applied in most of the subtests. In this way it is possible to test acute stroke patients for aphasic and dysarthric symptoms. The course of recovery in a typical case is described. PMID- 1709725 TI - [Current therapy of multiple sclerosis: value of cyclosporin A and FK 506]. PMID- 1709726 TI - Delayed hearing loss after surgery for acoustic neurinomas: clinical and electrophysiological observations. AB - In a series of 26 patients with medium-sized and large acoustic neurinomas and documented hearing before surgery, 7 patients had preservation of hearing initially after the procedure but then developed delayed hearing loss. The most prominent intraoperative electrophysiological finding in these cases was a gradual deterioration of brain stem auditory evoked potentials (BAEP), especially of Wave V. Four additional patients with a similar gradual intraoperative loss of BAEP and severe postoperative hearing deterioration received vasoactive treatment after surgery (low-molecular weight dextran). In all 4 patients, including 1 patient with documented total deafness after surgery, hearing was preserved. Initial preservation of cochlear nerve function after the removal of an acoustic neurinoma does not guarantee postoperative hearing. Intraoperative BAEP help to identify patients at risk for delayed postoperative hearing loss. The pathophysiological mechanism can be attributed to disturbances of the microcirculation in endoneurial vasa nervorum caused by the mechanical manipulation of the cochlear nerve. PMID- 1709727 TI - Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons. AB - The involvement of protein phosphorylation in cAMP-induced transmembrane current was tested electrophysiologically and pharmacologically in identified neurons of the Japanese land snail, Euhadra peliomphala. Intracellular injection of cAMP elicited a biphasic transmembrane current (cAMP current) which consisted of inward and outward components. The inward component was blocked with Na(+)-free, Ca2(+)-free saline and the outward component abolished by either application of tetraethylammonium or a long-lasting exposure to caffeine in Ca2(+)-free saline. The cAMP current was completely suppressed by the protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle or isoquinoline sulfonamide (H-8). The catalytic subunit of cAMP-dependent protein kinase transiently restored the cAMP current suppressed by H-8 nearly to pre-H-8 level. These findings suggest that protein phosphorylation may be an intermediate step in the activation process of the cAMP current. PMID- 1709728 TI - [Development studies during office hours]. PMID- 1709729 TI - Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation. AB - IGF-I, IGF-II, and their binding proteins (BP) were studied in sera obtained by direct puncture of umbilical cords in utero between 20 and 37 wk of gestation in 103 normal fetuses and in 16 fetuses with intrauterine growth retardation, as well as in the cord blood of 37 normal newborns of 38- to 42-wk pregnancies. In normal fetuses, IGF-I levels were approximately 50 ng/mL and IGF-II levels approximately 350 ng/mL up to the 33rd wk of pregnancy. Thereafter, both increased to reach values two to three times higher at term. Correlations were found between fetal placental lactogen levels and those of IGF-I and IGF-II, which is consistent with the hypothesis that placental lactogen is involved in the regulation of IGF synthesis in the fetus. With weight (either measured at birth or deduced from echographical data) as index of fetal size, IGF-I levels were significantly (p less than 0.001) higher in fetuses with weights above the mean for gestational age than in fetuses with weights below the mean, whereas IGF II levels were similar in the two groups. Similarly, IGF-I (but not IGF-II) levels in fetuses with intrauterine growth retardation were significantly lower than those in normal fetuses of the same age (p less than 0.01). These findings suggest that, during the latter months of intrauterine life, IGF-I (but not IGF II) is involved in the control of fetal size. Total fetal BP concentrations were approximately 1/3 those of adults. The fetal electrophoretic profile obtained by Western-ligand blotting bore a strong resemblance to that of subjects with growth hormone deficiency.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709730 TI - [Bronchial endoprostheses (stents) in inoperable bronchial carcinoma]. AB - Three patients suffering from bronchial carcinoma with severe bronchial obstruction have been successfully treated by implantation of metallic stents. Two of them suffered from peribronchial tumor growth while the third one complained about dyspnoea caused by the instability of the central airways. Stents were implanted into the left main bronchus or the intermediate bronchus either through a flexible bronchoscope under local anaesthesia and sedation or through a rigid bronchoscope under general anaesthesia. The successful dilatation of the stenosis could be demonstrated by bronchography. The patients tolerated the stents well. We found no dislocation, deformation or ulceration of the bronchial mucosa. The implantation of metallic stents thus seems to offer new possibilities for the palliative treatment of bronchial carcinoma. PMID- 1709731 TI - [The activity of trypsin-like proteinases and their inhibitors inpatients with tuberculosis of bones and joints]. PMID- 1709732 TI - A link between double-strand break-related repair and V(D)J recombination: the scid mutation. AB - We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNA-damaging agents that caused double-strand breaks (x irradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random double-strand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining [V(D)J] DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein. PMID- 1709733 TI - Expression of cardiac Na channels with appropriate physiological and pharmacological properties in Xenopus oocytes. AB - The objective of this study was to determine whether the Xenopus laevis oocyte can express an exogenous cardiac Na channel that retains its normal physiological and pharmacological properties. Cardiac Na channels were expressed in oocytes following injection of RNA from guinea pig, rat, and human heart and detailed analysis was performed for guinea pig cardiac Na channels. Average current amplitudes were -351 +/- 37 nA with peak current observed at -8 +/- 1 mV. Steady state inactivation was half-maximal at -49 +/- 0.6 mV for the expressed channels. All heart Na currents were resistant to block by tetrodotoxin compared to Na currents expressed from brain RNA with IC50 values for guinea pig, rat, and human heart of 651 nM, 931 nM, and 1.3 microM, respectively. In contrast, rat brain Na channels were blocked by tetrodotoxin with an IC50 value of 9.1 nM. In addition, the effects of the cardiac-selective agents lidocaine and DPI 201-106 were examined on Na currents expressed from brain and heart RNA. Lidocaine (10 microM) blocked cardiac Na current in a use-dependent manner but had no effect on brain Na currents. DPI 201-106 (10 microM) slowed the rate of cardiac Na channel inactivation but had no effect on inactivation of brain Na channels. These results indicate the Xenopus oocyte system is capable of synthesizing and expressing cardiac Na channels that retain normal physiological and pharmacological properties. PMID- 1709734 TI - Effects of metal ions, including Mg2+ and lanthanides, on the cleavage of ribonucleotides and RNA model compounds. AB - The cyclization/cleavage of 3',5'-uridyluridine to form 2',3'-cyclic uridylic acid is very effectively catalyzed by Eu3+, and the cyclization/cleavage of the 1 p-nitrophenyl phosphate ester of propane-1,2-diol also shows strong metal ion catalysis by Eu3+, Tb3+, and Yb3+. It also shows moderate catalysis by Mg2+, but not by Ca2+; Zn2+ and Pb2+ are also good catalysts. Various ligands activate these reactions further, and imidazole apparently acts as an additional base catalyst. Some cyclodextrin derivatives act to bind both the substrate and the metal ion but, contrary to what is reported elsewhere, there is no strong selectivity among nucleotides that can be ascribed to cyclodextrin binding. PMID- 1709735 TI - Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. AB - Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, stimulates immune responses by activating macrophages, B lymphocytes, and other cells of the immune system. The mechanisms by which LPS activates these cells are poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signaling event that mediates cellular responses, we examined whether LPS alters tyrosine phosphorylation in macrophages. We found that Escherichia coli K235 LPS increased tyrosine phosphorylation of several proteins in the RAW 264.7 murine macrophage cell line and in resident peritoneal macrophages from C3H/HeSNJ mice. Changes in tyrosine phosphorylation were detectable by 4-5 min, reached a maximum by 15 min, and declined after 30-60 min. Protein tyrosine phosphorylation increased following stimulation with LPS at 100 pg/ml and was maximal with 10 ng/ml. Similar changes in tyrosine phosphorylation were induced by Salmonella minnesota R595 LPS and by the biologically active domain of LPS, lipid A, but not by the inactive lipid A derivative N2 monoacylglucosamine 1-phosphate. Phorbol 12-myristate 13-acetate also stimulated protein tyrosine phosphorylation, but some of the modulated proteins were different than those phosphorylated by LPS. Treatment of RAW 264.7 cells with a tyrosine kinase inhibitor, herbimycin A, inhibited both LPS-stimulated tyrosine phosphorylation and LPS-stimulated release of arachidonic acid metabolites. Thus, increased protein tyrosine phosphorylation is a rapid LPS-activated signaling event that may mediate release of arachidonic acid metabolites in RAW 264.7 cells. PMID- 1709736 TI - Low temperature and peptides favor the formation of class I heterodimers on RMA-S cells at the cell surface. AB - RMA-S murine cells have a mutation that interferes with the assembly of class I major histocompatibility complex (MHC) heterodimers and are deficient in the expression of class I molecules on the cell surface. The mutant phenotype has been reported to be normalized upon incubation of RMA-S cells at 25 degrees C. We find that much of the increased expression of class I heterodimers is dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2 microglobulin. Furthermore, epitopes that are associated with class I MHC molecules that have bound xenogeneic beta 2-microglobulin are preferentially formed on RMA-S cells cultured at 25 degrees C. These heterologous class I molecules are thermolabile. Increased expression of class I molecules has also been observed on RMA-S cells incubated at 37 degrees C in the presence of class I restricted peptides. We find that the increased expression of Db molecules induced by influenza virus nucleoprotein residues 365-380 is similarly dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2 microglobulin. PMID- 1709737 TI - Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1. AB - Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that mediates endothelial leukocyte interactions by inducing expression of adhesion molecules. In this report, we demonstrate that human dermal mast cells contain sizeable stores of immunoreactive and biologically active TNF-alpha within granules, which can be released rapidly into the extracellular space upon degranulation. Among normal human dermal cells, mast cells are the predominant cell type that expresses both TNF-alpha protein and TNF-alpha mRNA. Moreover, induction of endothelial leukocyte adhesion molecule 1 expression is a direct consequence of release of mast cell-derived TNF-alpha. These findings establish a role for human mast cells as "gatekeepers" of the dermal microvasculature and indicate that mast cell products other than vasoactive amines influence endothelium in a proinflammatory fashion. PMID- 1709738 TI - Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation. AB - Ternary complexes of RNA polymerase II, bearing the nascent RNA transcript, are intermediates in the synthesis of all eukaryotic mRNAs and are implicated as regulatory targets of factors that control RNA chain elongation and termination. Information as to the structure of such complexes is essential in understanding the catalytic and regulatory properties of the RNA polymerase. We have prepared complexes of purified RNA polymerase II halted at defined positions along a DNA template and used RNase footprinting to map interactions of the polymerase with the nascent RNA. Unexpectedly, the transcript is sensitive to cleavage by RNases A and T1 at positions as close as 3 nucleotides from the 3'-terminal growing point. Ternary complexes in which the transcript has been cleaved to give a short fragment can retain that fragment and remain active and able to continue elongation. Since DNA.RNA hybrid structures are completely resistant to cleavage under our reaction conditions, the results suggest that any DNA.RNA hybrid intermediate can extend for no more than 3 base pairs, in dramatic contrast to recent models for transcription elongation. At lower RNase concentrations, the transcript is protected from cleavage out to about 24 nucleotides from the 3' terminus. We interpret this partial protection as due to the presence of an RNA binding site on the polymerase that binds the nascent transcript during elongation, a model proposed earlier by several workers in preference to the hybrid model. The properties of this RNA binding site are likely to play a central role in the process of transcription elongation and termination and in their regulation. PMID- 1709739 TI - CpG island in the region of an autosomal dominant polycystic kidney disease locus defines the 5' end of a gene encoding a putative proton channel. AB - In an attempt to isolate candidate genes for autosomal dominant polycystic kidney disease, a number of CpG-rich islands have been identified from a region defined genetically as the site of disease mutations. Genomic fragments adjacent to one of these islands were used to isolate cDNAs from both HeLa cells and cultured cystic epithelium that encode a 155-amino acid peptide having four putative transmembrane domains. The corresponding transcript was found in all tissues tested but was most abundant in brain and kidney. Potential control response elements were identified in the genomic region 5' to the initiation codon. The deduced amino acid sequence has 93% similarity to the 16-kDa proteolipid component that is believed to be part of the proton channel of the vacuolar H(+) ATPase. Possible roles for a mutated proton channel in the pathogenesis of cystic disease were considered. However, sequencing of cDNAs corresponding to both alleles of an affected individual revealed no differences in the deduced amino acid sequence. Moreover, transcript size and abundance were not altered in cystic kidney. PMID- 1709740 TI - Transcription of a subset of human class II major histocompatibility complex genes is regulated by a nucleoprotein complex that contains c-fos or an antigenically related protein. AB - Transcriptional regulation of the human major histocompatibility complex class II genes requires at least two upstream elements, the X and Y boxes, located in the 50- to -150-base-pair region of all class II promoters. The DRA and DPB promoters contain phorbol ester-responsive elements overlapping the 3' side of their X boxes. Mutation of this sequence down-regulates the efficiency of the DRA promoter, suggesting that a positive regulator(s) binds to this site. In this report, anti-sense c-fos RNA and an anti-c-fos antibody were used to show that the product of the protooncogene c-fos or an antigenically related protein is a component of a complex that binds to the X box and is required for maximal transcription from the DRA and DPB promoters. As c-fos (or its related proteins) cannot bind alone to DNA, these results suggest that it may dimerize with other members of the JUN/AP-1 family, such as hXBP1, to participate in the activation of a subset of class II major histocompatibility complex genes. PMID- 1709741 TI - Expression of mammalian gamma-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. AB - Gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is known to interact with two classes of GABA receptors denoted GABAA and GABAB. Using Xenopus oocytes, we compared the electrical and pharmacological properties of GABA receptors expressed by poly(A)+ RNA isolated from mammalian brain and retina. RNA from cerebral cortex expressed GABA responses with features characteristic of currents mediated by GABAA receptors. In contrast, RNA from retina expressed responses mediated by GABAA receptors and, in addition, GABA responses that were insensitive to the GABAA antagonist bicuculline and the GABAB agonist baclofen and showed no modulation by barbiturates or benzodiazepines. The bicuculline/baclofen-insensitive GABA response was a Cl- current that was blocked by picrotoxin but showed little desensitization or outward rectification. Our results suggest that mammalian retina contains RNAs encoding GABA receptors with distinct pharmacology. PMID- 1709742 TI - Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor. AB - The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4 hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. PMID- 1709743 TI - Barrel construction in rodent neocortex: role of thalamic afferents versus extracellular matrix molecules. AB - The rodent primary somatosensory cortex is characterized by aggregates of cellular and axonal elements that replicate the distribution of mystacial vibrissae on the face. The periphery-related cortical pattern ("barrels") is influenced by an amalgam of elements extrinsic (i.e., afferents) and intrinsic (i.e., neurons, glia, and their substrate) to the developing neocortex. To assign the role of some of these elements in cortical pattern formation, we have examined the temporal correlation between periphery-related patterns formed by thalamocortical axons and by extracellular matrix (ECM) molecules associated with neurons and glia in the cortex. Thalamocortical axons were labeled with the lipophilic tracer 1,1'-dioctydecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in aldehyde-fixed neonatal rat brains, and the same brains were also prepared for immunohistochemical localization of ECM molecules cytotactin and cytotactin-binding proteoglycan. We present evidence that thalamocortical axons form a periphery-related pattern well before such an organization is detectable in the distribution of ECM molecules. Furthermore, a patterned distribution of ECM molecules results from the down-regulation of these molecules from barrel centers, where thalamic axons have established vibrissa-specific patches. We conclude that thalamic axons convey the blueprint of the sensory periphery onto the neocortex and that ECM molecules do not participate in the initial formation of this pattern. PMID- 1709744 TI - Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions. AB - Galanin (GAL) is widely distributed in the peripheral and the central nervous systems. In the brain, the highest GAL concentrations are observed within the hypothalamus and, particularly, in nerve terminals of the median eminence. This location, as well as GAL actions on prolactin, growth hormone, luteinizing hormone (LH), and LH-releasing hormone (LHRH) secretion, suggest the possibility that GAL may act as a putative hypothalamic-hypophysiotropic hormone. To establish this, GAL and LHRH levels were measured in hypophyseal portal plasma samples using specific radioimmunoassays. Rat galanin (rGAL) concentrations in portal blood were approximately 7-fold higher than those observed in peripheral plasma in male and female (estrus, diestrus) rats, indicating an active secretory process of rGAL into the portal vasculature. Frequent (10 min) sampling revealed that rGAL and LHRH were secreted into the portal circulation in a pulsatile manner with a pulse frequency of one pulse per hour. Interestingly, both hormone series depicted a high degree of coincident episodes. In fact, the probability of random coincidence, calculated by the algorithm HYPERGEO, was less than 0.01. Moreover, the retrograde tracer Fluoro-Gold, when given systemically, was taken up by GAL neurons in the hypothalamus, including a subset of neurons expressing rGAL and LHRH, strengthening the notion of the existence of a GAL neuronal system connected to the hypophyseal portal circulation. These observations reinforce the concept that GAL regulates pituitary hormone secretion. To analyze this in further detail, the effects of rGAL on LH secretion were evaluated under basal and stimulated conditions. rGAL induced a small but dose-dependent increase in LH secretion from cultured, dispersed pituitary cells. Interestingly, rGAL enhanced the ability of LHRH to stimulate LH release. The tight link between GAL and LHRH neuronal systems is strengthened by the observation that during the estrous cycle of the rat, rGAL and LHRH contents in the median eminence show an identical profile (r = 1.00). These data indicate that GAL should be considered as a hypothalamic-hypophysiotropic hormone and as an important neuromodulator of LHRH secretion and action. The colocalization and cosecretion of GAL and LHRH and the cooperative action at the level of the anterior pituitary afford important evidence for the functional significance of coexistence of neurotransmitters in neurons of the central nervous system. PMID- 1709745 TI - [Angiogenesis and superficial venous insufficiency, the induced disease]. PMID- 1709746 TI - Concentrations of cerebrospinal fluid monoamine metabolites in suicides. PMID- 1709747 TI - Regulatory peptide and serotonin content and brush-border enzyme activity in the rat gastrointestinal tract following neonatal treatment with capsaicin; lack of effect on epithelial markers. AB - The possible trophic influence of the capsaicin-sensitive extrinsic innervation of the gastrointestinal mucosa was investigated. Rats were treated neonatally with capsaicin. The gastrointestinal content of serotonin and glucagon-like immunoreactivity were used as a measure of the effect on the endocrine gut mucosa and gastrointestinal aminopeptidase and alkaline phosphatase activities were used as a measure of the effect on the gut brush-border. The gastrointestinal content of the neuropeptides substance P, VIP and CGRP were used to monitor effects on the innervation of the gut. The depletion of substance P-immunoreactivity(-IR) and calcitonin gene-related peptide(CGRP)-IR in extracts of urinary bladder and lung from the capsaicin-treated rats is evidence of the efficacy of capsaicin treatment in affecting a loss of C-fibre sensory nerves. The significant depletion of CGRP-IR measured in the stomach and duodenum of capsaicin-treated rats indicated the loss of the C-fibre sensory innervation to the gastrointestinal tract. The gastrointestinal content of VIP and substance P, which are predominantly within intrinsic gut neurones, were unaffected by capsaicin treatment. In all regions of the gastrointestinal tract of capsaicin treated rats, the serotonin and glucagon-IR levels were not significantly different from those in controls. Similarly the levels of activity of the brush border enzymes were not significantly effected by capsaicin treatment. This suggest the absence of any major trophic influence of capsaicin-sensitive sensory nerves on the gut endocrine mucosa and the brush border. PMID- 1709748 TI - Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J. AB - Competitive inhibition binding studies on membranes from the rat pancreatic AR 4 2J cell line revealed the predominance (80%) of low selectivity CCK receptors (KD of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17I (G-17I] over selective receptors (20% with a KD of 1 nM and 1 microM for, respectively, CCK-8 and G 17I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17I and CCK-4. G-17I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2-17)I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [32P]ADP ribosylation of AR 4-2J cell membranes allowed to detect the presence of two Gs alpha (the 50 kDa form predominating over the 45 kDa form) and one Gi alpha (41 kDa). However, Gi and Gs may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17I-dependent amylase secretion. PMID- 1709749 TI - Effect of prolonged administration of long-acting somatostatin on caerulein, CCK 8 and GRP induced pancreatic growth in the rat. AB - This work investigates the effects of the long-acting somatostatin analogue, octreotide also named SMS 201-995 or Sandostatin, on pancreatic growth in function of the dose and duration of treatment. Octreotide was administered s.c. twice daily, while pancreatico-trophic peptides, caerulein and CCK-8 (1.8 nmol/kg b.wt.) or GRP (3.6 nmol/kg b.wt.) were administered s.c. three times daily. Octreotide (1,10,20 micrograms/kg b.wt.) administered for 4 days reduced pancreatic growth induced by caerulein in a dose-dependent manner. This effect, significant from 10 micrograms/kg, was more obvious with 20 micrograms/kg. At this latter dose, octreotide inhibited significantly the increase in pancreatic weight and protein, RNA, DNA and enzyme content induced by a 4- or 10-day treatment with GRP. A similar effect was observed after a 4-day treatment with CCK-8, but after a 10-day treatment only protein and enzyme contents were reduced. Octreotide by itself did not affect pancreatic size and composition after a 10-day treatment, but decreased enzyme content after a 4-day treatment. It is concluded that octreotide exerts an antitrophic effect on the rat exocrine pancreas which depends on the dose and duration of treatment and can be modulated by the trophic factor applied for a long-term. PMID- 1709750 TI - Mechanism of capsaicin action: recent learnings. AB - Capsaicin has long been known to act selectively on a subpopulation of neurons to produce initial excitation, followed by a prolonged neuroinhibitory action commonly referred to as 'capsaicin desensitization'. This property has been exploited extensively as a tool with which to investigate the role of these nerves in the normal and pathophysiology of the airways. However, the cellular basis for these effects is only now beginning to be understood. The purpose of this review is to summarize recent findings that provide new insights into the mechanisms by which capsaicin acts to exert its effects. These findings suggest that capsaicin acts at a receptor that is either tightly coupled to, or indeed is, a relatively non-selective cation channel. Binding of capsaicin to this receptor allows both sodium and calcium (and possibly potassium) ions to flow down their concentration gradients, causing initial depolarization and neurotransmitter release. Prolonged exposure to capsaicin produces a subsequent desensitization or neuroinhibition. The neuroinhibition has now been shown to be of two types: capsaicin-specific and non-specific. The former is characterized by a loss of the acute excitatory response evoked by application of capsaicin, but maintained responses to other stimuli. The latter is characterized by a loss of responsiveness to all stimuli and is probably associated with the neurotoxic effect of this agent. Despite these recent findings, many questions remain regarding the nature and physiological role of the capsaicin receptor. The availability of two new probes for this receptor, ruthenium red and resiniferatoxin, promises to provide at least some of the answers to these intriguing questions. PMID- 1709751 TI - [Granulo-monocyte growth factors. Their value in pneumonologic oncologic practice]. AB - Granulo-monocytic growth factors are important members of the family of growth factors which alter the biological response. These factors are glycoproteins and are hormones regulating the production of haematopoietic cells and show their adaptation to possible needs. There are four which are currently well recognised; they are now grouped collectively under the term colony-stimulating factor and of these two, GM-CSF and G-CSF are available thanks to genetic engineering and have reached the stage of therapeutic trials. It is essentially the treatment of solid tumours which should lead to the most profound changes in the current management of cytotoxic drugs because they may allow an increase of the dose and a shortening of the duration of the phase of neutropenia and thus the infection risk. In therapeutic pneumonology they would be able to be used as an adjuvant to chemotherapy in small cell cancers and available in protocols using polychemotherapy with a risk of marrow suppression, indeed even permitting the practice of intensified chemotherapy regimes with or without the support of bone marrow graft. However the clinical experience acquired using growth factors remains limited and is almost exclusively restricted to the haematological domain. Although the secondary effects appear tolerable their harmlessness--and notably the absence of a stimulating effect on tumour growth--remains to be shown before extension of their use will lead to any changes of therapeutic strategies. PMID- 1709752 TI - Width of thermal damage after using the YAG contact laser for cutting biological tissue: animal experimental investigation. AB - At the University Women's Clinic in Kiel, the YAG contact laser has been used as a cutting instrument in pelviscopic operations since 1987. When the laser cuts, it produces only a scant amount of mechanical trauma. The determining factor is the amount of thermal damage produced along the wound margins and in direct neighboring tissue. The extent of the tissue change seen in the uterus and liver parenchyma of rats and the striated muscle of rabbits after application of the YAG contact laser was demonstrated using various staining techniques and stains. Liver parenchyma proved to be the most sensitive to thermal damage. In the uterine horn, enzyme-histochemical ATPase and alkaline phosphatase demonstrations showed a significantly wider zone of thermal damage after laser incision than did hematoxylin-eosin and Goldner staining techniques. A good understanding of the extent of thermal damage is essential for atraumatic pelviscopic operations using the YAG contact laser and also for the preventing of complications. PMID- 1709753 TI - Metabolic and ultrastructural characteristics of biosynthetic processes in the cerebral cortex at 3 months following a prolonged hemorrhagic shock. AB - Biochemical indicators (the content of ATP, total RNA and DNA, acid and alkaline phosphatase activity) and ultrastructural changes in cells of the nerve tissue were studied in the cerebral cortex of anesthetized dogs 3 months after they had sustained a 4-h hemorrhagic shock (arterial pressure = 40 mmHg). A new variant of reconstruction of cell membranes and organelles that develops in neurons and gliocytes of the brain in the process of adaptation 3 months after resuscitation is identified. This reconstruction variant is described by (a) the presence of a monolayer of a substance with a medium electron density with the incorporated dense granules instead of the common three-layer organisation; (b) the ability displayed by the organellar structure to detect "errors" in its organisation; (c) changes in the character of intercellular relations. The development of this variant of cell structure in the postresuscitation period is likely to be based on the information disintegration of a cell as a system, resulting in distortion of biosynthesis of supracellular ensembles and biological membranes of nerve cells in particular. PMID- 1709754 TI - [Implantation of urological prostheses. Role of the nurse]. PMID- 1709755 TI - Arrhythmia review. Premature atrial contractions. PMID- 1709756 TI - Exaggerated intestinal histamine release by casein and casein hydrolysate but not whey hydrolysate. AB - Loops of rabbit distal small intestine received luminal acetic acid (pH 4.0) alone or in combination with bovine casein, casein hydrolysate, or whey hydrolysate. Blood-to-lumen movement of 51Cr-labeled ethylenediaminetetraacetic acid (EDTA) (an index of epithelial permeability) and loop fluid histamine levels were quantified after 45 min. Luminal acetic acid caused a marked increase in 51Cr-EDTA accumulation which was not modified by the addition of bovine casein or hydrolysates by of casein or whey. However, acetic acid-induced histamine release was potentiated by casein and casein hydrolysate (six- and four-fold respectively) but was not altered by whey hydrolysate. Casein hydrolysate dependent histamine release was evident in naloxone-pretreated rabbits, suggesting that beta-casomorphins were not solely responsible. We conclude that luminal casein or casein hydrolysate, but not whey hydrolysate. can activate intestinal mast cells under conditions of enhanced epithelial permeability. This effect appears to involve components other than beta-casomorphins. PMID- 1709757 TI - [Percutaneous echography-guided alcohol block of the celiac plexus as treatment of painful syndromes of the upper abdomen: study of 21 cases]. AB - Celiac plexus block is usually performed under fluoroscopic or tomodensitometric guidance. We report on a new procedure using sonographic guidance. the patient lies in supine position. We use a real-time sonograph with a 3.5 MHz probe. On a transverse plane, the celiac axis is localized emerging from the aorta. Under local anesthesia, the tip of the spinal needle (177 mm, 22 g) is placed close to the aorta (about 5 mm) on both sides. 5 to 10 ml of 1% lidocaine, then 10 to 20 ml of absolute alcohol, are injected on each side. 21 patients (10 males, 11 females, mean age: 61.4) underwent the procedure. They presented with cancer of the pancreas in 14 cases, metastatic nodes from an extra-pancreatic tumor in 5 cases and chronic calcifying pancreatitis (CCP) in 2 cases. No pain relief was secured in 3 patients (14%). One of these presented with CCP, but endoscopic cystic diversion of a small cyst was successful in eradicating pain. Partial pain relief was secured in 5 cases (24%) and total pain relief in 13 cases (62%). No treatment-related complication was observed. We conclude that sonography is a simple and safe method of guidance in performing alcohol block of the celiac plexus. The anterior approach may prevent neurologic complications occurring with other methods of guidance using a posterior approach. PMID- 1709758 TI - Retronphage phi R73: an E. coli phage that contains a retroelement and integrates into a tRNA gene. AB - Some strains of Escherichia coli contain retroelements (retrons) that encode genes for reverse transcriptase and branched, multicopy, single-stranded DNA (msDNA) linked to RNA. However, the origin of retrons is unknown. A P4-like cryptic prophage was found that contains a retroelement (retron Ec73) for msDNA Ec73 in an E. coli clinical strain. The entire genome of this prophage, named phi R73, is 12.7 kilobase pairs and is flanked by 29-base pair direct repeats derived from the 3' end of the selenocystyl transfer RNA gene (selC). P2 bacteriophage caused excision of the phi R73 prophage and acted as a helper to package phi R73 DNA into an infectious virion. The newly formed phi R73 closely resembled P4 as a virion and in its lytic growth. Retronphage phi R73 lysogenized a new host strain, reintegrating its genome into the selC gene of the host chromosome and enabling the newly formed lysogens to produce msDNA-Ec73. Hence, retron Ec73 can be transferred intercellularly as part of the genome of a helper-dependent retronphage. PMID- 1709759 TI - [Psychosocial rehabilitation. The mentally insufficient schoolchild]. PMID- 1709760 TI - Biopsy of the prostate guided by transrectal ultrasonography: early experience in a teaching community hospital. AB - Transrectal ultrasonography (TRUS) was used to guide 277 biopsies of the prostate in 182 patients. The Bard Biopty system was used. Of these 182 patients, 107 (59%) had benign changes, and 75 (41%) had malignant lesions. Of those with cancer, 40% (30/75) had negative results of digital rectal examination (DRE). The cancer detection rate by TRUS was 40.7%, and by DRE alone it was 24.2%. Use of TRUS before transurethral resection of the prostate (TURP) in 47 patients with negative findings on DRE yielded 22 cancerous lesions (47%). We advocate this method in patients with abnormalities detected on DRE or with elevated levels of prostate-specific antigen or prostatic acid phosphatase, and as preoperative screening in patients having TURP because of the potential impact of a preoperative diagnosis of cancer upon the choice of treatment. PMID- 1709762 TI - RNA editing fixes problems in plant mitochondrial transcripts. PMID- 1709761 TI - Effect of ischemia on protein synthesis in the septic liver. AB - The effect of ischemia on hepatic protein synthesis during sepsis is not known, but is of clinical relevance, since hepatic blood flow decreases during the late phase of sepsis. In this study, synthesis of acute-phase proteins was measured in perfused livers of rats 16 hours after sham operation or cecal ligation and puncture. Livers from each group had 45 minutes of complete ischemia or control perfusion. Protein synthesis was measured during two hour perfusion after the ischemia or control period, by determining incorporation of 3H-leucine into total secreted trichloracetic acid precipitated proteins, immunoprecipitated complement component C3 and albumin and phosphotungstenate-precipitated alpha 1-acid glycoprotein. Lactate, glutamine-oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels in the perfusate were measured during preischemic and postischemic perfusion. Tissue glutathione levels were measured at the end of the perfusion. Synthesis of alpha 1-acid glycoprotein was increased by 100 per cent and albumin synthesis decreased by 46 per cent in septic livers, consistent with an acute-phase response and apparent downregulation of albumin synthesis during early sepsis. Synthesis rates were reduced by 50 to 60 per cent after ischemia in perfused livers from sham operated rats and 70 to 80 per cent in livers from septic rats. Hepatic production of interleukin-1 was not different between the groups during perfusion. GOT and GPT levels increased significantly during ischemia of both nonseptic and septic livers and rapidly returned toward baseline during reperfusion. Lactate levels were higher in perfusate of septic than of nonseptic livers before ischemia and increased further during ischemia. The results suggest that ischemia inhibits production of secreted hepatic proteins similarly in nonseptic and septic livers, but perhaps to a slightly greater extent in septic livers. PMID- 1709763 TI - [Specific features of the pathogenesis of epichlorohydrin eye burns and their treatment (an experimental clinico-morphological study)]. AB - Specific features in the pathogenesis of ocular burns with a solution of an organic product, epichlorohydrin, are analyzed. Abnormalities in the vascular system anterior portion were revealed, related to the toxic effect of the agent on tissue vessels and to enhanced permeability and involvement of not only the cornea, but (and even more so) of the iris, where necrotic changes and hemorrhages eventuating in iris atrophy were detectable. Contrykal (hordox) therapy promoted a drastic inhibition of leukocytic and lymphocytic migration into burnt tissue, this resulting in a month in the formation of gentle cicatricial tissue with negligible residual inflammatory infiltration in the burn wound. Iris changes still persisted, since the drug appeared to have no effect on this process. PMID- 1709764 TI - HGH, PRL and beta HCG/beta LH gene expression in clinically inactive pituitary adenomas detected by in situ hybridization. AB - Within our surgical collection clinically inactive pituitary adenomas represent 30.7% of all pituitary tumours. To characterize their endocrine activity we studied 40 clinically inactive pituitary adenomas with in situ hybridization (ISH) using cRNA probes labelled with 35S encoding growth hormone (GH), prolactin (PRL) and chorionic gonadotrophin (beta HCG). No tumour was associated with clinical evidence of elevated hormone secretion. A mild hyperprolactinaemia not correlated with hormone or the mRNA content of the cells was interpreted to be incidental in 11 patients. By histological analysis, immunohistochemistry (IH) and electron microscopy the adenomas were diagnosed as small cell chromophobic (n = 16) and large cell chromophobic (n = 8) adenomas, and oncocytomas (n = 16). Gene expression of one or more hormones was identified by ISH in 18 of 40 adenomas in few cells. GH and PRL gene expression was rare (GH mRNA in 3 of 40 tumours and PRL mRNA in 8 of 40 tumours) whereas in 14 of 40 adenomas beta HCG/beta LH gene expression was identified in scattered cells. Five of 40 adenomas lacking hybridization signals revealed hormones by IH. The detection of mRNA was accompanied by positive immunostaining for the respective hormones in 72%. The combination of ISH and IH reveals good evidence that the hormones are synthesized in the tumours and not taken up from the serum and stored in the cells. The two methods used together permit a more precise analysis of tumour biology than each alone. PMID- 1709765 TI - Cytokeratin polypeptide expression in a cloacogenic carcinoma and in the normal anal canal epithelium. AB - The aim of the present study was to explore the origin of cloacogenic carcinoma in the anal canal by immunohistochemical methods. We compared cytokeratin polypeptide expression of a cloacogenic carcinoma to normal and epithelia, to anal squamous cell carcinoma and to basal and squamous cell carcinoma of the skin, using a battery of monoclonal anti-cytokeratin, polypeptide-specific antibodies. Our results indicate that cloacogenic carcinoma expresses cytokeratin polypeptides similar to those of the basal layer of anal squamous epithelium, of the anal transitional zone epithelium and of a layer of basal cells in the anal glands. Thus we concluded that each of the above cell types may be the cell of origin of cloacogenic carcinoma. PMID- 1709766 TI - [Molecular cloning of lipopolysaccharide genes of the Vibrio cholerae in E. coli HB101]. AB - A genomic library of the V. cholerae 178 (Eltor biotype, Ogawa serotype) was constructed by using cosmid pHC 79 as a cloning vector. We screened the library with immune agglutination test and colonies solid phase ELISA. 13 positive recombinants which could express the O antigen of the V. cholerae lipopolysaccharide (LPS) were acquired. The LPS was then extracted from a positive recombinant PMM-VO 38 by using hot phenol-water method. It was found that purified LPS specifically reacted to antisomatic serum against the V. cholerae. The restriction endonucleases analysis showed that the molecular weight of the recombination cosmid PMM-VO 38 was about 46 kb. PMID- 1709767 TI - [Studies on the serogroup of Yersinia enterocolitica. II. Selection and establishment on serogroup reference strains of Yersinia enterocolitica]. AB - Since 1980, we have collected 1120 strains of Yersinia enterocolitica, from the different parts of China. These strains have been obtained from various sources in man, animals and natural environment accompanied by their clinical or ecological information of Yersinia enterocolitica. The results of our tests have shown that the 747 strains have exhibited the clinical morphological and biochemical characteristics of Yersinia enterocolitica. Through comparing under the same conditions, out of the 747 strains 335 have been selected out with better antigenicity and have been produced antisera from their representative strains. This set of antisera is very satisfactory for its potency and specificity. This set of antisera is ready to supply and have good efficacy and application facilitated for control strains on identifying strains and their epidemiologic observation. PMID- 1709768 TI - Modifying effects of interferon and puromycin on cytogenetic damage induced by alkylating drug phopurine in human lymphocytes. AB - The induction of sister chromatid exchanges (SCEs) by the bifunctional alkylating antineoplastic drug phopurine (2-dimethyl-amino-6-diethyleneiminophosphamido-7 methylpurine) and its modification by human recombinant interferons alpha 2, beta and gamma (HuIFN alpha 2, HuIFN beta and HuIFN gamma) and puromycin (PM) were studied in human lymphocytes. Results demonstrated a striking similarity in the modifying action of HuIFN alpha 2 and PM: 1) both modifiers reduced SCE values induced by phopurine, 2) at high and low doses of phopurine the effect of both modifiers was minimal, and 3) both agents were able to convert DNA lesions from short-term to long-term. PMID- 1709769 TI - Can pregnancy-associated plasma protein A (PAPP-A) predict the outcome of pregnancy in women with threatened abortion and confirmed fetal viability? AB - Depressed pregnancy-associated plasma protein A (PAPP-A) concentrations have been found in patients with threatened abortion, often weeks before spontaneous abortion while the fetus was still alive. In order to extend these findings we have developed a highly sensitive PAPP-A radio-immunoassay and have established a reference range in early pregnancy for PAPP-A between week 7 and week 20 of pregnancy, based on blood samples from 240 pregnant women. The gestational age was determined by ultrasound. PAPP-A was measured in 128 women admitted to hospital because of vaginal bleeding in the 7th to 20th gestational week. The viability of the fetus was confirmed by ultrasonography. The serum values of PAPP A were significantly lower (p = 0.002) in the group of women with vaginal bleeding than in the group of normally pregnant women. However, with regard to abortion later on, the predictive value of an abnormal blood test on admission was only 18.7%. Serial determinations showed increased PAPP-A values corresponding to the centile expected in the 12 women who aborted, as well as in the 116 women who gave birth. Consequently, the test is of no clinical value in the assessment of the prognosis in patients with symptoms of threatened abortion. PMID- 1709770 TI - High-dose intravenous gammaglobulin therapy for neonatal immune haemolytic jaundice due to blood group incompatibility. AB - Three newborn infants who developed hyperbilirubinemia due to blood group incompatibility were treated with high-dose gammaglobulin. Hyperbilirubinemia was caused by Rhesus (Rh) incompatibility (anti-E + anti-c) in Infant 1 and ABO incompatibility (anti-B) in Infants 2 and 3. Hyperbilirubinemia was refractory to conventional phototherapy but responded well to intravenous gammaglobulin (IVGG) at a dose of 1 g/kg in all infants. No adverse effects were observed. These findings suggest that high-dose IVGG may be useful in the treatment of hyperbilirubinemia due to isoimmune haemolytic disease resistant to phototherapy. PMID- 1709771 TI - A follow-up study of children with neonatal herpes simplex virus infections with particular regard to late nervous disturbances. AB - Forty-five children with neonatal herpes simplex virus (HSV) infection, representing all known cases in the diagnostic records of four virological laboratories within a 15-year period, were followed up. Twelve children had died. Sixteen of the 33 survivors were healthy. Thirteen children had severe disabilities: all of them showed severe mental retardation; moreover, 11 were tetraplegic, one was hemiplegic with hydrocephalus and one had a pronounced behavioural abnormality. Four children had slight to moderate disabilities: one child was mildly mentally retarded and three, although mentally normal, had hemiplegia and delayed speech development, one of them having a learning disorder as well. Of these 17 neurologically impaired children 16 had ophthalmological abnormalities. EEG recordings were made in 29 patients in the neonatal period. They were markedly abnormal in 24 patients, 14 of whom had localized periodic complexes. An abnormal EEG was a bad prognostic sign. The neurological outcome was better in the HSV-1-infected children (10 cases) than in the HSV-2-infected ones (35 cases). Progressive or recurrent encephalitis was strongly suspected in two preterm children. PMID- 1709772 TI - Coordination of low birthweight seven-year-olds. AB - The coordination and laterality of a group of 171 seven-year-old children, free from major disability, with a birthweight of 2,000 g or less, were examined and compared with those of normal birthweight peers. More low birthweight children were left-handed and of mixed or undetermined hand, foot and eye dominance. Left handedness may adversely affect some areas of performance of normal birthweight but not of low birthweight children. Low birthweight children performed significantly less well in tests of both fine and gross motor coordination. Girls tended to perform better than boys in fine motor tests. In the low birthweight group there was a correlation between IQ and coordination. PMID- 1709773 TI - Children in sauna: electrocardiographic abnormalities. AB - We studied changes in ECG in 81 children and 20 adolescents and young adults when they were exposed to heat stress in a climatic chamber, which resembled an ordinary Finnish sauna. Changes were similar to those caused by increased sympathetic activity in 57% of the subjects during the bath and in 23% during the recovery period. Three children had extrasystoles during and four after sauna. One previously healthy 5-year-old girl with wandering pacemaker suffered a 3.3 sec sinus arrest and had reversible junctional rhythm during the heat exposure. We conclude that, although these changes were transient and benign, the sinus arrest suggest that heat stress in sauna is considerable and may be risky for those children who have disorders of the sinoatrial node. PMID- 1709774 TI - Relationship between redistribution on exercise thallium-201 scintigraphy and repetitive ventricular premature beats in patients with recent myocardial infarction. AB - The relationship between myocardial ischemia detected by exercise thallium-201 scintigraphy and repetitive ventricular premature beats (VPBs) during ambulatory monitoring was evaluated in 57 patients with recent myocardial infarction. Multivariate analysis was performed to obtain the relatively important factor related to repetitive VPBs with the use of the following variables: age, redistribution, left ventricular ejection fraction, serum potassium and magnesium concentration, QRS score, left ventricular aneurysm, and the number of diseased vessels. Thirty-five patients had redistribution, but only three of them had repetitive VPBs during exercise testing. The average heart rate before 79% of 398 episodes of repetitive VPBs during ambulatory monitoring was in the range of 56 to 70/min. These data indicate that most of repetitive VPBs during ambulatory monitoring were not provoked by exercise-induced acute myocardial ischemia. However, redistribution was found to be an important factor associated with repetitive VPBs. The electrical abnormality relating to a substrate characterized by chronic reversible ischemia may explain the association between redistribution and repetitive VPBs. PMID- 1709775 TI - Ventricular function during supine bicycle exercise in univentricular connection with absent right atrioventricular connection. AB - Fourteen patients with univentricular connection, absent right connection (tricuspid valve atresia) and normally related great arteries underwent rest and supine bicycle exercise equilibrium radionuclide blood pool studies. Ejection fraction, heart rate, systemic blood pressure and oxygen saturation (ear oximetry) were measured. There were 6 male and 8 female patients. Mean age +/- standard error of the mean was 14.5 +/- 1.1 years (range 6.3 to 21.1). Eight patients (group 1) had systemic to pulmonary shunts placed as palliation 8.2 +/- 2.2 years before study and 6 patients (group II) had caval to pulmonary shunts placed 11.8 +/- 1.6 years previously. Ejection fraction at rest was 54 +/- 2% and an abnormal response to exercise (failure of ejection fraction to increase greater than or equal to 5% from rest to peak exercise) was found in 9 of 14 patients. When analyzed separately, ejection fraction at rest in group I was higher than in group II; however, this difference disappeared at peak exercise. There was a significant negative correlation between ejection fraction at peak exercise and the interval since palliative surgery, although it was not apparent at rest. These data suggest that ventricular function is compromised during exercise and that abnormal performance is influenced by long-standing volume overload. Importantly, this abnormal state is concealed at rest and the choice of palliative shunting procedure appears to have little effect on normalizing pump performance. PMID- 1709776 TI - Lead poisoning in children with developmental disabilities. PMID- 1709777 TI - Brain and ocular abnormalities in infants with in utero exposure to cocaine and other street drugs. AB - We describe 10 infants with developmental delay and congenital cerebral anomalies who were found to have had in utero exposure to vasoactive drugs. Nine infants had ophthalmological abnormalities; these included strabismus, nystagmus, and/or hypoplastic optic discs. Six mothers used cocaine, one used cocaine and heroin, one used only heroin, one used amphetamine, and one used phenylpropanolamine. Each of these cerebral anomalies (agenesis of the corpus callosum, septo-optic dysplasia, schizencephaly, hydranencephaly, congenital hydrocephalus, porencephaly, and cerebral infarctions) can be attributed to insults at different stages of development. There appears to be a relationship between the time of prenatal drug exposure and the type of cerebral anomaly, evoking malformations, disruptions, or fetal strokes. Since many or possibly all of these anomalies are thought to have a vascular origin, it seems appropriate to implicate prenatal exposure to vasoactive drugs. PMID- 1709778 TI - Screening for non-delta F508 mutations in five exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Italy. AB - Analysis of exons 10, 11, 14a, 15, and 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing-gradient-gel electrophoresis (DGGE) allowed the identification of mutations causing cystic fibrosis (CF) in 25 of 109 non-delta F508 chromosomes, as well as identification of a number of polymorphisms and sequence variations. Direct sequencing of the PCR fragments which showed an altered electrophoretic behavior not attributable to known mutations has led to the characterization of four new mutations, two in exon 11, and one each in exons 15 and 20. Screening for the different mutations thus far identified in our patients by the DGGE analysis and other independent methods should allow detection of about 70% of the molecular defects causing CF in Italy. Mutations located in exons 11 and 20 account for at least 30% of the non-delta F508 mutations present in Italian CF patients. PMID- 1709780 TI - Update on maternal serum alpha-fetoprotein screening. PMID- 1709779 TI - G gamma and A gamma globin genes are identical from -471 of the promoter midway through gamma IVSII in a Benin beta s haplotype associated with elevated fetal hemoglobin. AB - In seven kindreds in which sickle cell (SS) patients had elevated (greater than 12%) fetal hemoglobin (Hb F), Milner and colleagues reported that a determinant for elevated Hb F and elevated F cells was linked to the beta s gene. Independently, the Senegal (SEN) beta s haplotype has been found in association with elevated Hb F in SS and beta-thalassemia patients. We have used the kindreds of Milner and colleagues to characterize further the association of haplotype and gamma gene DNA sequence variation with Hb F expression. For the largest kindred, Wi, all four SS had high (greater than 14%) Hb F and both SEN and Benin (BEN) haplotypes. Two AS cases carrying SEN had low Hb F and low F cells, while three AS and one CS carrying BEN had elevated Hb F and elevated F cells; only one AS carrying BEN had low Hb F and low F cells. In order to look for genetic alterations that could account for the elevated Hb F of kindred Wi, we sequenced both the G gamma and A gamma genes of the Wi BEN haplotype. The data showed largely identical G gamma and A gamma genes which may have been generated by two gene conversions: the A gamma promoter was like that of G gamma 3' to -471, while the G gamma IVSII was like that of A gamma in its 5' half. In addition, three new mutations were found in gamma IVSII.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709781 TI - Umbilical vein administration of oxytocin for the management of retained placenta: is it effective? AB - In a multicenter randomized controlled trial involving 220 women with retained placenta no beneficial effects could be established of intraumbilical vein administration of 10 IU of oxytocin in 20 ml of saline solution. A reduction was not gained in the rate of manual removal of the placenta and there was no decrease in the amount of blood loss. Oxytocin only induced a minor shortening of the median time interval from administration to the spontaneous expulsion of the placenta as compared with a placebo injection. Maternal serum alpha-fetoprotein levels before and after intraumbilical vein injection did not show evidence of fetomaternal transfusion. PMID- 1709782 TI - Fetal thyroid-stimulating hormone response to maternal administration of thyrotropin-releasing hormone. AB - Thyroid-stimulating hormone was measured in fetal blood samples obtained by cordocentesis before and after blood transfusion in Rh-affected pregnancies at 25 to 37 weeks' gestation. In eight of 26 study subjects thyrotropin-releasing hormone was given to the mothers immediately after the pretransfusion samples were obtained. In this group the fetal thyroid-stimulating hormone concentrations in the posttransfusion samples were significantly higher than those before the transfusion. PMID- 1709783 TI - Histamine-induced chloride channels in apical membrane of isolated rabbit parietal cells. AB - The electrical properties of the apical membrane of isolated rabbit parietal cells were studied using the patch-clamp technique. The apical membrane of the parietal cells plated on Matrigel and maintained in culture conditions was identified by lectin-binding studies. Cell-attached and excised inside-out patches from 10(-4) M cimetidine-treated parietal cells infrequently contained Cl channels (9% of the patches). A single class of voltage-dependent outwardly rectifying Cl- channels with 24 +/- 1-pS conductance was observed in 75% of the patches from cells stimulated (acid secreting) by 10(-4) M histamine. Other anions passed through these channels with a permeability sequence of I- (1.2) greater than Br- (1.1) greater than or equal to Cl- (1.0) greater than NO3- (0.7) greater than SO4(2-) (0.1), but there was a very low permeability for Na+ or K+ (PCl-/PNa+ or PCl-/PK+ greater than 5). In inside-out patch configurations the Cl channel was insensitive to Ba2+ and stilbene derivatives but was inhibited by diphenylamine-2-carboxylic acid in a manner characteristic of a reversible open channel blocker. It is concluded that H2-receptor agonist stimulation of acid secretion by rabbit parietal cells activates Cl- channels in the apical cell membrane. PMID- 1709784 TI - Tyrosine phosphorylation and oxygen consumption induced by G proteins in neutrophils. AB - In neutrophils, receptor-mediated activation of the respiratory burst requires ATP, possibly for phosphotransferase reactions. The oxidative response is only partially inhibited by blockers of protein kinase C, suggesting the involvement of other kinases. Recent evidence has demonstrated activation of tyrosine phosphorylation in chemoattractant-stimulated cells. This effect is likely mediated by G proteins because it is obliterated by pretreatment with pertussis toxin. In this report we have attempted to correlate the respiratory burst and phosphotyrosine accumulation induced by activation of G proteins, accomplished by treatment of electroporated cells with nonhydrolyzable analogues of GTP. In cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) both responses displayed similar time course and concentration dependence. The guanine nucleotide selectivity sequence and the divalent cation requirements were also similar for both responses. These similarities suggest a relationship between tyrosine phosphorylation and the activation of the NADPH oxidase. GTP gamma S induced phosphotyrosine accumulation was found to be inhibited by pretreatment of the cells with phorbol esters, underlining the existence of regulatory interactions between different signal transduction pathways in neutrophils. PMID- 1709785 TI - Norepinephrine and iloprost improve barrier function of human endothelial cell monolayers: role of cAMP. AB - The barrier function of human artery endothelial cells was improved by addition of agents that increase the cellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration. Together with a decrease in the passage rate of peroxidase, an increase in the transendothelial electrical resistance was observed. A direct correlation was found between the relative increases in cellular cAMP concentration and the relative decrease in peroxidase passage after incubation of the cells with forskolin (0.25 and 2.5 microM), the beta-adrenergic agonist isoproterenol (10 microM), and the stable prostacyclin analogue iloprost (10 microM). Norepinephrine (10 microM) reduced the peroxidase passage to a much larger extent (40% reduction) than might be expected on the basis of a small increase of cAMP concentration. This small increase in cAMP (44%) was the result of interactions of norepinephrine with beta-adrenergic receptors, which increase cAMP, and alpha-adrenergic receptors, which decrease cAMP. The relatively strong reduction in permeability (also found in the presence of the alpha-adrenergic antagonist phentolamine) suggests that an additional cAMP-independent mechanism underlaid the barrier-improving effect of norepinephrine. A marked elevation of cAMP by forskolin was accompanied by a disappearance of F-actin and myosin from stress fibers. They were found diffusely spread over the cell, and F-actin in the cell periphery became prominently visible. PMID- 1709786 TI - Simultaneous measurement of membrane potential, cytosolic Ca2+, and tension in intact smooth muscles. AB - Microelectrode techniques and the fluorescent Ca2+ indicator indo-1 were used to measure membrane potential, cytosolic Ca2+ ([Ca2+]cyt), and muscle tension simultaneously in canine antral smooth muscles. Responses of muscles from the myenteric and submucosal regions were compared, since electrical activity and excitation-contraction coupling in these regions differ. The upstroke phase of electrical slow waves in both regions induced an increase in [Ca2+]cyt. In myenteric muscles the plateau phase of slow waves often caused either a further rise in [Ca2+]cyt or maintenance of the level reached during the upstroke event. In submucosal muscles, the plateau phase was significantly smaller and did not induce a second phase in the Ca2+ transient. Contractions were related to the amplitudes of Ca2+ transients. Acetylcholine (ACh; 3 x 10(-8)-10(-6) M) increased the amplitude and duration of the plateau phase of slow waves in a concentration dependent manner. ACh also increased the second phase of Ca2+ transients and contractile responses associated with the plateau potential. In submucosal muscles ACh induced a significant increase in the plateau phase of the slow wave and increased the corresponding phase of Ca2+ transient. Nicardipine (10(-6) M) inhibited plateau phase of slow waves and the associated increases in [Ca2+]cyt and muscle tension. BAY K 8644 (10(-7) M) augmented the plateau potential and increased [Ca2+]cyt and muscle tension. These results suggest that dihydropyridine-sensitive Ca2+ currents participate in the plateau potential. Cholinergic stimulation modulates [Ca2+]cyt and therefore force by regulating the amount of Ca2+ entering cells through these channels. PMID- 1709787 TI - Characterization of gap junction channels in A7r5 vascular smooth muscle cells. AB - Recent evidence suggest that coordination of blood flow in the microcirculation involves cell-to-cell coupling via gap junctions. In this study, using A7r5 cells as a model of vascular smooth muscle, we have characterized the gap junctions in terms of the unitary conductances of the observed channels, the responses to second messengers, and subunit protein composition. The cells were typically well coupled several hours after plating, with junctional conductances on the order 20 40 nS. Channels with mean conductances of 36 and 89 pS were observed in low conductance cell pairs and in cell pairs whose macroscopic conductance was reduced by exposure to halothane. Connexin43 was the only known gap junction sequence detected by Northern blots (low and high stringency), immunoblots, or immunohistochemical studies. Junctional conductance was reduced 15% by 8 bromoadenosine 3',5'-cyclic monophosphate; 8-bromoguanosine 3',5'-cyclic monophosphate had no effect. The results suggest that connexin43 can form stable channels of at least two distinct conductances and gap junctions with differing responses to second messengers. PMID- 1709788 TI - Prostaglandin E2 inhibits secretagogue-induced enzyme secretion from rat pancreatic acini. AB - Prostaglandins of the E type may have a potential role in pancreatic physiology and pathophysiology. Because prostaglandins of the E type inhibit HCl secretion in parietal cells via a specific receptor by inhibition of adenylylcyclase, we studied whether a similar mechanism exists in the exocrine pancreas. Isolated rat pancreatic acini were incubated with various concentrations of secretagogues, such as cholecystokinin-octapeptide (CCK-8), bombesin, carbachol, and vasoactive intestinal peptide (VIP), in the absence or presence of prostaglandin E2 (PGE2), and amylase secretion was measured. For receptor binding studies, acini and pancreatic membranes were incubated with [3H]PGE2 and either unlabeled PGE2 or other types of prostaglandins. PGE2 (10(-13) to 10(-5) M) did not inhibit basal amylase secretion. However, CCK-8-stimulated secretion was significantly inhibited. Stimulation of secretion by bombesin, carbachol, VIP, and secretin was also inhibited by PGE2, but not as pronounced as CCK-8-stimulated secretion. The formation of inositol 1,4,5-trisphosphate induced by CCK-8 was markedly inhibited by simultaneous incubation with PGE2. Furthermore, PGE2 slightly but significantly reduced the CCK-8-induced efflux of 45Ca2+ from prelabeled acini. Intact acini and a membrane fraction bound [3H]PGE2 and this function could be equally competed by either unlabeled PGE2 or PGE1 in contrast to less-related prostaglandins such as PGF2 alpha, PGD2, and prostacyclin. We conclude that prostaglandins of the E type inhibit pancreatic enzyme secretion stimulated by various secretagogues. This function is mediated via specific receptors for PGE. With regard to CCK-8-stimulated secretion this function may be mediated by an inhibition of formation of inositol 1,4,5-trisphosphate. PMID- 1709789 TI - Abnormal epithelial transport in cystic fibrosis jejunum. AB - Abnormal epithelial electrolyte transport has been identified in a range of cystic fibrosis (CF) organs and appears to account for the various clinical manifestations of the disease. The aim of this study was to further define the Cl secretion defect in CF jejunum. Excised jejunum was obtained from 11 CF patients and 12 controls. Transport studies were performed on stripped epithelium in vitro under short-circuited conditions in Ussing Chambers. 3-Isobutyl-1-methylxanthine (IBMX) (300 microM) significantly increased Cl- secretion in control (-2.3 +/- 0.6 to -3.3 +/- 0.7 mueq.cm-2.h-1; P less than 0.01, paired t test; n = 5 subjects) but not in CF jejunum (-0.5 +/- 0.3 to -0.1 +/- 0.4; n = 4). However in contrast to control jejunum, net Na+ absorption in CF jejunum was higher in the IBMX (1.3 +/- 0.5 mueq.cm-2.h-1) compared with basal periods (0.6 +/- 0.3; P less than 0.05, paired t test). IBMX stimulation of tissue adenosine 3',5'-cyclic monophosphate (cAMP) was similar in both control and CF jejunum. A range of secretagogues known to induce secretion in mammalian intestine, including dibutyryl cAMP (DBcAMP), DBcGMP, Ca2+ ionophore A23187, and the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate, failed to induce secretion in CF jejunum. In conclusion, CF jejunum failed to exhibit Cl- secretion and also demonstrated abnormalities of Na+ absorption. These results support the view that the defect lies at a site distal to the intracellular messengers. Moreover, these abnormalities of intestinal electrolyte transport may account for some of the gastrointestinal manifestations of the disease such as meconium ileus and distal intestinal obstruction syndrome. PMID- 1709790 TI - Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells. AB - Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6 23 cells infected with the viral stock obtained from the supernatant of pSVXgas transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G 14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing. PMID- 1709791 TI - Glomerular dysfunction in nephrotic humans with minimal changes or focal glomerulosclerosis. AB - Fractional clearances (theta) of uncharged dextrans (radii 28-60 A) were used to characterize glomerular dysfunction in 34 nephrotic humans with either minimal change nephropathy (MCN) or focal, segmental glomerulosclerosis (FSGS). A theoretical analysis of theta of dextran with a heteroporous membrane model revealed a similar alteration, more marked in FSGS than MCN. The number of restrictive pores perforating the major membrane component was reduced in parallel with the prevailing glomerular filtration rate (GFR). Parallel shuntlike pores in the remaining membrane component were more prominent, pointing to impaired size selectivity. However, the theta of large (60 A) dextrans attributable to these shunts exceeded control in FSGS only, suggesting that coexistent impairment of charge selectivity contributed importantly to the proteinuria in MCN. Membrane properties returned toward normal when MCN remitted. Glomerular morphometry revealed the frequency of epithelial filtration slits to vary with the extent of membrane dysfunction. Despite offsetting hypertrophy of remnant glomeruli in FSGS, a loss of filtration surface due to sclerosis likely contributed to the more marked reductions in pore number and GFR observed in this disorder than in MCN. PMID- 1709792 TI - Heparin promotes the formation of extracardiac to coronary anastomoses in a canine model. AB - We developed a canine model for the in vivo utilization of angiogenesis factors to promote revascularization of a collateral-dependent area of the heart and assessed the potential of heparin in this preparation. Ameroids were placed on the proximal left anterior descending coronary artery (LAD) of 29 dogs, and the left internal mammary artery (IMA) was implanted in an intramyocardial tunnel in proximity to the LAD. A tube positioned in the distal IMA provided a continuous retrograde infusion directly into the vessel from an implanted pump. Heparin (15 or 150 U/h) or saline vehicle was infused. After 8 wk, regional myocardial blood flow was assessed in the anesthetized state during adenosine-induced vasodilatation, before and during occlusion of the IMA. The IMA provided a greater proportion of maximal collateral flow in heparin-treated dogs (22 +/- 5%, n = 17) than in saline-treated dogs (9 +/- 2%, n = 12, P less than 0.05). Thus continuous infusion of heparin promotes the formation of collaterals between the extracardiac artery and the myocardial circulation, establishing the feasibility of targeting angiogenic agents for myocardial revascularization. PMID- 1709793 TI - Interleukin 6 stimulates growth of vascular smooth muscle cells in a PDGF dependent manner. AB - We have investigated the effect of interleukin 6 (IL-6) on the growth of vascular smooth muscle cells (VSMC) isolated from rat aortas. Murine recombinant IL-6 significantly increased the number of VSMC and stimulated tritiated thymidine incorporation into VSMC in a dose-dependent manner. The IL-6-induced thymidine incorporation into VSMC was totally inhibited by the Ca2+ channel blocker verapamil; however, IL-6 showed no effects on the intracellular Ca2+ level ([Ca2+]i) in VSMC. Antibody against platelet-derived growth factor (PDGF) also totally inhibited the IL-6-induced thymidine uptake. PDGF caused a significant increase in the [Ca2+]i, which was totally inhibited by verapamil. IL-6 mRNA was not detected in unstimulated "quiescent" VSMC, but its expression was stimulated by exposure of VSMC to 10% fetal bovine serum. Immunohistochemical study using anti-PDGF antibody showed that IL-6 stimulated PDGF production in VSMC. These results support the premise that IL-6 is released by VSMC in an autocrine manner and promotes the growth of VSMC via induction of endogenous PDGF production. PMID- 1709794 TI - Inhibition of transient outward K+ current by DHP Ca2+ antagonists and agonists in rabbit cardiac myocytes. AB - The 1,4-dihydropyridine (DHP) Ca2+ antagonists and agonists can inhibit a time- and voltage-dependent, but intracellular Ca(2+)-independent transient outward K+ current (It), in myocytes from rabbit atrium. In the presence of 0.3 mM CdCl2, DHPs decreased the peak It slightly and markedly accelerated its apparent rate of inactivation. When the inhibition of It was measured from integrated It records, the 50% inhibitory concentrations (IC50) of nicardipine and BAY K 8644 were 630 nM and 7 microM, respectively, and the IC50 of nicardipine for inhibition of the Ca2+ current (ICa) was only approximately fourfold lower (160 nM). The inhibition of It by nicardipine was not affected by changing holding potential from -55 to 100 mV; in contrast, the inhibitory effect on ICa was significantly reduced by this hyperpolarization. We conclude that the DHP Ca2+ antagonist nicardipine blocks It at similar doses to those that block ICa and that nicardipine blocks this K+ current by mechanism different from that for ICa inhibition. This inhibitory effect on It is shared by other DHP compounds; the rank order for potency of It inhibition is nicardipine greater than benidipine greater than nisoldipine greater than BAY K 8644 greater than nitrendipine greater than nifedipine. PMID- 1709795 TI - Role of radiation after operative palliation in cancer of the proximal bile ducts. AB - Cancer of the proximal bile ducts continues to pose a formidable problem to even the most experienced biliary surgeon. From 1977 through 1985, 51 patients with histologically confirmed proximal bile duct cancers underwent surgical treatment. The lesion was confined to the hilar region in 30 patients; there was extensive hepatic infiltration or distant metastatic disease in 21 patients. One patient underwent resection. Biopsy only was performed in six patients. In the remaining 44 patients, transtumoral dilation and intubation were performed. These 44 patients were further analyzed with regard to how survival was affected by the presence of metastatic disease and by the adjunctive use of radiation therapy. Mean survival in those patients with metastatic disease (n = 16) was 6.1 months, and survival was not improved by the use of postoperative radiation. In the absence of metastatic or advanced local disease, however, the addition of external beam radiation did significantly extend the mean survival from 4.5 to 12.2 months and the median survival from 2.2 to 12.2 months. The operative mortality for the series was 14% and postoperative complications occurred in 18 patients. These findings suggest that the addition of external beam radiation improves survival in patients undergoing palliative treatment of hilar tumors. Further confirmation of the value of radiation awaits prospective investigation. PMID- 1709796 TI - Monitoring of irrigating fluid absorption during transurethral prostatectomy. A study in anaesthetised patients using a 1% ethanol tag solution. AB - A simple, reliable method to detect absorption of irrigating fluid during transurethral prostatectomy is to tag irrigating fluids with 1% ethanol and monitor expired breath ethanol concentrations. This method correlated well (n = 0.79) with other existing methods of absorption monitoring in 20 anaesthetised patients. Ethanol (1%) tagging does not alter the optical quality of the irrigating fluid and is harmless to the patient. The technique is non-invasive, repeatable, cheap and gives instant results. It can be used in anaesthetised or awake patients and can detect absorption of as little as 100-150 ml in any 10 minute period. PMID- 1709797 TI - Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample. AB - Chromatofocusing was utilized to characterize charge microheterogeneity of purified human alpha-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4.57 (52%), 4.27 (43%), and less than 4.00 (5%), respectively. In contrast, 10 micrograms of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With greater than or equal to 5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with greater than or equal to 5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins with pIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared. PMID- 1709798 TI - Microtubule bundles in fish cerebellar Purkinje cells. AB - The initial axon segments and the cell bodies of Purkinje cells were examined in electron microscopic serial sections and toluidine blue semithin sections of goldfish cerebellum. We observed two characteristic cytoplasmic features different from those of other vertebrate neurons, 1. The areas of Nissl substance and Golgi apparatus are sharply divided in the periphery and center of the cytoplasm, 2. Microtubules fasciculated by cross-bridges in the axon hillock and initial axon segment remain bundled in the perikaryon, pass near the eccentric nucleus, and enter into the Golgi area of the central cytoplasm, where they are surrounded by mitochondria. We suggest that the intracellular fasciculated microtubules may establish a prepared pathway for fast anterograde and retrograde transport to and from the Golgi area of the cell body. PMID- 1709799 TI - Changes in serum immunoglobulin values in kittens after ingestion of colostrum. AB - Immunoglobulin values were determined in fetal and kitten sera. In the fetal and precolostral kitten sera, only IgG was detected, except in 1 case in which IgM was detected. The IgG, IgA, and IgM were transferred to the kittens through colostrum ingestion with some selectivity. Concentration of the transferred IgG, IgA, and IgM decreased significantly with half-lives of 4.15 +/- 1.29 days, 2.03 +/- 0.33 days, and 2.2 +/- 1.2 days, respectively. As a result of this decrease and increase of de novo immunoglobulin synthesis, IgG, IgA, and IgM were at their lowest values when kittens were 20 to 25 days, 14 to 20 days, and 8 to 10 days old, respectively. After their nadir was reached, IgG values increased gradually, IgA slowly, and IgM rapidly, as a result of de novo immunoglobulin synthesis. When the kittens were 90 days old, their immunoglobulin values were 80% (IgG), 7% (IgA), and 100% (IgM), compared with those of adult cats. These findings suggest that kittens that receive inadequate colostrum from their mothers will be particularly susceptible to infection after they are 5 weeks old. PMID- 1709800 TI - Brain-behavior relationships in aphasia studied by positron emission tomography. AB - Positron emission tomography allows for the study of human brain physiology and chemistry including cerebral blood flow, oxygen or glucose metabolism. We applied PET to study glucose metabolism using aphasia as a model of neurobehavior. The most striking observation was that the extent of cerebral glucose metabolic changes in aphasic patients consistently involve brain regions that are not structurally damaged. The remote metabolic effects can be predicted depending on the location and extent of structural damage. Two observations were made: (1) In our experience, all right-handed aphasic patients with left hemisphere structural lesions have metabolic abnormalities in the left temporoparietal region, and (2) metabolic abnormalities are variably found in undamaged, left prefrontal lobe, basal ganglia, and thalamus. Variations in clinical aphasic syndromes were found to relate to these frontal metabolic changes, suggesting that aspects of the aphasia result from differences in prefrontal function rather than directly from structural damage to perisylvian or deep structures. PMID- 1709802 TI - Selective antiviral activity of synthetic soluble L-tyrosine and L-dopa melanins against human immunodeficiency virus in vitro. AB - Melanins are pigments found in hair, skin, irides of the eye, and brain. Their functions in mammals include protection from exposure to sunlight, camouflage from predators, sexual recognition within species, and possible electron transfer reactants. Most natural melanins exist in an insoluble form, which is one reason there is little information on the biological properties of soluble melanins. Here, synthetic soluble melanins were obtained by chemical oxidation of L tyrosine or spontaneous oxidation of L-beta-3,4-dihydroxyphenylalanine (L-dopa). Replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) was inhibited by soluble melanin in two human lymphoblastoid cell lines (MT-2 and H9) and in phytohemagglutinin-stimulated human T cells. Effective concentrations of 0.15-10 micrograms/ml had no cell toxicity. Melanin blocked infection by cell free virus and interfered with HIV-induced syncytium formation and cytopathic effects when fusion-susceptible, uninfected cells, were mixed with chronically infected cells. Melanin also impeded the HIV-1 envelope surface glycoprotein, and T cell specific monoclonal antibody leu-3a (CD4), but not leu-5b (CD2), from binding to the surface of MT-2 cells. No effect on HIV-1 reverse transcriptase activity in viral lysates was observed. These results identify a unique biological property of melanin, and suggest that soluble melanins may represent a new class of pharmacologically active substances which should be further investigated for potential therapeutic utility in the treatment of Acquired Immune Deficiency Syndrome (AIDS). PMID- 1709801 TI - Quantitative electroencephalography and anatomoclinical principles of aphasia. A validation study. AB - No single technology in isolation can provide a full view of the anatomoclinical principles evident in the clinical populations we study. The dynamic nature of quantitative electrophysiology makes it an ideal complement to anatomic and metabolic imaging. The statistical conundrum it has presented may be resolved by the approach incorporated in CART. The intent of this study was to examine QEEG and CART in the evaluation of the neurologic bases of a well-defined behavioral disorder like aphasia. The combined power of QEEG and CART yielded objective electrophysiologic methods to predict aphasia that rival the reliability of the language examination. Such success is unprecedented. This success allows us to incorporate QEEG and CART into our technological armamentarium and to return to the evaluation of less well-understood disorders with confidence in both our findings and anatomoclinical principles we derive from them. PMID- 1709804 TI - The use of a low molecular weight heparinoid (Org 10172) for extracorporeal procedures in patients with heparin dependent thrombocytopenia and thrombosis. AB - We report two cases of heparin induced thrombocytopenia (HIT), in patients who required anticoagulation for extracorporeal procedures (haemodialysis and cardiopulmonary bypass) one associated with recurrent thrombosis of the artificial circuits. Resolution of thrombocytopenia and successful anticoagulation were achieved using a low molecular weight heparinoid (LMWH) Org 10172. Anticoagulation was monitored using estimations of plasma anti-factor Xa activity. These cases demonstrate that LMWH provides safe and effective anticoagulation in patients in whom unfractionated heparin has caused HIT. PMID- 1709803 TI - [Diagnosis of hepatocellular carcinoma performed by searching for serologic tumor markers]. AB - Serum levels of alkaline phosphatase, gamma-glutamyltranspeptidase, -1 fucosidase and glutathione-S-transferase are increased in 60, 90, 75 and 64% of patients with hepatocellular carcinoma. In these patients the mean plasma fibrinogen levels is 461.78 mg/dl, while mean serum copper is 200.50 mg/dl. Serum levels of desgamma-carboxiprothrombin is over 900 mg/dl in 67% of the patients (60% of them have HB virus, mostly anti HBe positive). Forty to 95% of them have increased levels of -fetoprotein (AFP). The authors suggest that cirrhotic patients, with or without HB virus, specially those with increased AFP, should have ultrasound examination of the liver every 6 months. This method of imaging has been shown to be more sensitive than AFP (72% versus 25%) in the detection of hepatocellular carcinoma smaller than 2 cm in diameter. PMID- 1709805 TI - Differential effects of recombinant human colony stimulating factor (rh G-CSF) on stem cells in marrow, spleen and peripheral blood in mice. AB - Previously it has been hypothesized that the granulopoietic and erythropoietic lineages may compete for differentiating stem cells. According to this hypothesis one would expect that a stimulation of granulopoiesis by G-CSF administration would lead to a reduction of the stem cell pool and be followed by a decline of erythropoietic progenitor numbers. In addition one would expect an enhanced response of granulopoiesis if G-CSF administration were combined with suppression of erythropoiesis by red cell transfusion. To evaluate whether this hypothesis holds true C57bl mice were injected subcutaneously for 6 d with 3.75 micrograms rh G-CSF/mouse/d (150 micrograms G-CSF/kg body weight/d). Marrow CFU-S numbers showed an increase to 160% on day 2, followed by a decrease to 50% of control on day 6. Splenic and peripheral blood CFU-S increased 20-fold and 10-fold, respectively. Marrow CFU-E declined to 40% of the control value. Splenic CFU-E increased 10-fold. The increase in marrow CFU-GM numbers ranged between 140% and 180%. CFU-GM obtained from the spleen and the peripheral blood increased 60-fold and 15-fold, respectively. Regarding the CFU-S and CFU-GM a similar pattern of response was found in an experiment where rh G-CSF administration was combined with an additional red cell transfusion. These data do not provide convincing evidence for an exhaustion of haemopoietic stem cells during treatment with G CSF. They rather suggest that an important side effect of G-CSF treatment is a release of CFU-S and progenitors from the marrow to the peripheral blood and a reseeding in the spleen. PMID- 1709806 TI - Expression of the multiple drug resistance gene (mdr-1) and epitope masking in chronic lymphatic leukaemia. AB - Resistance to cytotoxic agents is a common clinical problem in the treatment of chronic lymphatic leukaemia (CLL). The multidrug resistant (MDR) phenotype characterized by increased levels of a specific cell membrane p-glycoprotein, confers cross resistance to a wide range of structurally dissimilar antineoplastic drugs. We have studied the expression of this p-glycoprotein in chronic lymphatic leukaemia measured by immunofluorescence using a monoclonal antibody MRK 16 by flow cytometry. Initial results showed that only 12% of lymphocyte samples from CLL patients showed increased p-glycoprotein, conflicting with a previous observation that 53% of CLL patients had an increased level of mdr-1 mRNA. Treatment of the cells with neuraminidase to remove sialic acid residues increased the proportion of patients showing increased p-glycoprotein to 52%. This suggest that in a subset of CLL patients post translational modification of the protein occurs masking the epitope recognized by MRK 16. Abnormal sialylation patterns associated with malignancy are a well-recognized phenomenon. PMID- 1709807 TI - Juvenile chronic myelogenous leukaemia: the only example of truly fetal (not fetal-like) erythropoiesis. PMID- 1709808 TI - Deficiency of DAF and CD16 on PNH neutrophils. PMID- 1709809 TI - Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate. AB - The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT. PMID- 1709810 TI - Human alpha-fetoprotein primary structure: a mass spectrometric study. AB - The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590. PMID- 1709811 TI - High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions. AB - Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(A ACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The DNA-RNA junction residues exhibit quite different COSY, chemical shift, and NOE behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras also display a mixture of characteristic A-type and B-type absorption bands. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but our results differ somewhat from previous theoretical model studies. PMID- 1709812 TI - Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides. AB - We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac (Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly. PMID- 1709813 TI - Synthetic analogues of alamethicin: effect of C-terminal residue substitutions and chain length on the ion channel lifetimes. AB - In a previous study, a synthetic analogue of the peptaibol alamethicin, in the sequence of which all alpha-aminoisobutyric acid (Aib) were substituted by leucine residues and the C-terminal residue modified, was shown to display the same single-channel behaviour as alamethicin in planar lipid bilayer, except that the sublevel lifetimes were much reduced. New analogues differing in their C terminal residue (Phe-NH2, Pheol, Trp-NH2) have now been tested for their single channel properties in neutral lipid bilayers. The conductance amplitudes and open channel lifetimes do not differ significantly from the previous analogue. Thus, the nature of the last residue, which may be located near the membrane interface, does not seem to play an important role in the destabilisation of the conducting aggregate observed after the Aib substitution by Leu. Since the deletion of one residue (Glu18) in the 14-20 moiety induces a slight decrease of the increment between the conductance levels, but has no effect upon the channel lifetimes, this residue and the length of this segment do not interfer much with the channel lifetime of peptaibols. In conclusion the factors influencing the aggregate stability may be sought in the helix-helix interactions. PMID- 1709814 TI - Estradiol increases the secretion of hepatic lipase by rat hepatocyte cultures. AB - Hepatic lipase (EC 3.1.1.3) is synthesized and secreted by parenchymal hepatocytes and binds to endothelial cells of liver sinusoids. The present study shows that the activity of hepatic lipase secreted by hepatocyte cultures from male rats in increased approx. 6-fold after 10 h culture with 10 microM 17 beta estradiol. The stimulatory effect of 17 beta-estradiol is biphasic and declines at higher concentrations. In hepatocytes from male rats: progesterone, unlike 17 beta-estradiol, had only a small stimulatory effect when present as the sole hormone and a small inhibitory effect in the presence of 17 beta-estradiol, while testosterone and dexamethasone had no effect. Hepatocyte cultures from female rats had a higher basal rate of hepatic lipase secretion than cells from male rats and showed a smaller stimulation by 17 beta-estradiol. These results suggest that 17 beta-estradiol might regulate the secretion of hepatic lipase by hepatocytes, and presumably the activity of the enzyme at either the endothelial surface of the liver sinusoids or at extrahepatic sites. PMID- 1709815 TI - A maximum likelihood procedure for the analysis of group and individual data in aphasia research. AB - The limitations inherent in group versus case studies appear to lie in a complementary distribution, underscoring the importance of combining both strategies within a single research program. However, this compromise approach requires analytic tools that permit us to combine and evaluate individual and group data in a common format. Maximum likelihood estimation (MLE) belongs to a family of procedures for determining goodness of fit. MLE can be used in conjunction with a linear or nonlinear model of the way that sources of information combine to determine a given behavioral outcome; such models can be used to estimate the distance between two groups, the degree to which an individual case deviates from a given empirically or theoretically defined group profile, and the degree to which one individual case resembles another. We offer a demonstration of how MLE can be used to evaluate group and individual profiles, in a cross-linguistic study of sentence comprehension in nonfluent aphasic speakers of English, Italian, and German. This includes a demonstration in which the MLE models for each language are "lesioned" to simulate several competing accounts of receptive agrammatism. PMID- 1709816 TI - Agrammatism is a theoretically coherent aphasic category. PMID- 1709817 TI - The effect of L-glutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta. AB - The effect of the putative amino acid transmitter, L-glutamate, on adenylate cyclase in crude membrane preparations of the rat tapeworm Hymenolepis diminuta was investigated to determine if glutamate effects the generation of the second messenger cAMP. Addition of glutamate at 10(-3) and 5.5 x 10(-9) M resulted in significant elevations in basal activity of adenylate cyclase, while concentrations in the 10(-5)-10(-7) M range caused significant depressions below basal activity. Assays with glutamate agonists and other acidic compounds showed glutamate to be the only amino acid, dicarboxylic acid, or acidic compound capable of this pattern of stimulation and inhibition. While the response of adenylate cyclase to glutamate agonists suggested that an N-methyl-D-aspartic acid (NMDA) type receptor may be present, glutamate agents acting as NMDA antagonists in vertebrate systems were agonists. Metabolic end products of glycolysis stimulated adenylate cyclase, suggesting that these, along with metabolic glutamate may regulate glycolytic enzymes. Only 10(-3) M L-glutamate significantly stimulated adenylate cyclase activity in tissue slices, and this response was restricted to those slices rich in nervous tissues. L-Glutamate eliminated the 5-hydroxytryptamine (5-HT) stimulated adenylate cyclase response suggesting that glutamate can modulate the 5-HT stimulated elevations in adenylate cyclase activity. The data support the hypothesis that L-glutamate is a neurotransmitter-modulator in the cestode. PMID- 1709818 TI - A study, by electron microscopy, of the specific uptake of alpha-fetoprotein by mouse embryonic fibroblasts in relation to in vitro aging, and by human mammary epithelial tumour cells in comparison with normal donors' cells. AB - A covalent conjugate of alpha-foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow the internalization pathway of this serum protein in early and late passages of primary cultures of mouse embryonic fibroblasts as well as in a spontaneously immortalized cell line. AFP, as transferrin (Tf) used in parallel as a control, are endocytosed through coated pits and vesicles and move then to endosomes in every case; in cells of the late passages, at least a part of the internalized proteins would be routed to lysosomes. Cells of three different established human mammary cancer lines (MCF-7, Evsa-T, T-47D) internalize AFP-HRP through coated pits and vesicles. Such localization of the conjugate is practically never detected in normal human mammary epithelial cells in primary culture. Taken together, these results are in agreement with the view that AFP receptors are expressed at the surface of proliferating mouse embryonic fibroblasts and human mammary epithelial cancer cells but absent from the surface of normal human mature cells of the same origin. PMID- 1709819 TI - Structure of the O-antigen of Actinobacillus pleuropneumoniae serotype 7 lipopolysaccharide. AB - The structure of the O-antigen polysaccharide of A. pleuropneumoniae serotype 7 was investigated by methylation analysis, partial acid hydrolysis, periodate oxidation, and 1H- and 13C-n.m.r. spectroscopy. The polysaccharide repeating-unit consists of a branched tetrasaccharide having the following structure. [formula: see text] PMID- 1709820 TI - Structure of a glucorhamnan from the lipopolysaccharide of Serratia marcescens strain S1254. PMID- 1709821 TI - Conformational analysis of the amino termini (5 residues) of human glycophorin AM and AN: differentiation of the structural features of the TN and T antigenic determinants in relation to their specificity. AB - The N-terminus of glycophorin A, the main transmembrane erythrocyte glycoprotein responsible for the MN blood-group specificity, has been modelled. As the minimum size of the protein recognised by the antiglycophorin A antibodies is the N terminal glycopentapeptide, attention was focused on the TN and T antigenic determinants of this size in order to determine wether differences in 3D structure exist and how a specific response with different antibodies is induced. PMID- 1709822 TI - Reactivity of an anti-(human gastric carcinoma) monoclonal antibody with core related peptides of gastrointestinal mucin. AB - A murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti (gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins. PMID- 1709823 TI - The effects of staphylococcal protein A on human lymphokine-activated killer cell induction. AB - Staphylococcal protein A (Cowan strain; SpA), a biologically active molecule capable of inducing augmented natural killer (NK) cell cytotoxicity, was studied in regard to its effects on lymphokine-activated killer (LAK) cell development. SpA, when co-cultured with interleukin-2 (IL-2) for 4 days, significantly augmented both LAK activity against NK-resistant M14 (melanoma) target cells and DNA synthesis of peripheral blood mononuclear cells (PBMC). This enhancement occurred with SpA concentrations of 1-100 micrograms/ml in a dose-dependent fashion; concentrations above 100 micrograms/ml were no more effective. When SpA (10 micrograms/ml) was added to PBMC cultures with various IL-2 concentrations, cytotoxicity was increased over controls with IL-2 alone. The peak cytotoxic effect reached a plateau at 80 U/ml IL-2. SpA alone induced early (day 1) cytotoxicity, which rapidly declined. SpA alone did not induce PBMC proliferation but it did increase expression of CD25 (Tac), IL-2 receptor alpha chain, on CD56(Leu19)-positive and -negative cells. The potentiating effect of SpA was significantly enhanced in serum-free medium. If either human AB serum or human IgG was added to cultures SpA-enhanced LAK cytotoxicity was diminished. The addition of anti-interferon gamma (anti-IFN gamma) antibody, but not anti-IFN alpha, inhibited (SpA+IL-2)-induced cytotoxicity, indicating that IFN gamma is partially responsible for the additive cytotoxic effect. PMID- 1709824 TI - Characterization of the 3',5'-cyclic adenosine monophosphate-mediated regulation of IL2 production by T cells and Jurkat cells. AB - The effect of cyclic AMP-elevating agents on mitogen-stimulated IL2 production was examined. Prostaglandin E2 (PGE2) inhibited IL2 production by human peripheral blood T cells stimulated with PHA. In contrast, PGE2 did not inhibit PHA-stimulated IL2 production by the human leukemic T cell line. Jurkat, and often slightly enhanced IL2 production by those cells. Other cyclic adenosine monophosphate (cAMP) elevating agents (forskolin, isoproterenol, and the cAMP analogue, dibutyryl cAMP) also inhibited lectin-stimulated IL2 production by T cells, but could not inhibit IL2 production by Jurkat cells. Of the cAMP elevating agents examined, only cholera toxin (CT) inhibited IL2 production by both Jurkat cells and peripheral blood T cells. Although phorbol myristate acetate (PMA) greatly enhanced PHA-stimulated IL2 production by Jurkat cells. CT remained markedly inhibitory. The combination of PMA and the calcium ionophore, ionomycin, also induced IL2 production by Jurkat cells, and this was similarly suppressed by CT, suggesting that a step after initial second messenger generation was inhibited. A prolonged increase in intracellular cAMP levels was induced by CT in both T cells and Jurkat cells, but the maximal level and the length of elevation achieved in T cells were much less than those observed in Jurkat cells. In contrast, PGE2 caused only a modest and transient increase in intracellular cAMP levels in Jurkat cells compared to that noted with T cells. PGE2 induced a more marked and sustained increase in cAMP levels in Jurkat cells treated with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Moreover, in the presence of IBMX, PGE2 caused a marked inhibition of IL2 production by PHA-stimulated Jurkat cells. Differences in the capacity of PGE2 to induce cAMP could not be explained by disparities in the level of cAMP phosphodiesterase activity as this was comparable in Jurkat cells and in T cells. Thus, these observations indicate that IL2 production by both peripheral T cells and Jurkat cells can be modulated by cAMP-elevating agents. The data suggest that the diminished capacity of PGE2 to inhibit IL2 production by Jurkat cells reflects both a diminished capacity of PGE2 to induce increases in cAMP levels in these cells and an increase in the threshold of cAMP required to inhibit Jurkat cells. PMID- 1709825 TI - Pentoxifylline and other methyl xanthines inhibit interleukin-2 receptor expression in human lymphocytes. AB - Addition of pentoxifylline to lymphocytes caused a dose-dependent decrease in PHA induced interleukin-2 receptor (IL-2R) expression. Expression of IL-2R protein and mRNA were inhibited by 60% at a concentration of 1 mM. Pentoxifylline also inhibited release of IL-2R into the medium by 85%. Treatment with recombinant IL 2 (50 U/ml) did not abrogate the effect of pentoxifylline. In addition to inhibition of IL-2R expression, pentoxifylline also decreased the expression of transferrin receptors and class I MHC antigens. Pentoxifylline also inhibited cell proliferation. However, aphidicolin, an inhibitor of DNA polymerase alpha inhibited cell proliferation to the same extent as pentoxifylline, but had no effect on IL-2R expression, indicating that inhibition of cell proliferation does not necessarily lead to inhibition of IL-2R expression. The inhibitory effect on IL-2R expression was also noted with other methylxanthines, theophylline and isobutylmethylxanthine, and with dbcAMP and forskolin. The inhibitory activity of pentoxifylline was prevented by W-13, a calmodulin antagonist, but not by HA 1004, a cyclic AMP-dependent protein kinase inhibitor. This suggests that pentoxifylline might act in part through a Ca2+/calmodulin-dependent mechanism. Pentoxifylline and other methylxanthines may prove useful in delineating the biochemical pathways involved in induction and expression of cell surface receptors. PMID- 1709826 TI - Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis. AB - Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis. PMID- 1709827 TI - Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells. AB - The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity. PMID- 1709828 TI - Intracellular routing rather than cross-linking or rate of internalization determines the potency of immunotoxins directed against different epitopes of sIgD on murine B cells. AB - Several variables influence the potency of an immunotoxin (IT) prepared with a monoclonal antibody (mAb) and ricin A chain (IT-A). These include the affinity of the mAb, the nature and density of the target antigen (Ag), the epitope on the target Ag bound by the mAb, the type of cell target, and the rate of endocytosis and route of internalization of the bound IT-A. In a previous report, we demonstrated that anti-delta mAbs directed against epitopes which are putatively more proximal to the plasma membrane make more effective IT-As than those directed against epitopes that are putatively more distal from the plasma membrane. It is known that the latter mAbs cross-link sIgD less effectively than the former. Therefore, in the present study, we determined whether the differential cytotoxicity of IT-As directed against these epitopes is related to their ability to cross-link their specific surface antigen (sIgD). We further determined whether they were internalized at different rates by normal B cells. Our results show that neither cross-linking nor rate of internalization account for the different potencies of anti-Fc vs anti-Fd IT-As. However, when these IT As were used in the presence of the lysosomotropic agent chloroquine, the less potent IT-A became 100-fold more potent and was as cytotoxic as the effective anti-Fc IT-A. Taken together with the results of other studies, these findings further support the hypothesis that the epitope specificity of a given mAb may be an important factor in determining the intracellular routing of an IT-A after internalization. PMID- 1709829 TI - Mast cell proteases liberate stable encephalitogenic fragments from intact myelin. AB - Protease-containing supernatants from activated rat mast cells were found to degrade purified rat myelin with a subsequent release of a stable encephalitogenic peptide. The two most abundant peptides were identified as residues 69-87 (GSLPQKSQRTQDENPVV) and residues 69-88 (GSLPQKSQRTQDENPVVH). While additional exposure to the mast cell supernatants removes the COOH terminal histamine from peptide 69-88 to yield peptide 69-87, additional proteolytic degradation of the 69-87 peptide was not detected. Immunization with this peptide emulsified in CFA caused the development of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In addition this 69-87 sequence was found to activate resting encephalitogenic myelin basic protein-reactive T cell lines to adoptively transfer clinical EAE. The release of stable encephalitogenic peptides from the myelin sheath by mast cell proteases may play a role in activation of encephalitogen-specific T cells during the progression of EAE. PMID- 1709830 TI - A termination codon is created by RNA editing in the petunia atp9 transcript. AB - Analysis of the cDNA of the atp9-1 gene transcript from petunia mitochondria has revealed that ten C residues of the gene sequence are edited into U in the mRNA. Seven of these edits result in amino acid changes and one introduces a stop codon before the end of the open reading frame predicted from the gene sequence. The resulting protein is better conserved when compared to the same protein in other organisms. Comparison of the edited petunia sequence with other plant mitochondrial atp9 gene sequences idicates variation in the number and positions of edits required to obtain the same amino acids in ATP9 polypeptides of higher plants. PMID- 1709831 TI - Prevalence of hepatitis C virus antibodies in renal transplant patients. PMID- 1709832 TI - [Inhibition of human immunodeficiency virus reverse transcriptase in vitro by extracts of Chinese medicinal herbs]. AB - Thirty-three chinese medicinal herbs reputed by ancient chinese folklore to have anti-infective properties were extracted. The extracts and their compounds were tested for inhibitory activity against the human immunodeficiency virus RT in vitro. 8 of the 33 extracts were found to be active. Of these, MBAA9045 was studied in greater depth. We found that it was a noncompetitive inhibitor against HIV-RT. The concentration of MBAA9045 reducing HIV-RT activity by 50% (IC50) was 22 mumol/L. Chinese medicinal herbs appear to be a rich source of potentially useful materials for the treatment of human immunodeficiency virus infection. PMID- 1709833 TI - How to select Plasmodium falciparum pre-erythrocytic antigens in an expression library without defined probe. AB - The restricted access to Plasmodium falciparum liver stages has greatly limited the analysis of the antigenic content of that stage. Due to the lack of material to perform immunochemical studies, of access to mRNA, and of monoclonal probes, we decided to screen a genomic library with stage-restricted human antibodies. This strategy led to the identification of a large number of DNA fragments encoding both sporozoite specific as well as liver-stage specific epitopes. Following the initial characterization of one liver-stage antigen, further screening was performed by using additional selective human antibodies. These were defined as having a high degree of reactivity with native antigens on either of the two stages while being negative with the already known molecules of the two stages. From this second screening and the study of cross-reactions, several subsets of DNA clones expressing antigens present on the surface of sporozoites, or in liver stages, or in both, could be identified. In exposed individuals a high prevalence of antibodies to several of these antigens was found. PMID- 1709834 TI - Active and passive immunization against Plasmodium yoelii sporozoites. AB - Three subunit vaccines based on the major repeat, (QGPGAP)n, and flanking regions of the Plasmodium yoelii circumsporozoite protein were designed, produced, and tested. All were immunogenic, but none gave consistent protection against a 40 200 sporozoite challenge. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of NYS1, an IgG3 MAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, indicating that the epitope on sporozoites recognized by this MAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by IFAT and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing QGPGAP but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not the absolute concentration, could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective MAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective MAbs and non-protective polyclonal antibodies will be important in the further development of malaria vaccines. PMID- 1709835 TI - The circumsporozoite protein of Plasmodium: a mechanism of immune evasion by the malaria parasite? AB - Sporozoites of malaria are covered with a repetitive surface antigen, the circumsporozoite (CS) protein. This antigen also appears to be a major target of the host immune response. The natural immunogenicity of the CS protein has led to attempts to develop the molecule as a vaccine candidate. It seems paradoxical, however, that a successful parasite should present to the host an immunogenic surface molecule which would induce protective immunity. In this paper we suggest that the CS protein is not the target of protective immunity under natural conditions, and that naturally immunogenic repetitive antigens in malaria and other parasites have evolved as a mechanism of immune evasion, via the induction of thymus-independent B-cell responses. PMID- 1709836 TI - Evidence implicating MHC genes in the immunological nonresponsiveness to the Plasmodium falciparum CS protein. AB - The circumsporozoite (CS) protein is a major candidate vaccine antigen for the sporozoite stage of malaria. Both cytotoxic T cells (CTL) and antibody specific for the CS protein are thought to be important in protection. By examining the immune response in mice and humans we have shown that genes mapping to the major histocompatibility complex (MHC) are important for immune responsiveness. F1 mice between high antibody responders and low antibody responders are high antibody responders, suggesting that in this model immune suppressor genes do not control the immune response. Using synthetic peptides to map epitopes for CTL and helper T cells (which are important for the antibody response) we have shown that the T cell epitopes are located in the polymorphic region of the protein, and we hypothesize that T cells have indeed selected the variation observed in the CS protein. The success of subunit vaccines will depend on the pattern of variation in different geographical locations, the ability to construct multivalent vaccines containing different variant epitopes from this protein, and on the existence of other sporozoite and liver-stage proteins involved in protection. PMID- 1709837 TI - An invariant, "universal" T-cell epitope in the P. falciparum circumsporozoite protein. AB - Although there are important obstacles to malaria vaccine development, we believe they might be overcome by a strategy of searching for conserved regions of a vaccine candidate that are recognized in association with many different HLA molecules and, if necessary, deliberately modifying the conserved sequences to improve their immunogenicity for T cells. This approach is illustrated by work on the circumsporozoite (CS) protein of Plasmodium falciparum, which covers the surface of the malaria sporozoite and is the best characterized of current vaccine candidates. PMID- 1709838 TI - A CTL epitope on the circumsporozoite protein of P. yoelii. AB - Humans are infected with malaria by the bite of anophelene mosquitos carrying plasmodia sporozoites. These sporozoites pass quickly from the blood into hepatocytes, where they develop into mature liver-stage parasites over several days. The clinical stage of the illness begins only when the liver-stage parasites rupture into the bloodstream and erythrocytes are invaded. The pre erythrocytic stages of malaria are inviting targets for vaccine development, because an effective immune response to these early stages would prevent symptomatic infections. PMID- 1709839 TI - A model of the ventricular cardiac action potential. Depolarization, repolarization, and their interaction. AB - A mathematical model of the membrane action potential of the mammalian ventricular cell is introduced. The model is based, whenever possible, on recent single-cell and single-channel data and incorporates the possibility of changing extracellular potassium concentration [K]o. The fast sodium current, INa, is characterized by fast upstroke velocity (Vmax = 400 V/sec) and slow recovery from inactivation. The time-independent potassium current, IK1, includes a negative slope phase and displays significant crossover phenomenon as [K]o is varied. The time-dependent potassium current, IK, shows only a minimal degree of crossover. A novel potassium current that activates at plateau potentials is included in the model. The simulated action potential duplicates the experimentally observed effects of changes in [K]o on action potential duration and rest potential. Physiological simulations focus on the interaction between depolarization and repolarization (i.e., premature stimulation). Results demonstrate the importance of the slow recovery of INa in determining the response of the cell. Simulated responses to periodic stimulation include monotonic Wenckebach patterns and alternans at normal [K]o, whereas at low [K]o nonmonotonic Wenckebach periodicities, aperiodic patterns, and enhanced supernormal excitability that results in unstable responses ("chaotic activity") are observed. The results are consistent with recent experimental observations, and the model simulations relate these phenomena to the underlying ionic channel kinetics. PMID- 1709840 TI - Reduction of canine myocardial infarct size by a diffusible reactive oxygen metabolite scavenger. Efficacy of dimethylthiourea given at the onset of reperfusion. AB - A number of scavengers of reactive oxygen metabolites reduce myocardial injury when given before ischemia and reperfusion, but few, if any, have proven to be effective when given near the onset of reperfusion. This is particularly true when infarct size is measured after at least 48 hours of reperfusion, when the full extent of myocardial damage has become apparent. Dimethylthiourea (DMTU) is an extremely diffusible, potent scavenger of hydroxyl radical, hydrogen peroxide, and hypochlorous acid, with a long half-life of 43 hours. Sixteen chloralose anesthetized dogs underwent 90 minutes of left anterior descending coronary artery (LAD) occlusion followed by 48 hours of reperfusion. Collateral flow was measured by radioactive microspheres. Infarct size and risk area were measured by a postmortem dual-perfusion technique using triphenyl tetrazolium chloride and Evan's blue dye. In eight dogs, therapy with DMTU (500 mg/kg i.v.) was given during the last 15 minutes of ischemia and the first 15 minutes of reperfusion. In eight control dogs, the same volume of 0.9% saline was given during the last 15 minutes of ischemia through the first 15 minutes of reperfusion. Infarct size as a percent of risk area was reduced in the DMTU-treated group compared with the saline-treated controls (DMTU = 42 +/- 4% versus saline = 59 +/- 4%, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709841 TI - Gonadotrophin releasing hormone agonist suppressive treatment of ovarian function decreases serum LH-beta and bioactive LH but maintains elevated levels of LH alpha. AB - Ten patients with endometriosis were treated by a continuous subcutaneous infusion of the GnRH agonist buserelin for 6 months. Although serum oestradiol decreased into the menopausal range within 2 weeks after starting treatment, serum LH levels as measured by immunoassay remained elevated at least fivefold over baseline during the entire treatment. However, the bioactivity of LH as determined by mouse Leydig cell assay was rapidly lost, changing the mean +/- SEM bioactivity/immunoassay ratio from 2.4 +/- 0.5 before treatment to 0.4 +/- 0.01 after only 1 week of medication. When LH-alpha and LH-beta immunoreactivities were assessed by specific antibodies, the serum LH-alpha profile was parallel to immunoreactive LH whereas the LH-beta profile corresponded to the pattern of bioactive LH. LH-alpha was elevated at least tenfold over baseline whereas LH beta decreased to less than 35% of pretreatment level. The alpha/beta ratio shifted from 1.3 +/- 0.2 before treatment to 0.04 +/- 0.06 after 2 weeks of buserelin infusion. Thus in response to continuous buserelin exposure, the gonadotrophin releases excessive amounts of LH having predominant LH-alpha immunoreactivity. The effective loss of LH bioactivity would be related to decreased LH-beta subunits. The significance of high levels of LH-alpha subunits. The significance of high levels of LH-alpha or possibly modified LH molecules remains to be evaluated during GnRH agonist treatment using well characterized assays. PMID- 1709842 TI - The direct early diagnosis of cystic fibrosis by the detection of the delta F508 CFTR gene mutation in a prematurely delivered boy. AB - The suspicion of prenatal meconium ileus syndrome was raised in a pregnancy in a family with no history of cystic fibrosis because of significantly higher maternal serum alpha-fetoprotein in the 16th and 19th week of gestation, dispersed areas with increased echogenity in the fetal abdomen, slight fetal ascites in the 24th-25th weeks of gestation, decreased amniotic fluid gamma glutamyltranspeptidase (GGT) activity and alpha-fetoprotein level in the 25th 26th weeks, and normal 46,XY karotype of the fetus. The detection of a homozygous deltaF508 cystic fibrosis transmembrane regulator (CFTR) gene mutation, by means of PCR from a small amount of white blood cells and urine sediment cells, substantiated the diagnosis of cystic fibrosis in a prematurely delivered boy in the 28th week of gestation. The repeated sweat test was unsuccessful. The autopsy examination confirmed the diagnosis of cystic fibrosis. Fetal meconium ileus syndrome was complicated by peritonitis and by formation of a meconium pseudocyst. Direct PCR typing improves postnatal diagnostic possibilities in the early neonatal period in prematurely delivered babies when the sweat test is difficult to perform. PMID- 1709843 TI - Characterization of the frequency of delta F508 mutation and CF haplotypes in Swedish families. PMID- 1709844 TI - [Morphology and function-based surgery in different forms of hyperthyroidism]. PMID- 1709845 TI - A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. AB - A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4 degrees C with 0.25% buffered paraformaldehyde then for 15 min at 37 degrees C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90 degrees angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7 AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of CD3 epsilon and on NALM-6 for expression of mu. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence. PMID- 1709846 TI - Clinical implications of new drugs for lowering plasma cholesterol concentrations. PMID- 1709848 TI - The control of seborrhoeic dermatitis and dandruff by antipityrosporal drugs. AB - For many years the exact nature of the pathophysiology of seborrhoeic dermatitis and dandruff was in doubt. Different schools of thought debated whether Pityrosporum yeasts were of primary pathogenic significance or a secondary phenomenon, with epidermal hyperproliferation as the primary pathology. Although effective therapy in seborrhoeic dermatitis and dandruff has for a long time been based on compounds whose only common link was antipityrosporal activity, proof of this relevance was lacking until the introduction of effective antifungal drugs, in particular ketoconazole. This article charts the swing of opinion towards the primary pityrosporal aetiology of seborrhoeic dermatitis and dandruff, reviews the evidence that antipityrosporal activity is the common link to various compounds which benefit these conditions, and compares the efficacy of these substances in treatment. PMID- 1709849 TI - A practical guide to the nonsurgical treatment of gallstones. AB - Until recently, cholecystectomy was the only treatment available for symptomatic gallstone disease. During the past 20 years, better understanding of the pathogenesis of cholesterol gallstone disease has led to alternative nonsurgical methods for treating gallstones in selected groups of patients. Use of 2 naturally occurring bile acids, chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), was reported in 1972 and 1975, respectively, for successful dissolution of cholesterol gallstones in humans. Both these bile acids act by reducing cholesterol secretion in bile, thus enabling it to solubilise more cholesterol from the stone surface. Micellar solubilisation is involved, together with liquid crystal formation in the case of UDCA. Having been extensively studied in clinical trials to assess efficacy and safety, both these compounds are now available for general use. The efficacy of CDCA can be enhanced by single bedtime dose administration and by taking a low cholesterol diet. Bedtime administration also enhances the effect of a suboptimal dose of UDCA. CDCA induces dose-related diarrhoea and hypertransaminaemia, and UDCA can induce calcification of gallstones, thus rendering them resistant to medical dissolution. A combination of the 2 bile acids at half the recommended dose for each has become an accepted practice for reducing adverse effects, and this may also enhance efficacy. One of the main problems of bile acid therapy is that dissolution of gallstones is a very slow process. Use of extracorporeal shockwave lithotripsy (ESWL) to break the stones into smaller fragments, with concurrent use of bile acids, has been shown to speed dissolution rate and to achieve complete gallstone dissolution in 78% of selected cases within 12 months.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709847 TI - Involvement of brain catecholamines and acetylcholine in growth hormone deficiency states. Pathophysiological, diagnostic and therapeutic implications. AB - A cohort of brain neurotransmitters, especially catecholamines and acetylcholine, play a crucial role in the control of neurosecretory growth hormone-releasing hormone (GH-RH)- and somatostatin (SS)-producing neurons, and hence growth hormone (GH) secretion. Stimulation of alpha 2-adrenoceptors or of muscarinic cholinergic receptors in the hypothalamus stimulates GH release, probably via stimulation of GH-RH and inhibition of somatostatin release, respectively. Additionally, stimulation of dopamine receptors is stimulatory to GH release, while activation of beta-receptors inhibits GH release via stimulation of hypothalamic somatostatin function. As a corollary, in GH deficiency states drugs affecting catecholaminergic and cholinergic functions may be exploited for diagnostic and/or therapeutic purposes, and may be useful for a better understanding of the underlying pathophysiology. Levodopa (L-dopa) [125 to 500mg orally], the physiological precursor of the catecholamines, administered either alone or in combination with carbidopa (50mg orally), to prevent its peripheral decarboxylation to dopamine, and/or the beta-adrenoceptor antagonist propranolol (0.75 mg/kg orally), and the alpha 2-adrenoceptor agonist clonidine (0.15 mg/m2 orally), are a fairly reliable stimulus of GH release. In normal subjects, however, false-negative GH responses and wide inter-individual variability may occur with these drugs. Additionally, the GH secretory response to these provocation tests is a poor predictor of endogenous 24-hour GH secretion, since levodopa or clonidine may elicit a response within normal limits in children of short stature with reduced 24-hour GH secretion and good responsiveness to GH therapy. The availability of GH-RH, a direct probe of pituitary somatotrophs, held out promise of unravelling the hypothalamic or pituitary origin of GH secretory disturbance. It soon became apparent, however, that this was not the case, because of the wide inter- and intraindividual variation in the GH response. However, the coadministration of GH-RH and muscarinic cholinergic agonists, for example pyridostigmine (which deprive the pituitary of hypothalamic SS inhibitory influences), is a useful diagnostic probe. In a large group of normal children and adolescents who received an intravenous injection of GH-RH, preceded by oral administration of pyridostigmine (60mg orally), none gave a false-negative response; this was also true for a group of short children with different forms of GH disturbances, in whom 8-hour nocturnal GH secretion was within normal limits. However, some false-negative responses occurred in children following testing with GH-RH, clonidine or pyridostigmine alone. Interestingly, the cut-off point for normality following pyridostigmine + GH-RH was as high as 20 ng/ml, while for the other provocation tests it is only 5 to 10 ng/ml. Responses lower than 20 ng/ml were present in all children with organic and most of the children with idiopathic GH deficiency.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709850 TI - Osteoarthritis 1991. Current drug treatment regimens. AB - Osteoarthritis, the most common rheumatic disease, presents the clinician with a major therapeutic and management task. The chronic and progressive course of the disease is punctuated by pain, declining function and escalating disability. This article attempts to deal with the pharmacological therapeutic options available to use in the management of osteoarthritis. Other non-pharmacological therapeutic modalities are also briefly discussed, including patient education, joint protection, physiotherapy, occupational therapy and surgery. Drug therapy as discussed focuses on the control of pain and inflammation. For this intermittently painful condition analgesic drugs assume a very important role. Non-steroidal anti-inflammatory drugs (NSAIDs) have combined analgesic and anti inflammatory properties which are particularly useful in many patients who manifest inflammation in addition to pain. While systemic steroids have no role in the treatment of osteoarthritis, intra-articular steroid injections may be of benefit. In trying to slow the degenerative process chondroprotective agents such as glycosaminoglycan may also assume increasing importance in the future. Choosing one drug over another must be done after consideration of many factors including the therapeutic and toxicity profile of the drugs involved, their pharmacology and dose regimen, the patient profile, cost and the desired effect. PMID- 1709851 TI - Levocabastine. A review of its pharmacological properties and therapeutic potential as a topical antihistamine in allergic rhinitis and conjunctivitis. AB - Levocabastine is a long acting, highly potent and selective histamine H1-receptor antagonist, which has been developed for nasal and ocular administration. In controlled trials performed to date levocabastine was effective and well tolerated in the treatment of allergic rhinitis and allergic conjunctivitis. Comparative studies have demonstrated that levocabastine is superior to placebo and at least as effective as sodium cromoglycate (cromolyn sodium) in alleviating symptoms associated with seasonal allergic conditions. Although levocabastine appears to be less effective than the topical corticosteroid beclomethasone with regard to relieving runny and blocked nose, further comparative trials between these 2 agents would be desirable. Similar to other antihistamines, levocabastine provides minimal relief of nasal blockage, but this symptom is believed to be mediated by receptors other than histamine H1. The prompt onset of antiallergic activity after application differentiates levocabastine from the reference topical antiallergic, sodium cromoglycate, which has an onset of efficacy characterised by a lag period, thereby necessitating maintenance treatment. The incidence of adverse effects associated with levocabastine therapy is low and is similar to that observed with placebo and sodium cromoglycate. Levocabastine provides prophylactic protection as well as acute relief from nasal and ocular symptoms in patients with seasonal allergic disorders. With the ever increasing trend towards topical therapy for the treatment of allergic rhinitis and allergic conjunctivitis, levocabastine is a useful addition to the range of drugs currently available. Possible avenues for additional research should include determining whether the antiallergic efficacy of topical levocabastine is superior to that of oral agents such as astemizole and terfenadine, and whether topical therapy is indeed preferred, considering the relative ease of administration of effective oral antiallergic agents. PMID- 1709852 TI - Paroxetine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in depressive illness. AB - Paroxetine is a potent and selective inhibitor of the neuronal reuptake of serotonin, thereby facilitating serotoninergic transmission; this action appears to account for the antidepressant activity observed with this drug. A mean terminal elimination half-life of approximately 24 hours permits once daily administration. Results of short term clinical trials have shown paroxetine to be significantly superior to placebo, and comparable to amitriptyline, clomipramine, imipramine, dothiepin and mianserin in relieving symptoms associated with major depressive disorders. Paroxetine has shown some preliminary promise in the treatment of depressive illness resistant to tricyclic antidepressant therapy but further studies are required before any conclusions can be drawn. Paroxetine in therapeutic doses has been very well tolerated, and the favourable tolerability profile of this agent appears to be its primary advantage over traditional antidepressant agents. Paroxetine causes minimal anticholinergic-type adverse effects, and unlike tricyclic antidepressants, it does not precipitate cardiovascular effects or provoke cardiac conduction disturbances. Nausea has been the most frequently reported adverse event during short term use of paroxetine, but it is generally mild and transient and subsides with continued use. With longer term use headache, sweating and constipation were the most frequently reported side effects but the incidence rate was not significantly different from that noted for comparator antidepressants. Furthermore, the frequency of withdrawal due to adverse effects is less with paroxetine than with tricyclic antidepressant agents. Overall, available data appear to indicate that while the efficacy of paroxetine is similar to that of traditional antidepressant drugs, the newer agent possesses much improved tolerability. In addition, the wide therapeutic index of paroxetine may be beneficial when treating patients with an increased risk of suicide. Thus, paroxetine clearly looks to become a valuable addition to the range of drugs currently available to treat depressive illness. Future research may help to further define the relative place of this newer agent in antidepressant therapy and determine how its overall therapeutic efficacy compares with that of other related antidepressant agents such as fluoxetine. PMID- 1709853 TI - Goserelin. A review of its pharmacodynamic and pharmacokinetic properties, and clinical use in sex hormone-related conditions. AB - Goserelin is a synthetic analogue of gonadotrophin-releasing hormone (GnRH) [luteinising hormone-releasing hormone (LHRH); or gonadorelin] which stimulates gonadotrophin and sex hormone release in the short term, and then causes suppression with continued administration. Goserelin is given as a subcutaneous biodegradable depot incorporating 3.6 mg of the drug, which is released continuously at an average rate of 120 micrograms/day over 4 weeks. Monthly goserelin depot therapy produces partial disease remission or stabilisation in about 75% of men with previously untreated prostatic cancer, a rate equivalent to that achieved with orchidectomy or diethylstilbestrol (stilboestrol). The response to goserelin is more rapid than to diethylstilbestrol, and goserelin is better tolerated. About 30 to 45% of premenopausal women with breast cancer responded to goserelin using objective assessment criteria, suggesting comparability to ovariectomy. In benign hormone-dependent conditions, preoperative goserelin aids surgical removal of uterine leiomyoma (fibroids) and reduces blood loss, and 6 months of therapy relieves the signs and symptoms of endometriosis. The elevation in testosterone at the beginning of goserelin therapy can result in disease 'flare' in men with prostate cancer, and sex steroid suppression with continued treatment results in hot flushes and loss of libido in most patients. Thus, goserelin is an effective alternative to surgery or estrogen therapy in prostatic cancer palliation, and possibly to ovariectomy in premenopausal breast cancer. Other gynaecological conditions reliant on the pituitary-gonadal axis also appear amenable to hormone manipulation with goserelin. PMID- 1709855 TI - Multihormonal regulation of insulin-like growth factor-binding protein-1 in rat H4IIE hepatoma cells: the dominant role of insulin. AB - Circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) and the abundance of hepatic IGFBP-1 mRNA are increased in streptozotocin-diabetic rats and are regulated in accordance with insulin and metabolic status. We recently purified rat IGFBP-1 from medium conditioned by well differentiated rat H4IIE hepatoma cells. Since this cell line provides a useful model for examining the effects of hormones on hepatocellular function, we used H4IIE cells to examine the relative role that insulin and other factors may play in the regulation of IGFBP-1 production. H4IIE cells were stabilized in serum-free medium, then treated with specific hormones. The availability of IGFBPs in conditioned medium was estimated by [125I]IGF-I binding assay, and specific BPs were assessed by Western ligand and immunoblot analyses. The abundance of IGFBP-1 mRNA was determined by Northern and slot blot analysis. Initial studies revealed that [125I]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P less than 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75 microM 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1 microM dexamethasone (P less than 0.001 vs. control for each), but these effects were prevented by insulin. Ligand and immunoblotting demonstrated that insulin decreased the production of 32K and 34K forms of IGFBP-1, while both 8-CPT-cAMP and dexamethasone increased the production of IGFBP-1; again, insulin prevented the effects of 8-CPT-cAMP and dexamethasone. Of note, 1 microM rat GH, testosterone, progesterone, or 17 beta estradiol had no effect on either IGF-binding activity or IGFBP-1 production. Northern and slot blot analyses revealed that 100 nM insulin profoundly lowered the abundance of IGFBP-1 mRNA in H4IIE cells (4 +/- 0.6% of control at 4 h; P less than 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75 microM 8-CPT-cAMP (P less than 0.001) and 9-fold with 1 microM dexamethasone (P less than 0.001). Once again, the effect of insulin was dominant; insulin both prevented and reversed the effects of maximally effective concentrations of 8-CPT cAMP and dexamethasone. To determine whether this effect of insulin reflected altered generation or stability of IGFBP-1 mRNA, H4IIE cells were incubated with 2.5 micrograms/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709856 TI - Alpha 2-macroglobulin is not an acute-phase protein in the rat testis. AB - Earlier studies from this laboratory have shown that Sertoli cells actively synthesize and secrete a nonspecific protease inhibitor in vitro; N-terminal sequence analysis, subunit structural analysis, and other biological studies revealed that this protein is the homolog of serum alpha 2-macroglobulin. We have now quantified the relative distribution of alpha 2-macroglobulin in the reproductive compartments and their comparison with nonreproductive organs. In serum and all nonreproductive tissues examined, the concentration of alpha 2 macroglobulin progressively decreased with advancing age. However, in both the testis and epididymis, the levels of this protein increased with the age of the animals. Serum alpha 2-macroglobulin levels were consistently higher than those in any other tissues until 60 days when the concentrations of this protein were the highest in the epididymis. The distribution of alpha 2-macroglobulin in various nonreproductive tissues from female rats was similar to that observed for male rats in that its levels tended to decrease with age. However, uterine levels of alpha 2-macroglobulin increased progressively with advancing age, whereas ovarian levels of alpha 2-macroglobulin remained relatively stable with an increase in animal age. As serum alpha 2-macroglobulin is an acute-phase protein in the rat, the response of this protein in the testis to induced inflammation was examined. The concentration of alpha 2-macroglobulin in serum rose about 150 fold after injection of fermented yeast. By contrast, the levels of this protein in rete testis fluid, which is derived exclusively from seminiferous fluid, did not change in response to inflammation. These results suggest that there might be distinctive mechanisms that regulate this protein in the systemic circulation vs. the microenvironment behind the blood-testis barrier in the seminiferous epithelium. PMID- 1709854 TI - Pamidronate. A review of its pharmacological properties and therapeutic efficacy in resorptive bone disease. AB - Pamidronate [aminohydroxypropylidene diphosphonate disodium (APD), disodium pamidronate] is an orally and intravenously active amino-substituted bisphosphonate which produces potent and specific inhibition of bone resorption at doses devoid of any significant detrimental effect on bone growth and mineralisation. Clinical trials indicate that pamidronate is effective in a variety of conditions characterised by pathologically enhanced bone turnover, including Paget's disease, hypercalcaemia of malignancy, osteolytic bone metastasis, steroid-induced osteoporosis and idiopathic osteoporosis. Pamidronate is highly effective in restoring normocalcaemia in patients with hypercalcaemia of malignancy associated with bone metastases but, in common with other bisphosphonates, is marginally less effective against humoral hypercalcaemia of malignancy. Comparative studies in this area have suggested that, at therapeutic doses, pamidronate has a more pronounced calcium-lowering action than etidronate (etidronic acid) and clodronate (clodronic acid) and provides a longer period of normocalcaemic remission. In Paget's disease arrest and, in some patients, reversal of the progression of osteolytic lesions by pamidronate is associated with a sustained reduction in bone pain, improved mobility and a possible reduced risk of bone fracture. In patients with osteolytic bone metastasis pamidronate reduces skeletal morbidity and slows the progression of metastatic bone destruction. Long term use of low-dose pamidronate in conjunction with conventional antiosteoporotic therapy may halt bone loss in steroid-induced and idiopathic osteoporosis. Pamidronate appears to represent a valuable addition to the drugs currently available for the treatment of symptomatic Paget's disease and cancer-associated hypercalcaemia, and shows promise in the treatment of osteolytic bone metastasis and osteoporosis. PMID- 1709857 TI - Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4. AB - The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709858 TI - Bombesin potentiates taurocholic acid-induced neurotensin release in rats. AB - The hypothesis of synergistic effects between luminal stimulants and the intramural neural network in the control of intestinal neurotensin (NT) release was investigated with an isolated vascularly perfused rat jejuno-ileum. Luminal administration of low doses of taurocholic acid (TC; 1, 5, and 10 mM) provoked only a small increase in NT-like immunoreactivity (NT-LI) in the portal effluent (50%, 100%, and 130%, respectively, above basal). Increasing the concentration of TC to 20 mM induced a strong and sustained release of NT-LI (700% above basal). The arterial infusion of bombesin (BOM; 10(-9) M) induced only a transient rise in NT-LI levels (200% above basal), with a rapid return to basal values. In contrast, 10(-9) M BOM synergistically enhanced NT-LI responses induced by 10 mM TC; the integrated response of NT-LI release was 3.2-fold higher than the sum of responses to 10(-9) M BOM and 10 mM TC alone. These cooperative effects were apparent with 10(-10) M BOM and TC concentrations over the physiological range 1 10 mM. Neither tetrodotoxin (10(-6) M) nor atropine (10(-5) M) was able to modify this synergy. Substance-P and methacholine, two other NT secretagogues, did not potentiate the TC-induced NT responses. In conclusion, BOM potentiated the release of NT induced by TC, thus suggesting that bombesinergic neurons, among other stimulatory neurons of the enteric nervous system, may modulate the sensitivity of N-cells to luminal stimulants. PMID- 1709859 TI - Differentiation of type II cells during explant culture of human fetal lung is accelerated by endogenous prostanoids and adenosine 3',5'-monophosphate. AB - Explant culture of the fetal lung, in the absence of serum or hormones, results in precocious biochemical and morphological differentiation of alveolar epithelial cells. In this study we tested the hypothesis that these maturational events are induced by endogenous cAMP. During culture of human fetal lung the content of tissue cAMP increased 140% from days 3-6. Treatment with isobutylmethylxanthine caused a further doubling of cAMP content, and indomethacin blocked most of the increase in cAMP. Isobutylmethylxanthine accelerated and indomethacin inhibited the increases during culture in surfactant protein-A (SP-A), SP-A mRNA, SP-B mRNA, phosphatidylcholine content, and activity of fatty acid synthetase. There was no effect of these treatments on the content of SP-C mRNA, which did not increase during culture. Increasing concentrations of prostaglandins E1 and E2 in the presence of indomethacin produced a parallel stimulation of cAMP content, SP-A, and fatty acid synthetase activity. The temporal increase in SP-A was also blocked by inhibitors of protein kinase-A. In morphological studies, indomethacin-treated explants appeared less mature, with decreased intralumenal volume and more columnar epithelial cells. We conclude that increased tissue cAMP levels, stimulated by endogenous prostaglandins, accelerate differentiation of alveolar epithelial cells during explant culture. PMID- 1709860 TI - The nitric oxide synthase II inhibitor NG-nitro-L-arginine stimulates pancreatic islet insulin release in vitro, but not in the perfused pancreas. AB - The effects of L-arginine and its derivatives NG-methyl-L-arginine (Meth-arg) and NG-nitro-L-arginine (Nit-arg) on the insulin release and glucose oxidation of isolated rat islets were investigated to evaluate possible influences of nitric oxide. Also, the insulin release and flow distribution within the perfused rat pancreas were studied in response to these agents. Arginine, Meth-arg, and Nit arg stimulated insulin release in cultured islets, but had no effect on glucose oxidation. Arginine and Meth-arg, an inhibitor of the enzyme nitric oxide synthase I (NS-I) stimulated insulin release from the perfused pancreas. Nit-arg, however, which inhibits nitric oxide synthase II (NS-II) had no such effect. Neither arginine nor Nit-arg or Met-arg changed the distribution of microspheres within the perfused pancreas. We conclude that interference with NS type I or II has no effect on insulin release from cultured islets, but inhibition of NS-II abolishes the insulin response in the perfused pancreas, which may be mediated by factors released from endothelial cells. These effects are not due to any changes in the flow distribution within the pancreas. The lack of inhibitory effect by Nit-arg in cultured islets may reflect the absence of endothelial or nervous cells in the cultured islets. PMID- 1709861 TI - Cloning and characterization of the rat complementary deoxyribonucleic acid and gene encoding the neuroendocrine peptide 7B2. AB - A 7B2 cDNA clone was isolated from a rat insulinoma cDNA library. The 1.1 kilobase (kb) cDNA insert contained 1 open reading frame of 630 nucleotides which encoded a 210-amino acid sequence. The deduced rat 7B2 precursor peptide of approximately 24 kDa would yield an approximately 21-kDa peptide upon cleavage of the signal sequence. The rat 7B2 nucleotide and amino acid sequences were highly homologous to those of mouse, human, pig, toad, and salmon. Northern analysis revealed 7B2 expression in rat pituitary and brain and in the PC12 and RIN5F cell lines. A DNA clone containing the 5' end of the rat 7B2 gene was isolated from a rat genomic DNA library. A 3.5-kb fragment isolated from this clone contained 1.5 kb of 5' nontranscribed DNA and 2 kb of the transcriptional unit. Nucleotide sequence and primer extension analyses revealed a TATA-like sequence approximately 25 basepairs up-stream of the transcription start site. Sequence analysis also revealed three putative cis-acting elements in the 5' flanking region. The second exon encoded the first 73 amino acids of the 7B2 prepeptide. Plasmids containing the 7B2 promoter fused with a reporter gene were transiently transfected into LAN-5 cells. Expression was evident only when the first intron was included with the 5' flanking sequences in the construct and only when cells were treated with forskolin or a phorbol ester. The rat 7B2 gene and cDNA will be valuable tools for the identification of factors that regulate 7B2 gene expression. PMID- 1709862 TI - Adhesion receptors involved in clustering of blood dendritic cells and T lymphocytes. AB - The relationship of dendritic cells (DC) isolated from the peripheral blood to those of lymphoid tissue is, in terms of maturation and function, incompletely understood. In our present study, we have explored the molecular basis of adhesion of T cells to blood DC. Analysis of the expression of adhesion receptors on the cell surface of blood DC revealed that these cells express lymphocyte function-associated antigen (LFA)-1 (CD11a/18), ICAM-1 (CD54), LFA-3 (CD58) and CD44, but are very late antigen (VLA)-4 (CD49d) and vascular cell-adhesion molecule (VCAM)-1 negative. The LFA-1 pathway was found to play a key role in T cells-blood DC adhesion; monoclonal antibodies (mAb) against both LFA-1 and ICAM 1 strongly inhibited adhesion between those cells. Moreover, a T cell clone from an LFA-1-deficient patient showed poor binding to blood DC. The important role of LFA-1 in T cell-blood DC adhesion was also supported by the metabolic energy and divalent cation dependence of the interaction. mAb against LFA-3 and CD2 did not inhibit T cell-blood DC binding. In contrast to the strong inhibition by antibodies to LFA-1 and ICAM-1, antibodies to CD44 enhanced conjugate formation between T cells and blood DC. Together, our results show that the LFA-1/ICAM-1 pathway plays a central role in T cell-blood DC adhesion, a situation like that in T cell adhesion to lymphoid DC. However, unlike lymphoid DC, blood DC do not express VCAM-1 nor use LFA-3 for T cell binding. PMID- 1709863 TI - Antibody specificity and immunoglobulin VH gene utilization of human monoclonal CD5+ B cell lines. AB - Human B lymphocytes that bear the CD5 antigen are relatively abundant in early ontogeny and comprise a small fraction of the B cell population in adults. The CD5 B cell subset has attracted much attention because of its possible involvement in autoimmune disease and certain B cell malignancies. To begin to understand the role of CD5 B cells in disease processes, we have generated a panel of ten human monoclonal B cell lines selected for expression of the CD5 antigen. These cell lines were obtained by Epstein-Barr virus transformation of B lymphocytes isolated from the spleen, liver and bone marrow of a 19-week-old fetus, from cord blood and from peripheral blood of healthy volunteers. In addition, one cell line was isolated from the spleen of a patient with chronic lymphocytic leukemia. Here, we describe the antibody and immunoglobulin VH gene repertoire of this panel of CD5 B cell lines. The results of these experiments show that (a) some but not all CD5 B cell lines secrete polyreactive antibodies that bind to a variety of self- and xenoantigens and (b) members of the small VH4, VH5 and VH6 gene families are overrepresented in this panel of cell lines. Nucleotide sequence analysis revealed the expression of VH gene elements that have been previously reported in the preimmune B cell repertoire, in CD5 B cell tumors and in polyreactive antibodies. PMID- 1709864 TI - CD77: an antigen of germinal center B cells entering apoptosis. AB - We have previously reported that a neutral glycolipid (globotriosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA-1, LFA-3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low-affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor alpha chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti-CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti-CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death. PMID- 1709865 TI - Identification and characterization of a protective immunodominant B cell epitope of pertactin (P.69) from Bordetella pertussis. AB - Epitopes defined by monoclonal antibodies (mAb) specific for the Bordetella pertussis outer membrane protein P.69 (pertactin) were mapped using a series of amino- and carboxy-terminal deletion mutants expressed in Escherichia coli. mAb were found to bind predominantly to a region of pertactin spanning a (Pro-Gln Pro)5 repeat motif and one mAb was found to bind to another region spanning a (Gly-Gly-Xaa-Xaa-Pro)5 repeat motif. To localize further the mAb-binding sites, a panel of synthetic peptides, a series of 94 overlapping hexameric peptides, and a P.69 30-amino acid fusion to a hepatitis B core protein (HBcAg-69), were synthesized. This combined approach has identified the binding site for the mAb BBO5: Pro-Gly-Pro-Gln-Pro-Pro; mAb BBO7, E4A8 and E4D7: Ala-Pro-Gln-Pro-Pro-Ala Gly-Arg; and mAb BPE3: Thr-Leu-Trp-Tyr-Ala-Glu-Ser-Asn-Ala-Leu-Ser-Lys-Arg. We have used a non-lethal murine respiratory model of B. pertussis infection to investigate the ability of a peptide containing the epitope of the mAb BBO5 to elicit protective immunity. Immunization of mice with the HBcAg-69 protein prevented growth of B. pertussis in the lungs compared to mice receiving HBcAg alone, and protection correlated with high titers of anti-P.69 antibodies. PMID- 1709866 TI - Major histocompatibility complex binding and T cell recognition of a viral nonapeptide containing a minimal tetrapeptide. AB - The primary immune response of cytotoxic T lymphocytes in H-2d and H-2q mice to infection with lymphocytic choriomeningitis virus is directed mostly towards the common major T cell epitope of amino acids 112-132 on the viral nucleoprotein (NP). The molecules responsible for presentation of the T cell epitope NP112-132 are in both haplotypes the MHC class I L antigens (Ld, Lq). Truncations of the amino and carboxy termini of the NP 112-132 sequence revealed the nonapeptide RPQASGVYM (NP118-126) as a most effective peptide antigen, but even the tetrapeptide GVYM was recognized by CTL of both haplotypes in a class I antigen restricted specificity. When tyrosine (Y) or methionine (M) were substituted with alanine, CTL recognition of the altered nonamer required 10(6) to 10(8) times higher peptide concentrations and in one case (Y----A on Ld) the peptide was not recognized at all. Up-modulation of the expression of Ld and Lq class I antigens as measured by flow cytometry correlated with the ability to present the peptide antigens. The only exception was peptide NP118-126 (M----A), which was recognized by T cells on L-Ld and L-Lq target cells but failed to up-regulate Ld and Lq antigens. PMID- 1709867 TI - Direct involvement of CD7 (gp40) in activation of TcR gamma/delta+ T cells. AB - In this study we reported that on T cell receptor (TcR) gamma/delta+ cells from three cell lines Peer, MOLT-13 and ICRF-1, the T cell antigen CD7 (gp40) can be directly involved in the activation process. This is shown by a rapid increase in cytoplasmic free calcium after stimulation of these cells with an anti-CD7 monoclonal antibody (mAb). Activation through CD7 was further confirmed by measuring the production of interleukin 2 in ICRF-1 cells stimulated with anti CD7 mAb. In addition induction of mRNA for tumor necrosis factor (TNF)-alpha and TNF-beta in Peer and for granulocyte-macrophage-colony-stimulating factor in MOLT 13 was observed in these anti-CD7-stimulated cells. The same anti-CD7 antibody was unable to activate TcR alpha/beta+ Jurkat cells or normal resting peripheral blood T lymphocytes. We further showed that normal resting TcR gamma/delta+ cells were likewise activated via the CD7 molecule. TcR gamma/delta+ cells obtained from a patient with acute lymphoblastic leukemia 3 months after autologous bone marrow transplantation were induced to proliferate, as measured by [3H]thymidine incorporation after stimulation with anti-CD7 mAb but not with anti-CD3 mAb. Interestingly TcR alpha/beta+ cells from the same donor tested in parallel were not stimulated by anti-CD7 but by anti-CD3 mAb. In essence these findings contribute to the idea that on TcR gamma/delta+ cell, the CD7 antigen could play an important role during T cell differentiation. PMID- 1709868 TI - A myeloma M 104E private idiotope is represented predominantly among anti dextran, peritoneal cavity Ly-1+ precursor B cells. AB - Public and private idiotopes (Id) had been defined in the BALB/c anti-dextran B1355 fraction S (Dex) response by means of syngeneic monoclonal anti-M 104E Id antibodies. Most of the primary anti-Dex antibodies shared the public Id-1 while the private Id-5 constituted a comparatively low, but highly variable proportion of humoral anti-Dex antibodies of individual mice. In the present work we have analyzed by limiting dilution the expression of these two Id by anti-Dex B cell precursors from the spleen and peritoneal cavity of BALB/c mice. Testing spleen cells we found about 65% of anti-Dex precursors to be Id-1+ and only a minority to be Id-5+. Treatment with anti-Ly-1 and complement was without any effect on anti-Dex precursors from spleen. Anti-Dex B cell precursors among peritoneal cavity lymphocytes differed from those found in the spleen in two ways. First, on average, 50% of them were Id-5+; second, about 80% carried the Ly-1 marker. Both markers are, therefore, expressed on at least 30% of anti-Dex B cell precursors from the peritoneal cavity but rarely on anti-Dex precursors from the spleen. PMID- 1709869 TI - Neonatal complement depletion results in predominant expression of a myeloma M 104E private idiotope among anti-dextran antibodies. AB - Myeloma M 104E (IgM, lambda 1) with specificity for the alpha(1----3) glucosidic linkage of dextran B 1355 S (Dex) carries two idiotopes (Id) as defined by isogenic anti-idiotype mAb. The public Id is not influenced by two amino acid replacements in CDR 2 nor by an alternative D region sequence. It is shared by all or nearly all humoral antibodies in the primary immune response against Dex of mice carrying haplotype Igha. The private Id-5' appears to depend on the integrity of the VH germ-line sequence and on the particular M 104E D region sequence. It is present on a highly fluctuating but usually small fraction of primary anti-Dex antibody. We report here that this situation is changed when mice are treated with Cobra venom anti-complement factor (CVF), after birth and thereby were deprived of complement for the first two weeks of life. When immunized with Dex as adults the majority of anti-Dex Ab carried the M 104E Id-5. Thus, humoral antibody in CVF-treated animals resembled the Ly-1+ anti-Dex precursor B cell population in the peritoneal cavity, while anti-Dex Ab in animals not treated with CVF more closely corresponded to the Ly-1- precursor B cell pool in spleen (H.-P. Lehmann and G. Lehle, Eur. J. Immunol. 1991. 21: 1201). PMID- 1709870 TI - Sequences of the mouse F protein alleles and identification of a T cell epitope. AB - F protein is found predominantly in the liver and is of unknown function. The protein has been of interest to immunologists in the areas of self tolerance and the immunogenetics of the anti-F protein response. In the mouse there are two alleles (F1 and F2), and although mice are completely tolerant to the self form of the protein, mice of responder strains make a good antibody response to immunization with the non-self form. This response cross-reacts with the self form, implying firstly, that autoreactive B cells are present and that tolerance is therefore maintained at the T cell level, and secondly, that the difference between the two allelic products defines a T cell epitope. Primers based on the published sequence for rat F protein were used in the polymerase chain reaction to amplify the cDNA for the two mouse alleles. Subsequent sequencing shows a high degree of sequence identity between the rat and mouse cDNA. The two mouse cDNA are identical apart from a single A to G base change which predicts an asparagine (F1 protein) to aspartate (F2 protein) amino acid residue change. Using allele specific oligonucleotide probes we confirmed that this base change has the same strain distribution as the previously determined F protein type. Isoelectric focusing shows that F1 protein migrates in a more basic position than F2 protein, as predicted by the asparagine to aspartate change. Finally, a synthetic peptide from the allovariable site of F2 protein will successfully restimulate T cells in vitro from an F1 type mouse primed in vivo with whole F2 protein, whereas the corresponding peptide from F1 protein will not. This is evidence that, as predicted, the allovariable site does indeed define a T cell epitope. Peptides covering the rest of the F2 protein molecule were not stimulatory. PMID- 1709871 TI - T cell reactivity to an epitope of the mycobacterial 65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses. AB - Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans. PMID- 1709872 TI - Analysis of T cell reactivities to phosphorylcholine-conjugated hen egg lysozyme in C57BL/6 mice: hapten-conjugate specificity reflects an altered expression of a major carrier epitope. AB - We have selected several different T cell hybridoma clones reactive to hen egg lysozyme (HEL) conjugated to phosphorylcholine (PC) after fusion of PC-HEL-primed C57BL/6 lymphocytes with BW5147 parent cells. These hybridoma clones preferentially recognize PC-HEL over unconjugated HEL, but not other carrier molecules conjugated with the same hapten. All the PC-HEL-reactive clones are similarly responsive to not only p-azobenzenearsonate (ABA)-conjugated HEL (ABA HEL) but also to a variety of other diazotized hapten-HEL conjugates. However, these clones are not stimulated by fluoresceinated or dinitrophenylated HEL beyond the level of HEL carrier alone. Therefore, the type of hapten linkage (diazonium) to the carrier molecule appears to affect T cell recognition. The hybridoma clones apparently recognize the carrier molecule alone, although the level of stimulation is relatively low compared to that induced by either PC-HEL or ABA-HEL. Interestingly, HEL unfolded by S-carboxymethylation is capable of stimulating the hybridomas to a level comparable to that obtained with PC-HEL. T cell recognition of the unfolded HEL is independent of antigen processing, which is different from that of PC-HEL. The peptide sequence corresponding to the amino acids 81-93 of HEL appears to contain the epitope region for the hybridoma clones based on testing stimulation activity with synthetic peptides. Previously, the peptides including this region (81-96) have been reported as the determinant recognized by T cells derived from C57BL/6 mice after immunization with an HEL peptide (HEL 13-105) but not with native HEL. These results suggest that a hapten conjugation via diazonium linkage modifies antigen presentation and consequently the presentation of the major T cell epitopes similar to that of the HEL fragment. PMID- 1709873 TI - 12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea. AB - Human and bovine corneal epithelial cytochrome P450 convert arachidonic acid to compound D [12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid], a metabolite with inflammatory properties including vasodilatation and breakdown of the blood aqueous barrier. Angiogenic properties of the endogenous compound D and the synthetic enantiomers DR and DS were examined using the corneal micropocket technique. The synthetic compound DR was as active as the endogenously formed compound D. Neovascularization of the cornea was found in all the implants containing as little as 0.5 micrograms of compound DR. In contrast, the stereoisomer DS at the same concentration (0.5 micrograms) was inactive. Since angiogenesis can be secondary to a local inflammatory response, we evaluated the effects of compound DR and its stereoisomer DS on human neutrophil chemotaxis by using a modified Boyden chamber technique. DR, but not DS, was found to be a potent chemotactic factor, exhibiting dose-dependent neutrophil chemotaxis with significant responses observed at doses as low as 10(-11) M, a concentration at which leukotriene B4 does not exhibit significant chemotactic activity. Therefore, compound D produced by the cornea may qualify as an intrinsic corneal angiogenic factor which, in association with other inflammatory mechanisms, account for the growth of new vessels in the cornea that appear in chronic inflammation or in the reparative stages of an acute process. PMID- 1709874 TI - DNA damage is involved in the induction of opacification and neovascularization of the cornea by ultraviolet radiation. AB - Studies were conducted to examine ultraviolet radiation (UVR)-induced alterations of the cornea of the gray, short-tailed opossum. Monodelphis domestica, and the effect of post-UVR illumination to photoreactivation light (PRL, 320-500 nm). As photoreactivation treatment specifically monomerizes pyrimidine dimers, an amelioration of the UVR-induced biological end-point would implicate DNA as being a primary chromophore for induction of that end-point. Corneas of anesthetized, four-month-old, opossums were exposed to 250 J m-2 (0.025 J cm-2) from a Westinghouse FS20 sunlamp either one or three times a week for up to 13 exposures. The corneas of 4-5 animals received either: (a) 90 min of PRL immediately prior to UVR; (b) PRL immediately following UVR; (c) PRL alone; or (d) UVR alone. Eyes were examined with a slit lamp microscope 24 hr following each exposure and scored for the appearance of opacification and neovascularization of the cornea. In animals exposed to UVR alone, 2-5 exposures, depending on whether the exposures were given once or three times per week, were required to obtain opacification and neovascularization in 50% of the irradiated corneas. The onset of both opacification and neovascularization in 50% of the corneas required 8-11 exposures when the UVR was immediately followed by PRL. Based on the specificity of photoreactivation repair to act solely on pyrimidine dimers, these observations suggest that UVR-induced pyrimidine dimers in corneal DNA are involved in UVR-induced opacification and neovascularization of the cornea of Monodelphis domestica. PMID- 1709875 TI - Rat NGF receptor is recognized by the tumor-associated antigen monoclonal antibody 217c. AB - The expression of the epitope recognized by the tumor-associated antigen monoclonal antibody 217c increased on cultured rat Schwann cells with time. The same phenomenon has previously been reported for the rat nerve growth factor (NGF) receptor by using monoclonal antibody 192-IgG. The distribution of 217c antibody immunoreactivity closely paralleled that observed for NGF receptors on NGF-primed pheochromocytoma (PC12) cells in culture and a number of central neurons in vivo. Immunoprecipitation of affinity-labeled NGF receptors was used to examine whether these two antibodies shared common or unique antigens. Both the quantitative data and the electrophoretic mobility of the immunoprecipitated 125I-NGF/receptor complex indicate that the antigen recognized by the 217c mAb monoclonal antibody is, in fact, the NGF receptor. Furthermore, binding studies indicated that 192-IgG and 217c recognize different epitopes on the same molecule. The characterization of the 217c antibody should provide a valuable new tool in the study of NGF receptors. PMID- 1709876 TI - Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. AB - Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3' azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid. PMID- 1709878 TI - Aminoacyl-tRNA synthetases and DNA replication. Molecular mimicry between RNAII and tRNA(Lys). AB - Recent data pertaining to different research areas, aminoacyl-tRNA synthetases and replication of ColE1 plasmids, have provided mutually attractive prospects. The gene encoding Escherichia coli lysyl-tRNA synthetase was first isolated as a host suppressor mutation that restores replication of a mutant Co1E1 replicon. Comparison of RNAII and tRNA(Lys) suggests that lysyl-tRNA synthetase is involved in the formation of the displacement loop required for ColE1 plasmids replication and provides major identity elements of tRNA(Lys). PMID- 1709879 TI - Bidirectional effects of Kupffer cells on hepatocyte proliferation in vitro. AB - The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast-like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor beta (TGF beta). After neutralization of activated TGF beta in these media, DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor alpha (TGF alpha) demonstrated by competitive TGF alpha/EGF-receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast-like cells. Northern blot hybridization confirms the expression of both TGF beta and TGF alpha in Kupffer cells and myofibroblast-like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast-like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGF alpha and TGF beta and on the activation status of TGF beta, of which a significant fraction is secreted in the latent form. PMID- 1709877 TI - Differential regulation of the two mRNA species of the rodent negative acute phase protein alpha 1-inhibitor 3. AB - Screening of two rat liver cDNA libraries, one of which was constructed using an alpha 1-inhibitor 3 (alpha 1-13) specific primer, yielded overlapping cDNA clones which correspond to the full length cDNA for alpha 1-13 mRNA. On the basis of sequence microheterogeneity existing throughout the cDNA sequence we identified two alpha 1-13 mRNA species whose sequences are so grossly different in their bait regions that the amino acid homology therein is only 30%. Using oligonucleotide probes derived from their respective bait regions we investigated the regulation of the two alpha 1 I3 mRNA species and demonstrated that only one of them, alpha 1-I3 variant I, is regulated pretranslationally following experimentally induced inflammation. PMID- 1709880 TI - Hyperreactivity of adenines and conformational flexibility of a translational repression site. AB - We have used a diethylpyrocarbonate (DEPC) modification [(1976) Prog. Nucl. Acids Res. 16, 189-262] to probe the accessibility of adenines essential for coat protein binding in the MS2 translational operator [(1983) Biochemistry 22, 2601 2610, 2610-2615, 4723-4730; (1987) Biochemistry 26, 1563-1568]. The essential adenines are apparently hyperreactive with this reagent relative to other sites within the same molecule. Variation of ionic strength, pH and divalent cation concentrations reveal the existence of two distinct conformers of the RNA operator as judged by DEPC reactivity. We propose that the hyperreactivity observed is due to the participation of neighbouring bases in the DEPC modification reaction and can be used as a novel structural probe. PMID- 1709881 TI - Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS. AB - A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp13 library by complementation of the previously characterized mutation cps1-1 [(1981) J. Bacteriol. 147, 418-426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N-terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxy-peptidase yscS as sole nitrogen source. PMID- 1709882 TI - Expression of functional receptors for interleukin-6 by human polymorphonuclear leukocytes. Downregulation by granulocyte-macrophage colony-stimulating factor. AB - Surface interleukin-6 receptors were identified on human polymorphonuclear leukocytes (PMNL) by monoclonal anti p80-chain antibody MT 18 Cytoplasmic RNA harvested from PMNL also contained IL-6-R transcripts. Binding of recombinant human (rh) interleukin-6 (IL-6) to IL-6-R bearing PMNL was identified by flow cytometry using phycoerythrin (PE)-conjugated ligand. Treatment of PMNL with rh granulocyte-macrophage colony-stimulating factor (GM-CSF) led to the inability of PMNL to bind MT 18 monoclonal antibody (moAb) and to display binding sites for PE conjugated rh IL-6. Levels of IL-6-R transcripts in PMNL exposed to GM-CSF were about 5-fold below those of PMNL cultured in medium only. Though a definitive role for IL-6 to modulate the function of PMNL was not found, treatment of PMNL with rh IL-6 clearly resulted in an enhancement of transcript levels of the early response genes c-fos and c-jun in these cells, thus indicating that IL-6 binding is followed by signal transduction. PMID- 1709883 TI - Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells. AB - Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes. PMID- 1709884 TI - Direct platelet effect of low molecular weight dextran in small calibre PTFE grafts. AB - The thrombogenicity of synthetic vascular grafts is a major factor in occlusion of grafts when they are used to bypass small calibre arteries. In this paper, the effect of low molecular weight dextran (LMWD, Dextran-40) on graft surface platelet interaction was studied using Indium-III-oxine labelled platelets. It was found that LMWD significantly reduced platelet deposition onto graft surfaces (P less than 0.001). Dextran had a direct antiplatelet effect independent of plasma volume expansion as dextran-soaked grafts significantly reduced platelet deposition when compared to systemic dextran administration (P less than 0.001). We therefore conclude that LMWD has a direct antiplatelet effect which is beneficial in reducing platelet deposition on synthetic PTFE grafts which may improve the early patency of such grafts. PMID- 1709885 TI - Apolipoprotein E synthesis by cultured human ovarian granulosa cells: regulation by human chorionic gonadotropin and cholesterol. AB - OBJECTIVE: Apolipoprotein E (Apo E) is a surface component of several classes of plasma lipoproteins and functions as a receptor ligand for the low-density lipoprotein (LDL) (B/E) receptor, the hepatic E receptor, and the LDL receptor related protein. In continuing studies of Apo E synthesis by extrahepatic steroidogenic tissue, we have examined Apo E production and its control in cultured human granulosa cells (GC). DESIGN: Human GC obtained at the time of oocyte aspiration for in vitro fertilization were cultured, and the following parameters were measured: progesterone secretion into the medium, Apo E synthesis and secretion, and Apo E messenger ribonucleic acid (mRNA) content of the GC. RESULTS: Human chorionic gonadotropin induces a decrease in Apo E protein synthesis and Apo E mRNA content. The addition of aminoglutethimide (which blocks cholesterol conversion to pregnenolone) and/or LDL (which provides the cell with cholesterol), both result in an increase in Apo E protein synthesis and Apo E mRNA content. These latter findings are in agreement with studies in nonsteroidogenic cells, which show that Apo E production is positively correlated with cell cholesterol content. PMID- 1709886 TI - The effects of the antiprogesterone RU486 (Mifepristone) on an endometrial secretory glycan: an immunocytochemical study. AB - OBJECTIVE: To examine the effects of progesterone (P) receptor blockade by RU486 (Mifepristone; Roussel-Uclaf, Paris, France) on a secretory endometrial glycan recognized by monoclonal antibody D9B1. DESIGN: Retrospective comparison of endometrial biopsies from treated and untreated women from 2 to 8 days after the luteinizing peak (LH) peak. SETTING: Infertility clinic, Jessop Hospital for Women, Sheffield. PATIENTS: Twenty-two normal fertile women received the RU486. A control group of 44 normal fertile women were also assessed. INTERVENTIONS: RU486 was administered to 22 normal women during the first half of the luteal phase and an endometrial biopsy examined 3 days later. MAIN OUTCOME MEASURES: Immunohistochemistry was used to assess the production and secretion of the D9B1 epitope. RESULTS: When the drug was given 2 days after the LH peak, it prevented appearance of the epitope. When RU486 was administered 5 days after the LH peak, epitope already present in gland cells was subsequently secreted. CONCLUSIONS: These data suggest that production of the sialo-oligosaccharide is P-dependent, but secretion through established intracellular pathways is P-independent. PMID- 1709887 TI - [Local bleomycin therapy in condylomata acuminata]. PMID- 1709888 TI - Antibodies to intracellular antigens in systemic autoimmune disease. PMID- 1709889 TI - Heat-shock proteins as antigens in autoimmunity. PMID- 1709890 TI - Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord. PMID- 1709891 TI - Analysis of expression of the human ryanodine receptor gene in malignant hyperthermia skeletal muscle tissue. PMID- 1709892 TI - Changes in fast axonal transport of proteins prior to demyelination in optic nerves of mice following Semliki Forest virus infection. PMID- 1709893 TI - A phylogenetic comparison of the 16S rRNA sequence of the fish pathogen, Renibacterium salmoninarum, to gram-positive bacteria. AB - The 16S rRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonids, was sequenced by reverse transcriptase to produce a nearly complete sequence (97%) of 1475 nucleotides. Phylogenetic comparisons to seventeen genera and signature sequence analysis indicated that R. salmoninarum was a member of the high G + C Gram-positive eubacterial subdivision although the reported G + C value is only 53%. A phylogenetic tree details the relationship of R. salmoninarum to ten actinomycetes from diverse environments. PMID- 1709894 TI - Characterization of an enterobacterial common antigen (ECA) epitope recognized by anti-ECA-tetanus toxoid conjugate serum. AB - The antigenic reactivity of both native and chemically modified enterobacterial common antigen (ECA) with anti-ECA-tetanus toxoid (TT) conjugate serum was investigated. The results obtained suggest that reduction of the carboxyl group of the mannosaminuronic acid component of ECA diminishes, but does not destroy its antigenic reactivity. Each of the sugar components were found to contribute to its reactivity with anti-ECA-TT conjugate serum, indicating that the trisaccharide repeating unit represents the ECA epitope. A nonasaccharide (trimer of the ECA repeating unit) inhibited antibody binding better than the hexasaccharide dimer, a finding which suggests that oligosaccharide conformation also makes a contribution to its inhibitory activity. PMID- 1709895 TI - Trichosanthin injection in tubal pregnancy. AB - .6 mg trichosanthin, a drug which is frequently used for midtrimester abortion in China, was applied laparoscopically in a case of tubal pregnancy. No side effects occurred and beta-human chorionic gonadotropin decreased within several days. PMID- 1709896 TI - Serum levels and molecular sizes of growth hormone during pregnancy in relation to levels of lactogens, insulin-like growth factor I and insulin-like growth factor binding protein-1. AB - Growth hormone (GH), placental lactogen (PL), prolactin (PRL), insulin-like growth factor I (IGF-I) and IGF binding protein-1 (IGFBP-1) were determined in serum by radioimmunoassays (RIAs) in 12 women during pregnancy. GH and PL were analyzed by two monoclonal antibodies (Mab 3 and Mab 1) raised against pituitary GH. Serum IGFBP-1 had reached maximum levels at midpregnancy while PRL, PL and IGF-I increased continuously during pregnancy. Mab 1, which cross-reacts with PL, measured consistently higher levels of PL in serum than a commercial PL RIA (p less than 0.01) due to interference of cross-reacting serum proteins in the Mab 1 RIA. The GH-specific Mab 3 showed decreasing GH levels in unfractionated serum throughout gestation, but detected GH-immunoreactive proteins of approximately 40 200 kD after molecular sieve chromatography of pooled serum from late pregnancy. It is suggested that the formation of GH complexes of large molecular mass account for the successive disappearance of monomeric GH during pregnancy. PMID- 1709897 TI - Inhibitor of granulopoiesis in human cyclic neutropenia. AB - Peripheral blood mononuclear cells of a patient with cyclic neutropenia release a high molecular weight substance (over 300,000) inhibiting normal CFU-GM cells to enter the S-phase of cell cycle. The inhibitor was released predominantly in the neutropenic phase of the disease, while in the period of normal granulocyte count the release was lower or undetectable. Also sensitivity of patient's bone marrow CFU-GM cells to similar high molecular weight inhibitor produced by ML-2 cell liner or to human placental ferritin varied within the disease cycle. CFU-GM in the normal granulocyte count period were sensitive to the inhibitors, but CFU-GM in the neutropenic phase were resistant. PMID- 1709898 TI - [Diagnosis and treatment of lymphogranulomatosis in children]. AB - Since more than 10 years there has been an informal pediatriconcological co operation between Bulgaria, Hungary, Poland, GDR, CSFR, and USSR. In regular intervals an exchange was made for the coverage and care for oncologically affected children, on epidemiological, diagnostical and therapeutic results. Prospective co-operative therapy studies, however, could not be accomplished, with the exception of this retrospective analysis of M. Hodgkin covering 750 children, 465 boys and 285 girls according to age of disease, primary clinical division of stages, histological variant, applied treatment and their total findings. Thus, the demand for prospective studies of treatment is emphasized. PMID- 1709899 TI - Activity of some lysosomal enzymes in peritoneal macrophages of patients with end stage renal failure treated by intermittent peritoneal dialysis. AB - Acid phosphatase, beta-D-Glucuronidase and N-acetyl-beta-D-glucosaminidase were assessed cytochemically in peritoneal macrophages obtained from 50 patients with end-stage renal failure treated by intermittent peritoneal dialysis and from 30 control subjects with normal renal function. A statistically significant increase in beta-D-glucuronidase activity accompanied by a decrease in acid phosphatase activity were observed in peritoneal macrophages of dialysed patients, as compared with the control group. In patients with dialysis-associated peritonitis, the activity of N-acetyl-beta-D-glucosaminidase was significantly higher than that observed in the same patients during the complication-free period of the treatment. PMID- 1709900 TI - [The value of parameters of iron metabolism in the differential diagnosis of anemias]. AB - As a result of investigations into the diagnostic validity of selected haematologic-morphological and clinical-chemical test factors of iron metabolism in the diagnosis of hypochromic anaemia, the examined test faktora are differently evaluated as individual parameters and in their combination. 1. Haematocrit (PCV) is equal to the determination of haemoglobin concentration as a search parameter. 2. The number of reticulocytes, copper and zinc as well as caeruloplasmin have a separating effect as individual parameters on the examined classes of iron deficiency and tumour and infect anaemia. 3. Iron has no value as a individual parameter. It is only in combination with TEBK and the haematologic test factors that is has a diagnostic value. 4. In contrast, ferritin as an individual parameter is of primary importance and should be used extensively in the laboratory diagnosis of hypochromic anaemia. 5. TEBK and transferrin may be supposed to be equal in their diagnostic value. 6. When used in combination, haemoglobin, MCV, TEBK, Transferrin, and ferritin have effective separating function. They permit hypochromic anaemia to be widely assigned to one or another kind of the examined classes. PMID- 1709901 TI - Human normal B lymphocytes produce a growth-promoting activity for granulocyte progenitors in diffusion chamber culture after stimulation with pokeweed mitogen. AB - We have measured the effect of normal B lymphocytes on more primitive granulocyte progenitors (CFU-dG) clonal growth in double-diffusion chamber culture in vivo. It was found that pokeweed mitogen (PWM) stimulated B cells produce a growth promoting activity which augments the CFU-dG--derived myeloid colony formation in a dose-dependent fashion. All colonies formed under the experimental conditions were composed exclusively of granulocytes at different stages of maturation. Unstimulated B lymphocytes did not effect the CFU-dG clonal proliferation. PMID- 1709902 TI - Platelet-tumor cell interaction. AB - Several reports have appeared with regard to the potential role of platelets in tumor cells metastasis. Our in vitro studies with colon tumor cells in normals and in patients give some evidence against platelet aggregation as the determinant factor in tumor cell invasion and metastasis. Dimethylhydrazine induced colon tumors in Wistar rats demonstrate characteristic features of tumor cell dissociation in the invasion line and changes in intercellular adhesion. PMID- 1709903 TI - Nebulized heparin and anabolic steroid in the prevention of postoperative deep vein thrombosis following elective abdominal surgery. AB - One hundred eighty three patients, all over 40 years old, who underwent major abdominal surgery, were randomized into 3 groups: Group I received a single dose of nebulized heparin (800 IU per kg b.w.) administered by inhalation one day prior to surgery. Group II besides the above, also received a single injection of 50 mg of long acting anabolic steroid (nandrolone phenylpropionate) intramuscularly. Group III received 5000 IU heparin subcutaneously on hr prior to surgery as well as every 12 h for the next 5 postoperative days. Postoperatively the patients were evaluated for deep vein thrombosis (DVT) using the 125-I fibrinogen test. The occurrence of DVT was determined as: in Group I--16%, in Group II--7.9%, in Group III--7.8%. Haemorrhagic complications (clinically important) were observed in 7.8% of patients from Group III, but only in 1.7% of patients in Group I and 1.6% in Group II. For DVT prophylaxis following abdominal surgery a single application of nebulized heparin and long acting anabolic steroid is as effective as conventional low-dose subcutaneous heparin administration, but gives less haemorrhagic complications. This method is also more advantagenous in term of acceptance by the patients and represents considerable saving of nursing time. PMID- 1709904 TI - "Anti-platelet antibodies" in HIV infected haemophiliacs. AB - We have studied anti platelet antibodies and circulating immunocomplexes in 16 haemophiliacs with mild thrombocytopenia eight of which were infected by human immunodeficiency virus (HIV). No difference in platelet count was observed between HIV+ (143 +/- 31 x 10(9)/l) and HIV- patients (148 +/- 30 x 10(9)/l). On the contrary, HIV+ haemophiliacs had serum platelet bindable IgG (SPBIgG), normal platelet associated IgG (PAIgG), high serum IgG and circulating immunocomplexes (CIC). Considering all 16 patients serum IgG correlated with CIC (r = 0.7 p less than 0.01) and SPBIgG (r = 0.6 p less than 0.01) respectively. We obtained also a positive correlation between serum CIC and SPBIgG (r = 0.51 p less than 0.05). Immunoblotting of patients' sera showed no specific binding to target platelet antigens. In conclusion there is no evidence of HIV related immune thrombocytopenia in our haemophiliacs but the study confirms the appearance of immunocomplexes in the HIV+ subjects. PMID- 1709905 TI - [Multivariate analysis for the understanding of typical diabetic hemostasis criteria. 1: Plasma markers]. AB - In 309 patients investigations were made into the functions of blood coagulation with special consideration of numerous metabolic criteria. They referred to 111 healthy control persons and 198 patients with diabetes mellitus, 67 of them being of type I and 131 of type II. From a variety of metabolic characteristics and haemostasis optimal criteria were determined by means of the statistical method of multivariance analysis, which enables a distinction to be made between diabetics of type I, type II and healthy persons. Among those 13 characteristics detected as optimal amount there were thrombin time, thrombin coagulase time as parameter of haemostasis both before and after venous congestion, reptilase time prior to venous congestion and fibrinogen concentration after it. Thus, these coagulation factors indicate a different behaviour in both types of diabetes and in healthy control persons. PMID- 1709906 TI - [Multivariate analysis for the understanding of typical diabetic hemostasis criteria. 2. Prevalent thrombocytic markers]. AB - Various thrombocyte functions and metabolic criteria were investigated in 309 test persons, among them 198 Patients affected with diabetes mellitus, 67 of type I and 131 of type II as well as 111 healthy control persons. From the variety of these factors an optimal amount was determined by means of the statistical method of multivariance analysis separating both types of diabetes and healthy control persons. In addition to haemoglobin A1 concentration, thrombocyte parameters have been found together with the degree of capillary fragility, adrenalin-induced aggregation, platelet aggregation test and clot retraction, which allow individuals to be assigned diagnostically to various groups of test persons. Thus, thrombocyte functions found in both types of diabetes and normal persons exhibit differences which may contribute to a diagnostic classification. PMID- 1709907 TI - [Multivariate analysis for the understanding of typical diabetic hemostasis criteria. 3. Determination of a typical retinopathy parameter-constellation]. AB - Features of metabolism and haemostasis which are different in diabetics of both types in comparison with normal subjects were covered by the statistical method of multivariance analysis depending on the severity of diabetic retinopathy. In 29 diabetics without retinopathy, 46 patients with stage I or II, and 36 patients with stage III the following parameters could be found as optimal criteria for characterizing the extent of vascular changes: blood sugar concentration, concentration of sialic acid and HDL cholesterol in the serum, serum protein, sialic acid per protein volume, total cholesterol in the serum and capillary fragility and number of large spreading forms of platelets features of hemostasis. Thus, diabetic retinopathy is characterized by a wide spectrum of different features containing the parameters of hemostasis. Thrombocytic vascular interactions are characterized by platelet spreading and capillary fragility which are significant for the development of diabetic retinopathy. PMID- 1709908 TI - The evaluation of prognostic factors for achieving complete remission and survival in ANLL of adults. The proposition of a prognostic scale. AB - The retrospective analysis has concerned 323 patients with acute nonlymphocytic leukaemia (ANLL). The comparable patients groups were treated since 1981 according to protocols used by the Polish Acute Leukaemia Group (induction; modified TAD or Adriamycin plus Ara-C, maintenance; rotatingly changed polychemotherapy for 3 years). The prognostic value for achieving complete remission (CR) and survival of 67 pre-treatment factors (42 quantitative and 25 qualitative) was evaluated. The most important 9 parameters were scored according to the prognostic value as follows: age, proportion of blasts in bone marrow, blast count in peripheral blood, morphological subtype, percentage of granulocytes in bone marrow, proportion of blasts with CD-15 antigen, thrombocyte count, spleen/liver enlargement, protein concentration in cerebro-spinal fluid. The scoring system has been elaborated allowing selection of ANLL patients to standard risk group and a high risk group. PMID- 1709909 TI - Variability of plasma viscosity in monoclonal IgM gammopathies. AB - Hyperviscosity syndromes can caused by both plasmatic and cellular factors. We have studied 20 patients affected by IgM gammopathy of different origin and 12 healthy subjects matched for sex and age, in order to evaluate the relation between paraprotein levels and plasma viscosity. We have observed a significant plasma viscosity increase only in 14 patients with monoclonal IgMk gammopathy. In the same patients was also evident an hyperviscosity syndrome. In the other 6 patients, with monoclonal IgM or polyclonal gammopathy and without clinical symptoms, plasma viscosity was only slightly increased. We have also observed a significant correlation between IgM and light chains (kappa, lambda) serum level and increased plasma viscosity. These results suggest that one can't consider all IgM gammopathies as cause of hyperviscosity syndrome. PMID- 1709910 TI - Effect of conditioned medium on recovery of circulating blood cells in irradiated mice. AB - The effect of supernatant of thymus-cell conditioned medium (TCCM) on blood element recovery in peripheral blood was followed in mice exposed to a single whole body dose of 5.8 Gy of gamma radiation. As follows from our results. TCCM administered 18 h before irradiation accelerated the recovery of the reticulocyte and, in part, thrombocyte and granulocyte counts. However, no effect on the rate of lymphocyte recovery was found. PMID- 1709911 TI - [Isolation and purification of myelin basic protein from human brain]. AB - A simplified procedure for isolation and purification of myelin basic protein (MBP) from human brain is described. Purified myelin from white matter was isolated at first, then delipidated with heated organic solvents. The pellet was washed with triethanolamine buffer and extracted with 0.01 mol/L HCl. Finally the protein in the acidic supernatant was purified with Sephadex G-150 column. By using three different PAGE and immunoelectrophoresis, the purified MBP was identified as a homogeneous component with an apparent molecular weight of 18.5 kd and pI 10.6. This procedure has the advantage of simplicity, rapidity, high yield and purity. PMID- 1709912 TI - Genetic polymorphism of inter-alpha-trypsin-inhibitor (ITI): formal genetic and linkage analyses. AB - The polymorphism of inter-alpha-trypsin-inhibitor, ITI, was demonstrated by isoelectric focusing in agarose gels (pH 5-8) followed by protein blotting and immunoassay. Segregation in 239 families with 677 children is consistent with the formal hypothesis that there are two common codominant alleles, ITI*1, ITI*2, and one rare codominant allele, ITI*3, at an autosomal locus ITI. Allele frequencies were calculated as ITI*1 = 0.600, ITI*2 = 0.393, ITI*3 = 0.007. Linkage analysis with 36 markers is presented. Slightly positive lod scores were obtained for PGM3 (zeta = 1.35, theta = 0.10) and AK1 (zeta = 1.34, theta = 0.10). PMID- 1709913 TI - Aldosterone deficiency II (CMO II deficiency) is not the result of a mutation of an MspI restriction site within the CYP11B gene. AB - We report our investigations of a German family with aldosterone deficiency (CMO II deficiency). Restriction fragment length polymorphism analysis using a P450c11 probe demonstrates that a MspI restriction site mutation within the CYP11B gene cannot be the underlying cause for this defect, as has been suggested previously. PMID- 1709914 TI - DNA sequence, genomic organization, and chromosomal localization of the mouse peripheral myelin protein zero gene: identification of polymorphic alleles. AB - We have cloned and characterized the mouse gene, P0, that encodes the predominant protein of peripheral myelin. Similar to the rat gene, the mouse P0 gene is encoded by six exons that span about 7 kb of DNA. The DNA sequence of the mouse gene is highly homologous with the rat gene, including the regions believed to be important in transcriptional control. Furthermore, the P0 protein appears well conserved throughout evolution. The gene was mapped to mouse chromosome 1 by Southern analysis of a Chinese hamster x mouse somatic cell hybrid panel. Several polymorphic restriction enzyme sites were identified within the P0 locus. Recombinant inbred strain mapping has linked the P0 gene to Ly-9/Ly/Sap in a region corresponding to band 1H3. PMID- 1709915 TI - Cellular immune response to the antigen administered as an immune complex. AB - Immune complexes are specifically bound to the Fc receptors on the surface of various types of cells capable of presenting antigens. It was therefore determined if human serum albumin (HSA), bound in an immune complex, is presented more efficiently to the HSA-specific T cells than HSA alone. Primed, polyclonal murine T cells were stimulated in vitro with either HSA alone or HSA bound at equivalence to syngeneic, polyclonal anti-HSA antibodies of IgG class. The stoichiometric composition of the insoluble immune complex was AgAb2.91. The present study shows that 10(2)-10(3)-fold lower doses of HSA are sufficient for the same T-cell response in vitro, if HSA is administered to the cultures in the form of the immune complex rather than in the soluble form. The enhancement of T cell proliferation in the presence of immune complex was blocked with monoclonal antibody (mAb) against Fc receptor. Our results indicate that binding of antigen in the immune complex could play an important role in enhancing an antigen specific cellular immune response. PMID- 1709916 TI - Inhibition of T and B lymphocyte proliferation by rapamycin. AB - The immunosuppressive macrolide rapamycin shows marked structural similarity to FK-506, and like FK-506 inhibits the activation of cultured T and B lymphocytes at concentrations as low as 10(-10) M. However, rapamycin blocks T-lymphocyte proliferation at a much later stage than FK-506. It also inhibits human, porcine and murine T- and B-lymphocyte activation by all pathways tested, including pathways which are insensitive to FK-506, such as interleukin-2 (IL-2)-mediated proliferation of IL-2-dependent T-cell lines, activation of human peripheral blood T lymphocytes by phorbol ester and anti-CD28 and activation of murine B lymphocytes by bacterial lipopolysaccharide. Thus these two macrolides that bind competitively to the same major intracellular receptor protein inhibit T- and B lymphocyte activation by quite distinct mechanisms. PMID- 1709917 TI - Penicillamine and penicillin can generate antigenic determinants on rat peritoneal cells in vitro. AB - Conjugation of the protein-reactive drugs D-penicillamine (PA) and benzylpenicillin (BP) to immune cells to generate drug-derived antigenic determinants has been implicated in drug-induced allergies and autoimmunity. We have therefore developed an in vitro system to demonstrate and characterize the formation of cellular antigens by these drugs. Binding of PA and BP to rat peritoneal exudate cells was detected by a cell ELISA, employing rabbit antisera specific for each drug, and an indicator system employing a second antibody coupled to biotin-streptavidin-beta-galactosidase. For both drugs, binding was detected over the concentration range 125-1000 micrograms/ml. PA bound cells rapidly (maximum binding within 10 min), whereas BP bound relatively slowly (maximum binding occurring later than 4 hr). A possible role for intracellular processing and cellular metabolic activity in the generation of these drug derived antigenic determinants was examined. Pretreatment of the cells with the fixative paraformaldehyde significantly enhanced binding of PA but not BP. Treatment of cells with the lysosomotropic agents ammonium chloride or chloroquine, or with the metabolic inactivator sodium azide, did not affect the binding of either drug compared with untreated control cells. However, treatment with the oxidising agent copper sulphate, or the cellular activator phorbol myristate acetate, did significantly enhance binding of both drugs to the cells. Therefore, binding of PA and BP to the cell surface appears not to require an intracellular processing event to generate a recognizable antigenic determinant, but is enhanced by treatments that stimulate oxidative processes. PMID- 1709919 TI - Surface exposure of O1 serotype lipopolysaccharide in Klebsiella pneumoniae strains expressing different K antigens. AB - Surface exposure of the O1 serotype lipopolysaccharide in encapsulated Klebsiella pneumoniae strains belonging to different serotypes was examined by using the O1 antigen-specific bacteriophages FC3-1 and FC3-2 in conjunction with immunogold electron microscopy and enzyme immunoassays with specific antisera. Despite the presence of the capsular polysaccharide, the O1 antigen was exposed at the cell surface in strains producing K2, K7, K8, K12, K19, K21, K22, K34, K35, K42, K45, K55, K57, K62, K66, K69, and K70 capsular polysaccharides. However, in strains producing K1, K10, and K16 capsular polysaccharides, the O1 antigen was masked by the K antigen. These results suggest that, since the O1 antigen is surface exposed in many different strains of K. pneumoniae with different capsular serotypes and is also able to immunoprotect, its potential as a useful vaccine component should not be overlooked. PMID- 1709918 TI - Bacteroides (Porphyromonas) gingivalis fimbriae activate mouse peritoneal macrophages and induce gene expression and production of interleukin-1. AB - The purpose of this study was to examine whether Bacteroides (Porphyromonas) gingivalis fimbriae, an important structure involved in attachment of the bacteria to periodontal tissues, activate macrophages and subsequently induce gene expression and production of interleukin-1 (IL-1) in the cells. The fimbriae increased glucose consumption and lysozyme activity in BALB/c macrophages, both criteria of macrophage activation of peritoneal macrophages, in a dose-dependent fashion. A marked increase in the mRNA level of the c-myc gene, an oncogene, in the cells was observed after a 1-h treatment with the fimbriae, and the level decreased rapidly after 3 h. The fimbriae (4 micrograms of protein per ml) markedly induced IL-1 alpha and IL-1 beta gene expression in the cells and IL-1 production. The expression of IL-1 alpha and IL-1 beta genes measured in terms of specific mRNA increased 1 h after the start of treatment and peaked at 6 h. Such increased expression of IL-1 beta was also observed in C3H/HeJ mice, a lipopolysaccharide low-responder strain. The fimbriae stimulated transcriptional activity of IL-1 beta in the cells, but not that of IL-1 alpha. We also observed that fimbriae-induced IL-1 gene expression was not regulated by endogenous prostaglandin triggered by the fimbriae. Therefore, these observations suggest that B. gingivalis fimbriae may be involved in the pathogenesis of adult periodontal disease via triggering of IL-1 production by monocytes/macrophages in periodontal diseases. PMID- 1709920 TI - The phenotypically variable surface protein of Trichomonas vaginalis has a single, tandemly repeated immunodominant epitope. AB - Trichomonas vaginalis is a sexually transmitted protozoan parasite that undergoes phenotypic variation for numerous surface proteins. A monoclonal antibody (MAb) was used to isolate an approximately 400-bp cDNA encoding a fragment of an important phenotypically varying immunogen of T. vaginalis (Mr = 270 kDa; P270). The MAb completely inhibited the binding of P270 by antibody in sera of patients and by antibody in monospecific antiserum obtained toward purified P270, indicating that P270 contained only one immunodominant epitope. Hydrophilicity plot analysis of the deduced amino acid sequence of the recombinant protein predicted the hexapeptide sequence DREGRD as the antigenic determinant of P270. Synthetic peptides synthesized to this region demonstrated that the amino acid sequence DREGRD is important for antibody binding. Seven adjacent amino acids also contributed substantially to maximal recognition of the epitope by the MAb. A single transcript of approximately 9.5 kb, a size compatible with the reported Mr of the immunogen, hybridized to the cDNA in Northern blots of total RNA from T. vaginalis. DNA sequence and Southern blot analysis determined the epitope to be encoded by a 339-bp unit, which was found to be tandemly repeated at least 12 times within the single-copy gene. This 12-mer unit would only constitute approximately 50% of the protein, yet it is responsible for all of the serum antibody to the immunogen produced by animals and humans. The epitope sequence was found in all fresh and long-term-grown organisms examined to date, demonstrating the stability and conservation of this gene. PMID- 1709921 TI - Production and characterization of a monoclonal antibody to the complement fixation antigen of Coccidioides immitis. AB - Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgG1) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Ag14 myeloma cells, and the fusion products were screened for IgG antibody to coccidiodin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgG1 Mab was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60 degrees C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, alpha-mannosidase, glucose oxidase, and endoglycosidase H. This heat labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin. PMID- 1709922 TI - Complete evaluation of the child identified as a poor listener. AB - With increasing awareness among educators of the importance of early identification of hearing impairment, growing numbers of children are being referred for evaluation when a teacher or day care supervisor perceives that a child is having difficulty listening. Some children who manifest difficulty listening in a pre-school play group or the classroom may have conductive or sensorineural hearing loss, while others have normal hearing with an underlying and yet-to-be-detected behavioral or psychoeducational disorder. This report presents suggestions for evaluation of the child referred for difficulty listening. The otologist should consider that a child may have an attention deficit disorder when results of initial audiologic assessment indicate there is no hearing loss or when the degree of hearing loss appears to be small in relation to the degree of inattentiveness that has been observed. The features of Attention-deficit Hyperactivity Disorder (ADHD) and Specific Developmental Disorder (SDD) are described, and illustrative case studies are presented. Clues to diagnosis are provided and a distinction between overlapping disorders is made. PMID- 1709923 TI - Neurological and developmental follow-up of a population at risk. PMID- 1709924 TI - Norfloxacin concentrations in plasma and human epididymal tissue after repeated oral administration. PMID- 1709925 TI - Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate. AB - Treatment of BC3H1 myocytes or 3T3-L1 fibroblasts with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42 kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from growth factor-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within 15 min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by growth factors in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase. PMID- 1709926 TI - Interleukin-1 enhances the response of osteoblasts to platelet-derived growth factor through the alpha receptor-specific up-regulation. AB - Both platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) are produced by activated macrophages and are thought to contribute to bone remodeling, but their precise roles remain to be clarified. The interaction between PDGF and IL-1 was, therefore, studied in normal osteoblast-like cells (MC3T3-E1). The expression of alpha- and beta-PDGF receptors on MC3T3-E1 cells was detected by RNA blot analysis and confirmed by immunoblot analysis. PDGF-induced chemotactic as well as mitogenic activities were synergistically enhanced by either IL-1 alpha or IL-1 beta (40 units/ml) pretreatment in serum-free medium, although IL-1 alone did not show any detectable chemotactic activities. This biological enhancement by IL-1 was accompanied by a selective increase of alpha-PDGF receptor expression, following the augmentation of alpha receptor autophosphorylation and inositol phosphate hydrolysis induced by PDGF-AA. These findings suggest that PDGF and IL-1 are jointly involved in the bone-remodeling microenvironment as local coupling factors. PMID- 1709927 TI - Rapid secretagogue-induced activation of Na+H+ exchange in rat parotid acinar cells. Possible interrelationship between volume regulation and stimulus secretion coupling. AB - We demonstrate a rapid activation of the Na+/H+ exchanger in intact rat parotid acini in response to muscarinic (carbachol; K1/2 = 0.4 microM) and alpha adrenergic (epinephrine; K1/2 = 0.1 microM) stimulation. This rapid activation is apparently distinct from the relatively "slow" activation of the exchanger (t1/2 greater than or equal to 5 min) reported previously (Manganel, M., and Turner, R. J. (1989) J. Membr. Biol. 111, 191-198). This rapid activation is not produced by treatment of acini with active diacylglycerol analogues nor prevented by protein kinase inhibitors, arguing against the involvement of protein kinase C-dependent processes. Stimulation of the exchanger is, however, produced by concentrations of ionomycin which yield intracellular calcium levels in the physiologic (secretagogue-induced) range. In addition, chelation of intracellular calcium with 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocks the effect of carbachol, but calmodulin antagonists are without effect. The possibility that the rapid activation of the Na+/H+ exchanger may be associated with cell shrinkage arising from carbachol-induced calcium mobilization is explored. In support of this suggestion we present evidence that: (i) the Na+/H+ exchanger is stimulated by shrinkage of these cells, (ii) the carbachol dose dependence of Na+/H+ exchange activation correlates well with that of shrinkage (but not with that of intracellular calcium levels), and (iii) maneuvers which blunt carbachol- or calcium-induced shrinkage also blunt activation of the exchanger. We suggest that this osmoregulatory response may play an important role in maintaining ionic homeostasis during the acinar fluid secretory process. PMID- 1709928 TI - Enhancement of transcription termination factor rho activity with potassium glutamate. AB - The efficiencies of rho action as a termination factor during transcription in vitro of several DNA templates were determined as a function of the concentration and type of electrolyte ions. The termination efficiencies with lambda-tR1 and the promoter proximal lacZ intragenic terminators were significantly higher with 0.1-0.2 M potassium glutamate as the major electrolyte than with the optimal concentrations of KCl (approximately 0.05 M) or potassium acetate (approximately 0.15 M). Similar high efficiencies were obtained with salts of other acidic amino acids but not with a salt of N-acetylglutamic acid or with a mixture of 0.15 M potassium acetate and 0.15 M glycine, and termination was inhibited completely when 0.12 M KCl was present along with 0.12 M potassium glutamate. The salts that give high termination efficiencies have two properties in common; they consist of anions that are also zwitterions, and they are weak chelators of Mg2+ ions. The increase in termination efficiency with potassium glutamate can be ascribed mainly to a facilitation of the reactions of rho with RNA that are coupled to ATP hydrolysis, as the rate of ATP hydrolysis with isolated transcripts as cofactors was about five times higher with 0.15 M potassium glutamate than with 0.05 M KCl, whereas the rates of chain elongation, the general stability of the transcription complexes, and the binding affinity of rho with the transcripts were all very similar under the two conditions. Further analysis revealed that the activation of ATP hydrolysis is an outcome of a shift in the optimum magnesium salt concentration from 0.5 mM with 0.05 M KCl to 4 mM with 0.15 M potassium glutamate. Since glutamate is a relatively weak counterion for cationic groups in proteins, potassium glutamate can be used at 0.15 M without inhibiting the binding of rho to RNA. At that concentration, it serves to buffer the level of free Mg2+ available to stabilize RNA secondary structures that are known to impede rho action on RNA. The two special properties of glutamate together create conditions that allow rho to terminate transcription in vitro at an efficiency that matches the in vivo efficiency with use of a physiological level of K+ ions. PMID- 1709929 TI - Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes. AB - The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4. PMID- 1709930 TI - The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells. AB - An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607 613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564). PMID- 1709931 TI - The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor. AB - All five functional domains of the low density lipoprotein (LDL) receptor were assembled in their modern form more than 350 million years ago, as revealed from the sequence of two cloned cDNAs from the frog Xenopus laevis. The two cDNAs appear to represent duplicated copies of the LDL receptor gene that arose when the entire genome of Xenopus duplicated approximately 30 million years ago. Both frog LDL receptors bound Xenopus LDL with high affinity and human LDL with lower affinity when expressed in monkey COS cells. The receptors also showed high affinity for rabbit beta-migrating very low density lipoprotein and canine apoE HDLc, both of which contain apolipoprotein E. Each of the seven cysteine-rich repeats in the ligand binding domain of the Xenopus receptors resembles its counterpart in the human, indicating that these repeats had already acquired their independent structures by the time of amphibian development. The cytoplasmic tail of both Xenopus receptors is 86% identical to the human, including the FDNPVY sequence necessary for internalization in coated pits. The attainment of a fully developed receptor structure in Xenopus suggests that earlier forms of the receptor may exist in animals that are older than amphibians. An accompanying paper demonstrates that expression of both Xenopus receptor genes is controlled by a sterol regulatory element that closely resembles the human sequence (Mehta, K.D., Brown, M.S., Bilheimer, D.W., and Goldstein, J.L. (1991) J. Biol. Chem. 266, 10415-10419). PMID- 1709932 TI - The low density lipoprotein receptor in Xenopus laevis. II. Feedback repression mediated by conserved sterol regulatory element. AB - The 5'-flanking regions of the two low density lipoprotein (LDL) receptor genes in Xenopus laevis contain three repeat sequences that are virtually identical to the repeats that mediate sterol-regulated transcription of the human LDL receptor gene. Like their human counterparts, Xenopus repeats 1 and 3, but not repeat 2, bind the transcription factor Sp1 and thus probably function as positive transcription elements. Xenopus repeat 2, like human repeat 2, contains all of the nucleotides that are required for sterol regulation. Administration of sterols repressed Xenopus LDL receptor mRNA in cultured A6 kidney cells and in the liver of intact frogs. In frogs this repression was associated with a 2-fold increase in plasma LDL levels. Xenopus LDL contains a protein corresponding in size to human apoB-100, a ligand for the LDL receptor. We found no evidence that frog plasma contains B-48, nor did we observe a clear-cut protein corresponding to apoE. We conclude that the structural gene for the LDL receptor has been under sterol-mediated regulation at least since the time of amphibian development more than 350 million years ago. PMID- 1709933 TI - Isolation and characterization of the rat chromosomal gene for a polypeptide (pS1) antigenically related to statin. AB - Increasing evidence shows the existence of nonproliferation-specific gene(s) whose expression is mostly present in growth-arrested cells. One member of this gene family has been identified by previous work as a nuclear protein of 57,000 Da, termed statin. Logical extensions of statin research are to identify the genomic and cDNA clones encoding for statin and to study the regulation of statin gene expression. During the search for the statin gene, we have identified a cDNA clone and a genomic clone named S1 and S10, respectively, by screening a rat brain lambda gt11 expression library with the statin antibody and subsequently using S1 cDNA as a probe to screen a rat genomic cosmid library. Here, we report the cloning and sequencing of the S1 cDNA and S10 genomic clones. Primary sequence analyses indicate that the derived amino acid sequence of S1 shares high homology (greater than 92.6%) with human elongation factor 1 alpha (EF-1 alpha), whereas the 5'- and 3'-untranslated regions are less than 20% homologous. Despite the unusually high degree of similarity between S1 and human EF-1 alpha at the amino acid sequence level, their protein products are different and immunologically distinct. The in vitro transcription and translation product of S1 (pS1), a 49,000-Da polypeptide, reacts only with the monoclonal antibody against statin; this antibody exhibits no antigenic reaction to the EF-1 alpha protein. Northern blot analysis shows that the S1 message is most abundant in G0 phase of 3T3 mouse fibroblasts, but becomes significantly reduced in G1 and S phase cells. EF-1 alpha messages do not show such dramatic changes during cell cycle phase transition. These findings suggest that the expression of the identified S1 cDNA clone is specific for nonproliferating cells and that the in vitro translation product of the S1 cDNA is recognized by the statin antibody. Genomic Southern blots indicate that S1 cDNA is encoded by a single copy gene in the rat genome and is a unique member of the EF-1 alpha/S1 supermultigene family. DNA sequence analysis demonstrates that the rat S1 transcription unit is 12 kilobase pairs in length and contains seven introns. The organization of exons is virtually identical between S1 and human EF-1 alpha. In contrast, neither a TATA box nor a CAAT box is located in the proximal 5'-flanking regions from positions 1 to -1359 of the S1 gene, where we could expect to find the regulatory region containing the elements controlling gene expression; no evident sequence homology to the human EF-1 alpha gene is detected in this region.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1709934 TI - Redox manipulation of DNA binding activity and BuGR epitope reactivity of the glucocorticoid receptor. AB - Treatment of the transformed glucocorticoid receptor with hydrogen peroxide promotes the formation of disulfide bonds and inhibits the ability of the receptor to bind to DNA (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). It has not been determined whether the inhibition of DNA binding activity is due to disulfide bonds formed within the DNA binding domain or between the DNA binding domain and another region of the receptor. In this paper, we examined the ability of hydrogen peroxide to inactivate the DNA binding activity of the mouse glucocorticoid receptor. We show that inhibition of DNA binding activity caused by hydrogen peroxide can be accounted for entirely by the formation of disulfide bonds between cysteine residues lying within the 15-kDa tryptic fragment containing the DNA binding domain of the receptor. Reversal of the peroxide-induced inactivation of DNA binding activity requires both zinc and a thiol-disulfide exchange reagent, such as dithiothreitol. Peroxide also eliminates recognition of the intact receptor and the 15-kDa tryptic fragment by the BuGR monoclonal antibody, and the reactivity of the BuGR epitope is restored by reduction without a requirement for zinc. Pretreatment of the receptor with methyl methanethiosulfonate inhibits much of the peroxide-mediated inactivation of the BuGR epitope but pretreatment with N-ethylmaleimide does not. Similarly, DNA binding activity of the receptor is inhibited by methyl methanethiosulfonate but not by N-ethylmaleimide. These results are consistent with the proposal that peroxide promotes the formation of disulfide bonds between thiols that lie spatially close to one another in the 15-kDa tryptic fragment, resulting in rapid elimination of zinc. Restoration of the zinc finger structure restores DNA binding activity but restoration of the BuGR epitope requires only reduction without restoration of the zinc fingers. PMID- 1709936 TI - Protein kinase C regulates both production and secretion of interleukin 2. AB - Inhibiting protein kinase C (PKC) activity abrogated interleukin 2 (IL2) production by mitogen-stimulated human T lymphocytes. This effect was due partially to a 50% decrease in IL2 gene expression. However, when PKC inhibitors were added after IL2 gene transcription had already proceeded for 3-4 h, the IL2 in the culture supernatants was still reduced by 30-80%, and intracellular IL2 was increased by up to 50%. The inhibition of PKC affected the expression of IL2 receptors by these cells differently; it had little effect on gene expression or on the membrane-bound form of the receptor, but it decreased soluble receptors in the supernatants by 50-80%. These data indicate that in addition to its previously defined role in gene expression, PKC can also regulate extracellular secretion of proteins critical for T cell proliferation. PMID- 1709935 TI - RNA polymerase II elongation complex. Elongation complexes purified using an anti RNA antibody do not contain initiation factor alpha. AB - Gene expression in eukaryotes can be regulated by controlling the efficiency of transcript elongation by RNA polymerase II. The composition of the elongation complex is, however, poorly understood. Previous work has identified DNA sequences which block RNA polymerase II transcription and factors which stimulate RNA chain elongation. Here, I have purified elongation complexes arrested at discrete template locations. Complexes were rapidly and efficiently precipitated from in vitro transcription reactions using a monoclonal antibody that binds RNA. The isolated complexes remained transcriptionally active. This technique enables the facile manipulation of transcription elongation complexes. Using this approach, I show that transcription initiation factor alpha is not associated with a RNA polymerase II elongation complex. Since others have shown that alpha associates stoichiometrically with DNA, RNA polymerase II, and other required factors in an initiation complex, this work suggests that alpha departs from the transcription complex after nucleotides are required but before extensive RNA chain synthesis. In this regard alpha resembles the bacterial promoter recognition factor sigma. PMID- 1709937 TI - Apolipoprotein E gene expression in mouse 3T3-L1 adipocytes and human adipose tissue and its regulation by differentiation and lipid content. AB - Apolipoprotein E (apoE) is an important constituent of plasma lipoproteins and a ligand for several lipoprotein receptors. It is produced mainly in the liver but also in several peripheral tissues like brain, adrenal glands, kidney, and macrophages. Some of these tissues also coexpress lipoprotein lipase (LPL), an important enzyme in the metabolism of lipids and lipoproteins. This suggested a possible coordinate expression of these genes and led us to analyze whether adipocytes, a major source of LPL, could also synthesize apoE. Northern blotting experiments showed that apoE mRNA is found in differentiated mouse 3T3-L1 adipocytes as well as biopsies of human adipose tissue maintained in organ culture but not in undifferentiated 3T3-L1 preadipocytes. [35S]Methionine pulse labeling experiments revealed that apoE protein is produced in human adipose tissue and differentiated mouse 3T3-L1 adipocytes but not in preadipocytes. In biosynthetic labeling experiments, most apoE was found to be cell associated even after prolonged chase periods. Heparin treatment of the cultured cells did not enhance apoE secretion. During differentiation of 3T3-L1 cells, the onset of apoE gene expression was later than that of LPL. The apoE mRNA and intracellular apoE protein concentrations increased linearly with time of differentiation, at least through day 11, whereas LPL showed highest expression at day 7 and then declined. The increase in apoE mRNA correlated with the cellular lipid content. Inhibition of lipid accumulation in differentiated cells by biotin deprivation decreased apoE expression. Cholesterol-loading experiments suggested that apoE mRNA expression is regulated by the intracellular free cholesterol content of 3T3-L1 adipocytes. In contrast, the LPL mRNA level was not influenced by biotin deprivation or cholesterol loading. Human recombinant tumor necrosis factor, a potent inhibitor of LPL gene transcription, had no effect on adipocyte apoE mRNA levels. Therefore, although apoE and LPL are both expressed in adipocytes in a differentiation-dependent manner, the time course of their expression differs as do their responses to cellular lipid content and tumor necrosis factor. We conclude that these genes are not coordinately regulated in adipocytes. PMID- 1709938 TI - Identification of five different insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human. AB - Five different insulin-like growth factor binding proteins (IGFBPs) were isolated from adult rat serum using gel filtration, ligand affinity chromatography, and two steps of reversed-phase high performance liquid chromatography. Three of them were identified as IGFBP-2, -3, and -4 by their amino-terminal amino acid sequences. One of the remaining two proteins was the rat homologue of the partially characterized IGFBP isolated originally from human cerebrospinal fluid, while the other appeared to be a novel member of the IGFBP family. IGFBP-1 was not found in the adult rat serum under our experimental procedures. cDNAs encoding the novel IGFBP were isolated and characterized from a rat ovary and a human placenta library. The mature protein predicted for both species contained 252 amino acids including 18 cysteines that were located in the homologous positions as IGFBP-1, -2, -3, and -4. We propose to name this protein IGFBP-5. Northern analysis of IGFBP-5 mRNA in rat tissues demonstrated that transcription of this gene is highly active in kidney, although the mRNA was detectable in all tissues examined. Alignment of the amino acid sequences of the five rat IGFBPs revealed a 47-60% similarity, indicating that their individual genes diverged from a single ancestral gene by successive gene duplication in a short time frame during evolution. The chromosomal localizations of IGFBP-1, -2, -3, -4, and -5 genes in human have been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that they were located on chromosomes 7, 2, 7, 17, and 5, respectively. PMID- 1709939 TI - Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator. AB - The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI 1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346 M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators. PMID- 1709940 TI - The half-lives of platelet-derived growth factor A- and B-chain mRNAs are similar in endothelial cells and unaffected by heparin-binding growth factor-1 or cycloheximide. AB - Platelet-derived growth factor (PDGF) is mitogenic and chemotactic for vascular smooth muscle cells cultured in vitro, and, thus, may play a role in the smooth muscle cell proliferation and migration that occurs during atherosclerotic lesion development. Two related PDGF polypeptides, designated as the A and B chains, form functionally active PDGF-AA, AB, or BB dimers. The PDGF A- and B-chain genes are both transcribed in human umbilical vein endothelial (HUVE) cells and their expression is regulated by cytokines, growth factors, endotoxin, and phorbol ester. We reported previously that the angiogenic polypeptide heparin-binding growth factor (HBGF)-1 induces PDGF A-chain gene expression, but does not affect PDGF B-chain gene expression. In this study, we determined whether mRNA stabilization contributed to this induction by measuring the half-life of PDGF A chain mRNA in quiescent, HBGF-1-stimulated, and proliferating HUVE cells. PDGF A chain mRNA levels increase when quiescent HUVE cells are treated with the protein synthesis inhibitor cycloheximide; therefore, the effect of cycloheximide on PDGF A-chain mRNA decay was also investigated. The half-life of PDGF A-chain transcripts in quiescent cells was approximately 2.4 h and neither HBGF-1 nor cycloheximide significantly altered this decay rate. We also estimated the half life of PDGF B-chain mRNA under the three different growth conditions and in the absence or presence of cycloheximide. The half-life in quiescent cells was approximately 1.8 h and was unaffected by HBGF-1 or protein synthesis inhibition. Therefore, the PDGF mRNAs have similar decay rates in HUVE cells, even though the 3' untranslated region of B-chain transcripts, but not A-chain transcripts, contains AU-rich sequence motifs postulated to confer rapid turnover in vivo. PMID- 1709941 TI - Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts. AB - We studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12 myristate 13-acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH-induced cAMP accumulation was not due to an effect on the PTH-receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G-protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)-induced cAMP accumulation; i.e., at concentrations greater than or equal to 10(-6) M, PMA potentiated the PGE2-induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 less than or equal to 5.10(77) M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2-induced cAMP accumulation. PMID- 1709942 TI - Positive-ion thermospray liquid chromatography-mass spectrometry: detection of organic acidurias. AB - Positive-ion thermospray liquid chromatography-mass spectrometry (TSP-LC-MS) is used to detect organic acids via the direct injection of untreated urine from newborns and infants. Two methods are reported for the separation of organic acids. The separation of urinary organic acids is effected in either an acidic, pH 2.5 sulfuric acid, or a non-acidic, 0.05 M ammonium acetate, pH 6.8, mobile phase. Use of pH 2.5 sulfuric acid and an HPX-87H organic acid column produces better separation but has less sensitivity than the use of 0.05 M ammonium acetate, pH 6.8 and a C18 column. Positive ion TSP-LC-MS has been used to detect methylmalonic aciduria, 3-hydroxy-3-methylglutaric aciduria, propionic aciduria, isovaleric aciduria and argininosuccinic aciduria. PMID- 1709943 TI - Sensitive method for the determination of pentamorphone in serum by liquid chromatography-mass spectrometry with thermospray interface. AB - A liquid chromatographic-mass spectrometric method for the determination of 14 beta-n-pentylaminomorphinone (pentamorphone) and 14-beta-n-pentylaminocodeinone (PAC) as internal standard is developed. Concentration levels in serum were calculated by the ratio of the peak areas of pentamorphone to PAC versus the concentration of pentamorphone. Peak areas were measured using selected-ion recording of the pseudo-molecular ions of pentamorphone and PAC (m/z 369 and m/z 383, respectively). Aliquots (50 microliters) of sample were injected on a C18 mu Bondapak column following solid-phase extraction. The lowest limit of quantitation observed was 43 pg/ml. The sensitivity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application in pharmacokinetic study of pentamorphone. PMID- 1709944 TI - Specific immunofluorescence staining of Treponema pallidum in smears and tissues. AB - To date, tissue sections prepared from Formalin-fixed tissues have not been successfully stained with Treponema pallidum subspecies-specific antibody in a direct fluorescent-antibody assay. While current methods stain T. pallidum, they do not distinguish T. pallidum from other spirochetes such as Borrelia burgdorferi (E. F. Hunter, P. W. Greer, B. L. Swisher, A. R. Simons, C. E. Farshy, J. A. Crawford, and K. R. Sulzer, Arch. Pathol. Lab. Med. 108:878-880, 1984). Because trypsin pretreatment of tissue sections has enhanced other immunofluorescent-antibody (IFA) applications, we compared the use of the trypsin digestion method with the current 1% ammonium hydroxide (NH4OH) method as a means to obtain specific staining of T. pallidum in tissues by both direct and indirect IFA techniques. Pretreated T. pallidum-infected tissues sections from rabbits, hamsters, and humans were quantitatively examined with the direct fluorescent antibody-T. pallidum test conjugate absorbed with Treponema phagedenis, the Reiter treponeme. For indirect staining, a serum specimen from a patients with syphilis absorbed by affinity chromatography with T. phagedenis was used as the primary reagent, and a fluorescein isothiocyanate-labeled rabbit anti-human globulin was used as the secondary reagent. Serum specificity was established first by examining antigen smears of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, B. burgdorferi, T. phagedenis, and Treponema denticola MRB and then by examining tissues infected with these pathogens plus those infected with four Leptospira serovars. When we stained tissue using the direct IFA method that is currently a standard method for the examination of chancre smears, we found it to be unsuitable for use with tissue. Trypsin digestion did not offer an improvement over the NH4OH pretreatment method in the specific identification of T. pallidum by direct IFA. However, specific identification of T. pallidum in tissue sections was obtained by the indirect IFA technique after either trypsin or NH4OH pretreatment. PMID- 1709945 TI - Reactivities of serotyping monoclonal antibodies with culture-adapted human rotaviruses. AB - Rotaviruses collected in Bangladesh during 1985 to 1986 were culture adapted and used in a comparative serotyping study with three groups of monoclonal antibodies, all of which reacted with the major neutralization protein (VP7) of serotype 1, 2, 3, or 4. The goals were to determine which monoclonal antibodies most accurately predicted the serotype and why large variations in serotyping efficiencies have occurred with these monoclonal antibodies in previous studies. The 143 rotavirus isolates used in this study belonged to 69 different electropherotypes; and 44, 23, 21, and 55 isolates were identified as serotype 1 through 4, respectively, by neutralization with serotype-specific hyperimmune antisera. Serotyping specificity by enzyme-linked immunosorbent assay with monoclonal antibodies was 100% consistent with results found by neutralization with polyclonal antisera, but large differences were observed in the sensitivities of the different monoclonal antibodies. Monoclonal antibodies 5E8 (serotype 1), 1C10 (serotype 2), 159 (serotype 3), RV3:1 (serotype 3), ST-3:1 (serotype 4), and ST-2G7 (serotype 4) reacted with all the isolates of the corresponding serotype for which there were sufficient infectious particles. Monoclonal antibody 2F1 (serotype 2) was much less sensitive and reacted with only five serotype 2 isolates, but these were among those with the highest titers. Monoclonal antibodies RV4:2 (serotype 1), KU6BG (serotype 1), RV5:3 (serotype 2), and S2-2G10 (serotype 2), on the other hand, failed to react with between one and three isolates of the corresponding serotypes which had high titers, apparently because of epitope changes in these isolates. Effects of epitope variation were, however, most apparent with monoclonal antibodies 2C9 (serotype 1) and YO-1E2 (serotype 3), which reacted with one and no isolates of the corresponding serotypes, respectively. Cross-neutralization of escape mutants indicated that the serotype 1 monoclonal antibodies 5E8, 2C9, and RV4:2 reacted with different but probably overlapping epitopes, as did serotype 2 monoclonal antibodies 2F1, 1C10, and RV5:3, finding that were consistent with the enzyme linked immunosorbent assay data. Because of epitope variations between rotavirus strains, serotyping with several monoclonal antibodies directed at different epitopes may increase the sensitivity of the method. PMID- 1709946 TI - Antibody response to serotype-specific and cross-reactive neutralization epitopes on VP4 and VP7 after rotavirus infection or vaccination. AB - By using a competitive solid-phase immunoassay with serotype-specific and cross reactive neutralizing monoclonal antibodies directed at VP4 and VP7, we tested the antibody responses to some neutralization epitopes on VP4 and VP7 in individuals infected or vaccinated with rotavirus. Antibody responses to VP7 epitopes of the infecting serotype of virus were found at a high frequency in both infants and children. In contrast, antibody responses to VP4 and heterotypic VP7 were observed only when the individuals possessed antibodies to any serotype of rotavirus in their acute-phase or prevaccination sera. PMID- 1709947 TI - Immune response to a major epitope of p24 during infection with human immunodeficiency virus type 1 and implications for diagnosis and prognosis. AB - A sequential inhibition enzyme-linked immunoassay (SIEIA) using a peroxidase conjugated monoclonal antibody reacting to the sequence AAEWDRVHP of p24HIV-1 (amino acids 209 to 217 of p55) was developed in order to detect and determine the titer of antibody to this epitope in various populations of human immunodeficiency virus type 1 (HIV-1)-positive patients. There was a good correlation between SIEIA and a commercially available competition assay that uses recombinant p24 protein and polyclonal antibody to HIV-1 antigen, demonstrating the importance of the described epitope. Analysis of sera from French patients showed a decline of antibody to the AAEWDRVHP sequence associated with the progression of AIDS. No decrease was observed with serum samples from African patients. An immune response to the epitope was detected by SIEIA early in the course of seroconversion. Although our SIEIA uses a single p24 epitope, these data are in accordance with previously published studies in which antibodies to the whole p24 were analyzed. Sera reacting to p24 only (indeterminate profiles by Western blot [immunoblot]) did not bind to AAEWDRVHP. This epitope, which is conserved between HIV-1 and HIV-2/simian immunodeficiency virus, appears to be a major antigenic domain of p24. The area containing the sequence AAEWDRVHP and the corresponding monoclonal antibody may serve as a convenient alternative to whole purified p24 and polyclonal antibody in diagnostic and prognostic assays. PMID- 1709948 TI - Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction. AB - A combined reverse transcriptase reaction-polymerase chain reaction (RT-PCR) was developed to achieve the sensitive detection of group B rotaviruses (GBR). Sequences derived from genomic segment 3 of the IDIR (intestinal disease of infant rats) strain of GBR permitted the detection of greater than or equal to 0.08 pg of purified IDIR genomic RNA (4,000 genome copies). Primers complementary to the terminal sequences of gene 11 of GBR strain ADRV (adult diarrhea rotavirus) allowed for the detection of as little as 0.008 pg of purified ADRV genomic RNA. Detection of heterologous strains of GBR was also observed with these primer pairs. IDIR gene 3 primers recognized greater than or equal to 8 pg of RNA from bovine GBR obtained from a variety of geographic locations. RNA from IDIR, but not bovine GBR, strains was detected by means of RT-PCR with ADRV gene 11 primers. Neither set of GBR primers was reactive in RT-PCR with fecal specimens containing group A rotaviruses or fecal specimens from uninfected controls. This RT-PCR assay permits the sensitive and specific detection of a variety of GBR in fecal specimens. PMID- 1709949 TI - Detection of antibodies to hepatitis C virus in U.S. blood donors. AB - An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed. PMID- 1709950 TI - Rapid detection of bovine viral diarrhea virus by polymerase chain reaction. AB - The polymerase chain reaction was used to detect genomic sequences of the positive-stranded RNA of bovine viral diarrhea virus (BVDV), a member of the family Togaviridae. Using a set of 20-bp primers located within the conserved 3' region of the BVDV genome, we were able to consistently amplify a 205-bp target sequence from BVDV cDNA. BVDV RNAs from cell culture-propagated BVDV reference strains, diverse unrelated cytopathic and noncytopathic field isolates, and clinical serum samples were transcribed to cDNA by using avian myeloblastosis virus reverse transcriptase and further specifically amplified by using the polymerase chain reaction assay. The amplification assay was sensitive enough to detect one molecule of cloned BVDV cDNA. Reconstitution experiments conducted by adding decreasing amounts of BVDV (NADL strain) to BVDV-free serum indicated that the threshold of sensitivity of the assay was less than or equal to 1 50% tissue culture infective dose. These results show that the polymerase chain reaction may be used for the rapid detection of diverse strains of BVDV in cell cultures, biological products, and clinical specimens from cattle. PMID- 1709951 TI - Comparison of monoclonal antibody and calcofluor white stains for the detection of Pneumocystis carinii from respiratory specimens. AB - Three monoclonal antibody staining kits, from Genetic Systems, Disease Detection International, and Meridian Diagnostics, were compared with calcofluor white for the direct detection of Pneumocystis carinii in respiratory specimens. Of the 150 specimens tested, 23 were found positive for P. carinii by any of the four stains; 13 were bronchoalveolar lavage, 7 were induced sputum and 3 were expectorated sputum specimens. All stains detected the positive bronchoalveolar lavage specimens, the Genetic Systems stain detected six induced sputum specimens, and the other stains each detected four induced sputum specimens. The monoclonal antibody stains detected all three expectorated specimens, while the calcofluor stain detected only one. Overall, the sensitivities of the stains were 96% for Genetic Systems, 87% for both Disease Detection International and Meridian Diagnostics, and 78% for calcofluor. PMID- 1709952 TI - Morphological assessment of neuronal aggregates in the striatum of the rat. AB - Regional variations in cell-packing density, culminating in the formation of cell clusters, is now a recognized morphological characteristic of the striatum that has been correlated, in some instances, with either regional histochemical variations or the distribution pattern of afferent fiber systems, or both. Within these cluster regions a further level of organization exists, in the form of discrete neuronal aggregates. The light microscopic morphology of these neurons and the nature of their intercellular contacts at the electron microscope level form the focus of this report. The neurons composing such aggregates are characterized by contiguous soma-somatic or soma-dendritic contact with extended regions of junctionlike symmetrical and consistent contacts where the distance between the cytoplasmic membranes of apposing neurons narrows to as close as 7 nm. Coated vesicles close to the contact areas are common. Three-dimensional computer reconstructions of serial 1 micron sections through aggregates in either the caudatoputamen or nucleus accumbens reveal "chains" of contiguous cells that frequently involve as many as 60 neurons. These contiguous cell aggregates are discrete entities within the larger clusters or islands. It is postulated that the cellular aggregates may represent the fundamental level of striatal organization and may be local modules for intrinsic information processing, modifying extrinsic data processed through the biochemical compartmentalization of the striatum imparted by striosomes, neuropeptides, and dopaminergic, thalamic and cortical afferents. PMID- 1709953 TI - Targets of horizontal connections in macaque primary visual cortex. AB - Pyramidal neurons within the cerebral cortex are known to make long-range horizontal connections via an extensive axonal collateral system. The synaptic characteristics and specificities of these connections were studied at the ultrastructural level. Two superficial layer pyramidal cells in the primate striate cortex were labeled by intracellular injections with horseradish peroxidase (HRP) and their axon terminals were subsequently examined with the technique of electron microscopic (EM) serial reconstruction. At the light microscopic level both cells showed the characteristic pattern of widespread, clustered axon collaterals. We examined collateral clusters located near the dendritic field (proximal) and approximately 0.5 mm away (distal). The synapses were of the asymmetric/round vesicle variety (type I), and were therefore presumably excitatory. Three-quarters of the postsynaptic targets were the dendritic spines of other pyramidal cells. A few of the axodendritic synapses were with the shafts of pyramidal cells, bringing the proportion of pyramidal cell targets to 80%. The remaining labeled endings were made with the dendritic shafts of smooth stellate cells, which are presumed to be (GABA)ergic inhibitory cells. On the basis of serial reconstruction of a few of these cells and their dendrites, a likely candidate for one target inhibitory cell is the small-medium basket cell. Taken together, this pattern of outputs suggests a mixture of postsynaptic effects mediated by consequence the horizontal connections may well be the substrate for the variety of influences observed between the receptive field center and its surround. PMID- 1709954 TI - A note on the organization of the amphibian olfactory bulb. AB - After horseradish peroxidase (HRP) injections were made in limited sectors of the main olfactory bulb in the adult frog Rana pipiens, the cellular morphology of mitral cells and granule cells impregnated with HRP were examined in uninjected regions of the bulb. Mitral cells were observed to possess glomerular dendrites and prominent secondary dendrites, both of which have smooth shafts. The glomerular dendrites may be multiple, are often branched, and may arise from secondary dendrites, as well as from the cell body. The axon may also arise from a secondary dendrite. Granule cells have simple or branched peripheral dendrites, and these are spiny, where they intermingle with the mitral cell secondary dendrites. The prominence of the secondary dendrites of frog mitral cells contrasts sharply with their reported insignificance in urodeles, as studied in earlier literature. The layers of the main olfactory bulb are not as fully concentric in the frog, as they are in mammals. The implantation cone and glomerular layer occupy a small part of the surface area of the olfactory bulb on its anteroventral aspect, while the perimeters of the subjacent layers extend farther posteriorly and dorsally in successive steps. The granule cell core extends well beyond the perimeter of the mitral cell layer in a posterior direction. Long secondary dendrites of mitral cells also extend posteriorly beyond the perimeter of the mitral cell-external plexiform layer and interlace with granule cell peripheral dendrites in a plexiform layer external to the posterior region of the granule cell core. This layer, the superficial plexiform layer, forms an apron around the posterior segment of the olfactory bulb and contributes to the interbulbar adhesion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1709955 TI - Differential projections of the main and accessory olfactory bulb in the frog. AB - The central projections of the main olfactory bulb and the accessory olfactory bulb of the adult leopard frog (Rana pipiens) were reexamined, by using a horseradish peroxidase anterograde tracing method that fills axons with a continuous deposit of reaction product. The fine morphology preserved by this method allowed the terminal fields of the projection tracts to be delineated reliably, and for the first time. Herrick's amygdala has been newly subdivided into cortical and medial nuclei on the basis of cytoarchitecture, dendritic morphology, and the differential projections of the main and accessory olfactory tracts. The main olfactory bulb projects through the medial and lateral olfactory tracts to the postolfactory eminence, the rostral end of the medial cortex, the rostral end of the medial septal nucleus, the cortical amygdaloid nucleus, the nucleus of the hemispheric sulcus, and both the dorsal and ventral divisions of the lateral cortex, including its retrobulbar fringe. The lateral olfactory tract overlaps the dorsal edge of the striatal plate along the ventral border of the lateral cortex, but it is not certain whether any striatal cells are postsynaptic to the tract fibers. The lateral cortex is the largest of these territories, and receives the terminals of the main olfactory projection throughout its extent. It extends from the olfactory bulb to the posterior pole, and from the striatum to the summit of the hemisphere, where it borders the dorsal cortex. The medial and lateral olfactory tracts combine in the region of the amygdala to form a part of the stria medullaris thalami. These fibers cross in the habenular commissure and terminate in the contralateral cortical amygdaloid nucleus and periamygdaloid part of the lateral cortex. Cells projecting to the main olfactory bulb are found in the diagonal band and adjacent cell groups, but there is no evidence of an interbulbar projection arising from either the olfactory bulb proper or a putative anterior olfactory nucleus. The accessory olfactory bulb projects through the accessory olfactory tract to the medial and cortical amygdaloid nuclei. A fascicle of the tract crosses in the anterior commissure to terminate in the contralateral amygdala. While the main and accessory olfactory projections may converge in the cortical amygdaloid nucleus, the medial amygdaloid nucleus is connected exclusively with the accessory olfactory bulb. PMID- 1709956 TI - Distribution, laminar location, and morphology of tectal neurons projecting to the isthmo-optic nucleus and the nucleus isthmi, pars parvocellularis in the pigeon (Columba livia) and chick (Gallus domesticus): a retrograde labelling study. AB - Retrograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), fluorogold, fast blue, rhodamine labelled microspheres, and horseradish peroxidase (HRP) was employed to study the distribution, laminar location within the optic tectum, and morphology of tectal cells projecting upon the isthmo-optic nucleus (ION) and the nucleus isthmi, pars parvocellularis (Ipc), in the pigeon and chick. Following injections into the ION, all retrograde markers labelled tecto-ION neurons and their dendrites in the ipsilateral tectum. The cells were located within a relatively narrow band at the border between layers 9 and 10 of the stratum griseum et fibrosum superficiale (SGFS). Retrogradely labelled neuronal somata were different in both dendritic branching and shape; however, tecto-ION neurons generally possessed non-spiny radially oriented and multi-branched dendrites. The apical processes extended into the retino-recipient layers (2-7) of the SGFS and basal dendrites extended into layers 12-14 of the SGFS. Positive neuronal somata were observed throughout the rostro-caudal extent of the optic tectum. The average distance between adjacent tecto-ION neurons varied from one region to another. Specifically, retrogradely labelled cells were more numerous in the caudal, lateral, and ventral tectum, and less numerous at rostro-dorsal levels. Approximately 12,000 tecto-ION neurons were labelled within the ipsilateral optic tectum following either PHA-L or fluorescent dye injections. While the regional distribution of tecto-Ipc neurons was not examined, the morphology indicated that the cells had a single radially oriented dendritic process. Therefore, the apical dendrites are more restricted than those of tecto-ION cells. Moreover, the dendrites were spiny and arborized within layers 3, 5, and 9 of the ipsilateral optic tectum. The axon of tecto-Ipc cells arise from the apical process as a shepherd's crook and descend into the deep layers of the optic tectum. These results indicate that 1) tecto-ION and tecto-Ipc neurons are possibly monosynaptically activated by retinal input, 2) tecto-ION neurons are heterogeneous in morphology, and 3) there is a differential distribution of the tecto-ION neurons throughout the rostro-caudal extent of the optic tectum, suggesting a greater representation of the caudo-ventral portion of the optic tectum within the ION. The discussion primarily concerns the organization of the retino-tecto-ION-retinal circuit in light of the distribution and morphology of tecto-ION neurons within the optic tectum. PMID- 1709957 TI - Pleomorphic adenoma of the lip. AB - A case of pleomorphic adenoma in the upper lip is reported. A 61-year-old male had a tumor at the left side of the upper lip for 8 years. The tumor was surgically excised without recurrence. Histopathologically, the tumor was a typical pleomorphic adenoma with keratinization and glandular differentiation of tumor cells and fibroblastic, mucinous, hyalinized, and cartilagenous stroma. PMID- 1709958 TI - Cognitive function after spinal or general anesthesia for transurethral prostatectomy in elderly men. AB - Cognitive functions in 53 elderly men who underwent a transurethral prostatectomy were assessed pre-operatively and 4 days and 3 months post-operatively. Thirteen patients had a preference for one particular type of anesthesia, and the remaining 40 were randomly allocated to receive either spinal or general anesthesia. Cognitive function was not different between the groups receiving different types of anesthesia at either time point and did not decrease post operatively. No pre- or perioperative variable could distinguish the subgroup of patients who had a post-operative decrease of 2 points or more on the Mini-Mental State Examination. No difference in post-operative performance was found in the patient groups with pre-operative Mini-Mental State Examination scores above or under their age-specific norm. It is concluded that neither hospitalization nor the two forms of anesthesia investigated cause a decrease in cognitive function in elderly men. PMID- 1709959 TI - Co-existence of tyrosine hydroxylase and dopamine beta-hydroxylase immunoreactivity in glomus cells of the cat carotid body. AB - Catecholamines are thought to play an important role in sensory transduction in the arterial chemoreceptors of the mammalian carotid body, and classical cytochemical techniques have demonstrated their presence in the type I (glomus) cells of this organ. However, it remains controversial whether dopamine (DA) and norepinephrine (NE) occur in the same or in different subtypes of glomus cells. In the present study, we have addressed this issue using immunocytochemistry to compare the localization of tyrosine hydroxylase (TH) and dopamine beta hydroxylase (D beta H) in the cat carotid body. Both pre- and post-embedding double-labelling immunohistochemical techniques were employed. TH and D beta H were found to co-exist in over 90% of the glomus cells, and they were co localized at equivalent levels in almost 80% of the cells; less than 5% contained only TH. The results suggest that DA and NE are synthesized and stored in a common cell population in the cat carotid body. PMID- 1709960 TI - Analysis of interstrain variation in cytomegalovirus glycoprotein B sequences encoding neutralization-related epitopes. AB - Nucleotide sequences of a part of the envelope glycoprotein B (gB) gene of human cytomegalovirus (CMV), encoding epitopes recognized by virus-neutralizing monoclonal antibodies, were determined for 12 distinct clinical strains of CMV after amplification of suitable templates using the polymerase chain reaction. Sequence analysis of this region (codons 384-717) revealed that the clinical strains and previously sequenced laboratory strains Towne and AD169 belong to one of four variant groups, each with a characteristic nucleotide and peptide sequence. Peptide homology was greater than 99% for strains within a group, and varied from 91% to 98% for strains in different groups. Variation was most frequent between codons 448 and 480. The gB group of a CMV strain could be determined by restriction analysis of a small target sequence amplified from viral genomic DNA, and an additional 28 clinical strains were grouped in this manner. The existence of a limited number of variants of gB among clinical strains facilitates analysis of biologic function and cross-reactivity of immune responses. PMID- 1709961 TI - Role of normal serum in the binding of lipopolysaccharide to IgG fractions from rabbit antisera to Escherichia coli J5 and other gram-negative bacteria. AB - Because lipopolysaccharide (LPS) bound to lipoprotein is less active than unbound LPS in multiple assay systems, the binding of radiolabeled LPS to lipoproteins in sera prepared from normal rabbits and rabbits made hyperimmune to Escherichia coli J5 were compared. LPS-lipoprotein binding in hyperimmune sera to E. coli J5 was not greater than that in normal serum as assessed by ultracentrifugation, but more LPS was precipitated from hyperimmune antisera than normal sera under conditions designed to precipitate LPS-lipoprotein complexes with calcium and dextran. Radiolabeled LPS was precipitated by delipidated antisera and fractions of IgG purified by anion exchange chromatography, but the precipitation was dependent on the presence of normal serum in the reaction mixture. These data suggest that a fluid-phase RIA done in the presence of normal serum may facilitate the detection of IgG in antisera raised to E. coli J5 that binds to heterologous smooth LPS. PMID- 1709962 TI - Influence of surrounding media on preservation of cell wall ultrastructure of Candida albicans revealed by low temperature scanning electron microscopy. AB - Influence of surrounding media on the surface structures of the cell wall of Candida albicans was discussed with respect to the preservation of ultrastructure during the specimen preparation for scanning electron microscopy. The fibrillar structure of the cell surface was distinctly identified by the rapid-freezing technique. It was difficult, however, to observe this structure by the conventional specimen preparation technique. The reason for the difference between these two preparation techniques was studied using a low temperature SEM. Through investigating the influence of each step of the conventional technique on the fibrillar structure, it was found that the fibrils were drastically deformed and disappeared during the dehydration step in ethanol above 80% in concentration. In order to study which physicochemical properties participated in this disappearance phenomenon, yeast cells were treated with various media: solutions in different pH ranges and at different salt concentrations, ionic solutions, surfactants, formamide, dimethyl sulfoxide, acetone and Fehling's solution. As a result, the fibrillar structure was found well preserved when the medium had an affinity for the constituent molecules of the fibrils. When without affinity, the fibrils suffered a remarkable deformation. The mechanism of this deformation is discussed in terms of molecular interaction of solute and solvent. PMID- 1709963 TI - The tannin-osmium conductive staining after dehydration: an attempt to observe the chromosome structure by SEM without metal coating. AB - The tannin-osmium conductive staining method was modified in order to observe chromosomal structures without metal coating. The treatment with tannic acid and OsO4, which has been used in aqueous solution in the original method, was performed in acetone solution after dehydration in the present study. The usefulness of this method is discussed showing ultrastructural images of chromosomes of Drosophila and Tradescantia, which can be achieved by the method. PMID- 1709964 TI - Focal axonal injury: the early axonal response to stretch. AB - The development of a model for axonal injury in the optic nerve of the guinea pig has allowed analysis of early morphological changes within damaged axons. We provide evidence that the initial site of damage after stretch is the nodes of Ranvier, some of which develop 'nodal blebs'. The development of nodel blebs is correlated with the loss of subaxolemmal density, disruption of the neurofilament cytoskeleton and aggregation of membranous profiles of smooth endoplasmic reticulum. Nodal blebs are numerous 15 min after injury but less so at later survivals. The glial-axonal junction is intact at early survivals in damaged nodes. Marked accumulation of membranous organelles occurs in the paranodal and internodal regions adjacent to damaged nodes between two and six hours and is correlated with disruption of the myelin sheath. Axotomy and the formation of degeneration bulbs occurs between 24 and 72 h. The area of axonal injury is invaded by phagocytic cells by 72 h and large numbers of myelin figures occur within the neuropil until 14 days. The results are compared with those of other studies of diffuse axonal injury and other neuropathies. The time course of axonal changes is more rapid than during Wallerian degeneration. Our data from longer surviving animals is exactly comparable with published data. We are confident that the principal site of axonal injury is the node of Ranvier. We suggest that damage at the node results in disruption of axonal transport, which in turn leads to a cascade of events, culminating in axotomy between 24 and 72 h after the initial insult. PMID- 1709965 TI - Transmembrane cytoskeletal modulation in preterminal growing axons. II. Limax flavus agglutinin-induced receptor redistribution, capping and internalization in varicosities of growing axons. AB - Growing retinal ganglion cell axons of the goldfish exhibit varicosities of varying sizes and smaller non-protruding phase-dense inclusions that are mobile and mediate rapid bulk redistribution of axoplasm. In fixed axons, Limax flavus agglutinin, a lectin specific for sialic acid which has been shown to inhibit organelle transport in these axons, preferentially labels surface membrane associated with varicosities and inclusions in preterminal axons. In viable axons, Limax flavus agglutinin causes: (1) agglutination of closely apposed axons, (2) redistribution of lectin-binding sites on varicosities to surfaces of interaxonal contact with other varicosities, forming 'fused' multivaricosity complexes, and (3) formation of vacuoles in many single varicosities and some multivaricosity complexes. Vacuoles contain Limax flavus agglutinin binding sites distributed circumferentially. On the basis of immunocytochemistry, actin, myosin, calmodulin and alpha-spectrin are co-localized with redistributed Limax flavus agglutinin binding sites. The agglutination, redistribution of lectin binding sites and changes in the cytoskeleton can be reversed by treatment with sialic acid. The lectin-induced vacuole formation and internalization of Limax flavus agglutinin receptors can also be blocked either by sodium azide in a glucose-free medium, or by pretreatment with cytochalasin D and indicate an energy and a cytoskeletal dependence. The Limax flavus agglutinin-induced structural rearrangements are not altered after limited digestion with pronase. Western blots after ultramicroelectrophoresis of retinal ganglion cell axons subjected to limited digestion reveal Limax flavus agglutinin labelling of bands with apparent Mr of 64 and 70 KDa. In undigested axons, some 70 KDa protein remains unextracted with Triton X-100 lysis of axonal fields, and more remains unextracted when axonal fields are pretreated with Limax flavus agglutinin before Triton lysis, suggesting increased association with the cytoskeleton in response to lectin binding. The results indicate that cross-linking of one or more sialoglycoconjugates on the surface of varicosities of preterminal growing retinal ganglion cell axons causes a constellation of transmembrane-mediated cytoskeletal and membrane changes that are akin to those described for capping in motile cells. PMID- 1709966 TI - The role of interferon and tumor necrosis factor in the pathogenesis of AIDS. AB - Cytokines including interferon (IFN) and tumor necrosis factor (TNF) are potent modulators of immune processes. They are synthesized in response to microbial infections and inflammation. TNF and IFN interact with other cytokines to elicit differentiation and cellular responses of specific target cells. In view of their multiple biological effects, we have postulated that dysregulation of IFN and TNF may contribute to the pathogenesis of HIV infection. Here, we review data showing that the expression of IFN-alpha receptors is down-regulated in patients with AIDS. As a consequence, HIV-infected cultured cells and cells from AIDS patients show hyporesponsiveness to IFN action. This could contribute to mechanisms by which HIV evades the antiviral activity of IFN-alpha in HIV-infected cells and raise the question of the usefulness of IFN-alpha in the treatment of end-stage AIDS. TNF is a major mediator of inflammation and sepsis and also is capable of inducing the replication of HIV. TNF synthesis and its receptor expression are upregulated by the acid-labile IFN-alpha subtype present in the sera of HIV infected individuals. In addition, the acid-labile IFN present in AIDS sera may contribute to the pathophysiological changes in sepsis by rendering the cells from AIDS patients hypersensitive to endotoxin stimulation resulting in further synthesis of TNF. Thus aberrant regulation of these cytokines and their cognate receptors are likely contributing factors to the pathogenesis of AIDS. PMID- 1709967 TI - Developmental psychology casebook. Nonverbal learning disability. PMID- 1709968 TI - [Immunohistochemical investigation on expression of cytokeratins in normal epithelium, precancerous lesions and carcinomas of the hypopharynx]. AB - In order to obtain a more objective method to evaluate epithelial disorders and carcinomas of the hypopharynx, the expression pattern of cytokeratins (CKs) was investigated by ABC technique using several kinds of monoclonal antibodies that react monospecifically with each subclass of CKs. In normal epithelia, CK-19 was strongly positive in the basal layer but apparently reduced in suprabasal layers and completely negative in superficial layers, while CK-13 showed a striking contrast to CK-19, being expressed within the whole thickness of epithelia except only in the basal layer. These 2 subclasses were also observed in "abnormal" epithelia, and characteristic changes of their combination were demonstrated in proportion to the histological gradings. In invasive carcinomas, CK-19 was strongly positive in all carcinoma cells of poorly differentiated carcinomas. It was sporadically positive in moderately differentiated carcinomas, the more differentiated the more sporadic. It was completely negative in well differentiated carcinomas. CK-13, on the other hand, was negative in poorly differentiated carcinomas but positive in keratinized cells of moderately or well differentiated carcinomas. Strong expression of CK-1 was observed only in well keratinized cells of hyperkeratotic epithelia and well differentiated carcinomas. These characteristic findings are consistently observed in all samples and, then, may be useful in evaluating epithelial disorders and carcinomas of the hypopharynx, when used in conjunction with standard histological techniques. It seems most likely that these results play a part in investigating normal and abnormal processes of cell differentiation. PMID- 1709969 TI - Lectin histochemistry of cystic jaw lesions: an aid for differential diagnosis between cystic ameloblastoma and odontogenic cysts. AB - The binding sites for Ulex europaeus agglutinin I (UEA-I), Bandeirea simplicifolia agglutinin I (BSA-I), and peanut agglutinin (PNA) were comparatively examined in the surgical materials from 41 cases of cystic and solid ameloblastomas and 42 cases of non-neoplastic odontogenic cysts including dentigerous cyst, odontogenic keratocyst, and radicular cyst. In non-neoplastic cysts, most of epithelial lining layers gave positive binding with UEA-I and BSA I. However, no positive reactions were obtained for these two lectins in the epithelial components of ameloblastoma, except for limited UEA-I binding to markedly keratinized tumor cells in four cases. PNA binding was irregular and did not make any clear distinction between ameloblastomas and cysts. The results suggest that the lectin staining for UEA-I and BSA-I is a useful histologic aid for differential diagnosis between cystic ameloblastoma and non-neoplastic jaw cysts. PMID- 1709970 TI - Expression of mucin type carbohydrates may supplement histologic diagnosis in oral premalignant lesions. AB - Recent studies have shown that changes within membrane bound carbohydrates may be essential for cellular differentiation and malignant transformation. We have therefore, by means of immunohistochemistry, studied the expression of T/Tn related (Thomsen-Friedenrich) carbohydrates in 13 oral lesions with squamous cell dysplasia. The epithelial grade of dysplasia was graded as mild, moderate or severe. The following carbohydrate structures were studied: Tn, T, mucintype 3 chain H, and the sialylated derivates, sialosyl-Tn and sialosyl-T. In general, short structures were detected on the basal cells and longer structures on the more mature spinous cells. In many cases, this sequential expression was more disturbed with increasing grade of epithelial dysplasia. However, our results also showed that some lesions with the same grade of epithelial dysplasia showed different carbohydrate expression. These findings indicate that expression of carbohydrates may supplement histologic diagnosis in the evaluation of the prognosis of premalignant lesions. PMID- 1709971 TI - Mucoepidermoid tumors of salivary glands: histoprognostic value of NORs stained with AgNOR technique. AB - A total of 31 patients surgically treated for salivary mucoepidermoid tumor had a comprehensive follow-up. Two groups were classified as: 16 with favorable outcome (no recurrence or late recurrences at 5 yr postoperatively and still alive; 15 with poor outcome (local recurrences before 5 yr, metastases, lethal evolution in 11 cases). We evaluated nucleolar organizer in every case, using AgNOR count. The AgNOR count seemed better than histologic criteria for establishing the prognosis of mucoepidermoid tumors. It correlated significantly with the clinical course: a high count was principally found in lethal forms, low counts (less than 1.8) were always detected in patients with a good outcome. PMID- 1709972 TI - Properties and Modulation of Ca channels in adult human atrial cells. AB - Ca-channel currents have been investigated in single cells isolated from adult human atrium using the whole-cell patch clamp technique. Ca-channel currents are activated at voltage positive to -40 mV, peak between -10 and 0 mV and inactivate with a slow decay when Ba2+ ions (5 mM) are used as charges carrier. These properties correspond to those of the high voltage activated, DHP-sensitive, (L type) Ca channel. No low voltage activated (T-type) currents have been evidenced. The present work also provides the first report about the modulation of Ca channels in adult human atrial cells by beta-adrenergic agonists and dihydropyridines (agonists and antagonists). Electrophysiological and pharmacological properties of these Ca channels are qualitatively similar to those of the L-type Ca currents recorded from cardiac animal cells. However, at a physiological calcium concentration (2 mM), basal Ca currents are often very small or even absent but are revealed following the addition of the dihydropyridine (DHP) agonist Bay K 8644. Whether the decrease of the basal Ca current amplitude may be related to the chronic pretreatment of the patients by Ca channel blockers or to the pathology is discussed. PMID- 1709974 TI - Ion channel redox model. AB - Na(+)- and Ca2(+)-channel blockers behave as electron donors in reactions with excited dye radicals, while agonists of these channels behave as electron acceptors in the same reactions. The opposite redox characteristics of channel blockers and agonists may reflect their opposite action on the channels. The observed regularities of channel-modulator reactions with free radicals, as well as, the ability of many proteins to influence long-range electron transfer, are the basis for a model of channel function and regulation by various agents in which labile electrons in the channel-forming protein acts as electric field sensors. PMID- 1709973 TI - Effects of 8-bromo-cyclic GMP on contraction and on inotropic response of ferret cardiac muscle. AB - The effects of guanosine 3':5'-cyclic monophosphate (cGMP) on cardiac contraction are not established. Using isolated electrically-stimulated ferret papillary muscle at 29 degrees C, 2.0 mM calcium we investigated the effects of 8-Bromo cGMP on (a) basal contraction for comparison with the effects of reduction in extracellular calcium or of reduction in resting muscle length; (b) contraction of preparations stimulated by isoprenaline, the dihydropyridine calcium agonist Bay K8644 or post-extrasystolic potentiation. 8-Bromo-cGMP (0.1 mM) induced a small significant reduction in isometric twitch tension (TT) (7%), isotonic shortening (PS) (6%) and in twitch duration, but had no effect on maximum unloaded shortening velocity (Vmax) or rate of tension development (+dT/dt). Reduction in muscle length induced a similar immediate effect on contraction. Reduction of extracellular calcium (2.0 mM to 1.25 mM) reduced TT by 24% and PS by 14% as well as Vmax (19%) and +dT/dt (29%), but did not alter twitch duration. Bay K8644 (0.01 to 10 microM) produced increases in TT, +dT/dt, PS and twitch duration each of which was significantly reduced in the presence of 8-Bromo-cGMP (0.1 mM). 8-Bromo-cGMP had no effect on the responses to isoprenaline 1 nM to 100 microM--which increased TT, +dT/dt and PS but markedly reduced twitch duration- nor on post-extrasystolic potentiation which increased TT and +dT/dt but slightly reduced twitch duration. These results show that 8-Bromo-cGMP induces changes similar to the immediate effects of reduction in resting muscle length, and reduces the positive inotropic effects of Bay K8644 but not those of isoprenaline or post-extrasystolic potentiation. PMID- 1709975 TI - [The efficacy of immunoenzyme assay in determining IgG in blood]. AB - The efficacies of two methods for measuring human blood serum IgG, heterogenic enzyme immunoassay (EIA) and radial immunodiffusion in gel (RIG), are compared. The accuracy of both the methods was verified by the data of IgG spectrophotometry; IgG were isolated by ion exchange chromatography from human blood serum. The results of all the three methods were in high correlation: correlation coefficient of spectrophotometry and EIA data was 0.992, p less than 0.01; that of spectrophotometry and RIG data 0.975, p less than 0.01; that of RIG and EIA 0.888, p less than 0.01. Since EIA has some advantages over RIG (it is more rapid, sensitive, accurate, and the investigation may be automated), it is recommended for measuring human blood serum IgG in mass screenings. PMID- 1709976 TI - [Supplementary fluorochrome staining of lymphocytes with rhodamine in work with monoclonal antibodies]. PMID- 1709977 TI - [A method of studying the absorbent phase of phagocytosis]. AB - A method has been developed for examining phagocytosis, making use of the Soviet latex RFD-48. Optimal conditions of the procedure were specified, and results of measurements in normal subjects and patients and laboratory animals presented. PMID- 1709978 TI - [The effect of hemodialysis on neutrophil enzyme activity in patients with uremia]. AB - Basing on measurements of the activities of 7 lysosomal enzymes, the authors have assessed the enzymic activities of neutrophils in 22 patients with protracted renal insufficiency treated by hemodialysis. Induction of some examined enzymes was observed, which remained virtually unchanged after hemoperfusion. PMID- 1709979 TI - [Determination of urinary protein using the biuret method]. AB - Interlaboratory inspection of the quality of laboratory investigations in the Saratov region, carried out in 1987-1988, has revealed a 9 to 50 percent share of unsatisfactory results of measurements of urinary protein by the Roberts Stolnikov method. This fault was not mended despite the measures taken and the errors detected in the activities of clinical diagnostic laboratories. Analysis of the relevant literature has led the authors to a conclusion that the biuret method, the most specific and sensitive of all the universal methods, should be given preference to. The final stage of the investigation is carried out with the use of a KFK-2M photoelectrocolorimeter. A standard curve based on albumin calibration solution was plotted to estimate the results. At present 15 clinical diagnostic laboratories of this region are using the biuret method. Their results have improved, as evidenced by interlaboratory quality control: the share of satisfactory results of urinary protein measurements makes up 83 to 100 percent. PMID- 1709980 TI - [Flow-type impulse cytofluorometry of human spermatozoids]. AB - The steps of human spermatozoa treatment for flow cytofluorometric analysis of their DNA content are described in detail. The authors demonstrate the possibility of simultaneous measurement of haploid, pathologically altered, and immature spermatozoa in the ejaculate and estimation of their ratio. The diagnostic value of the method is assessed. PMID- 1709981 TI - [Preparation of tumor cells from human lung cancer tissue for the purpose of cloning]. AB - Various approaches to isolation of tumor cells are analyzed on the basis of examination of 46 human pulmonary tumors of different histologic types. Mechanical treatment alone resulted in the death of the majority of tumor cells. Short trypsin treatment (for 10-15 min in several stages) proved to be the most suitable for disaggregation of small-cell carcinoma, adenocarcinoma, and carcinoid samples. A specific approach has been developed for isolation of cells from squamous-cell carcinoma, consisting in tumor treatment with 0.25 percent trypsin at 4 degrees C for 16-18 h, followed by DNAase, collagenase, and trypsin treatment, and then again trypsin treatment at 37 degrees C for 10-15 min. The suggested approaches permit a harvest of at least 6 x 10(6) tumor cells from 1 g of tissue, with more than 70 percent of these cells viable. PMID- 1709982 TI - [Evaluation of the functional activity of alveolar macrophages based on the results of a study of blood cells]. AB - NBT test basal values and those after cell stimulation with killed BCG culture were measured by spectrophotometry in alveolar macrophages isolated from bronchoalveolar lavage fluid, in peripheral blood neutrophils and monocytes of 49 patients with newly detected pulmonary tuberculosis. Stimulation coefficient was estimated as the ratio of induced to spontaneous NBT-test values. The findings evidence that spontaneous NBT-test values are regularly growing in all three cell types, and the stimulation coefficients lowers as the process grows in severity at the expense of pulmonary tissue disintegration and bronchial obstruction; these parameters adequately reflect the status of the body specific reactivity. Significant correlations were revealed between spontaneous NBT-test parameters in all 3 cell types and their stimulation coefficients. This permits a conclusion that spontaneous and induced NBT-test of the peripheral blood cells may help assess alveolar macrophage function. PMID- 1709983 TI - [Cytologic criteria for the formation of a risk group in Barrett esophagus]. AB - Oncologic screenings of the populations in the areas with increased incidence of esophageal cancer have revealed Barrett's ulcer in 1 percent of the examinees. Endoscopic and cytologic characteristics of this condition are presented. Precancer changes--severe dysplasia--are most frequent in male Kazakhs (14.1 percent) aged 50 to 59 (14.7 percent). Subjects with Barrett's ulcer developing severe dysplasia, as evidenced by cytograms, should be included in the group of subjects at risk for carcinoma of the lower third of the esophagus and cardia. PMID- 1709984 TI - [Biometric studies in the cytological diagnosis of dysplasia and cancer of the uterine cervix]. AB - Morphometric parameters were measured and specific light optic features of epitheliocyte nuclei examined in the cytologic preparations from 67 patients with various abnormalities of the cervix uteri multi-layer squamous epithelium by a present-day long-distance microimage analyzer. The most informative biometric parameters were detected, role of DNA cytophotometry defined, and algorithm of differential diagnosis between benign conditions, dysplasia, and carcinoma developed. PMID- 1709985 TI - [New methods and equipment for studying the hemostatic system (review of the literature)]. AB - The author analyzes the principles and reviews the ranges of application of conductometric, filtration rapid method for studies of formed element aggregation in stabilized blood, hemostographic, photodynamic method for blood clotting registration, superfusion method for assessment of hemocoagulation tissue factor release reaction. He comes to a conclusion that the diagnosis may become objective, rapid, reliable, and accurate if biophysical characteristics of the hemostasis are investigated, their informative value assessed, and measuring equipment for hematologic laboratories designed and commercially produced. PMID- 1709986 TI - [The use of lyophilized plasma as a reference sample for determining factor VIII]. AB - The activity of lyophilized Soviet reference plasma Factor VIII was measured by a single-stage Papayan and Khrolova's method. This factor is referent in respect of the prothrombin complex factors, stored for 18-43.5 months. Factor VIII activity was found to be within 55-135 percent. Differences in the values of antihemophilic globulin activities obtained in parallel studies in different days may be explained by the fact that the native control was not standard. The results are promising as regards the possibility of using lyophilized donor reference plasma as a reference reagent for estimation of Factor VIII activity. PMID- 1709987 TI - [Criteria for the evaluation of the biochemical composition of bile]. AB - The authors suggest additional criteria evidencing inflammations in the gallbladder and colloid stability of the bile: bile acid absorption and cholesterol sedimentation coefficients. Derivation of these coefficients is based on biochemical examination of the bile with measurements of bile acid, cholesterol, and bilirubin concentrations in both portions. The results of examinations of 99 subjects evidence the diagnostic value and specificity of the characteristics, and availability of this method for clinical practice. PMID- 1709988 TI - [Determination of sialic acids in the urine and feces of children]. AB - Hess's method for measurement of the blood serum sialic acids has been adapted to measurements thereof in the urine and feces. The modification is based on ethanol sedimentation of urinary and fecal proteins. Sialic acids are measured in the protein precipitate by the unified Hess's procedure. The examinations have revealed differences in the levels and excretion of sialic acids with feces in healthy infants and those suffering from acute intestinal infections. PMID- 1709989 TI - [Determination of the monoamine oxidase and the diamine oxidase activity of a single blood sample]. AB - The known methods for simultaneous measurements of blood histamine and serotonin have prompted the authors to search for approaches to simultaneous measurements of monoamine oxidase and diamine oxidase, the enzymes contributing to serotonin and histamine inactivation. The authors suggest a method for simultaneous measurements of the blood serum monoamine oxidase and diamine oxidase activities that permits cut down the time of the blood and substrate incubation to 10 min, essentially shortens the procedure and simplifies it. The method is recommended for research and practice. PMID- 1709990 TI - [Chemiluminescence of the blood components in normal subjects]. AB - Presents data on chemiluminescence of blood plasma, red cells, membranes, hemolysate in normal subjects, initiated by hydrogen peroxide, bivalent iron, UV irradiation. Emphasizes differences in chemiluminescence kinetics of various blood components after exposure to different initiators of luminescence. PMID- 1709991 TI - [A method of detecting phages in lysogenic strains of Vibrio cholerae]. PMID- 1709992 TI - [The use of stains for the detection of the pathogenetic factors of Escherichia and Salmonella]. AB - The authors have modified the technique for using three stains to detect enterobacterial pathogenicity factors. The methods have been tried with 306 Escherichia and Salmonella strains. The most accurate results were obtained when azure eosin was layered onto culture growth surface and when Congo red was added into solid medium. Crystal violet layering yielded less specific results. The techniques listed above helped detect pathogenicity factors of up to 25-30 percent of Escherichia and up to 17 percent of Salmonella strains. The methods are recommended to be used in complex with other tests in practical studies. PMID- 1709994 TI - [The interpretation of laboratory parameters in evaluating the immune status of man]. AB - At least 3 physiologic parameters appear to be useful for the assessment of the immunity status of a patient. These parameters are as follows: immunocyte ability to activation, proliferation, and differentiation; these characteristics may be directly examined with the use of novel products of immunobiotechnology. The suggested approach essentially simplifies interpretation of the findings obtained in studies of human immunity system. Besides lymphocytes, special attention is paid to immunoregulatory monocytes, macrophages, and granulocytes. Since these cells are known to secrete quite a number of mediators, an imbalance of their production and reception may contribute to the pathogenesis of many a disease. PMID- 1709993 TI - [Coagulase-negative staphylococci in pyoseptic pathology and methods for their identification]. AB - Analysis of clinical material from cardiosurgical and cardiotherapeutic patients and that from neonates with pyoseptic infections, hospitalized in Moscow and Gorky, has demonstrated the etiologic significance of coagulase-negative Staphylococci (CNS). Since many strains did not belong to S. aureus, S. epidermidis, or S. saprophyticus, they were species-identified in accordance with the classification by W. Kloos and K. Schleifer (1975). A variety of CNS species was observed, and their role in the development of pyoseptic infections shown, the contribution of S. epidermidis and S. haemolyticus being the greatest here. Besides S. aureus, S. xylosus was often isolated from purulent foci. A certain specificity in CNS spectrum, related to the hospital profile, was revealed. A brief modified scheme of CNS classification was developed, based on analysis of about 700 strains, in accordance with the classification of W. Kloos and K. Schleifer, that included the tests available for practical laboratories. PMID- 1709995 TI - [The use of polyvinylpyrrolidone as a stabilizer in the lyophilization of Brucella]. AB - Stabilizing effects of 5 percent aqueous polyvinylpyrrolidone (PVP) solution and sucrose-agar-gelatin medium (SAG) were examined in lyophilization of 4 Brucella types. The number of viable cells immediately after lyophilization in PVP has made up 48.1 percent on an average; this value was different with different Brucella species: 70.9 percent with B. abortus, 50.4 percent with B. melitensis, 41.2 percent with B. rangiferi, 30.2 percent with B. suis. Only 27.5 percent of cells were viable after Brucella lyophilization in SAG medium. Gradual death of lyophilized cells was observed in the course of strain storage at 5 degrees C. After two-year storage the number of viable cells after lyophilization in PVP was 41.8, after three-year storage 35.1 percent. The number of viable cells after SAG lyophilization was still lower. Biological characteristics of the strains lyophilized in both media corresponded to the initial ones over the three-year follow-up. PMID- 1709996 TI - [The activity of meningococcal agglutinating sera in coagglutination and precipitation reactions]. PMID- 1709997 TI - [The effect of human plasma and cattle blood serum on the culture properties of liquid nutrient media]. AB - Cultivation characteristics of Shkolnikova's medium and medium No. 23 with human plasma or cattle blood serum added were studied by the radioactive indicator method. Cattle blood serum was preinactivated at 60 and 70 degrees C or intact. Plasma and serum preheated at 70 degrees C enhanced the growth of Mycobacterium tuberculosis. Intact serum or serum heated at lower temperature inhibited cell mitosis. PMID- 1709998 TI - [An auxiliary electrode for gastric pH probes]. AB - The author has examined the possibility of employing pH tubes with defective auxiliary electrodes in complex with a commercial auxiliary EBL-1 M3 silver chloride electrode. Examinations of gastroduodenal patients permit recommending this commercial electrode as an available and accurate auxiliary electrode for intragastric pH-metry, for measurements with promising pH microtubes, 2 mm in diameter as well. PMID- 1709999 TI - [Apparatus for electrophoresis on cellulose acetate films]. AB - Type EFPA-30 apparatus for electrophoresis on cellulose acetate films was developed at the Specialized Design Office for Biophysical Equipment of the Moscow Research and Production Association BIOFIZPRIBOR. Three autonomic separation cameras are united in one block, that permits separation of 12, 24, and 36 samples; the apparatus is supplied with time transducer, acoustic and visual indication. Clinical trials of the apparatus were carried out at the Institute of Biological and Medical Chemistry of the USSR Academy of Medical Sciences, its commercial production is to be started. The apparatus is intended for protein separation and may be used for studies of hemoglobins, serum proteins, lipoproteins, enzymes, and other biologic compounds. Methods for hemoglobin and serum protein separation are presented. PMID- 1710000 TI - [The determination of alpha fetoprotein using radioimmunologic and immunoenzyme methods of analysis and its relation to the level of alanine aminotransferase in children with viral hepatitis]. PMID- 1710001 TI - [Blood serum lipids in patients with eczema]. PMID- 1710002 TI - [Hemostatic indices in stomach and duodenal diseases]. PMID- 1710003 TI - [The rational use in serologic laboratories of surplus blood from donors and pregnant women]. PMID- 1710004 TI - [Vulnerability of the proposed method (apropos of the article by V.L. Morozov and his co-authors)]. PMID- 1710005 TI - Primed neutrophils injure rat lung through a platelet-activating factor-dependent mechanism. AB - Bacterial lipopolysaccharide (LPS) promotes transient lung neutrophil sequestration. These LPS-primed neutrophils, when stimulated by an N-formyl peptide (FNLP), promote lung injury. We hypothesized that LPS-primed, FNLP stimulated neutrophils promote lung injury through a platelet-activating factor (PAF)-dependent mechanism. Rats were pretreated with either saline or WEB2170, a PAF receptor antagonist (10 mg/kg po). One hour after pretreatment, rats were administered intraperitoneal LPS (salmonella typhimurium lipopolysaccharide, 500 micrograms/kg) followed 6 hr later by intravenous FNLP (250 micrograms/kg infused over 30 min). Two hours after the initiation of FNLP infusion, rats were sacrificed and assays were performed to measure: (1) lung neutrophil sequestration with myeloperoxidase (MPO) activity; (2) circulating neutrophil activation with nitroblue tetrazolium (NBT) staining, and (3) lung microvascular leak with 125I-albumin flux. We found that lung myeloperoxidase, circulating neutrophil NBT staining, and lung 125I-albumin flux were increased (P less than 0.05) in saline-pretreated LPS/FNLP rats, relative to control. While lung MPO remained increased (P less than 0.05) in WEB2170-pretreated LPS/FNLP rats, circulating neutrophil NBT and lung 125I-albumin flux were decreased (P less than 0.05) relative to those in saline-pretreated rats. We conclude that PAF mediates LPS/FNLP-induced neutrophil activation and lung injury, but is independent from lung neutrophil sequestration. Thus, lung neutrophil sequestration does not inevitably produce lung injury. Rather, neutrophils can accumulate in the lung without causing lung injury if neutrophil activation can be blocked. PMID- 1710006 TI - Distribution of thymic tissue in the mediastinal adipose tissue. AB - The distribution of thymic tissue in the anterior mediastinal, retrocarinal, and preaortic fat was examined histologically in 27 autopsy subjects. Thymic tissue was found in the anterior mediastinal fat in 12 subjects (44.4%), in the retrocarinal fat in two (7.4%), and in the preaortic fat in none. The finding of ectopic thymic tissue in these areas has not been reported previously, would appear to be surgically inaccessible via a median sternotomy, and may be responsible in part for some of the failures of thymectomy in the treatment of myasthenia gravis. PMID- 1710007 TI - Invited letter concerning: aprotinin. PMID- 1710008 TI - Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement, and renal function after cardiopulmonary bypass. AB - The use of aprotinin to reduce blood loss after cardiopulmonary bypass is under debate. Concern has been raised about the renal effects of aprotinin. We administered a mean aprotinin dose of 4.2 x 10(6) kallikrein-inhibiting units to 13 patients with coronary disease undergoing cardiopulmonary bypass for 74 +/- 5 minutes (mean +/- standard error of the mean); 13 comparable patients having cardiopulmonary bypass served as control subjects, and all were studied postoperatively for 24 hours. Aprotinin reduced postoperative blood loss by 50% (p = 0.0082). Two of the 13 patients who received aprotinin needed one red cell unit each versus a total of 18 units in eight of 13 control patients (p = 0.0096). Blood pressure, hemoglobin value and serum protein concentration were higher after operation in the aprotinin group (p less than 0.05 to p less than 0.01). Platelet counts did not differ, but plasma thromboxane was lower in aprotinin recipients (p less than 0.001). In control patients fibrinogen degradation products (D dimer) doubled, and alpha 2-antiplasmin activity was halved during and after cardiopulmonary bypass (p less than 0.01 to p less than 0.001), whereas aprotinin patients showed no changes. The complement breakdown products C4a, C3a, and C3dg as well as C9 neoantigen increased from prebypass baseline in both groups (p less than 0.001); the increment of C3a and C3dg was greater in the aprotinin than in the control patients (p less than 0.001). Serum electrolytes, osmolality, and creatinine remained normal in both groups of patients. Creatinine clearance was normal or above normal and virtually identical in both groups. Osmolar clearance and fractional sodium excretion were higher in the aprotinin group than in the control group shortly after cardiopulmonary bypass (p less than 0.05 to p less than 0.01); renal function was unremarkable the next morning. No adverse clinical effects attributable to aprotinin were seen. In summary, aprotinin offers advantages for cardiopulmonary bypass. PMID- 1710009 TI - Effect of intraoperative aprotinin administration on postoperative bleeding in patients undergoing cardiopulmonary bypass operation. AB - To study the hemostyptic effect of aprotinin (Trasylol) in patients undergoing extracorporeal circulation for coronary artery bypass operations, we randomized 12 of 24 patients to receive aprotinin in high dosage (about 800 mg) during extracorporeal circulation. From the resulting two groups each, one patient was excluded from the study because of postoperative myocardial infarction (control group) and surgical hemorrhage (aprotinin group) leading to a second operation. Although heparin was used for anticoagulation in all 22 patients, all had a marked increase in plasma levels of thrombin-antithrombin III complexes during extracorporeal circulation, indicating an intravasal activation of coagulation. By monitoring the plasma levels of fibrin degradation products in patients without aprotinin therapy, we recorded a concomitant hyperfibrinolysis significantly less pronounced in patients receiving aprotinin (p less than 0.005). The mean total postoperative blood loss was lower in patients receiving aprotinin (620 ml) than in control patients (1000 ml; p less than 0.03). The results confirm previous reports of a hemostyptic effect of aprotinin in cardiac operations. This effect is probably due to a prevention of hyperfibrinolysis. PMID- 1710010 TI - Alteration of enzymes in ageing human fibroblasts in culture. V. Mechanisms of glutathione peroxidase modification. AB - Ageing of WI-38 fibroblasts in culture was used as a model in order to investigate the evolution and the alteration of the key antioxidant enzyme glutathione peroxidase. The activity of glutathione peroxidase is influenced by the presence of selenium in the culture medium and we have also shown that the specific activity of this enzyme does not decrease during ageing, but rather slightly increases. No alteration could be detected by immunotitration. Also the kinetic parameter Km for tert-butyl hydroperoxide has not changed. However, the heat resistance of the enzyme dramatically decreases with ageing. Dilutions of the enzyme preparations had the same influence on the thermosensitivity of the enzyme. This dilution effect is most probably linked to the dissociation of the enzyme subunits into dimers and monomers. Moreover, the kinetic of thermoinactivation curves are best explained by consecutive reactions of inactivation with an intermediary enzyme form. These observations strongly support the hypothesis that ageing is associated with an increased dissociation constant of the tetrameric glutathione peroxidase leading to an easier dissociation of the enzyme in old cells. PMID- 1710011 TI - [Clinical and pharmacological aspects of pancreatic enzyme substitution therapy]. AB - The adequate therapy of pancreatic enzyme replacement in patients with exocrine pancreatic insufficiency is still a difficult clinical problem especially in patients following pancreatectomys, with chronic alcoholic pancreatitis or cystic fibrosis. The substitution of lipase to eliminate steatorrhoea is the most important aim but due to its acid lability even the most serious problem in pancreatic enzyme replacement therapy. Various different medications are meanwhile available: conventional preparations from porcine pancreatin or fungal enzymes as rizolipase, enteric-coated tablets or even enteric-coated microspheres or adjunctive therapy with H2-receptor antagonists. While dosage requirements vary widely and therefore have to be tried out individually, the choice of the adequate preparation should be influenced by the realization of the physiological and pathophysiological characteristics of the individual patient and the pharmaceutical characteristics of the different supplements. The advantages and disadvantages of the various medications for enzyme replacement therapy in patients with exocrine pancreatic insufficiency are reviewed in this article. PMID- 1710012 TI - Monoaminergic regulation of proopiomelanocortin messenger RNA concentrations in primary cell cultures of rat hypothalamus. AB - The effect of monoaminergic neurotransmitters on a 1.1-kb proopiomelanocortin messenger RNA (POMC mRNA) detected in rat hypothalamic cells maintained in culture has been evaluated. Serotonin caused a 15% increase in POMC mRNA levels, an effect which was blocked by the 5-HT2 receptor antagonist ketanserin. Dopamine markedly decreased POMC mRNA levels in a dose related manner. Haloperidol and the selective D2 antagonist (+)-butaclamol prevented the inhibitory effects of both dopamine and the selective D2 agonist, 2-bromo-alpha-ergocryptine. The selective dopamine D1 receptor agonist, SKF 38393, as well as norepinephrine and acetylcholine did not affect POMC mRNA levels. It is concluded that serotonin exerts a positive control and dopamine a negative control on POMC mRNA concentrations in primary cultures of rat hypothalamic neurons. The negative effect of dopamine appears to be exerted via D2 receptor-mediated mechanism. PMID- 1710013 TI - The chicken GABAA receptor alpha 1 subunit: cDNA sequence and localization of the corresponding mRNA. AB - We report the sequence of a complementary DNA (cDNA) that encodes the chicken GABAA receptor alpha 1 subunit, which is extremely homologous to mammalian alpha 1 subunits. The distribution of alpha 1 subunit transcripts is shown to correlate mainly, but not completely, with the previously-reported pattern of benzodiazepine type I (BZI) binding sites in the avian brain. These results suggest that the alpha 1 subunit may not necessarily be restricted to receptors having BZI pharmacology. PMID- 1710014 TI - Comparison of mutagenicity results for nine compounds evaluated at the hgprt locus in the standard and suspension CHO assays. AB - The Chinese hamster ovary (CHO) assay, which measures newly induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, has been widely used for mutagenesis testing. The insensitivity of the standard assay to some genotoxic agents has been speculated to be due to the relatively small number of cells used in the assay. In the present study, we have compared the standard monolayer assay with a suspension adapted CHO assay that uses cell numbers comparable to that of the L5178Y mouse lymphoma assay. Nine compounds, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), 2-methoxy-6-chloro-9-[3 (ethyl-2-chloroethyl)-aminopropylamino]-acridine 2HCl (ICR 170), methyl acrylate, ethyl acrylate, tetraethylene glycol diacrylate, trimethylolpropane triacrylate, 2-ethylhexyl acrylate and dicyclopentenyloxyethyl methacrylate were evaluated in the monolayer and suspension assays. Both assays gave the same overall qualitative results for the test compounds. There were some quantitative differences in the mutant frequency for the three compounds found to be mutagenic (EMS, MMS and ICR 170). The acrylates (many of which appear to exert their genotoxic effect through a clastogenic mechanism) were negative in both test systems. The use of the suspension assay did not improve the ability of the hgprt locus to detect the genotoxicity of the acrylates. Thus, increasing the number of cells does not improve the ability of the CHO/HGPRT assay to detect compounds that act primarily by a clastogenic mechanism. PMID- 1710015 TI - [Postoperative change of serum protease inhibitor and C-reactive protein level- with special reference to surgical resection of liver cirrhosis]. AB - Changes in the activities of blood protease inhibitors and acute-phase reactive substances during surgical resection of liver cirrhosis were investigated by measuring the pre- and postoperative blood concentrations of alpha 1-antitrypsin (alpha 1AT), alpha 2-macroglobulin (alpha 2MG), pancreatic secretory trypsin inhibitor (PSTI), urinary trypsin inhibitor (UTI) and C-reactive protein (CRP), in patients with liver cirrhosis who underwent hepatectomy (Group A, n = 19), those without liver cirrhosis who underwent hepatectomy (Group B, n = 6) and those without liver cirrhosis who underwent surgeries other than hepatectomy (Group C, n = 5). On examining the preoperative blood levels of protease inhibitors, Group A had an increased level of alpha 2MG and a decreased level of UTI compared to Groups B and C. alpha 1AT and CRP began to increase on the first day following hepatectomy and formed peaks on the third postoperative day. The increases were significantly higher in Group B than Group A (p less than 0.01). To investigate factors causative of these differences, alpha 1AT and CRP on the third postoperative day were compared in relation to the time of operation, amount of intraoperative bleeding, weight of the resected liver and preoperative ICGR15. alpha 1AT and CRP were significantly correlated to only preoperative ICGR15. PSTI was increased postoperatively but showed no difference between Groups A and B. PMID- 1710016 TI - Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942. AB - In a temperature-sensitive, high CO2-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41 degrees C). In contrast, inorganic carbon uptake and ribulose-1.5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb PstI fragments. These 4 fragments overlapped only in a 0.4 kb HindIII PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1.5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis. PMID- 1710017 TI - Isolation of a GC-rich cDNA identifying mRNA present in human epidermis and modulated by calcium and retinoic acid in cultured keratinocytes. Homology with murine loricrin mRNA. AB - Differential screening of a human epidermal cDNA library led to the isolation of cDNA clones homologous to mRNAs specifically expressed in epidermis but weakly or not expressed in the undifferentiated squamous carcinoma cell line TR146. One of these 'differentiation-specific' cDNA clones, A8, hybridized with a 1.7 kb transcript among RNAs isolated from normal human epidermis, but with several transcripts ranging from 1.4 to 2.1 kb when mRNAs were isolated from cultured keratinocytes. We examined the effects of modulators of epidermal differentiation such as calcium and retinoic acid on the production of these transcripts. Their amount was found to increase in the presence of high calcium concentration, but to decrease in the presence of retinoic acid. These results strongly suggest that A8 messages are up-regulated during epidermal differentiation. The sequence of the 1371 bp of A8 cDNA shows a very high GC content. Because of its homology with the murine loricrin mRNA, A8 is likely to correspond either to the human loricrin or to a related protein. PMID- 1710018 TI - Inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate allows estimation of the primers affinity. AB - Template-primer dependent inactivation of human DNA polymerase alpha and Klenow fragment of E. coli DNA polymerase I by adenosine 2',3'-riboepoxide 5' triphosphate was used for quantitative analysis of the Kd values for oligonucleotide primers of different length. The Kd values are smaller by a factor of 2.5 than the Km values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase alpha. The Kd and Km values are nearly the same for Klenow fragment. Such approach to the determination of Km/Kd ratio can likely be used for detailed quantitative analysis of DNA polymerases. PMID- 1710019 TI - [Basic factors in the stability of RNA-duplexes. The contribution of dehydration]. AB - The enthalpy effect of hydrate envelope deformation was first taken into consideration for estimating the influence of aqueous medium on the stability of RNA duplexes. In addition two rarely used parameters (macroscopic value of dielectric water permeability and entropy effect of dehydration) were employed. According to the energy model described the contribution of the free energy of dehydration to the RNA duplexes stability is significant even in comparison with that of the interstrand stacking. PMID- 1710020 TI - [B2 RNA and 7SK RNA, transcripts of RNA-polymerase III, have a cap-like structure at the 5'-end]. AB - We found that hydrolysates of poly(A)RNA from Ehrlich ascites carcinoma cells which were transcribed by RNA polymerase III contained an unusual component designated as X. It was part of B2 RNA representing a transcript of B2 retroposon, typical of rodents. The component X possesses a cap-like structure, xPPP5'G, where x has al non-nucleotide structure. About half of all B2 RNAs contained this group at the 5'-end. Previously, Epstein et al. (Epstein P., Reddy R., Henning D., Busch H. parallel J. Biol. Chem. 1980. V. 255. P. 8901-8906) detected a similar structure at the 5'-end of small nuclear U6RNA. Later, Singh and Reddy (Singh R., Reddy R. parallel Proc. Natl. Acad. Sci. USA. 1989. V. 86. P. 8280-8283) showed methyl to be the blocking group in the component X of U6RNA. Besides B2 RNA, we found 5'-ends containing methyl groups in 7S K-RNA. PMID- 1710021 TI - [Suppression of the human immunodeficiency virus in cultured cells by 5' phosphites of 3'-azido-2',3'-dideoxynucleosides]. AB - 5'-Phosphites (5'-hydrogenphosphonates) of 3'-azido-2'-, 3'-dideoxynucleosides are shown to be effective inhibitors of the human immunodeficiency virus (HIV-1) in MT4 cell culture. 5'-Phosphite of 3'-azido-2', 3'-dideoxythymidine was the most active among these compounds and even a little more active as compared to the well-known anti-AIDS drug 3'-azido-2',3'-dideoxythymidine; at the same time 5'-phosphites of 3'-azido-2',3' -dideoxynucleosides with adenine, guanine and cytosine bases were more active than the corresponding nucleosides. The toxicity of all four phosphites was comparatively low and the equimolar mixture of all four phosphites was 2-3 fold less toxic than each of them separately. Data on the decreased toxicity of the phosphite mixture are explained from the viewpoint of a decreased pool disbalance of natural 2'-deoxynucleoside 5'-triphosphates in cells; a significant pool disbalance is developed in the case of 3'-azido-2',3' dideoxythymidine action. PMID- 1710022 TI - Alternative splicing generates isoforms of the met receptor tyrosine kinase which undergo differential processing. AB - The met proto-oncogene is a member of the family of tyrosine kinase growth factor receptors. We describe the isolation and characterization of a cDNA clone (pOK) for the met receptor from a gastric carcinoma cell line. This clone differs from the published cDNA clone by the absence of 54 bp predicted to encode 18 amino acids in the extracellular domain. The pOK cDNA corresponds to the most abundant met RNA species of 8 kb expressed in human cell lines and tissue, and we show that there are in fact two 8-kb met receptor tyrosine kinase (RTK) isoforms that are generated by alternative splicing. This newly described met isoform when transiently expressed in COS cells encodes a protein of 190 kDa which corresponds in size to the p190 met alpha beta heterodimer expressed in human cell lines. Furthermore, we show that the 190-kDa product of pOK consists of the 140-kDa met beta subunit associated with the 50-kDa met alpha subunit. This finding suggests that both the alpha and beta met chains are encoded by this construct and confirms the hypothesis that a single chain precursor is cleaved to produce both subunits of met. In contrast, the previously characterized met isoform corresponds to a minor met RNA species and encodes a protein of 170 kDa that is not cleaved yet is processed in a manner that allows cell surface expression. Both met RTK isoforms are autophosphorylated in the in vitro kinase assay. These results suggest that different isoforms of the met RTK may have distinct biological activities. PMID- 1710023 TI - The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor. AB - The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development. PMID- 1710024 TI - DNA repair in the c-myc proto-oncogene locus: possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice. AB - This report describes an unexpected difference in the efficiency of removal of UV induced DNA damage in the c-myc locus in splenic B lymphoblasts from two inbred strains of mice. In cells from plasmacytoma-resistant DBA/2N mice, 35% of UV induced damage in the regulatory and 5' flank of c-myc is removed by 12 h. However, in cells from plasmacytoma-susceptible BALB/cAn mice, damage is not removed from this region. In the protein-encoding region and 3' flank of c-myc as well as in two dihydrofolate reductase gene fragments, UV damage is repaired with similar efficiency in B lymphoblasts from both strains of mice. Furthermore, in the protein-encoding portion and 3' flank of c-myc, damage is selectively removed from only the transcribed strand. No repair is detected in the nontranscribed strand. In contrast, DNA repair in the 5' flank of c-myc is not strand specific; in DNA from DBA/2N cells, UV damage is rapidly removed from both the transcribed and nontranscribed strands. In BALB/cAn cells no repair was detected in either strand in the 5'flank, consistent with the results with double-stranded, nick translated probes to this region of c-myc. In addition to the repair studies, we have detected post-UV-damage formation: in most of the genes studied, we find that additional T4 endonuclease-sensitive sites are formed in the DNA 2 h after irradiation. Our findings provide new insights into the details of gene-specific and strand-specific DNA repair and suggest that there may be close links between DNA repair and B-cell neoplastic development. PMID- 1710025 TI - In vitro analysis of the tissue plasminogen activator promoter reveals a GC box binding activity present in murine brain but undetectable in kidney and liver. AB - Tissue plasminogen activator (t-PA) mRNA levels are high in murine brain, lower in kidney, and undetectable in liver. Differences in t-PA mRNA levels are regulated in part at the transcriptional level. Brain, kidney, and liver nuclear extracts direct regulated transcription from the murine t-PA promoter in a manner that reflects the relative levels of t-PA gene expression in these tissues in vivo. Analysis of mutants has defined two GC box motifs as important elements for regulated transcription in vitro. Upon investigation of protein-DNA binding, we detected an activity in brain extracts which was not detected in kidney or liver extracts. An Sp1-like factor also binds to this region in all three tissue types. DNA interference experiments show that the brain-enriched binding activity and the Sp1-like factor contact the same GC-rich sequences. These studies provide additional evidence that brain-enriched DNA-binding activities can interact with sequences also recognized by ubiquitous transcription factors. PMID- 1710026 TI - Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus. AB - Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39 nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition. PMID- 1710027 TI - Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes. AB - Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively. PMID- 1710029 TI - A liver protein fraction regulating hormone-dependent in vitro transcription from the vitellogenin genes induces their expression in Xenopus oocytes. AB - Xenopus laevis oocytes were used to assay for trans-acting factors shown previously to be involved in the liver-specific regulation of the vitellogenin genes in vitro. To this end, crude liver nuclear extracts obtained from adult estrogen-induced Xenopus females were fractionated by heparin-Sepharose chromatography using successive elutions with 0.1, 0.35, 0.6, and 1.0 M KCl. When these four fractions were injected into oocytes, only the 0.6-M KCl protein fraction significantly stimulated mRNA synthesis from the endogenous B class vitellogenin genes. This same fraction induced estrogen-dependent in vitro transcription from the vitellogenin B1 promoter, suggesting that it contains at least a minimal set of basal transcription factors as well as two positive factors essential for vitellogenin in vitro transcription, i.e. the NF-I-like liver factor B and the estrogen receptor (ER). The presence of these two latter factors was determined by footprinting and gel retardation assays, respectively. In contrast, injection of an expression vector carrying the sequence encoding the ER was unable to activate transcription from the oocyte chromosomal vitellogenin genes. This suggests that the ER alone cannot overcome tissue-specific barriers and that one or several additional liver components participate in mediating tissue-specific expression of the vitellogenin genes. In this respect, we present evidence that the oocyte germinal vesicles contain an NF-I-like activity different from that found in hepatocytes of adult frogs. This observation might explain the lack of vitellogenin gene activation in oocytes injected with the ER cDNA only. PMID- 1710030 TI - Assessment of the in situ tyrosine kinase activity of mutant insulin receptors lacking tyrosine autophosphorylation sites 1162 and 1163. AB - In the present studies mutant insulin receptors with regulatory tyrosine residues 1162 and 1163 changed to phenylalanines were tested for tyrosine kinase activity. In agreement with prior studies, this mutant receptor was found to exhibit almost no insulin-stimulated exogenous kinase activity when assayed in vitro. In contrast, this mutant receptor was found in situ to have a significant, albeit reduced, ability to mediate the tyrosine phosphorylation of various endogenous proteins, as assessed by Western blotting with antiphosphotyrosine antibodies. In addition, extracts of insulin-treated cells overexpressing this mutant receptor exhibited increased amounts of tyrosine phosphorylated phosphatidylinositol 3 kinase compared to control cells. Finally, this mutant receptor, like the wild type receptor, was found to mediate an increase in the activity of a membrane associated phosphatidylinositol 4,5-biphosphate kinase. These results indicate that 1) in vitro assessments of the tyrosine kinase activity of mutant insulin receptors may not accurately reflect their in vivo activities; and 2) the ability of the mutant receptor lacking tyrosine autophosphorylation sites 1162 and 1163 to mediate insulin-stimulated tyrosine phosphorylation of various endogenous substrates may account for the reported ability of this receptor to mediate various biological responses. PMID- 1710028 TI - Cloning by differential screening of a Xenopus cDNA that encodes a kinesin related protein. AB - By differential screening of a Xenopus egg cDNA library, we selected nine clones (Eg1 to Eg9) corresponding to mRNAs which are deadenylated and released from polysomes soon after fertilization. The sequence of one of these clones (Eg5) revealed that the corresponding protein has the characteristic features of a kinesin-related protein. More specifically, Eg5 was found to be nearly 30% identical to a kinesin-related protein encoded by bimc, a gene involved in nuclear division in Aspergillus nidulans. PMID- 1710031 TI - Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin alpha-subunit genes. AB - Primate and equine species are thought to be unique among mammals in synthesizing placental gonadotropin glycoprotein hormones. Human chorionic gonadotropin (CG) and equine pregnant mare's serum gonadotropin (PMSG) are produced in placenta by the specific activation of a glycoprotein hormone alpha-subunit gene and a corresponding beta-subunit gene. The evolutionary mechanisms for the apparently independent acquisition of tissue specificity were investigated by cloning the 5' flanking region of the equine alpha-subunit gene and comparing the DNA elements and trans-acting factors involved in placental expression. We find that though the equine gene is expressed and induced by cAMP, it does not contain the elements known to confer tissue-specific expression to the human gene, the cAMP response element (CRE) and the trophoblast-specific element (TSE), nor does it bind to the trans-acting factors CREB and TSEB. Instead, an additional factor (alpha-ACT) is found which binds to the equine and human, but not the murine, alpha-subunit genes in a region between the positions of the CRE and TSE and confers cAMP responsiveness. PMID- 1710032 TI - Effects of neonatal hypothyroidism on rat brain gene expression. AB - To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development. PMID- 1710033 TI - Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer. AB - Cell-specific expression of the insulin gene is dependent on a conserved 8 basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells. PMID- 1710034 TI - Molecular cloning of an Echinococcus granulosus protein expressing an immunogenic epitope of antigen 5. AB - cDNA was synthesized from RNA extracted from Echinococcus granulosus protoscoleces and cloned in the lambda gt11 expression vector. A pool of 5 E. granulosus patient sera was used to screen the library and allowed the selection of 13 clones. Ten of these were shown to be identical, among which clone 6 (Eg6) was chosen for further analysis. The nucleotide sequence (456-bp) presented an entire open reading frame coding for 152 amino acids. The fusion protein (FP6) was recognized by a mouse monoclonal antibody (EG 02 154/12) specific for E. granulosus antigen 5. Moreover, the presence of antibodies to FP6 seemed to be correlated to the ability of sera from hydatidosis patients to immunoprecipitate antigen 5. These results indicate that the cloned protein could be used as a standardized antigen for the diagnosis of hydatidosis. PMID- 1710035 TI - Distinct patterns of tyrosine phosphorylation during the life cycle of Trypanosoma brucei. AB - Regulation of tyrosine phosphorylation is a critical element in controlling growth and differentiation in higher eukaryotes. We have determined that the protozoan Trypanosoma brucei, which diverged early in the eukaryotic lineage, possesses multiple proteins which react with a specific anti-phosphotyrosine antiserum. Anti-phosphotyrosine immunoprecipitates of [32P]orthophosphate-labeled cells were shown to contain phosphotyrosine by two-dimensional electrophoresis. Western analysis of cells from different stages of the life cycle demonstrates the appearance of tyrosine-phosphorylated proteins at 40-42 kDa during the transition from slender to stumpy blood-forms. Growth of procyclic form cells in orthovanadate resulted in increased levels of specific tyrosine-phosphorylated proteins. The demonstration of phosphotyrosine-containing proteins in T. brucei and their differential regulation during the life cycle suggests that tyrosine kinases and phosphatases may play an important role in the biology of primitive protozoa. PMID- 1710036 TI - An antigenically distinct lipophosphoglycan on amastigotes of Leishmania major. AB - We show that lipophosphoglycan (LPG) on the surface of amastigotes of Leishmania major is antigenically and biochemically distinct from promastigote LPG. A rabbit antiserum raised against the amastigote integral membrane fraction detected LPG spanning the region of Mr 55,000-100,000 on Western blots of the amastigote integral membrane fraction, but did not recognize the promastigote integral membrane fraction. WIC 79.3, a monoclonal antibody which recognizes L. major metacyclic promastigote LPG, did not recognize the amastigote integral membrane fraction on Western blots. The antigen recognized by this rabbit antiserum was shown to be LPG by its migration pattern on SDS-PAGE, the presence of terminal galactose residues, recognition by a monoclonal antibody to LPG, WIC 108.3, the biosynthetic incorporation of label from [3H]glucose and [32P]phosphate, a hydrophobic chromatography elution profile similar to promastigote LPG, and the presence of a lipid anchor sensitive to phosphatidylinositol-specific phospholipase C. The temporal regulation of LPG expression during parasite differentiation was studied in vitro. During amastigote-to-promastigote transformation, the amastigote-specific form of LPG disappeared after subculture at 48 h. The WIC 79.3 epitope was not detected by Western blotting on transforming parasites until 48 h in culture. During promastigote-to-amastigote transformation, the amastigote-specific form of LPG was detected 12 h after infection. WIC 79.3 epitopes gradually diminished over 48 h. The results demonstrate the developmentally regulated expression of an antigenically distinct LPG on amastigotes of L. major. PMID- 1710037 TI - Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells. AB - In this study, we demonstrated that lipofectin-treated DNAs which were injected into mouse brain could be incorporated and expressed by brain cells. When L7RH beta gal plasmid DNA harboring E. coli beta-galactosidase gene fused with the nuclear location signal of SV40 T-antigen gene was injected into brains of 1-week old mice, cells whose nuclei appeared to be densely stained with the chromogenic substrate X-gal were detected in several portions of the brain till 9 days after injection. Injection of pMLV-CAT plasmid DNA which contains the E. coli chloramphenicol acetyltransferase (CAT) gene also resulted in cells immunoreactive to the anti-CAT antibody. PMID- 1710038 TI - Expression of genes for the myelin-specific proteins in oligodendrocytes in vivo demands the presence of axons. AB - The level and distribution of mRNAs for several myelin-specific proteins were analyzed by in situ hybridization histochemistry in the mouse optic nerves after severance by eye enucleation during the period of active myelination, viz. the 2nd postnatal week. The autoradiographic signals for 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP)-, myelin basic protein (MBP)- and myelin-associated glycoprotein (MAG)-mRNAs all decreased progressively in the postoperative time course and all the signals fell below the detectable level by the 7th postoperative day. These findings indicate that the levels of genes for all the three major myelin-specific proteins in oligodendrocytes in vivo are down regulated at the transcriptional level after axonal withdrawal. PMID- 1710039 TI - Collateral projections from the midbrain periaqueductal gray to the nucleus raphe magnus and nucleus accumbens in the rat. A fluorescent retrograde double labelling study. AB - Collateral projections of single neurons in the midbrain periaqueductal gray (PAG) to the nucleus accumbens (ACB) and nucleus raphe magnus (NRM) were observed in the rat by a fluorescent retrograde double-labelling technique. After injecting propidium iodide into the ACB and bisbenzimide into the NRM, doubly labelled PAG neurons were most frequently seen in the ventrolateral subnucleus and ventral part of the medial subnucleus at the middle and caudal levels of the PAG. PMID- 1710040 TI - Morphine does not reduce the intraspinal release of calcitonin gene-related peptide in the cat. AB - In anaesthetised cats, antibody microprobes were used to measure the release of immunoreactive calcitonin gene-related peptide (irCGRP) in the lumbar dorsal horn during stimulation of non-nociceptive or nociceptive primary afferent fibres. Release of irCGRP was detected in the substantia gelatinosa, where immunostaining for CGRP was subsequently observed. Intravenous administration of morphine did not affect release of irCGRP detected during either non-nociceptive or nociceptive afferent stimulation. The results suggest that the analgesic action of morphine does not involve reduced release of CGRP from the central terminals of nociceptive primary afferents. PMID- 1710041 TI - Capsaicin-evoked neuropeptide release is not dependent on membrane potential changes. AB - In high K(+)-depolarized spinal cord slices, capsaicin evoked the in vitro release of substance P and calcitonin gene-related peptide (CGRP) from central terminals of C-fibre afferents. This shows that capsaicin-induced release of neuropeptides from sensory afferents is not dependent on membrane potential changes, and that capsaicin-induced Ca2(+)-influx into nerve terminals does not require activation of voltage-sensitive calcium channels (VSCC). On the other hand, in the continuous presence of capsaicin, high K+ did not evoke release of CGRP, but only released substance P, which most likely originated from intrinsic substance P- containing neurones in the spinal cord. PMID- 1710042 TI - Pathways and terminations of axons arising in the fastigial oculomotor region of macaque monkeys. AB - The majority of axons from the fastigial oculomotor region (FOR) decussated in the cerebellum at all rostrocaudal levels of the fastigial nucleus (FN) and entered the brainstem via the contralateral uncinate fasciculus (UF). Some decussated axons separated from the UF and ran medial to the contralateral superior cerebellar peduncle and ascended to the midbrain. Uncrossed FOR axons advanced rostrolaterally in the ipsilateral FN and entered the brainstem via the juxtarestiform body. The decussated fibers terminated in the brainstem nuclei that are implicated in the control of saccadic eye movements. In the midbrain, labeled terminals were found in the rostral interstitial nucleus of the medial longitudinal fasciculus, a medial part of Forel's H-field, the periaqueductal gray, the posterior commissure nucleus, and the superior colliculus of the contralateral side. In the pons and medulla, FOR fibers terminated in a caudal part of the pontine raphe, the paramedian pontine reticular formation, the nucleus reticularis tegmenti pontis, the dorsomedial pontine nucleus of the contralateral side, and the dorsomedial medullary reticular formation of both sides. In contrast, FOR projections to the vestibular complex were bilateral and were mainly to the ventral portions of the lateral and inferior vestibular nuclei. No labeled terminals were found in the following brainstem nuclei which are considered to be involved in oculomotor function: oculomotor and trochlear nuclei, interstitial nucleus of Cajal, medial and superior vestibular nuclei, periphypoglossal nuclei, and dorsolateral pontine nucleus. Labeling appeared in the red nucleus only when HRP encroached upon the posterior interposed nucleus. PMID- 1710043 TI - The corticostriatal and corticotectal projections of the feline lateral suprasylvian cortex demonstrated with anterograde biocytin and retrograde fluorescent techniques. AB - The relationship between the visual cortex and the striatum (ST) of the cat is poorly understood. The present experiments were an attempt to determine if regions along the lateral suprasylvian cortex (LS), known to send dense visual projections to the superior colliculus (SC), also project to the striatum and, if so, to determine whether corticostriatal and corticotectal axons arise from the same neurons. Injections of the anterograde tracer, biocytin, into the posterior portion of the lateral suprasylvian cortex resulted in dense label in both ST and SC. In ST, labeled fibers and terminals were found predominantly in the caudal part of the head of the ipsilateral caudate nucleus and the caudal portion of the ipsilateral putamen. These injections also resulted in label in the superficial and deep laminae of SC. After paired injections of retrogradely transported fluorescent dyes (dextran tetramethylrhodamine and dextran fluorescein) into ST and SC, numerous labeled LS neurons were observed in layer V and modest numbers in layer III: the corticostriatal neurons were found in layers III and V whereas corticotectal neurons were seen only in layer V. Although labeled neurons from each injection were intermingled in layer V, very few of them were double labeled. These data suggest that while ST and SC receive substantial visual inputs from the same cortical area, the nature of the information they receive may be quite different. PMID- 1710044 TI - Immunoglobulin superfamily molecules in the nervous system. AB - Among the various types of membrane molecules involved in cell-cell interactions in the nervous system, we have focused in this review upon membrane proteins belonging to the immunoglobulin superfamily (IgSF). IgSF molecules are distinctive in that: (1) a large percentage of known neural adhesion molecules belongs to the IgSF; (2) they are homologous in structure (Ig domain), yet exhibit large variation of function in cell-cell interactions. The structure of IgSF molecules is briefly summarized in Section II, and each member of the IgSF which has been found in the nervous system is reviewed in Section III. In Section IV, we have discussed possible properties of yet-unknown nervous system IgSF molecules, on the assumption that nervous system IgSF molecules thus far discovered comprise only a small portion of those existing. Discussion is based upon an analogy with the immune system and upon knowledge of cell-cell interactions in the development of the nervous system. Our principal aims in this review are to summarize knowledge of neural IgSF molecules and to discuss the possibility that some IgSF molecules may encode in their structures instructions for recognizing, or for being recognized by, target neural cells. Further growth of knowledge of IgSF molecules may yield insights into the patterns of cell-cell interactions underlying the formation of neuronal circuits during development. PMID- 1710045 TI - Angle neovascularization as a marker for aqueous outflow after argon laser trabeculoplasty. PMID- 1710046 TI - Mechanism of action of some commonly used nasal drugs. AB - This article provides a brief description of the mechanism of action of drugs commonly used in the treatment of nasal disorders. These include oral and topical vasoconstrictors, antihistamines, atropine-like drugs, cocaine, topical corticosteroids, and mast-cell stabilizers. PMID- 1710047 TI - Identification of perilymph proteins by two-dimensional gel electrophoresis. AB - Perilymph has a total protein component that is quantitatively distinct from serum and cerebrospinal fluid (CSF). The goal of this research was to determine if perilymph contains any qualitatively unique protein constituents that will distinguish it from serum or CSF. To test this hypothesis, matched sets of perilymph, serum, and CSF were obtained from 18 guinea pigs and seven human subjects. The purity of each sample was assured by measurement of the protein concentration of each sample and comparison of this parameter to known normal values for perilymph, serum, and CSF. Each sample was then subjected to two dimensional gel electrophoresis, separating proteins by isoelectric point in the horizontal dimension and by relative molecular weight in the vertical dimension. All gels were processed under precisely identical physical conditions by use of a diamine silver stain. A small number of perilymph proteins not found in plasma were identified in both the guinea pig and the human specimens. The finding of unique perilymph proteins may permit the development of a sensitive marker that will aid in the diagnosis of perilymph fistula. PMID- 1710048 TI - Pleomorphic adenoma of cervical heterotopic salivary gland tissue: case report and review of neoplasms arising in cervical heterotopic salivary gland tissue. PMID- 1710049 TI - Trichinella spiralis: antigenic epitopes from the stichocytes detected in the hypertrophic nuclei and cytoplasm of the parasitized muscle fibre (nurse cell) of the host. AB - Monoclonal antibodies raised against antigens present in the excretions/secretions (E/S) of larval Trichinella spiralis, polyclonal antibodies raised against E/S and antisera from rabbits and pigs infected with T. spiralis were used in conjunction with immunocytochemical techniques to detect antigens in sections of muscle from mice that had been infected with T. spiralis for 15 or 30 days. The antibodies recognized epitopes in the stichocytes, on the surface of the cuticle, in the lumen of the oesophagus and in the lumen of the intestine of encysted larvae. Monoclonal antibodies 7C2C5 and 1H7 and the polyclonal antibodies recognized epitopes in the cavity occupied by the larva, in the cytoplasm of the nurse cell, and in the hypertrophic nuclei of the nurse cell, but did not recognize material in the smaller nuclei of the nurse cell, in the cyst wall or in the surrounding muscle. Monoclonals 3B2E6 and 1D11G8B2, which recognized epitopes in the stichocytes and on the surface of the cuticle of the larvae, gave negative results with the cytoplasm and nuclei of the nurse cell. A polyclonal antibody raised against Trichuris suis recognized epitopes in the muscle and hypodermis of encysted T. spiralis but gave negative results with material in the nurse cell and nurse cell nuclei. The possibility that the antigen detected in the cytoplasm and nuclei of the nurse cell is produced by the stichocytes of the nematode and that it is controlling genes of the altered muscle fibre, either directly or indirectly, is discussed. PMID- 1710051 TI - Normal and abnormal development of human intrahepatic bile ducts. An immunohistochemical perspective. PMID- 1710050 TI - Effect of acute phase proteins, especially alpha 2-macroglobulin, on granuloma formation around Schistosoma mansoni eggs in the rat. AB - A new model for the study of granuloma formation in the liver is described. Rats received an injection of 20,000 Schistosoma mansoni eggs in the portal vein and granuloma formation was evaluated at 3, 5 and 7 weeks post-injection. Liver collagen was estimated at the same time and serum procollagen III peptide, a marker of collagenesis, weekly. With this model, wherein the number of S. mansoni eggs and the time of injury are standardized, the effect of high levels of acute phase proteins especially alpha 2-macroglobulin on granuloma formation was studied. It appeared that in rats with high levels of alpha 2-macroglobulin the mean size of granulomas was significantly greater at 3 and 5 weeks compared with controls. In both groups an increase in liver collagen was observed during this period, reaching a peak at 5 weeks in the acute phase group. This model facilitates the study of the effects of S. mansoni eggs on granuloma formation. PMID- 1710052 TI - Sensors and pacemaker mediated tachycardias. PMID- 1710053 TI - Paradoxic embolism due to altered hemodynamic sequencing following transvenous pacing. AB - A young patient, who experienced a cerebral embolic event 30 days after transvenous pacemaker lead placement, is reported. This patient had previously been paced with an epicardial lead without evidence of right to left intracardiac shunt. However, hemodynamic evaluation post-embolism demonstrated a marked temporal disparity of the pulmonary and systemic ventricles. This phasic divergence resulted in a brief reversal of right and left ventricular pressure ratios, and a paradoxic intracardiac shunt at a small ventricular septal defect. The potential for similar events in the presence of any defect of the atrial or ventricular septum mandates caution in the use of transvenous pacemaker leads in such patients. PMID- 1710054 TI - Transvenous cryoablation of the bundle of His. AB - Cardiac dysrhythmias are a prominent cause of morbidity and mortality. Pharmacological treatment is ineffective in a large number of patients and is associated with many serious side effects. Thus, direct treatment of cardiac arrhythmias has been used with increasing frequency. Each form of direct treatment, such as surgical ablation, DC catheter ablation, radiofrequency catheter ablation, laser catheter ablation suffer serious drawbacks. Thus, we investigated the utility of transvenous catheter cryoablation of the bundle of His in five miniature swine, 40-60 lbs. in weight. Complete atrioventricular block was produced in each animal during cryothermia and persisted for 1 hour of observation in four out of five swine. In the fifth animal, 2:1 atrioventricular block within the atrioventricular node persisted for 1 hour of observation. Morphological and histologic examination revealed no dysfunction of capillaries and myofibriles in the atrioventricular node and proximal bundle of His. This potential mode of transcatheter therapy deserves further investigation. PMID- 1710055 TI - Sensing through the esophagus for temporary atrial synchronous ventricular VDD pacing. AB - The purpose of this study was to evaluate the effectiveness and safety of temporary VDD pacing using an esophageal electrode for sensing of the atrial electrogram. We studied 15 patients, 8 men and 7 women, aged 77 +/- 2 years (mean +/- SE, range 61-90), with severe atrioventricular (AV) conduction disturbances. A 24-hour beat-to-beat ECG analysis was used to evaluate the effectiveness of the pacing system and special tests were performed to test the stability of pacing and sensing. The system performed satisfactorily in 12 of the 15 patients. The 24 hour Holter ECG monitoring revealed the following percentages of beats: 96.32 +/- 0.5 VDD, 2.92 +/- 0.6 VVI, and 0.14 +/- 0.05 paced beats resulting from pseudosensing. All the latter were single, with no bigeminy or salvos. The results of the stability tests were as follows: the percentage of VDD beats was significantly lower than the 24-hour mean when the patient lay on his right side (92.8 +/- 0.5, P less than 0.001), during the swallowing of liquids (91.26 +/- 0.4, P less than 0.001) and soft foods (84.2 +/- 1.4, P less than 0.001), and during coughing (94.2 +/- 0.6, P less than 0.001). The percentage of VVI type beats increased in these four cases (6.7 +/- 0.5, 7.2 +/- 0.3, 13.2 +/- 1.2 and 4.8 +/- 0.4, respectively, P less than 0.001 in each case). The percentage of ectopic beats due to pseudosensing did not change significantly during any of the tests. These results indicate that the method described is a safe and effective technique for temporary VDD pacing. PMID- 1710056 TI - Long-term functional integrity of atrial leads. AB - The effectiveness and reliability of atrial leads has been questioned. We studied retrospectively, all atrial leads implanted at our center (n = 494; 438 Medtronic Model 6957J, 56 Medtronic Model 4512) over a 5-year period ending December 31, 1987, to determine the frequency of atrial lead failure (pacing, sensing, or both) and the median duration of proper pacing and sensing function for each lead model studied. Eighty-eight percent of the polyurethane atrial leads continued to function satisfactorily at 5 years, results somewhat better than those reported heretofore in the literature, as well as our own past results with a variety of different lead types. There were 29 failures of pacing, sensing, or both (6% of implants). The cumulative survival of the atrial leads at 5 years was 88%. Pacing and sensing survival were 91% +/- 2.4% and 88% +/- 2.9%, respectively. We conclude that the choice of pacing mode for a new pacemaker should be based solely on the clinical indication and not on the concern that atrial pacing and sensing will be unreliable. PMID- 1710057 TI - Loss of atrial signal detection with increasing sensitivity settings: a software problem and its practical solution. AB - A logic error in the software of Cosmos II results in apparent malsensing of atrial signals at high rather than low sensitivity settings. The paradoxical function was found in 54 out of 87 units tested (= 62%) if the atrial sensitivity was increased and in 54 out of 87 units tested (= 62%) if the atrial sensitivity was increased and the ventricular refractory period was lengthened beyond critical limits that could be defined individually. The problem inherent in both unipolar (66%) and bipolar systems (54%) is attributed to a summation of a physiological signal and internal noise, which has to be detected at the atrial level within a specific timing window to disable atrial sensing for the next pacing cycle. The malfunction virtually disappears if the ventricular refractory period does not exceed 210 msec. It is recommended, therefore, that Cosmos II pacemakers be left at their nominal ventricular refractory value (200 msec). PMID- 1710058 TI - Automatic reduction of stimulus polarization artifact for accurate evaluation of ventricular evoked responses. AB - The ventricular evoked response, the cardiac depolarization generated in response to a pacing stimulus, is potentially useful as a sensor for rate responsive pacing and automatic threshold tracking. It is necessary to minimize the polarization artifact that results from pacing in order to sense cardiac depolarizations from the same electrodes that pace the heart. To accomplish this, a triphasic stimulus waveform consisting of precharge, stimulus, and postcharge was used. An algorithm was developed that introduced pacing stimuli during the refractory period of sensed beats, when cardiac depolarization could not occur by definition and polarization artifact could be evaluated. Precharge duration was varied until the amplitude of the polarization artifact was small compared to the evoked response. In 18 patients with temporary electrode catheters, polarization artifact was reduced from 6.8 +/- 3.4 mV to 1.9 +/- 1.1 mV after balancing (P less than 0.005). Initial precharge duration was 3200 mu sec and the mean final precharge duration was 3551 +/- 516 mu sec. In 14 patients with permanent bipolar pacing leads, polarization artifact was reduced from 3.2 +/- 3.5 mV to 0.7 +/- 0.6 mV (P less than 0.025). Final precharge duration averaged 3440 +/- 310 mu sec. Under a wide variety of pacing conditions, this algorithm simply and quickly reduces polarization artifact to a minimum to allow accurate analysis of evoked responses. PMID- 1710059 TI - A three-dimensional display for cardiac activation mapping. AB - A three-dimensional display is described that allows activation sequences from the epicardium and endocardium to be shown simultaneously on the same image. Three electrode arrays (epicardial sock, left ventricular balloon, right ventricular balloon) are represented in a three-dimensional perspective by an array of dots that are intensified when activated. This arrangement requires fewer calculations and is easier to interpret than sliced-isochronal maps but cannot represent a complete heart cycle in one image. The three-dimensional display eliminates the distortion caused by two-dimensional diagrams and facilitates activation correlation between electrode arrays. A standard, low cost microcomputer has been used to implement the activation display. PMID- 1710060 TI - Antitachycardia pacemaker treatment of postoperative arrhythmias in pediatric patients. AB - An automatic antitachycardia pulse generator (Intertach 262-12) was implanted in each of six pediatric patients (mean age, 10 years) with drug-resistant and persistent postoperative supraventricular arrhythmias. Four had bradycardia tachycardia syndrome, two after a Mustard procedure for transposition of the great arteries, one after a Senning procedure for the same anomaly, and one after a Fontan procedure for univentricular heart with transposition of the great arteries. Of the two remaining patients, one had atrial flutter after a modified Fontan procedure for univentricular heart and one had intra-atrial reentry tachycardia after a modified Fontan procedure for double-outlet right ventricle with pulmonary stenosis. During a mean follow-up interval of 31 months after implantation, pacemakers were activated on multiple occasions and functioned appropriately in all six patients. Complications necessitated six invasive interventions in three patients: erosion or infection of the system, adaptor fracture, and connector block fracture on one occasion each and lead dislodgment on three occasions. Four of the six patients continued to take drugs at the end of this study; however, all patients had their drug therapy reduced and one was taking digoxin only. The number of hospital admissions decreased after implantation. Despite a number of technical challenges, this newer multiprogrammable antitachycardia pacemaker appears to be a valuable addition to the treatment of refractory postoperative supraventricular tachyarrhythmias in pediatric patients. PMID- 1710061 TI - Orthogonal electrode catheter array for mapping of endocardial focal site of ventricular activation. AB - Precise location of the endocardial site of origin of ventricular tachycardia may facilitate surgical and catheter ablation of this arrhythmia. The endocardial catheter mapping technique can locate the site of ventricular tachycardia within 4-8 cm2 of the earliest site recorded by the catheter. This report describes an orthogonal electrode catheter array (OECA) for mapping and radiofrequency ablation (RFA) of endocardial focal site of origin of a plunge electrode paced model of ventricular activation in dogs. The OECA is an 8 F five pole catheter with four peripheral electrodes and one central electrode (total surface area 0.8 cm2. In eight mongrel dogs, mapping was performed by arbitrarily dividing the left ventricle (LV) into four segments. Each segment was mapped with OECA to find the earliest segment. Bipolar and unipolar electrograms were obtained. The plunge electrode (not visible on fluoroscopy) site was identified by the earliest wave front arrival times of -30 msec or earlier at two or more electrodes (unipolar electrograms) with reference to the earliest recorded surface ECG (I, AVF, and V1). Validation of the proximity of the five electrodes of the OECA to the plunge electrode was performed by digital radiography and RFA. Pathological examination was performed to document the proximity of the OECA to the plunge electrode and also for the width, depth, and microscopic changes of the ablation. To find the segment with the earliest LV activation a total of 10 +/- 3 (mean +/- SD) positions were mapped. Mean arrival times at the two earlier electrodes were -39 +/- 4 msec and -35 +/- 3 msec. Digital radiography showed the plunge electrode to be within the area covered by all five electrodes in all eight dogs. The plunge electrode was within 1 cm2 area of the region of RFA in all eight dogs. The width and depth of ablation were 5 +/- 3.5 and 7 +/- 3.5 mm, respectively. Microscopic changes revealed coagulative necrosis, hemorrhage, and inflammatory changes in all RFAs. In conclusion, the OECA can map the endocardial focal site of origin of paced ventricular activation within 1 cm2 area in a canine model. RFA from the OECA can cause discrete ablations representing all five electrodes or cross shaped ablation connecting central electrode to all four peripheral electrodes. This catheter holds promise for extending surgical and clinical catheter ablation procedures. PMID- 1710062 TI - Dynamic cardiomyoplasty: an overview. PMID- 1710063 TI - Permanent atrial leads: they have a will of their own! PMID- 1710064 TI - Biophysics of high frequency ablation. PMID- 1710065 TI - Performance of implantable cardiac rhythm management devices. PMID- 1710066 TI - Human chorionic gonadotrophin and alpha-fetoprotein levels in matched samples of amniotic fluid, extraembryonic coelomic fluid, and maternal serum in the first trimester of pregnancy. AB - Separately identified samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 32 women between 7 and 12 weeks of pregnancy were analysed for human chorionic gonadotrophin (hCG) and alpha-fetoprotein (AFP). There was a highly significant difference between the hCG levels in amniotic fluid (median level 6.3 U/ml; range 1.6-310.0 U/ml) and those in extraembryonic coelomic fluid (median level 400.0 U/ml; range 135.0-2250.0 U/ml) (p less than 0.001; Mann-Whitney U-test). The levels of AFP were very similar in amniotic fluid (median 26.0 kU/ml; range 10.0 116.5 kU/ml) and extraembryonic coelomic fluid (median level 24.1 kU/ml; range 12.4-94.4 kU/ml). PMID- 1710067 TI - Alteration in age-specific risks for chromosomal trisomy by maternal serum alpha fetoprotein and human chorionic gonadotropin screening. AB - Maternal serum screening for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) increases the detection rate of Down's syndrome (DS) pregnancies. To estimate the risk of a DS pregnancy for a particular woman, Wald et al. combine a trivariate function of AFP, hCG, and unconjugated oestriol with age-specific risk. Calculation of independent likelihood ratios (LRs) for AFP and hCG has allowed us to examine the predictive value of each test alone and the combination. AFP and hCG were measured in stored serum samples from 672 normal, 8 trisomy 21 (DS), 9 trisomy 18, and 2 trisomy 13 pregnancies. AFP and hCG multiples of the median (MOM) were calculated for each sample. The LRs for AFP MOM and hCG MOM were calculated and combined with age-specific risk. Of eight DS pregnancies, six had increased risk based on age and AFP. Addition of hCG detected two additional DS pregnancies. Of nine trisomy 18 pregnancies, four (44 per cent) had hCG MOM under 0.25. Three out of nine would have been classified as high risk by AFP, but none by combined AFP and hCG. Amniocentesis would have been recommended in 74 per cent of aneuploid pregnancies if both age and serum screening were used. Abandonment of amniocentesis based on age alone would have excluded two abnormal pregnancies from detection. Screening programmes should note that combined risk figures are specific for DS and do not include other trisomies. Detection of other trisomies requires inclusion of low hCG level as a discriminator and continuation of age-based testing. PMID- 1710068 TI - Prenatal diagnostic procedure for leukocyte adhesion deficiency. AB - Leukocyte adhesion deficiency (LAD) is a rare autosomal recessive disorder which leads to recurrent severe infections due to impaired leukocyte functions. The disorder is caused by an absence or deficiency of leukocyte cell adhesion molecules (LeuCAMs) on the leukocyte membranes. The diagnosis is established with monoclonal antibodies against the LeuCAMs. We have carried out a prenatal diagnostic procedure by means of cordocentesis in a mother who was 20 weeks pregnant and had previously given birth to a child with LAD. This previous child had the severe form of LAD with undetectable mRNA for the beta chain, the common subunit of the LeuCAMs. We found that the fetal granulocytes expressed the LeuCAMs normally. At birth, the baby was physically normal and showed no signs of impaired leukocyte functions. PMID- 1710069 TI - Acoustic rhinometry: values from adults with subjective normal nasal patency. AB - The nose with normal feeling of nasal patency, and no gross structural changes has been described in 82 individuals by acoustic rhinometry. Curves for one and both sides of the nasal cavity and before and after decongestion have been recorded. We have found that the minimal cross-sectional area (MCA) is located anteriorly in the nasal cavity; in some subjects it is localized at the head of the inferior turbinate and in other subjects more anteriorly at the nasal valve. After decongestion MCA moves even more anteriorly. Beyond the MCA the dimension of the nasal cavity increases, with maximal effect of decongestion at 4 cm from nostrils. Decongestion increases the total volume of the nasal cavity by 35%. PMID- 1710070 TI - [Effect of hypothalamic stimulation on RMBF and SP content in IML of spinal cord in rabbits]. AB - Experiments were performed on 48 urethane-chloralose anaesthetized, gallamine triethiodide immobilized and vagotomized rabbits under artificial ventilation. Radiolabeled microspheres were injected into the left atrium during stimulation of the left or right dorsomedial hypothalamic nucleus (LDMH or RDMH) to measure regional myocardial blood flow (RMBF). RDMH stimulation produced relatively little flow reduction of the left ventricle, whereas LDMH stimulation produced an obvious flow reduction. No significant change occurred in the right ventricle. Meanwhile, LDMH or RDMH stimulation caused increases of mean arterial blood pressure (MABP) and changes of epicardial electrogram (EECG) ST segment, but no significant changes in heart rate. EECG ST segment was elevated in relation to the reduction of RMBF in left ventricle (r = -0.825, P less than 0.001). Linear regression analysis of MABP change in relation to left ventricle RMBF showed no significant correlation. In intact rabbits, it was demonstrated that DMH stimulation caused an decrease of substance P (SP) immunoreactive material in bilateral intermediolateral cell columns (IML) of T2-5 spinal cord concomitantly with an elevation in MABP and EECG ST changes. The results suggest that electrical stimulation of DMH can elicit coronary constriction, decrease in RMBF, elevation of EECG ST segment and increase in MABP, which may be mediated by the release of SP immunoreactive material in the IML of the spinal cord. PMID- 1710071 TI - [Treatment of pediculosis of the scalp]. PMID- 1710072 TI - The peroxisome and the eye. AB - Several childhood multisystem disorders with prominent ophthalmological manifestations have been ascribed to the malfunction of the peroxisome, a subcellular organelle. The peroxisomal disorders have been divided into three groups: 1) those that result from defective biogenesis of the peroxisome (Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum's disease); 2) those that result from multiple enzyme deficiencies (rhizomelic chondrodysplasia punctata); and 3) those that result from a single enzyme deficiency (X-linked adrenoleukodystrophy, primary hyperoxaluria type 1). Zellweger syndrome, the most lethal of the three peroxisomal biogenesis disorders, causes infantile hypotonia, seizures, and death within the first year. Ophthalmic manifestations include corneal opacification, cataract, glaucoma, pigmentary retinopathy and optic atrophy. Neonatal adrenoleukodystrophy and infantile Refsum's disease appear to be genetically distinct, but clinically, biochemically, and pathologically similar to Zellweger syndrome, although milder. Rhizomelic chondrodysplasia punctata, a peroxisomal disorder which results from at least two peroxisomal enzyme deficiencies, presents at birth with skeletal abnormalities and patients rarely survive past one year of age. The most prominent ocular manifestation consists of bilateral cataracts. X-linked (childhood) adrenoleukodystrophy, results from a deficiency of a single peroxisomal enzyme, presents in the latter part of the first decade with behavioral, cognitive and visual deterioration. The vision loss results from demyelination of the entire visual pathway, but the outer retina is spared. Primary hyperoxaluria type 1 manifests parafoveal subretinal pigment proliferation. Classical Refsum's disease may also be a peroxisomal disorder, but definitive evidence is lacking. Early identification of these disorders, which may depend on recognizing the ophthalmological findings, is critical for prenatal diagnosis, treatment, and genetic counselling. PMID- 1710073 TI - [IgG4 antibodies to the allergens of grass and tree pollens in pollinosis patients: their diagnostic and prognostic significance]. AB - Total IgE, IgE and IgG4 antibodies to offending allergens were studied in 118 hay fever patients with allergy to tree pollen or to grass pollen. No difference in the level of total IgE and IgE antibodies was found between these groups of patients while the IgG4 antibody level was significantly higher in tree pollen allergic patients. It has been established that in tree pollen allergy, the determination of IgG4 antibodies is of no less diagnostic value than the assay of IgE antibodies. The authors discuss the problems of the prognostic value of IgG4 antibody increase during the hyposensitization therapy and of a possible relationship between IgG4 antibodies and allergy to vegetable food often associated with tree pollen allergy. PMID- 1710074 TI - [Antiviral preparations in the therapy of chronic viral hepatitis]. AB - The author analyzes current data on the potentialities of antiviral therapy of patients suffering from chronic viral hepatitides. Provides the data on the short and long-term therapeutic effects of interferon drugs and other antiviral agents, describes side effects produced by such type drugs. Compares the clinico enzymatic, pathohistologic characteristics of the liver status with the degree of elimination of serum antigenic markers of virus B hepatitis in patients afflicted with chronic viral hepatitides. Relates the effects of antiviral drugs in patients suffering from chronic virus non-A non-B hepatitis. PMID- 1710075 TI - Suction drainage: a new approach to the treatment of empyema. AB - Thirteen patients with empyema thoracis were treated with a new suction drainage technique. The method entails passing a catheter into the empyema cavity under ultrasound guidance and using strong suction to drain loculated pus. Eight patients had no recurrence after a single treatment and one patient had no recurrence after two treatments. The procedure was a useful palliative measure in two patients with malignant disease who subsequently died. In one patient failure of the lung to expand after the procedure showed the need for thoracotomy. In one other patient the empyema recurred and decortication was required. PMID- 1710076 TI - Mapping of an HLA-DRw52-associated determinant on DR beta 1 molecules. AB - The molecular reaction patterns of the DRw52-specific mouse monoclonal antibodies UL-52 and 7.3.19.1 were investigated. Upon immunoprecipitation and two dimensional IEF-SDS polyacrylamide gel electrophoresis analysis (2D-PAGE) mAb UL 52 selectively isolated DR beta 1 molecules from DRw52-positive cell lines, whereas mAb 7.3.19.1 predominantly precipitated DR beta 3 molecules. Reduced mAb UL-52 binding affinity was observed to DRw8- and DRw12-positive cells, potentially resulting from structural modifications within the antibody binding site. Comparison of mAb UL-52 reactivity with published DR beta chain amino acid sequences demonstrates that the amino acid residues -S- in positions 11 and 13 on DR beta 1 molecules essentially contribute to the formation of the antibody binding site. mAb 7.3.19.1 reactivity, on the other hand, correlates with the expression of DR beta 3 chain amino acid residues K, G and N, in positions 71, 73 and 77, respectively. In contrast to other DRw52 monoclonal antibodies described so far, mAb UL-52 demonstrates a similar reactivity to DRw52 allosera, suggesting that mAb UL-52 and DRw52 allosera possibly recognize the same or a similar determinant on DR beta 1 molecules. PMID- 1710077 TI - Involvement of the CDw50 molecule in allorecognition. AB - The CDw50 differentiation antigen is defined by 101-1D2 and 140-11 monoclonal antibodies (mAb), both produced and characterized in our laboratory. This molecule is broadly expressed on hematopoetic cells but not on other cells. In this report we show that these 2 mAb recognize different epitopes of the same molecule, which are resistant to neuraminidase and proteases. We also demonstrate that the CDw50 antigen is expressed on thymocytes and T lymphocytes as an N glycosylated glycoprotein monomer with a relative molecular weight (Mr) of 130,000 daltons with intrachain disulfide bonds, and that this molecule is resistant to treatment with phosphatidylinositol (PI) phospholipase C and therefore probably not PI-anchored to the membrane. CDw50 is a poorly or non constitutively phosphorylated molecule that becomes phosphorylated by treatment with phorbol 12-myristate 13-acetate (PMA) of peripheral blood mononuclear cells (PBMC). The addition of affinity-purified CDw50 mAb inhibits primary mixed lymphocyte culture (MLC) but not secondary MLC, cytotoxicity or proliferation induced by mitogens. The inhibition of alloreactivity is mediated at the level of both responding and stimulator cells. PMID- 1710078 TI - Canine leukocyte integrins: characterization of a CD18 homologue. AB - The major characteristics of the canine CD18 leukocyte antigen family, defined by a murine monoclonal antibody CA1,4E9, are described. The overall molecular organization of this family of leukocyte integrins was similar to the human counterpart, and consisted of a common beta subunit of 95 kD (CD18) and non covalently linked alpha subunits of 150 kD (CD11C), 165 kD (CD11b) and 180 kD (Cd11a). Conservation of CD18 epitopes between dog, human, feline, equine and bovine was observed. CD18 expression in lymphoid tissue had a characteristic pattern which was based on differences in the intensity of expression observed in different cell types. The expression of LeuCAMs in canine epidermotropic lymphosarcoma (mycosis fungoides) was also explored since LeuCAMs might be expected to play a major role in the tissue tropism of this tumor. Intense CD18 expression was observed in the malignant T cells which infiltrated the epidermis; these cells differed phenotypically from dermal T cells in that they often lacked Thy-1 expression. The potential pathogenetic significance of LeuCAM expression in canine mycosis fungoides is discussed. PMID- 1710079 TI - Mirex inhibits bile acid secretory function in vivo and in the isolated perfused rat liver. AB - The insecticides mirex and chlordecone suppress the biliary excretion of a wide variety of non-bile acid organic anions in vivo in the rat, and mirex inhibits the uptake of taurocholate (TC), a common bile acid, in isolated rat hepatocytes. We have therefore investigated the effects of mirex and chlordecone on bile acid secretory function (bile flow, bile acid concentration, bile acid secretory rate) in vivo and in the single-pass isolated perfused liver. Male Sprague-Dawley rats were orally dosed with corn oil, mirex (50 mg/kg), or chlordecone (18.75 mg/kg; in vivo studies only) for 3 consecutive days and experiments performed on Day 6. Mirex significantly increased liver weight from 12.2 +/- 0.8 to 20.8 +/- 1.3 g with no change in body weight whereas chlordecone had no significant effect on liver weight (11.9 +/- 0.7 g) or body weight. Mirex significantly decreased while chlordecone increased bile flow per gram liver in vivo; both compounds, however, increased bile flow when expressed per kilogram of body weight. Mirex and chlordecone significantly decreased the bile acid concentration in bile and the bile acid secretory rate (nmol/min/g liver and mumol/min/kg body weight). Studies in the isolated perfused liver were designed to determine the effect of mirex on the ability of the liver to extract increasing concentrations of [3H]TC from the perfusate and excrete it in the bile. Mirex treatment significantly decreased the TC extraction ratio by 40-89% and the hepatic intrinsic clearance by 85-95%. Mirex also significantly decreased the TC-induced choleresis, the concentration of TC in the bile, and the TC secretory rate. The data indicate that mirex treatment markedly inhibits the ability of the liver to extract TC from the blood/perfusate and concentrate it in the bile. PMID- 1710080 TI - Immunohistochemical features of giant cell carcinoma of the lung: patterns of expression of cytokeratins, vimentin, and the mucinous glycoprotein recognized by monoclonal antibody A-80. AB - Giant cell carcinoma of the lung (GCCL) is an uncommon and extremely aggressive variant of lung cancer. Characteristic microscopic findings include marked pleomorphism, aggregates of mononucleated or multinucleated giant cells (or both), a general lack of architectural cohesiveness, extensive necrosis, and endocytosis by the giant cells. Although the epithelial character of GCCL has been confirmed by a number of studies, controversy persists as to whether it represents a variant of poorly differentiated adenocarcinoma or of squamous carcinoma. Histochemical studies for mucosubstances have yielded variable and conflicting results. This report describes conventionally fixed and processed samples from 10 cases of GCCL studied with a panel of monoclonal antibodies (Mabs) recognizing different cytokeratin polypeptides (AE1, AE3, AE1/AE3 cocktail, and CAM 5.2), vimentin, and Mab A-80, the last of which binds to a mucinous glycoprotein associated with exocrine differentiation. All 10 cases of GCCL reacted with all cytokeratin Mabs; the extent and intensity of the reaction varied notably. All cases stained strongly and diffusely with Mab AE1 and AE1/AE3, the reaction was less extensive and weaker with CAM 5.2. Significantly, 2 cases reacted focally with Mab AE3. Nine cases reacted extensively and intensely with the vimentin Mab, often showing prominent paranuclear globular profiles. All cases reacted with Mab A-80; the reaction was often strong, but the extent was variable. Findings indicate that all GCCL are indeed cytokeratin positive but that most express polypeptides toward the low-molecular weight end of the spectrum; a small subset also expresses heavier polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710081 TI - A demonstration of ultrasound-induced quartz wind streaming, standing waves and absorption within a standing wave field. PMID- 1710082 TI - Bladder outlet obstruction treated with transurethral ultrasonic aspiration. One year follow-up on 59 patients. AB - Fifty-nine males with bladder outlet obstruction were treated with transurethral ultrasonic aspiration of the prostate. Utilizing a 26.5F urethral sheath, surgery was accomplished with a 10F, 0-700-micron-vibration-level ultrasonic tip with an excursion rate of 39 kHz. Complete removal of the adenoma was accomplished followed by transurethral electrocautery biopsies of both lateral lobes to compare pathologic specimens. One year follow-up revealed satisfactory voiding patterns in 57 of 59 men (96%). Bladder neck contractures developed in 2 men. Pathologic comparisons showed 100 percent correlation between aspirated and TUR specimens (56 BPH, 3 adenocarcinoma). Forty-seven men were active sexually preoperatively (6 with inflatable penile prostheses). Post ultrasonic aspiration, 46 men had erectile function similar to preoperative levels with 1 patient suffering erectile dysfunction. Forty men (85%) had antegrade ejaculation while 7 (15%) experienced retrograde or retarded ejaculation. No patients were incontinent. PMID- 1710083 TI - Oxygen free radicals and corneal endothelium. PMID- 1710085 TI - Mental illness in nursing homes. AB - This report presents statistics on residents of nursing homes who had at least one condition that can be classified as a mental illness. Data for this major subgroup of nursing home residents are presented by length of stay since admission, source of payment in the month before the survey, functional dependencies in the activities of daily living, usual living arrangements prior to admission, and reasons for admission, according to major demographic and facility characteristics. This report also includes selected comparisons between residents with and without mental disorders. Estimates are based on data collected in the 1985 National Nursing Home Survey. PMID- 1710084 TI - Advances in corneal preservation. AB - The functional status of the endothelium and sustained corneal deturgescence after corneal preservation are of great clinical importance and have been primary goals in the development of corneal storage media. In our investigational studies we have specifically addressed the improvement of the quality of donor tissue after 4 degrees C storage, the extension of corneal preservation time, the enhancement of corneal wound healing, and the reduction of the normal progressive loss of endothelial cells postkeratoplasty. Specifically we have developed in vitro HCE cell and epithelial cell culture models that can accurately reflect the response of human corneal tissue in vivo. These models have been utilized to study the effects of growth factors and medium components in relation to their biocompatibility and efficacy in the development of improved corneal preservation solutions. Our laboratory investigated in vitro conditions that allowed human corneal endothelium to shift from a nonproliferative state, in which they remain viable and metabolically active, to a proliferative, mitotically active state. Isolation techniques developed in our laboratory have enabled the establishment of primary and subsequent subcultures of human corneal endothelium that retain the attributes of native endothelium. These in vitro conditions maintain HCE cells in a proliferative state, actively undergoing mitosis. A quantitative bioassay has been developed to determine the effects of various test medium in the stimulation or inhibition of DNA synthesis. In attempting to learn more about the events that occur during in vitro endothelial cell isolation, cell reattachment, extracellular matrix interaction and migrating during subculture, SEM was done on isolated HCE cells incubated in CSM. These studies suggest that the components of the extracellular matrix modulate the growth response of HCE cells, and play a role in regulating proliferation and migration. These observations are important in view of the fact that anterior chamber environment limits cell regeneration of the endothelium, and supports wound healing via cell migration. In vivo, it is the complex interaction of the HCE cell and the extracellular matrix that signal the cell to respond to cell loss in this manner. As our knowledge of human corneal endothelium has increased so has our anticipation of developing the "optimum" medium. Thus additional components have been added to this basic medium to address specific complications encountered with 4 degrees C corneal preservation. Antioxidants, additional energy sources, and other nutritive substrates have been used to supplement and further define a chondroitin sulfate-based medium. These changes have been a part of our new awareness that, even at 4 degrees C, the cornea is metabolically active.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1710086 TI - [Modern labor and stress on skeletal muscles]. AB - Introductorily the actual state in analysis and evaluation of physical work is characterized as a problem of changing chemical into mechanical energy in the skeletal muscle. Modern work is characterized in the view of the physiologist. Considering experimental results and epidemiological studies, problems of the K+ homeostasis during long lasting low level muscle activity are discussed as reasons of limiting performance. Changes in the potassium distribution between intra- and extracellular space have enormous consequences for excitability, contractili, metabolism, and arterial blood pressure. Finally references are given for evaluation and designing of modern work. PMID- 1710087 TI - [A receptor for qualitative well-stained differential blood formation smears and bone marrow puncture biopsies]. PMID- 1710088 TI - [Non-Hodgkin's lymphomas of the stomach. Therapy from the surgical viewpoint]. AB - About 2-5% of all gastric tumours are Non-Hodgkin Lymphomas (NHL). In the last years we treated 15 patients with NHL of the stomach. Six of these patients were classified to group IE according to Ann Arbor classification, four to group II (three II 1 E and one II 2 E). In group III E were two patients (one III S and one III E) and one patient was classified to group IV. Ten patients displayed low grade and five high grade malignancies with respect to the Kiel classification. We performed in group I three gastrectomies and three BI resections. In four patients of group II two gastrectomies and two BI resections were carried out and in group III two BI resection. Our treatment policy consisted of operation and radiation in IE, to operation and chemotherapy in high grade lymphomas of group II, and operation plus radiation in low grade malignancies of group II. In group III we treated radiation, chemotherapy and operation and in group IV radiation and chemotherapy. In group I five patients survived, in group II two out of four and in group III one of two. The patient of group IV died within two years after diagnosis. PMID- 1710089 TI - [Electric stimulation of the autonomic nervous system and vasoactive intestinal peptide and substance P release on the lower esophageal sphincter]. AB - The effect of electrical stimulation of splanchnic and vagal nerve supply to the in vivo isolated porcine LES on the lower esophageal sphincter pressure (LESP) and the secretion of VIP and SP into the venous effluent into the venous effluent of the LES was investigated. Functional integrity of the autonomic nerve supply was assessed by the effect of nerve stimulation on heart rate. Vagal nerve stimulation increased LESP (8-Fold) as well as VIP output (3 Fold) significantly (p less than 0.01) whereas the secretion of SP was unaffected of vagal nerve stimulation. Splanchnic nerve stimulation increased heart rate significantly but was without effect on LESP, VIP, and SP outputs. Atropine partially abolished the effect of vagal nerve stimulation on LESP but the VIP secretion was completely resistant to atropine blockade. Administration of guanethidine was without effect on LESP, VIP, and SP outputs during vagal as well as splanchnic nerve stimulation. It is concluded that VIP acts as a neuro-transmitter since vagal stimulation increase the release of VIP from LESP. However, the finding of a partial atropine resistance of the LESP despite an unchanged release of VIP and SP, suggests that other transmitters participate in vagal activation of LESP. PMID- 1710090 TI - Satellite RNA for the biocontrol of plant disease. PMID- 1710091 TI - Endoscopic therapy for gastric cancer in patients more than 80 years old. AB - We prospectively studied 17 gastric cancer patients who were more than 80 yr old, in whom endoscopic therapy was performed. The patients were divided into two groups: the first group included 13 patients in whom curative treatment was attempted, and in 11 (85%) a curative response was achieved. Two patients in this group showed a positive gastric biopsy after 1 and 2 yr, respectively, of therapy: one patient is under current treatment with laser therapy, and the other patient underwent surgery with resection of the stomach. The second group included four patients in whom treatment was aimed at prolonging survival or palliation, and in all four patients, a successful response was obtained. Therefore, our results demonstrate that endoscopic therapy is effective for the treatment of gastric cancer patients over 80 yr old. PMID- 1710092 TI - Celiac disease and macroamylasemia. PMID- 1710093 TI - Cytokine-induced histamine release from basophils of AIDS patients. Interaction between cytokines and specific IgE antibodies. AB - Basophil leukocytes obtained from AIDS patients, allergic patients and healthy controls were stimulated in vitro with interleukin 4, lymphotoxin, tumour necrosis factor alpha and interferon gamma to examine the histamine releasing effect. The cytokines caused histamine release from the basophils of approximately half the AIDS patients and from 8-17% of the allergic patients. No response was obtained in the control group. Removal of cell surface immunoglobulins abolished the response to cytokines, indicating an Ig-dependent mechanism. Passive sensitization with cell-derived Ig, with Ig deprived of IgE, or with IgG, indicated that cell-bound IgE was responsible for the cytokine induced histamine release in AIDS patients. This response may be mediated by cytokine-selective IgE antibodies. PMID- 1710094 TI - Virus, bacteria and lipopolysaccharide increase basophil cell response to histamine releasing stimulators and calcium. Examination of allergic and normal individuals. AB - Histamine release from human basophil leukocytes from allergic patients or controls was induced by specific antigens, anti-IgE or calcium ionophore A23187. Influenza A virus, S. aureus and lipopolysaccharide from S. typhimurium increased the maximum release of histamine and caused a shift to the left of the dose response curves showing increased cell sensitivity and lowering of the threshold to these stimuli. The mechanism of action was elucidated by examining the mediator release as a function of increasing extracellular concentration of calcium. In these experiments the dose-response curves were changed by the microorganisms and lipopolysaccharide as before. This indicates that the microorganisms and lipopolysaccharide change the basophil cell response to IgE dependent and non-immunological stimuli by causing a change in the subcellular handling of calcium. PMID- 1710095 TI - The hexabrachion gene as a candidate for a tuberous sclerosis gene. PMID- 1710096 TI - [Outcome of herpetic encephalitis. Apropos of 10 cases]. AB - Ten cases of herpetic encephalitis in children aged 2 months to 13 1/2 years at onset are reviewed retrospectively. There were four infants and six children 3 to 13 1/2 years old. Diagnosis was established on the basis of widely accepted electroclincal and neuroradiological criteria. Biologic confirmation was obtained in only half the cases. Two patients died during the initial episode. One patient was lost to follow-up two months after the first episode. Follow-ups ranged from 2 to 10 years for the seven remaining patients. Epilepsy and neurodevelopmental impairment occurred in a significant number of cases (5/7 and 6/7 respectively). Two patients had two episodes and one had more than two episodes of encephalitis. This poor outcome is discussed, as well as possible explanations for recurrent disease and particular neuropsychologic patterns, including Kluver-Bucy syndrome seen in two patients of this series. PMID- 1710097 TI - Palliation of univentricular heart without increasing ventricular work. PMID- 1710098 TI - Aprotinin reduces intraoperative and postoperative blood loss in membrane oxygenator cardiopulmonary bypass. AB - To determine whether aprotinin can provide a significant improvement of hemostasis in cardiopulmonary bypass using a membrane oxygenator, we tested this drug in a prospective, randomized, double-blind, placebo-controlled clinical trial. The subjects were 80 male patients undergoing cardiopulmonary bypass for coronary artery bypass grafting. Forty patients received aprotinin and 40 patients served as placebo controls. Aprotinin (4 x 10(6) KIU) was given as a continuous infusion, starting before operation and continuing until after cardiopulmonary bypass; additionally, 2 x 10(6) KIU aprotinin was added to the pump prime. Intraoperative and postoperative bleeding, respectively two thirds and one third of the total perioperative blood loss, were both significantly reduced in the aprotinin-treated group (p less than 0.01). The total average perioperative blood loss, corrected to a hemoglobin concentration of 7 mmol/L, was 550 mL in the aprotinin-treated patients versus 900 mL in the control patients. This reduction in blood loss, furthermore, significantly decreased the amount of postoperative blood transfusions (p less than 0.05) and increased the percentage of patients who did not receive postoperative donor blood from 42% to 68%. Aprotinin increased the activated clotting time significantly during cardiopulmonary bypass, which led to a reduction in heparin usage. The improved hemostasis during operation, despite the prolonged activated clotting time, might even abolish the need for heparin conversion with protamine at the end of cardiopulmonary bypass, thus allowing retransfusion through cardiotomy suction to be continued, which saves the blood that is currently lost with vacuum suction. PMID- 1710099 TI - Altered serotonin activity in anorexia nervosa after long-term weight restoration. Does elevated cerebrospinal fluid 5-hydroxyindoleacetic acid level correlate with rigid and obsessive behavior? AB - To avoid the confounding influences of malnutrition or weight loss, we studied patients with anorexia nervosa at normal weight and stable dietary intake. Compared with 15 controls, 17 long-term weight-restored anorectic subjects had elevated concentrations of cerebrospinal fluid 5-hydroxyindoleacetic acid, the major serotonin metabolite, whereas levels of cerebrospinal fluid homovanillic acid, the major dopamine metabolite, were normal. Elevated levels of cerebrospinal fluid 5-hydroxyindoleacetic acid may indicate increased serotonin activity. Such activity could contribute to pathological feeding behavior. Most importantly, this study raises the question as to whether increased cerebrospinal fluid 5-hydroxyindoleacetic acid levels are associated with overly inhibited, anxious, or obsessive traits. PMID- 1710100 TI - Heterogeneity of epithelial marker expression in routinely processed, poorly differentiated carcinomas. AB - The application of immunohistochemical markers against epithelial antigens has proved useful for studying tumor differentiation and in aiding tumor diagnosis. However, the reactivity of various epithelial markers with poorly differentiated carcinomas (the situation in which they are most often used) has not been well established. As a result, it is unclear how negative results should be interpreted and how often more than one antibody may be needed to document the epithelial nature of poorly differentiated neoplasms. We studied 98 poorly differentiated epithelial tumors with AE1, CAM 5.2, and EMA to assess the use of these markers in their diagnosis. Both CAM 5.2 and EMA provided support for epithelial differentiation in 71% (70/98) of the cases, while AE1 stained 50% (49/98) of the tumors; CAM 5.2 was the single most useful marker in the subset of poorly differentiated neuroendocrine carcinomas, staining 20 (77%) of 26 tumors. Use of these markers in pairs increased the recognition of epithelial differentiation (at least one marker showing positive staining) as follows: AE1/CAM 5.2, 80% (78/98); AE1/EMA, 87% (85/98); and CAM 5.2/EMA, 99% (97/98). Thirty carcinomas stained with all three markers, 34 with two markers, and in 34 cases only one antibody supported epithelial differentiation. Twelve (21%) of 58 tumors showed evidence of S100 reactivity. None of the 71 cases to which PD7 was applied showed staining This study indicates that poorly differentiated carcinomas are heterogeneous in their expression of antigens recognized by AE1, CAM 5.2, and EMA. Moreover, these results quantitate the probability of reactivity with poorly differentiated carcinomas for each marker and support the use of one or more antibodies in a "backup" panel when a negative result is obtained with a single antibody and the diagnosis of carcinoma is still suspected. PMID- 1710101 TI - Microglandular adenosis of the breast. An immunohistochemical comparison with tubular carcinoma. AB - Microglandular adenosis (MA) of the breast is a benign, disorganized proliferation of glands lined by a single layer of cells. As such, differential diagnosis between MA and tubular carcinoma may be challenging in selected cases. A panel of antibodies was applied to 10 cases of MA and 10 of tubular carcinoma to investigate the potential benefit of immunohistochemistry in the separation of these lesions and the possible role of myoepithelial cells in MA. The luminal cells in nine cases of MA were surrounded by a cuff of muscle-specific actin reactive cells, which also coexpressed cytokeratin and vimentin. The immunophenotype of these cells is characteristic of myoepithelial differentiation, which was heretofore thought to be lacking in MA. This finding demonstrates that myoepithelial cells are indeed present in MA subjacent to luminal epithelial cells; moreover, it distinghuishes MA from tubular carcinoma, all examples of which were actin negative in this analysis. In addition, circumferential type IV collagen deposition was observed around constituent glands of MA in nine cases but was lacking in all tubular carcinomas. Other markers included in this evaluation (S100 protein, gross cystic disease fluid protein 15, carcinoembryonic antigen, estrogen receptor protein) were of no differential diagnostic value. PMID- 1710102 TI - Carcinoma (malignant mixed mullerian [mesodermal] tumor) of the uterus and ovary. Correlation of clinical, pathologic, and immunohistochemical features in 29 cases. AB - We examined the histologic, immunohistochemical, and clinical features of a series of 23 endometrial, five cervical, and one ovarian carcinosarcomas (malignant mixed mullerian [mesodermal] tumors) and nine associated distant peritoneal metastases. The primary tumors all showed epithelial differentiation (cytokeratin and/or epithelial membrane antigen expression) of the carcinomatous component, while sarcomatous areas showed epithelial differentiation in all but one case. The metastases showed uniform staining for cytokeratin (eight of eight cases) and epithelial membrane antigen (eight of eight cases), including the spindle cell component that was present in four of nine cases. Desmin significantly changed the interpretation of rhabdomyosarcoma differentiation by refuting putative rhabdomyoblasts in two cases and identifying rhabdomyoblasts in two other cases where they were unrecognized on hematoxylineosin staining. S100 protein was positive in all five cases with chondrosarcoma differentiation. Muscle-specific actin and vimentin were positive in the sarcomatous component of all cases and in the carcinomatous component of seven and 10 cases, respectively. After immunostaining, heterologous elements were present in 18 of 29 cases (11 cases of rhabdomyosarcoma, three cases of chondrosarcoma, three cases of mixed rhabdomyosarcoma and chondrosarcoma, and one case of liposarcoma). Only six of 27 patients with follow-up were disease free for 12 months or longer (associated with stage I or II disease, smaller size, no lymphatic invasion in the resection specimen, and no invasion of the outer two thirds of myometrium). Presence and type of heterologous elements, grade of sarcomatous or carcinomatous components, histologic type of carcinomatous component, gross appearance, presence of necrosis, or use of chemotherapy or radiotherapy did not affect outcome. Carcinosarcomas are clinically aggressive distinctive mixed epithelial-stromal neoplasms with histologic and immunohistochemical features that overlap with metaplastic carcinoma in many cases. PMID- 1710103 TI - Intracranial metastases from malignant pleural mesothelioma. Report of three autopsy cases and review of the literature. AB - We report three cases of brain metastases from malignant pleural mesothelioma that were seen at autopsy. We present a summarized review of 15 similar reports that were previously published. Our study included three aged male patients with a long occupational history of heavy asbestos exposure. In two patients, the metastases were discovered incidentally at autopsy, and there were no neurologic symptoms referred to before death. In the other patient, who had clinically occult mesothelioma, the intracranial tumor was discovered ante mortem: in this patient, the clinical features, as well as a computed tomographic scan, suggested a primary tumor of the brain. Interestingly, the histologic features of the latter case that were seen at autopsy depicted a spindle cell tumor that focally exhibited pseudopalisading, necrosis, vascular buds, which deceptively recalled a glioblastoma. All the three cases shared a basic sarcomatous pattern of malignant pleural mesothelioma in both primary and metastatic tumors. The immunohistochemical profile was consistent with such interpretation. It was concluded that metastases to the brain from malignant pleural mesothelioma, although rare, are not exceptional even if their clinical relevance is not prominent. They are seen concomitantly with high-grade tumors, and by mimicking a primary tumor on a clinical, instrumental, and histologic ground, they may occasionally represent a potential source of diagnostic pitfall. PMID- 1710104 TI - Primary gastric mantle zone lymphoma. A report of two cases. AB - We report two cases of primary gastric mantle zone lymphoma. Histologic examination revealed numerous lymphoid vague nodules in the mucosa and submucosa of the resected stomach. The neoplastic cells in these nodules were slightly larger than small lymphocytes and had more or less cleaved nuclei. Immunostaining on paraffin-embedded sections showed that the neoplastic cells in the nodules of these cases were LN-1- and LN-2+ and had monotypic immunoglobulin (IgM-lambda and IgM-kappa, respectively). Immunostaining on frozen tissue specimens showed that the neoplastic cells or nodules were positive for surface IgM, surface IgD, alkaline phosphatase, and DRC-1. One third to two thirds of the cells were Leu-1+ (CD5+). These results indicated that these cases were primary gastric mantle zone lymphoma. More attention should be paid to primary gastric mantle zone lymphoma because this disease might be erroneously diagnosed as either pseudolymphoma or reactive lymphoid hyperplasia of the stomach. PMID- 1710105 TI - Neuropathologic and neurochemical correlates of psychosis in primary dementia. AB - Neuropathologic and neurochemical correlates of psychosis were determined using brain tissue from 27 autopsy-confirmed cases of Alzheimer's disease. The densities of senile plaques and neurofibrillary tangles were determined in the middle frontal and superior temporal cortex, the prosubiculum, and the entorhinal cortex of the hippocampus. The concentrations of norepinephrine, dopamine, and serotonin, the metabolites of these biogenic amines, and the specific activity of choline acetyltransferase were also determined in these four cortical regions as well as in the substantia nigra, thalamus, amygdala, and caudate nucleus. Psychosis was associated with significantly increased densities of senile plaques and neurofibrillary tangles in the prosubiculum and middle frontal cortex, respectively, with trends toward increased densities of these lesions in the other areas examined. This finding is consistent with the increased rate of cognitive decline that accompanies this behavioral disorder. Psychosis was also associated with the relative preservation of norepinephrine in the substantia nigra, with trends in this direction for five of the remaining seven brain regions examined, and a significant reduction of serotonin in the prosubiculum that was accompanied by trends toward reduced levels of serotonin and 5 hydroxyindoleacetic acid in the remaining regions. The profile of neuropathologic and neurochemical changes associated with psychosis is distinct from that previously reported for major depression in the context of primary dementia. PMID- 1710106 TI - Activation of smooth muscle myosin light chain kinase activity by a monoclonal antibody which recognizes the calmodulin-binding region. AB - The regulatory domain of smooth muscle myosin light chain kinase (MLCK) was studied using monoclonal antibodies. Of the 22 monoclonal antibodies tested, a monoclonal antibody designated LKH-18 was found to activate MLCK in the absence of Ca2+/calmodulin. This activation was even greater when an Fab fragment of LKH 18 was used. Consequently, the actin-dependent smooth muscle myosin ATPase activity and the superprecipitation of actomyosin were significantly activated by MLCK plus LKH-18, even in the absence of Ca2+/calmodulin. The antibody-binding site was studied using proteolytic fragments and synthetic peptide analogues of MLCK. Immunoblot analysis revealed that LKH-18 reacted with the 66 kDa calmodulin dependent active fragment but not with the 64 kDa inactive fragment or with the 61 kDa calmodulin-independent active fragment. Furthermore, LKH-18 reacted with MLCK-(796-815)-peptide but not with MLCK-(786-801)-peptide or with MLCK-(796-807) peptide. Therefore the LKH-18-binding site was assigned to amino acid residues 808-815 of MLCK, which are thought to be a part of the calmodulin-binding site. The present results suggest that the binding of ligand to this region induces a conformation change in MLCK and that this abolishes the action of the inhibitory region which exists next to the N-terminal side of the calmodulin-binding site. PMID- 1710107 TI - Cloning of the cDNA encoding human skeletal-muscle fatty-acid-binding protein, its peptide sequence and chromosomal localization. AB - A cDNA clone for the human skeletal-muscle fatty-acid-binding protein (FABP) was isolated by screening of a human adult muscle lambda gt11 expression library with an anti-(muscle FABP) serum. The identify of the clone was confirmed by transcription/translation in vitro in plasmid pSP6.5, followed by immunoprecipitation. The nucleotide sequence of the 551 bp cDNA insert showed an open reading frame of 399 nucleotides, coding for a protein of 133 amino acid residues with a calculated molecular mass of 14858 Da and a pI of 4.94. Only one cysteine residue was found, at position 125. Peptide sequence analyses of human skeletal-muscle and heart FABP, after carbamoylmethylation and lysyl endopeptidase digestion followed by automatic Edman degradation, showed that both proteins are identical. No evidence was found for the existence of isoproteins in muscle. The chromosomal localization of the human muscle FABP gene was determined by analysing 31 human x rodent somatic-cell hybrid lines. The human muscle FABP gene could be assigned to chromosome 1pter-q31. PMID- 1710108 TI - Homology of placental protein 11 and pea seed albumin 2 with vitronectin. AB - Vitronectin (complement S-protein), a plasma and tissue glycoprotein of 75 kDa, shares the amino-terminal somatomedin B domain with the membrane glycoprotein PC1 of plasma cells and several hemopexin-type repeats with hemopexin and the non catalytic carboxy-terminal domain of collagenases. It serves as a ligand for certain integrin receptors, binds to distinct members of the serpin family and inhibits the pore-forming cytolytic reaction of the terminal complement pathway. Computer-assisted data base searches revealed the presence of a single somatomedin B domain in the recently cloned placental protein 11, and four hemopexin-type repeats in the cytosolic plant protein PA2, the major albumin of pea seeds, whose function is unknown. Our finding shows that hemopexin-type repeats are present in extracellular as well as in cytosolic proteins and most likely originated before the divergence of the animal and plant kindoms. PMID- 1710109 TI - Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. AB - Inhibition of nitric oxide (NO) synthase activity by L-NG-Nitroarginine (NO2Arg) in brain preparations is not reversed by dialysis and is enhanced by prolonged preincubation of NO2Arg with the enzyme. By contrast, the weaker inhibition by NO2Arg of macrophage NO synthase is fully reversible. NO2Arg inhibits NO synthase activity in the brain after i.p. administration of 5 or 50 mg/kg. This in vivo inhibition also appears to be irreversible. The potent in vivo inhibition of central NO synthase by NO2Arg may facilitate studies of the physiologic function of NO as a neuronal messenger. PMID- 1710110 TI - Calcium modulation of mitochondrial inner membrane channel activity. AB - Protocols were defined that enable patch-clamp studies of the approximately 107 pS voltage dependent channel and a class of activity we refer to as MCC (multiconductance channel) which is characterized by multiple levels and transitions as high as 1 to 1.5 nS. If free calcium was kept at 10(-7) M or lower during mitochondrial isolation, no activity was observed at low voltage (+/- 60 mV). If free calcium levels were higher, MCC activity was observed in about 96% of the patches. The observation of approximately 107 pS channel was enhanced from 2% to 68% of patches by washing isolated mitoplasts (mitochondria stripped of outer membrane) with EGTA. Increasing matrix calcium from 10(-9) to 10(-5) M decreased the probability of opening for the MCC and approximately 107 pS activities. PMID- 1710111 TI - A voltage, calcium, and ATP sensitive non selective cation channel in human colonic tumor cells. AB - A non selective cation channel has been identified in the human colonic cell lines T84 and HT29D4 using the patch clamp technique. The channel is equally permeable to Na+ and K+, has a linear current-voltage relationship and a conductance of about 20 pS in symmetrical NaCl conditions. The channel is not permeable to chloride or to large organic cations such as N-methyl-D-glucamine. The open probability of the channel is voltage dependent. Cytosolic Ca2+ concentrations higher than 0.1 mM are required to activate the channel. The channel is blocked by cytosolic ATP (1 mM). 3',5-dichlorodiphenylamine-2 carboxylic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid inhibit the channel when present on the extracellular side. The block is not voltage dependent. 3',5-dichlorodiphenylamine-2-carboxylic acid is the most potent blocker and completely inhibits channel activity at a concentration of 50 microM. The channel is insensitive to amiloride and derivatives. PMID- 1710112 TI - Structure and localization of the human insulin-like growth factor-binding protein 2 gene. AB - Insulin-like growth factor binding proteins (IGFBPs) are extracellular proteins that specifically bind IGF and modulate their effects. The human IGFBP2 gene was studied and shown to be localized to chromosome 2 region q33-q34, by somatic cell hybrid analysis and in situ hybridization. Structural characterization of the gene showed that it consists of four exons with three introns of lengths 27.0, 1.0, and 1.9 kilobase-pairs. Comparison of the encoded protein sequence of each exon in IGFBP1, 2, and 3 reveals the highest amino acid identity, 28%, in exon 1, while the lowest was found in exon 2. However, pairwise sequence comparisons demonstrate 50% identity between the protein sequences encoded by exon 4 in IGFBP1 and 2, while their respective identities with IGFBP3 are only 25 and 30%. PMID- 1710113 TI - Genes for the insulin receptor and the insulin-like growth factor I receptor are expressed in the chicken embryo blastoderm and throughout organogenesis. AB - The early expression of insulin and insulin-like growth factor I (IGF-I) in the chicken embryo suggests that these peptides play an important role in early development. The receptors for insulin and IGF-I, however, had not been studied at the molecular level in this model. We report two chicken sequences that, by comparison with known tyrosine kinases, appear to correspond to the tyrosine kinase domain of the insulin receptor homologue (CTK-1) and the IGF-I receptor homologue (CTK-2). Using reverse-transcription of RNA, amplification with the polymerase chain reaction (RT-PCR), and gene-specific hybridization, we demonstrate that the two genes, CTK-1 and CTK-2, are expressed in embryos at least as early as the blastoderm (Day 0), during neurulation (Day 1), and in early (Days 2-3) and late (Day 9) organogenesis. PMID- 1710114 TI - Endothelial cells have a particulate enzyme system responsible for EDRF formation: measurement by vascular relaxation. AB - Endothelium-derived relaxing factor (EDRF) released from endothelial cells (EC) has been shown to be nitric oxide (NO) or a closely related molecule. In cultured EC, the enzyme responsible for the formation of EDRF, EDRF-synthase, was initially described as being cytosolic, but more recently we have found it to be predominantly particulate. In view of this discrepancy we have investigated the EDRF synthesizing activity of cytosolic and particulate fractions isolated from native bovine aortic EC. EDRF was measured by cGMP formation in rat fetal lung cultured fibroblasts (RFL-6) and by the ability of cell fractions to relax endothelium-denuded, preconstricted rabbit aortic strips. Cytosolic fractions from native EC (100 micrograms) had no effect on the tone of rabbit aortic strips and little effect on cGMP levels in RFL-6 cells in the presence of L-arginine and NADPH (100 microM). However, under the same conditions the 100,000 x g pellet fractions relaxed rabbit aortic strips and increased cGMP levels in RFL-6 cells. Thus EDRF synthase from native EC, like those grown in culture, is located mainly in the particulate fraction. PMID- 1710115 TI - Identity between the novel integrin beta 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine. AB - A cDNA clone encoding the N-terminal sequence of the murine integrin beta 7 subunit, a novel member of the leukocyte cell adhesion molecule subset (Leu-CAM), has been isolated. An N-terminal region of 13 contiguous amino acids deduced from the cDNA shows complete identity with the N-terminus of the 120 kDa subunit of the M290 antigen, a surface molecule found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This unexpected result focuses two previously unconnected areas of research and suggests that integrins may have a special role to play in the defence of the gut mucosa. PMID- 1710116 TI - Rat ribophorin II: molecular cloning and chromosomal localization of a highly conserved transmembrane glycoprotein of the rough endoplasmic reticulum. AB - We report here the complete nucleotide sequence of rat ribophorin II. The predicted amino acid sequence is highly homologous to the corresponding human protein and consists of 631 amino acid residues, including a 22 amino acid N terminal cleavable signal sequence, and a single 23 amino acid putative transmembrane domain. Northern blot analysis reveals a single -2.4 kb message expressed in a number of rat cell lines and in adult liver. The gene was mapped to mouse chromosome 2, close to the Src proto-oncogene. PMID- 1710117 TI - Molecular cloning and expression of mouse leukotriene A4 hydrolase cDNA. AB - A cDNA clone for mouse leukotriene A4 hydrolase encoding the full sequence of the enzyme was isolated from a mouse spleen lambda ZAP-II library. The identification was ascertained by expression of enzyme activity in Escherichia coli. The encoded protein has 610 amino acids and exhibits an extensive identity (93%) with the human leukotriene A4 hydrolase. A region spanning between residues 233 and 340, where the zinc binding site is located, was found to be perfectly conserved between the two species. We found six sites of polymorphism in the cDNA sequence of mouse LTA4 hydrolase, one of which leads to a difference in the encoded amino acid. The polymorphism of cDNA was confirmed by reverse transcription-PCR sequencing of mouse spleen total RNA, prepared as a mixture from ten different strains. PMID- 1710118 TI - Expression of nuclear retinoic acid receptors in rat adipose tissue. AB - Expression of nuclear retinoic acid receptors (RAR-alpha, beta and gamma) was examined by Northern blots in rat lung, liver, and adipose tissue. Three transcripts of approximate sizes 7.3, 3.7, and 2.8 Kb were detected in adipose tissue. In contrast to adipose tissue only 3.7 and 2.8 Kb mRNA species were detected in liver and lung. Two RAR-beta gene transcripts (3.3 and 3.0 Kb) were expressed in adipose tissue, liver, and lung. RAR-gamma mRNA (3.36 Kb) was detected in adipose tissue and lung. The distribution of three RARs in adipose tissue suggests the physiological role of retinoic acid in the regulation of specific genes via RARs in adipocytes. PMID- 1710119 TI - Broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at CTP synthetase. AB - Cyclopentenylcytosine (Ce-Cyd) is a broad-spectrum antiviral agent active against DNA viruses [herpes (cytomegalo), pox (vaccinia)], (+)RNA viruses [picorna (polio, Coxsackie, rhino), toga (Sindbis, Semliki forest), corona], (-)RNA viruses [orthomyxo (influenza), paramyxo (parainfluenza, measles), arena (Junin, Tacaribe), rhabdo (vesicular stomatitis)] and (+/-)RNA viruses (reo). Ce-Cyd is a more potent antiviral agent than its saturated counterpart, cyclopentylcytosine (carbodine, C-Cyd). Ce-Cyd also has potent cytocidal activity against a number of tumor cell lines. The putative target enzyme for both the antiviral and antitumor action of Ce-Cyd is assumed to be the CTP synthetase that converts UTP to CTP. In keeping with this hypothesis was the finding that the antiviral and cytocidal effects of Ce-Cyd are readily reversed by Cyd and, to a lesser extent, Urd, but not by other nucleosides such as dThd or dCyd. In contrast, pyrazofurin and 6 azauridine, two nucleoside analogues that are assumed to interfere with OMP decarboxylase, another enzyme involved in the biosynthesis of pyrimidine ribonucleotides, potentiate the cytocidal activity of Ce-Cyd. Ce-Cyd should be further pursued, as such and in combination with OMP decarboxylase inhibitors, for its therapeutic potential in the treatment of both viral and neoplastic diseases. PMID- 1710120 TI - Disruption of Plasmodium falciparum-infected erythrocyte cytoadherence to human melanoma cells with inhibitors of glycoprotein processing. AB - Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule 1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600 700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM 1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis. PMID- 1710121 TI - Cytostatic synergism between bromodeoxyuridine, bleomycin, cisplatin and chlorambucil demonstrated by a sensitive cell kinetic assay. AB - Bromodeoxyuridine/Hoechst flow cytometry was used to analyse the interference of common cytostatic agents with cell activation and cell cycle progression of human B-cell lines. Bleomycin impaired both cell activation and G2 transit, the latter effect being oxygen dependent. The DNA alkylating agents cyclophosphamide, chlorambucil and mitomycin C caused G2 arrest, whereas cisplatin arrested cells in both the S and G2 phase of the cell cycle. Vinblastin interfered with mitosis, but in addition arrested cells in all phases of the cell cycle. The growth inhibitory action of bleomycin, cisplatin and chlorambucil was dependent upon the bromodeoxyuridine (BrdU) concentration in the culture medium. No interaction was found between BrdU and cyclophosphamide, mitomycin C and vinblastin. The cell cycle kinetic mechanism of the interaction between BrdU and bleomycin, cisplatin and chlorambucil was a potentiation of the G2 arrest. In conclusion, BrdU may be useful in clinical chemotherapy as a chemosensitizer for selected cytostatic agents. PMID- 1710122 TI - Differential effects of propranolol on the IgE-dependent, or calcium ionophore stimulated, phosphoinositide hydrolysis and calcium mobilization in a mast (RBL 2H3) cell line. AB - Our previous studies demonstrated that propranolol, an inhibitor of phosphatidic acid phosphohydrolase (PAPase) (EC 3.1.3.4) blocks the IgE-dependent mediator release from a rat mast (RBL 2H3) cell line. To continue these studies, we examined the ability of propranolol to inhibit the IgE-dependent or ionomycin mediated phosphoinositide hydrolysis and calcium mobilization in RBL 2H3 cells. RBL 2H3 cells, sensitized with mouse monoclonal anti-trinitrophenol IgE (anti-TNP IgE), were stimulated to release both histamine and peptidoleukotrienes (LT) in response to a suboptimal concentration of trinitrophenol-ovalbumin conjugate (TNP OVA) or ionomycin. Preincubation of the cells with d,l-propranolol (300 microM) significantly (P less than 0.05) inhibited the effects of both TNP-OVA and ionomycin on histamine and LT release. There was no difference in potency for the different isomers of propranolol, indicating that these effects were not a consequence of an effect on beta 2-adrenergic receptors. TNP-OVA produced a rapid hydrolysis of phosphoinositides resulting in a time-dependent increase in mono- (IP1), di- (IP2), tri- (IP3), and total inositol phosphate production. Ionomycin also produced a rapid increase in total inositol phosphate production; however, this largely reflected an accumulation of IP1. Both secretagogues produced a rapid elevation in cytosolic free calcium ([Ca2+]i); however, the effect of ionomycin maximized within a much shorter time frame than the effect of TNP-OVA. The effects of TNP-OVA on phosphoinositide hydrolysis and increase in [Ca2+]i were inhibited by propranolol over exactly the same concentration range as the effects of this compound on TNP-OVA-stimulated mediator release. In contrast, propranolol had no effect on the increase in [Ca2+]i and phosphoinositide hydrolysis in response to ionomycin. Taken together, these results suggest that PAPase/phospholipase D (PLD) (EC 3.1.4.4) activation may be a prerequisite for both IgE-dependent and ionomycin-stimulated mediator release from RBL 2H3 cells. Although other explanations are possible, the data further suggest that receptor mediated, but not ionophore-stimulated, phosphoinositide hydrolysis and [Ca2+]i in RBL 2H3 cells may be regulated by a propranolol-sensitive pathway involving possible activation of PAPase. PMID- 1710123 TI - A comparative study of histamine and K+ effects on (Ca(2+)-Mg2+)-ATPase activity in synaptosomes. AB - Histamine (10(-4) M) and 60 mM K+, but not 60 mM Na+ or 60 mM choline+, increased the maximal synaptosomal (Ca(2+)-Mg2+)-ATPase activity by 15 and 36% respectively and decreased the extrasynaptosomal Ca2+ concentration necessary to reach it. Histamine and K+ enhanced the synaptosomal (Ca(2+)-Mg2+)-ATPase activity in a concentration-dependent manner. In synaptic plasma membranes histamine (10(-4) M) and 60 mM choline+ were not able to alter the enzymatic activity, however 60 mM K+ and 60 mM Na+ elevated (Ca(2+)-Mg2+)-ATPase activity by 20 and 15%, respectively, without altering the affinity for Ca2+. Histamine effects in synaptosomes were mediated by H2 receptor stimulation. 3-Isobutyl-1-methyl xanthine (10(-4) M) potentiated (15%) the maximal histamine effect. The slow Ca2+ channel antagonists verapamil and diltiazem, both at 10(-6) M, completely inhibited K+ effects in synaptosomes, however histamine effects were only blocked by verapamil. The data suggest that K+ and histamine effects on synaptosomal (Ca(2+)-Mg2+)-ATPase activity are mediated by increases of intrasynaptosomal Ca2+ levels. Moreover, histamine effects on synaptosomal enzyme activity were mediated by cAMP. PMID- 1710125 TI - Biochemical and histological findings on the effect of fibronectin in rabbits with experimental corneal disorders. AB - The effects of purified plasma fibronectin (FN) in corneal disorders were investigated. Histological findings, the levels of ascorbic acid (AA) and glutathione (GSH) in tear fluids were observed sequentially in rabbits with experimental corneal damage and the degree of corneal lesions was observed. The results indicated that healing of the tissue damage was more promoted in the FN group than in the controls and it was suggested that FN promoted the healing of corneal disorders. PMID- 1710124 TI - Analysis of the binding of fluorescent ligands to soluble proteins. Use of simultaneous non-linear least squares regression to obtain estimates of binding parameters. AB - The binding of three fluorescent ligands (warfarin, dansylsarcosine and 1-anilino 8-naphthalene sulphonate) to human albumin was analysed using simultaneous non linear least squares regression analysis. Both mock and actual fluorescence data were examined and the results indicated that reliable estimates of the binding parameters as well as the molar fluorescence of bound ligand could be obtained. The advantage of this method of analysis is that it makes full use of all the experimental data and it eliminates the need for the graphical procedures usually employed to estimate the molar fluorescence of bound ligand and its binding constants. This type of analysis can be extended to other systems where some physical property of the bound ligand varies with increasing protein concentration. PMID- 1710126 TI - [Restoration of the Sistine Chapel: a microbiological study aimed at the preservation of the work of art]. PMID- 1710127 TI - [Compulsory vaccination and immune status in a group of young people at the time of entering secondary school]. PMID- 1710128 TI - [Incidence of tumors of the large intestine in a Local Health Unit. Screening for early diagnosis and cost-benefit analysis]. PMID- 1710129 TI - [Screening for colorectal cancer in a Local Health Unit. Initial evaluation of different promotion methods]. PMID- 1710130 TI - The dynamical and psycho-behavioural adaptations among ancestral primates and the human-like ecology. PMID- 1710131 TI - [Findings on the prevalence of serum markers of hepatitis C (HCV) in a group of 778 blood donors]. PMID- 1710132 TI - [Regulation of clinical research in European countries]. PMID- 1710133 TI - [Hygienic-sanitary aspects relating to the use of swimming pools]. PMID- 1710134 TI - [Dental caries in schoolchildren in relation to variables of the nutrition type and social factors]. PMID- 1710135 TI - Neovascularisation: has the angiogenic factor already been found? AB - Recently many soluble growth factors capable of influencing neovascularisation (angiogenesis) have been isolated and molecularly cloned. As such they are now available in a highly purified and active form. One or several of these already quite well known molecules may be of importance in the control of ocular neovascularisation. This article reviews what is presently known about growth factor control of neovascularisation with particular emphasis on both the eye and those factors that have already been molecularly cloned. In addition several recently reported inhibitors of neovascularisation are discussed. Such research is of particular interest to the ophthalmologist as knowledge gained in this area may allow for the use of both growth factors as well as growth factor inhibitors in the management of several ocular diseases involving neovascularisation. PMID- 1710136 TI - Combined oral contraceptives containing chlormadinone acetate and breast cancer: results of a case-control study. AB - The main subject of this hospital-based case-control study was the possible relationship between use of combined oral contraceptives (OCs) containing chlormadinone acetate and breast cancer. Analyses were based on data from 490 cases with newly diagnosed breast cancer and 1,223 controls and were separately performed for combined OCs with and without chlormadinone. For either of the combined OCs, risk was not elevated in ever users, did not increase with duration of use and did not change with time since initial exposure or with time since most recent use. However, the relative risk was increased in current users: RR = 1.72 (0.88, 3.36) for combined OCs with chlormadinone and RR = 1.42 (1.01, 2.00) for combined OCs without chlormadinone, which is, however, explained as a screening effect. These results show that chlormadinone as a constituent of combined OCs does not influence breast cancer risk. PMID- 1710137 TI - Serum and urinary levels of beta human chorionic gonadotrophin in patients with transitional cell carcinoma. AB - Serum and early morning urine specimens were obtained from 62 patients. The levels of beta human chorionic gonadotrophin (BhCG) in both serum and urine were estimated simultaneously in all cases. At the time of estimation 43 patients had transitional cell carcinoma of the bladder, one had transitional cell carcinoma of the renal pelvis and three had carcinoma in situ (two of whom also had overt carcinoma). Raised serum and urinary levels were found in only three patients, all of whom had poorly differentiated or metastatic transitional cell carcinoma of the bladder. This observation is in accordance with previous studies. In one of these patients, who underwent transurethral resection of her bladder tumour, the urinary levels returned to within normal limits post resection. An additional three patients had elevations of serum BhCG. Two of these three patients had poorly differentiated transitional cell carcinoma present at the time of estimation and one had no sign of recurrence. Using immunoperoxidase staining a retrospective study was undertaken to stain all available sections belonging to patients studied to observe whether BhCG was being produced by the respective tumours. Twelve well differentiated, nine moderately well differentiated and seven poorly differentiated specimens were available. In no case was evidence of BhCG production demonstrated in these tumours despite its presence being demonstrable in positive controls. We confirm the production of BhCG associated with bladder tumours, a feature correlated with poorer differentiation. Studies employing larger patient numbers are necessary to clarify the role of this tumor marker in patients with well differentiated bladder tumours. PMID- 1710138 TI - Epitope analysis of cluster 1 and NK cell-related monoclonal antibodies. AB - By flow cytometric assays, we tested the antibodies of the Second International Workshop on Small Cell Lung Cancer Antigens against 20 normal peripheral leukocytes, four small cell lung cancer (SCLC) cell lines (one classic type, and three variant type) and one gastric cancer line (KATO 3). Thirteen antibodies (Code # 4, 12, 21, 31, 34, 41, 48, 58, 60, 61, 74, 77, 82) among 98 registered antibodies showed a very similar pattern to antibody NE150, which was previously characterised as SCLC cluster 1. Since NE150 showed a positive reaction to the natural killer (NK) cell population, the serological specificity was compared with NK cell-associated antibodies, NKH1 (CD56), Leu7 (CD57) and Leu11 (CD16). Only NKH1 antibody showed a similar pattern to NE150, when tested against various target cells including SCLC lines and peripheral leukocytes, suggesting that NKH1 is a cluster 1 antibody, although it was already classified as CD56 of hematopoietic cells. By sequential immunoprecipitation, the antigen detected by NE150 antibody was depleted by preincubation with NKH1 antibody, but the reactivity of NE150 was not inhibited by NKH1 antibody, suggesting that NE150 and NKH1 detect different epitopes on the same antigen molecule. Epitope analysis was also conducted with 13 antibodies of cluster 1. Ten were found to detect the same epitope as NE150. The other three did not inhibit the binding of NE150 or NKH1, suggesting that there are at least three epitopes. Since the cluster 1 antibodies were demonstrated to detect NCAM, the present results suggest the presence of at least three epitopes on this molecule. PMID- 1710139 TI - Expression of the myelomonocytic antigens L1 and CD36 in human epidermis. PMID- 1710140 TI - Variable keratin polypeptide profile in human stratum corneum. AB - Stratum corneum samples obtained from 46 members in three generations of seven families were analyzed for keratin pattern by gel electrophoresis. All these samples of apparently normal upper arm skin expressed the 55 kDa, 56.5 kDa, and 65 kDa keratin proteins; while only 28%, 20% and 48% of the samples expressed the 50 kDa, 58 kDa, and 67 kDa proteins, respectively. The keratin phenotype was identical in all members of two families (9 individuals) and variable in members of five other families (37 individuals), in whom the patterns were consistent with autosomal dominant inheritance. These results demonstrate inter-individual variations in stratum corneum keratin pattern and may reflect either polymorphism of genes coding for the various keratin polypeptides or a post transcriptional modification of mature keratins by proteolytic digestion. PMID- 1710141 TI - Immunohistochemical comparison of actinic reticuloid with allergic contact dermatitis. AB - Biopsy specimens of chronic lesions and ultraviolet-induced lesions from actinic reticuloid patients were examined by immunoperoxidase techniques and compared with those of allergic contact dermatitis skin, one of the delayed-type hypersensitivity conditions. Each lesion of actinic reticuloid showed a clear predominance of suppressor/cytotoxic T cells to helper/inducer T cells and an increase of Langerhans cells in the epidermis and the dermis. These findings are generally similar to those in the late phase (on day 7 and 11) but not in the early phase (on day 2) of allergic contact dermatitis and suggest that delayed type hypersensitivity might be involved in some parts of the pathogenesis of actinic reticuloid. CD36+DR+ epidermal cells were also observed in ultraviolet induced lesions from actinic reticuloid patients, suggesting a possible role in the modulation of the mechanism. PMID- 1710142 TI - Life and times of E. coli and its friends: small science at the cutting edge. Molecular genetics of bacteria and phages sponsored by Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA August 21-26, 1990. PMID- 1710143 TI - Reciprocal expression of NF-IL6 and C/EBP in hepatocytes: possible involvement of NF-IL6 in acute phase protein gene expression. AB - The initial phase of inflammation is accompanied by dramatic changes in the concentrations of certain plasma proteins. Interleukin-6 (IL-6) is an important inducer of these acute phase proteins at the transcriptional level. The recently cloned nuclear factor NF-IL6, a potent trans-acting regulator of IL-6 gene expression, has a region that is highly homologous to the liver-specific transcriptional factor C/EBP. Both factors recognize the same nucleotide sequence. In this study the recombinant NF-IL6 was shown to interact with the IL 6-responsive elements (IL-6REs) identified in the promoter region of several acute phase protein genes whose activity increases during the acute phase reaction. Furthermore, in competition experiments, formation of all the DNA protein complexes by the IL-6RE and IL-6-treated hepatoma cell extracts was specifically decreased by adding either the 14-bp NF-IL6 binding motif identified in the IL-6 promoter or the antibody against the recombinant NF-IL6. NF-IL6 was expressed at a minor level in mouse liver, but was dramatically induced after stimulation with IL-6. In contrast, the amount of C/EBP mRNA decreased considerably after IL-6 stimulation. These results indicate that the NF-IL6 that regulated IL-6 expression was also involved in regulation of expression of the acute phase protein genes. The ability of NF-IL6 to replace C/EBP may explain the positive and negative acute phase responses induced by IL-6. PMID- 1710144 TI - Hydrazide pharmaceuticals as conjugates to polyaldehyde dextran: syntheses, characterization, and stability. AB - The coupling of ellipticine and CI-921, two antineoplastic pharmaceuticals, to polyaldehyde dextran can be carried out under mild reaction conditions with novel acid hydrazides of the parent drugs. The resulting acyl hydrazones resist hydrolytic and enzymatic cleavage and offer a method for drug loading via dextran conjugates to monoclonal antibodies. PMID- 1710146 TI - Growth factors in human cancer. PMID- 1710145 TI - Photo-cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to single-stranded DNA. AB - The preparation of oligodeoxyribonucleoside methylphosphonates derivatized with 3 [(2-aminoethyl)carbamoyl]psoralen [(ae)CP] is described. These derivatized oligomers are capable of cross-linking with single-stranded DNA via formation of a photoadduct between the furan side of the psoralen ring and a thymidine of the target DNA when the oligomer-target duplex is irradiated with 365-nm light. The photoreactions of (ae)CP-derivatized methylphosphonate oligomers with single stranded DNA targets in which the position of the psoralen-linking site is varied are characterized and compared to results obtained with oligomers derivatized with 4'-[[N-(aminoethyl)amino]methyl]-4,5',8-trimethylpsoralen [(ae)AMT]. It appears that the psoralen ring can stack on the terminal base pair formed between the oligomer and its target DNA or can intercalate between the last two base pairs of the oligomer-target duplex. Oligomers derivatized with (ae)CP cross-link efficiently to a thymidine located in the last base pair (n position) or 3' to the last base pair (n + 1 position) of the target, whereas the (ae)AMT derivatized oligomers cross-link most efficiently to a thymidine located in the n + 1 position. The results show that both the extent and kinetics of cross-linking are influenced by the location of the psoralen-linking site in the oligomer target duplex. PMID- 1710147 TI - Biotherapy after marrow transplantation and the use of hematopoietic growth factors. PMID- 1710148 TI - Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. AB - We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells. PMID- 1710149 TI - Further examination of the effects of recombinant cytokines on the proliferation of human megakaryocyte progenitor cells. AB - The effect of several recombinant cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-1 alpha, on megakaryocyte (MK) colony formation by a normal human bone marrow subpopulation (CD34+ DR+), enriched for the MK colony-forming unit (CFU-MK), was studied using a serum-depleted, fibrin clot culture system. IL-3 and GM-CSF, but not IL-6 or IL-1 alpha, stimulated MK colony formation by CD34+ DR+ cells. However, the addition of IL-1 alpha to CD34+ DR+ cultures containing IL-6 resulted in the appearance of CFU-MK-derived colonies, suggesting that IL-6 requires the presence of IL-1 alpha to exhibit its MK colony-stimulating activity (MK-CSA). Addition of neutralizing antibodies to IL-3 and GM-CSF, but not to IL-6 and IL-1 alpha, specifically inhibited the MK-CSA of IL-3 and GM-CSF, respectively. The addition of either anti-IL-6, anti-IL-1 alpha, or anti-IL-3 antisera to cultures containing both IL-6 and IL-1 alpha totally abolished the MK CSA of the IL-6/IL-1 alpha combination. However, neither anti-IL-3 nor anti-GM CSF antisera could totally neutralize the additive effect of the combination of IL-3 and GM-CSF on MK colony formation, indicating that these two cytokines act by affecting distinct effector pathways. These results suggest that while IL-3 and GM-CSF can directly affect CFU-MK-derived colony formation, IL-1 alpha and IL 6 act in concert to promote de novo elaboration of IL-3 and thereby promote CFU MK proliferative capacity. PMID- 1710150 TI - Proliferative responses to interleukin-3 and granulocyte colony-stimulating factor distinguish a minor subpopulation of CD34-positive marrow progenitors that do not express CD33 and a novel antigen, 7B9. AB - Human hematopoietic colony-forming cells (CFC) express the CD34 antigen (CD34+) as well as differentiation antigens such as CD33 and HLA-DR. CD34+ cells that do not express these latter differentiation antigens have been shown to contain few CFC in direct culture, but generate increasing numbers of CFC when cultured over a marrow stromal cell layer in the long-term culture system. In this study we determined if CD34+ cells with low or absent expression of CD33 and a novel antigen, 7B9 (CD34+CD33-7B9-), could be distinguished from CD34+ cells expressing these antigens (CD34+CD33+7B9+) based on their proliferative responses to interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) in a short term liquid culture system. These two populations were separated by fluorescence activated cell sorting, cultured with IL-3 (10 ng/mL), G-CSF (100 ng/mL), or IL-3 and G-CSF, and 3H-thymidine uptake was measured. CD34+CD33-7B9- cells proliferated in the presence of IL-3, but not G-CSF. However, a synergistic response to the combination of IL-3 and G-CSF was seen in most experiments. In contrast, CD34+CD33+7B9+ cells proliferated in the presence of either IL-3 or G CSF but did not display an additive or synergistic response to the combination of IL-3 and G-CSF. In colony-forming assays performed before and after liquid culture, the CD34+CD33-7B9- cells in two experiments contained 0.3% and 2.2% of all sorted marrow CFC before liquid culture and generated 40-fold and ninefold increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), respectively, after liquid culture with IL-3 and G-CSF. In contrast, the CD34+CD33+7B9+ cells contained 99.7% and 97.8% of all sorted marrow CFC before liquid culture and had no change or a threefold increase in the number of CFU-GM, respectively, after liquid culture with IL-3 and G-CSF. Single-cell liquid cultures containing IL-3 and G-CSF with cells that were either CD34+CD33-7B9- and depleted of mature lymphoid cells (CD34+lin-) or were CD34+lin+ showed that a higher proportion of wells containing a CD34+lin- cell gave rise to one or more CFC (8.7%) than did wells containing a CD34+lin+ cell (2.9%), with the responding cells in the former population giving rise to an average of 2.9 +/- 0.6 CFC and in the latter population, 2.0 +/- 1.0 CFC.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1710151 TI - Comparative analysis of the in vivo radioprotective effects of recombinant granulocyte colony-stimulating factor (G-CSF), recombinant granulocyte-macrophage CSF, and their combination. AB - The purpose of the present study was to evaluate and compare the in vivo radioprotective effects of pre-total body irradiation (TBI) conditioning with recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF) in a large series of lethally and supralethally irradiated mice. Also analyzed were the radioprotective effects of simultaneous as well as sequential combinations of rG-CSF and rGM-CSF. Our findings in 1,180 mice provide direct evidence that in vivo administration of rG CSF or rGM-CSF before TBI protects a significant fraction of mice from the lethal effects of LD100/30 TBI. At equivalent doses, rG-CSF displayed a more potent radioprotective activity than rGM-CSF. Not only was rG-CSF radioprotective at much smaller doses than rGM-CSF, the survival rate after lethal TBI was also significantly higher in mice receiving optimally radioprotective doses of rG-CSF as compared with mice receiving optimally radioprotective doses of rGM-CSF. Pretreatment of mice with rGM-CSF markedly attenuated the radioprotective affects of rG-CSF in lethally as well as supralethally irradiated mice. Pretreatment with rG-CSF followed by rGM-CSF was slightly more effective than rG-CSF alone in supralethally irradiated mice but not in lethally irradiated mice. Notably, marked differences among different strains of mice were noted regarding the optimally radioprotective doses of rG-CSF or rGM-CSF as well as probability of survival and median survival time after lethal or supralethal TBI. This report confirms and extends previous studies concerning the potential of cytokines in prevention or therapy of lethal radiation injury. PMID- 1710152 TI - Expression of interleukin-6 and interleukin-6 receptor in Hodgkin's disease. AB - Interleukin-6 (IL-6) is a multipotent lymphokine that can mediate differentiation of B cells into Ig-secreting cells, stimulate the growth of plasmacytomas, hybridomas, and T cells, and induce acute-phase proteins in liver cells. It has been suggested that IL-6 is involved in the pathogenesis of several diseases by autocrine or paracrine pathways. To examine whether IL-6 is possibly involved in the pathophysiology of Hodgkin's disease (HD), we analyzed the expression of IL-6 and IL-6 receptor mRNA and protein in cell lines and primary specimens from patients with HD. IL-6-specific transcripts were detected in three of six HD derived cell lines by Northern blot analysis. In the culture supernatants of four HD-derived cell lines, IL-6 was detected by radioimmunoassay. Biologic activity of IL-6 was confirmed by proliferation of an IL-6-dependent cell line. In situ hybridization experiments showed IL-6-specific transcripts in Hodgkin (H) and Reed-Sternberg (RS) cells in primary tissues of two patients. In addition, mRNAs specific for the IL-6 receptor were detected in five HD-derived cell lines. Immunostaining experiments showed expression of IL-6 receptor molecules on H and RS cells in 8 of 16 cases with HD. Thus, our data suggest that IL-6 might be involved in the pathophysiology of HD. PMID- 1710153 TI - Point mutations in the beta-subunit of cytochrome b558 leading to X-linked chronic granulomatous disease. AB - The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron. PMID- 1710154 TI - The influence of growth factors on the proliferative potential of normal and primary breast cancer-derived human breast epithelial cells. AB - In previous studies, we developed serum-free, bovine pituitary extract (BPE)-free culture conditions for the growth of normal and neoplastic rat mammary epithelial cells. The present studies were aimed at determining if these culture methods could be used to study the influence of specific growth factors on the proliferative potential of normal human mammary epithelial (HME) cells and cells derived from human breast cancer (HBC) specimens. Our results indicate that normal HME cells in primary culture express stringent requirements for insulin (IN), epidermal growth factor (EGF), and cholera toxin (CT). Of these factors, EGF is most important, with essentially no proliferation taking place in the absence of this factor. By contrast, when cells are grown in serum-free primary culture in the presence of a full complement of growth factors and then subcultured, growth in secondary culture is not influenced by the removal of individual growth factors. Growth in secondary culture in the absence of EGF is mediated by autocrine factors secreted by the cells. However, there is no evidence for autocrine activity that mediates growth in the absence of IN in secondary cultures. Primary culture of HBC cells in serum-free, BPE-free medium revealed two patterns of growth factor requirements. One set of HBC cells expressed identical requirements for IN and EGF in primary culture as normal cells. Likewise, these cells grew in secondary culture in the absence of either factor. The second set of tumors expressed independence of IN for growth in primary culture. These cells grew to confluence in primary culture in the absence of IN and could be subcultured in this medium. All tumor cells examined expressed a requirement for EGF for primary culture growth, whereas none of the HBC cells examined expressed a significant CT requirement. In many cases, growth in the absence of CT exceeded that observed in its presence. Thus, our culture system allows analysis of the growth factor requirements of HME and HBC cells in primary culture. Our results indicate significant differences between HME and HBC cells in this regard. However, the results of secondary culture experiments indicate that the growth factor milieu from which cells are taken can have a profound effect on the requirements for growth factors in culture. PMID- 1710156 TI - Accumulation levels of organochlorine pesticides in human adipose tissue and blood. PMID- 1710155 TI - Loss of basal cell phenotype with acquisition of lung-colonizing capability in mouse mammary tumors. AB - A transplantable pregnancy-dependent mouse mammary tumor, TPDMT-4, and its ovarian-dependent (T4-OR26) and autonomous (T4-OI96, T4-OI145, T4-OI165, T4-OI320 and T4-OI320CY) sublines were examined immunohistochemically for the expression of keratin 14 and type IV collagen. T4-OI96, T4-OI145, and T4-OI165, but not T4 OR26, T4-OI320, or T4-OI320CY, formed lung colonies (metastasis) after intravenous injection as a single-cell suspension. Despite the similar morphology of TPDMT-4 and its six sublines, only TPDMT-4 and the nonmetastatic sublines revealed a basal cell phenotype as defined by keratin 14 expression. Staining for type IV collagen was complete at the peripheries of the glandular structures in TPDMT-4 and nonmetastatic sublines but was patchy in the metastatic tumors. PMID- 1710157 TI - Chlorpyrifos in the air and soil of houses four years after its application for termite control. PMID- 1710158 TI - Influence of slurry and ammonium nitrate fertilizations on soil and plant metabolism of chlorpyrifos in field cauliflower. PMID- 1710159 TI - Lindane toxicity to one year old calves. PMID- 1710160 TI - South American snake venom proteins antigenically related to Bothrops asper myotoxins. AB - 1. The presence of proteins antigenically related to Bothrops asper myotoxins in various snake venoms, mainly from South America, was investigated by using polyclonal and monoclonal antibodies. 2. Myotoxin-like components were detected in ten Bothrops venoms from South America, and in the venoms of Crotalus atrox (North America), Trimeresurus flavoviridis (Japan), and Micrurus alleni (Costa Rica). 3. Cross-reactive components detected in several Bothrops venoms show a common subunit of 15-16 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, although significant charge variations are evident by immunoelectrophoresis. 4. It is concluded that proteins antigenically related to B. asper myotoxins are relatively common in the genus Bothrops and, in the light of findings discussed, are likely to possess myotoxic activity. PMID- 1710161 TI - Comparison of alpha 1 adrenoceptors in the prostate capsule of men with symptomatic and asymptomatic benign prostatic hyperplasia. AB - The objective of this study was to compare the binding and functional properties of alpha 1 adrenoceptors in prostate capsules obtained from men with symptomatic and asymptomatic benign prostatic hyperplasia (BPH) undergoing simple retropubic prostatectomy and cystoprostatectomy respectively. Saturation experiments using 125I-Heat demonstrated that the density and binding affinity of alpha 1 adrenoceptors in the prostate capsules obtained from men with symptomatic and asymptomatic BPH were similar. Non-cumulative dose response experiments using phenylephrine demonstrated that the magnitude of the contractile response to phenylephrine was 4-fold greater in the prostate capsules from men with symptomatic BPH than from those with asymptomatic BPH. The EC50 of phenylephrine in the prostate capsules of men with symptomatic and asymptomatic BPH was similar. A correlation between alpha 1 adrenoceptor density and phenylephrine Emax was not observed, implying that either alpha 1 adrenoceptors are not localised exclusively to the prostate smooth muscle or that spare alpha 1 adrenoceptors exist. This study suggests that the neuropharmacological properties of the prostate capsule may play a significant role in the development of infravesical obstruction in the ageing male population. PMID- 1710162 TI - Indoramin in the treatment of prostatic bladder outflow obstruction. AB - A series of 40 patients took part in a double-blind, placebo-controlled trial of indoramin in prostatic bladder outflow obstruction. Patients were assessed clinically and urodynamically before and after 4 weeks' treatment. Significant improvement was seen in nocturia, volume voided, flow rates and residual urine. The drug was well tolerated, although 7 patients on treatment and 7 on placebo noted side effects. These results suggest that indoramin may be a useful agent in the symptomatic management of bladder outflow obstruction. PMID- 1710163 TI - Histological grading of squamous cell carcinoma of the penis: a new scoring system. AB - A system of histological grading based on retrospective analysis of 239 patients with squamous cell carcinoma of the penis is presented. A new scoring system with 4 histological grades was used. The results of this study confirm previous reports that penile cancer is usually a highly (50%) or moderately (29%) differentiated squamous cell carcinoma. Poorly differentiated carcinomas and cancers of other types are very rare. The new grading system was found to be practical and a correlation between histological grade, clinical findings and prognosis was established. Patients with grade 1 tumours had an exceptionally favourable prognosis, with more than 80% being long-term survivors; for these patients, treatment with delayed side effects should be avoided and new forms of treatment should be explored. PMID- 1710164 TI - c-fos and ornithine decarboxylase gene expression in brain as early markers of neurotoxicity. AB - An increase of proto-oncogene c-fos expression in cerebral cortex of rats treated with subconvulsant doses of the pesticide organochlorine lindane (gamma hexachlorocyclohexane) has been detected using Northern blots. Immunohistochemical studies show that Fos protein was already increased in neuronal nuclei 3 h after treatment. The administration of the benzodiazepine diazepam prior to lindane totally blocked the activation of this proto-oncogene expression. Parallel to this increased expression of c-fos an activation of the ornithine decarboxylase (ODC) gene and enzyme was also observed. High levels of ODC mRNA and increased enzyme activity in cortex were found in rats following lindane treatment. These changes were attenuated by prior treatment of animals with diazepam. The co-induction of c-fos and ODC suggests a potential link between the ODC/polyamine system and the short-acting proto-oncogenes in stimulus transcription coupling events. PMID- 1710165 TI - Detection of the membrane inhibitor of reactive lysis (CD59) in diseased neurons of Alzheimer brain. AB - The membrane inhibitor of reactive lysis (MIRL) protects host cells from complement-mediated lysis. It was detected immunohistochemically in tangled neurons and dystrophic neurites of Alzheimer disease (AD) tissue in a pattern highly similar to that observed for the membrane attack complex of complement, C5b-9. MIRL was also detected in cultured IMR-32 neuroblastoma cells. The mRNA for MIRL was detected in RNA extracts of both AD and normal brain. These data provide the first evidence of brain neuronal expression of MIRL and its upregulation in neurons exposed to complement attack. They are consistent with the previously advanced hypothesis that complement-mediated neuronal injury may play a role in AD. PMID- 1710166 TI - Comparison of oxidative damage to rat liver DNA and RNA by primary nitroalkanes, secondary nitroalkanes, cyclopentanone oxime, and related compounds. AB - The hepatocarcinogen 2-nitropropane causes oxidative damage to liver DNA and RNA after administration to rats; increases in 8-hydroxydeoxyguanosine and formation of an unknown moiety (DX1) in DNA, plus increases in 8-hydroxyguanosine and the appearance of two unidentified peaks (RX1 and RX2) in RNA were observed by high performance liquid chromatography of nucleosides from 2-nitropropane-treated rats using electrochemical detection (E. S. Fiala et al, Cancer Res., 49:5518-5522, 1989). In the present study, damage to Sprague-Dawley rat liver RNA and DNA was assessed to determine whether the characteristic pattern of oxidative nucleic acid damage caused by 2-nitropropane also occurred after i.p. administration of primary nitroalkanes, other secondary nitroalkanes, 2-methyl-2-nitropropane (a tertiary nitroalkane), and cyclopentanone oxime. All of the secondary nitroalkanes and cyclopentanone oxime significantly increased levels of 8 hydroxyguanine in both DNA and RNA and caused the appearance of DX1, RX1 and RX2. The primary nitroalkanes and the tertiary nitroalkane 2-methyl-2-nitropropane did not cause a similar pattern of nucleic acid damage. The rates of reprotonation of nitronates of the secondary nitroalkanes to the respective un-ionized neutral forms at pH 7.7 were more than 20-fold less than the rates of reprotonation of primary nitroalkane nitronates, suggesting that the anionic nitronates, rather than neutral compounds, are more immediately responsible for the DNA and RNA damage observed in vivo. Since 8-hydroxyguanine is a miscoding lesion in DNA, these results suggest the possibility, still to be rigorously tested, that hepatocarcinogenicity may be associated not only with 2-nitropropane but also with other secondary nitroalkanes as well as with those ketoximes that are capable of being converted to secondary nitroalkanes in vivo. PMID- 1710167 TI - Phase I/pharmacokinetic reevaluation of thioTEPA. AB - Because the initial evaluation of N,N',N''-triethylenethiophosphoramide (thioTEPA) preceded the standardized approach to the Phase I trials, uncertainty surrounds the recommended dose. Since it has recently been demonstrated that an almost 100-fold increase in dose can be administered in bone marrow transplant regimens, we conducted a Phase I reevaluation of thioTEPA. ThioTEPA was administered i.v. in 50 ml 5% dextrose in water over 10 min. Twenty-seven patients were entered at doses ranging from 30 to 75 mg/m2. The major toxic effect was myelosuppression; thrombocytopenia greater than or equal to grade 3 occurred in four of seven patients, and leukopenia greater than or equal to grade 3 in two of seven patients at 75 mg/m2. Among eight patients at 65 mg/m2 only two had greater than or equal to grade 3 myelosuppression making this the recommended new phase II dose for the majority of patients. Moderate (grade 2) easily controlled nausea and vomiting was the only other major side effect. There was no alopecia or mucosal or neurological toxicity. Three partial remissions were observed among nine previously treated ovarian cancer patients. Plasma concentrations of thioTEPA and its major active metabolite triethylenephosphoramide (TEPA) were measured by gas chromatography. The half life of thioTEPA ranged from 51.6 to 211.8 min, and its pharmacokinetics was dose dependent; total body thioTEPA clearance decreased with increasing dose. The half life of TEPA was considerably longer than that of the parent compound (3.0 to 21.1 h); as a result, the area under the plasma concentration-time curve (AUC) of TEPA was severalfold greater than that of the parent compound. The ratio of TEPA AUC to thioTEPA AUC decreased with increasing dose, suggesting that formation of TEPA is a saturable step in elimination. The AUC and total body clearance of thioTEPA, but not of TEPA, were closely correlated with neutrophil but not platelet toxicity. PMID- 1710168 TI - Monoclonal anti-idiotypic antibodies to human melanoma-associated proteoglycan antigen: generation and characterization of anti-idiotype antibodies. AB - We have characterized a number of monoclonal anti-idiotype antibodies (mAb2s) made against a monoclonal antitumor antibody, MEM136. The monoclonal antibody 1 (mAb1) MEM136 recognizes an epitope on human melanoma-associated proteoglycan and blocks melanoma cell interaction with basement membrane components in vitro. The anti-idiotype antibodies (Ab2s) made against MEM136 each cross-inhibited, to varying degrees, their binding to MEM136. Thus, the mAb2s recognized overlapping idiotopes on MEM136. In an attempt to identify potential internal image candidates we set up a cell migration inhibition assay. In this assay, migration of melanoma-associated proteoglycan-positive Colo38 cells was determined through a membrane barrier impregnated with Matrigel, which is composed of extracellular matrix components, i.e., collagen type IV, heparan sulfate, and laminin. Interestingly, only Ab2s IM06 and IM32 inhibited melanoma cell migration. Additional studies indicate that of eight mAb2s tested, only IM32 and IM06 induced anti-MPG responses in rabbits. The possibility that IM32 and IM06 bear images of melanoma cell surface-associated proteoglycan epitopes is discussed. PMID- 1710169 TI - Angiogenesis determines blood flow, metabolism, growth rate, and ATPase kinetics of tumors growing in an irradiated bed: 31P and 2H nuclear magnetic resonance studies. AB - Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased and growth rate is slowed, these tumors are assumed to be metabolically deprived with hypoperfusion. In this study, we directly measured the effect of tumor bed irradiation on blood flow, growth rate, rate of nucleoside triphosphate (NTP) turnover, and metabolic state using 31P and 2H nuclear magnetic resonance, and an intradermal assay for angiogenesis. (NTP turnover refers to ATP-synthetase mediated NTP turnover that is visible to 31P nuclear magnetic resonance using the technique of saturation transfer.) A decrease in the number of small blood vessels perfusing tumors in a preirradiated bed was found. Most of the decrease was due to a loss of vessels with diameters less than 0.04 mm. When tumors growing in preirradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed in most tumors. Consistent with these observations, the phosphocreatine/inorganic phosphate and nucleoside triphosphate/inorganic phosphate ratios were significantly lower in the tumors growing in a preirradiated bed compared with tumors in a nonirradiated bed. The blood flow rate was similar to control for tumors less than 100 mm3 (45.8 versus 40.5 ml/100 g/min, P = not significant), but was significantly lower than control for tumors greater than 100 mm3 (40.4 versus 12.2 ml/100 g/min, P less than 0.01). The NTP turnover rates correlated (P less than 0.005, r = 0.66) with the volume doubling rate (1/tumor volume doubling time), but for tumors approximately 100 mm3 in size neither the volume doubling rate nor the NTP turnover rate of tumors growing in an irradiated bed was statistically lower than control [NTP turnover: 14 +/- 3%/s versus 9 +/- 2%/s; volume doubling rate: 0.47 +/- 0.07/day versus 0.33 +/- 0.04/day (mean +/- SE)]. A large intertumor variability of all metabolic parameters was observed. PMID- 1710170 TI - Regulation of growth by a nerve growth factor-like protein which modulates paracrine interactions between a neoplastic epithelial cell line and stromal cells of the human prostate. AB - Nerve growth factor-like substance(s) were identified in both conditioned media of a human prostatic tumor epithelial cell line (TSU-pr1) and a human prostatic stromal cell line (HPS) by Western blot analysis and bioassay of neurite outgrowth of PC12 cells. Nerve growth factor-beta (NGF) immunofluorescence was also localized to secretory vesicles in the cytoplasm of both the TSU-pr1 and HPS cells. Western blot of the TSU-pr1 and HPS cell-secreted protein identified an Mr 65,000 major protein which immunoreacted with murine NGF antibody. NGF Western blot of HPS cell-secreted protein also identified an Mr 42,000 minor band under reduced and nonreduced conditions and an Mr 61,000 minor band under reduced conditions. The secreted protein from the TSU-pr1 cells (50 micrograms/ml) and HPS (50 micrograms/ml), as well as murine NGF (50 ng/ml) or human recombinant NGF (50 ng/ml), stimulated neurite outgrowth from PC12 cells. This neurite outgrowth activity was partially inhibited by treatment with NGF antibody. Neither the serum containing growth medium nor bovine serum albumin (50 micrograms/ml) stimulated neurite outgrowth. The NGF-like secretory protein appeared to play a role in the paracrine regulation of prostatic growth between TSU-pr1 cells and HPS cells. The relative growth of TSU-pr1 cells, as indicated by [3H]thymidine incorporation, in response to HPS secretory protein was stimulated 2.8-fold in a dose-dependent manner. In the converse interaction, the relative growth of HPS cells in response to TSU-pr1 secretory protein was stimulated 1.8-fold in a dose dependent manner. Immunoneutralization of TSU-pr1 and HPS secretory protein was performed with antibody against NGF, acidic fibroblast growth factor, and basic fibroblast growth factor. Removal of the NGF-like protein from the maximal stimulatory dose of TSU-pr1 secretory protein (100 micrograms/ml) with NGF antibody reduced HPS proliferation to 52% of maximal levels, and immunoneutralization of the NGF-like protein in the maximal stimulatory dose of HPS secretory protein (20 micrograms/ml) also reduced TSU-pr1 proliferation to 16% of maximal levels. Addition of normal rabbit serum or prior immunoprecipitation of either TSU-pr1 or HPS secretory protein with antibody against acidic fibroblast growth factor and basic fibroblast growth factor did not inhibit the proliferation of either cell type. These results suggest that TSU pr1 tumor cells and HPS cells secrete NGF-like protein(s) which modulate their paracrine interactive growth in vitro. PMID- 1710171 TI - Massive congenital intracranial teratoma diagnosed in utero. AB - The authors describe a case of congenital intracranial teratoma. The patient was diagnosed to be hydrocephalic at 29 weeks' gestation, and to have a huge intraventricular mass lesion at 34 weeks' gestation. Subsequently, the patient underwent subtotal resection of the mass, resulting in a significant decrease in the remarkably elevated alpha-fetoprotein in serum and cerebrospinal fluid. Histological analysis revealed a malignant teratoma, also with alpha-fetoprotein positive elements in the immunohistochemical study. PMID- 1710172 TI - Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. AB - unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc 104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally. PMID- 1710173 TI - Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. AB - Rolling of leukocytes on vascular endothelial cells, an early event in inflammation, can be reproduced in vitro on artificial lipid bilayers containing purified CD62, a selectin also named PADGEM and GMP-140 that is inducible on endothelial cells. Neutrophils roll on this selectin under flow conditions similar to those found in postcapillary venules. Adhesion of resting or activated neutrophils through the integrins LFA-1 and Mac-1 to ICAM-1 in a lipid bilayer does not occur at physiologic shear stresses; however, static incubation of activated neutrophils allows development of adhesion that is greater than 100 fold more shear resistant than found on CD62. Addition of a chemoattractant to activate LFA-1 and Mac-1 results in the arrest of neutrophils rolling on bilayers containing both CD62 and ICAM-1. Thus, at physiologic shear stress, rolling on a selectin is a prerequisite for activation-induced adhesion strengthening through integrins. PMID- 1710174 TI - trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor. AB - A variety of findings seem to functionally link brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), while distinguishing both of these factors from the third member of the neurotrophin family, nerve growth factor (NGF). Here we demonstrate that all three of these neuronal survival molecules bind similarly to the low affinity NGF receptor, but that BDNF and NT-3, unlike NGF, do not act via the high affinity NGF receptor. However, both BDNF and NT-3, but not NGF, bind to full-length and truncated forms of a receptor-like tyrosine kinase, trkB, for which no ligand had previously been identified. In addition to binding BDNF and NT-3, trkB can mediate functional responses to both of these neurotrophins when it is expressed in PC12 cells, although BDNF appears to be the more effective ligand. Thus trkB encodes an essential component of a functional receptor for BDNF and NT-3, but not for NGF. Further evidence predicts the existence of additional functional receptors for the neurotrophins. PMID- 1710175 TI - Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. AB - Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome. PMID- 1710176 TI - High-performance liquid chromatography assay of bleomycin in human plasma and rat hepatocytes in culture. AB - A sensitive and rapid linear-gradient, ion-paired, reversed-phase high performance liquid chromatography technique using fluorescence detection was developed to quantify bleomycin (BLM) metabolites in the plasma of patients undergoing BLM therapy and in rat hepatocytes that had previously been incubated with 5 x 10(-5) M BLM. We could detect about 70 ng/ml using this procedure. BLM metabolites were assayed in the supernatant fractions of precipitated human plasma and in pellets of rat hepatocytes. Metabolite concentrations were below the level of detection in human plasma samples. In hepatocyte pellets, metabolites such as deamido-BLM A2 and deamido-BLM B2 were detected, indicating that isolated rat hepatocytes in culture can metabolize BLM analogues to the corresponding deamido-BLMs. The high-performance liquid chromatography procedure developed during this work can be used to study the metabolism of BLM in cell culture systems. PMID- 1710177 TI - Central nervous system myelin: structure, function, and pathology. AB - Multiple sclerosis (MS) and a number of related distinctive diseases are characterized by the active degradation of central nervous system (CNS) myelin, an axonal sheath comprised essentially of proteins and lipids. These demyelinating diseases appear to arise from complex interactions of genetic, immunological, infective, and biochemical mechanisms. While circumstances of MS etiology remain hypothetical, one persistent theme involves recognition by the immune system of myelin-specific antigens derived from myelin basic protein (MBP), the most abundant extrinsic myelin membrane protein, and/or another equally susceptible myelin protein or lipid component. Knowledge of the biochemical and physical-chemical properties of myelin proteins and lipids, particularly their composition, organization, structure, and accessibility with respect to the compacted myelin multilayers, thus becomes central to the understanding of how and why these antigens become selected during the development of MS. This review focuses on current understanding of the molecular basis underlying demyelinating disease as it may relate to the impact of the various protein and lipid components on myelin morphology; the precise molecular architecture of this membrane as dictated by protein-lipid and lipid-lipid interactions; and the relationship, if any, between the protein/lipid components and the destruction of myelin in pathological situations. PMID- 1710178 TI - Selective neovascularization of the retinal pigment epithelium in rat photoreceptor degeneration in vivo. AB - Photoreceptor cell degeneration in rodents from a variety of causes results in neovascularization of the retinal pigment epithelium as a late stage phenomenon. Even though the vessels within the pigment epithelium arise from the retinal circulation, they can manifest the choroidal endothelial cell phenotype of fenestrated endothelial cells. In order to study the detailed cellular events which result in incorporation of retinal vessels within the retinal pigment epithelium, a morphological and morphometric analysis of the RPE and vasculature was performed in rats. Urethane, given subcutaneously to newborn rats, results in a photoreceptor degeneration but does not affect the RPE, choroid or inner retinal layers. Retinas were studied from rats of 8 to 24 weeks of age, the time period when vascularization of the RPE occurs. Loss of retinal vessels is first seen at 12 weeks, primarily in substantial dropout of vessel profiles in the outer plexiform layer (OPL) vessel bed. There is a gradient of loss from the OPL bed to the nerve fiber layer (NFL) bed and from the central to peripheral region. Total vessel density of the experimental retinas is greater than controls at 8 and 12 weeks. This occurs because there is marked loss of retinal thickness, due to photoreceptor degeneration, without a comparable loss of vessel profiles. The total retinal vessel density decreases from 8 to 20 weeks, and appears to stabilize at 20 and 24 weeks. Analysis of the separate vessel beds shows that this apparent stabilization is due to continued loss of vessels within the sensory retina, and increased presence of vascular profiles within the RPE. Total absence of the photoreceptor cell is necessary for incorporation of vessels within the RPE. Since new vessel profiles develop in the RPE but not the adjacent sensory retina, we speculate that the RPE may stimulate neovascularization of the RPE. A model of the cellular events leading to RPE neovascularization is proposed. PMID- 1710179 TI - Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis. AB - The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other. PMID- 1710180 TI - Effects of lead on the kidney: roles of high-affinity lead-binding proteins. AB - Lead-induced nephropathy produces both tubular and interstitial manifestations of cell injury, but the pathophysiology of these lesions is not completely understood. Delineation of the molecular factors underlying renal handling of lead is one of central importance in understanding the mechanisms of renal cell injury from this agent. Recent studies from this laboratory have identified several distinct high-affinity lead-binding proteins (PbBP) from rat kidney and brain that appear to play critical roles in the intracellular bioavailability of lead to several essential cellular processes in these target tissues at low dose levels. The PbBP from rat kidney has been shown to be a specific cleavage product of alpha 2-microglobulin, which is a member of the retinol-binding protein superfamily. Recent preliminary Western blot and immunohistochemical studies have shown that a polyclonal antibody to the renal PbBP does not recognize the brain PbBP, which appears to be a chemically similar, but distinct molecule. These studies have also shown that the renal PbBP is selectively localized in only certain nephrons and only specific segments of the renal proximal tubule. The striking nephror and cell-type specificity of the localization reaction could result from physiological differences in nephron functional activity or selective molecular uptake mechanisms/metabolism differences that act to define target cell populations in the kidney. In addition, other preliminary studies have shown that short-term, high-dose lead exposure produces increased excretion of this protein into the urine with concomitant decreases in renal concentrations. PMID- 1710181 TI - Differences in the renal handling of pancreatic and salivary amylase in the rat. AB - Plasma activity and excretion of pancreatic (P) amylase in the rat was found to be negligible. In contrast, the excretion rate of salivary (S) amylase was substantial and variable, depending on diuresis. P-amylase had a higher isoelectric point, a greater sieving coefficient, and a shorter half-life than S amylase. A bolus injection of 125I-labelled enzymes was followed by the appearance of 125I-labelled enzyme- as well as protein-free 125I activity in the urine. The enzyme loss was smaller and the fraction of protein-free 125I activity higher following injection of P-amylase. The affinity of P-amylase to paraffin oil exceeded that of S-amylase in partition experiments with water and paraffin oil in vitro. It is concluded that both renal filtration and reabsorption of P amylase exceed those of S-amylase. This might be due to the higher lipophility of P-amylase in comparison to the salivary type. PMID- 1710182 TI - Hybridization and polymerase chain reaction amplification of simple repeated DNA sequences for the analysis of forensic stains. AB - We have evaluated oligonucleotide hybridization and amplification techniques with regard to quantity and quality of genomic DNA that is under investigation in practical forensic case work. In order to obtain sufficient information from analyzing stain material, we use hypervariable simple repeat sequences for individualization, which occur in all eukaryotic genomes. For the analysis of larger amounts of stains (greater than 500 ng DNA) the multilocus probes (CAC)5/(GTG)5* are superior because of their discrimination potential--provided that the hybridizing DNA is of high molecular weight. The less discriminating probes (CT)8 and (GACA)4 are more sensitive (minimal amount: 100ng DNA) and still informative when the DNA is degraded. To increase the sensitivity of forensic stain analysis in special cases we have used the polymerase chain reaction technique to amplify hypervariable simple (gt)n/(ga)m repeat structures from the intron 2 of HLA-DRB genes. Largely independent of the starting amount of DNA and independent of the degradation status, we were able to generate discriminating DNA fragments, which can be used to type (i) microstains and (ii) totally degraded material including human mummy DNA. PMID- 1710183 TI - Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers. AB - To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+) dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+. PMID- 1710184 TI - N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase. AB - Photoautotrophic cells of Euglena gracilis can be adapted to N (phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40 fold accumulation of the target enzyme. 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethyl)glycine. The other is connected with a herbicide-insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N-(phosphonomethyl)glycine excreted shikimate and shikimate 3-phosphate into the medium: the amounts depended on the actual concentration of the herbicide. Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3 phosphate synthase activity in crude extracts, as well as after separation by non denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein. Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system. In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N (phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N (phosphonomethyl)glycine. PMID- 1710185 TI - Evidence for the localization of the nuclear-coded 22-kDa heat-shock protein in a subfraction of thylakoid membranes. AB - The precursor to the nuclear-coded 22-kDa heat-shock protein of chloroplasts (HSP 22) has been transported into isolated intact chloroplasts from heat-shocked plants. The localization of the mature protein in the chloroplast membrane was investigated. We have shown that the processed HSP 22 of pea was not bound to envelopes and found predominantly in thylakoid membranes. The binding of HSP 22 was stable in the presence of high salt concentrations. Solubilization of thylakoid membranes with Triton X-100 and phase partitioning with Triton X-114 indicate an intrinsic localization of HSP 22 or, alternatively, a non-covalent association with integral membrane protein(s). After fractionation into grana and stroma lamellae, HSP 22 was found mostly in the grana-membrane subfraction. PMID- 1710186 TI - Effects of endothelins, Bay K 8644 and other oxytocics in non-pregnant and late pregnant rat isolated uterus. AB - Endothelins 1, 2 and 3 (ET-1, ET-2 and ET-3; 1-30 nM) caused long-lasting concentration-dependent tonic contractions of uterine strips from non-pregnant rats. The potency of ET-1 (EC50 7 nM) was similar to that of angiotensin II (AII) and greater than that of ET-2 or ET-3 (EC50S greater than or equal to 10 nM), bradykinin, Bay K 8644, oxytocin (OT), 5-hydroxytryptamine, prostaglandin F2 alpha (PGF2 alpha) or acetylcholine. Strips from 21-day pregnant rats were 2- to 3-fold more sensitive to ET-1, AII, OT and PGF2 alpha and 200-fold more sensitive to Bay K 8644 than non-pregnant preparations. The development of tonic responses to ET-1 (30 nM) and of phasic-rhythmic ones to Bay K 8644 (300 nM) was fully prevented in strips from non-pregnant rats bathed in Ca2(+)-free medium, but stepwise reintroduction of Ca2+ (0.03-3 mM) to the solution allowed the manifestation of contractions in response to both agonists. Responses to ET-1 required less Ca2+ than those to Bay K 8644. Strips challenged with ET-1 while in Ca2(+)-free medium developed greater contractions upon reintroduction of Ca2+ than preparations stimulated with the peptide in normal medium. The reverse occurred with Bay K 8644-induced contractions. Nicardipine (10 nM) abolished the responses of strips from non-pregnant rats to Bay K 8644 (300 nM), but only attenuated ET-1-induced (30 nM) contraction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710187 TI - The central pressor actions of a novel tachykinin peptide, gamma-preprotachykinin (72-92)-peptide amide. AB - Intracerebroventricular (i.c.v.) injections of a novel tachykinin peptide, gamma preprotachykinin-(72-92)-peptide amide (neuropeptide gamma, NP gamma), caused dose-dependent increases in blood pressure. The NP gamma-induced pressor responses (1 microgram i.c.v.) were blocked by peripheral administration of pentolinium (10 mg/kg i.v.) and phentolamine (10 mg/kg i.v.), but were not inhibited by a vasopressin antagonist. These results suggest that central NP gamma increases the blood pressure via sympathetic nerve activity. PMID- 1710188 TI - Effects of endothelin-1 on the cerebrovascular bed of the goat. AB - The aim of the present study was to analyze the effects of endothelin-1 (ET-1) in the cerebrovascular bed of the goat, the importance of endothelial integrity and the contribution of extracellular Ca2+ to these responses. We measured isometric tension and 45Ca2+ uptake in isolated middle cerebral arteries, and cerebral blood flow (CBF) in unanesthetized animals. ET-1 elicited concentration-dependent contractions which were potentiated in arteries without endothelium. Ca2(+)-free medium and nicardipine inhibited, and Bay K 8644 potentiated the ET-1-induced contractions. ET-1 enhanced 45Ca2+ uptake in isolated arteries. Injections of ET 1 directly into the cerebral circulation decreased CBF and increased cerebrovascular resistance in a dose-dependent manner. Infusion of nicardipine inhibited the ET-1-induced reductions in CBF. These results suggest that ET-1 reduces CBF of goats because of contraction of cerebral arteries by a direct action on smooth muscle, which is modulated by the endothelium and depends partially on the activation of Ca2+ influx through dihydropyridine-sensitive channels. PMID- 1710189 TI - Autoradiographic localization of muscarinic acetylcholine receptors in the rat pulmonary vascular tree. AB - The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]Quinuclidinyl benzylate [( 3H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (Kd) of 0.41 +/- 0.3 nM in WKY rats and of 0.37 +/- 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [3H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [3H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle. PMID- 1710190 TI - Ovarian granulosa cell-derived insulin-like growth factor (IGF) binding proteins: release of low molecular weight, high-affinity IGF-selective species. AB - The ovarian granulosa cell has previously been shown to be a site of insulin-like growth factor (IGF) I production, reception, and action. It is the objective of this study to characterize in greater detail the soluble IGF binding activity released by this cell type. To this end, use was made of granulosa cells from immature diethylstilbestrol-treated rats. Serum-free media conditioned for 72 h by cultured untreated cells acquired polyethylene glycol (PEG)-precipitable [125I]IGF-I binding activity. The latter proved cell density-dependent, displaying a minimal inoculum requirement of less than or equal to 3 x 10(5) cells/culture. The daily elaboration of IGF-I binding activity appeared constant throughout the 72 h experimental period, the overall time-dependent accumulation of binding activity (over the same time period) proving virtually additive. Scatchard analysis of detailed competition studies with IGF-I suggests that the latter ligand binds to granulosa cell-derived IGF binding protein(s) (IGFBPs) with an apparent affinity of 3 x 10(-10) M. Qualitatively similar results were obtained when using [125I]IGF-II suggesting that the IGFBPs in question are not IGF-I-selective. In fact, specificity studies using either [125I]IGF-I or [125I]IGF-II revealed a rank order of competitive potencies compatible with that observed in other tissues so studied (IGF-II greater than IGF-I much greater than insulin). The proteinacious nature of the acid-stable IGF binding activity under study was indicated by its sensitivity to relatively low concentrations of cycloheximide, its apparent deactivation following repeated cycles of freezing and thawing, and its virtual elimination when subjected to boiling or trypsin treatment. Cycloheximide-induced blockade of protein biosynthesis also revealed that the IGF binding activity is subject to measurable turnover thereby suggesting that its accumulation represents the balance struck between synthetic and degradative processes. Western ligand blotting of sodium dodecyl sulfate polyacrylamide gel electrophoresis-fractionated media revealed a non-glycosylated major band doublet of 28-29 kDa. A single minor IGFBP species represented by a 23 kDa band was also appreciated in some but not all experiments. Taken together, these findings document the ability of ovarian granulosa cells to secrete a heterogenous mix of low molecular weight, high-affinity IGF-selective binding species. As such, these observations are in keeping with the concept of a complete intraovarian IGF system replete with ligands, receptors, and soluble binding activity. PMID- 1710191 TI - Regulation of insulin-like growth factor binding protein-3 expression by dexamethasone. AB - Dexamethasone (DXM), a potent long acting glucocorticoid results in growth retardation when administered to children and experimental animals. We have used ligand blotting and RNA blotting techniques to examine the effects of DXM on serum insulin-like growth factor binding protein 3 (IGFBP-3) levels and hepatic IGFBP-3 mRNA abundance. A time- and dose-dependent increase in the 39-42 kDa serum IGF binding proteins and in IGFBP-3 mRNA abundance was observed in DXM treated rats. A significant increase in serum IGF binding capacity was seen with as little as 0.1 microgram/100 g body weight. A significant increase in IGFBP-3 mRNA abundance was apparent as early as 1 h following DXM administration. IGFBP-3 mRNA levels reached a peak at 3 h (1.9 +/- 0.2 fold, p less than 0.005) and declined to normal levels in 6-12 h after DXM administration. Since the IGF binding proteins may be able to inhibit the action of the IGFs, enhanced expression of IGFBP-3 may be one of the mechanisms involved in DXM-induced growth retardation. PMID- 1710192 TI - Expression of adhesion molecules during the formation and differentiation of the avian endocardial cushion tissue. AB - The expression of cytotactin, the cytotactin-binding (CTB) proteoglycan, and the neural cell adhesion molecule, N-CAM, was examined during the development of the avian endocardial cushion tissue (ECT). N-CAM was present in the cardiac mesoderm from its earliest time of development. At the time when endothelial cells converted to mesenchyme and began to migrate, they ceased their expression of N CAM. Cytotactin and CTB proteoglycan were present in the cardiac jelly (into which the ECT cells migrate) in patterns that were correlated with cell migration. At early times of migration (stage 18), the region of the cardiac jelly near the endocardium contained cytotactin in the vicinity of the migrating cells. During later migration (stage 22), cytotactin remained associated with the leading zone of cell migration, but its expression began to decrease in areas where cells had accumulated. After ECT cell migration had ceased, cytotactin expression decreased, remaining high only in the peripheral portion of the aorticopulmonary septum and absent from its ridges. CTB proteoglycan was expressed during early migration at high levels in and adjacent to the myocardium. By stage 22, its distribution had become more uniform throughout the ECT regions and in the myocardium. The combined results of this study suggest that cytotactin, CTB proteoglycan, and N-CAM each play a distinct, critical role in pattern formation in the early heart. PMID- 1710193 TI - Detection of 2',5' oligoadenylate synthetase activity in acute viral hepatitis with special reference to histologic features in the acute stage. AB - We measured the sequential changes in 2',5' oligoadenylate synthetase activity in 21 patients with acute viral hepatitis (5 with type A, 6 with type B, and 10 with type non-A, non-B hepatitis) by radioimmunoassay. Liver biopsies were performed during the acute phase in all patients. Among patients with acute hepatitis A and B, the 2',5' oligoadenylate synthetase levels were transiently elevated at the time of the peak alanine aminotransferase level in the patients in whom a liver biopsy showed acute hepatitis or non-specific reactive hepatitis. Of 10 patients with acute non-A, non-B hepatitis, 4 showed a similar 2',5' oligoadenylate synthetase activity pattern and liver histology to those observed in acute hepatitis A and B. In the remaining 6, the 2',5' oligoadenylate synthetase levels remained elevated for 3.5 to 6 months while the alanine aminotransferase was elevated. Liver biopsy in these patients showed chronic hepatitis. Persistent detection of raised 2',5' oligoadenylate synthetase activity levels during the acute stage of non-A, non-B hepatitis may thus be an indicator of progression of the disease. PMID- 1710194 TI - [Radiotherapy of osseous metastases in breast cancer]. AB - 1121 patients were treated for breast cancer over a period of 10 years in the Department of Radiotherapy-Radio-Oncology-of the University of Munster. 959 of these patients were regularly cared for in the tumor outpatient department after the treatment. Of these, 354 patients had, or developed later, distant metastases. Metastases were distributed frequently in the skeleton in 167 cases (47.2%). Radiation therapy was carried out under high-voltage conditions. As a rule, pain-free condition was achieved after 15 to 30 Gy (86.3% of all cases). After 30 to 40 Gy, in cases of extreme osteolyses also after higher focal doses, recalcification was evident in most cases. The average survival time after diagnosis of bone metastases was 11.8 months and 11.2 months without therapy. PMID- 1710195 TI - Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis. AB - A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 was probed for the presence of 16S rRNA genes, using 32P 5' end-labelled 16S rRNA fragments. Cosmid DNA from positive clones responsible for hybridisation was subcloned into a high copy number vector and a restriction map was constructed. The location of the 16S, 23S and 5S rRNA genes was determined on this map. Transcriptional promoter activity was identified upstream of the 5' end of the 16S rRNA gene. By probing L. lactis 712 chromosomal DNA cut with a range of restriction endonucleases, with a conserved oligonucleotide to the 5' end of the 16S rRNA gene, 6 copies of rRNA genes were identified. PMID- 1710196 TI - Medical and nonmedical therapies for benign prostatic hypertrophy. AB - Benign prostatic hypertrophy (BPH) is a common disorder of elderly men, affecting about 80% of men by the age of 80. Until recently, therapeutic options were limited, and transurethral surgery was the most common remedy. In recent years, however, a number of alternatives to traditional surgery have evolved. This article reviews and evaluates current medical and nonmedical therapies for BPH. PMID- 1710197 TI - The presence in experimental animals of a colon specific Mr 40,000 protein(s) with relevance to ulcerative colitis. AB - In patients with ulcerative colitis a colon tissue bound IgG and serum antibodies against an Mr 40,000 colonic protein(s) has been identified. Using an anti-Mr 40,000 protein monoclonal antibody, 7E12H12, by an immunocytochemical method, the protein was localised in human tissue exclusively to colonic epithelial cells. In this study the presence of the Mr 40,000 protein was assessed in experimental animals by the direct and inhibition enzyme linked immunosorbent assays (ELISAs) using the anti-Mr 40,000 protein monoclonal antibody, 7E12H12 (IgM isotype). In addition, a total of 129 specimens including colon, small intestine, gall bladder, biliary tract, and kidney from nine strains of rats and mice, and from human tissue were studied by the immunocytochemical method using 7E12H12. All colon specimens from both humans and animals reacted with 7E12H12 in the immunocytochemical and ELISA assays. None of the non-colonic organs reacted with 7E12H12. While in human colon 7E12H12 recognised the absorptive epithelial cells, in all the animals it recognised mainly the colonic goblet cells. Extracts of animal colon but not of small intestine inhibited the binding of 7E12H12 to the human colon extract. This study shows the presence of an organ specific Mr 40,000 colonic epithelial protein(s) in humans and experimental animals. A differing cellular localisation of the Mr 40,000 protein(s) in human v animal tissue was also shown. Further characterisation of the Mr 40,000 protein(s) may provide important clues regarding the autoimmune mechanisms in ulcerative colitis. PMID- 1710198 TI - Effect of a new synthetic ascorbic acid derivative as a free radical scavenger on the development of acute pancreatitis in mice. AB - The therapeutic effects of CV 3611, a new synthetic free radical scavenger prepared from an ascorbic acid derivative, on choline deficient, ethionine enriched (CDE) diet induced acute pancreatitis in mice were evaluated and compared with those of superoxide dismutase. Time/course studies after subcutaneous injection of CV 3611 in normal mice showed a peak plasma concentration of mean (SEM) 0.54 (0.09) micrograms/ml at one hour, with a gradual decrease over the next 10 hours, while a peak concentration in pancreatic tissue of mean (SEM) 425 (33) ng/g tissue was achieved at three hours and the drug was undetectable at 12 hours. Survival rates and activities of pancreatic enzymes (amylase, lipase, elastase I) were compared in control mice and animals that received CV 3611 before or at the time of feeding the CDE diet. The survival rate was observed in a no treatment group and mice given pretreatment or treatment with CV 3611 or superoxide dismutase. The survival rate was significantly better in the treatment group given CV 3611 (p less than 0.02), but superoxide dismutase had no significant effect on survival. The increases in the three serum enzyme activities were significantly less at 48 hours in the groups given pretreatment or treatment with CV 3611 than in the no treatment group. These results indicate that CV 3611, which has been proved to pass through the cell membrane and to have a long half life in plasma and tissue, had an important therapeutic effect on the development of acute pancreatitis. They also suggest that oxygen derived free radicals may play an important role in the development of acute pancreatitis. PMID- 1710199 TI - alpha Fetoprotein producing early gastric cancer with liver metastasis: report of three cases. AB - Three cases of alpha fetoprotein producing early gastric cancer are presented. Liver metastases occurred in all patients shortly after curative gastrectomy and all died within two years. The incidence of liver metastasis was significantly higher than that in alpha fetoprotein negative early gastric carcinoma (p less than 0.001). The incidences of lymph node metastasis and invasion in lymph vessels and veins were also substantially higher in this group of patients. Two radical hepatic resections, including extended right lobectomy, were performed on one patient but the tumour recurred immediately. PMID- 1710200 TI - Selective deficiency of pancreatic amylase. AB - Two patients with specific pancreatic amylase deficiency are described. The greatly reduced pancreatic amylase activity was not due to an enzymatically inactive amylase molecule but to an almost complete absence of the molecule itself. The findings are of diagnostic importance as they show that low pancreatic amylase activity in serum or duodenal aspirates, or both, does not necessarily represent chronic exocrine pancreatic disease such as chronic pancreatitis, carcinoma of the pancreas, or cystic fibrosis but may be an isolated finding. In one of our patients a familial occurrence was shown, indicating a congenital deficiency. PMID- 1710202 TI - Intracavitary therapy in carcinoma of the esophagus. PMID- 1710201 TI - Plasma levels of insulin-like growth factor-I and its binding protein in polycystic ovary syndrome. AB - Patients with polycystic ovary syndrome (PCOS) had significantly higher levels of total insulin-like growth factor (IGF-I) than those in age- and weight-matched controls (PCOS, 230 +/- 21 ng/ml; control, 180 +/- 16 ng/ml; mean +/- SE, p less than 0.05) as well as free IGF-I (PCOS, 3.8 +/- 0.2 ng/ml; control, 3.0 +/- 0.2 ng/ml; p less than 0.05). These elevated levels of IGF-I were correlated slightly with levels of LH and LH/FSH ratio (r = 0.171, p less than 0.05 and r = 0.239, p less than 0.01, respectively). Elevated fasting levels of insulin and decreased levels of IGF binding protein (32K-BP) were also observed in PCOS, and the levels of 32K-BP in PCOS were negatively correlated with insulin (r = -0.39, p less than 0.01). These results suggest that elevated IGF-I levels and decreased 32K-BP levels in the circulation are one of the endocrinological features of PCOS and that insulin is responsible for the clinical manifestation of decreased 32K-BP levels in PCOS. PMID- 1710203 TI - High dose rate intracavitary therapy in advanced carcinoma esophagus. AB - Nine patients with advanced squamous cell carcinoma of the middle third of the esophagus were treated by high dose rate intracavitary therapy. The dose delivered was 12 Gy in two sessions at 1 cm from the center of the source. All nine patients were alive after 9 months. Six months after treatment, 4 patients had strictures which were dilated. At the end of nine months, 6 patients had dysphagia, four of whom had strictures and two had recurrence which was treated by further intracavitary irradiation. Intracavitary radiation using high dose rate, remote controlled afterloader has a significant role in palliation in patients with advanced esophageal carcinoma and avoids intubation. PMID- 1710204 TI - Carcinoma of the esophagus presenting with esophagocutaneous fistula--a case report. AB - An interesting manifestation of carcinoma of the esophagus, hitherto undescribed is reported. The patient at the time of diagnosis had presented with an esophagocutaneous fistula. He was treated by feeding jejunostomy and local palliative radiotherapy and showed good clinical improvement. The extreme rarity of such a presentation is highlighted. PMID- 1710205 TI - Prevalence of antibody against the core protein of hepatitis C virus in patients with hepatocellular carcinoma. AB - We have cloned the whole structural region of the hepatitis C virus (HCV) genome and transiently expressed the nucleocapsid protein in animal cells. Since the nucleotide sequences of this region of the HCV genome has been shown to be highly conserved among different HCV isolates, the assay detecting the antibody to this expressed protein is useful for studying the pathogenicity of HCV. In this work, we investigated the presence of antibodies to HCV nucleocapsid protein (p22) in patients with hepatocellular carcinoma (HCC) and compared its frequency with that of antibody to HCV non-structural protein (C-100), which is presently applied for blood screening for transfusion and diagnosis for chronic hepatitis C. By a sensitive Western blot analysis, 85 of 102 (83.3%) sera of hepatitis B virus surface antigen (HBsAg)-negative HCC patients were positive for the antibody to p22 (anti-p22), whereas 68 of the same 102 cases (66.7%) were positive for the anti-C100 by ELISA. The prevalence of anti-p22 in 23 HBV carrier HCC patients, 56 patients with non-HCC cancer and 100 healthy blood donors were 4.3, 12.5 and 1.0%, respectively. Thus, high prevalence of anti-p22 in non-B HCC confirmed that HCV infection is closely related to the development of HCC. Furthermore, the anti p22 assay can detect HCV-infected patients who could not previously be identified as such by the present anti-C100 assay. PMID- 1710206 TI - Oncofetal expression of the human intestinal mucin glycoprotein antigens in gastrointestinal epithelium defined by monoclonal antibodies. AB - A mucin preparation from a colonic adenocarcinoma was used to prepare monoclonal antibodies (MAbs) that reacted specifically either with normal adult small intestine mucin antigen(s) (SIMA), or normal adult large-intestine mucin antigen(s) (LIMA). Both SIMA and LIMA show a unique oncofetal pattern of expression. Thus SIMA was expressed in early fetal stomach, large and small intestines but thereafter only in the normal small intestine. SIMA expression was detected immunohistochemically in cancers of the colorectum (82/112) and stomach (48/86). LIMA was detected in the stomach of the early fetus but thereafter only in the normal large intestine. LIMA expression was detected in 61/86 cancers of the stomach. Moreover, both SIMA and LIMA were expressed inappropriately in mucosa adjacent to tumors, indicative of the detection of possible pre-malignant epithelium. We used a sandwich ELISA and biochemical procedures to show that the SIMA and LIMA molecules were large extensively glycosylated multi-unit mucin glycoproteins that differed markedly from each other. SIMA, whether extracted from normal small-intestine or colonic cancers, had a molecular weight above 1.000 kDa, a mean buoyant density 1.33 g/ml and s value of 4.8. LIMA had a molecular weight above 10.000 kDa, a mean buoyant density 1.45 g/ml and an s value 9.5. The SIMA and LIMA epitopes were judged to be carbohydrate in nature by reason of their resistance to harsh physical chemical treatments or protease digestion, and sensitivity to periodate oxidation, neuraminidase or beta elimination. Only the SIMA epitope was sensitive to neuraminidase. In conclusion, MAbs to carbohydrate-dependent epitopes on SIMA and LIMA identify the oncofetal pattern of expression of these distinct intestinal mucin glycoproteins in colonic and gastric carcinoma. These MAbs will be useful in further studies of the significance of oncofetal mucin expression during carcinogenesis. PMID- 1710207 TI - Proteinase-alpha 2 macroglobulin complexes are not increased in plasma of patients with cancer. AB - Alpha 2-macroglobulin, a major glycoprotein component of plasma, is unique in its capacity to bind and inhibit the proteolytic activities of all classes of proteinases. Since proteinases implicated in cancer dissemination (type-IV collagenase, plasminogen activator, cathepsins B) are normal constitutents of blood, we have explored the hypothesis that elevated tissue levels of activated proteinases bound to alpha 2M might be detected in plasma of patients with cancer. To test this premise, blood was collected from 149 subjects (33 healthy controls, 31 patients with infections and non-malignant diseases, 16 with myeloproliferative disease, 10 with gastrointestinal cancer, 7 with genito urinary cancer, 16 with lung cancer, 14 with lymphoma, 11 with miscellaneous cancers and 11 with chronic lymphocytic leukemia and myeloma). Plasma was assayed for alpha 2M-proteinase complexes using a sandwich ELISA which employs a mouse monoclonal antibody (MAb) that binds to a neo-antigenic determinant on complexed alpha 2M and a rabbit polyclonal anti-native human alpha 2M antibody. The concentration of complexed alpha 2M in healthy controls was 14.2 +/- 9.8 micrograms/ml (mean +/- standard deviation). No significant differences in complexed alpha 2M were noted between normal and cancer groups (range 7.4-14.6 micrograms/ml). On the basis of these data, we propose that, in patients with cancer, activated proteinases are bound locally to inhibitors in the tissues and are not available to form complexes with plasma alpha 2M. An alternative explanation is that proteinases are not secreted in excess by cancer cells in vivo. PMID- 1710208 TI - Analysis of abnormal expression of g-csf gene in a novel tumor cell line (KHC 287) elaborating G-CSF, IL-1 and IL-6 with co-amplification of c-myc and c-ki ras. AB - We established a human carcinoma cell line (KHC 287) from a patient with large cell-typing lung carcinoma associated with marked leukocytosis. The culture supernatant of KHC 287 cells promoted granulocytic colony formation of human bone marrow cells in semi-solid culture. Colony formation was almost completely suppressed by treatment of the supernatant with a monoclonal anti-granulocyte colony-stimulating factor (G-CSF) antibody. Not only G-CSF but also interleukin-1 alpha (IL-I alpha), IL-I beta and IL-6 were detected in the culture supernatant by an ELISA method. Northern blot analysis of KHC 287 cells revealed distinct expression of these cytokine genes. Southern blot hybridization of KHC 287 DNA showed 20- and 40-fold co-amplification of c-myc and c-ki-ras, respectively. The chloramphenicol acetyl transferase (CAT) activity was distinctly enhanced in the KHC 287 cells which were transfected with the 360 bp upstream region of G-CSF gene inserted into pSV00CAT, but not in non-G-CSF-producing tumor cell lines. These results suggest that overproduction of the transactivating factor(s) which binds to the 360 bp of the G-CSF upstream region is responsible for the abnormal expression of G-CSF gene in KHC 287 cell line. PMID- 1710209 TI - Acanthamoeba keratitis. PMID- 1710210 TI - Effects of poliovirus replication on undifferentiated and differentiated monocytic U937 cells: comparative studies with human macrophages. AB - Poliovirus infection of either undifferentiated or differentiated U937 cells produced a decrease in the percentage of cells positive for the surface expression of the CD4, CD11c, CD14, or 8E11 antigens. The number of 4F2 surface molecules per cell increased in infected normal U937 cells, but was unaffected in differentiated cells. The level of O2- production in infected differentiated U937 cells was approximately 50% of that found when not infected. Finally, poliovirus RNA levels and infectious particle production were similar in either cell type. PMID- 1710211 TI - Histochemical localization of transforming growth factor-beta 1 in developing rat molars using antibodies to different epitopes. AB - In this study transforming growth factor-beta 1 (TGF-beta 1) has been immunolocalized in developing rat molars using two well characterized polyclonal antibodies, Anti-CC and Anti-LC, that recognize extracellular and intracellular TGF-beta 1, respectively. With immunohistochemical methods and the ABC-peroxidase system of detection, the growth factor was immunolocalized within the ectodermally derived enamel organ and the neural crest-derived dental papilla at the early and advanced bell stages of development. With Anti-CC, widespread and abundant extracellular TGF-beta 1 was found associated with the stellate reticulum and within central and apical regions of dental papilla mesenchyme. In contrast, Anti-LC localized TGF-beta 1 intensely within the cells of the outer dental epithelium. Moderate immunostaining for TGF-beta 1 with Anti-LC was also evident within the apical cytoplasm of inner dental epithelial cells and odontoblasts. These findings support the hypothesis that TGF-beta 1 may play a paracrine role in tooth development by regulating the epithelial-mesenchymal interactions that influence growth and cytodifferentiation events. PMID- 1710212 TI - Visual excitation and recovery. PMID- 1710213 TI - Characterization of the human androgen receptor transcription unit. AB - A full length human androgen receptor (hAR) cDNA was constructed from cDNA and genomic clones. Structurally the 10.6-kilobase (kb) hAR cDNA consists of a long 5'-untranslated region (5'-UTR, 1.1 kb), a previously described open reading frame (ORF, 2.7 kb) (Trapman, J., Klaassen, P., Kuiper, G. G. J. M., van der Korput, J. A. G. M., Faber, P. W., van Rooij, H. C. J., Geurts van Kessel, A., Voorhorst, M. M., Mulder, E., and Brinkmann, A. O. (1988) Biochem. Biophys. Res. Commun. 153, 241-248; Faber, P. W., Kuiper, G. G. J. M., van Rooij, H. C. J., van der Korput, J. A. G. M., Brinkmann, A. O., and Trapman, J. (1989) Mol. Cell. Endocrinol. 61, 257-262), and a very long 3'-untranslated region (3'-UTR, 6.8 kb). The complete 5'- and 3'-UTRs were found to be encoded by the previously reported first and eight protein coding exons of the hAR gene, respectively (Kuiper, G. G. J. M., Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Ris-Stalpers, C., Klaassen, P., Trapman, J., and Brinkmann, A. O. (1989) J. Mol. Endocrinol. 2, R1-R4). Two major sites of transcription initiation were identified in a 13-base pair region. DNA fragments spanning these transcription initiation sites conferred promoter activity upon a promoterless chloramphenicol acetyltransferase reporter gene construct. Two equally effective, functional polyadenylation signals (ATTAAA and CATAAA) at a mutual distance of 221 base pairs were detected. The ATTAAA hexamer sequence gave rise to multiple sites of poly(A) addition, whereas only one position was used following the CATAAA hexamer. In LNCaP prostatic carcinoma cells an alternatively spliced hAR mRNA species was identified which lacks 3 kb of the 3'-UTR. PMID- 1710214 TI - The transcription of DNA in chicken mitochondria initiates from one major bidirectional promoter. AB - Transcription start sites of chicken mitochondrial DNA have been mapped in the control region by direct sequencing of in vitro capped mitochondrial RNA species, by primer extension and by S1 nuclease protection analysis. Transcription of the heavy strand initiates predominantly at a site 156 nucleotides upstream of the tRNA(Phe) gene, i.e. about 135 nucleotides further upstream than the corresponding sites in amphibia and mammals. On the opposite strand, transcription starts predominantly one nucleotide removed from the site in the heavy strand. The L-strand position start site is similar to that found in other vertebrates. The chicken mitochondrial DNA control region thus contains one major transcriptional promoter, whose bidirectional capacity is similar to the situation in amphibia but which contrasts to the mainly unidirectional capacity of mammalian promoters. In chicken mitochondria, the sequence comprising the start sites is A + T rich and contains an almost perfect inverted repeat which can be folded into a cruciform structure. The heavy and light strand initiation sites are flanked on their respective 3' ends by an octanucleotide sequence matching those surrounding the start sites in Xenopus laevis (5'-ACPuTTATA-3'). This motif is found associated with the H-strand start sites in mouse but is not present in human nor bovine mitochondrial DNA promoters. PMID- 1710215 TI - Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix. AB - Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III 1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin. PMID- 1710216 TI - A proline-rich structural protein of the surface sheath of larval Brugia filarial nematode parasites. AB - Both cDNA and genomic DNA sequences have been isolated which encode a proline rich precursor protein of the sheath from microfilariae, the first stage larvae of the filarial nematode parasites Brugia pahangi and Brugia malayi. This 22-kDa protein is soluble only under reducing conditions and is extensively cross-linked by both disulfide and nonreducible bonds. Immunogold electron microscopy shows that the protein is localized exclusively in the sheath, a vestigial remnant of the eggshell, which is retained by and encloses the mature microfilaria. Analysis by Western blotting confirms that the protein is expressed only in microfilariae and adult female worms, although transcripts are detectable only in adult females. The deduced amino acid sequence contains a short N-terminal hydrophobic putative leader sequence, a central repetitive domain that contains 14 copies of a degenerate 5-amino acid repeat with the consensus sequence Met-Pro-Pro-Gln-Gly, and a C-terminal proline-rich domain flanked by clusters of cysteine residues. These clusters can be aligned with cysteine residues implicated in cross-linking of a family of cuticular collagens originally identified in Caenorhabditis elegans but which extends to other nematodes. PMID- 1710217 TI - Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. AB - A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation. PMID- 1710218 TI - Glucose induces insulin gene transcription in a murine pancreatic beta-cell line. AB - The ability of insulin secretagogues to stimulate insulin gene transcription was analyzed in the murine insulinoma cell line beta TC3, which had been derived from a transgenic mouse expressing SV40 T antigen under control of the rat insulin II gene regulatory region. Glucose induced a 3-fold increase in the transcription of both the endogenous mouse insulin genes and the transgene. This effect was inhibited by D600, a calcium channel blocker, which also inhibited glucose induced insulin secretion in these cells. This suggests that similar signals may be involved in glucose-stimulated insulin secretion and insulin gene transcription. Agents that increase intracellular levels of cAMP did not have a significant effect on the transcription of either the insulin genes or the transgene. Stimulation of transcription of the RIP-Tag transgene by glucose suggests that the 695-base pair fragment of the insulin gene regulatory region that is included in the transgene contains the cis elements required for response to the glucose-induced signal. PMID- 1710219 TI - Isolation and characterization of cDNA clones for an inhibitor protein of cAMP dependent protein kinase. AB - Synthetic oligonucleotides were designed to amplify DNA sequences related to the heat-stable protein kinase inhibitor (PKI) isolated from rabbit skeletal muscle. Using these oligonucleotides, a 167-base pair fragment was isolated and shown to code for a portion of the mouse protein kinase inhibitor gene. This amplified DNA sequence was used to isolate three cDNA clones from a mouse brain cDNA library. A composite sequence was derived from these clones and contained a 228-nucleotide open reading frame encoding a protein of 76 amino acids. In addition, the sequence contained 29 nucleotides of 5'-untranslated and 2022 nucleotides of 3' untranslated regions of the mouse PKI mRNA. Northern blot analysis of various mouse tissues indicated that the 3.8-kilobase pair mRNA is present at high levels in skeletal muscle and brain but is present at lower levels in heart, testis, and liver. RNase protection experiments also suggested that skeletal muscle and brain represent tissues of highest expression and that similar nucleotide sequences are found in the skeletal muscle, brain, and testicular transcripts. Southern blot analysis indicated a single prominent species of genomic DNA sequence related to the mouse PKI cDNA clones but a minor species was also detected. PMID- 1710220 TI - Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. AB - The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2 fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation. PMID- 1710221 TI - High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies. AB - The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE. PMID- 1710222 TI - Limited proteolysis of the alpha-macroglobulin rat alpha 1-inhibitor-3. Implications for a domain structure. AB - Rat alpha 1-inhibitor-3 is a 180-kDa monomeric proteinase inhibitor found in high concentration in rat plasma. By several criteria it has been shown to be a member of the family of alpha-macroglobulin proteinase inhibitors often exemplified by the tetrameric human alpha 2-macroglobulin. We have used limited proteolysis of rat alpha 1-inhibitor-3 to probe the domain structure of this family of proteins. Proteinases of different specificities, including trypsin, chymotrypsin, thermolysin, and Staphylococcus aureus V8 proteinase, were employed and a common fragmentation pattern was observed when the reaction products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These fragments were electrotransferred to polyvinylidene difluoride membranes and subjected to NH2 terminal amino acid sequence analysis in order to position them within the context of the primary structure. The fragmentation pattern may define the domain structure of alpha 1-inhibitor-3 and serve as a model for the domain organization of the family of alpha-macroglobulin proteinase inhibitors. PMID- 1710223 TI - Phospholamban regulation of cardiac sarcoplasmic reticulum (Ca(2+)-Mg2+)-ATPase. Mechanism of regulation and site of monoclonal antibody interaction. AB - Monoclonal antibodies raised against canine cardiac sarcoplasmic reticulum phospholamban were used to study the structure-function relationship between phospholamban and the sarcoplasmic reticulum (SR) (Ca(2+)-Mg2+)-ATPase (Suzuki, T., and Wang, J. H. (1986) J. Biol. Chem. 261, 7018-7023). Additional monoclonal antibodies are characterized further. When five of these monoclonal antibodies were assessed for their ability to affect SR Ca2+ uptake three of these antibodies had no effect on SR Ca2+ uptake, whereas the other two monoclonals were able to stimulate SR Ca2+ uptake to levels similar to those caused by phosphorylation of phospholamban at different calcium concentrations. Using synthetic peptides corresponding to various portions of phospholamban in a competitive binding assay, it was possible to map the epitope site of monoclonals which stimulate the (Ca(2+)-Mg2+)-ATPase activity to phospholamban residues 7-16. These results implicate phospholamban residues 7-16 in the regulation of the (Ca(2+)-Mg2+)-ATPase. PMID- 1710224 TI - PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A. AB - Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug. PMID- 1710225 TI - Sorting out IF networks: consequences of domain swapping on IF recognition and assembly. AB - Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins. PMID- 1710226 TI - J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth. AB - The influence of J1/tenascin adsorbed to polyornithine-conditioned plastic (substrate-bound J1/tenascin) and J1/tenascin present in the culture medium (soluble J1/tenascin) on neurite outgrowth was studied with cultured single cells from hippocampus and mesencephalon of embryonic rats. Neurons at low density grew well on J1/tenascin substrates and extended neurites that were approximately 40% longer than on the polyornithine control substrate after 24 h in vitro. The neurite outgrowth promoting effect of substrate bound J1/tenascin was largely abolished in the presence of mAb J1/tn2, but not by mAb J1/tn1. In contrast to the neurite growth-promoting effects of substrate bound J1/tenascin, neurite outgrowth on polyornithine, laminin, fibronectin, or J1/tenascin as substrates was inhibited by addition of soluble J1/tenascin to the cultures. Neither of the two mAbs neutralized the neurite outgrowth-inhibitory properties of soluble J1/tenascin. In contrast to their opposite effects on neurite outgrowth, both substrate-bound and soluble J1/tenascin reduced spreading of the neuronal cell bodies, suggesting that the neurite outgrowth-promoting and antispreading effects are mediated by two different sites on the molecule. This was further supported by the inability of the mAb J1/tn2 to neutralize the antispreading effect. The J1/tn2 epitope localizes to a fibronectin type III homology domain that is presumably distinct from the putative Tn68 cell-binding domain of chicken tenascin for fibroblasts, as shown by electronmicroscopic localization of antibody binding sites. We infer from these experiments that J1/tenascin contains a neurite outgrowth promoting domain that is distinguishable from the cell binding site and presumably not involved in the inhibition of neurite outgrowth or cell spreading. Our observations support the notion that J1/tenascin is a multifunctional extracellular matrix molecule. PMID- 1710227 TI - Four molecular pathways of T cell adhesion to endothelial cells: roles of LFA-1, VCAM-1, and ELAM-1 and changes in pathway hierarchy under different activation conditions. AB - T cell adhesion to endothelium is critical to lymphocyte recirculation and influx into sites of inflammation. We have systematically analyzed the role of four receptor/ligand interactions that mediate adhesion of peripheral human CD4+ T cells to cultured human umbilical vein endothelial cells (HUVEC): T cell LFA-1 binding to ICAM-1 and an alternative ligand ("ICAM-X"), T cell VLA-4 binding to VCAM-1, and T cell binding to ELAM-1. Contributions of these four pathways depend on the activation state of both the T cell and HUVEC, and the differentiation state of the T cell. ELAM-1 plays a significant role in mediating adhesion of resting CD4+ T cells to activated HUVEC. LFA-1 adhesion dominates with PMA activated T cells but the strength and predominant LFA-1 ligand is determined by the activation state of the HUVEC; while ICAM-1 is the dominant ligand on IL-1 induced HUVEC, "ICAM-X" dominates binding to uninduced HUVEC. Adhesion via VLA-4 depends on induction of its ligand VCAM-1 on activated HUVEC; PMA activation of T cells augments VLA-4-mediated adhesion, both in the model of T/HUVEC binding and in a simplified model of T cell adhesion to VCAM-1-transfected L cells. Unlike LFA-1 and VLA-4, ELAM-1-mediated adhesion is not increased by T cell activation. Differential expression of adhesion molecules on CD4+ T cell subsets understood to be naive and memory cells also regulates T/HUVEC adhesion. Naive T cell adhesion to HUVEC is mediated predominantly by LFA-1 with little or no involvement of the VLA-4 and ELAM-1 pathways. In contrast, memory T cells bind better to HUVEC and utilize all four pathways. These studies demonstrate that there are at least four molecular pathways mediating T/HUVEC adhesion and that the dominance/hierarchy of these pathways varies dramatically with the activation state of the interacting cells and the differentiation state of the T cell. PMID- 1710228 TI - Macrophage membrane glycoproteins that bind Griffonia simplicifolia I-B4: effect on cytotoxicity and protein secretion. AB - Thioglycollate elicited peritoneal (TG-Mos) but not resident peritoneal Mos (R Mos) were found to bind the lectin Griffonia simplicifolia isotype I-B4 (GSI-B4). This was demonstrated by ultrastructural studies and FACS analyses. Membranes from TG-Mos were isolated, separated on SDS-PAGE, electrotransferred onto nitrocellulose, and exposed to peroxidase-labeled GSI-B4. These procedures revealed two major membrane glycoproteins of molecular weights 180,000 and 94,000 daltons that bound the lectin GSI-B4 which has a specificity for recognizing terminal alpha-galactosyl residues. The presence of these epitopes on the two membrane glycoproteins was further substantiated by the fact that treatment of the membranes with alpha-galactosidase destroyed their capacity to bind GSI-B4 and that alpha-D-galactopyranoside but not N-acetyl-D-glucosamine competitively inhibited GSI-B4 from binding to the glycoproteins. Treatment of TG-Mos with GSI B4 reduced the capacity of interferon (IFN) and lipopolysaccharide (LPS), or IFN alone, to induce Mo mediated cytotoxicity towards tumor cells by as much as 40%. GSI-B4 also caused alterations in the pattern of biosynthetically 35S-methionine labeled secreted proteins as early as 2 hours after contact with TG-Mos. Out of 35 discernible proteins on fluorograms of SDS-PAGE separated proteins, 5 were down-regulated and 9 were enhanced. It is suggested that the two novel Mo membrane proteins may play a role in regulating the response of Mo subpopulations to their humoral and cellular environments. PMID- 1710229 TI - Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas. AB - The appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic-type tissue in the adult rat liver can be experimentally induced (Reddy et al.: J. Cell Biol., 98:2082-2090, 1984; Rao et al., J. Histochem. Cytochem., 34:197-201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light- and electron-microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and beta-tubulin were present in all cell lines. In addition, simple epithelial type II cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type II cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth-inhibitory effects of TGF-beta 1. No expression of tissue- or cell-specific proteins such as alpha-fetoprotein, albumin, amylase, elastase, or gamma-glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a "precursor" or "stem" cell compartment in vivo. PMID- 1710230 TI - Culture-induced increase in acidic and basic fibroblast growth factor activities and their association with the nuclei of vascular endothelial and smooth muscle cells. AB - The activity of acidic and basic fibroblast growth factor-like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4-fold more bFGF-like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10-fold more bFGF like activity than the fresh cell lysates. We consistently find aFGF-like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3-fold more aFGF-like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF-like activity in cell and ECM extracts was 8 fold higher than in fresh cell extracts. The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin-sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti-aFGF (1.1 M), or anti-bFGF (1.5 M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF-like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125IaFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens. PMID- 1710231 TI - Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum free medium. AB - Insulin was observed to modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary cultures of rabbit renal proximal tubule cells in serum free medium. Insulin was stimulatory to primary proximal tubule cell growth at a concentration of 10(-8) M. In contrast, insulin was inhibitory to a proximal tubule function, PEPCK activity, following a 5-minute incubation period. An insulin dosage as low as 10(-10) M was inhibitory to PEPCK activity, suggesting the involvement of insulin receptors. Although insulin was required at a significantly higher dosage to stimulate the growth of the primary renal proximal tubule cells than to inhibit PEPCK activity, the elevated dosage required in order to observe a growth effect may be explained by the degradation of insulin by the primary renal proximal tubule cells. However the possible involvement of receptors for Insulin-like Growth Factor I (IGF-I) and Insulin like Growth Factor II (IGF-II) in mediating the effects of insulin cannot be excluded. Other effector molecules were also examined with respect to their effects on PEPCK activity. The possible involvement of cyclic AMP in the control of the PEPCK activity of the primary renal cells was indicated by the stimulatory effects of 8 bromocyclic AMP, isobutyl methylxanthine (a cyclic AMP phosphodiesterase inhibitor), and forskolin (an activator of adenylate cyclase). Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, was inhibitory. The actions of these effector molecules and insulin on the PEPCK activity of the primary renal cultures are remarkably similar to their effects on hepatic PEPCK. Several growth factors, fibroblast growth factor (FGF), and transforming growth factor beta (TGF beta) were also examined. FGF was observed to be stimulatory, whereas TGF beta was inhibitory to the PEPCK activity of the primary renal proximal tubule cells. PMID- 1710232 TI - Dr(a-) polymorphism of decay accelerating factor. Biochemical, functional, and molecular characterization and production of allele-specific transfectants. AB - The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on decay accelerating factor (DAF), a glycosyl-phosphatidylinositol anchored membrane protein that protects host cells from complement-mediated damage. We studied the rare inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface expression of DAF but normal levels of several other glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression, Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-) phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change. This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I restriction fragment length polymorphism. We created transfected Chinese hamster ovary cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed the specificity of anti-Dra alloantisera. The allele-specific transfectants could form the basis of a new serological approach to immunohematology. PMID- 1710233 TI - Relationship of the CD5 B cell to human tonsillar lymphocytes that express autoantibody-associated cross-reactive idiotypes. AB - We examined human tonsillar B cells for expression of autoantibody heavy-chain or kappa light-chain cross-reactive idiotypes (CRIs), respectively defined by murine MAbs G6 or 17.109. We find 17.109 or G6 each specifically binds a subpopulation of B cells, respectively reacting with 3.8 +/- 3% (mean +/- SD) or 2.0 +/- 1.2% of all tonsillar lymphocytes. Cells reactive with both 17.109 and G6 comprise only 0.4 +/- 0.3% of tonsillar lymphocytes. Although each tested specimen had 17.109-positive cells, 2 of 19 tonsils (11%) did not have any G6-reactive cells. We find that CRI-positive cells and CD5 B cells both co-express slgD but fail to bind peanut agglutinin or MAbs specific for CD10, indicating that both cell types reside in the mantle zones of secondary B cell follicles. However, less than half of the B cells bearing one or both of these CRIs express detectable levels of CD5. Nevertheless, we find that G6-reactive lymphocytes constitute a multiclonal population of cells that express homologous heavy chain variable region genes, each rearranged to one of several distinct and apparently nonmutated D and JH gene segments. Collectively, these studies indicate that expression of nondiversified autoantibody-encoding variable region genes may not be an exclusive property of B cells that bear detectable levels of the CD5 surface antigen. PMID- 1710234 TI - Direct vasoconstriction as a possible cause for amphotericin B-induced nephrotoxicity in rats. AB - In anesthetized rats we tested the hypothesis that amphotericin B (AmB) reduces glomerular filtration rate (GFR) by activating the tubuloglomerular feedback (TGF) mechanism. Infusion of 1 mg/kg AmB over 50 min was followed by a reduction in kidney GFR (from 0.47 +/- 0.03 to 0.39 +/- 0.02 ml/min per 100 g body wt during the second hour after infusion; P less than 0.05) and by an increase in urine flow and urinary chloride excretion. Single-nephron GFR (SNGFR) measured in proximal (TGF interrupted) or distal tubules (TGF intact) decreased to a similar degree from 33.4 +/- 1.8 and 30.6 +/- 1.2 nl/min in the control period to 19.7 +/ 1.9 and 21.2 +/- 1.6 nl/min during the second hour after AmB infusion (P less than 0.05). Distal chloride concentrations and TGF responses to changes in loop of Henle flow rate were not significantly altered by AmB. AmB at 10(-5) M reduced the diameter of isolated perfused afferent arterioles from rabbit kidneys. In isometrically contracting rings of rabbit aorta and renal artery in vitro AmB produced endothelium-independent constriction, with half-maximal contraction (EC50) being achieved by 1.8 x 10(-6) and 2.6 x 10(-6) M in intact vessels and 1.3 x 10(-6) and 1.7 x 10(-6) M in endothelium-denuded vessels respectively. Tension development did not occur in Ca-free media or in the presence of Ca channel blockers. Pretreatment with ouabain or Bay K 8644 potentiated the effect of AmB. The vasoconstrictive effect of AmB was counteracted by aminophylline and atrial natriuretic peptide. We conclude that the AmB-induced reduction in GFR is not caused by TGF activation and that AmB has a direct vasoconstrictor effect that is probably initiated by depolarization-induced opening of Ca channels. This effect may be an important component of the nephrotoxic actions of AmB. PMID- 1710236 TI - Organization of pyramidal neurons in area 17 of monkey visual cortex. AB - In sections of area 17 of monkey visual cortex treated with an antibody to MAP2 the disposition of the cell bodies and dendrites of the neurons is readily visible. In such preparations it is evident that the apical dendrites of the pyramidal cells of layer VI form fascicles that pass into layer IV, where most of them gradually taper and form their terminal tufts. In contrast, the apical dendrites of the smaller layer V pyramidal cells come together in a more regular fashion. They form clusters that pass through layer IV and into layer II/III where the apical dendrites of many of the pyramidal cells in that layer add to the clusters. In horizontal sections taken through the middle of layer IV, these clusters of apical dendrites are found to have an average center-to-center spacing of about 30 microns, and it is proposed that each cluster of apical dendrites represents the axis of a module of pyramidal cells that has a diameter of about 30 microns and contains about 142 neurons. The MAP2 antibody reaction also reveals that some pyramidal cells in layers IVA and IVB have their cell bodies arranged into cones. There are about 118 such cones beneath 1 mm2 of cortical surface and the apical dendrites of the pyramidal cells within them bundle together at the apex of each cone to pass into layer III. Surrounding the cones of neurons there are horizontally aligned, thin dendrites. The location of these dendrites coincides with the dark walls of the honeycomb pattern seen in layer IVA after cytochrome oxidase reactions, or after the parvocellular input from the lateral geniculate nucleus has been labeled. Thus the cones of pyramidal cells within upper layer IV fit into the pockets of the honeycomb pattern. Below the cones of pyramidal cells are the outer Meynert cells within layer IVB, and the cell bodies of these large neurons are disposed so that they preferentially lie beneath the neuropil between the cones of pyramids. It is suggested that pyramidal cell modules are a basic feature of the cerebral cortex, and that these are combined together by afferent inputs to the cortex to generate the systems of functional columns. PMID- 1710235 TI - Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells. AB - The propensity of Staphylococcus aureus to cause acute endovascular infections during transient bacteremia is poorly understood. To examine the events leading to the attachment of staphylococci to endothelium, adherence assays were developed to study the role of blood factors in the mediation of staphylococcal adherence to cultured human umbilical vein endothelium in vitro. Results indicate that the preferential attachment of S. aureus to endothelial cells is mediated by fibrinogen adsorbed from plasma onto the endothelial surface. The binding is apparently specific because it could be blocked by goat anti-human fibrinogen antibody in a dose-dependent fashion while nonimmune goat IgG, mouse MAb against AG-1 (a platelet antigen found on the endothelial cell surface), nonspecific mouse MAb and rabbit antibodies to human vitronectin and fibronectin were not inhibitory. Our data also indicate that fibrinogen is a necessary but not the only blood constituent in the mediation of binding of S. aureus to endothelium. This was supported by the finding that fibrinogen alone, at a concentration equivalent to that in plasma, did not potentiate staphylococcal adherence as much as plasma while afibrinogenemic plasma reconstituted with fibrinogen did. Because fibrinogen is known to bind to endothelial cells, our data is consistent with the hypothesis that fibrinogen and additional plasma factor(s), possibly activated during inflammation, promote staphylococcal adherence to endothelium. In addition, the role of the fibrinogen binding receptor of S. aureus as an adherence factor to native endothelium is also suggested. PMID- 1710237 TI - Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells. AB - We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction. PMID- 1710238 TI - CD59 functions as a signal-transducing molecule for human T cell activation. AB - The CD59 Ag is a 20-kDa protein that is widely expressed on most leukocytes and RBC, is coupled to the membrane by a phosphatidylinositol-glycan anchoring structure, plays a role in cell interaction between monocytes and T cells, and also functions as an inhibitor of cytolysis by the terminal C components C5b-9. Because this molecule is structurally related to the murine Ly-6 family of Ag, we have investigated whether anti-CD59 mAb might be capable of activating human T lymphocytes in a manner similar to that described for antibodies to the murine Ly 6 Ag. In the presence of the appropriate co-stimulators, mAb to one of the two epitopes on CD59 were capable of inducing both a rise in intracytoplasmic free Ca2+, inositol phosphate production, IL-2 production, and T cell proliferation. Anti-CD59-induced inositol phosphate turnover and IL-2 production were dependent on co-expression of the CD3/TCR complex. CD59-loss mutants of the Jurkat cell line were completely responsive to stimulation by anti-CD3 thereby demonstrating that CD59 does not play a role as a signal transducer downstream from the TCR. Taken together, these results demonstrate that the CD59 Ag can play multiple distinct roles in the regulation of the immune response. PMID- 1710239 TI - CD69 in resting and activated T lymphocytes. Its association with a GTP binding protein and biochemical requirements for its expression. AB - CD69 is a phosphorylated disulfide-linked homodimer that appears on the surface of human T, B cells and thymocytes in the early steps of activation; its molecular mass is 28 to 34 kDa under reducing conditions. This molecule is able to mediate positive signals to the lymphocytes as the anti-CD69 mAb (MLR3, AIM, Leu 23) in synergism with phorbol esters induce IL-2 production and proliferation of lymphocytes. Here we show that this molecule is associated to a GTP binding protein that is a substrate for Bordetella pertussis toxin. The relevance of CD69 in the activation process is also suggested by the broad range of signals able to modulate CD69 on T cells. In fact, not only the mitogens or the CD3-promoted activation, but also the alternative pathways mediated by CD2 or CD28 are accompanied by CD69 expression; moreover a very rapid and transient appearance of CD69 on the cell surface is observed also in response to a stimulus not specifically involved in T cell activation such as heat shock. Finally we demonstrate that CD69 is present in the cytoplasm of nonactivated T cells; accordingly its surface expression at the onset of activation is independent on a new RNA or protein synthesis. PMID- 1710240 TI - Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice. AB - To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody. PMID- 1710241 TI - Role of CD11/CD18 in adhesion and transendothelial migration of T cells. Analysis utilizing CD18-deficient T cell clones. AB - The role of leukocyte function-associated Ag-1 (LFA-1) (CD11a/CD18) in T cell endothelial cell (EC) interactions was assessed by utilizing CD11a/CD18-deficient T cell clones generated from a patient with leukocyte adhesion deficiency (LAD). The ability of these clones to bind to and migrate through monolayers of EC in vitro was compared with that of clones generated in a similar manner from normal controls. The LAD clones bound to EC to a similar extent as the controls. The contribution of other cell surface adhesion molecules was assessed with mAb blocking experiments. It was found that part of the EC binding by these CD11a/CD18-deficient clones was mediated by an interaction of very late Ag-4 (VLA 4) with vascular cell adhesion molecule-1 (VCAM-1) on the EC. In contrast to their normal ability to bind to EC, the capacity of the LAD clones to migrate through EC monolayers was significantly less than that of the control clones. This impairment in migration was not related to decreased intrinsic motility. Moreover, neither phorbol ester stimulation of the LAD clones nor IL-1 stimulation of the EC increased the capacity of the clones to migrate through EC monolayers, although binding to EC was augmented by both treatments. Only a minimal percentage of the migration of either control or LAD clones was inhibited by mAb to VLA-4 or VCAM-1. These data demonstrate that LFA-1 plays a central role in the transendothelial migration of T cells. In the absence of LFA-1, T cells retain the ability to bind to EC because of the activity of other receptor/ligand pairs, including VLA-4/VCAM-1. Finally, it is likely that, during both binding and transendothelial migration of T cells, additional cell surface molecules play a role. PMID- 1710242 TI - Limited T cell receptor diversity of transplacentally acquired maternal T cells in severe combined immunodeficiency. AB - Circulating maternal T lymphocytes were noted in the peripheral blood of six patients with severe combined immunodeficiency. Phenotypical analyses revealed the presence of both CD4 and CD8 subsets in some but not all cases. The maternal T cells could be stimulated by anti-TCR/CD3 mAb +/- rIL-2, but were virtually silent in the MLR and against the recall Ag purified protein derivative of tuberculin and tetanus toxoid, even in immunized patients engrafted with T cells from a responding mother. Using a panel of mAb against TCR V region gene encoded epitopes including V beta 5, V beta 6, V beta 8, V beta 12, and V alpha 2, we show that maternal T cells displayed a profoundly reduced TCR diversity, characterized by a lack of one or even several TCR V subsets in all six cases and a dramatic (5- to 25-fold) expansion of other TCR V subsets in three cases. In one patient analyzed, limited TCR diversity was also seen in T cells cultured from bone marrow and skin; restimulation experiments of these cells against cells expressing host MHC Ag were unsuccessful, as were attempts to exclusively allocate anti-host proliferative responses of maternal control T cells to the TCR V subsets that had undergone expansion in vivo. We conclude that a severely reduced TCR diversity is a common feature of maternal T cells engrafted in severe combined immunodeficiency patients. These novel findings provide a structural basis to understand the failure of these cells to protect the host from infections and may also help to understand their relative inefficiency to induce lethal, multi-organ, graft vs host disease. Moreover, as an experiment of nature, the reported phenomenon clearly illustrates the functional consequences in vivo of an insufficient TCR diversity. PMID- 1710243 TI - Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene. AB - The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope. PMID- 1710244 TI - Induction of IL-5 receptors on normal B cells by cross-linking surface Ig with anti-Ig-dextran. AB - Regulation of IL-5R expression in normal, non-Ly-1 (CD5) B cells was evaluated. Freshly isolated unfractionated spleen B cells express little or no detectable IL 5R. In contrast, B cells stimulated with anti-Ig-dextran conjugates express substantial numbers of IL-5R. Phenotypic analysis of the B cells responding to anti-Ig-dextran, and expressing IL-5R, demonstrates that these cells do not express Ly-1 or Mac-1. Scatchard analysis of B cells stimulated with anti-IgD dextran reveals two classes of IL-5R: a high affinity receptor with a Kd of 17 pM and approximately 300 receptors/cell, and a low affinity receptor with a Kd of 0.6 nM and approximately 1000 receptors/cell. Peak receptor expression in response to anti-IgD-dextran is seen 72 h after stimulation and with a dose of 10 ng/ml. The induced receptors are functional, because both proliferation and Ig secretion by B cells treated with anti-IgD-dextran are enhanced by IL-5. Other B cell mitogens such as LPS, soluble anti-Ig/IL-4, or phorbol esters/ionomycin are poor inducers of the IL-5R. Finally, not only does LPS fail to induce significant IL-5R expression on spleen B cells, it suppresses both high and low affinity IL 5R expression induced by anti-IgD-dextran. These data indicate that normal, non Ly-1 B cells are capable of expressing both high and low affinity IL-5R but that receptor expression is critically dependent on the type of stimulus provided to the B cell. A stimulus that produces extensive cross-linking of surface Ig on B cells, i.e., anti-Ig-dextran, is very effective in inducing IL-5R whereas a variety of other B cell mitogens are ineffective. PMID- 1710245 TI - A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies. AB - A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis. PMID- 1710246 TI - Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL. AB - T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro. PMID- 1710247 TI - In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein. AB - Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies. PMID- 1710248 TI - An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection. AB - A human mAb (HmAb) termed F105 was obtained by fusion of antibody-producing EBV transformed cells with the HMMA2.11TG/O cell line. F105 is an IgG1 kappa antibody that binds to the surfaces of cells infected with all HIV-1 strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP120 from all four strains. F105 does not react with denatured GP120 on Western blots, but does react with viral lysates and purified GP120 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP120 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F105 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (100%) inhibition at approximately 1 microgram/ml. F105 inhibits infection of HT-H9 cells by 100 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F105 HmAb reacts with a conformationally defined epitope on HIV-1/GP120 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F105 and the F105 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers. PMID- 1710249 TI - Rat CD16 is defined by a family of class III Fc gamma receptors requiring co expression of heteroprotein subunits. AB - Rat CD16 is defined here by a family of highly homologous class III Fc gamma receptor isoforms. Northern blot and polymerase chain reaction analysis indicate that multiple rtFc gamma RIII alpha isoforms are expressed by rat NK and macrophages contrasting the expression of a single class III receptor isoform by human and mouse NK and macrophages. Analysis of genomic Southern blots and splice variants identified by polymerase chain reaction suggests the existence of several homologous rat Fc gamma RIII alpha genes organized similar to human and mouse class III receptor genes. All rat Fc gamma RIII alpha isoform protein sequences have conventional transmembrane insertion sequences containing the unique LFAVDTGL motif conserved in all other class III Fc gamma and Fc epsilon RI alpha receptor sequences. A model is proposed for the protein-protein interactions between this sequence and the transmembrane sequences of two heteroprotein subunits, Fc epsilon RI gamma and CD3 zeta, known to interact with human and mouse class III receptors. Rat NK, monocytes, and neutrophils were all found to express transcripts for both of these subunits, whereas rat macrophages express only Fc epsilon RI gamma subunit transcripts. Furthermore, the Fc epsilon RI gamma subunit was found to promote the cell surface expression of rtFc gamma RIII alpha isoforms in transfected COS cells. PMID- 1710250 TI - Nucleotide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype. AB - Cold agglutinins are human autoantibodies, usually of the IgM class, which agglutinate RBC at low temperature. The major subset recognizes the I/i carbohydrate Ag, and many of these antibodies bear cross-reacting idiotypic determinants. An anti-idiotypic mAb that is specific for one of the idiotopes largely confined to cold agglutinins has been used to identify and monitor tumor cells that secrete these molecules in two patients. The tumor cells were immortalized with EBV and the idiotope-positive lines used to investigate the utilization of the VH and VL genes by these antibodies. Nucleotide sequence analysis of the two cold agglutinins (FS-1 and FS-2) revealed the utilization of a single common gene segment, VH4-21. Serologic analysis documented that only human antibodies utilizing the VH4-21 gene segment were reactive in the idiotope assay, other VHIV antibodies as well as a panel of antibodies derived from other VH families being negative. The DH, JH, VK, and JK gene segments of FS-1 and FS-2 were structurally distinct. These data suggest that the structural basis for the cross-reactive idiotope as well as cold agglutinin activity is the VH4-21 gene segment. A nucleotide change in H chain CDR1 of both cold agglutinins results in the substitution of an aspartic acid residue for glycine at position 31, suggesting that this amino acid might be critical to recognition of the red cell Ag. PMID- 1710251 TI - Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). AB - The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets. PMID- 1710253 TI - Purification of anti-epithelial mucin monoclonal antibodies by epitope affinity chromatography. AB - Monoclonal antibodies against the protein core of epithelial mucins have been found to react with the immunodominant sequence P D T R P A P (Burchell et al., 1989; Price et al., 1990a). Two immunoadsorbent matrices were prepared by linking the peptide A P D T R P A P G to CNBr-activated Sepharose and by linking the peptide C A P D T R P A P G to activated thiol-Sepharose, so that each immunoadsorbent contained the immunodominant motif. Anti-epithelial mucin antibodies (anti-breast carcinoma antibodies, anti-purified mucin antibodies and anti-human milk fat globule antibodies) were examined for reactivity with these preparations. The initial tests indicated that the substituted CNBr-activated Sepharose displayed lower non-specific antibody binding and this matrix was selected for further investigation. The anti-mucin antibodies were shown to react specifically with this affinity matrix and irrelevant antibodies failed to bind. A Sepharose-peptide immunoadsorbent column was examined for its capacity to purify several of these anti-mucin antibodies and it was determined that this procedure was highly efficient--purified IgG and IgM antibodies could be isolated from either hybridoma tissue culture supernatants or ascitic fluids. The capacity of the column was in excess of 40 mg antibody protein per ml of gel for the IgG3 antibody, C595 (anti-urinary mucin) and at least 10 mg antibody protein per ml of gel for the IgM antibody, NCRC-11 (anti-breast carcinoma). The procedure described permits the efficient purification of anti-mucin antibodies and provides a product which would be suitable for further investigations requiring highly immunoreactive antibodies (e.g., for radioimmunotherapy or immunoscintigraphy in patients with malignant disease). PMID- 1710252 TI - Stability of monoclonal antibody-defined epitopes. AB - Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and defines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42 degrees C and 65 degrees C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability. PMID- 1710254 TI - [Experimental study on histological changes of articular cartilage of femoral head under nonweight-bearing conditions]. AB - Histological changes of articular cartilage of the femoral head in the nonweight bearing conditions were investigated using 10-week-old Wistar rats whose hind limbs had been unilaterally transected. At 2 weeks after limb transection, decreased DNA synthesis in chondrocytes and depletion of glycosaminoglycan in the cartilage matrix in the intermediate zone of the articular cartilage were observed. After 8 weeks, articular cartilage thickness decreased significantly, and at 16 weeks the number of chondrocytes diminished in comparison with those of a weight-bearing femoral head. At 20 weeks, chondrocyte death and reduced width of articular cartilage became evident. No osteoarthritic changes, however, were observed under nonweight-bearing conditions. These findings suggest that weight bearing is very important in maintaining of both the structure and metabolism of the articular cartilage. PMID- 1710255 TI - The incidence of recurrence and hypothyroidism following treatment with antithyroid drugs, surgery or radioiodine in all patients with thyrotoxicosis in Malmo during the period 1970-1974. AB - The incidence of recurrence and of hypothyroidism was determined in all new patients treated for thyrotoxicosis during the period 1970-1974 in an unselected, well-defined urban population. A total of 309 patients were followed up for a median time period of 108 (1-192) months. There was a cumulative incidence of 51% recurrence in patients who were treated with antithyroid drugs for Graves' thyrotoxicosis, whereas after surgery or radioiodine treatment there were few recurrences, but 32% and 78% cumulative incidences of hypothyroidism. There were no recurrences after surgery or radioiodine treatment in patients with toxic multinodular goitre or solitary toxic adenoma, but 29% and 40% cumulative incidences of hypothyroidism following radioiodine treatment. Late hypothyroidism occurred after surgery for Graves' thyrotoxicosis, and in all groups treated with radioiodine. Thus it is advisable that all patients with Graves' thyrotoxicosis, regardless of treatment, and all patients with toxic multinodular goitre or solitary toxic adenoma treated with radioiodine, should be followed up for many years, and probably for life. PMID- 1710256 TI - Novel marine sponge amino acids, 10. Xestoaminols from Xestospongia sp. AB - The study of the anthelminthic components from a Fiji sponge Xestospongia sp. has yielded new amino alcohols, xestoaminols A-C. Xestoaminol B, (2S*)-aminotetradeca 11, 13-dien-(3R*)-ol [2], is isomeric to known Xestospongia products, (2S) aminotetradeca-5,7-dien-(3R)-ol [6] and (2S)-aminotetradeca-5,7-dien-(3S)-ol [7], recently reported by Gulavita and Scheuer. Xestoaminols A [1] and C [3] are, respectively, the dihydro and tetrahydro derivatives of xestoaminol B. A combined nmr and molecular mechanics study on the oxazolidinone of xestoaminol A provided the basis for the relative stereochemistry assigned at C-2 and C-3 in xestoaminol A. This compound was extremely active in assays testing for action against parasites, microbes, and reverse transcriptase. PMID- 1710257 TI - Art of the buy. PMID- 1710258 TI - Art of the sale. PMID- 1710259 TI - Loma Linda University: to make man whole. PMID- 1710260 TI - Dental student of 1990: UCLA similar but different. PMID- 1710261 TI - UCSF: then and now. PMID- 1710262 TI - UOP: the uncommon educational experience. PMID- 1710263 TI - USC: the contemporary student. PMID- 1710264 TI - Dentistry's demise? PMID- 1710265 TI - Genetic aspects of ion transport systems in hypertension. AB - Environmental factors, genetic polymorphism and differences in experimental design have been the main impediments to evaluating the evidence for a genetic association between cell membrane cation transport abnormalities and human essential or genetic hypertension. The present paper reviews the results obtained in the Milan hypertensive rat (MHS) and in its corresponding normotensive strain (MNS) in order to illustrate our approach to defining the role of cation transport abnormality in a particular type of genetic hypertension. Kidney cross transplantation between the strains showed that hypertension is transplanted along with the kidney. Proximal tubular cell volume and sodium content were lower in MHS rats while sodium transport across the brush border membrane vesicles of MHS rats was faster. Erythrocytes of MHS rats have a smaller volume, faster Na,K cotransport and a lower Km for internal sodium compared with MNS rats. The faster cotransport is also present in renal cells of the ascending limb and in vascular muscle cells. Moreover, these erythrocyte abnormalities are genetically associated in F2 hybrids and are primarily determined in the stem cells. These differences in ion transport between MHS and MNS rats are not present when studied in erythrocyte inside-out vesicles, which are deprived of membrane skeletal proteins, suggesting that a molecular abnormality underlies the functional one. We have identified a point mutation of one of these cytoskeletal membrane protein adducin genes in MHS rats. At present we are evaluating the possibility that mutation of the adducin gene in MHS rats might account for the differences in ion transport and, therefore, in blood pressure between the two strains. PMID- 1710266 TI - [The vasoactivity of endothelin in the nasal mucosa]. AB - Endothelin (ET), a novel endothelium-derived vasoconstrictor peptide with 21 amino acid residues, has recently been isolated from the supernatant of cultured porcine aortic endothelial cells. Several authors reported preliminary results demonstrating that ET has a potent contractile action in vascular smooth muscles, but its effect is slightly different in each target tissue. In the present study, we have examined the vasoactivity of ET in human and canine nasal mucosa with in vitro bioassay. The results are as follows. 1) ET induced a slow, and very sustained contraction from 10(-9) M. This response is stronger than that of noradrenaline (NA) and continued for 6-12 hours. 2) Under the sustained contraction by the administration of alpha 1 adrenoceptor agonist (methoxamine), ET induced an additional contractile response. 3) The response of ET depended on the concentration of Ca2+. 4) ET did not affect the contraction of vascular smooth muscle which was caused by the electrical stimulation. 5) ET increased the contraction of vascular smooth muscle which was caused by the administration of NA into the bath solution. PMID- 1710267 TI - Phenotypic heterogeneity in the syndromes of 3-methylglutaconic aciduria. AB - Combined 3-methylglutaconic and 3-methylglutaric aciduria, one of the more common urinary organic acid abnormalities, has been observed in at least three clinical syndromes. We studied an additional seven patients with 3-methylglutaconic aciduria, four of whom were best categorized as having the type II syndrome, two as having an "unspecified" syndrome, and one who may have had a primary urea cycle defect. In cultured cells and autopsy tissues derived from patients with the type II and unspecified syndromes, we were unsuccessful in identifying a defect in the leucine degradative pathway distal to 3-methylcrotonyl-coenzyme A carboxylase and in the cholesterol biosynthetic pathway between 3-hydroxy-3 methylglutaryl-coenzyme A reductase and diphosphomevalonate decarboxylase. Further assessment of the cholesterol biosynthetic pathway in several patients with one of the two types of disease also provided no defined abnormality. The primary metabolic defects in the type II and unspecified syndromes remain undefined. PMID- 1710268 TI - Putative new expression of genes in ixodid tick salivary gland development during feeding. AB - Poly(A+) mRNA-enriched fractions from salivary glands of partially fed Amblyomma americanum female ticks were translated in vitro with a rabbit reticulocyte translation system. Translated proteins were labeled with [35S]methionine, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified by autoradiography. Thirty major identifiable polypeptides with molecular weights ranging from 14 to 136 kDa were synthesized from mRNA isolated from salivary glands of ticks weighing less than 100 mg. Polypeptides that comigrated at the same molecular weight were translated by mRNA from ticks at a more advanced stage of feeding (more than 300 mg) as were 8 others with molecular weights of 31, 71, 91, 106, 113, 118, and 128 kDa. Results demonstrated that differential gene expression may be stimulated in the developing salivary glands as the tick feeds. PMID- 1710269 TI - Characterization of a recombinant Eimeria acervulina antigen expressed in sporozoite and merozoite developmental stages. AB - A cDNA from Eimeria acervulina encoding an immunogenic region of antigens shared between sporozoites and merozoites was cloned and expressed in Escherichia coli. Immunofluorescence staining of sporozoites, merozoites, and coccidial-infected intestinal tissue with monoclonal antibody used to detect the recombinant clone indicated that the epitope was present on internal parasite proteins. Immunostaining of nitrocellulose paper containing protein from both asexual stages revealed numerous sporozoite antigens (18-120 kDa) and only 1 merozoite antigen (150 kDa). Northern blot hybridization assays using this cDNA clone for probing sporozoite and merozoite RNA showed that distinct transcripts were present in both asexual stages. Similar to the immunofluorescence studies, many homologous RNAs were observed in sporozoites (3.7-13 kb) and only 1 prominent hybridizing species was found in merozoites (3.3 kb). The recombinant coccidial antigen, designated MA16, is a 125-kDa beta-galactosidase fusion protein, representing about 10 kDa of parasite protein. In blastogenesis assays, purified recombinant MA16 antigen is capable of activating T lymphocytes obtained from E. acervulina-immune inbred chickens. DNA sequencing of MA16 clone and analysis of the predicted amino acid sequence indicated several putative T cell epitopes that may be responsible for the observed in vitro blastogenic response. PMID- 1710270 TI - Isolation of an SBA lectin-reactive glycoprotein (gp50) and its identification in Schistosoma mansoni larval and adult worm secretions. AB - A major 50-kDa soybean agglutinin (SBA)-reactive component present in extracts of Schistosoma mansoni eggs was isolated by SBA lectin affinity chromatography. In polyacrylamide gel electrophoresis (PAGE), the SBA-reactive component was seen as a 100-kDa polypeptide band that after reduction and alcylation was substituted by a 50-kDa component. This suggests that it occurs in native form as a dimer. Monoclonal antibodies produced against gp50 reacted with miracidial and cercarial secretions and with adult worm components including tegumental structures suggestive of a secretory function. PMID- 1710272 TI - The art and heart of nursing. PMID- 1710271 TI - Alterations of the intestinal innervation in mice infected with Schistosoma mansoni. AB - Schistosomiasis mansoni is a parasitic disease in which granulomas form around schistosome eggs in the liver and intestines. The purpose of this study was to determine the alterations in the intrinsic innervation of the distal ileum and proximal colon resulting from schistosomiasis. Using murine schistosomiasis mansoni, we examined light microscopic preparations stained with osmium-zinc iodide or the dihydronicotinamide adenine dinucleotide: nitro BT oxidoreductase (NADH) method. We also examined specific populations of peptidergic nerves (vasoactive intestinal polypeptide and substance P) using an avidin-biotin complex (ABC) immunohistochemical technique. We found that granulomas focally destroyed the enteric nerves. Occasionally nerves were found within granulomas, particularly at the periphery of the lesions. Nerve cell bodies close to granulomas had altered staining, which included increased staining for vasoactive intestinal polypeptide. The distribution of nerve injury varied between the 2 enteric segments studied. In the distal ileum, the principal injury was to the myenteric plexus; whereas, the submucous and mucosal plexuses were predominantly damaged in the proximal colon. The physiologic significance of this injury to the enteric nerves requires elucidation. PMID- 1710273 TI - Stabilization of the sick infant or child. AB - Infants and small children are physiologically different than adults. Because of these differences, the smaller patient is prone to hypoxia, hypoglycemia, hypovolemia, and hypothermia. This article reviews the physiological characteristics of infants and small children. Pertinent information to obtain when assessing the smaller patient is discussed as is the manner in which interpretation of the data may change based on the patient's age. PMID- 1710274 TI - Time-saving formats for patient care planning in outpatient surgery units. AB - Due to the frequent patient turnover and the fast pace of an outpatient surgery unit, time is limited for developing an individual plan of care. Other factors making this task more complex are related to brief nursing observations and unclear patient expectations. Care plans are now mandated in the outpatient areas at Central Baptist Hospital in Lexington, KY. The plans need to be realistic, comprehensive, and timesaving, yet generic. A checklist format, which is described in this article, was the solution. PMID- 1710275 TI - Practical points in the care of the post-lumbar spine surgery patient. AB - Care of the patient who has undergone lumbar spine surgery is facilitated by knowledge of the procedure. The purpose of this article was to identify indications for lumbar surgery, review spinal anatomy, and identify postoperative nursing priorities for these patients. A firm knowledge base, as well as aggressive attention to postsurgical and postanesthetic priorities, help to maximize the patient's chances for a successful recovery. PMID- 1710276 TI - Risk management: a proactive process. AB - Successful risk management is a proactive process designed to prevent litigation and possible loss of assets. Some examples of standards of care and legal implications for nurses in ambulatory and PACU units are discussed. Suggestions are made regarding specific risk management issues. PMID- 1710277 TI - Boundaries: personal limits. PMID- 1710278 TI - Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved. AB - In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level. PMID- 1710279 TI - Folding and function of the myelin proteins from primary sequence data. AB - To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular. In this program, propensities for the secondary structure conformations as well as physical chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two-dimensional map of the correlation coefficients. Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the alpha or beta conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of beta propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed alpha-beta-alpha folding. To trace the immunoglobulin fold along the P0 sequence, we compared the beta propensity curve of P0 with that of the immunoglobulin M603, whose three-dimensional structure has been determined. We propose that the flat beta-sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra beta-fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x-ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2',3' nucleotides into corresponding 2'-nucleotides. PMID- 1710280 TI - Co-activation of insulin-like growth factor-I receptors and protein kinase C results in parasympathetic neuronal survival. AB - We have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin-like growth factor I (IGF-I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival-promoting activity. In combination with PdBu, however, IGF-I or insulin, at concentrations sufficient to act through the IGF-I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)-induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF-I. When added alone, aFGF-induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF-I, or insulin with PdBu under serum-free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF-I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo. PMID- 1710281 TI - Hypothyroidism reduces the rate of slow component A (SCa) axonal transport and the amount of transported tubulin in the hyt/hyt mouse optic nerve. AB - Thyroid hormone deficiency in the developing brain leads to disorders of neuronal process growth. This is evidenced by reduced axonal and dendritic size and complexity (Garza et al.: Developmental Brain Research 43:287-297, 1988; Ruiz Marcos: Iodine and the Brain. New York: Plenum Press, pp 91-102, 1989). These findings may be related to alterations in the neuronal cytoskeleton in hypothyroidism, such as reduced or abnormal microtubular number and density (Faivre et al.: Developmental Brain Research 8: 21-30, 1983), and altered assembly, stabilization, and composition of microtubule protein in the hypothyroid brain. Neurofilaments also contribute to axonal caliber and process stability. Similar to microtubules, certain properties of neurofilaments are altered in developing hypothyroid axons (Marc and Rabie: International Journal of Developmental Neuroscience 3: 353-358, 1985; Faivre et al.: Developmental Brain Research 8:21-30, 1983) that may affect axonal caliber and process stability. Normal process growth is predicted on formation of appropriate numbers of microtubules and on the normal synthesis and axonal transport of cytoskeletal components [tubulin, microtubule associated proteins (MAPs), and neurofilament proteins]. Hypothyroidism might alter the neuronal cytoskeleton and neuronal growth either by affecting the developmental programs for expression of specific isoforms of cytoskeletal proteins or by changing the delivery of cytoskeletal proteins via slow axonal transport, particularly slow component a (SCa). Previous studies had demonstrated changes in the amount of specific microtubule protein isoforms and mRNAs (Stein et al.: Iodine and the Brain. New York: Plenum Press, pp 59-78, 1989a). To further elucidate the molecular basis for process growth abnormalities in the hypothyroid brain, we investigated slow axonal transport in the mouse to determine the effects of thyroid hormone deficiency on the rate and composition of SCa. Comparisons of SCa in the optic nerve of hyt/hyt hypothyroid mouse and euthyroid hyt/+ littermates and euthyroid progenitor strain, BALB/cBY +/+ mice, indicated that the velocity of SCa was significantly reduced in hyt/hyt optic nerve relative to hyt/+ and +/+. The axonal transport rate for tubulin, which is carried in SCa, was 0.118 mm/day in the hyt/hyt optic nerves. This rate was significantly different for the tubulin rates for the hyt/+ optic nerves (0.127 mm/day) and for the +/+ optic nerves (0.138 mm/day). Neurofilament proteins, as measured by the 140,000 daltons component, NFM, also appeared to be reduced in velocity in the hyt/hyt versus the hyt/+ and +/+ optic nerves.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1710282 TI - Distribution of proteolipid protein and myelin basic protein in cultured mouse oligodendrocytes: primary vs. secondary cultures. AB - The distribution of proteolipid protein (PLP) and myelin basic protein (MBP) was examined in differentiating oligodendrocytes of primary and secondary mouse brain cell cultures by single- and double-label indirect immunofluorescence. In primary cultures, MBP and PLP were differentially located in oligodendrocytes. MBP became concentrated as fine punctate dots lining the edges of processes and as coarse grains in flattened sheet-like structures. PLP was distributed diffusely throughout cell bodies and processes but was limited to the perimeter of sheets and some processes within sheets. To compare the detailed distribution of PLP and MBP in the absence of underlying cells, a simple method for the growth of isolated oligodendrocytes in secondary cultures was developed. Cells were maintained in primary culture for 39-41 days, harvested by scraping, enriched for oligodendrocytes, and plated at low cell density. After 1 week, isolated oligodendrocytes had developed long processes and large flattened membranous sheets. MBP and PLP were differentially localized in these cell structures. The sheets contained fine-grained patches of MBP, which were surrounded by networks of MBP- processes. In contrast, PLP was initially seen throughout the cell bodies and processes. In older cultures, PLP became strikingly concentrated in curvilinear membranous profiles. The observations show that PLP and MBP are differentially located in cultured mouse oligodendrocytes. Furthermore, the precise distribution of these myelin-specific antigens is dependent on culture conditions. PMID- 1710283 TI - Expression of plasmolipin in oligodendrocytes. AB - Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602-610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligodendrocyte cultures. Double-label immunofluorescence showed that plasmolipin was expressed by galactocerebroside (GC)-positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC-positive cells. During the following 3 weeks in culture plasmolipin staining of oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmolipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenates from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by oligodendrocytes. PMID- 1710284 TI - Advances in the use of fine-needle aspiration cytology in the diagnosis of palpable lesions of the head and neck. AB - Our experience in performing fine-needle aspiration for the investigation of palpable lesions of the head and neck region is reviewed. Three hundred cases were performed over a period of approximately two years. In most circumstances, a diagnosis could be rendered simply on the basis of routine cytomorphologic interpretation. In some situations, it was necessary to employ ancillary techniques such as immunoperoxidase staining, electron microscopy, and flow cytometry to confirm the morphologic diagnosis. Several cases illustrating the clinical utility of the technique are presented in detail. Fine-needle aspiration is a relatively atraumatic, well-tolerated procedure which can be readily performed in the usual outpatient setting. This technique is an excellent first line method for investigating the nature of palpable lesions in the head and neck region. PMID- 1710285 TI - Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus. AB - We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported. PMID- 1710286 TI - Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope. AB - The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T lymphocyte-mediated protective immunity. PMID- 1710287 TI - Molecular location of a species-specific epitope on the hamster scrapie agent protein. AB - Scrapie is a transmissible neurodegenerative disease of sheep and goats. An abnormal host protein, Sp33-37, is the major protein component of the scrapie agent and the only known disease- or agent-specific macromolecule. Two monoclonal antibodies (MAbs), 4H8 (immunoglobulin G2b [IgG2b]) and 6B11 (IgG1), produced by immunizing mice with the intact hamster 263K scrapie agent protein, Sp33-37Ha, were found to have species specificity similar to that reported previously for MAb 3F4 (IgG2a), which was produced by using PrP-27-30 as the immunogen (R. J. Kascsak, R. Rubenstein, P. A. Merz, M. Tonna-DeMasi, R. Fersko, R. I. Carp, H. M. Wisniewski, and H. Diringer, J. Virol. 61:3688-3693, 1987). These antibodies all bound to Sp33-37 derived from hamster but not from mouse cells. Competitive binding assays demonstrated that all three MAbs bound to the same or overlapping sites on Sp33-37Ha. The molecular location of the epitope for these antibodies was determined to within 10 residues by using an antigen competition enzyme linked immunosorbent assay in which synthetic peptides spanning Sp33-37Ha residues 79 to 93 or 84 to 93 specifically inhibited binding of these antibodies to plates coated with purified Sp33-37Ha. A synthetic peptide with the mouse specific sequence (83 to 92) that differed from the hamster sequence by substitution at two positions (MetHa-87----LeuMo-86 and MetHa-90----ValMo-89) did not inhibit antibody binding to Sp33-37Ha. MAb 3F4 binding to hamster Sp33-37 was eliminated by chemical modification of Sp33-37Ha with diethylpyrocarbonate or succinic anhydride and by cleavage with CNBr or trypsin. The effect of diethylpyrocarbonate on MAb 3F4 binding was not reversed by hydroxylamine treatment. MAb 3F4 binding was not affected by prolonged exposure of Sp33-37Ha to 70% formic acid or by boiling in sodium dodecyl sulfate. We conclude that the epitope for these MAbs is a linear determinant that includes Met-87, Lys-88, and Met-90 and that Met-90 is probably the major species-specific determinant. PMID- 1710288 TI - An Epstein-Barr virus protein associated with cell growth transformation interacts with a tyrosine kinase. AB - Epstein-Barr virus (EBV) encodes two integral membrane proteins in latently infected growth-transformed cells. One of these, LMP1, can transform rodent fibroblasts and induce markers of B-lymphocyte activation. The second, LMP2, colocalizes with LMP1 in a constitutive patch in the EBV-transformed B-lymphocyte plasma membrane. The experiments reported here demonstrate that LMP2 may biochemically interact with LMP1 and that LMP2 closely associates with and is an important substrate for a B-lymphocyte tyrosine kinase in EBV-transformed B lymphocytes or in B-lymphoma cells in which LMP2 is expressed by gene transfer. LMP2 is also serine and threonine phosphorylated. LMP2 localizes to a peripheral membrane (presumably plasma membrane) patch in transfected B-lymphoma cells and colocalizes with much of the cellular tyrosine-phosphorylated proteins. LMP2 undergoes tyrosine phosphorylation in anti-LMP2 or antiphosphotyrosine immunoprecipitates from transfected B-lymphoma cells or EBV-transformed B lymphocytes. The first 167 of the 497 amino acids of LMP2 retain full ability to associate with and act as a substrate for a tyrosine kinase. A 70-kDa phosphotyrosine cell protein associates with LMP2 in transfected cells or in EBV transformed B lymphocytes and could be a mediator of the effects of LMP2. PMID- 1710289 TI - Immunodominant T-cell epitope on the F protein of respiratory syncytial virus recognized by human lymphocytes. AB - The lymphocyte proliferative responses to respiratory syncytial virus (RSV) were evaluated for 10 healthy adult donors and compared with proliferative responses to a chimeric glycoprotein (FG glycoprotein) which consists of the extracellular domains of both the F and G proteins of RSV and which is produced from a recombinant baculovirus. The lymphocytes of all 10 donors responded to RSV, and the proliferative responses to the whole virus were highly correlated with the responses to the FG glycoprotein. These data suggested that one or both of these glycoproteins of RSV were major target structures for stimulation of the human lymphocyte proliferative response among virus-specific memory T cells. The lymphocytes of four donors were evaluated further for their proliferative responses to a nested set of overlapping peptides modeled on the extracellular and cytoplasmic domains of the F protein of RSV. Strikingly, the lymphocytes of all 4 donors responded primarily to a region defined by a single peptide spanning residues 338 to 355, and the lymphocytes of 2 donors responded to an overlapping peptide spanning residues 328 to 342 also, thus defining a region of the F1 subunit within residues 328 to 355 that may circumscribe an immunodominant site for stimulation of human T cells from a variety of individuals. This region of the F protein is highly conserved among A and B subgroup viruses. As revealed by monoclonal antibody blocking studies, the lymphocytes responding to this antigenic site had characteristics consistent with T helper cells. Similar epitope mapping studies were performed with BALB/c mice immunized with the FG protein in which a relatively hydrophobic peptide spanning residues 51 to 65 within the F2 subunit appeared to be the major T cell recognition determinant. The data are discussed with respect to an antigenic map of the F protein and the potential construction of a synthetic vaccine for RSV. PMID- 1710290 TI - Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src. AB - The molecular mechanism by which retroviral Gag proteins are directed to the plasma membrane for the formation of particles (budding) is unknown, but it is widely believed that the MA domain, located at the amino terminus, plays a critical role. Consistent with this idea, we found that small deletions in this segment of the Rous sarcoma virus Gag protein completely blocked particle formation. The mutant proteins appear to have suffered only localized structural damage since they could be rescued (i.e., packaged into particles) when coexpressed with Gag proteins that are competent for particle formation. To our surprise, the effects of the MA deletions could be completely suppressed by fusing as few as seven residues of the myristylated amino terminus of the oncoprotein p60src to the beginning of the mutant Gag proteins. Particles produced by the chimeras were of the same density as the wild type. Two myristylated peptides having sequences distinct from that of p60src were entirely unable to suppress MA deletions, indicating that myristate alone is not a sufficient membrane targeting signal. We hypothesize that the amino terminus of p60src suppresses the effects of MA deletions by diverting the Rous sarcoma virus Gag protein from its normal site of assembly to the Src receptor for particle formation. PMID- 1710292 TI - Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription. AB - A secondary structure in the 5' noncoding region of avian retrovirus RNA, called the U5-leader stem, was shown previously to have a role in initiation of reverse transcription (D. Cobrinik, L. Soskey, and J. Leis, J. Virol. 62:3622-3630, 1988). We now show that an additional RNA secondary structure near the U5 terminus, called the U5-IR stem, is also important for reverse transcription. Mutations that disrupt the U5-IR stem cause a replication defect associated with both a decrease in synthesis of viral DNA in infected cells and a decrease in initiation of reverse transcription in melittin-permeabilized virions. Structure compensating base substitutions in the U5-IR restore reverse transcription efficiency. In viral DNA, U5-IR sequences are included in the U5 terminal region that functions as a viral integration donor site. When base substitutions are introduced into these sequences, a reduced efficiency of integration in vitro and in vivo is observed. These observations indicate that U5-IR sequences have a structural role in reverse transcription of viral RNA and a sequence-specific role in the integration of viral DNA. PMID- 1710291 TI - Identification of two T-cell epitopes on the candidate Epstein-Barr virus vaccine glycoprotein gp340 recognized by CD4+ T-cell clones. AB - Current efforts to develop an Epstein-Barr virus subunit vaccine are based on the major envelope glycoprotein gp340. Given the central role of CD4+ T cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human CD4+ T cells within the 907-amino-acid sequence of gp340. A panel of gp340-specific CD4+ T-cell clones from an Epstein-Barr virus-immune donor were first assayed for their proliferative responses to a series of truncated gp340 molecules expressed from recombinant DNA vectors in rat GH3 cells, by using an autologous B lymphoblastoid cell line as a source of antigen-presenting cells. The first four T-cell clones analyzed all responded to a truncated form of gp340 which contained only the first 260 N-terminal amino acids. These clones were subsequently screened for responses to each of a panel of overlapping synthetic peptides (15 mers) corresponding to the primary amino acid sequence of the first 260 N terminal amino acids of gp340. One clone (CG2.7) responded specifically to peptides from the region spanning amino acids 61 to 81, while three other clones (CG5.15, CG5.24, and CG5.36) responded specifically to peptides from the region spanning amino acids 163 to 183. Work with individual peptides from these regions allowed finer mapping of the T-cell epitopes and also revealed the highly dose dependent nature of peptide-induced responses, with inhibitory effects apparent when the most antigenic peptides were present at supraoptimal concentrations. Experiments using homozygous typing B lymphoblastoid cell lines as antigen presenting cells showed that the T-cell clones with different epitope specificities were restricted through different HLA class II antigens; clone CG2.7 recognized epitope 61-81 in the context of HLA DRw15, whereas clones CG5.15, CG5.24, and CG5.36 recognized epitope 163-183 in the context of HLA DRw11. The present protocol therefore makes a systematic analysis of CD4+ T-cell epitopes within gp340 possible; it will be necessary to screen gp340-specific T cell clones from a variety of donors to assess the wider influence of HLA class II polymorphism upon epitope choice. PMID- 1710293 TI - Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells. AB - Alpha interferon (IFN-alpha) is effective in preventing the release of human immunodeficiency virus (HIV) from chronically infected T-lymphocytic (ACH-2) and promonocytic (U1) cell lines stimulated with the phorbol ester phorbol-12 myristate-13 acetate (PMA). In the present study, we observed that together with particle production, shedding of HIV antigen (p24gag) occurs in the T-cell line ACH-2 both constitutively and after stimulation with PMA. IFN-alpha, although effective in suppressing the release of HIV particles, did not inhibit shedding of p24gag into the culture supernatants of either unstimulated or PMA-stimulated cells. These observations may be of relevance in the evaluation of the in vivo efficacy of IFN-alpha treatment of HIV-infected individuals as determined by levels of p24 antigen in plasma. PMID- 1710294 TI - [The effect of recombinant human granulocyte colony-stimulating factor (rG-CSF) on childhood neutropenias]. AB - The clinical effect of recombinant human granulocyte colony-stimulating factor (rG-CSF), produced by Chinese hamster ovary cells, was studied in 27 patients with childhood neutropenias. The sample consisted of 8 patients with congenital neutropenia (Kostmann type), 9 with neutropenia with miscellaneous causes (5 chronic benign, 2 associated with hypogammaglobulinemia, 1 drug-induced, and 1 hypoplastic type), 3 with cyclic neutropenia, and 7 with severe aplastic anemia. The rG-CSF was given subcutaneously (or in a few cases intravenously) at a dose of 2 micrograms/kg/day for 7 days and 5 micrograms/kg/day for additional 7 to 28 days in cases with poor response. The rG-CSF was effective in 18 of 27 cases (67%). Patients with congenital neutropenia and aplastic anemia responded less frequently and poorly. The mean level of absolute neutrophil counts of 8 congenital neutropenia cases increased from 88/microliters to 2,718/microliters. That of 9 miscellaneous cases changed from 189/microliters to 7,224/microliters at a dose of 2 micrograms/kg/day. In 7 aplastic anemia cases pretreatment level of 220/microliters rose to 851/microliters, usually after increasing the dose up to 5 micrograms/kg/day. The rG-CSF was apparently effective in 3 cases of cyclic neutropenia. In any type of neutropenia, the effect was largely transient; after the discontinuation of rG-CSF, the absolute neutrophil counts tended to decrease to pretreatment levels within 1 to 2 weeks. The G-CSF was well tolerated, and only one case with mild lumbago and another with minimal elevation of transaminases were observed. We conclude that the rG-CSF can be effective for treating various types of childhood neutropenia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710295 TI - [Clinical evaluation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in autologous bone marrow transplantation]. AB - We administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 5 micrograms/kg/day by intravenous drip infusion for 21 consecutive days in autologous bone marrow transplanted patients. The period of posttransplant neutropenia was markedly shortened by the rhG-CSF treatment; mean days required for neutrophil recovery (greater than 500/mm3) of 14.3 days in the rhG-CSF group (n = 21) versus 27.8 days in the historical control group (n = 11). More importantly, the numbers of febrile days between day 15 and day 28 were found to be fewer in the rG-CSF group than in control group. These effects were obtained without delay in the recovery of other blood cell series and without any side effect. We conclude that the posttransplant use of the rhG-CSF is beneficial for prevention and treatment of infectious complications after autologous bone marrow transplantation. PMID- 1710296 TI - [Congenital hemoglobin abnormalities--thalassemia syndrome]. PMID- 1710297 TI - Veratramine-induced behavior associated with serotonergic hyperfunction in mice. AB - The administration of veratramine produced generalized tremor, myoclonus, hindlimb abduction, backward gait and Straub tail, similar to the "5 hydroxytryptamine (5-HT) syndrome", in mice. Pretreatment with metergoline, methysergide, mainserin or cyproheptadine ameliorated veratramine-induced myoclonus and tremor. For suppression of other symptoms, mianserin and cyproheptadine were effective. Metergoline improved hindlimb abduction and Straub tail, but did not inhibit backward gait. Methysergide was ineffective for the remaining symptoms. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) enhanced all these symptoms except for Straub tail. 8-Hydroxy-2-[di-n-propylamino] tetralin hydrobromide (8-OH-DPAT) augmented tremor, hindlimb abduction and backward gait, but did not influence myoclonus and Straub tail. 5-Methoxy-3[1,2,3,6 tetrahydropyridin-4-yl] 1H-indole (RU 24969) did not modify the symptoms. Destruction of 5-HT neurons using 5,6-dihydroxytryptamine (5,6-DHT) resulted in suppression of the syndrome. The denervation supersensitivity caused by 5,6-DHT did not increase the response to veratramine. These findings indicate that part of the site of action of veratramine may be the presynaptic 5-HT neurons. PMID- 1710298 TI - Mechanisms of veratramine-induced 5-HT syndrome in mice. AB - Regional monoamine assays revealed that during veratramine-induced myoclonic movements, the contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the cerebral cortex were reduced with a slight increase in dopamine metabolites in the midbrain and brainstem. A similar tendency to decrease 5-HT and 5-HIAA contents was observed in the hypothalamus and hippocampus without increase in the contents of dopamine and its metabolites. Norepinephrine levels were not modified in any brain region at any time after the administration of the veratrum alkaloid. It was found that the veratramine evoked 3H-5-HT release from the frontal cortical slices was Ca+(+)-independent and persistent, and it continued approximately 20 min after the 2-min exposure to veratramine. The uptake of 3H-5-HT into the frontal cortical slices was inhibited competitively by veratramine. These results suggest that veratramine is both a releaser and uptake inhibitor of 5-HT and that the veratramine-induced involuntary movements may be mediated by serotonergic hyperfunction. PMID- 1710299 TI - Gastric motility changes in capsaicin-induced cytoprotection in the rat stomach. AB - We have examined the effect of orally administered capsaicin on gastric motility in the rat to investigate a possible relationship between motility change and cytoprotection induced by this agent. Capsaicin, given orally (1-30 mg/kg), dose dependently inhibited hemorrhagic band-like lesions induced by ethanol (60% in 150 mM HCl). This protection was significantly mitigated by desensitization of afferent neurons following capsaicin pretreatment 2 weeks before the experiment, and it was also significantly attenuated by prior administration of indomethacin, but not by spantide. Intragastric administration of capsaicin (30 mg/kg) significantly inhibited gastric motility and increased the mucosal blood flow, but had no effect on the transmucosal potential difference of the stomach. These functional changes induced by capsaicin were also less marked in the afferent neuronal desensitized rat, and they were significantly attenuated by indomethacin but not by spantide. These results suggest that the mucosal protection by intragastric capsaicin may be associated with the inhibition of gastric motility and the increase of mucosal blood flow. These responses may be induced by activation of primary afferent neurons which are probably sensitized by endogenous prostaglandins. PMID- 1710300 TI - Clonidine reduces substance P binding in rat spinal cord membrane preparation. AB - The effects of clonidine on substance P (SP) binding was investigated using rat brain and spinal cord membrane preparations preincubated with various concentrations of clonidine. [3H]SP specific binding in the spinal cord was significantly decreased with 10(-4) M clonidine, but no effect on binding was seen in the brain. Scatchard analysis of SP binding indicated that Bmax was significantly depressed without changing the affinity. The mechanism of clonidine induced analgesia includes a spinal neural component and action on the SP receptors. PMID- 1710301 TI - [Early changes of postpneumonectomy lung growth in premature rats]. AB - Following pneumonectomy in animals, the contralateral lung increases in volume, weight, collagen content, protein, and cell number, reaching levels approximate to those of both lungs of control animals. The volume and weight response in quicker and more complete in young animals compared to old animals. The increase in the amount of DNA was found to be greater in young rats compared to old ones. However, little is known about the effects of pneumonectomy in immature animals, in which combined effects of normal and the compensatory lung growth may be expected. The present studies were aimed at elucidating early changes in terms of morphology and biochemistry in the contralateral lung following pneumonectomy in premature rats. Male Sprague-Dawley rats (2 week-old) were subdivided into 3 groups. Group P, underwent left pneumonectomy, group S was sham operated, which group C was matched by age, sex, and weight with group P. Morphological studies consisted of light microscopic morphometry and immunohistochemistry using anti bromodeoxyuridine (BrdU) were performed. Biochemical studies included measurement of DNA polymerase activity, DNA and RNA content, collagen and elastin content. The wet lung weight in group P after one week reached approximately the same as that of bilateral lungs of groups S and C. The fixed lung volume of group P reached that of group S or group C at three weeks. The activity of DNA polymerase and BrdU positive alveoli were increased only during the early period following pneumonectomy. DNA content in group P reached the same range as group S and C at 4 weeks, suggesting the occurrence of cellular hyperplasia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710302 TI - [Hyperthermic treatment in patients with benign prostatic hypertrophy]. AB - Hyperthermic treatment was performed in 31 patients with benign prostatic hypertrophy (BPH). Eight patients of them had a urethral catheter because of urinary retention. The prostate was heated trans-rectally to 43-45 degrees C. The treatment consisted of 10 sessions of 60 min. each. To evaluate this treatment, the following parameters were determined before, during and one week after the last hyperthermia session: subjective symptoms score, and residual urine volume, uroflowmetry and transrectal ultrasound of the prostate as objective data. Symptoms score improve in all patients. Of 8 patients with a catheter, the catheter could be removed from 4 patients. There was no significant change in prostate volume, but significant decreases of residual urine volume, and increases of maximum flow rate and mean flow rate were observed. No adverse reactions were seen. Judging from the above results, this treatment is considered to be useful for patients with BPH. PMID- 1710303 TI - [Studies of sodium pentosan polysulphate (SPP) therapy on the urinary stone disease. Experimental studies on the calcium oxalate crystallization and the effects in healthy subjects]. AB - Concerning with the inhibitory activity of sodium pentosan polysulphate (SPP) on calcium oxalate stone formation, the following three experiments were carried out. 1) The inhibitory activities for calcium oxalate crystal formation, aggregation and growth in vitro, 2) the effects on the deposition of calcium oxalate crystals in rats treated with ethylene glycol, and 3) the effects of oral administration to human subjects on the metastable limit of urinary calcium oxalate and on the inhibitory activity of the urine for calcium oxalate crystal growth. The results were as follows: 1. The degree of the calcium oxalate crystal formation measured by an aggregometer was decreased by addition of more than 1 mM of SPP. 2. The inhibitory activity of SPP on calcium oxalate crystal aggregation was measured using a Coulter Counter. The aggregation was inhibited at a low concentration of SPP (0.02 microM). 3. Calcium oxalate crystal growth was measured using a seeded crystal method with [14C] oxalic acid. SPP inhibited the crystal growth proportionally to the concentration of SPP (0.1 microM-100 microM). 4. Renal tubular deposition of calcium oxalate crystals was marked in rats that orally received ethylene glycol (0.4%), which was expected to be suppressed by intramuscular administration of SPP (30 mg/kg/day or 60 mg/kg/day). 5. Oral administration of SPP (500 mg/day) to five healthy male subjects for five days resulted a tendency to increase of the metastable limit of the urinary calcium oxalate as measurement by an aggregometer, but the inhibitory activity of the urine for calcium oxalate crystal growth, which was measured by seeded crystal method with [14C] oxalic acid, showed no variable changes. PMID- 1710304 TI - [Use of contrykal in intensive care of myocardial infarct during the prehospital stage]. AB - The effect of contrykal was evaluated in 146 male patients with first myocardial infarction and 116 control patients in the prehospital period. Intravenous contrykal was given in a single dose of 20,000 IU within 30 to 360 minutes of onset of myocardial infarction, followed by intravenous administration of heparin, 10,000-15,000 U. The control patients received conventional therapy. Earlier application of contrykal contributed to attenuation of clinical manifestations of myocardial infarction. The drug was found to produce a clear cut antianginal effect. It also exerted a positive action on the abdominal syndrome. There was a rapid inverse dynamics in ECG changes, fermentaemia (aspartate aminotransferase and alanine aminotransferase), decreased myocardial necrotic mass. PMID- 1710305 TI - Correlation of the percentage of activated, CD3 + DR + lymphocytes to serum neopterin level in HIV-seropositive haemophiliacs. AB - The percentage of activated, CD3+ DR+ and CD8+ Leu7+ lymphocytes and the serum neopterin concentration were determined in 17 HIV-seropositive and 10 HIV seronegative haemophiliacs and in 11 healthy control subjects. All three parameters tested were found to be significantly higher in the seropositive patients than in the seronegative controls. In the seropositive group, a significant positive correlation was found between the neopterin levels and the percentage of CD3+ DR+ cells. By contrast, no significant negative or positive correlation was observed between the neopterin levels and the percentage of the CD8+ Leu7+ subset. These data suggest that in the HIV-infected patients the activated T cells responsible for the stimulation of macrophages to produce neopterin are those that do not carry CD8. PMID- 1710306 TI - Palliative therapy of an ectopic Cushing's syndrome due to a metastatic carcinoid tumor. AB - The case of a 39-year-old patient with ectopic Cushing's syndrome due to a metastatic carcinoid tumor is presented. Palliative therapy consisting of 800 mg ketoconazole and 0.3 mg SMS 201-995/die resulted in clinical remission and correction of hypokalemia and hypercortisoluria. Combined therapy was clearly superior to monotherapy with ketoconazole or SMS 201-995, respectively. Side effects were not observed, the tumor masses remained unchanged throughout the observation period of now 19 months. PMID- 1710307 TI - [Uncertain decision making in benign prostatic hyperplasia]. PMID- 1710308 TI - [Quality of care and use of the resources in prostatic surgery--a follow-up study]. PMID- 1710309 TI - Opiate tolerance-induced modulation of free intracellular calcium in synaptosomes. AB - Synaptosomes were prepared from morphine-tolerant and non-tolerant mice. Significantly higher levels of basal free intracellular calcium were observed in the synaptosomes from the opiate-tolerant mice compared to synaptosomes from non tolerant mice (468 nM versus 328 nM, respectively). In addition, morphine (1 microM) failed to attenuate KCl-induced rises in intracellular calcium in the synaptosomes from the tolerant mice. Conversely, morphine produced a concentration-related, naloxone-reversible attenuation of 50 mM KCl-induced rises in intracellular calcium in the synaptosomes from the non-tolerant mice. Omega conotoxin, which blocks both "L" and "N" type calcium channels, attenuated KCl stimulated rises in intracellular calcium only in synaptosomes from non-tolerant mice. BAY-K 8644, an "L-type" calcium channel agonist, produced nifedipine reversible increases in intracellular calcium in the synaptosomes from the tolerant animals only. These data suggest that chronic exposure to morphine results in an alteration in either the number of the activation state of calcium channels in the membrane. Changes in intracellular free calcium may be the final common pathway through which neurons adapt to the chronic exposure to morphine. PMID- 1710310 TI - Lecture handouts of projected slides in a medical course. AB - Part of the teaching of clinical haematology to our third year medical students involves lectures given to the entire class. For the past several years, we have provided, at the beginning of each lecture, handouts on which are reproduced all or most of the 35-mm slides that will be projected during that lecture. The frames are positioned vertically on the left side of each page, thus allowing the students to add notes on the right side. There are advantages and disadvantages to this pedagogical method, but we believe that the former outweigh the latter. PMID- 1710311 TI - The stringent response in Streptomyces coelicolor A3(2). AB - The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of actIII occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis alone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2). PMID- 1710312 TI - An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein. AB - Escherichia coli strains harbouring the Yersinia pseudotuberculosis inv gene are able to enter cultured mammalial cells. We show here that this property is not shared by all enteric bacteria, since Shigella flexneri 2a cured of its virulence associated plasmid and harbouring the inv gene is unable to enter mammalian cells efficiently. Mapping studies showed that the region of the chromosome responsible for this phenotype includes rfaB, a locus involved in the production of O antigen. S. flexneri 2a strains that express O antigen were unable to enter mammalian cells, even though invasin was efficiently expressed and localized, showing that this structure interferes with invasin activity. The O antigen either masks invasin or sterically hinders the ability of the mammalian cell receptor to bind this protein. PMID- 1710313 TI - Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli. AB - Expression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE-pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper-repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build-up of the full-length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation. PMID- 1710314 TI - Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes. AB - Each of the two mutants isolated from a fliC (= hag, flagellin-deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti-d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild-type antigen. In one mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild-type antigen d but able to confer motility on flagellin-negative Salmonella sp. (and used for insertion of epitope specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild-type or mutated antigen d showed that the fusion proteins specified by lambda gt11 with the N terminal part of gene lacZ joined to a restriction fragment coding for residues 145-391 of flagellin gave the same pattern of parent-specific and mutant-specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105 bp deletion as the first-isolated mutant; the fifth had a 72 bp deletion made possible by a 7-base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar-antigen alleles. PMID- 1710315 TI - The Bessis reticular cell, the hemoglobin 'switch' and the role of fetal hemoglobin: a hypothesis. AB - With the help of diverse factors, definitive erythropoiesis is induced by the junction of a pluripotential stem-cell with a specialized macrophage. In the normal adult marrow the latter becomes the reticular cell forming the core of an 'erythroblastic island'. It stays in close contact with all the erythroid cells descending from the stem-cell until their full maturation. While this contact lasts, only alpha and beta (delta) globin chains are produced. The production of gamma chains is inhibited. Stress forces the reticular cell to let the erythroid cells go. The stronger the stress, the earlier the separation. When free from the reticular cell, the erythroid cells stop producing beta chains and switch to form gamma ones. Increase in the volume of blood circulation is a potent stress. Since it is relatively enormous in the embryo and the fetus, most hemoglobin produced at these periods will be fatal. The important quality of fetal hemoglobin is its high oxidizability. It permits the senescent fetal red cell to be hemolysed intravascularly before any membrane alterations appear. The heme is dealt with by the placenta. Since no erythrophagocytosis has occurred, no bilirubin is formed. This process prevents bilirubin, which is toxic to the fetal liver, to reach it. PMID- 1710316 TI - Expression of heat shock protein epitopes in tubular aggregates. AB - Tubular aggregates may be found in a variety of conditions and have been associated with a wide range of chemical and ischemic insults. We report clinical and histological features in a case of myopathy with tubular aggregates. The structure of these tubular aggregates was examined using antibodies to cytoskeletal proteins and heat shock proteins. Epitopes of the 72 kD heat shock protein were expressed in the areas of abnormality in this case and in a case of hypokalemic periodic paralysis with tubular aggregates. Heat shock proteins have a role in the modulation of the tertiary structure of proteins and may be involved in the pathogenesis of tubular aggregates and other microtubular abnormalities in muscle. PMID- 1710317 TI - Immunosuppression. Binding by design. PMID- 1710318 TI - Mitochondrial base prediction. PMID- 1710319 TI - FK506 and protein kinase C. PMID- 1710320 TI - Cerebral blood flow changes during localized hyperthermia. AB - Hyperthermia is becoming a potent therapeutic method for malignant brain tumors, either alone or in combination with radiation therapy. The heat response of organized tissues includes other factors besides the inherent cellular thermosensitivity, that is, tissue pH, PaO2, and nutrient supply, all of which are largely influenced by the tissue blood flow. In this study, the regional cerebral blood flow (rCBF) changes in 15 Japanese normal monkey brains during interstitial microwave hyperthermia were investigated by the hydrogen clearance method. Under general anesthesia and controlled respiration, a parieto-occipital craniectomy, 4 x 4 cm, was performed. A microwave antenna was inserted into the brain to a depth of 2.0 cm, and the brain tissue was heated with 2450 MHz microwave irradiation. The intracerebral temperatures and rCBF were measured in the white matter 1 cm from the brain surface. During hyperthermia, the rCBF linearly increased at a rate of 10% per 1 degrees C temperature rise. Heating at 42 degrees C for 180 minutes resulted in a constant increase in rCBF. The perfusion rate returned to the control levels after the termination of heating. Above 45 degrees C, the rCBF transiently increased and then started to decline during heating. No consistent results were obtained with heating at 43 degrees C. These results show that normal monkey brain tissues respond to hyperthermia by an rCBF increase as long as the threshold values of tissue temperature (43 degrees C) and exposure time (40-60 minutes) are not exceeded. Excessive heating may lead to irreversible damages to normal tissue and vasculature. PMID- 1710321 TI - Partial callosotomy for Lennox-Gastaut syndrome--first cases in Japan. AB - Corpus callosotomy is a well established procedure for the treatment of intractable epilepsy. However, this is the first clinical report of surgical division of the corpus callosum in Japan. Four patients with refractory seizures suffering from Lennox-Gastaut syndrome underwent anterior partial corpus callosotomy. Their seizures consisted of absences, tonic, atonic, tonic-clonic attacks and were characterized by frequent falls. Electroencephalograms showed paroxysms of bilateral synchrony of slow spike and wave complexes. Postoperative follow-up during 12-27 months showed that partial callosotomy reduced the frequency and severity of seizures in all the patients, although they still require antiepileptic medication. This procedure was effective even in patients with mixed cerebral dominance and also in a patient with low intelligence quotient. Postoperatively, disconnection syndrome developed in three patients, which was transient in one and lasting in two. PMID- 1710322 TI - Surgical approach to arteriovenous malformation of the medial temporal lobe- report of three cases. AB - We report three cases of arteriovenous malformation (AVM) of the medial temporal lobe and the surgical approaches used. The AVM was fed by the anterior choroidal artery (AChA) in two cases (Cases 1 and 2) and by the posterior cerebral artery in one (Case 3). The trans-Sylvian approach was first used for cerebrospinal fluid aspiration to retract the brain in all cases, and for confirming the feeding arteries to prevent premature bleeding from the AVM in Cases 1 and 2. In Case 1, a corticotomy was then made in the fusiform gyrus via the subtemporal approach to avoid the development of speech disturbance and visual field defects, while in Cases 2 and 3, a cortical incision was made in the middle temporal gyrus because visual field defects were preoperatively present. Cases 1 and 2 achieved good recoveries, but Case 3 suffered postoperative speech disturbance and died of rebleeding from a recurrent AVM fed by the AChA 22 months after the operation. This AVM was not demonstrated on the postoperative angiograms. We emphasize the usefulness of the combination of trans-Sylvian and subtemporal approaches for this lesion, because the feeding arteries are easily identified and retraction of the temporal lobe is alleviated. A corticotomy in the fusiform gyrus is also recommended to avoid the development of not only visual field defects but also aphasia. PMID- 1710323 TI - Arteriovenous malformation associated with moyamoya disease--case report. AB - The authors report a case of moyamoya disease accompanied by arteriovenous malformation (AVM). Angiography demonstrated typical moyamoya vessels on the right and early changes of moyamoya disease on the left. A small AVM in the left frontal lobe supplied by a distal branch of the middle cerebral artery (MCA) was also revealed. The AVM was surgically resected simultaneously with contralateral encephaloaponeurotic synangiosis for the moyamoya vessels. One month after surgery, left MCA occlusion at the origin occurred probably due to hemodynamic changes after the resection of the AVM. PMID- 1710324 TI - Persistent primitive hypoglossal artery associated with arteriovenous malformation--case report. AB - A case of persistent primitive hypoglossal artery (PPHA) associated with arteriovenous malformation (AVM) is reported. A 46-year-old male suddenly developed severe headache followed by transient unconsciousness and was admitted to our hospital 2 hours later. A computed tomographic scan showed subarachnoid hemorrhage. Angiograms revealed an AVM in the left cerebellar hemisphere and an ipsilateral PPHA. The AVM was completely removed and he was discharged 1 month after surgery without neurological deficit. Only three cases of PPHA associated with intracranial AVM have been reported in the literature. One patient died of rebleeding from the AVM before surgery, and another was conservatively treated because the AVM was too large for resection. The remaining one was surgically treated only by ligation of the feeding arteries. Ours is the first case treated by total removal of the AVM. Since these four cases, including ours, account for 3.0% of 134 cases of PPHA reported, PPHA associated with AVM is considered rare. PMID- 1710325 TI - Subarachnoid hemorrhage and spinal root injury caused by acupuncture needle--case report. AB - The authors report a case of subarachnoid hemorrhage and spinal root injury caused by an acupuncture needle buried in the posterior neck about 30 years before onset. A 33-year-old female presented with sudden onset of severe occipital headaches. Plain x-ray films of the cervical spine revealed a fine gold needle, about 1.5 cm in length, between the C1 and C2 vertebrae. The needle was piercing the spinal nerve root through the dural vein, and was removed. Postoperatively, the pain exacerbated by neck movement disappeared. PMID- 1710326 TI - Multiple intracranial xanthogranulomas--case report. AB - A 58-year-old female was admitted to our hospital with nausea, vomiting, and gait disturbance of 1 year duration. Postcontrast computed tomographic scans demonstrated enhanced lesions in the left cerebellopontine angle (CPA), the retrosellar region, the right parasellar region, and the left parietooccipital convexity. The left parieto-occipital tumor was totally removed in the first operation and the left CPA tumor was partially removed in the second. The histological diagnosis of both tumors was xanthogranuloma. She also had cutaneous lesions (subcutaneous nodules without tenderness) and an ureteral stenosis possibly due to the retroperitoneal involvement. A skin biopsy demonstrated infiltration of xanthoma cells and foamy cells in the dermis. A gallium scintigram demonstrated an abnormal uptake in the thoracic cavity, liver, and bones. From these findings, systemic Weber-Christian disease was diagnosed. Another unique aspect was that the serum IgE levels were increased during postoperative hospitalization. This suggests that abnormal immunological conditions are related to this disease and that intracranial xanthogranulomas are a manifestation of systemic Weber-Christian disease. PMID- 1710327 TI - Holocord astrocytoma--case report. AB - A rare case of intramedullary holocord astrocytoma extending from the medulla oblongata to the conus medullaris is reported. A 27-year-old male who had been suffering from nuchalgia for a few years was admitted to our department because of lumbago and gait disturbance. Neurological examination revealed sensory disturbances in various locations, weakness of the right lower extremity, mild swallowing disturbance, and bowel and bladder difficulties. Magnetic resonance (MR) images and myelograms showed a long cystic lesion extending from the medulla oblongata to the thoracolumbar spinal cord. Gadolinium-diethylenetriaminepenta acetic acid (Gd-DTPA)-enhanced images detected a solid tumor located at the Th6-7 level. Two syrinx-subarachnoid shunts were placed at the upper cervical and Th12 levels. The solid neoplasm at the Th7 level was partially resected and histologically diagnosed as astrocytoma. Differentiation between cystic lesions and solid masses in the spinal cord is difficult. The usefulness of Gd-DTPA enhanced MR imaging in the diagnosis of holocord tumor and the pathogenesis of secondary syringomyelia are discussed. PMID- 1710328 TI - Massive hemorrhage in acoustic neurinoma after minor head trauma--case report. AB - Massive hemorrhage within an intracranial neurinoma occurs rarely. The authors describe a 62-year-old female with intratumoral bleeding which led to the discovery of an acoustic neurinoma. She developed a gait disturbance after a minor head injury. A computed tomographic scan obtained 2 months later demonstrated multiple high-density areas in the anterior portion of the left cerebellopontine angle. Preoperative diagnosis was acoustic neurinoma. The tumor had multiple cysts which contained a mixture of xanthochromic fluid and old, brownish hematomas, and was successfully removed. The intratumoral hemorrhage is thought to have resulted from traumatic rupture of the dilated vessels, although the trauma was slight. This is the first reported case of an acoustic neurinoma discovered through treatment for intratumoral hemorrhage occurring after a minor head injury. PMID- 1710329 TI - Epidermoid tumor with unusual CT and MR findings--case report. AB - The authors report a case of epidermoid tumor attached to the dura of the frontal convexity and extending into the cerebral parenchyma, with unusual computed tomographic (CT) and magnetic resonance (MR) findings. The tumor appeared as a mixed density intracerebral mass containing dense calcifications on the precontrast CT scan, and the capsule was intensely enhanced on the postcontrast scan. It appeared as a mixed intensity lesion on both the T1- and T2-weighted MR images. PMID- 1710330 TI - Substance P-containing neurons innervating LHRH-containing neurons in the septo preoptic area of rats. AB - Neuroanatomical attempts have been made to determine the synapses between luteinizing hormone-releasing hormone (LHRH)-containing neurons and substance P (SP)-containing neurons in the hypothalamus of female rats. Wheat germ agglutinin was injected into the septo-preoptic area (SPA) and found to be incorporated into certain SP-containing neurons within the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus. Hence, we used a preembedding double immuno staining technique in demonstrating LHRH and SP neurons in the SPA. In light microscopic preparations LHRH was labeled with 3,3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen while SP was labeled with silver-gold particles; brown LHRH cells appeared to be surrounded by black silver-gold dots. In electron-microscopic preparations, the labelings for LHRH and SP were made reversely; SP was localized with DAB chromogen, and SP-containing axonal terminals appeared to make synaptic contacts on silver-gold-labeled LHRH cell bodies and dendritic processes. The terminals contained numerous small clear vesicles and some large dense-cored vesicles, and the synaptic membrane specialization appeared to be symmetric and asymmetric. These findings indicate that certain SP neurons existing in the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus may project fibers to make synaptic contact with LHRH neurons in the SPA in the rat. PMID- 1710331 TI - Hypothalamic thyrotropin-releasing hormone regulates pituitary thyrotropin beta- and alpha-subunit mRNA levels in the rat. AB - It has been controversial whether thyrotropin-releasing hormone (TRH) may be involved in the regulation of biosynthesis of anterior pituitary thyroid stimulating hormone (TSH), whereas TRH is well known to control the secretion of TSH from anterior pituitaries. We therefore studied the effect of the destruction of paraventricular nuclei (PVN), containing TRH neuronal cell bodies, on anterior pituitary TSH beta- and alpha-subunit mRNA levels the thyroidectomized rat. Median eminence TRH contents and serum TSH levels significantly decreased even 1 day after PVN destruction, whereas anterior pituitary TSH beta- and alpha-subunit mRNA levels did not change. In contrast, these mRNA levels significantly decreased by 3 days after PVN destruction. Growth hormone mRNA levels were not affected during 7 days after PVN destruction. Although the involvement of other factors related to the PVN is not totally excluded, these results raise the strong possibility that hypothalamic TRH regulates anterior pituitary TSH mRNA levels. PMID- 1710332 TI - Interactions between vasopressin- and gonadotropin-releasing-hormone-containing neuroendocrine neurons in the monkey supraoptic nucleus. AB - Vasopressin (VP) is a hypophysiotropic hormone which is also implicated in the control of gonadotropin-releasing hormone (GnRH) secretion. We examined whether VP- and GnRH-immunoreactive (-IR) elements interact directly in the supraoptic nucleus (SON) of cynomolgus monkeys. Neuroendocrine (NEU) neurons in 4 juveniles were retrogradely labeled from the median eminence with wheat germ agglutinin apohorseradish peroxidase conjugated to gold before aldehyde perfusion. Frontal vibratome sections were immunostained for GnRH with peroxidase-antiperoxidase (PAP) and for VP with 5- or 15-nm gold. Many of the GnRH-IR and more than half the VP-IR cell bodies in the SON were NEU. VP-IR elements formed axodendritic and axosomatic symmetrical synapses with one another. In addition, VP-IR boutons also synapsed with NEU GnRH-IR neurons. Although GnRH axon terminals and dendrites contacted VP-IR dendrites and NEU cell bodies, we were unable to find convincing examples of GnRH/VP synapses through serial sections, perhaps due to the use of PAP-diaminobenzidine as the GnRH (afferent) immunolabel. In summary, our study demonstrates anatomical synapses between VP-IR and other VP and GnRH-IR neurons in the SON, in which postsynaptic VP or GnRH cell bodies were NEU. On the other hand, reciprocal GnRH/VP contacts but no true synapses were seen. However, the results suggest coordinated roles for VP and GnRH in NEU control of gonadotropin secretion. Whether VP itself and/or coexistent neuroeffectors act directly on NEU GnRH secretion remains to be determined. As such, VP neurons could help coordinate suppression of gonadotropins and augmentation of glucocorticoids during the stress response in primates. PMID- 1710333 TI - [The reparative potentials of human corneal endothelium from the aspect of age]. AB - Morphometric and cytospectrophotometric studies of the endothelium of human cornea in age aspect, from 20 to 80 years, has revealed the presence of nuclei with double content of DNA (4 c) irrespective of age, as well as binuclear cells. Their number increases with age. At the age of 20-40 years, 4 tetraploid nuclei and 3 binuclear cells were found, and after 80 years of age - 45 tetraploid nuclei and 11 binuclear cells per a cornea. This speaks about the ability of the endothelium cell of the human cornea to incomplete polyploidizing cellular mitosis-variant of a normal proliferation of the cells and is characteristic for long-living and slightly proliferating tissues. The increase of the number of polyploid cells in the corneal endothelium with age speaks about activation of adaptation mechanisms in the course of age involution. PMID- 1710334 TI - Patch-clamp studies of K+ and Cl- channel currents in canine pancreatic acinar cells. AB - K+ and Cl- channel currents in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp single-channel and whole-cell current recording techniques. In excised inside-out patches, we found a Ca(2+)-activated (control range 0.01-0.4 microM) and voltage-activated K(+)-selective channel with a unit conductance of approximately 40 pS in symmetrical K(+)-rich solutions. In intact cells, addition of acetylcholine (1 microM) or bombesin tetradecapeptide (0.1 nM) to the bath evoked an increase in frequency of K+ channel opening. In whole-cell recordings on cells dialyzed with K(+)-rich and Ca(2+)-free solution containing 0.5 mM EGTA, the resting potential was about -40 mV. Depolarizing voltage pulses activated outward K+ currents, which were blocked by 10 mM tetraethylammonium, whereas hyperpolarizing pulses evoked smaller inward currents. Acetylcholine or bombesin activated the K+ current and enhanced the inward current, which was reduced by a low Cl- (10 mM) intracellular solution at 90 mV holding potential. These results suggest that both Ca(2+)- and voltage activated K+ channels and Ca(2+)-activated Cl- channels exist in the plasma membrane of canine pancreatic acinar cells. PMID- 1710335 TI - Multiple types of Ca2+ channels in visceral smooth muscle cells. AB - Single-channel currents were recorded from two classes of Ca2+ channels in visceral smooth muscle cells isolated from the stomach of the toad, Bufo marinus: a class of small-conductance channels (approximately 11 pS) and a class of large conductance channels (approximately 26 pS). Small-conductance channels were present in a majority of patches and gave rise to a slowly inactivating current (t1/2 approximately 250 ms at 0 mV). Openings of large-conductance channels could be unequivocally resolved only in the presence of the dihydropyridine Ca2+ agonist Bay K 8644. Two subtypes of the large-conductance channels were found- those with a very slow rate of decay (greater than 500 ms) and those with a faster one (less than 100 ms). Large-conductance channels resemble L-type Ca2+ channels of other preparations. Small-conductance channels do not fit unambiguously into the other existing categories (i.e., N or T). Correspondence between single-channel and macroscopic Ca2+ currents is discussed. PMID- 1710336 TI - Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current. AB - The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca2+ concentration and 37 degrees C. ET (1-100 nM) induced cell shortening and membrane depolarization from a resting potential of -50 mV to about -15 mV. Ca currents (ICa, L-type) were transiently reduced by ET. At -50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl- ion concentration, but removal of extracellular Na+ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (ICl) at potentials negative to -30 mV; at more positive potentials K currents (IK,Ca) are superimposed. The effects of ET on ICa, ICl, IK,Ca and contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents. PMID- 1710337 TI - Characterization of chloride and cation channels in cultured human keratinocytes. AB - Patch-clamp experiments on human cultured keratinocytes revealed the presence of three types of ion channel. The first type was a Cl(-)-selective channel, the current/voltage relationship of which showed outward rectification, the mean conductance at positive and negative membrane potentials being 66 pS and 16 pS respectively. The second type of channel showed almost equal permeability to alkali ions but was impermeable to Cl- and to the large organic cation N-methyl-D glucamine. Its current/voltage relationship was linear with a mean unitary conductance of 18 pS in symmetrical 140 mmol/l NaCl. Finally, the third type was a large-conductance cation channel, which had in physiological ionic conditions a peculiar rectifying current/voltage relationship, the shape of which was strongly dependent on the concentration of divalent cations on both sides of the membrane. Lowering of Ca2+ and/or Mg2+ on either side of the patch led to a marked increase of the single-channel current. With identical solutions without Ca2+ Mg2+ on both sides of the patch the current/voltage relationship became ohmic and reached a conductance of 150-200 pS. In addition, channel activity was reversibly affected by changes of the external Ca2+ concentration. In particular, open-channel probability strongly increased at negative membrane potentials when the external Ca2+ was lowered from millimolar to micromolar values. Whole-cell experiments confirm the role of the extracellular Ca2+ as a modulator of the cation conductance. PMID- 1710338 TI - Potassium-selective channels in the basolateral membrane of single proximal tubule cells of frog kidney. AB - The membrane potential of proximal tubule cells is dominated by the potassium conductance of the basolateral membrane. In the present paper the nature of this conductance is investigated by the patch-clamp technique. Only one type of K channel was found in the basolateral membranes of freshly isolated proximal cells. In cell-attached patches, the current/voltage relationship is markedly non linear with much larger inward (30 pS) than outward (approximately 6 pS) conductances, even in the presence of roughly symmetrical K concentrations. Thus the channels show inward rectification. The determination of the conductance for outward current flow is complicated since the current/voltage curves show an area of negative conductance. Nevertheless, taking the conductance for outward current flow and the density of the channels it is possible to account for all of the previously reported potassium conductance of amphibian proximal tubule cells. The open probability of the channels was found not to depend upon the membrane potential. However, the non-linearity of the current/voltage relationships will confer upon the channel the same voltage dependence as that seen in intact proximal tubules, i.e. the conductance decreases with depolarisation. Incubation of cells in Ringer with no substrates or in the presence of alanine and/or glucose showed no change in the activity of the channels. These findings suggest that, although these channels may represent the basolateral conductance of frog proximal tubule cells, they are not involved in the well-established coupling between transport rate and potassium conductance. PMID- 1710339 TI - Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches. AB - The activity of single L-type Ca2+ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca2+ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent run-down as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca2+ channel regulation with free access to both sides of the membrane. PMID- 1710340 TI - 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. AB - We describe the synthesis of 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides and demonstrate their utility as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. These 2'-O modified compounds were designed to possess the binding affinity of an RNA molecule towards a complementary RNA target with an enhanced stability against nucleases. The 2'-O-methyl and 2'-O-ethyl antisense compounds function as potent inhibitors of the reaction at 1-10 nM, approximately 5-fold more effective than a natural antisense RNA molecule and requiring an approximate 5-fold excess over the target RNA for 80% inhibition of the processing reaction. PMID- 1710341 TI - An NF kappa B-like factor is essential but not sufficient for cytokine induction of endothelial leukocyte adhesion molecule 1 (ELAM-1) gene transcription. AB - The endothelial leukocyte adhesion molecule 1 (ELAM-1) is transiently expressed specifically on the surface of cytokine-induced endothelial cells. We demonstrate that the transient expression of the protein is paralleled by an increase and decrease in transcription of the ELAM-1 gene. To identify the cis-acting transcription control regions within the ELAM-1 gene that are responsible for this cytokine-induced expression, we isolated and analyzed an ELAM-1 genomic clone containing sequences upstream of the transcription start site. We constructed a series of ELAM-1 deletion mutants linked to a reporter gene and analyzed their expression in both endothelial and non-endothelial cells. Results show that a fragment of 233 bp upstream of the transcription start site is sufficient to confer cytokine inducibility upon the reporter gene in both endothelial and non-endothelial cells. Further analysis defined two elements within this region that are involved in the cytokine inducibility of the ELAM-1 gene. One element lies within the -233 to -117 region, the other element represents an NF kappa B consensus binding site between nucleotides -94 to -85. Gel shift analysis reveals increased binding of an NF kappa B-like factor to this consensus sequence in extracts prepared from IL-1-induced endothelial cells. The results suggest that cytokine induction of ELAM-1 gene transcription is imparted by a combination of positive factors, one being an NF kappa B-like transcription factor, interacting with cis-acting elements within the enhancer/promoter of the gene. PMID- 1710342 TI - A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates. AB - We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component. PMID- 1710343 TI - A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison. AB - This article describes the latest version of an RNA folding algorithm that predicts both optimal and suboptimal solutions based on free energy minimization. A number of RNA's with known structures deduced from comparative sequence analysis are folded to test program performance. The group of solutions obtained for each molecule is analysed to determine how many of the known helixes occur in the optimal solution and in the best suboptimal solution. In most cases, a structure about 80% correct is found with a free energy within 2% of the predicted lowest free energy structure. PMID- 1710344 TI - Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate. AB - In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (3) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21G* indicate that, under the Cs symmetry restricted conditions, the P-O(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (O(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-O(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app lp assistance from O(3). The dianionic intermediate, however, loses the TS1 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus [1990) J. Am. Chem. Soc., 112, 5872-5873) for attack by OH- on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691-701) of rate limiting transition state possessing extended P-O(5') bond breaking character (the TS2 transition state) in the base catalyzed hydrolysis of RNA. Finally, although the lack of 2',3' -migration of phosphate moieties in basic condition appears to be in accord with the short lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text. PMID- 1710346 TI - Intermediates in the degradation of mRNA from the lactose operon of Escherichia coli. AB - We have analyzed the processing of mRNA from the lac operon in an Escherichia coli strain carrying the lac on a multicopy plasmid. Messenger RNA was analyzed by hybridization and nuclease protection of pulse-labeled RNA and precursor product relationships were determined by quantitating radioactivity in primary and processed transcripts at various times after induction of the lac promoter or inhibition of transcription with rifampicin. Our results support the existence of two types of processed transcripts with endpoints in the lacZ-lacY intercistronic region. One of these carries lacZ sequences and has a 3' endpoint about 30 bases downstream of this gene. The other carries lacY sequences and has a 5' end in the translation termination region of the lacZ gene. Finally, we have found evidence that transcription is continued at least 268 bases beyond the last gene (lacA) and that this 3' non-translated region is shortened by post-transcriptional processing. PMID- 1710347 TI - Mono Q chromatography permits recycling of DNA template and purification of RNA transcripts after T7 RNA polymerase reaction. PMID- 1710345 TI - The promoter of the endo A cytokeratin gene is activated by a 3' downstream enhancer. AB - Mouse cytokeratin EndoA is an intermediate filament subunit of the type II cytokeratin class which initiates expression in trophectoderm cells of blastocyst during embryogenesis. To identify the regulatory elements of the endo A gene, we constructed a series of CAT expression vectors and transfected them into PYS-2 cells. We found an enhancer element locating 1 kb downstream from the endo A gene which acts on both the endo A and SV40 promoters. This enhancer consists of six direct repeated sequences with homology to the PEA3 motif in polyoma virus alpha enhancer core. In undifferentiated F9 embryonal carcinoma cells, expression of the construct containing the enhancer was not detected. These results indicate that one of the regulatory mechanisms of endo A gene expression is the 3' downstream enhancer. PMID- 1710348 TI - A new polymorphic probe on chromosome 3p:lambda LIB25-55(D3S602). PMID- 1710349 TI - A new polymorphic probe on chromosome 3p:lambda LIB28-62(D3S736). PMID- 1710350 TI - A new polymorphic probe on chromosome 3p:lambda LIB29-48(D3S624). PMID- 1710351 TI - Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate. AB - Streptomycin is an aminocyclitol glycoside antibiotic, which interferes with prokaryotic protein synthesis by interacting with the ribosomal RNA. We report here that streptomycin is also able to inhibit self splicing of the group I intron of the thymidylate synthase gene of phage T4. The inhibition is kinetically competitive with the substrate guanosine. Streptomycin and guanosine have in common a guanidino group, which has been shown to undergo hydrogen bonds with the ribozyme (Bass & Cech, Biochemistry, 25, 1986, 4473). The inhibitory effect of streptomycin extends to other group I introns, but does not affect group II introns. Mutating the bulged nucleotide in the conserved P7 secondary structure element of the td intron alters the affinity of the ribozyme for both guanosine and streptomycin. Myomycin, an antibiotic with similar effects on protein synthesis as streptomycin, is also able to inhibit splicing. In contrast, bluensomycin, which is structurally related to streptomycin, but contains only one guanidino group does not inhibit splicing. We discuss these findings in support of an evolutionary model that stresses the antiquity of antibiotics (J. Davies, Molecular Microbiology 4, 1990, 1227). PMID- 1710352 TI - A cell-type specific and enhancer-dependent silencer in the regulation of the expression of the human urokinase plasminogen activator gene. AB - A transcriptional silencer has been identified in the 5' regulatory region of the human urokinase plasminogen activator (uPA) gene. This region is able to block transcription from the human u-PA as well as the rabbit beta-globin promoters in a cell type specific and orientation independent way. The silencer is enhancer dependent and is active in two cell lines (HeLa and CV-1) which produce little if any uPA, but not in the high uPA producer PC3. Silencing activity and enhancer dependence can be separated: the silencing activity has been localized to the DNA fragment -660 to -536, while the enhancer dependence is located in the -536 to 308 fragment. The DNA sequence of the silencer region contains an element that closely resembles the TGF-beta responsive negative element TIE. PMID- 1710353 TI - An RNA molecule copurifies with RNase P activity from Xenopus laevis oocytes. AB - Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related. PMID- 1710354 TI - Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains. AB - Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements. PMID- 1710355 TI - Differential expression of Epstein Barr viral transcripts for two proteins (TP1 and LMP) in lymphocyte and epithelial cells. AB - Studies presented here show that some functions of the human herpesvirus, EBV, may be transcriptionally differentially expressed in two cell types which carry the same (C15) isolate of this virus. Of the 'latent' viral functions investigated, only one (TP2) of the episomally-specific genes that encode terminal proteins (TP1 and TP2) is found to be expressed in the C15 epithelial cell tumour environment, whereas both are transcribed--as different, but related, messengers--in a B-cell line generated with virus from the C15 tumour. The other gene investigated is that for latent membrane protein (LMP), which is found in the same region of the EBV genome but on the opposite strand. This gene, apparently transcriptionally silent in B-cell (Burkitt's) lymphomas, is expressed in the C15 epithelial tumour, as well as in other nasopharyngeal carcinomas investigated. Promoter usage in the carcinomas and B-cells appears, in some cases at least, to be cell-type specific. Expression may also be governed by methylation since a chromosomally silent region in the carcinoma (that encompassing TP1) is highly methylated on CpG residues, whereas the active region (encoding TP2 and LMP) is virtually free of such methylation. Our data suggest that there may be selective transcriptional regulation of EBV genes in the two types of cells investigated. Thus, it may be unnecessary to invoke different virus genotypes to account for the two distinct malignancies--Burkitt's lymphoma and nasopharyngeal carcinoma--associated with EBV. PMID- 1710356 TI - Translational frameshifting in the Escherichia coli dnaX gene in vitro. AB - Production of the gamma subunit of Escherichia coli DNA polymerase III holoenzyme is dependent on a very efficient translational frameshif in the dnaX gene. I used an E. coli in vitro translation system to analyze the mechanism of this frameshifting event. In this system, gamma was produced almost to the same extent as the inframe translation product, tau, suggesting that efficient frameshifting was reproduced in vitro. Coupling with transcription was not necessary for frameshifting. Addition of purified tau or gamma had no effect on the frameshifting process suggesting the absence of direct feedback regulation. By use of mutant genes, a strong pausing site was identified at or very close to the frameshift site. This pausing was apparently caused by a potential stem-loop structure which was previously shown to enhance frameshifting. Thus, enhancement of frameshifting by this putative stem-loop seems to be mediated by the translation pausing at the frameshift site. Despite the apparent structural similarity of the dnaX frameshift site to that of the eukaryotic retroviral genes, dnaX mRNA synthesized in vitro failed to direct the production of gamma in eukaryotic translation systems. This suggests that frameshifting in the dnaX gene depends on components specific to the E. coli translation system. PMID- 1710357 TI - Hybridization and dissociation rates of phosphodiester or modified oligodeoxynucleotides with RNA at near-physiological conditions. AB - We have used RNase H to study both the rates of oligonucleotide hybridization and dissociation at near-physiological conditions. We have studied the Effects of oligonucleotide length, mismatch, and chemical modifications on oligonucleotide association and dissociation with RNA. Dissociation results were compared with standard thermal melting curves to compare relative stabilities evaluated by the two techniques. Although generally the two techniques correlate for the compounds evaluated, we found several instances where the thermal melting curves failed to reflect the relative stability of different oligonucleotides at 37 degrees C using near-physiological conditions. This study suggests that direct measurement of hybridization and dissociation of an oligomer with RNA more accurately assesses the complicated kinetic scheme at 37 degrees C using near-physiological conditions than thermal melting curves would predict. PMID- 1710358 TI - The computer simulation of RNA folding involving pseudoknot formation. AB - The algorithm and the program for the prediction of RNA secondary structure with pseudoknot formation have been proposed. The algorithm simulates stepwise folding by generating random structures using Monte Carlo method, followed by the selection of helices to final structure on the basis of both their probabilities of occurrence in a random structure and free energy parameters. The program versions have been tested on ribosomal RNA structures and on RNAs with pseudoknots evidenced by experimental data. It is shown that the simulation of folding during RNA synthesis improves the results. The introduction of pseudoknot formation permits to predict the pseudoknotted structures and to improve the prediction of long-range interactions. The computer program is rather fast and allows to predict the structures for long RNAs without using large memory volumes in usual personal computer. PMID- 1710359 TI - Proteins involved in mitosis, RNA synthesis and premRNA splicing share a common repeating motif. PMID- 1710360 TI - MspI RFLP detected by a ZNF-40 cDNA sequence. PMID- 1710361 TI - In vitro phenotypic alteration of human melanoma cells induced by differentiating agents: heterogeneous effects on cellular growth and morphology, enzymatic activity, and antigenic expression. AB - Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G1 phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and gamma-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B8G3 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of gamma-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells. PMID- 1710362 TI - [Philosophy of the premature ventricular complex (an "electrocardiographic jewel"): what is it? how is it originated? what does it mean?]. PMID- 1710363 TI - Specificity and function of Qa-1 restricted gamma delta T cells. PMID- 1710364 TI - Are TCR alpha beta cells and TCR gamma delta cells that different? PMID- 1710365 TI - Human T lymphocytes expressing TCR gamma/delta. PMID- 1710366 TI - Analysis of antigen specificity of human TCR gamma delta + T cells. PMID- 1710367 TI - Gamma/delta T cells and bacteria. PMID- 1710368 TI - The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen. AB - Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures. The major allergen of cod fish, Allergen M "parvalbumins pI 4.75", is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and EF domains each coordinates one Ca2+. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96. A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64. Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction. Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant. Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length. The N terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope. Peptide OA 323 339, was demonstrated to be valuable in studies of T-cell recognition of protein antigens. Three analogous peptides of this region were prepared and clearly shown to be immunogenic in rabbits and to bind specific IgE from patients allergic to egg. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-lymphocytes. Eight peptides in the region 11-122 were similarly synthesized. A test battery was performed to study this region using rabbit polyclonal antibodies and human specific IgE. Some of these sites were involved in binding of particular Ig paratopes. Five immunogenic peptides from the major allergens of tree pollen extracts (segment 23-38), were synthesized. The selection of those peptides was setteled using two algorithms for providing the optimal hydrophobicity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1710369 TI - Aspects of the regulation of gastric histamine release. AB - Histamine is found in large amounts in the gastric mucosa and plays an essential role in the regulation of acid secretion. It is thought to stimulate acid secretion directly after being released by the other two major secretagogues (gastrin and acetylcholine) (the mediator hypothesis) or to potentiate the action of the other two secretagogues (the interaction hypothesis). Recent studies with isolated, vascularly perfused rat stomach have shown that gastrin in physiologic concentrations elicits a release of histamine sufficient to explain its acid stimulatory effect. Vagal nerve stimulation, on the other hand, only gives a faint histamine release, indicating that the vagal acid stimulation is mainly mediated by a direct stimulation of the parietal cell. Furthermore, the gastrin stimulated histamine release seems to be mediated by a calcium-dependent mechanism. Somatostatin inhibits gastrin-stimulated histamine release via a paracrine mechanism, and a prostaglandin E1 analogue (misoprostol) has been shown to be a potent inhibitor of base-line and gastrin-stimulated histamine release. These studies show that the modulation of histamine release may be a central regulatory mechanism of gastric acid secretion. Although these studies have been done in rats, there are indications that these results are of a general nature nd valid for other species as well. PMID- 1710370 TI - Trophic effects of gastrin. AB - Gastrin is an important trophic hormone for the acid-producing part of the stomach. There is no solid evidence that gastrin is physiologically important as a trophic agent outside the stomach. The trophic effects in the stomach are manifested as an increased weight and thickness of the oxyntic mucosa and can be induced by both exogenous and endogenous gastrin--that is, in situations of long lasting hypergastrinemia (treatment with effective antisecretagogues, partial fundectomy, or antrum exclusion). Removal of endogenous gastrin by antrectomy induces the opposite effects--that is, diminished weight and thickness of the oxyntic mucosa. Unlike all other peptide hormone-producing endocrine cells in the oxyntic mucosa, the so-called enterochromaffin-like (ECL) cells respond readily to gastrin. An acute gastrin challenge results in release of stored products from the ECL cells (such as histamine) and activation of cytoplasmic enzymes (such as histidine decarboxylase). Sustained elevation of circulating gastrin over days results in hypertrophy of the ECL cells and over weeks results in marked hyperplasia (at most a fivefold increase in the rat). The results in other species are similar but often somewhat less marked than in the rat. PMID- 1710371 TI - Enterochromaffin-like tumour cells in the diffuse but not the intestinal type of gastric carcinomas. AB - Gastrin may play a role in gastric carcinogenesis, as indicated by an increased frequency of gastric carcinomas in patients with pernicious anaemia and the fact some human gastric cancer cell lines carry the gastrin receptor. Recently, it has been shown that the acid-stimulatory effect of gastrin may be solely mediated by histamine release from the enterochromaffin-like (ECL) cell, on which gastrin has a specific trophic effect. We therefore found it of interest to examine human gastric carcinomas for the presence of ECL tumour cells by using silver staining and chromogranin immunohistochemistry. We found evidence of ECL cell-derived tumour cells in 40% of the diffuse gastric carcinomas but no such tumour cells in the intestinal type of gastric carcinoma. This may suggest that diffuse gastric carcinomas, like malignant gastric tumours of the mastomys, are in fact malignant ECLomas. PMID- 1710373 TI - Meeting on Research in Multiple Sclerosis. Zurich, November 24, 1990. PMID- 1710372 TI - MS research in Switzerland. PMID- 1710374 TI - Repair of central nervous system lesions by glial cells immortalised with an oncogene-carrying retrovirus. PMID- 1710375 TI - Optic nerve explant cultures of newborn rats: evidence for a neuron or common neuron-glia progenitor. PMID- 1710376 TI - Correlation between in vitro cytokine production and clinical evolution of multiple sclerosis patients. AB - In this preliminary study we demonstrated that cytokine production by short whole blood PHA stimulated culture is correlated with clinical evolution of MS patients. The TNF alpha production could forewarn the physician of a new relapse. PMID- 1710377 TI - Immunological confirmation of the presence of NA1/NA2 (cytolysis inhibitor) in cerebrospinal fluid. PMID- 1710378 TI - Interactions of the axonally secreted protein axonin-1 with neurons and glia. PMID- 1710379 TI - Developmental expression, purification and partial sequencing of myelin/oligodendrocyte glycoprotein. PMID- 1710380 TI - In vitro remyelination after demyelination induced by anti-myelin/oligodendrocyte glycoprotein antibody. PMID- 1710381 TI - Antibody-dependent cellular cytotoxicity (ADCC) in antimyelin antibody-induced oligodendrocyte damage in vitro. PMID- 1710382 TI - A molecular biological approach to the regulation of oligodendrocyte differentiation. PMID- 1710383 TI - Analysis of circulating immune complexes in plasma of MS patients. PMID- 1710384 TI - Simulation of action potential propagation in terminal arborizations. PMID- 1710385 TI - Noradrenergic mechanisms involved in muscle relaxation: significance for the treatment of spasticity. PMID- 1710386 TI - Regulation of expression of TGF-beta 2 in the nervous system. PMID- 1710387 TI - Therapeutic options in multiple sclerosis: present and future. PMID- 1710388 TI - Amyotrophic lateral sclerosis: a possible example of autosomal recessive inheritance. AB - A family is reported in which three out of four siblings of a consanguineous healthy couple developed adult onset Amyotrophic Lateral Sclerosis (ALS). All patients showed a similar clinical course with regard to disease progression and absence of cognitive deterioration. Laboratory findings included modification of Spinal Evoked Potential (SEP) and normal value of thiamine and thiamine monophosphate in cerebrospinal fluid (CSF). These data suggest an autosomal recessive mode of inheritance of ASL in this family. PMID- 1710389 TI - [Combined peroneal and tibial nerve paralysis in femoral phlegmon]. AB - The case of a patient with a combined paresis of the peroneal and posterior tibial nerve caused by a phlegmone of the upper thigh is reported. The pathologic histological findings after leg amputation are described. PMID- 1710390 TI - [Suicidal behavior of schizophrenic patients in a psychiatric hospital]. AB - Discussing the increase of inpatient-suicides in psychiatric hospitals affords an exact assessment of suicidality among schizophrenic patients, because this subgroup shows a great discrepancy between assumed actual suicidality and suicidal behavior. In a cros-sectional study we assessed the "Basissuizidalitat" in the adult wards of the Psychiatric State Hospital of Weissenau (1986). In this article we report the results concerning the schizophrenic inpatients. Like other studies we found a lower suicidality of schizophrenic patients compared with other subgroups. We try to examine and interpret this result and correlate it to the assessed data in the area of aggressive behavior. Suicidal patients globally and suicidal schizophrenic patients specially are showing in a significantly higher frequency verbal aggression, aggressive and indirectly self-destructive behavior. Consequences for diagnosis and therapy of suicidal schizophrenic patients are discussed. PMID- 1710391 TI - [Androcur (cyproterone acetate in sex offenses--follow-up of psychiatric admissions]. AB - This paper presents the follow-up study of 31 sexual offenders with diminished criminal responsibility or irresponsibility who were treated with Androcur (cyproterone acetate, CPA) in depot injection form connected with psychotherapy during and after their legal psychiatric hospitalisation. There were paedophiliac, exhibitionistic, hetero-sexual aggressive, and mixed sexual deviant acts. 30 patients were visited by the author in their environment, 1 patient had died. Half of the patients had a hetero-sexual relationship. 3 patients had children. 28 of the 31 patients were free. After the treatment with CPA 2 kinds of relapses could be distinguished: homologue r. (4 pat.) and residual r. (5 pat.). As to the duration of the treatment after discharge, 2 groups of patients can be distinguished: 1 group needed Androcur for a comparatively short time (up to c. 3 years), the other group for a longer time (up to c. 5 years). The anti androgenic treatment was accompanied by psychotherapy and social therapy as well as during the hospitalisation and after discharge. The mutual effect of Androcur and psychotherapy was successful as is to be seen by the final result of this follow-up study. PMID- 1710392 TI - Surgery in the treatment of patients with advanced ovarian cancer. PMID- 1710393 TI - The immunobiology and immunotherapy of ovarian cancer. AB - Small volume residual peritoneal disease in patients undergoing therapy for ovarian carcinoma remains an attractive, but elusive, target for immunobiological therapy. Hypothetical advantages and disadvantages of regional peritoneal therapy are being better defined through increased clinical experience and more sophisticated animal models. Developments in cytokine biology, adoptive cellular therapy, monoclonal antibody conjugation, and molecular biology continue to provide an exciting, and nearly overwhelming, array of reagents for clinical evaluation. Ongoing and anticipated investigational trials should provide intriguing data in years to follow. PMID- 1710394 TI - Local norms of maternal serum alpha-fetoprotein values in early second trimester: a prelude to a Down's syndrome screening programme incorporating maternal serum alpha-fetoprotein values. AB - Maternal alpha-fetoprotein in (MSAFP) has recently been proposed for screening of Down's syndrome. This is based on the association of a lowered MSAFP level at a given gestational age in Down's syndrome (DS) pregnancies. Most conventional 'screening' programmes in DS are based on the maternal age alone, selecting older mothers, who are at greater risk of having a DS infant born, for antenatal karyotyping. However, as 75% of DS infants are born to mothers less than 35 years old, a combination of blood tests including MSAFP and maternal age as independent risk factors would screen the younger mothers as a good cost-benefit ratio in selecting the 'at-risk' mothers for karyotyping. In this study, the gestational age was calculated from the first day of the last menstrual period (LMP) as well as from ultrasound cephalometry. The local MSAFP levels follow the same trend at a slightly higher level as those based on clinical evaluation of the Abbot AFP EIA Monoclonal kits. PMID- 1710396 TI - [Mortality among filling station attendants]. AB - At the Danish census on 9 November 1970, 4,055 men and 1,195 women aged 20-64 years indicated an employment that was coded as retail sale of oil and petrol; almost all individuals probably worked as petrol station attendants. Record linkage at Danmarks Statistik showed that 529 men had died during the following 17 years. Respiratory cancer (75 deaths) was the only cause of death that showed a significant excess (standardized mortality ratio, 1.58; 95% confidence interval, 1.25-2.00) when compared to all men gainfully employed at the time of the census. An increased mortality due to the group of cardiovascular disease could not be related to any particular subgroup. The mortality in women did not differ from expected rates. These results are in accordance with data from other countries on occupational groups exposed to high concentrations of exhaust fumes. PMID- 1710395 TI - Leucine dose response in the reduction of urea production from septic proteolysis and in the stimulation of acute-phase proteins. AB - The administration of branched-chain amino acids (BCAAs) has been proved useful in reducing both urea nitrogen production and muscle proteolysis in trauma patients with sepsis, but the optimum infusion rate to achieve these effects is still in question. In this prospective randomized study, a group of 16 posttrauma patients with sepsis received a branched chain-enriched (BCAA = 49.4%) amino acid mixture (8 patients; 120 observations) or a standard amino acid infusion (BCAAs = 15.5%; 8 patients; 227 observations). Total calories, percent lipid calories, and amino acid nitrogen administration were not different in the two groups. Each patient was studied at 8-hour intervals for the plasma levels of amino acids, six hepatic acute-phase proteins, albumin, and other metabolic parameters, including urinary urea nitrogen and 3-methylhistidine excretion. The total intake of each amino acid and its clearance were calculated and the dose of leucine during each 8-hour period was related to the leucine clearance, plasma acute-phase protein levels, and the urinary production of urea and 3-methylhistidine, as an indicator of proteolysis. The results show a significant (r2 = 0.691; p less than 0.0001) reduction of urea nitrogen production and proteolysis as a function of the increase in leucine dose. The identification of a critical mean rate of leucine infusion has been derived from the analysis of the significant linear correlation between leucine intake and leucine clearance (r2 = 0.594; p less than 0.0001). Significant positive correlations between the leucine intake dose and the platelet count (r2 = 0.402; p less than 0.0001), the plasma fibrinogen level (r2 = 0.218; p less than 0.0001), and the regression-derived sum of six acute-phase proteins plus albumin (r2 = 0.696; p less than 0.0001) were found. The increase in leucine clearance was progressively less marked above a mean daily leucine intake rate of 1.4 mumol/kg/min, which also appeared to be the dose level that maximized the acute-phase protein and coagulation effects and reduced proteolysis and urea nitrogen production, suggesting that this is a critical BCAA infusion rate at which an optimum leucine effect occurs. From these data a BCAA (leucine) dose nomogram has been derived. PMID- 1710397 TI - [Liver microsome monooxygenase activity in rats under the effects of organochlorine and organophosphorus pesticides and different nutrition background]. AB - The influence of acute and chronic intoxication with a mixture of lindane and trichlorometaphos-3 on monooxygenases of rat liver was studied in rats insufficiently provided with lysine and vitamins A, C and E. It has been found that in the presence of poly-nutrient insufficiency monooxygenase induction develops more rapidly and to a greater extent than in the presence of balanced nutrition. Chronic intoxication with the pesticide mixture leads to monooxygenase system induction under both regimens of nutrition. It has been noted that in most cases monooxygenase induction during intoxication in the presence of poly nutrient insufficiency is attended by an increase of the relative mass of the liver. A possible role of this phenomenon has been considered. PMID- 1710398 TI - [Current status of adjuvant therapy in patients with colorectal cancer: report and commentary on the Consensus Conference, 16-18 April 1990, National Cancer Institute, Bethesda, Maryland]. AB - This is a report on the Consensus Development Conference on adjuvant therapy for patients with colorectal carcinoma, which was held at the National Cancer Institute of the National Institutes of Health (NIH) in Bethesda, Maryland between April 16th and 18th 1990. Based on statistically significant results of a clinical trial adjuvant therapy with 5-fluorouracil (5-FU) and levamisole was recommended outside controlled clinical studies for patients with Dukes C colon carcinoma, but because no optimal form of adjuvant therapy yet exists there is still the need for further trials. No recommendations were given for patients with Dukes B colon carcinoma. For patients with Dukes B + C rectal cancer combined radiation therapy and 5-FU-based chemotherapy was considered "standard therapy" according to the consensus panel, but this recommendation seems to be still worth discussing. Since there is need of further evaluation of newly recognized prognostic factors and also to optimize adjuvant strategies the panel stated that further prospective randomized studies are warranted. Hence, the establishment of a multicentre study group also in Austria appears to be an essential application of such trials in order to contribute towards ameliorating the prognosis of patients with colorectal carcinoma. PMID- 1710399 TI - [Effect of the stable prostacyclin analog Iloprost on enzyme release during extracorporeal kidney perfusion]. AB - The influence of the stable prostacyclin-analogue Iloprost (Fa. Schering, Berlin) in the enzyme release from extracorporalic perfused kidneys was tested in an experimental investigation. The mechanic hypothermic perfusion was carried out on four different conditions. The estimation of specially cytosolic, lysosomal, and membrane-associated enzymes in the perfusat of the kidneys showed less activities in the Iloprost-protected kidneys. The results after the replantation of the kidneys support the interpretation of the enzyme determinations. PMID- 1710400 TI - [The relationship between free-running period and monoamines in the SCN]. AB - Free-running period of blinded rats that had kept in a cage with a running wheel varied markedly between 23.4-24.7 h. The free-running period correlated negatively with motor activity (r = -0.61, P less than 0.005). In the SCN, 5-HT concentration correlated positively with motor activity (r = 0.70, P less than 0.005), consequently correlated negatively to the free-running period (r = -0.79, P less than 0.005). Similarly, 5-HIAA content showed weakly negative correlation to the free-running period (r = -0.45, P less than 0.05) but such correlation was never observed in other monoamines and their metabolites examined (NE, DA, DOPAC and HVA). Further, no correlation was seen between free-running period and contents of amines and their metabolites, including 5-HT and 5-HIAA, in the areas tested other than SCN. These facts suggest that motor activity affects the activity of serotonergic system in SCN, resulting in the change in the free running period. PMID- 1710401 TI - [Prospective immunohistologic search for metastases using monoclonal anti cytokeratin antibodies in gynecologic malignancies]. AB - A higher prevalence of positive lymph node metastases can be found with immunohistological methods in comparison with conventional technique. We examined the lymph nodes from 20 patients with gynecological malignant tumors. We found in 304 lymph nodes with conventional technique 3.3% metastases. With immunohistological methods we showed in 9.9% of the lymph nodes metastases. PMID- 1710402 TI - Plasmacytoid transitional cell carcinoma. Report of a case with initial presentation mimicking multiple myeloma. AB - The case of a 63-year-old man with a previously undescribed morphologic variant of transitional cell carcinoma of the urinary bladder is reported. The patient initially presented with multiple lytic bony metastases of the ribs and skull. Aspiration biopsy of one of the lytic lesions of the skull showed tumor cells with a striking plasmacytoid appearance, similar to the plasma cells seen in myeloma, leading to an initial observer's diagnosis of multiple myeloma. Subsequently, a bladder tumor with the same cytomorphology was found; immunohistochemical and ultra structural studies performed on the aspirated material and on the bladder biopsy specimen clearly established the epithelial nature of this neoplasm. PMID- 1710403 TI - Malignant pleural effusion in Hodgkin's lymphoma. Report of a case with immunoperoxidase studies. AB - The cytologic and histopathologic findings in a patient with Hodgkin's lymphoma, mixed cellularity type, and a malignant pleural effusion are presented. The consistency of staining with a battery of immunoperoxidase monoclonal antibody stains, including leukocyte common antigen, Leu-M1, UCHL1 and L26, was examined on sections of formalin-fixed lymph nodes and alcohol-fixed pleural fluid cell blocks. In addition, these same tissues were stained with carcinoembryonic antigen, B72.3, cytokeratin and epithelial membrane antigen immunoperoxidase antibodies to differentiate the tumor cells from reactive mesothelial cells and adenocarcinoma cells. The results on the pleural fluid specimens were consistent with what is known of the immunohistochemical staining properties of Hodgkin's lymphoma cells in lymph nodes. PMID- 1710404 TI - Cytopathology of exocrine pancreatic carcinoma in effusions. AB - The cytomorphologic features were analyzed in 26 fluid samples (18 peritoneal and 8 pleural fluids) obtained in vivo from 20 patients with pancreatic carcinoma. All tumors were ductal adenocarcinomas, as proven histologically on autopsy samples. The basic cytomorphologic pattern in the smears was that of a malignant glandular tumor, consisting of cell groups with various degrees of cohesiveness. The most prominent feature was a linear arrangement (the so-called "Indian file") of tumor cells showing nuclear molding; these aggregates were frequently closely associated with the flat round clusters of cells. Other nonspecific features of adenocarcinoma included eccentric hyperchromatic nuclei, abundant, often well preserved vacuolated cytoplasm, a variable amount of fibrin and a reactive background. Review of the autopsy specimens also revealed the presence of an "Indian-file" pattern in most cases, especially when a conspicuous desmoplastic reaction was present. These findings suggest that pancreatic carcinoma should be included in the differential diagnosis of positive serous effusions showing these cytomorphologic features. PMID- 1710405 TI - Fine needle aspiration cytologic findings in pseudolymphoma cutis. AB - Skin nodules in three patients were sampled by fine needle aspiration. Cytologic study of the aspirated material showed a polymorphic cell population composed of small and large lymphocytes, eosinophils, plasma cells and tingible macrophages. Occasional giant cells and mast cells were also seen. These cytologic features suggested Hodgkin's lymphoma, lymphomatoid papulosis, large-cell non-Hodgkin's lymphoma and regressing atypical histiocytosis. However, because of the lack of typical Reed-Sternberg cells and due to the presence of polymorphic cells with fine chromatin, regular nuclear borders and inconspicuous nucleoli, these cases were diagnosed cytologically as a benign lymphoproliferative disorder, pseudolymphoma cutis. Biopsy of the lesions confirmed the cytologic diagnoses. PMID- 1710407 TI - Endometriosis in an inguinal crural hernia. Diagnosis by fine needle aspiration biopsy. AB - The fine needle aspiration (FNA) biopsy findings of endometriosis is an inguinal crural hernia in a 40-year-old woman are presented. The cytologic findings were similar to those previously reported in aspirates of solid endometriosis in other sites: nonatypical, small, epithelial groups in an inflammatory and proteinaceous background. The cytologic diagnosis of a benign epithelial lesion, possibly endometriosis, was confirmed by histologic study of the extirpated mass. This case shows that endometriosis must be included in the differential diagnosis of FNA samples of palpable lesions of the groin in women of reproductive age. PMID- 1710406 TI - Dysgerminoma of the ovary. Cytologic, histologic and electron microscopic study of a case. AB - A case of ovarian dysgerminoma is reported. Both histology and cytology showed cells with distinctive anisokaryosis and large, sometimes bizarre, nucleoli as the most striking feature. A lymphocytic infiltration was present. Electron microscopy showed large convoluted nucleoli, structures resembling so-called "annulated lamellae" and glycogen particles, features that are typical of a germ cell tumor. The cytologic, histologic and ultrastructural investigations revealed a dysgerminoma; that diagnosis was not certain on the frozen sections. PMID- 1710408 TI - Hereditary hemorrhagic telangiectasia: analysis of platelet aggregation and fibrinolytic system in seven patients. AB - Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder characterized by the presence of generalized mucocutaneous and visceral telangiectasias associated with recurrent bleeding. In order to analyze the mechanism of bleeding in HHT we studied the hemostatic system and platelet in vitro aggregation in 7 unrelated patients suffering from HHT. Unlike the authors of previous reports, we could not find any significant alteration of the parameters measured suggesting a possible local role of the affected endothelial cells. PMID- 1710409 TI - Treatment of idiopathic neutropenia in the elderly with recombinant human granulocyte colony-stimulating factor. AB - We administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) intravenously for 2 weeks to 2 elderly patients with severe neutropenia. The absolute neutrophil count (ANC) recovered promptly after the initiation of rhG CSF therapy and reached a peak (greater than 10 x 10(9)/l) on the 13th day. The ANC fell rapidly after rhG-CSF was discontinued, but it remained within the normal range after therapy. There were no side effects during the entire course of treatment. Therefore, rhG-CSF seems to be a most beneficial treatment in elderly patients with severe neutropenia. PMID- 1710410 TI - Immunological analysis of acquired factor VIII inhibitor in a case with immunologic disorder. AB - Coagulation factor VIII inhibitor arising in a patient with autoimmune disease was immunologically analyzed. A 63-year-old man who had been diagnosed as suffering from polyarteritis nodosa was treated with prednisolone for 10 years. Severe bleeding tendency developed and coagulation studies demonstrated a high titer of inhibitor to factor VIII:C. As a result of immunological analysis, the inhibitor was found to be IgG type autoantibody having both kappa and lambda light chains. The subclasses were IgG1 and IgG4. The inhibitor recognized the COOH-terminal light chain (72-kDa thrombin fragment) on the factor VIII molecule as an epitope. PMID- 1710411 TI - Dr. Lesley Degner: an interview. Interview by Eve Henderson. PMID- 1710412 TI - Transitional cell carcinoma with sarcomatous elements in the urinary tract. Six cases examined by immunohistochemistry. AB - We report six cases of carcinoma showing sarcomatous change in the urinary tract examined by conventional histochemistry and immunohistochemistry. All of the cases were transitional cell carcinoma with or without focal squamous cell carcinoma. Sarcomatous components resembling spindle cell sarcoma with a marked myxoid stroma or chondrosarcomatous element were also observed in all cases. The sarcomatous elements were closely associated with the areas of squamous cell carcinoma in three cases. Various histochemical staining procedures demonstrated mesenchymal features in the stroma of sarcomatous areas. By immunohistochemical examination, the epithelial components showed positive reactions for keratin, epithelial membrane antigen and, focally, carcinoembryonic antigen. The sarcomatous components revealed a positive immunoreaction for keratin but lacked other epithelial markers in all cases. Chondrosarcomatous elements in two cases were positive for both keratin and S-100 protein. These findings indicate that sarcomatous elements in carcinoma may represent mesenchymal metaplasia with partial or complete loss of epithelial features. However, further study will be necessary in order to determine whether heterogeneous elements, such as chondrosarcomatous areas, are epithelial or truly mesenchymal in origin. PMID- 1710413 TI - Diffuse malignant peritoneal mesothelioma in a young woman with a high serum level of CA125. AB - An autopsy case of diffuse malignant peritoneal mesothelioma in a young woman who showed a high serum level of CA125 is reported. Autopsy revealed extensive tumor involvement of the visceral and parietal peritoneum. The liver, spleen and other abdominal viscera were encased by tumor nodules. Histologically, the polygonal tumor cells were arranged mostly in a sheet-like fashion with a few tubular or papillary forms. No PAS reaction-positive mucin was recognized, but there was a strongly positive colloidal iron reaction. The colloidal iron positivity was effaced after combined treatment with hyaluronidase and sialidase. Immunohistochemically the tumor cells showed strongly positive reactions for CA125, epithelial membrane antigen (EMA) and cytokeratin, weak positivity for carcinoembryonic antigen (CEA) and focal positivity for vimentin. Ultrastructurally, the most characteristic feature was the expression of numerous long microvilli projecting from the tumor cell surfaces and abundant long desmosomes between the tumor cells. We consider that pretreatment using a combination of hyaluronidase and sialidase might be useful for the diagnosis of malignant mesothelioma. CA125 staining should be performed routinely in cases where this tumor is suspected. PMID- 1710414 TI - Ultrastructure of IL2-stimulated tumor-infiltrating lymphocytes showing cytolytic activity against tumor cells. AB - Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD8+/CD57- and CD8+/CD57+, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing. PMID- 1710415 TI - Effects of temperature on the biochemistry of the testis. PMID- 1710416 TI - [The effect of recombinant epidermal growth factor in corneal angiogenesis]. AB - Recent advances in genetic engineering techniques have enabled large-scale manufacture of human epidermal growth factor (hEGF), making possible the clinical use of this particular agent in treating a variety of corneal epithelial disorders. In view of future application to humans, it has to be determined whether hEGF could induce neovascularization in the cornea upon topical instillation, since the angiogenic effect of mouse EGF on the cornea in vivo has been reported. For this, a sheet of slow-release form polymer (EVA) containing hEGF was surgically implanted into the rabbit corneal stroma in search for subsequent corneal neovascularization. EVA sheets contained one of the following agents: (1) 250ng hEGF, (2) 500ng hEGF, (3) 250ng bFGF (positive control), (4) vehicle alone (negative control). On 5 and 14 days after implantation, the corneas were excised, sectioned, and stained with hematoxylin and eosin for histological evaluation. Slit lamp examination revealed that marked neovascularization developed in the corneas when EVA sheets containing bFGF were implanted. A number of polymorphonuclear leukocytes were accumulated around the implants. However, neovascularization did not occur in the corneas when EVA sheets containing either concentrations of hEGF or vehicle alone were implanted. Only a few polymorphonuclear leukocytes infiltrated. This result indicates that as much as 500ng hEGF does not induce corneal neovascularization. PMID- 1710417 TI - Prognostic value of serum proteins synthesized by breast carcinoma cells. AB - Several breast carcinoma cell lines or explants of such tumors, as well as examples of lactating or dysplastic breast tissue, synthesized three serum proteins (alpha 2-Zn-glycoprotein, alpha 1-antichymotrypsin, and alpha lipoprotein) in vitro. These proteins were detected by immunoperoxidase techniques in 126 breast carcinomas that had been evaluated clinically for more than six years. Alpha-2-Zn-glycoprotein was present in 58% of the carcinomas, whereas alpha 1-antichymotrypsin was seen in 55% and alpha-lipoprotein in 52%. These markers showed a relationship with clinical outcome. Alpha-1 antichymotrypsin and alpha-lipoprotein were unfavorable determinants, whereas alpha 2-Zn-glycoprotein was detected in lesions with a favorable evolution. Taken individually, these markers have similar but rather weak associations with five year survival rates; roughly 20% of patients in the "favorable" group died, compared with 33% in the "unfavorable" group. Alpha-2-Zn-glycoprotein in grade 1 tumors was a marker with marginally favorable significance, but alpha 1 antichymotrypsin significantly worsened the prognosis of grade 2 and 3 tumors. Furthermore, stratification of patients according to the number of positive unfavorable markers yielded striking results. Eight percent of patients with none of the unfavorable markers were dead at five years, compared with 55% of those whose lesions expressed three unfavorable markers. PMID- 1710418 TI - Argyrophilic nucleolar organizer regions and alpha-fetoprotein in adenomatous hyperplasia in human cirrhotic livers. AB - Recently, adenomatous hyperplasia (AH) of the liver has been suspected as a precancerous lesion in human hepatocarcinogenesis. The authors examined 75 cases of AH from 42 cirrhotic livers, using staining of argyrophilic nucleolar organizer regions (AgNORs). These reflect proliferative cell activity. Findings in AH were compared with those seen in hepatocellular carcinoma (HCC) and other chronic liver diseases. Expression of alpha-fetoprotein (AFP) was also examined immunohistochemically. The authors classified AH into three types: ordinary (OAH), atypical (AAH), and AH with focal malignancy (FM). OAH implies a lack of atypia; AAH represents AH with structural and cellular atypia but without the features of overt carcinoma; and FM denotes AH with foci of overt HCC. Forty of the 75 cases of AH were categorized as OAH, 19 as AAH, and 16 as FM. The noncancerous areas of FM had features of AAH. The mean number of AgNORs in AH was intermediate between that seen in cirrhosis (2.93) and HCC (6.18) and showed a step-wise increase in the following order: OAH (2.95), AAH (3.89), noncancerous areas in FM (4.58), and malignant foci in FM (5.71). There was no significant difference in AgNOR counts between OAH and cirrhosis. AgNOR counts in AAH and FM were significantly higher than those of OAH, and lower than those of HCC. AFP was positive in 12 of 25 HCCs and in malignant foci of 3 FM lesions, but it was absent in OAH and AAH. These data suggest that OAH has a limited capacity for proliferation but that AAH and FM are much more replicative. The latter two conditions are probably preneoplastic lesions or early forms of HCC. PMID- 1710419 TI - Immunocytochemical panel for the identification of malignant cells in serous effusions. AB - The cytologic diagnosis of malignancy in serous effusions can be challenging. An immunocytochemical (ICC) panel using commercially available antibodies (to carcinoembryonic antigen [CEA], epithelial membrane antigen [EMA], B72.3, Leu-M1, cytokeratin [CK], leukocyte common antigen [LCA], S-100 protein, and vimentin) was applied to cell blocks fixed in methyl Carnoy's solution that were from 55 consecutive pleural, peritoneal, and pericardial fluid specimens. The results were correlated with data from clinical records and routine cytologic studies. Final cytologic diagnoses included 26 of adenocarcinoma and 1 of mesothelioma. The remaining 28 cases were considered to be benign (reactive) proliferations. EMA, CEA, B72.3, and Leu-M1 were present in 96%, 77%, 58%, and 42% of adenocarcinomas, respectively. These determinants were absent in the mesothelioma and the reactive effusions, although anti-CEA yielded strong background staining of inflammatory cells. The CK markers identified malignant cells in 93% of cases, but consistently stained mesothelial cells as well. Antivimentin strongly labeled mesothelial cells in all cases, with weak to absent staining of malignant cells. In 3 of 26 carcinoma cases (12%), the ICC panel identified malignant cells that were not recognized initially on routine cytologic examination. In 1 of 26 cases (4%), the panel was falsely negative. Use of this approach can improve the diagnostic accuracy of cytologic examination of serous fluids. The ICC panel is especially helpful when atypical mesothelial proliferation is present, or in cases that are clinically suspect for malignancy, but cytologically negative because there are only a few malignant cells, or those that are cytologically bland. PMID- 1710420 TI - Mucocele-like tumors of the breast. Cytologic findings in two cases. AB - Mucocele-like tumors of the breast originally were reported by Rosen in 1986 as benign lesions that histologically resembled colloid carcinoma of the breast. The authors document two cases of mucocele-like tumors to illustrate the difficulty in separating these lesions from colloid carcinoma on the basis of fine-needle aspiration biopsy. Cytologically, mucocele-like tumors contained abundant mucin, few clusters, and sheets of regular epithelium that lacked nuclear atypia, and they contained no intact single cells. The authors recommend open surgical biopsy when fine-needle aspiration biopsy findings in such cases are equivocal. PMID- 1710421 TI - A survey of the health of homeless children in Philadelphia shelters. AB - We conducted a random-sample survey of homeless children and their mothers residing in Philadelphia (Pa) shelters. One hundred forty-six families were included in the final sample, resulting in an 80% response rate. The aims of the survey were to characterize the child's current and past health status, to determine access to and use of medical services, and to determine the serum erythrocyte protoporphyrin levels and tuberculin skin test status of the children. In addition, psychological tests were administered to both child and parent to assess developmental level and psychological problems. Finally, detailed questions were asked concerning the reasons for the homeless condition. The important reasons for homelessness cited in the survey included physical abuse, substance abuse, disagreements with landlords, and poor living conditions. The children's health problems included a high incidence of reported accidents and injuries, burns, and lead toxicity; the parents suffered from depression, physical abuse, and substance abuse. School-aged children tended to have low scores on tests of expressive vocabulary and word decoding, and preschoolers seemed to be below age expectations in receptive vocabulary and visual motor skills. The findings of this study suggest that homeless children tend to score poorly on developmental and psychological tests and tend to sustain serious burns and accidents. Policy implications of the survey include suggestions for health screening, rehabilitation, and education. PMID- 1710422 TI - Murine infantile polycystic kidney disease: a role for reduced renal epidermal growth factor. AB - Polycystic kidney disease (PKD) represents a form of renal epithelial hyperplasia. The C57BL/6J-cpk mouse, which has an infantile form of PKD, has a dramatically reduced expression of renal prepro-epidermal growth factor (EGF) mRNA and immunoreactive protein. Since EGF promoted maturation of epithelia in the neonate, the relative lack of renal EGF may contribute to the development of the collecting duct cysts by delaying epithelial maturation. PMID- 1710423 TI - Endothelium and glomerular growth. AB - The role of glomerular endothelium has been barely considered, let alone investigated, but this is in large part due to the extreme difficulty in isolating and growing it in culture. The glomerular capillary is part of the arterial system and is better perceived as a "hemiarteriole." The mesangium and its response to hemodynamic events in the glomerular capillary is equivalent to the smooth muscle cell wall of an arteriole. The capability of cytokines, derived from the endothelium, to act in an autocrine and paracrine manner is clearly demonstrated by angiogenesis. The glomerular endothelial cell is likely to play a crucial role in the glomerulus in health and disease. PMID- 1710424 TI - Acridine orange detection of Plasmodium falciparum malaria: relationship between sensitivity and optical configuration. AB - Blood samples collected from five volunteers participating in a P. falciparum infectivity trial were examined to determine the efficacy of the acridine orange technique. Several lens configurations were tested for efficiency in the diagnosis of malaria using this system. There was no significant difference in the sensitivity for detecting positive specimens or number of parasites among three lens configurations: a 50x long working distance objective (0.34 mm) with either a 10x ocular (total magnification 500x) or a 12.5x ocular (625x) and a 750x configuration using a 50x objective with a shorter working distance (0.24 mm). All three lens configurations were significantly better than the 1,000x configuration using a commonly available 100x oil immersion objective. The results achieved using this lens still exceeded the sensitivity of the thick blood film. PMID- 1710425 TI - New generation vaccines: does antibody play a directional role in antigen processing? AB - Analyses of recombinant proteins isolated from genomic libraries of pathogenic organisms represent the beginning of identifying immunologically-reactive epitopes. The induction of cell-mediated and humoral immune responses to any pathogen begins with the uptake and processing of antigen by antigen-presenting cells and the display of specific epitopes to the immune system of the host. Little emphasis is placed on the molecular mechanisms underlying transport of foreign proteins into antigen-presenting cells and factors that influence degradation to the peptides which represent the epitopes that associate with newly synthesized class II molecules of the major histocompatibility complex. These cellular processes are crucial to the design of any new generation vaccine. We describe our analysis of the 18 kDa protein antigen of Mycobacterium leprae and consider a possible role for antibody in antigen-processing. In both macrophage/dendritic cells and B lymphocytes, we suggest that antibody plays a directional role in antigen uptake, subcellular compartmentalization, and antigen degradation to yield peptides. These steps will all have an impact on the construction of new generation vaccines. PMID- 1710426 TI - Changes in mesenchymal cell-shape, matrix collagen and tenascin accompany bud formation in the early chick lung. AB - In the chick, lung branches arise as buds from the center of the pre-existing mesobronchial tube. Budding is known to be controlled by the mesenchyme. We have investigated mesenchymal properties in budding vs non-budding regions of the early chick lung, including sources of mesenchyme, cell shapes and densities, morphology and composition of the basement membrane, and distribution of the ECM components collagen, fibronectin and tenascin. We found that at points of outgrowth--the buds and the distal tip of the mesobronchus-mesenchymal cells adjacent to the lung epithelium are flattened, and the basement membrane is markedly thinned. In these basement membranes collagen is largely absent and tenascin redistributed into amorphous clumps. Of these characteristics only the cell-shape change, which results in the flattened mesenchymal cells at the bud tips, is correlated with initiation of the bud. We suggest that the cell-shape change leads to localized loss of collagen, which promotes emergence of buds, and that tenascin, which is found in the mesenchyme only in the budding region, promotes outgrowth and elongation of the bud. PMID- 1710427 TI - Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenii). AB - A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of FLG in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and alpha 2HS glycoprotein in human fetal brain. The sequence of appearance of FLG-positive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of FLG in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed. PMID- 1710428 TI - Heterogeneity of human mast cells and basophils in response to muscle relaxants. AB - The authors studied the effects of increasing concentrations 10(-5)-10(-3) M) of four muscle relaxants (succinylcholine, d-tubocurarine, vecuronium, and atracurium) on histamine release from peripheral blood basophils and mast cells isolated from human lung parenchyma, skin tissues, and heart fragments. Basophil granulocytes released less than 5% of their histamine content when incubated with any one of the muscle relaxants tested. In contrast, mast cells showed a significant heterogeneity in response to different muscle relaxants. Succinylcholine did not induce histamine release from any type of mast cell, and only high concentrations of d-tubocurarine (10(-3) M) caused histamine release from skin and lung mast cells. Vecuronium concentration-dependently induced histamine release from skin and lung--but not from heart mast cells--to a maximum of 7.2 +/- 2.1% and 4.9 +/- 1.4%, respectively. Atracurium concentration dependently caused significant histamine release from skin and lung mast cells to a maximum of 46.2 +/- 15.1% and 30.6 +/- 6.0%, respectively. Atracurium (5 x 10( 5) - 2 x 10(-4) M) also induced histamine release from heart mast cells. The histamine release process from both lung and skin mast cells caused by atracurium and vecuronium was extremely rapid (t1/2 = less than 1 min). The releasing activity of atracurium and vecuronium on lung and skin mast cells was not reduced, and not abolished, by lowering the temperature of the incubation buffer to 22 degrees C and 4 degrees C. Extracellular calcium did not affect the capacity of atracurium and vecuronium to induce histamine release from lung and skin mast cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710429 TI - Epidural opioid requirements. PMID- 1710430 TI - Preparation of a human DOPA decarboxylase cDNA probe by PCR and its assignment to chromosome 7. AB - The reverse transcription of mRNA of a human pheochromocytoma using an oligonucleotide complementary to rat DOPA decarboxylase (DDC) sequence as a primer, gave rise to a single strand cDNA. This resultant cDNA permitted by PCR amplification the preparation and cloning in PUC 19 of a human DDC probe of 747 base pairs. The choice of primer was dictated by the presence of tryptophan codons in the DOPA decarboxylase sequence which were conserved in different species throughout evolution. The presence of mismatches on the primers was not an obstacle to a specific amplification and the probe sequence was found identical to the human DDC cDNA sequence. This probe, labelled by nick translation detected on Southern blot of human-rodent hybrids, bands on human DNA after EcoRI, BamHI or HindIII digestion. The results with EcoRI were explicit and drove to the conclusion that DDC is located on chromosome 7. Five bands were obtained on human DNA digested with EcoRI, indicating that either numerous introns could interrupt the coding sequence of DDC gene or duplicated sequences could be present in chromosome 7. PMID- 1710431 TI - Tandem duplication of proximal 5q. AB - A 3.5-year-old boy with a de novo tandem duplication 5q11.1----5q15 is reported. Since the physical stigmata of seven liveborn cases with 5q proximal duplications are variable and inconspicuous, a recognizable syndrome could not be delineated. On the contrary, the associated developmental delay seems to be severe in duplications extending into 5q22 and mild in duplications 5q11----q13. PMID- 1710432 TI - Partial trisomy 17q and monosomy 9p due to a familial translocation. AB - Described is an infant with partial trisomy 17q and monosomy 9p [46,XX, 9,+der(9)t(9;17)(p21;q23)] due to adjacent-1 segregation of a maternal balanced reciprocal translocation. Characteristic clinical features of both partial 17q trisomy and monosomy 9p are present, but the former syndrome is less recognisable in this infant than in previously reported cases due to the concomitant 9p monosomy. PMID- 1710433 TI - The hypothalamic lateral tuberal nucleus in Alzheimer's disease. AB - The hypothalamic lateral tuberal nucleus was investigated in 5 young patients, aged 45 to 64 years, with Alzheimer's disease (AD) and in 5 age-matched control subjects. Combining conventional histopathological and immunocytochemical staining with neuronal counts, a peculiar form of neuronal pathology was characterized. Although neurons and neurites in the lateral tuberal nucleus of AD specimens were heavily stained by Alz-50, silver and thioflavine-S stains disclosed few neurofibrillary tangles or neurites. The numbers of neurons in the lateral tuberal nucleus of patients with AD (67,450; SEM = 5,050) were no different from those of control subjects (58,900; SEM = 2,450). In the AD patients, few plaques were present and were almost exclusively of the amorphous variety. We conclude that neurons in the lateral tuberal nucleus show an early stage of AD-related cytoskeletal pathology (Alz-50 positivity), but without plaques or neuronal death. PMID- 1710434 TI - Heterogeneity of the T-cell receptor beta gene rearrangements generated in myelin basic protein-specific T-cell clones isolated from a patient with multiple sclerosis. AB - Seventeen T-cell clones derived from the peripheral blood of a patient with multiple sclerosis and reactive with a synthetic peptide corresponding to residues 152-170 of the human myelin basic protein molecule were previously shown to be cytotoxic for myelin basic protein-coated target cells. Genetic restriction studies have now demonstrated that these clones recognize myelin basic protein in association with human leukocyte antigen DRw13. Studies of the T-cell receptor beta gene rearrangements generated by these clones demonstrated 12 different patterns, as evaluated by Southern blot analysis. Thus, the human T-cell response to myelin basic protein is exceedingly heterogeneous, even among T cells that recognize the same small fragment of the molecule in association with the same class II restriction element. PMID- 1710435 TI - Regulators of angiogenesis. PMID- 1710436 TI - Modulation of cardiac ion channels by magnesium. PMID- 1710437 TI - Ion channels and colonic salt transport. PMID- 1710438 TI - Surface charges and ion channel function. PMID- 1710439 TI - Regulation of ion transport across gallbladder epithelium. PMID- 1710440 TI - Strategies for studying permeation at voltage-gated ion channels. PMID- 1710441 TI - Is rheumatoid arthritis in Indians associated with HLA antigens sharing a DR beta 1 epitope? AB - HLA class II antigens were identified in a group of 44 patients with rheumatoid arthritis (RA) originating largely from the north or northeast of the Indian subcontinent and resident now in east London. Compared with 67 locally typed east London Asian controls, the prevalence of three HLA-DR antigens was raised in the patients: DR1 18.2% v 6.0% chi 2 = 3.99, DR4 20.5% v 11.9% chi 2 = 1.48, and DRw10 27.3% v 8.9% chi 2 = 6.56. These differences were also found when the patients with RA were compared with a larger control group of 110 northern Indians: DR1 18.2% v 7.2% chi 2 = 4.02, DR4 20.5% v 7.2% chi 2 = 5.56, and DRw10 27.3% v 8.1% chi 2 = 9.7. Twenty five (57%) of the patients expressed at least one of these antigens. All patients were also characterised for HLA-Dw types by mixed lymphocyte culture typing. The prevalence of the HLA-DR4 associated Dw types in the patients was: Dw4 2.3%, Dw10 0%, Dw14 11.4%, and Dw15 6.8%. The DR beta 1 chains of DR1 and DRw10 together with the Dw types of DR4 other than Dw10 share amino acid residues in a region of the third hypervariable region considered to be critical in antigen presentation. It is concluded that RA in Indians is associated with these HLA antigens, and data from this study support the hypothesis of a cross reactive epitope common to HLA specificities associated with RA. PMID- 1710442 TI - Anti-allergic effects of ketanserin on animal models of allergic reactions. AB - The aim of the present study is to investigate the effect of ketanserin, a potent and selective S2-serotonergic antagonist, on some anaphylactic reactions in mice, rats and guinea-pigs. Ketanserin (1 and 5 mg/kg) inhibited both IgE antibody mediated 48 hr homologous passive cutaneous anaphylaxis and IgG antibody-mediated 1.5 hr homologous passive cutaneous anaphylaxis in mice. In histamine-, serotonin and LTC4-induced skin reactions in mice, ketanserin inhibited an increase of capillary permeability caused by each mediator. Cyproheptadine, at doses of 0.1 and 0.5 mg/kg, inhibits 48 hr and 1.5 hr homologous passive cutaneous anaphylaxis and histamine-induced capillary permeability increase in mice ear. The inhibitory activity of cyproheptadine on these reactions is more potent than that of ketanserin. However, ketanserin showed a more potent inhibition than cyproheptadine for serotonin-induced capillary permeability. In rat passive peritoneal anaphylaxis, ketanserin had little effect on the release of histamine from peritoneal mast cells. Ketanserin inhibited an antigen-induced contraction of the sensitized guinea-pig trachea at an early period (within 5 min after treatment with antigen) but not at a late period (between 5 to 15 min). Cyproheptadine had little effect on this antigen-induced contraction of sensitized guinea-pig trachea. Moreover, ketanserin inhibited both histamine- and serotonin-induced contractions of the guinea-pig trachea, but not the contraction caused by carbachol and LTC4. When Forssman antibody was injected into the guinea pigs, a biphasic increase of airway resistance was observed. Ketanserin inhibited an increase of airway resistance at late phases. These results suggest that ketanserin inhibited some anaphylactic reactions in mice and guinea-pigs probably due to the antagonistic action of histamine and serotonin. PMID- 1710443 TI - Echographic diagnosis of anterior hyaloidal fibrovascular proliferation. AB - High-resolution contact B-scan echographic imaging of the ciliary body and peripheral retina was performed on five eyes with anterior hyaloidal fibrovascular proliferation and media opacity by means of a wide (58 degrees) scanning arc. This technique determined the circumferential and anteroposterior extent of peripheral traction retinal detachment associated with anterior hyaloidal fibrovascular proliferation, which correlated highly with findings at subsequent vitrectomy. Final visual acuity of 20/400 or better was achieved in the two eyes (50%) with more limited peripheral traction detachment. In the presence of media opacity, anterior echographic imaging may allow early detection of traction retinal detachment associated with anterior hyaloidal fibrovascular proliferation and may be useful in characterizing the severity of this condition. PMID- 1710444 TI - Optisol corneal storage medium. AB - Optisol is an investigational, intermediate-term corneal storage medium containing chondroitin sulfate and dextran to enhance corneal dehydration during storage. We used scanning electron microscopy to grade endothelial cell morphologic characteristics in terms of cell shape, cell borders, cell swelling, and apical holes in pairs of corneas stored in Optisol and Dexsol. Optisolstored corneas showed significantly fewer morphologic changes after 14 days at 4 degrees C than did Dexsol-stored corneas. No significant differences were seen after 1 to 4 days at 26 degrees C. Temperature-reversal analysis showed no significant change in corneal thickness with warming after 2-week storage at 4 degrees C in either medium, although Optisol-stored corneas were significantly thinner than those stored in Dexsol at all times. The results of scanning electron microscopy suggest that preservation at refrigerator temperature for 2 weeks in Optisol is superior to preservation in Dexsol. Both media may be useful in preserving endothelial structure for limited periods at room temperature, which could provide a measure of safety in shipping or storage where refrigeration is unreliable. PMID- 1710445 TI - Basic fibroblast growth factor is a calcium-mobilizing secretagogue in rat pancreatic acini. AB - Basic fibroblast growth factor (bFGF) induced a marked increase in the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and a rapid rise in cytosolic free calcium [Ca2+]i levels in rat pancreatic acini. The bFGF-mediated calcium transient was not dependent on the presence of extracellular calcium, and was abolished by pretreatment of acini with carbachol. bFGF stimulated amylase release in pancreatic acini in a monophasic, dose-dependent manner, and this effect was blocked by neutralizing anti-bFGF antibodies. At much higher concentrations, epidermal growth factor (EGF), but not insulin-like growth factor I (IGF-I), partially mimicked some of the actions of bFGF. These findings suggest that bFGF is a previously unrecognized calcium-mobilizing pancreatic secretagogue that may participate in the regulation of pancreatic exocrine function. PMID- 1710446 TI - Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3). AB - Phosphotyrosine-containing proteins were detected by western blotting of whole cell lysates of purified human neutrophils or rat basophilic leukemia cells (RBL 2H3) using a polyclonal anti-phosphotyrosine antibody. When either cell type was stimulated with the appropriate Fc crosslinking agent, heat-aggregated IgG for the neutrophil or DNP-HSA for the IgE-sensitized RBL-2H3, a rapid increase in the phosphotyrosine content of several proteins was observed. The kinetics and specificity of both responses suggest that Fc receptor crosslinking activates a receptor-associated tyrosine kinase, probably a member of the src family of tyrosine protein kinases. The subsequent tyrosine phosphorylation events are likely to be important in Fc receptor-mediated stimulus-response coupling in inflammatory cells. PMID- 1710447 TI - Tissue-specific methylation in the 5' flanking region of the gamma-glutamyl transpeptidase gene. AB - We have studied the relationship between the methylation and the expression of the gamma-glutamyl transpeptidase gene in adult rat liver and kidney. In the liver, where the level of expression is very low, the 5' flanking region of the gene appeared fully methylated, whereas in the kidney, where the gene is expressed at the highest level, it is undermethylated. In addition, kidney chromatin showed a DNase I hypersensitive site located near the origin of transcription. These results support a strong correlation between DNA undermethylation, DNase I sensitivity and tissue-specific gene expression. PMID- 1710448 TI - Bombesin activates large-conductance chloride channels in Swiss 3T3 fibroblasts. AB - Using the patch-clamp technique (cell-attached patches), we found that bombesin, a Ca-mobilizing peptide mitogen, activates large-conductance Cl channels in Swiss 3T3 fibroblasts. The channel activation required a lag period of about 50 s and was equally observed whether bombesin was applied to the patch-pipette or to the bath. A23187 (10(-6)M) in the bath induced the similar currents with almost identical current-voltage relationship as bombesin: their slope conductances were 292 +/- 15 (bombesin) and 318 +/- 42 (A23187) pS. In inside-out patches, the induced channels were selective to Cl over gluconate (11:1). These observations strongly suggest that in Swiss 3T3 fibroblasts bombesin activates the Cl channels through a mechanism involving an increase in the intracellular free Ca concentration. PMID- 1710449 TI - Human elongation factor 1 beta: cDNA and derived amino acid sequence. AB - From a cDNA library in lambda gt11 derived from poly (A+)RNA of human ovarian granulosa cells a cDNA clone lambda HGP34, containing an EcoRI insert of 829 bp, was identified. After subcloning of the insert into pUC18, the clone pHGP34 was obtained and sequenced. The derived amino acid sequence, corresponding to a protein of 225 amino acids, shows a high degree of homology to elongation factor 1 beta (EF-1 beta) of Artemia salina (57%) and known peptide sequences of Xenopus laevis EF-1 beta (86%). We therefore assume that the protein coded for by pHGP34 represents human EF-1 beta. Northern analysis reveals an EF-1 beta specific mRNA of 900 bp. Southern analysis indicates that EF-1 beta in the human genome, like EF-1 alpha, appears to be specified by more than one gene. A high degree of sequence homology for EF-1 beta specific sequences is observed for bovine, rat and mouse species. PMID- 1710450 TI - Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor. AB - A cDNA encoding non-selective type (ETB) of endothelin receptor was isolated from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 442 amino acid residues with a relative Mr of 49,643. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and bovine lung ET-1-specific (ETA) receptor, respectively, and contained a relatively long and proline-rich extracellular N-terminal region in addition to a significant similarity with the G protein-coupled receptor super family with seven transmembrane segments. PMID- 1710452 TI - Interaction of RNA polymerase II with acetylated nucleosomal core particles. AB - Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery. PMID- 1710451 TI - Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4). AB - In rat adipocytes, palmitate: a) increases basal 2-deoxyglucose transport 129 +/- 27% (p less than 0.02), b) decreases the insulin sensitive glucose transporter (GLUT4) in low density microsomes and increases GLUT4 in plasma membranes and c) increases the activity of the insulin receptor tyrosine kinase. Palmitate stimulated glucose transport is not additive with the effect of insulin and is not inhibited by the protein kinase C inhibitors staurosporine and sphingosine. In rat muscle, palmitate: a) does not affect basal glucose transport in either the soleus or epitrochlearis and b) inhibits insulin-stimulated glucose transport by 28% (p less than 0.005) in soleus but not in epitrochlearis muscle. These studies demonstrate a potentially important differential role for fatty acids in the regulation of glucose transport in different insulin target tissues. PMID- 1710453 TI - Nuclear matrix protein mitotin messenger RNA is expressed at constant levels during the cell cycle. AB - During the course of an investigation on nuclear matrix protein cDNAs, we have isolated a cDNA clone hybridizing with the messenger RNA encoding mitotin. Mitotin is a 125 kDa/pI 6.5 nuclear matrix protein present in proliferating but not in resting cells. This protein was shown to have a marked increase and characteristic redistribution in G2/M phase of the cell cycle. In this report, using synchronized Raji and WISH cells, we demonstrate that mitotin messenger RNA is expressed at the same level throughout the cell cycle. PMID- 1710454 TI - Demonstration of platelet activating factor receptor in guinea pig kidney. AB - Distribution of platelet activating factor (PAF) receptor was examined in the guinea pig kidney. Northern blot analysis showed a single band electrophoresed just below the 28S rRNA, and the mRNA was richest in the cortex with lesser amounts in the outer and then inner medulla. Scatchard analysis of membrane fraction using [3H]WEB 2086, a specific PAF receptor antagonist, revealed a single binding site with Bmax of 522, 228, 58 fmol/mg protein for the cortex, outer medulla and inner medulla, respectively. Kd values were in the same order of magnitude (10(-8) M). These results indicate the presence of a single class of PAF receptor in the guinea pig kidney which is most abundant in the cortex. PMID- 1710455 TI - Effect of cyclic RGD peptide on cell adhesion and tumor metastasis. AB - Several kinds of cyclic peptides containing an L-arginine-glycine-L-aspartic acid RGD sequence were synthesized by the liquid phase method, and we investigated their effects on the attachment of mouse B16 melanoma cells onto fibronectin coated well. Cyclo (GRGDSPA) inhibited the cell attachment at a 20-fold lower concentration than the linear form. The cell adhesion was inhibited by the synthetic peptides with the following relative order of activity: cyclo (GRGDSPA) much greater than cyclo (GRGD) greater than cyclo (RGDS), cyclo (GRGDSP) greater than cyclo (GRGDS) greater than cyclo (RGDSP), cyclo (RGDSPA). Cyclo (GRGDSPA) was more effective at inhibiting cell attachment to vitronectin than it was at competing with fibronectin attachment, as reported in the case of GRGDSP. Moreover, cyclo (GRGDSPA) significantly reduced the formation of colonies in mice injected with B16-FE7 melanoma cells. PMID- 1710456 TI - Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends. AB - Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions. PMID- 1710457 TI - Cysteine proteinase inhibitors and bleomycin-sensitive and -resistant cells. AB - We have isolated a new human head and neck carcinoma cell line (C-10E) that is highly resistant to BLM (40-fold) when compared to the parental (A-253) cell line. Consonant with BLM resistance in the C-10E cell line, we found that this cell line accumulated 2- to 3-fold less BLM A2 than A-253 cells. Kinetic analyses of BLM A2 association revealed a decreased Vmax for C-10E cells with little change in Ka. Furthermore, the BLM-resistant cell line (C-10E) metabolized BLM A2 to a greater extent than its sensitive counterpart (A-253). Thus, compared to A 253 cells, the C-10E cells exhibited both decreased cellular association and increased metabolism of BLM. Synergistic cytotoxicity was seen when BLM was combined with either E-64 or leupeptin, cysteine proteinase inhibitors known to block BLM metabolism in vitro. E-64 inhibited the metabolism of BLM A2 in both C 10E and A-253 cells, and cellular accumulation of radiolabeled BLM A2 was increased by leupeptin or E-64 in only A-253 cells. These results suggest that both inhibition of drug metabolism and increased drug accumulation contribute to this synergism. PMID- 1710458 TI - Time course of IL-6 and TNF alpha release during endotoxin-induced endotoxin tolerance in rats. AB - Development of endotoxin tolerance in rats induced by repeated application of low dosages of endotoxin is associated with repeatable IL-6 formation, reversible drop in white blood cells, pronounced consumption of platelets, gradual formation of alpha 2M as an example of acute phase proteins, and flattening TNF alpha formation. In the status of full tolerance the TNF alpha release is completely eliminated. Inhibition of TNF alpha biosynthesis and induction of acute phase protein formation by IL-6 are discussed as possible factors in the development of endotoxin tolerance. PMID- 1710459 TI - Induction of cytochromes P450IA1 and P450IA2 as determined by solution hybridization. AB - Two oligodeoxyribonucleotides were synthesized that were specific for the messenger RNAs for the polycyclic hydrocarbon-inducible cytochromes P450IA1 and P450IA2. The solution hybridization technique was modified for the use of these oligodeoxyribonucleotide probes so as to increase the sensitivity and specificity of this method. Using this technique, the steady-state levels of the mRNAs for cytochromes P450IA1 and P450IA2 in control rat liver were determined to be less than 3 and 6 molecules/cell, and 1.8 and 4.0 attomol/micrograms poly (A)+ RNA, respectively. At 15 hr after induction with 3-methylcholanthrene, the steady state levels of the mRNAs for P450IA1 and P450IA2 were 68 and 200 molecules/cell, and 41.6 and 123 attomol/micrograms poly (A)+ RNA. PMID- 1710460 TI - Changes in synaptosomal pH and rates of oxygen radical formation induced by chlordecone. AB - The resting pH of 7.14 +/- 0.02 within rat cortical synaptosomes is elevated in vitro by the insecticide chlordecone, in a dose-dependent manner. Chlordecone also reduces the rate of oxygen radical formation within synaptosomes. Both of these changes can also be demonstrated following in vivo treatment of rats with chlordecone (75 mg/kg body wt). Although chlordecone increases the permeability of the plasma membrane, the increase in pH observed is unlikely to be caused by this, since in vivo administration of chlordecone does not appreciably alter membrane order as evaluated by both a lipophilic probe, and a probe with an ionic segment. Another xenobiotic agent, methyl mercuric chloride, and a free radical generating system, an ascorbic acid-ferrous sulfate mixture, did not modulate synaptosomal pH, although membrane permeability was increased. Other evidence of the ability of synaptosomes to maintain homeostasis was the failure of mitochondrial inhibitors to significantly reduce pH. The drop in synaptosomal pH effected by amiloride, an inhibitor of Na+/H+ exchange, and the transient rise in pH caused by ammonium chloride further suggested that synaptosomes may be a good model in the study of the regulation of intracellular pH. The elevation of cytosolic pH, and depression of oxygen radical formation by chlordecone, may result from both the attenuation of respiratory metabolism and an impaired capacity of the plasma membrane to maintain ionic gradients. PMID- 1710461 TI - Expression of mRNA species encoding steroidogenic enzymes in the rat ovary. AB - We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha), aromatase cytochrome P-450 (P-450AROM) and 3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)(+)-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of approximately 1.9 kb was detected at low levels, while 3 beta-HSD mRNA of 1.7 kb was in relatively high abundance throughout the oestrous cycle. While P-450(17) alpha mRNA of 1.9 kb and P-450AROM of 2.7, 2.2 and 1.7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14-21 of gestation). Levels of P-450(17) alpha mRNA on day 10 of gestation were lower than at pro oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3 beta-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P 450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450(17) alpha mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1710463 TI - Expression of retinoic acid alpha and beta receptor genes in liver and hepatocellular carcinoma. AB - cDNA probes for human retinoic acid receptors alpha and beta (RAR alpha and RAR beta) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RAR beta mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RAR beta mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RAR alpha mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RAR alpha or RAR beta mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low alpha fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of alpha fetoprotein were markedly stimulated by RA. PMID- 1710462 TI - TGF-beta 1 and skin carcinogenesis: antiproliferative effect in vitro and TGF beta 1 mRNA expression during epidermal hyperproliferation and multistage tumorigenesis. AB - The role of transforming growth factor-beta 1 (TGF-beta 1) in multisage carcinogenesis in mouse skin was assessed by studying its growth inhibitory effects on nontumorigenic and tumorigenic keratinocytes and by examining its mRNA expression in vitro and during epidermal hyperproliferation and multistage carcinogenesis. While growth of primary basal keratinocytes was inhibited by TGF beta 1 in doses as low as 0.1 ng/mL, the immortal keratinocyte line MCA/3D ("putatively initiated" cells) responded to TGF-beta 1 with slightly reduced sensitivity, and the papilloma-producing keratinocyte line 308 was considerably less sensitive. In contrast, the squamous carcinoma cell line Carc B was completely nonresponsive, and two other tumorigenic cell lines (PDV and PDVC57) were sensitive to growth inhibition by TGF-beta 1. Steady-state levels of TGF beta 1 mRNA were high in all the malignant cell lines and in line 308 papilloma cells, but low in primary basal cells and in the nontumorigenic keratinocyte lines V2, Reb1, and MCA/3D. Our in vivo studies showed that tumor promoters, but not mitogenic or weak hyperplasiogenic agents, were able to induce transient expression of TGF-beta 1 mRNA in mouse epidermis. A constitutive overexpression of TGF-beta 1 mRNA was observed in malignant carcinomas but not in the benign premalignant lesions, indicating that overexpression may be associated with malignant progression. PMID- 1710464 TI - Tyrosine phosphorylation of three cellular proteins correlates with transformation of rat 1 cells by pp60src. AB - The analysis of phosphotyrosine-containing proteins in Rat 1 cells overexpressing either the tyrosine kinase pp60c-src or genetic variants containing alterations in functional and structural domains has led to the identification of three proteins whose tyrosine phosphorylation correlated with pp60src-induced cellular transformation. The tyrosine phosphorylation of one of these proteins, p120, has been previously shown by us and others to coincide with the presence of kinase activated, membrane-associated pp60src in chicken embryo cells. The second protein was identified as the ras-associated GTPase-activating protein (GAP). The third protein whose tyrosine phosphorylation was markedly elevated in Rat 1 cells expressing activated, membrane-bound forms of pp60src had an apparent molecular mass of 64-67 kDa. The electrophoretic mobility of this protein varied in cells expressing different pp60src variants. The tyrosine-phosphorylated form of p64-67 was present in immune complexes containing GAP, suggesting a stable interaction between these two cellular proteins. PMID- 1710466 TI - Polyamide profiles of porcine milk and of intestinal tissue of pigs during suckling. AB - Previous studies have suggested that luminal polyamines can directly influence intestinal differentiation of neonatal rats. The present investigation has demonstrated the presence of high levels of polyamines in porcine milk and in the intestinal tissues of suckling pigs. The quantities of polyamines in sow's milk sampled between wk 1 and 8 of lactation were determined using high performance liquid chromatography (HPLC). The concentration of milk spermidine (SPD) remained constant over the first 3 to 4 wk of lactation but increased 4-fold between wk 4 and 7. Neither putrescine nor spermine (SPN) were detected in any of the milk samples. During intestinal development the mucosal SPD/SPN ratio was elevated between wk 1 and 3 and wk 5 and 7. The latter period of increase corresponded with the surge in milk SPD concentration. It is suggested that milk SPD is taken up from the intestinal lumen and is involved in potentiating intestinal differentiation during the latter part of the suckling period. PMID- 1710465 TI - Effect of myogenic determination on tumorigenicity of chemically transformed 10T1/2 cells. AB - Transforming agents have been postulated to interfere with cellular differentiation programs, thus causing uncontrolled growth. Inducing transformed cells to differentiate can result in loss of the transformed phenotype since many end-stage differentiated cells are unable to divide. We attempted to bypass or suppress the tumorigenic phenotype of 3-methylcholanthrene (MCA)-transformed 10T1/2 cells (MCA Cl 15C1) by induction of myogenic determination. MCA Cl 15C1 cells were either treated with the hypomethylating drug 5-azacytidine (5-aza-CR) or were transfected with the muscle determination gene MyoD1, both of which induce a myogenic phenotype in 10T1/2 cells. Colonies containing myoblast-like cells were isolated and examined. Muscle markers were detected both in 5-aza-CR treated and in MyoD1-transfected myogenic clones by immunofluorescence and northern analyses. The myogenic clones did not show decreased tumorigenicities relative to that of the parental cells upon subcutaneous injection in nude mice. Some of the resulting tumors, however, were classified as rhabdomyosarcomas rather than fibrosarcomas. Although induction of myogenic determination was not sufficient to abolish the tumorigenic phenotype of MCA Cl 15C1 cells, several tumors showed decreased levels of MyoD1 mRNA, suggesting that growth in vivo either selected for or caused decreased determination gene expression. PMID- 1710467 TI - Angiotensin and the regulation of cellular growth. Pathophysiologic implications for cardiovascular and noncardiovascular tissues. AB - Components of the renin-angiotensin system can be found in the vasculature, although in most cases it is unclear how much, if any, of this renin in the vasculature is locally synthesized. Over recent years, a variety of novel actions of angiotensin II have been delineated which suggest that in appropriate physiologic or pathologic circumstances vascular angiotensin II can play an important role in determining vascular structure. Moreover, angiotensin II may play a role in neoplastic growth of vascular and nonvascular tissues. PMID- 1710468 TI - Pediatric pain intervention in the PACU. AB - The assessment and management of a child's pain in the postoperative period remain a challenge to the PACU nurse. Assessment of pain in children is often difficult; ongoing clinical research in this area continues to expand knowledge of how to improve children's communication of their pain to caregivers. Many pain management strategies are available, both pharmacologic and nonpharmacologic, to ease pain and distress postoperatively. Through understanding and knowledge of pain, its assessment and its management, the PACU nurse contributes positively to the child's surgical experience. PMID- 1710469 TI - Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry. AB - We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro opiomelanocortin 1-77 (N-POMC(1-77)) purified from porcine pituitaries. Antibody 1-244 recognizes an epitope located within the gamma 3-melanotropin (gamma 3-MSH or POMC(51-77)) sequence, whereas antibody 2-197 binds specifically to a determinant in the 1-49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1-77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1 77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2-197 and 1-244 antibodies could recognize a major peptide comigrating with purified N-POMC(1-77). In addition, antibody 2-197 also detected a peptide with a mobility similar to that of standard N-POMC(1-49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2-197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary. PMID- 1710470 TI - RNA transcription and translation in the hearts of normal and cardiomyopathic Syrian hamsters. AB - The cardiomyopathic Syrian hamster has an autosomal recessive defect that results in the development of an early onset cardiac myopathy leading to cardiac dysfunction and, eventually, complete heart failure. To assess the regulatory mechanisms modulating gene expression in the normal and myopathic myocardium, we investigated both RNA transcription and translation. Our results indicated that the incorporation of [3H]UMP into myocardial cell nuclear RNA decreased 10-fold from 7 to 210 days of age in the normal Syrian hamster. The incorporation of [3H]UMP was approximately 50% lower in the cardiomyopathic as compared with the normal Syrian hamster. RNA translation, as assessed by rabbit reticulocyte lysate in vitro translation, indicated that a coordinated 50% decrease in RNA translation occurred in normal Syrian hamster from 7 to 210 days of age. A further reduction of 20% in translation was found in cardiomyopathic Syrian hamster ventricular RNA translation as compared with matched random bred control groups. Two-dimensional polyacrylamide gel analysis of cell-free translated protein products demonstrated two myocardial peptides that were found to be consistently altered when the normal and cardiomyopathic Syrian hamsters were compared. These results indicate that transcription and translation decrease with age and that these processes are further downregulated, in an additive manner, with the genesis of the disease process. PMID- 1710471 TI - [Structure of the endosperm in the mature seed of Melilotus alba]. AB - The results of the exam at the light, the fluorescence and the scanning electron microscope of the endosperm of Melilotus alba mature impermeable seeds are reported. Cryostat sections, semithin sections and squashes are observed. Melilotus alba endosperm is variable in thickness and envelopes cotyledons and radicle. Its "aleurone" layer is one-cell thick, while the number of layers of its internal cells varies in relation to the location in the seed. In the aleurone cells, the cytoplasm and the outer portion of the wall are autofluorescent; tannic acid-ferric chloride stains the outer portion of the wall and allows to see clearly the inner thickenings, DAPI and haematoxylin demonstrate the presence of the nucleus. The cytoplasm of these cells is coloured by Sudan black b, and its fluorescence is enhanced by auramine and calcofluor white. Calcofluor white enhances the fluorescence of the outer portion of these walls, too, but is without effect on the non-autofluorescent thickening, indicating presence of cellulose only in the first case. Callose is absent. Also the thin autofluorescent walls of the endosperm inner cells react positively to calcofluor. These cells are very large, almost completely filled with "gelatinous" substances--the galactomannans--and very rarely contain a nucleus. PMID- 1710472 TI - Hyaluronic acid deposition in cardiac myxomas: localization using a hyaluronate specific binding protein. AB - Myxomas are the most common primary tumors of the heart. These lesions contain a gelatinous, ground-substancelike material which has been described as glycosaminoglycan in nature. Using a newly developed, cartilage-derived hyaluronic acid-binding protein and a modification of the avidin-biotin immunostaining procedure, we demonstrate that hyaluronic acid is contained in the jellylike material of cardiac myxomas. PMID- 1710473 TI - Histo-blood group antigens as differentiation markers in testicular germ cell tumours. AB - The distribution of histo-blood group antigens in a series of eleven human non seminomatous testicular germ cell tumours is described and the variable expression of these antigens is related to the patterns of differentiation that are reflected morphologically within these tumours. The results suggest that histo-blood group antigens of type 2 chain carbohydrate structures may be used as markers of differentiation. All the tumours contained binary 2-3 sialosyllactosamine structures. N-acetyllactosamine and Le(y)-negative tumors were the least differentiated morphologically, while N-acetyllactosamine and Le(y)-positive tumours exhibited more differentiated tumour patterns. Whether the occurrence of the latter antigens in poorly differentiated forms of germ cell tumours reflects a biological potential for differentiation remains to be proven in a larger material. PMID- 1710474 TI - Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells. AB - The distribution of immunostaining in normal major salivary gland and in 12 pleomorphic adenomas was studied using monospecific monoclonal antibodies to a number of cytokeratins, including cytokeratin 14, to smooth muscle actin and vimentin. A number of these antibodies enabled a distinction to be made between structural components of the normal gland, and to relate this to the different structures of pleomorphic adenomas. In the normal gland, the luminal duct cells expressed cytokeratins 7, 8, 18 and 19. Three antibodies were of particular value for the characterization of normal myoepithelial and basal cells; while the antibody to smooth muscle actin and the cytokeratin antibody Ks8.12 mutually exclusively stained the myoepithelial (basket) cells and the basal duct (light) cells, respectively, the recently established monospecific antibodies to cytokeratin 14 showed specific immunostaining with both cell types. These three antibodies left luminal cells virtually unstained. Ck 13 was found occasionally in single luminal excretory duct cells. Antibodies to cytokeratins 1/2, 10 and 10/11 did not show any staining in the normal gland. In the pleomorphic adenomas, the staining pattern of the two-layered tubular formation resembled that of the normal gland ducts: tumour luminal cells showed the characteristic, although more irregular, expression of cytokeratins 7, 8, 18 and 19; the outer cells resembled normal ductal basal cells with their anti-cytokeratin 14/Ks8.12-epitope staining and in that they virtually lacked staining for smooth muscle actin. Trabecular formations and cells in myxoid areas were reactive with Ks8.12 and for cytokeratin 14, occasionally also for cytokeratins 7, 18 and 19. Epidermoid cell islets expressed mainly cytokeratin 14 and inconsistently the squamous epithelial cytokeratin 13 and the epidermal cytokeratin 10/11. Vimentin was found in cells of myxoid areas. The results support the postulate that some of the normal duct basal cells act as reserve cells and can give rise to tumour formation with a primitive myxoid or trabecular pattern and a more differentiated tubular or epidermoid configuration. PMID- 1710475 TI - Cytokeratin intermediate filament pattern in uterine cervical biopsies. AB - Twenty-seven uterine cervical biopsies with histological diagnoses ranging from normal through dysplasia to invasive carcinoma were analysed for cytokeratin pattern in two-dimensional gel electrophoresis. No direct correlation between histological diagnosis and cytokeratin pattern was observed. PMID- 1710476 TI - New developments in Kawasaki disease. AB - Kawasaki disease is an acute vasculitis of infancy and early childhood. Left untreated, up to 25% of children with Kawasaki disease develop coronary artery abnormalities. Recent studies indicate that patients with Kawasaki disease have elevated cytokine production and increased expression of cytokine-inducible activation antigens on their vascular endothelium. Treatment with intravenous gammaglobulin significantly reduces the incidence of coronary artery abnormalities in this disease. Furthermore, patients who respond clinically to intravenous gammaglobulin demonstrate a marked reduction in immune activation. These recent insights into the immunology and treatment of Kawasaki disease may eventually provide new directions for research into the etiology of this fascinating disease. PMID- 1710478 TI - Diminished A gamma T fetal globin levels in Sardinian haplotype II beta 0 thalassaemia patients are associated with a four base pair deletion in the A gamma T promoter. AB - In Sardinia, the beta-39 nonsense mutation is the primary cause of beta 0 thalassaemia. This mutation is found mainly on beta-globin gene cluster haplotypes I and II, which differ in their A gamma globin types (A gamma I and A gamma T, respectively). This report presents data on G gamma, A gamma I and A gamma T levels, and the presence or absence of a 4 base pair (bp) deletion at 225 to -222 of the A gamma globin promoter, in 55 poly-transfused beta 0 thalassaemia major patients. Six patients were homozygotes for the normal (N) A gamma promoter lacking the 4 bp deletion, had no A gamma T globin, and their mean G gamma:A gamma I: A gamma T ratio was 52.9:47.1:0. Twenty-five patients were homozygotes for the mutant (M) A gamma promoter with the 4 bp deletion, had no A gamma I globin, and the mean G gamma:A gamma I: A gamma T ratio was 62.1:0:37.9. For M/M compared to N/N, the lower A gamma T than A gamma I was significant by the t-test (P less than 0.001). Twenty-four N/M cases had mean G gamma:A gamma I:A gamma T of 56:24.4:19.6, and the lower A gamma T than A gamma I was also significant (P less than 0.001). Partial haplotype analysis on these and 17 other beta 0-thalassaemia patients suggested that the 4 bp deletion was strongly associated with haplotype II. Of 33 M/M, 32 were haplotype II/II and one was II/5a; of 31 N/M, 29 were I/II and two were II/IX; of eight N/N, seven were haplotype I/I and one was I/IX. These data show a strong association of the 4 bp promoter deletion with decreased expression of the A gamma T globin gene on haplotype II. PMID- 1710477 TI - Extended visiting in a surgical ward. PMID- 1710479 TI - Molecular analysis of BCR/ABL products in a case of myelodysplastic syndrome with late appearing Philadelphia chromosome. PMID- 1710480 TI - Simultaneous expression and regulation of G-CSF and IL-6 mRNA in adherent human monocytes and fibroblasts. AB - The regulation of granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6) mRNA was studied in human adherent monocytes in response to the protein kinase C activator, oleolyl-acetylglycerol (OAG), the calcium-ionophore A23187 and the cyclic AMP elevating agents, dibutyryl c-AMP (DBcAMP), cholera toxin and isobutyl-methylxanthine (IBMX). G-CSF and IL-6 transcripts were simultaneously expressed in response to OAG, A23187, DBcAMP, IBMX and cholera toxin. However, the time course demonstrated a difference; a rapid induction by OAG and A23187 and a delayed pattern by cAMP elevating agents. In addition it appeared that the induction of CSFs by DBcAMP was independent of the adherence procedure or the presence of fetal bovine serum but could be counteracted by the simultaneous addition of H8, an inhibitor of the cAMP dependent kinases. Finally, experiments were performed to study in how far comparable mechanisms operate in other cell types. Human fetal lung fibroblasts were stimulated with A23187, DBcAMP and OAG. All these agents induced simultaneous expression of G-CSF and IL-6 mRNA and secretion of proteins, indicating that different signalling pathways exist in both cell types which regulate the expression of both genes. PMID- 1710481 TI - Cisplatin, etoposide, ifosfamide, vincristine and bleomycin combination chemotherapy for far advanced testicular carcinoma. AB - Forty-eight patients with advanced testicular cancer, defined as abdominal mass greater than 10 cm, mediastinal mass greater than 5 cm, more than 20 lung metastases, or visceral organ involvement were treated with an intensive, alternating five-drug regimen consisting of cisplatin 50 mg/m2 d 1-3, etoposide 170 mg/m2 d 1-3, ifosfamide 5 g/m2 d 15, vincristine 2 mg weekly, bleomycin 15 mg/m2 weekly, q d 28. Thirty-four (71%) of the patients attained tumor-free status. This was achieved by chemotherapy alone in 14 patients and by surgical resection of residual disease in the remaining 20 patients (histology of resected tissue: necrosis 12, mature teratoma 7, viable carcinoma 1). Patients with pure seminoma responded better than patients with nonseminoma (CR 100% vs. 67%, respectively). In a univariate analysis only the value of HCG (less than vs greater than 10,000 U/L) and the number of involved organ sites (less than or equal to 2 vs greater than to 2) had significant influence on the response rate. After a minimum follow-up of 24 months 3 patients (9%) have relapsed. The survival rate is 76% after 36 months, with 61% remaining disease-free. Though this intensive regimen might bestow some of the therapeutic advantages of standard three-drug protocols in far advanced testicular cancer, the results are still less than optimal and warrant the exploration of new therapeutic strategies. PMID- 1710482 TI - BOP/VIP--a new platinum-intensive chemotherapy regimen for poor prognosis germ cell tumours. AB - Ninety-one patients with poor prognosis non-seminomatous germ cell tumours (NSGCT) were treated with an initial intensive chemotherapy schedule. Suitable patients fulfilled one or more of the following criteria: lymph node metastases greater than 10 cm diameter; liver, brain or bone metastases; serum HCG level greater than 50,000 IU/L; and extragonadal primary tumours. Treatment consisted of 3 cycles of bleomycin, vincristine and cisplatin (BOP) administered at 10 day intervals, followed by 3 cycles of etoposide, ifosfamide and cisplatin (VIP) at 21 day intervals. A total of 64 patients (70%) achieved a complete remission. This comprised 46 patients who received BOP/VIP only, and 18 patients who received additional chemotherapy after BOP/VIP. Of these 64 patients, 51 underwent complete surgical resection of residual masses, including 11 in whom there was evidence of teratoma with cellular atypia or non-germ cell cancer in the resected tissue. A further 9 patients had persisting unresected radiological masses in the presence of marker complete remission. The overall response rate was 80%. Currently 57 patients (63%) remain alive and free from disease progression. The median follow-up period is 90 weeks (range 24-206 weeks), with a 2 yr actuarial progression-free survival of 66% (95% c.i. 55-77%). Major toxicity was myelosuppression, occurring during the VIP arm of therapy, with a median nadir WBC of 1.1 x 10(9)/L and median platelet count of 51 x 10(9)/L. Other toxicity included peripheral neuropathy (WHO Grade 2 and 3 in 22%). We conclude that treatment results with the BOP/VIP schedule in this poor prognosis patient group are at least comparable with other schedules, and toxicity is manageable.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710483 TI - Intrapleural palliative treatment of malignant pleural effusions with mitoxantrone versus placebo (pleural tube alone). AB - From 11/87 until 7/90 103 patients entered a prospective randomized trial on the treatment of malignant pleural effusions (MPE) with intrapleural mitoxantrone versus placebo (pleural tube alone with instillation of isotonic NaCl). Our data suggest no statistically significant difference between the two arms with respect to response and response duration. There is no influence on survival time. The toxicity is moderate, with only fever occurring more often in the mitoxantrone arm. We recommend performance of pleurodesis in patients with MPE first by sufficient drainage with a tube of 16-20 char. Only in instances of failure it is necessary to add sclerosing agents such as tetracycline, etc. PMID- 1710484 TI - Is there an effective salvage therapy for advanced Hodgkin's disease? AB - The availability of increasing numbers of active agents has led to the development of a succession of regimens for use as alternative and second-line therapy following relapse from or refractoriness to MOPP (mechlorethamine/vincristine/procarbazine/prednisone) or its variants. ABVD (doxorubicin/bleomycin/vinblastine/dacarbazine) has been the most widely used and has been considered non-cross-resistant. Other programs containing the nitrosourea lomustine have been used with results similar to those with ABVD. A relatively small fraction of relapsed patients remain failure free at 5 years (about 20% to 30%) despite a 30% to 60% second-line complete response (CR) rate. The few randomized trials (Cancer and Leukemia Group B [CALGB], European Organization for Research and Treatment of Cancer) evaluable to assess the efficacy of alternating MOPP/ABVD compared with MOPP alone have shown a small but significant advantage in freedom from progression and/or survival favoring the complex regimens over MOPP. The CALGB trial (8251) included a third arm of ABVD alone. The ABVD and MOPP/ABVD arms had a higher CR rate and superior failure-free survival (FFS) than did MOPP, but have thus far shown no difference between ABVD and alternating MOPP/ABVD, suggesting that full doses of a single regimen are equivalent to the more complex multidrug regimen. The next step in the CALGB program was to attempt to improve ABVD. The substitution of etoposide, an active single agent, for dacarbazine and bleomycin in ABVD resulted in a new regimen, EVA (etoposide/vinblastine/doxorubicin). This program has already demonstrated a 66% response rate in MOPP-resistant/relapsed patients (CALGB 8751).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710485 TI - MACOP-B and VACOP-B in diffuse large cell lymphomas and MOPP/ABV in Hodgkin's disease. AB - Between 1981 and 1986, 126 patients with diffuse large cell lymphoma were treated with MACOP-B (methotrexate/doxorubicin/cyclophosphamide/vincristine/prednisone/bleomy cin). All had advanced-stage lymphoma (Ann Arbor stage III or IV or stage I or II if the tumor mass was greater than 10 cm or B symptoms were present). The complete response (CR) rate was 84% and the toxic death rate was 6%. Actuarial overall survival at 3 years was 67% and at 8 years 62%; the failure-free survival at 8 years was 52%. The follow-up for MACOP-B is 39 to 106 months (median 76) for living patients. A multivariate prognostic factor analysis for this group of patients identified age greater than 60 years. B symptoms, more than one extranodal site of disease, and more than three nodal sites of disease as the four significant prognostic variables. From June 1986, 108 patients were enrolled on a modification of MACOP-B called VACOP-B (etoposide/doxorubicin/cyclophosphamide/vincristine/prednisone/bleomycin ). Their CR rate was 81%, and the toxic death rate was lower, at 3%. The 60% overall survival at 3 years is not statistically significantly different from that of MACOP-B. The incidence of moderate or severe mucositis and Cushingoid changes was much lower with VACOP-B. The MOPP/ABV (mechlorethamine/vincristine/procarbazine/prednisone- doxorubicin/bleomycin/vinblastine) hybrid chemotherapy regimen for advanced-stage Hodgkin's disease was standard therapy from April 1981 to June 1988 for untreated patients aged 16 to 65. Advanced stage was defined as stages IIB, IIIB, III2A, IVA, IVB, or stages IIA or IIIA with greater than four splenic nodules or a mediastinal mass greater than one third of the transthoracic diameter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710486 TI - Etoposide, ifosfamide, and methotrexate with or without bleomycin in refractory or recurrent lymphomas. AB - The prognosis of patients with refractory or relapsed malignant lymphoma is poor. To improve the outcome of such patients, a therapeutic regimen of VIM +/- B (etoposide/ifosfamide plus mesna/methotrexate/ with or without bleomycin) was administered. Of 47 patients treated, 15 had relapsed following complete remission (CR) after first-line chemotherapy, 28 had failed to achieve CR with first-line therapy, and four failed to respond to multiple salvage regimens. All patients had received extensive prior chemotherapy, and 36 had received combinations containing doxorubicin. Eight patients had low-grade non-Hodgkin's lymphoma (NHL), 28 had high-grade NHL, and 11 patients had Hodgkin's disease. Overall response rate was 87%, with 45% CR and 42% partial remission (PR). Median relapse-free interval was 8 months in patients with CR and 6 months in those with PR. Of patients with CR, 43% were predicted to be without relapse at 2 years and 31% at 5 years. Median survival time for all patients treated with 14 months-22 months for those with CR and 10 months for those with PR. Probability of survival at 2 years was 30% in all patients, 50% in patients with CR, and 15% in those with PR. VIM +/- B appears to be effective against refractory or recurrent lymphoma, resulting in response in a large number of patients and long-term survival and possible cure in a small but significant number. Results indicate that VIM +/- B is particularly effective in patients with high-grade NHL who have responded suboptimally to primary therapy. PMID- 1710487 TI - Long-term follow-up of ProMACE-CytaBOM in non-Hodgkin's lymphomas. AB - Initial results from studies with third-generation combination chemotherapy regimens for the treatment of aggressive non-Hodgkin's lymphomas (NHL) demonstrated complete remission (CR) rates higher than those reported with first generation regimens. Long-term follow-up of these studies is required to know if the increased number of CRs translates into an increased number of long-term disease-free survivors. In this report, results obtained with one of the third generation regimens, ProMACE (prednisone/methotrexate/doxorubicin/cyclophosphamide/etoposide)-Cyta BOM (cytarabine/bleomycin/vincristine/methotrexate) are described. From 1981 to 1988, 193 patients with stage II, III, or IV aggressive NHL treated at the National Cancer Institute were randomly assigned to receive either ProMACE (day 1)-CytaBOM (day 8) or ProMACE (day 1)-MOPP (mechlorethamine/vincristine/procarbazine/prednisone) (day 8). With a median follow-up of 5 years, the CR rate was 86% for ProMACE-CytaBOM v 74% for ProMACE MOPP (P = 0.048). A plateau in the disease-free survival curve is seen at 69% for ProMACE-CytaBOM v 54% for ProMACE-MOPP (P = 0.082). A plateau is also seen in the overall survival curves at 69% for ProMACE-CytaBOM v 53% for ProMACE-MOPP (P = 0.046). The Southwest Oncology Group also conducted a phase II study of ProMACE CytaBOM in 78 patients with stages II to IV intermediate- or high-grade NHL to determine the CR rate and long-term disease-free survival of this regimen in a national cooperative group setting. The CR rate was 65%. With a median follow-up of 38 months, disease-free survival is 50% at 3 years and overall survival is 57% at the same time point.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710488 TI - The place of third-generation regimens in the treatment of adult aggressive non Hodgkin's lymphoma. AB - Diffuse large cell lymphoma, the most common form of aggressive non-Hodgkin's lymphoma, has been known to be curable with combination chemotherapy for almost 20 years. Although occasional patients were cured with regimens like COP (cyclophosphamide/vincristine/prednisone) the development of four-drug regimens made cure possible in a significant percentage of patients. Subsequently, newer, so-called third-generation regimens incorporated even more drugs with an apparent increase in the cure rate. However, the identification of important clinical and biologic features in the patient or the tumor that predict for treatment outcome (prognostic factors) has complicated comparison between nonrandomized and nonconcurrent trials. Recently, the Southwest Oncology Group presented data from pilot studies showing that patients treated with ProMACE-CytaBOM (procarbazine/methotrexate/doxorubicin/cyclophosphamide/etoposide- cytarabine/bleomycin/vincristine/methotrexate), m-BACOD (methotrexate bleomycin/doxorubicin/cyclophosphamide/vincristine/dexa met hasone) , and MACOD-B (methotrexate/doxorubicin/cyclophosphamide/vincristine/dexamethasone- ble omycin) did not seem to do better than patients treated in the past with CHOP cyclophosphamide/doxorubicin/vincristine/prednisone). Also, the Eastern Cooperative Oncology Group has presented a study in which patients treated with a six-drug regimen did no better than those treated with a CHOP-like regimen. Some have interpreted these reports to mean that all patients with diffuse large cell lymphoma should receive CHOP, since they feel it to be safer. I believe this is not a good interpretation of the available data for the following reasons: (1) when administered at maximum tolerated doses, CHOP has the same approximate 5% treatment-related mortality seen with all regimens; (2) when CHOP is given at reduced (ie, safer) doses, it is not as effective; and (3) it is not yet clear that all patients with diffuse large cell lymphoma are the same in their response to treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710489 TI - Primary and salvage chemotherapy in advanced Hodgkin's disease: the Milan Cancer Institute experience. AB - This paper reviews the most relevant therapeutic results achieved by the Milan Cancer Institute through successive randomized studies in advanced Hodgkin's disease. The experience achieved confirms the therapeutic importance of an anthracycline-containing regimen such as ABVD (doxorubicin/bleomycin/vinblastine/dacarbazine) as salvage treatment and as primary chemotherapy, either when combined with irradiation or cyclically alternated with MOPP (mechlorethamine/vincristine/procarbazine/prednisone). Anthracycline-based combinations can be further refined to decrease toxicity. The biologic aspects of drug-sensitive v drug-resistant tumor cells and the accurate definition of prognostic categories at the time of first treatment failure are also discussed. PMID- 1710490 TI - Immunogenicity of epikeratophakia tissue lenses containing living donor keratocytes. AB - The purpose of this study was to compare the survival of epikeratophakia tissue lenses prepared with cryolathing and lyophilization (frozen lenses) and without (fresh lenses) in donor-sensitized recipients with vascularized corneas. Fresh lenses placed in vascularized corneas of immune recipients were subjected to immune attack. Frozen lenses placed in vascularized corneas of immunized recipients did not elicit an immune reaction. Neither fresh nor frozen lenses elicited immune reactions in nonvascularized corneas of immune recipients or in nonvascularized, nonimmune recipients. These results indicate that although the fresh lenses are more antigenic than the lenses in which the cells have been killed by freezing and lyophilization, the fresh lenses prepared using the BKS 1000 technique containing living stromal keratocytes are not likely to stimulate allograft immune reactions in unsensitized patients with avascular graft sites. PMID- 1710491 TI - [Molecular genetic analysis of neurologic diseases]. AB - With the recent progress in molecular genetics, our understanding of neurologic diseases on molecular basis has improved tremendously. Molecular analyses of gene mutations in hereditary neurologic diseases bring us not only the identification of mutations but also better understanding of molecular mechanisms involved in the neurologic diseases. We have identified a missense mutation (444Leu----Pro) in glucocerebrosidase gene of a type 2 Gaucher patient. The mutant glucocerebrosidase carrying 444Pro is unstable and degraded rapidly before the mutant protein is targetted to lysosomes. Detailed analysis on the 444Leu----Pro mutations among three phenotypes (types 1, 2 and 3) of Gaucher disease, it has been demonstrated that patients homozygous for the mutation frequently have the neurologic involvement (types 1 and 2). Very recently we have identified a mutation in tRNA(Lys) gene of mitochondrial genome of patients with myoclonus epilepsy associated with ragged-red fibers. The pathophysiologic mechanism with the tRNA(Lys) mutation is considered to be based on the impaired function of tRNA(Lys). PMID- 1710492 TI - [Detection of activated oncogenes in hematological malignancies using RT-PCR method and its clinical applications]. AB - Recently, a new and simple method of PCR has been developed and its application is extending widely including clinical research. We are using this method to detect various activated oncogenes within clinical samples from hematological malignancies. We now attempt to use these results as supporting evidences in clinical diagnosis. Here we report several successful results we have obtained through the study of activated RAS oncogenes. PMID- 1710493 TI - [Establishment and characterization of a human malignant fibrous histiocytoma cell line (K-MFH-1)]. AB - We established and characterized a cell line (K-MFH-1) of human malignant fibrous histiocytoma in vitro and in nude mice. The cells have been kept in culture for nearly 4 years in 121 subcultures in vitro and transferred serially in nude mice in 10 generations. The original tumor histology was epithelioid, angiomatoid variant of MFH, but cells in culture showed a variety of morphology. Chromosomal study disclosed that this tumor was monoclonal in origin. Electron microscopic study revealed that cells had some epithelial characteristics as well as histiofibroblastic features. Tumor cell morphology gradually shifted from epithelioid to myxomatous fibroblastic forms after serial transplantations in nude mice. Some tumors showed an extensive vascular proliferation like angioma suggesting that tumor cells have released angiogenic factors. PMID- 1710494 TI - Lipid-protein and protein-protein interactions in double recombinants of myelin proteolipid apoprotein and myelin basic protein with dimyristoylphosphatidylglycerol. AB - The integral proteolipid apoprotein (PLP) from bovine spinal cord has been reconstituted in dimyristoylphosphatidylglycerol (DMPG) bilayers, and the mutual interactions on binding the peripheral myelin basic protein (MBP) have been studied. Quantitation of protein and lipid contents in the MBP-PLP-DMPG double recombinants at different PLP:DMPG ratios led to the conclusion that MBP binds only to the DMPG lipid headgroups and is hindered from interaction with the first shell of lipids surrounding the PLP. No specific PLP-MBP association could be detected. Electron spin resonance (ESR) spectra of phosphatidylglycerol spin labeled at position n = 5 in the sn-2 chain showed that complexation of MBP with the PLP-DMPG recombinants leads to a decrease in lipid chain mobility to an extent which correlates with the degree of MBP binding. At low DMPG:PLP ratios, the perturbations of lipid mobility by both proteins are mutually enhanced. In single recombinants of PLP with DMPG, the ESR spectra of phosphatidylglycerol spin-labeled at position n = 14 in the sn-2 chain indicated that approximately 10 lipids/protein are motionally restricted by direct contact with the intramembranous surface of the protein. This number is in agreement with earlier results for reconstitutions of PLP in dimyristoylphosphatidylcholine (DMPC) [Brophy, P. J., Horvath, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865] and is consistent with a hexameric arrangement of the PLP molecules in DMPG bilayers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710495 TI - Evidence for a voltage-gated, non-selective cation channel in the human red cell membrane. AB - Using the patch clamp technique we have identified a voltage-dependent, non selective cation channel in the human red cell membrane. Basic properties of this channel are reported, and it is proposed that it may be involved in the increased transport of cations which is seen when intact human red cells are suspended in a depolarising media. PMID- 1710496 TI - An infrared spectroscopic study of specifically deuterated fatty-acyl methyl groups in phosphatidylcholine liposomes. AB - The region of the infrared spectrum corresponding to C-2H stretching vibrations (2050-2250 cm-1) has been examined for liposomes composed of dimyristoylphosphatidylcholine deuterated specifically at the methyl ends of either one (sn-2) or both the fatty acyl chains. This label is intended to provide information on lipid dynamics in the contact region between monolayers. The two most prominent bands observed correspond, respectively, to antisymmetric (2212 cm-1) and symmetric (2075 cm-1) C-2H stretching vibration. The antisymmetric band consists of two overlapping peaks, whose positions vary with the gel or liquid-crystalline state of the lipid. The separation between the peaks making up the antisymmetric band increases with temperature, and is maximum above the Tc transition temperature; this rules out the previously proposed assignment of these two peaks to different rotational modes of the methyl group relative to the adjacent methylene. The position and width of the symmetric band at 2075 cm-1 are also sensitive to the physical state of the lipid. The presence of cholesterol at an equimolar ratio with the phospholipid abolishes all the phase-dependent changes observed. The intrinsic polypeptide gramicidin A, at a 5:1 lipid/peptide mol ratio, is seen to enlarge the lipid thermotropic transition, with small effects above Tc. Cytochrome c, an extrinsic protein, at a 10:1 mole ratio, does not modify the phase-dependent behaviour of the terminal methyl groups, but consistently shifts all the observed bands to lower-frequency positions, which suggests a long-range effect of the protein along the phospholipid fatty acyl chains. PMID- 1710497 TI - Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains. AB - Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation. PMID- 1710498 TI - The manipulation of DNA with restriction enzymes in low water systems. AB - The cleavage of phage lambda (lambda) DNA by the restriction enzyme HindIII in low water systems has been investigated. Two types of low water systems have been studied--those which contain a surfactant in a reverse micelle environment and a surfactant-free system in which a solid support (celite) is used. The effect of the surfactants themselves in a normal aqueous environment has also been studied. Charged surfactants were found to greatly inhibit HindIII activity in aqueous buffer, while non-ionic surfactants did not affect either the activity or the specificity of the restriction enzyme. The rate of cleavage by HindIII in a reverse micelle system consisting of sodium dioctylsulphosuccinate is very slow, however, in a Triton B system the expected fragments are observed. In a surfactant-free low water environment, cleavage occurs at the expected sites but in a different order to that observed in normal aqueous systems. These results suggest that DNA tertiary structure in low water systems is different to that in aqueous solution and that this influences cleavage by the restriction enzyme HindIII. PMID- 1710499 TI - Serotonin in the control of blood pressure and ultrafiltration in CAPD. AB - Urinary 5-OH-Indoloacetic acid (5-HIAA) indicates the turnover of almost all circulating serotonin (5-HT), a potent vasoactive agent. We tried to investigate the possible excretion of 5-HIAA in the dialysate of CAPD patients and its possible effects on blood pressure control (BP) and/or ultrafiltration (UF). The 24 h excretion of 5-HIAA in the urine of 10 patients with chronic renal failure (CRF) and 8 healthy persons (CONTROL), and in the dialysate of 15 patients on CAPD was measured by FPIA. In CRF 5-HIAA excretion was less than that in control (p less than 0.01). In CAPD patients two distinct patterns of 5-HIAA excretion were found. Patients with high excretion (greater than 6 mg) had high BP and low UF, while those with low excretion (less than 3.14) had low BP, high UF and frequent hypotensive episodes. Excretion of 5-HIAA in the dialysate had a negative correlation with UF (r = -0.779, p less than 0.001) and a positive correlation with B.P. (r = 0.487, n.s.). A highly significant (p less than 0.01) correlation between BP and UF was also found. As no differences between the two groups in factors that could affect UF or BP were found, the implication that 5 HT affects ultrafiltration and/or BP regulation is suggested. PMID- 1710500 TI - The role of nutrition in neurologic health and development of infants with chronic renal failure. PMID- 1710501 TI - Gastric, esophageal, and pancreatic cancer, and rare tumors. PMID- 1710502 TI - Cancer pain. PMID- 1710503 TI - Supportive care in patients with cancer: quality of life and ethical issues. PMID- 1710504 TI - Supportive care. PMID- 1710505 TI - Recent advances in biology and treatment of myelodysplasia. AB - The myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by varying degrees of pancytopenia and often a progression to acute myeloid leukemia. Recent evidence has linked myelodysplastic syndromes to environmental and occupational genotoxic exposure. Specific cytogenetic abnormalities are well described in myelodysplastic syndromes and have been demonstrated to be useful diagnostic and prognostic tools. Activation of protooncogenes such as ras and fms have also been noted in myelodysplastic syndromes; however, their contribution to the pathogenesis of the syndrome remains to be determined. Aggressive leukemia-like induction therapy, differentiating agents (low-dose cytarabine, 13-cis-retinoic acid) have had little impact on overall survival in myelodysplastic syndromes. The recombinant hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor) may be of significant benefit to patients with myelodysplastic syndromes, although it remains to be determined whether they will have a substantial impact on survival. Allogeneic bone marrow transplantation is the only potentially curable treatment of myelodysplastic syndromes. The advanced age of these patients as well as the lack of histocompatible donors restricts this modality to only a small proportion of patients. PMID- 1710506 TI - Chronic lymphocytic leukemia and hairy-cell leukemia. AB - The chronic lymphoid leukemias are a heterogeneous group of disorders with different immunologic, biologic, and clinical features. The most common of these are the B-cell diseases, chronic lymphocytic leukemia and its variants, including prolymphocytic leukemia and hairy cell leukemia. The increased use of immunophenotyping has identified a number of other less common but related disorders. Despite being clonal disorders, the chronic B-cell leukemias exhibit immunologic abnormalities in multiple other lineages, the mechanism for which is not clear. Fludarabine, 2'-deoxycoformycin, and 2-chlorodeoxyadenosine are purine analogues that have advanced the treatment of chronic B-cell leukemias. Fludarabine appears to be the single most effective agent for chronic lymphocytic leukemia, while 2'-deoxycoformycin and 2-chlorodeoxyadenosine are both extremely effective in hairy cell leukemia. A recently completed comparison of alpha interferon with 2'-deoxycoformycin in hairy cell leukemia may redefine the standard therapy for this disorder. Continued interaction between laboratory and clinical scientists is essential for continued progress in these diseases. PMID- 1710507 TI - [Detection of human immunodeficiency virus in seronegative adults]. AB - The diagnosis of human immunodeficiency virus infection is sometimes difficult or nonspecific, both in early and late stages, as the patients may be seronegative at the time of testing. Although serologic testing usually suffices to identify infected individuals and to follow up the course of the infection, in some cases direct detection of the virus is required. The culture of the mononuclear cells of the patient permits, after stimulation with mitogens and interleukin-2, the expression of viral antigens even in asymptomatic patients with latent or apparently nonproductive infection. In this way we have recovered the virus in four patients without serological evidence of infection. The possibility that human immunodeficiency virus infection can be undetectable with the usual diagnostic techniques, at least in a small proportion of patients, supports the need to use other methods such as direct viral culture to permit the identification of a greater number of infected individuals and the adoption of the appropriate prophylactic or therapeutic measures. PMID- 1710508 TI - Two regions of M13 phage genome hybridizing with human DNA are similar to several keratin genes. AB - DNA from two regions of the phage M13 genome hybridizes with DNA restriction fragments from genomes of various species including man [15, 20]. As the pattern of hybridization is individual-specific, this phage M13 probe can be used for DNA fingerprinting. We demonstrate here that the regions of many keratin genes coding for glycine-rich parts of C and N end domains are very similar to the phage M13 probe, and this similarity may be responsible for hybridization. PMID- 1710509 TI - Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse oocytes. AB - The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1 1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II. PMID- 1710510 TI - Ovarian intrabursal administration of insulin-like growth factor-binding protein inhibits follicle rupture in gonadotropin-treated immature female rats. AB - The effects of an insulin-like growth factor-binding protein (IGF-BP) on rat follicular function were examined by using the technique of ovarian intrabursal (IB) injection. Immature female rats were injected with 15 IU of eCG followed immediately with IB injections of 4 micrograms IGF-BP3 (right ovary) and vehicle (left ovary). Forty-eight hours later, the same animals were either killed (eCG treated group) or injected with 1 microgram of hCG as an ovulatory stimulus. These animals were killed 24 h later (eCG/hCG-treated group). Intrabursal administration of IGF-BP3 inhibited ovulations in the eCG/hCG-treated rats by 55% when compared with the contralateral vehicle-treated ovary (p = 0.01). Examination of the ovaries exposed to IGF-BP3 revealed the presence of unruptured follicles containing a matured oocyte and a disintegrated basement membrane, in addition to normal follicles and corpora lutea. In contrast, IB injection of IGF BP3 had no effect on ovarian weights or circulating estradiol concentrations in the eCG-treated animals, and the ovaries appeared to be morphologically normal. Ligand blotting experiments using [125I]-labeled insulin-like growth factor I revealed that granulosa cells obtained from both untreated and eCG-treated rats synthesized and secreted two IGF-BPs of Mr 35,000 and 30,000. Equine chorionic gonadotropin treatment reduced the amount of the 30,000 Mr form of IGF-BP. These data suggest that locally produced ovarian IGF-BPs may modulate follicle functions in vivo. PMID- 1710511 TI - Ovarian surface epithelium: autonomous production of connective tissue-type extracellular matrix. AB - The ovarian surface epithelium (OSE) is known to contribute to postovulatory repair of the ovarian cortex by proliferation and migration over the site of follicular rupture, and by deposition of a basement membrane. We examined the production of other extracellular matrix components in culture by OSE cells of the rat (ROSE), using immunofluorescence microscopy, electron microscopy, and proline incorporation. We compared recently explanted cells in low passage, the immortal line ROSE 239, whose growth pattern resembles low passage cultures, and the immortal line ROSE 199, which forms ridges and papillae. The epithelial nature of all three cell types was confirmed by the presence of keratin and laminin. All three cell types secreted collagen types I and III and at least one (ROSE 199) produced highly polymerized banded fibrils, which are characteristic for stromal or interstitial extracellular matrix. Simultaneous production of collagen types I and III, keratin, and laminin by cloned subpopulations ruled out an origin of the lines in mixed epithelial/fibroblast populations. The results demonstrate that OSE has the capacity to synthesize major components of connective tissue stroma. They suggest that this epithelium, in addition to its postulated proteolytic role, may also express synthetic activity in the remodelling of the ovarian cortical stroma. A capacity of OSE cells to produce stromal components autonomously might be an important factor in the formation of ovarian surface papillae and in neoplastic progression of OSE-derived ovarian carcinomas. PMID- 1710512 TI - Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry. AB - Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM. PMID- 1710513 TI - Stimulatory effects of granulocyte colony-stimulating factor on colony-forming units-spleen (CFU-S) differentiation and pre-CFU-S proliferation in mice. AB - Granulocyte colony-stimulating factor (G-CSF) was reported to increase the number of colony-forming units-spleen (CFU-S) and multilineage colonies as well as myeloid-committed cells. We investigated the effects of G-CSF on myeloid progenitors and primitive stem cells in a mouse bone marrow transplantation (BMT) system. Lethally irradiated mice received BM cells from untreated or 5 fluorouracil-treated mice, and then were administered G-CSF or carrier buffer (control) for 5 days from immediately after BMT. A pre-CFU-S assay was performed by the repeated transplantation of BM cells from the first BMT recipients to other mice. By the method of polymerase chain reaction, most of the spleen colonies in the secondary recipients were confirmed to be derived from the first donors. G-CSF did not increase the peripheral white blood cell count significantly, but did increase the number of immature myeloid cells and granulocyte-macrophage colony-forming cells in the BM. The number of erythroid cells in the BM was initially suppressed and then increased by G-CSF treatment. In addition, the pre-CFU-S assay showed an increase in pre-CFU-S cells due to G CSF administration. The number of spleen colonies of first BMT recipients did not increase, but a higher percentage of them were committed to a certain lineage by G-CSF treatment. These findings suggest that G-CSF has important roles in the early stages of hematopoiesis. PMID- 1710514 TI - Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7. AB - We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7-producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis. PMID- 1710515 TI - Epithelial membrane glycoprotein PAS-IV is related to platelet glycoprotein IIIb binding to thrombospondin but not to malaria-infected erythrocytes. AB - Glycoprotein (GP) IIIb (also termed GPIV or CD36) is an integral platelet membrane protein, and has been identified as a binding site for thrombospondin, collagen, and malaria-infected erythrocytes. PAS-IV is an integral membrane protein found in lactating mammary epithelial cells and capillary endothelial cells. The N-terminal sequence of PAS-IV is nearly identical to that of GPIIIb and monospecific anti-PAS-IV antibody reacts with GPIIIb, indicating that PAS-IV is structurally related to GPIIIb. In this study, human platelet GPIIIb and bovine epithelial PAS-IV were compared in terms of structural, immunologic, and functional characteristics. The two-dimensional tryptic peptide map of both intact and deglycosylated PAS-IV was highly similar but not identical to that of GPIIIb. PAS-IV and GPIIIb reacted to an equal extent with monoclonal antibodies OKM5 and OKM8 by enzyme-linked immunosorbent assay. GPIIIb bound to surface immobilized thrombospondin (TSP) in a concentration-dependent and saturable manner, with approximately 60% reduction in binding in the presence of EDTA. PAS IV bound to TSP with similar characteristics except that maximum binding was consistently approximately 50% of that of GPIIIb and binding was not inhibited by EDTA. GPIIIb supported adhesion of Plasmodium falciparum-infected erythrocytes (PRBC) in a dose-dependent manner while no significant adhesion of PRBC to PAS-IV was observed. Our data demonstrate that while epithelial PAS-IV and platelet GPIIIb are structurally and immunologically related, there are significant differences in their functional properties. Whether this result is due to different posttranslational glycosylation modifications or that PAS-IV and GPIIIb represent a family of related cell adhesive protein receptors remains to be determined. PMID- 1710516 TI - Alpha 2-macroglobulin-kallikrein complexes detect contact system activation in hereditary angioedema and human sepsis. AB - Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2-macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M-Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks. PMID- 1710517 TI - Characteristics of quinine- and quinidine-induced antibodies specific for platelet glycoproteins IIb and IIIa. AB - Recent studies have shown that antibodies characteristic of quinine- and quinidine-induced thrombocytopenia sometimes recognize the platelet membrane glycoprotein (GP) complex IIb/IIIa in addition to their well known target, GPIb/IX. We have investigated the frequency with which drug-induced antibodies bind to GPIIb/IIIa and the nature of their target epitopes. In studies of sera from 13 patients sensitive to quinidine or quinine, we found that 10 contained IgG antibodies specific for both GPIb/IX and GPIIb/IIIa, two reacted with GPIb/IX alone, and one reacted with GPIIb/IIIa alone. In all cases, the presence of drug was required for binding of IgG to target GPs. By immunoabsorption, we found that each of five polyspecific sera contained at least two different antibodies, one reactive with GPb/IX and the other with GPIIb/IIIa. Further studies with eight drug-dependent antibodies (DDAb) specific for GPIIb/IIIa showed that three recognized the GPIIb/IIIa complex only, one recognized GPIIb alone, and three recognized GPIIIa alone. The eighth serum appeared to bind to both GPIIIa alone and to an epitope determined by the GPIIb/IIIa complex. The three antibodies specific for GPIIIa alone also reacted with GPIIIa deglycosylated with endo-H, and with the major (61 Kd) fragment obtained by chymotryptic digestion of GPIIIa but failed to react with reduced GPIIIa. These findings demonstrate that, in drug induced, immunologic thrombocytopenia, the anti-platelet immune response is typically directed against epitopes on both GPIb/IX and GPIIb/IIIa. The three DDAb we studied that were specific for GPIIIa alone recognize epitopes resistant to chymotrypsin and endo-H treatment that are dependent on intrachain disulfide bonding. PMID- 1710518 TI - Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity. AB - Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII. PMID- 1710519 TI - Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement. AB - To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement. PMID- 1710520 TI - Appraisal of a new scheme for prenatal screening for Down's syndrome. AB - OBJECTIVE: To appraise a new method of prenatal screening for Down's syndrome based on maternal serum concentrations of alpha fetoprotein, unconjugated oestriol, and human chorionic gonadotrophin combined with maternal age--the "triple test." DESIGN: Examination of the cost effectiveness of the triple test relative to screening only by maternal age over a range of population detection rates. SETTING: Leicestershire Health Authority. MAIN OUTCOME MEASURES: Costs per affected fetus detected. RESULTS: The triple test is more cost effective than screening only by maternal age for risk cut off points for amniocentesis, resulting in a detection rate over 45%. The most efficient detection rate is around 60-65%, for which the cost per case detected is around 29,000 pounds, through screening with higher detection rates is still likely to be cost beneficial. CONCLUSIONS: Prenatal screening for Down's syndrome based on the triple test should replace screening based only on maternal age. Individual women's preferences should be elicited by the use of structured decision analysis in order to maximise utility and so increase the benefits of the screening programme. PMID- 1710521 TI - Biochemical screening for Down's syndrome. PMID- 1710522 TI - Human hemopoietic progenitors and their signals. AB - A number of recent advances have resulted in the definition of human pluripotent hemopoietic progenitors that can be considered candidates for stem cells. These cells may be CD34 positive and HLA-DR negative. They are capable of producing colonies composed of cells with blast-like morphology and re-establish long term bone marrow cultures when placed on irradiated stromal layers. These cells appear to interact at short range with stromal cells and become responsive to signals that initiate their proliferation and differentiation. These signals can be provided by interleukins produced by various auxiliary hemopoietic cell populations and may act over long distances. In addition, these interleukins may be aberrantly produced by certain malignant hemopoietic cells. This may result in paramoplastic auto- or paracrine growth promotion of the underlying malignancy and occasionally in the development of paraneoplastic syndromes. PMID- 1710523 TI - Modes of hexamethonium action on acetylcholine receptor channels in frog skeletal muscle. AB - 1. The antagonism between hexamethonium and cholinoceptor agonists was investigated in frog skeletal muscle fibres with voltage-clamp techniques. Hexamethonium caused a voltage-dependent reduction in the amplitude of endplate currents. For neurally evoked endplate currents, the reduction increased e-fold with a 38 mV membrane hyperpolarization. 2. The effect of hexamethonium on the time course of endplate currents was small, and was most apparent as a slight prolongation of the decay phase at hyperpolarized potentials (more negative than 100 mV). A similar small prolongation of single channel lifetime was detected with fluctuation analysis techniques. Hexamethonium produced a voltage-dependent reduction in apparent single channel conductance as the membrane was hyperpolarized. 3. Log (concentration-response) curves for acetylcholine (ACh) induced currents, determined either from currents accompanying ramp changes in membrane potential or from steady state currents in voltage-jump experiments, were less steep for responses in the presence of hexamethonium. This reduction in slope became more pronounced at more negative membrane potentials. Observations at +50 mV suggested that the equilibrium constant for competitive antagonism was approximately 200 microM. 4. In voltage-jump experiments with a two microelectrode voltage clamp, the current evoked by ACh in the presence of hexamethonium differed from that recorded with ACh alone. In the presence of hexamethonium, the expected 'instantaneous' ohmic increase in membrane current in response to a hyperpolarizing step was not detected; instead a decrease in current was observed. This problem was further investigated with a vaseline-gap voltage-clamp technique which provides improved temporal resolution. With this method a rapid decrease in the ACh-induced inward current was observed with step hyperpolarizations in the presence of hexamethonium. 5. When the membrane potential was stepped back to its resting level from a more hyperpolarized potential in the presence of hexamethonium, there was a surge of ACh-induced inward current that decayed with a time constant of less than 100 microseconds. 6. The slow relaxation in the ACh-induced current that followed a voltage step recorded in the presence of hexamethonium was slower than that recorded with ACh alone. In the presence of hexamethonium the time constant of this relaxation increased e-fold for a 67 mV hyperpolarization. 7. The results are consistent with a rapid voltage-dependent block of ACh-activated channels by hexamethonium with hyperpolarization, and voltage-dependent unblock with depolarization. The voltagedependent block is combined with competitive antagonism at the ACh receptors. However, not all observations appear to be compatible with a simple sequential block of open ion channels, but rather suggest that occupation of the channel by hexamethonium may not prevent channel closure. PMID- 1710524 TI - Effects of a novel smooth muscle relaxant, KT-362, on contraction and cytosolic Ca2+ level in the rat aorta. AB - 1. Inhibitory effects of a novel smooth muscle relaxant, KT-362 (5-[3-([2-(3,4 dimethoxyphenyl)-ethyl]amino)-1-oxopropyl]-2,3,4,5- tetrahydro-1,5 benzothiazepine fumarate), on contraction and the cytosolic Ca2+ level ([Ca2+]cyt) in isolated vascular smooth muscle of rat aorta were examined. 2. KT 362 inhibited the contractions induced by high K+ and noradrenaline. The inhibitory effect was antagonized by an increase in external Ca2+ concentration. A Ca2+ channel activator, Bay K 8644, did not change the effect of KT-362 on high K+-induced contraction. 3. [Ca2+]cyt, measured with fura-2-Ca2+ fluorescence, increased during the contractions induced by high K+ or noradrenaline. KT-362 decreased [Ca2+]cyt and muscle tension stimulated by high K+ or noradrenaline. By contrast, a Ca2+ channel blocker, verapamil, inhibited the noradrenaline-induced increase in [Ca2+]cyt with only partial inhibition of the noradrenaline-induced contraction and KT-362 inhibited the verapamil-insensitive portion of the contraction without changing [Ca2+]cyt. 4. In a Ca2(+)-free solution, noradrenaline and caffeine induced a transient contraction following a transient increase in [Ca2+]cyt. KT-362 inhibited the increments due to noradrenaline but not those induced by caffeine. 5. These results suggest that KT-362 inhibits vascular smooth muscle contraction by inhibiting Ca2+ channels, receptor-mediated Ca2+ mobilization, and receptor-mediated Ca2+ sensitization of contractile elements. PMID- 1710525 TI - Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. AB - 1. The synthesis of nitric oxide (NO) from L-arginine by rat peritoneal neutrophils (PMN) and the murine macrophage cell-line J774 and the inhibition of this synthesis by N-iminoethyl-L-ornithine (L-NIO), NG-monomethyl-L-arginine (L NMMA), NG-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) were investigated. 2. L-NIO was the most potent inhibitor in both types of cells while L-NMMA was less active. L-NNA and L-NAME had no significant effect in PMN and L NNA produced only approximately 40% inhibition of the generation of NO in the J774 cells at the highest concentration tested (300 microM). 3. The inhibitory effect of L-NIO was rapid in onset, requiring 10 min pre-incubation to achieve its full inhibitory activity, while the other compounds required 20-60 min pre incubation to achieve their full effect. 4. The inhibitory effect of L-NIO (10 microM) on intact cells could not be reversed by L-arginine (300 microM) but could be prevented by concomitant incubation with this compound (300 microM), while the effect of the other inhibitors could be reversed by a 3-5 fold molar excess of L-arginine. 5. The NO synthase from both PMN and J774 cells was cytosolic and NADPH- but not Ca2(+)-dependent, with Km values for L-arginine of 3.3 +/- 0.8 and 4.2 +/- 1.1 microM respectively. 6. L-NIO was the most potent inhibitor of the neutrophil and J774 enzymes with IC50 values of 0.8 +/- 0.1 and 3 +/- 0.5 microM respectively. Furthermore, the effect of L-NIO was irreversible. The other three compounds were less potent, reversible inhibitors. 7. The inhibitory effects of all these compounds were enantiomerically specific. 8. These data indicate that L-NIO is a novel, potent, rapid in onset and irreversible inhibitor of NO synthase in phagocytic cells. The rapid uptake of L NIO compared with the other compounds indicates that phagocytic cells have different uptake mechanisms for L-arginine analogues. PMID- 1710526 TI - Octimibate, a potent non-prostanoid inhibitor of platelet aggregation, acts via the prostacyclin receptor. AB - 1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP inhibited phosphodiesterase (phosphodiesterase-ITI), which is the major cyclic AMP-degrading enzyme in human platelets.5. Octimibate inhibits, apparently competitively, the binding of [3H]-iloprost (a stable PGl2 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6. The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fold with no obvious difference between primates and non-primates. 7. Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non-prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes. PMID- 1710527 TI - The effects of calcitonin gene-related peptide on submucosal gland secretion and epithelial albumin transport in the ferret trachea in vitro. AB - 1. We have examined the effect of calcitonin gene-related peptide (CGRP) on basal mucus volume, lysozyme and albumin outputs from the ferret whole trachea in vitro, and on the outputs produced by methacholine and substance P (SP). We have also examined the effect of inhibiting neutral enkephalinase with thiorphan on the responses to CGRP. 2. CGRP (1-100 nM) produced small concentration-dependent increases in basal mucus volume, lysozyme and albumin outputs. These effect of CGRP were enhanced by thiorphan. The increases in basal outputs with CGRP and the potentiation by thiorphan were considerably less than previously observed with SP and neurokinin A (NKA). CGRP had no significant effect on potential difference (PD) across the trachea. 3. CGRP produced a concentration-dependent inhibition of methacholine- and SP-induced lysozyme output but a concentration-dependent increase in methacholine- and SP-induced albumin output. The effects of CGRP on methacholine-induced lysozyme and albumin outputs were enhanced by thiorphan. CGRP weakly inhibited methacholine-induced mucus volume output and weakly enhanced SP-induced mucus volume output. 4. Thus, CGRP weakly stimulates basal serous cell secretion and epithelial albumin transport, but does not alter epithelial integrity. CGRP inhibits the serous cell secretion due to methacholine or SP, but potentiates the epithelial albumin transport produced by these agents. The interaction between CGRP and other sensory neuropeptides or muscarinic agonists on airway submucosal glands and epithelium may be important in the normal airway and in inflammatory airway diseases where release of sensory neuropeptides is enhanced. PMID- 1710529 TI - IgE binding studies with large peptides expressed from Der p II cDNA constructs. AB - The major mite allergen Der p II shows marked resistance to denaturation and is expressed from cDNA in bacteria with almost all of its IgE binding activity. Despite these properties, the IgE binding activity appears to be dependent on maintaining the complete primary structure. Random fragment libraries of cDNA, able to code for up to 93 of the 129 amino acid residue protein, did not express IgE binding peptides. Large overlapping peptides 1-69, 69-129 and 42-117 expressed as the fusions from the glutathione transferase of pGEX vectors only had binding activity with IgE in 15 out of 57 sera, and this was typically weak. Sera from children with atopic dermatitis bound IgE in seven out of eight cases but this was also weak compared with their strong reactivity to intact recombinant Der p II. The inability of such large peptides to form IgE binding structures suggests that the antigenic determinants of Der p II are highly conformational and restricted. PMID- 1710528 TI - Experimental vitamin E deficiency in rats. Morphological and functional evidence of abnormal axonal transport secondary to free radical damage. AB - Morphological and functional studies have been performed on experimental vitamin E deficient rats. The predominant morphological change was axonal dystrophy and degeneration in the rostral parts of the dorsal columns, particularly in the gracile fasciculi. The dystrophic changes comprised focal axonal swellings containing accumulations of normal and abnormal organelles which included tubulovesicular structures probably derived from the smooth endoplasmic reticulum, mitochondria, dense lamellar bodies, neurofilaments, multifascicular bodies and lysosomes. Similar but lesser changes were observed in distal peripheral nerves. The appearances suggested a disturbance of axonal transport with a defect of 'turnaround' in the distal axons. Studies on the axonal transport of endogenous acetylcholinesterase showed an impairment both of fast anterograde and retrograde transport. The changes were considered to be secondary to the lack of the antioxidant effect of vitamin E as the neurological deficits could be reduced by the concomitant dietary administration of the synthetic antioxidant ethoxyquin and were markedly aggravated by the administration of polyunsaturated fatty acids. It is suggested that the neurological syndrome produced by vitamin E deficiency could be the result of damage to the function of mitochondria and other intra-axonal membranous structures which would interfer both with fast anterograde transport and 'turnaround' and lead to a distal axonal degeneration. PMID- 1710530 TI - Structural and secretory characteristics of bovine lung and skin mast cells: evidence for the existence of heterogeneity. AB - We have examined cells dispersed enzymatically from three different sites in the bovine lung (tracheal mucosa, bronchial mucosa and parenchyma) and the skin, in order to ascertain whether the bovine model could be used to study mast cell heterogeneity. Histochemically there were two sub-populations of mast cells present in both lung and skin (on the basis of toluidine blue staining and the sensitivity to formalin fixation), but their proportions were similar in all sites studied. Skin mast cells contained approximately twice the amount of histamine than their counterparts in the lung (P less than 0.05). Functional heterogeneity was examined by in vitro release of histamine following secretagogue challenge. Calcium ionophore induced a substantial release of histamine; skin mast cells releasing significantly more histamine than any of the lung mast cells (at 10 microM ionophore, 37.1% and 20.7% net histamine release, respectively, P less than 0.05), although the time-course of release from the two tissues was similar. The neuropeptides vasoactive intestinal peptide and somatostatin induced a modest but statistically significant release of histamine from both skin and lung mast cells, whilst substance P only induced histamine secretion from skin mast cells. A range of other potential immunological and non immunological secretagogues was unsuccessful in eliciting histamine release from mast cells in any of the tissues. We conclude that there were no convincing histochemical differences between mast cells from the sites examined in the lung or skin. Additionally, there was no discernable functional heterogeneity between mast cells within the lung, but functional differences were evident between mast cells of the bovine lung and skin. However, in the absence of a suitable immunological stimulus the bovine model cannot be regarded as a good model of mast cell heterogeneity. PMID- 1710531 TI - The American Cancer Society National Prostate Cancer Detection Project. Findings on the detection of early prostate cancer in 2425 men. AB - The American Cancer Society National Prostate Cancer Detection Project (ACS NPCDP) is a multidisciplinary, multicenter effort to assess the feasibility of early prostate cancer detection by digital rectal examination (DRE), transrectal ultrasound (TRUS), and prostate specific antigen (PSA) assay. By June 1990, 2425 men not previously suspected of having prostate cancer had been examined in ten participating clinical centers according to the project protocol. Three hundred ninety-six men (16.3%) were recommended for biopsy on the basis of TRUS or DRE. An analysis of the results of 330 completed biopsies showed 52 cancers detected by DRE and/or TRUS. Forty-four (84.6%) of the men with cancer had positive TRUS examination results compared with 33 (63.5%) with positive DRE. Five additional cancers were discovered as a result of elevated PSA levels. The overall detection rate was 2.4% and this rate varied by age. The detection rate in men 55 to 60 years of age was 1.3% and this rose to 3.3% in men older than 65 years of age. The estimated sensitivity was significantly greater for TRUS compared with DRE (77.2% versus 57.9%; P less than 0.05). The estimated specificity of DRE was greater than that of TRUS (96.3% versus 89.4%; P less than 0.01). The positive predictive value (PPV) for the tests varied as a function of patient and disease characteristics. The overall PPV was 28.0% for DRE and 15.2% for TRUS. The occurrence of elevated PSA levels significantly increased the PPV of both TRUS and DRE. The majority of cancers detected were at early stages. These preliminary data suggest the feasibility of using these techniques to promote cancer control, but additional data and follow-up are needed to assess the significance of the results. PMID- 1710532 TI - Prognostic significance of basement membrane deposition in operable squamous cell carcinomas of the lung. AB - Tumor histology, stage of disease, and performance status are the most important prognostic factors in squamous cell lung cancer (SqCLC). A potentially important descriptor is the pattern of tumor growth as reflected in the dissolution of preexisting or deposition of newly formed basement membrane (BM) around tumor cell nests. The possible correlation between the pattern of BM deposition and patient survival was investigated. Immunohistochemistry testing, using polyclonal antibodies to human type IV collagen, was done on tumor samples of 68 patients with operable Stages I or II SqCLC, and BM staining was scored semiquantitatively. More than 75% immunoreactivity was scored as extensive BM and less than 75% as moderate or limited BM. In six patients, no immunoreactivity could be detected. In 27 of 62 patients, extensive and, in 35 of 62 patients, moderate-limited BM deposition was found. This deposition had a significant effect on survival (P = 0.02). Cox regression analysis, including BM deposition and tumor stage, indicated that BM deposition might also have value as an additional independent prognostic indicator for survival (P = 0.02). Deposition of an appreciable amount of BM in the center of the SqCLC was a prognostically favorable sign, independent of tumor stage. PMID- 1710533 TI - DNA ploidy and prostate-specific antigen as prognostic factors in clinically resectable prostate cancer. AB - Prostate-specific antigen (PSA) and DNA ploidy as measured by flow cytometry were compared with conventional prognostic indicators in 112 patients who underwent radical prostatectomy for clinically resectable prostate cancer. The variables examined included age, race, prostatic acid phosphatase (PAP), Gleason score of the radical prostatectomy specimen, and pathologic stage. No significant relationships were found between DNA ploidy and age, mean PAP value, and absolute PAP value. Of the 112 patients, 65 (58.0%) had disease limited to the prostate (pathologic Stages A and B); 47 (42.0%) had extraprostatic disease (pathologic Stages C and D1). The stage was related to the Gleason score (P less than 0.0001) where extraprostatic disease was associated with a Gleason score of 6 to 10. Nineteen (17.0%) patients had aneuploid tumors, and 93 (83.0%) had diploid tumors. DNA ploidy significantly correlated with pathologic stage (P = 0.04); aneuploidy was identified more frequently in patients with Stages C and D1 tumors. Aneuploid tumors occurred more frequently than diploid tumors in patients with a Gleason score of 6 to 10 (P = 0.034). Mean PSA values were higher in patients with aneuploid tumors (P = 0.078), extraprostatic neoplasms (P = 0.00001), and cancers with a Gleason score of 6 to 10 (P = 0.0004). Furthermore, PSA values greater than 10.0 ng/ml were associated with extraprostatic disease and a Gleason score of 6 to 10 (P less than 0.05 and P less than 0.001, respectively). Significant racial differences were found with respect to DNA ploidy, mean DNA indices, and mean PSA values. The 18 black patients had more DNA aneuploid tumors (P = 0.043), a higher mean DNA index (P = 0.017), and a higher mean PSA value (P = 0.043) than the 94 white patients. Both PSA and DNA ploidy analysis by flow cytometry appear to be valuable indicators in the evaluation of patients with prostatic carcinoma. PMID- 1710534 TI - ABO(H) antigens and beta-2 microglobulin in transitional cell carcinoma. Predictors of response to intravesical bacillus Calmette-Guerin. AB - The response of patients with superficial transitional cell carcinoma of the bladder (STCB) to intravesical chemotherapy is variable; some patients enjoy a long period without recurrence, whereas others have recurrence of tumor within 2 years of removal of the primary lesion. Previously, others have demonstrated that the loss of normal cell surface antigens, such as ABO(H) blood group antigens or beta-2 microglobulin (B2M) has been correlated with more aggressive behavior by tumor. In this study, using immunohistochemical techniques, the authors evaluated the initial pretreatment biopsy specimen of bladder tumors for the presence of ABO(H) antigens and B2M. Data from this sample patient population, all with biopsy-proven STCB, indicate that expression of these two markers is predictive of a therapeutic response to prophylactic intravesical bacillus Calmette-Guerin (BCG) (Tice strain) after resection, and that expression of the two markers is of greater predictive value than expression of either antigen alone. PMID- 1710535 TI - Immunohistochemical study of thyroid peroxidase in normal, hyperplastic, and neoplastic human thyroid tissues. AB - An immunohistochemical study using two monoclonal antibodies (MoAb 30 and MoAb 47) against thyroid peroxidase (TPO) was performed on surgical specimens of human thyroid carcinoma (n = 65), adenoma (n = 70) and Graves' disease (n = 10). Normal adjacent thyroid tissue was used as positive control. Monoclonal antibody 30 reacted significantly with all adenoma and most carcinoma, whereas MoAb 47 reacted with 66 adenoma but only two carcinoma. Of the four adenomas that did not react with MoAb 47, three were of the fetal type. Both carcinoma reacting with MoAb 47 were of the well-differentiated follicular type. These findings further confirm the hypothesis that thyroid carcinoma is associated with changes in the quantity and antigenic properties of TPO. Although the alterations in antigenic behavior revealed by MoAb 47 are not 100% specific, they may allow more accurate diagnosis of malignancy in thyroid tumors. PMID- 1710536 TI - Hepatoid adenocarcinoma of the renal pelvis producing alpha-fetoprotein of hepatic type and bile pigment. AB - A right renal pelvic mass in a 72-year-old man was resected. The histologic appearance of the tumor was a mixture of tubular adenocarcinoma cells and hepatoid neoplastic cells, and there was a resemblance to hepatoid adenocarcinoma. The intraoperative level of serum alpha-fetoprotein (AFP) was calculated to be 2246 ng/ml, and the postoperative level ranges from 183.6 to 285.6 ng/ml. Lectin binding assays showed that the serum AFP was the hepatic carcinoma type. In a hepatoid portion, an iron-negative, brown to green pigment was positive for bile. Alpha-fetoprotein was immunohistochemically evident in the neoplastic cells. In addition to the hepatic differentiation, the tumor had differentiated into intestinal absorptive or pancreatobiliary tract cells, as deduced from the frequent presence of spicular bodies, a unique light microscopic feature equivalent to microvilli with an actin core. The hepatoid adenocarcinoma is a distinct type of AFP-producing carcinoma present in the organs with epithelium of endodermal origin. Hepatoid adenocarcinoma in the renal pelvis may arise from a metaplasia of neoplastic mesonephric cells into endodermal cells. PMID- 1710537 TI - Immunohistochemical study of androgen receptors in metastatic prostate cancer. Comparison of receptor content and response to hormonal therapy. AB - A longstanding goal has been to determine whether androgen receptor (AR) levels could be used to predict the clinical response of metastatic prostate cancer to androgen withdrawal therapy. A major limitation of previous studies was the use of homogenized tissue, which yields an average AR content for all cells. By AR immunohistochemical study using an antibody specific for AR the authors assessed nuclear AR content specifically in the malignant epithelial cells of prostate needle biopsy specimens of 17 patients with Stage D prostate cancer. The authors found that prostate cancer contains AR-positive and AR-negative malignant cells before androgen withdrawal therapy, but the percentage of AR-positive cells did not predict the time to tumor progression after therapy. There was no significant correlation between the percentage of AR-positive malignant cells and the time to tumor progression. When patients were divided into two groups based on the median time to progression, the percentage of AR-positive nuclei was not significantly different in poor responders versus good responders. When patients were divided into two groups based on the median percentage of receptor-positive nuclei, Kaplan-Meier estimates of the progression-free interval revealed no significant difference between the group of patients with AR-poor tumors and patients with AR rich tumors. Potential explanations for these results are discussed. The authors conclude that the percentage of AR-positive nuclei is not a sufficient criterion to predict tumor behavior. PMID- 1710538 TI - Determination of growth fraction in advanced prostate cancer by Ki-67 immunostaining and its relationship to the time to tumor progression after hormonal therapy. AB - Reliable predictors of response for prostate cancer patients undergoing hormonal therapy are lacking. This study investigates the possibility that tumor proliferation rates might predict tumor behavior for these patients. The growth fraction of metastatic prostate cancer biopsy specimens obtained before androgen withdrawal therapy was evaluated by Ki-67 antibody immunohistochemical study to determine whether a higher tumor growth fraction was associated with a shorter time to tumor progression after therapy. The percentage of Ki-67-positive malignant epithelial nuclei in the primary tumors of 17 patients ranged from 1.7% to 7.5% (median, 2.5%). When patients were divided into two response groups according to the median time to progression, poor responders (time to progression less than 20 months) and good responders (greater than or equal to 20 months) had similar growth fractions (3.5 +/- 0.5% versus 3.1 +/- 0.6% Ki-67-positive nuclei, respectively). However, when patients were divided into two groups based on the median growth fraction, patients with a high growth fraction (greater than 2.5% Ki-67-positive nuclei) tended to have a shorter time to progression (median, 10 months) than patients with a low (less than 2.5%) growth fraction (median time to progression, 25 months), although statistical significance was not reached. Despite this interesting trend, Ki-67 immunostaining was not accurate to predict the time to progression in individual patients undergoing hormonal therapy. Reliable prediction of growth rates may require measurement of both cell proliferation and cell death rates. PMID- 1710539 TI - Immunohistochemical study of childhood rhabdomyosarcomas and related neoplasms. Results of an Intergroup Rhabdomyosarcoma study project. AB - The authors assessed a panel of immunohistochemical stains against 109 pediatric solid tumors, primarily rhabdomyosarcomas, under the auspices of the Intergroup Rhabdomyosarcoma Study. Fresh tumor tissue received from participating organizations was divided into portions that were either frozen or fixed in formalin, alcohol, or B5. Immunostaining was performed by the avidin-biotin complex method using monoclonal antibodies to desmin, neurofilaments, vimentin, cytokeratin, and leukocyte common antigen on cryostat sections. Tissue was also embedded in paraffin and stained with antimuscle-specific actin (MSA) and polyclonal antibodies to desmin, creatine kinase M subunit (CKM), myoglobin, and neuron-specific enolase (NSE). Antidesmin staining of cryostat sections was the most sensitive indicator of rhabdomyosarcoma (58 of 62 specimens positive). Results with this reagent in alcohol-fixed and formalin-fixed tissue were similar (46 of 56 positive versus 43 of 56 positive, respectively) and comparable with results with anti-MSA in formalin-fixed tissue (43 of 55 positive). However, the proportion of cells stained by antidesmin was higher in alcohol-fixed tissue than in formalin-fixed tissue. Staining with antimyoglobin and anti-CKM was much less satisfactory, with positivity rates of 17 of 37 and 11 of 57, respectively, in formalin-fixed rhabdomyosarcomas. Immunostaining of muscle markers revealed evidence of myogenesis in six undifferentiated sarcomas and in two sarcomas with inadequate histologic study on hematoxylin-eosin-stained sections. However, positivity was also noticed in samples of fibromatosis, Wilms' tumor, ectomesenchyoma, peripheral primitive neuroectodermal tumor, renal rhabdoid tumor, myositis ossificans, malignant fibrous histiocytoma, and embryonal sarcoma of the liver. The authors conclude that combined use of antidesmin and anti-MSA enhances the diagnosis of childhood sarcomas, especially when employed with other techniques such as electron microscopic study. PMID- 1710540 TI - Quantitative determination of acid-labile DNA in cervical intraepithelial neoplasia. AB - Using a modified Feulgen hydrolysis procedure and integrating microdensitometry, the acid-labile nuclear DNA in exfoliated cervical epithelial cells was quantified in a range of histologically confirmed cervical intraepithelial neoplasia (CIN), invasive cancer, and normal controls. The mean relative optical densities obtained for each sample group showed an increase from normal epithelium, through CIN grades, to invasive cancer. Although there was some overlap between groups, the difference in the overall mean values between the adjacent groups was statistically significant. The sensitivity of the test was 87.1% with a specificity of 99.2% and a predictive value of 99.5%, with no false negatives in the severe dysplasia and cancer groups. Quantitative data allows the threshold value to be altered to vary the sensitivity and specificity according to prevailing requirements. This suggests the possibility of using quantitative acid-labile DNA measurements to improve existing screening for cervical precancer. PMID- 1710541 TI - Quality of life assessment. An independent prognostic variable for survival in lung cancer. AB - Improved quality of life has long been the goal of cancer treatment, but only recently have investigators begun to include a systematic assessment of quality of life in clinical trials. The major interest for its inclusion in clinical trials has been to assess treatment outcome. An evaluation of the relationship between patient-rated quality of life and survival is reported in a homogeneous sample of patients with advanced metastatic lung cancer participating in a clinical trial. Under the Cox proportional-hazards model (with quality of life, marital status, and their interaction in the model), a statistically significant relationship was observed between initial patient-rated quality of life and subsequent survival. In addition, being married led to a significantly improved survival. These findings suggest that nonmedical factors, such as quality of life assessment and marital status, play a role in survival and that they should be evaluated and described as potential predictors of survival in cancer patients in clinical trials. PMID- 1710542 TI - Beta-human chorionic gonadotropin-positive extragonadal germ cell neoplasia of the renal pelvis. AB - A 56-year-old woman showed renal obstruction on the right side. Further examinations revealed a tumor of the renal pelvis. Nephroureterectomy on the right side was done. This extragonadal germ cell neoplasia had an immunohistochemically detected beta-human chorionic gonadotropin (beta-hCG) expression. There were no other primary tumors or metastases. Although there were normal values after the operation, during the postoperative follow-up, the serum beta-hCG temporarily reached 2400 IU/l and then dropped to normal without detection of metastases. No further therapy was given. The patient was alive and well 1 year and 10 months after surgery. To the authors' knowledge, this is the first extragonadal germ cell neoplasia of the renal pelvis with secretion of beta hCG to the serum. PMID- 1710543 TI - Effects of anti-idiotype vaccine on tumour growth and on production of soluble factors modulating cell-mediated immunity in vitro. AB - The previous observation, that single i.p. doses of a monoclonal anti-idiotypic antibody (MAIA) injected into BALB/c mice induced suppressor factors, was extended to multiple i.v. doses. These induced enhancing factors, which were produced in spleen cell cultures, required L3T4+ cells for their formation, lacked the IJ marker, and bound to anti-immunoglobulin, showing them to be antibodies. Selective immunoabsorption demonstrated two separate enhancing antibodies; both bound to MAIA but they had different affinities for specific and non-specific tumour antigens. Subsequently, single and multiple MAIA doses were tested in vivo for their effects on tumour growth. The single doses had variable effects depending on time of administration, and these effects were tumour specific; the multiple doses strongly inhibited tumour growth when given before tumour challenge, but also had non-specific effects on another tumour as anticipated from the in vitro results. PMID- 1710544 TI - Transcription by T7 RNA polymerase using benzo[a]pyrene-modified templates. AB - Synthesis of T7 RNA polymerase is inhibited by the presence of bulky adducts in the DNA template. Of the types of adducts tested, those formed by the potent carcinogen benzo[a]pyrene (B[a]P) caused the greatest inhibition. M13 DNA molecules containing a single late T7 RNA polymerase promoter have been prepared containing B[a]P adducts in either the displaced or template strand and these have been used as templates for in vitro RNA transcription by the T7 RNA polymerase. We find that the level of inhibition of RNA synthesis is substantially greater (greater than or equal to 10-fold) when adducts are positioned specifically in the template strand. Polyacrylamide gel analysis of the products synthesized off these strand-specifically modified templates showed that adducts situated in the template strand gave rise to discrete bands which presumably represent the termination of synthesis at the adduct site while the product derived from a template containing adducts in the displaced resembled that obtained using a native template. PMID- 1710545 TI - Evidence for the covalent binding of aflatoxin B1-dichloride to cytosine in DNA. AB - In vitro studies of the effect of aflatoxin B1-dichloride (AFB1-Cl2) on the template function for RNA synthesis of several single- and double-stranded synthetic DNAs containing cytosine and/or hypoxanthine bases are reported. The results indicate: (i) AFB1-Cl2 strongly inhibits the template function of the single-stranded homopolymer polydC and has no effect on polydI, (ii) the inhibition is stronger when cytosine is in the double-stranded alternating copolymer poly[d(I-C)], and (iii) polydI directed RNA synthesis can be inhibited if it is in the double-stranded homopolymer polydI.polydC, although the template function of the polydC strand is still inhibited to a greater extent. The evidence that the selective inhibition of the DNA template function is a direct reflection of the binding specificities of AFB1-Cl2 is provided by the binding studies of [3H]AFB1-Cl2 to these DNAs. The binding of AFB1-Cl2 to polydC is substantiated by the dose-response template inhibition and by the dose-response template binding studies. Additionally, these results show that AFB1 per se has neither inhibitory nor binding activity. Auto radiography of [alpha-32P]GTP labeled RNAs suggests that the mechanism of inhibition of polydC template function by AFB1-Cl2 is mainly due to the inhibition of the elongation of RNA synthesis. Spectrum measurement of the products of enzyme digestion of the AFB1 Cl2 modified polydC reveals that the deoxycytidine fraction gives a typical cytosine absorption peak at 275 nm followed by a broad peak between 300 and 400 nm with a maximum at 390 nm. High performance liquid chromatography confirms the existence of a cytosine-AFB1 adduct which absorbs strongly in the regions between 250 and 400 nm with peaks identifiable at 260, 350 and 390 nm. These results strongly suggest that AFB1 in the activated form of AFB1-Cl2 is able to covalently bind to cytosine in DNA. PMID- 1710546 TI - An immunologic pathway for intravascular catabolism of creatine kinase subform MM3. AB - In vitro incubation of the MM3 subform of human creatine kinase (ATP:creatine N phosphotransferase, EC 2.7.3.2, CK) with fresh human serum resulted in the formation of a complex of high relative molecular mass (Mr 320 kDa). The formed complex (macro CK-MM3) consists of both CK-MM3 and immunoglobulin A (IgA), and its amount of the formed complex was proportional to CK-MM3 activity and IgA concentration. Two molecules of CK-MM3 combined with one molecule of IgA, and the immunoglobulin inhibited the enzyme activity. As IgA does not form complexes with other subforms (CK-MM2 and CK-MM1) or CK-MB, the antigen specificity of IgA to CK MM3 is definitely exacting. The circumstantial evidence suggests that macro CK MM3 is a specific antigen-antibody complex. Macro CK-MM3 was detected in all of the examined sera of adult patients with more than 2001 U/1 CK activity (the positive percentage of macro CK-MM3 in all adult patients was 73%), but not detected in sera of patients who were younger than 12 months old. No relationship was observed between macro CK-MM3 and the patients' underlying diseases. Macro CK MM3 formation suggested to be an immunologic pathway for intravascular catabolism of CK-MM3 when its activity increases. PMID- 1710547 TI - Constitutive IL-2 expression in HTLV-I-infected leukaemic T cell lines. AB - An IL-2 autocrine growth circuit has been proposed as a major mechanism in HTLV-I related leukaemogenesis. We have developed a polymerase chain reaction combined with reverse transcription RT-PCR to detect IL-2 transcripts and a sensitive immunostaining method for IL-2 protein. Combination of these two methods with in situ hybridization demonstrated that most cells of the T cell line IARC 301.5, whose proliferation is stimulated by autocrine IL-2, constitutively synthesize IL 2. This pattern was also found in the HTLV-I T cell lines HUT, MT2 and C 8166/45, and in the HTLV-I-negative T cell lines Jurkat and HSB2 but not MOLT4. Four non lymphoid cell lines and cultured fibroblasts were negative, in agreement with the T cell specificity of IL-2 synthesis. Hence, most T cell lines tested, whether HTLV-I-infected or not, constitutively synthesize IL-2, suggesting a possible common feature of leukaemic T cell lines. The presence of IL-2 transcript and protein in most cells of the reacting cell lines is consistent with an autocrine process possibly involved at some stage in acquiring growth autonomy. PMID- 1710548 TI - Characterization of human monoclonal antibodies directed against the pp65-kD matrix antigen of human cytomegalovirus. AB - Human monoclonal antibodies specific for human cytomegalovirus (CMV) antigens have been established using peripheral blood lymphocytes from a seropositive donor. Immortalization of antigen-specific B cells was achieved by Epstein-Barr virus transformation followed by somatic cell fusion of antigen-specific lymphoblastoid cells. Four clones producing high-affinity antibodies (0.2-7 x 10(9) M-1) specific for the viral matrix protein pp65 have been further characterized with respect to epitope specificity of secreted antibodies. The studied antigen represents a major protein produced by in vitro-cultivated virus, and is important in the serodiagnosis of CMV infection. The human monoclonal antibodies recognized different epitopes, some of which proved to be overlapping. The fine specificity of these antibodies was evaluated using synthetic peptides covering the sequence of pp65. The antibody MO58 recognized a linear epitope (residues 283-288) whereas antibody MO53 recognized a discontinuous epitope involving residues 208-216 and 280-285. Despite the close proximity of these epitopes, the antibodies did not compete with each other for the same binding site on intact antigen. PMID- 1710549 TI - Identification of T cell epitopes within a 23-kD antigen (P24) of Toxoplasma gondii. AB - Among the potentially vaccinating antigens, the products excreted/secreted by the parasite T. gondii have been demonstrated to be excellent candidates. The molecular cloning of one of these antigens (P24) present in excreted/secreted antigens (ESA) has recently been carried out in our laboratory. The recombinant antigen P24 corresponds to a native molecule of 23 kD. We were interested in determining the main epitopes of the P24 antigen eliciting a T lymphocyte response using synthetic peptides derived from the primary structure of P24. Five peptides: 64-79, 88-109, 170-193, 194-208 and 231-250 were synthesized according to their hydrophobicity, mobility and accessibility profiles. The presence of T lymphocyte epitopes in these peptides has been examined in the rat model. The determination of T cell epitopes was carried out using T lymphocytes from infected rats, and from ESA and P24 expression vaccine virus immunized rats. The results showed that the stimulation of T cells with these peptides varied according to the period after Toxoplasma infection. The main T cell stimulation was obtained with the 88-109, 170-193 and 194-208 peptides. When Fisher rats were immunized with ESA, a most significant stimulation was achieved with the 170-193 and 194-208 peptides. In addition, T lymphocytes primed with P24 expressed vaccine virus immunization were more stimulated with the 88-109 and the 194-208 peptides. This study showed that P24-derived peptide-specific T cells were elicited in the three experimental situations, although no antibody response against the 23-kD native antigen was evidenced in the Fisher rat model. However, the native antigen (presented by irradiated parasites) can induce a proliferative response of the 170-193 peptide-specific T lymphocytes, confirming that this peptide contains an important T cell epitope. The adoptive transfer into athymic rats of T helper cells recovered from 170-193 peptide-immunized Fisher rat conferred a significant protection to infected nude rats despite the fact that no antibody production was observed. PMID- 1710550 TI - The physiology of anti-idiotypic interactions: from clonal to paratopic selection. AB - On theoretical and experimental grounds, it has been proposed that the idiotypes of immunoglobulins and of T cell receptors are composed of multiple paratopes, as opposed to a single paratope and several idiotopes. This necessitates a revision of some of the basic principles of anti-idiotypic reactions. It is also possible to infer the presence of the same or similar paratopes on different idiotypes. A paratope cannot therefore be regarded as restricted to or unique on an idiotype. For these reasons, the perception of immunological specificity in terms of clonal units is misleading. This review proposes instead that the physiological unit of immunological specificity and regulation is the paratope. This essential alteration in the perception of the immune system is referred to as paratopic selection. The approach is assessed in terms of immunological regulation and specificity, and appears to allow new insights into these areas. PMID- 1710551 TI - Heterodimeric capping proteins constitute a highly conserved group of actin binding proteins. AB - The two subunits of the heterodimeric protein cap32/34, an actin-binding protein, are encoded by separate single-copy genes. We have established the genomic structure of both genes. A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences from Saccharomyces cerevisiae and Xenopus laevis show that heterodimeric capping proteins belong to a highly conserved group of actin-binding proteins. This conclusion is supported by the cross-reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes. In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34 promoter. PMID- 1710552 TI - Biochemical and genetic analysis of an antigenic determinant found on N-linked oligosaccharides in Dictyostelium. AB - Dictyostelium discoideum synthesizes many highly immunogenic carbohydrates of unknown structure and function. We have used monoclonal antibodies prepared against one of these called CA1 to investigate its structure and the consequences of its loss. CA1 is preferentially expressed on lysosomal enzymes as a specific arrangement of mannose-6-SO4 residues on N-linked oligosaccharides. Mutant strains HL241 and HL243 do not express CA1, and synthesize a truncated lipid linked oligosaccharide (LLO) precursor that lacks the critical mannose residues needed for expression. The lesion appears to result from the loss of mannosyl transferase activity involved in LLO biosynthesis. The truncated LLO is poorly transferred to an artificial peptide acceptor in a cell-free N-glycosylation assay, and this appears to result from improper topological localization of the LLO or to a lower affinity of the LLO for the oligosaccharyl transferase. Although both mutants share these lesions, they are biochemically and genetically distinct. Only HL243 is lower in N-glycosylation in intact cells, and this is not a result of an altered structure of the LLO. There are other differences between the strains. HL241 can form fruiting bodies at a slower rate than normal while HL243 cannot aggregate. Genetic analysis of defects shows that the CA1 lesion in HL241 is recessive, while the lesion in both CA1 and in development are dominant and co-segregate in HL243 and are, therefore, likely to be in the same gene. Lysosomal enzyme targeting is normal but enzyme processing proceeds at a 2-3 fold slower rate in HL241 and HL243 compared to wild-type. Strain HL244 does not express CA1 since it completely lacks protein sulfation, but lysosomal enzyme targeting and processing proceeds at a normal rate, showing that sulfate is not essential for these processes. Alterations in oligosaccharide structure can have individualized effects on the biosynthesis of lysosomal enzymes. The results presented here illustrate how this approach can be used to study both the structure and function of carbohydrate epitopes. PMID- 1710553 TI - [A case of chronic demyelinating polyneuropathy with benign IgM anti-myelin associated glycoprotein paraproteinenia--transient improvement with weekly plasmapheresis]. AB - We report a case of chronic demyelinating polyneuropathy accompanying benign IgM monoclonal gammopathy treated with plasmapheresis, which brought the improvement of the neurological signs and conduction velocities of peripheral nerves. A 60 year-old man developed numbness in the hands and legs and unstable gait. These symptoms became worse slowly. Three years after the onset, he was admitted to Matsuyama Red Cross Hospital because mild clumsiness in the hands was added. The neurological examinations revealed marked loss of both superficial and deep sensations of the glove-stocking type, mild weakness of distal muscles and hyporeflexia in the upper and lower extremities, and mild sensory ataxia. On the laboratory examinations, the serum showed a marked increase of IgM with a monoclonal IgM of lambda light chain. The cytological examination of the bone marrow showed no evidence of malignancy. Marked decrease of nerve conduction velocities was noted in the electrophysiological examinations of the peripheral nerves. Segmental demyelination and widely spaced major dense lines of myelin were observed in the histological examinations of the sural nerve. The immunological examination revealed the antibody activity of IgM against myelin associated glycoprotein in the patient's serum. He was treated with double filtration plasmapheresis of 2 liters once a week for 3 months as inpatient. During this treatment, the concentration of IgM in the serum was kept to be much lower than before the treatment, and the sensory disturbances, grasping powers and nerve conduction velocities were mildly improved at the end of the treatment. After discharge, he was treated with monthly plasmapheresis of 2 liters for 3 months.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710554 TI - Children of battered women: developmental and learning profiles. AB - Children in battered women's shelters have been shown to have more behavior problems than their peers but limited information is available about their development. A pilot survey was undertaken to determine the prevalence of developmental or academic problems in children of residents of a battered women's shelter. Demographic data, medical and school histories and responses to standardized developmental or behavioral surveys were obtained from 39 mothers of 76 children. Two-thirds of the children were victims of abuse. On the Minnesota Child Development Inventory, the mean General Developmental Quotient (DQ) of 28 preschool children was 98; however, 39% had developmental delays by test criteria. Of 46 school-aged children, 21 (46%) had evidence of academic problems, including grade repetition, failing grades and need for special educational services. On the Louisville Behavior Checklist, 75% of 48 children had behavior problems. Children in a battered women's shelter are likely to experience academic and behavioral problems; however, further study is needed to elucidate etiological factors. PMID- 1710555 TI - Tachykinin immunoreactivity in the European common frog, Rana temporaria: localization, quantification and chromatographic characterization. AB - 1. Tachykinin immunoreactivity has been localized, quantified and chromatographically-characterized in the brain, stomach, intestine and skin of Rana temporaria. 2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium. 3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain. 4. Chromatographic analysis of both frog tachykinins revealed that they possessed different physico-chemical properties than their mammalian counterparts. PMID- 1710556 TI - Effects of 5-azacytidine and 5-aza-2-deoxycytidine on alphafetoprotein levels in mice. AB - 1. Production of alphafetoprotein in adult C3H mice was monitored by radial immunodiffusion both in controls, and in animals treated with carbon tetrachloride, 5-azacytidine, or 5-aza-2-deoxycytidine, either alone or in combination. 2. Carbon tetrachloride routinely induced alphafetoprotein synthesis in our experiments, but neither of the cytidine analogues showed any effects on the serum levels of this protein when administered alone. 3. Treatment of mice with either cytidine analogue prior to carbon tetrachloride injection markedly reduced the consequent production of alphafetoprotein, whereas if carbon tetrachloride injection was followed by a subsequent injection with either cytidine analogue, a markedly enhanced level of serum alphafetoprotein was detected. 4. It is suggested that carbon tetrachloride induces alphafetoprotein production in adult mice by inducing liver damage, followed by synthesis of the protein in the dividing and differentiating cells during recovery. We also propose that the cytidine analogues ablate this response by a cytotoxic effect on the liver cells when they are administered prior to the CCl4, but enhance the alphafetoprotein levels when administered after the CCl4 because they inhibit the methylation of cytidine residues in the recovery cell population in the liver and thus prevent early cessation of synthesis of the protein. PMID- 1710557 TI - Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum. AB - 1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium. 2. Insulin (100 microU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum. 3. Dexamethasone inhibited the effects of 100 microU insulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 microU and 1 mU/ml respectively. PMID- 1710558 TI - Mercurochrome-induced allergic contact dermatitis. PMID- 1710559 TI - Allergic contact dermatitis from eosin. AB - Before 1960, eosin sensitivity was not rare and lipstick cheilitis was very common. We report 4 patients seen in 1988 and 1989 who were sensitized to eosin from topical bacteriostatic preparations. All 4 patients had positive patch tests to eosin. The allergen is probably an impurity rather than eosin itself. PMID- 1710560 TI - Capsaicin enhances allergic contact dermatitis in the guinea pig. AB - Guinea pigs were sensitized to dinitrochlorobenzene (DNCB) by the intracutaneous route and challenged epicutaneously on the flanks. The intensity of the allergic contact dermatitis was evaluated by inspection and palpation as well as by wet weight determination. With the purpose of diminishing tissue neuropeptides, and substance P in particular, the animals were treated with capsaicin either between induction and challenge, or before sensitization. In comparison with controls, the contact dermatitis was enhanced in both groups of animals treated with capsaicin. PMID- 1710561 TI - Speech and the Neanderthals. AB - The ability to communicate by speech was a crucial step in human evolution and there has been much controversy concerning the point at which it occurred. The recent discovery at Kebara of a well-preserved hyoid bone some 60,000 years old suggests that Neanderthal man had developed the anatomical structures necessary to articulate words. This in itself does not prove that such articulation occurred. But contributory evidence, such as endocranial casts indicates that the necessary brain differentiation had also developed. Further, what we know of the social organisation of Neanderthals suggests that some form of communication by speech was necessary. PMID- 1710562 TI - Intraglandular colloid induced nuclear proliferation of murine thymic cells as determined by flow cytometry. AB - The ability of intraglandular colloid, the holocrine secretion of cells of the marginal half of the bovine pituitary intermediate lobe, to enhance or suppress the proliferation of immature thymic cells of CH3/6JH mice, 4 to 6 weeks of age, was studied. The mice were given subcutaneous injections of colloid for 3 consecutive days at a dose of 20 mg/kg body weight in 0.2 ml of saline. The analysis of propidium iodide stained thymic cell preparations by means of flow cytometry showed that there was a significant increase in the percentage of cells in the S phase (proliferative phase) of the cell cycle of mice injected with colloid (33%) compared with saline injected (15%) and non-injected controls (7%). These findings are the first to suggest that various combinations and concentrations of intraglandular colloid neuropeptides play a role in the immune system and shed light on additional functions of the intermediate lobe. PMID- 1710563 TI - Identification of a major human immunodeficiency virus-1 reverse transcriptase epitope recognized by mouse CD4+ T lymphocytes. AB - Delineation of major T helper cell recognition sites of human immunodeficiency virus (HIV-1) proteins represents one important step in the design of an efficient acquired immune deficiency syndrome (AIDS) vaccine. Towards this end, we have explored the immunogenicity of HIV-1BRU proteins in the mouse model. Preliminary experiments revealed that inbred mice primed with whole inactivated HIV-1 developed strong CD4+ T cell proliferative responses to a variety of recombinant viral proteins including reverse transcriptase (RT). To characterize further the mouse T cell responses to this protein, several Ad- or Ed-restricted T hybridoma cells (THC) were established from BALB/c or DBA/2 mice. These THC were tested for their capacity to recognize a series of 15-mer synthetic overlapping peptides spanning three segments of HIV-1 RT that had been preselected on the basis of either alpha-helicity, amphipaticity, and/or for containing rare amino acid sequence patterns. Peptides corresponding to a C terminal region (residues 528-560) of RT were recognized by several of the THC established from RT-primed mice. Furthermore, a non-alpha-helical peptide from this region (A3, 528-543) was capable of priming mice with different H-2 haplotypes for both peptide A3 and native RT CD4+ T cell recognition. In addition to the recently identified RT determinant 203-219 capable of triggering both mouse and human CD8+ CTL, the present results identify a good candidate for an immunodominant RT epitope capable of eliciting RT-specific T helper cell responses. PMID- 1710564 TI - Mouse LFA-3 studied with chimeric soluble CD2 shows preferential expression on lymphoid cells. AB - Here we have used a soluble, polyvalent form of mouse CD2, i.e. CD2-hC mu, to detect its ligand LFA-3. Mouse LFA-3 is preferentially expressed on lymphoid cells unlike human LFA-3 which shows a wide tissue distribution. Mouse LFA-3 is one of the early surface molecules expressed on developing thymocytes appearing on the surface of fetal thymocytes on day 13 of gestation. Like human LFA-3, the mouse homolog could be shown to be a phosphatidyl inositol-linked membrane protein. Since mouse LFA-3 is preferentially expressed on lymphoid cells and since CD2 is expressed by both T and B lymphocytes, we would favor the view that the CD2/LFA-3 adhesion system in the mouse may play a role in interactions between lymphocytes (T-T and/or T-B) rather than in cell interactions between lymphocytes and non-lymphoid cells in the thymic, bone marrow or spleen microenvironments. PMID- 1710565 TI - T cell response to myelin basic protein epitopes in multiple sclerosis patients and healthy subjects. AB - T cell lines and clones specific for human myelin basic protein (BP) were selected from three multiple sclerosis (MS) patients and two healthy subjects and tested for their proliferative responses to a battery of synthetic peptides, 9 to 21 amino acid residues long. The combined amino acid sequence of the peptides spanned the complete sequence of the human BP. The results suggest the development of T cells sensitized to at least four independent regions of the human BP, indicating some diversity of the human T cell repertoire to BP. However, an immunodominant T cell epitope was located in the C-terminal region, defined by residues 149-162. This epitope was recognized by T cells from three subjects out of five (one MS patient and both healthy controls) in the context of different DR specificities. Another epitope (located in the 57-75 region) which triggered one MS patient's T cell response was also recognized by a mycobacteria specific T cell clone cross-reacting with BP. PMID- 1710566 TI - Allo-cross-reactivity of a human neuraminidase-specific T cell clone dependent on presentation of an endogenous B cell-specific antigen. AB - T cells specific for foreign antigen recognize a complex of peptides and self major histocompatibility complex (MHC) molecules and can also cross-react with allo-MHC molecules. It remains controversial, however, what alloreactive T cells exactly recognize. It has been proposed that alloreactive T cells recognize endogenous peptides presented by allo-MHC molecules. To test this hypothesis, we examined an influenza virus-specific T cell clone (6H5), specific for neuraminidase N2 and restricted by HLA-DR1. In the absence of influenza virus, this clone cross-reacted with HLA-DR1Dw1+ but not with HLA-DR1Dw20+ Epstein-Barr virus-transformed lymphoblastoid cells (B-LCL). Cold target inhibition experiments and the rearrangement pattern of the T cell receptor beta chain indicated that 6H5 was a monoclonal T cell population most likely using the same T cell receptor for both responses. To determine whether determinants other than HLA-DR1Dw1+ B-LCL or activated B cells, but, surprisingly, not to other cell types expressed HLA-DR1Dw1, including monocytes and transfected L cells. These experiments further support the concept that recognition of allogeneic MHC (in this case HLA-DR1Dw1) may result from a cross-reactivity of T cells specific for a complex of foreign antigen and self-MHC (neuraminidase N2 and HLA-DR1Dw20). Furthermore, allorecognition of T cell clone 6H5 appears to depend upon the recognition of a complex of allogeneic MHC and a cell-type specific endogenous peptide presented by activated B cells. PMID- 1710567 TI - Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. AB - The pathogenesis of multiple sclerosis (MS) could involve an autoimmune response to proteolipid protein (PLP). Immunization of experimental animals with this major myelin protein can lead to experimental allergic encephalomyelitis. To identify a possible role of PLP as target antigen in MS, we evaluated T cell immunity to PLP in blood and cerebrospinal fluid (CSF) from patients with MS and controls by counting cells which in response to PLP in short-term cultures secreted interferon-gamma. The PLP-specific B cell response was analyzed by counting cells secreting anti-PLP antibodies. PLP-reactive T cells were detected in blood of most MS patients (mean value 1 per 20,408 mononuclear cells), and at 41-fold higher numbers in CSF (mean 1 per 500 CSF cells). Anti-PLP IgG antibody secreting cells were detected in blood from most MS patients (mean 1 per 30,303 cells), but such cells were 49-fold more frequent in CSF (mean 1 per 625 cells). PLP-reactive T and B cells were also detected in blood and CSF from control patients, but at much lower numbers. A strong and persistent autoimmune response to PLP as well as to other myelin proteins, enriched in CSF, is proposed to be pathogenetically important in MS. PMID- 1710568 TI - Antibodies raised against peptide fragments of bovine alpha s1-casein cross-react with the native protein, but recognize sites distinct from the determinants on the protein. AB - Bovine alpha s1-casein (alpha s1-CN) and its peptides 61-110 and 91-110, which contain both T and B cell determinants on alpha s1-CN and can elicit peptide native protein cross-reactive antibodies, were selected as model antigens to study whether or not the immune response to the peptides is similar to that to the corresponding regions of the native protein, because they both have a similar disordered conformation in solution. Both alpha s1-CN- and peptide 61-110-primed T cells responded to peptides 61-80 and 91-100, but not to peptides 76-95 and 101 110. In addition, T cells immunized with peptide 91-110 were also stimulated by peptide 91-100, but not by peptide 101-110. These results suggest that the location of the T cell determinant was almost the same in alpha s1-CN and its peptides. On the contrary, antibodies raised against alpha s1-CN bound to peptides 76-95 and 91-100, but not to peptides 61-80 nor 101-110, while anti peptide 61-110 antibodies preferentially reacted with peptides 61-80 and 101-110, and anti-peptide 91-110 antibodies also bound to peptide 101-110 but not to peptide 91-100. These results indicate that the B cell epitopes were not similar between alpha s1-CN and its peptides. This difference may have arisen because the antigen-B cell or T-B interactions required for the development of a specific antibody response occurred in a different manner between alpha s1-CN and its peptides. These findings may be useful for basic studies on immunology, and could also be applied to the design of new peptide vaccines. PMID- 1710569 TI - A highly selected panel of anti-CD4 antibodies fails to induce anti-idiotypic antisera mediating human immunodeficiency virus neutralization. AB - Anti-CD4 antibodies directed to the N terminus of CD4 can inhibit human immunodeficiency virus (HIV) infection. Therefore, it has been proposed that some of these reagents may contain idiotypic determinants which conformationally model the binding site expressed on gp120. In this report, we have selected a panel of anti-CD4 monoclonal antibodies as idiotypic mimics of gp120 by employing cross blocking techniques, and CD4 epitope mapping using site-directed mutagenesis. These studies suggest that only 4 out of the original panel of 12 would be expected to represent suitable candidates for modelling the gp120 binding site. Nevertheless, anti-idiotypic antisera raised against these antibodies failed to inhibit gp120 binding to CD4. This negative result may reflect the incomplete modelling of the virus binding site by anti-CD4, or the lack of internal image antibody in the anti-idiotypic preparations. Alternatively, the binding site on gp120 may not be accessible to antibody neutralization, excluding the possibility of an idiotypic vaccine to HIV based on anti-CD4 antibody as surrogate antigen. PMID- 1710570 TI - Induction of antibodies against a peptide hapten does not require covalent linkage between the hapten and a class II presentable T helper peptide. AB - Following immunization with a complex antigen, a B cell internalizes this antigen through an interaction between its surface immunoglobulins and an epitope of the antigen. Enzymatic processing of the antigen frees one or more short peptide determinants (TD) which bind to class II molecules of the B cell. If the complex TD-MHC II is recognized by the receptor of a T helper cell, T cell help is provided leading to the expansion of an antibody-producing B cell clone specific for the epitope. We present experimental evidence proving that the induction of anti-peptide hapten antibodies does not require covalent linkage between the peptide hapten and the peptide behaving as TD. Indeed, high anti-peptide hapten antibody titers are induced if an emulsion of TD and hapten are injected in the same immunization site. This result suggests a way to manipulate antibody production with useful applications to research and therapy. PMID- 1710571 TI - Antigen-independent T cell activation mediated by a very late activation antigen like extracellular matrix receptor. AB - Cell-cell and cell-extracellular matrix (ECM) binding mediated by integrin molecules has been implicated in lymphocyte migration and adhesion. We describe here that ECM binding triggers antigen-independent activation of T cell functions. Fibronectin and vitronectin, when coated on plates, not only acted synergistically on anti-CD3-induced serine esterase release in a murine cytotoxic T lymphocyte clone and interleukin 2 production in a murine helper T cell hybridoma but also could trigger these responses alone. All these stimulatory effects of ECM were abrogated by a monoclonal antibody which reacts with a unique very late activation antigen-like integrin and this monoclonal antibody, when coated on plates, exhibited a similar synergistic effect to that of ECM proteins. Therefore, ECM receptors expressed on activated T cells appear to play an important role in triggering T cell effector functions in localized tissues abundant in ECM proteins. PMID- 1710572 TI - Specific outgrowth from neurons of ventral mesencephalic grafts to the catecholamine-depleted striatum of adult hosts. AB - Grafts of fetal ventral mesencephalon including substantia nigra have been used to correct some motor deficits produced by unilateral destruction of the dopaminergic nigrostriatal pathway in rats. Histochemical studies have shown that dopaminergic neurons within the graft send processes from the graft to the host neuropil, wherein they form synapses. The results of numerous immunocytochemical studies indicate, however, that a large proportion of neurons in grafts are not catecholaminergic. Whether or not the nondopaminergic neurons in grafts project to the host brain is unknown. The purpose of the present study was to combine immunocytochemistry and retrograde tracing with fluorogold to identify the cell types which project from grafts to the host striatum. Tissue from the ventral mesencephalon of E15 fetuses was placed into the 6-hydroxydopamine denervated striatum of graft recipients. Six weeks to 6 months following transplantation, fluorogold was pressure injected under stereotaxic control immediately adjacent to the ventral mesencephalic grafts; after 4 days CNS tissue was prepared for light microscopic immunocytochemistry. Ventral mesencephalic grafts contained cell bodies immunoreactive for enkephalin, GAD, substance P, and serotonin in addition to those immunoreactive for tyrosine hydroxylase. Some cells of each immunochemically defined type were retrogradely labeled by the fluorogold injection into the host brain. Nevertheless, more catecholaminergic and serotonergic cells projected from grafts to the fluorogold injection site than did other cell types. Since many of the nonmonoaminergic neurons in grafts are probably projection neurons, our results suggest that the extent of neurite outgrowth from grafted cells is influenced by the surrounding target tissue. PMID- 1710573 TI - The role of substance P and calcitonin gene-related peptide containing nerve fibers in maintaining fungiform taste buds in the rat after a chronic chorda tympani nerve injury. AB - Taste buds in the anterior part of the tongue of adult rats were denervated by unilateral resection of the chorda tympani nerve in the middle ear. Three months later one group of animals was perfused and their tongues were processed for demonstration of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity. Fungiform taste buds found on the denervated side showed increased numbers of intragemmal SP- and CGRP-immunoreactive (IR) fibers compared to the normal side. Compared to the normal side, the number of taste buds appeared to be fewer on the denervated side. Moreover, taste buds on this side seemed to be only partially restored. Another group of animals was given the neurotoxin capsaicin which causes a depletion of SP and CGRP from sensory axons. The animals were perfused 2 or 3 weeks after the capsaicin treatment, and their tongues prepared for SP and CGRP immunohistochemistry or for histological examination of taste buds. Very few SP- and CGRP-IR fibers were present in capsaicin-treated animals. In these animals almost all fungiform taste buds and papillae on the chorda tympani-injured side disappeared. In contrast, normal numbers of taste buds were still present on the contralateral side where the chorda tympani innervation remained intact. It is conceivable that taste buds on the chorda tympani-innervated part of the tongue, deprived of the normal chorda tympani-innervation, can regenerate and become reinnervated by SP- and CGRP containing fibers, and that these are essential for partially restoring and maintaining the structure of the denervated taste buds and the fungiform papillae. PMID- 1710574 TI - Pre- and postnatal lead exposure affects the serotonergic system in the immature rat brain. AB - The effect of pre- and postnatal lead exposure on the development of the serotonergic system in striatum and brain stem was investigated. Serotonin and its metabolite 5-HIAA where determined by HPLC-EC. A significant decrease of 5-HT was detected in the brain stem at postnatal day 28. At both days 6 and 28 postnatal, 5-HIAA was reduced in striatum and brain stem. The results provide support to the hypothesis that developing 5-HT neurons are sensitive to relatively low levels of lead exposure. PMID- 1710575 TI - Surgical restaging of advanced Hodgkin's disease after first line chemotherapy. AB - 47 patients with stages IIIB and IV Hodgkin's disease underwent laparotomy with splenectomy as restaging procedure after first-line chemotherapy (CT) which included 4 cycles of CT ABV/IMEP/PCAV/ABV. After surgical restaging (SR), all patients were scheduled to receive 2 additional cycles IMEP/PCAV followed by TNI 20 Gy and patients with residual clinical abnormality or positive restaging surgery received a 20 Gy boost to these areas. 11 patients (23.4%) were found to have active disease in the spleen (10 patients) and/or the lymph nodes (3 patients) after SR. In the spleen, foci of active disease had a size in millimeters and confirm the limits of clinical restaging. According to the response to CT, 5 of the 35 patients (14%) clinically restaged as good responders had active disease; 6 of the 12 patients (50%) clinically restaged as poor responders had active disease. SR is of interest in selected patients with slowly responding disease to determine the indication of an extended field RT for responding patients and salvage therapy for patients resistant to CT. PMID- 1710576 TI - Serum levels of G-CSF, M-CSF and GM-CSF in a patient with cyclic neutropenia. PMID- 1710577 TI - Increased responsiveness of rat mast cells to compound 48/80 due to removal of extracellular magnesium. Effects of ouabain and EGTA. AB - A decreased secretory response of mast cells to compound 48/80 (12% of control value) after preincubation of the cells with magnesium but without calcium was partially restored by removal of magnesium. EGTA (10 microM) blocked the restoration and decreased the restored secretory activity again, while this was further increased by ouabain (1 mM). Furthermore, ouabain completely restored the decreased secretion (50% of control value) due to preincubation without the divalent cations. This may indicate that magnesium influences a pool of cellular calcium that is involved in the stimulus-secretion coupling and is available to EGTA, and ouabain did not counteract the inhibitory mechanism of magnesium. PMID- 1710578 TI - Human galanin: primary structure and identification of two molecular forms. AB - From acid/ethanol extracts of surgical specimens of human large intestine we isolated two peptides, in approximately equal amounts, that reacted with an antiserum against porcine galanin. By amino acid analysis, sequence analysis and mass spectrometry, the larger of the two peptides was found to consist of 30 amino acid residues, the sequence of which was identical to that of porcine galanin except for the following substitutions: Val16, Asn17, Asn26, Thr29 and Ser30. Unlike porcine galanin, the carboxy-terminus was not amidated. The smaller peptide corresponded to the first 19 amino acid residues counted from the N terminus of the 30 residue peptide (again without amidation). The structural analysis was repeated on another batch of tissue with identical results. By HPLC analysis of extracts of specimens from a further 4 patients, the same peptides were identified. Thus, human galanin includes two peptides of 19 and 30 amino acids that share the sequence of the N-terminal 15 residues with other mammalian galanins, but exhibit characteristic differences in the remaining part of the molecules. PMID- 1710579 TI - Molecular cloning and sequence analysis of cDNA encoding delta 4-3-ketosteroid 5 beta-reductase of rat liver. AB - A cDNA clone encoding delta 4-3-ketosteroid 5 beta-reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981-base pair open reading frame encoding 327 amino acid residues (Mr 37,376) and an unusually long 3' untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains the sequences similar to the putative NADPH- and steroid-binding regions. PMID- 1710580 TI - Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope. AB - The C-termini of p66 and p51 forms of HIV-1 reverse transcriptase have been engineered to contain a Glu-Glu-Phe sequence recognized by a monoclonal antibody to alpha-tubulin, YL1/2. Mutated RTs were purified in a single step using peptide elution from columns of immobilized YL1/2. The known sequence requirements of the YL1/2 epitope are consistent with protein eluting from the column with an intact C-terminus. Kinetic parameters of these mutated RTs are essentially unchanged from wild-type enzyme. The p15 RNaseH domain has been purified using this method and shown to have low enzyme activity compared to the parental p66 subunit. PMID- 1710582 TI - [Health bulletins on AIDS]. PMID- 1710581 TI - Voltage-dependent cationic channels formed by a cytolytic toxin produced by Gardnerella vaginalis. AB - A cytolytic toxin produced by G. vaginalis was incorporated in artificial membranes and giant liposomes. The toxin formed ionic channels when incorporated in lipid bilayers. The electrical properties of such channels were studied. Current records revealed a unitary conductance of 126 pS (in symmetrical 150 mM KCl). The open state probability of the cytolysin formed channels was a function of the applied membrane potential. The permeability ratio of cations to anions was estimated to be 6.5. PMID- 1710583 TI - The possibility of participation of different protein kinases in the regulation of degradation of Glp6[125I]Tyr8SP6-11 with different endopeptidases in subcellular fractions of rat brain. AB - 1. Nuclear fraction of cortex and synaptosomal fraction of hippocampus of rat brain were preincubated with ATP and different, "second messengers" as well as some "classical" neurotransmitters and then incubated with Glp6[125I]Tyr8SP6-11 (JP). 2. It was found that different neurotransmitters and especially "second messengers" might be involved in the activation or inhibition of peptidase I and II. 3. Activation of different protein kinases results in substantial modulation of degradation of JP (SP C-terminal fragment). 4. The data confirm that products of one peptidase might be inhibitors of the second peptidase acting on JP, although the second enzyme might be affected additionally in conditions activating different protein kinase systems. PMID- 1710584 TI - Characterization of a hyaluronic acid-binding protein from sheep brain comparison with human brain hyaluronectin. AB - 1. A hyaluronic acid (HA)-binding glycoprotein from sheep brain was characterized. 2. The specific affinity for HA was shown in vitro by high performance liquid chromatography, polyacrylamide gel electrophoresis and ELISA methods. 3. The KD for high molecular weight HA was 5.4 10(-9) M at 37 degrees C and lower than 10(-10) M at 4 degrees C. 4. No link protein was found and HA molecules could bind up to 10 times their weight of the glycoprotein. 5. The specific site for interaction was the HA-derived decasaccharide HA10. 6. The protein is composed of one polypeptidic chain. Tryptophan and lysine play a prominent role in the conformation of the binding site to HA. 7. Enzyme analysis indicated that the protein different forms are due to differences in glycosylation and that N- and O-linkages coexist in the molecules. 8. Immunohistochemistry localized the glycoprotein at the nodes of Ranvier and at the periphery of neurons. The perineuronal labeling was seen around all neurons studied in the cerebellum whereas it was almost undetectable in the cerebral hemispheres. 9. HA is not saturated by hyaluronectin (HN) in the sheep nervous system. 10. The glycoprotein is largely similar to human brain HN, and different from the hyaluronate-binding protein characterized in the cartilage. PMID- 1710585 TI - Doctors' decisions and prognostications for infants with Down syndrome. AB - Random samples of pediatricians, family practitioners and pediatric surgeons were surveyed with a questionnaire, which included three questions regarding their decisions about treatment of duodenal atresia in two hypothetical infants with Down syndrome, one with complete trisomy 21 and one with mosaicism. Results showed that doctors are more likely now to advocate surgery than in the past. Pediatricians and family practitioners had similar views, but pediatricians were more assertive about treatment if parents refused. Pediatric surgeons had the most pessimistic outlook and a greater number would not encourage surgery. There was a relationship between the doctors' prognostications and their decisions about treatment. These results emphasize the importance of ensuring that doctors have accurate perceptions about the capabilities of individuals born with Down syndrome. PMID- 1710586 TI - Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells. AB - When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm like cells in this process. PMID- 1710587 TI - [Intestinal absorption of insulin with a new telemetric shuttle in dogs]. AB - Telemetric shuttles for the in vivo investigation of the gastrointestinal tract have been available for sometime. We describe herein the use of a new shuttle model whose original features include: a) continuous, real time transmission of its location in the small bowel and accurate measurement of the gut length, b) controlled release of 1 ml of a given substance at any chosen site, allowing detailed investigation of intestinal absorption at different levels of the small bowel under physiological conditions. Small bowel length was measured in dogs using the shuttle and was later compared to the actual small gut length measured in the same animals at laparotomy. The telemetric measurements appeared to closely match the direct operative measurements. Insulin absorption from the canine small bowel was then investigated releasing different dosages of insulin together with the pancreatic enzyme inhibitors Soybean and Aprotinine and a surfactant (5-methoxysalicylate). By adjusting the dose of insulin released, the type of adjuvant substance delivered with it and the site of release in the small bowel, we have been able to precisely define the conditions of insulin absorption. Insulin as such is exclusively absorbed in the ileum when released in doses of 500 IU or higher and mixed with aprotinine. For absorption to take place the solution delivered by the shuttle needs to have the correct pH and natremic concentration. PMID- 1710588 TI - Secretagogue response of azaserine-induced rat pancreatic acinar tumors in vivo. AB - This study investigates digestive and lysosomal enzyme secretion of azaserine induced pancreatic acinar carcinomas in response to cholecystokinin in rats. After 15-20 months of treatment, 95% of the animals developed pancreatic acinar carcinomas encompassing more than 90% of the tissue. In anesthetized rats basal trypsin output was significantly elevated in the tumor group despite diminished fluid secretion. There was a linear correlation between tumor size and basal amylase and trypsin secretion. Intravenous infusion of cholecystokinin (25 IDU.kg 1.h-1) induced a significantly lower secretion of fluid per gram pancreas, as well as decreased amylase and trypsin output, in the tumor group compared with the control group. Plasma amylase and lipase levels were significantly elevated in the tumor group under both basal and stimulated conditions. The output of the lysosomal enzymes beta-D-glucuronidase and alpha-D-glucosidase was significantly increased in the tumor group under background secretin infusion. Additional cholecystokinin infusion caused a sharp increase in glucuronidase output in this group with only minimal increase in controls. Glucosidase output increased similarly in both groups. Amylase, lipase, and trypsin tumor tissue concentrations were markedly reduced by 85%, 90%, and 87%, respectively. It was concluded that the decreased secretory response of digestive enzymes may result from decreased synthesis, lowered storage capabilities, and/or a decreased/increased responsiveness to cholecystokinin. Increased glucuronidase secretion may reflect an augmented cell turnover of malignant tissue. PMID- 1710589 TI - Efficacy of dextran-70 as a substitute for albumin. PMID- 1710590 TI - The role of reactive oxygen species in the antitumor activity of bleomycin. AB - Calf thymus DNA was incubated with bleomycin and FeCl3 in the presence of isolated rat liver microsomal NADH-cytochrome b5 reductase, cytochrome b5 and NADH which catalyze redox cycling of the bleomycin-Fe-complex. Furthermore, isolated rat liver nuclei were incubated with bleomycin, FeCl3 and NADH, a system in which redox cycling of bleomycin-Fe leads to DNA damage. In both systems free bases from DNA were released. Furthermore, 8-hydroxy-guanine was also found in the supernatant. On the other hand, 8-hydroxy-deoxyguanosine was detected in DNA of cell nuclei indicating that hydroxylation of the guanine molecule occurred in intact DNA. The release of bases correlated with the release of malondialydehyde as well as with NADH and oxygen consumption. These results indicate that NADH cytochrome b5 reductase catalyzes redox cycling of the bleomycin-Fe-complex which results in the formation of reactive oxygen species which oxidize deoxyribose as well as bases of DNA. Both mechanisms may contribute to the cytotoxic and cytostatic effects of bleomycin observed in intact cells. PMID- 1710591 TI - [Hemodilution in patients with central retinal vein thrombosis. A placebo controlled randomized study]. AB - A randomized placebo-controlled study was conducted in 40 patients with acute retinal vein occlusion, 19 of whom received iso-(Hct greater than or equal to 42%) or hypervolemic (Hct less than 42%) hemodilution over 10 days with daily infusion of 250 ml hydroxyethyl-starch (MW 200,000/0.5, 10% HAES-steril) in combination with pentoxifylline (oral: 1200 mg/day; i.v.: 300 mg/day; Trental). After this 10-day trial of hemodilution rheological therapy was continued with pentoxifylline (oral: 1200 mg/day; Trental). The control group of 21 patients received no hemodilution or rheological therapy. After 10 days 35 patients underwent laser coagulation. Clinical, hemodynamic and rheological data of all patients were recorded before therapy, after 10 days but before laser coagulation, and after 6 weeks. In the group treated with hydroxyethyl-starch in combination with pentoxifylline, 10 patients had an improvement of central vision by two or more lines after 6 weeks. In the control group only 4 patients showed central vision improved by two or more lines after 6 weeks. In the treated group the retinal circulation and rheological data were significantly improved after 10 days of hemodilution therapy. PMID- 1710592 TI - Reduction of [3H]6-nitroquipazine-labelled 5-hydroxytryptamine uptake sites in rat brain by 3,4-methylenedioxymethamphetamine. AB - 3,4-Methylenedioxymethamphetamine (MDMA; Ecstasy) is a known neurotoxin to 5 hydroxytryptamine (5-HT; serotonin) nerve terminals. It has recently been demonstrated that [3H]6-nitroquipazine is a new radioligand for studying the 5-HT transport system in brain. Therefore, we examined the effects of repeated systemic administration (10 mg/kg ip, twice daily for 3 d) of MDMA on [3H]6 nitroquipazine-labelled 5-HT uptake sites in rat brain. Marked reductions in the concentrations of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5 HIAA) were observed in the cerebral cortex 1 week after the last injection of MDMA. In addition, the density of [3H]6-nitroquipazine-labelled 5-HT uptake sites was significantly decreased by MDMA. Furthermore, the reduction of 5-HT and 5 HIAA content and the density of [3H]6-nitroquipazine-labelled 5-HT uptake sites by MDMA were significantly prevented by co-administration of 6-nitroquipazine (5 mg/kg), a very potent and selective 5-HT uptake inhibitor. The present results indicate that the 5-HT uptake carrier plays an important role in the neurotoxic action of MDMA. PMID- 1710593 TI - Serum bioactive and immunoreactive follicle stimulating hormone during chronic treatment with gonadotropin releasing hormone agonist in elderly men. AB - Chronic administration of GnRH agonists "down regulates" the pituitary and decreases LH and FSH serum levels. Changes in the bioactivity of FSH have not been adequately assessed under such treatment, for lack of a proper test. We examined serum changes under GnRH agonist treatment among 12 healthy elderly men suffering only from benign prostatic hypertrophy, for up to one year, using a modification of a granulosa cell bioassay for the determination of FSH bioactivity. While radioimmunoassay-FSH decreased, we noticed a significant increase in the bioactivity of this hormone. The clinical importance of this increase is discussed. PMID- 1710594 TI - Prolonged survival in resistant myeloma after treatment with interferon--a case report and review. PMID- 1710595 TI - Oral idarubicin as a palliative agent in leukemia. PMID- 1710596 TI - Lymphoepithelial cyst of the pancreas. AB - A case of lymphoepithelial cyst of the pancreas is reported. The unilocular cyst was filled with keratin, lined by mature, keratinizing squamous epithelium and surrounded by lymphoid tissue. PMID- 1710597 TI - Mapping nonisotopically labeled DNA probes to human chromosome bands by confocal microscopy. AB - A method for mapping nonisotopically labeled probes to human metaphase chromosomes that can be used with laser scanning confocal microscopy has been developed. Only a limited number of wavelengths are available from the argon ion lasers used in most commercial instruments and therefore a method that allowed the visualization of bands on human chromosomes stained with propidium iodide and, simultaneously, the detection of hybridization signals using FITC-labeled antibodies was developed. The confocal microscope was used to map single-copy probes to chromosome bands and the positions of the probes on the R-banded chromosomes corresponded to map positions previously determined on Hoechst 33258 stained chromosomes (G-banded). A comparison of confocal imaging of single-copy hybridization signals with conventional fluorescence microscopy and high sensitivity video cameras revealed little difference in sensitivity but greater resolution of chromosome bands with the confocal microscope. The polymerase chain reaction was used to prepare nonisotopically labeled probes for in situ hybridization and to amplify Alu and KpnI family repeats from cloned DNA to be used to suppress hybridization of these repeat sequences so that a cosmid probe could be mapped to a chromosome band. PMID- 1710598 TI - Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. AB - The gene responsible for cystic fibrosis, the most common severe autosomal recessive disorder, is located on the long arm of human chromosome 7, region q31 q32. The gene has recently been identified and shown to be approximately 250 kb in size. To understand the structure and to provide the basis for a systematic analysis of the disease-causing mutations in the gene, genomic DNA clones spanning different regions of the previously reported cDNA were isolated and used to determine the coding regions and sequences of intron/exon boundaries. A total of 22,708 bp of sequence, accounting for approximately 10% of the entire gene, was obtained. Alignment of the genomic DNA sequence with the cDNA sequence showed perfect colinearity between the two and a total of 27 exons, each flanked by consensus splice signals. A number of repetitive elements, including the Alu and Kpn families and simple repeats, such as (GT)17, (GATT)7, and (TA)14, were detected in close vicinity of some of the intron/exon boundaries. At least three of the simple repeats were found to be polymorphic in the population. Although an internal amino acid sequence homology could be detected between the two halves of the predicted polypeptide, especially in the regions of the two putative nucleotide-binding folds (NBF1 and NBF2), the lack of alignment of the nucleotide sequence as well as the different positions of the exon/intron boundaries does not seem to support the hypothesis of a recent gene duplication event. To facilitate detection of mutations by direct sequence analysis of genomic DNA, 28 sets of oligonucleotide primers were designed and tested for their ability to amplify individual exons and the immediately flanking sequences in the introns. PMID- 1710599 TI - Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. AB - Five different mutations have been identified in the gene causing cystic fibrosis (CF) through sequencing regions encompassing exons 1-8, including the 5' untranslated leader. Two of these apparent mutations are missense mutations, one in exon 3 (Gly to Glu at position 85; G85E) and another in exon 5 (Gly to Arg at 178; G178R), both causing significant changes in the corresponding amino acids in the encoded protein--cystic fibrosis transmembrane conductance regulator (CFTR). Two others affect the highly conserved RNA splice junction flanking the 3' end of exons 4 and 5 (621 + 1G----T, 711 + 1G----T), resulting in a probable splicing defect. The last mutation is a single-basepair deletion in exon 4, causing a frameshift. These five mutations account for the 9 of 31 non-delta F508 CF chromosomes in our Canadian CF family collection and they are not found in any of the normal chromosomes. Three of the mutations, 621 + 1G----T, 711 + 1G----T, and G85E, are found in the French-Canadian population, with 621 + 1G----T being the most abundant (5/7). There are two other sequence variations in the CFTR gene; one of them (129G----C) is located 4 nucleotides upstream of the proposed translation initiation codon and, although present only on CF chromosomes, it is not clear whether it is a disease-causing mutation; the other (R75Q) is most likely a sequence variation within the coding region. PMID- 1710600 TI - Detection of three rare frameshift mutations in the cystic fibrosis gene in an African-American (CF444delA), an Italian (CF2522insC), and a Soviet (CF3821delT). AB - We have identified three new frameshift mutations in the CFTR gene in patients with cystic fibrosis (CF). The first one involves the deletion of an adenine nucleotide in exon 4 in an African-American patient (CF444delA), the second involves the insertion of a cytosine nucleotide in exon 13 in an Italian patient (CF2522insC), and the third results from the deletion of a thymidine nucleotide in exon 19 in a Soviet patient (CF3821delT). Each mutation is predicted to result in premature termination of the CFTR protein. PMID- 1710601 TI - A deletion of two nucleotides in exon 10 of the CFTR gene in a Soviet family with cystic fibrosis causing early infant death. PMID- 1710602 TI - Antigen-specific modulation of murine IgE and IgG2a responses with glutaraldehyde polymerized allergen is independent of MHC haplotype and Igh allotype. AB - C57BL/6 mice treated with high Mr, glutaraldehyde-polymerized ovalbumin of highly restricted heterogeneity (termed OVA-POL) exhibit IgE responses upon later exposure to unmodified OVA which, at peak, are 1-3% of those observed in untreated controls. Concomitantly, anti-OVA IgG2a responses are elevated 250-1000 fold via an interferon-gamma (IFN-gamma)-dependent mechanism (ref. 4). Here, the impact of OVA-POL treatment on antigen-specific primary and secondary IgE responses is examined in 14 strains of mice. The data indicate that the capacity of this modified allergen to induce pronounced inhibition of IgE responses (75 99%), paralleled by up to 1000-fold increases in IgG2a responses, is not genetically restricted. Moreover, these changes in antibody production were (i) antigen-specific, (ii) isotype-specific and (iii) operated independently of the responder status, MHC or Igh haplotype of the responder mice. In contrast, treatment with unmodified OVA under the same conditions was without effect on IgE production and led to minor increases in anti-OVA IgG2a production. PMID- 1710603 TI - Suppression of experimental allergic encephalomyelitis by alpha 2-macroglobulin. AB - Lewis rats sensitized with guinea-pig spinal cord in Freund's complete adjuvant developed an acute-phase protein response. This was characterized by a marked increase in plasma alpha 2-macroglobulin (alpha 2 M) levels which, however, declined towards normal values before the onset of clinical signs of experimental allergic encephalomyelitis (EAE). In contrast, levels of two other acute-phase proteins, fibrinogen and caeruloplasmin, remained variably elevated over the entire study period. Recovery from EAE coincided with an increase in alpha 2 M levels. Infusion of purified alpha 2 M effectively protected the rats against clinical EAE and this was associated with a restimulation of the acute-phase response. The protected rats were shown to be sensitized to myelin basic protein and to have comparable mononuclear infiltration of the central nervous system with the diseased animals. It is postulated that the infusion of alpha 2 M leads to the inhibition of the effector pathways of the delayed type hypersensitivity response. PMID- 1710604 TI - Enhancement of neutralizing efficacy by combining three monoclonal antibodies to human interferon-alpha. AB - Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity. PMID- 1710605 TI - Suppression of rat deoxycorticosterone-salt hypertension by kallikrein-kinin system. AB - Brown Norway kininogen-deficient rats had very low levels of plasma kininogens and lower levels of plasma prekallikrein, compared with those of normal rats of the same strain. Systolic blood pressure, determined by the tail-cuff method, of 5-week-old kininogen-deficient rats (106 +/- 0.4 mm Hg, n = 7) and the rate of systolic blood pressure increase with age were not different from those in normal rats. Weekly injections of deoxycorticosterone acetate (5 mg/kg s.c.) with 1% sodium chloride solution in drinking water after uninephrectomy at 7 weeks of age caused a gradual increase in the blood pressure of normal rats, reaching a plateau at 18 weeks of age, whereas that of deficient rats rose rapidly to 158 +/ 6 mm Hg 2 weeks after the start of treatment and continued to increase slightly, becoming significantly higher than normal rats at 8, 9, 10, 11, and 12 weeks of age (p less than 0.05 or 0.01). The levels of urinary prokallikrein and active kallikrein were slightly higher in deficient rats before deoxycorticosterone acetate-salt treatment but were not significantly increased after this treatment, whereas these levels in normal rats were increased 3.6- and 4.7-fold by this treatment. Urinary free kinin, collected from the ureter in untreated deficient rats, was below the detection limit. The plasma level of low molecular weight kininogen, the substrate of glandular kallikrein, was decreased in normal rats during the treatment. Continuous subcutaneous injection of aprotinin by an osmotic pump to normal rats induced significant increase in blood pressure. These results indicate that glandular kallikrein may play a suppressive role in deoxycorticosterone acetate-salt hypertension. PMID- 1710606 TI - Functional interactions between angiotensin II and substance P in the dorsal medulla. AB - Low doses of either angiotensin (Ang) II or substance P (SP) microinjected into the medial nucleus tractus solitarii (NTS) produce hypotension and bradycardia, mimicking activation of the baroreceptor reflex. Anatomical evidence suggests that Ang II binding sites in the medial NTS are located presynaptically on vagal afferent fibers that may contain SP and are codistributed with SP binding sites located postsynaptically on intrinsic medial NTS neurons. To evaluate whether the similar cardiovascular effects of Ang II and SP in the medial NTS could involve Ang II-evoked release of SP, we compared the effects of these peptides on the spontaneous activity of medial NTS neurons recorded in vitro and determined whether Ang II evoked release of SP from rat medulla slices. Both Ang II and SP (1 microM in artificial cerebrospinal fluid) excited 11 of 40 medial NTS neurons. In these cells, the peak response latency was significantly longer to Ang II than to SP (59.5 +/- 4.7 versus 26.5 +/- 2.4 seconds, p less than 0.0001). When rat medulla slices were perfused with Ang II (2 microM in Krebs' bicarbonate), release of SP immunoreactivity was increased by 400% over control perfusion with Krebs' solution alone (p less than 0.05). We have provided the first evidence for an excitatory action of Ang II on neurons in the NTS of the rat and for excitation by both Ang II and SP of a subset of neurons in the medial NTS. Moreover, we have shown for the first time that Ang II can stimulate the release of SP immunoreactivity from the brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710607 TI - Aniline blue staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men. AB - A retrospective study of 49 men with proven fertility and 396 suspected infertile men was conducted with the primary objective of investigating the relationship between the nuclear maturity of sperm and male fertility. Acidic aniline blue staining was used to detect chromatin defects of sperm nuclei related to their nucleoprotein content as associated with DNA. The discriminant value of the percentage of unstained nuclei (= percentage of mature heads, MH) and of other semen characteristics, was analysed by a stepwise (forward) linear regression model. Semen characteristics that discriminated significantly between the two groups of subjects were, in descending order: (1) the percentage of normal sperm, (2) the percentage of amorphous heads, (3) the percentage of tapered heads, (4) semen volume, and (5) the percentage of MH. The discriminant value of each of the significant characteristics was studied by means of ROC-curves. MH had the best ROC-curve profile; its cut-off value was found to be equal to 70% (74.5 +/- 2.6% for the donor group versus 53.0 +/- 1.1% for the patient group). A simple infertility score (SIS) including MH, was built according to the cut-off values inferred from the ROC-curves. SIS allowed an overall satisfactory separation of the two groups (less than or equal to 4 = fertile, 5-6 = uncertainty zone, greater than 6 = infertile). Our results indicate that the addition of the evaluation of sperm head maturity to routine semen analysis improves the assessment of fertility in men. PMID- 1710608 TI - Expression of leucocyte adhesion molecules on 66 clinical neuroblastoma specimens. AB - LFA-3, ICAM-1, HLA.ABC and HLA.DR expression was analyzed on 66 neuroblastoma specimens. HLA.ABC was expressed on 26 specimens, HLA.DR on 2, LFA-3 on 20 and ICAM-1 on 10. HLA.ABC and LFA-3 were positive on ganglioneuroblastoma or ganglioneuroma, but they were negative on neuroblastoma, independently of the clinical staging; HLA.ABC and LFA-3 were induced in vivo by chemotherapy in parallel with tumoral cell differentiation, in both the primary and the metastases. The expression of ICAM-1 was restricted to 5 of the 10 low-grade stage-1 or stage-2 specimens, 1 stage-3 specimen, and the primary tumors of 2 patients with stage-4 disease, analyzed hence at diagnosis and after chemotherapy (4 specimens); metastatic cells obtained in 1 of these patients were negative. HLA.ABC and LFA-3 expressed on both mycN-negative and -positive specimens, whereas ICAM-1 was restricted to MYCN-negative specimens. LFA-3 diffusely stained partially differentiated neuroblasts, Schwann cells and ganglion cells. The expression of HLA.ABC on differentiated neuroblasts varied from one sample to another and within the same tumor; Schwann cells were strongly positive, but ganglion cells were negative. In positive samples, ICAM-1 was expressed on differentiated neuroblasts and Schwann cells, but negative on ganglion cells; however, most of the differentiated tumors were ICAM-1-negative, suggesting ICAM 1 induction by unknown local signal. The 4 markers were negative on undifferentiated neuroblasts. The distribution of these 4 markers on clinical specimens was in agreement with their reactivity on fetal tissues, as well as with results obtained on neuroblastoma cell lines before and after in vitro treatment with IFN-gamma. PMID- 1710609 TI - Altered expression of P-glycoprotein and cellular adhesion molecules on human multi-drug-resistant tumor cells does not affect their susceptibility to NK- and LAK-mediated cytotoxicity. AB - Drug resistance has been associated with resistance to NK- and LAK-cell-mediated cytotoxicity. We evaluated this issue in human cell lines, using multiple myeloma cells (8226) and 2 multi-drug-resistant (MDR) sublines selected using doxorubicin (8226/Dox40) and mitoxantrone (8226/MR40). In parallel, we studied the human breast carcinoma cell line series MCF7, MCF7/D40 and MCF7/Mitox. Unlike the sensitive parental cell lines, all 4 sublines display MDR-patterns of resistance, with the P-glycoprotein pump (P-170) detected only in the doxorubicin-selected sublines. Flow cytometric and immunocytochemical analyses showed expression of cellular adhesion molecules ICAM-I and LFA-3, and MHC-Class-I (MCF7/D40 only), to be decreased in the doxorubicin-selected MDR-sublines, whereas expression of CD56 (Leu 19) was strongly up-regulated in 8226/Dox40. Lysis of P-170-positive MDR tumor cells by NK or LAK cells was, however, unaffected by these alterations, suggesting redundancy in effector:target-cell adhesion pathways. Mitoxantrone selected tumor cells did not display P-170, nor did they show altered expression of cellular adhesion molecules. Their susceptibility to NK or LAK cytolysis was also unimpaired as compared to the parental cell lines. Clinically, these results imply that immunotherapeutic modalities aiming at increased natural killer functions deserve full consideration even in patients who have become refractory to further cytostatic drug treatment. PMID- 1710610 TI - Production of a recombinant human T-cell leukemia virus type-I trans-activator (tax1) antigen and its utilization for generation of monoclonal antibodies against various epitopes on the tax1 antigen. AB - A 42-kDa recombinant protein, PX141, consisting of the trans-activator protein encoded by human T-cell leukemia virus (HTLV-1) (tax1 antigen) and the amino terminal fusion peptide of 12 amino acid residues of the alpha-peptide encoded by the plasmid pUC19 was produced. In order to investigate the immunogenicity of the tax1 antigen, mice were immunized with the purified PX141 and 4 anti-tax1 monoclonal antibodies (MAbs) designated TAXY-1, TAXY-6, TAXY-7 and TAXY-8 were generated, and their reactivity was characterized along with another anti-tax1 MAb, Lt-4. Immunoblot assays showed that all the MAbs reacted with the PX141, the native tax1 antigen expressed in various HTLV-1-infected cell lines and the gp68 of MT-2 cells expressing the tax1 amino acids 94-353. Immunoblot assays using recombinant, truncated tax1 antigens, XD59 (expressing amino acids 180-338) and XD128 (expressing amino acids 1-47 and 286-353) showed that: (1) TAXY-1 and Lt-4 did not react with either antigen; (2) TAXY-6 and TAXY-8 reacted with only XD128: and (3) TAXY-7 reacted with both. In addition, TAXY-1, but not the other MAbs, reacted with a putative tax antigen of an STLV-I-infected cell line, designated RfM26-I. Competitive binding assays showed that TAXY-6 and TAXY-8 did not compete against each other. Sera from HTLV-I-infected humans interfered with the binding of all of these anti-tax1 MAbs. These results indicate that the tax1 antigen and the PX141 express at least 5 distinct epitopes recognized by human and mouse antibodies. PMID- 1710611 TI - Synthetic peptides for Plasmodium vivax malaria sero-epidemiology. Application of Fmoc-polyamide and displacement chromatography. AB - The immunodominant epitope of Plasmodium vivax, one of the major causative agents of malaria in man, consists of the tandem repetitions of a nonapeptide sequence, AspArgAlaAsp/AlaGlyGlnProAlaGly, with Asp (variant d) or Ala (variant a), in the fourth position. Synthetic peptides corresponding to the P. vivax epitope, containing a different number of nonapeptide sequences, were prepared by solid phase synthesis according to the Fmoc-polyamide method. Three peptides, containing 1, 2, and 4 copies of the d variant, were assembled on the gel polymer; none of these peptides, however, was suitable for P. vivax sero epidemiology. A 45-peptide containing both the d and a variants, ddaad, was prepared by continuous-flow Fmoc-polyamide (flow-polyamide). Among the cleavage procedures evaluated for the removal of the five Mtr groups only TFMSA/TFA/1,2 ethanedithiol (1:89:10 by vol) brought deblocking to completion; a substantial level of impurities originated, however, from these procedures. The product was purified by reversed-phase displacement chromatography, a technique only recently applied to peptides, which shows distinct advantages over conventional, linear elution chromatography. In a single experiment, 107 mg of the crude mixture were loaded onto an analytical column (250 x 4 mm), obtaining in purified form 85% of the desired material present in the sample. An ELISA test base on the ddaad peptide was developed and is being applied to the sero-epidemiology of P. vivax malaria. PMID- 1710612 TI - Of HLA, tryps, and selection: an essay on coevolution of MHC and parasites. PMID- 1710613 TI - The influence of national attitudes on the use of radiotherapy in advanced and metastatic cancer, with particular reference to differences between the United Kingdom and the United States of America: implications for future studies. AB - Recent publications have suggested significant variation in dose and number of fractions of radiotherapy given to palliate advanced and metastatic cancer. Four types of published study are considered: multicenter trials, retrospective studies, patterns of care studies, and surrogate studies. These suggest that there are significant differences in the characteristics of patients considered incurable, the aims and expectations of palliative treatment, the predicted survival of patients treated palliatively, and the influence of clinical trials. Future studies are proposed to confirm such variation. PMID- 1710614 TI - Pelvic palliation radiotherapy of advanced bladder cancer. PMID- 1710615 TI - In vivo tumor growth enhancement by granulocyte colony-stimulating factor. AB - The intraperitoneal administration of human recombinant granulocyte colony stimulating factor (G-CSF) enhanced the growth of intradermally inoculated tumor in mice; in a Meth A fibrosarcoma model, G-CSF administration significantly shortened the latency before tumor appearance, accelerated the increase of tumor size, shortened the survival time of tumor-bearing mice and increased the incidence of lethal tumor growth. A similar growth-enhancing effect of G-CSF was observed in models employing Meth 1 fibrosarcoma, colon carcinoma 26, and L1210 leukemia, although not all the effects were statistically significant. In vitro study showed that G-CSF did not enhance Meth A growth in suspension culture or in soft agar. These data suggest that G-CSF enhances the Meth A growth not directly but through the mediation of host factors. The accumulation of neutrophils was histologically observed in the tumor nodule, the blood, and the spleen in mice given G-CSF repeatedly. The spleen cells and the peripheral blood leukocytes of G CSF-injected mice enhanced Meth A growth in vitro as compared with those of mice injected with physiological saline. These results suggest the possibility that the in vivo growth of tumor cells was enhanced by G-CSF-induced overproduction of cells including neutrophils. PMID- 1710616 TI - Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans. AB - Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium. PMID- 1710617 TI - Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro. AB - pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light. PMID- 1710619 TI - Further characterizations of bleomycin-sensitive (blm) mutants of Saccharomyces cerevisiae with implications for a radiomimetic model. AB - Direct selection for 12 mutations (blm) conferring hypersensitivities to lethal effects of bleomycins in Saccharomyces cerevisiae resulted in mutants exhibiting cross-hypersensitivity to ionizing radiation and hydrogen peroxide. Remaining mutations did not confer cross-hypersensitivity to radiation. All blm mutations were recessive, except codominant blm3-1, and were assigned to seven complementation groups. PMID- 1710618 TI - A novel transcriptional response by the cat gene during slow growth of Escherichia coli. AB - A novel response to growth rate was found with expression of the chloramphenicol acetyltransferase (cat) gene in Escherichia coli. The amount of cat mRNA relative to total RNA increased about 11-fold as growth rates decreased 5- to 6-fold, without an increase in translation. The accumulation of cat mRNA was in contrast to decreased cellular concentrations of total RNA, trxA, ompA, or 23S rRNA as the growth rate decreased and was not due to changes in gene dosage or mRNA stability. Stability of the cat mRNA does not appear to be regulated by growth rate. No significant change in either chemical or functional stability was observed within a five- to sixfold range of growth rates when chemostat-grown cells were used. However, cat mRNA stability was affected by growth medium composition. The half-life of cat mRNA decreased about threefold, with an approximate fourfold increase in generation time due to changes in growth medium. Transcriptional studies have indicated that accumulation of cat mRNA at slow growth rates is the result of a specific transcriptional response to changes in cellular generation times. We propose that increases in the cellular concentration of a specific message at slow growth rates may reflect an additional type of survival response in E. coli. PMID- 1710620 TI - Effect of temperature reduction on responsiveness to cholinomimetics in the taenia caeci of the guinea-pig. AB - 1. The effect of temperature reduction on the interaction of carbachol (CCh) and McN-A-343 (McN) with muscarinic receptors in the guinea-pig taenia caeci was investigated. 2. McN, a partial agonist, acted on the smooth muscle to produce contraction. The response was unaffected by tetrodotoxin and the pKB for inhibition by pirenzepine was 6.8, indicating that ganglionic M1 receptors were not involved in the response. 3. Reduction in temperature from 37 degrees C to 18 degrees C for 3 h led to a marked reduction in the contractile response to McN (2 200 microM) but no reduction in the response to CCh (0.1-3 microM). 4. The reduction in temperature was not accompanied by any change in the affinity of McN or CCh for muscarine receptors in binding experiments with [3H]-QNB. 5. The KA value for CCh determined after irreversible receptor inactivation with propylbenzilylcholine mustard followed by ca 60-min wash-out was 7.6 microM, a value similar to that obtained in binding experiments. 6. The EC50 for McN in producing contraction at 37 degrees C (2.1 microM) was similar to the KA value for the partial agonist obtained in experiments with the irreversible antagonist phenoxybenzamine (2.5 microM). It was also similar to the KB value determined at 18 degrees C (3.4 microM) when McN could be used as an antagonist of contractions to CCh. 7. At 18 degrees C, phosphatidylinositol (PI) hydrolysis by CCh was reduced to 23% of that at 37 degrees C. 8. It is concluded that reduction of muscarinic receptor activation of the PI pathway by cholinomimetics with lowering of the temperature could account for the findings with McN on contractility. PMID- 1710621 TI - Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems. AB - Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins. PMID- 1710622 TI - Cyclic adenosine 3',5'-monophosphate responses to parathyroid hormone, prostaglandin E2, and isoproterenol in dermal fibroblasts from patients with familial benign hypercalcemia. AB - Plasma concentrations of PTH are much lower for a given calcium or phosphorus level in patients with familial benign hypercalcemia (FBH, or familial hypocalciuric hypercalcemia) than in those with primary hyperparathyroidism; these and other data suggest that there might be tissue hypersensitivity to PTH in FBH. To test this hypothesis, we have used cultured dermal fibroblasts from abdominal skin biopsies of six patients with FBH and six age- and sex-matched controls as surrogate PTH-responsive tissues. Cells in 24-well plastic plates were exposed to vehicle, human PTH-(1-34) (10(-10)-10(-7) M), prostaglandin E2 (10(-6) M), or isoproterenol (10(-4) M) for 10 min in the presence of isobutylmethylxanthine, and cellular cAMP was determined by RIA. All cells responded to PTH with dose-dependent increases in cAMP, and all responded strongly to prostaglandin E2 and isoproterenol. There were no consistent or significant differences between control and FBH fibroblasts in maximal responses to the three agonists, and half-maximal stimulation was achieved with about 10( 9) M PTH in both normal and FBH cells. These data are not consistent with increased tissue sensitivity to PTH in FBH. PMID- 1710623 TI - Androgens: risks and benefits. PMID- 1710624 TI - Short and long term effects of growth hormone on circulating levels of insulin like growth factor-I (IGF-I), IGF-binding protein-1, and insulin: a placebo controlled study. AB - The short and long term effects of GH on serum concentrations of insulin-like growth factor-I (IGF-I), IGF-binding protein-1 (IGFBP-1), and insulin were investigated in women participating in an in vitro fertilization program. In this placebo-controlled study, sterile saline (eight women) or 24 IU GH (eight women) were given im on alternate days, starting on cycle day 4, in combination with GnRH and human menopausal gonadotropin. IGFBP-1 levels decreased significantly during the first 4 h after GH administration, whereas no significant changes were seen in the placebo group. The concentrations of serum IGF-I and insulin did not change during 4 h after GH injection. During the 11-day follow-up period, serum levels of both IGF-I and insulin were significantly higher in GH-treated than in placebo-treated women. These results suggest that the serum concentration of IGFBP-1 is not completely GH independent. They also support the earlier findings that long term treatment with GH increases serum IGF-I and insulin levels. PMID- 1710625 TI - The secretion of human chorionic gonadotropin as well as the alpha- and beta messenger ribonucleic acid levels are stimulated by exogenous gonadoliberin pulses applied to first trimester placenta in a superfusion culture system. AB - It is well documented that the hypothalamic decapeptide gonadoliberin (GnRH) controls the biosynthesis and secretion of pituitary gonadotropins; however, it is still unclear whether GnRH synthesized by the placenta plays the same role with respect to hCG. In the current study we have investigated the acute response of placenta tissue to a single GnRH pulse as well as the influence of GnRH pulses on the secretion of hCG and hCG mRNA concentrations elicited several hours after application of the peptide hormone. For this purpose we have used a superfusion culture model of first trimester placenta tissue (8-12 weeks of gestation). In the first hour after explantation of the tissue, hCG secretion was decreased, and increasing amounts of free subunits were released. Afterward, the original hCG secretion rates were recovered and maintained for several days, attended by decreased levels of free subunits in the culture medium. The superfusion model was superior to static incubations, since it showed approximately a 4-fold higher amount of hCG to be secreted within 24 h (day 3 of cultures). A single GnRH pulse (1 mumol/L; 30 min) caused a significantly increased transient release of hCG (P less than 0.0001). Two GnRH pulses (concentration range, 0.01-10 mumol/L; 30 min) applied 24 h after (first pulse) and in the interval between 36-48 h after (second pulse) the start of the superfusion culture elicited a long-lasting 2 fold increase in the hCG secretion rate, which rose approximately 6 h after the second GnRH pulse. This was correlated with increased mRNA concentrations measured by means of Northern blots of total RNA. At 0.02 mumol/L GnRH, 4-fold higher beta mRNA levels were observed. The alpha mRNA levels were 2.5-fold elevated. GnRH pulses of 0.01 and 10 mumol/L, respectively, were ineffective. A further effect of GnRH pulses was augmentation of the episodic character of hCG secretion. Our results suggest that GnRH causes different specific acute and late effects on the amount and pattern of hCG secretion as well as on hCG biosynthesis at the levels of both hCG subunit mRNAs. PMID- 1710626 TI - A cocktail of three monoclonal antibodies significantly increases the sensitivity of an enzyme immunoassay for human granulocyte-macrophage colony-stimulating factor. AB - A sensitive and specific two-site ELISA was developed for human granulocyte macrophage colony-stimulating factor (huGM-CSF) based on monoclonal antibodies (mAbs) which have been selected for high affinities and different epitope specificities. Using a cocktail of three mAbs, both for coating and, in their labeled form, for detection, a major increase in sensitivity was achieved (20 fold) compared to a two-site assay employing two different mAbs (one for coating and one for detection). The assay is as sensitive as the most sensitive biological assays. Recombinant mammalian cell expressed and natural huGM-CSF can be reliably detected down to 100 pg/ml (7 pmol/l). In contrast to conventional bioassays, the ELISA is highly specific for huGM-CSF and does not detect other human lymphokines. Results from quantification of recombinant and natural huGM CSF in ELISA and bioassay correlate. PMID- 1710627 TI - Guinea pig pancreatic ganglia: projections, transmitter content, and the type specific localization of monoamine oxidase. AB - The ganglionated plexus of the guinea pig pancreas was investigated by using histochemical, immunocytochemical, and tract-tracing methods in order to determine whether pancreatic ganglia are analogous to the ganglia of the enteric nervous system (ENS). Three lines of evidence suggest that the ganglia of the pancreas appear to be interconnected with one another, as are enteric ganglia. First, microinjections of the retrograde tracer Fluoro-Gold into individual pancreatic ganglia labeled the perikarya of neurons in distant pancreatic ganglia, whereas no labeling of neurons was observed if injections were placed in the connective tissue adjacent to pancreatic ganglia. Second, when the intercalating dye DiI was microinjected into single pancreatic ganglia in fixed tissues, DiI-labeled terminals were found in additional pancreatic ganglia. Finally, microinjections of the beta subunit of cholera toxin into individual pancreatic ganglia yielded similar results. The ganglionated plexus of the pancreas also expresses a diversity of transmitter content and cell type-specific localization of monoamine oxidase (MAO) that is analogous to the ENS. In common with guinea pig enteric ganglia, pancreatic ganglia contain highly varicose 5 hydroxytryptamine (5-HT)-immunoreactive axons and intrinsic neuropeptide Y (NPY)- and substance P (SP)-immunoreactive neurons. The vast majority, but not all, of SP-immunoreactive fibers in the pancreatic parenchyma also contain calcitonin gene-related peptide (CGRP) immunoreactivity. MAO-B was the primary type of MAO found in the intrinsic elements of the pancreas where it was located in neurons and fibers in the pancreatic parenchyma. In common with serotoninergic enteric neurons, MAO-B immunoreactivity was not found at the LM level in pancreatic serotoninergic neurites. In contrast, NPY- and tyrosine hydroxylase (TH) immunoreactive perivascular axons were found to contain abundant MAO-A, but no MAO-B immunoreactivity. It is concluded that MAO-B immunoreactivity is characteristic of a portion of the intrinsic innervation of the pancreas, whereas MAO-A immunoreactivity is a marker for the extrinsic sympathetic innervation of the pancreas. Because of its receipt of a direct neural innervation from myenteric ganglia of the bowel (Kirchgessner and Gershon, '90: J. Neurosci 10:1626-1642), similar connections, transmitter content and localization of type specific MAO, the ganglionated plexus of the pancreas should be regarded as an extension or subset of the ENS. PMID- 1710628 TI - Lipid composition of teat canal keratin collected before and after milking from Holstein and Jersey cows. AB - In three experiments, keratin was collected from individual teats of 40 Holstein and 20 Jersey cows immediately before and after milking. In Experiments 1 and 2, keratin collected from teats of 20 Holstein cows before milking was compared with keratin collected after milking. In Experiment 3, keratin was collected from two teats of 20 Jersey and 20 Holstein cows before milking and compared to the other two teats of the same cows after milking. All three experiments yielded similar results. In Holsteins, keratin weight before milking was 1.6 times greater than keratin weight after milking (3.1 vs. 1.9 mg). In Jerseys, only small amounts of keratin were removed during milking (3.5 mg before vs. 3.1 mg after) In Holsteins and Jerseys, neutral lipid concentration was 1.6 times greater after milking than before, suggesting that when keratin was removed during milking, only moderate amounts of lipid were removed. In Holsteins, total lipid collected per teat was similar before or after milking (59.2 vs. 48.5 micrograms). Results demonstrate that keratin collected after milking had a different lipid composition than keratin collected before milking. PMID- 1710629 TI - Atypical structure of the 23S ribosomal RNA molecule in certain oral bacteria. AB - Ribosomal RNA (rRNA) isolated from Wolinella recta and seven related bacteria was examined by agarose gel electrophoresis. The 23S rRNA molecule could not be detected in W. recta, Wolinella curva, Bacteroides gracilis, or Bacteroides ureolyticus. In place of the 23S molecule, there were three smaller molecules of approximately 1700, 650, and 600 bases designated 23S alpha, 23S beta, and 23S delta, respectively. An intact 23S rRNA molecule could be isolated from Wolinella succinogenes, Campylobacter concisus, and Campylobacter sputorum. The cleavage sites of the W. recta 23S rRNA molecule were located by direct RNA sequence analysis and were found to be in similar locations, nucleotides 546 and 1180, as cleavage sites described in other prokaryotes. The presence or absence of the 23S rRNA molecule may be a useful marker for these micro-organisms. PMID- 1710630 TI - House dust mite-derived amylase: allergenicity and physicochemical characterization. AB - Amylase activity was found in extracts of both Dermatophagoides pteronyssinus whole mite (0.16 U/mg) and spent growth medium (0.01 U/mg) but not in unused growth medium. It was also detected in all extracts of house dust obtained from mattresses (n = 20; geometric mean, 1.95 U/gm) and in 18 extracts of dust obtained from lounge room carpets (n = 20; geometric mean, 0.54 U/gm). Although the origins of amylase in dust are unclear, enzyme activity correlated with mite counts (n = 40; r = 0.35; p less than 0.05) and Der p I concentrations (r = 0.41; p less than 0.01). Mite amylase was purified from spent growth medium by affinity chromatography, gel filtration, and chromatofocusing. It was physicochemically similar to mammalian amylase with regard to molecular weight (60,000), charge heterogeneity (isoelectric point, 5 to 7) and the capacity to bind to an organomercurial affinity matrix. The optimum pH for enzymatic activity was revealed to be 6.4. IgE immunoblot studies demonstrated that the enzyme was allergenic and that its expression was dependent on the integrity of intrachain disulfide bonds. Sera from 25% of mite-allergic children and 46% of mite-allergic adults contained specific IgE to mite amylase. IgE to amylase was associated (p less than 0.01) with increased concentrations of total mite-specific IgE determined with a direct RAST assay. PMID- 1710631 TI - Skin test reactivity in patients suffering from lung and breast cancer. AB - Mast cells and histamine-mediated reactions may be altered in patients with cancer. In an attempt to characterize the possible skin defects in patients with cancer, we tested 22 patients suffering from lung cancers, 30 from breast cancers, and 30 age-matched normal individuals, using several compounds, in investigating the pathophysiology of the skin response. Histamine hydrochloride (10 and 100 mg/ml) and codeine phosphate (9%) were tested by prick test. Substance P (50 and 500 ng per injection site), phentolamine (20 micrograms per injection site), and carbachol (1 microgram per injection site) were tested by intradermal skin tests. Skin mast cells were also microscopically examined in 10 patients with lung cancer, five with breast cancer, and 10 normal subjects. The mean wheal sizes induced by all the tested substances were similar in patients with cancer and chronic bronchitis and in normal individuals. The flare to histamine, codeine phosphate, and substance P was completely abolished in 7/22 patients with lung cancer, but the lack of flare was not related to the age of the patients, nor to the staging of cancer, nor to metastasis. The mean numbers of alcian blue-stained or toluidine blue-stained positive mast cells were similar in normal subjects and in subjects with cancer. This study does not confirm the skin hyporeactivity of patients with cancer. PMID- 1710632 TI - Bacterial meningitis: T cell activation and immunoregulatory CD4+ T cell subset alteration. AB - Meningitis is the most important cause of acquired postnatal deafness and neurologic disorders in children. To determine if cell-mediated immunity is casually related to the pathogenesis of bacterial meningitis, T cell subsets were quantitated from blood of the 29 children with clinical and bacteriologic diagnosis of Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis bacterial meningitis. The CD4+ T cells increased and CD8+ T cells decreased in patients with meningitis as compared to patient control subjects (bacterial infections without meningitis) and normal healthy control subjects. An elevated percentage of CD25+ (interleukin-2 receptors) and HLA-DR+ (immune response gene-associated antigen) T cells were detected from all patients with meningitis. All 29 patients with meningitis had highly elevated CD4+ CD45R+ (suppressor-inducer) cells and reciprocally depressed CD4+ CDw29+ (helper inducer) cells compared with healthy age-matched normal and patient control subjects. These findings indicate characteristic immunologic T cell abnormalities from meningitis. The abnormal increase in the CD4+ CD45R+ suppressor-inducer or "virgin" cells and expression of activation antigens on T cells may be of help in future understanding of abnormal immune reactions from bacterial meningitis. However, deficiency of the CD4+ CDw29+ helper-inducer or "memory" cells may contribute to the impaired helper function for B cell-induced protective antibody synthesis to bacterial capsular polysaccharides found in this disease. PMID- 1710633 TI - Influence of indomethacin and L-cysteine on histamine and peptidoleukotriene release from superfused tracheas taken from guinea pigs passively sensitized with IgG1 and IgE antibodies. AB - We have previously reported differences in mediator release during equivalent levels of antigen (Ag)-induced smooth muscle contraction of guinea pig pulmonary tissues after passive sensitization with IgG1 versus IgE antibodies (Abs). In the present study, we have examined the influence of indomethacin (5 x 10(-6) mol/L) and L-cysteine (3 or 10 mmol/L) on mediator release from superfused trachea taken from guinea pigs passively sensitized with IgG1 or IgE Ab 1 day before in vitro studies. Tissues were challenged with Ag (oxazolone-human serum albumin conjugate), and contractions and superfusate histamine and peptidoleukotrienes were monitored at discrete time intervals thereafter. Superfusate mediator contents were determined by spectrophotofluorimetry (histamine) and RAST (peptidoleukotrienes). The profiles of peptidoleukotrienes were examined with high-pressure liquid chromatography. At equivalent levels of contraction, significantly less histamine and peptidoleukotrienes were found in superfusate samples after sensitization with IgE Abs. None of the drug pretreatments significantly altered Ag-induced histamine release after IgG1 or IgE sensitization. Indomethacin resulted in an increase in total measurable peptidoleukotrienes found only after IgG1 receptor activation, but it did prolong tracheal contractions with both Abs. L-cysteine, 10 mmol/L, resulted in an increase in total measurable superfusate peptidoleukotriene content under all experimental conditions. The percentage increase in peptidoleukotriene content from that found without drug pretreatment was larger in the case of IgE compared to IgG1 sensitization. During early time periods, after Ag challenge, measurable peptidoleukotriene levels in superfusate samples were similar for both Abs in the presence of L-cysteine, 10 mmol/L. These data suggest that there is a differential pattern of peptidoleukotriene metabolism after activation of IgG1 versus IgE receptors in guinea pig trachea. PMID- 1710634 TI - A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. AB - Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX) treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes. PMID- 1710635 TI - IEF analysis of HLA molecules immunoprecipitated by putative anti-class I-like alloantisera. AB - Two human alloantisera, previously described as possibly detecting new beta 2 microglobulin associated proteins, selectively expressed on HLA-A2 and HLA-A1 phytohaemagglutinin (PHA)-activated lymphocytes, immunoprecipitated only molecules with the same isoelectric point as HLA-A1 and A2 products. This result suggests that the selected alloantisera do not react with the products of an HLA class I locus different from ABC but probably recognize a new epitope arising on HLA-A molecules upon conformational changes consequent to cell activation. PMID- 1710636 TI - Cyclosporine A metabolism by cytochrome P-450III occurs in microsomes from rat liver but not from normal epidermis or psoriatic lesions. AB - Cyclosporine A is efficacious in the treatment of psoriasis when taken orally or injected intralesionally but not topically. Lack of penetration to necessary locations or rapid metabolism during passage through the epidermis may account for the ineffectiveness. Cytochromes P-450III in the liver are known to be involved in cyclosporine metabolism and inactivation. This study was undertaken to determine if an epidermal cytochrome P-450III exists that can inactivate topical cyclosporine A. Rats were treated with the macrolide antibiotic erythromycin to induce the cytochrome P-450III family of enzymes. Microsomal fractions were prepared from liver and epidermis of rats and from lesional areas of psoriasis patients. NADPH cytochrome C reductase activity was determined as a positive control for microsomal enzymatic activity. Formation of metabolite 1, the predominant metabolite of cyclosporine A, by liver microsomes was increased 193% after 10 d erythromycin treatment. The cytochrome P-450 dependent activity in microsomes from the epidermis of control and erythromycin-treated rats and in microsomes from psoriatic tissue was at the detection limits of the assay system. Cytochrome P-450III gene family mRNA were detectable by polymerase chain reaction in liver but not in psoriatic or normal epidermis. The lack of detectable P 450III mRNA and the absence or minimal conversion of cyclosporine A to inactive metabolites by epidermal microsomes suggest that the ineffectiveness of topical cyclosporine A in psoriasis may not be due to inactivation of cyclosporine A by cytochrome P-450 in the skin. PMID- 1710637 TI - Extracellular localization of human connective tissue mast cell granule contents. AB - In early phases of cutaneous inflammation, connective tissue mast cell degranulation is associated with apparent secretion and externalization of immunoreactive chymotryptic serine proteinase. To determine whether this event is associated with structural evidence of granule externalization, we studied the sequential evolution of IgE-mediated hypersensitivity in vivo, as well as mast cell degranulation provoked by a variety of stimuli in cultured explants of human skin. By 1 min after intradermal antigen challenge with ragweed extract, mast cell degranulation was associated with apparent extrusion of intragranule constituents into the pericellular connective tissue. Similar features typified cultured skin explants exposed for 45 min to anti-IgE and other mast cell secretagogues (morphine sulfate, calcium ionophore A23187, compound 48/80, and substance P). Once externalized, granule constituents could be identified within the dermal matrix by their rounded contour and structural similarity to solubilized granule matrices remaining within actively secreting cells. These data indicate that externalization of connective tissue mast cell granule contents occurs early after secretagogue exposure, potentially accounting for infrequent documentation of this event in naturally occurring dermatoses. The ability to recognize externalized granule products at a morphologic level should facilitate the understanding of interactions between mast cell-derived mediators and target structures of the dermal microvasculature. PMID- 1710638 TI - The autologous mixed epidermal cell-T lymphocyte reaction is elevated in psoriasis: a crucial role for epidermal HLA-DR+/CD1a- antigen-presenting cells. AB - The objective of this study was to determine whether epidermal cells (EC) from psoriasis lesions and uninvolved skin could stimulate autologous T lymphocytes in the in vitro autologous mixed epidermal cell-T lymphocyte reaction (autologous MECLR). The functional role of antigen-presenting cell (APC) subsets was concurrently determined in this reaction. Mononuclear cells and purified T lymphocytes from peripheral blood of psoriasis patients showed a clear proliferative response to autologous unpurified epidermal cells from involved as well as uninvolved skin. The autologous mixed leukocyte reaction (MLR) was not elevated in psoriasis patients. In healthy controls and contact allergy patients, T-lymphocyte proliferation was not observed either in the autologous MECLR or in the autologous MLR. The level of proliferation in the autologous MECLR from psoriasis patients correlated to the number of epidermal cells that were added. To exclude the possibility that the observed proliferation in the autologous MECLR in psoriasis was due to the presence of epidermal T lymphocytes that were being stimulated and expanded in vitro, the stimulator EC were gamma irradiated (30 Gy) in some experiments. Preincubation of EC with cyclosporin A (CsA) significantly inhibited the autologous MECLR. The CsA-induced inhibition could be neutralized by the addition of fresh untreated EC to these cultures. This indicated that one of the modes of action of CsA in resolving psoriasis is, as some investigators have already shown, via inhibition of epidermal accessory cell function. In the autologous MECLR, APC from psoriasis skin could initiate this reaction, whereas APC from peripheral blood could not. This occurred in an MHC class II restricted fashion. Depletion experiments showed that Langerhans cells (HLA-DR+/CD1a+) were not the principal stimulators of autologous T lymphocytes in the MECLR. These results indicated that mainly HLA-DR+/CD1a- epidermal cells from psoriasis patients could stimulate autologous peripheral blood T lymphocytes in an MHC class II-restricted fashion. PMID- 1710639 TI - Mediators, initiating the inflammatory response, released in organ culture by full-thickness human skin explants exposed to the irritant, sulfur mustard. AB - Mediators released from injured human skin that initiate the inflammatory response have not been adequately identified. Organ culture of full-thickness skin explants enables us to do so, because injury to the skin can be made in vitro, eliminating the rapid leakage of serum and infiltration of leukocytes that occur in vivo. In our studies, the military vesicant sulfur mustard (SM) (10 microliters of a 0.01 to 1.0% dilution) was topically applied to injure the epidermis of the explant. Then, the explants were cultured in small Petri dishes, usually for 18 h at 36 degrees C, and the organ-culture fluids were assayed for various inflammatory mediators. We found that the culture fluids from SM-exposed and control explants contained similar amounts of angiotensin-converting enzyme, trypsin-like and chymotrypsin-like proteases, acid phosphatase, beta glucuronidase, beta-galactosidase, lysozyme, deoxyribonuclease, ribonuclease, interleukin 1, and lactic dehydrogenase. However, the culture fluids from SM exposed explants contained increased amounts of histamine and plasminogen activating activity, and often prostaglandin E2, when compared to culture fluids from control explants. After 3 to 4 d in culture, full-thickness human skin explants, when exposed to 0.2% SM (but not when exposed to 1.0% SM), sometimes showed separation of the epidermis and increased collagenase activity (i.e., hydroxyproline release). Thus, histamine (from local mast cells), and prostaglandin E2 and plasminogen-activating activity (probably from both mast cells and epidermal cells) are apparently involved in early mediation of the inflammatory response. PMID- 1710640 TI - Patterns of glycosaminoglycan/proteoglycan immunostaining in human skin during aging. AB - Proteoglycans and their component glycosaminoglycans are involved in such cell cell and cell-matrix interactions as cell adhesion and migration, processes that are essential for embryonic and fetal development. As definitive organs such as skin emerge, structurally different proteoglycans partition into highly defined compartments. In skin, these compartments correspond to morphologically and functionally distinct layers. However, during the normal aging process, the relative amounts of structurally distinct proteoglycans apparently varies independently in each of these layers. This was demonstrated, in an indirect immunocytochemical study, through the use of monoclonal antibodies that detect structurally distinct domains in glycosaminoglycan chains of proteoglycans. Using samples of normal human skin obtained from individuals ranging in age from 20 weeks of gestation to 98 years of age, we determined that a common distribution pattern existed in skin. The epidermis contained chondroitin 4- and keratan sulfates, the basal lamina was the only layer that contained chondroitin 6 sulfate, the papillary and reticular dermis contained principally dermatan sulfate. In addition, antibodies that recognize native domains in chondroitin sulfates identified proteoglycan subsets that partitioned into distinct layers. An important new finding was that the relative amounts of specific types of glycosaminoglycans varied in an age- and layer-dependent manner. In the epidermis there was a notable increase in keratan sulfate beginning at age 50. Chondroitin 6-sulfate, found principally in the basal lamina, decreased after age 60. In the papillary dermis, the amount of dermatan sulfate increased after age 50, whereas the amount of novel chondroitin sulfate epitope, detected by antibody 4C3, decreased with age. Thus, age-related changes in proteoglycan distribution exist and correlate with morphologic and functional changes that occur in the intrinsic process of aging in human skin. PMID- 1710641 TI - [Molecular heterogeneity of hCG in urine of the patients with trophoblastic diseases]. AB - In addition to whole hCG, hCG beta-related molecular species are also present in the serum and urine of patients with trophoblastic diseases. We simultaneously measured whole hCG, free hCG beta and beta-core fragment in the serum and urine of patients with hydatidiform mole and choriocarcinoma by the highly specific and sensitive sandwich enzyme-immunoassay (EIA) systems using two kinds of antibodies. During the clinical courses of patients, although the whole hCG levels in serum and urine were closely correlated, the concentrations in the serum were almost twice as high as those in the urine. The free hCG beta levels were also closely correlated in serum and urine, but the concentrations of free hCG beta were extremely low as compared with those of whole hCG. Furthermore, free hCG beta/whole hCG ratios were significantly higher in choriocarcinoma patients than in hydatidiform mole patients. While whole hCG and free hCG beta were contained in both serum and urine, the beta-core fragment could be detected only in the urine of the patients. The relative contribution of the beta-core fragment to the total urinary hCG beta-immunoactivity accounted for about 40% in hydatidiform mole and about 70% in choriocarcinoma. We conclude that whole hCG should be measured in the serum rather than in the urine as a tumor marker for trophoblastic diseases, and suggest that the ratios of whole hCG, free hCG beta and beta-core fragment to each other may be useful indices to employ in the differential diagnosis of trophoblastic diseases. PMID- 1710642 TI - [Intravascular platelet transfusion in utero for the treatment of immunologic thrombocytopenic purpura: a case report]. PMID- 1710643 TI - Radioprotective properties of DNA methylation-disrupting agents. AB - 5-Azacytidine and sodium butyrate, two DNA methylation-disrupting agents, were tested for radioprotective properties on V79A03 cells. Both compounds can activate genes not previously expressed (e.g. metallothionein). 5-Azacytidine treatment (3 microM, 24 h) caused a 50% decrease in the 5-methylcytosine content of V79A03 DNA whereas sodium butyrate treatment (1 mM, 24h) resulted in a 700% increase in 5-methylcytosine content. Additionally, 5-azacytidine treatment resulted in the increased survival of V79A03 cells, with treatment 24 h prior to exposure to gamma radiation providing a dose reduction factor of 1.8. Sodium butyrate treatment did not result in a significant increase in survival. These results indicate that the hypomethylation of genomic DNA prior to exposure to gamma radiation correlates with an increase in survival of V79A03 cells, possibly due to the activation of the enzymes involved in repair. PMID- 1710644 TI - Classification and identification of bacteria by Fourier-transform infrared spectroscopy. AB - This study describes a computer-based technique for classifying and identifying bacterial samples using Fourier-transform infrared spectroscopy (FT-IR) patterns. Classification schemes were tested for selected series of bacterial strains and species from a variety of different genera. Dissimilarities between bacterial IR spectra were calculated using modified correlation coefficients. Dissimilarity matrices were used for cluster analysis, which yielded dendrograms broadly equated with conventional taxonomic classification schemes. Analyses were performed with selected strains of the taxa Staphylococcus, Streptococcus, Clostridium, Legionella and Escherichia coli in particular, and with a database containing 139 bacterial reference spectra. The latter covered a wide range of Gram-negative and Gram-positive bacteria. Unknown specimens could be identified when included in an established cluster analysis. Thirty-six clinical isolates of Staphylococcus aureus and 24 of Streptococcus faecalis were tested and all were assigned to the correct species cluster. It is concluded that: (1) FT-IR patterns can be used to type bacteria; (2) FT-IR provides data which can be treated such that classifications are similar and/or complementary to conventional classification schemes; and (3) FT-IR can be used as an easy and safe method for the rapid identification of clinical isolates. PMID- 1710645 TI - Characterization of human antibody-binding sites on the external envelope of human immunodeficiency virus type 2. AB - Antibody-reactive peptide scanning (Pepscan) using overlapping nonapeptides and human sera as probes allows the identification of amino acids contributing significantly to antigen-antibody interaction. Five-hundred and two overlapping nonapeptides derived from the human immunodeficiency virus type 2 strain Rod (HIV 2Rod) external envelope glycoprotein gp125 were synthesized to serve as probes for reactivity with eight sera of HIV-2-infected individuals. Fifteen antibody binding regions were identified, among which two amino-terminal regions [E3, amino acids (aa) 118 to 132; E4, aa 125 to 141] and four carboxy-terminal regions (E11, aa 303 to 324; E12, aa 340 to 358; E14, aa 436 to 452; E15, aa 486 to 507) were the most antigenic. The amino acids in binding sites E3 and E4 were highly variable among simian immunodeficiency virus (SIV), HIV-2 and HIV-1. The antibody binding domains E14 and E15 were highly conserved among HIV-2 strains (94% and 86% identity of HIV-2Rod to HIV-2Isy and HIV-2NIHZ, respectively). Both domains had more amino acids in common with SIV (88% for E14, 64% for E15) than with HIV 1 (41% for E14, 45% for E15). Epidemiological studies revealed that the sera of African HIV-2-infected individuals bound the E11 and E15 peptides best (31%, 8/26). The sera of African HIV-1-infected individuals showed significant levels of cross-reactivity to the HIV-2 peptides, especially to the E15 peptide, whereas the sera of European HIV-1-infected individuals showed only moderate levels of cross-reactivity. If peptides covering the E15 epitope were used, African HIV-2 positive sera showed only a low level of cross-reactivity (4%) to E15 of HIV-1 and SIV. African HIV-1-positive sera bound the HIV-1 E15 peptide best (81%, 52/64), but showed high levels of cross-reactivity to SIV E15 (17%, 11/64) and HIV-2 E15 (25%, 16/64). European HIV-1-positive sera showed a high level of reactivity of HIV-1 E15 (91%, 50/55) and a low level of cross-reactivity to the HIV-2 (2%, 1/55), and none to the SIV E15 (0/55) peptide. These results indicate that the immunodominant regions of the HIV-2 external envelope (E11 and E15) align with the most immunodominant regions of HIV-1. PMID- 1710646 TI - Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins. AB - The immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines. PMID- 1710647 TI - Regulated M1 mRNA splicing in influenza virus-infected cells. AB - Influenza virus RNA segment 7 generates three poly(A)+ RNAs, M1 mRNA, M2 mRNA and mRNA3, the last of which has almost no coding capacity; M2 mRNA and mRNA3 derive from M1 mRNA by removal of a single intron. The kinetics of M1 and M2 mRNA accumulation in the cytoplasm of productively infected cells were studied by means of a quantitative RNA protection assay; the ratio of M2 mRNA to M1 mRNA increased 2.7-fold during the course of infection. To analyse the basis for this change, the kinetics of M1 and M2 mRNA synthesis and nuclear accumulation, their stability and nucleocytoplasmic transport were studied. Under the experimental conditions used, the synthesis of segment 7-specific RNA showed a peak at 4 h post-infection and continued later at a slower rate. The half-lives of M1 and M2 mRNAs were indistinguishable (2.73 h for M1 mRNA and 2.70 h for M2 mRNA) and the kinetics of nucleocytoplasmic transport in vivo or in vitro showed no preference for either mRNA early or late in infection. Consequently, regulation at the level of mRNA splicing is proposed. Using the mRNA synthesis and stability data, a simulation was performed to predict the change in splicing efficiency required to account for the mRNA accumulation results. The best fit was obtained when splicing efficiency changed about 20 times during a period in which viral gene expression was maximal. PMID- 1710648 TI - Fusion activity of flaviviruses: comparison of mature and immature (prM containing) tick-borne encephalitis virions. AB - The fusion activity of flaviviruses [tick-borne encephalitis (TBE) virus and Japanese encephalitis virus] was assessed by inducing fusion from without of C6/36 mosquito cells with purified virus preparations. Membrane fusion and polykaryocyte formation was observed only after incubating the viruses at acidic pH. Two groups of monoclonal antibodies reacting with distinct non-overlapping antigenic domains on the TBE virus protein E inhibited fusion from without. One of these domains contains the most highly conserved and putative fusion-active sequence of the flavivirus protein E. Of five TBE virus monoclonal antibody escape mutants, each defined by a single amino acid substitution in the envelope protein E, one revealed a reduced fusion activity and another one a lower pH threshold. TBE virus grown in the presence of ammonium chloride as well as Langat virus purified from the supernatant of infected chick embryo cells contained the precursor of protein M (prM) rather than M itself. These 'immature' virions did not cause fusion from without, suggesting that the proteolytic processing of prM may be necessary for the generation of fusion-competent virions. PMID- 1710649 TI - Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells. AB - The cDNA encoding the E and NS1 proteins of the yellow fever virus (YFV) was expressed in Spodoptera frugiperda cells via the recombinant baculovirus Ac-E. NS1 as a gp100 precursor which was cleaved to generate the recombinant proteins E and NS1 similar in size, folding and antigenicity to the authentic ones. Recombinant protein E exhibited immunodominant epitopes as judged by its reactivity with YFV-neutralizing MAbs. Using the Triton X-114 phase separation system, authentic and recombinant E proteins as well as the gp100 precursor exhibited hydrophobic properties similar to those of integral membrane proteins. Recombinant protein E was found neither in the extracellular medium nor on the cell surface, suggesting that it did not migrate within the secretory pathway of insect cells. Analysis of protein NS1 expressed in primate and insect cells revealed that the newly synthesized 48K NS1 glycoprotein was converted to a heat labile gp72 homo-oligomeric form. This phenomenon did not require the presence of carbohydrate groups. Using the Triton X-114 phase separation system, the oligomeric form of NS1 was shown to be associated with cellular membranes although it appeared less hydrophobic than protein E and gp100. A small fraction of YFV NS1 oligomers were transported throughout the secretory pathway to be shed into the extracellular medium of primate cells. YFV NS1 oligomers migrated from the endoplasmic reticulum to the Golgi complex, whereas their N-oligosaccharides of the high-mannose type are processed to the complex-mannose type. Protein NS1 expressed by recombinant baculovirus-infected insect cells was not found in the extracellular medium but associated with the plasma membrane of the cells. Two recombinant NS1 forms were detected in insect cells: a major one with an apparent Mr of 48K and a minor one of 47K in which N-linked glycans were probably processed to a trimannosyl core without further elongation. Thus, it appears that the transport strategy as well as the N-glycosylation of NS1 in insect cells infected with recombinant baculovirus were different from those of the NS1 in primate cells infected with YFV. PMID- 1710650 TI - Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides. AB - The entire amino acid sequence of human cytomegalo-virus (HCMV) 150K matrix phosphoprotein (pp150), consisting of 1048 amino acid residues, was divided into 95 overlapping 20 amino acid peptides which were synthesized on polyethylene rods. The rods were subjected to ELISA with pooled anti-HCMV-positive and anti HCMV-negative sera. Four peptides recognized by the anti-HCMV-positive pool only were synthesized by the solid-phase method and their reactivity in a conventional ELISA, using a panel of 14 individual anti-HCMV-negative and 20 anti-HCMV positive antisera, was evaluated; three peptides were found to be specifically reactive. Results obtained with one of these peptides (residues 595 to 614) in ELISA showed a good correlation with those obtained using a routinely performed complement fixation test. PMID- 1710651 TI - 17D yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein. AB - We have constructed a recombinant baculovirus containing cloned DNA encoding the membrane and envelope (E) proteins of 17D yellow fever vaccine virus. Spodoptera frugiperda cells infected with this recombinant baculovirus produced a 66K protein which corresponded to the estimated size of the protein encoded by the cloned inserted DNA, and a 54K protein with the same molecular size as that of the authentic 17D yellow fever virus E protein. This recombinant 54K protein was labile, producing E protein-specific breakdown products (45K to 36K). Indirect immunofluorescence, using a panel of E protein-specific monoclonal antibodies, showed that the recombinant protein was presented both inside as well as on the surface of cells and was antigenically indistinguishable from the E protein of 17D yellow fever vaccine virus. PMID- 1710652 TI - Insecticide susceptibility of Aedes aegypti from Santo Domingo, Dominican Republic. AB - The insecticide susceptibility of Aedes aegypti adults and larvae from Santo Domingo, Dominican Republic, was investigated using World Health Organization standard procedures. A field strain was more resistant to insecticides than a colony strain that originated from the same place. Larvae produced from ovitrap collected eggs were resistant to temephos (78.2% mortality on exposure to 0.025 mg/liter). Mortality rates after exposure of adults to discriminating concentrations showed that wild populations were resistant to DDT, malathion, propoxur, permethrin and deltamethrin. The problem of resistance was considered serious enough to warrant consideration of control measures other than the use of chemicals. PMID- 1710653 TI - Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. AB - Inhibition of human immunodeficiency virus reverse transcriptase is currently considered a useful approach in the prophylaxis and intervention of acquired immunodeficiency syndrome (AIDS), and natural products have not been extensively explored as inhibitors of this enzyme. We currently report that the reverse transcriptase assay developed for the detection of the enzyme in virions involving polyadenylic acid.oligodeoxythymidylic acid (poly rA.oligo dT) and radiolabeled thymidine 5'-triphosphate (TTP), can be applied as a simple method for screening the human immunodeficiency virus type 1 reverse transcriptase (HIV 1 RT) inhibitory potential of natural products. As reported herein, 156 pure natural products have been examined in this system. Benzophenanthridine alkaloids such as faragaronine chloride [1] and nitidine chloride, which are known inhibitors of avian myeloblastosis virus reverse transcriptase, demonstrated potent activity in the HIV-1 RT system, and 1 (IC50 10 micrograms/ml) was adopted as a positive-control substance. Additional inhibitors found were columbamine iodide [2] and other protoberberine alkaloids, the isoquinoline alkaloid O methylpsychotrine sulfate [3], and the iridoid fulvoplumierin [4]. A number of indolizidine, pyrrolizidine, quinolizidine, indole, and other alkaloids, as well as compounds of many other structural classes, were tested and found to be inactive. A total of 100 plant extracts have also been evaluated, and 15 of these extracts showed significant inhibitory activity. Because tannins and other polyphenolic compounds are potent reverse transcriptase inhibitors, methods were evaluated for the removal of these from plant extracts prior to testing. Polyphenolic compounds were found to be responsible for the activity demonstrated by the majority of plant extracts. After appropriate tannin removal procedures were established, the bioassay system was shown to be generally applicable to both pure natural products and plant extracts. The method also proved useful in directing an isolation procedure with Plumeria rubra to yield fulvoplumierin [4] as an active compound (IC50 45 micrograms/ml). PMID- 1710654 TI - New avarone and avarol derivatives from the marine sponge Dysidea cinerea. AB - Six new avarol and avarone derivatives, 3'-hydroxyavarone [3], 3',6' dihydroxyavarone [4], 6'-hydroxyavarol [5], 6'-acetoxyavarol [6], 6' acetoxyavarone [7], and 6'-hydroxy-4'-methoxyavarone [8], are reported from the Red Sea sponge Dysidea cinerea. The structures of the new compounds were determined by spectroscopic data, mainly 1D and 2D nmr measurements. The absolute configurations of 5, 6, and 7, and most likely also of 3, 4, and 8, were established on the basis of cd measurements to be the same as that of avarol. Several of the new compounds are cytotoxic, possess antimicrobial activities, and have anti-HIV-1 reverse transcriptase activities; the most active is compound 8. PMID- 1710655 TI - Randomized study of cisplatin dose intensity in poor-risk germ cell tumors: a Southeastern Cancer Study Group and Southwest Oncology Group protocol. AB - Between 1984 and 1989, 159 patients presenting with advanced germ cell cancer were entered on a randomized clinical trial comparing the efficacy and toxicity of etoposide and bleomycin and either standard-dose cisplatin (20 mg/m2 daily for 5 days) or high-dose cisplatin (40 mg/m2 daily for 5 days). Of the 159 patients, 153 were assessable for toxicity and response. As expected, patients receiving the high-dose cisplatin regimen experienced significantly more neurotoxicity, ototoxicity, nausea and vomiting, and myelo-suppression. Four patients (3%) died related to therapy. Despite the toxicity encountered, dose intensity was maintained. Overall, 84% of patients in the high-dose arm received 80% or more of the projected dose of cisplatin, etoposide, and bleomycin; and 90% of patients on the standard-dose arm received 80% or more of the projected dose. Of the 76 eligible patients randomized to receive the high-dose cisplatin regimen, 52 (68%) became disease-free with chemotherapy alone or with subsequent resection of residual teratoma or cancer. Of the 77 patients randomized to the standard-dose arm, 56 (73%) became disease-free with chemotherapy alone or with surgery. Median follow-up is now 24 months. Eleven patients (three high-dose and eight standard dose) relapsed from disease-free status. Overall, 74% of patients receiving the high-dose cisplatin regimen are alive, and 63% are continuously free of disease. Of the patients receiving the standard-dose cisplatin regimen, 74% are alive, and 61% are continuously free of disease. This randomized prospective trial in advanced germ cell cancer achieved dose intensity of the most active single agent in this disease. This dose intensity did not translate into an improved survival or cure. We conclude that dose escalation of cisplatin beyond standard doses results in excess toxicity with no accompanying therapeutic benefit. PMID- 1710656 TI - Value of follow-up procedures in patients with large-cell lymphoma who achieve a complete remission. AB - Salvage therapy for relapsed large-cell lymphoma (LCL) is more effective in patients with minimal disease, suggesting that early detection of relapse might increase the chance of long-term survival. To determine whether current follow-up procedures are effective in identifying preclinical disease, we analyzed patterns of relapse in 139 LCL patients who achieved a complete remission (CR) with high/moderate-dose methotrexate with leucovorin, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (M/m-BACOD). The timing and results of all posttreatment follow-up tests were examined in the 36 patients who relapsed from complete remission (CR) and 46 controls who remain in CR. Despite conscientious posttreatment follow-up, only two of the 36 relapses (6%) were detected before the development of symptoms. Sixty-seven percent of patients relapsed in new disease sites (42% in new and old sites, and 25% in new sites only). Consistent with this observation, the tests most sensitive to clinical relapse were those not targeted to specific sites of disease: gallium scan (sensitivity, 90%), physical examination (80%), and lactate dehydrogenase (LDH) (65%). Of screening tests performed, only LDH was successful in detecting preclinical relapse, with a sensitivity of 42% and specificity of 85% for impending symptomatic relapse. These results indicate that conventional screening was ineffective in detecting preclinical relapse in LCL patients. We recommend prospective evaluation of a strategy that (1) screens with a frequency appropriate to a patient's risk of relapse, (2) uses sensitive test(s) not targeted to specific sites, and (3) limits aggressive screening to those high risk patients eligible for potentially curative salvage therapy. PMID- 1710657 TI - Chronic quinolinic acid lesions in rats closely resemble Huntington's disease. AB - We previously found a relative sparing of somatostatin and neuropeptide Y neurons 1 week after producing striatal lesions with NMDA receptor agonists. These results are similar to postmortem findings in Huntington's disease (HD), though in this illness there are two- to threefold increases in striatal somatostatin and neuropeptide Y concentrations, which may be due to striatal atrophy. In the present study, we examined the effects of striatal excitotoxin lesions at 6 months and 1 yr, because these lesions exhibit striatal shrinkage and atrophy similar to that occurring in HD striatum. At 6 months and 1 yr, lesions with the NMDA receptor agonist quinolinic acid (QA) resulted in significant increases (up to twofold) in concentrations of somatostatin and neuropeptide Y immunoreactivity, while concentrations of GABA, substance P immunoreactivity, and ChAT activity were significantly reduced. In contrast, somatostatin and neuropeptide Y concentrations did not increase 6 months after kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) lesions. At both 6 months and 1 yr, QA lesions showed striking sparing of NADPH-diaphorase neurons as compared with both AMPA and KA lesions, neither of which showed preferential sparing of these neurons. Long-term QA lesions also resulted in significant increases in concentrations of both 5-HT and 5-hydroxyindoleacetic acid (HIAA), similar to findings in HD. Chronic QA lesions therefore closely resemble the neurochemical features of HD, because they result in increases in somatostatin and neuropeptide Y and in 5-HT and HIAA. These findings strengthen the possibility that an NMDA receptor-mediated excitotoxic process could play a role in the pathogenesis of HD. PMID- 1710658 TI - Temperature-dependent peptidergic feedback: potential role in seasonal egg laying in Aplysia. AB - alpha-Bag cell peptide (alpha-BCP), one of the secretory products of the neuroendocrine bag cells in Aplysia, has been reported by various investigators to have either excitatory or inhibitory feedback effects. Though conflicting, these results may be explained by the difference in temperature at which the experiments were performed. Because egg laying in this animal is temperature dependent, the alteration in function of this peptide by temperature may offer a possible molecular basis for the seasonal regulation of egg laying. This hypothesis was investigated by assessing the feedback actions of alpha-BCP at various temperatures. At 15 degrees C, alpha-BCP hyperpolarized bag cells, shortened the duration of synaptically evoked bag cell discharges, and reduced the number of action potentials per discharge. However, at 20 degrees C, the peptide depolarized bag cells, lengthened discharges, and increased the number of action potentials per discharge. A temperature-dependent influence on bag cell cAMP levels may underlie these effects, because alpha-BCP reduced basal cAMP levels in intact bag cells at temperatures of 15 degrees C and below, while at 17 22 degrees C it increased these levels. However, the inhibitory effects of alpha BCP on stimulated adenylate cyclase activity in bag cell homogenates were not temperature dependent. Moreover, a low-Ca2+/high-Mg2+ solution abolished alpha BCP's ability to increase bag cell cAMP levels at 20 degrees C. This suggests that the peptide may evoke the secretion of an excitatory modulator at the higher temperature. These results imply that alpha-BCP is autoinhibitory at typical winter temperatures, but becomes autoexcitatory as ocean temperature rises in the summer. Thus, the peptide may function in coordination with other factors to regulate egg laying in response to seasonal temperature variations. PMID- 1710659 TI - The segmentation of the leech nervous system is prefigured by myogenic cells at the embryonic midline expressing a muscle-specific matrix protein. AB - In the leech, adult muscle cells and embryonic mesodermal/myogenic cells express the cell-type-specific Laz 10-1 epitope on extracellular matrix-associated proteins. Using this muscle-specific epitope as a marker, we found the following correlations between the development of identifiable myogenic cells at the embryonic midline and the segmentation of the leech CNS into 32 reiterative ganglia. (1) During the production of mesodermal and ectodermal stem cells, cell bodies of midline myogenic cells create 32 anterior-posterior intervals along the midline of the embryonic germinal plate. The mesoblasts then rearrange themselves into 32 somites whose spacing follows the intervals between the midline myogenic cell bodies. (2) Bilateral segmental zones of myogenic differentiation originate in juxtaposition to the midline myogenic cells. (3) The first two types of muscle precursors develop from midline and adjacent bilateral myogenic cells; they are the precursors of the CNS muscles and of three groups of ventral blood sinus muscles. These two types of muscle precursors demarcate the boundaries of the territory within which neuroblasts proliferate and coalesce into segmental hemiganglionic primordia: Cell bodies of muscle precursors, like cornerstones, demarcate the anterior and posterior borders of the hemiganglionic primordia; their longitudinal processes surround the expanding hemiganglionic primordia on the medial, lateral, dorsal, and ventral aspect. The contours of muscle precursors and midline myogenic cells are sharply delineated by immunohistochemical staining of the Laz 10-1 epitopes. In contrast, undifferentiated mesoblasts are surrounded by diffuse layers of stained epitopes, which are expressed at fluctuating levels. Because elevated levels of this matrix epitope are associated with mesodermal/myogenic cells undergoing morphogenetic rearrangements, it may participate in the molecular mechanisms underlying the segmentation of the nervous system. PMID- 1710660 TI - New hope for children with Kawasaki disease. AB - Kawasaki disease is now the most common cause of acquired heart disease in America's children. It is an acute febrile illness that may cause coronary artery aneurysm formation in infected children. The results of a multicenter, randomized trial on the effect of intravenous administration of gamma globulin (IVGG) plus aspirin versus aspirin alone upon coronary aneurysm formation show a decrease in coronary aneurysm formation from the usual 20%-30% to 3%. Administration of IVGG presents some unique challenges for nurses. Also, the pediatric nurse must educate parents and children about this disease to prepare them for discharge and long-term follow-up care. PMID- 1710661 TI - Calcitonin gene-related peptide stimulates adenylate cyclase and relaxes intracerebral arterioles. AB - The effects of calcitonin gene-related peptide (CGRP) on lumen diameter and adenylate cyclase activity in isolated intracerebral arterioles were examined. CGRP produced a concentration-dependent relaxation of spontaneous tone developed by the arterioles with an EC50 value of 3.9 x 10(-9) M. Calcitonin, as well as substance P, which is frequently colocalized with CGRP, had no effect on arteriolar tone. CGRP also relaxed arterioles contracted with the thromboxane mimetic, U-44619, yielding an EC50 value of 3 x 10(-9) M. The CGRP fragment, human CGRP(8-37), antagonized CGRP-induced relaxation in a noncompetitive manner. Adenylate cyclase activity in single arterioles was stimulated by CGRP, but not by substance P, in a concentration-dependent fashion. Half maximal stimulation occurred at 8 x 10(-9) M, whereas maximum stimulation (2.5-fold over basal) occurred at 10(-7) M. CGRP(8-37) inhibited CGRP-stimulated adenylate cyclase activity over a concentration range of 10(-9) to 10(-5) M. The results demonstrate that CGRP stimulates adenylate cyclase activity and is a potent vasodilator of small parenchymal cerebral arterioles in vitro and may play an important role in the regulation of cerebral blood flow. PMID- 1710662 TI - In vivo evidence for tachykininergic transmission using a new NK-2 receptor selective antagonist, MEN 10,376. AB - We have studied the effects of two newly developed tachykinin antagonists, MEN 10,207 and MEN 10,376, which are highly selective for NK-2 tachykinin receptors on tachykinin-induced contraction in the rat urinary bladder and guinea pig airways in vivo. MEN 10,207 exhibited antagonism only at doses which produced agonist effects. By contrast, MEN 10,376 was devoid of significant agonist activity at i.v. doses (1-3 mumol/kg) which selectively antagonized the effects of an NK-2 agonist [beta-Ala8]-neurokinin A(4-10) (bladder contraction in rats, bronchoconstriction in guinea pigs) without affecting the response to an NK-1 agonist [Sar9]-substance P sulfone (hypotension, salivation and bladder contraction in rats, bronchoconstriction in guinea pigs). At these NK-2-selective blocking doses, MEN 10,376: 1) did not affect urodynamic parameters at cystometry in normal rats but reduced amplitude of micturition contractions following induction of chemical (intravesical xylene) cystitis and 2) reduced by a maximum of 50% the noncholinergic bronchoconstrictor response to vagal nerve stimulation. These findings provide the first evidence for involvement of NK-2 receptors in physiological responses in the urinary and respiratory tracts. PMID- 1710663 TI - Effects of agonist efficacy on desensitization of phosphoinositide hydrolysis mediated by m1 and m3 muscarinic receptors expressed in Chinese hamster ovary cells. AB - Muscarinic receptor agonist-induced desensitization of phosphoinositide (PI) hydrolysis and loss of receptors were studied in Chinese hamster ovary (CHO) cells transfected with the m1 and m3 muscarinic receptor genes. Long-term exposure to the full agonist carbamylcholine (CBC) resulted in a time-dependent attenuation of the maximal PI response and a decrease in agonist potency. This desensitization was accompanied by a parallel loss of maximal ligand binding without an alteration of the binding affinity. The time course of both receptor desensitization and down-regulation was similar in m1 and m3 CHO cells. The PI response to the partial agonist McN-A-343 (McN) in m1 cells was more sensitive to desensitization by CBC than the response to the latter agonist, and this desensitization was faster than receptor down-regulation. Desensitization of the PI response to McN was reflected as a decrease in the maximal response without a marked change in potency. McN induced slow desensitization of the PI response to CBC but a much faster desensitization of its own response. Our data provide evidence that although muscarinic agonist-induced desensitization of PI hydrolysis in CHO cells is due mainly to loss of receptors, there are other important factors which play a role in this process, e.g., receptor-effector uncoupling. The relative contribution of these different mechanisms depends on the efficacy of the agonists used for the receptor desensitization and activation steps. PMID- 1710664 TI - Family-centered nursing in the postanesthesia care unit: the evaluation of practice. AB - The philosophy of nursing care at the Medical Center Hospital of Vermont (MCHV) is family-centered. In the summer of 1989, an open family visitation policy was implemented. The goals of allowing family members into the PACU were to (1) enhance communication between staff and family members, and (2) evaluate whether patients and family members perceived the open visitation policy as beneficial. The specific focus of phase I of the study is the evaluation of the patients and their families who participated in the experience with the PACU staff. A questionnaire was developed to assess several dimensions of the experience. To date, 46 complete sets of patients and families have responded to the questionnaire. Results show that 89% of the patients and 96% of the families involved felt that the open visitation policy was beneficial. Data were also collected from the PACU nurses who participated and two thirds stated a perceived benefit to patients and family members. Phase II of the study will include implementing a PACU booklet and video preoperatively to prepare patients and family for the perioperative experience. Questionnaires will then be redistributed and responses of the prepared study group will be compared with the phase I control group. The objective of phase II will be to determine the impact of improved environmental awareness of the patient and family on their perception of the PACU visitation. PMID- 1710665 TI - Standard care plans for the postanesthesia care unit. PMID- 1710666 TI - Practical points in the assessment and management of postoperative pediatric pain. AB - Pain assessment is a complex yet essential aspect of holistic care of the pediatric patient during the postoperative period. Because children cannot verbalize pain, the PACU nurse is faced with the challenge of assessing pain. This article attempts to provide guidelines in assessing postoperative pediatric pain and reviews common analgesic drugs. PMID- 1710667 TI - Preparation for ambulatory surgery: a patient education program. AB - New experiences for patients and new role responsibilities for nurses emerge in the ambulatory surgical setting. Due to limited access to health care providers, individual patients must assume presurgical and postsurgical responsibility for their own well-being and self-care. Patient education that focuses on the perioperative experiences associated with ambulatory surgery is necessary to assist the patient's recovery efforts. This article will analyze the numerous steps involved in creating and evaluating a patient education program that prepares an adult patient for ambulatory surgery. The Systems Evaluation of Patient Education model developed by Rankin and Duffy will be used to illustrate how a PACU nurse systematically proceeded to examine needs, identify resources, implement the instructional program, and analyze outcomes. PMID- 1710668 TI - The career plateau--the differential diagnosis: Part III. AB - This is the last article of a three-part series on the issue of the career plateau. The first article defined the problem of career plateauing and the heightened awareness by hospital and nursing administrators of hospital nurses' career needs. The second article dealt with strategies for change in the hospital organization, the manager, and the employee. This article discusses eight basic career needs of hospital nurses that resulted from a research study the author conducted on the development of an inventory designed to measure the career specialty needs of hospital nurses. The article focuses on the career needs of PACU and surgical nurses compared with three other speciality areas. PMID- 1710669 TI - Cognitive-behavioral therapy in patients with ankylosing spondylitis in a German self-help organization. AB - A cognitive-behavioral treatment program for pain control was administered to 22 subjects with a diagnosis of ankylosing spondylitis (AS) in a self-help setting of the German Rheumatism League. A sample of 17 AS subjects from the same setting served as waiting-list controls. The program consisted of training in progressive muscle relaxation, cognitive restructuring, attention related techniques and pleasant activity scheduling, and was aimed at an improvement of self-control strategies. Ratings of pain severity, anxiety, depression, psychophysiological complaints, and sleep disturbances were used to evaluate the outcome. Follow-up assessments were conducted six months post treatment. A significant interaction between treatment condition and assessment period was demonstrated. Further analyses indicate a beneficial effect of the treatment in all outcome measures apart from general symptoms during pain attacks at the follow-up assessment. Reductions of pain intensity, anxiety, and psychophysiological symptoms were maintained at 12 month follow-up. Although pain reduction was statistically significant, it did not exceed 14% in the pain diary. The more important aspect of the treatment appears to be emotional stabilization and increased feelings of well-being. PMID- 1710671 TI - Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture. AB - Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2(+)-gated K+ current. The characteristics of the Ca2(+)-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 microM of the intracellular Ca2+, but it was independent of the membrane potential. Charybdotoxin reduced the activity of the Ca2(+)-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2(+)-activated K+ channels. PMID- 1710670 TI - Structure and function of channels and channelogs as studied by computational chemistry. PMID- 1710672 TI - Charybdotoxin-sensitive K(Ca) channel is not involved in glucose-induced electrical activity in pancreatic beta-cells. AB - The effects of charybdotoxin (CTX) on single [Ca2+]-activated potassium channel (K(Ca)) activity and whole-cell K+ currents were examined in rat and mouse pancreatic beta-cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured beta-cells and beta cells in intact islets were compared. K(Ca) activity was very infrequent at negative patch potentials (-70 less than Vm less than 0 mV), channel activity appearing at highly depolarized Vm. K(Ca) open probability at these depolarized Vm values was insensitive to glucose (10 and 20 mM) and the metabolic uncoupler 2,4 dinitrophenol (DNP). However, DNP blocked glucose-evoked action potential firing and reversed glucose-induced inhibition of the activity of K+ channels of smaller conductance. The venom from Leiurus quinquestriatus hebreus (LQV) and highly purified CTX inhibited K(Ca) channel activity when applied to the outer aspect of the excised membrane patch. CTX (5.8 and 18 nM) inhibited channel activity by 50 and 100%, respectively. Whole-cell outward K+ currents exhibited an early transient component which was blocked by CTX, and a delayed component which was insensitive to the toxin. The individual spikes evoked by glucose, recorded in the perforated-patch modality, were not affected by CTX (20 nM). Moreover, the frequency of slow oscillations in membrane potential, the frequency of action potentials and the rate of repolarization of the action potentials recorded from pancreatic islet beta-cells in the presence of glucose were not affected by CTX. We conclude that the K(Ca) does not participate in the steady state glucose-induced electrical activity in rodent pancreatic islets. PMID- 1710673 TI - Low-dose chemotherapy with central nervous system prophylaxis and zidovudine maintenance in AIDS-related lymphoma. A prospective multi-institutional trial. AB - OBJECTIVE: --To ascertain if low-dose multiagent chemotherapy, with central nervous system prophylaxis and antiretroviral therapy, might be associated with increased efficacy and decreased risk of intercurrent infection in patients with malignant lymphoma related to the acquired immunodeficiency syndrome (AIDS). DESIGN: --A phase II prospective clinical trial, with median follow-up of 33 months. SETTING: --Eight university hospitals, within the context of the AIDS Clinical Trials Units, sponsored by the National Institute of Allergy and Infectious Diseases. PATIENTS: --Forty-two patients with AIDS-related malignant lymphoma. All were evaluable for toxicity assessment, and 35 for response. INTERVENTION: --A low-dose modification of the M-BACOD regimen (day 1): cyclophosphamide, 300 mg/m2 intravenously (IV); doxorubicin, 25 mg/m2 IV; vincristine sulfate, 1.4 mg/m2 IV; bleomycin, 4 mg/m2 IV; dexamethasone, 3 mg/m2 orally on days 1 through 5; methotrexate, 500 mg/m2 IV on day 15, with leucovorin rescue. Intrathecal cytosine arabinoside (50 mg) to all on days 1, 8, 21, and 28, with radiation therapy to a helmet field to those with central nervous system involvement. Zidovudine for 12 months after completion of four to six cycles of chemotherapy. MAIN OUTCOME MEASURES: --Response rate and number of opportunistic infections. RESULTS: --Response rate was 51% with a complete response of 46%. Of 16 complete responses, relapse occurred in four, none isolated to the central nervous system. Opportunistic infections occurred in 21% of those receiving treatment. Median duration of survival among all 42 patients is 5.6 months, 6.5 months in 35 patients evaluable for response, and 15 months in patients with complete response. Lower concentration of CD4 cells, history of prior AIDS, bone marrow involvement, and stage IV disease were independently associated with decreased survival. CONCLUSIONS: --Low-dose chemotherapy with central nervous system prophylaxis and zidovudine maintenance may be associated with durable remissions in AIDS-related lymphoma with fewer opportunistic infections than noted in prior reports. PMID- 1710674 TI - [A prospective trial of the mass survey for pancreatic cancer using serum markers and ultrasonography]. AB - We designed a prospective trial of the mass survey for pancreatic disease, especially pancreatic cancer in consecutive 2576 subjects undergoing periodical health examination during 1989. In order to detect abnormality of the pancreas we measured serum amylase, elastase-1 and SPan-1 as serum markers and used ultrasonography (US). When abnormal elevation of serum markers or abnormal findings of US were obtained at the first screening, re-examination or further examination using ERCP, CT and endoscopic US were performed. Frequencies of elevated amylase, elastase-1 or SPan-1 were 0.9%, 3.4% or 1.7%, respectively. However, no pancreatic diseases were detected among the cases of elevated serum markers after subsequent further examination. Frequency of abnormal US findings of pancreas were 2.3% (59/2576), including dilatation of main pancreatic duct, cystic lesion, space occupying lesion and calcification. Subsequent further examinations on subjects with abnormal US revealed 8 cases of pancreatic diseases including one small pancreatic cancer. US may be an efficient tool of mass survey for pancreatic cancer in the health examination at present. PMID- 1710675 TI - [Localization of alpha 1-adrenoceptors in hypertrophic prostate]. AB - In order to determine the localization of alpha 1-adrenoceptors in human hypertrophied prostates, in vitro autoradiography was performed on the frozen specimens from 13 enucleated hypertrophied prostates with [125I]-HEAT (iodo-2 [beta-(4-hydroxyphenyl)-ethylaminomethyl] tetralone) and [3H]-prazosin. In vitro autoradiograms showed macroscopically the specific binding site on the areas seemed to the nodular area for [125I]-HEAT, but not so clear specific binding sites for [3H]-prazosin. Microscopic autoradiograms seemed to show binding sites located mainly on the interstitial beneath the gland, and partly on the basement membrane and epithelium of the prostatic gland. Further studies are needed to show clearer specific binding sites of alpha 1-adrenoceptors. PMID- 1710676 TI - [Immune response against BCG 65 kDa protein antigen in bladder cancer with BCG instillation therapy. Analyses carried out with the gene of this protein to develop a monoclonal antibody]. AB - The BCG 65 kDa protein is part of the 60 kDa heat-shock protein (hsp) family. It is clear that hsp has a huge homology among mammalian and bacterial cells. In our study, we have observed the relevance of this protein in the anti-tumor response. Antibody production against the BCG 65 kDa protein was determined by solid phase ELISA using the sera of 51 bladder cancer patients who have undergone BCG instillation therapy. The BCG 65 kDa protein was made by E. coli transfected with pTB12 (plasmid DNA) which encodes this protein. It was clearly observed that the level of serum antibody titer to this protein was raised by the above instillation therapy. In order to investigate the role of this protein in anti tumor response, we developed monoclonal antibodies against BCG 65 kDa protein. Four monoclonal antibodies were developed (B-20, B-97, B-108, B-167). We also estimated the epitope defined by each monoclonal antibody by using the truncated protein which was produced by E. coli transfected with the deletion mutants of pTB12. At this stage, we proceeded with observation of the cross epitope between mammalian cancer cells and BCG after using these monoclonal antibodies. There is no cross epitope defined by B-20, B-97 and B-167 in mammalian cells. However, the epitope defined by B-108 exists in normal tissue as well as in bladder cancer cells. PMID- 1710677 TI - [Evaluation of weight of the prostate by transrectal longitudinal ultrasonography]. AB - The correlation between the resected weight of prostate and the estimated weight of prostate by transrectal longitudinal ultrasound was evaluated for 53 patients with benign prostatic hypertrophy. Sonography was done with a real time linear scanner and 5.0 MHz transducer. Maximal prostatic dimensions were measured along the two major axes, namely the maximal length and the maximal thickness of total prostate and adenoma. By regarding benign prostatic hypertrophy as an ellipsoid, we calculated the weight of prostate according to the ellipsoid formula. The correlation coefficient was calculated as; r = 0.929, Y = 0.942Xt - 2.642 (Xt: total estimated weight), r = 0.962, Y = 0.965Xa + 0.028 (Xa: estimated weight of adenoma). The estimated weight of the prostate correlated with the resected weight of the prostate. These results suggested that rough estimation of the weight of benign prostatic hypertrophy was found to be possible by using this calculation technique. PMID- 1710678 TI - The Denver II replaces the Denver Developmental Screening Test. PMID- 1710679 TI - [The role of occupational and social stress in the development of non-ischemic arrhythmia in pilots]. AB - In the absence of coronary heart disease and ischemic responses to exercise, many highly skilled pilots have been shown to have arrhythmias of non-ischemic origin. The occupational stress-exercise on a flight trainer reveals such arrhythmias much more frequently than bicycle ergometric exercise. Under social stress, namely during medical- and flying examination which decides if a pilot is professionally fit, 24-hour monitoring in the out- and inpatient settings detects premature+ ventricular contractions of non-ischemic origin in high-grade pilots; the contractions regularly decreasing or ceasing during a true flight. Thus, the social stress in pilots is a stronger arrhythmogenic factor than is the routine occupational stress. PMID- 1710680 TI - [Effectiveness of single intravenous administration of cordarone, ethacizine and obsidan in ventricular extrasystole in patients with chronic alcoholism during withdrawal]. AB - Antiarrhythmic effects of cordarone, ethacizine, and obsidan were evaluated in 30 patients with chronic alcoholism in whom premature ventricular contractions were recorded at a rate of at least of 5 per minute by 30-minute ECG monitoring when they were admitted to a narcologic hospital, having the alcohol withdrawal syndrome. Cordarone, ethacizine, and obsidan were found to reduce the total number of premature ventricular contractions by 90% or more within an hour after their single intravenous injection in 80, 100, and 50% of the patients, respectively. PMID- 1710681 TI - [Use of cardiorespiratory synchronization in the differential diagnosis of arrhythmia]. AB - A method was proposed for differential diagnosis of functional and organic cardiac arrhythmias. It is based on the concept that the nervous system produces a trigger impact on the heart. Cardiorespiratory synchronization is one of the manifestations of the impact. The advantage of the method is that it takes only several minutes and requires no expensive equipment. In fact it may be used at any medical institution. It is particularly effective in mass surveys of persons with cardiac rhythm disturbances of unknown etiology, which are identified for the first time. PMID- 1710682 TI - Molecular biology of type A endogenous retrovirus. AB - Intracisternal A particles (IAPs) are retrovirus-like structures consistently observable in a variety of mouse tumor cells such as myeloma and hybridoma and in early embryonic cells derived from rodents but nothing is known of their infectivity. Mouse IAPs contain a gag-like protein, a reverse transcriptase and a polyadenylated RNA molecule (IAP RNA). DNA sequences complementary to IAP RNA (IAP genome) are interspersedly present in rodent such as mice, rats, Chinese hamsters and Syrian hamsters at several hundred to a thousand copies per haploid genome. Molecularly cloned IAP genomes from two species Mus and Syrian hamster were 6 to 8 kb in length with LTRs of about 0.4 kb long. The nucleotide sequence of the Syrian hamster IAP genome, H18, predicted a typical LTR-gag-prt-pol-env LTR structure, although many stop codons were present in the region corresponding to env. The comparison of the deduced amino acid sequences of the pol region showed IAP (type A), mouse mammary tumor virus (MMTV) (type B), and squirrel monkey retrovirus (SMRV) (type D) genomes to be closely related. By using a DNA fragment encoding the pol region of the Syrian hamster IAP genome, human endogenous retroviruses termed HERV-K, were cloned from a fetal human liver gene library. Typical HERV-K genome was 9.5 kb in length having LTRs of about 1.0 kb. The HERV-K provirus could encode gag (666 codons), prt (334 codons), pol (937 codons), and env (618 codons) genes. HERV-K was shown to be closely related to types A, B and D retroviruses. The HERV-K genomes are present at about 50 copies per haploid human genome. In several human tumor cell lines, the HERV-K genome was expressed as 8.8 kb poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression was observed following female steroids treatment. In a detailed investigation on the organization of HERV-K proviruses in human genome, we found repetitive sequences homologous to the LTR region of the HERV-K genome. They were about 630 bp in length with an A rich tail at 3' end and found to be a SINE type nonviral retroposon. These elements were present at 4,000 to 5,000 copies per haploid human genome. PMID- 1710683 TI - Histochemical and immunohistochemical study on the expression of alkaline phosphatase, gamma-glutamyl transpeptidase and alpha-fetoprotein during the process of rat hepatocyte proliferation. AB - The changes in the activity and the localization of alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GTP) and alpha-fetoprotein (AFP) were examined during cell regeneration in the galactosamine-injured rat liver. D galactosamine was injected i.p. into rats at a single dose level of 400 mg/kg. The biochemical activities of ALP and gamma-GTP in rat liver homogenate increased significantly in comparison with those in the control rats 3 days and 4 days after administration of D-galactosamine. In the histochemical analysis, 3 days, 4 days and 5 days after the administration of the amino sugar, a high level of activity of both ALP and gamma-GTP was seen along the cell borders between adjacent hepatocytes. AFP was detected by the enzyme-labeled antibody technique in the cytoplasm of a few small hepatocytes around Glisson's sheath and epithelial cells of small tubules within Glisson's sheath which show morphological features similar to bile duct 3 days, 4 days and 5 days after the administration of the amino sugar. AFP was detected in serum by the western blotting method 3 days and 4 days after the administration of D-galactosamine, whereas serum albumin decreased significantly in the same period. In this study, it was shown that ALP, gamma-GTP and AFP were proper markers to justify the degree of the differentiation of hepatocytes during the state of proliferation. PMID- 1710684 TI - [The optimal choice of multichannel analytical systems in clinical biochemistry]. PMID- 1710685 TI - [The measurement of NMR-1H relaxation time of blood serum as a method of laboratory diagnosis]. AB - Measurement of NMR-1H-relaxation of the blood serum cannot be employed in the differential diagnosis of individual diseases. This method may be useful as an additional test in examination of the time course of a disease in a patient and in mass screenings or prophylactic check-ups. PMID- 1710686 TI - [The use of liquid exclusion chromatography for evaluating methods of treating urologic diseases]. AB - Gel chromatography was used for measuring medium-molecular levels in the blood sera of urologic patients: (1) low-pressure chromatography in Sephadex G-15 packed columns with fractionation range less than 1500 dalton or in Toyopearl HW 40 packed columns with fractionation range from 100 to 100,000 dalton; (2) high pressure chromatography in LKB (Sweden) blue columns. Liquid exclusion chromatography was employed to assess the efficacy of blood extracorporeal detoxication of urologic patients by means of hemoperfusion, hemodiafiltration, hemodialysis, plasma perfusion, plasmapheresis. The highest correlation between patients' state of health and blood levels of medium molecules was observed in hemodiafiltration. PMID- 1710687 TI - [The determination of free and bound oxyproline in urine]. AB - A method of measuring urinary free, peptide- and protein-bound hydroxyproline is suggested. Adults were found to excrete 18 mumol of free, 155 mumol of peptide bound, and 8 mumol of protein-bound hydroxyproline daily. In children daily urinary levels of free and peptide-bound hydroxyproline were higher. Data are presented on changes in various hydroxyproline forms in pneumonia, rheumatic fever, and hemorrhagic fever with the renal syndrome. PMID- 1710688 TI - [Excretion of bile acids in feces in pathology of the liver and intestines]. AB - Intestinal excretion of free bile acids (BA), i.e. of cholic, chenodeoxycholic, deoxycholic, lithocholic, and of conjugated BA, i.e. of glycocholic, glycodeoxycholic together with glycochenodeoxycholic, taurocholic, taurodeoxycholic together with taurochenodeoxycholic acids, was examined in patients with viral hepatitides (VH), chronic hepatitis (CH), biliary cirrhosis of the liver (BCL), and acute dysentery (AD), as were the effects of therapy on these acid levels. The findings evidence that fecal levels of free BA are significantly reduced during the acute period of VH, CH, AD, and BCL, whereas the levels of conjugated acids are elevated in all the examinees except the BCL patients, in whom these acids are unchanged. Study of BA excretion in VH patients treated with prednisolone has demonstrated a normalizing effect of this therapy on the spectrum of excreted free BA. Furazolidone and erythromycin therapy resulted in disordered transformation of 'primary' BA into 'secondary' ones and to deconjugation of BA, this being possibly related to these drugs effects on intestinal microflora. PMID- 1710689 TI - [Gas chromatographic analysis of fatty acids in the blood of alcoholic patients]. PMID- 1710690 TI - [A comparative analysis of the chemiluminescence of capillary and venous blood granulocytes]. AB - The parameters of zymosan-stimulated luminol-dependent chemiluminescence of whole capillary and venous blood were compared in patients with chronic nonspecific diseases of the lungs. The parameters of a chemiluminescence response were in good correlation in general, but the mean values of luminescence intensity of granulocytes from the blood collected from the finger were 1.6 times lower that may be explained by the inhibiting effects of the lymph and intercellular fluid. PMID- 1710691 TI - [The informativeness of the luminescent analysis of blood lymphocytes in the assessment of health status]. AB - The author emphasizes the informative value and high sensitivity of fluorescent analysis of blood lymphocytes, fluorochromium-treated with acridine orange, to changes in the body status. Use of blood microvolumes makes this method promising for screenings of the population and for monitoring the patients. PMID- 1710692 TI - [The biochemical diagnosis of lysosomal storage diseases at medical genetic centers. II. Glycoproteinoses, sphingolipidoses (review of the literature]. PMID- 1710693 TI - [Laboratory diagnosis of anemias involving disorders of iron metabolism]. PMID- 1710694 TI - [Cytometric analysis of neoplastic processes in the large intestine]. AB - Examinations of macroscopically intact mucosa, of adenomas with various dysplasia degrees, and of adenocarcinoma have helped single out cytometric parameters and a number of morphologic characteristics describing the proliferation processes in the large intestine. Variability of the nuclear shape and size, the mitotic activity of the glandular epithelium, and the volumic fraction of proliferating DNA-synthesizing cells are characteristic of malignant processes. PMID- 1710695 TI - [The use of immunochemical reactions in determining a cytological diagnosis]. AB - Cytologic preparations of 56 samples of pleural fluid, of 31 samples of ascitic fluid, and 13 puncture biopsy specimens were examined in immunocytochemical tests. Carcinoembryonic antigen and trophoblastic beta 2-globulin were detected in the cytograms. Antibodies to carcinoembryonic antigen and trophoblastic beta 2 globulin employed in the tests did not react with the cells from benign exudation, because tumor cells, particularly glandular carcinoma ones, contain these markers in high concentrations. PMID- 1710696 TI - [Immunoenzyme analysis of the Willebrand factor using monoclonal antibodies]. AB - A method for measuring Willebrand's factor protein has been developed, based on indirect solid-phase enzyme immunoassay with Soviet monoclonal antibodies to this factor. Normal Willebrand's factor level in the plasma has been found 55-161 percent. The method has been tried in patients with Willebrand's disease and with oncologic diseases. PMID- 1710697 TI - [Plasma ceruloplasmin in patients with hepatorenal insufficiency]. AB - Changes in plasma ceruloplasmin levels were studied by enzyme immunoassay in 81 patients with the acute hepatorenal insufficiency syndrome (AHIS). Changed plasma ceruloplasmin concentration was detected in the first days of the disease, this change depending on AHIS severity and unrelated to the disease cause. A lowering of plasma ceruloplasmin level below 0.75 mg/ml is considered to be a poor prognostic sign. Measurement of plasma ceruloplasmin is a test available for clinical laboratories, it is highly sensitive, accurate, and reproducible. PMID- 1710698 TI - [A method of determining heparin in blood plasma based on its antithrombin activity]. AB - The suggested method for measuring blood plasma heparin is based on heparin ability to enhance antithrombin activity of antithrombin III (AT-III), the major Xa and thrombin inhibitor. The method consists in measurement of blood plasma AT III activity in the presence and absence of protamine sulfate that destroys the heparin--AT-III complex. Heparin content in U/ml is determined from the difference in the activities of heparin--AT-III complex and AT-III proper activity represented on the calibration curve. The method is sufficiently sensitive, it permits registration of heparin concentrations in a wide band (from 0.01 U/ml to 0.75 U/ml of plasma). PMID- 1710699 TI - [Impaired production and reception of interleukin in human diseases]. PMID- 1710700 TI - [Morphometric analysis of immunocompetent cells]. AB - The authors have developed the technique of morphometric analysis of human peripheral blood immunocompetent cells by means of Laborscal PSA-1, PSL-1 conductometric cytoanalyzer and DBK-010 computer-processing of the resultant histograms. The method was tried in examinations of more than 400 patients with various diseases and in studies of lymphocyte activation changes. Formulae for calculation of cellular diameters and volumes are offered, as is the program for histogram processing. PMID- 1710701 TI - [The informativeness of determining circulating immune complexes and immunoglobulins during immunization]. AB - The authors suggest using measurements of the concentrations of circulating immune complexes (CIC) by the PEG precipitation method and of immunoglobulins by radial immunodiffusion technique for monitoring the patients after immunization with rhesus antigen (n = 20), with staphylococcal antitoxin (n = 59), and after renal allograft transplantation (n = 18). The results evidence that CIC level measurements by the PEG precipitation method are valuable in the monitoring of rhesus-immunized patients. The authors suggest including such measurements in the complex of tests for examination of rhesus-immunized donors. PMID- 1710702 TI - [A comparative evaluation of culture media for isolating bifidobacteria, lactobacillus and enterococci]. PMID- 1710703 TI - [A method of determining lipase in bacteria of the intestinal group]. AB - A method for detection of lipase in intestinal bacteria is suggested with fatty acid esters used as enzyme substrates. This modification not only allows the enzyme detection but is more rapid that the known procedures. Preliminary cultivation of bacteria in the accumulation media rich in organic substances helps shorten the time of investigation and simplifies reading the reaction. PMID- 1710704 TI - [Diagnosis of opisthorchiasis using the membrane filtration technique]. AB - Filtration method for the detection of Opisthorchis eggs in the bile is by an order more sensitive than the routine method. It is recommended for diagnostic studies for cases with a low invasion level and in assessment of the efficacy of chemotherapeutic agents. This technique may be useful in the diagnosis of other helminthiases as well. PMID- 1710705 TI - [A highly sensitive method of detecting the structural proteins of hepatitis A virus]. AB - A modified technique of protein staining with silver nitrate was employed in electrophoretic analysis of hepatitis A virus structural proteins. The modifications of the original technique, i.e. thorough washing of the gel, increased formaldehyde concentration and a more intensive lighting of the gel, have elevated the method sensitivity 10-fold permitting the detection of up to 0.1 ng protein. The modification has allowed detection of VP1, VP2, and VP3 structural proteins with molecular masses 33, 29, and 27 kD, respectively, in virus preparations of low concentrations. PMID- 1710706 TI - [Cytomorphology of malignant histiocytosis of the bones]. AB - Cytomorphologic signs of malignant histiocytosis of the bones are described in detail, that have not been reported in the Soviet literature before. Cytologic signs that permit a differential diagnosis between this condition and plasmacytoma and osteogenic sarcoma are presented. PMID- 1710707 TI - [Morphologic characteristics of tumor cells in the cystic fluid of oligodendroglioblastoma]. PMID- 1710708 TI - [Nuclear magnetic resonance relaxation of the blood in patients with burns]. AB - Blood plasma of patients with burns was examined by NMR relaxation over the course of exchange plasmapheresis and delayed reinfusion of sorbed defrosted autoplasma. Prolonged time of aqueous proton relaxation was revealed, evidencing a positive time course. A hypothetical model of this mechanism is suggested. PMID- 1710709 TI - [The determination of phospholipase activity using the 31P-NMR spectroscopic method]. AB - The authors have developed a rather rapid and convenient method for testing and measurement of phospholipase C (EC 3.1.4.3) activity, based on continuous recording of the signal reduction in 31P-NMR spectrum of lecithin phosphate group (chemical sigma shift = 0.2 parts per million as regards H3PO4) or of phosphocholine signal augmentation (sigma = -4 parts per million). This method permits a quantitative estimation of lecithin loss or phosphocholine accrual from the kinetics of integral intensity changes in the course of an enzymic reaction and then calculate phospholipase C activity without resorting to thin-layer chromatography traditionally used for this purpose. PMID- 1710710 TI - [An index to the antioxidant activity of biological materials]. AB - A new method for measuring serum and tissue antioxidative activity is based on a relationship between accumulation of lipid peroxidation (LPO) products in incubation medium and the amount of the substrate during LPO activation. The ratio of malonic dialdehyde accumulation during LPO activation in two samples of different volumes is proposed as an index of antioxidative activity. PMID- 1710711 TI - [The dynamics of the blood level of phosphatidylinositol in patients with myocardial infarction]. AB - Phospholipids, particularly inositol-containing ones, that serve as secondary messengers and bioregulators of fundamental biochemical processes, essentially contribute to the origination of cardiovascular diseases. These compounds are highly labile and the data on their levels in the blood and its components in various cardiovascular conditions are contradictory. This may be explained by the fact that phosphatidylinositol levels change immediately after blood collection. Therefore the authors found it interesting to investigate the time course of this phospholipid levels, that may to a certain measure reflect the pattern of the metabolic disorders present in this case. They suggest a method of multiple measurements of phosphatidylinositol levels, carried out in short intervals, this yielding additional data on the function of biologic membranes, that may be useful in the diagnosis of coronary disease and assessment of the efficacy of treatment of this condition. PMID- 1710712 TI - [Analytical and clinical evaluation of methods of determining the pancreatic isoenzyme alpha-amylase]. PMID- 1710713 TI - [The content of fibronectin in the blood of patients with psoriasis]. AB - Fibronectin, a high-molecular glycoprotein, one of acute-phase proteins, contributes to cellular proliferation and macrophagal activity regulation, and its blood plasma levels are an indicator of the reticuloendothelial system functional activity. Blood plasma fibronectin levels were measured in psoriasis patients before and after hemoperfusion. These levels were found significantly increased in the course of hemoperfusion treatment, this being associated with disappearance of psoriatic elements. A correlation between blood plasma fibronectin level and NBT test parameters, on the one hand, and severity of psoriasis course and efficacy of therapy, on the other, has been revealed. PMID- 1710714 TI - [A study of the enzymes in citrated blood plasma]. AB - Elevated blood serum, vs. the plasma, levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities are explained by these enzymes discharge from red cells into the liquid fraction of the blood during its in vitro coagulation. To eliminate the "tube" hemolysis, the authors recommend measurements of these enzymes activities in the blood citrate plasma. PMID- 1710715 TI - [An electrochemical method of determining peroxidase in saliva]. AB - Electrochemical method has been developed for measuring human salivary peroxidase. Salivary peroxidase levels were measured in donors in various hours of the day, in girls in various periods of the cycle, and in myocardial infarction patients. The results may be used to develop rapid methods for assessment of ovarian functional activity and for prediction of myocardial infarction outcome. Electrochemical analysis productivity is 360 samples per hour, the minimal peroxidase concentration detectable is 5.10(-12) M. PMID- 1710716 TI - [The determination of nitrites in saliva]. AB - A method for measuring salivary nitrites, developed by the authors, is based on the Griess-Iloswai test. Salivary microflora was found to synthesize and transform nitrites to other substances, their concentration passing its peak. If salivary excretion is stimulated, nitrite synthesis is accelerated. Nitrites are recommended to be measured in stimulated saliva over the course of its incubation starting from the first and up to the thirtieth minutes. PMID- 1710717 TI - [A micromodification of the method of determining the activity of processes of free radical oxidation]. AB - A micromodification of the method for registration of bivalent iron ion-initiated chemiluminescence of the apo-beta-lipoprotein total fraction isolated from the blood serum is described. A significant elevation of lipid peroxide levels and of lipid ability to oxidation, paralleled by reduced levels of endogenous antioxidants as against the norm were revealed in the patients with acute disorders of brain circulation with the use of this method. Chemiluminescent studies of lipid peroxidation processes permit monitoring lipid peroxidation reactions and thus obtain a complex of parameters that characterize the status of these processes in the studied system. Due to its high sensitivity and specificity, chemiluminescent analysis may become one of the principal methods in studies of lipid peroxidation processes. PMID- 1710718 TI - [Atomic absorption determination of mercury in the urine]. PMID- 1710719 TI - [Determination of coproporphyrin in urine]. AB - Methods for urinary coproporphyrin measurements are analyzed. Data obtained by two methods, spectrophotometry and fluorescent method, are presented, the advantages and shortcomings of both are discussed. The authors recommend the fluorescent technique as a universal rapid method for wide medical practice. The problem of the units of coproporphyrin measurement is also discussed. The authors suggest replacing measurements per g of creatinine or daily portion by estimation of the coproporphyrin index in the morning portion of the urine. Study of the effects of various factors (climatic conditions, nutrition, patient's age and sex) on normal coproporphyrin excretion from the body has demonstrated that these factors do not noticeably influence porphyrin metabolism; for normal subjects the coproporphyrin index varies from 30 to 110 micrograms/day. PMID- 1710720 TI - [The physicochemical evaluation of the bile using infrared spectroscopy]. AB - Native bile, inspissated bile, and cholesterol choleliths were examined by infrared spectroscopy. Model compounds spectra were compared with native bile spectra. The spectra were divided into 3 groups: with acid fragments or cholesterol groups predominating and intermediate group. The peak of 3620 cm-1, absent in normal human bile and liable to augment if acid fragment level reduced was detected. The authors suggest a technique of infrared spectroscopy permitting early detection of disordered physicochemical characteristics of the bile in the course of cholesterol cholelithiasis development. PMID- 1710721 TI - [The Willebrand factor in the diagnosis of hemostatic disorders in patients with rheumatoid arthritis]. AB - Studies of coagulation hemostasis in 108 patients with rheumatoid arthritis have revealed that high blood coagulation is characteristic of this disease, and a number of coagulation parameters (antithrombin III, fibrinogen, fibrinolytic activity, ethanol test) point to intravascular microthrombi formation (disseminated intravascular coagulation). Along with hemostasis investigations, Willebrand's factor levels were measured in the blood plasma of 44 patients. Enhancement of the inflammatory process activity was found to be associated with elevation of this factor level and augmentation of the blood coagulation activity. These results permit considering Willebrand's factor as one of the diagnostic criteria of thrombophilic conditions in patients with rheumatoid arthritis. PMID- 1710722 TI - [Determination of the anticoagulant activity of the low and middle molecular fractions of the blood plasma]. AB - A method for measuring the anticoagulant activities of plasma low- and medium molecular fractions is described. Use of this method permits differentiation between bleedings induced by high blood levels of these substances. PMID- 1710723 TI - [A spectrophotometric method of determining the myeloperoxidase activity in phagocytic cells]. AB - A spectrophotometric method has been developed for measurements of myeloperoxidase activity in phagocytes, and conditions of measurements specified. Contribution of mononuclear cells to myeloperoxidase activity was found negligible, the major role here was played by neutrophils and eosinophils. Myeloperoxidase activity was found reduced in the patients with chronic granulomatous disease, agammaglobulinemia, and elevated in hyper-IgE-syndrome; this parameter was unchanged in ataxia telangiectasia and chronic dermatomucosal candidiasis. PMID- 1710724 TI - [Lysosomal storage diseases. Early prenatal diagnosis (review of the literature)]. PMID- 1710725 TI - [A comparative evaluation of methods for detecting duodenogastric reflux in patients with peptic ulcer]. AB - The duodenal contents were examined in 81 patients with gastroduodenal ulcer. Bile acid concentrations, alkaline phosphatase activity, and sodium ion concentration were measured for the detection of duodenogastric reflux. Measurements of sodium ion concentration permitted estimation of the immediate volume of the duodenogastric reflux in the gastric contents. No methods for duodenogastric reflux detection should be given preference in examinations of peptic ulcer patients. Multiple-modality studies appear to be the most effective. PMID- 1710726 TI - [Immunologic indices for the diagnosis of the initial stages of chronic alcoholism]. AB - Immunologic examinations of 60 patients suffering from common hard drinking and of 24 chronic alcoholics, stage I, has revealed a clear-cut difference in the levels of circulating immune complexes and theophylline-sensitive lymphocytes in these populations. These data are recommended as additional tests for the differential diagnosis of the initial forms of alcoholism. PMID- 1710727 TI - [A study of the initial suppressor function of lymphocytes]. AB - The new method for analysis of initial suppressor lymphocyte function in newly isolated monocultures is based on quantitative cytofluorometric analysis of early mitogen-induced activation of B lymphocytes in the presence of prednisolone that switches off a T lymphocyte immunoregulatory effect. Suppressor lymphocytes monitor B lymphocyte exit into proliferative pool and early processes of nuclear chromatin activation. The levels of initial suppressor lymphocyte function and lymphocytic proliferative activity are in inverse correlation. Suppressor lymphocyte function has been essentially reduced in rheumatoid arthritis patients as against normal subjects. A reduction as low as 70 percent of the normal value (recorded in 1/3 of the patients) is compensated with unchanged regulatory control over B cells, and reduction as low as 40 percent of the normal value (in 2/3 of the patients) is characterized by loss of regulatory control and B cell hyperactivation. PMID- 1710728 TI - [Discrete dynamic analysis of the functional activity of the leukocytes in predicting the nature of the course of viral hepatitis]. AB - Lymphocytic populations were examined by the rosette formation technique and lymphocytic and neutrophilic functional and metabolic activities by cytochemical methods in 87 patients with viral hepatitides A and B. None of the parameters alone permitted a prediction of the disease course. To solve this problem, the authors have undertaken a discrete dynamic computer-aided analysis. Analysis of 351 pairs of parameters helped single out several combinations informative as regards hepatitis prognosis. PMID- 1710729 TI - [Venous blood plasma for the serodiagnosis of syphilis]. AB - Microtest and enzyme immunoassay with venous blood plasma, provided the ratio 0.1 ml of 2 percent EDTA and 2 ml of venous blood is observed, are sufficiently sensitive and specific. These results may be useful in practical laboratories, during prophylactic check-ups, etc. PMID- 1710730 TI - [The advantages of using anti-A and anti-B TsOLI-clones for determining blood groups]. PMID- 1710731 TI - [The use in in vitro reactions of a house dust allergen prepared by a rapid method]. AB - The author suggests a method for preparing home dust allergen within 24 hours. Biochemical and trace-element characteristics of raw material for the preparation of this allergen are presented. The resultant allergen was effective in initiation of basophil degranulation test and of leukocytic synthesis of leukotrienes; the leukocytes were isolated from patients (adults and children) suffering from asthma and preasthma. Hypersensitivity diagnosis can be made more accurate if home dust allergen homologous to patient's home, prepared by the rapid method, is used in the studies. PMID- 1710732 TI - [The use of growth stimulants in the bacteriologic diagnosis of Yersinia]. AB - The effects of K. S. Karpuzidi's stimulants (0.5-1 percent) and of para-amino benzoic acid (0.1-05 mM) on the growth and multiplication of 3 Yersinia pseudotuberculosis, 1 Y. enterocolitica strains, and E. coli nonpathogenic strain were examined. Karpuzidi's stimulant was found to selectively stimulate Yersinia cell growth and inhibit Escherichia development in conditions unfavorable for these bacteria. Para-amino benzoic acid intensively stimulated the growth of both Yersinia and Escherichia. When Karpuzidi's stimulant was added to the accumulation medium after frosting-defrosting, it increased Yersinia harvest in cultures isolated from humans 2-3.5 times and that in cultures from the environmental objects 6-7 times. The results of bacteriologic investigation may be obtained 3-5 days sooner. PMID- 1710733 TI - [A differential diagnostic medium for the isolation of bacteria of the Yersinia genus]. AB - A formula of a solid differentiating diagnostic media for the isolation of Yersinia genus bacteria is suggested. The medium does not include components hard to get and is simple to prepare. No additional sterilization is necessary after the preparation, after the medium is poured into Petri dishes, it is dried in the air. The medium inhibits the growth of cocci and gram-negative contaminant microflora. Yersinia bacteria may be easily differentiated by the color and typical morphology of the colonies. Dishes with primary inoculation may be examined with a magnifying glass in 48 and 72 hrs incubation. The medium is highly sensitive. The results of its trials in examinations of washings off the environmental objects recommend it for practical studies. PMID- 1710734 TI - [A nutrient medium for isolating Lactobacilli]. AB - The composition of and method for preparation of nutrient medium for the isolation of Lactobacilli from biologic material are described. The medium is simple to prepare, consists of only Soviet reagents, this making it available for laboratories in this country. PMID- 1710735 TI - Chromogranin A-like immunoreactive neurites are major constituents of senile plaques. AB - The distribution of chromogranin A (CgA), a soluble protein in dense-core synaptic vesicles expressed by a variety of neuronal cell types, was studied immunocytochemically in Alzheimer's disease and normal aging. In addition to its presence in neuronal perikarya and process, CgA-like immunoreactivity (CgA-li) was demonstrated in multiple dystrophic neurites forming the crown of senile plaques. Two different monoclonal antibodies, LK2H10 and PHE5, gave identical results. In the two regions of the brain studied--the calcarine cortex and the molecular layer of the dentate gyrus--the areal density of plaques associated with CgA-like immunoreactive neurites was greater than the density of Congo red stainable amyloid cores, but smaller than the density of beta amyloid peptide deposits identified by the Campbell silver stain. By comparison, other synaptically released peptides--somatostatin 28, somatostatin 14, substance P, cholecystokinin, neurotensin, vasoactive intestinal peptide, and leu-enkephalin- were immunocytochemically detected in less than 30% of plaques. Thus CgA appears unique among known synaptically released substances in being present in dystrophic neurites in virtually all classic (i.e., Congo red stainable) plaques and additionally in a subpopulation of preamyloid plaques. PMID- 1710736 TI - Approaches to the prediction of language abilities in a sample of children who have developmental delays. AB - Prediction of the quality of language was explored using planned comparisons of three approaches, one cognitive, one neurodevelopmental, and one a combination of the two. Subjects were 37 children, ages 5-9 years, whose significant developmental delays included language and speech skills. The cognitive predictors were mental age (MA) and IQ from the Stanford-Binet Intelligence Scale. Neurodevelopmental predictors consisted of fine motor skill quotients (MQs) and dichotic speech processing scores. Chronological age (CA) was also evaluated as a predictor. A composite language ability score constituted the dependent variable. Results of regression analyses showed that CA and MQ, and MA and MQ, were nearly equal in their predictive strengths and were substantial predictors of composite language scores. Larger multiple correlations (low .8 range) were found when combinations of MA, IQ, and MQ or CA, IQ, and MQ were used as predictors. Statistical control over the 4-year age range revealed that approximately equal amounts of prediction of language scores were attributable to CA and a combination of MA, IQ, and MQ. Each of the latter variables contributed important amounts of unique variance to the language score prediction. Dichotic ear scores did not relate to cognitive or language scores and were ineffectual as predictors in regression analysis. Results indicated that children of the type studied have language and speech delays that show substantial relationships to their verbal cognitive abilities and MQs, in addition to their CAs. PMID- 1710738 TI - Lipid disorders in diabetes. Satellite symposium. Vienna, May 23, 1990. PMID- 1710737 TI - The role of target word stress in auditory comprehension by aphasic listeners. AB - The present investigation was designed to determine the influence of stressed word prosody on auditory comprehension by listeners with aphasia. Paragraph length narratives were computer-edited to yield two conditions. In one condition, both the target words and the surrounding context were prosodically neutral; in the second condition, target words were stressed and the surrounding contexts were prosodically neutral. The paragraph-length stimuli were presented to 10 aphasic listeners and their comprehension was tested. Analysis revealed that prosodic information carried only by stressed target words, within paragraph length stimuli, did not provide significant comprehension benefits to aphasic listeners. The comprehension improvement typically observed when paragraph-length narratives are stressed is, therefore, most likely due to prosodic cues that precede stress-bearing target words. PMID- 1710739 TI - Pathophysiology of hyperlipidemia in diabetes mellitus. AB - Many lipoprotein abnormalities are seen in the untreated, hyperglycemic diabetic patient. The non-insulin-dependent diabetic (NIDDM) patient with mild fasting hyperglycemia commonly has mild hypertriglyceridemia due to overproduction of TG rich lipoproteins in the liver, associated with decreased high-density lipoprotein (HDL) cholesterol levels. The more hyperglycemic untreated NIDDM and insulin-dependent diabetic (IDDM) patient have mild to moderate hypertriglyceridemia due to decreased adipose tissue and muscle lipoprotein lipase, (LPL) activity. These patients also have decreased HDL cholesterol levels associated with defective LPL catabolism of TG-rich lipoproteins. Treatment of diabetes with oral sulfonylureas or insulin corrects most of the hypertriglyceridemia and some of the decrease in HDL cholesterol. The abnormality in adipose tissue LPL activity corrects slowly over several months of therapy. The treated IDDM patient often has normal lipoprotein levels. The treated NIDDM patient may continue to have mild hypertriglyceridemia, increased intermediate density lipoprotein levels, small dense low-density lipoproteins (LDL) with increased apoprotein B, and decreased HDL cholesterol levels. The central, abdominal distribution of adipose tissue in IDDM is associated with insulin resistance, hypertension, and the above lipoprotein abnormalities. Improvement in glucose control, in the absence of weight gain, leads to lower triglyceride and higher HDL cholesterol levels. In addition, the diabetic patient is prone to develop other defects that, in themselves, lead to hyperlipidemia, such as proteinuria, hypothyroidism, and hypertension, treated with thiazide diuretics and beta-adrenergic-blocking agents. When a diabetic patient independently inherits a common familial form of hypertriglyceridemia, he might develop the severe hypertriglyceridemia of the chylomicronemia syndrome. PMID- 1710740 TI - HDL metabolism and atherosclerosis. AB - Epidemiologic studies of recent years have demonstrated an association between low plasma high-density lipoprotein (HDL) cholesterol levels and the development of atherosclerosis. The PROCAM (Prospective Cardiovascular Munster) study has identified HDL cholesterol as the single parameter predictor with the highest potency. Also, in multiple logistic function analysis of these data, which included the eight best independent predictors, HDL cholesterol contributed the largest portion to the overall predictive power of the algorithm. Further evidence for the important role of HDL in the atherosclerotic process was provided by the Helsinki Heart Study, in which the lipid-lowering and HDL increasing drug gemfibrozil was effective in reducing CAD incidences. In cohort studies, we have shown that HDL cholesterol concentrations largely differ between CAD patients and sex- and age-matched unaffected controls and that this difference increases with age. The reverse cholesterol transport hypothesis is the most widely used to explain the biochemistry of HDL-mediated atherosclerosis. In fact, it has been shown that HDL mediates the disposal of excess cellular cholesterol. There is evidence for the existence of two different mechanisms by which HDL can take up cellular cholesterol: both involve specific cellular recognition sites and probably also different HDL subpopulations. At least two different mechanisms have been identified by which the HDL cholesterol is finally targeted to the liver: attachment of apolipoprotein E for direct recognition by liver receptors or lipid transfer to lipoproteins of lower density that are subsequently also recognized by internalizing liver receptors. This knowledge gives rise to many different candidate genes for the formation of HDL deficiencies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710741 TI - Effect of bezafibrate on metabolic profiles in non-insulin-dependent diabetes mellitus. AB - Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by hyperglycemia secondary to increased hepatic glucose output and impaired extrahepatic insulin sensitivity. Many NIDDM patients also have raised serum triglyceride and low density lipoprotein (LDL) cholesterol levels, which may require drug therapy, as well as increased plasma nonesterified fatty acid concentrations. Some hypolipidemic agents such as bezafibrate lower fatty acid levels as well as LDL cholesterol and triglycerides. It has been suggested that raised fatty acid levels may be responsible in part for the increased hepatic glucose output and insulin insensitivity of NIDDM. Several groups have therefore sought to examine the effects of fibrates on glycemic control in NIDDM. In our own studies, we used bezafibrate (200 mg t.i.d.) in a double-blind, placebo-controlled design for 3 months. Fasting blood glucose, serum insulin, C-peptide, plasma nonesterified fatty acids, blood lactate and alanine, serum triglycerides, and LDL cholesterol were all lowered. HbA1 showed no significant change. Meal hormone and metabolite profiles were all improved although this reflected mainly the lower premeal values. The results of our and other studies are sufficiently encouraging to suggest that bezafibrate could provide alternative first-line drug therapy in hyperlipidemic NIDDM patients when diet alone has failed. PMID- 1710742 TI - Bezafibrate retard in type II diabetic patients: effects on hemostasis and glucose homeostasis. AB - A double-blind, placebo-controlled trial assessed the effect of a slow-release formulation of bezafibrate (Bezalip Mono, 400 mg daily for 3 months) on lipid profile, glucose homeostasis, platelet function, and plasma fibrinogen concentration in non-insulin-dependent (type II) diabetics. Twenty-four patients completed the trial. There was a significant improvement in the cholesterol (p less than 0.02), triglyceride (p less than 0.01), and nonesterified fatty acid (p less than 0.05) concentrations and in the fasting blood glucose (p less than 0.03) and glycosylated hemoglobin (p less than 0.01) levels of those (n = 11) who received the active preparation but not in those (n = 13) who received placebo. Treatment, but not placebo, also resulted in a significant (p less than 0.01) fall in plasma fibrinogen concentration and a trend towards inhibition of platelet aggregation. Bezafibrate was well tolerated; only one patient (not included in the analysis of results) withdrew from the trial possibly because of side effects of the drug. A larger study is needed to establish whether bezafibrate can reduce nonlipid risk factors (e.g., plasma fibrinogen concentration, glucose intolerance, and hyperinsulinemia) in normo- and hyperlipidemic subjects. PMID- 1710743 TI - Bezafibrate retard in patients with insulin-dependent diabetes: effect on serum lipoproteins, fibrinogen, and glycemic control. AB - The effects of a sustained-release preparation of bezafibrate (Bezalip Mono) 400 mg once daily and placebo administered for 3 months were compared in 36 patients with stable type 1 diabetes and hypercholesterolemia and/or hypertriglyceridemia. There was a significant decrease in fasting glucose levels with bezafibrate, but not in glycosylated hemoglobin. The serum cholesterol concentration decreased on bezafibrate [from 7.1 +/- 0.2 (mean +/- SEM) to 6.3 +/- 0.3 mmol/L; p less than 0.05] predominantly due to a reduction in low-density lipoprotein (LDL) cholesterol [from 4.8 +/- 0.3 to 4.2 +/- 0.3 mmol/L; p less than 0.05. There was also a decrease in fasting serum triglycerides with bezafibrate [1.82 to 1.26 mmol/L (geometric mean)] and in very-low-density lipoprotein (VLDL) cholesterol. Plasma fibrinogen decreased significantly with bezafibrate (from 4.1 +/- 0.2 to 2.9 +/- 0.2 g/L; p less than 0.001). Serum apolipoproteins B and A showed no statistically significant changes. Overall, there was no change in high-density lipoprotein (HDL). However, in patients who were initially hypertriglyceridemic, there was a significant increase in the cholesterol content of total HDL and the HDL2 subfraction (both p less than 0.05). It is concluded that in insulin dependent diabetic patients with hyperlipidemia, bezafibrate is effective in lowering both serum VLDL and LDL. In addition, it has a potentially important action in decreasing plasma fibrinogen levels. PMID- 1710744 TI - Effects of various lipid-lowering treatments in diabetics. AB - The frequency of all forms of atherosclerotic vascular disease is much greater in diabetics than in nondiabetics. Abnormalities in both the quantity and the quality of lipoproteins are among the many factors that can contribute to this. The most frequent quantitative lipoprotein abnormality in diabetics is hypertriglyceridemia. High-density lipoprotein (HDL) levels may be reduced, normal, or increased depending on the type of diabetes, its treatment, and the presence or absence of obesity. Low-density lipoprotein (LDL) levels in diabetics generally are not different from those in nondiabetics. The qualitative changes in lipoprotein composition may include alterations in the C apolipoproteins that could retard very low-density lipoproteins (VLDL) catabolism, enrichment of LDL and HDL with triglyceride, and modifications of LDL (e.g., glycosylation or oxidation) that makes it more atherogenic. The present rationale for the treatment of LDL abnormalities in diabetics is based on extrapolation from intervention trials in nondiabetics. These trials have suggested targets for plasma and LDL cholesterol levels. To date, no similar trials have been conducted in diabetics. Hence, it is not known whether the same or even lower LDL targets should apply to diabetics. Primary intervention trials have also shown the benefits, at least in nondiabetics, of increasing HDL levels. There is increasing evidence to support a cardiovascular risk effect of hypertriglyceridemia and one secondary intervention trial has demonstrated benefit associated with the correction of this. An added benefit of triglyceride reduction, at least in the milder diabetics, appears to be an improvement in insulin sensitivity. Fibrates are the drugs of choice for the correction of hypertriglyceridemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710745 TI - Diabetes and coronary artery disease: what a coincidence? AB - The incidence of coronary artery disease (CAD) is markedly increased in both insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). The background for this coincidence is as yet incompletely understood. In uncomplicated IDDM, the levels of cardiovascular risk factors do not show any substantial abnormalities if the metabolic control is good. However, when diabetic nephropathy ensues, even in its early microalbuminuric stage, blood pressure tends to become elevated and multiple atherogenic plasma lipid abnormalities appear. In juvenile-onset IDDM, increased occurrence of clinically manifest CAD emerges after the age of 30 years and becomes particularly marked in patients with diabetic nephropathy. Premenopausal female patients with IDDM develop CAD almost as often as male diabetics with IDDM of the same age--a situation in sharp contrast to that in nondiabetics, with a large excess of CAD in men. IDDM may act as a promoter of the progression of atherosclerotic lesions in subjects who are otherwise prone to develop them. This could explain why patients with IDDM have an increased risk of CAD, even in the absence of diabetic nephropathy, which enhances atherogenesis through several mechanisms. NIDDM is associated with multiple changes in cardiovascular risk factors, including abnormalities in the levels and composition of plasma lipids and lipoproteins and increased frequency of hypertension. These changes in cardiovascular risk factors are already present in subjects with impaired glucose tolerance (IGT), the precursor stage of NIDDM. In patients with NIDDM, the incidence of CAD is markedly increased compared to that in nondiabetic subjects of the same age, and more markedly in women than in men.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710746 TI - Induction of granulocytic maturation in acute myeloid leukemia by G-CSF and retinoic acid. AB - AML cells were cultured free of serum with G-CSF in combination with all-trans retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only. PMID- 1710747 TI - [Stress in health professionals and palliative care]. PMID- 1710748 TI - [The determination of amylase in pleural effusions due to neoplasms]. PMID- 1710749 TI - [Medical approach to the preterminal patient in oncology]. PMID- 1710750 TI - Alfuzosin for treatment of benign prostatic hypertrophy. The BPH-ALF Group. AB - To assess the long-term efficacy and safety of alfuzosin, a selective alpha 1 adrenergic antagonist, 518 symptomatic patients with benign prostatic hypertrophy (BPH) were randomised to received either alfuzosin (daily dose 7.5-10 mg) or placebo for 6 months. Obstructive and irritative symptoms, assessed according to the Boyarsky scale, significantly improved in the alfuzosin group compared with the placebo group (p = 0.0004). Fewer patients in the alfuzosin group than in the placebo group dropped out due to lack of efficacy (6.8% vs 14.6%, p = 0.004) and the prevalence of spontaneous acute urine retention was lower in the alfuzosin group (0.4% vs 2.6%, p = 0.04). By 6 months, mean urinary flow rates had increased (p less than 0.05) and residual volume had decreased (p = 0.017) in the alfuzosin group, although the two groups were broadly similar with respect to increase in peak flow rate. The overall incidence of adverse events was similar in the two groups, which led to the withdrawal of 10.8% and 9.0% of patients, respectively. The findings emphasise the magnitude of the placebo response in symptomatic patients with BPH and show that treatment with alpha 1 adrenergic antagonist drugs provides long-lasting improvement in such patients. PMID- 1710751 TI - In vivo pupillary constrictor effects of substance P in man. AB - The ocular effects of substance P (SP) were studied in 13 normal volunteers. Various concentrations of SP (0.135, 1.35 and 135 micrograms per 100 microliters) were instilled into the conjunctival sac and pupillary area changes were evaluated by means of an electronic pupillometer. The ability of SP to modify the mydriasis induced by pretreatment with 1% homatropine eyedrops was also studied. The instillation of SP produced miosis in a dose-dependent manner without provoking any ocular disturbances. Furthermore, the highest concentration tested was unable to reduce the homatropine-induced mydriasis. These findings indicate that SP exerts a pupillokinetic action in humans which probably occurs via a receptor mechanism. Since muscarinic blockade is not overcome by the peptide instillation, the results do not clarify whether SP causes miosis acting on iris muscles and/or cholinergic fibres. PMID- 1710752 TI - The stimulatory effect of estradiol 17-beta on prolactin mRNA is inhibited by anti-calmodulin drugs. AB - The stimulatory action of estrogens on prolactin (PRL) secretion and synthesis is well known; on the other hand, anti-calmodulin drugs have recently been shown to inhibit prolactin in vitro release induced by estrogens. Based on these data, we decided to evaluate the in vivo effect of anti-calmodulin drugs (trifluoperazine and W7) on basal and estradiol-17 beta stimulated levels of PRL mRNA in anterior pituitary lobes obtained from adult male rats. Total RNA was isolated from pooled pituitaries recovered from animals under the same treatment and, from it, hybridizable PRL mRNA was detected. Estradiol-17 beta consistently stimulated PRL mRNA levels by 3-4 fold. The utilization of either trifluoperazine or W7, invariably inhibited estradiol-17 beta stimulated PRL mRNA. Metoclopramide, a drug with antidopaminergic activity, potentiated the stimulatory effect of estradiol-17 beta on PRL mRNA levels. These results suggest that anti-calmodulin drugs have an in vivo antiestrogenic effect on PRL mRNA levels confirming previous in vitro studies. Although, it is difficult to be conclusive about the mechanism through which these drugs act, one possibility is that the calcium calmodulin system may be involved. PMID- 1710754 TI - Cellular immune responses to hepatitis B virus antigens in man. PMID- 1710753 TI - Platelet-activating factor stimulates expression of IL-1 beta mRNA in THP-1 cells. AB - A human monocytic cell line (THP-1) was used to study the effects of PAF (platelet-activating factor) on the expression of IL-1 beta mRNA. THP-1 cells were incubated with 10 pM PAF in the presence or absence of 0.1 microgram/mL endotoxin for 4 hr, after which cytoplasmic RNA was extracted and subjected to Northern hybridizations. PAF, alone and in combination with endotoxin, caused an increase in mRNA levels for IL-1 beta. The magnitude of the effects of PAF on IL 1 beta mRNA levels matched closely the effects seen at the level of protein synthesis, suggesting that the effects of PAF on IL-1 beta release may result largely from its effects on IL-1 beta mRNA levels. PMID- 1710755 TI - Evolutionary origin and diversification of the mammalian CD1 antigen genes. AB - CD1 antigens are cell-surface glycoproteins which have a molecular structure which is similar (consisting of extracellular domains alpha 1, alpha 2, and alpha 3, a transmembrane portion, and a cytoplasmic tail) to that of class I MHC molecules. Phylogenetic analysis of mammalian CD1 DNA sequences revealed that these genes are more closely related to the class I major histocompatibility complex (MHC) than to the class II MHC and that mammalian genes are more closely related to avian class I MHC genes than they are to mammalian class I MHC genes. The CD1 genes form a multigene family with different numbers of genes in different species (five in human, eight in rabbit, and two in mouse). Known CD1 genes are grouped into the following three families, on the basis of evolutionary relationship: (1) the human HCD1B gene and a partial sequence from the domestic rabbit, (2) the human HCD1A and HCD1C genes, and (3) the human HCD1D and HCD1E genes plus the two mouse genes and a sequence from the cottontail rabbit. The alpha 1 and alpha 2 domains of CD1 are much less conserved at the amino acid level than are the corresponding domains of class I MHC molecules, but the alpha 3 domain of CD1 seems to be still more conserved than the well-conserved alpha 3 domain of class I MHC molecules. Furthermore, in the human CD1 gene family, interlocus exon exchange has homogenized alpha 3 domains of all CD1 genes except HCD1C. PMID- 1710756 TI - Intervening sequences (IVSs) in the 23S ribosomal RNA genes of pathogenic Yersinia enterocolitica strains. The IVSs in Y. enterocolitica and Salmonella typhimurium have a common origin. AB - The 23S ribosomal RNA (rRNA) was shown to be in two fragments in pathogenic Yersinia enterocolitica. The cleavage site in the structural gene of the 23S rRNA was occupied by an intervening sequence (IVS) of about 100 nucleotides, analogous to IVSs found in salmonellae (Burgin et al., 1990). Nucleotide sequences of IVSs of several Y. enterocolitica strains revealed that the IVSs of the highly virulent Y. enterocolitica serotypes strains, and the IVS of Salmonella typhimurium were about 90% similar. On the other hand, the IVSs of the highly and the poorly virulent Y. enterocolitica serotypes were only about 60% similar. These results give the impression that at some point during the IVS evolution, the highly virulent Y. enterocolitica and S. typhimurium both received their IVSs at about the same time from the same source, and that the poorly virulent serotypes received their IVSs earlier. We also found that strain LB5010, derived by extended mutagenization of S. typhimurium LT2, had lost the IVSs originally present in LT2, and that this loss had created a new 'hairpin loop' which substituted for the original 'hairpin loop'. PMID- 1710757 TI - Error-prone EF-Tu reduces in vivo enzyme activity and cellular growth rate. AB - Mutations in Salmonella typhimurium encoding error-prone EF-Tu (tufA8, tufB103) enhance translational error levels and also cause a reduced growth rate. The relative changes in error level and growth rate are inversely related and dependent on the status of the two tuf genes. Possible causes of the reduced growth rate were investigated. Several important parameters with the potential to alter growth rate (the EF-Tu-ribosome interaction, the in vivo elongation rate and the processivity of translation), are all relatively unaffected by the tuf mutations. The small reduction in processivity observed in some strains is not quantitatively related to the growth rate reduction. Instead, the error-enhancing mutations are associated with a large reduction in the specific activity of a test protein, beta-galactosidase, suggesting by inference that the reduced growth rate is a consequence of the synthesis of error-containing proteins. PMID- 1710758 TI - A copy-number mutant of plasmid pSC101. AB - Copy-number mutants of plasmid pSC101 were isolated by u.v. mutagenesis and selection for elevated expression of ampicillin resistance. Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA. The mutated plasmids were maintained at a level four to five times higher than that of the wild type. For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild-type protein. We postulate that the copy-number effect is due to a combination of these two effects, i.e. higher protein concentration and increased affinity of the protein for the repeated sequences. PMID- 1710759 TI - Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). AB - The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G + C ratio of the rfb cluster extended the area of low-G + C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G + C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon. PMID- 1710760 TI - Osmotic induction of the periplasmic trehalase in Escherichia coli K12: characterization of the treA gene promoter. AB - The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase. A strain carrying a treA-lacZ transcriptional fusion was constructed. The beta-galactosidase activity produced in this strain growing exponentially in a medium of high osmotic pressure was 10-fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible. treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild-type E. coli strains. The treA promoter was identified by S1 nuclease protection. Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide -40 with respect to the transcription start. Transcription initiation at treAp required the presence of a functional sigma 70 subunit of RNA polymerase. treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon. PMID- 1710761 TI - Granulocyte colony-stimulating factors. PMID- 1710762 TI - Behavioral and neurochemical effects of prenatal methylenedioxymethamphetamine (MDMA) exposure in rats. AB - MDMA is a hallucinogenic drug that is used by the general public as a recreational drug of abuse. The neurobehavioral consequences of prenatal MDMA exposure are unknown. Groups of pregnant rats were gavaged with 0, 2.5, or 10 mg/kg MDMA during gestation on alternate gestational days 6-18. Gestational duration, litter size, neonatal birth weights and physical appearance at birth were unaffected by MDMA treatments. Pregnancy weight gain was significantly reduced by MDMA treatment. Progeny growth, maturational parameters (eye opening and incisor eruption times), surface righting reflex, swimming performance, forelimb grip strength, milk-induced behaviors, passive avoidance behavior, figure-8 maze activity over 48 hours, the density of brain serotonin (5-HT) uptake sites, and brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were unaffected by MDMA treatments. Olfactory discrimination on postnatal days (PND) 9 11 was enhanced in both male and female MDMA-treated progeny, while negative geotaxis (PND 7-10) was delayed in female pups. In contrast to progeny, MDMA caused dose-dependent decreases in 5-HT and 5-HIAA levels in discrete brain areas of the dam. It is concluded that prenatal exposure to MDMA at the levels used here produces only subtle behavioral alterations in developing rats. The dam is more at risk for MDMA-induced 5-HT depletion than is the conceptus. PMID- 1710763 TI - Developmental toxicity of desbromoleptophos in chicks: enzyme inhibition, malformations and functional deficits. AB - The relationship among inhibition of acetylcholinesterase (AChE), inhibition of neuropathy target enzyme (NTE), and developmental toxicity of the organophosphorus ester desbromoleptophos (DBL) was evaluated in chicks exposed on day 3 or day 15 of incubation or 10 days posthatching. DBL induced prolonged inhibition of AChE and NTE when administered either early or late in incubation, structural malformations if administered before organogenesis, posthatching paresis if administered after organogenesis, and delayed deficits of gait if administered after hatching. The posthatching paresis and abnormal gait are not determined solely by either AChE inhibition of NTE inhibition, since they occur in the absence of the latter and are not invariably seen in the presence of the former (Toxicology 49: 253-261; 1988). PMID- 1710764 TI - In utero PCB/PCDF exposure: relation of developmental delay to dysmorphology and dose. AB - In 1979, there was an outbreak of food poisoning in central Taiwan due to cooking oil contaminated with polychlorinated biphenyls and their thermal degradation products. Starting in 1985, we studied 128 children born to exposed women after the oil was removed from the market; the exposure of these children was transplacental or through breast milk. We also studied matched controls. The exposed children exhibited developmental delays as measured by parental report, by neurologic examination, and by standard cognitive tests; delay was seen at all ages and persisted over time. Delay was greater in children who were smaller in size and in children who had exhibited neonatal symptoms of intoxication. Children with a history of nail deformity also were delayed. However, there was little relationship between other physical findings or measures of maternal exposure and developmental delay. There was some indication that the child's prenatal exposure was more important to developmental delay than was exposure through breast milk. PMID- 1710765 TI - Lead exposure and the cognitive development of urban preschool children: the Cincinnati Lead Study cohort at age 4 years. AB - The purpose of this analysis was to determine if significant associations could be observed between prenatal/postnatal blood lead (PbB) levels and the cognitive development of 258 urban, inner-city children at 4 years of age. These children have been followed since birth with frequent assessments of general health, PbB, and neuropsychological status. The Kaufman Assessment Battery for Children (K ABC) was administered at approximately 4 years of age. Higher neonatal PbB levels were associated with poorer performance on all K-ABC subscales. However, this inverse association was limited to children from the poorest families. Maternal PbB levels were unrelated to 4-year cognitive status. Few statistically significant associations between postnatal PbB levels and K-ABC scales could be found. However, the results did suggest a weak inverse relationship between postnatal PbB levels and performance on a K-ABC subscale which assesses visual spatial and visual-motor integration skills. In these results we note both contradiction and accord with previously published prospective studies. PMID- 1710766 TI - Alternatively spliced murine lyn mRNAs encode distinct proteins. AB - Two lyn proteins of 56 and 53 kDa have been observed in immunoprecipitates from a variety of murine and human cell lines and tissues. We report the cloning and nucleotide sequence of two distinct murine lyn cDNAs isolated from an FDC-P1 cDNA library. One of the cDNAs, designated lyn11, encodes a protein of 56 kDa which shares 96% similarity with human lyn. The other cDNA, designated lyn12, encodes a protein of 53 kDa. The proteins differ in the presence or absence of a 21-amino acid sequence located 24 amino acids C terminal of the translational initiation codon. Using RNase protection analysis, we have identified mRNAs corresponding to both cDNAs in murine cell lines and tissues. Sequence analysis of murine genomic clones suggests that the distinct mRNAs are alternatively spliced transcripts derived from a single gene. Expression of both cDNAs in COS cells leads to the production of lyn proteins with the same molecular weight as the two forms of lyn proteins immunoprecipitated from extracts of FDC-P1 cells and mouse spleen. Subcellular fractionation studies and Western immunoblotting analysis suggest that both isoforms of lyn are membrane associated. The association of both lyn isoforms with the membrane fraction supports the notion that lyn, like other src related kinases, may interact with the intracellular domain of cell surface receptors. PMID- 1710767 TI - An endoplasmic reticulum-specific cyclophilin. AB - Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression. PMID- 1710768 TI - Identification of cis sequences controlling efficient position-independent tissue specific expression of human major histocompatibility complex class I genes in transgenic mice. AB - We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon. PMID- 1710769 TI - The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides. AB - Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly. PMID- 1710771 TI - Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice. AB - Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region. PMID- 1710770 TI - The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts. AB - A set of genes is rapidly inducible when quiescent fibroblasts are stimulated by growth factors or by the activation of temperature-sensitive retroviral protein tyrosine kinases. Most of these so-called immediate-early genes were cloned by differential cDNA hybridization. DNA sequence analysis identified many of them as putative members of the growth factor or of the transcription factor gene family, suggesting a role in signal transmission during the G0-to-G1 transition. In this study, we identified one of the genes that are rapidly inducible by the retroviral protein-tyrosine kinases v-Src and v-Fps of Rous sarcoma virus and Fujinami sarcoma virus, respectively, as the rhoB gene, a member of the ras gene superfamily whose products are GTP-binding proteins, rhoB is transiently activated at the transcriptional level by v-Fps and by epidermal growth factor. Its labile RNA is inducible in the presence of cycloheximide but not of actinomycin D. rhoB is strongly induced by epidermal growth factor and by platelet-derived growth factor both in subconfluent, serum-starved and in density arrested Rat-2 fibroblasts. Fetal calf serum is a poor inducer, particularly in density-arrested cells, and phorbol esters do not increase rhoB expression at all. These data suggest that rhoB is inducible by protein-tyrosine kinases through a pathway not involving the activation of protein kinase C. Neither the closely related rhoC and rhoA genes nor the distantly related c-H-ras gene is rapidly inducible by mitogens. Thus, rhoB is the first known member of the small GTP-binding proteins among the immediate-early genes. PMID- 1710772 TI - Secretion and transcriptional regulation of transforming growth factor-beta 3 during myogenesis. AB - Transforming growth factor-beta 3 (TGF-beta 3) mRNA is differentially expressed in developing and mature mouse tissues, including high-level expression in developing and adult cardiac tissue. We show now that TGF-beta 3 mRNA is also expressed highly in skeletal muscle as well as in the mouse skeletal myoblast cell line C2C12. We also show that C2C12 cells secrete TGF-beta 3, and that this TGF-beta is able to inhibit C2C12 myoblast fusion after activation. In order to begin to understand how the TGF-beta 3 promoter is regulated in specific tissues during development, we therefore studied the regulation of TGF-beta 3 during myoblast fusion. After fusion of C2C12 cells into myotubes, TGF-beta 3 mRNA levels increased eightfold as a result of increased TGF-beta 3 transcription. TGF beta 3 transcriptional regulation was studied in myoblasts and myotubes by transfection of chimeric TGF-beta 3/CAT promoter plasmids. Chloramphenicol acetyltransferase (CAT) activity was stimulated in myoblasts by several upstream regions between -301 and -47 of the TGF-beta 3 promoter and by the TGF-beta 3 5' untranslated region. CAT activity directed by the TGF-beta 3 promoter in myotubes was stimulated by a distinct upstream region located between -499 and -221. Therefore, the high level of TGF-beta 3 mRNA expression in muscle cells appears to be dependent on multiple regulatory events during different stages of myogenesis. PMID- 1710773 TI - The new steroids: can there be potency without toxicity? PMID- 1710775 TI - Effect of residual splenic function and folate levels on the frequency of micronucleated red blood cells in splenectomized humans. AB - Frequencies of micronucleated erythrocytes in the peripheral blood of splenectomized individuals can be used as an index of genetic damage to erythrocyte precursor cells in the bone marrow. This is in contrast to non splenectomized humans, whose micronucleated erythrocytes are removed by the spleen. Many subjects whose spleen has been removed surgically have residual spleen tissue and consequent residual spleen function (RSF), which can be measured by the percentage of 'pitted' peripheral red blood cells. In this study evidence of RSF was associated with decreased frequencies of micronucleated erythrocytes. Analysis of data limited to subjects with minimal spleen function suggested an inverse association between the incidence of micronucleated erythrocytes and serum folate levels that was not apparent in the absence of stringent control for RSF. PMID- 1710774 TI - The new steroids: clinical experience in ulcerative colitis. PMID- 1710776 TI - Biochemical evidence for two different mechanisms for bleomycin-induced cell killing. AB - Using pulsed-field gel electrophoresis, we have measured the ability of two bleomycin-sensitive mutants, XR-1 and BL-10, to repair DNA double-strands breaks (DSB). XR-1 was originally isolated by its hypersensitivity to killing with ionizing radiation, but we have also shown that it is sensitive to killing with bleomycin. In contrast, BL-10 was isolated by its extreme sensitivity to killing with bleomycin, and it is not cross-sensitive to other DNA breaking agents. A 1-h treatment of bleomycin induces a similar number of DNA double-strand breaks in XR 1, BL-10 and CHO cells. However, XR-1 is unable to repair bleomycin-induced DNA double-strand breaks, whereas BL-10 possesses the same kinetics of repair as parental CHO. These data lead us to conclude that at least two mechanisms of killing exist for bleomycin; one of them is DNA DSB-dependent, and the other seems to be DNA DSB-independent. PMID- 1710777 TI - Pain in sickle cell disease. Rates and risk factors. AB - BACKGROUND AND METHODS: Acute episodes of pain are the principal symptom of sickle cell disease, but little is known about the epidemiologic features of these episodes or risk factors for them, nor is it known whether patients with high rates of such episodes die prematurely. We prospectively studied the natural history of sickle cell disease in 3578 patients ranging from newborns to persons up to 66 years old who were followed at clinical centers across the United States. RESULTS: There were 12,290 episodes of pain in 18,356 patient-years. The average rate was 0.8 episode per patient-year in sickle cell anemia, 1.0 episode per patient-year in sickle beta 0-thalassemia, and 0.4 episode per patient-year in hemoglobin SC disease and sickle beta(+)-thalassemia. The rate varied widely within each of these four groups--e.g., 39 percent of patients with sickle cell anemia had no episodes of pain, and 1 percent had more than six episodes per year. The 5.2 percent of patients with 3 to 10 episodes per year had 32.9 percent of all episodes. Among patients with sickle cell anemia who were more than 20 years old, those with high rates of pain episodes tended to die earlier than those with low rates. High rates were associated with a high hematocrit and low fetal hemoglobin levels. alpha-Thalassemia had no effect on pain apart from its association with an increased hematocrit. CONCLUSIONS: The "pain rate" (episodes per year) is a measure of clinical severity and correlates with early death in patients with sickle cell anemia over the age of 20. Even when the fetal hemoglobin level is low, one can predict that small increments in the level may have an ameliorating effect on the pain rate and may ultimately improve survival. This outcome is particularly encouraging to investigators studying hydroxyurea and other treatments designed to increase the fetal hemoglobin level. PMID- 1710778 TI - Maternal serum alpha-fetoprotein screening. PMID- 1710779 TI - Second-trimester maternal serum alpha-fetoprotein levels and the risk of subsequent fetal death. AB - BACKGROUND: The finding of an elevated level of maternal serum alpha-fetoprotein during the second trimester of pregnancy may indicate that the fetus has died or is about to die. It is uncertain, however, whether the finding is associated with an increased risk of fetal death later in gestation independent of known causes of elevation, such as the presence of neural-tube defects or multiple gestation. METHODS: To address this question, we performed a case-control study of 612 women whose pregnancies ended in fetal death and 2501 women who gave birth to live infants, using reports from California vital statistics for 1987. All the women had signleton pregnancies and alpha-fetoprotein screening in the second trimester. RESULTS: Women with elevated levels of serum alpha-fetoprotein in the second trimester of pregnancy had an increased risk of fetal death, and the risk was increased until term. Women with the highest levels of serum alpha fetoprotein--greater than or equal to 3.0 times the median value--had a very high risk of fetal death (odds ratio, 10.4; 95 percent confidence interval, 4.9 to 22.0) as compared with women who had normal levels of alpha-fetoprotein. Maternal serum alpha-fetoprotein levels that were 2.0 to 2.9 times the median were also associated with an elevated risk of fetal death (odds ratio, 2.4; 95 percent confidence interval, 1.7 to 3.4). Elevated levels of alpha-fetoprotein were especially likely to be associated with fetal death in cases in which maternal hypertension or placental infarction was also present. CONCLUSIONs. An unexplained elevated level of maternal serum alpha-fetoprotein in the second trimester of pregnancy is associated with an increased risk of subsequent fetal death, up to four to five months after alpha-fetoprotein screening. PMID- 1710780 TI - Lower receptor avidity required for thymic clonal deletion than for effector T cell function. AB - Clonal deletion in the thymus plays a major part in T-cell tolerance to self antigens. But the mechanism of negative selection, its fine specificity and the threshold of affinity and avidity remains unknown. We have now examined these aspects of negative selection with mice expressing a transgenic T-cell receptor with specificity for lymphocytic choriomeningitis virus (LCMV) glycoprotein in association with the class I H-2Db molecule. These mice were rendered tolerant to LCMV by neonatal infection with mutant LCMVs bearing point mutations in the T cell epitope recognized by the transgenic T-cell receptor. Variant LCMVs were also tested for their ability to elicit antiviral responses in transgenic mice in vivo and in vitro. Comparison in vivo revealed that a low-avidity receptor interaction, which was unable to induce effector T cells in the periphery, was still sufficient for clonal deletion in the thymus. PMID- 1710781 TI - Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. AB - The transforming proteins of several DNA tumour viruses, including adenovirus E1a and simian virus 40 large T antigen, complex with the retinoblastoma (Rb) tumour suppressor gene product. This requires regions in these viral proteins necessary for transformation and is thought to inactivate the growth-suppressing properties of the Rb protein by disrupting its interaction with cellular targets. Indeed, regions of Rb required to form a complex with E1a and large T antigen are often mutated in transformed cells. The level at which the Rb protein regulates proliferation is unknown, although one possibility is transcription. We have previously characterized a sequence-specific transcription factor, DRTF1, the activity of which is downregulated as embryonal carcinoma stem cells differentiate. DRTF1 is found in several discrete protein complexes (a, b and c) which are of different sizes but have the same DNA specificity. We now show that one of these also contains the Rb protein and, further, that the adenovirus E1a protein causes the dissociation of the Rb protein from this complex. This requires conserved regions 1 and 2 of E1a that are known to be required for efficient transformation. These results demonstrate that the Rb protein forms a complex with a DNA-bound transcription factor, and suggests that the Rb protein might act by regulating transcription. PMID- 1710782 TI - Relationship of FKBP to PKCI-2. PMID- 1710783 TI - Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation. AB - The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation. PMID- 1710784 TI - Sympathetic regulation of cardiac calcium current is due exclusively to cAMP dependent phosphorylation. AB - The positive inotropic effect of the sympathetic nervous system on the heart is partly mediated by an increase in the voltage-gated Ca2+ current (ICa). This increase is generally attributed to beta-adrenergic receptor-stimulated cyclic AMP-dependent phosphorylation of the Ca2+ channel. It has been suggested that cAMP-dependent phosphorylation cannot explain all the effects of beta-adrenergic agonists on ICa and that a parallel membrane-delimited pathway involving the 'direct' action of the G protein Gs also stimulates ICa. A precedent exists for such a membrane-delimited pathway in the activation of a K+ channel by acetylcholine in heart. A membrane-delimited pathway for stimulation of ICa might be important in rapid beat-to-beat regulation of contraction by the sympathetic nervous system, because isoproterenol may produce a biphasic increase in ICa with the rapid phase (tau = 150 ms) putatively mediated by the direct pathway and the slow phase (tau = 35 s) by cAMP-dependent phosphorylation. Here we report that in frog, rat, and guinea pig ventricular myocytes ICa increases slowly and monophasically in response to isoproterenol. The increase is completely blocked by inhibitors of cAMP-dependent phosphorylation. Furthermore, the time course of the increase in ICa closely parallels the increase in contractile force produced by sympathetic nerve stimulation. These data refute earlier suggestions that regulation of Ca2+ channels by the sympathetic nervous system involves or requires a direct G-protein pathway. PMID- 1710785 TI - Inhibitory prejunctional muscarinic receptors at sympathetic nerves do not operate through a cyclic AMP dependent pathway. AB - In mouse atria previously incubated with [3H]-noradrenaline, carbachol (1.0 mumo1/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the alpha-adrenoceptor antagonist phentolamine (1.0 mumol/l), which by itself significantly increased the S-I outflow of radioactivity. In both cases the inhibitory effect of carbachol was blocked by atropine (0.3 mumol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 mumol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (IBMX, 100 mumol/l), was used to maximally enhance the S-I outflow of radioactivity through the cyclic AMP mechanism. The inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was not reduced in the presence of 8-bromo cyclic AMP and IBMX. Similar results with carbachol in the presence of 8-bromo cyclic AMP and IBMX were also found in rat right atrial strips which had been incubated with [3H]-noradrenaline. These results suggest that the effects through inhibitory prejunctional muscarinic receptors are not mediated by cyclic AMP. The protein kinase inhibitor, staurosporine (0.1 mumol/l), significantly blocked the enhancing effects of 8 bromo cyclic AMP (270 mumol/l) plus IBMX (100 mumol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 mumol/l) however, was not reduced in the presence of staurosporine, suggesting that protein kinases affected by staurosporine (protein kinase A, protein kinase C) are not involved in the post-receptor mechanism for inhibitory prejunctional muscarinic receptors. This finding further rules out the involvement of cyclic AMP in muscarinic inhibition. The inhibitory effect of carbachol either by itself or in the presence of phentolamine, was not reduced in atria from mice that had been pretreated with pertussis toxin (1.5 or 3.0 micrograms). Furthermore, in rat atrial strips, the inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was also not altered by pretreating the rats with pertussis toxin (8.4 micrograms). The results suggest that in both tissues the major mechanism for inhibition of noradrenaline release through muscarinic receptors does not involve a pertussis toxin sensitive G protein. PMID- 1710787 TI - Smooth muscle of rabbit isolated aorta contains the NK-2 tachykinin receptor. AB - Neurokinin A, neurokinin B and substance P caused concentration-related contractions of rabbit isolated aorta with pD2 values of 8.1, 6.9, and 6.0, respectively. [D-Pro2, D-Trp7, 9]-substance P, a competitive tachykinin antagonist, had pA2 values of 5.3 against neurokinin A, 5.1 against neurokinin B and 5.2 against substance P indicating that tachykinin receptors mediated responses to the agonists. [pGlu5,MePhe8,-MeGly9]-substance P 5 - 11 (DiMe-C7), senktide and septide did not contract the aorta. It is concluded that of the known tachykinin receptors smooth muscle of the rabbit isolated aorta contains only the NK-2 type. PMID- 1710786 TI - Comparison of cyclic nucleotide phosphodiesterase isoenzymes in rat and rabbit ventricular myocardium: positive inotropic and phosphodiesterase inhibitory effects of Org 30029, milrinone and rolipram. AB - The species dependent variation in the cardiotonic activity of selective cyclic nucleotide phosphodiesterase (PDE) isoenzyme inhibitors was examined by comparing the inotropic and PDE inhibitory effects of Org 30029 (N-hydroxy-5,6-dimethoxy benzo[b]thiophene-2-carboximidamide HCl), 3-isobutyl-1-methyl-xanthine (IBMX), milrinone and rolipram in rat and rabbit ventricular myocardium. The relative activities of PDE isoenzymes in rat and rabbit cardiac ventricle were also examined to assess the role of the different PDE subtypes in modulating contractile force in the two species. In rabbit papillary muscles, IBMX, Org 30029 and milrinone increased contractile force whilst rolipram was inactive. The rank order of potency of the active compounds was Org 30029 greater than IBMX greater than milrinone. Only Org 30029 and IBMX produced significant positive inotropic responses in rat papillary muscles, milrinone and rolipram being inactive. However, large positive inotropic responses were obtained in rat papillary muscles when milrinone and rolipram were tested in combination. In rabbit papillary muscles, the positive inotropic action of milrinone was markedly potentiated by rolipram. Four main types of PDE (I, II, III, IV) isoenzymes were resolved, by DEAE-sepharose or Mono-Q ion-exchange chromatography, from both rat and rabbit cardiac ventricular tissue. In rabbit, Ca2+/calmodulin dependent PDE (PDE I) and cyclic GMP inhibited PDE (PDE III) were the dominant cAMP activities. In contrast, cyclic GMP stimulated PDE (PDE II), PDE III and cGMP insensitive PDE (PDE IV) represented the main cAMP activities in rat cardiac ventricle. The inhibitory effects of Org 30029, IBMX, milrinone and rolipram on PDE isoenzymes from rat and rabbit cardiac ventricle were essentially similar.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710788 TI - Sex differences in urinary alpha-1-microglobulin value in normal individuals. PMID- 1710789 TI - Immunocytochemical localization of border disease virus in the spinal cord of fetal and newborn lambs. AB - The peroxidase-antiperoxidase technique was used to determine the cellular localization of Border Disease (BD) virus in cryostat sections of fetal and newborn lamb spinal cord following experimental infection by maternal inoculation in early gestation. Viraemic fetuses and lambs with hypomyelinogenesis showed BD viral antigen in neurons, glia, ependymal cells, vascular endothelial cells and fibrocytes within the dura mater. Double immunolabelling demonstrated co expression of BD viral antigen and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) in both fetal and newborn lamb glia. In fetal lambs there was a pia-associated population of glia in which viral antigen was also co expressed with GFAP or MBP. The results suggest that BD virus infects myelinating oligodendroglia, astroglia and probably also transitional cells and pluripotential glioblasts. The relationship between infection of specific cell types and hypomyelinogenesis was not resolved but infection of transitional cells and oligodendroglia may affect oligodendroglial function and permit morphologically inapparent perturbations leading to hypomyelinogenesis. A single nonviraemic lamb with a precolostral antibody titre to BD virus and cystic cerebral cavities but no hypomyelinogenesis showed BD viral antigen confined to glia of the spinal cord white matter. This suggests that oligodendroglia may require to be infected before a critical period in their development or factors additional to oligodendroglia infection are necessary for hypomyelinogenesis. PMID- 1710790 TI - A useful technique for improving the diagnostic yield from single stereotactic biopsies of brain. PMID- 1710791 TI - Possible involvement of hypothalamic monoamines in mediating the action of alpha 2u-globulin on the pituitary-testicular axis in rats. AB - A major androgen-dependent urinary protein of male rodents, alpha 2u-globulin, has been shown to influence adenohypophyseal hormone release. In an attempt to elucidate the mechanisms of its action, we have examined several parameters of hypothalamic and pituitary function in adult male rats treated for 2 weeks with two injections daily of 0.75 mg alpha 2u-globulin and sacrificed 16 h after the last injection. This treatment led to an increase in plasma luteinizing hormone levels, a decrease in plasma prolactin levels, an increase in testosterone concentrations in both plasma and testicular tissue, and increases in testicular weights. The norepinephrine turnover in median eminence and anterior hypothalamus was increased in alpha 2u-globulin-injected animals, while the norepinephrine turnover in the remaining medial basal hypothalamus was reduced. alpha 2u Globulin-treated animals had a significantly decreased dopamine turnover in the anterior hypothalamus, while in the medial basal hypothalamus the dopamine metabolism was increased. These data suggest that alpha 2u-globulin-induced changes in gonadotropin and prolactin secretion are mediated by changes in catecholamine metabolism in several hypothalamic regions. Increased testosterone secretion appears to be due to increased secretion of gonadotropins from the pituitary. PMID- 1710792 TI - Thyrotropin alpha- and beta-subunit responses to thyrotropin-releasing hormone and domperidone in normal subjects and in patients with microprolactinomas. AB - Microprolactinoma is a particular pathological situation characterized by the presence of increased hypothalamic dopaminergic tone reactive to tumoral hyperprolactinemia. Since dopamine (DA) is a physiological regulating factor of the secretion of thyroid-stimulating hormone (TSH), we investigated the responses of serum TSH (holo-TSH) and its subunits (alpha-subunit: alpha-sub, and TSH-beta) to thyrotropin-releasing hormone (TRH) and domperidone (DOM; an antidopaminergic drug acting outside the blood-brain barrier) in 36 euthyroid subjects (20 controls and 16 patients with microprolactinoma) in order to evaluate the possible in vivo effects of DA excess on TSH subunit secretion. No significant difference in serum TSH increase after TRH (200 micrograms i.v.) was observed between patients with microprolactinoma and controls (TSH net incremental area under the curve, nAUC: 146 +/- 9, mean +/- SE, and 143 +/- 7.7 micrograms/l/60 min, respectively), while serum alpha-sub and TSH-beta responses were markedly reduced in patients with microprolactinoma as compared to those found in normals (alpha-sub nAUC: 3.0 +/- 0.5 vs. 19.8 +/- 2.2 micrograms/l/60 min, p less than 0.001; TSH-beta nAUC: 5.0 +/- 0.8 vs. 9.5 +/- 0.9 micrograms/l/60 min, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710793 TI - Differential effects of d-fenfluramine and p-chloroamphetamine on H75/12-induced depletion of 5-hydroxytryptamine and dopamine in the rat brain. AB - The effects of the two 5-HT-releasing drugs, p-chloroamphetamine and d fenfluramine, on central serotoninergic and dopaminergic systems were compared in adult rats. Both drugs (0.5-5.0 mg/kg i.p., 2 hr before death) produced a dose dependent reduction in levels of 5-HT, but only p-chloroamphetamine decreased the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus, striatum and cerebral cortex. Within the dose range tested, d-fenfluramine did not affect the levels of DA and of its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in brain. By contrast, p-chloroamphetamine significantly increased the levels of DA and HVA and decreased the levels of DOPAC, notably in the striatum. As expected of a 5-HT uptake inhibitor, d-fenfluramine at small doses (0.2-0.5 mg/kg) prevented the depletion of 5-HT due to 4-methyl-alpha-ethyl meta-tyramine (H75/12, 40 mg/kg i.p.), whereas at large doses (1.0-5.0 mg/kg) d fenfluramine, like p-chloroamphetamine (0.2-1.0 mg/kg), slightly enhanced the effect of H75/12. Neither d-fenfluramine (0.5 mg/kg) nor p-chloroamphetamine (0.5 mg/kg) affected the depletion of DA due to H75/12. These data indicate that p chloroamphetamine is a 5-HT-releasing drug, at any dose between 0.2 and 5.0 mg/kg, whereas d-fenfluramine acts as a 5-HT uptake inhibitor at 0.2-0.5 mg/kg and as a 5-HT releasing drug at larger doses. On account of the potential neurotoxicity of 5-HT-releasing drugs but not 5-HT uptake inhibitors, it can be inferred that d-fenfluramine is very probably devoid of any neurotoxic action in the dose range (less than 1.0 mg/kg) required for its anorectic action. PMID- 1710794 TI - Serotonin inhibits release of substance P evoked by tooth pulp stimulation in trigeminal nucleus caudalis in rabbits. AB - The influence of the serotonin (5-HT) system on the release of immunoreactive substance P after electrical stimulation of the lower incisor pulp was examined by perfusion of the superficial layers of the subnucleus caudalis of the brain stem trigeminal sensory nuclear complex of rabbits in situ. Increased release of immunoreactive substance P was observed after electrical stimulation of the pulp at 40 V. Stimulation of the nucleus raphe magnus significantly increased the release of 5-HT and completely inhibited the release of immunoreactive substance P, evoked by stimulation of the tooth pulp. Local application of 5-HT (10(-6) M) inhibited the release of immunoreactive substance P induced by stimulation and this inhibition was antagonized by methysergide (10(-4) M) applied concomitantly to the superficial layers of the trigeminal nucleus. These results suggest a functional interaction between substance P and 5-HT in the superficial layers of the trigeminal nucleus for regulation of transmission of dental pain. PMID- 1710795 TI - Influence of neurotransplantation on search for food in rats following electrolytic lesion of the amygdala. PMID- 1710796 TI - Features of the ultrastructure of the cerebellar cortex during postnatal development of animals under conditions of protein-energy insufficiency and subsequent rehabilitation. PMID- 1710797 TI - The role of ultrasound in evaluation of patients with elevated maternal serum alpha-fetoprotein: a review. AB - Accumulated experience with the sonographic detection of spina bifida and its associated fetal cranial changes indicates that nearly all these fetuses may be identified by ultrasound examination alone. A review of data from multiple centers shows that a complete and detailed, normal ultrasound is an appropriate basis for a reduction of at least 95% in the maternal serum alpha-fetoprotein (MSAFP)-based risk for neural tube defects. The use of this adjusted risk in patient counseling before amniocentesis may lower the rate of the procedure with no significant loss of diagnostic sensitivity. Among patients with elevated MSAFP, the rate of abnormal cytogenetic findings in fetuses with no abnormalities detected at ultrasound appears to be near 0.61%. Furthermore, the spectrum of abnormal cytogenetic results appears to differ from that associated with increased maternal age, in that the incidence of sex chromosome abnormalities is higher and that of Down syndrome is lower. PMID- 1710798 TI - Ocular pathology in disialotransferrin developmental deficiency syndrome. AB - Disialotransferrin developmental deficiency (DDD) syndrome is a recently described disease consisting of hepatopathy, mental retardation and neuropathy. The biochemical findings indicate a defect in the assembly of the carbohydrate moiety that is common to the secretory glucoproteins. It is believed to be of autosomal recessive inheritance. An ophthalmological examination of ten children suffering from this syndrome showed that all had ocular involvement. Esotropia (and deficient abduction) was found in all ten patients. Seven children had retinitis pigmentosa which was verified by an ERG in three. One patient had retinal signs suggestive of retinitis pigmentosa. The high incidence of ocular findings in the DDD syndrome, which are reported for the first time, indicate that an ophthalmological examination is a helpful diagnostic tool in this disease. PMID- 1710799 TI - Modified Blalock Taussig shunt anastomosis in a three month old child with pulmonary stenosis: embolization therapy. AB - A case of transcatheter occlusion of a modified Blalock-Taussig (BT) shunt with a detachable balloon is described. A three month old boy with pulmonary atresia with intact ventricular septum had a repair consisting in valvotomy and a modified BT. This palliative aorticopulmonary shunt created congestive heart failure. As an alternative to surgery, a detachable balloon was used to occlude the BT shunt. PMID- 1710800 TI - Interaction of progesterone derivatives with the digitalis target enzyme: impact of glycosidation on inhibitory potency. AB - As a function of the structural modification of the steroid nucleus, the inhibitory interaction of 11 progesterone derivatives with human Na/K-ATPase (Na+/K(+)-transporting ATPase, EC 3.6.1.37), through C3-O-rhamnosylation, is either much decreased or weakly up to strongly increased, so that the rhamnosyl residue contributes to the complementary Gibbs energy of interaction, at the most, the same Gibbs energy increment as realized in ouabain. After C3 beta-O rhamnosylation, the activity of some progesterone derivatives considerably surpasses that of 3 beta-O-rhamnosyl-chlormadinolacetate, which has been known to elicit positive inotropy in cats. The progesterone derivatives (aglycons and glycosides), that have been analysed more closely, produce their effects by the same molecular mechanism of interaction with Na/K-ATPase as characteristic for digitalis aglycons and glycosides. The results promise to pave the way for the identification of the chemical nature of endogenous digitalis and for the design of novel inotropic drugs. PMID- 1710801 TI - Chlordecone inhibits three types of ion channels in a neural cell line. PMID- 1710802 TI - [Synthetic antithyroid drugs and Basedow's disease or the choice of a therapeutic strategy]. AB - We present the conclusions of two prospective studies of patients examined at their first manifestation of Graves' disease and treated with antithyroid drugs (ATD). The purpose of the first study was to investigate the effects of long-term treatment: the patients were given carbimazole in degressive doses without hormone replacement for 18 months, the followed up for 2 to 6 years after drug withdrawal. The second study was designed to determine the effect of treatment duration on the prognosis: the patients were given an ATD according to the same protocol for a duration randomly set at either 6 or 18 months, then seen again 2 years after ATD withdrawal. The results showed that after 18 months of treatment at least 50 percent of the patients could be expected to remain in remission for 6 years. Remissions were less frequent when treatment was shorter (41.7 percent after the 6 month treatment versus 61.8 percent after the 18 month treatment, with a 2 years' follow-up; P less than 0.05). The relapses that occurred came early: 70 percent of them took place within the first post-treatment month. This article also provides evidence of high T3 and/or T4 levels without signs of thyrotoxicosis during the post-treatment clinical course; these exclusively biochemical relapses spontaneously disappeared and may have been expressing epidoses of active thyroiditis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710803 TI - Detection of species-specific and cross-reactive epitopes in Sarcocystis cystozoites by monoclonal antibodies. AB - A total of 24 hybridoma cell lines producing monoclonal antibodies (mAbs) to Sarcocystis muris were established from 3 fusions performed on mice that had been immunized with total protein derived from cystozoites. In all, 19 mAbs were of the IgG1 isotype, 3 were of the IgG2a isotype, and 2 were of the IgG3 isotype. The reactivity of the mAbs with homologous and heterologous antigens was examined by an indirect fluorescent antibody test, an enzyme-linked immunosorbent assay (ELISA), and a dot ELISA. MAbs were directed against various structures of the cystozoite as revealed by different immunofluorescence patterns. In all, 12 mAbs recognized only antigens of S. muris cystozoites, whereas the other 12 mAbs also recognized various antigens of cystozoites of S. gigantea, S. tenella, S. arieticanis, S. capracanis, S. miescheriana, or S. suihominis. No cross-reactions with endozoites of Toxoplasma gondii were observed. PMID- 1710804 TI - Antigenic characterisation of monoclonal antibodies against Sarcocystis muris by western blotting and immuno-electron microscopy. AB - The antigenic specificities of 13 monoclonal antibodies (mAbs) raised against Sarcocystis muris cystozoites were examined by Western blotting and immuno electron microscopy against homologous S. muris and heterologous S. gigantea, S. tenella, S. arieticanis, S. capracanis, S. miescheriana and Toxoplasma gondii antigens. Four mAbs reacted in Western blots against S. muris antigens: SM-4 and 17 recognized single antigenic bands (31,000 and 34,000 MW, respectively) and SM 2 and -3 reacted against multiple bands (ranging from 12,500-30,000 and 13,000 50,000 MW, respectively). Similar antigens were also recognized by polyclonal immune sera from chronically infected mice. None of the mAbs cross-reacted with heterologous Sarcocystis spp. or T. gondii. Ultrastructural studies performed with colloidal-gold conjugates demonstrated that three mAbs reacted with specific antigenic elements in S. muris cystozoites: SM-3 and -4 labelled pellicular determinants and SM-19 labelled micronemes. None of the mAbs cross-reacted with heterologous Sarcocystis spp., whereas polyclonal immune sera from chronically infected sheep, goats and pigs cross-reacted with a variety of antigens in all Sarcocystis spp. except the primary cyst-wall determinants. PMID- 1710805 TI - Hyaline globules in Kaposi's sarcoma: a light microscopic and immunohistochemical study. AB - Hyaline globules (HG) were detected in 51 of 54 Kaposi's sarcoma (KS) lesions (94.4%), including all four non-acquired immunodeficiency syndrome (AIDS) cases of cutaneous KS in this group of cases. Thus, there was no correlation between the presence of HG and the presence or absence of AIDS, nor could we demonstrate any relationship between the presence or prominence of HG and either the histologic pattern or anatomical distribution of KS. HG were located mainly in the cytoplasm of perivascular cells, histiocytoid cells, and spindle-shaped cells and occasionally in endothelial cells lining vessels or slit-like spaces. Extracellular HG were also seen. HG stained positively with periodic acid-Schiff with and without diastase digestion and with phosphotungstic acid-hematoxylin. HG were immunohistochemically negative for alpha 1-antitrypsin, alpha 1 antichymotrypsin, lysozyme, and Factor VIII-related antigen, but the cells containing HG were often positive for alpha 1-antichymotrypsin and occasionally for alpha 1-antitrypsin and Factor VIII-related antigen. HG were also detected in five of six angiosarcomas, two of ten pyogenic granulomas, and seven of 32 inflammatory granulation tissues. These were immunohistochemically similar to HG in KS. Thus, HG are not specific for KS. We support the interpretation that HG are most likely digested erythrocytes. PMID- 1710806 TI - Cytokeratin immunohistochemistry as a diagnostic tool for distinguishing malignant from benign epithelial lesions of the prostate. AB - The basal cell layer (BCL) is believed to be absent in malignant but present in nonmalignant epithelial lesions of the prostate. Using the avidin-biotin peroxidase complex (ABC) immunoperoxidase method, we examined the value of the monoclonal antibody cocktail MA-903, which stains selectively the prostatic BCL layer, in the distinction between benign and malignant epithelial lesions of the prostate. We immunostained histologic sections of 63 prostates, containing 235 morphologic appearances: normal prostate glands, 43; benign prostate hyperplasia (BPH), 59; basal cell hyperplasia (BCH), 24; adenosis, seven; prostatic intraductal neoplasia (PIN 1), 21; PIN 2, 25; PIN 3, 16; and cancer, 40. Some degree (continuous, continuous with focal disruption, and disrupted patterns) of basal cell staining was demonstrable in all normal and BPH, BCH, and PIN 1 lesions, but was absent in 39 of 40 cancers. However, not every gland in benign lesions stained positively. Further, two of 25 PIN 2 and six of 16 PIN 3 lesions failed to reveal BCL. Our results suggest that the presence or absence of BCL, predicated on cytokeratin MA-903 immunoreactivity, may be a useful indicator in the distinction between benign and malignant epithelial lesions of the prostate. PMID- 1710807 TI - Immunohistochemistry as a diagnostic aid in the interpretation of unusual mesenchymal tumors of the uterus. AB - Sixty-three pure mesenchymal tumors of the uterus were studied to explore the value of immunostaining in the diagnosis of unusual mesenchymal tumors encountered in the uterus, some not reported previously. Each tumor was evaluated using a panel of immunostains including actin, desmin, vimentin, S-100 protein, and cytokeratin. The final classification, which incorporated the immunohistochemical findings, resulted in the identification of 33 relatively common pure mesenchymal tumors (13 benign and malignant endometrial stromal tumors and 20 benign and malignant smooth muscle tumors) and 30 uncommon tumors (five leiomyosarcomas with osteoclastic giant cells, two xanthomatous leiomyosarcomas, one melanotic schwannoma, one pure rhabdomyosarcoma, one neurofibroma, five plexiform tumorlets, and 15 combined smooth muscle-stromal tumors). The normal endometrial stroma, present in 14 cases, invariably showed a negative reaction for all antibodies. With rare exceptions, the pure endometrial stromal tumors displayed a negative immunoreaction for all antibodies utilized, while the pure smooth muscle tumors consistently showed a positive reaction for actin. Only the two tumors of neural origin (a neurofibroma and a melanotic schwannoma) reacted with S-100 protein. Immunostaining influenced most the final classification of neoplasms initially interpreted as uterine tumors with a sex cord stromal pattern, endometrial stromal tumors that diverged from the classic lesions by having a spindle cell component, and intravascular leiomyomas with areas of compact proliferation of small round cells with prominent vascularity. All tumors in these three groups were reclassified as combined smooth muscle stromal tumors following immunohistochemical studies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710808 TI - Differential influence of various calcium-modulating compounds on ouabain intoxication in isolated rat left atria. AB - Various calcium-modulating compounds were tested with respect to their protective action against cardiac glycoside toxicity. At the concentrations applied, control force of contraction was reduced by nifedipine and verapamil and slightly attenuated by flunarizine, R 56865, and cimetidine, while it was strongly enhanced by Bay K 8644. The positive inotropic response to ouabain (stimulation rate: 1 Hz) was impaired by nifedipine and verapamil. The increment in contractile force induced by Bay K 8644 was not enhanced by ouabain. The increase in diastolic tension during toxic conditions of ouabain (stimulation rate: 3 Hz) was attenuated by nifedipine, verapamil, bepridil, flunarizine, cimetidine, phenytoin, and R 56865 but not by diltiazem, amiodarone, and amiloride. K loss was prevented by nifedipine, verapamil, diltiazem, bepridil, flunarizine, cimetidine, phenytoin, and R 56865. The increase in cellular Na content was inhibited by R 56865 only. Ca gain was prevented by verapamil, bepridil, flunarizine, R 56865, and cimetidine but not by nifedipine, diltiazem, phenytoin, amiodarone, and amiloride. Ionic deterioration was enhanced by Bay K 8644. These results suggest that pretreatment with various calcium-modulating compounds protects against mechanical and ionic changes during ouabain intoxication induced by Na-Ca overload through different mechanisms. PMID- 1710809 TI - Changes in ion channel properties related to gravity. PMID- 1710810 TI - Interaction between high-risk infants and their mothers: the NCAST as an assessment tool. AB - Using the NCAST Feeding, Teaching, and HOME Scales, we tested 37 high-risk infants matched with 37 healthy infants on gender, race, and socioeconomic status. All infants were 8 months old. A one-to-one matched case-control design was used to determine whether increased risk of impaired mother-infant interaction was associated with case status. Conditional logistic regression was used to control for possible confounding and to evaluate interaction. Of the 37 high-risk infant-mother dyads, 25 had a low score on one or more scales while only 10 of the control dyads had a low score on one or more of the three scales. The Feeding scale was the only assessment in which the association found in the univariate analysis persisted after adjusting for other variables. Because of its low cost and high efficiency, the NCAST battery appears to be valuable for directing more specialized intervention services in a high-risk infant population. PMID- 1710811 TI - Non oat cell lung carcinoma: is there a role for combined brachytherapy and external radiotherapy? PMID- 1710812 TI - [Left subclavian-pulmonary trunk fistula with a vascular prosthesis as palliative treatment in Fallot conditions]. AB - In this paper, our experience with a new systemic-to-pulmonary artery shunt: subclavian-pulmonary artery trunk shunt with PTFE (central shunt) is presented. Between November 1985 and March 1990 this central shunt was employed in 8 children with ages ranged between 4 days and 3 years, and weights between 2 and 12 kg. Diagnosis were Fallot's tetralogy in three; pulmonary atresia with intact septum in three; complete AV canal and Fallot's tetralogy in one, and univentricular heart and pulmonary atresia in 1 patient. There were no surgical deaths. Hospital mortality was present in 1 case (AV canal and Fallots tetralogy) at the moment of reoperation 1 month later. Follow-up ranged between 2 and 46 months. One case, pulmonary atresia with intact septum, has undergone total correction. The remainder 6 cases are in good situation with O2 saturation above 70%. This central shunt has the advantages to provide a bidirectional blood flow to both pulmonary branches avoiding the risk of direct damage in the pulmonary arteries found with the conventional aorto-pulmonary shunts. PMID- 1710813 TI - [Intestinal microbial pathology in AIDS. A clinical case series]. AB - Microbial isolates from 60 diarrheic AIDS patients hospitalized to the Infectious Disease Division of Careggi hospital (Florence) are described. Clinical, microbiological and diagnostic features of each case are discussed with emphasis to some rare or underestimated entities in Europe: Campylobacter laridis bacteremia, Whipple-like disease by atypical Mycobacteria, Schistosoma mansoni proctocolitis. Results regarding newly AIDS-related microorganisms are also stressed. PMID- 1710814 TI - DNA damaging activity of methyl parathion. AB - 14C-methyl parathion was covalently bound to DNA, RNA and proteins of various rat and mouse organs 22 hr after i.p. injection. Covalent binding index (CBI) to liver DNA was low in both species and typical of weak initiators. The labelings of RNA and proteins from different organs of both species was slightly higher than DNA binding. No interaction with brain nucleic acids was observed (CBI detection limit: 2.8). The in vitro enzyme-mediated interaction of methyl parathion with calf thymus DNA was mainly performed by rodent liver microsomes and, to a lesser extent, by microsomes from mouse kidney and lung whereas brain microsomes were inefficient. Activation of methyl parathion by cytosolic fractions from different organs of both species to form(s) capable of binding to DNA was negligible. When microsomes and cytosolic fractions from rodent liver and lung or mouse kidney were simultaneously present in the incubation mixture, a synergistic effect in catalyzing DNA binding was observed. The extent of DNA binding was reduced by adding SKF 525-A to the microsomal standard incubation mixture, whereas it was enhanced by adding GSH to liver or lung murine microsomes or to mouse kidney microsomes. These results suggest that methyl parathion is bioactivated by P450-dependent microsomal mixed function oxidase system and by microsomal GSH-transferases. By contrast, cytosolic GSH- transferases play a detoxificant role in the metabolism of this compound. PMID- 1710815 TI - [Angiogenesis and gene]. PMID- 1710816 TI - The lipopolysaccharide of Shigella bacteria as a virulence factor. AB - The virulence factors of the lipopolysaccharide of Shigella species bacteria include the endotoxic activities of the lipid A component of the molecule and the ability of the polysaccharide chain--the core and the O-antigenic polysaccharide- to provide the bacterium with resistance to host defense mechanisms such as opsonization, phagocytosis, and intracellular killing. Structural features of the lipopolysaccharides of four Shigella species-S. dysenteriae, S. flexneri, S. boydii, and S. sonnei--are described. PMID- 1710817 TI - Introduction: the increasing importance of Acanthamoeba. PMID- 1710818 TI - Effects of corticosteroids in experimental Acanthamoeba keratitis. PMID- 1710819 TI - The adjuvant action of cholera toxin is associated with an increased intestinal permeability for luminal antigens. AB - This study addresses the question of whether cholera toxin (CT) increases gut permeability for molecules greater than 3000 Da and whether such an effect is associated with an adjuvant function by CT on the gut immune response. We found that CT after oral administration gives rise to strikingly increased gut permeability for Dextran (Mw 3000) concomitantly with a strong enhancing effect on the anti-keyhole limpet hemocyanin (KLH) specific immune response in the lamina propria after oral immunization with KLH plus Dextran and CT. In contrast, the B-subunit of the holotoxin, which lacks the adenylate cyclase/cAMP-activating property of CT, failed to increase gut permeability as well as local anti-KLH immune responses. These results might suggest a causal linkage between the ability of CT to increase gut permeability and its adjuvant property on gut mucosal immune responses. In addition this finding supports the notion that the adenylate cyclase/cAMP system plays a regulatory role in gut permeability and is important in enhancing mucosal immune responses. Based on previous studies and the present data we propose that the mechanism for CT's adjuvant function on mucosal immune responses is by affecting antigen-presenting cells, T and B cells in the gut to give a net enhancing effect on the stimulation of local immunity, and that the CT-induced increase in gut permeability might be part of the adjuvant mechanism by facilitating luminal antigens to access the gut mucosal immune system. PMID- 1710820 TI - The complex of alpha 2-macroglobulin with CD2 in the plasma of gastric carcinoma patients. AB - alpha 2-macroglobulin (alpha 2M) preparations purified from the plasma of healthy persons and gastric carcinoma patients were further analysed by immunochemical techniques. It was found that only alpha 2M from the plasma of gastric carcinoma patients gave a positive reaction with E-receptor sheep antiserum in rocket immunoelectrophoresis. The alpha 2M fraction giving the positive reaction with E receptor sheep antiserum was isolated and analysed by the immunoblotting technique with monoclonal antibodies to T11 antigen. The presence of the alpha 2M CD2 complex in the plasma of gastric carcinoma patients was demonstrated. This finding may explain the immunosuppressive effect of the plasma alpha 2M in cancer patients. PMID- 1710821 TI - Effect of Salmonella bacteria on the interaction of human NK cells with endothelial cells. AB - Leukocyte-endothelial cell adhesion and its regulation are essential and complex initial aspects of lymphocyte migration. Various factors (IL-1, TNF-alpha, IFN gamma etc.) have been shown to increase the endothelial adhesiveness for human lymphocytes, including natural killer cells (NK cells). In this work we have demonstrated that pretreatment of either the target endothelial cell monolayers or the binding LGL-cells with mR595 Salmonella Minnesota bacteria results in a substantial increase in the adhesiveness of LGL-cells to endothelial cells. The increase was more prominent when the endothelial cells were treated than when the adhering LGL-cells were similarly pretreated. The adhering cell population was significantly enriched with CD56 (Leu19) and CD16 positive cells, i.e. cells with NK cell phenotype, when the lymphocyte population was pretreated. However, the pretreatment of EC resulted in a non-specific increase in EC adhesiveness since the relative proportion of CD56+ (Leu19), CD16+ and CD3+ cells among the adhering cells did not significantly differ from the starting population. The bidirectional enhancement of adhesiveness of human NK cells to endothelium by mR595 Salmonella bacteria may be significant in the host defense responses against microbial infections. PMID- 1710822 TI - Primary biliary cirrhosis (PBC): characterization of a monoclonal antibody (PBC MoAb) having specificity identical with disease-associated autoantibodies. AB - We have raised a monoclonal antibody (PBC-MoAb) directed against mitochondria which resembles patent anti-mitochondrial autoantibodies (AMA) (M2 type) in several respects. The reaction pattern of PBC-MoAb was characterized by western blot experiments, immunoaffinity purification and enzyme inhibition studies. PBC MoAb reacts specifically with an epitope on the E2 subunit of pyruvate dehydrogenase (dihydrolipoamide acyltransferase) which is essential for enzymatic activity. This was shown as follows: (1) PBC-MoAb, like PBC-AMA, completely inhibited PDH enzyme activity and reacted weakly with OGDH; (2) PBC-MoAb bound strongly to the E2 subunit in western blots, with weaker binding to a doublet of about 56 kDa; and (3) in immunosorbent experiments, PBC-MoAb absorbed most (greater than 95%) of the AMA reactive material found in solubilized mitochondria. The present data together with earlier findings that the majority of PBC patient autoantibodies bind to epitopes defined by the PBC-MoAb, makes this antibody a valuable tool for characterizing the major PBC-associated epitope on PDH-E2 and localizing this epitope in liver tissue. PMID- 1710823 TI - Efficacy and side-effects of prazosin as a symptomatic treatment of benign prostatic obstruction. AB - This randomized double-blind crossover trial was conducted to assess the effects of prazosin, an alpha 1-adrenoceptor blocking drug, on the voiding of 35 patients with benign prostatic obstruction. Maximum and mean flow rates, residual urine, blood pressure and heart rate were measured at baseline and 2, 4, 6, and 8 weeks after starting the treatment with placebo or prazosin. At 4 weeks the treatments were switched over. The patients filled micturition charts at home and scored their voiding associated feelings. The maximum and mean flow rates increased significantly during prazosin treatment, as also did the maximum and mean voided volumes. Residual urine decreased and voiding improved subjectively but these changes were not statistically significant. Blood pressure was lowered and heart rate increased. Prazosin caused postural dizziness more often than placebo. Prazosin seems to offer an alternative to improve voiding in some patients with prostatic obstruction. PMID- 1710824 TI - Long term experience with the prostatic spiral for urinary retention due to benign prostatic hyperplasia. AB - Twenty high risk patients with benign prostatic hyperplasia and urinary retention were treated by insertion of an intraprostatic spiral. The device was positioned under fluoroscopic guidance using a technique developed by us. The procedure required only local anaesthesia and successfully relieved the obstruction in all patients. At the six-month follow-up full continence had been achieved in 15 patients (75%). Three patients (15%) complained of mild stress incontinence but this was not severe enough to require removal and repositioning of the spiral. A 92-year-old patient reported severe incontinence despite correct positioning of the spiral. Removal of the spiral was necessary in one of the continent patients who was receiving anti-coagulant drugs because of sudden, severe macrohaematuria with subsequent acute urinary retention. The spiral became displaced and required repositioning in one patient. Median residual urine volume was constantly lower than 50 ml and the median maximal flow rate at six-month follow-up was 13.2 ml/s. Our long term follow-up study shows the effectiveness and safety of the prostatic spiral as an alternative treatment for selected patients with urinary retention caused by benign prostatic hyperplasia. PMID- 1710825 TI - Long term survival after transurethral resection of the prostate. Influence of preoperative bacteriuria and indwelling catheter treatment on late mortality. AB - In this report we have analysed the long term survival after transurethral resection of the prostate in patients with cancer and benign hyperplasia, with special reference to the effect of bacteriuria. One hundred and eighty-nine men were followed for seven years after operation. Life tables according to the Kaplan-Meier method indicated a decreased survival rate for patients with preoperative catheter treatment and/or bacteriuria (p = 0.004 and p = 0.013, respectively). In order to evaluate the influence on the long-term survival of each of these factors alone as well as of other factors like diagnosis, age at operation and perioperative antibiotic treatment, a multivariate analysis, according to Cox proportional hazards method was made. This displayed a two-fold increase of mortality in the patients attributed to the catheter treatment per se, whereas bacteriuria alone was not associated with an increased risk of earlier death. PMID- 1710826 TI - [Osteonecrosis following short-term, high-dosage steroid therapy]. AB - Osteonecrosis of the femoral head is one of many well documented side effects of long term steroid (glucocorticoid) use. Studies have reported on the skeletal effects of short term, high dose steroid therapy. This paper illustrates that short term (up to 6 weeks), high dose steroid therapy, utilized in neurotraumatology or central nervous system disease, can lead to necrosis of bone at multiple sites. This retrospective review of 6 patients supports the 18 previously reported cases: there were 5 male and one female patient, with a mean age of 32.2 years. The patients received an average dose of 5100 mg methylprednisolone for 23 days. The average interval between steroid administration and the onset of symptoms was 28 months. There was radiographic evidence of osteonecrosis of both femoral heads in all 6 patients. The female patient also had osteonecrosis of both humeral heads and both femoral condyles. Five of twelve hip joints were healed by intertrochanteric osteotomy and revascularization. One patient underwent total hip replacement. We were unable to find risk factors in these 6 patients which would make them susceptible to belated osteonecrosis of the femoral head. It is reasonable to conclude that there is a substantial risk of osteonecrosis in patients treated with high dose steroids even on a short term basis. PMID- 1710827 TI - Recognition by class II alloreactive T cells of processed determinants from human serum proteins. AB - Alloreactive T cells recognize a complex composed of an allogeneic major histocompatibility complex (MHC) molecule and a peptide derived from the processing of nonpolymorphic proteins. A sizable fraction of MHC class II alloreactive T cells is shown to recognize peptides derived from constitutive processing of human serum proteins. One such epitope is a fragment of human serum albumin. This epitope bound selectively to the human class II molecule DRw11 and was constitutively present on antigen-presenting cells in vivo. These data indicate that, in the case of MHC class II, peptides involved in allorecognition may originate from exogenous proteins. PMID- 1710828 TI - Prevention of xenograft rejection by masking donor HLA class I antigens. AB - Destruction of target cells by cytotoxic T lymphocytes requires the presence of HLA (human lymphocyte antigen) class I antigens on the target cells for adhesion as well as for triggering of the antigen-specific T cell receptor. Rejection of xenogeneic human pancreatic islets and liver was circumvented by masking, before transplantation, donor antigens with F(ab')2 antibody fragments to HLA class I or tissue-specific epitopes. This strategy eliminated the need for recipient immunosuppression and allowed islet xenograft survival beyond 200 days, as demonstrated functionally by C peptide secretion as well as by histology. These in vivo observations are consistent with the importance of donor HLA class I in eliciting graft rejection and have potential applicability to the successful transplantation of other HLA class I-bearing donor tissues. PMID- 1710829 TI - Structural determinants of ion flow through recombinant glutamate receptor channels. AB - Functional glutamate receptor (GluRs) were transiently expressed in cultured mammalian cells from cloned complementary DNAs encoding GluR-A, -B, -C, or -D polypeptides. The steady-state current-voltage (I-V) relations of glutamate- and kainate-induced currents through homomeric channels fell into two classes: channels composed of either the GluR-A, -C, and -D subunits showed doubly rectifying I-V curves, and channels composed of the GluR-B subunits displayed simple outward rectification. The presence of GluR-B subunits in heteromeric GluRs determined the I-V behavior of the resulting channels. Site-directed mutagenesis identified a single amino acid difference (glutamine to arginine) in the putative transmembrane segment TM2 responsible for subunit-specific I-V relationships. The properties of heteromeric wild-type and mutant GluRs revealed that the dominance of GluR-B is due to the arginine residue in the TM2 region. PMID- 1710830 TI - Histamine-releasing factors and heterogeneity of IgE. AB - The duration and severity of the allergic response are variable. Even though antigens are rapidly cleared from the individual, an acute allergic response is frequently followed by a recrudescence of symptoms hours or even days after the initial exposure. Experimentally, the cellular infiltrates and mediators released during this late response resemble those associated with chronic inflammatory disease. Although basophils are present in this late reaction, the stimuli for their activation remain unknown. A heterogeneous group of unique cytokines called histamine-releasing factors (HRF), discovered over a decade ago, may well play a role in stimulating basophils during this late-phase reaction. These factors have been reported from a variety of cell sources including alveolar macrophages, platelets, vascular endothelial cells, B and T lymphocytes, mononuclear cell cultures, the U937 monocyte/macrophage-like cell line and the RPMI 8866 B cell line. These ubiquitous factors cause non-cytotoxic, calcium-dependent mediator release from human basophils in vitro and are also present and active in vivo. Purification attempts have revealed that HRF exists in at least three forms, based on molecular weight. In our hands, the mechanism of mediator release by one of the forms of HRF is IgE dependent. Since only about 50% of allergic donors' basophils respond to HRF, a heretofore unappreciated heterogeneity of IgE was revealed. The presence of HRF has been shown to correlate with severity of allergic disease in children with food allergies, with symptoms in the late-phase response in adults and with severity of the allergic response to an inhaled antigen. Thus, the study of HRF has evolved over the last decade and may lead to better understanding of the complex allergic response. PMID- 1710831 TI - [Importance of carcinoembryonic antigen, alpha-fetoprotein and cholylglycine in differential diagnosis of viral hepatitis B and mechanical jaundice]. AB - Fifty-four patients with viral hepatitis B (VHB) were examined at the height of the disease as were 30 patients with mechanical jaundice (MJ) of tumorous etiology and 19 normal persons. Mechanical jaundice was mainly characterized by a considerable growth of the concentration of carcinoembryonic antigen, whereas VHB by an increase of the cholylglycine level. Concomitant detection of those markers can be used in differential diagnosis of parenchymatous jaundice and mechanical jaundice of tumorous etiology. Alterations of the alpha-fetoprotein level were of no information content. PMID- 1710832 TI - Low prevalence of coagulation and fibrinolytic activation in patients with primary untreated cancer. AB - The prevalence of subclinical coagulation abnormalities greatly differs in the various studies due to selection of patients and differences in study design. We performed coagulation studies in 69 consecutive patients with primary untreated cancer of various origin. The control group consisted of 42 sex and age matched healthy volunteers. Plasma coagulation tests included thrombin-antithrombin-III complex (TAT), plasmin-alpha 2-antiplasmin-complex (PAP) and tissue-plasminogen activator-antigen (t-PA-ag). These tests were performed once, prior to any anti cancer treatment. We evaluated if activation of the coagulation system (elevated TAT-complexes) and the fibrinolytic system (elevated PAP-complexes and t-PA-ag) correlated with the tumor-type or the extent of the tumor. To document clinical manifest haemorrhage or thromboembolic disease (TED) we performed a 6 months follow-up study. In 8 patients (12%) and in 3 control subjects (7%) an elevated TAT-complex level was observed (this difference is not significant). An increased plasma level of PAP-complex was seen in 8 patients (12%) versus none in the control group (p less than 0.05). In one patient both TAT and PAP-complex concentrations were elevated. Consequently, 15 of the 69 patients (22%) showed activation of the coagulation and/or fibrinolytic system. Fibrinolysis seems to be enhanced in a subset of cancer patients in contrast to blood coagulation. In 10 patients (14%) we found raised t-PA-ag levels. Three patients had both elevated levels of PAP-complex and t-PA-ag. These findings suggest that in a minority of patients increased PAP-complex levels may be a result of t-PA induced plasminogen activation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710833 TI - In vitro effects of urokinase--prevention by different inhibitors. AB - The present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activator (u-PA, urokinase) on normal citrated plasma. When 10 micrograms/ml u-PA were added to pooled normal plasma and incubated for 30 min at an ambient temperature (25 degrees C), alpha 2 antiplasmin decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control, whereas alpha 2-antiplasmin was fully consumed at 37 degrees C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25 degrees C. Thrombin time prolonged to 190% of control. Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KIU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase induced plasmin generation without interfering with haemostatic assays. The anti u-PA-antibody afforded full protection of alpha 2-antiplasmin at therapeutic levels of u-PA. It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody. PMID- 1710834 TI - Identification of platelet antigens by immunoprecipitation of unlabelled platelet glycoproteins. AB - A time-saving and sensitive method for the identification of antigens on unlabelled platelet glycoproteins (GP) based on immunoprecipitation has been developed. Platelet solubilisates were incubated with antibodies to form GP antibody complexes that were precipitated with Protein G Sepharose 4 Fast Flow (PGS). PGS-bound material was eluted, separated by SDS-PAGE under reducing conditions and visualized by silver staining. The method was designed as an alternative to radioimmunoprecipitation for laboratories not equipped to work with radioactive substances. It is proposed as a complementary procedure for the characterization of platelet GP antigens by murine monoclonal antibodies and human alloantibodies. PMID- 1710835 TI - Selectins: novel receptors that mediate leukocyte adhesion during inflammation. PMID- 1710836 TI - The characterisation of thrombus development in an improved model of arterio venous shunt thrombosis in the rat and the effects of recombinant desulphatohirudin (CGP 39393), heparin, and iloprost. AB - An existing arterio-venous shunt thrombosis model in the rat has been modified to increase its usefulness for the testing of anti-thrombotic agents and characterised using morphological and radiometric techniques. The thrombus formed on the cotton thread held in the shunt was found to be composed of platelet aggregates surrounded by red thrombus. After 30 min of blood flow there was a 15 fold increase in the platelet content of the thrombus and a 4-fold increase in the fibrin(ogen) content compared with an equivalent weight of whole blood. Use of the anticoagulants recombinant desulphatohirudin (CGP 39393, 4 mg/kg, s.c.) and unfractionated heparin (800 IU/kg, s.c.) showed that approx. 90% inhibition of thrombus weight and approx. 80% inhibition of fibrin(ogen) content could be achieved without significant effect on the platelet content. Conversely, using the platelet inhibitor Iloprost (1 microgram kg-1 min-1), a reduction in thrombus weight of 50% was associated with 75% inhibition of platelet content and only 20% inhibition of fibrin(ogen). These observations suggest that the growth of this type of thrombus is largely the result of continued fibrin formation rather than continued platelet recruitment and activation. PMID- 1710837 TI - Synergism between tissue-type plasminogen activator and a genetically engineered variant lacking the finger domain, the growth factor domain and the first kringle domain. AB - A modified variant of human tissue-type plasminogen activator (t-PA) lacking the finger domain (F), the growth factor domain (G) and the first kringle domain (K1), has an extended plasma half-life in vivo, compared to that of t-PA. When the variant (denoted K2P) was tested in vitro for its ability to lyse human plasma clots we found that the activity was characterized by a time lag phase and a sigmoidal dose-response curve. However, an attenuation of the lag phase in vitro was observed both when K2P was mixed with t-PA in a w/w ratio of 4:1 and when K2P was allowed to lyse a clot that had been pre-exposed to t-PA i.e. submitted to a limited plasmic digestion. Dosis that in vitro caused 50% lysis within 6 h were calculated from individual dose-response curves and were for K2P, t-PA and K2P/t-PA (4:1 w/w) 540 ng/ml, 360 ng/ml and 310 ng/ml, respectively. These results indicated a synergistic effect between K2P and t-PA. However, the data from individual dose-response curves showed that the effect of the K2P/t-PA mixture never was better than that of t-PA alone, and the synergistic effect in vitro is therefore considered to be of limited use. The thrombolytic activity in vivo was evaluated in a rabbit jugular vein thrombus model. Despite the lag phase observed in vitro, K2P was approximately 3 times as effective as t-PA in vivo (bolus injection). The thrombolytic effect of K2P was further potentiated when it was administered together with a small amount of t-PA (4:1 w/w).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710838 TI - Feline hyperthyroidism. PMID- 1710839 TI - Differential sensitivities of avian and mammalian neuromuscular junctions to inhibition of cholinergic transmission by omega-conotoxin GVIA. AB - Nerve stimulation-induced contractions of the chick biventer cervicis muscle were slowly reduced by omega-conotoxin. However, omega-conotoxin had no effect on skeletal muscle function after i.v. injection in mice or on nerve stimulation induced contractions of focally innervated muscle of the rat diaphragm or the rabbit proximal oesophagus, or the multiply innervated extra-ocular rectus muscle from rabbit. The lack of effect of omega-conotoxin on mammalian neuromuscular junctions was not due to the high safety factor in transmission or to a high local concentration of Ca2+ originating from the muscle, and could not be accounted for in terms of the operation of facilitatory or inhibitory feedback modulation of transmitter release from motoneurone terminals. It is concluded that the Ca2+ channels of mammalian motoneurone terminals differ from those of avian motoneurone terminals and other omega-conotoxin-sensitive nerve terminals. PMID- 1710840 TI - Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion. AB - Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha-granule membrane protein 140 (GMP-140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium-labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP-140, progressing from a mean of 4 +/- 2 percent (SD) on the day of collection to a mean of 25 +/- 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = 0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/- 15 percent of the number predicted by the absolute platelet increment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710841 TI - Acceleration of the hemopoietic reconstitution in mice undergoing bone marrow transplantation by recombinant human granulocyte colony-stimulating factor. AB - We have investigated the effects of recombinant human granulocyte colony stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 micrograms/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT. PMID- 1710842 TI - Percutaneous biopsy of bladder-drained pancreas transplants. AB - Percutaneous biopsy is a valuable investigation in the management of allograft rejection for all solid organs. Pancreas transplants have not been biopsed percutaneously, though open and percystoscopic biopsies have proved useful. We have compared percutaneous needle core biopsy with fine-needle aspiration cytology for the diagnosis of rejection in 18 patients receiving combined kidney and pancreas transplants and in one who was transplanted with the pancreas alone. Percutaneous needle core biopsy was successful in 37 of 40 attempts (93%), while fine-needle aspiration yielded diagnostic material on 33 of 47 attempts (70%). Transient hyperamylasemia occurred in 29%, returning to baseline in three days. One patient twice developed transient macroscopic hematuria. There was agreement between needle core biopsy and fine-needle aspiration on the diagnosis of rejection on six occasions and for the absence of rejection on 16. There was an 8% false-positive rate for fine-needle aspiration. In 13 instances of histologically proved renal rejection, concurrent pancreas biopsy revealed rejection in 69%. Pancreas rejection was not, however, seen in the absence of renal rejection. In this pilot study, percutaneous biopsy of the bladder-drained pancreas allograft was shown to be a practicable and valuable investigation without major complications. PMID- 1710843 TI - The effects of immunosuppressive agents on in vitro production of human immunoglobulins. AB - We have evaluated the effects of CsA, methylprednisolone (MP), 6-mercaptopurine (6-MP), and FK506 on T cell-dependent and T cell-independent immunoglobulin production. FK506 and 6-MP were potent inhibitors of IgG and IgM production by PWM-stimulated peripheral blood mononuclear cells, which depend on the presence of T cells. CsA was less effective in this system and MP actually enhanced IgG and IgM production. In order to assess the direct effects of these various immunosuppressive agents on B cells, we utilized human B cell lines representing different stages of B cell differentiation. The B cell lines CESS and SKW6.4 exhibit increased production of IgG and IgM, respectively, in response to interleukin-6. These cells represent activated, but not fully differentiated, B cells. CsA inhibited IL-6-induced IgG production by CESS cells by 64% at 100 ng/ml and 6-MP inhibited this response by 82% at 250 ng/ml. Neither CsA nor 6-MP effectively inhibited IL-6-induced IgM production by SKW6.4 cells. MP at 250 ng/ml inhibited IL-6-induced IgG production by 89%, but enhanced IL-6-induced IgM production more than two-fold. FK506 did not inhibit IL-6-induced IgG or IgM production, suggesting that it has no direct effect on the ability of B cells to respond to this differentiation factor. These experiments clearly demonstrate that CsA, MP and 6-MP have direct inhibitory effects on the response of human B cells to IL-6. In contrast, FK506 has no direct effect on these B cells lines, but is more potent than the other agents at inhibiting T cell-dependent immunoglobulin production. PMID- 1710844 TI - Evidence that FK506 and rapamycin block T cell activation at different sites relative to early reversible phosphorylation involving the protein phosphatases PP1 and PP2A. PMID- 1710845 TI - Horizontal and vertical pathways in neural induction. PMID- 1710846 TI - The possible role of adhesion in synaptic modification. PMID- 1710847 TI - Neuroscience in the Republic of Georgia. AB - The current uncertain status of the republics of the USSR poses special problems for the future development of their scientific communities. The system of Institutes within an Academy, on which most Eastern European research was modelled, was developed in the USSR. Because of the Republic of Georgia's relatively small population, its research might be especially at risk if collaborative integration with other Republics and the West is not achieved. PMID- 1710848 TI - Neurological disease and mitochondrial genes. AB - Mitochondria contain 2-10 copies of a small, double-stranded, circular DNA molecule that is exclusively maternally transmitted. Until recently, the only function of mitochondrial DNA that had any possible significance for clinicians was the fact that the mutation conferring chloramphenicol resistance occurs in one of the mitochondrial ribosomal RNA genes. It is now clear that major deletions and point mutations of mitochondrial DNA cause human diseases, chiefly mitochondrial myopathies and encephalopathies, and Leber's hereditary optic neuropathy. PMID- 1710849 TI - Hippocampal RSA and DLSN neurons. PMID- 1710850 TI - Hippocampal RSA in humans. PMID- 1710851 TI - The neurobiology of blindsight. AB - Some patients can respond to visual stimuli presented within their clinically absolute visual field defects that have been caused by partial destruction of striate cortex. This puzzling phenomenon of looking, pointing, detecting and discriminating without seeing has been called blindsight, and has fascinated philosophers and neuroscientists alike as a spotlight on the nature of unconscious or covert awareness, and the means it provides of studying the visual information carried by pathways other than the major route through the striate cortex. PMID- 1710852 TI - Is interleukin 2 a neuromodulator in the brain? AB - A bidirectional flow of information exists between the CNS and the neuroendocrine and immune systems, representing an important homeostatic mechanism in the body. Lymphokines and other products of immunocompetent cells seem to play a crucial role in this communication and seem to exert powerful effects on neurones in the brain. In this article, Giuseppe Nistico and Giovambattista De Sarro describe the central effects following interleukin 2 (IL-2) microinfusion into several areas of the rat brain. The locus coeruleus seems to be the main site in the brain through which IL-2 exerts soporific effects. In addition, the possible transducing mechanisms coupling IL-2 receptor stimulation and the electroencephalogram (EEG) spectrum power responses elicited from the locus coeruleus seem to involve stimulation of specific receptors coupled to adenylate cyclase through a Gi protein. PMID- 1710853 TI - The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium. AB - Cation channels that are directly gated by guanosine 3', 5'-cyclic monophosphate (cGMP) control the flow of ions across the surface membrane of vertebrate rod and cone photoreceptor cells. A similar channel, gated by adenosine 3',5'-cyclic monophosphate (cAMP), exists in vertebrate olfactory sensory neurons. The channel polypeptide of rod photoreceptors has been identified and the amino acid sequence of the channel polypeptide in rod and olfactory cells has been determined by cloning cDNA. Although the cyclic nucleotide-gated channels functionally belong to the class of ligand-gated channels, they share some structural features with voltage-gated channels. PMID- 1710854 TI - FK506 and rapamycin: novel pharmacological probes of the immune response. AB - The immune system is an intricate network of circuitry which is incompletely understood. Novel tools are needed to unravel the relevance of even the smallest components of this network. While the clinical potential of FK506 and rapamycin as selective immunosuppressants is the major reason for their current importance, preclinical studies described here by Joseph Chang and colleagues have already suggested several provocative ideas which may revise our biochemical concepts of T-cell activation. With the combination of molecular and cellular studies, the insights gained with FK506/rapamycin may lead to a better understanding of the biochemical circuits that are involved in the immune response. Ultimately, studies may lead to the identification of an endogenous immunosuppressive ligand that mimics FK506 or rapamycin. PMID- 1710855 TI - Altered tenascin expression during spontaneous endometrial carcinogenesis in the BDII/Han rat. AB - The BDII/Han rat develops spontaneous endometrial adenocarcinoma, which appears virtually identical histologically to human endometrial adenocarcinoma. The incidence rate of cancer formation in the rat is 90% and the mean lifetime of the animals is 22 months. This animal model therefore, is useful in the study of molecular aspects of spontaneous transformation as well as mammalian neoplastic progression. In this study we address the in-situ expression of tenascin, an extracellular matrix glycoprotein, during normal cyclic growth, during development of proliferative states, and during malignant transformation of the endometrium. Trace amounts of immunocytochemically detectable tenascin were found in 10% of young BDII/Han rats with a normal estrus cycle. In these inbred animals no tenascin was detectable in uteri without neoplastic progressive alterations of the endometrium. Tenascin immunoreactivity first appeared during proliferation in one of three uteri with cystic glandular hyperplasia. Prominent tenascin expression was detectable in all adenomatous hyperplasia, but restricted to the stromal mesenchyme, that surrounded the glands. In all endometrial adenocarcinomas tested, essentially the entire extracellular space of the stromal mesenchyme was immunoreactive with anti-tenascin antibodies while the epithelial glands themselves were negative. This staining pattern was observed independent of the degree of tumor differentiation or extent of myometrial invasion. The tenascin staining pattern was not significantly altered in tumors transplanted into the soft tissues of the neck of female BDII/Han rats. From our studies we conclude that tenascin may be a marker for the early detection of proliferative endometrial states.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710857 TI - Detailed diagnoses and procedures, National Hospital Discharge Survey, 1988. AB - This report presents statistics on conditions diagnosed and surgical and nonsurgical procedures performed in non-Federal short-stay hospitals. The statistics are based on data collected through the National Hospital Discharge Survey from a national sample of the hospital records of discharged inpatients. Estimates of first-listed diagnoses, all-listed diagnoses, days of care for first listed diagnoses, and all-listed procedures are shown by sex and age of patient and geographic region of hospital. PMID- 1710856 TI - Drug-induced lysosomal storage of sulfated glycosaminoglycans in cultured bovine and human fibroblasts. AB - Several di-cationic amphiphilic compounds are known to cause lysosomal accumulation of sulfated glycosaminoglycans (sGAG) in intact rats and in cultured rat fibroblasts. The purpose of the present investigation was to examine whether this drug side effect also occurs in bovine and human cells. Cultured fibroblasts from both species were exposed to tilorone (3 microM and 5 microM) for 72 h; lysosomal sGAG-storage was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The cytological alterations as well as the radiochemical results in both species were in good agreement with previous data from rat fibroblasts. The present findings indicate that the drug-induced lysosomal storage of sGAG is a species-independent phenomenon. Thus, cultured bovine and human fibroblasts are a suitable model for further studies concerning the as yet unknown molecular mechanisms underlying this adverse drug action. PMID- 1710858 TI - [Physioprophylaxis and rehabilitation in patients with Sudeck's atrophy]. PMID- 1710859 TI - Different lectin-binding patterns of the male and female harderian gland in the golden hamster. AB - Ten lectin-binding patterns were examined with the PAP method for the detection of sugar residues on the Harderian glands of golden hamsters of both sexes. Each lectin showed a specific binding pattern. The most characteristic pattern was exhibited by DBA which intensely stained cells with small lipid vacuoles, but not cells with large lipid vacuoles in the male gland. In the female gland, both cell types showed no reaction with the DBA. The PNA had almost the opposite binding pattern. From the lectin-binding patterns, it can be derived that, not only do the two cell types in same sex glands have different glycoconjugate distribution, but also the same type of cells in different sex glands has a different glycoconjugate distribution. Therefore, the sexual dimorphism of the golden hamster Harderian gland also occurs in its carbohydrate moieties. In this study, the duct of the Harderian glands revealed no sexual dimorphism in the lectin binding patterns. PMID- 1710860 TI - Control of locomotion in vitro: II. Chemical stimulation. AB - Previous studies have described the presence of alternating activity induced in left and right ventral roots of the neonate rat in vitro brainstem-spinal cord preparation, following application of certain neuroactive substances to the bathing solution. The present findings show the presence of chemically induced, adult-like coordinated airstepping demonstrated by electromyographic recordings in the hindlimb-attached in vitro brainstem-spinal cord preparation. Analysis of muscular activity demonstrated alternation between antagonists of one limb and between agonists of different limbs, as well as a proximodistal delay in agonists active at different joints of the same limb. Neuroactive agents were applied independently to either the brainstem or spinal cord bath. The substances surveyed in the present studies included some of those used previously, as well as additional compounds: bicuculline and picrotoxin (gamma-aminobutyric acid ergic antagonists), N-methyl-D-aspartic acid (excitatory amino acid agonist), substance P, acetylcholine, carbachol (cholinergic agonist), and serotonin. Application of these substances to the brainstem bath produced rhythmic airstepping. Application of dopamine, aspartate, glutamate, and N-methyl-D aspartic acid to the spinal cord bath also produced rhythmic airstepping, while application of acetylcholine produced tonic, long-lasting co-contractions. These findings reveal the presence of several neurochemical systems in the central nervous system that can be activated at birth to induce coordinated airstepping in the neonate rat in vitro brainstem-spinal cord preparation. PMID- 1710861 TI - Effect of PUVA radiation on anaphylactic histamine release from rat dermal tissues. AB - We have devised a new in vitro model of type I cutaneous anaphylaxis. Male albino rats were sensitized with DNP-Ascaris. Abdominal skin was shaved, and thin, split thickness slices of skin were cut with a dermatome. The dermis was excised and cut into 100 mg pieces. The dermal tissue was incubated with antigen in Tyrode's solution for 30 min at 37 degrees C. Antigen-induced histamine release from dermal tissue was measured fluorimetrically. Using this system, we measured histamine release from PUVA-irradiated and non-irradiated dermal tissues. A single PUVA irradiation inhibited type I cutaneous anaphylaxis, but did not affect spontaneous histamine release or total dermal histamine. Our model is considered to be useful for investigation of the mechanism of suppression of type I cutaneous anaphylaxis by PUVA. PMID- 1710862 TI - [Laser treatment of rectosigmoidal tumors: long-term results]. AB - Over a 10 year period, 530 patients were treated at the Lille Laser Center for a rectosigmoid tumour. Both argon and Nd:YAG lasers were used. Two hundred patients were treated for palliation of symptoms from a rectosigmoid cancer, 313 patients were treated for a rectosigmoid villous adenoma, and 17 patients with a familial polyposis were treated for the rectal polyposis remaining after total colectomy. The immediate success rate and complication rate were 88% and 2.7%, respectively, for patients with advanced cancer, 92% and 2% for those with a villous adenoma, and 100% and 0% for those with polyposis. Patients with an advanced cancer remained functionally improved during a 10.1 month average period after initial improvement. The recurrence rate after initial treatment for villous adenomas was 14% during a 3.1 year average follow-up. Immediate results were influenced by reason for treatment, initial symptoms and circumferential extension for patients with a cancer, and only by circumferential extension for patients with a villous adenoma. Long term results were influenced by reason for treatment and circumferential extension for patients with a cancer, and by reason for treatment, initial histology and localization for patients with a villous adenoma. PMID- 1710863 TI - [Expression of intermediate filaments in cutaneous cylindroma]. AB - Using different monoclonal anti-cytokeratin antibodies (broad spectrum, cytokeratin 7, 8, 18, 19) and the antibody A55-A/A9 until now characterized only by immunohistology the staining of ductal structures and some cells within the lobulae of dermal cylindromata was demonstrated. In regard to the cytokeratin polypeptide patterns of the adnexes in uninvolved skin an exact histogenetic correlation by our findings is not possible, but the pattern is consistent with the conception of the glandular origin of the tumours. PMID- 1710864 TI - Immunohistochemistry of porcine skin. AB - The present paper reports immunohistological findings in porcine skin, which were obtained by use of mono- and polyclonal antihuman antibodies and either alkaline phosphatase anti-alkaline phosphatase (APAAP) or peroxidase (POX) technique. Epidermal staining was observed with antibodies to keratins (K 8.12, RSKE 60), filaggrin, and calmodulin (ACAM). Staining of connective tissue and vessels was achieved using antibodies to vimentin (V9(1)), collagen type IV, and fibronectin. In general, these antibodies gave a staining pattern similar to that of normal human skin. The similarities of immunoreactivity to poly- and monoclonal antihuman antibodies in porcine and human skin render porcine skin a reliable model in biomedical research. PMID- 1710865 TI - Effect of anti-androgen (TZP-4238) on steroid-induced canine prostatic hyperplasia. Light and electron microscopic investigations. AB - The effect of a synthetic steroidal anti-androgen, TZP-4238, on steroid-induced canine prostatic hyperplasia was studied by light and electron microscopy. Male beagle dogs (1-2 years old) were divided into four experimental groups. Group 1 consisted of intact controls. The other animals were castrated. The castrated animals were treated for 25 weeks with 1) 5 alpha-androstane-3 alpha, 17 beta diol (A-diol) plus 17 beta-estradiol (E2) (Group 2), 2) A-diol plus E2 + TZP-4238 0.5 mg/kg (Group 3) and 3) A-diol plus E2 + chlormadinone acetate (CMA) 2.5 mg/kg (Group 4). TZP-4238 and CMA were administered orally for 21 weeks after 4 weeks treatment with A-diol plus E2. In group 2, glandular hyperplasia of the prostate was clearly noted. In contrast, combined treatment with TZP-4238 (Group 3) or CMA (Group 4) produced marked atrophy of the glandular epithelium. Loss of secretory and metabolic activities was confirmed by ultrastructural investigations. Our data indicate that TZP-4238 is a potent anti-androgen for the prevention of canine prostatic hyperplasia in the steroid-induced benign prostatic hyperplasia (BPH) model. PMID- 1710866 TI - On the non-adrenergic, non-cholinergic contribution to the parasympathetic nerve evoked secretion of parotid saliva in the rat. AB - A secretion of parotid saliva in the anaesthetized rat in response to stimulation of the parasympathetic nerve occurs in the presence of atropine and adrenoceptor antagonists, albeit reduced and transient in the face of continuous high frequency stimulation (40 Hz). In non-atropinized rats prolonged stimulation of the parasympathetic nerve at a high frequency (40 Hz, 40 min), aiming at depletion of the neuronal stores of transmitters thought to be responsible for the non-adrenergic, non-cholinergic (NANC) secretion of parotid saliva, was performed. The magnitude of the secretory response to various stimulation frequencies (0.2-60 Hz) applied to the parasympathetic nerve was assessed before and after the period of high-frequency stimulation. The second time, the frequency-response curve was shifted to the right and, moreover, the initial maximal secretory response was not reached. Control experiments suggested that this reduction in secretory responses could be attributed neither to impaired cholinergic neurotransmission nor to decreased responsiveness of the secretory cells. The secretory responses to parasympathetic nerve stimulation after high frequency stimulation are thought to be evoked by acetylcholine predominantly. When these responses were compared with (1) those obtained before high-frequency stimulation and thought to be evoked by acetylcholine and non-adrenergic, non cholinergic transmitters in conjunction and (2) those depending on non adrenergic, non-cholinergic transmitters only it appears that the non-adrenergic, non-cholinergic transmission contributes to the parasympathetic secretory response at a frequency (0.2 Hz) far below threshold frequency (5 Hz) for the non adrenergic, non-cholinergic evoked secretory response. At frequencies below 20 Hz it appears that acetylcholine and non-adrenergic, non-cholinergic transmitters interact positively thereby enhancing the secretory responses. PMID- 1710867 TI - Insulinlike growth factor II and transforming growth factor beta regulate collagen expression in human osteoblastlike cells in vitro. AB - Insulinlike growth factor II (IGF-II) and transforming growth factor beta (TGF beta) are the most abundant polypeptide growth factors found in human bone matrix and are produced by human bone cells in vitro. IGF-II and TGF-beta 1 increased total protein synthesis, collagenous protein synthesis, and the steady-state level of type I procollagen mRNA in a time-dependent manner in osteoblastlike cells isolated from human bone. Type III procollagen mRNA expression was low in untreated cultures and was not affected by IGF-II or TGF-beta. TGF-beta 1 elevated type I procollagen mRNA rapidly, with the maximal observed change at 10 h. In contrast, procollagen mRNA levels increased more slowly in response to IGF II and reached a lower maximal level than with TGF-beta, but the response was sustained through 24 h. Collagenous protein synthesis in IGF-II- and TGF-beta treated cells increased in parallel with increases in procollagen mRNA levels and was higher at 21 h for TGF-beta 1 and at 36 h for IGF-II. The difference in the time course and magnitude of change in type I procollagen mRNA levels in response to IGF-II and TGF-beta 1 suggests that these two growth factors work through distinct mechanisms that provide both a rapid transient response and a later sustained response in bone matrix biosynthetic activity. PMID- 1710868 TI - Interaction between organic phosphates and sheep hemoglobins. AB - Measurements have been made of the effect of the organic phosphate 2,3-DPG and inositol hexaphosphate (IP6) on the OEC of sheep hemoglobins A, B, and F. A thin film method (HEM-O-SCAN) was used and the hemoglobin concentration was about one quarter to one third of that normally found in red cells. All experiments were in bis-tris-saline buffer and in the absence of CO2. At low concentrations of IP6 and at concentrations of 2,3-DPG similar to red cell concentrations, the OEC was right-shifted in all cases, P50 being increased by a factor of 1.58-1.84 by IP6 and by a factor of 1.41-1.59 by 2,3-DPG. The value of Hill n, the index of cooperativity was not generally changed by 2,3-DPG but was decreased by IP6. PMID- 1710869 TI - Oxygenation of tumors derived from ras transformed cells. AB - In order to gain insight into mechanisms governing the development of tumor hypoxia, malignancies derived from spontaneously tumorigenic or ras-transformed cell lines were grown in nude mice. As a rule, tumors with ras oncogenes exhibited rapid growth rates and large areas with low pO2 readings even at small tumor sizes. The slow proliferation rate of a spontaneously tumorigenic cell line was consistent with more adequate tissue oxygen levels. In all lines, hypoxia was accentuated at larger tumor sizes. These results demonstrate that ras transformation can lead to accelerated proliferation rates and is then concomitant with the development of pronounced tumor hypoxia. PMID- 1710870 TI - Isolation and characterization of cDNA clones from human placenta coding for phospholipase A2. PMID- 1710871 TI - [Retrospective clinical evaluation of prognosis factors in stage D2 prostatic cancer treated with endocrine therapy]. AB - Of 91 patients with prostatic cancer treated at our Department of Urology, from January 1976 through December 1987, 42 cases of stage D2 cancer treated with endocrine therapy were evaluated retrospectively with regard to clinical parameters and prognoses. The patients with marked increase in the level of the prostatic acid phosphatase (PACP), and the lactate dehydrogenase (LDH) in serum, and marked decrease in volume of hemoglobin (Hb) had a poor prognosis. The patients who took a long time to obtain a favorable response to the therapy had a poor prognosis. The response grade in NPCP criteria at 3 months after the initiation of therapy reflected the prognosis, and showed good correlation to the grade of favorable response. PMID- 1710872 TI - [Two cases of spermatocytic seminoma]. AB - Two cases of spermatocytic seminoma are reported. The first case was a 58-year old man who visited our hospital with a complaint of painless swollen left scrotal content. Left orchiectomy was performed under the diagnosis of testicular tumor. Since the pathological diagnosis first made was anaplastic seminoma, he was treated with combined chemotherapy (PVB, 1 course). However, since the pathological diagnosis after re-examination of the specimen, was spermatocytic seminoma, he underwent prophylactic radiation therapy. The second case was a 64 year-old man who visited our hospital with a complaint of painless swelling of right scrotal content. Right orchiectomy was performed under the diagnosis of testicular tumor. The pathological diagnosis was spermatocytic seminoma. He underwent prophylactic radiation therapy. Postoperatively these two patients have been well with no evidence of recurrence. These are the 14th and 15th cases of spermatocytic seminoma reported in Japan. PMID- 1710873 TI - Cytokines in tumor therapy (GM-CSF, G-CSF, and IL-3). Proceedings of the satellite symposium to the 15th UICC Congress. Hamburg, Federal Republic of Germany, August 18, 1990. PMID- 1710874 TI - The roles of ultrasonography and amniocentesis in evaluation of elevated maternal serum alpha-fetoprotein. AB - A total of 681 pregnant women were referred for evaluation of elevated maternal serum alpha-fetoprotein levels. Ultrasonographic examination yielded an explanation for the elevation of maternal serum alpha-fetoprotein in 42% of patients. Diagnoses made by ultrasonography included incorrect fetal dating, multiple gestation, fetal death, open neural tube defect, abdominal wall defect, placental abnormalities, cystic hygroma, renal anomalies, and oligohydramnios. Optimal prenatal diagnosis of fetal anomalies also requires the use of amniocentesis in many patients. Amniocentesis may be obviated if fetal dating is incorrect, if an unsuspected multiple gestation is discovered, or if there is a clear anomaly and the parents do not desire genetic counseling based on karyotype information. If the fetus appears normal, the ultrasonographic results are equivocal, or the parents desire more detailed genetic counseling when an anomaly is found by ultrasonography, then amniocentesis should be performed. Thirteen abnormalities were diagnosed by amniocentesis alone in this group. PMID- 1710875 TI - Follow-up of elevated maternal serum alpha-fetoprotein levels: ultrasonography only or amniocentesis? PMID- 1710876 TI - Partition of free and monoclonal-antibody-bound horseradish peroxidase in a two phase aqueous polymer system--novel procedure for the determination of the apparent binding constant of monoclonal antibody to horseradish peroxidase. AB - The principle that the antigen and the antibody prefer different phases in an aqueous two-phase system is the analytical basis of the work presented here. The antigen horseradish peroxidase, which is bound to a monoclonal antibody (mAb), is separated from free Ag in an aqueous phase system (polyethylene glycol (PEG)/dextran) as a function of the concentration of mAb. The plot of the partition coefficient kappa of horseradish peroxidase versus the concentration of mAb yields a sigmoidal curve similar to the curve obtained by enzyme-linked immunosorbent assay (ELISA). Comparing the plots normally used for ELISA in order to determine the apparent binding constant of mAb and the number of epitopes on the Ag we derived a relationship between the difference in partitioning of the free Ag and the bound Ag (delta kappa) and the concentration of mAb. The new linear plot of reciprocal delta kappa versus reciprocal concentration of mAb gives the apparent binding constant of mAb, which is evaluated from the slope. From the intercept at the ordinate the maximum difference of the partition coefficient of the free and bound antigen is derived and the apparent partition coefficient of the free monoclonal antibody can be calculated. PMID- 1710877 TI - Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes. AB - A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure. PMID- 1710878 TI - Rearrangement of the metaphyseal vasculature of the rat growth plate in rickets and rachitic reversal: a model of vascular arrest and angiogenesis renewed. AB - The morphology of the metaphyseal microvasculature at the epiphysis was examined at both the light and electron microscopic level in rickets and rachitic reversal. The animals studied were normal, rachitic, and rachitic reversed at 8, 24, and 96 hours post-vitamin D administration. The overall architecture of the metaphyseal vessels was significantly altered throughout the intervals examined. In the rachitic animal, arterioles, venules, and capillaries were found adjacent to the growth plate, either directly apposed to the hypertrophic chondrocytes or separated from them by bone-forming cells. These vessels are in many ways similar to the larger arterioles and venules that normally supply the metaphyseal capillary sprouts, but in the normal growing animal are usually located 350-500 microns from the epiphyseal cartilage. The rachitic capillaries appear relatively well differentiated with a partial basement membrane and a perivascular cell lining. In early rachitic reversal, small vascular projections are induced to grow from the large diameter venules that border upon the hypertrophic chondrocytes. These vascular sprouts that invade the epiphyseal cartilage are quite undifferentiated, with no basement membrane or pericyte lining at the sprout apex and occasional abluminal endothelial cell projections. Within 96 hours, the metaphyseal microvasculature has returned to an apparently normal state with only capillaries at the cartilage-vascular interface and larger vessels (arterioles and venules) located several hundred microns deeper into the metaphysis. The sequential processes of differentiation and cessation of capillary growth followed by dedifferentiation and reinitiation of microvascular growth make the rachitic system a unique one in which to study angiogenesis. PMID- 1710879 TI - Upper respiratory tract environmental tobacco smoke sensitivity. AB - Some patients report rhinitis symptoms after exposure to environmental tobacco smoke (ETS), but objective assessments of this response have been lacking. Furthermore, the mechanism of this response is unknown. We assessed the frequency of ETS-related symptoms by administering a questionnaire to 77 healthy nonsmoking young adults who were participating in an unrelated study. Of the subjects 34% (26 of 77) reported one or more rhinitis symptoms (congestion, rhinorrhea, or sneezing) following ETS exposure. We then exposed 10 historically ETS-sensitive (ETS-S) and 11 historically ETS-nonsensitive (ETS-NS) subjects to 15 min of clean air followed by 15 min of sidestream tobacco smoke (CO concentration of 45 parts per million). At selected time points during these procedures we recorded symptoms, posterior nasal resistance, and spirometry and performed nasal lavages. ETS-S but not ETS-NS subjects reported significant (p less than 0.01) increases in nasal congestion, headache, chest discomfort or tightness, and cough following exposure to sidestream tobacco smoke. Rhinorrhea symptoms were greater and more prolonged in ETS-S subjects compared to ETS-NS subjects. Significant (p less than 0.01) increases in perception of odor and in eye, nose, and throat irritation occurred in both study groups, but ETS-S subjects reported significantly more nose and throat irritation. No significant changes in posterior nasal resistance occurred in the ETS-NS group but a significant increase occurred in the ETS-S subjects, with the resistance rising from 3.8 +/- 0.5 cm H2O/L/s (mean +/- SE) preexposure to a peak of 8.0 +/- 2.7 cm H2O/L/s 20 min after completion of the smoke exposure (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710880 TI - Vitronectin in bronchoalveolar lavage fluid is increased in patients with interstitial lung disease. AB - Vitronectin, also known as S-protein, is a 75,000-dalton serum glycoprotein that has a variety of functions, including the capacity to interact with the terminal components of the complement cascade, the coagulation system, and cell surfaces. By virtue of its ability to interact with cells, vitronectin is capable of mediating cell-spreading and adhesion and may also influence cell differentiation and cell growth. To investigate the possibility that vitronectin might contribute to the pathogenesis of interstitial lung disease, vitronectin was measured in bronchoalveolar lavage fluid from patients with sarcoidosis, idiopathic pulmonary fibrosis, and, for comparison, normal volunteers. Vitronectin was detected in lavage fluid and serum of all study subjects. Increased lavage fluid concentrations were found in patients with interstitial lung disease when compared with normal subjects (p less than 0.005), and glucocorticoid-treated patients with interstitial lung disease had lower vitronectin levels than did untreated patients. Furthermore, on SDS-PAGE and Western blot analysis lavage fluid vitronectin comigrated with serum vitronectin, suggesting similar molecular size. Thus, vitronectin is a normal constituent of the epithelial lining fluid, and lavage fluid vitronectin is similar to serum vitronectin. The increase of vitronectin concentrations in the epithelial lining fluid of patients with interstitial lung disease suggests that vitronectin may contribute to the pathogenesis of interstitial lung disease. PMID- 1710881 TI - Enkephalinase inhibitor potentiates substance P- and capsaicin-induced bronchial smooth muscle contractions in humans. AB - To determine the roles of endogenously released tachykinins (substance P, neurokinins A and B) in human bronchial tissues, and to determine the roles of enkephalinase (neutral endopeptidase, E.C. 3.4.24.11) in regulating the effects of the tachykinins, we studied the effects of substance P and capsaicin, which releases tachykinins, on human bronchial smooth muscle contraction in the presence or absence of enkephalinase inhibitor phosphoramidon in vitro. Substance P alone caused human bronchial smooth muscle contraction at 10(-6) M or more. Phosphoramidon (10(-7) to 10(-5) M) potentiated the substance P-induced contraction in a dose-dependent fashion, and phosphoramidon shifted the dose response curve to lower concentrations. Capsaicin (10(-5) or 10(-4) M) alone caused bronchial smooth muscle contraction in four tissues from nine patients. After the contraction by capsaicin reached a plateau, phosphoramidon (10(-5) M) increased and prolonged the contraction significantly. Furthermore, pretreatment of bronchial tissues with phosphoramidon (10(-5) M) potentiated capsaicin-induced contraction in all tissues from five patients. Phosphoramidon (10(-5) M) shifted the dose-response curve to capsaicin to lower concentrations more than 1 log unit. Captopril did not alter the contractile response to substance P, suggesting that angiotensin-converting enzyme does not regulate the contractile response to substance P in human bronchial smooth muscle in vitro. These results suggest that enkephalinase regulates the contractile effects of exogenous substance P and endogenous substances, probably tachykinins, released by capsaicin in the human bronchus. PMID- 1710882 TI - [Whipple's disease with sarcoidosis-like cutaneous manifestations]. PMID- 1710883 TI - Entrapment of proteins by aggregation within sephadex beads. AB - When a protein is aggregated by chemical crosslinking inside Sephadex beads of appropriate pore size, it gets trapped inside the beads. The above approach was used for immobilization of beta-galactosidase, acid phosphatase, trypsin, and concanavalin A. It was found to be a simple, convenient, and fast method for immobilization of proteins. PMID- 1710884 TI - [Usefulness of radiotherapy in the treatment of infiltrating bladder cancer]. PMID- 1710885 TI - Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae. AB - Several species of Enterobacteriaceae were investigated for their ability to synthesize selenium-containing macromolecules. Seleniated tRNA species as well as seleniated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobically induced formate dehydrogenase isoenzymes of E. coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunological and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hybridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of conservation of the seleniating system throughout the enterobacteria. PMID- 1710886 TI - Corneal vital staining with gentian violet. AB - We have used 0.5% gentian violet solution as a corneal vital stain in 112 patients with variable degrees of corneal involvements and in 40 normal eyes as control. Gentian violet stained the epithelial defects and degenerated epithelial cells of cornea. The stain persisted 3-5 minutes and disappeared by 8-10 minutes. There was no cross infection from dye use. The dye did not hamper usual process of repair of corneal lesions. It is found highly feasible, with a sensitivity about 100% and specificity about 95%, with some minor side effects. Thus it could be an excellent method in community ophthalmology for early diagnosis of corneal affections, thereby commencing prompt and appropriate treatment in early stage. PMID- 1710887 TI - Cloning of microbial epitopes relevant for T- and B-cells. AB - This review summarises, and illustrates, the technology that is available for the molecular cloning and precise identification of T-cell and B-cell epitopes, particularly those of bacteria and parasites. Methods include: selective cloning following subtractive hybridization of nucleic acids; selective screening of expression libraries; analysis of subcloned "epitope libraries" or "deletion constructs"; scanning of multiple synthetic peptides, and computer enhanced prediction. The direct sequencing of polymerase chain reaction products allows the rapid analysis of epitope heterogeneity occurring among natural populations. Multiple epitopes can be assembled either by synthesis or by the expression of polymeric epitope-bearing peptides. Prospects for probing expression libraries with T-cells are bleak due to the complexities of antigen processing, presentation and T-cell recognition in vitro. Elucidation of the enzymic steps involved in processing, resolution of peptide/MHC II co-crystals, and pairing of a large number of known epitopes with their functional restriction elements will significantly improve the ability to predict T-cell epitopes. PMID- 1710888 TI - Hypomethylation of the decorin proteoglycan gene in human colon cancer. AB - We have previously reported that the connective tissue stroma of human colon carcinoma contains elevated amounts of decorin, a small proteoglycan involved in the regulation of matrix formation and cell proliferation. These biochemical changes were correlated with increased mRNA levels and general hypomethylation of the decorin gene in human colon cancer DNA. In this report we use a quantitative polymerase chain reaction method coupled with digestion of the DNA template by methylation-sensitive restriction endonucleases to investigate in detail the location of hypomethylated sites in decorin gene. We demonstrate that a specific site in the 3' region of the gene, encompassing codons 360-361, is specifically hypomethylated in both colon carcinoma and benign polyp. In contrast, three HpaII sites, clustered in the 5' untranslated region, show full methylation in normal and neoplastic DNA. The lack of such changes in ulcerative colitis DNA suggests that chronic inflammation alone is not sufficient to alter cytosine methylation in the decorin gene. These results suggest the possibility that the 3' region of the decorin-coding sequence may be involved in the control of decorin gene expression. PMID- 1710889 TI - Human sputum cathepsin B degrades proteoglycan, is inhibited by alpha 2 macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C. AB - The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2 macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation. PMID- 1710891 TI - Evidence for involvement of glycoprotein-CD45 phosphatase in reversing glycoprotein-CD3-induced microtubule-associated protein-2 kinase activity in Jurkat T-cells. AB - Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a 43 kDa serine kinase which itself is a substrate of an unidentified tyrosine kinase (pp43). The reversibility of the MAP-2K response agrees with removal of tyrosine phosphates from pp43. Since both activity as well as tyrosine phosphorylation of MAP-2K could be prolonged by Na3VO4, a phosphotyrosine phosphatase inhibitor, we studied the effect of the common CD45 isoform, which is a member of the CD45 phosphatase family, on MAP-2K activity in vivo and in vitro. We demonstrate the ability of purified CD45 phosphatase to remove tyrosine phosphates from partially purified lymphoid MAP-2K. Utilizing the approach of heterologous receptor aggregation, we also showed that CD45 could inhibit the induction of MAP-2K activity in intact Jurkat cells during CD3 or CD3 + CD4 stimulation. We therefore suggest that this phosphatase may control the activity of lymphoid MAP-2K in vivo. PMID- 1710890 TI - Heterogeneity of chromogranin A-derived peptides in bovine gut, pancreas and adrenal medulla. AB - Chromogranin A is produced in many endocrine cell types, and is widely used as a marker in endocrine-cell pathology and secretory-cell biology. There is some evidence that it may be proteolytically processed to yield the putative pancreatic regulatory peptide, pancreastatin, and, in order to characterize the relevant pathways in gastrointestinal and pancreatic endocrine cells, we have used, in radioimmunoassay, site-directed antibodies to pancreastatin itself (L331) and to a sequence of chromogranin A immediately C-terminal to pancreastatin (L300). The latter antibody revealed three major forms of immunoreactivity of 8 kDa and five peptides of approx. 3 kDa in bovine pancreas and gut extracts. The 8 kDa peptides were characterized as chromogranin A-(248 313)-peptides, i.e. C-terminally extended forms of pancreastatin; two of the 8 kDa variants differed in two positions, confirming a polymorphism predicted from cDNA sequencing. One of the 3 kDa peptides was characterized as chromogranin A (297-313)-peptide, i.e. the C-terminal heptadecapeptide of the 8 kDa peptide that would be liberated after cleavage to yield pancreastatin. On the basis of chromatographic studies, immunohistochemistry and the stoichiometry of different immunoreactive peptides, three different pathways of chromogranin A processing were identified: in adrenal chromaffin cells chromogranin A existed mainly as the unmodified intact protein, in pancreatic islet and gastric antral endocrine cells pancreastatin and the 3 kDa peptides were major products, but in small intestine and gastric corpus endocrine cells there was little nor no pancreastatin and the 8 kDa cleavage product predominated. There are therefore important differences in the distribution of chromogranin A-derived peptides between quite closely related populations of endocrine cells that are attributable not only to variable post translational cleavage but also to the expression of different primary sequences. It seems possible that in different cell types chromogranin A-derived peptides might subserve a variety of different functions. PMID- 1710893 TI - The analysis of underivatized oligosaccharides by matrix-assisted laser desorption mass spectrometry. AB - Matrix Assisted Laser Desorption Mass Spectrometry is shown to provide a rapid and sensitive technique for the analysis of underivatized oligosaccharides. Typical sample loading is 1 pmol and analysis time is around 5 minutes. Through the use of an internal standard, mass measurements are generally accurate to within 0.5 Da. The technique is particularly useful for the analysis of oligosaccharide mixtures released from glycoproteins. PMID- 1710892 TI - Increased weight gain, nitrogen retention and muscle protein synthesis following treatment of diabetic rats with insulin-like growth factor (IGF)-I and des(1 3)IGF-I. AB - We have examined the effects of infusing recombinant human growth hormone (hGH), insulin-like growth factor-I (IGF-I), the truncated IGF-I analogue, des(1-3)IGF I, and insulin over a 7-day period in streptozotocin-induced diabetic rats. IGF-I at a dose of 1.05 or 1.08 mg/kg per day in two experiments increased body weight and nitrogen retention above those of vehicle-infused controls to about 30% of the improvement achieved with 25 or 30 units of insulin/kg per day, but only in the second experiment were the differences statistically significant (P less than 0.05). A 2.5-fold higher IGF-I dose, or des(1-3)IGF-I at 1.08 mg/kg per day, gave effects that were approx. 70% of those obtained with insulin. hGH at 1.38 mg/kg per day was not effective. The IGF peptides, unlike insulin, did not ameliorate the diabetic glucosuria. The improvements in nitrogen balance could be accounted for in part by increases in muscle protein synthesis. Muscle protein breakdown, as assessed by 3-methylhistidine excretion, was inhibited by insulin, but not by the IGF peptides. Carcass fat increased substantially following insulin administration. This did not occur with the IGF peptides, suggesting that IGF predominantly stimulates the growth of lean tissue. IGF-I concentrations and IGF I-binding proteins in plasma were increased by IGF-I, especially at the higher dose, whereas hGH produced only a transient increase in IGF-I. Des(1-3)IGF-I induced binding proteins, but had only a slight effect on measured IGF-I concentrations. We conclude that IGF peptides stimulate muscle protein synthesis and improve nitrogen balance in diabetes without obviously influencing the abnormal carbohydrate metabolism. Moreover, des(1-3)IGF-I is at least as potent as the full-length IGF-I. PMID- 1710894 TI - Antigen specificity of anticolipase monoclonal antibodies: characterization of colipase-Fab complexes using gel filtration high-performance liquid chromatography. AB - The epitope specificity of eight mouse monoclonal antiporcine procolipase antibodies (MAbs) was characterized on the basis of their competitive binding with antigen. Binary and ternary Fab-colipase complexes formed between antibody and porcine procolipase or its trypsin activated derivative were identified using gel filtration HPLC. The eight MAbs were divided in two groups that recognized overlapping epitopes located in distinct antigenic regions on procolipase. The gel filtration HPLC technique allowed to characterize two MAbs which did not react with solid-phase coupled antigen. Three MAbs formed Fab-antigen complexes with procolipase and not with activated colipase which suggests that epitopes recognized by these MAbs involve residues of the N-terminal pentapeptide of procolipase. PMID- 1710895 TI - [Cyclic analogs of bradykinin, containing a carboxyl group]. AB - 1 alpha-beta-carboxypropionyl-cyclo(9----1 epsilon)-[Lys1, Gly6]bradykinin (Suc c[Lys1, Gly6]B), 1 alpha-beta-carboxypropionyl-cyclo(10----1 epsilon)kallidin (Suc-cK), cyclo(10 gamma----1 epsilon)-[Glu10]kallidin (c[Glu10]K) and cyclo(11 gamma----1 epsilon)kallidylglutamic acid (cKG) were synthesized. Suc-c[Lys1, Gly6]B and Suc-cK were prepared by acylating the appropriate cyclopeptides with succinic anhydride. c[Glu10]K and cKG were obtained by the classic peptide synthesis, the cyclization being carried out with 61 and 42% yields, respectively. The protecting groups were then eliminated by catalytic hydrogenation. c[Glu10]K and cKG exerted myotropic action on isolated rat uterus (alpha 0.73 and 0.89, pD2 6.61 and 8.61, respectively). cKG displayed direct myotropic activity with respect to electrically stimulated rat vas deferens and guinea-pig ileum, potentiating the contractions (by 100%) in response to electric stimuli. c[Glu10]K and cKG elicit histamine release in isolated rat mast cells (EC30 4.91.10(-5) and 1.47.10(-6) M, respectively). Both cyclopeptides alter arterial pressure following intravenous administration to anaesthetized rats, cats and dogs and affect heart rate. In all assays cKG is more active than c[Glu10]K. Suc-c[Lys1, Gly6]B and Suc-cK do not possess myotropic, histamine releasing or hypotensive activity, though they were found to elicit a transient increase of bloodflow in cats and dogs. PMID- 1710896 TI - Investigation on the dosage/efficacy relationship of iron dextran in veal calves. AB - The efficacy of a single dose of 800 mg resp. 1600 mg iron in the form of an intramuscularly administrable iron (III)-dextran complex (Anaemex, CAS 9004-66-4) has been tested. On 3 groups of 13 calves each, 0 ml (comparing group), 4 ml resp. 8 ml iron dextran 20% have been applied. All calves received iron containing food during the test period of 10 weeks. At the beginning of the therapy, 5 weeks and 8 weeks after application, the parameters: weight, hemoglobin, erythrocytes, hematocrit, mean corpuscular hemoglobin (MCH), MCH concentration, mean corpuscular volume, plasma protein, fibrinogen, leukocytes and serum iron were measured. After 10 weeks the dead weight has been determined and the spleen of some calves tested histologically. The study shows that, by the administration of 1600 mg iron as a depot injection, a better growth results with the same quality of veal. The red-coloring of the veal was not significantly different from that of the comparing group. The histological findings show especially that the iron depots of the spleen were empty in all three groups and thereby in this collective no connection exists between the color of the veal and the tested dosage of iron dextran 20%. It is considered meaningful and economic to renounce in future the iron-containing food and in its place to apply intramuscular a single dose of 1600-2400 mg iron per calf. The results are compared with a study on full term infants, which has shown that a intramuscular single dose of 150 mg of iron as iron dextran at birth affords a nutritional advantage in iron status for up to 15 months.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710897 TI - Influence of some natural and semisynthetic agents on elastase and cathepsin G from polymorphonuclear granulocytes. AB - The in vitro effect of eglin C, glycosaminoglycan-peptide complex "DAK 16", glycosaminoglycan polysulfate and sodium pentosan polysulfate on the PMN-enzymes elastase and cathepsin G was investigated. The activity of elastase and cathepsin G was measured with the highly specific chromogenic substrates methoxysuccinyl-l ala-l-ala-l-pro-l-val-p-nitroaniline and succinyl-l-ala-l-ala-l-pro-l-phenyl alanin-p-nitroaniline. Eglin C and DAK 16 displayed a pronounced inhibition of PMN-elastase and PMN-cathepsin G. Surprisingly a higher amount of product in the elastase containing assays were found in the presence of glycosaminoglycan polysulfate and sodium pentosan polysulfate. The higher cleavage of the elastase substrate is related back to an electrostatic interaction of the polysulfates with this substrate. Both polysulfates strongly inhibited cathepsin G. The qualitatively and quantitatively different behavior of the drugs on elastase and cathepsin G are discussed with regard to their in vivo contribution to the therapy of chronic inflammatory joint diseases. PMID- 1710898 TI - Hemodynamic and mitochondrial parameters during hypoxia and reoxygenation in working rat hearts. AB - Hypoxia and reoxygenation in working rat hearts were investigated in this study. Cardiac hemodynamic parameters which decline immediately under hypoxic conditions, recover during reoxygenation. Biochemical and ultrastructural alterations exhibit a more complicated pattern. There is a primary phase in hypoxic perfusion up to 15 min with a steep increase of ADP contents and ATPase activities, and a severe fall of ATP/ADP ratios in mitochondria, as well as in tissue. High CAT (carboxyatractyloside) sensitivity of the ATPase is observed at 5 min of hypoxia. Furthermore, the number of ATPase particles visible at the inner mitochondrial membrane decreases. During the ensuing second phase of hypoxic perfusion (from 30 min on) the damage of mitochondrial ultrastructure becomes more evident. The amount of ATPase particles visible at the inner mitochondrial membrane further decreases. ATPase activities fluctuate, however, they remain connected with the membrane during hypoxia. ATP/ADP ratios attain values of almost 1. During reoxygenation (after 30 min of hypoxia) the levels of mitochondrial adenine nucleotides, oxidative phosphorylation rate and respiratory control index increase within 20 min and then slightly decline again. The ATP/ADP ratio is diminished in the course of reoxygenation. ATPase activity also decreases within 20 min of reoxygenation and the ADP/O ratio reaches control values. The ATPase activity gains its highest sensitivity towards CAT at 10 min of reoxygenation attaining a value similar to that of 5 min of hypoxic perfusion. It is suggested that hypoxia and reoxygenation under our conditions result in reversible derangement of ATPase and mitochondrial membrane structure. PMID- 1710899 TI - Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. I. Purification and some properties of AP DNA endonucleases in rat liver chromatin. AB - Three kinds of apurinic/apyrimidinic (AP) DNA endonuclease, APcI, APcII, APcIII, were purified from rat liver chromatin through 1M KCl extraction, DEAE-trisacryl ion exchange chromatography. Sephadex G-150 gel filtration and AP DNA cellulose affinity chromatography. Activities of the purified APcI, APcII and APcIII were 62.5, 83.3 and 52.0 EU/mg of protein, respectively. Molecular weights of APcI, APcII and APcIII, each consisting of a single polypeptide, were 30,000, 42,000 and 13,000, and isoelectric points of them were 7.2, 6.3 and 6.2, respectively. Three enzymes showed different substrate specificities; APcI acted only on AP DNA, and APcII acted on both AP DNA and UV DNA, while APcIII acted on 3'-methyl-4 monomethylaminoazobenzene (3'-Me MAB) DNA adduct as well as AP DNA and UV DNA. These results indicate that three kinds of AP DNA endonuclease present in rat liver chromatin have structural and functional diversities. PMID- 1710900 TI - Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin. AB - An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides. PMID- 1710901 TI - A study on nesidioblastosis in hyperinsulinemic hypoglycemia--diagnosis, treatment, and neurologic sequelae. AB - The medical records of six cases of nesidioblastosis were examined to determine the diagnostic approach, treatment, and neurologic sequelae. All six patients were male, and their ages at the onset of the disease ranged from one day to six months (mean 3.36 +/- 2.5 mo.). Initial clinical features were seizure, cyanosis, poor feeding, and apnea. Other subsequent symptoms were developmental delay, hyperactivity, and cold sweating. The Birth weight of the neonatal onset group was heavier than the postneonatal onset group (4.4 +/- 0.3 vs 3.26 +/- 0.04 kg). Before the diagnosis of hyperinsulinism, steroids of ACTH proved effective for seizure control. Initially, hyperinsulinemia (serum insulin greater than 10 microU/ml) was detected in four cases, but another two cases also showed hyperinsulinism by insulin/glucose(I/G) ratio greater than 0.3 during the fasting test. The glucagon response performed in 2 cases, showed normal and partial responses. Euglycemia was obtained by near total pancreatectomy (95% pancreatic resection)without malabsorption or persistent diabetes. In one case, nesidioblastoma coexisted with nesidioblastosis. Developmental delay was noted in three cases. In this group, the mean duration between symptom onset and operation was longer than the group without developmental delay (1.25 +/- 0.47 vs 0.38 +/- 0.19 yr). PMID- 1710902 TI - [Trans-membrane cationic flow and hemodialysis]. AB - Abnormalities of intracellular ion concentrations and transmembrane fluxes were reported in uremia. In RBC from 12 chronically hemodialyzed patients (age 41 + 12, 7 men, 5 women; mean dialysis duration 31 + 24 months), we evaluated the acute effects of hemodialysis on intracellular Na and K concentrations, ouabain sensitive Na/K pump, furosemide sensitive Na/K cotransport, Na/Li countertransport, and passive permeability to Na. Six patients were normotensive and 6 were taking antihypertensive drugs which were withdrawn before the study. When compared to our normal reference group, uremic patients showed a significant increase in intracellular K concentration and a significant decrease in ouabain sensitive Na/K pump. Intracellular sodium was not increased. No correlation was found between the activity of sodium-potassium pump and the duration of hemodialysis. The other transport systems were comparable to normal. No significant change was observed between the values measured before and after dialysis. Ouabain sensitive Na/K pump was lower in hypertensive as compared to normotensive patients, but this difference was not significant. Our data support the existence of ion transport derangements in uremia, which are not acutely affected by hemodialysis. PMID- 1710903 TI - Comparative quantitation of anti-viral titers of polyclonal human IgG and (Fab')2 preparations by an enzyme-linked immunosorbent assay (ELISA): pitfalls of using appropriate second antibodies. AB - When comparing anti-viral antibody titers of intravenous immunoglobulins (i.v. Igs) comprising two 7S IgG preparations and one corresponding (Fab')2 product, the specificity of the second ab-peroxidase conjugate employed in the ELISA was found to be decisive for the experimental results. Abs specific for the whole IgG molecule, in contrast to the (Fab')2 specific ones, obviously contain populations recognizing epitopes localized on C gamma 2 and C gamma 3 domains, and, therefore, cause an underestimation of titers of the (Fab')2 preparation. PMID- 1710904 TI - [Changes in the behavior of drug addicts after preventive intervention in drug addiction in several areas of Northern Italy, 1987-1989]. PMID- 1710905 TI - [Evaluation of the efficacy of information and health education interventions concerning AIDS. I. Methodological aspects of the research]. PMID- 1710906 TI - [Evaluation of the efficacy of information and health education interventions concerning AIDS. II. Knowledge among secondary school teachers in the Province of Rieti]. PMID- 1710907 TI - [Evaluation of the efficacy of information and health education interventions concerning AIDS. III. Knowledge among students of 3 technical institutes in Locride]. PMID- 1710908 TI - [Brucellosis control program in the Campania Region]. PMID- 1710909 TI - [Trends and some characteristics of female genital neoplasm mortality in the Campania Region]. PMID- 1710910 TI - Immunization strategies in rural areas. Examples from Somalia. PMID- 1710911 TI - [Nisin in food preservation: a bibliographic update and review of related international legislation]. PMID- 1710912 TI - Effect of FGFs on adult bovine Muller cells: proliferation, binding and internalization. AB - A new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS), alpha cristallin (alpha C) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC50 = 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of 125I-bFGF bound to the Muller cell receptors did not occur at 4 degrees C. At 37 degrees C, however, there was a rapid decrease in receptor-bound 125I-bFGF due to the downregulation of bFGF receptors. Concomitantly 125I-bFGF appeared inside the Muller cells. After 2 h, 125I-bFGF began to be degraded and after 6 h three fragments of 16 kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these factors may be the mediators involved in the communication between glial cells and neurons. PMID- 1710913 TI - Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping. AB - Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments. PMID- 1710914 TI - Porin interaction with hexokinase and glycerol kinase: metabolic microcompartmentation at the outer mitochondrial membrane. AB - Porin is the pore-forming protein involved in the movement of adenine nucleotides across the outer mitochondrial membrane (OMM). Hexokinase and glycerol kinase interact with porin on the outer surface of the OMM in a manner which provides these enzymes with preferred access to the ATP generated in the mitochondrion. We review recent evidence which permits refinement of our knowledge of these proteins and their interactions at the OMM. The involvement of this system in metabolic microcompartmentation is discussed, as well as possible pathological consequences of its disruption in malignancy and genetic deficiencies of hexokinase, glycerol kinase, and porin. PMID- 1710915 TI - Magnesium deficiency may be an important determinant of ventricular ectopy in digitalised patients with chronic atrial fibrillation. AB - Digitalised patients with chronic atrial fibrillation (AF) have a high prevalence of ventricular premature beats (VPB); magnesium deficiency may be a contributory factor. We have used a magnesium loading-test to examine the relationship between ventricular ectopy and magnesium status in 14 digitalised patients with chronic AF. Among seven patients with infrequent VPB (less than 250 24 h-1; mean 107 24 h 1) mean magnesium retention was 10.1% and four subjects retained no significant quantities of magnesium, indicating magnesium repletion. Among the remaining seven patients, mean magnesium retention was significantly higher (33.1%, P less than 0.02) and all patients retained 20% or more of the load given. There was an overall relationship between Mg retention and numbers of VPB (rs = 0.54; P less than 0.05). Magnesium deficiency may be determinant of ventricular ectopy in digitalised patients with chronic AF. PMID- 1710916 TI - New growth factor identified at Sloan-Kettering. PMID- 1710917 TI - A possible new adhesive site in the cell-adhesion molecule uvomorulin. AB - The calcium-dependent cell-adhesion molecule uvomorulin is a member of the cadherin gene family. Recent studies on the homophilic binding of molecules from neighbouring cells have shown that the amino-terminal part of these proteins plays an important role in the adhesive mechanism. We show here that the epitope for monoclonal antibody DECMA-1, capable of blocking uvomorulin function, is located close to the membrane proximal part of the extracellular domain. To test the effect of structural changes in this membrane proximal region on the adhesive function of uvomorulin, we have studied the cluster of cysteine residues located in the vicinity of the DECMA-1 epitope. Treatment of cells with the reducing agent dithiothreitol (DTT) cleaved the di-sulphide bonds in uvomorulin and affected the adhesive properties of cells. Close cell-cell contacts accompanied by cell flattening and changes in cell shape were blocked by DTT; however, cell aggregation was not inhibited. Consistent with this, uvomorulin became more susceptible in its membrane proximal part to trypsin digestion after treatment with DTT, indicating that conformational changes in this region of the molecule affect the adhesive function. These results suggest that the membrane proximal region of uvomorulin is involved in the adhesive mechanism. PMID- 1710918 TI - Stage I-II low-grade lymphomas: a prospective trial of combination chemotherapy and radiotherapy. AB - Between 1984-1989, 44 patients with stage I-II low grade lymphoma were treated prospectively with sequential chemotherapy and involved-field radiotherapy. The chemotherapy was cyclophosphamide, vincristine, prednisone, and bleomycin (COP Bleo); doxorubicin was included (CHOP-Bleo) for patients with adverse prognostic features (high LDH; extranodal sites; bulky nodes). Of the 44 patients, 37 had measurable disease and all have responded. With a median follow-up of 32 months, the 5-year survival and failure-free survival were 89% and 74%, respectively. Compared to past experience with involved-field radiotherapy alone, the failure free survival is significantly better with COP-Bleo plus radiotherapy. The potentially cured fraction has risen from 40% to 74%. PMID- 1710919 TI - Prospective multicenter trial for the response-adapted treatment of high-grade malignant non-Hodgkin's lymphomas: updated results of the COP-BLAM/IMVP-16 protocol with randomized adjuvant radiotherapy. AB - In a prospective multicenter trial the efficiency of the response-adapted COP BLAM/IMVP-16 protocol to induce complete remissions (CR) in high-grade malignant non-Hodgkin's lymphomas as well as the prognostic relevance of adjuvant radiotherapy were investigated. From 1986-1989, 548 patients (median age 56 years) with stage II-IV (Ann Arbor) disease were treated with five cycles of COP BLAM followed by two cycles of IMVP-16. If only a partial remission was obtained at the time of first restaging (RS) after three cycles (delayed response), treatment was switched to IMVP-16 (two to five courses) immediately. Patients achieving CR by the second RS after chemotherapy were randomized to adjuvant radiotherapy or observation. Responses to chemotherapy were 63% CR in patients completing the second RS (N = 350) or 72% if patients achieving late CR by consolidating radiotherapy are added; responses were 58% or 65% if all deaths prior to the second RS are included (N = 50). Overall and relapse-free survival were 71% and 68% at one year and 63% and 61% at two years. Multivariate risk factor analysis proved the early (by first RS) CR response to possess predominant prognostic relevance for survival. A significant advantage of adjuvant radiotherapy over no further treatment for duration of CR is not yet discernible. These results emphasize the importance of a rapidly achieved CR, thus contributing to the design of future trials. PMID- 1710920 TI - Hodgkin's disease in 63 intravenous drug users infected with human immunodeficiency virus. Gruppo Italiano Cooperativo AIDS & Tumori (GICAT). AB - Sixty-three cases of Hodgkin's disease in intravenous drug users (IVDUs) have been collected by the Italian Cooperative Group on AIDS-Related Tumors (GICAT). In most patients (74%) the histological pattern was that of mixed cellularity and lymphocyte depletion. In 39% of patients the initial symptom was a persistent lymph node enlargement due to persistent generalized lymphadenopathy (PGL). Unusual presentations included Waldeyer's ring, skin, meninges, colon, and pleura. After MOPP alternated or followed by ABVD, MOPP alone, or ABVD alone, 15 of 32 patients (47%) had a complete remission (CR) and 15 of 32 (47%) had a partial remission (PR). The median duration of CR was 14 months, while the median survival of patients with CR has not been reached; the median survival of patients treated with chemotherapy who had CD4 levels at presentation greater than or equal to 400/mm3 was significantly superior to that of those who had CD4 less than 400/mm3. The overall median survival was only 14 months. Forty-four percent of patients receiving chemotherapy, with or without radiotherapy, developed opportunistic as well as nonopportunistic infections. Lethal hepatic toxicity was observed in one patient. Among IVDUs, unusual presentations of Hodgkin's disease occurred at a lower rate than was previously reported for homosexuals. Complete remissions could be achieved in almost half the patients, but non opportunistic infections, in addition to parenchymal function impairment due to drug abuse, may limit treatment administration in IVDUs. PMID- 1710921 TI - Alternating versus hybrid MOPP-ABVD in Hodgkin's disease: the Milan experience. AB - The long-term therapeutic results achieved in a previous randomized study on stage IV Hodgkin's disease confirm the superiority of MOPP monthly alternated with ABVD compared to MOPP alone. To more closely meet the requirements of the Goldie and Coldman hypothesis, we activated a randomized study testing MOPP-ABVD through two different sequences in July 1982. One arm consisted of monthly alternating one cycle of MOPP and one cycle of ABVD; in the other arm, one half cycle of MOPP was alternated with one half cycle of ABVD within a one-month period (hybrid regimen). Each regimen was given to complete remission plus two consolidation cycles (minimum six cycles). After maximal tumor shrinkage, moderate doses of radiotherapy (25-30 Gy) were delivered to the lymphoid region(s) if bulky at the start of chemotherapy. A total of 300 patients with stage IB, IIA bulky, IIB, III (A + B) and IV Hodgkin's disease previously untreated with chemotherapy or failing after extensive irradiation were evaluated. At a median follow-up of five years, alternating and hybrid regimens yielded superimposable treatment outcomes: complete remission 89 versus 88%, freedom from first progression 65 versus 70%; relapse-free survival 72 versus 78%, overall survival 81 versus 80%, respectively. Tumor cell burden expressed as number of involved nodal sites and presence of pulmonary hilus involvement were the prognostic variables able to significantly influence treatment outcome. Conversely, stage, constitutional symptoms, and histology had no impact on the five-year results.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710922 TI - Management of relapse and survival in advanced stage Hodgkin's disease: the EORTC experience. AB - The EORTC Lymphoma Cooperative Group and the Pierre and Marie Curie Group conducted a multicentre randomised trial on clinical stages IIIB-IV Hodgkin's disease from 1981-1986. Two hundred seven patients were registered and 192 randomised. Actuarial survival at five years for the whole group was 68%. Induction chemotherapy with eight cycles of MOPP resulted in more patients with progressive disease and fewer partial responders than a combination of MOPP and ABVD, for an equal complete remission rate. Half of the partial responders went into complete remission after radiotherapy. At five years there was no significant survival difference between the arms. Progression was recorded in 39 patients of whom only 4 survived. Relapses were most frequent in previously involved unirradiated areas. For 46 relapsed patients, including 21 early relapses within 18 months of start of treatment, the four-year survival rate was 53%. When complete remission was reached, whether early or late with combination chemotherapy or after additional radiotherapy, prognosis was independent of the way in which it was achieved. All efforts should be taken to reach a complete remission for initially progressing patients and for partial responders. PMID- 1710923 TI - Cardiopulmonary toxicity after three courses of ABVD and mediastinal irradiation in favorable Hodgkin's disease. AB - The combination of chemotherapy and radiotherapy in Hodgkin's disease has been associated with iatrogenic effects. Forty adult patients were studied to evaluate the early toxicity following three courses of ABVD (cumulative dose of doxorubicin [Adriamycin] 150 mg/m2, and bleomycin 60 mg) and mediastinal irradiation at 40 Gy. Cardiopulmonary toxicity was assessed from six months to three years after completion of irradiation. Of the 40 patients, all of whom were in complete remission from Hodgkin's disease, 6 experienced dyspnea on exertion. In studies related to Cardiac toxicity, the left ventricular ejection fraction ranged from 50 to 77% (mean 63%); 8 patients had a minor pericardial effusion, 4 had valvular calcification, and 6 had minimal cardiac abnormalities. With regard to pulmonary toxicity, CT scan showed a small pleural effusion with pleural thickening in 19 patients and mediastinal or apical fibrosis in 15 patients. The total pulmonary capacity value was low (less than 80%), in 19 patients, and decreased carbon monoxide diffusion capacity (less than 70%) was found in 10 patients. We conclude that early cardiac toxicity was absent despite the use of Adriamycin and mediastinal irradiation. Pulmonary toxicity was present but minor, and it may decrease with the use of smaller fraction sizes for mantle field irradiation. PMID- 1710924 TI - High-molecular-weight Ly-5 isoforms expressed on T cells: activation-dependent expression. AB - Interleukin 2 (IL-2)-dependent granular T lymphocyte (GTL) lines derived from murine spleen were shown to express a Ly-5 antigen with the so-called 'B220 epitope' recognized by 6B2 mAbs, which were normally exhibited on B-lineage cells. Immunoprecipitation analysis revealed that the Ly-5 isoform on GTL lines represented the highest-molecular-weight Ly-5 so far described (260 kd), distinct from the B220 isoform on a pre-B cell line (240 kd). The size difference between them was also evident after the endoglycosidase treatment (210 versus 190 kd), strongly suggesting that these Ly-5 isoforms had core proteins of distinct size. Sequential immunoprecipitation by 6B2 and 14.8 mAbs further indicated that the 260 kd Ly-5 on GTL lines predominantly expressed '6B2 epitope' while '14.8 epitope' dominated in the B220 (240 kd) on a pre-B cell line. Northern blot analysis of the Ly-5 transcripts using probes for the known alternative exons failed to show any evidence for mRNA of the 260 kd Ly-5 distinct from that of B220. Polymerase chain reaction analysis, however, suggested that the 260 kd isoform mRNA might be transcribed from a distinct promoter with a yet undefined exon(s). The 6B2+ Ly-5 isoform was hardly detected on normal splenic T cells, but was shown to be induced rapidly on the majority of T cells following IL-2 stimulation in vitro, indicating that this particular Ly-5 isoform behaved as an 'activation antigen' on T cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710925 TI - In vivo primed mouse T cells selectively express T cell-specific serine proteinase-1 and the proteinase-like molecules granzyme B and C. AB - Previous studies have shown that mouse CD8+ T lymphocyte clones (TLC) produce T cell-specific serine proteinase-1 (MTSP-1) as well as a family of six homologous molecules, termed granzymes B - G, which are structurally related to serine proteinases. Of these proteins, only MTSP-1 has been studied in detail. It has been shown to occur in the majority of CD8+ and a fraction of CD4+ T effector cells in vivo and in vitro and has demonstrable enzyme activity in these cells. The presence of the other serine proteinase-like molecules in T cells is less well defined. We have now analyzed the expression of mRNA species specific for granzymes B - G in activated T cell populations using the sensitive polymerase chain reaction which allows the detection of mRNA species from as little as 2 pg of total cytoplasmic RNA. We demonstrate that MTSP-1 and all six serine proteinase-like transcripts are expressed in a panel of four CD8+ and six CD4+ long-term-cultured TLC, though at greatly differing concentrations. In contrast, in vivo primed T cells of both phenotypes, CD4+ and CD8+, and in vitro activated T cells derived from short-term cultures only express mRNA species specific for MTSP-1 and CCP1 and little of those for CCP2, but no transcripts for granzymes D G. These findings argue against the participation of granzymes D - G in T cell mediated functions in vivo. PMID- 1710926 TI - Inhibition of in vitro differentiation of human monocytes to macrophages by lipopolysaccharides (LPS): phenotypic and functional analysis. AB - Blood monocytes (MO) undergo maturation into macrophages (MAC) upon migration from the capillary bed to tissue sites of inflammation where they are exposed to environmental signals. Functional competence and phenotype heterogeneity is the result of both differentiation-inducing and -activating events. In vitro, MO to MAC maturation is induced by serum factors, can be followed by the expression of specific maturation-associated antigens and is accompanied by a characteristic change in the secretory repertoire of MAC in comparison to MO. Here we report that bacterial lipopolysaccharides (LPS) at subnanogram quantities very effectively inhibited the serum-induced maturation of human MO in vitro. At the same time LPS induced the up-regulation of CD14 antigens. The lipid A moiety was shown to be responsible for this novel biological activity of the LPS molecule. Inhibition of maturation was not due to secondary LPS-induced signals like interleukin (IL)-1, IL-6, tumor-necrosis factor (TNF)-alpha or interferon (IFN) alpha--even though the latter by itself suppressed MAC maturation in vitro. The inhibitory activity of IFN-alpha could be abolished by neutralizing anti-IFN alpha antibodies whereas these antibodies had no effect on LPS-induced suppression of MAC maturation. Functional analysis of LPS-treated MO long-term cultures showed that the pattern of secretory products released was similar to that of freshly-isolated immature blood MO: compared with mature MAC, LPS-treated MO released high amounts of IL-6 but significantly less TNF-alpha, neopterin, lysozyme and beta-2-microglobulin. At the same time, in LPS-treated MO cultures the MAC maturation-associated molecules alpha-2-macroglobulin and fibronectin could be detected only in trace amounts. The ability to secrete IL-1, however, was lost both in control as well as in LPS-treated MO cultures. The results indicate that endotoxins may influence the biology of the MO/MAC system distinctively: they not only induce a functional activation but also interfere with the ontogeny of this cell family. PMID- 1710927 TI - Hydrogen peroxide effects on ionic and non-ionic permeability of the rabbit corneal endothelium. AB - Perfusion of the isolated rabbit corneal endothelium with 0.3 mM hydrogen peroxide (H2O2) caused an increased passive permeability to bicarbonate relative to control tissues. This was accompanied by a reduction in the active flux that resulted in a reduced net bicarbonate flux. Perfusion with 0.3 mM H2O2 resulted in a marked increase in the active and net flux of sodium beginning at two hours. By four hours the net sodium flux had increased by nine-fold over control values. Perfusion with 0.3 mM H2O2 resulted in a 16% and 30% increase in endothelial permeability to inulin and dextran, respectively. Suppression of catalase activity by in vivo pretreatment with intravenous 3-aminotriazole (3AT) did not result in an increased sensitivity of the corneal endothelium to 0.2 mM H2O2: both bicarbonate and sodium fluxes were normal. Inhibition of glutathione synthesis with intravitreal buthionine sulfoximine (BSO) increased the sensitivity of the corneal endothelium to 0.2 mM H2O2 only in the case of sodium flux, with a 4.8-fold increase in net sodium flux at 3 hours after initiation of perfusion. Bicarbonate fluxes were unaffected after BSO pretreatment. The data show that ionic and non-ionic fluxes are altered by H2O2, that pretreatment with 3AT has a minimal effect on ion fluxes while BSO markedly alters sodium flux without changing bicarbonate fluxes, and that sodium and bicarbonate movement are not locked in a symport. PMID- 1710928 TI - Effect of chronic ethanol consumption on hepatic mitochondrial transcription and translation. AB - Liver mitochondria from ethanol-fed rats display an impaired ability for protein synthesis in vitro. Studies were conducted to explore the possible mechanisms which might account for this impaired capacity of ethanol mitochondria for protein synthesis. The present studies did not demonstrate any significant ethanol-induced lesion in mitochondrial nucleic acid metabolism in organelles isolated from ethanol-fed rats for any of the parameters investigated (mtDNA content, steady-state mtRNA concentration, mtRNA polymerase activity, concentration of specific mRNAs and rRNAs, mtRNA processing). An investigation of ribosome function in isolated mitochondria demonstrated significant decreases in the number of active ribosomes (55% fewer) in mitochondria from ethanol-fed rats. Initiation of protein synthesis was also significantly depressed (46%) in ethanol mitochondria. In addition, the yield of ribosomal particles from ethanol mitochondria was decreased 32% as compared to the yield of ribosomal particles from control mitochondria. However, isolated ribosomes from ethanol mitochondria were determined to be fully functional in a poly(U)-directed phenylalanine polymerization system. Soluble translation factors from ethanol mitochondria were also found to support full activity of control ribosomes in a poly(U)-directed phenylalanine polymerization system. These results suggest strongly that the ethanol-induced depression of mitochondrial protein synthesis is due to a decrease in the number of competent ribosomes in hepatic mitochondria from chronically ethanol-fed rats. PMID- 1710929 TI - Characterization and purification of the hyaluronan-receptor on liver endothelial cells. AB - In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents. PMID- 1710930 TI - Kinetic analysis of the interaction between type 1 plasminogen activator inhibitor and vitronectin and evidence that the bovine inhibitor binds to a thrombin-derived amino-terminal fragment of bovine vitronectin. AB - The interaction between guanidine-activated bovine type 1 plasminogen activator inhibitor (PAI-1) and bovine vitronectin was investigated. Activated PAI-1 bound to vitronectin in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 3.10(-10) mol/l by Scatchard analysis. Complexes of activated PAI-1 and vitronectin were relatively stable at 4 degrees C (T1/2 greater than 24 h), but dissociated with a T1/2 of 4 h at 37 degrees C. The half-life of PAI-1 activity was increased from 2.5 to 4.5 h upon binding to immobilized vitronectin. In order to identify the binding domain(s) in vitronectin for activated PAI-1, the ability of PAI-1 to bind to vitronectin fragments was assessed. Vitronectin was cleaved by thrombin in a dose- and time-dependent manner, generating fragments of Mr 60,000, 54,000 and 38,000. The PAI-1 binding domain(s) were not destroyed by this treatment, since the digested vitronectin competed with immobilized vitronectin for PAI-1 binding to the same extent as uncleaved vitronectin. The thrombin digested vitronectin fragments were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 38,000) retained PAI-1 binding function, and sequence analysis demonstrated that this fragment contained the NH2-terminus of bovine vitronectin. These results suggest that the high-affinity binding site for activated PAI-1 is located in the NH2-terminal region of the bovine vitronectin molecule. PMID- 1710931 TI - The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha 2-macroglobulin and thrombin. AB - A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2 macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study. PMID- 1710932 TI - Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes. AB - Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin. PMID- 1710933 TI - Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells. AB - Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells. PMID- 1710934 TI - Essential role for polyamine biosynthesis in thyroxine stimulated pancreatic development in neonatal rats. AB - Administration of thyroxine to rat pups leads to precocious development of the pancreas. The role of ornithine decarboxylase (ODC) and polyamines in thyroxine induced pancreatic maturation was examined. Rat pups (aged 5 days) were given daily subcutaneous injection of thyroxine (0.1 micrograms/g body wt.) until the day before death. Serial ODC activities were measured in pancreatic homogenates after 1, 2, 3, 4, 5, 6, 7 and 10 days of thyroxine treatment. There was a biphasic induction of ODC activities by thyroxine: an early peak appeared on day 2 of treatment followed by a decrease on day 4; a second peak was evident on day 5 and then a decrease to control values by day 7. Significant increases in tissue concentrations of putrescine and spermidine were observed concomitant with two peaks of ODC activity. Pancreatic amylase concentration, DNA and protein also showed a significant increase after thyroxine treatment. Difluoromethyl ornithine (DFMO), a specific ODC inhibitor, given orally (8% in drinking water) to nursing dams at postnatal day 5 for 5 days caused an 83% inhibition of pancreatic ODC activity in thyroxine-treated pups when compared to thyroxine-treated pups not exposed to DFMO. Concomitantly, the thyroxine-induced increases in pancreatic weight, protein and amylase activity were suppressed. Our results suggest that increases in ODC activities and polyamine levels are critical intermediary steps in the precocious induction of pancreatic development by thyroxine. PMID- 1710935 TI - Preparation and characterization of immunoconjugates for antibody-targeted photolysis. AB - Monoclonal antibody (MAb)-dextran-tin(IV) chlorin e6 (SnCe6) immunoconjugates were prepared by a new technique involving the use of reducing, terminal-modified dextran carriers and site-specific modification of the Fc oligosaccharide moiety on the antibodies. Dextran carriers were synthesized to increase the number of SnCe6 molecules attached to a MAb. The dextran carriers were coupled to the MAb via a single, chain-terminal hydrazide group to prevent aggregation of MAbs. Conjugates were prepared with antimelanoma MAb 2.1 containing up to 18.9 SnCe6 molecules per MAb. Under neutral conditions, no hydrolysis of the hydrazone bond between the MAb and the dextran carrier could be detected, and the hydrazone was not stabilized by reduction with NaCNBH3 or NaBH4. Analysis of the purified immunoconjugates showed that approximately two dextran carrier chains were attached to a MAb regardless of the number of SnCe6 molecules linked to a dextran carrier. Site-specific covalent attachment of the SnCe6-dextran chains to the MAb was confirmed by SDS-PAGE. HPLC analysis of the conjugates gave a single species eluting in the range of 200-240 kDa. As determined by a competitive inhibition radioimmunoassay using viable SK-MEL-2 human malignant melanoma cells, the conjugates showed excellent retention of antigen-binding activity relative to unconjugated MAb. PMID- 1710936 TI - Quantitation of whole molecule human chorionic gonadotropin and free beta-human chorionic gonadotropin subunit in the Abbott IMx analyser. AB - An assay for the quantitation of the serum levels of free and whole molecule associated beta-subunit of human chorionic gonadotropin on the Abbott IMx analyzer is described. The assay detects human beta-chorionic gonadotropin with a sensitivity of approximately 0.5 IU/l while showing no cross-reactivity with follitropin (1000 IU/l) or thyrotropin (2.0 IU/l), and 0.02% cross-reactivity with lutropin (1000 IU/l). Haemoglobin (7.50 milligrams), bilirubin (0.50 milligrams), and triacylglycerols (10.6 milligrams) did not interfere with the assay. Pooled within-run, between-run, and total assay CVs were less than or equal to 5.2%, and less than or equal to 2.7%, and less than or equal to 6.3%, respectively. Values obtained with this assay correlated well (r = 0.98, n = 228) with those values obtained using the Hybritech TandemR-R HCG (Total Beta-HCG) IRMA. The normal range of the assay was found to be less than or equal to 5 IU/l (n = 311). The assay protocol provides results for up to 23 serum samples in approximately 47.5 minutes with the ability to report the human beta-chorionic gonadotropin concentrations of 5 specimens in approximately 15.5 minutes. We conclude that this is an acceptable assay for monitoring human beta-chorionic gonadotropin levels associated with normal pregnancy, reproductive pathology, and reproductive technology such as in vitro fertilization or embryo transfer. PMID- 1710937 TI - Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1. AB - The structure of the membrane bound state of the 178-residue thermolytic COOH terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin. PMID- 1710938 TI - Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine. AB - Antagonism of glutamate-receptor responses activated by N-methyl-D-aspartic acid (NMDA) was studied using whole cell voltage clamp recording from mouse dissociated hippocampal neurons cultured for 10-15 d. The kinetics of onset of and recovery from NMDA receptor block during continuous application of NMDA together with either glycine, or L-alanine, were recorded in response to concentration jump application of NMDA- and glycine-binding site directed competitive antagonists, applied with a multibarrel flow pipe under conditions which allowed rapid solution changes around the cell less than 10 ms. Mathematical solutions for both one- and two-equivalent site models for competitive antagonism were determined according to the differential equations outlined by Colquhoun and Hawkes (1977. Proc. R. Soc. Lond. B. 199:231-262). The kinetics of action of D-CPP and D-AP5, NMDA binding site antagonists, and 7Cl kynurenic acid, a glycine binding site antagonist, were examined for each model. For all these antagonists, the kinetics for the onset of and recovery from antagonism were better fit by the two-equivalent site model, which yielded antagonist microscopic kBoff/kBon values which closely approximated Ki values determined from analysis of equilibrium dose response curves. These results suggest that two molecules of NMDA and two molecules of glycine must bind to the NMDA receptor for activation of ion channel gating. PMID- 1710939 TI - Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation. AB - Membrane patches from chick skeletal muscle were stretched by applying controlled suction or pressure to the pipette. From images of the patch, the patch dimensions (area and radius of curvature) were computed by nonlinear regression of the images to a geometric model. With no applied pressure, patch membranes are nearly planar and normal to the wall of the pipette. With increasing pressure gradients, the patch bulges, the radius of curvature decreases, and the area increases. The patch capacitance changes in exact proportion to the change in area at a rate of 0.7 microF/cm2. The increase in area is due to a flow of lipid (with perhaps small amounts of diffusible protein) along the walls of the pipette into the patch. The flow is reversible with a relaxation of the pressure gradient. The area elastic constant of the membrane is approximately 50 dyn/cm, insensitive to cytochalasin B and probably represents the elasticity of the underlying spectrin/dystrophin network. Simultaneous measurements of stretch activated (SA) ion channel activity in the patch showed that the sensitivity of channels from different patches, although different when calculated as a function of applied pressure, was the same when calculated as a function of tension. Because patch lipid is free to flow, and hence stress-free in the steady state, SA channels must be activated by tension in the cytoskeleton. PMID- 1710940 TI - Assessment of red blood cell aggregation with dextran by ultrasonic interferometry. AB - Aggregation of human red blood cells (RBCs) induced by dextrans of various molecular weight has been studied by using a new ultrasonic interferometry method. This method, based on A-mode echography, allowed for the measurement of the accumulation rate of particles on a solid plate which is related to their sedimentation rate (i.e., to their mean size). The initial aggregation process, the mean and the maximum sedimentation rate of aggregates and the packing of the sedimented RBCs have been investigated. Effects of hematocrit, molecular weight of dextrans and inhibition by dextran 40 on the RBC aggregation induced by dextran of higher molecular weight have been determined by analysing variations of the aggregate size. Results obtained confirm the aggregation effect of dextrans of molecular weights equal or higher than 70,000 dalton and disaggregation effect of dextran 40,000 dalton on aggregation by dextrans of higher molecular weight. PMID- 1710941 TI - The effect of the solvent viscosity on the migration of small molecules through the structure of myoglobin. AB - The migration rate of small molecules through the structure of proteins can be monitored by quenching the light emitted from an excited optical probe located within the protein. In the present study we examined the influence of the solvent viscosity on the migration rate of the quencher anthraquinone sulfonate through myoglobin towards an excited Zn protoporphyrin molecule at the binding site of the protein. The solvent viscosity was increased by adding dextrans of different molecular weight but forming isoviscous solutions. The results demonstrate that the migration rate in the protein decreases with increasing solvent viscosity. This suggests that the fluctuations on the protein structure, which make the above migration possible, are affected by the solvent macroviscosity. PMID- 1710942 TI - In vitro induction of early mouse embryo intracisternal particles (epsilon particles) in cultured cell lines. AB - The experimental induction of epsilon particles, retrovirus-like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent-cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycin , deoxy fluorouracil or 5-azacytidine. However, only two mouse-derived cell lines: Ki BALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed epsilon particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previously shown to attenuate IAP production, had no effect on that of epsilon particles. PMID- 1710943 TI - Hematopoietic recovery in a patient with acute lymphoblastic leukemia after an autologous marrow graft purged by combined hyperthermia and interferon in vitro. AB - We describe a patient with acute lymphoblastic leukemia (ALL) in whom hemopoiesis recovered after an autologous marrow graft purged by in vitro hyperthermia. A 17 year-old woman was diagnosed as having ALL in April 1985. After clinical remission was induced, marrow cells were harvested. The marrow cells were treated with hyperthermia at 42.0 degrees C for 1 h in the presence of alpha-interferon to eliminate residual leukemic cells, and then cryopreserved. In January 1990, during her fourth remission she was treated with busulfan and cyclophosphamide, and then received the thawed autologous marrow. Her hematopoietic recovery was prompt with normal trilineage regeneration without any life-threatening complications. She is in good health without evidence of a leukemic relapse at 6 months after autologous bone marrow transplantation. This case suggests that human multilineage progenitor cells retain self-renewal capacity in vivo even after treatment with heat and alpha-interferon in vitro followed by the freezing and thawing procedures. PMID- 1710944 TI - Metabolic and anatomical adaptations of heavy-bodied chicks to intermittent feeding. 2. Pancreatic digestive enzymes. AB - 1. Activities of digestive enzymes in meat-type chickens under ad libitum or alternate-day feeding were determined from 14 to 83 d of age. 2. Final body weight of intermittently fed birds attained 75% of that of the ad libitum-fed controls. 3. When compared with the ad libitum-fed counterparts, a marked increase in the relative weight of the pancreas and intestinal contents were found on repletion days. On depletion days the relative weights of the pancreas and of the intestinal contents were about half those found in ad libitum-fed birds. 4. The activity of the digestive enzymes in the pancreas, expressed as U/g pancreas or U/kg body weight, was not affected consistently by the feeding regime. In the small intestine a marked increase in relative activity (U/kg body weight) was observed on repletion days and a marked decrease on depletion days as compared with ad libitum-fed controls. The activities per g intestinal contents following food restoration did not differ significantly from those of ad libitum fed controls except for trypsin, which was higher in the former. On depletion days the activities per g intestinal contents were lowest, lipase excepted. PMID- 1710945 TI - Modified staining techniques for avian blood cells. AB - 1. Two routine staining methods for domestic fowl blood cells have been modified with superior results. 2. Type of anticoagulant had a major effect on staining quality: EDTA gave excellent results whereas lithium heparin was unsatisfactory. 3. Unfixed blood smears were preferred to smears fixed in methanol before staining with May Grunwald and Giemsa's stain. Intact heterophil and basophil granules were clearly demonstrated. 4. Staining for 60 min in Natt and Herrick's haemocytometer diluent improved the differentiation between small lymphocytes and thrombocytes. PMID- 1710947 TI - What value is given to quality of life assessment by health professionals considering response to palliative chemotherapy for advanced cancer? AB - A study was designed to obtain information on the importance of quality of life assessment (QL) during palliative chemotherapy. A questionnaire was answered by 542 health professionals (392 general practitioners, 20 specialist oncologists, and 130 oncology nurses). In both simulated patient situations and multiple choice questions, all groups rated QL higher than other standard methods of assessment. General practitioners and oncologists appeared to weight the assessment criteria more equally than nurses who gave strong emphasis to QL. In the simulated patient situation, there was a small degree of interaction between QL and other assessment criteria. However, the analysis showed that QL was regarded as an independent variable and was considered to be the most important objective of palliative chemotherapy for advanced cancer. PMID- 1710946 TI - Spindle cell stromal tumors of gastrointestinal tract: a histological and immunohistochemical study. AB - Twenty-one consecutive cases of gastrointestinal spindle cell stromal tumors (SCSTs) were studied histologically and immunohistochemically. They consisted of 18 smooth muscle tumors, 2 neurilemmomas (schwannomas), and 1 unclassified malignant tumor designated as stromal sarcoma. Slender, spindle and wavy nuclei with palisading associated with peripheral tumor aggregation of lymphocytes are the pathological hallmarks of neurilemmoma. With peroxidase-antiperoxidase method, antibodies to Glial Fibrillary Acidic Protein (GFAP), Leu-7, S-100, desmin and HHF35 were applied. Antibodies to Leu-7 and GFAP could only be demonstrated in neurilemmomas (2 cases). Antibody to S-100 was observed strongly in 2 neurilemmomas and 1 stromal sarcoma, and focally in a leiomyoma, while the other SCSTs were negative. One neurilemmoma disclosed focal positivity of desmin. Six of 10 leiomyomas revealed varied degrees of positive staining of desmin and HHF35. One epithelioid leiomyoma, two leiomyosarcomas and five smooth muscle tumors of undetermined malignant potential (STUMP) disclosed no immunoreactivity. The study suggests that panel of immunostaining should be applied. Coexpression of GFAP, Leu-7 and S-100 as well as negative staining of HHF35 is characteristic of neurilemmoma. On the contrary, coexpression of desmin and HHF35 while negative for GFAP, S-100 and Leu-7 are suggestive of smooth muscle tumor. In poorly differentiated SCST, histological features and immunostains are always disappointing. Diagnosis of those tumors as stromal tumor is more appropriate or electron microscopic observation should be included for accurate classification. PMID- 1710948 TI - An immunohistochemical study of sarcomatoid liver carcinomas. AB - Six cases of primary hepatic carcinomas with a significant amount of sarcomatoid elements were examined by using immunohistochemical stainings. Four of the six cases were associated with ordinary hepatocellular carcinoma (HCC), one with cholangiocellular carcinoma (CCC), and one with mixed HCC and CCC. Alpha fetoprotein and alpha-1-antitrypsin were negative in sarcomatoid cells of all cases; vimentin stained positively in sarcomatoid tumor cells in two of the six cases; and cytokeratin (CK8) was detected in five cases. The CK8 was not detected in tumor cells of two cases of hepatic angiosarcoma, two of metastatic leiomyosarcomas, and one of metastatic fibrosarcoma, although vimentin stained positively in all these true sarcomas. It was concluded that sarcomatoid dedifferentiation of liver carcinomas might derive from both HCC and CCC. In addition CK8 might be an excellent marker to make a differential diagnosis of sarcomatoid cancers from true metastatic or primary sarcomas of the liver. PMID- 1710949 TI - Stimulation of growth of human breast cancer cells (T47D) by platelet derived growth factor. AB - The human breast cancer cell lines T47D and MCF-7, respond to the mitogenic action of exogenously added PDGF with a 2-3-fold increase in cell number. The maximal response was obtained at a concentration of 1.25 half maximal units/ml (125 ng/ml). PDGF-AA was even more effective than PDGF-AB while PDGF-BB was without effect. The growth-enhancing activity of PDGF was completely abolished by Fab fragments of anti PDGF. Within 7 min of addition of PDGF to cultures of T47D cells, specific phosphorylation of a 65-kDa protein was observed. T47D cells contain specific receptors for PDGF with approximately 4-7 x 10(4) sites (kDa of 3-4 x 10(-10) M) per cell. PMID- 1710950 TI - Effect of lethal scald on the mechanisms of acute-phase protein synthesis in rat liver. AB - Acute-phase protein (APR) synthesis was studied under conditions in which the acute-phase response to a sublethal scald was interrupted at the onset of APR synthesis by the infliction of a second scald that overwhelmed the defense mechanisms. The rate of APR synthesis increased shortly after the second scald and then declined rapidly to the control level. At this time point, APR messenger RNA (mRNA) concentrations exceeded severalfold the control values, whereas the APR gene transcription rates fell to the control level. These mRNAs were active in APR synthesis in a cell-free system, as well as in hepatocytes grown in a standard culture medium. These results and those demonstrating a drop in the free amino acid pool level in the liver after the second scald suggested that the lethal outcome was preceded by an impaired supply of liver cells with amino acids and resulting inhibition of APR mRNA translation. Changes in amino acid transport were considered to occur secondarily to those causing hypovolemia and circulatory shock. PMID- 1710951 TI - Variants of prostate-specific antigen separated by concanavalin A. PMID- 1710952 TI - Evaluation of an assay of the free beta-subunit of choriogonadotropin and its potential value in screening for Down's syndrome. AB - In this study I investigated the analytical and clinical performance of the measurement of the free beta-subunit of choriogonadotropin (hCG) in normal pregnancies and in pregnancies affected by Down's syndrome. Free beta-hCG in maternal serum has been shown to be increased in Down's syndrome-affected pregnancies and is proportionally increased in more cases than is total hCG. This study confirms previous findings of low concentrations of unconjugated estriol and alpha-fetoprotein in maternal serum in Down's syndrome-affected pregnancies. Using a multivariate risk analysis of maternal age and concentrations of alpha fetoprotein, unconjugated estriol, and hCG in maternal serum, I determined that, at a risk cutoff value of 1 in 300, 52% of Down's cases could be detected with total hCG in the calculation, compared with 66% with the free beta-hCG concentration. The false-positive rate was 5.9% in both cases. Therefore, free beta-hCG can be used effectively in a screening program for Down's syndrome; however, further studies are required to ascertain whether the measurement of free beta-hCG has any advantages over the use of total hCG for detecting Down's syndrome. PMID- 1710953 TI - Measurement of prostate-specific antigen and prostatic acid phosphatase concentrations in serum before and 1-42 days after transurethral resection of the prostate and orchidectomy. AB - Preoperative intra-individual variation for determinations of prostate-specific antigen and prostatic acid phosphatase concentrations, 15-30% in 92 patients with benign prostatic hyperplasia, limits the diagnostic usefulness of both tumor markers. In benign prostatic hyperplasia (214 patients), concentrations of these tumor markers increased in the initial postoperative period. Prostatic acid phosphatase concentration then decreased by the third postoperative day. Prostate specific antigen concentration remained above normal in the first postoperative week but had decreased by 42 days. In prostatic carcinoma (46 patients), the concentrations of these tumor markers did not increase postoperatively. During the first week, the concentrations of prostatic acid phosphatase began to fall, but prostate-specific antigen showed a decrease only at 42 days. After orchidectomy (11 patients), the concentrations of both markers had decreased by five days. Concentrations of prostate-specific antigen but not of prostatic acid phosphatase were significantly increased in patients with metastases at 42 days postoperatively. When the concentration of tumor marker did decrease, the magnitude of change was greater for prostatic acid phosphatase than for prostate specific antigen. These changes were accentuated after an orchidectomy. PMID- 1710954 TI - The presence and measurement of secretory component in human bile and blood. AB - Five monoclonal antibodies which recognized three separate epitopes on the free secretory component molecule were produced using free secretory component obtained from human colostrum. Two-site immunoradiometric assays were developed to measure free secretory component and secretory IgA. Monoclonal antibody M9 was used on coated plates as the capture antibody. Monoclonal antibody M7 was used as the labelled signal antibody for the assay of free secretory component and a commercially available monoclonal anti-IgA antibody was used as the labelled signal antibody for the assay of secretory IgA. Free secretory component was found in human serum and bile. In serum, its concentration was raised in patients with high serum alkaline phosphatase due to liver disorders but not in patients with high serum alkaline phosphatase due to non-liver disorders. In bile from bile duct drains collected during the first week after liver transplantation, free secretory component was found in concentrations of up to 33 mg/l, in vast excess of that found in bile from gallstone patients (up to 0.3 mg/l). Bile from gallstone patients but not from liver transplant patients produced proteolytic degradation of free secretory component when incubated in vitro. The finding of large amounts of free secretory component, the free cleaved fragment of the polymeric IgA receptor in human bile, further supports the existence of the blood to bile transhepatocytic pathway in humans. PMID- 1710955 TI - Doppler assessment of right ventricular filling dynamics during volume loading in ischemic heart disease. AB - To assess the effects of volume loading on right ventricular (RV) filling dynamics, the RV inflow pattern was recorded using pulsed Doppler echocardiography (PDE) during dextran infusion in 7 normal subjects (Group I) and 24 patients with ischemic heart disease. The patients with ischemic heart disease were divided into three groups according to the left ventricular (LV) and RV ejection fractions (EF). Group II consisted of 11 patients whose LVEF and RVEF exceeded 50%. Group III consisted of 7 patients whose LVEF was lower than 50% and RVEF was higher than 50%. Group IV was comprised of 6 patients whose LVEF and RVEF were lower than 50%. Peak flow velocity of the RV rapid filling wave in early diastole [R(T)] and that of the atrial contraction wave [A(T)] were measured, and the ratio of A(T) to R(T) [A(T)/R(T)] in each cardiac cycle was calculated. In some of the subjects, simultaneous right-sided cardiac catheterization was performed with a Swan-Ganz thermodilution catheter. The LV and RV function during volume loading were calculated according to the following formulae: delta Stroke volume index (SVI)/delta pulmonary artery wedge pressure (PAWP) and delta SVI/delta right atrial pressure (RAP), respectively. After dextran infusion, R(T) increased significantly in Groups I, II, and III, but not in Group IV. The A(T) and A(T)/R(T) ratio remained unchanged in all groups. The percent change of the R(T) correlated well with delta SVI/delta RAP (r = 0.56, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1710956 TI - The effect of iron deficiency and iron overload on the evolution of Chagas disease produced by three strains of Trypanosoma cruzi in CFW mice. AB - 1. CFW mice were fed either on control diet or on iron-deficient diet. 2. After 5 months the mice were infected with CL, Y or YuYu strain of Trypanosoma cruzi. 3. On the fifth day after the infection, the mice on control diet were divided in three groups: one group remained as controls, two groups were injected either with desferrioxamine or iron-dextran. 4. The severity of the disease was evaluated by parasitemia and mortality. 5. The experimental groups were compared with the infected group fed on the control diet. 6. In mice fed on the iron deficient diet, the disease was more severe for CL strain and less severe for Y and YuYu strains. 7. Treatment with desferrioxamine produced a less severe disease with YuYu strain and no difference with the other strains. 8. On Treatment with iron-dextran, the disease became more severe with Y and CL strains; no effect was observed with YuYu strain. 9. These findings may be due to intraspecific differences among the strains. PMID- 1710957 TI - A sensitive enzyme immunoassay for the quantitation of human C5a/C5a(desArg) anaphylatoxin using a monoclonal antibody with specificity for a neoepitope. AB - The direct quantitation of C5a/C5a(desArg) in human plasma was achieved by a specific and highly sensitive ELISA which is based on the neoepitope-specific monoclonal antibody C17/5. The error-prone removal of C5 from plasma prior to the assay is therefore not required. With a detection limit of 20 pg C5a/ml plasma, the sensitivity of this assay allowed to define normal ranges (1.94 +/- 1.49 ng C5a/nl, mean +/- SD) of C5a/C5a(desArg) in human blood plasma. This assay will also be applicable to the quantitation of C5a in specimens with low protein content where precipitation-based assays fail to accurately determine C5a. In addition, the mAb C17/5 turned out to efficiently block the binding of C5a/C5a (desArg) to its cellular receptor and therefore provides a valuable tool in the delineation of C5a effects in complex biologic systems in the presence of native C5, such as under in vivo conditions. PMID- 1710958 TI - DNA histogram debris theory and compensation. AB - In this paper we describe a theory of DNA histogram debris generation and compensation that can be applied to paraffin-embedded frozen tissue preparations. The theory predicts the distribution of fragments generated from single and multiple random sectioning of three-dimensional ellipsoids representing nuclei. The fragment distribution is assumed to be a major component of the underlying debris in DNA histograms. A comparison of S-phase fractions (SPF) from matched tissue prepared by frozen and formalin-fixed paraffin-embedded DNA methods demonstrates the usefulness of the theory. PMID- 1710959 TI - Method for kinetic analysis of drug-induced cell cycle perturbations. AB - A method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0, 1/3Ts, 2/3Ts, Ts, and Tc + TG1 recovery times, where Ts, TG1, Tc are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of 1-(2-chloroethyl)-1-nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 microgram/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase. PMID- 1710960 TI - Detection and analysis by dual-laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli. AB - Bacteriophage T4 DNA was detected and analyzed inside E. coli by dual-laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A-T- and G-C-rich regions of DNA, respectively. An exponentially-growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanol-fixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty-five minutes post-infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA-containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E. coli. A value of 35.6 +/- 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that dual laser flow cytometry can be used to study viral DNA inside the bacterial host. PMID- 1710961 TI - Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry. AB - 7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen. PMID- 1710962 TI - Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342. AB - By halogenation of methylfluorescein-diacetate (MFDA) or eosin-diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)-excitation system as required by bimane-based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37 degrees C, and fluorescence is sensitive to pretreatment with diethylmaleate but not to buthionine sulfoximine. In a dual-laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position. PMID- 1710963 TI - [The role of viruses in the induction of allergic encephalomyelitis]. PMID- 1710964 TI - Fibrillation potentials and positive sharp waves: are they the same? PMID- 1710965 TI - Recording single motor unit activity of human nasal muscles with surface electrodes: applications for respiration and speech. AB - A method is presented for recording nasal single motor unit (SMU) potentials from the skin surface using a 3-pole 'branched' bipolar electrode. Stable, high quality recordings of single motor unit activity were obtained for up to 3 h. Branched electrode arrays were capable of locating an SMU's maximal voltage point within 5 mm. We examined nasal SMU discharge patterns in relation to respiration in 9 adult humans. The majority of SMUs which discharged during quiet breathing began firing late in expiration and ceased firing in mid-inspiration, other SMUs discharged only during expiration, and a few fired continually with frequency modulation during breath cycles. With increased ventilation, new SMUs were recruited, and previously active SMUs increased the frequency and duration of their discharge. We examined the discharge of 13 units (5 adults) which discharged during speech but were never active during quiet or moderately increased breathing. Some of these SMUs fired during production of nasal consonants, and others were active for articulations involving facial movements (bilabial stops, labio-dental fricatives, and vowels produced with lip movement). By providing information about motor neuron recruitment which cannot be obtained from gross EMG recordings, surface recording of unit potentials may be useful in studying the central nervous control of the nasal upper airway, face, and neck for respiration and speech. PMID- 1710966 TI - Compound activity in sensory nerve fibers is related to intensity of sensation evoked by air-puff stimulation of the index finger in man. AB - An analysis of a population response recorded from the primary afferents was undertaken to study the neural signs of intensity coding in the peripheral somatosensory system. Short air-puff stimuli were applied to the volar aspect of the tip of the index finger to obtain both neural and psychophysical responses. The detection threshold (So) was first determined and 6 levels of stimulus intensity above threshold (So + 0.25 kg/cm2, So + 1.25 kg/cm2, So + 2.50 kg/cm2, So + 3.75 kg/cm2, So + 5.00 kg/cm2, and So + 6.25 kg/cm2) were adopted for magnitude estimation using the stimulus level of So + 2.50 kg/cm2 as the standard stimulus. The subject was asked to give a numerical estimate of the intensity of a series of stimuli randomly presented. Compound sensory nerve action potentials (SNAPs) were also recorded from surface electrodes over the median nerve at the wrist for the 6 stimulus intensities. Eight SNAP components (4 positive and 4 negative) were recorded within 10 msec following stimulation. A simple power function with an exponent of 0.85 provided an adequate description of the magnitude estimation function, as was verified by the high correlation coefficient (r = 0.89, P less than 0.001). Similarly, stimulus-amplitude functions of individual SNAP components were well represented by straight lines in double logarithmic plots. The function of the late N2-P3 component had the highest power exponent (0.80) and also the highest correlation coefficient (r = 0.59, P less than 0.001). The functions of other SNAP components had considerably lower power exponents with lower correlation coefficients. Thus, a mismatch between neural and psychophysical functions was obvious when individual neural functions were compared with the psychophysical function. Conversely, it was found likely that the total number or time-integral of peaks in the population response was a more pertinent parameter of neural activity and, thus, had a closer correlation with the psychophysical response. PMID- 1710967 TI - Variability of cortically evoked motor responses in multiple sclerosis. AB - The calculated central motor conduction time (CMCT), onset latency variability (expressed as the mean consecutive difference; MCD) and amplitude (expressed as percentage of maximum peripheral M wave size) of electromyographic (EMG) responses in the first dorsal interosseous (FDI) muscle following magnetic motor cortex stimulation were investigated in 20 normal subjects and 21 patients with multiple sclerosis (MS). EMG responses were present in all patients studied. CMCT was prolonged (greater than 8.1 msec; the mean CMCT for normals plus 3 S.D.) in 19 out of 42 muscles (12 patients). Onset latency variability was increased (greater than 1.1 msec; mean plus 3 S.D. for normals) in 20 out of 42 muscles (14 patients). Maximal response amplitudes varied between 5% and 67% and were not significantly different from the normal group (range 16-64%). In 3 patients, increased onset latency variability was the only neurophysiological abnormality. Prolonged CMCT was the sole abnormal finding in only 1 patient. Abnormally large onset latency variability was associated with the clinical finding of both impaired fine finger movements and increased finger jerks. Abnormal CMCT was associated with increased finger jerks only. This study confirms the findings of prolonged CMCT in multiple sclerosis. The additional finding of abnormal variability in response latencies which correlates with the clinical signs suggests that this variability may also be a useful measure of pyramidal tract function. PMID- 1710968 TI - Standardization of facilitation of compound muscle action potentials evoked by magnetic stimulation of the cortex. Results in healthy volunteers and in patients with multiple sclerosis. AB - To establish the importance of standardization of the facilitation of central motor conduction measured by magnetic stimulation we studied the effect of increasing voluntary muscle contraction on the central motor conduction time (CMCT) and motor evoked potential (MEP) amplitudes for 3 upper and 2 lower limb muscles. MEPs were elicited by magnetic stimulation of the cortex and the spinal roots. Muscle force was indirectly assessed from the integrated electrical muscle activity and expressed as the root mean square (RMS) and was varied from 0 to 40% of maximal activity. The central motor conduction time (CMCT) decreased during increasing muscle contraction, reaching constant values at approximately 10-20% RMSmax. Similarly, the increases of MEP amplitude tapered off at about the same RMS level. For each muscle an optimal RMS level was defined. The shortening of the CMCTs at the optimal RMS levels were: the brachial biceps, 3.4 msec; the radial carpal flexor of the wrist, 2.7 msec; the first dorsal interosseus muscle of the hand, 2.9 msec; the anterior tibial, 4.2 msec; and the abductor hallucis, 2.4 msec. The standardizing procedure was applied to 10 patients with multiple sclerosis. The stimulus thresholds were higher in these patients compared with those of the normals. Only the CMCT reduction of the BB was significantly larger (8.1 msec) than in the controls. Using standardized facilitation the diagnostic value of the amplitudes seems to be only a little less than that of the CMCTs. PMID- 1710969 TI - Motor evoked potentials (MEPs) in lacunar syndromes. AB - Motor evoked potentials (MEPs) evoked in the biceps, thenar and tibialis anterior muscles by electrical stimulation of the scalp and of the spinal regions were recorded in 32 patients with focal deficits due to minor cerebral ischemia of the lacunar type and in a control group. Somatosensory evoked potentials (SEPs) to median nerve stimulation were also recorded. The central motor conduction times (CMCTs) and the threshold intensities for eliciting MEPs in the relaxed muscles were significantly increased on the affected side. Central motor conduction, for at least one muscle, was altered in 18 patients. MEP abnormalities were related to pyramidal signs (though they could be observed also in a patient without any motor impairment) and occurred independently of a specific clinical picture or a radiologically confirmed lacunar lesion. SEPs were less frequently altered than MEPs. PMID- 1710970 TI - Age-dependent decline in motor evoked potential (MEP) amplitude: with a comment on changes in Parkinson's disease. AB - Peak-to-peak measurement of the maximum amplitude motor evoked potential (MAXMEP) elicited by 20 consecutive transcranial magnetic stimuli recorded from the contracting thenar and hypothenar muscles measured 9.8 +/- 2.0 mV and 7.25 +/- 2.9 mV respectively (P less than 0.01). The ratio of MAXMEP/CMAP measured 92.6 +/ 25.8% and 54.8 +/- 12.3% respectively (P less than 0.001). Repeat studies showed good individual reproducibility. Amplitudes declined linearly with age (r = 0.836 for thenar MAXMEP P less than 0.001). It is argued that MAXMEP related to age is more meaningful than the MEP/CMAP wave ratio and is proportional to the number of fast conducting cortical motor neurons excited. In 7/18 patients with Parkinson's disease (PD) MAXMEP was increased; in 2 other patients MAXMEP was decreased for their age. PMID- 1710971 TI - Modification of cortical somatosensory evoked potentials during tactile exploration and simple active and passive movements. AB - The present study compares the effects of different types of movement on median nerve somatosensory evoked potentials (SEPs) recorded from frontal, central and parietal electrodes. Test conditions included tactile exploratory movements, repetitive active and passive thumb movements and isometric contraction. All these conditions modified the SEPs in a similar manner. Parietal N20, P25, and N60, central P22 and N32, and frontal N25, N30 and P40 deflections were diminished, while later centro-parietal P40 and fronto-central N60 were unchanged. A small frontal P35 emerged during movement. The subcortical P14 was not changed in any of the conditions. The similar modulatory effects of simple active movements and of tactile exploration indicate that the modification of SEPs does not depend on the importance of proprioceptive feedback information for movement execution. As all modulatory effects were present also during passive movement, these observed effects are most likely to be caused by afferent occlusion in the ascending thalamo-cortical pathways or sensorimotor cortical cell populations. PMID- 1710972 TI - Measurement of the electric field induced into inhomogeneous volume conductors by magnetic coils: application to human spinal neurogeometry. AB - We measured the electric fields induced by round and figure "8" magnetic coils (MCs) in homogeneous and inhomogeneous volume conductors. In homogeneous media, the round MC held tangential (i.e., flat) to the volume conductor induced an annular electric field. When the round MC was held on-edge (i.e., orthogonal) to the volume conductor, the induced electric field consisted of two loops mainly parallel to the surface of the volume conductor and which approximated each other directly under the contacting edge of the MC. The tangentially oriented figure "8" MC similarly induced two electric field loops which approximated one another maximally under the region of the junction in its long axis. In a complex inhomogeneous volume conductor, such as a segment of human cervical-thoracic vertebral spine located eccentrically within a large cylindrical tank and submerged in isotonic saline, the direction of electric fields within the spinal canal and across the intervertebral neuroforamina was similar to that observed in the homogeneous volume conductor. However, in and near a single neuroforamen, the electric field and especially its first spatial derivative were markedly elevated compared to that recorded within the long central axis of the vertebral canal. Motor unit and compound muscle action potentials elicited in limb muscles by MC stimulation of human cervical spine confirmed predictions derived from the physical model. The predictions included: (1) absence of spinal cord stimulation compared to relative ease of nerve root stimulation by current that is most likely concentrated at the neuroforamina. When stimulating current is directed towards the periphery, the most likely low threshold site of stimulation is inferred to be just distal to the neuroforamina. It is emphasized that with supramaximal stimulation, more distal sites of excitation may occur; (2) invariant latency shifts at threshold intensities when moving the MC along the rostrocaudal axis of the cervical vertebral column; (3) significant effect (on motor unit activation thresholds) of the direction of induced current flow across the neuroforamina; (4) reduced stimulation when the targeted nerve roots are close to the null point of the electric field, i.e., between locations of high electric field intensity, of opposite polarity; and (5) relatively focal nerve root stimulation by the junction of a transversely orientated figure "8" MC, i.e., parallel to the nerve roots. PMID- 1710973 TI - The measurement of electric field, and the influence of surface charge, in magnetic stimulation. AB - A circular magnetic stimulator coil placed perpendicularly to the surface of a large uniform conductor induces surface charge. The resulting electrostatic field reduces the total electric field within the conductor to 58% of the value in the absence of surface charge. The properties of 3 kinds of probe for measuring the effect of a magnetic stimulator are considered. A short dipole electric field probe is the only one which correctly measures the total electric field, including the contribution from any surface charge. A search coil generally gives incorrect results, since it is insensitive to the electrostatic field. PMID- 1710974 TI - Multiple firing of motoneurones is produced by cortical stimulation but not by direct activation of descending motor tracts. AB - In the present report we have tested whether stimulation of the motor descending tracts at the brain-stem level could set up repetitive motor unit discharges in a similar manner to that described for motor cortical stimulation. We have seen that a large descending motor volley, evoked by brain-stem stimulation, cannot produce repetitive firing of motor units. Repetitive motoneurone firing is therefore produced by multiple excitatory volleys set up by single cortical shocks. PMID- 1710975 TI - Detection of beta-glucanase activity on various beta-1,3 and beta-1,4-glucans after native and denaturing polyacrylamide gel electrophoresis. AB - beta-Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various beta-1,3- and beta-1,4-glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley beta-glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for beta-1,3 linkages and Calcofluor White M2R for beta-1,4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed beta-(1,3)(1,4)-glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of beta-1,3-glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of beta-1,3-glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various beta-glucans displayed lysis bands migrating between 32 and 35 kDa. PMID- 1710976 TI - Electrophoretic characterization of posttranslational modifications of human parotid salivary alpha-amylase. AB - Human salivary alpha-amylase displays multiple bands upon native polyacrylamide gel electrophoresis. In fresh saliva, due to posttranslational modifications, a pattern of 5-6 isozymes is observed. The isozymes are designated 1-6, in the order of increasing anodal mobility. As a result of the development of a rapid and sensitive electrophoresis system, with markedly higher resolution than previously reported, we concluded that a previously proposed model (Karn et al., Biochem. Genet. 1973, 10, 341-350) is inadequate to explain the origin of the various bands. We propose an alternative model that fits in with our new and previously made observations. According to this model, band 2 is the primary gene product and band 1 is its glycosylated counterpart--with only one neutral oligosaccharide present on each molecule. Band 3 originates from band 1 by the transialidase-catalyzed incorporation of sialic acid into the biantennary chain. Bands 4 and 6 originate from bands 2 and 4, respectively, by deamidation; band 5 is the deamidation product of amylase with an acidic oligosaccharide (band 3). Only a minor part of band 3 consists of the deamidation product of band 1. Peptide Asn-Gly-Ser (residues 427-429) is the most probable candidate for glycosylation; literature data suggests that deamidation occurs in the stretch Glu-Asn-Gly-Lys-Asp (residues 364-368) and Asn-Gly-Asn-Cys (residues 474-477). Both glycosylation and deamidation might play a role in the clearance of amylase from the systemic circulation. The electrophoresis system described is a powerful tool to determine amylase isozyme distributions in health and disease, especially for the screening of alterations seen in ectopically produced amylase. PMID- 1710977 TI - Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution. AB - P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5' mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule. PMID- 1710978 TI - Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations. AB - African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies. PMID- 1710980 TI - In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells. AB - The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo. PMID- 1710979 TI - The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. AB - Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both. PMID- 1710981 TI - Specific expression of the tobacco Tnt1 retrotransposon in protoplasts. AB - The Tnt1 transposable element of tobacco belongs to the retrotransposon family and shares the structural features of viral retroelements including two long terminal repeats (LTRs) which are known to contain promoter regions. We show that two Tnt1 RNAs of 5.2 and 6.5 kb can be found. The 5.2 kb RNA matches with the size of the Tnt1 elements so far isolated (5.3 kb), whilst the evidence suggests that the 6.5 kb RNA could be a chimaeric RNA initiated in a gene in which Tnt1 has inserted. The Tnt1 5.2 kb RNA starts in the LTR, and the LTR can promote the expression of a translational LTR-beta-glucuronidase (GUS) fusion at a high level in transient expression assays. The Tnt1 5.2 kb RNA and the LTR-GUS fusion of transgenic tobacco plants are specifically expressed in leaf-derived protoplasts whereas they are not expressed in leaf tissue. The 5.2 kb RNA is also transcribed at low levels in roots. This RNA is induced after 2 h of maceration in the protoplast isolation medium, and its level declines rapidly after protoplast isolation. The induction requires only the presence of cell wall hydrolases, and is independent of wounding and plasmolysis. The induction of Tnt1 expression is not mediated by typical oligosaccharide elicitors released from the cell wall known to mediate defense gene responses. Tnt1 transcription features provide a first example of tissue culture-induced mutagenesis in plants and a molecular basis for some of the somaclonal variation events. PMID- 1710982 TI - An indicator gene for detection of germline retrotransposition in transgenic Drosophila demonstrates RNA-mediated transposition of the LINE I element. AB - We have marked a cloned Drosophila transposable element--the I element--with an engineered neomycin-containing indicator gene, whose expression is conditioned by passage of the transposon through an RNA intermediate. Mobility of the marked element introduced into Drosophila as a transgene could be detected in vivo, upon in toto selection of developing embryos on G418-containing medium. For resistant individuals, Southern blot analysis and nucleotide sequencing after PCR amplification disclosed transposition of the marked element into new loci, with target site duplications and splicing out of the intron in the indicator gene. It demonstrates that the I element, which is closely related to the widespread mammalian LINEs, transposes through an RNA intermediate, as up to now only conjectured from sequence singularities of this class of 'non-viral retrotransposons'. The developed indicator gene provides a potent new genetic tool for detection and quantitative analysis of retrotransposon mobility and its regulation as it occurs in vivo. PMID- 1710983 TI - Isolation and characterization of cDNAs encoding viscotoxins of mistletoe (Viscum album). AB - Viscotoxins have been isolated from leaf homogenates of European mistletoe (Viscum album L.) and purified to apparent homogeneity. Antisera raised against these polypeptides were used to screen a cDNA expression library in lambda gt11. Two positive clones have been isolated, one encoding a full-length preprotein of viscotoxin A3 and the other encoding the precursor of viscotoxin B. Besides the viscotoxin domain the precursor contained a signal sequence and an acidic polypeptide domain. Similar higher molecular mass precursor proteins have been described for thionins of leaves and seeds of barley. Even though the acidic part of the viscotoxin precursor is much shorter than the corresponding domain of the precursors of the leaf and seed thionins of barley, both the negative charge and the number and the relative position of cysteine residues have been conserved within the acidic domain. This result is consistent with our proposal that the acidic domain of the thionin precursor may play an important role in keeping the thionin inactive within the plant cell. PMID- 1710984 TI - Mechanism of the permissive action of dexamethasone on the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes. AB - Rat hepatocytes were cultured for 24 h in the presence or absence of 100 nM dexamethasone (DX). After a medium change, phosphoenolpyruvate carboxykinase (PCK) was induced by addition of glucagon at different concentrations, from physiological 0.1 nM to hyperphysiological 10 nM, again in the presence or absence of 100 nM dexamethasone. 1. With dexamethasone addition during the culture and induction phase (DX+/+), 10 nM glucagon increased PCK mRNA abundance (Northern blot analysis) and activity (in vitro translation) synchronously to the same extent with maxima after 2 h and PCK enzyme activity after a time lag with a maximum after 6 h. The total detectable PCK mRNA amount was apparently also translationally active. 10 microM N6,2'-O-dibutyryladenosine 3',5' (cyclic)phosphate (Bt2cAMP) as the second messenger had essentially the same effect as 10 nM glucagon. 2. In the absence of dexamethasone during the preculture and the induction phase (DX-/-), 10 nM glucagon and 10 microM Bt2cAMP could enhance PCK mRNA only about half-maximally. Glucagon or dexamethasone added alone in physiological concentrations of 0.1 nM and 100 nM, respectively, were unable to increase PCK mRNA. However, treatment of the cells with dexamethasone also enabled 0.1 nM glucagon to enhance PCK mRNA to a maximum after 2 h, independent of the presence of dexamethasone during the induction period (DX+/+ and DX+/- cells). Thus, dexamethasone was a permissive agent in that it shifted the sensitivity of the cells towards glucagon into the physiological concentration range. 3. In the presence of dexamethasone during the culture and induction phase (DX+/+) 0.1 nM glucagon maximally enhanced the transcription of the PCK gene (nuclear run on) fourfold after 30 min; in the absence of dexamethasone during both phases (DX-/-) glucagon was without any effect. The overall transcriptional rate was not significantly different in cells with and without dexamethasone during the culture and induction phase (DX+/+ vs. DX-/-). Thus, dexamethasone acted permissively mainly on the transcription of the PCK gene. 4. With culture in the presence of dexamethasone over decreasing periods of time, 1 nM glucagon could induce submaximal PCK mRNA amounts already after 1-3 h steroid culture. This restitution by dexamethasone of the PCK mRNA inducibility by glucagon was inhibited by cycloheximide. This suggested that ongoing protein synthesis was required for the permissive action of dexamethasone on the expression of the PCK gene. The results allow the following conclusions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1710985 TI - Rat liver dimethylglycine dehydrogenase. Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone. AB - Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8 alpha position to N pi of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) J. Biol. Chem. 259, 12475-12480]. Subcellular fractionation of [14C]riboflavin-labelled hepatocytes and immunoprecipitation with Me2GlyDH-specific antiserum identified a [14C]riboflavin labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [35S]Met-labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction. These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD-X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) Methods Enzymol. 154, 3-28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of [35S]Met-labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease-recognition site Arg( 2)-Ala(-1)-Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH [Wittwer, A.J. & Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108], and a 43-amino-acid leader peptide. The N-terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor. PMID- 1710986 TI - The inhibitory action of okadaic acid on mechanical responses in guinea-pig vas deferens. AB - Okadaic acid and nifedipine inhibited contractions induced by noradrenaline (NA), KCl and ATP in guinea-pig vas deferens. NA, KCl and ATP induced initial spike like changes followed by a sustained increase in cytosolic Ca2+ levels ([Ca2+]cyt) and tension. Okadaic acid inhibited the sustained increments in [Ca2+]cyt and muscle tension due to NA and ATP more than the initial spike-like changes, whereas nifedipine more strongly inhibited the initial spike changes. Okadaic acid also inhibited the KCl-induced contraction with only a small inhibition of the stimulated [Ca2+]cyt. By contrast, nifedipine (10(-7) M) inhibited the increments in both [Ca2+]cyt and muscle tension due to KCl. Okadaic acid markedly inhibited the maximal contractile response to Bay K 8644 but nifedipine only shifted the response curve to the right without affecting the maximum responses. In a Ca2(+)-free medium containing EGTA and nifedipine, okadaic acid did not inhibit the residual phasic contractile response to NA (10( 4) M) but the contractile response to a subsequent addition of Ca2+ (1.2 mM) was suppressed. These results suggest that, in guinea-pig vas deferens, okadaic acid has an inhibitory effect on smooth muscle contraction but not on the intracellular Ca2+ mobilization. The inhibitory effect may be due to the inhibition of Ca2+ influx and the possible interference of contractile elements. PMID- 1710987 TI - Endothelin receptor: a profoundly desensitizing receptor expressed in Xenopus oocytes. AB - Xenopus oocyte expression studies can help to define the physiological properties of a receptor and can aid in receptor cloning. Expression of an endothelin receptor in oocytes injected with brain mRNA is inferred from depolarizations induced by several endothelin-related peptides. The response is abolished by intracellular EGTA injection but not in the absence of extracellular Ca2+. It is not present in non-injected oocytes, and reverses at a potential indicating that it is mediated by an increased Cl-1 conductance. Responses display striking, long lasting desensitization. The expressed endothelin receptor thus displays properties characteristic of several receptors coupled to changes in phosphoinositide turnover, several of which have been successfully cloned using this response as a reporter. PMID- 1710988 TI - Comparison of the effects of endothelin and Bay k 8644 on cardiohemodynamics in anesthetized pigs. AB - The effects of endothelin on cardiohemodynamics in anesthetized open-chest pigs were compared with those of the Ca2+ channel agonist Bay k 8644. I.v. administration of endothelin (0.1-1 microgram/kg) dose relatedly decreased coronary blood flow velocity (CFV), cardiac output, the maximal rate of rise in left ventricular pressure (LV dP/dtmax) and heart rate, and increased systolic and diastolic blood pressure and systemic vascular resistance. Bay k 8644 (0.1-30 micrograms/kg) showed similar effects on cardiohemodynamics, except that it caused a substantial increase in LV dP/dtmax and did not change stroke volume. I.c. administration of endothelin (0.1-1 microgram) into the left circumflex coronary artery (LCX) produced a dose-dependent and sustained decrease in CFV followed by a marked increase in the ST segment of epicardial electrocardiograms and a depression in myocardial shortening in the region supplied by LCX. An increase in left ventricular end-diastolic pressure and a reduction in LV dP/dtmax were also observed after endothelin. In contrast, Bay k 8644 caused a transient decrease in CFV and an increase in LV dP/dtmax, without causing any changes indicative of myocardial ischemia. Although the reduction in CFV after i.c. endothelin was not affected by i.v. infusion of nicardipine (1 microgram/kg per min), the reduction in CFV observed after i.c. Bay k 8644 was significantly suppressed (P less than 0.05). We conclude that endothelin is a more potent coronary vasoconstrictor than Bay k 8644 and provokes marked myocardial ischemia. PMID- 1710989 TI - Effects of porcine galanin on the mesenteric microcirculation and arteriolar smooth muscle in the rat. AB - The actions of porcine galanin on the mesenteric circulation at the arteriolar level and on the isolated mesenteric small artery were studied in the rat. Male Wistar rats were anesthetized then laparotomized. Microscopic observation of the mesenteric microvascular area was made with a video camera and changes in arteriolar diameter were measured continuously with a width analyzer. Galanin (0.03-300 pmol), given intra-arterially into the mesenteric arteriole, caused an intermittent interruption of blood flow within 40 s and finally stopped the blood flow within a few minutes. The diameter of arterioles was not changed or was slightly widened. Galanin also relaxed the preconstricted small mesenteric artery in an endothelium-independent manner. Furthermore, the relaxing action of galanin was not antagonized by glibenclamide, indicating that activation of ATP-sensitive K+ channels was not involved. The present results suggest that galanin plays a modulatory role in the mesenteric circulation. PMID- 1710990 TI - Effects of idazoxan on dorsal raphe 5-hydroxytryptamine neuronal function. AB - The effects of the alpha 2-adrenoceptor antagonist idazoxan on 5 hydroxytryptamine (5-HT) neuronal firing and release have been investigated. Idazoxan, administered i.v. (10 micrograms/kg and 0.5 mg/kg) increased dorsal raphe nucleus (DRN)-5-HT neuronal firing rate in a dose-dependent fashion. At the higher dose, a voltammetric study revealed increases in extracellular 5-HT and 5 hydroxyindole acetic acid (5-HIAA) levels, there was no effect with the lower dose. Intra-raphe administration of idazoxan (1 ng) also elevated the firing rate of 5-HT neurones in the dorsal raphe, suggesting that idazoxan may produce the increase in firing by a direct effect in the DRN. However, microiontophoretic application of idazoxan did not increase the firing rate of 5-HT neurones in the DRN. Thus the increase in the firing rate of 5-HT neurones in the DRN observed with systemic and local administration of idazoxan is probably not due to a direct action of idazoxan on the 5-HT neurone. Possibly the idazoxan acted at alpha 2-adrenoceptors located on noradrenergic terminals thus stimulating noradrenaline release and consequently increased 5-HT activity. Chronic administration of idazoxan (0.8 mg/kg per h for 14 days), using osmotic mini pumps, caused an elevation in basal firing rate and an attenuation of the inhibitory response of DRN 5-HT neurones to the 5-HT1A agonist, 8-hydroxy-2-(di-n propylamino) tetralin (8-OHDPAT) (10 micrograms/kg i.v.). This finding suggests that chronic infusion with idazoxan leads to desensitisation of the 5-HT1A somatodendritic autoreceptor. PMID- 1710991 TI - Aspirin blocks 5-azacytidine- and hydroxyurea-induced changes in hemoglobin proportions in adult rats. AB - The effects of 5-azacytidine and hydroxyurea on their independent ability to change adult hemoglobin proportions toward newborn proportions in adult rats were examined. The results revealed that both the chemotherapeutic agents were capable of switching certain hemoglobin components toward newborn values and required similar time-span to express their actions. However, the switching effect of these drugs was totally lost if aspirin was simultaneously administered into the rats, reflecting the need for concurrent prostaglandin synthesis. PMID- 1710992 TI - Opioid and neurokinin activities of substance P fragments and their analogs. AB - Newly developed substance P (SP) analogs with altered N-terminal sequences which equalize the lipophilicity of the N-terminal and C-terminal elements and of their fusion product were examined using i.t. injection in mice. I.t. injection of either the full length analog or the C-terminal hexapeptide (CP) produced biting and scratching behavior similar to that elicited by SP. SPF was approximately 5 fold and CP 14-fold less potent than native SP. The N-terminal peptide (NP) was inactive by itself but inhibited CP-elicited behavior. Naloxone antagonized this action of NP and shifted the SPF dose-response curve 4-fold to the left. However, naloxone had no effect on the action of CP or on the action of any of the native neurokinins. The results are consistent with the hypothesis that N- and C terminal analogs of SP can have opioid and SP-like actions, respectively, in the CNS of rodents. Furthermore, analogs of SP which include at least the terminal tetrapeptide retain neurokinin activity. PMID- 1710994 TI - Localization of 5S DNA by in situ hybridization in metaphase chromosomes of Vicia faba L. AB - The tritium-labelled cloned 5S DNA from Lupinus luteus was used for localization of 5S RNA genes in Vicia faba subsp. minor metaphase chromosomes. In situ hybridization sites were found to be localized in chromosomes I and VI. In chromosome I the probe hybridized to the region adjoining NOR whereas in chromosome VI silver grains were found in the median part of the long arm. After prolonged exposure the autoradiographic grains expanded in the proximal part of that chromosome arm. PMID- 1710993 TI - Calcitonin and somatostatin immunoreactive cells are present in human bone marrow and bone marrow cells are responsive to calcitonin and somatostatin. AB - The presence of calcitonin and somatostatin immunoreactive cells has been determined in semithin sections of human bone and bone marrow samples by immunocytochemical techniques. Calcitonin and somatostatin immunoreactive cells were demonstrated at the interface between bone and bone marrow, in close contact with vessels. It has also been shown that exogenous calcitonin and somatostatin affect the ligand-receptor internalisation, depress the incorporation of 3H thymidine into acid-insoluble material, and exert opposite effects on 35S methionine incorporation to proteins of bone marrow cells in vitro. The evidence for calcium-dependent action of calcitonin and somatostatin on bone marrow cells has been presented. The regulatory role of calcitonin and somatostatin in bone marrow hemopoiesis is suggested and the implication of these findings for local hormonal regulation of hemopoiesis and bone structure is discussed. PMID- 1710995 TI - The effect of the typical peptidases (proteases) inhibitors on the degradation of Glp6 (125I) Tyr8SP6-11 hexapeptide in the nuclear and synaptosomal fraction of the cortex and hippocampus of rat brain. AB - The main peptidase PN/cutting Tyr8-Gly9 or Gly9-Leu10 bond (of sequence Glp6-Phe7 Tyr8-Gly9-Leu10-MetNH2) seems to be, at least in part, cysteine type enzyme. Cutting of Phe7-Tyr8 bond with PC enzyme is apparently negligible. Further degradation of labelled PN products seems to be accomplished with PI, being serine enzyme at least in part. Metalloenzymes, including "enkephalinase", seem to be of minor importance in hexapeptide degradation, at least in its very low concentration. Some typical inhibitors enhance the degradation what might be explained assuming that products of action of one peptidase strongly inhibit the other peptidase's action. Namely, products of PN and PI seem to inhibit PC except the hippocampal synaptosomes where the opposite is true. PMID- 1710996 TI - Malignant trophoblastic cell tumor localized to the fallopian tube: a case report. PMID- 1710997 TI - Neuropeptides synthesised in the anterior pituitary: possible paracrine role. PMID- 1710998 TI - Human endometrial adenocarcinoma cell lines HEC 1B and KLE secrete insulin-like growth factor binding protein-1 and contain IGF-I receptors. AB - The production of insulin-like growth factor binding proteins (IGFBP) with special reference to human IGFBP-1 was evaluated in five endometrial adenocarcinoma cell lines (HEC 1A, HEC 1B, KLE, RL952 and AN3CA) in continuous culture. Two of the cell lines (HEC 1B and KLE) produced immunoreactive IGFBP-1. The production was inhibited by clomiphene and progesterone, whereas estrogen, cortisol and insulin had no effect on IGFBP-1 secretion. The two cell lines which secreted immunoreactive IGFBP-1 also had IGF-I receptors, whereas the cell lines RL952 and AN3CA, not producing IGFBP-1, had no saturable IGF membrane binding sites. IGF-I receptor binding to HEC 1B and KLE cells was inhibited in the presence of purified IGFBP-1. In addition to IGFBP-1, the endometrial cancer cells secreted several other forms of IGFBPs as determined by cross-linking. Immunoprecipitation of IGF-BP complexes with a polyclonal antiserum against IGFBP 3 indicated that all cell lines secreted binding proteins antigenically related to IGFBP-3 with molecular weights ranging from 20 to 39 kDa. PMID- 1710999 TI - The effects of cocaine upon 5-hydroxyindole levels of the maturing mouse brain. AB - 1. Mice at various ages between birth and adulthood were injected with cocaine hydrochloride (30 mg/kg) and then their brains were assayed for 5 hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA). 2. Changes in the levels of both metabolites were in keeping with 5-HT uptake inhibition at all ages. PMID- 1711000 TI - Effects of interferon on intrahepatic human leukocyte antigens and lymphocyte subsets in patients with chronic hepatitis B and C. AB - We investigated the effects of interferon therapy on hepatocyte human leukocyte antigen class I and class II antigen expression and intrahepatic lymphocyte subsets in patients with chronic viral hepatitis B (n = 11) and C (n = 10). Interferon-alpha was administered intramuscularly in doses ranging from 3 to 18 million international units daily for 4 wk. Liver biopsy specimens were obtained just before and immediately after treatment, and the specimens were stained by the indirect immunoperoxidase method for evaluation of human leukocyte antigen expression and lymphocyte subsets. Before therapy, no significant difference was noted between hepatitis B and C in human leukocyte antigen class I antigen expression on hepatocytes or in the lymphocyte subsets in the intralobular and portal areas. After interferon-alpha treatment, hepatocyte expression of human leukocyte antigen class I antigens and serum beta 2-microglobulin levels were virtually unchanged in chronic viral hepatitis C patients, but both were increased in chronic viral hepatitis B patients. Human leukocyte antigen class II antigens were not expressed during treatment. The mean number of intralobular CD3+ and CD8+ cells and the mean serum ALT level decreased significantly in chronic viral hepatitis C patients (p less than 0.05) but not in chronic viral hepatitis B patients. The mean number of intralobular CD4+ cells was unaffected by interferon therapy in both groups. In all 21 patients, the changes in CD8+ cell numbers paralleled the changes in serum ALT levels. Our findings suggest that T-cell cytotoxicity may play an important role in hepatocyte damage in both chronic viral hepatitis C and chronic viral hepatitis B and that the response to interferon-alpha differs in these two types of hepatitis. PMID- 1711001 TI - Effect of interferon administration on serum hepatitis C virus RNA in patients with chronic hepatitis C. AB - Hepatitis C virus RNA as detected by reverse transcription and nested polymerase chain reaction was monitored in 16 patients with chronic hepatitis C treated with interferon. Hepatitis C virus RNA became undetectable after 4 to 8 wk of interferon administration in 13 of the 16 patients. During 6 mo of follow-up, 5 of the 13 patients who became negative for hepatitis C virus RNA after interferon administration remained negative, and all five continued to have normal ALT levels. Repeat liver biopsy in these five patients revealed histological improvement. Antibody to hepatitis C virus, which was initially positive in all treated patients, fell to undetectable levels in three of the five patients. In contrast, aminotransferase levels rose again in all eight patients who had become hepatitis C virus RNA negative but had again exhibited hepatitis C virus RNA after completion of therapy. In 16 untreated patients, hepatitis C virus RNA remained detectable. These results indicate that detection of hepatitis C virus RNA may be useful as a marker of viral replication in chronic hepatitis C; they also suggest that interferon should again be administered to patients who become hepatitis C virus RNA negative on treatment but again exhibit this marker of viral replication when treatment is stopped. PMID- 1711002 TI - Eosinophil cationic protein's role in human hepatic allograft rejection. AB - Although it is known that eosinophils consistently infiltrate rejecting human liver allografts, their function is unknown. Infiltrating eosinophils can release a cytotoxic substance, eosinophil cationic protein. Furthermore, eosinophil cationic protein may be identified in biopsy specimens using immunoperoxidase staining of an eosinophil cationic protein-specific monoclonal antibody. To study a possible effector role of eosinophils in rejecting liver allografts, 38 serial allograft biopsy specimens from 12 patients with acute rejection, 54 biopsy specimens from 11 patients with allograft dysfunction caused by other causes and 22 biopsy specimens from 8 patients without allograft dysfunction were stained for extracellular eosinophil cationic protein. In addition, the absolute blood eosinophil counts and the portal tract eosinophil percent of the total number of portal tract inflammatory cells were tabulated in these patients until 30 days after transplantation. The blood absolute eosinophil count, portal tract eosinophil percent and incidence of positive extracellular eosinophil cationic protein staining were significantly increased in patients with rejection compared with patients with dysfunction from other causes (p less than 0.05 to 0.005, t test). Furthermore, each of these parameters predicted rejection with excellent sensitivity (75% to 92%) and specificity (91% to 100%). Of patients with rejection, 59% had significant elevation of all three parameters before or during rejection, and 92% had at least two parameters elevated. Conversely, of the patients with dysfunction from other causes, 0% had elevations of any parameter. The recognized cytotoxic properties of eosinophil cationic protein and its almost exclusive presence, along with eosinophils in the blood and allograft biopsy specimens of patients during acute rejection, support the role of the eosinophil as an effector cell in tissue injury during acute liver allograft rejection. PMID- 1711003 TI - Human fetal hepatocytes respond to inflammatory mediators and excrete bile. AB - Under strict observation of the ethical guidelines of the 1975 Declaration of Helsinki Human Research Committee, primary hepatocyte cultures were prepared from second-trimester fetal liver specimens. We have shown for the first time that fetal hepatocytes have the capacity to produce an acute-phase response on treatment with inflammatory mediators. Addition of interleukin-6 to the cultures resulted in strong induction of C-reactive protein and alpha-1-antichymotrypsin expression, whereas albumin expression was repressed. In contrast to interleukin 6, transforming growth factor-beta did not induce C-reactive protein expression. However, as in adult hepatocytes, fetal cells responded to transforming growth factor-beta by reduced albumin synthesis. We were able to show by virtue of fluorescein excretion into sealed clefts that fetal hepatocytes have the functional capacity to form bile. Our findings indicate that second-trimester hepatocytes can be regarded as fairly mature liver cells. PMID- 1711004 TI - A rat model of acute liver necrosis induced by a monoclonal antibody to liver specific antigen and complement. AB - Acute massive hepatic injury was induced in rats by a monoclonal antibody against a rat liver-specific membrane antigen, and its histological characteristics were investigated. A single intravenous injection of murine ascites containing a monoclonal antibody produced numerous hemorrhagic foci of degenerated and necrotic liver cells predominantly in zones 1 (the periportal area) and 2 (the area of transition between the periportal zone and the perivenular zone) of the liver lobule within 10 min. Massive hepatocellular necroses were observed 1 hr later, but no inflammatory cell infiltration occurred in and around the necrotic foci. Immunohistological study demonstrated marked deposition of the third component of the complement system in the necrotic area. Serum complement activity was sharply decreased immediately after the injection of the antibody, suggesting that the hepatic necrosis is ascribable to a complement-mediated immune attack on the liver cell membrane induced by the antigen-antibody reaction. The hepatic necrosis in response to monoclonal-antibody injection did not progress to a chronic disease and healed almost completely, changing to scar tissues within 2 wk. Although it is not clear whether this hepatic injury has any clinical relevance, this antibody/complement model may be useful for investigating the cause and therapy of hepatic diseases such as fulminant hepatitis. PMID- 1711005 TI - FK 506--a promise of good things to come? PMID- 1711006 TI - Mapping of the T and B cell epitopes of the Mycobacterium bovis protein, MPB70. AB - A clone coding for the entire gene for the Mycobacterium bovis protein antigen MPB70 was used to produce a series of overlapping subclones by making a series of deletions from the 3' end of the gene. The subclones expressed incomplete MPB70 proteins as fusions with glutathione-S-transferase. The insert DNA was sequenced to determine the extent of the deletion and the proteins expressed by the clones were examined for the presence of T cell and B cell epitopes. T cell epitopes were mapped by measuring the ability of recombinant antigens to stimulate gamma interferon (gamma-IFN) production in a whole blood culture system. gamma-IFN production was measured using a sandwich enzyme immunoassay specific for bovine gamma-IFN. B cell epitopes were mapped with a series of anti-MPB70 monoclonal antibodies using an indirect enzyme immunoassay. PMID- 1711007 TI - Development of human anti-murine antibody (HAMA) response in patients. AB - Human anti-mouse antibody (HAMA) response was determined in the serum of 67 patients who received subcutaneously administered radiolabelled murine monoclonal antibodies (MoAb) (50 micrograms-3 mg) for immunolymphoscintigraphy and of 10 patients with advanced colorectal cancer who received murine MoAb-N-acetyl melphalan (MoAb-N-AcMEL) conjugates (amount of MoAb ranged from 120 mg/m2 body surface area to 1000 mg/m2 body surface area) as therapy. A pre-existing low level of apparent human anti-mouse antibody reactivity could be detected in the serum of normal subjects and patients prior to administration of murine MoAb. Subcutaneous administration of low doses of murine MoAb, as used in immunolymphoscintigraphy, was associated with a low incidence (4/67 or 6%) of elevated HAMA response; the use of F(ab')2 fragments was associated with the development of elevated HAMA response in one of three patients. By contrast, therapy with hepatic artery infusion of murine MoAb-N-AcMEL conjugates in three repetitive daily doses (each infusion lasting 2 h) elicited elevated HAMA responses in 10/10 (100%) patients, usually 1-3 weeks after the start of therapy. The HAMA response of patients in the therapy group was higher than those in the immunolymphoscintigraphy study and the use of steroids did not prevent the development of the HAMA response. Further administration of MoAb-N-AcMEL conjugates to a patient, who had already developed HAMA, led to 'serum sickness' type symptoms and a transient reduction in the HAMA titres. The elevated HAMA response was polyclonal, containing increased levels of both immunoglobulin M and G (IgM and IgG) and was directed against mouse-specific determinant, the isotype (presumed to be the Fc portion), the F(ab')2 and the 'idiotype' of mouse immunoglobulins. PMID- 1711008 TI - Nomenclature for factors of the HLA system, 1990. The WHO Nomenclature Committee for factors of the HLA system. PMID- 1711009 TI - Molecular cloning of the gene coding for the human T cell differentiation antigen CD7. AB - The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites -122 base pairs (bp) to -38 bp from ATG translation initiation site were demonstrated. The promoter region had high G + C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs. PMID- 1711010 TI - HLA antigens and risk for development of pemphigus foliaceus (fogo selvagem) in endemic areas of Brazil. AB - Endemic pemphigus foliaceus (EPF), is an autoimmune disease associated with production of IgG antibodies against epidermal antigens. We have tested 38 patients and 50 control subjects living in endemic areas to investigate whether HLA genes are associated with host factors that determine whether or not exposed individuals will develop this disease. A variant of HLA-DR1, an antigen common in Blacks (DRB1*0102), was found to be the main susceptibility factor (relative risk = 7.3, P less than 0.0002). Two amino acids, in positions 85 and 86 of DRB1, distinguish DRB1*0102 from DRB1*0101. These residues appear to be involved in the formation of a functional epitope that causes T cell recognition and determines disease susceptibility. Moreover, subjects having DQw2 did not develop the disease, while the frequency of DQw2 in controls was 22% (RR = 0.04, P less than 0.006). Thus HLA genes appear to play a crucial role in the response to an environmental factor which in this setting frequently leads to the development of autoimmune disease. An HLA-DQ allele, DQw2, appears to be associated with factors that prevent the development of the disease in exposed individuals. PMID- 1711011 TI - The contribution of beta-strand residues to serologic epitopes on the A beta k polypeptide. PMID- 1711012 TI - Double-labeling in situ hybridization analysis of mRNAs for carbonic anhydrase II and myelin basic protein: expression in developing cultured glial cells. AB - We applied in situ hybridization to analyze the location and the developmental changes in the distribution of the transcripts for carbonic anhydrase II (CAII) and myelin basic protein (MBP) in mouse primary cultured glial cells. Both mRNAs were localized to the oligodendrocyte using double-labeling in situ hybridization. No evidence for CAII transcripts in astrocytes was obtained, indicating that CAII is expressed only by oligodendrocytes in normal rodent glia. As early as 48 h after plating, CAII and MBP mRNAs are present in a few, small round cells. Message is present 2-4 days before levels of these proteins can be detected in similar primary glial cultures. The intensity of labeling for MBP and CAII mRNA positive cells increases significantly during the second week but then decreases after the end of the third week. Only the oligodendrocyte perikaryon and a few processes are positive during the first week. In contrast, at 14 days, a large number of cell processes in addition to the cell bodies are heavily stained for both mRNAs. Both mRNAs could be detected far away from the cell body, up to 250 microns in some cell processes. Some segments on a cell process accumulate higher levels of mRNA than other areas. These areas may correspond to the accumulation of free ribosomes and to starting points for the membrane sheets elaborated by cultured oligodendrocytes. The developmental profile for timing and distribution of these two messages mimics closely their in situ pattern. PMID- 1711013 TI - Identification of B- and T-cell epitopes within the MTP40 protein of Mycobacterium tuberculosis and their correlation with the disease course. AB - Synthetic peptides derived from the amino acid sequence of MTP40, a recently characterized Mycobacterium tuberculosis protein, were tested by two different immunological assays in 91 individuals. For the purposes of this study, the population was distributed in four groups: active tuberculosis (TBC) patients with elevated bacillus loads (BK+), active TBC patients with low bacillus loads (BK-), healthy individuals living in the same household with tuberculous patients (HH), and normal individuals, who had presumably never been in contact with the bacilli (control). We found that T cells of individuals belonging to the HH group showed the highest and most frequent recognition of these peptides in a T-cell proliferation assay, while their antibodies showed the lowest recognition of these peptides when tested by enzyme-linked immunosorbent assay. In contrast, TBC patients revealed an inverse pattern of immune response. Interestingly, one of these peptides (P7) was recognized by T cells of 64% of the HH individuals and by 4.5% of normal donors. Another peptide (P4) was recognized by 55% of sera from BK+ patients and by 5.5% of normal donors. The results presented here indicate the existence of T- and B-cell epitopes within the MTP40 protein. Given the particular recognition pattern of this protein, added to the fact that it appears to be a species-specific antigen of M. tuberculosis, a detailed study of the immune response to it may be useful in the design of more accurate diagnostic tests and an improved vaccine against human TBC. PMID- 1711014 TI - Characterization and immunogenicity of EX880, a Salmonella typhi Ty21a-based clone which produces Vibrio cholerae O antigen. AB - EX645 is a derivative of Salmonella typhi Ty21a which carries a plasmid specifying production of Vibrio cholerae O antigen. When cultured with exogenous galactose to overcome the galE defect of the vector, EX645 also synthesizes S. typhi O antigen, and this can result in the masking of the shorter V. cholerae O antigen on the bacterial surface. To determine whether the potential for such masking at least partly underlies the inconsistency of anti-V. cholerae responses elicited by EX645, a derivative of this strain has been isolated, characterized, and tested for immunogenicity in human volunteers. EX880 has an rfb defect which prevents synthesis of S. typhi O antigen, and consequently V. cholerae O antigen is still detectable on the surface of the clone following growth in the presence of galactose. Compared with EX645, EX880 more consistently elicited significant rises in serum bactericidal antibody levels, although individual responses within a cohort still varied widely. PMID- 1711015 TI - Failure of recombinant vaccinia viruses expressing Plasmodium falciparum antigens to protect Saimiri monkeys against malaria. AB - Saimiri sciurus monkeys were immunized at multiple sites with recombinant vaccinia viruses expressing Plasmodium falciparum antigen genes and boosted 4 weeks later. Control monkeys were immunized with a thymidine kinase-negative vaccinia virus mutant. Two weeks later, all of the monkeys were challenged by intravenous inoculation of P. falciparum (Indochina strain) parasites. A group of unimmunized monkeys was challenged in parallel. All of the monkeys that received vaccinia virus recombinants or the control virus produced good anti-vaccinia virus antibody responses. However, those that received a single construct containing ring-infected erythrocyte surface antigen (RESA) given at eight sites did not produce significant antibody to any of the three major RESA repeat epitopes after immunization but were primed for an enhanced antibody response after challenge infection with P. falciparum. Most of the monkeys produced detectable antibodies to the RESA epitopes after challenge infection. One group of monkeys was immunized with four constructs (expressing RESA, two merozoite surface antigens [MSA-1 and MSA-2], and a rhoptry protein [AMA-1]), each given at two sites. While these monkeys failed to produce significant antibody against MSA 2 or AMA-1 after immunization, they produced enhanced responses against these antigens after challenge infection. Immunization involved an allelic form of MSA 2 different from that present in the parasite challenge strain, so that the enhanced responses seen after challenge infection indicated the presence of T cell epitopes common to both allelic forms. No groups of monkeys showed any evidence of protection against challenge, as determined by examination of the resulting parasitemias. PMID- 1711016 TI - A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites. AB - Babesiosis is a tick-borne hemoparasitic disease affecting horses worldwide. To investigate mechanisms of immunity to this parasite, the antibody response of infected horses to Babesia equi merozoite proteins was evaluated. Immunoprecipitation of B. equi merozoite antigens with sera from infected horses revealed 11 major proteins of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28, and 25 kDa. Monoclonal antibody (MAb) 36/133.97, which binds to live merozoites, immunoprecipitated proteins of 44, 36, 34, and 28 kDa. When immunoprecipitations were performed with in vitro translation products of merozoite mRNA, MAb 36/133.97 immunoprecipitated proteins of 38, 28, 26, and 23 kDa which comigrated with proteins immunoprecipitated by sera from infected horses at 10(-3) to 10(-4) dilutions. In Western blot analysis, MAb 36/133.97 recognized proteins of 44, 36, 34, and 28 kDa, and a 28-kDa protein was identified by sera from infected horses at a dilution of 10(-4). MAb 36/133.97 bound to B. equi isolates from Florida and Europe. Furthermore, the binding of MAb 36/133.97 to merozoite proteins was inhibited by sera of infected horses from 19 countries. Collectively, these data indicate MAb 36/133.97 binds to a geographically conserved peptide epitope on multiple B. equi merozoite proteins, including a merozoite surface protein, and MAb 36/133.97 reacts with a B. equi protein immunodominant in infected horses. PMID- 1711018 TI - Hepatitis C virus antibodies among different groups at risk and patients with suspected non-A, non-B hepatitis. AB - 4000 sera were tested for antibodies against hepatitis C virus (HCV) by means of an ELISA using the C100-3 antigen. 38.9% of patients with non-A, non-B hepatitis following blood transfusion (n = 108) had HCV antibodies. Among patients with chronic liver damage of unknown origin (n = 316) 30.4% were anti-HCV positive, and in 2,506 patients with transitional or chronic elevation of transaminases 14.8% showed HCV antibodies. Haemophiliacs (n = 26) with 65.4% anti-HCV positives and drug addicts (n = 46) with 56.5% anti-HCV positives had the highest prevalence among high risk groups. Addicts dying from drug abuse (n = 216) and HIV 1 positives (n = 127) were anti-HCV positive in 37.5% and 26.0%, respectively. Patients on haemodialysis (n = 331) had antibodies against HCV in 12.4%. Health care workers (n = 217) appear to be at a comparably low risk with only 2.8% anti-HCV positives. Up to now we could not find a single case of intrafamilial spread of HCV in 46 examined cases. We suggest that HCV infectivity of contaminated body fluids and blood is lower than that of hepatitis B virus or human immunodeficiency virus type 1 carriers. In suspected non-A, non-B hepatitis negative test results should be confirmed in a second sample because it may take three to six months after infection before HCV antibodies occur. However, about 10% of chronic HCV infections are not detectable with the presently available test. This may change when new tests become available using HCV specific antigens other than C100-3. PMID- 1711017 TI - Immunogenicity of Vibrio cholerae O1 toxin-coregulated pili in experimental and clinical cholera. AB - A functional tcpA gene, encoding the major subunit of toxin-coregulated pili (TCP), is necessary for Vibrio cholerae O1 Ogawa strain 395 to colonize the human intestine and confer protective immunity to virulent challenge. The immunogenicity of TCP and other antigens in experimental and naturally acquired cholera was determined. Seroconversion to cholera toxin (CT), whole cell preparations, and to Ogawa lipopolysaccharide but not to purified native TCP or to a TcpA mimiotope was found in volunteers. Local intestinal secretory immunoglobulin A from volunteers showed conversions to cholera toxin and lipopolysaccharide but not to TCP. Cholera patients in Indonesia showed a seroconversion rate to TCP of 3 of 6 and a seroconversion to a TcpA mimiotope of 1 of 6. Volunteer and patient sera showed similar vibriocidal seroconversions when assayed against either TCP-positive and TCP-negative V. cholerae O1 strains, suggesting that TCP do not contribute demonstrably to the vibriocidal antigen. We conclude that although seroconversion to TCP can occur in naturally acquired cholera, solid long-term protection can be engendered in the absence of a detectable anti-TCP immune response. PMID- 1711019 TI - Disseminated malignant melanoma. New therapeutic approaches. PMID- 1711020 TI - [Early and late results after surgical treatment of pulmonary atresia with intact ventricular septum]. AB - Between 1970 und 1989 30 children were admitted with the diagnosis of pulmonary atresia with intact ventricular septum (PA/IVS). Before palliation 4 children died. According to the grade of right heart hypoplasia the patients were divided into 3 groups of mild, moderate or severe hypoplasia. Palliative operations were performed in 25 children (17 m, 9 f) with a mean age of 10 days: 13 valvotomies (V), 5 aortopulmonary shunts (S), 7 V plus S. One patient had total correction as primary procedure. A total of 17 reoperations was necessary in 12 of 26 patients (10 palliations, 7 total corrections). Total corrections were: 2 conduits and 5 patches of the right ventricular outflow tract (RVOT). Total mortality was 14/30 (54%) children: early 10/26 (38%), late 4/16 (25%) children. After total correction mortality was 3/7 (43%) children. Actuarial survival after palliation was 46% after 5 and 10 years. For patients with PA/IVS we recommend the following surgical strategy: 1. mild hypoplasia: V plus S for palliation; 2. moderate hypoplasia: S plus patch of RVOT; 3. severe hypoplasia: after initial ballon septostomy S and antegrade decompression of the right ventricle (RV). For total correction in a well developed RV we prefer ASD-closure and patch of RVOT if possible with homograft monocusp. In moderate or severe hypoplasia a Fontan operation is done with closure of the ASD and tricuspid orifice with a single patch. PMID- 1711021 TI - New investigations on hematoxylin, hematein, and hematein-aluminium complexes. I. Spectroscopic and physico-chemical properties of hematoxylin and hematein. AB - Analytically pure hematoxylin (Htx), pentaacetylhematoxylin (PAHtx), and hematein (Hm) were isolated and characterized by 1H-NMR spectroscopy. The VIS/UV spectra of Htx and Hm were recorded in MeOH and in H2O at various pH values. The molar extinction coefficients of the long wavelength absorption bands are reported. The pKa value for the 1st acidic dissociation step of Hm has been determined from the pH dependency of the absorption spectra of Hm in aqueous buffer solutions. Finally, the absorption spectra are qualitatively discussed. PMID- 1711022 TI - Demonstration of elastic fibres with reagents for detection of magnesium. AB - Investigation of elastic fibres in various human and animal tissues with the reagents quinalizarin, magneson II, and titan yellow for the detection of magnesium revealed striking positive results. After pretreatment of skin and ligamentum flavum with elastase the tests were negative. The results support the supposition that the amount of magnesium in elastic fibres is sufficient for histochemical detection. It is speculated that the marked chelate-forming property of magnesium, or its antagonistic function to calcium, is associated with the elastic property of the fibres. PMID- 1711023 TI - The fix vital stain method. Simultaneous determination of viability and acrosomal status of bovine spermatozoa. AB - A rapid, accurate and precise method for simultaneous determination of acrosomal status and viability of bull spermatozoa is introduced and evaluated. The method involves fixation of semen with glutaraldehyde and subsequent addition of the fluorescent dye Hoechst bisbenzimide 33258 (H33258). Wet mounts were examined using a combination of phase-contrast and fluorescence microscopy (x500) for simultaneous visualization of the acrosomal apical ridge, which is indicative of the presence of an intact acrosome, and H33258-labeled nuclei, which is indicative of membrane-damaged cells. This fix-vital stain method allows differentiation between true acrosome reactions and degenerative postmortem loss of acrosomal membranes. Incubation of frozen-thawed spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 resulted in an increase in the percentage of true acrosome-reacted spermatozoa. The fix-vital stain method does not contain any processing steps that result in loss, selection, or damage of spermatozoa and therefore allows evaluation of representative semen samples. PMID- 1711025 TI - Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism. AB - To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed. PMID- 1711024 TI - Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli. AB - The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae. PMID- 1711026 TI - Effect of ermC leader region mutations on induced mRNA stability. AB - Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression. PMID- 1711027 TI - The exoR gene of Rhizobium meliloti affects RNA levels of other exo genes but lacks homology to known transcriptional regulators. AB - Rhizobium meliloti strains mutant in the exoR gene overproduce an exopolysaccharide called succinoglycan or EPS I. Protein fusions to several different exo genes required for EPS I biosynthesis are expressed at a higher level in an exoR strain than in a wild-type strain, showing that the overproduction of EPS I in exoR strains results at least in part from increased gene expression. This regulation is important to nodulation, since exoR mutants fail to invade alfalfa nodules unless secondary suppressor mutations that cause a decrease in EPS I production occur. Here, we show that an exoR strain contains higher levels of mRNA for other exo genes than does the wild-type parental strain. ExoR therefore most probably exerts its regulatory effect at the level of transcription. In addition, we have localized, subcloned, and sequenced the exoR gene. A newly constructed insertion allele of exoR has the same phenotype as the original mutant. The deduced sequence of ExoR is 268 amino acids long but does not show homology to other sequenced genes. PMID- 1711028 TI - Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. AB - We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria. Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites. A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome. Passing shuttle vectors through E. coli dam mutants effectively avoided this barrier. Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment. Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA). These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants. Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb. This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed. PMID- 1711029 TI - Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase. AB - L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspH1 mutation at 215 degrees. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t1 to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2- to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspH1 mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by t0 of sporulation but then showed a small increase, which was mirrored by changes in the level of beta-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulation-specific RNA polymerase tested yielded a -73 transcript in vitro. PMID- 1711030 TI - Phylogenetic analysis and evolution of RNase P RNA in proteobacteria. AB - The secondary structures of the eubacterial RNase P RNAs are being elucidated by a phylogenetic comparative approach. Sequences of genes encoding RNase P RNA from each of the recognized subgroups (alpha, beta, gamma, and delta) of the proteobacteria have now been determined. These sequences allow the refinement, to nearly the base pair level, of the phylogenetic model for RNase P RNA secondary structure. Evolutionary change among the RNase P RNAs was found to occur primarily in four discrete structural domains that are peripheral to a highly conserved core structure. The new sequences were used to examine critically the proposed similarity (C. Guerrier-Takada, N. Lumelsky, and S. Altman, Science 246:1578-1584, 1989) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia coli. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous. PMID- 1711031 TI - The omega subunit of Escherichia coli K-12 RNA polymerase is not required for stringent RNA control in vivo. AB - Igarashi et al. (K. Igarashi, N. Fujita, and A. Ishihama, Nucleic Acids Res. 17:8755-8765, 1989) reported that the omega (omega) subunit of Escherichia coli RNA polymerase was required for stringent control as judged by in vitro transcription assays in the presence and absence of guanosine 3',5' bispyrophosphate (ppGpp). This conclusion predicts that a deletion of the omega gene (designated rpoZ or spoS) should show a relaxed RNA control phenotype in vivo. However, we find that wild-type stringent control of stable RNA accumulation is unaffected by a spoS null allele that abolishes cellular production of the omega protein. We conclude that omega protein is not necessary for the operation of the stringent RNA control response. PMID- 1711032 TI - Labeling of integrin alpha v beta 3 with 58Co(III). Evidence of metal ion coordination sphere involvement in ligand binding. AB - Integrin-mediated cell adhesion to the extracellular matrix is divalent metal ion dependent; however, a demonstration of the interaction between native integrins and divalent metal ions is lacking. Here we provide direct evidence that the vitronectin receptor (VNR) is a metalloprotein. The unique electron shell of Co(II), an ion which we show supports ligand recognition by VNR, enables its oxidative conversion to inert Co(III). This property facilitated "affinity labeling" of VNR with 58Co(III) by oxidation of the metal ion in situ (i.e. in position). An average of 3.5 +/- 0.5 mol of cobalt were incorporated per mol of VNR. The ability of VNR to bind metal ions was independently confirmed by examining the interaction between VNR and Mn2+ under native conditions. The apparent high affinity between VNR and Mn2+ allowed us to observe the specific binding between 54Mn2+ and VNR by equilibrium gel filtration studies. Interestingly, the oxidative incorporation of Co(III) into VNR specifically blocked ligand binding, suggesting that the coordination sphere of metal ion bound to VNR is a critical determinant in integrin-ligand recognition. Furthermore, Mn2+ abolished the oxidative affinity labeling of VNR with Co(III) and consequently blocked the inactivation of VNR by in situ incorporation of Co(III). Thus, Mn2+ and Co2+ bind to the same or mutually exclusive sites on VNR. These observations provide the first demonstration that an integrin, specifically VNR, is a metalloprotein and demonstrate a functional link between the coordination sphere of the bound metal ion and ligand recognition by this receptor. PMID- 1711033 TI - CD4+ lipid bilayers. A model for human immunodeficiency virus type 1 coat protein binding. AB - gp120, the coat glycoprotein of the human immunodeficiency virus type 1 (HIV1) binds to a molecule on the surface of a class of T-lymphocytes, CD4, which is also the receptor for major histocompatibility complex class II (MHCII). To study the events that follow the interaction of gp120 with CD4, we have incorporated CD4 into lipid bilayers and recorded the electrical changes which occur after the addition of gp120. Interaction of gp120 to CD4-containing bilayers induces multistate ion-permeable channels with a maximum conductance of 380-400 picosiemens. When CD4+ bilayers were preexposed to either MHCII or to OKT4A antibody, no channels were formed after the addition of gp120. These results indicate that CD(4+)-containing bilayers bind gp120, MHCII, and OKT4A, that binding of gp120 produces ion-permeable channels, and that CD4+ bilayers can be used to assay for gp120 in the solution bathing the bilayer. PMID- 1711034 TI - Carbohydrate-binding specificity of Tetracarpidium conophorum lectin. AB - The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans. PMID- 1711035 TI - The cecropin locus. Cloning and expression of a gene cluster encoding three antibacterial peptides in Hyalophora cecropia. AB - Cecropins A, B, and D are antibacterial peptides of 35-37 amino acids that are synthesized in pupae of the Cecropia moth (Hyalophora cecropia) as a response to a bacterial infection. cDNA cloning has shown that the cecropins are made as preproproteins that are processed in four steps to the mature peptides. We have now cloned the genes for preprocecropins A and D, data that together with earlier work on the B gene has made it possible to deduce the arrangement of the cecropin locus. The genes for the three cecropins are organized in a large cluster spanning 20 kilobases of DNA and for each gene there is one copy/haploid genome. The size of the cluster is in part due to long distances between the genes and to the presence of insertion elements in the introns of the A and D genes. The cecropin genes are not expressed in parallel. Transcripts for cecropins A and B appear within 2 h after injection of live bacteria, they reach a maximum after 48 h, and they are continuously expressed at this level for several days. The D gene has a delayed pattern of expression where transcripts appear within 48-96 h and reach a maximum after 144 h. In consonance is also the production of the mature cecropins A, B, and D where the active cecropins A and B are detected in the hemolymph within 10-24 h while the D form is not detected until 48 h post infection. Control injections with sterile saline produced only a weak induction of the cecropin genes. PMID- 1711036 TI - Reduced uptake of cholesterol esterase-modified low density lipoprotein by macrophages. AB - Changes in low density lipoprotein (LDL) lipid composition were shown to alter its interaction with the LDL receptor, thus affecting its cellular uptake. Upon incubation of LDL with 5 units/ml cholesterol esterase (CEase) for 1 h at 37 degrees C, there was a 33% reduction in lipoprotein cholesteryl ester content, paralleled by an increment in its unesterified cholesterol. CEase-LDL, in comparison to native LDL, was smaller in size, possessed fewer free lysine amino groups (by 14%), and demonstrated reduced binding to heparin (by 83%) and reduced immunoreactivity against monoclonal antibodies directed toward epitopes along the LDL apoB-100. Incubation of CEase-LDL with the J-774 macrophage-like cell line resulted in about a 30% reduction in lipoprotein binding and degradation in comparison to native LDL, and this was associated with a 20% reduction in macrophage cholesterol mass. Similarly, CEase-LDL degradation by mouse peritoneal macrophages, human monocyte-derived macrophages, and human skin fibroblasts was reduced by 20-44% in comparison to native LDL. CEase-LDL uptake by macrophages was mediated via the LDL receptor and not the scavenger receptor. CEase activity toward LDL was demonstrated in plasma and in cells of the arterial wall such as macrophages and endothelial cells. Thus, CEase modification of LDL may take place in vivo, and this phenomenon may have a role in atherosclerosis. PMID- 1711037 TI - Phosphatidylazidothymidine. Mechanism of antiretroviral action in CEM cells. AB - Dimyristoylphosphatidylazidothymidine has been shown to inhibit human immunodeficiency virus (HIV) replication in CEM or U937 cells infected with the LAV-1 strain in vitro, but its metabolism and mechanism of antiretroviral activity have not been determined (Hostetler, K. Y., Stuhmiller, L. M., Lenting, H. B. M., van den Bosch, H., and Richman, D. D., J. Biol. Chem. (1990) 265, 6112 6117). We synthesized phosphatidylazidothymidine labeled with tritium in the 5' position of dideoxyribose and incubated the phospholipid prodrug with CEM cells for 24 h. The radioactive products were analyzed by high pressure liquid chromatography and thin layer chromatography. Phosphatidylazidothymidine is deacylated by cellular phospholipases A to lysophosphatidylazidothymidine, which is subsequently hydrolyzed to glycero-3-phospho-5'-azidothymidine by lysophospholipases. Phosphodiesteratic cleavage of glycero-3-phospho-5' azidothymidine occurs with formation of azidothymidine (AZT) or AZT-5' monophosphate. Anabolic phosphorylation leads to the formation of AZT-diphosphate and AZT-triphosphate. To evaluate the possibility of direct inhibition of HIV reverse transcriptase by phosphatidyl-AZT, lysophosphatidyl-AZT or glycero-3 phospho-5'-AZT, we synthesized these compounds and found them to lack the ability to inhibit HIV recombinant reverse transcriptase in vitro. AZT-triphosphate was greater than 10,000 times more potent in inhibiting reverse transcriptase activity. Thus, phosphatidyl-AZT exerts its antiviral activity by metabolic conversion to AZT-triphosphate. Phospholipid prodrugs of this type may be useful in treating the macrophage reservoir of HIV infection. PMID- 1711038 TI - Evidence for the facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Basic properties and specificity for polyglutamate chain length. AB - Evidence is presented outlining basic properties of a previously undescribed facilitative transport system mediating transfer of methotrexate (MTX) polyglutamates from the cytoplasmic to the lysosomal compartment of the cell. These experiments were conducted using purified lysosomes prepared from murine S180 cells, and a model substrate ([3H]MTX + G1; methotrexate with 1 additional glutamyl residue) to examine biological aspects as well as pharmacological significance of this process in a tumor cell model. The data, expressed as a function of latent beta-hexosaminidase activity, a measure of lysosomal integrity, show that [3H]MTX + G1 uptake in lysosomes is temperature-dependent, is stimulated specifically by magnesium chloride and potassium chloride with maximal enhancement observed in the presence of both agents together, exhibits Michaelis-Menten saturation kinetics with Km and Vmax values of 346 +/- 39 microM and 2.8 +/- 0.3 pmol/min/unit of beta-hexosaminidase activity, respectively, and is competitively inhibited by longer chain polyglutamates with increasing effectiveness as shown by Ki values of 334 +/- 19, 201 +/- 16, 106 +/- 13, and 42 +/- 8 microM, for MTX + G1, MTX + G2, MTX + G3, and MTX + G4, respectively. In addition, uptake is inversely related to medium osmolarity indicating that the phenomenon we observe represents internalization of the [3H]MTX + G1 and not adsorption to a possible surface binding site. As a whole, the data are consistent with a single mediated transport system shared by all MTX polyglutamates for entry into lysosomes. It is our view that this transport system represents the initial step in the degradation of polyglutamates in the cell. In addition, based on a comparative analysis of the kinetics for hydrolysis and transport, we suggest that it is also the limiting step in this process and, as such, regulates the extent of degradation of the free cellular pools of these compounds. PMID- 1711039 TI - Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling. AB - We have isolated several Chinese hamster ovary cell lines with temperature sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2 macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling. PMID- 1711040 TI - Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback. AB - The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes. PMID- 1711041 TI - Structure of the murine fifth complement component (C5) gene. A large, highly interrupted gene with a variant donor splice site and organizational homology with the third and fourth complement component genes. AB - To understand fifth complement component (C5) gene regulation, splicing, and C5 protein deficiency at the molecular level, the organization of the murine C5 gene was determined. The C5 structural gene is present as a single copy in the mouse genome as demonstrated by Southern blot analysis. Accordingly, three cosmid clones were isolated from a genomic library that was prepared from mouse strain B10.D2/nSnJ. These clones overlapped and contained the structural gene encoding the complete C5 alpha-chain and 90% of the beta-chain. The 5'-flanking region of the C5 gene was obtained from a clone isolated from a genomic lambda-MOPC-41 library. Unique restriction fragments were prepared from the genomic clones and subcloned, and the exons were sequenced. All introns were sized by sequencing or Southern analysis. The C5 structural gene was found to be a highly interrupted gene of approximately 78 kilobases containing 42 exons and 41 introns. The exons ranged in length from 58 to 247 base pairs, with an average length of 131 base pairs. The introns ranged in size from 100 base pairs to 4 kilobases with an average length of 1.5 kilobases. The C5 alpha-chain was encoded by 49 kilobases containing 26 exons; the beta-chain was encoded by 29 kilobases containing 16 exons. The C5a coding sequence was split between two exons. All intron/exon junctions followed the normal consensus rule except at intron 35 in which the 5' donor GT was substituted by GC. The 2-base-pair gene deletion and HindIII and PvuII restriction fragment length polymorphisms associated with murine C5 deficiency were localized to exon 7, exon 16, and intron 20, respectively. Comparison of the intron-exon junctions of the murine C5, human C3, and mouse C4 genes indicated that these genes are nearly identical in structural organization. However, the rat alpha 2-macroglobulin gene showed only moderate genomic organizational similarity to the murine C5 gene. A major and a minor transcriptional initiation site in the C5 gene were identified by primer extensions and confirmed by RNase protection assays. Sequence analysis of the 5' flanking region (760 base pairs) revealed a TATA-like and CAAT box upstream of the major transcriptional initiation site at positions -274 and -303, respectively, suggesting an atypical promoter. The 5'-flanking region also contained sequences identical with several cis-acting motifs known to bind the liver-specific nuclear protein LF-A1 and the nuclear protein NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711042 TI - An ORF323 with homology to crtE, specifying prephytoene pyrophosphate dehydrogenase, is encoded by cyanelle DNA in the eukaryotic alga Cyanophora paradoxa. AB - Carotenoids are essential constituents of the light-harvesting and light protective systems of photosynthetic organisms. The biochemistry of carotenoid biosynthesis in eukaryotes is known, whereas evidence for the genes specifying this biosynthetic pathway is scant. We report here the nucleotide sequence and expression of a gene likely encoding crtE (prephytoene pyrophosphate dehydrogenase). The reaction product of this enzyme is phytoene, a C40 carotenoid precursor common to all organisms. The gene is found in the cyanelle (plastid) DNA of an eukaryotic alga, Cyanophora paradoxa. The expression into protein of cyanelle crtE has been demonstrated in vitro. The identity and similarity scores of CrtE from cyanelles with the corresponding protein from the photosynthetic bacterium Rhodobacter capsulatus are 28.6 and 68.5%, respectively. PMID- 1711043 TI - Keratin expression in rat intestinal crypt and villus cells. Analysis with a panel of monoclonal antibodies. AB - Seven monoclonal antibodies were prepared against cytoskeletal components of rat intestinal brush borders. In the following paper (Chandler, J. S., Calnek, D., and Quaroni, A., J. Biol. Chem. 266, 11932-11938), three of them were shown to be specific for, respectively, keratin 8 (RK4), keratin 19 (RK7), and a newly identified type I keratin (keratin 21) (RK5). With these antibodies we have investigated the changes in keratin gene expression accompanying intestinal cell differentiation. Keratin 21 was detected exclusively in differentiated villus cells and in goblet, enteroendocrine, and Paneth cells in the crypts; in the proliferative crypt cells keratin 19 was predominant. Analysis of keratins expressed by cultured rat crypt cells (IEC cells) confirmed the absence of keratin 21 in undifferentiated intestinal cells. Changes in keratin's expression similar to those observed with cell differentiation in the adult intestinal mucosa were also demonstrated during early fetal intestinal development: the stratified epithelium present at 15-16 days of gestation contained predominantly keratin 19 with only a small amount of keratin 8; keratin 21 was first detected at 18-19 days of gestation, concomitant with the appearance of a well formed brush border and an apical cytoplasmic terminal web. These results suggest that keratin tonofilaments may play a role in the morphological and structural alterations accompanying intestinal cell differentiation in vivo. PMID- 1711044 TI - Identification and characterization of rat intestinal keratins. Molecular cloning of cDNAs encoding cytokeratins 8, 19, and a new 49-kDa type I cytokeratin (cytokeratin 21) expressed by differentiated intestinal epithelial cells. AB - In the previous paper (Quaroni, A., Calnek, D., Quaroni, E., and Chandler, J.S. (1991) J. Biol. Chem. 266, 11923-11931) we describe the use of a panel of "antikeratin" monoclonal antibodies to study cytokeratin distribution in rat intestinal epithelium. In the present paper we describe the use of three antikeratin monoclonal antibodies to identify and recovery cDNA clones expressing immunologically specific fusion proteins from a rat intestinal cDNA library. DNA sequence analysis identified each cDNA encoded epitope including the carboxyl terminal portions of cytokeratins 8 and 19 (as cataloged by Moll, R., Franke, W.W., and Schiller, D.L. (1982) Cell 31, 11-24) recognized by antibodies RK4 and RK7, respectively. In addition, antibody RK5 was used to recover a cDNA clone (pRK5) encoding a portion of a 48-kDa keratin-related protein with unique tissue and cellular distribution, designated cytokeratin 21. Translation of cDNA selected mRNAs yielded individual proteins which could be resolved and identified by their specific immunoreactivities. The pRK5 cDNA was used to recover a larger (approximately 1.3 kilobase pairs) cDNA clone (KB2) from an independent cDNA library for DNA sequence analysis and for the recovery of additional overlapping cDNA clones. The resulting cDNA sequence (1519 base pairs) contains the complete coding region of cytokeratin 21 (49,387 daltons). The predicted amino acid sequence of cytokeratin 21 confirms its identity as a novel type I cytokeratin expressed predominantly in the intestinal epithelium. PMID- 1711045 TI - The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. AB - Vascular endothelial growth factor (VEGF) is an apparently endothelial cell specific mitogen that is structurally related to platelet-derived growth factor. By Northern blot and protein analyses, we show that VEGF is produced by cultured vascular smooth muscle cells. Analysis of VEGF transcripts in these cells by polymerase chain reaction and cDNA cloning revealed three different forms of the VEGF coding region, as had been reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indicates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing: the 165-amino-acid form of the protein is missing the residues encoded by exon 6, whereas the 121-amino-acid form is missing the residues encoded by exons 6 and 7. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 factor binding sites. The promoter region also contains several potential binding sites for the transcription factors AP-1 and AP-2; consistent with the presence of these sites, Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured vascular smooth muscle cells after treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. PMID- 1711046 TI - Epidermal growth factor activation of vinculin and beta 1-integrin gene transcription in quiescent Swiss 3T3 cells. Regulation through a protein kinase C independent pathway. AB - Growth activation of quiescent Swiss 3T3 fibroblasts leads to a rapid induction of vinculin and beta 1-integrin gene expression. Addition of serum, epidermal growth factor (EGF), or platelet-derived growth factor to serum-starved, density arrested cells resulted in a rapid increase in vinculin and beta 1-integrin mRNA levels and a corresponding increase in vinculin synthesis. The increase in vinculin and beta 1-integrin mRNA expression by serum or EGF was not blocked by the inhibition of protein synthesis by cycloheximide. The kinetics of induction of vinculin and beta 1-integrin mRNAs by EGF are different: vinculin mRNA levels reached a peak of expression 4-5-fold greater than that measured in quiescent cells by 2 h after addition of growth factor, whereas beta 1-integrin mRNA levels increased more slowly and to a lesser extent, reaching peaks of 2-3-fold induction at 5 h poststimulation. Down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol 1,2-myristate 1,3-acetate had no effect on the ability of EGF or platelet-derived growth factor to activate vinculin or beta 1-integrin mRNA expression. Furthermore, direct activation of protein kinase C with 1,2-myristate 1,3-acetate did not induce the expression of vinculin or beta 1-integrin mRNA, but did activate c-fos expression. In vitro nuclear "run-on" transcription assays demonstrate a greater than 7-fold increase in vinculin and beta 1-integrin transcription at 40-60 min after addition of EGF when compared with levels in quiescent cells. This activation was rapid and transient, but appeared to occur later than the increase in c-fos and actin transcription. These results demonstrate that vinculin and beta 1-integrin, important components of the cell adhesion apparatus, are members of a group of immediate early growth-responsive genes, along with c-fos, c-myc, actin, and fibronectin. In addition, regulation of these cell adhesion genes occurs exclusively through a protein kinase C-independent pathway in serum-deprived, density-arrested Swiss 3T3 cells. PMID- 1711047 TI - Molecular cloning of a human serum protein structurally related to complement factor H. AB - Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H. PMID- 1711048 TI - Heterozygous mutation in the G+5 position of intron 33 of the pro-alpha 2(I) gene (COL1A2) that causes aberrant RNA splicing and lethal osteogenesis imperfecta. Use of carbodiimide methods that decrease the extent of DNA sequencing necessary to define an unusual mutation. AB - Cultured skin fibroblasts from a proband with osteogenesis imperfecta were found to synthesize normal and shortened alpha 2(I) chains of type I procollagen. A cDNA library was prepared using mRNA isolated from the proband's fibroblasts. Partial nucleotide sequencing of five clones demonstrated that two clones lacked the 54 base pairs (bp) of coding sequences found in exon 33 of the pro-alpha 2(I) gene (COL1A2). To reduce the amount of nucleotide sequencing required, heteroduplexes were prepared from two of the clones, one normal and the other lacking exon 33, and reacted with a water-soluble carbodiimide under conditions in which nonbase-paired G and T nucleotides are specifically modified by the reagent. Analysis of the heteroduplexes by immunoelectron microscopy suggested that the sequence variation near the codons of exon 33 was the only sequence difference in the cDNA clones. Amplification of cDNA from the proband by polymerase chain reaction gave products of two sizes, one of the expected size for the normal sequence and the other of the expected size for a product lacking the 54 bp in exon 33. To define the mutation in genomic DNA, a 1.6-kilobase region spanning exons 32 and 34 was amplified by the polymerase chain reaction and DNA heteroduplexes were prepared from the products. The heteroduplexes were treated with a water-soluble carbodiimide and then used as templates for primer extension under conditions in which extension terminates at the site of a carbodiimide-modified base. The results suggested a mismatch near the exon-intron boundary of exon 33 and a second mismatch near the 3' end of intron 33. Nucleotide sequencing of the polymerase chain reaction products revealed a single base substitution in one allele that changed the moderately conserved G at position +5 of the 5' splice site of intron 33 to an A. In addition, there was an apparently neutral single-base substitution that placed both a G and T at position +661 of intron 33. The results provide only the third example of a mutation in the G at the +5 position of an intron that causes aberrant RNA splicing. Also, the results demonstrate that use of techniques involving carbodiimide modification of DNA heteroduplexes can reduce the amount of nucleotide sequencing necessary to define mutations in large and complex genes. PMID- 1711049 TI - Interaction of plasma proteins with heparinized gel particles studied by high resolution two-dimensional gel electrophoresis. AB - In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding. PMID- 1711050 TI - The role of an endothelial cell lining in limiting distal anastomotic intimal hyperplasia of 4-mm-I.D. Dacron grafts in a canine model. AB - The effect of an endothelial cell (EC) lining on intimal hyperplasia at the distal anastomosis of Dacron grafts was assessed in a canine model. Enzymatically derived autologous EC were used to seed 14 to 17 cm long, 4 mm I.D., knitted Dacron aortoiliac grafts implanted in an end-to-side manner in six dogs (Group I). Unseeded grafts were similarly implanted in six control dogs (Group II). All animals received acetylsalicylic acid (325 mg po qd) 24 h prior to graft placement and for 2 weeks postoperatively. Distal anastomotic intimal hyperplasia (AIH) and luminal surface EC coverage were quantitated at the conclusion of a 16 week study period. Patency for Group I and Group II grafts were 90% and 55%, respectively (p = 0.07). Maximum AIH, defined as the maximum reduction of luminal cross-sectional area at the distal anastomosis, was not significantly different between Group I (13.1 +/- 8.0%) and Group II (15.1 +/- 7.3%) conduits. However, AIH was inversely related to the extent of luminal EC coverage (r = -0.6, p less than 0.05), thus greater endothelialization was associated with decreased AIH. These data support the idea that EC coverage of the luminal surface of prosthetic vascular grafts may limit the development of AIH. PMID- 1711051 TI - Tachykinins (substance P, neurokinin A, neuropeptide K, and neurokinin B) in the cerebral circulation: vasomotor responses in vitro and in situ. AB - The vasomotor responses of tachykinins have been studied in the cerebral vasculature of human, pig, cat, and guinea pig. Substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide K (NPK) induced concentration dependent relaxations of precontracted cerebral arteries in all species when examined by a sensitive in vitro technique. In addition, the relaxant responses to SP, NKA, and NKB were studied in cat pial arterioles by peptide microapplication in situ. In human pial vessels, the order of relaxant potency was SP greater than NKB greater than NKA greater than NPK; in the pig middle cerebral artery, there was no difference in potency between the tachykinins; in the cat middle cerebral artery, SP = NKB greater than NKA = NPK; and in the guinea pig basilar artery, SP much greater than NPK = NKA greater than NKB. Responses induced by SP, NKA, and NKB in the cat were comparable in vitro and in situ. Removal of the endothelium abolished relaxation induced by all four tachykinins. The relaxant responses of guinea pig basilar arteries to SP, NKA, and NPK were competitively antagonized by the SP antagonist Spantide. However, Spantide lowered the Imax of the NKB concentration-response curve without any rightward shift, suggesting action at a different site than the other tachykinins. In the guinea pig basilar artery, the relaxation seems to be exerted via a NK-1 receptor subtype while the receptor subtype is more unclear in cerebral arteries from human, cat, and pig.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711052 TI - High-performance liquid chromatographic determination of bleomycins. AB - A rapid and specific ion-pair reversed-phase high-performance liquid chromatographic method was developed for the determination of bleomycins. The use of 5-microns particles of less adsorptive reversed-phase packings and sodium perchlorate as ion-pairing reagent permitted a short analysis time and the transferability of the separations on different batches of the reversed-phase materials. The detection sensitivity and precision of the method demonstrated that the system is suitable for routine analysis. PMID- 1711053 TI - Organization of the cerebellum in the pigeon (Columba livia): I. Corticonuclear and corticovestibular connections. AB - The projections of the cerebellar cortex upon the cerebellar nuclei and the vestibular complex of the pigeon have been delineated using WGA-HRP as an anterograde and retrograde tracer. Injections into individual cortical lobules (II-IXa) produce a pattern of ipsilateral terminal labeling of both the cerebellar and vestibular nuclei. The pattern of corticonuclear projections indicates both a rostrocaudal and a mediolateral organization with respect to the lobules and is consistent with a division of the cerebellar nuclei into a medial (CbM) and a lateral (CbL) nucleus. The retrograde experiments indicate that these nuclei receive projections, respectively, from Purkinje cells within medial (A) and lateral (C) longitudinal zones, which alternate with longitudinal zones (B, E) projecting upon the vestibular complex. Purkinje cells in (vestibulocerebellar) lobules IXb-X show only limited projections upon the cerebellar nuclei, but do project extensively upon the cerebellovestibular process (PCV), as well as upon the medial, superior, and descending vestibular nuclei. As the injection site shifts from medial to lateral, there is a corresponding shift in focus of the projection within PCV from areas bordering CbM to those abutting CbL. The topographic organization of corticovestibular projections is less clear-cut than those of the corticonuclear projections. Lobules II-X project upon the lateral vestibular nucleus (anterior lobe) or the dorsolateral vestibular nucleus (posterior lobe). These projections originate from either side of the lateral (C) zone. Projections originating from the medialmost (B) zone are interrupted in lobules VI and VII. The anterior and posterior portions of the lateralmost (E) zone overlap along lobules VI and VII. In addition, the E zone of the anterior lobe is the source of projections upon the medial, the descending, and the superior vestibular nuclei. Projections from the auricle and adjacent lateral unfoliated cortex (F zone) focus upon the infracerebellar nucleus, the medial tangential nucleus, and the medial division of the superior vestibular nucleus. The data suggest that the cerebellar cortex of the pigeon, like that of mammals, may be subdivided into a mediolaterally oriented series of longitudinal zones, with Purkinje cells in each zone projecting ipsilaterally to specific cerebellar nuclei or vestibular regions. For cortical regions exclusive of the auricle and lateral unfoliated cortex, three such zones (A, B, and C) are defined that are comparable in their efferent targets with the A, B, and C zones of mammals. There does not appear to be a D zone in the pigeon. The results are discussed in relation to comparative data on amphibians, reptiles, and mammals. PMID- 1711054 TI - Organization of the cerebellum in the pigeon (Columba livia): II. Projections of the cerebellar nuclei. AB - The projections of the deep cerebellar nuclei in the pigeon have been delineated using autoradiographic and histochemical (WGA-HRP) tracing techniques. A medial (CbM) and lateral (CbL) cerebellar nucleus are recognized and CbM may be further partitioned into internal, intermediate, and intercalate divisions. As in mammals, most extracerebellar projections of CbM travel in the fasciculus uncinatus (FU); the rest travel with those of CbL in the brachium conjunctivum (BC). In the pigeon, both of these pathways are bilaterally but primarily contralaterally projecting systems. FU is a predominantly descending tract, with terminations within (1) the vestibular complex, (2) a column of contiguous medial reticular nuclei from pontine to caudal medullary levels; (3) the plexus of Horsley portion of the parvicellular reticular formation, continuing through the nucleus centralis medullae oblongatae, pars dorsalis, into intermediate layer VII of the cervical spinal cord, down to cervical segment 8-9; (4) the lateral reticular nucleus and the paragigantocellular reticular nucleus; (5) the dorsal lamella of the inferior olive. Rostrally FU terminals are found in the locus ceruleus and dorsal subcerulean nucleus. Minimal FU projections are also seen to the motor trigeminal nucleus and the subnucleus oralis of the descending trigeminal system. A small projection from the intercalate division of CbM travels in BC and projects upon the midbrain central grey, the intercollicular nucleus, the lateral tectal periventricular grey, the stratum cellulare externum and, sparsely, upon the dorsolateral thalamus. The bulk of BC originates from the lateral cerebellar nucleus and consists of a massive ascending and a small descending branch. The ascending system projects upon the red nucleus and the dorsally adjacent interstitial nucleus of Cajal and midbrain central grey, the prerubral fields continuing into the stratum cellulare externum, the nucleus intercalatus thalami, the ventrolateral thalamic nucleus, the medial spiriform nucleus, the nucleus principalis precommissuralis, the nucleus of the basal optic root, the nucleus geniculatus lateralis pars ventralis, the dorsolateral thalamus, including the dorsal intermediate posterior, and the dorsolateral intermediate and anterior nuclei. BC also contains axons from the infracerebellar nucleus, which projects upon the trochlear and the oculomotor nuclei. The descending branch of BC distributes to the papilioform nucleus, the medial pontine nucleus, the gigantocellular and paramedian reticular nuclei, and, minimally, the rostral portions of the medial column and ventral lamella of the inferior olive. Taken in conjunction with data on amphibia and reptiles the present findings suggest that the fundamental ground plan of vertebrate cerebellar organization involves a medial and lateral cerebellar nucleus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711055 TI - Organization of the cerebellum in the pigeon (Columba livia): III. Corticovestibular connections with eye and neck premotor areas. AB - The connections of the cerebellar cortex with vestibular premotor neurons of the oculomotor and collimotor systems in the pigeon were delineated in experiments using WGA-HRP as an anterograde and retrograde tracer. Putative premotor neuron pools were identified by injections into the oculomotor (mIII) and trochlear nuclei (mIV) and into the most rostral portion of the cervical neck motor nucleus, nucleus supraspinalis (SSp). The retrograde data indicate that ipsilateral projections upon oculomotor neurons arise from the medial portions of the superior (VeS) and tangential (Ta) nuclei. Contralateral projections originate from the infracerebellar nucleus, the interstitial vestibular region including the main (lateral) portion of the tangential nucleus, and from the descending and medial vestibular nuclei (VeD, VeM). These projections were confirmed in anterograde studies that also defined the connections of these vestibular premotor regions with specific subnuclear divisions of the pigeon's "oculomotor" nuclei (mIII, mIV, mVI). The organization of projections from the vestibular nuclei to the pigeon's extraocular motoneurons is similar to that reported in mammals. Projections upon neck premotor neurons arise primarily from neurons in the interstitial region of the vestibular nuclear complex. After injections in SSp, retrogradely labeled neurons were found, contralaterally, in the lateral part of the tangential and superior vestibular nuclei and in the dorsolateral vestibular nucleus (VDL). Ipsilateral labeling was seen in the medial interstitial region (VeM, VeD, and medial Ta). These projections were confirmed in anterograde experiments. With the exception of VDL, vestibular nuclei projecting to neck motoneurons also project to extraocular motoneurons. Thus the infracerebellar nucleus projects exclusively, and the superior vestibular nucleus predominantly, upon oculomotor (mIII, mIV) nuclei; VDL projects predominantly upon the neck motor nucleus, whereas the interstitial vestibular regions (medial Ta, rostral VeD, intermediate VeM) project upon both collimotor and oculomotor neurons. The pattern of retrograde labeling seen in the cerebellar cortex after injections into vestibular premotor nuclei was used to define the projections of specific cerebellar cortical zones upon vestibular eye and neck premotor neurons. Corticovestibular projections upon these regions arise from the auricle and lateral unfoliated cortex, the posterior lobe components of cortical zones B and E, and from the vestibulocerebellum. Each of these cortical zones projects upon components of the vestibular nuclear complex, which are premotor to either oculomotor nuclei or collimotor nuclei. The hodological findings are related to the functional organization of the oculomotor and collimotor systems in the pigeon and compared with the mammalian data. PMID- 1711056 TI - Ultrastructural localization of substance P, met-enkephalin, and somatostatin immunoreactivity in lamina X of the primate spinal cord. AB - The ultrastructural localization of substance P (SP), met-enkephalin (MENK), and somatostatin (SS) in the lamina X area surrounding the central canal of the macaque monkey was examined by the indirect peroxidase-antiperoxidase method. The most common synaptic terminals in lamina X were simple terminals (S) with small rounded or pleomorphic clear vesicles; one to two dense-core vesicles were occasionally also present. These were found on soma, dendrites, and dendritic spines, in all regions of lamina X. A second class of terminal with round or oval clear vesicles was glomerular (G) in shape, with scalloped edges, and contained many mitochondria. These large terminals had several synaptic contacts onto dendrites, spines, and small terminals and were found mainly in the lateral region. The third class (L) contained small clear vesicles and several vesicles with large, dense cores (100-125 nm), and also contacted dendrites, mainly lateral to the canal. The fourth class of terminal (D) contained small clear vesicles and several vesicles with small, dense cores (75-100 nm); these contacted dendrites and somata in all areas. Very few terminals with flat vesicles were identified. There was an unequal distribution of immunoreactivity among the several terminal classes identified in lamina X. Most SP terminals were S terminals, but SP L terminals were also common; few were D terminals. MENK terminals were usually either S terminals or D terminals; L terminals were rarely MENK positive. SS terminals were commonly D terminals or S terminals; L terminals were also rarely SS positive. Only SP terminals were identified as G terminals. Synaptic targets of SP, MENK, and SS terminals were most commonly dendrites. In addition to unlabelled neurons, peptidergic neurons and their processes were also synaptic targets of terminals containing the same peptide. The distributions of these peptides in primate lamina X differ from that of the same peptides in primate superficial dorsal horn. These differences are important, in consideration of some of the parallels that may be drawn between the lamina X area and the superficial dorsal horn; both areas have high concentrations of the same peptides, receive nociceptive primary afferents, and contain spinothalamic and other projection neurons. Nevertheless, comparison of the distribution of immunoreactivity among terminal classes indicates that neurochemical organization at the ultrastructural level is quite distinct in each of the two areas. This may also reflect other roles of the lamina X area, including its involvement in visceral functions, although it would be expected that this element might be less prominent at the cervical levels we investigated. PMID- 1711057 TI - Dendritic morphology of pyramidal neurones of the visual cortex of the rat: I. Branching patterns. AB - The aim of this study was to provide quantitative descriptions of the branching patterns of basal and apical dendrites of pyramidal neurones from the visual cortex of the rat. Thirty-nine neurones from cortical layers 2/3 and 5, that had been injected with horseradish peroxidase, reconstructed, and measured with the light microscope as part of an earlier study (Larkman and Mason, '90; J. Neurosci. 10:1407-1414), were used. The cells had previously been divided into three classes, layer 2/3 cells and thick and slender layer 5 cells, on the basis of their dendritic morphology. The branching pattern of the basal and apical oblique dendrites was similar for all the cells. Between 3 and 9 basal trees arose from the soma and the number of tips in each tree varied widely, between 1 and 13. The path lengths of all the basal dendrites of a given cell were relatively constant, however. Most basal dendritic branching occurred close to the soma, such that terminal segments were much longer than intermediate segments and contributed approximately 90% of the total dendritic length of each tree. Terminal segments showed only a narrow range of diameters. Most apical oblique trees arose from the proximal part of the apical trunk. They tended to be less highly branched but were otherwise extremely similar to basal trees. Distal oblique trees were unbranched or branched only once, and their terminal segments tended to be shorter and thinner than those of basal trees. The branching pattern of the apical terminal arbors was different, with many longer intermediate segments. The terminal segments tended to be thinner than those of basal or proximal oblique trees. Slender layer 5 cells were without obvious terminal arbors. The basal and proximal oblique dendrites jointly sampled a roughly spherical volume of cortex centred about the soma, and together they accounted for the substantial majority of the cell's total dendritic shaft membrane area. Comparisons with previous studies suggest that intracellular HRP injection can yield a more complete visualization of dendritic morphology than is obtained using Golgi-based methods (unless cells are reconstructed across tissue slabs), and can therefore result in a different view of the relative importance of the various components that make up the cell's dendritic system. PMID- 1711058 TI - Dendritic morphology of pyramidal neurones of the visual cortex of the rat: II. Parameter correlations. AB - This study concerns the correlations between the various morphometric parameters obtained for the dendrites of neocortical pyramidal cells. The primary aims were to uncover underlying design principles in dendritic morphology, to see if these differed between different types of dendrite, and to see if estimates of parameters such as total dendritic shaft membrane area could be obtained from a limited number of measurements, avoiding the need to measure every dendritic segment. The data were from a sample of 39 pyramidal neurones, from layers 2/3 and 5 of the visual cortex of the rat, that had been injected with horseradish peroxidase, reconstructed, and measured with the light microscope as part of an earlier study (Larkman and Mason, '90: J. Neurosci. 10:1407-1414). Correlations between the somal area or the combined diameters of the stem segments and measures of the overall size of the dendrites were generally weak. For basal dendrites, the size of a tree was correlated with both its number of tips and the diameter of its stem segment, but these correlations were weaker for apical dendrites. Within individual cells, the diameter of any basal segment was closely related to the size of the tree arising from it, and quantitatively similar relations applied to apical oblique trees from the same cell. Terminal arbor trees showed relations that were similar in pattern but differed quantitatively, whereas apical trunk segment diameter correlations were often weak. In all cases, the number of tips in a tree was closely related to its size. Segment lengths, however, were not closely related to the size of the trees arising from them. It appears that at least some aspects of pyramidal dendritic morphology obey simple design rules. There was heterogeneity between trees of different types, although basal and oblique trees were very similar in most respects. It should prove possible to make use of correlations to estimate the sizes of basal, oblique, and terminal arbor trees from a limited number of measurements, but this does not seem to be possible for apical trunks. PMID- 1711059 TI - Dendritic morphology of pyramidal neurones of the visual cortex of the rat: III. Spine distributions. AB - The vast majority of excitatory synaptic inputs to neocortical pyramidal cells terminate on dendritic spines, which can thus serve as markers, visible by light microscopy, for the locations of these synapses. The aim of this study was to provide estimates of the total numbers and distributions of spines on the dendrites of individual pyramidal neurones from layers 2/3 and 5 of the visual cortex of the rat. High magnification camera lucida drawings were made of dendritic segments lying close to the plane of section and the number of spines per unit length of dendrite calculated for each. These spine densities were used to estimate the numbers of spines on the other dendritic segments and the results were entered to a computer program that calculated various statistics. Mean total numbers of spines per cell were 7,965 +/- 2,723 (S.D.) for layer 2/3 cells, 8,647 +/- 3,097 for slender layer 5 cells, and 14,932 +/- 3,371 for thick layer 5 cells; these figures are in good agreement with previous stereological estimates. For all cell classes, 70% or more of spines were located on the basal and apical oblique dendrites. The distribution of spines with respect to cortical layers was also explored. Most cells had most of their spines in the layer containing the soma, but there were differences within and between cell classes. Layer 2/3 cells showed a progressive reduction in the proportion of their spines in layers 1 and 2 with increasing depth of their soma in the cortex. Thick layer 5 cells had substantial contributions from layers 4, 3, 2, and especially layer 1. Slender layer 5 cells had small contributions from layers 6 and 4, but relatively few spines in layers 3 and 2. The distribution of spines with path distance from the soma was explored by estimating the numbers of spines contained within a series of concentric shells centred on the soma. All cells showed a rapid increase in the number of spines per shell for the proximal 100 micrograms or so, followed by a sharp decline to approximately 250 micrograms, beyond which the number remained relatively constant until the end of the terminal arbor. In each case, the majority of spines were located within a path length of 150 micrograms from the soma. PMID- 1711060 TI - Retinohypothalamic tract in the female albino rat: a study using horseradish peroxidase conjugated to cholera toxin. AB - There are several anatomically and functionally distinct retinofugal pathways, one of which is the retinohypothalamic tract (RHT). In this study, horseradish peroxidase conjugated to cholera toxin (CT-HRP), a sensitive neural tracer, was employed to describe the RHT in the female albino rat. Following uniocular injection of CT-HRP, both medial and lateral components of the RHT were evident. The medial component swept caudally into and through the suprachiasmatic nucleus (SCN) and dorsally to the subparaventricular zone. Terminal label was seen in the medial preoptic region, peri-SCN area, retrochiasmatic area, periventricular nucleus, anterior and central parts of the anterior hypothalamic area, and the subparaventricular zone. In contrast to the more focused and symmetrical medial component, the lateral component was diffuse with light terminal label in the lateral preoptic region, olfactory tubercle, lateral hypothalamus, supraoptic nucleus, and medial and posteroventral medial amygdaloid nuclei. The striking exception to this diffuse pattern of the lateral component was an extremely dense columnar terminal field over the dorsal border of the supraoptic nucleus. Whereas the intensity of label in terminal fields of the medial component was often similar on the sides ipsilateral and contralateral to the injection, the lateral component was consistently asymmetrical with greater labeling on the side contralateral to the injection. In addition, a light projection arrived at several thalamic nuclei by returning toward the thalamus from the tectal or pretectal areas via stria medullaris, and thus was not a part of the RHT. Implications for circadian as well as noncircadian photobiologic effects are discussed. PMID- 1711061 TI - Evaluation of antiarrhythmic drug effects with simultaneous analysis of single ventricular premature contractions, couplets and salvos. AB - To improve the clinical value of ambulatory Holter electrocardiographic (ECG) monitoring as a tool of antiarrhythmic therapy control, a new statistical model was developed. In a patient group at increased risk of sudden cardiac death, the spontaneous variability of ventricular arrhythmias was assessed, with simultaneous consideration of single ventricular premature complexes, couplets and salvos. The study included 100 patients who suffered from coronary heart disease or idiopathic dilated cardiomyopathy and for whom greater than 30 ventricular premature complexes/h and couplets had been demonstrated on the last Holter ECG before the study. Between 3 and 12 Holter recordings were made for each patient in a drug-free state; the mean follow-up period was 260 days (maximum 1,403). The mean hourly values of the ectopic events (EE) were assessed separately for ventricular premature complexes, couplets and salvos. The spontaneous variability (SV) was calculated for single ventricular premature complexes, couplets and salvos as SV = log (EEday 2 + 0.01/EEday 1 + 0.01) and linked in one, two and three dimensions. Compared with the consideration of only one type of arrhythmia (one-dimensional model), the simultaneous use of two or three types of arrhythmia (two- or three-dimensional model) resulted in considerably lower reduction and aggravation rates as sufficient proof of drug effects. With control intervals up to 1 week, the one-dimensional model yielded reduction rates for ventricular premature complexes, couplets and salvos of -63%, -90% and -95%, respectively. In contrast, with the three-dimensional model, the rates were -28%, -72% and -88%. The corresponding aggravation values were +370, +1,114% and +2,189% versus +38%, +256% and +747%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711062 TI - Results of balloon pulmonary valvuloplasty as a palliative procedure in tetralogy of Fallot. AB - Balloon pulmonary valvuloplasty was attempted in 67 patients with tetralogy of Fallot at a median age of 5 months (range 0.03 to 52 months) for relief of cyanosis. In three patients, the valve could not be crossed and an aortopulmonary shunt was performed. In 35 patients, follow-up angiography was performed 3 to 30 months (average 12) after valvuloplasty. In 24 of these 35 patients (group A), the stenosis had been adequately palliated by valvuloplasty; the other 11 patients (group B) had required an aortopulmonary shunt 1 month (range 0 to 3 months) after valvuloplasty. The two groups were similar (p greater than 0.1) with respect to age at valvuloplasty, pulmonary anulus diameter, ratio of pulmonary artery to descending aorta diameter before valvuloplasty and interval to follow-up angiography. In contrast to patients in group B, patients in group A had a significant immediate improvement in systemic arterial oxygen saturation (p less than 0.01) and a significant increase in pulmonary anulus diameter at follow up angiography (p less than 0.001). The growth of the branch pulmonary arteries was similar (p greater than 0.1) in the two groups. Among 42 patients who have had surgical correction, a transannular patch for right ventricular outflow tract reconstruction was used in 27 (64%); there was no difference between groups A and B with respect to its use. Eight patients died (three after repair) and death could not be directly attributed to valvuloplasty in any. Balloon valvuloplasty promotes growth of the pulmonary valve anulus and pulmonary arteries and is a useful alternative to an aortopulmonary shunt in patients with small pulmonary arteries or associated complex intracardiac defects. PMID- 1711063 TI - Is there a choice of palliation for tetralogy of Fallot? PMID- 1711064 TI - Serum type I collagen and N-terminal peptide of type III procollagen in chronic hepatitis. Relationship to liver histology and conventional liver tests. AB - The aim of this study was to compare serum N-terminal peptide of type III procollagen to aminotransferases and gamma-globulins as a marker for histological activity in patients with chronic hepatitis and to assess the role of type I collagen, a new serum marker, as a marker of fibrosis in these patients. Sixty patients with biopsy-proven chronic hepatitis were included in this study. Liver disease was virus B-related in 29, autoimmune in five, drug-induced in five, and of unknown etiology in 21. Each biopsy was independently assessed by two liver pathologists. Two histological scores, a score of activity and a score of fibrosis, were established. Serum N-terminal peptide of type III procollagen and type I collagen were assayed by liquid phase RIA. Significant correlations were noted between serum N-terminal peptide of type III procollagen and scores of activity (r = 0.70, p less than 10(-4)) and fibrosis (r = 0.45, p = 0.0005), and between serum type I collagen and scores of activity (r = 0.46, p = 0.0004) and fibrosis (r = 0.67, p less than 10(-4)). When the correlation between scores of activity and fibrosis (r = 0.52, p = 10(-4)) was considered by partial correlation, serum N-terminal peptide of type III procollagen was correlated with the score of activity (r = 0.63, p less than 10(-3)) but not with the score of fibrosis, and serum type I collagen was correlated with the score of fibrosis (r = 0.58, p less than 10(-3)), but not with the score of activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711065 TI - Detection of pre-S1 proteins in peripheral blood mononuclear cells from patients with HBV infection. AB - The presence of pre-S1 proteins in peripheral blood mononuclear cell (PBMC) samples from 115 patients with different forms of hepatitis B virus (HBV) infection was investigated by Western blot. Among 67 chronic HBsAg carriers, HBV antigens were detected in the PBMC in 80% for HBsAg, 27% for HBc/e Ag and 34% for pre-S1 proteins. The detection of pre-S1 proteins in PBMC was significantly associated with the presence of serum markers of HBV replication (HBV DNA and/or DNA polymerase). In the group of 48 consecutive patients negative for serum HBsAg, but positive for anti-HBc with or without anti-HBs, HBsAg and pre-S1 proteins could be detected in PBMC. This finding was more frequent among anti-HIV positive patients (77 and 23% of the cases, respectively) than in the negative ones (23 and 4% of the cases, respectively). The detection of HBV DNA and polyadenylated RNA in some of the PBMC samples positive for HBV proteins suggests that these proteins may be expressed in PBMC, especially during intense HBV replication. In patients negative for serum HBsAg, PBMC may constitute a reservoir of HBV. PMID- 1711066 TI - Effect of a new monoclonal antibody, TA-2, that inhibits lymphocyte adherence to cytokine stimulated endothelium in the rat. AB - An important event in the migration of lymphocytes out of the blood is their adherence to endothelial cells (EC). In inflammatory sites cytokines activate EC and promote lymphocyte EC adherence and migration. Small peritoneal exudate lymphocytes (sPEL) preferentially migrate from the blood to cutaneous delayed type hypersensitivity reactions and to sites injected with IFN-gamma, IFN alpha/beta, and TNF-alpha, rather than to peripheral lymph nodes. The basis of this migration is sPEL adherence to cytokine-activated EC. To study this adhesion mAb to rat sPEL were screened for inhibition of sPEL adherence to IFN-gamma stimulated EC. One mAb, TA-2, inhibited IFN-gamma-stimulated adherence to EC by 60%. This antibody had no effect on the baseline adherence of sPEL to unstimulated EC. Treatment of sPEL, but not EC, with TA-2-inhibited adhesion. TA 2 also inhibited adhesion to EC activated with mIL-1 alpha, TNF-alpha, and LPS, and the adhesion of spleen T cells to activated EC. The TA-2 Ag was expressed on virtually all lymph node, spleen, and sPEL lymphocytes but sPEL expressed two to three times higher levels than lymph node lymphocytes, and the highest levels were found on CD4+ and CD45R- memory T cells. TA-2 immunoprecipitated a group of four polypeptides with molecular mass of 150, 130, 83, and 66 kDa. Finally, TA-2 inhibited sPEL adhesion to TNF-alpha and IL-1 stimulated human umbilical vein EC to the same extent as an anti-human VCAM-1 mAb, and combinations of TA-2 and anti VCAM-1 were not different from treatment with either antibody alone. Thus, TA-2 appears to recognize rat VLA-4 based on immunoprecipitation, immunofluorescence, and lymphocyte EC studies. VLA-4 mediates the adhesion of rat lymphocytes to rat microvascular EC stimulated with IFN-gamma, mIL-1 alpha, TNF-alpha, and LPS. VLA 4 is important in the increased adhesion of sPEL to EC and the enhanced sPEL migration to inflammation may in part be explained by increased expression of VLA 4 on these cells. PMID- 1711067 TI - Expression and functional characterization of a soluble form of endothelial leukocyte adhesion molecule 1. AB - Endothelial leukocyte adhesion molecule 1 (ELAM1) is a leukocyte adhesion molecule induced on human venular endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for ELAM1 has been constructed, stably expressed in Chinese hamster ovary cells, and the secreted recombinant soluble form of ELAM1 (rsELAM1) purified to homogeneity by immunoaffinity chromatography. rsELAM1, when immobilized on plastic, is fully functional as an adhesion protein, and selectively binds only cells known to bind cell-surface ELAM1 expressed on human endothelial cells, including the myelomonocytic cell line HL60 and the colon carcinoma cell line HT29. Immobilized rsELAM1 also binds human PMN, monocytes, NK cells, and T cells. T cell subset analyses indicate preferential binding of CD4+ T memory cells. However, rsELAM1 is only a weak inhibitor of ELAM1-mediated adhesion. rsELAM1 should prove valuable for the further study of the role of ELAM1 expressed on the vascular wall during the inflammatory response. PMID- 1711068 TI - Structural and functional studies of the endothelial activation antigen endothelial leucocyte adhesion molecule-1 using a panel of monoclonal antibodies. AB - We have produced a panel of mAb to the endothelial activation Ag endothelial leucocyte adhesion molecule-1 (ELAM-1), using both a conventional immunization protocol and one involving immunosuppression. By constructing ELAM-1 mutants we have demonstrated that seven of these antibodies recognize epitopes within the lectin domain of ELAM-1 and that one binds within the complement regulatory protein domains. These studies also suggest that the EGF-like domain is important in maintaining the conformation of the neighbouring lectin domain. In functional studies, U937 cells bound to Cos cells expressing either ELAM-1 or ELAM-1 with the complement regulatory protein domains deleted. No adhesion was observed to Cos cells expressing ELAM-1 mutants lacking either the lectin or EGF-like domains. The fact that antibodies directed against the lectin domain can inhibit adhesion suggest that this domain is directly involved in cell binding. PMID- 1711069 TI - FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes. AB - In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP 140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1.2), suggesting a role in locomotion. On dividing multinegative thymocytes, FMC46 was found almost exclusively along the cleavage furrow, implicating it in detachment processes. We conclude that the properties of the LAM-1 molecule recognized by FMC46 are consistent with a role in detachment phases of motility and of cell interactions. PMID- 1711070 TI - Interaction of CD2 with its ligand lymphocyte function-associated antigen-3 induces adenosine 3',5'-cyclic monophosphate production in T lymphocytes. AB - CD2 (T11, the T cell E receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, is a T cell-specific surface protein that plays an important role in T lymphocyte adhesion, signal transduction, and differentiation. A natural ligand of CD2 is lymphocyte function associated Ag-3 (LFA-3 (CD58)), a widely expressed glycoprotein of 50 to 70 kDa. The physiologic interaction of CD2 with LFA-3 functions to increase intercellular adhesion and plays a role in T cell activation. This interaction, however, in the absence of other stimuli, has not previously been shown to induce intracellular signals such as Ca2+ mobilization or IL-2 production. To investigate whether cAMP may play a role in ligand triggered CD2-mediated signal transduction, we have studied the ability of purified LFA-3 and anti-CD2 mAb to induce changes in intracellular cAMP content in murine Ag-specific T cell hybridomas that stably express wild-type and mutated human CD2 molecules. By using a RIA sensitive to the femtomolar range and specific for cAMP, we demonstrate that purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3 alone to CD2-expressing hybridoma cells, however, did not stimulate phosphatidylinositol turnover nor IL 2 production. The cytoplasmic domain of CD2 is necessary for these ligand-induced cAMP changes, demonstrating that LFA-3 binding to CD2 transduces a signal to the cell. Experiments using the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine showed that CD2-mediated regulation of cAMP levels occurs primarily by the stimulation of cAMP production rather than by the inhibition of cAMP degradation. These results demonstrate that the interaction of LFA-3 with CD2, in the absence of other stimuli, is capable of initiating intracellular biochemical changes and suggest that CD2/LFA-3 interactions may regulate T cell function at least in part through the generation of intracellular cAMP. PMID- 1711071 TI - CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production. AB - The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production. PMID- 1711072 TI - A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain. AB - There are six known HLA-B alleles that share the HLA-B27 allospecificity, yet differ by one to six amino acid substitutions. Each of these B27 alleles can be readily assigned by one of the six representative IEF patterns. Two unrelated individuals, LH and HS, express B27 Ag that appear to be identical by IEF, but an HLA-B27 alloreactive CTL clone I-73 was found to react differently with these cells, suggesting these B27 molecules are not identical. We sequenced polymerase chain reaction-amplified B27 cDNA clones obtained from HS and compared its deduced amino acid sequence (B27-HS) with the B27 sequence of LH (B27-LH) which was previously designated the B*2701 allele. B27-HS and B27-LH differ by eight amino acids; three in alpha 1 domain and five in alpha 2 domain. These amino acid substitutions of B27-HS altered T cell recognition but not the B27 serologic epitope or IEF pattern. B27-HS differs from the six known B27 alleles by five to eight amino acid substitutions, and thus it represents the seventh allele of the HLA-B27 Ag family. This novel B27 allele might have been derived from a gene conversion event. Previously, two amino acid residues at positions 70 and 97 were suggested to be specific for B27 Ag family. B27-HS now reveals that Lys at position 70 is specific for B27 but Asn at position 97 is not. We propose that the region around position 70 might be crucial in determining the B27 serologic epitope and possibly in peptide Ag binding. This study also demonstrates that class I molecules of the same Ag specificity sharing an indistinguishable IEF pattern are not necessarily identical, and indicates that only the definitive determination of primary structure would identify all the class I alleles that are functionally relevant in regard to alloreactivity, T cell restriction, and disease association. PMID- 1711073 TI - Conformational and structural characteristics of peptides binding to HLA-DR molecules. AB - A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein. PMID- 1711074 TI - Comparison of structural requirements for interaction of the same peptide with I Ek and I-Ed molecules in the activation of MHC class II-restricted T cells. AB - We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107 116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules. PMID- 1711075 TI - Antibody MT3 is reactive with a novel exon B-associated 190-kDa sialic acid dependent epitope of the leukocyte common antigen complex. AB - MT3 is a new antibody reactive with a restricted isoform of the leukocyte common Ag (CD45) family. The Ag is mainly expressed on T lymphocytes and thymocytes. It is differentially expressed on B cell subpopulations, with no staining of the majority of small follicular mantle zone B cells but positive staining of the majority of marginal zone B cells of spleen. Like most CD45 antibodies, reactivity can be demonstrated in fresh frozen as well as in formalin-fixed, paraffin-embedded tissues. This reactivity clearly differs from all other published anti-CD45 antibodies. In immunoprecipitation and Western blot procedures, the antibody reacts with a major band with a molecular mass of 190 kDa and weak bands with molecular masses of 205 and 220 kDa. Compared to antibody PD7 that reacts with exon B-encoded sequences, the reactivity with the 205 and 220 bands is much weaker. This is reflected in MT3 reactivity with leukocyte common Ag transfectants that include exon B-encoded sequences, such as AB and B, but not with those that also include C-encoded sequences, such as ABC or BC. It can be concluded that MT3 recognizes additional heterogeneity in the leukocyte common Ag complex, that is based on the differential expression of sialic acid dependent determinants associated with exon B-encoded sequences. PMID- 1711076 TI - Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. AB - Murine bone marrow cells expressing the cell surface Ag RB6-8C5 were identified by fluorescence-activated cell-sorting analysis using a rat IgG mAb. The fluorescent intensity of RB6-8C5 was variable on bone marrow cells. This made it possible to separate bone marrow cells into distinct subpopulations, RB6-8C5neg, RB6-8C5lo, and RB6-8C5hi cells. Morphologic analysis of the sorted populations demonstrated that the Ag was expressed on myeloid cells. The expression of RB6 8C5 increases with granulocyte maturation, whereas expression is transient on cells in the monocytic lineage. The RB6-8C5hi sorted cells were enriched for end stage neutrophils (75%), whereas the RB6-8C5lo sorted cells contained more immature myeloid cells and myelocytes (75%). Lymphocytes and macrophages were less than 5% in any RB6-8C5+ population, whereas the erythroid precursors were RB6-8C5neg. The colony forming unit culture (CFU-C) (greater than 90%) were found in the RB6-8C5neg and RB6-8C5lo populations, and all the CFU-granulocyte, erythroid, megakaryocyte, and macrophage (CFU-GEMM) and burst-forming units erythroid (BFU-E) were in the RB6-8C5neg population. Granulocyte-macrophage-CSFR (GM-CSFR) and IL-1 alpha R were expressed on RB6-8C5hi bone marrow cells, whereas no receptors could be detected on RB6-8C5neg and RB6-8C5lo cells. The expression of the RB6-8C5 Ag can be induced on RB6-8C5neg cells in liquid culture by IL-3 and granulocyte-macrophage CSF. Thus, RB6-8C5 is a myeloid differentiation Ag whose expression can be regulated by cytokines. PMID- 1711077 TI - Platelet-activating factor induces mediator release by human basophils primed with IL-3, granulocyte-macrophage colony-stimulating factor, or IL-5. AB - The phospholipid platelet-activating factor (PAF) is a potent cell-derived bioactive molecule thought to be involved in diverse inflammatory processes. It has been shown that PAF can activate different leukocyte types and platelets, particularly in synergy with other agonists. In this study we examined the effect of PAF upon the release of histamine and leukotriene (LT) C4 by basophils when added alone and in combination with different agonists and cytokines. PAF by itself did neither induce histamine release nor the generation of LTC4 by basophils. However, basophils primed by the hematopoietic growth factors (hGF) IL 3, granulocyte-macrophage (GM)-CSF, or IL-5 (10 ng/ml) released preformed and de novo synthesized mediators in response to PAF at 10 to 100 nM concentrations. The extent of mediator release by hGF primed basophils in response to PAF was similar to that induced by an optimal concentration of monoclonal anti-IgE. Thus, similar to NAP-1/IL-8 and C3a, PAF efficiently stimulates mediator release in hGF-primed basophils only. However, PAF was clearly a more potent trigger of LTC4 formation in IL-3-primed cells than NAP-1/IL-8 or C3a. When PAF was used as a second trigger, the priming effect of IL-5 was less than that of IL-3 or GM-CSF, whereas the response for other IgE-independent agonists (i.e., C5a or FMLP) was augmented equally by all three hGF. IL-1 beta-pretreated basophils released minimal amounts of histamine in response to PAF. Neither TNF-alpha nor PAF, nor the combination thereof, was able to induce basophil mediator release. The efficiency of the different cytokines to prime for PAF responsiveness was strikingly similar to their capacity to enhance anti-IgE-induced mediator release. Similar to other IgE independent agonists, the kinetic of mediator release in response to PAF was very rapid. PAF pretreatment of basophils did not enhance mediator release in response to diverse agonists, such as C5a and FMLP, in contrast to the capacity of PAF to augment the response of other leukocyte types to appropriate stimuli. Thus, depending on the presence of IL-3, GM-CSF, or IL-5, PAF is a potent basophil agonist capable of inducing histamine release as well as de novo synthesis of LTC4. PMID- 1711078 TI - Vaccination with the major secretory protein of Legionella induces humoral and cell-mediated immune responses and protective immunity across different serogroups of Legionella pneumophila and different species of Legionella. AB - In a previous study, we demonstrated that immunization of guinea pigs with the major secretory protein (MSP) of Legionella pneumophila, serogroup 1 induced humoral and cell-mediated immune responses to MSP and protective immunity against lethal aerosol challenge with this serogroup of L. pneumophila. Although serogroup 1 L. pneumophila cause most cases of Legionnaires' disease, other serogroups of L. pneumophila and species of Legionella cause many cases. In this study, we have examined if immunization with MSP induces humoral and cell mediated immune responses and protective immunity across different serogroups of L. pneumophila and species of Legionella. By immunoblot analysis, MSP from L. pneumophila serogroup 1 (Lp1 MSP), L. pneumophila serogroup 6 (Lp6 MSP), and Legionella bozemanii (Lb MSP) shared common epitopes recognized by guinea pig anti-Lp1 MSP antiserum. These MSP molecules, however, were not identical as they had different apparent m.w. Immunization of guinea pigs with MSP induced strong cell-mediated immune responses across the different serogroups and species, as indicated by splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity in response to both homologous and heterologous MSP. Immunization with MSP induced strong protective immunity across two serogroups of L. pneumophila; overall, 9 survived aerosol challenge with L. pneumophila serogroup 1 compared to 0 of 12 (0%) sham-immunized control animals (p = 3 x 10( 4), Cochran-Mantel-Haenzel chi 2 statistic for pooled data). Immunization with MSP also induced protective immunity across species of Legionella but protection was species-specific. Whereas immunization with Lb MSP induced protective immunity against L. pneumophila, neither immunization with Lp1 MSP nor immunization with Lb MSP induced protective immunity against L. bozemanii, which produces MSP. Not surprisingly, immunization with MSP did not induce protective immunity against MSP-negative Legionella micdadei. In the case of both L. bozemanii and L. micdadei, immunization with a sublethal dose did confer protective immunity to aerosol challenge indicating that these species do contain immunoprotective components. This study demonstrates that immunization with MSP induces humoral and cell-mediated immune responses across different serogroups of L. pneumophila and species of Legionella, but that the capacity of MSP immunization to induce protective immunity is species-specific. Nevertheless, an MSP vaccine has the potential to induce protective immunity against the great majority of cases of Legionnaires' disease. PMID- 1711079 TI - Effect of genistein, a tyrosine kinase inhibitor, on latent EBV activation induced by cross-linkage of membrane IgG in Akata B cells. AB - The activation of phosphoprotein tyrosine kinases was studied in the regulation of EBV activation in Akata cells after cross-linking membrane IgG with anti-IgG. Protein tyrosine phosphorylation was induced in Akata cells after stimulation with anti-IgG, as determined by immunoblotting with the PY20 anti-phosphotyrosine mAb. The frequency of phosphotyrosine-activated cells was also measured by immunofluorescence with the PY20 antibody. Genistein, an inhibitor of tyrosine kinases, at non-cytotoxic doses blocked EBV activation, as measured in the induction of EBV Ag, EBV immediate early BZLF1 mRNA, and its protein product ZEBRA. Such inhibitions were reversed upon removing genistein from the cultures. Genistein inhibition of early Ag induction depended upon the time of addition of genistein after stimulation with anti-IgG. These findings indicate that activation of tyrosine kinase is required for EBV activation after cross-linking membrane IgG in Akata cells. PMID- 1711080 TI - Lysis of Neisseria gonorrhoeae initiated by binding of normal human IgM to a hexosamine-containing lipooligosaccharide epitope(s) is augmented by strain specific, properdin-binding-dependent alternative complement pathway activation. AB - We studied the specificity of naturally acquired IgM bactericidal for strains of Neisseria gonorrhoeae that varied in sensitivity to the lytic action of normal human serum (NHS) and the relative ability of these strains to deplete the classical (CP) and alternative (ACP) C pathways. Lysis of both highly sensitive and relatively insensitive strains was inhibited by the same gonococcal lipooligosaccharides (LOS), as well as by Salmonella minnesota Re LOS and three hexosamine-containing glycose polymers. A polymer of N-acetylgalactosamine phosphate was the most inhibitory; a polymer of N-acetylglucosamine phosphate only partially inhibited. Neither 3-deoxy-D-manno-octulosonic acid (dOc1A) nor a polymer that contained dOc1A but not hexosamine inhibited NHS lysis. A co-polymer of N-acetylgalactosamine-dOc1A inhibited both bactericidal activity and the binding of IgM to the LOS of a highly serum-sensitive (sers) gonococcal strain. Carboxyl reduction of the dOc1A in this polymer did not affect its inhibitory capacity for gonococcal antibody, but abolished its binding to homologous antibody induced by vaccination. CP activity was not affected by vaccination. CP activity was not affected by absorption of NHS with gonococcal strains, whereas ablation of CP activity markedly but variously diminished lytic activity for highly sers strains. ACP activity was variously depleted by gonococcal strains, and the proportion of bacteria that could be lysed through the ACP varied among strains and among different populations of a given strain. The titer at which a strain was sensitive to NHS lysis was a function of its ACP consumption (p = 0.006), which accounted for 70% of the differences in titer among strains. Analyses of the absorbed sera revealed that the gonococci had variously depleted properdin from NHS as assessed by using an Ag-capture solid-phase RIA. Addition of purified properdin to absorbed sera restored ACP activity to normal levels. Western immunoblots of gonococcal lysates showed that purified properdin bound directly to a 39-kDa outer membrane protein. We conclude that both CP activation by IgM binding to LOS epitopes, one of which contains hexosamine, and ACP activation, which is a function of strain-specific direct binding of properdin, can initiate lysis of sers strains and that ACP activation, also enhances lysis and accounts for variations in sensitivity of sers strains. PMID- 1711081 TI - Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys. AB - Infection of macaque monkeys with the simian immunodeficiency virus of macaques (SIVmac) results in disease similar to human AIDS. Therefore, the macaque monkey is proving to be an important model for testing the effectiveness of various AIDS vaccine approaches. A detailed analysis of the cellular immune responses is necessary for the evaluation of candidate vaccines. However, this has not been possible in macaques, due, in part, to the unknown nature of the MHC molecules that restrict their T lymphocytes. In our report we demonstrate that a particular MHC class I molecule involved in the rhesus monkey's effector T lymphocyte response to SIVmac is expressed at a high frequency in a colony of rhesus monkeys. SIVmac-infected monkeys that express this MHC class I molecule all develop CTL that are restricted by that molecule and recognize an identical nine amino acid epitope of the SIVmac gag protein. This MHC class I molecule has been defined as an HLA-A homolog by cDNA cloning and sequencing. It has also been expressed in an MHC class I-deficient cell line to demonstrate directly the cloned molecule's capacity to bind and present peptide Ag to CTL. These studies illustrate that AIDS virus-specific CTL can be characterized in detail in the rhesus monkey and lay the foundation for exploring novel approaches to AIDS virus vaccination in this species. PMID- 1711082 TI - Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization. AB - We have generated a number of mAb against various epitopes on the external envelope glycoprotein, gp46, of human T cell leukemia virus type I (HTLV-I) from a WKA rat immunized with a recombinant vaccinia virus containing the HTLV-I env gene. Among these mAb, one group of mAb, represented by a mAb designated LAT-27, could neutralize the infectivity of HTLV-I, as determined by a HTLV-I-mediated cell fusion inhibition assay. LAT-27 also interfered with transformation of normal T lymphocytes by HTLV-I in vitro. An antibody-binding assay using overlapping synthetic oligopeptides showed that LAT-27 bound specifically to 10 mer peptides that contained the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser Asn-Leu). Antibodies from HTLV-I+ humans interfered with the binding of LAT-27 to gp46 Ag. Sera from rabbits immunized with a LAT-27-reactive peptide, 190-199, conjugated with OVA, but not sera from OVA-immunized rabbits, reacted with gp46 Ag and neutralized infectivity of HTLV-I. These results show that the HTLV-I neutralization epitope recognized by LAT-27 locates to the gp46 amino acids 191 196, and that immunization with a peptide containing the LAT-27 epitope can elicit an HTLV-I neutralizing antibody response. PMID- 1711083 TI - Recurrent utilization of genetic elements in V regions of antinucleic acid antibodies from autoimmune mice. AB - Two IgG anti-DNA and two IgG anti-RNA autoantibodies derived from lupus prone NZB/NZW F1 mice have been analyzed for their Ag fine specificities and for their H and L chain V-region sequences. A remarkable similarity of VH gene sequences with previously sequenced antinucleic acid autoantibodies (Eilat, D., D. M. Webster and A. R. Rees. J. Immunol. 141:1745, 1988) was noted. This finding indicates that a small number of unique VH genes is involved in this autoimmune response and that the sequences of these genes are correlated with the different specificities for the autoantigen. The VK sequences appeared, by computer search, to be selected nonrandomly, but their use was not restricted to autoantibodies. An additional striking feature was evident in the construction of the D region elements, giving rise to CDR3 peptides that can interact with DNA and RNA. These constructs probably include D-D fusion products, which are relatively rare in Ig rearrangements. PMID- 1711084 TI - Endothelial leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 mediate the adhesion of eosinophils to endothelial cells in vitro and are expressed by endothelium in allergic cutaneous inflammation in vivo. PMID- 1711085 TI - Anti-CD40 monoclonal antibodies or CD4+ T cell clones and IL-4 induce IgG4 and IgE switching in purified human B cells via different signaling pathways. AB - IL-4 induces IgE and IgG4 synthesis, but in addition to IL-4, a second signal provided by CD4+ T cells is required. Here we demonstrate that the signal provided by CD4+ T cells can be replaced by anti-CD40 mAb. Highly purified surface (sIgM+) human B cells cultured with soluble anti-CD40 mAb in the presence of IL-4 produced IgM, total IgG, IgG4, and relatively high levels of IgE, indicating that production of these isotypes represented H chain switching and was not the result of a selective outgrowth of isotype committed B cells. No IgA was produced in these cultures. However, the T cell signal was different from the signal provided by anti-CD40 mAb, because in contrast to CD4+ T cells, anti-CD40 mAb failed to induce germ-line epsilon transcripts. However, anti-CD40 mAb strongly enhanced germ-line epsilon mRNA expression induced by IL-4. In addition, IFN-gamma, IFN-alpha, and anti-CD23 mAb, which block IL-4-induced IgE production by PBMC, or B cells cocultured with CD4+ T cell clones, failed to inhibit IgG4 and IgE synthesis induced by anti-CD40 mAb. Finally, anti-CD40 mAb and CD4+ T cell clones had strong synergistic effects on IgG4 and IgE synthesis. These results indicate that different B cell activation pathways can result in IgG4 and IgE switching in the presence of IL-4. PMID- 1711086 TI - Trichophyton tonsurans allergen. I. Characterization of a protein that causes immediate but not delayed hypersensitivity. AB - Fungal infections of skin or nails are extremely common and often caused by dermatophyte fungi of the genus Trichophyton. These fungi are unusual in that they can give rise to delayed hypersensitivity (DH) or immediate hypersensitivity (IH) responses. Recently, IH to Trichophyton tonsurans has been demonstrated in patients by skin tests, serum IgE antibody test (RAST), and positive nasal and bronchial challenges. To further investigate the immunology of Trichophyton, a 30 kDa T. tonsurans allergen was isolated by gel filtration and hydrophobic interaction chromatography. This protein, Tri t I, gave a single band on SDS PAGE, and the 30 amino-terminal amino acids have been determined. Among patients with positive IH skin tests, 34 of 48 (71%) had IgG antibody and 26 of 48 (54%) had IgE antibody to Tri t I. Among those who had positive responses to both skin tests and RAST, 22 of 30 (73%) had IgE antibodies to Tri t I; thus, this protein represents a major allergen. Twelve clones of murine IgG mAb antibodies were produced. Two clones, 2F2-F7 and 6B11-C2, were found to define separate epitopes on Tri t I and were used to develop an immunometric assay for the quantitation of Tri t I. Twenty-three of 38 volunteers with a history of athlete's foot were found to have either IH and/or DH to Trichophyton mix and underwent further testing with purified Tri t I. Of the nine found to have IH to the mix, eight were sensitive to Tri t I. Seven of these eight had IgG and IgE antibodies to Tri t I, by Ag-binding RIA, and all were RAST positive to the unpurified extract. An additional 14 had either DH alone (n = 7) or a wheal and flare response followed by DH at 48 h (n = 7). Of these 14 who had DH responses to Trichophyton mix, only one showed DH to an equivalent quantity of purified Tri t I; among this group, none showed IH or serum IgE antibodies and only one had detectable IgG antibody to Tri t I. The results suggest that the majority of subjects with DH to Trichophyton are responding to a protein other than Tri t I and that the wheal that precedes DH reactions is some patients is not associated with IgE antibodies. PMID- 1711087 TI - Effects of allopurinol on ischemic experimental pancreatitis. AB - The effect of allopurinol, a xanthine oxidase inhibitor, on canine experimental ischemic pancreatitis was studied. The animals were divided into nine groups: 1. Group 1. Control with pancreatic ischemia; 2. Group 2. Received allopurinol once, previous to ischemia; 3. Group 3. Received allopurinol once, immediately after ischemia; 4. Group 4. Received allopurinol immediately after ischemia and then daily; and 5. Groups 5, 6, and 7 were controls for the operation, allopurinol, and its vehicle, respectively; 6. Group 8 (pancreatic ischemia) and Group 9 (that received allopurinol after ischemia and daily) were also studied histologically. Serum amylase was determined in all animals. In Groups 1 and 5, following the ischemic period, hyperamylasemia developed and a peak was reached 24 h after ischemia. In Group 2, a significant decrease of amylase levels was found, compared to matched controls immediately after ischemia and then rose, reaching on the fifth day a peak that was less than the controls at 24 h. In Group 3, the serum amylase level increased immediately to values similar to controls; later, there was a drop to levels lower than those found in controls, followed by a peak on the fifth day. In Group 4, there was no significant elevation in the amylase values. Groups 6 and 7 showed no changes of amylasemia. In this experimental model, allopurinol blocked or ameliorated significantly cellular injury, as shown by a decrease of amylase levels in blood, and of histopathological changes, depending on dose and time of administration. These results offer the possibility of a prophylactic therapy for chronic relapsing and idiopathic pancreatitis. PMID- 1711088 TI - Heating characteristics of a helical microwave applicator for transurethral hyperthermia of benign prostatic hyperplasia. AB - A new applicator for intraurethral hyperthermic treatment of benign prostatic hyperplasia is described. The applicator uses an insulated helical antenna wound on the outer surface of a silicone urological (Foley) balloon catheter. The balloon catheter assures rapid and reproducible localization of the antenna in the prostatic urethra. Two small cannulae are fixed to the exterior surface of the applicator. One holds a temperature control sensor at a fixed location, the other is used to map temperature along the applicator. Two-dimensional SAR and steady-state temperature distributions measured in a plane tangent to the applicator in a tissue-equivalent phantom are presented, as well as longitudinal temperature distributions measured in situ at the applicator-urethral interface. Prostatic temperatures were also measured intraoperatively. The applicator appears to be capable of elevating temperature to greater than 42 degrees C in a cylindrically symmetric volume of about 4 cm length and about 0.5 cm radial penetration surrounding the antenna. The heating characteristics of this applicator are similar to an earlier design that employed an array of three dipoles. The helical applicator is narrower, more flexible and simpler to use than the earlier design. PMID- 1711089 TI - Combination radiotherapy of urinary bladder carcinoma with chemohyperthermia. AB - The combination therapy of urinary bladder cancer with radiation and hyperthermia with bleomycin was investigated. Immediately following daily external irradiation (40 Gy/4 weeks), patients were irrigated with a solution of warmed saline (intravesical temperature, 42-43 degrees C) containing 30 micrograms/ml bleomycin. Of a total of 56 patients, complete responses were observed in 25, and partial responses in 21. Among T2-T3 cases, an 84% response rate was noted in combination therapy, whereas a 56% response rate was observed after radiation alone (50-70 Gy). The side-effects of the combination therapy were limited to reversible bladder irritation, and bladder capacity could be maintained within normal limits. These results suggest that combination therapy represents an effective conservative therapy for the management of bladder cancer. PMID- 1711091 TI - Effect of gamma-aminobutyric acid on intracellular pH in the crayfish stretch receptor neurone. AB - The effect of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) was examined in the crayfish stretch-receptor neurone using H(+)-selective microelectrodes and a two-microelectrode voltage clamp. In the presence of 30 mmol l-1 HCO3- (pH 7.4), application of GABA (0.5 mmol l-1) produced a mean fall in pHi of 0.26 units. The initial rate of fall of pHi was attributable to a net influx of acid equivalents of 6.3 mmol l-1 min-1. In the nominal absence of HCO3 , GABA had little effect on pHi. The HCO3(-)-dependent acidosis caused by GABA was inhibited by picrotoxin (0.1 mmol l-1) but not by depletion of extracellular and intracellular Cl-. Acetazolamide (0.1 mmol l-1) decreased the rate of fall of pHi caused by a step increase in CO2 partial pressure as well as by GABA, which indicates that the neurone contains carbonic anhydrase. In the presence of both Cl- and HCO3-, the reversal potential of the GABA-activated current was more positive than under nominally HCO3(-)-free conditions. In line with this, GABA induced a marked HCO3(-)-dependent depolarization, and this depolarizing action was enhanced in the absence of Cl- so as to lead to triggering of action potentials. All these observations support the conclusion that the GABA-induced fall in pHi is due to a net efflux of HCO3- through the inhibitory anion channels. PMID- 1711090 TI - Trans-catheter hepatic arterial injection of lipiodol soluble anti-cancer agent SMANCS and ADR suspension in lipiodol combined with arterial embolization and local hyperthermia for treatment of hepatocellular carcinoma. AB - The clinical effect and safety of Lp-TAE alone and combined with radiofrequency (RF) capacitive hyperthermia (HT) were evaluated in 20 patients with hepatocellular carcinoma (HCC) associated with cirrhosis of the liver. After the oily carcinostatic agents were administered by Lp-TAE, HT, at a temperature of greater than 42.5 degrees C, was induced for 40 min, twice a week by an RF of 8 MHz for a total of 10 to 38 times. The response rate was 40% in the 10 cases that were treated with Lp-TAE combined with HT and 20% in the 10 cases that were treated with Lp-TAE. The patients who were treated with Lp-TAE combined with HT had a tendency to have better survival rates than those of the Lp-TAE group (p less than 0.099). The main side-effects of Lp-TAE combined with HT were low-grade fever, localized pain, myelo-suppression and liver dysfunction, but these were transient and eventually disappeared. PMID- 1711092 TI - Intrathecal IgG synthesis and specificity of oligoclonal IgG in patients infected with HIV-1 do not correlate with CNS disease. AB - The CSF/serum immune response to HIV 1 was studied in 24 patients admitted for investigation. The level of antibody to HIV-1 and specificity of oligoclonal IgG were determined in blood and cerebrospinal fluid (CSF). The majority of patients demonstrated elevated levels of intrathecal IgG synthesis, with levels of HIV-1 specific antibody frequently being significantly higher in CSF than in serum. In 16 of 21 patients the CSF/serum antibody ratio indicated active intrathecal synthesis. Oligoclonal banding was present in CSF from all 24 patients. Immunoprinting of serum and CSF demonstrated antigenic specificity (p24, gp 160, RT) of the clonal antibodies in all of 12 patients though the patterns of reactivity in CSF did not necessarily correspond with that of serum. Although a specific association of particular patterns with HIV CNS disease was not found we feel that these markers should be included in longitudinal studies of HIV-related diseases of the CNS. The specificity of oligoclonal antibodies, both in CSF and in serum was demonstrated, and this specificity may be a useful marker for longitudinal studies in HIV-1 antibody-positive asymptomatic patients. PMID- 1711093 TI - Intrafamilial transmission of hepatitis C virus in Japan. AB - To clarify the intrafamilial transmission of hepatitis C virus (HCV), the prevalence of antibody to HCV (anti-HCV) in 107 index patients with type C chronic liver disease was studied and compared with the prevalence of anti-HCV antibody in their 296 family members. Of the 85 index patients who were positive for anti-HCV, 15 (8%) of 196 of their family members were also HCV antibody positive, whereas of the 22 index patients who were anti-HCV antibody negative, none of the family members of the 100 evaluated was positive for anti-HCV antibody, a statistically significant difference between groups (P less than 0.02). No specific relative (spouse, child, parent, and sibling) was linked to HCV positivity in the index cases making it difficult to identify the route of infection that is believed to occur via the parenteral route in the home or community. PMID- 1711094 TI - Significance of antibody to hepatitis C virus in Japanese patients with viral hepatitis: relationship between anti-HCV antibody and the prognosis of non-A, non B post-transfusion hepatitis. AB - In a retrospective study, antibody to hepatitis C virus (anti-HCV antibody) was measured in 80 patients with acute viral hepatitis (type A, 18; type B, 21; type non-A,non-B, 41). Anti-HCV antibody was found in 12 of 20 patients (60%) with non A,non-B post-transfusion hepatitis (NANB-PTH) and in 9 of 21 patients (43%) with sporadic NANB hepatitis (NANB-SPO). Patients with acute hepatitis type A or type B did not have anti-HCV antibody. The number of patients who developed chronic hepatitis was greater in the group with anti-HCV antibody than in the anti-HCV negative group in both NANB-PTH and NANB-SPO. The difference was significant in those with NANB-PTH (P less than 0.05). To investigate the relationship between the long-term prognosis of NANB-PTH and the course of anti-HCV, we studied anti HCV antibody in 12 patients who developed chronic type C hepatitis (C-CH) after PTH and followed them for more than 5 years after the development of PTH. One year after the development of PTH, all 12 had anti-HCV antibody. Five lost anti HCV antibody (group 1) while 7 remained positive (group 2) at the final examination. Four of the 5 patients in group 1 had normal serum transaminases; however, abnormal transaminase persisted in all 7 patients in group 2 until the end of follow-up (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711095 TI - Cerebral polyamine metabolism in reversible hypoglycemia of rat: relationship to energy metabolites and calcium. AB - Thirty minutes of insulin-induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20-microns thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p less than or equal to 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p less than or equal to 0.01 and p less than or equal to 0.05, respectively). Twenty-four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1-subfield.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711096 TI - Differential effects of acidic and basic fibroblast growth factors on spinal cord cholinergic, GABAergic, and glutamatergic neurons. AB - When spinal cord cultures from embryonic day 12 rats were cultured at low density, both acidic and basic fibroblast growth factors significantly increased neuronal survival and neurite outgrowth in a dose-dependent manner. The effects of acidic fibroblast growth factor were independent of heparin, in contrast to its mitogenic effects on both NIH3T3 cells and cerebral cortical astrocytes. In high-density cultures, acidic fibroblast growth factor increased choline acetyltransferase activity by 57%, glutamic acid decarboxylase activity by 58%, and aspartate aminotransferase activity by 65%. Basic fibroblast growth factor increased choline acetyltransferase activity by 73% and glutamic acid decarboxylase activity by 200% but decreased aspartate aminotransferase activity by 40%. Growing these cultures in the presence of a mitotic inhibitor did not significantly alter the effect of acidic or basic fibroblast growth factor on these enzyme activities. These results demonstrate that acidic and basic fibroblast growth factors differentially affect neurotransmitter enzyme levels of multiple classes of neurons, rather than having effects on a single neuronal population. PMID- 1711097 TI - Solubilization of myelin membranes by detergents. AB - The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included. PMID- 1711098 TI - Hypoxia increases the cyclic AMP content of the cat carotid body in vitro. AB - The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 +/- 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 +/- 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 microM) nor by propranolol plus haloperidol (27 microM). Inhibition of the cyclic nucleotide phosphodiesterase with 1-methyl-3-isobutylxanthine (0.8 mM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hypoxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N-(cis-2 phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (MDL 12330A; 20-1,000 microM) under zero-Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2(+)-calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50-200 microM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the PO2-sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone-receptor interactions. PMID- 1711099 TI - Expression of transferrin mRNA in the CNS of normal and jimpy mice. AB - Both the iron mobilization protein transferrin and iron itself are found predominantly in oligodendrocytes in the brain and consequently have been hypothesized to have a role in myelination. This study is designed to begin to understand the mechanism(s) that control the expression of transferrin at the gene level in the nervous system using a hypomyelinating murine mutant (jimpy mouse). With this animal model it is possible to determine if transferrin gene expression in the nervous system is dependent on the presence of a mature oligodendrocytic population. The results demonstrate that normally expression of the transferrin gene increases from postnatal day 5 to 22-25 and then levels off in the adult. In the jimpy mouse, the relative amount of transferrin gene expression is less than that of littermate controls at 5 days of age. Furthermore, transferrin gene expression does not increase with age beyond the level observed at postnatal day 5 in the jimpy mouse. It is concluded from this study that the majority of the transferrin mRNA in the mouse brain is expressed by and/or requires the presence of a mature oligodendrocytic population. PMID- 1711101 TI - Acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in a model of rat designated a poor metabolizer of debrisoquine. AB - The relationship between oxidative polymorphisms and the cause of Parkinson's disease is controversial. The drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces parkinsonism in humans and in some animal models, is metabolized by cytochrome P450 db1 isozyme (the same enzymatic system implicated in 4-hydroxylation of debrisoquine). In this study, we treated females of three rat species, which differ in their ability to hydroxylate debrisoquine, with MPTP (three doses of 30 mg/kg s.c. at 12-h intervals), and we measured their motor activity and brain monoamine levels. Female dark-adapted rats (poor metabolizers of debrisoquine) showed a more pronounced and more maintained reduction of their motor activity after treatment with MPTP. MPTP-treated, dark-adapted rats also had a depletion of noradrenaline in the diencephalon and a depletion of dopamine and serotonine and their respective metabolites in the limbic system when compared with the other two species. These results suggest that oxidative polymorphism of debrisoquine plays a role in the acute effects of MPTP. PMID- 1711100 TI - Structure of the mouse myelin P2 protein gene. AB - Myelin P2 protein is a small (14,800 Da) protein found in peripheral and central nervous system myelin. To investigate the regulation of expression of this myelin protein, a mouse genomic library was screened with a rabbit P2 cDNA (pSN2.2-2), and a single positive phage clone containing a 20-kb insert was obtained. This insert contained a single internal SalI restriction site and several EcoRI sites. The EcoRI fragments from this insert were subcloned into Bluescript. The rabbit P2 cDNA (pSN2.2-2) hybridized with a 4-kb EcoRI fragment, and this 4-kb fragment was then sequenced after the creation of nested deletions. The mouse gene contained four exons: exon 1 coded for amino acids 1-24, exon 2 for amino acids 24-81, exon 3 for amino acids 82-115, and exon 4 for amino acids 116-131. The three introns were 1.2 kb, 150 bp, and 1.3 kb in length. Primer extension analysis revealed two transcription start sites at +1 and +47, consistent with the presence of two P2 mRNAs, with the larger transcript appearing more abundant. The amino acid sequences predicted from the mouse DNA indicate that the mouse protein differs from the rabbit protein at 16 different positions, with most of the differences located in exon 3. When the gene structure of fatty acid binding protein (FABP) genes were compared, the P2 gene had the same overall structure as others in the FABP family, suggesting a common ancestral gene for members of this family. The 5'-flanking region contains candidate TATA and CAAT sequences, as well as two AP-1 binding sites that may be important in modulation of the expression of this gene. PMID- 1711102 TI - A pi form of glutathione-S-transferase is a myelin- and oligodendrocyte associated enzyme in mouse brain. AB - The pi form of glutathione-S-transferase (GST), previously found to be oligodendrocyte associated, has also been found in myelin. In the brains of adult mice, immunocytochemical staining for a pi form of GST was observed in myelinated fibers, as well as oligodendrocytes. In contrast, and as previously found in rats, positive immunostaining for mu forms occurred in astrocytes and not in oligodendrocytes or myelinated fibers. Double immunofluorescence staining strengthened the conclusion that pi was in oligodendrocytes and myelin in mouse brains. The results of enzyme assays performed on brain homogenates and purified myelin indicated that GST specific activities in mouse brain myelin were almost as high (0.8-fold) as those in mouse brain homogenates. Low, but reproducible, GST activities were also observed in myelin purified from rat brains, in which pi had been demonstrated in oligodendrocytes and mu in astrocytes. The pi form was also stained by the immunoblot technique in myelin purified from mouse brain. It was concluded that pi is a myelin associated, as well as oligodendrocyte associated, enzyme in mouse brain, and possibly also in rat brain. This is the first report of localization of GSTs in mouse brain and of GST in myelin. PMID- 1711103 TI - Cellular relationships of paranodal Marchi-positive bodies studied with monoclonal antibodies against partially degraded CNS myelin fragments. AB - Histochemical and electron microscopical studies have shown that Marchi-positive bodies in the normal mammalian CNS are associated with paranodal regions and it has been proposed that the formation of Marchi-positive bodies represents a step in the catabolic events of normal myelin turnover. After a two-step density gradient ultracentrifugation a light 'floating fraction' highly enriched in these structures can be collected and in the present study two monoclonal antibodies, FC4 and 3B5, were produced against proteins present in the floating fraction but absent from the myelin fraction. Immunohistochemical studies showed that these antibodies bound preferentially to the PNS-CNS transitional region and the glia limitans. Double-staining experiments demonstrated an extensive overlap in these regions with cells stained by antibodies against the astrocyte marker glial fibrillary acidic protein. Centrally in the white matter, FC4 and 3B5 mainly stained cells which also stained with the microglia lectin marker Bandeiraea simplicifolia isolectin B4. Three-dimensional reconstructions made from confocal microscopic scans showed that FC4/3B5-positive cells in the white matter extend processes enveloping paranodal Marchi-positive bodies and nodes of Ranvier. It is suggested that astrocyte-like and microglia-like cells both are participants in paranodal myelin turnover and that a division of labour with respect to, for example, protein degradation and immunological functions, may be present between the two cell types. PMID- 1711104 TI - Synaptic potentials evoked in spiny neurons in rat neostriatal grafts by cortical and thalamic stimulation. AB - 1. Fetal rat striatal primordia were implanted into the neostriatum of adult rats 2 days after kainic acid lesion. Two to 6 mo after transplantation, in vivo intracellular recording and staining were performed to study the responses of spiny neurons in the grafts to the cortical and thalamic stimuli. The physiological characteristics and synaptic responses of 27 cells recorded in the grafts were compared with a sample of 23 neurons recorded from the surrounding host neostriatum in the same animals. Nineteen of the graft neurons and 19 of the host neurons were identified as spiny neurons by intracellular staining with biocytin. The responses of the remaining neurons were the same as those of identified spiny cells. 2. The spontaneous synaptically driven membrane potential shifts and long-lasting responses to afferent stimulation that are characteristic of neostriatal cells in normal animals were greatly reduced or absent in graft neurons. Presumably this reflects the reduction in synaptic input to the grafts and the lack of convergence of inputs from diverse sources. 3. Short-latency synaptic responses to cortical and thalamic stimulation were present and could consist of either excitatory postsynaptic potentials (EPSPs) or inhibitory postsynaptic potentials (IPSPs). The IPSPs were accompanied by a membrane conductance increase, and their reversal potentials could be altered by injection of chloride ions. Several minutes after impaling the cell, the IPSPs gradually disappeared, and the same stimuli could then evoke EPSPs. The disappearance of the IPSPs was independent of the presence of chloride in the electrodes. Most of the EPSP responses appeared to be monosynaptic but occurred at longer latencies than those seen in host neurons of the same type. 4. In cells not exhibiting IPSPs, or after the IPSP responses disappeared, cortical or thalamic stimulation could evoke slow depolarizing potentials and bursts of action potentials. These could not be evoked by current injection. They could be prevented or delayed by an exaggerated action potential after hyperpolarization that developed in neurons maintained in a depolarized state for several seconds, but could not be prevented by passage of hyperpolarizing current from the recording electrode. 5. The input resistance of graft spiny neurons was higher than that of the host cells, and time constants were longer. Both of these properties appeared to be due to the absence of the strong inward rectification that is usually present at resting membrane potentials in neostriatal neurons. PMID- 1711105 TI - Differentiation of NG108-15 cells alters channel conductance and desensitization kinetics of the 5-HT3 receptor. AB - 1. NG108-15 cells undergo morphological differentiation in response to appropriate culture conditions. We have used patch clamp techniques to compare responses mediated by the 5-HT3 receptor in differentiated and undifferentiated NG108-15 cells. 2. In differentiated cells, desensitization of 5 hydroxytryptamine (5-HT) responses was much slower than in undifferentiated cells. Desensitization in differentiated cells was also highly variable, with half-times varying by greater than 40-fold. Rapidly desensitized responses in differentiated cells were qualitatively similar to the responses of undifferentiated cells. 3. In outside-out patches from undifferentiated cells, single channel currents could be seen after 5-HT application. These channels had a conductance of 12 pS. The 5-HT-activated channels in differentiated cells were too small to observe at the single-channel level. Noise analysis indicated that the channel conductance was approximately 4 pS. In differentiated cells, both rapidly and slowly desensitized responses were generated by channels with essentially the same conductance. 4. The 5-HT responses of differentiated cells were also distinguished from those of undifferentiated cells on the basis of the voltage dependence of desensitization and the curvature of the current-voltage curve. 5. NG108-15 cells can produce different receptor subtypes, which may be expressed in different tissues or at different stages of development. These variations in receptor behavior suggest that there are at least two distinct mechanisms for regulation of the 5-HT3 receptor. PMID- 1711106 TI - Ectopic excitability of injured nerves in monkey: entrained responses to vibratory stimuli. AB - 1. The responses to mechanical stimulation of myelinated fibers that originate from an acutely cut nerve or a neuroma were studied in the anesthetized monkey. The superficial radial or sural nerve was tightly ligated and cut. Either immediately (acute experiment) or 2-6 wk later (chronic experiment), single-unit recording techniques were used to record the evoked neural activity after vibratory mechanical stimulation (5-100 Hz; 50-800 microns) near the injury site. 2. The 30 myelinated afferents studied in the chronic experiments displayed an entrained response (1 action potential for each stimulus cycle) to vibratory stimuli applied at or near the nerve injury site. For 19 fibers, the minimum amplitude for entrainment was determined as a function of frequency (tuning curve). For 11 others, complete tuning curves were not obtained, although the frequency range over which they were most sensitive could be estimated. The afferents could be classified into three groups on the basis of the frequency range over which they were most sensitive: 1) a low-frequency group that was most sensitive to frequencies less than or equal to 5 Hz (n = 7), 2) a mid-frequency group that was most sensitive to a broad range of frequencies (i.e., 20-75 Hz, n = 13), and 3) a high-frequency group that was most sensitive to frequencies greater than or equal to 100 Hz (n = 10). These three response classes are similar to the three classes of response associated with the different low threshold mechanoreceptors (i.e., slowly and rapidly adapting and Pacinian-like mechanoreceptors).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711107 TI - Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system. AB - 1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+) K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions. PMID- 1711108 TI - Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones. AB - 1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells. PMID- 1711109 TI - Orthodontics: questions and answers. PMID- 1711110 TI - Prosthodontics. PMID- 1711111 TI - Pediatric dentistry. PMID- 1711112 TI - Oral pathology. PMID- 1711113 TI - Threat to peer review. PMID- 1711114 TI - For whom the bell tolls. PMID- 1711115 TI - Inhibitory effect of acyclic retinoid (polyprenoic acid) on the secretion of alpha-fetoprotein in CCl4-treated rats. AB - A study was conducted to examine the inhibitory effect of acyclic retinoid (polyprenoic acid) on the secretion of alpha-fetoprotein (AFP) in rats with chronic liver damage induced by CCl4. Oral administration of the compound brought about a significant reduction of serum AFP levels at the time when liver cirrhosis was formed. Acyclic retinoid also decreased the activities of serum aminotransferases and ornithine carbamyl transferase, while it increased serum albumin levels, demonstrating the reduction of hepatic parenchymal damage. Significant negative correlation was observed between serum AFP and albumin levels. This cytoprotective effect of the retinoid on the parenchymal cell may well be related to the inhibition of the synthesis and/or secretion of AFP. No significant side effect was observed, despite a long-term administration of the compound. The present finding will provide a potential scope for the future use of acyclic retinoid for the treatment of chronic liver damage. PMID- 1711116 TI - Reflected light microscopy of the human mandibular condyle--a study of a post mortem material. AB - The surface characteristics of the articulating surfaces of 11 human mandibular condyles removed post-mortem were determined by the use of reflected light dark field microscopy. Good agreement was obtained between macroscopical grading of degenerative change and appearance as observed by microscopy. Microscopical appearances believed to correspond to normal surface, fibrillation and degenerative changes are described. PMID- 1711117 TI - The developmental accumulation of gamma-glutamyl transferase in the pancreas of the rat. AB - The developmental accumulation of pancreatic exocrine secretory enzymes is well defined, but little is known of the development of other enzymes in the pancreas. This report focuses on the developmental accumulation of gamma-glutamyl transferase (GGT), a membrane-bound ectoenzyme whose specific activity in the pancreas is the second largest among rat organs. GGT activity is large in organs with active glutathione metabolism. Pancreatic GGT specific activity increased 100-fold from prenatal day 14 to birth, decreased 3-fold until about postnatal week 2, and then increased until the adult value was reached 4 weeks after birth. There was a 500-fold increase in specific activity from prenatal day 14 to the adult. The developmental accumulation pattern of GGT was very similar to that of the exocrine secretory enzyme amylase, which increased 1,300-fold from prenatal day 14 to birth, decreased 8-fold by postnatal week 1, and then increased to the adult level soon after week 4. The overall increase in amylase specific activity was 1,100-fold. The similar developmental accumulation patterns of GGT and amylase suggested that their accumulation might be regulated in a similar fashion. Although the thymidine analogue 5-bromodeoxyuridine inhibited the prenatal accumulation of amylase, as previously reported, it did not inhibit prenatal GGT accumulation. Therefore, the prenatal accumulation of GGT appears to be regulated differently than amylase. On the other hand, the postnatal levels of GGT appear to be controlled by glucocorticoids in a fashion similar to the previously reported control of amylase levels, since both enzymes could be induced to rise prematurely to adult levels by a series of three injections of the glucocorticoid dexamethasone beginning on days 7, 8, 9, 10, 11, or 12.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711118 TI - Phosphorothioate oligodeoxycytidine interferes with binding of HIV-1 gp120 to CD4. AB - In addition to their properties as sequence-specific inhibitors of gene expression, sequence nonspecific phosphorothioate oligodeoxynucleotides have been shown to protect against the cytopathic effects of HIV-1. Although these compounds are effective inhibitors of HIV-1 reverse transcriptase in vitro, it is not certain that they exert their cytoprotective effect only in this manner. Initial binding of the HIV-1 virion to cells involves the interaction of the viral envelope protein gp120 with CD4. In this report, we describe flow cytometric data and a solid-phase ELISA assay that document the ability of a phosphorothioate deoxycytidine 28-mer to interfere with this interaction by competing with gp120 binding to CD4. The biological importance of this interaction is demonstrated by the fact that phosphorothioate oligodeoxycytidine inhibits syncytium formation resulting from HIV-1-induced cell fusion. These data suggest that phosphorothioate oligodeoxynucleotides may exert their cytoprotective effects, perhaps at least in part, by interfering with the binding of HIV-1 to the target cells. PMID- 1711119 TI - Sensitive and specific radioimmunoassays for opiates using commercially available materials. I: Methods for the determinations of morphine and hydromorphone. AB - Sensitive and specific radioimmunoassay (RIA) methods for the analysis of morphine and hydromorphone in human plasma samples using commercially available materials were developed. The limit of quantitation was 0.3 ng/mL of plasma for morphine and 50 pg/mL of plasma for hydromorphone. An extraction step preceding the RIA quantitatively removed morphine, leaving 99% morphine-3-glucuronide (M3G) and 95% hydromorphone-3-glucuronide (H3G) in the aqueous phase. The specificity of the morphine RIA method for the analysis of clinical samples was confirmed by HPLC quantitation. The antiserum used in the hydromorphone RIA method cross reacted slightly with H3G at 0.66%. However, analysis of clinical samples using the direct versus the extraction RIA showed that the extraction step was necessary for the specific determination of hydromorphone in pharmacokinetic studies. After extraction, only 0.033% of the H3G present in plasma samples would be observed as interference for the free hydromorphone. The RIA methods were shown to be accurate and reproducible with almost 100% recovery of morphine and hydromorphone. They offer convenient alternatives to chromatographic methods for pharmacokinetic studies. PMID- 1711120 TI - A simple two-step purification of human and monkey alpha fetoprotein from amniotic fluid and serum. AB - We developed a two-step purification system to characterize alpha fetoprotein (AFP) in early gestation amniotic fluid and late gestation fetal serum or cord blood from monkey and human. It involves only two chromatographic steps, allows preparative purification using up to 12 ml of starting sample, can purify up to 350 micrograms of AFP at one time, and can be used to purify both fetal serum or amniotic fluid AFP from two different species. This procedure will allow detailed biochemical analysis of purified AFP from different stages of fetal development. PMID- 1711121 TI - Major Myelin proteolipid: the 4-alpha-helix topology. AB - Several conflicting models have been proposed for the membrane arrangement of the major myelin proteolipid (PLP). We have compared features of the sequence of PLP with those of other eukaryotic integral membrane proteins, with the view of identifying the most likely transmembrane topology. A new, simple model is suggested, which features four hydrophobic alpha-helices spanning the whole thickness of the lipid bilayer. Its orientation may be such that both the N- and C-termini face the cytosol. None of the biochemical, biophysical or immunological experiments hitherto reported provides incontrovertible evidence against the model. The effect or absence thereof of various PLP mutations is discussed in the frame of the proposed 4-helix topology. PMID- 1711122 TI - Decreased rotational diffusion of band 3 in Melanesian ovalocytes from Papua, New Guinea. AB - Melanesian ovalcytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson, H.M., Wesche, D., Saul, A. 1990. Biochim. Biophys. Acta 1096:33-40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy. PMID- 1711123 TI - Splice site selection and role of the lariat in a group II intron. AB - The structural elements involved in 5' and 3' splice site (SS) selection in a group II intron were analyzed. While 5' SS selection appears to be defined by only one element, the EBS1-IBS1 pairing, four distinct structural components contribute to 3' SS selection, one of which being analogous to the "internal guide sequence" described for group I introns. Moreover, some of the mutants analyzed during this study induce efficient 5' SS hydrolysis and suggest how 5' SS transesterification is selected against hydrolysis. Finally, the lariat structure was found to accelerate both steps of splicing, suggesting that it "locks" the ribozyme in an active configuration. PMID- 1711124 TI - Carbohydrate epitopes involved in neural cell recognition are conserved between vertebrates and leech. AB - We are reporting on the evolutionary conservation of carbohydrate epitope families from vertebrate to leech. 1) The sulfated L2/HNK-1 carbohydrate epitope (Abo T, Balch CM (1981): J Immunol 127:1024-1029; Kruse J, Mailhammer R, Wernecke H, Faissner A, Timpl R, Schachner M (1984): Nature 311:153-155) is detected on glycoproteins of leech neurons using monoclonal antibodies (mAbs) L2 (336) and HNK-1. 2) Three rat mAbs, L3, L4, and L5, bind to leech nerve and muscle. The L3, L4, and L5 epitopes are localized to a group of mannosidic leech glycoproteins originally identified through mAbs Lan3-2 (Hogg N, Flaster M, Zipser B (1983): J Neurosci Res 9:445-457 and Laz6-189 (McRorie JW III, Zipser B (1988): "Cell Culture Approaches to Invertebrate Neuroscience." London: Academie Press, pp 33 52. MAb Lan3-2, which binds to a mannosidic epitope of the 130 kD sensory protein, has recently been shown to perturb the penetration of sensory afferents into the synaptic area of the central neuropile (Zipser B, Morell R, Bajt ML (1989): Neuron 3:621-630). The L3, L4, and L5 mAbs have been described to recognize different mannosidic epitopes on glycoproteins, some of which have been identified as neural cell adhesion molecules, and on astrocyte-specific proteoglycan from mouse brain (Kucherer A, Faissner A, Schachner M (1987): J Cell Biol 104:1597-1602; Fahrig T, Schmitz B, Weber D, Kucherer-Ehret A, Faissner A, Schachner M (1990): Eur J Neurosci 2:153-161; Streit A, Faissner A, Gehrig B, Schachner M (1990): J Neurochem In Press). The superposition of five different mannosidic epitopes on the axons of sensory afferents suggests complex, concerted participation of mannosidic epitopes in neuronal pathfinding and target recognition. PMID- 1711125 TI - Recombinant peripheral myelin protein P0 confers both adhesion and neurite outgrowth-promoting properties. AB - To probe into the functional properties of the major peripheral myelin cell surface glycoprotein P0, its ability to confer adhesion and neurite outgrowth promoting properties was studied in cell culture. To this aim, P0 was expressed as integral membrane glycoprotein at the surface of CV-1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin like extracellular domain of P0 (P0-ED) was expressed as soluble protein in a bacterial expression system and used as substrate coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth-promoting assays. The adhesion of P0-expressing CV-1 cells to P0-ED substrate was specifically inhibitable by polyclonal P0 antibodies (54% +/- 6%). In addition, the specific interaction between P0 molecules could be reduced (49% +/- 8%) by adding soluble P0-ED to the culture medium, demonstrating that the homophilic interaction between recombinant P0 molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig-like domain. Substrate-coated P0-ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 microns. This neurite outgrowth was specifically inhibitable by soluble P0 (74% +/- 14%) and P0 antibodies (65% +/- 9%). These observations indicate that P0 is capable of displaying two different types of functional roles in the myelination process of peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interaction may indicate its role in the self-recognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath. PMID- 1711126 TI - Phagocytosis of peripheral nerve myelin in vitro: effect of antibody. AB - We have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C-labeled PNS myelin increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole P0 and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to almost the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self-associate to form large clumps which were found to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711127 TI - Novel NGF-induced proteins in PC12 cells: immunological evidence for their presence in brain nerve endings using a single monoclonal antibody. AB - A monoclonal antibody, S-11D9, detected a group of novel polypeptides whose expression was induced 6 hr after incubation of PC12 cells with nerve growth factor. The antigens also were visualized by immune precipitation and Western blotting in synaptic vesicles, clathrin-coated vesicles, and synaptic plasma membrane prepared from bovine brain. A large polypeptide was detected on the synaptic plasma membrane; an intermediate size protein was visualized on the plasma membrane and on both synaptic and clathrin-coated vesicles, while a smaller molecule was found only on brain Golgi-enriched membrane preparations. Immunofluorescence labeling with S-11D9 on nerve growth factor-stimulated PC12 cells showed the antigens distributed in the cytoplasm and concentrated in discrete areas on the tip of most neurites. The particular distribution of these proteins in vesicles and synaptic plasma membrane and the finding that the different antigens share a common epitope recognized by a single monoclonal antibody open the possibility that these molecules are related markers for organelle transport pathways in nerve cells. PMID- 1711128 TI - Immunohistochemical studies on cross-transplantations between jimpy, shiverer, and normal newborn mice. AB - Cross-transplantations of neural tissue have been performed between jimpy (jp), shiverer (shi), and normal mice. Taking advantage of the absence of immunodetectable myelin basic protein (MBP) in the shi brain, jp myelin has been identified in the shi recipient by using an anti-MBP antiserum. By contrast, shi as well as normal myelin have been identified in the jp brain by using an anti-C terminal hexapeptide of the proteolipid protein (PLP) (this PLP hexapeptide being absent in the jp PLP). When transplanted under homochronic conditions (newborn into newborn), jp oligodendrocytes (ODC) express their usual phenotype in a normal or a shi environment, suggesting that at birth the jp ODCs phenotype is strictly established and cannot be modified by environmental conditions. The reverse transplantations (newborn shi or normal into newborn jp brain) demonstrate that the jp environment does not modify the phenotype of normal or shi ODCs. Finally, these experiments demonstrated a normal timing of differentiation of jp axons and of jp ODCs. PMID- 1711129 TI - The health status of rural primary schoolchildren in Central Zambia. AB - In a study of 528 rural primary schoolchildren in Central Zambia, it was found that the health status of the schoolchildren was not good as indicated by inadequate nutrition, a high prevalence of S. haematobium (18%), hookworm (33%), and malaria (43%) infections. There were no statistically significant differences in prevalence of undernutrition between girls and boys and there were no significant trends with age. The treatment and control of hookworm disease, urinary schistosomiasis and malaria deserve a high priority in this area. As for malaria, until an international programme on its control can be developed, the acquisition of protective immunity is of paramount importance. This study shows how the use of 'simple' screening procedures can provide information to direct health education and other disease control measures in school health programmes. As the economic situation in Zambia is not good, the best hope for improvement of the children's health lies with environmental improvement in sanitation, water supplies and provision of basic health education. PMID- 1711130 TI - [Cytoplasmic staining pattern characteristic for anti Jo-1 antibody in HEp-2 cells]. AB - We wished to confirm the staining pattern of HEp-2 cells by the anti Jo-1 antibody, because we found antibodies in serum with positive anti Jo-1 antibody which showed either a fibrilar cytoplasmic staining or a nuclear speckled staining pattern in indirect immuno fluorescence+ examinations using HEp-2 cells. Sera available from eight patients with PM DM (polymyositis and/or dermatomyositis) showed positive anti Jo-1 antibody in the double immuno diffusion technique but had various staining patterns of HEp-2 cells in the immunofluorescent examination. We examined these eight sera with the immuno blotting method utilizing whole cell extract of HeLa cells, and found the 50 kDa band from all sera tested, to which Jo-1 antigen had been reported to move. We eluted the antibody which formed the 50 kDa band from the nitrocellulose membrane and applied it on HEp-2 cells. This maneuver gave us the fine granular cytoplasmic staining of anti Jo-1 antibody on HEp-2 cells. We therefore concluded that the anti Jo-1 antibody should have cytoplasmic staining on HEp-2 cells although observers might miss it due to other types of associated antinuclear antibodies. PMID- 1711131 TI - [Prediction of remission in Graves' disease after thionamide therapy by technetium-99m early uptake]. AB - In the clinical management of Graves' thyrotoxicosis, one of the most important subject is when to stop antithyroid drugs after achieving an euthyroid state. T3 suppression test and other methods have been used to forecast the outcome after drug cessation, but the results were not always satisfactory. We have attempted to predict remission of Graves' disease by single measurement of early technetium uptake without administration of triiodothyronine. Drugs were discontinued in the seventy-five patients with Graves' disease on maintenance doses of either methimazole or propylthiouracil who showed normalized uptake (4.0% or less). Of 64 patients evaluable after twelve months, 55 (86%) remained euthyroid, 8 relapsed, and 1 became hypothyroid. With its accuracy in prediction of short-term remission comparable or superior to T3 suppression test, this rapid and simple method seemed suitable for routine use in clinical practice. PMID- 1711132 TI - [Palliation of airway obstruction under a T-tube stent in a patient with thyroid carcinoma invading the trachea: a case report]. AB - Invasion of the neoplasma to the upper airway tract is the life threatening factor. We experienced a 74-year-old female with thyroid carcinoma invading the trachea, for whom radical resection was infeasible. So for the palliation of airway obstruction we inserted the silicone rubber T-tube stent from 0.5 cm above the vocal cord to 2 cm before the carina after endotracheal tumor resection with Nd-YAG laser. As the result, she has led an active life for the last 6 months after the operation. We consider that the T-tube stent is effective tool in the management of airway obstruction for the slow growing neoplasma. PMID- 1711133 TI - [Discrepancy between the serum levels of gamma seminoprotein and prostate specific antigen in patients with prostatic neoplasms. Both true or either untrue]. AB - Serum levels of prostatic acid phosphatase (PAP), gamma-seminoprotein (gamma-Sm) and prostate-specific antigen (PSA) were determined simultaneously in 57 patients with benign prostatic hyperplasia (BPH) and in 50 untreated patients with prostatic cancer (adenocarcinoma, N = 47 and non-adenocarcinoma, N = 3). The correlations between the serum levels of gamma-Sm and PSA in these patients were assessed by linear regression analysis. Some fundamental studies were added for explaining the causes of discrepancy between the serum levels of gamma-Sm and PSA. All of BPH group underwent transurethral resection of the prostate (TURP) and the sera were obtained for measurements before, immediately after and 18 hours after TURP. The gamma-Sm correlated well with the PSA in the sera obtained before (r = 0.76) and 18 hours after (r = 0.73) TURP. However, there was no correlation (r = 0.26) between them in the sera obtained immediately after TURP. In 47 untreated patients with adenocarcinoma of the prostate, no significant correlation (r = 0.19) between serum levels of gamma-Sm and PSA was observed, although there was correlation (r = 0.51) between those of PAP and PSA. When these patients were classified into two groups, M0 (stage A-C; N = 26) and M1 (stage D; N = 21), however, the serum gamma-Sm correlated with the serum PSA in M0 group (r = 0.57), but didn't in M1 group (r = 0.11). Furthermore, the differences in the means of PAP (p less than 0.05) and PSA (p less than 0.001) between M0 group and M1 group were statistically significant, although the serum gamma-Sm failed to distinguish M0 from M1. The anti-PSA antibody of "PSA Kit" reacted against the standard gamma-Sm adopted from "gamma-Sm Kit". Surprisingly, the anti-gamma-Sm antibody of "gamma-Sm Kit" also reacted against the standard PSA adopted from "PSA Kit". The gamma-Sm and PSA apparently cross-reacted each other. The quantitative analyses with serial dilution of the sera were done by using each assay in 3 patients whose serum levels of gamma-Sm were markedly different from those of PSA. The dilution curve for PAP appeared to be rectilineal, and that for PSA also appeared to be approximately rectilineal. However, the gamma-Sm assay failed to be proportional. In conclusion, the correlation between serum levels of gamma-Sm and PSA was absent in certain circumstances, when the true values of them were expected to be much higher than those determined.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711134 TI - [Clinical and prognostic value of monitoring the acute phase protein levels in patients with myocardial infarction]. AB - The value of evaluating the protein levels at the acute stage was examined to define the clinical course and prognosis of myocardial infarction. The time course of hospital myocardial infarction was studied in 117 patients. The Manchini radial immunodiffusion with reference monospecific antisera was employed to determine individual proteins during the acute stage (orosomucoid, C-reactive protein, alpha 1-antitrypsin, C3 complement, ceruloplasmin). The level of circulating immune complexes was measured by polyethylene glycol precipitation. There was a close correlation between the circulating immune complex levels and some acute phase parameters, suggesting the relationship between immune and inflammatory processes. The changes in the magnitude of some acute phase parameters and circulating immune complexes were found to be significant in predicting the development of complications and the fatal outcome of the disease. PMID- 1711135 TI - [Anti-arrhythmic effect and kinetic parameters of bonnecor in the treatment of extrasystole in patients with acute myocardial infarction]. AB - Bonnecor was tested for its antiarrhythmic activity in patients with acute myocardial infarction in the first 24 hours of the disease complicated by arrhythmias as ventricular premature beats (VPB). Proceeding from the comparison of its clinical efficiency and pharmacokinetic parameters in 38 patients, the optimal dosage of the drug was formulated. When given intravenously to the patients in dose of 25-37.5 mg bonnecor produced 92% antiarrhythmic responses, failed to shorten heart rate and to lower blood pressure; the agent led to a prolonged P-Q interval as evidenced by ECG. The antiarrhythmic activity, the dose of the drug, and its plasma concentration were found to be closely related. The mean effective bonnecor concentration was ascertained to be 0.45 +/- 0.24 micrograms/ml. PMID- 1711136 TI - Alterations in lysosomal enzymes of the proximal tubule in gentamicin nephrotoxicity. AB - Gentamicin accumulates in proximal tubule lysosomes, increases their number, and changes their structure. An important lysosomal function is degradation of intracellular proteins. To evaluate the effect of gentamicin on this lysosomal function, we measured the activity of the key lysosomal proteinases, cathepsin B and L, in microdissected S1, S2, and S3 segments of rat proximal tubules by means of a fluorometric microassay. The cathepsin activities were decreased in S1 and S2 following one and four gentamicin injections of 100 mg/kg body weight. The lysosomal enzyme, acid phosphatase, was also measured and was not decreased by gentamicin. The urine excretion of cathepsins B and L was decreased after gentamicin. This excludes an increase in urinary loss of cathepsins as the cause of decreased tubule activity. Structural changes of the lysosomes per se were excluded as the factor responsible for the reduced cathepsin activity by demonstrating increased cathepsin B and L activity in proximal tubule segments from rats injected with dextran, since dextran induces an increase in number and size of proximal tubule lysosomes. In vitro incubation of urine and tubule segments with gentamicin demonstrated a concentration-dependent reversible inhibition of cathepsin B and L. We conclude that gentamicin per se decreased cathepsin B and L activities in proximal tubule segments as early as 24 hours following one injection due to either enzyme inhibition or reduced generation of active intralysosomal cathepsin B and L. Gentamicin may, therefore, reduce renal protein catabolism by decreasing the activity of the key proteolytic enzymes, cathepsin B and L. Since cathepsin B and L are proteolytic activators of other lysosomal enzymes, their reduced activity may also decrease the activities of other lysosomal enzymes. PMID- 1711137 TI - [Retroperitoneal tumors--the surgical treatment procedures]. PMID- 1711138 TI - Enzyme-inhibiting antibodies and immunoglobulin combining site functional perspectives. AB - Advances in the understanding of antibody-antigen interactions and in the production of monoclonal antibodies have outlined possibilities for the broad application of natural immune phenomena to biologic processes occurring beyond the functional realm of the immune response. PMID- 1711139 TI - [Fever, rash, arthralgia and uveitis in a 50-year-old man]. PMID- 1711140 TI - Role of take-off potential and second plateau response in generation of early afterdepolarization in arterial fibers of mouse heart. AB - Take-off potentials (TOPs) of triggered bursts were studied on aconitine- 3.0 mM K+ and quinidine-induced early afterdepolarization (EADs) in mice atrial fibers. TOPs varied from -40 to -66 mV (-53.4 +/- 6.4 mV, n = 14) depending on the cycle length of stimulation. TOPs were cycle length-dependent and the relationship between TOP and cycle length was exponential. Before the generation of EAD, a second plateau appeared following the rapid repolarization phase of action potential (AP). In some preparations, EAD could not be induced, especially at a shorter cycle length, leaving a definite second plateau following the AP under treatment with effective agents. By application of a premature stimulation on the second plateau, only one burst could be observed. However, two or more bursts could be induced when a stimulation was applied on the rapid repolarization phase at -50 +/- 6 mV (n = 17) level. We defined this phenomenon as "second plateau response" which was studied in the present work under treatment with quinidine (4 cases), 3.0 mM K+ (4), ryanodine (6) and Bay K 8644 (3). The second plateau response was abolished by tetrodotoxin (1.0 microM), nifedipine (2.0 microM) or rapid driving. All of these were similar to the properties of the EAD. It is suggested that the second plateau response may be taken as an indicator of the capability of the EAD generation in myocardiac cells. PMID- 1711141 TI - Fetal brain serotonin synthesis and catabolism is under control by mother intake of tryptophan. AB - Previous morphological studies reported that serotonergic neurons appear in rats in the second half of prenatal life. Initially the biochemical differentiation of these neurons before birth was studied. Both serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) was detected in the fetal brain on day 15 of gestation. During prenatal development an increase was detected in the brain levels of 5-HT (200% higher on day 19 than on day 15) and 5-HIAA (700% higher on day 19 than on day 15). Oral administration of tryptophan to pregnant rats induced a dose related increase of tryptophan concentration in different fetal tissues, including brain. The increase in tryptophan tissue concentration was detected for low doses (50 mg/kg) and remained unsaturated after administration of high doses (1000 mg/kg). This observation suggests that the placental barrier is not effective to block the influx of high levels of tryptophan to the fetus. Tryptophan concentration in the brain is 300% higher than in the carcass and 600% higher than in the placenta. These data suggest a mechanism to assume a role in concentrating of tryptophan in the brain. Finally, it was found that an increase in brain tryptophan induced changes in both serotonin and 5-HIAA brain levels, but did not modify tyrosine, dopamine or norepinephrine levels. Thus, under physiological conditions, tryptophan hydroxylase activity in prenatal brain is probably not saturated by its substrate tryptophan. PMID- 1711142 TI - Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells. AB - Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, alpha-galactosyl-1,4-beta-galactose (Gal alpha 1,4Gal beta), also found on human cells. PMID- 1711143 TI - Substance P mechanisms in the regulation of striated muscle microcirculation. AB - The effects of substance P (SP) on microvessels in the cremaster of sodium pentobarbital-anesthetized male, Sprague-Dawley rats were investigated using closed-circuit television microscopy. Topically added SP (bath concentrations of 10(-13) to 10(-8) M) caused significant dilation of small arterioles. SP vasodilation was sensitive to pretreatment of the muscle tissue with SP analogues, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-substance P (APTL-SP) and [D-Pro2, D Trp7,9]-substance P (PT-SP). Attenuation of the responses by pyrilamine suggests that part of the SP-induced dilation involves the release of histamine. The vasodilation was abolished by hydroquinone, suggesting an intermediate role for endothelium-derived relaxing factor in the response to SP. PMID- 1711144 TI - Convection of macromolecules is the dominant mode of transport across horizontal 0.4- and 3-microns filters in diffusion chambers: significance for biologic monolayer permeability assessment. AB - The purpose of the present study was to evaluate the permeability characteristics of fluorescein-labeled hydroxyethyl starch (FITC-HES, 20 less than molecular radius, aE less than 111 A) across large pore (0.4- and 3-microns polycarbonate) filters used in endothelial cell monolayer diffusion studies. Although the apparent permeability (P) of the FITC-HES macromolecules across these filters declined as the molecular radius increased, this decline was less than that associated with each solute's free diffusion (D0). Thus, the P/D0 anomalously increased as aE increased, a pattern not seen for free diffusion (flat P/D0 with increased aE) or restricted diffusion (decline in P/D0 with increased aE). Substantial natural convection across the porous filters produced the anomalous P/D0 curve for the following reasons: (1) this effect was elevated with positive pressure and ameliorated by zero driving pressure, and (2) the effect was much greater across filters with large (3 microns) vs small (0.4 microns) pore sizes. In addition, we estimated that less than half of macromolecular transport above 50 A probe radius at zero transmural pressure arrives by diffusion. The findings suggest that the property of restricted diffusion of biologic layers on these filters will be artifactually exaggerated when the measured resistance of the filter is subtracted from that of the filter plus biologic monolayer. Remedies for this problem may include using smaller pore filters, filters layered with extracellular material, or structural methods to determine true filter permeability. PMID- 1711145 TI - The use of digital image processing to quantitate angiogenesis induced by basic fibroblast growth factor and transplanted pancreatic islets. AB - We have used digital image processing as a technique to quantify the formation of blood vessels in situ in response to the application of either an angiogenic peptide, basic fibroblast growth factor, or isolated pancreatic islets. The peptide or the islets were placed under the rat kidney capsule. For in vivo microscopy fluorescein-labeled dextran was injected intravenously and video images were made as deemed appropriate. Images to be analyzed were selected manually, digitized, and stored in a computer for processing. The calculation of the total vessel length per area was called the microvascular index. Three weeks after the transplantation of 200 islets, the microvascular index was fairly constant and did not change significantly when the same measurements were performed 1 week later. In another experiment, pellets containing 0.5 micrograms of basic fibroblast growth factor produced an angiogenic response no different from that of a control pellet. At a dose of 1 microgram, however, the response was statistically significant after 1 week. These data indicate that digital image processing may be a useful method to quantitate the angiogenesis induced by local administration of angiogenic stimulants. PMID- 1711146 TI - [The safety of the preparation viroden for vertebrate animals]. AB - Toxicity-pathogenicity test of viroden, a new preparation, and its acting agent- a mosquito densonucleosis virus (MDV) has been carried out on warm-blooded animals. It is shown that the preparation is not toxic for laboratory animals (white common mice, rats, guinea pigs, rabbits), chicken embryos and cell cultures of warm-blooded animals. The MDV is not adapted to a warm-blooded organism with different ways of introduction and in passages. Using electron and luminescent microscopy, serological reactions, specific test systems and a biological test for sensitive insects no explicit or latent infection was found in animals, chicken embryos and cell cultures of vertebrates with primary infection and in passages. Sensibilized animals shown an immunological rearrangement of the organism proceeding by the retarded hypersensitivity type. PMID- 1711147 TI - [Experimental studies on targeting chemotherapy using adriamycin entrapped in liposomes conjugated with anti-human alpha-fetoprotein monoclonal antibody]. AB - The in vivo tissue distribution of adriamycin (ADM) entrapped in liposomes conjugated with anti-human alpha-fetoprotein (AFP) monoclonal antibody (Lip-ADM = Ab) and the therapeutic effects of the conjugates on the AFP producing human hepatoma xenograft, Li-7, transplanted in BALB/c nu/nu murine liver were studied. The tissue distribution studies of Lip-ADM = Ab revealed that ADM levels were augmented in tumors, when Lip-ADM = Ab was administered intravenously as compared with free ADM or ADM entrapped in liposomes (Lip-ADM) alone. In addition, cardiac distribution of ADM was decreased in mice using ADM entrapped in liposomes. The therapeutic effects of Lip-ADM = Ab were tested in vivo on the experimentally transplanted Li-7 in liver. The anti-tumor effects of Lip-ADM = Ab were greater than those of Lip-ADM or free ADM as assessed by tumor weight. This experiment also indicated that the toxicity of ADM was reduced when the drug was injected using liposomes entrapped form by the body-weight losses and spleen weights. PMID- 1711148 TI - Highly potent and selective inhibition of human immunodeficiency virus type 1 by a novel series of 6-substituted acyclouridine derivatives. AB - In the search for novel derivatives of 1-[2-(hydroxyethoxy)methyl]-6 (phenylthio)thymine (HEPT), we have found that several 5-ethyl-6-(3,5 dimethylphenylthio)uracil and 5-ethyl-6-(3,5-dimethylbenzyl)uracil analogues are exquisitely potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in a variety of cell culture systems. Of this series, 5 ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil (E-EBU-dM) emerged as the most active congener. Its 50% inhibitory concentration for HIV-1 (HTLV-IIIB) in MT-4 cells and peripheral blood lymphocytes is 2.2 and 0.45 nM, respectively. These concentrations are more than 10(5)-fold lower than the 50% cytotoxic concentrations of E-EBU-dM for the host cells. All compounds proved equally inhibitory to a number of clinical HIV-1 isolates, including a 3'-azido-2',3' dideoxythymidine-resistant variant. However, as previously noted for HEPT, they do not inhibit human immunodeficiency virus type 2 replication. Reverse transcriptase assays have revealed that these HEPT derivatives act specifically on HIV-1 reverse transcriptase, according to a mechanism that is different from that of the dideoxynucleosides. PMID- 1711149 TI - Specificity of mutational DNA sequence changes induced by X-rays in the cloned Escherichia coli crp gene. AB - Plasmid DNA carrying the adenosine 3',5'-cyclic monophosphate receptor protein (crp) gene of Escherichia coli was irradiated, in solution, with X-rays, and the mutations produced in the crp gene were assayed by transforming the recipient E. coli cells. Ninety-six mutant clones were isolated, and mutational changes were determined by DNA sequencing. Of the 92 mutations thus detected, 74 represented base substitution mutations and the remaining 18 were frameshifts. The base substitutions included 56 G:C to A:T transitions, 10 G:C to T:A transversions and 7 G:C to C:G transversions. An A:T to G:C transition was found only once, and neither an A:T to T:A nor an A:T to C:G transversion was detected. The frameshift mutations consisted of 11 one-base deletions and 7 one-base insertions. Accordingly, G:C to A:T transition was the predominant type of mutation, which constituted 76% (56/74) of the total base substitutions and 60% (56/92) of all detected mutations. Furthermore, of the 56 transitions, about three-quarters (41 clones) clustered at an identical site, a cytosine residue at the 706 position, demonstrating that this site is a distinct hot spot for X-ray mutagenesis. These results raise the possibility that radiation-induced mutations may not necessarily occur randomly, at least in certain cases. PMID- 1711150 TI - Partial complementation of the UV sensitivity of Deinococcus radiodurans excision repair mutants by the cloned denV gene of bacteriophage T4. AB - Deinococcus radiodurans has 2 endonucleases that incise UV-irradiated DNA. UV endonuclease-alpha and UV endonuclease-beta, that are believed to functionally overlap. Both endonucleases must be mutationally inactivated to yield an incisionless, markedly UV-sensitive phenotype. denV, the bacteriophage T4 gene encoding pyrimidine dimer-DNA glycosylase (PD-glycosylase), was introduced and expressed via duplication insertion in D. radiodurans wild-type, and single and double UV endonuclease mutants. The strain deficient in UV endonuclease-alpha has wild-type UV resistance, and the expression of PD-glycosylase exerted no survival effect on this strain or wild-type. Expression of denV increased survival of both the markedly UV-sensitive double mutant and the moderately UV-sensitive strain deficient only in UV endonuclease-beta. In endonuclease-beta-deficient cells phenotypic complementation by denV was almost complete in restoring UV resistance to wild-type levels. These results suggest that UV endonuclease-alpha (which is present in the endonuclease-beta-deficient cells) does not recognize one or more types of cyclobutane dimer incised by the PD-glycosylase or UV endonuclease-beta. PMID- 1711151 TI - Activation of RecA protein in recombination-deficient strains of Escherichia coli following DNA-damaging treatments. AB - Activation of the RecA protein following UV-irradiation or bleomycin (BM) treatment was measured in rec mutants of E. coli by monitoring beta-galactosidase activity. We provide evidence here that the defect in the recN mutant results in high constitutive and induced levels of activated RecA protein. In all rec mutants studied, with the exception of the recN mutant, induction of enzyme activity, following DNA-damaging treatments, was reduced relative to the wild type. The kinetics of induced sfiA expression indicates that the DNA-unwinding activity of the RecBCD enzyme plays a major role in SOS-signal formation. The RecF protein is not needed for BM induction in strains with a functional RecBCD pathway of recombination. However, a functional product of recF gene is implied in the formation of an efficient inducing signal after UV-irradiation, as well as in the additional processing of BM-induced lesions after exposure to the drug. A fully expressed RecF pathway of recombination does not provide a high level of activated RecA protein following DNA-damaging treatments. PMID- 1711152 TI - AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans. AB - An endonuclease specific for apurinic/apyrimidinic (AP) sites was identified and purified from extracts of Deinococcus radiodurans. The enzyme is 34.5 kD, has no activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA, and is active in the presence of EDTA. The addition of up to 10 mM Mg2+ or Mn2+ did not affect activity, but higher concentrations were inhibitory. There is no associated exonuclease activity, either in the presence or absence of divalent cation. Optimal reaction conditions were 150 mM NaCl and pH 7.5. A uracil DNA glycosylase was also detected, active in the presence of EDTA, selectively removing uracil from DNA without generating other byproducts. The optimal reaction conditions were 50 mM NaCl and pH 7.5. Implications for base excision repair in D. radiodurans are discussed. PMID- 1711153 TI - Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli. AB - Toward the future development of a defined subunit vaccine against leishmaniasis is, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli. Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions. The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E. coli. When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E. coli. Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania. Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L. major GP63 expression clone as antigen. PMID- 1711154 TI - Molecular characterisation of a protective, 11-kDa excretory-secretory protein from the parasitic stages of Trichostrongylus colubriformis. AB - An 11-kDa protein occurring as a major component of the non-glycosylated fraction of 4th larval stage (L4) and adult Trichostrongylus colubriformis excretory secretory (ES) fluid has been found to be highly protective in guinea pigs, an alternate host for T. colubriformis. The protein has been purified, characterised and partly sequenced. With a reverse-complement oligonucleotide based on the carboxy-terminal sequence of the protein, recombinant lambda gt11 clones were detected in an L4 cDNA library. The DNA sequence from one clone has a single extended open reading frame coding for a highly charged 11-kDa protein which lacks a leader sequence and contains a potential N-glycosylation site. Expression of the cloned DNA in Escherichia coli was detected with an antibody, raised in rabbits against gel-purified 11-kDa protein. PMID- 1711155 TI - Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct. PMID- 1711156 TI - Reduction by granulocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with small-cell lung cancer. AB - BACKGROUND: Neutropenia and infection are major dose-limiting side effects of chemotherapy. Previous studies have suggested that recombinant methionyl granulocyte colony-stimulating factor (G-CSF) can reduce chemotherapy-related neutropenia in patients with cancer. We conducted a randomized clinical trial to test this hypothesis and the clinical implications. METHODS: Patients with small cell lung cancer were enrolled in a multicenter, randomized, double-blind, placebo-controlled trial of recombinant methionyl G-CSF to study the incidence of infection as manifested by fever with neutropenia (absolute neutrophil count, less than 1.0 x 10(9) per liter, with a temperature greater than or equal to 38.2 degrees C) resulting from up to six cycles of chemotherapy with cyclophosphamide, doxorubicin, and etoposide. The patients were randomly assigned to receive either placebo or G-CSF, with treatment beginning on day 4 and continuing through day 17 of a 21-day cycle. RESULTS: The safety of the study treatment could be evaluated in 207 of the 211 patients assigned to either drug, and its efficacy in 199. At least one episode of fever with neutropenia occurred in 77 percent of the placebo group, as compared with 40 percent of the G-CSF group (P less than 0.001). Over all cycles of chemotherapy, the median duration of grade IV neutropenia (absolute neutrophil count, less than 0.5 x 10(9) per liter) was six days with placebo as compared with one day with G-CSF. During cycles of blinded treatment, the number of days of treatment with intravenous antibiotics, the number of days of hospitalization, and the incidence of confirmed infections were reduced by approximately 50 percent when G-CSF was given, as compared with placebo. Mild-to moderate medullary bone pain occurred in 20 percent of the patients receiving G CSF. CONCLUSIONS: The use of G-CSF as an adjunct to chemotherapy in patients with small-cell cancer of the lung was well tolerated and led to reductions in the incidence of fever with neutropenia and culture-confirmed infections; in the incidence, duration, and severity of grade IV neutropenia; and in the total number of days of treatment with intravenous antibiotics and days of hospitalization. PMID- 1711157 TI - The B-cell surface protein CD72/Lyb-2 is the ligand for CD5. AB - The glycoprotein CD5 is expressed on the surface membrane of all mature T cells and a small proportion of B lymphocytes. Its exact role in immune interactions is still unknown. Studies indicate that CD5 functions both in mice and humans as a receptor, delivering co-stimulatory signals to T cells in a manner similar to CD2 (ref. 11) and CD28 (ref. 12). Anti-CD5 antibodies stimulate both T-cell proliferation mediated by CD3 in association with the T-cell receptor and secretion of interleukin-2 and expression of its receptor, as well as inducing an increase in intracellular Ca2+ concentration (refs 5-10). To identify the ligand for CD5 we purified the human CD5 protein, labelled it with biotin and used it as a probe. Here we report that CD5 specifically interacts with the cell-surface protein CD72 exclusive to B cells. This interaction is blocked by anti-CD72 antibodies, but not by any other anti-B-cell antibodies. Moreover, non-B cells (mouse L-cell fibroblasts and human Jurkat T cells) expressing a transfected human CD72 complementary DNA could bind to the CD5-biotin conjugate. The results demonstrate that the B-cell surface protein CD72 (Lyb-2 in mice) is the ligand for CD5. PMID- 1711158 TI - [The role of cAMP in generating mollusk neuron responses, related to negative slope conductance, to dopamine application]. AB - Dopamine (DA)-induced steady-state inward current has been studied in isolated mollusc neurons through two different mechanisms: an increase in the slow potential-dependent Na(+)-conductance and a decrease in the K(+)-conductance. Dibutyryl-cAMP mimicked the both types of DA-responses. 3-Isobutyl-1 methylxanthine (IBMX) in low concentration potentiated DA-responses and in high concentration mimicked them. It was concluded that most of DA-induced inward current responses associated with a negative slope conductance are mediated by cAMP. PMID- 1711159 TI - Age-related decrease in proopiomelanocortin gene expression in the arcuate nucleus of the male rat brain. AB - The decline in reproductive function with aging is due in part to decreased gonadotropin-releasing hormone (GnRH) secretion. beta-Endorphin (beta E), an endogenous opioid peptide derived from proopiomelanocortin (POMC), is thought to exert a tonic inhibitory effect upon hypothalamic GnRH secretion. We tested the hypothesis that the age-related decrease in GnRH secretion in male rats is due to increased beta E synthesis, by comparing POMC mRNA levels in the arcuate nucleus (ARC) of intact young, middle-aged and old male rats. In an initial study (Study 1), sixteen 20-microns coronal sections each from the ARC of 3- (n = 5) and 23 month-old (n = 4) male Fischer 344 rats were anatomically matched and analyzed. In a second study (Study 2), four anatomically matched sections of caudal arcuate nucleus from 3- (n = 4), 11- (n = 7) and 23-month-old (n = 5) male rats were compared. POMC mRNA levels were quantitated by in situ hybridization histochemistry, using a 35S-labeled oligodeoxynucleotide probe complementary to a portion of rat POMC cDNA and computerized image analysis. The number of grains per cell and cells per section were used as indices of cellular POMC mRNA content and the number of neurons expressing the POMC gene, respectively. Cellular POMC mRNA content was significantly lower in old compared to young animals (Study 1: 54 +/- 3 vs. 74 +/- 2 grains/cell, p less than 0.01; Study 2: 59 +/- 2 vs. 71 +/- 2 grains/cell, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711160 TI - Retrograde response to axotomy of motoneurons in the thoracic spinal cord of the aging cat. AB - The retrograde response of HRP-labelled intercostal motoneurons was compared in adult (1-2 years) and aging (10-15 years) cats, 64-68 days following crush of intercostal nerves or following nerve transection with proximal ligation. A comparison of the neuroglial response to these two lesions was also made. In both adult and aging cats, 64-68 days following nerve crush, most labelled motoneurons had a normal appearance. In contrast, 64-68 days following nerve transection and ligation the Nissl bodies of labelled motoneurons lacked the highly ordered ultrastructure characteristic of normal and control motoneurons. No axotomy induced neuronal loss was found in aging cats. A three-fold increase in numbers of microglial cells was quantified in the ipsilateral ventral horn of aging cats following nerve transection and ligation. This increase was not seen following nerve crush in aging cats, nor following either type of nerve injury in adult cats. Numbers of astroglia and oligodendroglia were unaffected by axotomy in adult and aging animals. PMID- 1711161 TI - Accumulation of amyloid precursor fragment in Alzheimer plaques. AB - Regenerative and degenerative neurites are components of classical senile plaques found in brain tissue of patients with Alzheimer's disease (AD). Amyloid beta/A4 protein derived from its precursor, amyloid beta/A4-protein precursor (APP/ABPP), constitutes the major portion of the amyloid core of senile plaques. A large N terminal portion of APP (approximately Mr 100,000) is released from cells, leaving a minor C-terminal portion (approximately Mr 15,000) behind. A series of antisera against various sequences of APP were prepared and used to study the localization of each sequence in brain tissue. Plaque neurites stained as intensely as neuronal cell bodies with three antisera against the N-terminal portion of APP (N-terminal to a.a. 225), whereas five other antisera directed against the other C-terminal portions of APP (a.a. 284 to C-terminal) and antisera against the Kunitz-type protease inhibitor portion of APP stained plaque neurites less intensely than neuronal cell bodies in the hippocampus. These results suggest that a major part of the APP present in the neuritic component of senile plaques is a fragment representing the N-terminal one-third of the molecule. PMID- 1711162 TI - Effect of acute and chronic cholinesterase inhibition on biogenic amines in rat brain. AB - The effects of five cholinesterase inhibitors on forebrain monoamine and their metabolite levels, and on forebrain and plasma cholinesterase (ChE) activity in rat were studied in acute and chronic conditions. Acute tetrahydroaminoacridine (THA) dosing caused lower brain (68%) and higher plasma (90%) ChE inhibition than the other drugs studied and increased levels of brain dihydroxyphenylacetic acid (DOPAC) (236%), homovanillic acid (HVA) (197%) and 5-hydroxyindoleacetic acid (5 HIAA) (130%). Acute physostigmine (PHY) administration caused a 215% increase in brain DOPAC content. Despite high brain ChE inhibition induced by metrifonate (MTF), dichlorvos (DDVP) or naled no changes in brain noradrenaline (NA), dopamine (DA) or serotonin (5-HT) occurred due to treatment with the study drugs in the acute study. In the chronic 10-day study THA or PHY caused no substantial ChE inhibition in brain when measured 18 hours after the last dose, whereas MTF induced 74% ChE inhibition. Long-term treatment with THA or MTF caused no changes in monoamine levels, but PHY treatment resulted in slightly increased 5-HT values. These results suggest that MTF, DDVP and naled seem to act solely by cholinergic mechanisms. However, the central neuropharmacological mechanism of action of THA and PHY may involve changes in cholinergic as well as dopaminergic and serotoninergic systems. PMID- 1711163 TI - Large scale purification and immunological characterization of human placental nerve growth factor. AB - Nerve growth factor (NGF) is a protein which plays a critical role in the development and survival of not only peripheral neurons, but possibly also cholinergic brain neurons. The present study describes a procedure for large scale isolation of human NGF of placental origin, and its immunological characterization. A protein species of approximately 26 kDa was obtained, which cross-reacted with antibodies to mouse NGF. Polyclonal and monoclonal anti-mouse NGF antibodies appeared to recognize different bands within this human NGF preparation. Although these polyclonal antibodies recognized both the dimeric and monomeric forms of mouse NGF, the monoclonal antibody recognized only a band corresponding to the dimeric form of mouse NGF. PMID- 1711164 TI - Evidence for electrogenic sodium-dependent ascorbate transport in rat astroglia. AB - The dependence of ascorbate uptake on external cations was studied in primary cultures of rat cerebral astrocytes. Initial rates of ascorbate uptake were diminished by lowering the external concentrations of either Ca2+ or Na+. The Na(+)-dependence of astroglial ascorbate uptake gave Hill coefficients of approximately 2, consistent with a Na(+)-ascorbate cotransport system having stoichiometry of 2 Na+:1 ascorbate anion. Raising external K+ concentration incrementally from 5.4 to 100 mM, so as to depolarize the plasma membrane, decreased the initial rate of ascorbate uptake, with the degree of inhibition depending on the level of K+. The depolarizing ionophores gramicidin and nystatin slowed ascorbate uptake by astrocytes incubated in 5.4 mM K+; whereas, the nondepolarizing ionophore valinomycin did not. Qualitatively similar results were obtained whether or not astrocytes were pretreated with dibutyryl cyclic AMP (0.25 mM for 2 weeks) to induce stellation. These data are consistent with the existence of an electrogenic Na(+)-ascorbate cotransport system through which the rate of ascorbate uptake is modulated by endogenous agents, such as K+, that alter astroglial membrane potential. PMID- 1711165 TI - Dopamine dependent decrease in enkephalin and substance P levels in basal ganglia regions of postmortem parkinsonian brains. AB - This study examined whether a relationship exists between the degree of dopamine (DA) loss and the changes in opioid (Met5-enkephalin, ME; dynorphin A (1-8) (DYN)) or tachykinin (substance P, SP) peptidergic systems in basal ganglia (caudate and putamen) and limbic (frontal cortex) regions of postmortem tissue samples derived from patients who died of Parkinson's disease (PD). The levels of ME, SP and DYN were determined by radioimmunoassays. The levels of DA and 5 hydroxytryptamine (5-HT) and their metabolites were determined by HPLC with electrochemical detection. The degree of loss of DA in PD tissues was classified into two major categories, those with less than 80% and those with more than 80% loss as compared to control. The results reveals that only the category with greater than 80% DA loss exhibited lower levels of ME in caudate and SP in putamen whereas no differences were observed in the levels of DYN in these regions. The frontal cortical region exhibited no changes in the levels of peptides. In other studies, experimental DA deficiency in rodents induced by neurotoxin such as 6-hydroxydopamine (6-OHDA) produced an increase in ME and a decrease in SP in basal ganglia. However, the levels of both peptides were lower in postmortem Parkinsonian basal ganglia in the present study. It appears that there is a DA-dependent, secondary loss of enkephalin and tachykinin peptides in PD. In view of the involvement of these peptidergic systems in the regulation of behaviour, movement, memory and other functions, derangements in these systems should be considered as additional factors in the progression of symptoms of PD. PMID- 1711166 TI - Pharmacological actions of substance P in the rat vas deferens. AB - SP produces two different muscular responses in the isolated vas deferens of the rat. 1) A myotropic, post-synaptic effect that results from stimulation of one subtype of SP receptor, located on the membrane of the smooth muscle (NK-3 receptor). 2) A neurogenic effect at the pre-synaptic level that results from stimulation of another subtype of SP receptor, located on the nerve terminals (NK 1 receptor). PMID- 1711167 TI - Induction of somatic sensory inputs to the lateral geniculate nucleus in congenitally blind mice and in phenotypically normal mice. AB - The incidence of aberrant innervation of the lateral geniculate nucleus by ascending somatic sensory axons was examined following injections of wheat germ agglutinin conjugated with horseradish peroxidase into the dorsal column nuclei of adult mice which were: (1) normal; (2) normal, but bilaterally enucleated on the day of birth; (3) normal, but received a large unilateral lesion of the rostral cortex on the day of birth; (4) normal, bilaterally enucleated, as well as unilaterally lesioned in the rostral cortex on the day of birth; (5) homozygous for an ocular retardation mutation (orj/orj); or (6) homozygous for the orj mutation and received a large unilateral lesion of the rostral cerebral cortex on the day of birth. In the phenotypically normal animals which were untreated, no somatic sensory inputs enter into the dorsal lateral geniculate nucleus. A few labeled axons enter into and arborize within the dorsal lateral geniculate nucleus in normal animals which received bilateral enucleations or unilateral rostral cortical lesions on the day of birth. However, in congenitally blind animals and in phenotypically normal animals which received bilateral enucleations as well as unilateral rostral cortical lesions on the day of birth, a significant number of labeled axons enter into and arborize within the dorsal lateral geniculate nucleus. Among all these experimental groups, the densest innervation of the lateral geniculate nucleus occurred in congenitally blind animals which received rostral cortical lesions on the day of birth. In these, robust arborizations of labeled somatic sensory axons occupy a substantial extent of the lateral geniculate nucleus. These results not only demonstrate that ascending somatic sensory axons can be rerouted to the lateral geniculate nucleus, but also indicate that the ability of a thalamic afferent pathway to undergo extensive reorganization and to innervate inappropriate thalamic targets following early perturbations is not unique to the retinal projection (in which this has previously been demonstrated), and may be a more general characteristic of the major thalamic afferent systems. PMID- 1711168 TI - Segregation and heterogeneity of thalamic cell populations projecting to superficial layers of posterior parietal cortex: a retrograde tracer study in cat and monkey. AB - The thalamic neurons projecting to the superficial layers of areas 5 and 7 in the cat, and area 5 in the monkey, were investigated by using superficial deposits of either horseradish peroxidase or Fast Blue in one hemisphere. In the contralateral hemisphere injections of the same tracer involving the full cortical depth were made in homotopical locations, and the distribution and soma size of retrogradely labeled thalamocortical neurons in each side of the thalamus were compared. It was found that, in the cat, labeled neurons in the lateral posterior pulvinar complex, and in paralaminar regions of the ventrolateral complex, were fewer in number and smaller in size in cases of superficial deposits than in cases of deep injection. In more lateral portions of the ventrolateral complex, however, there were no size differences. In the monkey, similar differences in number and size appeared in the caudal division of the ventrolateral complex and in the lateral posterior and pulvinar nuclei, whereas no such differences were found for neurons labeled in the oral and medial divisions of the ventrolateral complex, and in the ventral posteroinferior nucleus. In all cases the intralaminar and midline nuclei exhibited retrogradely labeled neurons only when deep layers were injected. These and previous findings point to the existence of a widely distributed layer I-projecting system of neurons which, in most nuclei, are interspersed among neurons projecting mainly to middle or deep layers. In some nuclei, however, as is the case with the ventromedial nucleus proper, layer I-projecting system neurons would make up the whole nucleus. The cell groups located in a paralaminar position, which would be but a part of this system, could provide through their projections to layer I in the posterior parietal and frontal cortical regions a final path for recruiting responses and spontaneous spindling activities. PMID- 1711169 TI - Convergence of cortical and cerebellar projections on single basilar pontine neurons: a light and electron microscopic study in the rat. AB - A protocol that involved a combination of two orthogradely transported tracer substances, wheat agglutinin-horseradish peroxidase and Phaseolus vulgaris leucoagglutinin injected at separate locations in the same animal was utilized to investigate the possible congruence of axonal projection fields formed by the cerebral cortical and cerebellar afferents to the basilar pontine nuclei. When large placements of tracer material were made in the cerebellar nuclei to label the cerebellopontine projections and a second tracer was injected in one of several cerebral cortical areas to visualize certain corticopontine projections, it was noted that axon terminal zones of the cortical and cerebellar systems occupied greater or lesser amounts of the same basilar pontine territory depending on the location of the cerebral cortical injection. Cerebellopontine terminal fields exhibited their greatest congruency with projections from the motor cortex containing the representation for facial musculature and with projections from the forelimb sensorimotor cortex. A lesser degree of overlap was observed when cerebellar projection zones were visualized in combination with basilar pontine projections from sensory face cortex, hindlimb sensorimotor cortex, visual cortex and auditory cortex. In addition, it was apparent that portions of the cerebellopontine and corticopontine terminal fields did not overlap at all. A related series of electron microscopic experiments was undertaken to establish that within the zones of overlapping cerebellar and cortical projections, there was in fact a convergence of the two afferent systems on single basilar pontine neurons. Boutons of the corticopontine system were labeled by the orthograde transport of wheat germ agglutinin horseradish peroxidase injected into the sensorimotor cortex while cerebellopontine terminals were marked for electron microscopic identification in the same animal by transecting the brachium conjunctivum and allowing sufficient time for boutons in the pontine nuclei to exhibit degeneration. Although the number of definitive examples of convergence was small, nonetheless it was possible to observe single basilar pontine neuron dendrites receiving synaptic contacts from both the cortical and cerebellar afferents systems. Taken together these observations indicate that some basilar pontine neurons receive a dual or convergent input from the cerebral cortex and cerebellar nuclei. It is difficult to estimate the prevalence of such convergence since cortical and cerebellar inputs typically contact distal and proximal pontine neuron dendrites, respectively, thus limiting the chances that both types of boutons can be observed in contact with a single basilar pontine neuron dendrite.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711170 TI - Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system. AB - The rat brain and spinal cord have been mapped for Type II glucocorticoid receptor-like immunoreactivity in neurons and glia, using a monoclonal antibody, BUGR2, which recognizes an epitope close to the DNA-binding domain of the rat Type II receptor. The study revealed a widespread distribution of Type II-like immunoreactive neurons and glia, and a heterogeneity of densities and intensities of immunoreactive elements. Our results corresponded to a large extent with previous immunocytochemical mapping using Ig2a, a monoclonal antibody against a different epitope in the variable domain, with some notable differences in the hippocampus, hypothalamus and cerebellum. There was also a good correlation between immunocytochemical mapping and binding studies, [3H]steroid autoradiography and mRNA localization of the Type II receptor. PMID- 1711171 TI - Circling behavior induced by intranigral administration of ruthenium red and 4 aminopyridine in the rat. AB - We have studied the effects of the unilateral intranigral microinjection of Ruthenium Red and 4-aminopyridine in the rat, as compared with that of muscimol. The three drugs produced contralateral turning when injected into the central nigra reticulata. Muscimol was the most effective but its effect disappeared in 3 4 h, whereas that of Ruthenium Red lasted for up to 3 days. When injected into the caudoventromedial nigra, Ruthenium Red produced intense ipsiversive turning, 4-aminopyridine weak ipsiversive turning and muscimol intense contraversive turning. Pretreatment with haloperidol (i.p.) abolished the effect of Ruthenium Red after injection into the caudoventromedial nigra but only partially reduced it after administration into the central nigra. The effect of muscimol, when injected into either of the nigral regions studied, was only slightly diminished by haloperidol. The release of [3H]GABA in slices of the Ruthenium Red-injected substantia nigra was not altered. Histological examination showed that the microinjected Ruthenium Red was located mainly inside the soma of nigral neurons. It is concluded that alterations of transmitter release are probably responsible for the circling behavior induced by 4-aminopyridine, but the effects of Ruthenium Red seem to be secondary to its penetration into the neuronal somas. Dopaminergic neurons seem to play an important role in the ipsilateral turning induced by Ruthenium Red when injected into the caudoventromedial nigra. PMID- 1711172 TI - Intrathecal somatostatin, somatostatin analogs, substance P analog and dynorphin A cause comparable neurotoxicity in rats. AB - Rats chronically implanted with intrathecal catheters received intrathecal injections (10 microliters followed by 10 microliters saline flush) of either saline (n = 5), somatostatin (100 micrograms, n = 10), the somatostatin analog BIM 23003 (100 micrograms, n = 5), the somatostatin analog SMS 201-995 (100 micrograms, n = 5), the substance P analog [D-Pro2, D-Trp7,9] SP (10 micrograms, n = 10), or dynorphin A (1-17) (20 nmol, n = 8). These doses (somatostatin, substance P and dynorphin A) were selected based on previous studies in which they caused significant motor deficits. Effects on thermal cutaneous nociception, behavior, motor function and spinal cord histopathology were evaluated. All peptides caused severe neurotoxicity, evidenced by flaccid hind leg paralysis and lumbar spinal neuronal degeneration, which was accompanied by an inflammatory reaction in meninges and spinal gray matter. Histopathological changes had developed within 24 h after injection of somatostatin, substance P analog and dynorphin A, showing mild to severe neuronal degeneration and mild inflammatory responses in spinal cord and meninges. Significant antinociceptive effects, due to severe neurotoxic effects, were only observed following intrathecal injection of SMS 201-995 and the substance P analog. Potential neurotoxic mechanisms of the different peptides are discussed. PMID- 1711173 TI - Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves. AB - The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide immunoreactive neurons. PMID- 1711174 TI - Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities. AB - Physiological studies have established that preganglionic sympathetic nerve fibers innervating the rat superior cervical ganglion release a second transmitter, in addition to acetylcholine. Based on pharmacological and histochemical investigations, possible candidates for this non-cholinergic neurotransmitter include vasoactive intestinal peptide and peptide histidine isoleucine amide. For example, previous immunohistochemical studies have demonstrated that antisera raised against both of these peptides stain neural processes in the rat preganglionic cervical sympathetic trunk and in the superior cervical ganglion. In the present study, it was found that, when the cervical sympathetic trunk was ligated, vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities built up on both sides of the ligature. In addition, examination of the thoracic spinal cord in colchicine treated animals revealed vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivies in neuronal cell bodies in the intermediolateral cell column and in the region of the lateral funiculus adjacent to it. In a second group of animals in which retrograde tracing techniques were used, these two regions of the spinal cord were shown to contain most of the cell bodies of the preganglionic neurons that project to the superior cervical ganglion. Smaller numbers of retrogradely labeled neurons were found dorsal to the central canal and in the nucleus intercalatus. When either vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunostaining and retrograde labeling were examined in the same animals, double-labeled neurons were found in the intermediolateral cell column and in the lateral funiculus. These data demonstrate that vasoactive intestinal peptide- and peptide histidine amide-like immunoreactivities are present in certain of the preganglionic neurons that project to the superior cervical ganglion, supporting the hypothesis that vasoactive intestinal peptide and peptide histidine isoleucine amide are released in the ganglion when these preganglionic neurons are activated. PMID- 1711175 TI - The topography of magnocellular projecting ganglion cells (M-ganglion cells) in the primate retina. AB - The projection from the retina to the dorsal lateral geniculate nucleus in the primate arises from two morphologically distinct types of ganglion cells. The P ganglion cells project to the parvocellular layers, the M-ganglion cells to the magnocellular layers. We have developed a neurofibrillar stain which stains the M ganglion cell population with a high degree of selectivity allowing us to map their distribution across the retina. As with other ganglion cell types the M ganglion cell density peaks close to the fovea and declines towards the periphery. At 1 mm from the fovea the proportion of M-ganglion cells ranges from 6 to 10% and then increases to about 8-10% over much of the retina except along the nasal horizontal meridian. Along the nasal horizontal meridian the percentage increases from 10% at 7 mm eccentricity to 20% or more at higher eccentricities. The increased percentage of M-ganglion cells in the nasal quadrant of the retina correlates with the relatively smaller dendritic trees of M-ganglion cells in this region. PMID- 1711176 TI - Mapping the development of the rat brain by GAP-43 immunocytochemistry. AB - Growth-associated protein-43 (GAP-43) is a phosphoprotein of the nerve terminal membrane which has been linked to the development and restructuring of axonal connections. Using a monospecific antibody prepared in sheep against purified GAP 43, we examined the temporal and spatial changes in the distribution of this protein from embryonic stage day 13 (E13) to adulthood. At stages in which neurons are still dividing and migrating, levels of GAP-43 are extremely low, as is seen in the cortical plate throughout the embryonic period. With the onset of process outgrowth, intense GAP-43 immunoreactivity appears along the length of axons: by E13, such staining is already strong in the brainstem, where it continues up through the first postnatal week and then disappears. In the neocortex, intense fiber staining first appears several days later but ends at the same time as in the brainstem. At the end of the period of intense axonal staining there is a brief interval in which high levels of GAP-43 immunostaining are seen in the neuropil. In regions of the brain in which specific developmental events have been characterized anatomically and physiologically, the period of dense neuropil staining coincides with the formation of axonal end-arbors, the beginning of synaptogenesis, and the time at which synaptic organization can be modified by the impingent pattern of activity (i.e. the critical period). Over the next few days, staining in neuropil declines sharply in most regions except for certain structures in the rostral neuraxis which may be sites of ongoing synaptic remodeling. PMID- 1711177 TI - Ultrastructural visualization of glutamate and aspartate immunoreactivities in the rat dorsal horn, with special reference to the co-localization of glutamate, substance P and calcitonin-gene related peptide. AB - Antisera raised against the fixation products of L-glutamate and L-aspartate were used, singly or in combination, to study the ultrastructural localization of the amino acids in the rat dorsal horn, with post-embedding immunogold techniques. Immunostaining for each of the amino acids was also combined with immunolocalization of GABA, an important inhibitory neurotransmitter in the spinal cord, or synaptophysin, a synaptic vesicle glycoprotein. In addition, we examined the localization of glutamate immunoreactivity in relation to that of calcitonin-gene related peptide and substance P, two neuropeptides present in high concentrations in the dorsal horn. Glutamate- and aspartate-immunoreactive neuronal cell bodies, dendrites, axons and terminals were apparent in the first three laminae of the dorsal horn. In somatic and dendritic profiles, the immunolabel was present over the general cytoplasm and mitochondria; in the terminals, it was found over small, agranular vesicles, mitochondria and, at times, synaptic densities. Quantitative estimation indicated that the colloidal gold density in the glutamate-immunoreactive terminals was five-fold more than in any other neuronal profile. Both glutamate- and aspartate-immunopositive terminals made asymmetric synaptic contacts onto unlabelled dendrites; glutamate positive terminals often formed the core of type I and II glomeruli. After double labelling of the same sections, glutamate and aspartate immunoreactivities consistently occurred in different axonal and terminal profiles. In these preparations, it was clearly seen that glutamate-immunoreactive terminals were far more numerous than (more than 10-fold) those immunoreactive for aspartate. Double labelling for glutamate or aspartate and GABA also revealed distinct staining of different terminals. Simultaneous immunolocalization of each of the amino acids and synaptophysin showed the amino acid and glycoprotein immunoreactivities co-localized in small, agranular vesicles in immunoreactive terminals. Finally, triple labelling of the same sections for glutamate, calcitonin gene-related peptide and substance P revealed that glutamate was often co-localized with either of the two neuropeptides in the same axonal boutons; terminals that showed simultaneous labelling for glutamate, calcitonin gene related peptide and substance P were also noted. In all cases, the glutamate immunoreactivity was restricted to small, clear vesicles whereas the neuropeptide immunoreactivities were present in larger, dense-cored vesicles. Our observations demonstrate that there is an abundant glutamate immunoreactivity in the superficial layers of the rat dorsal horn, localized in neuronal profiles distinct from those containing aspartate or GABA.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711178 TI - [Brain infarcts in humans during middle age and senility. I. Defects of blood brain barrier permeability in immunocytochemical studies]. AB - Brains of the deceased in the age from 45 to 57 years (6 cases) and from 80 to 101 years (9 cases) with ischemic brain damage were studied. Technique of the peroxidase-antiperoxidase (Sterberger et al. 1970) was used for visualization of the brain tissue reactivity for albumin, IgG, alpha 1-antitrypsin and alpha 2 macroglobulin. In all cases the disturbances of the blood vessels permeability were found. The BBB deterioration within the first days of brain infarct seems to be smaller in senile age than in middle age. However, after some days senile blood vessels permeability increases and become similar in both age groups. PMID- 1711179 TI - Occurrence of galanin-like immunoreactivity in growth hormone-releasing factor (GRF)-containing neurons of the monkey (Macaca fascicularis) infundibular nucleus and median eminence. AB - The distribution of growth hormone-releasing factor (GRF)- and galanin (GAL) immunoreactive (-IR) neurons in the mediobasal hypothalamus of the monkey (Macaca fascicularis) was studied with immunohistochemistry using a direct double labelling method. GRF- and GAL-IR cell bodies were demonstrated in the ventral part of the infundibular nucleus and dense aggregations of GRF- and GAL-IR fibers were seen in the external layer of the median eminence, closely surrounding portal vessels. Double-staining revealed that GRF and GAL were colocalized in cell bodies of the infundibular nucleus and in nerve fiber varicosities in the external layer of the median eminence. GAL has been reported to stimulate the secretion of growth hormone in both rats and humans, most likely via hypothalamic mechanism(s). PMID- 1711180 TI - Abnormal distribution of phosphorylated neurofilaments in neuronal degeneration induced by kainic acid. AB - An abnormal distribution of phosphorylated neurofilaments is present in some human neurodegenerative disorders including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. This study reports the changes of phosphorylated neurofilaments observed in rat spinal cord after intrathecal injection of kainic acid. This receptor agonist of excitatory amino acid produces abnormal phosphorylation of neurofilaments in the cell body and proximal neurites of degenerating neurons. These immunocytochemical modifications observed 2 and 10 days after injections are predominantly located in ventral horn neurons. This study indicates that one of the neuronal responses to excitatory amino acid toxicity is the pathological distribution of phosphorylated neurofilaments in affected neurons. Pathological findings are comparable to those observed in neurodegenerative diseases such as amyotrophic lateral sclerosis. PMID- 1711181 TI - ATP-activated single-channel currents recorded from cell-free patches of pheochromocytoma PC12 cells. AB - Single-channel recordings were made using cell-free membrane patches (outside-out configuration) isolated from pheochromocytoma PC12 cells. ATP (50 microM) activated single channel currents in the isolated patches and the currents inactivated with a half-decay time of about 5s. The single channel conductance was about 13 pS in external solution with 140 mM Na. The amplitudes of the single channel currents were decreased when external Ca was increased from 1.8 to 16.2 mM, suggesting that Ca blocks ion permeation through the channel. These properties of single-channel currents may underlie those of the macroscopic current. PMID- 1711182 TI - Acute phase proteins are present in amorphous plaques in the cerebral but not in the cerebellar cortex of patients with Alzheimer's disease. AB - Using immunohistochemical staining for beta-amyloid proteins in the brains of Alzheimer patients two basic types of plaques can be found: 'amorphous' or 'diffuse' non-congophilic plaques and classical plaques. Acute phase proteins alpha 1-antichymotrypsin, complement factors and P-component ('plaque-associated molecules') have been reported in both types of plaques in the cerebral cortex. In the present study we could not find immunoreactivity for these acute phase proteins in the amorphous plaques in the cerebellar cortex. These proteins seem to be essential for formation of classical plaques. PMID- 1711183 TI - Lipotrope supplementation prolongs survival of leukemic mice. PMID- 1711184 TI - Strategies for assessing neurotoxicity. AB - The ultimate end point among the various measures of neurotoxicity is the death of neurons, since they do not regenerate. Experiments that accurately assess this degree of neurotoxicity must utilize a sensitive means of detection and control for the factors that are variables in the expression of neurotoxicity. Neurotoxic substances have the characteristic trait that each selectively destroys specific populations or subpopulations of neurons. The mechanisms underlying this selectivity are generally unknown. Without a priori knowledge of where in the brain to look, it is prudent to employ a histological procedure that imparts an easily detected marker to degenerating neuronal components. For example, the so called "reduced silver" stains specifically impregnate degenerating neural elements. From studies of known neurotoxins, several factors have been shown to influence neurotoxicity: 1) species and strains; 2) age; 3) sex; 4) dose rate (acute vs. chronic); 5) survival time; and 6) sampling interval in the brain. Failure to include these factors in a screening paradigm could yield false negative results and, thereby, invalidate extrapolations made to man. PMID- 1711185 TI - Allergy, cold sufferers must cope with effects of OTC medications. PMID- 1711186 TI - HMB-45 immunohistochemical staining of conjunctival melanocytic lesions. AB - HMB-45 is a monoclonal antibody recently described as being highly specific for melanocytic proliferations of the skin and in metastases of melanotic lesions. To determine a possible role for HMB-45 in ophthalmic pathology, 45 conjunctival lesions, including 23 melanomas, were analyzed using immunohistochemical techniques with anti-S-100 and HMB-45 as primary antibodies. Nineteen (82.6%) of the melanomas exhibited HMB-45 positivity, and 19 (82.6%) contained S-100 protein, with concordance of all but two cases. Seven cases of primary acquired melanosis were studied; one (33%) of three with atypia was HMB-45 positive, as were two (50%) of four without atypia. Among nevi, 1 (9.1%) of 11 showed faint staining with HMB-45. Fifteen conjunctival epithelial dysplasias were negative with HMB-45. At present, HMB-45 appears to offer no great advantage over S-100 protein in the analysis of conjunctival melanomas. Its role in the distinction of benign from atypical or malignant junctional melanocytic proliferations remains unclear. PMID- 1711187 TI - Polymorphous low-grade adenocarcinoma of minor salivary gland. A comparative histologic and immunohistochemical study. AB - Sixteen polymorphous low-grade adenocarcinomas were reviewed and compared with 17 adenoid cystic carcinomas and with 21 other histologically similar minor salivary gland neoplasms. The polymorphous low-grade adenocarcinomas were for the most part distinctive in their microscopic appearance. Typically they exhibited infiltrative growth by small uniform cells in single-layered ducts. A syncytium of tumor cells was also characteristic, although solid and cribriform patterns were seen, making definitive diagnosis difficult with some tumors. Immunohistochemical staining for S-100 protein, glial fibrillary acidic protein, actin, vimentin, and keratins resulted in relatively distinctive antigenic profiles for the tumors studied. Of significance was strong S-100 protein and weak actin staining of polymorphous low-grade adenocarcinomas, moderate actin staining of adenoid cystic carcinomas, moderate glial fibrillary acidic protein staining of monomorphic adenomas and pleomorphic adenomas, and nonreactivity of monomorphic adenomas for vimentin. It is believed that the immunoprofiles could be useful in the microscopic diagnosis of salivary gland tumors. The identification of antigens found normally in myoepithelial and epithelial cells supports the concept that these tumors are derived from pluripotential reserve cells. PMID- 1711188 TI - Effects of overexpression of ornithine decarboxylase (ODC) on growth control and oncogene-induced cell transformation. AB - The enzyme ornithine decarboxylase (ODC) has been implicated in the control of cell growth, differentiation and tumor promotion. To further elucidate its precise role a murine ODC cDNA was inserted into the retrovirus-derived vector pMV7 and retrovirus-like particles were used to infect NIH3T3 and rat 6(R6) fibroblasts. Derivatives were obtained that stably express a 5-40 fold increase in ODC enzyme activity. Despite these high levels of enzyme activity the cells retained a normal morphology and displayed no major changes in growth properties in monolayer culture or in agar suspension. On the other hand, R6 cells that expressed high levels of ODC displayed a marked increase in susceptibility to morphologic transformation by an activated c-H-ras oncogene. These results provide the first evidence that ODC can cooperate with an activated oncogene in the process of cell transformation. Although the mechanism is not known, these findings may be relevant to the multistage carcinogenic process. PMID- 1711189 TI - Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways. AB - A variety of signal transduction pathways contribute to the regulation of transcription in mammalian cells. Several of these pathways ultimately rely upon the interaction of transcription factors with genetic sequences termed response elements in the promoter regions of some genes. The biochemical mechanisms that control the levels and state of activation of transcription factors are poorly understood. However, specific phosphorylation events mediated by protein kinase C, growth factor receptor-linked tyrosine kinases, and protein kinase A clearly participate in the regulation of these signal transduction pathways. To understand the relationship between activation and/or inhibition of these pathways and regulation of gene expression controlled by specific response elements, cell lines were prepared containing the TPA response element (TRE), serum response element (SRE), or cyclic AMP response element (CRE) fused to a gene encoding a secretable form of alkaline phosphatase (SEAP). These TRE-SEAP, SRE-SEAP, and CRE-SEAP cells exhibit dramatic increases in alkaline phosphatase (AP) activity following exposure to TPA, PDGF, or forskolin. Down regulation of protein kinase C or inhibition of tyrosine kinase activity blocked the stimulation of AP activity caused by TPA or PDGF. These cell lines can be used to characterize existing inhibitors, and to identify new agents that affect specific signal transduction pathways in mammalian cells. PMID- 1711190 TI - PCR-based identification of new receptors: molecular cloning of a receptor for fibroblast growth factors. AB - Transmembrane tyrosine kinases are involved in the control of cell growth and differentiation by extracellular signals. To enable identification of new receptor tyrosine kinases we developed a method that selectively amplifies segments of receptor genes. The method is based on a combination of polymerase chain reaction (PCR) and hybridization screening and it employs three oligonucleotide primers derived from conserved domains of receptor tyrosine kinases. It yields amplification of receptors' genes and appears to ignore cytoplasmic tyrosine kinases. When applied to RNA from 12.5 days post coitum mouse placenta, this methodology resulted in the detection of several putative or established receptors. Molecular cloning of one of these genes, which is identical to the partially characterized bek gene, identified a transmembrane tyrosine kinase with three immunoglobulin-like domains in the extracellular portion, and a cytoplasmic tyrosine kinase sequence. The isolated cDNA shows remarkable homology to the murine flg gene that encodes a receptor for fibroblast growth factors. Indeed, an antibody directed to the carboxy terminus of the deduced bek protein specifically recognized a receptor for acidic and basic fibroblast growth factors in murine hepatoma cells. We therefore expect that the methodology we developed will enable the study of new receptors in hardly accessible biological systems such as early mammalian embryos or stem cells. PMID- 1711191 TI - Tumor progression following transformation of murine monocytes by v-myc: acquisition of immortalization and tumorigenicity. AB - Monocyte transformation by the v-myc oncogene has been used to study myelomonocytic tumor progression in vitro. Murine monocytes transformed by a recombinant retrovirus containing MC29 v-myc were found to exhibit a proliferative burst to day 28-40 post-infection. There-after growth slowed and cell number remained relatively static to day 80-90 post-infection. During both the proliferative and quiescent periods, the cells were dependent on the myelomonocytic growth factor CSF-1 for growth and viability. Analysis of this transformation revealed that the initial transformants were polyclonal, non immortal, and non-tumorigenic in syngeneic mice. At day 80-90 post infection, a fresh round of cellular proliferation occurred and, in contrast to the initial burst, growth was sustained allowing the establishment of cell lines. These lines were found to be monoclonal, immortal, growth factor independent and, in certain cases, tumorigenic in syngeneic mice. Associated with the establishment of growth factor independent cell lines was the constitutive synthesis of the myelomonocytic growth factor, CSF-1. Proto-oncogene screening of the initial transformants and the cell lines also revealed the expression of c-raf and the CSF-1 receptor, c-fms. Our results indicate that, following transformation by v myc, monocytes can progress in vitro to become growth factor independent and immortal and that both monocyte transformation and immortalization can be dissociated from tumorigenicity. PMID- 1711192 TI - Post-herpetic neuralgia: further post-mortem studies of cases with and without pain. AB - The pathological features associated with post-herpetic neuralgia require further study. We report here 5 cases, 3 with severe post-herpetic neuralgia (PHN) and 2 with no persistent pain. The findings of dorsal horn atrophy and cell, axon and myelin loss with fibrosis in the sensory ganglion were found only in patients with persistent pain. Marked loss of myelin and axons in the nerve and/or sensory root were found in cases with and without pain. Some evidence is presented for a more generalized subacute or chronic inflammatory process which may explain the clinical features of some patients. Further studies will be necessary to fully describe the morbid anatomy of this disorder. PMID- 1711193 TI - Neurokinin and NMDA antagonists (but not a kainic acid antagonist) are antinociceptive in the mouse formalin model. AB - While much evidence implicates substance P (SP), an endogenous neurokinin (NK), as a primary sensory transmitter of acute pain in mammalian spinal cord, its role in continuous (tonic) pain is less clear. Although glutamate is co-localized with SP in dorsal root ganglion neurons, its role in nociceptive processing is uncertain. While antagonists of NKs and excitatory amino acids (EAAs) have been found to be antinociceptive in some acute assays, they have not been tested against tonic pain. We hypothesize that: (1) NKs and EAAs contribute to signaling of tonic chemogenic nociception; and (2) interaction between NK and EAA systems is important in determining the perceived intensity of a continuous noxious stimulus. We therefore evaluated two NK antagonists ([D-Pro2,D-Trp7,9] SP (DPDT SP, 0.26-6.6 nmoles, non-specific) and [D-Pro4, D-Trp7,9,10,Phe11]-SP(4-11) (DPDTP-octa, 1.6-12.3 nmoles, somewhat NK-1 selective], as well as DL-2-amino-5 phosphonovalerate (DL-AP5, NMDA antagonist, 0.05-1 nmole) and urethane (a kainic acid (KA) antagonist at 2.5 mumoles) for antinociceptive activity in the mouse formalin model. Administered intrathecally (i.t.), DL-AP5 and both NK antagonists were significantly antinociceptive while urethane (2.5 mumoles) and naloxone (2.7 nmoles) were inactive. A50 values for mean % analgesia, nmoles/mouse i.t. (95% CLs) were: DPDT-SP, 1.1 (0.79-1.6); DPDTP-octa, 3.9 (2.4-6.1); DL-AP5, 0.29 (0.16 0.71). The antinociception associated with 1.3 nmoles of DPDT-SP was not reversed by co-administering 2.7 nmoles of naloxone. Co-administration of 0.1 nmoles of DL AP5 with either 1.3 nmoles of DPDT-SP or 3.3 nmoles of DPDTP-octa did not lead to additive antinociception.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711195 TI - The cellular and molecular basis of the human T cell response to Dermatophagoides spp. (house dust mite). PMID- 1711194 TI - Intrathecal injections of galanin and its antiserum affect nociceptive response of rat to mechanical, but not thermal, stimuli. AB - To ascertain the involvement of galanin in nociceptive transmission in the spinal dorsal horn, we examined the effects of intrathecal injections of porcine galanin and its antiserum on behavioral nociceptive responses of the rat, using the paw pressure and paw-radiant heat tests. An intrathecal injection of antiserum against porcine galanin reversed the decreased nociceptive threshold for mechanical, but not thermal, stimulation of the carrageenin-treated hind paw of rat, with no effect on the nociceptive threshold of the non-inflamed hind paw. Rat galanin as well as porcine galanin suppressed the binding of [125I]porcine galanin to the antiserum, a finding suggesting that the antiserum can bind rat galanin. An intrathecal injection of porcine galanin (0.1 and 1 nmol, but not 0.01 nmol) decreased the mechanical nociceptive threshold of the rat hind paw with no effect on thermal nociception. These results suggest that galanin present in the dorsal horn is involved in the facilitation of mechanical, but not thermal, nociceptive transmission. PMID- 1711196 TI - Analysis of peripheral leucocyte populations in N'Dama and Boran cattle following a rechallenge infection with Trypanosoma congolense. AB - Monoclonal antibodies, flow cytometry and routine haematological techniques were used to analyse circulating leucocyte populations in trypanotolerant (N'Dama) and trypanosusceptible (Boran) cattle following a homologous rechallenge with Trypanosoma congolense clone IL13-E3. The N'Damas developed a low, transient parasitaemia and did not develop anaemia. The Borans became parasitaemic and developed chronic anaemia but three of the five animals eventually self-cured, whilst, a group of primary-challenged Borans experienced a severe infection characterized by high levels of parasitaemia and acute anaemia. During infection the numbers of circulating B-cells increased in all three groups from day 21 onwards. The proportion of B-cells expressing the CD5 antigen increased from pre infection levels of 5-10% of B-cells to 49-90% by day 19 post infection in all three groups. The neutrophil count declined in both Boran groups but not in the N'Damas. The CD4+ T-cell and gamma delta T-cell populations decreased in both Boran groups but did not alter significantly in the N'Damas. Although it was not possible to infer from the data, that the CD4+, gamma delta T-cell, neutrophil and erythrocyte populations were directly responsible for the differential control of the disease by the two breeds, it was possible to correlate alterations in these cell populations with the severity of the disease. PMID- 1711197 TI - Heterogeneity in the expression of surface-exposed epitopes among larvae of Ascaris lumbricoides. AB - Quantitative immunofluorescence was used to examine differences in the binding of antibody to the surfaces of individual living infective stage larvae of Ascaris lumbricoides. Using rabbit antisera, it was first established that larvae cultured for 48 h after artificial hatching were relatively uniform in their levels of antibody binding and in minimal exposure of epitopes expressed by later larval stages. Aliquots from a pool of larvae were probed with serum from individual infected people living in an endemic area of Nigeria. The larvae used were derived from parasites collected in the same geographical area in which serum donors were living. Two principal points emerged. First, serum donors varied considerably in the degree to which their antibody bound to the larvae. Secondly, the binding of antibody from a given donor revealed remarkable heterogeneity in surface epitope expression. Such intra-specific variability in antigen expression has considerable implications for the development of immunity to parasitic nematodes. PMID- 1711198 TI - Antinociceptive action of the SP1-4 tetrapeptide and of some tuftsin analogs. AB - The N-terminal tetrapeptide of substance P (SP1-4) was found to produce analgesia, after the icv injection to the rat brain, which is lower in its intensity than that produced by tuftsin (Thr-Lys-Pro-Arg tetrapeptide). Among investigated tuftsin analogues Thr-Lys-Pro-Thr and Thr-Lys-Pro-Thr-Asp (partial sequences of S-protein of HB virus) were weakly active, Thr-Arg-Pro-Arg was inactive, and Thr-Lys-Pro-Gly-Arg produced a weak hyperalgesia 30 min after the icv injection. The obtained results were compared with those obtained previously in the phagocytosis stimulation test. In the control experiments the effects of free amino acids of the tuftsin molecule (Thr, Lys, Pro, Arg) were also studied. PMID- 1711199 TI - Substance P pretreatment modifies morphine-induced hypotension in anesthetized rats. PMID- 1711200 TI - Different effects of prostacyclin and its stable analogues iloprost and cicaprost on isolated guinea pig atria. PMID- 1711201 TI - Determination of degree of substitution of formyl groups in polyaldehyde dextran by the hydroxylamine hydrochloride method. AB - Colorimetric or potentiometric titration of the aldehyde residues in polyaldehyde dextran by the hydroxylamine hydrochloride/sodium hydroxide method has been found to be a convenient and accurate method to determine formyl content. Nitrogen combustion analyses on the isolated oximes confirmed the titrametric results. PMID- 1711202 TI - Retinoid-mediated transcriptional regulation of keratin genes in human epidermal and squamous cell carcinoma cells. AB - Vitamin A and other retinoids profoundly inhibit morphological and biochemical features of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, we examined retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. Our studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5' upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors alpha and gamma, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes. PMID- 1711203 TI - Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products. AB - Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity. PMID- 1711205 TI - Computation of ionic distributions around charged biomolecular structures: results for right-handed and left-handed DNA. AB - We introduce an efficient computational methodology employing the potentials of mean force approach for estimating the detailed three-dimensional ionic distributions around arbitrarily complex charged biomolecular structures for all monovalent salt concentrations of practical interest (e.g., 0.1-5.0 M NaCl). Such distributions are required for specifying thermodynamic and structure-specific features of ion-mediated interactions of charged proteins, DNA and RNA, membranes, and macromolecular assemblies. As a first application, we present results for distributions around the B and ZI conformers of the DNA oligomer d(C G)18.d(C-G)18. The ionic microenvironment depends strongly on the DNA conformation, sequence, and bulk salt concentrations. PMID- 1711204 TI - Cytolytic and ion channel-forming properties of the N terminus of lymphocyte perforin. AB - Perforin lyses cells by binding to the target cell membrane, where it polymerizes into large nonspecific pores. It is shown here that the first 34 amino acids of the N-terminal region of either human or murine perforin are soluble in aqueous medium and spontaneously insert into membranes. The N-terminal peptides lyse liposomes and nucleated cells, and they form ion channels in planar bilayers, some of which are comparable to those previously described for perforin. The lytic activity of the N-terminal domains does not require calcium, is independent of the lipid headgroup composition, and can be inhibited by heparin. Tumor cells incubated with the N-terminal peptides undergo the same morphological changes as those induced by native perforin. None of the peptides corresponding to the putative membrane-spanning domains from the central region of perforin is cytolytic. Taken together, these results suggest that the N-terminal region is an important part of the pore-forming domain of perforin. PMID- 1711206 TI - Self-assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunogenically similar to native virions. AB - B19 parvovirus is pathogenic in humans, causing fifth disease, transient aplastic crisis, some cases of hydrops fetalis, and acquired pure red cell aplasia. Efforts to develop serologic assays and vaccine development have been hampered by the virus's extreme tropism for human bone marrow and the absence of a convenient culture system. We constructed recombinants containing either the major (VP2) or minor (VP1) structural proteins of B19 in a baculovirus-based plasmid, from which the polyhedrin gene had been deleted; these recombinant plasmids were used to generate recombinant infectious baculovirus. Subsequent infection of insect cells in vitro resulted in high-level expression of either B19VP1 or VP2. Parvovirus capsids were obtained by self-assembly in cell cultures coinfected with either VP1- and VP2-containing baculoviruses or, surprisingly, VP2-containing baculoviruses alone. Empty B19 capsids composed of VP1 and VP2 could replace serum virus as a source of antigen in a conventional immunoassay for detection of either IgG or IgM antiparvovirus antibodies in human serum. Immunization of rabbits with capsids composed of VP1 and VP2 resulted in production of antisera that recognized serum parvovirus on immunoblot and neutralized parvovirus infectivity for human erythroid progenitor cells. Baculovirus-derived parvovirus antigen can substitute for scarce viral antigen in immunoassays and should be suitable as a human vaccine. PMID- 1711207 TI - Steel-Dickie mutation encodes a c-kit ligand lacking transmembrane and cytoplasmic domains. AB - Mice homozygous for the viable Sl allele steel-Dickie (Sld) are sterile, severely anemic, and black-eyed white. The nature of the Sld mutation was investigated at the molecular level and was found to be due to a 4.0-kilobase intragenic deletion in mast cell growth factor (MGF) genomic sequences, providing conclusive evidence that Sl encodes MGF. As a consequence of this deletion, Sld is only capable of encoding a soluble truncated growth factor that lacks both transmembrane and cytoplasmic domains. Northern analysis indicates that Sld mRNA is expressed at approximately wild-type levels in adult tissues, and yeast expression studies suggest that the Sld protein is as biologically active as wild-type soluble MGF. These studies provide a molecular basis for explaining the Sld phenotype, a description of a germ-line mutation in the transmembrane and cytoplasmic domains of a membrane-bound growth factor, and in vivo evidence for the importance of membrane-bound forms of growth factors in mammalian development. PMID- 1711208 TI - Characterization of the decay-accelerating factor gene promoter region. AB - Decay-accelerating factor (DAF) expression modulates susceptibility of cells to autologous complement attack. To characterize the regulatory region controlling DAF gene transcription, genomic DNA extending from 815 base pairs (bp) upstream to approximately 4 kilobases downstream of DAF's AUG codon (designated +1) was cloned and sequenced. The 5' flanking sequence showed 59-76% G + C content (-355 to +1), at least one GC box(es) (-135 to -131), and variable length sequences (from -629 to -285) conforming to the motifs TCCTCC and TCn. Nuclease S1 digestions and primer extensions localized a major transcriptional start site to 82/-81, 38 bp downstream of a possible TATA variant, (A)TTTAA. In COS cell transfections, the sequence encompassing -815 to -67 functioned 2.5% as efficiently as the Rous sarcoma virus 3' long terminal repeat, but following deletion upstream of -355 its activity increased approximately 4-fold. Two octanucleotides exhibiting partial homology to phorbol 12-myristate 13-acetate (PMA) and cAMP responsive elements (PREs and CREs, respectively) were detected, and the respective modulators enhanced transcriptional efficiency 2- and approximately 10-fold, respectively. Thus, the DAF gene promoter (i) exhibits sequences resembling both conventional and unconventional transcriptional control elements, (ii) possesses a region with negative regulatory activity, and (iii) responds to PMA and cAMP induction presumably via PRE- and CRE-like enhancer elements. PMID- 1711209 TI - Altered expression of insulin receptor types A and B in the skeletal muscle of non-insulin-dependent diabetes mellitus patients. AB - The human insulin receptor exists in two isoforms, HIR-A and HIR-B, which are generated by alternative splicing of a primary gene transcript and differ by a 12 amino acid insertion sequence in the alpha-subunit. The two receptor isoforms bind insulin with different affinities and are differentially expressed in human tissues. We report here a tissue-specific alteration of the insulin receptor RNA splice pattern in non-insulin-dependent diabetes mellitus (NIDDM) patients. Whereas skeletal muscle of healthy individuals contains exclusively high-affinity HIR-A-encoding RNA, we consistently find low-affinity HIR-B RNA expression in NIDDM muscle tissue at levels similar to HIR-A. PMID- 1711210 TI - Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules. AB - Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilament system as in the zonula adherens or with intermediate filaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended family, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of approximately 29 amino acid residues predicted to have an antiparallel beta-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2(+)-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu. PMID- 1711211 TI - Localized cytosolic domains of the erythropoietin receptor regulate growth signaling and down-modulate responsiveness to granulocyte-macrophage colony stimulating factor. AB - Erythrocyte development in mammals depends in part upon the interaction of the glycopeptide hormone erythropoietin (EPO) with cell surface receptors on committed erythroid progenitor cells. Both this factor and an EPO receptor polypeptide previously have been cloned, yet little is presently understood concerning molecular mechanisms of receptor activation and signal transduction. To identify cytosolic receptor domains necessary for signaling, we have compared the activities of a series of deletionally mutated EPO receptor constructs by their expression in interleukin 3-dependent, myeloid FDC-P1 cells. EPO-induced growth was transduced efficiently in these cells by the full-length receptor (507 amino acids), and no measurable loss in activity resulted from the deletion of up to 111 carboxyl-terminal residues. In contrast, the deletion of 44 additional residues led to a dramatic loss (86.3% +/- 7.8%; mean +/- SD) in the ability of this receptor to mediate EPO-induced growth, thus indicating that residues between Gly-352 and Met-396 constitute a functionally critical cytosolic subdomain. Interestingly, the expression of full-length EPO receptors in FDC-P1 cells also led to a selective inhibition of normal proliferative responsiveness to the alternative hematopoietic factor granulocyte-macrophage colony-stimulating factor. Moreover, this inhibition was progressively reversed in forms of the EPO receptor in which distal cytosolic residues were sequentially deleted. These results suggest that EPO receptors normally may trans-modulate components in the pathway of granulocyte-macrophage colony-stimulating factor-induced proliferation and that this down-modulation, as exerted by intact EPO receptors, may play a role in promoting erythroid commitment during myeloid blood cell development. PMID- 1711212 TI - Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr. AB - Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr. PMID- 1711213 TI - Characterization of a member of the immunoglobulin gene superfamily that possibly represents an additional class of growth factor receptor. AB - We have screened cDNA libraries prepared from embryonic chicken tissues to isolate additional genes encoding growth factor receptors. Nucleotide sequencing of a cDNA encoding a gene, which we have termed klg, revealed it to represent an additional member of the immunoglobulin gene superfamily, which also possesses extensive sequence similarity to the protein-tyrosine kinase growth factor receptor genes. The klg gene was shown to encode a 140-kDa glycoprotein. However, the sequence of the tyrosine kinase domain is unusual in that the aspartate residue located within the highly conserved Asp-Phe-Gly triplet is replaced by an alanine residue. The presence of this aspartate has previously been found to be essential for tyrosine kinase activity. Consistent with the replacement of this aspartate, we were unable to detect any evidence of an associated kinase activity with the klg-encoded protein. These observations raise the possibility that the klg gene product represents a newly discovered class of receptor that plays a role in signal attenuation rather than signal propagation. PMID- 1711214 TI - 9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] derivatives of purines: a class of highly selective antiretroviral agents in vitro and in vivo. AB - A new class of compounds, 9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] [(RS) FPMP] derivatives of purines, is described that has selective activity against a broad spectrum of retroviruses [including human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)] but not other RNA or DNA viruses. This activity spectrum is completely different from that of the parental compounds, 9-[(2S)-3 hydroxy-2-phosphonylmethoxypropyl] [(S)-HPMP] derivatives of purines, which are active against a broad range of DNA viruses. The racemic (RS)-FPMP derivatives of adenine and 2,6-diaminopurine, termed (RS)-FPMPA and (RS)-FPMPDAP, respectively, are markedly more selective as in vitro antiretroviral agents than their 9-(2 phosphonylmethoxyethyl) (PME) counterparts, PMEA and PMEDAP. Also, (RS)-FPMPA and (RS)-FPMPDAP have a substantially higher therapeutic index in mice in inhibiting Moloney murine sarcoma virus-induced tumor formation and associated death and are markedly less inhibitory to human bone marrow cells than PMEA and PMEDAP. The diphosphate derivative of (RS)-FPMPA [(RS)-FPMPApp] is a potent and selective inhibitor of HIV-1 reverse transcriptase but not of HSV-1 DNA polymerase or DNA polymerase alpha. (RS)-FPMPApp, akin to PMEA diphosphate (PMEApp), acts as a DNA chain terminator. The DNA chain-terminating properties of PMEApp and (RS)-FPMPApp seem to be a prerequisite for acyclic nucleoside phosphonates to exhibit antiretrovirus (i.e., anti-HIV) activity. PMID- 1711215 TI - Kinetics of expression of multiply spliced RNA in early human immunodeficiency virus type 1 infection of lymphocytes and monocytes. AB - The genome of human immunodeficiency virus type 1 (HIV-1) encodes at least six proteins involved in regulation as well as the structural proteins Gag, Pol, and Env. The interplay of the various regulators generates early and late transcriptional phases in the HIV-1 life cycle; the earliest RNA is enriched in subgenomic species, and the genomic transcript appears at the later stage of infection. We investigated the nature of the mRNAs expressed in the early stages of infection when the 2 kilobase subgenomic species predominate. RNA was analyzed in the early phase of a one-step growth cycle of HIV-1 infection in T-lymphoid and monocytic cell lines by using PCR amplification of in vitro-synthesized viral cDNAs. In both cell lines, expression of Tat-, Rev-, and Nef-specific messages appeared simultaneously and could be detected within 8-12 hr of infection but in different amounts with a predominance of Nef-specific message. The Env-specific message could be detected as early as the Rev-specific message, indicating that expression of at least small amounts of the singly spliced message could occur before the accumulation of Rev. PMID- 1711216 TI - Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro. AB - The response of endothelial cells to angiogenic stimuli has been shown to be influenced by the extracellular microenvironment. We tested whether thrombospondin, an extracellular matrix protein, modulated the spontaneous formation of cords by endothelial cells in vitro. Despite continued proliferation, a decrease in secreted thrombospondin was detected in cord containing, as compared with subconfluent, cultures of both aortic and microvascular endothelial cells. Consistent with this trend, mRNA levels of thrombospondin decreased by factors of 16 in aortic and 60 in microvascular cultures that contained endothelial cords. Since thrombospondin was immunolocalized to fibrillar arrays that appeared to be associated with endothelial cords, we added anti-thrombospondin IgG to cord-forming cultures to limit the availability of the protein during this process. In the presence of anti-thrombospondin antibodies, there was a 33-50% increase in cord formation. These results suggest that thrombospondin is an inhibitor of angiogenesis in vitro and are consistent with its proposed roles as a destabilizer of endothelial cell focal contacts and as an inhibitor of endothelial cell proliferation. PMID- 1711217 TI - Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21. AB - PTPG, the gene for protein-tyrosine phosphatase gamma (PTP gamma), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP gamma allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP gamma allele was lost in three lung tumors that had not lost flanking loci. PTP gamma mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP gamma gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21. PMID- 1711218 TI - Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. AB - Studies on cell lines have greatly improved our understanding of many important biological questions. Generation of cell lines is facilitated by the introduction of immortalizing oncogenes into cell types of interest. One gene known to immortalize many different cell types in vitro encodes the simian virus 40 (SV40) large tumor (T) antigen (TAg). To circumvent the need for gene insertion in vitro to generate cell lines, we created transgenic mice harboring the SV40 TAg gene. Since previous studies have shown that TAg expression in transgenic mice is associated with tumorigenesis and aberrant development, we utilized a thermolabile TAg [from a SV40 strain, tsA58, temperature sensitive (ts) for transformation] to reduce the levels of functional TAg present in vivo. To direct expression to a broad range of tissues, we used the mouse major histocompatibility complex H-2Kb promoter, which is both widely active and can be further induced by interferons. tsA58 TAg mRNA was expressed in tissues of all animals harboring the hybrid construct. Development of all tissues was macroscopically normal except for thymus, which consistently showed hyperplasia. Fibroblast and cytokeratin+ thymic epithelial cultures from these mice were readily established without undergoing crisis and were conditionally immortal in their growth; the degree of conditionality was correlated with the levels of tsA58 TAg detected. One strain of H-2Kb-tsA58 mice has been bred through several generations to homozygosity and transmits a functional copy of the transgene. PMID- 1711219 TI - Hormone-dependent transcriptional regulation and cellular transformation by Fos steroid receptor fusion proteins. AB - The protooncogene c-fos has been implicated in the control of proliferation and transformation of fibroblasts, and its protein product is an essential component of transcription factor AP1. The important target genes and, hence, the molecular mechanism of Fos function are, however, still unknown, partly due to the lack of a tightly regulated Fos-induction system. Here we show that different activities of the Fos protein can be controlled hormonally by fusing the mouse c-Fos protein to the ligand-binding domain of either the rat glucocorticoid or the human estrogen receptor. These fusion proteins stimulate AP1-dependent transcription and repress endogenous fos mRNA synthesis in a strictly hormone-dependent manner. Expression of these chimeric proteins in rat fibroblasts results in fast, reversible, and tightly controlled transformation in response to hormone. A Fos estrogen receptor expressing cell line was used to isolate Fos-responsive genes by subtractive cDNA cloning. Run-on analysis of one of these genes showed that its transcription is rapidly and directly regulated by the hormone-activated Fos estrogen receptor protein, demonstrating the potential of this induction system for identifying Fos target genes. PMID- 1711220 TI - A cluster of transcribed sequences between the Pb and Ob genes of the murine major histocompatibility complex. AB - The region of the murine major histocompatibility complex (MHC) between the Pb (A beta 3) and Ob (A beta 2) genes controls the expression of an intracellular complex named the LMP (low molecular weight polypeptide) complex. DNA probes for at least seven different genes mapping to this region were isolated. These hybridize to a minimum of eight different transcripts ranging from approximately 1.3 to 3.7 kilobases (kb). The deduced amino acid sequences of the corresponding cDNAs indicate that three of these genes are new members of the MHC class II gene family. These genes are transcribed in a tissue-specific pattern similar to that of the traditional class II genes. Two of the remaining four genes, HAM1 and HAM2, are homologous to one another and to a family of eukaryotic and prokaryotic transport proteins and may be involved in antigen processing. The tissue distribution of HAM1 transcripts is consistent with its proposed role in class I restricted antigen processing, whereas HAM2 transcription appears more restricted and may be involved in antigen processing for class II-restricted T cells. The HAM2 gene may produce two differentially spliced transcripts. The identity of the remaining two genes is not known. Analyses of transcript sizes, tissue distribution, sequence, and genetic mapping data suggest that none of these genes code for LMP antigens. PMID- 1711221 TI - Developmental expression of the 25-kDa synaptosomal-associated protein (SNAP-25) in rat brain. AB - The developmental expression and subcellular distribution of the neuron-specific 25-kDa synaptosomal protein (SNAP-25) were investigated by using Northern (RNA) blots, immunoblots, and immunocytochemistry. Both SNAP-25 protein and mRNA were present at low levels in embryonic day 15 rat brain, and levels of both increased during early postnatal maturation. Developmental immunoblots with antipeptide antisera demonstrated that a 25-kDa peptide was the major isoform in brain, and this form increased steadily from embryonic day 15 through adulthood. A second 27 kDa immunoreactive isoform was present in brain only during early development. Immunoblots of two-dimensional SDS/polyacrylamide gels revealed the presence of a predominant 25-kDa isoform of SNAP-25 in adult brain. Immunocytochemical studies indicated that as immunoreactivity for SNAP-25 increased during development, the cellular localization of SNAP-25 immunoreactivity concomitantly shifted from axons and cell bodies to presynaptic terminals. These data suggest that the SNAP 25 protein shifts in subcellular localization during development and may play a role in the establishment and stabilization of specific presynaptic terminals in brain. PMID- 1711222 TI - The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity. AB - The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication. PMID- 1711223 TI - A single base insertion in the putative transmembrane domain of the tyrosinase gene as a cause for tyrosinase-negative oculocutaneous albinism. AB - We have determined a molecular defect to be the likely basis for inactivity of the tyrosinase (EC 1.14.18.1) from a patient with tyrosinase-negative oculocutaneous albinism. A single base (thymine) was inserted in exon 5 of the tyrosinase gene following codon 471 in the putative transmembrane coding region. This insertion caused a shift in the reading frame of 19 amino acids at the 3' end and introduced a premature termination signal that would be expected to truncate the protein by 21 amino acids at the carboxyl terminus. The albino tyrosinase was not recognized by antibodies directed to the carboxyl terminus of tyrosinase. Furthermore, as shown by gel electrophoresis of the immunoprecipitated protein, the tyrosinase was approximately 3 kDa smaller than normal. Similar immunoprecipitation data were obtained when cloned normal and mutant tyrosinases were expressed in COS-1 cells. PMID- 1711224 TI - Cystic fibrosis gene expression is not correlated with rectifying Cl- channels. AB - Cystic fibrosis (CF) involves a profound reduction of Cl- permeability in several exocrine tissues. A distinctive, outwardly rectifying, depolarization-induced Cl- channel (ORDIC channel) has been proposed to account for the Cl- conductance that is defective in CF. The recently identified CF gene is predicted to code for a 1480-amino acid integral membrane protein termed the CF transmembrane conductance regulator (CFTR). The CFTR shares sequence similarity with a superfamily of ATP binding membrane transport proteins such as P-glycoprotein and STE6, but it also has features consistent with an ion channel function. It has been proposed that the CFTR might be an ORDIC channel. To determine if CFTR and ORDIC channel expression are correlated, we surveyed various cell lines for natural variation in CFTR and ORDIC channel expression. In four human epithelial cell lines (T84, CaCo2, PANC-1, and 9HTEo-/S) that encompass the full observed range of CFTR mRNA levels and ORDIC channel density we found no correlation. PMID- 1711225 TI - Polarized intestinal hybrid cell lines derived from primary culture: establishment and characterization. AB - A cell culture system has been developed that produces stable gastrointestinal (GI) polarized cell lines capable of maintaining hormone secretion. A spontaneously transformed rat mucosal epithelial cell was selected for hypoxanthine/guanine phosphoribosyltransferase deficiency and transfected with a plasmid conferring hygromycin resistance (BRIE 291 cells). Fusion of these cells with dispersed small intestinal epithelia cells resulted in hybrid cell lines that retained characteristic properties of the native GI cell more effectively than the transformed tumorigenic parental cell line. Hybrid hBRIE 380 cells are uniformly cuboidal with microvilli, contain villin, are contact inhibited, are anchorage dependent, require serum supplementation for growth, and are more sensitive to virus infection than the parental BRIE 291 cells. Fusion of BRIE 291 with dispersed pancreatic islet cells has resulted in a variety of pancreatic hormone-producing cell lines. One of these, hybrid hBRIE 291-i2, forms confluent monolayers capable of synthesizing insulin-like immunoreactivity. These studies demonstrate that functionally polarized GI cells can be generated from primary cultures of nondividing committed epithelial cells by somatic cell hybridization and make feasible the selection and maintenance of specific GI epithelial cell types in confluent monolayer cultures. PMID- 1711226 TI - Newly synthesized RNA: simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes. AB - The levels of several RNA transcripts in cultured hepatocytes are regulated by transcriptional and post-transcriptional mechanisms and are affected by growth hormone and insulin. We assessed the effects of these hormones on transcription rates and the stability of insulin-like growth factor I, actin, and albumin transcripts in intact cells of primary cultures of rat hepatocytes by analyzing thiol-labeled, newly synthesized RNA isolated by mercurated agarose affinity chromatography. The application of this concept to the measurement of transcript stability is presented in detail. The data indicate that growth hormone stimulates the transcription rates of insulin-like growth factor I, actin, and albumin genes. The stability of all three transcripts, particularly albumin, appears to be lower in growth hormone-containing medium than it is in insulin containing medium. The experiments indicate that the rates of transcription and/or degradation of albumin mRNA are influenced by hormonal treatment. However, the cells maintain roughly constant albumin transcript levels independent of hormone treatment by compensatory changes in the rates of transcription and degradation. PMID- 1711227 TI - Crystal structure of [Leu1]zervamicin, a membrane ion-channel peptide: implications for gating mechanisms. AB - Structures in four different crystal forms of [Leu1]zervamicin (zervamicin Z-L, Ac-Leu-Ile-Gln-Iva-Ile5-Thr-Aib-Leu-Aib-Hyp10-Gln-Aib-Hyp-Aib-P ro15-Phol, where Iva is isovaline, Aib is alpha-amino isobutyric acid, Hyp is 4-hydroxyproline, and Phol is phenylalaninol), a membrane channel-forming polypeptide from Emericellopsis salmosynnemata, have been determined by x-ray diffraction. The helical structure is amphiphilic with all the polar moieties on the convex side of the bent helix. Helices are bent at Hyp10 from approximately 30 degrees to approximately 45 degrees in the different crystal forms. In all crystal forms, the peptide helices aggregate in a similar fashion to form water channels that are interrupted by hydrogen bonds between N epsilon H(Gln11) and O delta (Hyp10) of adjacent helices. The Gln11 side chain is folded in an unusual fashion in order to close the channel. Space is available for an extended conformation for Gln11, in which case the channel would be open, suggesting a gating mechanism for cation transport. Structural details are presented for one crystal form derived from methanol/water solution: C85H140N18O22. 10H2O, space group P21, a = 23.068(6) A, b = 9.162(3) A, c = 26.727(9) A, beta = 108.69(2) degrees (standard deviation of last digit is given in parentheses); overall agreement factor R = 10.1% for 5322 observed relfections [magnitude of Fo greater than 3 sigma (F)]; resolution, 0.93 A. PMID- 1711228 TI - The secretin gene: evolutionary history, alternative splicing, and developmental regulation. AB - The gene encoding the hormone secretin has been isolated and structurally characterized. The transcriptional unit is divided into four exons spanning 813 nucleotides. Comparison of the rat secretin gene to the other members of the glucagon-secretin gene family reveals that similarities are restricted to the exons encoding the biologically active peptides. Analysis of RNA from porcine intestine indicates that at least two transcripts are generated from the porcine secretin gene as a result of differential splicing. The longer and more abundant transcript appears to be identical to a previously isolated cDNA, which encodes a precursor that includes a 72-amino acid C-terminal extension peptide. The shorter transcript does not contain the third exon and, as a result, encodes only 44 residues beyond the C terminus of secretin. The amino acid sequence deduced from the shorter transcript is identical to a precursor form of secretin recently isolated from porcine duodenum [Gafvelin, G., Jornvall, H. & Mutt, V. (1990) Proc. Natl. Acad. Sci. USA 87, 6781-6785]. Developmental studies reveal that both secretin mRNA and peptide levels in the intestine are highest just before birth, prior to the onset of gastric acid secretion and feeding. This observation implies that secretin biosynthesis in developing animals is controlled independently of the principal factors known to regulate secretin release in adult animals. PMID- 1711229 TI - Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits. AB - 1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. & Van Montagu, M. Proc. Natl. Acad. Sci. USA (1990) 87, 4859-4863]. We report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5' and 3' ends. Gene specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes. PMID- 1711230 TI - Monoclinic uncomplexed double-stranded, antiparallel, left-handed beta 5.6-helix (increases decreases beta 5.6) structure of gramicidin A: alternate patterns of helical association and deformation. AB - A comparison of the monoclinic and orthorhombic crystal structures of the uncomplexed double-stranded, antiparallel, left-handed beta-helix (5.6 amino acid residues per turn) (increases decreases beta 5.6) conformers of gramicidin A reveals marked differences in the tryptophan side-chain orientations and the degree of helical uniformity of the dimer and in the manner in which these helical dimers associate with one another in the crystal. The helix of the orthorhombic dimer exhibits a regular pattern of bulges and constrictions that appears to be induced by crystal packing forces affecting tryptophan side chains that are aligned parallel to the helix axis. The monoclinic dimer is more uniform than the orthorhombic dimer as a consequence of pi stacking interactions between dimers in which orientation of tryptophan side chains is normal to the helix axis to relieve the lateral crystal packing forces that may locally twist and deform the helix. It may be inferred from these observations that lipid interactions may be expected to destabilize the increases decreases beta 5.6 helix when it is inserted into a membrane bilayer. PMID- 1711231 TI - Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation. AB - Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA from primary cultures of unstimulated human fetal adrenal cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1 24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process. PMID- 1711233 TI - Reverse-transcriptase-associated RNaseH activity mediates template switching during reverse transcription in vitro. AB - During the first steps of reverse transcription of the retroviral genome, sequences present at the extremities of the RNA are used to reconstitute a host cell PolII promoter. The assembly of the promoter occurs by template switching, which takes advantage of a direct repeat at the ends of the RNA molecule. These steps are catalysed by the viral reverse transcriptase, which carries an intrinsic RNaseH activity that is probably also involved therein. To study the role of the RNaseH activity in this first template-switching event, an in vitro system has been developed based on primer extensions of synthetic RNAs. When an RNA was reverse transcribed with wild-type reverse transcriptase in the presence of a second RNA the 3' part of which was repeated at the 5' end of the first one, extension products could be observed corresponding to a chimeric cDNA comprising both RNA species. This template switching could not be detected when a mutant reverse transcriptase lacking the RNaseH activity was used. The results show that the RNaseH activity is needed to remove the 5' RNA sequences from the cDNA:RNA hybrid thereby enabling its translocation to another RNA containing an appropriate complementary target sequence. PMID- 1711232 TI - Identification of an immunodominant epitope within the capsid protein of hepatitis C virus. AB - We have isolated cDNA clones from the 5' end of the Hutchinson strain of hepatitis C virus. Sequences encoding various segments of the HCV structural region were fused to the gene for glutathione S-transferase and analyzed for the expression of hepatitis C virus-capsid fusion proteins. With a set of these fusion proteins, both human and chimpanzee immune responses to capsid were studied. An immunodominant epitope was located within the amino-terminal portion of capsid that is preferentially recognized by antibodies in both human and chimpanzee hepatitis C virus-positive sera. In addition, analyses of sequential serum samples taken from humans and chimpanzees with either chronic or apparently self-limited infections revealed that a strong anti-capsid response develops rapidly after onset of infection. PMID- 1711234 TI - Pain relief for the Hispanic burn patient using cultural metaphors. PMID- 1711235 TI - Effect of azelastine on activation and release stages of allergic histamine secretion in rabbit basophils. AB - In this study the effect of azelastine on the activation (antigen-dependent, Ca(2+)-dependent, Stage I) and release (antigen-independent, Ca(2+)-dependent, Stage II) phases of allergen-induced histamine secretion in rabbit mixed leukocytes (basophils) was investigated. Azelastine (5 microM, 10-min) and diltiazem (5 microM: a Ca2+ antagonist, 10 min) inhibited ragweed extract-induced histamine secretion during the Stage II (release) phase. Theophylline (100 microM), a phosphodiesterase inhibitor, added immediately before antigen challenge, inhibited allergic histamine secretion during the Stage I (activation) phase. The data obtained in this study suggest that diltiazem and azelastine act on the Ca(2+)-dependent and antigen-independent phase (Stage II) of allergic histamine secretion in rabbit basophils. PMID- 1711236 TI - Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus. AB - Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28 degrees C. When the infected cells were shifted from 28 to 37 degrees C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28 degrees C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells. PMID- 1711237 TI - Increased susceptibility to mouse hepatitis virus type 3 (MHV3) infection induced by a hypercholesterolaemic diet with increased adsorption of MHV3 to primary hepatocyte cultures. AB - The administration of a hypercholesterolaemic (HC) diet rendered genetically resistant A/J mice susceptible to mouse hepatitis virus 3 (MHV3) infection. The animals died of acute hepatitis with high viral titres in the liver accompanied by many necrotic foci and high serum transaminase levels. Resistance to virus was re-established by refeeding HC mice with a normal diet for 2 weeks. This modification of pathogenesis was accompanied by an increase in the susceptibility of hepatocyte cultures from HC mice to MHV3 and could be explained by an enhancement in virus adsorption. We hypothesize that the incorporation of cholesterol into the plasma membranes of hepatocytes of HC mice, thereby decreasing the membrane fluidity, may lead to an increase in the availability of virus receptors. PMID- 1711238 TI - Rene Jules Dubos. PMID- 1711239 TI - [Post-traumatic alveolar changes in lung contusion]. AB - Bronchoalveolar lavage (BAL) specimens taken from nine patients with lung contusion following multiple trauma were compared with specimens from different control groups. Early interstitial and intra-alveolar reactions are PMN degranulation, mediator release and high protein leakage. The alveolar reactions are similar in extent to the reaction found in post-traumatic ARDS. PMID- 1711240 TI - Extubation after transsternal thymectomy for myasthenia gravis: a prospective analysis. AB - Recommendations concerning postoperative extubation after thymectomy for myasthenia gravis are presently based upon retrospective chart reviews. We present the results of a prospective investigation of time to extubation after thymectomy for 14 patients over a 12-month period based upon a protocol that included preoperative immunologic therapy, combined epidural and general anesthesia, postoperative epidural narcotic analgesia, and a standardized approach to discontinuation of ventilatory support. After a neurologist took measures to optimize preoperative neuromuscular function, all 14 patients received agents to produce lumbar epidural anesthesia and light general anesthesia. Muscle relaxants were avoided in all but one patient. Postoperative analgesia was initially maintained with epidural hydromorphone, then therapy was switched to patient-controlled intravenous morphine sulfate. Criteria for weaning from mechanical ventilation, first measured at the end of anesthesia, were partial pressure of oxygen (arterial) greater than or equal to 90 mm Hg (fraction of inspired oxygen = 0.40), partial pressure of carbon dioxide (arterial) less than or equal to 50 mm Hg, pH greater than or equal to 7.30, and respiratory rate less than or equal to 30 breaths/min. If these criteria were not met, ventilatory support was continued postoperatively with intermittent mandatory ventilation, and the patient was weaned gradually from this support. Criteria for extubation included meeting the criteria for weaning, vital capacity greater than or equal to 10 mL/kg, and inspiratory pressure better than -30 cm H2O. Criteria for reintubation included tachypnea (respiratory rate greater than 40 breaths/min), respiratory acidosis not due to narcotics, or vital capacity less than or equal to 8 mL/kg. The mean time to extubation was 9 hours (range, 0.75 to 25 hours). Mean preoperative vital capacity was 2.59 +/- 0.64 L (range, 1.90 to 4.20), which decreased approximately 50% to 1.19 +/- 0.39 L (range, 0.70 to 2.0) at the time of extubation. No patient required reintubation. Half of the patients required postoperative anticholinesterase therapy based upon serial neurologic examinations; there were no instances of cholinergic crisis. Thirteen patients returned to the ward on the first postoperative day, and one on the second day. Thirteen patients preferred epidural analgesia to patient-controlled analgesia. The time to extubation and average length of stay in an intensive care setting were markedly reduced compared to those reported in previous retrospective studies. We conclude that a multidisciplinary approach that optimizes neuromuscular function and decreases poststernotomy pulmonary insult will shorten the time to extubation and decrease the length of stay in the intensive care or recovery room after thymectomy. PMID- 1711241 TI - Maternal serum placental alkaline phosphatase as a marker for low birth weight: results of a pilot study. AB - In a pilot study, maternal serum was analyzed for placental alkaline phosphatase (PLAP) to evaluate the possibility of using PLAP values as a prenatal marker to predict low birth weight in neonates. Study subjects were selected from among women whose newborns were of low birth weight. These women were screened for maternal serum alpha-fetoprotein (MSAFP) between 15 and 20 weeks of gestation. Of the mothers of low-birth-weight neonates, 43% had PLAP values of 2.0 multiples of the median (MoM) or higher; only 22% of the mothers in the control group had such high values. The adjusted odds ratio was 2.6 (95% confidence interval, 1.1 to 6.5) and was not confounded by race, age, or weight. Odds ratios were improved by selecting more extreme cutoff values, with fewer cases identified as positive. In 14% of the cases of low birth weight, both MSAFP and PLAP values were elevated, compared with 5.6% of those in the control group. These data suggest that PLAP elevations, with or without measurements for MSAFP, may be a useful indicator during the second trimester of pregnancy for the risk of low birth weight. PMID- 1711242 TI - Age changes to the anulus fibrosus in human intervertebral discs. AB - This study deals with age changes in the anulus fibrosus of the lumbar intervertebral discs of human individuals 21-83 years of age. The anular laminas from individuals less than 40 years of age consisted of obliquely orientated collagen fibers exhibiting a pennate arrangement. These fibers were intensely argyrophilic after silver nitrate impregnation. The fibers and surrounding substance appeared light pink after exposure to the periodic-acid-Schiff (PAS) reaction and blue with alcian blue complex. Beginning during middle age and continuing into the eighth decade, there was a progressive degeneration of the laminas. The breakdown of the intact laminas was characterized by the fraying, splitting, and loss of collagen fibers. The newly formed spaces became filled with intense PAS-positive material. In addition, there was a continual deposition of chondroid substance in the anuluses of the aging discs. This phenomenon was not seen in the young disc. These age related changes lead to a loss of integrity to the disc, which may be a factor in disc pathology. PMID- 1711243 TI - The effect of sensory deprivation in the reduction of pain in patients with chronic low-back pain. AB - Patients who suffer from persistent pain for prolonged periods of time (6 months or more) are often influenced to an increasing extent by psychological factors. Patients begin to focus on their pain as the problem rather than its physical origin. This study evaluated the effectiveness of sensory deprivation in reducing pain in patients with chronic low-back pain. Sixty patients were divided into two groups of 30 patients each: One group underwent 1 hour of sensory deprivation; the other received a lecture on relaxation skills. In the group receiving sensory deprivation, statistically significant decreases in pain and stiffness were noted. Sensory deprivation is an effective treatment to reduce pain and thus interrupt the pain cycle in patients with chronic low-back pain. PMID- 1711244 TI - [Comparative study of treatment of keratocysts by enucleation, enucleation combined with cryotherapy or fixation of the cyst membrane with Carnoy's solution followed by enucleation: a preliminary report]. AB - Comparative study of treatment of keratocysts by enucleation, enucleation combined with cryotherapy or treatment with Carnoy's solution followed by enucleation: a preliminary report. The aim of this preliminary report was to compare the effect on the recurrence rate of keratocysts treated by enucleation (group E, 14 cysts), enucleation combined with cryotherapy (group EK, 14 cysts) or a fixing agent (Carnoy's solution) followed by enucleation (group CE, 10 cysts). A total of 32 patients with 38 keratocysts are included in the study. In group E 5 recurrences (36%) was developed after 17, 34, 36, 37 and 39 months postoperatively, whereas 5 recurrences (36%) after 22, 24, 24, 25 and 59 months was registered in group EK. Due to a rather high incidence of side effects to cryotherapy this treatment technique can not be recommended. No recurrences has been found in group CE, and usually complete bone healing has occurred after 6 to 12 months postoperatively. Due to the use of the strong fixing agent we recommend treatment by this method performed under general anaesthesia to secure an immobile surgical field. In accordance with recent studies, the results indicate that fixation before enucleation has revolutionized the art of treating keratocysts with respect to recurrence rate. PMID- 1711245 TI - PAI-1 synthesis in the human hepatoma cell line HepG2 is increased by cytokines- evidence that the liver contributes to acute phase behaviour of PAI-1. AB - The acute phase behaviour of the fast inhibitor of tissue-type plasminogen activator (PAI-1) in vivo has been attributed to increased synthesis by endothelial cells. However, most other acute phase proteins in vivo are synthesized in the liver, which process is regulated by cytokines and can be studied in the hepatoma derived cell line HepG2. In this study, we investigated whether the synthesis of PAI-1 by HepG2 cells is regulated by the cytokines recombinant IL-1, rIL-6 and rTNF. Recombinant IL-1 and rTNF each increased PAI-1 synthesis by HepG2 cells two to three fold, whereas rIL-6 hardly had an effect. Mixtures of rIL-1, rIL-6 and rTNF increased PAI-1 synthesis up to eleven fold. The effects observed were not due to non-specific effects on HepG2 cell metabolism, since synthesis of alpha-2-antiplasmin was not effected by any of those cytokines, whereas fibrinogen synthesis was increased three to four fold by rIL-6, but was unaffected by rIL-1. Thus, our results demonstrate that synthesis of PAI-1 by HepG2 cells is regulated by cytokines and implicate that the acute phase behaviour of PAI-1 in vivo at least in part may be due to an increased synthesis by the liver. PMID- 1711246 TI - Blood group A and B determinants are expressed on platelet glycoproteins IIa, IIIa, and Ib. AB - We report the localization of A, B blood group determinants on intrinsic glycoproteins using anti-A, B IgG antibodies. By radioimmunoprecipitation, anti-A and anti-B precipitated three bands with apparent molecular masses in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of 159, approximately 120 and 85 kDa under non-reduced conditions and 145, approximately 126 and 97 kDa under reduced conditions. In two-dimensional gel electrophoresis (isoelectric focussing/SDS-PAGE) these three bands could be resolved into six spots and fulfilled previously defined criteria for platelet membrane glycoprotein complexes I a/IIa, Ic/IIa, I b/IX and II b/IIIa. The results were supported by data obtained by an assay employing antibody-specific immobilization of platelet antigens (MAIPA). By this technique, blood group A, B determinants were shown to be immobilized by monoclonal antibodies specific for GPIa, Ic', IX, IIb/IIIa and strongly by mab specific GPIIa, but not by mab specific for HLA class I molecules. The more precise localization on platelet glycoproteins was achieved by immunoblotting technique by which blood group A determinants could be assigned to GPs IIa, IIIa and Ib. PMID- 1711247 TI - Using appropriate nomenclature. PMID- 1711248 TI - Retroelements in bacteria. AB - A peculiar type of satellite DNA, called msDNA, has been discovered in myxobacteria and some natural isolates of E. coli. These molecules are characterized by the presence of single-stranded DNA branching out from an internal guanosine residue of an RNA molecule by a unique 2',5'-phosphodiester linkage. Reverse transcriptase is required for the synthesis of msDNA. The discovery of retroelements in bacterial populations raises many intriguing questions concerning the evolutionary origin of reverse transcriptase, the function and the biosynthesis of msDNA, and the nature of the mechanisms generating the extensive diversity found in msDNA and reverse transcriptase genes among different bacterial strains. PMID- 1711249 TI - [Prostatic hypertrophy: natural history. Subjective and objective changes during a 6-month period]. AB - Out of 30 patients, referred consecutively, with symptomatic and urodynamic signs of benign hypertrophy of the prostate, 22 were observed for six months as regards symptom scoring, urine flow measurements, serum creatinine and culture from the urine. After the period of observation, 1/3 of the patients no longer wanted operation on account of subjective improvement. The symptom scoring in this group was significantly lower (p less than 0.01) than in the group which was subsequently submitted to operation, on the other hand, no difference in urine flow was demonstrated. No statistically significant alteration in symptom scoring or urine flow was observed during the period of observation but both parameters showed fluctuations. The variations in flow were no greater than in men with normal micturition. None of the patients developed acute retention or involvement of upper urinary tracts during the period of observation. This investigation speaks for a more observing attitude towards treatment of benign hypertrophy of the prostate. In cases where no absolute indications for operative treatment are present (episodes of retention of urine or involvement of the upper urinary tracts), the patient's subjective symptoms constitute an important factor in the indications for operation. PMID- 1711250 TI - Treatment of symptomatic metastatic prostatic cancer with cyproterone acetate versus orchiectomy: a prospective randomized trial. AB - During a 2-year period, 37 patients with symptomatic metastatic prostatic cancer were included in a prospective randomized phase-III trial. Nineteen patients were randomized to subcapsular orchiectomy, and 18 to cyproterone acetate (CPA) treatment with a dose of 50 mg b.i.d. The median age of the patients was 74 years (range 48-88 years), with no differences between the treatment groups. At 3, 6, and 12 months after initiation of the therapy and then every 6 months, patients were clinically and biochemically examined, and isotope scans and X-rays were performed. All patients were followed until death. Relief from symptoms was found following 3 months of treatment in 70.6% (95% confidence limits = 44.0-89.7%) of the patients treated with CPA, and in 83.3% (95% confidence limits = 58.6-96.4%) of the orchiectomized patients. The median time to relapse was 9 months in the CPA group, and 11 months in the orchiectomy group (p greater than 0.05). The median survival time was 13 months, with no differences between the groups. The treatment of advanced prostatic cancer with CPA is found to be a valuable alternative to orchiectomy. PMID- 1711251 TI - Intraprostatic spiral during waiting time for transurethral prostatectomy. AB - When benign prostatic hyperplasia results in voiding problems such as acute or chronic urinary retention, transurethral prostatectomy is often necessary. We have evaluated 24 patients, treated with an intraprostatic spiral, during waiting time for transurethral prostatectomy. The spiral treatment was a success in 21 patients. Three patients had a transurethral prostatectomy before scheduled time due to spiral failure, 2 with urinary retention, 1 with severe local discomfort. We conclude that spiral treatment of symptomatic benign prostatic hyperplasia while waiting for transurethral prostatectomy has a high success rate and is recommended as a favorable alternative to an indwelling urethral catheter. PMID- 1711252 TI - Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period. AB - To determine if periparturient immunosuppression in dairy cattle might be due to an alteration in total numbers of percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks preparatum through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCD8, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4+ cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period. PMID- 1711253 TI - Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. AB - Considerable progress has been made in the understanding of the molecular biology of the human interferon system. The genes encoding the interferons, their receptors, and the proteins that mediate many of their biological effects have been molecularly cloned and characterized. The availability of complete cDNA clones of components of the interferon systems has contributed significantly to our understanding of both the biology and the biochemistry of the antiviral actions of interferons. At the biological level, the antiviral effects of interferon may be viewed to be virus-type nonspecific. That is, treatment of cells with one type or even subspecies of interferon often leads to the generation of an antiviral state effective against a wide array of different RNA and DNA animal viruses. However, at the biochemical level, the antiviral action of interferon is often virus-type selective. That is, the apparent molecular mechanism which is primarily responsible for the inhibition of virus replication may differ considerably between virus types, and even host cells. For example, the IFN-regulated Mx protein selectively inhibits influenza virus but not other viruses when constitutively expressed in mouse cells. The IFN-regulated 2',5' oligoadenylate synthetase selectively inhibits EMC and mengo viruses, two picornaviruses, but not viruses of other families when constitutively expressed in transfected cells. Some viruses are typically insensitive to the antiviral effects of interferon, both in cell culture and in intact animals. This lack of sensitivity to IFN may result from a virus-mediated direct antagonism of the interferon system. For example, in the case of adenovirus, the activation of the IFN-regulated RNA-dependent P1/elF-2 protein kinase is blocked by the virus associated VA RNA. The relative sensitivity to interferon of different animal viruses varies appreciably. All three of the basic components required to measure an antiviral response may play a role in determining the relative effectiveness of the antiviral response: the species of interferon administered; the kind of cell treated; and, the type of virus used to challenge the interferon-treated host cell. Thus, the relative sensitivity to interferon observed for a particular interferon-cell-virus combination is likely the result of the equilibrium between the many agonists and antagonists which contribute to the overall response. That is, the relative sensitivity of a virus to the inhibitory action of IFN is governed by the qualitative nature and quantitative amount of the individual IFN regulated cell proteins that may collectively contribute to the inhibition of virus replication.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711254 TI - In vitro macrophage tropism of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac). AB - The significance of infection of mononuclear phagocytes by immunodeficiency lentiviruses of primates is not clearly established. To explore the relationship of macrophage tropism and pathogenesis, conditions to culture and infect monocyte derived macrophages from rhesus macaques were established and the growth properties of two molecular proviral clones of simian immunodeficiency virus (SIVmac) were studied. Rhesus macrophages supported productive infection of the nonpathogenic SIVmac-1A11 clone; extensive cytopathology characterized by formation of multinucleated giant cells and release of particle-associated reverse transcriptase activity in culture supernatant were observed. In contrast, the pathogenic SIVmac-239 did not establish a productive infection of macrophages and no cytopathology was observed. Both SIVmac-1A11 and SIVmac-239 replicated and induced cytopathic effects in cultures of rhesus peripheral blood lymphocytes and the Cemx174 lymphoid cell line. These results, together with the previously published reports on the pathogenic potential of these two clones of SIVmac, suggest that macrophage tropism measured in vitro does not correlate with in vivo virulence. PMID- 1711255 TI - Localization of common cytotoxic T lymphocyte recognition epitopes on simian papovavirus SV40 and human papovavirus JC virus T antigens. AB - Human papovavirus JC virus (JCV) and Simian virus 40 (SV40) tumor or T antigens demonstrate considerable sequence homology which is reflected by antibody cross reactivity. This similarity raised the possibility that JCV and SV40 T antigen also might contain common cytotoxic T lymphocyte (CTL) recognition epitopes. In this study we identified and mapped such sites on the JCV T antigen. C57Bl/6 cell lines transformed by JCV/SV40 T antigen chimeras were generated and tested for susceptibility to lysis by five H-2b restricted SV40-specific CTL clones: Y-1, Y 2, Y-3, Y-4, and Y-5. These CTL clones recognize specific epitopes within amino acids 205-219 (site I), 220-233 (sites II and III), 369-511 (site IV), and 489 503 (site V) on SV40 T Ag, respectively. The results show that sites I, II, III, and IV (recognized by CTL clones Y-1, Y-2, Y-3, and Y-4, respectively) represent common epitopes on SV40 and JCV T antigens. CTL clone Y-5 failed to recognize JCV T antigen indicating that CTL can discriminate between the two antigenically related T antigens. PMID- 1711256 TI - Characterization of a membrane-associated phosphoprotein of murine cytomegalovirus (pp50) and its immunological cross-reactivity with a human cytomegalovirus protein. AB - We have identified an abundant 50K phosphoprotein (pp50) in MCMV-infected 3T3-L1 cells and shown by immunofluorescence microscopy and surface-iodination experiments that pp50 is localized to the plasma membrane of the infected cell. Furthermore, the kinetics of its synthesis suggests that it belongs to the early late class of herpesvirus proteins. Using monoclonal antibodies specific for pp50 to screen a lambda ZAP II expression library constructed from poly(A)+ mRNA of MCMV-infected cells, we have isolated a cDNA clone that synthesizes a truncated form of pp50 as a beta-galactosidase fusion protein. This allowed us to localize the partial pp50 transcript to a region between map coordinates 0.228 and 0.260 of the MCMV genome (Smith strain, Vancouver). Finally, we demonstrated that the MAb 5H10.21A recognizes an antigenic determinant that is conserved between pp50 and a 50K human cytomegalovirus (HCMV) nonstructural protein. PMID- 1711257 TI - Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein. AB - The S glycoprotein of transmissible gastroenteritis virus (TGEV) has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. The residues that contribute to these sites were localized by sequence analysis of 21 mutants that escaped neutralization or binding by TGEV-specific monoclonal antibodies (MAbs), and by epitope scanning (PEPSCAN). Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. In addition, mar mutant 1B.H6 with residue 586 changed had partially altered both subsite Aa and Ab, indicating that these subsites overlap in residue 586; i.e. this residue also is part of site A. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). The location of site D is in agreement with PEPSCAN results. Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus. Its accessibility can be modulated by treatment at pH greater than 11 (at 4 degrees) and temperatures greater than 45 degrees. Sites A and B are fully dependent on glycosylation for proper folding, while sites C and D are fully or partially independent of glycosylation, respectively. Once the S glycoprotein has been assembled into the virion, the carbohydrate moiety is not essential for the antigenic sites. PMID- 1711258 TI - Nonmutant forms of the avirulent satellite D of turnip crinkle virus are produced following inoculation of plants with mutant forms synthesized in vitro. AB - The avirulent satellite RNA D (sat D) of turnip crinkle virus (TCV) has been cloned as a copy DNA. RNA transcripts synthesized in vitro from the cloned cDNA are infectious when coinoculated with RNA transcripts of the cloned TCV genome. Sat D is the smallest and most likely the progenitor of the other TCV satellites and, in contrast to the virulent satellite RNA C (sat C), does not intensify viral symptoms. Mutant forms of sat D including internal deletions up to 50 bases yielded sat D in infected plants. However, sat D mutants were not recovered in mutant form, but reverted to normal size and sequence in infected turnip plants. Mutations at a site with homology to the catalytic strand of self-cleaving sequences in certain viroids and satellites appeared to confer virulence on sat D in that test plants showed severe crinkling and stunting normally associated with sat C. However, sat C appeared along with a restored form of sat D in the progeny RNAs of these severely infected plants. Sat C was presumably generated by recombination between sat D and the TCV genome. In contrast, when plants were inoculated with transcripts containing the equivalent mutations in sat C, sat C was recovered from infected plants in mutant form. These findings demonstrate the tendency for mutant forms of the avirulent satellite, sat D, to revert, but raise questions about the source of information used in the reversion process. PMID- 1711259 TI - Vaccinia virus-encoded eIF-2 alpha homolog abrogates the antiviral effect of interferon. PMID- 1711260 TI - Non-A, non-B hepatitis and the anti-HCV assay. AB - The successful cloning of a non-structural antigen from the genome of what is now designated as the 'hepatitis C virus' (HCV) has transformed an erstwhile diagnosis of exclusion for non-A, non-B hepatitis (NANBH). The assay has been validated against panels of known infectivity for NANBH and sera from haemophiliac patients treated either with virally inactivated or uninactivated factor VIII. The predictive value of the assay is being assessed clinically in prospective studies of post-transfusion hepatitis and by using laboratory techniques such as polymerase chain reaction. While the assay shows good predictability in high-risk subjects, an appreciable number of false-positive results are likely in blood donor populations. Furthermore, the extent of infectivity of seropositive blood donors is still the subject of active research. The prevalence of anti-HCV in blood donors varies from approximately 0.2 to 1.5% around the world, based on repeat reactivity in the Ortho antiglobulin ELISA assay. These rates may be appreciably reduced following supplementary testing with recombinant immunoblot assay (RIBA). Prevalence data in African sera are as yet unreliable, pending assessment by RIBA, presumably because of high levels of IgG interfering with the assay. Presence of anti-HBc or elevated alanine aminotransferase associates to a greater or lesser extent with seropositivity, especially when both surrogate markers are present, but conversely many (unconfirmed) seropositive subjects lack these surrogate markers. An understanding of the modes of transmission of HVC is of obvious importance to transfusion practice. Intravenous drug use is a striking risk factor, but the contribution made by sexual transmission is not so clear.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711261 TI - [Perioperative prevention of thrombosis in cesarean section: results of a randomized prospective comparative study with 6% hydroxyethyl starch and 0.62 low dose heparin]. AB - In 207 consecutive randomized women undergoing cesarean section, of whom 104 received 3 X 5000 IE unfractionated heparin and 103 3 X 500 ml hydroxyethylstarch 6% 0.62, the frequency of deep vein thrombosis was evaluated with a non-invasive diagnostic technique-impedance plethysmography. 5.9% of the patients in the hydroxyethylstarch group and 7.8% in the heparin-group developed deep vein thrombosis. Activation of blood coagulation at cesarean section results in an increasing of factor VIIIR:Ag, Fibrinogen and Reptilase clotting time. Hydroxyethylstarch 6% 0.62 produces minor abnormalities of coagulation test results. After 1500 ml infusions HES the following effects were noted: 1. Factor VIIIR:Ag fell by about 20%, a greater decrease than could be explained simply by hemodilution. 2. Reptilase clotting times shortened without a different increase in fibrin activation products (resulting in an increased lysability of the thrombus). 3. Plasma fibrinogen decreased by approximately 7% due to hemodilution caused by plasma volume expansion. For patients with a low risk of deep vein thrombosis (Cesarean section) HES has an excellent safety record in doses not exceeding 1500 ml/24 h. PMID- 1711262 TI - [The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]. AB - The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization. PMID- 1711263 TI - [The specificity of Campylobacter jejuni antigens in immunoenzyme analysis]. AB - The surface antigen has been obtained from C. jejuni culture and its immunochemical analysis has been carried out by the methods of immunoelectrophoresis, double diffusion with homologous and heterologous rabbit sera. The protein profile of this antigen has been studied by means of vertical electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, and the molecular weight of the protein components of the antigen has been determined. The presence of anti-Campylobacter antibodies has been revealed in a number of rabbit sera by enzyme immunoassay techniques. The results thus obtained indicate the possibility of developing the enzyme immunoassay for the determination of antibodies in the sera of patients with Campylobacter infection. PMID- 1711264 TI - [Current methods for the immunological diagnosis of typhoid fever]. PMID- 1711265 TI - [Retinal projections into the diencephalon in the fowl (Gallus gallus domesticus)]. AB - The localization of the primary visual centers in the hen diencephalon was determined by anterograde transport horseradish peroxidase (HRP) techniques. Twelve fowls (Gallus gallus domesticus) were used for HRP study and four were used for cytoarchitectural study (Nissl and Kluver-Barrera stained preparation). One-hundred microliter of 30% HRP solution in physiological saline was injected into the vitreous body of one eye of each hen under anesthesia of sodium pentobarbital (30 mg/kg body wt). After a postoperative period of 48 hours, the animals were deeply anesthetized and perfused with an injection of 1,000 ml of Ringer solution which was followed by 2,000 ml of 1% paraformaldehyde and 1.25% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) which was then followed by 1,000 ml of 10% sucrose in the same buffer. The brain was cut into serial transverse sections of 60 microns on freezing microtome. Every section was treated with tetramethylbenzidine (TMB). Retinal projections were found in the hypothalamic area, lateral geniculate nucleus (GL), lateral part of dorsolateral thalamus (DLL), medial part of dorsolateral thalamus (DLM), ventrolateral thalamus (VLT), rostrolateral part of dorsolateral anterior thalamus (DLAlr), magnocellular part of dorsolateral anterior thalamus (DLAmc), lateral anterior thalamus (LA), ectomammillary nucleus (EM), external nucleus (NE), and nucleus superficial synencephalica (SS), contralaterally. No labeled terminals were found in the ipsilateral brain stem. In the hypothalamic region, terminals were found to be just lateral to the rostral part of the third ventriculus and the bottom of the lateral margins of the hypothalamus, which we termed medial (MRH) and lateral (LRH) retinorecipient hypothalamic nucleus. LRH had high density terminals compared with MRH and caudal MRH continued into rostral LRH so that there was no boundary between MRH and LRH in HRP preparation. MRH is contained large cells (25 35 microns in diameter) and occupied rostral 1/4 of retinorecipient of hypothalamus (RH), whereas LRH contained small type cells (about 15 microns in diameter) and occupied caudal 3/4 of RH. In the retinorecipient nuclei of the thalamus, high density terminals were found in GL, LA, DLAlr, NE, SS and EM. In DLAlr and EM, granulae of HRP product were bigger than in other terminal nuclei and also the density of those terminals was high. GL and LA have large nuclei which receive retinal afferents. Labeled terminals of those nuclei were distributed homogeneously throughout the whole nucleus except for the inner layer of GL. Cytoarchitectonically, GL was divided into two layers. PMID- 1711266 TI - Chronic imipramine treatment increases the affinity of [125I]galanin binding sites in the tel- and diencephalon of the rat and alters the 5-HT1A/galanin receptor interaction. PMID- 1711267 TI - Tachykinins mediate changes in spinal reflexes after activation of unmyelinated muscle afferents in the rat. AB - The effect of intrathecally applied tachykinin antagonist D-NicLys1, 3-Pal3, D Cl2Phe5, Asn6, D-Trp7.9, Nle11-substance P, spantide II, on the long-term increase of spinal cord excitability after activation of unmyelinated muscle afferents was studied in decerebrate, spinalized, unanaesthetized rats. A conditioning stimulus train (1 Hz, 20 s) that activated unmyelinated fibres in the gastrocnemius muscle nerve facilitated the flexor reflex for about 1 h, which was strongly blocked by pretreatment with spantide II (3 micrograms). The present results indicate that the facilitation of the flexor reflex by conditioning stimulation of a muscle nerve is mediated by tachykinins and possibly other neuropeptides which may be released from the central terminals of these unmyelinated afferents. PMID- 1711268 TI - Evidence for a link between mechanical and electrical alternans in acutely ischaemic myocardium of anaesthetized dogs. AB - In order to examine the relation between mechanical alternans and associated electrical alternans during acute myocardial ischaemia, we determined the effect of a ventricular premature beat and calcium antagonists on mechanical and electrical alternans during acute coronary occlusion in anaesthetized dogs. Isometric contractions and unipolar electrocardiograms were recorded from ischaemic myocardium. During coronary occlusion, mechanical alternans was accompanied by electrical alternans, which was an alternate change in the ST segment elevation, i.e. the higher ST and the lower ST. Electrical alternans was frequently discordant and in some cases accompanied by discordant mechanical alternans. Both discordant electrical and mechanical alternans became concordant and were potentiated after the ventricular premature beat. In all cases, concordant mechanical alternans was accompanied by concordant electrical alternans and vice versa. In this situation, the higher and the lower ST corresponded to the larger and the smaller contractions respectively. Thus, a fixed correspondence was observed between mechanical and electrical alternans. A fixed correspondence was also observed between mechanical alternans and the variation in the time taken for repolarization of the monophasic action potential. Verapamil and diltiazem inhibited both electrical and mechanical alternans. The present results support the idea that a common mechanism, such as a beat-to-beat cycle of the transmembrane and intracellular movement of calcium ions, may play a role in the mechanisms of electrical and mechanical alternans. PMID- 1711269 TI - [The anti-influenza effect of rimantadine and isoprinosine when used in combination in mice]. AB - The antiviral effect of rimantadine and isoprinosine applied in combination against fowl plague virus (FPV) in cell cultures of chick embryo fibroblasts and in experimental influenza infection in mice has been studied. Isoprinosine does not inhibit the reproduction of FPV when given alone and does not increase the antiviral activity of rimantadine. After oral application of both substances according to an appropriate scheme in mice infected with influenza virus A/Aichi (N3N2), an increase in the antiviral effect with an index of protection by 12% higher than the theoretically calculated one for additive effect was established. The results suggest that the combined effect is synergic. PMID- 1711270 TI - Membrane electrophysiology of epileptiform activity in the hippocampus. AB - Several different types of Na+ and Ca++ channels in the membrane of neurones provide the driving force for excitation. Whilst some of these may be activated by the transmembrane voltage or ionic concentrations, others are mediated by neurotransmitters and neuromodulators. The role of these ionic mechanisms in epileptiform activity are discussed, with particular reference to the involvement of the NMDA receptor mediated channel. Multiple K+ and Cl- mediated mechanisms provide the stabilizing influence on the electrophysiological behavior of the cells. Loss or reduction in activity of one or more of these conductances may lead to the expression of epileptiform activity. The role of the extracellular concentration of K+ in burst firing of populations of cells is discussed. The examples are primarily chosen from studies of the hippocampus in animal models of epilepsy, however wherever possible experiments on human tissue have been discussed. These studies on the membrane and synaptic mechanisms that contribute to epileptiform activity provide us with the necessary insights to allow the development of new methods for controlling such activity. PMID- 1711271 TI - Selection criteria for epilepsy surgery psychometric evaluation. PMID- 1711272 TI - Osmotic stress induces aldose reductase in glomerular endothelial cells. PMID- 1711273 TI - Endocrine regulation and methylation patterns of rat class I alcohol dehydrogenase in liver and kidney. PMID- 1711274 TI - Controlled clinical trials and cross-sectional studies with plasma histamine measurements and histamine receptor antagonists: solving the problem of preoperative H1- + H2-prophylaxis by asking new questions? AB - The problem of a preoperative histamine H1- + H2 - prophylaxis was tackled by a group of new studies including randomized controlled clinical trials and cross sectional studies with plasma histamine measurements and administration of H1- + H2 - antagonists to a control group. The first study demonstrated serial histamine release in the induction of anaesthesia up to 4 times in a single patient. Basal plasma histamine levels in resting subjects fell below 100 pg/ml during the time necessary for preparation of the surgical patient. Hence, spikes of elevated plasma histamine concentrations corresponded to histamine release. Although this histamine release very often was less than 1 ng/ml plasma histamine, it created systemic reactions after atracurium. The cut-off point of 1 ng/ml for such anaphylactoid reactions does no longer exist, also lower plasma levels are of patho-physiological significance. The clinical signs of histamine release in the induction of anaesthesia vary from drug to drug. Sometimes tachycardia and hypertension produce the highest likelihood ratio, sometimes tachy- and bradycardia, but no changes in blood pressure as in the case of atracurium. It is concluded that the reasons why histamine release in anaesthesia and surgery is so much underreported and under-estimated include the present paradigms about plasma histamine levels and the "classical picture" of histamine release. Both are no longer valid and need a re-assessment. PMID- 1711275 TI - Novel chiral H3-receptor agonists. AB - Several alkyl-substituted histamine derivatives have been synthesized and investigated for their agonistic potency on three classes of histamine receptors. While all investigated compounds are full agonists at H3-receptors their relative potency vs. histamine is strongly varying and culminates in alpha R, beta S dimethylhistamine which is a highly potent and selective H3-receptor agonist. PMID- 1711276 TI - Calcium signal and histamine secretion induced by aggregation of immunoglobulin E receptors in rat basophilic leukemia (RBL-2H3) cells. AB - The changes in cytosolic Ca concentration in rat basophilic leukemia cells (RBL 2H3) were determined using a fluorescent Ca probe, fura-2, after aggregation of IgE receptors. By means of a superfusion system, both histamine secretion and changes in cytosolic Ca concentration were determined simultaneously. In addition, the microscopic observation revealed the oscillatory Ca signal after stimulation with an antigen. PMID- 1711277 TI - Rat mast cells inhibit platelet aggregation by releasing a nitric oxide-like factor: influence of histamine release. AB - In th present paper we report the ability of rat serosal mast cells (MC) to release a nitric oxide (NO)-like factor by the inhibition of platelet aggregation and the effect of sodium nitroprusside (NaNP, a NO-generating drug) on MC histamine release by different stimuli. PMID- 1711278 TI - Pentagastrin-stimulated histamine release and acid secretion from the totally isolated vascularly perfused rat stomach. AB - This study was performed to examine the effect of cAMP levels on histamine release in the isolated rat stomach. Pentagastrin-induced histamine release was unaffected by phosphodiesterase inhibition, and pentagastrin itself had no phosphodiesterase-like effect. The results support previous observations showing that histamine is likely to be the mediator of the acid secretagogue effect of (penta) gastrin. PMID- 1711279 TI - [Detection of destruction of anionic sites in the outer blood-retinal barrier and damage caused by iron]. AB - We studied the alteration of the anionic charge in the outer blood-retinal barrier in ocular siderosis by electron microscopy using the cationic probe, polyethyleneimine (PEI). The eyes of four days after injection of iron powder and control rabbit eyes demonstrated numerous PEI-positive sites at the basement membrane of the retinal pigment epithelium, collagen fiber of Bruch's membrane, and the basement membrane of the choriocapillaris. Seven days after injection of iron powder, no change was observed in the retina by light microscopy, but PEI positive sites at the basement membrane of the retinal pigment epithelium were decreased. At 14 days, it was seen that the outer layer of the retina disappeared and was replaced by pigment containing macrophages and Muller cells. Berlin blue reaction became positive in some of those macrophages and retinal pigment epithelia. PEI positive sites decreased. At 28 days, the retina was completely disorganized, and became thin, while the retinal pigment epithelium proliferated and became double layered. Berlin blue reaction was strongly positive in pigment laden macrophages and retinal pigment epithelium. PEI-positive sites became scarce. As a result of these methods, anionic sites decreased, despite negative iron reaction of 7 days after injection of iron powder. It was shown that in ocular siderosis at an early stage, the barrier function of anionic sites between choroid and retina decreased before histological change. PMID- 1711280 TI - [A morphological study of experimental intravitreal proliferative tissues]. AB - Experimental tractional retinal detachments in rabbits' eyes was produced several weeks following aspiration of vitreous gels through a posterior small window of the eye wall. Histological examination showed that intravitreal proliferative tissues contained many fibroblasts, derived from the optic disc a small amount of migrated RPE, macrophages and other cells associated with proliferative tissues, Glial cells also grew especially at the epi-retina, causing shrinkage of the sensory retina. Vitreous neovascularization deriving from the retinal vessels was also found in these proliferative tissues. Our experimental data indicated that the extent of proliferative tissues mainly depend upon the amount of vitreous loss and vitreous hemorrhage, and the duration of retinal detachment. This experiment may be useful as an animal model of vitreous neovascularization. PMID- 1711281 TI - Does ketotifen act by directly stimulating beta-adrenergic system? AB - Through this study we proved that the inhibitory effect ketotifen has on antigen specific histamine release (ASHR) does not have any relation with beta-adrenergic system stimulation. However, it increases the inhibitory effect of histamine release caused by isoproterenol. Therefore, although ketotifen does not directly stimulate beta-adrenergic receptors to inhibit mediator secretion, its action correlates with the response produced through the stimulation of these receptors. PMID- 1711282 TI - Effect of ketotifen on the methyltransferase activity of asthmatic patients. AB - The results of this study have revealed that asthmatic individuals have less methyltransferase activity, compared to healthy individuals. This is reflected in a less fluidity of the plasmatic membrane and thus, presents with less beta receptors compared to healthy subjects. Ketotifen is capable of partially reversing this situation where it stimulates methyltransferase activity of asthmatic individuals approaching the enzyme activity to healthy individuals. PMID- 1711283 TI - Influence of iloprost and various prostaglandin derivatives on mouse skin allograft survival, HLA-DR antigen expression and eicosanoid metabolism by human leukocytes. AB - Treatment of mice bearing allogeneic tail skin grafts with iloprost, a stabilized prostacyclin derivative, as well as dexamethasone prolonged graft survival. Nalador and flunoprost, stabilized prostaglandin E analogues, had similar but weaker effects. The thromboxane agonist U 46619 had no effect on graft rejection. An incubation of human monocytes with iloprost or prostaglandin E2 led to a dose dependent reduction of HLA-DR antigen expression by these cells. Furthermore, a suppressive effect of these prostaglandin derivatives on the calcium ionophore stimulated release of arachidonic acid metabolites by human polymorphonuclear leukocytes has been shown, which demonstrates an antiinflammatory action of these drugs. Additionally, the eicosanoids were determined in the tail skin after complete allograft rejection. The importance of thromboxane for the rejection of skin grafts has not been confirmed. These data, along with the known antiaggregatory and antiischemic cytoprotective effects of iloprost, suggest that this newly developed drug may be all the more important in clinical organ transplantation. PMID- 1711284 TI - [Coupling of bovine gamma globulin to carbonochloridate-activated cellulose beads, CNBr-activated Sepharose CL-4B and NaIO4-oxidized Sepharose 6B and use of the coupled products for immunoaffinity chromatography]. AB - For coupling 25 mg of bovine IgG (BGG) were given to 5 ml volumes of packed bead cellulose activated by 5-norbornene-2.3-dicarboximido carbonochloridate and CNBr activated Sepharose CL-4B, respectively. Thus, BGG-immunosorbents were obtained with 4.6 to 4.9 mg BGG/ml matrix. 5 ml volumes of packed NaIO4-oxidized Sepharose 6B coupled 27% from 25 mg of BGG, only. In this case, immunosorbents with 1.35 mg BGG/ml matrix were produced. All BGG-immunosorbents were chemically relatively stable. The use of these immunosorbents for affinity chromatography results in the isolation of one milligram of pure rabbit anti-BGG antibodies by means of about 4.6 mg of BGG coupled to the cellulose or the Sepharose-CL-4B matrices. On the other side, only 3.4 mg of BGG coupled to the NaIO4-activated Sepharose 6B were necessary in order to isolate one milligram of antibodies in an immunoelectrophoretically pure state. PMID- 1711285 TI - [Monoclonal antibodies against determinants on human MHC class II antigens]. AB - Monoclonal antibodies against different non-polymorphic determinants of HLA class II antigens are described. The epitopes are present on HLA-DR and HLA-DQ gene products as detected by monoclonal antibody BL-Ia/l, and at HLA-DR and HLA-DP gene products which are recognized by monoclonal antibody BL-Ia/4. Another epitope recognized by the monoclonal antibody BL-Ia/DQ is only found on DQ molecules. The monoclonal antibodies BL-Ia/1, BL-Ia/4, and BL-Ia/5 are directed against the beta-subunit of 29 kDa/35 kDa class II heterodimer molecules. The recognized epitopes are shown to be not dependent on the association of the alpha and beta-chains. PMID- 1711286 TI - Mortality among sulfide ore miners. AB - Lung cancer mortality was studied during 1965-1985 in Outokumpu township in North Karelia, where an old copper mine was located. Age-specific lung cancer death rates (1968-1985) were higher among the male population of Outokumpu than among the North Karelian male population of the same age excluding the Outokumpu district (p less than .01). Of all 106 persons who died from lung cancer during 1965-1985 in Outokumpu township, 47 were miners of the old mine, 39 of whom had worked there for at least three years and been heavily exposed to radon daughters and silica dust. The study cohort consisted of 597 miners first employed between 1954 and 1973 by a new copper mine and a zinc mine, and employed there for at least 3 years. The period of follow-up was 1954-1986. The number of person-years was 14,782. The total number of deaths was 102; the expected number was 72.8 based on the general male population and 97.8 based on the mortality of the male population of North Karelia. The excess mortality among miners was due mainly to ischemic heart disease (IHD); 44 were observed, the expected number was 22.1, based on the general male population, and the North Karelian expected number was 31.2 (p less than .05). Of the 44 miners who died from IHD, 20 were drillers or chargers exposed to nitroglycerin in dynamite charges, but also to several simultaneous stress factors including PAHs, noise, vibration, heavy work, accident risk, and working alone. Altogether 16 tumors were observed in the cohort. Ten of these were lung cancers, the expected number being 4.3. Miners who had died from lung cancer were 35-64 years old, and had entered mining work between 1954 and 1960. Five of the ten lung cancer cases came from the zinc mine (1.7 expected). Three of them were conductors of diesel-powered ore trains. The slight excess mortality from lung cancer could be explained by exposure to radon daughters and by the combined effect of silica dust and diesel exhaust gases in the zinc mine. PMID- 1711287 TI - Localization of nuclear matrix proteins (NMPs) in multiple tissue types with NM 200.4 (an antibody strongly reactive with NMPs found in breast carcinoma). AB - Nuclear matrix proteins (NMP) are nonhistone proteins found in the nucleus of many eukaryotic cells. Furthermore certain NMPs are reported to be cell-type specific and expressed differentially by malignant cells. To study the specificity of NM-200.4 (an antibody reactive to NMPs extracted from cultured breast carcinoma cells of the T-47D line), cancers and benign tissues from multiple body sites were surveyed. All 17 breast carcinomas showed strong reactivity to tumor cell nuclei. Also nuclei from one of two lung carcinomas, a papillary thyroid carcinoma, an ovarian fibroma, and a lymphoma were strongly reactive. One leiomyosarcoma and a dermoid cyst were negative. Although 1 benign breast with duct hyperplasia showed moderate reactivity, only 1 of 10 benign breast biopsies without hyperplasia showed reactivity. Three of 4 skin biopsies, 2 liver biopsies, 6 of 9 kidney biopsies, and 5 of 10 gastrointestinal mucosal biopsies showed reactivity in benign nuclei. It is concluded that, although breast carcinoma nuclei showed the most consistent reactivity for NM-200.4, both benign and malignant nuclei from other body sites also show reactivity. PMID- 1711288 TI - Differential expression of markers for endothelial cells, pericytes, and basal lamina in the microvasculature of tumors and granulation tissue. AB - The structure and function of the tumor microvasculature is of great interest for cancer biology, diagnosis, and therapy. The distribution of endothelial cells, pericytes, and basal lamina in tumors is not well documented. In this study, the authors investigated the distribution of markers for these different components in a series of malignant human tumors and in human granulation tissue, both situations with extensive angiogenesis. Their results show a striking heterogeneity in the expression of markers for pericytes and endothelial cells between different tumors, but also within a single tumor lesion. To be able to distinguish between these two adjacent cell types decisively, all marker studies were carried out both on the light and the electron microscopical level and compared with staining results in granulation tissue of cutaneous wounds in healthy volunteers and of decubitus lesions. In granulation tissue of decubitus lesions, well-defined zones with increasing levels of maturation can be delineated. It was found that antibodies recognizing von Willebrand factor often failed to stain the tumor capillaries. Of the pericyte markers, alpha-smooth muscle actin was only locally expressed by pericytes in the tumor vasculature, whereas the high-molecular-weight melanoma-associated antigen, a chondroitin sulfate proteoglycan, stained the microvasculature broadly. Staining of the basal lamina components collagen type IV and laminin was, within the tumor, not restricted to the microvasculature. From their findings the authors conclude that 1) for the visualization of the tumor vasculature, antibodies recognizing endothelial markers, especially monoclonal antibodies PAL-E and BMA 120, are preferable to those recognizing pericytes or basal lamina; 2) within the microvasculature of tumors and granulation tissue, a heterogeneity of expression of endothelial and pericyte markers is observed; 3) during the formation of granulation tissue, all three microvascular components can be demonstrated already in the histologically earliest stage, suggesting not only an involvement of endothelial cells but also of pericytes and basal lamina in the initial steps of angiogenesis in wound healing. PMID- 1711289 TI - Cytokeratin expression and vimentin content in large cell anaplastic lymphomas and other non-Hodgkin's lymphomas. AB - The immunophenotypes of 74 malignant lymphomas (9 Hodgkin's disease, 19 low-grade B-cell, 20 high-grade B-cell, 8 T-cell, and 18 large cell anaplastic lymphomas [LCAL]) have been characterized with antibodies against leucocyte differentiation antigens, keratin, and vimentin. All the non-LCAL were CD45 positive and keratin negative. The LCALs had a more varied immunophenotype, with CD45 present only in 11 of 18 cases and keratin present in 5 of 18 of these rare lymphomas. The lymphoid origin of these latter cases was proven by gene rearrangement studies. All LCALs were CD30+, and, where tested, vimentin positive. Of four different vimentin monoclonal antibodies tested, V9 and MVI stained the highest number of lymphomas. Positive staining of tumor cells was seen in 61 of 71 cases. Vimentin negative cases included Burkitt's as well as some follicular lymphomas. PMID- 1711290 TI - Alterations in proteoglycan synthesis common to healing wounds and tumors. AB - Wound healing and tumor stroma generation share several important properties, including hyperpermeable blood vessels, extravasation of fibrinogen, and extravascular clotting. In both, the deposits of fibrin gel serve initially as provisional stroma and later are replaced by granulation tissue. Proteoglycans (PG) are also important constituents of the extracellular matrix, but their composition and role in healing wounds and tumor stroma generation are poorly understood. The authors used immunohistochemical and biochemical methods to investigate the dermatan sulfate proteoglycan (DSPG) and chondroitin sulfate proteoglycan (CSPG) composition of healing skin wounds and solid tumors. By immunohistochemistry, the great majority of normal guinea pig and human dermis stained weakly for CSPG and strongly for decorin. In contrast, the granulation tissue of healing skin wounds and scars stained intensely for CSPG and weakly or not at all for decorin; however decorin staining was restored to normal intensity after digestion with chondroitin ABC lyase, suggesting that decorin antigenic sites had been masked by glycosaminoglycan (GAG) chains. Like wounds, the stroma of several carcinomas (line 1 guinea pig, human breast, colon, basal cell, and squamous) stained strongly for CSPG and weakly or not at all for decorin, but decorin staining developed after chondroitin ABC lyase digestion. Thus healing wounds and tumor stroma express a common pattern of altered PG staining, adding another to the properties these pathologic entities share. Proteoglycans extracted from healing wounds after in situ labelling with [35S] Na sulfate contained more CSPG than normal dermis with significantly longer GAG chains. Granulation tissue also synthesized more DSPG than normal skin, with greater heterogeneity and longer GAG chains. These alterations in PG synthesis correlate with the cell proliferation, migration, and collagen synthesis that accompany wound healing and may provide clues to the mechanisms responsible for both wound healing and tumor stroma generation. PMID- 1711291 TI - CD45 epitope mapping of human CD1a+ dendritic cells and peripheral blood dendritic cells. AB - The authors studied the pattern of leukocyte common antigen (CD45) epitope expression on dendritic cells in sections of human epidermis, tonsillar epithelium, dermatopathic lymph nodes, and in isolates from blood. The monoclonal antibodies (MAb) used were specific for all known CD45 epitopes, including the seven different CD45 common epitopes as well as the four known CD45R epitopes (two CD45RA, one CD45RB, and one CD45RO). Dendritic cells in all sites were uniformly reactive for the CD45 common epitopes tested except 2B11, which may recognize a CD45R rather than CD45 epitope. By single-label immunoperoxidase and double-label immunofluorescence epitope mapping of CD1a+ dendritic cells in tissue sections, it was generally difficult or impossible to detect expression of CD45RA, CD45RB, CD45RO, or 2B11. In blood dendritic cells, however, low levels of these CD45R epitopes were detected consistently using single-label immunoperoxidase staining of cytocentrifuge preparations. Monocytes were similar to blood dendritic cells except that the staining with MAb to CD45RO and 2B11 was slightly stronger. The authors conclude that dendritic cells differ from most subpopulations of lymphocytes in that CD45 common epitopes are readily detectable but the existing RA, RB, and RO epitopes are either undetectable or expressed at relatively low levels. These studies raise the possibility that CD1a+ dendritic cells may express a novel dominant CD45 isoform. PMID- 1711292 TI - Restricted V gene usage in T-cell lymphomas as detected by anti-T-cell receptor variable region reagents. AB - Monoclonal antibodies reactive with variable regions of the human T-cell receptor (TCR) may be used to detect populations of T lymphocytes with restricted V gene usage. The authors have studied a series of 44 T-cell lymphomas with a panel of seven reagents reactive with four different TCR beta-chain variable region families. The authors found that, with this limited panel, restricted V gene usage of the neoplastic cells could be demonstrated in 29% of TCR-positive lymphomas. Whereas this is somewhat higher than expected, no preferential use of specific families could be demonstrated. An additional, unexpected finding was that most large cell T-cell lymphomas did not express TCR despite the presence of cytoplasmic CD3. The findings indicate that with a somewhat expanded panel it should be feasible to demonstrate restricted V gene usage as an indicator of clonality in a majority of T-cell lymphomas in a manner similar to the application of anti-immunoglobulin subclass antibodies in the diagnosis of clonal B cell proliferations. PMID- 1711293 TI - Inhibition of myosatellite cell proliferation by gamma irradiation does not prevent the age-related increase of the number of dystrophin-positive fibers in soleus muscles of mdx female heterozygote mice. AB - In skeletal muscles of young mdx female heterozygote mice, there is a mosaic of dystrophin-positive and dystrophin-negative fiber segments. In older animals, there is a marked decline in the number of dystrophin-negative fiber segments. This phenomenon might be due to a fusion of dystrophin-competent satellite cells into the originally dystrophin-negative fiber segments during growth. To study this possibility, soleus muscles of 10-day-old mdx female heterozygotes were gamma irradiated (2000 rads) to inhibit subsequent myosatellite cell proliferation and fusion. In the irradiated soleus muscles of animals at 60 days, the relative amount of dystrophin measured by quantitative immunoblots was not significantly different from that of the contralateral nonirradiated muscles. The prevalence of dystrophin-negative fibers in the 60-day-old irradiated solei was not higher than in the nonirradiated contralateral muscles, implying that dystrophin-competent satellite cell fusion was not a significant factor in the observed conversion. A longitudinal expansion of the cytoplasmic domain of the original dystrophin-competent myonuclei during growth could explain the observed conversion phenomenon. PMID- 1711294 TI - Expression of neu protein, epidermal growth factor receptor, and transforming growth factor alpha in breast cancer. Correlation with clinicopathologic parameters. AB - The major objectives of this study were twofold: to determine 1) if growth factors or growth factor receptors were expressed similarly or differently in a clinically well-characterized group of breast cancer patients and 2) if these phenotypic characteristics were associated with any of the commonly used prognostic parameters. Formalin-fixed paraffin-embedded tumor tissue from 51 node positive breast cancer patients were analyzed for the expression of neu, epidermal growth factor-receptor (EGF-R), and transforming growth factor alpha (TGF alpha) using immunoperoxidase staining. Positive membranous staining for neu was observed in 15 (29%) tumors. Over-expression of neu was observed in high grade, estrogen-receptor-negative tumors (P less than 0.05). Epidermal growth factor receptor was expressed in 22 (43%) of the tumors analyzed and found to a greater degree in estrogen-receptor-negative and high-grade tumors (P less than 0.025). A significant correlation between neu and EGF-R expression was also noted. Tumors expressing membranous staining of neu had a greater than 70% chance of expressing EGF-R (P less than 0.01). Expression of TGF alpha was found in 68% of tumors and TGF alpha was detected in grade 1 and 2 tumor to a greater degree than EGF-R. The authors conclude that assaying tumors for these antigens may give additional phenotypic characteristics that can give further insight into the biology of breast cancer. PMID- 1711295 TI - Endothelial cell damage by Walker carcinosarcoma cells is dependent on vitronectin receptor-mediated tumor cell adhesion. AB - The transport of cancer cells from blood vessels to extravascular tissue is a critical step in metastasis, where endothelial cells and the vascular basement membrane act as barriers to cell traffic. Because endothelial injury can facilitate the metastasis of intravascular cancer cells in vivo, the authors have studied in vitro the free-radical-mediated endothelial damage caused by the rat Walker 256 carcinosarcoma (W256) cell after stimulation with 10(-6) mol/l (molar) phorbol ester. Here the authors have examined the hypothesis that W256 cell mediated endothelial injury is dependent on adhesion between the effector and target cells. Attachment of phorbol 12-myristate, 13-acetate (PMA)-stimulated W256 cells to endothelial monolayers was increased 1.8 +/- 0.1-fold and damage (3H-2-deoxyglucose release from labeled endothelium) 1.4 +/- 0.1-fold after 4 hour pretreatment of the endothelium with 10 ng/ml recombinant human interleukin 1 alpha (rIL-1 alpha). Under various assay conditions, the release of 3H-2 deoxyglucose correlated directly with tumor cell adhesion (r = 0.98, P less than 0.005). In the presence of a polyclonal anti-vitronectin receptor antiserum, adhesion of stimulated W256 cells to rIL-1 alpha-treated monolayers was inhibited by 39% +/- 2%, and 3H-2-deoxyglucose release was inhibited by 53% +/- 13%. Immunoblot analysis and immunofluorescence flow cytometry demonstrated that the endothelial cells but not the W256 cells expressed vitronectin receptor (VnR) on their cell surface. The surface expression of VnR by endothelial cells was increased 1.9 +/- 0.1-fold after 4 hours' incubation with rIL-1 alpha. The authors conclude that W256 cell-mediated endothelial damage is dependent on cell adhesion, which, in turn, is partly regulated by the expression of VnR on the endothelial cell surface. PMID- 1711296 TI - Regional differences in the distribution of nerve fibers showing substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat larynx. AB - The distribution of substance P (SP) - and calcitonin gene-related peptide (CGRP) - containing nerve fibers in the rat larynx was studied using immunohistochemistry. Double-labeling studies revealed a high degree of co existence of SP- and CGRP-like immunoreactivity (LI) in the nerve fibers in the larynx. There was a considerable regional difference in the number of immunoreactive nerve fibers in the epithelium and lamina propria. Richly supplied sites were the laryngeal side of the epiglottis and the ventral recess, whilst there was no evidence of nerve fibers in the squamous epithelium of the vocal cords. However, where the squamous epithelium of the vocal cords changed into a cuboidal epithelium, a moderate number of nerve fibers was present, and a large number of fibers was seen where the squamous epithelium of the cords was in close contact with cartilage. Nerve fibers showing SP- and CGRP-LI were also observed close to the acini and ducts of the glands, in the blood vessel walls, close to the perichondrium of all the cartilages, and outside the cricothyroid and cricoarytenoid joints. CGRP-LI was detected in epithelial cells facing the lumen of the airway and in cells in the acini and ducts of glands in the subglottic region and trachea. Unilateral sympathectomy did not affect the pattern of SP- and CGRP-innervation in the larynx, whereas after vagotomy, the SP- and CGRP innervation almost disappeared ipsilaterally in the upper parts of the epiglottis and aryepiglottic folds. PMID- 1711298 TI - Immunocytochemical study of tissues from clinically normal dogs and of neoplasms, using keratin monoclonal antibodies. AB - Three commonly used keratin monoclonal antibodies (MAB)--AE1:AE3, CAM 5.2, and MAK-6--were compared with routinely used cytokeratin antibody. The expression of these antibodies was analyzed in several tissues obtained from clinically normal dogs and in a variety of neoplasms from dogs. Using appropriate enzymatic digestion, paraffin-embedded tissues processed in routine manner retained their typical keratin expression. Differentiated and poorly differentiated epithelial neoplasms, lymphomas, and melanomas were studied by use of the avidin-biotin peroxidase technique. All 4 of the aforementioned antibodies had similar staining profiles. Of 3 anaplastic carcinomas, 2 had positive reaction to all 4 antibodies. All lymphomas, plasma cell tumors, and amelanotic melanomas had negative reaction to MAK-6, CAM 5.2, AE1:AE3, and cytokeratin MAB. Three basal cell epitheliomas had positive reaction to all 4 antibodies, whereas 1 basal cell tumor with a solid pattern had negative staining reaction. Two carcinoids had negative reaction to all markers and 1 of 2 malignant chemodectomas and 1 transitional cell carcinoma had staining reaction to only AE1:AE3 MAB. Comparing the 4 antibodies, use of AE1:AE3 MAB produced the strongest staining intensity followed by cytokeratin, MAK-6, and CAM 5.2 MAB. All 4 antibodies had low background staining. In conclusion, AE1:AE3 and MAK-6 MAB are as useful as cytokeratin MAB for identification of poorly differentiated epithelial neoplasms in dogs and cats. PMID- 1711297 TI - Afferent nerve endings in the tracheal muscle of guinea-pigs and rats. AB - The trachea of guinea-pigs was stained as a whole-mount preparation with the zinc iodide-osmium technique. A distinct class of nerve endings was observed associated with the tracheal muscle. The endings, issued from myelinated fibres of the vagus nerve via the recurrent laryngeal nerve, are distributed on either side of the midline and ventral to the tips of cartilages. They are interpreted as afferent nerve endings that may correspond to slow adapting stretch receptors identified by physiological studies. Each nerve contributes predominantly, but not exclusively, to the receptors of the ipsilateral side. There are 120-180 receptors along the full length of the guinea-pig trachea, their density being higher at the cranial end. The receptors are variable in size and structural complexity, and, to some extent, also in spatial orientation, but distinct subtypes are not recognizable. Receptors of similar morphology and distribution are found also in the rat trachea. The receptors can also be visualized with a cytochrome oxidase method for nerve endings, but they do not stain with immunohistochemistry for the neuropeptides substance P, calcitonin gene-related peptide, vasointestinal polypeptide and neurotensin. PMID- 1711299 TI - Serum amylase determination and blunt abdominal trauma. AB - To determine the value of serum amylase sampling as an indicator of intra abdominal injury, the records of 940 consecutive victims of blunt trauma were retrospectively reviewed. The sensitivity, specificity, and predictive value were poor in the determination of intra-abdominal injury, whether accompanied by craniofacial injury or not. It was concluded that routine serum amylase determination is of no value in the clinical management of the patient suffering blunt injury. PMID- 1711300 TI - Hemostasis activation during esophageal variceal sclerotherapy with thrombin in cirrhotics. AB - Sclerotherapy of bleeding esophageal varices in liver cirrhotics is a common procedure, but little is known about the possible entry of sclerosants into the systemic circulation. We injected a mixture of thrombin, sodium tetradecyl, and cefazolin and studied the effect of this sclerosant on selected hemostasis parameters. Twenty-four patients with liver cirrhosis (Child's Classification C) were studied 29 times. Blood samples were drawn before and immediately after the injection of the sclerosant. In seven patients we collected a sample 30 minutes and 24 hours after treatment. Before injection, almost all patients had elevated D-dimer, t-PA and PAI-1 levels. Fibrinogen, antithrombin, alpha-2 antiplasmin, and protein C were decreased. Only thrombin/antithrombin III complex (TAT) levels were within normal ranges. Immediately after the injection, TAT, D-dimer, and t PA levels rose significantly (P less than 0.001, P less than 0.01, P less than 0.001), PAI-1 and PC levels decreased (P less than 0.01), while antithrombin, alpha-2 antiplasmin, and fibrinogen concentrations were unchanged. TAT and D dimer levels were still elevated after 24 hours (P less than 0.05). These data indicate that thrombin entered the systemic circulation (elevated TAT) and that the hemostasis system was briefly systemically activated (elevated D-dimer). In spite of these changes in the hemostasis system, clinically there were no detectable thrombotic or hemorrhagic complications. PMID- 1711301 TI - Safe and rapid palliation of dysphagia for carcinoma of the esophagus. AB - Patients with carcinoma of the esophagus continue to present late when their tumors are inoperable. This makes palliation of their dysphagia the main therapeutic aim. The Nd-YAG laser has been used in our department to treat dysphagia resulting from cancer of the esophagus since 1986. Our rapid, one-stage cannulation technique using the Nd-YAG laser in both contact and noncontact modes was applied to 35 cases of carcinoma of the esophagus with the aim of achieving rapid and safe palliation of dysphagia. During the treatment we aimed not to coagulate the tumor and await sloughing, but to vaporize the tumor and ablate as much as possible in a single session. In this way there was less need for repeat sessions to create an adequate lumen. In a small number of patients (9) who had tight strictures with no visible lumen, a pre-laser dilation was required to allow visualization of the lumen and tumor vaporization. For nondilated patients (26) we achieved a 15-mm lumen in an average of 1.6 sessions, and in the dilated patients (9) this was achieved in one session in all patients. Functional improvement occurred in 28 patients (80%). There were four minor complications and no mortality associated with the procedure. PMID- 1711302 TI - Endoscopic contact Nd:YAG laser resectional vaporization (ECLRV) and esophageal dilatation (ED) in advanced malignant obstruction of the esophagus. AB - Malignant esophageal obstruction in patients with advanced and metastatic carcinoma is unsuitable for surgery. Palliative treatment must provide adequate swallowing with minimum complications in these often seriously ill patients. Twenty consecutive patients underwent endoscopic Nd:YAG contact laser resection and vaporization (ECLRV) and esophageal dilatation (ED) for advanced esophageal carcinoma since August, 1985. Average duration of the disease when first referred was 7.2 months. Tumor cell type was either squamous cell carcinoma (n = 11) or adenocarcinoma (n = 9). Tumor location was distal (n = 14), middle (n = 5), or upper (n = 2). Mean tumor length was 7.5 cm. Mean preoperative luminal diameter was 1 mm, with total obstruction in ten (50%) patients. The operative procedure in all patients was under general anesthesia with endotracheal tube intubation. Rigid and flexible endoscopes were both used as indicated. Mean postoperative luminal diameter was 15 mm. All but four were able to swallow fluids on the first postoperative day, followed by semisolids the next day without discomfort. Minor perforation was noted in three cases and managed in two conservatively. One more patient had difficulty in swallowing due to extra-esophageal compression, in spite of a technically successful laser therapy. Percutaneous endoscopic gastrostomy (PEG) was carried out in eight cases. Eleven patients were retreated successfully for recurrent obstruction and two were treated more than twice, at a mean of six-week intervals. Endoscopic contact laser resectional vaporization with esophageal dilatation was relatively safe and provided an improved quality of life in this preliminary study group, providing a mean survival of 18.5 weeks (range 2-50 weeks). PMID- 1711303 TI - [Salvage chemotherapy with IMV-triple P for relapsed or refractory malignant lymphoma]. AB - Twelve patients with relapsed or refractory malignant lymphoma were treated with IMV-triple P regimen consisting of ifosfamide (IFM), mitoxantrone (MIT), vindesine (VDS), pepleomycin (PEP), procarbazine (PCZ) and prednisolone (PDN). Three of 12 patients achieved complete remission (CR), and 5 patients achieved partial remission (PR). Hence, the overall response rate was 66.7% (8/12). Of 9 relapsed patients who had attained CR after the former chemotherapy, 3 had CR and 4 had PR. The overall response rate was 77.8% (7/9). Side effects were relatively mild, including leukopenia (less than 1,000/microliters) (33.3%) and thrombocytopenia (less than 5 X 10(4)/microliters) (8.3%). There was no severe cardiac toxicity such as heart insufficiency and severe arrythmia. These results suggest that IMV-triple P regimen is effective in the treatment of relapsed or refractory malignant lymphoma. PMID- 1711304 TI - [Tumor markers--personal experience. Multidisciplinary treatment of disseminated germ cell tumor in full use of tumor markers]. AB - The ideal course of multidisciplinary treatment of disseminated germ cell tumors was shown with special reference to the role of the tumor markers for 1) diagnosis of pathological elements and risk factors, 2) appropriate selection of the protocol before and during the chemotherapeutic treatment, 3) timely resection of residual tumor and 4) monitoring of the recurrence after treatment. The practical role of tumor markers for evaluation of chemotherapeutic effectiveness, the following reconsideration of the appropriate protocol and timely indication of surgical intervention for residual mass after chemotherapy were emphasized by demonstrating the clinical courses of 2 patients with advanced nonseminomatous testicular germ cell tumors. PMID- 1711305 TI - [The role of growth factors in cancer treatment]. PMID- 1711306 TI - Miller-Dieker syndrome with ring chromosome 17. AB - A girl presented at 6 weeks of age with failure to thrive and arching of the back. She had various dysmorphic features, hepatosplenomegaly, and developmental delay. The electroencephalogram and cranial ultrasound were abnormal, and a computed tomogram showed lissencephaly and apparent agenesis of the corpus callosum. Because of frequent aspiration she became oxygen dependent. She later developed intractable convulsions and died at the age of 9 months. PMID- 1711307 TI - [Experimental study of protease C--proteolytic enzyme of microbial origin]. AB - The specific activity of protease C, a proteolytic enzyme isolated from Acremonium chrysogenum was studied under experimental conditions. Protease C was shown to lyse necrotic biological substrates (dry crusts of burn wounds) and blood clots. By the nature of the effect protease C was analogous to terrilytin and by the level of the effect it was superior in some experiments. Protease C was low toxic and had no mutagenic action. PMID- 1711308 TI - Hepatotoxicity and lethality of halomethanes in Mongolian gerbils pretreated with chlordecone, phenobarbital or mirex. AB - The hepatotoxic and lethal effects of CBrCl3, CCl4 and CHCl3 were investigated in gerbils with or without prior exposure to dietary chlordecone (CD), phenobarbital (PB) and mirex (MX) at 10, 225 and 10 ppm, respectively, for 15 days. Gerbils were quite sensitive to these halomethanes (48 h LD50: 20, 80 and 400 microliters/kg, respectively). CD, known to potentiate hepatotoxic and lethal effects of halomethanes in rats, failed to potentiate the toxic effects of any of these three halomethanes in gerbils. PB and MX were also ineffective. Since stimulation of early hepatocellular regeneration has been shown to be responsible for the recovery from the toxicity of a low dose of CCl4, liver cell regeneration and tissue repair were studied in gerbils after CCl4 administration. The objectives of these studies were to investigate the possible reasons for the high sensitivity of gerbils to halomethane toxicity and to investigate the mechanism for their refractoriness to CD-potentiated halomethane toxicity. A low and a high dose of CCl4 (15 and 80 microliters/kg, i.p. respectively) were used to study the time-course of liver injury in gerbils pretreated with or without CD. The low dose of CCl4 stimulated cellular regeneration as indicated by the increase of 3H thymidine (3H-T) incorporation in hepatic nuclear DNA. The cellular regeneration and tissue repair activities resulted in complete recovery from the limited liver injury in both CD-pretreated and control gerbils. In contrast to rats, however, the process of cell division in gerbils occurred much later, 2 days after CCl4 administration. Evidence from histomorphometric studies was consistent with serum enzyme and 3H-T incorporation data.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711309 TI - [Histamine release test of patients allergic to the house dust mite, Dermatophagoides farinae--analysis according to various clinical backgrounds]. AB - Since the histamine release test is more complicated than some of the other tests, there are smaller number of clinical studies employing this method. In this study, we selected 25 males and 20 females, allergic to the house dust mite, to undergo the histamine release test challenged by 7 serial concentrations of the mite allergen diluted from 10 times to 10 million times the original allergen at a concentration of 0.01%. Histamine release was expressed as a percentage of the total histamine concentration contained in the cells. Allergen induced dose response curve of histamine release was drawn for each patient, and in order to characterize each curve, 5 different indices were selected and were averaged according to the clinical backgrounds of the patients. Significant increases in histamine release were observed in males under 25 and females above 26 years of age. Histamine release rate in patients with RAST score of above 3+ was significantly more enhanced than in those with RAST score of below 2+, though this result was significant only for those under 25 years of age. Blood samples taken in June-August, and September-November, showed significant increases in the histamine release, though the indices for enhancement differed in both seasons. When analyzed according to the numbers of years the patient was desensitized, the results showed, contrary to our expectations, a more increased release in the desensitized group than in the non-desensitized group. PMID- 1711310 TI - [Effect of tranilast (N-5) on cellular immunity--delayed type hypersensitivity]. AB - Tranilast (N-5) as a mast-cell stabilizer is widely used in cases of bronchial asthma and nasal allergy. Some reports showed that N-5 is effective against keloids and ulcerative colitis. So, it is considered that N-5 inhibits cell mediated immunity. N-5 inhibited BCG-CWS-stimulated lymphocyte proliferation which was proved to be mainly operated by OKT4 positive T cells. Production of the lymphokine from BCG-CWS-stimulated lymphocytes was suppressed by the addition of N-5. Thus, it is demonstrated that N-5 inhibits delayed type hypersensitivity in vitro. PMID- 1711311 TI - [Genetic, immunological and functional studies on lymphocytes with OKT4-epitope deficiency]. AB - Although it is well-known that Leu3a-epitope on CD4 molecule functions as a receptor for human immunodeficiency virus (HIV), the function of OKT4-epitope is still obscure. In order to learn the significance of OKT4-epitope, we performed immunological and functional studies on lymphocytes obtained from individuals with incomplete/complete OKT-4 epitope deficiency. Their lymphocytes 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 or the expression of IL-2R (alpha/beta-chain) by PHA-stimulation. By flow cytometric analysis it was demonstrated that quantity of OKT4-epitopes in the incomplete deficiency was approximately one-half less than that of normal individuals. Coupled with this fact and DNA analysis previously reported, individuals with incomplete/complete OKT4-epitope deficiency were considered to be heterozygote and homozygote, respectively. These results led us to the conclusion that OKT4 epitope deficiency was inherited as an autosomal codominant trait. Individuals with complete OKT4-epitope deficiency were found in 7 cases out of 1486 random samples (0.47%), from which individuals with incomplete OKT4-epitope deficiency were estimated to account for 12.8%. PMID- 1711312 TI - [Cellular composition of tracheal lymphoid tissue in children in their first year of life]. AB - By means of morphometrical methods in histological preparations the quantitative relations of various cell types of lymphoid formations has been studied in newborns and in suckling children. The trachea in the newborns practically does not possess any morphological substrate in the form of lymphoid accumulations, responsible for immune defense of the organ from any external influence. Development of the lymphoid tissue begins in the suckling age and its cytoarchitectonics depends on their localization in the organ's wall. A special place among the accumulations of the lymphoid cells occupy connective tissue spaces of the tracheal glands, where besides lymphocytes and fibroblasts a great amount of plasma cells is situated. Under epithelium these cells are in the least amount, in the prenoduli they are of an intermediate amount. In all the lymphoid structures investigated reproductive function is absent; this is proved by absence of blasts and mitotically dividing cells. Increase in amount of the tracheal lymphoid formations in all age groups studied takes place at the expense of cells migration across blood vessels. PMID- 1711313 TI - [Characteristics and structural variants of human lumbar intervertebral disks]. AB - By means of macro- and microscopical methods structure of 412 lumbar intervertebral discs (ID), obtained from 84 corpses of persons died at the age 16 90 years have been studied, as well as 30 macerated lumbar vertebra. During these periods the lumbar ID are formed by hyalin laminae and a connective tissue wall, surrounding the cavity. According to the tissue type, in the ID wall three main layers are differentiated. The external layer is determined as a fibrous ring, the middle one--as fibrous-cartilagenous and the internal one--as a chondromucoid ring. The three layers gradually and consequently turn one into another. According to manifestation degree of these layers, development of the internal layer and size of the cavity three main variants of the ID structure are nominated: intervertebral synarthrosis, intervertebral hemiarthrosis and intervertebral "diathrosis" (real articulations have not been revealed between the vertebral bodies). The occurrence rate of the ID structural variants revealed in the lumbar part of the spinal cord is demonstrated. Absence of chordal mucous in the cavity is specific for the ID during the age periods investigated. The role of the "nucleus" is performed by the internal layer of the ID wall, which possesses a system of processes and a forming peculiar pulpous complex, which ensures the ID adaptation to various changes in position of the vertebral locomotor segment. The pulpous complex is surrounded with a united fibrillar carcass of hyalin laminae and fibrous-cartilagenous ring; together with the carcass it forms an elastic layer between the vertebral bodies. PMID- 1711314 TI - [Characteristics of tissue basophils associated with the structures of the general immune system of mucous membranes]. AB - By means of a complex histochemical staining methods human and rat mast cells (MC) have been studied in the organs, where lymphoid tissue makes the common immune system of mucous membranes. The mucous membranes of the stomach body (its glandular part in rats), intestine, bronchi, renal pelvis and calyces, uterine tubes, as well as intralobular ducts of the parotid and mammary glands have been studied in men and rats. As a control--lip, tongue and esophagus have been taken. In the man and rat MC, associated with structures of the common immune system, possess atypical cytochemical characteristics: in their cytoplasm (unlike in MC of the control organs) only sulfated glycosaminoglycans are revealed, while histochemically detected protein, neutral and sialic acid carbohydrate components are not revealed. MC are absent in single lymphoid follicles and in cupola follicular complexes of grouped lymphoid nodules++ and they are found in all other areas of the objects investigated, interfollicular zones including. PMID- 1711315 TI - [The pavement lining of the vessels in the earthworm (Lumbricus terrestris)]. AB - Effect of some preparatory methods to make ready earthworm tissues for electron microscopical investigation has been studied in order to reveal structural components of the vascular wall. The method for an immediate fixation of the earthworm tissues in liquid nitrogen with a subsequent additional fixation in cooled 2.5% glutar aldehyde, applied in combination with OsO4-thiocarbohydroside OsO4 method is optimal. This method of samples preparation gives a reliable information concerning existence of an internal--endothelial--lining in the earthworm vessels. A conclusion is made on a qualitatively new level of cell differentiation of the internal vascular lining in the earthworm, which is called phase of blast transformation of endotheliocytes. PMID- 1711316 TI - [The reaction of somatic lymph nodes to experimental exposure to sulfide baths]. AB - Influence of strong sulfide baths of the sanatorium "Talgi" to the popliteal and superficial inguinal lymph nodes (LN) has been studied in 35 rats (140-150 g of body mass) and 15 animals have been used as the control. The slices are stained with hematoxyli-eosin, after van Gieson, Romanovsky-Giemsa, Kurnik and silver nitrate impregnation after Foot. The section area of the LN decreases, but amount of lymphoid nodules increases (especially in deep layers of the cortex). In the germinative centers amount of middle lymphocytes and mitoses becomes larger. In the internodular zones of the popliteal nodes amount of macrophages and eosinophiles enlarges significantly, and in the inguinal-amount of mast cells and eosinophiles. In the medullary sinuses amount of macrophages declines. In the medullary cords amount of immature plasma cells and blasts increases, while amount of mature cells decreases. PMID- 1711317 TI - [The theory of the staining of tissue basophils with the PAS method]. PMID- 1711318 TI - [A macro- /microscopic method of demonstrating lymphatic vessels and nodes]. PMID- 1711319 TI - [A combined method for staining the goblet and mast cells in slices of small intestine fixed in Carnoy's solution]. PMID- 1711320 TI - Characterization of the fine specificity of peptide antibodies to HLA-DQ beta chain molecules. AB - In an attempt to produce epitope specific antisera which could distinguish two closely associated HLA-DQ beta-chain alleles, we immunized 20 rabbits with synthetic peptides representing sequences from the first domain of the HLA-DQw8 and -DQw7 beta-chain molecules, differing only by one amino acid in position 57. Several of the antisera in immunoblotting specifically recognized either the HLA DQw7 or the HLA-DQw8 beta-chain allele as previously reported. The fine specificity of the antisera was tested in ELISA using synthetic peptides of varying length as solid phase antigen. Two out of the 20 antisera specifically recognized DQw7 beta peptides and two antisera bound only to DQw8 beta peptides from the region containing the amino acid in position 57. To analyze whether the antisera bound to native HLA-DQ beta-chain molecules, FACS analysis was carried out. Seven of the 20 antisera bound to intact EBV-transformed B-lymphocytes, and one antiserum bound preferentially to HLA-DQw8 positive cells. PMID- 1711321 TI - Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2. AB - Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we have produced peptide antisera to an antigenic epitope which is unique to mouse and rat C-peptide 2. Antisera recognizing this epitope may be effective tools to study differential expression of the two insulin genes in mouse and rat. PMID- 1711322 TI - Identification and characterization of opsonic fibronectin in synovial fluids of patients with active rheumatoid arthritis. AB - A cofactor that selectively opsonizes particulate activators of the human alternative complement pathway and enhances their phagocytosis by human monocytes was identified in synovial fluids of patients with rheumatoid arthritis. The active material was present in fluids treated with protease inhibitors, was heat stable, and was unaffected by incubation with hyaluronidase. Chromatographic isolation of synovial fluid fibronectin by gelatin affinity and by immunoaffinity on antifibronectin monoclonal antibody BD4 yielded similar quantities of protein for each of 3 fluids. Synovial fluid proteins with the BD4 fibronectin epitope accounted for essentially all of the phagocytosis-enhancing activity and expressed this activity by opsonizing target activators. Additional chromatographic analyses of synovial fluid fibronectin with the BD4 epitope were carried out using Sepharose-bearing gelatin and 4 additional antifibronectin monoclonal antibodies. The opsonic materials were characterized as having 2 distinct fibronectin epitopes, which always mapped from the cell adhesive domain to the carboxyl-terminus of plasma fibronectin, but only rarely contained the gelatin binding domain. PMID- 1711323 TI - Sera from patients with autoimmune disease recognize conformational determinants on the 60-kd Ro/SS-A protein. AB - Anti-Ro antibodies are found in a large proportion of patients with systemic lupus erythematosus and primary Sjogren's syndrome. These antibodies also characterize neonatal lupus, subacute cutaneous lupus erythematosus, and vasculitis associated with Sjogren's syndrome and rheumatoid arthritis. Anti-Ro positive sera may contain either or both of 2 sets of antibodies, recognizing either a 60-kd or a 52-kd polypeptide component of the Ro particle. We found in this study that the immune response to the 60-kd Ro antigen is heterogeneous. Some sera specifically recognize the native Ro antigen but fail to bind the corresponding denatured polypeptides. In addition, after immunodepletion using the denatured 60-kd Ro polypeptide, all anti-Ro-positive sera tested still contained high titers of antibodies recognizing conformational determinants on the Ro antigen. The frequent immunodominance of anti-Ro antibodies targeted to conformational determinants suggests that native autoantigens may directly drive the autoimmune response. PMID- 1711324 TI - Alpha2-macroglobulin and related plasma proteins. Structure, function, regulation. 87th Congress of the Society for Biological Chemistry. Aachen, October 24-27, 1990. Abstracts. PMID- 1711325 TI - Carrier bound templates for single tube reverse transcriptase assays and for combined purification and activity analyses, with special reference to HIV. AB - Polyriboadenosine (prA) was coupled to polycarbonate macrobeads or magnetic beads. The efficiency of the beads and of prA-Sepharose, after priming with odT, as templates in activity assays of purified AMV- and HIV-reverse transcriptase (RT), using [125I]iododeoxyuridine-triphosphate as substrate, was studied. Although the use of immobilized templates, compared with soluble template, resulted in a decreased total molar turnover, it did not affect the sensitivity of the assay for detecting RT. The utility of the new assay was analyzed by mixing purified AMV- or HIV-Rt with different dilutions of the untreated clinical specimen. This showed that RT activity was unaffected by 100 microliters of an extract of whole blood cells resuspended to their original blood volume and diluted 1/64, and also by 100 microliters of serum diluted 1/64. To improve the utility of the assay at the inhibitory concentrations of clinical specimens, the following procedure was adopted: the sample to be analyzed was incubated with the carrier bound template in order to allow the RT to bind, the carrier was washed to remove inhibitory factors, and the reaction components were then added to determine the amount of bound RT. This procedure greatly enhanced the recovery of RT activity from crude specimens and made the direct detection of HIV-RT possible. The assay is easily automated and useful for RT determination in multiple samples and for determining RT-inhibiting substances such as substrate analogs and antibodies. PMID- 1711326 TI - Role of nitric oxide synthesis in macrophage antimicrobial activity. AB - Research over the past 5 years has demonstrated that immunologic activation of mouse macrophages induces the activity of nitric oxide synthase, which oxidizes a guanidino nitrogen of L-arginine, yielding citrulline and the reactive radical, nitric oxide. A review of the biochemistry and immunologic regulation of this pathway in macrophages provides a backdrop against which to evaluate its effector functions. Reports published in the past 2 years suggest that synthesis of NO mediates much of the antimicrobial activity of mouse macrophages against some fungal, helminthic, protozoal and bacterial pathogens. PMID- 1711327 TI - Multiple receptors for endotoxin. AB - Recent studies have identified three classes of receptor molecules involved in recognition of endotoxin. Two classes of receptors, the CD18 antigens and the scavenger receptor, recognize lipopolysaccharide directly and function principally in its catabolism. A third molecule, CD14, recognizes lipopolysaccharide with the aid of a serum protein, lipopolysaccharide-binding protein, and may be a principal mediator of secretory responses of leukocytes. PMID- 1711328 TI - An antigenic determinant is shared by psoriasis-associated p27 antigen and the Fc part of human IgG. AB - Rabbit antisera against the major internal protein, p27, of retrovirus-like particles from psoriatic urine, and against the serologically cross-reacting antigen, pso p27, from psoriatic scale, reacted with the Fc part of human IgG. Evidence indicating that the p27 antigen and the pso p27 antigen are identical has been presented in previous reports. A commercial antiserum against human IgG recognized a component in the pso p27-containing solution used as the source of antigen for immunization of the rabbits. By means of monoclonal antibodies against the pso p27 antigen, it was demonstrated that the Fc-reacting antibodies, and the antiserum against human IgG, recognized an epitope on the pso p27 antigen. The data indicated that an antigenic determinant is shared by the p27 antigen(s) and human IgG, suggesting that p27 antigen(s) may act as antigen(s) eliciting the production of antibodies with rheumatoid factor activity in psoriatic patients. PMID- 1711329 TI - [Productivity in planning administration. A proposal of a planning project: the aged. Technical and health considerations]. PMID- 1711330 TI - New patterns and control of food-borne infections in industrialized countries. PMID- 1711331 TI - [Role of environmental factors in the development of acute respiratory diseases]. PMID- 1711332 TI - [Volatile anesthetic agent pollution in operating rooms in a large Roman hospital. A preliminary note]. PMID- 1711333 TI - [Microclimate survey of operating rooms in a Roman hospital. A preliminary note]. PMID- 1711334 TI - [Seroepidemiology of hepatitis B in pediatric age. Studies conducted in the city of Rome]. PMID- 1711335 TI - [Reduction of infective and mutagenic risks of waste waters: action of chlorine and ozone]. PMID- 1711336 TI - Influence of algae on ultrafiltration for enteroviruses recovery from seawater. PMID- 1711337 TI - [Cigarette smoking habits and opinions in a sample of students of the Province of Potenza: notes for an antismoking campaign]. PMID- 1711338 TI - [Knowledge about AIDS and attitudes in comparisons of persons at high risk of contracting the disease: a survey of high school students of Brescia]. PMID- 1711339 TI - [Poliovirus inactivation in sea water treated with ozone]. PMID- 1711341 TI - [Epidemiological approach to osteoporotic pathology and retrospective notes on the situation in the Veneto Region (1977-1987)]. PMID- 1711340 TI - [Action of alkyl-dimethylbenzylammonium saccharinate (onyxide 3300)]. PMID- 1711342 TI - [Young population and AIDS: a study on knowledge and beliefs of 690 university students]. PMID- 1711343 TI - [Incidence of Gardnerella vaginalis infections in relation to age in a sample of 161 women of the Province of Teramo]. PMID- 1711344 TI - [Prevalence of Listeria monocytogenes infection in a Rome population group]. PMID- 1711345 TI - [Gram-negative flora of horticulture products prevalently to be consumed fresh. II, Acinetobacter calcoaceticus]. PMID- 1711346 TI - [Hormonal contraception in Family Counseling Services in Rome. II. Verification of the situation in 1989]. PMID- 1711347 TI - Agrin induces phosphorylation of the nicotinic acetylcholine receptor. AB - Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution. PMID- 1711348 TI - EGF and TGF-alpha stimulate retinal neuroepithelial cell proliferation in vitro. AB - Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-alpha, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina. PMID- 1711349 TI - Role of the neurotrophic factors BDNF and NGF in the commitment of pluripotent neural crest cells. AB - Since trophic factors are increasingly recognized as playing a role in some decision-making steps during development, the influence of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) on the commitment of pluripotent neural crest cells was investigated by in vitro clonal analysis. BDNF caused an increase of up to 21-fold in the number of sensory neuron precursors per colony without a corresponding increase in the total number of cells. By contrast, BDNF treatment caused an equivalent decrease in the number of undifferentiated cells per colony. The data suggest that BDNF, but not NGF, directs pluripotent neural crest cells to differentiate along the primary sensory neuron lineage. PMID- 1711350 TI - Two alpha-herpesvirus strains are transported differentially in the rodent visual system. AB - Uptake and transneuronal passage of wild-type and attenuated strains of a swine alpha-herpesvirus (pseudorabies [PRV]) were examined in rat visual projections. Both strains of virus infected subpopulations of retinal ganglion cells and passed transneuronally to infect retino-recipient neurons in the forebrain. However, the location of infected forebrain neurons varied with the strain of virus. Intravitreal injection of wild-type virus produced two temporally separated waves of infection that eventually reached all known retino-recipient regions of the central neuraxis. By contrast, the attenuated strain of PRV selectively infected a functionally distinct subset of retinal ganglion cells with restricted central projections. The data indicate that projection-specific groups of ganglion cells are differentially susceptible to the two strains of virus and suggest that this sensitivity may be receptor mediated. PMID- 1711351 TI - The elastolytic activity of cathepsin G: an ex vivo study with dermal elastin. AB - To determine whether human neutrophil cathepsin G can act by itself or in concert with human neutrophil elastase to destroy elastic fibers in vivo, we used cryostat sections of human skin as an ex vivo substrate for these leukoproteinases. Specifically stained dermal elastic fibers were quantitated using an accurate and almost entirely automatic morphometric procedure that included computerized threshold selection and elimination of non-elastic dark elements. AA, the area fraction occupied by the dermal elastic fibers, was found to be 0.100 +/- 0.014 (mean +/- SD) for 21 control skin sections originating from a single donor. Measurement of the fiber diameters in these control sections (2.4 +/- 0.8 microns [mean +/- SD]) allowed calculation of the Weibel factor used to convert AA into Vv, the volume fraction occupied by the elastic fibers: Vv was 0.028 +/- 0.004 (mean +/- SD). Incubation of skin sections with elastase, cathepsin G, or mixtures of the two enzymes resulted in an important decrease in AA accompanied by a slight increase in the average fiber diameter. The largest increase (14%) was noticed for cathepsin G and was due to a preferential attack of thin fibers and to fiber fragmentation. The AA of fibers remaining after elastolytic activity of cathepsin G was 20 to 30% that of elastase in this ex vivo assay. On the other hand, cathepsin G stimulated the elastolytic activity of elastase. For instance, the activity of a mixture of 1.1 microM elastase and 1.5 microM cathepsin G was 1.9-fold higher than the sum of the activities of the individual proteinases. The stimulation increased with the cathepsin G concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711353 TI - Palliative surgery of metastatic bone disease: a review of 83 cases. AB - Of a total of 83 patients with metastatic bone disease, surgery was performed in 17 cases at the prefracture stage, in 54 cases after complete fracture and in 10 cases to decompress the spinal cord. Positive short-term results were obtained in 75% of cases. 7 patients presented mild complications. In 2 cases, the patients had to be reoperated. 55% of the patients were still alive after 6 months, 31% after 12 months and 10% after 2 years. PMID- 1711352 TI - Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha-galactosyl end groups. AB - We used Griffonia (bandeiraea) simplicifolia I (GS I) lectin and flow cytometry to isolate subsets of rat tracheal epithelial cells based on the presence or absence of cell surface alpha-galactosyl end groups. These fractions were designated GS I-positive and -negative, respectively. Ninety-eight percent of the cells in the GS I-positive fraction expressed cell surface alpha-galactosyl end groups; 95% had immunocytochemically detectable keratin 14-related protein (a basal cell marker) and 98% lacked alcian blue-periodic acid-Schiff (AB-PAS) stained cytoplasmic granules. More than 90% of the GS I-positive cells had a high nuclear-to-cytoplasm ratio, had tonofilaments, and lacked organelles characteristic of other differentiated cell types; they were thus classified as basal cells. In bioassays, the GS I-positive fraction had a colony-forming efficiency greater than or equal to that of native tracheal cell suspensions, and the cells were able to repopulate denuded tracheal grafts with ciliated, secretory, and basal cells. More than 99% of the cells in the GS I-negative fraction lacked cell surface alpha-galactosyl end groups, 98% did not stain for keratin 14-related protein, 54% had significant numbers of AB-PAS-stained cytoplasmic granules, and 16% were identified as ciliated cells. The GS I negative fraction had a lower colony-forming efficiency than the GS I-positive fraction but, it too, was able to repopulate denuded tracheal grafts with a complete mucociliary epithelium. These results show that both GS I-positive and negative cells had the potential to proliferate and differentiate into the major tracheal cell types. PMID- 1711354 TI - Cell mediated cytotoxicity and cytokine production in peripheral blood mononuclear cells of glioma patients. AB - A mannoprotein preparation (MP) from Candida albicans induced MHC-unrestricted cytotoxicity in peripheral blood mononuclear cells (PBMC) from healthy subjects, but not in those from glioma-bearing subjects. The two groups of subjects did not significantly differ in the number of cells bearing typical natural killer (NK) markers (both in resting and MP stimulated PBMC) and NK activity. However, interferon gamma (IFN-gamma) production was in tumour patients minimal or significantly reduced, as compared to healthy subjects, following PBMC stimulation by MP or phytohaemoagglutinin, respectively. In addition, minimal, if any, stimulation of interleukin-2 (IL-2) production was achieved in MP stimulated PBMC from glioma patients. Considering the pivotal role of the above cytokines in immune responses, particularly in those concerning generation of antitumour effectors, our results consistently suggest that defective cytokine production is one possible mechanism of immunological impairment in glioma patients. They also provide indirect support for a possible clinical use of IFN-gamma as an immunopotentiating agent in gliomatous subjects. PMID- 1711355 TI - Prostate-specific antigen: problems in analysis. AB - We have compared an "in-house" Tenovus Institute prostate-specific antigen (PSA) assay with four different commercial kits (ELSA-PSA, IRMA-Count PSA, PROS-CHECK PSA and TANDEM-R PSA) that are available in the UK. There was only good correlation and linear regression parameters between the in-house assay and one of the kit methods. The difference in values for the same sample ranged from 2 to 100-fold. These discrepancies are due, in part, to the specificity of the polyclonal and monoclonal antibodies used in the procedures and the differing "hook effects" caused by the binding capacity of the antibody pairs in the immunometric assays. Discrepancies will, however, result from the differing potencies of the standards used for the calibration curves. This data highlights the urgency for the introduction of an internationally accepted reference standard for PSA. PMID- 1711356 TI - Pneumocystis pneumonia in a patient treated with fludarabine for chronic lymphocytic leukemia. PMID- 1711357 TI - Discrimination between strontium and calcium in suckling rats. AB - Discrimination between strontium (Sr) and calcium (Ca) was examined in suckling rats and compared with that in older rats after weaning. Concentrations of Sr and Ca and the Sr/Ca ratios in serum and femur of 10-d old and 21-d old rats were determined. The Sr concentrations and Sr/Ca ratios in the serum and femur of 10-d old rats were lower than those of 21-d old rats, that could be explained by the fact that 10-d old rats ingested only maternal milk in which the Sr/Ca ratio was much lower than the laboratory diet. The relative ratios of Sr/Ca in serum and femur to that in the diet were found to be higher in 10-d old rats compared with those in 21-d old rats, and also higher than those in the older rats after weaning, as described in our previous publication. This result may reflect that discrimination between Sr and Ca during intestinal absorption is lacking in very young animals before weaning and develops after this age. Renal discrimination between Sr and Ca in the suckling rats at 10 d of age was evaluated by determining the relationship between the relative clearances of Ca and Sr. The mathematical model proposed by Walser and Robinson was applied on these results and the parameter for the equation, that is, the discrimination constant, was shown to be higher in 10-d old rats compared to those in young (7 wk of age) and adult (25 wk of age) rats. This result suggests that the discrimination of Sr in favor of Ca during the tubular reabsorptive process may not be fully developed in the very young rats before weaning. PMID- 1711358 TI - A comparison of phenotype and copper distribution in blotchy and brindled mutant mice and in nutritionally copper deficient controls. AB - The murine mottled mutants brindled, Mo br, and blotchy, Mo blo, are valuable animal models for the study of mammalian copper metabolism. In this paper, we present data showing that a nutritionally copper deficient suckling mouse, Cu-, with strong phenotypic similarities to the brindled mutant can be produced by feeding genetically normal dams a copper deficient diet (0.1-0.4 ppm Cu2+) from the day of mating. Comparisons of copper distribution between the Cu- mice and brindled mutants indicate that when a small dose of copper (0.5-0.9 micrograms Cu2+) was administered by intracardiac injection, the copper was abnormally distributed, and that the pattern of tissue distribution was very similar in Cu- mice and brindled mutants 24 h after injection. When, however, a treatment dose (50 micrograms Cu2+) was injected subcutaneously, and tissues assayed 3 d after injection, copper distribution in Cu- mice and brindled mutants was clearly different. Copper deficiency in Cu- suckling mice is entirely derived from maternal effects. Evidence that maternal effects may also influence the survival and phenotype of the brindled and blotchy mutants was obtained by comparing the viability of mutants born to dams carrying mottled mutations on one or both X chromosomes. PMID- 1711359 TI - Determination of eight elements in six human cancer cell lines and two human normal cell lines by PIXE. AB - Gastric, hepatic, and pulmonary cancer cell lines, and the third passage of normal gastric and pulmonary cell lines were analyzed by proton induced X-ray emission (PIXE) method. The contents of element Sr, Ca, Fe, Zn, P, K, Cu, and As in the cell lines were determined. The Sr, Ca, Fe, Zn, and As contents in cancer cell lines were significantly lower than those in the normal cell lines (p less than 0.05), whereas there were no significant differences for the P, K, and Cu contents (p greater than 0.1). The results suggest that the need of some essential elements has been diminished in cancer cell proliferation. PMID- 1711360 TI - Distribution of structural and trace elements in human temporal bone. AB - This study was undertaken to evaluate a systematic analysis of mineral and trace elements of individual functionally determined parts of adult temporal bone. Marked differences were observed in basic structural elements (Ca, P, Mg, and Zn) among different bone regions. The more so, molar Ca/P ratio was significantly different in various regions, being highest in the hammer and vestibular regions. Taxonomic analysis revealed specific differences in the mineral ratio between the two petrous bone regions believed to develop from various embryonal bases. According to results, the observed differences in mineral trace element composition of particular regions of human temporal bone might be explained by their developmental specificities and functional adaptation. PMID- 1711361 TI - Copper and zinc in experimental hypertension. AB - The role of the trace minerals, copper (Cu) and zinc (Zn) are important in maintaining blood pressure. Copper has been found to inhibit the activity of angiotensin's converting enzyme. An interrelationship has been found to exist between Cu and Zn. Data in renal (RH) and spontaneous hypertensive rates (SHR) regarding Cu and Zn is lacking. The purpose of this report was to measure Cu and Zn levels in two types of experimental animal models of hypertension compared to normotensive (NT) rats. Blood samples were drawn to measure serum levels of Cu and Zn in three types of animals, RH, SHR, and NT. Serum Cu values were found to be lower, whereas Zn levels were elevated in the SHR animals. Serum levels of Cu and Zn in the RH animals were similar to those found in the NT animals. Further study of the interaction of those trace minerals is documented, and extends over knowledge of the role of minerals in blood pressure control. PMID- 1711362 TI - Minor and trace elements in human milk from Guatemala, Hungary, Nigeria, Philippines, Sweden, and Zaire. Results from a WHO/IAEA joint project. AB - Concentrations of As, Ca, Cd, Cl, Co, Cr, Cu, F, Fe, Hg, I, K, Mg, Mn, Mo, Na, Ni, P, Pb, Sb, Se, Sn, V, and Zn were determined in human whole milk samples from Guatemala, Hungary, Nigeria, Philippines, Sweden, and Zaire; in most of these countries, three groups of subjects representing different socioeconomic conditions were studied. Analytical quality control was a primary consideration throughout. The analytical techniques used were atomic absorption spectrophotometry, atomic emission spectrometry with an inductively coupled plasma, colorimetry, electrochemistry, using an ion-selective electrode and neutron activation analysis. The differences between median concentrations of Ca, Cl, Mg, K, Na, and P (minor elements) were lower than 20% among the six countries. Among trace elements, concentrations observed in Filipino milk for As, Cd, Co, Cr, Cu, F, Fe, Mn, Mo, Ni, Pb, Sb, Se, and V were higher than for milk samples from other countries. The remaining five countries showed a mixed picture of high and low values. In the case of at least some elements, such as, F, I, Hg, Mn, Pb, and Se, the environment appears to play a major role in determining their concentrations in human milk. The nutritional status of the mother, as reflected by her socioeconomic status, does not appear to influence significantly the breast milk concentrations of minor and trace elements. Significant differences exist between the actual daily intakes observed in this study and current dietary recommendations made by, for example, WHO and the US National Academy of Sciences. These differences are particularly large (an order of magnitude or more!) for Cr, F, Fe, Mn, and Mo; for other elements, such as, Ca, Cu, Mg, P, and Zn, they amount to at least a factor 2. In the opinion of the present authors, these findings point to the need for a possible reassessment of the dietary requirements of young infants with respect to minor and trace elements, particularly for the elements Ca, Cr, Cu, F, Fe, Mg, Mn, Mo, P, and Zn. PMID- 1711363 TI - Vasculature as a target for anti-cancer therapy. PMID- 1711365 TI - Cholera in 1990. PMID- 1711364 TI - Structure of solid tumors and their vasculature: implications for therapy with monoclonal antibodies. AB - Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternative approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. PMID- 1711366 TI - Bladder cancer chemotherapy: is response of squamous tumours different to that of transitional cell tumours? PMID- 1711367 TI - Modelling of protein adsorption on polymeric surfaces. AB - A new method of calculation of protein adsorption on polymeric surfaces is presented. The method considers the thermodynamic equilibrium of a three component system--water, protein, and polymer surface--and describes the protein concentration profile based on the interaction energy parameters for protein polymer, water-polymer, and protein-water contacts. Calculation of these parameters calls for introduction of the energies arising from the dispersive forces, the hydrophobic effect, and the Drago et al. (1971) electron donor acceptor interactions. Comparison with experimental results of protein adsorption on various polymeric surfaces gave satisfactory agreement. PMID- 1711368 TI - HIV reverse transcriptase structure-function relationships. AB - HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented. PMID- 1711369 TI - Thermodynamic study of internal loops in oligoribonucleotides: symmetric loops are more stable than asymmetric loops. AB - Thermodynamic parameters for internal loops of unpaired adenosines in oligoribonucleotides have been measured by optical melting studies. Comparisons are made between helices containing symmetric and asymmetric loops. Asymmetric loops destabilize a helix more than symmetric loops. The differences in free energy between symmetric and asymmetric loops are roughly half the magnitude suggested from a study of parameters required to give accurate predictions of RNA secondary structure [Papanicolaou, C., Gouy, M., & Ninio, J. (1984) Nucleic Acids Res. 12, 31-44]. Circular dichroism spectra indicate no major structural difference between helices containing symmetric and asymmetric loops. The measured sequence dependence of internal loop stability is not consistent with approximations used in current algorithms for predicting RNA secondary structure. PMID- 1711370 TI - Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase. AB - We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro 76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively. PMID- 1711371 TI - Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes. AB - The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions. PMID- 1711372 TI - Analysis of streptomycin-resistance of Escherichia coli mutants. AB - We previously reported about Escherichia coli transformation experiments yielding streptomycin-resistant cells carrying a C912 to T transition in a plasmid-born 16S rRNA gene. These experiments were based on results obtained with streptomycin resistant Euglena chloroplasts bearing an equivalent mutation in the single chloroplast 16S rRNA gene. We extended this study and transformed E. coli with plasmid constructs having a mutated 16S rRNA gene at position 914 (A to C) or a double mutation at positions 912 and 888 (C to T:G to A) or a mutation in the S12 gene (Lys-42 to Thr). We tested the transformed cells before and after a screening procedure in the presence of streptomycin. We find that the plasmid born mutations protect colonies against a short streptomycin exposure, but ribosomes carrying mutated 16S rRNA do not significantly reduce codon misreading in vitro. However, ribosomes isolated from transformed cells after the screening procedure resist misreading. These ribosomes have acquired a second mutation in the S12 protein as shown in one case by sequencing and by transformation experiments. Furthermore, we show that the A914 to C mutation prevents (strongly reduces) base methylation in the central domain of 16S rRNA. PMID- 1711373 TI - Molecular cloning, structure and expression analysis of a full-length mouse brain glutamate dehydrogenase cDNA. AB - We isolated and analysed a full-length mouse brain glutamate dehydrogenase (GLUD) cDNA as a preliminary step to use the mouse model for the investigation of GLUD function in neurotransmission and neurodegeneration. GLUD coding sequences were found highly conserved among mouse, human and rat. Northern blots revealed two transcripts with different ratios in different mouse organs implying some mechanism of tissue-specific expression. In contrast to human, mouse GLUD gene family appears not to contain an intronless member. PMID- 1711374 TI - Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts. AB - A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis. PMID- 1711375 TI - Acid depurination after field inversion agarose gel electrophoresis reduces transfer of large DNA molecules. AB - Field-inversion gel electrophoresis (FIGE) is an economical method for the resolution of DNA fragments 20 to 1000 kilobase pairs (kbp) in length, sizes beyond the resolving capabilities of normal agarose gel electrophoresis. A theoretical limitation to FIGE is the subsequent transfer of large DNA molecules to membrane supports for hybridization. After normal electrophoresis, uniform and rapid capillary transfer of DNA fragments from agarose gels has been previously reported to be facilitated by brief depurination of DNA with 0.25 N HCl. However, after FIGE we have found that brief treatment of large (greater than 200 kbp) linear DNA fragments with 0.25 N HCl reduces the extent of subsequent transfer by capillary methods. After FIGE and transfer, ethidium bromide staining of DNA suggests that acid treatment causes trapping of DNA within the agarose matrix. PMID- 1711376 TI - Effects of inhibitors on proteinase activities in gels following isoelectric focussing. AB - Homogenates of rat submandibular glands were electrophoresed on isoelectric focussing gels and bands of proteinase activity demonstrated by means of overlay membranes impregnated with fluorogenic oligopeptide substrates. Inhibition characteristics of individual proteinase bands were tested within the gels and after excision and subsequent elution from the gels. Some bands of activity were resistant to inhibition by pre-incubation of the gel with certain inhibitors, but were subsequently found to be inhibited when tested with the same substrate after elution. These inconsistencies were influenced by the use of different substrates. The results indicate that, though some useful screening information may be obtained by applying inhibitors to the gels, this system does not permit a reliable assessment of inhibition characteristics which require subsequent elution and assay. PMID- 1711377 TI - Antibody conjugates with morpholinodoxorubicin and acid-cleavable linkers. AB - Antibody-morpholinodoxorubicin conjugates were prepared for targeted immunotherapy of human melanoma. Spacer molecules that differ in hydrolytic stability were employed between the C-13 of the drug and amino residue of lysine on the monoclonal antibody. Antibody-drug conjugates were made with five structurally different morpholinodoxorubicin derivatives including oxime, phenylhydrazone, (sulfonylphenyl)hydrazone, and acylhydrazone moieties. Hydrolytic stability of the antibody conjugates directly correlated with their in vitro cytotoxicity against melanoma cells. Derivatives or conjugates with the greatest hydrolytic stability showed the least cytotoxicity. PMID- 1711378 TI - Improved detection of cancer of the body or tail of the pancreas. AB - Ultrasonography (US), computed axial tomography (CT) and endoscopic retrograde cholangiopancreatography (ERCP) have not improved detection or prognosis of carcinoma of the pancreatic head. We investigated the influence of these imaging techniques on detection, and consequently prognosis of carcinoma of the pancreatic body or tail, where the symptoms are less specific (seldom jaundice or vomiting) and imaging techniques may be more important. Of 139 patients, 29 were treated in 1972-1977, when US, CT and ERCP were not used, 27 in 1978-1980, when US was occasionally performed, and 83 in 1981-1989, when all three methods were common. In 1978-1980 and 1980-1981 correct ante-mortem diagnosis was more common than in 1972-1977, and the diameter and stage of tumour were significantly reduced at laparotomy. The resectability rate was not increased, however, and the incidence of exploratory laparotomy was not reduced. The survival time in the last study period was significantly longer only in the non-operatively treated patients. The reason was not earlier diagnosis, but possibly better general management. PMID- 1711379 TI - Palliation of colorectal carcinoma with the Nd-YAG laser. AB - The Nd-YAG laser used for endoscopic palliation of carcinoma of the rectum (n = 16) or the sigmoid colon (n = 5). Indications were cardiorespiratory insufficiency, advanced disease, severe concomitant illness or patient's refusal of surgery. The treatment was repeated every 2-3 (range 1-5) months, with about half the sessions on an out-patient basis. Symptomatic relief was excellent in 16 of 20 patients, moderate in three and poor in one case (1 patient was asymptomatic). The treatment was effective against bleeding, mucous discharge and signs of intestinal obstruction, but not against incontinence. In the absence of incontinence, palliation was excellent in patients with noncircumferential tumours and in four of the five with circumferential growth. In the latter group frequent treatment sessions were required for long tumours. There were no major complications. Endoscopic laser therapy is concluded to provide rapid, safe and excellent control of local symptoms in most patients with inoperable colorectal carcinoma, to be less useful when the tumour is large and circumferential and not effective in patients with incontinence. PMID- 1711380 TI - [The effect of substance P on the individual and group behaviors of rhesus monkeys]. AB - The tendencies in the substance P influence on the expression of individual and group behaviour of adult males of rhesus macaques have been studied on the background of cardiopathogenic emotional stress (CES) and without it. CES stimulated the general activity of rhesus macaques in the individual cages, while the substance P injection without CES increased the frequency and duration of pathologic behavioral patterns. The maximal influence of substance P was expressed on the 3-4 day of experiments. On the 5th day the intensification of locomotion and social activity was found during the settling of all individuals in the large cage. The definite regularity of connection between the value of arterial pressure before and after experiments and the individual's social ranks is found. The injection of substance P on the CES background and without it the second-ranking individuals stood more hard, the leader and the most subordinated individuals stood easier. PMID- 1711381 TI - Microinjection of apomorphine into the prefrontal cortex of the rat reduces dopamine metabolite concentrations in microdialysate from the caudate nucleus. PMID- 1711382 TI - The effects of typical and atypical neuroleptics on mitogen-induced T lymphocyte responsiveness. PMID- 1711383 TI - [Participation of the prostacyclin-thromboxane system in the mechanisms of prevention of arrhythmia caused by occlusion of the coronary artery in adapted rats]. AB - It was found, that adaptation of rats to cold and physical exercise prevented ventricular fibrillation, caused by the occlusion of the left anterior coronary artery. An adaptation to cold only or to physical exercise do not prevent ventricular arrhythmias. An significant increase of prostacyclin/thromboxane index in plasma and heats was estimated in rats adapted to cold and physical exercise in relation to control non-adapted group in condition of functional rest or acute myocardial ischemia. It was assumed that an increase of prostacyclin/thromboxane ratio has a significant role in antiarrhythmic action of adaptation. PMID- 1711384 TI - [Identification, isolation and physico-chemical properties of neurospecific alpha2-globulin]. AB - In the immunization process of rabbits with the protein fraction of the water salt extract from human brain partially and concentrated at 500-600 times was received antiserum revealed brain specific alpha 2-globulin that is not identical to the known cytoplasmatic brain specific antigens. This antigen has got electrophoretic mobility of alpha 2-globulins, molecular weight 90 +/- 10 kD and isoelectric point 4.1-5.4. Develop the procedure for purification of this antigen on the basis of the combination ion change, affinity, hydrophobic chromatography gel-filtration and isochromatofocusing. PMID- 1711385 TI - [A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]. AB - Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids. PMID- 1711386 TI - [Keratin phenotype of the human thymus gland epithelium in the pre- and postnatal periods]. PMID- 1711387 TI - [The inhibition by strongly acidic polypeptides of the repair of double-stranded DNA breaks induced by the gamma irradiation of Chinese hamster fibroblast cells]. AB - The ability of polypeptides consisted of aspartic and glutamic acids to inhibit the repair and to promote the formation of unrepaired double-strand DNA breaks and chromosomal aberrations in gamma-ray induced Chinese hamster cells was shown. A complete inhibition of the double-strand DNA breaks repair was observed at the concentrations of 20 mu M/l (polyglutamic acid with molecular weight 2000-15,000 daltons) and 100 mu M/l (aspartylglutamic acid with molecular weight 1500-4500 daltons). Both polypeptides were low toxic at the given concentrations. PMID- 1711388 TI - [The structure and physicochemical properties of human hyaline cartilage in the disorganization of the carbohydrate-protein complexes of the ground substance]. AB - In the experiments in vitro we studied the influence of the process of disorganization of the carbohydrate-protein complexes of the ground substance on the structure, water content and biomechanical properties of the human hyaline cartilage. It was shown that the disorganization process of the cartilage ground substance and the subsequent removal of the formed products resulted in the increasing porosity of the cartilaginous tissue. This is expressed in the exposure of the fibrillar frame of the cartilage and formation of cavities of various volumes between its elements. The mentioned changes of the cartilage structure are followed by the reduction of the amount of monomolecular-bound water and simultaneous increase in swelling in water and water vapor sorption at maximal relative humidity. The removal of about 25% of glycosaminoglycanes of the ground substance resulted in the reduction of the rigidity of the cartilagenous tissue, and the increase in the residual deformation. The examination of the hyaline cartilage did not reveal any interrelation between the contents of proteoglycanes and the water content of the cartilagenous tissue. PMID- 1711389 TI - [Enhancement of the resistance of the escape reaction to ethanol after substance P]. AB - An ability of substance P (30 micrograms/kg intravenously) to prevent deleterious effects of ethanol (E) (0.5 g/kg intravenously) on central mechanisms of escape reaction elicited by threshold electrical stimulation of the ventromedial hypothalamus was investigated in chronic experiments upon rabbits. Substance P was found to prevent E effects on excitability of the ventromedial hypothalamus (VMH) and on facilitatory influences of the midbrain reticular formation on this emotional centre which were observed in intact animals. Inhibitory effects of the dorsal hippocampus on the VMH could not be evaluated due to its alterations in response to previous substance P administration. The authors suggest that substance P can be considered to be a possible endogenous factor to increase a tolerance of emotional behavioural reactions of an organism to alcohol. PMID- 1711390 TI - AIDS in the Americas. PMID- 1711391 TI - Electrophysiological and morphological properties of rat motor cortex neurons in vivo. AB - This paper describes results obtained from intracellular recordings and stainings of motor cortex neurons in the rat in vivo. Rats were anesthetized with phenobarbital. Neurons were intracellularly recorded with micropipettes filled with K+-methylsulphate + 4% HRP in phosphate buffer (pH 7.4). Successful recordings and stainings were obtained from 31 neurons. Intracellular recordings were distinguished as either intrasomatic or intradendritic. Action potentials (APs) recorded from somata were distinguished by their fast hyperpolarizing afterpotential from those recorded within dendrites. Dendritic APs were broader and often followed by an afterdepolarization. The firing patterns elicited by depolarizing current pulses allowed to distinguish 3 groups of neurons. (a) Group A neurons with a moderate firing-rate of up to 17 APs during a 100 ms depolarizing current pulse of 3.5 nA comprised small and large pyramidal cells and one aspiny multipolar neuron, probably a large basket neuron. (b) Group B neurons generated bursts, which either occurred spontaneously or during low intensity current injection. These neurons were classified as small pyramidal neurons and spiny star cells. (c) Group C neurons had a firing rate 3 times as high as group A neurons. These neurons were small aspiny cells with radial dendritic fields, which were classified as local interneurons. Intradendritic recordings were characterized by the occurrence of broad APs, most likely generated within the dendritic tree. Intracellular current injections produced burst-like potentials consisting of several APs with different amplitude and duration. In 3 penetrations of one apical dendrite up to 4 neurons were stained. In these recordings APs activated by intracellular current injection were particularly broad (up to 40 ms). The results suggest that neuronal firing patterns observed in in-vitro neocortical slices are also observed in in-vivo conditions. PMID- 1711392 TI - Phylogeny of tachykinin receptor localization in the vertebrate central nervous system: apparent absence of neurokinin-2 and neurokinin-3 binding sites in the human brain. AB - Binding of [125I]Bolton-Hunter labeled tachykinins substance P (BHSP), neurokinin A (BHNKA) and eledoisin (BHELE) to brain sections from several vertebrates was investigated by receptor autoradiography. Densities of BHSP binding sites were low in fish brain, increased in lower vertebrates, were high in birds and rodents, and relatively constant in cat, monkey and human. In contrast, BHELE binding site densities were moderate in fish brain and high in frog, snake, chick, pigeon, mouse and rat brain. Low and very low densities were localized in guinea pig and cat, while no significant BHELE specific binding was found in monkey and human brain. BHSP and BHELE binding sites were distinctly distributed in the vertebrate brains analyzed. Each ligand showed a characteristic regional distribution which was similar from species to species. The affinity profiles of tachykinins for BHSP and BHELE binding sites as analyzed on frog, chick and rat brain sections, corresponded to the NK1 and NK3 receptor types, respectively. No BHNKA binding sites could be detected in any vertebrate brain investigated. In conclusion, marked species variations exist in the density and distribution of tachykinin receptor types in the vertebrate brain. Thus, neurokinin A receptors (NK2 type) seem to be absent in the vertebrate central nervous system and, while substance P receptors (NK1 type) appear to be preserved and increase in density during evolution, the contrary seems to happen for the eledoisin receptors (NK3 type) which are more abundant in lower vertebrates and apparently absent in primate, particularly human brain. PMID- 1711393 TI - Projections of the paraventricular nucleus of the hypothalamus to the sexually dimorphic lumbosacral region of the spinal cord. AB - Lumbar regions L5-L6 of the spinal cord of the male rat contain the sexually dimorphic motor nuclei, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), which innervate perineal muscles, the bulbocavernosus and the ischiocavernosus, respectively. This neuromuscular system controls penile reflexes which are essential to male reproductive success. Oxytocin has been shown to induce penile reflexes and the site of action for these effects is the PVN. Since PVN is known to project to cervical and thoracic levels of spinal cord, the present study examined projections of the PVN to the L5-L6 region of the spinal cord. WGA-HRP was injected into the region of L5-L6, aimed at the SNB and DLN and their dendritic extents, in intact male, castrated male and female rats. WGA-HRP-labelled cells bodies were found in the parvocellular subnuclei of PVN, as well as regions of the lateral hypothalamus and the dorsal area of the hypothalamus. These results demonstrate that the PVN projects to lumbar levels of the spinal cord that are sexually dimorphic and androgen-dependent. This suggests that PVN may modulate the activity of these motoneurons. PMID- 1711394 TI - Projections of neurons in the ventromedial medulla to pontine catecholamine cell groups involved in the modulation of nociception. AB - Stimulation of neurons in the nucleus raphe magnus (RMg) or the adjacent gigantocellular nucleus pars alpha (Gi alpha) and paragigantocellular nucleus (PGi) produces antinociception which is partially mediated by bulbospinal noradrenergic neurons. Since no norepinephrine-containing neurons are located in either the RMg or the Gi alpha/PGi, it is likely that neurons located in these nuclei have axonal connections with the spinally-projecting catecholamine neurons located in the A5, A6 (locus coeruleus), or A7 catecholamine cell groups. To provide evidence for such connections, the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was injected into the RMg or Gi alpha/PGi and labeled axons were identified near catecholamine-containing neurons labeled with dopamine beta-hydroxylase-immunoreactivity (D beta H-ir). A dense field of PHA-L-positive terminals was seen within the A7 cell group which was mainly ipsilateral to PHA-L injections made into either the RMg or the Gi alpha/PGi. Many PHA-L-positive terminals were closely apposed to D beta H-ir A7 perikarya or proximal dendrites. A modest number of terminals was seen within the A5 and LC cell groups. In the second experiment, a unilateral injection of the retrograde tracer, Fluoro-Gold, was made into the A7 cell group and brainstem sections were processed for serotonin (5-HT) immunocytochemistry. Many neurons retrogradely labeled with Fluoro-Gold were seen in the RMg, but a much larger number were found in the Gi alpha/PGi. Less than 5% of these Fluoro-Gold-labeled cells contained 5-HT immunoreactivity. The results of these experiments indicate that the RMg and Gi alpha/PGi have a substantial population of non-serotonergic neurons which project to the A7 noradrenergic cell group. PMID- 1711395 TI - Regional effects of ceruletide, a cholecystokinin-8 analog, on the striatal monoaminergic systems in food-deprived mice. AB - To clarify the pharmacological mechanism by which ceruletide affects involuntary movements, we used 20-h food-deprived mice to examine the acute effects of ceruletide (600 micrograms/kg, i.p.) on the histochemistry of striatal dopaminergic and serotonergic systems. The latter was unchanged but a reduction in catecholamine fluorescence was seen which was restricted to the ventrolateral (VL) portion of the striatum. Biochemical assays also indicated decreased levels of dopamine (DA) and its metabolites in this restricted region of the striatum with little or no change in noradrenaline, serotonin or their metabolites. In all regions examined, except the dorsomedial part of the mid-striatum, the ratio of dopamine metabolites to dopamine was higher in the ceruletide-treated group than in controls, suggesting increased DA release. Further pharmacological experiments showed that, compared to results in mice receiving only ceruletide: the ceruletide-induced decreases in DA and dihydroxyphenylacetic acid (DOPAC) in VL were less after alpha-methyl-p-tyrosine treatment; ceruletide caused no significant decrease in either DA or DOPAC after pargyline pretreatment, although the low levels of homovanillic acid (HVA) were still further significantly reduced; and the ceruletide-induced decrease of DA was reduced, that of DOPAC was abolished and that of HVA enhanced by nomifensine pretreatment. These results suggested that ceruletide might induce a more rapid degradation of DA in VL and increased efflux of HVA through the blood-brain barrier. This evidence suggests that ceruletide has a regionally specific effect on the striatal dopaminergic system which may relate to the amelioration of involuntary movements. PMID- 1711396 TI - Persistent blockade of potassium-evoked serotonin release from rat frontocortical terminals after fluoxetine administration. AB - We examined 5-HT and 5-HIAA release from frontal cortex evoked by high potassium chloride concentrations in rats pretreated for 3 days with high doses of the 5-HT uptake blocker fluoxetine or of dexfenfluramine, which both releases 5-HT and blocks its reuptake. The standard fluoxetine dose (30 mg/kg i.p.) was about 4 times the drug's ED50 in producing a serotonin-related behavioral effect, anorexia, while the dexfenfluramine dose (7.5 mg/kg i.p.) was about 6 times its ED50. These high doses were chosen in order to elucidate the mechanism by which similar doses of fluoxetine and dexfenfluramine had been found to produce long term changes in serotonin dynamics. Fluoxetine decreased the basal release of both compounds; dexfenfluramine decreased basal 5-HIAA efflux without affecting the release of 5-HT release. Potassium-evoked 5-HT release was unchanged after dexfenfluramine pretreatment but was suppressed by fluoxetine doses as low as 7.5 mg per kg per day. Basal release of 5-HT and 5-HIAA returned to normal after 7 days of fluoxetine pretreatment, but evoked release continued to be suppressed. These data suggest that long-term changes in brain serotonin dynamics after high doses of dexfenfluramine or fluoxetine are related to the drug's mechanisms of action, specifically their blockade of 5-HT reuptake. PMID- 1711397 TI - Inhibition of chronic hindlimb flexion in rat: evidence for mediation by 5 hydroxytryptamine. AB - Prolonged high-intensity stimulation of the rat hindlimb produces a persistent unilateral flexion. 5-Hydroxytryptamine (5-HT) has been implicated in the modulation of spinal cord mechanisms. Electrical stimulation across the upper hindlimb was used to induce a persistent hindlimb flexion. The flexion was measured after stimulation and at 72 h, both before and after spinal transection at T7. Transection of the spinal cord typically resulted in an increase in flexion of 3-5 g (rebound). Pretreatment with para-chlorophenylalanine (pCPA) to deplete 5-HT, or the administration of metergoline, a non-specific 5-HT antagonist, had no significant effect on flexion at 72 h in the intact rat but abolished rebound. The 5-HT1A agonist, (+-)-8-hydroxy-2-(di-N propylamino)tetralin hydrobromide (8-OH-DPAT) and 5-HT1B agonist, m trifluoromethylphenylpiperazine-HCl (TFMPP), had no effect on flexion at 72 h in the intact rat but reduced rebound. The 5-HT2 agonist, 1-(2.5-dimethoxy-4 iodophenyl)-2-aminopropane-HCl (DOI), suppressed post-stimulation flexion and flexion subsequent to spinal section. Furthermore, ketanserin, a 5-HT2 antagonist, restored flexion suppressed by DOI in the acutely spinalized rat. These results suggest that chronic hindlimb flexion is suppressed in the intact rat by descending, serotonergic fibers which exert an effect through spinal 5-HT2 receptors. Moreover, 5-HT1 agonist suppression of rebound implicates these receptors as well in the modulation of chronic hindlimb flexion. PMID- 1711398 TI - Preliminary studies on sensitization of Lewis rats with sulfated glucuronyl paragloboside. AB - A large number of patients with peripheral neuropathy and IgM paraproteinemia have IgM monoclonal antibodies which recognize a carbohydrate determinant shared by myelin-associated glycoprotein (MAG) and sulfated glucuronyl glycolipids (SGGLs). There is considerable evidence that these IgM monoclonal antibodies are responsible for demyelination in this disorder. To study the pathogenic role of SGGLs in this type of neuropathy, we sensitized Lewis rats with sulfated glucuronyl paragloboside (SGPG), a major SGGL. Fifty percent of the animals (8/16) developed neurological symptoms such as mild to moderate distal tail tone loss, with or without abnormal posture, along with development of anti-SGPG antibodies. These antibodies reacted with SGGLs, but not with rat MAG. Morphological studies showed: (1) axonal change in the lateral aspects of the dorsal columns in the spinal cord; and (2) damage to the endothelial cells in the spinal cord which suggested a breakdown of the blood-brain barrier. There was no obvious change in the peripheral nerve. Since no marked cellular infiltration was detected in these lesions, the clinicopathological findings observed could be induced by humoral mechanism, most likely anti-SGPG antibodies. PMID- 1711399 TI - Promotion of neuronal survival in vitro by thermal proteins and poly(dicarboxylic)amino acids. AB - Evaluating molecules for their ability to promote survival and growth of neurons, we tested thermal proteins on cultures of dissociated fetal rat forebrain neurons. (Thermal proteins are polyamino acids formed when mixtures of amino acids with minimal proportions of glutamic or aspartic acid are heated.) Thermal proteins, added to low-density cultures in serum-free medium, stimulated neurite outgrowth and induced the formation of neuronal networks which survived for 6-10 days. Neurons in control cultures failed to grow and degenerated completely within 2-4 days. Effective concentrations (EC50) of thermal proteins ranged from 3 to 100 micrograms/ml. They were equally effective when present in the medium during the culture time or after precoating of the culture dishes. A single preparation which contained only aspartic and glutamic acid was effective, and similar survival promoting actions were then found for polyglutamic acid and mixed polyamino acids containing glutamic or aspartic acid. Thermal proteins and polyglutamic acid acted in a specific manner since, under the same experimental conditions, many control peptides, proteins and growth hormones failed to promote survival of neurons. Furthermore, their effects were antagonized by heparin, but not heparan sulfate nor chondroitin sulfate. These findings suggest that sequences of successive dicarboxylic amino acid residues are able to promote survival and neurite elongation of cultured neurons and that such sequences are responsible for the survival promoting action of thermal proteins. They invite the speculation that sequences of successive dicarboxylic amino acids, while occur in many proteins and show a high degree of evolutionary conservation, may have functional role in molecular recognition processes during neuronal development. PMID- 1711400 TI - Intrathecal galanin blocks the prolonged increase in spinal cord flexor reflex excitability induced by conditioning stimulation of unmyelinated muscle afferents in the rat. AB - The effect of intrathecal (i.t.) galanin (GAL) on the prolonged increase of spinal reflex excitability produced by conditioning stimulation (CS) of unmyelinated muscle afferents was studied in decerebrate, spinalized, unanesthetized rats. A CS train (1 Hz, 20 s) applied to the unmyelinated fibers in the gastrocnemius muscle nerve facilitated the ipsilateral flexor for about 1 h. Pretreatment with GAL (0.1-10 microgram) decreased reflex facilitation induced by the gastrocnemius nerve CS. The present results indicate that GAL is capable of blocking the prolonged increase in spinal cord excitability after stimulation of unmyelinated muscle afferents, possibly by antagonizing the facilitatory effect of tachykinins and calcitonin gene-related peptide released at the intraspinal terminals of these fibers. PMID- 1711401 TI - Electronmicroscopic detection of the axonal coexistence of serotonin and substance P in B1-B2 raphe cells transplanted into the transected spinal cord of adult rats. AB - One week after a complete spinal cord transection at the thoracic (T8) level in adult rats, a suspension of rhombencephalic embryonic (day 14) cells containing the B1-B2 serotonergic groups was injected below the section. After a survival period of one month, the spinal cord was processed for an ultrastructural dual immunocytochemical detection of serotonin (5-HT) and substance P (SP). It was shown by ultrastructural dual immunolabeling that 5-HT and SP coexist in the same axon terminals of transplanted cells. PMID- 1711402 TI - Retinoic acid potentiates the neurotrophic but not the mitogenic action of the class 1 heparin-binding growth factor (HBGF-1). AB - Retinoic acid promotes the neuronal survival properties of the class 1 heparin binding growth factor (HBGF-1) on 8-day-old chick ciliary and sensory neurones. It has little or no effect on the survival promoting properties of class 2 heparin-binding growth factor (HBGF-2) on these cell types. Nerve growth factor (NGF) promoted survival of sympathetic and sensory neurones, and was unaffected at any concentration of retinoic acid. The mitogenic effect of both HBGF-1 and HBGF-2 was unaltered at any concentration of retinoic acid. It is suggested that naturally occurring gradients of retinoic acid, for example, as occur in the developing limb bud, could have a role in the development of normal innervation patterns through selective interaction with neurotrophic molecules. PMID- 1711403 TI - Reciprocal connections between the 'nucleus rotundus' and the dorsal lateral telencephalon in the weakly electric fish Gnathonemus petersii. AB - Injections of horseradish peroxidase into the dorsal lateral telencephalon of the weakly electric mormyrid fish Gnathonemus petersii resulted in retrogradely filled cells and anterogradely labelled terminals in the 'nucleus rotundus' of the ipsilateral rostral diencephalon. This connection courses via the lateral part of the lateral forebrain bundle. The present results suggest a particularly close relationship between the Dla cell group of the lateral telencephalon and the 'nucleus rotundus'. PMID- 1711404 TI - Descending adrenergic input to the primate spinal cord and its possible role in modulation of spinothalamic cells. AB - The present study focuses on 3 different aspects of the descending adrenergic system in the primate: (1) the distribution of adrenergic fibers and terminals in the spinal cord, (2) the source of this input and (3) the possible physiological effects of this system on spinal nociceptive processing. Antibodies to the enzyme phenylethanolamine-N-methyltransferase (PNMT) were employed to map the distribution of epinephrine-containing axonal profiles in the primate spinal cord. Smooth longitudinally oriented fibers were localized to the outer edge of the lateral funiculus. PNMT-containing axonal enlargements were distributed to the superficial dorsal horn, intermediate gray matter and the region surrounding the central canal at all spinal cord levels. PNMT-immunostained profiles were also observed in the intermediolateral cell column. A double labeling study employing retrograde transport of HRP from the spinal cord and PNMT immunohistochemistry identified a small population of HRP-PNMT-labeled neurons in the 'C1' region at the levels of the medulla and ponto-medullary junction. Thus, these cells are a probable source of adrenergic input to the spinal cord. Electrophysiological studies demonstrated that iontophoresis of epinephrine onto identified primate spinothalamic tract neurons in the lumbar dorsal horn resulted in inhibition of the glutamate-induced firing of these cells. The data from these studies support the hypothesis that adrenergic (PNMT-containing) cells in the caudal brainstem project to all levels of the cord and may contribute to descending modulation of nociceptive processing at these levels. PMID- 1711405 TI - [Comparative study in aged rats of behavioral deficiencies and morphometrical modifications of galanin containing neurons in the medial septal area]. AB - Age-related alterations in cued-training and place-training tasks were evaluated and compared to alterations in the galanin-like (GAL-LI) immunoreactive neurons in the medial septal area of the rat. Young adult (4 months) and aged (25-26 months) male Sprague-Dawley rats performed a modified version of the Morris water maze task. Immunohistochemical and morphometric techniques were used to analyse the distribution, density and morphology of GAL-LI neurons in the medial septum diagonal band of Broca (MS-DBB) complex. A large variability in performance of the aged rats was evident, with the majority of aged rats exhibiting impaired performance on both parts of the task as compared to the young rats. In addition, there was a significant loss of GAL-LI cells in the MS-DBB complex, a significant reduction of the immunolabeling intensity of the subsisting GAL-LI cells and a non-statistically significant loss of septo-hippocampal GAL-LI neurons. There were no statistically significant correlations between the behavioral performances and the quantitative and qualitative modifications of GAL-LI cell bodies. PMID- 1711406 TI - Determinants of atrial natriuretic factor secretion in dogs expanded with isotonic saline or colloid solutions. AB - Through increments in blood volume and atrial pressure are thought to be the primary stimuli for ANF secretion, plasma levels of this peptide do not always behave as a simple function of volume status. To outline the relationship between the latter and cardiac ANF release, we used five different volume-expansion protocols in anesthetized dogs. A stepwise expansion of plasma volume (PV) was achieved by two consecutive infusions: 0.9% saline followed or preceded by 4 or 25% bovine serum albumin (BSA), 4 or 25% dextran (Dx), or homologous plasma. Saline expansion led to a two- to four-fold increase in arterial plasma ANF level in all five protocols. Both 4 and 25% BSA caused no or very modest increase in plasma ANF, while all other colloid expanders caused the expected ANF release. In all protocols, plasma ANF closely correlated with central venous pressure (CVP). BSA expansion was the only protocol with no correlation between PV and ANF release. Changes in serum Ca2+ could not explain this finding. During BSA expansion, the lack of atrial response was related to the absence of increment (or even fall) in CVP despite the expanded PV. Similarly, urinary Na+ excretion was correlated both with CVP and ANF level but not with PV in BSA expansion. When the dogs were depleted of histamine before BSA infusion, the atrial secretory response was restored, suggesting that this colloid was associated with augmented capillary leakiness and vascular fluid efflux. These results show that the expansion of PV leads neither to ANF release nor to Na+ excretion if it is not accompanied by an expanded central blood volume with elevated atrial pressure. PMID- 1711407 TI - Effect of vasopressin on myocardial capillary recruitment and coronary blood flow in the anesthetized rabbit. AB - The effects of infusion of arginine vasopressin (20 mU.kg-1.min-1) on coronary blood flow and the proportion of the coronary microvasculature perfused was studied in rabbit myocardium. Fluorescein isothiocyanate--dextran was injected into anesthetized open-chest rabbits to identify the perfused vessels and an alkaline phosphatase stain was employed to locate the total microvasculature. Coronary blood flow (radioactive microspheres) was studied in separate groups of rabbits. Vasopressin infusion caused bradycardia (243 +/- 19 to 165 +/- 22 beats/min, mean +/- SD) and an increase in mean blood pressure (92 +/- 18 to 104 +/- 12 mmHg) (1 mmHg = 133.32 Pa). Coronary blood flow decreased significantly with vasopressin from 209 +/- 68 to 97 +/- 36 mL.min-1.100 g-1. The proportion of the arteriolar bed per millimeter squared perfused decreased significantly after vasopressin from 54 +/- 13 to 44 +/- 21%, while the percentage of capillaries per millimeter squared increased significantly from 57 +/- 6 to 67 +/- 11%. There were no subepicardial versus subendocardial differences in any measured parameter. Thus, both coronary blood flow and the proportion of the arteriolar bed perfused decreased with vasopressin. However, compensation occurred in that the proportion of capillaries perfused increased. This indicated an independent level of control of the coronary arteriolar and capillary beds. These microvascular changes may help to maintain oxygen supply-demand balance with vasopressin in the heart. PMID- 1711408 TI - Hypothyroidism increases serotonin turnover and sympathetic activity in the adult rat. AB - Adult, male Sprague-Dawley rats underwent surgical thyroidectomy (Tx) or sham surgery. In all three experiments from which data are reported, a 3-week recovery period was allowed. In experiments I and II, baseline measurements of colonic temperature (Tc) and urinary norepinephrine excretion (NE) were obtained, and both variables were monitored daily for the duration of the studies. After baseline measurements, half of each surgical group was given either triiodothyronine (T3) or vehicle injections subcutaneously; in experiment I replacements continued for 1.5 days, while in experiment II T3 replacement continued for 3.5 days. Rats were decapitated at the end of each experiment and serotonin (5-HT) turnover was measured in brainstem. Serotonin turnover in rostral and caudal brainstem was increased with Tx (p less than 0.05). Increased turnover in caudal brainstem was normalized by T3 only in experiment II. Similarly, decreased Tc and elevated NE with Tx were normalized in experiment II but not in experiment I. In experiment III, NE measurements normalized on a creatinine excretion basis indicated that increased NE is evident with Tx, irrespective of normalization procedure. Significant correlations between 5-HT in caudal brainstem and metabolic correlates of sympathetic function, concurrent normalization of NE and 5-HT in caudal brainstem, plus work from other laboratories describing sympathoexcitatory serotonergic neurons located in the caudal brainstem suggest that the central and peripheral changes in the hypothyroid rat are causally related. PMID- 1711409 TI - Inhibition of influenza A virus hemagglutinin and induction of interferon by synthetic sialylated glycoconjugates. AB - Multivalent forms of neoglycoproteins and polyacrylamides containing sialic acid were prepared and shown to be potent inhibitors of influenza A virus (H3N2) hemagglutinin with chick red blood cells. The synthetic sialylated glycoconjugates, although they were neuraminidase substrates, did not suppress viral neuraminidase and did not reduce infectivities in chick embryos. The copolyacrylamide conjugate containing a spacer group of approximately 11 A (1 A = 0.1 nm) between the polymer backbone and the sialic acid residues was the best hemagglutinin inhibitor. Moreover, it exhibited promising interferon-inducing properties. PMID- 1711410 TI - Supplemental calcium suppresses colonic mucosal ornithine decarboxylase activity in elderly patients with adenomatous polyps. AB - Epidemiological and animal studies suggest a role for calcium in the chemoprevention of colorectal neoplasia. This study was designed to investigate whether supplemental oral calcium has a suppressant effect on colonic mucosal ornithine decarboxylase (ODC) and tyrosine kinase activities in patients with adenomatous polyps or a history of adenomatous polyps and whether this is affected by age. ODC and tyrosine kinase activities were measured in rectal mucosal biopsies of 19 male patients (age, years 46-85 years; mean, 66 years) with adenomatous polyps or a history of adenomatous polyps before and after 1 week of calcium supplementation p.o. (CaCO3; 2500 mg/day) and 2 weeks after cessation of calcium treatment. The basal rectal mucosal ODC activity of patients greater than or equal to 64 years old was nearly 4-fold higher than that of patients less than 64 years old (P less than 0.005). In patients greater than or equal to 64 years old, there was a significant decrease in rectal mucosal ODC activity following 1 week of calcium p.o. compared to those age less than 64 years (P less than 0.05). Overall tyrosine kinase activity did not differ significantly in either patient group before or after calcium supplementation p.o. However, the concentration of phosphotyrosine membrane proteins with molecular weights between 40,000 and 60,000 and between 80,000 and 100,000 were suppressed in patients age greater than or equal to 64 years after 1 week of calcium treatment p.o. These patients also had a corresponding decrease in their rectal mucosal ODC activity. Alternatively, patients whose ODC was not affected by calcium showed no apparent change in the relative concentration of rectal mucosal phosphotyrosine membrane proteins. Our data indicate that there is an age related increase in basal rectal mucosal ODC activity in patients with adenomatous polyps which can be suppressed with calcium supplementation p.o., suggesting a role for dietary calcium in the chemoprevention of colorectal neoplasia. PMID- 1711411 TI - Peripheral hormone levels in controls and patients with prostatic cancer or benign prostatic hyperplasia: results from the Dutch-Japanese case-control study. AB - The possible relationship between changes in peripheral hormone levels and the occurrence of prostatic pathology was studied in a case-control study, involving estimation of various plasma hormones in 368 Dutch and 258 Japanese men, who were grouped as controls and patients with benign prostatic hyperplasia, focal prostatic carcinoma, or clinically evident prostatic carcinoma. Results of a number of previous, smaller studies concerning interrelationships between hormone levels in elderly men were confirmed within the Dutch and Japanese groups. Plasma levels of testosterone and estradiol were significantly lower in the Japanese men, when compared with those in Dutch men. Probably as a result of this difference in testosterone levels, the ratio between serum levels of testosterone and sex hormone-binding globulin (SHBG) was decreased in the Japanese men, while the ratio between the concentrations of dihydrotestosterone and testosterone was increased. These differences were also found when results from Japanese subgroups (controls and patients with prostate pathology) were compared with those from the Dutch subgroups. There were no significant differences in plasma androgen levels between Japanese or Dutch prostate cancer cases and their respective control subgroups. These findings do not support a correlation between the lower plasma testosterone levels and a lower incidence of prostate cancer in the Japanese men. Furthermore, no significant differences were found between salivary levels of testosterone or the ratio between testosterone and SHBG in the various Dutch subgroups. In Japanese benign prostatic hyperplasia patients, the testosterone to SHBG ratio was significantly increased. In conclusion, the results of this retrospective, cross-sectional study do not indicate that hormonal levels play a primary role in the origin or promotion of prostatic abnormalities. The finding of a lower plasma testosterone in the Japanese men, however, remains suggestive, warranting a more extensive prospective study. PMID- 1711412 TI - The presence of alpha 2u-globulin is necessary for d-limonene promotion of male rat kidney tumors. AB - In a 2-year carcinogenesis bioassay, d-limonene (dL) induced kidney tumors in male F344 rats, but not in female F344 rats or either sex of mice, d-Limonene-1,2 oxide, a metabolite of dL, has been shown to bind reversibly the male rat specific urinary protein, alpha2u-globulin (alpha 2u-G), lysosomal degradation than alpha 2u-G alone. This reduced degradation of alpha 2u-G-chemical complex leads to an accumulation of this protein in the proximal convoluted tubules of the male rat kidney and to the morphological changes characteristic for alpha 2u globulin nephropathy. The only male rat strain known to be resistant to this renal disease is the alpha 2u-G deficient NCI-Black-Reiter (NBR) rat. The objectives of this study were to determine whether or not dL causes sustained increases in cell proliferation and has promoting activity for renal adenomas in male rats and if the male rat-specific urinary protein, alpha 2u-G, is required. In a 32-week initiation-promotion assay, male F344 and NBR rats were treated with either 0 or 500 ppm N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water for 2 weeks. Experimental groups of 31 to 38 rats then received 0 or 150 mg d-limonene/kg/day in corn oil for 30 weeks by p.o. gavage 5 days/week. Cell proliferation in the proximal tubules was assessed via 5-bromo-2'-deoxyuridine filled osmotic mini-pumps and immunohistochemistry after 7 weeks (2 weeks EHEN + 5 weeks dL) and at the end of the study (2 weeks EHEN + 30 weeks dL). Preneoplastic and neoplastic lesions were quantified in perfusion-fixed kidneys. A 5-fold increase in the labeling index of P2-cells was found after 5 weeks and 30 weeks of promotion in all dL-treated F344 rats, whereas no difference between treatment groups was detected in NBR rats. No increase in tumors or preneoplastic lesions was detected in dL-treated NBR rats, whereas a 10-fold increase in renal adenomas and atypical hyperplasias was found in the EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil. d-Limonene treatment alone caused a significant increase in the number of atypical tubules and atypical hyperplasias in F344 rats when compared with the F344 vehicle control. On the other hand, a significantly lower incidence of liver tumors was found in EHEN-dL treated F344 rats compared with F344 rats treated with EHEN-corn oil, suggesting a chemopreventative effect of dL on EHEN-induced liver carcinogenesis in F344 rats.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711413 TI - Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. AB - Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3 methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to less than or equal to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G1 and not in the G0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [3H]uridine, [3H]leucine, and initial [3H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase. PMID- 1711414 TI - Multiple neuropeptides stimulate clonal growth of small cell lung cancer: effects of bradykinin, vasopressin, cholecystokinin, galanin, and neurotensin. AB - We tested whether Ca(2+)-mobilizing neuropeptides can function as growth factors for small cell lung carcinoma cells. The neuropeptides bradykinin, neurotensin, cholecystokinin, and vasopressin at nanomolar concentrations stimulated a rapid and transient increase in the intracellular concentration of Ca2+. Crucially, these peptides in the same concentration range also caused a marked increase in colony formation in semisolid medium in responsive small cell lung carcinoma cell lines. At optimal concentrations bradykinin, neurotensin, cholecystokinin, vasopressin, galanin, and gastrin-releasing peptide were equally effective in promoting clonal growth. These findings support the hypothesis that small cell lung carcinoma growth is sustained by an extensive network of autocrine and paracrine interactions involving multiple neuropeptides. PMID- 1711415 TI - A comparison of the immunofluorescent localization of collagen types I, III, and V with the distribution of reticular fibers on the same liver sections of the snow monkey (Macaca fuscata). AB - Localizations of collagen types I, III, and V in monkey liver, as determined by the indirect immunofluorescence method, were photographically superimposed on the fibers revealed by silver-staining in the same tissue sections. Immunofluorescence for type I collagen was found to correspond with the brown collagen fibers and with some of the coarse reticular fibers, while that for type III collagen was found to correspond with most, but not all, reticular fibers of the liver as well as with the brown collagen fibers. The distribution of type V collagen coincides not only with the collagen fibers in the stroma of portal triads and around the central veins, but also with the coarse and fine reticular fibers in the liver lobules. By immuno-electron microscopy, reaction products with anti-type III and V collagens antibodies were demonstrated on cross-striated collagen fibrils, about 45 nm in diameter, in the space of Disse. From these observations, it is concluded that: (1) the fine reticular fibers are mainly composed of type III and type V collagens, and (2) the collagen fibers and coarse reticular fibers in the periphery of liver lobules are composed of type I, type III and type V collagens. PMID- 1711416 TI - An interplexiform cell in the goldfish retina: light-evoked response pattern and intracellular staining with horseradish peroxidase. AB - The light-evoked response pattern and morphology of one interplexiform cell were studied in the goldfish retina by intracellular recording and staining. The membrane potential of the cell spontaneously oscillated in the dark. In response to a brief light stimulus, the membrane potential initially gave a slow transient depolarization. During maintained light, the oscillations showed a tendency to be suppressed; the response of the cell to the offset of the stimulus was not so prominent. The perikaryon of the interplexiform cell was positioned at the proximal boundary of the inner nuclear layer. The cell had two broad layers of dendrites; one was diffuse in the inner plexiform layer, the other was more sparse in the outer plexiform layer. The morphological and electrophysiological characteristics of the cell are discussed in relation to dopaminergic interplexiform cells and the light-evoked release pattern of dopamine in the teleost retina. PMID- 1711417 TI - Actin pegs and ultrastructure of presumed sensory receptors of Beroe (Ctenophora). AB - We have investigated the actin content and ultrastructure of two kinds of presumed sensory projections on the lip epidermis of beroid ctenophores. Transmission electron microscopy showed that conical pegs contain a large bundle of densely packed, parallel microfilaments. Rhodamine-phalloidin brightly stained the pegs, confirming that they contain filamentous actin. Epidermal cells with actin pegs also bear a single long cilium with an onion-root structure, previously described as arising from a different type of cell. The actin peg and onion-root cilium project side-by-side, defining a polarized axis of the cell which is shared by neighboring cells. The onion-root body is surrounded by a flattened membrane sac which lies immediately below the plasma membrane. The perimeter of the membrane sac is encircled by aggregates of dense material. An extra layer of dense material is found along the side of the membrane sac facing the peg; this material often makes direct contact with the adjacent actin filament bundle. Cells with actin pegs and onion-root cilia synapse onto adjacent neurites and secretory gland cells, indicating that one or both types of projections are sensory elements. Since the feeding responses of beroids are reported to depend on chemical and tactile stimuli to the lips, the cells bearing pegs and cilia may function as both mechanoreceptors and chemoreceptors, that is, as double sensory receptors. PMID- 1711419 TI - Palliative dental therapy of postsurgical side effects in trigeminal neuralgia: a case report. PMID- 1711418 TI - Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti). AB - The hagfishes (cyclostomes) are known to secrete copious amounts of mucus mainly by the holocrine mode from the slime glands. Stressed animals release two types of cells (gland thread cells, GTCs; gland mucous cells. GMCs) which rupture on contact with water and rapidly form a mass of viscous mucus. Herein we report some key sequential events of this process and document a novel role for cytoskeletal polymers. After electrostimulation of Pacific hagfish (Eptatretus stouti), the exudate was collected in a stabilization buffer and GTCs segregated from GMC vesicles. Water was added progressively to mixtures of known quantities of these entities. The changing mucous composition and properties were monitored by light- and electron microscopy, viscometry and immunogold assay. Sequentially, the threads uncoil from GTCs, aggregate with the vesicles, the vesicles rupture and release mucin-like substances, at least some of which adhere to the thread. It was found that the intermediate filament (IF)-rich threads markedly facilitate hydration and modulate the viscoelastic and cohesive properties of the resultant mucus. It was speculated that the thread abets localization of mucus in an aqueous environment and promotes adhesion of mucus to surfaces such as the fish integument. As judged by immunostaining in situ, GTCs, as well as several cell types in the epidermis, contain keratin-like components. The role of biopolymers on the properties of teleost and mammalian mucus is discussed. PMID- 1711420 TI - Polymorphic phospholipid phase transitions as tools to understand peptide-lipid interactions. AB - The effect of peptides on bilayer----non-bilayer phase transitions can be used as a tool to investigate the molecular aspects of peptide-lipid interactions. In this contribution the action on membranes of the peptide antibiotic gramicidin A and the bee venom component melittin are compared. Although the known structures and locations of these peptides upon membrane binding are very different, their actions on membranes show striking parallels. A general model is proposed that explains the seemingly complex peptide-lipid interactions by making use of simple concepts. PMID- 1711421 TI - The identification and treatment of motor/learning difficulties: parents' perceptions and the role of the therapist. AB - The diagnostic experience of 31 children, assessed as having motor/learning difficulties, attending an occupational therapy department within a children's hospital, was investigated. The study demonstrates the difficulty of specific identification of perceptuomotor problems. Close examination of the children's individual diagnostic pathways revealed a high number of health-care professional contacts and the fact that frequently there were lengthy gaps between parental (and sometimes professional) suspicions and final confirmation. The children's diagnostic experiences prior to starting treatment were varied and involved, and no one single route was predominant. The rationale for occupational-therapy assessment and treatment of this disorder is described, and parental perceptions of its effect are discussed. The findings suggest that an important part of therapeutic intervention may be increasing children's self-confidence and reducing intra and extra family tensions. Fewer behavioural problems were reported once treatment had commenced. It was concluded that an important part of the therapist's role was to provide information and support for parents and to liaise with school teachers, in addition to treating the children themselves. PMID- 1711422 TI - Diminished tolerance of prehypertrophic, cardiomyopathic Syrian hamster hearts to Ca2+ stresses. AB - Although abnormal myocardial calcium homeostasis in the cardiomyopathic hamster (CMH) has been documented in the hypertrophic stage of the disease, the Ca2+ tolerance before the hypertrophic stage has not been investigated. We studied isovolumic contractile function in response to a variety of Ca2+ stresses including increases in perfusate [Ca2+] (Cao), the Ca2+ channel agonist Bay K 8644, and alpha- or beta-adrenergic agonists of isolated perfused hamster hearts from 24-45-day-old male CMH, BIO 14.6 strain, and age- and sex-matched F1B strain controls. The coronary flow at a constant perfusion pressure did not differ between two groups at baseline or after any Ca2+ stress. At a Cao of 1.0 mM, neither end-diastolic pressure (EDP) nor developed pressure (DP) nor half relaxation time (RT1/2) during stimulation at 1-3 Hz differed between the two groups; as Cao was increased up to 10 mM, CMH hearts showed a lower threshold for the occurrence of a Ca2+ overload profile: EDP and RT1/2 increased to a greater, and DP to a lesser, extent in CMH than in control hearts. To determine whether calcium influx via Ca2+ channels mediates the lower threshold for Ca2+ overload in CMH hearts, we measured resting pressure and scattered laser light intensity fluctuation (SLIF) in unstimulated hearts. Prior studies have shown that SLIF is generated by microscopic tissue motion caused by diastolic spontaneous sarcoplasmic reticulum Ca2+ release and that SLIF amplitude reflects the extent of cell and sarcoplasmic reticulum Ca2+ loading. The Ca(2+)-dependent increase in resting pressure in unstimulated hearts was highly correlated with an increase in SLIF, and this relation was steeper in CMH than in control hearts. CMH hearts also showed a reduced threshold for the occurrence of a Ca2+ overload profile in response to the adrenergic receptor agonists and the Ca2+ channel agonist during electrical stimulation in a Cao of 2.0 mM: maximum DP achieved with each agonist was significantly less and the dose-response curves to each agonist were shifted leftward in CMH versus control hearts. In CMH hearts EDP began to increase at a significantly lower concentration of each agonist, and the maximum extent of increase in EDP in response to all agonists was significantly enhanced compared with control hearts. In response to beta-adrenergic or Ca2+ channel agonists, neither resting pressure nor SLIF in unstimulated hearts increased in control or in CMH hearts. In contrast, in response to alpha-adrenergic stimulation, both SLIF and resting pressure increased to a greater extent in CMH than in control hearts.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711423 TI - Effects of acidic fibroblast growth factor on normal and ischemic myocardium. AB - We sought to determine the effects of acidic fibroblast growth factor (FGF) on ischemic and normal myocardium and to determine whether direct application of acidic FGF to the heart could promote angiogenesis. Eighteen dogs underwent placement of an ameroid constrictor on the left anterior descending coronary artery (LAD). Three weeks later, a left internal mammary artery (IMA) pedicle was positioned over the LAD territory, with a sponge saturated with acidic FGF (n = 12) or saline (n = 4) interposed between the pedicle and the heart. Polytetrafluoroethylene fiber or collagen I sponges were used to deliver the acidic FGF. Weekly angiography of the IMA was performed in all dogs, but significant IMA to coronary collaterals were not demonstrable in any dog. Eight dogs had histological evidence of subendocardial infarction in the LAD territory (five acidic FGF, three control, p = NS). Striking smooth muscle cell hyperplasia was present in arterioles and small arteries exclusively in areas of subendocardial infarction in all of the acidic FGF-treated dogs but in none of the control dogs (p less than 0.05). Noninfarcted myocardium appeared normal in all dogs. In two additional dogs, ameroid constrictors were not placed on the LAD, such that acidic FGF-treated sponges were placed on normally perfused myocardium of the LAD territory. Histological evaluation of those hearts revealed normal myocardium, without evidence of myocardial infarction or smooth muscle cell hyperplasia. Thus, when acidic FGF is delivered to the myocardium via an epicardial sponge in dogs whose coronary flow is compromised, acidic FGF does not cause an angiogenic response in viable myocardium but causes vascular smooth muscle cell hyperplasia in areas subjected to ischemic injury. PMID- 1711424 TI - Alizarin red S staining of synovial fluid in inflammatory joint disorders. AB - In 153 patients with mainly inflammatory joint disorders alizarin red S staining was used to detect calcium-containing crystals in synovial fluid (SF). The reproducibility of the results of this staining technique in 207 SF samples proved to be fairly good (65% concordant results after two observations). Electron-microscope studies confirmed the presence of apatite or calcium pyrophosphate dihydrate (CPPD) crystals in 92% of the alizarin red S positive samples, and in 27% of the weakly positive samples. Based on these figures it can be estimated that approximately 16% of SF samples obtained from patients with inflammatory joint disorders contain apatite and/or CPPD crystals. In contrast with other studies, mainly involving patients with degenerative joint disorders, no correlation was found between alizarin red S staining results and either radiological joint destruction (n = 130) or age. PMID- 1711425 TI - Diversity of the immune response to bovine type II and XI collagens in rats. AB - Immunisation of rats with native type II or type XI collagen produced an inflammatory arthritis in certain strains of rats. Antibodies to the native and denatured whole molecules, to the individual alpha chains and to the cyanogen bromide derived peptides of these chains were studied. Inter- and intra-strain variation in the specificity of the antibodies produced was seen and there were also changes with time, especially with the rats immunised with type XI collagen. Both collagen type-specific and cross-reacting antibodies were produced following immunisation with either type II or type XI collagen. Although no specific pattern of antibodies was unique to the presence or absence of arthritis in all rats, antibodies that bound to the CB-11 peptide or antibodies that bound to the CB-9,7 peptide of type II collagen occurred at the time of onset of arthritis in type II or type XI immunised, arthritic rats. Antibodies to these peptides occur in many patients with rheumatoid arthritis (RA) who also have antibodies to type II collagen. Therefore these findings suggest that epitopes on these peptides may be important in the continued production of antibodies to these collagens in patients with RA and that these antibodies may indeed be pathogenic. PMID- 1711426 TI - Hepatitis C virus antibodies in mixed cryoglobulinemia. PMID- 1711427 TI - Cryoglobulinaemia and hepatitis C virus (HCV) infection. PMID- 1711428 TI - AIDS education for patients with chronic mental illness. AB - Despite the AIDS epidemic's impact, development of prevention and risk-reduction programs has been slow, especially for patients with chronic mental illness. These patients may be at particular risk for HIV transmission and acquisition due to characteristics of their illness. Despite a paucity of such program descriptions in the literature and widespread concern that exposure of such patients to educational material related to sexuality or AIDS would be overstimulating, an effective and safe curriculum to teach risk-reduction can be designed. This paper describes such a program at the Massachusetts Mental Health Center, in Boston. PMID- 1711429 TI - Quantification and partial characterisation of regulatory peptides in the liver fluke, Fasciola hepatica, from different mammalian hosts. AB - 1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP immunoreactivity was detected by RIA of HPLC fractions. PMID- 1711430 TI - Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors MDL12330A and SQ22536. AB - 1. The effects of both adenyl cyclase inhibitors (MDL12330A and SQ22536) have been studied on the ionic transport induced by vasopressin and isoprenaline across the frog skin. 2. MDL12330A inhibits the vasopressin action on the short circuit current (SCC), confirming that this effect is cAMP-mediated. 3. On the other hand, isoprenaline action on the SCC is unaffected by MDL12330A. However, this lack of effect is not a sufficient argument against the role of cAMP in this action; in fact, as MDL12330A is also an inhibitor of cAMP phosphodiesterase, this action could mask the inhibitory effect of the drug on adenyl cyclase. 4. By using the other adenyl cyclase inhibitor (SQ22536), probably deprived of effect on the cAMP phosphodiesterase, we obtained a strong inhibition of isoprenaline action on the SCC. Thus we conclude that the actions of isoprenaline on the ionic transport across the frog skin are also cAMP-mediated. PMID- 1711431 TI - Retrobulbar anesthesia and eyelid closure--effect on corneal angiogenesis. AB - We evaluated the effect of corneal anesthesia produced by retrobulbar injections of bupivicaine and epinephrine on corneal neovascularization in the rat. The eyelids were sutured closed to prevent dessication and ulceration of the insensitive corneas. The amount of corneal neovascularization induced in animals receiving a retrobulbar anesthetic did not differ from those receiving control solutions. However, rats receiving retrobulbar injections exhibited greater corneal neovascularization than those that did not. Rats that had their eyelids sutured or patched closed also had more neovascularization than animals that did not. Likewise, rats that had sutures placed in the upper and lower eyelids, without palpebral immobilization, manifested more neovascularization than rats without sutures. These studies suggest that corneal anesthesia does not affect neovascularization induced by silver/potassium nitrate cauterization and that retrobulbar injections and eyelid closure enhance corneal neovascularization. PMID- 1711432 TI - Hypertonic saline dextran resuscitation during the initial phase of acute endotoxemia: effect on regional blood flow. AB - BACKGROUND AND METHODS: Small-volume resuscitation by means of bolus application of hypertonic saline solutions has been demonstrated to restore central hemodynamics and regional blood flow in severe hemorrhagic and traumatic shock. The aim of this study was to elucidate the potential of this new concept for treatment of profound hypovolemia and microcirculatory deterioration associated with sepsis and endotoxic shock. In a porcine model of acute hyperdynamic endotoxemia (elicited by continuous iv infusion of Salmonella abortus equi endotoxin for 3.5 hrs), small-volume resuscitation applying hypertonic hyperoncotic solutions was analyzed for its effect on central hemodynamics, oxygen delivery (Do2), and regional blood flow. Fluid therapy was initiated when the pulmonary artery occlusion pressure (PAOP) tended to decrease (at 43 to 52 mins of endotoxemia), and consisted of 4 mL/kg bolus infusion of either 7.2% sodium chloride, 10% dextran, or 10% dextran in 7.2% sodium chloride; thereafter, PAOP was maintained by controlled infusion of 6% dextran-60. In a control group, 6% dextran-60 was given without preinjection of hypertonic-hyperoncotic solutions. RESULTS: On small-volume resuscitation, cardiac index significantly increased within 5 mins in all groups, while mean arterial pressure remained unchanged. Fluid requirements were significantly reduced after small-volume resuscitation and the hyperdynamic circulatory state was maintained until the end of the observation period; Do2 as well as blood flow to heart, kidneys, and splanchnic organs remained high. CONCLUSION: Small-volume resuscitation by means of hypertonic saline-dextran proved the most effective, and seems to be an attractive supportive therapy to prevent microcirculatory failure in sepsis and endotoxemia. PMID- 1711433 TI - Stress-induced proteins in immune response to cancer. AB - Chemically induced tumors of inbred mice elicit immunity in animals in which the tumors are induced and in other animals of the same inbred stock. The immunity is specific for each tumor: even two tumors induced in one animal with the same carcinogen are not cross-reactive. Immunity to cancer has since been observed in the case of sarcomas and carcinomas induced by a number of chemical and physical carcinogens and in several species, including mice, rats, and guinea pigs. The nature of molecules which mediate immunity to tumors is a central question in cancer immunology. A small number of such molecules have been biochemically defined. Of these, some are viral antigens expressed in tumor cells, while the relationship of some others to viral antigens is unclear. A surprising majority of nonviral tumor antigens have turned out to bear homology with stress-induced proteins. Four families of such molecules are discussed: the gp96 (hsp100) and p84/86 (hsp90) antigens of chemically induced mouse sarcomas, hsp70 antigens of tumors obtained by transfection of normal rat fetal fibroblasts with an H-ras oncogene, and the albuminoid antigens of murine melanomas and a rat histiocytoma. (Albumin-like antigens are included among the stress-induced proteins because albumin, though constitutively expressed in adult tissues, is heat shock inducible in fetal liver.) Each of these antigens is a moderately abundant protein, present not only in tumors but also in normal tissues. Administration of each of these antigen preparations from the tumor, but not from normal tissue, renders the animal immune to challenge with live cells of the tumor from which the antigens are prepared. And yet, no structural differences in the antigens have been observed between normal tissues and tumors. It is suggested that these stress-induced proteins may not be tumor antigens per se, but may be carriers of immunogenic moieties such as short peptides. The stress-induced proteins may therefore serve either as antigen-presenting molecules like the MHC-encoded molecules or as accessory molecules in the presentation of antigens by MHC molecules. The ability of stress-induced proteins to bind to a variety of molecules, including peptides, is consistent with this possibility. PMID- 1711434 TI - Glial fibrillary acidic protein, J1-31 antigen and vimentin in adult hamster brain: an immunohistochemical study. AB - Different regions of the prosencephalon and mesencephalon of the adult hamster brain displayed differences in the immunofluorescence expression of astrocytic proteins, namely glial fibrillary acidic protein and J1-31 antigen (30 kD protein). Neither of these proteins could be detected in layers II-VI of the cerebral cortex. However, varying degrees of immunostaining were detectable in perivascular glia, stria medullaris thalamus, the basal cerebral peduncle and the dentate molecular layer of the hippocampus. Vimentin was conspicuous in neurons, particularly in the cerebral cortex and hippocampus, and in glial fibrillary acidic protein-positive astrocytes in major fibre tracts. These observations are discussed in relation to interspecies differences in the expression of intermediate filament proteins. PMID- 1711435 TI - Chromosomal localization of the gene for AA-type platelet-derived growth factor receptor (PDGFRA) in humans and mice. AB - The full-length cDNA of the receptor for human AA-type platelet-derived growth factor (PDGF) was used to assign the PDGFRA gene to region q11----q21 of human chromosome 4 and to mouse Chromosome 5 by somatic cell hybrid analysis. Since the same region also contains the c-kit oncogene homolog KIT, we carried out pulsed field gel electrophoresis to determine the physical distance between the two genes in human DNA. The two probes, when successively applied to the same filters, hybridized to a 450-kb EagI-fragment but not to other common restriction fragments. The genes are separated by at least one NotI, one XhoI, and one SalI site. PMID- 1711436 TI - Localization of the gene encoding myelin basic protein to mouse chromosome 18E3-- -4 and rat chromosome 1p11----p12. AB - The location of a gene encoding myelin basic protein in rat (MBP) and mouse (Mbp) was determined by in situ hybridization using the mouse Mbp cDNA labeled with biotin-11-dUTP as a specific probe. The localization of biotin signals in the mouse was found on Chromosome 18E2----3. The result is consistent with the previous report that the Mbp gene is located on the distal half of Chromosome 18. In the rat, the signals localized on chromosome 1p11----p12, suggesting homology between mouse Chromosome 18 and the short arm of rat chromosome 1. PMID- 1711437 TI - The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli. AB - The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis. PMID- 1711438 TI - [Immunoregulation disturbance in experimental allergic neuritis. The cellular and humoral mediated immunological damage]. AB - The cellular and humoral mediated immune responses were investigated in the rabbits with experimental allergic neuritis (EAN) induced by the myelin basic protein (MBP) purified from the human sciatic nerve. The cellular mediated immunity to MBP were assayed by skin testing and lymphocytes transformation. The antibodies to myelin and muscle, the immunoglobulin deposits in the nerve and tissues respectively were detected by indirect and direct immunoenzyme assays. In rabbits with EAN, the skin tests became positive at the onset of EAN. There was a significantly higher index of the lymphocytes transformation in the EAN than in normal animals. At the height of the disease, the anti-muscle antibody titres were high. The granular deposits containing IgG were found in sciatic nerve and heart. The results favour cellular and humoral mediated immunological damage as the most important pathogenetic mechanism in EAN. PMID- 1711439 TI - Use of photodynamic therapy in the palliation of massive advanced rectal cancer. Phase I/II study. AB - Photodynamic therapy (PDT) is a relatively new form of cancer therapy utilizing a photosensitizer such as hematoporphyrin derivative. We conducted a pilot study to determine the efficacy of its use in palliating advanced rectal cancer, to determine toxicity, and to establish objective outcome criteria. Six patients with very advanced, usually recurrent rectal cancer were treated with PDT after being photosensitized with Photofrin II. A protocol was established to measure clinical and radiologic response to therapy. A new intraluminal delivery system was incorporated. Five patients had both clinical and radiologic responses to therapy. In two patients we observed such significant responses that they cannot be accounted for on a photobiologic basis alone. One patient developed a significant sunburn after discharge. There was no major toxicity of bleeding or sepsis even at maximum doses (200 J/cm2). We are confident that PDT has a role to play in rectal cancer and speculate as to future applications. PMID- 1711440 TI - Prophylaxis for genital herpes. Should it be used routinely? PMID- 1711441 TI - Opioid agonist-antagonist drugs in acute and chronic pain states. AB - The agonist-antagonist opioid analgesics are a heterogeneous group of drugs with moderate to strong analgesic activity comparable to that of the pure agonist opioids such as codeine and morphine but with a limited effective dose range. The group includes drugs which act as an agonist or partial agonist at one receptor and an antagonist at another (pentazocine, butorphanol, nalbuphine, dezocine) and drugs acting as a partial agonist at a single receptor (buprenorphine). These drugs can be classified as nalorphine-like or morphine-like. Meptazinol does not fit into either classification and occupies a separate category. Pentazocine, butorphanol and nalbuphine are weak mu-antagonists and kappa-partial-agonists. All three drugs are strong analgesics when given by injection: pentazocine is one sixth to one-third as potent as morphine, nalbuphine is slightly less potent than morphine, and butorphanol is 3.5 to 7 times as potent. The duration of analgesia is similar to that of morphine (3 to 4 hours). Oral pentazocine is closer in analgesic efficacy to aspirin and paracetamol (acetaminophen) than the weak opioid analgesics such as codeine. Neither nalbuphine nor butorphanol is available as an oral formulation. At usual therapeutic doses nalbuphine and butorphanol have respiratory depressant effects equivalent to that of morphine (though the duration of such effects with butorphanol may be longer). Unlike morphine there appears to be a ceiling to both the respiratory depression and the analgesic action. All of these 3 drugs have a lower abuse potential than the pure agonist opioid analgesics such as morphine. However, all have been subject to abuse and misuse, and pentazocine (but not the others) is subject to Controlled Drug restrictions. Buprenorphine is a potent partial agonist at the mu-receptor, and by intramuscular injection is 30 times as potent as morphine. A ceiling to the analgesic effect of buprenorphine has been demonstrated in animals and it is also claimed in humans. However, there are no reliable data available to define the maximal dose of buprenorphine in humans. A practical ceiling exists for sublingual use in that the only available formulation is a 2 micrograms tablet and few patients will accept more than 3 or 4 of these in a single dose. The duration of analgesia is longer than that of morphine, at 6 to 9 hours. There have been suggestions that buprenorphine causes less respiratory depression than morphine, but viewed overall it appears that in equianalgesic doses the 2 drugs have similar respiratory depressant effects.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711442 TI - Cardiovascular therapies in the 1990s. An overview. AB - Cardiovascular medicine has evolved steadily over the past two decades. Inspired by progressive declines in the overall incidence and mortality rates from cardiovascular diseases, emphasis has been placed on 3 specific areas: prevention, early diagnosis, and aggressive intervention. During the decade spanning the 1980s, impressive strides were made in many areas--diagnostic and therapeutic alike. However, an observed reduction in patient mortality stemming from acute myocardial infarction was particularly gratifying. Clearly, the large scale use of thrombolytic therapy and postinfarction strategies designed to prevent reinfarction, limit ventricular dilation, and reduce cardiac death figured prominently. Despite these encouraging facts, however, coronary heart disease remains the leading cause of death in Western society, and thrombolytic therapy is still not being utilised by the medical community to its full potential. Furthermore, adjuvant therapy, during both the early and the late phases of acute myocardial infarction, is being instituted inconsistently, and at times haphazardly. Arrhythmia management and the prevention of sudden cardiac death require further investigation, as does the treatment of chronic congestive heart failure, and the prevention of coronary atherosclerosis. This overview provides a state-of-the-art review and look into the future of 5 critical areas: acute myocardial infarction, adjuvant treatment strategies for acute myocardial infarction, cardiac arrhythmias, chronic congestive heart failure, and hyperlipidaemias. PMID- 1711443 TI - Current concepts in the management of pancreatitis. AB - Acute pancreatitis is often a mild, self-limiting illness that responds to simple supportive therapy in the form of intravenous fluids and analgesics. More severe attacks may result in organ failure or pancreatic necrosis. Such patients should be identified early in the course of an attack and actively monitored within an intensive care unit or high dependency area. Supportive therapy remains the basis of management. Attention to the adequacy of the fluid balance and oxygenation are of prime importance and supportive therapy may include inotropic support, assisted ventilation and renal dialysis. Pancreatic necrosis should be sought by contrast-enhanced computed tomography (CT) scanning, and surgical intervention may be required if the patient's clinical condition continues to deteriorate. Surgery should ideally be delayed until the second or subsequent week when necrosectomy (debridement of necrotic pancreatic tissue) may be possible rather than formal pancreatic resection. The role of various drugs to suppress pancreatic secretion and inhibit pancreatic enzymes, although shown to be consistently effective in experimental pancreatitis, has not been established by controlled clinical trials in humans. Recent controlled studies examining peritoneal lavage in humans have failed to confirm the beneficial results suggested in earlier studies. Early endoscopic sphincterotomy for patients with severe gallstone pancreatitis and ductal calculi has been reported to reduce mortality and morbidity in one controlled clinical trial and may prove to be an important advance. PMID- 1711444 TI - Current perspectives on drug therapies for anorexia nervosa and bulimia nervosa. AB - Anorexia nervosa and bulimia nervosa are eating disorders that strike a significant number of adolescent and young adult women and carry a significant morbidity and mortality. Over the last 30 years, virtually every class of psychiatric drug as well as atypical agents have been tested in the management of these vexing and often chronic disorders. For anorexia nervosa, there is in 1991 little if any role for pharmacotherapy. Drugs used to promote food intake and weight gain, such as cyproheptadine, amitriptyline, clonidine and opiate antagonists, have provided disappointing results. For bulimia nervosa, double blind placebo-controlled clinical trials have confirmed an antibulimic effect for tricyclic antidepressants, such as imipramine and desipramine. Monoamine oxidase inhibitors, while having a significant antibulimic effect, require careful adherence to a dietary regimen which may limit patient compliance. Newer antidepressants such as trazodone, amfebutamone and fluoxetine may prove useful if further study substantiates preliminary beneficial results. Other agents such as anticonvulsants, benzodiazepines, lithium, fenfluramine and opiate antagonists also require additional study, indicating that the optimal integration of pharmacotherapies with other treatments awaits further research. PMID- 1711445 TI - Quinapril. A review of its pharmacological properties, and therapeutic efficacy in cardiovascular disorders. AB - Quinapril is a monoethyl ester which is hydrolysed after absorption to form an active metabolite, quinaprilat, which is a more potent angiotensin converting enzyme (ACE) inhibitor than the parent drug. Quinaprilat has a short elimination half-life but a potent binding affinity for ACE which enables once daily administration. Data from clinical studies indicate that quinapril 10 to 40 mg daily, given as a single dose, is an effective antihypertensive agent, suitable as monotherapy for reducing high blood pressure and maintaining satisfactory control during long term treatment of mild to severe hypertension. Dosages of 80 mg daily have been used in some patients. Concomitant diuretic therapy usually elicits a response in patients who fail to respond adequately to monotherapy. Initial studies suggest that quinapril also has a role in the treatment of mild to severe congestive heart failure. In the few long term studies conducted the beneficial acute haemodynamic effects were maintained during long term treatment and were accompanied by symptomatic and functional improvement. The majority of these patients responded to twice daily administration. Adverse effects associated with the antihypertensive action of quinapril are generally mild, well tolerated and are similar to those of other ACE inhibitors. Thus, quinapril appears to be a useful alternative ACE inhibitor for the treatment of mild to severe hypertension and congestive heart failure. PMID- 1711447 TI - Citalopram. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in depressive illness. AB - Citalopram is an antidepressant belonging to a new class of drugs which enhance serotoninergic neurotransmission through potent and selective inhibition of serotonin reuptake. Preliminary trials suggest that its short term therapeutic efficacy is significantly greater than that of placebo and mianserin, and comparable to that of amitriptyline, maprotiline and imipramine. It appears to be a weaker antidepressant agent than clomipramine, but better tolerated. Its elimination half-life of 33 hours permits once daily oral administration. Symptomatic improvement obtained with short term treatment has been maintained when therapy has been extended for up to 1 year; in the few patients studied for this extended period, the relapse rate was lower than with fluvoxamine, fluoxetine or imipramine. Compared to standard antidepressant agents, citalopram is well tolerated. It does not appear to be cardiotoxic, has not been associated with seizures in humans, and is relatively nonsedating. Unlike the tricyclic antidepressants, citalopram has minimal anticholinergic effects. Mild and transient nausea, with or without vomiting, is the most frequent adverse effect- occurring in 20% of patients--and increased perspiration, headache, dry mouth, tremor and insomnia are experienced by 15 to 18% of patients. Citalopram thus offers similar therapeutic efficacy and a more favourable tolerability profile than the tricyclic antidepressants. Preliminary data suggest that it may be particularly useful in patients who cannot tolerate the anticholinergic or cardiovascular side effects of tricyclic antidepressants and in those for whom sedation is not indicated. PMID- 1711449 TI - Comparative study of myelin basic protein isoforms in developing vertebrate central nervous system: absence of 21.5- and 20.2-kilodalton myelin basic proteins in chicken may point to their importance in mammalian myelinogenesis. AB - Developmental appearance and accumulation pattern of myelin basic protein (MBP) isoforms were analyzed by quantitative immunoblotting in central nervous system of three mammalian (guinea pig, rabbit and bovine) and one avian (chicken) species. In these four species, myelination onset occurred in the spinal cord well before birth. In addition to the 18.5-kD MBP observed in all species studied, a 21.5- and a 20.2-kD MBPs were detected in the three mammalian species but not in chicken. In calf and chicken, a 17.3-kD MBP was also observed. The 18.5-kD and 17.3-kD MBPs were the major MBP isoforms of chicken central nervous system where a faint MBP-related 14.5-kD protein could also be seen. The major difference between mammalian and avian MBP profiles was indeed the presence only in mammals of the 21.5-kD and the 20.2-kD MBPs. The development patterns of these two isoforms as well as their rate of accumulation as compared to the 18.5-kD MBP suggest that their role in mammalian myelination may be of greater importance at early rather than late stages of this process. Differences in quantity as well as type of MBP isoforms present may indicate that in diverging animal species, the process of myelination may follow a different pathway and possibly involve different MBP isoforms at different stages. PMID- 1711448 TI - Amlodipine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in cardiovascular disease. AB - Amlodipine, a basic dihydropyridine derivative, inhibits the calcium influx through 'slow' channels in peripheral vascular and coronary smooth muscle cells, thus producing marked vasodilation in peripheral and coronary vascular beds. Short to medium term clinical trials indicate that amlodipine is effective as both an antianginal agent in patients with stable angina pectoris and an antihypertensive agent in patients with mild to moderate hypertension. In small comparative studies amlodipine was at least as effective as 'standard' agents, including atenolol, verapamil, hydrochlorothiazide or captopril in hypertension, and diltiazem or nadolol in angina pectoris. Amlodipine is well tolerated, and does not appear to cause some of the undesirable effects often associated with other cardiovascular agents (e.g. adverse changes in serum lipid patterns, cardiac conduction disturbances, postural hypotension). The most common adverse effects associated with amlodipine therapy--oedema and flushing--are related to the vasodilatory action of the drug, and are generally mild to moderate in severity. Thus, amlodipine seems to provide a useful alternative to other agents currently available for the treatment of essential hypertension and chronic stable angina pectoris, with certain pharmacodynamic and tolerability properties that should be advantageous in many patients. PMID- 1711450 TI - [A structural and ultrastructural study of the inferior olivary complex in ground squirrels (Citellus citellus L.)]. AB - Morphological characteristics of neurons in the inferior olivary complex of ground squirrels at various ages were studied by a light and electron microscope. This nuclear complex consists of three nuclei: medial accessory olivary nucleus, dorsal accessory olivary nucleus and main olivary nucleus. Its cytoarchitectonics cytoarchitecture was studied by serial sections at the coronary level, stained by kresyl-violet. Two types of olivary neurons were established by silver impregnation in accordance with dendrite ramifications. Nematosomes, special forms of granulated endoplasmic reticulum, which could be probably connected with advancement of age and the process of aging were observed by an electron microscope together with organelles and intranuclear inclusions typical for the relay neurons. PMID- 1711451 TI - Quantitative EEG monitoring for patients with subarachnoid hemorrhage. AB - We evaluated the sensitivity of continuous quantitative EEG in 11 patients with subarachnoid hemorrhage (SAH). We correlated compressed spectral array (CSA) and trend analysis (TA) of total power (1-30 Hz), frequency centroid (1-30 Hz), alpha ratio and percent delta power with clinical and radiological findings. For all ischemic events (n = 11), the most sensitive TA parameter was a change in total power (91%), followed by changes in alpha ratio (64%), frequency centroid (55%), and percent delta (45%). Comparable CSA features were changes in power (44%) and slowing (39%). Total power and frequency varied independently. In 4 cases, EEG findings on TA appeared before clinical changes. Continuous quantitative EEG may be useful for monitoring and predicting ischemia following SAH. TA of individual EEG parameters is more sensitive than CSA, and total power is the most sensitive. PMID- 1711446 TI - Mitoxantrone. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in the chemotherapy of cancer. AB - Mitoxantrone is a dihydroxyanthracenedione derivative which as intravenous mono- and combination therapy has demonstrated therapeutic efficacy similar to that of standard induction and salvage treatment regimens in advanced breast cancer, non Hodgkin's lymphoma, acute nonlymphoblastic leukaemia and chronic myelogenous leukaemia in blast crisis; it appears to be an effective alternative to the anthracycline component of standard treatment regimens in these indications. Mitoxantrone is also effective as a component of predominantly palliative treatment regimens for hepatic and advanced ovarian carcinoma. Limited studies suggest useful therapeutic activity in multiple myeloma and acute lymphoblastic leukaemia. Regional therapy of malignant effusions, hepatic and ovarian carcinomas has also been very effective, with a reduction in systemic adverse effects. Mitoxantrone inhibits DNA synthesis by intercalating DNA, inducing DNA strand breaks, and causing DNA aggregation and compaction, and delays cell cycle progression, particularly in late S phase. In vitro antitumour activity is concentration- and exposure time-proportional, and synergy with other antineoplastic drugs has been demonstrated in murine tumour models. Leucopenia may be dose-limiting in patients with solid tumours, whereas stomatitis may be dose-limiting in patients with leukaemia. Other adverse effects are usually of mild or moderate severity although cardiac effects, particularly congestive heart failure, may be of concern, especially in patients with a history of anthracycline therapy, mediastinal irradiation or cardiovascular disease. Mitoxantrone displays an improved tolerability profile compared with doxorubicin and other anthracyclines, although myelosuppression may occur more frequently. Thus, mitoxantrone is an effective and better tolerated alternative to the anthracyclines in most haematological malignancies, in breast cancer and in advanced hepatic or ovarian carcinoma. Further studies may consolidate its role in the treatment of these and other malignancies. PMID- 1711452 TI - Propagation of human complex-partial seizures: a correlation analysis. AB - Inter- and intrahemispheric propagation of ictal discharges was analysed in 7 epileptic patients having chronic intracranial electrodes. Five had temporal foci and two had frontal mesial foci. Correlation analysis of seizure discharge was used to detect the emergence of linear relationships between different structures both intra- and interhemispherically. This analysis included mesial and lateral temporal sites, cingulate gyri, anterior ventral and dorsomedial thalamic nuclei during the discharges of 13 ictal episodes. Correlation between temporal mesial structures was found to be low throughout seizure onset. Propagation of paroxysmal activity through the anterior ventral thalamic nuclei and cingulate gyri was observed in all cases with temporal or frontal mesial focus. Propagation of the discharge from the temporal lateral cortex to the contralateral homologue area was observed with a short delay during 4 complex-partial seizures originating in the temporal lobe. These results show that, even though the commissural pathways may have a role in propagation, it is a relatively unimportant one and they suggest that ictal activity during mesial temporal seizures may spread preferentially through the thalamic nuclei and cortical structures. PMID- 1711453 TI - Moving dipole inverse solutions using MEGs measured on a plane over the head. AB - Since magnetoencephalograms (MEGs) are measurements of the magnetic field produced in the air around the head by electrical sources in the brain, it is possible to measure MEGs on some regular surface over the head such as a plane. Such measurements are easier to make than traditional measurements at a fixed distance from the head. This paper presents results of computer modeling studies of source localization errors caused by using MEGs measured on a plane over the head. It was found that non-spherical head shape does not have a greater effect on localization accuracy for measurements on a plane than for traditional fixed distance measurements. The source localization errors were less than 1 cm for both types of measurement for sources in the cortical region of the brain. Localization errors were found to increase for sources at greater depth in the brain, but the errors using measurements on a plane were not found to be significantly larger than those using traditional measurements. Hence, because of the ease with which measurements on a plane can be made, more wide-spread use of such measurements should be considered. PMID- 1711454 TI - A neuromagnetic study of selective auditory attention. AB - Auditory event-related magnetic fields and electrical potentials were recorded from subjects who were instructed to attend to a sequence of constant pitch tones presented to one ear and ignore a concurrent sequence of tones with a different pitch presented to the other ear. Subjects' task was to detect and count longer duration 'target' tones (P = 0.1) interspersed with 'standard' tones (P = 0.4) in the attended ear and to ignore tones (both standards and targets) in the other ear. All stimuli, both attended and ignored, elicited a prominent response approximately 100 msec after tone onset (N1m). Beginning approximately 150 msec following stimulus onset, an attention-dependent modulation of the magnetic response (Ndm) was observed for each subject. In 2 subjects whose magnetic field patterns were mapped in detail, equivalent current dipole (ECD) modeling was used to estimate the sources of N1m and Ndm activity. By transforming the coordinate systems for magnetic resonance images (MRIs) and ECD solutions, the locations and orientations of ECDs were determined relative to each subject's brain structures. ECDs for both N1m and Ndm were located in auditory cortex along the posterior regions of the sylvian fissure. Monte Carlo error analyses indicated that the ECD for Ndm is near, but significantly anterior to that for N1m. PMID- 1711455 TI - Event-related potentials and eyeblink responses in automatic and controlled processing: effects of age. AB - Seventeen young (mean age = 20.2 years old) and 16 elderly (mean age = 72.6 years old) women were tested with event-related potential (ERP) paradigms designed to elicit responses in reaction time tasks and to a startling noise burst. EEG was analyzed from 17 standard 10-20 electrode sites. Reaction time and performance data suggested that the elderly did not perform worse than the young. Nevertheless, the physiological responses of the elderly differed significantly from those of the young. While the task-dependent P3s at Pz were smaller in the elderly than in the young, the automatic P3 was smaller yet. The distribution of both types of P3 across the scalp was more uniform in the elderly than in the young. Single-trial analyses revealed that the P3 amplitude differences at Pz were not due to latency dispersal of single trials. Single-trial startle eye blink responses to intense noise bursts during the automatic paradigm were considerably less frequent in the elderly, although their individual startle blinks were actually larger. The data demonstrate that the electrophysiological responses of the elderly are different from the young both in tasks eliciting automatic responses and in tasks requiring controlled processing. PMID- 1711456 TI - A new statistic for steady-state evoked potentials. AB - Steady-state evoked potentials are often characterized by the amplitude and phase of the Fourier component at one or more frequencies of interest. We introduce a new statistic for the evaluation of these Fourier components. This statistic, denoted T2circ, is based on the same physiologic assumptions concerning the sources of variability of a Fourier component that are made in the use of the Rayleigh phase-coherence statistic as well as the standard T2 statistic (Hotelling 1931) for multivariate data. However, the T2circ statistic also exploits the relationship between the real and imaginary components of Fourier estimates, which is not exploited by T2, and utilizes amplitude information, which is ignored by the Rayleigh criterion. For these reasons, the T2circ statistic is more efficient than previously used criteria for detection and quantitation of steady-state responses, both in principle and in practice. PMID- 1711457 TI - Fluctuations of steady-state VEPs: interaction of driven evoked potentials and the EEG. AB - We performed a detailed analysis of the variability of a steady-state human evoked potential (EP) and the spectral properties of the simultaneously recorded electroencephalogram (EEG). This allowed us to determine whether the background EEG was influenced by the evoked potential stimulus, and to what extent variability of evoked potential estimates is simply due to the addition of the background EEG. Steady-state visual evoked potentials (VEPs) were elicited by a checkerboard undergoing contrast-reversal modulation at 1 of 3 fundamental frequencies f: 5.0 Hz, 7.5 Hz, and 10.0 Hz. To a first approximation, the evoked potential (at frequency 2 f) and the undriven components of the EEG combined linearly. However, two kinds of interactions were present: (i) patterned visual stimulation decreased the power in the undriven EEG in the 5-17 Hz range by as much as a factor of 2; (ii) superimposed on this overall effect of pattern stimulation, there were changes in the EEG power at specific harmonics of the input frequency. Power increased by as much as 6-fold at the stimulus reversal rate (2 f) and its second harmonic (4 f). These findings imply a complex non linear interaction between the visual input and the EEG. PMID- 1711458 TI - Evidence for slow brain waves: a dynamical approach. AB - Various techniques of non-linear dynamics have been applied with success to EEG data and have provided a new insight into brain dynamics. Among these the 'recurrence plot' is a powerful tool for revealing the presence of drift or periodicities and is easily obtained in the framework of a non-linear dynamical analysis. When this analysis is applied to the EEG recorded from a patient suffering from Creutzfeldt-Jakob disease one observes the presence of slow periodicities of the order of 58 sec. We suggest that the recurrence plots are powerful tools for discovering hidden periodicities of EEG as well as the degree of stationarity of brain activity. PMID- 1711459 TI - Differential regulation of the insulin-like growth factors (IGF-I and -II) and IGF binding proteins during malnutrition in the neonatal rat. AB - Insulin-like growth factor (IGF)-I and -II are known to play a major role in fetal and early postnatal growth. The IGF binding proteins (IGFBPs) are thought to be important in modulating the actions of the IGFs. In this paper, the effect of malnutrition in the neonatal rat on serum IGFs and IGFBPs and hepatic IGFBP messenger (m) RNA was examined. Control (C) dams (n = 9) were allowed ad libitum intake, whereas restricted (R) dams (n = 9) were limited to 50% of ad libitum intake throughout lactation, which results in decreased milk production and malnutrition of pups suckling on restricted dams. A subset of pups were cross fostered from the R-dams to the C-dams from days 15-19 postpartum (PP) to investigate the effect of nutritional repletion (refed). Pups were killed on days 8, 12, 15, and 19 PP and liver and blood collected. Serum IGF-I and -II concentrations were measured by RIA after acid-chromatography to remove IGFBPs. Serum IGFBPs were characterized by Western ligand blot. Hepatic mRNA for IGFBP-1, -2, and -3 were determined by northern analysis. Body weight (BW) of R-pups was significantly less than C-pups by day 10 PP (P less than or equal to 0.05), and mean BW at day 19 was 56% of the C-pups. Refeeding from days 15-19 resulted in a significantly greater rate of growth vs. R-pups (3.2 vs. 0.9 g/day), and mean BW of refed pups at day 19 PP was 75% of C-pups. Malnutrition caused a significant reduction in both serum IGF-I and -II after day 12 PP, while causing an elevation in serum IGFBP-2. IGFBP-1 and IGFBP-2 mRNA expression were not significantly affected at days 8 and 12, but were elevated in livers of day 15 and 19 pups. Malnutrition caused a delay in the development shift from IGFBP-2 to IGFBP-3, which normally occurs between day 15 and 19 in the rat. Refeeding raised serum IGF-I and -II levels to those found in the C-pups and a trend toward normalization of IGFBP profiles. In conclusion, IGFs and IGFBPs are differentially regulated during neonatal malnutrition. The decrease in IGF peptide and induction of IGFBP-1 and -2 may provide protective mechanisms by inhibiting growth during malnutrition. PMID- 1711460 TI - Synergistic action of purified alpha-fetoprotein and growth factors on the proliferation of porcine granulosa cells in monolayer culture. AB - alpha-Fetoprotein (AFP) is present in high levels in fetal fluids, certain neoplasias, and regenerating liver. Although AFP's physiological role remains an enigma, we have recently demonstrated mitogenic activity for AFP. Using a primary monolayer culture system, we have further investigated the proliferative activity of purified AFP. Porcine granulosa cells from small ovarian follicles were attached for 2 days in Ham's F-12-Dulbecco's Modified Eagle's Medium (1:1) and 5% fetal calf serum, followed by 6 days of culture in medium containing 0.25% plasma derived serum plus 25 micrograms/ml low density lipoprotein with or without growth factors and/or purified human AFP. In this system AFP alone does not stimulate proliferation. However, when combined with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I; 10 ng/ml each), AFP (5 micrograms/ml) significantly (P less than 0.01) enhanced growth factor-mediated proliferation 4.5-fold over that of medium controls. Equivalent doses of purified human serum albumin or transferrin demonstrated no effect. The effects of AFP were dose dependent, with significant (P less than 0.05) enhancement of proliferation (2.7-fold over controls) observed with as little as 0.313 micrograms/ml AFP. Increased proliferation was noticed as early as 24 h after the addition of AFP and by 48 h AFP, EGF, and IGF-I had significantly (P less than 0.05) increased proliferation over that seen in medium controls, cells treated with EGF plus IGF-I, or cells treated with 10% fetal calf serum plus EGF, and this trend continued linearly over 5 days of culture. AFP (5 micrograms/ml) significantly increased the proliferative response observed with increasing doses of EGF, IGF-I, or EGF plus IGF-I, but did not appear to alter the dose-response curves. AFP dose-dependently (1.25-5 micrograms/ml) and significantly (P less than 0.05) increased proliferation of porcine granulosa cells in response to platelet-derived growth factor (PDGF) and EGF (25 and 10 ng/ml, respectively), but not to PDGF alone. In contrast, AFP produced no further proliferation of porcine thecal cells in response to PDGF plus EGF. Binding of EGF, IGF-I, or PDGF to purified AFP could not be demonstrated. These results demonstrate that physiological levels of AFP, although not mitogenic alone, can significantly enhance the mitogenic activity of EGF plus IGF-I/PDGF and may function to modulate growth factor-mediated cell proliferation during development and neoplasia. PMID- 1711461 TI - Cation-induced restoration of insulin action in insulin-desensitized HTC cells. AB - Insulin desensitization of amino acid uptake in HTC cells was induced by preincubation with 4 or 10 micrograms/ml insulin. Insulin binding after desensitisation was decreased by both insulin concentrations due to a 45-49% decrease in insulin receptor numbers. Desensitization with 4 micrograms/ml insulin increased the ED50 for half-maximal stimulation of amino acid uptake from 19.5 +/- 9.2 ng/ml in control cells to 84.0 +/- 8.3 ng/ml (P less than 0.0001). It also decreased the maximal insulin response of amino acid uptake from 1.40 +/- 0.10 to 1.14 +/- 0.10 nmol/mg protein, indicating the production of a mild postreceptor defect. Desensitization with 10 micrograms/ml insulin completely abolished this insulin response. When cellular receptors were down-regulated with 4 micrograms/ml insulin and restimulated with insulin in the presence of 0.03 mM ruthenium red (RR) or 10 mM Ca2+, both the insulin response and insulin binding were increased. Insulin binding was restored to levels comparable to those observed in control cells by an increase in receptor affinity. The ED50 of amino acid uptake decreased to 20.5 +/- 7.3 ng/ml insulin in the presence of RR and to 42.2 +/- 8.9 ng/ml in the presence of 10 mM Ca2+ (both P less than 0.0001 from down-regulated cells). In addition, the maximal insulin response increased from 1.14 +/- 0.10 to 1.40 +/- 0.10 and 1.45 +/- 0.10 nmol/mg protein, respectively. Preincubation with 10 micrograms/ml insulin prevented the effect of RR and Ca2+ on the recovery of insulin responses. These experiments suggest that insulin desensitized cells undergo a progressive loss of their insulin response and that RR and Ca2+ provide useful reagents to investigate the mechanisms of this process because they can counteract the decrease in insulin response by increasing receptor affinity and receptor-effector coupling. PMID- 1711462 TI - Adrenocorticotropin activates barium-conducting channels from bovine adrenocortical zona fasciculata cells in lipid bilayers. AB - Plasma membranes from bovine adrenal zona fasciculata cells were incorporated into lipid bilayers, and the activity of individual calcium channels was monitored under voltage clamp conditions. Using barium as the permeant ion, two distinct channels with conductances of approximately 8 and 20 picoSiemens (pS) were characterized. The 20 pS conductance corresponds to conductances found in L type calcium channels. This channel displayed voltage dependency and a sensitivity to BayK 8644. Addition of ACTH (10 nM) to the bilayer preparations promoted an increased open time for the 20-pS channel. These data confirm the presence of dihydropyridine-sensitive calcium channels in bovine adrenocortical cells and support a potential role for these channels as a site of modulation by ACTH. PMID- 1711463 TI - Colocalization of galanin and prolactin within secretory granules of anterior pituitary cells in estrogen-treated Fischer 344 rats. AB - Galanin is localized within specific cell types of the rat anterior pituitary gland (AP). Immunocytochemical studies at the light microscope level have shown that lactotrophs, somatotrophs, and thyrotrophs contain galanin in the intact female rat, whereas lactotrophs in the male AP do not. We recently reported that galanin and PRL release from estrogen-treated male and female pituitary cells in culture are coregulated by dopamine, TRH, and somatostatin. This suggested that galanin is stored within secretory granules, conceivably with PRL. Using postembedding immunocytochemistry at the ultrastructural level, the objectives of this study were to: 1) determine the subcellular location of galanin in the AP; 2) elucidate if galanin and PRL are colocalized within the same secretory granules; and 3) compare the cellular localization of galanin in the male and female AP. Male and ovariectomized female (OVEX) Fischer 344 rats were implanted with estradiol-containing or empty Silastic capsules for 2 weeks. Postembedding immunogold labeling was performed using rabbit (for galanin) and guinea pig (for PRL) generated antisera. Two different sizes of colloidal gold spheres were used to localize the hormones in the same tissue section. Galanin was primarily localized in secretory granules of adenohypophyseal cells. Based upon immunocytochemical results and morphological criteria, galanin was contained in somatotrophs but not lactotrophs in the male and OVEX AP. The AP of estrogen treated rats contained more specific immunogold labeling for galanin than untreated rats. The increased immunoreactivity for galanin was notably associated with lactotrophs. After exposure to estrogen, galanin and PRL were colocalized within the same secretory granules of the male and OVEX pituitary cells. We conclude: 1) galanin is localized within secretory granules of the rat AP; 2) galanin and PRL are colocalized within secretory granules of the male and OVEX AP after estrogen treatment; and 3) galanin is localized in similar cell types in the male and OVEX AP, before and after estrogen treatment. These data provide a morphological basis for the coregulation of galanin and PRL secretion by hypothalamic factors. PMID- 1711464 TI - Insulin and insulin-like growth factors stimulate in vivo receptor autophosphorylation and tyrosine phosphorylation of a 70K substrate in cultured fetal chick neurons. AB - Insulin and insulin-like growth factors (IGFs) have been shown to regulate neuronal growth and differentiation. To investigate the possible role of tyrosine phosphorylation in the neurotrophic actions of these peptides, we examined the effects of insulin, IGF-I, and IGF-II on tyrosine phosphorylation in cultured fetal chick neurons. Tyrosine phosphorylation was detected by immunoblot analysis using antiphosphotyrosine antibodies. Under basal conditions five major phosphoproteins (170K, 140K, 115K, 103K, and 44K) and a number of minor proteins were detected by three separate antisera. In response to insulin, IGF-I, or IGF II, an 87K membrane-associated protein (pp87) became phosphorylated on tyrosine in a rapid, dose-dependent manner. The Mr of pp87 was identical to that of the insulin and IGF-I receptor beta-subunits. Comparison of the dose-response curves for pp87 phosphorylation by insulin and IGF-I suggests that each peptide stimulated autophosphorylation of its own receptor beta-subunit. The maximal response obtained with IGF-I was approximately 10-fold higher than that obtained with insulin (3- to 5-fold), consistent with the larger number of IGF-I receptors in these cells. The maximal response obtained with IGF-II was 4- to 7-fold higher than that with insulin, suggesting that it acts through the insulin receptor as well. Tyrosine that IGF-II acts through the insulin receptor as well. Tyrosine phosphorylation of other proteins by the activated receptor kinases was not detected in neurons cultured for 5 days, however neurons cultured for only several h contained a predominant 70K protein (pp70) that was phosphorylated on tyrosine in response to all three hormones. Tyrosine phosphorylation of pp70 was maximal after 2-5 h in culture and was undetectable in neurons cultured longer than 24 h. The time course and dose dependence of pp70 phosphorylation in response to hormone paralleled that of insulin and IGF-I receptor autophosphorylation. These results demonstrate that both insulin and IGF-I receptors are active tyrosine kinases in fetal neurons, and that pp70 represents a potential endogenous substrate for the insulin and IGF-I receptor kinases. The transient phosphorylation of pp70 may be involved in the neurotrophic effects of insulin and insulin-like growth factors. PMID- 1711465 TI - Rat thecal/interstitial cells express transforming growth factor-beta type 1 and 2, but only type 2 is regulated by gonadotropin in vitro. AB - We previously demonstrated that granulosa cells from diethylstilbestrol-primed immature rats expressed transforming growth factor-beta 2 (TGF beta 2), but not TGF beta 1, mRNA and that its expression was regulated by FSH in vitro. The present studies were designed, therefore, to establish whether thecal/interstitial cells (TIC) from diethylstilbestrol-primed immature rats express more than one subtype of TGF beta mRNA and gene product and whether the levels of expression/production in vitro were regulated by LH/hCG. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of total RNA indicated that TIC expressed both TGF beta 1 and TGF beta 2 mRNA. In response to hCG (200 ng/ml), TIC expressed TGF beta 2 mRNA levels that were 51% of control levels; TGF beta 1 mRNA levels were not altered. In response to cholera toxin (10(-9) M), TIC expression of TGF beta 2 and TGF beta 1 mRNA levels was 10% and 55% of control values, respectively. Western blot analysis established that both TGF beta 1 and TGF beta 2 were secreted in vitro. hCG and cholera toxin reduced secretion of TGF beta bioactivity by 55% and 90%, respectively. In summary, these results indicate: 1) TIC express both TGF beta 1 and TGF beta 2 mRNA; 2) TIC expression of TGF beta 2, but not TGF beta 1, mRNA is regulated by hCG in vitro; 3) TIC secrete both TGF beta 1 and TGF beta 2, and 4) TIC secretion in vitro of TGF beta activity is gonadotropin regulated. PMID- 1711466 TI - Immunolocalization of transforming growth factor-beta 1 in the bovine adrenal cortex using antipeptide antibodies. AB - Eight- to 12-amino acid long peptides, representing fragments of transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 were selected on the basis of their potential immunogenicity and were used to generate polyclonal antibodies. Anti-TGF beta 1-(91-103) antibodies recognized specifically TGF beta 1, prevented TGF beta 1 binding to NRK-49F cells, and neutralized the biological activity of TGF beta 1 in adrenocortical cells (consisting in the inhibition of angiotensin II-induced cortisol production). Antibodies raised against TGF beta 2-(65-73) appeared to recognize TGF beta 2 with a better affinity than TGF beta 1, but were unable to block the binding of either TGF beta 1 or TGF beta 2 to their receptors or to inhibit their biological activity. These observations are in line with a prominant role of the C-terminal domain of TGF beta 1 in its interaction with its receptor(s) and, hence, in its biological activity. Using anti-TGF beta 1-(91 103) antibodies, we could localize immunoreactive TGF beta 1-like material in the cortex of adult bovine adrenal glands. No reactivity was detected in the capsule or adrenal medulla. The reactivity was maximal in the zona fasciculata/reticularis and weaker in the zona glomerulosa. TGF beta-like material was present in a latent form in the conditioned medium from primary cultures of bovine adrenocortical cells. These cells secreted about 5 ng heat activatable TGF beta-like material/24 h of culture.10(6) cells. Taken together with our previous reports that bovine adrenocortical cells possess high affinity TGF beta 1 receptors and secrete a TGF beta 1-like molecule under a latent form, the present observations further support the hypothesis that TGF beta 1 or a closely immunologically related protein acts as an autocrine regulator of adrenocortical steroidogenic functions. PMID- 1711467 TI - A clinico-pathological study of cervical carcinoma through histopathology, SEM and tumour marker expression. AB - Carcinoma of the uterine cervix is the commonest tumour in India. Our study on the biological behaviour of cervical carcinoma was made with respect to oncofetoprotein expression, histopathology and scanning electron microscopy. A clinical picture was compared with the above three parameters to note the embryonic degeneration of the tumour. The objective of this study was to establish a new grading system of tumour pathology which might be an adjunct to clinical staging. This should be helpful for the formulation of different treatment protocols. PMID- 1711468 TI - Comparison of the effects of endothelin-1 and Bay k 8644 on twitch contractions of the field-stimulated rat vas deferens. AB - Endothelin-1 (ET-1) potentiated both the fast and the slow components of the twitches of the isolated field-stimulated rat vas deferens, whereas Bay k 8644 potentiated only the fast component. Nicardipine tended to decrease the potentiation. ET-1, but not Bay k 8644, caused membrane depolarization and resting tension elevation, which were suppressed by nicardipine. The excitatory junction potentials (EJPs) were augmented by Bay k 8644 and the effect was not changed by nicardipine. ET-1, however, apparently did not augment the EJPs, because of the accompanying depolarization. These results indicate a difference in mechanism of the potentiating effects of ET-1 and Bay k 8644. PMID- 1711469 TI - Immortalization of human fetal sinusoidal liver cells by polyoma virus large T antigen. AB - Fetal sinusoidal liver cells were isolated from human liver explant cultures and transfected with pCMVLT, a plasmid containing the immediate early promotor of cytomegalovirus (CMV) and the large tumor antigen (LT) coding part of the polyoma virus (py) genome. Whereas nontransfected cells stopped proliferating after 4 weeks, the transfected sinusoidal cells were stimulated to divide more quickly without changes in their morphology. Up to now, cells have been permanently cultured for more than 18 months and passaged over 130 times, corresponding to around 400 generations. This allows them to be regarded as "immortalized" cells. The presence of LT protein in the cells has been documented by means of immunoprecipitation and immunofluorescence. Expression of the v. Willebrandt factor VIII was the main criterion for classifying the cell population as endothelial cells. The presence of cytokeratins 7, 8, and 18 in these cells underlines their close ontogenic and functional relationship to mesothelial cells. Sinusoidal endothelial cells (SECs) synthesize vimentin and the typical extracellular matrix components collagen IV and fibronectin, but are negative for laminin and entactin. We used immortalized SEC's in co-culture experiments with fresh fetal human hepatocytes and adult mouse hepatocytes. They promoted survival of both types of hepatocytes over a period of 8-10 weeks. Control human fetal liver explant culture cells survived for only 3-4 weeks, whereas control adult mouse liver cells retained vitality for 8-10 days only. PMID- 1711470 TI - Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I. AB - Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2. PMID- 1711471 TI - Aberrant in vitro expression of keratin K13 induced by Ca2+ and vitamin A acid in mouse epidermal cell lines. AB - Normally the expression of the murine type I keratin K13 is restricted to differentiating cells of internal squamous epithelia which line the oral cavity and the upper digestive tract. Recently, however, we were able to show that K13 is aberrantly but constitutively expressed without its normal type II partner K4 also in differentiating parts of 7,12-dimethylbenz(a)anthracene (DMBA/TPA) 12-O tetradecanoylphorbol-13-acetate-induced squamous cell carcinomas of mouse back skin, whereas its likewise suprabasal expression in papillomas is variable (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988). In an attempt to reproduce the aberrant expression of K13 in a mouse in vitro system, we have investigated eight established murine epidermal cell lines for their putative ability to express K13. The cell lines differed distinctly in their derivation and comprised cell lines originating from DMBA/TPA induced papillomas (line SP1) or DMBA treated adult mouse epidermis (line 308) as well as cell lines derived from DMBA or DMBA/TPA-treated primary epidermal keratinocytes (lines PDV and MCA 3D) and cell lines which arose spontaneously by long-term culture of normal epidermal keratinocytes (lines HEL 30 degrees HEL 37 degrees, HELP I and HELP III). We show that, independent of their derivation, all cell lines possess the intrinsic property to aberrantly express K13. Invariably the K13 gene is not expressed when the lines are cultured under low Ca2+ conditions (0.05 mM) and thus prevented from differentiation. Its expression can, however, be induced either by increasing the extracellular Ca2+ concentration or by the addition of physiological concentrations of vitamin A acid to low Ca2+ medium. Whereas in the latter case, K13 expression occurs without concomitant induction of morphological differentiation of the cells, Ca2+ elevation in the culture medium induces squamous differentiation and K13 expression occurs only in differentiating cells, thus reflecting the situation observed in in vivo tumors. All cell lines exhibit a concentration optimum for the stimulatory agents; however, the degree of maximal K13 expression varies considerably among the individual cell lines and shows a striking correlation with the reported tumorigenicity of the lines after transplantation to animals. In contrast, a tentatively suggested correlation between the activation of the Ha-ras gene and the aberrant expression of K13 (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988) could not definitely be confirmed since we observed K13 expression also in three cell lines which did not carry a mutation in codon 61 of the Ha-ras gene.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711472 TI - Secretagogue induction of cell differentiation in pancreatic acinar cells in vitro. AB - The effect of two different types of secretagogues on rat pancreatic acinar cells cultured onto a reconstituted basement membrane was studied. Cells cultured without any secretagogue were able to reaggregate but did not form monolayer patches. Most of them lost their differentiated ultrastructural characteristics but regained their polarity. In contrast, when CCK, caerulein, or carbamylcholine was added to the culture medium cells developed both acini-like structures and cell monolayer patches. The cells retained the differentiated ultrastructural appearance and polarity resembling their in situ morphology. Furthermore, secretagogue-conditioned cells presented higher amylase contents. The use of secretagogue antagonists such as L-364,718 and L-365,260 for caerulein, or atropine and mecamylamine for carbamylcholine, did not profoundly modify the cultures and the morphological effects triggered by the secretagogues alone. However, both CCK antagonists and cholinergic antagonists inhibited to a certain degree the secretory stimulation. Our data support the theory that a major role is played by secretagogues in conjunction with the basement membrane for the maintenance of differentiation in pancreatic acinar cells in vitro which appears to be independent from their secretory effect. PMID- 1711473 TI - Characterization of trans-immortalized hepatic cell lines established from transgenic mice. AB - Hepato-specific regulatory (promoter/enhancer) DNA sequences were used for targeting the expression of onc genes, such as murine c-myc and Simian Virus 40 T Antigen, to hepatocytes of transgenic mice which subsequently developed hepatocellular carcinomas after a variable period of time (depending on the type of onc gene employed). Several trans-immortalized cell lines were established and compared with respect to the expression of adult hepatic markers and response to growth factors. Despite the morphological differences observed between trans hepatomas, owing to the expression of the two different onc genes, all tumor derived cell lines behaved in a comparable fashion during long-term culture displaying an adult hepatic phenotype for at least 40 passages. They differed, however, in response to epidermal growth factor. When the gene coding for human alpha 1-antitrypsin was placed under the control of the same hepato-specific promoter/enhancer, high levels of the human recombinant protein could be harvested from the supernatants of trans-hepatoma-derived cell lines. PMID- 1711474 TI - Gastrin affects enzyme activity and gene expression in the aging rat pancreas. AB - The structural and functional properties of the pancreas are known to be affected by a number of hormones, particularly those of the gastrin-CCK family, yet little is known about the responsiveness of the pancreas to gastrin-CCK peptides during the latter stages of life. The present investigation examined the changes in pancreatic growth, the activity, and the steady-state mRNA levels of some of the digestive enzymes during advancing age and after administration of gastrin. Groups of 3-, 6-, 12-, and 16-month-old male Fischer-344 rats were infused (osmotic minipump) with either gastrin G-17 (250 ng/kg/h) or saline (controls) for 14 days. In control pancreas, aging resulted in slight progressive reduction in pancreatic DNA, RNA, and protein concentrations. This decrease was markedly enhanced by gastrin treatment in 16-month-old rats. Pancreatic amylase and trypsin (TRP) activities in these animals were also slightly decreased with aging, whereas the steady-state mRNA levels of both enzymes were significantly higher in 16-month-old rats than in their 3-month-old counterparts. However, in 16-month-old rats, the steady-state mRNA levels of amylase and TRP were significantly reduced after gastrin administration, when compared with the corresponding controls. Chymotrypsin (CHY) activity in the pancreas remained essentially unchanged between 3- and 12-month-old rats, but in 16-month-old animals it was markedly decreased. CHY activity was further reduced by gastrin treatment only in the 16-month-old group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711475 TI - Interleukin 4 alone or in combination with interleukin 1 stimulates 3T3 fibroblasts to produce colony-stimulating factors. AB - Colony-stimulating activity (CSA) can be produced by fibroblasts when stimulated by interleukin 1 (IL-1). We show that like IL-1, interleukin 4 (IL-4) can stimulate 3T3 fibroblasts to produce CSA. Biological and molecular analyses show that a significant portion of the CSA is colony-stimulating factor 1 (CSF-1) and granulocyte colony-stimulating factor (G-CSF). CSF-1 production in cells stimulated with a combination of both IL-1 and IL-4 was greater than that observed when cells were stimulated with either cytokine alone. However, the data show a synergistic induction of the expression of high levels of G-CSF mRNA and protein in cells incubated in the presence of both IL-1 and IL-4. The concentration of G-CSF in supernatants from cells stimulated with both IL-1 and IL-4 was at least tenfold higher than that measured in supernatants harvested from cells stimulated with either IL-1 or IL-4 alone. Previous investigations have shown that IL-4 had direct effects on hematopoietic progenitor cell growth. The studies described herein indicate that IL-4 is also involved in the regulation of hematopoiesis in an indirect manner, that is, by playing a role in the regulation of hematopoietic growth factor production. PMID- 1711476 TI - Functional dichotomy in the CD4+ T cell response to Schistosoma mansoni. PMID- 1711477 TI - Hymenolepis diminuta: changes in the levels of certain intestinal regulatory peptides in infected C57 mice. AB - The levels of 10 regulatory peptides in acid-alcohol extracts of three regions of the small intestine (0-20%, 30-60%, and 70-100%, with respect to distance from the pylorus) have been monitored radioimmunometrically in sham-infected male (6-8 week old) C57 mice and mice given a 5-cysticercoid infection of the rat tapeworm Hymenolepis diminuta and autopsied 10 days postprimary infection and 5 days postsecondary infection (administered 28 days postprimary infection). The regulatory peptides examined were gastrin, gastrin-releasing peptide (GRP), glucagon (= enteroglucagon), motilin, neurotensin (NT), pancreatic polypeptide (PP), peptide histidine isoleucine (PHI), somatostatin (SRIF), substance P (SP), and vasoactive intestinal peptide (VIP). Statistical analyses revealed significant deviations from control values of five of the peptides (enteroglucagon and SP, both elevated; NT, PHI and VIP, all lowered) in intestinal tissue from infected mice; measurement of the same peptides in colonic extracts revealed no significant differences between infected and sham-infected mice. Parallel changes in peptide levels between normal infected and immunosuppressed infected mice were not evident, although elevations in the tissue levels of enteroglucagon and SP were found in infected Wistar rats (normal host). Results are discussed with respect to a peptidergic involvement in the pathology and host immune response to an intestinal tapeworm. PMID- 1711478 TI - Schistosoma mansoni: ingestion of dextrans, serum albumin, and IgG by schistosomula. AB - Schistosomula of Schistosoma mansoni develop the ability to ingest and digest red blood cells after the fourth day post-transformation. Here, we have used fluorescently-labeled dextrans and two plasma proteins, albumin and IgG, to test whether day-old schistosomula can ingest and process macromolecules prior to the time that they eat red cells. Worms ingested dextrans of molecular weights 4,000, 70,000 and 2 x 10(6) in a time- and concentration-dependent manner. The dextran remained in the cecal lumen for up to 2 days after feeding. Parasites ingested both fluorescein-conjugated bovine serum albumin and rabbit IgG, but neither of these proteins remained confined to the cecum over time. Instead, fluorescence redistributed to the acetabular glands within a few hours. Thin-layer chromatography indicated that schistosomula degraded fluorescein-conjugated albumin to fluorescein-conjugated peptides approximately 10-15 amino acids long. The volume of the cecum was estimated to be 2431 microns 3 and the surface area 299 microns 2. These results demonstrate that larval schistosomes can ingest both proteins and complex carbohydrates shortly after transformation, before they can ingest red cells. Further, the gut apparently releases proteases that cleave plasma proteins, but not saccharidases that cleave dextran. PMID- 1711479 TI - Evaluation of the carcinogenic potency of 4 environmental polycyclic aromatic compounds following intrapulmonary application in rats. AB - The carcinogenic potential of 4 highly purified polycyclic aromatic compounds (PAC) was studied in the respiratory tract of rats. Using a beeswax/trioctanoin mixture as vehicle, 10, 3 and 1 mg phenanthrene (PHE), 3 and 1 mg chrysene (CHR), 0.1 mg dibenz(a,h)anthracene (DBahA) and 6, 3 and 1 mg benzo(b)naphto(2,1 d)thiophene (BNT) were injected into the lungs of 35 female Osborne-Mendel rats per group. Benzo(a)pyrene (BaP, 0.3, 0.1 and 0.03 mg) was used as the reference substance. Whereas only one squamous cell carcinoma developed at the highest PHE dose, a dose-dependent tumor incidence was found for CHR. BNT showed a carcinogenic effect similar to CHR, but an increasing incidence of neoplasms was not seen between the median and high dose. DBahA induced carcinomas in even more than half of the animals at the dose level of 0.1 mg and, therefore, has to be classified as the most potent PAC under investigation. BaP resulted in a clear dose-response relationship. According to probit analysis of the results, the carcinogenic potencies of the PAC relative to BaP (1.00) rank as follows: DBahA, 1.91; CHR, 0.03; BNT, 0.02; and PHE, 0.001. The estimated ED10- values were 0.031 mg for BaP, 0.016 mg for DBahA, 1.02 mg for CHR, 1.65 mg for BNT and 22.84 mg for PHE. PMID- 1711480 TI - Transplantable mouse hepatoblastoma: histologic, ultrastructural and immunohistochemical study. AB - Hepatoblastoma found adjacent to a liver cell adenoma in an aged (CBA x C57Bl/6)F1 male mouse was transplanted to the syngeneic host and passed through 30 generations. Histologically, tumours that grew on transplantation retained principal morphological features of the primary tumour. Transplanted tumours were negative for AFP and for antigen A6. This antigen in the normal mouse liver is found in epithelial cells lining bile ducts and ductules including the terminal Hering canals. Some of the Hering cells were consistently A6 negative under normal conditions, and the suggestion is made that these A6 negative cells might be the cells of hepatoblastoma origin. PMID- 1711481 TI - [Effect of emotional-pain stress on RNA and protein synthesis in rats of different age]. AB - The intensity of RNA and protein biosynthesis is studied in different tissues as well as in active and low-active fractions of liver chromatin, when adult and old rats are subjected to emotional-painful stress during 3 days. Significant stimulation of RNA and protein biosynthesis in chromatin fractions in liver and total RNA and protein in adrenals and hypothalamus is observed. PMID- 1711482 TI - [The use of thin slices of myocardium for recording the currents across single ion channels]. AB - Thin cardiac slices (100-200 microns) from newborn (1-14 days old) rat heart ventricles were used for patch clamp recordings. High resistance seals (10-50 GOhms) between patch-clamp pipettes and the membrane of cardiac cells as well as classical patch-clamp configurations can be achieved on this preparation without any enzymatic treatment of tissue. Resisting potential for cardiac cells measured in whole-cell configuration ranged between -30 and -65 mV. Averaged sodium currents and single inward rectifying potassium elementary currents recorded in cell-attached mode displayed basic features similar to those previously reported for isolated rat ventricular cells. Application of the method described here in cardiac electrophysiology will allow patch-clamp studies on heart cells without the complicated procedures of cell isolation. In addition, the uncertainty associated with enzyme treatment can be avoided. In future, this technique could be a new tool for studying electrophysiological properties of heart cells in situ. PMID- 1711483 TI - [The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]. AB - In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+) carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested. PMID- 1711484 TI - [The identification and properties of chloride potential-dependent channels in the membrane of secretory cells]. AB - Outward current of the salivary gland cells membrane of chironomus larva activated by the displacement of the membrane potential to the region of positive values has been registered by the voltage-clamp method under conditions of intracellular dialysis in the presence of the chloride transmembrane gradient. Activation threshold of the current is about +20 mV. Subsequent displacement of the membrane potential to the region of positive values causes an increase of the current. Time constant of the current activation is (573 +/- 34.4) ms. The current decreases with the reduction of extracellular chloride concentration, under the influence of tannin acid and temperature lowering, under conditions of alkaline medium. The current increases due to Hg2+ ions and lowering of the outward solution pH. Thus, the membrane of secretory cells contain high-threshold potential-dependent chloride channels which are characterized by the following selectivity series: Br- greater than Cl- greater than NO3- greater than SO4(2-) greater than F- greater than HCOO- greater than CH3COO-. PMID- 1711485 TI - Cell type-specific and efficient synthesis of human cytokeratin 19 in transgenic mice. AB - In studies designed to identify cis-regulatory elements involved in the cell-type specific expression of human cytokeratin (CK) genes we have dissected from the major type I CK gene locus on chromosome 17 a region containing the gene that encodes CK 19, with flanking segments of different lengths, and have examined the expression of related gene constructs in transgenic mice. Adult transgenic mice have been characterized by immunohistochemistry, gel-electrophoretic analyses of cytoskeletal proteins and genomic DNA (Southern blots). We have found that a construct harbouring the transcriptional unit plus approximately 0.7 kb downstream from the polyA-addition site and an immediately adjacent 5' upstream segment of approximately 3.6 kb, when combined with a further 5' upstream element of -6.4 - -8.6 kb, is sufficient to guarantee the synthesis of human CK 19 in the same cells and to a similar extent as the murine genome expresses its endogenous CK 19 gene. The findings demonstrate that all cis-elements necessary for the specific and efficient expression of a single type I CK gene, in the context of epithelial differentiation, can be located in the vicinity of the gene itself and that more-distant elements are not required. PMID- 1711486 TI - Activation of the interferon system during myogenesis in vitro. AB - Differentiation of skeletal muscle involves withdrawal of myoblasts from cell replication, fusion to form multinucleated myotubes, coordinate appearance of a variety of muscle-specific proteins and the disappearance of a set of other proteins responsible for cell growth. The possible activation of the interferon (IFN) system in this process was studied. Thus, the activity of two IFN-induced enzymes known to be part of the system-(2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA-activated protein kinase as well as the expression of 2-5A synthetase coding genes were examined during myogenesis. It is demonstrated that the activity of the enzymes is transiently increased in cultured myoblasts, reaching a peak activity on the 3rd day in culture and then declining to a basal level. This peak activity precedes both cell fusion and the appearance of muscle-specific proteins--acetylcholine receptors (AChR) and creatine kinase. The same kinetics of 2-5A synthetase activity was evident in myoblasts from chick, rat or mouse origin. The enzymatic product appears to be primarily the trimer form of 2-5A, rather than a set of oligomers observed in enzymatic reactions performed on IFN-treated cells, including muscle cultures. The kinetics of 2-5A synthetase gene expression revealed that the largest amount of specific RNA transcripts appeared on the 1st day after seeding, followed by a reduction thereafter. In addition, a decrease was also observed in expression of c-myc, a cell-growth-associated protooncogene. However, an increase towards the 2nd day of both AChR and myosin light chain gene expression was evident, indicating selective regulation of gene expression during myogenesis. PMID- 1711487 TI - Coexpression of the c-myc protooncogene with alpha-fetoprotein and albumin in fetal mouse liver. AB - The level of mRNAs for the c-myc protooncogene and the serum proteins alpha fetoprotein (AFP) and albumin in liver, visceral yolk sac and gut between day 9 and day 19 of mouse gestation was studied by in situ hybridization employing single-stranded RNA probes. In the prehepatocyte population, c-myc was coexpressed with albumin and AFP. No heterogeneity was noted within this cell population with respect to the expression of these mRNAs up to day 15. AFP expression was high in the liver primordium and rose further until day 15. Albumin mRNA was expressed weakly but distinctly in the hepatic bud and increased throughout fetal life. C-myc expression in prehepatocytes exhibited a maximum around day 13 and a dramatic decline after day 15, but was much lower in other cell types of the fetal liver. In the visceral yolk sac, AFP was strongly expressed, with albumin expression first becoming detectable at day 13, while c myc mRNA was detected up to day 9. In the endodermal gut epithelium, c-myc expression was high, albumin mRNA was not detected and AFP message was restricted to individual loops of the gut. These results suggest that a period of high c-myc expression in the developing liver may allow rapid expansion of the prehepatocyte population at a specific stage of differentiation. PMID- 1711488 TI - [Percutaneous aortic valvuloplasty in adults: prediction of the immediate result and current indications]. AB - Here we present a series of 45 patients (21 M and 24 F) between the ages of 36 and 91 (average age: 71 +/- 8), who underwent Percutaneous Aortic Valvuloplasty (PAV) between Oct. 1986 and Dec. 1989. We used the traditional retrograde technique with balloon catheters sized 20 or 23 mm, with the exception of the first stage in which the kissing balloon technique was used in 7 cases. The calculated mean increase in aortic valve area (AVA) was 55.6 +/- 38% (from 0.49 +/- 0.11 cm2 to 0.74 +/- 0.07 cm2) and the peak gradient was reduced from 83 +/- 16 to 41 +/- 13 mmHg. We could observe only two relevant complications, i.e., two pulsating femoral artery haematomas at the site of catheter insertion. This artery underwent elective surgical resection two weeks after PAV. The dishomogeneity of the survey, due not only to the complexity of the valvular stenosis functional anatomy, but also to the changes in the PAV indications observed during the three-year period, led us to appraise our results by using a score based on the following features: valvular calcification degree (0-2); commissural fusion extent (0-4); bicuspid of tricuspid valve (0-2); and predilatation valve area less than 0.5 or greater than or equal to 0.5 cm2. In this way we were able to identify two groups of patients, one having a score of less than or equal to 6 (group I, 25 patients) and the other having a score of greater than or equal to 8 (group II, 20 patients). Mean AVA increase was 29% in group I and 84% in group II. At 24 +/- 6 months clinical follow-up, a significant discrepancy was maintained; the two groups showed a 5% and a 37.5% improvement, respectively. The score we suggest seems to single out cases with a high likelihood of success, i.e. the achievement of an AVA higher than 0.9 cm2. This seems to be helpful for a better selection of patients. Using this score as the basis for such an immediate result predictability, we believe that PAV could be advisable in the following cases: a) palliation for elderly patients (greater than 80 years) or patients with contraindications for valve replacement; b) as a bridge to surgical intervention; c) emergency procedures such as bailout valvuloplasty; d) diagnostic clarification in the most complex cases where a severe reduction in ventricular function and cardiac output, together with a low transvalvular gradient are present. PMID- 1711489 TI - [Surgical therapy of progressive or recurrent malignant ovarian tumors]. AB - Out of a total of 120 patients operated on for recurrent ovarian cancer, two at the very best, but possibly not even one, will have a definitive chance of cure. Despite the poor long-term prognosis, as well as the lengthy operation and postoperative treatment involved, it does not seem justified to withhold surgery for recurrent disease totally. In some cases, symptoms can be treated with surgery, such as tumour pain or an impending ileus. In other cases, patients live for 10 years and longer, after multiple operations for relapse, without suffering severe physical symptoms. These are mainly patients with circumscribed, solitary, and very slowly growing tumours, in which cases, it is possible to remove the tumour again and again by surgery. The most relevant prognostic factors include the size of the residual tumour left at the primary operation, the time between the primary operation and the recurrence of the tumour, the type of growth of the recurrent tumour, as well as the extent of the tumour size reduction achieved at the first recurrence operation. PMID- 1711490 TI - [Discolored amniotic fluid--results of prenatal diagnosis and clinical significance]. AB - 7000 pregnancies were analysed after genetic amniocentesis in respect of the further course and results of prenatal diagnosis (observation period 1975-1988). In 3.1% (217 cases) samples of amniotic fluid were discoloured. Vaginal haemorrhages prior to amniocentesis were recorded with significantly higher incidence (18%) in patients with discoloured amniotic fluid than in a control group (n = 217) with normal colour of the amniotic fluid (4.6%) (p less than 0.001). Miscarriages and chromosome anomalies occurred more often in the study group (3.7%/2.8%, control group: 0.9%/1.8%, n.s.). The risk of miscarriages was increased in cases with sanguineous amniotic fluid if the amniotic fluid alpha fetoprotein values were enhanced at the same time. Significant differences were observed in respect of the incidence of foetal malformations in patients with discoloured amniotic fluid (7.8%) and in the control group (2.3%) (p less than 0.01). Borderline or definitely pathological amniotic fluid AFP concentrations were found often if the amniotic fluid was discoloured (6.9%, control group 3.2%). If discoloured amniotic fluid was sampled, foetal malformations should be excluded sonographically. In 82% of all cases with discoloured amniotic fluid and foetal malformations pathological sonographic findings and/or enhanced MS-AFP values were recorded even prior to amniocentesis. PMID- 1711491 TI - [Effect of infusion hemodilution on the indicators of the blood coagulation system in children during planned thoracic operations]. AB - The blood coagulation system and fibrinolysis were studied in 16 children with chronic bronchopulmonary diseases, aged from 4 to 15 years, before surgery, immediately, and on the first day after the operation. The method of infusion hemodilution, under the control of hematocrit, hemoglobin and total protein, was used for the treatment of operation hemorrhage. The investigations conducted have revealed hypercoagulation signs in the patients before the beginning of the operation (as compared to normal children). Operative intervention has intensified many signs of hypercoagulation, however, in the postoperative period no clinical signs of hypercoagulation were observed in all the cases, that was associated with the positive effect of hemodilution. PMID- 1711492 TI - The effects of acute and extended monoamine oxidase inhibition upon 5 hydroxyindoles in maturing mouse brain. AB - 1. Mice of four ages between newborn and adult were exposed to the monoamine oxidase inhibitor amiflamine both acutely and in an extended (5 day) regimen. Brains were then assayed at various times following amiflamine for changes in the levels of serotonin (5-HT, 5-hydroxytryptamine) and 5-hydroxyindole acetic acid (5-HIAA). 2. Although both metabolites initially changed as might be expected, with 5-HT elevating and 5-HIAA decreasing, the younger brains recovered their 5 HT levels slower than older brains and eventually young brains had levels of 5 HIAA that were in excess of normal. At some times both metabolites were in excess of normal at younger ages. 3. These results are compared to changes seen with the 5-HT uptake inhibitor citalopram and it is concluded that in young brain 5-HIAA levels lack firm regulatory control and are not passive reflections of 5-HT changes. PMID- 1711493 TI - Calcitonin gene-related peptide immunoreactivity in the hypothalamo-hypophysial system of the green frog, Rana esculenta. AB - The distribution of calcitonin gene-related peptide (CGRP) has been investigated in the hypothalamus of the frog Rana esculenta L. by means of different immunohistochemical techniques. A few immunopositive cell bodies and several fibers have been demonstrated in the preoptic area and in the caudal hypothalamus. Some CGRP-like fibers were also recognized in the outer zone of the median eminence. Simultaneous double immunofluorescence methods showed CGRP-like immunoreactivity to be often contiguous to substance P-like positive structures, but separated from them. PMID- 1711494 TI - Immunological relationships between neuropeptides from the sinus gland of the lobster Homarus americanus, with special references to the vitellogenesis inhibiting hormone and crustacean hyperglycemic hormone. AB - Antisera raised in guinea pigs against four major neuropeptides purified from sinus glands of the lobster, Homarus americanus, were used to study the immunological relationships between several sinus gland peptides. On the basis of their behavior in ELISA and in absorption procedures, three groups of peptides are defined. Two groups may be related to the crustacean hyperglycemic hormone (CHH groups); the third one is composed of three immunologically identical peptides and, since one of these peptides was characterized in previous studies as a vitellogenesis inhibitor, is referred to as VIH group. This closely meets our present knowledge about the physiological effects and biochemical characteristics of these neuropeptides and gives immunological insights on the question of molecular polymorphism of lobster neurohormones. PMID- 1711495 TI - Identification and initial characterization of glucose-repressible promoters of Streptococcus mutans. AB - Three catabolite-repressible promoters from Streptococcus mutans have been isolated. These promoters were identified by utilizing the vector pRQ200 which contains a promoterless amylase-encoding gene, a Gram- origin of replication, and an erythromycin-resistance determinant. A library of S. mutans DNA was constructed in pRQ200, amplified in Escherichia coli and integrated by Campbell type insertion into the S. mutans chromosome following transformation. Colonies exhibiting amylase production on media lacking an extraneous carbohydrate source were screened for diminished amylase production on media containing glucose. The effect of glucose on these promoters has been characterized using a quantitative spectrophotometric assay of amylase activity. PMID- 1711496 TI - Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen. AB - A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies. PMID- 1711497 TI - Cellular transcripts encoded at a locus which permits retrovirus expression in mouse embryonic cells. AB - Three independent recombinant retroviruses have been activated on insertion into the F2 locus of mouse F9 embryonal carcinoma cells. Each provirus has integrated downstream from the cellular F2 promoter, which is active in transient transfection assays using a chloramphenicol acetyltransferase reporter enzyme. The F2 promoter drives expression of a series of related transcripts in F9 and 3T3 cells, and a single 450-nt transcript in mouse tissues. F2 homologous sequences have been detected in the genomes of all mammalian species tested, and the 450-nucleotide (nt) F2 transcript is expressed in rat and human cells. Three pairs of differently sized F2 cDNA clones have been isolated and analyzed. The largest clones possess two 199-nt 98.5% identical repeats, one of which is present in the smaller clones, as well as the major 450-nt transcript. Activated proviral integration sites map to introns of the largest F2 cDNA clone. While none of the F2 cDNA contains a long open reading frame or homology to databank sequences, evidence suggests that the F2 locus encodes a constitutive function required at high levels, or represents an expressed but nonfunctional, single copy element, conserved among mammals. PMID- 1711498 TI - The structure and expression of a gene encoding chick claw keratin. AB - A cDNA library was constructed from embryonic chick claw mRNA and a claw keratin (cKer)-encoding clone was isolated and sequenced. Subsequently, a genomic clone, containing four cKer-encoding genes (cKer) was isolated and one of the genes (cKer1) was completely sequenced. The cKerl gene appears to be differentially expressed in the keratinizing tissue appendages of the embryonic chick, being abundantly expressed in the claw and at a low level in feather tissue. Comparison of the deduced amino acid (aa) sequence of the cKer to those of feather (fKer) and scale keratins (sKer) showed that the regions conserved between fKer and sKer are also found in the cKer. The glycine-rich as repeat region characteristic of sKer is also present in a shortened form in the cKer sequence. Like the fKer genes (fKer) and the feather histidine-rich protein-encoding gene (HRP), the cKer1 gene also contains one intron which interrupts the 5'-noncoding region at an equivalent position to that found in the fKer and HRP genes. Genomic Southern analysis using the cKer cDNA as a probe indicated the presence of several related genes in the chick genome. PMID- 1711499 TI - Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen. AB - Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV. PMID- 1711500 TI - [Comparative evaluation of the effects of sulfocarbathione on the gonads of male and female rats]. PMID- 1711501 TI - [Visual aids in health education of the population in outpatient clinics]. PMID- 1711502 TI - [Knowledge and ethics in gynecologic oncology]. AB - The ethical problems of prospectively randomized clinical studies are discussed. It is concluded that such studies are necessary even for ethical reasons. Furthermore the problem of long-term treatment of cancer patients is reviewed; the question arises as to when withdrawal of curative chemotherapy is ethically justifiable. PMID- 1711503 TI - Alpha-interferon therapy in advanced unresponsive immunoblastic lymphoma evolved from Waldenstrom's macroglobulinemia. PMID- 1711504 TI - ACL automated method for plasma inhibitors. PMID- 1711505 TI - MN (N = Novantrone) COP-B therapy on high and intermediate grade non-Hodgkin's lymphoma: an alternative to MA (A = adriamycin) COP-B? AB - BACKGROUND AND METHODS: From February, 1987 to August, 1988, fifty-one patients with high and intermediate grade non-Hodgkin's lymphoma (NHL) entered a chemotherapy program MNCOP-B (M = Methotrexate, N = Novantrone, C = Cytoxan, O = Oncovin, P = 6 methyl-prednisolone, B = Bleomycin). Forty patients received MNCOP B as first-line therapy and 11 patients as salvage therapy after relapse resistance to other schemes or radiotherapy. RESULTS: The overall remission rate was 51% with a similar distribution of remissions between the two groups under study. At a mean follow-up of 28 months, 20 patients (40%) remain in CR with a disease-free probability of 83% for patients treated with MNCOP-B as up-front therapy and 67% for patients treated with MNCOP-B as salvage therapy. The overall survival at 3 years is 45%; bulky, disease stages III-IV and symptoms are not correlated with poorer survival or a lower remission rate. Patients who received less than 70% of the scheduled doses paradoxically figure better than the others, showing an 87% remission rate, as compared to those who received 100% of the scheduled doses and obtained a 35% remission rate. Major toxicity consisted of mucositis, recorded in 15% and 9% of previously untreated and treated patients, respectively; severe neutropenia was recorded in 27% and 63%, respectively. Despite the number of recorded side effects, only one toxic death due to sepsis occurred. CONCLUSIONS: MNCOP-B is an effective regimen in intermediate and high grade NHL and easily manageable in the out-patient clinic. The incidence of mucositis is sensibly reduced as compared to the parental regimen MACOP-B reported in the literature; this result allows the use of MNCOP-B in older patients. PMID- 1711506 TI - Evaluation of factors predisposing to arterial thrombosis in coronary artery disease. AB - Estimation of antithrombin III, alpha 2 macroglobulin and alpha 1 antitrypsin in patients with stable and unstable angina and acute myocardial infarction (15 cases each) were carried out. Twenty age, sex and weight matched healthy subjects were included as controls. Mean platelet factor 4(PF4) levels measured in 10 cases of each subgroup were significantly elevated in myocardial infarction (MI) (48.4 +/- 15.16 ng/ml) and III unstable angina patients (44.7 +/- 15.9 ng/ml) as compared to controls (25.42 +/- 12.47 ng/ml; P less than 0.01). Mean antithrombin III (AT III) levels were markedly reduced in all patients with MI (39.65 +/- 12.8% of normal pooled plasma) and unstable angina (37.9 +/- 16.6% of normal pooled plasma) and in 9 patients with stable angina. Alpha I antitrypsin and alpha 2 macroglobulin levels in these cases showed no significant difference compared to normals. Reduced AT III in coronary artery disease suggests a prethrombotic tendency in these patients. Raised PF4 levels in acute phase of the disease suggests heightened platelet activation. PMID- 1711507 TI - Conformation of alginate and pectate chains monitored by the binding of the dye stains-all. AB - The carbocyanine dye Stains-all displays spectral colour shifts or metachromasia upon binding to proteins, polysaccharides and other anionic substrates. The conformational status of the binding region of the substrate appears to govern the metachromatic features of the bound Stains-all. We have used this property to derive information about the conformational differences between the two anionic polysaccharides alginate and pectate. The stronger induction of circular dichroism in the 500 nm region of the dye by pectate is indicative of a greater extent of helical order in this polymer in solution than in the alginate or perhaps even hyaluronate chains. PMID- 1711508 TI - T cells regulate the IgM antibody response of BALB/c mice to dextran B1355. AB - The IgM antibody response of BALB/c mice to bacterial (Leuconostoc) dextran B1355 is influenced in a positive and negative manner by regulatory CD4+ and CD8+ T cells, respectively. Treatment with concanavalin A (ConA) at the time of immunization or 2 days later caused suppression and enhancement of the antibody response, respectively. Priming of mice with a sub-immunogenic dose of dextran resulted in profound suppression upon subsequent immunization 3 days later. None of these effects were demonstrable in athymic mice. Transfer of T cells from mice primed 18 h previously with a subimmunogenic dose of dextran suppressed the antibody response in immunized recipients; such suppression was abolished by the treatment of transferred cells with anti Thy 1.2 or anti Lyt 2.2 (CD8) antibody in the presence of complement. By contrast, the transfer of T cells from mice, which had been given an immunogenic dose of dextran 4 days previously, increased the antibody response in immunized recipients; such enhancement was abolished by treating transferred cells with anti Thy 1.2 or anti L3T4 (CD4) antibody in the presence of complement. These findings indicate that the immune response to dextran B1355 is regulated by CD4+ T-amplifier cells (Ta cells) and by CD8+ T suppressor cells (Ts cells) which are activated during the course of a normal antibody response. PMID- 1711509 TI - The effect of alpha 2 macroglobulin-proteinase complexes on macrophage Ia expression in vivo. AB - Alpha-2-Macroglobulin (alpha 2M) is a major plasma proteinase inhibitor. It can also regulate the function of cells of the immune system, including macrophage expression of Ia antigens in tissue culture systems. The present work was done to assess the effect of alpha 2M-trypsin complexes (alpha 2M-t) on macrophage Ia expression in vivo. Bacillus Calmette-Guerin-infected mice were injected intraperitoneally with 100nM alpha 2M-t, phosphate buffered saline (PBS), or bovine serum albumin (BSA) in PBS. The peritoneal cells were harvested by lavage from 3 to 6 days after injection. Differential cell counts were performed, and macrophage Ia antigen expression determined by indirect immunofluorescence. Injection of either alpha 2M-t or BSA solutions tended to increase the number of total cells and lymphocytes harvested, without changing the number of macrophages harvested. alpha 2M-t injection reduced the proportion of macrophages which were Ia positive from 60 to 37% on day 3 after injection, and to 20% Ia positive on day 6. The reduction in Ia positive macrophages was statistically significant when compared to either PBS or BSA injected groups. In summary, in vivo exposure to alpha 2M-t can alter macrophage function. alpha 2M-proteinase complexes formed during the course of coagulation or inflammation may play a physiologic role as regulators of the immune response. PMID- 1711510 TI - Patterns of lymphokine production by primary antigen-specific/MHC restricted murine helper T cell clones. AB - In this study we examined a panel of CD4+ antigen specific/MHC restricted T cell clones for their ability to secrete IL-2, IL-4, and IFN-gamma upon stimulation with con A, three lymphokines which are diagnostic for the TH1 and TH2 subtypes of helper T cells. Eight of the twelve clones we analyzed did not fit the classical TH1/TH2 patterns of lymphokine secretion. Seven of these clones secreted both IL-2 and IL-4 and two of these also produced IFN-gamma. The remaining non-classical clone secreted IL-4 and IFN-gamma but not IL-2. Data from the subcloning of the IL-2/IL-4/IFN-gamma triple producers were not consistent with the parental lines being a mixture of TH1 and TH2 cells. The IL-2/IL-4 double producers (IFN-gamma negative) cannot be explained by the parental lines being a mixture of the TH1 and TH2 subtypes. Nevertheless, these double producers were subcloned and the results provided convincing evidence that clones which secrete both IL-2 and IL-4 do exist. Lymphokine loss variants involving IL-2, IL 4 or IFN-gamma were observed among subclones derived from the double and triple producers as well as in several parental lines maintained in continuous culture. We also observed the appearance of inducible IFN-gamma production in some subclones derived from parental clones where production of IFN-gamma was not detectable. The phenotypes of these variants failed to indicate an obvious trend toward the TH1 and TH2 subtypes. Thus, our results suggest that more heterogeneity in the population of CD4+ helper T cells exists than can be explained by the TH1 and TH2 subtypes of these cells. PMID- 1711511 TI - Major acute-phase reactant synthesis during chronic inflammation in amyloid susceptible and -resistant mouse strains. AB - Hepatic levels of mRNA specific for total serum amyloid A (SAA), the SAA1 and SAA2 isotypes, serum amyloid P (SAP), C-reactive protein (CRP), and fibronectin, as well as the plasma concentrations of SAA and SAP were examined in amyloid resistant (A/J) and amyloid-susceptible (CBA/J) mice during azocasein-induced chronic inflammation. In both strains hepatic SAA and SAP mRNA levels and plasma SAA and SAP protein concentrations increased dramatically during the early stages of inflammation; this was followed by a decrease to concentrations that were maintained at levels considerably higher than background. The ratios of SAA1 and SAA2 mRNA and plasma protein were 1:1 throughout. This indicated that there was no preferential accumulation of mRNA specifying a particular isotype and no preferential synthesis or clearance of a particular isotype during chronic inflammation and the early stages of amyloidogenesis in either strain. Similarly, hepatic SAP mRNA levels in both strains increased dramatically during the early stages of inflammation and were subsequently maintained at elevated levels. Plasma SAP concentrations increased rapidly during the first three days of the study in both A/J and CBA/J mice; however, during the later stages of inflammation, A/J plasma SAP levels decreased to a steady-state concentration that was approximately half that observed in CBA/J mice. Our results identify differences in the hepatic mRNA and plasma protein levels of the major mouse acute-phase reactants (APR) in the amyloid-resistant A/J and amyloid-susceptible CBA/J mouse strains. These findings are consistent with circulating inflammatory APR concentrations contributing, together with other factors, to the onset and pathogenesis of secondary amyloidosis. PMID- 1711512 TI - Effect of alpha-2-macroglobulin on cytokine-mediated human C-reactive protein production. AB - Alpha-2-macroglobulin (alpha 2-M), a serum protease inhibitor that also binds cytokines, neutralized the inhibitory effect exerted by transforming growth factor-beta (TGF-beta) on IL-6-induced C-reactive protein (CRP) production by the human hepatoma cell line PLC/PRF/5. alpha 2-M was found to bind noncovalently with TGF-beta to form a complex that, upon acidification, released TGF-beta inhibitory activity as detected by IL-6-induced CRP production. Although alpha 2 M also binds IL-6, it did not alter IL-6-induced CRP production by the hepatoma cells. The interaction between alpha 2-M and TGF-beta may influence the production of acute-phase proteins by liver hepatocytes. PMID- 1711514 TI - Baroda development screening test for infants. AB - A screening test for the assessment of the motor-mental development of infants was developed by selecting items from the Bayley Scales of Infant Development (BSID). Baroda norms as a simple and quick test for use in the door to door survey by health workers. The reason for choosing BSID and the criteria for the selection of items are described. The method of using the screening test in community surveys (by health workers) and in office practice is discussed. Some aspects of the development of our screening test and the Denver Development Screening Test (DDST) are compared. A routine use of our test is recommended for following the development of normal children as well as for screening from the community children with possibility of development delay. The latter must be referred for detailed testing on the full scales. PMID- 1711513 TI - Distribution and localization of inter-alpha-trypsin inhibitor and its active component acid-stable proteinase inhibitor: comparative immunohistochemical study. AB - Inter-alpha-trypsin inhibitor (ITI) is a complex that consists of three components. One of these is the acid-stable proteinase inhibitor (ASPI), which is an acute-phase reactant and a broad-spectrum inhibitor. The tissue distribution of ITI and ASPI were investigated and compared using immunohistochemical methods. ITI immunoreactivity was revealed only in the liver and plasma, while ASPI immunoreactivity was found to be distributed in the brain, liver, kidney, gastrointestinal tract, plasma, and urine. Both immunoreactivities were demonstrated in Kupffer cells of the liver, which is thought to be an ITI producing organ. From these results, it seems unlikely that ASPI is distributed as a part of the ITI molecule. The residual component of ITI may act as a carrier protein of ASPI, or ASPI in the tissues may be produced independently of ITI. PMID- 1711515 TI - Bombesin-induced pancreatic secretion and growth in rats: effect of proglumide, spantide and ranitidine. AB - The effect of proglumide (400 mg/kg), spantide (400 g/kg) and ranitidine (20 mg/kg) on pancreatic secretory and trophic response to bombesin (10 micrograms/kg) was studied in the rat. Drugs were administered alone or combined with bombesin three times daily for 5 days. Saline-treated rats were used as controls. At the end of treatment, animals were anaesthetized and pancreatic juice was collected for 1 h after caerulein stimulation (1 microgram/kg intraperitoneally). Afterwards, rats were sacrificed and the weight and composition of pancreatic tissue were determined. As compared with control (saline) values, the volume of pancreatic juice and the output of trypsin and amylase were increased by treatment with bombesin. Neither proglumide nor spantide affected basal and caerulein-stimulated pancreatic exocrine secretion. Ranitidine, although unable to modify protein and enzyme content of pancreatic secretion, significantly reduced the volume of pancreatic juice in both basal conditions and after caerulein stimulation. Bombesin increased pancreatic weight as well as the protein and enzymatic contents of the gland. Neither the weight of the pancreas nor its composition were significantly affected by CCK-antagonist proglumide, the putative bombesin antagonist spantide or the H2-receptor antagonist ranitidine. These results show that bombesin has a trophic effect on rat pancreas and concomitantly increases its secretory capacity. Both effects are likely to be mediated through a direct action of the peptide on the pancreatic gland. PMID- 1711516 TI - A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium. AB - The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/ 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells. PMID- 1711517 TI - The effect of total-body irradiation on corneal neovascularization in the Fischer 344 rat after chemical cauterization. AB - Previous investigations of corneal neovascularization after irradiation yielded discordant results. Most studies indicated that new blood vessel formation in the cornea is inhibited by irradiation. However, others reported that angiogenesis after corneal cauterization is similar in both irradiated and nonirradiated animals. To assess the effect of total-body irradiation on neovascularization further, the amount of angiogenesis was determined in irradiated rats after chemically induced corneal injury. Corneal tissue was evaluated quantitatively with computerized image analysis 2, 3, or 4 days postcautery in rats perfused with India ink and gelatin immediately after death. The rats were exposed to a single dose (9 Gy) of total-body irradiation 6 days before corneal cauterization. In both the nonirradiated and irradiated rats, neovascularization increased with the duration of the postcautery interval. The amount of corneal neovascularization was not significantly different in the irradiated and nonirradiated rats at any of the postcautery intervals studied. This investigation suggests that endothelial cell migration plays a more important role than cell replication in the pathogenesis of corneal angiogenesis in the Fischer 344 rat. Moreover, the suppression of corneal angiogenesis by irradiation may be dependent on the experimental conditions and species examined. PMID- 1711518 TI - Polymorphism of the human CD46 gene in normal individuals and in recurrent spontaneous abortion. AB - Restriction fragment length polymorphism analysis of the human CD46 gene, which encodes a cell surface complement regulatory protein, has been performed using a 1.5-kb cDNA probe containing a complete CD46 coding sequence. The sum of the invariant band sizes indicates that this gene encompasses at least 35-kb genomic DNA. Polymorphisms were detected in normal individuals with both HindIII and EcoRI. Several of the polymorphic bands occurred significantly less frequently in recurrent spontaneous abortion individuals. Northern blot analysis has shown the predominant RNA transcript size to be 4.2 kb in trophoblast-derived cell lines and peripheral blood mononuclear cells, indicating these transcripts may contain an unusually long 3'-untranslated region. PMID- 1711519 TI - Differentiation of the genus Listeria from other gram-positive species based on low molecular weight (LMW) RNA profiles. AB - Low molecular weight RNA (LMW RNA; 5S rRNA and tRNAs) profiles of several Gram positive species were generated on 9% denaturing polyacrylamide gels. The profiles of five Listeria spp. (L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri) were identical when cultured in three different media (APT, BHI and TSBYE), either shaken or statically, both at 30 and 37 degrees C. Twenty-six strains from 15 other species representing seven different genera were also compared. Each species exhibited a characteristic profile. Strain variants of the same species gave identical profiles. The technique represents a simple, reproducible approach to the identification of species and possibly of relationships between species. The taxonomic and phylogenetic implications, particularly with respect to Listeria spp., Brochothrix thermosphacta and the lactic acid bacteria, are considered. PMID- 1711520 TI - Anaphylactoid reactions to glycopeptide antibiotics. AB - Anaphylactoid reactions to vancomycin have been reported consistently since the earliest clinical trials. Signs and symptoms of the reaction, which usually occur during the first dose, can range in severity from mild pruritus and upper body flushing, to dramatic hypotension and cardiovascular arrest. The frequency of the reaction, and its severity, is proportional to the total dose administered and the infusion rate. Recent prospective investigations report that when 1 g of vancomycin is administered over 60 min to normal volunteers and infected patients, between 50-90% of adults will have a reaction, although most are mild and of little clinical consequence. Vancomycin causes a release of histamine into blood, and the severity of the reaction is proportional to the amount of histamine released. Tachyphylaxis rapidly develops in most persons, although decreasing the dose, or increasing the infusion time will also help alleviate the signs and symptoms. Antihistamine H1 blocking agents are also effective in preventing the reaction. Teicoplanin, a new glycopeptide antibiotic, does not appear to cause histamine release or related symptoms, even when administered at a more rapid rate than vancomycin. PMID- 1711521 TI - Antioxidants attenuate endotoxin-induced microvascular leakage of macromolecules in vivo. AB - The purpose of this study was to examine whether antioxidants attenuate endotoxin induced microvascular hyper-permeability for macromolecules in the hamster cheek pouch. Twenty-two adult male Syrian hamsters were anesthetized, and a removable plastic chamber was placed in the cheek pouch to observe and collect suffusate from the microvasculature. Fluorescent-labeled dextran (FITC-D; mol wt 150,000) was injected intravenously, and changes in the number of microvascular leaky sites and microvascular clearance of FITC-D were measured in five groups: saline control (group 1, n = 4), endotoxin (0.1 mg/ml) suffusion for 120 min (group 2, n = 6), endotoxin plus dimethyl sulfoxide (1.0 g/kg iv; group 3, n = 4), endotoxin plus allopurinol (30 mg/kg ip; group 4, n = 4), and endotoxin plus dimethyl sulfoxide and allopurinol (group 5, n = 4). The number of leaky sites and the FITC-D clearance were significantly higher in group 2 [45 +/- 18 (SD) sites/cm2 and 20 +/- 6 X 10(-6) ml/min, respectively; P less than 0.01] than in group 1 (7 +/- 6 sites/cm2 and 7 +/- 5 X 10(-6) ml/min), group 3 (9 +/- 5 sites/cm2 and 8 +/ 2 X 10(-6) ml/min), group 4 (11 +/- 7 sites/cm2 and 9 +/- 4 X 10(-6) ml/min), and group 5 (11 +/- 6 sites/cm2 and 7 +/- 1 x 10(-6) ml/min). The leaky sites appeared predominantly in postcapillary venules. There was a positive and significant correlation between the number of leaky sites and FITC-D clearance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711522 TI - Functional assessment of pulmonary capillary surface area in the 2-mo-old lamb. AB - We recently reported that measurements of the maximal velocity of pulmonary endothelial angiotensin-converting enzyme (Vmax) in vivo provide information regarding microvascular surface area in the developing lamb. To obviate any subtle influences of development on Vmax aside from simple increases in surface area, we correlated Vmax with postmortem stereological assessments of alveolar surface area in the relatively mature lung of the 2-mo-old lamb (n = 14). We attempted to increase the range of surface area beyond its normal variability by injecting nine of the lambs with bleomycin, an antineoplastic agent with significant pulmonary toxicity in other species. Vmax, measured shortly after birth and then weekly, increased monotonically in all lambs. Despite their wide dispersion, Vmax and the stereological determinations correlated strongly at 2 mo of age, confirming that Vmax is a robust indicator of the surface area of the air blood barrier. There was no significant difference in either measurement between the control lambs and those treated with bleomycin, suggesting that the newborn lamb is resistant to the effect of this agent. PMID- 1711523 TI - Effects of substance P and calcitonin gene-related peptide on the pulmonary circulation. AB - To assess the in vivo effects of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) on the pulmonary vascular bed, the hemodynamic responses to both CGRP and SP were examined in the in situ-perfused lung lobe of open-chest anesthetized pigs. Peptides were infused into the lobar artery under conditions of elevated pulmonary vascular tone by prostaglandin F2 alpha (PGF2 alpha, 20 micrograms/min). Pulmonary airway lobar dynamic compliance (Cdyn) and airway resistance (Re) were computed from simultaneously measured airway pressure and airflow entering the lobe through a Carlens endobronchial divider. PGF2 alpha infusion slightly reduced Cdyn (-20%) and increased Re (+11%) while lobar arterial pressure rose from 14 +/- 1 to 31 +/- 2 mmHg (n = 12). In these conditions, lobar artery infusion of SP (0.5-50 pmol/min) or CGRP (15-5,000 pmol/min) produced a dose-dependent decrease in the pressor response to PGF2 alpha, reaching -54 +/- 3 and -64 +/- 7%, respectively, without alterations in lung mechanics. On a molar basis, SP was more effective than CGRP; its vasodilatory effect was more rapid and of shorter duration. Higher CGRP infusion rates were not studied because of marked systemic hypotension. SP infused at 150, 500, and 1,000 pmol/min significantly reduced Cdyn by 12 +/- 2, 24 +/- 4, and 62 +/- 7%, respectively, but also induced a rise in lobar arterial pressure and a fall in systemic arterial pressure. The results show that both SP and CGRP are potent pulmonary vasodilators. In contrast to CGRP, which did not affect lung mechanics, high infusion rates of SP decreased Cdyn and increased Re.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711524 TI - Cognitive and social cognitive development of depressed children and adolescents. AB - Depressed juveniles show evidence of functional impairment in various cognitive and social domains. Actual school performance seems to be more consistently affected by depression than cognitive and intellectual abilities. In addition, depressed youth appear to be less socially adept than nondepressed peers, although depression does not consistently impair social-cognitive abilities. Indications that depressed youth show mild declines in tested verbal performance over time and that residual problems in social functioning persist after symptomatic recovery suggest that major depression may have negative effects on development in childhood. PMID- 1711525 TI - Effects of soluble factors and extracellular matrix components on vascular cell behavior in vitro and in vivo: models of de-endothelialization and repair. AB - Vessel walls are comprised of several different cell populations residing in and on complex extracellular matrices. Each of the vascular cell types has diverse and sometimes unique functions and morphologies, and each has roles in repair processes following injury. Large vessel endothelial cells are known to respond to denudation injury by sheet migration and proliferation. This is in contrast to the migration through soft tissues with tube formation and subsequent lumen formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells of larger vessels respond to injury by migration from the arterial media into the intima, proliferation, and matrix biosynthesis, ultimately causing intimal thickening. Both these cell types exhibit "dysfunctional" phenotypes during their responses to injury. Microvascular cell responses to injury, while extremely variable, are less well documented. Specifically, responses to injury by microvascular endothelial vascular cells appear to be modulated, in part, by the composition and organization of the surrounding matrix as well as by the various soluble factors and cytokines found at sites of injury, suggesting that the extracellular matrix and soluble factors modulate each other's effects on local vascular cell populations following injury. PMID- 1711526 TI - Structure-function studies of heparin-binding (acidic fibroblast) growth factor-1 using site-directed mutagenesis. AB - The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-tupe HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711527 TI - Lectin cell adhesion molecules (LEC-CAMs): a new family of cell adhesion proteins involved with inflammation. AB - The means by which leukocytes, including lymphocytes, monocytes, and neutrophils, migrate from the circulation to sites of acute and chronic inflammation is an area of intense research interest. Although a number of soluble mediators of these important cellular interactions have been identified, a major site of great importance to the inflammatory response is the physical interface between the white cell and the endothelium. This critical association is mediated by an array of cell surface adhesion molecules. Previous data have demonstrated that the integrin subfamily of heterotypic adhesion molecules was a major component of these adhesive interactions, although it was clear that other, non-integrin-like molecules of unknown identity also seemed to be involved during the inflammatory process. A number of these other cell-surface glycoproteins which may be involved with inflammation have recently been characterized by molecular cloning. These glycoproteins, including the peripheral lymph node homing receptor (pln HR), the endothelial cell adhesion molecule (ELAM), and PADGEM/gmp140, are all members of a family of proteins which are unified by the inclusion of three characteristic protein motifs: a lectin or carbohydrate recognition domain, an epidermal growth factor (egf) domain, and a variable number of short consensus repeats (scr) which are also found in members of the complement regulatory proteins. The appearance of lectin domains in all of these adhesion molecules is consistent with the possibility that these glycoproteins function by binding to carbohydrates which are expressed in a cell and/or region specific manner, and the members of this adhesion family have been given the generic name LEC-CAM (lectin cell adhesion molecules).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711528 TI - GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium. AB - GMP-140 is a membrane glycoprotein located in secretory granules of platelets and endothelium. When these cells are activated by agonists such as thrombin, GMP-140 is rapidly translocated to the plasma membrane. GMP-140, along with ELAM-1 and the peripheral lymph node homing receptor, defines the selectin family of structurally related molecules that regulate interactions of leukocytes with the blood vessel wall. Each of these molecules contains an N-terminal lectin-like domain, followed by an EGF-like region, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The genomic structures of the selectins suggest that they arose by duplication and modification of exons encoding specific structural domains. GMP-140 is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to endothelium at regions of tissue injury as well as platelet leukocyte interactions at sites of inflammation and hemorrhage. Like other leukocyte adhesion molecules, GMP-140 may also participate in pathologic inflammation, thrombosis, and tumor metastasis. Confirmation of such pathologic roles may lead to design of new drugs that block adhesive receptor function in human disease. PMID- 1711529 TI - Extracellular matrix-resident basic fibroblast growth factor: implication for the control of angiogenesis. AB - Despite the ubiquitous presence of basic fibroblast growth factor (bFGF) in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues, suggesting that the extracellular matrix (ECM) may serve as a reservoir for bFGF. Moreover, functional studies indicated that bFGF is an ECM component required for supporting endothelial cell proliferation and neuronal differentiation. We have found that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by heparanase expressed by normal and malignant cells (i.e. platelets, neutrophils, lymphoma cells). It is proposed that restriction of bFGF bioavailability by binding to ECM and local regulation of its release provide a novel mechanism for neovascularization in normal and pathological situations. The subendothelial ECM contains also tissue type- and urokinase type-plasminogen activators which participate in cell invasion and tissue remodeling. These results and studies on the properties of other ECM-immobilized enzymes (i.e. thrombin, plasmin, lipoprotein lipase) and growth factors (GM-CSF, IL-3, osteogenin), suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized and persistent mode of action, as compared to the same molecules in a fluid phase. PMID- 1711530 TI - Taurine release associated to volume regulation in rabbit lymphocytes. AB - Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4 degrees C, but was unchanged at 15 degrees C or 25 degrees C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested. PMID- 1711531 TI - The polarized epithelial phenotype is dominant in hybrids between polarized and unpolarized rat thyroid cell lines. AB - We have studied the expression of cell polarity in hybrids between two rat thyroid epithelial cells: FRT and FRTL-5. FRT cells are polarized but do not express tissue-specific properties, FRTL-5 are unpolarized and express many thyroid-specific genes. A and express many thyroid-specific genes. A pool of 170 hybrid clones and five independent clones were characterized. The chromosome complement was that expected from 1:1 fusion of the parental cells. No chromosome loss was observed for several generations. All hybrids were polarized as judged from: (1) morphology, (2) transepithelial resistance, (3) preferential secretion of several proteins either through the apical (e.g. thyroglobulin) or through the basolateral pole, and (4) basolateral trapping of iodide. On the other hand, the expression of thyroid-specific markers: thyroglobulin synthesis and secretion, trapping of iodide, thyrotropin-dependent growth and expression of specific membrane antigens, were greatly reduced or inhibited in the pool and in the isolated clones. We also found that reduction of thyroglobulin synthesis was correlated with the loss of activity of the trans-acting factor TgTF1. We conclude that cell polarity, a property of FRT cells, is dominant in the hybrids whereas thyroid differentiation is recessive. PMID- 1711532 TI - Immortalization of male genital duct epithelium: an assay system for the cystic fibrosis gene. AB - The epithelia lining the vas deferens and epididymis are directly involved in the pathology of the autosomal recessive disease cystic fibrosis (CF). We have established culture systems for these epithelial cells. Long-term cell lines have now been generated from these primary epithelial cells by transformation with a plasmid containing an origin-defective simian virus 40 (SV40). Lines have been established from vas deferens and epididymis and both maintain expression of the CF gene. PMID- 1711533 TI - Cytokeratins in mesenchymal cells: impact on functional concepts of the diversity of intermediate filament proteins. PMID- 1711535 TI - Design of a gas chromatograph with parallel radioactivity and mass spectrometric detection. Application to the identification of the major metabolite of d limonene associated with alpha 2u-globulin. AB - A Perkin Elmer 3920 gas chromatograph, equipped with a versatile inlet system (i.e. an injector/trap), was interfaced to a radioactivity detector and a mass selective detector (H/P 5970B) to identify 14C-labeled compounds. The use of a pre-trap as a demountable, programmable-temperature injector, in conjunction with the injector/trap, allowed the introduction of 0.5-ml samples of rat kidney cytosol extracts to 0.32 mm I.D. capillary columns. The instrumentation greatly facilitated the identification of the major radiolabeled metabolite of d-limonene associated with the male rat-specific protein alpha 2u-globulin as 1,2-cis-d limonene oxide. PMID- 1711534 TI - A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix. AB - Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality. PMID- 1711536 TI - Interleukin-1 beta decreases HLA class II expression on a glioblastoma multiforme cell line. AB - Antigens encoded within the major histocompatibility complex (MHC) are not normally expressed in the central nervous system (CNS), but can be induced by treatment with interferon-gamma (IFN-gamma). Other cytokines released during an inflammatory process can potentially influence MHC expression as well. One cytokine of interest is interleukin-1 (IL-1), an immunoregulatory polypeptide that is produced by macrophages and also by cells in the CNS. In this study, the effect of IL-1 beta on MHC expression in a human glioblastoma multiforme cell line, U-105 MG, has been examined. Treatment of U-105 MG with 10 U IL-1 beta/ml for a period of 5 days resulted in a decrease in constitutive cell surface HLA class II expression and limited the induction of class II by IFN-gamma. This effect was also observed on steady-state levels of class II RNA and could be neutralized with antibodies to IL-1 beta. All class II transcripts examined (HLA DR, -DQ, and -DP alpha and beta) were affected. Class I expression was only marginally changed by IL-1 beta treatment. A minimal concentration of 1 U IL-1 beta/ml was required to reduce class II expression and a kinetics experiment indicated that U-105 MG must be treated for at least 4 days with IL-1 beta for a decrease in class II expression to be observed. This study suggests that IL-1 may play a role in limiting immunoreactivity in the CNS by limiting class II induction. PMID- 1711537 TI - Expression of immunoreactive growth hormone in leukocytes in vivo. AB - In the present study, we investigated the production of growth hormone (GH) related RNA and protein in vivo by rat leukocytes after intraperitoneal treatment with different inducing agents including bacterial lipopolysaccharide (LPS) and Freund's complete adjuvant (FCA). The data showed that in rats after exposure to LPS or FCA leukocytes obtained from the spleen, thymus, and peritoneum all showed a dose-dependent increase in GH-related RNA content. The peak production of GH related RNA was observed 48 h after treatment in the spleen and thymus and 96 h after treatment in the peritoneum. We also evaluated the ability of LPS-sensitive (C3HeB/FeJ) and resistant (C3H/HeJ) inbred mice treated with LPS to produce GH related RNA. The LPS-sensitive mice presented with a typical pathophysiologic response pattern and higher levels of GH-related RNA in the spleen and thymus than the LPS-resistant mice. An increase in the production of immunoreactive GH (irGH) was also observed by direct immunofluorescence with specific antibodies to rat GH. We validated that the GH-related RNA produced in vivo by leukocytes was similar in structure to pituitary GH RNA using reverse transcription and the polymerase chain reaction (PCR). A sample of the PCR reaction, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5'-ends of the two GH-specific primers that was specifically detected with a GH-specific probe after Southern transfer. In other studies with normal nontreated animals, the GH RNA levels are higher in the evening hours and early on in the first month of life. Taken together, our data are the first demonstration that GH RNA and immunoreactive protein can be detected in leukocytes in vivo both in normal and stimulated animals and support the idea that GH may be active in an immune response. PMID- 1711538 TI - Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides. AB - Myelin proteolipid protein (PLP) can induce a T cell-mediated chronic relapsing autoimmune encephalomyelitis in animals and therefore is a candidate for an antigen involved in the pathogenesis of multiple sclerosis. In this report, evidence is presented that peripheral blood mononuclear cells from certain multiple sclerosis (MS) patients recognize the intact PLP molecule as well as certain synthetic PLP peptides in proliferation assays. PLP-specific T cell lines could be obtained from six of ten MS patients with early relapsing-remitting disease. These lines recognized more than one PLP peptide and the relevant peptides differed among patients. The relevance of these observations to the pathogenesis of MS remains to be determined. PMID- 1711539 TI - T cell sensitization to proteolipid protein in myelin basic protein-induced relapsing experimental allergic encephalomyelitis. AB - (SJL/J x PL/J)F1 mice immunized with myelin basic protein (MBP) develop an autoimmune demyelinating disease termed relapsing experimental allergic encephalomyelitis (rEAE). The acute state of disease is mediated by CD4+ T cells specific for MBP amino acids 1-9. To determine the immunologic bases for disease relapse, host sensitization to additional autoantigens of the central nervous system was measured. Results indicate that most animals develop T cell reactivity to endogenous myelin proteolipid protein (PLP) during rEAE. However, PLP-specific immunity does not appear to account for expression of relapse episodes of demyelination. PMID- 1711540 TI - Mechanisms of eosinophil adherence to cultured vascular endothelial cells. Eosinophils bind to the cytokine-induced ligand vascular cell adhesion molecule-1 via the very late activation antigen-4 integrin receptor. AB - We have examined the mechanisms involved in the adherence of normal peripheral blood eosinophils to cultured human umbilical vein endothelial cells (HEC) under three conditions: (a) adherence in the absence of treatment of HEC or eosinophils with activating agents (basal adherence); (b) adherence induced by stimulation of eosinophils with phorbol ester (eosinophil-dependent adherence); and (c) adherence induced by pretreatment of HEC with LPS, tumor necrosis factor (TNF), or IL-1 (endothelial-dependent adherence). A mechanism was identified that was equally active in basal, eosinophil-dependent, and endothelial-dependent adherence. This mechanism was optimally active in the presence of both Ca++ and Mg++, and reduced in the presence of Ca++ only or Mg++ only. Furthermore, like the other mechanisms of eosinophil adherence, it was active at 37 degrees C but not at 4 degrees C. A second mechanism of adherence was involved in eosinophil- and in endothelial-dependent adherence. This mechanism was dependent on the CD11/CD18 adhesion complex of eosinophils (i.e., inhibited by anti-CD18 MAb) and it was active in the presence of Ca++ and Mg++ or Mg++ only, but not Ca++ only. The third mechanism of adherence was specific for endothelial-dependent adherence. It involved the endothelial ligand vascular cell adhesion molecule-1 (VCAM-1) and the eosinophil receptor very late activation antigen-4 (VLA-4, CD49d/CD29, i.e., inhibited by anti-VCAM-1 MAb or anti-VLA-4 MAb). This mechanism was active in the presence of Ca++ and Mg++ but not of Ca++ only or Mg++ only, and was not up- or downregulated when eosinophils were stimulated with phorbol ester. In contrast, the endothelial leukocyte adhesion molecule-1 (ELAM-1), that binds neutrophils and monocytes, was not involved in eosinophil adherence to LPS , TNF-, or IL-1-stimulated HEC (i.e., not inhibited by anti-ELAM-1 MAb). We conclude that eosinophils, like monocytes and lymphocytes, bind to the cytokine induced endothelial ligand VCAM-1 via the integrin receptor VLA-4. PMID- 1711541 TI - Identification of immunodominant T cell epitopes of the hepatitis B virus nucleocapsid antigen. AB - Several lines of experimental evidence suggest that inclusion of core sequences in the hepatitis B vaccine may represent a feasible strategy to increase the efficacy of the vaccination. In order to identify immunodominant core epitopes, peripheral blood T cells purified from 23 patients with acute hepatitis B and different HLA haplotypes were tested with a panel of 18 short synthetic peptides (15 to 20 amino acids [AA]) covering the entire core region. All patients except one showed a strong T cell proliferative response to a single immunodominant 20 amino acid sequence located within the aminoterminal half of the core molecule. Two additional important sequences were also identified at the aminoterminal end and within the carboxyterminal half of the core molecule. These sequences were able to induce significant levels of T cell proliferation in 69 and 73% of the patients studied, respectively. T cell response to these epitopes was HLA class II restricted. The observations that (a) polyclonal T cell lines produced by PBMC stimulation with native HBcAg were specifically reactive with the relevant peptides and that (b) polyclonal T cell lines produced with synthetic peptides could be restimulated with native HBcAg, provide evidence that AA sequences contained within the synthetic peptides represent real products of the intracellular processing of the native core molecule. In conclusion, the identification of immunodominant T cell epitopes within the core molecule provides the molecular basis for the design of alternative and hopefully more immunogenic vaccines. PMID- 1711542 TI - An acquired antithrombin autoantibody directed toward the catalytic center of the enzyme. AB - Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin. PMID- 1711543 TI - Characterization of keratocalmin, a calmodulin-binding protein from human epidermis. AB - Using affinity-purified calmodulin-binding proteins from human epidermis we have developed a monoclonal IgM antibody, ROC 129.1, to a human desmosomal calcmodulin binding protein. This antibody reacts with a submembranous 250-kD protein from human keratinocytes and stains human epidermis in a "cell-surface pattern". Permeability studies indicated that the epitope with which this monoclonal reacts is on the inner surface of the cell membrane. Immunoelectronmicroscopy localized the antigen to the desmosome. The epitope is restricted to stratified squamous epithelia and arises between 8-12 wk of fetal development. This desmosomal calmodulin-binding protein, which we have termed keratocalmin, may be involved in the calcium-regulated assembly of desmosomes. PMID- 1711544 TI - Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors. AB - We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino acids 261-418) were unresponsive. The lack of response of the chimera with TSH receptor domains DE was anticipated because it fails to transduce a signal with TSH stimulation, unlike the other two chimeras. A different spectrum of responses occurred when the TSH-LH/CG chimeras were examined in terms of autoantibody competition for TSH binding. IgG with TSH binding-inhibitory activity when tested with the wild-type TSH receptor also inhibited TSH binding to the chimera with TSH receptor domains DE. Dramatically, however, these IgG did not inhibit TSH binding to the chimera with TSH receptor domains CD, and had weak or absent activity with the chimera with TSH receptor domains ABC. Chimeras with TSH receptor domains ABC and DE were equally effective in affinity-purifying IgG with thyroid-stimulatory and TSH binding-inhibitory activities. Nonstimulatory IgG with TSH binding-inhibitory activity inhibited the action of stimulatory IgG on the wild-type TSH receptor, but not with the chimera containing TSH receptor domains ABC. In summary, TSH receptor autoantibodies and TSH bind to regions in both domains ABC and DE of the TSH receptor extracellular region. Stimulatory and inhibitory TSH receptor autoantibodies, as well as TSH, appear to bind to different sites in domains ABC, but similar sites in domains DE, of the receptor. Alternatively, TSH and the different TSH receptor antibodies bind with differing affinities to the same site in the ABC region. PMID- 1711546 TI - Laboratory evaluation of some larvicidal agents against Anopheles culicifacies in Pune. AB - A laboratory study to evaluate some larvicidal agents against Anopheles culcifacies was carried out. The findings of this study brought out that the larvae of this species were highly susceptible to temephos, fenthion, Paris green and Mosquito Larvicidal Oil (MLO) in that order. The LC50 values in respect of these larvicides were 0.0009 ppm, 0.0081 ppm, 0.029 ppm and 0.015 ml respectively and LC90 values were 0.0018 ppm, 0.022 ppm, 0.11 ppm and 0.046 ml respectively. PMID- 1711545 TI - Neutral endopeptidase and kininase II mediate glucocorticoid inhibition of neurogenic inflammation in the rat trachea. AB - Glucocorticoids inhibit plasma extravasation induced in the rat tracheal mucosa by substance P and other tachykinins released from sensory nerves. This study was performed to determine whether this antiinflammatory effect of glucocorticoids is mediated by the tachykinin-degrading enzymes neutral endopeptidase (NEP) and kininase II (angiotensin converting enzyme, ACE). In addition, we studied the effect of dexamethasone on a nonpeptide inflammatory mediator, platelet activating factor (PAF), which is not degraded by NEP or ACE. Adult male pathogen free F344 rats were treated for 2 d with dexamethasone (0.5 mg/kg per d i.p.), or with the vehicle used to dissolve the steroid. The magnitude of plasma extravasation produced by an intravenous injection of substance P (5 micrograms/kg) or PAF (10 micrograms/kg) was then assessed by using Monastral blue pigment as an intravascular tracer. The role of NEP and ACE activities in the changes produced by dexamethasone was investigated by examining the effect of the selective inhibitors of these enzymes, phosphoramidon and captopril. Dexamethasone reduced the substance P-induced extravasation by 57% but did not affect the PAF-induced extravasation. The suppressive effect of dexamethasone on substance P-induced extravasation was completely reversed by simultaneously inhibiting NEP and ACE activities, but the inhibition of these enzymes had no effect on PAF-induced extravasation, regardless of whether the rats were pretreated with dexamethasone or not. These results suggest that NEP and ACE mediate a selective inhibitory effect of glucocorticoids on neurogenic plasma extravasation. PMID- 1711547 TI - Are CD1a antigens on Langerhans cells empty class I-like molecules that come out in the cold? PMID- 1711548 TI - Organization of the monocyte/macrophage system of normal human skin. PMID- 1711549 TI - Regional development of Langerhans cells and formation of Birbeck granules in human embryonic and fetal skin. AB - The regional development of Langerhans cells (LC) and the formation of Birbeck granules (BG) were examined in human embryonic and fetal skin. Samples were obtained from multiple anatomic sites and stained with anti-CD36, anti-CD1a, and anti-HLA-DR antibody as well as Lag antibody specifically reactive to BG and some vacuoles of human LC. In the first trimester, CD36+ dendritic epidermal cells were identified before the appearance of CD1a+ cells and Lag+ cells. Some of the former co-expressed HLA-DR antigens but not CD1a antigens. In the second trimester, regional variations in LC development were observed. Epidermal LC of palms and soles reached a peak in number in the first trimester but were rarely detected after 18 weeks estimated gestation age (EGA), whereas, in other regions, their number increased with age. In the second trimester, CD1a+ cells and Lag+ cells were also identified in the epidermis, although Lag+ cells appeared later than CD1a+ cells. The Lag+ cells until 17 weeks EGA showed a variety of staining intensities and immunoelectron microscopy revealed that they contained various amounts of Lag-reactive BG. Flow cytometric analysis showed that relative amounts of Lag antigens in LC increased during the second trimester and that fetal LC of 18 weeks EGA expressed the same amounts of HLA-DR, CD1a, and Lag antigens as did adult human LC. In the dermis, in the second trimester, numerous CD36+ cells and HLA-DR+ cells were found, whereas CD1a+ cells and Lag+ cells were rarely detected. Taken together, it is suggested that HLA-DR+ dendritic cells acquire CD1a+ antigens first and then form BG after migration to the epidermis and that fetal LC are phenotypically mature in the second trimester. PMID- 1711550 TI - Upper keratinocytes of psoriatic skin lesions express high levels of NAP-1/IL-8 mRNA in situ. AB - In order to better understand the factors regulating disease promotion and activity in psoriasis (PS), we searched for the in situ expression of mRNA for various cytokines in long-standing PS skin lesions. Specific hybridization with a NAP-1/IL-8 anti-sense RNA probe was keratinocyte associated and yielded strong and specific signals exclusively in the upper layers of the lesional epidermis, but not in uninvolved skin from psoriatic patients or normal skin from non psoriatics. Interestingly, NAP-1/IL-8 transcripts were focally clustered in a spotty pattern predominantly between the tips of elongated papillae, but were absent in the lower epidermal region and the dermal compartment. We consistently failed to detect appreciable numbers of TNF-alpha and/or IL-6 mRNA-containing cells in psoriatic lesions. These results support the notion that IL-8, rather than IL-6, is an important disease-promoting cytokine in PS. In view of the known in vitro and in vivo effects of IL-8, it is conceivable that this substance greatly contributes to the major pathologic changes seen in psoriatic skin, i.e., keratinocyte hyperproliferation and leucocyte infiltration. In this case, local pharmacologic down-regulation of NAP-1/IL-8 activity could be a promising therapeutic strategy in PS. PMID- 1711551 TI - Ribotype stability of serial pulmonary isolates of Pseudomonas cepacia. AB - Eighty-three isolates of Pseudomonas capacia were recovered from respiratory secretions from 12 chronically colonized cystic fibrosis patients and examined by ribotype analysis. In 9 patients, the ribotype of the cultured P. cepacia remained unchanged throughout the entire period of observation, indicating chronic pulmonary colonization with a single strain. In each of the remaining 3 patients, two genetically distinct strains were detected among serial P. cepacia isolates. No significant change in clinical condition was correlated with the change in identity of the colonizing strain. In control experiments, the stability of strain ribotype was demonstrated among isolated that had been subcultured 100 times in vitro and among isolates recovered from chronically colonized mice. These data demonstrate the utility of ribotype analysis and indicate that most chronically colonized cystic fibrosis patients harbor a single strain of P. cepacia for prolonged periods. PMID- 1711552 TI - Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1. AB - The CD36 and ICAM-1 glycoproteins on vascular endothelial cells have been implicated as cytoadherence receptors for Plasmodium falciparum-infected erythrocytes (IRBC). Adhesion of IRBC from Thai patients with uncomplicated and severe falciparum malaria to purified CD36 or ICAM-1 and to C32 melanoma cells was compared. All malaria isolates bound to solid phase-adsorbed CD36 and to fluid-phase 125I-labeled CD36. IRBC adhesion to purified ICAM-1 varied widely, and no correlation with clinical severity of disease was observed. The cytoadherent phenotype of IRBC was modulated by selective panning on plates coated with purified CD36 or ICAM-1. IRBC selected by panning on CD36+, ICAM-1+ melanoma cells bound to cells that express surface CD36 but not to CD36-deficient cells, indicating that CD36 exerts a strong selective pressure on the IRBC cytoadherent phenotype. IRBC adhesion to CD36 and ICAM-1 suggests that P. falciparum parasites may use these receptors in vivo to promote parasite survival and immune evasion. PMID- 1711553 TI - Morphologic and staining characteristics of a cyanobacterium-like organism associated with diarrhea. AB - A spherical organism 9-10 microns in diameter, seen in three outbreaks of diarrhea in Southeast Asia and the United States during the past 2 years, bore characteristics of a cyanobacterium when observed in formalin-preserved stool specimens and by electron microscopy. Organisms in freshly passed stool specimens showed an internal morula of lipid-containing globules. In fresh water, the morula divided into two sausage-shaped structures resembling the sporocysts of an isosporid coccidian. After 7 months, the organisms had not developed the crescentic sporozoites seen in the Coccidia but had begun to multiply slowly in culture. It was impossible to stain the internal structures of the organisms because the outer cyst wall ruptured during desiccation, releasing the contents of the cysts. The organisms were readily identified by their intense blue autofluorescence under UV light, but they were also recognizable by bright-field microscopy and by a modified acid-fast stain. Almost all infected persons suffered intermittent diarrhea for 2-3 weeks and many emphasized a feeling of intense fatigue during the course of their illness. PMID- 1711554 TI - Effect of the calcium channel blocker verapamil on human immunodeficiency virus type 1 replication in lymphoid cells. AB - Cell signaling events are known to affect human immunodeficiency virus type 1 (HIV-1) replication. Treatment of lymphoid CEM cells with the calcium channel blocker verapamil (25-75 microM) enhanced HIV-1 expression in acute, whole virus infection experiments, despite lowering intracellular calcium levels, ablating the acute rise in intracellular calcium normally seen with infection, and lengthening the doubling time of cell replication. Verapamil had no effect on cell surface CD4 expression. Transfection of CEM cells with plasmids containing the HIV-1 long terminal repeat linked to the chloramphenicol acetyltransferase reporter gene showed that verapamil enhanced expression of the HIV-1 long terminal repeat in a dose-dependent fashion. This effect was abolished by mutations in the binding sites for nuclear factor kappa-B. Electrophoretic mobility shift assays confirmed that verapamil induced nuclear factor kappa-B activity in CEM cells. Thus, verapamil, in high concentrations, can potentiate HIV-1 replication in lymphoid cells, and this effect may be mediated by induction of nuclear factor kappa-B. PMID- 1711555 TI - [Treatment of advanced or recurrent cervical cancer by a new BOMP regimen consisting of bleomycin, vincristine, mitomycin-C, and low-dose consecutive cisplatin]. AB - Twenty-five patients with cervical cancer (4 post-operative cases with FIGO stage Ib or IIb, 2 with stage IV, and 19 recurrence) were treated with a new BOMP consisting of bleomycin (5 mg/body, drip, i.v., days 1-7), vincristine (0.7 mg/m2, bolus, i.v., day 7), mitomycin-5 (7 mg/m2, bolus, i.v., day 7) and cisplatin (10 mg/m2, drip, i.v., days 1-7). The mean age of the patients was 54 years (range 30-77). Prior therapy included radiotherapy (13 cases), radical hysterectomy (11), and none (1). Fifteen (79%) of the 19 evaluable patients responded, including 6 with a complete response (CR) lasting over 15 months. Almost all the disease located in lung, liver, bone, and vulva showed a response. In particular, lesions confined to the lung had a 100% CR when the size of each tumor was under 2 cm in diameter even in the case of multiple metastasis. In contrast, 9 patients with pelvic disease had a 56% response with only 1 CR who had no previous radiotherapy. Such a poor response in the pelvic disease was considered to be due to vascularity reduced by prior radiotherapy. The important factors affecting the response to a new BOMP were found to be lesion size, prior radiotherapy, and the site of lesion. Patient age, performance status (PS), and the interval from a previous treatment to BOMP were not of significance with regard to response. The dose limiting factor was hematologic toxicities. Other toxicities including nausea, renal dysfunction, pulmonary fibrosis, and loss of hair were acceptable. Thus, the decrease in the PS of patients due to BOMP was minimal. It is suggested that this regimen will be useful as a neoadjuvant chemotherapy for advanced cervical cancer. PMID- 1711556 TI - Topical steroid induced chronic demodicidosis. AB - We report a 39-year-old female patient who developed pruritic erythematous telangiectatic patches with scaly follicular papules on the neck and upper chest for 4 years. Ten per cent potassium hydroxide preparation of skin scrapings revealed Demodex folliculorum. Histology showed three Demodex mites in one of the hair follicles. She was treated with a topical steroid without improvement. The skin lesions and Demodex mite disappeared after a single application of 1 per cent gamma benzene hexachloride but twice daily application of 1 per cent gamma benzene hexachloride for 2 weeks was needed to prevent recurrence. PMID- 1711557 TI - Selective expression of a VHIV subfamily of immunoglobulin genes in human CD5+ B lymphocytes from cord blood. AB - Human B lymphocytes expressing the CD5 surface antigen (CD5+ B cells) constitute a subset capable of producing polyspecific antibodies recognizing a variety of self antigens. The repertoire of antibodies produced by CD5+ and CD5- B cells is different. However, it is not yet established whether this distribution is reflected in different immunoglobulin variable region gene (IgV) use. Rearrangement of heavy chain IgV (IgVH) genes represents one of the first identifiable stages in the maturation of B cells, and occurs in a developmentally ordered fashion. The repertoire of IgVH gene expression is highly restricted during fetal life but diversifies progressively after birth. A high frequency of VH gene use from the relatively small VHIV gene family has previously been demonstrated in human fetal liver B cells. In the present study, 102 B cell lines established by Epstein-Barr Virus-transformation of separated CD5+ and CD5- cord blood B cells, were examined for the frequency of IgV expression using monoclonal antibodies to cross-reactive idiotypes (CRI). The results demonstrate a relatively high frequency of VHIV gene use (30%) in B cells from cord blood. Furthermore, two mutually exclusive CRI associated with distinct subgroups of the VHIV family are segregated in their association with either subset of B cells. One CRI is exclusively expressed in lines established from CD5+ B cells while the other is associated with lines established from CD5- B cells. PMID- 1711558 TI - Junctional sequences of fetal T cell receptor beta chains have few N regions. AB - T cell receptors (TCRs) and immunoglobulins (Igs) derive a large fraction of their repertoire from diversity generated at the junctions of the V, D, and J coding segments. This diversity is derived both from the random deletion of nucleotides from the ends of coding regions and from the subsequent addition of nontemplated N region nucleotides. While the vast majority of TCRs and Igs from adult mice have N regions, less than 5% of both TCR-gamma/delta and Ig from fetal and neonatal mice have N regions. This study analyzed the ontogeny of junctional diversity of TCR-alpha/beta. Genomic DNA or C beta-primed cDNA was prepared from thymocytes of mice at varying stages in ontogeny, and the rearranged V beta 8 or V beta 5 sequences were amplified by polymerase chain reactions. Sequencing of the V beta-D beta-J beta junctions showed few N regions early in ontogeny, although the fraction of sequences with N regions exceeded that previously reported for Ig and for TCR-gamma/delta. N regions were found in 13% of V beta junctional sequences from day 18-19 fetal thymocytes, 33% of sequences from newborn thymocytes, 76% of sequences from day 4 postnatal thymocytes, and 88% of sequences from 5-wk-old thymocytes. In addition, nonrandom usage of the D beta and J beta segments was observed in both fetal and adult TCR sequences. While the usage of each of the six J beta 2 segments was different, the same pattern of usage was seen regardless of whether D beta 1 or D beta 2 was used, suggesting that a factor controlling the rate of usage of each J segment is intrinsic to the J gene itself. Since TCRs derive so much of their diversity from N regions, the relative paucity of N regions in fetal alpha/beta T cells would create a fetal TCR-alpha/beta repertoire that would be quite different from, and smaller than, the adult repertoire. The lack of N regions might be predicted to limit the range of affinities of TCR-MHC + peptide interactions, which may have important consequences for positive and negative selection of fetal and newborn T cells. PMID- 1711559 TI - The rat c-kit ligand, stem cell factor, induces the development of connective tissue-type and mucosal mast cells in vivo. Analysis by anatomical distribution, histochemistry, and protease phenotype. AB - Mast cell development is a complex process that results in the appearance of phenotypically distinct populations of mast cells in different anatomical sites. Mice homozygous for mutations at the W or S1 locus exhibit several phenotypic abnormalities, including a virtual absence of mast cells in all organs and tissues. Recent work indicates that W encodes the c-kit tyrosine kinase receptor, whereas S1 encodes a c-kit ligand that we have designated stem cell factor (SCF). Recombinant or purified natural forms of the c-kit ligand induce proliferation of certain mast cell populations in vitro, and injection of recombinant SCF permits mast cells to develop in mast cell-deficient WCB6F1-S1/S1d mice. However, the effects of SCF on mast cell proliferation, maturation, and phenotype in normal mice in vivo were not investigated. We now report that local administration of SCF in vivo promotes the development of connective tissue-type mast cells (CTMC) in the skin of mice and that systemic administration of SCF induces the development of both CTMC and mucosal mast cells (MMC) in rats. Rats treated with SCF also develop significantly increased tissue levels of specific rat mast cell proteases (RMCP) characteristic of either CTMC (RMCP I) or MMC (RMCP II). These findings demonstrate that SCF can induce the expansion of both CTMC and MMC populations in vivo and show that SCF can regulate at least one cellular lineage that expresses c-kit, the mast cell, through complex effects on proliferation and maturation. PMID- 1711560 TI - Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity. AB - C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL 4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy. PMID- 1711561 TI - The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin. AB - Trypanosoma cruzi expresses a developmentally regulated neuraminidase (TCNA) implicated in parasite invasion of cells. We isolated full-length DNA clones encoding TCNA. Sequence analysis demonstrated an open reading frame coding for a polypeptide of 1,162 amino acids. In the N-terminus there is a cysteine-rich domain containing a stretch of 332 amino acids nearly 30% identical to the Clostridium perfringens neuraminidase, three repeat motifs highly conserved in bacterial and viral neuraminidases, and two segments with similarity to the YWTD repeats found in the low density lipoprotein (LDL) receptor and in other vertebrate and invertebrate proteins. This domain is connected by a structure characteristic of type III modules of fibronectin to a long terminal repeat (LTR) consisting of 44 full length copies of twelve amino acids rich (75%) in serine, threonine, and proline. LTR is unusual in that it contains at least 117 potential phosphorylation sites. At the extreme C-terminus is a hydrophobic segment of 35 amino acids, which could mediate anchorage of TCNA to membranes via a glycosylphosphatidylinositol linkage. This is the first time a protozoan protein has been found to contain a YWTD repeat and a fibronectin type III module. The domain structure of TCNA suggests that the enzyme may have functions additional to its catalytic activity such as in protein-protein interaction, which could play a role in T. cruzi binding to host cells. PMID- 1711562 TI - In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors. AB - Purified CD3-4- thymocytes were obtained by depletion of CD3+ and CD4+ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20-40% of thymic CD16+ cells coexpressed CD1, while approximately half were cyCD3+. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3-4- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3-4- or CD3-4-16- thymocytes in the presence of irradiated H9 cells resulted in large proportions (approximately 50%) of CD16+ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16+ cyCD3+; CD16+ cyCD3-; CD16- cyCD3+; and CD16- cyCD3-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-gamma and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16+ cells expressed transcripts for CD16 and for CD3 epsilon (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long, 1988. J. Immunol. 140:1685.) and zeta chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Lond.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3- CD16+ NK cells may arise from immature thymocytes. PMID- 1711563 TI - Highly restricted expression of the thymus leukemia antigens on intestinal epithelial cells. AB - The TL region of the major histocompatibility complex of the mouse contains dozens of tandemly arranged class I genes, including those encoding the thymus leukemia (TL) antigens. TL antigens have been thought to be expressed only on the surface of some T lineage cells, namely immature thymocytes of some mouse strains (TL+ strains), some leukemia cells, and activated T cells. While the function of TL antigens is unknown, recent studies have implicated the products of at least some TL region class I genes as molecules that present antigens to gamma/delta T cells. Since some gamma/delta T cells are known to be specifically associated with certain epithelial tissues, we have investigated the expression of some TL region class I genes in a variety of epithelium-containing tissues. Our results show that the TL antigen gene of C57BL/6 mice, T3b, and the TL antigen genes of BALB/c mice, T3d (previously T3c) and T18d (previously T13c), are highly expressed in the epithelium of the small intestine. In the case of T3b, we further show, using a T3 product-specific antibody, that its product is expressed on the surface of the columnar epithelial cells. In addition, we demonstrated that two other TL region class I genes of C57BL/6 origin, T9b and T21b, are also expressed nearly exclusively in intestinal epithelial cells. These results are consistent with the hypothesis that the products of these TL region class I genes are recognized by gamma/delta T cell receptors of intestinal intraepithelial lymphocytes, a subset of gamma/delta T cells that is localized in the intestinal epithelium and has a restricted V gamma repertoire. Finally, our study indicates that the relative levels of expression of the two homologous TL antigen genes, T3d and T18d, differ widely between the thymus and the intestine. PMID- 1711564 TI - T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein. AB - M proteins, the major virulence factor of group A streptococci, have been implicated in the pathogenesis of acute rheumatic fever (ARF) and other streptococcal related autoimmune diseases. A 22-kD fragment of M type 5 protein is a potent stimulant of human T cells and has recently been shown by our laboratory to belong to the newly designated family of superantigens. Using flow cytometry and the polymerase chain reaction, we demonstrate that this molecule reacts with subsets of human T cells expressing specific T cell receptor (TCR) V beta elements, namely V beta 2, 4, and 8. We employed similar techniques to analyze the TCR V alpha usage of pep M5-stimulated T cells. These studies revealed that the preferential usage of particular V alpha elements is not specific for the superantigen; rather, it may reflect the repertoire of the individual being tested. The expansion of a large number of T cells bearing specific TCR V beta sequences by M protein may account for its role in mediating the pathogenesis of post-streptococcal diseases. Furthermore, the preferential usage of TCR V alpha elements in certain individuals may be an important factor that predisposes them to development of self-reactivity. PMID- 1711565 TI - Phospholipid-anchored and transmembrane versions of either decay-accelerating factor or membrane cofactor protein show equal efficiency in protection from complement-mediated cell damage. AB - Decay-accelerating factor (DAF) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that protects cells from complement-mediated damage by regulation of the C3 convertase. To investigate the role of the GPI anchor in the function of DAF, the cDNA encoding human DAF was expressed by transfection in Chinese hamster ovary (CHO) cells. Testing of these DAF transfectants in an antibody plus human complement-mediated cytotoxicity assay demonstrated that DAF protects these cells from cytotoxicity, and that the level of protection increases with expression of surface DAF. A cDNA construct encoding a transmembrane version of DAF (DAF-TM) protects CHO transfectants from cytotoxicity with equal efficiency to DAF. This DAF-TM construct used the TM and cytoplasmic domains of membrane cofactor protein (MCP); an alternate TM version of DAF constructed with the TM and cytoplasmic domains of HLA-B44 showed equivalent protection. The protection from cytotoxicity involved a decrease in the deposition of C3 on the cell, consistent with the effect of DAF on the C3 convertase. A second pair of anchor variants, MCP and a GPI-anchored construct, MCP-PI, were also equivalent in their complement protection. The equivalent function of GPI-anchored and TM versions of a protein was not expected based on the hypothesized increased lateral mobility of GPI-anchored proteins, which should confer a functional advantage in contacting ligand, in this case, C3b or C4b, on the cell surface. These data suggest either that GPI-anchored and TM versions of a protein have equal lateral mobility in the membrane, or else that increased lateral mobility is not advantageous to DAF or MCP in carrying out their complement inhibitory roles. Furthermore, DAF and MCP demonstrated approximately equal protection of cells from complement-mediated cytotoxicity, suggesting that DAF and MCP provide overlapping levels of protection to cells against damage mediated by the complement system. PMID- 1711566 TI - VH gene family expression in mice with the xid defect. AB - Preferential use of particular VH gene families in the response to specific antigens has been demonstrated in several systems. The lack of responses to certain types of antigens, therefore, could be the result of deletion of or failure to express some VH genes. Because CBA/N mice, which carry the X-linked immunodeficiency (xid) gene defect, have been shown to be unresponsive to thymus independent polysaccharide antigens, it was of interest to examine if this unresponsiveness could be accounted for by abnormal expression of particular VH gene families. Using in situ hybridization on B cell colonies, we determined the expression of nine VH gene families in CBA/CaHN females (genotypically normal), CBA/N males (xid) and females (xid), and (CBA/N x CBA/CaHN)F1 males (xid) and females (phenotypically normal). Our results indicate that VH gene family expression, including the S107 family, in CBA/N males and F1 males, is similar to that of CBA/CaHN and F1 females with predominant expression of J558, the largest gene family, in all individuals. Interestingly, CBA/N female mice, which carry two defective X chromosomes, as a group expressed significantly reduced levels of the J558 gene family, and as individuals showed variation in which family was predominantly expressed. We conclude that the unresponsiveness of mice with the xid defect to polysaccharide antigens can not attributed to a failure to express the nine VH gene families that we examined. Our findings do not support previous studies (Primi, D., and P.-A. Cazenave 1986. J. Exp. Med. 165:357), which found an absence of expression of the S107 family in xid mice. PMID- 1711567 TI - Human immunodeficiency virus type 1 gp120 mimics a hidden monomorphic epitope borne by class I major histocompatibility complex heavy chains. AB - Murine monoclonal antibodies (mAbs) M38 and L31 define two epitopes of a surface protein of activated lymphocytes and monocytes. It has been shown that M38 also defines a crossreactive epitope of human immunodeficiency virus type 1 (HIV-1) gp120 (Beretta et al., 1987. Eur. J. Immunol. 17: 1793). The mAb inhibits syncytia formation driven by HIV-1-infected cells. The surface protein was demonstrated to be a class I MHC alpha chain, by sequence analysis of the corresponding cDNA and by immunological means. The epitopes defined by mAbs M38 and L31 are monomorphic and hidden (i.e., inaccessible to antibodies) on native HLA molecules expressed by resting cells, but can be evidenced on denatured proteins by Western blot analysis. The two epitopes become accessible after activation processes have been implemented, likely reflecting a conformational alteration of alpha chains (such as that described by Schnabl et al. 1990. J. Exp. Med. 171:1431). Consistent with molecular data are the results of functional analysis, which indicate that the molecule recognized by M38 and L31 is a gate for pleiotropic negative signals, since the two mAbs were shown to inhibit monocyte antigen presentation and lymphocyte mitogenic proliferation, respectively. PMID- 1711568 TI - Expression and function of c-kit in hemopoietic progenitor cells. AB - The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation. PMID- 1711569 TI - Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells. AB - The effect of nerve growth factor (NGF) on proliferation/differentiation of mast cells was investigated in vitro. Although NGF alone neither supported colony formation of bone marrow-derived cultured mast cells (BMCMC) nor induced development of mast cell colonies from nonadherent bone marrow cells (NBMC), addition of NGF to the suboptimal dose of interleukin 3 (IL-3) significantly increased the numbers of mast cell colonies produced by BMCMC or NBMC in methylcellulose. When stimulated by IL-3 alone, cells in mast cell colonies were not stained by berberine sulfate, a fluorescent dye. In contrast, mast cells developing in methylcellulose cultures obtaining both IL-3 and NGF were stained by berberine sulfate. The fluorescence was abolished by the treatment of heparinase but not of chondroitinase ABC, suggesting that mast cells stimulated by IL-3 and NGF produced and stored heparin proteoglycan. The histamine content of BMCMC maintained by IL-3 was also increased by addition of NGF. Since BMCMC showed mucosal mast cell-like phenotype, NGF appeared to induce the phenotypic change to connective tissue-type mast cells (CTMC). In the culture containing BMCMC, 3T3 fibroblasts, and IL-3, the phenotypic change of BMCMC to CTMC was observed as well. Since NGF was detected in this coculture and since addition of anti-NGF monoclonal antibody suppressed the phenotypic change, NGF produced by fibroblasts appeared to induce the phenotypic change. Neither BMCMC alone nor IL 3 alone increased the concentration of NGF. Therefore, there is a possibility that BMCMC stimulated by IL-3 may induce the production and/or release of NGF by fibroblasts. PMID- 1711570 TI - Membrane cofactor protein of the complement system: alternative splicing of serine/threonine/proline-rich exons and cytoplasmic tails produces multiple isoforms that correlate with protein phenotype. AB - Membrane cofactor protein (MCP) is a complement regulatory protein that is expressed on human cells and cell lines as two relatively broad species with Mr of 58,000-68,000 and 48,000-56,000. The structure of a previously reported cDNA clone indicated that MCP was a type 1 membrane glycoprotein and a member of the regulators of complement activation gene/protein cluster. However, it did not provide an explanation for the unusual phenotypic pattern of MCP. Therefore, in parallel with an analysis of the gene, additional cDNAs were cloned and characterized. Six different MCP cDNA classes were identified. All encode the same 5' untranslated signal peptide, four SCRs, transmembrane domain, and basic amino acid anchor. However, they differ in the length and composition of an extracellular serine/threonine/proline (STP)-rich area, a site of heavy O glycosylation, and cytoplasmic tail. Analysis of the MCP gene demonstrated that the variation in cDNA structure was a result of alternative splicing. Peripheral blood cells and cell lines predominantly expressed four of the six isoforms. These varied by the presence or absence of an STP-rich segment of 15 amino acids (STPB) and by the use of one of two cytoplasmic domains. Analysis by polymerase chain reaction, Northern blots, and transfection indicated that the predominance of MCP cDNA isoforms with STPB correlated with the high molecular weight protein phenotype, while the predominance of isoforms without STPB correlated with the lower molecular weight phenotype. The expression in a single cell of four distinct protein species with variable STP-rich regions and cytoplasmic tails represents an interesting example of the use of alternative splicing to provide variability in a mammalian protein. PMID- 1711571 TI - Noninvasive detection of Chlamydia trachomatis urethritis in men by a rapid enzyme immunoassay test. AB - BACKGROUND: The purpose of this investigation was to evaluate the ability of a rapid enzyme immunoassay test to noninvasively detect Chlamydia trachomatis urethritis in men from a urine specimen. METHODS: Urethral samples and urine from 207 patients were evaluated. Urethral and urine sediment Gram stains, leukocyte esterase dipstick tests, and enzyme immunoassay analyses of centrifuged and uncentrifuged urine were compared with urethral C trachomatis culture. RESULTS: The prevalence of infection in this population was 10.3%. Sensitivity and specificity of the enzyme immunoassay on the centrifuged urine specimen were 70% and 96%, respectively. The positive and negative predictive values were 67% and 97%, respectively. The uncentrifuged urine enzyme immunoassay sensitivity was 35.7% and specificity was 98.9%. Leukocyte esterase test sensitivity compared with that of the Neisseria gonorrhoeae and/or C trachomatis cultures was 83.3%, and specificity was 52%. CONCLUSIONS: The rapid enzyme immunoassay clinically complemented the screening urine sediment Gram stain and the leukocyte esterase test. The judicious use of a noninvasive C trachomatis rapid enzyme immunoassay test to identify organism-specific urethritis may improve patient management of sexually transmitted disease. PMID- 1711572 TI - Contractions of dysgenic skeletal muscle triggered by a potentiated, endogenous calcium current. AB - The dihydropyridine (DHP) receptor of normal skeletal muscle is hypothesized to function as the voltage sensor for excitation-contraction (E-C) coupling, and also as the calcium channel underlying a slowly activating, DHP-sensitive current (termed ICa-s). Skeletal muscle from mice with muscular dysgenesis lacks both E-C coupling and ICa-s. However, dysgenic skeletal muscle does express a small DHP sensitive calcium current (termed ICa-dvs) which is kinetically and pharmacologically distinct from ICa-s. We have examined the ability of ICa-dys, or the DHP receptor underlying it, to couple depolarization and contraction. Under most conditions ICa-dys is small (approximately 1 pA/pF) and dysgenic myotubes do not contract in response to sarcolemmal depolarization. However, in the combined presence of the DHP agonist Bay K 8644 (1 microM) and elevated external calcium (10 mM), ICa-dys is strongly potentiated and some dysgenic myotubes contract in response to direct electrical stimulation. These contractions are blocked by removing external calcium, by adding 0.5 mM cadmium to the bath, or by replacing Bay K 8644 with the DHP antagonist (+)-PN 200-110. Only myotubes having a density of ICa-dys greater than approximately 4 pA/pF produce detectible contractions, and the strength of contraction is positively correlated with the density of ICa-dys. Thus, unlike the contractions of normal myotubes, the contractions of dysgenic myotubes require calcium entry. These results demonstrate that the DHP receptor underlying ICa-dys is unable to function as a "voltage sensor" that directly couples membrane depolarization to calcium release from the sarcoplasmic reticulum. PMID- 1711573 TI - Color opponency in cone-driven horizontal cells in carp retina. Aspecific pathways between cones and horizontal cells. AB - The spectral and dynamic properties of cone-driven horizontal cells in carp retina were evaluated with silent substitution stimuli and/or saturating background illumination. The aim of this study was to describe the wiring underlying the spectral sensitivity of these cells. We will present electrophysiological data that indicate that all cone-driven horizontal cell types receive input from all spectral cone types, and we will present evidence that all cone-driven horizontal cell types feedback to all spectral cone types. These two findings are the basis for a model for the spectral and dynamic behavior of all cone-driven horizontal cells in carp retina. The model can account for the spectral as well as the dynamic behavior of the horizontal cells. It will be shown that the strength of the feedforward and feedback pathways between a horizontal cell and a particular spectral cone type are roughly proportional. This model is in sharp contrast to the Stell model, where the spectral behavior of the three horizontal cell types is explained by a cascade of feedforward and feedback pathways between cones and horizontal cells. The Stell model accounts for the spectral but not for the dynamic behavior of the horizontal cells. PMID- 1711574 TI - Effects of sublethal dosages of Abate upon adult fecundity and longevity of Aedes aegypti. AB - Sublethal concentrations of Abate (temephos) were applied to F2 generation Aedes aegypti larvae, and fecundity and longevity were recorded in the emerged adults. Females exposed to Abate oviposited only in the first 2 gonotrophic cycles, meanwhile control females laid a few eggs after taking the third blood meal. Dosages of 0.009, 0.013 and 0.015 mg/liter of Abate decreased the mean egg production per gonotrophic cycle 37, 47 and 69%, respectively, in relation to the control. Females that were exposed as larvae to Abate lived longer than the control females. PMID- 1711575 TI - Glioma malignancy and its biological and histological correlates. AB - Neoplastic transformation is a multiphasic process developing in several subsequent steps. Many things are known on oncogene activation mechanisms and oncogene cooperation in neoplastic transformation, growth factors (EGF, PDGF, etc.), antioncogenes and emerogenes. Tumor progression from benign to malignant growth has diagnostic importance. Morphologically tumor progression is represented by increased anaplasia based on genotypic heterogeneity; among human gliomas the main characteristic is represented by the lack of GFAP expression in a variable percentage of anaplastic cells. Increased cellular density and mitotic index are direct consequence of the growth fraction enhancement, whereas nuclear polymorphism and necroses are less reliable parameters. Finally tumor angiogenesis and the different meaning of endothelial proliferations in different oncotypes is discussed. PMID- 1711576 TI - Applications of confocal microscopy to the study of myelin development and neuron structure. AB - Confocal laser scanning microscopy has been used to study the localization of myelin basic proteins expressed in nonglial cells, and to probe the three dimensional structure of central auditory neurons in the lateral superior olive. The paper focuses on the techniques used to obtain the results. The key roles of confocal microscopy and computer image processing of the images obtained are emphasized as they relate to the discovery of essential structural information about these specimens. PMID- 1711577 TI - Measurement of the tissue distribution of immunoperoxidase staining with polyclonal anti-BCG serum in lung granulomata of mice infected with Mycobacterium tuberculosis. AB - Mice inoculated with Mycobacterium tuberculosis, strain H37Rv were used as a model of human tuberculosis. The microanatomical location of immunoperoxidase staining with a polyclonal anti-BCG serum was within macrophages and appeared granular rather than delineating whole bacilli. Immunoperoxidase staining appears to demonstrate degraded mycobacterial antigens from disrupted organisms and so reflects prior turnover of bacilli. On Ziehl-Neelsen staining, intact or almost intact bacilli are seen and so the extent of this form of staining reflects the current bacillary load. Both methods have limited sensitivity, but with larger mycobacterial loads the area of immunoperoxidase stain measured on a semi automated image analyser correlated with the numbers of bacilli observed. The immunoperoxidase method will be useful in the evaluation of residual antigen in studying the pathogenesis of experimental murine tuberculosis. In human mycobacterial granulomata, this immunohistochemical technique should provide an alternative method of estimating the extent of bacillary load: this approach may also provide evidence of mycobacterial infection from residual antigen deposits in the tissue when whole bacilli have been successfully cleared. PMID- 1711578 TI - Chronic administration of sublethal doses of carbaryl increases pineal N acetyltransferase and hydroxyindole-O-methyltransferase activities and serum melatonin levels. AB - The purpose of this study was to examine the effects of chronic administration of sublethal doses of carbaryl on pineal melatonin synthesis. N-methyl 1 naphthylcarbamate (carbaryl) (8.33 mg/kg B.W. daily) was administered orally to adult male albino rats for 6 successive days. Nocturnal (0100) N acetyltransferase and hydroxyindole-O-methyltransferase activities were increased (roughly 75% and 60%, respectively) by carbaryl administration; likewise, carbaryl augmented serum melatonin levels at 2300. Pineal tryptophan. 5 hydroxytryptophan, serotonin, and 5-hydroxindole acetic acid levels were unaffected at all three time points. The results indicate that the carbamate pesticide, i.e., carbaryl, modifies pineal melatonin synthesis in vivo. PMID- 1711579 TI - Differential effects of pinealectomy on amygdala and hippocampus serotonin metabolism. AB - The purpose of the present study was to determine the effects of long-term pinealectomy on serotonin metabolism in the amygdala and the hippocampus of male rats. Pinealectomy did not significantly alter either tryptophan or serotonin concentrations in the amygdala or the hippocampus. However, statistically significant decreases in 5-hydroxyindole-3-acetic acid levels and tryptophan hydroxylase activity were found in the amygdala. Monoamine oxidase activity was unchanged in both regions. These results support the involvement of the amygdaloid serotoninergic system in mediating the functions of the pineal gland. PMID- 1711580 TI - The effect of immunization with protein-sulphanilic acid conjugate on sulphanilic acid disposition in the rat. AB - Rats were immunized with bovine gamma-globulin-sulphanilic acid conjugate and the plasma concentration of sulphanilic acid examined after an intravenous injection of this drug. There was a significant increase in plasma half-life and AUC and a significant decrease in clearance of sulphanilic acid in the immunized rats compared with the control. In the immunized rats, binding of sulphanilic acid to macromolecules in serum, determined by ultrafiltration, decreased with increase of sulphanilic acid concentration. At low concentrations, there was a significant increase in % binding of the drug in the serum of immunized rats compared with controls. There was also a significant reduction in urinary excretion of total drug in the immunized rats compared with controls. These findings suggest that sulphanilic acid-specific antibodies in the serum of immunized animals bind [14C]sulphanilic acid, giving rise to higher serum levels thereby making it unavailable for normal excretory processes. PMID- 1711581 TI - Nucleic acid levels in the developing ovaries of Hyalomma (Hyalomma) dromedarii (Acari: Ixodidae). AB - Protein and nucleic acid levels from the ovaries of Hyalomma dromedarii Koch were determined during different stages of oogenesis. The concentrations of total protein, DNA, and RNA increased during oogenesis, reflecting the rapid developmental changes taking place in this tissue. Peak protein and DNA levels were reached in the fully fed females, whereas RNA level peaked slightly earlier. Ribosomal RNA (rRNA) was found to be composed of 27.0s, 17.0s, and 4.1s particles. The ratio of 27.0s to 17.0s varied within the developing ovary, yet the 27.0s/4.1s ratio remained constant. The nucleotides of total RNA and rRNA were determined, and the ratio of purine/pyrimidine equaled approximately 1 and remained unchanged during oogenesis. PMID- 1711582 TI - Developmental changes in regulation of embryonic chick heart gap junctions. AB - Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14 day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of +/- 100 mV. At all ages, the instantaneous junctional current (or conductance = current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steady-state junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue. PMID- 1711583 TI - Dose response characteristics of hypertonic saline dextran solutions. AB - In an effort to find the best hypertonic saline-dextran solution (HSD) for prehospital use, 33 chronically catheterized sheep were bled using a fixed pressure shock model (50 mm Hg x 2 hours) and resuscitated with 4 ml/kg of HSD solution (2-minute bolus). In the first set of experiments colloid was varied and sodium chloride was held constant, as 7.5% NaCl was paired with either 0%, 6%, or 12% dextran 70. A dose-response relationship existed, with cardiac output increasing 20% with each sequential dextran 70 concentration. Mean arterial blood pressure was higher in animals that were resuscitated with either the 7.5% NaCl/6% dextran 70 or 7.5% NaCl/12% dextran 70 solution (p less than 0.05). Using the optimal dextran 70 concentration from the first set of experiments (i.e., 12%), solute was varied in a second set of experiments comparing 0.9%, 3.8%, 7.5%, or 10% NaCl/12% dextran 70. Again, dose-response features were demonstrated, as cardiac output increased as a function of NaCl concentration. However, this response plateaued with the 7.5% NaCl concentration and no advantage was obtained by increasing the NaCl concentration to 10%. We conclude that a 4-ml/kg bolus of 7.5% NaCl/12% dextran 70 solution may be a more effective form of therapy than those previously evaluated. This new solution is now being included in our ongoing clinical trials. PMID- 1711584 TI - [Effect of pituitrin or phentolamine alone or in combination on WHVP and systemic hemodynamics in patients with liver cirrhosis]. AB - We observed the effect of pituitrin and phentolamine alone or in combination on wedged hepatic venous pressure (WHVP) and systemic hemodynamics in 28 patients with cirrhosis. The results showed that either of these drugs used separately could lead to reduction in WHVP, each of them could also cause by-effects on systemic hemodynamics. When pituitrin in combination with phentolamine was administered, no change could be found in inferior vena cava pressure, mean arterial pressure, pulse rate and cardiac index. This suggested that pituitrin in combination with phentolamine could not only efficaciously decrease WHVP, but also counteract side effects on systemic hemodynamics of each other and improve hepatic microcirculation. Our study provided evidence for the usefulness of the combination of the two drugs in controlling bleeding from esophagus varices. PMID- 1711585 TI - Preparation of AHTG-DNR conjugates and their antitumor effect in vitro. AB - 100%, 75%, 50%, 25% and 12.5% oxidized dextran T10 (Dex T10) were used as intermediate carriers for conjugating drug daunorubicin (DNR) and antibody anti human thymocytic globulin (AHTG), to form different immunoconjugates, AHTG:Dex:DNR. It was demonstrated that the conjugate with 25% oxidized Dex T10 as intermediate carrier linked more DNR molecules than the others. The degree of its substitution was 10-11 moles of DNR per mole of AHTG. Moreover, because the amount to reducing agent sodium borohydride (NaBH4), required for the reduction reaction, was relatively small, its damaging effect on AHTG and DNR was lessened accordingly. The antitumor effect of AHTG:Dex:DNR in vitro was tested by using 24 h cytotoxicity assay, with CEM as target cell. Cytotoxic effect of the conjugate was proven and the LD50 was 10.68 micrograms/ml. However, it showed only slight cytotoxic effect on non-target cell K562. When 10 min cytotoxicity assay was performed to show the specific tumor-killing effect of the conjugate, it revealed an obvious cytotoxic activity toward CEM, with the LD50 being 14.79 micrograms/ml, but hardly toward K562. These results suggest that AHTG:Dex:DNR possesses specific cytotoxic effect. PMID- 1711586 TI - The role of alkaline phosphatase isoenzymes as tumor markers for testicular germ cell tumors. AB - The role of serum alkaline phosphatase as a tumor marker for testicular germ cell disease was investigated in 26 patients with testicular seminoma and 13 with nonseminomatous germ cell testis tumors. Placental alkaline phosphatase-like enzyme was elevated in 50% of the stage I seminoma patients and in all patients with stages II to III disease. In addition, liver (tissue unspecific) alkaline phosphatase was elevated in 10 and 83% of the patients, respectively. Lactic dehydrogenase and beta-human chorionic gonadotropin (beta-HCG) were detected in 50 to 60% of the patients with stage I seminoma. By combining placental alkaline phosphatase-like enzyme, lactic dehydrogenase and beta-HCG, 75% of the stage I and 100% of the stages II and III seminoma patients could be identified correctly. Placental alkaline phosphatase-like enzyme in serum also occurred with nonseminomatous germ cell tumor but less frequently, while liver alkaline phosphatase was not detected at all. Thus, placental alkaline phosphatase-like enzyme and liver alkaline phosphatase were predominantly determined in the serum of patients with seminoma. In studies of tumor tissues from 31 of these patients, those with normal serum placental alkaline phosphatase-like enzyme levels had significantly lower tissue placental alkaline phosphatase-like enzyme levels than patients with elevated serum levels (p less than 0.01). Seminoma tissues showed significantly higher levels of placental alkaline phosphatase-like enzyme and liver alkaline phosphatase than nonseminomatous germ cell tumors (p less than 0.01), explaining the infrequent elevation of serum placental alkaline phosphatase-like enzyme and liver alkaline phosphatase found in patients with nonseminomatous germ cell tumors. PMID- 1711587 TI - Characterization of prostate cancer, benign prostatic hyperplasia and normal prostates using transrectal 31phosphorus magnetic resonance spectroscopy: a preliminary report. AB - We assessed the ability of 31phosphorus (31P) transrectal magnetic resonance spectroscopy to characterize normal human prostates as well as prostates with benign and malignant neoplasms. With a transrectal probe that we devised for surface coil spectroscopy we studied 15 individuals with normal (5), benign hyperplastic (4) and malignant (6) prostates. Digital rectal examination, transrectal ultrasonography and magnetic resonance imaging were used to aid in accurate positioning of the transrectal probe against the region of interest within the prostate. The major findings of the in vivo studies were that normal prostates had phosphocreatine-to-adenosine triphosphate (ATP) ratios of 1.2 +/- 0.2, phosphomonoester-to-beta-ATP ratios of 1.1 +/- 0.1 and phosphomonoester-to phosphocreatine ratios of 0.9 +/- 0.1. Malignant prostates had phosphocreatine-to beta-ATP ratios that were lower (0.7 +/- 0.1) than those of normal prostates (p less than 0.02) or prostates with benign hyperplasia (1.1 +/- 0.2, p less than 0.01). Malignant prostates had phosphomonoester-to-beta-ATP ratios (1.8 +/- 0.2) that were higher than that of normal prostates (p less than 0.02). Using the phosphomonoester-to-phosphocreatine ratio, it was possible to differentiate metabolically malignant (2.7 +/- 0.3) from normal prostates (p less than 0.001), with no overlap of individual ratios. The mean phosphomonoester-to phosphocreatine ratio (1.5 +/- 0.5) of prostates with benign hyperplasia was midway between the normal and malignant ratios, and there was overlap between individual phosphomonoester-to-phosphocreatine ratios of benign prostatic hyperplasia glands with that of normal and malignant glands. To verify the in vivo results, we performed high resolution magnetic resonance spectroscopy on perchloric acid extracts of benign prostatic hyperplasia tissue obtained at operation and on a human prostatic cancer cell line DU145. The extract results confirmed the differences in metabolite ratios observed in vivo. We conclude that transrectal 31P magnetic resonance spectroscopy can characterize metabolic differences between the normal and malignant prostate. PMID- 1711588 TI - The ability of systematic transrectal ultrasound guided biopsy to detect prostate cancer in men with the clinical diagnosis of benign prostatic hyperplasia. AB - Multiple directed and systematic ultrasound guided biopsies of the prostate were performed in 73 men with the clinical diagnosis of benign prostatic hyperplasia (BPH). Seven men (10%) had prostate cancer. Of the 67 patients with benign biopsies 40 underwent subsequent transurethral prostatectomy and 2 (5%) had prostate cancer. Multiple directed and systematic biopsies of the prostate detected 78% of the nonpalpable prostate cancers diagnosed in the study population. Radical prostatectomy was performed in all 9 men with prostate cancer: there were 3 small organ-confined tumors, 5 large organ-confined tumors and 1 stage C tumor with 1 focus of microscopic capsular penetration. Our results suggest that multiple directed and systematic ultrasound guided biopsies are capable of detecting low volume nonpalpable prostate cancer in men with BPH. However, the exact indication for pre-treatment ultrasound guided biopsy of the prostate in men with symptomatic BPH remains unclear. It may be that use of this modality is most appropriate for patients undergoing pharmacological therapy or balloon dilation of BPH rather than for those undergoing transurethral prostatectomy. PMID- 1711589 TI - Efficacy of transrectal ultrasound for identification of clinically undetected prostate cancer. AB - A total of 51 patients underwent transrectal ultrasound of the prostate before radical cystoprostatectomy for transitional cell carcinoma of the bladder. Each had a normal prostate by digital rectal examination, no history of prostatic adenocarcinoma and no invasion of the prostate by transitional cell carcinoma. Real-time and step-sectioned ultrasound images were interpreted at the time of sonography and results were compared to pathological examination of the step sectioned prostate specimen. When adenocarcinoma was identified in the specimen, cancer volume was determined. Positive ultrasound scans consisted of those exhibiting hypoechoic lesions. Hypoechogenicity due to transurethral resection defects, benign hyperplasia, vascular structures or shadowing from calcifications was not considered positive. Of 51 patients 27 (52.9%) exhibited no abnormality on ultrasound and were free of cancer in the prostate specimen, while 8 (15.7%) demonstrated a hypoechoic lesion that was proved to be prostate cancer. Seven patients (13.7%) with normal transrectal ultrasound scans had adenocarcinoma of the prostate, while 9 (17.6%) had lesions on ultrasound but no cancer. Based on these results, transrectal ultrasound has a sensitivity of 53.3% and a specificity of 75%. Further analysis reveals that transrectal ultrasound is more accurate in the detection of cancers of greater than 0.20 cc in volume than those of 0.20 cc or less. Transrectal ultrasound also is more accurate in the detection of peripheral zone than transition zone cancers. PMID- 1711590 TI - Preoperative prediction of pathological tumor volume and stage in clinically localized prostate cancer: comparison of digital rectal examination, transrectal ultrasonography and magnetic resonance imaging. AB - Accurate preoperative staging is important for proper selection of patients for radical retropubic prostatectomy. Preoperative staging by digital rectal examination, transrectal ultrasound, magnetic resonance imaging (MRI), Gleason grade and prostate specific antigen was compared to pathological stage for 25 patients who underwent radical retropubic prostatectomy. The predictive value for tumor confinement was 36% by rectal examination, 37% by ultrasound and 30% by MRI. The predictive value for extracapsular disease was 100% by rectal examination, 83% by ultrasound and 66% by MRI. Preoperative determinations of tumor volume by any modality did not correlate with pathological tumor volume. Digital rectal examination, ultrasound and MRI clinically understage the disease in most patients but they may be reliable to predict extracapsular disease. PMID- 1711591 TI - An improved method for computerized tomography-planned transperineal 125iodine prostate implants. AB - Transperineal 125iodine implants of the prostate can be performed with ultrasound guidance, a simple technique that has met with widespread acceptance. However, ultrasound does not allow good visualization of the pubic bones in relation to the pelvic outlet, and the pubic bones may interfere with needle placement in the anterior peripheral aspect of the prostate. Adequate irradiation of the entire periphery of the prostate is important to assure tumor control, since most tumors are multicentric and may involve the anterior aspect of the prostate. A computerized tomography-based treatment planning procedure that allows for angulation of transperineal needles to avoid the pubic bones and still reaches the most peripheral aspects of the gland is described. The technique also allows for the use of transrectal ultrasound and fluoroscopy to verify correct needle placement in the prostate at the procedure. Early treatment results, based on prostate specific antigen and regression of palpable tumors, are encouraging. PMID- 1711592 TI - Long-term followup results after expectant management of stage A1 prostatic cancer. AB - A total of 132 patients with stage A1 adenocarcinoma of the prostate was followed for 5 to 23 years (mean 8.2 years). Of these patients 52 underwent a second staging transurethral resection of the prostate between 1977 and 1986. Progressive disease developed in 3 of the 12 patients (25%) in whom residual foci of well differentiated cancer were detected by the second transurethral resection and who did not undergo further treatment. Of the 38 patients in whom the second transurethral resection did not detect residual cancer 3 (8%) also had progressive disease. From April 1989 to December 1989, 44 patients were re evaluated by transrectal ultrasonography and ultrasonographically guided biopsies. Of these patients 3 had locally progressive disease. Progressive disease also developed in 4 more patients. Thus, 13 of the 132 patients (10%) had either locally or systemically progressive disease after long-term followup. The interval from diagnosis of stage A1 disease to detection of progression ranged from 6 months to 20 years (mean 7 years). Ten patients underwent definitive treatment for what was believed to be locally progressive disease, 2 underwent palliative therapy and 1 had no therapy due to poor physical condition. Of the 10 patients who underwent definitive therapy 6 are alive without evidence of disease, 2 died of unrelated causes without evidence of disease and 2 are alive with stage D1 disease. These data suggest that patients in whom a second staging transurethral resection of the prostate detects residual cancer have a high probability of progressive disease. Also, negative findings from a second staging transurethral resection may not exclude the possibility of disease progression. Expectant management of stage A1 disease is warranted but regular and long-term followup is mandatory. PMID- 1711593 TI - From the Food and Drug Administration. PMID- 1711594 TI - Involvement of the renin-angiotensin system in ischemic damage and reperfusion arrhythmias in the isolated perfused rat heart. AB - We have investigated the contribution of the renin-angiotensin system to the damage caused by 40-min global ischemia in the isolated rat heart. A converting enzyme inhibitor, enalaprilat (70 nM), an angiotensin II receptor antagonist, compound 89 (2 microM), and an inhibitor of rat renin, CGP 44099A (20 nM), given before ischemia reduced the median duration of ventricular fibrillation on reperfusion to a similar extent (5.53, 5.72, and 5.14 min, respectively, compared to 13.98 min in the control group) but had no effect on creatine phosphokinase release (22.2 +/- 2.6, 22.1 +/- 6.8, and 24.1 +/- 3.6, IU/30 min, respectively, compared to 19.9 +/- 1.9 IU/30 min) or recovery or left ventricular developed pressure (67 +/- 6, 73 +/- 7 and 71 +/- 6%, respectively, compared to 66 +/- 3% after 30 min reperfusion). The increase in coronary resistance and left ventricular diastolic pressure on reperfusion was not affected by any of the agents. All three agents also tended to reduce the duration of ventricular fibrillation when given only on reperfusion. We conclude that angiotensinogen is present in the rat heart and it is converted to angiotensin I by a renin or a renin-like aspartic proteinase. The angiotensin I is converted to angiotensin II by converting enzyme. The angiotensin II formed is an important mediator of postreperfusion ventricular fibrillation in the isolated rat heart but does not contribute to the reduction in mechanical function produced by global ischemia in this preparation. PMID- 1711595 TI - Transport of beta-blockers and calcium antagonists by diffusion in cat myocardium. AB - Beta-blockers and calcium antagonists have been claimed to possess cardioprotective properties. This study addresses the question of whether a significant amount of these drugs will reach the cardiac myocytes during no-flow ischemia, where solute transport depends solely on diffusion. In anesthetized cats the hearts were excised. Apparent diffusion coefficients in cat myocardium at 37 degrees C (D'37) for 14C-verapamil (protein bound), 3H-metoprolol (lipophilic), 3H-atenolol (hydrophilic), and 3H-propranolol (lipophilic and protein bound) were determined by means of a "true transient diffusion" method and compared with the free diffusion coefficients in water (D37). D'37 of 14C verapamil, 3H-metoprolol, 3H-atenolol, and 3H-propranolol (in cm2 s-1 10(5)) were (mean +/- SEM) 0.025 +/- 0.002, 0.055 +/- 0.003, 0.041 +/- 0.007, and 0.025 +/- 0.002, respectively. The mean diffusive progression of the concentration profile of 3H-metoprolol and 3H-atenolol into the tissue during 20 min was calculated to be 0.36 and 0.31 mm, respectively. The protein binding of 14C-verapamil and 3H propranolol caused a significant fall in the progression to 0.24 mm for both drugs. These results indicate that, by diffusion, these drugs traverse the tissue too slowly to reach a significant amount of myocardium before myocyte necrosis occurs during conditions of noflow. Cardioprotective drugs are probably most effective, provided sufficient amounts are present in the tissue prior to the ischemic episode or sufficient supply via collateral blood flow is achieved. PMID- 1711596 TI - Different mechanisms involved in the positive inotropic effects of benzimidazole derivative UD-CG 115 BS (pimobendan) and its demethylated metabolite UD-CG 212 Cl in canine ventricular myocardium. AB - UD-CG 115 BS produced a positive inotropic effect in a concentration-dependent manner (EC50 = 9.2 x 10(-5) M, efficacy = 0.65) in isolated canine ventricular muscle. UD-CG 212 Cl also elicited a positive inotropic effect (EC50 = 1.9 x 10( 7) M, efficacy = 0.23); its potency was higher, but its efficacy was much less than that of UD-CG 115 BS. Although the effect of UD-CG 115 BS was not altered by a beta-adrenoceptor antagonist, bupranolol (3 x 10(-7) M), the response to UD-CG 212 Cl in high concentrations became transient in the presence of bupranolol: After reaching a peak, the force decreased gradually to the control level at greater than or equal to 10(-4) M. Both UD-CG 115 BS and UD-CG 212 Cl elevated the cyclic AMP level, but to a much smaller extent than other newly developed cardiotonic agents such as amrinone, milrinone, enoximone, and piroximone. Carbachol (3 x 10(-6) M) abolished the accumulation of cyclic AMP produced by these agents while it suppressed the maximum contractile response to UD-CG 115 BS by only 30%. The positive inotropic effect of UD-CG 212 Cl was converted to a negative effect by carbachol. Both UD-CG 115 BS and UD-CG 212 Cl produced a leftward shift in the concentration-response curve for the positive inotropic effect of isoproterenol. These results suggest that an elevation of cyclic AMP levels owing to cyclic AMP phosphodiesterase inhibition may be predominantly responsible for the positive inotropic effect of UD-CG 212 Cl but that a cyclic AMP-independent mechanism may contribute significantly to the positive inotropic effect of UD-CG 115 BS. UD-CG 212 Cl (greater than 3 x 10(-6) M) elicits a negative inotropic effect that is unmasked by beta-adrenoceptor blockade. PMID- 1711597 TI - Electrophysiologic properties of UK-66,914, a novel class III antiarrhythmic agent. AB - A class III antiarrhythmic agent that preferentially increases the effective refractory period without altering conduction velocity holds considerable promise for the treatment of life-threatening cardiac arrhythmias dependent on a reentrant mechanism. In the present study, the cellular electrophysiologic effects of a novel class III antiarrhythmic agent, UK-66,914, were evaluated. UK 66,914 prolonged action potential duration and extended the effective refractory period in isolated canine ventricular muscle and Purkinje fibers in a concentration-dependent manner, beginning at a threshold concentration of 0.1 microM. Analogous effects were found in isolated rabbit atrium beginning at a threshold concentration of 2 microM. At concentrations of UK-66,914 up to 20 microM there was no effect on the maximum rate of phase 0 depolarization (Vmax) or the amplitude of the action potential. In guinea pig papillary muscles. UK 66,914 at concentrations from 0.1 to 20 microM increased the effective refractory period at stimulation frequencies of 1 or 5 Hz, but did not slow conduction velocity. Therefore, UK-66,914 exhibits high selectivity for a class III antiarrhythmic effect in normal tissue. To elucidate the mechanisms responsible for the increase in effective refractory period, voltage clamp procedures were used in guinea pig ventricular myocytes. UK-66,914 reduced the amplitude of outward tail currents following depolarizing clamp steps with little effect either on the background K+ current or calcium currents, indicating that UK 66,914 selectively blocked the time-dependent potassium current. In anesthetized dogs, UK-66,914 (10 micrograms/kg to 1 mg/kg i.v.) prolonged both atrial and ventricular effective refractory periods, but in contrast to the studies performed in vitro, the minimum effective doses required to increase the effective refractory period in atria and ventricle were the same. Therefore, UK 66,914 is a potent selective class III antiarrhythmic agent, which owes its electrophysiologic profile to blockade of the time-dependent potassium current. PMID- 1711598 TI - Effect of salt loading on aldosterone response to long-term infusion of angiotensin II in rats. AB - We examined changes in plasma aldosterone level and blood pressure (BP) after long-term (12 days) intravenous infusion of angiotensin II (Ang II; 15 and 60 ng/min) with two different sodium intakes (0.66% and 8% salt-containing diet) in Wistar rats. Without salt loading, 15 ng/min of Ang II did not increase mean BP (112 +/- 1 vs. 112 +/- 2 mm Hg), but 60 ng/min of Ang II increased mean BP (144 +/- 3 mm Hg, p less than 0.01). However, the 8% salt diet not only enhanced the pressor effect of 60 ng/min Ang II (168 +/- 9 mm Hg, p less than 0.05), but also elevated mean BP with 15 ng/min of Ang II (159 +/- 3 mm Hg, p less than 0.01). On a normal-salt diet (0.66%), plasma aldosterone was increased by Ang II infusion from 11.3 +/- 0.3 ng/dl to 12.7 +/- 0.5 ng/dl (NS) at a rate of 15 ng/min Ang II, and to 15.7 +/- 0.4 ng/dl (p less than 0.01) at 60 ng/min Ang II. However, salt loading reduced Ang II-induced elevation of aldosterone: 11.3 +/- 0.5 ng/dl (less than 0.01) at a rate of 60 ng/min Ang II. Thus, salt loading diminished aldosterone response to Ang II, which might compensate salt-induced enhancement of the pressor effect of Ang II. PMID- 1711599 TI - Persistent cardioprotection by azapropazone in a canine model of coronary artery thrombosis and thrombolysis. AB - Activated neutrophils and possibly xanthine oxidase-derived free radicals are believed to be mediators of ischemia and reperfusion-induced myocardial damage. We studied the cardioprotective effect of the neutrophil stabilizer and xanthine oxidase inhibitor azapropazone in dogs subjected to thrombotic occlusion of the left anterior descending coronary artery (LAD), induced by intracoronary introduction of a copper coil, followed 60 min later by thrombolytic treatment with intracoronary streptokinase and 4-day reperfusion; we then determined infarct size by triphenyltetrazolium stain. Azapropazone [100 mg/kg intravenously (i.v.) followed by a 24-h i.v. infusion of 10 mg/kg/h, n = 8] or vehicle (n = 10) treatments were started immediately before the streptokinase infusion. Steady state plasma levels of azapropazone ranged from 97 to 163 micrograms/ml during the infusion. Myocardial blood flow and underperfused area at risk were determined using radiolabeled microspheres. Results were as follows (mean +/- SEM): area at risk (percentage of left ventricle) azapropazone 22.7 +/- 3.16 and vehicle 21.8 +/- 4.13; infarct size (percentage of area at risk), azapropazone 45.1 +/- 11.8 and vehicle 75.7 +/- 10.6, p less than 0.03; collateral blood flow (ml/min/g), azapropazone 0.27 +/- 0.02 and vehicle 0.23 +/- 0.02; total ischemic period (min), azapropazone 106 +/- 5.9 and vehicle 91.5 +/- 4.9. Azapropazone had no effects on heart rate (HR), blood pressure (BP), or rate/pressure product (RPP). These dta show that azapropazone limits infarct size in a canine model of coronary thrombosis and long-term reperfusion and that this cardioprotection is independent of cardiovascular parameters. PMID- 1711600 TI - Effects of beta-adrenergic blockade on the glomerular and tubular response to acute renal denervation. AB - Using micropuncture techniques in euvolemic adult male Munich-Wistar rats, we assessed the functional role of renal beta-adrenoceptors in mediating neural control of glomerular filtration and proximal tubular reabsorption. The determinants of nephron filtration and rate of proximal tubular reabsorption were measured in two groups of animals before and after acute surgical renal denervation (DNX). Group A animals (n = 6) were pretreated with the beta adrenoceptor antagonist propranolol (25 mg/kg body weight per day for 4-6 days). Group B animals (n = 7) served as non-beta-blocked controls. Acute renal DNX resulted in no significant change in nephron filtration rate or any of its determinants in either group. Acute DNX caused similar decrements in the rate of fluid reabsorption from the proximal convoluted tubule of beta-blocked and control rats. Loop of Henle fluid reabsorption did not appear to be affected by DNX in either group. Because the effect of denervation on proximal tubular reabsorption was not conditioned by prior beta-blockade, the beta-adrenoceptors present within the proximal convoluted tubule do not appear to be the primary mediators of the adrenergic influence on fluid transport in that segment of the nephron. PMID- 1711601 TI - Biological actions of cleaved atrial natriuretic factor (ANF101-105/106-126) in conscious sheep. AB - Atrial natriuretic factor (ANF) cleaved between Cys105 and Phe106 is the primary metabolite of ANF and circulates in human plasma. Because the role of this metabolite in vivo and its possible interaction with intact ANF are unclear, we studied the biologic effects of a 2-h infusion of rat cleaved ANF101-105/106-126 (15 pmol/kg/min) or vehicle alone in six normal sheep. Infusions of cleaved ANF increased venous plasma levels of cleaved ANF from less than 5 to 260 pmol/L and induced a progressive and significant increase in plasma cyclic GMP (p = 0.025) without significantly affecting plasma ANF levels. These changes were associated with a small (nonsignificant) decrease in arterial pressure and a significant increase in heart rate (HR) and sympathetic nervous activity and were followed by activation of the renin-angiotensin-aldosterone (RAA) axis after infusions were terminated. Unlike ANF itself, cleaved ANF was not natriuretic and did not reduce plasma volume or right atrial pressure. Calculated metabolic clearance rate (MCR) (1.47 +/- 0.4 L/min) and disappearance rate of cleaved ANF from plasma (4.8 +/- 0.37 min) were similar to values reported previously for intact ANF in sheep. These studies show that cleaved ANF stimulates guanylate cyclase and alters hemodynamics and the RAA system in vivo. PMID- 1711602 TI - Captopril-induced reversal of nitroglycerin tolerance: role of sulfhydryl group vs. ACE-inhibitory activity. AB - The angiotensin-converting enzyme (ACE) inhibitor captopril has been shown to reverse vascular tolerance to nitroglycerin (NTG). Whether captopril reverses NTG tolerance by providing sulfhydryl (SH) groups or by inhibiting ACE is not clear. To examine this issue, we treated rat aortic rings with buffer, captopril (SH +, ACE inhibitory activity +), enalaprilat (SH-, ACE inhibitory activity +), or N acetylcysteine (NAC, SH+, ACE inhibitory activity-) prior to their contraction with epinephrine and subsequent relaxation with NTG. Previous exposure of NTG treated rings resulted in marked resistance to the vasorelaxant effect of a subsequent exposure to NTG in buffer-treated rings. Both NAC and captopril, but not enalaprilat, potentiated the vasorelaxant effects of NTG during the first exposure of vascular rings to NTG and also prevented the development of tolerance to NTG during a second exposure. Buffer-treated rings showed an inability to accumulate cyclic guanosine monophosphate (GMP) in response to a second exposure to NTG. In contrast, both NAC and captopril-pretreated rings demonstrated a persistence of cyclic GMP accumulation during the second NTG exposure. The endothelium-dependent vasodilator acetylcholine (ACh) caused relaxation of the NTG-tolerant rings and also induced cyclic GMP accumulation in these rings. In other experiments, we found that prior exposure of vascular rings to ACh did not cause resistance to the subsequent vasorelaxant effects of ACh. NAC, captopril, and enalaprilat did not modulate the effects of ACh during either the first or subsequent exposures to ACh. In addition, indomethacin did not influence the "protective" effects of NAC or captopril against NTG tolerance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711603 TI - Plasma angiotensins in anephric humans: evidence for an extrarenal angiotensin system. AB - The recent identification of messenger RNAs encoding renin and angiotensinogen in nonrenal tissues raises the possibility that angiotensins (Ang) may be formed extrarenally and released into the plasma. The aim of this investigation was to test the hypothesis that plasma angiotensins may originate from extrarenal sites. Twenty-five patients with chronic renal failure (six surgically anephric and 19 with kidneys in situ) were studied prior to and after a standard hemodialysis treatment. Angiotensins were measured by extraction, high-pressure liquid chromatography (HPLC) separation, and radioimmunoassays. In patients with kidneys present, plasma renin activity (PRA) was 3.1 +/- 0.7 ng Ang I/ml/h. Ang I, Ang II, and Ang III levels were 70.6 +/- 9.0, 44.0 +/- 9.8, and 20.2 +/- 3.6 pg/ml, respectively. In all six anephric patients PRA was undetectable (less than 0.1 ng Ang I/ml/h). Ang I and Ang II were detected in four anephric patients, and Ang III was detected in three anephric patients (Ang I, 10.4 +/- 5.2; Ang II, 2.6 +/- 1.2; Ang III, 2.7 +/- 1.5 pg/ml, n = 6). At the completion of dialysis treatments, which reduced body weight by 2.5 +/- 0.2 kg in patients with kidneys and by 2.1 +/- 0.3 kg in anephric patients, there were no significant changes in PRA or plasma angiotensins in either group. Reduction in body water by hemodialysis did not increase the concentration of angiotensins in plasma. We conclude that there is a small but definite component of plasma angiotensin that is produced by nonrenal mechanisms and that is not stimulated by volume depletion. PMID- 1711604 TI - Effects of nitrovasodilators on platelet cyclic nucleotide levels in rabbit blood; role for cyclic AMP in synergistic inhibition of platelet function by SIN 1 and prostaglandin E1. AB - Nitrovasodilators increase both cyclic GMP and cyclic AMP in isolated platelets (Maurice DH, Haslam RJ. Mol Pharmacol 1990;37:671-81). To determine whether this occurs in blood, platelet cyclic[3H]GMP and cyclic [3H]AMP were measured in prelabeled rabbit platelets resuspended in modified Tyrode's solution or citrated blood. In the former medium, increases in cyclic [3H]nucleotides in response to nitroprusside (NP) and 3-morpholinosydnonimine (SIN-1) were maximal by 1 min; in blood, maximal increases were observed only after 10 min and were much smaller. In blood, SIN-1 was more effective than the same concentration of NP. After 10 min, 100 microM SIN-1 increased platelet cyclic[3H )GMP by 475 +/- 58% and cyclic[3H]AMP by 29 +/- 7% (means +/- SEM, 18 experiments). Supraadditive increases in platelet cyclic [3H]AMP in blood were observed when SIN-1 was combined with prostaglandin E1 (PGE1). Thus, after 10 min, SIN-1 (100 microM), PGE1 (20 nM), and SIN-1 + PGE1 increased cyclic[3H]AMP by 25 +/- 7, 35 +/- 6, and 130 +/- 17%, respectively (four experiments). In the same experiments, release of platelet [14C]serotonin by platelet-activating factor (PAF) was inhibited by 22 +/- 5, 2 +/- 2, and 61 +/- 5%, respectively. Increases in platelet cyclic[3H]GMP with SIN-1 were unaffected by PGE1. These results suggest that although cyclic GMP may mediate the effects of SIN-1 alone on platelet function, cyclic AMP mediates the synergistic action of SIN-1 and PGE1. M&B 22,948 (a selective cyclic GMP phosphodiesterase inhibitor) enhanced the increases in platelet cyclic[3H]GMP and cyclic[3H]AMP caused by SIN-1 and also increased the associated inhibition of [14C]serotonin release. M&B 22,948 also augmented the synergistic increases in cyclic[3H]AMP and inhibition of platelet function caused by SIN-1 + PGE1. The results show that a selected nitrovasodilator (e.g., SIN-1), a prostaglandin and a cyclic GMP phosphodiesterase inhibitor can exert synergistic effects on platelets in blood. This may be relevant to the pharmacologic management of thromboembolic disease. PMID- 1711605 TI - Cocaine depresses cardiac sympathetic efferent activity in anesthetized dogs. AB - Increased use of cocaine has increased the incidence of sudden cardiac death concomitantly, likely due to life-threatening arrhythmias and/or myocardial ischemia. The effects of cocaine on regulation of the heart and circulation by the autonomic nervous system (which is active during arrhythmias and myocardial ischemia) are not fully understood, however. Therefore, we wished to evaluate the influence of intravenous (i.v.) cocaine on spontaneous thoracic cardiac sympathetic efferent nerve activity in anesthetized dogs. In six pentobarbital anesthetized dogs, blood pressure (BP), heart rate (HR), and two cardiac sympathetic nerves (6 right-sided, 6 left-sided; 3 preganglionic, 9 postganglionic) were simultaneously recorded. Cocaine was infused for 15 min to a total dose of 6 mg/kg. Sympathetic multifiber efferent activities, HR, and BP were recorded continuously throughout the infusion and quantified at 5-min intervals during the infusion and for 45 min after infusion. Neural activities declined sharply to 54.7% of control (p less than 0.01) after only 5 min of infusion. After 15 min of infusion, nerve activity decreased to 39.4% of control (p less than 0.01). Cardiac nerve activity remained depressed (44.9%; p less than 0.01) 45 min after cocaine infusion. Cocaine caused a slight decrease in both HR and BP at 15 min. The rate-pressure product (RPP) decreased significantly during cocaine infusion. Comparable administration of lidocaine (6 mg/kg i.v. in 15 min) failed to influence cardiac sympathetic efferent activities significantly. We conclude that i.v. cocaine significantly depresses spontaneous cardiac sympathetic efferent neural activities in anesthetized dogs. PMID- 1711606 TI - Vascular smooth muscle relaxation by alpha 1-adrenoceptor blocking action of denopamine in isolated rabbit aorta. AB - We investigated the mechanism of vascular relaxation by denopamine (Deno), an oral positive inotropic agent that has selective beta 1-adrenergic action. Deno relaxed, dose-dependently (0.1-30 microM), ring segments of rabbit aorta, which were partially precontracted with 1 microM phenylephrine (Phe) or norepinephrine (NE), but did not relax those precontracted with 5 microM prostaglandin F2 alpha or 40 mM K+. The relaxation was not significantly inhibited by pretreatment with 10 microM propranolol or metoprolol. Deno produced parallel shifts in concentration-response curves to Phe, but this was not true for clonidine. The Schild plot analysis resulted in a linear regression of a slope of 1.075 +/- 0.063, which was not significantly different from unity, and the pA2 value of Deno against Phe was 5.57 +/- 0.02. The specific binding of [3H]prazosin to a rabbit aorta membrane preparation was displaced in a concentration-dependent manner by the simultaneous addition of Deno. The slope of a Hill plot was not significantly different from unity (1.102 +/- 0.147). The pK1 value for Deno calculated from the displacement curve was 5.29 +/- 0.17, which was not significantly different from the pA2 value of Deno. In conclusion, vascular smooth muscle relaxation by Deno was mediated by the blocking effect of alpha 1 adrenoceptors. Thus, these findings suggest that Deno may be effective in the treatment of congestive heart failure because it elicits a positive inotropic effect by beta 1-adrenergic action and vasodilation by alpha 1-adrenergic blocking action. PMID- 1711607 TI - MDL 27,032 relaxes vascular smooth muscle and inhibits protein kinase C. AB - The smooth muscle relaxant effect of MDL 27,032, 4-propyl-5-(4-pyridinyl)-2(3H) oxazolone, was studied in vitro using strips of femoral arteries and saphenous veins from dogs and trachea from guinea pigs. MDL 27,032 (10(-6)-10(-3) M) produced a concentration-dependent relaxation of arterial and venous strips contracted by carbachol. MDL 27,032 also antagonized contractions of arterial and venous strips produced by phorbol 12,13-dibutyrate (PDB), a protein kinase C activator, both in normal-Ca2+ and zero-Ca2+ medium. The compound inhibited protein kinase C in soluble extracts prepared from saphenous veins of dogs, with an IC50 value of 36.6 microM. MDL 27,032 was more effective against the contractions produced by phenylephrine and serotonin than by KCl in arteries, but no such selectivity was noted in veins. MDL 27,032 (10(-3) M) also inhibited accumulation of inositol phosphates in femoral artery but not in saphenous vein, and this effect may have contributed to the arterial-relaxant effect. Because the vascular smooth muscle relaxant effect of MDL 27,032 was endothelium independent, did not involve blockade of alpha-adrenoceptors or inhibition of cyclic nucleotide phosphodiesterases, stimulation of beta-adrenergic receptors, stimulation of adenosine A2-receptors, or activation of K+ channels, these data suggest that the relaxant effects of MDL 27,032 primarily involve inhibition of protein kinase C. PMID- 1711608 TI - Antihypertensive activity during inhibition of neutral endopeptidase and angiotensin converting enzyme. AB - The specific neutral endopeptidase (NEP) inhibitor, SQ 29,072 (7-[2 (mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid), was studied in conscious spontaneously hypertensive rats (SHRs) and in DOCA/salt hypertensive rats during inhibition of angiotensin-converting enzyme (ACE) activity with captopril or SQ 27,519 (the free acid of fosinopril). In the SHR, the maximal depressor responses to the combination of SQ 29,072 and SQ 27,519 (-44 +/- 4 mm Hg) were greater than the responses to any of the inhibitors given alone (-26 +/- 5, -40 +/- 10, and -28 +/- 6 mm Hg for SQ 29,072, captopril, and SQ 27,519, respectively). In contrast, the maximal antihypertensive activities of SQ 29,072 were the same in conscious DOCA/salt hypertensive rats infused with saline, captopril, or SQ 27,519 (-54 +/- 10, -51 +/- 8, and -58 +/- 11 mm Hg, respectively), indicating a lack of synergism in this model. In agreement, SQ 28,133 [N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine], a compound that inhibits both NEP and ACE, elicited significant depressor activities in both SHR and DOCA/salt hypertensive rats. In conclusion, a selective NEP inhibitor enhanced the depressor activity of ACE inhibitors in the conscious SHR, indicating that these agents may be effectively combined for treatment of some types of hypertension. PMID- 1711609 TI - Effect of inosine on function and adenine nucleotide content of the isolated working rat heart: studies of postischemic reperfusion. AB - Inosine added to the perfusion medium for isolated working rat heart induced a dose-dependent coronary vasodilator and positive functional effect. Inosine increased coronary flow (ml/min) from 11.8 +/- 0.5 (control, n = 16) to 15.6 +/- 0.6 (0.1 mM, n = 10), to 18.2 +/- 1.1 (0.5 mM, n = 6), and to 18.4 +/- 0.8 (1.0 mM, n = 14). Left ventricular systolic pressure was increased (LVSP, mm Hg) from 72.5 +/- 1.1 (control) to 75.4 +/- 1.7 (0.1 mM) to 78.7 +/- 2.1 (0.5 mM) and to 82.6 +/- 1.5 (1.0 mM). and cardiac output (CO, ml/min) from 26.4 +/- 2.0 (control) to 29.6 +/- 2.3 (0.1 mM), to 30.2 +/- 3.5 (0.5 mM), and to 37.4 +/- 2.0 (1.0 mM). At the end of 30-min reperfusion after a 15-min period of global total ischemia (n = 9), the functional parameters were significantly reduced (coronary flow 8.5 +/- 0.4 ml/min, LVSP 63 +/- 1.9 mm Hg, CO 13.6 +/- 1.9 ml/min). When inosine was present in the perfusion medium during the entire experimental protocol, coronary flow and heart function were also enhanced in a dose-dependent manner. The content of cardiac ATP (mumol/g) was decreased at the end of the 15 min ischemic period (1.89 +/- 0.17, n = 7; control 3.85 +/- 0.05, n = 4), and inosine had no effect. At the end of the 30-min postischemic reperfusion period, the ATP pool had recovered to an appreciable extent (3.01 +/- 0.11, n = 9) and was higher when 1.0 mM inosine had been infused (3.44 +/- 0.11, n = 7). Thus, inosine increased coronary flow dose dependently and, as a consequence, the function of the isolated perfused working heart both under control conditions and during the postischemic reperfusion period. PMID- 1711610 TI - Altered alpha 1-adrenoceptor-mediated responses in atria of rats with chronic left ventricular infarction. AB - Phosphoinositide (PI) turnover, chronotropic and inotropic responses to alpha 1 adrenoceptor activation, and alpha 1-adrenoceptor density were studied in atria from rats with left ventricular myocardial infarction (LVMI) and noninfarcted rats. LVMI was produced after surgical ligation of the left coronary artery in 8 week-old Wistar rats. Rats were killed 4 weeks after this operation when rats with LVMI had developed significant hypertrophy of both ventricles and atria. Phenylephrine 0.1 mM to 1 mM, with propranolol 0.3 mM, produced a concentration dependent increase in heart rate (HR) in right atria from noninfarcted rat hearts, and this response was significantly reduced in rats with LVMI. In electrically driven left atria, the concentration-dependent, phenylephrine induced positive inotropic responses observed with propranolol added were also significantly impaired in rats with LVMI as compared with those of noninfarcted rats. In contrast, neither PI turnover in response to phenylephrine in the presence of propranolol nor alpha 1-adrenoceptor density was reduced in rats with LVMI. These results suggest that the impaired alpha 1-adrenoceptor-induced chronotropic and inotropic responses in atria from rats with LVMI are not due to downregulation of alpha 1-adrenoceptors or to impaired activation of PI turnover after alpha 1-adrenoceptor stimulation, but to impairment of one or more biochemical responses distal to PI hydrolysis or changes in coupling mechanisms other than hydrolysis of PIs. PMID- 1711612 TI - Systemic and regional hemodynamic effects of the 5-hydroxytryptamine1A receptor agonists flesinoxan and 8-hydroxy-2(di-N-propylamino)tetralin in the conscious rat. AB - The systemic and regional hemodynamic effects of the centrally acting 5 Hydroxytryptamine1A (5-HT1A) receptor agonist flesinoxan (0.5 and 2.5 mg kg-1, intraarterially, i.a.) were investigated in conscious freely moving spontaneously hypertensive rats (SHR) and compared with those of 8-hydroxy-2(di-N propylamino)tetralin (8-OH-DPAT) (0.1 and 0.5 mg kg-1, i.a.). In one group of animals, cardiac output (CO) was measured with a precalibrated electromagnetic flow probe around the ascending aorta. In another group, regional vascular conductances were measured using radioactive microspheres. Flesinoxan and 8-OH DPAT dose dependently decreased blood pressure (BP) (22 +/- 5 and 13 +/- 4%, respectively, at the highest dose) mainly resulting from an increase in total peripheral vascular conductance (TPC) (34 +/- 12 and 16 +/- 3%, respectively) since there was no effect on CO. Both drugs reduced heart rate (HR) (17 +/- 4 and 20 +/- 4% for flesinoxan and 8-OH-DPAT, respectively, at the highest dose). Flesinoxan and 8-OH-DPAT showed a qualitatively similar pattern with regard to their effects on vascular conductances, causing increases in vascular conductances in the heart and skeletal muscles in contrast to results in a saline treated group. Vascular conductances in the lungs were markedly increased by both flesinoxan and 8-OH-DPAT, which may indicate that the conductance in the arteriovenous shunt vessels was enhanced. These results demonstrate that flesinoxan and 8-OH-DPAT elicit a qualitatively similar systemic and regional hemodynamic profile in conscious SHR. Furthermore, the increase in TPC appears to be due mainly to vasodilatation in the skeletal muscles. PMID- 1711611 TI - Effects of the new class III antiarrhythmic drug E-4031 on myocardial contractility and electrophysiological parameters. AB - The effects of the new class III antiarrhythmic agent E-4031 were investigated in different guinea pig cardiac preparations. In left atria, E-4031 (10(-8)-10(-5) M) prolonged the functional refractory period up to 45% and reduced the frequency of spontaneously beating right atria by 32%. In papillary muscles, E-4031 (3 x 10(-8)-3 x 10(-7) M) reversibly prolonged the action potential duration (APD70) of fast and slow APs by 68 and 51%, respectively. Vmax, resting potential, and AP amplitude (APA) were not altered. In isolated ventricular myocytes, E-4031 reversibly prolonged the APD90 from 275 ms (control) to 1,496 ms (10(-6) M), pD2 value 6.5. The current changes that underlie the AP-prolonging effect were also studied in ventricular myocytes: in concentrations up to 10(-5) M), E-4031 did not affect the Na+ or Ca2+ inward current but reduced the delayed rectifier (IK) tail current by 76% (10(-7) M). Contractility was enhanced by E-4031 in isolated atria by 20% (3 x 10(-7) M) and increased cell shortening in ventricular myocytes. Thus, the class III antiarrhythmic action of E-4031 is due to a selective reduction of outward currents. PMID- 1711613 TI - Modification of circadian blood pressure and heart rate variability by five different antihypertensive agents in spontaneously hypertensive rats. AB - Effects of captopril, clonidine, hydralazine, metoprolol, and prazosin on the circadian variability of blood pressure (BP) and heart rate (HR) as well as on BP and HR rises at awakening were studied in spontaneously hypertensive rats (SHR). An on-line computerized system was used for continuous intraarterial (i.a.) recording of BP and HR. Drugs were infused intravenously (i.v.) with osmotic minipumps. Circadian BP patterns were more pronounced and variable in SHR than in Wistar-Kyoto normotensive control rats. HR patterns were similar in both groups. Antihypertensive treatment resulted in differential effects on the expression of the circadian BP and HR patterns in SHR. Reductions in BP variability and reductions in the awakening increase in BP were not correlated with the BP lowering efficacy of agents. Treatment with clonidine reduced both circadian variability of BP and HR and the awakening increase. Prazosin reduced circadian BP variability and its awakening increase, whereas metoprolol reduced circadian HR variability only. Hydralazine and captopril did not cause any major effects on the circadian variability of BP and HR. These data indicate that circadian rhythms of BP and HR are under sympathetic control in SHR and are not influenced by nonsympatholytic vasodilators. PMID- 1711614 TI - Potassium supplementation versus bendrofluazide in mildly to moderately hypertensive Kenyans. AB - Eighty-four consecutive black patients who had confirmed but untreated hypertension participated in a double-blind randomized clinical trial involving potassium supplement (64 mmol/day) versus bendrofluazide (10 mg/day). Diastolic blood pressure (DBP) of the 42 patients receiving potassium supplementation (group A) decreased from a mean (+/- SD) of 108 +/- 3 to 88 +/- 4 mm Hg (p less than 0.001; paired t test) after 28 weeks of medication. DBP of the 42 patients receiving bendrofluazide (group B) decreased from a mean of 108 +/- 2 to 84 +/- 4 mm Hg (p less than 0.001; paired t test). There was no statistically significant difference between the magnitude of decrease in DBP in group A and B patients (unpaired t test). No clinical, biochemical, or ECG abnormalities occurred in group A patients. Eight group B patients showed hyperuricemia; 4 patients in the same group had hyperglycemia and 3 other patients had hypokalemia. The results support the notion that potassium supplementation may be an effective therapeutic approach to mildly hypertensive blacks. PMID- 1711615 TI - Characteristics of the vasorelaxing action of (3E)-4-ethyl-2-hydroximino-5-nitro 3-hexamide FK409, a new vasodilator isolated from microbial sources, in isolated rabbit arteries. AB - We examined the vasoinhibitory effect of (3E)-4-ethyl-2-hydroximino-5-nitro-3 hexamide FK409, a new vasodilator, on contractile responses in isolated rabbit arteries. FK409 (10(-8)-10(-5) M) inhibited contractile responses to norepinephrine (NE), histamine (His), and 5-hydroxytryptamine (5-HT) in rabbit aorta. The pattern of inhibition by FK409 was not competitive. The inhibitory effect of FK409 on the 5-HT response was much greater than that of nitroglycerin (NG). A high concentration of FK409 (10(-5) M) was necessary to inhibit the response to KCl (10-70 mM). The effect of combined treatment with FK409 (10(-5) M) and a subthreshold concentration of nifedipine (10(-9) M) on the KCl response was much greater than a single treatment with either agent. In addition, 3 x 10( 6) M D600, but not FK409 (10(-6) or 10(-5) M), inhibited the increase in the rate of 45Ca influx stimulated by a 40-mM KCl substituted solution. In a Ca2(+)-free medium containing EGTA and nifedipine, FK409 (10(-9)-10(-5) M) inhibited phasic responses to NE, His, and 5-HT, and subsequent sustained responses owing to addition of Ca2+. The response to caffeine in rabbit iliac arteries incubated in Ca2(+)-free medium was also inhibited by FK409 (10(-6) and 10(-5) M). In rabbit aorta precontracted with NE (10(-5) M) and partially inhibited by prior exposure to NG (10(-5) M), the relaxing effect of FK409 was slightly attenuated. Pretreatment of tissues with FK409 (10(-6) M) inhibited the relaxing action of NG much more than prior NG inhibited the relaxing action of FK409. Methylene blue (10(-5) M), but not hemoglobin (10(-6) M), inhibited the relaxing action of FK409, whereas M&B 22,948 (3 x 10(-4) M) potentiated it. FK409 caused a relaxation of precontracted aorta without endothelium that was inhibited by methylene blue. In rabbit aorta precontracted with NE, FK409 (10(-6) M) increased cyclic GMP but not cyclic AMP content. FK409 (10(-5) M) had no effect on the NE mediated increase in tissue inositol monophosphate (IP). These results suggest that FK409 inhibits the responses attributed to both intracellular Ca2+ release and Ca2+ influx through receptor-operated channels. The inhibitory effect of FK409 on both the KCl contractile response and KCl-stimulated 45Ca influx appears to be different from that of nifedipine or D600. Furthermore, the inhibitory action of FK409 may be partially mediated by cyclic GMP. PMID- 1711616 TI - Differential effects of central and peripheral desipramine on sympathoadrenal function and heart rate in conscious rabbits. AB - The effects of the tricyclic antidepressant, desipramine, on the baroreflex regulation of renal sympathetic nerve activity (SNA) and heart rate (HR), the nasopharyngeal reflex, plasma epinephrine and blood pressure (BP) were studied in conscious rabbits. Renal SNA and HR were recorded during slow ramp changes in mean arterial pressure (MAP) and during inhalation of cigarette smoke. Intracisternal (i.c.) and intravenous (i.v.) drug administration were compared, using doses which produced similar total central nervous system (CNS) concentrations. After a brief sympathoexcitation, i.c. desipramine inhibited renal SNA and MAP and increased plasma adrenaline and HR. The renal sympathetic baroreflex was substantially attenuated, with reflex range and gain reduced by 46 and 31%, respectively, but the cardiac baroreflex and nasopharyngeal reflex were affected minimally. Sixty-four percent of the desipramine remaining in the brain was concentrated in the medulla oblongata and spinalis; levels in cortex, thalamus, midbrain, lower spinal cord, and peripheral tissues were minimal. Treatment with i.v. desipramine decreased renal SNA and increased HR without altering MAP or epinephrine release. There was a slight attenuation of the nasopharyngeal reflex, a slight baroreceptor-independent reduction in renal SNA at most MAP levels, and an augmentation of the cardiac baroreflex. The drug was uniformly distributed throughout the CNS; only 20% of the centrally accumulated dose was in the medulla. Thus, i.c. desipramine produces a differentiated pattern of sympathoadrenal effects, probably by increasing norepinephrine (NE) concentrations at several sites within the medulla. The effects of i.v. desipramine were different, owing to poorer access to the medulla and the consequences of peripheral neuronal uptake blockade, which may include a modest inhibition at the sympathetic ganglia and an excitation at cardiac and vasoconstrictor neuroeffector junctions. PMID- 1711618 TI - Selective 5-lipoxygenase inhibitor BW A4C does not influence progression of tissue injury in a canine model of regional myocardial ischaemia and reperfusion. AB - The effects of BW A4C, a selective arachidonate 5-lipoxygenase (5-LO) inhibitor, on the progression of myocardial tissue injury were examined in anaesthetised, open-chest beagle dogs subjected to 90-min occlusion of the left anterior descending coronary artery (LAD) followed by 120-min reperfusion. Regional myocardial blood flow (RMBF, microspheres), segment shortening (sonomicrometry), and infarct size (tetrazolium stain) as an index of tissue injury were measured. Control animals (group 1, n = 11) received an infusion of vehicle [50% vol/vol glycofurol and distilled water, 47 ml at 12 ml h-1, intravenously (i.v.)] beginning 15 min before ischaemia and continuing until the end of reperfusion. Treated animals received either 10 (group 2, n = 11) or 50 micrograms kg-1 min-1 (group 3, n = 5) BW A4C i.v. in the same period. The infarct/risk zone ratio (I/R) in group 1 (24.1 +/- 6.0%) was not significantly different from that of group 2 (28.0 +/- 8.4%) or group 3 (46.1 +/- 6.7%). The close inverse relationship observed in controls between I/R ratio and collateral flow was not altered by either dose of BW A4C. Segment shortening during ischaemia (-0.2 +/- 2.7, -2.4 +/- 1.7, and -1.5 +/- 1.7%) and reperfusion (4.9 +/- 2.8, 1.0 +/- 1.8, and -1.0 +/- 1.9%) and during an isoprenaline infusion to unmask stunned myocardium (14.7 +/- 3.0, 14.7 +/- 2.6, and 7.4 +/- 1.7%) were not significantly different between groups 1, 2, and 3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711617 TI - Myocardial uptake and pharmacodynamics of quinidine and propafenone in isolated rabbit hearts: metabolic versus respiratory acidosis. AB - The influence of metabolic and respiratory acidosis on the myocardial accumulation and pharmacodynamics of quinidine and propafenone was studied in isolated perfused rabbit hearts. Three pH groups were evaluated: physiologic buffer, pH 7.4; metabolic acidosis, pH 7.0; and respiratory acidosis, pH 7.0. Myocardial accumulation of quinidine and propafenone was significantly reduced during acidosis. Although myocardial quinidine concentrations were similar in the metabolic acidosis group (14.4 +/- 1.2 micrograms/g) and the respiratory acidosis group (14.5 +/- 1.3 micrograms/g), the myocardial propafenone concentration was significantly less during metabolic acidosis (8.9 +/- 2.0 micrograms/g) as compared with respiratory acidosis (12.7 +/- 2.4 micrograms/g, p less than 0.05). The myocardial concentration-effect relationships were linear over the observed myocardial concentration ranges. The slopes of the linear concentration-effect relationships describing QRS duration were increased twofold by both types of acidosis as compared with normal pH (p less than 0.05). In contrast, the slopes of the concentration-effect relationships describing changes in ventricular repolarization and refractoriness were increased only during metabolic acidosis as compared with pH 7.4 (p less than 0.05). Thus, for any given concentration of drugs, the effects of quinidine and propafenone on ventricular conduction time are dependent on the pH of the perfusate, whereas these drug effects on ventricular repolarization and refractoriness are dependent on the buffer composition. PMID- 1711619 TI - Isradipine, a calcium-entry blocker, decreases vascular [125-I]low-density lipoprotein entry in hypercholesterolemic rabbits. AB - In 72 male rabbits aged 6 months, the endothelium of the abdominal aorta was abraded by a Fogarthy catheter. The animals were then fed a 1% cholesterol supplemented diet for 4 weeks. In addition, half of the animals were treated for the entire period with isradipine (0.3 mg/kg daily), a dihydropyridine calcium antagonist; the other 36 animals served as controls. One hour and 3, 6, 12, 24, and 48 hours before the animals were killed, [125-I]low-density lipoprotein (LDL 10 microCi) was administered intravenously (i.v.) to six animals in each group. The [125-I]LDL entry was quantified in the abdominal aorta according to the type and presence of endothelial lining. Isradipine significantly reduced the [125 I]LDL entry at most time intervals. In parallel, an increase in vascular prostaglandin (PGI2) synthesis was noted, which might be the underlying mechanism for the decreased LDL entry. PMID- 1711620 TI - Cicletanine and reperfusion injury: is there any correlation between arrhythmias, 6-keto-PGF1 alpha, thromboxane B2, and myocardial ion shifts (Na+, K+, Ca2+, and Mg2+) induced by ischemia/reperfusion in isolated rat heart. AB - We studied the effects of cicletanine, an anti-hypertensive drug, on reperfusion arrhythmias in relation to 6-keto-PGF1 alpha, thromboxane B2 (TXB2), and ion shifts (Na+, K+, Ca2+, and Mg2+) induced by ischemia and reperfusion in hearts isolated from spontaneously hypertensive rats. Hearts were subjected to 30-min global ischemia followed by 10-min reperfusion. 6-keto-PGF1 alpha and TXB2 concentrations were determined by radioimmunoassay (RIA) from coronary effluents, and myocardial Na+, K+, Ca2+, and Mg2+ contents were measured by atomic absorption spectrophotometry from myocardial tissue. Two basic protocols were used: (a) acute administration when 3, 10, 30, or 100 mg/L cicletanine was included in the perfusion buffer; and (b) chronic application, in which rats received cicletanine 3, 10, 30, or 100 mg/kg/day orally for 14 days. Acute administration of the drug in low concentrations (3 or 10 mg/L), significantly increased endogenous 6-keto-PGF1 alpha production before ischemia and during reperfusion, whereas higher doses of cicletanine (30 or 100 mg/L) as well as chronic application of the drug failed to increase production of 6-keto-PGF1 alpha in the myocardium. TXB2 production was not influenced by either acute or chronic treatment with the drug. Neither treatment changed myocardial ion contents in comparison with control values (Na+ = 45 +/- 4, K+ = 252 +/- 7, Ca2+ = 1.4 +/- 0.1, and Mg2+ = 12.5 +/- 0.3 mmol/kg dry weight) in nonischemic hearts. Thirty-minute ischemia resulted in a two- and fourfold accumulation of myocardial Na+ and Ca2+ and a 50% decrease in both K+ and Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711621 TI - A novel orally active dopamine (DA) prodrug TA-870. IV. Renal vasodilatory and negative chronotropic effects in anesthetized dogs: influence of DA1 and DA2 dopamine receptor selective antagonists. AB - We investigated the involvement of the activations of dopamine (DA) receptors DA1 and DA2 in the effects of a novel DA prodrug TA-870 in anesthetized dogs and compared the effects with those of DA. Intravenous (i.v.) administration of TA 870 in anesthetized dogs produced a dose-dependent increase in free DA in blood and caused a decrease in renal vascular resistance (RVR) and an increase in renal blood flow (RBF). It also produced a bradycardia at high doses. In phenoxybenzamine-treated anesthetized dogs, the renal vasodilatory action of intrarenally administered TA-870 was less than or equal to that of i.v. TA-870, whereas that of intrarenally administered DA was greater than that of i.v. DA. The renal vasodilatory effects of TA-870 and DA were almost completely inhibited by the DA1 selective antagonist SCH-23390 but were not affected by the DA2 selective antagonist domperidone. In bilaterally vagotomized dogs, both TA-870 and DA at high doses partially reduced the positive chronotropic responses to the right postganglionic cardioaccelerator nerve stimulation; this effect was abolished after treatment with domperidone. In isolated guinea pig right atria, unchanged TA-870 and deethoxycarbonyl metabolite of TA-870, the intermediate from TA-870 to DA, did not affect the spontaneous atrial rate even at high concentrations, whereas DA produced a concentration-dependent increase in the rate. In addition, neither compound showed antagonistic action to the positive chronotropic effect of DA, which was competitively inhibited by propranolol. We conclude that the renal vasodilatory effect of TA-870 results simply from the activation of DA1 receptors by DA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711623 TI - The effect of captopril on pharmacokinetics of digoxin in patients with mild congestive heart failure. AB - The effect of captopril on steady-state pharmacokinetics of digoxin was studied in 12 patients with mild congestive heart failure (CHF; New York Heart Association functional class 1 or 2). Serum and urine digoxin concentrations were determined before and after a repeated administration of captopril in the patients on chronic digoxin therapy. The patients were taking digoxin, 0.25-0.375 mg/day, once daily, and were concurrently administered captopril, 37.5 mg/day, three times daily, for seven days. Peak serum concentration of digoxin (SCD) before and after captopril was 2.1 +/- 0.2, mean +/- SEM, and 2.0 +/- 0.1 ng/ml; the time to peak was 1.1 +/- 0.2 and 1.8 +/- 0.3 h; the terminal half-life (t1/2 alpha) was 10.9 +/- 1.0 and 8.7 +/- 0.9 h, and the area under the concentration time curve to 24 h was 26.9 +/- 2.4 and 27.6 +/- 2.0 ng.h/ml. There was no significant difference between patients without and with captopril in SCD and its pharmacokinetic parameters. Renal digoxin clearance and creatinine clearance also showed no significant difference. After an administration of captopril, angiotensin-converting-enzyme (ACE) activity was well suppressed. These results suggest that captopril does not increase SCD in patients with CHF, and effectively suppresses ACE activity. Thus, modification in the dosage regimen of digoxin may be unnecessary in the case of coadministration with captopril. PMID- 1711622 TI - Mr 40,000 and Mr 39,000 pertussis toxin substrates are increased in surgically denervated dog ventricular myocardium. AB - To test the general hypothesis that cardiac innervation may participate in myocardial G protein regulation, we examined the effects of complete intrapericardial surgical denervation or sham operation in dogs. In particulate fractions of dog left ventricular (LV) myocardium harvested 28-33 days after denervation or sham operation, Mr 40,000 and Mr 39,000 pertussis toxin-sensitive substrates (G proteins) were increased by 31% (1.31 +/- 0.084 vs 1.00 +/- 0.058 OD, arbitrary units, p less than 0.01) and 40% (1.40 +/- 0.117 vs. 1.000 +/- 0.084 OD, arbitrary units, p less than 0.02), respectively, as compared with sham operated controls. The Mr 40,000 pertussis toxin-sensitive band comigrated with a pertussis toxin-sensitive substrate in human erythrocyte membranes known to contain an alpha Gi species. In these same preparations basal, GTP and GppNHp stimulated adenylate cyclase activities were decreased in denervated heart by 20, 26, and 19%, respectively, consistent with increased activity of an inhibitory G protein. In contrast, Gs function was not altered, because cyc(-) membranes reconstituted with membrane extracts and fluoride and beta-receptor-stimulated adenylate cyclase activity were not different between groups. Furthermore, adenylate cyclase catalytic subunit function as assessed with forskolin and manganese stimulation was not different between preparations of control and denervated heart. We conclude that in preparations of surgically denervated dog myocardium Mr 40,000 and Mr 39,000 pertussis toxin-sensitive G proteins are increased by 31 and 40%, respectively, and that functional alterations in adenylate cyclase activity exist, consistent with increased inhibitory G-protein function. PMID- 1711624 TI - Effects of the new class-III antiarrhythmic drug D-sotalol on contractile function of postischemic myocardium. AB - The hemodynamic effects of the new class-III antiarrhythmic drug D-sotalol (2 mg/kg i.v.) on postischemic myocardium were investigated in comparison with the actions of the racemic D,L-sotalol (2 mg/kg i.v.) in rats. Drug infusion (7 min) was started 20 min after 3 x 4 min of global ischemic injury (oxygen deficiency). The left ventricular isovolumic pressure-generating capacity at the beginning of infusion was reduced by 15%, indicating postischemic dysfunction. Infusion of D sotalol caused a highly significant (p less than 0.01) reduction of the left ventricular pressure-generating capacity (to 58 +/- 5%), as well as of the dp/dtmax (to 21 +/- 3%). Stroke volume (to 66 +/- 6%), ejection fraction (to 62 +/- 7%), and cardiac output (to 51 +/- 6%) were also decreased (p less than 0.01). Fifteen minutes after infusion a partial renormalization of the hemodynamic measures (dp/dtmax 41 +/- 5%; stroke volume 94 +/- 8%) was observed. Infusion of the racemic D,L-sotalol caused similar hemodynamic changes (left ventricular pressure-generating capacity 60 +/- 2%, dp/dtmax 25 +/- 2%), indicating its cardiodepressant action. In contrast to prior findings on normal myocardium, our present results indicate that D-sotalol, a potential new class III antiarrhythmic drug, has a considerable negative inotropic effect on postischemic myocardium. PMID- 1711625 TI - (-)[3H]amlodipine binding to rat cardiac membranes. AB - Amlodipine is a newly developed long-acting dihydropyridine-based calcium antagonist. To characterize the binding properties of this compound, saturation binding studies were undertaken, using (-)[3H]amlodipine and rat cardiac membrane fragments. (-)[3H]Amlodipine bound to a single population of high-affinity binding sites with a KD of 1.68 +/- 0.12 nM, a Bmax of 0.34 +/- 0.08 pmol/mg protein, and a Hill coefficient approaching unity. Binding required up to 5 h to reach asymptote, and was pH- and temperature-sensitive. The specific binding was totally inhibited by (-) amlodipine and (-) D600 (IC50 values of 9.20 +/- 5.56 and 6.58 +/- 6.57 nM, respectively) and only partially inhibited by (+) PN 200 110, (-) Bay K 8644, (+) D600, and d-cis diltiazem (IC50 values of 60 +/- 10, 160 +/- 20, 250 +/- 40, and 200 +/- 30 nM, respectively). These results indicate that in addition to its ability to bind to the dihydropyridine and benzothiazepine recognition sites in rat cardiac membrane fragments, (-)[3H]amlodipine also binds strongly to the recognition sites for the phenylalkylamine-based calcium antagonists. The results also show that the inhibition of (-)[3H]amlodipine binding by D600 is stereospecific with (-) greater than (+)D600. Dissociation of bound (-)[3H]amlodipine was slowed under acidotic (pH 6.0) and accelerated under alkalotic (pH 10.0) conditions. PMID- 1711626 TI - The effect of the partial beta-1-adrenoceptor agonist xamoterol on hemodynamics and myocardial energetics in patients with idiopathic dilated cardiomyopathy. AB - Xamoterol, a new partial beta 1-adrenoceptor agonist, was evaluated in respect to its hemodynamic and energetic effects when given acutely i.v. (0.2 mg/kg body weight) to 10 patients with idiopathic dilated cardiomyopathy. Hemodynamically, a small drop in left ventricular end-systolic volume index (from 105 +/- 38 to 91 +/- 48 ml/m2; p less than 0.05) and end-diastolic volume index (from 168 +/- 44 to 152 +/- 41 ml/m2; p less than 0.05) and an increase in maximum rate of left ventricular pressure rise (from 1,003 +/- 358 to 1,283 +/- 398 mm Hg/s; p less than 0.001) was observed. The systolic stress-time integral was only slightly reduced after injection of xamoterol, from 107 +/- 28 to 94 +/- 35 x 10(3) dyn.s/cm2 (NS). Other conventional hemodynamic variables did not change significantly. Myocardial oxygen consumption per beat increased from 125 +/- 39 microliters/beat/100 g to 153 +/- 62 microliters/beat/100 g (p less than 0.05). The ratio of myocardial oxygen consumption per beat and stress-time integral--as an inverse measure of left ventricular contraction economy--increased significantly, from 132 +/- 62 to 192 +/- 115 microliters/beat/10(3) dyn.s/cm2 (p less than 0.02) after injection of xamoterol, whereas left ventricular efficiency decreased from 17.6 +/- 7.8 to 13.8 +/- 6.5% (p less than 0.05). Thus, in idiopathic dilated cardiomyopathy, xamoterol exhibited only minor hemodynamic effects whereas myocardial contraction economy and efficiency were significantly reduced. PMID- 1711627 TI - In vivo effect of bradykinin during ischemia and reperfusion: improved electrical stability two weeks after myocardial infarction in the pig. AB - In this study, the effect of bradykinin or saline infusion during ischemia and reperfusion on electrical stability, 2 weeks after myocardial infarction, was assessed. Acute myocardial infarction was induced in 21 pigs by a transluminal occlusion of the left coronary artery with a catheter balloon, inflated for 45 min. Bradykinin was administered by a 30-min infusion that started after 30 min of coronary occlusion and was continued until 15 min after reperfusion. Although creatine kinase levels in bradykinin-treated animals were significantly lower (p less than 0.001), 2 week survival was not different between groups. In survivors, the filtered QRS (ventricular deflection) duration (detected using signal averaged electrocardiography) was significantly prolonged in saline-treated pigs, whereas in bradykinin-treated pigs this prolongation was prevented. The terminal voltage of the QRS complex was significantly lower in saline-treated pigs than in bradykinin-treated pigs. These two parameters signify an improved electrical stability after bradykinin treatment. Refractory periods in saline-treated hearts were longer than in bradykinin-treated hearts (106 +/- 10% vs. 95 +/- 13%, p less than 0.05). Also, current thresholds in the infarct border zones showed a greater variance in saline-treated hearts (p less than 0.001), pointing toward more tissue heterogeneity of the infarct border zone. Programmed electrical stimulation showed a trend toward reduced inducibility of sustained ventricular tachycardia in bradykinin-treated hearts. Therefore, bradykinin improves electrical stability weeks after experimental myocardial infarction. PMID- 1711628 TI - Hemodynamic response to intracoronary infusion of atrial natriuretic factor in patients with normal or altered left ventricular function. AB - To assess the effects of atrial natriuretic factor (ANF) on cardiac function, synthetic human ANF was infused directly into the left main coronary artery of eight patients with congestive heart failure (CHF) and six subjects with normal left ventricular (LV) function (controls) who underwent cardiac catheterization. ANF infusion at the incremental rates of 60, 125, 400, and 800 ng/min induced a dose-related increase in plasma ANF concentrations in the coronary sinus, from 1,223 +/- 590 to 3,923 +/- 1,123 pg/ml in patients with CHF (p less than 0.01) and from 1,041 +/- 605 to 2,710 +/- 1,741 pg/ml in controls (p less than 0.01). Peripheral plasma ANF concentrations (femoral artery) increased from 538 +/- 278 to 752 +/- 262 pg/ml (p less than 0.01) in patients with CHF and from 193 +/- 63 to 401 +/- 147 pg/ml (p less than 0.01) in controls. The increase in peripheral or coronary sinus plasma ANF concentrations did not differ between patients with CHF and controls. At the three lowest ANF infusion rates, cardiac index (CI), systemic vascular resistance (SVR), and LV contractility assessed by peak positive dP/dt remained unchanged both in patients with CHF and in controls. At the highest ANF infusion rate, CI increased from 2.18 +/- 0.53 to 2.54 +/- 0.49 L/min/m2 (p less than 0.01) and SVR decreased from 14.6 +/- 3.6 to 12.8 +/- 4.5 mm Hg.min/L (p less than 0.01) in patients with CHF. There was no associated change in heart rate (HR), mean arterial blood pressure (MAP), cardiac filling pressures, or peak positive dP/dt.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711629 TI - Contribution of splanchnic and peripheral vascular tissues to the disposal of angiotensin-II and to regional conversion rates of angiotensin-I: a pilot study in humans. AB - The present study investigates angiotensin-II (A-II) balance across the splanchnic vascular bed and leg vascular tissues in humans, at baseline conditions and following a systemically ineffective femoral arterial angiotensin I (A-I) infusion, both before and during inhibition of angiotensin-converting enzyme (ACE) with enalaprilat. In six healthy men, net regional A-II balance was calculated from the transfemoral or transsplanchnic arteriovenous differences in plasma concentrations and the corresponding regional plasma flow. Systemic and splanchnic hemodynamics remained unchanged throughout the trial, indicating an absence of major hemodynamic effects during A-I infusion or concomitant ACE inhibition. In contrast, regional leg plasma flow significantly decreased following A-I infusion (165.2 +/- 15.7 vs. 304.7 +/- 43.7; p less than 0.01) and again gradually returned to about baseline values after ACE inhibition. Baseline net transfemoral A-II balance was equilibrated at slight formation rates in four subjects and at minimal extraction rates in two of the six subjects. Following A I infusion, a shift toward net femoral A-II formation (average increase 2,774% above baseline) was observed in all subjects (p less than 0.01). After concomitant ACE inhibition, femoral A-II balance again returned to baseline levels. Across the splanchnic vascular bed a net baseline. A-II extraction was observed in all subjects. Following A-I infusion an average increase by 285% (p less than 0.05) in splanchnic A-II extraction was observed. During concomitant ACE inhibition splanchnic A-II extraction tended to decrease toward baseline values in four subjects, and remained rather unchanged in two subjects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711630 TI - Effects of R 56,865, a preventer of cellular calcium overload, on left ventricular diastolic properties during pacing-induced ischemia in dogs. AB - The effects of R 56,865, a compound with unusual calcium antagonistic properties, on altered left ventricular (LV) diastolic properties were studied during pacing induced ischemia in dogs with coronary stenosis. Severe coronary artery stenosis was produced on both the left anterior descending (LAD) and circumflex (Cx) coronary arteries in anesthetized beta-blocked dogs. The right atrium was paced at 200 beats/min for 3 min. In the post-pacing period and before drug intervention, the most characteristic observation was a significant increase in LV end-diastolic pressure (LVEDP) and in the time constant of (tau) LV pressure decrease; after return of LV hemodynamics to baseline values (15 min), R 56,865 (0.16 mg.kg-1) or solvent (control) was injected and four subsequent pacing runs were performed at 30-min intervals. In the postpacing period of these four subsequent runs, there was a steep increase in LVEDP and tau-values while HR returned more slowly to baseline values. After R 56,865 infusion, the increase in LVEDP and tau-values was significantly lower than in the pretreatment run and all LVEDP values were significantly lower than in the control group. We conclude that R 56,865 effectively attenuates ischemia-induced changes in LV diastolic stiffness. PMID- 1711631 TI - Dual mechanism of the relaxing effect of nicorandil by stimulation of cyclic GMP formation and by hyperpolarization. AB - In addition to previous results from our laboratory showing that nicorandil relaxed vascular smooth muscle by increasing cyclic GMP levels, it was shown to activate K-channels as well, an effect that also leads to relaxation. In the present study, we attempted to differentiate quantitatively between these two effects in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclic GMP. When the strips were contracted by the thromboxane A2 analogue U 46619 (1 microM) with 10 microM methylene blue added, nicorandil produced 30-50% relaxation without significant changes in cyclic GMP. When in U 46619-contracted strips the hyperpolarizing effect of nicorandil was suppressed by increasing extracellular K+ to 80.4 mM (30-fold), nicorandil caused only 52% relaxation, whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechanisms of relaxation by nicorandil was further achieved through calculation of the cyclic GMP-mediated component from a correlation between increases in cyclic GMP and percentage of relaxation as produced by nicorandil under conditions of inhibited hyperpolarization, i.e., in strips contracted with 1 microM U 46619 or 26.8 mM K+ (10-fold) and exposed to either 30-fold K+ or 10 mM Ba2+. Under both conditions, similar correlations between cyclic GMP and relaxation were obtained. Because U 46619, in addition to its contractile effect, partially antagonized the relaxation by nicorandil without changing cyclic GMP, the correlation was corrected for this effect and indicated a participation of cyclic GMP in the overall relaxant response of approximately 30-40% at low and less than or equal to 80-90% at high concentrations of nicorandil. PMID- 1711632 TI - Cardiovascular responses to the stereoisomers of dobutamine in isolated rat hearts 48 hours after acute myocardial infarction. AB - Effects of acute myocardial infarction (48 h) on cardiovascular responses to (+/ )-, (-)-, and (+)-dobutamine were studied in isolated perfused rat hearts. The effects of the racemate and the isomers on ventricular pressure were measured simultaneously in infarcted left ventricle and noninfarcted right ventricle. Administration of (+/-)-, (-)-, and (+)-dobutamine resulted in dose-dependent increases in inotropic parameters and coronary flow (CF) in both control and in infarcted hearts. As compared with the (+)-isomer, the racemate and the (-) isomer in both control and infarcted hearts were approximately 1.5 and 15 times weaker with respect to inotropic parameters. For (+/-)-, (-)-, and (+) dobutamine, there was no significant difference in pD2 values in any of the inotropic measurements between the infarcted group and the corresponding control group. The maximal obtainable responses were significantly lower in the infarcted groups as compared with their corresponding control group. In control hearts, effects of isoproterenol and methoxamine as compared with (+/-)-dobutamine were approximately 2 log units more and 2 log units less in potency, respectively. The inotropic effects of (-)-dobutamine but not those of methoxamine were completely antagonized by propranolol (0.3 microM). Although the results provide evidence for the existence of myocardial alpha 1-adrenoceptors, all forms of dobutamine exerted positive inotropic effects through activation of beta-adrenoceptors both in control and in infarcted in rat hearts. PMID- 1711633 TI - In vivo enhancement of platelet activating factor-induced prostacyclin production by OKY-046, a selective inhibitor of thromboxane A2 synthase. AB - Platelet-activating factor (PAF), a likely mediator of endotoxin action, causes thromboxane A2 (TXA2) release and pulmonary hypertension in pigs. We examined the effect of selective TXA2 synthase inhibition with OKY-046 on cyclooxygenase metabolites during PAF-induced pulmonary hypertension. Six closed-chest pigs received PAF in escalating doses (0.1, 0.3, 1.0, and 3.0 nmol intravenously, i.v.) before and after (E)-3[4-(1-imidazolyl methyl) phenyl]-Z-propenoic acid hydrochloride monohydrate OKY-046, 10-mg/kg i.v. bolus plus 20-mg/kg/h infusion. Plasma samples at peak PAF effect had radioimmunoassay (RIA) for the stable metabolites of TXA2 (TXB2) and prostacyclin (6-keto-PGF1 alpha). Tachyphylaxis was not noted in 5 control pigs given sequential repeats of the PAF dosing series. Pulmonary vascular resistance (PVR) was 240 +/- 30 (SE) dyne s cm-5 at baseline and increased to 3,100 +/- 1,300 after 1.0 nmol PAF (p less than 0.05). When the same amount of PAF was given after OKY-046, PVR increased only to 820 +/ 280 dynes/s/cm-5. TXB2 was 34 +/- 7 pg/0.1 ml at baseline and increased to 70 +/ 4 pg/0.1 ml with PAF 1.0 nmol (p less than 0.001). TXB2 levels were unchanged from 34 +/- 4 pg/0.1 ml when PAF 1.0 nmol was administered after OKY-046 (NS vs. pre-OKY-046). In contrast, 6-keto-PGF1 alpha, 6 +/- 2 pg/0.1 ml at baseline, increased to 24 +/- 4 pg/0.1 ml after PAF 1.0 nmol and increased further to 50 +/ 8 pg/0.1 ml when PAF 1.0 nmol was given after OKY-046 (p less than 0.05 vs. pre OKY-046).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711634 TI - Alpha-adrenoceptor-mediated inotropism and beta-adrenoceptor-mediated inotropism in isolated rabbit ventricles: a comparison in mechanical effects and energetic efficiency. AB - We assessed alpha-adrenoceptor-mediated inotropism in comparison with beta adrenoceptor-mediated inotropism in whole left ventricle. We used an isolated perfused isovolumic rabbit heart preparation, which enables one to assess contractility and time course of contraction as well as energetic efficiency. Left ventricular pressure (LVP) and volume were measured with an intraventricular fluid-filled balloon. Myocardial oxygen consumption was assessed from the oxygen tension of coronary effluent. Both isoproterenol (n = 7) and phenylephrine (n = 7) increased LV developed pressure (LVDP). Isoproterenol shortened the time to peak pressure (Tmax) from 171 +/- 6 to 142 +/- 7 ms (p less than 0.01) and the time constant of LV pressure decay (TBF) from 44.8 +/- 2.7 to 32.8 +/- 1.9 ms (p less than 0.01). In contrast, phenylephrine slightly but significantly prolonged Tmax from 178 +/- 4 to 184 +/- 4 (p less than 0.05) and did not alter TBF. Both isoproterenol and phenylephrine increased O2 consumption, whereas only phenylephrine increased the force-time integral from 14.0 +/- 1.5 to 17.2 +/- 2.1 g.s (p less than 0.01). These results confirmed in the whole heart preparation that alpha- and beta-adrenoceptor-mediated inotropisms had qualitatively different effects on the time course of contraction and energetic efficiency. PMID- 1711635 TI - Chronic effects of metoprolol on myocardial beta-adrenergic receptors in doxorubicin-induced cardiac damage in rats. AB - This study was designed to clarify whether chronic administration of metoprolol had any influence on cardiac beta-adrenergic receptors (BAR) in doxorubicin (DOX) induced cardiac damage. DOX was injected through the tail vein into rats (3 mg/kg/week, n = 22) for 5 weeks. One week after the final injection, the rats were randomly divided into two groups, M (with metoprolol, 10 mg/kg/day subcutaneously, s.c.; n = 11) and D (without metoprolol; n = 11). After 3-week infusion, plasma norepinephrine (NE) levels and BAR density [[125I]iodocyanopindolol (ICYP) binding on crude membranes] were measured. Left and right ventricular end-diastolic pressure (LVEDP, RVEDP) and myocardial NE levels were also measured, and the results were compared with those from an age matched control group (C n = 11). Decreased BAR density and increased plasma NE levels were evident in group D, indicating downregulation. In group M, BAR density and plasma NE levels were similar to those in group C. The myocardial NE levels were decreased in group D, but were higher in group M than in group D. The LVEDP and RVEDP were increased in group D, but were almost normal in group M. These results suggest that metoprolol is a promising drug for treatment of DOX induced cardiac damage. PMID- 1711636 TI - Effects of intravenous endothelin on hemodynamics and cardiac contractility in conscious Milan normotensive rats. AB - The effects of endothelin-1 (ET-1) on hemodynamics and cardiac contractility were compared with the responses to angiotensin I (AI) and phenylephrine (PE) in Milan normotensive rats. Intravenous (i.v.) injection of ET-1 (0.8 nmol/kg) initially decreased mean blood pressure (MBP), total peripheral resistance (TPR), and dP/dt (-28 +/- 2, -34.8 +/- 3.7, and -9.4 +/- 1.3%, p less than 0.01, respectively), and increased heart rate (HR), cardiac output (CO), and the velocity of myocardial anterior wall shortening (dL/dt) (11.8 +/- 2.1, 10.4 +/- 2.9, and 28.3 +/- 8.3% p less than 0.05, respectively). These effects were followed by a sustained increase in MBP and TPR and a decrease in CO. As compared with AI (0.25 nmol/kg) and PE (35 nmol/kg), which produced a similar degree of increase in TPR, the reduction in CO induced by ET-1 was more prominent (-25 +/- 2 by ET-1 vs. -14 +/- 2 by AI and -14 +/- 3% by PE, p less than 0.05, respectively). Moreover, ET-1 induced a significant decrease in dP/dt, shortening fraction (SF), and dL/dt ( 6.1 +/- 1.6, -35.0 +/- 3.8, and -38.6 +/- 5.5%, p less than 0.01, respectively), whereas these indexes of left ventricle performance were not affected by either AI or PE. Furthermore, the reduction in SF induced by ET-1 was mainly due to the decrease in myocardial diastolic segment length, suggesting reduction in diastolic filling volume.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711637 TI - Enhanced clearance of specifically bound digoxin from human myocardial and skeletal muscle samples by specific digoxin antibody fragments: subsequent complete digitalis glycoside receptor (Na,K-ATPase) quantification. AB - The effect of digoxin antibody fragments (Fab) on clearance of specifically bound digoxin from its specific receptor (Na, K-ATPase) was studied in human heart left ventricle (LV) and vastus lateralis skeletal muscle (SK) samples obtained postmortem. Initially, [3H]digoxin was bound to samples at conditions giving high relative occupancy of receptor. Half-life (t1/2) for its net release from LV in buffer was 32.2, 6.7, and 0.9 h at 0 degrees, 30 degrees, and 37 degrees C, respectively. For SK, t1/2 was 5.4 h in buffer at 30 degrees C. Inhibition of rebinding of digoxin by addition of specific digoxin Fab (5 x 10(-7) M) or excess unlabeled digoxin (1 x 10(-4) M) to buffer at 30 degrees C increased net release rate for specifically bound digoxin 2.5- to 3.0-fold in heart and SK. [3H]Digoxin was also bound to samples at conditions giving low relative occupancy. Samples were subsequently washed in buffer containing 5 x 10(-7) M specific digoxin Fab for 16 h at 30 degrees C. This wash reduced occupancy of receptors by digoxin from 10 to 0.5% in LV and from 9 to 0.3% in SK, respectively. At variance with wash at 37 degrees C, this procedure allowed subsequent vanadate-facilitated complete quantification of Na,K-ATPase by [3H]ouabain binding; values were 378 +/ 13 and 370 +/- 12 pmol/g wet weight (p greater than 0.6) in LV and 309 +/- 19 and 315 +/- 16 pmol/g wet weight (p greater than 0.7) in SK with and without previous wash, respectively (mean +/- SEM, n = 12).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711638 TI - Cardioprotective effects of recombinant human extracellular-superoxide dismutase type C in rat isolated heart subjected to ischemia and reperfusion. AB - The cardioprotective effect of recombinant human extracellular-superoxide dismutase type C (rh-EC-SOD C) was studied in isolated perfused rat heart subjected to left coronary artery ligation for 30 or 60 min followed by 30-min reperfusion. A comparison was made with the effects of bovine CuZn-SOD. Reperfusion after 30-min coronary artery ligation was associated with a release of creatine kinase (CK) into the coronary effluent (71 +/- 5.2 IU/30 min), which was markedly reduced (39 +/- 5.5 IU/30 min) in hearts perfused with rh-EC-SOD C (28 mg/L). CuZn-SOD (4 or 20 mg/l) or a lower concentration of rh-EC-SOD C (5.6 mg/l) did not significantly attenuate CK outflow during reperfusion, however. In both vehicle- and SOD-treated hearts, the left ventricular developed pressure (LVDP) and the coronary flow recovered to 80-90% of baseline at the end of the reperfusion period. Increasing the ischemic period from 30 to 60 min caused a much more pronounced cardiac injury measured after 30-min reperfusion. In the hearts that received vehicle, recovery of LVDP (in percentage of baseline values) at the end of reperfusion was 58 +/- 2%, which was increased to 84 +/- 3 and 83 +/- 5% after treatment with rh-EC-SOD C (28 mg/L) and CuZn-SOD (20 mg/L), respectively. The corresponding values for recovery in coronary flow were 54 +/- 3% (vehicle), 69 +/- 4% (rh-EC-SOD C), and 74 +/- 3% (CuZn-SOD).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711639 TI - De novo acute B-cell leukemia with translocation t(14;18): an entity with a poor prognosis. AB - Three cases of de novo acute B-cell lymphoblastic leukemia are presented, all with an unusual phenotype, involvement of translocation t(14;18) and additional chromosomal abnormalities, including translocation t(8;14) and deletion of chromosome 9. In contrast to normal FAB-L2 or FAB-L3 acute lymphoblastic leukemia (ALL), these leukemias did not express cytoplasmatic and membranous immunoglobulin. The combination of translocation t(14;18) and additional chromosomal events on the other chromosome 14 account for the lack of immunoglobulin expression. In one case a low grade follicular lymphoma was found next to a high grade Burkitt type ALL. The translocation t(14;18) takes place as a mistake in the VDJH joining in pre-B cells in the bone marrow. It is proposed that some cases of de novo ALL may arise as a blast crisis induced by genetic events, secondary to the primary t(14;18) translocation. This type of ALL seems to have a poor prognosis. PMID- 1711640 TI - Mast cell growth factor, a ligand for the receptor encoded by c-kit, affects the growth in culture of the blast cells of acute myeloblastic leukemia. AB - The c-kit proto-oncogene encodes a transmembrane receptor with a tyrosine kinase internal domain. C-kit has been mapped to the W locus in the mouse, and the gene encoding the ligand has been shown to be the product of the murine SI locus. Previous genetic studies have shown that the murine W and SI loci play important roles in the normal function of hemopoietic stem cells. As these stem cells have been identified as the origins of abnormal clones in acute myeloblastic leukemia (AML), a study was begun of c-kit in AML. By Northern blot analysis, it was shown that all of 21 blast populations from AML patients were kit expression positive, but some AML cell lines did not transcribe detectable c-kit mRNA. This study is now extended to the responses of freshly obtained AML cells and cell lines to the ligand, mast-cell growth factor (MGF). In culture, fresh cells usually responded to added ligand with increases in both self-renewal and terminal divisions. The most obvious effects were seen when MGF was combined with either IL-3 or G-CSF. The response of cell lines to MGF mirrored their expression of c-kit; expression positive lines responded in culture with patterns similar to those seen for fresh cells. C-kit expression negative cells did not respond to MGF. RNA prepared from the cells giving rise to one such line, OCI/AML-5, was available for study. mRNA for c-kit could not be detected in this RNA sample by Northern blot analysis or the polymerase chain reaction. Thus the heterogeneity found in AML blast populations extends to the involvement of c-kit and its ligand in growth regulation, although blast populations without this regulatory apparatus appear to be rare. PMID- 1711641 TI - Cytosine arabinoside (ara-C) and cis-dichlorodiammineplatinum II (cisplatin) alone and in combination: effects on acute myeloblastic leukemia blast cells in culture and in vivo. AB - Cytosine arabinoside (ara-C) and cis-dichlorodiammineplatinum II (cisplatin) are lethal to mammalian cells by very different mechanisms; however, they share interactions with the biology of blast cells in acute myelogeneous leukemia (AML). Both agents are more toxic to AML blasts in suspension than when a clonogenic assay in methyl cellulose is used; both agents are more toxic in suspension in the presence of rG-CSF than with rGM-CSF. Accordingly, preclinical tests were undertaken of cisplatin and ara-C in combination. At the same time, a phase I/II clinical trial of the combination was conducted, using AML patients refractory to treatment or in relapse. In the laboratory, blasts from eight AML patients were tested against each agent singly and in combination. The observed survival values for the mixture were compared with those predicted by assuming either an additive effect or a more general effect that allows synergism or antagonism. Blasts from two patients were tested with this design in the presence of rG-CSF or rGM-CSF. In most instances the toxic effects of ara-C and cisplatin were additive. Evidence of synergism was seen in blasts from three patients. PMID- 1711642 TI - Histochemical study of RNA content of neurones in the dorsal lateral geniculate nucleus during postnatal development. AB - Nuclear and cytoplasmic dLGN neurons were investigated by cytophotometric measurements of RNA. This study has been carried out in rats from birth to adulthood. In order to quantify the RNA content a cytophotometer was used. Extinction mean values were obtained which indicated RNA concentrations per surface unit. The nuclear and cytoplasmic surface were calculated simultaneously and from the product of the mean extinction and the surface the RNA total content was calculated. Our results have suggested that the changes are age-related. From day 1 to day 21 the neuronal size and RNA content increase; this may somehow be involved with the differentiation process. Around post-natal day 21 neuronal maturation may begin, reaching its optimal phase around day 42, on which the RNA concentration per surface unit, surface neuronal content and RNA total content are stable. PMID- 1711643 TI - [Prostate-specific antigen (PSA) is a valuable marker--if correctly used]. PMID- 1711644 TI - Impairments, disabilities, and handicaps of very preterm and very-low-birthweight infants at five years of age. The Collaborative Project on Preterm and Small for Gestational Age Infants (POPS) in The Netherlands. AB - The Project On Preterm and Small for gestational age infants (POPS) was started in the Netherlands in 1983 to investigate the relation between prenatal/perinatal factors and mortality/morbidity in very preterm and very-low-birthweight infants. Of the 1338 liveborn infants (less than 32 weeks and/or less than 1500 g) 966 were enrolled in the five-year (chronological age) follow-up programme; 96% of these children were assessed during a home visit. The overall outcome was expressed as impairments, disabilities, and handicaps according to World Health Organisation criteria. Of the assessed children, 13% had a disability and 14% were handicapped, which are much higher frequencies than those found in the general population. Handicaps were due mainly to abnormalities of neuromotor function, mental development, or language and speech development. Compared with the handicap frequency in the same cohort at two years of age, a more favourable outcome at five years of age was seen in 10%, and a less favourable outcome in 7% of the children. The findings show that most of those high-risk children survived without handicap or serious disability at preschool age. PMID- 1711645 TI - Evidence for a colocalization of oxytocin mRNA and galanin in magnocellular hypothalamic neurons: a study combining in situ hybridization and immunohistochemistry. AB - The possible colocalization of oxytocin (OT) and galanin (GAL) was studied by combining, on the same cryostat sections, in situ hybridization (ISH) for OT mRNA with a tritiated oligonucleotide probe and immunohistochemistry (ICC) of GAL. Many cells were either labelled by ISH (OT mRNA containing cells), or by ICC (GAL containing cells). Moreover, some magnocellular neurons in the supraoptic and paraventricular nuclei were labelled for both OT mRNA and GAL. These results demonstrate that some magnocellular neurons of the rat hypothalamus contain both GAL and OT. This approach is suitable for studying the intracellular distribution of OT gene expression and mature GAL under different physiological or experimental conditions. PMID- 1711646 TI - Quantification of keratinolytic activity from Dermatophilus congolensis. AB - The bacterium Dermatophilus congolensis is the causative agent of pitted keratolysis, a skin disease. Infection occurs mainly in keratinized tissues and it is necessary for the organism to produce and excrete exoenzymes which are able to degrade keratin. We investigated the amount of keratinase liberated using Keratinazure as substrate and the fungal protease XI as standard. When compared with uninoculated samples, D. congolensis liberated significant amounts of keratinase during a 12-day incubation period with this substrate. An equivalent of 15 units of protease (keratinase) was produced by 10(7) colony-forming units of D. congolensis during a 12-day period at 37 degrees C. We consider the extracellular proteolytic activity of this bacterium to be responsible for keratinized tissues being the main sites of infection. PMID- 1711648 TI - Ultrastructural localization of factor VIII-related antigen in endothelial proliferations of malignant gliomas. AB - The ultrastructural localization of Factor VIII-related antigen (FVIII/RAg) in the endothelial cells of malignant gliomas was studied with immunogold labelling on thin sections of LR White-embedded tissue. FVIII/RAg was localized in Weibel Palade bodies (WPBs) as well as in cytoplasmic vacuoles and vesicles, and in extracellular spaces. Stained WPBs were found both in cells surrounding vascular channels and in several proliferating cells of solid buds and glomeruli. Our results confirm previous ultrastructural observations on non-neural tissues that FVIII/RAg is produced by endothelial cells and stored in WPBs. The presence of FVIII/RAg-stained WPBs in capillary buds and tufts indicates that at least some of the proliferating cells are endothelial in origin rather than pericytes. Thus, the contribution of endothelial cells to new vessel formation seems to be more substantial than previously reported. PMID- 1711647 TI - Germ cell tumors in children: gonadal and extragonadal. AB - Sixty-three pediatric patients with germ cell tumors are presented with details of symptoms, histological findings, staging, serological markers, treatment, and response to therapy. The primary sites were: ovarian 32, testicular 17, presacral 7, mediastinal 3, intraabdominal 2, vaginal 1, and right inguinal canal 1. These patients were treated with T2 (sequential use of dactinomycin, doxorubicin, vincristine, and cyclophosphamide, with or without radiation), T6 (combination chemotherapy with cyclophosphamide, bleomycin, dactinomycin, doxorubicin, methotrexate, vincristine), or VAB treatment protocols (velban, dactinomycin, bleomycin, cisplatin). The cure rate for stage I ovarian and testicular germ cell tumors was 100%; for stage III, all primary sites, 82% and for stage IV, all primary sites, 75%. Histology was prognostic in ovarian tumors of the immature malignant teratoma type; the neural type immature teratoma, grades II and III, had the worst prognosis. Initial debulking surgery in combination with chemotherapy and radiation plays an important role in germ cell tumors. Stages II, III, and IV germ cell tumors require aggressive treatment with surgery, radiation, and chemotherapy. For stage I patients, with primary ovarian malignant tumor, cure with surgery alone can be achieved in 50% of the cases and in testicular tumors in about 70% of the patients. For those with stage I and elevated serological markers, it is feasible to follow these markers and give no treatment until there is evidence of persistent elevation or a rise in titers after an initial fall. In those without elevated serological markers, one should take into consideration the size of the tumor and the histological type before taking the "wait and see" approach. These stage I tumors are highly curable when they first present but, if allowed to recur, chemotherapy may not offer the patient such a favorable response and cure rate. PMID- 1711649 TI - A GABAergic projection from the pretectum to the dorsal lateral geniculate nucleus in the cat. AB - To study the projection from the pretectum to the lateral geniculate nucleus, we placed wheat-germ agglutinin conjugated to horseradish peroxidase into the lateral geniculate nuclei of six cats, allowed this marker to be retrogradely transported by afferent axons to their parent somata in the pretectum, and revealed the label in these cells with stabilized tetramethylbenzidine histochemistry. In three cases we made large pressure injections that completely infiltrated the lateral geniculate nucleus and extended into neighboring thalamic nuclei; in the other three we made smaller iontophoretic injections largely confined to the A- and C-laminae of the lateral geniculate nucleus. In both types of injection we found labeled pretectal cells mainly in the nucleus of the optic tract but also found some cells labeled in the olivary pretectal nucleus and the posterior pretectal nucleus. After one of the larger injections we analysed both sides of the pretectum and found that 11% of the labeled cells were located contralaterally and were distributed in the same three nuclei. We analysed only the ipsilateral side in the remaining five cats. In those five experiments we also immunohistochemically stained the pretectal sections with an antibody directed against the neurotransmitter, GABA. Of the retrogradely labeled pretectal cells, 40% were also labeled for GABA, and those were similar in soma size (350 microns 2 in cross-sectional area) to those labeled only with the retrograde marker (331 microns 2). GABA-positive cells not labeled by retrograde transport were smaller (246 microns 2) than either of these other cells populations. These results indicate that at least 40% of the cells involved in the projection from the pretectum to the lateral geniculate nucleus are GABAergic. We suggest that this extrathalamic projection may serve to inhibit thalamic GABAergic cells. This, in turn, would disinhibit geniculate relay cells, thereby facilitating the geniculate relay of retinal information to cortex. PMID- 1711650 TI - Topographical organization of the tecto-olivo-cerebellar projection in the cat. AB - The superior colliculus sends a climbing fiber output to cerebellar vermal lobules VI-VII through the inferior olive. The present study in cats morphologically clarified the existence of a topographical organization in the tecto-olivo-cerebellar projection. A horseradish peroxidase study on the tecto olivary projection showed that the rostral and caudal superior colliculus projected mostly contralaterally to the caudal and rostral areas of the caudomedial part of the medial accessory olive, respectively. The lateral superior colliculus was found to project more laterally than was the medial superior colliculus. Investigation on the olivocerebellar projection demonstrated that the rostral and caudal areas of the caudomedial part of the medial accessory olive sent climbing fiber terminals contralaterally to the lateral and medial parts of vermal lobules VI-VII, respectively. Thus, it was revealed that the rostral superior colliculus projected mostly to the medial part of ipsilateral vermal lobules VI-VII while the caudal superior colliculus projected mostly to the lateral part of ipsilateral vermal lobules VI-VII. PMID- 1711651 TI - Extracellular alkaline-acid pH shifts evoked by iontophoresis of glutamate and aspartate in turtle cerebellum. AB - The effect of glutamate and aspartate iontophoresis on extracellular pH was investigated in the turtle cerebellum in vitro. Both amino acids produced a rapid alkaline transient, typically followed by a prolonged acidification. These responses could be evoked in all layers of the cerebellum. Transition from bicarbonate to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered media amplified the pH shifts. Similar alkaline-acid transients could be evoked in the molecular layer by electrical stimulation of the parallel fibers or the ipsilateral peduncle, or by superfusion of glutamate or aspartate. However, no alkaline shifts were evoked in the granular layer by either parallel fiber or peduncle stimulation. In contrast, the iontophoretically induced alkaline shifts were largest in the granular layer. Compared with the stimulus-evoked alkalinizations, the iontophoretic alkaline shifts were relatively insensitive to Mn2+ or Cd2+. These data suggest that the activity-dependent alkalinization of brain extracellular space is generated by a bicarbonate-independent mechanism related to excitatory synaptic transmission. The results are consistent with a flux of hydrogen ions through cationic channels, but do not support a direct role for voltage-dependent Ca2+ channels. In view of the sensitivity of ion channels to changes in external pH, and the magnitude of the amino acid-induced pH shifts, these results indicate that extracellular pH could play an important modulatory role in excitatory synaptic transmission. PMID- 1711652 TI - Local depletion of monoamines induced with in vivo voltammetry in the cat brain. AB - A significant depletion of the electroactive monoamines and their metabolites in the vicinity of a carbon fiber microelectrode may be induced by in vivo staircase voltammetry in the brain, even if the duration of the voltammetric scans is relatively short (approximately 5 s). The variation of this depletion was determined in the extracellular fluid of the cat thalamus at different durations of the pauses separating consecutive measurements. Pauses not shorter than 5 min ensured a nearly full relaxation, so that at the beginning of each subsequent scan a virtually undisturbed environment surrounded the electrode. With pauses shorter than 5 min, it is still possible to monitor major changes in the monoamine concentration. Staircase scans separated with 45 s pauses, for example, were suitable to study the increase in monoamine levels after administration of reserpine, and release phenomena stimulated with KCl were monitored with frequently repeated voltammetric pulses. The electrochemically induced depletion, on the other hand, can be used for characterizing the dynamics of mass transport in the studied brain structure. This was demonstrated with staircase voltammetry alternated with pauses of 1-100 s, and with quasi-chronoamperometry. In vivo brain voltammetry is generally used for monitoring extracellular monoamine (including dopamine) levels. These may be significantly altered by the voltammetric measurement itself through depletion in the vicinity of the electrode. This effect can be minimized with appropriate selection of sampling intervals and other parameters of staircase voltammetry. Conversely, depletion and the following relaxation can be used for determining dynamic characteristics of the studied brain structure which would be difficult to obtain otherwise. PMID- 1711653 TI - Low pH-induced release of calcitonin gene-related peptide from capsaicin sensitive sensory nerves: mechanism of action and biological response. AB - Protons can release in a Ca(2+)-dependent manner, calcitonin gene-related peptide (CGRP)-like immunoreactivity from peripheral endings of capsaicin-sensitive afferents. Here we have studied the mechanism by which proton promotes CGRP-like immunoreactivity release and whether the neuropeptide released might exert a biological action. In muscle slices of guinea-pig urinary bladder high pH (pH 8 or 9) media neither enhanced CGRP-like immunoreactivity outflow nor affected the capsaicin-evoked CGRP-like immunoreactivity release. The CGRP-like immunoreactivity release evoked by superfusion with pH 5 medium was not affected by tetrodotoxin (0.3 microM) indomethacin (10 microM) or the protein kinase C inhibitor H-7 (30 microM). However, it was reduced by 35% in the presence of the voltage-sensitive Ca(2+)-channel antagonists nifedipine (1 microM) and omega conotoxin (0.1 microM) and by 80% in presence of the capsaicin "antagonist" Ruthenium Red (10 microM). The CGRP-like immunoreactivity release by capsaicin (10 microM) was reduced by 80% in the presence of Ruthenium Red, and not affected by voltage-sensitive Ca(2+)-channel blockers, while that evoked by 80 mM K+ was decreased by 82% in the presence of nifedipine and omega-conotoxin. The Ca(2+) channel agonist Bay K 8644 enhanced the high K(+)-evoked CGRP-like immunoreactivity release but not that induced by capsaicin or pH 5 medium. Exposure to pH 6 solution of one half of the neck of guinea-pig urinary bladder induced a slowly developing inhibition of electrically evoked contractions, that was absent in the half pre-exposed in vitro to a desensitizing dose of capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711654 TI - Substance P-like immunoreactive neurons are depleted in Alzheimer's disease cerebral cortex. AB - We studied the morphology and distribution of substance P-like immunoreactive elements in normal and Alzheimer's disease brain with a monoclonal anti-substance P antibody. Bands of prominent terminal-like staining were found in the dentate gyrus of normal brain. Multipolar substance P-immunoreactive neurons were seen in dentate polymorphic layer and CA4 and prominent fiber staining was present in the CA fields of the hippocampus and adjacent allocortex. Reactive perikarya, concentrated in deep cortex and infracortical white matter, were found in all isocortical regions. Greatest density was in frontal and parietal association cortex; lowest in visual cortex. Fiber density was generally greatest in layers I and II. In Alzheimer's disease, staining intensity was reduced in the dentate gyrus. Hilar neurons were unaffected but other CA field neurons were distorted with pruned dendritic trees. Isocortical perikarya and fibers were significantly depleted and distorted in all regions. Globular deposits consisting of distorted neurites or dissolving perikarya were frequently seen. Double staining methods showed that the vast majority of isocortical, but not hippocampal, substance P like immunoreactive neurons are nicotinamide adenine dinucleotide phosphate diaphorase-positive. Despite the modest quantitative depletion of substance P in Alzheimer's disease cortex as measured by radioimmunoassay compared to somatostatin, there is a significant depletion of substance P-like immunoreactive perikarya. This disparity may be due to persistence of afferent projections which make a major contribution to substance P concentrations in cerebral cortex or to the high substance P content of dystrophic fibers in Alzheimer's disease cortex. PMID- 1711655 TI - Evaluation of the Quantitative Buffy Coat (QBC) method to detect malaria-infected red blood cells. AB - The conventional thin blood film method of detecting malaria is a long and tedious procedure, requiring significant technical expertise. In this study, we compared the Quantitative Buffy Coat (QBC) capillary tube method, which requires only minimal technical training, to the thin blood film method, and found it to be not only more rapid but also more sensitive than the thin blood film method in the detection of parasitized red blood cells. We do not suggest that the QBC capillary method can replace the conventional thin blood film method for the detection of malaria since it does not identify or quantitate the parasite species, only that it would serve as a valuable screening test. PMID- 1711656 TI - The binding of basic fibroblast growth factor to Alzheimer's neurofibrillary tangles and senile plaques. AB - Brain sections from Alzheimer's disease (AD) patients and controls were treated with basic fibroblast growth factor (bFGF) and then immunostained with anti-bFGF. Additional sections were treated with biotinylated bFGF without using the anti bFGF. Labelling was visualized by the ABC method. Both protocols above intensely labelled neurofibrillary tangles, senile plaques and amyloidotic vessels in AD brains. Omission of the bFGF treatment abolished the staining of the AD lesions. The pretreatment of sections with heparitinase also reduced their staining. These results indicate that AD lesions contain bFGF-binding sites and that the chemical substrate for bFGF binding to AD lesions was heparan sulfate. PMID- 1711657 TI - Mouse trisomy 16 neurons, a model of human trisomy 21 (Down syndrome), can be maintained by intracerebral transplantation. AB - The trisomy 16 mouse is considered to be a model of human trisomy 21 (Down syndrome) due to genetic homology between parts of human chromosome 21 and mouse chromosome 16. Additionally, and because older Down syndrome individuals develop neuropathology indistinguishable from that of Alzheimer's disease, trisomy 16 tissue may provide a model of some pathological processes occurring in Alzheimer's disease. However, trisomy 16 fetuses die in utero or shortly after birth, preventing exploitation of this model. We therefore sought to examine trisomy 16 brain tissue over an extended period of time. We report that neural transplantation to normal hosts allows the maintenance of cortical and hippocampal neurons for at least 8 months, thus providing a model in which to examine pathological processes related to Down syndrome, and perhaps to Alzheimer's disease. PMID- 1711658 TI - Object recognition deficit in Alzheimer's disease: possible disconnection of the occipito-temporal component of the visual system. AB - An early object recognition impairment was observed in a patient presenting with a slowly progressive Alzheimer's disease. At autopsy, the cerebral cortex showed increased neuritic plaque and neurofibrillary tangle counts, particularly in the occipital and temporal lobes as compared to Alzheimer's disease cases with the usual clinical presentation and distribution of the neuropathological lesions (i.e., no early visual deficits and fewer pathological profiles in the occipital cortical areas than frontal or posterior parietal areas). This observation further supports the hypothesis that long corticocortical projections are selectively damaged in Alzheimer's disease and that the resulting disconnections may underlie specific neurological symptoms. PMID- 1711659 TI - Ethanol potentiation of 5-HT3 receptor-mediated ion current in NCB-20 neuroblastoma cells. AB - The effect of acute ethanol (EtOH) exposure on 5-HT3 receptor-mediated ion current was examined in whole-cell patch-clamp recordings from NCB-20 neuroblastoma cells. The physiologic and pharmacologic properties of 5-HT activated ion current in NCB-20 cells indicated that it was mediated by 5-HT3 receptors. EtOH potentiated 5-HT3 receptor-mediated current in a concentration dependent manner at concentrations (25-100 mM) which are achieved during EtOH intoxication in vivo. PMID- 1711660 TI - Neurocatin induces changes in release and level of serotonin in synaptosomal fraction from rat brain. AB - A newly isolated factor from mammalian brain, neurocatin, is shown to increase both the level and release of serotonin in suspensions of synaptosomes isolated from rat brain. Incubation of synaptosomes for 10 min with approximately 20 nM neurocatin resulted in release of about 50% of the total pool of serotonin. Quantification of serotonin and its major metabolite, using an electrochemical detector, indicated that the presence of neurocatin also caused an increase in the absolute level of serotonin and decrease in its catabolism to 5 hydroxyindoleacetic acid (5-HIAA). The effect is dependent on the time of incubation and concentration of neurocatin. At a concentration of 20 nM, neurocatin increased about 60% the level of serotonin and decreased about 50% the level of 5-HIAA. Depolarization conditions--50 microM veratridine and 50 mM K+ medium--increased release of serotonin by 50% and 30% respectively without affecting the level of serotonin or its catabolism to 5-HIAA. PMID- 1711661 TI - Galanin increases potassium evoked release of [3H]5-hydroxytryptamine from rat hypothalamic synaptosomal preparations. AB - The effects of galanin (GAL) have been evaluated on the depolarization evoked release of [3H]5-HT (serotonin or 5-hydroxytryptamine) from rat hypothalamic synaptosomal preparations, using low concentrations of potassium (15 mM). In the same preparation effects of GAL were also evaluated on [3H]5-HT uptake, using kinetic analysis to determine effects on Vmax values and Km values. GAL concentrations of 0.1-10 nM caused a concentration-related increase of the depolarization-evoked release of [3H]5-HT without influencing [3H]5-HT uptake. The results indicate the existence of high affinity GAL receptors on the hypothalamic 5-HT nerve terminals, exerting a facilitatory influence on depolarization-evoked [3H]5-HT release. PMID- 1711662 TI - Fine structure of substance P-containing nerve fibers in the rat lateral septum; simultaneous localization in pre- and postsynaptic elements. AB - The fine structure of nerve fibers with substance P (SP)-like immunoreactivity in the rat lateral septum (LS) was investigated by preembedding immunoelectron microscopy. SP axon terminals frequently made synapses with non-immunoreactive neuronal soma and dendrites in the LS. Occasionally, two closely apposed nerve endings with SP immunoreactivity were presynaptic to the soma. A small number of immunopositive axon terminals formed synapses not only with neuronal perikarya but also with small dendrites in the vicinity of the perikarya. There were also some SP dendrites in contact with immunoreactive as well as non-immunoreactive nerve endings. These findings may provide a morphological basis for the complexity of SP actions on septal neurons. PMID- 1711663 TI - Treatment of pediculosis capitis. PMID- 1711664 TI - [Operative therapy in tumor involvement of the spine]. AB - We report on 56 patients, who underwent operative treatment of tumorous lesions of the spine. We indicated dorsal instrumentation in 14 cases, ventral tumorectomy in 11 cases and a combined dorsal and ventral instrumentation in 31 cases. The median survival time was 14.8 months. Reduction of pain and the improvement of the preexisting neurological symptoms were the most important postoperative factors. According to our follow-up study, the combined dorsal and ventral instrumentation seems to be the best method for an operative treatment of tumorous spine lesions. PMID- 1711665 TI - Methylene blue stain guiding layered excision in Mohs' micrographic surgery. AB - Resection of a continuous layer of tissue by means of Mohs' micrographic surgery is problematic at several periorbital sites. We describe a technique in which methylene blue is used to identify instances of incomplete specimen removal and thus facilitate subsequent complete removal. PMID- 1711666 TI - [Endoscopic intubation and intracavitary irradiation in the palliative treatment of inoperable esophageal tumors]. AB - The authors have applied at first in Hungary the intracavital after loading radiotherapy in patients suffering from esophageal tumor. A new method was elaborated for the combined use of endoscopic tube insertion and intracavital irradiation. Out of the 155 patients admitted on the 1st Surgical Department of Semmelweis University, Budapest, 63 had been treated only by tube insertion, 44 were intubated and irradiated, whereas 48 patients had intracavital radiotherapy without intubation. Radiotherapy was performed at the Radiological Unit of Semmelweis University. The irradiation dose ranged from 26,9 to 32,2 Gray in average. The mean survival of group I. was 4,0 months, that of group II. 6,3 months, and in group III. 7,0 months respectively. The expected survival is proportional to the radiation dose. No correlation exists between tumor localisation and survival. Respiratory fistula, the most common complication, occurred in different groups as follows: 3,4%, 6,2%, and 9,0%; but could have been treated by tube insertion, or adequate positioning of the tube. The combined use of intubation and intracavital after loading radiotherapy has been proven to be suitable for palliative treatment of inoperable esophageal tumors. PMID- 1711667 TI - Neurobiology of parasitic platyhelminths: possible solutions to the problems of correlating structure with function. AB - This paper provides an overview of research on the nervous system of parasitic platyhelminths. We have emphasized studies concerned with the physiological, pharmacological and biochemical nature of the major small molecule neurotransmitters of these parasites. We have attempted to provide a critical review of the work by focusing on important unresolved issues. Finally, we have focused on some recent work in our laboratory, using patch-clamp recording techniques and quantitative fluorescence cytometry, as an example of newer methods that will hopefully resolve some of the unanswered questions concerning the nervous system of these parasites. PMID- 1711668 TI - Evolutionary aspects of transmitter molecules, their receptors and channels. AB - Classical transmitters are present in all phyla that have been studied; however, our detailed understanding of the process of neurotransmission in these phyla is patchy and has centred on those neurotransmitter receptor mechanisms which are amenable to study with the tools available at the time, for example, high affinity ligands, tissues with high density of receptor protein, suitable electrophysiological recording systems. Studies also clearly show that many neurones exhibit co-localization of classical transmitters and neuropeptides. However, the physiological implications of this co-localization have yet to be elucidated in the vast majority of examples. The application of molecular biological techniques to the study of neurotransmitter receptors (to date mainly in vertebrates) is contributing to our understanding of the evolution of these proteins. Striking similarities in the structure of ligand-gated receptors have been revealed. Thus, although ligand-gated receptors differ markedly in terms of the endogenous ligands they recognize and the ion channels that they gate, the structural similarities suggest a strong evolutionary relationship. Pharmacological differences also exist between receptors that recognize the same neurotransmitter but in different phyla, and this may also be exploited to further the understanding of structure-function relationships for receptors. Thus, for instance, some invertebrate GABA receptors are similar to mammalian GABAA receptors but lack a modulatory site operated by benzodiazepines. Knowledge of the structure and subunit composition of these receptors and comparison with those that have already been elucidated for the mammalian nervous system might indicate the functional importance of certain amino acid residues or receptor subunits. These differences could also be exploited in the development of new agents to control agrochemical pests and parasites of medical importance. The study of the pharmacology of receptor proteins for neurotransmitters in invertebrates, together with the application of biochemical and molecular biological techniques to elucidate the structure of these molecules, is now gathering momentum. For certain receptors, e.g. the nicotinic receptor, we can expect to have fundamental information on the function of this receptor at the molecular level in both invertebrates and vertebrates in the near future. PMID- 1711669 TI - Emerging developmental sequelae in the 'normal' extremely low birth weight infant. AB - Thirty-six extremely low birth weight (less than 1000 g birth weight) children received neurodevelopmental testing in infancy (mean age = 19.1 months), and again in early childhood (mean age = 46.5 months). Children were categorized into a high-risk group (n = 20) if bronchopulmonary dysplasia and/or Grades III or IV intracranial hemorrhage were present or a low-risk group (n = 16) if neither were present. Using standardized testing and neuromotor examination, 24 (67%) of 36 children showed normal infant development. Only 11 (31%) of 36 children (P less than .005) had normal development upon reassessment in early childhood. A decline in developmental status occurred in both groups. This indicates that for the extremely low birth weight population, normal infant development is poorly predictive of continued normal development. With or without major complications, extremely low birth weight places children at substantial risk for ongoing and emerging developmental problems with age. PMID- 1711670 TI - Chloride channel regulation in the skeletal muscle of normal and myotonic goats. AB - External intercostal muscle biopsies from normal and congenitally myotonic goats were studied in vitro at 30 degrees C using a two-microelectrode square-pulse cable analysis assisted by computer. The resting chloride conductance (Gcl) was estimated from the difference between the mean membrane conductance in chloride containing and chloride-free bathing media. The protein kinase C (PKC) activator, 4-beta-phorbol-12,13-dibutyrate. (0.1-2.0 microM) blocks a maximum of 76% of Gcl in normal goat fibers and induces myotonic hyperexcitability similar to that of congenitally myotonic goat fibers. The Gcl block was partially antagonized by pretreatment with the PKC inhibitor, staurosporine (10 microM). The "inactive" 4 alpha-phorbol-12,13-didecanoate had no effect at 50 microM, whereas the "active" 4-beta isomer blocked 41% Gcl at 1 microM. The nearly absent Gcl of congenitally myotonic goat fibers was not restored by treatment with high concentrations of the PKC inhibitors staurosporine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), or tetrahydropapaveralone (THP). Also, forskolin and cholera toxin, which may increase cyclic adenosine monophosphate (cAMP) levels, or the R(+) clofibric acid enantiomers and taurine, which increase Gcl in normal fibers, were also unable to restore Gcl in myotonic goat fibers. The data suggest that PKC may be a chloride channel regulator in normal goat skeletal muscle fibers, however the molecular defect of congenitally myotonic fiber does not appear to be due to excessive activity of PKC. PMID- 1711671 TI - Bicarbonate permeability of epithelial chloride channels. AB - Bicarbonate permeability of epithelial chloride channels has been studied using the patch-clamp technique. The experiments were performed in excised inside-out oriented membrane patches from three different cultured cell types: (a) HT29 colon carcinoma cell line, (b) T84 colon carcinoma cell line, and (c) respiratory epithelial cells (REC) in primary culture. In all three preparations we observed outwardly rectifying chloride channels with similar conductances with 145 mmol/l NaCl solution in the pipette and in the bath (Cl- pipette/Cl- bath). When Cl- was replaced by HCO3- in the bath (Cl-/HCO3-) the conductance of the channel at negative clamp voltages was reduced significantly by 40% for HT29 (n = 6), 39% for T84 (n = 7), and 38% for REC (n = 6). Similarly, the zero-current potential (V1 = 0) was shifted from 0 mV (Cl-/Cl-) to negative values (Cl-/HCO3-) revealing permeability ratios PCl/PHCO3 of 2.4 +/- 0.1 for HT29 (n = 6), 2.0 +/- 0.1 for T84 (n = 7), and 1.8 +/- 0.1 for REC (n = 7). With NaHCO3 as the pipette solution and NaCl in the bath, the V1 = 0 was positive and a PCl/PHCO3 value of 2.3 +/- 0.1 was determined for HT29 (n = 5). Replacement of Cl- in the bath by HCO3- reduced V1 = 0 to values close to 0 mV. In another series of experiments, the pipette was filled with 145 mmol/l NaCl and the bath contained 35 mmol/l NaCl to which 35 mmol/l NaHCO3 were added. We found that neither the conductance for the inward current nor V1 = 0 was changed significantly with the addition of NaHCO3 (HT29, n = 6). We conclude that the HCO3- permeability and HCO3- conductance of these channels is about half of that for Cl-. PMID- 1711672 TI - The housekeeping promoter from the mouse CpG island HTF9 contains multiple protein-binding elements that are functionally redundant. AB - The mouse CpG-rich island HTF9 harbours the divergent RNA initiation sites shared by two genes that are both expressed in a housekeeping fashion. In this work we have analyzed the architecture of the HTF9 promoter. Gel shift assays were first employed to locate nuclear factor-binding sites within HTF9. Multiple protein binding sites were identified across a 500 bp-long region, two of which appear to interact with novel factors. Deletion analysis was used to determine the requirements for the different sites in transient expression of a CAT reporter gene. Although multiple elements contributed to the overall promoter strength in each orientation, extensive deletions failed to affect the basal level of transcription from HTF9 in either direction. Thus, only a subset of elements is necessary to activate transcription from HTF9. Functional redundancy may be a general feature of housekeeping CpG-rich promoters. PMID- 1711673 TI - Novel upstream elements and the TATA-box region mediate preferential transcription from the uteroglobin promoter in endometrial cells. AB - To understand the mechanisms responsible for endometrium-specific expression of the uteroglobin gene, we have compared transcription from the uteroglobin promoter in a human endometrial cell line (Ishikawa) and in HeLa cells. In transient transfection experiments and in nuclear extracts, sequences from -395 to +14 of the uteroglobin gene are able to promote transcription of a reporter gene more efficiently in Ishikawa cells than in HeLa cells relative to the RSV or the SV40 early promoter. Analysis of progressive 5'-deletion mutants identifies three promoter regions, -258/-220, -205/-177, and -96/-35, that are important for preferential transcription in endometrial cells. DNase I footprinting experiments with nuclear extracts from Ishikawa and HeLa cells reveal a series of defined protections overlapping these regions. The relative intensity of individual protections differs between the two cell lines. Oligonucleotide competition experiments suggest that similar factor(s) bind(s) to the two relevant upstream regions of the promoter that share no homology to known regulatory elements. A protection over the TATA-box is detected only with extracts from Ishikawa cells. Band shift experiments show that an Ishikawa-specific factor binds to sequences overlapping the TATA-box region that are partially conserved in other endometrium expressed genes. We propose that novel transcription factors mediate endometrium specific expression of the uteroglobin gene in conjunction with a tissue-specific factor that binds to the TATA-box region. PMID- 1711674 TI - Nucleic acid-binding specificities of tobacco chloroplast ribonucleoproteins. AB - Many ribonucleoproteins (RNPs) are involved in the regulation of gene expression at the post-transcriptional level. We previously isolated three nuclear-encoded RNPs from tobacco chloroplasts. Here we report their binding specificities for various nucleic acids. The three RNPs synthesized in vitro were subjected to binding assays using ssDNA, dsDNA and four ribonucleotide homopolymers. The affinities for ribonucleotide homopolymers are higher than those for ssDNA and dsDNA, suggesting that they bind preferentially to RNA in vivo. All three RNPs show high specificities for poly(G) and poly(U) in the presence of 1.2-1.8 M NaCl, providing additional evidence for the similarity to HeLa hnRNP proteins. Possible involvement of these RNPs in the post-transcriptional regulation of chloroplast gene expression is discussed. PMID- 1711675 TI - A gene encoding 22 highly related zinc fingers is expressed in lymphoid cell lines. AB - A cDNA was isolated from a T cell library using an oligonucleotide probe corresponding to a sequence conserved in proteins with multiple zinc fingers of the C2H2 type. The predicted protein structure of this cDNA (ZNF43) showed that it contained 22 of the Kruppel type of zinc finger motifs in tandem. The amino acid sequence was strongly conserved between each of the finger domains of this cDNA, except for variable residue positions within the putative DNA binding site. Within the zinc finger domain the amino acid sequence of the four zinc fingers 6 to 9 was very similar to the amino acid sequence of fingers 10 to 13, the DNA sequence bound by this group of eight fingers may include a short repeat. Southern blotting showed that ZNF43 was one of a closely related family of proteins with 10 to 20 members. The members of the ZNF43 family did not appear to be clustered at the chromosomal level. The transcription of many members of this gene family was increased in lymphoid cell lines. After in vitro induced terminal differentiation of the human HL60 cell line the expression of the ZNF43 family was reduced. The expression of the ZNF43 gene was mainly limited to T and B cell lines. The gene was differentially spliced and different cell lines expressed different combinations of transcripts. PMID- 1711676 TI - Interaction of protein SRP19 with signal recognition particle RNA lacking individual RNA-helices. AB - Derivatives of human SRP-RNA were constructed by site-directed mutagenesis and tested for their ability to interact with protein SRP19. An RNA missing helix 6 barely interacts with SRP19, while the helix 8-deletion mutant retains much binding capability. A mutant RNA consisting just of helix 6 also binds the protein, but not as well as the unaltered molecule. SRP19 interacts to a full extent with the fourth mutant RNA composed of helices 6, 7, 8 and a portion of helix 5. It is concluded that helix 6- and not helix 8- is the major SRP19 binding site. Helices 7, 8 and portions of helix 5 contribute to the formation of a functional site. These results agree with data suggesting a proximity of helix 6 and the conserved part of SRP-RNA. PMID- 1711677 TI - Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers. AB - The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2' OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations. PMID- 1711678 TI - Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template. AB - Steady state kinetics and inhibition by a dipyridodiazepinone of the reverse transcriptase from human immunodeficiency virus type 1 (HIV) were studied using a heteropolymeric RNA template with a sequence from the authentic initiation site on the HIV genome. For addition of the first deoxynucleotide to primer, kcat/KM is 0.05 (nM-min)-1 and KM is 10 nM. When all 4 deoxynucleotide triphosphates are present and processive synthesis occurs, catalysis is less efficient; kcat/KM = .0077 (nM-min)-1 and KM = 100 nM for dATP. These results are consistent with a rate determining conformation change involved in translocation of the enzyme along the template. Inhibition by the dipyridodiazepinone BI-RG-587 is noncompetitive with respect to both nucleotide and template-primer; this compound decreases Vmax but does not affect KM. Thus, this inhibitor binds to a site distinct from the substrate binding sites with Ki of 220 nM. Inhibition by BI-RG 587 results in a uniform decrease in amount of products of all lengths rather than a shift from longer to shorter products, suggesting the inhibitor does not affect processivity of reverse transcriptase. PMID- 1711679 TI - The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide. AB - Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick. PMID- 1711680 TI - Hybridization properties of deoxyoligonucleotides containing anthraquinone pseudonucleosides. AB - Achiral pseudonucleosides bearing an anthraquinone moiety have been incorporated into deoxyoligonucleotides in internal, 3' and 5' positions. The ability of these modified deoxyoligonucleotides to hybridize to complementary RNA and DNA was investigated using Tm measurements. The anthraquinone was shown to enhance binding to a complementary RNA when linked to the 3' and 5' end. Pseudonucleoside substitutions on the 3' end of an oligonucleotide are also shown to confer serum stability to the oligonucleotide. PMID- 1711682 TI - Trace metals determination in microquantities of nucleic acid samples. PMID- 1711683 TI - An MspI polymorphism in the human acid sphingomyelinase gene (SMPD1). PMID- 1711681 TI - Testis-specific expression of the human MYCL2 gene. AB - We have characterized the expression of MYCL2, an intronless X-linked gene related to MYCL1. RNase protection analysis of a panel of human normal and tumor tissues has revealed that MYCL2 is expressed almost exclusively in human adult normal testis; much lower levels of transcript were detected in one human lung adenocarcinoma. No MYCL2 transcript was found in human testis RNA obtained from second trimester fetuses. This observation suggests a germ cell rather than somatic cell origin of the transcript and possible developmental regulation of MYCL2. Northern blot analysis of poly(A)+ RNA from adult human normal testis with an antisense riboprobe revealed a transcript of approximately 4.8-kb, which is in agreement with the size predicted from the MYCL2 nucleotide sequence. Antisense transcripts were found spanning regions of MYCL2 corresponding to all three exons of MYCL1. No sizable open reading frame was seen for the MYCL2 antisense transcripts suggesting that they may represent either regulatory sequences or an intron of a gene encoded by the complementary strand. RNase protection assays and the 5' RACE protocol (Rapid Amplification of cDNA Ends) were used to address the localization of the transcription start site of the MYCL2 sense transcript and different putative promoters and transcription regulatory elements have been identified. PMID- 1711684 TI - [Usefulness of testing histamine release from isolated human basophils in anti allergic and anti-asthmatic drug assessment]. AB - Histamine release from isolated human basophils test was used to evaluate an activity of: histamine receptors H1 and H2 blockers, agonists of beta-receptors, calcium channel blocking agents, hydrocortisone, and disodium cromoglycate (Intal). The study involved 84 patients hospitalized for the bronchial asthma. Basophils were isolated with Day's technique modified by Shov and Norn. Histamine was measured with Shov's spectrofluorimetric technique. It was found that histamine release from isolated human basophils may be used in both evaluation of the mechanism of action and efficiency of drugs used in allergic diseases therapy. PMID- 1711685 TI - Synthesis and some biological properties of the hexapeptide analog of substance P with a C-terminal thioamide group. AB - A new hexapeptide analog of Substance P, containing a C-terminal thioamide group in the molecule [( Glp6, Mett11]SP6-11) was synthesized: Glp-Phe-Phe-Gly-Leu-Mett NH2. Conversion to thioamide was accomplished from tert-butoxycarbonyl-L methionine amide (Boc-Met-NH2) using Lawesson's Reagent. Its contracting activity on isolated guinea-pig ileum was considerably lower than that of [Glp6]SP6-11. PMID- 1711686 TI - Prostate--an extrapituitary source of follicle-stimulating hormone (FSH): occurrence, localization, and de novo biosynthesis and its hormonal modulation in primates and rodents. AB - A comparative study on the immunocytochemical localization, de novo biosynthesis, and hormonal modulation of follicle-stimulating hormone (FSH) was carried out in the prostates of man, monkey, dog, guinea pig, hamster, rat, and mouse. FSH was localized in the cytoplasm of the prostatic epithelial cells. In some specimens, staining was also observed in the nucleus. Both pituitary as well as prostatic FSH were coeluted on a Sephadex G-100 column and high-performance liquid chromatography (HPLC) indicating physicochemical similarities of FSH in both the tissues. Surprisingly, the modulation of pituitary and prostatic FSH by inhibin and its related peptides were comparable. The intensity and grandularity of FSH staining was stronger in the case of benign prostatic hyperplasia as compared with normal prostatic specimens. In view of the well-known effects of FSH on the cellular growth, differentiation, and function of gonadal tissues, a similar role for FSH in pathophysiology of prostate is postulated. PMID- 1711687 TI - Differential expression of specific cytokeratin polypeptides in the basal and luminal epithelia of the human prostate. AB - The purpose of the present study was to identify cytokeratin polypeptides that are specifically associated with the basal and luminal epithelia of the human prostate. This aim was accomplished by immunohistochemical and immunoblot analysis of human prostate using cytokeratin-specific monoclonal antibodies. In immunohistochemical studies, monoclonal anticytokeratin 8.12 exhibited immunoreactivity with the basal, but not luminal, epithelial cells of fetal, juvenile, normal adult, and hyperplastic prostate. The 8.12 antibody did not stain prostate cancer tissues. Epithelia of 30 and 36 week fetal prostate contained only basal cells whereas both luminal and basal cells were noted in 7 month and 1 year old juvenile prostate. This finding suggests a stem cell function for the prostatic basal cells. Immunoblot analysis of proteins separated by two-dimensional electrophoresis showed that cytokeratins 5 and 15 were basal cell-specific cytokeratins that were absent from prostatic carcinoma while cytokeratins 8 and 18 appear to be luminal-cell-specific. These results indicate that antibodies to specific cytokeratin polypeptides can be used not only to differentiate between prostatic basal and luminal cells but also to study the biological processes of prostatic organogenesis and carcinogenesis. PMID- 1711688 TI - Treatment response with transurethral microwave hyperthermia in different forms of benign prostatic hyperplasia: a preliminary report. AB - During a 7-month period, 79 patients with benign prostatic hyperplasia (BPH) were treated with 915 MHz transurethral hyperthermia (TUHT). All patients had obstructive and irritative signs and symptoms which warranted surgical treatment considerations. Of the 79 patients treated, 31 had follow-ups of 12 months or longer and seven additional patients experienced treatment failure requiring surgical management. These 38 patients were studied to evaluate the relationship of treatment response to the pretreatment prostatic morphology assessed during cystoscopy. There was a well-balanced distribution of patients regarding the important pretreatment characteristics in different morphological types of prostatic hypertrophy. These important characteristics included: prostate volume, postvoiding residual volume, mean peak flow rate, and mean symptom score on the FDA scale. The study patients were scheduled to receive five 60-min TUHT sessions with temperature controlled on the urethral surface at 45.5 degrees C. The treatment were well tolerated and administered on an outpatient basis without sedation or anesthesia. There was a significant difference in the incidence of major improvement in patients with lateral lobe hyperplasia and those with median lobe enlargement, 73 vs. 30%, P = 0.018. From this study, it appears that BPH patients who are found at cystoscopy to have a predominance of median lobe hypertrophy should, perhaps, be selected for treatment other than TUHT. A Phase I study utilizing a modified transurethral applicator to accommodate the specific problem of patients with median lobe hyperplasia is currently being planned. PMID- 1711689 TI - The efficacy and safety of terazosin for the treatment of symptomatic BPH. AB - At least 16 clinical investigations have documented the effectiveness of alpha blockade for BPH. In the present review, four clinical studies evaluating the efficacy and safety of terazosin, a selective long-acting alpha 1 blocker, for symptomatic BPH are reviewed. The unique features of these clinical investigations are: the study designs established detailed inclusion and exclusion criteria, the outcome assessments were based upon quantitative outcome parameters, large cohorts of homogeneous patients were enrolled, and appropriate statistical methods were utilized. The dose of terazosin was titrated to maximal doses ranging between 5-20 mg. Only four of the 163 patients developed orthostatic hypotension. Overall, the peak and mean uroflow rates increased 50% and 46%, respectively (P less than 0.001). The cumulative improvement in the mean obstructive, irritative, and total symptom scores was 67%, 35%, and 54%, respectively (P less than 0.001). The present review of terazosin in males with symptomatic BPH supports the following conclusions: (1) the dose of terazosin can be safely titrated to 10 mg in normotensive and hypertensive patients with symptomatic BPH; (2) the adverse events associated with doses of terazosin up to 10 mg are relatively mild and reversible; and (3) the improvements in the outcome parameters (symptom scores and urinary flow rates) are clinically and statistically significant. Although the ultimate role of terazosin for symptomatic BPH will be determined by multi-center randomized placebo-controlled studies, the present review provides further evidence that selective alpha 1 blockers are effective and safe for the treatment of symptomatic BPH. PMID- 1711690 TI - Horizontal transmission of hepatitis B virus amongst British 2nd World War soldiers in South-East Asia. AB - Infection with hepatitis B virus (HBV) is much more common in tropical than in temperate countries. Visitors to the tropics are thus at risk from HBV, though the degree of risk, and the routes of infection involved are uncertain. We report serological markers of HBV in two groups of 2nd World War soldiers, who served in the Thai/Burma jungles. The groups comprised 100 ex-prisoners of the Japanese (POW), and 100 Burma Campaign Veterans (BCV). Surface antigen to HBV (HbsAg) was positive in 0% of POW and 2% of BCV (P = not significant). Surface antibody (anti HBs) and core antibody (anti-HBc) were both positive in 40% POW and 13% BCV (P less than 0.001). Quoted UK prevalence rates for these markers are 0.1% for HBsAg, 1.5% for anti-HBs and 0.7% for anti-HBc. Both groups thus show very high rates of past HBV infection. For the POW there were many possible reasons, including contaminated surgical instruments and needles, blood transfusions, and multiple beatings with common weapons. None of these factors operated significantly for BCV. Malarial transmission was, however, intense in both groups, though more so in POW. The data thus again raise the possibility of horizontal transmission of HBV by biting insects in tropical countries. PMID- 1711691 TI - [Hyalin globuliform deposits with distinct AZT reactivity in the brain and spinal cord of AIDS cadavers]. PMID- 1711692 TI - [Comparative immunohistochemical studies of the histopathology of the breast using monoclonal antibodies Lu-5 and b-12]. PMID- 1711693 TI - [Early diagnosis of cancer of the prostate]. PMID- 1711694 TI - The characterization of EIAV reverse transcriptase and its inhibition by 5' triphosphates of 2'-deoxyuridine analogs, PFA and PAA. AB - A characterization of equine infectious anemia virus reverse transcriptase (EIAV RT) and its inhibition by 5'-triphosphate analogs was undertaken to explore the possibility of using EIAV RT as an in vitro model for studying human immunodeficiency virus (HIV). EIAV RT activity was found to be dependent on the bivalent cations Mg++ and Mn++. The optimal pH for enzyme reaction was pH 8.2. EIAV RT preferred a 70 mmol/L concentration of monovalent salts. Phosphonoformic acid (PFA) was an active inhibitor of EIAV RT, but phosphonoacetic acid (PAA) and N-ethylmaleimide (NEM) were not. The inhibition of EIAV RT activity by 5' triphosphates of nucleoside derivatives was in the following decreasing order: FLTTP greater than AZTTP greater than nPrearaUTP greater than nPredUTP = CEdUTP greater than EtdUTP greater than nPrdUTP greater than HMdUTP. nPrearaUTP was a linear competitive and PFA a linear noncompetitive inhibitor of EIAV RT with respect to dTTP. Apparent Kis and Kii were 1.5 and 2.2 mumol/L respectively. The susceptibility pattern of EIAV RT to inhibitors was similar to that of HIV RT. PMID- 1711695 TI - Inhibition of human immunodeficiency virus reverse transcriptase by Chinese medicines in vitro. AB - Thirty-five Chinese medicines, including extracts of traditional medicines or clinically useful recipes, herbs and isolated compounds, were tested for inhibitory activity against human immunodeficiency virus reverse transcriptase in vitro. Fourteen of the 35 medicines were found to be active. Baicalin, in particular, was studied in great depth. We found it to be a noncompetitive inhibitor of HIV RT, with an effective concentration (IC50) of 22 mumol/L. Chinese medicines were shown to be a rich source of potentially useful materials for the treatment of human immunodeficiency virus infection. PMID- 1711696 TI - The role of substance P of the central nervous system in the pathogenesis of spontaneously hypertensive rats. AB - In comparing the involvement of substance P (SP) in the central regulation of vasomotor tone in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), we found that: the degree of SP-like immunoreactivity (SPLI) in the hypothalamus, rostral ventral surface of the medulla oblongata (RVM) and intermediolateral cell column (IML) of the thoracic spinal cord of SHR rats was significantly higher than in WKY rats; intrathecal injection (ith) of SP receptor antagonist D-Pro2, D-Phe7, D-Trp9-SP (D-SP) caused a decrease of mean blood pressure (MBP) in both SHR and WKY rats. These results indicate that SP might participate in the pathogenesis of hypertension and the regulation of normal blood pressure. PMID- 1711698 TI - Trabecular architecture of the mandibular condyle of the rat as revealed by vital staining. AB - The variability of the internal architecture of bone was studied in the mandibular condyle of young rats with the aid of vital staining capable of revealing bone trabeculae. The effect of season and age was observed in Spring- and Fall-born rats 28 and 56 days of age. There was no clear left-right asymmetry in the average direction of the condylar trabeculae, but their ranges of direction within the condyles exhibited such asymmetry. Both the average directions and the internal variability were different in the medial and the lateral sides of the condyles. There were also small but in most respects significant seasonal differences. These findings indicate that even the relatively small mandibular condyle of the rat is a very complex structure. PMID- 1711697 TI - Relationship between mandibular incisor crowding and nasal mucosal swelling. AB - In this study, cephalometric and dental cast variables relating to 30 male and 20 female children, 8 to 13 years old with chronic nasal mucosal swelling, were compared with those relating to age- and sex-method controls. These controls were orthodontically untreated subjects with no histories of airway obstruction. The children with chronic nasal mucosal swelling had been referred because of chronic difficulties with nasal breathing to the Department of Otolaryngology Airflow Laboratory at the Hospital for Sick Children in Toronto. Previously active posterior rhinomanometry with a head-out volume displacement plethysmograph had been used to measure nasal resistance in 1000 consecutive subjects. Participants in the study reported here were selected from subjects whose nasal resistance fell markedly following administration of a decongestant spray. The subjects selected were found to have significantly (p less than 0.001) more mandibular incisor crowding, significantly (p less than 0.01) smaller mandibular arch widths than the controls, and significantly (p less than 0.001) smaller maxillary arch widths than the controls. The male subjects had significantly (p less than 0.01) smaller mandibular arch widths than the male controls. PMID- 1711699 TI - Toxicokinetics of Ro 5-4864, lindane and picrotoxin compared. AB - The effects produced by IP administration of these three agents in the rat were compared because of in vitro evidence that each modulates the picrotoxinin site of the GABAA receptor. For each, hypothermia had the lowest threshold and convulsions the next, with hypophagia produced only by the highest dose of either Ro 5-4864 or lindane. Convulsant effects had a shorter latency and a shorter duration than did hypothermia. Hypophagia, when present, lasted the longest. Myoclonus was the seizure type with the lowest threshold for all three agents. At the highest dose, lindane produced a high incidence of maximal clonic (hopping) seizures, whereas Ro 5-4864 and picrotoxin produced a high incidence of maximal tonic seizures instead. On a mole/kg basis, picrotoxin was 40 times more effective than the other two agents and produced seizures which started later, peaked later, and persisted longest. Ro 5-4864 and lindane were effective at equimolar concentrations and, in combination, produced effects which suggested either dose-addition or synergism. The data are consistent with the hypothesis that the toxic effects of both Ro 5-4864 and lindane may be attributable, at least in part, to an action at a subpopulation of GABAA receptors. PMID- 1711700 TI - Ion channels in leukocytes. PMID- 1711701 TI - Nuclear pore complex: structure, function, and regulation. PMID- 1711702 TI - In vivo neurochemical effects of electroconvulsive shock studied by microdialysis in the rat striatum. AB - The present study examined the effects of electroconvulsive shock (ECS) on interstitial concentrations of dopamine (DA), its metabolites DOPAC and HVA and the serotonin metabolite 5-HIAA in the striatum of freely moving rats using on line microdialysis. DA increased sharply following a single ECS. Interstitial concentrations of DOPAC. HVA and 5-HIAA also increased significantly. The ECS induced increase in DA varied as a function of days following implantation of the microdialysis probe, being 1300%, 305% and 300% of baseline 24, 48 and 72 h after surgery, respectively. In contrast, the response of the metabolites to ECS did not differ across days following surgery, being approximately 130%, 140% and 110% of baseline for DOPAC, HVA and 5-HIAA, respectively. Seizure activity induced by the convulsant agent flurothyl did not influence dialysate DA concentrations, suggesting that the ECS-induced DA release was related to the passage of current and not to the seizure activity. Interstitial concentrations of acetylcholine and choline in the striatum increased by approximately 20% and 140%, respectively, in response to a single ECS. The DA (but not the DOPAC or HVA) response to ECS was refractory to a second ECS delivered 2 h after the first. A second ECS delivered 24 h after the first produced the normal increase in DA. The ECS-induced increase in DA was attenuated following repeated ECS (eight treatments, one every second day). Baseline DOPAC and HVA concentrations were significantly elevated by repeated ECS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711703 TI - Long-term effects of neonatal exposure to isobutylmethylxanthine. I. Retardation of learning with antagonism by mianserin. AB - Pregnant women regularly ingest the methylxanthines, caffeine and theophylline, during pregnancy and lactation. Also, theophylline is used to treat apnea in premature infants. In this study, rat pups were treated with 3-isobutyl-1 methylxanthine (IBMX), on days 7-10 of life. Transient IBMX treatment during infancy caused a retardation of acquisition of a delayed reinforced autoshaped lever touch response in adulthood. Treated rats required more trials to learn the task, but did not show altered exploratory activity in the operant chambers. Coadministration of the serotonin (5-HT) antagonist mianserin with IBMX was able to attenuate significantly the effects of IBMX in both males and females, even though mianserin treatment alone caused an apparent learning deficit in the males. The results indicate that 5-HT and 5-HT receptors are important during development for normal expression of a specific cognitive function later in life. Furthermore, a 5-HT system appears to play a role in the mechanism whereby perinatal methylxanthine exposure could lead to learning impairments or other undesirable behavioral consequences. The use of IBMX in developing rats may also offer a model for studying the long-term consequences of the expression of opioid withdrawal during the neonatal period, since this agent induces a quasi-morphine withdrawal syndrome (QMWS) in mature rats. It is of interest that mianserin can block or attenuate effects of both quasi- and true morphine withdrawal. PMID- 1711705 TI - [Current theories on the pathogenesis and treatment of nocturnal paroxysmal hemoglobinuria (NPH)]. PMID- 1711704 TI - Long-term effects of neonatal exposure to isobutylmethylxanthine. II. Attenuation of acute morphine withdrawal in mature rats. AB - An acute model of morphine withdrawal was used to determine if neonatal exposure to 3-isobutyl-1-methylxanthine (IBMX) would cause alterations in the expression of withdrawal in the adult rat. IBMX induces a quasi-morphine withdrawal syndrome (QMWS), which is almost identical to true morphine withdrawal both behaviorally and neurochemically. Transient IBMX treatment during infancy (on days 7-10 of life) caused an attenuated suppression of fixed ratio (FR) responding during acute morphine withdrawal in adulthood; however, there appeared to be no attenuation of withdrawal-induced hypothermia. The attenuated behavioral response was not due to an altered ability to express withdrawal, as these rats did not react differently to various doses of IBMX plus naloxone (i.e., varying severities of quasi-morphine withdrawal) in adulthood. Coadministration of the serotonin (5-HT) antagonist mianserin with IBMX in the neonate prevented the effects of IBMX. Both the mianserin-treated and the IBMX plus mianserin-treated groups had increased levels of [3H]naloxone binding in brainstem, while IBMX treatment alone apparently had no significant effect. None of the neonatal drug treatments affected [3H]naloxone binding in frontal cortex. Thus, the long-term effects of IBMX on the opioid withdrawal response cannot be explained by changes in the number of opioid binding sites (labelled with [3H]naloxone) within the brain. The results indicate that exposure to a methylxanthine, and thus quasi morphine withdrawal, during development results in long-lasting alterations of a system which is involved in opioid withdrawal. Because coadministration of mianserin prevented the effects of IBMX, 5-HT and 5-HT2 receptors are implicated in these effects. PMID- 1711706 TI - [Visual communication in the prevention of cross infection: study performed at the intensive care unit of the Evangelic Hospital on Londrina]. AB - The influence of posters with illustrations, instructions, and appeals for hand washing was evaluated in terms of their effect on the frequency of occurrence this procedure, before the contact with the patients, in the Intensive Therapy Unit of the Hospital Evangelico, Londrina, Parana, Brazil. The influence of posters was statistically significant on the frequency of hand-washing by physicians, trained-nurses and laboratory technicians, but not in respect to physical-therapists, or blood-bank and X-ray technicians. PMID- 1711707 TI - Lineage relationships and functions of CD4+ T-cell subsets in the rat. PMID- 1711708 TI - [Proteinases, protease inhibitors and antirheumatic therapy]. PMID- 1711709 TI - BPH. Treating older men's most common problem. PMID- 1711710 TI - [Indolent arthropathies of the lower limbs]. AB - The didactic lecture deals with four questions: What is the origin of an isolated indolent arthropathy? diabetes, amylose, leprosis. What diagnosis in an adult familial form? Thevenard's disease when amylosis has been excluded. What are the varieties of congenital indolent arthropathies? An early recessive form of Thevenard's disease and the congenital analgesia. How to deal with a unilateral indolent arthropathy? First of all, look for dysraphism. PMID- 1711711 TI - [Embolization of metastases of the mobile spine]. PMID- 1711712 TI - [Synovial angiogenesis]. AB - Synovial angiogenesis, the formation of new capillaries from pre-existent capillaries, is a constant feature of synovial inflammation. Strictly regulated, it normally disappears after recovery from the acute episode. However it may persist during chronic synovial inflammation and then participates in pannus development in RA. This is the result of biochemical events which have contributed to breakdown of the extracellular matrix and cartilage in association with activation or secretion into this micro-environment of angiogenic factors. Relations with immunocompetent cells (lymphocytes and monocytes) suggest that this final common pathway may be partially dependent upon stimulation by the antigen. The development of treatment aimed at inhibiting angiogenesis could offer additional therapeutic hope in rheumatoid arthritis. PMID- 1711713 TI - [Serum beta 2-microglobulin and HLA alloantigens in primary Gougerot-Sjogren syndrome. A possible relation with HLA-DR3 specificity]. AB - The relationships between some allo-antigens of the HLA system and beta 2 microglobulin (beta 2m) serum level were examined in a group of 24 subjects with primitive Sjogren's syndrome (pSS). While the beta 2m serum level of all the patients with pSS were higher at the limits of significance (p congruent to 0.05), compared to the values of the 14 control subjects, the division of the patients into two sub-groups of 14 and 10 subjects, according to the presence or absence of the haplotypes DR2 and/or DR3, pointed up a beta 2m serum level which was significantly higher in the first compared to the second (p less than 0.02) and to the group of normal subjects (p less than 0.01). Among the individual haplotypes studied, only the DR3 was observed with a significantly greater frequency (p less than 0.01) in the patients compared to the control group. The haplotype DR3 and also the B8, although at a lesser level, were found to be correlated with a high value of the serum beta 2m: p less than 0.004 and p less than 0.05 respectively. A similar association was not found for the DR2 and DRW52 specificities. PMID- 1711715 TI - Balloon dilatation in the treatment of BPH. PMID- 1711714 TI - [Hepatitis C antibodies in acute Non-A, Non-B hepatitis]. AB - Stored serum samples of 20 patients with clinically and bioptically proven non-A, non-B hepatitis (NANBH) in the acute stage were tested for the presence of antibodies to hepatitis C virus (anti-HCV) by means of the Ortho ELISA system. After a mean period of 8 weeks from onset of the disease, 8 of 20 patients (40%) had anti-HCV. Our follow-up study included 14 patients. Of 9 primarily anti-HCV negative patients, 2 became positive after 2 and 7 months respectively, whereas 7 patients remained anti-HCV-negative up to 52 months (range 1-128) after the onset of hepatitis. The prevalence of anti-HCV was 71% in 7 patients with parenteral hepatitis related to transfusions (n = 2) or drug abuse (n = 5), and 38% in 13 patients with sporadic NANBH. Of the 8 anti-HCV-negative patients with sporadic NANBH, 5 had stayed in one of the countries where enterically transmitted NANBH is endemic 3 to 6 weeks before the onset of their disease. Our results show that at present the anti-HCV-test supplies an etiologic basis for approximately half of all cases of NANBH in acute stage. Nevertheless, in most cases the acute NANBH remains a diagnosis of exclusion. PMID- 1711716 TI - Analysis of Japanese encephalitis (JE) virus genome and implications for recombinant JE vaccine. AB - From the information of nucleotide sequences and deduced amino acid sequences of flaviviruses including JEV, we can postulate processing mechanisms of a polyprotein translated from single long open reading frame of the genome and mechanisms of construction of antigenic structures of structural proteins with biologically active forms after these proteins are translated. The results of comparative analysis of amino acid sequences among flaviviruses and epitope analysis on the E proteins which are the most important antigens for protective immunity suggest that the E protein of flaviviruses may have a similar structure closely related to each other. PrM and E proteins which had predictable signal sequences upstream on the N terminals were expressed with antigenically active form and molecular size the same as the authentic ones by the recombinant viruses. However, the recombinant viruses which had no such signal sequence expressed unprocessed proteins with antigenically denatured forms. These results suggest that normal proteolytic processing is needed to construct biologically active structures of JEV structural proteins. The E proteins which were expressed by the recombinant viruses as antigenically active form could elicit nutralizing and HI antibodies in animals and protective immunity in mice. The recombinant vaccinia viruses which express the E protein could induce strong immunologic memory against the E protein in mice. These results indicate that the development of a new type of vaccine against JEV will become possible in future. PMID- 1711717 TI - [Catecholamines and thyrotoxicosis]. PMID- 1711718 TI - [Palliative embolic treatment of synchronous bilateral renal carcinoma with metastasis]. AB - The paper reports the use of embolic treatment in a case of synchronous bilateral renal carcinoma with caval thrombosis extending to the right atrium. Embolic treatment was also used for a large metastatic mass in the right iliac wing. The problems associated to palliative embolic treatment are discussed together with the relative histopathological aspects and the use of intrarterial IFN. PMID- 1711719 TI - Western blot analyses of prekallikrein and its activation products in human plasma. AB - Prekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAL-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G11. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88 kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with C1 inhibitor (C1 INH) and alpha 2 macroglobulin (alpha 2 M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-alpha 2 M complexes. Complexes of KAL-antithrombin III (ATIII) and the ratio of KAL-alpha 2 M/KAL-C1 INH were higher in activated C1 INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KAL-alpha 2 M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and alpha 2 M. Immunoblots of activated plasma also showed bands at the position of KAL-C1 INH and KAL-ATIII complexes. When alpha 1-antitrypsin Pittsburgh (alpha 1-AT. Pitts) was added to plasma before activation, KAL-alpha 1-AT. Pitts was the main complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711720 TI - Detection of GPIV (CD36) mRNA in Naka- platelets. PMID- 1711721 TI - Staining of light-cured aesthetic resin restorative materials by different staining media: an in vitro study. AB - Aesthetic restorative resins have been characterised by clinical discolouration. New light-cured anterior and posterior resins are currently available to the profession and this study investigated the in vitro colour stability at 24 hours and 7 days of ten such resins in three different staining media. Disc specimens were prepared and stained at 37 degrees C and 100 per cent R. H. and then evaluated by double blind visual assessment. All the materials undergo some staining in coffee and red wine. Coca-Cola, however, does not cause any staining of the restorative resins. Adaptic II is the most stain-resistant material and most of the staining occurs within the first 24 hours after immersion in the staining media. PMID- 1711722 TI - [The dynamics of the healing of an infected fracture of mandibular bone exposed to andekalin and kontrikal]. AB - Infected mandibular fracture was simulated in rats. 10 percent calcium chloride solution (control) or andecalin (10 U/Kg) and contrykal (2.5000 U/kg) were administered to fracture site by vacuum electrophoresis. Histologic examination has revealed that andecalin activated the exudative phase of inflammation and decelerated the proliferative processes. A contrary effect was induced by contrykal: rapidly decreasing exudation, activation of osteoclastic resorption of necrotic bone and of osteogenesis processes. PMID- 1711723 TI - Sequences of wool keratin proteins: the CSIRO connection. AB - Wool, a dead tissue of epithelial origin, derives many of its properties as a textile fibre from the structure and arrangement of the proteins from which it is comprised. Much of the progress in the elucidation of wool protein structures, as a step towards understanding this relationship between structure and properties, has been made in the Division of Protein Chemistry of Australia's Commonwealth Scientific and Industrial Research Organization. PMID- 1711724 TI - Nitric oxide (NO): a versatile second messenger in brain. PMID- 1711725 TI - Carbohydrate differentiation antigens: probable ligands for cell adhesion molecules. PMID- 1711726 TI - [Antikeratin antibodies in rheumatoid arthritis]. AB - On the basis of the literature and the author's own investigations the present knowledge about antikeratin antibodies (AKA) in rheumatoid arthritis (RA) is summarized. The specificity of AKA for RA is described between 90% and 100%, but the sensitivity is reported as varying from 36% to 80%. In conclusion, AKA have great diagnostic value in RA and, high titre AKA, were found to be pathognomonic for RA. Further studies are necessary to confirm if AKA are also of prognostic and pathogenetic value in RA. PMID- 1711727 TI - [Beta-HCG positive seminoma--incidence with special reference to tumor marker concentration in testicular venous blood]. AB - The treatment of so-called "marker-positive" pure seminoma is still a controversial subject. We report on 32 cases of seminoma treated within 2 years. In these we measured beta-HCG and AFP in a cubital vein and in the spermatic cord veins. While 78% of the patients affected had elevated beta-HCG levels in the spermatic cord, in only 25% elevated beta-HCG levels were found in a cubital vein. This shows the markedly higher sensitivity of marker evaluation in spermatic cord veins. Furthermore, we conclude that seminoma generally produces beta-HCG and that therefore no change of the usual principles of therapy is required. PMID- 1711728 TI - [Local hyperthermia in benign prostatic hyperplasia]. AB - In recent years, hyperthermia has been used for the treatment of benign prostatic hyperplasia (BPH). The preliminary results reported were promising. Except for patients with total urinary retention, however, objective voiding parameters have not been reported in detail for patients with "prostatism". In a phase II study we treated 30 patients with BPH by local microwave hyperthermia (915 MHz). The prostate was heated transrectally to 42-43 degrees C in eight sessions of 60 min each. The sessions were given twice a week. To assess the results of the treatment the following parameters were determined before and 4 weeks after hyperthermia therapy: transrectal ultrasound of the prostate with volumetry, urinary flow rate, and residual volume. In all, 28 patients were evaluatable. Only 2/28 showed clinical improvement. Neither the voiding parameters nor the size of the prostate were significantly changed by hyperthermia. The success rate of 7.1% is even lower than the spontaneous temporary regression rate of BPH. Thus, to our mind, hyperthermia cannot be regarded an effective treatment comparable to TUR for BPH. PMID- 1711729 TI - [The value of interferons, interleukin-2 and tumor necrosis factor in the therapy of renal cell cancer]. AB - The treatment of patients with metastasized renal cell carcinoma by biological response modifiers such as interferon (IFN), interleukin-2 (IL-2) and tumor necrosis factor (TNF) should at present only be carried out in prospective studies since there are still no generally accepted treatment regimens for these substances. In addition, one must remember that only interleukin-2 has been approved for the treatment of renal cell cancer by the Bundesgesundheitsamt (Federal Health Authority) in Berlin. Regarding interferons, IFN-alpha seems to be the most suitable substance for the treatment of renal cell cancer. However, objective response rates (almost exclusively partial responses) can only be expected in about 15% of the cases. By combining IL-2 with lymphokine-activated killer cells or IFN-alpha, objectively assessed remissions can be found in 35%. The approximate complete-response rate using this form of treatment, however, is in the range of 10%. PMID- 1711730 TI - [Localization and extent of tissue damage caused by extracorporeal lithotripsy (ESWL)]. AB - Extracorporeal shock wave lithotripsy (ESWL) causes proteinuria. In our study we investigated the protein fractions and the electrolyte composition of the urine in patients who had been treated with ESWL. The aim was to obtain information on the degree and the localisation of the glomerular, tubular or vascular destruction caused by ESWL in humans. A total of 34 patients with stones had been treated with ESWL. As parameters we used: urine output, creatinine clearance, total protein, albumin, immunoglobulin G, N-acetyl-beta-D-glucosaminidase (beta NAG), alpha-1-microglobulin, the fractional excretion of Na+ and apolipoprotein-A 1. After ESWL treatment proteinuria and albuminuria are found. Our parameters show no deterioration of the glomerula or the tubulus. The increase in apolipoprotein-A-1, a postglomerular parameter, however, is interpreted as a manifestation of vascular destruction after ESWL; this is normally temporary, leaving no permanent damage. PMID- 1711731 TI - Cardiac myxomas: immunohistochemical study of benign and malignant variants. AB - Immunohistochemical investigation of 11 cardiac myxomas (CMs) including one malignant metastasizing CM showed a co-expression of epithelial (lu-5 and CAM 5.2), mesenchymal (vimentin) and neuroendocrine antigens (neuron-specific enolase) in all tumour cells. Factor VIII was found in the endothelial cells of capillaries only. In the subendocardium of fetal heart tissue close to the foramen ovale myofibroblasts reacting with the panepithelial antibody lu-5 were detected. We conclude that CMs are neoplasms that may develop from embryonic cell remnants. PMID- 1711732 TI - Immunohistochemical demonstration of keratins 8 and 14 in benign tumours of the skin appendage. AB - The expression of keratins 8 and 14 was investigated immunohistochemically by the avidin-biotin-peroxidase (ABC) method using formalin-fixed paraffin-embedded specimens from 42 tumours of human skin appendages. Results were compared with the staining of 34 specimens from normal skin and skin appendages adjacent to the tumours. Keratin 14 was detected by the monoclonal antibody (mAb) 312C8-1, and was found in the basal cells of the epidermis, the outer root sheaths of hair follicles, and the peripheral cells of sebaceous glands. It was also detected in the inner and outer layers of cells in the ductal portion and the myoepithelial cells in the secretory portion of apocrine and eccrine sweat glands. Keratin 8 was detected by mAb 35BH11, and was present in the secretory cells of eccrine and apocrine sweat glands but not in myoepithelial or ductal cells. The pilosebaceous apparatus and the epidermis were uniformly negative. In benign skin appendage tumours, the staining patterns for both keratins generally resembled their distribution in the corresponding normal tissues. The demonstration of keratins 8 and 14 may be useful in the recognition, classification and diagnosis of skin appendage tumours. PMID- 1711733 TI - Well-differentiated hepatocellular carcinoma associated with long-term survival. Report of two cases. AB - Two cases of well-differentiated hepatocellular carcinoma (HCC) with focal biliary differentiation are presented. The distinct histological features of these neoplasms and the unusually protracted clinical course of 8 and 10 years distinguish them from previously described pathological categories of primary hepatic tumors. Electron microscopic and immunohistochemical findings support a dual hepatic and bile duct differentiation of the tumor cells. If additional examples of this tumor are found to be associated with a similarly prolonged symptom-free survival, the distinction of this entity from traditional, rapidly fatal HCC becomes important. Less aggressive therapeutic options may be entertained. PMID- 1711734 TI - [The diagnosis of diseases of the prostate by bioelectrolytic data on its secretion]. PMID- 1711735 TI - [Natural killer cells of different immunological phenotypes (CD16+, CD56+ and CD57+) in the blood of patients with insulin-dependent diabetes mellitus]. AB - The method of flow cytometry employing monoclonal antibodies a Leu 7 (CD57), a Leu 11 (CD16) and a NKH-1 (CD56) was used to examine to content of different subpopulations of natural killer cells in the blood of primary patients with insulin-dependent diabetes mellitus and in healthy subjects. Patients with insulin-dependent diabetes mellitus showed as compared with healthy persons a lower content of natural killer cells carrying all the examined markers, in particular, CD56. The degree of reduction of the number of natural killer cells of different subpopulations is individual for each patient that, possibly, reflects the diversity of causes leading to development of the disease. PMID- 1711736 TI - [The significance of the specific sensitization of immunocytes in assessing the immune response in viral hepatitis B]. AB - A study is presented of 235 patients with viral hepatitis and 35 patients with other diseases of the liver. The method of antigen-specific indirect rosette formation was used. Sensibilization of immunocytes to superficial B hepatitis virus antigen was revealed in 84% of patients with viral B hepatitis. The author established a dependence of the clinical course of viral B hepatitis on the character and time appearance of the specific reaction of immunocytes. The determination of specific sensibilization of immunocytes improves the diagnosis and possibilities of viral B hepatitis prognosis. PMID- 1711737 TI - [The morphofunctional changes in the perivascular and lymphoepithelial nodes of the respiratory organs following antigenic stimulation]. AB - Morphological, experimental-immunological methods with mathematical analysis were used in 108 Wistar line rats in the pre- and postnatal ontogenesis to study the lymphoid structures of the trachea, bronchi and stroma of the lungs. Two types of lymphoid nodules were detected in the respiratory organs of rats and man: 1. perivascularly located in the wall of the trachea, bronchi and stroma of the lungs; 2. lymphoid nodules of the mucous membrane of the respiratory ways which have a different structure. The former are related to a common immune system of the internal milieu, the latter--to a common immune system of the mucous membranes. Antigenic stimulation revealed that formation of lymphoid structures is enhanced and more rapidly terminated as to functional maturation. Lymphoid structures play a decisive role in local immunity. PMID- 1711738 TI - [Anti-arrhythmia effectiveness of potassium-magnesium-aspartate infusion]. AB - In 21 patients with ventricular arrhythmias we analysed the effect of an intravenous infusion of potassium-magnesium-aspartate. It could be demonstrated that the frequency of ventricular ectopic beats significantly declined 1 hour after starting the medication. The maximum effect occurred at the 6th and 7th hour and continued until the 10th hour after starting the medication. PMID- 1711739 TI - [Long-term treatment with amiodarone]. AB - The aim of this study was to investigate the efficacy and the side effects of a long-term treatment with amiodarone. We analyzed the data of 41 patients in whom amiodarone therapy had been initiated between 1974 and 1984. Twenty-one patients had dilative cardiomyopathy, 14 patients had chronic myocardial infarction, four patients suffered from WPW syndrome with intermittent atrial fibrillation, one patient had aortic valve surgery, whereas in one patient there was no clinical evidence of a heart disease. All patients had salvos of ventricular extrasystoles, ventricular tachycardia or documented intermittent ventricular fibrillation. There have been seven drop-outs up to the present time. In each patient, the lowest antiarrhythmically effective dose was applied, which was generally higher in patients with low ejection fraction. Effective treatment of the ventricular tachycardia was achieved in 55-92% of patients and did not depend on the duration of treatment. In 10 patients in whom amiodarone therapy had to be stopped for various reasons. Sudden cardiac death was slightly more frequent than in the 24 patients treated with amiodarone, though the difference was not significant. In cases with a history of syncope the prognosis was poor, even with amiodarone therapy. Due to side effects, a dosage reduction or discontinuation of amiodarone treatment became necessary in 14 patients. Amiodarone proved to be an effective drug also for the long-term treatment of ventricular tachycardia, and possibly for the prevention of sudden cardiac death. With the exception of blue skin color, there was no accumulation of side effects, even during long-term treatment of several years. PMID- 1711740 TI - [Effect of amiodarone on signal-averaged and long-term ECG]. AB - This prospective study examined the influence of long-term amiodarone therapy on the parameters of the signal-averaged ECG and their relation to simultaneously derived Holter monitoring data. For this purpose, 23 patients with angiographically confirmed dilated cardiomyopathy or coronary heart disease and high-grade ventricular arrhythmias, in whom an average of four class I antiarrhythmic drugs had proven ineffective, were stabilized on amiodarone. Before the beginning of therapy, as well as after 2 months and, subsequently, every 3 months, a resting ECG, a signal-averaged ECG by Simson's method, and Holter monitoring were performed. Compared to the initial measurement, we found a significant increase in the duration of the total filtered QRS complex from an average of 114 +/- 24 ms to 127 +/- 35 ms, while the change in voltage did not reach the significance level. The incidence of late potentials remained largely constant under amiodarone; 10 patients showed a constant late potential, 12 patients had no late potential, and one patient with coronary heart disease developed a new late potential. In the long-term follow-up, we ascertained a relatively high responder rate under amiodarone between 41% and 81%. No relation could be detected between the results of the signal-averaged ECG and those of 24 h Holter monitoring. PMID- 1711741 TI - Mapping of a fusion related epitope of the respiratory syncytial virus fusion protein. AB - The region of the fusion glycoprotein of respiratory syncytial virus which reacts with a neutralizing and fusion inhibiting monoclonal antibody, was mapped using a deductive method derived from analysis of Western blot reactivity of proteolytic fragments. Reaction of the whole fusion protein was found to be so conformationally dependent, that complete digestion of the protein with a variety of proteases resulted in fragments which were not sufficiently reactive to permit mapping. For this reason, polyclonal antibodies to synthetic peptides which spanned the fusion protein sequence, were used to map the position of large peptides derived from partial digests, and these peptides were then analysed for their ability to react with the monoclonal antibody. Comparison of the peptides which were reactive with the monoclonal antibody to those which were not, identified a region of non-overlap between residues 283 and 327 in the F1 subunit of the fusion protein. Synthesis of a peptide within this region confirmed the placement of the epitope. PMID- 1711742 TI - Neutralizing epitopes of human parainfluenza virus type 3 are conformational and cannot be imitated by synthetic peptides. AB - The possibility that linear epitopes on the haemagglutinin-neuraminidase (HN) surface glycoprotein of human parainfluenza virus type 3 (PIV-3) might induce neutralizing antibodies after virus infection was investigated. Thirty-seven peptides, representing 64% of the extramembranous portion of the HN molecule of PIV-3, were synthesized. Their ability to bind to 14 neutralizing murine monoclonal antibodies (mAbs) specific for HN or 26 high-titre human serum samples were tested in a direct enzyme-linked immunosorbent assay (ELISA) and in an indirect competition ELISA. None of the synthetic peptides reacted with any of the mAbs or serum samples in the direct test and none of 11 synthetic peptides tested blocked mAbs from binding to HN in the competition ELISA. These findings suggest that synthetic peptides cannot be used to imitate the known neutralizing epitopes on the HN. Analyses of reduced and non-reduced HN in ELISA and immunoblot assays confirmed that protein folding and tertiary structure are essential for epitope formation in these neutralizing sites. However, some children's sera analysed by immunoblotting contained antibodies to an uncharacterized linear epitope(s) not recognized by our panel of mAbs, raising the possibility that a neutralizing linear epitope does exist on the HN of PIV-3. PMID- 1711743 TI - [The facilitation of the reproduction of a conditioned avoidance reaction by substance P in rats]. PMID- 1711744 TI - [Surgical therapy of bronchial cancer. Healing or palliation?]. AB - In spite of the revolutionary innovations concerning diagnostic and intensive care techniques as well as better understanding of tumour biology during the last decade, rates of resection and long-term survival in bronchogenic carcinoma could not be improved. Nevertheless there is no reason for therapeutic nihilism. On the contrary all efforts are to be done in investigation on practicable, suitable, and supportable screening programmes to improve the patients outcome. PMID- 1711745 TI - [Results of cryosurgery in the treatment of inoperable tumor stenoses of the anus and rectum]. AB - Inoperable tumor stenosis of anus and rectum may be treated surgically only by implantation of artificial anus. Cryosurgery proves to be a feasible alternative in order to maintain free passage of the gut. The cryoprobe is introduced through a rectoscopy under direct view. Visible tumor is touched with the tip of the cryoprobe and subsequently temperature is lowered to approximately -19 degrees C. Usually anesthesia is not required. From 1976 to 1989, 213 patients had been treated by cryosurgery in our department. In 23 cases the use of an artificial anus became inevitable afterwards. PMID- 1711746 TI - [Therapeutic protocols in the treatment of liver metastases]. AB - The liver is the main site for metastatic spread from many cancer, particularly those of the colorectum. Surgical treatment of liver metastases is nowadays a safety therapeutical approach, which has been improved by the development of modern imaging procedures, clear indications and standardized surgical techniques. In carefully selected patients the 5-year survival ranges between 30 40%. Unfortunately only 20% of the patients with liver metastases are candidates for this potentially curative therapy. Palliative modalities remain for the majority of patients with unresectable metastases limited to the liver. Although various types of intrahepatic arterial chemotherapy, sometimes in combination with whole liver irradiation or embolization shows a higher response rate than systemic chemotherapy no significant impact on survival time has been proven. One of the main unsolved problem is the extrahepatic spread. For getting better results--median survival of responders ranges between 18 to 24 months versus 8 months of non-responders--a more exact selection is needed. PMID- 1711747 TI - [Pancreatic ductal carcinoma. Intra- and postoperative complications in palliative and curative interventions]. AB - During 1982 and 1989 we treated 95 patients with ductal pancreatic carcinoma. Of these, 32.6% (31/95) had resection. The operation lethality was 3% (1/31), the clinical lethality 12% (4/31). The median length of survival was 6,8 months. Almost half of the potential curatively operated patients showed complications. Remarkable, that patients, operated in stages III and IV have been involved over proportionally often. Complications are the intra- and postoperative bleeding, the pancreatic fistulae, the intra-abdominal abscess, the sepsis and cardiopulmonary insufficiencies. 33 Patients underwent palliative operation, where the complication rate was significantly lower. Indeed, 5 patients who got a biliodigestive anastomosis, subsequently developed a pyloric or duodenal stenosis and had to be reoperated. The 30-day-lethality of the palliatively operated patients was 13% (4/33), the clinical lethality was 15% (5/33) and the median length of survival was 5,6 months. 24 patients underwent exploratory laparotomy and 7 patients were inoperable. Due to the higher complication rate in the curatively operated patients and only little higher life expectancy, an extensive surgical treatment of pancreatic carcinoma should only be considered effective in selected cases and in an early stage of carcinoma. PMID- 1711748 TI - [Rectal carcinoma. Analysis of 10-year results]. AB - Between 1974 and 1979 139 patients with rectal cancer have been operated. Operative mortality was 14.4%. The fate of all patients was followed during 10 to 16 years. 46.8% of of patients died from recurrencies and metastases after a medium of 27.5 months. 3.6% died from another malignancy, 15.8% from other unrelated diseases. The 10-year survival rate is 22.3%; 19.4% of all patients are still alive with a mean survival time of 155 months. Prognosis depends mainly on tumour stage. No patient with recurrencies or metastases survived 10 years or longer. PMID- 1711749 TI - [Significant effects on alpha-fetoprotein (AFP) concentration in maternal serum in pregnancy]. AB - The influence of oral contraceptives, nicotine, analgesics, thyroid hormones as well as the age of pregnant women, parity and later birth weight on the concentration of maternal serum AFP was examined in 200 patients to check the reliability of AFP determinations in serum of pregnant women. There were correlations only with nicotine consumption. The results confirmed an insignificant influence of these factors on maternal serum AFP and underlined the reliability and stability of this prenatal screening method. PMID- 1711750 TI - [A new method for the rapid diagnosis of acute streptococcal infection]. AB - The work deals with the development of the rapid method of the identification of acute streptococcal infection on the basis of the coagglutination test. The rapid method of the extraction of group-specific polysaccharide antigen from the cell walls of group A streptococci is proposed. The data on the use of native sera and their fractions in the development of coagglutination diagnostica have been described and analyzed. The advantages of the new method of the diagnosis of acute streptococcal infection in comparison with the traditional microbiological method are shown. PMID- 1711751 TI - Middermal elastolysis. Report of a case and immunohistochemical studies on the dermal distribution of fibrillin, vitronectin and amyloid P component. AB - A 39-year-old woman with demarcated wrinkled areas, histologically characterized by absence of elastic fibers in the middle and upper reticular dermis, is described. Immunoreactivity of vitronectin and amyloid P component, present at the periphery of elastic fibers in normal skin in adults, was absent from the middermis of lesional skin as were orcein stained fibers. C9 neoantigen immunoreactivity, associated with elastic fibers in sun-exposed skin of middle aged and elderly individuals, was present in conjunction with elastic fibers in papillary and lower reticular dermis in lesional skin but was absent in the middermis. In contrast, a fibrillin immunoreactive network was present throughout the dermis, indicating that the elastin-associated microfibrils are retained in the absence of amorphous elastin in lesional skin of middermal elastolysis. PMID- 1711752 TI - Neurogenic inflammation induced by capsaicin in patients with psoriasis. AB - Increasing doses of capsaicin were applied topically to the forearm skin of 30 patients with psoriasis, 16 patients with systemic scleroderma and 16 healthy volunteers. Only one-third of the patients with psoriasis responded with neurogenic inflammation to capsaicin doses of 0.125 and 0.25 microgram/cm2 in contrast to 81% of scleroderma patients and all the normal controls, who showed a positive cutaneous reaction. Higher doses of capsaicin (0.5-4 micrograms/cm2) were required to induce erythema and flare in patients with late-onset psoriasis (after 21 years of age) as well as in patients with more than 40% of skin surface involved with psoriatic lesion. PMID- 1711753 TI - Intralesional injection of bleomycin sulphate into resistant warts in renal transplant recipients versus non-transplant warty patients. AB - Sixteen adult renal transplant patients and 20 non-transplant patients with warts underwent intralesional therapy with bleomycin sulphate. One unit/ml bleomycin sulphate was injected in 93 warts in renal transplant recipients and 100 warts in non-transplant patients with proven resistance to conventional treatment for at least 6 months. The treatment was compared with a normal saline placebo injected into the paired warts in the same patient. Thirty-four out of 93 warts (37%) in renal transplant recipients vs. 59 out of 100 warts (59%) in non-transplant patients were completely cured after one to three injections. We found bleomycin completely ineffective in 56 warts (60%) in renal transplant recipients, but ineffective in only 17 warts (17%) in non-transplant warty patients. None of the patients treated experienced any side effects except for local pain which was well tolerated, especially by non-transplant patients. PMID- 1711754 TI - New O antigens of Morganella morganii and the relationships between haemolysin production. O antigens and morganocin types of strains. AB - A collection of 142 strains of Morganella morganii, principally from unrelated patients' faeces was examined to determine the relationship, if any, between haemolysin production, O antigen and morganocin production/sensitivity type. Only 55 (38.7%) were agglutinable with the existing 44 O antisera. However, when O antisera were raised to some of the non-typable strains 11 new O antigens were found and 126 (88.7%) of the strains were typable. The number of O antigenic groups in M. morganii is now 55. It was confirmed that the O antigenic characteristics of strains were independent from morganocin producer types. An epidemiological retrospective survey showed that finer strain recognition in M. morganii can be achieved by using both methods than either method alone. Approximately 30% of strains were haemolytic. The ability to produce haemolysin was more common in strains of certain O serogroups and morganocin producer types than in others. PMID- 1711755 TI - Alzheimer's disease affects limbic nuclei of the thalamus. AB - Sensitive silver techniques for amyloid and neurofibrillary changes were applied to examine the pathological changes revealed by limbic nuclei of the thalamus in Alzheimer's disease. Large numbers of extracellular amyloid deposits occurred in almost all thalamic nuclei. The antero-ventral nucleus harbored numerous large globular patches, other areas contained more densely packed and smaller deposits, while narrow zones of gray matter subjacent to the ependymal lining of the third ventricle remained virtually devoid of amyloid. Intraneuronal neurofibrillary changes were encountered in the form of distended argyrophilic processes covering the medial convexity of the antero-ventral nucleus. Similar structures, although in considerably lesser density, occurred in the laterally adjoining reticular nucleus. The anterior nuclear complex, the latero-dorsal nucleus, portions of the intralaminar complex, the paraventricular and reuniens nucleus contained numerous neurofibrillary tangles and neuropil threads. The antero-dorsal nucleus showed the most severe involvement. At first glance, the thalamus appeared to be only mildly affected by Alzheimer's disease. Closer inspection revealed that severe changes were confined to only a few limbic nuclei. These changes were virtually identical in amount, type and location in all cases of severe Alzheimer's disease studied. It is assumed that these changes considerably hamper the transport of information through limbic circuits. PMID- 1711756 TI - Striatonigral degeneration, olivopontocerebellar atrophy and "atypical" Pick disease. AB - A 75-year-old woman with parkinsonism plus was found at autopsy to have striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA) and intracytoplasmic neuronal inclusions, mostly confined to the hippocampus and pontine nuclei. These inclusions were intensely argyrophilic, ubiquitinated and expressed variable immunoreactivity for neurofilament but not for tau-1 and Alz 50 proteins. Ultrastructurally, they were formed of skeins of intermediate filaments averaging 11 nm in diameter. They were considered to represent Pick bodies. There was no cortical atrophy, gliosis or sponginess. To our knowledge, SND and OPCA in association with Pick's disease has not been previously reported. In addition, intracytoplasmic oligodendroglial inclusions were present in the deeper layers of the cortex, especially the pericentral gyri, the striatum and the white matter of certain regions of the cerebral hemispheres, as well as in the cerebellum. These inclusions which have been previously reported in multisystem atrophy, had to be distinguished from cortical Lewy bodies, Pick bodies, and the nonspecific ubiquitinated bodies in the white matter of the aged brain, mainly by their topographical distribution and immunostaining properties. PMID- 1711757 TI - Permanent alterations of the dendritic tree of cerebellar Purkinje neurons in the rat following postnatal exposure to cis-dichlorodiammineplatinum. AB - The aim of this study was to characterize further the effect of cis dichlorodiammineplatinum (cisDDP) on cerebellar Purkinje neurons of the immature rat. Ten-day-old rats were treated with cisDDP subcutaneously and killed after 1, 7, 20 or 65 days. The cerebellar vermis was impregnated by the Golgi-Cox method to evaluate the extent of morphological maturation of the Purkinje cell dendritic tree. One day after treatment, the dendritic network of Purkinje cells of treated animals was poorly developed and the cell somata still showed numerous perisomatic processes. This indicates that cisDDP interferes with the organization of microtubules and microfilaments by the cell. Later, several abnormal shapes of the Purkinje cell dendritic tree were observed. These included: (1) elongated primary dendrites; (2) asymmetrical dendrites; (3) sprouting of secondary and spiny branches in two planes of the molecular layer; and (4) damming of spiny branchlets at the pial surface. Moreover, all the dendritic networks of Purkinje cells in treated animals were of a lower Strahler order than in controls. All these data suggest that the late anomalies of the dendritic trees are secondary to the general cisDDP-induced damage of the cerebellar cortex, rather than being a primary effect of the drug on the dendritic tree growth. PMID- 1711758 TI - Subtypes and differential laminar distributions of beta A4 deposits in Alzheimer's disease: relationship with the intellectual status of 26 cases. AB - beta A4 immunoreactivity was studied in temporal neocortex, area 22, of 26 cases with graded intellectual status. Sampling was performed in psychometrically assessed women over 75 years, either intellectually normal or affected by senile dementia of Alzheimer type of various degrees of severity. beta A4 antibodies labelled various types of beta A4 deposits in 22/26 cases: (1) small, stellate deposits; (2) diffuse deposits, (3) primitive, (4) classic and (5) compact, or burn-out, plaques. The densities of the stellate deposits, primitive and classic plaques were always positively linked with the severity of the intellectual status, whereas those of the diffuse deposits were not. This was due to a single case with normal mental status and numerous beta A4 deposits. Densities of stellate and diffuse deposits were higher in layers I, III and IV, whereas densities of primitive, classic, and neuritic plaques observed with Bodian's technique were higher in layers II and III. Topographical distribution of each subtype did not vary as a function of the severity of the intellectual status. These data suggest that deposits of beta A4 protein appear a necessary but not a sufficient condition for inducing neuritic plaque formation, in the neocortex as in other brain areas. beta A4 proteins could accumulate either as diffuse deposits, which do not cause an intellectual deficit, or as dense deposits, associated with argyrophilic neurites, i.e., classic neuritic plaques, highly correlated to the intellectual impairment. This evolution could depend on factors which are laminarily distributed in the neocortex. PMID- 1711759 TI - Endoneurial proliferation of perineurial cells in leprosy. AB - In leprous neuropathy the perineurium has very often an abnormal multilayered appearance and is infiltrated by many different types of inflammatory cells. We report here 13 cases characterized by an abnormal endoneurial proliferation of fibroblasts which seems to differentiate in perineurial cells. In several instances there is formation of many intrafascicular microcompartments. Such aspects have been described in various, but infrequent, cases of experimental and human neuropathies. It seems that severe Wallerian degeneration, diffuse endoneurial macrophage infiltration and lesion of the perineurium might lead to such a process. PMID- 1711760 TI - Delayed institution of hypertension during focal cerebral ischemia: effect on brain edema. AB - The effect of induced hypertension instituted after a 2-h delay following middle cerebral artery occlusion (MCAO) on brain edema formation and histochemical injury was studied. Under isoflurane anesthesia, the MCA of 14 spontaneously hypertensive rats was occluded. In the control group (n = 7), the mean arterial pressure (MAP) was not manipulated. In the hypertensive group (n = 7), the MAP was elevated by 25-30 mm Hg beginning 2 h after MCAO. Four hours after MCAO, the rats were killed and the brains harvested. The brains were sectioned along coronal planes spanning the distribution of ischemia produced by MCAO. Specific gravity (SG) was determined in the subcortex and in two sites in the cortex (core and periphery of the ischemic territory). The extent of neuronal injury was determined by 2,3,5-triphenyltetrazolium staining. In the ischemic core, there was no difference in SG in the subcortex and cortex in the two groups. In the periphery of the ischemic territory, SG in the cortex was greater (less edema accumulation) in the hypertensive group (1.041 +/- 0.001 vs 1.039 +/- 0.001, P less than 0.05). The area of histochemical injury (as a percent of the cross sectional area of the hemisphere) was less in the hypertensive group (33 +/- 3% vs 21 +/- 2%, P less than 0.05). The data indicate that phenylephrine-induced hypertension instituted 2 h after MCAO does not aggravate edema in the ischemic core, that it improves edema in the periphery of the ischemic territory, and that it reduces the area of histochemical neuronal dysfunction. PMID- 1711761 TI - Hereditary diabetes insipidus: an immunohistochemical study of the hypothalamus and pituitary gland. AB - We report the histological findings in a case of hereditary diabetes insipidus (HDI) using vasopressin (VP) immunohistochemistry. The hypothalamus displayed a marked loss of magnocellular VP neurons, with preservation of the smaller cells. The neurohypophysis was severely atrophic with scanty immunoreactivity. Our results support the hypothesis that HDI results from a selective degeneration of VP neurons affecting chiefly the magnocellular elements projecting to the neurohypophysis. The sparing of the parvocellular component may reflect the projection of these neurons to non-pituitary targets. PMID- 1711762 TI - Morphology and histochemistry of rabbit submandibular glands. AB - The rabbit submandibular glands are of the heterocrine type. The most distal part of the ductal system consists of secretory endpieces, which are constructed mainly of two morphologically and histochemically different and distinct structures: distally the seromucous acini and proximally the serous tubuli. The cells of the seromucous acini are strongly AB pH 2.5 positive, AB pH 1.0 negative and PAS negative, indicating production of acid glycoproteins--mainly sialomucin. The cells of the serous tubuli are AB pH 2.5 negative and strongly PAS positive, indicating the presence of neutral glycoproteins. Glycol methacrylate embedded sections (Historesin) stained with a modified Trichrome stain were superior to standard paraffin sections regarding resolution and clarity of morphological details. Quantitative studies of intralobular structures on AB-PAS-stained paraffin sections compared with Trichrome-stained glycol methacrylate sections gave corresponding results. The latter method is well suited for study of structural changes in submandibular glands, as in assessing the effect of ionizing irradiation. PMID- 1711763 TI - The keratinizing tympanic epithelium: an enigma. AB - Both the centrifugal keratin dispersion on the pars tensa as well as the centripetal proliferative properties at the inferior annular tympanic region are discussed. There is evidence, histological and clinical, that these features are two distinct and different phenomena which both have clinical implications. While the centrifugal keratin dispersion is a physiological cleaning mechanism, the cytokeratin expression demonstrates the unusual but for years clinically noticed proliferative nature of the lower annular epithelium and provides biochemical evidence for a relationship between this epithelium and cholesteatoma formation. PMID- 1711764 TI - Immunomodulators interfere with angiopathy but not vasospasm after subarachnoid haemorrhage in rabbits. AB - The present study deals with the effects of immunomodulators on the morphology of intracerebral arterial walls in rabbits with experimental subarachnoid haemorrhage (SAH). Immunostimulators:thymostimuline and inosine dimethylamino isopropanol-p-acetamido-benzoate were found to aggravate the angiopathic changes, whereas immunosuppressive drugs-cyclosporine A and azathioprine appeared to prevent the damage. The authors consider the possibility of using immunosuppressive drugs in patients with ruptured intracranial aneurysms. PMID- 1711765 TI - The role of dipeptidylpeptidase IV positive T cells in wound healing and angiogenesis. PMID- 1711766 TI - Proliferative and synthetic activity of cells from reoccluded distal anastomosis and pharmacological therapy of this postoperative complication. PMID- 1711767 TI - Lipoxygenase-inhibitory azomethines and benzoylhydrazones. I. Inhibition of the lipoxygenase activity and of the degranulation of mast cells in vitro by phenolic azomethines. PMID- 1711768 TI - Influence of cyclooxygenase- (COX-) and lipoxygenase- (LOX-) inhibition on the degranulation of activated peritoneal rat mast cells (pRMC) in vitro. PMID- 1711769 TI - Influence of some prostaglandin-analogues on mouse skin allograft survival compared to dexamethasone. Possible role of thromboxane. PMID- 1711770 TI - Catecholaminergic modulation of rat acute phase reactants. PMID- 1711771 TI - Benign prostatic hypertrophy. AB - By age 75, between 10 and 25 percent of men require intervention for problems caused by benign prostatic hypertrophy. Symptoms of bladder outlet obstruction include hesitancy, terminal dribbling, postvoid fullness and double voiding. Symptoms of bladder irritability include frequency, urgency, dysuria and nocturia. Urinary retention, hydronephrosis, azotemia and worsening obstructive symptoms are indications for treatment. In addition to catheter drainage or surgical resection, new treatment options include the use of alpha-adrenergic blockers and antiandrogens, as well as balloon dilatation of the prostate. Transurethral resection of the prostate remains the mainstay of treatment, providing effective relief in 85 percent of patients. Since only the enlarged portion of the prostate is removed, prostatic cancer or recurrence of benign prostatic hypertrophy is possible. PMID- 1711772 TI - Morphological innervation pattern of the developing rabbit heart. AB - The morphological innervation pattern of developing fetal and neonatal rabbit hearts was delineated histochemically by a cholinesterase/silver procedure and immunohistochemically with the monoclonal antibody HNK1, an antibody which recognizes some cells derived from neuroectoderm. Cholinesterase-containing nerves appeared distally on the outflow tract by gestational day 15 (G15). Isolated cells with cholinesterase-stained fine processes were present near the base of the pulmonary trunk. HNK1 antibody stained the same nerves and ganglia revealed by the cholinesterase reaction and other nerves in the rabbit heart. It was used to confirm that cells with fine neuron-like processes were present before nerve ingrowth. The G14 heart contained many HNK1 staining cells in the right atrium, outflow, and inflow tracts; cells with fine processes were few but increased at G16. By G17, a plexus of interweaving nerves and associated cells began to form at the base of the pulmonary trunk. Fine nerves encircled the base of the aorta, and others crossed the intercaval region dorsally. At G19, nerves 1) extended downward from a rich "bulbar" plexus along the front ventricular surface, 2) grew near the epicardial surface at the base of the heart along the atrial floor and ventricular roof, 3) traversed the vena cavae and intercaval region to enter the atrial roof, and 4) crossed the coronary sinus to reach the back ventricular walls. By G23, cholinesterase-staining nerves and ganglia in the atria and, epicardially, in the ventricles formed the general innervation pattern of the newborn and adult rabbit heart. PMID- 1711773 TI - Neutropenia in an extremely premature infant treated with recombinant human granulocyte colony-stimulating factor. AB - Neutropenia in the newborn is often associated with sepsis, maternal hypertension, or prematurity. We describe a 654-g infant born at 30 weeks' gestation by cesarean section due to severe maternal hypertension. His course was complicated by five episodes of sepsis, including three with group B streptococcus. The results of hematologic and immunologic studies were normal except that absolute neutrophil counts were low (less than 1 x 10(9)/L) with intermittent increases during sepsis. Human recombinant granulocyte colony stimulating factor administered subcutaneously (10 micrograms/kg per day initially) resulted in an absolute neutrophil count of greater than 30 x 10(9)/L within 2 weeks. The dosage was lowered and the absolute neutrophil counts were maintained at 8 to 12 x 10(9)/L with no further septic episodes. The human recombinant granulocyte colony-stimulating factor therapy was discontinued after 7 months, and the patient remained healthy with an absolute neutrophil count of greater than 2 x 10(9)/L. Thus, treatment with human recombinant granulocyte colony-stimulating factor may be useful as a temporary measure for neonatal neutropenia associated with sepsis. A controlled, clinical trial is warranted. PMID- 1711774 TI - Predictors of neurodevelopmental outcome following bronchopulmonary dysplasia. AB - In infants with bronchopulmonary dysplasia, the influence of the severity of their pulmonary disease on neurodevelopmental outcome is unknown. Neurodevelopmental outcomes at a mean age of 36 months were assessed in 27 premature subjects who had bronchopulmonary dysplasia. Subjects had a mean birth weight of 940 g (range, 540 to 1690 g) and a mean gestational age of 27 weeks (range, 25 to 31 weeks). The duration of mechanical ventilation ranged from 22 to 128 days, and the duration of requirement of supplemental oxygen ranged from 34 to 1033 days. No significant correlations were found between duration of mechanical ventilation or oxygen therapy and overall neurodevelopmental outcome. In contrast, cranial ultrasound findings of intracranial hemorrhage and/or periventricular echodensity related specifically to poorer cognitive outcome. By age 3 years, severity of bronchopulmonary dysplasia is not a sufficient predictor of neurodevelopmental outcome. Intracranial hemorrhage and periventricular echodensity continue to be important predictors. PMID- 1711775 TI - Lectin-reactive patterns of markedly elevated serum alpha-fetoprotein in patients with chronic active hepatitis. AB - Four cases of chronic hepatitis associated with high serum levels of alpha fetoprotein (AFP) without hepatocellular carcinoma are reported. All showed transient elevations of serum AFP, with peak levels of 13,500, 8,000, 4,450, and 3,000 ng/ml shortly after aggravation resulting from liver function tests. Liver biopsies revealed severe parenchymal damage in all the cases with piece-meal necrosis, bridging necrosis or bridging fibrosis. In two of four cases, there was a lobular distortion. AFP stain by an immunoperoxidase method showed a positive result in surviving hepatocytes. Lectin affinity electrophoresis of AFP in the four cases, together with an additional 12 patients with chronic hepatitis and cirrhosis and 44 patients with hepatocellular carcinoma, all having AFP levels above 1,000 ng/ml, revealed that the chronic hepatitis patients had a benign pattern of AFP bands, in contrast with the pattern of hepatocellular carcinoma with increased proportions of lentil lectin-reactive AFP-L3 and/or erythroagglutinating phytohemagglutinin-reactive AFP-P4, indicating that the analysis of lectin reactivity of AFP has a great value in differentiating the benign and malignant conditions with increased serum levels of AFP above 1,000 ng/ml. PMID- 1711776 TI - Regulation of epithelial chloride channels by protein phosphatase. AB - A combination of planar bilayer and patch-clamp techniques was used to determine whether apical membrane Cl- channels of shark (Squalus acanthias) rectal gland (SRG) were regulated by a phosphorylating and dephosphorylating cycle. In channel reconstitution studies, apical membrane vesicles of SRG were purified, incubated in ATP-Mg2+ and the presence or absence (control) of catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAMP-PK) and incorporated into planar lipid bilayers. In the presence of cAMP-PK, two distinct Cl- channels were found when imposing either 450/50 or 300/50 mM KCl (cis/trans) gradients. The most frequently observed channels (G beta 1) were open greater than 80% at all potentials between -60 and +20 mV (trans ground) and were inactivated by alkaline phosphatase added to the cis chamber. The single-channel conductance of G beta 1 was 42 pS between -60 and +20 mV with a 300/50 mM KCl gradient. The second channel (G beta 2) was always observed in pairs of 62-pS subchannels and was not affected by alkaline phosphatase, but the open probability increased with depolarizing potentials. G beta 2 was observed once, but G beta 1 was never observed in the absence of cAMP-PK. In parallel patch clamp studies of the apical membrane of cultured SRG, a 50-pS channel similar to G beta 1 was noted after incubating cells with either forskolin, an activator of adenylate cyclase, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A. It is concluded that G beta 1 of SRG can be studied in both patch-clamp and bilayer preparations and that G beta 1 is regulated by reversible phosphorylation by cAMP-PK and dephosphorylation by a protein phosphatase. PMID- 1711777 TI - cAMP-dependent protein kinase regulates renal epithelial cell properties. AB - Previous studies have implicated adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase (PKA) in regulation of both growth and expression of differentiated function in the pig renal epithelial cell, LLC-PK1. To investigate this possible regulatory mechanism, we compared growth behavior, morphology, and appearance of two differentiated functions, Na-hexose symport (SYMP) and gamma glutamyl transpeptidase (gamma-GT), in the LLC-PK1 line and two PKA-deficient mutants (FIB4 and FIB6). Compared with the wild-type cell line, the mutant lines continued to proliferate at higher population densities and exhibited altered cell morphology, poorer formation of the brush-border structure, and decreased or lack of expression of SYMP and gamma-GT activities. Wild-type and mutant cells exhibit an identical logarithmic growth rate. Both lines form cell-cell junctions and exhibit identical kinetic properties of expressed SYMP activity. These results strongly support the hypothesis that PKA modulates a defined subset of cellular processes, including aspects of growth control and expression of the differentiated phenotype, in this renal epithelial cell line. PMID- 1711778 TI - Responses of protein synthesis in different skeletal muscles to fasting and insulin in rats. AB - The rate of protein synthesis (Ks) was measured in nine skeletal muscles from young rats (100 g body wt) after fasting and subsequent insulin infusion. An overnight fast (12 h) resulted in a fall in Ks in all muscles, but this ranged from a small nonsignificant decrease in soleus (Sol) to one-half of the fed rate in tensor fascia latae (TFL). After fasting for 36 h, Ks was significantly reduced in all muscles, although there was still a range apparent between different muscles. The response of the individual muscles to fasting was related to fiber type composition, and, in particular, the responses of the commonly studied Sol were characteristic of the highly oxidative fiber composition rather than being peculiar to Sol itself. The magnitude of the increase in Ks during an infusion of insulin (0.5 h) in the 12-h fasted rats was proportional to the magnitude of the initial response to fasting and these changes were largely reflected in changes in the rate of synthesis expressed per unit RNA (KRNA). After 36 h of fasting, Ks in the muscles responded little to the insulin stimulus, and a similar small effect on KRNA, compared with that in the 12-h fasted animals, suggests an apparent insensitivity to insulin after this longer fasting period. PMID- 1711779 TI - Intracellular mediators of bombesin action on rat pancreatic acinar cells. AB - The effects of bombesin on physiological responses (amylase secretion, protein synthesis) and intracellular mediators [inositol 1,4,5-trisphosphate (1,4,5-IP3), [Ca2+]i, and diacylglycerol] were studied in isolated rat pancreatic acini and compared with the actions of cholecystokinin (CCK). Bombesin stimulated amylase secretion to the same extent as CCK. However, it failed to reproduce the inhibition of amylase secretion by high concentrations of CCK and likewise did not inhibit incorporation of [3H]leucine into protein in contrast to high concentrations of CCK. Low concentrations of bombesin (1-100 pM) induced repetitive oscillations in [Ca2+]i, whereas higher concentrations of bombesin (1 10 nM) induced a large transient increase in [Ca2+]i followed by a small sustained plateau. Bombesin (1-100 nM) induced an early peak of 1,4,5-IP3 at 5-15 s but was without measurable effect at lower concentrations. These effects on [Ca2+]i and 1,4,5-IP3 were similar to those seen with CCK except that bombesin was approximately 10-fold less potent than CCK. Bombesin induced an increase in acinar 1,2-diacylglycerol with a biphasic time course similar to CCK. However, the magnitude of the response to bombesin was much smaller than the response to CCK. The results suggest that bombesin receptors initiate similar intracellular messengers as does CCK. However, CCK induces a larger increase of diacylglycerol and probably an as yet unidentified messenger responsible for its inhibitory effects. PMID- 1711780 TI - CCK-B (gastrin) receptor regulates gastric histamine release and acid secretion. AB - To study the interdependence between gastric histamine release and acid secretion, we examined the effects of gastrin-(1-17) [G-(1-17)] or cholecystokinin-(1-33) [CCK-(1-33)] alone or combined with the gastrin (CCK-B) antagonist L365,260 or the CCK-A antagonist L364,718 in the isolated vascularly perfused rat stomach. G-(1-17) and CCK-(1-33) gave concentration-dependent increases in acid secretion and histamine release. Gastrin or CCK-A antagonist alone did not stimulate histamine release or acid secretion. Maximally G-(1-17) or CCK-(1-33) stimulated histamine release and acid secretion was unchanged by the CCK-A antagonist, while the gastrin antagonist induced a parallel and concentration-dependent decrease in stimulated histamine and acid secretion. We conclude that G-(1-17) and CCK-(1-33) stimulate histamine and acid secretion by a CCK-B (gastrin) receptor. The present results indicate that gastrin, at least in this species, stimulates acid secretion by releasing histamine. PMID- 1711781 TI - Expression of a liver fatty acid binding protein/human decay-accelerating factor/HLA-B44 chimeric gene in transgenic mice. AB - The intestinal epithelium is characterized by the rapid and continuous renewal of its four principal cell types and by its ability to establish and maintain remarkably complex spatial differentiation along its crypt-to-villus and duodenal to-colonic axes. We have previously used transgenic mice containing liver fatty acid binding protein/human growth hormone (L-FABP/hGH) fusion genes to analyze the molecular mechanisms responsible for encoding positional information in this epithelium. Because these studies could not distinguish whether cis-acting sequences in the L-FABP promoter or hGH structural gene were responsible for the observed cellular and regional patterns of transgene transcription in the gut, a second model fusion gene has now been constructed. It consists of nucleotides 596 to +21 of rat L-FABP linked to a cDNA encoding a chimeric protein, human decay-accelerating factor (DAF, minus the site of attachment of its COOH-terminal glycophospholipid anchor), coupled to the transmembrane (TM) and cytoplasmic domains of human HLA-B44. RNA blot hybridization and immunocytochemical analyses revealed that the cell-specific and region-specific expressions of DAF-TM and hGH in adult mice appear identical along both axes of the gut, indicating that cis acting elements contained within the 5' nontranscribed region of the L-FABP gene rather than in the reporter are largely responsible for these observed patterns of transgene expression. Unlike pre-hGH, a prototypical secreted protein, DAF-TM is a membrane protein. The ability to direct its expression along the length of both axes of the gut provides an opportunity to analyze in vivo the sorting pathways of membrane-associated proteins in normal epithelial cells as a function of their location and differentiation. Light microscopic studies indicate that DAF-TM is targeted to the basolateral and apical surfaces of villus-associated enterocytes. PMID- 1711782 TI - GLP-1-(7-36) amide, -(1-37), and -(1-36) amide: potent cAMP-dependent stimuli of rat parietal cell function. AB - We investigated the effect of glucagon-like peptide 1 (GLP-1)-(7-36) amide and its molecular variants GLP-1-(1-37) and GLP-1-(1-36) amide on enzymatically dispersed enriched rat parietal cells using [14C]aminopyrine accumulation as a measure of H+ production. GLP-1-(7-36) amide was 100 times more potent than GLP-1 (1-37) and GLP-1-(1-36) amide in stimulating [14C]aminopyrine accumulation. At their maximally effective concentrations, GLP-1-(7-36) amide (10(-8) M), GLP-1-(1 37) (10(-6) M), and GLP-1-(1-36) amide (10(-6) M) reached 80-90% of the response to 10(-4) M histamine. However, the peptides were 100-10,000 times more potent than histamine, which induced maximal [14C]aminopyrine accumulation at 10(-4) M. Stimulation by GLP-1 was dependent on the presence of a phosphodiesterase inhibitor and was not altered by pertussis toxin. Ranitidine failed to affect the response to the GLP-1 variants. Stimulation of H+ production by GLP-1 was accompanied by an increase in the formation of adenosine 3',5'-cyclic monophosphate (cAMP) but not by changes in phosphoinositol breakdown. In stimulating [14C]aminopyrine accumulation, the GLP-1 variants acted additively to threshold but not to maximal concentrations of histamine, suggesting that histamine and GLP-1 activate the same cAMP pool. In contrast, in anesthetized rats GLP-1-(7-36) amide (10-500 ng.kg-1.h-1) had no effect on basal and pentagastrin-stimulated acid secretion in vivo. We conclude that GLP-1 exerts a direct stimulatory effect on rat parietal cells. This potent effect is mediated by cAMP and is independent of H2 receptors. In vivo direct stimulation by GLP-1 of the parietal cells might be counterbalanced by indirect inhibitory mechanisms that are excluded in the in vitro cell system. PMID- 1711783 TI - Immunohistochemistry of central respiratory neurotransmitters. AB - Significant progress has been made in identifying the nuclei and pathways involved in central control of respiration in mammals. This knowledge provides a framework from which to focus on the question of which putative neurotransmitters may be involved in central respiratory control. Application of chemical neuroanatomical methods, particularly immunohistochemistry, is a powerful approach to this important question. This commentary discusses how immunohistochemical approaches have been used so far to extend our knowledge of central respiratory neurotransmitters and also how they can be further applied to gain new information. The results of immunohistochemical studies provide a rationale for subsequent physiological and pharmacological studies of respiratory neurons where specific actions of putative neurotransmitters can be evaluated. PMID- 1711784 TI - Identification and structural characterization of Fc gamma-receptors on pulmonary alveolar macrophages. AB - Little is known about the structure of the cell surface receptors for the Fc portion of immunoglobulin G (Fc gamma R) on tissue macrophages. Studies on leukocytes indicate the existence of three classes of Fc gamma R, denoted I, II, and III. The purpose of this study was to structurally characterize Fc gamma R on alveolar macrophages obtained by bronchoalveolar lavage, comparing them with Fc gamma R of monocytes, neutrophils, and U937 cells. Flow-cytometric evaluation, utilizing anti-Fc gamma R class-specific monoclonal antibodies, showed that alveolar macrophages expressed three Fc gamma R classes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the immunopurified Fc gamma R molecules revealed the following apparent molecular mass for each Fc gamma R class: Fc gamma RI, 70 kDa; Fc gamma RII, 44 kDa; and Fc gamma RIII, 57-65 kDa. RNA blot analysis demonstrated a 1.7-kb transcript for Fc gamma RI, 2.5 and 1.6 kb transcripts for Fc gamma RII, and a 2.2 kb mRNA for Fc gamma RIII. Fc gamma RII displayed the high-responder/low-responder polymorphism. Fc gamma RIII did not express the neutrophil antigen-type specific structural polymorphism of the deglycosylated Fc gamma R molecule and appeared to be a transmembrane molecule. PMID- 1711785 TI - Perinatal rat lung catalase gene expression: influence of corticosteroid and hyperoxia. AB - Dexamethasone accelerates the late gestational rise in rat lung catalase activity; neonatal hyperoxia elevates rat lung catalase activity. We studied the regulation of catalase gene expression in these instances. Catalase mRNA/mg DNA increased to gestation day 22 and then fell to the concentration in adult lungs. The rate of transcription of catalase mRNA was higher on gestation day 22 than gestation day 19, whereas the half-life of catalase mRNA (approximately 7 h) was the same on both days. Dexamethasone given 48 and 24 h before expected birth (gestation 22 days) increased catalase mRNA concentration at days 20 and 22 without a change in catalase mRNA stability. Early postnatal hyperoxia (greater than 95% O2, 72 h) elevated catalase mRNA/mg DNA and doubled its half-life without changing its rate of transcription. We conclude the normal late gestational elevation of catalase activity and the increase of activity during prenatal dexamethasone treatment are regulated at the level of gene transcription. By contrast, the elevation of catalase activity during neonatal hyperoxia is mediated posttranscriptionally by increased catalase mRNA stability. PMID- 1711786 TI - Developing fetal alveolar epithelial cells secrete fluid in primary culture. AB - The developing pulmonary epithelium secretes a Cl(-)-rich fluid during fetal life by a process involving active transport. To determine if alveolar cells contribute to this fluid production, we studied developing fetal rat alveolar epithelial cells (18-day gestation) in primary culture. Fetal alveolar epithelial cells aggregated to form cystic, alveolar-like structures with a fluid-filled lumen. On light and transmission electron microscopy, the cells were polarized with microvilli facing the lumen. Dexamethasone and triiodothyronine stimulated lamellar body production in many of the epithelial cells of the cyst wall. The transepithelial voltage in the cyst was -2.4 +/- 0.4 mV (lumen negative), suggesting the presence of active electrolyte transport. Bumetanide, an inhibitor of Cl- secretion in other systems, decreased the size and number of cysts. A membrane-permeant analogue of adenosine 3',5'-cyclic monophosphate (cAMP) and 3 isobutyl-1-methylxanthine increased the size of cysts, an effect that was blocked by coincubation with bumetanide. The increased size of the cysts did not result from stimulation of cell growth; in fact, [3H]thymidine incorporation was inhibited to 40% control values by cAMP, suggesting that growth was inhibited rather than stimulated. These results suggest that fetal alveolar epithelial cells secrete fluid via a cAMP-mediated Cl(-)-secretory process. Secretion of fluid by the developing alveolar epithelium may play an important role in lung development. PMID- 1711787 TI - Syncytial organization of cultured rat mesangial cells. AB - To investigate communication competence of cultured rat mesangial cells, Lucifer yellow transfer was studied using microinjection and scrape-loading techniques. Both methods yielded results indicating considerable gap junctional communication between cultured mesangial cells. Gap junctional communication between mesangial cells was upregulated by adenosine 3',5'-cyclic monophosphate (cAMP). Conversely, cell-to-cell communication was attenuated by exposure to the tumor promoter phorbol myristate acetate, the Ca ionophore ionomycin, reduced oxygen intermediates, and cell acidification. Expression of voltage gated calcium channels by mesangial cells was studied microspectrofluorimetrically using fura-2 fluorescence. KCl-induced depolarization, BAY-K 8644, and readdition of calcium to Ca-free depolarizing medium all produced a nifedipine-inhibitable increase in cytosolic calcium concentration. The existence of voltage-gated calcium channels in communication-competent cells suggests the possibility of propagation of depolarizing signals across the syncytium. This was studied by microapplication of KCl to the microenvironment of a single cell and monitoring fura-2 fluorescence in remote cells. This maneuver resulted in propagating calcium waves in communication-competent monolayers; calcium waves could not be evoked in monolayers exposed to an alkanol-type gap junction uncoupler, octanol. It is concluded that cultured rat mesangial cells form a syncytium capable of propagating calcium transients from a single depolarized cell to its coupled neighbors. PMID- 1711788 TI - Calcium-activated potassium channels from coronary smooth muscle reconstituted in lipid bilayers. AB - This work is the initial characterization of Ca(2+)-activated K+ (KCa) channels from coronary smooth muscle reconstituted into lipid bilayers. The channels were obtained from a surface membrane preparation of porcine coronary smooth muscle. KCa channels were the predominant K+ channels in this preparation. The conductance histogram (n = 137 channels) revealed two main populations of "maxi" KCa channels with conductances of 245 and 295 pS. Each population could be subdivided in two "isoforms" or "isochannels" with different functional properties (voltage and Ca2+ sensitivities and kinetics). The analysis of "burst" probability of opening showed that at pCa 4 the two isochannels of 245 pS (KCa-1 and KCa-1') had half-activation potentials (V1/2) of -80 and 6 mV, respectively. The isochannels of 295 pS (KCa-2 and KCa-2') had V1/2 of -28 and -66 mV, respectively. KCa-1 had the highest Ca2+ sensitivity; at -60 mV, the concentration of half-activation value for Ca2+ was 1.2 +/- 0.3 microM (n = 5). External tetraethylammonium reduced channel amplitude in a voltage-dependent manner; dissociation constant was 180 +/- 6 and 466 +/- 41 microM at -40 and +80 mV, respectively (n = 5). Charybdotoxin (5-50 nM) produced typical long closings. These effects were similar in all the channels. We conclude that coronary smooth muscle possesses isoforms of maxi KCa channels with Ca2+ and voltage sensors with different properties, which may confer to each channel a specific functional role. PMID- 1711789 TI - Limited cardiac preload reserve in conscious cirrhotic rats. AB - In conscious rats with experimental cirrhosis without ascites, we have studied whether there is a limited cardiac preload reserve by performing cardiac output (CO) curves. CO was determined by thermodilution at basal, 5, 7.5, and 10 cmH2O of right atrial pressure (RAP). RAP was elevated by dextran infusion (1 ml/min iv, 30 min). CO curves were performed by plotting changes in CO with changes in RAP. In the basal state, cirrhotic rats showed a hyperdynamic circulation defined by increased CO and stroke volume, decreased total peripheral resistances, and normotension without changes in heart rate. Blood volume was also elevated in cirrhotic rats compared with the control animals. Between the limits of RAP studied, the CO curve of control rats presented a typical ascending limb. In contrast, the CO curve of the cirrhotic animals showed first an ascending shifted upward limb and afterward a descending limb. These alterations were accompanied by changes in the inotropic state as measured as left ventricular (LV) peak dP/dt in hexamethonium-pretreated animals submitted to the same volume loads described above. With the same increases in RAP, LV dP/dt changed, in every group, in a manner similar to CO. The results of the present study indicate that cirrhotic rats with high blood volume and hyperdynamic circulation show, in the steady state, a limited preload reserve. The partial utilization of the preload reserve can make the cirrhotic heart unable to modulate cardiac performance with changes in loading conditions, thus determining a state of heart failure. PMID- 1711790 TI - Increased negativity of interstitial fluid pressure during the onset stage of inflammatory edema in rat skin. AB - Interstitial fluid pressure (Pif) was measured in skin of pentobarbital anesthetized rats during anaphylaxis toward dextran and after subdermal injection of histamine by using sharpened glass capillaries (tip diam 5-7 microns) connected to a servo-controlled counterpressure system. Control Pif averaged -1.5 mmHg (SD = 1.0). After intravenous dextran Pif in the rat paw fell transiently to -3 mmHg up to 20 min and thereafter increased to +1-2 mmHg when edema had developed. To study the full magnitude of the increased negativity of Pif, circulatory arrest was induced 1 min after dextran injection. This procedure prevents accumulation of edema that will cause underestimation of Pif. In this group Pif fell to about -10 mmHg in 20 min and remained at this level throughout the observation period of 90 min. Subdermal injection of 1-10 micrograms histamine in 10 microliters saline reduced Pif to about -6 mmHg within 5 min after injection. Injection of 10 microliters saline increased Pif by +2 mmHg. Indomethacin or cyproheptadine did not alter the response in the above situations. The increased negativity in Pif of about 6-8 mmHg will add directly to normal transcapillary net filtration pressure of 0.5 mmHg and increase the latter pressure 10-20 times. PMID- 1711791 TI - Undergraduate teaching project of the American gastroenterological Association: a pathophysiology resource. AB - The Undergraduate Teaching Project was founded as a committee of the American Gastroenterological Association to improve medical school teaching of the pathophysiological basis of gastrointestinal and liver diseases. Twenty-six units of colored illustrations have been produced in 18 years, which are designed to be used by instructors to supplement their own material. The slides are the result of extensive reviews and revisions, a process that has created a unique repository of educational concepts utilized by most US medical schools and over 50 foreign schools. This brief summary of the genesis and operation of this venture may be of interest to other professional societies concerned with the development of teaching materials. PMID- 1711792 TI - Undifferentiated (embryonal) sarcoma of the liver. A tumor of uncertain histogenesis showing divergent differentiation. AB - Pathologic features of eight cases of undifferentiated (embryonal) sarcoma of the liver (USL) in childhood were studied. Light microscopic examination showed a diffuse growth of spindle cells with occasional polygonal cells and multinucleated giant cells and also revealed focal areas of storiform pattern in four tumors, cambium layer formation in one tumor, and alveolar arrangement in one tumor. Immunohistochemical study showed positive staining of proliferating cells for suggestive histiocytic markers (A1AT in 6/6, A1ACT in 5/6, lysozyme in 4/6, and KP1 in 4/6) and for muscle markers (desmin in 4/6 and HHF35 in 3/6). Ultrastructural examination demonstrated that the individual tumors were composed of a mixture of cells having fibroblastic, histiocytoid, fibrohistiocytoid, myofibroblastic, and undifferentiated (primitive mesenchymal) morphologies. Also identified were cells with definite myoblastic morphology in three tumors: leiomyoblastic in one and rhabdomyoblastic in two. In conclusion, the tumor cells in USL show phenotypical diversity comparable to those of malignant fibrous histiocytoma with or without additional rhabdomyosarcomatous or leiomyosarcomatous differentiation. PMID- 1711793 TI - Clear cell tumor of the lung. Immunohistochemical and ultrastructural evidence of melanogenesis. AB - Clear cell tumors of the lung (CCTL) are rare neoplasms of uncertain differentiation. A previous study of eight CCTL demonstrated a lack of epithelial features, but their exact nature remained unknown. In the current study of nine CCTL, immunohistochemistry using preliminary enzymatic digestion showed strong reactivity with the antimelanocytic markers HMB-45 (seven cases) and HMB-50 (six cases) and focal positivity for S-100 (nine cases), neuron-specific enolase (three cases), synaptophysin (one case), and Leu-7 (one case). Staining for cytokeratin, epithelial membrane antigen, chromogranin, and glial fibrillary acid protein was uniformly negative. Frozen-section immunoreactivity for vimentin and the antimelanocytic monoclonal preparation NKI/BETEB was noted in the one CCTL for which snap-frozen tissue was available. Ultrastructural examination of three glutaraldehyde-fixed CCTL showed rare neoplastic cells containing the full spectrum of melanosomes in two, one of which also contained neurosecretory-type granules. Aberrant melanosomal forms were identified in the third case. Melanosomes were not identified in the remaining five CCTL studied from formalin- or paraffin-retrieved material. The findings indicate that CCTL exhibits melanocytic differentiation. This feature may be of considerable value in distinguishing CCTL from other clear cell neoplasms. PMID- 1711794 TI - Sclerosing adenosis of the prostate. Histopathologic and immunohistochemical analysis. AB - A prostatic lesion, histologically identical to sclerosing adenosis of the breast, was found in five (1.9%) of 263 patients who underwent transurethral resection, open prostatic adenectomy, radical prostatectomy, or total cystoprostatectomy. This uncommon lesion was a localized proliferation of crowded small glands, small solid nests, and individual cells embedded in a cellular stroma, mimicking a small acinar prostatic adenocarcinoma. The proliferating glands were lined by a single layer of secretory cells surrounded by an eosinophilic membranous structure. Basal cells were disclosed in individual glands or as small nests and even individual cells with immunostainability for basal cell-specific cytokeratin (EAB903), S-100 protein, and muscle-specific actin (HHF35). These findings indicate the benign nature of the lesion with myoepithelial differentiation of the basal cells. In contrast, all 25 small acinar adenocarcinomas examined as controls lacked positive stains for the above three antibodies, verifying the usefulness of these antibodies to distinguish between this benign lesion from adenocarcinoma. PMID- 1711795 TI - Spindle-cell argyrophilic mucin-producing carcinoma of the breast. Histological, ultrastructural, and immunohistochemical studies of two cases. AB - We report two cases of neuroendocrine carcinomas of the breast displaying unusual histological features: numerous spindle cells and argyrophilic signet-ring cells. Both patients were older than 70 years, and both presented with a bloody nipple discharge. The tumor in both cases was predominantly intraductal. The tumor cells showed little pleomorphism or cytological atypia; because of the presence of spindle cells, benign diagnoses, such as ductal epithelial hyperplasia and intraductal papilloma, were considered for the in situ component. Recognition of the palisading arrangement of the peripheral cells, intracytoplasmic lumina, mitotic figures, and mucin permitted the diagnosis of intraductal carcinoma. Invasive nests composed of identical cells confirmed the diagnosis of malignancy in both cases. Our cases, along with those previously reported, suggest that neuroendocrine carcinoma with mucin production is a distinct breast tumor that usually occurs in older patients who experience bloody nipple discharge. The prognosis may be more favorable than that of the usual type of breast carcinoma. Common histological features include predominantly intraductal growth, an absence of desmoplasia, and low-grade atypia. Awareness of morphological variants of this tumor, such as those reported here, is necessary to avoid erroneous diagnoses. PMID- 1711796 TI - [Effect of trental and rheopolyglucine infusions on the temperature of female rabbits and their fetuses (normally growing and with growth retardation)]. PMID- 1711797 TI - HIV antigen-induced release of histamine from basophils from HIV infected patients. Mechanism and relation to disease progression and immunodeficiency. AB - Basophil leukocytes from 39 HIV-infected patients with various degrees of immunodeficiency and disease progression were stimulated with an HIV antigen preparation. Cells from 19 of 22 patients with AIDS and all of six patients with milder degrees of HIV-related disease showed significant histamine release. In contrast, cells from 11 asymptomatic HIV-infected patients and 11 healthy control persons released no histamine. The histamine release induced by HIV antigen was found to be inversely correlated to the number of CD4 positive T lymphocytes. These results indicate that the histamine release was related to both the clinical stage of disease and the degree of immunodeficiency. Passive sensitization experiments showed that IgE, but not IgG, was responsible for the induction of histamine release, indicating the reaction to be type 1 allergic. The histamine release caused by HIV might be involved in the development of disease because of the immunomodulating properties of this mediator. PMID- 1711799 TI - [PSA and surveillance of radical prostatectomy in cancer. Value of a technique with a low detection limit]. PMID- 1711798 TI - Identification of mast cells in bronchoalveolar lavage fluid. Comparison between different fixation and staining methods. AB - The present study was carried out to compare the effectiveness of different fixation and staining methods in the identification of mast cells obtained by bronchoalveolar lavage from patients with interstitial lung diseases. Cell preparations were fixed with formaldehyde or methanol. Mast cells were identified by metachromatic staining with May Grunwald Giemsa, Toluidine blue or Gallamine blue Giemsa. After formaldehyde fixation only a few mast cells were identified, regardless of the stain method used. In contrast, a significantly higher number of mast cells were observed after methanol fixation. Using this fixative, Toluidine blue stain showed a higher number of mast cells than May Grunwald Giemsa. However, there was no difference between Toluidine and Gallamine blue Giemsa in the number of cells observed. The easy identification of mast cells after staining with toluidine, combined with its easy application, suggest that Toluidine blue stain after methanol fixation is the most useful method for determining the presence of mast cells in lavage fluid. PMID- 1711800 TI - [Fibrinolytic activity in traumatic hemothorax fluids]. AB - Twenty-nine patients, 15 to 85-year-old (mean: 50 years) who presented with a pleural effusion after trauma were studied. The blood content of pleural fluid was confirmed by thoracocentesis. None of the patients had been taking anticoagulant drugs during the fortnight preceding the trauma. Thoracocentesis was always carried out less than 90 min after the trauma. The following parameters were measured in the haemothorax liquid samples: clotting fibrinogen fraction (Fg C), fibrin degradation D-dimers, functional plasminogen, alpha 2 antiplasmin, alpha 2-macroglobulin, plasminogen tissue activator (tPA Ag), type 1 tPA plasma inhibitor (PAI), and haematocrit. Haemothorax liquid haematocrit values ranged from 13 to 35% (25 +/- 7%, with a mean peripheral venous haematocrit of 34 +/- 6%). Only three patients had some Fg C (0.05-0.13 g.l-1). The D-dimer level was very high (0.23 +/- 0.22 g.l-1). The other factors involved in fibrinolysis were also present. Moreover, there was a statistically significant inverse correlation between D-dimer and alpha 2-macroglobulin levels (r = -0.64, p less than 0.0025). These data suggest two possible mechanisms to explain the fibrinogen levels: coagulation is activated, followed by an important fibrinolytic reaction elicited by the large amounts of plasminogen and tPA present in the haemothorax liquid. PMID- 1711801 TI - Cellular mechanisms of anesthesia. PMID- 1711802 TI - Molecular and Cellular Mechanisms of Alcohol and Anesthetics. A conference. Calgary, Alberta, Canada, June 25-28, 1990. PMID- 1711803 TI - Mechanism of volatile anesthetic action on ion channels. AB - Single channel recording techniques have been used to study effects of halothane and isoflurane on the properties of nicotinic channels activated by 250 nM acetylcholine in cell-attached patches of BC3H1 mouse tumor cells grown in culture. Halothane and isoflurane were diluted in air and delivered as vapors in the atmosphere above the cells. Both halothane and isoflurane shortened the duration of individual opening events and caused openings to appear grouped together in bursts. The slower time constant of channel open-time distributions was decreased 50% by approximately 0.25% isoflurane (0.12 mM) or 0.30% halothane (0.15 mM) at room temperature. Total open time per burst was also decreased by each agent, an effect that is not consistent with a sequential channel blocking model. Effects of halothane and isoflurane can be explained by a sequential blocking model only if anesthetics also enhance the rate at which open channels normally close. Alternatively, results are also consistent with a cyclic blocking model in which blocked channels may close directly without having to pass back through the open state. PMID- 1711804 TI - Allosteric actions of central nervous system depressants including anesthetics on subtypes of the inhibitory gamma-aminobutyric acidA receptor-chloride channel complex. PMID- 1711805 TI - General anesthetic action on gamma-aminobutyric acid-activated channels. PMID- 1711807 TI - Effects of alcohols on ion channels of cultured neurons. PMID- 1711806 TI - Structure and modulation of voltage-gated ion channels. PMID- 1711808 TI - Isoflurane depresses both glutamate- and peptide-mediated slow synaptic transmission in neonatal rat spinal cord. PMID- 1711809 TI - Actions of intermediate chain-length n-alkanols on single channel NMDA currents in rat hippocampal neurons. PMID- 1711810 TI - Effects of isoflurane on N-methyl-D-aspartate gated ion channels in cultured rat hippocampal neurons. PMID- 1711811 TI - Actions of n-alcohols on nicotinic acetylcholine receptor ion channels in cultured rat muscle cells. AB - The effects of the n-alcohols from pentanol to dodecanol on nAChR channel function were resolved at the single channel level. ACh-activated channel activity was recorded from isolated membrane patches using the patch clamp method. The intermediate-chain alcohols (C5-C8) had two main effects: (1) They caused channel openings to be interrupted by brief shut or blocked periods, the duration of which was dependent on chain length of the alcohol but independent of concentration. (2) They caused a reduction in the duration of bursts of openings. The long-chain alcohols (C9-C11) produced only the second effect, and there was a decline in activity beyond undecanol. Results were consistent with a mechanism of channel blockade and were analyzed in terms of an open channel block model with a long-lived closed-blocked state beyond the blocked state. Affinity for the binding site increased with chain length up to octanol. The standard free energy per methylene group for adsorption to the site was calculated to be -3.3 kJ/mol, indicating the very hydrophobic nature of the site. PMID- 1711812 TI - Anesthetic actions in the hippocampal formation. PMID- 1711813 TI - Role of signal transduction in anesthetic action. Alpha 2 adrenergic agonists. AB - The molecular mechanism for general anesthetic action is not known. The alpha 2 adrenergic agonists represent a novel class of "anesthetic-like" agent because of their selectivity for receptor binding sites and because the transmembrane signaling systems mediating their biologic responses in non-CNS systems are known. We have begun to characterize the signal transduction pathway involved in the anesthetic-like action of the alpha 2 adrenergic agonists. The alpha 2 adrenergic agonists potently decrease both central noradrenergic neurotransmission and halothane anesthetic requirements (MAC). Since MAC is only reduced by 30-40% when noradrenergic neurotransmission is totally abolished and since the reduction in MAC with the highly selective alpha 2 adrenergic agonists exceeds 90%, factors in addition to noradrenergic neurotransmission must be contributing to the anesthetic action of the alpha 2 agonists. Studies with the superselective alpha 2 agonist dexmedetomidine confirmed this, as the alpha 2 agonist could still reduce the MAC for halothane in rats depleted of their central norepinephrine stores. The profound reduction in anesthetic requirements with dexmedetomidine raised the possibility that alpha 2 adrenergic agonists may be considered an anesthetic hypnotic agent by itself. This sole anesthetic hypnotic response was established together with the confirmation that a central alpha 2 adrenoceptor mediated this action. Subsequently, data using molecular biologic techniques suggested that the alpha 2 C4 isoreceptor was the probable receptor that mediated the anesthetic response. We further explored the postreceptor effector mechanism for the signal transduction pathway for alpha 2 anesthetic action and identified the participation of two other molecular components, namely, a pertussis-toxin-sensitive G protein and a 4-aminopyridine sensitive ion channel. Whether the signal transduction pathway for alpha 2 anesthetic action mediates the further response to other non-alpha 2 anesthetic agents needs to be defined. PMID- 1711814 TI - Sensitivity of N-methyl-D-aspartate (NMDA) and nicotinic acetylcholine receptors to ethanol and pyrazole. PMID- 1711815 TI - GABAA receptor function and expression following chronic ethanol and barbiturate administration. PMID- 1711816 TI - The nicotinic acetylcholine receptor in its membrane environment. PMID- 1711817 TI - Actions of volatile anesthetics and alcohols on cholinergic receptor channels. PMID- 1711818 TI - A common binding site for channel-permeant cations and local anesthetics on the nicotinic acetylcholine receptor. PMID- 1711819 TI - Increased permeability of the gut to polyethylene glycol and dextran in rats fed alcohol. PMID- 1711820 TI - Viewing anesthesia research 1954-1990. PMID- 1711821 TI - Alcohol and anesthetic actions on excitatory amino acid-activated ion channels. AB - The actions of alcohol and anesthetics have been studied on excitatory amino acid activated ion channels in mammalian neurons. Ethanol inhibits NMDA-activated current over a concentration range that produces intoxication, and the potency of several alcohols for inhibiting the NMDA-activated current is correlated with their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication. Studies on the mechanism of ethanol inhibition of NMDA-activated current indicate that ethanol does not appear to block the ion channel, alter the ion selectivity of the channel, or interact with previously described binding sites on the NMDA receptor/ionophore complex. The linear relation between the potency of several alcohols for inhibiting the NMDA activated current and the hydrophobicity of the alcohols suggests that ethanol may inhibit the NMDA-activated ion current by a novel type of interaction with a hydrophobic site associated with the NMDA channel. In addition, different types of general anesthetic agents exhibit different inhibitory actions on NMDA-, kainate-, and quisqualate-activated currents, suggesting that differences in the profile of inhibition of excitatory amino acid neurotransmission in the CNS among different classes of general anesthetics may contribute to the differences in their behavioral and physiological effects. PMID- 1711822 TI - Insulin-like growth factor I as an intraovarian regulator: basic and clinical implications. AB - Although much remains to be learned with respect to the possible relevance of IGF I to ovarian physiology, it may be possible at this time to tentatively formulate possible functions of IGF-I in this connection: 1. Amplification of gonadotropin hormonal action--a key requirement given the exponential nature of follicular development. 2. Integration of follicular development--an essential facet concerned with the coordination of granulosa-theca cooperation. 3. Selection of dominant follicle(s)--a speculative proposition assuming timely and selective activation of the IGF-I system in "chosen" follicles. Aside from its possible role(s) in the course of established follicular cycles, IGF-I (and/or IGF-II) may also participate in the very formation of the follicular apparatus during the late fetal/early neonatal period. Although the ovary is gonadotropin-independent at that time, we previously showed that IGF-I may well interact with VIPergic input now implicated in the morphodifferentiation of the follicular apparatus. Similarly, IGF-I may be concerned with the promotion of juvenile and early pubertal follicular gonadotropin (FSH) levels; ovarian IGF-I may have a bearing on the puberty-promoting effect of growth hormone. Indeed, an association appears to exist between isolated growth hormone deficiency and delayed puberty in both rodents and human subjects, a process reversed by systemic growth hormone replacement therapy. Given that ovarian IGF-I and its receptor may be growth hormone-dependent, it is tempting to speculate that the ability of growth hormone to accelerate pubertal maturation may be due, at least in part, to the promotion of ovarian IGF-I production and reception with the consequent local potentiation of gonadotropin action. PMID- 1711823 TI - Insulin-like growth factor-binding protein-1 (IGFBP-1) in the human ovary. PMID- 1711824 TI - Protooncogenes in endometriotic and endometrial tissue. PMID- 1711825 TI - Molecular cloning of complementary DNAs for two human endometrial proteins and cellular localization of their messenger RNAs. AB - Insulin-like growth factor binding protein-1 (IGFBP-1) and a human beta lactoglobulin homologue (beta LG/PP14) are two major secretory proteins of the human endometrium. The genes coding for these two proteins are expressed in separate types of the endometrial cells, with the IGFBP-1 gene being expressed in the stromal and the beta LG/PP14 gene in the glandular epithelial cells. Although the biological reasons for the presence and expression of IGFBP-1 and beta LG/PP14 in human endometrial cells remain to be elucidated, the fact that these gene products are expressed in different endometrial cell types provides a unique opportunity to employ them as markers in studies on epithelial-to-stromal cell communication in the endometrium. The primary structures of these proteins have been deduced from their cloned cDNAs. beta LG/PP14 is highly homologous to all known beta-lactoglobulins from various species. For example, horse beta lactoglobulin I monomer exhibits a 53% protein sequence identity with beta LG/PP14; they have the same number of amino acid residues, and their three dimensional structures are predicted to be similar. This latter conclusion is inferred from the fact that the four cysteinyl residues that are responsible for the formation of intramolecular bridges in beta-lactoglobulins are spatially conserved in beta LG/PP14. The human protein is encoded by a 900-base pair-long mRNA that is expressed in the glandular epithelial cells of the endometrium in a cyclic manner; in addition, it is found in the mucosal epithelial cells of the fallopian tubes. Several lines of circumstantial evidence suggest that the expression of the beta LG/PP14 gene is regulated by progesterone; however, whether this regulation is elicited by the progesterone receptor at the transcriptional level has not so far been demonstrated. The IGFBP-1 protein sequence contains 259 amino acid residues, with the propeptide possessing a 25 amino acid-long signal peptide. The NH2-terminal sequence of this and other IGFBPs is very cysteine-rich, suggesting the possibility that this domain is involved in the binding of IGF-I and IGF-II ligands. A PEST region, a sequence that is found in proteins with short intracellular half-lives, is included in the middle half of the IGFBP-1 polypeptide. Among the IGFBPs, IGFBP-1 appears to be the only one with a PEST sequence. The carboxy-terminal end of IGFBP-1 contains an Arg-Gly-Asp tripeptide that is also found in IGFBP-2 and may function as a cell attachment recognition signal in these proteins.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711826 TI - Insulin-like growth factor-II (IGF-II) and IGF binding proteins in human endometrium. AB - The insulin-like growth factor autocrine/paracrine system is believed to play a role in steroid-mediated endometrial differentiation. It is constituted of the mitogenic peptides (IGF-I and IGF-II), membrane receptors, and a family of high affinity binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. We have investigated expression of the mRNAs encoding the three major IGFBPs (IGFBP-1, IGFBP-2, and IGFBP-3) in human endometrium and have found, by Northern analysis, differential expression of all three mRNAs in secretory compared to proliferative endometrium, different steroidal milieux. IGF-II mRNAs were also detected in secretory endometrium. Finally, we found that human endometrial stromal cells in culture synthesize and secrete IGFBP-2 and IGFBP-3, and that the synthesis of IGFBP-2 is regulated by steroid hormones. PMID- 1711827 TI - Endometrial proteins as local regulators of human endometrial function and their appearance in serum: clinical applications. PMID- 1711828 TI - Synthetic peptides in HIV antibody screening and typing. AB - We have defined continuous native epitopes of HIV proteins by using a systematic epitope-scanning technology. We have demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 that is immunoreactive with all studied HIV-1 antibody-positive sera. The corresponding region in HIV-2 gp34 behaves similarly. There is a clear difference, however, between HIV type 1 and type 2 transmembrane proteins in the number of highly immunoreactive regions, when presented properly as synthetic antigens in solid-phase EIA, can provide tests unusually suitable for early and reliable diagnosis of HIV-1 and HIV-2 infections and for type-specific distinction of the two types of HIV infections. PMID- 1711829 TI - Biochemistry and endocrinology of the Down's syndrome pregnancy. PMID- 1711830 TI - Alpha 2-macroglobulin: a multifunctional protein of the seminiferous tubule. PMID- 1711831 TI - Wartime nursing. One nurse's experience in Vietnam. PMID- 1711832 TI - 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA. AB - Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences from three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type. PMID- 1711833 TI - Abnormalities of T lymphocyte subsets in systemic sclerosis demonstrated with anti-CD45RA and anti-CD29 monoclonal antibodies. AB - T cell subpopulations were assessed by two colour flow cytometry with phycoerythrin conjugated anti-CD45RA and anti-CD29 and fluorescein conjugated anti-CD4 and anti-CD8 monoclonal antibodies, on peripheral blood lymphocytes from 12 patients with systemic sclerosis and from nine control subjects. The percentage of CD4+CD29+ cells was significantly higher in patients with systemic sclerosis than in controls (mean (SEM) 68.8 (3.1) v 47.9 (4.1) respectively). CD4+CD45RA+ cells were not significantly different in patients and controls. CD8+CD29+ and CD8+CD45RA+ subpopulations were significantly higher in patients with systemic sclerosis than in controls (83.0 (3.2) v 58.7 (6.8) and 80.2 (3.0) v 66.9 (3.2) respectively). The increase in the percentage of CD29+ cells suggests an activation of memory cells in patients with systemic sclerosis, which may play an important part in the pathogenesis of the disease. PMID- 1711834 TI - Retrospective comparison of iloprost with other treatments for secondary Raynaud's phenomenon. AB - One hundred and twenty seven patients who had Raynaud's attacks secondary to connective tissue disease received intravenous infusions of iloprost in controlled clinical trials. Results of previous treatments for Raynaud's attacks had been recorded by clinicians in 84 of these cases, allowing a comparison to be made with the response to iloprost treatment. Iloprost was reported by the patients as beneficial in 49 (58%) of 84 cases, whereas only 36 (43%) of the 84 patients had previously found any other treatment to be useful. Twenty four of 48 (50%) patients who had not responded to any previous treatment found iloprost to be of benefit. Success or failure of treatment with iloprost was not accurately predicted by the result of treatment with any other drug, except prostacyclin. This survey suggests that iloprost is a useful treatment for patients with severe secondary Raynaud's phenomenon and can be effective in patients unresponsive to other treatments. PMID- 1711835 TI - Oxygen derived free radicals and synovial fluid hyaluronate. AB - High performance liquid chromatography with TSK 5000 PW or TSK 6000 PW size exclusion columns combined with a 125I labelled hyaluronic acid binding protein assay was used to study the effects of oxygen derived free radicals on synovial fluid hyaluronate. A continuous flux of free radicals was generated by the xanthine oxidase/hypoxanthine system. When the free radical flux was generated with xanthine oxidase/hypoxanthine in the presence of the iron chelator desferrioxamine and the hydroxyl radical scavenger mannitol a 30-50% decrease in hyaluronate peak was detected, but the molecular weight of synovial fluid hyaluronate remained almost unchanged as a result of reaction with superoxide radicals and hydrogen peroxide. When trace amounts of iron and EDTA were present in the reaction mixture depolymerisation of synovial fluid hyaluronate occurred, and it reached a final molecular weight of about 13,500 daltons. These results suggest that superoxide and hydroxyl radicals may have a different mode of action on synovial fluid hyaluronate. Superoxide radicals and hydrogen peroxide do not induce depolymerisation but, rather, change the molecular configuration of synovial fluid hyaluronate. PMID- 1711836 TI - E-3123, a new synthetic protease inhibitor, protects against deoxycholic acid induced acute pancreatitis in dogs. AB - The effects of intravenous infusion of 4-(2-succinimidoethylthio)phenyl 4 guanidinobenzoate (E-3123), a new synthetic protease inhibitor, on deoxycholic acid-induced acute pancreatitis in a canine pancreas were investigated. The retrograde injection of 1% deoxycholic acid into the pancreatic duct significantly and time-dependently increased venous serum amylase and lipase activities. E-3123 significantly decreased these increased amylase and lipase activities. Two hours after deoxycholic acid injection, the amylase activity was reduced by 23 +/- 3% and 34 +/- 4% after infusion of 0.1 and 1 mg/kg/hr of E 3123, respectively, compared to the corresponding values in the control group, and the lipase activity was reduced by 13 +/- 3% and 29 +/- 3%, respectively. The potency of 1 mg of E-3123 to decrease these enzymes was almost the same as that of 5 mg of gabexate, a novel protease inhibitor. Pancreatic wet weight/body weight also decreased after treatment with E-3123 in dogs injected with deoxycholic acid. Histologic examination revealed that injection with deoxycholic acid caused marked interstitial edema, hemorrhage, vacuolization and inflammation as well as focal acinar cell necrosis, while in the E-3123-treated group, only mild changes in these parameters were seen. These results suggest that E-3123 may be useful in suppressing the progression of acute pancreatitis induced by deoxycholic acid, at least in part, through its protease-inhibiting ability. PMID- 1711837 TI - Chlorpyrifos-induced delayed polyneuropathy. AB - Chlorpyrifos [0,0-diethyl 0-(3,5,6-trichloro-pyridyl) phosphorothioate] caused delayed polyneuropathy in man. Contrary to previous studies, we report here that it also causes delayed polyneuropathy in the hen, the animal model for this toxicity. The minimal neuropathic dose was 60-90 mg/kg p.o., corresponding to 4-6 times the estimated LD50. Consequently, pralidoxime (2-PAM) in conjunction with atropine was necessary to reverse acetylcholinesterase (AChE) inhibition and cholinergic toxicity in hens given high enough doses of chlorpyrifos to cause neuropathy. Chlorpyrifos was slowly absorbed after single oral doses and the threshold of inhibition (greater than 70%) of neuropathy target esterase (NTE), the putative target for delayed neuropathy, was reached within 5-6 days. High AChE inhibition (greater than 90%), however, was measured within hours after dosing because of the higher potency of chlorpyrifos to inhibit this enzyme. In vitro studies showed that chlorpyrifos-oxon, the active metabolite of chlorpyrifos, was 10-20 times more active against AChE than against NTE, confirming the clinical observation. No differences were seen between human and hen enzymes in this respect. Hen and human brain homogenates contain A-esterases which hydrolysed chlorpyrifos to about the same extent in both species. In conclusion, chlorpyrifos causes delayed polyneuropathy in the hen, as was reported in man. The reasons for previous negative data in the hen are probably due to the relatively lower doses which were used. Judging from in vitro studies with hen and human enzymes, there are no differences in the two species as far as their relative sensitivity to delayed polyneuropathy. It is likely that delayed polyneuropathy would develop in both species only after severe cholinergic toxicity requiring aggressive antidotal treatment. PMID- 1711839 TI - The effect of dietary consistency on bone mass and turnover in the growing rat mandible. AB - Hard and soft diets were fed to weanling rats for up to 8 weeks. Some animals were switched after 4 weeks to the opposite diet. A histomorphometric study of bone formation activity at the mandibular ramus, body, and condyle was made after in vivo fluorochrome labelling. Mineral apposition rates at the lateral and inferior periosteal surfaces of the ramus were lower in the soft diet than in the hard diet animals. The rate of bone formation at the lateral periosteal surface of the ramus was significantly lower in soft than in hard diet animals. The medial periosteal surface of the ramus sometimes changed to bone formation in the soft diet groups. Condylar cartilage zones were somewhat thinner in soft diet groups. In the mandibular body, differences due to dietary consistency were less marked than near the gonial angle. Adaptation of periosteal bone and condylar cartilage to a new dietary consistency occurred within 4 weeks of switching. These results suggest that lateral and inferior periosteal bone growth of the ramus and condylar elongation were slowed in rats consuming soft diets. Decreased functional force during rapid mandibular bone growth causes changes in shape. The changes are due to regional decreases in osteoblast function, realignment of bone formation surfaces in the ramus area, and slowed growth in the condylar cartilage. PMID- 1711840 TI - Immunohistochemical localization of tenascin and fibronectin in the dentine and gingiva of Canis familiaris. AB - Specific, well-characterized antisera to the extracellular matrix glycoprotein tenascin were used. Immunoreactivity was detected in association with dentinal tubules; it was particularly prominent in the tooth crown, and was stronger within matrix than within predentine. In contrast, there was no anti-fibronectin staining in dentinal tubules. In periodontium, anti-tenascin immunoreactivity was stronger in the oral and sulcular gingival epithelia than in the underlying connective tissue, in contrast to the strong staining of connective tissue by anti-fibronectin. The appearance of tenascin immunoreactivity in gingival epithelia indicates that this protein is not exclusively a component of mesenchymal extracellular matrix. PMID- 1711838 TI - Mechanisms of bleomycin-induced lung damage. AB - Bleomycins are a family of compounds produced by Streptomyces verticillis. They have potent tumour killing properties which have given them an important place in cancer chemotherapy. They cause little marrow suppression, but pulmonary toxicity is a major adverse effect. The mechanisms of cell toxicity are well described based on in vitro experiments on DNA. The bleomycin molecule has two main structural components: a bithiazole component which partially intercalates into the DNA helix, parting the strands, as well as pyrimidine and imidazole structures, which bind iron and oxygen forming an activated complex capable of releasing damaging oxidants in close proximity to the polynucleotide chains of DNA. This may lead to chain scission or structural modifications leading to release of free bases or their propenal derivatives. The mechanisms are well described based on in vitro experiments on DNA, but how they relate to intact cells in whole animals is more tenuous. Bleomycin is able to cause cell damage independent from its effect on DNA by induction lipid peroxidation. This may be particularly important in the lung and in part account for its ability to cause alveolar cell damage and subsequent pulmonary inflammation. The lung injury seen following bleomycin comprises an interstitial oedema with an influx of inflammatory and immune cells. This may lead to the development of pulmonary fibrosis, characterized by enhanced production and deposition of collagen and other matrix components. Several polypeptide mediators capable of stimulating fibroblasts replication or excessive collagen deposition have been implicated in this, but the precise role of these in bleomycin-induced fibrosis is yet to be demonstrated. Current therapy for bleomycin-induced lung damage is inadequate, with corticosteroids most often used. Given the mechanism of action described above, antioxidants and iron chelators might be beneficial. Although, studies to date are equivocal and there is insufficient evidence to promote their use clinically. Novel drugs are currently being developed and it is hoped these may be more useful. PMID- 1711841 TI - Cloning and characterization of bovine insulin-like growth factor binding protein 3 (bIGFBP-3). AB - We report for the first time the isolation of a cDNA encoding the complete amino acid sequence for bovine growth hormone-dependent insulin-like growth factor binding protein-3 (bIGFBP-3). The deduced amino acid sequence from the cDNA revealed a mature polypeptide consisting of 264 amino acids and a 27 amino acid putative signal peptide. The amino acid sequence is over 80% homologous with human IGFBP-3 with complete conservation of the 18 cysteine residues and the 3 Asn-linked glycosylation sites. Between the two species there are 44 amino acid substitutions. Northern analysis of the bIGFBP-3 mRNA in bovine tissue revealed a single mRNA species of 1.65 kilobases. PMID- 1711843 TI - Developmental regulation in the expression of rat heart glucose transporters. AB - Antibodies against human erythrocyte glucose transporters (GLUT-1) were used to determine if the transporters of embryonic and adult rat hearts have similar reactivity. On the basis of immunoblotting, these antibodies react more strongly with embryonic transporters than with adult ones. To determine if this phenomenon may be correlated with changes in the expression of transporter types during development, RNA isolated from either the embryonic or the adult rat heart was amplified by polymerase chain reaction (PCR) to identify the transporter species. Both GLUT-1 and GLUT-4 fragments were obtained among the PCR products. They were used for Northern blot analysis. The results indicate that the embryonic heart is rich in GLUT-1 mRNA; whereas the adult heart contains predominantly GLUT-4 mRNA. Thus, it appears that the major type of glucose transporter in rat heart switches from GLUT-1 to GLUT-4 during development. PMID- 1711842 TI - Cyclic AMP stimulation of transferrin secretion by breast cancer cell grown on extracellular matrix or in two-compartment culture chambers. AB - Extrahepatic synthesis and secretion of transferrin (Tf), the major iron-carrying protein, have been described in normal and tumoral tissues suggesting a potential role for paracrine or autocrine function. In breast tumor cell MCF-7, we have previously shown a Tf secretion stimulated by estradiol which might confer selective growth advantages of these rapidly proliferating cells. The present work refers to possible additional Tf functions related to differentiation of breast tumor cells. We induced MCF-7 cell differentiation by the cyclic AMP derivative, dibutyryl cAMP (dB cAMP) and studied Tf secretion in different culture conditions after labeling with [35S] methionine. Our results demonstrate that dB cAMP stimulates Tf secretion only in culture environment that permits access to the basolateral surface and caters to the polarity requirements of the cell. These results suggest that Tf may also act as a modulator of cellular differentiation in breast cancer cells. PMID- 1711844 TI - Analysis of the human type I insulin-like growth factor receptor promoter region. AB - We isolated genomic fragments containing the 5' region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5'-untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5'-flanking region. The region surrounding the transcription start site was similar to a recently described "initiator" sequence. The 5'-flanking and 5'-untranslated regions were highly GC rich, with numerous potential Sp1 binding sites. A potential AP-2 binding site was identified in the 5'-flanking region and a potential thyroid response element was identified in the 5'-untranslated region. The 5' region of the human gene was very similar to that of the rat gene, with conservation of many of the potential regulatory elements. PMID- 1711845 TI - Impairment of the L-arginine-nitric oxide pathway in mast cells from spontaneously hypertensive rats. AB - Serosal mast cells (MC) from 6 month old spontaneously hypertensive rats (SHR) were compared to MC from 6 month old Wistar Kyoto rats (WKYR) for their ability to release nitric oxide (NO). The relationship between histamine release and NO like activity from these cells was also investigated. MC from SHR released less NO-like factor than MC from WKYR as assessed by the use of platelet aggregation and soluble guanylate cyclase activation as bioassays for NO. Sodium nitroprusside elevated the concentrations of cGMP to a similar extent in MC from SHR or WKYR. No changes in the levels of cAMP were observed. The release of histamine from MC induced by compound 48/80 or the calcium ionophore A23187 was greater in MC from SHR than in MC from WKYR. Thus, MC from SHR show a decreased production of NO-like activity which is reflected by a decreased ability to inhibit platelet aggregation. The decreased production of cGMP in the MC leads to an increased stimulated release of histamine. PMID- 1711846 TI - A chimeric glutamate receptor subunit: discrete changes modify the properties of the channel. AB - GluR1 and GluR2 are two highly homologous subunits of the glutamate AMPA receptor but with different functional properties. In ligand gated channels the transmembrane domain II is thought to form the wall of the ionic pore and determine the electrical properties. A chimeric AMPA receptor subunit was constructed by replacing the region comprising transmembrane domains I and II in GluR1 by the corresponding region of GluR2. Alone or forming an heteromer with GluR1, the resulting chimera has the properties of GluR2. Sequence comparison suggests that an arginine at position 600 in the chimera instead of a glutamine in GluR1 is responsible for these properties. PMID- 1711847 TI - Molecular cloning and characterization of a ras p21-like GTP-binding protein (24KG) from rat liver. AB - We have isolated cDNA clones from a rat liver cDNA library that encode a ras p21 like small GTP-binding protein (24KG) which was purified from the microsomes Golgi complex fraction of the rat liver. The cloning was accomplished using polymerase chain reaction amplified with a set of oligonucleotide primers which were designed from the partial amino acid sequences for 24KG. The cDNA contained an open reading frame encoding a 216 amino acid protein with a calculated Mr weight of 24,397. This Mr weight was similar to that of the purified 24KG estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sequence analysis of 24KG revealed that a 24KG cDNA is the rat counterpart of a rab11 cDNA cloned from a Madin-Darby canine kidney cell cDNA library. The 1.0 kilobase 24KG mRNA corresponding to the isolated cDNA was also detected in various rat tissues, such as brain, testis, spleen, and heart. PMID- 1711848 TI - Evidence for autophosphorylation of hyaluronate binding protein and its enhanced phosphorylation in rat histiocytoma. AB - This report documents for the first time the in vitro autophosphorylation of purified 68 kDa hyaluronate binding protein in presence of [32P] ATP. The rate of phosphorylation is proportional to the concentration of protein and to the time of incubation up to 5 min. By both phosphoamino acid and western blot analysis with antiphosphotyrosine antibodies, we have confirmed that the phosphorylation occurs at tyrosine residues. Immunoprecipitation with anti HA binding protein antibody shows a 5 fold increase in the phosphorylation in macrophage histiocytoma compared to normal macrophage. Supplementing hyaluronate with hyaluronate binding protein in the medium is further shown to enhance total protein phosphorylation in rat histiocytoma. PMID- 1711849 TI - Complete cDNA and gene sequence of the developmentally regulated arylphorin of Calliphora vicina and its homology to insect hemolymph proteins and arthropod hemocyanins. AB - Two cDNA libraries were prepared from poly(A)+ RNA isolated from fat bodies of last instar larvae of the blowfly Calliphora vicina. The libraries were probed with a genomic clone containing the coding sequence for an arylphorin subunit. Two cDNA clones as well as the genomic clone were mapped and their nucleotide sequences were determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 759 amino acid residues. The deduced primary structure of Calliphora arylphorin and hemolymph proteins of other insect species and arthropod hemocyanine show nearly 30% identity. Highly conserved regions could be also identified. PMID- 1711850 TI - Some yeast proteins are recognized by antibodies to reverse transcriptases from several mammalian retroviruses and display reverse transcriptase activity. AB - Antibodies directed against reverse transcriptases (RT) of the mammalian retroviruses SSV and RD114 recognize specifically on Western Blots yeast cytoplasmic soluble proteins of 31 and 45 kDa respectively and inhibit RT activity in this fraction. Anti RLV RTIgG recognizes a protein of 40-43 kDa in the particulate (VLP) fraction and inhibits RT activity in it. No inhibition of RT activity was seen with normal serum anti AMV RTIgG or antisera against yeast DNA polymerase I and DNA polymerase III. These yeast RTs display RNase H activity and are not inhibited by aphidicolin. They prefer Mn+2 as cofactor over Mg+2, display an optimum temperature of 25-30 degrees C and are expressed in a diploid as well as 2 haploid strains and are thus distinct from yeast Ty encoded RTs. PMID- 1711851 TI - Elevated level of hyaluronic acid binding protein in diabetic rats. AB - Hyaluronic acid binding protein (HABP), an extracellular matrix glycoprotein which interacts specifically with hyaluronic acid (HA) has been purified to homogeneity by HA affinity chromatography. Antibody was raised against it and the specificity of antibody towards HABP was confirmed by western blot analysis. A specific and sensitive assay method has been developed adopting solid phase non competitive double sandwich method of ELISA. This new assay method enabled us to determine the levels of HABP in different tissue extracts of normal, diabetic and reverse diabetic rats. A significant increase in the levels of HABP was observed in diabetic animals, which however attained normal levels with insulin treatment. PMID- 1711852 TI - Ro/SS-A antigen in human platelets. Different distributions of the isoforms of Ro/SS-A protein and the Ro/SS-A-binding RNA. AB - Ro/SS-A antigen is present in human platelets at a concentration that is approximately one-twentieth to one-thirtieth its concentration in lymphocytes, but the distribution of the Ro/SS-A isoforms is different from that in lymphocytes or red blood cells. The Ro/SS-A in platelets is largely the 52-kd form. Moreover, the 52-kd isoform in platelets is antigenically different from that in lymphocytes. In addition to the varied isoform distribution, a different distribution of the Ro/SS-A-binding RNA (hY RNA) is seen in platelets. While there are 4 hY RNA (hY1, hY3, hY4, and hY5) in lymphocytes and other nucleated cells, we have found that platelets contain only 2 hY RNA: hY3 and hY4. This hY RNA distribution is also different from that in red blood cells, which contain only hY1 and hY4. The different distributions of the Ro/SS-A components in lymphocytes, red blood cells, and platelets suggest different functional roles for the various protein and RNA components, as well as the possibility of different tissue-specific events related to the Ro/SS-A-autoanti-Ro/SS-A system. PMID- 1711853 TI - Relevance of a positive crossmatch in liver transplantation. AB - We studied 27 liver transplants in 24 patients performed between November 1984 and January 1988. We investigated retrospectively the importance of donor reactive HLA class I and class II and of non-HLA antibodies for graft survival in these patients. In order to determine the specificity and class of the antibodies, we used monoclonal antibodies to HLA-A, -B, -C and DR and DQ antigens to block cytotoxicity of sera and the reagent dithiothreitol to characterize the immunoglobulin class. We found that humoral immunity to HLA antigens in liver grafted patients, demonstrable as the presence of cytotoxic antibodies reactive with donor splenic T and/or B cells in the pretransplantation period, is associated with significantly lower graft survival as compared with patients without demonstrable preformed HLA antibodies (P = 0.01). In addition we found that a substantial proportion of patients had donor-reactive cytotoxic antibodies which were not HLA specific. Thus, our study shows that HLA immunity can influence liver allograft survival, and that it is useful to have patient cytotoxic antibodies characterized with regard to HLA reactivity prior to transplantation. PMID- 1711854 TI - The distinctive specificity of antigen-specific suppressor T cells. AB - Although suppressor T cells have been cloned in only a few instances, the existence of a functional cadre of T cells that acts to downregulate the immune response is well documented. In this review Eli Sercarz and Urszula Krzych describe studies on suppressor T-cell (TS-cell) specificity that provide some support for the conclusion that the TS cell is a distinctive cell type with an expressed repertoire that is different from that expressed by helper T (TH) cells. They go on to explore the interaction between cells recognizing TS-cell inducing determinants (SDs) and TH-cell-inducing determinants (HDs), and their relationship to immunogenicity and Ir gene effects. PMID- 1711855 TI - Antigen presentation: structural themes and functional variations. AB - T cells recognize nonnative processed fragments of antigens presented in association with major histocompatibility complex (MHC) class I or class II molecules. Recently, an accumulating body of evidence has provided a functional linkage between antigen presentation events and the cell biology of MHC molecule assembly and transport. In this review Thomas and Vivian Braciale synthesize these developments into a cohesive model of MHC assembly and antigen presentation pathways. PMID- 1711856 TI - Sodium and potassium ATPase of the teleost fish Catostomus commersoni. Sequence, protein structure and evolutionary conservation of the alpha-subunit. AB - The alpha-subunit of a Na+/K+ ATPase has been cloned by analysing a lambda gt11 library constructed from polyA+ RNA from the hypothalamic region of the teleost fish Catostomus commersoni (white sucker). The cDNA clone consists of 3853 bp and predicts a protein of 1027 amino-acid residues. Alignment of the sucker sequence with protein sequences previously published for alpha-subunits from various species reveals a high degree of homology throughout the entire sequence containing five potential sites for N-glycosylation, a phosphorylation site and a site for binding fluorescein 5'-isothiocyanate (FITC). A hydropathy profile predicts a secondary structure of the Na+/K+ ATPase alpha-subunit with at least eight membrane-spanning domains. Northern and southern blot analyses suggest the existence of two distinct Na+/K+ ATPase alpha-subunit genes in the sucker genome. PMID- 1711857 TI - Explicit distance geometry: identification of all the degrees of freedom in a large RNA molecule. AB - An alternative approach to distance geometry ("explicit" distance geometry) is being developed for problems, such as the modeling of RNA folding in the ribosome, where relatively few distances are known. The approach explicitly identifies minimal sets of additional distances that can be added to a distance matrix in order to calculate structures that are consistent with all the known information without distorting the original input data. These additional distances are bounded to the extent possible by the known distances. These explicitly added distances can be treated as degrees of freedom and used to explore the full range of alternative foldings consistent with the original input in an organized way. The present paper establishes that it is practical to explicitly determine such degrees of freedom for even very large RNAs. To demonstrate the feasibility of the approach tRNA was represented as a simple undirected graph containing all relevant information represented in the usual cloverleaf secondary structure and nine base-base tertiary interactions. Using a three atom representation for each residue a total of 206 degrees of freedom are explicitly identified. To accomplish this a graph theoretic approach was used in which a minimal covering cycle basis was determined. PMID- 1711858 TI - Evidence for calcitonin gene-related peptide contacts on a population of lamina I projection neurons. AB - Using double-labeling techniques, we evaluated small diameter primary afferent input, as indicated by calcitonin gene-related peptide-immunoreactive varicosities, to a population of lamina I projection neurons in the rat lumbar spinal cord. About one third of the lamina I neurons labeled after injections of a retrograde tracer into the region surrounding the brachium conjunctivum received contacts from immunoreactive varicosities. Significantly fewer immunoreactive varicosities were in apposition to fusiform neurons than pyramidal or flattened neurons. A positive correlation was found between the size of the retrogradely labeled neuron and the number of contacts received. This study demonstrates that a known population of nociceptive lamina I neurons received direct input from presumed nociceptive primary afferents. PMID- 1711859 TI - Arcuate nucleus projections to brainstem regions which modulate nociception. AB - Anterograde tracing studies were conducted in order to identify efferents from the arcuate nucleus, which contains the hypothalamic opiocortin neuronal pool. Phaseolus vulgaris leucoagglutinin (PHA-L) was stereotaxically iontophoresed into the arcuate nucleus and the terminal fields emanating from the labelled perikarya were identified immunocytochemically. PHA-L-immunoreactive (-ir) fibers were identified in nucleus accumbens, lateral septal nucleus, bed nucleus of the stria terminalis, medial and lateral preoptic areas, anterior hypothalamus, amygdaloid complex, lateral hypothalamus, paraventricular nucleus, zona incerta, dorsal hypothalamus, periventricular gray, medial thalamus and medial habenula. In the brainstem, arcuate terminals were identified in the periaqueductal gray (PAG), dorsal raphe nucleus (DRN), nucleus raphe magnus (NRM), nucleus raphe pallidus, locus coeruleus, parabrachial nucleus, nucleus reticularis gigantocellularis pars alpha, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve. Dual immunostaining was used to identify the neurochemical content of neurons in arcuate terminal fields in the brainstem. Arcuate fiber terminals established putative contacts with serotonergic neurons in the ventrolateral PAG, DRN and NRM and with noradrenergic neurons in periventricular gray, PAG and locus coeruleus. In the PAG, arcuate fibers terminated in areas with neurons immunoreactive to substance P, neurotensin, enkephalin and cholecystokinin (CCK) and putative contacts were identified with CCK-ir cells. This study provides neuroanatomical evidence that putative opiocortin neurons in the arcuate nucleus influence a descending system which modulates nociception. PMID- 1711860 TI - Report of a WHO workshop on synthetic peptides in HIV diagnosis and AIDS-related research, Moscow 24-26 May 1989. World Health Organization Global Programme on AIDS. PMID- 1711861 TI - Immunocytochemical determination of antigen and epitope specificity of HIV-1 specific B cells in lymph-node biopsies from HIV-1-infected individuals. AB - Knowledge about B-cell dysfunction and HIV-specific antibody production is necessary for the understanding of both HIV-1-related immunopathology and the (vaccine-induced) humoral immunity involved in protection against AIDS. This paper describes the application of recently developed methods to detect epitope specificity of B cells in lymph-node biopsies with antigen-enzyme conjugates. Cryosections of five lymph-node biopsies from HIV-1-infected individuals and four control tissues were stained with a panel of HIV-1 antigen-enzyme conjugates: recombinant HIV-1 proteins (gp 160, gp 120 and p24), labelled with peroxidase, and synthetic peptides representing neutralizing epitopes from gp120 and gp41, labelled with alkaline phosphatase. Antibody-forming cells (AFCs) were detected in all the HIV-1-infected biopsies with gp160, gp120 and/or p24, in numbers up to 350 per section. AFCs producing specific antibodies against peptide 101 (SP 101), representing the neutralizing epitope 586-608 of gp41, were detected in one patient. These techniques allow correlation of in vivo function of B cells with lymph-node pathology, clinical stage of the disease and serological data. Their potential for the elucidation of HIV-related immunopathogenesis and the development of vaccines is discussed. PMID- 1711862 TI - Antisera raised against recombinant soluble CD4s can have reactivity to recombinant HIV-1 envelope glycoproteins. AB - Antisera from sheep immunized with two different recombinant soluble CD4 preparations both exhibited cross-reactive antibodies specific for recombinant gp120s and gp160s prepared in different expression systems in immunoblots and radiobinding assays. The cross-reactivity was broadly mapped to the amino terminal 70K cleavage fragment of gp120 and more closely identified to residues 204-274 using a series of truncated recombinant gp120s. However, this reactivity could not be detected against virally encoded gp160/120. PMID- 1711863 TI - Differential inhibition of retroviral reverse transcriptase by poly(2 fluoroadenylic acid), a template analogue. PMID- 1711864 TI - Antibody to histo-blood group A antigen neutralizes HIV produced by lymphocytes from blood group A donors but not from blood group B or O donors. AB - Three virus isolates HTLV-IIIB/lyA, HTLV-IIIB/lyB and HTLV-IIIB/lyO, obtained by passaging and propagating the HTLV-IIIB/H9 isolate in three separate cultures of mixed peripheral blood mononuclear cells (PBMC) from donors of blood type A, B or O, respectively, were tested for susceptibility for virus neutralization by the monoclonal antibody (MAb) AH16 directed against the blood group A epitope. MAb AH16 was previously shown to inhibit cell-free virus infection using HTLV-IIIB propagated in H9 cells. AH16 showed a concentration-dependent inhibition of the HTLV-IIIB/lyA isolate but did not inhibit the HTLV-IIIB/lyB or the HTLV-IIIB/lyO isolate. Specificity of the MAb-mediated inhibition was shown using A-antigen (tetrasaccharide). Thus, HIV infection of PBMC from donors with blood type A appears to induce expression of host-cell-encoded carbohydrate blood group A epitope on HIV which can be a target for MAb-mediated virus neutralization. PMID- 1711865 TI - Cerebrospinal fluid markers for the monitoring of AIDS dementia complex severity: usefulness of anti-myelin basic protein antibody detection. PMID- 1711866 TI - Effects of substance P on norepinephrine release from vascular adrenergic neurons in spontaneously hypertensive rats. AB - This study was performed to investigate the role of substance P in the vascular adrenergic transmission in hypertension. In perfused mesenteric vasculatures prepared from spontaneously hypertensive rats (SHR, 7 to 10 weeks old) and age matched Wistar-Kyoto rats (WKY), we have examined the effects of substance P on vascular responsiveness as well as on norepinephrine release from the vascular adrenergic neurons. In preliminary studies with normotensive Wistar rats, pressor responses and endogenous norepinephrine release during electrical nerve stimulation were inhibited by substance P in a dose-dependent manner. However, vasoconstrictor responses to exogenous norepinephrine were not affected by the peptide. In SHR, the stimulation-evoked pressor responses and norepinephrine release were enhanced compared with WKY. Alternatively, the suppression of these responses by substance P was significantly less in SHR than in WKY. These results demonstrate that substance P could have a modulatory effect on noradrenergic activity and cause a decrease in stimulation-evoked norepinephrine release from the vascular adrenergic neurons. The attenuated reduction of pressor responses and norepinephrine release by substance P in SHR might suggest insufficient regulation of vascular adrenergic transmission by the peptide in hypertension. PMID- 1711867 TI - Role of endogenous endothelin in the development of hypertension in rats. AB - To further elucidate the pathophysiologic role of endogenous endothelin (ET), we have studied the chronic effect of anti-ET gamma-globulin on the development of hypertension in spontaneously hypertensive rats (SHR) and stroke prone SHR (SHR SP) for three weeks. In neither SHR nor SHR-SP did repetitive bolus injection of anti-ET gamma-globulin suppress the rise in blood pressure. The present data suggest that endogenous ET is not likely to play a key role in the development of hypertension, although ET may induce a local vasoconstriction at the injured vascular wall. PMID- 1711868 TI - Actions of halothane on single-channel currents evoked by acetylcholine in rat myoballs. AB - The actions of halothane on single-channel currents evoked by acetylcholine in myoballs cultured from neonatal rat skeletal muscle have been investigated using the patch clamp technique. Halothane at concentrations of 0.5-4.0 mM was applied to inside-out cell-free membrane patches in the presence of 1.0 microM acetylcholine. Halothane produced concentration-dependent decrease in the mean duration of the single-channel current events; 3.0 mM-halothane halved the slow time constant of channel closure. There was no effect on the amplitude of the single-channel currents, and no increase in the number of closing events per burst was observed. The distribution of closed times was complex but halothane did not markedly alter the frequency of channel opening events. Halothane therefore decreased the time-averaged acetylcholine-evoked membrane current, largely through an increase in the rate of channel closure. PMID- 1711869 TI - Fast isolation of recombinant baculovirus by antibody screening. AB - The use of a rapid and efficient scheme for the detection and purification of recombinant baculoviruses is described. The method is based on the detection of foreign proteins in cellular lysates of baculovirus-infected insect cells by antibody screening. The recombinant virus is purified by repeated serial dilutions. The method allows the identification and purification of recombinant viruses within two to three weeks. PMID- 1711870 TI - A fluorometric microtiter assay for the rapid enumeration of adherent bacteria. PMID- 1711871 TI - A reliable method for northern blot analysis using synthetic oligonucleotide probes. AB - We have developed a method for using short (30-42 base pair) synthetic oligonucleotide DNA probes in Northern blot assays. The method involves labeling the probes to high specific activity, very stringent hybridization and wash conditions, and the presence of several inhibitors of nonspecific binding in the hybridization buffer. We have tested this method with several probes obtained from local and commercial sources. The results with every probe used were high signal-to-noise ratios in an exposure time range of 30 min to 7 days. PMID- 1711872 TI - A novel non-enzymatic procedure for removing DNA template from RNA transcription mixtures. AB - A novel, simplified procedure for the removal of DNA template from newly synthesized RNA is described. The method avoids the use of DNase and involves extraction of the RNA transcription products at acid pH using either phenol or phenol: chloroform:guanidinium thiocyanate mixture. Both procedures are as effective as DNase digestion in removing DNA template, but are free of the hazard of RNA degradation, are highly reproducible and remove proteins at the same time. PMID- 1711873 TI - The role of different types of corticosteroids on the inflammatory mediators in cardiopulmonary bypass. AB - In a placebo-controlled double-blind study on patients undergoing cardiopulmonary bypass (CPB) we studied the inhibiting effects of dexamethasone, a high dose of methylprednisolone, and a low dose of prednisolone on the inflammatory reaction induced by CPB. During CPB two episodes of blood activation were noticed. First, the blood-material interaction caused a significant increase in complement C3a and elastase concentrations after the start of bypass (p less than 0.01). Secondly, the reperfusion of the ischemic heart, lungs, and peripheral tissue, after release of the aortic cross-clamp, caused an additional increase in C3a and elastase concentration and a statistically significant increase in leukotriene B4 (LTB4) concentration and tissue plasminogen activator (t-PA) activity (p less than 0.01, p less than 0.05, respectively). Dexamethasone treatment effectively inhibited the increase in LTB4 concentration and t-PA activity after release of the cross-clamp (significant differences to the placebo group, p less than 0.01, p less than 0.05, respectively). High-dose methylprednisolone treatment was almost as effective as dexamethasone treatment, whereas low-dose prednisolone treatment was less effective than methylprednisolone in the inhibition of the inflammatory mediators (DM greater than MP greater than P). None of the corticosteroid regimens was able to inhibit the increase in complement C3a and elastase. We therefore conclude that corticosteroids do not have an effect on complement activation during CPB. However, leukocyte activation and t-PA activity after release of the aortic cross-clamp are effectively inhibited by corticosteroid treatment, in a dose-dependent way. The inhibition of this inflammatory reaction will have a favourable effect on the postoperative course in patients who have undergone CPB. PMID- 1711874 TI - Saccharide-protein covalent conjugates: immunochemical characterization of Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates. AB - Core oligosaccharides (complete and incomplete) isolated from Citrobacter 036 lipopolysaccharide were covalently conjugated with tetanus toxoid. Serological examination of the Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates showed that they are strong immunogens. The monospecific anti-conjugate sera prepared by immunization of rabbits, were used to study the antigenic relations between lipopolysaccharide core regions of 8 strains of Citrobacter. Immunoelectrophoresis, immunoblotting and quantitative microprecipitation were performed in the experiments. PMID- 1711875 TI - Characterisation of the urease from Helicobacter pylori and comparison with the ureases from related spiral gastric bacteria. AB - The urease enzyme of Helicobacter pylori was partially purified from whole cell extracts and found to have a molecular weight of 484 +/- 12 kDa. Ten monoclonal antibodies (mAbs) were produced against four different epitopes of the native enzyme. These mAbs also recognised the ureases of H. pylori-like organisms isolated from monkeys and pigs and the H. mustelae urease from ferrets. The urease enzymes of each of these organisms were found to be of the same molecular weight. The urease enzyme of H. pylori consisted of two subunits of 68.2 and 31.3 kDa. PMID- 1711876 TI - Evidence for (lipo) oligosaccharides in Borrelia burgdorferi and their serological specificity. AB - SDS-PAGE and Western immunoblot profiles have been determined for different strains of Borrelia burgdorferi. Major proteins of 60 kDa, 41 kDa corresponding to flagellin, 34-36 kDa and 30-31 kDa corresponding to OspB and OspA respectively, and 18-20 kDa corresponding to 'pC' fractions were detected. A "rough" lipopolysaccharide which we called lipooligosaccharide (LOS) of 8-11 kDa appeared to be present, being detected by specific silver staining, as in crude Borrelia lysates as in proteinase K digested Borrelia strains, quite similar in shape among the different strains examined. The LOS reacted in Western blotting with immune anti-B. burgdorferi rabbit serum and also with sera collected from humans affected by Lyme borreliosis. The LOS did not react with sera positive for syphilis or leptospirosis, and their immunological specificity is discussed. PMID- 1711877 TI - Identification of a novel B-cell epitope of restricted specificity on the hsp 65 kDa protein of Mycobacterium tuberculosis. AB - A B-cell epitope on the carboxy-terminal region of the mycobacterial 65-kDa heat shock protein that distinguishes Mycobacterium tuberculosis/Mycobacterium bovis BCG from Mycobacterium leprae was identified by two novel monoclonal antibodies (mAbs), Ne5 and Nd4. These mAbs also showed a limited cross reactivity with mycobacterial species belonging to M. tuberculosis complex and Mycobacterium avium complex with the exception of Mycobacterium vaccae. Characterization of the epitope recognized by these mAbs was done with M. bovis BCG 65-kDa fusion proteins expressed in Escherichia coli encoding various segments of the 65-kDa protein. Our results together with those reported in literature indicated that this epitope resides in the highly divergent region of amino acid residues 525 to 540. This B-cell epitope on the 65-kDa protein of M. tuberculosis/M. bovis BCG has not been recognized by previously reported mAbs, although the analogous epitope sequence of M. leprae 65-kDa has been identified by a known mAb (IIIC8) reported in the literature. Therefore Ne5/Nd4 epitope could be considered important in studying the differential immune response of the host against infections with M. tuberculosis complex/M. avium complex and M. leprae. PMID- 1711878 TI - Antigenic epitopes on Mycobacterium tuberculosis recognized by antibodies in tuberculosis and mouse antisera. AB - The effect of sodium periodate and proteolytic enzyme treatments on the antibody binding capacity of Mycobacterium tuberculosis antigen (Ag) was studied by ELISA. Treatment with sodium periodate resulted in a marked decrease in the capacity of M. tuberculosis Ag to bind antibodies in human TB sera, but had no effect on the reactivity with antibodies in mouse. In contrast, treatment with proteolytic enzymes (trypsin and chymotrypsin) had no effect on the reactivity of M. tuberculosis Ag with human TB sera but reduced substantially the reactivity to antibodies in mouse antisera. These results indicate that anti-M. tuberculosis antibodies in human TB sera react predominantly with carbohydrate determinants and not with protein epitopes sensitive to trypsin and chymotrypsin. The bulk of murine antibodies on the other hand were directed against protein determinants and not the carbohydrate epitopes. PMID- 1711879 TI - [Whipple's disease with gastric and duodenal involvement. A new case]. AB - Whipple's disease (WD) is a chronic multisistemic process in which the gastrointestinal tract is almost invariably affected. It is believed to be bacterial in origin, although a causative agent has not yet been isolated. Antibiotic treatment is usually quickly effective. We report a case of Whipple's disease in a 69-year-old man with typical clinical history and laboratory data. However, an associated allergic vasculitis not yet described in WD, the characteristical endoscopic appearance, and a partial response to treatment with persistence of histological lesions, made us consider of interest the report of the case. PMID- 1711880 TI - Localization of the proteinases in the human alpha 2-macroglobulin-chymotrypsin complex by image processing of electron micrographs. AB - Human alpha 2-macroglobulin (alpha 2M), a large tetrameric plasma glycoprotein, inhibits a wide spectrum of proteinases by a particular "trapping" mechanism resulting from the proteolysis of peptide bonds at specific "bait" regions. This induces the hydrolysis of four thiol esters triggering both the possible covalent bonding of the proteinases and a considerable structural change in the alpha 2M molecule, also observed following direct cleavage of the thiol esters by methylamine. By subtracting average images of electron micrographs from two populations of alpha 2M molecules in the same biochemical state (with both the four cleaved bait regions and thiol esters), but containing either two or zero chymotrypsins, we are able to demonstrate the position of the two proteinases inside the tetrameric alpha 2M molecule. The comparison of the alpha 2M molecules transformed either by immobilized chymotrypsin or methylamine shows that the proteolysis of the bait regions seems of minimal importance for the general shape of the molecule and provides a direct visualization of the actual role of the thiol esters in the conformational change. PMID- 1711881 TI - Microwave-stimulated fixation for preembedding immunoelectron microscopy. PMID- 1711882 TI - Routine use of microwave technology in Scotland: retic, mucin, myelin and fungi stainings. PMID- 1711883 TI - Forrest H. Nielsen recipient of Klaus Schwarz Medal for 1990. PMID- 1711884 TI - Examination of the roles of selenium in the Kaschin-Beck disease. Cartilage cell test and model studies. AB - The Kaschin-Beck Disease, an endemic disease in China, occurs in low-selenium areas. Using human embryonic cartilage cell as a system, the effect of selenite and another etiological factors, such as, organic matters in water, and grain from disease regions, were studied. It was shown that Se(IV), as well as superoxide dismutase, could prevent the cells from damage by organic matters, and increase the activity of GSHpx and decrease the production of lipid peroxide. A model test of adrenalin autooxidation was carried out, and it was found that the oxy-radical can be eliminated by Se(IV). Thus, it was assumed, that selenium was a protective factor and free radical scavenger for Kaschin-Beck Disease. PMID- 1711885 TI - Study of immune function of cancer patients influenced by supplemental zinc or selenium-zinc combination. AB - Since hair Zn and serum Zn are usually decreased in cancer patients, and Zn deficiency may reduce the function of T-cells, granulocytes, and Nk cells, we observed in cancer patients the influences of the Zinc or Selenium-Zinc on DNCB skin delayed hypersensitivity mediated by T cell, and the effects of Zinc on oxidative metabolic function of neutrophils and level of serum interferon that potentiate NK cell activity. The results showed that DNCB skin reaction was strengthened, the oxidative metabolic function of neutrophils and serum interferon level were increased by the drugs. It is reasonable to expect that Zinc or Selenium-Zinc is instrumental in restoring failing immunocompetence of cancer patient. PMID- 1711886 TI - Selenium and Behcet's disease. AB - Behcet's disease is an inflammatory disorder of unknown etiology, characterized by recurrent oral and genital aphthous ulcers, ocular inflammation, and skin lesions of erythema nodosum and acneiform eruptions. Selenium (Se) affects all components of the immune system, i.e., the development and expression of nonspecific, humoral, and cell-mediated responses. In general, a deficiency in Se appears to result in immunosuppression, whereas supplementation with low doses of Se appears to result in augmentation and/or restoration of immunologic functions. In this study, the distribution of Se and IgG, IgM in serum were compared in samples from healthy adult control and Behcet's disease patients. The serum Se levels were measured by AA-30-40 Varian Spectra, and immunoglobulins were measured by immunodiffusion technique. The mean (SD) serum Se level of 54.24 +/- 8.06 ng/mL among Behcet's disease subjects was significantly different (P less than 0.01) from that in the control subjects (90.01 +/- 9.94 ng/mL). We also measured IgG and IgM as 10.01 +/- 2.74 mg/mL and 1.26 +/- 0.29 mg/mL, respectively for patients, and 15.08 +/- 4.73 mg/mL and 1.58 +/- 0.43 mg/mL for controls. The mean values of IgG and IgM for patients were significantly (P less than 0.05) different from the values of controls. It seems, therefore, that a deficiency in selenium impedes the humoral immune response. PMID- 1711887 TI - Milk transfer of inorganic mercury to suckling rats. Interaction with selenite. AB - The transport of mercury into rat milk, and uptake in the suckling offspring was studied after peroral administration of inorganic mercury to lactating control rats, and to rats fed selenite in the diet. On day 8, 9, 10, or 11 of lactation, dams were administered a single oral dose of 0.1, 0.4, 0.7, 1.3, or 5.8 mg Hg/kg bw labeled with 203mercuric acetate. There was a linear relationship between mercury concentrations in dam's plasma and milk. The level of mercury in milk was approximately 25% of the level in plasma. After 3 d, milk levels were reduced to half the levels at 24 h. In the suckling offspring, exposed to mercury via milk during 3 d, the mercury level in blood was approximately 1% of the level in maternal blood. Mercury concentration in milk was linearly correlated to the levels in kidney, liver, and brain in the suckling offspring after 3 d exposure to mercury via milk. Selenite treatment of rats, 1.3 micrograms Se/g diet for 5 mo, resulted in increased transport of mercury to milk, probably because of increased plasma levels of mercury. However, selenite treatment of the dams did not cause any increased tissue levels of mercury in the suckling offspring. PMID- 1711888 TI - Uptake and distribution in rat brain of organic and inorganic selenium. AB - Polyunsaturated fatty acids (PUFAs) occur in relatively high amounts in phospholipids of the synapses. PUFAs may thus determine the fluidity of the synaptosomal membrane and, hereby, they may regulate the neuronal transmission. It was therefore tempting to suggest a system in the brain, that inhibits autooxidation of PUFAs. In order to trace such a protection system, Wistar rats were equally loaded with 4500 kBq of 75-Se either as selenite or as L-Se methionine. By means of gradient ultracentrifugation, particulate fractions of the brains were isolated, and the radioactivity as well as the glutathione transferase and -peroxidase activities were estimated. The distribution of the two selenium components among the particulate fractions was different. Thus, selenite gave higher radioactivity in myelin, then followed by the light synaptosomal and the vesicular fraction. L-Se-methionine was more equally incorporated in all particulate fractions, although highest activity was found in the mitochondrial fraction. Myelin and synaptic vesicles were devoid of transferase activity. On the other hand, the synaptosomal fraction showed highest specific transferase activity. The glutathione peroxidase activity was highest in the myelin fraction, followed by the vesicular and the synaptosomal fractions. The data obtained thus support the idea that the PUFAs of the synaptic compartment are protected against peroxidation, at least in part, by the selenium containing glutathione peroxidase. PMID- 1711889 TI - Influence of selenium on the efficiency of fungicide action against certain fungi. AB - Aspergillus funiculosus was isolated from rotted banana fruits, whereas Alternaria tenuis and Fusarium sp. were isolated from rotted tomato fruits. The isolated fungi tolerated relatively high levels of the fungicide, Dithane, up to 2560 ppm on solid medium, but grew well at 40 ppm when supplemented with liquid medium. They are able to tolerate selenite up to 2% (w/v) sodium selenite. A. funiculosus showed no growth in the presence of mixture of 2.5 ppm selenium and 20 ppm Dithane, whereas Fusarium sp. failed to grow at 2.5 ppm selenium and 10 ppm Dithane, or at 10 ppm of each. Nevertheless, Alternaria tenuis is more tolerant; it showed growth in the presence of relatively high levels of selenium and Dithane; up to 10 ppm selenium and 40 ppm Dithane, however, its growth was inhibited by the presence of a mixture of both. The results suggested new form of highly active fungicides. Selenium as an essential nutrient at such very low concentrations, as well as the application of very low concentrations of the fungicide, would certainly reduce the hazardous effect of such pollutant in the environment. PMID- 1711890 TI - Metabolism, cellular actions, and cytotoxicity of selenomethionine in cultured cells. AB - Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way. PMID- 1711891 TI - Visuotopic organization of the lateral suprasylvian area and of an adjacent area of the ectosylvian gyrus of cat cortex: a physiological and connectional study. AB - We have explored the visuotopic organization of the territory surrounding the middle suprasylvian sulcus (MSS) of cat cerebral cortex by electrophysiological mapping, and by tracing the topography of its cortical and subcortical connections using wheatgerm-agglutinin horseradish peroxidase (WGA-HRP). Observations from the two approaches were concordant, and confirmed the presence of two separate visual areas in the MSS that approximate, but do not exactly correspond, to the location and internal organization of the posterior medial and posterior lateral lateral suprasylvian (PMLS, PLLS) areas of Palmer et al. (1978). We define as part of the lateral suprasylvian (LS) area the territory on the medial bank and caudal end of the lateral bank of the MSS that receives a topographically organized projection from the region of area 17 representing the lower visual quadrant. This territory is connected with other structures that are themselves striate-recipient (cortical areas 18 and 19, and the lateral division of the lateral posterior (LPl) nucleus), and with a variety of nuclei that receive direct retinal input, such as the C-laminae of the LGd, the medial interlaminar nucleus (MIN), and the superficial layers of the superior colliculus (SC). Its connections with the LPl, LGd, MIN, and SC correspond topographically with the input from area 17. Revised maps of area LS were produced from the physiological and connectional data: its rostral border is formed by a representation of lower visual elevations with the horizontal meridian represented caudally, and its lateral border is formed by the vertical meridian; area LS shares a representation of the center of gaze with the visual area of the lateral bank at its caudal end. The adjacent lateral bank area has larger receptive fields than area LS, and very different connectivity. It receives no input from area 17 and little input from striate-recipient structures, including area LS, but instead is connected to more remote extrastriate visual areas, such as the anterior ectosylvian visual (AEV) area in insular cortex, and to zones of the thalamus in receipt of tectal input (LPm and the lateromedial-suprageniculate nuclear complex). According to both mapping approaches, the lateral bank area contains representations of both the upper and lower visual quadrants but a rather limited degree of visuotopic order. We refer to it as the posterior ectosylvian visual (PEV) area, because it appears to be functionally and connectionally dissociated from area LS, but is possibly a functional antecedent of area AEV. PMID- 1711892 TI - Organization of reciprocal connections between area 17 and the lateral suprasylvian area of cat visual cortex. AB - The lateral suprasylvian (LS) area (or Clare-Bishop area) is a region of visual cortex in the cat which has been defined as an isolated projection zone of area 17 (V1 or striate cortex) within the suprasylvian sulcus. We have studied the overall topography and detailed pattern of connection between these two visual areas following injections of WGA-HRP into one or the other. The projection from area 17 to LS is formed largely (approximately 90%) from supragranular layer neurons that are distributed, in the coronal plane, in multiple regularly spaced patches. These patches are especially prominent in regions of area 17 representing central vision along and around the horizontal meridian. In reconstructions of serial coronal sections, and in flatmounts of the same region, the patches are seen to align so that in the plane tangential to the cortical surface they appear as a system of parallel bands whose main axis of elongation is rostro-ventral to caudo-dorsal, or near parallel to the area 17/18 border. The mean periodicity of the bands is about 1.0 mm. The projection from area 17 terminates mainly in layers 4, 3, and 2 of area LS, and also appears patchy in the coronal plane. Reconstruction of the cortical surface view again reveals a system of rostrocaudal bands, but with a mean periodicity of 2 mm. The back projection is less periodically organized, arising predominantly (approximately 80%) from a continuous sheet of infragranular neurons in area LS and terminating mainly in layer 1 of area 17, across the underlying patch and interpatch zones of the supragranular projection cells. However, neurons in layers 2 and upper 3 of area LS, which form the minority origin of the back projection, are mostly located in columnar registration with the patches of area 17 terminals. The bands of supragranular layer neurons projecting to area LS are aligned obliquely to the iso-orientation domains of area 17, indicating a further component to its organization. It is suggested that this may correspond to a segregation of the X and Y channels in area 17, with outputs to area LS selectively arising from the Y pathway, in accordance with previous reports. PMID- 1711893 TI - Three-stranded alpha-fibrous proteins: the heptad repeat and its implications for structure. AB - Amino acid sequence data have been collected for the coiled-coil rod domains of three-stranded alpha-fibrous proteins--fibrinogen, laminin, tenascin, macrophage scavenger receptor protein and the leg fibre protein from bacteriophage. Such domains are characterized by a heptad substructure in which apolar residues occur alternately three and four residues apart. The distribution of residues in each position of the heptad has been analysed, and the results compared with those obtained for the two-stranded alpha-fibrous proteins, which include the intermediate filament and myosin families. Distinctions can be drawn between the sequences in two- and three-stranded coiled-coil structures and these provide criteria that will prove useful in predicting secondary and tertiary structure purely from sequence data. PMID- 1711894 TI - Hydrogen bonding monitored by deuterium isotope effects on carbonyl 13C chemical shift in BPTI: intra-residue hydrogen bonds in antiparallel beta-sheet. AB - Deuterium isotope effects on carbonyl 13C magnetic shielding were measured for the backbone carbonyl groups in BPTI (basic pancreatic trypsin inhibitor), and interpreted as a measure of hydrogen bond energies. The effects originate from peptide amide proton deuterium substitution and were observed on carbonyl carbons separated by two or three covalent bonds from the amide H/D. Two-bond isotope effects depend on the energy of the hydrogen bond donated by NH/D. Calibration of the effect with model compound data leads to hydrogen bond enthalpies less than 4.7 kcal/mol. Isotope effects over three bonds from the amide H/D to the carbonyl carbon of the same amino acid residue are observed for seven carbonyl resonances in BPTI. The three-bond isotope effects are highly related to the various backbone conformations. The largest effects are observed for residues with an approximate syn- periplanar conformation of the H-N-C alpha-C = O atoms, as realized for many residues in the BPTI antiparallel beta-sheet. The residues that show measurable three-bond effects have unusually short distances between H and O. The size of this effect decreases rapidly with increased O..H distance in the open five-membered ring. This observation suggests appreciable interactions in these rings. PMID- 1711895 TI - The specificity of sea urchin hatching enzyme (envelysin) places it in the mammalian matrix metalloproteinase family. AB - The sea urchin hatching enzyme (HEz) is a protease capable of dissolving the fertilization envelope that surrounds the embryo as a protective coat during early development. We have now purified a 37-kDa active enzyme from the supernatant fluid of hatched blastula medium of the species Hemicentrotus pulcherrimus. The purified enzyme was completely inhibited by alpha 2 macroglobulin and the chelating agents EDTA, EGTA, and 1,10-phenanthroline and was slightly inhibited by chymostatin and pepstatin, but was not inhibited by various other serine and thiol protease inhibitors. These results suggest that HEz is a metalloproteinase. Quantitative analyses of the products of HEz's action on various peptides revealed that the enzyme preferentially cleaved the peptide bonds on the amino side of bulky hydrophobic residues, -Leu, -Ile, and -Phe as well as -Tyr, in a similar but more limited fashion than thermolysin. Furthermore, although substance P was a good substrate of HEz, snake venom alpha protease, and thermolysin, the analogue [Sar9]substance P was a poor substrate for HEz. This analogue was a good substrate for thermolysin and alpha-protease, but the scissile bonds were altered from those of substance P. The failure of HEz and alpha-protease to cleave the P1-P1 bond that meets the primary specificity is ascribed to the presence of an imino acid residue (proline or sarcosine) or the absence of any amino acid at the P2h or P3' position, in contrast to the simple P2' restriction of thermolysin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711896 TI - Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system. AB - A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM. PMID- 1711897 TI - A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA. AB - The well-characterized RNA binding site of the bacteriophage R17 coat protein has been used to investigate the cross-linking of protein to 5-bromouridine (BrU) substituted RNA using medium-wavelength UV light. We have demonstrated a specific RNA-protein cross-link and identified the site on the RNA of protein attachment. Formation of the covalent complex is dependent upon the presence of BrU at position -5 of the RNA and specific binding of the RNA by coat protein. The amount of cross-linking increases with time and depends on the light source and conditions used. Irradiations using a broad-spectrum UV transilluminator (peak at 312 nm) or monochromatic XeCl excimer laser (308 nm) gave levels of cross-linking exceeding 20 and 50%, respectively. The quantum yield of photo-cross-linking, determined with 308-nm excitation, was 0.003. While little strand breakage or debromination of the RNA occurred, significant protein photodamage was observed. PMID- 1711898 TI - GTP is required for the integration of a fragment of the Neurospora crassa H(+) ATPase into homologous microsomal vesicles. AB - The integration of a fragment of the Neurospora crassa plasma membrane H(+) ATPase was examined to determine if insertion of the fragment into homologous microsomal vesicles is obligatorily dependent on a nucleoside triphosphate. RNA transcripts that encoded the amino terminal 344 amino acids of the Neurospora crassa plasma membrane H(+)-ATPase(pma(344)+) were translated in a N. crassa in vitro system. The pma(344)+ integrated post-translationally into homologous microsomal vesicles independent of the associated ribosomes and dependent on the presence of GTP or guanylyl imidodiphosphate, a nonhydrolyzable analogue of GTP. ATP or analogues thereof did not support the integration of pma(344)+ into nRM post-translationally. These results were interpreted to suggest that a GTPase plays an essential role in the integration of the amino terminal portion of the pma+ into the endoplasmic reticulum. PMID- 1711899 TI - Isolation and partial characterization of the human erythrocyte band 7 integral membrane protein. AB - Monoclonal antibodies to the Mr 31,000 major integral membrane protein of the human erythrocyte band 7 region were used to identify the corresponding polypeptide chain and epitope-carrying fragments on immunoblots. Analysis of the erythrocyte membrane, membrane fractions, and cytosol revealed that the Mr 31,000 band 7 integral membrane protein is unique and not related to any of the other water-soluble or membrane-bound band 7 components. Cross-reacting proteins were identified in the membranes of other mammalian erythrocytes and in cell lines of epithelial and lymphoid origin. Proteolytic digestion of intact human erythrocytes or erythrocyte membranes demonstrated that the band 7 integral membrane protein has an intracellular domain larger than Mr 12,000; it does not have an extracellular one. One of the monoclonal antibodies was employed for the isolation of band 7 integral membrane protein by immunoaffinity chromatography; subsequent Edman degradation revealed a blocked N-terminus. PMID- 1711900 TI - Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels. AB - The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells. PMID- 1711901 TI - Visualization of alkaline phosphatase-labelled antibodies on immunoblots by means of formazan staining using indoxyl phosphate and thiazolyl blue. AB - Alkaline phosphatase-conjugated secondary antibodies on blotting membranes were visualized by means of a modified formazan staining method (Hodson & Skillen 1988). It is based on the standard 5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium method, but employs different buffer conditions and thiazolyl blue instead of Nitroblue. It gave a sensitivity of 1 ng albumin in a 12 mm2 dot and less background staining on dot blots than the standard method. The new protocol provides an advantage with regard to ease of staining and preparation of reagents while it equals the former method with respect to sensitivity. PMID- 1711902 TI - Detection and recovery of proteins from gels following zinc chloride staining. AB - This report describes a method for detecting proteins in SDS polyacrylamide gels using ZnCl2 and their recovery using passive elution. Washing unfixed gels in a dilute solution of ZnCl2 produces two desirable effects. First, it makes the proteins easily visible as clear zones in a white background, and second, it prevents loss of proteins due to diffusion into the wash solution. Compared to other commonly used methods of protein detection such as Coomassie, KCl, copper or silver staining, the zinc stain offers some distinct advantages. Zinc staining can be completed in 15 min for most applications, making it much faster than Coomassie or most silver stains. The zinc stain is more sensitive than Coomassie, KCl or copper stain. Since no harsh chemical conditions are used in the zinc staining procedure, recovery of proteins from the gel is facilitated. More than 90% of selected proteins were recovered from 2-D gels by simple elution from the gel pieces with a buffer containing 10 mM EDTA. PMID- 1711903 TI - Polymeric contrast agents for magnetic resonance imaging: synthesis and characterization of gadolinium diethylenetriaminepentaacetic acid conjugated to polysaccharides. AB - The synthesis and characterization of polysaccharides esterified with gadolinium diethylenetriaminepentaacetic acid (GdDTPA) are described. The results of several synthetic methods are presented for esterification of dextrans and inulin with DTPA. One method results in highly conjugated products labeled with an average of 0.4 mol of GdDTPA/mol of glucopyranose unit in dextrans of up to 70,800 average molecular weight and 0.5 mol of GdDTPA/mol of fructofuranose unit in inulin. Chromatographic and potentiometric evidence supporting the absence of significant chelate cross-linking of the conjugated polysaccharides is presented. The thermodynamic stability constant, log K (Gd3+ + L4(-)----GdL-), of the complexes was 18.0 +/- 0.2 based on an independent chelate model. In vitro ester hydrolysis of the GdDTPA-dextran 70,800 (at 37 degrees C, pH = 7.4 phosphate buffer) occurs with a half-life of 21 h. The agents exhibit T1 relaxivities ranging from 1.5 to 2.3 times that of GdDTPA at 100 MHz, and decreasing in vitro relaxivity with increasing molecular weight of the dextran carrier was observed. Phantom MRI studies indicate that the T1 and T2 effects of the complexes differ from those of GdDTPA, with the polysaccharide-bound complexes exhibiting a considerably faster drop in relative signal intensity with increased concentration in T1 and T2 weighted pulse sequences. PMID- 1711904 TI - Long-term preservation of whole blood samples for flow cytometry analysis in normal and HIV-infected individuals from Africa. AB - A whole blood method requiring less than 4 ml of heparinized blood was developed to assess the practicality of preparing whole blood samples that could be easily stored, transported and readily used to determine the lymphocyte phenotypes and proliferation responses of individuals from remote areas who are infected with the human immunodeficiency virus. Minor modifications in standard whole blood procedure for lymphocyte phenotyping have significantly increased the stability of light scatter and fluorescence intensity of the cells for subsequent flow cytometry (FC) analysis. These changes include removal of lysis solution prior to fixation, fixation of monoclonal antibody-stained cells in 1% paraformaldehyde for 30 minutes and storage of fixed samples in medium containing 1% bovine serum albumin. Lymphocyte subsets and their functional subsets could reliably be determined on samples stored for up to 4 weeks. Further, blood samples could be kept at room temperature for up to 96 hours or at ambient temperature during transportation from Africa before staining for FC without affecting their quantitation. While samples could be processed for FC analysis under field laboratory conditions, proliferation assays could only be performed on samples that were transported within 48 hours of their collection. The whole blood method saves time and expense and decreases the volumes of blood required to perform phenotypic analysis and functional assays on specimens collected in remote areas. PMID- 1711905 TI - The effect of organic cosolvents on the oxygen affinity of fetal hemoglobin. Relevance of protein-solvent interactions to the functional properties. AB - We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen affinity of human fetal hemoglobin stripped of phosphates and have compared them with the effects of the same cosolvents on the oxygen affinity of human adult hemoglobin under the same experimental conditions. Our results confirm that, in fetal hemoglobin, the T in equilibrium R conformational equilibrium is more displaced toward the T conformation than in the adult form and indicate that increased electrostatic and hydrophobic protein solvent interactions contribute to this effect. The data reported are discussed in terms of the known amino acid substitutions between the beta- and gamma-chains and an attempt is made to rationalize the results with a molecular mechanism based on the crystallographic structure of fetal deoxyhemoglobin. PMID- 1711906 TI - On the morphology of the terminal microvasculature during endochondral ossification in rats. AB - The investigation was carried out on the proximal tibia metaphysis by means of India ink-injection, reconstruction, corrosion casting combined with scanning electron microscopy, and transmission electron microscopy of thin sections. The terminal vasculature during endochondral ossification proved to be a labyrinth of interconnected sinusoidal complexes with bulbous buds invading the chondrocyte cavities of the growth plate. This system of vessels was canalized throughout. A special feature was the existence of endothelial pockets. The formation of the terminal microvasculature as a labyrinthine cavity during endochondral ossification, together with the still incompletely developed bone could be a weak point for compression injuries. PMID- 1711907 TI - Therapeutic ratio and defined phases: proposal of ethical framework for palliative care. PMID- 1711908 TI - Morphological changes in the vascularisation of delayed flaps in rabbits. AB - The effect of the delay phenomenon on the vascularisation of the pectoralis major myocutaneous flap was studied in 32 rabbits. In 36 flaps, submitted to different methods of delay, vascularisation was studied by making the tissue transparent and comparing the results with a control group of 16 undelayed flaps. In another 12 flaps, 6 of them previously delayed, the vascularisation of the skin was studied by light microscopy and through a morphometric analysis (image digitalizing process). Delayed flaps showed a significant (P less than 0.0001) increase of their vascularisation related to the undelayed flaps (average vascular area in the slides of 0.04 mm2 and 0.01 mm2 respectively). In addition, there were a nonspecific inflammatory reaction and angiogenesis, that could contribute to the increase of the vascularisation. PMID- 1711909 TI - The oblique rat groin flap. AB - Neovascularisation from the bed can result in the partial survival of failing rat groin flaps. This can interfere with the interpretation of pharmacological augmentation and ischaemia experiments. We describe the development of a modified rat groin flap which utilises the inguinal fat pad to diminish the effects of the bed on the skin component of the flap. The behaviour of this model is compared to that of standard rat groin flaps and flaps which have had polythene sheeting placed under them. PMID- 1711910 TI - Role of interstitial therapy in the treatment of liver cancer. AB - Conventional palliative management of inoperable focal hepatic tumours remains unsatisfactory. Interstitial techniques such as cryotherapy, alcohol injection, low power laser hyperthermia and interstitial radiotherapy offer alternative approaches. Cryotherapy is an effective and precise technique for inducing tumour necrosis. It can only be performed at laparotomy making it relatively invasive and retreatment impractical. Alcohol is cheap and can be injected percutaneously. However, inhomogeneous distribution produces imprecise and nonreproducible lesions. Low power laser hyperthermia produces precise and reproducible areas of necrosis that are roughly spherical in shape. At present, this technique is most effective for small tumours. Interstitial radiotherapy remains the least evaluated of all the interstitial techniques. Unlike cryotherapy and low power laser hyperthermia, the biological effect of ethanol injection and interstitial radiotherapy cannot be monitored in real time by ultrasound. With the exception of cryotherapy, all methods can be applied percutaneously with low morbidity and mortality. None of these techniques is established, but they may offer the prospect of cure in cases where all areas of tumour can be positively identified and fully treated. However, in most instances the intention is to control the growth of relatively small discrete volumes of tumour within the hepatic parenchyma. PMID- 1711911 TI - In vitro studies of multiple impact injury to mammalian CNS neurons: prevention of perikaryal damage and death by ketamine. AB - We have developed an in vitro model of rapid acceleration injury (RAI) to study the effects of multiple impact (220 g/impact, 3-5 s intervals) trauma on cultures of mammalian CNS cells. Our initial investigations have shown that: (1) multiple impacts delivered tangential to the plane of growth caused neuronal death while normal impacts did not; (2) glia were not affected by tangential or normal RAI; (3) most neuronal death occurred within 15 min; (4) the threshold for neuronal death was above 440 g (cumulative); (5) neuronal death reached a maximum of 50% at cumulative accelerations greater than or equal to 1100 g; (6) somal swelling and increased nuclear prominence were often observed after tangential RAI, and the frequency of these changes increased with the cumulative acceleration; and (7) ketamine prevented neuronal death and morphological changes during tangential RAI. We hypothesize that neuronal sensitivity to multiple impact RAI depends on the density of N-methyl-D-aspartate (NMDA) complexes in the dendrosomatic membranes. We also hypothesize that the events leading to neuronal death during multiple impact injury are: (1) calcium leakage through NMDA channels causes weakening of the cytoskeleton; (2) loss of cytoskeletal integrity allows nuclear shifting during impact; and (3) nuclear pressure disrupts the plasmalemma causing a lethal influx of calcium. PMID- 1711912 TI - Glutathione levels in olfactory and non-olfactory neural structures of rats. AB - Olfactory receptor neurons are a CNS entry point for a wide variety of airborne substances. Therefore, it is probable that detoxification mechanisms are present in these neurons to neutralize such agents. Glutathione (GSH) is an essential component of several detoxification schemes, and in this study we examined the distribution and levels of GSH in the olfactory epithelium, olfactory bulb, cortex, hippocampus and cerebellum in neonatal, weanling, adult and aged rats. We report that GSH is primarily localized to the olfactory receptor neurons and their oxons within the olfactory epithelium. It is also localized within the glomerular neuropil and granule cells of the olfactory bulb. Levels of GSH in the olfactory epithelium and hippocampus do not change as a function of age, although GSH levels decrease in several brain regions, including the olfactory bulb, cerebellum and cortex. PMID- 1711913 TI - Estimation of the chloramphenicol and cycloheximide inhibition of protein synthesis in brain cholinergic synaptosomes. AB - Cholinergic synaptosomes have been prepared from sheep brain cortex by means of an immunoaffinity method using a specific anti-(Chol I) antiserum. The [14C]leucine incorporation into proteins of this preparation shows a low cycloheximide and a high chloramphenicol sensitivity. This fact suggests that the mitochondrial protein synthesis system is the only one present in this fraction. PMID- 1711914 TI - Activation of 5-HT3 receptor by 1-phenylbiguanide increases dopamine release in the rat nucleus accumbens. AB - The serotonin-3 (5-HT3) agonist 1-phenylbiguanide (0.1-1.0 mM in perfusate) caused a robust, dose-dependent enhancement of extracellular dopamine content in nucleus accumbens as measured by in vivo microdialysis. This action was antagonized by co-perfusion of the 5-HT3 antagonists zacopride and GR38032F (1 mM in perfusate). Similar effects were observed in 5-HT-denervated rats. These findings suggest that there is a potent modulation of dopamine (DA) release in the nucleus accumbens mediated via 5-HT3 receptors, which appear to be located presynaptically on DA terminals of the mesolimbic DA pathway. PMID- 1711915 TI - Interactions between type 1 plasminogen activator inhibitor, extracellular matrix and vitronectin. AB - Regulation of plasminogen activation is a key process in controlling proteolytic events in the extracellular matrix (ECM) and this regulation is achieved through the action of specific plasminogen activator (PA) inhibitors (PAIs). Type I PAI (PAI-1) is the physiological inhibitor both of urinary-type PA (u-PA) and tissue type PA (t-PA) (Loskutoff et al., 1989) and is a major component of the ECM of cultured cells. This inhibitor may protect ECM constituents against cellular proteases and thus influence the cell migration and tissue destruction that occurs during development, inflammation and tumor metastasis. In this review, we discuss the properties of PAI-1 and the evidence that the binding of PAI-1 to ECM is mediated by serum-derived vitronectin (Vn). PMID- 1711916 TI - Growth factor control of extracellular proteolysis. AB - The involvement of proteases and growth factors in angiogenesis is complex. The angiogenic factor basic fibroblast growth factor (bFGF) induces increased synthesis of both plasminogen activator and collagenase in endothelial cells. In addition, bFGF increases the number of plasminogen activator receptors on the cell surface. Increased production of plasmin may be responsible for the release of soluble complexes of heparan sulfate-bFGF which may be the active form of bFGF. The activity of a negative regulator of angiogenesis, transforming growth factor beta (TGF-beta), is also regulated by proteases since the released latent form of TGF-beta is activated by a surface proteolytic assembly plasminogen activator and plasmin. Since TGF-beta induces an inhibitor of plasminogen activator, the activation reaction is self-regulatory. PMID- 1711918 TI - Syndecan and tenascin: induction during early tooth morphogenesis and possible interactions. PMID- 1711917 TI - Proteolytic balance and capillary morphogenesis. AB - Angiogenesis is the process by which new capillary blood vessels are formed from preexisting vessels. A number of components of this morphogenetic process, including endothelial cell invasion and capillary lumen formation, are believed to be dependent on tightly controlled proteolytic degradation of the extracellular matrix. The critical importance of an appropriate balance between proteases and protease inhibitors in these processes is suggested by two sets of observations. Firstly, that extracellular matrix invasion and capillary lumen formation are inhibited in the presence of an excess of protease inhibitors. Secondly, that when unchecked by protease inhibitors, excessive proteolysis is incompatible with normal capillary morphogenesis. These results clearly suggest that a precisely regulated proteolytic balance is necessary for normal capillary morphogenesis. PMID- 1711919 TI - Expression of tenascin and of the ED-B containing oncofetal fibronectin isoform in human cancer. AB - Tenascin (TN) and the oncofetal ED-B containing fibronectin isoform (B-FN) have been reported to be stromal markers of a number of malignancies. Here we report on studies of the distribution of TN and B-FN in normal adult tissues and in benign and malignant tumors, as well as on the levels of the B-FN mRNA in cultured fetal and non-fetal human fibroblasts originating from different tissues. B-FN has an extremely restricted distribution in normal adult tissues, is not expressed in benign tumors, but is greatly expressed in a high percentage of malignant tumors. On the contrary, human TN in normal adult tissues is less restricted than what has previously been reported and it is largely expressed in a number of both benign and malignant tumors. Moreover, we observed a great variability in the relative amount of B-FN mRNA among the 17 normal human fibroblast cell lines tested. We found very low levels in non-fetal skin fibroblasts and higher levels in fetal lung fibroblasts. We also found differences in the relative amounts of B-FN mRNA between fibroblast cell lines originating from the skin and the lung of the same subject. PMID- 1711920 TI - Extracellular matrix proteins and their receptors in the normal, hyperplastic and neoplastic breast. AB - We studied by immunohistochemistry, the distribution of tenascin (Ten), cellular fibronectin (cFn), laminin and certain pertinent extracellular matrix protein receptors in normal human female breast, variants of fibrocystic disease (FCD), benign tumors, and ductal and lobular carcinomas. Monoclonal antibodies (mAb) to Ten, extradomain A containing cFn (EDAcFn), A and B chains of laminin, and beta-1 (beta-1) and different alpha subunits of intergrins were used. In in-situ ductal and lobular carcinomas, laminin staining had focal gaps, Ten-immunoreactivity displayed periductal or periacinar bands, and cFn showed broad and intense periductal staining; strong reactions for beta-1 and alpha-6 were noted in the basal cytoplasm of non-neoplastic myoepithelial cells while few tumor cells stained weakly. In infiltrating ductal and lobular carcinomas (IDC, ILC), laminin reactivity was weak, uneven or absent around neoplastic clusters whereas stromal staining for Ten and cFn was extensive and strong. In most IDC, moderate beta-1 and alpha-6 staining involved variable subpopulations; one mucinous carcinoma stained strongly and diffusely. In 20-40% of cells in ILC, beta-1 and alpha-6 were localized in delicate, ramified cytoplasmic processes. Indirect immunofluorescence studies with mAbs to other alpha-integrin subunits suggest that in various breast carcinomas only alpha-3 is expressed in tumor cells and that the vessels contained alpha-1 integrin. As compared with the normal breast, FCD and benign tumors, reactivity for Ten and cFn is increased in breast carcinomas while laminin is attenuated and decreased or absent; yet, Ten cannot be regarded as a carcinoma marker since it can be detected in benign tumors, FCD, and even in the normal breast.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711921 TI - Differential expression of tenascin splicing variants in the chick gizzard and in cell cultures. AB - Tenascin is a large disulfide-linked hexameric extracellular matrix glycoprotein. It is a multidomain protein containing many repeated structural units such as heptad-, EGF-like-, and fibronectin type III repeats, as well as a homology to the globular domains of beta- and gamma-fibrinogen. In the chick embryo three major tenascin variants exist. They arise from one gene by alternative splicing of three of its 11 fibronectin type III repeats. Monoclonal antibodies against the alternatively spliced domains allowed us to study the expression of tenascin variants in tissue sections and in cell cultures. In the gizzard, the largest tenascin variant was only detected in the smooth muscle layer and the connective tissue below the epithelium of the villi, whereas the shortest tenascin variant was predominant in the tendons and the intramuscular connective tissue. Differential expression of tenascin variants was also obtained in cell cultures of chick embryo fibroblasts. Fetal calf serum equally stimulated the accumulation of all three tenascin variants, whereas after transformation with polyomavirus middle-T only the secretion of the largest tenascin variant was greatly enhanced. PMID- 1711922 TI - [Fibronectin and laminin changes in rat lungs with interstitial fibrosis]. PMID- 1711923 TI - [Clinical significance of determining free and bound myelin basic proteins and their antibodies in the cerebrospinal fluid]. PMID- 1711924 TI - Colicinogeny of O157:H7 enterohemorrhagic Escherichia coli and the shielding of colicin and phage receptors by their O-antigenic side chains. AB - Twenty O157:H7 enterohemorrhagic Escherichia coli strains from patients with different clinical conditions were tested for colicinogeny and the presence of Verotoxin (VT) genes. From bloody diarrhea cases, 7/8 isolates and from hemolytic uremic syndrome cases 3/5 isolates all synthesized what appeared to be colicin D. The remaining strains, which included 7 from asymptomatic sources, were noncolicinogenic. The plasmid determining the colicin was found to be 1.4 kb larger than the 5.2-kb pColD. The colicin D protein had a molecular weight of about 90,000, whereas the O157 colicin was 87,000. The plasmid was designated pColD157 to reflect these differences. Of O157:H7 isolates 17/20 had genes for both of the Verotoxins VT1 and VT2, and the remaining 3/20 for VT1 only. There was no correlation between the presence of VT determinants and colicinogeny or symptoms. The O157:H7 strains exhibited significant resistance to other colicins and bacteriophages. PMID- 1711925 TI - Acute phase reactants and suppressive E-receptor factor in various groups of patients receiving autolymphocyte therapy. AB - The levels of selected acute phase proteins (APP) and of tumor-associated suppressive E-receptor factor (SER) were determined in sequential serum specimens from (1) cancer patients under treatment with autolymphocyte therapy (ALT) or plasmapheresis, (2) patients with acute infections, (3) patients with autoimmune disorders, and (4) other nonmalignant diseases. The absolute serum levels of APP, including SER, and their patterns of change over time are shown to differ significantly between cancer and non-cancer patients. The absolute levels of APP and the patterns of change over time appear to be useful indicators of immune status and predictors of antitumor response to autolymphocyte (and possibly other anti-cancer) therapy. PMID- 1711926 TI - Palliative care--the fourth phase of cancer prevention. AB - The prevention of suffering represents the fourth phase of a comprehensive cancer control program. The palliative care movement is responsible for the current development of programs which address the suffering of patients with advanced cancer and their families. Palliative care should be integrated with the other elements of cancer controls thus ensuring the continuity of excellent care for patients and strengthening the research and educational base of the palliative care movement. PMID- 1711927 TI - Differential growth inhibition and enhancement of major histocompatibility complex class I antigen expression by interferons in a small-cell lung cancer cell line and its doxorubicin-selected multidrug-resistant variant. AB - Expression of class I and class II major histocompatibility complex antigens on a human small-cell lung cancer cell line and its multidrug-resistant variant was examined before and after exposure to interferon alpha (IFN alpha) and IFN gamma by flow cytometry. Neither IFN alpha nor IFN gamma induced class II antigen expression on the drug-sensitive or resistant cell line. Induction of class I antigen expression along with an inhibition of proliferation was observed in both cell lines after IFN alpha treatment. On the other hand, IFN gamma treatment resulted in growth inhibition and enhancement of class I antigen expression in the sensitive cell line but not the resistant cell line. The differential response of the two cell lines to IFN gamma cannot be directly attributed to the acquisition of drug resistance but it suggests that further investigation of the possibility that drug-sensitive and resistant small-cell lung tumors may respond differently to immunotherapies that include IFN gamma is warranted. PMID- 1711928 TI - Ecdysterone decreases the transcription level of the retrotransposons 1731 and 412 in a Drosophila cell line. AB - Cultured Drosophila melanogaster cells are responsive to the steroid hormone 20 hydroxyecdysone (ecdysterone). The transcripts of two copia-like transposable elements, 412 and 1731 were examined in cells treated for different lengths of time by the hormone. Using dot-blot and Northern-blot analysis, we have shown that the transcripts of these two retrotransposons were rapidly decreased by ecdysterone treatment. PMID- 1711929 TI - The relationship between circulating CD5+ B lymphocytes and in vitro autoantibody synthesis in normal individuals. AB - Recent observations suggest that a subpopulation of B lymphocytes bearing the phenotype CD5+ may be enriched for cells committed to the synthesis and secretion of autoantibodies. We had previously shown that a subset of normal individuals has an expanded subpopulation of B lymphocytes committed to the synthesis of IgM and IgM-rheumatoid factor (RF), and that this condition was associated with HLA DR4 (4). In these studies, we performed simultaneous quantitation of the size of the circulating CD5+ B lymphocyte subpopulation in a group of 20 normal donors, and of the pokeweed mitogen-induced in vitro synthesis of a panel of autoantibodies by the same peripheral blood cells depleted of CD8+ suppressor lymphocytes in 18 of the 20 individuals. The culture supernatants were assayed for total IgM and IgG, RF, IgM, and IgG anti-single-stranded DNA, anti-human thyroglobulin, and anti-tetanus toxoid. The mean percentage CD5+ B cells was 13.5 +/- 2.5%. There was no significant correlation between total B lymphocytes and CD5+ B cells (R = 0.25, P greater than 0.20. Positive correlations were found between the proportion of circulating CD5+ B lymphocytes and synthesis of RF (R = 0.73, P less than 0.001), and IgM anti-DNA (R = 0.58, P less than 0.03). Significant correlations were not found between CD5+ B cells and secreted IgM or IgG antibodies against the exogenous antigen tetanus toxoid, measured in the same supernatants. The antibodies produced in vitro by T cell-dependent B cell activation appear to have limited or no polyspecificity. These results indicate that the size of the circulating CD5+ B cell subpopulation in any given individual contributes importantly to the magnitude of autoantibody synthesis in cultures where T cell-mediated B lymphocyte activation takes place in the absence of suppressor signals. PMID- 1711930 TI - Functional effects of a monoclonal antibody directed against a distinct epitope on 4F2 molecular complex in human peripheral blood mononuclear cell activation. AB - The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation. PMID- 1711931 TI - Variant cDNAs encoding proteins similar to the alpha subunit of chicken CapZ. AB - Chicken adult muscle and liver cDNA libraries were screened with a cDNA, alpha 1, previously isolated from a chicken embryo library by screening with antibodies against the alpha subunit of chicken CapZ. cDNAs with a new coding region, called alpha 2, were found in addition to ones with the alpha 1 coding region. alpha 2 predicts a protein sequence that matches exactly the N-terminal sequence of 5 peptides prepared from CapZ alpha purified from chicken muscle, while the protein sequence predicted by alpha 1 matches the peptides well, but not exactly. The predicted protein sequences of alpha 1 and alpha 2 are very similar to each other, and they are similar to those of the alpha subunit of capping protein from Dictyostelium [Hartmann et al., J. Biol. Chem. 163:5254-5254, 1989] and an actin binding protein from Xenopus [Ankenbauer et al., Nature 342:822-824, 1989]. Other conserved features of the predicted primary and secondary structures are noted. Chicken alpha 1 and alpha 2 are transcribed in all of 7 adult chicken muscle and non-muscle tissues in comparable amounts by Northern analysis. alpha 2 has four poly(A)+RNA transcripts, one of which is rare in liver. alpha 1 has two transcripts. alpha 1 and alpha 2 are encoded by different single-copy genes by Southern analysis of chicken genomic DNA. PMID- 1711932 TI - Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration. AB - Experiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of microfilaments, intermediate filaments, and tight junction-specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub-apical region of the dying hair cell. This region becomes cytokeratin-positive. The two supporting cells meet at the mid-line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens-type junction, but desmosomes are absent. PMID- 1711933 TI - Multivesicular liposomes containing bleomycin for subcutaneous administration. AB - Optimal cancer treatment with cell-cycle-specific agents requires maintenance of a cytotoxic drug level for a prolonged period. We explored the use of multivesicular liposomes as a slow-release depot of bleomycin for systemic administration via the s.c. route. The average volume-adjusted liposome size was 19.1 microns, the half-life of leakage in human plasma was 32.1 h, and the half life of s.c. liposomal bleomycin was 31.8 h. When tested against the s.c. B-16 melanoma model in BDF1 mice, the therapeutic index of single-dose bleomycin given s.c. was significantly improved when the drug was encapsulated in multivesicular liposomes. The efficacy was improved as assessed by both inhibition of tumor growth and increased life span, and the toxicity appeared to be decreased. PMID- 1711934 TI - m-BACOD chemotherapy for intermediate- and high-grade non-Hodgkin's lymphoma. AB - A total of 92 patients with previously untreated intermediate- or high-grade non Hodgkin's lymphoma attending the University Department of Medicine, Queen Mary Hospital, Hong Kong, were treated with the m-BACOD chemotherapy regimen (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone). Additional involved-field radiotherapy was given to 32 (35%) patients. Myelosuppression was the major toxicity, and 5 (5%) treatment-related deaths occurred due to pneumonia, bleomycin sensitivity, doxorubicin cardiotoxicity and reactivation of hepatitis B infection. The overall complete response (CR) rate was 65/92 (71%) and the relapse rate was 22/65 (34%). The disease-free survival of the 65 CR patients at 2 years was 52% and the overall survival of all 92 patients at 3 years was 56%. The CR rate of stage I and II patients was significantly better than that of those with stage III and IV disease (87% vs 59%; P = 0.01), and the CR rate of stage III patients was superior to that of those with stage IV disease (86% vs 50%; P = 0.05). The overall survival of stage III and IV patients was significantly worse than that of subjects with stage I and II disease (31% vs 73%; P = 0.02). Multivariate analysis revealed that the independent prognostic variables significantly determining the CR rate and survival included the clinical stage and the serum lactate dehydrogenase level. From this study, the results of treatment with the m BACOD regimen in patients with advance disease appeared to be similar to those obtained using the conventional CHOP regimen (cyclophosphamide, doxorubicin, vincristine and prednisone). PMID- 1711935 TI - Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells. AB - The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S] methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen specific antibodies demonstrated no antigenic differences between HL-60/S and HL 60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL 60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins. PMID- 1711936 TI - [Construction of a cDNA library of Schistosoma japonicum]. AB - The template mRNA was extracted from Schistosoma japonicum. The first strand of cDNA was synthesized by AMV-reverse transcriptase. The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse transcriptase, T4-DNA polymerase. Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb. Homopolymeric tailing of vector (pUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions. After annealing, the plasmids with cDNA were transformed into E. coli MC1061. The efficiency of cloning was about 10(4)/micrograms mRNA with 30% of the transformants having the inserts of cDNA (Figs. 1-2). PMID- 1711937 TI - Disparate effects of substance P on systemic and coronary beds in conscious dogs. AB - BACKGROUND: Previous studies in anesthetized animals indicated that substance P is a coronary and peripheral vasodilator. However, coronary vasodilation was only transient perhaps because of tachyphylaxis. In the present study, the steady state effects of intravenous substance P on systemic and coronary beds were investigated in conscious, instrumented dogs. METHODS AND RESULTS: With intact autonomic reflexes, 5 ng/kg/min i.v. substance P resulted in increases (p less than 0.01) in cardiac output by 22 +/- 5%, in decreases (p less than 0.01) in mean arterial pressure by 9 +/- 2%, and in total peripheral resistance by 23 +/- 4% 7-9 minutes after the beginning of substance P infusion. Heart rate increased (p less than 0.01) by 35 +/- 7% and left ventricular dP/dt (p less than 0.05) by 13 +/- 4%. In this situation, coronary blood flow decreased (p less than 0.01) by 19 +/- 4% and coronary vascular resistance increased (p less than 0.05) by 13 +/- 5%. Myocardial oxygen delivery was reduced (p less than 0.05) by 13 +/- 5% and the arteriovenous oxygen difference widened (p less than 0.01). After ganglionic blockade, increases in cardiac output, heart rate, and left ventricular dP/dt with substance P administration were abolished, but total peripheral resistance and mean arterial pressure decreased (p less than 0.01) by 12 +/- 3% respectively. Under these conditions, coronary blood flow decreased (p less than 0.01) by 37 +/- 5% and coronary vascular resistance increased (p less than 0.01) by 47 +/- 8%, which were more (p less than 0.01) than control responses. In this situation, myocardial oxygen delivery was reduced (p less than 0.01) by 37 +/- 4% and the arteriovenous oxygen difference widened (p less than 0.01). Intracoronary infusion of substance P (0.4 ng/kg/min) resulted in significant and sustained decreases in coronary blood flow, which were similar before and after ganglionic blockade. CONCLUSIONS: Thus, in conscious dogs, systemic vasodilation is the prevailing effect of substance P, but paradoxically, this peptide simultaneously elicits coronary vasoconstriction. PMID- 1711938 TI - Immunohistochemical localization of basic and acidic fibroblast growth factors in the developing rat heart. AB - BACKGROUND: We used biochemical and immunohistochemical techniques to investigate the expression and distribution of immunoreactive basic and acidic fibroblast growth factors (bFGF and aFGF, respectively) in the hearts of rat embryos (11-20 days of gestation) and of postnatal rats (1-35 days after birth). Our purpose was to assess the relation between the cellular distribution of these growth factors and histogenetic and morphogenetic events in the developing heart. METHODS AND RESULTS: Western-blot analysis of heparin-bound material from neonatal heart extracts identified a single band with a molecular weight of approximately 18 kD for both bFGF and aFGF. Five antibodies for bFGF and three for aFGF showed superimposable distribution of immunoreactive bFGF and aFGF in the heart at each stage examined. At the cellular level, these peptides were localized in the cytoplasm and extracellular matrix. In the myocytes, immunostaining was positive throughout the embryonic and neonatal periods. In the majority of the mesenchymal cells of the cushions and endothelial cells of endocardium and vessels, staining was also positive. In the smooth muscle cells of the aorta, other large arteries, and coronary arteries, immunostaining was intensely positive at early stages of development but became faint or negative with increasing cell differentiation. CONCLUSIONS: The wide distribution of immunoreactive bFGF and aFGF that we identified in the developing rat heart suggests that these growth factors play an important role in heart cytodifferentiation and morphogenesis. Their superimposable distribution may reflect functional interaction. The progressive changes in their distribution suggest a changing role for these peptides during organogenesis. PMID- 1711939 TI - The mechanism of action of cyclosporine: a perspective for the 90's. AB - The introduction of cyclosporine (CyA) as a pharmacological agent has resulted not only in a dramatic improvement in the clinical management of transplant recipients but also in a better understanding of the molecular basis of the immune response, especially T cell function. Knowledge of the mechanism of action of CyA has led to exciting areas of study. Among these are the sequence of regulatory events leading to T cell activation, the potential relevance of isomerases in signal transduction pathways (as the receptor for CyA, cyclophilin has been shown to be an isomerase), the blocking effect of CyA on the development of multidrug resistance, and the striking parallelism between CyA and the newer immunosuppressive agent FK-506. These fields promise to be relevant in solving some of the crucial questions in transplantation immunology, and developing better strategies for immunosuppression. PMID- 1711940 TI - Purification and characterization of cyclosporine and FK-506 binding proteins from a human T-helper cell line. AB - Cytosolic proteins that specifically bind cyclosporine A and FK-506 were isolated and purified from the JURKAT human T-helper cell line. These binding proteins were purified by affinity, molecular weight exclusion and weak cation exchange column chromatography. Radiolabeled cyclosporine A specifically bound to a approximately 17 kDa molecule which is cyclophilin and also bound to a approximately 50 kDa protein(s). Radiolabeled FK-506 did not bind to the approximately 17 kDa molecular weight protein, but specifically bound to soluble approximately 10 kDa and approximately 50 kDa proteins. PMID- 1711941 TI - Office therapy for human papillomavirus infection in nongenital sites. AB - The many different treatment possibilities for the eradication of warts provide evidence that no single method that is completely effective has been found. Although the various methods described herein are usually successful therapies for warts, they are all associated with treatment failures and side effects. Until the perfect cure for warts is discovered, the physician must evaluate every wart carefully before deciding on a course of action. PMID- 1711942 TI - Interferon treatment of anogenital human papillomavirus-related diseases. AB - Because of their antiviral, antiproliferative, and immunoregulatory properties, IFNs are potentially well suited for use in the management of benign HPV-related anogenital diseases. Parenteral and intralesional therapy of condylomata with various natural and recombinant IFN preparations has consistently resulted in beneficial response rates ranging between 40% and 60%, often in patients in whom other therapeutic measures have repeatedly failed. Adverse effects from IFN are dose dependent and generally tolerable at concentrations of IFN found to be effective in the treatment of condylomata, and they are not associated with any known long-term sequelae. When combined with conventional medical and surgical treatment modalities, IFN offers real promise for the control of both extensive primary and recalcitrant HPV-related conditions. PMID- 1711943 TI - Serum amyloid A concentrations in giant-cell arteritis and polymyalgia rheumatica: a useful test in the management of the disease. AB - A prospective clinical study of 23 patients with giant-cell arteritis (GCA) and/or polymyalgia rheumatica (PMR) was undertaken in order to assess the behaviour of the non-specific markers of the disease activity, the erythrocyte sedimentation rate (ESR) and other acute phase markers, particularly the C reactive protein (CPR) and serum amyloid A apolipoprotein (apo SAA) levels during induction of disease remission by prednisone therapy, and possible further recurrence of GCA and/or PMR. The apo SAA measurement is more sensitive than the CRP measurement in determining disease activity (97% and 61%, respectively). The specificity of apo SAA is greater than ESR in the determination of inactive disease (86% and 77%, respectively). In some cases with clinically active disease the ESR and CRP were normal, whereas the apo SAA was always elevated. We conclude that the apo SAA measurement in combination with clinical data and other laboratory parameters may be useful in the management of GCA and/or PMR. PMID- 1711944 TI - Growth retardation in juvenile chronic arthritis patients treated with steroids. AB - Steroids are widely used for treating chronic connective tissue diseases in both adults and children. Unfortunately steroid treatment may produce many side effects and growth retardation is one of these. In juvenile chronic arthritis patients (JCA) oral steroids are mainly administered in severe systemic or polyarticular onset or in JCA complicated by chronic uveitis which is unresponsive to mydriatic and steroid eye drops. Intraarticular steroid treatment is used in pauciarticular onset JCA. This paper will review the literature on steroid treatment and growth rate, and report 35 cases of children with JCA treated with steroids. PMID- 1711945 TI - A cross-sectional study of platelet cyclic AMP in healthy and hypertensive pregnant women. AB - 1. Platelet activation in vivo occurs in healthy pregnant women and is more marked in women with preeclampsia. During pregnancy platelets have also been shown in vitro to be less susceptible to the inhibitory effects of prostacyclin. The cyclic nucleotide cyclic AMP has a key role as an inhibitory second messenger in platelets and mediates the inhibitory effects of prostacyclin. 2. We have studied cyclic AMP in relation to platelet behaviour in healthy pregnant women in the third trimester and in women with pregnancy-induced hypertension and pre eclampsia. Non-pregnant young women were used as controls. 3. Pharmacological agents which increase levels of cyclic AMP were significantly less effective as inhibitors of platelet activation during pregnancy, but there was no difference between the healthy and hypertensive pregnant subjects. 4. Basal platelet cyclic AMP levels were the same in all three groups. However, the production of cyclic AMP in response to a range of adenylate cyclase stimulators was reduced during pregnancy, but again there was no difference between healthy and hypertensive pregnant subjects. 5. The reduction in platelet cyclic AMP levels in pregnancy occurred not only with those adenylate cyclase stimulators which operate via surface receptors, but also on direct stimulation of the enzyme with forskolin. 6. The most likely explanation of these observations is a reduction in the ability of the platelet adenylate cyclase enzyme to respond to stimulation of the third trimester of pregnancy. The consequent reduction in formation of the inhibitory second messenger cyclic AMP may in part be responsible for platelet activation in vivo during pregnancy. There does not appear to be a further difference in platelet cyclic AMP production in hypertensive pregnant women. PMID- 1711946 TI - Comparative gene frequencies between the Canchim breed of Brazil beef cattle and their foundation breeds. AB - 1. Four hundred and seventy-five animals of the Canchim breed from EMBRAPA-UEPAE de Sao Carlos were analyzed by starch gel electrophoresis. 2. Albumin, amylase, transferrins, carbonic anhydrase, hemoglobin and nucleoside phosphorylase gene frequencies were compared with the same frequencies in the foundation breeds, Charolais and Indubrazil. 3. Theoretical values obtained considering the 5/8 European, 3/8 Zebu admixture are near the means calculated from our results and data from literature. PMID- 1711947 TI - Characterization of monoclonal antibodies to the globular domain of collagen IV. AB - Monoclonal antibodies were produced against NC1, the globular noncollagenous domain of collagen IV, isolated from bovine glomerular basement membrane. Cells from eight positive wells were cloned and the resulting monoclonal antibodies were studied in detail by immunofluorescence on human kidney sections, by Western blot and by ELISA against denatured subunits from NC1 hexamers and against native NC1 hexamers from different tissues. The monoclonal antibodies could be divided into two groups. Firstly, those monoclonal antibodies that, in ELISA and Western blot, reacted with peptides related to the alpha 1 chain of collagen IV and stained all basement membranes in the kidney. Secondly, a monoclonal antibody that, in ELISA and Western blot, reacted with peptides related to the Goodpasture antigen, the alpha 3 chain of collagen IV. When this antibody was applied to human kidney sections it stained the glomerular basement membrane very intensively. Bowman's capsule and some tubular basement membrane were also stained, although to a lesser extent. This staining pattern is the same as that observed with sera from patients with Goodpasture's syndrome. An attempt was made to separate different subtypes of the NC1 hexamer. A monoclonal antibody from the first group was used to make an affinity chromatography column. Glomerular basement membrane digested with collagenase was separated on this column and the collected fractions were analyzed by ELISA and SDS-PAGE. The result from this study support the idea that glomerular basement membrane is composed of at least two different subtypes of type IV collagen. PMID- 1711948 TI - Immunorecognition of chondrocytes in articular cartilage activated by synovial interleukin 1. AB - A polyclonal antiserum which recognizes surface epitopes on IL1-activated pig chondrocytes has been used to immunolocalize chondrocytes responding to IL1 produced during co-culture of pig synovium and articular cartilage. Activation of the chondrocytes by the cytokine was restricted to the articular and subarticular region of the cartilage adjacent to the synovium. Chondrocyte activation was also seen when human rheumatoid synovium was co-cultured with the cartilage. The presence of IL1 in some synovial cells was confirmed by immunolocalization using antisera specific for IL1 alpha and IL1 beta. PMID- 1711949 TI - A histological and histochemical study on glycosaminoglycans in epiphysial cartilage of osteochondrodysplasia rat (OCD/OCD). AB - The osteochondrodysplasia rat, inherited by a single autosomal recessive lethal gene ocd, shows a typical dwarfing syndrome with systemic subcutaneous edema. The skeletal system is most severely affected. The affected neonates are associated with cleft palate, abnormal kidney position and central nerve system malfunction. The present study describes histological and histochemical appearances of the affected epiphysial cartilage. Irregular columnization, thinner hypertrophic zone, swelled chondrocytes, tightly packed chondrocytes with a poor amount of cartilage matrix was found in the affected. The defining characteristic was a wide-spread degenerating area from the resting to hypertrophic zone. The extracellular matrix (ECM) reacted weakly for the glycosaminoglycans (GAGs). A reduced content of sialic acid in the ECM was suggested. It is concluded that the cartilage abnormalities in the ocd/ocd is a new type disease of osteochondrodysplasia possibly due to some defects in GAGs and/or sialic acid metabolism. PMID- 1711950 TI - Medial collagen organization in human arteries of the heart and brain by polarized light microscopy. AB - The mechanical properties of collagen as a biopolymer ensures that collagen has a significant influence on the mechanical behavior of the host tissue. Structural organization is a key to that influence. We have assessed this relationship quantitatively in the tunica media of arteries from the heart and brain, using the polarizing light microscope and Universal stage. Arteries from 22 autopsies were isolated, cannulated and fixed with 10% buffered formalin, at a distending pressure spanning normal values in vivo. We prepared the tissue for light microscopy, with paraffin embedding, sectioning at 7 microns, and staining with picrosirius red to enhance the natural birefringence of medial collagen. Individual measurements, 30 to 50 per arterial section, referenced against the central axis of the vessel segment, revealed a coherent organization, with an average orientation which was within 1 to 2 degrees of being perfectly concentric for all artery segments. Analysis was done with Lambert projections and circular statistics. We calculated the circular standard deviation, which was 5.2 degrees for 27 brain arteries (S.D. 1.9 degrees) and 5.6 degrees (S.D. 2.1 degrees), for 5 coronary arteries sectioned at less than 15 degrees. Our interpretation is that medial collagen can be strained even though highly aligned, revealing a mechanical property which contrasts that of type I collagen. PMID- 1711951 TI - Pharmacologic and other chemical causes of interstitial lung disease. PMID- 1711952 TI - Effect of antiallergic agents and bronchial hypersensitivity in short-term bronchial asthma. AB - The effect of antiallergic agents with no antihistamine activity on bronchial hypersensitivity to histamine inhalation was studied in 37 asthmatic patients. Improvement in bronchial hypersensitivity to histamine was observed in 11 out of the 24 (46 percent) antiallergic agents-treated patients, but in none of the 13 (0 percent) untreated patients. The 11 patients whose bronchial hypersensitivity improved with antiallergic agents consisted of eight short-term cases of less than one year's duration and three long-term cases of more than one year's duration. Thus, improvement in bronchial hypersensitivity was observed in 8 of 11 (73 percent) short-term cases, and 3 of 13 (23 percent) long-term cases. A significant improvement in %FEV1 was observed only in the short-term cases treated with antiallergic agents, but the improvement of baseline FEV1 did not seem to explain entirely the improvement in bronchial hypersensitivity seen. The decrease in bronchial hypersensitivity was in parallel with that of other asthmatic symptoms. These results suggest that antiallergic agents might be most effective in the treatment of asthmatic patients with a short-term disease duration. PMID- 1711953 TI - Glomerulopathy in patient with Donohue syndrome (leprechaunism). AB - OBJECTIVE: To evaluate renal structure in a child with Donohue syndrome (leprechaunism), who at 10 yr of age was noted to have hypertension, microalbuminuria, and enlarged kidneys, a renal biopsy was performed. RESEARCH DESIGN AND METHODS: The renal biopsy tissue was evaluated by light and electron microscopy with standard stereological techniques to measure glomerular volume, glomerular basement membrane width, fractional mesangial volume, and peripheral capillary filtering surface density. RESULTS: On renal biopsy, there was a marked increase in glomerular volume, glomerular basement width, and mesangial volume, findings similar to those seen in patients with diabetic nephropathy. CONCLUSIONS: This patient with marked insulin resistance associated with Donohue syndrome demonstrates renal and glomerular enlargement and morphometric glomerular changes similar to those seen in patients with diabetic nephropathy. In unusual syndromes with hyperglycemia and hyperinsulinemia, renal structural and functional changes typical of traditional diabetes mellitus may be seen. PMID- 1711954 TI - [The role of plasma substance P and catecholamines in hypertension]. AB - In the present study, the contents of plasma substance P (SP) and noradrenaline (NA) and adrenaline (AD) in human normotensive subjects and patients with essential hypertension as well as Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured. The results showed that: (1) The levels of plasma SP in both hypertensive subjects were lower than that in both normotensive subjects. (2) The concentration of plasma NA and AD were significantly higher in patients with essential hypertension than that in normotensive subjects. (3) The levels of plasma SP increased and the concentrations of NA and AD decreased after antihypertensive drug treatment. These results suggest that both plasma SP and catecholamines were involved in essential hypertensive pathogenesis. PMID- 1711955 TI - [The force-interval relationship of the heart]. PMID- 1711956 TI - Gastric cytoprotection. What does it really mean for the prescriber? PMID- 1711957 TI - New developments in the drug treatment of glaucoma. AB - This article reviews standard treatment modalities for patients with glaucoma and describes 3 classes of drugs which are undergoing development: apraclonidine (aplonidine, ALO 2145), an alpha 2-adrenergic agonist which has been released for clinical use; topical carbonic anhydrase inhibitors, a modification of the systemic carbonic anhydrase inhibitors currently in use; and prostaglandins (PGs), a new class of drugs with topical ocular hypotensive activity. Standard treatment modalities include parasympathomimetic agents such as pilocarpine, carbachol, and phospholine iodide, which lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork. A newer form of pilocarpine as a gel produces a longer action. Adrenergic agonist medications, such as epinephrine (adrenaline) and its prodrug dipivefrine (dipivalyl epinephrine), function by increasing uveoscleral outflow and trabecular outflow facility. A decrease in aqueous formation by the ciliary processes is thought to be the mechanism of action of beta-adrenoceptor antagonists, but the physiological basis for this action has not been clearly demonstrated. A newer beta-blocker, betaxolol, has relatively selective beta 1-blocking activity. Carbonic anhydrase inhibitors are nonbacteriostatic sulphonamide derivatives which decrease aqueous formation by the ciliary body. Almost 50% of patients taking these medications are unable to tolerate them because of their adverse effects, and there is thus much interest in the development of a topical carbonic anhydrase inhibitor with the potential for fewer adverse effects. MK 507 is the most recent and most potent compound in the series of topically active carbonic anhydrase inhibitors. Apraclonidine hydrochloride is a derivative of clonidine hydrochloride, an alpha 2-adrenergic agonist. Clonidine has previously been shown to lower IOP significantly, but has the potential to produce marked lowering of both systolic and diastolic blood pressures. Its major ocular effect appears to be a decrease in aqueous production. The structural modification to apraclonidine decreases corneal absorption and the drug's ability to cross the blood-brain barrier, minimising the risk of centrally mediated cardiovascular side effects. Apraclonidine may also influence secondary avenues of aqueous outflow, such as uveoscleral outflow, and may also affect conjunctival and episcleral vascular flow. It produces a mean decrease in IOP of 25% for as long as 12 hours. Adverse effects include blanching of the conjunctiva, minimal mydriasis and eyelid retraction. This drug has been approved in the US for use in prevention of elevated IOP after argon laser trabeculoplasty and iridotomy, and has potential uses in preventing an IOP rise after YAG laser posterior capsulotomy and cataract surgery in patients already on other antiglaucomatous medications.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711958 TI - Dissociation between the antinociceptive and anti-inflammatory effects of the nonsteroidal anti-inflammatory drugs. A survey of their analgesic efficacy. AB - The authors challenge the general view that the analgesic effect of the nonsteroidal anti-inflammatory drugs (NSAIDs) can be universally attributed to their inhibitory effects on the synthesis of peripherally formed prostaglandins. Analgesic activity by some of these compounds in the reduction of physiological pain elicited by a single noxious stimulus, or the treatment of acute pain which results from sudden trauma to otherwise healthy tissue, is better described as an antinociceptive effect. Single-dose studies in the dental pain model that have been conducted in double-blind conditions and included a placebo control group have been reviewed; those NSAIDs which are significantly superior to the reference compound aspirin 650mg and those which could represent real alternatives to the use of narcotics in certain situations for the management of acute pain have been identified. Azapropazone, diflunisal, naproxen, oxaprozin and tolmetin are all weak inhibitors of prostaglandin synthesis, yet they have been shown to be more effective than aspirin. In a model of joint pain, azapropazone 600mg has been shown to be as effective as pethidine (meperidine) 100mg despite being the weakest inhibitor of prostaglandin synthesis. Whether the antinociceptive effect of azapropazone acts at a peripheral or a central level, or both, is not clear; evidence for the effects of NSAIDs on the central nervous system (CNS) is discussed. Historically, the antinociceptive character of some NSAIDs is apparent in several studies in both animals and humans. More recently, experimental algesimetry models designed to distinguish the antinociceptive effects of NSAIDs include the use in humans of photoplethysmography and computer supported infrared thermographic imaging. PMID- 1711959 TI - The management of diabetes mellitus in older individuals. AB - Nearly 50% of individuals with type II diabetes mellitus are over the age of 65 years. There are numerous reasons to maintain blood glucose levels below 11.1 nmol/L (200 mg/dl) in older persons, and there are a number of changes often seen with advancing age that persons, and there are a number of changes often seen with advancing age that may interfere with the management of diabetes mellitus, e.g. hypodipsia, anorexia, visual disturbance, altered renal and hepatic function, depression, impaired basoreceptor response and multiple medications. Hyperglycaemia appears to produce cognitive impairment which may lead to poor compliance. It is often difficult to manipulate diet in older people, and in fact dietary changes can lead to severe protein energy malnutrition. High maximum voluntary oxygen intake has been correlated with increased glucose disposal, but there is little evidence that physical exercise can improve diabetic control in the elderly. Oral sulphonylurea hypoglycaemic agents are extremely useful in the treatment of diabetes in these patients, but it should be remembered that they are more liable to develop hypoglycaemia than are younger diabetics. The role of metformin in the management of older diabetic patients is poorly studied. Many older persons can cope well with insulin therapy, but those with visual disturbances often make errors when drawing up insulin and require special attention. Combination therapy of insulin with oral hypoglycaemic agents is not recommended in this group of patients, and serum fructosamine is preferred to glycated haemoglobin to monitor control. Successful management of elderly diabetic patients thus requires an interdisciplinary team approach. PMID- 1711960 TI - Use of oral rehydration therapy in acute watery diarrhoea. A practical guide. AB - Various foods and fluids have been used in traditional treatments for diarrhoeal illnesses in infants and children for centuries. During the last 2 decades, however, with the advent of an improved scientific understanding of oral rehydration, effective treatment of dehydrating diarrhoea has been improved, expanded and simplified. The appropriate use of oral rehydration solutions depends on an appreciation of the physiological mechanisms of diarrhoeal illness. Since dehydrating diarrhoea is such a common cause of morbidity and mortality, and because oral rehydration therapy is inexpensive, effective and adaptable, it has become a powerful intervention for improvement in health care for all ages. Newer formulations using starches, cereals and/or amino acids promise to make oral rehydration therapy even more efficacious and acceptable. Nearly all developing countries now have active national diarrhoeal control programmes which facilitate rehydration therapy as the first treatment of diarrhoea while discouraging the use of other diarrhoea medicines (e.g. kaolin and pectin, antispasmodics, etc.). Industrialised countries are also increasingly using oral rather than intravenous fluids. For most patients with lesser degrees of dehydration (up to about 8%) or no detectable dehydration, oral rehydration therapy is the only form of hydration needed. The 'standard' oral replacement solution recommended by the World Health Organization has the advantage of wide experience, demonstrated safety and effectiveness and wide availability. However, rehydration is only part of the management of diarrhoea, and nutritional management (including electrolytes and glucose, alternative substrates to glucose, inclusion of starches and proteins in the solution if possible, etc.) must also be integrated into programmes for diarrhoea control. PMID- 1711961 TI - Ondansetron. Therapeutic use as an antiemetic. AB - Ondansetron (GR 38032F) is a highly selective 5-HT3 receptor antagonist, one of a new class of compounds which may have several therapeutic applications. Animal and clinical studies show that ondansetron reduces the 24-hour incidence and severity of nausea and vomiting induced by cytotoxic drugs, including cisplatin, and by single exposure, high dose radiation. Ondansetron is more effective than high dose metoclopramide in the 24 hours following chemotherapy, and preliminary clinical evidence suggests that it is equally effective in the following 4 days. It is also more effective than the 'moderate' doses of metoclopramide used to suppress emesis following radiotherapy. The antiemetic efficacy of ondansetron is enhanced by dexamethasone in cisplatin-treated patients. Importantly, extrapyramidal effects have not been reported with ondansetron. Further comparisons are required with standard combination antiemetic therapy to complement the data presently available. Thus, ondansetron is a promising new agent for prophylaxis against nausea and vomiting in chemotherapy and radiotherapy. It may be particularly useful in young and elderly patients who are more susceptible to extrapyramidal symptoms induced by high dose metoclopramide. With its improved tolerability and clinical response profiles, ondansetron represents an important advance in a difficult area of therapeutics. PMID- 1711962 TI - Adenosine. An evaluation of its use in cardiac diagnostic procedures, and in the treatment of paroxysmal supraventricular tachycardia. AB - Adenosine (adenine riboside), administered either as the free base or as the 5' triphosphate (ATP) by rapid intravenous bolus, depresses atrioventricular (AV) nodal conduction, resulting in transient AV block. Adenosine is the active agent and ATP is rapidly converted to adenosine after exogenous administration. By blocking the anterograde AV nodal limb of a re-entrant circuit, adenosine 6 to 12 mg (or ATP 10 to 20 mg) converts almost all episodes of paroxysmal supraventricular tachycardia (PSVT) involving the AV node within 30 seconds of administration. This is at least equivalent in efficacy to verapamil in adults, and superior to lanatoside C in children, with a considerably more rapid onset of action. Furthermore, if a dose of adenosine is ineffective, the exceptionally short plasma half-life of the adenyl nucleosides (less than 10 sec) allows rapid upward dosage titration until PSVT is terminated. Because the induced conduction block primarily affects the AV node, adenosine is a useful diagnostic tool in patients with broad or narrow QRS complex tachycardia; it terminates arrhythmias dependent on the AV node, unmasks other supraventricular mechanisms during transient AV block, but almost always has no effect on ventricular tachycardia. Noncardiac adverse effects, i.e. flushing, dyspnoea and chest pain, may occur during acute arrhythmia termination or diagnosis with adenosine, and arrhythmias may develop; however, these effects are usually transient (lasting less than 1 minute). Adenosine has also been used to induce coronary vasodilation in patients undergoing thallium-201 single photon emission computed tomography (201Tl SPECT), 2-dimensional echocardiography or positron emission tomography to evaluate suspected coronary artery disease. Intravenous infusion of adenosine 140 micrograms/kg/min for 6 minutes was generally associated with only mild adverse effects. These usually resolved within 1 to 2 minutes of discontinuing adenosine, although occasionally patients required aminophylline and/or nitroglycerin (glyceryl trinitrate). Diagnoses based on the results of scintigraphy were of a sensitivity, specificity and predictive accuracy comparable to those achieved with exercise- or dipyridamole-201Tl SPECT. Adenosine is therefore particularly suitable for the diagnosis of tachycardias and the acute management of PSVT involving the AV node in all age groups, without the risks of cardiac arrest and hypotension associated with verapamil. Furthermore, intravenous adenosine infusion may be used to induce coronary vasodilation in patients unable to perform exercise stress tests for 201Tl scintigraphy, and is well tolerated. PMID- 1711963 TI - Tenoxicam. An update of its pharmacology and therapeutic efficacy in rheumatic diseases. AB - Tenoxicam administered orally, rectally or parenterally is an effective analgesic and anti-inflammatory agent for the symptomatic treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis and various rheumatic conditions such as tendinitis, bursitis, sciatica, back pain and gouty arthritis. In clinical trials its efficacy is at least equivalent to that of other NSAIDs and it is at least as well tolerated as piroxicam and probably better tolerated than diclofenac, indomethacin and ketoprofen. Compared with many other NSAIDs, tenoxicam offers certain advantages in that it is conveniently administered once daily and dosage adjustment is not required in the elderly or in patients with renal or hepatic impairment. PMID- 1711965 TI - Sudden cardiac death after myocardial infarction. AB - Recent advances in the understanding of the mechanisms of sudden cardiac death have been paralleled by technical advances in diagnosis and treatment, involving ambulatory Holter monitoring and the use of implantable defibrillators. Risk factors predisposing toward sudden cardiac death in the postmyocardial infarction setting and in patients with congestive heart failure include the presence of ventricular ectopy [greater than 10 premature ventricular contractions (PVC) per hour], frequent episodes of ventricular pairs and nonsustained ventricular tachycardia on 24-hour Holter monitoring, and a depressed left ventricular ejection fraction. Additional risk factors for sudden cardiac death in coronary artery disease include arterial stenosis in coronary vessels supplying intact myocardium remote from the infarction site, the presence of late potentials on the signal averaged ECG, and attenuation of the normal variation in heart rate. The ability to induce sustained ventricular tachycardia (SVT) on electrophysiological testing is highly predictive of sudden cardiac death after myocardial infarction. Conversely, the ease of suppression of the induced tachycardia with antiarrhythmic agents is correlated with the risk of subsequent lethal ventricular arrhythmia. The detrimental effect of frequent ventricular ectopy (greater than 10 PVC/h) on survival in coronary artery disease is particularly pronounced in patients with moderately well preserved left ventricular function [ejection fraction (EF) greater than 30%], thereby suggesting that these patients may be better served by antiarrhythmic therapy than those with severely depressed left ventricular function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1711964 TI - Olsalazine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in inflammatory bowel disease. AB - Olsalazine (sodium azodisalicylate; azodisal sodium) is an anti-inflammatory agent designed to deliver its active moiety, mesalazine (5-aminosalicylic acid; mesalamine), to the colon while avoiding the adverse effects associated with the use of a sulfapyridine carrier. As a prodrug, olsalazine is an effective oral treatment for both active ulcerative colitis and for maintenance of disease remission and may possibly be of benefit in patients with Crohn's colitis. Findings from both short and long term noncomparative and comparative studies demonstrate that olsalazine 1 to 3g daily in divided doses improves clinical signs and symptoms of colitis in approximately 60 to 80% of patients with acute ulcerative colitis of mild to moderate severity. This improvement rate was similar to that obtained with sulfasalazine. Lower doses of olsalazine, usually 1g daily in divided doses, also maintained remission in patients with chronic ulcerative colitis. While olsalazine effectively delivers mesalazine to the colon, the prodrug itself increases net luminal water secretion and accelerates gastrointestinal transit of a meal. The resulting diarrhoea (occurring in approximately 17% of patients and resulting in withdrawal from therapy in 6% of patients) is distinguishable from that associated with inflammatory bowel disease by the high water content and the absence of blood. Olsalazine-induced diarrhoea usually occurred soon after initiation of olsalazine therapy or dosage increase, was more frequent with higher doses and was usually transient. Dosage reduction, increases in frequency of dosing and concomitant administration with food reduced the severity in many patients with persistent olsalazine-induced diarrhoea. With the exception of diarrhoea, olsalazine was generally well tolerated. Fewer than 14% of patients allergic to or intolerant of sulfasalazine had similar reactions to olsalazine. Olsalazine appears to be a suitable therapy for the treatment of first attacks as well as acute exacerbation of mild to moderate acute ulcerative colitis, and for the maintenance of remission in patients with chronic ulcerative colitis. PMID- 1711966 TI - Mechanisms of sudden cardiac death. AB - Although coronary artery disease is the most frequent cause of sudden cardiac death, it may be caused by a heterogeneous group of disorders. Acute ischaemia is responsible for about half the cases of sudden death after acute myocardial infarction, and is manifested through ventricular fibrillation or polymorphic ventricular tachycardia. Several factors affect the haemodynamic consequences of a ventricular arrhythmia. Re-entry is the mechanism involved in patients with a history of myocardial infarction and therapy should be individualised and directed to the arrhythmia. Simple decision trees are available that can help to find the most appropriate therapy; implantable defibrillators are the most effective modality in certain very high risk subsets. PMID- 1711968 TI - The value of oral amiodarone in the treatment of ventricular arrhythmias in heart disease. AB - The antiarrhythmic properties of amiodarone at the ventricular level were discovered in the early 1970s. The unanimously recognised efficacy of amiodarone includes a weak negative inotropic effect and compensatory vasodilatory properties, making amiodarone particularly suitable for treating the potentially malignant arrhythmias associated with organic disease. In a review of 611 hospitalised patients on amiodarone, and 353 patients in whom the drug had been prescribed, over a 52-month period in our 60-bed department, we noted that amiodarone was prescribed in 53% of patients for arrhythmias and in 47% of patients for coronary insufficiency. Ventricular arrhythmias represented 13% of the rhythmic indications. These indications differ from those in the USA. The efficacy (70 to 90%) of amiodarone in ventricular extrasystoles has been shown in open studies. In coronary patients, the antiarrhythmic activity of amiodarone is superior to that of propranolol. However, there has been no controlled study because the need for a loading dosage, and the electrocardiographic effects render such studies difficult. After myocardial infarction, ventricular arrhythmias constitute a significant risk factor independently of prognosis; amiodarone may be useful in this indication, and studies of the European Myocardial Infarction Amiodarone Trial (EMIAT) type will examine its value here. Since 1973, it has been recognised that amiodarone can prevent ventricular tachycardia in 55 to 89% of patients in the clinical situation. After a long standing controversy, the positive predictive value of programmed stimulation has finally been agreed on. In hypertrophic cardiomyopathy, retrospective studies suggest a reduction in mortality in patients treated with amiodarone. By contrast, the value of amiodarone in dilated cardiomyopathy requires more intensive investigation. We consider amiodarone to be indicated in ventricular arrhythmic complexes, particularly if they are associated with an ejection fraction of less than 35% and/or atrial fibrillation. The value of amiodarone in arrhythmias associated with heart failure needs to be evaluated. In conclusion, amiodarone is a powerful antiarrhythmic agent but, because of the possibility of dose- and duration-dependent side effects, evaluation of the risk: benefit ratio in each indication is needed. PMID- 1711967 TI - When is drug therapy warranted to prevent sudden cardiac death? AB - Sudden arrhythmic death is an important contributor to the mortality rate in patients with cardiac disease, accounting for over 450,000 deaths per year in the USA alone. About 80% of such patients, particularly those survivors of acute myocardial infarction with low ventricular ejection fractions, have coronary artery disease. The remainder have cardiomyopathy or valvular disease and the common denominator in all these subsets of patients is the association with complex and frequent premature ventricular contractions (PVCs). The most common mechanism of death is ventricular tachycardia (VT) deteriorating into ventricular fibrillation (VF); the initiating factor is a PVC (the trigger mechanism). Thus, if an effective antiarrhythmic (? antifibrillatory) regimen could be identified, these subsets of patients clearly constitute targets for mortality reduction by pharmacological suppression. The question that has arisen is whether suppression of PVCs will reduce the incidence of sudden death (the PVC hypothesis). The alternative approach is to modify the arrhythmogenic substrate in the ventricle by eliminating the source of ischaemia, extirpating the ectopic focus, dividing re-entry circuits, or pharmacologically prolonging the refractory period so that VT does not deterioate into VF (the antifibrillatory approach). Whether sudden death in postinfarct survivors could be reduced has been the subject of study. These patients are at high risk of sudden death or reinfarction, the risk being greatest in those with a low ventricular ejection fraction, continuing myocardial ischaemia and asymptomatic high density and complex PVCs. Numerous trials have been performed to determine whether beta-blockers, calcium antagonists and antiarrhythmic agents reduce the incidence of sudden death and reinfarction in survivors of myocardial infarction. beta-Blockers remain the only agents which, when given prophylactically, have been found to reduce the incidence of sudden death and reinfarction (by 18 to 45% in the first 2 years after infarction). The reduced incidence of sudden death appears to be associated with a reduction in VF, but not in PVCs. A meta-analysis of data from trials with calcium antagonists found that these drugs either had no effect or tended to increase mortality (by an average of 6%), indicating that an effect on ischaemia alone is unlikely to be the sole mechanism mediating the effect of beta-blockers. The divergent effects of beta-blockers and calcium antagonists are unexplained, but may be partly due to a lack of a significant bradycardiac effect of calcium antagonists. There were no differences in effect between different calcium antagonists.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1711970 TI - Practical aspects of the use of amiodarone. AB - Amiodarone is a class III antiarrhythmic drug with sympatholytic properties, which prolongs the refractory period of all cardiac tissues, depresses sinus node automaticity and atrioventricular nodal conduction. It is active on all cardiac arrhythmias, its use being limited by the risk of side effects, mainly extracardiac, which are dependent upon dosage and duration of treatment. The use of an oral loading dose of intravenous infusion to rapidly obtain an efficacious blood concentration may minimise the delay in onset of drug action. Amiodarone progressively accumulates in cardiac muscle and is eliminated slowly, allowing drug free periods (2 days a week) on long term therapy. Pharmacokinetic interaction with digoxin, class I antiarrhythmic drugs and warfarin must be considered during combination therapy, as well as the potentiation of electrophysiological effects when amiodarone is co-administered with combined calcium antagonists and beta-blockers. PMID- 1711969 TI - Amiodarone in long term prophylaxis. AB - Amiodarone is predominantly a potassium channel inhibitor which prolongs repolarisation and refractoriness, and thus qualifies as a Group III antiarrhythmic agent. In addition, it possesses a variety of electrophysiological actions such as sodium channel blockade, calcium channel blockade and noncompetitive inhibition of adrenergic receptors. In the studies reported below, the incidence of successful treatment of refractory ventricular arrhythmias with amiodarone appears to range between 50 to 60% in the first year. However, there are very few prospective randomised studies which assess its efficacy in controlling ventricular tachycardia in comparison with placebo or another antiarrhythmic compound. As there have been no controlled studies to examine the impact of amiodarone in preventing ventricular tachycardia in survivors of cardiac arrest compared with either no treatment or with alternative therapies, the actual efficacy of amiodarone in patients who have survived a cardiac arrest is virtually unknown. Although there are indications that amiodarone reduces the incidence of sudden death in patients with malignant arrhythmias, definitive evidence based on controlled trials is not available. PMID- 1711971 TI - Can we predict sudden cardiac death? AB - Up to now only 30 to 40% of patients who die suddenly can be identified as likely candidates before the event. Risk factors in these asymptomatic subjects include a familial history of coronary artery disease, high blood cholesterol levels, hypertension, smoking and, more importantly, an abnormal ECG at rest or during exercise. The predictive value of these abnormalities is too low to justify more detailed clinical investigations in most of these asymptomatic subjects. Exceptions might be the group of patients with multiple risk factors and competitive sportsmen. Sudden cardiac death is a well known complication in patients with hypertrophic and dilated cardiomyopathy. Risk factors in hypertrophic cardiomyopathy include a familial history of this disease, syncope and increasing age. Furthermore, in the adult, the presence of nonsustained episodes of ventricular tachycardia during Holter monitoring seems to indicate an increased risk of sudden cardiac death. In idiopathic dilated cardiomyopathy, the presence of frequent episodes of ventricular pairs and/or episodes of ventricular tachycardia during Holter monitoring, together with a reduced left ventricular ejection fraction, characterises the patient at risk of sudden cardiac death. In patients with coronary artery disease, the patient at risk of sudden cardiac death can be identified by investigating the following: coronary anatomy; global and regional left ventricular function; the presence of ischaemia during rest and/or exercise; the presence of late potentials, by means of the signal-averaged ECG; the presence of spontaneous ventricular arrhythmias (especially sustained and nonsustained ventricular tachycardia); and the results of electrophysiological testing. On the basis of these investigations, 3 subgroups can be distinguished: patients at low risk, medium risk, and high risk of sudden cardiac death. PMID- 1711972 TI - The interaction between cells in different phases of the cell cycle and aminocarb. AB - Synchronous cultures of Chlamydomonas segnis Ettl. were treated with aminocarb (0.5, 5.0, 10.0, or 50.0 micrograms ml-1) at each specific phase of the cell cycle, namely, G1, S, G2, and M phases. The subsequent effects of some macromolecular products were assayed at the end of the M phase. Aminocarb treatments of 0.5 microgram ml-1 were at the no effects level on the parameters monitored. However, the higher concentrations of aminocarb tested, namely, 5, 10, and 50 micrograms ml-1, dramatically affected not only macromolecular syntheses but also some cell cycle events. Algistatic and algicidal effects were obtained with some treatments. PMID- 1711973 TI - Drastic change of reverse micellar structure by protein or enzyme addition. AB - On addition of cytochrome c to a AOT reverse micellar solution, the percolation process usually observed at high temperatures and surfactant concentrations, occurs at room temperature. This is observed either at relatively high water content at a given cytochrome c concentration or at low content on increasing the cytochrome c concentration. On increasing the water content a phase transition is observed with two optically transparent phases. A similar phase transition is observed on solubilizing various enzymes. The temperature of the transition appears to be strongly dependent on the location of the macromolecule in the reserve micelle. PMID- 1711974 TI - Functional expression of the endogenous Thy-1 gene and the transfected murine Thy 1.2 gene in rat basophilic leukemia cells. AB - An interaction of MRC OX7 monoclonal antibody with Thy-1.1 antigen of rat peritoneal and pleural mast cells has been previously shown to induce rat mast cell activation (L. Draberova, Eur. J. Immunol. 1989, 19: 1715). In the present study we analyzed the expression and function of the Thy-1 antigen in rat basophilic leukemia cells, clone RBL-2H3. Two RBL-2H3-derived cell lines with stable expression of murine Thy-1.2 antigen, after transfection of a genomic clone of murine Thy-1.2 or Thy-1.2 cDNA under the control of simian virus 40 promoter, were also analyzed. Direct radioantibody binding assays, indirect immunofluorescence studies and flow cytometry analyses revealed that both endogenous Thy-1.1 and transfected murine Thy-1.2 gene products were expressed on the surface of the cells under study. Analysis of the distribution of the Thy-1 antigen in situ in cells grown attached to tissue culture vessels located the Thy 1 predominantly in regions of cell-cell contacts. Incubation of RBL-2H3 cells with Thy-1.1-specific antibodies, or of the transfected cells with both Thy-1.1- and Thy-1.2-specific antibodies, induced a rapid early increase in the concentration of intracellular free calcium [( Ca2+]i) released from internal stores. Sustained increase of [Ca2+]i required the presence of Ca2+ in the extracellular medium. The increase in [Ca2+]i was followed by histamine release from the target cells. The combined data indicate that RBL-derived cells can be used as a useful model system for analysis of Thy-1 antigen-mediated activation of rat mast cells. PMID- 1711975 TI - Characterization of the CAMPATH-1 (CDw52) antigen: biochemical analysis and cDNA cloning reveal an unusually small peptide backbone. AB - The CAMPATH-1 (CDw52) antigen has been purified from human spleen. The antigenic epitope is heat stable but sensitive to mild alkali treatment. Experiments with phosphatidylinositol-specific phospholipase C indicate that it is anchored by a glycosylphosphatidylinositol (GPI) anchor. An N-terminal sequence of 11 amino acids was determined, followed by an abrupt stop. Using short overlapping mixed oligonucleotide primers, cDNA synthesized from the mRNA of a human B cell line was amplified by the polymerase chain reaction. The product was used to isolate cDNA clones and the full amino acid sequence of the CAMPATH-1 antigen was deduced. It consists of 37 amino acid residues plus a 24-residue signal peptide. It has all the features expected for a GPI-anchored membrane protein except that the predicted mature protein is remarkably short, comprising no more than 18 residues and possibly as few as 12 (depending on the GPI linkage site). Potential attachment sites for carbohydrate are present and it is shown that the antigen contains N-linked oligosaccharide(s). This structure accounts for the known properties of the antigen, though the exact reasons why it is such a good target for cell lysis in vitro and in vivo are not yet clear. PMID- 1711976 TI - Identification of a murine monoclonal antibody specific for an allotypic determinant on mouse CD3. AB - A murine monoclonal antibody (mAb; 7D6) that was mitogenic for T cells was derived from 129/Sv animals immunized with a T helper clone from C57BL/6 origin. Fluoresceinated 7D6 labeled T cells from most common mouse strains but not from 129/Sv and LP/J animals, and this labeling was inhibited by the anti-CD3 epsilon mAb 145-2C11. The mitogenicity of 7D6 for T cells had a similar strain specificity. The antibody immunoprecipitated the T cell receptor (TcR) complex from a T cell hybridoma. After dissociation of this immunoprecipitate with detergents, the CD3 gamma and epsilon chains were retained by the 7D6 antibody. Immunoprecipitation data were also obtained with COS cells transfected with the CD3 gamma, delta or epsilon chains alone, in pairs or together. They confirmed that 7D6 bound the CD3 gamma epsilon pair, suggesting that the antibody recognizes a conformational epitope formed by gamma epsilon pairing, whereas 145 2C11 bound both gamma epsilon and delta epsilon pairs. These results, therefore, add to current information about TcR structure and subunit stoichiometry. We have demonstrated that the 7D6 mAb specifically binds to a CD3 dimer comprised of gamma and epsilon chains. We thus provide additional evidence that indicates that two CD3 epsilon chains are found within the receptor, one linked to CD3 gamma and the other to CD3 delta. PMID- 1711977 TI - Remote T cell co-stimulation via LFA-1/ICAM-1 and CD2/LFA-3: demonstration with immobilized ligand/mAb and implication in monocyte-mediated co-stimulation. AB - Proliferative response of resting T cells generally requires not only cross linking of the T cell receptor (TcR) but also co-stimulatory signals from accessory molecules. We here have used a "three-cell" model consisting of: (a) resting human CD4+ T cells as responders; (b) CD3 monoclonal antibody (mAb) OKT3 on latex beads as surrogate stimulators; (c) autologous monocytes as source of co stimulation. As described by Kawakami et al. (J. Immunol. 1989, 142: 1818), T cell proliferation in this system is observed with paraformaldehyde-fixed monocytes if they have been activated and interleukin (IL) 1 beta/IL 6 is supplied. Since this three-cell system provides TcR cross-linking at a site spatially "remote" from co-stimulation, they help distinguish adhesion from signal transduction but the molecules that mediate co-stimulation in this system have not been identified. Our studies now demonstrate that co-stimulation by the monocytes is dependent on each of two receptor/ligand pathways CD2/LFA-3 and LFA 1/ICAM-1 since it is inhibited by each relevant mAb but not a variety of control mAb. The hypotheses that CD2 and LFA-1 could each mediate co-stimulation was tested in simplified model systems in which the monocyte was replaced with immobilized CD2 mAb or purified ICAM-1 presented on a separate surface from the CD3 mAb. The results in these simplified models demonstrate that on resting T cells either CD2 or LFA-1 molecules alone can mediate "remote" co-stimulation unlike most other T cell surface molecules. Co-stimulation requires IL 1 beta/IL6 both in the weaker LFA-1 ligand-mediated co-stimulation and at lower CD2 mAb concentrations in the stronger CD2 mAb-mediated co-stimulation. Thus: (a) the accessory cell function of stimulated fixed monocytes in T cell proliferation requires both the LFA-1/ICAM-1 and CD2/LFA-3 pathways; and (b) the T cell molecules CD2 and LFA-1 can give co-stimulatory signals that can act in a "remote" fashion. PMID- 1711978 TI - Effects of Bay K 8644, a Ca2+ channel agonist, on [3H]norepinephrine release in hypothalamus of spontaneously hypertensive rats. AB - We describe the effects of Bay K 8644, a dihydropyridine-sensitive Ca2+ channel agonist, on [3H]norepinephrine (NE) release from the hypothalamus of spontaneously hypertensive rats (SHR). The electrical stimulation-evoked [3H]NE release was greater in hypothalamic slice of SHR than in those of Wistar Kyoto (WKY) rats. Bay K 8644 (10(-6) M) significantly increased the stimulation-evoked [3H]NE release in SHR. However, the agonist showed no significant effects in normotensive WKY rats. The results suggest that the dihydropyridine-sensitive Ca2+ channels might participate actively in the regulation of the central sympathetic tone of hypertension. PMID- 1711979 TI - Slow inward current induced by achatin-I, an endogenous peptide with a D-Phe residue. AB - Following a preliminary report on the isolation of a neuroactive tetrapeptide, achatin-I (Gly-D-Phe-L-Ala-L-Asp) that has a D-phenylalanine residue, from the Achatina fulica ganglia, the pharmacological features of this peptide on Achatina giant neurones were now worked out in detail. Of the eight possible stereoisomers, only achatin-I markedly, and [D-Ala3]achatin-I slightly, induced a slow inward current (Iin) with an increase in membrane conductance (g) of the identifiable neurones, tonically autoactive neurone (TAN), dorsal-right cerebral distinct neurone (d-RCDN) and periodically oscillating neurone (PON) which had been tested previously. Of 23 types of neurones tested, 10 types including the three mentioned were excited by achatin-I, whereas no neurone was inhibited. The ED50 of achatin-I for the neurones tested were 0.2-2.7 x 10(-5) M, and that for PON was the lowest. The Hill coefficients of achatin-1. 0.62-0.80, derived from 1.0 Emax values of achatin-I for producing Iin, 4.2-6.3 nA, were significantly greater than those of [D-Ala3]achatin-I, 1.8-3.4 nA. Iin of TAN and d-RCDN induced by achatin-I was blocked in the Na(+)-free state, but unaffected in the Ca2(+)-free (replaced with Co2+), Cl(-)-free or K(+)-enriched (3.0X) state, indicating that the current was produced by the g increase in response to Na+. However, the Iin was partially blocked by tetrodotoxin 10(-4) M. We propose that achatin-I is an excitatory neurotransmitter on Achatina neurones. PMID- 1711980 TI - Interactions between endothelin-1 and other chronotropic agents in rat isolated atria. AB - In isolated spontaneously beating right and left atria and in electrically driven left atrium from rat, endothelin-1 increased the rate and force of contraction, but significantly decreased the positive chronotropic and inotropic responses to sympathetic nerve stimulation. The decrease may be partly dependent on the positive cronotropic and inotropic effects of endothelin-1, since other agents with chronotropic activity (noradrenaline, isoprenaline, serotonin and Bay k 8644) also decreased stimulation-induced chronotropic responses. Endothelin-1 caused a significant rightward shift of the linear portion of the log concentration-response curve for the chronotropic actions of noradrenaline and isoprenaline. The changes in the log concentration-response curve were not a consequence of the direct chronotropic effect of endothelin-1, since they were still evident when the chronotropic action of endothelin-1 was offset by carbachol. Furthermore, the chronotropic agent, Bay k 8644, did not shift the linear portion of the log concentration-response curves for noradrenaline and isoprenaline. The mechanism of the effects of endothelin-1 in rat atria is not known, but they were not changed by blockade of alpha-adrenoceptors or of L-type voltage-sensitive Ca2+ channels. PMID- 1711981 TI - Effects of actinomycin D on airway constriction induced by tachykinins and capsaicin in guinea-pigs. AB - The effects of actinomycin D on airway constriction induced by tachykinins was studied in guinea-pigs in vitro and in vivo. Actinomycin D significantly inhibited the constriction of isolated guinea-pig trachea induced by neurokinin A (NKA) and eledoisin. Conversely, substance P (SP)- and physalaemine-induced constrictions were not affected by actinomycin D. The same selectivity in the inhibitory action of actinomycin D against tachykinins was also observed in in vivo. Actinomycin D given i.v. specifically inhibited the increase in airway resistance induced by NKA. I.v. injection of NKA caused not only airway constriction but also transient systemic hypotension. Interestingly, actinomycin D injected i.v. inhibited only airway constriction and the systemic hypotension induced by NKA was not affected. These results clearly suggest that actinomycin D specifically inhibits NKA-induced airway constriction in guinea-pigs. Actinomycin D also had an inhibitory action on the airway constriction induced by capsaicin. In the case of capsaicin-induced constriction, actinomycin D was more effective on the later phase of constriction than on the acute phase. The airway constriction induced by capsaicin is thought to be mediated by the release of SP and NKA from sensory nerve endings, and the persistent increase in airway resistance induced by capsaicin is thought to be due mainly to NKA. PMID- 1711982 TI - A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli. AB - The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing. The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria (Escherichia coli) or to those that have been reported from eukaryotes. PMID- 1711983 TI - Reduction in cab and psb A RNA transcripts in response to supplementary ultraviolet-B radiation. AB - The cab and psb A RNA transcript levels have been determined in Pisum sativum leaves exposed to supplementary ultraviolet-B radiation. The nuclear-encoded cab transcripts are reduced to low levels after only 4 h of UV-B treatment and are undetectable after 3 days exposure. In contrast, the chloroplast-encoded psb A transcript levels, although reduced, are present for at least 3 days. After short periods of UV-B exposure (4 h or 8 h), followed by recovery under control conditions, cab RNA transcript levels had not recovered after 1 day, but were re established to ca. 60% of control levels after 2 more days. Increased irradiance during exposure to UV-B reduced the effect upon cab transcripts, although the decrease was still substantial. These results indicate rapid changes in the cellular regulation of gene expression in response to supplementary UV-B and suggest increased UV-B radiation may have profound consequences for future productivity of sensitive crop species. PMID- 1711984 TI - Monoclonal antibody to chick embryo hyaluronan-binding protein: changes in distribution of binding protein during early brain development. AB - A monoclonal antibody, MAb IVd4, that recognizes hyaluronan-binding protein (HABP) from chick embryo brain has been produced and characterized. By immunoblotting, MAb IVd4 was shown to recognize three proteins in chick embryo brain of molecular weight 93, 90, and 69 kDa; this interaction was inhibited by addition of hyaluronan hexasaccharides. Overlay of transblots with [3H]hyaluronan showed binding to proteins of similar molecular weight. MAb IVd4 blocked binding of [3H]hyaluronan to brain HABP and to simian virus-transformed 3T3 cells, indicating a possible relationship with the 85-kDa hyaluronan receptor of these cells. The distribution of HABP during early brain development was analyzed by immunohistochemistry. Immunoreactivity was uniform in newly formed neuroectoderm but became more concentrated in the roof of the brain during the second day of embryonic development. As the neuroectoderm becomes layered, the HABP was increasingly restricted to the forming plexiform layer, an area enriched in neural cell processes. Immunoreactivity was greatly enhanced by pretreatment of tissue with hyaluronidase, presumably due to removal of hyaluronan bound to the HABP, and was abolished on treatment with hyaluronan hexasaccharide, presumably due to inhibition of HABP-antibody interaction. These results suggest that a hyaluronan receptor is involved in early cellular events in brain development. PMID- 1711985 TI - Coordinate inactivation of class III genes during the Gastrula-Neurula Transition in Xenopus. AB - We have identified a period during early Xenopus development when several different genes transcribed by RNA polymerase III (class III genes) are coordinately inactivated. During the late gastrula stage a major reduction in the number of active transcription complexes gives rise to a pattern of class III gene activity typical of adult somatic cells. This event is referred to as the Gastrula-Neurula Transition and involves the inactivation of genes encoding oocyte-type tRNAs and 5S RNA, along with several heterogeneous RNAs expressed during the blastula and gastrula stages of embryogenesis. PMID- 1711987 TI - Localization of intermediate filament proteins in various structures of the human placenta as revealed by immunoperoxidase technique. AB - Formalin-fixed, paraffin embedded fragments of normal human placenta were subjected to immunoperoxidase technique with the use of Dako Kits appropriate for detection of vimentin, desmin, and cytokeratins (K 660, 530, and 518 respectively). Distribution of the intermediate filament proteins in syncytiotrophoblast, connective tissue of villous stroma, walls of blood vessels as well as in cells of basal plate and placental islets was compared. General pattern of staining and its localization was characteristic for each of the 3 types of antibodies. In spite of that, most structures observed revealed the presence of more than one type of intermediate filament proteins, with the exception of syncytiotrophoblast containing cytokeratins only. Staining of blood serum and of connective tissue matrix with anti-desmin and anti-cytokeratin antibodies was also observed, whereas fibrinoid remained always negative. PMID- 1711986 TI - Nutrition and somatomedin. XXV. Regulation of insulinlike growth factor binding protein 1 in primary cultures of normal rat hepatocytes. AB - Although mRNAs encoding insulinlike growth factor (IGF) binding proteins (BPs) are present in adult rat liver and IGF BP-1 circulates at elevated levels in diabetic animals, there is little knowledge of the metabolic regulation of IGF BPs in normal tissues. We examined the release of IGF BPs by adult rat hepatocytes maintained in primary culture. When cultured for 2 days in the absence of added insulin, hepatocytes released a BP identified as BP-1 on the basis of approximately 30,000-Mr on ligand blotting and reactivity with antiserum to human BP-1 in immunoblotting and immunoprecipitation studies. Release of BP-1 was sensitive to insulin with suppression of 24 +/- 4, 73 +/- 5, and 64 +/- 14% at 10(-10), 10(-8), and 10(-6) M insulin, respectively; ED50 was approximately 1.7 x 10(-9) M, which is within the physiological range. Suppression by insulin was reversible and began within 3 h. Because normal hepatocytes in primary culture exhibit insulin-responsive release of both BP-1 and IGF-1, this system may be an ideal model for studies of molecular mechanisms of metabolic regulation. PMID- 1711988 TI - Characterization of bovine rotavirus VP6 and VP7 as glycoproteins using monoclonal antibodies. AB - Bovine rotavirus proteins were analysed by a panel of monoclonal antibodies. Glycosylated epitopes were identified on both inner and outer capsid proteins (VP6 and VP7 respectively). VP7 possessed a periodate insensitive epitope which was, however, sensitive to endoglycosidase H, mixed glycosidases and to protease treatment. This epitope was not detected on viruses grown in the presence of 2 deoxy-D-glucose or tunicamycin. An epitope was detected on VP6 which was sensitive to periodate oxidation. The blotted protein reacted with a glycan assay kit; yet the epitope was not affected by endoglycosidase H and was found on viruses grown in the presence of 2-deoxy-D-glucose or tunicamycin. These results suggest that VP7 and VP6 epitopes are carbohydrate dependent. The VP7 epitope contains an N-linked carbohydrate moiety in contrast to the VP6 epitope which appears to contain O-linked glycosyl units. PMID- 1711989 TI - An unusually heavy contamination of honey products by Clostridium botulinum type F and Bacillus alvei. AB - Many spores (1-60/g) of Clostridium botulinum type F were detected in different containers of honey products of the same brand. Microbiological and physicochemical properties of the contaminated honey were compared with those of the negative one. No difference in pH, hydroxymethyl furfural contents or diastase activity was found between them. The total counts of anaerobes other than C. botulinum and of yeast were also similar, whereas the aerobe counts, which were proportionally related with the C. botulinum counts, were higher in the positive honey than in the negative one. Motile colony-forming Bacillus alvei was predominant among the aerobes. B. alvei stimulated the toxin production by C. botulinum type F in culture medium incubated under aerobic conditions. The high count of C. botulinum in the honey might have been due to the possible stimulation of growth by B. alvei or some other microorganisms at some stage of honey ripening. PMID- 1711990 TI - Non-radioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile. AB - A typing method for Clostridium difficile based on restriction fragment length polymorphisms (RFLP) is described. The technique utilizes commercially available Escherichia coli ribosomal ribonucleic acid (rRNA) as probe material. Probe labelling, hybridization and detection was performed using the Enhanced Chemiluminescence (ECL) gene detection system. The probe labelling procedure was easy to perform, taking only 20 min. The complete typing method was comparatively simple, reproducible and readily adaptable to most bacterial genera. PMID- 1711991 TI - Mechanisms of inflammation and potential role in the pathogenesis of asthma. AB - Neurogenic inflammation produces potent responses in multiple cells in the airways. These responses normally are limited by the presence on the surface of target cells of neutral endopeptidase (NEP), an enzyme that cleaves and inactivates neuropeptides that come in close contact with the cell and thereby limits neurogenic inflammatory responses. Inhibition of NEP by drugs, respiratory viruses, cigarette smoke, and toluene diisocyanate result in exaggerated neurogenic responses, while upregulation of NEP (e.g., by corticosteroids) may suppress the responses. Exogenously delivered recombinant human NEP also inhibits the responses. The novel system whereby sensory nerve stimulation results in the release of inflammatory neuropeptides provides a potentially important mechanism in the pathogenesis of airway inflammation. The strategies discussed here provide tools for the investigation of this system and suggests methods for therapeutic intervention. PMID- 1711992 TI - Cloning, sequence and chromosomal location of a MEL gene from Saccharomyces carlsbergensis NCYC396. AB - Yeast strains producing alpha-galactosidase (alpha Gal) are able to use melibiose as a carbon source during growth or fermentation. We cloned a MEL gene from Saccharomyces carlsbergensis NCYC396 through hybridization to the MEL1 gene cloned earlier from Saccharomyces cerevisiae var. uvarum. The alpha Gal encoded by the newly cloned gene was galactose-inducible as is the alpha Gal encoded by MEL1. A probable GAL4-protein recognition sequence was found in the upstream region of the NCYC396 MEL gene. The gene was transcribed to a 1.5-kb mRNA which, according to the nucleotide sequence, encodes a protein of 471 amino acids (aa) with an Mr of 52,006. The first 18 aa fulfilled the criteria for the signal sequence, but lacked positively charged aa residues, except the initiating methionine. The enzyme activity was found exclusively in the cellular fraction of the cultures. The deduced aa sequence was compared to the aa sequences of other alpha Gal enzymes. It showed 83% identity with the S. cerevisiae enzyme, but only 35% with the plant enzyme, 30% with the human enzyme and 17% with the Escherichia coli enzyme. With pulsed-field electrophoresis, the MEL gene was located on chromosome X of S. carlsbergensis, whereas the S. cerevisiae var. uvarum MEL1 gene is located on chromosome II. PMID- 1711993 TI - Growth factors and the liver. PMID- 1711994 TI - Palliation of proximal malignant biliary obstruction by endoscopic endoprosthesis insertion. AB - For four years up to December 1987, 190 patients (median age 73 years) with proximal malignant biliary obstruction were treated by endoscopic endoprosthesis insertion. Altogether 101 had cholangiocarcinoma, 21 gall bladder carcinoma, 20 local spread of pancreatic carcinoma, and 48 metastatic malignancy. Fifty eight patients had type I, 54 type II, and 78 type III proximal biliary strictures (Bismuth classification). All patients were either unfit or unsuitable for an attempt at curative surgical resection. A single endoprosthesis was placed initially, with a further stent being placed only if relief of cholestasis was insufficient or sepsis developed in undrained segments. The combined percutaneous endoscopic technique was used to place the endoprosthesis when appropriate, after failed endoscopic endoprosthesis insertion or for second endoprosthesis placement. Full follow up was available in 97%. Thirteen patients were still alive at the time of review and all but one had been treated within the past six months. Initial endoprosthesis insertion succeeded technically at the first attempt in 127 patients, at the second in 30, and at a combined procedure in a further 13 (cumulative total success rate 89% - type I: 93%; type II: 94%; and type III: 84%). There was adequate biliary drainage after single endoprosthesis insertion in 152 of the 170 successful placements, giving an overall successful drainage rate of 80%. Three patients had a second stent placed by combined procedure because of insufficient drainage, giving an overall successful drainage rate of 82% (155 of 190). The final overall drainage success rates were type I: 91%; type II: 83%; and type III: 73%. The early complication rates were type I: 7%; type II: 14%; and type III: 31%. The principle early complication was clinical cholangitis, which occurred in 13 patients (7%) and required second stent placement in five. The 30 day mortality was 22% overall (type I: 14%; type II: 15%; and type III: 32%) but the direct procedure related mortality was only 3%. Median survival overall for types I, II, and III strictures were 21, 12, and 10 weeks respectively but survival was significantly shorter for metastatic than primary malignancy (p<0.05). Endoscopic insertion of a single endoprosthesis will provide good palliation of proximal malignant biliary obstruction caused by unresectable malignancy in 80% of patients. Second stents should be placed only if required. Extensive structuring because of metastatic disease carries a poor prognosis and careful patient selection for treatment is requires. PMID- 1711996 TI - [Causal treatment of refractory pain]. PMID- 1711995 TI - Gastrin-histamine sequence in the regulation of gastric acid secretion. PMID- 1711997 TI - [Changes in the intracellular free calcium of cultured human epidermal keratinocytes]. AB - Changes in the intracellular free calcium ([Ca2+]i) of cultured normal human epidermal keratinocytes (NHEK) were investigated in order to determine whether the adenylate cyclase cAMP (AC) system and phospholipase C activating system are involved in increasing [Ca2+]i. NHEK were obtained from neonatal foreskin and grown in serum-free medium (K-GM) supplemented with 2% bovine pituitary extract. [Ca2+]i was measured by fluorescence ratio imaging microscopy using Fura-2 as the indicator. In the case of the AC system, transient increases in [Ca2+]i were observed in response to stimulation with epinephrine, norepinephrine, isoproterenol and salbutamol. Methoxamine, clonidine and dobutamine did not induce any [Ca2+]i increase. The [Ca2+]i increase evoked by epinephrine was inhibited by pretreatment with propranolol, but not by prazosin or yohimbine, indicating that epinephrine-induced [Ca2+]i elevation via beta 2-adrenergic stimulation. Similar changes were observed when NHEK were stimulated with histamine, adenosine, GTP gamma S, forskolin and dibutyryl cAMP respectively. The absence of extracellular Ca2+ had no effect on the epinephrine-induced [Ca2+]i increase. It appears that activated protein kinase A, based on cAMP accumulation via stimulatory GTP binding protein, elicited the release of Ca2+ from intracellular stores. On the other hand, when drugs known to activate phospholipase C in a wide variety of cell types were tested, a transient increase in [Ca2+]i was demonstrated in response to the addition of thrombin, bradykinin and substance P. This reaction was not affected by the presence of EGTA, suggesting that these drugs raise [Ca2+]i via phosphatidylinositol breakdown. Vasopressin, angiotensin II, serotonin and acetylcholine did not induce any increase in [Ca2+]i. On the basis of these studies, it was concluded that NHEK possess the mechanism which increase [Ca2+]i via AC system and phospholipase C activating system. It seems probable that this rise in [Ca2+]i initiates a calcium-dependent cellular response, such as activation of calcium/calmodulin dependent kinase, and subsequently regulates the proliferation and differentiation of human epidermal keratinocytes. PMID- 1711998 TI - Interferon induction in rabbits after intraduodenal administration of a phosphorylated glucomannan-protein fraction of the cell wall of Candida albicans. AB - The aim of this work was to demonstrate whether a glucomannan protein fraction (GMP) of Candida albicans cell wall could induce interferon after intraduodenal administration in normal rabbits and rabbits immunized against C. albicans. For this purpose we collected simultaneously plasma and abdominal lymph for 10 h after the administration of the inducer. We observed a peak of antiviral activity in the lymph 4 h after intraduodenal administration of 20 mg GMP dissolved in saline to 6 normal rabbits. Immunized rabbits (anti-GMP titres greater than 1024) responded earlier (peak after 2 h) and more intensely; analysis of the values of the areas under the curve indicated that the IFN response in the lymph of immunized rabbits was significantly higher (P less than 0.0025) than in normal rabbits. Antiviral activity was absent in plasma in all cases. Preliminary characterization of the IFN activity has shown it to be trypsin-sensitive, acid and heat stable, and species-specific. PMID- 1711999 TI - Drimarene brilliant blue--a better pre-stain in protein separation and visualization. AB - Prestaining of human serum proteins with a new reactive dye Drimarene Brilliant Blue (DBB), was standardized employing 940 separations and examining 30 variables. Under the critical condition, the serum and the soluble dye (0.1 g/100 ml in working Tris-glycine buffer, pH, 8.3), was mixed in equal proportion, conjugate warmed at 40 degrees C for 2 hr and a 30 microliter of the sample electrophoresed by disc electrophoresis. The method when compared with prestaining by Remazol Brilliant Blue (RBB) and postelectrophoretic staining by Amido Black (AB) in 50 normal sera, revealed that the discs stained with DBB were intense and well defined and appeared in 2 hr on a sparkingly clear gel. Quality of resolution was better than RBB and AB. Protein bands eluted from the DBB prestained gels retained their immunoreactivity. The dye-protein complex of albumin and transferrin produced high-titre monospecific antisera in rabbits. PMID- 1712000 TI - Skin reactivity to substance P, not to neurokinin A, is increased in allergic asthmatics. AB - The tachykinins substance P (SP) and neurokinin A are believed to be major mediators of neurogenic inflammation. To determine whether the skin reactivity to tachykinins is increased in asthmatics, we examined the erythemas and wheals induced by intradermal injections of SP, the C-terminal peptide SP6-11, the N terminal peptide SP1-9 and neurokinin A (10(-7)-10(-5) M) in 10 allergic asthmatics and 9 normal subjects. SP and SP1-9 induced both erythemas and wheals in a concentration-dependent manner in allergic asthmatics and in normal subjects, whereas SP6-11 and neurokinin A induced only wheals in both groups. SP induced greater erythemas and wheals in allergic asthmatics than in normal subjects. However, the wheals induced by neurokinin A were not significantly different between the two groups. SP1-9 also induced greater erythemas and wheals in allergic asthmatics than in normal subjects, whereas the wheals induced by SP6 11 were not significantly different between the two groups. Therefore, the increased skin reactivity to SP was dependent on the N-terminal peptide but not on the C-terminal peptide. We conclude that the skin reactivity to SP but not to neurokinin A is increased in allergic asthmatics. PMID- 1712001 TI - Neutral endopeptidase modulates substance P-induced activation of human neutrophils. AB - Neutral endopeptidase (NEP; EC 3.4.24.11) is well recognized as a regulatory peptidase for substance P (SP)-induced responses in various tissues. To determine whether NEP regulates SP-induced activation of human neutrophils, we examined the effect of the NEP inhibitor phosphoramidon on SP-induced superoxide generation and chemotaxis in human blood neutrophils. SP (10(-6)-10(-4) M) induced superoxide generation and chemotaxis in the neutrophils dose dependently. The NEP inhibitor enhanced the SP-induced responses. Thus, phosphoramidon (10(-6) M) shifted the dose-response curves of SP-induced superoxide generation and chemotaxis of the neutrophils to the left by 0.5-0.6 log. Phosphoramidon prevented the hydrolysis of SP by the neutrophils, the NEP activity of the neutrophils being assessed as 125 +/- 13 pmol of SP/min/10(6) cells. The N terminal peptide SP (up to 3 x 10(-4) M), which was a major degrading product by NEP of the neutrophils, did not activate the neutrophils. We conclude that NEP modulates SP-induced activation of human neutrophils. PMID- 1712002 TI - Modulation of rat peritoneal mast cell and human basophil histamine release by estrogens. AB - This study was undertaken to investigate the effect of estrogens on the histamine release mediated by IgE in rat peritoneal mast cells (PMC) and in sensitized human basophils. The estrogens were found to enhance the histamine release of either rat PMC and sensitized human basophils upon stimulation with anti-IgE. The enhancement was estrogens dose-dependent reaching the maximum value of 23% for rat PMC and 41% for sensitized human basophils stimulated with anti-IgE upon preincubation with 10(-8) M estrogens. Moreover, when purified PMC were used, the enhancing effect was still detected, suggesting a direct interaction between estrogens and mast cells. The enhancing effect took place quite rapidly reaching plateau levels in about 60 min. Basophils preincubated at 4 instead of 37 degrees C did not give any appreciable enhancement, suggesting that it was temperature dependent and that the effect observed was not due to cytotoxicity. Incubation of PMC or human basophils with estrogens alone, without challenge with anti-IgE, did not give any detectable histamine release. The enhancement of histamine release by estrogens is probably mediated by IgE molecules present on the cell membrane, since this effect was not observed on challenge with substance P or compound 48/80, two segretagogues known to induce histamine release not via IgE. PMID- 1712003 TI - Inhibition of histamine release from rat peritoneal mast cells by MY-1250, an active metabolite of Repirinast (MY-5116). AB - MY-1250, an active metabolite of Repirinast (MY-5116), strongly and dose dependently inhibited the in vitro histamine release from rat peritoneal mast cells induced by antigen. The IC50 value of MY-1250 for histamine release was in the order of 0.3 microM. Moreover MY-1250 strongly inhibited calcium ion mobilization from the intracellular Ca2+ store. In the presence of 3.6 x 10(-5) M of MY-1250, the initial rise in [Ca2+]i reached a maximum of only 13 nM, and the inhibitory effect was 81% for the initial rise. The IC50 value for this event was 0.25 microM. The inhibitory effect of MY-1250 on the initial rise in [Ca2+]i was closely correlated with that on histamine release. PMID- 1712004 TI - CI-959, a new, potential antiallergic drug, inhibits mediator release from lung and contractions of human airways in vitro. AB - Many symptoms of the immediate allergic response can be attributed to the synthesis/release and subsequent actions of histamine and metabolic products of arachidonic acid oxidation. CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazole-5 yl-benzo(b) thiophene-2-carboxamide], a new, potential antiallergic drug, inhibited the release of histamine, immunoreactive sulfidopeptide leukotrienes C4, D4 and E4 and immunoreactive thromboxane B2 from immunologically activated guinea-pig and human lung cells in vitro. The IC50s of CI-959 using guinea-pig lung were: histamine, 0.8 +/- 1.4 microM; leukotriene, 0.7 +/- 1.6 microM and thromboxane, 9.6 +/- 3.3 microM. Using human lung the IC50s were: 2.3 +/- 1.3 microM for histamine; 0.3 +/- 5.1 microM for leukotriene, and 0.3 +/- 2.6 microM for thromboxane. CI-959 caused a concentration-dependent inhibition of anti-IgE induced contractions of human bronchial muscle. Mean percent inhibitions were 45, 65 and 96 at 1, 3 and 10 microM, respectively. Cromolyn, 10 microM, inhibited bronchial contractions only 42%. The ability of CI-959 to inhibit these immunologically induced contractions indicates that the release of all mediators responsible for bronchoconstriction was effectively inhibited. These data suggest that CI-959 may be effective in preventing the development of symptoms directly related to inflammatory mediator release in a variety of allergic and inflammatory states. PMID- 1712005 TI - Multipotent and committed CD34+ cells in bone marrow transplantation. AB - In order to study the role of CD34+ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34+ cells were also performed to evaluate their in vitro hematopoietic function. CD34+ cells were detectable in bone marrow cells on day 14. More than 80% of CD34+ cells co expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34+ cells. In normal bone marrow cells, CD34+, CD33+ cells amounted to about 40% of CD34+ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34+, CD33- cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34+, CD33+ cells as committed progenitors and CD34+, CD33- cells as multipotent stem cells have distinctive biological behaviors in BMT. PMID- 1712006 TI - Suppressive activity of interleukin 4 on the induction of antigen-specific cytotoxic T cells in humans. AB - The effect of interleukin 4 (IL 4) on the induction of cytotoxic T cells (CTL) was studied by using human peripheral blood lymphocytes in vitro. IL 4 suppressed the induction of CTL specific for allogeneic antigens in a concentration dependent manner. However, IL 4 did not suppress proliferative responses induced with allogeneic antigens or mitogens. The suppressive effect of IL 4 on CTL induction was observed when IL 4 was added at the early period of the CTL induction culture, but not at the later period. Furthermore, IL 4 did not suppress the effector function of CTL to target cells. IL 4 suppressed the production of IL 1 by monocytes/macrophages and the production of IL 2 and the expression of IL 2 receptors on T cells. Moreover, IL 4 suppressed the induction of lymphokine-activated killer cells. These results suggest that IL 4 has a suppressive activity on the induction of killer cells in humans. PMID- 1712007 TI - Pyrrolomycin group antibiotics inhibit substance P-induced release of myeloperoxidase from human polymorphonuclear leukocytes. AB - In order to search for microbial modulators of the activity of neuropeptide, we established a screen based on substance P (SP)-induced myeloperoxidase (MPO) release from human polymorphonuclear leukocytes (PMN). SP induced MPO release in a dose-dependent manner at concentrations ranging from 1 approximately 10 x 10( 4) M. In comparison at 1 x 10(-4) M, induction was also observed with SP derivatives but not with other neuropeptides such as neurokinin and enkephalin. Based on this, we searched for microbial inhibitors against SP-induced MPO release. An actinomycete metabolite designated HS3, which turned out to be identical with dioxapyrrolomycin or A1-R2081, and structurally related pyrrolomycins were found to inhibit SP-induced MPO release. In addition, these compounds inhibited the f-Met-Leu-Phe (FMLP)-induced MPO release from PMN. Pyrrolomycin derivatives with an N-methylated pyrrole ring showed, however, a selective inhibition of the SP-induced MPO release. This was in contrast to results with aseanostatin P5 which selectively inhibited FMLP-induced MPO release. PMID- 1712008 TI - Effect of FK-506 on replication of human cytomegalovirus in vitro. PMID- 1712009 TI - Expression of intermediate filament proteins in the mature inner ear of the rat and guinea pig. AB - The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, desmin, neurofilament proteins and glial fibrillary acidic protein (GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins, suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and 19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the cochlear and vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neurofilament proteins. Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP. The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates a close similarity between the different epitopes. PMID- 1712010 TI - Hypertrophy and hyperplasia of bovine fetal tissues during development: fetal liver insulin-like growth factor I mRNA expression. AB - Tissue growth of crossbred fetal beef calves was examined by measuring RNA, DNA, and protein concentrations in liver, heart, and biceps femoris. Furthermore, liver insulin-like growth factor I (IGF-I) mRNA expression and mRNA species size during fetal development was observed. Tissue samples were collected from six fetuses every 42 d of gestation, from d 106 to d 274. In the liver, protein and DNA concentrations decreased, whereas RNA levels remained constant throughout fetal growth. The RNA/DNA and protein/DNA ratios in liver increased with fetal age. Heart DNA and RNA levels decreased, whereas protein concentration and protein/DNA ratios increased with fetal age. Protein and protein/DNA ratios decreased in biceps tissue, whereas DNA and RNA concentrations were constant. IGF I mRNA was seen at 4.4, 2.5, and 1.2 kb in adult and 4.4, 2.5, and 1.7 kb in fetal bovine liver. Relative expression of liver IGF-I mRNA did not vary during fetal development. The current study shows that during the last 2 and 3 mo of gestation, heart and liver were undergoing hypertrophic growth, whereas biceps tissue did not exhibit the same trend. Elevated ratios of RNA and protein to DNA in liver above that of the heart and biceps suggest extensive hepatic cellular hypertrophy as well as increased transcriptional and translational activity. Insulin-like growth factor I mRNA levels were not related to the changes in RNA, DNA, and protein seen in hepatic tissue. PMID- 1712011 TI - Caffeine administered during pregnancy augments subsequent lactation in mice. AB - Mice were administered caffeine (500 mg/liter of drinking water) from d 1 until d 18 of pregnancy. Before and after receiving caffeine, mice were given distilled water for drinking, as were controls. Litter size, birth weight, and offspring survival were not affected by caffeine, but litter weight on d 15 of lactation was increased by caffeine 84.0 +/- 3.1 g (n = 8) in controls vs 98.3 +/- 3.7 g (n = 7) in caffeine-treated mice (P less than .05). Mammary gland cell number, measured by the DNA content of mammae, was increased by giving caffeine during pregnancy (P less than .05). On d 18 of pregnancy, mammary DNA was .47 +/- .07 mg (n = 6) in controls vs .71 +/- .11 mg (n = 6) in caffeine-treated mice. On d 15 of lactation, mammary DNA was .96 +/- .12 mg (n = 8) in control vs 1.26 +/- .11 mg (n = 7) in caffeine-treated mice. RNA increased (P less than .05) parallel to DNA. In additional experiments, litters were cross-fostered and standardized to eight pups per litter. Mice were bred and caffeine was administered as described previously. At birth, eight pups from mice treated with caffeine or as controls during the preceding pregnancy were fostered to a control or caffeine-treated mother. In these experiments, litter weight on d 15 of lactation was 82 g for control litters nursing control mothers, 84 g for caffeine litters nursing control mothers, 96 g for control litters nursing caffeine mothers, and 99 g for caffeine litters nursing caffeine mothers (n = 7 per groups, pooled SE = +/- 3.4 g).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712012 TI - Association of a retroelement with a P4-like cryptic prophage (retronphage phi R73) integrated into the selenocystyl tRNA gene of Escherichia coli. AB - A new multicopy single-stranded DNA (msDNA-Ec73) was found in a clinical strain of Escherichia coli. Retron-Ec73, consisting of an msDNA-coding region and the gene for reverse transcriptase (RT), was found to be a part of a 12.7-kb foreign DNA fragment flanked by 29-bp direct repeats and integrated into the gene for selenocystyl-tRNA (selC) at 82 min on the E. coli chromosome. Except for the 2.4 kb retron region, the integrated DNA fragment showed remarkable homology to most of the bacteriophage P4 genome. Among the phage genes found in this element, however, the integrase gene had very low identity (40%) to P4 integrase, indicating that the cryptic prophage associated with the retroelement has its own unique site-specific integrase different from P4 integrase. Recently, we have shown that P2 phage can act as a helper to excise the cryptic prophage and to package its genome into an infectious virion. The newly formed phage (retronphage phi R73) can also lysogenize a new host strain, reintegrating its genome into the selC gene and enabling the newly formed lysogen to produce msDNA-Ec73 (S. Inouye, M. G. Sunshine, E. W. Six, and M. Inouye, Science 252:969-971, 1991). PMID- 1712013 TI - The major outer membrane protein of Acidovorax delafieldii is an anion-selective porin. AB - The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldii) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70 degrees C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% beta sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth. PMID- 1712014 TI - Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction. AB - The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7. PMID- 1712015 TI - Interrelationships between mevalonate metabolism and the mitogenic signaling pathway in T lymphocyte proliferation. AB - Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25 hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression. PMID- 1712016 TI - Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor. AB - We have previously reported that polypeptide growth factors had an anti inflammatory effect by decreasing the cytokine-enhanced expression of factor B (FB), an activator of the alternative complement pathway, in human fibroblasts. To further characterize the role of cytokines and growth factors in the inflammatory/repair continuum, we have studied the effects of interleukin-1 (IL 1) and platelet-derived growth factor (PDGF) on the expression of metalloproteinases/antiproteinases of the extracellular matrix in cultured human fibroblasts. Co-incubation of IL-1 and PDGF synergistically increased the expression of stromelysin and interstitial collagenase to 23-fold (for both proteins) over background, while PDGF decreased the IL-1-enhanced expression of FB by 82%. PDGF, but not IL-1, alone or in combination, increased the synthesis of tissue inhibitor of metalloproteinases. RNA blot analysis indicated that the changes in protein synthesis were regulated at a pretranslational level. Cycloheximide treatment indicated that the effects of PDGF on the metalloproteinases/antiproteinases were not protein-dependent, in contrast to results obtained for FB. The effect of the three dimeric forms of PDGF (AA, AB, and BB) on the synthesis of metalloproteinases and FB was also analyzed. The effects were qualitatively similar for each of the dimeric forms; however, the BB and AB isoforms had considerably greater effects than PDGF-AA. It has been reported that the PDGF receptors found in human fibroblasts have higher binding affinity for the BB and AB isoforms of the growth factor. The results presented in this paper are in accord with the possibility that differences in the biological activity of the three isoforms of PDGF are due to differences in the number or affinity of the binding sites of the target cells, rather than to different activation pathways of the receptor. Thus, PDGF increased cytokine effects on metalloproteinases, while decreasing cytokine effects or complement activator FB. The net effect of these changes may be to decrease inflammation and enhance remodeling early in repair and to enhance matrix stability later in the repair process. PMID- 1712017 TI - Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin. AB - The purpose of this investigation was to characterize the reaction of alpha 2 antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface. PMID- 1712018 TI - Cloning, primary sequence, and chromosomal mapping of a human flavin-containing monooxygenase (FMO1). AB - cDNA clones that code for a pig and human flavin-containing monooxygenase (FMO) have been isolated. The full-length sequence of the human cDNAs revealed that they encode a polypeptide of 532 amino acid residues containing putative FAD- and NADP-binding sites. The deduced amino acid sequence has 88 and 86% identity, respectively, with the pig and rabbit "hepatic" forms of FMO, but is only 58% similar to the rabbit "pulmonary" FMO, and thus represents the human ortholog of the "hepatic" form of FMO. However, as this FMO is present in low abundance in human adult liver, the general term "hepatic" for this form of the enzyme is misleading, and thus we propose the name FMO1 to describe this human FMO and its mammalian orthologs. Northern blot analysis demonstrated that human FMO1 mRNA is more abundant in fetal than in adult liver, indicating that in man the enzyme is subject to developmental regulation. Southern blot hybridization of human genomic DNA suggests that the protein is encoded by a single gene, which has been designated FMO1 and mapped to chromosome 1. PMID- 1712019 TI - Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128). AB - To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr* Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc) Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies. PMID- 1712020 TI - Duplicated variable region genes account for double isotype expression in a human leukemic B-cell line that gives rise to single isotype-expressing cells. AB - The SSK41 cell is an immunoglobulin IgM+ human neoplastic B-cell line that switches to IgG+ cells (SSK gamma) spontaneously. We isolated a derivative of SSK41 (SSKWB) that expressed both IgM and IgG. Studies on the Ig heavy chain gene organization have shown that the SSKWB cell had two identical VDJ genes on the same chromosome; one linked to the C mu gene and the other to the C gamma 1 gene, both of which are transcribed to produce mu- and gamma-mRNAs with the same VDJ sequence. We also isolated the two switch variants derived from the SSKWB cell by cell sorter: 1G (IgG+) and 11M (IgM+). The 1G cells contained two populations; one had a similar genetic organization as SSK gamma and expressed only IgG1, and the other carried the same genetic organization as the SSKWB cell but produced aberrantly spliced mu-chain mRNA, in which the hydrophobic signal sequence exon is directly joined to the C mu exon. Te 11M cell deleted the VDJ-C gamma 1 segment of the SSKWB cell and expressed IgM. These results raise the interesting possibility of another mechanism for class switching. PMID- 1712021 TI - Purification of nitric oxide synthase from rat macrophages. AB - Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and gel filtration chromatography on a Superose 12 HR 10/30 column. The apparent molecular weight is 300,000 by gel filtration. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-biopterin, and dithiothreitol. Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme. The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme). PMID- 1712022 TI - Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a new family of cell adhesion junction plaque proteins. AB - Bullous pemphigoid is a subepidermal blistering disease in which patients have autoantibodies against the plaque of the hemidesmosome. Starting with a previously isolated 2-kilobase (kb) cDNA for bullous pemphigoid antigen (BPA), we used primer extension of keratinocyte mRNA to isolate overlapping cDNAs with a combined open reading frame of 6.3 kb, encoding most (243 kDa) of the BPA, but lacking the far amino terminus. Analysis of this amino acid sequence revealed a carboxyl-terminal domain containing two regions of 174 and 176 residues with high sequence identity. Most of the amino-terminal two-thirds of BPA is predicted to be in an alpha-helical conformation in which two chains would aggregate into a coiled-coil rod structure. BPA and desmoplakin I, a desmosome plaque protein, show remarkable sequence and structural homology. In its carboxyl-terminal domain, desmoplakin I also has 176 residue repeats with 40% sequence identity to those in BPA. The repeats in both molecules have a regular linear distribution of acidic and basic residues with a period of 9.5, the same as that found in the 1B segment of keratin filaments, suggesting a means of ionic interaction between keratin and these plaque proteins. Also, desmoplakin I, like BPA, is predicted to have a rod domain, which in both proteins has similar regular charge periodicities, suggesting a means of ionic self-aggregation. These findings extend those of Green et al. (Green, K. J., Parry, D. A. D., Steinert, P. S., Virata, L. A., Wagner, R. M., Angst, B. D., and Nilles, L. A. (1990) J. Biol. Chem. 265, 2603-2612) which show that BPA and desmoplakin I represent the first members of a new family of adhesion junction plaque proteins. PMID- 1712023 TI - Functional expression of the human formyl peptide receptor in Xenopus oocytes requires a complementary human factor. AB - Human phagocytic cells express receptors for bacterial N-formyl peptides (formyl peptide receptor or FPR) which mediate chemotaxis, degranulation, and the respiratory burst. Although cDNA encoding a human phagocyte formyl peptide binding protein has been reported recently (Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990) Biochem. Biophys. Res. Commun. 168, 1103-1109), functional coupling to signal transduction processes was not demonstrated. We describe corresponding full-length cDNA clones and prove that they encode the calcium mobilizing human formyl peptide receptor by demonstrating functional reconstitution in the Xenopus oocyte. We further demonstrate that in contrast to all other cloned guanine nucleotide-binding regulatory protein (G-protein) coupled receptors expressed in this system, microinjection of FPR transcripts is not sufficient to confer ligand responsiveness to the oocyte: co-injection of phagocyte RNA encoding a complementary human factor that is not the alpha subunit of the heterotrimeric G-proteins Gi1, Gi2 or Gi3 is also required. Whereas a 1.4 kilobase FPR transcript is expressed exclusively in differentiated phagocytic cells, the complementary factor activity localizes to a 3.5-kilobase RNA fraction and is expressed in both differentiated and undifferentiated myeloid cells as well as in liver. The deduced human FPR protein possesses seven hydrophobic putative membrane spanning segments, three sites for N-linked glycosylation, and a short 18-amino acid predicted third cytoplasmic loop. Surprisingly, the human FPR possesses only 28% amino acid identity with the rabbit FPR reported recently by Thomas and co-workers (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20065). Moreover, the rabbit FPR does not require a complementary factor for calcium mobilization in the oocyte. Structural alignment reveals at most 20% amino acid identity of the human FPR with other G-protein coupled receptors, indicating a common ancestral gene. Functional reconstitution of the recombinant FPR will now permit precise delineation of its functional and regulatory domains. Moreover, discovery of a complementary factor for oocyte expression of the human FPR establishes a novel approach to the qualification by ligand screening of cDNA encoding other suspected G-protein coupled receptors. PMID- 1712024 TI - Post-translational regulation of IgM expression in B lymphocytes. Selective nonlysosomal degradation of assembled secretory IgM is temperature-dependent and occurs prior to the trans-Golgi. AB - B cells express on their surface the membrane form of IgM (mIgM). Upon differentiation, the resulting plasma cells synthesize and secrete large amounts of the secretory form of IgM (sIgM). Surprisingly, B lymphocytes synthesize an excess of secretory mu chain over the expressed membrane mu chain. However, the sIgM is degraded intracellularly, indicating regulation of IgM expression at the post-translational level. In the present report, we show that the assembly, maturation, and degradation of IgM in 38C B lymphocytes are highly accelerated above a certain threshold temperature. Furthermore, the degradation of sIgM is delayed and takes place by the time the maturation of mIgM in the trans-Golgi is almost completed. Neither chloroquine nor monensin has any effect on this degradation, demonstrating a nonlysosomal pre-trans-Golgi process. In addition, the degradation is of endoglycosidase H-sensitive assembled sIgM molecules. We conclude that the degradation of sIgM in 38C B lymphocytes is a postendoplasmic reticulum, pre-trans-Golgi process. We suggest that this degradation process plays a role in the post-translational regulation of expression of soluble lumenal sIgM. PMID- 1712025 TI - A 25-hydroxycholesterol-resistant cell line deficient in acyl-CoA: cholesterol acyltransferase. AB - We describe a line of mutant Chinese hamster ovary cells, SRD-4 cells, that lacks acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and fails to synthesize cholesteryl esters when stimulated with 25-hydroxycholesterol or low density lipoprotein. The cells also have a partial defect in their ability to repress transcription of three sterol-regulated genes, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and the low density lipoprotein receptor. The cells were selected by mutagenesis followed by growth in the presence of 25-hydroxycholesterol, which kills the parental cells by cholesterol depletion, owing to an inhibition of cholesterol synthesis and a stimulation of cholesterol esterification. Treatment of parental cells with compound 58-035 (3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1- phenylethyl]propanamide), an inhibitor of ACAT, abolished cholesterol esterification but did not reproduce the defect in gene repression seen in the SRD-4 cells, and it only partially reproduced the resistance to the killing effect of 25-hydroxycholesterol. We conclude that the SRD-4 cells most likely have two independent defects, one in ACAT and the other in a factor that mediates sterol-dependent transcriptional repression. The SRD-4 cells thus resemble a line of hamster cells previously isolated (Cadigan, K.M., Heider, J.G., and Chang, T. Y. (1988) J. Biol. Chem. 263, 274-282), which has similar independent defects. The results raise the possibility that a partial resistance to sterol repression provides a growth advantage to cells that lack ACAT. PMID- 1712026 TI - Improved detection of human pancreatic proteins separated by two-dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis using a combined staining procedure. AB - In order to assess secretory pancreatic proteins in a two-dimensional isoelectric focusing/sodium dodecyl sulphate electrophoresis gel, a highly sensitive double staining method with Coomassie Brilliant Blue followed by silver stain was used. This combined procedure afforded more distinct spots and additional bands, particularly glycoproteins, than either silver or Coomassie Blue staining alone. As measurements of dye volumes by densitometry have shown, double staining of two dimensional separated pancreatic proteins is up to twenty times more sensitive than the usual Coomassie Brilliant Blue staining. PMID- 1712027 TI - Scabies resistant to lindane 1% lotion and crotamiton 10% cream. PMID- 1712028 TI - Genetic polymorphism of PAS-I, the mucin-like glycoprotein of bovine milk fat globule membrane. AB - A sensitive analytical method based on gel electrophoresis and silver staining was developed to detect PAS-I, a glycoprotein of bovine milk fat globule membrane. Application of the method to samples of milk from individual animals demonstrated that PAS-I is polymorphic and established that it also occurs in the skim milk phase. This polymorphism consisted of two bands showing variable mobility among samples of individual animals. Band patterns for an individual persisted from one lactation to the next. Comparison of 12 dam-daughter pairs for PAS-I patterns indicated that each pair had at least one band matching in mobility. Milk from identical twins had identical PAS-I patterns. Two out of three sets of fraternal twins had one band that did not match. Based on these data and genetics established for a similar protein of human milk, two codominant alleles, one from the sire and the other from the dam, account for the two bands of PAS-I. The PAS-I band patterns may be related to inheritance of milk production and composition factors. PMID- 1712029 TI - Molecular biology of aging. Part II: A synopsis of current research. PMID- 1712030 TI - Liposomes as carriers of antigens and adjuvants. AB - Liposomes have been widely used as carriers of protein or peptide antigens. Antigenic materials can be attached to the outer surface, encapsulated within the internal aqueous spaces, or reconstituted within the lipid bilayers of the liposomes. The natural tendency of liposomes to interact with macrophages has served as the primary rationale for utilizing liposomes as carriers of antigens. Liposomes also serve as carriers of a variety of adjuvants and mediators, including lipid A, muramyl dipeptide and its derivatives, interleukin-1, and interleukin-2. Research utilizing in vitro cell culture models has demonstrated that liposomes containing both appropriate antigens and major histocompatibility gene complex molecules can induce antigen-specific genetically restricted cytotoxic T lymphocytes. Liposomes induce immune reactions through classical interactions with antigen presenting cells. However, modelling experiments have also demonstrated that liposomes can even substitute for antigen presenting cells, and cell-free genetically restricted and nonrestricted presentation of antigens by liposomes to helper T lymphocytes has been demonstrated. Liposomes are successful for inducing potent immunity in vivo and they are now being employed in numerous immunization procedures and as vehicles for candidate vaccines. PMID- 1712031 TI - Detection of tumor necrosis factor alpha from porcine alveolar macrophages using an L929 fibroblast bioassay. AB - Tumor necrosis factor plays a central role in the mediation of the pathophysiological sequelae of infection and inflammation in animals and humans. The elucidation of its role in respiratory disease of swine has not been investigated, due in part to the lack of a sensitive and specific quantitative assay for its presence in tissue and fluid samples. Here we describe the detection of porcine tumor necrosis factor utilizing L929 murine fibroblast cells and characterize various parameters affecting assay sensitivity. Plating cell density and length of exposure time to test supernatants were the most critical factors. Using standard assay conditions as described here, porcine tumor necrosis factor was detected in alveolar macrophage conditioned media diluted more than 400-fold. Specificity of the assay for porcine tumor necrosis factor was shown by inhibition of cytotoxicity with neutralizing polyclonal antibodies for human recombinant tumor necrosis factor. Furthermore, comparisons of bioactivity with tumor necrosis factor mRNA levels from lipopolysaccharide stimulated porcine alveolar macrophages indicated that the L929 bioassay was specific for porcine tumor necrosis factor. PMID- 1712032 TI - Anti-myelin-associated glycoprotein antibodies in patients with a monoclonal IgM gammopathy and polyneuropathy, and a simplified method for the preparation of glycolipid antigens. AB - The two sulfated glucuronic acid containing glycolipids, sulfate-3-glucuronyl paragloboside (SGPG) and sulfate-3-glucuronyl lactosaminyl paragloboside (SGLPG), were prepared by ion exchange chromatography of lipid extracts from bovine cauda equina. Thin layer chromatography (TLC) immunostaining and an enzyme-linked immunosorbent assay (ELISA) were used to show that the SGPG/SGLPG fraction was free of crossreactive antigenic components such as gangliosides. The results obtained by ELISA demonstrate that sera of all patients were validated IgM monoclonal anti-myelin-associated glycoprotein (MAG) demyelinating neuropathy display high titer antibodies with high affinity type titration curves. These antibodies were absent from the sera of 16 patients with other neurological diseases and ten normal controls. ELISA with SGPG/SGLPG as an antigen appears to be a valuable and highly specific immunodiagnostic test for determining anti-MAG antibody titers in the sera of patients. The amount (approximately 0.4 mg) of SGPG/SGLPG partially purified from 10 g of fresh tissue is sufficient for the preparation of approximately 100 96-well plates for ELISA procedures. PMID- 1712033 TI - [A study of chemical mediators in patients with allergic rhinitis. 3). Release of histamine and leukotrienes from in vitro nasal mucosa]. AB - Responses to mite antigen and nonimmunological stimuli, substance P, of chopped fragments of nasal mucosae were studied from eleven patients with perennial allergic rhinitis who were sensitive to mite antigen. Amount of released histamine significantly increased by either stimulation. However, the release of leukotrienes (LTs) increased only by the stimulation of mite antigen, which correlated with that of histamine. By either stimulation, amount of histamine release tended to be higher in patients with severe nasal symptoms than in those with moderate nasal symptoms. Only by mite antigen stimulation, amount of the release of peptide LTs and LTB4 tended to be higher in patients with severe nasal symptoms. We concluded that the amounts of histamine and LTs released from nasal mucosa in allergic reaction were closely related with the severity of nasal symptoms in patients with allergic rhinitis. PMID- 1712034 TI - Guidelines for the care of children and adolescents with HIV infection. Approach to neurodevelopmental and neurologic complications in pediatric HIV infection. PMID- 1712035 TI - Monitoring of serum alpha-fetoprotein levels in children with chronic hepatitis B virus infection. AB - Changes in serum alpha-fetoprotein (alpha FP) levels were investigated by radioimmunoassay during the follow-up (17 +/- 12 months, two to three times per year) of 50 children with chronic hepatitis B virus infection (mean age of 8 years, 30 males) and of 35 healthy age- and sex-matched controls. Eleven of 50 were healthy carriers; 7 had chronic persistent hepatitis, 29 had chronic active hepatitis, and 3 had cirrhosis-associated chronic active hepatitis. Serum alpha FP levels in controls were found to be always lower than 5 ng/ml (0.1-4.4 ng/ml, mean +/- SD of 1.34 +/- 1.32 ng/ml). Statistical analysis after logarithmic transformation showed a significant difference between mean levels (ng/ml) in controls and in patients [geometric mean = 0.83 C.L. (95% confidence limits of 1.19/0.58) vs. 3.43 (95% C.L. of 4.79/2.45); p = 0.0001]. Mean values of serum alpha FP levels at entry were higher than those found at the end of the follow-up period [geometric mean = 3 (95% C.L. of 4.69/1.92) vs. 1.48 (95% C.L. of 2.13/0.95); p = 0.038]. Only three patients repeatedly showed high alpha FP levels (76.7, 122.8, and 1,600 ng/ml at entry): alpha FP values became normal after a mean follow-up of 17 +/- 7.8 months as well as liver enzymes, with no changes in serum "e" antigen-antibody and anti-delta antibody status being observed. Mean values of serum alpha FP levels in HBeAg-positive patients were significantly higher than in HBeAg-negative patients both at entry and during the follow-up (p = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712036 TI - New insights into peptidergic abnormalities in Hirschsprung's disease by wholemount immunohistochemistry. AB - In a pilot study previously reported, we showed that individual nerves could be traced in the different layers of the gut in Hirschsprung's disease (HD) using wholemount immunohistochemistry (WI). Little is known about the course of the important nonadrenergic, noncholinergic nerves containing neuropeptides in HD. Therefore, we studied the distribution of neuropeptides in 9 HD patients and 5 controls using WI. The new findings include the following: (1) there were two populations of substance P (SP) nerves--in aganglionic gut, SP-efferent nerves were decreased but SP-afferent fibres innervating blood vessels and mucosa remained unchanged; (2) met-enkephin was present only in efferent nerves to muscle and was decreased in aganglionic gut; and (3) peptidergic nerves have a disorganised pattern in HD affecting not only aganglionic gut but also "normal" gut at the colostomy site. These peptidergic abnormalities may play an important role in the pathophysiology of HD. In particular, the imbalance of afferent and efferent innervation, a finding not previously described in HD, warrants special attention in future studies. PMID- 1712037 TI - Effects of neuropeptides, ruthenium red and neuraminidase on chemoreflexes mediated by afferents in the dog epicardium. AB - 1. Experiments were performed on anaesthetized, open-chest dogs to determine reflex effects on blood pressure and heart rate produced by stimulation of neural afferents of the left ventricular epicardium by local application of capsaicin, bradykinin, nicotine and the neuropeptides substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and calcitonin gene-related peptide (CGRP). 2. Studies also included assessing whether reflexogenic actions of capsaicin, bradykinin and nicotine are influenced by epicardial treatment with either neuropeptides, Ruthenium Red or neuraminidase. 3. Epicardial application of either capsaicin (0.1-10 micrograms) or bradykinin (0.1-1 micrograms), consistently resulted in dose-related increases in blood pressure and heart rate, whereas reflex bradycardia and hypotensive effects were initiated by the application of nicotine (30-50 micrograms). 4. SP, NKA, NKB and CGRP caused marked hypotensive effects and tachycardia when injected intravenously (1 microgram), but failed to produce any cardiovascular response when applied to the epicardium of the left ventricle (0.1-1 microgram). Treatment of the heart surface with these neuropeptides (0.05 0.5 micrograms min-1) was also without any effect on the magnitude of reflex responses evoked by epicardial application of either capsaicin, bradykinin or nicotine. 5. Superfusion of the ventricular epicardium with Ruthenium Red (10-30 microM), a cationic dye known to have sialic acid as a molecular target, antagonized the reflexogenic effects of capsaicin but not those of bradykinin or nicotine. The reflex effects of capsaicin, but not those of bradykinin, were also sensitive to inhibition by epicardial treatment with neuraminidase, an enzyme which cleaves sialic acid residues from glycosides and sialoglycoproteins. 6. We conclude that neuropeptides which may be released from the peripheral endings of some cardiac sensory neurons neither directly activate nor sensitize spinal sympathetic and vagal afferents in the dog heart to the reflexogenic action of bradykinin, nicotine or capsaicin. 7. We further suggest that activation of the cardiac sympathetic chemoreflex by capsaicin involves its interaction with calcium-binding sialic acid moieties present on the surface of axons and/or terminals of chemosensitive sympathetic afferents distributed in the dog ventricular epicardium. PMID- 1712038 TI - Regional heterogeneity of endothelium-dependent vasodilatation in the rabbit kidney. AB - 1. Regional heterogeneity of endothelial function exists but its role in the local regulation of vascular tone is uncertain. This heterogeneity may be very important in the control of the glomerular filtration rate (GFR) in which the differential tone in the afferent and efferent arterioles is crucial. 2. When an endothelium-independent vasodilator, prostacyclin (PGI2) or nitroprusside, was infused into anaesthetized rabbits there were dose-dependent falls in both mean arterial pressure (MAP) and GFR; PGI2 (0.4 nmol kg-1 min-1) altered MAP and GFR by -18.5 +/- 3.6% (mean +/- S.E.M.) and -37.7 +/- 13.3% respectively and nitroprusside (30 nmol kg-1 min-1) by -29.7 +/- 3.1% and -67.0 +/- 2.4%. In contrast infusion of an endothelium-dependent vasodilator, acetylcholine (ACh) or substance P, produced dose-dependent decreases in MAP but dose-dependent increases in GFR; ACh (10 nmol kg-1 min-1) -15.1 +/- 2.0% and +43.8 +/- 16.5% and substance P (30 nmol kg-1 min-1) -18.7 +/- 1.9% and +45.3 +/- 23.1% respectively. The effects of endothelium-dependent and independent vasodilators on GFR was significantly different (p less than 0.005). 3. Simultaneous administration of indomethacin, Methylene Blue or NG-monomethyl-L-arginine (L-NMMA), inhibitors of cyclo-oxygenase and endothelium-derived relaxing factor (EDRF) respectively, attenuated or reversed the effect of ACh (10 nmol kg-1 min-1) on MAP and GFR. 4. These data suggest that endothelium-dependent vasodilatation in the kidney has a heterogeneous effect on the renal microvasculature, exerting a preferential effect on afferent glomerular arterioles and thereby preserving GFR despite the fall in MAP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712039 TI - Muscarinic receptor is coupled with a cation channel through a GTP-binding protein in guinea-pig chromaffin cells. AB - 1. The ionic current evoked by muscarinic receptor agonists was investigated in dispersed chromaffin cells of the guinea-pig adrenal medulla using the whole-cell version of the patch-clamp procedure. 2. Muscarine or oxotremorine (0.03-10 microns) produced an inward current associated with an increase in current noise at a holding potential of -40 mV. The relationship between current and oxotremorine concentration fitted well to a rectangular hyperbole with an apparent dissociation constant (KA) of 0.23 micron. 3. The muscarinic antagonists pirenzepine (0.1 micronM) and AF-DX 116 (0.3 microM) shifted the dose-response curve to the right in a parallel manner. Dissociation constants (KB) for pirenzepine and AF-DX 116 were estimated to be 50 and 70 nM, respectively. 4. The current-voltage relation for the current induced by muscarine had a negative slope below -30 or -20 mV, and the current reversed its polarity at 0.4 +/- 0.8 mV (n = 4) in standard salt solution. Removal of Mg2+ or Ca2+ from the perfusate did not modify the muscarinic current-voltage relationship. 5. When Na+ in the bath solution was replaced with Tris, the muscarinic current-voltage relationship shifted to the left (the hyperpolarizing direction); the current reversed its polarity at -18.7 +/- 1.2 mV in a solution containing 72 mM-Na+ (three cells) and at -57.5 +/- 2.7 mV in nominally Na(+)-free solution (three cells). When Ca2+ was replaced by Mg2+, in Na(+)-free solution, an inward current was not evoked by muscarinic stimulation. 6. Tetraethylammonium (TEA; 0.03-3 mM) reduced the muscarinic current at -40 mV, and the KD value was 0.34 mM with a Hill coefficient of 1. Barium (4 mM) reduced the current to 0.69 +/- 0.06 of control (n = 3). 7. When the recording electrodes contained guanosine-5'-O-(3 thiophosphate) (GTP gamma S, 100 microM), an inward current developed at -55 mV during the first few minutes after breaking into the cell interior. This inward current was associated with an increase in noise and was not larger at -70 mV than at -55 mV; it reached a peak value about 3-4 min after breaking into the cell interior and then declined over the next 2-3 min. 8. Muscarinic agonists had no effect in those cells in which intracellular GTP gamma S had first evoked a transient inward current. The inward current caused by nicotine was unaffected by intracellular GTP gamma S.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712040 TI - Characterization of ion channels seen in subconfluent human dermal fibroblasts. AB - 1. Ion channels expressed in human dermal fibroblasts are characterized using the patch-clamp technique. 2. A number of different ion channels were found but their expression occurred at various frequencies. The most commonly found phenotype was the expression of voltage-gated K+ current. This 'typical' K+ current was seen in about 60% of the cells recorded. 3. Subtypes of voltage-gated K+ channels could be discerned by differences in gating kinetics. One has fast inactivation and resembles the 'A' K+ current. Additional subtypes were sometimes discerned based on activation kinetics. 4. The large-conductance Ca(2+)-activated K+ channel (maxi-K+) could be found in nearly every cell but required large depolarizations to activate using the standard Ca(2+)-buffered pipette solution (10(-8) M [Ca2+]i). 5. Inward rectifier K+ channels were seen in a low percentage of cells. The inward rectifier K+ current was sensitive to 'wash-out' if guanosine 5'-O-(3 thiotriphosphate) (GTP gamma S) was included in the pipette solution dialysing the cell. 6. Tetrodotoxin (TTX)-sensitive voltage-gated Na+ channels were seen but in a lower number of cells recorded, about 20%. Evidence for subtypes of Na+ channels were sometimes seen based on differences in gating kinetics. 7. An ATP dependent osmotically activated Cl- current was also found. This current showed some outward rectification but was otherwise voltage independent. 8. In addition, a cell-to-cell contact-associated K+ current was described. This current was linear over the voltage ranges used and whose gating correlated with the existence of gap junctions. 9. These currents were characterized to determine the baseline behaviour of unstimulated cells and to compare to bradykinin-stimulated cells described in the following paper. As unexcitable cells, human dermal fibroblasts are capable of expressing a surprising diversity of ion channel phenotypes and of ion channel modulations. PMID- 1712041 TI - Novel chloride conductance in the membrane of bovine chromaffin cells activated by intracellular GTP gamma S. AB - 1. The effects of introducing the non-hydrolysable GTP analogue guanosine 5'-O-(3 thiotriphosphate) (GTP gamma S) into perfused bovine chromaffin cells were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence [Ca2+]i measurements. 2. GTP gamma S (5-300 microM) induced a slowly developing transient current (inwardly directed at the holding potential 60 to -70 mV) and [Ca2+]i oscillations. The current activated with a 10-50 s delay after the start of whole-cell dialysis, peaked at 70-120 s and decayed almost to its initial level during the next 150-300 s. Calcium oscillations were observed within the first 100-150 s of cell perfusion. 3. GTP competitively lowered the probability of current activation by GTP gamma S. At low GTP gamma S/GTP ratio (5 and 300 microM, respectively) activation of the current was observed only rarely. 4. The activation of the current was accompanied by an increase in conductance but not by changes in the current reversal potential. The changes in the conductance did not depend on the membrane potential; no time dependent relaxation of the current was induced by steps in the membrane voltage. 5. The current reversal potential was close to the Cl- equilibrium potential; changes in the extracellular Cl- concentration induced corresponding changes in the current amplitude and shifted its reversal potential. The permeability to larger anions--aspartate, glutamate and isethionate--was about one-tenth of that for chloride. 6. Single-channel conductance, estimated from the ratio of the mean current and its variance, was about 1-2 pS. 7. The current could be reversibly blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 10 microM), chlorpromazine (5 microM) and tolbutamide (0.5-5 mM). 8. It is suggested that the GTP gamma S-induced increase in the permeability to Cl- ions is due to a G protein-mediated production of an as yet unidentified second messenger. PMID- 1712042 TI - Second messengers mediating activation of chloride current by intracellular GTP gamma S in bovine chromaffin cells. AB - 1. Intracellular mechanisms and second messengers involved in chloride current activation by intracellular GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] in bovine chromaffin cells were studied using the whole-cell patch-clamp technique combined with measurements of intracellular calcium [Ca2+]i. 2. No correlation between the time of current activation and the appearance of [Ca2+]i transients was observed; intracellular introduction of sufficient EGTA (10 mM) to suppress the [Ca2+]i transients did not affect the current activation by GTP gamma S. 3. The cyclic nucleotides, cyclic AMP or cyclic GMP, did not activate the current when introduced intracellularly (50-250 microM). The ability of GTP gamma S to activate the current decreased when cyclic GMP (250 microM), together with MgATP (2 mM), was added to the perfusate. 4. Neomycin (0.5-1 mM), a presumed inhibitor of phospholipase C effectively prevented the current activation by GTP gamma S but it did not prevent [Ca2+]i transients. 5. Modulation of protein kinase C activity using specific inhibitors (H-7, 300 microM; polymyxin B, 400 U/ml) or activators (phorbol ester PMA, 100 nM, 20-90 min at 37 degrees C) did not affect the current activation by GTP gamma S nor did it cause current activation in the absence of GTP gamma S. 6. Activation of the current by GTP gamma S could be prevented by incubating the cells for 10-15 min with 2.5 microM p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10 microM), applied extracellularly or intracellularly, neither activated the current itself nor did it interfere with its activation by GTP gamma S. 7. Activation of the current by GTP gamma S could also be prevented by incubating the cells with 1 microM-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2 microM), an inhibitor of cyclo oxygenase pathway of arachidonic acid metabolism. 8. It is suggested that chloride current activation by GTP gamma S in bovine chromaffin cells involves G protein-mediated stimulation of phospholipase A2 activity and subsequent formation of lipoxygenase product(s) of arachidonic acid metabolism. PMID- 1712044 TI - Altered patterns of keratin expression in oral hairy leukoplakia: prognostic implications. AB - To establish why the lateral border of tongue is the site of predilection for the development of hairy leukoplakia (HL) and to understand its likely behavior, the pattern of keratin expression was compared in 8HL lesions with matched controls in an immunocytochemical study. Keratins 7, 8, 18 were absent in HL and normals; uniform basal keratin 19 was present in normals but much reduced in HL. Loss of conformationally sensitive epitopes of keratin 14 in lower epithelial layers was seen in HL. Overall expression of non-cornifying keratins 4/13 was reduced in HL and completely lost in the parakeratin zone. Expression of the high-turnover keratins 6/16 was reduced in HL. The HL keratin phenotype suggests that no dysplastic change is likely, but in contrast there is enhanced differentiation, which suggests a benign course for the condition. PMID- 1712043 TI - Characterization of ionic currents of circular smooth muscle cells of the canine pyloric sphincter. AB - 1. The ionic currents of circular muscle cells from canine pyloric sphincter were characterized using the whole-cell patch clamp technique. 2. Subpopulations of circular muscle cells from the myenteric and submucosal halves of the circular layer were isolated and studied separately to determine whether differences in the currents expressed by these cells could explain differences in electrical behaviour observed in situ. 3. Resting potentials of isolated cells were about 20 mV positive to cells in intact muscles. Polarization under current clamp to the level of tissue resting potentials caused spontaneous discharge of action potentials in many cells. 4. Outward current measured under voltage clamp could be divided into a voltage-dependent component and a voltage- and Ca(2+)-dependent component. The latter was affected by manipulations of external [Ca2+], nifedipine and dialysis of cells with EGTA. 5. A few cells exhibited a channel that was activated with hyperpolarization. These channels produced inward current at potentials positive to the potassium reversal potential, EK, and reversed at 13 mV. 6. Inward currents, recorded from Cs(+)-loaded cells, were characterized by a transient phase and a sustained phase that persisted throughout the test depolarization. The inward current was reduced by nifedipine but in some cells a nifedipine-resistant component was observed. 7. There were no fundamental differences in the ionic currents recorded from circular muscle cells from the myenteric and submucosal regions, suggesting that the electrical activity of the tissue must be dependent upon structural characteristics (i.e. electrical coupling, fibre bundle dimensions, etc.) of the tissue. 8. The ionic conductance characterized can be related to many of the excitable events recorded from pyloric muscles. PMID- 1712045 TI - Experimental epithelial dysplasia produced by excisional wounding and application of Trp-P-2 in the hamster tongue. AB - All 10 groups of hamsters received excision of the tip of tongue. Subsequently, 3 groups in which tongues were treated with Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3 b] indole) and then again excised in their tips followed by no treatment or by additional applications of DMSO or Trp-P-2, showed moderate to severe epithelial dysplasia. One group in which tongues were not treated and then again excised followed by applications of Trp-P-2, exhibited very slight epithelial dysplasia. However, one group in which tongues were treated with DMSO and then again excised followed by additional applications of DMSO, did not show any pathological changes. The remaining 5 groups in which tongues were treated with DMSO or Trp-P 2 or received no treatment and then not excised followed by no treatment or by additional applications of DMSO or Trp-P-2, also did not show any pathologic changes. These results clearly indicated that Trp-P-2 treatment together with two times of excisional wounding could produce lingual epithelial dysplasia in hamsters. PMID- 1712046 TI - Pancreatic enzyme activity in pregnancy. AB - Serum amylase activity and the amylase:creatinine clearance ratio (Cam:Ccr%) are two of the most commonly used indicators for the diagnosis of pancreatitis. However, published data on the effect of pregnancy on these indicators are conflicting. Furthermore, there are no published data on the effect of pregnancy on serum lipase activity, which is considered one of the most sensitive and specific indicators of pancreatitis. A study was undertaken to determine the effect of pregnancy and gestational age on serum amylase, serum lipase and Cam:Ccr% levels and to establish a baseline of normal values for use in the diagnosis of pancreatitis in pregnant women. Serum amylase, serum lipase and Cam:Ccr% levels were determined on a sample population consisting of 175 pregnant women with gestational ages ranging from 5 to 40 weeks and on a control group of 44 reproductive-age, nonpregnant women. The study results indicated that there is no significant difference in serum amylase, serum lipase and Cam:Ccr% levels between pregnant and nonpregnant women. Cam:Ccr% showed a small but statistically significant increase in the third trimester of pregnancy. PMID- 1712047 TI - Lipophilic glycosyl phosphotriester derivatives of AZT: synthesis, NMR transmembrane transport study, and antiviral activity. AB - Phosphate derivatives of AZT esterified with a carbohydrate (D-glucose, D mannose, and ethyl D-mannopyranoside) and a hexadecyl chain were prepared from glucose 6-phosphate and D-mannose precursors. The 31P NMR study of the mannosyl phosphotriester series in the presence of large unilamellar vesicles demonstrated either an interaction with the external lipid layer or a transmembrane transport into the intravesicular interface. The antiviral activity, measured by the inhibition of cytopathogenicity on different infected cells and of reverse transcriptase activity in the supernatant of cultures, appeared to be comparable to that of AZT, in the micromolar range. PMID- 1712048 TI - Case for prostate therapy wanes despite more treatment options. PMID- 1712049 TI - [Modulation of the surface antigens of Salmonella-phagocytized U937 cells]. AB - In this study, modulation of the surface antigens of Salmonella enteritidis phagocytized U937 cells and morphology of the bacteria in these cells were analyzed by the indirect immunofluorescence technique. The results are as follows: (1) Morphological studies revealed that the bacteria phagocytized by the U937 cells were transformed to a small coccoid form. (2) The expression of CD14 antigen was observed 24 to 48 h after phagocytosis. (3) The levels of CD11b and CD23 antigens were clearly enhanced 48 h after phagocytosis. (4) No modulation of HLA-class II (DR, DQ and DP) antigens was observed after phagocytosis. PMID- 1712050 TI - [Effect of heat-staining procedure on the gram staining properties of mycobacteria]. AB - Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure. PMID- 1712051 TI - [Kinetics of immune complex deposition and influence of decomplementation on their clearance in cationized antigen induced acute serum sickness]. AB - Using a murine acute serum sickness model caused by cationized bovine gamma globulin (CBGG), the histological findings, accumulation of CBGG in the organs and the effect of decomplementation for the clearance of immune complex (IC) were investigated. The accumulation of CBGG increased in the lungs in the presence of anti-CBGG antibody 1 hour after injection of CBGG, but did not change in the kidneys. This suggests that CBGG forms IC in situ in the kidneys, and that circulating IC accumulates in the lungs. Decomplementation did not influence the uptake of CBGG immediately after challenge but delayed the clearance of CBGG in the kidneys, lungs, liver and spleen. A beneficial role of the complement system in the clearance of IC even for those formed in situ was recognized. Histological changes, cellular infiltration and microvascular destruction, evoked in the lungs in this experimental model immediately after challenge, were not seen in the kidney, suggesting the different modes of complement activation in these organs. PMID- 1712052 TI - [Acute pancreatitis in primary and metastatic tumors of the pancreas]. AB - Malignant tumours both of the pancreatoduodenal zone and of other organs situated close to or remote from it occupy a certain place among various etiopathogenetic factors of acute pancreatitis. Complication of the neoplastic process of these organs by acute carcinogenic pancreatitis (ACP) has an effect on the clinical picture of the disease as well as on the therapeutic tactics and the outcomes of the treatment. According to the authors' data (30 patients), ACP occurs in primary carcinoma of the pancreas and in its secondary involvement (metastases and growth of tumours of other organs into the pancreatic tissue). The article discusses the causative factors and clinical forms of ACP, the specific features of their diagnosis and therapeutic tactics. Purposeful nonoperative and operative treatment of ACP makes it possible to reduce the mortality in malignant diseases and prolong the patients' survival. PMID- 1712053 TI - Major amputations done with palliative intent in the treatment of local bony complications associated with advanced cancer. AB - Palliative amputations were performed on 11 patients (7 men, 4 women) with disseminated disease to control local bony complications. The average patient age was 54 years (range 14-78 years). The primary diseases were melanoma/sarcoma (seven patients) and carcinoma (four patients). All had pain; eight had intractable pain that could not be controlled by analgesics. All 11 patients had additional severe local complications, which included recurrent pathological fracture (4), sepsis (2), hemorrhage (2), radiation necrosis (2), and iliofemoral thrombosis secondary to tumor (1). Previous attempts of palliation had been made in all 11 patients, and 8 had undergone previous operative procedures (5 had undergone two or more) prior to amputation. Three anterior hemipelvectomies, five posterior hemipelvectomies, two hip disarticulations, and one forequarter amputation were performed. All patients survived the surgery, and there were no intraoperative complications. All patients received dramatic relief of pain. Postoperative complications included two cases of flap necrosis and two infections; all resolved satisfactorily. The six patients who were nonambulatory before surgery ambulated postoperatively, and two eventually ambulated with a prosthesis. Six of 11 patients survived 1 year or longer, with a median postoperative survival period of 13 months (average 16 months). Although major amputations are viewed at times as offering little to already-compromised patients, they can improve dramatically the quality of life in selected patients. PMID- 1712054 TI - Automated image analysis for counting unstained cultured neurones. AB - A fully automated image analysis technique was developed for counting the number of live or fixed, unstained neurones present in a representative region of a cell culture dish. A dish containing cultured mouse hippocampal neurones was placed on the motorized stage of an inverted microscope, and the neurones were visualized using Hoffman modulation contrast optics. The resulting image was digitized, and processed by subtracting the background illumination, low pass filtering, thresholding, then deleting objects whose areas fell outside a specified range. Two threshold levels were used, each with its own area range, and the two resulting binary images were combined. The number of objects in the combined image was counted. The number of cells in each field was also counted manually, and the processing was repeated on a series of 100 fields covering a representative region of the dish. The automated counts were highly correlated with the manual counts for each of the 12 culture dishes examined in this study. The correlation coefficient was calculated for the manual and automated counts from each dish, and the values ranged from 0.91 to 0.97. Six of the dishes were treated with the envelope protein of the human immunodeficiency virus (gp120), which reduces survival of neurons in this system. The six treated dishes were found to have significantly fewer neurones than the six control dishes, using either manual or automated counting techniques. PMID- 1712055 TI - A method using DiI to study the connectivity of cortical transplants. AB - Carbocyanine DiI is described as a suitable fluorescent tracer to investigate the connectivity of cortical transplants. DiI was applied in 2 ways: in tissue previously fixed with aldehydes, and in vivo for labelling the donor tissue prior to transplantation. When applied in fixed tissue DiI travelled anterogradely, allowing the study of efferent connections from the transplant to the host. When DiI was applied in vivo, it travelled retrogradely showing the afferents to the transplant from the host. PMID- 1712056 TI - GABA release from mouse striatal neurons in primary culture as a test for the functional activity of N-methyl-D-aspartate (NMDA) antagonists. AB - N-Methyl-D-aspartate (NMDA)-induced release of [3H]GABA from mouse striatal neurons in primary culture has been evaluated as a screening method for demonstrating the functional activity of potential NMDA antagonists with respect to a cellular response. Antagonists were chosen for their specificity towards each of the three principal binding sites which have been characterised on the NMDA-receptor complex: the glutamate site, the ion-channel and in particular the glycine regulatory site where several novel halogenated derivatives of kynurenic acid have been tested. All the compounds were effective in blocking [3H]GABA release and their activity was related to their potency in displacing the binding of specific ligands for each of the three sites in rat cortex membrane preparations. This was confirmed by a correlation curve for the series of kynurenate derivatives (correlation coefficient r = 0.96). The specificity of these latter compounds for the glycine site was demonstrated by the addition of excess glycine which totally reversed their inhibition but not that of antagonists acting at the glutamate or ion-channel sites. Within the kynurenate series the 5,7-dichloro derivative was shown to be more active than the 7-chloro derivative, the most active glycine antagonist previously described. These results show that this is a simple and reliable system for demonstrating a functional effect of NMDA antagonists. PMID- 1712057 TI - A fluorescent labeling strategy for staining the enteric nervous system. PMID- 1712058 TI - Co-localization of proenkephalin mRNA using cRNA probes and a cell-type-specific immunocytochemical marker for intact astrocytes in vitro. AB - To determine whether cultured astrocytes express opioid gene mRNA, a method was developed for co-localizing a cell-type specific immunocytochemical marker for astrocytes, glial fibrillary acidic protein (GFAP), and proenkephalin mRNA in situ hybridization signal using high affinity cRNA probes. GFAP immunoreactivity and proenkephalin mRNA hybridization reaction were examined in intact glial cell preparations from neonatal mice that were cultured for 4-6 days prior to fixation. The double labelling method described herein permits the unambiguous identification of mRNA expression in specific populations of intact cultured cells using cell type-specific markers. PMID- 1712059 TI - A new rapid silver impregnation for neuronal bodies on methacrylate sections. AB - A simple and rapid method for the impregnation of neuronal bodies applicable to methacrylate embedded sections is described in the present paper. Sections of 10 12 microns in thickness were attached to slides, placed in mordant for 1 min, rinsed in distilled water and impregnated in ammoniacal silver solution for 1 min. They were then rinsed in absolute ethanol for 30 s and developed in 50% formalin. Sections were toned in 0.25% gold chloride, reduced in 10% oxalic acid and fixed in 5% sodium thiosulfate. After washing, the sections were dehydrated through 90% and absolute ethanol, cleared in eucalyptol, and mounted in the usual way. When this method is used most of the neuronal somata and proximal dendritic trees are impregnated. Frequently some glial cell are also weakly impregnated but their density does not obscure the neurons. PMID- 1712060 TI - A note on the use of biocytin in anterograde tracing studies in the central nervous system: application at both light and electron microscopic level. AB - A new neuroanatomical method based on the anterograde transport of biocytin has recently been reported. The validity of this method is examined closely on a number of well established pathways in the central nervous system, as well as the experimental parameters necessary for its effective use. iontophoretic application of this amino acid-biotin complex allows the placement of very discrete injections. At the injection sites neurones appear to be completely filled, whereas fibres of passage are left unlabelled. From the injection site axons can be traced at the light microscopic level to their terminal fields, where their pattern of termination and morphology can be clearly visualised. These anterogradely labelled fibres can be examined further by electron microscopy to identify synaptic specializations. The uptake and transport of biocytin, following iontophoretic injection, appears to be similar to that observed using techniques employing radiolabelled amino acids or Phaseolus vulgaris-leucoagglutinin (PHA-L), in that it is transported predominantly in the anterograde direction. However, biocytin offers some advantages over these techniques in that its detection is relatively easy at electron microscopic levels. Furthermore, biocytin appears to be transported rapidly making it possible to study short pathways under acute experimental conditions. PMID- 1712061 TI - Dual fluorescence combined with a two-color immunoperoxidase technique: a new way of visualizing diverse neuronal elements. AB - A method is described that allows an estimation of the neurotransmitter-related immunoreactivity, morphology and relationship to other immunoreactive elements, of single functionally identified neurons in the central nervous system. First, neurons are identified electrophysiologically using intracellular recording and labelled by iontophoresis of lucifer yellow (LY). After fixation and sectioning of the brain tissue, the location of the labelled neuron is determined by fluorescence microscopy. Sections are then processed using an indirect immunofluorescence procedure in order to determine the antigen content of the labelled neurons. Antisera to LY and an avidin-biotin immunoperoxidase technique is then used to localize LY in a permanent form, while the other previously localized antigen is permanently visualised by using the fluorescent-labelled second antibody as a bridge antibody in a peroxidase anti-peroxidase technique. The method is illustrated by an examination of neurons in the medulla oblongata of the rat, that have been stained intracellularly with LY, their content of tyrosine hydroxylase assessed, and their relationship to other tyrosine hydroxylase immunoreactive neurons determined. PMID- 1712062 TI - Prediction of lymph node involvement in breast cancer by detection of altered glycosylation in the primary tumour. AB - Axillary lymph node metastases at the time of diagnosis of breast cancer is the most accurate predictor of long-term prognosis. However, in patients treated by conservative surgery lymph node status often remains unknown. We have investigated the relation between changes in glycosylation of primary breast cancer cells, as judged by lectin binding, and the presence of axillary lymph node metastases. In a 24-year retrospective study, paraffin-embedded sections of 373 primary breast cancers were stained for the binding of Helix pomatia lectin (HPA). There was a strong association between HPA binding and presence of lymph node metastases, but no association with tumour size, histological grade, S-phase fraction, or patient age at diagnosis. This relation was confirmed by multiple regression analysis (in both survival and relapse free survival models) in which the prognostic significance of HPA binding was lost once nodal status had been introduced into the models. Life tables calculated for lymph-node positive versus lymph-node negative and HPA staining versus non-staining patients were almost identical over 15 years of follow-up. We propose that HPA recognises a glycoprotein that is associated with metastasis (to axillary lymph nodes and elsewhere) and poor prognosis in breast cancer. HPA binding to paraffin sections of primary tumour could aid difficult treatment decisions by providing an additional assessment of staging and likely long-term patient prognosis. PMID- 1712063 TI - A staining plate for electron microscopy. PMID- 1712064 TI - Use of paramagnetic chelated metal derivatives of polysaccharides and spin labeled polysaccharides as contrast agents in magnetic resonance imaging. AB - Soluble and insoluble polysaccharides were derivatized with diethylenetriaminepentaacetic acid (DTPA) and/or spin-labeled with 2,2,6,6 tetramethylpiperidine-1-oxyl (TEMPO). Polysaccharides derivatized with DTPA were prepared via cyanogen bromide activation, coupling to a diamine linker, and to DTPA anhydride. Spin-labeled polysaccharides were also prepared via cyanogen bromide activation. The extent of derivatization for dextran (18 kDa) was about 120 glucose units per DTPA, and for cellulose and starch about 15-30 units per DTPA. For spin-labeled polysaccharides, the average loading ranged from 1 nitroxide per 16 glucose units for starch to 181 for dextran (82 kDa). These derivatized paramagnetic polysaccharides were shown to be more effective relaxants than the small paramagnetic molecules alone. Both soluble and insoluble polysaccharide-linker-DTPA-Gd(III) complexes were effectively cleared from the body (rats) after oral administration. After intravenous administration, the biodistribution of dextran-linker-DTPA-Gd(III) complexes differed significantly from that of GdDTPA. Reduction of the nitroxide by ascorbic acid was retarded in the polysaccharide derivatives, particularly in starch derivatized with both nitroxide and linker-DTPA-Cu(II). These agents showed contrast enhancement in the gastrointestinal tract of rabbits. PMID- 1712065 TI - Characterization of basement membranes of rat choroid plexus using the critical electrolyte concentration technique. AB - Using the critical electrolyte concentration technique, with ruthenium red as a strain for polyanionic macromolecules, we examined the basement membranes of the rat choroid plexus. Concentrations of Na+ exceeding 3.0 M were required to reversibly inhibit discrete staining of endothelial and epithelial basement membranes by ruthenium red, whereas 2.5 M Na+ inhibited staining of renal pertitubular capillary basement membranes. The findings are consistent with recent evidence that basement membranes underlying fenestrated capillaries are more polyanionic that those underlying continuous capillaries, and suggest that basement membranes of the choroid plexus are more polyanionic than those of peritubular capillaries. PMID- 1712066 TI - Endothelial cells are required for inhibition of contractile responses induced by reduction in extracellular magnesium and sodium ions in rat aortic smooth muscle [corrected]. AB - The possible importance of facilitation of sodium-calcium (Na(+)-Ca2+) exchange by removal of extracellular magnesium ions ([Mg2+]o) in expression of endothelium dependent relaxation was investigated in aortic rings isolated from female rats. Simultaneous [Mg2+]o withdrawal (0 mM Mg2+) and reduction in extracellular Na+ (Total [Na+]o = 84 mM), by replacement of NaCl with isosmolar amounts of sucrose in normal Krebs-Ringer bicarbonate (NKRB), induced significant increases of basal tone of denuded rat aortic rings, but not in tissues with intact endothelium. These vascular effects were not affected by indomethacin, phentolamine or atropine in any of the tissues tested. Reintroduction of 1.2 mM Mg2+ or removal of extracellular Ca2+ ([Ca2+]o) from the Mg2+ and Na(+)-deficient incubation media induced complete relaxation of the denuded tissues. Methylene blue (10(-5) M), an inhibitor of endothelium-derived relaxant factor (EDRF), potentiated tension development in intact tissues. These results suggest that: (1) as in vascular muscle, Mg2+ plays an important role in Ca2+ homeostasis in endothelial cells (EC), probably via Na(+)-Ca2+ exchange; and (2) such Mg(+)-regulated internal Na(+)-dependent Ca2+ entry participates in the expression of endothelium dependent relaxation. PMID- 1712067 TI - The cps gene cluster of Salmonella strain LT2 includes a second mannose pathway: sequence of two genes and relationship to genes in the rfb gene cluster. AB - We report the presence in Salmonella enterica strain LT2 (serovar thyphimurium) of duplicate genes for two steps in the synthesis of GDP-mannose. The previously known genes, rfbK (phosphomannomutase) and rfbM (mannose-1-phosphate guanyltransferase), are part of the gene cluster for the O antigen. The two new genes, cpsB and cpsG, respectively, are thought to be part of the gene cluster for the M antigen capsular polysaccharide, present in many Enterobacteriaceae. The two genes have been sequenced and have a GC content of 0.61, suggesting an origin outside of Salmonella. Comparison of the inferred protein sequences for cpsB and rfbM shows 57% identity of amino acids whereas for cpsG and rfbK there is only 19% identity. It is suggested that the greater divergence between cpsG and rfbK may be due to a period of accelerated evolution, perhaps precipitated by transfer of the genes from another species. PMID- 1712068 TI - Studies on the biochemical structure of the major cat allergen Felis domesticus I. AB - The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences. PMID- 1712069 TI - Covalent disulfide binding of human IL-1 beta to alpha 2-macroglobulin: inhibition by D-penicillamine. AB - Secreted human IL-1 beta is known to have two free SH groups due to unpaired cysteines (positions 8 and 71). Alpha 2-Macroglobulin (alpha 2-M) has internal thioester bonds between cysteine and glutamate residues. Free SH groups may be generated at these alpha 2M residues through the action of proteinases, amines such as methylamine, or at a slow rate, by H2O ("aging" of alpha 2M). Thus, the possibility that IL-1 beta forms a disulfide bond with alpha 2M was investigated. 125I-labeled human rIL-1 beta (15 kDa) was incubated with fresh normal human serum or with purified alpha 2M, treated or not with methylamine. The mixtures were submitted to nondenaturing and denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. IL-1 beta bound to commercially purified "aged" alpha 2M and to alpha 2M in methylamine-treated serum but not to native serum alpha 2M. It did not bind detectably to any other serum proteins. The addition of D-penicillamine (D-pen) during the reaction of [125I]rIL-1 beta with serum or purified alpha 2M blocked the covalent binding of rIL-1 beta to alpha 2M. [125I]rIL-1 beta was removed from alpha 2M by 2-mercaptoethanol in SDS. Thus, disulfide bonds were formed between the free SH groups on [125I]rIL-1 beta and those resulting from the cleavage of the internal thioester bonds of alpha 2M. "Cold" rIL-1 beta and a Cys71----Ser71 rIL-1 beta mutant effectively competed with [125I]rIL.1 beta for binding sites on alpha 2M. When complexes of rIL-1 beta or the mutant rIL-1 beta and alpha 2M were subjected to nonreducing SDS-PAGE and subsequent Western blot analysis, the rIL-1 beta molecules were found to be present in the alpha 2M bands in a dose-dependent manner. rIL-1 beta attached to alpha 2M in the presence or absence of D-pen showed similar biological activity in the mouse thymocyte-assay. Thus, rIL-1 beta attached noncovalently to alpha 2M is biologically active. The lack of inhibition of rIL-1 beta activity by binding to methylamine-treated alpha 2M in the absence of D-pen suggests, but does not prove, that the covalently bound rIL-1 beta is also active. We concluded that human rIL-1 beta binds to alpha 2M through the Cys at position 8 and that D-pen inhibits this binding. We speculate that this inhibitory effect may contribute to the therapeutic benefits of D-pen in patients with rheumatoid arthritis. PMID- 1712070 TI - An antigenic region of topoisomerase I in DNA polymerase chain reaction-generated fragments recognized by autoantibodies of scleroderma patients. AB - Topoisomerase cDNA and various fragments thereof generated by the DNA polymerase chain reaction were cloned into plasmid expression vectors (pET series) and the expressed polypeptides were probed with scleroderma sera from seven different patients immunoreactive with topoisomerase I. All sera reacted selectively with a region between amino acid residues 405 and 484 of human topoisomerase I. This conclusion is based on loss of reactivity when this region was omitted from larger pieces. Other portions of topoisomerase I were not reactive with these autoantibodies. At least two different epitopes appear to be recognized within this region by different sera based on differences in immunoreactivity of the 405 484 region when expressed as C-terminal, N-terminal or internally within a peptide. PMID- 1712071 TI - Quantitation of cytokine mRNA levels utilizing the reverse transcriptase polymerase chain reaction following primary antigen-specific sensitization in vivo--I. Verification of linearity, reproducibility and specificity. AB - The role of cytokines in vivo has been difficult to assess. This difficulty is due, in part, to the limited number of producer cells and the strict regulation of cytokine production. In order to address this situation, we have developed assays which allow us to quantitate both protein production and steady state mRNA levels from specific in vivo sites. In this report, we present data utilizing these assays on cells obtained from draining LN following specific sensitization with antigen in vivo. In order to determine the relative quantities of cytokine mRNA, we modified the reverse transcriptase-polymerase chain reaction which had been previously described. The modified assay is (1) linear over a large concn range of input template (2) demonstrates a high degree of reproducibility (SE approximately 13%) and (3) is very sensitive. Utilizing this assay, we have measured a constitutive mRNA (DHFR), quantitated both the presence of lymphokine mRNA (IL-2) and the induction of cytokine mRNA (TNF alpha). In this report we have examined the kinetics of TNF alpha mRNA expression and have demonstrated that following epicutaneous sensitization with picryl chloride, there is rapid induction (within 24 hr) of TNF alpha mRNA in the draining LN and that the levels of mRNA remain detectable through d7. In addition, we determined the time course of production of TNF protein by the draining LN cells and found that it was similar to that of the mRNA levels. A potential pathologic role for immune response generated TNF alpha is also discussed. We believe these experiments demonstrate that cytokine production following antigen-specific sensitization in vivo can be analyzed at both the cellular and molecular level. The data suggests that this approach can be used to study cytokine regulation in vivo. PMID- 1712072 TI - A monoclonal antibody recognizing the C-terminal region of chicken egg white riboflavin carrier protein terminates early pregnancy in mice. AB - In order to identify the functionally relevant epitopes on chicken riboflavin carrier protein, we have raised monoclonal antibodies to the vitamin carrier. One of these, 6B2C12, was found to interact specifically with a synthetic oligopeptide corresponding to the C-terminal 17 amino acid residues of the chicken egg white riboflavin carrier protein, which is missing in part in the egg yolk riboflavin carrier protein. This epitope is conserved through evolution in mammals including humans. Administration of the ascites fluid of 6B2C12 to pregnant mice intraperitoneally, resulted in the termination of pregnancy indicating that this epitope is involved in or closely associated with the transplacental transport of the vitamin from the maternal circulation to the growing fetus. PMID- 1712073 TI - Presentation of insulin and insulin A chain peptides to mouse T cells: involvement of cysteine residues. AB - The requirements for insulin presentation and recognition by A alpha b A beta b- and A alpha b A beta k-restricted mouse T cells were studied using a variety of derivatives of the insulin A chain. It was found that A chain peptides with irreversibly blocked Cys residues are non-stimulatory for the T cells. This suggests that at least one of the Cys residues is essential for recognition. On the other hand, all A chain peptides containing Cys residues modified in a way reversible by reaction with thiols are stimulatory yet differ in antigenic potency. All these A chain derivatives including a 14 amino acid fragment require uptake by antigen presenting cells (APC) for efficient presentation. Differences in stimulatory potency between the A chain peptides derived from the same insulin appear to be mainly due to the efficiency of uptake and/or processing by APC. Based on these findings we propose that processing in the case of insulin and its A chain derivatives involves the reductive deblocking of Cys residues or the rearrangement of disulfide bonds apart from a possible proteolytic cleavage. The same may apply to other proteins if Cys residues in the presented peptides are important for the interaction with Ia or the T cell receptor. PMID- 1712074 TI - Specificity and variable region cDNA sequence of an isogeneic monoclonal antiidiotype to an anti-alpha(1----6)dextran. AB - We have characterized a monoclonal isogeneic antiidiotype, IdB5.7, from a BALB/c mouse immunized with the anti-alpha(1----6)dextran C57BL/6 45.21.1. It defined a hapten-inhibitable idiotope expressed on four of the 2 myeloma and 37 hybridoma anti-alpha(1----6)dextrans tested. Sequence comparison of Id+ and Id- anti alpha(1----6)dextrans suggested that two extra amino acids at VH 100A and 100B and different residues at VH 101 abolish the expression of the idiotope in the Id anti-alpha(1----6)dextrans. Sequence analysis of the VH of IdB5.7 showed a CDR1 longer than usual and a D segment in CDR3 formed by the fusion of two D minigenes. The IdB5.7 V kappa uses the V kappa 1 germline gene K5.1 with a few substitutions. The D-D fusion in VH CDR3 is a feature which has been reported in several other antiidiotypic antibodies. PMID- 1712075 TI - Inhibitory activity of monoclonal antibody F35.25 on the interaction between hepatocytes (HepG2 cells) and preS1-specific ligands. AB - The capacity of a preS1-specific monoclonal antibody (McAb) F35.25 to block the attachment of preS1-specific ligands to human hepatoma HepG2 cells was studied. In order to define more precisely the fine epitope specificity of McAb F35.25, its reaction with synthetic peptides derived from the preS1 sequence (12-53) was investigated. McAb F35.25 was found to recognize better synthetic peptide preS(21 47) from the adw 2 and ayw sequences than the synthetic peptide preS(32-53) adw 2. The shortest sequence recognized by McAb F35.25 among the peptide sequence studied was preS(32-47). The corresponding amino acid sequence (for HBV subtype adw 2) is PAFGANSNNPDWDFNP. As expected, it was found that McAb F35.25 inhibited the attachment of HepG2 cells to HBsAg-cellulose, as well as to preS(21-47) cellulose, corresponding to two HBV subtypes adw 2 and ayw. Finally, the inhibitory effect of different peptides on the interaction of McAb F35.25 with HBsAg particles containing the preS1 sequence was also studied. The peptide preS(12-47) appeared to be the most effective inhibitor. Therefore, the McAb F35.25 is specific for the sequence preS1(X to 47), where (12 less than or equal to X less than 32). These results indicate that McAb F35.25 is probably virus neutralizing and represents a reagent of great value to study the interaction between HBV and hepatocytes independently of d/y subtype changes. PMID- 1712076 TI - Risk factors for pancreatic cellular injury after cardiopulmonary bypass. AB - BACKGROUND: Pancreatitis is a known complication of cardiac surgery with cardiopulmonary bypass. Although ischemia is believed to be a factor, the cause of pancreatitis after cardiopulmonary bypass remains unknown. METHODS: We prospectively studied 300 consecutive patients undergoing cardiac surgery with cardiopulmonary bypass. Serum amylase, pancreatic isoamylase, and serum lipase were measured on postoperative days 1,2,3,7, and 10. Pancreatic cellular injury was defined as the presence of hyperamylasemia (greater than 123 U per liter) with an increase in either the serum level of lipase (greater than 24 U per liter) or the peak level of pancreatic isoamylase. Trypsinogen-activation peptides, which indicate intrapancreatic enzyme activation, were measured in the urine of the last 101 patients studied. RESULTS: Evidence of pancreatic cellular injury was detected in 80 patients (27 percent), of whom 23 had associated abdominal signs or symptoms and 3 had severe pancreatitis (2 with pancreatic abscess and 1 with necrotizing hemorrhagic pancreatitis). Two of 19 postoperative deaths were secondary to pancreatitis. In multivariate analyses, the development of pancreatic cellular injury was significantly associated with preoperative renal insufficiency, valve surgery, postoperative hypotension, and perioperative administration of calcium chloride. The administration of more than 800 mg of calcium chloride per square meter of body-surface area was an independent predictor of pancreatic cellular injury, and the increase in risk was dose related. No differences were found in the level of trypsinogen-activation peptides between patients who had pancreatic cellular injury and those who did not. CONCLUSIONS: Pancreatic cellular injury, as indicated by hyperamylasemia of pancreatic origin, is common after cardiac surgery. The administration of large doses of calcium chloride is an independent predictor of pancreatic cellular injury and may be a cause of it. PMID- 1712077 TI - Cloned and expressed nitric oxide synthase structurally resembles cytochrome P 450 reductase. AB - Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase. PMID- 1712078 TI - Nanovid microscopy. AB - By combining small colloidal gold probes with video-enhanced quantitative microscopy, the intracellular dynamics of specific proteins in living cells can now be studied. PMID- 1712079 TI - Cystic fibrosis. Acidification indication. PMID- 1712080 TI - Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor. AB - Ligand-gated ion channels, a major class of cell-surface proteins, have a pseudosymmetric structure with five highly homologous subunits arranged around a central ion pore. The correct assembly of each channel, whose subunit composition varies with cell type and stage of development, requires specific recognition between the subunits. Assembly of the pentameric form of the acetylcholine receptor from adult muscle (AChR; alpha 2 beta epsilon delta) proceeds by a stepwise pathway starting with the formation of the heterodimers, alpha epsilon and alpha delta. The heterodimers than associate with the beta subunit and with each other to form the complete receptor. We have now determined which parts of the subunits mediate the interactions during assembly of the adult form of the receptor from mouse muscle by using a chimaeric subunit in which the N-terminal and C-terminal extracellular domains are derived from the epsilon subunit with the remainder from the beta subunit. The epsilon and beta subunits were chosen because the epsilon subunit forms a heterodimer with the alpha subunit in the pathway for assembly of the receptor, whereas the beta subunit does not. The epsilon beta chimera can substitute for the epsilon but not the beta subunit in the oligomeric receptor, indicating that the alpha subunit specifically recognizes an extracellular domain of the epsilon subunit. PMID- 1712081 TI - Defective acidification of intracellular organelles in cystic fibrosis. AB - The phenotype of cystic fibrosis (CF) includes abnormalities in transepithelial transport of Cl- (refs 1-5), decreased sialylation and increased sulphation and fucosylation of glycoproteins, and lung colonization with Pseudomonas. It is not apparent how these abnormalities are interrelated, nor how they result from loss of function of the CF gene-encoded transmembrane regulator (CFTR). We have previously shown that that the pH of a secretory granule is regulated by the vesicular conductance for Cl- (ref. 11). Here we find defective acidification in CF cells of the trans-Golgi/trans-Golgi network, of prelysosomes and of endosomes as a result of diminished Cl- conductance. Sialytation of proteins and lipids is reduced and ligand traffic altered. These abnormalities can result from defective acidification because vacuolar pH regulates glycoprotein processing and ligand transport. The CF phenotype is similar to that of alkalinized cells and acidification-defective mutatants. PMID- 1712082 TI - [Peritoneovenous shunt in the treatment of ascites caused by a malignancy]. PMID- 1712083 TI - Regulation by calcium of short-term plasticity of the cholinoreceptors of RPa3 and LPa3 neurons of the edible snail. AB - The pharmacological influences which change the intracellular content of free Ca2+ reversibly change the rate and depth of the extinction of the inward current of RPa3 and LPa3 neurons of the edible snail, elicited by repeated iontophoretic applications of acetylcholine to the soma. Suppression by a calcium-free extracellular medium and by verapamil (100-150 mumoles/liter) of the influx of Ca2+ into the cell, induced by the activation of cholinoreceptors, reversibly attenuates the extinction. An increase in the level of intracellular Ca2+ by ruthenium red blockade (5-10 mumoles/liter) of the specific transport of Ca2+ by the mitochondria and by caffeine mobilization (1-4 mmoles/liter) of Ca2+ deposited by the endoplasmic reticulum increases and intensifies extinction. The results obtained indicate that the short-term plasticity of the cholinoreceptors of these neurons is positively regulated by Ca2+ which has entered the cell along chemoregulated ion channels, and has been mobilized from the intracellular Ca depots. PMID- 1712084 TI - Influence of quinolinic acid, an endogenous neurotoxin, on a passive avoidance conditioned reflex in rats. AB - Quinolinic acid, an endogenous neurotoxin, was introduced into the cerebral ventricles of a rat in doses of 0.5 microgram and 30 micrograms. The influence of the substance on the reproduction of a passive avoidance conditioned reflex (CR) was evaluated. It was found that a small dose of the preparation decreases extinction, while a large dose disrupts the reproduction of a passive avoidance CR. The disruption of the reproduction of the skill was also observed after bilateral electrolytic destruction of the dorsal hippocampus. Quinolinic degeneration of this structure exerted a substantial influence on the reproduction of a passive avoidance CR. PMID- 1712085 TI - Neuromorphological aspects of experimental herpes infection. PMID- 1712086 TI - Production of antisera to acidic fibroblast growth factor and their application to immunohistochemical study in rat brain. AB - Antisera against acidic fibroblast growth factor purified from bovine brain were produced in rabbits and used for immunohistochemical study of the rat brain. When examined in an immunospot assay using a nitrocellulose membrane, the best antibody was capable of detecting 80 fmol of acidic fibroblast growth factor but failed to react even with up to 5 pmol of basic fibroblast growth factor. Using this antiserum, the immunohistochemical distribution of acidic fibroblast growth factor was examined in rat brain. Acidic fibroblast growth factor-like immunoreactivity was localized mainly in a subpopulation of ependymal cells and tanycytes, as well as in some glial cells. Positive ependymal cells were observed throughout the walls of ventricles, including the third ventricle and cerebral aqueduct. Immunoreactive processes of tanycytes were found extending from the ventral wall of the third ventricle to the brain parenchyma and surface. The most intense immunostaining was observed in circumventricular organs such as the organum vasculosum laminalis terminalis and the subfornical organ. Particularly in the latter organ, there was an extremely dense plexus of immunoreactive fibers and processes around the wall of capillaries. The present results suggest that the effects of acidic fibroblast growth factor on brain functions may be exerted through the circumventricular organs and/or ependymal cells. PMID- 1712087 TI - Retrograde, trans-synaptic and transneuronal transport of fragment C of tetanus toxin by sympathetic preganglionic neurons. AB - The atoxic binding fragment of tetanus toxin, Fragment C, was injected into paravertebral ganglion 14, the avian homologue of the mammalian stellate ganglion. Postinjection survival intervals were varied from 2.5 h to 33 days. Experiments performed at the shortest survival time of 2.5 h showed that Fragment C was retrogradely transported by sympathetic preganglionic axons at a rate greater than or equal to 10 mm/h. At survival times ranging from 5 to 15 h. Fragment C-positive, retrogradely labeled sympathetic preganglionic neurons were observed within the last cervical spinal segment and throughout the first three thoracic spinal cord segments. Sporadic retrograde labeling of sympathetic preganglionic neurons was evident within the fourth and fifth thoracic spinal cord segments. Fragment C-labeled perikarya and dendrites exhibited both diffuse cytoplasmic immunostaining as well as intracellular, perinuclear accumulations of small. Fragment C-positive granules. Retrogradely labeled preganglionic neurons were found within both autonomic subnuclei within avian thoracic spinal cord; the column of Terni and the nucleus intercalatus spinalis. The distribution and numerical density of retrogradely labeled sympathetic preganglionic neurons indicated further that: (a) both myelinated and unmyelinated preganglionic axons appear to be capable of intra-axonally transporting Fragment C; and (b) it is unlikely that there is differential Fragment C labeling of a morphologically distinct population of sympathetic preganglionic neurons within or across subnuclei. Fragment C is transferred out of sympathetic preganglionic somas and dendrites into the surrounding neuropil at an aggregate rate greater than or equal to 5 mm/h. Trans-synaptic transport was evident at postinjection survival times as short as 5 h and continued to increase in density within the sympathetic preganglionic neuropil for 24 h. Fragment C-positive terminal labeling persisted for at least 20 days. At survival times greater than or equal to 1 day. Fragment C-positive puncta and weak intracellular labeling of neurons were evident in areas of the spinal gray outside of the nuclear boundaries of the column of Terni and nucleus intercalatus. The regions showing evidence of trans-synaptic and transneuronal labeling included: (a) a group of small cells dorsal to the column of Terni, (b) lamina V and (c) lamina VII. This expansion of Fragment C-labeled neuronal elements was segmental in organization and co-extensive with the retrograde labeling pattern of sympathetic preganglionic neurons. Spinal interneurons in these regions may provide segmental, monosynaptic input to sympathetic preganglionic neurons. Fragment C leaked into the systemic circulation from the site of injection in paravertebral ganglion 14.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712088 TI - Neurochemical and behavioural features induced by chronic low dose treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the common marmoset: implications for Parkinson's disease? AB - Protracted long-term treatment of common marmosets with 15 doses (0.5-4.5 mg/kg, i.p.) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; total dose 25 mg/kg, given over 29 days) caused transitory changes in motor behaviour reminiscent of human Parkinson's disease. 16 days from the start of MPTP administration, all animals showed motor impairment, consisting of profound akinesia and a rigid posture, but in no case resting tremor. Biogenic amines were measured in nigrostriatal regions one month after finishing MPTP treatment. There was a profound loss of dopamine and serotonin in the substantia nigra and in the striatum; noradrenaline was only reduced in the putamen. Continuous analyses of the concentrations of biogenic amine metabolites in the CSF during this study revealed persistent dopaminergic disturbances and temporary alterations in serotoninergic and noradrenergic function. PMID- 1712089 TI - Pallidocortical projections to the tempolar polar gyrus in the cat. AB - Marked projections from the globus pallidus (GP) to the temporal polar gyrus (TPG) of the cat were found by means of the PHA-L (Phaseolus vulgaris leucoagglutinin) and WGA-HRP (horseradish peroxidase conjugated to wheat germ agglutinin) methods. Pallidocortical fibers to the TPG originate mainly from the middle levels of the GP, and terminate all layers of the TPG cortex, especially in layers I, II and III. The GP neurons projecting to the TPG are large multipolar or small-bipolar neurons. Almost all of these GP neurons show choline acetyltransferase-like immunoreactivity. PMID- 1712090 TI - Regulation of MHC molecules on MBP positive oligodendrocytes in mice by IFN-gamma and TNF-alpha. AB - The expression of class I and class II histocompatibility antigens by myelin basic protein (MBP)-positive oligodendrocytes, in response to exogenous cytokines, has been investigated in vitro. It has been found that interferon gamma (IFN-gamma), although capable of class I induction, does not induce class II on oligodendrocytes. Furthermore, tumour necrosis factor-alpha (TNF-alpha), which was shown to induce class I MHC on other neural cells, failed to induce class I on oligodendrocytes. A combination of IFN-gamma and TNF-alpha also failed to facilitate the expression of class II antigens on oligodendrocytes, nor did it amplify the expression of class I seen with IFN-gamma alone. Thus it appears that MBP+ murine oligodendrocytes are refractory to class II induction, and express class I in response to IFN-gamma but not TNF-alpha. The differential regulation and class of MHC expression may have implications in terms of the initiation and targeting of immune responses directed toward the oligodendrocyte. PMID- 1712091 TI - Autoradiographic distribution of galanin binding sites in the brain and pituitary of the sea bass (Dicentrarchus labrax). AB - Specific binding sites for galanin (GAL) were detected in brain and pituitary of a marine teleost fish, the sea bass, after in vitro incubation of tissue sections with [125I]GAL and light microscopic autoradiography. Binding conditions were optimized and as a result the binding was saturable and specific. In the brain, [125I]GAL binding was found to occur in all parts of the dorsal and ventral telencephalon, in the anterior, tuberal and posterior hypothalamus, in the thalamus and in the tectum opticum, in the inferior lobe and in the ventral medulla oblongata. In the pituitary dense [125I]GAL binding was confined to the area occupied by the prolactin cells in the rostral part of the adenohypophysis. These findings provide the first anatomical evidence for the presence of GAL specific binding sites in the teleost brain and pituitary. PMID- 1712092 TI - [Metastatic dissemination of cancer cells]. AB - During the natural history of a tumor, cancer cells become more and more aggressive and their increasing malignancy leads usually to the patient's death. The expression of malignant properties by tumor cells is manifested by the occurrence of metastases and is the result of an overexpression of molecules that are normally or barely non expressed by the normal cell progenitors. These molecules can be involved in cell attachment (receptor to the extracellular matrix), in proteolysis (collagenases), in angiogenesis (b FGF), in adhesion to endothelial cells, in resistance to the immune system. The genetic instability of tumor cells favors the amplification, mutation and gene translocation events, resulting in the activation of some genes or/and oncogenes which might direct the expression of the malignant properties. Finally, metastatic cells have been shown to have a growth advantage over non metastatic cells, so that metastatic cell population becomes ultimately numerously dominant in the primary tumor. The current knowledge about the malignant cell properties allow us to begin to understand how a cancer cell becomes metastatic and how the metastatic dissemination is usually an ineluctable process. PMID- 1712093 TI - Langerhans cell depletion in gliotoxin-treated murine epidermis. AB - Langerhans cells (LC) are dendritic antigen presenting cells of bone marrow origin which reside in the suprabasal layer of the epidermis. They express high concentrations of Class II MHC glycoproteins on their plasma membrane and transport cutaneous antigen to local lymph nodes for presentation to helper T cells. They are thus essential for the induction of cutaneous immunity. Gliotoxin is a member of the epipolythiodioxopiperazine (ETP) group of fungal metabolites, derived from the human pathogen Aspergillus fumigatus. It has been shown to have immunomodulating properties in vivo and in vitro, and has been proposed as a potential immunosuppressant for transplantation therapy. Epicutaneous application of gliotoxin reduced the numbers of epidermal LC by 30-35 per cent with an associated morphological change from highly dendritic to a more rounded form. Electron microscopic studies showed selective damage to LC at very low (nM) concentrations of gliotoxin, with no obvious effect on adjacent keratinocytes. LC numbers remained depleted for 13 weeks after initial treatment, suggesting that systemic suppression or prolonged retention of gliotoxin within the skin may play a role in its mechanism of action. PMID- 1712094 TI - Iatrogenic developmental effects of pediatric intensive care. AB - Critically ill children in the pediatric intensive care environment are at particular risk for experiencing health care interventions that hinder them from progressing through their normal developmental milestones. Knowledge of the factors that influence iatrogenic developmental insults can help the nurse develop the skills and sensitivity to meet the complex needs of these children and their families. PMID- 1712095 TI - Peripheral polyneuropathies associated with monoclonal IgM. Antibody activity of monoclonal IgM and therapeutic implications. AB - Monoclonal IgM from patients with peripheral neuropathy react in nearly 80% of the cases with some components of myelin. The main target is myelin associated glycoprotein (50% of the cases); several types of glycolipids or gangliosides have been identified as the reactive antigen in the other cases. Anti-MAG IgM share little cross reactive idiotopes. Only primate antisera identified a combining site related public idiotope. In fact, these IgM use various light and heavy chains belonging to different variability subgroups. The preliminary results of an ungoing trial aimed to establish the benefit of plasmapheresis in these patients are discussed. PMID- 1712096 TI - Leader region of mdg1 Drosophila retrotransposon RNA contains 3'-end processing sites. AB - Transient expression of the mdg1 deletion mutants revealed sites of 3'-end processing in the leader region of the transcribed RNA. The efficiency of the processing is regulated in different types of cells. The sequences within the mdg1 body and the 3'-LTR are involved in its regulation. We have also shown, that one of the small open reading frames in the mdg1 leader region in principle might be translated. PMID- 1712097 TI - Polyinosinic acid as a carrier in the microscale purification of total RNA. AB - Three different RNA carriers were compared for use in microscale RNA isolation and subsequent cDNA synthesis and amplification via the polymerase chain reaction. E.coli rRNA alone gave considerable cDNA synthesis which under standard carrier conditions overwhelmed cDNA synthesis from lymphocyte mRNA. Yeast tRNA caused inhibition of mRNA primed cDNA synthesis, giving low levels of cDNA synthesis when used without cellular RNA. In contrast, commercially available poly I alone did not prime detectable cDNA synthesis nor did it inhibit such synthesis primed by cellular mRNA. When RNA preparations were made using these three carriers and decreasing numbers of starting lymphocytes, poly I allowed the detection of cDNA from two orders of magnitude fewer lymphocytes than the other carriers. Thus poly I was found to be a superior carrier molecule for microscale RNA preparations suitable for reverse transcription and subsequent amplification using the polymerase chain reaction. PMID- 1712099 TI - Pharmacology of spinal peptides affecting sensory and motor functions: dynorphins, somatostatins and tachykinins. AB - In recent years, the pharmacological activity of dynorphins and somatostatins on spinal sensory transmission has been intensively investigated with a view to developing new agents for pain control. Similarly, a series of tachykinin-related peptides with apparent receptor antagonist activity on endogenous substance P and neurokinins has been investigated. However, a number of observations suggest that these peptides, injected intrathecally in laboratory animals, not only exert a direct effect on nociceptive transmission but also affect a broader range of spinal somatomotor and autonomic functions and may cause peculiar neurotoxic effects that are not elicited by a large number of peptides affecting spinal neurotransmission. This article makes a critical review of their pharmacological activity on spinal sensory and motor functions and briefly touches on their anatomical and functional organization in the spinal cord. PMID- 1712098 TI - RNA editing of the transcript coding for subunit 4 of NADH dehydrogenase in wheat mitochondria: uneven distribution of the editing sites among the four exons. AB - The wheat mitochondrial (mt) NADH dehydrogenase subunit 4 gene (nad4) has been localized and sequenced. This gene, about 8 kb long, is composed of four exons separated by three class II introns. The nad4 gene exists as a single copy in the wheat mitochondrial genome and it is transcribed into one abundant mRNA of 1.8 kb, whose extremities have been mapped. The complete cDNA sequence corresponding to the nad4 transcript has been determined by combining the direct sequencing of uncloned cDNA and a method involving cDNA synthesis and PCR amplification using specific oligonucleotides as primers, followed by cloning and sequencing of the amplification product. Comparison of the genomic sequence with that of the cDNA shows that all nad4 transcripts are fully edited at 23 positions, with an uneven distribution of the editing sites between the different exons: While exon 1 and exon 4 are extensively edited (with a change of 11% of the amino acid sequence), exon 2 is not edited at all and exon 3 is 0.5% edited. This uneven distribution is discussed. PMID- 1712100 TI - Prenatal screening for chromosome abnormalities using maternal serum chorionic gonadotrophin, alpha-fetoprotein, and age. AB - Human chorionic gonadotrophin (hCG) levels were assayed retrospectively in stored maternal serum samples from 78 chromosomally abnormal pregnancies and 410 controls matched for gestation and maternal age. The median serum hCG concentration in 49 pregnancies with Down's syndrome was significantly elevated, at 2.18 multiples of the normal median. Significantly reduced hCG concentrations were found in a group of four trisomy 18 pregnancies (all less than 0.4 multiples of the median). Eight cases of unbalanced chromosome rearrangements appeared to show some lowering of hCG levels, while there was no significant difference in the levels in the cases of trisomy 13, balanced translocations, and sex chromosome abnormalities. Maternal serum hCG alone is a better indicator of Down's syndrome pregnancies than maternal age or maternal serum alpha-fetoprotein (AFP), either individually or in combination, and provides a further virtually independent measure of risk. On the basis of our findings, screening for Down's syndrome using hCG and AFP results combined with maternal age risks is predicted to result in a higher detection rate (57 per cent) for a lower false-positive rate (5.0 per cent) than would be attainable by combined AFP and age screening (37 per cent detection at a 6.6 per cent false-positive rate). PMID- 1712101 TI - Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1. AB - Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nucleotide binding protein but does not induce the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Western blot analysis reveals that PLC-gamma 1 is tyrosine-phosphorylated in response to TCR stimulation. Nearly all of the PLC activity recovered in eluates from anti phosphotyrosine immunoprecipitates was depleted by anti-PLC-gamma 1 antibodies. Stimulation of the TCR on mutants derived from Jurkat that are defective in TCR induced PLC activation results in markedly reduced, if any, PLC activity recovered in phosphotyrosine immunoprecipitates and in no detectable PLC-gamma 1 tyrosine phosphorylation. Thus, the TCR functions like PTK growth factor receptors, but through an indirect interaction, to induce tyrosine phosphorylation of PLC-gamma 1. Since other studies have implicated two members of the src family of PTKs in TCR-mediated signal transduction, our findings suggest that the induction of tyrosine phosphorylation of PLC-gamma 1 by a mechanism involving a src-like kinase may be the means by which the TCR regulates PLC activity in T cells. PMID- 1712102 TI - Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system. AB - We present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles. PMID- 1712103 TI - Phylogeny of viroids, viroidlike satellite RNAs, and the viroidlike domain of hepatitis delta virus RNA. AB - We report a phylogenetic study of viroids, some plant satellite RNAs, and the viroidlike domain of human hepatitis delta virus RNA. Our results support a monophyletic origin of these RNAs and are consistent with the hypothesis that they may be "living fossils" of a precellular RNA world. Moreover, the viroidlike domain of human hepatitis delta virus RNA appears closely related to the viroidlike satellite RNAs of plants, with which it shares some structural and functional properties. On the basis of our phylogenetic analysis, we propose a taxonomic classification of these RNAs. PMID- 1712104 TI - Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene. AB - Nerve growth factor (NGF) promotes the survival and differentiation of specific populations of neurons. The molecular mechanisms by which cells respond to NGF are poorly understood, but two clues have emerged recently. First, NGF rapidly stimulates tyrosine phosphorylation of several unidentified proteins in the NGF responsive pheochromocytoma cell line PC12 [Maher, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791]. Second, the protein-tyrosine kinase encoded by the protooncogene trk (p140trk), a member of the receptor class of tyrosine kinases, becomes activated and phosphorylated on tyrosine after NGF treatment of PC12 cells [Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature (London) 350, 158-160]. We now report that NGF rapidly induces tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), and we present evidence that the responsible tyrosine kinase is either p140trk or a closely associated protein. Treatment of responsive cells with NGF elicited phosphorylation of PLC-gamma 1 on tyrosine and serine. PLC-gamma 1 immunoprecipitated from NGF-stimulated cells was phosphorylated in vitro by coprecipitating protein kinase activity, and the phosphorylations occurred principally on tyrosine. The responsible kinase could be depleted from cellular lysates by antibodies specific for p140trk. This procedure also depleted a 140-kDa protein that normally coprecipitated with PLC gamma 1 and became phosphorylated on tyrosine in vivo in response to NGF. Analysis of tryptic peptides from PLC-gamma 1 indicated that the residues phosphorylated in vitro by p140trk-associated kinase activity were largely congruent with those phosphorylated in vivo after NGF treatment. Our findings identify PLC-gamma 1 as a likely substrate for the trk-encoded tyrosine kinase, and they provide a link between NGF-dependent activation of p140trk and the stimulation of intracellular second messenger pathways. PMID- 1712105 TI - Identification of type-specific linear epitopes in the glycoproteins gp46 and gp21 of human T-cell leukemia viruses type I and type II using synthetic peptides. AB - Synthetic peptides of 20-25 amino acids were employed in enzyme-linked immunosorbent assays to identify linear epitopes in the external glycoprotein gp46 and the transmembrane glycoprotein gp21 of human T-cell leukemia/lymphotropic viruses type I (HTLV-I) and II (HTLV-II). Ten linear epitopes were identified in the HTLV-I glycoproteins, seven in gp46 and three in gp21. Three major linear epitopes were identified in the gp46 of HTLV-II. Peptides representing linear epitopes of gp46 were found to be sensitive and specific for the detection of antibody and permit serological identification and differentiation of HTLV-I and HTLV-II infections. PMID- 1712106 TI - Vascular endothelial cell growth factor (VEGF) produced by A-431 human epidermoid carcinoma cells and identification of VEGF membrane binding sites. AB - A distinct family of endothelial cell mitogens that are homologous to platelet derived growth factor has recently been identified. Unlike other known endothelial cell mitogens, these vascular endothelial cell growth factors (VEGFs) are secreted and appear to act specifically on endothelial cells. We have purified VEGF 2083-fold to apparent homogeneity from protein-free culture medium conditioned by A-431 human epidermoid carcinoma cells. This A-431-derived VEGF was characterized as a homodimer composed of 22-kDa subunits with an N-terminal sequence that was similar to VEGFs produced by human HL-60 leukemic and U-937 histiocytic lymphoma cells. A-431 VEGF was used to identify specific and saturable binding sites for VEGF on human umbilical vein endothelial cells (HUVEC). By affinity cross-linking, VEGF-binding site complexes of 230, 170, and 125 kDa were detected on HUVEC. VEGF specifically induced the tyrosine phosphorylation of a 190-kDa polypeptide, which was similar in mass to the largest binding site detected by affinity cross-linking. PMID- 1712107 TI - Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51. AB - The microtubule-associated protein tau that is incorporated into paired helical filaments (PHFs) undergoes some form of aberrant posttranslational processing in Alzheimer disease. Difficulties in deciding which changes are critical for PHF formation stem in part from the lack of immunochemical markers specific for PHF tau. The only monoclonal antibody (mAb) that is known to react with PHF tau but not with the predominant normal adult tau species is mAb 423. Another mAb (7.51, described in this paper) recognizes a segment of tau that is included in the minimal recognition unit required by mAb 423. Unlike 423, which is PHF tau specific, mAb 7.51 recognizes all PHF core-derived tau as well as native soluble tau and recombinant tau expressed in bacteria and so serves as a generic tau marker. Both epitopes are in the 12-kDa fragment released from the Pronase resistant core of the PHF (which encompasses the tandem repeat region). The mAb 7.51 epitope requires segments located in the last two repeats, which are common to all tau isoforms. The mAb 423 epitope requires sequences located near both the N and the C terminus of the 12-kDa fragment common to three- and four-repeat tau isoforms. Fragments denatured by concentrated formic acid and SDS regain 423 reactivity when denaturing agents are removed. Since the primary amino acid sequences of PHF tau and normal tau are identical in the repeat region, we conclude that 423 reactivity also requires a modification(s) occurring within an approximately 90-residue segment that are not present in tau proteins so far described in the human brain. PMID- 1712108 TI - Absence of the 40-kDa form of (2'-5')oligoadenylate synthetase and its corresponding mRNA from skin fibroblasts derived from Alzheimer disease patients. AB - Cultured skin fibroblasts derived from Alzheimer disease patients fail to express the 1.6-kilobase (kb) mRNA and the corresponding 40-kDa form of (2' 5')oligoadenylate (2-5A) synthetase when exposed to interferon. In addition, the 3.6-kb mRNA, which is present in normal fibroblasts, is barely detectable in the Alzheimer disease counterpart. The deficiency of the 2-5A synthetase 1.6-kb mRNA and its corresponding protein is not related to an impairment of interferon receptors but most probably represents an alteration in the expression of the 2 5A synthetase gene. The data have potential implications for the diagnosis of Alzheimer disease and demonstrate that the absence of a specific form of 2-5A synthetase is linked to a disease state. PMID- 1712109 TI - Tyrosine kinase activity coupled to the high-affinity nerve growth factor receptor complex. AB - Antibodies directed against nerve growth factor (NGF) immunoprecipitate a tyrosine kinase activity from NGF-treated PC12 rat pheochromocytoma cells. Based on several criteria, this activity has been correlated with the high-affinity and not the low-affinity NGF-receptor complex. The in vitro kinase activity and the tyrosine phosphorylation of the high-affinity complex can be blocked by an agent that inhibits NGF (and not epidermal growth factor)-induced tyrosine phosphorylation in PC12 cells, as well as NGF-induced neuronal differentiation of PC12 cells. These observations suggest that the high-affinity NGF-receptor complex is a substrate of tyrosine kinase activity. Phosphorylation reactions by the complex, performed in the absence of added substrate, label a single phosphopeptide of 130-135 kDa. This observation suggests that this phosphopeptide may represent the phosphorylation of the receptor kinase or the phosphorylation of a coimmunoprecipitating substrate, and possible signal-transducing molecule, of the high-affinity NGF-receptor complex. PMID- 1712110 TI - A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes. AB - We have identified a major T-cell epitope, amino acids 48-54 (DRAHYNI, in one letter code) in the E7 open reading frame protein of human papillomavirus (HPV) type 16. Lymph node cells from mice immunized with synthetic peptides containing DRAHYNI proliferated and produced interleukin when challenged in vitro with peptide or whole HPV-16 E7 fusion protein. The T epitope was recognized in association with all five major histocompatibility complex class II I-A and I-E alleles tested. Synthetic peptides consisting of DRAHYNI linked to major B-cell epitopes on the E7 molecule formed immunogens capable of eliciting strong antibody responses to HPV-16 E7. The T epitope could provide help for the production of antibody to several B epitopes simultaneously, including a B epitope of HPV-18 E7 protein. Mice immunized with a peptide containing DRAHYNI and B epitope and, at a later date, infected with recombinant vaccinia E7 virus, displayed secondary antibody responses to E7. Because E7 has a role in cell transformation and is the most abundant viral protein in HPV-associated neoplastic cervical epithelial cells, the data have implications for vaccine strategies. PMID- 1712112 TI - Autoradiographic localization of the GABA-A-receptor agonist (3H)-muscimol in the rat intestinal musculature. AB - Radioreceptor-binding assay and autoradiography were used to study the pharmacological profile and the anatomical localization of GABA-A-receptor sites in sections of rat duodenum, jejunum and ileum. (3H)-Muscimol, used as a ligand, was bound by sections of the intestinal portions investigated in a manner consistent with the labeling of GABA-A-receptor sites. The dissociation constant (Kd) was about 12.5 nmol/l in the three different intestinal portions. The maximum density of binding sites (Bmax) was highest in the duodenum (118.9 +/- 7.4 fmol/mg tissue followed, in descending order, by the jejunum (105.8 +/- 6.3 fmol/mg tissue) and the ileum (67.8 +/- 5.9 fmol/mg tissue). Light microscope autoradiography revealed a dense accumulation of specific silver grains within intestinal smooth muscle. In the duodenum (3H)-muscimol-binding sites were rather homogeneously distributed both in circular and longitudinal smooth muscle. In the jejunum the density of silver grains was similar to that seen in the duodenum in the circular musculature and lower in the longitudinal musculature. The ileum displayed the lowest accumulation of (3H)-muscimol-binding sites, with no significant differences in the density of silver grains between the two muscular layers. The possible significance of the GABA-A-receptor sites observed in the intestinal musculature is discussed. PMID- 1712111 TI - Evidence for regulation of the human ABL tyrosine kinase by a cellular inhibitor. AB - Phosphotyrosine cannot be detected on normal human ABL protein-tyrosine kinases, but activated oncogenic forms of the human ABL protein are phosphorylated on tyrosine in vivo. Activation of ABL can occur by substitution of the ABL first exon with breakpoint cluster region (BCR) sequences or by deletion of the noncatalytic SH3 (src homology region 3) domain. An alternative mode for the activation of the ABL kinases is hyperexpression at greater than 500-fold over endogenous levels. This is not a consequence of transphosphorylation of the hyperexpressed ABL molecules. ABL proteins translated in vitro lack phosphotyrosine, but tyrosine kinase activity is uncovered after immunoprecipitation and removal of lysate components. The rates of dephosphorylation of ABL and BCR-ABL fusion protein by phosphotyrosine-specific phosphatases are approximately the same. These combined results indicate that inhibition of ABL activity is reversible and suggest that a cellular component interacts noncovalently with ABL to inhibit its autophosphorylation. PMID- 1712113 TI - Decrease in repetitive extrasystole threshold during epinephrine infusion is enhanced in conscious dogs with perinephritic hypertension. AB - Hypertension is associated with myocardial hypertrophy as well as increased adrenergic responsiveness, both of which can predispose to malignant ventricular arrhythmias. This study was designed to test the effects of subpressor doses of epinephrine (0.15 and 0.3 micrograms/kg/min x 30 min) on vulnerability to ventricular arrhythmia in normotensive and perinephritic hypertensive dogs. Two groups of 6 dogs each were chronically instrumented with aortic catheters to measure mean arterial pressure and bipolar pacing catheters in the apex of the right ventricle to measure repetitive extrasystole threshold, an index of vulnerability to ventricular fibrillation. In the normotensive dogs, the low dose of epinephrine (0.15 micrograms/kg/min IV) had no significant effects on mean arterial pressure, heart rate of repetitive extrasystole threshold. However, in the hypertensive dogs, the same dose caused a significant 39% increase in heart rate (p less than 0.05) and 41% decrease in repetitive extrasystole threshold (p less than 0.05). These findings suggest that electrophysiological vulnerability of the myocardium caused by epinephrine infusion is enhanced in the hypertensive animal. PMID- 1712114 TI - Cerebrospinal fluid levels of monoamine metabolites in panic disorder. AB - The cerebrospinal fluid (CSF) levels of the serotonin metabolite 5 hydroxyindoleacetic acid (5HIAA), the noradrenaline metabolite 3-methoxy-4 hydroxyphenylglycol (MHPG), and the dopamine metabolite homovanillic acid (HVA) did not differ significantly in a group of patients with panic disorder (n = 17) as compared to age- and sex-matched normal controls (n = 17). While CSF concentrations of HVA and 5HIAA were significantly correlated in both patients and controls, CSF MHPG levels were significantly correlated with the concentrations of CSF 5HIAA and HVA only in patients. In a small number of subjects (n = 5), successful reduction of anxiety attacks by administration of clomipramine or imipramine (50-150 mg/day) for at least 2 months was associated with a significant decrease in CSF concentrations of 5HIAA and MHPG, but not HVA. PMID- 1712115 TI - CSF monoamine metabolite concentrations vary according to age, rearing, and sex, and are influenced by the stressor of social separation in rhesus monkeys. AB - In humans, CSF monoamine metabolite concentrations have been shown to vary as a complex function of age, sex, psychiatric diagnosis, and stress. To test for such relationships in rhesus monkeys, 28 subjects, reared either in anxiety producing peer-only groups or in mother-infant dyads, were studied at 6, 18 or 50 months of age. Each monkey underwent a series of four 4-day social separations, each followed by 3 days of reunion. Prior to and during the first and fourth separations, CSF was obtained from the cisterna magna and assayed for the serotonin metabolite 5-HIAA, the dopamine metabolite HVA, and the norepinephrine metabolite MHPG. CSF 5-HIAA showed an age-related decline which was greater in the mother-reared subjects. Peer-only-reared males had an increased 5-HIAA concentration relative to females, and higher 5-HIAA levels than mother-reared males. MHPG was also higher in peer-only-reared monkeys than in mother-reared subjects at all ages. In both groups HVA declined across the three ages, and MHPG increased from the 18- to the 50-month measurements. Both MHPG and 5-HIAA concentrations increased during the initial social separation, although only MHPG remained elevated across the repeated separations; HVA, on the other hand declined during social separation. These results are discussed in terms of established anxiety and aggression differences between peer-only and mother reared monkeys. PMID- 1712116 TI - [The role of c-kit on intra-marrow hemopoiesis]. PMID- 1712117 TI - [Structural variants of laminin and cell function]. PMID- 1712118 TI - Ca2+ activates glycogenolysis in isolated mantle storage cells of Mytilus galloprovincialis Lmk. AB - Glycogenolytic activity (GA) in isolated mantle storage cells (MSC) from Mytilus galloprovincialis was studied, while glycogen and free-glucose content, as well as glucose released from cells were tested. In the period studied (November December), the glucose releasing activity measured can be considered as an output of GA. In both, whole cells system (WCS) and crude cell-free system (CFS), a non stimulated GA was detected. In WCS, dopamine and 5-hydroxytryptamine (5-HT) stimulated glycogenolysis, while epinephrine, norepinephrine and isoproterenol did not show any effect. Furthermore, mellitin and the Ca(2+)-ionophore, A23187, had a stimulating effect on the GA. In CFS, the absence of Ca2+ ions was a sufficient condition to depress GA. These and other findings suggest that: 1) GA in MSC may be stimulated by dopamine and 5-HT and not by adrenergic agonists; 2) cytosolyc Ca2+ signalling may have become an absolute requirement for activation of the glycogenolytic cascade in MSC; 3) a rapid high-affinity glucose transport may occur in these cells. PMID- 1712119 TI - [Low-grade polymorphic adenocarcinoma of papillary type. Morphological and immunohistochemical study]. AB - Two cases of polymorphous low-grade adenocarcinoma of the papillary type, from minor salivary glands were studied by light microscopy and immunohistochemistry. One case exhibited a predominance of the papillary pattern, whereas the other presented the following patterns of histological appearance: papillary, solid, pseudocystic and tubular. Utilizing the peroxidase-antiperoxidase (PAP) method, the intermediate filament vimentin, keratin and S100 protein were observed in tumor cells. The immunohistochemical analysis revealed two types of neoplastic cells: myoepithelial and luminal. PMID- 1712120 TI - [Haemobartonella detection in cat blood smears]. AB - Two useful staining methods for the diagnosis of haemobartonellosis on blood smears and distribution of Haemobartonellae on erythrocyte surfaces are shown. PMID- 1712121 TI - A new role for gases: neurotransmission. PMID- 1712122 TI - Triplex RNA. PMID- 1712123 TI - The myeloid colony-stimulating factors: introduction and overview. PMID- 1712124 TI - The role of granulocyte colony-stimulating factor in cancer chemotherapy. PMID- 1712125 TI - The impact of myeloid growth factors on engraftment following autologous bone marrow transplantation for malignant lymphoma. PMID- 1712126 TI - Psychoanalytic perspectives on substance abuse: implications for treatment, program planning and social policy. AB - Current clinical and socio-political interventions with substance abusers are based on two suppositions regarding the addict: a cognitive ability to discern harm to oneself, and a corresponding capacity to discipline oneself in recognition of the punitive consequences of behavior. A psychoanalytic understanding of the addict's intrapsychic needs provides clarification of these assumptions: offering a psychodynamic explication of why the addict can not "just say no." A comparative analysis of early psychoanalytic thought and contemporary ego psychology elucidates the relationship between hysteria and the addictions, demonstrated in clinical studies with addicted patients. The clinician's use of self is delineated as a treatment tool, mirroring the need for self-exploration on societal and political levels, with implications for treatment, program planning and social policy. PMID- 1712127 TI - [Effect of active and passive smoking on the course of pregnancy in women and on the establishment of the erythrocytic system in their children]. AB - As a result of a clinicohematological examination of the tobacco-smokers' families, it has been revealed that passive and active tobacco-smoking exert an extremely unfavorable effect on the course and outcome of pregnancy as well as on lactation in women. The children coming from the tobacco-smokers' families manifested disorders of iron metabolism, processes of hemoglobin formation, red blood cell metabolism, leading to the development in them of anemia during the first year of life. The slow rate of the replacement of fetal hemoglobin by hemoglobin A, carboxyhemoglobinemia, strenuous metabolism of SH-groups, 2,3-DPG in red blood cells are pathognomic for erythron impairment in children exposed to tobacco smoke. PMID- 1712128 TI - The CD1 system. PMID- 1712129 TI - Initial pathophysiological changes in chronic pancreatitis induced by pancreatic ductular obstruction model. AB - An experimental chronic pancreatitis model was made in five dogs with chronic pancreatic fistula by injection of microspheres into the peripheral pancreatic duct. Sequential changes of pancreatic exocrine and endocrine functions with morphology were studied. Significant decreases in volume bicarbonate output and amylase output were detected in each sample collected separately on secretin and secretin cerulean stimulation. While the viscosity of pancreatic juice was significantly increased with a concomitant increase in hexosamine concentration, chronic pancreatitis was demonstrated morphologically. These results suggest that concentrated pancreatic juice caused by a decrease in volume and an increase in viscosity of pancreatic juice with a concomitant increase in hexosamine concentration brings about the progression of chronic pancreatitis in this experimental model. PMID- 1712130 TI - Distribution of anti-keratins and anti-thymostimulin antibodies in normal and in Down's syndrome human thymuses. AB - The localization of three monoclonal (A,B,C) anti-cytokeratin antibodies and of an anti-thymostimulin antibody were studied in normal children's thymuses, aged from 2 months to 10 1/2 years and in Down's children thymuses, aged from 5 months to 6 1/2 years. Two anti-cytokeratins were positive in the thymus: the anti-B was found in the epithelial cells of all thymic zones, the anti-C only in the external cells of Hassall's corpuscles. The distribution and the intensity of immuno-reactions were the same in normal and in Down's thymuses. The distribution of anti-thymostimulin was superimposed to the distribution of anti-cytokeratin B and was similar in normal and in the youngest Down's thymuses, whereas in the 6 1/2 years-old Down's thymuses there was a loss of anti-TS reaction in the subcapsular zone. A relationship between the reduction of anti-thymostimulin immuno-reaction and the beginning of an eventual loss of T-lymphocyte differentiation was supposed. PMID- 1712131 TI - Purification and partial characterization of an avian thymic hormone. Avian thymic hormone. AB - An avian thymic hormone, originally designated the T1-antigen, was purified from chicken thymus by Sephadex G-75-40 chromatography and affinity chromatography, following enrichment by heat and pH treatments. It was characterized as an acidic polypeptide rich in phenylalanine, alanine and serine, lacking in histidine, tryptophan, methionine and cysteine, and having a blocked N-terminal amino acid. The hormone also was rich in hydrophobic amino acid residues, which gave it a propensity to form aggregates. Its molecular weight was estimated by gel electrophoresis and low speed sedimentation equilibrium to be 12-13 Kd, and by molecular sieving chromatography to be 15-16 Kd. The hormone was lacking in carbohydrates and amino sugars. PMID- 1712132 TI - Inhibition of human T-cell activation by FK 506, rapamycin, and cyclosporine A. PMID- 1712133 TI - Current status of FK 506 in liver transplantation. PMID- 1712134 TI - Intraoperative use of aprotinin (Trasylol) in orthotopic liver transplantation. PMID- 1712135 TI - Aprotinin inhibits tissue plasminogen activator-mediated fibrinolysis during orthotopic liver transplantation. PMID- 1712136 TI - In vitro study of the effects of aprotinin on coagulation during orthotopic liver transplantation. PMID- 1712137 TI - Fibrinolytic changes and the influence of the early perfusate in orthotopic liver transplantation with intraoperative aprotinin treatment. PMID- 1712138 TI - Nitric oxide as a neuronal messenger. PMID- 1712139 TI - Inhibition of the induction of nitric oxide synthase by glucocorticoids: yet another explanation for their anti-inflammatory effects? PMID- 1712140 TI - [A hemostatic method in extravesicular retropubic adenomectomy]. AB - To improve hemostasis in extrabladder retropubic adenomectomy, use was made of catgut nonremovable sutures and displacement by pi-shape sutures of the back semicircular mucosa of the neck of the urinary bladder into the urethral lumen fixing it to the prostatic adenoma bed walls. Surgical results were compared for 317 patients after extrabladder retropubic adenomectomy against 501 patients following transbladder adenomectomy. In the former group early complications were registered in 16.4%, in the former in 22.8% of the cases. The above approach to hemostasis management permitted hemorrhage incidence reduction to 2.1%; postoperative lethality to 2.5% and hospital stay to 16.5 patient days. PMID- 1712141 TI - Cutaneous plasmacytomas with amyloid in six dogs. AB - Cutaneous plasmacytomas associated with local deposition of amyloid were diagnosed by light microscopy in a series of six older dogs (mean age 10.7 years) consisting of two Cocker Spaniels, a Poodle, a Weimeraner, and two mixed-breed dogs. The neoplasms occurred on the digits (2 dogs), forelimb (2 dogs), lip (1 dog), and ear (1 dog). In most cases, groups of neoplastic plasma cells were widely separated by large homogeneous islands of amyloid. The neoplastic cells had characteristic plasmacytoid features, but the degree of pleomorphism varied greatly between different neoplasms. In four of the six tumors, the diagnosis of plasmacytoma was confirmed by the demonstration of a monoclonal plasma cell population using immunofluorescent staining for anti-canine immunoglobulins. In these tumors, the neoplastic cells reacted with only one class of immunoglobulins (IgG). The amyloid did not react with any of the reagents used. The suspicion that the amyloid was of immunoglobulin origin (primary amyloid) was supported by its retention of birefringence under polarized light after treatment with potassium permanganate and staining with Congo red. PMID- 1712142 TI - Cowdria ruminantium is recognized by a monoclonal antibody directed against the major outer membrane protein of Chlamydia trachomatis. AB - The relationship between Cowdria ruminantium and Chlamydia trachomatis was studied by immunofluorescence. A monoclonal antibody directed against the major outer membrane protein of C. trachomatis recognized rickettsial colonies of C. ruminantium in infected goat brain. No specific fluorescence was observed in non infected brain. Two commercial Chlamydia-specific monoclonal antibodies as well as polyvalent anti-Chlamydia rabbit serum recognized C. trachomatis, but did not recognize Cowdria. Moreover, polyvalent Cowdria antiserum failed to recognize C. trachomatis cultivated in HeLa cells. It is concluded that Cowdria and Chlamydia are to a certain extent related, confirming similarities in ultrastructure and developmental cycle. PMID- 1712143 TI - Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with Pasteurella haemolytica. AB - The lymphocyte subpopulations in peripheral blood obtained from eleven lambs experimentally infected with Pasteurella haemolytica were compared with those obtained from eight control lambs by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with P. haemolytica was characterized by a transient but significant reduction in SBU-T1+ (CD5+) T cells and SBU-T4+ (CD4+ or helper) T lymphocytes (P less than 0.05) and a significant rise in lymphocytes which did not express the LCA p220 epitope and the pan T cell surface marker (CD5-LCA p220-) ("null"). The reductions in CD5+ and CD4+ lymphocytes occurred 24 h after experimental infection, returning to preinoculation levels 5 days post inoculation (DPI). Five to 9 days after experimental infection, there was a significant increase in the number of lymphocytes, which expresses the pan T cell surface marker (CD5+) but which were CD4-CD8-. Lymphocyte transformation responses to the mitogen phytohaemagglutinin (PHA) were significantly reduced 24 h after experimental infection with P. haemolytica (P less than 0.05). PMID- 1712144 TI - [Possibilities of therapy in chronic pain conditions]. PMID- 1712145 TI - [Treatment and rehabilitation of disabilities caused by leprosy at a rural hospital (Kapolowe, Zaire)]. AB - Since 1985, we contribute to treat and rehabilitate the leprosy infirmities at Kapolowe District Hospital (Shaba, Zaire). The setting up of a permanent autochtonous Department (Tschibangu), has taken the place of the temporary expeditions. A first follow-up refers to 138 patients, on which 259 interventions have been done: 165 refer to foot ulcerations and theirs septic osteoarticular complications, 75 relate to neuritis, 8 only have been restorative interventions. 75% of our patients have had a good social reinsertion. The 25% relapses show the importance of a good limb prosthesis, of sanitary education of the patients and their supervision when they go back to brushwood. In the initial period, it is necessary to start by performing in cleanliness surgery, otherwise bright restorative interventions would be inefficient. PMID- 1712146 TI - [Effect of several factors on enzymic activity and antigenicity in chemical modification of L-asparaginase]. AB - Acetic anhydride, dextran and monomethoxypolyethylene glycol and different modification methods were used for modification of L-asparaginase to maintain enzyme activity and completely remove its antigenicity. The results showed that the macromolecular modifiers PEG and dextran were better than the small molecular modifier acetic anhydride. For maintenance of enzyme activity and removal of antigenicity modification in the presence of substrate was better than absence of substrate and activated PEG2 was better than activated PEG1. When PEG2-L asparaginase was modified in the presence of substrate, its antigenicity was completely removed, while more than 30% of native enzyme activity were still retained. PMID- 1712147 TI - Physicochemical and immunological properties of the hepatitis B surface antigen containing the preS2 9 amino acid sequence produced by a recombinant yeast. AB - The hepatitis B virus surface antigen containing the preS2 nine amino acid sequence produced by a recombinant Saccharomyces cerevisiae (yHBsAg) was purified and its physicochemical properties were determined. Ultrastructurally, the yHBsAg was found to be a homogeneous spherical particle with a diameter of 24 +/- 4 nm. The homogeneity of the yHBsAg particles was also demonstrated by analyses of their buoyant density and isoelectric point. They consisted of protein (53%), lipid (36%) and carbohydrate (11%), and the alpha-helix content was estimated to be 32%, differing from the reported values for human plasma-derived HBsAg (hHBsAg). Immunodiffusion analysis showed that the antigenic specificity of yHBsAg was identical to that of hHBsAg. Immunization of mice demonstrated that the immunogenicity of the yHBsAg was significantly higher than that of hHBsAg. PMID- 1712148 TI - Ascaridia galli: effect of some anthelmintics on amino acid uptake and macromolecular synthesis in vitro. AB - L-(U-14C) aspartic acid, L-(U-14C) alanine and L-(U-14C) leucine uptake by Ascaridia galli was found to be a non-linear function of time and limiting substrate concentration. The uptake was rapid initially but achieved steady state thereafter, possibly owing to the saturation of transport loci. Linear transformations of substrate saturation kinetics by Lineweaver-Burk plots of L-(U 14C) aspartic acid, L-(U-14C) alanine and L-(U-14C) leucine gave Kt values of 4.76, 3.03 and 2.0 mM and Jmax of 5.0, 3.57 and 2.08 m moles/100 mg dry weight/2 min, respectively. D1-tetramisole and 1-tetramisole (levamisole) inhibited the uptake of amino acids. The uptaken amino acids were readily metabolized into different tissue fractions. D1-tetramisole and levamisole significantly inhibited the incorporation of the three amino acids into the nematode's total protein, RNA and lipid fractions in an in vitro incubation system. PMID- 1712149 TI - Immunization with nitrocellulose strips from western blots. PMID- 1712150 TI - Dermal ridge development on the volar pads of the rat (Rattus norvegicus) and comparative study of pattern formation using inbred strains. AB - The development of dermal ridges, ridge configurations, and volar pad contours was investigated in the volar skin of the rat (Rattus norvegicus). The ridged structures corresponding to the epidermal ridges of primates exist only at the epidermal-dermal junction in the rat. Dermal specimens were prepared by treatment with alkaline solution and examined by toluidine blue staining and scanning electron microscopy, together with histological sections. Differentiation of dermal ridges began on day 18 of gestation on the palm followed by the sole. Ridges increased in number with advancing age. The process was complete approximately 2-3 days after birth, and sweat ducts began to develop simultaneously. As dermal ridges present various configurational patterns on palmar interdigital pad III, pattern formation on this pad was inspected in fetuses of three inbred strains possessing different pattern types, and in the hybrid progeny derived from them. Patterns and pad forms appeared to be under genetic control. It was revealed that the ridge arrangements, i.e., whorls, triradii, comb-like patterns, and others, are closely related to the pad contours during the developmental period, as hypothesized in primates. PMID- 1712151 TI - Enalapril can treat the proteinuria of membranous glomerulonephritis without detriment to systemic or renal hemodynamics. AB - The effect of enalapril on renal hemodynamics and glomerular permselectivity was studied in eight patients with nephrotic syndrome secondary to biopsy-proven membranous glomerulonephritis. The patients received the drug in incremental doses (median, 5 mg) until 24-hour urinary protein excretion had decreased persistently by 30%. Median treatment duration was 6 weeks. Patients were studied three times: (I) after a 4-week run-in period, (II) on the final day of treatment, and (III) after a 4-week wash-out. Median 24-hour urinary protein excretion decreased on treatment from 10.45 g/d to 5.25 g/d and increased to pretreatment levels after the drug was stopped (P less than 0.05 for both changes). Fractional clearance of dextrans greater than 4.1 nm decreased on treatment, indicating both a reduction of macromolecules passing through the shunt pathway of the glomerular basement membrane (GBM) and a possible decrease in ultrafiltration coefficient. There were no significant changes in glomerular filtration rate (GFR), effective renal plasma flow (ERPF), or mean arterial blood pressure (MAP) throughout the study. The effect of enalapril in treating proteinuria appears therefore to be due to a specific intraglomerular action. PMID- 1712152 TI - Case report: dextran-induced acute anuric renal failure. AB - Acute renal failure is an infrequent adverse reaction following the administration of dextran-40. We report a case of anuric acute renal failure in a 59-year-old female following the administration of 90 gm of dextran-40 and radiocontrast. An increased risk secondary to radiocontrast-induced ischemia is discussed in relationship to the pathogenesis of the dextran-induced acute renal failure. In addition, plasmapheresis is demonstrated to be of potential therapeutic benefit. PMID- 1712153 TI - Septic shock. AB - Septic shock (SS) is the most common type of shock encountered by internists, and its prevalence appears to be increasing. SS complicates all types of infections. The hemodynamic characteristics of SS include a low systemic vascular resistance and an elevated, but relatively inadequate, cardiac output. A cardiomyopathy frequently occurs. The major endogenous mediator of SS is tumor necrosis factor, and interleukins-1 and -2 may also contribute. Important secondary phenomena include release of platelet activating factor, vasodilator prostaglandins, and upregulation of adhesion molecules on polymorphonuclear leukocytes and endothelial cells. Current therapy is often ineffectual, and potentially promising new therapeutic approaches are reviewed. PMID- 1712154 TI - Hydroxyethyl starch macromolecule and superoxide dismutase effects on myocardial reperfusion injury. AB - Myocardial reperfusion injury may be due to biophysical changes (e.g., endothelial cell junctional separations), as well as biochemical mechanisms (e.g., oxygen free radical activity). Superoxide dismutase (SOD), a free radical scavenger, may be effective in reducing chemical injury. Fractions of hydroxyethyl starch (HES-Pz), a large macromolecule, have been shown to decrease microvascular permeability associated with reperfusion-induced biophysical alterations. A comparison of SOD to HES-Pz was performed using a canine model of 1-hour left anterior descending coronary artery (LAD) clamping followed by 24 hours of reperfusion. Amounts of the test solution equal to 10% of the dog's blood volume were administered intraatrially to the animals just before release of the LAD clamp. Six dogs received Ringer's lactate, 7 were given 600,000 IU of SOD, 13 received 6% HES-Pz, and 9 were given SOD and HES-Pz. The ratio of infarct to area at risk was 20 +/- 3% in the control dogs receiving Ringer's lactate, 16 +/- 4% in animals receiving SOD alone (p = NS), 6 +/- 3% in dogs receiving HES-Pz alone (p less than 0.05), and 8 +/- 3% in dogs given a combination of SOD and HES Pz (p less than 0.05). HES-Pz alone and with SOD significantly reduced reperfusion injury, although addition of SOD to HES-Pz did not have an additive effect. Appropriate-sized macromolecules may act by reducing ischemia-induced microvascular permeability. PMID- 1712155 TI - The effects of dietary protein deficiency on rat testicular function. AB - Diets containing 0%, 5% and 10% protein were used for treatment periods of 30, 50, and 90 days respectively. Control rats were fed a diet containing 20% protein. Protein deficient rats failed to gain weight during the experiment. In addition, the weights of the testis, epididymis, prostate and seminal vesicle also decreased, with the 10% group less affected than the 0% and 5% groups. Testicular histology indicated retarded germ cell maturation in the 0% and 5% groups only. Overall testicular cell number and size were reduced in treated rats and there was a reduction in the diameter of the seminiferous tubule in these groups. Epididymal epithelial height was also reduced in protein deficient rats with a concomitant increase in the number of epididymal duct cross sections devoid of sperm. Protein deficiency caused significant reductions in testicular DNA, RNA and protein content. The proportion of motile epididymal sperm decreased in the 0% and 5% groups by 90% and 35% respectively. Epididymal sperm number decreased in both the 0% and 5% groups by 90% while the proportion of abnormal sperm increased by 65% and 61% respectively. Circulating androgen levels were also lowered by more than 50% on average in protein deficient animals. PMID- 1712156 TI - The insulin-like growth factor binding proteins--the endometrium and decidua. PMID- 1712157 TI - On the activity of MR 889, a new synthetic proteinase inhibitor. PMID- 1712158 TI - Ten-year changes of protease inhibitors in the sons of patients with COPD. PMID- 1712160 TI - [Surgery of mammary hypertrophy and ptosis using the dermal vault technique. 5 years' use (apropos of 230 surgically-treated patients]. AB - Using the simplest possible criteria, the author reviews the results obtained in 230 patients who underwent breast reduction using the dermal vault technique. A part from the usual criticisms of the technique defined by J.P. Lalardrie, the analysis reveals: 44% of "good" or "very good", 42% of "acceptable" results and 14% of "unacceptable results". The increase of "good" cases and decrease of "unacceptable" cases with time and the number of cases confirm the author in his opinion that it is worthwhile to continue with operative procedure. PMID- 1712159 TI - [Chronic lupus erythematosus and epidermoid carcinoma]. PMID- 1712161 TI - [Reduction mammaplasty using the postero-inferior glandular pedicle without de epithelialization]. AB - The author describes the technique which he has used for the last two years and in 140 cases of breast reduction. This technique uses an essentially posterior glandular pedicle, the blood supply of gland is essentially derived from the thoracic wall and there is no de-epithelialisation for vascular purposes. This simple technique provides patients with the certainty of normal lactation and almost certain restoration of nipple sensitivity. Moreover, as the skin is not placed under tension, the scars appear to be of better quality than when they are placed under tension as with many other techniques. PMID- 1712162 TI - [Mammaplasty or the war of scars?]. AB - The author of the total dermoglandular pedicle mammaplasty discusses the controversy concerning the length of the incisions to be used in mammaplasties. He geometrically compares the scars of pre-established drawings with those obtained by trimming incisions at the end of the operation and draws interesting conclusions about the two approaches. PMID- 1712163 TI - [Treatment of recurrent Dupuytren's disease by scalar incision and firebreak graft]. AB - We report our experience of the use of a scalar type incision associated with a total skin graft in the treatment of recurrences of Dupuytren's contracture. This is not an original technique, but one described by Hueston in 1984, which consists of a "Fire Break" skin graft after a simple transverse incision of recurrent Dupuytren's contracture. We attribute the absence of recurrence with this graft to the impossibility of the disease to affect the thin tissue between the skin graft and the underlying tendons. Our series is composed of 25 patients, all male. The majority of these patients had undergone surgery on a single occasion before treatment of recurrences with an average time interval of seven years. In a great majority of cases the little finger was deformed and generally severely (stage III or IV). All of our patients were reviewed with a mean follow up of 28 months after surgery, and we did not observe any recurrences under the graft. In this series, which remains too small and too recent, 67% of cases presented an acceptable result with nearly complete extension and satisfactory grasp. We do not apply this technique to the treatment of all cases of recurrent Dupuytren's contracture, but we reserve it preferentially for elderly patients, operated on several occasions for ulnar fingers especially the little finger, in digital or digito-palmar forms in which the deformity predominates on the proximal interphalangeal joint with marked digital infiltration. PMID- 1712164 TI - [Current techniques and indications of pedicled groin flap for hand surgery. Apropos of 100 cases]. AB - Based on an analysis of the anatomical and physical data reported in the literature, the authors describe their personal technique based on an experience of 100 pedicled inguinal flaps. The authors define the current indications for this flap in reconstructive hand surgery. The quality of pedicled inguinal flaps makes this procedure a technique of choice in the emergency treatment of wounds of the hand. PMID- 1712165 TI - [Morbidity of iliac bone grafts. A study apropos of 100 consecutive cases]. AB - A study of the real morbidity after iliac bone graft harvesting was conducted on a homogenous series of 100 consecutive cases. Functional and aesthetic consequences were evaluated in relation to the immediate post-operative course and the long-term follow-up and in relation to the indication, the technique used and the amount of bone removed. Considering the small number of sequelae observed, autogenous iliac bone graft remains the best material for craniomaxillofacial reconstruction. The main disadvantage consists of the resorptions noted; which raises the possibility of using other types of bone grafts or bio-materials in some indications. PMID- 1712166 TI - [Escalator flap in the treatment of claw nail]. AB - Nail horn deformity can be corrected by a proximal withdrawal of the nail complex on the distal remaining skeleton. The main problem is the flap coverage and we have combined an "in bloc" O'Brien island flap with the withdrawal of the nail. This "escalator" technique allowed a good correction with a satisfactory palmar skin cover and a decreased "wave" of the dorsal skin. PMID- 1712167 TI - [Vaginal and perineal reconstruction following excision for cancer. Apropos of 4 cases]. AB - Vaginal reconstruction at a Cancer Treatment Institute is discussed on the basis of 4 personal cases, using either myocutaneous flaps [gracilis (1 case), gluteus maximus (2 cases)] for the lower portion and the perineum, or a sigmoid graft when vulvo-perineal structures are conserved. A review of techniques and published cases is presented. PMID- 1712168 TI - [Giant sacrococcygeal teratoma. A method of perineal reconstruction]. AB - A method to reconstruct the perineum, after excision of a giant sacrococcygeal teratoma, using one anterior V and multiple posterior V incisions, is described. A baby weighing 2,400 g with a 1,000 g sacrococcygeal teratoma was operated using this technique. PMID- 1712169 TI - [Contribution of a 3% solution of boric acid in the treatment of deep wounds with loss of substance]. AB - The correct application of antiseptics to major surgical wounds must comply with appropriate protocols. Compliance with the protocol established by the CHRU of Nancy was evaluated by means of a questionnaire assessing the understanding of the protocol by the nursing staff and by a survey in the wards in which it was applied. The defects observed cannot be explained by a lack of efficacy for superficial wounds and deep wounds, as a randomised study of 42 wounds demonstrated the superiority of the protocol in relation to the use of another product which is widely used in the wards. In contrast, for deep wounds with loss of substance, the proposed protocol does not always achieve therapeutic success. This finding has led the authors to propose the use of a 3% boric acid solution based on a case-control study which demonstrated a significantly superior efficacy. All of the epidemiological and clinical elements are summarised in order to demonstrate the solid basis for compliance with antiseptic protocols of surgical wounds which can only be beneficial in terms of therapeutic success, length of hospital stay and cost savings. PMID- 1712170 TI - [Cystic chondromalacia of the auricle. A case report. Review of the literature]. AB - Cystic chondromalacia is a clinical and histopathological entity which can be clearly distinguish from all other cystic lesions of the auricle. We report a case which was clinically asymptomatic and involved the scaphoid fossa of the anterior surface of the pinna. The pathological process consisted of degenerative changes of the auricular cartilage which produced a cavity containing a serous fluid. No etiologic factor was found, in particular no trauma. The posterior wall of the cyst was excised under local anesthesia. This case allowed us to review the clinical, histopathological features and surgical difficulties of this rare lesion. PMID- 1712171 TI - [Beta-hemolytic streptococcal periorbital necrotizing fasciitis in a child]. AB - The authors report a case of beta-haemolytic streptococcal periorbital necrosing fasciitis in a two old girl. Extensive cutaneous necrosis of the four eyelids developed after the installation of a major septic syndrome. Excision of the necrotic tissues required removal of the palpebral part of the orbicularis muscle and opening of the orbital septum. A graft was performed on the 23rd day. Active palpebral occlusion was retained by means of the orbital portion of the orbicularis muscle. Two complementary grafts had to be performed to ensure satisfactory palpebral occlusion. The periorbital localization, exceptional in children, must not be confused with periorbital cellulitis, a common disease, which never progresses towards necrosis. The authors stress the necessity of an early diagnosis in view of the importance of medical treatment and surgical drainage with opening of the septum, which is the only way of decompressing the oedema and preventing palpebral necrosis due to tissue ischaemia. PMID- 1712172 TI - [Esthetic rhinoplasty in the elderly]. AB - The classical refusal to perform rhinoplasty in elderly subjects needs to be revised. In fact, this operation gives satisfactory results provided the patients are well selected on the basis of psychological and anatomical criteria. Apart from the repair of accidental or surgical skin defects, the delayed request from rhinoplasty candidates should be carefully assessed and, when in doubt, the patient may require psychiatric consultation. The major technical problem is that of the lack of elasticity of the skin requiring very moderate modifications of the osteocartilaginous skeleton in every case and occasionally skin resections to allow skin cover of the revised structures. Various techniques for the root of the nose have been proposed. The classical difficulty for elderly people to assume their new body image is more theoretical than real provided "minimal" rhinoplasties are performed. This operation warrants a place in the surgery of ageing. PMID- 1712173 TI - [Bacterial population on the site of loss of cutaneous substance. Its role on the success of skin graft]. AB - The bacterial population of 53 skin defects was studied together with its influence on the success of split skin grafts. All of the skin defects were found to be infected. Coagulase + Staphylococcus, group A beta-haemolytic Streptococcus and Pseudomonas aeruginosa were the micro-organisms most frequently isolated. The number of microorganisms does not influence the success of the graft. Streptococcus almost always causes failure of the graft in contrast with the other microorganisms which do not appear to have any specific influence. The anti infectious strategy must therefore be selectively directed against this microorganism. PMID- 1712174 TI - [Plastic surgery of the eyelids after tumoral excision. Apropos of 299 cases]. AB - Plastic surgery of the peri-orbital area after tumor resection is a special and interesting field of reconstructive surgery. Our experience concerns the surgical treatment of 299 malignant tumors of the skin of the peri-orbital area in 291 patients. Basal cell carcinomas were the most common but we also had 8 squamous cell and 4 mixed carcinomas and 1 malignant melanoma. Among these tumors, 277 were primary and 22 were recurrences: 5 after X-Ray therapy, 9 after surgery elsewhere, 4 after electrocautery and 4 after combined treatments. The lower eyelids and the inner canthus areas were the most frequently involved in our series with relative infrequency in the outer canthus and upper eyelids. We used direct closure and local flaps in the repair of most of our patients, but we also used some full thickness skin grafts by preference for the upper eyelid. Complications consisted of 8 cases of ectropion and 2 flap necroses for which a revision was performed in some cases. Concerning our carcinological results among the 123 patients with one year minimum follow up we noticed 4 recurrences which represents a percentage of 3.2%. PMID- 1712175 TI - Inhibition of growth of HeLa cell tumours in nude mice by 125I-labeled anticytokeratin and antiPLAP monoclonal antibodies. AB - The radiommunotherapeutic potential of 125I-labeled monoclonal antibodies was investigated in 48 nude mice (BALB/c, nu/nu) inoculated s.c. with the HeLa Hep 2 human adenocarcinoma cell line. This isotope, 125I, which is not commonly used for therapeutic purposes caused significant decrease in tumour growth from day 10 to day 42, when coupled to monoclonal antibodies directed against placental alkaline phosphatase (H7) or cytokeratins (TS1). The average growth rate was approximately 50-60% of that observed in the untreated control group after 42 days. The specific radioactivity in each organ 42 days after injection of radiolabeled monoclonal antibodies, indicated that these target antigens retain significant amounts of radiolabeled antibody in the tumours for at least 6 weeks after injection. No weight loss was seen in the animals during this experiment. By use of autoradiographic techniques, the labeled monoclonal antibodies were visualized deep in tumours in characteristic patterns representative of viable tumour cells (H7) and necrotic areas (TS1). The therapeutic approach using 125I labeled antibodies is encouraging and may offer new dimensions in radioimmunotherapy. PMID- 1712176 TI - Inhibition by 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil of hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene in rats. AB - It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). The administration of 3'-MeDAB in combination with 1-(2-tetrahydrofuryl)-5-fluorouracil and uracil (UFT) delayed the appearance of oval cells and the formation of hyperplastic nodules, which were observed in the liver from 3 and 5 weeks, respectively, after the onset of 3'-MeDAB feeding, and also delayed the transient increase of serum alpha-fetoprotein level, which transiently peaked at 5 weeks, and completely suppressed the transient increase of tissue thymidylate synthetase activity, but not thymidine kinase, which were induced by 3'-MeDAB at 5 weeks, and finally reduced markedly the incidence of hepatocarcinomas. These results indicate that the suppression of de novo synthesis in pyrimidine metabolism prevents hepatocarcinogenesis. PMID- 1712177 TI - Stroma formation in Ehrlich carcinoma. I. Oedema phase. A mitosis burst as an index of physiological reoxygenation? AB - Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels leads to diffuse oedema. It is here reported that cell loosening and a remarkably high mitotic burst were observed in Ehrlich carcinoma in regions in contact with the exudate, particularly at the perinecrotic (hypoxic) region. This suggests both an enhanced cell detachment from the tumour parenchyma and an improvement of the microenvironment, the exudate thus appearing as beneficial to the malignant cells contributing to the reoxygenation of formerly hypoxic regions. The temporary and well-localized concentration of mitoses in inner tumour areas has perhaps been disregarded by the pathologists engaged in mitosis counting for tumour grading. Peripheric and intraparenchymal concentrations of mast cells, lipid pools and platelets were seen in apparently key geometric disposition for controlling fibrin deposition and angiogenesis. Hypoxia is known to cause resistance to oxygen-dependent treatments and to facilitate cell detachment; normal fibroblasts respond and survive under hypoxic conditions by exhibiting features of the malignant phenotype. During reoxygenation, gene instability, cellular heterogeneity and increased drug resistance and metastatic spread have been reported. A reoxygenation process can also be deduced from several other histochemical and morphological patterns observed in this study. The findings here reported thus suggest that the oedema phase is a crucial phase regulating growth, invasion and dissemination of tumour cell populations, that should be specifically addressed therapeutically. PMID- 1712178 TI - Immunocytochemical staining of cells in 153 pleural effusions with a panel of monoclonal antibodies and lectins. AB - A panel of markers, including MAbs to different epitopes of CEA, B 6.2, detection of AP activity and lectin receptors (PNA, HPL, SBA, LAL, LcL) for cell identification in pleural effusions, is proposed. Using immunocytochemical methods cancer cells were determined in all 80 cytological positive and in 13 from 21 cytologically negative cancer effusions. The reaction was not observed in 45 benign effusions. The panel was unsuccessful to determine the tumor cell origin. The immunophenotypic features of reactive transformed lymphocytes in effusions were described and the criteria for diagnosis of B-cell and T-cell NHL were present. By means of these criteria lymphomatous cells in serous effusions of seven patients were revealed. PMID- 1712179 TI - Lectin histochemistry of normal and neoplastic nasopharyngeal epithelium. AB - The cell surface carbohydrate profile of formalin-fixed paraffin-embedded tissue sections of normal and neoplastic epithelium was evaluated using 9 plant lectins. Three lectins, namely Con A, RCA and WGA, showed a similar pattern and staining intensity from normal epithelium to metaplastic squamous epithelium and nasopharyngeal intraepithelial neoplasia (NPIN). However, a decrease in staining reactivity was observed in undifferentiated nasopharyngeal carcinoma. Significant differences in intensity and distribution were seen in UEA and cryptic PNA residue (after neuraminidase pretreatment) from normal nasopharyngeal epithelium to NPIN. Infiltrative undifferentiated carcinomas showed a heterogenous lectin binding pattern and altered intensity of lectin binding in one case of DBA and three cases of PNA (no neuraminidase pretreatment), suggesting a variation in expression of carbohydrate by tumour cells. These results indicate that neoplasia in nasopharyngeal epithelium is associated with alterations in terminal sialic acid, -Fucose residues and -Gal-D-GalNac residues present in the outer parts of glycoconjugates. SBA, VVL and BSL failed to stain any types of epithelia. Desialylation of tissues by preincubation with neuraminidase did not expose DBA, SBA, VVL and BSL binding sites. These findings may be used as a baseline for evaluation of lectin binding in preinvasive and invasive lesions of the nasopharynx. PMID- 1712180 TI - Establishment, characterization and determination of cell surface sugar receptor (lectin) expression by neoglycoenzymes of a human myeloid marker-expressing B lymphoblastoid cell line. AB - Mediation of cellular interactions by protein (lectin)-carbohydrate recognition presupposes the expression of respective surface determinants. Due to the importance of cellular contacts between bone marrow stromal cells, recently shown to express cell surface lectins, and tumor or normal progenitor cells for biosignaling and marrow egress, quantitation of cell surface sugar receptor expression by a panel of chemically glycosylated enzymes (tetrameric E. coli beta galactosidase) for human leukemia/lymphoma cells was initiated. Cells of the new B lymphoblastoid line Croco II that are partially positive for the CD15-specific epitope expressed receptors for various sugar specificities on their surface, fulfilling an indispensable prerequisite for establishment of glycobiological interactions. Binding studies with increasing neoglycoenzyme concentrations up to saturation in four cases disclosed values for apparent affinity constants in the range of 25-200 nM with 0.25-3 x 10(5) bound probes per cell. The presence of receptors for constituents of carbohydrate chains of cellular glycoconjugates was also ascertained biochemically, namely for beta-galactosides, alpha-mannosides, alpha-fucosides and N-acetylgalactosaminides. Expression of this property was modulated by changes in the culture conditions, as revealed by binding studies with cells, derived from growth in medium containing different serum concentrations. These findings indicate that cell surface sugar receptors of tumor cells warrant further attention with respect to recognitive interactions. PMID- 1712181 TI - Immunocytochemical differential diagnosis of diffuse malignant pleural mesotheliomas--a clinicomorphological study of 158 cases. AB - Formalin-fixed paraffin-embedded material of 158 diffuse malignant pleural mesotheliomas (DMPMs) was used in order to determine the differential diagnostic value of immunocytochemical probes against 9 different antigens. While vimentin expression was found in only 50% of cases, regardless of their histological subtype all tumours were found to be cytokeratin-positive when an antibody with broad-spectrum cytokeratin reactivity was used. Conversely, none of the cases was immunostained by antisera against carcinoembryonic antigen (CEA), Leu-M1 antigen, chromogranin, S-100 protein, lysozyme and a T-cell associated antigen. The density of inflammatory cell infiltrates reactive with antisera against the three latter antigens was not associated with the clinical behaviour of the neoplasms examined. Eight DMPM cases showed immunoreactivity with HEA-antibodies against Egp 34, an antigen previously supposed only to be expressed by carcinomas. On the basis of these findings, the consistent cytokeratin reactivity, also of the sarcomatous type of DMPM, may help to exclude metastatic involvement of the pleura by a mesenchymal neoplasm of other origin. CEA and Leu-M1 staining of a given pleural tumour, on the other hand, is indicative of a carcinoma secondarily afflicting the pleura, thus making the diagnosis of primary DMPM unlikely. PMID- 1712182 TI - Potentiation of bleomycin cytotoxicity by high molecular weight polyacrylic acid. II. Involvement of rapid conformational change of polyacrylate. AB - We report that bleomycin (BLM) cytotoxicity is dramatically potentiated in the presence of high molecular weight polyacrylic acid (A119). For potentiation, an appropriate amount of divalent cations such as Ca++, Mg++, or Ba++ was required, in addition to vortex - stirring of the reaction mixture. There was no potentiation when A119 alone was pre-stirred or left standing for several days in the presence of divalent cations prior to use. Taking into account of kinematic viscosity of A119 observed during the treatment, a rapid conformational change of A119 in the presence of divalent cations might be involved in the potentiation mechanism. PMID- 1712183 TI - Potentiation of bleomycin cytotoxicity in cultured mammalian cells by polyacrylic acid. III. Induction of DNA strand breakage and suppression of cellular heat production. AB - The 2E cytotoxicity of bleomycin toward cultured mammalian cells was synergistically enhanced by vortex-stirring in the presence of a low dose of high molecular weight polyacrylic acid. Cellular DNA isolated immediately after the above treatment suffered from severe single strand breaks. Heat production of the treated cells also decreased sharply immediately after the treatment, indicating that some functional disorder was probably induced on the cell membrane leading to cell death, possibly resulting from enhanced DNA strand breaks. PMID- 1712184 TI - Bleomycin iontophoretic therapy for verrucous carcinoma. PMID- 1712185 TI - [Alternatives to transurethral resection of benign hypertrophy of the prostate]. PMID- 1712186 TI - [Use of the urologic resectoscope in the treatment of rectal pathology. Analysis of our experience and review of the literature]. AB - Treatment of rectal polypoid disease, particularly villous adenoma, by electrocoagulation has proven its efficacy in a large number of cases. However, it does not permit adequate control or analysis of the specimen because the morbid excrescence is destroyed. Transsphincteric or transanal surgery, anterior resection or abdominoperineal amputation are generally too aggressive for a recurrent but benign condition. The enhanced efficacy of other therapeutic modalities such as chemo-, immuno- or radiotherapy will undoubtedly be beneficial to conservative surgical management of a malignant rectal pathology. Our initial experience in 15 patients with different rectal tumors is reported. These patients were treated by endoscopic transanal resection (ETAR) utilizing the urologic resectoscope. Local control of the disease process was achieved in 66.6% of the cases with scant morbidity and a mortality rate of 7.3% (1 case). The foregoing results and those reported in the literature indicate that ETAR is a valid alternative surgical procedure for definitive treatment of sessile, villous, or mixed adenomatous polyps and certain cases of rectal adenocarcinoma. Moreover, this procedure has proved effective as palliative treatment in advanced rectal adenocarcinoma and obviates the need for stomas. PMID- 1712187 TI - Low dose epirubicin for hypercalcemia associated with renal pelvis carcinoma. AB - We present a case of hypercalcemia associated with a renal pelvis squamous neoplasm. Severe hyperleukocytosis was also present, resembling a leukaemoid reaction. After failure of standard therapy, we performed a trial of low dose epirubicin in this patient, obtaining a short-term clinical remission. Although a palliative non-toxic effect seems to be achievable with this approach, further studies are needed to ascertain the role of chemotherapy in the long-term control of this condition. PMID- 1712188 TI - [Open adenomectomy: review of 223 cases]. AB - A retrospective study was undertaken to determine the results achieved in 223 patients with prostatic hypertrophy who underwent open surgery during a period spanning 5 years. Good results were achieved in 88.34% of the cases, the mortality rate was 0.44%, and the post-operative morbidity rate was 31.3%. Urinary infection was the most common complication during this period (21.01%). Late post-operatively, we observed the following low incidence of urethral stenosis and lodge sclerosis, 1.79% and 2.24%, respectively. Six patients (2.69%) were permanently incontinent and one required surgery for recurrent obstruction of prostatic origin. PMID- 1712189 TI - [Specific prostatic antigen in prostatic carcinoma: its relationship with tumor differentiation and clinical course]. AB - Carcinoma of the prostate is a tumor with a variable clinical course and a high incidence of local progression and/or metastasis. This study was undertaken to evaluate tissue prostate specific antigen (PSA) in patients with carcinoma of the prostate, its correlation with Gleason's grading and its value in predicting the clinical course of these patients. We studied 28 transurethral biopsies of patients with prostatic carcinoma utilizing HE and peroxidase-antiperoxidase staining techniques. These were given a score of 2 to 10 using Gleason's grading. PSA was determined according to percent positivity. The clinical course was considered favourable (F) when the lesion remained stable and unfavourable (U) when peri-prostatic spread was evidenced, metastasis and/or death from the disease. Statistical analysis was performed with the linear discriminatory test. PSA percentages ranged from 0 to 95 and the Gleason score from 3 to 11. There was an indirect correlation between these methods (r = 0.74): high Gleason scores corresponded to low PSA values and viceversa. PSA was highly positive in patients with F and U clinical courses whereas low positive values (less than 40%) were observed only in patients with U clinical course. High Gleason (8 to 10) and low (less than 5) scores were observed only in patients with a clinical course of U or F, respectively, while intermediate values (5 to 8) were not predictive of the clinical course. Discriminatory analysis gave Z values of -2.446 (P = 0.014) for PSA, -2.90 (P = 0.004) for the Gleason score in predicting prognosis, conferring a greater value overall to the latter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712190 TI - [Occult cancer in patients with symptomatic benign prostatic hyperplasia]. AB - The results of a prospective study undertaken in 29 patients with symptomatic benign prostatic hyperplasia (BPH) are presented. Transrectal ultrasound, ultrasound-guided biopsy and prostate specific antigen (PSA) were utilized in the search for hidden cancer of the prostate. However, no cancer was detected in any patient. Very high values of PSA were found, particularly in patients with an indwelling catheter. Transrectal ultrasound yielded no false negatives and no complications were observed. PMID- 1712191 TI - Small-volume hypertonic saline dextran resuscitation from canine endotoxin shock. AB - This study evaluated resuscitation of endotoxin shock with 7.5% hypertonic saline dextran (HSD 2400 mOsm) by measuring hemodynamic and regional blood flow responses. Endotoxin challenge (1 mg/kg) in adult dogs caused a significant decrease in mean arterial blood pressure (MABP), cardiac output (CO), left ventricular +/- dP/dt max, and regional blood flow (radioactive microspheres). Cardiocirculatory dysfunction and acid-base derangements persisted throughout the experimental period in untreated endotoxin shock (group 1, n = 10). In contrast both regimens of fluid resuscitation (group 2, n = 11: bolus of 4 mL/kg HSD followed by a constant infusion of lactated Ringer's [LR] to maintain MABP and CO at baseline values; group 3, n = 10; LR alone given as described for group 2) improved regional perfusion and corrected acid-base disturbances similarly in all dogs. Hypertonic saline dextran enhanced all indices of cardiac contraction and relaxation more than LR alone. The total volume of LR required to maintain MABP and CO at baseline values was less in the HSD group (59.2 +/- 6.8 mL/kg) than in the LR alone group (158 +/- 16 mL/kg, p = 0.01). The net fluid gain (infused volume minus urine output and normalized for kilogram body weight) was five times greater in the LR (24.8 +/- 6.2 mL/kg) than in the HSD group (4.6 +/- 1.2 mL/kg, p = 0.01). Lung water was similar in all dogs, regardless of the regimen of fluid resuscitation. Hypertonic saline dextran effectively resuscitates endotoxin shock in this canine model. PMID- 1712192 TI - The medical therapy of hemorrhagic complications following coronary artery bypass grafting. PMID- 1712193 TI - [Comparison of the efficacy of moricizine and disopyramide in the treatment of ventricular extrasystoles]. AB - Moricizine chlorhydrate (Ethmozine), a relatively unknown antiarrhythmic agent in France, is a derivative of Phenothiazine, related to the Vaughan-Williams Class IB drugs. A randomised, double-blind, crossover trial with Disopyramide 600 mg/day after a placebo period in 10 patients with ventricular extrasystoles, half of whom had underlying cardiac disease, showed that moricizine 750 mg/day significantly reduced (p less than 0.05) the overall number of ventricular extrasystoles by 81 +/- 46% (disopyramide 72 +/- 69%; NS) and that this drug is effective in 2/3 of patients by suppressing 70 to 100% of ventricular extrasystoles, whereas disopyramide was effective in only 40% of the same patients and never gave better results than Moricizine. Cardiac and extracardiac tolerance of Moricizine was good in this study, confirming previously reported results and its superiority when compared with disopyramide (20% of unwanted effects in this series). PMID- 1712194 TI - Influence of laboratory test volume and geographic location on maternal alpha fetoprotein results. Information from the College of American Pathologists Maternal alpha-fetoprotein Survey. AB - Using 1989 College of American Pathologists Maternal alpha-fetoprotein (AFP) Survey data, we found virtually no association between a laboratory's AFP test volume and the reliability of reported multiples of the median uncorrected for maternal weight, race, or diabetes. We also found no differences in AFP gestational age-specific medians across geographic regions of the United States. We did find that clinically important adjustments for maternal weight, race, and diabetes were made more frequently by laboratories with higher clinical AFP test volumes. Consequently, we believe that a minimum AFP test volume for satisfactory performance of AFP testing, as proposed by several professional organizations, cannot be justified on inability of laboratories with low AFP test volumes to quantify AFP in clinical specimens or calculated uncorrected multiples of the medians accurately. However, high-volume laboratories enhance clinical utility of the reported AFP results by making clinically important adjustments of their reported multiples of the medians, and they tend to provide more accurate clinical interpretations. Therefore, we recommend that efforts to improve the quality of AFP testing nationally should focus on improving the ability of laboratories to provide appropriate adjustments to and clinical interpretation of their AFP results, in addition to more traditional efforts directed toward quality control of the analytical aspects of the AFP assay. PMID- 1712195 TI - Neuropeptide Y- and substance P-like immunoreactivities in the guinea pig parotid gland. AB - These immunoreactivities were examined by double immunofluorescence, which demonstrated that many nerve fibres around the glandular acini and blood vessels contain neuropeptide Y and substance P. It appears that these substances may coexist in nerve fibres around acini, but that there may be two populations of fibres containing these peptides around the blood vessels. PMID- 1712196 TI - Axonal transport and anterior ischemic optic neuropathy. PMID- 1712197 TI - Solitary metastatic ovarian carcinoma of the spleen: a case report. AB - Carcinomatous metastatic involvement of the spleen usually indicates a widespread malignant disease. Solitary metastatic lesions in the spleen are exceedingly rare. The literature contains fewer than 16 cases. In this paper we report a case of a solitary metastatic lesion of the spleen arising from a serous cystadenocarcinoma of the ovary 5 years after the initial operation. A splenectomy was performed followed by smooth postoperative course. PMID- 1712198 TI - Mechanism of vanadate-induced activation of tyrosine phosphorylation and of the respiratory burst in HL60 cells. Role of reduced oxygen metabolites. AB - Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+) OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover. PMID- 1712199 TI - Purification and characterization of metabolically active capillaries of the blood-brain barrier. AB - Microvessels were isolated from bovine and rat cerebral cortex by simple procedures involving mechanical homogenization, differential and density-gradient centrifugation, and chromatography on a column of glass beads. The preparations were composed of short capillaries with a diameter of 1-10 microns. Both purifications were monitored by assaying the activity of the marker enzyme gamma glutamyl transpeptidase (gamma-GTase). The final bovine and rat preparations were enriched 20- and 14-fold over the homogenate respectively. gamma-GTase activity was measured in different fractions after bovine and rat membranes were solubilized with 0.5% and 0.3% Triton X-100 respectively. Measurement of 5' nucleotidase and acetylcholinesterase activities indicated very low levels of contamination of the microvessel preparations by glial cells and neurons. The integrity of the capillary membranes was confirmed by the assay of a cytosolic marker enzyme, lactate dehydrogenase. Viability of the microvessels was demonstrated by the presence of detectable levels of adenylates and by tissue respiration induced by glucose and succinate. Comparison of the proteins of homogenized bovine and rat brain cortex with those of purified capillaries separated by SDS/PAGE revealed enrichment of at least three predominant proteins of 14, 16 and 18 kDa in the capillary preparations. It is concluded that these methods allow rapid isolation of small blood vessels of the blood-brain barrier which are suitable for metabolic and structural studies in vitro. PMID- 1712200 TI - [Antigenic polysaccharides of Shigella bacteria. Structure of the polysaccharide chain of the lipopolysaccharide from Shigella boydii, type 11]. AB - On mild acid degradation of the Shigella boydii, type 11 lipopolysaccharide, the corresponding O-specific polysaccharide composed of D-glucuronic acid, 2 acetylamino-2-deoxy-D-glucose, D-ribose and L-rhamnose residues in the ratio 1:1:1:3 was obtained. Methylation, partial acid hydrolysis and 13C-NMR spectral data for the polysaccharide led to the structure of the oligosaccharide repeating unit as a branched hexasaccharide: [formula: see text]. Numerous O-acetyl groups attached non-stoichiometrically to the residues of D-glucuronic acid, L-rhamnose and 2-acetylamino-2-deoxy-D-glucose were located with the use of 13C-NMR spectroscopy. PMID- 1712201 TI - [Localization of the antigenic segment of the tick-borne encephalitis virus envelope protein using monoclonal antibodies]. AB - The largest cyanogen bromide fragment (GP-14,5; coordinates 78-176) of E protein belonging to the envelope of the tick-borne encephalitis (TBE) virus (Far Eastern subtype, strain Sofjin) interacted with five out of twelve E-specific monoclonal antibodies (MAbs). Having compared; efficiencies of some MAbs binding to the antigens of TBE viruses of Far Eastern and West European subtypes and primary structures of analogous peptides of these viruses, we suggested the epitopes of these MAbs to be located in the vicinity of 89 and/or 116-th amino acid residues of E protein. Effect of denaturing agents and reduction followed by carboxymethylation on the protein E antigenic properties was studied. PMID- 1712202 TI - [Epitope specificity of hemagglutinating monoclonal anti-A-antibodies]. AB - Fine epitope specificity of three anti-A monoclonal antibodies (MA) 1H410, 3F9, and 44F9 was studied by: 1) direct MA binding to synthetic oligosaccharides (OS) linked to polyacrylamide matrix, and 2) inhibition of MA binding to natural antigen by synthetic OS and their polyacrylamide conjugates. It has been established that the antigen binding site of MA 1H10 is specific for tetrasaccharide A (type 3), whereas MAs 3F9 and 44F9 recognize trisaccharide A, the contribution of alpha-L-fucosyl residue being insignificant in the case of 44F9 binding. The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells of A1 and weak A subgroups is discussed. PMID- 1712203 TI - [Technique for automating HLA-DR typing]. AB - Two types of anti-class II coated magnetic beads were compared for the use in isolation of B lymphocytes from whole blood. The HLA DR typing was performed in parallel after separation of the cells by the rosette technique. The depletion was monitored by Flow cytometry using CD2 and CD20 antibodies. Typing was performed using classic lymphocytotoxicity test of Propidium-Iodide Carboxyfluorescein diacetate fluorescence. The results were scored with an ordinary microscope or by photometric evaluation. These techniques lead to simplification in cell isolation from small amounts of blood with an important gain of time and a less subjective interpretation of DR phenotyping was until now long and difficult. PMID- 1712204 TI - Lipid-bound, native-like, myelin basic protein. Batch-wise preparation and perspectives for use in demyelinating diseases. AB - Batch purification of the myelin basic protein (MBP) in the lipid-bound form was obtained from bovine brain white matter by using the slightly polydisperse nonionic detergent, n-octyl-pentaoxyethylene (octyl-POE) and hydroxyapatite. This large-scale procedure can also be carried out in laboratories without chromatographic equipment, and is applicable to small amounts of myelin. More interestingly, removal and inhibition of the proteolytic activity associated with myelin allowed us to obtain more stable and intact forms of the protein when compared with MBP isolated in the lipid-bound form by our previous method. Since it retains binding to all myelin lipids, this purified MBP may be considered as being in a native-like form. In this article, we suggest why this more intact form of MBP could be used to advantage as an alternative to lipid-free, water soluble MBP in the study, detection, and treatment of myelin damage in pathology. PMID- 1712205 TI - Suppression of adenovirus type 5 E1A-mediated transformation and expression of the transformed phenotype by caffeic acid phenethyl ester (CAPE). AB - Viral transformation and DNA-transfection assays were employed to investigate the differential toxic effect of caffeic acid phenethyl ester (CAPE), an extract of the honeybee hive product propolis, on adenovirus type 5 (Ad5)-transformed cloned rat embryo fibroblast (CREF) cells. CAPE inhibited, in a dose-dependent manner, both de novo and carcinogen-enhanced transformation of CREF cells by H5hr1, the cold-sensitive (cs) host-range mutant of Ad5. CAPE had a selective inhibitory effect on Ad5-induced transformation when a wild-type (wt) Ad5 E1A gene or a cs Ad5 E1A gene (at 37 degrees C, but not at 32 degrees C) was cotransfected into CREF cells with a dominant-acting bacterial hygromycin-resistance gene. A requirement for the expression of Ad5 E1A-encoded mRNAs and transforming proteins and sensitivity to CAPE was demonstrated using CREF cells stably transformed by a cs Ad5 E1A gene and an Ad5 E1A gene under the transcriptional control of a mouse mammary tumor virus promoter. To distinguish between the effects of the two Ad5 E1A-encoded proteins of 289 amino acids (aa) and 243 aa, CREF cells were stably transformed with cDNAs encoding either the 13S or the 12S E1A mRNA. CREF cells expressing the 13S E1A-encoded 289-aa protein were more sensitive to the growth suppressing effect of CAPE than cells producing only the 12S E1A-encoded 243-aa protein. However, the growth-suppressing and toxic effects of CAPE were greatest in cells expressing both E1A-encoded transforming proteins. Analysis of the effect of CAPE on E1A and beta-actin gene expression in wt and cs E1A and H5hr1 transformed CREF cells indicated that low levels of CAPE, which were growth suppressive, did not selectively suppress E1A expression. These results demonstrated that cellular changes induced in CREF cells by the 13S E1A-encoded 289-aa protein of Ad5, when expressed alone or in combination with the 12S E1A encoded 243-aa protein, rendered transformed cells sensitive to the growth suppressing and toxic effects of CAPE. PMID- 1712206 TI - Monoamines and related metabolite levels in the cerebrospinal fluid of patients with dementia of Alzheimer type. Influence of treatment with L-deprenyl. AB - An impairment of the monoaminergic systems has frequently been reported for Alzheimer's disease (AD) as well as an overactivity of cerebral monoamineoxidase B (MAO-B). L-deprenyl (LD), a selective and irreversible MAO-B inhibitor, has recently been proposed for the treatment of AD. The cerebrospinal fluid (CSF) levels of norepinephrine (NE), epinephrine (E), dopamine (DA), 3-methoxy-4 hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) were studied in 14 patients suffering from dementia of Alzheimer type (DAT) and in 14 controls. A three-month double-blind study comparing LD with placebo was carried out, in the DAT group, and the influence of the treatment on neurotransmitter levels and cognitive performance was evaluated. The basal study revealed a significant reduction in CSF NE and HVA levels in DAT patients when compared with controls; the treatment with LD determined a significant decrease in HVA levels only and, as to neuropsychological investigation, a global amelioration of cognitive performances. PMID- 1712207 TI - Lewy bodies in parkinsonism share components with intraneuronal protein bodies of normal brains. AB - Histochemical characteristics of the Lewy bodies, in catecholamine neurons of 10 Parkinsonian patients, were compared to those of the spherical protein bodies, the basic protein-rich markers of catecholamine neurons in man. Special methods for proteins and lipids showed that the core of the Lewy bodies, in the neurons of the locus coeruleus and the substantia nigra, contains basic proteins and lipids normally found in the protein bodies. Acid fuchsin and the lipid-soluble fluorescent dye rhodamine B stained the entire core of the Lewy body in the parkinsonian brains and the entire sphere of the protein body in the control brains. Bromsulfophthalein, another acidic dye, which selectively binds to the enzyme gluthathione-S-transferase, had affinity only for a ring-like lamina at the outer layer of the core of the Lewy body and for the outer rim of the protein body. These results demonstrate that Lewy bodies and protein bodies contain similar macromolecular components, that is lipids and two different types of proteins, which also show similar stratification in the two structures. On the other hand, the presence in several neurons of the Parkinsonian patients, of aggregates representing transitional forms between protein bodies and Lewy bodies, indicates that abnormalities of protein bodies precede, and are somehow linked to Lewy body production. PMID- 1712208 TI - Control of buffer pH during agarose gel electrophoresis of glyoxylated RNA. AB - There is a shift in buffer pH routinely encountered during electrophoresis. If one is running glyoxylated RNA on agarose gels, this pH shift can have potentially detrimental effects on experimental objectives if the shift is allowed to proceed unabated; this is due to the fact that the RNA will deglyoxylate if the pH is allowed to rise above 8.0. In order to counteract the shift, the buffer may be continuously recirculated or totally replaced at predetermined intervals. Because constant recirculation may be technically bothersome to achieve and because repeated total buffer replacement may require large amounts of highly purified water, we have studied (a) the time course of the pH change encountered during the electrophoretic process and (b) whether or not simple manual mixing of the buffer system at specific intervals was sufficient to maintain pH within acceptable bounds. We found that, under the given conditions, the cathode pH had nearly reached 8.0 at 25 min and had soared to 10.5 at 40 min. We further found that manual mixing at 20-min intervals not only prevented the cathode pH from rising above 7.8, but also restored the buffer to its initial pH. PMID- 1712209 TI - A simple and convenient way of blotting nucleic acids. PMID- 1712210 TI - x121: a localized maternal transcript in Xenopus laevis. AB - We describe the cloning and characterization of a partial cDNA, x121, that represents an RNA, which is localized in the animal hemisphere of Xenopus oocytes. This RNA is also detected in an animal to vegetal gradient during early cleavage stages. The x121 RNA titer decreases from fertilization through the gastrula stage, after which it is not detectable on northern blots. The amino acid composition of the x121 conceptual protein derived from cDNA sequencing reveals a large number of acidic residues similar in distribution to proteins that function as transcriptional activators. PMID- 1712211 TI - Epidermal differentiation. PMID- 1712212 TI - Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization. AB - The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory. PMID- 1712213 TI - An improved and rapid procedure for isolating RNA-free Escherichia coli plasmid DNA. AB - We describe a simple, rapid, and inexpensive procedure for the isolation of plasmid DNA in high yields from Escherichia coli cultures. The procedure entails two main steps, which involve treating intact bacterial cells with phenol/chloroform in the presence of Triton X-100 and LiCl followed by polyethylene glycol precipitation. Plasmid DNA preparations isolated by this method are highly pure and virtually devoid of RNA. The DNA is suitable substrate for restriction mapping, DNA-modifying enzymes, and in vitro transcription with SP6 and T7 RNA polymerases. PMID- 1712214 TI - Molecular modelling of HIV-1 reverse transcriptase inhibitors. PMID- 1712215 TI - Comparison of linear antigenic sites in the envelope proteins of human immunodeficiency virus (HIV) type 2 and type 1. AB - The occurrence of dominant linear antigenic sites in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2) was evaluated. Twenty-five peptides representing different regions of HIV-2, strain SBL-6669, were synthesized. For comparison the corresponding peptides of HIV-1, strain BRU, were also prepared. The peptides were tested in enzyme-linked immunosorbent assay (ELISA) with human sera from individuals with proven HIV-1 or HIV-2 infection and simian sera from animals infected with HIV-2 or simian immunodeficiency virus of sooty mangabay monkey origin (SIVsm). Four major antigenic regions were identified. Peptides representing parts or the whole V3 (neutralizing loop) region and an additional stretch of amino acids located at the carboxy terminal of this region showed considerable reactivity. This reaction was predominantly type specific, but some heterotypic reactivity was also seen. Peptides representing the carboxy terminal 21 amino acids of the V3 region of the type related viruses HIV-2 and SIVsm allowed selective identification of strain specific antibodies. A second major antigenic region was found close to the carboxy terminal end of the large glycoproteins. This region was cross-reactive between the two types. The two additional dominating antigenic regions were located in the amino terminal region of the transmembrane glycoprotein. One region has previously been shown to be a uniquely antigenic type-specific site. The other region was also type-specific, but was identified only in HIV-2, amino acids Glu634-Lys649. Excellent facilities are available for the design of not only type-unique site-specific serological tests but potentially also type-cross reactive and strain-specific assays. PMID- 1712216 TI - Characterization of an HIV-1 isolate displaying an apparent absence of virion associated reverse transcriptase activity. AB - In characterizing a group of independent human immunodeficiency virus (HIV-1) isolates, we noted that certain isolates had anomolously low levels of virion associated reverse transcriptase activity. In an attempt to understand the basis of this phenomenon, we examined in detail one such isolate, HIV-1G. We found correctly processed forms of the viral reverse transcriptase in virions as well as processed forms of other viral proteins, suggesting that viral proteins are both expressed and properly processed. We have detected a nuclease activity associated with the outer face of the HIV-1G envelope. This nuclease degrades the DNA product generated during the reverse transcription assay. The nuclease activity is more sensitive to mild protein denaturation than is the viral reverse transcriptase, and it is stimulated by the presence of Ca2+. The amount of virion associated nuclease activity relative to reverse transcriptase activity varies between virus isolates and can vary also for one isolate during virus spread through a culture. The origin of the nuclease activity is unknown but is presumed to be cellular. The variability in amount of nuclease activity may reflect variability in the interaction of the virus with different cellular components during maturation. PMID- 1712217 TI - Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products. AB - Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot. PMID- 1712218 TI - [The pharmacological treatment of postoperative pain in childhood]. PMID- 1712219 TI - Modulation of leucocyte adhesion molecules, a T-cell chemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) in allergic contact dermatitis (rhus dermatitis). AB - To understand the molecular events which are important in leucocyte trafficking in cutaneous inflammation, poison ivy/oak extract was applied topically to the skin, and the simultaneous assessment of a variety of clinical and immunopathological parameters performed. The clinical response of subjects was divided into three main groups: I, 2-24h after application, before the onset of erythema; II, 48 h-1 week after application during maximal clinical changes; III, 2-3 weeks after application when the inflammation had subsided. Six different biopsies per subject were evaluated over the study period and the density of dermal cellular infiltrate, and the distribution of intercellular adhesion molecule-1, (ICAM-1), endothelial leucocyte adhesion molecule-1, (ELAM-1), vascular cell adhesion molecule-1, (VCAM-1), interleukin 8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha), determined. Eight hours after exposure, before lymphocytes and monocytes had entered the dermal interstitium or epidermis, the keratinocytes expressed TNF-alpha and ICAM-1, whilst the endothelial cells expressed ELAM-1, VCAM-1 and ICAM-1. Group II biopsies revealed increasing keratinocyte expression of TNF-alpha and ICAM-1 with the appearance of IL-8, which correlated with the onset of epidermal T-cell trafficking. The endothelium was strongly positive for ELAM-1 and VCAM-1, but there was no influx of neutrophils. Group III biopsies showed a decrease in the expression of ICAM-1, VCAM-1 and ELAM-1 by both keratinocytes and endothelium with a reduction in epidermal/dermal inflammation, although the endothelial cell staining of VCAM-1 and ELAM-1 did not completely disappear. These results suggest that on exposure to poison ivy/oak, keratinocytes rapidly produce TNF-alpha which leads to an early autoinduction of ICAM-1, and later IL-8. There is also a paracrinemediated induction and augmentation of underlying endothelial cell ELAM-1, VCAM-1 and ICAM 1. PMID- 1712220 TI - Expression of a cell adhesion protein (VLA beta) in normal and diseased skin. AB - Biopsies from normal skin (n = 17) and various cutaneous disorders (n = 83) were examined immunohistologically for reactivity with an antibody (CD29) against the common beta chain of the VLA integrin family. In normal skin, CD29 recognized a number of cell types, i.e. endothelial cells, fibroblasts, T lymphocytes and basal keratinocytes. Similar cells were positive in diseased skin, but the expression of VLA beta was upregulated on keratinocytes. The phenotype of the VLA beta-positive T cells was examined in more detail by staining with anti-T-cell antibodies, i.e. CD3, CD4, CD8, CD45RO (UCHL1) and CD45R (2H4). These studies showed that most of the T cells in normal skin, benign cutaneous conditions and early cutaneous T-cell lymphomas (CTCL) expressed a similar phenotype and resembled antigen committed 'memory' (helper/inducer) cells (CD4+, CD29+, CD45RO+, CD45R-). In advanced CTCL, expression of these antigens was more variable, and many of these infiltrates showed aberrant (or unusual) expression of CD29, CD45RO, CD45R and other T-cell antigens. It is concluded that several cells involved in cutaneous immune reactions express a molecule (VLA beta) which acts as a receptor for extracellular matrix components. This molecule is important for the attachment of cells to connective tissue constituents and may act to facilitate the migration of lymphocytes (and other cells) during immune reactions in normal and diseased cutaneous conditions. Advanced CTCL differ from the early lesions and it is possible that there is a progressive accumulation of increasingly malignant (or transformed) cells in these conditions. PMID- 1712221 TI - Neuropeptides in skin disease: increased VIP in eczema and psoriasis but not axillary hyperhidrosis. AB - The neuropeptides vasoactive intestinal polypeptide (VIP), substance P and somatostatin were studied in skin biopsies from patients with eczema, psoriasis and axillary hyperhidrosis. VIP concentrations were elevated in skin affected by eczema and psoriasis, whereas substance P and somatostatin levels did not differ from controls. There was a higher concentration of VIP, but not of substance P or somatostatin, in normal axillary skin when compared to adjacent trunk skin, with abundant VIP-containing fibres surrounding eccrine sweat glands. The VIP concentration was unchanged in skin affected by axillary hyperhidrosis. VIP may increase local blood flow in eczema and psoriasis, but does not appear to play a role in axillary hyperhidrosis. PMID- 1712222 TI - Myelodysplastic syndrome with increased marrow fibrosis: a distinct clinico pathological entity. AB - Seventeen cases of myelodysplastic syndrome (10 primary and seven secondary to previous radio-chemotherapy), characterized by trilineage dysplasia, severe bone marrow fibrosis and a high number of megakaryocytes, are described. All of these patients had similar clinical and prognostic features consisting of pancytopenia, modest or absent visceral enlargement and poor survival. The use of CD61 antibodies, which recognize megakaryocytic cells at all stages of maturation, confirmed that these patients had a higher number of these cells than either normal subjects or patients affected by myelodysplastic syndrome (MDS) without fibrosis. Furthermore, primary and secondary MDS with fibrosis, although clinically and histopathologically similar, differed in terms of the number of megakaryoblasts which were significantly higher in primary forms (P less than 0.02). We conclude that MDS with fibrosis may represent a clinicopathological entity which needs to be distinguished from other MDS subtypes as well as from idiopathic myelofibrosis or malignant myelosclerosis. PMID- 1712223 TI - Effects of recombinant interleukin 4 on the growth and differentiation of blast progenitors stimulated with G-CSF, GM-CSF and IL-3 from acute myeloblastic leukaemia patients. AB - The effects of human recombinant interleukin 4 (rIL-4) on the growth of leukaemic blast progenitors were investigated. Cells obtained from 20 acute myeloblastic leukaemia (AML) patients were evaluated using the blast colony assay in methylcellulose and suspension cultures. While rIL-4 by itself did not show any colony stimulatory activity in the blast colony assay, it suppressed the blast colony formation in methylcellulose stimulated with G-CSF, GM-CSF or IL-3 in 14 patients. In another six patients, rIL-4 enhanced blast colony growth in four patients or did not show any significant effect with any CSF in two patients. In suspension cultures of 12 cases studied, the effects of rIL-4 on the clonogenic cell recoveries were essentially similar to the results of the blast colony assay in each case. In three patients, rIL-4 augmented the differentiation of the leukaemic cells to monocyte lineage. Further, the clinical outcome was significantly different between the patients whose blast progenitors were stimulated by rIL-4 and the patients whose blast progenitors were suppressed by rIL-4 (P less than 0.05); three out of four patients in the former group failed in achieving complete remission (CR), while 12 out of 14 patients in the latter group achieved CR. The results show that the effects of IL-4 on leukaemic blast progenitors were diverse and the responsiveness to IL-4 may be correlated with the prognoses of the patients. PMID- 1712224 TI - In vitro assessment of marrow 'stem cell' and stromal cell function in aplastic anaemia. AB - An in vitro model system is described that allows separate assessment of 'stem cell' and stromal cell function in aplastic anaemia (AA). Seven patients with non severe AA, who had responded to immunosuppressive therapy and had haematological evidence of residual marrow function, were studied. Of these, three with otherwise typical AA had an acquired clonal cytogenetic marker. Purified bone marrow haemopoietic progenitors labelled with CD34 monoclonal antibody were positively selected using the fluorescence activated cell sorter (FACS) from both normal subjects and from patients with AA. The generative capacity of the CD34 positive cells was assessed by monitoring the output of granulocyte/macrophage colony forming cells (CFU-GM) in the non-adherent layer after inoculation onto irradiated performed long-term marrow culture (LTBMC) stromas. Stromal function in AA was assessed by inoculating CD34 positive cells from normal bone marrow onto performed irradiated stromas from patients with AA. Haemopoietic cell ('stem cell') function in AA was assessed by inoculating CD34 positive cells from AA patients onto confluent irradiated normal marrow stromas. Using these crossover/LTBMC experiments, all patients exhibited severe defects in haemopoietic cell function with normal functioning stroma. The proportion of CD34 positive cells present in bone marrow from these patients was reduced compared with controls, they comprised fewer small primitive 'blast-like' cells which in normal bone marrow are known to possess marrow repopulating ability, and demonstrated reduced clonogenic potential in short-term colony assays. PMID- 1712225 TI - Antiviral effect of interferon: the interferon in viral hepatitis. AB - A study was made of the effects of interferons on the evolution of different forms of viral hepatitis and on the possibility to use them to treat the diseases. A review of researches in this field conducted in Cuba is given. PMID- 1712226 TI - Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor. AB - The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation. PMID- 1712227 TI - The macrophage-colony stimulating factor gene is a growth factor-inducible immediate early gene in fibroblasts. AB - Polypeptide growth factors rapidly induce the expression of a group of genes during the onset of cell proliferation. We report that one of these genes, which is induced by several mitogens in NIH 3T3 cells, is identical to the gene for macrophage-colony stimulating factor (M-CSF). In contrast to other immediate early genes, the expression of the M-CSF gene lasted for several hours. Run-on assays demonstrated that the increased level of M-CSF mRNA following stimulation was mainly due to transcriptional activation. Our results support the notion that the products of the immediate early genes are not all mediators of fibroblasts growth but that some play an important role in other physiological responses such as wound repair. PMID- 1712228 TI - The rise and fall of the RNA world. AB - It is generally believed that there was a time when life on earth was based on RNA rather than on DNA and protein. Considering the relevant evidence from geophysics, geology, paleobiology, and molecular biology, it is possible to set the time frame for the existence of RNA-based life to a 400 million year interval beginning 4.0 to 4.2 billion years ago and ending 3.6 to 3.8 billion years ago. The minimum level of biochemical complexity that existed during this time consists of those functions necessary for the establishment and maintenance of an RNA-based evolving system, namely, an RNA unwinding activity, an RNA replicase activity, and a primitive biosynthetic apparatus leading to enrichment of the local environment with activated D mononucleotides. PMID- 1712229 TI - Modulation of extracellular matrix proteins by endothelial cells undergoing angiogenesis in vitro. AB - Angiogenesis results in part from the response of endothelial cells to the integrated action of morphogenic factors and extracellular matrix proteins. In this study we identified specific components of the extracellular matrix that were modulated in endothelial cells derived from bovine aorta and rat cerebral microvessels, both of which spontaneously form cords and tubes under standard culture conditions. SPARC (secreted protein, acidic and rich in cysteine) was upregulated 4.2-fold in aortic and 10-fold in microvascular cultures that had organized into cords and/or tubes. This Ca(2+)-binding glycoprotein was synthesized primarily by endothelial cells in the process of cord formation. Transcription of type I collagen was initiated in aortic endothelial cells undergoing angiogenesis in vitro and showed a 12-fold increase in similar cultures of microvascular cells. Type VIII collagen protein was upregulated to a lesser degree (4.3-fold in aortic and 1.8-fold in microvascular cells). Dense cytoplasmic staining for these two collagen types was seen in cells directly participating in the organization of cords. In contrast, the disparate levels of fibronectin observed in both types of endothelium indicated an indirect or secondary role for this glycoprotein in cord/tube formation in vitro. These results identify SPARC, type I collagen, and type VIII collagen as extracellular matrix components that are actively synthesized by endothelial cells undergoing angiogenesis in vitro. Moreover, expression of these proteins during the formation of tubes and cords appears to follow a biosynthetic program that is common to endothelial cells from both the macrovasculature and microvasculature. PMID- 1712230 TI - Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli. AB - The primary structure of a multifunctional protein, the large alpha-subunit of the Escherichia coli fatty acid oxidation complex, was determined by sequencing the fadB region of the fadBA operon. The amino-terminal sequence of this protein had been established by Edman degradation. The transcription start site of the fadBA operon was located 42 nucleotides upstream of the initiator codon of the fadB gene by primer extension analysis. Sequences of -10 and -35 regions of the promoter responsible for interaction with RNA polymerase were found to be CACACT and TTTGCA, respectively. The location of the promoter of the fadBA operon was defined, and the transcription direction of this operon, from fadB to fadA, as previously proposed [Yang, S.-Y., et al. (1990) J. Biol. Chem. 265, 10424-10429], was corroborated. The multifunctional protein is composed of 729 amino acid residues and has a calculated Mr of 79,593. A putative NAD-binding beta alpha beta-fold necessary for L-3-hydroxyacyl-CoA dehydrogenase function was found in the central region of the fadB gene product. Sequence analyses suggest that the functional domains of the multifunctional protein are arranged in the order enoyl CoA hydratase:L-3-hydroxyacyl-CoA dehydrogenase: delta 3-cis-delta 2-trans-enoyl CoA isomerase and suggest that the genes of the E. coli multifunctional protein and rat peroxisomal trifunctional beta-oxidation enzyme evolved from a common ancestral gene. PMID- 1712231 TI - Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium rubrum L. AB - Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L. The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes. PMID- 1712232 TI - Radiation inactivation of ion channels formed by gramicidin A. Protection by lipid double bonds and by alpha-tocopherol. AB - The conductance induced by the channel-forming peptide gramicidin A in lipid membranes is reduced by many orders of magnitude on exposure of the membrane and its aqueous environment to ionizing radiation. This results from an interaction of free radicals of water radiolysis with the tryptophan residues of gramicidin A. The sensitivity of the ion channels towards irradiation is strongly reduced in the presence of either vitamin E or of highly unsaturated lipids. An increase of the D37 dose up to a factor of 50 was found. The phenomena are interpreted via a reduction of the effective concentration of free radicals (such as OH.) in the membrane by reaction with unsaturated fatty acid residues or with vitamin E. PMID- 1712233 TI - Isolation of two forms of decay-accelerating factor (DAF) from human urine. AB - Decay-accelerating factor (DAF) was purified from human pooled urine by conventional techniques. The urine DAF was separated into two peaks, pool I and pool II, by gel chromatography. DAF-U1 was isolated from pool I by hydrophobic chromatography, and DAF-U2 from pool II by anti-DAF IgG column. The specific activities of DAF-U1 and DAF-U2 to decay membrane-phase C5 convertase were about 3% and 70% of membrane form DAF, respectively. However, both urine DAFs revealed a similar activity to each other and slightly higher activity than that of membrane form DAF in decay-accelerating fluid-phase C3 convertase of the alternative pathway. PMID- 1712234 TI - The presence of glycoproteins in the cell membrane complex of a variety of keratin fibres. AB - Cell membrane complex preparations have been extracted using formic acid from human hair and nail, and from the hair of sheep, alpaca, rabbit, rat, cat, and dog. On analysis they were found to have similar amino acid compositions and they all contained carbohydrate. The sugars were typical of those found in membrane glycoproteins and all preparations reacted with peroxidase-conjugated lectins. PMID- 1712236 TI - Colony-stimulating factors. PMID- 1712235 TI - Antiproliferative function of glia maturation factor beta. AB - Recombinant human glia maturation factor beta (GMF-beta) reversibly inhibits the proliferation of neoplastic cells in culture by arresting the cells in the G0/G1 phase. This phenomenon is not target-cell specific, as neural and nonneural cells are equally inhibited. When tested simultaneously, GMF-beta suppresses the mitogenic effect of acidic fibroblasts growth factor (aFGF), but the two are synergistic in promoting the morphologic differentiation of cultured astrocytes. GMF-beta also counteracts the growth-stimulating effect of pituitary extract and cholera toxin on Schwann cells. The results underscore the regulatory role of GMF beta and its intricate interaction with the mitogenic growth factors. PMID- 1712237 TI - Effective pore radius of the gramicidin channel. Electrostatic energies of ions calculated by a three-dielectric model. AB - Electrostatic calculation of the gramicidin channel is performed on the basis of a three-dielectric model in which the peptide backbone of the channel is added as a third dielectric region to the conventional two-dielectric channel model (whose pore radius is often referred to as the effective pore radius reff). A basic principle for calculating electrostatic fields in three-dielectric models is introduced. It is shown that the gramicidin channel has no unique value of reff. The reff with respect to the "self-image energy" (i.e., the image energy in the presence of a single ion) is 2.6-2.7 A, slightly depending upon the position of the ion (the least-square value over the whole length of the pore is 2.6 A). In contrast, the reff with respect to the electric potential due to an ion (and hence the reff with respect to the interaction energy between two ions) is dependent upon the distance s of separation; it ranges from 2.6 to greater than 5 A, increasing with an increase in s. However, for the purpose of rough estimation, the reff with respect to the self-image energy can also be used in calculating the electric potential and the interaction energy, because the error introduced by this approximation is an overestimation of the order of 30% at most. It is also shown that the apparent dielectric constant for the interaction between two charges depends markedly upon the positions of the charges. In the course of this study, the dielectric constant and polarizability of the peptide backbone in the beta-sheet structure is estimated to be 10 and 8.22 A3. PMID- 1712238 TI - "Reversed" alamethicin conductance in lipid bilayers. AB - Alamethicin at a concentration of 2 micrograms/ml on one side of a lipid bilayer, formed at the tip of a patch clamp pipette from diphytanoyl phosphatidylcholine and cholesterol (2:1 mol ratio) in aqueous 0.5 M KCl, 5 mM Hepes, pH 7.0, exhibits an asymmetric current-voltage curve, only yielding alamethicin currents when the side to which the peptide has been added is made positive. Below room temperature, however, single alamethicin channels created in such membranes sometimes survive a sudden reversal of the polarity. These "reversed" channels are distinct from transiently observed states displayed as the channel closes after a polarity reversal. Such "reversed" channels can be monitored for periods up to several minutes, during which time we have observed them to fluctuate through more than 20 discrete conductance states. They are convenient for the study of isolated ion-conducting alamethicin aggregates because, after voltage reversal, no subsequent incorporation of additional ion-conducting aggregates takes place. PMID- 1712239 TI - Ionic channels in Langmuir-Blodgett films imaged by a scanning tunneling microscope. AB - The molecular structure of channels formed by gramicidin A in a lipid membrane was imaged by a scanning tunneling microscope operating in air. The mono- and bimolecular films of lipid with gramicidin A were deposited onto a highly oriented pyrolitic graphite substrate by the Langmuir-Blodgett technique. It has been shown that under high concentration gramicidin A molecules can form in lipid films a quasi-regular, densely packed structure. Single gramicidin A molecules were imaged for the first time as well. The cavity of 0.4 +/- 0.05 nm in halfwidth was found on the scanning tunneling microscopy image of the gramicidin A molecule. The results of direct observation obtained by means of scanning tunneling microscope are in good agreement with the known molecular model of gramicidin A. It was shown that gramicidin A molecules can exist in a lipid monolayer as individual molecules or combined into clusters. The results demonstrate that scanning tunneling microscope can be used for high spatial resolution study of ionic channel structure. PMID- 1712240 TI - Small iminium ions block gramicidin channels in lipid bilayers. AB - Guanidinium and acetamidinium, when added to the bathing solution in concentrations of approximately 0.1M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels. PMID- 1712241 TI - Fast atom bombardment mass spectrometric analysis of arginine-containing neuropeptides. AB - A new procedure using fast atom bombardment mass spectrometry for the determination of arginine residues in neuropeptides is described. The technique is based on the modification of the arginine side-group with 1,2 cyclohexanedione. This novel procedure may have a potential use in the study of neuropeptides and their processing and degrading enzymes. PMID- 1712242 TI - Differences in the leucine aminopeptidase activity in extracts from human prostatic carcinoma and benign prostatic hyperplasia. AB - Extracts of tissue showed that prostatic carcinomas contain less leucine aminopeptidase activity than benign prostatic hyperplasia. This is true when activity is expressed as specific activity (P = 0.0033), specific activity/% epithelium (P = 0.0007), activity/wet weight of tissue (P = 0.0028), or activity/wet weight of tissue/% epithelium (P = 0.0005). Almost all histochemically demonstrable activity is located in the epithelium. Enzymatic activities in extracts and in histochemical preparations showed similar differences between carcinoma and benign prostatic hyperplasia and were related (R = -0.38, P = 0.0400) to Gleason's grades. The transurethrally resected prostate cancers studied contained no well-differentiated tumors and a high proportion of poorly differentiated tumors. Histochemical activity is absent in most prostatic carcinomas and decreased in others. This observation is particularly interesting in view of the growing knowledge of tumor suppressor genes. PMID- 1712243 TI - Calculation of bleomycin doses. PMID- 1712244 TI - Human carcinoma cells bind thrombospondin through a Mr 80,000/105,000 receptor. AB - The metastatic 11B squamous carcinoma cell line synthesizes and secretes high levels of the extracellular matrix glycoprotein thrombospondin (TSP) and displays aggressive invasiveness in a nude mouse model forming highly undifferentiated tumors. The importance of adhesion events involving extracellular matrix proteins and the tumorigenic cell surface in metastasis led us to investigate the nature of the 11B cell surface receptor for TSP. Using TSP affinity chromatography, a cell surface complex of molecular weight 80,000 and 105,000 was isolated that appears to function as a receptor for TSP. Binding was specific for the COOH terminal Mr 140,000 fragment of TSP. TSP and the Mr 140,000 fragment competed for the binding of the 125I-labeled Mr 80,000/105,000 cell surface complex to TSP coated microtiter wells in a dose-dependent manner with half-maximal inhibition observed at 16 and 40 micrograms/ml, respectively. In contrast, the NH2-terminal heparin-binding domain did not inhibit binding in a dose-dependent manner. Other extracellular matrix proteins, such as laminin, vitronectin, or type I collagen, were also unable to inhibit the binding of the 125I-labeled Mr 80,000/105,000 cell surface complex to TSP. The specificity of the Mr 80,000/105,000 receptor for the Mr 140,000 fragment of TSP was further confirmed through the use of monoclonal antibodies. Monoclonal antibody C6.7 specific for the distal COOH terminus of TSP, but not monoclonal antibody A2.5 specific for the heparin binding domain, inhibited binding. Binding was observed to be strongly Ca2+ dependent, slightly Mg2+ dependent, and independent of Mn2+. Immunoprecipitation analyses demonstrated no apparent cross-reactivity between the Mr 80,000/105,000 TSP receptor and members of the beta 1 or beta 3 integrin receptor families. Additionally, V-8 protease mapping demonstrated that the Mr 80,000 and 105,000 polypeptide bands are not related to each other through proteolytic processing. This initial identification and characterization of a carcinoma cell TSP receptor should allow a more detailed examination of the role of TSP in metastatic adhesion and motility. PMID- 1712245 TI - Transduction and expression of the human carcinoembryonic antigen gene in a murine colon carcinoma cell line. AB - A cell line derived from the mouse colon adenocarcinoma, MC-38, has been transduced with a retroviral construct containing complementary DNA encoding the human carcinoembryonic antigen (CEA) gene. MC-38, which forms tumors in syngeneic C57BL/6 mice, has been extensively studied as a target for active immunotherapy. Individual transduced clones that express high levels of cell surface CEA were isolated, and two clones, termed MC-38-ceal and MC-38-cea2, were extensively characterized. The levels of CEA found on the surface of these clones were considerably higher than that found in a moderately differentiated human colon carcinoma cell line (WiDr) and were comparable to those found on the human colon carcinoma cell lines GEO and CBS (among the highest CEA-expressing cells reported). Further analysis demonstrated that the CEA expressed in the MC-38-cea1 clone had a similar molecular weight to native CEA (Mr 180,000), but the MC-38 cea2 cell line expressed a single Mr 70,000 glycosylated immunoreactive product. Seven anti-CEA monoclonal antibodies were found to react with both clones. The CEA gene present in the MC-38-cea2 clone was partially sequenced and was found to contain a deletion of two of the three repeated domains present in CEA. These results provide a basis for future studies to map immunodominant epitopes of CEA and to develop a syngeneic model system that may aid in the design of reagents and protocols to study active and passive immunotherapy directed against a carcinoma expressing human CEA. PMID- 1712246 TI - Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats. AB - The pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol (PEG-rhG-CSF) and rhG-CSF were studied in male Sprague-Dawley rats. The serum concentration after i.v. administration at a dose of 100 micrograms protein/kg was investigated by a bioassay. The serum rhG-CSF concentration decreased steadily after injection with a terminal half-life of 1.79 h. The PEG-rhG-CSF concentration after injection decreased much more slowly with a half-life of 7.05 h. The slower disappearance of PEG-rhG-CSF resulted in a greater area under the concentration-time curve. The neutrophil count after 100 micrograms of protein/kg of rhG-CSF administration reached a peak 12 h after injection and returned to the control level 48 h after injection. The neutrophil count after 100 micrograms of protein/kg of PEG-rhG-CSF administration was identical to that of rhG-CSF after 12 h but the highest level was maintained for 24 to 72 h after injection and returned to the control level after 168 h. These data indicated that PEG-rhG-CSF administration exerted a sustained biological effect on peripheral blood neutrophils. It is expected that PEG-rhG-CSF may contribute greatly to human G-CSF treatment because it has a prolonged neutrophil proliferating activity enabling fewer administrations. PMID- 1712247 TI - Cellular pharmacology of cyclopentenyl cytosine in Molt-4 lymphoblasts. AB - The toxicity, uptake, and metabolism of the oncolytic nucleoside cyclopentenyl cytosine (CPEC) have been examined in the Molt-4 line of human lymphoblasts. This compound is known to be converted to its 5'-triphosphate, which inhibits CTP synthetase and depletes the pools of cytidine nucleotides. In the Molt-4 system, the concentration of drug reducing proliferation by 50% in a 24-h incubation was between 50 and 100 nM. Cytidine, uridine, and nitrobenzylthioinosine almost fully prevented the cytotoxicity of CPEC when introduced shortly before or together with the drug, but only cytidine was effective as an antidote when added 12 h after 200 nM CPEC. Studies of the cellular entry of CPEC revealed that nitrobenzylthioinosine fully blocked this process over a 60-s interval and for as long as 2 h, suggesting that the initial interiorization was mediated by facilitated diffusion. In Molt-4 cells incubated with tritiated CPEC, 9 metabolites could be distinguished: prominent among these was cyclopentenyl uridine (CPEU), the deamination product of CPEC; other major metabolites included the 5'-mono-, di-, and triphosphates of CPEC, and of CPEU, along with two phosphodiesters provisionally identified as CPEC-diphosphate choline and CPEC diphosphate ethanolamine. When the accumulation of CPEC-5'-triphosphate was measured as a function of concentration of the drug in the medium, the process was found not to be saturable by levels of CPEC up to 1000 nM. In cells incubated with 200 nM drug, CPEC-5'-triphosphate accumulated rapidly and linearly for approximately 4 h, the time for doubling of the concentration being 2 h. After a 16-h incubation with 100 nM CPEC, the concentration of CPEC-5'-triphosphate was 50-fold that of the parent drug in the medium and could be readily monitored spectrophotometrically in high-pressure liquid chromatography effluents without recourse to radiolabeled nucleoside. In 2-h incubations, the concentration of free CPEC required to reduce CTP by 50% was 150 nM; this corresponded to a CPEC 5'-triphosphate level of 750 nM. After washout of extracellular CPEC, CPEC-5' triphosphate decayed with a half-life that ranged from 9 to 14 h. Twenty-four h after washout of 200 nM CPEC (the concentration of drug reducing proliferation by 80%), cells had not resumed proliferation, and CTP pools were still depressed by 90%. Cytidine, uridine, and nitrobenzylthioinosine all strongly repressed the anabolic phosphorylation of CPEC when added to Molt-4 cells along with the drug.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712248 TI - Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP. AB - Prostate-specific antigen (PSA) is a member of the kallikrein gene family and is expressed exclusively in human prostatic epithelial cells. PSA protein has been an important biological marker for prostate cancers. Until now, very little was known about the regulation of PSA expression in prostatic cells. In this study, we have developed a specific oligonucleotide probe which recognizes PSA but not the human glandular kallikrein. This is crucial because both PSA and human glandular kallikrein are expressed in the prostate at relatively high levels and have high nucleotide sequence homology (greater than 82%). Utilizing a S-labeled PSA-specific probe, PSA mRNA was localized within the glandular epithelium of the prostate. Northern blot analysis detected a single 1.6-kilobase transcript in LNCaP cells, a cell line derived from a human prostate adenocarcinoma metastasis. Therefore, LNCaP cells were used to study the androgenic effects on PSA mRNA expression. A time course study demonstrated that PSA mRNA was induced by mibolerone (a nonmetabolizable synthetic androgen) and reached maximal levels after 9 h. The induction of PSA mRNA required as little as 0.3 nM mibolerone. In addition to mibolerone, PSA mRNA could be induced by the natural androgen, dihydrotestosterone, but not by the synthetic glucocorticoid, dexamethasone, or the synthetic estrogen, diethylstilbestrol. Moreover, in the presence of dihydrotestosterone, PSA mRNA was depressed by hydroxyflutamide (an antiandrogen). These results suggest strongly that the androgenic effects on PSA mRNA in LNCaP cells may be via the function of the androgen receptor. PMID- 1712249 TI - Acceleration of human prostate cancer growth in vivo by factors produced by prostate and bone fibroblasts. AB - Prostate cancer, the most prevalent cancer affecting men, frequently metastasizes to the axial skeleton where it produces osteoblastic lesions with growth rates often exceeding that of the primary tumor. To evaluate the role of tumor cell host stromal interaction and stromal specific growth factors (GFs) in prostate cancer growth and progression, we coinoculated athymic mice with human prostate cancer cells (LNCaP) and various nontumorigenic fibroblasts s.c. LNCaP tumor formation was most consistently induced by human bone (MS) fibroblasts (62%), followed by embryonic rat urogenital sinus mesenchymal (rUGM) cells (31%) and Noble rat prostatic fibroblasts (17%), but not by NIH-3T3, normal rat kidney, or human lung CCD16 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. The human prostate component of these tumors was confirmed with immunohistochemical staining for prostate-specific antigen (PSA), Northern analysis for PSA expression, and Southern analysis for human repetitive Alu sequences. Elevations in serum PSA paralleled the histomorphological and biochemical findings. LNCaP and fibroblast cell conditioned media (CM) was used to determine whether autocrine and paracrine mitogenic pathways exist between LNCaP and fibroblast cells in vitro, and various defined GFs were tested to identify possible active factors. Mitogenic assays revealed a 200-300% bidirectional stimulation between LNCaP and bone or prostate fibroblast-derived CM. Lung, normal rat kidney, and 3T3 fibroblast CM were not mitogenic for LNCaP cells. Among the purified GFs tested basic fibroblast growth factor (bFGF) was the most potent mitogen, stimulating LNCaP growth 180% in a concentration-dependent manner. Transforming growth factor alpha and epidermal growth factor were both minimally mitogenic. Coinoculation of LNCaP cells with a slowly absorbed matrix (Gelfoam) absorbed with bFGF or dialyzed and concentrated rUGM or MS CM was also capable of inducing LNCaP tumor formation in vivo. These observations illustrate that fibroblasts differentially modulate prostate cancer growth through the release of paracrine-mediated GFs, possibly including bFGF, and that tumor-stromal cell interactions play an important role in prostate cancer growth and progression. PMID- 1712250 TI - Human bronchial epithelial cells transformed by the c-raf-1 and c-myc protooncogenes induce multidifferentiated carcinomas in nude mice: a model for lung carcinogenesis. AB - We have previously described the neoplastic transformation of immortalized human bronchial epithelial cells (BEAS-2B) by the combination of the c-raf-1 and c-myc protooncogenes and the concomitant induction of neuron-specific enolase mRNA expression (A. Pfeifer et al., Proc. Natl. Acad. Sci. USA, 86: 10075-10079, 1989). In this paper we describe the morphological, biochemical, and immunohistochemical characteristics of the primary c-raf-1/c-myc tumors, xenografts of these tumors, and tumors that originated from cell lines of the primary neoplasm. The tumors were morphologically characterized by the appearance of desmosomes and tonofilaments, microvilli, and dense core granules representing markers of squamous, glandular, and neuroendocrine differentiation, respectively. A total of 11 of 13 tumors were positive by immunohistochemical techniques for neuron-specific enolase, serotonin (nine of 13), and calcitonin (six of 13). Keratins were expressed in 11 of 13 tumors, and while specific keratins (K5, K7, K16/K17) decreased, there was an increase of vimentin in the tumor cells. Gastrin releasing peptide immunoreactivity was detectable in a small number of tumors (five of 13). BEAS-2B cells transfected with the c-raf-1 and c-myc protooncogenes and cell lines established from the primary tumors expressed major histocompatibility Class II antigen which has been found on small cell lung carcinoma cells. The tumors induced by the c-raf-1 and c-myc protooncogenes resemble the multidifferentiated phenotype of small cell lung cancer frequently detected in vivo and present a defined model to study the relation between molecular markers, phenotypical appearance, and response to chemotherapeutic agents and radiation. PMID- 1712251 TI - A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage specific epitope required for invasion of mammalian cells. AB - When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition. PMID- 1712252 TI - The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo. AB - We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1 type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth responsive control of plasmid replication and copy number. PMID- 1712253 TI - Kinetics and requirements for activation of macrophages for fungicidal activity: effect of protein synthesis inhibitors and immunosuppressants on activation and fungicidal mechanism. AB - Peritoneal-and pulmonary macrophages can be activated in vitro with lymphokines (LK) or IFN-gamma, without exogenous lipopolysaccharide, for fungicidal activity against several pathogenic fungi. However, neither the biochemical nor metabolic events of the activation process or of the effector phase have been defined. In the present work we sought to elucidate these events with time-course studies using inhibitors of protein synthesis as well as immunosuppressive agents. We found that protein synthesis inhibitors abrogated the activation process, because cycloheximide (CHX) (1-2 micrograms/ml) prevented activation of macrophages for fungicidal activity against Candida albicans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. Blocking of the activation process by CHX was not due to macrophage cytotoxicity, and CHX did not impair the ability of nonactivated macrophages to kill Candida parapsilosis. In kinetic studies we showed that activation of macrophages was induced in 4 hr of LK treatment and that CHX had no effect if added after this time. In contrast to CHX, therapeutic concentrations of hydrocortisone (HC), such as less than or equal to 5 micrograms/ml, or cyclosporin A (CsA), 5 micrograms/ml, did not significantly inhibit LK activation of macrophages for killing of fungi. In the effector phase, the fungicidal capacity of activated macrophages in short-term (less than or equal to 4 hr) killing assays could not be abrogated by CHX (5 micrograms/ml), HC (100 micrograms/ml), or CsA (10 micrograms/ml). These results demonstrate that the activation but not the effector mechanism of macrophages for fungicidal activity is blocked by inhibition of protein synthesis. In contrast, therapeutic concentrations of HC or CsA may not interfere with activation of macrophages or their killing mechanisms, thus providing a rationale for antifungal immunotherapy in certain clinical situations (e.g., infection in the immunosuppressed patient). PMID- 1712254 TI - Analysis of polyadenylated RNA from human heart mitochondria. AB - The content of the mitochondrial poly(A)-RNA fraction of human heart has been analyzed by electrophoresis through agarose slab gels in the presence of methyl mercury hydroxide and staining with ethidium bromide. It is possible to identify all the heavy strand coded mRNAs in the electrophoretic pattern. This pattern is qualitatively similar to that obtained from the mitochondria of cells cultured in vitro (HeLa cells), although differences in the relative amount of some components have been detected. PMID- 1712255 TI - [Demonstration of intrahepatic HBsAg and HBcAg in chronic liver disease by double staining]. AB - Expression and distribution of intrahepatic HBsAg and HBcAG were studied by double staining immunohistochemical assay in chronic liver disease. HBsAg was homogenously cytoplasmic in typing in 41 and HBcAg was cytoplasmic-membranous in 31 of 46 cases which were known to be positive for both HBsAg and HBcAg intrahepatically. The expressing sites of both HBsAg and HBcAg in 40 of the 46 cases were found to be positive in the same areas of the sections. Interestingly, it was found that intrahepatic HBsAg and HBcAg were often coexistent in the same hepatocyte, and these hepatocytes were considered to be further classifiable into type I and type II according to their distribution. The detective rate of type II cell was significantly higher during the HBV replication stage serologically. HBsAg and HBcAg coexpression in cytoplasm of hepatocyte was noticed to be closely related to active pathological change of the liver cells. PMID- 1712256 TI - [Immunohistochemical demonstration of neurohormonal polypeptides in primary carcinoid tumor of testis]. AB - A primary carcinoid tumor of testis was studied. The tumor cells showed a strong positive reaction to argyrophil or argentaffin stainings, and neuroendocrine granules were identified by electron microscopy. Immunohistochemically, tumor cells expressed various markers such as those for NSE, synaptophysin, CG, Leu-7, 5-HT, HCG, cytokeratin, EMA, CEA and PACP, which indicated the special multiple directions of differentiation of cells possessing neuroendocrinal, epithelial or carcinoembryonic behavior. PMID- 1712257 TI - [A preliminary study on substance P and vasoactive intestinal polypeptide contents in intestinal mucosa of chronic diarrhea with splenic weakness]. AB - By means of radioimmunoassay, the mucosal substance P (SP) and vasoactive intestinal polypeptide (VIP) concentration in distal ileum, transverse colon and sigmoid colon in 30 patients of chronic diarrhea with splenic weakness was determined and compared with that in 28 patients of chronic diarrhea without splenic weakness and in 15 controls without chronic diarrhea. In patients of chronic diarrhea with splenic weakness, the SP contents in the mucosa of distal ileum (120.95 +/- 90.70 pg/mg wet weight) was significantly increased compared with that in controls without diarrhea (47.86 +/- 35.49 pg/mg wet weight) and in chronic diarrhea without splenic weakness (52.50 +/- 42.49 pg/mg wet weight), P less than 0.01 and 0.01 less than P less than 0.05 respectively. The VIP contents of sigmoid mucosa in patients of chronic diarrhea with splenic weakness (510.63 +/- 265.22 pg/mg wet weight) was markedly increased in comparison of that in controls without chronic diarrhea (308.67 +/- 204.49 pg/mg wet weight), 0.01 less than P less than 0.05, and was not significantly augmented compared with that in chronic diarrhea without splenic weakness (398.97 +/- 240.80 pg/mg wet weight), but the tendency of increase was present. Our results suggested that the increased SP and VIP in patients of chronic diarrhea with splenic weakness might be closely related to the symptom of chronic diarrhea. According to the general function of VIP, the authors predicted that VIP might play a more important role in the pathogenetic action of chronic diarrhea with splenic weakness. PMID- 1712258 TI - Idiopathic orthostatic hypotension, midodrine, and anaesthesia. AB - A patient with idiopathic orthostatic hypotension receiving chronic oral midodrine therapy required anaesthesia for coronary artery bypass grafting. A perioperative infusion of phenylephrine was substituted for midodrine, an alpha-2 agonist, enabling hypotension resulting from low systemic vascular resistance to be controlled easily. Anticipated adrenergic receptor denervation hypersensitivity was noted. The only significant perioperative problem was one episode of syncope from orthostatic hypotension during the reambulation period. PMID- 1712259 TI - Stimulus-secretion coupling of arginine-induced insulin release: significance of changes in extracellular and intracellular pH. AB - The possible relevance of changes in extracellular and/or intracellular pH to the insulinotropic action of L-arginine and L-homoarginine was investigated in rat pancreatic islets. A rise in extracellular pH from 7.0 to 7.4 and 7.8 augmented the secretory response to these cationic amino acids whilst failing to affect the uptake of L-arginine by islet cells and whilst decreasing the release of insulin evoked by D-glucose. Under these conditions, a qualified dissociation was also observed between secretory data and 45Ca net uptake. Moreover, at high extracellular pH, the homoarginine-induced increase in 86Rb outflow from prelabelled islets rapidly faded out, despite sustained stimulation of insulin release. The cationic amino acids failed to affect the intracellular pH of islet cells, whether in the absence or presence of D-glucose and whether at normal or abnormal extracellular pH. These findings argue against the view that the secretory response to L-arginine would be related to either a change in cytosolic pH or the accumulation of this positively charged amino acid in the beta-cell. Nevertheless, they suggest that the yet unidentified target for L-arginine and its non-metabolized analogue in islet cells displays pH-dependency with optimal responsiveness at alkaline pH. PMID- 1712260 TI - Pentafraction-Du Pont versus albumin for resuscitation of a lethal intestinal ischemic shock in rats. AB - This study compares the effect of pentafraction-Du Pont 6% (PF), albumin 5% (ALB), and Ringer's lactate (RL) on plasma volume (PV) expansion and survival in a lethal intestinal ischemic shock model. Shock was induced by exteriorizing the small intestines and occluding the mesenteric vessels for 75 min. Changes in PV were estimated using hematocrit (Hct). The solutions were administered continuously for 6 hr in volumes to maintain a stable Hct, or 15 ml/100 g body weight (bwt) for PF and ALB and 44 ml/100 g bwt for RL. Hct and bwt were measured hourly during the infusion and at 24 h. Untreated animals in shock developed hemoconcentration (Hct 62%) corresponding to a PV of 41% of baseline preshock level, with 9% (3/35) of the animals surviving 72 hr. RL expanded PV to 87% of preshock level, with a 57% (20/35) 72 hr survival rate and 46% (16/35) surviving greater than 7 days. Only 34% as much ALB was needed to induce a greater PV expansion of 101% with a 72 hr survival rate of 51% (18/35) and 46% (16/35) surviving greater than 7 days. When PF was used, PV expanded to 109% of preshock level with survival rates improving to 80% (28/35) at 72 hr and 71% (25/35) greater than 7 days compared to RL and ALB (P less than 0.05). There were no significant differences in survival rates between RL and ALB treated animals. We conclude that PF improves survival following intestinal ischemic shock compared to ALB and RL. PF is a safe and effective alternative to albumin for resuscitation in this shock model. PMID- 1712261 TI - [Does circumscribed ischemic white matter lesion produce neuropsychological symptoms?]. AB - In order to determine a possible role of ischemic white matter lesion in producing neuropsychological symptoms, we conducted a retrospective survey. Subjects were 206 consecutive patients who had been admitted to our neurology service under the diagnosis of ischemic stroke from April 1988 to November 1989 and had received brain CT as well as SPECT scans. From this cohort only patients that had circumscribed white matter lesion on CT scans were selected. Patients who had lesion in basal ganglia or thalamus were rigidly excluded. Eight subjects (3.9%) fulfilled above criteria, i.e. 5 with left hemispheric, 2 with right hemispheric and 1 with bilateral white matter lesions. Then we supplemented MRI information which had happened to be available for all the eight subjects. Exclusion of grey matter lesion based on MRI reduced the number of cases with exclusive white matter lesion into 4, i.e. 3 with left hemispheric and 1 with right hemispheric lesion. Of these, only one case with a discrete lesion in the genu of the left internal capsule had manifested mild neuropsychological impairments in naming, comprehension and writing. I123 amphetamine SPECT study revealed low uptake in the anterior half of the left middle cerebral artery in this case. We conclude that the role of a circumscribed white matter lesion as a cause of neuropsychological symptom seems to be much less clear than has been postulated. PMID- 1712262 TI - Children with Apert syndrome: developmental and psychologic considerations. AB - Innovations in craniofacial surgery have enabled children with Apert syndrome to achieve their full potential by maximizing their opportunities for intellectual growth, physical competence, and social acceptance. However, at each developmental phase they are confronted with different challenges to their emotional and social adaptation that must be overcome. Children with Apert syndrome continue to face erroneous assumptions of mental retardation and social stigmatization because of their appearance. Families and medical care providers play a critical role in fostering their adjustment and supporting them during these emotionally stressful periods. Most importantly, by understanding the unique issues of each developmental period, parents and professionals also have the capacity to maximize their opportunity for psychologic well-being. PMID- 1712263 TI - Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry. AB - Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from true B-cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non-specific binding of isotypic control mouse Ig and non-immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas. PMID- 1712264 TI - The effect of improvements in cytometer sensitivity on the detection of CD5 positive B cells with dim fluorescence. AB - When antigen density on the surface of a cell population is low and variable, the percentage of that population determined to express the antigen (i.e., to be positively stained) depends directly on the sensitivity of the flow cytometer for resolving particles which are dimly fluorescent from those which are unstained. In this study, the sensitivity of a commercial flow cytometer has been improved by changes in the photomultiplier tube, the fluorescence filter, and the amount of stray light entering the fluorescence channel. In a model system with human lymphocytes, modifications to these factors increased the percent of the B lymphocyte population found to express the CD5 antigen. PMID- 1712265 TI - [The egg--symbol and myth]. AB - The egg is one of the most ancient symbols in mankind. This is exemplified on the basis of various myths--cosmogony, theogony, magic witchcraft for healing and defence--originating from different pre-Christian and Christian cultures. PMID- 1712266 TI - Clinical pharmacodynamic studies with cilazapril and a combination of cilazapril and propranolol. AB - We evaluated the degree of inhibition of angiotensin converting enzyme (ACE), principally by cilazapril, by assessing the blood pressure response to a continuous infusion of increasing doses of angiotensin I, and assessed the possible pharmacokinetic and pharmacodynamic interactions between cilazapril and propranolol in healthy volunteers and patients with mild to severe essential hypertension. The specificity of the angiotensin I infusion method was verified when a single dose of cilazapril 30mg antagonised the increase in diastolic blood pressure induced by angiotensin I but did not influence the response to angiotensin II. Using this method, we showed that single oral doses of cilazapril 4 mg, captopril 25 mg and enalapril 10mg shifted the angiotensin I dose-effect curve to the right and determined a pharmacological half-life of about 4 hours for cilazapril. Increasing single oral doses (1.25, 3.75, 10 and 30mg) of cilazapril reduced diastolic blood pressure dose-dependently and shifted the angiotensin I dose-response curves to the right. The dose representing 50% inhibition of ACE activity (apparent Ki-dose) was about 0.6mg, 3 hours after cilazapril administration. Cilazapril and propranolol did not exhibit any clinically significant pharmacokinetic interaction in healthy volunteers; each drug reduced diastolic and systolic blood pressure by about 7 mm Hg, and this was doubled by the combination. Cilazapril had no significant effect on heart rate, in patients with essential hypertension whereas both propranolol and the combination of cilazapril and propranolol reduced it. Monotherapy with each drug reduced blood pressure, and combined administration enhanced the antihypertensive effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712267 TI - Cilazapril. A review. AB - Cilazapril is a new nonthiol group containing angiotensin converting enzyme (ACE) inhibitor, which was designed by a computer-modelling technique in order to obtain a compound with high specificity and selectivity for the target enzyme. Cilazapril has been investigated in more than 4000 patients with all degrees of hypertension, as well as in the special patient groups such as the elderly, renally impaired, and patients with concomitant diseases, such as congestive cardiac failure or chronic obstructive pulmonary disease. In these studies, the blood pressure-lowering effect of a single daily dose has been clearly established. The tolerability profile is similar to other frequently prescribed antihypertensive drugs, such as sustained-release propranolol, enalapril, captopril, atenolol and hydrochlorothiazide. Recently, investigations have revealed that cilazapril, in addition to its blood pressure-lowering abilities, can moderate the proliferative response seen in vessels after vascular injury caused by techniques such as ballooning. Clinical studies to verify these findings are currently ongoing. PMID- 1712268 TI - Effects of ACE inhibition on renal haemodynamics in essential hypertension and hypertension associated with chronic renal failure. AB - Angiotensin II has many actions in the kidney, including regulation and distribution of renal circulation and glomerular filtration, as well as effects on mesangial contraction and on the filtration coefficient. The reduction in circulating and intrarenal angiotensin II by angiotensin converting enzyme (ACE) inhibitors in essential hypertension is associated with a significant increase in renal blood flow and a decrease in filtration fraction, without changes in glomerular filtration rate. In addition, administration of ACE inhibitors can reduce proximal sodium reabsorption via changes in peritubular hydrostatic and oncotic forces resulting from the fall in postglomerular capillary resistance. In severe hypertension the state of the renal vasculature does not allow ACE inhibition to induce similar haemodynamic changes and, therefore, it cannot contribute to renal sodium handling that requires the recruitment of alternate mechanisms. In spite of this, ACE inhibitors may exert a protective effect on the renal function of patients with severe hypertension as well as in those with renal impairment, by lowering systemic and, probably, intraglomerular pressure, reducing proteinuria and slowing the progression of renal failure. PMID- 1712269 TI - Clinical pharmacology of cilazapril. AB - In clinical pharmacology studies, cilazapril, after its bioactivation to cilazaprilat, was characterised as a potent, reversible angiotensin converting enzyme (ACE) inhibitor with a terminal half-life of 30 to 50 hours, which is consistent with saturable binding to ACE. Despite the arterial vasodilatation, only slight increases in heart rate occurred during cilazapril administration. Cilazapril had no acute effect on cardiovascular reflexes, and increased effective renal plasma flow slightly. Glomerular filtration rate remained unaltered. A close positive correlation was found between the cilazaprilat plasma concentration and degree of ACE inhibition. The potency of cilazaprit, defined as the concentration of cilazaprilat causing 50% inhibition of ACE, was approximately 1 microgram/L plasma. In short term studies in patients with hypertension, it appeared that more than 90% inhibition of plasma ACE was needed to obtain blood pressure reduction. Results of various dose-response studies established the indirect relationship between dose, the plasma concentration of the drug, and the blood pressure response, and identified the dose producing the maximal effect to be 5mg. Cilazapril inhibited ACE for a relatively long period which was extended in patients with severe chronic renal impairment or hepatic failure. In these patients a reduction of the dose and/or less frequent administration is recommended. There was no clinically relevant interaction of cilazapril with food, furosemide (frusemide), digoxin or coumarins. The effects of hydrochlorothiazide on sodium and chloride excretion were potentiated by cilazapril, and an additive effect of propranolol and nitrendipine on the blood pressure response to cilazapril was observed. An interaction with indomethacin and cilazapril might occur, potentially reducing the blood pressure-lowering effect of cilazapril. In general, cilazapril was well tolerated. PMID- 1712270 TI - Antihypertensive duration of action of cilazapril in patients with mild to moderate essential hypertension. AB - The efficacy of cilazapril monotherapy was evaluated in 2 multicentre double blind dose-response trials. After 4 weeks of a single-blind placebo run-in period, patients with uncomplicated mild to moderate essential hypertension and a sitting diastolic blood pressure of 100 to 115 mm Hg, 24 hours after the last placebo dose (trough), were randomised to take either placebo or cilazapril 2.5 mg or 5 mg for 4 weeks (study 1, 86 patients) or 8 weeks (study 2, 78 patients). Sitting diastolic blood pressure was checked every 2 weeks at trough in both studies and at peak in study 2. The reductions in sitting diastolic blood pressure from baseline at trough, and the difference from placebo, were clinically and statistically significant for both cilazapril groups in the 2 studies. The reduction in blood pressure in both active treatment groups was similar, but the response rate with cilazapril 5 mg was greater than that with 2.5 mg. More than 50% of the peak effect was still present at trough for both cilazapril groups. It is concluded that both dosages of cilazapril are effective and reduce blood pressure compared with placebo over a 24-hour period. PMID- 1712271 TI - First experience with cilazapril in the treatment of sleep apnoea-related hypertension. AB - Sleep-related breathing disorders are associated with considerably impaired vitality and reduced life expectancy. In this respect, a particularly important role is played by obstructive snoring and obstructive or mixed sleep apnoea. Because of the often underestimated prevalence of sleep-related breathing disorders and their association with hypertension in greater than 50% of patients, it is important to introduce antihypertensive drug therapy that does not exacerbate the effects and the degree of these disorders, and that above all produces an adequate lowering of blood pressure by night. In the present study in 12 patients, it has been shown that the new ACE inhibitor, cilazapril, achieves a good reduction in blood pressure over 24 hours and during all stages of sleep, without any negative influence on the ratio of REM: nonREM sleep. The apnoea index was reduced from 40 (range 12 to 84) to 27 (range 0 to 72) apnoeas per hour of sleep (p less than 0.01). It will be increasingly important to take into account the effects of antihypertensive therapy on other clinical parameters, especially the nocturnal blood pressure profile and its association with sleep related breathing disorders. PMID- 1712272 TI - Enalaprilat, but not cilazaprilat, increases inflammatory skin reactions in guinea-pigs. AB - The inflammatory effects of enalaprilat and cilazaprilat were tested in an experimental model of ovalbumin-sensitised guinea-pigs. Enalaprilat, but not cilazaprilat, enhanced the ovalbumin-induced inflammatory skin responses. The effect of enalaprilat was dose-dependent. Enalaprilat significantly increased the skin content of substance P and histamine. Cilazaprilat did not alter the level of these inflammatory mediators. Enalaprilat, applied locally, but not cilazaprilat, enhanced the inflammatory reactions caused by intradermal injections of allergen and substance P. Both angiotensin converting enzyme (ACE) inhibitors enhanced the inflammatory skin response evoked by bradykinin. Our study strongly indicates that enalaprilat has pro-inflammatory properties, whereas the new long-acting ACE inhibitor cilazaprilat does not. This might give a better safety profile of cilazaprilat. PMID- 1712274 TI - Vascular protection with cilazapril. AB - The hypertrophy of the media of coronary arteries associated with hypertension reduces cross-sectional area and limits vascular reserve. Cilazapril 10 mg/kg daily decreased cardiac hypertrophy, and decreased minimal coronary vascular resistance by 40% when administered to spontaneously hypertensive rats (SHR) at the onset of hypertension. After hypertension had developed, cilazapril restored arterial pressure to normal and increased the maximal coronary blood flow in isolated perfused hearts by 96%, which was probably a result of a marked decrease in medial hypertrophy of the coronary arteries. Similarly, cilazapril improved cerebral vascular reserve in the mesenteric and renal arteries of SHR. In the rat model of vascular injury produced by ballooning, cilazapril 10 mg/kg daily demonstrated a marked preventive effect on the myointimal proliferation that resulted in untreated controls, a phenomenon responsible for restenosis in humans after arterial angioplasty. Although this effect occurred with usual antihypertensive dosages in rats, it appeared to be independent of the decrease in arterial pressure since effective antihypertensive dosages of verapamil did not prevent neointima formation. In view of the clinical potential for preventing restenosis after coronary angioplasty, 2 multicentre trials of cilazapril are ongoing to test this hypothesis. PMID- 1712273 TI - Cilazapril in congestive heart failure. A pilot study. AB - The haemodynamic effects of a single dose of cilazapril 2.5 or 5 mg were studied in 33 patients with stable chronic congestive heart failure who were receiving digitalis and diuretics. Subsequently, a double-blind comparison of the haemodynamic and clinical effects of 3 months' treatment with cilazapril 1.25 to 5 mg daily or placebo in 24 evaluable patients revealed that the acute haemodynamic improvement produced by a single dose of cilazapril was maintained in patients receiving repeated administration of the drug, but not in those randomly allocated the placebo. Acute cilazapril significantly decreased mean arterial pressure, systemic vascular resistance, pulmonary capillary wedge pressure, pulmonary artery pressure and right atrial pressure, while cardiac index and stroke volume index increased at rest and during submaximal exercise. After 3 months' treatment 11 of 13 cilazapril recipients improved their New York Heart Association (NYHA) class compared with 2 of 11 patients treated with placebo. This functional improvement was paralleled by a patient-perceived improvement in general well-being. PMID- 1712275 TI - Relative biochemical reactivity of three hexachlorocyclohexane isomers. AB - The rate and mechanisms of degradation of three hexachlorocyclohexane (HCH) isomers were studied with rat liver cytochrome P450, which is known to react with chlorinated compounds in the presence and absence of molecular oxygen. In addition, P450 is produced from HCH metabolites which are found aerobically and anaerobically during microbial biodegradation of this compound in a complex soil system. Conversion assays with P450 were carried out with alpha-, beta-, and gamma-HCH. Hexachloroethane served as a reference compound for bioconversion kinetics. Under anaerobic conditions, gamma-HCH was readily dechlorinated (r0 = 0.31 nmol/min.nmol cyt.P450) to tetrachlorocyclohexene and monochlorobenzene. gamma-HCH was also reductively dechlorinated, but at a much slower rate (r0 = 0.03 nmol/min.nmol cyt.P450). Both substrates followed Michaelis-Menten kinetics (gamma-HCH: Vmax = 1.87 nmol/min.mg and Km = 47 mumol/liter; gamma-HCH: Vmax = 0.24 nmol/min.mg and Km = 201 mumol/liter). Under aerobic conditions, bioconversions of gamma-HCH (r0 = 0.05 nmol/min.nmol cyt. P450) and alpha-HCH (r0 smaller than detection limit) were slower. beta-HCH proved to be recalcitrant under both aerobic and anaerobic conditions. The differences in bioconversion rates between the three HCH isomers could be related to the differences in spatial chlorine configuration of the three isomers. beta-HCH recalcitrance was explained by the absence of axial-orientated chlorines. PMID- 1712276 TI - The value of sleep recording in evaluating somnambulism in young adults. AB - Somnambulism (SOM) is a benign childhood sleep disorder which may persist until young adulthood. The diagnosis relies heavily on the history, and no polysomnographic (PSG) criteria have yet been defined. The present study attempts to evaluate the role of whole-night polysomnographic recording in the investigation of SOM. The PSG records of 24 sleepwalkers, 18-25 years old, and 12 age-matched controls, were analysed. Sleepwalkers had remarkably more epochs containing hypersynchronous delta waves (HSD) (59.6 +/- 60.1 vs. 1.7 +/- 3.2; P less than 0.0001), a higher proportion of HSD/total time spent in stage 3-4 (24.9 +/- 21.1% vs. 1.1 +/- 2.0%, P less than 0.0002), and more stage 3-4 sleep interruptions (8.4 +/- 5.7 vs. 3.7 +/- 1.7, P less than 0.004). They also tended to have a larger proportion of their sleep time in stage 3-4 (30.6 +/- 11.7% vs. 22.6 +/- 6.8%; P less than 0.07). Although their sensitivity and specificity have yet to be more fully investigated, these seem to be quantitative, easy-to-use variables which may characterize adult SOM and may aid in its proper diagnosis. PMID- 1712277 TI - Different susceptibilities of the geniculate and extrageniculate visual pathways to human Creutzfeldt-Jakob disease (a combined neurophysiological neuropathological study). AB - Flash evoked visual potentials (FEPs) of 7 patients with advanced Creutzfeldt Jakob disease (CJD) were compared with those recorded in 7 patients with senile dementia of the Alzheimer type (SDAT), in 13 age- and sex-matched healthy volunteers and in 7 neuropsychiatrically normal subjects whose occipital evoked responses were increased in amplitude (amplitude controls). Post-mortem examination was performed in 4 of 7 CJD patients in order to map pathological changes along the visual pathways, including the retino-geniculo-striate and extrageniculate pathways. Normal FEPs were typified by 2 constant early components (P1 and N2) followed by several (3 or more) late components that were characterized by marked interindividual variability. Amplitude controls had enlarged (from 14 to 44.8 microV, mean 25.7) P1 component. Both SDAT and CJD patients had normal early FEP waves (P1 and N2) and important alterations of the late FEP components. Moreover, a late positive component was responsible for abnormally enlarged FEPs (52.6 and 58.2 microV) in 2 CJD patients. Finally, electroretinograms, recorded in 1 CJD patient, were normal. These findings suggested relative functional integrity of the retino-geniculo-striate pathway associated with important dysfunction of the cortical visual processing in both SDAT and CJD patients. Pathological studies disclosed preservation of optic nerves, chiasmas, lateral geniculate nuclei and Gennari's strip of the striate cortex but associated with important spongiform change, neuronal loss and gliosis in the superior colliculi (layer II), pulvinar, extrastriate cortex and layers II III, V and VI of the striate cortex. We conclude that different visual pathways have different susceptibilities to CJD: important functional and anatomical alterations of the intracortical and extrageniculate pathways contrast with relative preservation of the retino-geniculo-striate pathway. PMID- 1712278 TI - Event-related potential correlates of the serial position effect in short-term memory. AB - Event-related potential (ERP) correlates of the serial position effect in short term memory were investigated using a memory scanning task. Nine normal young adults (18-39 years) indicated whether a probe item was a member of a previously presented 5-item memory set by pressing 1 of 2 reaction-time buttons. Three types of stimuli were used: verbal digits presented both auditorily and visually, and musical notes presented auditorily. The ERPs to the probes were separately averaged according to the serial position of the probe (1, 2, 3, 4 or 5) in the memory set. The ERPs to the memory set items in positions 1, 3 and 5 also were separately averaged. Both baseline-to-peak and average amplitudes of a late positive parietal potential to the probes were larger to probe items presented in the last position in the memory set than to probes presented in the middle positions (2, 3 and 4), showing a significant recency effect, but only for auditory digits. Reaction time reflected significant recency effects for both auditory digits and notes, but not for visual digits. Response accuracy (percent correct) showed a significant recency effect only for notes. For each stimulus type, both the baseline-to-peak and average amplitudes of a late frontal component to the memory set items became more negative (in the case of the visual digits, less positive) in the third and last serial position of the memory set compared to the first. These findings provide electrophysiological evidence of serial position effects in short-term memory, which, during memory scanning, are dependent on stimulus modality (auditory, visual) and type (verbal, non-verbal). PMID- 1712279 TI - Effects of crossmodal divided attention on late ERP components. I. Simple and choice reaction tasks. AB - We studied several effects of dividing attention between visual and acoustic inputs on different processing stages. Simple and choice responses were required to single letter stimuli. RTs and P300 latencies were delayed for divided attention (variable stimulus modality) as compared to focused attention (constant stimulus modality). In all but one condition, RT and P300 delays were similar. The exception was choice tasks to auditory stimuli, in which the RT delay was far larger than the P300 delay. Since the amplitude of the late ERP was larger in choice tasks than in simple tasks, the differences between the ERPs of choice and simple tasks were computed. They revealed that an additional late positive wave ("P-CR") occurred in all choice ERPs. In the divided attention condition the auditory (but not the visual) P-CR showed a longer delay compared to focused attention. We interpret the P-CR to be time-related to the response selection process. Our results suggest that the division of attention causes a slight impairment of stimulus evaluation (shown in P300 latency) and, after auditory stimuli only, a strong impairment of response selection (shown in P-CR latency). We therefore conclude that the observed RT effects are due to a bias of processing resources towards the visual modality, which mainly affects response selection. The results are in accordance with the theory of visual dominance. PMID- 1712280 TI - Effects of crossmodal divided attention on late ERP components. II. Error processing in choice reaction tasks. AB - Reaction times and event-related potentials in correct and incorrect trials were studied in a bimanual choice reaction task. In a focused attention (FA) condition, the stimulus modality was constant (visual or auditory); in a divided attention (DA) condition, the modality was varied at random from trial to trial. Stimulus- and response-triggered averages were computed from the midline EEG leads. In error trials, the ERP amplitude was reduced in the P300 range (300-500 msec) and enhanced in the slow wave range (500-700 msec) compared to correct reaction trials. Difference plots between the ERPs (incorrect minus correct reaction trials) revealed a large fronto-central negativity ("NE") and a parieto occipital "slow wave." These components appeared larger in the response-triggered averages. We believe that they reflect two different stages of error processing. After auditory stimuli the NE peaked much later for DA than for FA, which supports the idea of an asymmetrical allocation of processing resources to the disadvantage of the auditory modality in our DA condition. PMID- 1712281 TI - Intensity-amplitude relationships in monkey event-related potentials: parallels to human augmenting-reducing responses. AB - In human, the amplitudes of specific event-related potential (ERP) components can increase or decrease in response to increasing stimulus intensity depending on the location of the recording site. Large increases characterize components presumably generated by modality-specific sites, while smaller increases or even decreases are associated with those originating in associational areas. Comparable data from non-human primates, which would permit invasive studies of the neural substrates underlying these intensity-amplitude differences, are limited. To more fully characterize these relationships, auditory ERPs were recorded from chronically implanted epidural electrodes in 5 squirrel monkeys (Saimiri sciureus) in response to tones (500 Hz, 300 msec duration) of varying intensities (50, 60, 70, 80 dB SPL). Squirrel monkey ERPs recorded at Fz exhibited 3 peaks during the 200 msec post-stimulus interval. These peaks included a positivity (P1), followed by a negativity (N1), and then another positivity (P2). At posterior sites, the frontal P1-N1 configuration was recorded as an N1-P1 complex. At these sites, a small negativity (N2) preceded the last positive peak (P2). Changes in polarity were independent of reference site and posterior N1-P1 peaks exhibited latencies similar to those of the frontal P1-N1 components. Amplitudes at Fz, Cz, and Pz increased substantially with increasing stimulus intensity ('augmenting'). In contrast, only small increases or even decreases in amplitude ('reducing') were evident at T3 and T4. On the other hand, peak latencies decreased with higher stimulus intensities at most sites. The site specific amplitude responses exhibited considerable temporal stability. In one subject, for example, similar 'augmenting' profiles were recorded at Fz in 8 sessions over a 6-month period. The topography of monkey intensity-amplitude response profiles, their temporal stability, and peak latency shifts resemble observations made in humans. The data show that 'augmenting' characterizes monkey vertex potentials, which, like the analogous human potentials, may originate in primary auditory cortex. In contrast, potentials recorded over temporal cortex, which may originate in auditory association cortex, exhibit 'reducing.' Thus, the data support the hypothesis that differences in amplitude with increasing intensity may reflect differences in cortical origin. PMID- 1712282 TI - Right hemisphere dominance of different mismatch negativities. AB - Auditory stimulus blocks were presented to 10 reading subjects. Each block consisted of 2 types of stimulus, standard (P = 90%) and deviant (P = 10%), delivered in a random order with short constant inter-stimulus intervals. The standard stimuli were 600 Hz. 80 dB SPL 50 msec sine wave bursts. In different blocks, the deviant stimuli differed from the standards either in frequency (650 Hz), intensity (70 dB) or duration (20 msec). Left- and right-ear stimulations were used in separate blocks. Event-related brain potentials (ERPs) were recorded with 16 electrodes over both hemispheres. All the different types of deviant stimuli elicited an ERP component called the mismatch negativity (MMN). The MMN was larger over the right hemisphere irrespective of the ear stimulated whereas the N1 component, elicited by both standards and deviants, was larger over the hemisphere contralateral to the ear stimulated. The results provide further evidence for the view that the MMN reflects a neural mismatch process with a memory trace which automatically codes the physical features of the repetitive stimuli. PMID- 1712283 TI - Persistence of a P3 component in severe amnestic syndrome. AB - Event-related potentials (ERPs) to an odd-ball paradigm were recorded from 19 scalp derivations in a patient (47 years old, housewife) affected by complete amnestic syndrome following limbic encephalitis. CT scans and magnetic resonance imaging showed severe bilateral lesions of anterior, mid-temporal lobes and frontal lobes. A P3 component, with a peak latency inside normal limits for age matched controls, was recorded to 'target' stimuli from all leads except Fp1, Fp2, F7, F8, T3 and T4. PMID- 1712284 TI - Comments on article by Amassian et al. PMID- 1712285 TI - Guanidino compounds that are increased in hyperargininemia inhibit GABA and glycine responses on mouse neurons in cell culture. AB - The effects of arginine, homoarginine, alpha-keto-delta-guanidinovaleric acid and argininic acid (guanidino compounds that were found to be increased in hyperargininemia) were evaluated on responses to gamma-aminoburtyric acid (GABA) and glycine (Gly) on mouse neurons in primary dissociated cell culture. GABA and Gly were applied iontophoretically and intracellular microelectrode recording techniques were used. The guanidino compounds rapidly and reversibly inhibited both GABA and Gly responses. The guanidino compounds inhibited GABA responses in a concentration-dependent manner and inhibited Gly responses at a concentration of 10 mM. Argininic acid was the most potent in reducing inhibitory amino acid responses, followed in decreasing potency by alpha-keto-delta-guanidinovaleric acid, homoarginine and arginine. The guanidino compounds were equally potent in decreasing Gly and GABA responses. Co-application of CGS 9896, a benzodiazepine receptor antagonist, did not antagonize the guanidino compound-induced inhibition of GABA responses. These findings suggest that the guanidino compounds inhibited responses to the inhibitory neurotransmitters GABA and Gly by blocking the chloride channel. This effect might underlie the in vivo epileptogenicity of some of the guanidino compounds and might contribute to the pathogenesis of seizures in hyperargininemia. PMID- 1712286 TI - Guanidino compound levels in the serum of healthy adults and epileptic patients. AB - Some guanidino compounds are known to be convulsants and to change in the brain during seizures. In this study, we examined the serum levels of guanidino compounds in healthy adults (controls), non-epileptic neurological patients (NENP) and epileptic neurological patients (ENP). In healthy adults, serum levels of guanidinoacetic acid (GAA), creatinine (CRN) and homoarginine (HArg) were significantly lower in women than in men. Serum levels of GAA in ENP and NENP were significantly lower than in controls, with the exception of female NENP. In the male patients, CRN levels were significantly lower in ENP and NENP compared to the controls. Significantly higher arginine (Arg) levels were observed in both male and female ENP and NENP. HArg levels in the male patients were significantly lower in ENP compared with both controls and NENP. With regard to serum levels of guanidino compounds in ENP with symptomatic generalized epilepsy and with symptomatic partial epilepsy, significantly lower levels of HArg were observed in male ENP with symptomatic generalized epilepsy than in NENP. Serum levels of GAA and HArg in uncontrolled female ENP were significantly lower than those in controlled ENP. Furthermore, Arg and HArg levels in uncontrolled male ENP were significantly lower than in controlled ENP. Serum levels of Arg in male ENP and HArg in both sexes of ENP taking valproic acid were significantly lower than those in ENP not taking valproic acid. These results suggest that some metabolic disorder of guanidino compounds may exist in ENP and NENP and that guanidino compounds may be affected by seizure types, seizure severity and anticonvulsants. PMID- 1712287 TI - Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger. AB - A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29 18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger. PMID- 1712288 TI - Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I. AB - The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse-chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described. PMID- 1712289 TI - In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development. AB - Previous studies on mice bearing various mutations within the c-kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c-kit gene and a respectable understanding of the molecular nature of c-kit, however, it is not clear at what stage of gestation c-kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti-c kit antibody, ACK2, as an antagonistic blocker of c-kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid-gestation, c-kit is functionally required during a restricted period around day 14.5 post-coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c-kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair. PMID- 1712290 TI - Structure-activity relationship study of human interleukin-3: role of the C terminal region for biological activity. AB - A structure-activity relationship study of human interleukin-3 (huIL-3) was performed by functional analysis of huIL-3 deletion and substitution variants combined with epitope mapping of huIL-3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL-3 variants was accomplished by defining their capacity to compete with wild-type huIL-3 for binding to the huIL-3 receptor and to induce the proliferation of the huIL-3 dependent cell line M-O7. HuIL-3 variants with either 14 amino acids (aa) deleted from the N-terminus or eight aa from the C-terminus retained full biological activity in vitro. An huIL 3 variant, with 18 N-terminal aa deleted, exhibited a greater than 7-fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C-terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C-terminal region. Computer-aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10-fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL-3 molecule can be constructed with two active sites in close proximity. PMID- 1712291 TI - Molecular requirements for the mu-induced light chain gene rearrangement in pre-B cells. AB - During B cell differentiation rearrangement of immunoglobulin (Ig) genes is partially regulated by the Ig proteins. Rearrangement of heavy (H) chain genes is inhibited, whilst that of light (L) chain genes is induced by the membrane form of the mu H chain. In order to analyse additional structural requirements of mu induced L chain gene rearrangement we transfected wild-type mu and mutant mu constructs lacking functional exons encoding the first or second constant domains into Abelson murine leukemia virus (AMuLV) transformed pre-B cells. All mu chains are expressed on the surface of the pre-B cell and all associate with omega and iota, two proteins forming a surrogate light chain, necessary for mu membrane expression. Nevertheless, only wild-type mu and not the mutant mu proteins promote L gene rearrangement. A heterodimer of proteins with Mr of 33 kd and 36 kd was found associated with wild-type but not with the mutant mu proteins. Continuous presence of mu is required for L chain gene recombination since loss of mu stopped and readdition of mu started L gene rearrangement. We propose that the protein complex composed of mu and the 33 kd/36 kd protein heterodimer is responsible for the activation of the L chain gene locus and its rearrangement. PMID- 1712292 TI - The path of mRNA through the Escherichia coli ribosome; site-directed cross linking of mRNA analogues carrying a photo-reactive label at various points 3' to the decoding site. AB - mRNA analogues approximately 40 bases long were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 3'-side of these coding triplets. The thio U residue was either substituted with 4-azidophenacyl bromide to introduce a photo-reactive group, or was left unsubstituted for direct UV cross-linking. After binding to Escherichia coli 70S ribosomes in the presence of tRNA-Thr or tRNA-Ala, the thio-U residue or azidophenyl group was photo-activated and the products of cross-linking (which was exclusively to the 30S subunit) were analysed. Immunological analysis of the cross-linked proteins showed that S5 and S3, together with S1, were the targets of cross-linking at positions close to the decoding site, with the cross-linking to S3 and S1 persisting at positions further away. Analysis of the 16S RNA showed cross-links to the region of bases 1390-1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross-linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three-dimensional models of the 30S subunit. PMID- 1712293 TI - A functional pseudoknot in 16S ribosomal RNA. AB - Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watson-Crick interactions between these nucleotides are essential for ribosomal function. Moreover, we find that certain mild perturbations of the structure, for example, creation of G-U wobble pairs, generate resistance to streptomycin, an antibiotic known to interfere with the decoding process. Chemical probing of mutant ribosomes from streptomycin resistant cells shows that the mutant ribosomes have a reduced affinity for streptomycin, even though streptomycin is thought to interact with a site on the 30S subunit that is distinct from the 530 region. Data from earlier in vitro assembly studies suggest that the pseudoknot structure is stabilized by ribosomal protein S12, mutations in which have long been known to confer streptomycin resistance and dependence. PMID- 1712294 TI - The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development. AB - As defined by dominant and recessive ovo mutations, the ovo gene is required for development of the Drosophila female germ line, and does not exert any function in males or in somatic tissues. However, reversion of dominant ovo mutations can result in new phenotypes that are not related to the female germ line: the svb and lzl mutations affect cuticle and eye development, respectively. We have identified a 7.2 kb genomic fragment that rescues ovo mutations in transgenic Drosophila and thus contains all sequences necessary for ovo+ function. This fragment has been sequenced almost in its entirety, defining the ovo locus at the molecular level. Multiple copies of the same fragment also rescue the lzl mutation. They do not rescue svb mutations, in agreement with genetic evidence that the svb function requires additional, more distal sequences. Nevertheless, a number of transposable element insertions that induce a svb phenotype interrupt the coding sequence of ovo. Taken together, the genetic and molecular data indicate the existence of a complex locus, where the ovo and svb functions depend on overlapping coding sequences but distinct regulatory elements. The data also suggest a model for the lzl phenotype. Expression of ovo at the RNA level is detectable at stage 8 of oogenesis in nurse cells and persists through the rest of oogenesis and in early embryogenesis. The ovo transcript encodes a protein of at least 1209 amino acids with four zinc fingers, suggesting that ovo might be a transcription factor required for female germ line maintenance and gametogenesis. PMID- 1712295 TI - Core I protein of bovine ubiquinol-cytochrome-c reductase; an additional member of the mitochondrial-protein-processing family. Cloning of bovine core I and core II cDNAs and primary structure of the proteins. AB - Core I and core II proteins are the largest nuclear-encoded subunits of the mitochondrial ubiquinol-cytochrome-c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of lambda ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase-chain reaction-amplified fragment. The core I precursor protein consists of 362 amino acids with a 34-amino-acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14-amino-acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter E., Neupert, W. & Weiss, H. (1989) Nature 339, 147 - 149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol-cytochrome-c reductase. PMID- 1712296 TI - Analysis and nucleotide sequence of the genes encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis. AB - The genes (slgA) encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methano-thermus pervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences. In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T-rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3' end of the 16S rRNA of these methanogens. Both slgA genes encode for a precursor of the mature surface-layer protein containing 593 amino acid residues with a putative N terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate-binding sites. In comparison with other surface-layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of beta-sheet structure (44%) and only 7% alpha-helix structures. PMID- 1712297 TI - Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane. AB - Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic alpha subunit of Na+/K(+)-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005 1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the alpha subunit of pork kidney Na+/K(+)-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K(+)-ATPase. When the antibodies were used to probe the sidedness of the sequences in right side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 1005-1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles. PMID- 1712298 TI - The C-terminal domain of plant catalases. Implications for a glyoxysomal targeting sequence. AB - A castor bean (Ricinus communis cv. Hale) cDNA encoding catalase was cloned and sequenced. The cDNA encoding the carboxy-terminal domain of catalase was compared to the corresponding sequences of six other plant catalases. The deduced amino acid sequences were compared according to the chemical attributes of each amino acid within each carboxy-terminal domain. A tripeptide sequence having the chemical attributes of the peroxisomal targeting sequence [Gould, S.J., Keller, G.-A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell Biol. 108, 1657 1664] was common to all the glyoxysomal/peroxisomal plant catalases. This sequence motif was located six amino acids from the carboxy terminus of each of the plant catalases. An identical motif was also found within the carboxy terminal domain of three mammalian catalases previously sequenced. We hypothesize that these motifs are at least part of the targeting mechanism for catalase entry into plant glyoxysomes/peroxisomes. PMID- 1712300 TI - Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance. AB - A 70-kDa heat-shock-protein (hsp 70) expression vector which contains the human hsp 70 gene linked to the human beta-actin promoter, was constructed and used to transfect CV1 monkey cells. Stably transfected CV1 clones were isolated which constitutively synthesized increased amounts of hsp70 at normal temperature. It is shown that these clones are resistant to elevated temperature. This finding indicates that hsp70 is involved in the protection of the cells against a lethal heat treatment and maybe responsible for the phenomenon of thermotolerance. PMID- 1712299 TI - A family of bombinin-related peptides from the skin of Bombina variegata. AB - A peptide fraction was isolated from the skin of Bombina variegata that showed antimicrobial activity. This fraction contained several molecular species, all of them consisting of 27 amino acid residues, with a constant C-terminal region (from residues 14-27), including an amidated carboxyl end and a variable N terminal segment. These peptides are related but not identical to bombinin [Csordas, A. & Michl, H. (1970) Monatsh. Chem. 101, 182-189]. By using synthetic oligonucleotides corresponding to the C-terminal region of the peptides, a cDNA library from the skin of B. variegata was screened and several positive clones coding for the corresponding peptide precursors were isolated and sequenced. Each clone contained the genetic information for a different bombinin-like peptide. The antimicrobial activity towards different bacterial species of a synthetic peptide corresponding to one of the variants deduced from cDNA sequences was tested. This peptide was found to be mainly active against different isolates of Staphylococci and Escherichia coli. PMID- 1712301 TI - Assembly of the surfactant protein SP-A. Deletions in the globular domain interfere with the correct folding of the molecule. AB - The C-terminal non-collagenous domain of the surfactant glycoprotein SP-A was shown to be essential for its correct folding and assembly, as judged by the secretion of various deletion mutants transiently expressed in COS cells. A deletion mutant coding for this domain was successfully secreted while the expression of the collagenous domain only did not lead to any detectable secretion. Deletion mutants lacking small parts of the non-collagenous domain interfered more or less with the correct folding and assembly of the molecule, thus either reducing or inhibiting the secretion. These data suggest that three prefolded non-collagenous domains register and act as a nucleation center for the folding of the collagenous triple helix which proceeds in a zipper-like fashion towards the N-terminus. PMID- 1712302 TI - Ligand binding at membrane mimetic interfaces. Human serum albumin in reverse micelles. AB - The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2 ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine 411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels. PMID- 1712303 TI - Product inhibition of alpha-chymotrypsin in reverse micelles. AB - The alpha-chymotrypsin-catalyzed hydrolysis of succinyl-L-alanyl-L-alanyl- L prolyl-L-phenylalanyl p-nitroanilide has been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that alpha-chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis-Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (kcat) at low water content is lower than in water and increases as the water content in the system is lower than in water and increases as the water content in the system (wo = [H2O]/[AOT]) increases, kcat reaching the value in water at high wo. If however, initial velocity data, as obtained under conditions where the enzyme is not saturated with substrate, are plotted against wo, the curves are bell-shaped, with a maximum around wo = 15. PMID- 1712304 TI - Signal averaging of pre- and post-extrasystolic beats in patients with ventricular arrhythmias. AB - To evaluate the effects of premature ventricular beats on the impulse conduction of adjacent sinus cycles, we compared the high amplification signal-averaged electrocardiogram parameters of the pre- and post-extrasystolic beats with those of the remaining sinus cycle. According to the duration of filtered QRS (fQRS), to the voltage of root mean square of the terminal 40 ms (RMS 40) and to the duration of low amplitude terminal components of the sinus cycles, ventricular late potentials were detected in nine out of 29 subjects. Patients with an abnormal signal-averaged electrocardiogram exhibited a longer fQRS (146 +/- 6 versus 116 +/- 2 ms), a reduced RMS40 voltage (18 +/- 2 versus 80 +/- 10 microV) and a prolonged duration of less than 40 microV components (42 +/- 4 versus 17 +/ 2 ms). Analysis of the pre-extrasystolic beats did not reveal any significant variation in the above parameters, showing a mean difference of 0.44 +/- 2.4 ms; 0.02 +/- 1.14 microV; 1 +/- 1.9 ms and of -1.45 +/- 1.02 ms; 3.5 +/- 8.6 microV; 0.7 +/- 0.84 ms respectively, for patients with and without ventricular late potentials. In addition, no significant variation was observed when the post extrasystolic beats were considered. These results indicate that the sinus cycles adjacent to premature ventricular discharges do not present variations of signal averaged electrocardiogram parameters that may suggest an influence of the ectopic beats on their intramyocardial impulse propagation. PMID- 1712305 TI - Injection of tachykinins and selective neurokinin receptor ligands into the substantia nigra reticulata increases striatal dopamine and 5-hydroxytryptamine metabolism. AB - Bilateral injection of endogenous tachykinins (substance P (SP), neurokinin A (NKA)) and selective neurokinin receptor ligands (senktide, [Sar9,Met(O2)11]SP, [MePhe7]NKB) into the substantia nigra reticulata increased striatal dopamine and serotonin metabolism. The increase in dopamine metabolism in the dorsal striatum at a low dose of the substances may be a direct effect on dopamine neurons in the substantia nigra reticulata via NK1 and NK3 receptors. The lack of effect at intermediate doses may be due to inhibitory mechanisms or desensitization. The changes after high doses in the dorsal and ventral striatum may be due to actions on dopaminergic neurons in the substantia nigra as well as in the ventral tegmental area, since there was considerable diffusion from the site of injection. An apparent rapid degradation of injected SP or NKA indicates that N terminal SP fragments may participate in the SP response. The increased serotonin metabolism that occurs only at a high dose may involve all three neurokinin receptors. PMID- 1712306 TI - Anti-inflammatory steroids inhibit cytokine induction of nitric oxide synthase in rat renal mesangial cells. PMID- 1712307 TI - Interactions of dihydropyridine Ca2+ channel agonists with the human platelet thromboxane A2/prostaglandin H2 receptor. AB - The specific interactions at the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor by four 1-4 dihydropyridine (DHP) agonists were studied. Using competition equilibrium binding assays with the TXA2/PGH2 receptor agonist [125I]BOP and the antagonist [125I]PTA-OH, the affinities of racemic BAY K 8644 (BAY), racemic CGP 28392 (CGP) and (+) and (-) SDZ 202-791 (SDZ) for the TXA2/PGH2 receptor were determined. The rank order potencies for competition were BAY greater than (-)SDZ greater than CGP greater than or equal to (+)SDZ. Bay, CGP and SDZ (stereoselectively) inhibited specific incorporation of the TXA2/PGH2 receptor photoaffinity probe [125I]PTA Azido into three proteins associated with the TXA2/PGH2 receptor with Mr of 43, 39 and 27 kDa as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. Using the fluorescent Ca2+ probe Fura-2, it was observed that I-BOP failed to stimulate classical divalent cation channels. However, SDZ stereoselectively inhibited the rise in [Ca2+]i induced by I-BOP while not affecting that induced by thrombin. Although DHPs specifically and stereoselectively interact with the TXA2/PGH2 receptor on human platelets, the TXA2/PGH2 receptor-mediated rise in [Ca2+]i is not through stimulation of a classical divalent cation channel. PMID- 1712308 TI - Site-specific acylation of GABA-gated Cl- channels: effects on 36Cl- uptake. AB - Radioligand binding studies indicate that p-isothiocyanato-t butylbicycloorthobenzoate (p-NCS-TBOB) specifically acylates GABA-gated chloride channels. Preincubation of synaptoneurosomes with p-NCS-TBOB followed by washing resulted in a concentration dependent (63-500 nM) inhibition of both muscimol stimulated chloride uptake and [355]t-butylbicyclophosphorothionate (TBPS) binding. The extent of acylation (assessed by inhibition of [35S]TBPS binding) was highly correlated (r = 0.89; p less than 0.001) with the inhibition of muscimol-stimulated Cl- uptake. Neither basal Cl- uptake nor [3H]muscimol binding to GABAA receptors were affected by p-NCS-TBOB. Preincubation with the nonacylating 'cage' convulsant t-butylbicycloorthobenzoate (500 nM) followed by washing had no effect on either muscimol-stimulated Cl- uptake or [35S]TBPS binding. These findings indicate that p-NCS-TBOB interferes with the efficacy of muscimol promoted channel openings, but does not affect the recognition qualities of GABAA receptors. p-NCS-TBOB should prove useful in electrophysiological and biochemical studies examining the properties of GABA-gated Cl- channels. PMID- 1712309 TI - Three regulation mechanisms of nitric oxide synthase. PMID- 1712310 TI - Isobutylmethylxanthine enhances adrenergic-induced ocular hypotension in rabbits and beagles. AB - Isobutylmethylxanthine (IBMX), a phosphodiesterase/adenosine receptor inhibitor, was combined with norepinephrine (nE), epinephrine (Epi) and isoproterenol, respectively, to evaluate their effect on intraocular pressure (IOP). Application of topical IBMX alone had no measurable effect on IOP. When IBMX was combined with nE or Epi the ocular hypotension in rabbits and beagles increased. The Emax for nE alone was 2.9 +/- 0.4 mmHg, for Epi alone 7.3 +/- 0.5 mmHg and for isoproterenol alone 5.1 +/- 0.3 mmHg. The EC50 was 0.2 +/- 0.05% (nE), 0.05 +/- 0.01% (Epi) and 0.003 +/- 0.001% for isoproterenol. When given in combination with 1% IBMX the Emax for nE was 7.4 +/- 1.7 mmHg, for Epi 9.0 +/- 0.8 mmHg and for isoproterenol 6.1 +/- 0.3 mmHg. The corresponding values for EC50 were 0.07 +/- 0.03% (nE), 0.02 +/- 0.006% (Epi) and 0.002 +/- 0.001% for isoproterenol. Combining 1% IBMX with 0.1% Epi increased the aqueous humour cyclic AMP-levels at 1, 3 and 5 hr in rabbits. The results of this study demonstrate that a phosphodiesterase/adenosine receptor inhibitor such as IBMX enhances the reduction in IOP induced by adrenergic agonists. PMID- 1712311 TI - The capsaicin-induced inflammatory reaction in the cat eye: antagonism by ruthenium red. AB - The neurogenic ocular inflammatory response in the cat was investigated by means of intracameral injections of capsaicin. At a dose of 200 micrograms intracamerally the eye responded with a breakdown of the blood-aqueous barrier (BAB) and a transient ocular hypertension. A response of the same magnitude was observed when a dose of 1 microgram capsaicin was given. Most of the response to this lower dose was prevented by ruthenium red, an inorganic dye thought specifically to antagonize the effects of capsaicin on sensory nerve endings. Following injection of 200 micrograms capsaicin there was a transient increase in pupil size, but this response was not seen after 1 microgram. Repeated injections of 200 micrograms capsaicin when the effects of the first dose had vanished resulted in tachyphylaxis of the mydriatic response. A dose of 200 micrograms of capsaicin had no effect on resistance in the outflow routes for aqueous humour. PMID- 1712312 TI - Sequence analysis, expression and chromosomal localization of a gene, isolated from a subtracted human retina cDNA library, that encodes an insulin-like growth factor binding protein (IGFBP2). AB - The metabolic functions of insulin-like growth factors (IGFs) I and II are modulated by a family of binding proteins which are present in biological fluids and are synthesized by a variety of cell types. A cDNA clone, isolated at random from a subtracted human retina library, has been identified to code for a novel IGF-binding protein (IGFBP2) by its sequence homology to the peptide sequence of IGF binding proteins purified from bovine MDBK and rat BRL-3A cells. The complete nucleotide sequence of the IGFBP2 cDNA is 1406 bp long, contains 66% G-Cs and an open reading frame of 328 amino acids with a putative signal or pro-peptide of 39 residues. The mature polypeptide of 289 amino acids has 18 cysteines, a putative ATP-binding site and an RGD tripeptide. The 1.4 kb IGFBP2 transcript is expressed in several human tissues including fetal eye and fetal brain, but not in the human lymphoblastoid cell line against which the retinal cDNA library was subtracted. In situ hybridization to sections of mouse retina localized the mRNA for IGFBP2 primarily in the outer nuclear layer of photoreceptors. Southern blot analysis of DNA from human x rodent and mouse x rodent somatic cell hybrids assigned the gene for IGFBP2 to human chromosome 2q33-qter and mouse chromosome 1 in a known conserved syntenic region. PMID- 1712313 TI - Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment epithelium. AB - Insulin-like growth factors (IGFs) I and II are mitogenic polypeptides structurally homologous to insulin, which are thought to mediate important neurobiologic actions in the CNS. The purpose of this study was to determine if cultured bovine retinal pigment epithelial cells (RPE) express IGF receptors and secrete soluble IGF binding proteins, and to characterize these receptors and binding proteins. We also characterized the soluble IGF binding proteins present in juvenile and adult rat vitreous and serum, as well as those in fetal bovine vitreous and serum, in order to facilitate identification of the RPE IGF binding protein, and to determine potential destinations for this protein once produced. Affinity labeling was used to characterize insulin, IGF-I and IGF-II receptors. Western radioligand blotting and immunoprecipitation were used to characterize IGF binding proteins. We found that RPE cells in culture express virtually no insulin receptors, and only modest amounts of IGF-I receptors. IGF-II receptors were abundantly expressed. Additionally, RPE cells secrete a soluble IGF binding protein which is immunologically related to IGFBP-2, the primary IGF binding protein produced in the central nervous system. Bovine vitreous was found to contain a mixture of IGF binding proteins (IGFBP). The most prominent IGFBP in this mixture is immunologically related to IGFBP-2. Likewise, juvenile and adult rat vitreous contained only one IGF binding protein that was shown to be immunologically related to IGFBP-2. Juvenile rat vitreous contained more binding activity corresponding to IGFBP-2 than did adult vitreous, suggesting developmental regulation. These data suggest that IGF's and their binding proteins may have important, and as yet undefined, roles in retinal neurophysiology. PMID- 1712315 TI - The local domain for divergence of subcortical afferents to the striate and extrastriate visual cortex in the common marmoset (Callithrix jacchus): a multiple labelling study. AB - In the common marmoset (Callithrix jacchus), the cortical projection from the pulvinar and other diencephalic structures into the striate and prestriate cortex was investigated with various fluorescent retrograde tracers. Single cortical injections as well as multiple injections at distances of 1-2 mm with one tracer into an extended but coherent cortical region were applied. Fields with multiple injections were placed so that they touched each other (minimal distances 2 to 3 mm). Retrogradely labelled cells in the LGN and/or the pulvinar were arranged in coherent columns, volumes or slabs, but cell volumes resulting from neighbouring cortical injections overlapped at their border (for details of the thalamo cortical topography see the companion paper Dick et al. (1991]. Double labelled cells (dl) were only found in the zones of overlap of the cell volumes labelled by the respective tracers. The relative number of dl-cells in these overlap zones was 6.2 +/- 3.1%. The dl-frequency was the same in the various nuclei of the pulvinar and the LGN. In the main layers of LGN, dl-cells were found only in the overlap zone of two injection fields into area 17, but a few dl-cells were found in interlaminar cells after injections into area 17 and 18. Maximal cortical distances between injection fields which produced dl in the pulvinar, were 3 to exceptionally 4 mm but dl was highest at injection distances less than or equal to 2.5 mm and decreased sharply at wider distances. Such overlap zones were concerned with identical or overlapping regions of visual field representation in the cortex and probably also in the pulvinar. Although in individual experiments up to four different tracers were injected into different striate/prestriate regions, often embracing the same visual field representation, individual cells in the pulvinar showed dl from maximally only two tracers injected into neighbouring cortical regions. We conclude that dl in the posterior thalamic projection nuclei is determined essentially by cortical distance and thus reflects the local domain of branching of thalamo-cortical afferents. Pruning of such branches during development may further restrict bifurcating axons to identical visual field representations, but representation of identical visual field regions in different visual areas is not, per se, a sufficient condition for dl. It is not found if such regions are further apart from each other than the typical local domain of 2-3 mm, exceptionally up to 4 mm in one experiment after injections into area 17 and MT.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712314 TI - Topographical and topological organization of the thalamocortical projection to the striate and prestriate cortex in the marmoset (Callithrix jacchus). AB - In eleven hemispheres of nine marmoset monkeys (Callithrix jacchus), we have investigated the thalamo-cortical organization of the projections from the pulvinar to the striate and prestriate cortex. In each experiment, single or multiple injections of various retrograde fluorescent tracers were injected into adjacent regions or areas. In two experiments, horseradish peroxidase (HRP) was injected into the lateral geniculate nucleus (LGN) and the lateral pulvinar, respectively. The results show that the thalamo-cortical projection from LGN to striate cortex and from pulvinar to the prestriate cortex are similarly organized, but the geniculo-striate projection is more precise than the pulvinar prestriate projection. The pulvinar-prestriate projection is topographically organized and preserves topological neighbourhood relations. Projection zones to the various visual areas are concentrically wrapped around each other. The projection zone to area 18 constitutes a central core region. It begins ventro laterally in PuL where the pulvinar is in contact with the LGN. This contact zone we called the hilus region of the pulvinar. The area 18-projection zone stretches as a central cone into the posterior pulvinar through PuL and into PuM. It is surrounded by the projection zone to the posterior belt of area 19 and this in turn is surrounded by the projection zone to the anterior belt of area 19. The projection zones to area 19 are then surrounded medially and dorsally by zones projection to the temporal and parietal association cortex, respectively. The projection zone to area MT is located medio-ventrally in the posterior pulvinar (PuIP and surrounding nuclei) and coincides with a densely myelinated region. Area 17 also receives input from the pulvinar but probably predominantly in the region of the central visual field. The pulvinar zone projecting to area 17 is located ventrolaterally from the central core region projecting to area 18 and is contiguous laterally with the LGN. If the positions of the vertical and the horizontal meridian in the pulvinar correspond to those in the respective cortical projection zones, a second order visual field representation such as found in area 18, with the horizontal meridian split at an eccentricity of about 7-10 degrees, can also be recognized in the pulvinar. PMID- 1712316 TI - Interaction of HIV-1 with susceptible lymphoblastoid cells. 1H NMR studies. AB - Different strains of HIV susceptible lymphoblastoid cells have been infected by HIV-1 and examined by means of 1H NMR spectroscopy at different times after infection, taking advantage of the presence of high resolution lipid signals from the plasma membrane of tumor cells. A transient decrease in intensity of fatty acid signals, originated by changes in membrane structure, has been observed early after viral infection. Marked alterations in membrane-dependent steps of phospholipid synthesis can also be inferred by the observed transient depression in peaks from choline-based metabolites. Spectral modifications deriving from changes in lipid metabolism are also produced both in infected cells a few days after infection and in permanently infected cells. 1H NMR can, therefore, monitor structural and metabolic effects induced by HIV infection. PMID- 1712317 TI - Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices. AB - Recent studies indicated that the formation of a major constituent of Alzheimer's disease (AD) senile plaques, called beta A4-peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its beta A4 sequence was found to be part of APP processing the formation of the beta A4-peptide seems to be prevented under normal conditions. We considered whether in AD one of the endogenous proteinase inhibitors might interfere with APP processing. After we had recently found that cultured human neuronal cells synthesize the most potent of the known human proteinase inhibitors, alpha-2-macroglobulin (alpha 2M), upon stimulation with the inflammatory mediator interleukin-6 (IL-6) we now investigated whether alpha 2M and IL-6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong alpha 2M and IL-6 immunoreactivity while no such immunoreactivity was found in age-matched control brains. Strong perinuclear alpha 2M immunoreactivity in hippocampal CA1 neurons of Alzheimer's disease brains indicates that neuronal cells are the site of alpha 2M synthesis in AD brains. We did not detect elevated IL-6 or alpha 2M levels in the cerebrospinal fluid of AD patients. Our data indicate that a sequence of immunological events which seem to be restricted to the local cortical environment is part of AD pathology. PMID- 1712318 TI - Okadaic acid inhibits amylase exocytosis from parotid acini stimulated by cyclic AMP. AB - To evaluate the role of protein phosphorylation in amylase exocytosis, we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation in rat parotid acini. Although okadaic acid by itself weakly stimulated amylase release, it did not potentiate amylase release stimulated by half-maximum doses of isoproterenol or cAMP, and markedly inhibited their maximum effects. Okadaic acid dose-dependently increased cAMP-independent phosphorylation of some proteins and enhanced cAMP dependent phosphorylation of 21- and 26-kDa proteins. These results indicate that increase in protein phosphorylation does not necessarily enhance the exocytosis of amylase from parotid acini. PMID- 1712319 TI - Characterization of a discontinuous epitope on annexin II by site-directed mutagenesis. AB - Recombinant annexin II mutants were generated to identify amino acids involved in the formation of the discontinuous epitope of the monoclonal antibody H28. Analysis of the various mutant proteins by immunoblotting and enzyme-linked immunosorbent assay revealed that residues Lys27, Arg62, Glu65, and Arg67 are indispensable for H28 reactivity. Residues in equivalent positions are also in close proximity in the recently determined X-ray structure of annexin V, a different member of the same family of Ca2+/lipid-binding proteins. Thus annexins II and V show a similar three-dimensional folding in this region of the molecule. Consequently, the Ca2+ binding sites and the residues phosphorylated by pp60src (Tyr23) and protein kinase C (Ser25) most likely reside on opposite sides of the annexin II molecule. PMID- 1712320 TI - The induction of adipose conversion in 3T3-L1 cells is associated with early phosphorylation of a 60-kDa nuclear protein. AB - The induction of adipose conversion in 3T3-L1 cells by bezafibrate has been shown to be enhanced by dibutyryl-cAMP. We here report that the induction of adipose conversion in 3T3-L1 cells, by either bezafibrate and dibutyryl cAMP, or isobutylmethyxanthine alone, is associated with a very early phosphorylation of a 60-kDa acidic protein found in the nuclear fraction. PMID- 1712321 TI - Selective effect of inhibitors on inner mitochondrial membrane channels. AB - The effect of amphiphilic cationic drugs on the channel activity of the mitochondrial inner membrane was examined with patch-clamp techniques. The therapeutic drugs amiodarone, propranolol and quinine reduced the probability of being open for the multiconductance channel (MCC) activity (levels from 30 pS to over 1 nS). While amiodarone decreased the probability of being open for the voltage dependent approximately 100 pS channel, it increased the conductance 42 +/- 20% (mean +/- SD, n = 6) with no significant change in mean open time. Similar results were obtained with propranolol. These data indicate that the approximately 100 pS channel is distinct from MCC activity. PMID- 1712322 TI - Intrabursal administration of protein kinase or proteinase inhibitors: effects on ovulation in the rat. AB - OBJECTIVE: To test the hypothesis that inhibition of protein kinase (PK) activity or proteolysis inhibits ovulation. DESIGN: Rats were injected intrabursally with protein kinase (H9 or staurosporin) or proteinase (alpha 2-macroglobulin) inhibitors and oocyte release was evaluated. SETTING: Clinical Research Laboratory, Center for Reproductive Medicine, University of Kentucky Medical Center. PARTICIPANTS: Immature rats stimulated with pregnant mare serum gonadotropin. INTERVENTIONS: Staurosporin (1 or 10 microM), H9 (1 mM), alpha 2 macroglobulin (835 microIU of activity); or vehicle was injected into the right ovarian bursa. The left ovarian bursa remained intact. Animals immediately received human chorionic gonadotropin (hCG). MAIN OUTCOME MEASURES: Analysis of oocyte release and ovarian morphology. RESULTS: Oocyte release from the inhibitor treated side decreased for the H9 group (8.1 +/- 1.9 fewer oocytes released, P less than 0.001) and the 10 microM staurosporin group (5.5 +/- 0.6, P less than 0.001). No change in oocyte release was observed in the 1 microM staurosporin, alpha 2-macroglobulin, or vehicle injected groups. Histologic examination of vehicle treated ovaries demonstrated numerous developing corpora lutea (CL; 20.5 +/- 2.1 CL/ovary) and a lack of preovulatory follicles. In contrast, ovaries treated with PK inhibitors contained unruptured preovulatory follicles coincident with fewer forming CL (11.5 +/- 3.5 CL/ovary). CONCLUSIONS: Inhibition of PK activity in vivo suppresses ovulation, demonstrating that protein phosphorylation plays an important intermediary role in the ovulatory process. PMID- 1712323 TI - Pregnancy termination with mifepristone and gemeprost: a multicenter comparison between repeated doses and a single dose of mifepristone. World Health Organization. AB - OBJECTIVE: To compare two regimens of mifepristone plus gemeprost for early pregnancy termination. DESIGN: A prospective, randomized, multicenter trial. SETTING: Ten gynecological services, mostly in academic hospitals. PARTICIPANTS: Three hundred eighty-five healthy women up to 35 years of age with amenorrhea less than or equal to 49 days requesting pregnancy termination. TREATMENT: Mifepristone, 25 mg five times at 12-hour intervals (n = 192) or 600 mg as a single dose (n = 193) followed by 1 mg gemeprost 60 hours after the start of mifepristone. MAIN OUTCOME MEASURES: Pregnancy outcome, time of onset and duration of vaginal bleeding, subjective complaints, and hormone changes during treatment and 6-week follow-up. RESULTS: Treatment outcome was identical in both groups with an overall complete abortion rate of 92.7% among the 385 women included in the analysis. The frequency of complaints, bleeding patterns, and changes in hemoglobin, beta-human chorionic gonadotropin, estradiol, and progesterone were also similar in both groups. Cortisol (at 12 and 36 hours after mifepristone) and prolactin (at 12 hours) were significantly higher in the single 600-mg dose group. CONCLUSION: When used for early pregnancy termination with prostaglandin, a lower dose of mifepristone than the currently recommended single 600-mg dose may suffice. PMID- 1712324 TI - The prognostic value and significance of preclinical abortions in in vitro fertilization-embryo transfer programs. AB - Preclinical abortions occur in natural conceptions as well as in in vitro fertilization-embryo transfer (IVF-ET) cycles. Nevertheless, although known, this entity is ill defined. OBJECTIVE: The purpose of this study was to propose a classification of these pregnancies on the basis of pathophysiological evidence and to evaluate their future clinical impact. DESIGN: Of 970 IVF-ET cycles, 114 cycles (11.7%) terminated in preclinical abortions. Abortions were divided according to peak beta human chorionic gonadotropin (beta-hCG) values into chemical abortions (52%) occurring 2 weeks after ET with beta-hCG values not higher than 21 mIU/mL and peri-implantation abortions (47%) terminating spontaneously 4 weeks after ET; the latter had higher beta-hCG values for a longer period of time but without any sonographic evidence of gestational sac. No woman in the two groups needed curettage. RESULTS: After a chemical abortion, the pregnancy outcome had better ongoing pregnancy rates (24.7%) in comparison with the 17% achieved in the total IVF-ET cycles. CONCLUSIONS: It is concluded that these two groups most probably have different pathophysiological backgrounds and concomitantly different future clinical impacts. PMID- 1712325 TI - Regulation of HIV-1 gene expression. AB - The quantity and quality of HIV-1 gene expression is temporally controlled by a cascade of sequential regulatory interactions. Basal HIV-1 transcription is determined by interaction of cellular regulatory proteins with specific DNA target sequences within the HIV-1 long-terminal repeat. The most notable of these protein:DNA interactions involves NF-kappa B, a transcription factor that plays a pivotal role in the activation of genes important for cellular responses to infection and inflammation. A second level of control involves the virally encoded Tat trans-activator. Tat, in combination with as yet unidentified cellular proteins, activates HIV-1 gene expression through a specific interaction with the viral TAR RNA stem-loop target sequence. A final level of regulation is mediated by the viral Rev protein. Rev acts posttranscriptionally to induce the expression of HIV-1 structural proteins and thereby commits HIV-1 to the late, cytopathic phase of the viral replication cycle. Rev activity appears to require a critical, threshold level of Rev protein expression, thus preventing entry into this late phase in cells that are unable to support efficient HIV-1 gene expression. In total, this cascade of regulatory levels allows the HIV-1 provirus to respond appropriately to the intracellular milieu present in each infected cell. In activated cells, the combination of Tat and Rev can stimulate a very high level of viral gene expression and replication. In quiescent or resting cells, in contrast, these same regulatory proteins are predicted to maintain the HIV-1 provirus in a latent or nonproductive state. PMID- 1712326 TI - Targeted therapy of human immunodeficiency virus-related disease. AB - Since the discovery of human immunodeficiency virus (HIV) as a pathogenic retrovirus linked to acquired immunodeficiency syndrome (AIDS), a number of potentially useful strategies for antiretroviral therapy of AIDS and its related diseases have emerged. One such strategy involves use of the broad family of 2',3'-dideoxynucleosides, to which 3'-azido-2',3'-dideoxythymidine (AZT) belongs. AZT has been shown to reduce the replication of HIV in vivo and to confer significant clinical benefits in patients in both early and advanced stages of infection. Other members of the family, 2',3'-dideoxycytidine (ddC), 2',3' dideoxyinosine (ddI), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), have also been reported to be active against HIV in short-term clinical trials. The armamentarium of antiretroviral agents is rapidly growing. Various nonnucleoside agents have recently been identified to be active against HIV in vitro. HIV-1 protease inhibitors are notable as possible new therapies for HIV-1-related diseases. However, we have faced several new challenges in the antiretroviral therapy in AIDS. These include long-term drug-related toxicities; emergence of drug-resistant HIV variants; and development of various cancers, particularly as effective therapies prolong survival. Progress in understanding structure activity relations and clinical effectiveness will continue with dideoxynucleoside analogs. However, it seems certain that a variety of nonnucleoside analogs affecting multiple steps in viral replication will become available before long, and combination therapies using multiple antiretroviral drugs will be available. Such therapies will exert major effects against the moribidity and mortality caused by HIV. PMID- 1712327 TI - Development of artificial vaccines against HIV using defined epitopes. AB - HIV may not follow the paradigm that has been used successfully for developing most viral vaccines, namely, that the best vaccine is the one that most closely mimics natural infection. This approach is based on the premise that natural infection leads to long-lasting protective immunity, which may not be applicable to HIV. Also, some immune responses elicited by infection with HIV may enhance infection or contribute to the development of immune deficiency. To overcome these problems, an artificial vaccine could be constructed using only antigenic epitopes that elicit neutralizing antibodies, helper T cells, and CD8+ cytotoxic T cells, and avoiding epitopes that elicit deleterious responses. Progress has been made in identifying all three of these types of epitopes, in characterizing their activity in animals, and in demonstrating that at least two of these can be linked to induce neutralizing antibodies without a carrier. Methods have also been developed to induce cytotoxic T cells. It is therefore feasible to construct an artificial vaccine for HIV that should be safer and more effective than a natural whole viral or subunit vaccine. PMID- 1712328 TI - Neutralization of HIV-1: a paradox of humoral proportions. AB - The production of immunoglobulin capable of neutralizing the infectivity of a virus represents one of the most remarkable molecular accomplishments of the host's available immune defenses. It should be no surprise that a virus that has existed in the parenchyma of the immune system has evolved as an equally dynamic molecule (i.e., viral envelope) for survival. Neutralizing immunoglobulin (Ig) can best serve the host under conditions where the invading pathogen requires a well-defined cell-free state for establishing an infection or transmission. Evidence for a controlling and therefore protective role of neutralizing Ig against lentiviruses has been defined in natural and experimental infections with equine infectious anemia virus of ungulate members in the family equidae. Rapid replication of the virus immediately after infection and its release in a cell free state leads to the production of neutralizing Ig and subsequent control of the primary viremia. A similar cause-effect relationship exists in humans between the high-titered viremia, observed shortly after HIV-1 infection, and the subsequent production of neutralizing Ig. Partially controlling this acute stage of viral replication by neutralizing Ig and thus preventing an otherwise acute form of immunosuppression or immune complex disease may be viewed paradoxically as a survival property of the virus. Immunologically mediated control, in a Darwinian sense, selects for viruses that have optimized the parameters of longevity and transmission from host to host. This paradox of neutralization in HIV-1 infection appears to be mediated by the convergence of structural and functional roles of the third variable domain (V3) of the external envelope glycoprotein. During infection or envelope-based vaccination, antibody to this cross-reactive, immuno-dominant epitope dominates the antigenic repertoire. Once this occurs, the host is less able to respond to emerging viruses containing closely related V3 structures. Thus a relatively restricted clonal-dominance of the neutralization response results. The V3 domain, apparently in concert with the rest of the molecule, provides an epitope that can tolerate and utilize its conformational flexibility to allow immune escape while maintaining its functional role in infectivity. Sixteen other putative epitopes have been described as being involved in the induction of neutralizing Ig. Currently the biologically functional role of neutralizing Ig to these other epitopes are complicated by a prior lack of knowledge and appreciation of the in vitro variables affecting their measurements.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712329 TI - Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth. AB - The authors have established a long-term tissue culture cell line (BON) derived from a metastatic human carcinoid tumor of the pancreas. The cells have been in continuous passage for 46 months. Tissue culture cells produce tumors in a dose dependent fashion after SC inoculation of cell suspensions in athymic nude mice. BON tumors, grown in nude mice, are histologically identical to the original tumor; they possess gastrin and somatostatin receptors, synthesize serotonin and chromogranin A, and have a doubling time of approximately 13 days. The antiproliferative effects of the long-acting somatostatin analogue, SMS 201-995 (300 micrograms/kg, t.i.d.), and 2% alpha-difluoromethylornithine on BON xenografts in nude mice were examined. Tumor size was significantly decreased by day 14 of treatment with either agent and at all points of analysis thereafter until the animals were killed (day 33). In addition, tumor weight, DNA, RNA, and protein contents were significantly decreased in treated mice compared with controls. Establishment of this human carcinoid xenograft line, BON, provides an excellent model to study further the biological behavior of carcinoid tumors and the in vivo effect of chemotherapeutic agents on tumor growth. PMID- 1712330 TI - Activation of mast cells by bile acids. AB - To test whether bile acids interact with mast cells, dilute, aqueous solutions of five pure unconjugated natural bile acids and their corresponding glycine or taurine conjugates were incubated with murine PT-18 cells (a mast cell line functionally and cytochemically similar to mucosal mast cells) or with freshly isolated rat peritoneal mast cells. Bile acid solutions ranged in concentration from 0.3 to 10 mmol/L; histamine release was assessed by a fluorimetric assay, and cell lysis by cytosolic enzyme (lactate dehydrogenase) release. Lipophilic, dihydroxy bile acids (chenodeoxycholic acid and deoxycholic acid as well as their glycine and taurine conjugates) caused histamine release in a dose-related manner; cholic acid and its conjugates caused much less or no histamine release. Two hydrophilic bile acids (ursodeoxycholic acid and ursocholic acid and their conjugates) were virtually devoid of activity. Histamine release, which was independent of extracellular Ca2+, occurred at 0.3 mmol/L, well below the critical micellization concentration. For a given concentration, unconjugated bile acids and glycine-conjugated bile acids induced more histamine release than taurine-conjugated bile acids; maximal release was observed at 3 mmol/L for lipophilic, dihydroxy bile acids. To test whether bile acids could also cause histamine release from cutaneous mast cells in vivo, rats were injected intradermally with bile acid solutions and histamine release assessed by capillary leakage of Evan's blue dye. Cutaneous blueing was greater with cytotoxic bile acids, chenodeoxycholyglycine or deoxycholylglycine, than with ursodeoxycholylglycine and was inhibited by prior antihistamine treatment. Histamine release correlated highly and positively with lipophilicity and with bile acid surface activity. It was concluded that lipophilic but not hydrophilic bile acids possess concentration-dependent cytotoxicity toward mast cells causing histamine release, that unconjugated and glycine-conjugated bile acids are more potent than taurine-conjugated bile acids, and that mast cell histamine release is highly correlated with lipophilicity of bile acids as well as their surface activity. PMID- 1712331 TI - [Mechanisms of the anticoagulation activity of new semi-synthetic heparinoids]. AB - Compounds representing chemically modified derivatives of natural polysaccharides and possessing heparin-like properties have been obtained basing on new methods of sulphatization++ of hydroxyl-containing polymers. The study of the anticoagulation activity of mannan, starch, dextran sulfates permits a suggestion that these compounds realize their anticoagulant potential not through antithrombin-III. A relationship has been established between the properties of semi-synthetic heparinoids and sulfur percentage, molecular mass and the type of polymeric matrix. The investigations conducted have made possible the development of effective thrombosis-preventing drugs and further thorough study of their effects. PMID- 1712332 TI - [The HLA system]. PMID- 1712333 TI - Effect of RNA secondary structure on polyadenylation site selection. AB - Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout. PMID- 1712334 TI - [Toxicological substantiation of safe use of xenobiotics]. PMID- 1712335 TI - Studies on the mechanism of arterial vasodilation produced by the novel antihypertensive agent, carvedilol. AB - The mechanism(s) responsible for arterial vasodilation observed following acute administration of racemic carvedilol, a novel vasodilator/beta adrenoceptor antagonist, has been investigated in rats. In conscious spontaneously hypertensive rats, carvedilol (0.03-3.0 mg/kg, iv) produced a dose-dependent reduction in blood pressure with no significant effect on heart rate. Because cardiac output was relatively unaffected, the antihypertensive response of carvedilol was associated with a dose-dependent reduction in total peripheral vascular resistance. Submaximal antihypertensive doses of carvedilol were chosen for mechanism of action studies in pithed rats. Carvedilol (0.3 mg/kg, iv) produced a significant inhibition of the beta 1 adrenoceptor mediated positive chronotropic response to isoproterenol. This same dose of carvedilol also inhibited, but to a lesser degree, the beta 2 adrenoceptor mediated vasodepressor response to salbutamol in pithed rats whose blood pressure was elevated by a constant intravenous infusion of angiotensin II. Thus, carvedilol blocks both beta 1 and beta 2 adrenoceptors at antihypertensive doses, with modest selectivity being observed for the beta 1 adrenoceptor subtype. Carvedilol produced significant inhibition of the alpha 1 adrenoceptor mediated pressor response to cirazoline in the pithed rat, but had no effect on the alpha 2 adrenoceptor mediated pressor response to B-HT 933, suggesting that carvedilol is also an alpha 1 adrenoceptor antagonist at antihypertensive doses. Carvedilol had no effect on the pressor response elicited by angiotensin II, indicating a lack of nonspecific vasodilator activity. The vasopressor response to the calcium channel activator, BAY-K-8644, which is mediated through the opening of voltage dependent calcium channels and the subsequent translocation of extracellular calcium, was significantly inhibited by carvedilol (1 mg/kg, iv), suggesting that carvedilol is also a calcium channel antagonist, consistent with our previous in vitro studies. In anesthetized spontaneously hypertensive rats, the antihypertensive activity of carvedilol was nearly abolished by combined pretreatment of the rats with high doses of the alpha 1 adrenoceptor antagonist, prazosin (1 mg/kg, iv), and the nonselective beta adrenoceptor antagonist, propranolol (3 mg/kg, iv), suggesting that the majority of the antihypertensive response produced by carvedilol may be accounted for by blockade of beta and alpha 1 adrenoceptors. We therefore conclude that carvedilol, at antihypertensive doses, is an antagonist of beta 1, beta 2, and alpha 1 adrenoceptors, and also of calcium channels in vascular smooth muscle.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712336 TI - Immunophenotyping of lymphocytes in liver tissue of patients with chronic liver diseases by flow cytometry. AB - Immunological factors are important in the pathogenesis of a spectrum of hepatobiliary diseases. To characterize the nature of specific immunological responses in liver disease, we determined lymphocyte changes in liver tissue and in blood using flow cytometry. A total of 113 liver biopsy specimens was collected from patients with the following diseases: 19 chronic hepatitis B; 39 chronic non-A, non-B hepatitis; 27 alcoholic liver disease; 10 hepatic malignancy; 8 autoimmune hepatitis; 6 fatty liver and 4 primary biliary cirrhosis. The lymphocytes were isolated from the liver biopsy specimens by mechanical and enzymatic methods. The lymphocyte yield was 7,901 +/- 575 cells/mg of liver tissue. The viability of lymphocytes was 97.7% +/- 0.3%. Lymphocytes were stained with four pairs of two-color mixed fluorescein-conjugated monoclonal antibodies, including T4-T8 (CD4/CD8), T11-B1 (CD2-CD20), NKH1-T8 (CD56-CD8), IL 2R1-T11 (CD25-CD2), and the ratios were determined by an Epics Profile flow cytometer. Immunophenotyping of lymphocytes in whole blood samples was simultaneously analyzed. Variability in lymphocyte yield and different patterns of lymphocyte subsets were found in the liver biopsy specimens. The yields of lymphocytes from patients with chronic non-A, non-B and autoimmune hepatitis were highest, and the lowest yield was from patients with fatty liver. Patients with primary biliary cirrhosis, fatty liver and hepatic malignancy had relatively high ratios of CD4/CD8, CD56/CD8 and CD25/CD2; whereas patients with chronic hepatitis B, autoimmune hepatitis and non-A, non-B hepatitis had lower ratios of CD4/CD8, CD56/CD8 and CD25/CD2. No difference in lymphocyte ratios between the patients with cirrhotic and noncirrhotic alcoholic liver disease was found.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712337 TI - Studies on mechanisms of augmentation of liver regeneration by cyclosporine and FK 506. AB - Evidence could not be found of immune modulation of liver regeneration. The powerful immunosuppressive drug FK 506, which augments the response after partial hepatectomy in normal rats, had the same effect in T cell-deficient nude rats. The cytotoxicity of natural killer cells in treated nude rats was not significantly changed by FK 506 therapy. However, the serum of FK 506-treated nude rats increased hepatocyte proliferation when added to third-party hepatocyte cultures, suggesting that FK 506 had induced a serum growth factor in the nude rats or had suppressed an inhibitory factor. A hypothesis was advanced that FK 506 (and cyclosporine) affects hepatic growth by nonimmunological pathways. PMID- 1712338 TI - Adenine arabinoside monophosphate and acyclovir monophosphate coupled to lactosaminated albumin reduce woodchuck hepatitis virus viremia at doses lower than do the unconjugated drugs. AB - The woodchuck was selected to study the efficacy of liver-targeted antiviral drugs on hepadnavirus replication. Nineteen woodchucks chronically infected with woodchuck hepatitis virus were treated with adenine arabinoside monophosphate or acyclovir monophosphate, either free or conjugated with the liver-targeting molecule lactosaminated human serum albumin. Circulating woodchuck hepatitis virus DNA levels remained unchanged in untreated animals and in those receiving the carrier lactosaminated human serum albumin alone; in contrast, they were consistently lower after 5 days of treatment with the antiviral drugs. Free and conjugated adenine arabinoside monophosphate were active at doses of 10 and 0.75 mg/kg, respectively, and free and coupled ACVMP were active at doses of 20 and 2.6 mg/kg, respectively. These results indicate that the dosages of adenine arabinoside monophosphate and acyclovir monophosphate required to inhibit hepadnavirus growth can be sharply reduced by coupling the drugs to lactosaminated human serum albumin. PMID- 1712339 TI - HBxAg in the liver from carrier patients with chronic hepatitis and cirrhosis. AB - Formalin-fixed, paraffin-embedded specimens from 110 cases of chronic hepatitis and 108 cases of cirrhosis were stained for HBxAg by the avidin-biotin complex technique using specific antisera made against full-length HBxAg polypeptide or derived synthetic peptides. These tissues were also stained for the HBsAg and HBcAg by the peroxidase-anti-peroxidase method. Among patients with chronic hepatitis, 86% were HBsAg positive in liver cells, 60% were surface antigen positive and 32% were core antigen positive. Among patients with cirrhosis, 97% were HBsAg positive in liver cells, 72% were surface antigen positive and 17% were positive for core antigen. Staining specificity was demonstrated, in part, by using preimmune sera in the place of primary antibody, by blocking of the primary antibody with the appropriate antigen before assay and by testing uninfected liver controls. The persistence and high frequency of HBxAg in liver cells from patients with chronic liver disease suggest that it may play one or more important roles in the pathogenesis of chronic infection. It is possible that detection of HBxAg in the liver could be an additional new diagnostic marker for hepatitis B virus infection. However, the function(s) of HBxAg in the pathogenesis of the chronic liver disease, if any, remains to be explained. PMID- 1712340 TI - IgM antibody to hepatitis C virus in acute and chronic hepatitis C. AB - To assess possible role of testing for IgM-specific antibody in the diagnosis and monitoring of patients with hepatitis C, we tested sera from 14 patients with acute and 97 patients with chronic non-A, non-B hepatitis for IgG and IgM antibody to hepatitis C virus. IgG antibody to hepatitis C virus was detected in 93% of acute cases and 91% of chronic cases. Of the 101 patients with IgG antibody to hepatitis C virus, 57% had IgM antibody to hepatitis C virus. None of the 20 healthy subjects or 40 patients with acute or chronic hepatitis A or hepatitis B had IgM antibody to hepatitis C virus. At the onset of clinical symptoms in acute hepatitis C, IgG antibody to hepatitis C virus was detected in 8 (57%) and IgM antibody to hepatitis C virus in 9 of 14 patients (64%). Eventually, both IgG and IgM antibody to hepatitis C virus became detectable in 13 of 14 patients with acute hepatitis C. Seven patients with antibody to hepatitis C virus resolved the acute infection within 6 mo and all seven cleared IgM antibody to hepatitis C virus, whereas two cleared IgG antibody to hepatitis C virus. Six patients had a chronic outcome of the acute infection and IgM antibody to hepatitis C virus persisted in detectable amounts for more than 6 mo in all (mean = 15.5 mo). Among 88 patients with chronic non-A, non-B hepatitis with IgG antibody to hepatitis C virus, IgM antibody to hepatitis C virus was detected in 45 (51%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712341 TI - Specificities of serum alpha-fetoprotein in HBsAg+ and HBsAg- patients in the diagnosis of hepatocellular carcinoma. AB - Serum alpha-fetoprotein level is often elevated in patients with chronic liver disease and patients with hepatocellular carcinoma. One of the most difficult problems frequently encountered in practice is differentiating hepatocellular carcinoma from chronic liver disease. This study investigated the specificity and predictive value positive of serum alpha-fetoprotein at various levels in the diagnosis of hepatocellular carcinoma, using 54 patients with histologically proven hepatocellular carcinoma and 200 patients with chronic liver disease (40 patients with chronic active hepatitis and 160 patients with cirrhosis) as nontumor controls. Among 254 patients, 170 (66.9%) were HBsAg+. A wide range of overlap (from 0 to 6,400 ng/ml) in the distribution of serum alpha-fetoprotein levels between hepatocellular carcinoma and chronic liver disease patients was observed mainly among HBsAg+ patients. In contrast, the overlapping range of serum alpha-fetoprotein levels between HBsAg- patients with hepatocellular carcinoma and chronic liver disease was remarkably narrow (from 0 to 200 ng/ml). Therefore the specificity and predictive value positive of alpha-fetoprotein at a given level were significantly lower in HBsAg+ than in HBsAg- patients, especially when alpha-fetoprotein was between 25 and 200 ng/ml. The specificities of alpha-fetoprotein at 200 ng/ml and 400 ng/ml in HBsAg+ patients were 79.8% and 91.5%, respectively, whereas these specificities were both 100% in HBsAg- patients. The predictive values positive at 200 ng/ml and 400 ng/ml in HBsAg+ patients were 53.6% and 72.5%, respectively, in contrast to 100% at both levels in HBsAg- patients. The serum alpha-fetoprotein level, which showed a predictive value positive of 95% in HBsAg+ hepatocellular carcinoma patients, was 3,200 ng/ml, whereas that in HBsAg- hepatocellular carcinoma patients, was 200 ng/ml. We conclude that serum HBsAg status should be considered when serum alpha fetoprotein is measured as an independent test to diagnose hepatocellular carcinoma, and suggest that regular serum alpha-fetoprotein determination may be more useful in HBsAg- patients with chronic liver disease for the early diagnosis of hepatocellular carcinoma than in HBsAg+ patients. PMID- 1712342 TI - Ehlers-Danlos syndrome type VII: a single base change that causes exon skipping in the type I collagen alpha 2(I) chain. AB - We have examined the procollagens and collagens produced by skin fibroblasts from a patient with Ehlers-Danlos syndrome type VII. The patient was heterozygous for an abnormal alpha 2(I) chain migrating with the approximate size of pN alpha 2(I) chains after pepsin digestion. Peptide mapping suggested that the abnormality was located at the amino-terminus of the alpha 2(I) chain. Quantitative analysis of the alpha 2(I) mRNA indicated loss of the exon 6 sequences, and subsequent polymerase chain reaction amplification of cDNA demonstrated a deletion of the 54 bp of exon 6 from some of the alpha 2(I) mRNA. Analysis of genomic DNA from the patient revealed a single base change in one COL1A2 allele, substituting an A for a G as the first base of intron 6. This change mutates the obligate GT dinulceotide splicing signal to AT and leads to exon skipping with splicing from exon 5 to exon 7. Loss of exon 6 sequences results in the loss of the procollagen N-propeptidase cleavage site and a lysine residue that normally participates in covalent intermolecular crosslinking within collagen fibres. PMID- 1712343 TI - Automatic segmentation and classification of ionic-channel signals. AB - Identification of ionic-channel types and their selectivity depends critically on the open channel current that can be resolved. In this paper, an automatic channel detection algorithm is proposed that is based on sequential minimization of an index which is usually used in cluster analysis. The algorithm consists of two stages, namely segmentation and classification. In the first stage, the signal samples are segmented based on the assumption that the samples in each segment should be sequentially connected. In the second stage, the resultant segments are classified with no regard to their connectivities. Results on synthetic and real channel currents are very encouraging and they suggest that this algorithm will substantially increase the productivity of many laboratories involved in ionic-channel research. PMID- 1712344 TI - Different epitopes on the dendritic cell-associated NLDC-145 molecule during ontogeny. AB - A monoclonal antibody, 6D2, is described that recognizes a different epitope on the NLDC-145 dendritic cell associated molecule in the mouse. During ontogeny the epitope appears on interdigitating cells in lymphoid organs only around birth, whereas the NLDC-145 antigen can be detected as early as day 16 of gestation. No differences can be observed in the expression of the two antigenic determinants on the epithelial cells of the thymus during ontogeny. Evidence is presented that the two antibodies recognize different epitopes on the same molecule. PMID- 1712345 TI - Variations in 3H-diisopropylfluorophosphate binding proteins in human seminal plasma. AB - We have characterized the electrophoretic pattern and variations in 3H diisopropylfluorophosphate (DFP)-binding proteins in human seminal plasma from normal men and from 103 patients attending the infertility clinic of our hospital. This study shows that 34 kDa prostate-specific antigen (PSA) is the major 3H-DFP-binding protein and that two other ubiquitous bands of 100 and 60 kDa are also present in seminal plasma from all the men studied. Additional bands of 92, 50-54 (doublet) and 38 kDa were also observed in some patients. The 38 kDa band was shown to be a highly glycosylated form of PSA. Further complexity was demonstrated by two-dimensional gel electrophoresis in the 27-30 kDa range of the gels since at least 10 major spots and rows of spots were seen. The concentration of these spots, including PSA, was extremely variable, as was their pattern of inhibition by various active site inhibitors of serine-proteases; these variations were not correlated with any specific sperm characteristics. With the exception of PSA, the proteins have not been identified. Their distribution suggests that most of them are exclusively of prostatic origin although a few could also derive from the seminal vesicles or blood. Future studies will be aimed at determining the nature of these proteins and their potential usefulness in andrology. PMID- 1712346 TI - Peptide-mediated allo-recognition of HLA-B27 by cytotoxic T lymphocytes. AB - The molecular basis of the recognition of class-I HLA antigens by allo-reactive cytotoxic T lymphocytes (CTL) remains obscure. This article reviews our work in which HLA-B27-specific allo-reactive CTL clones were obtained and their fine specificity was analyzed with a panel of structurally defined HLA-B27 natural variants and site-directed mutants expressed on human and mouse cells. The results have implications for the involvement of endogenous peptides in determining the clonal diversity of HLA-B27 allogeneic responses and the fine specificity of T-cell recognition when HLA-B27 is expressed on different cell types. PMID- 1712347 TI - Frequency of abnormal expression of HLA-A,B,C and HLA-DR molecules, invariant chain, and LFA-3 (CD58) in colorectal carcinoma and its impact on tumor recurrence. AB - HLA-A,B,C and HLA-DR molecules are involved in cognate LFA-3 (CD58) in antigen independent T-cell/target-cell interaction. T-cell-mediated host-versus-tumor response might therefore depend on the presence of both types of molecules on the surface of the target cell. To investigate whether presence or absence of these molecules in colorectal carcinoma influences the recurrence rate, 149 patients who underwent curative surgery were surveyed for a maximum of 65 months (mean, 48 months). As determined by immunohistochemistry, aberrant reduction of HLA-A,B,C determinants was observed in 34.9 and a complete loss in 8.7% of the tumor specimens. An induction of HLA-DR molecules was found in 55.0 and of the HLA-DR associated invariant chain (Ii) in 81.9%. An abnormal reduction of LFA-3 was detected in 43.6%, while a complete loss of this structure was observed in 6.7%. Reduction/loss of HLA-A,B,C was correlated with reduction/loss of LFA-3 (p = 0.03). In contrast to the prognostic role of tumor stage and grade, the presence vs. absence of all these structures was not correlated with the recurrence rate. We conclude that, although encoded on different chromosomes, an abnormal reduction/loss of HLA-A,B,C and LFA-3 might be the consequence of one transacting down-regulating signal. However, the resulting deviant immunophenotypes do not profoundly influence survival and growth potential of residual tumor cells. PMID- 1712348 TI - Five-decade international trends in the relation of perinatal mortality and congenital malformations: stillbirth and neonatal death compared. AB - The relation between long-term temporal trends in stillbirth and neonatal death rates and the congenital malformation frequencies in such deaths were analysed, using data from hospital-based European, USA, and Canadian reports published from 1950. In the last 50 years the overall perinatal mortality rate has fairly steadily improved, decreasing by 65-80%. This was accomplished by the control of some serious problems of early life. However, lingering disorders form an ever larger proportion of the causes of perinatal mortality. Among the prominent of these are congenital malformations, accounting for nearly 30% of perinatal deaths at present. However, this figure conceals important differences between stillbirths and early neonatal deaths. For example, although stillbirth and early neonatal mortality rates have decreased to similar extents during these years, congenital malformations, which were almost equally frequent causes of death in both of them at the beginning of this period, are now about twice as common in early neonatal (one week) deaths as in stillbirths. Other differences between them are in birthweight-related malformation frequencies and in characteristic arrays of malformations. The significance of these patterns and of some geographical variations, and the likelihood of continuing improvement in the stillbirth and early neonatal mortality rates are discussed. PMID- 1712349 TI - Hemipelvectomy in malignant neoplasms of the hip region. AB - The authors report the results of 76 hemipelvectomies performed from 1978 to 1988 in malignant neoplasms of the hip region. Several surgical techniques were employed, including King and Steelquist's "classic" technique (77%), the technique involving the anterior flap of the thigh (9%), and the technique involving the subcutaneous gluteal flap (14%). In 8 cases palliative surgery was performed. Of the remaining 68 patients, 31 (45%) are alive and show no signs of the disease after an average of 44 months. Postoperative complications are discussed in relation to surgical technique and previous adjuvant therapy; the subcutaneous gluteal flap technique exposes the patient to the greatest risk of major complications (54%), while the "classic" technique is the most reliable, although there was superficial infection in 18% of cases. Fifty percent of the patients previously treated with radiotherapy suffered local postoperative complications. In order to reduce local recurrence, special care is advised in the preoperative stages and in the execution of the pelvic osteotomies. PMID- 1712350 TI - Identification of the amino acid residues contributing to monoclonal antibody defined DQw1 epitopes. AB - The reactivity of monoclonal antibodies (mAbs) R1, S1, and S5, shown previously to recognize polymorphic epitopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQB1 alleles. mAb S5 reacts with cells expressing DQB1*0503, 0601, 0602, 0603, or 0604 alleles while R1 and S1 react with all DQB1 alleles except *0201 and 0301. In the case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitopes. This prediction has been confirmed by showing that a G ---E mutation in position 45 of the DQB1*0302 gene eliminates binding of both mAbs. PMID- 1712351 TI - Production of two human hybridomas secreting antibodies to HLA-DRw11 and- DRw8+w12 specificities. AB - In this study we describe the production of two human monoclonal antibodies (mAbs) HMP12 and HMP14, that recognize polymorphic HLA-DR specificities. These mAbs have been produced by hybridization of antibody-secreting Epstein-Barr virus transformed cells with SHM-D33 human-mouse heteromyeloma. By microcytotoxicity assay HMP12 mAb was found to react with all DRw11-positive cells and HMP14 mAb with all cells bearing the DRw8 or the DRw12 specificity. Cytotoxic activity of HMP14 was completely removed after absorption with DRw8- or DRw12-positive cells and unaffected by absorption with cells carrying different DR specificities. The HLA specificity was further analyzed by cytofluorometry on mouse transfectant cells. The reactivity of the two mAbs was correlated with the presence of a particular polymorphic amino acid residue in the DR beta chain and by this approach the epitopes possibly involved in the antibody binding sites were predicted. PMID- 1712352 TI - Effects of age and castration on activities of calpains and calpastatin in sheep skeletal muscle. AB - The objectives of this experiment were to assess effects of animal age and castration on activities of calpain I, calpain II, and calpastatin in sheep skeletal muscle. Ten newborn male lambs (2.9 kg), six weaned wethers (23.2 kg), six weaned rams (22.2 kg), six market wethers (55.4 kg), and six market rams (60.2 kg) were slaughtered and samples of biceps femoris were taken for assay of calpain I (micromolar calcium-dependent proteinase), calpain II (millimolar calcium-dependent proteinase), and calpastatin. Preweaning weight gain was similar for rams and wethers; however, postweaning ram growth exceeded (P less than .05) that of wethers. Ram biceps femoris weights at market were greater than those of wethers (P less than .05). Irrespective of age or gender, activity of calpain II was two- to threefold greater than that of calpain I. Muscle calpastatin activity was severa fold higher than calpain I and II activities. Activities of calpains and calpastatin declined (P less than .05) between birth and weaning. A portion of these losses were due to a dilution effect caused by accumulation of muscle proteins. Neonatal attenuation of calpain activities may underlie age-related attenuation of fractional rates of muscle protein degradation. Although ram muscle growth exceeded that of wethers, no differences (P greater than .05) in activities of muscle calpains or calpastatin were detected between these two groups at weaning or at market weight. Hence, castration did not influence lamb muscle growth by altering muscle calpain or calpastatin activities. PMID- 1712353 TI - Maternal and fetal influences on uterine and conceptus development in the cow: I. Growth of tissues of the gravid uterus. AB - Objectives of this study were to evaluate maternal and fetal influences on development of gravid uterine tissues of cows. Brahman cows with Brahman or Charolais fetuses and Charolais cows with Brahman or Charolais fetuses were used. Cows were killed 232 +/- .5 or 271 +/- .7 d after mating. The gravid uterus of each cow was weighed and dissected into its component parts. Weights of the fetus, fetal membranes, cotyledons, caruncles, and uterus were recorded as were weights of the fetal liver, heart, kidneys, spleen, lungs, stomach complex, intestines, and semitendinosus muscle. Ribonucleic acid, DNA, and protein concentrations in caruncles, cotyledons, liver, heart, kidney, and semitendinosus muscle were determined. Data were analyzed by analysis of variance; breed of cow (C), breed of fetus (F), day of gestation (D), and all interactions were included in the model as fixed effects. Fetal weights were influenced (P less than .003) by C, F, D, and C x D and tended (P = .07) to be influenced by C X F X D. Weight, RNA, DNA, and protein contents of selected fetal tissues followed similar patterns of significance. Thus, both maternal and fetal genotype influenced fetal growth. Greater influences of the maternal system and interrelationships between maternal and fetal systems were observed at the latter stage of gestation. Placentomal (caruncle + cotyledon) weights were greater for Charolais than for Brahman cows (P less than .02) or fetuses (P less than .001) and were greater (P less than .01) at 271 than at 232 d. Caruncular weights followed similar patterns; however, fetal genotype was the only significant source of variation in cotyledonary weight, RNA, DNA, or protein content.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712354 TI - Effect of fusaric acid on brain regional neurochemistry and vomiting behavior in swine. AB - Fusaric (5-butylpicolinic) acid is a phytotoxin produced especially by Fusarium moniliforme, a mold commonly found in Canadian-grown corn. Experiments were conducted to determine the effects of acute doses of fusaric acid on brain neurochemistry and behavior in swine. A total of 40 crossbred barrows (initial weight 10 kg) were orally dosed with 0 or 200 mg of fusaric acid/kg of BW and five animals from each treatment were killed 4.5, 9, 18, or 36 h after dosing. All brains were dissected, and concentrations of indoleamine and catecholamine neurotransmitters and metabolites were determined. Animals in the group killed 36 h after dosing were observed for behavioral changes. Vomiting was noted in 60% of the pigs dosed with fusaric acid. These pigs also seemed more lethargic than controls and appeared sedated. The major neurochemical changes due to exposure to fusaric acid were seen in the hypothalamus 18 h after dosing. Brain tryptophan, serotonin, and 5-hydroxyindoleacetic acid all tended to be elevated by the action of fusaric acid. Brain catecholamine concentrations were largely refractory to treatment. It was concluded that exposure to acute doses of fusaric acid can cause vomiting and neurochemical changes in swine. Fusaric acid may, therefore, be acting synergistically with trichothecene mycotoxins to cause vomiting and feed refusal in pigs consuming trichothecene-contaminated feedstuffs. PMID- 1712355 TI - Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae. AB - The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteriods compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two- to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown, negative regulation mechanism. PMID- 1712356 TI - Isolation, transcription, and inactivation of the gene for an atypical alkaline phosphatase of Synechococcus sp. strain PCC 7942. AB - The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein. Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake. PMID- 1712357 TI - Role and expression of the Bacillus subtilis rodC operon. AB - The role of the rodC operon in Bacillus subtilis was investigated. The operon encodes two genes (rodD and rodC) necessary for the synthesis of the cell wall teichoic acid. Transcription of this operon is responsive to levels of phosphate and to concentrations of magnesium ions in the growth medium. This regulation of mRNA production corresponds to conditions that dictate the type of polymer that will be synthesized for the cell wall, i.e., teichoic or teichuronic acid. While the introduction of multiple copies of rodC was tolerated by the cells, multiple copies of rodD appeared to be lethal. The lethality of the rodD fragment was not exhibited if multiple copies of rodC were also present. PMID- 1712358 TI - Nucleotide sequence of the Spiroplasma citri fibril protein gene. AB - Electron microscopic observation of spiroplasmas lysed by detergent (sodium deoxycholate) revealed the release of bundles of fibrils from the cells. Individual fibrils are 4 nm in diameter and possess a 9-nm periodicity along their length. These fibrils are thought to function as cytoskeletal structures involved in the shape and motility of spiroplasmas. Polyacrylamide gel electrophoresis of density gradient-purified fibrils showed a protein of approximately 55 kDa. Oligonucleotide probes were constructed from the N-terminal amino acid sequence of two peptides obtained after V8 protease hydrolysis of the fibril protein. The probes were used to identify the clones in a genomic DNA library of Spiroplasma citri that contained inserts carrying the probe sequence. Sequencing of a 3.3-kbp fragment yielded the full open reading frame of the fibril protein gene and the start of a second open reading frame of an unknown protein. The fibril protein is composed of 515 amino acids, which have a computed molecular mass of 59 kDa. Northern (RNA) blot hybridization and primer extension experiments showed that transcription of the fibril protein gene starts from a promoter located 100 nucleotides upstream of the initiation codon and stops at a rho-independent type terminator, leading to a 1.7-kbp transcript. Southern blot hybridization of genomic DNA using the fibril protein gene as the probe showed that a single copy of the gene is present in the chromosomes of both S. citri and Spiroplasma melliferum. The genotypic symbol fib is proposed for the spiroplasma fibril protein gene. PMID- 1712359 TI - Novel combination of epirubicin, bleomycin, vinblastine and prednisone (EBVP II) before radical radiotherapy in localized stages (I-IIIA) of Hodgkin's disease. Early results in 100 consecutive patients. Pierre-et-Marie-Curie Group. AB - A novel combination of epirubicin, bleomycin, vinblastine and prednisone (EBVP II) was scheduled to reduce the toxicity of chemotherapy and to improve its application in treatment of Hodgkin's disease. This combination followed a previous regimen given every 15 days (EBVP I) by the same cooperative group. EPVP II is given every 21 days with increased dosage and increased intensity of epirubicin. This regimen was given to 100 consecutive patients with favourable or unfavourable limited-stage disease (clinical stages I-IIIA) excluding very favourable stages I and II and stages IIIB and IV. Such patients first received three injections of EBVP II and were then radically irradiated; those with unfavourable prognosis factors received three subsequent injections of EBVP II. The present analysis reports the early results of such treatment and considers particularly toxicity and the obtention of complete remission, which is pre eminent for a cure. EBVP II was given in full dosage in 99% of the primary set of three injections. The main toxicity was alopecia and to a lesser degree nausea and vomiting and veinitis. Complete remission was obtained in 76 patients before radiotherapy and in 20 others after radiotherapy. With a median follow-up of 30 months 1 patient died from Hodgkin's disease, 9 are alive after relapse and 90 with no evidence of disease. This treatment appears to be as efficient as previous chemotherapy, well tolerated and particularly easy to give. It deserves further comparison with other proved regimens taking into consideration the survival and quality of life of patients. PMID- 1712360 TI - Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: identification of heparinoids as likely candidates. AB - Conditioned medium from Sertoli cells, prepared from testes of 20-day-old rats, contains component(s) that inhibit the incorporation of [3H]-thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin-resistant, heat-stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose-dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum-rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin-like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co-culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo. PMID- 1712361 TI - Competition ELISA using a human monoclonal antibody for detection of antibodies against human immunodeficiency virus type 1. AB - A novel competition ELISA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. coli-produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-1 antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV 1. PMID- 1712362 TI - Detection of human immunodeficiency virus type 1 transcripts in peripheral blood lymphocytes by the polymerase chain reaction. AB - A simplified application of the polymerase chain reaction (PCR) to the routine detection of human immunodeficiency virus type 1 (HIV-1) transcripts from peripheral lymphocytes of infected subjects is described. This technique is simpler than previously described assays and was shown to be highly sensitive after ethidium bromide staining of polyacrylamide gel electrophoresis of amplified material. The method can be used for the virologic evaluation of HIV-1 infected subjects, thus allowing early identification of seropositive patients with signs of active infection. PMID- 1712363 TI - The use of PCR for cloning of large cDNA fragments of turnip mosaic potyvirus. AB - A method is described whereby turnip mosaic virus RNA (TuMV RNA) was reverse transcribed and the resulting cDNA amplified enzymatically using the Taq DNA polymerase and degenerate oligonucleotide primers. Two degenerate oligonucleotide primers based on regions of homology in the amino acid sequence of the cytoplasmic inclusion protein and the nuclear inclusion b protein from five potyviruses were synthesized. Polymerase chain reactions utilizing these degenerate primers in association with specific primers amplified a 1.2 kb and a 3.3 kb fragment. These amplified fragments were dC-tailed and cloned into pUC9. Their partial sequence, when compared to potyvirus sequences, showed that they were derived from TuMV RNA and approximately 4.4 kb of viral genome was cloned. PMID- 1712364 TI - Autoantibodies to glycoprotein antigens mediate subacute demyelinating encephalomyelitis in the Lewis rat. AB - Serum autoantibodies were induced in Lewis rats by immunization with a mixture of lentil lectin-binding glycoproteins isolated from bovine brain myelin. Intraperitoneal administration of 2-10 million syngeneic myelin basic protein activated spleen cells to these rats led within 4-5 days to paralysis which, in most cases, persisted for several weeks. The major neuropathological features of the disease were numerous macrophages in both brain and spinal cord and large areas of demyelination, generally with axon preservation, particularly adjacent to the pial surfaces of the cord. This model is easily induced and will be useful for studies of demyelination and remyelination. PMID- 1712365 TI - Protection against experimental autoimmune encephalomyelitis requires both CD4+ T suppressor cells and myelin basic protein-primed B cells. AB - Suppressor cells that regulate experimental autoimmune encephalomyelitis (EAE) are present in rats that recover from the disease and can protect against the development of active EAE when transferred to normal recipients. Both CD4+ T suppressor cells, known to regulate EAE effector cell lymphokine production, and myelin basic protein (MBP)-primed B cells are required to transfer protection against EAE to normal recipients. Neither CD4+ T suppressor cells nor MBP-primed B cells alone could transfer protection. Moreover, the co-transfer of normal B cells with CD4+ T suppressor cells did not provide protection against EAE. These results suggest that the regulation of EAE and perhaps the recovery from acute clinical disease requires the interaction of two specific subpopulations of regulatory lymphocytes. PMID- 1712366 TI - CD21+ (B2 antigen+) cell decrement and CD4+CD29+ (helper-inducer) cell increment suggest an activation of cell immune reactivity in multiple sclerosis. AB - Two-color flow cytometric analysis on peripheral blood lymphocytes of 35 untreated multiple sclerosis (MS) patients, 17 other medical disease (OMD) patients and 14 healthy control (HC) subjects was performed to evaluate the levels of different T and B cell subpopulations. In MS patients we observed an increase in CD4+CD29+ helper-inducer cells but this increase was not related to the different phases of the disease. We hypothesize that this change is related to the reduction of CD21+ cells expressing B2 antigen, a 140 kDa molecule disappearing after B cell activation. An increased level of CD4+CD45RA- (helper inducer-like cells) and a reduction of CD4+CD29- (suppressor-inducer-like cells) were also present in our patients. These findings demonstrate an immune 'disequilibrium' in MS, which is linked with an increased level of CD25+ cells expressing the interleukin-2 (IL-2) receptor. IL-2, besides being a T cell growth factor, is also a B cell growth factor. These data let us hypothesize that an activation of the immune response is present in MS. PMID- 1712367 TI - ACP Broadsheet 128: June 1991. Laboratory methods for diagnosing cryptosporidiosis. PMID- 1712368 TI - Analysis of adhesion molecules in the immunopathogenesis of giant cell arteritis. AB - To explore the role of adhesion molecules in mediating mononuclear cell localisation, development of the granulomatous reaction, and cell mediated damage to the arterial wall in giant cell arteritis, 17 temporal artery biopsy specimens were examined. Eleven showed the histological features of giant cell arteritis and six showed no evidence of arteritis. All were examined for the expression of LFA-3, ICAM-1 and its receptor LFA-1, and HLA-DR. Temporal arteries with early features of arteritis, as well as histologically unaffected skip areas, showed a regional induction of ICAM-1 expression, but not HLA-DR, on smooth muscle cells of the media. ICAM-1 expression was detected in areas where a clinically important mononuclear cell infiltrate had not yet developed. In more florid cases of giant cell arteritis there was an additional widespread induction of ICAM-1 expression on intimal myofibroblasts. Strong expression of ICAM-1, HLA-DR, and LFA-3 was found on macrophages, epithelioid cells, and giant cells comprising the granulomatous lesion. The pattern of expression of these adhesion molecules suggests that they have a role in leucocyte traffic into the vascular lesion as well as in mediating the intercellular interactions which constitute the granulomatous response. PMID- 1712369 TI - Almost eleven million special children. AB - A review of the recent report on national rates for children with delayed development, learning disabilities and emotional problems is provided. The concerns of pediatric dentists are considered. PMID- 1712370 TI - Residue levels of chlorinated hydrocarbon compounds in water and sediment samples from Nile branches in the Delta, Egypt. AB - Water and sediment samples were collected from eight different locations along the River Nile and its branches. Residues of hexachlorocyclohexane (HCH's), hexachlorobenzene (HCB), DDT's, cyclodienes and polychlorinated biphenyls (PCB's) were analyzed by GLC. Data on Grand Total (GT) concentration values pointed out that Rosetta Branch was more polluted with all components than Demietta Branch. Kafr El-Ziate was the most polluted location showing 1355.8 ng/L for water and 7396.9 ng/g for sediments, while Delta Barrage was the least polluted site. The concentrations of gamma-HCH were higher than the other isomers (alpha- and beta HCH) in all studied sites. The results showed that HCB was the smallest pollutant at all locations on comparison with other chlorinated hydrocarbons. El-Mansoura, Rosetta and Kafr El-Ziate sites contained the highest concentrations of DDT's in both water and sediment samples. P,P'-DDE was dominate in all locations of water samples, but P,P'-DDT was in sediment samples. Also, the results showed the prominent presence of cyclodienes when compared with the other OC's compounds in sediment samples, especially Aldrin. Kafr El-Ziate was the most polluted location by PCB's, particularly the Ar1242. However, there were increasing levels of chlorinated hydrocarbons in the sediment samples parallel to percentage extractable organic matter (% EOM). Sediment/water ratios were calculated for all locations. PMID- 1712371 TI - Short-term antibiotic treatment in Whipple's disease. AB - We report the results of short-term antibiotic treatment in 19 patients with Whipple's disease (WD). The diagnosis was based on clinical features and on a characteristic small bowel biopsy. Patients received treatment for a mean of 7.9 weeks (range 4-20). Fourteen were treated with de-methyl-chlortetracycline (600 mg/day), and 1 also received chloramphenicol (1 g/day); 1 was treated with ampicillin (2 g/day), and 4 were treated with amoxicillin (1.5 g/day). In all patients, the clinical response was rapid and excellent, body weight increased significantly, diarrhea subsided, and fecal fat values returned to normal. Intestinal biopsies obtained after treatment was completed showed significant improvement based on a decrease in the number of macrophages staining positive with periodic acid-Schiff (PAS), normalization of villous structure, and decreased dilatation of lymphatic channels; free bacilli were absent, as shown both by light and electron microscopy. Seventeen patients have been followed for a mean of 99.4 months (range 6-300). Two died 30 and 72 months after diagnosis of Whipple's disease, 1 of laryngeal carcinoma and the other of colonic carcinoma. Fifteen patients are in excellent health. Three patients treated with tetracycline have had clinical and/or histologic relapses. In our experience, short-course antibiotic treatment with tetracycline or ampicillin and derivatives can be effective in WD, with few relapses and excellent outcome. No neurologic symptoms, either initially or during follow-up were observed. PMID- 1712372 TI - A crossreactive antipeptide monoclonal antibody with specificity for lysyl lysine. AB - Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable. PMID- 1712373 TI - [Molecular heterogeneity of alpha-fetoprotein in ovarian or extragonadal yolk sac tumor]. AB - The molecular heterogeneity of alpha-fetoprotein (AFP) in yolk sac tumor (YST) was investigated. The study included 14 sera from YST, 78 sera from primary hepatocellular carcinoma (PHC), 5 fetal yolk sac (YS) culture fluids and 4 fetal liver culture fluids. The microheterogeneity of AFP was assessed by differences in reaction with AFP carbohydrate chain and lectins, and concanavalin A (Con A), Lens culinaris hemagglutinin (LCH) and phytohemagglutinin E (PHA-E) affinity crossed-line immunoelectrophoresis was used to fractionate AFP. It was found that AFP in YST or YS had similar subfraction patterns and differed clearly from AFP in PHC or fetal liver. Characteristic features of AFP subfraction in YST or YS were the presence of a LCH weakly-reactive subfraction and a high proportion of both Con A non-reactive and PHA-E strongly-reactive subfractions. The LCH weakly reactive subfraction was specifically found in YST or YS, but was not found at all in PHC or in fetal liver. This variant is known to exist in amniotic fluid at an early stage of gestation, and is assumed to have a carbohydrate chain with both fucose and bisecting N-acetyl-glucosamine. The present findings therefore suggest that glycosylation of AFP in YST takes place by retro-genetic differentiation toward fetal yolk sac, but not toward fetal liver, and these studies confirm the suggested yolk sac origin of ovarian, as well as extragonadal, yolk sac tumor. PMID- 1712374 TI - Cu2(+)-mediated oxidation of dialyzed plasma: effects on low and high density lipoproteins and cholesteryl ester transfer protein. AB - We previously reported that the expression of an epitope of apolipoprotein B (apoB), mapped to the C-terminus and defined by antibody Bsol7, increased during Cu2(+)-mediated oxidation of isolated low density lipoprotein (LDL). We describe now the properties of Bsol7 as a marker of LDL oxidation in whole plasma in relation to other effects of oxidative treatment of plasma, such as the distribution of apoA-I and cholesteryl ester transfer protein (CETP). In dialyzed plasma, no LDL oxidation was detected at Cu2+ concentrations (5 microM) sufficient for extensive oxidation of isolated LDL. At a higher Cu2+ concentration (50 microM), an increased expression of the Bsol7 epitope was observed; at 250 microM Cu2+, other evidence of LDL oxidation was found. The pattern of LDL response to Cu2+ observed in dialyzed plasma could be reproduced by adding 3% bovine serum albumin to isolated LDL. We demonstrate that the effect of albumin most likely results from its ability to bind copper ions. Incubation of plasma with increasing concentrations of Cu2+ resulted first in the disappearance of alpha 2-migrating HDL, the usual carrier of CEPT; free CETP and high molecular weight apoA-I-containing particles were also generated during oxidation. Addition of oxidized, but not native, LDL to plasma resulted in a transfer to LDL of some of the CETP initially associated with apoA-I. In conclusion, the increased immunoreactivity of the Bsol7 epitope was the most sensitive parameter of LDL oxidation, but other parameters, such as the presence of alpha 2-HDL and CETP-lipoprotein associations were even more sensitive evidence of lipoprotein oxidation. PMID- 1712375 TI - Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen. AB - Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4 like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon aminocaproic acid affected the binding of any of our monoclonal antibodies. PMID- 1712376 TI - Half-body irradiation in multiple myeloma. PMID- 1712377 TI - Actin dynamics in growth cones. AB - The mechanism of actin incorporation and turnover in the nerve growth cone was examined by immunoelectron microscopy and low-light-level video microscopy of cultured neurons injected with biotin-labeled actin or fluorescently labeled actin. We first determined the sites of actin incorporation into the cytoskeleton of growth cones by immunoelectron microscopy of cultured neurons injected with biotin-labeled actin and reacted with an anti-biotin antibody and a gold-labeled secondary antibody. Shortly after the injection, biotin-actin molecules incorporated into the cytoskeleton were localized in the distal part of actin bundles in the filopodia and at the membrane-associated fringe of the actin filament network. With longer incubation, most actin polymers in the growth cones were labeled uniformly, suggesting that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. We then determined whether actin filaments translocate within the growth cones by low light-level video microscopy of living neurons injected with fluorescently labeled actin and photobleached with a laser beam. When actin fluorescence at the leading edge of a growth cone was bleached, a rearward translocation of the bleached spot toward the base of the growth cone was observed. This observation suggests the presence of a rearward flow of actin polymers within growth cones. Taken together, these results indicate that there is a continuous addition of actin monomers at the leading edge of the growth cone and a successive rearward translocation of the assembled filaments. PMID- 1712378 TI - Selectivity of the effects of guanosine-5'-O-(2-thiodiphosphate) on agonist inhibition of the M-current in amphibian sympathetic neurons. AB - In bullfrog sympathetic neurons, luteinizing hormone-releasing hormone, muscarine, and substance P act as agonists at specific membrane receptors to decrease a potassium current, IM. The receptors are coupled to guanine nucleotide binding proteins (G-proteins). Whole-cell recordings of IM were made from isolated bullfrog sympathetic neurons to examine the effects of intracellularly applied guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) on agonist inhibition of IM. Successive responses to a given agonist were decreased in the presence of GDP beta s. Subsequent responses to the other agonists were then measured to determine the degree of overlap of the effect of GDP beta S for the different agonists. GDP beta S selectively inhibited successive responses to one agonist such that a subsequent application of a different agonist was still effective. If GDP beta S acts at the level of the G-protein, this suggests that each receptor is coupled to a separate population of G-proteins. Alternatively, GDP beta S may act at the receptor level to block receptor coupling to IM. PMID- 1712379 TI - Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts. AB - A culture system of "giant" Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiated into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers (Wu et al., 1990). Here, we report that these giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibited distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca(2+)-activated K+ currents. In addition, we found a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke (slo), which is known to lack a component of the Ca(2+)-activated K+ currents in muscles, exhibited prolonged action potentials associated with defects in the Ca(2+)-activated K+ current. This abnormality appeared to be more severe in the neurites than in the soma. PMID- 1712380 TI - Catechol oxidation by peroxidase-positive astrocytes in primary culture: an electron spin resonance study. AB - In rodents, chronic estrogenization has been shown to induce degeneration of dendrites and myelin figures in the hypothalamic arcuate nucleus adjacent to peroxidase-positive astrocyte processes. Because in this brain region estradiol is metabolized to 2-hydroxyestradiol (catecholestrogen), we hypothesized that the latter may be oxidized by the astrocytic peroxidase activity to cytotoxic ortho semiquinones as occurs in peripheral tissues. Cysteamine induces nonenzymatic peroxidase activity in cultured astroglia identical to that observed in vivo. Using electron spin resonance, we demonstrate robust peroxidase-catalyzed oxidation of 2-hydroxyestradiol and dopamine by cysteamine-pretreated astrocyte cultures relative to untreated controls. These results implicate the peroxidase positive astrocytes in the pathogenesis of estradiol-related hypothalamic damage, parkinsonism, and other free-radical-related neurologic disorders. PMID- 1712381 TI - Characterization of dopamine release in the substantia nigra by in vivo microdialysis in freely moving rats. AB - Dopamine (DA) is released not only from the terminals of the nigrostriatal projection, but also from the dendrites of these neurons, which arborize in the substantia nigra pars reticulata (SNR). Although striatal DA release has been extensively studied by in vivo microdialysis, dendritic DA release in the SNR has not been characterized by this technique. Extracellular DA was monitored simultaneously in the ipsilateral striatum and SNR. The nigral probe was implanted at a 50 degree angle, permitting 2.5 mm of SNR to be dialyzed. Delivery of the tracer Fluoro-Gold into the striatal probe retrogradely labeled tyrosine hydroxylase-positive cell bodies and dendrites in the vicinity of the nigral probe. Hence, it could be demonstrated that dopaminergic neurons near the nigral probe projected to the vicinity of the striatal probe. Addition of 50 mM KCl to the SNR perfusion solution produced a 3.5-fold increase in DA and a 50% reduction in dihydroxyphenylacetic acid (DOPAC) in the SNR; in contrast, this manipulation in the SNR caused DA release in the striatum to be decreased by 20%, while striatal DOPAC was increased by 50%. Local administration of nomifensine (10 microM) in the SNR produced a sevenfold increase in SNR DA but had no effect on striatal DA. Systemic injection of d-amphetamine (2 mg/kg, s.c.) elevated DA in the SNR and striatum five- to sevenfold, while DOPAC was decreased in both structures by at least 40%. To determine the effect of tetrodotoxin (TTX), basal concentrations of DA in the SNR were first elevated threefold by including nomifensine (1 microM) in the nigral perfusion solution.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712382 TI - Voltage-dependent calcium currents in Purkinje cells from rat cerebellar vermis. AB - Whole-cell patch clamp recording was used to characterize calcium currents in Purkinje cells dissociated from the cerebellar vermis of 1-3-week postnatal rats. A subset of Purkinje cells had a low-threshold, transient current similar to the T-type current in peripheral neurons. All Purkinje cells had a high-threshold, slowly inactivating current. Only a small component of the high-threshold current was sensitive to dihydropyridine (DHP) antagonists or to the dihydropyridine agonist BAY K8644. omega-Conotoxin had very little effect on the high-threshold current. The results suggest that these Purkinje cells have at least three types of calcium channels: T-type channels (present in only a fraction of cells), DHP sensitive L-type channels (contributing a small fraction of the high-threshold current), and a predominant type of high-threshold channel that is pharmacologically distinct from L-type and N-type channels characterized in peripheral neurons. PMID- 1712383 TI - The clinical utility of prostate-specific antigen and bone scintigraphy in prostate cancer follow-up. AB - To assess the value of serum prostate-specific antigen (PSA) in prostate cancer follow-up, we prospectively studied 107 consecutive patients with: (1) pathologically confirmed prostate cancer; (2) definitive prostatectomy and/or radiation therapy greater than or equal to 3 mo prior to bone scanning; and (3) one bone scan and serum PSA sampling within 3 mo of each other. The mean and range of patient follow-up since definitive therapy was 1.6 and 0.5-8 yr, respectively. Abnormal bone scans were correlated with pertinent radiographs. Of 107 bone scans, 16 demonstrated metastatic bone disease. A PSA value of less than or equal to 8 ng/ml excluded bone metastases with a predictive value of a negative test of 98.5%. Without radiographic correlation, abnormal bone scans rarely represented metastases if the PSA value was less than or equal to 8 ng/ml. In summary, serum PSA concentration determines the need for follow-up bone scanning and assists in scan interpretation in patients status post definitive therapy for prostate cancer. PMID- 1712384 TI - Evaluation of housestaff physicians' preparation and interpretation of sputum Gram stains for community-acquired pneumonia. AB - OBJECTIVE: To evaluate the preparation and interpretation of sputum Gram stains by housestaff physicians in the assessment of patients with community-acquired pneumonia. DESIGN: A prospective, multicenter study. SETTING: Two university affiliated hospitals in Pittsburgh. PATIENTS: Ninety-nine cases of clinically and radiographically established pneumonia occurring in 97 patients. Diagnostic test assessment: Housestaff and microbiology personnel prepared a Gram stain for each case of pneumonia. Housestaff assessed the presence and identity of a predominant microbial organism on the slides they prepared. Two senior staff microbiologists, blinded to patient and preparer, evaluated all slides for preparation, sputum purulence, and identification of the predominant organism. Two reference standards were used to assess the sensitivity, specificity, and predictive values of housestaff's Gram-stain interpretations: 1) senior staff microbiologists' determinations of the microbes present using the slides without benefit of culture results, and 2) the etiologic agent derived from results of sputum culture, blood culture, or serology. MEASUREMENTS AND MAIN RESULTS: Housestaff physicians completed a Gram stain in 58% of the pneumonia episodes. Gram stains were not made in 42% of cases, primarily because patients were unable to produce sputum. Fifteen percent of housestaff's smears were judged inadequately prepared, compared with 3% for the laboratory personnel (p less than 0.01). Housestaff obtained purulent sputum samples significantly more often than did nursing personnel (58% versus 38%; p less than 0.01). Housestaff's Gram stains were 90% sensitive for detecting pneumococcus, with a 50% false-positive rate. The sensitivity of the Gram stain was less for identification of Haemophilus influenzae than for identification of Streptococcus pneumoniae. A single antimicrobial agent was chosen as initial therapy for 50% of the patients in whom housestaff identified a predominant organism, compared with 30% in whom a predominant organism was not identified (p less than or equal to 0.05). CONCLUSIONS: Although housestaff obtained purulent sputum samples more frequently than did nursing personnel, they made systematic errors in the preparation and interpretation of Gram-stained slides. Housestaff physicians should receive formal training in the preparation and interpretation of Gram stains; the specific defects elucidated in this study warrant special attention. PMID- 1712385 TI - The sputum Gram stain. PMID- 1712386 TI - Histological findings in rabbit lymph nodes after endolymphatic injection of liposomes containing blue dye. AB - Liposomes produced from phosphatidylcholine and cholesterol and containing Patent Blue V have been injected endolymphatically in the rabbit. The lymph nodes stained dark blue and retained this colour until termination of the experiment after 28 days. No toxic effects were observed histologically. The liposomes were deposited within the macrophages in a fine granular pattern. Because endolymphatic injection of such liposomes produces excellent contrast between retroperitoneal lymph nodes and their surrounding fatty tissue, it can improve both the radicality and the selectivity of lymphonodectomy in oncological surgery. PMID- 1712387 TI - A fluorescence method for the determination of the molecular weight cut-off of alginate-polylysine microcapsules. AB - Several applications of microcapsules for the encapsulation of living cells or macromolecules require well defined pore sizes. The molecular weight cut-off of alginate-polylysine microcapsules has been determined using a range of fluorescent labelled dextran molecules. The diffusion of the fluorescein isothiocyanate labelled (FITC)-dextrans into the microcapsules was followed by fluorescence and confocal laser scanning microscopy. The permeability of microcapsules for FITC-dextrans with a molecular weight of 4,700 daltons and the impermeability for FITC-dextrans with a molecular weight of 40,500 daltons was confirmed with both techniques. Determination of the molecular weight cut-off, using confocal laser scanning microscopy was more reliable and required a smaller sample than fluorescence measurements. PMID- 1712388 TI - [Pharmacological study on citrus fruits. II. Anti-allergic effect of fruit of Citrus unshiu MARKOVICH (2). On flavonoid components]. AB - A study was carried out to clarify the active component of green fruit of Citrus unshiu MAKOVICH on the type I allergic reaction. The flavonoid component, isolated from its methanolic extract, hesperidin, was found to inhibit histamine release from peritoneal mast cells of rats induced by compound 48/80. Forty eight hour homologous passive cutaneous anaphylaxis (PCA) in intact rats was significantly inhibited by the oral administration of hesperidin. However, the anti-allergic action on PCA was not observed in adrenalectomized rats. These results suggests that hesperidin is an effective component of the fruit of Citrus unshiu with the anti-allergic action against the type I reaction. PMID- 1712389 TI - Demonstration of Epstein-Barr virus in scrape material of lateral border of tongue in heart transplant patients by negative staining electron microscopy. AB - Scrape material from the lateral border of the tongue of 50 heart transplant patients and 20 controls was studied for the presence of EBV by negative staining electron microscopy. Mild oral hairy leukoplakia was observed in two cases. Particles of the herpes virus were found in 20% of the specimens. Controls were negative for EBV. The study has shown that EBV may be expressed at the lateral border of the tongue during immunosuppression, occasionally resulting in the clinical appearance of hairy leukoplakia. PMID- 1712390 TI - Epithelial lining of sinus tracts associated with periapical disease: an immunocytochemical study using monoclonal antibodies to keratins. AB - We examined 30 specimens of mucosal sinus tracts arising in association with periapical inflammation. Immunocytochemistry and a panel of monoclonal antibodies to keratins were used to demonstrate the epithelial lining and its pattern of keratin expression. Half of the specimens were found to have an epithelial lining that was continuous with the mucosal epithelium and showed various degrees of extension into the sinus. The presence of an epithelial lining appeared to correlate with the duration of the sinus tracts. The immunostaining patterns of the epithelial linings were similar to those of the mucosal epithelium near the sinus opening although some differences were found. Proliferating strands of epithelium, presumably derived from the rest of Malassez, were observed deep in the tissues but were not continuous with the epithelial lining. The results suggest that the epithelial lining of sinus tracts is derived not from epithelial rests in the periapical region but from the mucosal epithelium adjacent to the opening of the sinus tract and that the observed changes in the pattern of keratinization are due to the influence of inflammation or of the connective tissue substrate. PMID- 1712391 TI - Effects of temperature on color stability of porcelain stains. AB - The effects of oven firing on the color stability of extrinsic stains used for characterization and color modification of metal ceramic restorations were studied by comparing the color of the stain as initially applied with that observed after it was fired to the fusion temperature. Perceptible changes were noted for all stains studied. Multiple firings of the stain showed only minor effects on the observed color. The effects at fusion temperatures of 1700 degrees F and 1775 degrees F appeared similar. Finally, there appeared to be no marked differences between the color change observed with the use of autoglazing and overglazing techniques. PMID- 1712392 TI - An NMR and theoretical study of the conformation and internal flexibility of butaclamol hydrochloride. AB - A theoretical (MM2) and experimental (1H and 13C NMR) study of butaclamol hydrochloride in CDCl3 has been done in order to determine preferred conformations and internal molecular flexibility of this molecule. The theoretical calculations suggest the presence of four low-energy conformations, two of which involve a trans junction of the D and E rings, with the other two involving a cis I ring junction. An alternative cis junction (cis II) was excluded on energetic grounds. The 1H NMR data strongly suggest the presence of a trans D-E ring junction and are consistent with a chair conformation of the E ring. 13C spin-lattice relaxation time measurements show that most of the molecule is rigid, although there is some degree of mobility in the seven membered B ring, associated with rapid flipping of the bridging C8 and C9 carbons between two skewed conformations, which have previously been referred to as conformer A and conformer B (Laus et al. Heterocycles 1984, 22, 311). PMID- 1712393 TI - NMR studies of the conformational interconversion of butaclamol in solution. AB - 1H NMR experiments at 300 MHz have been carried out to determine the identity and study the interconversion of two conformations of butaclamol in solution. The hydrochloride salt in DMSO exists as an equilibrium mixture of two conformations, which differ in their stereochemistry about the ring junction that contains the single nitrogen atom in butaclamol. The trans form has a relative population of 80% and the cis I form 20%. In CDCl3 only the trans form is observed, while in CDCl3-DMSO mixtures, both forms are detected in a ratio (trans:cis I) that decreases as the percentage of CDCl3 decreases. For the free base in either CD2Cl2 or DMSO, only a single set of resonances is observed at room temperature, but as temperature is lowered, peaks from methine protons H4a and H13b near the ring junction broaden and (for samples in CD2Cl2) eventually split into two resonances corresponding to the cis and trans forms. It is suggested that nitrogen inversion is the dynamic process responsible for the interconversion of the two forms. Line shape analysis as a function of temperature yielded an energy barrier of 9.6 +/- 0.5 kcal/mol for the interconversion, in good agreement with values obtained from saturation transfer experiments. In the hydrochloride salt, the barrier in DMSO was somewhat higher, i.e., 17.3 +/- 0.9 kcal/mol, as determined by saturation transfer and variable-temperature measurements. PMID- 1712394 TI - Novel thiophene-, pyrrole-, furan-, and benzenecarboxamidotetrazoles as potential antiallergy agents. AB - The synthesis and antiallergic activity of a series of novel thiophene-, pyrrole , furan-, and benzenecarboxamidotetrazoles are described. A number of compounds inhibit the release of histamine from anti-IgE-stimulated human basophils. Optimal inhibition is exhibited in compounds with a 3-alkoxy, a 4-halo, and a 5 methyl, 5-methoxy, or 5-bromo on a thiophene-2-carboxamidotetrazole. PMID- 1712395 TI - Novel non-nucleoside inhibitors of HIV-1 reverse transcriptase. 1. Tricyclic pyridobenzo- and dipyridodiazepinones. AB - Novel pyrido[2,3-b][1,4]benzodiazepinones (I), pyrido[2,3 b][1,5]benzodiazepinones (II), and dipyrido[3,2-b:2',3'-e][1,4]diazepinones (III) were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in vitro at concentrations as low as 35 nM. In all three series, small substituents (e.g., methyl, ethyl, acetyl) are preferred at the lactam nitrogen, whereas slightly larger alkyl moieties (e.g., ethyl, cyclopropyl) are favored at the other (N-11) diazepinone nitrogen. In general, lipophilic substituents are preferred on the A ring, whereas substitution on the C ring generally reduces potency relative to the corresponding compounds with no substituents on the aromatic rings. Maximum potency is achieved with methyl substitution at the position ortho to the lactam nitrogen atom; however, in this case an unsubstituted lactam nitrogen is preferred. Additional substituents on the A ring can be readily tolerated. The dipyridodiazepinone derivative 11 cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2',3'-e] [1,4]diazepin-6-one (96, nevirapine) is a potent (IC50 = 84 nM) and and selective non-nucleoside inhibitor of HIV-1 reverse transcriptase, and has been chosen for clinical evaluation. PMID- 1712396 TI - Potent inhibitory effect of a series of modified cyclodextrin sulfates (mCDS) on the replication of HIV-1 in vitro. PMID- 1712397 TI - Characterization and sequence of the Escherichia coli stress-induced psp operon. AB - We describe a new Escherichia coli operon, the phage shock protein (psp) operon, which is induced in response to heat, ethanol, osmotic shock and infection by filamentous bacteriophages. The operon includes at least four genes: pspA, B, C and E. PspA associates with the inner membrane and has the heptad repeats characteristic of proteins that can form coiled coils. The operon encodes a factor that activates psp expression, and deletion analyses indicate that this protein is PspC; PspC is predicted to possess a leucine zipper, a motif present in many eukaryotic transcription factors. The pspE gene is expressed in response to stress as part of the operon, but is also transcribed from its own promoter under normal conditions. In vitro studies suggest that PspA and C are modified in vivo. Expression of the psp genes does not require the heat shock sigma factor, sigma32. The increased duration of psp induction in a sigma32 mutant suggests that a product (or products) of the heat shock response down-regulates expression of the operon. PMID- 1712398 TI - Morphometric mapping of regional myocyte diameters after healing of myocardial infarction in cats. AB - Myocyte diameters were measured in two models of healed myocardial infarction to test the hypothesis that myocyte hypertrophy is a function of proximity to the infarct. Left ventricular transmural and non-transmural myocardial infarctions were produced in cats by multiple ligatures of the distal tributaries of the left coronary artery system. Thirteen to twenty months after surgery the left ventricular free wall was cut longitudinally, embedded in plastic and stained for reticulum with modified silver stain. Myocardial cell diameters were measured from apex to base through the infarct. No regional differences were found in non operated control hearts. In the transmural infarct hearts, all cell diameters were significantly increased in comparison to controls (P less than or equal to 0.05). In the hearts with non-transmural infarcts, cell diameters were significantly increased in tissues adjacent to the infarct, but as distance from the infarct increased the cell diameters were not different from controls. Cells from the transmural infarctions had a greater percent increase in diameter, compared to controls, than did cells from the non-transmural infarctions. There is a gradient increase in myocyte diameters in transmural and non-transmural healed myocardial infarctions; this increase is greatest in the tissues adjacent to the infarct. We conclude that cells close to a healed myocardial infarction hypertrophy because they are contracting against a non-compliant scar. PMID- 1712399 TI - Workshop on screening for hepatocellular carcinoma. PMID- 1712400 TI - Symptom control studies: an important part of cancer control research. PMID- 1712401 TI - [Cellular responses by cytokines--gene regulation in the IFN system]. AB - Interferon (IFNs), as a class of antiviral cytokines, are also known as "negative growth regulators," they inhibit the growth of a variety of normal and malignant cells. Normally, Type I IFNs (i.e. IFN-alpha, -beta) are not induced, but viruses and a number of other cytokines transiently activate the IFN genes. In order to elucidate the molecular mechanisms of cellular responses by viruses and cytokines, we have identified two nuclear factors, IRF-1 and IRF-2, both bind to the regulatory cis-elements of IFN and IFN-responsive genes. The genes encoding IRF-1 and IRF-2, have been cloned and extensively characterized. The IRF cDNA expression studies in factor-negative cells have revealed IRF-1 and IRF-2 to function as transcriptional activator and repressor, respectively. In normal cells, the IRF genes are subject to induction through stimuli such as viruses and cytokines including IFNs per se. The findings provide evidence for the presence of an elaborate network of cytokines system wherein the IRFs play a crucial role for the cytokine-mediated cellular responses. PMID- 1712402 TI - [Recent progress in the treatment of acute leukemia]. AB - Acute leukemia has become a curable disease. In 3 studies for adult AML (BHAC DMP, BHAC-DMP (II) and M-85) at Nagoya University Hospitals from 1979 to 1987, intensive induction resulted in higher cure rate, and the reduction of the blasts in bone marrow at 2 weeks after the initiation of therapy to less than 20% was the most important prognostic factor to predict the long CR. However, it seemed impractical to give very intensive chemotherapy during the induction because of high frequency of complications due to prolonged myelosuppression. Thus, consolidation should be as intensive as possible. In M-85 protocol, the predicted 5-years survival and disease-free survival (DFS) of CR cases are 70 and 53% respectively. The result of JALSG-AML 87 study seems to confirm the above result. As for the indication of bone marrow transplantation (BMT) at the first CR for adult AML, only a prospective randomized study will answer this important question. In case that DFS of chemotherapy will exceed 40 to 45%, it seems be wise to give chemotherapy first, and then BMT when the leukemia relapse. Differentiation induction therapy seems to be indicated in acute promyelocytic leukemia, although a confirmative study is awaited. PMID- 1712403 TI - [FK 506, a new immunosuppressant produced by a Streptomyces]. PMID- 1712404 TI - Effects of pretreatment with basic fibroblast growth factor, epidermal growth factor and nerve growth factor on neuron survival and neovascularization of superior cervical ganglion transplanted into the third ventricle in rats. AB - Effects of short-term treatment with basic fibroblast growth factor (FGF), epidermal growth factor (EGF) and nerve growth factor (NGF) on neurite outgrowth of superior cervical ganglia (SCG) in culture or on neuron survival and neovascularization of SCG in a transplantation system were examined in rats. SCG were preincubated with FGF, EGF and/or NGF for 30 min and cultured with drug-free medium for 2 days. FGF or EGF neither promoted neurite outgrowth of SCG nor potentiated the NGF-induced neurite elongation in culture. In the transplantation study, SCG were exposed to these factors for 30 min and grafted into the third ventricle of adult rats for 14 days. Although pretreatment of NGF, FGF, EGF or the combination of NGF and EGF were not effective, there was better neuron survival in SCG grafts pretreated with 1 micrograms/ml NGF and 1 micrograms/ml FGF together. In addition, density of capillaries in these grafts was significantly greater than in the other group tested. These results suggest that the synergistic interaction between NGF and FGF cause rapid neovascularization, which can prevent neuron death after transplantation. PMID- 1712405 TI - Muscarinic receptor subtype in isolated dog and monkey facial vein. AB - Using the cannula inserting method, we investigated vascular responses to ACh and McN-A-343 (a M1-agonist) in isolated and perfused canine and simian facial veins in non-preconstricted conditions. ACh usually induced only a vasoconstriction, but McN-A-343 did not induce any significant vasoconstriction. It is concluded that canine and simian facial veins contain very few receptors of the muscarinic M1-subtype; and according to previous studies, these vessels have abundant M3 receptors. PMID- 1712406 TI - [Rationale for using kinin system inhibitors in patients with acute ischemia and infarction of the myocardium during the prehospital phase]. AB - The paper discusses if it is advisable to use kallikrein-kinin system inhibitors earlier. A total of 122 patients with acute ischemia and infarction of the myocardium were studied. Contrykal and heparin infused in the prehospital period, followed by hospital treatment are optimal to prevent vascular wall lesions, maximally retain retrograde blood flow, and substantially reduce the size of myocardial infarction. Therapy with protease inhibitors proved to be most beneficial within the first 2 hours of the disease, as evidenced by a profound improvement in the clinical course of myocardial infarction, a decrease in ECG and precordial mapping signs of ischemia, a positive change in myoglobin and MB creatine phosphokinase levels, and a significant reduction in the rehabilitative period. PMID- 1712407 TI - Expression of homeobox genes in a proximal tubular cell line derived from adult mice. AB - We have been studying the expression of several homeobox genes in cultures of proximal tubular epithelium (MCT cells) harvested from adult mus musculus. Hox genes 2.1, 2.3, and 3.3, in particular, are all expressed at low levels in resting MCT cells. The expression of Hox 2.1 and 3.3 were not influenced by mitogenic (epidermal growth factor: EGF, and platelet-derived growth factor: PDGF) nor by hypertrophogenic cytokines (angiotensin II: Ang II) in serum-free media. Transcripts for Hox 2.3, however, were elevated in MCT cells by Ang II. EGF, and serum treatment, as early as 30 minutes after their addition, whereas no change, or slight reductions were observed with transforming growth factor beta (TGF beta), PDGF, and gamma-interferon (gamma IFN). Hox 2.3 was also super induced by serum, in the presence of cycloheximide, in cells rested previously in serum-free media, suggesting that new protein synthesis was not required for expressive augmentation. The induction of Hox 2.3, moreover, was not specific for tubular epithelium, since the gene could be activated in tubulointerstitial fibroblasts after treatment with EGF. These experiments collectively represent a first report regarding the characterization of transcripts encoding homeoboxes in adult cells derived from renal tissue. The putative DNA-binding properties of homeobox proteins in general, the prompt and rapid induction of Hox 2.3 by morphogenic cytokines in tubulointerstitial cells, and the observed effect of cycloheximide on this gene, all indicate that Hox 2.3 might have a role in the general activation of mature somatic cells, as an immediate early event. probably in the capacity of a nuclear trans-acting factor. PMID- 1712408 TI - Proto-oncogene expression in peripheral blood mononuclear cells in IgA nephropathy. AB - We have investigated the expression of proto-oncogenes in mononuclear cells obtained from patients with IgA nephropathy using a RNA hybridization technique. Patients with IgA nephropathy expressed more c-myc, c-raf, c-fos, and c-jun proto oncogene RNA than did normal controls. However, no significant expression of c-N ras, c-mos or c-myb genes was found in the mononuclear cells of these patients. When the amount of urinary protein excretion was used as an indicator of disease activity (greater than 1 g/day), a positive correlation was found between c-myc, c-raf, c-fos, and c-jun expression and urinary protein excretion (P less than 0.01). The expression of these genes correlated also with the serum IgA concentration (P less than 0.01), IgA immune complex (P less than 0.01), and histopathological changes in renal tissues obtained from patients with IgA nephropathy (P less than 0.01). The results of this survey suggest that abnormally regulated proto-oncogene expression in mononuclear cells may play an important role in the progression of IgA nephropathy and may be useful as an indicator of disease activity and/or prognosis. PMID- 1712409 TI - [Advisability of using donor blood immunoglobulin preparations for treating infections caused by Pseudomonas aeruginosa]. AB - Resulting from the studies of 623 series of the donor blood immunoglobulins, it was established that 19.2% of the series of a commercial preparation put on the market by the Republican blood service contain the natural antibodies to O antigen and antigens of the Pseudomonas aeruginosa extracellular mucus at the titer of 1:80 and more. Series of the immunoglobulin preparation with a high content of specific antibodies can be used for the treatment of purulent-septic complications caused by Pseudomonas aeruginosa. PMID- 1712410 TI - [Change in nucleic acid metabolism in patients with acute purulent processes]. AB - In 127 patients with purulent-inflammatory processes of different location, the DNA concentration in the blood serum increased at the period of pronounced local manifestations of an inflammatory process and intoxication of an organism, in elimination of a focus of inflammation--decreased sharply. RNA concentration in the blood serum increased after the operation in intensive development of granulation tissue. The level of inorganic phosphorus decreased. Concentration of the uric acid changed inconsiderably. This evidenced in favour of intensity of the regulatory mechanisms, which control the nucleic metabolism. PMID- 1712411 TI - [Use od visual teaching aids at the department of surgery]. PMID- 1712412 TI - [Status of the proteinase-protease inhibitor system in patients with pathological scars]. AB - In patients with pathological scars, especially in children, the activity of proteolytic enzymes (prekallikrein, acid and neutral proteinases) is increased. Summary inhibitory activity and content of the alpha 2-macroglobulin in the serum and scar tissue is also increased. This is indicative of continued inflammation. PMID- 1712413 TI - [A flip-flop model of the chloride channel complex explains the dysregulation of the chloride flow in the plasmalemma of cells in cystic fibrosis]. AB - The basic defect in cystic fibrosis is the chloride impermeability of the plasmalemma in different cells. A candidate for the chloride channel, thought to be affected in the syndrome, is "Porin 31HL" recently described by us. The molecule is i) expressed in the plasmalemma of different cells, it has ii) a molecular mass of 31,000 Daltons, it shows iii) high conductance in artificial membranes and it can be iv) modified by 4,4'-Diisothiocyanatostilbene-2,2' disulfonate. A porin in the outer membrane of cells should furthermore v) be regulated by modulators. All these characters of "Porin 31HL" correspond to those given in literature for chloride channels. The regulation of the channels can be explained by a two component flip flop model. PMID- 1712414 TI - High rate of false positives in blood donor screening for antibodies to hepatitis C virus. Cause of underestimation of virus transmission rate? AB - Anti-hepatitis C virus antibody screening of blood donors in different countries revealed prevalences ranging from 0.4-1.4%. These results were obtained with an enzyme immunoassay based on a recombinant hepatitis C virus antigen. We applied a specific inhibition assay (neutralization assay) and a recombinant immunoblot assay to determine the specificity of positive reactions in the enzyme immunoassay. Of 2836 blood donor sera tested, 10 (0.35%) were reactive in the enzyme immunoassay, however, only 3 sera (0.1%) proved to be specifically anti HCV positive in the inhibition assay. The recombinant immunoblot assay gave similar results. The prevalence of anti-hepatitis C virus antibodies among blood donors has been overestimated in recent publications. Furthermore the high rate of false positives in the enzyme immunoassay may explain reports claiming that only a minor part of EIA positive blood units transmitted the hepatitis C virus to recipients. The inhibition assay was also applied to sera of haemophiliacs and of patients with hepatopathy which had reacted positively in the anti-hepatitis C virus antibody enzyme immunoassay. The anti-hepatitis C virus specificity was confirmed for all sera from the haemophiliacs group (100%) and for 77% of the hepatopathy patients group. Thus, the anti-hepatitis C virus enzyme immunoassay has a high predictive value when it is used to screen groups with high risks of parenteral hepatitis C virus infection, however, its predictive value is very low when it is used for blood donor screening. PMID- 1712415 TI - Endoscopic gastrointestinal laser therapy. PMID- 1712416 TI - A cell culture assay for the detection of cardiotoxicity. AB - An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. We propose the use of "shock protein" formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing "shock protein" formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of "shock proteins." Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes "shock protein" formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce "shock protein" synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive. PMID- 1712417 TI - Purity of ruthenium red used in pharmacological research. AB - The inorganic dye, ruthenium red, is an ammoniated form of ruthenium oxychloride. The purity of commercial samples of ruthenium red has been confused as commercial vendors often provide a "dye content" figure for their product, which some investigators have equated with purity. However, dye content is the same as ruthenium content and is not related to purity, although it will affect the relative molecular weight of this compound. As a result, concentrations of ruthenium red used in various biochemical studies have been calculated based on the supposed impurity of the commercial product, a product purified in the laboratory of the investigator, or by simply ignoring this purity question. Purity of four different commercial products as well as two different "purified" materials was determined by comparison of absorbance spectra and extinction coefficients. Taking into account differences in the relative ruthenium content of each preparation of ruthenium red, the results demonstrate no significant differences between these materials indicating that commercially available samples of ruthenium red are essentially pure. PMID- 1712418 TI - Aprotinin in cardiac surgery. PMID- 1712419 TI - [High-dose immunoglobulins for the treatment of rheumatoid arthritis: pilot study of 7 cases]. AB - High intravenous doses (400 mg/kg) of gammaglobulin (IVIG) were administered monthly for six months to 7 patients with severe rheumatoid arthritis (RA). In all cases, previous treatment with NSAIDs and corticosteroids and in 3 of them with gold and/or methotrexate had been ineffective. A 50 per cent improvement of Ritchie index was obtained in 6/7 patients, morning stiffness was reduced from greater than 2 hours to less than 30 minutes in 6/7 patients. Swollen joints and Lee index improved in all patients. ESR did not show any change but RCP improved in 6/7 patients. The study of lymphocyte subpopulation showed no substantial changes in CD20+, CD3+, CD4 and CD8 cells as well as in CD4/CD8 ratio and a significant increase in 2H4+T cells without changes in 4B4+ subpopulation. IVIG improved the clinical and laboratory features of patients with severe RA. The major problem raised by IVIG therapy is its high cost suggesting that this therapy should only be applied in well selected patients with RA. PMID- 1712420 TI - [Macroglobulin alpha-2 in synovial fluid: relationship with reactants of the acute phase of rheumatoid arthritis]. AB - Metalloproteinases (e.g. collagenase, elastase, stromelysin) are present in large amount in synovial fluid (SF) during rheumatoid arthritis (RA) and are actively involved in articular tissue damage. alpha 2-Macroglobulin (alpha 2M) functions as a "molecular trap" for proteinases and is considered the major inhibitor of metalloproteinases. We found increased concentrations of alpha 2M in SF of RA patients, significantly related to acute phase reactants, local inflammatory parameters and joint damage. The alpha 2M ratio between, RA SF and control SF, was found higher than between RA serum and control serum, indicating a selective localization and activity of alpha 2M in inflamed joint. The relationship between alpha 2M and the inflammatory parameters, including IL-6, is discussed. PMID- 1712421 TI - The use of gradient flow compensation to separate diffusion and microcirculatory flow in MRI. AB - This paper describes a new MR imaging technique termed Modified Stejskal Tanner versus Flow Compensation (MST/FC) for the separation of diffusion and microcirculatory flow. The theory behind the sequence is explained, along with a five-component model of microcirculation applicable to any "perfusion" imaging technique. Phantom data is presented showing that (1) diffusion effects can be matched between MST and FC (suggesting the possibility of flow-compensated diffusion imaging), and (2) the technique is a quantitative method of separating diffusion and slow (less than 0.25 mm/s) tortuous flow through a Sephadex column. Furthermore, animal images show the technique to be feasible and quantitative in measuring rat brain microcirculation under normal, vasodilated (hypercarbia), and no-flow (post mortem) conditions. PMID- 1712422 TI - The present role of prostatic specific antigen (PSA) as a tumor marker in carcinoma of the prostate. AB - Prostatic specific antigen (PSA) is an important tumor marker for prostate cancer and is being used more frequently in men suspected of having this disease. Although PSA cannot be recommended by itself for either screening or staging of prostate cancer, its present role is discussed by the author. PMID- 1712423 TI - Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair. AB - The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of DNA repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of DNA repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3 aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced DNA damage leads to specific types of mutations. PMID- 1712424 TI - Human cells (normal and ataxia telangiectasia) transfected with pR plasmid are hypersensitive to DNA strand-breaking agents. AB - Ataxia telangiectasia (AT) cells are known to be hypersensitive to ionizing radiations and to drugs such as bleomycin and epipodophyllotoxin VP16, a topoisomerase II poison. Both of these produce DNA double-strand breaks even if through different mechanisms. In this work we analyzed the sensitivity to bleomycin and to epipodophyllotoxin of AT cells after transfection with pR plasmid. This plasmid, interacting with bacterial SOS repair pathways, expresses itself in mammalian cells conferring cell resistance to the SOS inducers UV and 4NQO and cell sensitivity to different drugs such as bleomycin. This effect is presumably due to the interaction of pR products with double-strand breaks. Our findings indicate that pR plasmid, in both AT lines tested (AT5BIVA fibroblasts and ATL6 lymphoblasts), expresses itself (increasing UV protection) and amplifies the already enhanced AT cell sensitivity to both bleomycin and VP16. PMID- 1712425 TI - Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells. AB - HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4 specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein. PMID- 1712426 TI - Investigation of selected toxicological parameters of gamma pentachlorocyclohexene (gamma-PCCH). AB - The toxic effects of the gamma-hexachlorocyclohexane metabolite gamma pentachlorocyclohexene (gamma-PCCH) were studied by acute and subacute (6 weeks) experiments. The investigations included cerebral convulsibility with chemoshock (Tetrazolium), reactivity with hot plate method, the learning ability with learning tests, and peripheral nervous activity (EMG). Nociceptive reaction time was not influenced, the learning process (6 weeks) was inhibited by gamma-PCCH. The conduction velocity of the peripheral nerve was decreased. At the end of the 6th week liver enlargement was found. PMID- 1712427 TI - Acceleration of activation and inactivation by the beta subunit of the skeletal muscle calcium channel. AB - The L-type voltage-dependent calcium channel is an important link in excitation contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current. PMID- 1712428 TI - In vivo effects of some histamine H1-receptor antagonists on monoamine metabolism in the mouse brain. AB - The in vivo effects of four H1-antagonists, diphenhydramine, chlorpheniramine, mepyramine, and promethazine, on the metabolism of noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) were investigated in the whole mouse brain. Diphenhydramine and chlorpheniramine had no significant effect on levels of NA, 3 methoxy-4-hydroxyphenylethyleneglycol (MHPG), DA, and 5-HT, but they significantly decreased levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA). In particular chlorpheniramine markedly decreased 5-HIAA levels at doses as low as 1 mg/kg, i.p. Mepyramine significantly decreased 5-HIAA levels but not those of other substances. High doses of promethazine significantly decreased NA levels but markedly increased those of MHPG, DOPAC, HVA, 5-HT, and 5-HIAA. The DA reduction induced by alpha-methyl-p-tyrosine (alpha-MT) was significantly inhibited by diphenhydramine, chlorpheniramine, and promethazine, but the alpha-MT-induced NA decrease was significantly enhanced by promethazine. The 5-HIAA accumulations induced by probenecid were significantly inhibited by chlorpheniramine and mepyramine. These results suggest: (1) Diphenhydramine and chlorpheniramine inhibit the turnover of both DA and 5-HT by blocking their neuronal uptake. (2) Promethazine and mepyramine inhibit DA and 5-HT turnover, respectively, as a result of the inhibition of the uptake mechanism. (3) Promethazine increases NA turnover by enhancing NA release. The discriminative effects of these drugs on the monoamine systems may be related to some differences in their CNS actions. PMID- 1712429 TI - In vivo release of neuronal histamine in the hypothalamus of rats measured by microdialysis. AB - Using an in vivo intracerebral microdialysis method coupled with an HPLC fluorometric method, we investigated the extracellular level of endogenous histamine in the anterior hypothalamic area of urethane-anaesthetized rats. The basal rate of release of endogenous histamine in the anterior hypothalamic area measured by this method was 0.09 +/- 0.01 pmol/20 min. When the anterior hypothalamic area was depolarized by infusion of 100 mM K+ through the dialysis membrane or electrical stimulation at 200 mu A was applied through an electrode implanted into the ipsilateral tuberomammillary nucleus, histamine release increased to 175% and 188%, respectively, of the basal level. These increases were completely suppressed by removal of extracellular Ca2+. The basal release of histamine was also suppressed after infusion of 10(-6) M tetrodotoxin or i.p. administration of 100 mg/kg of alpha-fluoromethylhistidine. On the other hand, 3 fold increase in the basal release was observed after i.p. administration of 5 mg/kg thioperamide. These results clearly indicate that both the basal and evoked release of histamine measured by our method are of neuronal origin. PMID- 1712430 TI - Characterization of NK-3 binding sites in rat and guinea pig cortical membranes by the selective ligand [3H]Senktide. AB - We have used [3H]Senktide, a selective Neurokinin B receptor ligand, for the characterization of NK-3 receptors in rat and guinea pig CNS membranes. Scatchard analysis of saturation binding studies in cerebral cortex membranes indicated that this ligand bound to a single site with apparent high affinity (KD = 4.6 +/- 1.6 and 3.1 +/- 0.37 nM, Bmax = 13.7 +/- 1.6 and 21.8 +/- 2.2 fmol/mg protein in rat and guinea pig membranes, respectively). However, in competition studies with a group of neurokinins and related peptides two different rank orders of affinities were obtained, as follows: NKB greater than [MePhe7]-NKB greater than or equal to Arg0-NKB greater than or equal to Senktide much greater than NKA greater than SP, in rat membranes, and [MePhe7]NKB greater than Senktide = NKB greater than Arg0-NKB much greater than SP greater than NKA, in guinea pig membranes. PMID- 1712431 TI - Modulation of neural bronchoconstrictor responses in the guinea pig respiratory tract by vasoactive intestinal peptide. AB - Vasoactive intestinal peptide (VIP) is localised to cholinergic nerves in airways. We have investigated the effects of VIP on both cholinergic and non adrenergic, non-cholinergic (NANC) neuronal bronchoconstrictor responses to electrical field stimulation (EFS) in guinea pig airways and on cholinergic neurotransmission following sensory nerve depletion. VIP significantly attenuated the cholinergic bronchoconstrictor responses to EFS in trachea (EC50 values in upper and lower trachea of 3.7 +/- 0.2 x 10(-9) M and 8.6 +/- 0.3 x 10(-9) M, respectively) and bronchi (31.2 +/- 1.6% inhibition in main and 15.1 +/- 3.3% in hilar bronchi at 10(-7) M VIP) and the NANC bronchoconstrictor responses to EFS in bronchi (with maximum inhibitions of 93.1 +/- 1.8% at 3 x 10(-8) M VIP in main and 40.2 +/- 5.3% at 10(-8) M in hilar bronchi). VIP at 10(-7) M, but not at 10( 10) M, significantly attenuated the contractile responses to exogenously applied ACh in trachea (EC50 values of 4.9 +/- 0.2 x 10(-6) M in the absence and 8.4 +/- 0.4 x 10(-5) M in the presence of VIP 10(-7) M VIP) to SP in main bronchi (EC50 values of 5.7 +/- 0.2 x 10(-8) M in the absence vs. 7.3 +/- 0.3 x 10(-7) M in the presence of 10(-7) M VIP). Since the inhibition of these neural responses is greater than the inhibition of the equivalent responses elicited by the exogenous transmitters, this indicates that VIP may modulate release of acetylcholine from cholinergic nerves and of neuropeptides from sensory nerves, in addition to a post-junctional functional antagonist action. PMID- 1712432 TI - Substance P and substance K in the rat hypothalamus following monosodium glutamate lesions of the arcuate nucleus. AB - Adult rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Following MSG lesioning, dopamine content in median eminence/arcuate nucleus (ME/AN) tissue extracts declined by 60 70%. Substance P (SP) content as determined by radioimmunoassay was significantly decreased in the paraventricular nucleus (PVN) (531 +/- 30 pg, mean +/- SEM) compared to controls (871 +/- 110 pg) but was unchanged in ME/AN extracts. Substance K (SK) content decreased to 257 +/- 20 pg in the PVN of lesioned animals compared to controls (367 +/- 31 pg) and the ME/AN content of SK was also significantly decreased (236 +/- 36 pg compared to control levels of 619 +/- 65 pg). The CRF-41 content of the PVN and ME/AN was unchanged by MSG lesioning, indicating that these areas are not affected by MSG. The partial depletion of SP and SK in the PVN following MSG treatment provides evidence that at least some of the neurokinin content of the PVN may originate in cell bodies of the arcuate nucleus. However, the lack of response of ME/AN SP to MSG treatment may suggest that the arcuate nucleus is not the major source of SP in the median eminence. PMID- 1712433 TI - Both intravenous lidocaine and morphine reduce the pain of postherpetic neuralgia. AB - We studied the analgesic efficacy of an intravenous infusion of lidocaine and morphine in 19 adults with well-established postherpetic neuralgia in a three session, randomized, double-blind, placebo-controlled trial. Compared with saline placebo, both lidocaine and morphine reduced pain intensity. Reductions in pain did not correlate with side effects produced by the infusions. For morphine, there was a significant correlation between reductions in pain intensity and blood level achieved. In the majority of subjects who reported definite pain relief, allodynia also disappeared. The results show that neuropathic pain can respond to opioids and to systemically administered local anesthetic drugs. PMID- 1712434 TI - [Behavior of serum angiotensin converting enzyme in hyperthyroidism correlated to that of TSH]. AB - The study examined the relations between serum levels of angiotensin-converting enzyme and those of thyroid-stimulating hormone in a group of hyperthyroid patients, and the respective therapy. The study continued for 12 months, from the onset of disease until remission. From an analysis of the results it was seen that levels of SACE and TSH during the first 4 months were significantly different to those in normal subjects: levels of SACE were increased, whereas TSH levels had fallen. This difference gradually diminished over the course of the following 8 months, and SACE and TSH values returned to within normal limits. The increment of SACE may depend on the presence of damage to the vascular endothelium within and surrounding the thyroid, in addition to intense vasodilation caused by the hyperthyroid state. The correlation between increased SACE levels and the course of disease is further confirmed by the fact that levels returned to within normal values during thyrostatic therapy; this was also observed in the case of TSH. PMID- 1712435 TI - Retrograde neuronal labeling of motoneurons in the rat by fluorescent tracers, and quantitative analysis of oxidative enzyme activity in labeled neurons. AB - Extensor digitorum longus motoneurons in the rat spinal cord were identified by retrograde labeling with two fluorescent tracers, Fast blue (FB) and Nuclear yellow (NY). Labeled motoneurons had a blue fluorescent cytoplasm at 360 nm excitation wavelength with FB, and a golden-yellow fluorescent nucleus with NY on the cryostat section. Labeled motoneurons were further examined for succinate dehydrogenase activity on the same section used for identification of the motoneurons. This study demonstrates that fluorescent dyes are useful for neuroanatomical studies by the retrograde axonal transport method, and that quantitative analysis of metabolic activity in labeled motoneurons is also possible. PMID- 1712436 TI - Pharmacological blockade of Cl- pumps or Cl- channels reduces the adenosine mediated depression of stimulus train-evoked Ca2+ fluxes in rat hippocampal slices. AB - Stimulus train-evoked decreases of the extracellular Ca2+ concentration (delta Ca) were measured with ion-sensitive electrodes in the CA1 area of rat hippocampal slices. The adenosine receptor antagonist, theophylline, led to a marked increase of delta Ca in the synaptic and in the soma layer reflecting an increased activation and enhanced frequency potentiation of pyramidal neurons in the absence of endogenous adenosine action. The theophylline effect was significantly reduced in the presence of the C1 channel blocker, 4,4' diisothiocyano-2,2'-stilbenedisulfonate (DIDS) or of the Cl- pump blocker, furosemide. The data indicate that the adenosine-mediated modulation of the repetitive input strength is related to the function of Cl- ions. PMID- 1712437 TI - Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy. AB - The effects of peripheral axotomy (sciatic nerve transection) on the presence and distribution of neuropeptide Y (NPY) in rat dorsal root ganglion (DRG) and spinal grey matter were examined using immunocytochemistry. In normal rats and on the sham-operated side of experimental rats, NPY-like immunoreactivity (NPYir) was observed in all laminae of the lumbar spinal cord, with an especially dense concentration of immunostained axons and axonal varicosities in laminae I-II of the dorsal horn. There was no detectable NPYir in L4-L5 DRG cells from normal rats or from the sham-operated side of experimental rats. At 14 days after axotomy, there was a large ipsilateral increase in the density of NPYir axons and varicosities in the lumbar spinal cord on the side of the nerve injury; this was especially apparent in laminae III-V. In the same rats, NPYir was observed in many small, medium, and large neurons in the L4-L5 DRGs on the side of the severed nerve. PMID- 1712438 TI - Evidence that extremely low frequency Ca(2+)-cyclotron resonance depresses pineal melatonin synthesis in vitro. AB - The effects of ion cyclotron resonance (ICR)-type magnetic fields on pineal glands were investigated. Both the synthesis and the release of pineal melatonin, the gland's major hormone, were significantly (P less than 0.001 in each case) reduced by Ca(2+)-ICR-exposure, presumably caused by a reduced activity (P less than 0.05) of the enzyme N-acetyltransferase. It is concluded that this type of exposure may help to explain some of the effects of electromagnetic fields on biological systems. An extension to the existing ICR theory is presented which explains that ICR-like conditions may occur under various environmental circumstances. PMID- 1712439 TI - Cellular localization of gap junction mRNA in the neonatal rat brain. AB - Cellular localization of grap junction mRNA was examined in the neonatal rat brain by in situ hybridization histochemistry using cDNA of rat liver gap junction (connexin 32). Hybridizable gap junction mRNA was localized on neural cells in the hippocampus. Gap junction mRNA was also found to be localized on neural cells of the parenchyma and ependymal layers in many other regions. Immunohistochemical and ultrastructural observations of the hippocampus supported the presence of gap junctions in the examined region. These results are the first demonstration of morphologically identified gap junctions and cellularly localized gap junction mRNA in the neonatal rat brain. PMID- 1712440 TI - 3-((+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) more potently antagonizes the high-affinity Mg2+ binding site on the N-methyl-D aspartate/phencyclidine receptor ion channel complex than the L-glutamate recognition site. AB - Using frozen-thawed and extensively washed rat cortical membranes, the effects of 3-((+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) on [3H]N-(1-[2 thienyl]cyclohexyl)-3,4-piperidine ([3H]TCP) binding stimulated by either 1 microM L-glutamate or 300 microM Mg2+ were examined. CPP much more potently inhibited Mg(2+)-stimulated [3H]TCP binding than [3H]TCP binding stimulated by L glutamate, suggesting that CPP preferentially acts at Mg2+ recognition sites with high affinity, which may be anatomically and/or functionally associated with a recognition site for N-methyl-D-asparate (NMDA) antagonists. PMID- 1712441 TI - Extremely high maternal serum alpha-fetoprotein levels at second-trimester screening. AB - Maternal serum alpha-fetoprotein (MSAFP) screening is widely used for the detection of open neural tube defects (NTDs) and a variety of other anomalies and complications. We examined the outcomes of 44 pregnancies with MSAFP elevations of 8 or more multiples of the median (MoM) from among 40,676 screened pregnancies. At the initial evaluation by ultrasound, 82% of the patients had at least one finding that may have accounted for the elevation. Approximately 45% of the fetuses had a major fetal anomaly, 25% died, 16% had an identifiable placental abnormality, and 5% had an underestimation of gestational age; 18% of the elevations remained unexplained after ultrasound. In follow-up of the pregnancies, all of those with an unexplained elevation after initial ultrasound had at least one obstetric complication or placental abnormality. The overall positive predictive value of an MSAFP value of 8 or more MoM for NTDs was 22.7%. The proportion of infants born alive in the overall group was low, with only 16 live births among 46 fetuses. The majority of the nonviable outcomes were associated with a fetus with a major anomaly that was terminated or died before 20 weeks. Of the live-born infants, 31% had a major anomaly, 19% had intrauterine growth retardation (IUGR) and an anomaly, 12.5% had IUGR without an anomaly, and 25% were preterm. Eighty-eight percent of those pregnancies with a live-born infant had at least one obstetric complication. Among pregnancies with MSAFP of 8 or more MoM, the majority are associated with large structural fetal anomalies or fetal death before 20 weeks.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712442 TI - Elevated maternal serum alpha-fetoprotein and mild fetal uropathy. AB - Sixty-one consecutive patients referred because of elevated maternal serum alpha fetoprotein (MSAFP) levels and 80 referred for second-trimester ultrasound for other reasons were examined. Ultrasound examination of the genitourinary tract and assignment of phenotypic sex was done by ultrasonographers blinded to the MSAFP results. Among male fetuses with elevated MSAFP, 33% had pyelectasis compared with only 5% of controls. Among female fetuses, pyelectasis was seen in 16% of cases and no controls. Increased MSAFP not caused by an open neural tube defect may be seen in conjunction with mild benign uropathy in the second trimester. PMID- 1712443 TI - The involvement of protein kinase C in mediating growth suppressive signals of interferons in hematopoietic cells. AB - The possible involvement of protein kinase C in transducing the growth suppressive signals of interferons was studies in this work in two different hematopoietic cell lines. Chronic exposure of human Burkitt lymphoma and mouse M1 myeloblastic cell lines to phorbol myristate acetate (PMA), reduced by more than 90% the PKC protein levels and enzymatic activity in cell extracts. The depletion of PKC from cells abrogated the ability of IFN (alpha + beta) to arrest cell growth at the G0/G1 resting phase of the cell cycle. In contrast, other responses to IFN such as the induction of (2'-5') oligoadenylate synthetase gene, continued to take place at the same dose response pattern thus excluding the possibility that early targets in the pathway, such as the number or affinity of IFN cell surface receptors might be affected by PMA. The same prolonged treatment of M1 cells with PMA did not interfere with the ability of another cytokine, transforming growth factor beta (TGF-beta), to induce the normal type of G0/G1 arrest further supporting the specificity of the effect towards IFN responses. Unexpectedly, depletion of PKC from cells did not interfere with the negative effects of IFN on c-myc mRNA and protein expression in spite of the direct involvement of this molecular event in growth responses to IFN. The putative PKC dependent molecular event could therefore function either downstream to or in combination with the reduction in c-myc protein levels, providing a necessary but not a sufficient step to arrest cell cycle progression at the G0/G1 phase. PMID- 1712444 TI - Effect of the myb gene product on expression of the PCNA gene in fibroblasts. AB - Tk-ts13 cells are BHK-derived fibroblasts that have a G1-specific temperature sensitive (ts) mutation so that the cells arrest in the G1 phase of the cell cycle at the restrictive temperature of 39.6 degrees. We have introduced into these cells a plasmid carrying the human c-myb cDNA under the control of the early SV40 promoter. The resulting cell line, ts13 myb cells, express the c-myb RNA and protein and, when serum-stimulated, they undergo one round of DNA replication even at the restrictive temperature. Under the same conditions, the parent cell line (tk-ts13 cells) are blocked in G1 and fail to replicate DNA. We have investigated the expression of two late growth-regulated genes, PCNA and histone H3, in tk-ts13 and in ts 13 myb cells, both at permissive and restrictive temperature. At the permissive temperature of 34 degrees, the mRNA levels for PCNA and histone H3 increase markedly after serum stimulation in both cell lines, reaching a peak at the time of DNA synthesis. At the restrictive temperature of 39.6 degrees, the mRNA's for PCNA, and histone H3 are detectable in serum stimulated ts13 myb cells while they are not detectable in the parent tk-ts13 cell line. Run-on transcription assays indicate that these 2 genes are transcribed in tk-ts13 cells equally well at permissive and nonpermissive temperatures, and that the presence of the myb product does not significantly increase their rates of transcription. The stability of the respective mRNA's is roughly the same at either temperature and in both types of cells. These results indicate: (1) a constitutively expressed c-myb gene product confers to fibroblasts the ability of temporarily by-passing a ts block in the G1 phase of the cell cycle and (2) the myb product, under these conditions, regulates the mRNA levels of PCNA and histone H3 either directly or indirectly by a post transcriptional mechanism. PMID- 1712445 TI - A 43 kD c-mos protein is only expressed before meiosis during rat spermatogenesis. AB - We have investigated the RNA and protein expression pattern of the rat c-mos proto-oncogene during spermatogenesis. In mouse testis a 43kD c-mos protein is expressed throughout spermatogenesis, which is in agreement with one report detecting a 1.7 kb c-mos RNA in pachytene spermatocytes and in early spermatids. However, several other reports show that the mouse 1.7 kb c-mos RNA is exclusively expressed in post-meiotic male germ cells. We report that in rat male germ cells three c-mos RNA species of 5, 3.6 and 1.7 kb are detectable by Northern blotting analysis both before and after meiosis, with highest levels in early spermatids. However, western immuno-blot analysis reveals the presence of a 43 kD c-mos protein in total testis and pachytene spermatocytes, but not in post meiotic cells. These findings combined with those made in the mouse system strongly suggest that c-mos protein may be a regulator of meiosis during spermatogenesis. PMID- 1712446 TI - [Interdisciplinary "developmental pediatrics" management principle--comments on the future of "social pediatrics"]. AB - Preconditions for social pediatrics are derived from the principles of developmental pediatrics. Own experience through the last 15 years have shown that social problems with their solutions con be an important basis for the improvement of a complete social pediatrics. PMID- 1712447 TI - [Use of zinc and parlodel for treatment of physical and sexual development retardation in kidney diseases and rheumatoid arthritis]. PMID- 1712449 TI - In memorium: Michel Mirowski, M.D. 10/14/24-3/26/90. PMID- 1712448 TI - Michel Mirowski: a man with a mission. PMID- 1712450 TI - Bibliography of publications Mieczyslaw (Michel) Mirowski. PMID- 1712451 TI - Personal comments written about Michel Mirowski on behalf of the family. PMID- 1712452 TI - Michel Mirowski and the Department of Medicine at the Sinai Hospital of Baltimore. PMID- 1712453 TI - Michel Mirowski's thesis on mitral commissurotomy. PMID- 1712454 TI - Regulatory issues in the development and future of the AICD. PMID- 1712455 TI - Future studies with the implantable cardioverter defibrillator. PMID- 1712456 TI - Coalition for the prevention of sudden cardiac death. PMID- 1712457 TI - Sudden cardiac death, implanted defibrillation, and clinical electrophysiology. AB - A mere 25 years ago, the technique of external defibrillation became the starting point for the development of clinical electrophysiology by permitting routine use of endocavitary programmed electrical stimulation of the heart without undue risk. Major advances in knowledge of clinical arrhythmias and the understanding of their mechanisms were, thus, permitted. Mirowski's implanted defibrillator also constituted a major breakthrough therapeutically; unfortunately, however, some 10 years later, it has not yet induced similarly hoped for consequences in terms of progressing knowledge concerning lethal arrhythmias, largely due to the absence of Holter functions in the implanted devices. As a result of this, in our opinion, better established therapeutic indications are still needed. The reasons for the present situation, we believe, may be partly technical but are conceptual as well. The key point is that even the clear demonstration of the great practical efficacy of a therapeutic tool does not exempt us from the obligation of determining the mechanisms of this effect. PMID- 1712458 TI - Potential interactions between antiarrhythmic medication and the automatic implantable cardioverter defibrillator. PMID- 1712459 TI - The industrialization of the AICD. PMID- 1712460 TI - Collaboration with Michel Mirowski on the development of the AICD. PMID- 1712461 TI - Abnormal atrial rhythms, an early interest of Michel Mirowski. PMID- 1712462 TI - Multicenter automatic defibrillator implantation trial (MADIT): design and clinical protocol. MADIT Executive Committee. PMID- 1712463 TI - Building the AICD with Michel Mirowski. PMID- 1712464 TI - Pathophysiology of sudden cardiac death. PMID- 1712465 TI - The role of signal averaged electrocardiography in identifying patients at high risk for lethal ventricular tachyarrhythmias. PMID- 1712467 TI - State-of-the-art of the AICD. PMID- 1712466 TI - The surgical aspects of automatic implantable cardioverter-defibrillator implantation. AB - Since February 1980, the automatic implantable cardioverter-defibrillator (AICD) has been implanted in over 12,000 patients. This report examines the surgical aspects of AICD implantation. The various surgical techniques, their indications, and our recent clinical experience in over 250 patients are reviewed. PMID- 1712468 TI - Elevated levels of substance P in intraocular fluid in proliferative vitreoretinopathy. AB - Proliferative vitreoretinopathy is the most common reason for failure in retinal reattachment surgery. Since both substance P (SP) and SP receptors were found to be present in the human eye, and as pharmacological studies suggest an importance of SP for ocular functions, we investigated intraocular fluids for the presence of SP in eyes elected for cataract surgery, retinal detachment surgery and retina surgery for severe proliferative vitreoretinopathy (PVR) as well as in eyes with proliferative diabetic retinopathy (PDR). High performance liquid chromatography and radioimmunoassay (RIA) for SP immunoreactivities were performed. The SP mean concentration in intraocular fluid (IOF) of patients for cataract surgery (CS) was 2.2 fmol/ml, for retinal detachment (RD) was 2.7 fmol/ml and for PDR was 1.9 fmol/ml; significantly higher levels (mean concentration of 26.9 fmol/ml) were measured in eyes with PVR. HPLC analysis revealed two immunoreactive peaks coeluting with synthetic SP and SP-sulfoxide, indicating that RIA values represent authentic SP. We conclude that SP may play an important role in PVR. Since SP antagonists are known to inhibit a variety of SP effects in the eye, there might be a useful tool to reveal the importance of SP in this disease and, in this instance, a new possible treatment. PMID- 1712469 TI - Calcitonin gene-related peptide, neurokinin A and substance P: effects on nociception and neurogenic inflammation in human skin and temporal muscle. AB - Calcitonin gene-related peptide (CGRP) was injected alone and in combination with substance P (SP) or neurokinin A (NKA) into the forearm skin and temporal muscle of human volunteers. In the skin, 50 pmol of CGRP induced a wheal response and a delayed erythema. No pain was recorded. No interaction between CGRP and SP or NKA was observed. In the temporal muscle, 200 pmol of CGRP alone did not induce pain or tenderness but, in combination with SP or NKA, CGRP elicited a significant pain sensation. It is concluded that CGRP may be involved in neurogenic inflammation and that only SP, of the three peptides present in nociceptive C fibers, seems to be of major importance in relation to cutaneous nociception. Simultaneous neurogenic release of CGRP and other neuropeptides in skeletal muscle may induce myofascial pain. PMID- 1712470 TI - Immunohistochemical mapping of galanin-like immunoreactivity in the brain of the dogfish Scyliorhinus canicula. AB - The distribution of galanin-like immunoreactivity in the brain of the dogfish Scyliorhinus canicula was investigated using the indirect immunofluorescence technique. In the telencephalon, positive cells and fibers were located in the mid-caudal part of the area superficialis basalis, the n. septi caudoventralis and in the n. interstitialis commissurae anterioris. Most of the galanin containing neurons observed in the hypothalamus were located in the magnocellular preoptic nucleus. Positive perikarya were also found in the n. lobi lateralis hypothalami and in the n. lateralis tuberis. A dense network of positive nerve processes was noted in the caudal part of the median eminence. In the dorso caudal part of the diencephalon numerous immunoreactive neurons were seen in the recessus posterioris. A large bundle of galanin-containing fibers, which divided in two branches, was observed in the basal midbrain tegmentum. The widespread distribution of galanin-like material suggests that, in the dogfish, galanin may be involved in various brain functions including neuroendocrine regulations. PMID- 1712471 TI - Correlation of blood group antigen expression and oncogene-related proteins in malignant prostatic tissues. AB - The use of multiple tumor markers could better predict the behavior of malignant tumors. In prostate cancer, there are no reliably predictive markers for metastatic behavior but the histologic grade and clinical stage of tumor do influence prognosis. We have determined the expression of blood group antigens A, B, H, Lewis-a and Lewis-b and the proto-oncogene proteins v-erbB, c-fos and v-H ras in both benign and malignant prostate tissues by immunohistochemistry. We determined the relationship between these markers and the grade of malignancy and by inference, clinical behavior. There was reduced expression of blood group antigens A, Lewis-a and Lewis-b in all grades of prostatic adenocarcinoma. We also found that the expression of v-erbB was greater in tumors of high grade. We suggest that loss of blood group antigens may be correlated with elevated v-erbB oncoprotein expression (related to epidermal growth factor receptor) and increasing grade of prostatic malignancy. PMID- 1712472 TI - Quantitation of the prostatic intra-epithelial neoplasia. Analysis of the nucleolar size, number and location. AB - Correlation has previously been demonstrated between qualitative and quantitative architectural and cytological features of the prostatic intra-epithelial neoplasia. In the present study, a standard Zeiss microscope and a horizontal eyepiece micrometer were used in an attempt to quantitatively define the size, number and location of nucleoli in relation to the degree of prostatic intra epithelial neoplasia and associated benign prostatic hyperplasia and adenocarcinoma. In total, 20 prostatectomies, where features of benign prostatic hyperplasia (20 cases), prostatic intra-epithelial neoplasia of grade 1 (10 cases) and grade 2 (10 cases), and adenocarcinoma (20 cases) were present, were studied. The quantitative analysis showed that, going from benign prostatic hyperplasia to prostatic intra-epithelial neoplasia of grade 1 and 2, and to adenocarcinoma, the values of the size-related nucleolar features, are progressively greater; when considering the frequency-related nucleolar features, the percentage of nucleolated nuclei increased sharply. This was associated with the decrease in the proportion of mononucleolated nuclei and increase in nuclei with two and three nucleoli. When considering the location-related nucleolar features, the degree of shift of the nucleoli to the periphery of the nucleus increased. Among all the quantitative features, the most evident changes in the nucleolar size were expressed by the nucleolar hypertrophy index after including in the calculation only those nucleoli with a diameter larger than 3 arbitrary units, whereas the percentage of nucleolated nuclei and the proportion of nuclei with two and three nucleoli best represented the modifications to the frequency.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712473 TI - Placental vascular anomaly with diffuse mesenchymal stem villous hyperplasia. A new clinico-pathological entity? AB - Diffuse mesenchymal hyperplasia of placental stem villi produces a pathological increase of placental volume, gives images of partial hydatidiform mola on ultrasound examination and is associated with elevated levels of alpha feto protein. On gross examination, the placenta is enlarged, the vessels on the fetal plate show aneurysmal and varicose dilatations, and the stem villi appear as distinct, semitranslucent lobulated structures. At microscopy level, stem villi show excessive proliferation of mesenchymatous tissue with foci of myxoid degeneration and large macrophages containing alcian blue positive material within cytoplasmic vacuoles. Involved areas show blood vessels with abnormally thin walls, which are negative to factor VIII and alpha feto-protein immunohistochemical reactions. Pathological trophoblastic proliferation or stromal trophoblastic inclusions are not part of this condition. Although there is a clear predominance of a female genotype in this cases, the true sex incidence in placental vascular anomalies with diffuse mesenchymal stem villus hyperplasia has yet to be defined. PMID- 1712474 TI - Surgical palliation for pancreatic carcinoma. AB - A review of 122 patients treated for pancreatic adenocarcinoma from January 1978 through December 1984 was accomplished to determine patient survival and the effect of surgical palliation. One hundred patients underwent laparotomy, including biopsy only (n = 42), biliary bypass (n = 30), gastric bypass (n = 1), biliary and gastric bypass (n = 14), and curative resection (n = 13). Total patient median survival was 3.6 months and no patient lived 5 years. No significant difference in survival was found between the biliary bypass and combined biliary-gastric bypass groups. Only 1 of 30 patients (3.3%) undergoing biliary bypass alone without evidence of pre-operative gastric outlet obstruction developed late gastric outlet obstruction requiring gastrojejunostomy. Operative time and postoperative morbidity were greater in the biliary-gastric bypass group. These results do not support routine prophylactic use of gastrojejunostomy at the time of biliary bypass for patients with unresectable carcinoma of the pancreas. PMID- 1712475 TI - Characterization by image cytometry of duct epithelial proliferative disease of the breast. AB - To develop a morphometric model of premalignant breast epithelium, we evaluated 120 lesions classified as nonproliferative disease (n = 20), hyperplasia (n = 20), moderate hyperplasia (n = 20), atypical hyperplasia (n = 20), carcinoma in situ (n = 20), and carcinoma (n = 20) in tissue from surgical biopsy or mastectomy. Atypical hyperplasia, a component of duct epithelial proliferative disease, has frequently been described in breasts with carcinoma. Atypical hyperplasia is generally viewed as premalignant or as a marker of increased risk for breast cancer. Measurements of nuclei in breast lesions were obtained with the Leitz TAS Plus on 4-microns sections stained for DNA with the Azure A Feulgen reaction. Nuclei of duct epithelial lesions had morphometric features that displayed changes from nonproliferative disease to carcinoma. The morphometric data from each lesion were compared among the six disease groups. Means of nuclear area, perimeter, maximum and minimum diameter, and large dark and large light intranuclear areas increased with higher degrees of proliferative abnormality. When the six groups of lesions were compared using the means of the first four nuclear features, atypical hyperplasia was significantly different (P less than 0.05) from carcinoma and non-proliferative lesions, but not from hyperplasia, moderate hyperplasia, or carcinoma in situ. These findings suggest that objective morphometric descriptors for characterizing significant proliferative lesions can be established using image cytometry. The progressive increases also suggest that proliferative breast disease is a continuum that includes premalignant lesions. PMID- 1712476 TI - Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues. AB - We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases. PMID- 1712477 TI - Molecular mimicry by Trypanosoma cruzi: the F1-160 epitope that mimics mammalian nerve can be mapped to a 12-amino acid peptide. AB - Antigenic mimicry by Trypanosoma cruzi antigens that share epitopes with mammalian tissues may drive autoreactive B- or T-cell clones to expand and cause autoimmune pathogenesis. We have been studying one of these antigens, F1-160, a 160-kDa protein on the surface of T. cruzi that antigenically mimics a 48-kDa protein found in mammalian axonal and myenteric plexus cells. The F1-160 antigen has been characterized by cloning and expression of T. cruzi DNA encoding F1-160 in Escherichia coli. Recombinant peptides from various regions of the F1-160 gene were expressed and used to compete with affinity-purified polyclonal anti-F1-160 antibodies binding to nerve. Recombinant 48-amino acid peptide (48X) derived from expression of base pairs 611-761 of the DNA sequence completely inhibited anti-F1 160 binding to nerve. Recombinant peptides expressed from DNA lacking this region did not inhibit anti-F1-160 binding to nerve. Three peptides were synthesized to encompass the 48X peptide, a 12-amino acid peptide and two 18-amino acid peptides. The 12-amino acid peptide TPQRKTTEDRPQ (12X), corresponding to bases 615-651, completely inhibited the binding of anti-F1-160 antibodies to nerve at a concentration of 80 ng/ml (30 microM). The two 18-residue peptides did not inhibit, even at 10 micrograms/ml. Thus, the epitope of F1-160 crossreactive with nervous tissue can be mapped to a 12-amino acid peptide. Some humans with T. cruzi infection make antibodies to F1-160 and to the 48X and 12X peptides. Control sera from uninfected persons did not react with these antigens. Anti-48X antibodies, immunoselected from human serum with 48X peptide, bind to human nerve axons. This demonstrates that some individuals infected with T. cruzi make antibodies to the F1-160 epitope crossreactive with nervous tissues. PMID- 1712479 TI - Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase. AB - Substrate inhibition was observed with the heterodimeric (p66/p51) and the homodimeric (p66/p66, p51/p51) forms of human immunodeficiency virus type 1 reverse transcriptase (RNA-dependent DNA polymerase, EC 2.7.7.49). An apparent Ki value of 195 +/- 37 microM was determined for dTTP using the bacterial cloned and expressed heterodimer. Similar values were obtained with the homodimeric and the virus-encoded enzymes. When poly-(rC).p(dG)10 was used as template-primer, dGTP exhibited substrate inhibition with an apparent Ki value of 189 +/- 32 microM. Substrate inhibition was not observed with dTTP when DNA.DNA template-primers were used. Hill coefficients for substrate binding determined in the presence of saturating concentrations of template-primer were equal to 1.0, suggesting that substrate inhibition of the heterodimer is not the result of an allosteric mechanism involving the p51 subunit. Furthermore, UV crosslinking experiments with [gamma-32P]dTTP showed crosslinking only to the p66 subunit. Substrate inhibition was not as pronounced with other retroviral reverse transcriptases as it was with human immunodeficiency type 1 reverse transcriptase. PMID- 1712478 TI - Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. AB - The genetic disease cystic fibrosis (CF) causes decreased Cl- transport in several epithelia. cAMP-dependent regulation of apical membrane Cl- channels is defective in CF airway epithelia; as a result, CF epithelia fail to secrete Cl-. In contrast, Ca(2+)-stimulated Cl- secretion is intact in CF airway epithelia and thus has the potential to bypass the CF Cl- secretory defect. For a Cl- channel to govern Cl- secretion, it must be located in the apical membrane. To specifically investigate apical membrane Cl- channels, we studied cells grown on permeable filter supports and measured Cl- currents across the apical membrane. We found that Ca2+ and cAMP activate different Cl- channels in the apical membrane. (i) Ca(2+)-activated Cl- channels were present in the apical membrane of airway but not in intestinal epithelia. (ii) cAMP- but not Ca(2+)-activated Cl channels were defective in CF airway epithelia. (iii) Ca(2+)- but not cAMP activated Cl- channels were blocked by 4,4'-diisothiocyanato-2,2' stilbenedisulfonate. (iv) Ca(2+)- and cAMP-activated apical channels had different anion permeabilities. (v) An increase in both second messengers produced an additive increase in Cl- current. These results also explain the puzzling observation that Ca(2+)-stimulated Cl- secretion is defective in CF intestine: the Ca(2+)-activated Cl- channels that could circumvent the Cl- secretory defect in CF airway are missing from the apical membrane of intestinal epithelia. PMID- 1712480 TI - Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. AB - Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation. The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues. PMID- 1712481 TI - Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas. AB - Insulin-like growth factor I (IGF-I) mRNA expression was studied after 90% partial pancreatectomy in the rat to determine whether IGF-I was associated with pancreatic regeneration. The level of IGF-I mRNA was maximally increased (4-fold above control value) 3 days after pancreatectomy, but thereafter gradually decreased, returning to control levels by 14 days after surgery. By in situ hybridization, IGF-I mRNA in both pancreatectomized and sham-operated rats was localized to capillary endothelial cells, indicating that this is the site of IGF I expression in the normal rat pancreas. However, enhanced IGF-I mRNA expression was localized to focal areas of regeneration unique to pancreatectomized rats. In these areas, epithelial cells of proliferating ductules and individual connective tissue cells expressed IGF-I, suggesting that IGF-I may play an important role in the growth or differentiation of pancreatic tissue. PMID- 1712482 TI - The Barr body is a looped X chromosome formed by telomere association. AB - We examined Barr bodies formed by isodicentric human X chromosomes in cultured human cells and in mouse-human hybrids using confocal microscopy and DNA probes for centromere and subtelomere regions. At interphase, the two ends of these chromosomes are only a micron apart, indicating that these inactive X chromosomes are in a nonlinear configuration. Additional studies of normal X chromosomes reveal the same telomere association for the inactive X but not for the active X chromosome. This nonlinear configuration is maintained during mitosis and in a murine environment. PMID- 1712483 TI - CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) recognize the same carbohydrate ligand, sialyl-Lewis x. AB - The leukocyte receptor CD62, which is expressed on activated platelets and endothelial cells, is shown to mediate cell adhesion by binding a sialylated carbohydrate structure, sialyl-Lewis x, found on neutrophils, monocytes, and tumor cells. This structure has previously been identified as the ligand for another member of the LEC-CAM family of cell adhesion molecules, endothelial cell leukocyte adhesion molecule 1, which also binds neutrophils and monocytes. The results demonstrate that although the two LEC-CAMs differ in their biological activities by their distribution and mode of expression, they are capable of mediating cell adhesion by recognition of the same carbohydrate ligand. PMID- 1712484 TI - Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. AB - Investigations of the actions and interactions of the immunophilin ligands FK506, cyclosporin A (CsA), rapamycin, and 506BD suggest that complexes of FK506 with an FK506-binding protein or of CsA with a cyclophilin (CsA-binding protein) inhibit the T-cell receptor-mediated signal transduction that results in the transcription of interleukin 2. Now we report an identical spectrum of activities of FK506, CsA, rapamycin, and 506BD on IgE receptor-mediated signal transduction that results in exocytosis of secretory granules from the rat basophilic leukemia cell line RBL-2H3, a mast cell model. Both FK506 and CsA inhibit receptor mediated exocytosis (CsA IC50 = 200 nM; FK506 IC50 = 2 nM) without affecting early receptor-associated events (hydrolysis of phosphatidylinositol, synthesis and release of eicosanoids, uptake of Ca2+). In contrast, rapamycin and 506BD, which share common structural elements with FK506, by themselves have no effect on IgE receptor-mediated exocytosis. Both compounds, however, prevent inhibition by FK506 but not by CsA. Affinity chromatography with FK506, CsA, and rapamycin matrices indicates that the same set of immunophilins present in RBL-2H3 cells have been found in Jurkat T cells and calf thymus; however, the relative amounts of these proteins differ in the two cell types. These results suggest the existence of a common step in cytoplasmic signaling in T cells and mast cells that may be part of a general signaling mechanism. PMID- 1712485 TI - Direct proliferative actions of stem cell factor on murine bone marrow cells in vitro: effects of combination with colony-stimulating factors. AB - Stem cell factor (SCF), the ligand for the c-kit protooncogene product, was able to stimulate blast cell and granulocytic colony formation by precursors from normal murine bone marrow. The blast cell colonies contained a high content of progenitor cells able to form macrophage and/or granulocyte colonies. Clone transfer studies, the secondary culture of colony cells, and the culture of populations freed of accessory cells all indicated a direct proliferative action of SCF. SCF receptors were present in high numbers on blast cells and in lower numbers on immature granulocytic, monocytic, and eosinophilic cells. Combination of SCF with granulocyte, granulocyte-macrophage, or multipotential colony stimulating factors, but not macrophage colony-stimulating factor, resulted in enhancement of colony size. Granulocyte colony-stimulating factor enhanced cell proliferation initiated by SCF, but not vice-versa, and resulted in a 10-fold increase in colony cell numbers and a 7-fold increase in progenitor cells in blast colonies. No evidence was obtained that SCF, alone or in combination with granulocyte colony-stimulating factor, could stimulate self-generation by blast colony-forming cells. PMID- 1712486 TI - Direct observation of brownian motion of lipids in a membrane. AB - Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 10(-8) cm2/sec, and paucivalent probes had an average D of 0.73 x 10(-8) cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 x 10(-8) cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 x 10(-8) cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results. Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes. PMID- 1712487 TI - Depolarizing stimuli regulate nerve growth factor gene expression in cultured hippocampal neurons. AB - Although trophic factors and neuronal activity have been implicated in regulating functional synaptic circuits, the relationship of trophic interaction to impulse activity in synaptogenesis remains unclear. Using cultured hippocampus as a model system, we provide direct evidence that depolarization and impulse activity specifically increase nerve growth factor gene expression in neurons. Depolarizing stimuli, such as a high K+ concentration or the Na+ channel agonist veratridine, elicited a 3-fold increase of nerve growth factor mRNA levels in both explant and dissociated cultures. Blockade of depolarization by tetrodotoxin prevented the increase of neuronal nerve growth factor mRNA. Further, nerve growth factor gene expression was stimulated by picrotoxin, a gamma-aminobutyric acid antagonist frequently used to enhance hippocampal neuronal activity. Impulse regulation of trophic gene function may be relevant to developmental synaptogenesis and synaptic strengthening in learning and memory. PMID- 1712488 TI - Multiple overlapping homologies between two rheumatoid antigens and immunosuppressive viruses. AB - Amino acid (aa) sequence homologies between viruses and autoimmune nuclear antigens are suggestive of viral involvement in disorders such as systemic lupus erythematosus (SLE) and scleroderma. We analyzed the frequency of exact homologies of greater than or equal to 5 aa between 61 viral proteins (19,827 aa), 8 nuclear antigens (3813 aa), and 41 control proteins (11,743 aa). Both pentamer and hexamer homologies between control proteins and viruses are unexpectedly abundant, with hexamer matches occurring in 1 of 3 control proteins (or once every 769 aa). However, 2 nuclear antigens, the SLE-associated 70-kDa antigen and the scleroderma-associated CENP-B protein, are highly unusual in containing multiple homologies to a group of synergizing immunosuppressive viruses. Two viruses, herpes simplex virus 1 (HSV-1) and human immunodeficiency virus 1 (HIV-1), contain sequences exactly duplicated at 15 sites in the 70-kDa antigen and at 10 sites in CENP-B protein. The immediate-early (IE) protein of HSV-1, which activates HIV-1 regulatory functions, contains three homologies to the 70-kDa antigen (two hexamers and a pentamer) and two to CENP-B (a hexamer and pentamer). There are four homologies (including a hexamer) common to the 70-kDa antigen and Epstein-Barr virus, and three homologies (including two hexamers) common to CENP-B and cytomegalovirus. The majority of homologies in both nuclear antigens are clustered in highly charged C-terminal domains containing epitopes for human autoantibodies. Furthermore, most homologies have a contiguous or overlapping distribution, thereby creating a high density of potential epitopes. In addition to the exact homologies tabulated, motifs of matching sequences are repeated frequently in these domains. Our analysis suggests that coexpression of heterologous viruses having common immunosuppressive functions may generate autoantibodies cross-reacting with certain nuclear proteins. PMID- 1712489 TI - Coordinate regulation of HOX genes in human hematopoietic cells. AB - Hematopoiesis is a continuous process in which precursor cells proliferate and differentiate throughout life. However, the molecular mechanisms that govern this process are not clearly defined. Homeobox-containing genes, encoding DNA-binding homeodomains, are a network of genes highly conserved throughout evolution. They are organized in clusters expressed in the developing embryo with a positional hierarchy. We have analyzed expression of the four human HOX loci in erythroleukemic, promyelocytic, and monocytic cell lines to investigate whether the physical organization of human HOX genes reflects a regulatory hierarchy involved in the differentiation process of hematopoietic cells. Our results demonstrate that cells representing various stages of hematopoietic differentiation display differential patterns of HOX gene expression and that HOX genes are coordinately switched on or off in blocks that may include entire loci. The entire HOX4 locus is silent in all lines analyzed and almost all the HOX2 genes are active in erythroleukemic cells and turned off in myeloid-restricted cells. Our observations provide information about the regulation of HOX genes and suggest that the coordinate regulation of these genes may play an important role in lineage determination during early steps of hematopoiesis. PMID- 1712490 TI - Molecular cloning and primary structure of Kell blood group protein. AB - The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity. PMID- 1712492 TI - CD5+ B lymphocytes. PMID- 1712491 TI - Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor. AB - We investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF164 (rrSCF164) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of "connective tissue-type mast cells" (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF164 induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC. BMCMC maintained in rrSCF164 not only proliferated but also matured. Prior to exposure to rrSCF164, the BMCMC were alcian blue positive, safranin negative, and berberine sulfate negative; had a histamine content of 0.08 +/- 0.02 pg per cell; and incorporated [35S]sulfate into chondroitin sulfates. After 4 wk in rrSCF164, the BMCMC were predominantly safranin positive and berberine sulfate positive, had a histamine content of 2.23 +/- 0.39 pg per cell, and synthesized 35S-labeled proteoglycans that included substantial amounts (41-70%) of [35S]heparin. These findings identify SCF as a single cytokine that can induce immature, IL-3 dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation. PMID- 1712493 TI - Serologic demonstration of the albuminoid nature of the B700 murine melanoma antigen. AB - Limited available evidence indicates that the B700 murine melanoma antigen is related to serum albumin, but potential relationships to other members of the serum albumin protein family have not yet been established. Using specific antibodies raised against each of the members of the albumin family, we have studied cross-reactivity by solid phase enzyme-linked immunosorbent assay and Western immunoblotting. We demonstrate that B700 is serologically cross-reactive to members of the serum albumin family, which includes alpha-fetoprotein and vitamin D binding protein. Therefore, B700 is part of the serum albumin family of proteins, although the mechanism underlying its specific expression by transformed melanocytes remains unknown. PMID- 1712494 TI - Modulation of protein expression associated with chemically induced differentiation of neuroblastoma cells. PMID- 1712495 TI - Cell adhesion molecules expression and modulation on human neuroblastoma cells. PMID- 1712496 TI - Intranigral tachykinin NK3 receptor agonist elicits oral movements in rats. AB - Synthetic agonists for the tachykinin NK1 and NK3 receptors were bilaterally infused at three dose levels (4.2, 0.17, and 0.007 nmol) into each substantia nigra of freely moving rats and oral behaviors were monitored for 30 min postinfusion. It was found that all doses of senktide, an agonist at the NK3 receptor, induced a significant increase of nonobject-directed chewing, vacuous chewing movements (VCM). The highest dose of senktide produced the greatest effect (p less than 0.001) and precipitated wet shakes for about 15 min after infusion. Septide, selective at the NK1 receptor, was without effect on oral behavior. The present results suggest that NK3 receptor-active peptides might be symptom inducers in oral dyskinesia. PMID- 1712497 TI - Minipump clorgyline administration and CSF amine metabolites in unrestrained monkeys. AB - The irreversible MAO-A inhibitor clorgyline was administered in doses of 0.5 mg/kg (N = 1), 1 mg/kg (N = 3), and 2 mg/kg (N = 1) to 5 young (age 5.5 to 23.9 months) pigtail (M. nemestrina) monkeys using a 28-day (Alza 2ML4) osmotic minipump. CSF MHPG, 5-HIAA, HVA, and plasma MHPG were measured before and at approximately weekly intervals after pump implantation. Implants were well tolerated. CSF MHPG decreased about 75%, 5-HIAA 30%, and HVA from 30-50% with a tendency to plateau by the second week. Plasma MHPG decreased to undetectable levels. The findings demonstrate that long-term inhibition of MAO-A can be produced in unrestrained monkeys by minipump administered clorgyline. There is an apparently greater effect on the norepinephrine system relative to the serotonin and dopamine systems. PMID- 1712498 TI - Effects of split-dose X irradiation on rat salivary gland function. AB - The effect of a single local dose of 15 Gy on salivary gland function in male Albino Wistar rats was compared with the effect of two doses of 7.5 Gy. The intervals chosen were 0-24 h and 1 week. Before and 1-30 days after the last radiation dose, samples of parotid and submandibular saliva were collected simultaneously after stimulation of the glands with pilocarpine. Irradiation with the single dose resulted in an increased lag phase and potassium concentration, and a decreased flow rate and sodium concentration. The rate of secretion of amylase was decreased during Days 1-6, increased at Day 10, and was decreased again at Day 30. With two dose fractions, substantial dose-sparing effects on lag phase, flow rate, and secretion of amylase were observed for both the very early (0-6 days postirradiation) and later (6-30 days postirradiation) effects. These effects were maximal when the interval between the fractions was 6 h. A significant dose-sparing effect on electrolytes was observed for the later effects only, again with a maximum for the 6-h interval. The dose-sparing observed for the very early effects cannot be explained satisfactorily by repair of sublethal damage (SLD), redistribution of cells over the cell cycle, or repopulation of salivary gland tissue between the doses. In contrast to the earlier dose-sparing effects, the split-dose recovery seen for later damage may be attributed, in part, to SLD repair in providing for greater reproductive survival of intercalated ductal cells and enhanced tissue regeneration. PMID- 1712499 TI - Small hepatocellular carcinoma treated with percutaneous ethanol injection: MR imaging findings. AB - Fifty-seven magnetic resonance (MR) imaging examinations were obtained at 0.5 T in 19 patients before and after percutaneous ethanol injection (PEI) for 23 hepatocellular carcinoma (HCC) lesions less than 3.5 cm in diameter. Seventeen patients also underwent MR imaging 6 months after completion of therapy. In 11 patients, computed tomography was performed before and after treatment. After PEI, fine-needle biopsy specimens were obtained in all cases. Before treatment, HCC lesions had low signal intensity on T1-weighted images in 13 cases, had the same signal intensity as normal liver parenchyma in six, and had high signal intensity in four; all 23 tumors had high signal intensity on T2-weighted images. After treatment and at 6-month follow-up, all 21 lesions that contained no malignant cells at fine-needle biopsy had high signal intensity on T1-weighted images and had low signal intensity on T2-weighted images. The remaining two HCC lesions in which tumor necrosis was not achieved with PEI displayed a different MR pattern, since the residual neoplastic tissue showed no change in signal intensity on either T1- or T2-weighted images. The authors conclude that MR imaging may be useful for evaluating the effectiveness of PEI in achieving tumor regression. PMID- 1712500 TI - Esophagogastric neoplasms: palliation with a modified gianturco stent. AB - Self-expanding metallic stents of a modified Gianturco design were used for palliative treatment of malignant esophagogastric strictures. Over a 10-month period, 10 stents were placed in nine patients. All patients with severe dysphagia due to malignant strictures in whom all other treatment options had failed were candidates for these stents. Neither extensive length of esophageal involvement nor complete esophageal obstruction was a contraindication. All stents were placed with fluoroscopic guidance without any technical failures or procedural morbidity or mortality. Mild reflux occurred in three patients in whom the stent tubes straddled the distal esophageal sphincter. Five patients were still alive after 1-8 months. The remaining four patients died 6-28 weeks after stent placement; all stents were patent at the time of death. These stents are easy to insert, safe, and reasonably effective for short-term palliative treatment of esophagogastric neoplasms. PMID- 1712501 TI - [Sudeck's disease--MRT as a new diagnostic procedure]. AB - In this prospective study, we determined the value of MR as an imaging technique in diagnosis and therapy with the option of simultaneous assessment of bone and soft tissue. 20 patients with clinical diagnosis of reflex sympathetic dystrophy syndrome (RSDS) and positive results in radiogram and three-phase radionuclide bone scanning were examined by MR, using T1- and T2-weighted images before and after Gadolinium-DTPA i.v. Soft tissue and bone signal intensity changes can be classified in our diagnostic-therapeutic scheme and proven by histopathological changes. The differentiation between clinical stages is possible and allows an evaluation of course and therapy. The higher costs are justified by shorter examination time without using radiation. PMID- 1712502 TI - Role of major and minor histocompatibility antigens in the suppression of alloreactive cytotoxic responses induced by alloantigen pretreatment. AB - We have recently shown that priming mice with allogeneic strain A spleen cells before immunization with (A x B)F1 spleen cells strongly suppresses the cytotoxic T-lymphocyte (CTL) response directed against linked strain B alloantigens. This specific decrease in the CTL responses against the second immunizing alloantigen is associated with a high CTL response against the first priming alloantigen. The suppression of CTL responses against the strain B alloantigens is, however, not due to killing of F1 spleen cells by anti-A CTL, since it was observed after immunization of primed mice with a mixture of (A x B)F1 and B cells. In the present study, attempts were made to determine the relative contribution of H-2 and minor histocompatibility background antigens towards induction of suppression. Our results demonstrate that priming and immunizing spleen cells have only to share H-2 antigens in order to induce a downregulation of CTL responses directed against the linked alloantigens. This indicates that immunity against H-2 antigens is sufficient to induce suppression. However, priming against minor histocompatibility antigens also induces suppression, but only if spleen cells used for priming and immunization share H-2 antigens with the recipient strain. Therefore, the suppression can be induced by priming with non-H 2 antigens but is H-2-restricted. This study has also demonstrated that suppression can be induced by intraperitoneal or subcutaneous administration of allogeneic cells. PMID- 1712503 TI - Conformational change in the N-terminal domain of the Escherichia coli tryptophan synthase beta 2 subunit induced by its interactions with monoclonal antibodies. AB - A fluorescence energy transfer signal was used to follow conformational changes occurring in two types of protein-protein complexes. The first complex studied was the native-like beta 2 subunit of Escherichia coli tryptophan synthase reconstituted by reassembly of the N- and C-terminal proteolytic domains of the beta chain. The other complexes were formed by the association of the N-terminal fragment (F1) with a monoclonal antibody that recognizes the native dimeric protein; four such complexes, obtained with different antibodies that recognize four distinct antigenic sites on native beta 2, were investigated. It was shown that a structural readjustment, which the isolated F1 domain was unable to undergo alone, was imposed upon F1 by interdomain interactions. Furthermore, with three of the four antibodies studied, the same conformational change in F1 also occurred after formation of the F1-antibody complex. These results demonstrate that, through an "induced fit mechanism", antigen-antibody stereospecific assembly can force the polypeptide chain to adopt a structure more closely resembling the conformation it has in the native protein. PMID- 1712504 TI - Intrageneric relationships of Enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA. AB - The 16S ribosomal ribonucleic acid (rRNA) sequences of eleven Enterococcus species were determined by reverse transcription in an attempt to clarify their intrageneric relationships. Comparative analysis of the sequence data revealed the presence of several species groups within the genus. The species E. avium, E. malodoratus, E. pseudoavium and E. raffinosus formed a distinct group as did E. durans, E. faecium, E. hirae and E. mundtii and the pair of species E. casseliflavus and E. gallinarum. Of the remaining species, E. cecorum, E. columbae, E. faecalis and E. saccharolyticus formed distinct lines of descent within the genus, whereas E. solitarius displayed a closer affinity with Tetragenococcus halophilus than with other enterococcal species. PMID- 1712505 TI - [Free radicals of oxygen and their effect on inflammation mediators in patients with systemic lupus erythematosus]. AB - The active forms of oxygen (AFA) participate in modulation of mediation processes in patients with systemic lupus erythematosus. A direct correlation of AFA with serotonin and kallikrein was noted, a reverse one with the inhibitory systems. Reactivity of AFA proved to be most marked in patients with lupus nephritis. The role of polymorphonuclear leukocytes in the activation of vasculo-thrombocytic hemostasis is discussed. PMID- 1712506 TI - [Comparative study of the activity of immunoglobulin G and therapeutic preparations of normal human immunoglobulin in patients with systemic lupus erythematosus and rheumatoid arthritis and in healthy persons]. AB - The authors consider that the development of immunopathological reactions in SLE, AIDS depends on the reduction in normal gamma-globulin level in the patient's serum. The sera with anti-DNA, anti-HIV antibodies show a decreased binding of corresponding antigens and decreased concentrations of gamma-globulin with normal features. PMID- 1712507 TI - [Induced scleroderma]. PMID- 1712508 TI - Human alpha-fetoprotein and its purification by chromatography on immobilized estrogens. AB - Human alpha-fetoprotein (AFP) was isolated from human abortive tissue by biospecific chromatography on immobilized estrogens. The most effective sorbents were: estrone-0-3-hemisuccinyl-hexamethylenediamine-Sepharose CL 4B and diethylstilbestrol-diasoanisole-sulfonyl-oxyethyl-Sepharose CL 4B. As elution solution the most optimum was 10% buffered aqueous butanol. Taking into consideration the data obtained, one can conclude that AFP in human biological fluids is bound to immobilized estrogens. Butanol extraction deestrogenizes AFP, and as a result human AFP acquires affinity to immobilized estrogens. During rechromatography on immobilized diethylstilbestrol, it was possible to obtain AFP preparations of about 95% purity. The present results provide the opportunity to work out new methodological approaches to human AFP isolation using biospecific chromatography on immobilized estrogens. PMID- 1712509 TI - Novel epitopes on the CA50-carrying antigen: chemical and immunochemical studies. AB - Novel tumour-associated epitopes showing elevated levels in sera from patients with colon carcinoma were studied by means of monoclonal antibodies with respect to co-expression with the tumour-associated epitope CA50 on the cancer antigen (CanAg) glyco-conjugate complex. Co-expression of the different epitopes and CA50 was found on CanAg both from COLO 205 spent medium and sera from patients with colorectal cancer. In a few sera from patients with non-mucinous ovarian tumours, the CanAg complex was also found to express the CA125 epitope. The chemical characterisation showed that all of the novel epitopes on CanAg were of carbohydrate nature, and the results may suggest a structural relationship between several of them. PMID- 1712510 TI - The MHC class I associated beta 2-microglobulin (beta 2m) light chain is expressed in a molar excess over HLA-ABC and CD1 on the membrane of leukaemic B cells but not leukaemic T cells: evidence for further beta 2m-associated molecules. AB - Beta 2-microglobulin (beta 2m) constitutes the common light chain of both the MHC encoded HLA-ABC molecules and a group of structurally related glycoproteins recognized by antibodies of the first cluster of differentiation (CD1a, CD1b and CD1c). These CD1 antigens appear similar to murine T1 and Qa molecules in terms of structure and tissue distribution, although the question of inter-species homology is controversial. A further group of alloantigens expressed predominantly on T cells has been reported however, with immunogenetic characteristics more closely analogous to the murine T1/Qa system than the CD1 antigens, although their precise identity remains ill-defined. Having previously shown that malignant B cells may express membrane CD1c, we examined leukaemic B cells corresponding to early lymphoblastic differentiation (null- and common acute lymphoblastic leukaemia) through to the terminal plasma cell stage for the expression of other non-HLA class I beta 2m-associated molecules. It was found that leukaemic B-cells at intermediate/late stages of differentiation, represented by non-Hodgkin's lymphoma (B-NHL) and 'hairy-cell' leukaemia (HCL), had significantly higher beta 2m:HLA-ABC ratios than did the cells from other types of B-cell malignancy. Although leukaemic B cells with a demonstrable non HLA-ABC-associated beta 2m component expressed detectable levels of CD1c, and insignificant levels of CD1a and CD1b, the antigen density was insufficient to account for the excess beta 2m. In vitro stimulation of leukaemic B cells by phorbol ester substantially increased the expression of HLA-ABC and CD1c, but also accentuated further the difference between the expression of these molecules and that of beta 2m. There was no detectable beta 2m other than that associated with HLA-ABC and CD1 on the surface of malignant T cells by contrast. Our findings strongly support the existence, at certain stages of leukaemic B-cell differentiation, of an additional beta 2m component(s) other than that associated with HLA-ABC and CD1. PMID- 1712511 TI - Heparin, non-heparin glycosaminoglycans, and heparinoids: an overview of their application in atherosclerosis. PMID- 1712512 TI - Interaction of heparinoids with platelets: comparison with heparin and low molecular weight heparins. AB - Heparin interacts with platelets to impair collagen-induced aggregation and adhesion to collagen. Low molecular weight heparin and heparinoids have little or no inhibitory activity in these platelet-collagen interactions. Thrombin-induced aggregation of platelets is inhibited by any of the glycosaminoglycans that will block the action of thrombin on fibrinogen. However, heparin is a much more potent inhibitor than low molecular weight heparins or heparinoids at equigravimetric concentrations in these reactions. The inhibition of ristocetin or asialo-von Willebrand factor aggregation of platelets is partially blocked by high dose heparin but not by low molecular weight heparin or heparinoids. The heparin-induced IgG antibody, produced to a heparin-platelet complex, aggregates platelets strongly in the presence of heparin, less strongly in the presence of low molecular weight heparins and pentosan polysulfate and not at all with dermatan sulfate or the pentasaccharide. Heparan sulfate does interact with platelets and this antibody, which is of interest because of the heparan sulfate on endothelial cells. Clinical information to date suggests a low incidence of heparin antibodies in patients receiving only low molecular weight heparin of the depolymerized type. Whether long-term clinical use of low molecular weight heparins, heparan sulfate, and dermatan sulfate will give rise to specific antibodies that would cause a similar problem as heparin remains to be seen. PMID- 1712513 TI - [Ionic channels formed in the lipid bilayer membranes by aureofuscin, a polyene antibiotics]. AB - A chamber filled with salt solution and separated into two compartments by a Teflon partition with a pore of 700 microns diameter was used to investigate the action of aureofuscin, a polyene antibiotics, on a planar lipid bilayer. The pore was covered with a bilayer formed by a N-decane solution of lecithin and cholesterol (20 mg/ml and 5 mg/ml). The electrical property and ionic permeability of the bilayer were studied by using voltage clamp method. Decrease of the bilayer resistance and the channel-like activity could be observed 20 min after the addition of aureofuscin (10-20 micrograms/ml) to one compartment. The existence of a transmembrane voltage and ionic gradient were not necessary for the channel-forming activity of aureofuscin. Discrete current steps were recorded at a concentration of 1.4 micrograms/ml aureofuscin, with a predominant unit conductance of 4-6 pS in a symmetric KCl, solution of 100 mmol/L. By using Goldmann-Hodgkin-Katz voltage equation the ionic selectivity of the channel formed by aureofuscin was estimated by the reversal potential measured in the asymmetrical solution system. The results showed that aureofuscin channels were more permeable to potassium ion than to chloride ion (PK/PCl approximately 5.2). These data may be used to explain the action of aureofuscin on neurotransmitter release and muscle membrane potential in addition to providing some explanation on its curing effect in clinical use. PMID- 1712514 TI - Application of estrogen receptor immunocytochemical assay to aspirates from mammographically guided fine needle biopsy of nonpalpable breast lesions. AB - The importance of hormone receptors in the management and prognosis of breast cancer is well established, but difficult to apply to the growing numbers of very small breast cancers being detected. To assess the feasibility of applying estrogen receptor immunocytochemical assay (ER-ICA) to cytologic specimens, we prospectively studied 100 patients who had fine needle aspiration biopsy (FNAB) of mammographically detected nonpalpable breast lesions. All 100 patients also had surgical excision of these nonpalpable lesions immediately after cytologic aspiration. Twenty malignancies were ultimately diagnosed by histology; 17 of them had been cytologically diagnosed. Using specific monoclonal antibody for estrogen receptor, we applied ER-ICA to cytologic preparation of 15 malignant neoplasms with sufficient cellular material available for the assay. Positive immunostaining was demonstrated in nine cases. No ER expression was seen in six cases. Immunocytochemical assay was also done on frozen tissue of the corresponding surgically removed tumors, with 86.6% concordance between the two results. This study is the first to demonstrate that ER-ICA can be effective in assessing hormone receptor content of mammographically directed cytologic aspirates. PMID- 1712515 TI - [The effect of plasmasorption on the content of natural antibodies to thrombin and alpha 2-macroglobulin in stenocardia patients]. AB - A study was made of changes in the content of natural antibodies (N-AB) to thrombin and alpha 2-macroglobulin in patients with angina pectoris under the influence of plasma perfusion. The content of N-AB being increased, plasma perfusion makes it possible to reduce their content to normal. Provided the content of N-AB is initially normal, plasma perfusion does not produce any changes in their level. In 78% of the patients, a beneficial clinical effect was attained, being more pronounced in initially high content of N-AB. PMID- 1712516 TI - Endoscopic palliation of tracheobronchial malignancies. AB - The prognosis for tracheobronchial tumours remains poor. Most patients can be offered only palliation. When the main symptom is breathlessness or refractory haemoptysis from a large airway tumour endoscopic treatment may be very effective. Over the last decade most attention has focused on the neodymium YAG laser. This often produces dramatic effects but has some important limitations. In the last few years better techniques for stenting and intrabronchial radiotherapy (brachytherapy) have also been developed. This article discusses the range of techniques now available and aims to help clinicians decide which patients may benefit from referral to centres providing these techniques. PMID- 1712518 TI - The polymerase in its labyrinth: mechanisms and implications of RNA recombination. AB - The wide variety of RNA viruses, and the diseases associated with them, may result in part from the capacity of RNA genomes to evolve through genetic recombination. Here we address the mechanism of RNA recombination, and ask questions about its prevalence and purpose in nature. PMID- 1712517 TI - Minor role of hepatitis B virus in the causation of chronic liver disease in Somalia indicated by a case-control study. AB - Chronic liver disease (CLD) is frequent in Somalia. In a case-control study, 116 in-patients with CLD were compared with the same number of age and sex matched controls. Demographic variables, use of drugs, symptoms and signs, serological markers for hepatitis B virus (HBV) and serum alpha-foetoprotein (AFP) were assessed. Hepatitis B surface antigen (HBsAg) was found in 44 cases of which 17 had antibodies to hepatitis D virus (anti-HD) and 7 had hepatitis B e antigen (HBeAg). Twenty-three controls were HBsAg-positive, of whom 3 had anti-HD and one HBeAg. Increased relative risks (95% confidence intervals in parentheses) were 2.5 (1.3-4.5) for HBsAg, 6.5 (1.7-21.5) for anti-HD, and 7.4 (0.9-66.5) for HBeAg. Despite the association between the presence of HBV markers and CLD, 62% of the cases had no markers indicating current HBV infection. This was reflected in the low risk attributable to chronic HBV infection (22.6%), which was lower than that in patients with CLD in other African populations with a high HBsAg carrier rate. The prevalence of HBV markers did not differ between cases with AFP greater than 100 ng/ml and those with AFP less than 100 ng/ml. The former were characterized by male predominance, shorter duration of symptoms, and larger mean liver size, indicative of malignancy. The mean age of HBsAg-positive cases with AFP greater than 100 ng/ml was significantly lower (by 7.7 years) than that of HBsAg-negative cases with AFP greater than 100 ng/ml. Among the CLD patients with AFP less than 100 ng/ml, 48 were HBsAg-negative. These cases differed significantly from the other 68 cases in that more were females (35% against 16%), more originated from an agricultural area (56% against 30%), and more were regular consumers of drugs (48% against 28%). In conclusion, factors as yet undefined play a considerable role in the causation of CLD in Somalia. The possibility of determining the role of hepatitis C virus (HCV) awaits the development of more specific assays for anti-HCV antibodies. PMID- 1712519 TI - Immunohistochemical, ultrastructural and biochemical studies of an amylase producing breast carcinoma. AB - We describe a breast cancer with ectopic production of amylase, found in the patient's serum, urine and in the tumour. Clinically, serum amylase levels reflected both the progression of the disease and regression induced by various therapies. Using agarose gel electrophoresis and a wheat protein inhibitor assay, the predominant serum amylase appeared to be identical to pancreatic-type isoenzyme. However, the action mode analysis using a new fluorogenic substrate revealed that the serum contained non-salivary, non-pancreatic amylase. The tumour had microscopic features of invasive ductal carcinoma with some argyrophilic differentiation. The component cells stained positively for amylase, and ultrastructurally numerous secretory granules were seen. PMID- 1712521 TI - Clinical use of biologic response modifiers in cancer treatment: an overview. Part II. Colony-stimulating factors and interleukin-2. AB - Colony-stimulating factors (CSFs) are hematopoietic growth hormones that stimulate the production, maturation, and function of white blood cells. The best studied are granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), both of which can be produced by recombinant DNA technology. Clinical indications for these agents include bone marrow failure secondary to administration of chemotherapeutic drugs or radiation, bone marrow transplantation, and a variety of congenital or iatrogenic neutropenias. Toxicity in usual clinical doses is mild, and consists mainly of bone pain and constitutional symptoms such as fever, headache, and myalgias. Interleukin-2 (IL-2) is a lymphokine that stimulates that multiplication of several types of killer cells. These cells can recognize and destroy foreign substances, such as tumors, without destroying normal cells. Major applications of IL-2 include treatment of patients with renal cell carcinoma, in whom the overall objective response rate is 15-30 percent, and malignant melanoma with response rates of about 18 percent. Combination therapy with other biologics and conventional cytotoxic drugs may increase IL-2's efficacy against these tumors. Toxicity is generally severe, but reversible. Hemodynamic toxicity, consisting of hypotension, edema, weight gain, and decreased renal function, is most characteristic. Suggestions are given for pharmacologic management of these and other IL-2 toxicities. PMID- 1712520 TI - Towards the phenotyping of soft tissue tumours by cell surface molecules. AB - This study is aimed at the characterization of soft tissue tumours (STT) by means of cell surface molecules. To achieve this, normal mesenchymal tissues were extensively examined for expression of leucocyte differentiation (CD) antigens and HLA molecules. The panel of antigens finally examined in STT comprised CD10, CD13, CD24, CD34, CD36, CD56, CD57, HLA-A,B,C, beta 2-microglobulin, HLA-DR, -DP, and -DQ and the HLA-D-associated invariant chain (Ii). STT were determined by conventional histomorphological and immunohistochemical criteria. The immunohistological analysis was based on serial frozen sections, one of which was used to demonstrate CD53 antigen. This very broadly distributed leuco/histiocyte restricted antigen allowed for the distinction between the background of interstitial "stromal" cells and the neoplastic population. In some STT, the expression pattern of the cell surface molecules corresponded to that in their non-neoplastic counterparts. The majority of STT, however, showed considerable changes in the cell surface immunophenotype compared to their cells of origin. These alterations consisted mainly in an aberrant induction/neoexpression and, to a much lesser extent, in an aberrant down-regulation/loss of cell surface antigens. Nevertheless, some immunophenotype configurations are described which, for the time being, can be considered to be useful supplements in the differential diagnosis of this complex class of tumours. The data also indicate considerable changes in cell surface antigen expression occurring in the course of neoplastic transformation of mesenchymal cells. Detailed analysis of alterations in the functional repertoire of neoplastic mesenchymal cells might provide new insights into the biology of STT, possibly leading to new concepts for therapeutic intervention. PMID- 1712522 TI - [Tetramethylbenzidine--a chromogenic substrate for peroxidase in enzyme immunoassay]. AB - 3,3' 5,5'-tetramethylbenzidine (TMB) is a high sensitive chromogenic substrate for horseradish-peroxidase as a marker enzyme. In an enzyme immunoassay (EIA) for alpha-1-fetoprotein and in an rapid EIA for myoglobin it reveals higher sensitivity compared to o-phenylenediamine, the increase depends on reaction time. The optimal peroxide concentration depends on reaction time of enzyme chosen in different assays. TMB used for histochemistry is also suitable for EIA if hydrochloric acid is used as stopping reagent. TMB lacks in mutagenic properties and it should preferred for peroxidase rather than all other chromogenic substrates applied up to now. PMID- 1712523 TI - [Experiences with ORWOANALYT base material ET 250 in the production of tissue supported ultrathin polyacrylic amide gel for IEF- and SDS-electrophoresis]. AB - The "VEB Filmfabrik Wolfen" offers a textile material for sale, which is well suited for manufacturing of ultrathin reinforced polyacrylamide gels. In contrast to the glass- or polyester-stabilized gels such fabric-reinforced gels allow diffusion- or electroblotting methods. First experiences with ultrathin SDS- and IEF-gels, manufactured using this new supporting material are represented. PMID- 1712524 TI - [Zinc, calcium and sodium values in secretions of patients with benign prostatic hyperplasia]. AB - In two randomized patients groups suffering from benign prostatic hyperplasia (BPH) two exprimate samples were drawn at a seven-day interval. In the untreated control group no changes in the values of Ca, Na and Zn were found. In the other group the patients have been treated with ERU capsules (Radicis urticae) for 7 days and thereafter a significant decrease of Zn values were found. PMID- 1712525 TI - [Phyllodes type of atypical prostatic hyperplasia]. AB - The author describes one case of phyllodes type of atypical prostatic hyperplasia. This is a benign prostatic lesion characterised by atypical epithelial hyperplasia and pleomorphism of the stromal elements. There is a resemblance with cystosarcoma phyllodes of the female breast. There have been described only a few cases of such atypical hyperplasia. PMID- 1712526 TI - Late recurrence following treatment of anal canal carcinoma. AB - A late pelvic recurrence of a cloacogenic anal canal carcinoma, occurring eleven years after an abdominoperineal resection, is reported in a 61-year old female patient. The primary tumour, 2.5 cm in diameter had infiltrated the rectal wall but did not show any evidence of local lymph node involvement on pathological examination. Recurrence of this disease is frequently considerably delayed, with several cases recurring after 5 or more years. This tendency to late recurrence clearly limits the reliability of short-term survival data. Current concepts concerning the management of malignant tumours of the anal canal are discussed from this point of view. PMID- 1712527 TI - Immunoreactive insulin-like growth factor binding protein-3 in the culture of human luteinized granulosa cells. AB - Granulosa cells from human preovulatory follicles were cultured under serum-free conditions to investigate the presence of immunoreactive insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 levels were measured by a radioimmunoassay developed against the acid-stable subunit of the protein. The antiserum had no cross-reactivity to the low molecular weight GH-independent IGFBP-1. Granulosa luteal cells exhibited a continuous release of IGFBP-3 into the culture medium during the whole time (6 days) of the incubation. A dose dependent increase in IGFBP-3 was observed when the cells were treated by dibutyryl cAMP. Cycloheximide suppressed almost completely both the basal and the stimulated production of IGFBP-3. The smallest effective dose of dibutyryl cAMP enhancing the progesterone release was lower than that for IGFBP-3. The different time course of IGFBP-3 and progesterone secretion to dibutyryl cAMP treatment, as well as the failure of progesterone to elicit IGFBP-3 increase alone, do not support the participation of progesterone in the IGFBP-3 production of granulosa cells. It is concluded that 1. immunoreactive IGFBP-3 is produced by cultured granulosa luteal cells; 2. its synthesis is regulated by physiological intracellular mechanisms. PMID- 1712528 TI - The alpha-galactosyl epitope on mammalian thyroid cells. AB - Studies on the distribution of alpha-galactosyl epitopes with the structure Gal alpha 1----3Gal beta 1----4GlcNac-R on mammalian thyroid cell membranes are of interest, since a natural antibody interacting with this carbohydrate antigen (i.e. the natural anti alpha-galactosyl IgG antibody) was found to increase in its titre in patients with autoimmune thyroid disorders. By using a radioimmunoassay for quantification of the alpha-galactosyl epitope, we found variable concentrations of the alpha-galactosyl epitope, we found variable concentrations of this epitope on thyroid cell membranes of all nonprimate mammals and New World monkeys studied, but not in Old World monkeys and human thyroid. The absence of the identifiable alpha-galactosyl epitopes on human and Old World monkey thyroid cells correlates with diminished activity of the enzyme, alpha 1-3 galactosyltransferase, which, in other species, synthesizes the alpha galactosyl epitopes within the Golgi apparatus. It is argued that a proportion of anti-thyroid reactivity in human normal and pathologic sera, when assayed with mammalian thyroid cells, may be attributed to natural anti alpha-galactosyl IgG antibody, which interacts with alpha-galactosyl epitopes on thyroid tissues used for the bioassay. PMID- 1712529 TI - Human leukemic T and B lymphoblasts produce insulin-like growth factor binding proteins 2 and 4. AB - The production of insulin-like growth factors and insulin-like growth factor binding proteins by twelve human leukemic lymphoblast cell lines was evaluated by radioimmunoassay, affinity cross-linking, ligand blot, and immunoprecipitation of conditioned media. In all cell lines, detectable IGF-I and IGF-II levels were less than 0.1 micrograms/l and less than 0.3 micrograms/l, respectively. IGF binding proteins were identified in 6/12 of the lymphoblast cell lines studied. A pair of IGF binding proteins at 31 and 33 kD, immunoprecipitated with an antibody recognizing IGF binding protein 2 but not by an IGF binding protein 3 antibody, was produced by both T and B cells. A 24 kD IGF binding protein, presumably IGF binding protein 4, since it was not recognized by the antibodies for IGF binding proteins 1, 2, and 3, was produced by B cell precursor cells and faintly by one T cell line. These IGF binding proteins were not altered by deglycosylation. Neither IGF binding protein 1 nor IGF binding protein 3 was identified in any of the conditioned media. We speculate that local production of IGF binding proteins 2 and 4 regulates access of the IGFs to lymphoblasts and to hematopoietic precursors in general. PMID- 1712530 TI - Luminal non-specific cationic channels in cultured strial marginal cells of guinea pig and gerbil as determined by patch clamp technique. AB - Using primary cultures of marginal cells of stria vascularis from guinea pig and gerbil, ionic channels located on the luminal membrane were investigated by means of patch clamp technique. Recordings were performed in cell-attached and inside out configurations. In cell-attached configuration, single channel activity was identified with a conductance of about 25 pS. I-V curve was linear. The probability of opening was increased upon depolarization. Up to 7 channels could be present in the same patch, indicating a rather high density. In inside-out configuration, the reversal potential was 0 mV, suggesting a non-specific cationic channel. These luminal non-specific cationic channels would allow the passive K+ efflux and Na+ influx across the apical membrane of marginal cells. This finding is consistent with the "one-pump" model of strial activity. The present study suggests that culture of strial marginal cells may be a suitable model for in-depth investigation of endolymph physiology. PMID- 1712531 TI - Mast cells and histamine in adenoid tissue and middle ear. AB - Biopsy specimens from middle ear mucosa of patients with secretory (SOM) and chronic (COM) otitis media as well as specimens of adenoid and tonsil tissue were studied for mast cells. Effusion fluid, nasopharyngeal secretion and supernatant of crushed adenoid tissue were analyzed for histamine with a radioenzymatic method. Astra blue (AB) safranine stained highly significantly more mast cells than did toluidine blue. Mast cell counts in SOM and COM were similar. There were significantly more mast cells in adenoid subepithelial tissue than in middle ear mucosal subepithelial layer. For epithelium the counts were within the same range in adenoids and middle ear mucosa. Histamine concentrations were significantly higher than plasma levels for SOM fluid and nasopharyngeal secretion. Crushed adenoid tissue showed values over 100 times higher than the histamine level in the secretion. PMID- 1712532 TI - Regulatory peptides and general neuroendocrine markers in human nasal mucosa, soft palate and larynx. AB - Various peptide immunoreactivities in the respiratory system have been reported, indicating complex physiological mechanisms. There is only little information on the upper respiratory system of man. The present study was carried out to demonstrate regulatory peptides in the nasal mucosa, larynx (vocal cords and ventricular folds) and soft palate of man using highly efficient immunocytochemical methods. In addition, some peptide immunoreactivities were measured by use of radioimmunoassay (RIA). Using indirect immunofluorescence and immunogold-silver staining (IGSS) with silver acetate autometallography, a series of peptides could be detected, including vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), galanin, calcitonin gene-related peptide (CGRP), substance P, neuropeptide tyrosine (NPY), C-flanking peptide of NPY (CPON) and somatostatin. In addition, antibodies to protein gene-product (PGP) 9.5, neuron-specific enolase (NSE), S-100, PHE-5 and neurofilament proteins gave positive reactions in tissue sections. Using RIA, CGRP, substance P, and neurokinin A were measured. Our results demonstrate a complex network of regulatory peptide-containing nerve fibers and the possible existence of endocrine cells regulating various functions of the upper respiratory system, which need to be further investigated. PMID- 1712533 TI - Parotid salivary secretory pattern in bulimia nervosa. AB - Parotid gland enlargement occurs in about 25% of patients with the binge eating syndrome of bulimia nervosa. The parotid salivary secretory patterns in 28 bulimics were determined in order to investigate the functional abnormality in the glands. Bulimia patients had a reduced resting flow rate. Bulimics who developed sialadenosis (4 patients) had reduced resting and stimulated flow rates. The salivary amylase activity was increased in both the resting and stimulated states in bulimics and the sialadenosis group. The resting total protein levels were greater in the bulimics. The electrolyte and immunoglobulin levels were within normal limits. The possibility of protein and enzymatic secretory disturbances due to autonomic nerve disorders as an explanation for the development of sialadenosis in bulimia nervosa is discussed. PMID- 1712534 TI - Antigen-specific plaques formation of cultured mononuclear cells in head and neck cancer. AB - Antigen-specific antibody production in vitro from peripheral blood mononuclear cells was studied for the assessment of the immune competence of patients with head and neck cancer. Optimal culture conditions were studied using inactivated Staphylococcus aureus and a saturation with IL-1 during B cell activation and pooled human AB serum on day 2. After passage of the mononuclear cells through sephadex G-10 columns, a significant increase in the antigen-specific antibody production was observed. In healthy donors a significant reduction of the antigen specific antibody production according to the abuse of alcohol and/or cigarette smoking was detectable. Interestingly, high alcohol consumption resulted in a more pronounced decrease of the antigen-specific antibody production in vitro than excessive cigarette smoking. Patients with squamous cell carcinoma of the head and neck who are considered to be most immunodeficient did not show any antigen-specific antibody production in vitro upon activation with sheep red blood cells in the presence of Interleukin-1 (IL-1). After filtration of mononuclear cells from peripheral bloodover sephadex G-10 beads, two thirds of the patients studied became stimulable. This increase in the antigen-specific antibody production in vitro was significant, though not as dramatic as in the age- and sex-matched control groups. Interestingly, the antigen-specific antibody production raised almost to the same level as that measured in healthy donors with high alcohol abuse and cigarette consumption. PMID- 1712535 TI - Development of liver metastasis in colorectal carcinoma. With special reference to venous invasion and basement membrane laminin. AB - The development of hepatic metastases in 344 patients with colorectal carcinoma was examined for correlation with the presence of both venous invasion by the primary tumor and basement membranes in the tumor tissue. The former was detected by Victoria blue and hematoxylin-eosin staining and the latter by antilaminin antibody. A significant difference in the incidence of venous invasion was noted between patients with and those without liver metastasis at surgery. Basement membrane deposition was found in half of all cases of well differentiated adenocarcinoma, which was significantly high compared with other tumor types. This was more distinct in metastatic foci in the liver and lymph nodes than in the primary lesion, but less marked in intravascular tumor tissue. Basement membrane deposition was seen more frequently in Dukes' A tumors than in B tumors, although this was not statistically significant. No relationship was found between basement membrane laminin positivity and five-year survival, nor was there any correlation between the incidence of liver metastasis and tumor histologic type. Venous invasion was considered to be intimately related to the development of liver metastasis. Deposition of laminin-positive basement membrane was dependent on the grade of tumor differentiation, whereas it had no direct relation to the development of liver metastasis. PMID- 1712536 TI - Selective alteration of cytokeratin intermediate filament by cyclosporine A is a lethal toxicity in PTK2 cell cultures. AB - The cytoplasm of eukaryotic cells contain a series of three filamentous structures, microtubules, microfilaments, and intermediate filaments that are termed the cytoskeleton. Cytokeratin, one type of intermediate filament, has no known physiological function, yet, can comprise up to 30% of the total cytoplasmic protein content. As there are no selective toxins to cytokeratins, it is not known if alterations to these hydrophobic filaments is a lethal event. Cyclosporine A, a novel hydrophobic immunosuppressant compound used to prevent allograft rejection, may show a selective toxicity to the cytokeratin filaments. This effect is seen in PtK2 cell cultures as a single large perinuclear aggregate of collapsed cytokeratin filaments (5 mM, 72 hr). Microtubules and microfilaments are not affected in PtK2 cell cultures (5 mM, 72 hr). Increased LDH levels into cell culturing media occur soon after cyclosporine exposure to PtK2 cell cultures (5 mM, 2 hr). Cytokeratin filaments show no changes at 12 hr exposure but show thickening, decreased plasma membrane attachments and some peri-nuclear ring formations at 24 hr (5 mM, 24 hr). Cyclosporine G, an analog of cyclosporine A, does not exhibit the cytokeratin filament collapse (5 mM, 72 hr). The effect of cyclosporine A on DNA binding protein (Mr 64 kd), believed to be a nuclear scaffolding protein related to intermediate filaments, exhibited an early invagination and folding of the nuclear membrane (5 mM, 4 hr). Due to a hydrophobic bonding potential between cyclosporine A and cytokeratin and cytokeratin-like intermediate filaments, cyclosporin A may be a selective cytokeratin toxin. Alteration of the cytokeratin filaments in PtK2 cell cultures may be a lethal event. PMID- 1712537 TI - Selective inhibition by magnosalin and magnoshinin, compounds from "Shin-i" (Flos magnoliae), of adjuvant-induced angiogenesis and granuloma formation in the mouse pouch. AB - Inhibitory effects of magnosalin and magnoshinin, compounds from the crude drug "Shin-i" (Flos magnoliae), on angiogenesis and pouch granuloma formation in mice induced by an adjuvant containing croton oil were investigated. The anti-chronic inflammatory effect of "shin-i" was caused by selective inhibition of angiogenesis by magnosalin and of granuloma formation by magnoshinin. PMID- 1712538 TI - Comparison of an isoelectric focusing technique and high-performance liquid chromatography for determination of fetal hemoglobin levels. AB - The authors previously have reported measurements of fetal hemoglobin in infants in blood samples taken at autopsy using an isoelectric focusing (IEF) procedure. The current study was undertaken to compare this methodology with a high performance liquid chromatography (HPLC) procedure. The correlation coefficient between the IEF and HPLC procedures was 0.938. The HPLC method is technically easier and has fewer disadvantages than the IEF procedure and is recommended for the determination of fetal hemoglobin levels. PMID- 1712539 TI - Auer rod-like inclusions in circulating lymphoma cells. AB - Circulating malignant lymphocytes from a 55-year-old woman with small cleaved follicular center cell lymphoma contained azurophilic splinter-shaped cytoplasmic inclusions. By light microscopic and ultrastructural criteria, these structures closely resembled Auer rods found in acute myeloid leukemia; however, the authors could not find cytochemical evidence of lysosomal origin (results were negative for myeloperoxidase, Sudan black B, acid phosphatase, and periodic acid-Schiff). Immunostaining and flow cytometric analysis confirmed a monoclonal IgM-kappa immunophenotype of the circulating malignant lymphoid cells. The inclusions did not show specific immunoglobulin staining by light microscopic or electron microscopic immunostaining techniques. The authors conclude that these membrane bound inclusions probably represent aberrant lysosomes in the malignant cells. PMID- 1712540 TI - Anaplastic thyroid carcinoma. Immunocytochemical study of 32 cases. AB - To study the histogenesis of and determine the most useful markers for diagnosing anaplastic thyroid carcinoma (ATC), 32 cases, including 2 with numerous osteoclast-like cells, were stained with a battery of antibodies to epithelial (keratin, epithelial membrane antigen [EMA], carcinoembryonic antigen [CEA]), mesenchymal (vimentin, desmin, muscle-specific actin [MSA], Factor VIII-related antigen [FVIII:RAg]), endocrine (thyroglobulin, calcitonin, chromogranin [Cg]), lymphocytic (leukocyte common antigen [LCA]), histiocytic (alpha-1-antitrypsin [alpha 1AT], alpha-1-antichymotrypsin [alpha 1AChy], KP1), melanocytic (HMB-45), and Schwann cell (S-100 protein) markers. Five tumors were associated with papillary carcinoma. In one of these cases, a morphologic continuum between the well-differentiated carcinoma and the ATC was visualized by their positive immunostaining for both vimentin and keratin, thus supporting the hypothesis that the latter tumor originated from the former. Twenty-five (78.1%) tumors expressed keratin, 10 (31.3%) reacted for EMA, and 3 (9.4%) expressed CEA, confirming the epithelial nature of this neoplasm. Reactivity for thyroglobulin was seen in a small number of cells in five (15.6%) thyroglobulin was seen in a small number of cells in five (15.6%) ATCs. Because all of the cases that expressed keratin also stained positively for EMA, CEA, or thyroglobulin, it is believed that keratin is the most useful epithelial marker for diagnosis of ATC. A lack of reactivity for calcitonin and Cg indicates that these tumors are not derived from C cells, as has been proposed by some authors. Reactivity for KP1 (CD68), a monoclonal antibody that reacts with a macrophage-associated antigen, occurred in the osteoclast-like cells but not in the anaplastic tumor cells. This finding, together with negative keratin staining of the osteoclast-like cells, indicates that these cells are not epithelial in nature and therefore should be considered reactive rather than neoplastic. Thirty tumors (93.8%) expressed vimentin, 15 (46.9%) marked for alpha 1AChy, 11 (34.4%) exhibited alpha 1AT, and 11 (34.4%) expressed S-100 protein. Because all of these markers can be seen in a wide variety of tumors of different histogeneses, they have no value in the diagnosis of ATC. Although immunostaining for FVIII:RAg, desmin, and MSA was negative in all of these tumors, these markers can help to differentiate between ATCs and some soft tissue sarcomas with which they can be confused. PMID- 1712541 TI - The human hematopoietic progenitor cell antigen (CD34) in vascular neoplasia. AB - The human hematopoietic progenitor cell antigen CD34 is synthesized and expressed by early normal hematopoietic progenitor cells and by many acute leukemias. Anti CD34 antibodies also have been reported to stain blood vessels in tissue sections, and, more recently, CD34 mRNA has been detected in vascular endothelial cells. Therefore, the authors studied the diagnostic utility of immunohistochemical CD34 antigen detection in tumors of endothelial cell derivation and compared the results with stains for von Willebrand (vW) factor. A wide variety of epithelial and mesenchymal neoplasms also were examined to assess the specificity of CD34 for vascular neoplasia. Seven cases of angiosarcoma (seven of seven), five cases of Kaposi's sarcoma (five of five), and eight cases of epithelioid hemangioendothelioma (eight of eight) were moderately to strongly positive for CD34. This reactivity was equally intense in frozen sections, alcohol-fixed tissue, and formalin-fixed specimens. In many cases, the malignant endothelial cells stained more strongly than adjacent benign endothelium. Moreover, in most cases CD34 positivity was quantitatively and qualitatively stronger than staining for vW factor. Two cases of hemangiopericytoma (two of two) were CD34 positive but stained less intensely than the angiosarcomas, Kaposi's sarcomas, or hemangioendotheliomas. Five of six cases of hemangioma also stained positively for CD34; the nonreactive tumor in this group was the only one among 28 vascular neoplasms studied that was not reactive for CD34. In comparison, 9 of the 28 vascular tumors did not stain for vW factor. Three hundred fifty-seven tumors of nonvascular derivation also were examined for CD34 antigen expression. Focal light staining was seen in one pulmonary squamous cell carcinoma; moderate to intense staining was observed in half of the epithelioid sarcomas studied (8 of 16) and in a minority of leiomyosarcomas (3 of 22). These findings indicate that CD34 is a sensitive and relatively specific marker for neoplasms of vascular origin. PMID- 1712542 TI - Comparative immunohistochemical staining for desmin and muscle-specific actin. A study of 576 cases. AB - Muscle-specific actin (MSA) and desmin are considered to be sensitive and specific markers for muscle differentiation. The authors compared staining patterns for these markers in 576 samples of normal, reactive, and neoplastic tissues. The standard avidin-biotin-peroxidase complex technique was performed with the use of two commercial antibodies against MSA (HHF35; Enzo Biochemical, Inc., New York, NY) and desmin (DER11; DAKO Corporation, Santa Barbara, CA), respectively, on consecutive paraffin-embedded tissue sections from these cases. Both MSA and desmin were found in all 80 normal muscle samples. Although MSA appeared diffusely in all vascular smooth muscle samples, desmin was demonstrated focally in vascular smooth muscle cells in 100 of 196 samples. MSA but not desmin always was found in myoepithelial cells (25 samples), pericytes (286 samples), and decidual cells (7 samples). Among 76 cases of myofibroblast-containing lesions, 14 and 54 were found to have desmin and MSA, respectively. MSA and desmin were found in 4 of 4 cardiac rhabdomyomas, 34 of 34 rhabdomyosarcomas, and 5 of 6 leiomyomas. Among 22 leiomyosarcomas, 7 displayed either MSA or desmin and 7 showed both markers. In general, more tumor cells showed staining for MSA than desmin, but the reverse was true in some cases. Tissue fixed in Zenker's solution seemed to show a significant decrease in MSA immunoreactivity, but no significant change for desmin staining was observed. None of the 154 normal tissues and 22 benign nonmyogenic tumors expressed MSA or desmin. Among 133 malignant nonmyogenic tumors, positive staining for both desmin and MSA was found in 3 of 8 cases of glioblastoma multiforme, 1 of 10 malignant schwannomas, and 1 of 14 malignant fibrous histiocytomas; staining for only MSA was found in 3 of 14 malignant fibrous histiocytomas, 1 of 10 malignant schwannomas, 6 of 6 fibromatoses, 1 of 1 mammary myofibroblastoma, and 1 of 7 malignant mesotheliomas; and staining for desmin only was seen in 1 of 7 malignant mesotheliomas.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712543 TI - Expression of a polymorphic epithelial mucin antigen defined by the monoclonal antibody BC2 in ovarian carcinoma. Use of the BC2 antibody for the detection of micrometastases. AB - The BC2 monoclonal antibody, which binds to an epitope on the peptide backbone of polymorphic epithelial mucin, was tested immunohistochemically for reactivity with epithelial ovarian carcinoma. This epitope was expressed in 90 of 91 malignant ovarian tumors; in 88% of these, more than 50% of the tumor cells expressed the epitope. In 94% of the positive tumors, the epitope was expressed on the cell membrane; in 56%, cytoplasmic expression was evident; and in 39%, secreted extracellular antigen was detected. Differences were not clearly discernible between dissimilar histotypes with respect to the percentage of cells expressing antigen and antigen localization. Thirteen of 19 benign ovarian cystadenomas also expressed the epitope, but staining was weak and restricted to the luminal surface of the cell membrane. A blind retrospective immunohistochemical analysis of all second-look laparotomy biopsy specimens from 20 patients also was performed. All four patients in whom microscopic disease was detected by standard pathologic assessment had BC2-positive metastases. Of seven patients in whom recurrent disease subsequently developed despite negative pathologic findings, four had biopsy specimens containing BC2 antigen-positive adenocarcinoma-like cells. Of the nine patients with negative results on operation and no recurrence, one had biopsy specimens containing BC2 antigen positive adenocarcinoma-like cells. Mesothelial cells, although typically negative, expressed the epitope in one biopsy specimen, necessitating caution in the interpretation of positive cells. The BC2 antibody is reactive with most epithelial ovarian carcinomas and appears to be a useful tool for the detection of micrometastases. PMID- 1712544 TI - Immunocytochemical analysis of progesterone receptors in breast cancer. AB - Breast cancer specimens from 116 patients were assayed for the presence of progesterone receptor (PR), with the use of a highly specific monoclonal antibody and the peroxidase-antiperoxidase technique on cryostat and permanent sections. Results were compared with those obtained by the conventional PR determination by dextran-coated charcoal (DCC) assay; they were in concordance in 90% of cryostat sections and 85% of paraffin-embedded tissue. The sensitivity and specificity of the PR immunocytochemical assay (PR-ICA) were 91% and 89% for frozen sections and 83% and 89% for permanent sections, respectively. The immunostained slides also were evaluated for several semiquantitative features, including staining intensity, heterogeneity of staining, and the proportion of positive tumor cells. A statistically significant correlation was found between the percentage of tumor cells stained with the PR immunocytochemical technique and the PR-cytosol levels (P less than 0.05). These results suggest that the PR-ICA is an effective tool in the evaluation of PR content in breast cancer and can be applied in paraffin as well as frozen sections. This technique provides excellent morphologic detail, as well as tissue localization for PR. It also offers an alternative for assessment of PR when fresh tissue is not available for conventional hormone receptor analysis. The immunocytochemical assay can be performed easily at community hospitals. Because it requires only a small amount of tissue, PR-ICA is an ideal method for analyzing specimens of insufficient size for the DCC assay. This technique also is suited to the evaluation of fine-needle aspiration biopsy specimens. PMID- 1712545 TI - Automated procedure for dewaxing and rehydration of paraffin-embedded tissue sections for DNA flow cytometric analysis of breast tumors. AB - Flow cytometric DNA analysis of paraffin-embedded tumors is an important diagnostic and prognostic tool in clinical pathology. The technique is limited, however, by the time-consuming multistep procedure for dewaxing and rehydrating tissue. The authors developed an automated procedure to complete the dewaxing and rehydration of tissue using a routine histologic tissue processor with a 24-hour timer. This technique provided excellent tissue recovery and reproducible DNA histograms comparable to those obtained by manual methods. Subsequently, the authors analyzed the DNA content of 93 paraffin-embedded breast cancer tissues. The automation of a significant portion of the routine processing required for paraffin-embedded tissue makes cytometric DNA analysis a more practical procedure in the laboratory. PMID- 1712546 TI - Aberrant MT2 positivity distinguishes follicular lymphoma from reactive follicular hyperplasia in B5- and formalin-fixed paraffin sections. AB - Often, it is difficult to distinguish follicular lymphoma from reactive follicular hyperplasia histologically. Immunotypic evidence of monoclonality cannot be demonstrated routinely or reliably in routine paraffin-embedded sections. To determine whether a panel of monoclonal antibodies reactive with lymphoid cells in paraffin-embedded sections might be useful in distinguishing these confusing proliferations, the authors examined 45 follicular lymphomas and 30 follicular hyperplasia with the following antibodies; L26, B2, MT1, MT2, and UCHL-I. All sections were routine paraffin preparations from formalin- or B5 fixed tissue and were immunostained with the avidin-biotin immunoperoxidase technique. Ninety-two percent of the B5-fixed and 77% of the formalin-fixed lymphomas were MT2 positive. None of the reactive hyperplasias stained positively, and none of the other antibodies demonstrated consistent differences between these benign and malignant proliferations. MT2 marks interfollicular T cells and mantle-zone B cells in normal lymph nodes but does not mark normal germinal centers; this staining pattern is retained in reactive hyperplasia. However, paradoxically, in most follicular lymphomas the neoplastic germinal centers show aberrant MT2 positively. The authors conclude that MT2 may be useful in distinguishing follicular lymphoma from follicular hyperplasia in paraffin sections. PMID- 1712547 TI - The predictive value of bone marrow morphologic characteristics and immunostaining in primary (AL) amyloidosis. AB - The authors previously demonstrated that bone marrow plasmacytosis in primary (AL) amyloidosis may be monoclonal or polyclonal. However, the clinical implications of the degree of plasmacytosis and its clonality have not been studied. The authors evaluated 62 patients with AL amyloidosis, 40 of whom had monoclonal medullary plasma cells. There was complete concordance between the light chain class of the plasma cells in the monoclonal cases and that of the circulating paraprotein in the 22 cases associated with a paraprotein. The remaining 22 patients had polyclonal plasma cells, although a paraprotein was detected in 6. The degree of plasmacytosis was significantly higher among patients with monoclonal plasma cells and correlated inversely with length of survival. The authors' findings indicate that the quantitation of bone marrow plasma cells in AL amyloidosis by immunoperoxidase studies may predict the clinical course. PMID- 1712548 TI - Evaluation of continuous infusion low-dose 5-azacytidine in the treatment of myelodysplastic syndromes. AB - The treatment of myelodysplastic syndromes (MDS) is both difficult and controversial. In the current study, we evaluated the efficacy of low-dose 5 azacytidine in the treatment of this disorder. Fifteen patients with MDS were entered on study to be treated with 5-azacytidine by continuous intravenous infusion for 14 days. Doses of the drug ranged from 10 mg/m2/day to 35 mg/m2/day, with most patients receiving 16.5 mg/m2/day. In two patients, the drug was stopped early in the course of treatment because of thrombocytopenia. Thirteen patients completed therapy according to protocol. Three of 13 patients demonstrated a partial response to therapy. Of these three patients, one had an increase in platelet and absolute neutrophil counts, while the other two no longer required support with red cell transfusions. The drug was well tolerated and significant myelosuppression did not occur in most patients. Low-dose 5 azacytidine appears to have activity in the treatment of primary MDS and future studies should consider evaluation of this drug in combination with hematopoietic growth factors. PMID- 1712549 TI - Prostatic acid phosphatase in carcinoid tumors. Immunohistochemical and immunoblot studies. AB - The immunohistochemical demonstration of prostatic acid phosphatase (PAcP) and/or prostate-specific antigen (PSA) has been accepted as being reliable in identifying metastatic adenocarcinoma of prostate origin. However, islet cell tumors, especially hindgut-derived carcinoid tumors, have occasionally been reported to be positive for PAcP. We therefore studied a series of carcinoid tumors of the lung and gastrointestinal tract immunohistochemically for PAcP expression by using two polyclonal antibodies and one monoclonal antibody. Thirty three carcinoid tumors were examined. All five rectal carcinoids in the series showed convincing PAcP positivity with at least two of the three anti-PAcP antibodies. No significant PAcP positivity was observed in the remaining 28 foregut- and midgut-derived carcinoid tumors, except for weak focal positivity in one lung carcinoid. PSA antibody reacted negatively in all cases. Western blots of an aqueous cell lysate from one rectal carcinoid revealed protein bands in the region of 45-55 kd that immunoreacted with anti-PAcP antibodies, confirming the validity of the immunostains. These results suggest that PAcP positivity is common in rectal carcinoid tumors and that it most likely represents true PAcP expression. This seemingly aberrant protein expression may be explained by the shared cloacal derivation of the rectum and prostate, giving rise to cells with both endocrine and partial prostatic epithelial differentiation. PMID- 1712550 TI - Selection of normal human hematopoietic stem cells for bone marrow transplantation using immunomagnetic microspheres and CD34 antibody. AB - Complete yet nontoxic removal of tumor cells from autologous marrow grafts has proved difficult. New methods for separating normal stem cells from tumor cells are needed. The CD34+ cells in bone marrow, 1-2% of the low-density leukocytes, include precursors of all lymphohematopoietic lineages and probably also the primitive cells responsible for engraftment. A nontoxic, inexpensive, reproducible, and clinically applicable method for positive selection of CD34+ cells was developed. Paramagnetic microspheres coated with goat anti-mouse IgG1 are used to partition the cells; brief incubation with chymopapain is used to release them from the beads. Chymopapain exposure does not injury colony-forming cells or delay engraftment in rodents. Clinical volumes of bone marrow can be processed rapidly. In pilot experiments, the resulting grafts have a purity of 85 99% CD34+ cells and 40% median recovery of the assayable colony-forming cells. These studies form the background for a Phase I trial of autologous BMT using CD34+ stem cells. PMID- 1712551 TI - Antibodies to specific cell surface antigens of a human leukemia cell line, K 562, transduce negative growth signals. PMID- 1712552 TI - Control of HILDA/LIF gene expression in activated human monocytes. AB - In the present study we have investigated some of the mechanisms involved in HILDA/LIF gene expression in activated human monocytes and compared them to those of G-CSF gene expression, another monocyte-derived cytokine. In the absence of added stimuli, HILDA/LIF mRNA was barely detectable in monocytes. HILDA/LIF mRNA accumulation was weakly induced by stimuli such as LPS or phorbol ester alone, and in a synergistic manner when they were used in combination with 1,25 dihydroxyvitamin D3. Nuclear run-on transcription analysis in U937 cells did not detect an increase of HILDA/LIF gene transcription upon phorbol ester and 1,25 dihydroxyvitamin D3 stimulation. Posttranscriptional control of HILDLA/LIF mRNA levels by an increase in mRNA half-life was demonstrated in the synergy between phorbol ester and 1,25-dihydroxyvitamin D3 and in the superinduction of HILDA/LIF transcript accumulation when CHX was added to stimulated cells shortly before cell harvesting. HILDA/LIF mRNA expression was largely inhibited when U937 cells were treated with CHX either at the onset or 4 h after the beginning of the stimulation period. When CHX was added 2 h before cell harvesting, a superinduction of mRNA accumulation was obtained. G-CSF mRNA accumulation showed a different pattern of response to the same stimuli, in particular a higher rate of response to LPS. In contrast to HILDA/LIF, an augmentation of G-CSF gene transcription was detected in activated monocytic cells when compared to controls. These studies indicate that HILDA/LIF gene expression by phorbol ester- and 1,25-dihydroxyvitamin D3-treated human monocytes has a relatively specific regulation, as compared to G-CSF gene expression, and that it is largely dependent on posttranscriptional mechanisms probably acting through labile, newly synthesized proteins. PMID- 1712553 TI - Interleukin-1 and tumor necrosis factor-alpha inhibit erythropoietin production in vitro. AB - The effects of several immunomodulatory peptides (recombinant, human) on the in vitro production of erythropoietin (Epo) were studied in cultures of the human hepatoma cell line Hep G2. A dose-dependent decrease of up to 60% in Epo production was induced by interleukin-1 beta, interleukin-1 alpha, and tumor necrosis factor-alpha (in that order of potency). In contrast, moderately increased Epo levels resulted with interleukin-6 or interferon-gamma treatment at high concentrations. Concomitant measurements of the production of alpha fetoprotein indicated that the observed effects were specific for Epo. Hence, we suspect a modulating role of the immune system in the in vivo control of Epo production and postulate that interleukin-1 and tumor necrosis factor-alpha are involved in some of the cases of lowered blood Epo levels in association with renal diseases, chronic inflammation, and malignancies. PMID- 1712554 TI - Negative control of the number of F cells by the immune system of the adult. PMID- 1712555 TI - Effect of human immunodeficiency virus type 1 on CD34+ cells. AB - The effect of HIV-1 on the in vitro growth of enriched hematopoietic stem cells (CD34+ cells) obtained from normal peripheral blood samples was studied. In comparison to untreated controls, the number of viable CD34+ cells progressively and significantly decreased in liquid cultures containing interleukin-3 (IL-3, 100 U/ml) after inoculation with HIV-1. In inoculated samples there was a significant reduction of all the hematopoietic progenitors (CFU-GM, BFU-E, CFU Meg) starting from the second day of culture, CFU-GM being the most affected. In spite of these findings, no evidence of viral replication was observed: the total amount of p24 in HIV-1-inoculated CD34+ cell cultures showed a plateau, slightly declining towards the end of the experimental observation period. Moreover, erythroid and granulomacrophage colonies harvested from inoculated CD34+ cell cultures were unable to infect susceptible cells. PMID- 1712556 TI - Molecular analysis of primitive hematopoietic cell proliferation control mechanisms. AB - Cells at two distinct early stages in the development of mature human blood cells from primitive totipotent hematopoietic stem cells can now be defined and quantitated by separate in vitro assays. Current evidence suggests that most, if not all, colony-forming cells--that is, cells that give rise to colonies of mature progeny within one to three weeks in semisolid culture systems, represent an intermediate stage of hematopoietic progenitor. These cells are not self sustaining; if they are used to initiate hematopoiesis on competent marrow stromal layers, they rapidly disappear as they differentiate or die. However, clonogenic cells can be generated in such cultures from another cell type over a period of four to eight weeks. We have, therefore, assigned the term long-term culture initiating cell (LTC-IC) to this latter type of clonogenic precursor cell. The production and differentiation of cells in both of these compartments in LTC are dependent on, and regulated by, nonhematopoietic "stromal" cells that form a heterogeneous adherent layer in which close-range interactions with hematopoietic cells take place. The use of separate endpoints to monitor the maintenance, differentiation, and reversible activation or arrest of cycling of these cells has recently revealed different molecular mechanisms regulating their respective functions. However, an important common feature appears to be the relative local concentration of positive and negative regulators to which the target hematopoietic cell is exposed. Both gene expression and growth factor release measurements as well as results obtained using genetically engineered stroma and repeated soluble growth factor addition implicate G-CSF as an endogenous positive regulator of primitive hematopoietic cells. Similarly, gene expression, factor production, factor addition, and neutralizing antibody experiments implicate TGF-beta as an endogenous inhibitor of primitive hematopoietic cells. PMID- 1712557 TI - Studies of stromal fibroblastic progenitors and hematopoietic progenitors in patients with acute graft-versus-host disease. PMID- 1712558 TI - Inhibition of interleukin-3-dependent growth of CD34+ acute myelogenous leukemia cells by recombinant soluble CD23. PMID- 1712559 TI - Feedback suppression of normal and leukemic B cell colony growth by CD5+ B cells. PMID- 1712560 TI - On the possible role of I-J+ suppressor cells in murine hematopoiesis. PMID- 1712561 TI - [Nasal reconstruction: a comparative study between the Converse scalping flap and expanded median forehead flap]. AB - The authors, reviewing a series of 16 at least subtotal nasal reconstructions over the past 4 years, discuss the respective advantages of the two methods, which both give good results for this purpose. Nowadays, the balance favors expansion despite its duration and complexity for the very significant aesthetic benefits in terms of forehead scarring. The guidelines concerning this technique employed in 6 cases are described and reviewed in detail. Nevertheless, the Converse scalping flap remains a simple and quick solution especially after cancer resection in the elderly; some details seem important to diminish its sequelae. PMID- 1712562 TI - [Gastro-omental free flap: technique, indications and results in buccopharyngeal reconstruction after cancer excision. Apropos of 7 cases]. AB - The gastro-omental flap is centered around the midportion of the greater curve of the stomach, with the attached omentum. The flap is based on the right gastro epiploic pedicle. The size of the mucosal patch is adaptable. This flap offers several practical advantages: great plasticity (including the omentum), large length and size of the pedicle, mucus secretion which may improve xerostomia, and less morbidity. Disadvantages include the propensity for excessive mucus production (needing tracheotomy), the requirement for an abdominal operation, and the possibility of peptic ulceration (no case reported in the literature). Seven patients have been treated, with a good result. This flap is suitable for repair of complex soft-tissue defects after extensive cancer surgery. PMID- 1712563 TI - [Purpose and effects of mammaplasty...]. AB - Mammaplasties (following amputation or for the treatment of hypertrophy) involve the femininity of each patient, but at different moments in her life. The patient's request for plastic surgery reflects her discomfort with her body related to a mutilating operation or to breasts which interfere with her interpersonal and sexual life. The two types of operation have multiple psychological effects, generally beneficial as they allow the woman to regain the elements of her identity disturbed by the amputation or enable her to more fully explore her potential in interpersonal relationships. PMID- 1712564 TI - [Reduction of mammaplasty scars: from a short inframammary scar to a vertical scar]. AB - A better understanding of the vascular anatomy of the breast has drastically reduced the risk of postoperative necrosis in breast reduction. Scars however remain a major concern, and techniques to reduce these have often been considered to be less satisfactory in terms of the shape and stability of the result. Our experience with more than 1,000 breasts operated on between 1984 and 1989 with a short inframammary scar technique has proved the contrary. The next step was to eliminate the inframammary scar, as proposed by Lassus, and to leave just a periareolar scar and a lower vertical scar which does not cross the inframammary fold. One hundred and four breasts, in sixty four patients--17 to 60 years old- have been operated on according to this vertical technique between April and September 1989. Twenty seven cases of ptosis correction in seventeen patients, and seventy seven reductions in forty seven patients, with a median excision weight of 460g, have been performed. By means of an individualized preoperative drawing and several technical devices, the results have proved that vertical mammaplasty is an excellent technique particularly indicated for women with elastic skin and a firm gland. Recent experience with liposuction at the beginning of the operation, has given new possibilities for breast modelling. In fatty juvenile hypertrophies, liposuction alone may even be adequate to reduce the volume, retaining a satisfactory shape for the breast with minimal scarring. PMID- 1712565 TI - [Changes in the peroneal flap and its use in reconstructive surgery of the lower limb. Apropos of an experience of 10 cases]. AB - A peroneal fasciocutaneous flap supplied by the peroneal septocutaneous vessels and raised from the lateral side of the lower leg was reported by Yoshimura in 1983. This flap which can be used as a proximally or distally pedicled or free flap is very useful for leg skin coverage. This flap has a great potential for skin cover and composite reconstruction of the lower limb due to its multiple structural facets (cutaneo-aponevrotic or composite flap), its possible extensions to other vascular territories and the variable geometry of its mode of transfer. 8 reconstructions have been performed. Their indications are described: 4 proximally pedicled flaps (3 with the fibula), 4 reverse-flow island flaps (1 with Soleus and Peroneus longus muscles). The authors stress the importance of preoperative assessment of the feasibility of a given flap which may be limited by post-traumatic, surgical or anatomic modifications. In particular, the uppermost septocutaneous artery which corresponds inconstantly to the "circumflex peroneal artery" can only be visualized by preoperative arteriography. This artery supplies a proximal peroneal flap which can be used as an island or a free flap. We have used this new variety as a free flap in 2 cases and were satisfied with the results. These various clinical applications without any significant complication or flap failure confirm the biological performance and the safe procedure of peroneal flaps. PMID- 1712566 TI - [High pedicle scalp flaps]. AB - A retrospective study of 207 transposition flaps with a high pedicle of various sizes and locations allows a better understanding of the esthetic effects, the reliability, the complications and the indications of this hair replacement surgery. Four kinds of flaps with a high pedicle have been transposed separately or jointly for an immediate correction of pattern baldness. The long flaps are delayed but the short flaps are transposed in one session without delay. PMID- 1712567 TI - [Surgical treatment of blepharoptosis in patients with mitochondrial myopathies. Apropos of 4 cases]. AB - The authors briefly describe the commonest forms of mitochondrial myopathies particularly those associated with disorders of ocular motility. They report their series of four patients suffering from ptosis of the eyelid caused by mitochondrial myopathies. The ptosis was corrected surgically in all cases by means of Muller's muscle adaptation technique. The authors discuss their results and the indication for this technique. PMID- 1712568 TI - [Glaucoma as etiopathogenic hypothesis of amaurosis after blepharoplasty. Apropos of a clinical case]. AB - Amaurosis after blepharoplasty is a complication which is considered to be rare, but which nevertheless deserves extreme attention. Although various hypotheses have been proposed (and sometimes proved) its etiopathogenesis is still uncertain. The observation of a case of temporary loss of vision after blepharoplasty, which promptly regressed after medical therapy, led us to a revision of the most common causes of amaurosis, and to formulate, for this case, the etiopathogenetic hypothesis of acute glaucoma, which has already been reported in scientific literature, but without any case report. PMID- 1712569 TI - [Cleft lip and palate. Guarded approach]. PMID- 1712570 TI - [Apropos of a case of infection after esthetic rhinoplasty]. AB - Infection after rhinoplasty is infrequent occurring in less than 1% of cases. When it does occur it may be due to devascularised spicule of bone or in a hematoma. Of much less frequent occurrence is the toxic shock syndrome associated or not with nasal packing and due to staphylococcus aureus. When administering prophylactic antibiotic in nasal surgery one must take into consideration their own hazards: drug reaction, candida infection or resistant staphylococcus aureus. PMID- 1712571 TI - [Hand injuries caused by snake bites. Apropos of 9 cases treated in a West African regional hospital]. AB - The author reports 9 cases of snake bite hand injuries in a regional hospital in the ivory coast. This study presents a number of particular points: patients often consult quite late; the precarious hygiene induces microbia contamination; patients often have a very poor conception of their body schema, which sometimes creates problems of understanding during reeducation. Treatment consists of emergency debridement under antibiotic cover of Iselin's "emergency with delayed surgery". The good blood supply of the hand allows satisfactory functional results. PMID- 1712572 TI - [Review of cranio-maxillo-facial malformations characterized by abnormal bone density]. AB - This paper is a review of cranio-maxillo-facial bone malformations, characterized by an abnormal cortical bone density. For each lesion, clinical and radiological cranio-maxillo-facial features are described. Whenever possible, recent etiopathogenic and differential diagnostic elements are included, on the basis of a review of the literature. PMID- 1712573 TI - [Cytokine regulation of hemopoietic stem cell proliferation]. AB - The maintenance of mature blood cells requires the presence of hemopoietic stem cells, whose characteristics are their ability both to self-renew and to make differentiated progeny. We proposed a stochastic model for the mechanism of self renewal and commitment of hemopoietic stem cells using in vitro clonal culture techniques. Recent progress in the molecular biology facilitated isolation and characterization of a number of cytokines that regulate the proliferation of hemopoietic stem cells. In this review, I summarize the current understanding of the mechanisms on interaction between several cytokines including IL-1, IL-3, IL 6, IL-11, G-CSF and stem cell factor (SCF). PMID- 1712574 TI - A chymotrypsin-like proteinase that may be involved in desquamation in plantar stratum corneum. AB - We have recently reported that unipolar cell shedding from plantar stratum corneum incubated in vitro, and the associated degradation of the desmosomal protein desmoglein I, are dependent on the activity of a proteinase that can be inhibited by aprotinin, chymostatin and zinc ion. The aim of this work was to find a proteinase in plantar stratum corneum that fulfils the criteria for being the responsible enzyme. Dissociated plantar corneocytes were incubated with the chymotrypsin substrate 3-carbomethoxypropionyl-L-Arg-L-Pro-L-Tyr-p-nitroanilide hydrochloride (S-2586) and H-D-Ile-Pro-Arg-p-nitroanilide dihydrochloride (S 2288), a substrate for a wide range of serine proteinases with arginine specificity. There was a significant rate of hydrolysis of S-2586, but S-2288 was hydrolysed only very slowly. Extraction of dissociated corneocytes with buffers containing KCl or sodium dodecyl sulphate released one major proteinase that could be detected by electrophoresis in polyacrylamide gels with copolymerized casein and subsequent incubations of the gels. Both the caseinolytic activity and the S-2586-hydrolysing activities were inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. The S-2586-hydrolysing activity was also inhibited by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride. Both activities were optimal at pH 7-8 but were also significant at pH 5.5. On gel exclusion chromatography, the S-2586-hydrolysing and caseinolytic activities were eluted with an apparent molecular weight of around 18 kDa. When analyzed by electrophoresis in the presence of sodium dodecyl sulphate under non-reducing conditions the caseinolytic enzyme had an apparent molecular weight of around 25 kDa.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712575 TI - The expression of skin-specific gene K51 in the epidermal layer of human skin and in basal cell carcinoma cells. AB - Gene K51 probe isolated previously from the rat genomic library has been used to study the expression of its human counterpart by in situ hybridization and Northern blot analysis. A polyA-containing transcript of human gene K51 of 3 kb size has been detected in embryonic skin. The gene is also expressed in the epidermis of newborn humans and adults, but not in the adjacent mesenchymal tissues. Immunostaining with keratin antisera revealed predominantly earlier stage expression of K51 than cytokeratin markers. Sebaceous and sweat glands also contain cells expressing K51 gene. K51 expression was found in the cells of eight individual basal cell carcinomas tested, with the level of expression lower than in keratinocytes from normal human epidermis. We propose that K51 gene expression could serve as a convenient marker for the study of the process of skin keratinocyte development and the changes in this process associated with skin cancers and dysplasia. PMID- 1712576 TI - Regulation of epidermal growth factor receptor expression in normal and transformed keratinocytes. AB - Transformed keratinocytes (SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation programme were used to study the regulation of EGF-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We were able to demonstrate that EGF-receptor expression in normal and transformed keratinocytes depends upon the cell type and one or more levels of regulatory control. At the DNA level, EGF-receptor gene amplification occurred in poorly differentiating cells. At the mRNA level, cells showing EGF-receptor gene amplification expressed elevated mRNA and protein levels when cultured under low Ca2+ conditions. Cells not exhibiting EGF-receptor gene amplification showed equal mRNA expression, regardless the Ca2+ concentration in the culture medium. At the protein level, EGF-receptor protein was decreased in cells exhibiting EGF receptor gene amplification when extracellular Ca2+ was increased (to 1.6 mM) to stimulate differentiation, the decrease in protein being comparable to mRNA expression. Cells not exhibiting EGF-receptor gene amplification showed equal protein expression, regardless of the Ca2+ concentration in the culture medium. Under the same conditions, SV40 transformed keratinocytes showed equal mRNA but elevated protein expression in cells grown under low Ca2+ conditions. At the membrane level, normal keratinocytes and SCC-17F2 cells showed elevated numbers of cell surface exposed EGF-receptors in cells grown under low Ca2+ conditions, but equal mRNA and protein expression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712577 TI - Characterization of grass pollen reactive T-cell lines derived from lesional atopic skin. AB - Since it has been hypothesized that atopic dermatitis represents a cellular immune reaction to exogenous aeroallergens, we investigated whether lesional skin contains allergen-specific T-cells and which lymphokines they might secrete. Using phytohaemagglutinin or grass pollen for the cloning procedure, we established a series of T-cell lines from the skin of two patients. When rechallenged with the allergen, three out of 12 dermal lines which had been cloned with the pollen extract and three out of 20 epidermal lines cloned with PHA were found to proliferate specifically. With one exception, allergen-specific lines were CD4+, CD8-, alpha/beta receptor +. The reaction pattern to the single components of the grass allergen extract was assessed with the line UH-D3. Further, the proliferative response to Lolium perennis was inhibited by HLA-DR antibody, indicating its dependence on structures of the MHC class II complex. Only one out of four CD4+ allergen-reactive lines secreted considerable interferon-gamma activity but all secreted interleukin-4. The relative predominance of IL-4 points to a possible role of skin-derived T-cells in the synthesis of IgE. The identification of allergen-specific T-cells in lesional skin of patients with atopic dermatitis is consistent with the hypothesis that their dermatitis represents a T-cell-mediated immune reaction. PMID- 1712578 TI - Expression of tenascin in perifollicular connective tissue: comparison of normal scalp and alopecia areata. AB - Tenascin, a recently discovered extracellular matrix protein, was demonstrated in perifollicular connective tissue of normal human scalp using immunohistochemistry. Its localization was different from other well-known extracellular matrix components, like fibronectin, laminin and heparan sulphate proteoglycan. A comparison between alopecia areata and normal scalps did not reveal major qualitative differences, except for an increased expression near heavily infiltrated follicles. PMID- 1712579 TI - Atrioventricular septal defect repair in infants. AB - From September 1984 through August 1989, 33 consecutive infants (mean age, 9 months; 13 male) received a single-stage intracardiac repair of complete atrioventricular septal defect. Preoperative evaluation of valvar morphology and function involved echocardiograms in 21% (7/33) and echocardiograms with cineangiograms in 79% (26/33). All infants operated on were included in the analysis. Patients with other complicating abnormalities were not excluded. All operations used a two-patch technique for closure of the atrioventricular septal defect in association with mitral valve repair. The newly formed septal leaflet of the mitral valve was repaired using unpledgeted interrupted sutures. Preoperative and postoperative echocardiograms were used to evaluate mitral valve regurgitation and left ventricular dysfunction as mild, moderate, or severe. The 30-day mortality was 6% (2/33). Follow-up ranged from 1 month to 60 months. Postoperative mitral valve insufficiency was mild in 84% versus 6% preoperatively, moderate in 3% versus 52% preoperatively, and severe in 13% versus 42% preoperatively. Mitral valve dysfunction necessitating reoperation occurred in 6% (2/31). Mitral valve function postoperatively was improved compared with preoperatively (p less than 0.001). The low 30-day operative mortality and the excellent late postoperative valvar function demonstrate the value of single-stage two-patch repair of atrioventricular septal defect early in life. PMID- 1712580 TI - Cellular oncogene activation by human cytomegalovirus. Lack of correlation with virus infectivity and immediate early gene expression. AB - The contribution of expression of human cytomegalovirus (HCMV) immediate early (IE) genes to the rapid and transient increase in cellular (c)-oncogene (fos, jun, myc) transcription following HCMV infection was investigated. A partial temporal overlap was observed between the increases in c-oncogene RNA levels and the increase in either transcripts from HCMV IE genes or the number of cells in which HCMV IE proteins were detected. The increases in c-oncogene RNA levels, however, slightly preceded the increase in the detection of HCMV IE transcripts or proteins. To distinguish between the temporal coincidence and a direct relationship between expression of HCMV IE genes and the increased transcription of c-oncogenes, the number of cells synthesizing HCMV IE proteins was reduced by infecting with virus stock enriched in defective particles. Alternatively, the synthesis of HCMV IE proteins was essentially eliminated by ultra-violet (UV) irradiation of virus stock or by inhibitors of protein synthesis. Virus stocks enriched in defective particles demonstrated a substantially reduced capacity to direct the synthesis of HCMV IE proteins, but were more efficient in activating c oncogene expression than infectious virus stocks. Elimination of expression of HCMV IE genes by UV-irradiation of virus stock or by inhibiting de novo viral and/or cellular protein synthesis with cycloheximide (100 micrograms/ml) or anisomycin (100 micrograms/ml) did not eliminate the HCMV-induced increase in RNA levels of c-oncogenes. These data indicate that activation of these early response cellular genes is independent from de novo expression of HCMV IE proteins, and possibly involves biologically active virion proteins that are related to the induction of a cascade of cellular events associated with the binding of HCMV to its cellular receptor. PMID- 1712581 TI - Cleavage of the HIV-1 p66 reverse transcriptase/RNase H by the p9 protease in vitro generates active p15 RNase H. AB - The reverse transcriptase/RNase H of HIV-1 is composed of a p66/p51 heterodimer when analyzed from virus particles. A recombinant reverse transcriptase (RT)/RNase H which after purification consisted mainly of p66 was analyzed as substrate of the purified recombinant HIV-1 protease p9 in vitro. The p66 protein if treated with the protease is processed to a stable p66/p51 heterodimer. A p15 protein is a prominent cleavage product which was identified as the carboxyterminal portion of p66 by means of a monoclonal antibody. It exhibits RNase H activity when tested by activated gel analysis. Presence of SDS during the incubation allowed complete degradation of p66 depending on the conditions, which indicates that conformation of a substrate is relevant for cleavage by the HIV-1 protease. A synthetic heptapeptide AET-FYVD derived from the region between RT and RNase H is cleaved efficiently in vitro by the HIV-1 protease at the F'Y junction, and may mimick a natural cleavage site. P66/p51 heterodimers exhibit higher RT and RNase H activities than p66 when renatured from polyacrylamide gels. PMID- 1712582 TI - Transmission of human T-cell leukemia virus type I from a patient with HTLV-I associated myelopathy/tropical spastic paraparesis and an asymptomatic carrier to rabbits. AB - Rabbits were infected successfully with two strains of human T-cell leukemia virus type I (HTLV-I), one isolated from a Colombian patient with HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the other from an asymptomatic carrier. HTLV-I was repeatedly demonstrated in peripheral blood mononuclear cells (PBMNC) of infected rabbits, and the rabbits had elevated antibodies against the various structural proteins of HTLV-I. Four rabbits inoculated with HTLV-I-infected autologous lymphoid cells intravenously (i.v.) and intracerebrally (i.c.) had virus present in their PBMNC for more than 40 weeks, while those that were inoculated either with HTLV-I-infected human lymphoid cells or with autologous rabbit lymphoid cells intraperitoneally (i.p.) had episodes during which virus was not recovered from their PBMNC. The one rabbit inoculated i.p. developed antibodies to viral envelope glycoproteins earlier than did those inoculated i.v. and i.c. Rabbit lymphoid cell lines persistently infected with HTLV-I were established by cocultivating the rabbit PBMNC with HTLV-I-infected human lymphoid cells that had been irradiated or by inoculation with cell-free supernatant fluids of HTLV-I infected non-irradiated lymphoid cell cultures. HTLV-I-infected rabbit cell lines were of T-cell origin and expressed HTLV-I antigens by immunofluorescence. Electron microscopy revealed type-C retrovirus particles. PMID- 1712583 TI - [Eosinophil cationic protein in patients with bronchial asthma]. AB - To evaluate the role of eosinophils in the pathogenesis of bronchial asthma, we measured eosinophil cationic protein (ECP), one of the eosinophil granule proteins. Serum ECP levels were measured by radioimmunoassay in asthmatic (n = 59) and non-asthmatic (n = 47) patients. Preliminary study showed that ECP levels were time-dependently increased in the blood samples until 3 hr. Based on the findings, we determined to measure serum ECP levels at 30 min after blood sampling. Serum ECP levels and blood eosinophil counts in asthmatic patients were significantly higher than those in non-asthmatic patients (p less than 0.01). There was also a positive correlation between serum ECP levels and blood eosinophil counts in patients with asthma (r = 0.46, p less than 0.001). No significant difference was observed in either serum ECP levels or blood eosinophil counts in asthmatic patients classified by clinical type and severity. Blood eosinophil counts in patients with asthma attacks were significantly greater than in those in remission (p less than 0.05), but no significant difference was observed in serum ECP levels between these groups, suggesting an enhanced elimination of ECP during attack. Serum alpha-2 macroglobulins, which bind to ECP and may function as scavengers for ECP, were not significantly different in these group. These results suggest that serum ECP levels may not be a direct indicator of eosinophil activation or degranulation in the pathogenesis of asthma. PMID- 1712584 TI - Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells. AB - We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line. PMID- 1712585 TI - Antibody to a zinc finger protein in a patient with paraneoplastic cerebellar degeneration. AB - In some patients with paraneoplastic cerebellar degeneration (PCD), autoantibodies against neural components have been identified. Here, we demonstrate a major 58 kd protein antigen in an immunoblot of human cerebellum by serum from a patient with PCD. Immunohistochemically, the serum recognized neural cells especially Purkinje cells in a human brain. To identify the details of the target antigens for the antibody, we isolated a cDNA clone from a human cerebellar library. Homology searches revealed a similarity with the zinc finger proteins. PCD related proteins reported here may be important to maintain neural cells especially those in the cerebellum, and further studies on this molecule may help us elucidate the causes of degenerative or autoimmune diseases in the cerebellum. PMID- 1712586 TI - Structure of mouse myelin-associated glycoprotein gene. AB - The mouse myelin-associated glycoprotein gene was isolated from a mouse gene library. This gene was split into 13 exons distributed about 15 kb in length. Each extracellular immunoglobulin-related domain was encoded by a single exon, and RNA splicing between those exons occurred between the first and second nucleotides of the junctional codon, the features of which are conserved in most of the genes of the immunoglobulin superfamily. The sequence of the 5'-flanking region appeared to have some regions homologous to other myelin proteins, which suggested that they were possible cis-elements for specific expression of oligodendrocytes. PMID- 1712587 TI - Comparison of spantide II and CP-96,345 for blockade of tachykinin-evoked contractions of smooth muscle. AB - CP-96,345, a quinuclidine, is a potent inhibitor of substance P for the NK1 receptor of bovine brain, but has reduced potency for the corresponding receptor of the rat and mouse, and none for NK2 or NK3 receptors. A related quinuclidine showed similar but lower potency than CP-96,345 for NK1. CP-96,345 was more potent than the spantide I of 1984, D-Arg1,Pro2,Lys3,Pro4,Gln5,Gln6,D-Trp7,Phe8,D Trp9, Leu10,Leu11,NH2. Our continued designs for antagonists of substance P led to spantide II in 1990 which is: D-NicLys1,Pro2,3-Pal3,Pro4,D-Cl2Phe5,Asn6,D-Trp7 ,Phe8,D-Trp9,Leu10,Nle11-NH2. The pA2 values of spantide II and CP-96,345 for guinea pig taenia coli were 7.6 and 6.8, respectively. The pIC50 values for blockade of tachykinin-mediated neurotransmission in the rabbit iris sphincter were 6.1 and 5.4, respectively. Spantide II was nearly 10 times more potent than CP-96,345 in these two assays. PMID- 1712588 TI - Demonstration and characterization of the specific binding of growth hormone releasing peptide to rat anterior pituitary and hypothalamic membranes. AB - Since the growth hormone-releasing peptide (GHRP), His-D-Trp-Ala-Trp-D-Phe-Lys NH2, was found to specifically release growth hormone by a complementary but yet not clearly defined action on the pituitary as well as the hypothalamus, in vitro studies have been performed to demonstrate and characterized GHRP binding sites on peripheral membranes of both the rat anterior pituitary and hypothalamus. Optimum binding assay conditions were established using [125I]Tyr-Ala-GHRP as the radioligand. The membrane binding sites were specific, reversible, saturable and time, temperature, pH and concentration dependent. Computerized analyses of competition experiments suggested two classes of binding sites in both pituitary and hypothalamic membranes. The maximum specific binding was observed at pH 5.0 than the physiological pH in both tissues. Pretreatment of the membranes with trypsin prevented specific binding. The increase in Bmax was statistically significant and showed a 2.0- to 8.9-fold and 5.8- to 11.2-fold in pituitary and hypothalamus, respectively, whereas the affinity constants (Kds) were not significant. Of the synthetic and natural neuropeptides that influence the release of GH from somatotrophs, only (D-Lys3)GHRP, substance P antagonists and growth hormone-releasing factor analog were potent inhibitors of GHRP binding in both tissues. PMID- 1712589 TI - The human liver and reticulocyte cytochrome b5 mRNAs are products from a single gene. AB - Using a combination of standard cDNA library screening techniques and the Polymerase Chain Reaction (PCR) we have isolated and sequenced DNA fragments corresponding to the human reticulocyte cytochrome b5 mRNA. The reticulocyte specific sequence codes for amino acids 97 and 98 only, with a TAA stop codon. The reticulocyte specific 3'non-translated sequence has 15 new nucleotides then utilizes the liver mRNA sequence from amino acid 97 onward. This indicates that the reticulocyte specific exon has 24 base pairs (bp). In addition, we have isolated sequences that are derived from a transcribed cytochrome b5 pseudogene. This transcript contains multiple mutations which prevent the synthesis of any functional protein. PMID- 1712590 TI - Complementary DNA cloning and nucleotide sequence of rabbit serum amyloid A protein. AB - A complementary DNA clone encoding serum amyloid A protein has been isolated from an acute rabbit liver cDNA library. Complete nucleotide sequence analysis reveals that the cloned gene contains a 24 bases 5' untranslated region, 369 bases coding region and a 106 bases 3' untranslated region. Primer extension analysis indicates that the full-length 5' untranslated region contains 80 nucleotides. Northern blot analysis of mRNA from normal and acute rabbit livers demonstrates that this gene is expressed constitutively at a low level and undergoes induction of transcription in response to acute inflammation by the administration of turpentine. PMID- 1712591 TI - Murine strain differences in pulmonary bleomycin metabolism. PMID- 1712592 TI - The involvement of TNF, IL-1 and IL-6 in the immune response to protozoan parasites. AB - One early reaction of the host to infection with protozoan parasites is the secretion of an array of potent cytokines including tumor necrosis factor (TNF), interleukin (IL-1) and IL-6. The combined action of these cytokines causes fever, leukocytosis and the production of acute phase proteins such as C-reactive protein (CRP). These early responses contribute significantly to the outcome of infection by influencing the course of infection directly and by regulating the specific immune response to the parasite. PMID- 1712593 TI - Nonspecific defence mechanism: the role of nitric oxide. AB - There is a marked contrast between the extraordinary complexity and specificity of the adaptive immune response and the limited number of effector mechanisms that it can direct. Recently, a great deal of interest has focused on the possible role of nitric oxide (NO) in one of these mechanisms. Here F.Y. Liew and Frank Cox examine the evidence supporting a role for NO in parasitic disease and suggest possible mechanism of NO-mediated parasite damage. PMID- 1712594 TI - The acute phase protein response during parasitic infection. AB - There is a paucity of comprehensive studies of the APP response in parasitic infection. This should be remedied for two reasons. First, as there is a limited number of cytokines with hepatocyte-stimulating activities, and as each one elicits a unique spectrum of protein changes, examination of the APP response during infection could provide insight into the cytokines involved. Second, the presence of IL-1, IL-6 and TNF, the mediators of the APR, in tissues and circulation have important implications for subsequent immune responses. PMID- 1712595 TI - [Loratadine inhibits mediator liberation from a suspension of human pulmonary mast cells]. PMID- 1712596 TI - [The so-called "idiopathic" anaphylaxis: allergic and pseudo-allergic reactions]. AB - The anaphylaxis that is called idiopathic (A.I.) forms less than 1% of the publications that are concerned with anaphylaxis. The clinical picture associates all the symptoms of anaphylaxis, with particular frequency of Quincke's laryngeal oedema. A vital risk is supposed. No abnormal biological factor can be found. There is an associated, variable pathology in 20% of subjects, 58% are atopic. A.I. effects women more--69%. Quincke's hereditary angioneurotic oedema, the carcinoid syndrome, and the capillary hyperpermeability syndrome, paroxysm with monoclonal gammopathy, systemic mastocytosis must be eliminated as well as false anaphylaxis. The authors review the exceptional causes that may not be considered: drug anaphylaxis, to foods, hymenoptera, effort anaphylaxis, to hydatic antigens, to toboggans, to progesterone. Pathogenic hypotheses incriminate sensitization to unknown allergens, functional anomalies of mastocytes, heterogeneity of IgE. Addition of allergic and non-allergic factors is possible. Release of mediators other than histamine is one hypothesis proposed, to account for the inefficiency of anti H1. Prevention requires avoidance of aspirin, non-steroid anti-inflammatory drugs and beta blockers. Basic treatment is always corticosteroids, with anti H1 and sympathomimetic amines where the A.I. is severe. PMID- 1712597 TI - The effect of immunostimulatory drugs on sulfhydryl compounds in plasma, liver and brain after ethanol-induced liver injury in rats. AB - Sulfhydryl compounds in plasma, liver and brain of rats treated with two immunostimulant drugs, isoprinosine and levamisole, after alcoholic liver injury have been investigated. After use of both drugs for 6 days we found partially beneficial effect on the SH-groups in plasma and liver. No changes in nonprotein SH compounds were observed in rat brain after treatment with isoprinosine, levamisole or ethanol. Furthermore, levamisole shortens the time necessary for the return of AlAT activity to normal value. PMID- 1712598 TI - The polymerase chain reaction and related techniques. AB - The polymerase chain reaction, and other methods for rapidly amplifying DNA or RNA are versatile tools that can be used in diverse applications that include HLA typing, analyzing the T-cell repertoire, constructing chimaeric antibodies and quantifying cytokine expression. The sensitivity of the polymerase chain reaction has allowed many cellular events to be investigated in vitro and in vivo. Many currently emerging adaptions of the basic technique are of interest to both the research and the clinical immunologist. PMID- 1712599 TI - Modulation of tumor-induced angiogenesis by proteins of extracellular matrix. AB - The formation of new vessels is a known event in enlarging tumors. Furthermore, the metastatic potential is abrogated or reduced markedly in the absence of neovascularization. Shedding of tumor cells into the circulation is not observed until vascularization has occurred. As a result, the interruption of neovascularization could be a good target for cancer control. This research was an attempt to see if two proteins present in extracellular matrix, collagen and fibronectin (FN), could modify the tumor-induced angiogenesis. The strong angiogenic response induced by S13 tumor cells in the skin of BALB/c mice was blocked by treatment with FN and FN-derived peptides. In contrast, collagen did not modify tumor-induced angiogenesis. PMID- 1712600 TI - Complex formation between Escherichia coli lipopolysaccharide O antigen and capsular K antigen as detected by immunoelectrophoresis. AB - Many Escherichia coli of serotypes commonly found in the normal intestine and in extraintestinal diseases, and having capsular antigens of the low molecular group called group II, will, in simple saline extracts, produce complexes between some or all of the lipopolysaccharide molecules and some of the polysaccharide K molecules. Non-complex-forming and complex-forming strains with the same O and K can be found. The complexes are thermostable but are disrupted by some detergents. O-K complex formation may lead to misinterpretation of immunoprecipitation results; one example is the counter current technique used for K determination of E. coli. In this technique O antigen lipopolysaccharide may, when complexed to K polysaccharide, mimic a K antigen. The possible implications of O-K complex formation during the infection process, especially for antibody formation need to be examined. PMID- 1712601 TI - Computer localization of some Gm markers on the surface of the Fc region of human immunoglobulin G. AB - The surface localization of some Gm markers on the Fc fragment of IgG has been identified from previously published amino acid sequences associated with known Gm markers using the atomic coordinates described by Deisenhofer, INSIGHT software and a Digital VAX 11/785 computer, which together permit a study of the three-dimensional structure of the Fc fragment. The G1m(x)-associated amino acid residue 431, the G3m(s)- and G3m(u)-associated residue 435 and the nG4m (a)- and (b)-associated residue 309 are all localized in the interface between the CH2 and CH3 domains. Furthermore, it is postulated that the G1m(a)-associated residue 356 (Asp, Glu) influences the interface formation through an ion pair interaction to Lys 439. Finally, G3m(b) and G3m(g) are associated with the interface via residues 435 and 436. The data explain why sera from patients with rheumatoid arthritis are useful tools for the detection of some Gm markers and support the view that rheumatoid factors from these patients are internal images of microbial Fc-binding proteins. PMID- 1712602 TI - MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain. AB - Microtubule-associated protein 1B (MAP1B), an abundant developmentally regulated neuronal protein, is a stoichiometric complex of a heavy chain and two light chains (light chain 1 and light chain 3). We find that light chain 1 is encoded within the 3' end of a previously reported MAP1B heavy chain cDNA. Amino acid sequencing, epitope mapping, Northern blotting, and Southern blotting indicate that the light chain and heavy chain are encoded by the same mRNA within the same open reading frame. In addition, amino acid sequencing of a 120 kd microtubule binding and light chain-binding fragment of the heavy chain reveals that light chain 1 binds near the heavy chain N-terminus. Together these data indicate that the heavy chain and light chain 1 are produced by proteolytic processing of a MAP1B polyprotein and form a complex microtubule-binding domain. PMID- 1712603 TI - Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit. AB - Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol. PMID- 1712604 TI - R-cadherin: a novel Ca(2+)-dependent cell-cell adhesion molecule expressed in the retina. AB - cDNAs encoding a novel member of the cadherin cell adhesion receptor family were cloned. This cadherin is expressed in the retina of the chicken and is termed R cadherin. It is similar to other cadherins in its primary structure, but most resembles N-cadherin, showing 74% amino acid identity. Cells expressing R cadherin can adhere to those expressing N-cadherin when mixed, but they form homotypic clusters within their chimeric aggregates. In the development of the neural retina, R-cadherin begins to be expressed around embryonic day 8 in both neuronal and glial cells, and this expression continues up to the hatching stage. The pattern of the expression of R-cadherin was different from that of N cadherin, suggesting distinctive roles in retinal morphogenesis. PMID- 1712605 TI - Tumour angiogenesis and metastasis. PMID- 1712607 TI - Carboplatin dose in combination chemotherapy for testicular cancer. AB - Carboplatin was given in escalating doses in combination with etoposide and bleomycin (CEB) to 36 patients with testicular cancer. The platelet nadirs but not the white cell nadir correlated significantly with the dose of carboplatin administered. The best correlation was seen with area under the curve (AUC) calculated from a knowledge of the glomerular filtration rate (GFR). A further 40 patients were treated with a carboplatin dose calculated to give an AUC of 4.6 or 5.0 mg.min/ml. From the first part of the study it was predicted that 5-10% of the patients would have significant thrombocytopenia with the first course of treatment. The observed incidence was in fact 5%. When dose escalation and reduction were carried out for platelet nadirs falling outside the range 50-100 x 10(9)/l the average cumulative dose after four courses of carboplatin was very similar to four times the starting dose. Furthermore, as many reductions as escalations were carried out. Thus a starting dose for carboplatin calculated to give an AUC of 5.0 mg.min/ml in the CEB combination is one which will produce an acceptable level of thrombocytopenia. The CEB combination was found to produce a cumulative suppression of platelet nadirs. A mean net fall in haemoglobin of 7.5 9.5% was seen with each cycle. PMID- 1712606 TI - Combination chemotherapy with bleomycin, etoposide and cisplatin (BEP) for metastatic testicular teratoma: long-term follow-up. AB - 127 men with previously untreated non-seminomatous germ cell tumours (NSGCT) of the testis were given BEP chemotherapy (bleomycin, etoposide and cisplatin) between 1979-1986. Long-term follow-up (median 65 months) has shown an overall 5 year survival of 87.2% (95% confidence limits 81.1%-93.3%). Outcome was related to both tumour volume and serum marker levels of alpha-fetoprotein (alpha FP) and beta human chorionic gonadotropin (HCG), with 5 year actuarial survivals of 97.8%, 72.2% and 26.7% respectively for small, large and very large volume disease defined by Medical Research Council criteria, and 91.2% and 60.8%, respectively, for men with low (alpha FP less than or equal to 500 kU/l and HCG less than or equal to 1000 iU/l) or high serum marker levels. 79 men (62%) had a complete radiological and serum marker response to chemotherapy alone; residual masses postchemotheraphy were resected in 39 patients (31%), showing undifferentiated tumour in only 6 (15%). 23 of the 127 patients (18%) failed to respond or developed recurrent disease after BEP; only 5 were successfully salvaged. Myelotoxicity of treatment was mild with grade 4 toxicity in 2% of chemotherapy courses and 3 episodes of neutropenic sepsis. Mean glomerular filtration rates fell by 15.6% between courses 1 and 4 of BEP. Bleomycin pneumonitis developed in 13% of cases with 1 fatality. So far 21 men have had children following chemotherapy, but semen analysis 12 months or more (median 36 months) after treatment showed azoospermia in 11 out of 54 (20%) men tested. BEP chemotherapy can be regarded as standard treatment for patients with metastatic NSGCT in low-risk categories, but more intensive therapy is required for advanced presentations. Strategies to develop "risk related" treatment are under investigation. PMID- 1712608 TI - Novel growth regulatory factors and tumour angiogenesis. PMID- 1712609 TI - [Repair of the flexor digitorum profundus and the flexor pollicis longus by the "rope down" technique. Results in a series of 77 cases]. AB - Seventy-seven flexor tendon lesions in zone I have been reinserted by the "rope down" technique using the Jennings barb-wire. They included 20 cases of repair of FPL. The patients were reviewed with an average follow-up of 4 years (minimum 3 months, maximum 10 years). By means of this technique, immediate active mobilisation was possible in 70 of the 77 cases. Mobility of the MCPJ and the PIPJ was maintained in all but one case. At DIPJ level active flexion was recovered at an average of 42.8 degrees with an average extension deficit of 5.5 degrees. This corresponds to a 1.3% (D1) and 2% (D2-D5) handicap as assessed by the International Federation of Hand Surgery. Complications were recorded in 12 of the 77 cases. Two cases required a secondary tenolysis. The factors which influence on the result were analysed. This analysis demonstrates the frequency with which the association of other lesions worsens an outcome which, traditionally, is otherwise good. The average time off work was 6.9 weeks (minimum 0, maximum 6 months). IN CONCLUSION: this simple, rapid technique achieves secure reinsertion allowing immediate active mobilisation. The use of barb-wire demands meticulous surgical technique and close post-operative surveillance. These requirements indicate why this method does not readily lend itself to the management of these lesions in infants. PMID- 1712610 TI - [Role of scapho-trapezo-trapezoidal arthrodesis in the treatment of Kienbock disease. 11 cases]. AB - 11 patients with Decoulx stage III Kienbock's disease with rotatory subluxation of the scaphoid were treated with scapho-trapezo-trapezoid arthrodesis. The associated procedures were lunate implant resection arthroplasty in 6 cases, lunate resection in 3 cases, shortening of the radius in 1 case. The average follow-up was 40 months (12 to 84 months). There was no complication of the arthrodesis. Relief of pain is satisfactory in 7 of the 11 patients. The average grip strength is 66% of that observed on the affected side. The range of movement was decreased especially for radial deviation. There was a positive correlation between, the exact scaphoid reduction, a wrist without preoperative degenerative arthritis and the good clinical results. No differences were observed in the results between the associated lunate procedures. STT fusion seems to be a useful procedure in the treatment of stage III Kienbock's disease with carpal malalignment as it removes compressive stress from the diseased lunate and treats, the accompanying rotatory subluxation of the scaphoid. PMID- 1712611 TI - [Segmental arterial sympathectomies at the level of the fingers]. AB - The authors relate their experience of thirty digital artery sympathectomies in twenty-four fingers. Ten men and two women were operated: six crush injuries to fingers, three Raynaud's disease and two Buerger disease and one scleroderma. All presented digital vascular insufficiency. The pre-operative arteriogram always revealed more extensive thrombosis than suspected by the clinical symptoms. In this way we performed a segmental arterial sympathectomy depending on the site of the thrombosed artery. The follow-up was five years. The immediate postoperative results were excellent. Five years later the results depended on the nature of the initial disease. PMID- 1712612 TI - [Three-dimensional CT study of the carpus under pronation-supination constraints]. AB - By studying 3D imaging of the wrist under pronation-supination strain, we found that the simple comparison of a series of two corresponding cuts may provide a great deal of useful information on how the carpus transmits the longitudinal torque from the forearm to the hand. A special wooden trestle was made to fix the subject in the CT scanner in a permanent effort of pronation or supination. In the first group of scans, this effort was said to be "free" because the hand was simply maintained in a fixed window without any muscular contraction, except pronation or supination muscles. In the second group of scans, this effort was said to be "constrained" because the hand gripped a fixed bar with contraction of the flexor muscles. The thickness of the cuts was 1.2 millimeters and they were separated by 1.5 millimeters. Four levels were specially studied: the lower radio ulnar joint (LRUJ), the proximal row of the carpus, the distal one and the metacarpal bases. Many elementary movements occur in the carpus in constrained supination: the triquetrum "supinates" (7 degrees), the scaphoid flattens and "pronates" (2 degrees) around the capitum the ridges of the carpal anterior concavity approximate (3 mm). In constrained pronation, the anterior concavity of the carpus flattens emphasizing the role of the anterior retinaculum. The LRUJ is very unstable: in free pronation, the ulnar head moves dorsally, firmly pressing the posterior part of the sigmoid notch, responsible for fracture of a postero medial fragment in Colles fracture. The quadratus pronatus is a very important muscle to coapt this joint. We propose the "screwing (or unscrewing) test" in the diagnosis of arthrosis or instability of the LRUJ. We define the notion of "rotational shift" to appreciate the quality of the pronation/supination torque transmission. In constrained pronation/supination, this rotational shift is 5 degrees in the radio-carpal joint. This is very important to appreciate the quality of the wrist prosthesis. In free pronation/supination, the rotational shift is 45 degrees between radius and metacarpal bases. In constrained pronation/supination, it becomes 10 degrees. The wrist ligaments are unable to resist the wrist rotational shift and favor the torque transmission. The tendinous caging of the wrist is the main factor for maintaining rigidity of the carpus and transmitting the torque as muscles are contracted. The wrist can be compared with a fluid drive clutch, whose pedal is muscular contraction. PMID- 1712613 TI - [Presentation of a new homodigital, countercurrent sensitive island flap]. AB - The authors present new sensitive homodigital island flap which can be used to cover large finger pulp defects when classical techniques cannot be applied. An anatomical study consisting of the meticulous dissection of the branches and anastomoses of the digital arteries formed the basis for this new flap. The precise but safe surgical technique is described with the presentation of the first five clinical cases. The authors consider the following advantages of this flap: unrestricted coverage of large pulp defects, the flap is innervated after repair of the transposed digital nerve, tension is avoided allowing immediate mobilization. They also consider the disadvantages related to the necessary section of the digital nerve and artery. However the authors consider that the benefits outweight the disadvantages when compared with conventional techniques. PMID- 1712614 TI - [Luxation-fractures of the radiocarpal joint. Clinical study of 6 cases and general review]. AB - The authors present a series of six cases of radio-carpal fracture-dislocation, which occurred between 1978 and 1989. The results were analysed with a follow-up of between 3 months and 11 years. This rare lesion (0.2% of all joint dislocations) is often caused by a severe injury, although the mechanism remains obscure. Radiological lesions consist of a combination of anterior or posterior carpal dislocation, fractures of the radial styloid and ulnar styloid processes, fracture of the anterior or posterior lip of the radius. This association of radiological lesion was also found in review of literature so that radio-carpal fracture-dislocation can be considered to be a real and a distinct entity. The authors recommend a surgical treatment, after immediate joint reduction, including stabilization with cancellous screw, sometimes Kirschner wires. However, anatomic reduction must be obtained, to ensure a good functional result. PMID- 1712615 TI - [Surgical treatment of congenital brachymetacarpus by dynamic elongation osteosynthesis. Report of a clinical case]. AB - Dynamic elongation osteosynthesis is a new technique studied and applied by us in the treatment of congenital brachymetacarpal deformity. The anatomical, functional and aesthetic results of this operation in a young woman were excellent. We used a microtractor according to Ilizarov's in order to achieve methodology 22 mm of lengthening of the fourth metacarpal bone of the left hand. All soft tissue structures were elongated such as arteries, veins, nerves, intrinsic muscles, flexor and extensor tendons without any adverse effects. We therefore recommend this technique for the treatment of this congenital disease. PMID- 1712616 TI - ["Desensitization" technique in the rehabilitation of the painful hand]. AB - Hand injury patients fairly frequently develop, soon after the injury, hypersensitivity phenomena on contact of the hand with the environment, which may lead to exclusion of the hand. The authors attempt to explain this syndrome on the basis of the pathophysiological theories concerning pain. American authors have proposed an original desensitization treatment based on simple and varied techniques, which provides extremely encouraging results. PMID- 1712617 TI - [Supraepitrochlear process and median nerve compression]. AB - The supra-condylar process exists in a small percentage of the population. Infrequently this process may cause compression of the median nerve. The authors such a case associated with compression of the ulnar nerve. Excision of the bony process led to immediate resolution of the symptoms. In the presence of compressive symptoms with neurologic deficit, it is important to always to look for a supracondylar process by palpation. PMID- 1712618 TI - [Edema in surgery of the hand]. PMID- 1712619 TI - [The hand symbol]. PMID- 1712620 TI - The three-dimensional structure of frozen-hydrated Nudaurelia capensis beta virus, a T = 4 insect virus. AB - The three-dimensional structure of Nudaurelia capensis beta virus (N beta V) was reconstructed to 3.2-nm resolution from images of frozen-hydrated virions. The distinctly icosahedral capsid (approximately 40-nm diameter) contains 240 copies of a single 61-kDa protein subunit arranged with T = 4 lattice symmetry. The outer surface of unstained virions compares remarkably well with that previously observed in negatively stained specimens. Inspection of the density map, volume estimates, and model building experiments indicate that each subunit consists of two distinct domains. The large domain (approximately 40 kDa) has a cylindrical shape, approximately 4-nm diameter by approximately 4-nm high, and associates with two large domains of neighboring subunits to form a Y-shaped trimeric aggregate in the outer capsid surface. Four trimers make up each of the 20 planar faces of the capsid. Small domains (approximately 21 kDa) presumably associate at lower radii (approximately 13-16.5 nm) to form a contiguous, non-spherical shell. A T = 4 model, constructed from 80 trimers of the common beta-barrel core motif (approximately 20 kDa) found in many of the smaller T = 3 and pseudo T = 3 viruses, fits the dimensions and features seen in the N beta V reconstruction, suggesting that the contiguous shell of N beta V may be formed by intersubunit contacts between small domains having that motif. The small (approximately 1800 kDa), ssRNA genome is loosely packed inside the capsid with a low average density. PMID- 1712621 TI - Divergent effects of flavone acetic acid on established versus developing tumour blood flow. AB - Flavone Acetic Acid (FAA) exerts much of its effect by reducing tumour blood flow. Previous studies on FAA-induced changes in blood flow have used established tumours with a functional microvasculature. Using radioactive Xenon(133Xe) clearance to monitor local blood flow we show that the effects of FAA are dependent on the presence of this functional microvasculature with no evidence that FAA inhibits the actual development of tumour microcirculation. Thus, administration of multiple doses of FAA around the time of tumour cell injection failed to diminish t1/2 values of 133Xe (e.g. t1/2 16 min for FAA vs 14 min for saline controls at 10 days) or to affect tumour volumes (5.55 +/- 0.06 cm3 in FAA treated animals vs 5.7 +/- 1.3 cm3 in controls at 25 days). In marked contrast a single dose of FAA (200 mg kg-1 body weight) 2 weeks after tumour cell injection dramatically extended t1/2 times (47 min for FAA vs 7 min for controls; P less than 0.001) and significantly reduced tumour burden. This effect is specific for tumour microvasculature and is not directed simply at new vessels since a similar treatment of animals with implanted-sponge-induced granulation tissue had no effect on t1/2 times (6.8 +/- 1.1 min for FAA at 200 mg kg-1 vs 7.2 +/- 1.0 min for saline-treated controls. PMID- 1712622 TI - Characterisation and properties of a small cell lung cancer cell line and xenograft WX322 with marked sensitivity to alpha-interferon. AB - Controversy exists as to whether interferons usefully influence the growth of epithelial carcinomas. A small cell lung carcinoma (SCLC) cell line, WX322, has been derived which is greater than 1000-fold more sensitive to alpha-interferon (IFN) when grown in agar than other reported SCLC cell lines. The WX322 line has been characterised to prove its epithelial origin and its chemosensitivity compared with that of the NCI-H69 small cell line. The WX322 cell line expresses neuroendocrine and epithelial markers and possesses a morphology consistent with SCLC origin. A concentration of 5 IU ml-1 of IFN produced 50% inhibition of colony formation in agar in the WX322 line, whereas a concentration of greater than 10(5) IU ml-1 was required to produce a comparable effect with the NCI-H69 cell line. In contrast, WX322, possessed similar sensitivity to NCI-H69 cells when exposed to a range of cytotoxic agents. Analysis of the cell cycle indicated that IFN increased the percentage of cells in the G0/G1 phase for the WX322 cell line but increased the percentage in S phase for the NCI-H69 line. Growth of the xenograft, from which the cell line was derived, was also inhibited by IFN at doses greater than 10(5) IU/mouse/day. The WX322 cell line whether grown in agar or as a xenograft shows an unusually high sensitivity to IFN and provides an interesting model for studying mechanisms of IFN cytotoxicity to epithelial cells. PMID- 1712623 TI - Intraperitoneal cytostatics impair healing of experimental intestinal anastomoses. AB - We investigated the effect of two doses of cytostatics, administered intraperitoneally during 5 consecutive days, on the healing of ileal and colonic anastomoses constructed on the third day. The cytostatics regimen consisted of a combination of 5-fluorouracil, bleomycin and cisplatin at 10, 2 and 0.35 mg kg-1d 1, respectively, or at twice higher doses. The lower dose was similar to that given intravenously in previous experiments. Rats were sacrificed 3 or 7 days after operation. No effects of cytostatics were observed after 3 days, neither on anastomotic bursting pressure nor on hydroxyproline concentration (microgram/mg dry weight) or content (microgram cm-1). Profound effects were seen at 7 days. In the high dose group, bursting pressures in both anastomoses were greatly reduced with respect to the control group. Concurrently, collagen synthesis was severely impaired, as indicated by sustained decreased hydroxyproline concentrations and content. The lower dose of cytostatics showed essentially similar effects on hydroxyproline parameters, but affected anastomotic strength less dramatically. The data indicate that, while intraperitoneal chemotherapy may show less detrimental systemic toxicity and thus allow higher doses, its application as an adjunct to gastrointestinal surgery may be limited because of its severe effects on anastomotic repair. PMID- 1712625 TI - A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes. AB - Keratins K1 and K10 represent the major differentiation products of the maturing epidermal keratinocytes. Primary epidermal cell cultures from newborn K1 transgenic mice containing a 12-kilobase human K1 genomic fragment were established in order to examine the expression of both human and mouse K1 in the presence of known modulators of epidermal differentiation. Elevated levels of Ca2+ in the culture medium induced both mouse K1 and human K1. Supplementing the medium with retinoic acid or 12-O-tetradecanoylphorbol-13-acetate or introducing a Harvey viral ras oncogene (v-rasHa) into the cells completely suppressed mouse K1 but not human K1. Our results suggest that: (a) the human 12-kilobase insert contains all the necessary cis-acting elements to respond to the Ca2+ signal, and (b) other cis-acting elements, not present within this insert, may function independently to regulate the response of K1 to retinoids, 12-O tetradecanoylphorbol-13-acetate, and v-rasHa transformation. This transgenic model provides an approach to identify elements required for the regulation of an epidermal differentiation-specific gene. PMID- 1712624 TI - Expression of mutant p53, c-erbB-2 and the epidermal growth factor receptor in transitional cell carcinoma of the human urinary bladder. AB - Expression of the p53, the epidermal growth factor receptor (EGFr; c-erbB-1) and c-erbB-2 proteins was studied in 82 patients with primary transitional cell carcinoma of the bladder using an immuno-histochemical method. Strong or moderate staining was found in 18% of tumours for p53 with weaker staining in a further 36% giving a total of 54% of tumours stained for p53. Strong staining was found in 15% of tumours for c-erbB-2 and in 31% for the EGFr. Tumours invading the bladder muscle were significantly more likely to be strongly stained positively for p53 and/or EGFr compared with superficial tumours: only 15% of invasive tumours were stained negatively for both p53 and EGFr. No statistical association was found between p53 and EGFr expression. Weakly positive associations were found between the expression of c-erbB-2 and p53 and between muscle invasive tumours and increased expression of c-erbB-2. Alterations in the expression of p53, c-erbB-1 and c-erbB-2 were found frequently in human transitional cell carcinoma of the urinary bladder and may be of clinical use in defining patient sub-groups of differing prognosis. PMID- 1712626 TI - Failure of salvage treatment in metastatic testicular teratoma. AB - From January 1978 to March 1989, 92 consecutive patients with metastatic testicular teratoma have been treated with cisplatin-based chemotherapy. Thirty seven failed to achieve a complete response, and another four subsequently relapsed. These 41 have required further treatment, consisting of surgery (16 patients), radiotherapy (n = 13) and chemotherapy (n = 12). Surgery was generally used for residual masses where tumour markers were normal, radiotherapy for masses where surgery was not possible or for palliation, and second line chemotherapy was used in patients with raised serum tumour markers or in the presence of multiple inoperable pulmonary metastases. Nine of 16 (56%) patients treated surgically are disease-free, including two who had malignant teratoma in the resection specimen. Three of 13 patients irradiated are disease-free, although two of these three had subsequent excision of residual masses. All 12 patients treated with second-line chemotherapy have died. Surgical excision of residual masses appears to be the most effective way of rendering patients disease-free, providing serum tumour markers are normal. Most of these residual masses will consist of differentiated teratoma or necrosis, but it may be possible to salvage patients with residual malignant disease, providing complete clearance can be achieved. Incompletely resected malignant disease carries a poor prognosis, and incompletely resected disease that is histologically benign will run the risk of subsequent relapse. Radiotherapy provides good palliation but is much less effective than surgery as treatment for residual masses, and should only be used if complete excision cannot be accomplished.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712627 TI - Haematological toxicity compromises MOPP/ABVD chemotherapy in Hodgkin's disease. AB - A total of 23 patients with previously untreated Hodgkin's disease received MOPP/ABVD hybrid chemotherapy and response to treatment and toxicity were assessed. Of these 14 (61% (95% confidence limits 38.5%-80%] achieved complete remission with chemotherapy alone, six (26% (10.2%-48.4%)) achieved partial remission and there were three treatment failures (13%). Toxicity was mainly haematological resulting in treatment delays and dose reductions. Those in partial remission after chemotherapy achieved complete remission with additional radiotherapy. So far five of the 20 who remitted (25%) have relapsed. We conclude that the haematological toxicity from this regimen compromises dose intensity. The results from using this hybrid regimen are not superior to those using MOPP or ABVD alone in our experience. PMID- 1712628 TI - Neurotransmitter organization of the nucleus of Edinger-Westphal and its projection to the avian ciliary ganglion. AB - Two morphologically distinct types of preganglionic endings are observed in the avian ciliary ganglion: boutonal and cap-like. Boutonal endings synapse on ciliary ganglion neurons (called choroidal neurons) innervating choroidal blood vessels, while cap-like endings synapse on ciliary ganglion neurons (called ciliary neurons) controlling the lens and pupil. Some of both types of preganglionic endings contain the neuropeptides substance P (SP) and/or leucine enkephalin (LENK). Although both types of preganglionic terminals are also known to be cholinergic, there has been no direct evidence that SP and LENK are found in cholinergic endings in the ciliary ganglion. The present studies in pigeons, which involved the use of single- and double-label immunohistochemical techniques, were undertaken to examine this issue, as well as to (1) determine the relative percentages of the boutonal and cap-like endings that contain SP, LENK, or both SP and LENK; and (2) determine if the two different types of terminals in the ciliary ganglion arise from different subdivisions of the nucleus of Edinger-Westphal (EW). Single- and double-label immunohistochemical studies revealed that all neurons of EW, regardless of whether they contained immunohistochemically detectible amounts of SP or LENK, are cholinergic. In the medial subdivision of EW (EWM), which was found to contain approximately 700 neurons, 20.2% of these neurons were observed to contain both SP and LENK, while 11.6% were observed to contain SP only and 10.7% were observed to contain LENK only. In contrast, in lateral EW (EWL), which was found to contain approximately 500 neurons, 16.2% of the neurons were observed to contain both SP and LENK, while 19.2% of the neurons were observed to contain SP only and 12.6% were observed to contain LENK only. Retrograde-labeling studies involving horseradish peroxidase injections into the ciliary ganglion revealed that EW was the sole source of input to the ciliary ganglion and all, or nearly all, neurons in EW innervate the ciliary ganglion. Immunohistochemical labeling of the ciliary ganglion neurons with an antiserum against choline acetyltransferase revealed that approximately 900 choroidal neurons and approximately 600 ciliary neurons are present in the ganglion, all of which receive cholinergic preganglionic endings. Of the choroidal neurons, 94% receive butonal terminals containing both SP and LENK, while only 2% receive SP+ only boutonal endings and 2% receive LENK+ only butonal endings. Of the ciliary neurons, 25% receive cap-like endings containing both SP and LENK, 30% receive cap-like endings containing only SP and 3% receive cap-like endings containing only LENK.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712629 TI - SCE-measurements as tool for evaluation of drug-induced chromosomal disorders. AB - A staining procedure that detects sister chromatid exchanges has been used to examine the response of chromosomes in cultured human fibroblasts to a series of chemical and physical mutagens. The test represents a sensitive and reproducible method for detecting mutagenicity on the chromosome level. It provides a powerful method for the detection of environmental mutagens and can be used in ocular toxicity studies. PMID- 1712630 TI - Effects of silicone oils on corneal endothelial permeability. AB - Silicone oils, varying by viscosity and manufacturer, were infused into rabbit anterior chambers. Polydimethyl-siloxane oil, 5000 cps, increased corneal endothelial permeability to inulin (mw 5000) and dextran (mw 60000) when measured in vitro at 1, 4 and 7 days after ocular infusion. The effects of five other oils were measured at 7 days after infusion. Four of the oils increased endothelial permeability and induced similar morphological changes. Dow Corning Medical Fluid 360 had no effect on either permeability or morphology of the endothelium. These results show that contact of most silicone oils with corneal endothelium rapidly induces physiological and morphological changes. If these oils, when used as a retinal tamponade, gain access to the cornea they should be removed quickly to avoid the rapid initiation of physiologic changes. PMID- 1712631 TI - Removal of salt from a salt-induced protein crystal without cross-linking. Preliminary examination of "desalted" crystals of phosphoglucomutase by X-ray crystallography at low temperature. AB - A model procedure for removing salt from relatively fragile salt-induced protein crystals is proposed. The procedure is based on physical principles and is validated by using millimeter-size crystals of rabbit muscle phosphoglucomutase grown from a 2.1 M solution of ammonium sulfate. Three types of operations are included in the procedure: initial transfer to salt solutions of reduced concentration; transfer to the organic-rich phase of an equilibrium biphasic mixture obtained with aqueous solutions of polyoxyethylene and the salt; and addition of various replacement cosolutes in aqueous solutions of polyoxyethylene to reduce osmotic stress on the crystal as the remaining salt is removed. A critical feature of the overall procedure is maintenance of near equilibrium throughout by using a large number of steps involving small changes in solute concentration. The conditions used in the actual transfer were adjusted to eliminate the fracturing of crystals by visually distinguishing between two opposing types of fracture patterns: those produced by osmotic crushing as opposed to osmotic expansion. Basic requirements for a successful procedure with other protein crystals are a high permeability toward small solutes and a relatively slow dissolution rate at salt concentrations for which biphasic mixtures can be obtained. Desalted crystals of phosphoglucomutase have no visible fractures, are stable in the final solution for at least a week, and exhibit no noticeable change in the resolution of their X-ray diffraction pattern. In fact, desalted crystals can be rapidly cooled to 160 K, whereas untreated crystals are almost completely disordered by the same cooling procedure. The component of the desalting mixture whose presence is crucial to the success of the cooling process is polyoxyethylene, which apparently impedes the formation of ice within the protein crystal. Diffraction data obtained with an area-detector diffractometer did not differ significantly, either in terms of quality or resolution range, between crystals in 2.3 M ammonium sulfate at room temperature and crystals at 160 K in which ammonium sulfate had been replaced by glycine. The successful use of the following replacement solutes, instead of glycine, also is documented: sucrose, glycerol, and a low molecular weight poly(ethylene glycol) (PEG-400). PMID- 1712632 TI - Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase. AB - Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity. PMID- 1712633 TI - Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion. AB - Several human melanoma cell lines produced tissue-type plasminogen activator (t PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion. PMID- 1712634 TI - Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes. AB - In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters. PMID- 1712635 TI - Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells. AB - Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism. PMID- 1712636 TI - The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated. AB - The functional activity of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes can be regulated by T-cell receptor (TCR) stimulation and pharmacologic agents. It was of interest to determine if functionally active LFA 1 could be reconstituted on a nonhematopoietic, LFA-1-negative cell line. We report the expression of LFA-1 and diethylaminoethyl (DEAE) Mac-1 alpha beta heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the alpha and beta subunit cDNAs. Immunoprecipitation studies demonstrated that the alpha and beta subunit was expressed in heterodimers. The alpha or beta subunit was expressed at lower levels after transfection with the alpha or beta subunit cDNA alone. Cotransfection of the alpha and beta subunit cDNAs, but not transfection of alpha or beta alone, was sufficient to reconstitute intercellular adhesion molecule-1 (ICAM-1) binding activity. Consistent with this observation, LFA-1 on the fibroblastoid cells possesses the activation epitope defined by the L16 monoclonal antibody (mAb). This epitope marks the conversion of LFA-1 from the low to high avidity state on peripheral blood T lymphocytes (PBLs) and is constitutively present on activated cell lines. In contrast to LFA-1 on leukocytes, the functional activity of LFA-1 on fibroblastoid cells was not influenced by phorbol ester treatment. Furthermore, the use of agents that interfere with intracellular signaling, a protein kinase C inhibitor, cAMP analogue, or the combination of a phosphodiesterase inhibitor and adenyl cyclase activator, did not affect the binding of COS cells expressing LFA-1 to purified ICAM-1. PMID- 1712637 TI - Evaluation of an automated commercial immunoluminometric assay for alpha foetoprotein. AB - We evaluated a two-site immunoluminometric assay (AFP LIA-mat Byk Sangtec) for the determination of alpha-foetoprotein (AFP). The assay, involving two monoclonal antibodies which recognize two different alpha-foetoprotein epitopes, is rapid (4 h) with a wide working range (0-600 x 10(3) IU/l), a good lower limit of detection and good reproducibility (CV less than 10%). The regression equation for the AFP LIA-mat (y) and the immunoradiometric assay AFP Bridge Serono (x) was y = 1.175x - 2.27 (n = 95, r = 0.996) for serum and y = 1.16x + 479.2 for amniotic fluid. It did not display a hook effect, and when we assessed the linearity by assaying a high concentration of alpha-foetoprotein, it gave a linear response down to 3.1 x 10(3) IU/l. We also evaluated the clinical response of AFP LIA-mat in 278 patients with different diseases. Eighteen of the 19 patients with hepatocellular carcinoma had alpha-foetoprotein levels greater than 100 x 10(3) IU/l. In contrast, only 5 of the 47 patients with cirrhosis showed values above 50 x 10(3) IU/l, demonstrating that this assay discriminates fairly well between hepatocellular carcinoma and cirrhosis. Data in agreement with the literature were obtained for testicular tumours; all of seven seminomatous tumours presented values below 5 x 10(3) IU/l, whereas 50% of the non seminomatous tumours (n = 8) presented values above 20 x 10(3) IU/l. PMID- 1712638 TI - Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. AB - Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL 1 beta), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM-CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells. PMID- 1712639 TI - Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment. AB - Erythrocytes promoted platelet reactivity in a plasma medium, as demonstrated in an in vitro system that independently evaluated the biochemistry of platelet activation and recruitment. The prothrombotic erythrocyte effects were metabolically regulated, as evidenced by lack of activity of ATP-depleted or glutaraldehyde-fixed erythrocytes. They occurred in the absence of cell lysis as verified by lactate dehydrogenase assays, and had an absolute requirement for platelet activation. The presence of erythrocytes induced a twofold increase in platelet thromboxane B2 (TXB2) synthesis upon collagen stimulation, indicating that erythrocytes modulated platelet eicosanoid formation. Cell-free releasates from stimulated platelet-erythrocyte suspensions, which exhibited increased recruiting capacity, contained 6.9-fold more ADP and 4.9-fold more ATP than releasates from stimulated platelets alone. Following aspirin ingestion, TXB2 formation was blocked, but erythrocyte promotion of platelet reactivity persisted at those doses of collagen that reinduced platelet activation. Moreover, when platelet mixtures consisted of as little as 10% obtained before aspirin plus 90% obtained post-aspirin ingestion, significant erythrocyte enhancement of platelet reactivity occurred, even at low agonist concentrations. These erythrocyte effects would decrease the therapeutic potential of inhibition of platelet cyclooxygenase by aspirin. The erythrocyte-induced modulation of platelet biochemistry and function emphasizes the importance of cell-cell interactions in stimulus-response coupling. PMID- 1712640 TI - Effects of interleukin-4 and interleukin-6 on the proliferation of CD34+ and CD34 blasts from acute myelogenous leukemia. AB - We studied the effects of interleukin-4 (IL-4) and IL-6 on the growth of leukemic blasts from 40 patients with acute myelogenous leukemia (AML). Patients were selected on the basis of negativity for a series of B-cell antigens including CD10 and CD19. Twenty-one cases were CD34-positive (CD34+) (greater than 15% of blasts) and the remaining 19 were CD34-negative (CD34-) (less than 3% of blasts). IL-4 alone (100 U/ml) could stimulate either DNA synthesis (with greater than 2.0 stimulation index) or leukemic blast colony formation in 24 of 40 AML patients. In the presence of other growth factors, IL-4 showed divergent effects on IL-3-, granulocyte-macrophage colony-stimulating factor-, granulocyte colony-stimulating factor-, or erythropoietin-dependent colony formation. These effects of IL-4 were observed in both CD34+ and CD34- AML cases. IL-6 (100 U/mL) alone could not stimulate DNA synthesis and blast colony formation except for one CD34+ case. On the other hand, IL-6 showed synergistic effects on IL-3- and IL-4-dependent blast colony formation in 10 of 12 and 7 of 9 CD34+ AML cases, respectively. Among CD34 AML cases, such synergism was seen only in 1 of 12 cases for IL-3-dependent colony formation and in 3 of 7 cases for IL-4-dependent colony formation. The divergent effect of IL-4 and the synergistic effect of IL-6 were also observed in purified CD34+ leukemic blast populations, indicating that these phenomena are not mediated by accessory cells. The present study suggests that IL-4, alone or in combination with other growth factors, has divergent effects on the growth of AML progenitors irrespective of the CD34 expression, and that IL-6 acts synergistically with IL-3 or IL-4 on the growth of leukemic progenitors preferentially in CD34+ AML. PMID- 1712641 TI - Effects of hydroxyurea on hemoglobin F and water content in the red blood cells of dogs and of patients with sickle cell anemia. AB - A rationale for clinical trials of hydroxyurea (HU) treatment in sickle cell disease is that the agent increases red blood cell (RBC) fetal hemoglobin content. However, an additional effect of HU is to raise the mean corpuscular volume (MCV). To investigate the action of HU in a species that makes no electrophoretically distinguishable fetal hemoglobin, we treated dogs with the drug and compared their response to that of five patients with sickle cell anemia. Both dogs and patients had an increase in MCV, but the effect of HU treatment on the mean corpuscular hemoglobin concentration (MCHC), density, and water content of the RBCs differed in the two species. The dog RBCs became low in MCHC, high in ion and water content, and low in mean density. Thus, HU can raise MCV and lower MCHC without influencing fetal hemoglobin synthesis. A different pattern was seen in the sickle cell patients during HU treatment. Although the MCV of their RBCs increased, there was no change in MCHC, ion content, or mean density. A notable change in the sickle cell patients' blood was that two subpopulations of cells were nearly eliminated during HU treatment; the hypodense reticulocyte fraction and the hyperdense fraction that contains irreversibly sickled cells. These findings lead us to suggest that trials of HU in sickle cell disease must recognize the possibility that any beneficial effect of this agent might be due not only to an increase in hemoglobin F alone, but perhaps also to the associated increase in MCV or the altered RBC density profile. PMID- 1712642 TI - Rapid increase in red blood cell density driven by K:Cl cotransport in a subset of sickle cell anemia reticulocytes and discocytes. AB - We have previously demonstrated that young normal (AA) and sickle cell anemia (SS) red blood cells are capable of a volume regulatory decrease response (VRD) driven by a K:Cl cotransporter that is activated by low pH or hypotonic conditions. We now report on the characteristics of young SS cells (SS2, discocytes) capable of rapid increase in density in response to swelling. We have isolated cells with high VRD response (H-VRD) and low VRD response (L-VRD) cells by incubation and density-gradient centrifugation under hypotonic conditions. Comparison of these cells in patients homozygous for hemoglobin (Hb)S indicated that H-VRD cells have 91% more reticulocytes (P less than 9 x 10(-9) than L-VRD cells, 25% less HbF (P less than 5.5 x 10(-5), 106% more NEM (N-methylmaleimide) stimulated K:Cl cotransport activity (P less than 2 x 10(-4), and 86% more volume stimulated K:Cl cotransport activity (P less than 1.8 x 10(-3). H-VRD and L-VRD cells have similar G-6-PD and Na+/H+ antiport activity. In agreement with the reduced percent HbF in H-VRD cells, F cells (red blood cells that contain fetal Hb) are depleted from the H-VRD population; however, F reticulocytes are enriched in the H-VRD population to the same extent as non-F reticulocytes, which suggests that both F and non-F reticulocytes have a similar initial distribution of volume sensitive K:Cl cotransport activity but that it may be more rapidly inactivated in F than in S reticulocytes. We find that H-VRD cells consist of 20% reticulocytes (or 79% of all reticulocytes in SS2) and 80% more mature cells. This study demonstrates the role of K:Cl cotransport in determining red blood cell density, the heterogeneity of K:Cl cotransport activity in reticulocytes, and the capacity for rapid change in the density of reticulocytes with high K:Cl cotransport activity. We speculate that the H-VRD population may be more susceptible to generation of dense and irreversibly sickled cells. PMID- 1712643 TI - Asymmetrically primed selective amplification/temperature shift fluorescence polymerase chain reaction to detect the hemoglobin Constant Spring mutation. AB - Hemoglobin (Hb) Constant Spring is an alpha-thalassemic hemoglobinopathy that is a major cause of severe alpha-thalassemia in Southeast Asians. The difficulty of diagnosing Hb Constant Spring using standard electrophoretic methods has led to interest in DNA-dependent diagnostic methods. The methods developed have had to contend with the high degree of homology of the alpha 2-globin gene (the site of the Hb Constant Spring mutation) and the alpha 1-globin gene. We have developed a single reaction polymerase chain reaction-based method that uses asymmetric priming and a temperature shift to accomplish dual ends, selective amplification of alpha 2 but not alpha 1 DNA and discrimination of normal and Hb Constant Spring alpha 2 genes by allele-specific fluorescence polymerase chain reaction. Advantages of this method over previous approaches include avoiding radioisotopes, precluding the need for electrophoresis, and serving as its own control for successful amplification. It is readily applicable to routine diagnosis, population screening, and prenatal diagnosis. PMID- 1712644 TI - Expression of the YB5.B8 antigen (c-kit proto-oncogene product) in normal human bone marrow. AB - The c-kit proto-oncogene product is a member of the family of growth factor receptors with intrinsic tyrosine kinase activity. In the mouse c-kit maps to the W locus, which is known to be of central importance in hematopoiesis. Monoclonal antibody (MoAb) YB5.B8, which was raised against peripheral blood blast cells from a patient with acute myeloid leukemia (AML), was recently shown to bind to the extracellular domain of the c-kit product. This antibody does not bind detectably to normal peripheral blood cells and identifies a sub-group of AML patients with poor prognosis. We have used MoAb YB5.B8 to study the expression of c-kit by normal human bone marrow cells by immunofluorescence and flow cytometry, and to isolate multipotential and erythroid colony-forming cells. In a series of 11 normal adult bone marrow specimens, MoAb YB5.B8 bound to 4.0% +/- 1.8% of the cells in the low-density fraction. Dual-labeling experiments were performed with YB5.B8, and CD33, CD34, and CD10 MoAbs. Three populations of cells binding YB5.B8 could be identified based on their pattern of coexpression of the other markers; ie, YB5.B8+/CD34+/CD33-, YB5.B8+/CD34+/CD33+ and YB5.B8+/CD34+/CD33+. These populations had distinctive two-dimensional light scatter characteristics and are likely to correspond to precursor colony-forming cells, colony-forming cells, and maturing mast cells, respectively. No cells binding both YB5.B8 and an MoAb to the early lymphoid marker CD10 were found, implying that most early lymphoid cells do not express c-kit. MoAbs to the c-kit protein should prove valuable in multimarker studies of human hematopoietic stem and progenitor cells. Definition of a reference range of c-kit expression in normal human bone marrow will provide a sound basis for further studies of this marker in diagnosis and prognosis in AML. PMID- 1712645 TI - CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules. AB - Preliminary results from the IVth Leucocyte Culture Conference have classified the monoclonal antibody (MoAb), YTH 71.3.2, as CD66. Two other MoAbs, YPC 2/12.1 and CE6/2D3.1, share a common cellular specificity, reacting with cells of the neutrophil series and colonic epithelium. The YTH 71.3.2 and CE6/2D3.1 MoAbs both recognize a similar CD66 defined epitope that is distinct from that identified by YPC 2/12.1. By Western blotting, these antibodies react with different molecular species from cells of different lineages. The antibodies identify 50- to 55-Kd, 80- to 100-Kd, and 130- to 200-Kd components present in a semi-purified carcinoembryonic antigen (CEA) preparation from colonic adenocarcinomas and a 90- to 130-Kd molecule from HL-60 cells. With the colonic cell line, LS174T, YPC2/12.1 stains diffuse bands of 160 to 200 Kd and 90 to 130 Kd with equal intensity, whereas the binding of CE6/2D3.1 and YTH 71.3.2 is biased toward the lower molecular weight set of molecules. Remarkably, all three antibodies recognize CEA-related molecules. Defined analyses using HeLa cells transfected with CEA, NCA(NCA-50/90), and CGM6(NCA-95) cDNAs show that the three MoAbs identify CEA to varying degrees. While YTH 71.3.2 and CE6/2D3.1 also bind to NCA 50/90, YPC 2/12.1 recognizes an epitope expressed by both the NCA-50/90 and NCA 95 molecular species. PMID- 1712646 TI - Hepatitis C infection in children with hemophilia A and B. AB - Antibodies to hepatitis C virus (anti-HCV) were quantitated in stored sera from selected groups of hemophilic children (less than or equal to 18 years of age). During the period 1987 to 1989, seropositivity rates were as follows: untransfused hemophiliacs 0% (0 of 11 cases), hemophiliacs treated exclusively with vapor-heated factor VIII or IX concentrates 0% (0 of 9 cases), hemophiliacs treated only with cryoprecipitate or single donor blood products 0% (0 of 9 cases), and hemophiliacs regularly treated with unheated or dry heat-treated factor VIII or IX concentrates 95% (21 of 22 cases). Corresponding alanine aminotransferase (ALT) results were similar: values were always below the upper limit of laboratory normal (40 U/L) in untransfused hemophiliacs, hemophiliacs treated with vapor-heated factor concentrates, or those who received only cryoprecipitate or single donor blood products. By contrast ALT values were greater than 40 U/L in 82% (18 of 22 cases) of hemophilic children regularly infused with unheated or dry heat-treated factor concentrates. Three conclusions are drawn from this data: (1) HCV is a major cause of chronic hepatitis in multitransfused hemophilic children, (2) unheated and dry heat-treated clotting factor concentrates carry a very high risk of transmitting HCV infection, and (3) clotting factor concentrates inactivated by vapor heating carry a very low and perhaps zero risk of transmitting HCV infection. These findings are of therapeutic significance for previously untransfused hemophiliacs susceptible to HCV infection. PMID- 1712647 TI - Selective expression of two homeobox genes in CD34-positive cells from human bone marrow. AB - Proteins coded by homeobox-containing genes are sequence-specific DNA-binding proteins that have been implicated in the control of gene expression both in developing as well as in adult tissues. Two recently characterized human homeobox genes, HB24 and HB9, were found to be highly expressed in bone marrow cells enriched for CD34-positive cells, present at low levels in unfractionated bone marrow cells, and essentially undetectable in bone marrow cells depleted of CD34 cells. Treatment of CD34-enriched cells with recombinant interleukin-3 (IL-3) and granulocyte macrophage-colony-stimulating factor for 24 hours increased expression of HB24 threefold and HB9 fourfold. Based on studies with actinomycin D, the HB24 and HB9 transcripts in human CD34-positive cells have short half lives, estimated to be 30 to 45 minutes. Downregulation of HB24 and HB9 expression was found following the treatment of in vitro cultures of CD34 positive cells with IL-3. Thus, the differentiation of CD34-positive cells along a specific cell lineage likely requires downregulation of both HB24 and HB9. PMID- 1712648 TI - The stable prostacyclin analog, iloprost, and prostaglandin E1 inhibit monocyte procoagulant activity in vitro. AB - Exposure of human peripheral blood to 100 ng/mL of bacterial endotoxin for 2 hours resulted in a 20-fold increase in monocyte procoagulant activity. The activity was functionally identified as tissue factor, because it was not expressed in plasma deficient in factor VII and was specifically inhibited by a monoclonal antibody directed against human tissue factor. When the stable prostacyclin analog, iloprost, was added to blood 30 minutes before endotoxin, a dose-dependent inhibition of monocyte procoagulant activity occurred, with an I50 of 20 nmol/L. Prostaglandin E1 (PGE1) produced similar effects, with an I50 of 150 nmol/L. Exposure of THP-1 monocytic cells to 100 ng/mL endotoxin resulted in a threefold increase in procoagulant activity after 2 hours and a 20-fold increase after 6 hours. A 30-minute pretreatment with iloprost or PGE1 again inhibited development of procoagulant activity, with I50 values of 5 nmol/L and 150 nmol/L, respectively. Treatment of THP-1 cells with iloprost 2 hours after exposure to endotoxin significantly inhibited further increases in procoagulant activity. Iloprost was less potent under these conditions, 30% inhibition being obtained at 100 nmol/L and 70% at 1 mumol/L. These results suggest that prostacyclin may be a physiologic modulator of monocyte tissue factor expression; in addition, its stable analog, iloprost, may have clinical potential for the treatment of thrombotic disorders in which elevated monocyte procoagulant activity plays a role. PMID- 1712649 TI - The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance. AB - Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct "linear" areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA. PMID- 1712650 TI - Colocalization of immunoreactive oxytocin, vasopressin and interleukin-1 in human thymic epithelial neuroendocrine cells. AB - Monoclonal antibodies to oxytocin (OT) and vasopressin (VP) revealed some positively staining stromal cells in the subcapsular cortex and in the medulla of the human thymus. We further demonstrated that these cells are a subset of epithelial endocrine cells and also contain immunoreactive interleukin-1 together with the neuropeptides. In addition, the thymic cells stained by monoclonal antibodies directed to the cyclic part of oxytocin or vasopressin also contained some immunoreactive neurophysins. These data support the concept of intrathymic synthesis of neurohypophyseal-like peptides fitting the hypothalamic model. However, we observed that, contrary to the situation in the brain, OT- and VP like peptides colocalized in the same thymic cells. Furthermore, one monoclonal antibody, specific for the tail part of oxytocin, did not label thymic cells. Therefore, thymic neuropeptide(s) could be related to, but distinct from, authentic OT and VP. These observations suggest some molecular differences between hypothalamic and thymic oxytocin biosynthetic pathways which need to be further investigated. PMID- 1712651 TI - The neuro-B cell link of peptidergic innervation in the Bursa Fabricii. AB - The Bursa Fabricii, restricted to birds, specifically provides the microenvironment for B-cell maturation. The presence of nerve fibers containing immunopotent neuropeptides in immune organs opens interesting perspectives on the understanding of neuroimmune communication. As an organ for the development of only B-lymphocytes is not known in mammals, the contribution of a peptidergic innervation to the microenvironment of B-cells is not known. Therefore, we studied the peptidergic innervation of the Bursa Fabricii as an organ of B-cell maturation. Four different neuropeptides were found in nerve fibers of the Bursa Fabricii: tachykinins (TK), vasoactive intestinal peptide (VIP), galanin (GAL), and calcitonin gene-related peptide (CGRP). All these neuropeptides were distributed throughout the different bursal compartments, with the exception of the medulla of the bursal follicles, where the early stages of lymphocyte maturation are found. Double immunofluorescent studies revealed three different fiber populations: One containing coexisting VIP and GAL, the second TK and CGRP, and the last TK and no CGRP. Some of the TK-ir fibers may contain coexisting VIP. As demonstrated by double labeling for neuropeptides and lymphocytes, these heterologous fiber populations were found adjacent to B-cells in the follicle cortex. In addition, VIP-ir fibers were seen in association with macrophages. The origins of the fiber populations and the possible functional implications of our findings are discussed. PMID- 1712652 TI - Substance P innervation of spleen in rats: nerve fibers associate with lymphocytes and macrophages in specific compartments of the spleen. AB - We investigated the distribution of SP+ nerve fibers in the spleen of adult male Fischer 344 rats. SP+ nerve fibers entered the spleen with the splenic artery in the hilar region, arborized along the venous sinuses, and extended from these larger plexuses into trabeculae and the surrounding red pulp. In the white pulp, SP+ nerve fibers were found in the marginal zone, and in the outer regions of the PALS among T lymphocytes. No SP+ nerve fibers were observed in association with the splenic capsule, the central arteries of the white pulp, or the follicles. SP levels in rat spleen were 5.7 +/- 0.4 ng/g wet wt. On the basis of the present findings of SP presence in nerve fibers in the spleen, and published evidence for SP receptors on lymphocytes and macrophages, we suggest that SP derived from nerve fibers in the spleen can act as a neurotransmitter with cells of the immune system as targets. These SP nerve fibers may be an important neural link between the nervous system and the immune system and may participate in modulation of immune reactivity and inflammatory responses. PMID- 1712653 TI - Tachykinin receptor cross-talk. Immunological cross-reactivity between the external domains of the substance K and substance P receptors. AB - In the present study, we have chemically synthesized peptides which correspond to the four putative extracellular domains of the predicted substance K (SK) receptor protein and raised specific polyclonal antibodies against these peptides. These antibodies were then tested for both structural and functional recognition of epitopes on the substance P (SP) receptor on rat AR42J pancreatic cells and human IM9 lymphoblasts, which express the SP receptor, but not the SK receptor. Antibodies directed against the first, second, and fourth external domains of the predicted SK receptor recognized a 58-kDa protein on AR42J cells. This protein has a molecular weight similar to the previously demonstrated SP receptor on both AR42J cells and IM9 cells. Furthermore, antibodies against the second and fourth extracellular domains significantly inhibited specific 125I-SP binding on both AR42J and IM9 cells, and also significantly inhibited SP-induced mobilization of [Ca2+]i on AR42J cells. These data suggest that the second and fourth extracellular domains of the SK and SP receptors may share common structural motifs for ligand binding and signaling mechanism. PMID- 1712654 TI - Clinical experience with subcutaneous urinary diversion: new approach using a double pigtail stent. AB - Short-term results in 8 patients with ureteric obstruction and hydronephrosis who underwent subcutaneous urinary diversion show this procedure to be a simple and useful method of urinary diversion for uraemic cancer patients. This method has the additional advantage of avoiding the complications and social implications of other methods of palliative treatment. PMID- 1712655 TI - Prostatic specific antigen related to clinical status 1 to 14 years after radical retropubic prostatectomy. AB - Serum prostatic specific antigen (PSA by Tandem-R immunoassay) was measured in 190 patients after radical prostatectomy for prostate cancer. Serial measurements were made in all patients operated on in the past 3 years; 131 had undetectable levels and 59 had levels in the detectable range. Only 10% of the patients with undetectable PSA following surgery had seminal vesical involvement or positive lymph nodes. None of the patients with undetectable PSA have had clinical recurrence within the 41 months of mean follow-up. In 14 patients who had PSA serially measured since surgery, detectable levels were found within 6 months of operation and 7 of these patients had clinical progression within 2 years; 39 patients had detectable PSA after radical prostatectomy with no clinical evidence of recurrence. Biopsy of the anastomosis was performed in 11 patients with isolated detectable PSA after surgery and local recurrence was established in 4. PSA was detectable later in the follow-up of 45 patients operated on before the PSA assay became available but the date when PSA actually became detectable is not known. A relatively new method of estimating that date and constructing a corresponding Kaplan Meier curve is presented. PSA is an effective marker for monitoring patients after radical prostatectomy as it often detects early persistent disease in patients with detectable measurements 6 months post operatively or recurrent disease in patients with later rising levels. PMID- 1712656 TI - Devine exclusion for unresectable carcinoma of the stomach. AB - Between July 1986 and July 1988, Devine exclusion was performed in 20 patients with unresectable carcinoma of the gastric antrum. All 20 patients presented with repeated vomiting. On endoscopy, 16 patients had complete gastric outlet obstruction while the remainder manifested significant gastric outlet stenosis. There was no hospital mortality. All except two patients could take an oral diet after surgery until their demise. Devine exclusion is safe and effective in relieving gastric outlet obstruction and is not associated with prolonged delay in return of gastric emptying. PMID- 1712657 TI - Comparative aspects of the distribution of substance P and dopamine immunoreactivity in the substantia nigra of amniotes. AB - In the present account, the relation between the descending, substance P (SP) containing projection fibers from the striatum and the dopaminergic cell bodies in the midbrain tegmentum was investigated in several species of reptiles and the domestic chicken by means of antisera against SP and dopamine. It was found that in all species studied an almost complete match of the SP and the dopamine immunoreactivity occurs in the ventral tegmental area. In contrast, considerable differences were observed in the substantia nigra and the retrorubral area. Two different patterns were recognized: one pattern in which there is little overlap between the dopaminergic cells and SP fibers (lizards, turtles) and another with an extensive overlap (snakes, chickens). Similar patterns were found in mammalian species. On the basis of an out-group comparison with anamniotes, it is suggested that the pattern with little overlap represents the primitive character. The pattern with considerable overlap, found in the three classes of amniotes, should be considered an independent development from the same primitive character and should, therefore, be defined as an example of parallel homoplasy. PMID- 1712658 TI - Enhanced in vivo release of substance P in the nucleus tractus solitarii during hypoxia in the rabbit: role of peripheral input. AB - In the adult, pentobarbitone-anaesthetized rabbit, the in vivo release of substance P-like immunoreactivity was measured in the nucleus tractus solitarii using microdialysis and radioimmunoassay. Increased 160 +/- 16%) extracellular concentrations of substance P-like immunoreactivity were observed during hypoxic provocations of 9% O2 in N2 which also resulted in an increase in phrenic nerve activity. In bilateral carotid sinus nerve-denervated animals no enhanced release of substance P was seen in response to hypoxic challenges (105 +/- 6%) and the phrenic nerve activity was not significantly affected. Perfusion of the nucleus tractus solitarii region with the dopamine agonist, apomorphine (10(-5) M) resulted in a significant decrease in the extracellular level of substance P. These results provide further evidence that substance P is involved in the mediation of the hypoxic drive inputs from the peripheral chemoreceptors. The interactions of apomorphine with substance P release might also suggest a presynaptic modulation of substance Pergic neurons by dopamine in the nucleus tractus solitarii. PMID- 1712659 TI - Mechanism of modulation of GABA-activated current by internal calcium in rat central neurons. AB - GABA-activated currents in Purkinje cells isolated from rat cerebellum were investigated. Increase of intracellular Ca2+ in the physiological range of concentrations caused a decrease in GABA-activated chloride currents. This effect resulted from a decrease of both the maximal values of GABA-activated currents and possibly from the affinity of GABAA receptors. Therefore, the mechanism of Ca2+ effect on GABAA receptors in central rat neurons differs from that in bullfrog sensory neurons. PMID- 1712660 TI - Analgesic effects of vibration and transcutaneous electrical nerve stimulation applied separately and simultaneously to patients with chronic pain. AB - The analgesic effects of transcutaneous electrical nerve stimulation (TENS) and vibratory stimulation (VS), used both separately and simultaneously, were compared in 24 patients suffering from chronic pain. We tested the hypothesis that these combined procedures might improve the pain reducing effects obtained with a single type of stimulation, since they make it possible to recruit a larger number of large diameter afferents and/or to increase the discharge frequencies. Four 35-minute treatment sessions (VS, TENS, VS + TENS, Sham stimulation) were run with each patient. The vibrations (100 Hz) and TENS (100 Hz) were applied to the surface of the painful region. The sham stimulation treatment consisted of positioning the TENS electrodes without actually delivering any current. The short form of the McGill pain questionnaire was used to assess the subjects' pain levels. The assessments took place immediately after any treatment (0h.), and again 4 hours and 24 hours later. The results showed that dual stimulation not only alleviated pain in more cases than either VS or TENS alone, but also had stronger and more long-lasting analgesic effects. On the other hand, all three types of stimulation used produced stronger analgesic effects than those obtained with the sham stimulation. PMID- 1712661 TI - Treatment of neuroendocrine carcinomas with combined etoposide and cisplatin. Evidence of major therapeutic activity in the anaplastic variants of these neoplasms. AB - Forty-five patients with metastatic neuroendocrine tumors were treated with a regimen of etoposide 130 mg/m2/d for 3 days plus cisplatin 45 mg/m2/d on days 2 and 3. Both drugs were given by continuous intravenous infusion. Among 27 patients with well-differentiated carcinoid tumors or islet cell carcinomas, only two partial objective tumor regressions were observed (7%). Among 18 patients prospectively classified as having anaplastic neuroendocrine carcinomas, however, there were nine partial regressions and three complete regressions, an overall regression rate of 67%. For anaplastic disease, the median duration of regression was 8 months (range to 21 months). Tumor response was unrelated to primary site, endocrine hyperfunction, or prior therapy experience. The median survival of all patients with anaplastic tumors was 19 months; this seemed favorable when considering the small experiences with these rare tumors reported in the literature. Toxicity, which was severe for most patients, consisted primarily of vomiting, leukopenia, thrombocytopenia, anemia, alopecia, and neuropathy. The anaplastic neuroendocrine tumor is strongly responsive to therapy with combined etoposide and cisplatin. Patients with undifferentiated carcinomas, originating in typical neuroendocrine tumor sites (small and large bowel, pancreas, and stomach) or of unknown origin, who have consistent histologic findings by light microscopy should be evaluated for this possibility with appropriate immune staining or electron microscopy. PMID- 1712662 TI - Results of treatment with high intensity, brief duration chemotherapy in poor prognosis non-Hodgkin's lymphoma. AB - A novel chemotherapeutic approach was designed for the treatment of intermediate and high-grade histology non-Hodgkin's lymphoma using augmented (but subtransplantation) doses of chemotherapy administered at frequent intervals in the inpatient setting. For the initial evaluation of this regimen, poor prognosis patients were treated with a projected long-term survival rate of less than 25% in response to standard therapy. Between March 1982 and May 1988, 56 previously untreated patients were entered into this study; all patients had either high grade histology (20 patients) or predominantly large cell lymphoma (36 patients). Median age was 41.5 years (range, 18 to 69 years). Poor prognosis features included: Stage IV, 71%; poor performance status (Eastern Cooperative Oncology Group scale, 2 to 4), 55%; multiple extranodal sites of disease, 52%; elevated lactic dehydrogenase (greater than 300 IU/l), 43%; and bulky (greater than 10 cm) tumor masses, 30%. Thirty-three of 56 patients (59%) were in Shipp's Category 3. During the 6-year study, the chemotherapy regimen was modified in an attempt to improve efficacy and reduce toxicity. However, most patients received a 2-month course of therapy as follows: cyclophosphamide 1500 mg/m2 intravenously (IV) on days 1, 2, and 29; etoposide 400 mg/m2 IV on days 1, 2, and 3 and 100 mg/m2 on days 29, 30, 31; doxorubicin 45 mg/m2 IV on days 29, 30; vincristine 1.4 mg/m2 IV on days 8, 22, 36, and 50; bleomycin 10 units/m2 IV on days 8, 22, 36, and 50; methotrexate 200 mg/m2 IV on days 15 and 43 followed 24 hours later by leucovorin 15 mg/m2 IV every 6 hours for six doses; and prednisone 60 mg/m2 orally on days 1 to 7 and 29 to 35. The complete response (CR) rate was 77% (95% confidence interval, 64% to 86%). There were ten relapses, only one of which occurred after 18 months of follow-up. Overall event-free survival (EFS) was 52% (95% confidence interval, 36% to 68%), with a median follow-up of 36 months. Eleven of 13 patients with small noncleaved lymphoma had CR; actuarial EFS in this subgroup was 61%. Myelosuppression occurred in all patients, with severe leukopenia (less than 1000/microliters) lasting a median of 12 days (range, 3 to 29 days); toxic deaths occurred in five patients (9%; 95% confidence interval, 4% to 19%). This intensive approach improved the response and survival of very poor risk non Hodgkin's lymphoma patients. PMID- 1712663 TI - The role of second-look surgery in the management of advanced germ cell malignancies. AB - The need for second-look surgery after chemotherapy in children with advanced germ cell tumors is controversial, particularly when levels of the tumor markers alpha-fetoprotein (AFP) or beta-human chorionic gonadotropin (beta HCG) are elevated at diagnosis. The authors evaluated the outcome of second-look surgery in relationship to tumor marker status in 27 patients with Stage III to IV disease who had completed four courses of chemotherapy. Markers were elevated at diagnosis in 19 patients. After chemotherapy, markers normalized in 12 of these patients. Second-look surgery confirmed complete response (CR) in these 12 patients, two of whom had residual masses on computed tomography (CT) scan (mature teratoma and necrotic tumor). The AFP decreased but did not normalize in seven patients; five had residual disease at second look and the other two later developed measurable disease. Of the eight patients with normal AFP at diagnosis, second look confirmed clinical CR in four. The other four patients had CT evidence of residual masses: surgery showed necrotic tissue in two cases, mature glial elements in one, and mature teratoma with glial elements in one. Thus second-look surgery added no information for treatment planning in children with elevated tumor markers at diagnosis and might best be reserved for patients without tumor markers at diagnosis and residual masses on CT scan, and those with persistent elevation of tumor markers and potentially resectable residual disease. Because of the possibility of small amount of residual tumor, second look surgery may also be useful in patients whose markers normalize but who have residual masses on CT scans. PMID- 1712664 TI - Oral contraceptive-associated liver cell adenoma and hepatocellular carcinoma. Cytomorphology and mechanism of malignant transformation. AB - From January 1976 to May 1990, 1673 patients with a liver mass or masses detected by imaging techniques underwent percutaneous fine-needle aspiration biopsy of the liver. Of these, 99 were diagnosed cytologically as "hepatocellular carcinoma" and 9 as "consistent with liver cell adenoma." The cytologic diagnoses were confirmed in the follow-up of all cases. Among the 99 patients with hepatocellular carcinoma, 3 had taken oral contraceptives for a period of 10, 11, and 12 years, respectively. The nine patients with liver cell adenoma were all users of oral contraceptives over a period ranging from 5 to 10 years. Of these, two who had taken oral contraceptives for a period of 8 and 10 years, respectively, had foci or areas of liver cell dysplasia within the adenomas. The cytologic criteria for the diagnosis of liver cell dysplasia included cytoplasmic and nuclear enlargement, nuclear pleomorphism together with prominent nucleoli, hyperchromasia and multinucleation. The cytologic features of liver cell dysplasia strikingly mimic hepatocellular carcinoma. From this study, the foci or areas of liver cell dysplasia arising within the liver cell adenomas appear to be the missing link responsible for the transformation of liver cell adenoma to carcinoma. It is believed that liver cell adenomas are not premalignant and may undergo reversible change after withdrawal of causative agents, whereas liver cell dysplasia is an irreversible, premalignant change and will eventually progress to hepatocellular carcinoma. PMID- 1712665 TI - A model of prostatic carcinoma tumor kinetics based on prostate specific antigen levels after radiation therapy. AB - Serial prostatic specific antigen values (PSA) were determined on 42 patients receiving definitive radiation therapy for localized adenocarcinoma of the prostate. The PSA declined exponentially in 25 patients. None of these patients experienced metastases. The PSA initially declined then increased exponentially in 17 patients. The rate of decline was similar to the rate of rise in all 17 patients. Five of these patients had distant metastases (P less than 0.02) within 2 years of treatment. The PSA values after radiation therapy were employed to formulate a model of tumor kinetics. This model predicts the mean duration of G0. This parameter is correlated with the development of distant metastases within 2 years of treatment. For those patients at low risk for relapse, the mean G0 is calculated to be 22.5, and 13.6 weeks for those who have relapsed or are at high risk for relapse. PMID- 1712666 TI - Bullous pyoderma gangrenosum after granulocyte colony-stimulating factor treatment. AB - The hematopoietic growth factors are under investigation for the treatment of patients with chemotherapy-induced bone marrow suppression. One such trial at the University of California, Los Angeles involves chemotherapy with or without granulocyte colony-stimulating factor (G-CSF) in patients with small cell lung cancer. The authors report a case of a patient who had bullous pyoderma gangrenosum at the site of previous eczema during treatment with G-CSF. The lesions resolved promptly when the drug was discontinued. Other investigators have recently reported inflammatory complications of G-CSF and granulocyte macrophage colony-stimulating factor (GM-CSF) but this is the first case report of biopsy-proven neutrophilic dermatosis associated with administration of a hematopoietic growth factor. Patients should be monitored for development of inflammatory processes during G-CSF therapy and this therapy should be given with caution to those patients with existing inflammatory conditions. PMID- 1712667 TI - Interstitial cells of Cajal and Auerbach's plexus. A scanning electron microscopical study of guinea-pig small intestine. AB - Interstitial cells of Cajal (ICC) appear to be involved in the regulation of intestinal motility, probably as pacemaker cells. We investigated the complex organization of ICC associated with Auerbach's plexus of guinea-pig small intestine in the scanning electron microscope. The plexus was exposed by microdissection of zinc iodide/osmic acid stained tissue. After separation of the muscle layers by microdissection alone, the exposed Auerbach's plexus was seen to be covered by a smooth mat of reticular fibrils, thin enough to allow the detailed examination of the intact nerve plexus and interstitial tissue. ICC were distinguished as small, ovoid cell bodies from which 2-5 long, branching, roughly cylindrical processes emerge, associating to form a complex network. Characteristically, ICC processes participated in the formation of small bundles along their course, individual processes passing from one bundle to another. Cell bodies and processes of ICC were intimately associated with tertiary nerves of Auerbach's plexus. Axons were identified and distinguished these from ICC processes by their varicose structure and by th smaller diameters as compared with ICC processes. We found no single axons in our material. The characteristic morphology of ICC clearly distinguished these as a separate cell population different from neurons, glial cells, fibroblasts, and smooth muscle cells. After removal of the mat of reticular fibrils by chemical digestion the detailed organization of the interstitial tissue was preserved. Macrophage-like cells were previously demonstrated by other techniques to constitute a constant and rather dense population of cells in the studied location. As an indication that we preserve the full complement of interstitial cells by our technique, these macrophage-like cells wer for the first time identified in material processed for scanning electron microscopy. The cells had characteristically irregular cell surfaces with short, veil-like, folded extensions intertwined between the other cells in the interstices. Our study establishes an improved correlation between results obtained by the application of scanning electron microscopy to dissected tissue and results from light and transmission electron microscopy of the intact tissue. PMID- 1712668 TI - Further cytological observation on 'activation' by superimposed antigen of inflammation-mediated macrophages. AB - Compact cell aggregates were induced by an injection of a superimposed antigen, sheep erythrocytes (SRBC) into PSK (a protein-bound polysaccharide)-'prepared' mouse footpad. Major cell types in the cell aggregate were macrophages which rapidly digested superimposed SRBC but not PSK substances which were retained over seven days within their phagosomes. Macrophages without PSK like substances containing phagosomes occurred invariably outside the cell aggregate. The macrophages in the cell aggregate were interlocked via their cytoplasmic projections. Ruthenium red, a specific dye for extracellular proteoglycans, clearly revealed their surface coats and also closely contacted zones of adjoining macrophages. The surface coats were not uniform in distribution and became sporadically thickened deposits at invaginations of the plasma membrane. Ladder-like structures among adjacent cytoplasmic projections also showed a strong affinity to the dye. It is suggested that a primary function of these structures is the maintenance of close contact of aggregating macrophages. Another observation was that binucleate macrophages occurred in the cell aggregates. Careful inspection on sections of well-preserved tissues concludes that such cells were not formed by a cell fusion between mononucleate macrophages in the cell aggregates. Formation of such a binucleate cell from the macrophage in the aggregates is also discussed. PMID- 1712669 TI - [The effect of amino acids on neurotransmitter metabolites in the cerebrospinal fluid]. AB - Metabolites of neurotransmitters of dopamine, homomovanillic acid (HVA), and of serotonin, 5-hydroxyindole acetic acid (5-HIAA), were assessed in cerebrospinal fluid by the method of high pressure liquid chromatography with electrochemical detection. The HVA concentration in cerebrospinal fluid rose markedly in a two month-old infant with intracranial hypertension caused by a communicating hyporesorptive hydrocephalus following administration of tyrosine, the precursor of dopamine. The 5-HIAA concentration in cerebrospinal fluid rose significantly in a 20-month-old boy with epilepsy and arrested psychomotor development after administration of 5-hydroxytryptophan, the precursor of dopamine. Biochemical normalization of concentrations of neurotransmiteed metabolites did not lead to changes in the clinical condition of the children. PMID- 1712670 TI - Nuclear import-export: in search of signals and mechanisms. PMID- 1712671 TI - BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner. AB - BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways. PMID- 1712672 TI - Staufen, a gene required to localize maternal RNAs in the Drosophila egg. AB - The posterior group gene staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of staufen and show that staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, staufen protein is also concentrated at the anterior pole, in the same region as bicoid RNA. PMID- 1712673 TI - Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. AB - Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy. PMID- 1712674 TI - Drug-induced scleroderma. PMID- 1712675 TI - The inhibitory effect of 3,3',4,5'-tetrahydroxystilbene, a constituent of Cassia garrettiana, on anti-IgE-induced histamine release from human basophils in vitro. AB - 3,3',4,5'-Tetrahydroxystilbene (I), a constituent of Cassia garrettiana, strongly inhibited the anti-IgE-induced histamine release from human basophils in vitro at concentrations of 3 to 30 microM. Considering that disodium cromoglycate showed no significant inhibitory activity in this assay method, the strong effect of I should be emphasized. PMID- 1712676 TI - L-arginine-dependent formation of N-nitrosamines by the cytosol of macrophages activated with lipopolysaccharide and interferon-gamma. AB - The cytosol fraction of J774-1 murine macrophages activated with lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) was found to nitrosate a wide range of secondary and tertiary amines. The reaction was dependent on L arginine and NADPH. The optimal pH for nitrosation was 7.2-7.3. Nitrosation was inhibited by arginine derivatives such as NG-monomethyl-L-arginine and NG-nitro-L arginine, well-known inhibitors of nitric oxide (NO) synthase. These results indicate that nitrosation is mediated by NO synthase, which catalyzes formation of NO and L-citrulline from L-arginine. Nitrosamine formation also required oxygen and was inversely correlated with the basicity of nitrosatable amines. The nitrosation was inhibited by oxyhemoglobin, an NO trapping agent, and enhanced by superoxide dismutase, which stabilizes NO. LPS + IFN-gamma induced approximately 500-600 times greater nitrosation activity than that of non-activated macrophages. Macrophages treated with LPS alone exhibited 3-4 times greater nitrosation activity than untreated macrophages, whereas macrophages treated with IFN-gamma alone did not show enhanced nitrosation activity. A combination of the cytosols from macrophages treated with LPS alone and IFN-gamma alone did not nitrosate morpholine as rapidly as the cytosol of macrophages treated with both compounds together. The activity for forming L-citrulline and nitrite/nitrate from L-arginine was markedly induced by treatment with either LPS alone or LPS + IFN-gamma but not with IFN-gamma. Those results suggest that some other factor(s) in addition to NO synthase is involved for efficient nitrosation by the macrophage cytosol. This factor(s) was not induced in macrophages by either LPS- or IFN-gamma alone, but was induced only in the presence of the two compounds. PMID- 1712677 TI - Glutathione S-transferase (mu class) as an early marker of azaserine-induced foci in the rat pancreas. AB - The alpha, mu and pi classes of glutathione S-transferase (GST) were evaluated as early immunocytochemical markers for the development of atypical foci within the pancreases of azaserine treated rats. Changes detected with haematoxylin and eosin (H&E) were compared with those detected by immunocytochemistry using antibodies raised against each class of GST. All foci detected with H&E staining were classified as acidophilic atypical acinar cell nodules (AACN), which have previously been reported in this model. All of these AACN overexpressed GST mu. However, 64% of foci detected with GST mu staining had not been identified as AACN during a prior examination with H&E. Re-evaluation of the H&E sections revealed that some of these foci showed subtle morphological changes which are indicative of AACN. In many cases, however, no morphological difference could be seen with H&E staining. We conclude that immunocytochemical staining for GST mu is a more reliable and sensitive method than H&E for detecting the early stages of azaserine-induced foci. Furthermore, we suggest that studies on the incidence and growth of these foci can be shortened considerably if GST mu staining is used in conjunction with H&E. PMID- 1712678 TI - Induction of glutathione content in murine melanocytes after transformation with c-H-ras oncogene. AB - Malignant melanoma tumors are inherently resistant to anticancer drugs, yet the mechanism of this resistance is not understood. B16 melanoma, a spontaneous tumor which arose in the C57BL/6 mouse; BL6 melanoma, a highly invasive variant; and Mel-ab melanocytes, isolated from C57BL/6 mouse skin, were examined for intracellular glutathione (GSH) content. GSH was higher in BL6 and B16 cells than in Mel-ab cells, with the highest concentration in BL6 cells. Since GSH is thought to be involved in the resistance of many cells, including melanoma, to cytotoxic drugs, we determined whether intracellular GSH content was altered during transformation of Mel-ab cells. After transfection with pHO6T1 plasmid DNA, containing an activated c-H-ras oncogene flanked by transcriptional enhancers, 1.3% of successfully transfected Mel-ab melanocytes formed distinct colonies in soft agar, compared to 0.2% of cells transfected with control pHO6 plasmid without H-ras. Approximately 53% of the pHO6T1-transfected colonies isolated from soft agar grew in 5% CO2 in the absence of phorbol-12-myristate-13 acetate, a requirement for the extended growth of Mel-ab cells. Cells transfected with control pHO6 plasmid and non-transfected Mel-ab cells did not survive under these conditions. All of the isolated pHO6T1 transfected cells formed tumors when inoculated into C57BL/6 mice. Transformed cells had higher GSH content than non transfected Mel-ab cells, whether expressed on a cellular or cell volume basis. Although the amount of oxidized glutathione was greater in the tumorigenic cells, this could not account for the overall increase in GSH. Neither glutathione S transferase nor gamma-glutamyl transpeptidase activities were increased in the H ras-transfected cells. Northern blot analysis confirmed H-ras-specific RNA in pHO6T1-transformed cells. PMID- 1712679 TI - The nature of the epithelium in acquired cholesteatoma. AB - Monoclonal antibodies with defined specifications for individual cytokeratins were used to stain the epithelia of the external auditory meatus, the middle ear and cholesteatoma. The observed staining indicated that the epithelium of the external auditory meatus has a pattern of keratin expression typical of epidermis in general and the epithelium of the middle ear resembles simple columnar epithelia. The pattern of staining of cholesteatoma closely resembled that of the skin of the external auditory meatus. PMID- 1712680 TI - The delta F508 mutation in mild adult forms of cystic fibrosis (CF). AB - Twenty CF chromosomes from ten patients with mild adult form of cystic fibrosis were tested for delta F508. This mutation was found to be significantly less frequent than in the severe form of the disease. PMID- 1712681 TI - Inhibition of lymphocyte proliferation by a monoclonal antibody (RFT2) against CD7. AB - The effect of the monoclonal antibody RFT2, directed against CD7, on T cell proliferation in unseparated peripheral blood mononuclear cell populations induced by various stimulants was investigated. The addition of RFT2 to cell cultures inhibited the T cell proliferation induced by tuberculin PPD and OKT3 but not by phytohaemagglutinin, concanavalin A or phorbol myristic acid; RFT2 had to be present during the first 24 h of culture in order to elicit inhibition; inhibition of proliferation was not due to down regulation of interleukin-2 receptor on the surface of T cells; and suppressive effects could be transferred by mononuclear cells pre-treated with RFT2. These results are of particular relevance in view of the known in vivo suppressive effect of RFT2 in humans. PMID- 1712682 TI - Expression of CD34 on human B cell precursors. AB - CD34 is a 110-kD glycoprotein previously shown by a variety of monoclonal antibodies (MoAbs) to be expressed selectively on immature hematopoietic cells. However, more detailed characterization of CD34+ cells has been hampered by lack of anti-CD34 MoAbs that can be labelled directly with fluorochromes to facilitate subpopulation analysis by multi-parameter flow cytometry. We have recently isolated a murine anti-CD34 MoAb, designated as 8G12, that can be directly labelled with fluorochromes such as FITC. In this study, we have exploited this property of 8G12 to compare the reactivity of 8G12 and My10 with normal and leukaemic human marrow cells and to characterize normal early human B cell precursors by two- and three-colour immunofluorescence analysis. Comparison of three-colour staining profiles of normal bone marrow cells incubated with both 8G12 and MY10, and either anti-CD10 or anti-CD19 MoAb revealed the reactivity patterns of 8G12 and MY10 to be indistinguishable. This conclusion was confirmed by a similar comparative analysis of 8G12 and MY10 staining of blood and bone marrow cells from 4 patients with B lineage acute lymphoblastic leukaemia (ALL). Of interest, both 8G12 and MY10 detected a CD34+CD10+CD19- population in normal adult bone marrow. To determine whether a CD34+CD10+CD19- precursor population previously reported by others to exist in fetal liver could also be identified, CD10+CD16- marrow cells were first isolated by FACS and the sorted cells then re analysed for expression of CD19 and CD34. These studies showed that all of the sorted CD10+ cells that expressed CD34 appeared to coexpress CD19. No CD34+CD10+CD19- cells were detected (at a sensitivity of less than or equal to 0.1%). Further studies will be required to determine whether a very minor population of CD34+CD10+CD19- cells may still be generated in the normal development of B cells in adult human marrow. PMID- 1712683 TI - Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases. AB - A new monoclonal antibody (MoAb) HA58 (IgG1) was prepared, which recognizes the binding site on the intercellular adhesion molecule-1 (ICAM-1) antigen to the lymphocyte function-associated antigen-1 (LFA-1). The double-determinant immunoassay (DDIA) was established with use of MoAb HA58 and another anti-ICAM-1, MoAb CL207, to detect the soluble, shedding ICAM-1 antigen. Human recombinant interferon-gamma (IFN-gamma) induced not only the expression of cell surface ICAM 1, but also the shedding ICAM-1 antigen in an IFN-gamma concentration-dependent and incubation-time-dependent manner. DDIA was applied to detect the shedding ICAM-1 antigen in the sera of patients with malignant or benign diseases. The incidence of positivity for ICAM-1 antigen in malignant diseases was higher than that in benign diseases or in healthy controls. Furthermore, the sera of cancer patients with liver metastasis showed higher levels of the shedding ICAM-1 antigen. These findings suggest that serum ICAM-1 antigen may be a useful marker to monitor tumor burden in cancer patients. PMID- 1712684 TI - T cell interactions in active rheumatoid arthritis: insights from the human autologous mixed lymphocyte reaction as a model of T cell activation cascade. AB - The autologous mixed lymphocyte reaction (AMLR) represents the activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Using monoclonal anti-Leu8 antibody to isolate subpopulations of human CD4+ and CD8+ T cells, we have investigated the role of these subpopulations in the T cell activation cascade during the course of AMLR. In normal subjects, CD4+Leu8+ cells are necessary for the initiation of the AMLR response, and sequentially lead to activation and proliferation of both CD4+Leu8- cells and CD8+Leu8+ cells. The activated CD8+Leu8+ cells, in turn, induce CD8+Leu8- cells to generate proliferation of the latter cells. Soluble mediators could be involved in the T cell activation cascade induced by the AMLR. Patients with active rheumatoid arthritis have a profound defect in the AMLR. Further analysis indicates that rheumatoid arthritis CD8+ T cells are markedly defective as responding cells in the AMLR. The impaired AMLR response by CD8+ cells cannot be reconstituted with AMLR-derived supernatants from normal T cells. The data suggest that the defective CD8+ T cell function may contribute to the pathogenesis of the disease. PMID- 1712685 TI - HIV induces modulation of functionally important cellular antigens. AB - Infection of T lymphoblastoid CEM cells with the IIIB isolate of HIV-1 results in modulation of the expression of several cellular antigens in addition to the CD4 molecule. The intercellular adhesion receptor LFA-1 (CD11a/CD18) and HLA-DR are markedly induced in the cytoplasm and at the cell surface, and the CD7 antigen is down-regulated, being virtually undetectable by sensitive immunocytochemical techniques in the infected cell population. These modulatory effects are to some degree dependent on the virus isolate examined, as the CBL-1 British isolate did not induce comparable phenotypic changes in the CEM cell line. Furthermore, these effects are not reproduced by recombinant gp120 (IIIB isolate) or p24 added exogenously to uninfected CEM cells. The CD7 molecule appears to play a regulatory role in T cell proliferation, and the LFA-1 integrin molecule is involved in a wide range of immunologically important cell-cell interactions, as well as HIV-induced syncytium formation. The possible contributions of such effects to the pathogenesis of HIV infection are considered. PMID- 1712686 TI - Inhibition of induction of natural killer activity in mice by general anesthesia (Avertin): role of interferon. AB - We have previously reported that general anesthesia (Avertin) inhibits the induction of natural killer (NK) cell activity following administration of Poly I:C in vivo. Since Poly I:C has been shown to increase NK activity through the induction of interferon (IFN), the current study examines the role of IFN in this inhibition. The data suggest that (i) anesthesia does not affect the ability of Poly I:C to induce endogenous IFN synthesis (serum IFN levels are unaltered by anesthesia) and (ii) anesthesia is capable of inhibiting stimulation of NK activity induced directly by treatment with IFN either in vivo or in vitro. The duration of the former effect was at least 10 days, with complete recovery by Day 14 after anesthesia. Interestingly, NK activity stimulated by IFN prior to anesthesia was not significantly altered. In view of the increasingly evident role of NK cells in anti-tumor and anti-infectious host defenses, their inertness to stimulation in the post-anesthesia period may be a significant contributing factor to the clinically observed postoperative morbidity. Thus, preanesthesia stimulation of NK activity with IFN may be of therapeutic value. PMID- 1712687 TI - Lymphocyte subset reference ranges in adult Caucasians. AB - We report here the distributions of lymphocyte populations bearing the following antigens: CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic and some NK cells), and CD3-, CD16+, and/or CD56+ (NK cells). At four sites, analyses were performed on healthy, normal subjects between the ages of 18 and 70, using identical flow cytometry systems and techniques. Reference ranges (unadjusted for sex differences and age variation) are CD3 (61 to 85%), CD19 (7 to 23%), NK (6 to 29%), CD4 (28 to 58%), and CD8 (19 to 48%). The lymphocyte subpopulation distributions for all antigens were found to be similar at all sites. By combining data from all sites, it has been possible to estimate age variation and sex differences for each of these subpopulations. Age and sex associated differences are substantial for some lymphocyte subsets (CD3, CD4, NK cells), and proper accounting of these effects is essential in evaluating the individual patient, if further disease-related variation is to be accurately and consistently assessed. It appears possible to recommend reference ranges for lymphocyte population parameters applicable across national and laboratory boundaries. These ranges provide a basis for comparing results from different institutions and for combining such results on subjects and patients from several institutions, provided the methodology and equipment are identical at all sites. PMID- 1712688 TI - Comparative studies of the effects of FK506 and cyclosporin A on passively transferred collagen-induced arthritis in rats. AB - We investigated the effect of a novel immunosuppressive agent, FK506, in comparison with cyclosporin A (CsA) on the development of passive arthritis induced by anti-type II collagen (CII) antisera in rats. FK506 pretreatment shortly before serum transfer markedly suppressed the incidence and the severity of passive arthritis, while CsA pretreatment had no observable effects on this disease when used in doses sufficient to suppress the development of active arthritis induced by CII immunization. In an additional study, we examined whether these agents affect antibody-mediated tolerance induction. CII-specific immunological tolerance was induced by serum transfer, but was unaffected by either FK506 or CsA pretreatment in our regimen. While its precise mechanism of the immunosuppressive activity remains to be elucidated, FK506 can act on the antibody-mediated effector phase of arthritis and may offer new insights into the possible role of potential therapeutic utility in human autoimmune diseases. PMID- 1712689 TI - Donor type natural killer cells after haploidentical T cell-depleted bone marrow stem cell transplantation in a patient with adenosine deaminase-deficient severe combined immunodeficiency. AB - T cell-depleted haploidentical (parental) bone marrow stem cell transplants are given to most infants with the syndrome of severe combined immunodeficiency (SCID) because they have no available HLA-identical sibling potential donors. Since they usually do not undergo cytoreduction prior to transplantation, these children later demonstrate mixed hematopoietic chimerism. Most often, T cells (but usually not B lymphocytes, macrophages, or other hematopoietic cells) can be shown to be of donor type. The origin of natural killer (NK) cells in such chimeras has not been reported. Two lymphocyte lines derived from the CD16+ fraction of an adenosine deaminase (ADA)-deficient male SCID's blood mononuclear cells (MNC) 13 months following maternal marrow stem cell transplantation demonstrated typical phenotypic and functional characteristics of NK cells after expansion. Karyotyping showed both lines to be XX. Thus, NK cell engraftment can occur in SCID infants who have not been conditioned, even when significant NK cell function is present before transplantation. PMID- 1712690 TI - Influence of oxytocin on integration of postsynaptic potentials in molluscan neurons. AB - 1. Intracellular recordings were made from identified and non-identified neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc, Helix pomatia. The influence of oxytocin (OXT) on neuronal integration: space and temporal summations of postsynaptic potentials (PSPs) in various neurons was investigated. The obtained data indicated that these PSPs were cholinergic PSPs. 2. Ten minute exposure to 10(-8) M OXT had no effects on the resting membranes, but triggered secondary mechanisms, which lead to enhancement of the excitatory PSP (EPSR) amplitudes and the decrease of the decay time constant (tau EPSR) obtained from the falling phase of the EPSP. 3. The enhancement of the EPSP amplitude and the decrease of tau EPSP after OXT application evoked the appearance of action potential under space summation of two spontaneous EPSPs and made easier the appearance of action potential under temporal summation of EPSPs produced by paired afferent stimuli, when the corresponding interstimuli interval was smaller than tau EPSP in the presence of OXT. 4. Ten minute exposure to 10( 8) M OXT made the integrated amplitude of the excitatory acetylcholine response and the inhibitory dopamine response in the neuron E5 more positive only when the interval between applications of these mediators was smaller than the time constant of desensitization of acetylcholine receptors in the presence of OXT. 5. The pharmacological studies showed that drugs, which elevate intracellular cyclic AMP levels, mimicked the influence of OXT on integration of PSPs in the investigated neurons. PMID- 1712691 TI - Inhibition by oxytocin of voltage-activated calcium influx in cultured PC12 pheochromocytoma cells. AB - 1. Voltage-activated dihydropyridine-sensitive Ca2+ influx was measured in PC12 pheochromocytoma cells using 45Ca. 2. It has been found that oxytocin inhibits voltage-activated dihydropyridine-sensitive Ca2+ influx with ED50 about 0.30 x 10(-6) M. 3. Tolbutamide (1.3 x 10(-3) M) has no visible effect on both Ca2+ influx itself and on the inhibitory oxytocin effect. 4. External application of Li+ (10 mM) causes a slight shift of ED-curve to lower oxytocin concentrations. 5. It is suggested that the hydrolysis of phosphoinositides may play a role in oxytocin action on Ca2+ influx in PC12 cells. PMID- 1712692 TI - Biogenic amines in newly-ecdysed cockroaches. AB - 1. Simultaneous quantification (HPLC and electrochemical detection) of biological extracts have shown dopamine, N-acetyl dopamine, tryptophan, 5-hydroxytryptamine, a 5-hydroxyindolacetic acid-like substance in nervous tissue and hemolymph of Blaberus craniifer and Periplaneta americana. 2. 5-Hydroxytryptophan was only detected in head and thoraco-abdominal nerve cord. 3. Octopamine, but not N acetyl-5-HT was quantified in the hemolymph. PMID- 1712693 TI - Indolamines and onset of vitellogenesis in the imaginal molt-decapitated cockroach Blaberus craniifer Burm. AB - 1. The effects of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and N-acetyl-5 hydroxytryptamine on oocytes of Blaberus craniifer, in which vitellogenesis was prevented by imaginal molt decapitation, were investigated. 2. Sites binding anti egg-protein antibodies were detected in the periphery of basal oocytes of treated females, with individual variability. 3. In this ovoviviparous cockroach, the onset of vitellogenesis may thus not be triggered solely by juvenile hormone, and indolamines may play a role in the uptake of haemolymphatic proteins by oocytes. PMID- 1712694 TI - Structure-activity relationship of philanthotoxins--I. Pre- and postsynaptic inhibition of the locust neuromuscular transmission. AB - 1. Like the natural toxin, synthetic delta-philanthotoxin, now called PTX-4.3.3 acts as a reversible postsynaptic open ion-channel blocker of the glutamatergic neuromuscular system of the locust. 2. It also inhibits the high-affinity re uptake of glutamate in the nerve endings and glial cells. 3. To study the structure-activity relationship, three parts of the PTX-4.3.3 molecule were changed. 4. One of these PTX-analogues, trifluoromethyl-PTX-4.3.3, proved to be a more potent postsynaptic blocker. 5. Moreover, compared with PTX-4.3.3 a delayed recovery period is seen with trifluoromethyl-PTX-4.3.3. 6. A number of PTX analogues were equipotent to PTX-4.3.3 regarding the inhibition of iontophoretically evoked, postsynaptic glutamate potentials. 7. However, complete inactivation was achieved by reducing the length of the polyamine chain, moreover dideaza-PTX-12 was nearly completely inactive and a reduced activity was seen with dephenol-PTX-4.3.3. 8. A decrease of the decay time constant of glutamate potentials, normally seen by open ion-channel blockers in Con A pretreated preparations, was unaffected during application of the latter two analogues. 9. Possibly these two toxins act as weak receptor antagonists. 10. The presynaptic inhibition of the glutamate re-uptake, seemed to be a very specific property of PTX-4.3.3. Only one of the tested analogues (dehydroxy-PTX-4.3.3) exhibited this capacity. PMID- 1712695 TI - Structure-activity relationship of philanthotoxins--II. Effects on the glutamate gated ion channels of the locust muscle fibre membrane. AB - 1. PTX-4.3.3, the synthetic product of the wasp toxin, delta-philanthotoxin, blocks the open, glutamate gated, ion channels of the locust muscle. 2. This block is accompanied by the appearance of double exponential distribution of the closed times frequency, and a decrease of the inverse relationship between the open and closed states. 3. The analogues with the shortened polyamine chain, PTX 4.3 and PTX-3.3, are less potent, trifluoromethyl-PTX-4.3.3 is a more potent analogue. 4. Yet, they still behave like PTX-4.3.3 as open ion-channel blockers. 5. Another analogue, without the aromatic nucleus, dephenol-PTX-4.3.3, proved to act in a different way. 6. This less potent analogue, doesn't block the open ion channels. 7. Most likely, the not-activation induced, non-competitive antagonism, caused by dephenol-PTX-4.3.3, has a modulatory effect on glutamate receptors. 8. Methyl-PTX-4.3.3 is also an open ion-channel blocker. However, at relatively low concentrations it has an agonistic-like effect, probably of a modulatory origin as well. 9. A long-term inhibition of PTX-4.3.3 applied at low concentrations also indicates that beside an open ion-channel block, also other inhibitory processes are involved. PMID- 1712696 TI - TGF-beta 1 and fibroblast growth factors selectively up-regulate tissue-specific fetal genes in cardiac muscle cells. AB - TGF-beta 1, like basic and acidic fibroblast growth factor (FGF), inhibits differentiated gene expression in skeletal myoblasts. It potentiates FGF-beta 1 down-regulated expression of the alpha-myosin heavy chain gene and the sarcoplasmic reticulum calcium ATPase gene, yet up-regulated expression of the genes for beta-myosin heavy chain, atrial natriuretic factor, and both skeletal and smooth muscle alpha-actin-four transcripts associated with the embryonic heart. TGF-beta 1 did not affect cardiac alpha-actin gene expression. These responses resemble the generalized 'fetal' phenotype seen during hypertrophy triggered by a haemodynamic load. Chick skeletal and cardiac alpha-actin promoter driven reported genes were transfected into neonatal rat cardiac myocytes. TGF beta 1 stimulated skeletal alpha-actin transcription, but not transcription from the cardiac alpha-actin promoter. Basic FGF produced the same results as TGF-beta 1, but acidic FGF suppressed expression of both alpha-actin genes; these results were true for purified and recombinant FGFs. Modulation of alpha-actin transcription by growth factors corresponded accurately to control of the endogenous genes. Three positive cis-acting elements were critical for skeletal alpha-actin transcription in cardiac, as well as skeletal, myocytes, particularly the downstream CCAAT box-associated repeat. Thus, TGF-beta 1 and FGFs selectively induce an ensemble of 'fetal' genes and differentially regulate alpha-actin transcription in cardiac muscle cells. PMID- 1712697 TI - The role of TGF-beta in pulmonary fibrosis. AB - Pulmonary fibrosis is an irreversible accumulation of connective tissue in the interstitium of the lung. The pathogenesis of pulmonary fibrosis is not well understood. Research on animal models and studies of human lung disease suggest the initiating events may be a combination of pulmonary injury and the recruitment of inflammatory cells, mainly macrophages. A number of well characterized cytokines, including TGF-beta, have been either found in the injured lung or produced by inflammatory cells removed from the lung. In an animal model of pulmonary fibrosis, TGF-beta production is increased prior to collagen synthesis and is mainly produced by alveolar macrophages. In advanced idiopathic pulmonary fibrosis, a human fibrotic lung disease, extensive TGF-beta deposition can be detected by immunohistochemical staining, primarily in epithelial cells in areas of lung regeneration and remodelling. This suggests that the pathogenesis of the progressive fibrosis characteristic of this lung disease may be an aberrant repair process. PMID- 1712698 TI - Influence of in situ neural isolation of jejunoileum on postprandial pancreatobiliary secretion and gastric emptying. AB - Our aims were to examine the influence of neural isolation of the jejunoileum on postprandial pancreatobiliary secretion. In four dogs, duodenal perfusion and aspiration catheters were implanted, and serosal electrodes were placed along the proximal small bowel. Control studies of gastric emptying, output of bile acids and amylase, and plasma concentrations of peptide YY and neurotensin were performed on three occasions following ingestion of a 340-kcal mixed-nutrient liquid meal. The dogs then underwent our model of in situ jejunoileal neural isolation, and the meal studies were repeated. Neural isolation, when compared to control, did not affect either postprandial conversion of intestinal myoelectric activity to the "fed" pattern, gastric emptying (T1/2, X +/- SE of the liquid meal (74 +/- 6 vs 79 +/- 7 min; P greater than 0.05), or cumulative amylase output (373 +/- 59 vs 305 +/- 66 kU; P greater than 0.05). Neural isolation decreased cumulative postprandial bile acid output from 6.6 +/- 0.9 mM to 3.4 +/- 1.1 mM (P less than 0.05) and increased postprandial plasma concentrations of peptide YY and neurotensin. Our findings suggest that the jejunoileal denervation that accompanies the in situ neural isolation of the jejunoileum is not associated with changes in postprandial motility patterns, gastric emptying, or pancreatic amylase secretion. Loss of this innervation, however, may decrease postprandial output of bile acids and lead to a compensatory increase in the postprandial release of neurotensin and peptide YY. PMID- 1712699 TI - Serum alpha-L-fucosidase. A more sensitive marker for hepatocellular carcinoma? AB - Forty-nine liver disease patients (7 chronic persistent hepatitis, CPH; 10 chronic active hepatitis, CAH; 13 liver cirrhosis, LC; 9 primary hepatocellular carcinoma, PHC, without LC; and 10 PHC with associated LC) and 20 controls were assessed for their serum alpha-L-fucosidase (ALF) and alpha-fetoprotein (AFP) levels and several routine liver injury parameters. Tumor diameter in those with hepatic cancer was assessed by angio-CT. Only ALF and AFP were significantly greater in patients with PHC and PHC + LC patients as compared to patients with LC alone. At an accepted cutoff level of 500 ng/ml, the AFP level provided 43% false negative tests. On the other hand, an ALF level exceeding 740 mumol/hr/ml provided a sensitivity of 84% with a specificity of 94%. No relationship between the ALF level and Child's criteria or with any liver injury parameter was evident. Considering all individual values, the ALF, rather than the AFP, correlated with tumor size. This finding suggests the ALF level may be of value in the early detection of PHC as well as in the follow-up of patients treated for PHC. PMID- 1712700 TI - [Intensive care of children after heart surgery. Risk groups and possible complications]. PMID- 1712701 TI - Gonadal expression of c-kit encoded at the W locus of the mouse. AB - Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation. PMID- 1712702 TI - Somatostatin receptors are restricted to a subpopulation of osteoblast-like cells during endochondral bone formation. AB - Specific binding sites for the peptide hormone somatostatin have previously been demonstrated in long bones from neonatal rats. In the present study, the distribution of somatostatin receptors during embryonic bone formation has been investigated using the stable radioiodinated somatostatin analogue, SDZ 204-090. Somatostatin receptors in rat long bones were first detectable at the time of invasion of the cartilage model by osteogenic cells. Initially, receptors were detectable throughout the region occupied by osteogenic cells. As bone growth proceeded, however, receptors were restricted to the region of most recent invasion of the hypertrophic cartilage, where osteoid had not yet been deposited. In vivo labelling studies in neonatal rats were carried out to identify the cells bearing somatostatin receptors. Receptors were present in a restricted region of the metaphysis, immediately adjacent to the hypertrophic cartilage. Chondrocytes, osteoclasts, and mature osteoblasts were not labelled by the radioligand. The labelled cells were often apposed to remnants of cartilage matrix and stained positively for the osteoblast marker, alkaline phosphatase. Thus the cells with specific somatostatin-binding sites were probably osteoblast precursor cells. Specific binding was detectable in all endochondral bones examined, including those of the skull, but no specific binding was found in the membrane bones of the skull. These data suggest that somatostatin is involved in the regulation of osteoblast differentiation during endochondral bone formation. PMID- 1712703 TI - The current role of platelet-active drugs in ischaemic heart disease. PMID- 1712705 TI - Current pharmacological treatment approaches to central nervous system leukaemia. AB - Significant advances in the treatment and prevention of meningeal leukaemia have been made in the past 3 decades. This progress has resulted from the development of innovative approaches to treatment as well as a better understanding of the pharmacokinetics and pharmacodynamics of the commonly used antileukaemic agents. Intrathecal therapy, via the intralumbar or intraventricular route, is a form of regional therapy that results in the delivery of very high drug concentrations to the principle target tumour site (the meninges) using a relatively small drug dose, thereby minimising both systemic drug exposure and systemic toxicity. The dosage and schedules, clinical pharmacology and toxicities of the commonly used intrathecal agents, methotrexate and cytarabine (cytosine arabinoside; Ara-C) are discussed in detail. Another approach which has been used to overcome the poor penetration of antileukaemic drugs into the CNS has been the use of high-dose systemic therapy. This strategy has been successfully applied in the treatment of meningeal leukaemia using both high-dose methotrexate and high-dose cytarabine. The clinical pharmacology, toxicities, and potential limitations of this approach are outlined. Finally, new agents that are currently undergoing clinical evaluation and future directions for research are also discussed. PMID- 1712706 TI - Drug treatment in precocious puberty. AB - Precocious puberty, as defined by the onset of pubertal development before the age of 8 years in girls or 9 years in boys, can be classified into central and peripheral aetiologies. Central precocious puberty (CPP) results from early activation of the hypothalamic-pituitary-gonadal axis and has similar physical and hormonal characteristics to normal puberty. Extrapituitary gonadotrophin secretion or independent sex steroid secretion results in peripheral precocious puberty (PPP). Precocious puberty is characterised by rapid growth and advancement of skeletal age. The skeletal advancement is greater than the growth increase, so that final adult height is compromised. Long-acting gonadotrophin releasing hormone (GnRH) agonists are the current therapy of choice for central precocious puberty, having demonstrated effectiveness in halting the precocious development associated with this condition with minimal side effects. GnRH agonists are not effective as therapy for peripheral precocious puberty, but a number of other agents have been used with some success. These include androgen antagonists, testolactone, ketoconazole, and medroxyprogesterone acetate. The use of GnRH agonists has been associated with an increase in predictions of final height; however, continuing studies in treated cohorts are necessary to determine the true benefit of any of these agents on increasing ultimate height. PMID- 1712704 TI - Antiarrhythmic drug classifications. A critical appraisal of their history, present status, and clinical relevance. AB - Classifications of antiarrhythmic drugs have developed because of a need to organize the large number of agents available according to pharmacological properties of clinical relevance. The current classification is a hybrid of classification systems developed in the early 1970s. It subdivides drugs according to 4 major pharmacological actions: (a) depression of phase 0 sodium current; (b) antagonism of adrenergic effects on the heart; (c) prolongation of of action potential duration; and (d) calcium channel blockade. Further subdivision of sodium channel blockers is based on the kinetics of sodium channel blockade and drug effects on action potential duration. A critical analysis of selected aspects of the clinical actions of antiarrhythmic drugs indicates the value of the current classification, as well as some limitations in its ability to separate drugs into distinct groups with characteristic clinical properties. The strengths of the current classification are due to the clinical importance of the pharmacological properties on which it is based. These results in electrophysiological actions, indications, and adverse effects that are typical for each group of drugs. The limitations of the current system relate to the propensity of individual drugs to have actions of more than one class simultaneously, the way that the various actions of a given drug are dependent on concentration, rate, and tissue type, and to problems in subclass definition. Some of these shortcomings could be alleviated by returning to the concept, originally put forward by Singh and Vaughan Williams, of classes of drug action rather than classes of drug per se. This approach would be pharmacologically more realistic than trying to assign each antiarrhythmic agent to a single unique class, would be better able to incorporate the complexities of drug action, and would potentially be more flexible. The wide use of antiarrhythmic drug classifications attests to their value, and suggests that they are likely to continue to be important in the future. PMID- 1712707 TI - Rational approaches to the treatment of culture-negative infective endocarditis. AB - The microorganism responsible for infective endocarditis may not be grown on blood culture in as many as 25% of cases. While this is to be expected with such relatively uncommon organisms as Coxiella burnetti, in most cases failure to grow the organism is likely to be due to either a low concentration of bacteria in the blood or because antibiotics were given before blood was taken for culture. The antibiotic treatment of culture-negative cases should be based on the assumption that the organisms responsible are the same as those found in cases with positive cultures, covering the most likely possibilities in such different circumstances as spontaneous infections of natural valves, endocarditis following cardiac surgery, early and late prosthetic valve endocarditis and infections associated with intravenous drug abuse. PMID- 1712709 TI - Azelaic acid. A review of its pharmacological properties and therapeutic efficacy in acne and hyperpigmentary skin disorders. AB - Azelaic acid is a naturally occurring saturated dicarboxylic acid which, on topical application (usually as a 20% cream), has been shown to be effective in the treatment of comedonal acne and inflammatory (papulopustular, nodular and nodulocystic) acne, as well as various cutaneous hyperpigmentary disorders characterised by hyperactive/abnormal melanocyte function, including melasma and, possibly, lentigo maligna. In addition, azelaic acid has an antiproliferative and cytotoxic effect on the human malignant melanocyte, and preliminary findings indicate that it may arrest the progression of cutaneous malignant melanoma. The mechanism of this selective cytotoxic action of azelaic acid is unclear, but may possibly be related to its inhibition of mitochondrial oxidoreductase activity and DNA synthesis. In controlled studies, topical azelaic acid demonstrated comparable anti-acne efficacy to topical tretinoin, benzoyl peroxide, erythromycin and oral tetracycline, while in patients with melasma azelaic acid proved at least as effective as topical hydroquinone. On topical application azelaic acid is well tolerated, with adverse effects apparently limited to a generally mild and transient local cutaneous irritation. Thus, topical azelaic acid, employed either as monotherapy or in combination with other treatments, is likely to prove of value in the management of acne and several hyperpigmentary disorders, most notably melasma. PMID- 1712710 TI - Cilazapril. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in cardiovascular disease. AB - Cilazapril is an orally active angiotensin converting enzyme (ACE) inhibitor which lowers peripheral vascular resistance without affecting heart rate. Like enalapril and ramipril it is a prodrug, and is hydrolysed after absorption to cilazaprilat, which has a long terminal phase elimination half-life permitting once daily administration. Given once daily at doses between 2.5 and 5 mg, cilazapril reduces arterial blood pressure in patients with mild to moderate essential and renal hypertension. Patients who do not respond adequately to cilazapril monotherapy usually respond with the addition of a diuretic such as hydrochlorothiazide. Preliminary data suggest that cilazapril is of comparable antihypertensive efficacy to usual therapeutic dosages of hydrochlorothiazide, slow release propranolol, nitrendipine, captopril and enalapril. In small studies cilazapril has produced sustained beneficial haemodynamic effects in patients with congestive heart failure. Cilazapril has been well tolerated and exhibits tolerability typical of ACE inhibitors as a class, including their lack of detrimental effect on glucose or lipid metabolism. Cilazapril should provide an effective alternative in the treatment of hypertension and, if preliminary data are confirmed, in congestive heart failure. PMID- 1712711 TI - How do loop diuretics act? AB - In the thick ascending limb of the loop of Henle, NaCl reabsorption is mediated by a Na+/2Cl-/K+ cotransport system, present in the luminal membrane of this nephron segment. Loop diuretics such as furosemide (frusemide), piretanide, bumetanide and torasemide bind reversibly to this carrier protein, thus reducing or abolishing NaCl reabsorption. This leads to a decrease in interstitial hypertonicity and thus to a reduced water reabsorption. In nephron segments other than the thick ascending limb, loop diuretics have no quantitative importance with respect to their saluretic and diuretic activities. Loop diuretics also reduce Ca++ and Mg++ reabsorption in the thick ascending limb in a way which is still not clear. Furthermore, these drugs increase the urinary K+ excretion by enhancing distal tubular K+ secretion and reducing K+ reabsorption in the loop of Henle. Finally, by reduction of active NaCl transport, loop diuretics drastically reduce the substrate requirement and oxygen dependence of the thick ascending limb cells. This renders these cells, which are characterised by high transport rates and only limited substrate reserves, less vulnerable in acute renal failure. PMID- 1712708 TI - Sustained release nifedipine formulations. An appraisal of their current uses and prospective roles in the treatment of hypertension, ischaemic heart disease and peripheral vascular disorders. AB - Nifedipine antagonises influx of calcium through cell membrane slow channels, and sustained release formulations of the calcium channel blocker have been shown to be effective in the treatment of mild to moderate hypertension and both stable and variant angina pectoris. Preliminary findings also indicate that these formulations are effective in the treatment of Raynaud's phenomenon and hypertension in pregnancy, and that they reduce the frequency of ischaemic episodes in some patients with silent myocardial ischaemia. The exact mechanism of action of nifedipine in all of these disorders has not been defined. However, its potent peripheral and coronary arterial dilator properties, together with improvements in oxygen supply/demand, are of particular importance. A major goal of sustained release therapy is to permit reductions in the frequency of nifedipine administration, preferably to once daily, and thus improve patient compliance. Two new once-daily formulations--the nifedipine gastrointestinal therapeutic system (GITS) and a fixed combination capsule comprising sustained release nifedipine 20 mg and atenolol 50 mg--have exhibited marked antihypertensive efficacy. The GITS preparation has also been used effectively in the treatment of stable angina pectoris, and both formulations appear to be well tolerated. Sustained release nifedipine formulations are generally better tolerated than their conventionally formulated counterparts, particularly with regard to reflex tachycardia. Adverse effects seem to be dose related, are mainly associated with the drug's potent vasodilatory action, and include headache, flushing and dizziness. Generally, these effects are mild to moderate in severity and transient, usually diminishing with continued treatment. Thus, sustained release nifedipine formulations are useful and established cardiovascular therapeutic agents which have demonstrable efficacy in various forms of angina, mild to moderate hypertension and Raynaud's phenomenon. Further, promising results shown by the nifedipine GITS formulation, with its advantage of once daily administration suggest that it is likely to become one of the preferred nifedipine formulations for the treatment of hypertension and the various forms of angina. PMID- 1712712 TI - Clinical pharmacology of loop diuretics. AB - The clinical pharmacology of torasemide, bumetanide, piretanide and furosemide (frusemide) is discussed. These drugs share a similar mechanism of action in inhibiting Na(+)-K(+)-2Cl- reabsorption at the thick ascending limb of the loop of Henle. They differ in their routes of metabolism, pharmacokinetics, and potency. Whether such differences are clinically important requires further study. Bumetanide and torasemide are metabolised by cytochrome P450 pathways, whereas furosemide is glucuronidated. These different routes of metabolism may have clinically important implications. Bumetanide, furosemide, and piretanide have similar pharmacokinetics, whereas the clearance of torasemide is less and the half-life concomitantly longer than the other 3 agents. Thus, torasemide has a longer duration of action. The rank order of potency is bumetanide greater than piretanide identical to torasemide greater than furosemide, although efficacy appears the same. Despite much being known about these diuretics, many clinically important questions remain. PMID- 1712713 TI - The loop diuretic torasemide in chronic renal failure. Pharmacokinetics and pharmacodynamics. AB - The pharmacodynamic effect of a diuretic agent is essentially dependent on its renal elimination characteristics. The influence of renal function on the pharmacodynamic and pharmacokinetic characteristics of a diuretic should therefore be considered. The results of a study with torasemide given intravenously in healthy subjects and in patients with stable chronic renal failure of various degrees are reported and discussed. After a single dose of torasemide 20mg, a marked diuresis was observed and electrolyte excretion was increased, whereas the glomerular filtration rate was unchanged. Throughout the duration of action (tau) of torasemide, drug-induced excretion of Cl-, Na+, K+, Ca++ and Mg++ was linearly related to the creatinine clearance (CLcr), and excretion of K+ and Ca++ were closely related to that of Na+ over the entire range of CLcr. Similarly, excretion of Mg++ was related to that of K+. As occurs with furosemide (frusemide), torasemide induced a kaliuresis which amounted to 12% of natriuresis. This kaliuretic effect of loop diuretics is less than that of thiazides. Beyond tau, e.g. over a 24-hour period, kaliuresis was no longer correlated with natriuresis. The extent to which a rebound effect occurred was diminished with increasing renal impairment. tau averaged 6 hours and was independent of CLcr. The mean half-life (t1/2) of torasemide was approximately 5 hours and was independent of renal function, since renal clearance accounted for only around 25% of total body clearance. In contrast to the parent drug, however, the active minor metabolite M1 and the inactive main metabolite, M5, were found to accumulate in patients with chronic renal failure. PMID- 1712714 TI - Effects of diuretics on outputs and flows of urine and urinary solutes in healthy subjects. AB - The effects of single oral doses of common formulations of diuretics (i.e. formulations on the market or designed to be marketed) on 24-hour diuresis and natriuresis in healthy subjects are considered as a measure of the renal excretory potency of diuretics. Common formulations of distal tubular diuretics (e.g. hydrochlorothiazide 25mg, xipamide 10mg and 20mg) are more potent diuretics and natriuretics than common formulations of loop diuretics [e.g. furosemide (frusemide) 40mg, torasemide 2.5, 5 and 10mg]. Indeed, some common formulations of loop diuretics, such as torasemide 2.5, do not increase 24-hour diuresis or natriuresis in healthy subjects. 24-hour kaliuresis and magnesiuresis are elevated by common formulations of distal tubular diuretics, but they are only slightly increased or (more usually) not affected by common formulations of loop diuretics, when single doses are administered to healthy individuals. Common formulations of loop diuretics have lower diuretic and natriuretic potency and lower kaliuretic and magnesiuretic effects than common formulations of distal tubular diuretics, because the pronounced elevations in urinary excretions caused by loop diuretics during the first 6 hours after dosing are followed by rebounds, with respect to post-placebo excretions, between 6 and 24 hours after dosing. These rebounds, which affect the urinary flows of fluid, chloride, sodium, potassium and magnesium, do not occur after administration of common formulations of distal tubular diuretics, at least during the first 24 hours after administration of single doses to healthy subjects. The time courses of urinary excretions after loop diuretics are dose dependent. Higher doses produce more rapid changes in the urinary flows of fluid, chloride, sodium, potassium and magnesium than lower doses, to the extent that single administration of torasemide 2.5 or 5mg to healthy subjects is followed by urinary fluid and solute flows whose time courses resemble those after administration of hydrochlorothiazide 25mg. PMID- 1712715 TI - Acute and long term effects of loop diuretics in heart failure. AB - Diuretics, together with digitalis glycosides and vasodilators are of prime importance in the medical treatment of patients with congestive heart failure (CHF). Diuretics provide quick symptomatic relief in these patients. Their beneficial effect is related to the promotion of sodium and water excretion via the kidney, thus reducing extracellular fluid volume expansion and mitigating the increase in preload and afterload caused by sodium and water retention. Loop diuretics administered intravenously are indispensable in the management of pulmonary oedema; thiazides and loop diuretics in low doses are effectively used in the oral treatment of mild to moderate heart failure. Torasemide is a new loop diuretic which differs from furosemide (frusemide) and related loop diuretics by virtue of its longer elimination half-life and longer duration of action, with almost complete bioavailability. The efficacy and tolerability of torasemide have been compared with furosemide in several studies. Once daily oral administration of torasemide (starting with 5mg) or furosemide 40mg reduce bodyweight, oedema and symptoms of heart failure to a similar extent. Mean New York Heart Association class is consistently reduced by 0.5 to 0.7. Intravenous administration attenuates the increase in intracardiac pressures during exercise in patients with CHF, and produces acute improvements in cardiac haemodynamics in patients with high grade left heart failure. A beneficial effect on both pulmonary and cardiac haemodynamics has been demonstrated during chronic oral treatment of patients with previously untreated CHF. Torasemide was well tolerated with only mild and transient adverse effects reported in a small number of patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712716 TI - The efficacy of diuretics in acute and chronic renal failure. Focus on torasemide. AB - Loop diuretics in high doses are the drugs of choice in the treatment of both acute renal failure (ARF) and chronic renal failure (CRF). Their pharmacokinetic and pharmacodynamic properties give them a high efficacy, even in severely compromised renal function. The serum elimination half-life and duration of action of most loop diuretics are dependent on the glomerular filtration rate and are therefore prolonged in renal failure. Torasemide, a new high ceiling and long acting loop diuretic, is as potent as furosemide (frusemide) in patients with advanced renal failure. Unlike other loop diuretics, the half-life and duration of action of torasemide are not dependent on renal function and the parent drug does not accumulate in renal failure. The extent of metabolism is clinically negligible. A number of studies have demonstrated the efficacy of furosemide, bumetanide, piretanide and torasemide in patients with ARF and CRF. When compared with the other loop diuretics, torasemide has the following advantages: a longer half-life independent of renal function, no indications of toxic side effects and apparently less influence on calciuresis. PMID- 1712717 TI - Low dose loop diuretics in essential hypertension. Experience with torasemide. AB - Diuretics belong to the class of antihypertensive drugs recommended for first line therapy of essential hypertension. Although they are widely and effectively used for the treatment of hypertension, the question remains whether their possible negative influence on metabolic and electrolyte parameters could partly offset the benefit of blood pressure reduction with respect to reduction of coronary artery disease. Recently published data demonstrate that much lower doses of thiazides exert the same antihypertensive effect as the higher doses used in the past and even prescribed today. These lower doses produce relatively little change in biochemical parameters. Thus, the postulated risks can be avoided by using the lowest effective dose. Traditionally, loop diuretics of the furosemide (frusemide) type are rarely used as first-line antihypertensive agents. They seem to display less efficacy coupled with an intense diuresis when used in standard available doses. However, there is evidence that newly developed loop diuretics, in lower doses than used in congestive heart failure, are effective antihypertensive agents. For example, torasemide 2.5mg once daily, which does not exert a significant diuresis over 24 hours compared with placebo, lowers elevated blood pressure to a similar extent than thiazides or related compounds. This antihypertensive effect is accompanied by less, if any, changes in metabolic or electrolyte parameters compared with widely used standard diuretics such as hydrochlorothiazide, indapamide or chlorthalidone. The influence on serum potassium and magnesium is similar to or even less than fixed combinations of hydrochlorothiazide and triamterene or amiloride. Thus, low-dose torasemide constitutes an alternative to established thiazide antihypertensive therapy. PMID- 1712718 TI - In vitro hypothalamic neurogenesis: morphological maturation of mouse hypothalamic cultures and in vitro versus in situ biochemical analysis. AB - The morphological and biochemical changes that occur during in vitro neurogenesis of the mouse hypothalamus were studied in rotary cultures prepared from mice between 4 and 16 days of postnatal age. After 6 days of in vitro growth, histotypic cultures with a high degree of morphological differentiation were obtained in cultures prepared from 8- to 10-day-old mice. Before day 8, the cultures showed immature neurons, while after day 12 most of them exhibited an undesired number of degenerated cells. Light-microscopic, Golgi and ultrastructural studies clearly showed the stages of development of the neurosecretory cells in culture. The particular organization of the hypothalamic cells in these cultures can be homologized to its equivalent region in vivo, as demonstrated by their morphological similarities as well as by the fact that the majority of the neurons orient their axons toward the external part of the culture in order to release the neurosecretory material outside the in vitro grown neuronal population, as is the case in situ since hypothalamic neurons release their neurosecretory products at the vascular system. Biochemical parameters such as DNA, RNA and protein contents were determined during the period of in situ development used for the preparation of histotypic cultures and compared to the biochemical changes that occurred during in vitro maturation. The changes in the in vitro DNA and protein contents showed the same variation pattern as in situ. The DNA/protein and RNA/protein ratios also had comparable characteristics, having peak values at days 10 and 16 in situ and in the histotypic cultures prepared from 10-day-old mice. These studies have demonstrated the correlation between the in vitro biochemical and morphological development and the significance of the critical period during hypothalamic neurogenesis for successful organotypic preparations. PMID- 1712719 TI - Degrees of cooperativity between triiodothyronine and hydrocortisone in their regulation of the expression of myelin basic protein and proteolipid protein during brain development. AB - Cultures of cells dissociated from embryonic mouse cerebra were used to demonstrate: (1) that the developmental expression of the mRNA of proteolipid protein is dependent on thyroid hormone; (2) that the expression of the mRNA of proteolipid protein is stimulated not only by triiodothyronine but also by hydrocortisone, which achieve their respective stimulations by an additive and uncompetitive mechanism; (3) the stimulation of the net accumulation of the mRNA of myelin basic protein by hydrocortisone and triiodothyronine is also cooperative, additive, and uncompetitive, and (4) the stimulation of the net accumulation of myelin basic protein, during development by hydrocortisone, is completely dependent on the presence of thyroid hormone. These results suggest that the regulation of the synthesis of myelin basic protein by hydrocortisone requires the presence of triiodothyronine at a posttranscriptional event, but not for transcription itself. PMID- 1712720 TI - Quantitative differences between homozygous 'USA' and 'Swiss' mld mutant mice. AB - Parallel developmental studies of central nervous system myelin proteins and morphology (postnatal days 15-118; P15-118) confirm qualitative similarities but substantial quantitative differences between homozygous mld mice with Billings Gagliardi and Wolf's 'USA' versus Matthieu's 'Swiss' genetic backgrounds. The USA mld/mld have fewer convulsions and significantly longer life span. While whole brain homogenates from both Swiss and USA mld/mld show increases in myelin basic protein (MBP) and in 2',3'-cyclic nucleotide 3'-phosphohydrolase specific activity with age, at P50 and older the levels of both proteins are approximately twice as high in the Swiss. The number of optic nerve axons myelinated is always greater in Swiss mld/mld, and they have approximately twice as many myelin sheaths showing any apposition of cytoplasmic membrane faces (the location of the major dense line in normal myelin), except at the youngest age. Evidence is presented which suggests that these quantitative differences between Swiss and USA mld stocks most likely reflect different regulatory genes influencing the expression of the same (mld) allele, rather than the presence of a different allele at the MBP locus. PMID- 1712721 TI - Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system. AB - The Ferguson plot in polyacrylamide gel electrophoresis (PAGE)(15%CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4 degrees C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3% T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest % T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3% T have not as yet been measured. As previously suggested, the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched, "reptating" DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5% T when 15% DATD rather than 2.5% Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4 degrees C to 25 and 50 degrees C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a "reptating" to a randomly distributed molecule, due to increased Joule heat.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712722 TI - Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele-specific restriction fragment length polymorphism. AB - A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory. PMID- 1712723 TI - Two-dimensional electrophoresis in small gels for applications in veterinary medicine. AB - A versatile method for two-dimensional electrophoresis that can be performed easily, even in small routine laboratories, is described. The procedure combines a first-dimensional isoelectric focusing run in a PhastSystem with denaturing electrophoresis in a small vertical electrophoresis chamber. The described arrangement of two first-dimensional gel strips in the second dimension allows direct comparison of two related samples, eliminating most of the artifacts that usually lead to misinterpretations. The presented procedure can be conveniently adapted to the needs of each laboratory; at present, it is being extensively used to establish protein patterns of healthy individuals of different species, a prerequisite with regard to help and support diagnosis. PMID- 1712724 TI - Structural basis of electrophoretic variants of rat alpha-fetoprotein. AB - The molecular basis for electrophoretic variations of rat alpha-fetoprotein was studied. Stepwise deglycosylations of proteins and radiolabeling of the sugars indicated that the number of such chains per molecule is two and one for the slow and fast variants, respectively. PMID- 1712725 TI - Computer analysis of DNA and protein sequences. AB - Some recent trends in the development of theoretical methods for DNA and protein sequence analysis are reviewed, with particular emphasis on the design of new databases, motif searches, sequence alignment algorithms and applications of neural networks. PMID- 1712726 TI - Cell-cycle-regulated expression of thymosin beta 4 in thymocytes. AB - Thymosin beta 4 belongs to a family of ubiquitous peptides present at a high cellular content but still with an unknown intracellular function. The expression of this peptide was studied in concanavalin-A-stimulated, proliferating rat thymocytes during cell cycle progression. An early, transient 10-fold increase of the peptide occurred 1 h after stimulation without elevation of the corresponding mRNA level. This increase coincided with that of thymosin beta 4 biosynthesis. The sharp decline of the thymosin beta 4 content was not due to a secretion of the peptide into the medium. During S phase and mitosis, the biosynthetic rates as well as mRNA content, but not the cellular thymosin beta 4 concentration, increased again. After 96 h of culture the values returned to those of quiescent cells. PMID- 1712727 TI - Enhancement of vitronectin expression in human HepG2 hepatoma cells by transforming growth factor-beta 1. AB - Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the regulation of vitronectin by transforming growth factor-beta 1 (TGF beta 1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGF beta-regulated gene, plasminogen activator inhibitor (PAI 1). Rabbit antibodies raised against human plasma-derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGF beta 1-induced production and extracellular deposition of vitronectin. Accordingly, TGF beta 1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2-20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGF beta induction profiles of vitronectin and PAI-1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet-derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGF beta 1 enhanced the expression of PAI-1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI 1 in these cells. The results indicate that TGF beta 1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI-1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the alpha subunit of the receptor in response to TGF beta 1. These effects of TGF beta are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair. PMID- 1712728 TI - Isolation of chick tenascin variants and fragments. A C-terminal heparin-binding fragment produced by cleavage of the extra domain from the largest subunit splicing variant. AB - The extracellular-matrix glycoprotein, tenascin, consists of disulfide-linked subunits of 190, 200 and 230 kDa (the three splicing variants reported in chicken) and usually exists as a six-armed structure under the electron microscope. We used monoclonal antibodies to isolate and characterize different splicing variants and proteolytic fragments obtained from the native protein. Purified monomeric tenascin has a native molecular mass of 216 kDa and is structured as single arms. Tenascin fragments obtained by pepsin digestion bind to monoclonal antibody (mAb) TnM1 which is directed against epidermal-growth factor-like repeats in the N-terminal half of all subunits. These fragments represent the thin proximal part of the tenascin arms and they are still partially linked to dimers and trimers via disulfide bridges. Using mAb Tn68, that reacts with a fibronectin-type-III repeat towards the C-terminus, a tenascin fragment, generated by treatment with pronase, can be isolated. Ultrastructurally, this fragment looks like the thicker distal part of the tenascin arms. Only the 230-kDa variant of tenascin gives rise to this distal fragment after cleavage within the alternatively spliced fibronectin-type-III repeats. Native tenascin and all fragments containing the distal part of its arms bind to heparin-agarose, whereas the proximal fragments do not. Oligomeric and monomeric tenascin inhibit fibronectin-mediated fibroblast spreading with comparable efficiency when added to the culture medium, while the proximal fragment has no effect. The distal fragment as well as reduced and alkylated tenascin are active in this assay, but only at higher molar concentrations when compared to the native protein. PMID- 1712729 TI - Expression of post-translational processing of preprocecropin A using a baculovirus vector. AB - A cDNA fragment encoding preprocecropin A was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in recombinant-infected last instar larvae of Trichoplusia ni and in diapausing pupae of Hyalophora cecropia. The identity of the recombinant product was established by electrophoresis with detection of antibacterial activity and mass spectrometry. The prepropeptide had been correctly processed including removal of signal peptide and pro-part. Biologically active and amidated cecropin A was exported to the hemolymph. The yield of recombinant protein in H. cecropia reached a level of 600 micrograms/ml hemolymph and about 70% of the material was amidated. PMID- 1712731 TI - Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osteoclasts. AB - Studies with a range of monoclonal and polyclonal antisera to components of the human, rat, and chick vitronectin receptor, alpha V beta 3, and the VLA beta 1 chain show that chick and rat osteoclasts express similar integrin receptors to those described in man. Biochemical analysis with monoclonal antibody 23C6 confirmed the presence on chick osteoclasts of a vitronectin receptor heterodimer of similar size (110/95 kDa reduced) to that immunoprecipitated from human osteoclastoma giant cells. The synthetic peptide GRGDSP, corresponding to the cell adhesion sequence in fibronectin, but not GRGESP peptide, induced significant (P less than 0.005) osteoclast retraction in chick and rat osteoclasts at IC50s (+/- SEM) of 210.0 +/- 14.4 and 191.4 +/- 13.7 microM, respectively; monoclonal anti-vitronectin receptor alpha V beta 3 complex antibody, 23C6, produced similar changes in chick osteoclasts (IC50 = 1.45 +/- 0.22 microM). Antibody 23C6 inhibited the number of pits resorbed in dentine by chick osteoclasts over a concentration range of 4.4 to 88 micrograms/ml; a significant 76% reduction (P = 0.03) was observed at a final concentration of 88 micrograms/ml (6 microM). The effect of peptides upon dentine resorption was less dramatic. No consistent inhibition was seen using chick osteoclasts. Inhibitory effects on resorption by rat osteoclasts were, however, observed; significant reduction in resorption occurred with both GRGDSP (78%; P less than 0.01) and GRGESP (67%; P = 0.02) peptides at 400 microM peptide concentration. These data demonstrate that osteoclast function can be disrupted by low concentrations of the anti-vitronectin receptor antibody, 23C6. The inhibitory effects of the peptides used in this study produced effects on dentine resorption which were generally weaker and variable, although osteoclast cell adhesion was consistently inhibited in an Arg-Gly-Asp (RGD)-dependent manner. We conclude that the vitronectin receptor may play an important role in effecting resorption of mineralized tissues by osteoclasts. PMID- 1712730 TI - Taxol protects the microtubules of concanavalin A-activated lymphocytes from disassembly by methylmercury, but DNA synthesis is still inhibited. AB - We have shown previously that there is a good correlation between the degree of microtubule disassembly by methylmercury (MeHg) and the extent of inhibition of DNA replication in Concanavalin A (Con A)-stimulated mouse splenic lymphocytes. The purpose of this study was to determine if these two events are causally related and to examine the effects of MeHg-induced microtubule disassembly on earlier events of the stimulation process. We show that early steps constituting the activation pathway, such as the Con A-induced increase in Ca2+ influx and the expression of interleukin 2 receptor, are not inhibited by concentrations of MeHg that disassemble microtubules. RNA synthesis is not affected by short-term (3 h) treatment with MeHg, but longer treatment (24 h) inhibits RNA synthesis. In contrast, DNA synthesis is effectively inhibited by a 3-h treatment with MeHg. In lymphocytes treated with taxol, microtubules are not disassembled by MeHg; however, the inhibition of RNA and DNA synthesis persists. We conclude that the inhibition of nucleic acid synthesis by MeHg is not causally related to MeHg induced microtubule disassembly. PMID- 1712732 TI - Possible role of hyaluronectin on cell adhesion in rat histiocytoma. AB - Distribution of hyaluronectin, a 68-kDa cell surface glycoprotein, has been demonstrated in normal peritoneal, alveolar macrophages as well as in macrophages of the AK-5 tumor cell line. AK-5, a transplantable histiocytic tumor cell line, is a mixture of four different populations and can be grown in both ascites and solid tumors. We are able to demonstrate a differential expression of hyaluronectin on the cell surface of these subpopulations of AK-5 when studied by immunocytochemical staining followed by cytofluorometric analysis. Cell fractions responsible for developing both ascites and solid tumors contain higher amounts of hyaluronectin than fractions which are capable of producing only ascites, suggesting its involvement in solid tumor formation. Furthermore, we established a secretory nature of hyaluronectin as it can be detected in the serum-free medium of AK-5 cells. Since it is localized on the cell surface and secreted into the medium, the cell adhesiveness of hyaluronectin has been examined. Hyaluronectin coating on the plates allowed more cells to attach, which could be specifically blocked by the antibody raised against hyaluronectin, indicating its possible role in cell attachment. The adhesive property of hyaluronectin and its role in tumor formation was further confirmed. The pretreatment of AK-5 cells with hyaluronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascites tumor. We discuss our observations of hyaluronectin as a cell attachment protein and its specific role on tumor formation. PMID- 1712733 TI - Correlations between hyaluronan and epidermal proliferation as studied by [3H]glucosamine and [3H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes. AB - The rates of keratinocyte proliferation and synthesis of Hyaluronan (HA) were studied in human whole-skin organ culture by labeling with [6-3H]glucosamine and [3H]thymidine, respectively, to reveal possible correlations between the two functions of the cell. HA distribution in epidermis was examined by staining with a specific probe prepared from cartilage proteoglycan. The keratinocyte proliferation rate was low on the first 2 culture days, but showed a tenfold increase on the third and fourth days while the synthesis of HA proceeded at a relatively stable level throughout the same period. The most intensive staining of HA occurred in the uppermost spinous cell layer, whereas mitotic cells resided in the basal and suprabasal layers. The keratinocytes under various stages of mitosis were surrounded by a HA staining not more intense than that around nondividing basal cells, but a thick pad of HA appeared rapidly between the daughter cells. These findings suggest that newly synthesized HA is associated with the separation of keratinocytes following mitosis but the majority of the synthesis and content of HA in epidermis is involved in other keratinocyte activities such as maintenance of the extracellular space and cell--cell interactions during migration and differentiation. PMID- 1712734 TI - Multiple integrins share the ability to induce elevation of intracellular pH. AB - Previous work has shown that adhesion of anchorage-dependent cells to fibronectin via integrin alpha 5 beta 1 leads to activation of the Na-H antiporter and a rise in intracellular pH (pHi). We now show that adhesion of bovine capillary endothelial cells (BCE) to fibrinogen; collagens type III, IV, and V; laminin; and vitronectin; ligands that bind other members of the integrin family, resulted in significant elevations in pHi. Other ligands (basic fibroblast growth factor, concanavalin A, and thrombin), which bind cells when immobilized on plastic, but that do not bind integrins and do not support cell growth, do not elevate pHi. Adhesion to an antibody against integrin alpha v beta 3 also elevates pHi. Adhesion of peripheral human T lymphocytes to an antibody against the integrin LFA-1 induced a rise in pHi. Antibodies to CD2 or ICAM-2 had only slight effects on pHi, whereas an antibody to the T cell receptor complex that strongly activates T cells induced a large increase in pHi. We conclude that elevation of pHi by integrins is specific and is a property shared by many members of the integrin family. PMID- 1712735 TI - Pharmacodynamics and pharmacokinetics after subcutaneous and intramuscular injection of human chorionic gonadotropin. AB - OBJECTIVE: The pharmacokinetics and efficiency of human chorionic gonadotropin (hCG) after subcutaneous (SC) injection was to clarify in comparison with the intramuscular (IM) mode of administration. DESIGN: In a prospective study, the pharmacokinetics of hCG and the response of serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) after an IM and SC injection of 5,000 IU hCG were evaluated up to 144 hours in two randomized groups. SETTING: The study was carried out in a clinical dermatology department providing tertiary care. PARTICIPANTS: Twenty-four healthy male volunteers with a mean age of 22.7 +/- 4.3 years were divided into two groups. INTERVENTIONS: Human chorionic gonadotropin (5,000 IU) was injected IM or SC. MAIN OUTCOME MEASURE: Serum concentration of /b-hCG, T, LH, and FSH were evaluated after IM and SC administration of hCG. Differences between the two groups were determined by t test. RESULTS: Compared with IM administration of hCG, peak serum drug concentration was significantly delayed (P = 0.01) and serum half-life was prolonged (P = 0.01) after SC injection; however, T, LH, and FSH responses were identical. CONCLUSIONS: Subcutaneous application of 5,000 IU hCG is as effective as IM administration in terms of steroidogenesis. PMID- 1712736 TI - Neovascular growth factors. AB - Neovascularisation is the biological process of forming new blood vessels. Many conditions can initiate neovascularisation including trauma or chronic ischaemia produced by diseases such as diabetes. Neovascularisation proceeds through a series of steps beginning with destruction of the basement membrane surrounding the microvascular endothelial cells, which allows endothelial cells to extend cytoplasmic buds in the direction of chemotactic factors. Migrating endothelial cells elongate, divide and eventually form tube structures which join to form mature new capillaries. Results of in vitro experiments, in vivo experiments, and clinical studies suggest that peptide growth factors can play key regulatory roles in each step of neovascularisation through both direct and indirect actions. At sites of vascular injuries, degranulating platelets release PDGF, IGF I, EGF, and TGF-beta. Macrophages and neutrophiles drawn into the ischaemic or injured areas synthesise and release TGF-alpha, TGF-beta, and PDGF, and wounded endothelial cells secrete FGF. These peptide growth factors can stimulate migration, mitosis and differentiation of endothelial cells in culture and can induce neovascularisation in animal models. Clinical correlations suggest that peptide growth factors in the vitreous such as IGF-I and bFGF may promote diabetic retinopathy. As the biological mechanisms of neovascular growth factors become better understood, it may be possible to develop therapeutic approaches to selectively inhibit the peptide growth factors which regulate neovascular diseases. PMID- 1712738 TI - Microsurgical management of neovascularisation secondary to posterior segment ischaemia. AB - Structural, metabolic and functional rehabilitation of eyes affected by the haemorrhagic and tractional sequelae of neovascularisation secondary to posterior segment ischaemia is discussed. Microsurgical management must pay due attention not only to the mechanical but also to the underlying cell-biological implications of the surgical pathology. The appropriateness of case selection for surgery rests upon the likelihood of successful technical and functional outcomes and also upon the overall visual status of the patient, issues of particular relevance to diabetes. PMID- 1712737 TI - Assessment of the eye at risk of neovascularisation. PMID- 1712740 TI - [Communicating effectively: an art or a technique?]. PMID- 1712739 TI - Mathematical model of antiviral immune response regulation. I. Conceptual description of the modelled processes. AB - The report covers the initial steps in construction of a mathematical model, namely a conceptual description of antiviral immune response regulation. The model describes cellular and molecular levels of the basic mechanisms: interaction of virus with a sensitive cell; action and activation of nonspecific resistance factors (phagocytosis, antiviral action of interferon, humoral inhibitors, and natural killer cells in the course of the immune response; humoral and cellular immune response induction and the specific antiviral defence mechanisms. Two helper cell subpopulations are considered explicitly: cellular immune response T helper cells, also named inflammatory cells (TH1) and humoral immune response T helper cells (TH2). Regulation of interleukin 1-, 2- and 4 induced lymphocyte progression through cell cycle phases is described. PMID- 1712741 TI - Galanin: distribution and effect on contractile activity and release of vasoactive intestinal polypeptide from the isolated perfused porcine ileum. AB - In the pig ileum galanin (GAL)-like immunoreactivity was identified in nerve cell bodies of the submucous plexus and in nerve fibers of the circular and longitudinal muscle layer. Infusion of 5.10(-10)-10(-8) M of GAL into the arterial line of the isolated perfused porcine ileum decreased the frequency of spontaneous phasic contractions in a dose-dependent manner. The frequency of phasic contractions during maximal inhibition by GAL 10(-8) M was 13 +/- 4% (mean +/- SE) of basal frequency (p less than 0.05). The recovery from inhibition by GAL 10(-8) M lasted 16 +/- 1 min. Tonic contractions were not observed in this experimental set-up, neither by standard perfused catheter manometry nor by measurement of cross-sectional area of an intraluminally located balloon. Infusion of GAL 10(-8) M decreased the venous release of vasoactive intestinal polypeptide to 80 +/- 8% of basal release (p less than 0.05). It is concluded that GAL may participate in the regulation of small intestinal motility in the pig. PMID- 1712742 TI - [Hepatoid adenocarcinoma of the stomach]. AB - One year after gastric resection for cancer, a 67 year old patient was hospitalized because of a large hepatic tumor with extremely high serum alpha foetoprotein levels (13,245 ng/ml). Histological and immunohistochemical studies of the gastric tumor revealed hepatoid foci with alpha-foetoprotein and protease inhibitor-producing cells. Histopathological and immunohistochemical features of this and the 8 previously reported cases of hepatoid adenocarcinoma of the stomach are analyzed. PMID- 1712743 TI - Bleomycin-detectable iron in brain tissue. AB - The normal brain contains regions with high concentrations of iron, part of which appears to be in a low molecular mass chelatable form. Iron complexes with a molecular mass of below 10,000, were measured in ultrafiltrates of homogenized gerbil brains using the bleomycin assay, and were found to average 20.5 +/- 3.5 microM (n = 8). As expected, no bleomycin detectable iron was found in the plasma of these animals. No obvious difference in the tissue levels of bleomycin detectable iron was recorded following ischaemia and reperfusion. This is probably due to the already abundant presence of iron in the brain and the likely release of iron from protected sites due to structural damage inherent in the preparative procedures used. PMID- 1712744 TI - [Hemisphere dominance of cortical speech processing and its dynamics in the course of aphasia therapy. A psychophysiologic contribution to the discussion of the neuronal plasticity hypothesis]. AB - Examinations of aphasic patients by using cognitive tasks were based on the hypothesis that semantically evoked potentials correlate to the processing of information in the cortical areas of Broca and Wernicke. Some of the examined right-handed patients with ischemic lesions of the left hemisphere produced characteristic potentials in the right temporal lobe, and not in the dominant left lobe as was expected. These case histories suggest that in these patients speech processing moved into the subdominant hemisphere as a result of compensation after cerebral damage by using the faculty of neuroplasticity. Furthermore an extended classification of aphasias is presented, illustrated by a three-parameter model. PMID- 1712745 TI - [Amiodarone for long-term treatment of ventricular arrhythmias]. AB - As one of the causes of sudden cardiac death, recurrent ventricular arrhythmia represents a potentially life-threatening rhythm disorder. amiodarone is an effective anti-arrhythmic drug, the effectiveness of which is based on a prolongation of the action potential and thus a lengthening of the refractory period. In a multicenter, noncontrolled observational study of 482 patients aged between 21 and 89, it was found that amiodarone effectively suppressed life threatening or crippling arrhythmias in a majority of the patients, even when they had previously been unresponsive to other anti-arrhythmic agents. Physical performance improved under the treatment. An analysis of the side effects observed revealed good tolerance at a low maintenance dose. PMID- 1712746 TI - Tachykinins in the rat substantia nigra: effects of selective dopamine receptor antagonists. AB - The effects of sustained blockade of dopamine receptors by selective dopamine antagonists on the tachykinin (substance P and neurokinin A) content in the substantia nigra were examined. The treatment of rats for 14 days with D-1/D-2 dopamine receptor antagonist haloperidol (2 mg/kg) or selective D-2 antagonist sulpiride (100 mg/kg) produced a similar and significant decrease in nigral substance P and neurokinin A-like immunoreactivity content, about 32-36% and 27 28% of control respectively. In contrast, administration of SCH 23390 (1 mg/kg), a selective and potent D-1 dopamine receptor antagonist, failed to affect the levels of substance P and neurokinin A in the substantia nigra and did not change the sulpiride-induced reduction of the nigral tachykinin peptides. These results indicate that the D-1 dopamine receptors are not involved in the modulation of nigral substance P and neurokinin A content and suggest that the blockade of the D-2 dopamine receptor subtype exerts the same regulation of the tachykinin gene expression, in spite of the existence of three mRNAs encoding substance P and neurokinin A. PMID- 1712748 TI - Microcarcinoma in the prostate: its association with duct-acinar dysplasia. AB - In a series of 100 prostatectomy specimens obtained for adenocarcinoma, 107 additional incidental microscopic (less than 0.05 cm3) carcinomas were identified. Their morphologic features including location, histologic grade, and associated premalignant changes were documented. In 51 cases there was strong evidence of transition between microcarcinoma and the premalignant lesion, duct acinar dysplasia. Invasive cancer was usually related to dysplasia through a characteristic intermediate morphologic stage of transitive glands. These glands were smaller than prostatic ducts; they appeared to arise by budding from dysplastic duct walls and showed the same distinctive lining epithelium. They were distinguished from invasive glands by their pseudo-stratified epithelial lining and by consistent association with a sparse, discontinuous basal cell layer. Cytoplasmic differentiation at the point of junction of invasive cancer with transitive or dysplastic glands was studied by immunohistochemical staining for the differentiation markers prostate-specific antigen and pepsinogen II, and staining for mucin. Markedly reduced cytoplasmic differentiation was common in dysplastic and transitive glands. Invasion often coincided with an abrupt increase in cytoplasmic differentiation with expression of ectopic differentiation products. This sequence of biologic changes should be tested in other carcinomas where the exact point of invasion can be identified. PMID- 1712747 TI - Tenascin in normal, reactive, hyperplastic, and neoplastic tissues: biologic and pathologic implications. PMID- 1712749 TI - Rhabdoid tumors of soft tissues: a clinicopathologic study of 26 cases enrolled on the Intergroup Rhabdomyosarcoma Study. AB - Twenty-six cases of malignant soft tissue tumors with features similar to renal rhabdoid tumors were identified among approximately 3,000 childhood sarcomas entered on Intergroup Rhabdomyosarcoma Studies I-III. The tumors consisted of polygonal cells with vesicular nuclei and prominent nucleoli and cytoplasmic intermediate filament inclusions as identified by electron microscopy and immunohistochemistry. The growth pattern was predominantly solid or solid trabecular. Immunohistochemistry showed vimentin, wide spectrum keratin, and epithelial membrane antigen to be the most consistent antigenic phenotypes. Eleven patients were infants less than 1 year of age. The tumors affected predominantly soft tissues of proximal extremities, trunk, and retroperitoneum/pelvis/abdomen. Nineteen patients died within 1 to 82 months (median, 6 months) from the start of treatment. Five patients have survived the disease for 2 to 13 years. When compared with the survival analysis of 991 Intergroup Rhabdomyosarcoma Study II patients, it was obvious that this group of tumors fares very poorly (P less than .001). The tumor belongs to the group of soft tissue neoplasms showing mesenchymal and subtle epithelial differentiation, similar to epithelioid sarcoma. Because of its identifiable histology, site and age distribution, and poor outcome, it warrants a status as an independent entity. PMID- 1712750 TI - Atypical mesothelial hyperplasia associated with bronchogenic carcinoma. AB - Atypical mesothelial hyperplasia encountered in pleural fluid or in a pleural biopsy specimen raises the suspicion that one may be dealing with a diffuse malignant mesothelioma of the pleura. We studied eight cases with cytologic or histologic changes of mesothelial atypia thought to be suspicious for diffuse malignant mesothelioma. In each case, the hyperplasia was associated with a bronchogenic carcinoma in the lung subjacent to the mesothelial hyperplasia. Bronchogenic carcinoma should be added to the list of causes of atypical mesothelial hyperplasia. This combination of reactive and malignant processes should be appreciated, since pleural carcinomatosis and diffuse malignant mesothelioma must be separated for clinical and epidemiologic reasons. PMID- 1712751 TI - A rapid stain for the diagnosis of granuloma inguinale. PMID- 1712752 TI - Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene. AB - The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse". PMID- 1712753 TI - A family of retrotransposons and associated genomic variation in wheat. AB - A family of related retroelements was characterized in the genomes of some Graminease species. The structure of these retroelements indicates that they are retrotransposons containing reading frames with sequence similarity to the polyproteins of copia and Ty. This family of retroelements (termed WIS-2) occurs in the genomes of barley, wheat, rye, oats, and Aegilops species. Ongoing genomic variation both within individual plants of a wheat variety and within and between varieties of wheat is associated with some members of the WIS-2 family. PMID- 1712755 TI - Lessons from the past. PMID- 1712754 TI - Site-specific deletions of the mitochondrial genome in the Pearson marrow pancreas syndrome. AB - The Pearson marrow-pancreas syndrome is a fatal disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a widespread defect of oxidative phosphorylation. Here, we describe deletions of the mitochondrial (mt) genome between repeated 8- to 13-bp sequences as consistent features of the disease. Studying a series of nine unrelated children, including the patient originally reported by H. Pearson, we found five different types of direct repeats at the boundaries of the mtDNA deletions and we provided evidence for conservation of the 3'-repeated sequence in the deletions. In addition, we found a certain degree of homology between the nucleotide composition of the direct repeats and several structures normally involved in mtDNA replication and mtRNA processing. These results are consistent either with the recognition and cleavage of a particular DNA sequence with a factor of still unknown origin or with a homologous recombination between direct-repeat mtDNA sequences in the Pearson syndrome. PMID- 1712756 TI - Effect of temperature of thawing and diluent on the post--thaw physiological changes of buffalo frozen semen. AB - The effect of thawing was studied in buffalo semen diluted in three diluents (Tris egg-yolk, Egg-yolk citrate and Citric Acid whey) at three temperatures (5 degrees C, 35 degrees C and 75 degrees C) on motility, eosin staining, morphological and acrosomal changes, hyaluronidase, glutamic oxalacetic transaminase and glutamic pyruvic transaminase activities. The motility and lack of staining of sperm by eosin were maximum on thawing at 35 degrees C and in tris egg-yolk diluent followed by egg-yolk citrate and citric acid whey. Hyaluronidase, glutamic oxalacetic transaminase and glutamic pyruvic transaminase increased significantly in the extra-cellular fluid on thawing of semen diluted with all the three diluents. The buffalo semen diluted in tris egg-yolk and thawed at 35 degrees C for 30 seconds gave the best results. PMID- 1712757 TI - Downward regulation of neutrophil infiltration by endogenous histamine without affecting vascular permeability responses in air-pouch-type carrageenin inflammation in rats. AB - The role of histamine in neutrophil infiltration and vascular permeability response in carrageenin air pouch inflammation in rats was examined. Injection of carrageenin solution into an air pouch induced a gradual increase in histamine content in the pouch fluid and histidine decarboxylase activity of pouch wall tissues, with a maximum attained at 24 h. Local administration of the H2 antagonists cimetidine and famotidine, but not the H1 antagonist pyrilamine, induced an increase in neutrophil infiltration at 24 h. Both types of histamine antagonists failed to suppress the vascular permeability response. In addition, H2 antagonists attenuated the inhibitory effect of indomethacin on neutrophil infiltration without affecting the indomethacin-induced suppression of vascular permeability response. These results suggest that histamine produced in the inflammatory locus exerts a downward regulation of neutrophil infiltration through H2 receptors but does not play any significant role in the vascular permeability response. Furthermore, the inhibition by indomethacin of neutrophil infiltration might be ascribed to the increase in histamine level in the pouch fluid. PMID- 1712758 TI - Comparison of serological expression of different epitopes on the CA50-carrying antigen CanAg. AB - C203 and C242 are mouse monoclonal antibodies (MAbs) generated using a human colon carcinoma cell line. They recognize novel tumour-associated epitopes present in elevated levels in sera from patients with colon and pancreatic cancer. These epitopes were found to be co-expressed with sialylated Lewisa on the CanAg molecule. To study the association and distribution of the epitopes of CanAg in sera, these new antibodies, together with C50, were used in different combinations in time-resolved fluoroimmunoassays. Relative serum concentrations were examined in patients with various types of carcinoma and in patients with ulcerative colitis and benign pancreatic, and hepatobiliary diseases. A double determinant assay using C50 and C242 was shown to distinguish carcinoma from benign biliary and hepatocellular diseases better than a single-determinant assay based on C50 as both catching and tracing antibody. The number of sera with elevated CanAg levels from patients with benign obstructive biliary disease was 17 out of 29 using the single-determinant CA50 assay. This was reduced to 4 out of 29 in the double-determinant assay. When sera from patients with liver cirrhosis were analyzed, 16 of 23 patients showed elevated CanAg levels with the C50-C50 combination, but only 4 of 23 patients had elevated antigen values using the C50-C242 assay. The increased specificity was obtained without loss of sensitivity. MAb C203 was evaluated both in a double-determinant combination with C50 and in an homologous assay, but did not contribute either increased sensitivity or specificity as compared with C50-C50. PMID- 1712759 TI - A human bronchial epithelial cell strain with unusual in vitro growth potential which undergoes neoplastic transformation after SV40 T antigen gene transfection. AB - Bronchial epithelial cells were cultured from an individual with no evidence of malignant disease. These cells, designated HB56B, had a greatly extended in vitro life-span, being able to undergo 50 passages and 200 population doublings in contrast to the usual 3 to 4 passages and 20 to 30 population doublings characteristic of normal human bronchial epithelial cells. HB56B cells had karyotypic evidence of an amplified region on the short arm of chromosome II. Unlike normal bronchial epithelial cells, which undergo terminal squamous differentiation in vitro in response to fetal bovine serum, HB56B cells were only minimally affected by serum. These cells were readily established as an immortalized cell line, HB56B/5T, following transfection with a plasmid containing SV40 early region DNA. HB56B cells were non-tumorigenic in athymic nude mice, but HB56B/5T cells within a few passages of transfection with the SV40 plasmid formed tumors of which 28/37 regressed. HB56B cells may offer an experimental system for the study of proliferation, differentiation, and senescence control in human bronchial epithelial cells. PMID- 1712760 TI - Differential expression of two lck transcripts directed from the distinct promoters in HTLV-I+ and HTLV-I- T-cells. AB - The lck gene encodes a lymphocyte-specific tyrosine protein kinase, p56lck, the expression of which is almost exclusive in T-cells. The expression of lck in human T-cell leukemia virus type I (HTLV-I)-transformed T-cell lines is closely associated with interleukin-2 (IL-2) dependence for their growth. That is, IL-2 dependent HTLV-I-transformed cell lines contain the lck message abundantly as HTLV-I-negative T-cell lines, whereas IL-2-independent HTLV-I-transformed cell lines express either no or little lck mRNA, although they are derived from T cells. The lck gene contains 2 distinct promoters which direct 2 types of lck transcript with different 5' untranslated regions. In this study, we show that HTLV-I-transformed IL-2-dependent T-cell lines contain the upstream promoter initiated lck transcript exclusively, in contrast to HTLV-I-negative transformed T-cell lines which express the down-stream promoter- as well as the upstream promoter-initiated lck transcript. In addition, lck mRNA disappears transiently in IL-2-dependent HTLV-I-transformed T-cell lines after stimulation for T-cell activation, which is also observed in peripheral blood T lymphocytes. These results indicate that the disappearance of lck mRNA in HTLV-I-transformed, IL-2 independent cell lines is caused by a mechanism which down-regulates the upstream promoter-initiated lck transcript and this IL-2-independent state may represent a further "activated" condition of the IL-2-dependent state by the stimulation which mediates T-cell activation. PMID- 1712761 TI - Neurotropin inhibits experimental allergic encephalomyelitis (EAE) in Lewis rats. AB - The effects of Neurotropin, a substance extracted from the inflammatory dermis of rabbits inoculated with Vaccinia virus, for experimental allergic encephalomyelitis (EAE) in Lewis rats, a model for human multiple sclerosis (MS), was studied. The peptide defined by residues 68-84 (MB 68-84) which corresponds to the encephalitogenic portion of the guinea pig myelin basic protein (MBP) in complete adjuvant H37Ra (CFA) was injected into the hind foot pad of each rat. Neurotropin significantly suppressed the clinical and histological expression of actively induced EAE when administered i.p. daily from day 0 to day 6 after immunization. In addition, passive EAE induced by precultured spleen cells from rats immunized with MB 68-84 in CFA was also suppressed by daily administration of Neurotropin after cell transfer. Neurotropin treatment significantly suppressed the delayed-type hypersensitivity (DTH) response to MB 68-84. Furthermore, the ability of spleen cells from Neurotropin-treated rats to transfer EAE was significantly lower than that of saline-treated rats. It seemed that the suppression may be due to the inhibition of the activation by MB 68-84 of sensitized spleen cells, as demonstrated by proliferative response to MB 68 84. However, no difference was observed in Con A-induced proliferative response of the spleen cells between Neurotropin- and saline-treated rats. These findings indicate that Neurotropin inhibits EAE by suppressing the immune responses to encephalitogenic MBP with little non-specific suppression. PMID- 1712762 TI - The effect of lentinan on proliferative processes in parenchymal organs of rats- I. The effect on pyrimidine and nucleic acid syntheses. AB - The present study investigated the effect of Lentinan on the biochemical events associated with the pyrimidine and nucleic acid syntheses in the liver, kidney, thymus and spleen of rats. Lentinan was used at a dose of 4 mg/kg/day (twice) and in a single dose of 20 mg/kg. The following changes were observed. (1) The utilization of (14C)orotic acid for the synthesis of uridine components of liver acid-soluble extract and RNA uracil was activated after the administration of both doses of the drug. The specific activity of cytidine components of the acid soluble extract and RNA were, on the other hand, not affected. The same holds true for the kidney. The ratio of the specific activity of cytidine:uridine components of the acid soluble extract as well as RNA decreased after the administration of both doses of the drug. The specific activity of DNA cytosine and thymine are slightly suppressed in the liver after the administration of a high dose of Lentinan; no effect was observed in the kidney. (2) The uptake of (14C)cytidine by the liver was not affected; the specific activity of DNA cytosine and thymine were increased after the administration of a high dose of Lentinan. (3) The uptake of (14C)thymidine by the liver was not affected; the specific activity of liver DNA thymine was increased after the administration of both doses of the drug. In the thymus an increase of specific activity of DNA thymine has also been observed. (4) Repeated doses of the drug (4 mg/kg for 6 consecutive days) increased the weight of the spleen. The specific activity of DNA thymine of the liver and spleen were significantly increased. PMID- 1712763 TI - Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. AB - Limbal basal epithelium is thought to possess corneal epithelial stem cells that are the ultimate source of corneal epithelial proliferation and differentiation during corneal epithelial wound healing. Destruction of the limbal epithelium results in corneal conjunctivalization and vascularization, suggesting that the limbal epithelium also may be a barrier between corneal and conjunctival epithelia. In this experiment, a total corneal epithelial debridement using combined n-heptanol and mechanical scraping was created immediately (one-step) or 5 weeks (two-step) after 15 or 30 sec n-heptanol treatment at the limbus. All defects healed in 1-2 weeks. The severity of corneal vascularization, as judged by external photography, followed the ascending order of 30-sec two-step and 15 sec two-step less than 15-sec one-step less than 30-sec one-step (P less than 0.005). Immunofluorescence studies using monoclonal antibodies AM-3 and AE-5 showed mixed expression of corneal and conjunctival epithelial phenotypes on the corneal surface in the one-step subgroups. By contrast, the two-step subgroups had a normal corneal epithelial phenotype. Impression cytology was used to map goblet-cell distribution on the perilimbal corneal surface. The specimens taken from superior, temporal, and inferior bulbar areas were evaluated by a scoring system at different times. The extent of goblet cells invading onto the corneal surface also followed the same ascending order (P = 0.005). A transient goblet cell surge was noted, and the extent was related to the extent of corneal vascularization. It is thus evident that in vivo n-heptanol treatment for different durations can result in different extents of corneal conjunctivalization and vascularization. The authors concluded that the capability of the remaining limbal basal epithelium to recover its original full thickness stratified layers determines the strength of the limbal barrier. PMID- 1712764 TI - Histamine and prostacyclin. Primary and secondary release in allergic conjunctivitis. AB - The relationship between the release of histamine, a major mast cell mediator of conjunctival type I reactions, and the production of a prostanoid, prostacyclin (prostaglandin I2, PGI2), was examined in a guinea pig model of allergic conjunctivitis. Guinea pigs were sensitized topically and challenged by repeated conjunctival instillation of fluoresceinyl ovalbumin. Histamine and 6-keto-PGF1 alpha, the stable product of the spontaneous degradation of PGI2, were measured in tears by radioimmunoassays. Clinical type I reactions and tear histamine appeared by 8 days and increased up to 22 days during the initial sensitization, with notable variations between animals. The kinetics of histamine and 6-keto PGF1 alpha release in tears were examined over a 24-hr period after the antigen challenge. Histamine release was maximal during the first 10 min and returned to baseline values by 1 hr in all instances. The 6-keto-PGF1 alpha release also peaked during the first 10 min but continued for an extended period. The ratio of tear 6-keto-PGF1 alpha to histamine increased more than 16-fold over the 2 hr after antigen challenge. Late-phase reactions with second peaks of histamine or 6 keto-PGF1 alpha in the tears were observed in two different guinea pigs 4-8 hr after antigen challenge. Histamine applied to the eyes of naive guinea pigs also induced the release of 6-keto-PGF1 alpha in tears. Histamine appeared to act as a primary mediator, stimulating the secondary production and release of PGI2 by constitutive (eg, vascular) and possibly infiltrating inflammatory cells during an allergic conjunctival reaction. PMID- 1712765 TI - Discordant hypothyroxinemia and hypertriiodothyroninemia in treated patients with hyperthyroid Graves' disease and toxic multinodular goiter. AB - Hypothyroxinemia and hypertriiodothyroninemia may occur in the course of antithyroid drug or 131I treatment for hyperthyroid Graves' disease and toxic multinodular goiter. To determine the frequency of this discordance, we reviewed 62 patients with hyperthyroidism. Among 41 Graves' disease patients treated with antithyroid drugs and/or 131I, discordant serum triiodothyronine (T3) and thyroxine (T4) levels occurred in 26.8%. Of these patients 72.8% had a high serum T3 level associated with a normal serum T4 level, whereas in 27.2% a high serum T3 level was associated with a low serum T4 level. Among 21 toxic multinodular goiter patients, 14.3% had discordant T3 and T4 levels. We conclude that discordance of serum T4 and T3 concentrations is frequently encountered in patients with hyperthyroid Graves' disease and less frequently in toxic multinodular goiter during or after therapy. PMID- 1712766 TI - Revised NMR assignments for rapamycin. PMID- 1712767 TI - Effects of a parenteral supplement of folic acid and its interaction with level of feed intake on hepatic tissues and growth performance of young dairy heifers. AB - Forty-seven dairy heifers of approximately 10 d of age were assigned to a factorial experiment in which a supplement of folic acid (0 or 40 mg) administered weekly by i.m. injection and level of feed intake were the two factors studied. The heifers were weaned after 5 wk of experimentation. Following weaning, and until the end of the experiment, 11 wk later, they had ad libitum access to grass hay and concentrates at two different levels, ad libitum or restricted, to allow a body weight gain of 700 g/d. A supplement of folic acid (P less than .05) and ad libitum access to feed (P less than .05) increased the mean concentration of serum folates. Blood hemoglobin and packed cell volume were not affected by the level of feed intake. However, they were both increased (P less than .05) by the supplement of folic acid. Average daily gain was analyzed over three different periods: 0 to 5 wk (before weaning), 5 to 10 wk, and 10 to 16 wk. Average daily gain was increased by the supplement of folic acid during the second period (P less than .05) and by ad libitum access to feed during the last two periods (P less than .05). Ad libitum access to feed increased (P less than .05) weight of the liver, decreased the (P less than .05) concentrations of RNA and DNA, and increased (P less than .05) the ratios of protein/DNA and RNA/DNA. The supplement of folic acid decreased (P less than .05) weight of the liver and increased the ratio RNA/DNA (P less than .05). These effects of supplement of folic acid on growth performance and on hematological cells may reflect a lack of folic acid during the weeks after weaning. PMID- 1712768 TI - Dynamics of differentiation in human epidermoid squamous carcinoma cells (A431) with continuous, long-term gamma-IFN treatment. AB - We investigated the long-term effects of continuous gamma interferon (gamma-IFN) treatment on A431, a human squamous carcinoma cell line. Cells were grown in an in vitro culture system, which over time produces cohesive cell masses ("tumoroids") exhibiting three-dimensional, histotypically differentiated structures, e.g., keratin "pearls", intercellular bridges (desmosomes), elongated flattened cells (squames) and stratification. The effects of gamma-IFN on cell growth, morphology and stage of differentiation were assessed at different treatment times by light and electron microscopy and by immunohistochemical staining using antibodies to keratins 1 and 14 and to filaggrin, markers of specific stages of keratinocyte differentiation. Our results show that A431 cells have the capacity for spontaneous differentiation, that this capacity is significantly enhanced and accelerated by gamma-IFN treatment leading to terminal differentiation and extensive cell death by 2 wk. Despite continuous exposure to IFN, a small number of viable, undifferentiated cells remain. Their proliferation, evident by 3 wk, reconstitutes the tumoroid which once again contains the full range of differentiating cell types. PMID- 1712769 TI - Aortic valve replacement and splenectomy in a patient with chronic idiopathic thrombocytopenic purpura--preoperative management with high-dose gamma-globulin. AB - We report the management of a patient with chronic idiopathic thrombocytopenic purpura and severe aortic valvular disease. Preoperative intravenous high-dose gamma-globulin administration was employed, and aortic valve replacement combined with splenectomy were performed during the same operation. The platelet count at admission was 34,000/mm3 and increased to 146,000/mm3 after the gamma-globulin therapy. Platelet transfusion at the end of the cardiopulmonary bypass was considered no longer necessary in the postoperative period, because the platelet count increased quickly after the procedure. The postoperative course was uneventful. We believe that open heart surgery and splenectomy can successfully be performed simultaneously in a patient with chronic idiopathic thrombocytopenic purpura treated with high-dose gamma-globulin therapy. PMID- 1712770 TI - Isolation and characterization of the 160,000-Da phosphotyrosyl protein, a putative participant in insulin signaling. AB - The 160,000-Da protein (pp 160) which is rapidly phosphorylated on tyrosine in response to insulin and thus is a putative participant in signaling from the insulin receptor has been purified to homogeneity from 3T3-L1 adipocytes. Isolation of this protein was accomplished by chromatography on an immobilized monoclonal antibody against phosphotyrosine, followed by gel electrophoresis. Sufficient protein was obtained to allow the determination of the sequences of several peptides, which in turn enabled the development of anti-peptide antibodies that specifically recognize pp 160. Immunoblotting of 3T3-L1 adipocyte lysates, together with the purified pp 160 as a standard, indicate that an insulin-treated 3T3-L1 adipocyte possesses about 230,000 copies of tyrosine phosphorylated pp160 and that this amount is approximately 25% of the total pp160 in the cell. The number of tyrosine-phosphorylated pp160s per cell is approximately the same as that of insulin receptor beta subunits. These results provide further evidence for a role of pp160 in insulin signaling. Moreover, the availability of purified protein and knowledge of peptide sequences will allow the elucidation of the structure and function of this protein. PMID- 1712771 TI - A second, expressed thrombospondin gene (Thbs2) exists in the mouse genome. AB - The diverse and occasionally conflicting properties described for the extracellular, cell surface-associated protein thrombospondin (TSP) have raised the possibility that functionally distinct forms of the protein exist in the same organism. We have isolated and characterized a partial cDNA clone for mouse TSP that is clearly homologous to, but distinct from, the coding sequence for mouse TSP deduced from a mouse genomic clone (Bornstein, P., Alfi, D., Devarayalu, L., Framson, P., and Li, P. (1990) J. Biol. Chem. 265, 16691-16698). This second TSP, which we term thrombospondin 2, is the product of a separate gene (Thbs2) and is expressed in a variety of mouse tissues in a pattern that differs from that for TSP1. Based on their translated amino acid sequences, it seems likely that TSP1 and TSP2 will be found to have both common and unique properties and that the functional consequences of TSP production will reflect the ratio of the levels of these two related proteins. PMID- 1712772 TI - TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. AB - TIS10 is a primary response gene whose cDNA was cloned as a result of its rapid, superinducible expression in Swiss 3T3 cells in response to 12-O Tetradecanoylphorbol-13-acetate. The 5'-untranslated region of the 3.9-kilobase TIS10 message contains only 124 nucleotides, whereas the 3'-untranslated region is almost 2 kilobases in length. Within this long 3' region, there are multiple repeats of the sequence ATTTA, a sequence often present in rapidly degraded mRNA species. Primer extension revealed that the TIS10 cDNA begins 16 base pairs downstream of the transcription start site for the TIS10 gene. The TIS10 cDNA encodes a predicted protein of 604 amino acids. A computer search identified striking similarities between the predicted TIS10 protein product and the murine, sheep, and human prostaglandin synthase/cyclooxygenase proteins. The TIS10 protein has many of the same conserved amino acids that are thought to be important for cyclooxygenase function. TIS10 mRNA is undetectable by Northern analysis in quiescent 3T3 cells. The TIS10 gene is rapidly and transiently induced by forskolin and serum, as well as by 12-O-tetradecanoylphorbol-13 acetate, in Swiss 3T3 cells. These agents elicit far more dramatic changes in TIS10 mRNA levels than in cyclooxygenase mRNA levels. The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS10 gene. PMID- 1712773 TI - Crystallographic refinement of the three-dimensional structure of the FabD1.3 lysozyme complex at 2.5-A resolution. AB - The three-dimensional crystal structure of the complex between the Fab from the monoclonal anti-lysozyme antibody D1.3 and the antigen, hen egg white lysozyme, has been refined by crystallographic techniques using x-ray intensity data to 2.5 A resolution. The antibody contacts the antigen with residues from all its complementarity determining regions. Antigen residues 18-27 and 117-125 form a discontinuous antigenic determinant making hydrogen bonds and van der Waals interactions with the antibody. Water molecules at or near the antigen-antibody interface mediate some contacts between antigen and antibody. The fine specificity of antibody D1.3, which does not bind (K alpha less than 10(5) M-1) avian lysozymes where Gln121 in the amino acid sequence is occupied by His, can be explained on the basis of the refined model. PMID- 1712774 TI - Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases. AB - We have examined the properties of reverse transcriptases (RTs) required for strand transfer synthesis on poly(rA). In this process, a primer is elongated on one template and then switches to other templates for additional elongation until it is much longer than the templates on which it was made. Models of retrovirus replication require the RT to catalyze two distinct strand transfers. Additionally, they propose that the RT ribonuclease H (RNase H) activity is involved in both transfers. RTs from human immunodeficiency virus (HIV), avian myeloblastosis virus, and murine leukemia virus differ in molecular mass and subunit composition. However, they all catalyzed strand transfer synthesis on (rA)300, generating characteristically long products. An RNase H-deficient enzyme, HIV-RTRD, catalyzed strand transfer synthesis to the same degree as native HIV-RT, indicating that a functional RNase H activity is not required. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity of HIV-RT, did not diminish strand transfer synthesis. Highly processive DNA synthesis by each RT was found to be required for the strand transfer reaction. RNase H- murine leukemic virus RT has a structural modification that not only eradicates RNase H, but also makes the polymerase much less processive for DNA synthesis. However, conditions that allow this modified enzyme to bind repeatedly to the same primer during synthesis, i.e. conditions that simulate higher processivity, allow strand transfer synthesis. Catalysis of strand transfer synthesis is not a property of all DNA polymerases, since the Klenow fragment of Escherichia coli DNA polymerase I is unable to catalyze this reaction even if high processivity is simulated. These results suggest that strand transfer synthesis relies on an unidentified functional activity present in RTs. PMID- 1712775 TI - SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells. AB - SPARC, a Ca(2+)-binding glycoprotein that is expressed during tissue morphogenesis and functions as an inhibitor of cell spreading in vitro, was found to induce the secretion of an Mr = 45,000 protein in bovine aortic endothelial (BAE) cells. This protein was identified as type 1 plasminogen activator inhibitor (PAI-1) on Western blots with anti-PAI-1 antiserum. SPARC stimulated the secretion of PAI-1 protein into the medium of subconfluent BAE cells, but not confluent BAE cells, in a dose- and time-dependent manner. Secretion of PAI-1 into the culture medium was progressive and exhibited an increase of 3- to 7-fold over control values within 24 h after the addition of SPARC. Levels of PAI-1 mRNA were elevated 2-fold within 4 to 24 h after the addition of SPARC and did not increase with higher concentrations of SPARC. Since the induction of PAI-1 mRNA by SPARC was not blocked by cycloheximide, de novo protein synthesis was apparently not required for this stimulation. Control experiments showed that the induction of PAI-1 was not due to contamination of the SPARC preparations with endotoxin. These data demonstrate that SPARC induces the biosynthesis of PAI-1 in BAE cells and suggest a role for SPARC in the regulation of fibrinolysis and in the control of proteolytic events in remodeling tissues. PMID- 1712776 TI - Glucocorticoids inhibit the transcriptional induction of JE, a platelet-derived growth factor-inducible gene. AB - Macrophages and monocytes have essential roles in normal wound healing, in the immune response, and in the pathogenesis of atherosclerosis. Platelet-derived growth factor (PDGF) stimulates the transcription of the early response gene, JE, and its human homolog, macrophage chemotactic protein-1 (MCP-1) in fibroblasts. JE/MCP-1 encodes a cytokine which is a member of a superfamily of small inducible genes that include platelet factor 4, beta-thromboglobulin, 310-C/NAP-1/IL-8, IP 10, KC/gro/MGSA, and others which may play important roles in the inflammatory and immune response. We now report that glucocorticoids inhibit the transcriptional induction of the JE gene by PDGF and serum in a dose-dependent manner. The glucocorticoid response followed the expected anti-inflammatory rank order of potency and was not due to a shift in the time course of induction. Nonsteroidal anti-inflammatory agents were ineffective in reducing JE mRNA levels. Dexamethasone inhibited the accumulation of JE transcripts induced by PDGF, 12-O-tetradecanoylphorbol-13-acetate, and double-stranded synthetic RNA. Nuclear runoff assays demonstrated that the negative regulation occurred by decreasing the transcriptional induction of the JE gene. No effects on JE message stability could be detected in the presence of dexamethasone. The protein synthesis inhibitors cycloheximide and puromycin reversed the glucocorticoid mediated inhibition and suggested that new protein synthesis was necessary. These results suggest that the transcriptional inhibition of glucocorticoids is mediated by the expression of a labile transcriptional repressor for the JE gene. PMID- 1712777 TI - Isoenzymes of horse liver alcohol dehydrogenase active on ethanol and steroids. cDNA cloning, expression, and comparison of active sites. AB - Horse liver alcohol dehydrogenase occurs as isoenzymes: E is active on ethanol but not steroids; S is active on ethanol and steroids. The cDNAs for these isoenzymes were cloned; both were 1.8-kilobase long and contained complete coding sequences. Both enzymes were expressed in Escherichia coli, and the purified proteins had properties similar to those of the natural enzymes. The amino acid sequence deduced from the open reading frame of the E-type cDNA agreed with the amino acid sequence of the E isoenzyme determined by protein sequencing and x-ray crystallography. When compared with the E-type cDNA, the coding region of the S type cDNA contains 24 substitutions and 3 deletions, giving rise to an amino acid sequence for the S. isoenzyme that differs from that of the E isoenzyme at 10 positions: nine conservative substitutions and one deletion, of Asp-115. These changes can be accommodated in the three-dimensional structure of the E isoenzyme, and models of the E and S isoenzymes complexed with a 3 beta-hydroxy-5 beta-steroid were built. The modeling shows that Leu-116 apparently sterically hinders binding of steroids in the E isoenzyme, and deletion in the S isoenzyme of Asp-115 moves Leu-116 and relieves the hindrance. The human gamma and rat liver enzymes are also active on steroids, but they have a different constellation of amino acid residues in the substrate pocket. Thus, there are multiple bases for the activity on steroids. PMID- 1712778 TI - Discrete functional stages of vaccinia virus early transcription during a single round of RNA synthesis in vitro. AB - We have developed a system for analysis of discrete steps in vaccinia virus early mRNA synthesis during a single round of transcription in vitro. A synthetic early promoter is used to direct transcription by vaccinia RNA polymerase of a G-less cassette in linear duplex DNA. Omission of GTP from transcription reactions leads to the formation of ternary elongation complexes paused stably at the end of the G-less cassette. These complexes can be induced to elongate by provision of GTP. While initiation of transcription is sensitive to low concentrations of salt and Sarkosyl, elongation is relatively resistant to these agents. Termination can be studied in a single synthetic cycle by forming transcription complexes paused just proximal to the termination signal TTTTTNT that can subsequently elongate and terminate. By selectively incorporating the termination-inhibiting analog BrUMP into proximal and distal portions of the nascent transcript, we localize the termination signal within or near the sequence UUUUUNU in the nascent RNA. We show that access of the vaccinia termination factor (VTF/capping enzyme) to the transcriptional apparatus can occur subsequent to initiation and synthesis of a 390-nucleotide nascent RNA. Termination is more sensitive to inhibition by salt and Sarkosyl than in elongation. This sensitivity is not reversed by preincubation of VTF with the transcription complex. Finally, we confirm the identity of VTF and vaccinia mRNA capping enzyme by demonstration of VTF activity associated with capping enzyme expressed in Escherichia coli. PMID- 1712780 TI - Pretranslational mechanisms determine the type of potassium channels expressed in the rat skeletal and cardiac muscles. AB - We have cloned a cDNA (RMK2) coding for a Shaker type delayed rectifier K+ channel from a rat skeletal muscle cDNA library. The clone encodes a putative protein of 602 amino acids, identical with a rat brain K+ channel Kv1 (Swanson, R., Marshall, R., Smith, J. S., Williams, J. B., Boyle, M. B., Folander, K., Luneau, C. J., Antanavage, J., Oliva, C., Burhow, S. A., Bennet, C., Stein, R. B., and Kaczmarek, L. K. (1990) Neuron 4, 929-939). Northern blot analysis showed that RMK2 is expressed in skeletal and cardiac muscle. RNase protection analysis showed that the 3'-noncoding regions of the brain, cardiac, and skeletal muscle RMK2 transcripts are identical. Cloning of the gene confirmed that the protein is encoded by a single exon (Swanson et al. (1990) Neuron 4, 929-939). We expressed RMK2 in Xenopus oocytes and showed that it encodes noninactivating delayed rectifier K+ channels, resistant to block by external tetraethylammonium, with a small unitary conductance of 8.0 picosiemens. Coinjection of RMK2 and RCK1 (RMK1) (Baumann, A., Grupe, A., Ackermann, A., and Pongs, O. (1988) EMBO J. 7, 2457 2463; Koren, G., Liman, E. R., Logothetis, D. E., Nadal-Ginard, B., and Hess, P. (1990) Neuron 4, 39-51) into Xenopus oocytes resulted in the expression of currents that have tetraethylammonium inhibition curves that differ from the linear combination of inhibition curves of the two types expressed individually. Thus, RMK2 and RCK1 (RMK1) can form heteromultimers. RNA blot hybridization analysis revealed that the RMK2 transcript is developmentally regulated in a different manner in the rat skeletal muscle, ventricle, and atrium. PMID- 1712779 TI - Molecular cloning of cDNA for lipopolysaccharide-binding protein from the hemolymph of the American cockroach, Periplaneta americana. Similarity of the protein with animal lectins and its acute phase expression. AB - A previous paper described the purification of a calcium-dependent lipopolysaccharide-binding protein from the hemolymph of Periplaneta americana (Jomori, T., Kubo, T., and Natori, S. (1990) Eur. J. Biochem. 190, 201-206). This paper describes the molecular cloning and characterization of cDNA for the LPS binding protein. This protein was found to have a carbohydrate-recognition domain at its carboxyl terminus containing amino acid sequences that are conserved in various mammalian C-type lectins. It was also shown to contain an N-linked carbohydrate chain, and the amino acid residue carrying this chain was assigned as Asn at position 56 (23rd amino acid residue from the amino terminus). Northern blot analysis revealed the presence of multiple mRNAs that hybridized with this cDNA and transient increases in their content after injection of Escherichia coli into adult Periplaneta, suggesting that the LPS-binding protein plays a role in the acute phase response of this insect. PMID- 1712781 TI - Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins. AB - Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56 53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor. PMID- 1712782 TI - Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen. AB - The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119 4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains. PMID- 1712783 TI - Construction and characterization of cyanobacterial mutants lacking the manganese stabilizing polypeptide of photosystem II. AB - Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition. PMID- 1712784 TI - Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement. AB - Protection against the pore-forming activity of the human C5b-9 proteins was conferred on a nonprimate cell by transfection with cDNA encoding the human complement regulatory protein CD59. CD59 was stably expressed in Chinese hamster ovary cells using the pFRSV mammalian expression vector. After cloning and selection, the transfected cells were maintained in media containing various concentrations of methotrexate, which induced surface expression of up to 4.2 x 10(6) molecules of CD59/cell. Phosphatidylinositol-specific phospholipase C removed greater than 95% of surface-expressed CD59 antigen, confirming that recombinant CD59 was tethered to the Chinese hamster ovary plasma membrane by a lipid anchor. The recombinant protein exhibited an apparent molecular mass of 21 24 kDa (versus 18-21 kDa for human erythrocyte CD59). After N-glycanase digestion, recombinant and erythrocyte CD59 comigrated with apparent molecular masses of 12-14 kDa, suggesting altered structure of asparagine-linked carbohydrate in recombinant versus erythrocyte CD59. The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming activity of human C5b-9. Induction of cell surface expression of CD59 antigen inhibited C5b-9 pore formation in a dose dependent fashion. CD59 transfectants expressing greater than or equal to 1.2 x 10(6) molecules of CD59/cell were completely resistant to human serum complement. By contrast, CD59 transfectants remained sensitive to the pore-forming activity of guinea pig C8 and C9 (bound to human C5b67). Functionally blocking antibody against erythrocyte CD59 abolished the human complement resistance observed for the CD59-transfected Chinese hamster ovary cells. These results confirm that the C5b-9 inhibitory function of the human erythrocyte membrane is provided by CD59 and suggest that the gene for this protein can be expressed in xenotypic cells to confer protection against human serum complement. PMID- 1712785 TI - Relaxant effects of alpha-human atrial natriuretic peptide on venous smooth muscle. AB - 1 The present study has examined the relaxant effects of alpha-human atrial natriuretic peptide (alpha-hANP) and sodium nitroprusside on dog saphenous vein ring preparations; responses were tested on a background of tone corresponding to 30% and 80% of maximal contraction. Also tested on the lower background of tone were the responses to carbachol, M&B 22,948, forskolin and 3-isobutyl-1 methylxanthine (IBMX). The vascular responses to alpha-hANP and carbachol were also examined in dog splenic artery ring preparations. 2 On a background of tone corresponding to 30% of maximal contraction, both alpha-hANP and sodium nitroprusside were found to relax venous ring preparations with IC50 values of 6 x 10(-8) and 2 x 10(-7) M, respectively. When the background of tone was raised to 80% of maximal contraction, the relaxant effect of sodium nitroprusside was significantly reduced and that of alpha-hANP became almost completely abolished; the IC50 value for sodium nitroprusside in these experimental conditions was 2 x 10(-6) M. 3 The cGMP phosphodiesterase inhibitor M&B 22,948 was found to produce a concentration-dependent relaxation, with an IC50 value of 2 x 10(-6)M in saphenous vein rings with a background of tone corresponding to 30% of maximal contraction. By contrast, saphenous vein rings were found to be insensitive to carbachol. Forskolin and IBMX were also found to produce concentration-dependent relaxing responses of venous preparations, with IC50 values of 2 x 10(-7) and 7 x 10(-7) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712786 TI - Nedocromil sodium inhibits substance P-induced potentiation of cholinergic neural responses in the isolated innervated rabbit trachea. AB - 1. Sensory neuropeptides such as substance P may be implicated in the pathophysiology of asthma. 2. It has been proposed that nedocromil sodium may inhibit the effects of neuropeptides. 3. In this study, using an isolated innervated preparation of rabbit trachea, substance P, 10(-6) M, potentiated contractions induced by parasympathetic stimulation. The effect of substance P at the preganglionic site (307 +/- 38% of control, n = 5), was similar to that at the postganglionic site (307 +/- 61% of control, n = 5). 4. Nedocromil sodium, 10(-7) M, significantly inhibited the substance P-induced potentiation preganglionically (199 +/- 44%, n = 4, P less than 0.05) but not postganglionically (356 +/- 118%, n = 4). 5. These results suggest that nedocromil sodium may modify neuropeptide action selectively at a preganglionic site and that this may contribute to its therapeutic efficacy. PMID- 1712787 TI - The revascularization of healing flexor tendons in the digital sheath. A vascular injection study in dogs. AB - The role of revascularization in the nutritional support of repair of the flexor tendons is not completely understood. To explore the extent to which intrasynovial flexor tendons revascularize after transection and suture, a vascular injection study was carried out in a canine model. The tendons to the second and fifth digits of the forepaw in twelve adult mongrel dogs were transected and repaired. There were twenty-four experimental tendons and twenty four normal tendons. The limb was placed in a polyurethane shoulder-spica cast, and the paw was treated with immediate protected passive mobilization. At three, seven, ten, seventeen, and twenty-eight days, the animals were killed and the major arteries supplying both the paw that had been operated on (left) and the contralateral normal paw (right) were injected with 200 milliliters of India ink. Segments of repaired and normal tendons were then clarified by a modified Spalteholz technique. The normal tendons demonstrated a well developed mesotenon that provided vascularization of the proximal portion of the flexor digitorum profundus tendon. A consistent three-cubic-millimeter avascular intrasynovial portion of tendon was noted. Distally, vessels arose from the vinculum breve, supplying the terminal twenty millimeters of tendon substance. In the experimental tendons, longitudinal and transverse clarified sections showed consistent revascularization of the site of repair by proximal vessels in the absence of ingrowth of peripheral adhesions. Vessels in the epitenon progressively extended for a distance of ten millimeters, through normally avascular regions, to reach the site of repair by the seventeenth postoperative day. Intratendinous vessels about the site of repair consistently originated from surface vessels, rather than from extensions of pre-existing intratendinous vessels. New vessels penetrated all areas, including the normally avascular volar segments of tendon, irrespective of previous topical zones of avascularity. Proximal vascular plexi were characterized by large tortuous vessels with frequent circuitous branches. More distal vessels had a longitudinally oriented, feathery appearance. PMID- 1712788 TI - Neovascularisation of the meniscus with angiogenin. An experimental study in rabbits. AB - Angiogenin, a potent blood vessel inducing protein, was implanted into experimentally injured menisci of 75 New Zealand white rabbits. Localised neovascularisation occurred in 52% of the angiogenin-treated animals, and in 9% of the controls. Neovascularisation induced by angiogenin may enhance healing of injuries within the poorly vascularised meniscal fibrocartilage, and improve the results of meniscal repair. PMID- 1712789 TI - Kinesin associates with anterogradely transported membranous organelles in vivo. AB - Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo. PMID- 1712791 TI - Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1. AB - The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAM1-1, LAM1-3, and LAM1-6 mAb each strongly inhibit the binding of lymphocytes to HEV in the in vitro frozen section assay, and defined three independent epitopes on LAM-1. Blocking of PPME or fucoidin binding by LAM1-3 indicated that this site is identical, or in close proximity, to the carbohydrate binding site, and analysis of the binding of LAM1-3 to chimeric selectins showed that the epitope detected by LAM1-3 is located within the lectin domain. Although the LAM1-6 epitope is also located in the lectin domain, LAM1-6 did not affect the binding of PPME or fucoidin. The LAM1-1 epitope was located in, or required, the EGF domain, and, importantly, binding of LAM1-1 significantly enhanced the binding of both PPME and fucoidin. These results suggest that adhesion mediated by LAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV. PMID- 1712790 TI - The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor. AB - The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin. PMID- 1712792 TI - Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. AB - The number of identifiable stages and expression of differentiation markers in cells of the osteoblast lineage are not well understood. In the present study, a mAb, designated rat bone marrow (RBM) 211.13, was prepared that stained selectively the osteogenic and preosteoblastic cells along the surfaces of bone in calvariae, femurs, and metatarsals. The staining was cell surface associated and coincided with that for alkaline phosphatase (APase) detected histochemically. Only cells positive for APase activity by biochemical assay and not those without APase activity (e.g., fetal rat skin) stained with RBM 211.13. By immunoblotting, RBM 211.13 recognized a band coinciding with APase activity on nonreducing/nondenaturing gels, and RBM 211.13 precipitated a protein which on reduced gels migrated with an apparent molecular mass of approximately 80 kD. RBM 211.13 labeling was abolished by phosphatidylinosital-specific phospholipase C, known to release APase from the cell surface. All of these data support the concept that RBM 211.13 recognizes the bone isoenzyme of APase. RBM 211.13 was used to sort by flow cytometry the APase-positive and APase-negative cells from mixed fetal rat calvaria (RC) cell populations. The osteoprogenitors we identified earlier that form bone nodules in vitro (Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143-154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132-138) were found within the APase-positive pool. By immunopanning, RC cells were separated into APase-enriched (APase-positive, adherent) and APase-depleted (APase-negative, nonadherent) populations. The APase-positive fraction was enriched two-to-threefold for bone-forming osteoprogenitors compared to unfractionated cells, while the APase-negative population formed very few nodules under the same conditions. Both populations responded to the glucocorticoid dexamethasone (DEX) with an increase in bone nodule formation. However, the fold stimulation in bone formation in the APase-negative population was approximately 30-fold, while the fold stimulation in the APase-positive population was only approximately 5-fold. These data suggest that APase expression can be used for immunoselection to fractionate osteoblastic populations into an APase-positive population and a population initially APase-negative, that virtually all osteoprogenitors forming bone in vitro in the absence of added glucocorticoids reside in the APase-positive pool, and that the only osteoprogenitors present in the APase-negative pool are those requiring DEX to differentiate. PMID- 1712793 TI - Position of axons in the cat's optic tract in relation to their retinal origin and chiasmatic pathway. AB - The positions of the crossed and uncrossed optic axons of distinct diameter classes has been examined in the optic tract of the adult cat. In addition, the retinal origin of axons occupying different positions within the tract has been studied. Since the position of a fibre within the optic tract reflects its time of arrival during development, we have used axonal position as an indicator of age and have related this to the chiasmatic pathway choice of the axons. Cats were either monocularly enucleated, to reveal the position and diameter of surviving crossed and uncrossed optic axons in semithin and thin sections, or implants of horseradish peroxidase (HRP) were placed so as to retrogradely label the ganglion cells giving rise to axons within the deep (early arriving), or superficial (later arriving) parts of the tract selectively. This was accomplished by either 1) surgically implanting HRP into the superficial portion of the optic tract, via a transbuccal approach, or 2) making such a transbuccal transection of the superficial fibres, followed by intracerebral injections of HRP to retrogradely label the surviving, deeper, optic axons from their target nuclei. The deep parts of the optic tract contain fine and medium, crossed and uncrossed axons arising from mainly medium sized cells in the contralateral nasal and the ipsilateral temporal retina; there is a clear line of decussation. In contrast, the superficial parts of the tract contain mainly fine diameter axons arising from small cells in the whole contralateral retina, and a small proportion of large diameter axons arising from large, alpha cells in the whole contralateral retina and in the ipsilateral temporal retina. The likelihood that axons from the temporal retina will project contralaterally therefore increases as development proceeds, since these axons are found in the superficial parts of the tract only. This suggests that a time-dependent signal that weakens with age is responsible for directing early arriving optic axons from the temporal retina to take an exclusively uncrossed course. PMID- 1712794 TI - Visual topography of area TEO in the macaque. AB - Previous studies have mapped the visuotopic organization of visual areas from V1 through V4 in the occipital cortex and of area TE in the temporal cortex, but the cortex in between, at the occipito-temporal junction, has remained relatively unexplored. To determine the visuotopic organization of this region, receptive fields were mapped at 1,200 visually responsive sites on 370 penetrations in the ventral occipital and temporal cortex of five macaques. We identified a new visual area, roughly corresponding to cytoarchitectonic area TEO, located between the ventral portion of V4 and area TE. Receptive fields in TEO are intermediate in size between those in V4 and TE and have a coarse visuotopic organization. Collectively, receptive fields in TEO appear to cover nearly the entire contralateral visual field. The foveal and parafoveal representation of TEO is located laterally on the convexity of the inferior temporal gyrus, and the peripheral field is represented medially on the ventral surface of the hemisphere, within and medial to the occipitotemporal sulcus. Beyond the medial border of TEO, within cyteoarchitectonic area TF, is another visually responsive region, which we have termed VTF; this region may also have some crude visual topography. Bands of constant eccentricity in TEO appear to be continuous with those in V2, V3v, and V4. The upper field representation in TEO is located adjacent to that in ventral V4, with a representation of the horizontal meridian forming the boundary between the two areas. The lower field representation in TEO is located just anterior to the upper field but is smaller. In contrast to the orderly representation of eccentricity in TEO, we found little consistent representation of polar angle, other than the separation of upper and lower fields. The results of injecting anatomical tracers in two animals suggest that TEO is an important link in the pathway that relays visual information from V1 to the inferior temporal cortex. TEO is thus likely to play an important role in pattern perception. PMID- 1712795 TI - Reorganization of the area dentata serotoninergic plexus after lesions of the median raphe nucleus. AB - Serotoninergic projections from the dorsal and median raphe nuclei to the area dentata of the hippocampal formation terminate mainly in the molecular layer and hilus, respectively. Consequently, a reduction in the density of the hilar serotoninergic plexus is seen by immunocytochemistry 2 weeks after lesions of the median raphe nucleus. Hippocampal serotonin concentration and serotonin high affinity uptake are also significantly reduced. Six weeks after lesion, surviving serotoninergic axons form a dense band in the inner molecular layer of the dorsal area dentata, a region that usually contains a sparse serotoninergic plexus. Moreover, serotoninergic fibers transverse the molecular layer and pass through the granule cell layer to reinnervate the hilus. Serotonin concentration and high affinity uptake have recovered to near normal levels by 6 weeks postlesion. Changes in the anatomical distribution of the area dentata serotoninergic plexus have not been reported in cases in which serotoninergic sprouting follows axotomy of serotoninergic projections. Thus direct lesions of serotoninergic neurons can produce a homotypic compensatory response that is qualitatively different from that generated by axotomy. The mechanistic basis for this reorganization is unclear, but the apparent extension of serotoninergic axon collaterals toward the hilus suggests that the denervated hilar neuropil is guiding reinnervation. Finally, anatomical evidence from animals studied 10 weeks postlesion suggests that the compensatory proliferation of serotoninergic axons observed 6 weeks after median raphe lesion is a transient event. PMID- 1712797 TI - Isolation and characterization of monoclonal antibodies monospecific for bovine alpha-casein and beta-casein. AB - Two monoclonal antibodies monospecific for bovine alpha s1-casein, which recognize three genetic variants of alpha s1-casein, have been isolated and their binding properties characterized. Antibodies 57-115 and 57-310 recognize different antigenic determinants on the alpha s1 protein with affinity constants of 1.63 x 10(11) and 2.13 x 10(11) M-1, respectively. Five monoclonal antibodies, 58-409, 58-416, 58-488, 58-504, and 58-557, monospecific for bovine beta-casein with affinity constants greater than 10(9) M-1, which recognize a similar epitope(s) also were isolated. All seven antibodies are of isotype IgG1 and recognize both the denatured and undenatured forms of their antigen, making them suitable for qualitative and quantitative radioimmunoassay. PMID- 1712796 TI - Differential innervation patterns of three divisions of frog auditory midbrain (torus semicircularis). AB - The connectivity pattern of the laminar, principal, and magnocellular nuclei of the frog torus semicircularis (TS) was investigated. A small amount of horseradish peroxidase was injected focally into individual divisions of the TS and anterograde and retrograde transport patterns were observed. Results of our tracing study showed that these divisions of the TS possessed distinct innervation patterns. The principal nucleus appeared to be the primary input port of the TS receiving extensive inputs from all caudal brainstem auditory nuclei bilaterally, but especially from the contralateral dorsal medullary nucleus and the ipsilateral superior olivary and lateral lemniscus nuclei. Descending projection to this nucleus was limited to that from the posterior thalamic nucleus. In contrast, the laminar nucleus, but even more markedly the magnocellular nucleus, received extensive descending inputs from numerous structures in the dorsal thalamus and less pronounced ascending afferents from caudal brainstem auditory nuclei. Similar to the afferent connection patterns, the efferent projections originating from these three toral divisions differed substantially. The principal nucleus gave restricted ascending projections, limited mainly to the caudal region of the posterior thalamic nucleus, a region important in processing spectral information of complex sounds, and the pretectal gray. Its descending projection was also somewhat restricted, being limited to the superior olivary and lateral lemniscus nuclei. The laminar nucleus and especially the magnocellular nucleus gave robust descending as well as ascending projections; these nuclei serve as the main output paths for the TS and provide the main routes by which auditory input reaches the central thalamic nucleus, a structure previously shown to be involved in temporal information processing. PMID- 1712798 TI - Ruminal digestion and microbial utilization of diets varying in type of carbohydrate and protein. AB - Three ruminally and duodenally cannulated, lactating Holstein cows were used in a 3 x 3 Latin square experiment to study the effects of differing levels of nonstructural carbohydrate and degradable intake protein on ruminal digestibility and microbial protein production. Three diets were formulated to contain 1) 38 and 13.2%, 2) 31 and 11.8%, and 3) 24 and 9% nonstructural carbohydrate and degradable intake protein as percentages of the DM, respectively. Dry matter intakes were similar for all diets (21.9, 21.1, and 18.3 kg/d for diets 1, 2, and 3, respectively). Likewise, microbial efficiency, as estimated from purine analysis, was unaffected by diet and averaged 24 g of microbial N/kg of OM digested for all treatments. Ruminal digestion of OM averaged 66.6, 65.1, and 55.7% for diets 1, 2, and 3, respectively, resulting in lower microbial N flow per day for diet 3 (317, 333, and 202 g, respectively). Digestion of nonstructural carbohydrate and CP followed similar trends as did OM digestion, whereas NDF digestion remained similar across all diets. These results indicate that nonstructural carbohydrate greater than 24% and ruminally degradable protein greater than 9% of DM will enhance microbial protein flow from the rumen. PMID- 1712799 TI - Experimental cutaneous protothecosis in mice: epithelioid cell granuloma formation. AB - Prototheca wickerhamii, isolated from skin biopsy specimens of a patient with cutaneous protothecosis, were cultured in Sabouraud's medium and inoculated in the skin of 8 ICR albino mice and 3 BALB/c mice. Only in 3 ICR albino mice and 3 BALB/c mice were organisms found in skin tissue, as confirmed by identification of organisms by staining with periodic acid-Schiff (PAS) stain and culture. Histologic findings from affected nodular skin lesions indicated epithelioid cell granuloma and histiocytic cell infiltrates in the dermis and subcutaneous tissue. PMID- 1712800 TI - [The direct stimulating action of thyroliberin on the biosynthesis of total protein in cultured liver cells of rat fetuses]. AB - In experiments using fetal rat liver cultured cells TRH was shown to stimulate total protein synthesis but not RNA synthesis during long incubation. Somatostatin affected neither protein synthesis nor RNA synthesis in cultured liver cells. Possible physiological role of peripheral TRH during perinatal period in the rat is discussed. PMID- 1712802 TI - Prolongation of filter life in continuous arterio-venous haemodialysis. PMID- 1712803 TI - The pathophysiology of rhinitis. V. Sources of protein in allergen-induced nasal secretions. AB - Allergic rhinitis is characterized by a profuse rhinorrhea in addition to paroxysms of sneezing, nasal congestion, and pruritus. To define better the sources of nasal secretion produced during rhinitis, nasal allergen challenges were performed on nine atopic subjects with seasonal rhinitis. A single dose of allergen was sprayed into one side of the nose, and nasal lavages were collected bilaterally for 7 hours. Nasal lavages were assayed for protein (total protein, albumin, lactoferrin, and lysozyme) and mediator (histamine and prostaglandin D2) content. Protein concentrations increased and remained elevated above baseline levels in both ipsilateral and contralateral secretions for up to 3 hours after allergen challenge. The proportion of albumin relative to total protein (the albumin percent) increased on the ipsilateral side, whereas the relative proportions of lactoferrin and lysozyme (the lactoferrin percent and lysozyme percent) increased on the contralateral side. Prostaglandin D2, but not histamine, increased selectively on the ipsilateral side. These data suggest that the ipsilateral protein secretory response is due to allergen-induced mast cell mediator release causing increased vascular permeability, whereas the contralateral protein secretory response is primarily a reflex-induced glandular secretion. PMID- 1712804 TI - Fel d I allergen distribution in cat fur and skin. AB - Immunohistochemical procedures were performed to ascertain Fel d I antigen (Ag) distribution in cat fur and skin biopsy specimens and to analyze Fel d I allergen concentrations in fur. One hundred strands of fur and 24 skin biopsy specimens (6 by 4 by 3 mm) from shaved areas were collected from 11 different cats. Freshly depilated hairs were immunostained by free-floating monoclonal anti-Fel d I, avidin-biotin-peroxidase complex, and either processed for scanning electron microscopic examination or mounted on glass slides for computer-assisted densitometric analysis (SAMBA system). Skin biopsy specimens were promptly frozen and sectioned just before the immunohistochemical processing. Densitometric analysis of fur demonstrated that immunoprecipitate concentrations were tenfold higher at the root than at the tip. However, this finding may be explained by decrease of the thickness of the hair cortex that varied in similar proportions. The Ag accumulated on the strand surface but may focally penetrate into the medulla through the scale-like cortical interstices. In skin biopsy specimens, Fel d I Ag was found in epithelial squamous cells, within the epidermis and hair follicles, on the surface of the epidermis and hair follicles, and in sebaceous gland cells. These data suggest that Fel d I Ag is produced by sebaceous cells and, to a lesser extent, by basal squamous epithelial cells and that it is stored mainly on the surface of the epidermis and fur. PMID- 1712801 TI - Low molecular weight hydroxyethyl starch 6% compared to albumin 4% during intentional hemodilution. AB - Intentional normovolemic hemodilution was chosen as the model to compare a 6% low molecular weight hydroxyethyl starch (LMW HES) to 4% albumin. The study ran over the plasma exchange period for 24 h. Nine patients, scheduled for abdominal aortic surgery, were included in each group. After basal measurements, blood was withdrawn and simultaneously replaced by either 4% albumin (Group 1) or 6% LMW HES (Group 2) to achieve a final hematocrit of approximately 30%. Hemodynamic blood oxygen gas and hormonal plasma levels were determined before hemodilution then at 30 min, 1, 2, 3, and 24 h after the end of hemodilution. Basal value for total blood volume was 4377 +/- 162 ml in group 1 and 4138 +/- 315 ml in group 2. As in both groups the decrease in blood cell volume was exactly compensated by the increase in plasma volume, no significant change in total blood volume (respectively 4432 +/- 159 and 4305 +/- 267 ml) was observed. Throughout the study, in both groups, no significant change in mean arterial and right atrial pressures was observed. In group 2 (LMW HES), a significant increase of pulmonary capillary wedge pressure was noted 120 min after hemodilution. After hemodilution, despite a significant decrease in arterial oxygen O2 content, systemic oxygen transport did not significantly vary until 24 h in relation to the increased cardiac index. An increase in O2 extraction was observed after the exchange but no further increase was observed until the 24 h. No significant changes either in global O2 consumption or in lactate concentration were detected.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712805 TI - [The value of immunohistochemistry in the analysis of axillary node dissection]. AB - Immuno-histochemistry (IHC) was used and compared with Hemalum-Eosin-Safran (HES) in the analysis of the products of axillary lymphatic clearance in 42 women had breast cancer. In 32 patients who were negative by the HES test there was no lymph metastasis found by the IHC test in the 365 lymph nodes which were examined. In 3 out of 10 patients who were positive which the HES test 2 extra lymph node invasions were found, and one breach in the lymph node capsule that had not been diagnosed through HES testing were found. A review of the literature shows that the IHC test has never been show to be inferior to the HES test. The number of additional nodes that have been invaded varies between 0.9-11% in infiltrating canalicular carcinomas. The changes of the IHC test is greater in infiltrating lobular carcinomas. It varies between seven and 33%. Although the technique for using IHC is longer, it is easier to read the result and this method should be set up in current practice. PMID- 1712806 TI - Role of intrastructural/intermolecular help in immunization with peptide phospholipid complexes. AB - The design of effective subunit vaccines requires the inclusion of both B and T cell epitopes. The best mechanism for including both types of epitopes within an Ag is dependent upon how the Ag is processed by the APC for presentation to a responsive Th cell. If it is more efficient to process a single molecule for both helper and primary epitopes, than covalent linkage of B cells and T cell epitopes for intramolecular presentation of help would be recommended. If however, separate peptides containing either B or Th cell epitopes could be included within a single complex for the elicitation of intermolecular/intrastructural help, more antigenically diverse structures could be designed. This paper reports that it is possible to generate intermolecular/intrastructural help within an antigenic peptide-phospholipid (PL) complex. These peptide-PL complexes use well defined epitopes from Plasmodium falciparum as Ag. In addition to generating intrastructural help, we have shown that the Ir to these peptide-PL complexes is controlled by Ir genes and is similar to the Ir to the circumsporozoite protein of this pathogen. PMID- 1712807 TI - Evidence that thymocyte-activating molecule is mouse CD26 (dipeptidyl peptidase IV). AB - We previously described a developmentally regulated, Mr 115,000 (reduced) and 110,000/128,000 (nonreduced) mouse T cell-activating molecule (THAM) also expressed on a variety of epithelial cell surfaces, and associated with neutral exoaminopeptidase activity. In the present study, we show that THAM is the mouse counterpart of the human T cell-activating ectoenzyme CD26 (dipeptidyl peptidase IV, DPP IV) and that highly purified THAM lacks neutral exoaminopeptidase activity. This conclusion is based on the following: 1) the N-terminal segments of the THAM Mr 110,000 and 128,000 components shared the same amino acid sequence with the rat DPP IV. These N-termini comprised a short intracytoplasmic tail of six residues followed by a downstream hydrophobic transmembrane segment. 2) THAM specific mAb H194-112-Affi-Gel immunoadsorbent was capable of removing DPP IV enzymatic activity from mouse thymoma cell detergent extracts. 3) H194-112 reactivity pattern on developing thymocytes was found to parallel that previously reported for membrane-bound DPP IV enzymatic activity. The extent of THAM N glycosylation, as measured by N-glycanase treatment of H194-112 immunoprecipitates, was found to be similar to that of human and rat DPP IV (i.e., approximately 20 kDa). Cross-linking experiments indicated that THAM was expressed at the cell surface as a dimer of approximately 220 kDa. Its two subunits were found to be structurally related but not identical as shown by their different Mr under nonreducing conditions and by their slightly distinct peptide profiles after proteolytic cleavage. We conclude from these data that DPP IV, in addition to its extracellular matrix receptor and ectoenzymatic functions, is a T cell-activating structure in both human and mouse species. PMID- 1712808 TI - In vitro unresponsiveness to autologous sequences of the immunopathogenic autoantigen, S-antigen. AB - Previous analyses of T cell recognition sites on immunopathogenic neural autoantigens have demonstrated, using LEW rats, the functional dissociation of in vitro proliferative responses and the ability to actively induce autoimmune diseases. In experimental autoimmune uveoretinitis, immunization of LEW rats with bovine retinal S-Ag reveals the presence of three immunodominant T cell recognition sites located in regions containing sequence differences between bovine and rat S-Ag. Immune responses of LEW rats to self (rat) and nonself (bovine and human) peptide homologues representing these three sites were compared. The immunodominant sequences of heterologous S-Ag were found to predict new pathogenic T cell recognition sites in the corresponding autologous rat sequence. Furthermore, in vitro proliferative responses to the pathogenic autologous sequences are dramatically diminished relative to the responses of lymphocytes raised to the non-self homologues. A pathogenic T cell line, R858, efficiently transferred disease, but was unresponsive to the autologous S-Ag peptide in proliferation assays. However, responses to autologous peptides were readily detected using nonirradiated splenic APC. Detection of responses to non self peptides was independent of this radiosensitive Ag-presenting activity. The lack of in vitro proliferative responses to pathogenic autologous sequences by T cells bearing self-specific receptors, contrasted with the strong proliferation induced by non-self peptide homologues, suggests a mechanism of unresponsiveness to self. PMID- 1712809 TI - Cerebral vascular endothelial cells are effective targets for in vitro lysis by encephalitogenic T lymphocytes. AB - Lysis of cerebral vascular endothelial cells (EC) by CD4-positive, myelin basic protein-specific encephalitogenic T cell lines was investigated. Unstimulated EC were not lysed, but culture in the presence of murine rIFN-gamma resulted in the expression of class II MHC (Ia) molecules and the concomitant ability to function as effective target cells for lysis. The possible requirement for Ia molecules was further demonstrated by antibody-blocking experiments. Lysis of EC targets also required the presence of specific Ag (myelin basic protein); PPD-specific T cell lines also lysed the PPD-pulsed EC. In all cases, lysis was directly proportional to E:T ratios. In addition, continuous passage of T cell lines resulted in the concomitant loss of encephalitogenicity and ability to affect EC lysis, indicating a possible relationship between these two factors. These results demonstrate that CD4+ T cells interact with cerebral vascular EC. It is suggested that such interactions may be important in the pathogenesis of diseases involving migrations of these cells across the blood-brain barrier. PMID- 1712810 TI - Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy. AB - Normal mice were injected with IL-7 (500 ng, twice daily) for various periods of time up to 6 days and the cellularity and phenotypic composition of the thymus, spleen, lymph node, and bone marrow was assessed. After 6 days of treatment, significant increases in the cellularity of the spleen, lymph node, and bone marrow were observed which returned to the normal range within 6 days after cessation of treatment. After 3 days of IL-7 treatment, increased numbers of B220+/surface(s) IgM- bone marrow cells were observed. After 6 days of treatment, these numbers were still further increased and a significant population of B220+/sIgM- cells were observed in the spleen. The numbers of c mu+/sIgM- cells were also increased in the IL-7-treated mice. Analysis of the expression of B220 and BP-1 on the sIgM- bone marrow cells revealed that the B220+/BP-1+ population was dramatically increased after IL-7 treatment and the size of the B220+/BP-1- population did not differ from control mice. The pre-B cell numbers declined rapidly after the cessation of IL-7 treatment. After 6 days of IL-7 treatment, a twofold increase in the number of B cells in the spleen and lymph node was observed. The B cell numbers declined to normal values within 6 days after the cessation of IL-7 administration. In the spleens of the IL-7-treated mice, there was a significant increase in the number of B cells with an immature phenotype (e.g., sIgMhi/sIgDlo, decreased levels of Ia and FcR expression). The numbers of CD8+ and CD4+ T cells were also increased in the lymph node and spleen of the IL 7-treated mice. These numbers declined to normal levels after the cessation of IL 7 treatment. PMID- 1712812 TI - HLA-binding regions of HIV-1 proteins. II. A systematic study of viral proteins. AB - To detect HLA-binding peptides in 10 HIV-1 proteins (Rev, Tat, Vif, Vpr, Vpu, Gag p18, Gag p24, Gag p15, Env gp120 and Env gp41), the peptide binding assay (PBA) has been performed using three HLA class I molecules. Correlations have been searched between the PBA results and the peptide competitor activity in a functional test using HLA-A2-restricted CTL and target cells. A correlation between the data found in the PBA and well-defined CTL epitopes could be attempted only for the three Gag proteins. For these proteins, our results are in agreement with the known existence of epitopes reacting with human CD8+ CTL, with some exceptions. Together with the results reported with a panel of Nef peptides, these experiments showed that at least 18/20 of the already reported CTL epitopes from HIV-1 Gag, Nef, and Env proteins could be detected by the PBA, most (17/18) corresponding to strong reactivities. Perhaps more important, the regions of HIV 1 Gag p24 or Nef proteins that contain multiple associated CTL epitopes, with different HLA restrictions, were clearly identified by the reactivities in the PBA of several overlapping peptides and the major practical interest of the PBA might be the detection of such polyepitopic regions. Prediction are proposed in this report for 10 proteins, including several proteins for which CTL epitopes remain presently unknown. PMID- 1712811 TI - HLA-binding regions of HIV-1 proteins. I. Detection of seven HLA binding regions in the HIV-1 Nef protein. AB - The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4 18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose. PMID- 1712813 TI - Eosinophil activation on biologic surfaces. Production of O2- in response to physiologic soluble stimuli is differentially modulated by extracellular matrix components and endothelial cells. AB - Production of O2- in response to FMLP, TNF, IFN-gamma, platelet activating factor, LPS, substance P, and PMA by human eosinophils in suspension and in contact with polystyrene ELISA plastic (PL) or biologic surfaces was studied. Monolayers of human endothelial cells (HEC) or PL coated with FCS, fibronectin, laminin, collagen types I and IV, fibrinogen, or fibrin were used as biologic surfaces. Only PMA and FMLP stimulated O2- generation by eosinophils in suspension. Eosinophils residing on HEC monolayers, either untreated or treated with LPS, were unresponsive to all stimuli except PMA. PMA induced O2- generation by eosinophils on all surfaces; FMLP on all surfaces but HEC monolayers; TNF and platelet-activating factor only on PL, fibrinogen, and fibrin; LPS and substance P only on PL. PMA was equally effective on eosinophils on surfaces and in suspension, whereas the effect of FMLP was greater on eosinophils on surfaces than on eosinophils in suspension. IFN-gamma was ineffective on any of the surfaces tested. These results indicate that biologic surfaces may profoundly affect the ability of eosinophils to respond with a respiratory burst to physiologically relevant soluble stimuli, the effect varying according to the nature of both the stimulus and the surface. Since the respiratory burst generates products of oxygen reduction that are toxic to several tissue components, it follows that biologic surfaces may modulate eosinophil-induced tissue injury. PMID- 1712814 TI - Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates. AB - Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate. PMID- 1712815 TI - Activation of human neutrophils increases thrombospondin receptor expression. AB - Thrombospondin (TSP), a 450-kDa extracellular matrix protein secreted by platelets may be instrumental in triggering polymorphonuclear leukocyte (PMN) activation and mediating PMN-endothelial cell interactions. TSP alone had no effect on O-2 generation but caused a significant increase in the chemoattractant FMLP-mediated O-2 generation. Purified HBD, but not the 140-kDa COOH-terminal fragment of TSP, retained the priming activity indicating that the priming effect was mediated through the HBD of TSP. The priming of FMLP-mediated O-2 generation by TSP, and our recent studies demonstrating that TSP stimulates PMN adhesion and motility suggested the presence of specific receptors for TSP on PMN. Binding studies on unactivated PMN, using 125I-TSP and competition with excess unlabeled TSP, demonstrated 2.4 x 10(3) binding sites/cell with an apparent Kd of 7 nM. Heparin did not compete for binding as effectively as unlabeled TSP. There were 1.5 x 10(3) heparin-inhibitable binding sites/cell with an apparent Kd of 8 nM that represented approximately 60% of the TSP-specific sites. Therefore, two distinct TSP receptors appeared to exist on unactivated PMN; one interacting with the heparin-binding domain of TSP and one interacting with a different site. Treating PMN with cytochalasin B followed by FMLP caused a 30-fold increase in TSP receptor expression. Binding studies on activated PMNs revealed 7.6 x 10(4) sites/cell; 60% of which were heparin inhibitable. The majority (5.3 x 10(4) sites/cell) of receptors expressed had an affinity of approximately 20 nM. About 50% of these sites were heparin inhibitable. In addition, there were 2.3 x 10(4) higher affinity sites/cell with an apparent Kd of 6 nM. Heparin-inhibitable sites comprised 70% of the higher affinity sites. The existence of a subset of TSP receptors that were heparin-inhibitable on PMN suggests that binding of TSP may trigger functionally independent responses. Increased receptor expression and expression of two high affinity binding sites following PMN activation may modulate PMN-endothelium or PMN-basement membrane interactions localized at the blood vessel wall. PMID- 1712816 TI - Hydrocortisone inhibits rat basophilic leukemia cell mediator release induced by neutrophil-derived histamine releasing activity as well as by anti-IgE. AB - We determined the ability of hydrocortisone to inhibit rat basophilic leukemia cell mediator release induced by anti-IgE and by neutrophil-derived histamine releasing activity (HRA-N). Serotonin release induced by HRA-N and anti-IgE was inhibited by 78 +/- 5 and 70 +/- 4%, respectively (IC50 7.5 x 10(-7)M) by hydrocortisone (10(-5)M). HRA-N does not cause arachidonic acid metabolism, however, anti-IgE induced the generation of PGD2 and leukotriene (LT)C4, and the generation of both mediators was inhibited by 10(-5)M hydrocortisone (IC50 = 4.8 x 10(-7)M, and 3.6 x 10(-9)M, respectively). Inhibition required at least 5 to 6 h of hydrocortisone exposure and was maximal after 22 h. The observed effects of hydrocortisone could be reproduced by human recombinant lipocortin-I (5 x 10( 7)M). Hydrocortisone, 10(-5)M, was a less potent inhibitor of calcium ionophore A23187-mediated serotonin release and PGD2 and LTC4 generation (inhibition of 20 +/- 2, 17 +/- 10, and 37 +/- 10%, respectively). Inasmuch as A23187-induced stimulation is not dependent on receptor coupling, the enhanced ability of hydrocortisone to inhibit IgE- and HRA-N-mediated events as compared with A23187 suggests that one possible site of action of hydrocortisone may be interruption of receptor-effector signals. In the presence of arachidonic acid, hydrocortisone treated cells released as much LTB4 and PGD2 as control cells, however, serotonin release and LTC4 generation were inhibited 50 and 55%, respectively. Thus, these data suggest that hydrocortisone has three possible sites of action: 1) inhibition of phospholipase A2 activity, 2) inhibition of glutathione-s transferase, and 3) inhibition of serotonin release by a third mechanism, possibly by interrupting the coupling of receptor and effector systems. PMID- 1712817 TI - A single peptide from the major outer membrane protein of Chlamydia trachomatis elicits T cell help for the production of antibodies to protective determinants. AB - The protective immune response to infection with Chlamydia trachomatis is associated with antibody reactivity to serovar-specific determinants on the major outer membrane protein (MOMP). Because this immunity is T cell dependent, it is essential to define those Th cell determinants that promote natural boosting of the protective antibody response. The gene for MOMP of serovar B was separated into nine overlapping fragments that represent the five C and four V regions. These fragments were expressed as fusion peptides with GST and used to identify the regions of the MOMP that contain T cell determinants recognized in BALB/c mice. We identified peptides that elicit a T cell response to Chlamydia by immunizing mice with the fusion peptides and testing the proliferative response of T cells in vitro to intact organism. For analysis of determinants seen after infection, animals were inoculated with live organism and the T cell proliferative response to each fusion peptide was measured in vitro. In contrast to proliferative analysis in which several regions of the MOMP elicited T cell responses, functional analysis demonstrated that a single fusion peptide, containing V segment three, elicited T cell help in vivo for the production of high titered antisera, specific for protective determinants on the MOMP. PMID- 1712818 TI - Molecular characterization of the mouse mannose-binding proteins. The mannose binding protein A but not C is an acute phase reactant. AB - Mannose-binding proteins play a role in first line host defense against a variety of pathogens. We report the molecular cloning of two mouse mannose-binding proteins designated A and C based on their close identity with their rat homologues. The deduced amino acid sequence of the mouse mannose-binding proteins, as with rat and the human forms, have an NH2 terminus that is rich in cysteine that stabilizes a collagen alpha helix followed by a carboxyl- terminal carbohydrate binding domain. We further show that the mouse mannose-binding protein A mRNA, as with the human, is induced like the acute phase reactant serum amyloid P protein, yet the expression of mouse mannose-binding protein C mRNA is not regulated above its low baseline level. The expression of both mannose binding proteins A and C mRNA is restricted to the liver under basal and stress conditions. PMID- 1712819 TI - Immunocytochemical analysis of the cellular infiltrate in primary regressing and non-regressing malignant melanoma. AB - Spontaneous regression occurs in a small proportion of malignant melanomas, and it is important to understand the processes involved in its induction as this may give a guide to future therapies for this disease. We have examined 36 primary malignant melanomas (19 regressing, 17 non-regressing) and identified the cellular phenotypes and activation states of the cells infiltrating regressing and non-regressing primary melanomas by immunochemistry. We have found a significantly increased number of CD3-positive cells and an increased ratio of CD4/CD8-positive cells infiltrating regressing compared to non-regressing tumors. In addition, the expression of the interleukin 2 receptor, an activation marker for T cells, was increased. However, there were no significant differences in class II MHC, CD1, intercellular adhesion molecule 1 (ICAM1), or melanoma associated differentiation-antigen expression in these tumors. These data are consistent with melanoma regression being induced by activated CD4 T cells and do not seem to be related to the differentiation markers we have examined on these tumors. PMID- 1712820 TI - C5a, cutaneous mast cells, and inflammation: in vitro and in vivo studies in a murine model. AB - To evaluate further the interactions of C5a and mast cells in cutaneous inflammation, the ability of human native C5a (nC5a) (10 to 500 ng/ml) and human recombinant C5a (rC5a) (10 ng/ml to 100 ng/ml) to induce histamine release from purified BALB/c cutaneous mast cells (CMC) and peritoneal mast cells (PMC) was analyzed. It was found that nC5a induced histamine release from CMC but not from PMC, with a maximal net release at 250 ng/ml nC5a (22.8 +/- 2.6%). Kinetic experiments demonstrated that nC5a-induced maximal net histamine release occurred 5 min after the presentation of this stimulus (25.8 +/- 6.0%). Using rC5a and CMC, dose-response studies indicated a maximal net release of 7.0 +/- 1.7% at rC5a of 10 ng/ml, and kinetic studies showed a maximal net release at 5 min of incubation (12.9 +/- 1.6%). Release induced by rC5a was calcium-dependent, and peaked at 30 degrees C. These results indicate that functional heterogeneity exists between the CMC and the PMC of BALB/c mice, that C5a is a relevant stimulus for characterization of this heterogeneity, and that CMC from these animals can serve as a convenient in vitro model for the study of human C5a-mast cell interactions. In vivo, injections of nC5a (25-100 ng) and rC5a (25-100 ng) into the skin of BALB/c mice induced an increase in cutaneous vasopermeability, as assessed by the extravasation of intravenously injected 125I-bovine serum albumin. nC5a induced a dose-dependent increase in vasopermeability, whereas alterations induced by rC5a plateaued at 50 ng. The C5a-induced vasopermeability was markedly enhanced in animals that had been previously treated with an inhibitor of serum carboxypeptidase, which converts C5a to the less potent derivative, C5a des Arg. These findings suggest that carboxypeptidase plays an important role in vivo in the modulation of C5a-induced cutaneous inflammation in murine skin. PMID- 1712821 TI - Identification of basement membrane zone antigens defined by antibodies that react to both the epidermal and dermal side of 1 M sodium chloride split skin. AB - Some individuals have basement membrane zone (BMZ) antibodies that react to both the epidermal and dermal side of skin split with 1 M NaCl. To examine the significance of this combined staining pattern, we tested sera from 185 different, sequential, patients with BMZ antibodies for reactivity to normal human skin split with 1 M NaCl. Six sera (3.2%) stained both the epidermal and dermal sides of split skin, 173 (93.5%) stained only the epidermal side, and 6 (3.2%) only the dermal side. All six sera with a combined staining pattern yielded the same pattern when tested with three different specimens of skin, indicating that this pattern is reproducible. By immunoblot analysis, five (83%) of the six combined staining sera reacted to a 160-kD antigen present only in epidermal extracts of normal skin, one reacted in addition to a 230-kD epidermal antigen, and one did not react to either epidermal or dermal extracts. In contrast, five (83%) of the six sera with dermal staining reacted to a 290-kD antigen present only in dermal extracts. Eighteen (90%) of twenty representative epidermal staining sera reacted to a 230-kD epidermal antigen and seven (35%) sera (five with the 230-kD antibody and two without) also reacted to the 160-kD epidermal antigen. Affinity purified antibody to the 160-kD antigen defined by combined staining sera reacted to the BMZ of normal human skin. These results indicate that the combined staining pattern on 1 M NaCl split skin is due to the presence of a distinctive antibody response directed predominantly to a 160-kD BMZ antigen located on the epidermal side of the split skin and to an as yet unidentified BMZ antigen located on the dermal side. PMID- 1712822 TI - Expression of c-jun, jun-B, and c-fos proto-oncogenes in human primary melanocytes and metastatic melanomas. AB - Analysis of the regulation of c-jun, jun-B, and c-fos RNA transcript expression was performed in human primary melanocytes and metastatic melanoma cell strains. The medium requirements for human melanocyte in vitro growth are phorbol esters, agents that elevate intracellular cAMP levels, hormones, and growth factors. Cellular jun, jun-B, and c-fos gene expression are known to be affected by growth promoting agents. In primary melanocytes, the expression of c-jun, jun-B, and c fos RNA transcripts was dependent on the growth-promoting agents present in the medium. Uniformly high c-jun, jun-B, and c-fos RNA transcript levels were observed in melanocytes cultivated in complete medium. Higher levels of c-jun RNA transcripts and low levels of c-fos RNA transcripts were observed in melanocytes cultivated in plain medium. In contrast, a range of c-jun, jun-B, and c-fos RNA transcript levels was detected in metastatic melanoma cell strains cultivated in medium with or without serum. In general, an increase in jun-B and c-fos RNA transcript expression and a decrease in c-jun RNA transcript expression was observed in metastatic melanomas compared to neonatal melanocytes. These data suggest a potential role for c-jun, jun-B, and c-fos genes in the transformation of melanocytes to malignant melanoma. PMID- 1712824 TI - Effects of interferons on cultured human melanocytes in vitro: interferon-beta but not-alpha or -gamma inhibit proliferation and all interferons significantly modulate the cell phenotype. AB - The effects of human recombinant interferon-alpha-2a (rIFN-alpha), natural interferon-beta (nIFN-beta) and recombinant interferon-gamma (rIFN-gamma) on the proliferation, morphology and antigen expression of cultured human melanocytes were studied in vitro. The investigations were performed in 12-O tetradecanoylphorbol-13-acetate (TPA)- and serum-containing melanocyte growth medium (MGM), in TPA- and serum-free complete melanocyte medium (CMM) and its mitogen reduced variant (RMM). In MGM, none of these interferons inhibited the growth of normal melanocytes at concentrations 1-10,000 international units (IU)/ml over a period of 5 d. Only nIFN-beta, dose dependently, inhibited melanocyte proliferation in CMM and RMM in a 6- and 12-d assay (growth inhibition at 10,000 IU/ml; 77-80% of the controls, p less than 0.001). In contrast, rIFN alpha and rIFN-gamma exerted no (RMM), or minor effects (CMM) on melanocyte proliferation (only in 12-d assays at 10,000 IU/ml: 24% and 21% of the controls respectively, p less than 0.01). In parallel experiments performed on melanoma cells, all three interferons were potent inhibitors of proliferation in a 5-d serum-free assay (growth inhibition at 10,000 IU/ml; rIFN-alpha 59%, nIFN-beta 78%, rIFN-gamma 56%, all p less than 0.001). In addition, nIFN-beta and also rIFN gamma caused striking morphologic changes of normal melanocytes in vitro. Especially under greater than or equal to 10 IU/ml rIFN-gamma cytoplasmic spreading and flattening of the cultured melanocytes and their nuclei were seen, thus resembling melanoma cells in vitro. Untreated human melanocytes grown in MGM showed high expression of the melanoma-associated antigens HMB-45 (95-100%) and K.1.2 (40-100%), whereas the progression marker A.1.43 was present only on less than 5% of the cells. Cultured melanocytes were 95-100% positive for histocompatibility antigen class I (HLA-I), 30-75% were positive for ICAM-1, whereas they were negative for HLA-DR. After treatment with rIFN-alpha, increased expression of HLA-I antigens was found; nIFN-beta and rIFN-gamma decreased the labeling with HMB-45 (75-100%) and with K.1.2 (25-80%), whereby the expression of A.1.43 was found slightly increased (5-15%). The HLA class I antigens were upregulated by both nIFN-beta and rIFN-gamma, nIFN-beta being the most potent agent. Also, both nIFN-beta and rIFN-gamma increased the expression of ICAM-1 (nIFN-beta, 75-90%; rIFN-gamma, 90-95%) and induced de novo expression of HLA-DR antigen (nIFN-beta, 15-20%; rIFN-gamma, 65-95%).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712823 TI - Transient expression of mouse hair keratins in transfected HeLa cells: interactions between "hard" and "soft" keratins. AB - Although it has been shown previously that an acidic (type I) "soft" keratin can interact with many basic (type II) "soft" keratins to form 10-nm intermediate filaments, it has been unclear whether "soft" keratins are compatible with the "hard" keratins typically found in hair and nail. To address this issue and to generate more structural information about hard keratins, we have isolated and sequenced a cDNA clone that encodes a mouse hair basic keratin (b4). Our sequence data revealed new information regarding the structural conservation of hard keratins as a group, being significantly different from soft keratins. Using expression vectors containing appropriate cDNA inserts, we studied the expression of this basic (b4) as well as an acidic (a1) mouse hair keratin in HeLa cells. The expression of these alien hair keratins in the transfected cells was surveyed using a panel of monoclonal and polyclonal antibodies. Our results indicated that the basic and acidic hair keratin readily incorporated into the existing endogenous soft keratin network of HeLa cells. Overproduction of hair keratin, however, occasionally led to the formation of cytoplasmic aggregates containing both hard and soft keratins. These data suggest that although small amounts of newly synthesized hair keratins can incorporate into the "scaffolding" of the preformed soft keratin filament network, possibly through dynamic subunit exchange, overproduction of hard keratins can lead to the partial collapse of the soft keratin network. These observations, along with the deduced amino acid sequence data, support and extend the concept that hard and soft keratins, although closely related, are divergent enough to justify their being divided into two separate subgroups. PMID- 1712825 TI - Correlation between dermal interstitial immunoglobulin G and hypergammaglobulinemia. AB - The diffuse dermal immunofluorescence (DDIF) observed in human skin biopsies that is produced by fluorochrome-conjugated antisera reactive with human immunoglobulin G (IgG) has commonly been viewed in the past as an artifact of direct immunofluorescence microscopy. However, it has been our belief that DDIF, which has been observed in 14% of the biopsies processed in our laboratory, might represent an in vivo phenomenon of excess unbound dermal interstitial immunoglobulin G reflective of elevated serum IgG levels. To examine this hypothesis, we carried out a blinded prospective study of the frequency of DDIF in two groups of age- and sex-matched patients that differed with respect to the presence or absence of serum hypergammaglobulinemia (gamma globulin greater than 2.5 mg/100 ml). The subjective intensity of DDIF and the fluorescein isothiocyanate (FITC) conjugated anti-human IgG antibody titer at which point DDIF disappeared were both compared with serum gamma globulin levels. Also, the amount of IgG that could be extracted from biopsy specimens by an overnight phosphate-buffered saline (PBS) elution procedure was quantified by solid-phase immunoassay. We noted a trend of increased DDIF intensity with increasing serum gamma globulin levels. In addition, there was a relatively weak but significant positive correlation between DDIF endpoint titers and serum gamma globulin levels (p = 0.05, r = 0.429). The strongest positive statistical correlation observed in the study was between the quantities of IgG that could be eluted from skin biopsies and serum gamma globulin levels (p = 0.01, r = 0.599). These findings suggest that DDIF is a true biologic phenomenon reflective of elevated serum gamma globulin levels and demonstrate that simple overnight elution of unbound dermal IgG can unmask the presence of disease-related, specifically-bound IgG. PMID- 1712826 TI - [Selective staining of DNA fragments on gels depending on their AT contents]. PMID- 1712828 TI - Retrograde axonal transport of locally synthesized phosphoinositides in the rat sciatic nerve. AB - Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo [3H]inositol into the rat sciatic nerve (50-55 mm distal to L4 and L5 dorsal root ganglia), a time-dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10-12-fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water-soluble label in the dorsal root ganglia still remained approximately 1.0, whereas the corresponding ratio in the chloroform/methanol soluble fraction was approximately 20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injection site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia. PMID- 1712827 TI - Effect of ethanol on gamma-aminobutyric acid and glycine receptor-coupled Cl- fluxes in rat brain synaptoneurosomes. AB - Chloride fluxes in synaptoneurosomes in response to additions of gamma aminobutyric acid, glycine, and ethanol were measured using a chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). The Cl- gradient was directed outward by bathing cells in a medium low in Cl- concentration. The synaptoneurosomes responded to both gamma-aminobutyric acid and glycine by outflow of Cl- ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of Cl- ions from the synaptoneurosomes. Both picrotoxin and strychnine inhibited this effect. When the antagonists were used together, the inhibiting effect was additive. These results indicate that ethanol affects both gamma-aminobutyric acid and glycine receptor-linked chloride fluxes in the rat brain. PMID- 1712830 TI - Molecular cloning and expression of biologically active human glia maturation factor-beta. AB - Glia maturation factor-beta, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor beta, we screened a human brainstem cDNA library in lambda gt11. A 0.7-kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides. This polypeptide, human recombinant glia maturation factor-beta, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein. PMID- 1712829 TI - Effects of surgical sympathetic denervation on myo-inositol trisphosphate production and contraction in the dilator and sphincter smooth muscles of the rabbit iris: evidence for interaction between the cyclic AMP and calcium signaling systems. AB - The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-1 methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced. (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation. CCh-stimulated IP3 production and contraction are attenuated. (c) The increase in tension by a maximal effective dose of NaF (209 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation. (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased. in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively. (e) Isoproterenol (5 microM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively. (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by approximately 30% of their respective controls. (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are caused by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to the hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle. PMID- 1712831 TI - K(+)-evoked depolarization stimulates cyclic AMP accumulation in photoreceptor enriched retinal cell cultures: role of calcium influx through dihydropyridine sensitive calcium channels. AB - The effect of membrane depolarization on cyclic AMP synthesis was studied in glia free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures. PMID- 1712832 TI - Effects of inhibitors of oligosaccharide processing on P0 protein synthesis and incorporation into PNS myelin. AB - Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin. PMID- 1712833 TI - Fetal hemoglobin of transfused neonates and spectrophotometric measurements of oxyhemoglobin and carboxyhemoglobin. AB - The records of 32 neonates in an intensive care unit were examined retrospectively to determine if fetal hemoglobin concentrations could be predicted on the basis of gestational or postnatal age, or on the volume of red blood cell transfusions. In nontransfused neonates, the correlation between measured concentrations of fetal hemoglobin and post-natal age was r = 0.53 with a 17.2 standard error of prediction. In these same neonates, the correlation between measured fetal hemoglobin divided by birth weight and gestational age was r = 0.70, with a 9.6 standard error of prediction. A three-variable regression equation (the latter two variables plus calculated fetal hemoglobin) was found to have a high correlation with data for measured fetal hemoglobin (r = 0.97) and a relatively low 8.4 standard error of prediction. In transfused neonates, however, measured hemoglobin concentrations divided by birth weight correlated poorly with gestational age (r = 0.30 and a 12.4 standard error of prediction). In addition, the transfused neonates had low correlations when fetal hemoglobin concentrations alone were compared with the total volume of red blood cell transfusions (r = 0.35) and with postnatal age (r = 0.18) and the standard errors of prediction were all approximately 17. The correlations found between concentrations of fetal hemoglobin and age in transfused neonates were poorer than those reported in earlier nontransfused infant studies. Previous studies have also shown that neonatal blood containing fetal hemoglobin interferes with the spectrophotometric measurements of carboxyhemoglobin and oxyhemoglobin. Because of the imprecision in the predictions of fetal hemoglobin using age, weight, or the volume of transfusion, we conclude that fetal hemoglobin should be measured if accurate spectrophotometric determinations of carboxyhemoglobin and oxyhemoglobin are desired. PMID- 1712834 TI - The effects of neurokinin A, neurokinin B, and eledoisin on substance P analysis. AB - Commercial sources for neuropeptide radioimmunoassays have made this sensitive tool available to clinical investigators for monitoring the potential involvement of neuropeptides in pain modulation. We measured substance P-like immunoreactivity in the plasma, saliva, and pericardial fluid of subjects with and without pain (chronic and acute) to determine if substance P levels are altered. Some recent studies have suggested that substance P in various body fluids may be a correlate of chronic pain. To test this correlation it is important to ensure that the assay is measuring what it was designed to measure. Therefore, the influence of three tachykinins on the analysis of substance P concentrations was assessed with a commercially available radioimmunoassay kit. A small (approximately 2 to 6%), apparently nonspecific elevation in measured substance P was found when alpha-neurokinin, beta-neurokinin, or eledoisin was incubated with substance P and its antibody. Our results also indicate an apparent specific affinity of the substance P antibody for alpha-neurokinin (above 1,000 pg/ml) and beta-neurokinin (above 5,000 pg/ml). Substance P levels in the body fluids we tested ranged from 0.47 to 62.88 pg/mg protein (47.4 to 230.8 pg/ml). Levels of the tested tachykinins have not been determined in body fluids. If alpha-neurokinin or beta-neurokinin is found to be present in high concentrations in these fluids, this commercially available substance P kit may overestimate substance P levels. The concentrations of tachykinins necessary to interfere specifically with the assay are 10- to 100-fold higher than substance P in body fluids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712835 TI - Effect of daily etidronate on the osteolysis of multiple myeloma. AB - Progressive bone disease in multiple myeloma frequently leads to osteolysis, bone resorption, pathologic fractures, vertebral compression, and hypercalcemia. We conducted a double-blind study in 173 newly diagnosed multiple myeloma patients of etidronate disodium (EHDP), a diphosphonate compound that reduces bone resorption by inhibiting osteoclastic activity. The patients were randomly assigned to receive oral EHDP 5 mg/kg/d or placebo until death or discontinuation due to intolerance or refusal. The extent of vertebral deformity was measured by a vertebral index as well as height. The frequency of pathologic fractures, hypercalcemia, and bone pain was regularly assessed, as well as size and number of osteolytic lesions. All patients received melphalan and prednisone daily for 4 days every 4 weeks as the primary chemotherapy for their disease. Although the repeated measures analysis showed a significant height loss, there was no difference between treatment arms (P = .98). There was no significant difference in bone pain, episodes of hypercalcemia, or development of pathologic fractures. Patients on EHDP showed less deterioration in their vertebral index, but this difference only approached statistical significance (P = .07). We conclude that EHDP therapy used in this dosage schedule does not have a clinically significant impact in multiple myeloma. PMID- 1712836 TI - Treatment of advanced-stage Hodgkin's disease: alternating noncrossresistant MOPP/CABS is not superior to MOPP. AB - One hundred twenty-five assessable patients with advanced-stage Hodgkin's disease were randomized to receive mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) or MOPP alternating with lomustine (CCNU), doxorubicin, bleomycin, and streptozocin (CABS). The median follow-up is 7.7 years. The complete response rate was 60 of 66 MOPP-treated patients (91%) and 54 of 59 MOPP/CABS-treated patients (92%) (difference not significant). The level of the disease-free survival curve at longest follow-up is 65% for MOPP-treated patients and 72% for MOPP/CABS-treated patients (difference not significant). The overall survival at 12 years is projected at 68% for MOPP-treated patients and 54% for MOPP/CABS-treated patients (difference not significant). Thus, there were no significant differences in efficacy between MOPP and MOPP/CABS. However, MOPP/CABS was more emetogenic than MOPP, and four MOPP/CABS-treated patients went on to develop secondary acute leukemia. No MOPP-treated patients developed leukemia. High initial erythrocyte sedimentation rate (ESR) and high platelet counts adversely affected treatment outcome. MOPP-treated patients who received greater than 81% of the projected dose intensity of vincristine over the first three cycles had significantly improved disease-free survival rates over those receiving less than 81%. MOPP/CABS-treated patients who received greater than 82% of the projected dose intensity of vincristine had significantly better overall survival than those who received less than 82%. Disease-free survival on both arms was significantly better in patients who received greater than 84% of the projected dose intensity of all agents. The effect of dose intensity was particularly apparent in patients with poor prognostic factors where those who received greater than 84% of the projected dose intensity of all agents had significantly improved disease-free and overall survival. PMID- 1712837 TI - Similar outcome of treatment of B-cell and T-cell diffuse large-cell lymphomas: the Stanford experience. AB - Although previous studies have suggested a relatively poor prognosis for some patients with peripheral T-cell lymphoma, the clinical significance of immunologic phenotype in diffuse large-cell lymphoma (DLCL) remains controversial. One hundred one patients with a uniform morphologic diagnosis of DLCL treated at Stanford between 1975 and 1986 with cyclophosphamide, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), vincristine, and prednisone (CHOP), methotrexate, bleomycin, Adriamycin, cyclophosphamide, vincristine, and dexamethasone ([M]BACOD), or methotrexate, Adriamycin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) chemotherapy were studied with regard to immunologic phenotype. Immunologic analysis, performed on frozen or paraffin-embedded tissue, identified 77 cases of B-cell origin, 21 cases of T cell origin, and three cases that lacked B-cell or T-cell markers. Analysis of complete remission (CR) rates (84% v 95%), 5-year actuarial freedom from disease progression (38% v 53%), and 5-year actuarial overall survival (52% v 79%) showed no statistically significant differences in prognosis between B- and T-cell patients, respectively. The 5-year actuarial survival of patients with stage IV T cell DLCL (56%) also did not differ in a statistically significant way from stage IV B-cell patients (36%). We conclude that treatment selection for DLCL should not be based on immunologic phenotype alone. PMID- 1712838 TI - Expression of different cytokeratin subclasses in human chordoma. AB - A detailed immunohistochemical characterization of different cytokeratin subclasses was performed on frozen tumour tissue from three classical chordomas. Simple epithelium cytokeratins Nos 8, 18, and 19 were detected in all tumour cells while cytokeratin No. 7 was not found. Cytokeratins characteristic of squamous differentiation, including keratinization, were generally lacking, with the exception of the varying expression of cytokeratin No. 4. Vimentin was found in all the tumours, while they lacked desmin immunoreactivity. The present study indicates the co-expression of vimentin and cytokeratins, predominantly of the simple epithelium type. In addition, chordoma cells have the ability to express cytokeratins characteristic of squamous differentiation. This finding corresponds well to the electron microscopic findings of tonofilament bundles ending in well developed desmosomes. PMID- 1712839 TI - Characterization of stretch-activated ion channels in Xenopus oocytes. AB - 1. The gating and permeation properties of endogenous stretch-activated (SA) ion channels in Xenopus oocytes have been studied using the patch-clamp single channel recording technique. 2. As estimated from the probability of being open (Po), SA channels were equally sensitive to suction or pressure. The Po was also weakly sensitive to voltage, increasing with depolarization. Channel activation did not require Ca2+. 3. Kinetic analysis of single-channel records indicated that there are three closed states and one open state. Among three closed-time distributions, the longest was the most sensitive to both pipette pressure and membrane voltage. The open time was independent of both pressure and voltage under a wide variety of ionic conditions, but was sensitive to the species of extracellular ion as follows: Na+ greater than Cs+ greater than K+ greater than Rb+ greater than Li+. The open time had a monotonic mole fraction relationship in mixtures of Li+ and K+. 4. The SA channels were cation-selective inward rectifiers. The selectivity for permeation, based on slope conductance, was: K+ greater than NH4+ greater than Cs+ greater than Rb+ greater than Na+ greater than Li+ greater than Ca2+. 5. Tetraethylammonium (TEA+) was impermeable but was not a channel blocker. 6.Open-channel current amplitude saturated with increasing extracellular K+, and was a monotonic function of the mole fraction of Li+ and K+ in mixtures of the two ions. 7. The channel has at least two separate ion binding sites: an intra-channel site suggested by the permeation data, and an allosteric site suggested by the voltage-independent effects of permeant ions on open time. A symmetric two-barrier, one-site model can quantitatively describe the permeation data. A kinetic model is proposed to quantify the gating kinetics and the effect of ion binding at the allosteric site. PMID- 1712840 TI - Membrane depolarization and intracellular Ca2+ increase caused by high external Ca2+ in a rat calcitonin-secreting cell line. AB - 1. Calcitonin secretion is regulated by the external Ca2+ concentration ([Ca2+]o) via a rise in intracellular Ca2+ concentration ([Ca2+]i). The mechanism which couples an increase in [Ca2+]o to an increase in [Ca2+]i was explored in a rat calcitonin-secreting cell line (rMTC 44-2). [Ca2+]i was monitored using Fura-2 AM, and the membrane potential or current was simultaneously measured. 2. Using the conventional whole-cell clamp, tetrodotoxin-sensitive voltage-gated Na+ channels, T- and L-type Ca2+ channels, and three types of K+ channels, the delayed K+ channel, the A-channel and the inward-rectifying channel were observed. 3. Using the nystatin-perforated whole-cell-clamp technique, the resting potential measured under current clamp in standard extracellular medium was -59.0 +/- 5.0 mV (mean +/- S.D., n = 25), and the input resistance was 3.9 +/ 1.9 G omega (n = 10). In 0.5 mM [Ca2+]o most cells (22/25) showed spontaneous action potentials. 4. An increase in [Ca2+]o depolarized the cell membrane and elevated [Ca2+]i even in the presence of 10 microM-tetrodotoxin. The rise in [Ca2+]i was greatly reduced when action potentials were inhibited by applying hyperpolarizing current. The increase in [Ca2+]i saturated with 3-4 mM [Ca2+]o. In 3 mM [Ca2+]o, [Ca2+]i was 188.9 +/- 40.5% (n = 12) of that in 0.5 mM [Ca2+]o. 5. In high [Ca2+]o the duration of action potentials was prolonged, but the action potential frequency did not always increase. In some cases it even decreased in high [Ca2+]o. 6. Two types of action potential were observed in high [Ca2+]o, one with a shorter duration and the other with a longer duration. [Ca2+]i transiently increased in association with the long-duration action potentials. These long-duration action potentials were also accompanied by a larger after-hyperpolarization. 7. Under voltage clamp, high [Ca2+]o caused a membrane conductance increase to Na+ ions. 8. Even when the membrane potential was clamped at a level below the threshold for Ca2+ channel activation, high [Ca2+]o provoked an increase of [Ca2+]i which was composed of an initial transient increase followed by a sustained increase, indicating an involvement of mechanisms other than Ca2+ influx through voltage-gated channels. The sustained increase was more frequently observed than the initial transient increase. The amplitude of the sustained phase was dependent on [Ca2+]o, and in 5 mM [Ca2+]o it was 120.9 +/- 18.9% (103-194%) (n = 58) of that in 0.5 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712841 TI - M-current noise and putative M-channels in cultured rat sympathetic ganglion cells. AB - 1. Whole-cell recordings of M-currents and single-channel recordings have been made in cultured rat sympathetic ganglion (SCG) neurones using the patch clamp technique. 2. Muscarine caused a reduction in macroscopic M-current relaxations, induced by voltage steps, and a concomitant reduction in whole-cell current noise. Power spectra of the muscarine-sensitive component of current noise were fitted with two Lorentzian components corresponding, on average, to 162 and 15 ms. The longer time constant was very similar to that of deactivation tail currents measured at the same potential. 3. The single-channel conductance at -30 mV was estimated from power density spectra and whole-cell current-variance relationships to be 1-2 pS. 4. Putative single M-channels, activated by depolarization, were identified in cell-attached and outside-out patches from cultured SCG neurones. In particular, the ensemble average of a small amplitude channel (estimated to be ca4 pS in physiological [K+]) in a cell-attached patch, exhibited a similar time dependence to whole-cell M-current. PMID- 1712842 TI - Effects of phorbol ester on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the rat. AB - 1. A comparative study was made of the effect of the phorbol ester, 12-O tetradecanoylphorbol-13-acetate (TPA) on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the anaesthetized rat and isolated permeabilized pancreatic acinar cells. 2. Cholecystokinin octapeptide (CCK8; 0.10-6.40 nmol (kg body weight)-1) induced dose-dependent increases in pancreatic juice flow, total protein output and amylase release in the anaesthetized rat. 3. Administration of TPA (10(-8) mol (kg body weight)-1) in combination with CCK8 resulted in marked attenuation of the CCK8-evoked secretory response. 4. Simultaneous injection of polymyxin B (10(-8) mol (kg body weight)-1), an inhibitor of protein kinase C, with TPA and CCK8 reversed the inhibitory effect of the phorbol ester on CCK8 induced pancreatic juice flow, total protein output and amylase release. 5. In permeabilized rat pancreatic acini CCK8 (10(-13)-10(-9) M) elicited dose dependent increases in [3H]leucine-labelled protein secretion (3H-labelled protein release). Combining TPA (10(-8) M) with CCK8 resulted in an inhibition of the CCK8-induced 3H-labelled protein release especially at lower concentrations of CCK8. At higher concentrations of CCK8, TPA was unable to inhibit the CCK8 evoked 3H-labelled protein release. Again, polymyxin B reversed the TPA-induced inhibition of CCK8-evoked 3H-labelled protein output. 6. The results indicate that protein kinase C activation may play an important physiological role in modulating the CCK8-evoked secretory response in rat pancreas in vivo and in vitro. PMID- 1712843 TI - Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones. AB - 1. The physiological and functional features of time-dependent anomalous rectification activated by hyperpolarization and the current which underlies it, Ih, were examined in guinea-pig and cat thalamocortical relay neurones using in vitro intracellular recording techniques in thalamic slices. 2. Hyperpolarization of the membrane from rest with a constant-current pulse resulted in time dependent rectification, expressed as a depolarizing sag of the membrane potential back towards rest. Under voltage clamp conditions, hyperpolarizing steps to membrane potentials negative to approximately -60 mV were associated with the activation of a slow inward current, Ih, which showed no inactivation with time. 3. The activation curve of the conductance underlying Ih was obtained through analysis of tail currents and ranged from -60 to -90 mV, with half activation occurring at -75 mV. The time course of activation of Ih was well fitted by a single-exponential function and was strongly voltage dependent, with time constants ranging from greater than 1-2 s at threshold to an average of 229 ms at -95 mV. The time course of de-activation was also described by a single exponential function, was voltage dependent, and the time constant ranged from an average of 1000 ms at -80 mV to 347 ms at -55 mV. 4. Raising [K+]o from 2.5 to 7.5 mM enhanced, while decreasing [Na+]o from 153 to 26 mM reduced, the amplitude of Ih. In addition, reduction of [Na+]o slowed the rate of Ih activation. These results indicate that Ih is carried by both Na+ and K+ ions, which is consistent with the extrapolated reversal potential of -43 mV. Replacement of Cl- in the bathing medium with isethionate shifted the chloride equilibrium potential positive by approximately 30-70 mV, evoked an inward shift of the holding current at -50 mV, and resulted in a marked reduction of instantaneous currents as well as Ih, suggesting a non-specific blocking action of impermeable anions. 5. Local (2-10 mM in micropipette) or bath (1-2 mM) applications of Cs+ abolished Ih over the whole voltage range tested (-60 to -110 mV), with no consistent effects on instantaneous currents. Barium (1 mM, local; 0.3-0.5 mM, bath) evoked a steady inward current, reduced the amplitude of instantaneous currents, and had only weak suppressive effects on Ih. 6. Block of Ih with local application of Cs+ resulted in a hyperpolarization of the membrane from the resting level, a decrease in apparent membrane conductance, and a block of the slow after hyperpolarization that appears upon termination of depolarizing membrane responses, indicating that Ih contributes substantially to the resting and active membrane properties of thalamocortical relay neurones.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712844 TI - Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones. AB - 1. Modulation of the hyperpolarization-activated cation current, Ih, by noradrenaline (NA) and serotonin (5-HT) was examined in guinea-pig and cat medial and lateral geniculate relay neurones using the in vitro slice technique. 2. In the absence of pharmacological antagonists, local application of NA resulted in a slow depolarization and decrease in apparent input conductance, a response which was blocked by local or bath application of the alpha 1-adrenoceptor antagonist prazosin. Application of NA after pharmacological block of alpha 1- and alpha 2 adrenoceptors, or application of 5-HT in all conditions, induced a 1-3 mV slow depolarization which was associated with a pronounced increase in apparent input conductance. This response to NA and 5-HT persisted during blocked synaptic transmission and was present in both the guinea-pig and cat medial and lateral geniculate nuclei. 3. The increase in membrane conductance elicited by NA was mimicked by the beta-specific agonist isoprenaline and blocked by the beta antagonists propranolol and atenolol, indicating that it is mediated by beta adrenoceptors. The response to 5-HT was blocked by the 5-HT1 and 5-HT2 antagonist methysergide, but not by the 5-HT2 antagonist ritanserin. Applications of either the 5-HT1A agonist ipsapirone or the partial agonist 8-hydroxy dipropylaminotetralin (8-OHDPAT) were without effect. 4. Current versus voltage relationships obtained under voltage clamp revealed NA and 5-HT to cause a voltage-dependent inward shift at membrane potentials negative to approximately 60 mV. This response appeared to be shared by NA and 5-HT since maximal application of 5-HT greatly reduced or abolished the response to NA. 5. Application of NA and/or 5-HT during hyperpolarizing voltage steps in voltage clamp resulted in a marked increase in amplitude of the hyperpolarization activated cation current, Ih. In addition, the rate of activation of Ih was strongly increased during activation of beta-adrenoceptors. 6. The activation curve of the conductance underlying Ih (Gh) was shifted by 4-6 mV on the voltage axis with NA and/or 5-HT. The positive shift of Gh activation in the voltage domain resulted in an increase in the amplitude of Gh which is active at resting, and more hyperpolarized, membrane potentials. The subsequent increase in resting membrane conductance decreased the responsiveness of thalamic neurones to hyperpolarizations of all durations. 7. Local or bath application of caesium blocked both Ih and the increase in membrane conductance in response to NA and 5 HT. By contrast, barium blocked neither Ih nor the responses to NA and 5 HT.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712845 TI - Prolonged inhibition of cardiac vagal action following sympathetic stimulation and galanin in anaesthetized cats. AB - 1. Stimulation of the right cardiac sympathetic nerve for 3 min at 16 Hz in the presence of effective beta-adrenoceptor blockade evoked prolonged attention of cardiac vagal action in the cat: 40.8 +/- 5.4% maximum inhibition of cardiac vagal action on prolonging pulse interval, with half-time to recovery of 8.3 +/- 1.4 min. 2. Intravenous injection of galanin (1.6-3.1 nmol/kg) evoked prolonged attenuation of cardiac vagal action: 40.9 +/- 8.2% maximum inhibition with a half time to recovery of 13.6 +/- 2.6 min. This effect of galanin was not significantly different from the action of sympathetic nerve stimulation. A slight depressor response (-14.4 +/- 1.9 mmHg) was seen in nine of sixteen cats. 3. Intravenous injection of neuropeptide Y (NPY) (2.8-6.3 nmol/kg) evoked slight attenuation of cardiac vagal action: 11.9 +/- 4.5% maximum inhibition of cardiac vagal action on pulse interval, with a half-time to recovery of 4.1 +/- 1.7 min. Blood pressure increased by 68.6 +/- 5.7 mmHg. 4. Following administration of guanethidine (1 mg/kg I.V.) the inhibitory effect of sympathetic nerve stimulation on cardiac vagal action was significantly reduced (P less than 0.001). The responses to exogenous NPY and galanin on vagal action were unchanged after guanethidine. 5. The prolonged attenuation of cardiac vagal action can be mimicked by exogenous galanin in the cat but not by exogenous NPY. PMID- 1712846 TI - Mechanism of action of substance P in guinea-pig ileum longitudinal smooth muscle: a re-evaluation. AB - 1. A proposed mechanism of contractile action of substance P in guinea-pig ileum longitudinal smooth muscle involving a decrease in membrane K+ permeability (PK) has been re-examined. 2. Potentiation of responses to substance P by the K+ channel blocker tetraethylammonium (TEA) was originally proposed as evidence for a mechanism of action of substance P involving a decrease in PK. Potentiation was confirmed; however this was found not to be specific to substance P since a similar potentiation of responses was seen with agonists not thought to act via a decrease in PK. 3. Antagonism of contractile responses to substance P by noradrenaline was similarly confirmed. However, this antagonism was found to represent a non-specific functional interaction through the inhibitory actions of beta-adrenoceptors rather than the proposed specific interaction with an increase in PK by noradrenaline which is normally alpha 1-adrenoceptor mediated. 4. Experiments were made measuring 86Rb efflux, in depolarized guinea-pig ileum longitudinal smooth muscle, to estimate PK. These studies confirmed a reported decrease in PK with TEA, but failed to detect the previously reported decrease with substance P. 5. These results, although not disproving a suggested mechanism of direct contractile action of substance P in guinea-pig ileum longitudinal smooth muscle involving a decrease in PK, do throw doubt on either the evidence, or its interpretation, as proposed by the original authors in support of such a mechanism. PMID- 1712847 TI - Capsaicin and sensory neuropeptide stimulation of goblet cell secretion in guinea pig trachea. AB - 1. We studied the effect of capsaicin and sensory neuropeptides on tracheal goblet cell secretion in anaesthetized guinea-pigs using a semi-quantitative morphometric technique whereby the magnitude of discharge of stained intracellular mucus, expressed as a mucus score (MS), was related inversely to discharge. 2. Capsaicin (i.v.) induced goblet cell secretion: a decrease of 50% in MS below control (indicative of increased secretion) was maximal at 3.3 x 10( 9) mol/kg. 3. Capsaicin-induced secretion was unaffected either by prior vagus nerve section or by pre-treatment with atropine, propranolol and phentolamine which suggests that local axon reflexes with release of sensory neuropeptides are involved in the response. 4. Intravenous substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and calcitonin gene-related peptide (CGRP) produced dose related increases in goblet cell secretion, with SP the most potent. Doses (mol/kg) causing a 50% decrease in MS from control were 3.5 x 10(-12) for SP; 72 x 10(-10) for NKA; 1.6 x 10(-9) for NKB; and 1.2 x 10(-8) for CGRP. The maximal increase in goblet cell secretion was 75% of control and occurred with SP at 10( 10) mol/kg. 5. SP-induced mucus discharge was not inhibited by atropine or the histamine receptor antagonists mepyramine or cimetidine. 6. We conclude that in guinea-pig trachea, goblet cell secretion is under the control of capsaicin sensitive sensory nerves and release of neuropeptides from these nerves may induce mucus discharge via tachykinin receptors of the NK-1 subtype (indicated by an order of potency of SP greater than NKA greater than NKB). PMID- 1712848 TI - Effect of platelet activating factor on formation and composition of airway fluid in the guinea-pig trachea. AB - 1. We studied the effect of platelet activating factor (PAF) on leakage of albumin, and secretion of fucose (a marker for mucus glycoprotein) and protein into the tracheal lumen of the guinea-pig isolated in situ, and on bioelectric properties and fluxes of mannitol in vitro. We also studied the effect of PAF on mucus secretion in human bronchi in vitro. 2. In guinea-pig, intravenous PAF markedly increased the luminal concentration of protein but did not significantly increase fucose concentrations. Increased albumin leakage (274% above controls at a dose of 50 ng/kg PAF) was associated with the increased luminal content of protein (248% above controls at the same dose of PAF). 3. Leakage of albumin was maximal 10 min after PAF, was significantly reduced by 20 min and had returned to baseline by 30 min. This pattern of leakage could be repeated with successive administrations of PAF. 4. PAF induced small but significant biphasic changes in bioelectric properties in vitro. The initial response was rapid in onset and characterized by maximal increases in short-circuit current (Isc) of 6.5% above controls at 7.5 min and in conductance (G) of 7% at 20 min. Both responses were blocked by the PAF receptor antagonist WEB 2086. Amiloride blocked the increase in Isc. Permeability of the tissue to mannitol (Pmann) was unaltered. The delayed response was characterized by maximal increases in Isc and G of 10% above controls at 60-90 min which were not significantly affected by WEB 2086 or amiloride. Pmann was increased by 38% at 90 min. 5. PAF increased fucose secretion in human bronchi in vitro. 6. Lyso-PAF in vitro caused changes similar to those induced by PAF on bioelectric properties and mucus secretion, but had no significant effects in vivo. 7. Light microscopy showed no evidence of epithelial disruption in animals given intravenous PAF at a dose causing significant albumin transudation. 8. We conclude that PAF increases the protein content of guinea-pig tracheal fluid principally by inducing plasma leakage rather than mucus secretion and that the small changes in ion transport and epithelial conductance may reduce the tendency to epithelial disruption during plasma leakage. PMID- 1712850 TI - Counselling and supporting parents of children with developmental delay: a research evaluation. AB - This paper describes an evaluation study of a home-based, family-focussed counselling scheme providing support for English-speaking and Bangladeshi families of children with intellectual or multiple disabilities. Mothers and children in the intervention groups showed significant and positive changes compared to randomly allocated controls. The greatest benefits were derived by the more deprived and initially less well-supported Bangladeshi families. Mothers changed positively in ratings of perceived support and family functioning, and in their constructions of their child, themselves, husbands and family relationships. Although systematic teaching was not included, their children also showed improvements in developmental progress and behaviour problems. PMID- 1712849 TI - Microleakage at the resin-alloy interface of chemically retained composite resins for cast restorations. AB - New retentive mechanisms between veneering resins and casting alloys are claimed to have a chemical bond that results in a high bond strength combined with low microleakage between the veneering resin and cast restoration. This study compared the microleakage of four chemical bonding mechanisms when three veneering resins were bonded to two dental casting alloys. Resin-veneered alloy disks were immersed in red India ink and kept at 37 degrees C for 72 hours. The disks were then bench dried for 24 hours. The resin veneer was sectioned into eight sectors in an engineering milling machine and these resin sectors were removed to display the microleakage pattern. It was concluded that (1) no microleakage was found in two combinations, and (2) the highest microleakage was with Sr-Isosit-N/Panavia EX/Firmilay combinations. PMID- 1712851 TI - Serotonergic response to spinal distraction trauma in experimental scoliosis. AB - The effects of distraction injury to the spinal cord on serotonin (5HT) content and metabolism in a rat model of scoliosis were studied. Previous studies in this laboratory (Salzman et al., 1987a) have identified the 5HT response as a major component of the posttraumatic progression of spinal injury after impact trauma in the rabbit. The present study was designed to determine the universality of this response by examining a different model of injury in a different species. The results demonstrate that distraction trauma in the rat, like impact injury in the rabbit, is associated with a rapid and robust increase in the local spinal cord content and metabolism of 5HT and a long-term depletion of 5HT below the site of injury. The roles of the blood platelet and the raphe-spinal tract in the acute response and the disruption of axoplasmic transport during the chronic phase of injury are discussed. PMID- 1712852 TI - Prostatectomy and inguinal hernia repair: a comparison of the sexual consequences. AB - This study investigated whether psychosexual changes found after surgery for benign prostatic enlargement relate specifically to the prostatectomy procedure or to the stresses of surgery in general. The sexual adjustment of 91 married men (ranging in age from 51 to 77) who had undergone either transurethral prostatectomy or inguinal hernia repair was compared using the same measures and experimental design. Results show that both surgeries appeared to result in relatively minor but widespread negative consequences for sexual adjustment and expression. Findings on both individual and couple sexual adjustment suggest that the psychosexual consequences of the two procedures do not differ substantially. As expected, the one exception was retrograde ejaculation, which was more likely to be experienced by men who had undergone prostate surgery. The results illustrate the necessity of conducting comparative studies when evaluating the sexual consequences of surgical procedures and highlight the importance of taking age into consideration when conducting research on the effects of surgery on older men. PMID- 1712853 TI - Direct sequencing from touch preparations of human carcinomas: analysis of p53 mutations in breast carcinomas. AB - A new technique for characterizing somatic mutations in very small samples of cellularly heterogeneous human cancer tissue was developed and tested using mutations in the p53 gene in breast carcinomas as a model system. The technique combines touch preparation of specimens to obtain homogeneous clusters of carcinoma cells free of normal cells with a nested pair of polymerase chain reaction (PCR) amplifications of DNA to increase the amount of target gene sequence sufficiently to permit direct sequencing of the p53 gene. Touch preparations of fresh or previously frozen tissue from human adenocarcinomas derived from several organs were stained, and clusters of 10-50 malignant cells were transferred by pipette into microfuge tubes for PCR amplification. Exons 5-9 of the p53 gene, which contain the major mutational hot spots associated with most human cancers, were sequenced by the following steps: 1) two rounds of PCR amplification using DNA Taq polymerase and two sets of oligonucleotide primers, the second set being nested within the segment amplified by the first set and having attached T7 and SP6 phage promoter sequences, 2) transcription of the amplified DNA sequences with T7 and SP6 RNA polymerases, and 3) dideoxy sequencing of single-stranded RNA transcripts with reverse transcriptase and with additional oligonucleotide primers to achieve specificity for this unique region of the genome. The utility of this approach is illustrated by our success in detecting and analyzing point mutations in cell clusters from four of 11 primary adenocarcinomas of the human breast. PMID- 1712854 TI - 1971-1991: molecular oncology comes into its own. PMID- 1712855 TI - Monoclonal antibody-defined epitope map of expressed rubella virus protein domains. AB - An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2 6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen. PMID- 1712856 TI - Isolation of an arenavirus from a marmoset with callitrichid hepatitis and its serologic association with disease. AB - Callitrichid hepatitis (CH) is an acute, often fatal viral infection of New World primates from the family Callitrichidae. The etiologic agent of CH is unknown. We report here the isolation of an arenavirus from a common marmoset (Callithrix jacchus) with CH by using in vitro cultures of marmoset hepatocytes and Vero-E6 cells. Enveloped virions 67 to 133 nm in diameter with ribosomelike internal structures were seen in infected cultures. Immunofluorescence and Western immunoblot analysis using CH-specific antisera (principally from animals exposed to CH during zoo outbreaks) revealed three antigens in cells infected with this CH-associated virus (CHV). These antigens had the same electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels as did the nucleocapsid, GP2, and GPC proteins of lymphocytic choriomeningitis virus (LCMV). Monoclonal antibodies specific for these arenavirus proteins also reacted with the three CHV antigens. Conversely, the CH-specific antisera reacted with the nucleocapsid, GP2, and GPC proteins of LCMV. CHV thus appears to be a close antigenic relative of LCMV. The serologic association of CHV with several CH outbreaks implicate it as the etiologic agent of this disease. PMID- 1712857 TI - An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag. AB - A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV 1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses. PMID- 1712858 TI - Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains. AB - A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3' terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes. PMID- 1712859 TI - Antigenicity of rabies virus glycoprotein. AB - Although the number of antigenic sites on the rabies virus glycoprotein that have been described regularly increases with time, no attempt has been made to carefully evaluate the relative importance of each of these sites. Here we provide a more precise description of the antigenicity of the protein in mice of the H-2d haplotype; we developed this description by using 264 newly isolated monoclonal antibodies (MAbs) and a collection of neutralization-resistant (MAR) mutants. Most of the MAbs (97%) recognized antigenic sites previously described as II and III. One minor antigenic site separated from site III by three amino acids, including a proline, was identified (minor site a). Despite their proximity, there is no overlap between site III and minor site a; i.e., site III specific MAR mutants were neutralized by the six MAbs defining minor site a, and vice versa. One of our MAbs, 1D1, reacted with sodium dodecyl sulfate-treated glycoprotein in Western blots (immunoblots) under reducing conditions and was therefore probably directed against a linear epitope, A MAR mutant selected with this MAb was still neutralized by MAbs of other specificities. This linear epitope was called G1 (G, Gif). As a general rule, we propose to reserve the term "antigenic site" (either major or minor) for regions of the protein which are defined by several MAbs originating from different fusions and to describe regions of the protein which are defined by a single MAb as epitopes. It would be interesting to test whether the same regions of the rabies virus glycoprotein are antigenic in mice of different haplotypes or in other species. PMID- 1712860 TI - Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells. AB - The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Muller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells. PMID- 1712861 TI - Lack of enhancing effect of human anti-human immunodeficiency virus type 1 (HIV 1) antibody on HIV-1 infection of human blood monocytes and peritoneal macrophages. AB - The influence of human anti-human immunodeficiency virus type 1 (HIV-1) antibody on HIV-1 infection of freshly isolated normal human peritoneal macrophages and blood monocytes was examined. Each of 14 HIV antibody-positive human serum samples was found to block the infection of four virus isolates (human T-cell lymphotropic virus type IIIBa-L [HTLV-IIIBa-L], HTLV-IIIB, D.U. 6587-7, and D.U. 7887-8) at serum dilutions ranging from 10(-1) to 10(-2). Three of these isolates (HTLV-IIIBa-L, D.U. 6587-7, and D.U. 7887-8) infected cultures of monocytes and macrophages rapidly and produced high levels of virus reverse transcriptase and p24 antigen. A fourth virus isolate (HTLV-IIIB) infected the monocytes and macrophages more slowly and produced low levels of viral protein. More dilute HIV antibody-positive sera had no significant effect on the overall level or rate of virus infection or expression. Complement did not appear to influence the course of infection by any combination of antisera or virus examined. Successful HIV-1 infection of the peritoneal macrophages and blood monocytes under the conditions tested showed strict dependence on CD4 since a recombinant CD4 polypeptide and an anti-CD4 monoclonal antibody effectively blocked the process. PMID- 1712862 TI - Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function. AB - The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer binding sites near the 5' ends of VL30 RNAs. PMID- 1712863 TI - Characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types 1 and 2. AB - Monoclonal antibodies (MAbs) raised against the core proteins of human immunodeficiency virus type 1 (HIV-1; laboratory strain HTLV-IIIB) and HIV-2 (strain ROD) were investigated in a variety of tests, e.g., enzyme-linked immunosorbent assay (ELISA), immunostaining of Western immunoblots, immunofluorescence, immunoprecipitation, and alkaline phosphatase anti-alkaline phosphatase assay. The MAbs were grouped according to their cross-reactions. Seven HIV-1-specific MAbs reacted exclusively with HIV-1, and five showed cross reactivity with HIV-2 and simian immunodeficiency virus of macaques in ELISA. Four of the 15 MAbs against HIV-2 reacted only with the HIV-2 protein p26. Six showed cross-reactivity with HIV-1, and five showed a broad reaction with all three viruses. Overlapping 30-amino-acid-long peptides derived from the p24 protein sequence of HIV-1 were used in an epitope-mapping system. Three different immunogenic regions (A, B, and C) could be defined. Specific regions where anti HIV-1 and -HIV-2 MAbs cross-reacted were mapped with shorter oligopeptides. PMID- 1712865 TI - Unintentional carbon monoxide-related deaths in the United States, 1979 through 1988. AB - OBJECTIVE: To describe the epidemiology of recent unintentional carbon monoxide poisoning deaths in the United States. DESIGN: Descriptive analysis of carbon monoxide-related deaths in the United States from 1979 through 1988, based on death certificate reports compiled by the National Center for Health Statistics. POPULATION STUDIED: All US deaths, 1979 through 1988. RESULTS: We reviewed data from 56,133 death certificates that contained codes implicating carbon monoxide as a contributing cause of death. Of these, 25,889 were suicides, 210 were homicides, 15,523 were associated with severe burns or house fires, and 11,547 were classified as unintentional. The number of unintentional deaths decreased steadily by about 63 deaths per year, from 1513 in 1979 to 878 in 1988. The highest death rates occurred in winter and among males, blacks, the elderly, and residents of northern states. Motor vehicle exhaust gas caused 6552 (57%) of the unintentional deaths; 5432 (83%) of these were associated with stationary automobiles. CONCLUSIONS: The rate of unintentional death from carbon monoxide poisoning is decreasing. This may be attributable to improvements in automobile pollution control systems and improved safety of cooking and heating appliances. Prevention programs should target young drivers, males, and the elderly. PMID- 1712864 TI - Host genetic background effect on the frequency of mouse mammary tumor virus induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors. AB - The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV. PMID- 1712866 TI - Coccidioidomycosis diagnosed from bone marrow smear. PMID- 1712867 TI - [Rapid assay of hyaluronic acid in serum]. AB - A method for measurement of hyaluronic acid (HA) level in serum was developed based on using "hyaluronic acid binding protein" (HABP)-coated polystyrene beads. After the beads and test serum being mixed, the mixture was incubated together with reaction buffer for 2 hours, and then the beads were washed. Subsequently, biotinylated HABP was added to the washed beads and incubated for 1 hour. Then peroxidase-conjugated avidin was added to the mixture and incubated again for 1 hour. After the beads being washed, a substrate solution was added to the washed beads and left for 1 hour. Then the reaction was stopped by adding 2N-H2SO4. The absorbance at 492 nm was recorded. The analytical range of HA in serum by this method was found to be between 10-800 micrograms/l, and the precision of the HA assay (CV%) was between 3.0-8.4 in the "with-in" assay (n = 10), and 4.8-8.9 in the "between" assay (n = 5). The analytical recovery of HA assay was between 92 115%. In this study, the results in screening of the serum HA level in RA patients (n = 107), OA patients (n = 16) and healthy subjects (n = 30) showed that the HA level of RA patients was demonstrated significantly higher than that of healthy subjects and OA patients. PMID- 1712868 TI - [Chronic lymphocytic leukemia with peripheral T lymphocytes expressing CD 2+, CD 3+, CD 4-, CD 8-, CD 16+, and CD 56+ and lymph-node lymphocytes expressing CD 2+, CD 3-, CD 4-, CD 8-, CD 16+, CD 38+, and CD 56+]. AB - A 33-year-old man was hospitalized because of thrombocytopenia and severe splenomegaly. On admission 78% of peripheral lymphoid cells were abnormally large, with pale cytoplasm. Flow cytometry of the abnormal lymphocytes showed that they expressed CD 2, CD 3, CD 11, CD 16, and CD 56, but not CD 4 nor CD 8, so they were T-cell large granular lymphocytes (T-LGL). Abnormal lymphocytes obtained from a lymph node expressed CD 2, CD 16, CD 38, and CD 56, but not CD 3, CD 4, and CD 8, so they were natural killer(NK) cells. Splenectomy was performed and the operative specimen showed diffuse infiltration of pleomorphic lymphocytes, probably chronic lymphocytic leukemia cells. After splenectomy, the platelet count returned to normal but the lymphocytosis continued. Two years after discharge, chemotherapy was done because of thrombocytopenia and hepatomegaly. The patient died of disseminated intravascular coagulation arising from sepsis. The differences and similarities between peripheral and lymph-node lymphocytes suggest that LGL and NK cells may be differentiated from the same kind of cell, somewhat differentiated from stem cells. PMID- 1712869 TI - [Immunotherapy and urological malignancy]. AB - Immunotherapy gained popularity as a treatment modality for malignant diseases in the 1960s. A number of trials, using tumor vaccines, immunopotentiators, interferons, cytokines and others, demonstrated antitumor effects in several urological malignancies, and, to date, immunotherapy plays a major role in treatment of advanced renal cell carcinoma and superficial bladder carcinoma. Interferon or interleukin-2, which became available for large scale clinical trial with the development of bioengineering, however, were shown to be not effective as initially expected, by single agent. Rational design of new strategies with multiple agents in combination based on basic and clinical research, should provide progress in treatment of urological malignancies. PMID- 1712870 TI - [Application of new intraurethral stent for higher risk patients with benign prostatic hypertrophy]. AB - We reviewed our experience of the application of a new urethral stent (PROSTAKATH) for 8 patients with prostatic outflow obstruction from December 1989 to March 1990. 6 patients had required the Foley catheter for several months because of chronic urinary retention. 2 were dys-uric patients having a higher bladder residual urine volume. All were in a high risk group for surgery. 7 out of the 8 patients were treated successfully. The stent was not placed in one. All 7 patients in whom the urethral stent was placed voided freely after placement of the stent. Bladder residual urine was not detected by the ultrasound sonography except in one patient. The urethral stent used in this study was a spring-like spiral with an outside diameter of 21 Fr, it is made of gold-plated stainless steel. Under local anesthesia, it can be easily inserted using a 6-7 Fr. ureteral catheter as a guide wire under ultrasonic scanning guidance. During the follow-up period of 5-8 months, 1 patient had an episode of migration of the stent to the bladder 2 months later, which was removed endoscopically, and a new stent was placed. Side effects were observed in 2 patients; one complained of strong discomfort and the other suffered from urge incontinence. Both symptoms were ameliorated during the follow-up period. We conclude that the urethral stent is an effective device as a non-invasive treatment of prostatic outflow obstruction. PMID- 1712871 TI - [Study on frequency of prostate carcinoma and benign prostatic hypertrophy by mass screening in Hokkaido]. AB - We conducted mass screenings for prostate diseases on male subjects over fifty years of age in three separate areas in Hokkaido. Prostate carcinoma and benign prostatic hypertrophy were searched in the 1,764 participants. Histopathologically proven prostate carcinoma was found in twenty-two (1.25%) of the 1,764 participants. This frequency of carcinoma was higher than any other carcinoma found in the mass screenings for gastric, uterine, breast and lung carcinoma in Hokkaido. Of the 22 prostate carcinomas found, 68% were in the early stage (stage B). This stage distribution was clearly distinct from that of prostate carcinoma found on the hospital visit, most of which had already progressed to an advanced stage. These results indicate that mass screening for prostate carcinoma on greater than or equal to 50 year old-male subjects is efficient in finding carcinoma of all stages but, in particular, carcinoma of early stage, when compared with mass screening for other carcinomas. BPH, defined as a moderately or markedly enlarged prostate on rectal palpation, was found in 10% of the participants. Questionnaire on subjective symptoms of voiding disturbance in the participants has confirmed that these symptoms, mainly elicited by BPH, become manifested in fifties and more frequent with age. Of thirteen patients with prostate carcinoma who received both rectal examination and prostate-related markers measurement in serum at the time of mass screening, three without induration of the prostate were diagnosed as having carcinoma from an abnormal value of the serum markers. This result suggests that the marker(s) is one of the useful screening tests for detecting carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712872 TI - [Significance of transrectal hyperthermia in the treatment of benign prostatic hyperplasia]. AB - Though the local hyperthermia for the management of benign prostatic hyperplasia has drawn much attention as one non-surgical treatment, no definite conclusion could yet be obtained in terms of the efficacy. In this study local hyperthermia was induced in evaluable 33 cases with benign prostatic hyperplasia using Primus, and the effectiveness of this modality of treatment was investigated by analysing the subjective and objective response following hyperthermia. The prostate was heated trans-rectally up to 43 degrees C with 915 MHz microwave for one hour. Hyperthermia was carried out twice a week for ten times for the sake of thermotolerance. Urinary obstructive symptoms were divided into diurnal and nocturnal frequency, urinary urgency, the degree of urinary stream, hesitancy and dribbling. Each symptom was described before and after the treatment according to the scoring system. Moreover, objective changes of urinary flow and prostatic size were estimated by the residual urine volume, uro-flowmetry, rectal palpation of the prostate and echography. Hyperthermic treatment improved urinary flow markedly, but no appreciable alteration could be observed as to the size of the prostate. The overall efficacy, including subjective and objective response, could be summarized as 37% of effectiveness, and 33% of slight effectiveness, that is, 70% of effective ratio. As to the side effect, anal pain was noted in few cases of the present series. Therefore, transrectal hyperthermia may be a suitable modality for non-surgical treatment of benign prostatic hyperplasia. PMID- 1712873 TI - [Clinical evaluation of serum basic fetoprotein for prostatic cancer--comparative study with PAP, gamma-Sm and PSA]. AB - The clinical significance of serum basic fetoprotein (BFP) in prostatic cancer was investigated together with serum prostatic acid phosphatase (PAP), gamma seminoprotein (gamma-Sm) and prostate specific antigen (PA). Investigated in this study were 40 patients with prostatic cancer, ranging in age from 50 to 85 years (mean age: 69.5 years). According to clinical staging, 3 cases (7.5%) had a stage A disease, 10 cases (25.0%) a stage B disease, 7 cases (17.5%) a stage C disease, and 20 cases (50.0%) a stage D disease. The positive rates for serum BFP, PAP, gamma-Sm, and PSA were 60.0, 45.0, 63.6, and 68.4%, respectively, and these rates increased as the stage advanced. The above results suggest that BFP is the most useful marker of the four for monitoring prostatic cancer. In a combination assay of these four markers, 29 (87.9%) of 33 patients with prostatic cancer could be diagnosed by observing an elevated serum level in one of the markers. This suggests that a combination assay of BFP, PAP, gamma-Sm and PSA in patients with prostatic cancer is useful for diagnosis and monitoring of the disease. PMID- 1712874 TI - Cytokine binding and clearance properties of proteinase-activated alpha 2 macroglobulins. AB - Multiple mechanisms are necessary to spatially and temporally restrict and direct the effects of potent mediators of inflammation, immune reactions and tissue repair. Recent studies implicate alpha 2-macroglobulin (alpha 2M) as a protein that regulates the distribution and activity of many cytokines, including transforming growth factors-beta (TGFs-beta), tumor necrosis factor-alpha (TNF alpha), platelet derived growth factor (PDGF), interleukin-6 (IL-6), nerve growth factor (NGF), fibroblast growth factor (b-FGF), and interleukin-1 beta (IL-1 beta). Some cytokines, including PDGF, NGF, and IL-6 bind preferentially to the native secreted form of alpha 2M, whereas the TGF-beta s, TNF-alpha and IL-1 beta bind preferentially to forms of alpha 2M that have been modified by proteinases such as plasmin. Cytokines bound to native alpha 2M retain much of their biologic activity in various bioassays, whereas cytokines bound to "activated" alpha 2Ms have decreased activity in some cell systems. Because native alpha 2M in circulation can escape into extravascular fluid during tissue injury and inflammation, alpha 2M is a putative cytokine carrier, especially in the presence of heparin or specific cytokine receptors that can displace non-covalently bound cytokines from native alpha 2M. However, proteinase or chemically modified alpha 2Ms become activated for receptor-mediated endocytosis (RME) when they undergo conformational alterations that expose a latent alpha 2M receptor-recognition domain. Circulating activated alpha 2Ms, together with bound cytokines, are rapidly removed by hepatic alpha 2M-receptors (alpha 2M-R) but also bind to other cells expressing alpha 2M-R. This suggests that diseases resulting from an apparent change in the production of one or several different cytokines might represent changes in either the production of alpha 2M "cytokine scavengers" or their alpha 2M-receptor-mediated clearance/targeting mechanisms. The sequence identity between the LDL-receptor related protein and the alpha 2M receptor (115) provides a theoretical basis for interference with cytokine clearance by association of competing lipoprotein ligands with this cytokine clearance pathway. Furthermore, activated alpha 2Ms or augmentation of alpha 2M-receptor dependent cytokine clearance might be novel strategies for preventing the harmful systemic or local effects of excess cytokines such as TGF-beta s and TNF-alpha in vivo. PMID- 1712875 TI - Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue. AB - The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors. PMID- 1712876 TI - Role of manganese in MG-63 osteosarcoma cell attachment to fibrinogen and von Willebrand factor. AB - Some integrin receptors have been reported to be functionally distinct in different cell types. In endothelial and melanoma cells, the vitronectin receptor, alpha v beta 3 binds fibrinogen (fg) and von Willebrand factor (vWf) in addition to vitronectin itself, whereas it fails to do so in MG-63 osteosarcoma cells. In this report, it is shown that, in the presence of Mn2+, MG-63 cells attach more efficiently to vitronectin and acquire the de novo capacity to adhere to fg- and vWf-coated surfaces. The latter phenomenon occurs with full cell spreading, F-actin microfilament organization, and alpha v and beta 3 clustering at focal contacts. In contrast, beta 1 and beta 5 do not localize to adhesion plaques under the same experimental conditions. An antiserum to the beta 3 chain and a synthetic peptide containing the sequence Gly-Arg-Gly-Asp-Ser-Pro block MG 63 attachment to fg and vWf in the presence of Mn2+. The minimal active concentration of Mn2+ is in the range of 0.1 to 1 microns. These data suggest that the acquired capacity of MG-63 to attach to fg and vWf in the presence of Mn2+ is mediated by alpha v beta 3 and that differences in alpha v beta 3 receptor specificity may be modulated by exogenous factors. PMID- 1712877 TI - Language and gesture in late talkers: a 1-year follow-up. AB - A 1-year follow-up of relationships between language and symbolic gesture in 10 children with delayed onset of early lexical skills (Thal & Bates, 1988a) is reported. Original data are reevaluated in light of new knowledge about which late talkers continued to be significantly delayed 1 year later (truly delayed) and which ones had "caught up" (late bloomers). Results showed that all 4 children who were truly delayed at follow-up had been delayed in language comprehension at the first visit, but the 6 late bloomers had been at the same level as their age-matched controls. In addition, truly delayed late talkers had been significantly poorer on all of the gesture tasks than late talkers who caught up. PMID- 1712878 TI - Carcinoma of the pancreas: a personal experience with 100 cases. AB - One hundred patients with pancreatic cancer were evaluated between March 1981 and December 1989. This study showed that 61 were not candidates for definitive surgery because of nonoperability (28 patients) or nonresectability (33 patients). An additional 25 patients had cancers that were unresectable because of metastases (13 patients) or local spread of disease (12 patients) discovered at laparotomy. Fourteen patients had resectable cancers. Ten were treated by total pancreatectomy, three by distal pancreatectomy and one by pancreatoduodenectomy (Whipple). There were two operative mortalities. The median patient survival time was 20.5 months. Two patients survived 5 years. Five patients are alive at 3, 14, 18, and 47 months. Palliative surgical procedures performed in 18 patients included 10 biliary bypasses, 9 gastrojejunostomies, and 6 T-tube placements. This was associated with an operative mortality rate of 11%. The median survival time was 5 months. Other palliative measures included endoscopic placement of biliary and pancreatic stents (47 patients, 2.7% mortality rate), endoluminal radiation therapy, interstitial radiation therapy and external beam radiation therapy. The median survival time of patients so treated was 4.5 months. PMID- 1712879 TI - Immunohistochemical detection of a monoclonal antibody directed against the NGF receptor in basal forebrain neurons following intraventricular injection. AB - It has been shown by autoradiography that, following intraventricular administration, a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor is specifically accumulated bilaterally by numerous cholinergic neurons of the basal forebrain. This is consistent with the evidence that cholinergic basal forebrain neurons have NGF receptors and respond to NGF under a variety of experimental conditions. The present study demonstrates that the immunohistochemical detection of unmodified monoclonal antibody in cholinergic forebrain neurons following transport from CSF is feasible, although injection of larger amounts of the antibody is required to obtain an image equivalent to the one obtained with the autoradiographic method. The location of the immunohistochemical product clearly indicates that the antibody has been internalized, probably in an endosomal compartment. PMID- 1712880 TI - New developments in an "expanded stick" model for coding, graphic representation and metric analysis of tracer-filled or Golgi-impregnated neurons, including spines and varicosities. AB - A new data model allowing the coding, graphic representation and metric analysis of dendritic and axonal processes including spines and varicosities, is here described. The model is implemented in an interactive light microscope-computer system and stores the three-dimensional coordinates of the selected neuronal points, their topological identifiers, and the width of the processes. In addition codes for "nature", and "shape" are stored in the data array. The "nature" code identifies structures such as perikaryon, axon, apical dendrite, basal dendrite, etc. The "shape" code defines varicosities and spines and allows their graphic representation. At present, the coding for metric analysis is made at a final magnification of x1875, with a resolution of 0.11 microns in the objective plane. The graphic representation of spines and varicosities is an ellipse, whose major axis is the length of spines and varicosities and the minor axis the width of these structures. From this "expanded stick" model a computer program calculates the length, area and form factor of the perikaryon; the mean length, width and area of each neuronal branch; the distribution of varicosity and spine number and their size (length and width) per length interval; the total number of processes, varicosities and spines; and the total length and area of the processes. PMID- 1712881 TI - Home treatment of hypogammaglobulinaemia with subcutaneous gammaglobulin by rapid infusion. AB - Intramuscular and intravenous gammaglobulin treatment for hypogammaglobulinaemia is often associated with systemic adverse reactions in some patients. Subcutaneous infusions of gammaglobulin are usually given at a slow rate. To assess the safety of home treatment with subcutaneous gammaglobulin, rapid infusions (34-40 ml/h) given by small portable pumps were used to treat twenty five patients with hypogammaglobulinaemia. Fifteen patients had previously had adverse reactions to intramuscular or intravenous gammaglobulin treatment. After the patients had been taught how to use the pumps during 6 months of treatment in hospital, in which they initially received 100 mg of an intramuscular gammaglobulin preparation/kg per week, they went on to use the pumps at home or at work. So far, the patients have given themselves 3232 rapid subcutaneous infusions (2308 in home therapy). A median pre-infusion serum IgG concentration of 8.1 g/l resulted after 6 months of treatment. There were only 30 (0.93%) mild systemic adverse reactions; there were fewer reactions with subcutaneous gammaglobulin than with previously given intramuscular injections (n = 21, p less than 0.001) or intravenous infusions (n = 9, p less than 0.001) in this group of patients. Overall, the patients spent 0.2 days a year in hospital due to respiratory tract infections. The findings show that the method for subcutaneous administration is very easy to learn and is appreciated by the patients; moreover, the infusions can be given much faster than previously reported without any pronounced local reaction. PMID- 1712882 TI - Alfuzosin for benign prostatic hypertrophy. PMID- 1712883 TI - Mild cystic fibrosis in child homozygous for G542 non-sense mutation in CF gene. PMID- 1712884 TI - Megadose methylprednisolone or IVIG for idiopathic thrombocytopenic purpura. PMID- 1712885 TI - Analysis of free radical damage within single cells using flow cytometry. AB - The data presented here illustrates the additional information that can be gained on single cell biological effects by using a method of damage estimation based on single cells. The experiments involving primarily free radical damage carried out using H2O2 and the radioprotectors cysteamine and WR 1065, both revealed data that could not have been obtained from a macroscopic study of free radical-DNA chemistry and analysis of reaction products. This serves to emphasise the difficulty in extrapolating both free radical based and other chemical reactions to effects seen in living systems. PMID- 1712886 TI - Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues. AB - Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene. PMID- 1712887 TI - Study of pro-opiomelanocortin mRNA expression in human postmortem pituitaries. AB - Complementary oligonucleotide probes specific for the human pro-opiomelanocortin (POMC) mRNA were used to analyze the expression of POMC gene in 56 human postmortem pituitaries by in situ hybridization histochemistry. POMC transcripts were visualized by autoradiography in anterior lobe of the pituitary where their distribution was in a 'patchy-like' pattern. No hybridization could be observed in the posterior lobe of the pituitary. We examined pituitaries from several controls and from patients dying with schizophrenia, Parkinson's disease. Alzheimer's disease, Wernicke's encephalopathy and depressive illness. Computer assisted microdensitometric semiquantification of POMC mRNA using a complementary oligonucleotide as hybridization standard, revealed no statistically significant effect of postmortem delay (between 2.5 and 66 h), of gender, age (between 22 and 103) or cause of death in 56 human pituitary glands. A large variation in POMC levels was already observed among all 30 control cases. The levels of POMC mRNA observed in pituitaries from different pathologies did not show a significant variation when compared with control cases. PMID- 1712888 TI - Recent evolutionary origin of the expression of the glial fibrillary acidic protein (GFAP) in lens epithelial cells. A molecular and genetic analysis of various mouse species. AB - We have investigated the phylogenetic distribution of the glial fibrillary acidic protein (GFAP) in lens epithelial cells (LEC) of various mouse species within the genus Mus. We have shown that lens GFAP is expressed in mice of the Mus musculus complex and in Mus spicilegus and Mus macedonicus species (L.GFAP(+) phenotype) while it is absent in Mus spretus, Mus caroli and Mus cooki species (L.GFAP(-) phenotype). Our results argue in favour of one of the phenograms illustrating the probable phylogenetic relationships between these species in the genus Mus. In animals where lens GFAP was immunodetected, Northern blots of lens RNA extracts hybridized with a mouse GFAP cDNA probe, revealed a single 2.7 kb band. Comparative Northern blot analysis of lens tissue from L.GFAP(+) mice or of brain tissue from L.GFAP(+) or L.GFAP(-) mice did not show any size heterogeneity of the GFAP mRNA. The pattern of the GFAP immunostaining of astroglial cells in brain was identical in both L.GFAP phenotypes. Analysis of interspecific crosses showed that the L.GFAP(+) character is transmitted in a dominant fashion and seems to be linked to the Mus musculus Gfap gene. In this study we have also confirmed the localization of the mouse Gfap gene on chromosome 11. PMID- 1712890 TI - Distribution of hobo transposable elements in natural populations of Drosophila melanogaster. AB - Forty-six strains derived from American and French natural populations of Drosophila melanogaster were tested for the presence and activity of hobo elements by using Southern blotting and a gonadal dysgenesis assay. The oldest available strains exhibited weak detectable hybridization to the hobo-element probe and revealed neither hobo-activity potential nor hobo-repression potential. In contrast, all recently collected strains harbored hobo sequences and revealed a strong hobo-repression potential but no strong hobo-activity potential. On the basis of restriction-enzyme analysis, old strains appear to have numerous fragments hybridizable to hobo sequences, several probably conserved at the same locations in the genome of the tested strain and others dispersed. In recently isolated strains, and unlike the situation in the published sequence of the cloned hobo108 element, a PvuII site is present in the great majority of full sized hobo elements and their deletion derivatives. When the genetic and molecular characteristics are considered together, the available evidence is consistent with the hypothesis of a worldwide hobo-element invasion of D. melanogaster during the past 50 years. Comparison of data from the I-R and P-M systems suggests that the putative invasion followed the introduction of the I element but preceded that of the P element. This hypothesis poses the problem of the plausibility of three virtually simultaneous element invasions in this species. Such a possibility might be due to a modification of the genetic structure of American populations of D. melanogaster during the first part of the 20th century. PMID- 1712889 TI - [Prognosis and treatment of chronic viral hepatitis]. AB - Prognosis of chronic viral hepatitis can be severe. One have to keep in mind the possibility of cirrhosis and hepatocellular carcinoma. Thanks to anatomopathological and immunological tests, it is possible nowadays to evaluate the extent of chronic viral hepatitis and its prognosis. The most efficient therapies are actually the antiviral drugs (vidarabine, interferon): against chronic viral hepatitis B: vidarabine or interferon preceded by a cure of corticoids lead to about 35% of H Be seroconversion--against chronic viral hepatitis B-Delta: interferon Has a suppressive effect in about 50% of the cases with an inevitable relapse when the treatment is stopped--against chronic viral hepatitis C: interferon is efficient in about 50% of the case when given for six months. The high proportion of relapse justified some treatments expended up to 12 or 18 months. PMID- 1712891 TI - Fluorescent imaging in vivo of developing blood vessels on the optic tectum of Xenopus laevis. AB - The growth and development of individual living capillaries, venules, and endothelial sprouts on the pial surface of the brain were examined with video microscopy and intravascular FITC-dextran in anesthetized tadpoles of pigment deficient Xenopus laevis, stages 42-50. The fluorescent tracer, injected intracardially through glass micropipets, was well tolerated by the tadpoles and improved the visibility of vessels compared to transmitted light. Case histories of vascular development on the optic tectum confirmed the sprouting of new capillaries during angiogenesis. The caudal tectum and its vascular domains grew faster than the rostral, but the densities of caudal surface vessels were at least as high as rostral densities, indicating that angiogenesis was well matched to neural development. Internal capillary branches were further elaborated and pial venules increased in diameter in premetamorphic tadpoles and in Xenopus frogs. PMID- 1712892 TI - Altretamine for ovarian cancer. PMID- 1712893 TI - Indirect angiogenic agents do not release fibroblast growth factors from extracellular matrix. AB - Vascular growth factors are categorized as either primary or secondary angiogenic factors. Primary angiogenic agents such as fibroblast growth factors, not only induce the complete angiogenic response, but also stimulate the individual components of vascular growth. Secondary angiogenic agents can induce vascular growth, but they do not act through the direct stimulation of endothelial proliferation, migration, and protease production. Since fibroblast growth factors are known to bind to components of the extracellular matrix, we assessed whether secondary agents act through liberating growth factors from matrix storage sites. The study utilized L6 skeletal myoblasts in culture, which we demonstrated were capable of synthesizing extracellular matrix containing heparin binding endothelial mitogens. The heparin-binding mitogenic activity accumulated in a time-dependent fashion, and matrix extracts contained a protein with immunologic identity to acidic fibroblast growth factor. The ability of secondary angiogenic agents and related compounds including adenosine, inosine, hypoxanthine, nicotinamide, lactic acid, phorbol esters, prostaglandin E2, and copper (at concentrations of 1 microM and 1 mM) to release heparin binding mitogenic activity from the matrix was evaluated. The results demonstrate that although heparin is capable of releasing heparin-binding growth factors from extracellular matrix storage sites in a dose dependent fashion, none of the known secondary angiogenesis factors are capable of functioning in a similar fashion. Thus these secondary angiogenic factors do not appear to exert their effect through increasing the bioavailability of preformed heparin-binding growth factors sequestered in the extracellular matrix. The mechanism(s) whereby these agents induce vascular growth remains to be elucidated. PMID- 1712894 TI - Immunolocalization of types V and XI collagen in cartilage using monoclonal antibodies. AB - Monoclonal antibodies produced against pepsin-solubilized newborn rat skin type V collagen [alpha 1(V)]2 alpha 2(V), and chondrosarcoma type XI collagen [alpha 1(XI) alpha 2(XI) alpha 3(XI)] are used to localize the collagens in sections of the chondrosarcoma as well as the normal rat knee joint by indirect immunofluorescence. Immunostaining for type V collagen shows strong cellular staining of chondrocytes; while the interstitial matrix as well as the lacunae are not stained. In contrast, antitype XI stains not only chondrocytes, but the extracellular compartments as well. In ELISA, rat anti-type XI collagen reacts with its native antigen, but does not cross-react with native types I, II, III, or V collagen from rat. The distinct locations of type V and XI collagens in cartilaginous tissue suggest varied functional roles for these constituents in the tissue. PMID- 1712895 TI - Oxygen regulated 80 kDa protein and glucose regulated 78kDa protein are identical. AB - Ischemic stress of cells within solid tumors arises from inadequate perfusion of regions of the tumor and results in microenvironments which are hypoxic and deficient in nutrient delivery and waste product removal. Stressed cells within these microenvironments show growth inhibition and synthesize unique sets of proteins referred to as glucose and oxygen regulated proteins (GRPs and ORPs respectively). The commonality of proteins induced by glucose-starvation and hypoxia has not been proven. To this end, ORPs were induced in Chinese hamster ovary cells in the presence of high glucose concentration in the media and ORP 80 isolated from two dimension gels. Eleven tryptic peptides of the 80 kDa ORP were sequenced and found to be identical to GRP 78 sequences. The data demonstrate that GRP 78 and ORP 80 have the same primary amino acid sequence and suggest that glucose-starvation and hypoxia can induce the same cellular responses. PMID- 1712896 TI - A mammalian sperm lectin related to rat hepatocyte lectin-2/3. Purification from rabbit testis and identification as a zona binding protein. AB - In rat liver the asialoglycoprotein receptor is composed of three polypeptides, RHL-1, RHL-2 and RHL-3. In rat testis and spermatozoa a galactosyl receptor (RTG r) which is immunologically related to RHL-2/3 has been described. We now report that in addition to its presence in the rat, an antigenic species of 54 kDa related to RHL-2/3 is present on rabbit, human, pig and mouse spermatozoa. Purified rabbit testis galactosyl receptor (RbTG-r) consists of two major proteins of 54 and 49 kDa, while purified rabbit liver galactose lectin consists of two major proteins of 43 and 40 kDa. In an ELISA the purified rabbit testis galactosyl receptor was shown to bind biotinylated heat solubilized rabbit zonae, while the purified liver galactose lectin did not. We conclude that one of the mammalian sperm's zona binding proteins is a galactose lectin of 54 kDa related to rat liver RHL-2/3. PMID- 1712897 TI - Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein. AB - We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia. PMID- 1712898 TI - Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2. AB - One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients. PMID- 1712899 TI - Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence. AB - Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases. PMID- 1712900 TI - Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution. AB - The mammalian signal recognition particle (SRP) is a small cytoplasmic ribonucleoprotein required for the cotranslational targeting of secretory proteins to the endoplasmic reticulum membrane. The heterodimeric protein subunit SRP9/14 was previously shown to be essential for SRP to cause pausing in the elongation of secretory protein translation. RNase protection and filter binding experiments have shown that binding of SRP9/14 to SRP RNA depends solely on sequences located in a domain of SRP RNA that is strongly homologous to the Alu family of repetitive DNA sequences. In addition, the use of hydroxyl radicals, as RNA-cleaving reagents, has revealed four distinct regions in this domain that are in close contact with SRP9/14. Surprisingly, the nucleotide sequence in one of these contact sites, predicted to be mostly single stranded, was found to be extremely conserved in SRP RNAs of evolutionarily distant organisms ranging from eubacteria and archaebacteria to yeasts and higher eucaryotic cells. This finding suggests that SRP9/14 homologs may also exist in these organisms, where they possibly contribute to the regulation of protein synthesis similar to that observed for mammalian SRP in vitro. PMID- 1712901 TI - The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1. AB - The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL 2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin. PMID- 1712902 TI - SHI, a new yeast gene affecting the spacing between TATA and transcription initiation sites. AB - In a genetic selection for Saccharomyces cerevisiae genes involved in transcription start site specification, two mutant genes which restore alcohol dehydrogenase activity to a functionally defective S. pombe ADH gene were recovered. Examination of S. pombe ADH initiation sites showed that mutations in the SHI gene shift the location of the transcription initiation window closer to TATA. The shi mutant also affected initiation site selection for two S. cerevisiae genes that were tested. For H2B mRNA, initiation occurred in the shi mutant at a series of initiation sites located 43 to 80 bp 3' of the histone H2B TATA sequence and at the usual initiation sites 102 and 103 bp downstream of the TATA sequence. Weakly used initiation sites ranging from 51 to 80 bp downstream of the TATA sequence were observed for the S. cerevisiae ADH1 gene in shi strains, in addition to the normal ADH1 initiation sites 89 and 99 bp from the TATA sequence. Restoration of function to the defective S. pombe ADH gene occurs only when this gene contains a TATA sequence; a single-base-pair TATA-to-TAGA change is sufficient to prevent this restoration of function. Genetic mapping placed the SHI locus on the left arm of chromosome VII, 22.3 centimorgans from cyh2; it does not correspond to any previously mapped gene. PMID- 1712903 TI - The mitochondrial uncoupling protein gene in brown fat: correlation between DNase I hypersensitivity and expression in transgenic mice. AB - The mitochondrial uncoupling protein gene is rapidly induced in mouse brown fat following cold exposure. To identify cis-regulatory elements, approximately 50 kb of chromatin surrounding the uncoupling protein gene was examined for its hypersensitivity to DNase I. Seven DNase I-hypersensitive sites were identified in the 5'-flanking DNA, and one site was identified in the 3'-flanking DNA. Transgenic mice with an uncoupling protein minigene were generated by microinjection of fertilized eggs with a transgene containing 3 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. Expression of the transgene is restricted to brown fat and is cold inducible. Four additional transgenic lines were generated with a second transgene containing a 1.8-kb deletion in the 5'-flanking DNA, and expression of this minigene is absent in all tissues analyzed. A DNase I hypersensitive site located in the 1.8-kb deletion contains a cyclic AMP response element that binds a brown fat tumor enriched nuclear factor. On the basis of these observations, we propose that a cis-acting regulatory sequence between -3 and -1.2 kb of the 5'-flanking region, possibly at a DNase I-hypersensitive site, is required for controlling uncoupling protein expression in vivo. PMID- 1712904 TI - Functional characterization of the human hypoxanthine phosphoribosyltransferase gene promoter: evidence for a negative regulatory element. AB - The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the metabolic salvage of the purine bases hypoxanthine and guanine. We previously characterized the genomic structure of the human HPRT gene and described its promoter sequence. In this report, we identify cis-acting transcriptional control regions of the human HPRT gene by linking various 5'-flanking sequences to the bacterial chloramphenicol acetyltransferase gene. The sequence from positions -219 to -122 relative to the translation initiation site is required for maximal expression of this gene, and it functions equally in both normal and reverse orientations. In addition, a cis-acting negative element is present in the region spanning from positions -570 to -388. This negative element can also repress promoters of heterologous genes, such as those of adenosine deaminase and dihydrofolate reductase, which are structurally and functionally similar to the human HPRT promoter. Furthermore, this repressor element functions independently of its orientation but appears to be distance dependent. In vivo competition assays demonstrated that the trans-acting factor(s) that binds to this negative element specifically inhibits human HPRT promoter activity. Taken together, these data localize cis-acting sequences important in the regulation of human HPRT gene expression and should allow the study of protein-DNA interactions which modulate the transcription of this gene. PMID- 1712905 TI - Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5' exons and possible mechanism for the genesis of the 3' end of v-src. AB - To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2. PMID- 1712906 TI - The herpes simplex virus type 1 thymidine kinase is expressed in the testes of transgenic mice under the control of a cryptic promoter. AB - We reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase reporter gene (tk) was expressed in the testes of transgenic mice when coupled to the promoter of a liver-specific mouse major urinary protein (MUP) gene. Here we show that HSV-1 tk is also expressed in the testis when coupled to a MUP pseudogene promoter, to a truncated MUP promoter that is not active in the liver, and to the promoter of the bovine thyroglobulin gene. Furthermore, HSV-1 tk itself was expressed in the testis, although its normal expression had been disabled by removing an upstream regulator of transcription. In every case, the same multiple transcripts were observed, with their 5' ends located downstream of the normal HSV-1 tk translation initiation codon. We conclude that the transcription of HSV-1 tk in the testis is directed by a cryptic TATA box independent promoter located in the coding region of the gene. The longest HSV-1 thymidine kinase (TK) polypeptides synthesized in the testis were shorter than full-length TK and probably result from translational initiation at Met46 and Met60, the second and third ATG codons of the tk reading frame. Male mice of most transgenic lines were sterile, and the severity of the lesion in spermatogenesis was directly related to the level of TK expression. In the most highly expressing lines, sperm counts were low and morphologically defective sperm were common. In other sterile lines, TK was expressed at a lower level and sperm counts were normal but sperm motility was greatly reduced. Lines with the lowest levels of HSV-1 TK expression were fertile. HSV-1 TK was expressed in germ line cells, mainly in the haploid spermatids. However, low-level HSV-1 TK activity was found in the testis before the first germ cells entered meiosis, showing that if expression is confined to the germ cells, it also occurs in spermatogonia. PMID- 1712908 TI - RNA editing intermediates of cox2 transcripts in maize mitochondria. AB - Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process. PMID- 1712909 TI - Glucocorticoid induction of the adipocyte clone 5 gene requires high cell density. AB - We have previously reported the identification of a glucocorticoid and cell density inducible gene (clone 5) that appears to play a regulatory role in adipocyte differentiation. In the current studies we have more carefully investigated the interplay between cell growth or differentiation and the glucocorticoid responsiveness of clone 5 RNA. We find that inducibility by steroid hormone is independent of the differentiated state but surprisingly, occurs only when cells are at high cell density. In subconfluent TA1 cells clone 5 RNA is refractory to induction whereas well characterized glucocorticoid inducible promoters such as the those from mouse mammary tumor virus and alpha-1 acid glycoprotein are induced under all conditions. These results point to the necessity of ancillary factors, absent in low density cell culture, that are required for glucocorticoid responsiveness of the clone 5 gene. PMID- 1712907 TI - Editing of pre-mRNAs can occur before cis- and trans-splicing in Petunia mitochondria. AB - Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription. PMID- 1712910 TI - Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor. AB - Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined. PMID- 1712911 TI - Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes. AB - A clone expressing a surface exposed, conserved epitope of a 60-kDa merozoite polypeptide was identified in a cDNA library constructed from a cloned Mexico strain of Babesia bovis. Sequencing of the 1.9-kb insert (pBv60) revealed an open reading frame encoding a 65-kDa polypeptide with a signal peptide and a tandemly repeated region. Monoclonal antibody 23/56.156, which binds a surface exposed epitope on the native polypeptide, specifically immunoprecipitated [35S]methionine-labeled polypeptides ranging from 60-30 kDa from pBv60 directed transcription and translation. Antibodies raised in rabbits against recombinant polypeptide reacted with the live merozoite surface in a polar immunofluorescence pattern, immunoprecipitated the native 60-kDa polypeptide, and were used to deplete the polypeptide by adsorption from a preparation of native [35S]methionine-labeled merozoite antigen. Restriction enzyme analysis indicated a single gene copy and the absence of introns. Hybridization demonstrated the presence of the gene in Mexico, Australia 'L', and Texas strains of B. bovis, but not in Babesia bigemina. A slightly different hybridization pattern was present in uncloned Australia 'L' B. bovis, indicating sequence diversity in the Bv60 gene among isolates. Cloning and structural analysis of pBv60 provides a source of defined antigen for determining the role of conserved merozoite surface epitopes in protective immunity against babesiosis. PMID- 1712912 TI - Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2. AB - The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1 8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine. PMID- 1712913 TI - Differential effects of electrical stimulation, blockade of neuronal amine uptake and activation of alpha 2-adrenoceptors on the release of endogenous noradrenaline and 5-hydroxytryptamine from the isolated rat pineal gland. AB - Isolated rat pineal glands were incubated in vitro and the release of endogenous noradrenaline or 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5 HIAA) was determined by HPLC with electrochemical detection. In the absence of test drugs, the spontaneous outflow of noradrenaline was about 10 fmol/10 min and electrical stimulation (5 Hz, 1500 pulses) evoked the release of about 70 fmol noradrenaline. Nomifensine enhanced the spontaneous outflow of noradrenaline about threefold and the electrically evoked release of noradrenaline about sixfold. In the presence of nomifensine, the alpha 2-adrenoceptor antagonist yohimbine markedly increased the electrically evoked release of noradrenaline, whereas the alpha 1-adrenoceptor antagonist prazosin had no effect. Clonidine inhibited the electrically evoked release of noradrenaline by about 65%, and this was antagonized by yohimbine in a competitive manner. In the absence of drugs, the initial spontaneous outflow of 5-HT was (compared with noradrenaline) very high 64 pmol/10 min). It declined by 80% within 1 h of incubation in vitro. The outflow of 5-HIAA amounted initially to 38 pmol 10 min and declined by 40% within 1 h of incubation. Addition of L-tryptophan (10 mumol/l) after 1 h of incubation in vitro largely enhanced the outflow of 5-HT and 5-HIAA within 30 min of incubation (about ten- and fourfold, respectively). When L-tryptophan was present from the onset of incubation the initial outflow of 5-HT and 5-HIAA was only slightly elevated, but the decline was largely attenuated. Neither omission of calcium nor addition of nomifensine, clonidine or yohimbine significantly affected the spontaneous outflow of 5-HT or 5-HIAA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712914 TI - Rat hind-paw swelling effect of an edema-producing protein isolated from Trimeresurus mucrosquamatus snake venom. AB - TMV F-IV, isolated from the venom of Trimeresurus mucrosquamatus (TMV), caused rat hind-paw edema in a dose-dependent manner. The maximum hind-paw swelling was reached at 1.5-2 h after subplantar injection of TMV F-IV. The edematous response caused by TMV F-IV was suppressed by the s.c. pretreatment with diphenhydramine, methysergide, acetylsalicylic acid or dexamethasone, and by the subplantar co injection with FPL 55712, a SRS-A antagonist, and BN 52021 or L 652731, both PAF antagonists. Polymorphonuclear (PMN) leukocyte infiltration appeared within 1 h and gradually increased in the rat paw 3-6 h after edema induction. Compound 48/80 or methotrexate pretreatment also inhibited paw edema caused by TMV F-IV. In isolated mast cells, TMV F-IV increased the formation of PGE2 and LTB4 and caused a dose-dependent release of histamine and beta-glucuronidase. Since there are no significant differences in paw edema and mast cell degranulation responses between TMV F-IV and its DFP-modified analogue, the esterase activity may not be necessary in these models. These results indicate that mast cells. PMN leukocytes and some inflammatory mediators such as histamine, serotonin, arachidonate metabolites and PAF are involved in TMV F-IV induced paw edema. PMID- 1712915 TI - Ruthenium red inhibits tail skin vasodilatation evoked by intracerebroventricular injection of capsaicin in the rat. AB - The effect of Ruthenium red on the tail skin vasodilatation evoked by an intracerebroventricular injection of capsaicin was studied in the anesthetized rat. Injection of capsaicin into the lateral ventricle resulted in a marked elevation of the tail skin temperature, indicative of peripheral vasodilatation. Ruthenium red, given by intracerebroventricular injection, significantly inhibited this response, which is known to be mediated by central warmth sensitive neuronal structures. The findings suggest that the sensitivity to Ruthenium red, reportedly characteristic of the capsaicin-sensitive neurons in the peripheral nervous system, is also a trait of the capsaicin-sensitive nerve cells in the central nervous system. This is the first evidence indicating that similar molecular mechanisms, presumably involving changes in cellular calcium metabolism, contribute to the capsaicin-induced activation of neurons in both the peripheral and central nervous systems. PMID- 1712916 TI - Microvascular decompression for hypertension--clinical and experimental study. AB - Balloon compression of the left IXth and Xth cranial nerves and ventrolateral medulla oblongata in dogs resulted in statistically significant increases in blood pressure without obvious changes in cardiac output. Five of 21 hypertensive patients with trigeminal neuralgia or hemifacial spasm demonstrated normalized blood pressure following microvascular decompression for arterial compression of the brainstem in the area of the IXth and Xth cranial nerves on the left. Our experimental and clinical results suggest that neurogenic hypertension caused by the vascular compression of the medulla oblongata can be relieved by microvascular decompression. PMID- 1712917 TI - Intraoperative monitoring of somatosensory evoked potentials in patients with cerebral aneurysm--correlation between central conduction time and postoperative neurological status. AB - Somatosensory evoked potentials (SEPs) were monitored during 55 aneurysm procedures in 49 patients to investigate the relationship between central conduction time (CCT) and neurological status in the immediate postoperative period. Significant CCT prolongation was observed in 15 cases. The postoperative neurological status deteriorated in nine of the 15 cases, compared with three of 40 cases without appreciable CCT changes. This difference was significant at p less than 0.05. The CCT prolongation was ascribable to temporary vascular occlusion in half of the cases. Intraoperative SEP monitoring is useful in predicting neurological morbidities in the immediate postoperative period in patients with cerebral aneurysm. PMID- 1712918 TI - Surgical results of brain metastasis from lung cancer--prognostic factors. AB - Twenty-five patients receiving surgical treatment for brain metastasis from lung cancer were retrospectively studied to evaluate the prognostic factors for survival time. Twenty-two patients had died of respiratory distress by April, 1989. Favorable prognostic factors derived from the median survival time (MST) in these patients included; 1) resection of primary tumor (MST 10 months); 2) total or subtotal removal of metastatic tumor (MST 6.5 months); 3) adenocarcinoma (MST 13 months); 4) metachronous onset of brain metastasis (MST 12 months); 5) single metastasis (MST 8 months). These results suggest that therapy for the primary lung cancer is important before surgery for metastatic brain tumor. PMID- 1712919 TI - Effect of high-dose methylprednisolone on vasospasm after subarachnoid hemorrhage. AB - The effects of high-dose methylprednisolone (MP) on vasospasm following subarachnoid hemorrhage (SAH) were investigated. In a double hemorrhage canine model, administration of high-dose MP (10 mg/kg, every 12 hours) reduced angiographic narrowing of the basilar artery and prevented morphological changes in the arterial wall. In addition, increased platelet aggregation observed from days 4 to 7 in untreated SAH dogs was inhibited by the MP treatment. An in vitro experiment showed that MP inhibited platelet aggregation dose-dependently. High dose MP had a nonspecific vasodilatory effect on the smooth muscle of the basilar artery. In another SAH model with dysautoregulation of cerebral blood flow (CBF), intravenous MP administration markedly attenuated the decrease in both blood pressure and CBF caused by exsanguination. These results indicate that MP has beneficial effects in normalizing CBF dysautoregulation following SAH. High-dose MP has several advantages for preventing and improving the multiple pathological status in cerebral vasospasm following SAH. PMID- 1712920 TI - Coagulopathy in chronic subdural hematoma. AB - Coagulation factors were studied in 30 fluids aspirated from 25 patients with chronic subdural hematoma. Compared with the normal range for plasma, the hematoma fluids demonstrated a marked reduction in factors II, V, VII, VIII, and X, moderate reduction of factors IX and XI, and slight reduction of XII. Factor VII and IX inhibitors were not present or negligible. Activated protein C and antithrombin III were decreased and fibrinopeptide A was markedly increased. No case had a basic disorder causing these abnormal data spontaneously. The decrease in activated protein C possibly caused the marked reduction of factor VIII, therefore the intrinsic and extrinsic clotting pathways were affected differently. The results show excessive activation of coagulation, predominantly via the extrinsic clotting pathway in hematoma, suggesting its importance in the growth of chronic subdural hematoma. PMID- 1712921 TI - Giant middle cerebral artery aneurysm with parent artery occlusion--case report. AB - A 54-year-old female was admitted with consciousness disturbance and right hemiparesis. Computed tomographic (CT) scans and angiograms revealed diffuse subarachnoid hemorrhage, a partially thrombosed, giant middle cerebral artery aneurysm (5 x 5 x 4 cm), and occlusion of the parent artery at the aneurysm site. Despite conservative treatment, a generalized convulsion occurred. Emergency CT scans revealed irregular enlargement of the left temporal high-density mass and severe mass effect due to cerebral infarction. Barbiturate coma therapy was administered, but she did not recover and died 9 days after admission. Only two cases of ruptured aneurysm with simultaneous occlusion of the major cerebral vessels have been reported, both with poor outcome. In this case, the mechanism of parent artery occlusion is unclear, but thrombus protrusion from the giant aneurysm into the parent artery may have been involved. PMID- 1712922 TI - Dural arteriovenous malformation associated with occlusion of the superior sagittal sinus--case report. AB - The authors report a rare case of dural arteriovenous malformation (AVM) associated with occlusion of the superior sagittal sinus. A 78-year-old female developed transient aphasia, followed by generalized convulsion. Common carotid angiography showed a dural AVM fed by the bilateral middle meningeal arteries, draining to the superior sagittal sinus, and sinus occlusion. 123I-single photon emission computed tomography demonstrated decreased blood flow in the bifrontal parasagittal regions. Transcatheter embolization via the feeding arteries improved the cerebral blood flow around the lesion, and the symptoms disappeared. PMID- 1712923 TI - Bilateral oculomotor nerve palsies due to posterior cerebral arterial compression relieved by microvascular decompression--case report. AB - A 59-year-old male developed peripheral oculomotor nerve paresis due to compression by the left posterior cerebral artery (PCA), which was successfully treated by microvascular decompression. Two months later, a similar oculomotor nerve paralysis due to the same mechanism occurred contralaterally and was also treated by microvascular decompression. The previous condition was probably caused by arteriosclerotic changes in the PCA, and the following condition by postsurgical adhesion of the arachnoid membrane. The possibility of vascular compression should be considered when oculomotor nerve palsy rapidly develops, although not proven by angiography. PMID- 1712924 TI - Subarachnoid and intracerebral hemorrhage associated with necrotizing angiitis due to methamphetamine abuse--an autopsy case. AB - The authors report an autopsy case of methamphetamine-related intracranial hemorrhage and vasculitis. A 22-year-old female was comatose after an intravenous injection of an unknown dose of methamphetamine. Computed tomographic scans demonstrated massive subarachnoid hemorrhage and hematoma in the corpus callosum. Cerebral angiography revealed nonfilling of bilateral intracranial carotid arteries and extravasation of contrast medium from the right pericallosal artery which was visualized retrogradely via the vertebral artery. Postmortem studies found cerebral edema, subarachnoid, intraventricular, and intracerebral hemorrhage, and intracranial vasculitis, but no aneurysm or arteriovenous malformation. Necrosis of vessel walls with destruction of the smooth muscle layer, but no leukocytotic infiltration of the vessel walls were observed in all major cerebral arteries. The hemorrhage probably resulted from medial necrosis in the large intracerebral vessels, and a sudden drug-induced rise in blood pressure. PMID- 1712925 TI - Drug-induced hypotension SEP test and acetazolamide test using 133Xe SPECT in patients with occlusive carotid disease--selection of candidates for extracranial intracranial bypass. AB - The correlation between the drug-induced hypotension somatosensory evoked potential (SEP) test and regional cerebral blood flow changes after acetazolamide administration was studied. Fourteen patients presenting with transient ischemic attack, reversible ischemic neurological deficits, or minor completed stroke were evaluated. All patients had no or only localized low-density areas on computed tomographic scans, and unilateral occlusion or severe stenosis of the internal carotid or middle cerebral artery on cerebral angiograms. The Diamox asymmetry enhancement (DAE) was studied to detect reduced cerebral perfusion reserve in the affected hemispheres. The DAE was 7.9 +/- 5.8% in seven patients positive in the SEP test, significantly higher than -1.5 +/- 2.9% in patients negative in the SEP test. Postoperative SEP tests were negative in all five patients who underwent extracranial-intracranial (EC-IC) bypass surgery, suggesting that the EC-IC bypass improved the cerebral perfusion reserve in the affected hemispheres. The DAE decreased significantly in four of these patients. This study disclosed a significant correlation between the drug-induced hypotension SEP test and DAE. These parameters are considered important for evaluating patients with hemodynamic compromise and/or suitable candidates for EC-IC bypass. PMID- 1712926 TI - Differential interactions between benzodiazepines and the dihydropyridines, nitrendipine and Bay K 8644. AB - The effects of the dihydropyridine calcium antagonist, nitrendipine and the calcium channel activator, Bay K 8644, have been compared on the anaesthetic, ataxic and anticonvulsant effects of benzodiazepines. Possible interactions between the peripheral benzodiazepine receptor antagonist, PK11195, and the classical benzodiazepines were also examined. Nitrendipine considerably potentiated the anaesthetic effects of benzodiazepines and increased their ataxic effects but had no effect on the anticonvulsant actions. Clonazepam did not produce anaesthesia, at doses up to 1 g kg-1 or when given with nitrendipine. When given alone, nitrendipine did not cause general anaesthesia. Nitrendipine did not appear to alter the metabolism of midazolam. The calcium channel activator, Bay K 8644, reduced the anaesthetic potency of midazolam and, when given alone, produced ataxia. It did not significantly alter central concentrations of midazolam. The "peripheral" benzodiazepine antagonist, PK11195, did not affect the ataxic or anaesthetic actions of benzodiazepines. These results suggest that dihydropyridine-sensitive calcium channels may be more important to the general anaesthetic than to the anticonvulsant actions of benzodiazepines. The "peripheral" benzodiazepine site did not appear to play a role in either of these properties. PMID- 1712927 TI - Cholinergic hyperactivity in the lateral septal area of spontaneously hypertensive rats: depressor effect of hemicholinium-3 and pirenzepine. AB - In the lateral septal area of spontaneously hypertensive rats, but not in Wistar Kyoto rats, the selective M1 antagonist, pirenzepine, and the depletion of acetylcholine storage, by hemicholinium-3 (HC-3), decreased blood pressure. The selective M1 agonist McNeil-A-343, produced a pressor response only after treatment of the lateral septal area with HC-3 in spontaneously hypertensive rats. Carbachol, at doses that mainly affect M2 muscarinic receptors, caused no cardiovascular changes in either strain, pointing to the main intervention of the M1 subtype of muscarinic receptor in the hypertensive condition. In addition, increases in the density of binding sites for [3H]QNB and in Vmax of sodium dependent, HC-3-inhibitable, high affinity uptake of choline were demonstrated, without significant changes of the activity of choline acetyltransferase in the lateral septal area of spontaneously hypertensive rats. These results suggest that a hyperactivity of the cholinergic system of this area could play a role in the development and/or maintenance of hypertension in spontaneously hypertensive rats. PMID- 1712928 TI - A prospective evaluation of anterior retinal cryoablation in neovascular glaucoma. AB - We report a prospective evaluation of the effect of anterior retinal cryopexy on 62 eyes with neovascular glaucoma. Pain was relieved and anterior chamber inflammatory reaction regressed dramatically in 95%. Iris neovascularization was reduced or regressed in 93.5%. Control of intraocular pressure was clinically significant in 82.3% 1 year after the procedure, especially in patients with pretreatment pressures less than 40 mm Hg on maximal medical therapy. Anterior retinal cryopexy is recommended in eyes with media opacities and as a preliminary procedure for filtering surgery in eyes with neovascular glaucoma. PMID- 1712929 TI - Characteristics and specificity of hybridoma antibodies against oocyst antigens of Cryptosporidium parvum from man. AB - Hybridoma antibodies (HAbs) against oocyst antigens of a human isolate of Cryptosporidium parvum were developed by fusion of SP2/0 mouse myeloma cells and spleen cells from BALB/c mice immunized with oocyst homogenates. In an indirect immunofluorescence antibody test (IFAT), using as antigen a mixture of air-dried sporozoites and oocysts, HAbs labelled either the oocyst wall or areas of the sporozoite, including the whole organism, the entire surface, a polar region or the interior. Most of the HAbs were specific for the sporozoite surface, and few of them recognized the oocyst wall. In Western blot analysis of oocyst antigens, sporozoite surface-reactive monoclonal antibodies (MoAbs) recognized one or more of seven polypeptide bands with molecular weights in the range 47- greater than 200 kD, and all reacted with the 47 kD band. Each of four heterologous parasite isolates had a unique recognition pattern with a panel of MoAbs in IFAT, suggesting antigenic differences may exist between strains of C. parvum. The ability to differentiate between parasite isolates by immunological methods might be of value in epidemiological studies of cryptosporidiosis. PMID- 1712931 TI - Antibody responses to Plasmodium falciparum gametocyte antigens during and after malaria attacks in schoolchildren from Madang, Papua New Guinea. AB - Sera from 49 school children in Madang, Papua New Guinea with malaria and follow up sera from 40 of these cases were tested by competitive ELISA for antibodies capable of inhibiting binding of eight monoclonal antibodies (MoAbs) to Plasmodium falciparum gametocytes. The proportion of sera inhibiting each MoAb ranged from 31.2% to 85.7%. At follow-up, the proportion of inhibitory sera decreased for 3 MoAbs, did not change significantly for 4 MoAbs and increased for one MoAb. When sera were grouped according to whether the follow-up blood slide was positive or negative, further trends emerged for MoAbs against the gamete surface antigen Pfs 48/45. Antibody levels to the IA3-B8 epitope decreased in follow-up positive cases, but remained unchanged for follow-up negative cases. The converse was observed for the IIC5-B10 epitope with an increase of antibody in follow-up positive cases and no change in the negative cases. Amount of antibody to the 3G12/58 epitope decreased when the follow-up was negative but not when it was positive. Increase in antibody to the 3E12/58 epitope occurred at the follow-up sample irrespective of the blood slide result. Thus four distinct patterns of longitudinal antibody response were observed against four epitopes on the same molecule. PMID- 1712930 TI - MHC-restriction of antibody responses to an 86 kilodalton antigen of Schistosoma mansoni. AB - The previously observed MHC-restriction of the antibody response to an 86kDa S. mansoni antigen has been investigated in more detail. The I-A locus of the H-2 complex has been implicated as conferring responder or non-responder status on mice expressing the k and b alleles respectively. Inheritance of responsiveness was dominant over non-responsiveness. An additional level of complexity was observed in the p86 antibody responder status of individuals within a responding inbred strain. This could not be accounted for directly by the level of patent infection, but showed an inverse correlation with the level of egg output in H-2k mice. Differential antibody responsiveness to other antigens, between individuals of the same strain, occurred independently of the differential responsiveness to p86. PMID- 1712932 TI - Production of interferons by bovine and ovine cell lines infected with Theileria annulata or Theileria parva. AB - Three bovine cell lines and four ovine cell lines infected with Theileria parva or Theileria annulata were examined for the production of interferon (IFN). Biologically active IFN was detected in the tissue culture supernatants of four of the cell lines. Only one, a bovine cell line infected with T. parva, produced IFN-gamma as measured by specific neutralization with a monoclonal antibody to bovine IFN-gamma. This observation was confirmed by analysing RNA from the cell lines on Northern blots using an IFN-gamma cDNA probe. The other three cell lines which produced IFN were infected with T. annulata. The IFN produced by those lines was not IFN-gamma. PMID- 1712933 TI - Prenatal cocaine use: impact on infants and mothers. AB - Prenatal cocaine use does not allow a mother to provide an environment that promotes her infant's normal development. Pediatric nurses and other health care professionals need to support and assist the mother in the recovery process for the benefit of the child's health and development. PMID- 1712934 TI - Birth outcomes, health problems, and neglect with prenatal exposure to cocaine. AB - Thirty children exposed prenatally to maternal use of cocaine were compared to 30 nonexposed subjects on maternal variables, birth outcomes, health problems in early childhood, and issues related to child maltreatment. Cocaine-exposed infants were more likely to have mothers who received inadequate prenatal care, have adverse birth outcomes including prematurity and retarded intrauterine growth, and have health problems beyond the newborn period including small stature and hypertonia. More cocaine-exposed children were placed in foster homes due to maternal neglect. PMID- 1712935 TI - Neonatal Listeria monocytogenes infection is refractory to interferon. AB - Despite aggressive treatment, early onset neonatal Listeria monocytogenes infection continues to have high morbidity and mortality. We recently showed that pretreatment of newborn L. monocytogenes-infected rats with interferon (IFN) alpha/beta or recombinant rat IFN-gamma dramatically improves survival. However, in the present experiment, when newborn rats were treated with IFN-alpha/beta or recombinant rat IFN-gamma after intraperitoneal injection with Listeria there was no benefit. Because most deaths occurred at or before 3 d in this animal model, we reasoned that the effect of interferon may be evident if animals survived longer. To accomplish this and test this hypothesis, ampicillin (20 mg/kg/d) was given 48 h after bacterial challenge. When ampicillin-treated Listeria-infected rats were randomized to receive PBS, IFN-alpha/beta, or recombinant rat IFN gamma, mortality rates were 79, 76, and 69%, respectively (p greater than 0.05 versus PBS). Animals treated in a similar fashion after a lower bacterial inoculum (25% lethal dose) were killed 5 d after bacterial challenge. Bacterial concentrations in the spleen were higher for IFN-treated animals than controls. We conclude that no direct benefit of IFN is found if it is given after bacterial infection has been established. PMID- 1712936 TI - Passive immunization to prevent mother-infant transmission of human immunodeficiency virus: current issues and future directions. AB - A definitive conclusion regarding the potential for the presence of neutralizing antibody to epitopes of the V3 loop to attenuate or prevent vertical transmission of HIV infection cannot be made based on the results of these four studies. However, the studies provide an intriguing suggestion that it may be possible to identify an epitope or array of epitopes from the V3 loop of judiciously selected HIV isolates that could induce protective immunity against HIV. Future collaborative studies are needed to standardize and independently validate the various assays. A recent conference, Early Diagnosis of HIV Infection in Infants, sponsored by the National Institute of Child Health and Human Development, the National Institute of Allergy and Infectious Diseases and the Centers for Disease Control, provided a forum to facilitate exchange of information among researchers in this field, and encourage future collaborative efforts. If one or more subpopulations of anti-gp120/V3 loop or other neutralizing antibodies are indeed associated with a decreased risk of maternal-infant transmission, it becomes important to determine whether these antibodies are protective per se or are merely surrogate markers of a different mechanism. The former conclusion seems to be supported by the finding of HIV-specific antibodies in seropositive persons' saliva and breast milk, both of which have relatively low infectivity, although the potential neutralizing activities of these antibodies have not been adequately evaluated. The development and testing of a highly specific (monoclonal antibody-derived) passive immunotherapy will probably be required to answer this question.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712937 TI - The three-dimensional folding of ribosomal RNA; localization of a series of intra RNA cross-links in 23S RNA induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine. AB - Intact 50S ribosomal subunits from E.coli were cross-linked with the symmetrical bifunctional reagent bis-(2-chloroethyl)-methylamine. After deproteinization, selected regions of the 23S RNA were excised by treatment with ribonuclease H in the presence of appropriate complementary decadeoxynucleotides, and screened for the presence of intra-RNA cross-links by two-dimensional gel electrophoresis. Individual isolated cross-linked RNA fragments were analysed by our established procedures. Sixteen intra-RNA cross-links were identified, three of which corresponded to those previously published. The thirteen 'new' cross-links were localized in the 23S RNA at positions 774-78 linked to 792-94, 876-79 linked to 899-900, 979-81 or 983-84 to 2029, 1715 to 1743-46, 1911-21 to 1964, 1933 to 1966, 2032 to 2054-55, 2112 to 2169-71, 2116-17 to 2163-67, 2128-32 to 2156-59, 2392-93 to 2422-23, 2737-38 to 2763-66, and 2791 to 2890. These results are discussed in the context of three-dimensional model-building studies with the 23S RNA, with particular reference to the environment of the 'active centre' of the 50S subunit. PMID- 1712938 TI - Termination of transcription in an 'in vitro' system is dependent on a polyadenylation sequence. AB - Using HeLa cell nuclear extract as a source of the different transcription and polyadenylation factors and reverse transcription to analyze the levels of RNA 5' and 3' to the cleavage-polyadenylation site, an in vitro assay has been established to study polyadenylation coupled to transcription directed by different adenovirus promoters. The levels of transcription 5' and 3' to the cleavage site in the L3 polyadenylation region are practically the same as described previously, however, the level of transcription 3' to the cleavage site in the SV40 early polyadenylation region decreases immediately after the cleavage site indicating a termination of the transcription. PMID- 1712939 TI - Preparation and hybridization properties of oligonucleotides containing 1-alpha-D arabinofuranosylthymine. AB - A pentadecanucleotide was prepared from 1-alpha-arabinofuranosylthymine. This novel oligonucleotide was found to hybridize to oligodeoxyadenylate, although not a s strongly as pentadecathymidylate. It formed duplex hybrids with both DNA and RNA complements, and triplex structures with a duplex containing a (dT)15-(dA)15 tract within a more complex strand. The Tm of the duplex with polyadenylate was almost the same as that of (dT)15 and polyadenylate, while its Tm with (dA)15 was substantially lower than that of the natural counterpart. A selective benzoylation of the 2'-O of a 5'-blocked alpha-ara-thymine was developed to greatly simplify the preparation of suitable blocked material for use in preparation of oligonucleotides on the automated DNA synthesizer. PMID- 1712940 TI - Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe. AB - The structure in solution of an RNA fragment (218 nucleotides long) containing part of E. coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U). In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules. An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content. Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U. The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size. The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing. 17 of the 41 possible s4U positions lead to detectable bridges. These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed. The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected. PMID- 1712941 TI - A novel 3' extension technique using random primers in RNA-PCR. PMID- 1712942 TI - Polymerase chain reaction (PCR) for detection of MspI polymorphism at the D3S30 locus. PMID- 1712943 TI - Polymerase chain reaction (PCR) for detection of MspI polymorphism at the D3S3 locus. PMID- 1712944 TI - Polymerase chain reaction (PCR) for detection of MspI polymorphism at the D3S6 locus. PMID- 1712945 TI - Polymerase chain reaction (PCR) for detection of MspI and DraI polymorphism at the THRB gene. PMID- 1712946 TI - Polymerase chain reaction (PCR) for detection of MspI polymorphism at the D3S2 locus. PMID- 1712947 TI - Removal of implanted hardware. PMID- 1712948 TI - The effect of posture on the response to atrioventricular synchronous pacing in patients with underlying cardiovascular disease. AB - In order to determine whether the hemodynamic benefit of atrioventricular synchronous pacing is maintained in the upright position, 14 patients with dual chamber pacemakers were paced in VVI mode and DDD mode in both the supine and standing position. The hemodynamic response was assessed by measuring the velocity time integral derived from the pulsed-wave Doppler signal in the left ventricular outflow tract during VVI pacing and dual chamber pacing at three different AV delays (125, 200, 250 ms). In the supine position, the velocity time integral during VVI pacing was 14.6 +/- 3.0 cm and this increased during DDD pacing at all three AV delays (17.7 +/- 3.3, 17.9 +/- 3.0, 17.5 +/- 3.5 cm). In the upright position, the velocity time integral during VVI pacing was 12.9 +/- 3.5 cm and this increased with DDD pacing (15.5 +/- 3.3, 15.1 +/- 4.0, 15.1 +/- 3.9 cm). It was concluded that although stroke volume decreases when assuming the upright position, the beneficial response to dual chamber pacing is maintained and equals that observed in the supine position. PMID- 1712949 TI - Fusion or confusion on Holter recording. AB - Holter recording of a patient with an implanted dual chamber rate responsive pacemaker revealed an electrocardiogram, where ventricular depolarization seemed to be initiated by the atrial stimulus. In a second patient with a VVI pacemaker, Holter recording showed delay of the pacemaker impulse that was registered after the onset of ventricular depolarization. Misalignment in one of the recorder heads of the display system was responsible for this phenomenon, which in case of dual chamber pacing could have been easily misinterpreted as pacemaker malfunction. PMID- 1712950 TI - Incessant ectopic atrial tachycardia and sudden death. AB - A patient with refractory and incessant ectopic atrial tachycardia (IEAT) is reported in whom it was possible to document, during ECG (Holter) the occurrence of aborted sudden death by spontaneous ventricular fibrillation (VF). Following the second of two attempts at surgical ablation of the origin of the IEAT, the patient has been asymptomatic without antiarrhythmic drugs and in sustained sinus rhythm for 24 months. Although we cannot exclude the residual action of amiodarone and flecainide (proarrhythmia) or the residual peripartum cardiomyopathy it is probable that the observed VF was a true complication of a cardiomyopathy induced by a chronically increased heart rate (HR). Although unclear, this VF might be considered as a form of adrenergic-dependent long QT syndrome due to early afterdepolarization in the presence of predisposing myocardial conditions. PMID- 1712951 TI - Ventricular pacing threshold and time to capture postdefibrillation in patients undergoing implantable cardioverter-defibrillator implantation. AB - To assess the effect of defibrillation and amiodarone on ventricular pacing threshold and time to capture in patients undergoing automatic implantable cardioverter-defibrillator (AICD) implantation, 28 patients were prospectively evaluated. The patients were entered into one of two protocols: Ia--epicardial ventricular pacing threshold measured at baseline (preventricular fibrillation induction) and 10 and 60 seconds postdefibrillation with 20 J, or Ib--two fibrillation-defibrillation sequences were performed 3 minutes apart and ventricular pacing thresholds were measured for each sequence at baseline and at 10 and 60 seconds postdefibrillation with 20 J. Ten patients also underwent asynchronous pacing at 1.1 times baseline threshold during ventricular fibrillation with measurement of time to capture postdefibrillation. All patients were randomly assigned to receive either amiodarone or no antiarrhythmic drug therapy. Ventricular fibrillation was induced with AC (applied for 1-2 seconds), and standard epicardial bipolar and epicardial patch electrodes of the AICD were used for pacing and defibrillation, respectively. Ventricular pacing threshold at baseline, 10 seconds, 60 seconds, and 3 minutes postdefibrillation did not differ significantly. There were no significant differences in patients with or without amiodarone therapy. Furthermore, there was no transient loss of ventricular capture postdefibrillation or significant difference in time to capture with amiodarone (less than or equal to 2 seconds). We conclude that following internal defibrillation with 20 J: (1) ventricular pacing threshold at 10 seconds, 60 seconds, and 3 minutes were not significantly different from baseline with one or two fibrillation-defibrillation sequences, (2) time to capture was short, and (3) there was no significant difference in no drug versus amiodarone. These findings have direct clinical importance in considering device therapy with both pacing and defibrillating capabilities. PMID- 1712952 TI - Day case permanent pacing. AB - We have previously reported our preliminary experience of day-case permanent pacing in the United Kingdom. The study has now been extended to 50 patients with follow-up of 22 +/- 4 months. During the study period, all patients referred for permanent pacing, either to the senior author, or as in-hospital transfers, were considered for the study. Forty two percent of patients considered fulfilled inclusion and exclusion criteria, resulting in a total of 50 patients being randomized either to day case or conventional in-patient management. In the first month postimplantation, one patient in each group developed a complication requiring revision of system. Only one further pacing related complication occurred over the follow-up period, percutaneous extrusion of a fixation sleeve with spontaneously healing of the wound. This was in a day-case patient. Mean duration of in-patient stay was 5.7 hours in day-case patients, compared with 70.0 hours in those managed conventionally. Postimplantation local physician consultation rates were equal in both groups. Questionnaires were used to determine the relative acceptability to patients of the two management protocols; on a ten point score of acceptability, the mean score for both groups was 8.8. The difference in cost per patient using day-case management was approximately 430 ($817) pounds. We conclude that day-case permanent pacing in the United Kingdom is feasible, acceptable to patients, and has considerable economic benefits. PMID- 1712953 TI - What is the ideal pulse frequency for skeletal muscle stimulation after cardiomyoplasty? AB - Routinely the latissimus dorsi (LD) muscle is stimulated with bursts of pulses at 30 pulses/sec after cardiomyoplasty to assist the failing heart. At a lower pulse frequency the contractile force decreases and at higher frequencies the energy demand of the pacemaker increases rapidly. We investigated the effect of the stimulus frequency variation on contractile force in untrained LD muscles and in muscles after 12 weeks of continuous cyclic electrical stimulation. In six dogs, two electrodes (Medtronic SP5528) were implanted in the left LD muscle together with an Itrel muscle stimulator. The LD muscle was stimulated with 30 pulses/sec in bursts to deliver initially 30 and after 10 weeks 80 contractions per minute. Both before and after training of the LD muscle maximum force was observed by stimulating with a frequency of 36 to 130 pulses/sec in a burst. However, training induced a shift in the steep part of the force-frequency relation to lower frequencies. In particular, at 15 pulses/sec 60% of the maximal force was obtained in contrast to 40% before training. A fatigue test of 8 minutes duration was performed specified by 100 bursts/min and a burst duration of 0.25 sec at pulse frequencies of 30, 36, 50, and 85 pulses/sec. The contractile force decreased significantly during the course of the test at all frequencies. However, the force obtained with 30 pulses/sec stimulation was lower during the initial phase and approximately 10% higher at the end of the fatigue test as compared to 36, 50, and 85 pulses/sec stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712954 TI - Malignant ventricular tachycardia during propafenone treatment in a child with junctional automatic tachycardia: effectiveness of intravenous molar sodium lactate. AB - Propafenone may aggravate the preexisting arrhythmia or induce another one. Usually, such proarrhythmic effects occur in patients with spontaneous ventricular arrhythmias and/or coronary heart disease with poor left ventricular function. We report the case of a 5-year-old girl with junctional automatic tachycardia and no structural heart disease, in whom malignant ventricular tachycardia occurring during propafenone treatment could be terminated by molar sodium lactate (MSL) infusion. The serum propafenone level obtained before MSL infusion was within the therapeutic range. Two hypothesis could explain the beneficial effects of MSL in our patient: (1) alkalinization facilitates the cell membrane hyperpolarization and thus can decrease the voltage-dependent effect of Class Ic drugs, (2) alkalinization could displace propafenone from its tissue receptor sites by an increase in the nonionized fraction. PMID- 1712955 TI - Influence of propranolol on the ventricular depolarization gradient. AB - Sensing of the ventricular depolarization gradient (VDG) has recently been used as the basis of a closed-loop rate responsive pacemaker. Factors influencing this aspect of the evoked response have not been fully evaluated although previous reports have suggested that sympathetic stimulation and circulating catecholamines are primarily responsible for the observed changes during stress and exercise. In five patients (Table I), four males and one female (mean age 60.4 +/- 10.1 years) implanted with the Prism pacemaker, the pacing response to exercise and tilting was assessed before and after the infusion of propranolol. There was an increase in the pacing rate in all patients during the infusion of the drug (mean 27 +/- 12.9 beats/min) suggestive of a direct drug effect on the VDG. The rate control parameter (RCP) of the pacemaker, the numerical equivalent of the VDG, was significantly different after the administration of propranolol (P less than 0.01). However, exercise performance and pacing rate behavior were not different after beta blockade. The pacing rate increase observed when tilting patients to the supine position was not altered by propranolol. Out date suggest that factors other than adrenergic stimulation may be of importance in affecting the ventricular evoked response and accordingly the rate adaptation of the Prism pacemaker. PMID- 1712956 TI - The relationship between heart rate and QT interval during atrial stimulation. AB - The relationship between heart rate and QT interval was investigated during atrial stimulation (intrinsic effect of heart rate) in ten healthy male volunteers prior to and after administration of sotalol. The QT interval in the ECG (paper speed 200 mm/s) was determined at rates of 70, 85, 100, 115, 130, 145, and 160 beats/min and at pacing periods of 180 s each at 30, 60, 120, and 180 s. After a 15-minute period, 2.0 mg sotalol/kg body weight were administered iv and the stimulation protocol was repeated. The analysis of QT interval behavior reveals contradictions to the mathematical implications of Bazett's equation QT = QTc square root of 60/HR, so that the relationship between heart rate and QT interval is not adequately described under the given conditions. After examination of approaches reported in the literature and our own approaches, the expression QT = a e-b (HR-60) is used as a possibility differentially to describe the data by nonlinear regression. The parameters a and b may be interpreted as QT reference value and shortening parameter. The QT reference value a, a parameter in reference to heart rate of 60 beats/min, has a comparable significance to the expression QTc in the Bazett equation. A reduction in the shortening parameter b indicates whether substances influencing the QT interval additionally produce overproportional shortening of the QT interval with increasing heart rate. After administration of sotalol, an increase can be observed in both the QT reference value and also in the shortening parameter. The suggested approach is an attempt to provide a more precise assessment of the QT interval under different conditions. PMID- 1712957 TI - Quality of life in patients with rate responsive pacemakers: a randomized, cross over study. AB - Eleven patients with rate responsive pacemakers (7 men, 4 women, mean age 41 years with a range of 23-60) were randomly assigned to a cross-over study in order to assess their overall exercise capacity and quality-of-life (QOL) scores. All of the pacemakers were implanted for complete AV block or sick sinus syndrome. The pacemakers were randomly programmed into VVI or rate responsive (VVIR) pacing modes for 3-week study periods in each mode. At the end of each period, an exercise test was performed and the QOL was evaluated by the "Hacettepe Quality-of-Life Questionnaire". All patients exercised longer in the VVIR mode (mean 10.54 +/- 0.73 min) than in the VVI mode (mean 7.81 +/- 0.62 min) (P less than 0.05). QOL scores were also found to be significantly higher in the VVIR mode (mean 173.81 +/- 16.22 points) compared to the VVI mode (mean 156.27 +/ 21.22 points) (P less than 0.01). In conclusion, our results suggest that VVIR pacing offers a better QOL in addition to an improved exercise capacity, compared to the single chamber nonrate modulated pacing (VVI). PMID- 1712958 TI - Assessment of effects of a radiofrequency energy field and thermistor location in an electrode catheter on the accuracy of temperature measurement. AB - Accurate measurement of temperature at the interface of the delivery electrode and the tissue during transcatheter delivery of radiofrequency energy (RFE) for ablation would provide better control of lesion production. Electromagnetic energy fields can affect the accuracy of temperature measurement with thermistors. An electrode probe was fabricated with a thermistor and an optical sensor in the center of the delivery electrode. Simultaneous temperature measurements during RFE delivery to cardiac tissue in the 37 degrees C bath showed good agreement between the sensors, indicating that the RFE field did not cause errors in thermistor temperature measurements with the electrode probe used. A second electrode probe was designed to determine optimal thermistor location. It was constructed using two thermistors with identical temperature resistance curves. One thermistor protruded through a hole in the side of the delivery electrode and was thermally isolated from it. The other thermistor was bonded to the inner surface of the electrode with heat conductive epoxy. The electrode was placed in contact with cardiac tissue in a 37 degrees C bath of flowing saline with the protruding thermistor centered in the area to be heated. Temperatures measured at steady state during RFE delivery with the protruding thermistor were consistently higher than those of the inner wall thermistor, ranging from 1.8 degrees C difference at 46 degrees C to 8.3 degrees C difference at 75 degrees C interface temperature. The thermistor must be in contact with the tissue and thermally isolated from the delivery electrode for accurate determination of electrode/tissue interface temperature. PMID- 1712959 TI - Long-term efficacy of an antitachycardia pacemaker and implantable defibrillator combination. AB - Antitachycardia pacemakers and implantable cardioverter defibrillators (ICD) were implanted in 14 patients to control recurrent hemodynamically stable ventricular tachycardia (VT). All patients underwent extensive preimplant testing in the electrophysiology laboratory documenting that in each patient at least 50 episodes of VT could be reliably terminated by an external model of the antitachycardia pacemaker. The burst scanning mode of antitachycardia pacing was used in all patients. ICDs were implanted solely as a back up should acceleration of VT occur, and all had high nonprogrammable rate cutoffs (mean 191 +/- 12 beats/min). During a mean follow-up of 25 +/- 6 months, 6,029 episodes of VT were treated in the 14 patients. Only 103 ICD discharges were required (approximately one discharge per 60 episodes of VT). Ten of the 14 patients received discharges from their ICDs. No deaths have occurred. All devices remain active and in the automatic mode. Thus, an antitachycardia pacemaker and ICD combination can safely and effectively terminate VT in highly selected patients who are subjected to extensive preimplant testing. In such patients, the vast majority of episodes of VT can be terminated with antitachycardia pacing, and only rarely is a discharge required from the ICD. PMID- 1712960 TI - A comparison of QRS complexes resulting from unipolar and bipolar pacing: implications for pace-mapping. AB - To examine differences in QRS configuration produced by bipolar versus unipolar pacing, 12-lead electrocardiograms recorded during bipolar (distal cathode) pacing with 5- and 10-mm interelectrode distances were compared to electrocardiograms recorded during unipolar cathodal pacing from the distal catheter pole. Pacing was performed at a cycle length of 500 msec using each of the two bipolar configurations at current strengths equal to late diastolic threshold, twice threshold and 10 mA. The pacing site was at the right ventricular apex in 15 patients and at various left ventricular locations in 14 patients. The electrocardiograms recorded during bipolar and unipolar pacing were compared by two independent observers for minor QRS configuration changes, major configuration changes and amplitude changes. Minor configuration differences between unipolar and bipolar pacing occurred occasionally when the interelectrode distance during bipolar pacing was 5 mm (mean +/- S.D. 0.5 +/- 1.2 leads per electrocardiogram). However, when the interelectrode distance was 10 mm, minor configuration differences were seen more commonly (1.3 +/- 2.0 leads per electrocardiogram; P less than 0.05 vs 5-mm distance). Major configuration differences were uncommon with either configuration at all current strengths. Pacing at 10 mA produced a larger number of configuration differences than pacing at either threshold or twice threshold (P less than 0.05). Amplitude differences were seen in a mean of 1.9 +/- 2.1 leads per electrocardiogram with the 5-mm interelectrode distance and a mean of 2.9 +/- 2.1 leads using the 10-mm interelectrode distance (P less than 0.05). IN CONCLUSION: (1) bipolar ventricular pacing can result in QRS complexes that are different from those obtained with unipolar pacing at the same catheter location, presumably due to an anodal contribution during bipolar pacing; (2) increasing the interelectrode distance and stimulus intensity increases these differences; and (3) because the proximal electrode's contribution to depolarization can alter the QRS configuration during pacing in a variable way, the use of bipolar pace-mapping to localize sites of origin of ventricular tachycardia may result in less spatial resolution than unipolar pace-mapping. PMID- 1712961 TI - Determinants of subsidiary ventricular pacemaker suppression in man. AB - To investigate the relative contribution of the duration and rate of overdrive to subsidiary ventricular pacemaker suppression, in six patients with complete heart block after His-bundle ablation, ventricular overdrive stimulation studies were performed. The studies, which were spread over a mean follow-up period of 745 days, were carried out invasively with a temporary lead (one patient) as well as noninvasively with the implanted pacemakers and chest wall inhibition (five patients). The overdrive pacing rate was increased in steps of 10 beats/min, and the pacing duration was 15, 30, 60, 90, and 120 seconds at each level. A recovery period of 2 minutes was allowed after each overdrive stimulation. Incremental ventricular overdrive stimulation at increasing pacing durations consistently caused progressive suppression of ventricular impulse formation. Nonparametric variance analysis demonstrated a significant (P less than 0.0001) influence of both the pacing rate and duration on ventricular recovery time. Nonlinear regression showed an exponential increase in recovery time with incremental pacing rate and a biphasic increase in recovery time with incremental pacing duration. Beyond a pacing duration of 60 seconds ventricular impulse suppression was primarily dependent upon the pacing rate. A nonlinear regression model was applied to predict the number of beats required for return of the escape rhythm toward prepacing control values. The predicted maximum mean number of beats was 15.4 +/- 5.9 and independent of the rate and duration of pacing, although, the initial temporary instability of the escape rhythm was directly related to the degree of overdrive. PMID- 1712962 TI - Effects of atrial impulse timing on AV concealed conduction in the rabbit heart. AB - The influence of the timing of a nontransmitted or transmitted atrial impulse on the atrioventricular (AV) conduction time of the subsequent impulse was studied in nine isolated rabbit hearts. AV conduction curves were determined by applying the atrial extrastimulus test. The extrastimulus was delivered preceded or not by an interposed atrial impulse whose coupling interval with respect to the last atrial beat of a basic train was kept constant at 100, 120, 140, 160, 175, 200, 225, 250, and 300 msec. In all experiments, there was a "concealment interval," i.e., the AV effective refractory period was longer than the atrial functional refractory period, and in seven experiments was comprised between 100 and 160 msec. For any given extrastimulus coupling interval in the presence of an interposed nontransmitted atrial impulse, AV conduction time was significantly greater than in its absence; the increase was greater than the longer the nontransmitted atrial impulse coupling interval, i.e., the shorter the subsequent transmitted impulse coupling interval with respect to the previous interposed nontransmitted impulse. The AV conduction curves relating the extrastimulus AV conduction time to its coupling interval with respect to the last atrial impulse of the basic train fitted to a hyperbolic model both in the absence of the interposed atrial impulse and in its presence (mean square residual 31 +/- 23 msec2), and the interposed atrial impulse modified the constants of the functions; the slope of the linear transformations was progressively more negative as the interposed atrial impulse was delayed. Furthermore, the effects of the interposed atrial impulse--transmitted or not--on AV conduction time of the subsequent impulse were qualitatively similar, their magnitude depending on the time elapsed were qualitatively similar, their magnitude depending on the time elapsed between the two. PMID- 1712963 TI - Pacemaker generator pseudomalfunction: an artifact of Holter monitoring. PMID- 1712964 TI - Removable or nonremovable endocardial electrodes: do not accept erroneous conclusions. PMID- 1712965 TI - Feasibility of long-term electrocardiographic monitoring with an implanted device for syncope diagnosis. PMID- 1712966 TI - Ventricular pacing from the atrial channel of a DDD pacemaker. PMID- 1712967 TI - Palliation of oesophageal cancer--endoscopic intubation and laser therapy. PMID- 1712968 TI - Developmental patterns of selected characteristics of the gastrointestinal tract of young turkeys. AB - Developing embryos and hatchling poults were sampled (n = 4) at Days 22, 24, 26, and 28 of incubation and at 1, 2, 4, 6, and 8 days after hatching, and selected characteristics of the gastrointestinal tract (GIT) were measured. Body weight increased linearly up to day of hatching and also from 2 to 8 days posthatching. Residual yolk weight decreased rapidly starting on Day 26 of incubation and was nearly depleted by 4 days posthatching. Changes in weight of segments of the GIT nearly paralleled the increase in body weight until day of hatching. Thereafter, weights of the proventriculus, small intestine, and pancreas increased more rapidly than body weight until 6 days after hatching. At this time, change in weight of small intestine and pancreas seemed to parallel that of body weight, whereas proventriculus weight continued to increase more rapidly. Gizzard weight, as a percentage of body weight, increased until Day 4 posthatching and then remained relatively constant through 8 days. Specific activities (SA) of pancreatic amylase, lipase, and trypsin were low until after hatching. Subsequently, amylase SA increased nearly threefold by Day 6. Lipase SA remained nearly constant between Days 1 and 8, and trypsin SA increased only slightly. Total activities of pancreatic enzymes, however, increased substantially after hatching, mainly because of increased pancreas weight. Jejunal maltase SA was high at hatching but decreased markedly by Day 4. This decrease in SA resulted in a notable reduction in total maltase activity of the jejunum despite an increase in jejunum weight. PMID- 1712969 TI - Effects of calcium-channel blockers on cytosolic free calcium and amylase secretion in rat pancreatic acini. AB - We investigated the effects of verapamil and diltiazem on cytosolic free calcium and amylase secretion in rat pancreatic acini. Verapamil and diltiazem reduced a rise in cytosolic free calcium and amylase release stimulated by the maximal concentration (10(-5) M) of carbachol in a dose-dependent manner. High concentrations (500 microM) of verapamil and diltiazem inhibited both the initial and the sustained amylase secretion stimulated by 10(-5) M carbachol. However, at low concentration (1 microM), they showed no effect on amylase secretion by 10( 5) M carbachol. These calcium-channel blockers did not affect calcium mobilization and amylase secretion stimulated by either caerulein or neuromedin C. Binding of 3H-N-methylscopolamine to pancreatic acini was inhibited by verapamil and diltiazem in a dose-dependent manner. These findings suggested that verapamil and diltiazem reduced carbachol-induced amylase secretion probably not due to their calcium-channel blocking activities but due to their non-competitive effects on the level of muscarinic receptors. PMID- 1712970 TI - Angiotensin II receptors in Xenopus oocytes. AB - Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1712971 TI - The protection of insulin against proteolytic processes after rectal application to normoglycemic rabbits. AB - In order to improve the bioavailability of insulin after rectal application to rabbits, the influence of surface-active amino acid-fatty acid condensate on absorption and, the effects of a protease inhibitor (aprotinine), of a disinfectant (methyl-hydroxybenzoate) and of chemotherapeutics (chloramphenicol, ambazone and metronidazole) were investigated. Only the application of methylhydroxybenzoate, ambazone and of metronidazole, which inhibit the anaerobic bacterial flora, improved bioavailability significantly. The examinations show that the proteolytic activity of the anaerobic bacterial flora causes the loss of the biological activity of peptides in the rectum. PMID- 1712972 TI - Alpha-fluoromethylhistidine decreases the Leu-enkephalinamide- and morphine induced corticosterone response in rats. AB - The effect of inhibition of brain histamine synthesis by alpha fluoromethylhistidine (alpha-FMH) on the pituitary adrenocortical activity stimulated by D-Ala-D-Leu-enkephalinamide (DADL) and morphine was investigated indirectly through corticosterone secretion in conscious rats. alpha-FMH (20 mg/kg i.p.) drastically reduced the whole brain histamine content, measured 2 h later. The same pretreatment also considerably reduced the corticosterone response to morphine given intraperitoneally. When alpha-FMH was administered intracerebroventricularly (50 micrograms), the maximum inhibition of the corticosterone response to DADL and morphine occurred 4 h after administration, which may suggest a weaker accessibility of alpha-FMH from the cerebral ventricle to the brain structures involved in pituitary-adrenocortical stimulation. The corticosterone responses were not related to the core temperature changes. These results indicate that inhibition of brain histamine synthesis by alpha-FMH considerably impairs the pituitary-adrenocortical response to the opioid delta- and mu-receptor agonists DADL and morphine. They also suggest that neuronal histamine is significantly involved in the central stimulation of the pituitary adrenal axis by opioids. PMID- 1712973 TI - Angiogenesis in arteries: review. AB - The inner parts of the walls of large blood vessels do not normally contain intrinsic vasculature. In pathologic conditions such as arteriosclerosis or thrombosis, angiogenesis occurs, and may have significant clinical consequences. This review attempts to relate the little that is known about the factors specific to vascular walls which regulate angiogenesis to more general knowledge of the phenomenon. PMID- 1712974 TI - Low molecular weight angiogenesis factors. AB - A number of substances have been proposed for the role of angiogenesis factors. Many of these are of protein origin and are therefore amenable to the tools of the molecular biologist. However a number of low molecular weight angiogenesis factors are emerging as important initiators and/or cofactors of neovascularization. Of these a number are known to stimulate angiogenesis indirectly, possibly through an inflammatory response. Some putative angiogenic factors stimulate microvessel endothelial cells nonspecifically, also causing migration and proliferation of large vessel cells. Others are specific for microvessel cells either for stimulating migration, proliferation or both. The nature and action of the low molecular weight factors in vivo and in vitro are reviewed. PMID- 1712976 TI - The tetrazolium-formazan system: design and histochemistry. AB - Our increasing knowledge about the chemistry and the correlations between chemical structure and histochemical properties of the tetrazolium/formazan system is resulting in: a better understanding of existing histochemical tetrazolium techniques; the selection of optimal tetrazolium salts for qualified use in histochemistry, cytochemistry and biochemistry; both qualitative and quantitative improvements in histochemical techniques for purposes demonstrating the activities of various dehydrogenating enzymes; an extended insight into the "state" of the tested biological object by means of tetrazolium indicators with special properties; and the combination of histochemical enzyme determination with further morphological techniques. This article has attempted to illustrate the progress in the use of the tetrazolium/formazan-system for histochemical purposes. PMID- 1712975 TI - Enhancement of anticancer agent activity by selective inhibition of rapidly proliferating tissues of the host. AB - Most cytotoxic drugs used in cancer therapy do not discriminate between neoplastic and normal proliferating cells. To avoid irreversible damage to vital host tissues, such as bone marrow and intestine, drugs must be administered at dosages which usually prove insufficient to eradicate all of the neoplastic cells present. This review focuses on an approach to improve cancer chemotherapy by selectively protecting normal, proliferating cells during treatment, thereby permitting the administration of otherwise lethal doses of drug. Preclinical in vivo studies of cytokinetic modulation with interferon, or L-histidinol, as well as recent clinical studies of interferon modulation of the activity of 5 fluorouracil are reviewed. PMID- 1712977 TI - High-dose-rate intraluminal brachytherapy for bile duct carcinoma after surgery. AB - Five patients with recurrent or residual bile duct carcinoma after surgery were treated with high-dose-rate intraluminal brachytherapy (HDRIBT) using a remote afterloader. External radiotherapy was also given in three cases. HDRIBT is considered to be an effective mode of radiotherapy for residual or recurrent bile duct tumors. PMID- 1712978 TI - Conservative therapy of interstitial cystitis. PMID- 1712979 TI - Experimental model for the study of bladder mast cell degranulation and smooth muscle contraction. PMID- 1712980 TI - [Autoantibodies and the immunopathological profile of lupus erythematosus]. PMID- 1712981 TI - Pancreatic disturbances and typhoid fever. AB - During an 8-year period, 14 adult patients were hospitalized with typhoid fever confirmed by positive blood cultures for Salmonella typhi. Among these patients, we have retrospectively (n = 7) and prospectively (n = 7) evaluated pancreatic disturbance by serum amylase and lipase measurements at the time of admission. In 7 (50%) biological signs of pancreatitis were noted: mean amylase level 81 IU (range 30-201 IU, normal value less than 40 IU), mean lipase level 949 IU (range 468-2,000 IU, normal value less than 300 IU). Clinical signs of pancreatitis were observed in 4 cases, one of whom had a concomitant salmonella biliary tract infection and gall stones demonstrated by laparotomy and the others a normal biliary ultrasonographic examination with a swelling of the pancreas. No alcohol or drug use or other infection were noted before admission. This study suggests that biological or clinical pancreatitis should be considered as a frequent complication of typhoid fever. S. typhi should therefore be added to the list of pathogens implicated in the pathogenesis of non-alcoholic or non-lithiasic pancreatitis. PMID- 1712982 TI - RNA editing: what's in a mechanism? PMID- 1712983 TI - Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. AB - The most frequently occurring RNA hairpins in 16S and 23S ribosomal RNA contain a tetranucleotide loop that has a GNRA consensus sequence. The solution structures of the GCAA and GAAA hairpins have been determined by nuclear magnetic resonance spectroscopy. Both loops contain an unusual G-A base pair between the first and last residue in the loop, a hydrogen bond between a G base and a phosphate, extensive base stacking, and a hydrogen bond between a sugar 2'-end OH and a base. These interactions explain the high stability of these hairpins and the sequence requirements for the variant and invariant nucleotides in the GNRA tetranucleotide loop family. PMID- 1712984 TI - Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. AB - Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity. PMID- 1712985 TI - Effect of deleting the R domain on CFTR-generated chloride channels. AB - The cystic fibrosis transmembrane conductance regulator (CFTR), which forms adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, is defective in patients with cystic fibrosis. This protein contains two putative nucleotide binding domains (NBD1 and NBD2) and an R domain. CFTR in which the R domain was deleted (CFTR delta R) conducted chloride independently of the presence of cAMP. However, sites within CFTR other than those deleted also respond to cAMP, because the chloride current of CFTR delta R increased further in response to cAMP stimulation. In addition, deletion of the R domain suppressed the inactivating effect of a mutation in NBD2 (but not NBD1), a result which suggests that NBD2 interacts with the channel through the R domain. PMID- 1712986 TI - Vaccination, immunopathology, and immunity. PMID- 1712987 TI - [Hemoglobin F Catalonia. A new variant of fetal hemoglobin]. PMID- 1712988 TI - Pregnancy associated with lupus anticoagulant and heparin induced thrombocytopenia: management with a low molecular weight heparinoid. AB - The management of pregnant patients with coagulopathies and heparin induced thrombocytopenia is difficult and poorly defined. We report the case of a patient who was treated with a low molecular weight heparinoid. The treatment was complicated by the delayed occurrence of thrombocytopenia and a thrombotic event. This is the first report of thrombocytopenia caused by heparinoid. It is possible that this complication could have been avoided by a shorter duration of treatment with heparinoid and the use of Vitamin K antagonists during the second trimester of pregnancy. PMID- 1712989 TI - [Endoscopy simulator. A new aid for the training of endoscopy specialists]. PMID- 1712990 TI - Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency. AB - Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (less than 4 hr) and later organizes into keratin 'plaques' (4 days) associated with cell-cell contacts; post confluent (greater than 7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of 35S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 x 10(-3) M EGTA, 1 microM cytochalasin B, 1 microM bradykinin, 1 microM A23187, and 1 microM PMA) exhibit border retraction and altered keratin filament staining. From these studies we conclude: 1) cytokeratin 8 and 19 containing intermediate filaments are present in confluent PMV cultures with vimentin but without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modulation of cytokeratin assembly in PMV may be involved in regulation of pulmonary microvascular structure and function. PMID- 1712991 TI - The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells. AB - This study is concerned with the use of freshly harvested bovine endothelial cells attached to microcarrier beads in the production of the endothelium-derived relaxing factor (EDRF). The results are compared to production of EDRF by endothelial cells grown in tissue cultures. We found that freshly harvested cells attach themselves to microcarrier beads within minutes. This results in large surface/area volume ratio and permits superfusion of cells suspension on a filter (pore size of 25-30 microns), resulting in cell free filtrate. When superfusing an endothelium-deprived pulmonary artery strip, the effluent causes relaxation; the response depends on the number of superfused endothelial cells. The number of viable freshly harvested cells attached to microcarrier beads in 5 ml Krebs Henseleit solution is small (30%), as compared to almost 100% for cultured cells. Despite this difference, percent relaxation induced for the same number of viable cells is identical for both groups. Scanning electromicrographs confirm anchorage of endothelial cells to microcarrier beads. While cultured cells cover the entire surface and are individually attached, freshly harvested cells are anchored as cell aggregates leaving some of the surface free. Attachment of freshly harvested endothelial cells to microcarrier beads offers an alternative for the study of the role of endothelial cells in the production of vasoactive substances. PMID- 1712992 TI - PB-PK derived metabolic constants, hepatotoxicity, and lethality of BrCCl3 in rats pretreated with chlordecone, phenobarbital, or mirex. AB - Pharmacokinetic modeling has been very useful in examining the complex relationships between exposure concentration and target tissue dose. This study utilizes a physiologically based pharmacokinetic (PB-PK) modeling approach for assessing the metabolism of BrCCl3 and to investigate its relationship with hepatotoxicity and lethality. Male Sprague-Dawley rats maintained for 15 days on normal diet (control), or on diets containing either chlordecone (CD, 10 ppm), phenobarbital (PB, 225 ppm) or mirex (M, 10 ppm), were used in gas uptake studies to determine the kinetic constants of BrCCl3 metabolism. Four initial concentrations of BrCCl3 at approximately 30, 200, 700, and 3000 ppm were used for each group. The uptake data were analyzed by computer simulation using a PB PK model containing relevant tissue solubilities and physiological parameters as well as an equation describing the behavior of BrCCl3 in the closed chamber atmosphere. Liver injury was assessed by serum enzyme elevations (alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase) and histopathological examination, at 24 hr after the exposure to BrCCl3. Another group of similarly pretreated rats was exposed to BrCCl3 and observed over a 14 day period for mortality. Dietary exposures resulted in increased Vmaxc value for BrCCl3 metabolism as compared to control (3.55 +/- 0.14 mg/hr/kg) for PB (8.52 +/ 0.28 mg/hr/kg) and M (5.06 +/- 0.19 mg/hr/kg) but not for CD (3.92 +/- 0.19 mg/hr/kg). Kfc, the first-order rate constant for BrCCl3 metabolism, was decreased after PB (12.9 +/- 0.5 hr-1/kg) and increased after M (17.6 +/- 0.5 hr 1/kg), but unchanged after CD (15.5 +/- 0.6 hr-1/kg) exposure as compared to control (15.0 +/- 0.3 hr-1/kg). The total amount of BrCCl3 metabolism at any initial concentration employed remained unchanged in all the pretreated groups as compared to control. However, the amount of BrCCl3 metabolized through saturable pathway only, at higher initial concentrations, was increased in the PB and M pretreated groups, but not in the CD pretreated group. It is concluded that the rates of metabolism of BrCCl3 were unchanged after CD pretreatment as compared to control, while PB and M pretreatment alter both the saturable and first-order rates. Serum enzymes were significantly increased in all the groups after exposure to BrCCl3 at 200 and 700 ppm concentrations. The increase was more pronounced in PB and M pretreated groups as compared to control and CD pretreated groups. Similarly, histopathological examination of liver showed alterations in the lobular architecture, the extent of alterations being dependent on the dose of BrCCl3 and the pretreatment.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1712993 TI - [Continuous epidural treatment of cancer pain using a portable infusion pump]. AB - Treatment of cancer pain with opioids through an epidural catheter, with a portable infuser has been recorded in 20 patients retrospective. In general there was a good acceptance of the treatment, which often could liberate the patients from close contacts to the pain clinic. There were not recorded adverse effects in relation to the infuser treatment. One patient had systemic adverse effects because of a very high daily opioid dose. The Pharmacia Deltec infuser employed has proven very secure and easy to handle. PMID- 1712994 TI - Trends in the methods used for suicide in Northern Ireland. AB - As domestic gas was made less toxic in Northern Ireland during the period 1960 1988, it was used less often for suicide. During the same period, as car ownership increased, the use of car exhaust for suicide increased in popularity without there being a corresponding decrease in the use of other methods. Part of the temporal variation in suicide rates in Northern Ireland may be accounted for by the relative availability of lethal methods for suicide. PMID- 1712995 TI - Selecting a sensitive immunoradiometric method of maternal serum alphafetoprotein for prenatal screening of abnormalities in the fetus. PMID- 1712996 TI - The response of the detrusor muscle to acetylcholine in patients with infravesical obstruction. AB - We previously examined the effects of overdistension on the neuromuscular system of canine urinary bladders and reported that bladder overdistension led to nerve degeneration and subsequent supersensitivity through a decrease of blood supply to the bladder. We have accordingly in this study evaluated these changes in human subjects with infravesical obstruction. The responses to acetylcholine of bladder strips obtained from patients with detrusor instability were not significantly different from those of bladder strips from patients without detrusor instability, but the dose-response curve of these groups showed a shift to the right compared to that of the unobstructed control patients. As compared with the response of bladder strips in patients without an episode of retention, the response in patients who received prostatectomy within 30 days demonstrated no significant difference, although in patients who received prostatectomy after more than 30 days there was a statistical difference. These results indicated a significant decrease in sensitivity of the detrusor muscle in patients with infravesical obstruction and suggest that bladder overdistension caused by infravesical obstruction may lead to supersensitivity of the detrusor muscle secondary to denervation. PMID- 1712997 TI - UNME/K1: an IgG2a monoclonal antibody specific to cytokeratin of human urinary bladder squamous cell carcinoma. AB - The main distinctive feature of carcinoma in schistosomal bladder is keratinized squamous cell carcinoma. Keratins/cytokeratins constitute a multigeneic family of structurally related polypeptide markers for the malignant state of epithelial cells. A monoclonal antibody (UNME/K1) regognizing keratins associated with squamous cell carcinoma of the human urinary bladder was generated at the Urology and Nephrology Center, Mansoura, Egypt (UNME), by fusion of spleenocytes from a BALB/c mouse immunized with a keratin extract (K1) of human squamous cell carcinoma and P3X63Ag8/U1 syngeneic myeloma cells. UNME/K1 was purified by a protein-A affinity column and was of the IgG2a type, as determined by immunoelectrophoresis and gel diffusion techniques. When tested against keratins of different types of urinary bladder tumors using enzym linked immunosorbent assay (ELISA), UNME/K1 reacted only with the high molecular weight keratin of squamous cell carcinoma and showed selectivity towards specific histopathological grades of tumors. PMID- 1712998 TI - Pulsed carbon dioxide laser for cartilage vaporization and subchondral bone perforation in horses. Part II: Morphologic and histochemical reactions. AB - A pulsed carbon dioxide laser was used to vaporize articular cartilage in four horses, and perforate the cartilage and subchondral bone in four horses. Both intercarpal joints were examined arthroscopically and either a 1 cm cartilage crater or a series of holes was created in the third carpal bone of one joint. The contralateral carpus served as a control. After euthanasia at week 8, the treated and control joints were examined for gross changes, and samples of cartilage and subchondral bone, synovial membrane, and peripheral lymph nodes were examined histologically. Depletion of cartilage matrix glycosaminoglycan was assessed by safranin-O histochemical staining of the laser site and adjacent cartilage. Cartilage removal by laser vaporization resulted in rapid regrowth, with fibrous and fibrovascular tissue and occasional regions of fibrocartilage at week 8. The subchondral bone, synovial membrane, and draining lymph nodes appeared essentially unaffected by the laser cartilage vaporization procedure. Conversely, carbon dioxide laser drilling of subchondral bone resulted in poor penetration, extensive areas of thermal necrosis of bone, and significant secondary damage to the apposing articular surface of the radial carpal bone. PMID- 1712999 TI - Formation of multimers of linear satellite RNAs. AB - A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat RNAs revealed the deletion of bases corresponding to the 3' and 5' ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). EMBO J. 9, 1709-1715). PMID- 1713000 TI - Mutations in a satellite RNA of turnip crinkle virus result in addition of poly(U) in vivo. AB - Turnip crinkle virus (TCV) is associated with many subviral RNAs including satellite (sat-) RNAs which require a helper virus for infectivity. When plants were inoculated with TCV and transcripts of TCV sat-RNA C containing deletions of 3 to 8 nucleotides beginning at position 100 and extending toward the 5' end, some of the sat-RNA isolated from plants migrated more slowly than expected on denaturing polyacrylamide gels. Cleavage of the sat-RNA into two segments by digestion with RNase H following hybridization to an oligonucleotide complementary to internal sat-RNA sequence indicated that the 5' one-third of the molecule was involved in the abnormal gel migration. Sat-RNAs derived from transcripts with a deletion of bases in position 96-100 were cloned. Sequencing of the cDNAs revealed that the aberrant migration of the sat-RNAs was due to the presence of variable lengths of poly(U) 10 nucleotides downstream from the deletion at a position which already contained five U residues. Deletions extending toward the 3' end in the same region did not result in poly(U) additions. Mutations in the original five U residues along with the 5' deletions also did not lead to poly(U) additions. The insertion of poly(U) in TCV sat-RNA C may be a new example of replicase stuttering with the distinction that it only occurs following specific upstream mutations. PMID- 1713001 TI - A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain. AB - An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotide long, single stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus "hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudo-knot. PMID- 1713002 TI - Palliative operative procedures for carcinoma of the gallbladder. AB - Gallbladder cancer afflicts predominantly women, the elderly, and persons with gallstones. Despite its producing symptoms of abdominal pain, nausea and vomiting, weight loss, jaundice, and anorexia, this disease remains difficult to detect. Even with contemporary imaging techniques, most gallbladder cancers escape diagnosis until the time of laparotomy. The aggressive character of this malignancy permits an overall 5-year survival rate of 3-5%. Although cures occur, the majority of operations performed for gallbladder cancer are for palliation. The objects of palliation include relief of pain, relief of jaundice, relief of intestinal obstruction, and the restoration of normal food intake. Resection of the tumor should be performed whenever possible; however, extensive operations including large liver resections and pancreaticoduodenectomy should be avoided in the presence of distant metastases. In the presence of large unresectable hilar masses, internal biliary bypass may relieve jaundice. Biliary-enteric anastomosis using the segment III duct exposed via the umbilical fissure may offer satisfactory relief of jaundice in selected cases. PMID- 1713003 TI - Binding proteins for growth hormone and somatomedins. Proceedings of a workshop. PMID- 1713004 TI - Biochemical characterization of insulin-like growth factor binding proteins. PMID- 1713005 TI - Molecular forms of human IGF binding proteins: physiological implications. PMID- 1713006 TI - IGFBP-1. Production and control mechanisms. PMID- 1713007 TI - The physiological role of IGFBP-1. AB - It appears that a readily available pool of IGF is achieved by IGFBPs, primarily IGFBP-3, that maintain a relatively constant high level of IGF. A circulating enzyme is present in the circulation which can modify IGFBP-3. When IGF levels are low this enzyme may be activated to alter the availability of the limited reserves of IGF to the tissues. In conditions where fuel supplies are compromised IGF-actions can be modulated by acute changes in IGFBP-1 levels. The extent to which IGFBP-1 acts by affecting IGF transport to the tissues or by affecting its activity in the tissues has still to be determined. However, this is still a simplistic concept of a system where there is much more yet to be understood. There are several IGF-binding proteins whose physiology is largely uncharacterized, and possible interactions between the various components of the system are still largely unexplored. PMID- 1713008 TI - Paracrine actions of IGF binding proteins. AB - The paracrine mode of action for IGFs has been hypothesized on the basis of the finding that these peptides are synthesized ubiquitously. As IGFs circulate in very high concentrations (nearly 1 mg/l) they should have an endocrine function; moreover since the liver seems to be responsible for most of the circulating IGF I it should be recognized that the basis of the "hormonal" IGF-I residues in the liver, whereas the extrahepatic synthesis provides support for local paracrine or autocrine actions. According to the endocrine hypothesis, the liver, which lacks IGF-I receptors, produces both IGF-I and IGF binding protein (IGFBP) and, possibly, the acid-labile subunit. All these components combine in the circulation, and only free IGF-I or small molecular weight complexes cross the capillary wall and reach the target cells. The paracrine hypothesis is based on the fact that fibroblasts are able to synthesize both IGF peptides and IGFBP, whereas keratinocytes are not, although they possess IGF receptors and are sensitive to IGF action. Since affinity of BP3 (which seems to be produced by peripheral fibroblasts) for IGF-I is higher than that of fibroblasts and, to a lesser degree, of keratinocytes, it is likely that IGF-I released from fibroblasts preferentially binds to BP3; the BP3-IGF-I complex reduces the autocrine effects on fibroblasts and preferentially directs the peptide binding to keratinocytes. The secretion of both IGFs and of IGF carrier proteins has been reported in several neoplastic epithelial cell lines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713009 TI - Assays for IGF binding proteins. AB - Growing interest in IGF binding proteins and their roles in the modulation of IGF actions has prompted several laboratories to establish immunological assays to measure these proteins. Three distinct IGF binding proteins, referred as IGFBP-1, IGFBP-2 and IGFBP-3, have so far had their cDNAs cloned and sequenced. The availability of purified proteins and the development of polyclonal and monoclonal antibodies now allow specific quantitation of different IGFBP classes. This brief review will address on the current progress in the immunological assays of IGFBPs. The importance of the sensitivity, the reproducibility, and the standardization of the assays is discussed. Some additional information, like levels in amniotic fluid and serum of healthy adults and children, is given. PMID- 1713010 TI - Clinical aspects of insulin-like growth factor binding proteins. PMID- 1713011 TI - Direct comparison of peripheral T-cell lymphomas with their B-cell counterpart. AB - The aim of this study was to determine the effect of immunophenotype on the clinical characteristics and prognosis of 144 adult Chinese patients with non Hodgkin's lymphomas. Those entities with well-recognised immunophenotype and clinical characteristics were excluded. Significantly more patients with T-cell lymphomas had B symptoms (52 vs. 30%, p = 0.05) and fewer had bulky disease (7 vs. 25%, p = 0.04). Extranodal involvements of liver, spleen, marrow, nasal region and skin were significantly more common in T-cell lymphomas. On the other hand, gastro-intestinal involvement was more commonly seen in B-cell tumours. Induction chemotherapy of comparable intensity was used in treating these patients. The immunophenotype did not appear to affect significantly their prognosis. PMID- 1713012 TI - Polymorphism of cyclic 3',5'-adenosine monophosphate stimulation in rat erythrocytes. AB - Significant differences were found in the cyclic 3',5'-adenosine monophosphate (cAMP) levels in (-)-isoproterenol-stimulated rat erythrocytes. The BN strain had the highest level (13.1 +/- 1.29 pmol cAMP/10 mg Hb) and the LEW strain had the lowest cAMP level (3.29 +/- 1.76 pmol cAMP/10 mg Hb) in the erythrocytes. The high levels were inherited in three intercrosses in a dominant fashion. The results of the backcross breeding suggested diallelic inheritance. However, the polygenic effect was not ruled out. PMID- 1713013 TI - RNA metabolism in relation to amyotrophic lateral sclerosis. PMID- 1713014 TI - Monoclonal gammopathy and motor neuron disease. PMID- 1713015 TI - Neurofibrillary proliferation revisited. PMID- 1713016 TI - The development of rational strategies for selective immunotherapy against autoimmune demyelinating disease. PMID- 1713017 TI - The management of occluded metallic self-expandable biliary endoprostheses. PMID- 1713018 TI - Tumor necrosis factor-alpha and interleukin-1 beta production by human fetal Kupffer cells. AB - This study describes the isolation and characterization of human fetal Kupffer cells. We demonstrated that these cells have the potential to respond to cytokines and lipopolysaccharide with an increased production of tumor necrosis factor-alpha and interleukin-1 beta. Kupffer cells were characterized by: (1) morphologic characteristics after adherence to plastic, (2) staining for alpha naphthyl acetate esterase, (3) immunofluorescence with monoclonal antibodies, and (4) phagocytosis of latex beads. More than 90% of the adherent cells were identified as macrophages. Kupffer cells cultured with lipopolysaccharide were able to produce interleukin-1 beta and tumor necrosis factor-alpha in a time- and dose-dependent fashion and maximal secretion was observed with the use of 10 micrograms of lipopolysaccharide per milliliter within 8 hours of treatment. We have demonstrated mature functional activity of human fetal Kupffer cells at an early gestational age (13 to 19 weeks) and discussed the roles that these cells may play in development and protection of the fetus. PMID- 1713019 TI - Relationship of epidermal growth factor receptor detectability with the A, B, H blood group antigens. Emphasis on normal and neoplastic urothelium. AB - The epidermal growth factor receptor (EGFR) is a glycoprotein that carries on its extracellular domains oligosaccharide chains, some of which are similar to blood group determinants. The author examined the expression of the EGFR using immunohistochemistry and three anti-EGFR antibodies with different fine specificities. These were applied to frozen sections from 120 tissue biopsies, including normal and neoplastic urothelium. The expression of blood group A, B, H determinants was also evaluated in the same tissues. Significant differences were noted, depending on the fine specificity of the anti-EGFR antibody, cellular differentiation, and the tissue donor's blood group. The most interesting finding was a reciprocal relationship between the reaction patterns obtained using antibodies with carbohydrate specificity and those with peptide specificity. These patterns were characteristic of cellular differentiation and suggested that the EGFR peptide determinants are detected in excess in immature and malignant cells that, however, appear deficient in oligosaccharide determinants. The combination of 'loss' of blood group antigen and an apparent overexpression of EGFR peptide determinants is indicative of invasive growth in transitional cell carcinomas. PMID- 1713020 TI - Expression of immunoreactive E-cadherin adhesion molecules in human cancers. AB - E-cadherin (E-CD), a Ca(2+)-dependent adhesion molecule, plays a major role in the maintenance of intercellular junctions in normal epithelial cells in most organs. The expression of E-CD in human carcinoma samples (esophagus, stomach, and breast) was investigated using immunohistochemical staining, which was performed on surgical specimens using a monoclonal antibody for human E-CD. E cadherin was strongly expressed in all normal epithelium examined. However E-CD expression in primary tumors of esophagus (11 of 15: 73%), stomach (5 of 20: 25%), and breast (9 of 20: 45%) was reduced, and 68% of these (esophagus: 8 of 11, stomach: 4 of 5, breast: 5 of 9) displayed heterogeneous E-CD expression. In some tumor cells with reduced E-CD expression, E-CD molecules were located in the cell cytoplasm. These results indicate that there are human cancer cells in which E-CD-related intercellular adhesion is impaired. PMID- 1713021 TI - Epitope map of neurofilament protein domains in cortical and peripheral nervous system Lewy bodies. AB - A subset of demented elderly patients exhibit large numbers of cortical intraneuronal inclusions similar to the neurofilament (NF)-rich Lewy bodies (LB) found in pigmented subcortical neurons of patients with Parkinson's disease (PD). Because these cortical inclusions may contribute to the emergence of cognitive impairments in afflicted individuals, the authors mapped the distribution of NF epitopes in these so-called cortical LBs. This was done using ethanol-fixed tissues and a large library of monoclonal antibodies (MAbs) with well characterized binding specificities to various regions of each NF triplet protein. Cortical LBs were examined by light, confocal, and electron microscopy, and they were compared with the subcortical LBs of PD and LBs in the peripheral nervous system (PNS). Monoclonal antibodies specific for the rod regions of each of the three NF subunits, or for phosphate-dependent and independent antigenic sites in the tail region of the high- (NF-H) and middle- (NF-M) molecular weight (Mr) NF subunits as well as other MAbs to the extreme COOH terminus of NF-L and NF-M or the head region of NF-M labeled a variable number of cortical LBs. Remarkably one of these anti-NF MAbs, RMO32, which recognized a phosphorylated epitope in the tail region of NF-M, immunolabeled nearly all cortical LBs, whereas each of the other anti-NF MAbs never labeled more than 10% of ubiquitin- or RMO32-positive cortical LBs. Further LBs in the PNS resembled those in the central nervous system (CNS) in their immunologic properties, and LBs in both sites were dominated by filamentous aggregates at the ultrastructural level. These findings suggest that NF proteins are profoundly altered during their incorporation into cortical and PNS LBs. Further the authors here identified immunologic and ultrastructural properties common to cortical LBs, PNS LBs, and classic substantia nigra LBs in PD. The accumulation of filamentous, perikaryal inclusions rich in NF proteins at diverse sites in the CNS and PNS of patients with a variety of neurodegenerative disorders suggests a widespread disruption of NF metabolism or transport. PMID- 1713022 TI - Unexpected immunoreactivities of intermediate filament antibodies in human brain and brain tumors. AB - Immunoreactivities of 35 different monoclonal antibodies (MAbs) that detect intermediate filaments were studied systematically on serial cryostat sections of 14 well-defined human gliomas (five astrocytomas, three oligodendrogliomas, six glioblastomas) and on normal brain. Glial fibrillary acidic protein (GFAP), vimentin, desmin, neurofilaments, and broad-specificity keratin MAbs, as well as MAbs that recognize several or only single keratin polypeptides, were used. Unexpected reactivities were surprisingly frequent. As these may lead to diagnostic confusion and misinterpretation on this material, the authors investigated these phenomena more thoroughly. Four major sources of artifactual staining were found: 1) positive staining attributable to the rabbit gamma G immunoglobulins used in the alkaline phosphatase anti-alkaline phosphatase technique; 2) certain desmin and keratin MAbs cross-reacted with astrocytic glia and with other brain-specific epitopes; 3) technical difficulties; 4) some MAbs directed against neurofilaments and keratins showed unexpected reactivities only on individual anaplastic gliomas. The implications of these findings for intermediate filament typing of neuropathologic material are discussed. PMID- 1713024 TI - Serum levels of Hyskon during hysteroscopic procedures. AB - Hyskon hysteroscopy fluid is used with the hysteroscope as an aid in distending the uterine cavity and in visualizing its surfaces. We correlated the amount of Hyskon used during hysteroscopy with both the instillation pressures generated within the uterine cavity and with serum levels of Hyskon in 11 healthy subjects. Serum levels in excess of 1000 mg% were measured at 30 min when an amount of Hyskon greater than 300 mL was used. Intrauterine Hyskon absorption and/or injection during hysteroscopy were found even after volumes of Hyskon as small as 50-100 mL were used. Serum levels were significantly higher with the larger amounts of Hyskon used in ablative procedures (P less than 0.01), as compared with serum levels seen with the smaller amounts of Hyskon used for diagnostic procedures. The injection pressures should be low to minimize the amount of intravascular injection. In cases in which extensive ablative procedures are necessary, we suggest that a two-stage procedure be considered. Because of the hypertonicity of Hyskon, intravascular volume expansion that may be larger than that seen during transurethral prostatectomy occurs. PMID- 1713023 TI - Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. AB - Capillary occlusions are characteristic features of the early diabetic retinopathy and are presumed to initiate neovascularization. Activated leukocytes can cause microvascular occlusions and cell damage by release of cytotoxic products. To explore the role of leukocytes in capillary occlusion, nonperfusion, and neovascularization of diabetic retinopathy, a rat model was used, in which a diabetic state was induced by alloxan. Retina flat preparations were differentially stained for monocytes and granulocytes. Capillary occlusion, nonperfusion, and neovascularization were assessed microscopically in the center, midperiphery, and periphery of the retina. In contrast to control retinas, 2- to 9-month diabetic rats showed many capillary occlusions by leukocytes, especially monocytes, endothelial cell damage, extravascular macrophage accumulation, and tissue damage. The percentage of activated monocytes and granulocytes in the circulating blood of diabetic rats was greatly increased, and areas of capillary 'loss' and neovascularization in the retina coincided with sites of extravascular leukocytes. The authors' results suggest a potential role of monocytes and macrophages in the pathogenesis of diabetic retinopathy. PMID- 1713025 TI - L-644,711, a novel anion transport inhibitor, increases isoflurane MAC in rats. AB - The effect of the anion transport inhibitor L-644,711 on isoflurane MAC was determined in rats (n = 24). After baseline MAC determination, each rat received one of the following drug protocols: (a) control, vehicle only; (b) L-644,711IT, a 3-mg/kg intrathecal bolus of L-644,711 followed by an infusion at 1.5 mg.kg-1.h 1; or (c) L-644,711IV, a 6-mg/kg intravenous bolus of L-644,711 followed by an infusion at 3 mg.kg-1.h-1. MAC was again determined. The baseline isoflurane MAC was not different between groups (control, 1.52% +/- 0.15%; L-644,711IT, 1.51% +/ 0.24%; L-644,711IV, 1.54% +/- 0.13% [mean +/- SD]). After drug or vehicle administration, isoflurane MAC was larger for the L-644,711IT group (2.25% +/- 0.17%) versus the control (1.38% +/- 0.13%) and L-644,711IV (1.39% +/- 0.15%) groups (P less than 0.05). These data are consistent with the hypothesis that isoflurane anesthesia is influenced by anion channels and that blocking these channels may reduce the pharmacodynamic potency of isoflurane. PMID- 1713026 TI - Contributions of peritoneal lavage enzyme determinations to the management of isolated hollow visceral abdominal injuries. AB - STUDY OBJECTIVE: To determine the role of lavage amylase (LAM) and lavage alkaline phosphatase (LAP) in the identification of intraperitoneal hollow visceral injuries. DESIGN: Retrospective. SETTING: Level I trauma center, city/county institution. TYPE OF PARTICIPANTS: Patients with hollow visceral organ injury following major blunt or penetrating trauma whose diagnostic peritoneal lavage was negative by lavage red blood cell and lavage white blood cell were negative. MEASUREMENTS AND MAIN RESULTS: Fifty-one patients with injury isolated to one or more hollow visceral structures underwent diagnostic peritoneal lavage; 28 were positive based on aspirate, lavage red blood cell count (greater than 100,000/mm3), or lavage white blood cell count (greater than 500/mm3). Of the remaining 23 patients, each of 11 with isolated small bowel injury had LAM greater than or equal to 20 IU/L and six of these had LAP levels greater than or equal to 3 IU/L. All six patients with colon injury and two of the patients with gallbladder injury had LAM less than 20 IU/L and LAP less than 3 IU/L. CONCLUSIONS: In patients with hollow visceral injury and otherwise normal diagnostic peritoneal lavage, elevation in LAM is highly specific for isolated small bowel injury. Lavage enzyme determinations appear unhelpful in the detection of colonic injury. We recommend routine enzyme determinations of lavage effluent as a marker for isolated small bowel injury. PMID- 1713028 TI - [Naturally occurring aliphatic polyamines-induced histamine release from rat peritoneal mast cells]. AB - Rat peritoneal mast cells obtained by Percoll gradient were incubated with different concentrations of naturally occurring aliphatic polyamines, spermine and spermidine, at 0.1-10 mM and the amount of histamine release into the supernatant solutions was measured. The addition of each polyamine to the suspensions of the mast cells caused a histamine release in a dose-dependent manner. The histamine release from the cells incubated with each polyamine was rapid and the amount of the histamine release into the supernatant solutions reached maximum at one minute with the incubations. PMID- 1713027 TI - Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp. cremoris. AB - Lactococcus lactis subsp. cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors. The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits. Our purpose was to identify unique rRNA sequences that could be used to discriminate L. lactis subsp. cremoris from related subspecies. The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L. lactis subsp. cremoris. All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus. In this region, all L. lactis subsp. lactis biovar diacetylactis strains examined had a sequence identical to that of L. lactis subsp. lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J. Brosius, J. L. Palmer, J. P. Kennedy, and H. F. Noller, Proc. Natl. Acad. Sci. USA 75:4801-4805, 1978). Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells. Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested. No hybridization was seen with the control species. All five strains of the subspecies cremoris hybridized to the subspecies-specific probe. PMID- 1713029 TI - Spontaneous regression of "dermal squamous cell carcinoma" in young chickens. AB - Eight young chickens with lesions characteristic of those described as dermal squamous cell carcinoma were obtained before slaughter. Lesions were measured; representative lesions were biopsied and examined microscopically; and gross changes were monitored. Lesions appeared to originate as cystic, keratin-filled proliferations of the feather-follicle epithelium. These cysts progressed into raised, keratin-filled, and eventually ulcerated, nodules. Loss of the keratin core resulted in a shallow ulcer that became progressively flattened and regressed into a fibrous dermal scar. All lesions in the broilers regressed in 4 to 16 days (mean 14 days). Twenty roaster lesions completely regressed. For roasters, the mean time to regression was 20 days, with a range of 6 to 93 days. Although there was limited invasion of the dermis by atypical keratinocytes, the overall architecture and biological behavior were more consistent with a benign lesion. PMID- 1713030 TI - Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens. AB - Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV. PMID- 1713031 TI - Noradrenaline stimulates glucose transport in rat brown adipocytes by activating thermogenesis. Evidence that fatty acid activation of mitochondrial respiration enhances glucose transport. AB - The mechanisms by which noradrenaline, lipolytic agents and long-chain fatty acids stimulate glucose transport were investigated in rat brown adipocytes. Glucose transport was evaluated with tracer D-[U-14C]glucose and cell respiration was measured polarographically. Noradrenaline increased basal oxygen consumption (8-10-fold) and glucose transport (4-5-fold) in a dose-dependent manner, with a maximal stimulation at 100 nM. The stimulatory effects of noradrenaline on respiration and glucose transport were selectively mimicked by dibutyryl cyclic AMP (DBcAMP), 3-isobutyl-1-methylxanthine, cholera toxin and physiological concentrations of palmitic acid. Cytochalasin B completely blocked the effects of these agents on glucose transport. The beta-adrenergic antagonist propranolol inhibited noradrenaline-induced glucose transport, but did not affect the action of DBcAMP, palmitic acid or cholera toxin on this process. The specific inhibitor of mitochondrial carnitine palmitoyltransferase, 2-tetradecylglycidic acid (McN 3802) (50 microM), inhibited the stimulatory effects of noradrenaline (100 nM) and palmitic acid (0.5 mM) on both glucose transport and mitochondrial respiration. Significantly, McN 3802 failed to affect insulin (1 nM) action under identical experimental conditions. These results demonstrate that (a) the stimulatory effects of noradrenaline on brown-adipocyte respiration and glucose transport can be dissociated from those induced by insulin, and (b) noradrenaline increases glucose transport indirectly, by activating adenylate cyclase via beta adrenergic pathways and by stimulating mitochondrial oxidation of fatty acids. PMID- 1713032 TI - Involvement of the C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analysed using region-specific monoclonal antibodies. AB - We have studied the effects of monoclonal antibodies that recognize different epitopes of the cerebellar Ins(1,4,5)P3 receptor on Ins(1,4,5)P3-induced Ca(2+) release activity. Ins(1,4,5)P3 stimulated Ca2+ flux from cerebellar microsomes, and half-maximal Ca2+ release occurred at 112 +/- 8 nM-Ins(1,4,5)P3 [concentration causing half-maximal effect (EC50) = 112.8 nM]. The minimum concentration of Ins(1,4,5)P3 necessary to initiate Ca2+ release (threshold concentration) was 20 +/- 5 nM. A monoclonal antibody (mAb) 18A10 (50 micrograms/ml), which recognizes the C-terminal region of the Ins(1,4,5)P3 receptor, suppressed Ins(1,4,5)P3-induced Ca2+ release: the EC50 and threshold concentration shifted to 460 +/- 56 nM and 61 +/- 6 nM respectively. On the other hand, the antibody at the same concentration raised the affinity of the receptor for binding to Ins(1,4,5)P3, and the Kd value decreased from 43 +/- 12 nM to 25 +/- 4 nM without a change in the number of Ins(1,4,5)P3-binding sites. However, mAbs that recognize the N-terminal domain affected neither Ca2+ release nor Ins(1,4,5)P3 binding. Among the various synthetic peptides, only the 12-residue long peptide from the most C-terminal portion of the receptor (amino acid residues 2736-2747) reacted strongly with mAb18A10. From these findings, combined with the Immunogold localization of the cerebellar Ins(1,4,5)P3 receptor [Otsu, Yamamoto, Maeda, Mikoshiba & Tashiro (1990) Cell Struct. Funct. 15, 163-173], we concluded that the C-terminus of the Ins(1,4,5)P3 receptor is exposed to the cytoplasmic side of the smooth endoplasmic reticulum and plays an important role in the regulation of both Ins(1,4,5)P3-binding affinity and channel gating. PMID- 1713033 TI - Potentiation of hyperthermia-induced haemolysis of human erythrocytes by photodynamic treatment. Evidence for the involvement of the anion transporter in this synergistic interaction. AB - Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4'-di-isothiocyano 2,2'-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakage were decreased significantly by preceding photodynamic treatment, suggesting that hyperthermia and photodynamic treatment have a common target for the induction of K+ leakage. Several lines of reasoning indicate that this common target is band 3. A model is thus proposed for the observed potentiation of hyperthermically induced K+ leakage by photodynamic treatment, in which photo-oxidation of band 3 results in increased sensitivity to subsequent thermal denaturation. These phenomena may be of more general significance, as photodynamic treatment and hyperthermia interacted synergistically with respect to K+ leakage with L929 fibroblasts also. PMID- 1713034 TI - Lack of 5-methylcytosine in Dictyostelium discoideum DNA. AB - We find no evidence for the presence of 5-methylcytosine in the DNA of Dictyostelium discoideum. Methylation was absent from CCGG sites in repetitive DNA and in DNA from the actin multigene family. Nor was 5-methylcytosine detected in total DNA when base composition was determined by means of h.p.l.c. PMID- 1713035 TI - Stability of ketoprofen-dextran ester prodrugs in homogenates of various segments of the pig GI tract. AB - Initial velocities of ketoprofen formation from ketoprofen-dextran ester prodrugs incubated in homogenates of various segments of the pig GI-tract were determined. Enzyme-mediated drug release was found in caecum and colon homogenates with their contents, whereas release rates in the stomach, duodenum, jejunum and ileum homogenates were comparable to those determined in pure buffer solutions of identical pH. In colon homogenates adjusted to various pH values between 6.0 and 7.9, little variation in release rates was observed. However, the contribution of enzyme-catalyzed drug regeneration to the overall initial velocity of ketoprofen formation increased significantly as a function of decreasing pH. The presence of several antibiotics and betamethasone in colon homogenates did not affect the drug activation process, whereas the addition of various enzyme inhibitors slowed down the ketoprofen release rates. During incubation in colon homogenates the average molecular weight of the dextran esters decreased. The drug release may therefore involve an initial fragmentation of the drug-liganded dextran chains carried out by dextranases secreted from the microflora which reside in the pig's large bowel. PMID- 1713036 TI - Conformational analysis of the tachykinins in solution: substance P and physalaemin. AB - A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties. PMID- 1713037 TI - Modulation of immune function by intestinal neuropeptides. AB - Direct regulatory control of the immune system by the central nervous system has been postulated. In support of this view is a large body of literature describing immunoregulatory activities of neuropeptides isolated from the gastrointestinal tract. In this review we examine the evidence for expression of specific receptors for gut peptides on immune effector cells and further explore the regulatory effects of these peptides on immune function. Peptides to be discussed include substance P, somatostatin, vasoactive intestinal peptide (VIP), the opioid peptides leu and met enkephalin, calcitonin gene related peptide (CGRP), neuropeptide Y, and cholecystokinin (CCK). PMID- 1713038 TI - Treatment of gastrointestinal endocrine tumours with interferon-alpha and octreotide. AB - This paper reports on the results of two controlled therapeutic trials on patients with endocrine tumours of the gastrointestinal tract. Seventeen patients were treated up to 18 months with recombinant interferon-alpha 2c (2 x 10(6) IU/m2 s.c. daily) and 16 patients are treated in an ongoing study with octreotide (3 x 200 micrograms daily). Objective response (greater than 50% reduction of hormone secretion) was observed in one of 15 evaluable patients on IFN-alpha and in 12 of 16 patients on octreotide. Reduction of tumour size was not observed in these two trials. However, the majority of patients had stable tumour size during IFN-alpha and octreotide treatment despite progressive disease before. Subjective improvement due to reduction of symptoms such as flushing, diarrhea, and dermatitis was significantly more frequent after octreotide than after IFN-alpha. Of five endocrine tumour patients with progressive disease on IFN-alpha, three responded to subsequent treatment with octreotide while one had stable disease and one progressed. Two cases are reported from the authors' series of patients treated with octreotide before start of these trials. Complete remission of the tumour by low-dose (2 x 100 micrograms daily) octreotide was observed in one carcinoid patient. This remission has now lasted for four years. In one patient with liver metastasis of a VIPoma, who had become resistant to streptozotocin, his watery diarrhoea is now completely controlled with 100 micrograms octreotide s.c. every second day. PMID- 1713039 TI - [Oromaxillofacial cancer. Development of therapeutic approach and results]. AB - We review the various theories, teams and lines of research pursued throughout history for knowledge of cancer, concentrating specifically in the analysis of prognostic variables and the evolution of therapy from major surgery to multifaceted treatment. We conclude with a demonstration of our results in the last few years while illustrating our method employed in the evaluation of patients. PMID- 1713040 TI - Cadmium, selenium, and tellurium chelators in Aspergillus terreus. AB - Aspergillus terreus was cultivated on Harrold's medium supplemented with 0.1% (w/v) cadmium chloride as well as on sulfur free medium amended with 0.1% (w/v) sodium selenite and potassium tellurite separately. The cell free extract of the fungus for each treatment was fractionated on a column packed with Sephadex G 75. The results demonstrated the ability of the fungus to synthesize several cadmium, selenium, and tellurium-binding proteins as well as metallothionein. The results suggested the biosynthesis of heavy metals chelators as well. The amino acids composition of a cadmium-binding metallothionein revealed the presence of high levels of both aromatic and sulfur amino acids in the hydrolysate. PMID- 1713041 TI - Dietary calcium and blood lead levels in women. AB - Nutritional factors are known to influence metabolism and toxicity of several metals in animal experiments, but relevant human data are scarce and inconclusive. In this work, we tested the hypothesis that dietary calcium influences lead metabolism in humans. Blood lead concentrations were used as indicators of lead exposure and metabolism. Two groups of peasant women living in similar conditions in two different regions in Yugoslavia (100 in each) were chosen as subjects for this purpose. In region A, the dietary calcium intake was about 940 mg, and in region B about two times lower, i.e., 450 mg/day. The average blood lead concentration was significantly lower in women from region A (69 micrograms/L) than from region B (83 micrograms/L). Our results support the assumption that adequate calcium intake might be one of the preventive measures for decreasing lead absorption. This new evidence, sought for some time by nutritionists and toxicologists, needs further international confirmation. PMID- 1713042 TI - Biological discrimination between calcium and strontium in kidneys and bone of young and adult rats. AB - Effects of aging on the biological discrimination between calcium (Ca) and strontium (Sr) by the kidneys and bone were studied in male and female rats of 5 to 50 wk of age by examining Sr/Ca ratios in the plasma, urine, and bone. The Ca Sr discrimination at the reabsorption process in the kidneys was not affected by aging in male or female rats. On the other hand, discrimination between the two elements was shown to be age-related at the absorption process in the digestive tract, and became more strict with age. The reverse situation was observed in the discrimination of Ca and Sr in the femur; younger rats discriminated the two elements more strictly than older animals. PMID- 1713043 TI - Copper deficiency and hyperlipoproteinemia induced by a tetramine cupruretic agent in rabbits. AB - The effectiveness of a cupruretic agent, N,N'-bis-(2 amino ethyl)-1,3 propanediamine HCl or 2,3,2-tetramine HCl (TETA), in the induction of copper (Cu) deficiency and the ability of a Cu-deficient diet in the maintenance of the depressed Cu status 10 wk after TETA treatment were examined in this study. In the first experiment, 42 male New Zealand White rabbits, 35 d of age, were randomly divided into three dietary treatments: a copper (Cu)-deficient (2.3 mg Cu/kg diet), a Cu-adequate (13.5 mg Cu/kg diet), and a commercial ration (21.6 mg Cu/kg diet) group. A single oral dose of 100 mg of 2,3,2-tetramine HCl TETA/kg body wt/d were administered to half of the rabbits from each treatment group for 10 d while the remaining rabbits were untreated. In the second experiment, 10 similar rabbits were assigned to three treatments: Cu-deficient plus TETA (n = 4); Cu-adequate plus TETA (n = 3); and Cu-adequate alone (n = 3). The rabbits were fed a TETA dose of 100 mg/d for three 4-d periods over 3 wk, and thereafter maintained on the diets for another 10 wk. Rabbits from the first experiment fed Cu-deficient diet and treated with TETA demonstrated cardiac hypertrophy and markedly reduced plasma and liver Cu concentrations that indicated that the animals were Cu-deficient. Significant elevations (twofold) in low density lipoprotein (LDL) protein, cholesterol, triglyceride, and apolipoprotein B (apo B) concentrations were observed in TETA treated rabbits fed Cu-deficient diet. In the second experiment, the plasma LDL protein level remained elevated, the plasma Cu level was reduced 45%, and the Cu level of the heart when expressed as microgram/g dry tissue was reduced, 10 wk post TETA treatment in rabbits maintained on Cu-deficient diet. Thus, Cu deficiency and hyperlipoproteinemia was rapidly induced by TETA and was still evident 10 wk posttreatment in rabbits maintained on a Cu-deficient diet. PMID- 1713044 TI - Effect of nickel on oxygen free radical metabolism. Inhibition of superoxide dismutase and enhancement of hydroxydopamine autoxidation. AB - The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 microgram/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10 4M to 1.1 x 10 4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 micrograms/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in whom increased serum nickel levels were reported to be in a similar range to those inhibiting SOD. PMID- 1713045 TI - Reevaluation of a sensitive indicator of early lead exposure. Measurement of porphobilinogen synthase in blood. AB - A principal target for the environmental toxin lead (Pb) is porphobilinogen synthase (PBGS), a Zn-metalloenzyme necessary for heme biosynthesis. Measurement of blood Pb inhibited PBGS is the most sensitive indicator of subclinical Pb intoxication, but problems with the assay have diminished its use. This report identifies Pb as a slow acting inhibitor of PBGS. The activity of PBGS could change up to sixfold during an hourlong clinical assay of Pb contaminated blood, and activity is profoundly effected by the presence of serum proteins, such as albumin. When PBGS catalyzed PBG production is allowed to reach a steady state rate, kinetic data on purified PBGS support the hypothesis that Pb inhibition of PBGS results from direct substitution for Zn. PMID- 1713046 TI - Sequential changes in propionate metabolism during the development of cobalt/vitamin B12 deficiency in sheep. AB - The changes in propionate metabolism that accompany cobalt deficiency in sheep are described. Two groups of sheep, fed either a cobalt sufficient or deficient diet, were given an iv propionate load at intervals during a 14 w experiment. There was a tendency towards increased propionate half-life as the animals became cobalt deficient. However, significant changes in the area under the plasma methylmalonic acid-time curve occurred very early, indicating significant impairment of propionate metabolism. Despite this, the area under the plasma glucose-time curve was unaffected by cobalt deficiency, suggesting that the impairment of propionate metabolism, although significant, is not extensive. PMID- 1713047 TI - Effect of boron on vitamin D deficient rats. AB - The effects of different levels of dietary boron were determined in vitamin D deficient rats. Vitamin D deficient diets containing either 0.158 ppm or 2.72 ppm of boron were fed to rats for 11 w, and calcium, magnesium, and phosphorus apparent absorption and balance were measured in the twelfth week. Higher apparent absorption and balance values for calcium and phosphorus were observed in the rats with higher dietary boron, but very few differences were seen in body wt, organ wt, and bone parameters. Balance measurements represented the present status of the rats after 12 w on the diets, but other measurements represented an accumulation over the lifetime of the rat, including a suckling period with ample vitamin D and boron. The data demonstrated that when rats are vitamin D deficient, as indicated by hypocalcemia, the level of boron in the diet affects mineral balance. PMID- 1713048 TI - Search for immunobiological parameters predictive of clinical effects of OK-432 in patients with malignant ascites. AB - Although OK-432, a potent BRM, has been known to induce the remarkable improvement of clinical conditions in cancer patients through its strong effects on their immune capabilities, no specific immune parameters have been identified to best predict the clinical outcome after the OK-432 treatment. In an attempt to identify early parameters indicative of the clinical effects, we have administered 0.1 mg of OK-432 intraperitoneally to a total of 12 patients with malignant ascites and examined peritoneal fluid and peripheral blood obtained on 4 days before, 1, 3, and 7 days after the OK-432 injection using various immunobiological assays. Four weeks later, clinical improvements were evaluated by the disappearance of malignant cells from and/or substantial decrease in ascites. Four patients (responders) showed the improvements while 8 patients (nonresponders) showed no clinical evidence for improvement. In a few parameters among the many examined, significantly different patterns of changes were noted between responders and nonresponders. Thus, in nonresponder patients MO and T cell population returned to an initial low level after early increases (on days 1 and/or 3), while they remained increased day 1 through 7 in responders. In responder patients, the cytotoxicity of peritoneal mononuclear cells against K562 and Daudi cells were augmented on day 7, but not in nonresponder patients. The in vitro stimulation of the mononuclear cells with OK-432 enhanced the cytotoxic activity and induced the interferon (IFN) production in the responders but not in nonresponders. These parameters will be useful for the early prediction of the expected clinical effects of OK-432. PMID- 1713049 TI - Fludarabine phosphate for the treatment of low grade lymphoid malignancy. AB - Thirty-four patients with previously treated, advanced, low grade NHL were treated with Fludarabine, a deamination-resistant analogue of adenosine arabinoside, at a dose of 25 mg m-2 intravenously, daily for 5 days (median number of cycles = 3, range 1-10). Complete remission (CR) was achieved in six and partial remission (PR) in a further seven. Overall, responses were seen in 11/23 patients (48%) with follicular lymphoma and in 2/11 (18%) with low grade, diffuse NHL. Fifteen patients with previously treated CLL and one patient with prolymphocytic leukaemia (PLL) were also treated as above (median no. of cycles = 3, range 1-6). A partial response was seen in three of the 11 evaluable patients with CLL and CR was achieved in the patient with PLL. There were four deaths due to infection and 19 further episodes requiring admission to hospital. No other significant toxicity was reported in a total of 164 cycles of Fludarabine. This agent is active in advanced low grade lymphoid malignancy. Further studies are required to assess its role in newly diagnosed patients. PMID- 1713050 TI - The antileukaemic activity of 5-Aza-2 deoxycytidine (Aza-dC) in patients with relapsed and resistant leukaemia. AB - In the present study we demonstrate that Aza-dC in combination with Amsacrine has major antileukaemic properties in patients who have not already received extensive Ara-C therapy. Eight out of 11 patients in their first relapse of acute leukaemia achieved complete remission. Cross resistance between Ara-C and Aza-dC was revealed by the lack of antileukaemic activity in five patients with with Ara C resistant leukaemia. Combination therapy with Aza-dC/Ams-acrine induced a considerable period of a granulocytopenia (28-35 days), while the toxic effect on erythro- and megakaryopoiesis was comparable to that reported for high dose Ara C/Amsacrine chemotherapy. Remarkable is the long disappearance time for leukaemic blast cells in bone marrow, i.e. 3-5 weeks in some cases. Analysis of cell membrane markers showed a loss of the early differentiation antigens CD34 and CD33 from leukaemic bone marrow cells after 7 days of Aza-dC treatment, which is suggestive of leukaemic cell differentiation. In the small group of patients tested for DNA hypomethylation no association existed between the degree of hypomethylation and clinical response. Non-haematologic side effects were considerable in patients receiving the highest dosages of Aza-dC and consisted of severe, although usually reversible, gastrointestinal and neurological complications. In comparison with Ara-C, Aza-dC causes less nausea and vomiting and is therefore better tolerated. PMID- 1713051 TI - The effect of a somatostatin analogue on the release of hormones from human midgut carcinoid tumour cells. AB - The use of a somatostatin analogue (SMS 201-995) has greatly facilitated the treatment of patients with the midgut carcinoid syndrome. Clinical studies have shown that SMS reduces the peripheral levels of tumour-produced serotonin (5-HT) and tachykinins, e.g. neuropeptide K (NPK), basally and after pentagastrin provocation. Some studies have indicated an inhibitory effect of SMS on tumour cell growth as well. In the present study we have investigated the effects of SMS on four different human midgut carcinoid tumours maintained in long term culture. Media levels of 5-HT and NPK-LI in tumour cell cultures decreased rapidly during incubation with SMS (10(-8)-10(-10) M) in all four tumours studied without evidence for tachyphylaxis (up to 6 weeks observation period). SMS treatment (10( 8) M) during 4 days reduced the media concentrations of 5-HT by 56%, while the intracellular contents of 5-HT were decreased by 27% indicating dual inhibitory effects on synthesis and secretion of 5-HT from tumour cells. The DNA contents of cultures were not affected by SMS (10(-8) M or 10(-10) M) treatment for 4 or 14 days. When tumour cell cultures were challenged with isoprenaline (IP) (10(-6) M) no reduction of the IP induced release of 5-HT could be detected after pretreatment of tumour cell cultures with SMS (10(-8) M) for 1 h, 4 h or 4 days. These studies provide evidence for a direct action of the somatostatin analogue on midgut carcinoid tumour cells, reducing both synthesis and secretion of hormones from tumour cells. This effect appears not to be related to inhibition of tumour cell growth. The inhibition of 5-HT secretion from tumour cells by SMS seems to operate via a second messenger system different from the one mediating the beta-adrenoceptor stimulated release of 5-HT. PMID- 1713052 TI - Multiple epitopes of the human ovarian cancer antigen 14C1 recognised by human IgG antibodies: their potential in immunotherapy. AB - We have defined a novel ovarian cancer-associated membrane antigen, 14C1, using human monoclonal antibodies derived by EBV-transformation of in situ sensitised patients' B-cells. The pattern of recognition of this antigen by these antibodies suggests that at least three epitopes are discernable. These antibodies can be used to promote the in vitro killing of ovarian cancer cells by activated macrophages and cytokines, implying a role for this antigen in the immunotherapy of ovarian malignancies. Evidence is presented that the 14C1 antigen may have some transmembrane signalling function. PMID- 1713053 TI - Synthetic androgens suppress the transformed phenotype in the human prostate carcinoma cell line LNCaP. AB - Experiments have been designed to investigate hormonal effects on the human prostatic carcinoma cell line LNCaP in the presence of complete foetal calf serum. At physiological concentrations (3.3 x 10(-9)M), several derivatives of 17 alpha-methyl-testosterone led to a significant reduction of cell proliferation, inhibition of colony formation in soft agar, change of morphology, induction of a prostate specific mRNA and down-regulation of c-myc RNA. Two different antiandrogens, hydroxyflutamide and cyproterone acetate, were capable of reversing the effects exerted by the synthetic androgens on growth properties. The proliferation rate of control cells devoid of androgen receptor was not inhibited by synthetic androgens. Our results indicate that the cellular androgen response mechanism of LNCaP cells is intact and that synthetic androgens elicit androgen receptor mediated suppression of the transformed phenotype. Rare cases of remission of prostatic cancer on androgen treatment have been reported. LNCaP cells may be a model of an uncommon class of prostatic cancer which responds favourably to androgen treatment. PMID- 1713054 TI - Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA. AB - The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae. PMID- 1713055 TI - Transfer of Brevibacterium divaricatum DSM 20297T, "Brevibacterium flavum" DSM 20411, "Brevibacterium lactofermentum" DSM 20412 and DSM 1412, and Corynebacterium glutamicum and their distinction by rRNA gene restriction patterns. AB - The results of DNA-DNA hybridization and chemotaxonomic studies indicated that the glutamic acid producers Brevibacterium divaricatum DSM 20297T (T=type strain), "Brevibacterium flavum" DSM 20411, "Brevibacterium lactofermentum" DSM 1412 and DSM 20412, Corynebacterium lilium DSM 20137T, and Corynebacterium glutamicum DSM 20300T and DSM 20163 are members of the same species. It is proposed that all of these strains should be classified in the species Corynebacterium glutamicum. Another glutamic acid-producing strain, Corynebacterium callunae DSM 20147T, was not related at the species level to C. glutamicum and should retain its separate species status. A restriction fragment length polymorphism analysis in which oligonucleotides targeted against conserved regions of 16S and 23S rRNA genes were used as hybridizing probes distinguished the individual strains. This method may be a helpful tool for strain identification. PMID- 1713056 TI - Preventive effects of OK432 on murine acute myocarditis due to encephalomyocarditis virus. AB - OK432, one of the immunomodulators used for cancer treatment in Japan was examined for its effects on murine myocarditis due to encephalomyocarditis (EMC) virus. The survival rate of mice administered with 1 KE of OK432 intraperitoneally every other day starting 2 days before viral inoculation was significantly higher than that of the control mice administered virus alone on days 10-21 (16/20 vs. 4/20, p less than 0.001), and the viral titer in the heart, the heart weight/body weight ratio, and the scores for myocardial inflammation and necrosis were significantly lower. The natural killer (NK) cell activity and interferon (IFN) titer on day 1 after infection were increased in comparison with the control group (NK cell activity: 51.1 vs. 37.9%, p less than 0.05. IFN: 1205 vs 512 U/ml), and the cytotoxicity of peritoneal macrophages was increased (50.6 vs. 29.1%). Thus, OK432 given before viral inoculation significantly improved survival and reduced both the viral titer in the heart and myocardial damage in this experimental model of acute EMC viral myocarditis. Accordingly, the stimulation of the host immunologic response by OK432 may be important for elimination of virus of the heart. PMID- 1713057 TI - Influence of culturing temperature and proteinase inhibitors on the spontaneously occurring changes in the organ culture of psoriatic skin. AB - Skin explants from involved psoriatic lesions showed dissociation of keratinocytes, dermal-epidermal separation and degenerative changes such as cytoplasmic swelling of subcorneal prickled cells within 24 h after culture initiation at 37 degrees C in the absence of fetal bovine serum (FBS). These histological changes developed time dependently, while normal skin explants did not exhibit such phenomena. Some of the uninvolved psoriatic skin explants showed only degenerative change 48 to 72 h after culture initiation at 37 degrees C. To determine the nature of these spontaneously occurring changes in psoriatic skin explants and then to approach the pathogenesis of psoriasis, the effects of FBS, various proteinase inhibitors (PIs) and culturing temperature (37, 31, 24 degrees C) were examined in skin organ culture of normal and involved psoriatic skin. At 37 degrees C, only serine PIs (5 or 10 mg/ml of soybean trypsin inhibitor (SBTI), 1000 KIU/ml of aprotinin, or 2 mg/ml of camostat mesilate in the medium) or FBS (20% in the medium) could suppress the occurrence of dissociation of keratinocytes and dermal-epidermal separation but not the degenerative change in involved psoriatic skin explants, while other types of PIs did not exhibit any such inhibition. When the culturing temperature was reduced from 37 degrees C to 31 or 24 degrees C, the formation of dissociation of keratinocytes and dermal epidermal separation was almost non-existent and only moderate degenerative change was observed. The addition of FBS or serine PIs to the culture at 31 degrees C revealed the formation of very weak degenerative change.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713058 TI - Occlusive hydrocolloid dressings decrease keratinocyte population growth fraction and clinical scale and skin thickness in active psoriatic plaques. AB - Clinical studies suggest a therapeutic role for occlusion in the treatment of psoriasis. Previous studies, using multiparameter RNA/DNA flow cytometric analysis of epidermal suspensions obtained from active plaques, demonstrated increased keratinocyte growth fraction which reversed with successful medical treatment. Because keratinocyte growth fraction reflected disease activity, it was used in this study in addition to clinical evaluations in order to determine the efficacy of occlusion in the treatment of psoriatic plaques. In each of 9 patients, scale, skin thickness and erythema were compared in one occluded and one control plaque using an analog scale. Both scale and skin thickness, but not erythema, were decreased after 2 weeks of occlusion. However after 10 weeks, no additional differences were seen when compared with assessments made after 2 weeks, suggesting that the benefits of occlusive therapy occurred early. After 10 weeks of occlusion, the keratinocyte growth fraction was significantly decreased in occluded plaques. This study demonstrates that occlusion plays a synergistic role with other therapeutic modalities in ameliorating psoriatic plaques. PMID- 1713059 TI - Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNA(Lys3)-primer excision. AB - Two model substrates were prepared to examine the mechanism of tRNA-primer excision catalyzed by reverse transcriptase associated ribonuclease H (RT-RNase H). The first model substrate contained sequences from the HIV genome and was designed to be structurally similar to the DNA-extended tRNA created by initiation of minus-strand DNA synthesis during retroviral replication. The DNA extended RNA was a template and was annealed to a DNA oligonucleotide that primed reverse transcription of the RNA in the template. The second model substrate was structurally similar the first substrate but contained sequences unrelated to the HIV viral genome. The RT-RNase H catalyzed excision of the RNA from the template of the two model substrates was examined. Human immunodeficiency virus (HIV) and Moloney murine leukemia virus RT-RNase H hydrolyzed the substrates to leave a single ribonucleotide 5'-phosphate at the 5'-terminus of the model DNA genome. In contrast, avian myeloblastosis virus RT-RNase H hydrolyzed the phosphodiester bond at the DNA-RNA junction. These hydrolytic specificities were not highly dependent on substrate sequence. The importance of these specificities to retroviral integration is discussed. Additional data indicated that the HIV polymerase and RNase H active sites are separated by a distance equivalent to the length of a 15-nucleotide RNA-DNA heteroduplex. PMID- 1713060 TI - Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade. AB - Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha T GTP gamma S was labeled with eosin-5 isothiocyanate (EITC-alpha T GTP gamma S) and served as the energy acceptor. The EITC-alpha T GTP gamma S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater than or equal to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha T GTP gamma S species was dose-dependent, saturable (Kd = 21 nM), and specific for the GTP gamma S-bound form of the alpha T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma subunit (gamma PDE), completely eliminates the quenching of the IAF fluorescence by the EITC-alpha T GTP gamma S complex. Although the EITC-alpha T GTP gamma S complex competes with the unlabeled alpha T GTP gamma S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha T GTP gamma S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme. PMID- 1713061 TI - Fluorescence studies on the interactions of myelin basic protein in electrolyte solutions. AB - This paper examines the influence of electrolytes on fluorescence spectral properties of the single tryptophanyl residue, Trp-115, within the 18.5-kDa species of myelin basic protein from bovine brain. Steady-state fluorescence spectra and intensities and time-correlated fluorescence lifetimes increased in the presence of increasing concentrations of mono- and divalent electrolytes (Li+, Na+, K+, Mg2+, Ca2+, Cl-, ClO4-, SO4(2-), and PO4(3-)). In all cases, the increases closely paralleled the ionic strength of the bulk aqueous medium and resembled that observed upon immersion of the protein in solutions of urea. This behavior was therefore concluded to reflect changes in the solution conformation of myelin basic protein. Bimolecular quenching of Trp-115 by acrylamide was rapid (10(9) M-1 s-1), approaching the diffusion limitation, and markedly dependent on the viscosity of the bulk aqueous medium. Rotational depolarization of myelin basic protein was rapid (phi less than or equal to 1 ns), occurring at rates exceeding those predicted for a rigid particle of revolution, and markedly dependent on the viscosity of the surrounding medium. Whereas the bimolecular quenching constants were unaltered in the presence of electrolytes, rotational depolarization of myelin basic protein underwent substantial slowing as indicated by the appearance of an additional decay component characterized by a correlation time of 5-10 ns. These studies indicate that Trp-115 of myelin basic protein is readily accessible to the bulk aqueous medium and is associated with a highly mobile segment of the protein. The slowing of rotational depolarization upon immersion of myelin basic protein in electrolyte solutions is consistent with an electrolyte-induced self-association of myelin basic protein molecules and indicates a relationship between the lability of solution conformation on the one hand and the capacity for self-association on the other. PMID- 1713062 TI - Effects of lindane on fluidity and lipid composition in rat renal cortex membranes. AB - The influence of lindane upon dynamic properties of plasma membranes from rat renal cortex has been investigated using a fluorescence polarization technique. Preincubation with lindane increased membrane fluidity in a manner that is dose dependent. This increase was higher in brush border membranes than in basolateral membranes. However, a significant decrease of the membrane fluidity was found in brush border membranes when rats were injected with lindane for 12 days. A possible solution to this difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the amount of cholesterol and phospholipid classes in the renal cortex membranes of lindane-injected rats. PMID- 1713063 TI - Planar bilayer membranes from photoactivable phospholipids. AB - Planar bilayer membranes formed from photoactivable phospholipids have been characterized by low frequency voltametry. Cyclic voltametric measurements were applied for simultaneous registration of planar membrane conductivity and capacitance. The procedure has been utilized to characterize the formation and stability of planar bilayer membranes. Bilayer membranes were formed from N'-(1,2 dimyristoyl-sn-glycero-3-phosphoethyl)-N-((m-3- trifluoromethyldiazirine)phenyl)thiourea (C14-PED), a head-group photosensitive phospholipid. In situ photoactivation of C14-PED at wavelengths greater than or equal to 320 nm altered neither the mean conductivity nor the capacitance of the bilayer. Ionophore (valinomycin) and ion channel (gramicidin) activities were not impaired upon photoactivation. In contrast, bilayer membranes formed from 1,2 bis(hexadeca-2,4-dienoyl)-sn- glycero-3-phosphocholine (C16-DENPC) revealed short life times. In situ photopolymerization of the diene fatty acids significantly increased the membrane conductivity or led to membrane rupture. PMID- 1713064 TI - [Nature of the fluorescence spectra of the DNA-specific dye "Hoechst 33258" in solvents and in DNA complexes]. AB - Fluorescence spectra of the DNA-specific dye Hoehst 33258 are obtained in a number of solvents. These spectra are interpreted as superpositions of monomeric and dimeric fluorescence bands of this compound. We show that abnormal Stokes' shift in the Hoehst fluorescence occurs only for the dimer form of this dye. It is suggested that formation of the A--T-specific complex is realized by dimers of the Hoehst 33258 located in the narrow groove of DNA. PMID- 1713065 TI - Prostate cancer-associated markers. AB - Immunodiagnosis of prostate cancer is at a more advanced stage than that of most other tumors. Two well-known markers, prostatic acid phosphatase and prostate specific antigen, have been used in the clinical management of patients. Prostate specific antigen is a more sensitive and reliable marker than prostatic acid phosphatase. Serum prostate-specific antigen is effective in monitoring disease status, predicting recurrence, and detecting residual disease. Prostate-specific antigen is a tool for the histological differential diagnosis of metastatic carcinomas, especially in the identification of metastatic prostate tumor cells in distant organs and in the differentiation of primary prostate carcinoma from poorly differentiated transitional cell carcinoma of the bladder. Few data on biological function are available. Prostatic acid phosphatase functions as a phosphotyrosyl-protein phosphatase and prostate-specific antigen as a protease. Physiological function in the prostate remains to be elucidated. Several of the prostate-specific and prostate-tumor-associated antigens, as well as a putative prostate tumor-specific antigen, as recognized by monoclonal antibodies are available. Clinical evaluation of these potential markers is not yet available. PMID- 1713066 TI - Testicular tumors. PMID- 1713067 TI - Markers for hepatocellular carcinoma. AB - Markers for hepatocellular cancer include the best and worst of cancer detection. Although hepatocellular cancer is relatively infrequent compared to other cancers in the western world, HCC has a very high incidence in parts of Asia and Africa. It is estimated to be one of the most common cancer worldwide. High risk factors for HCC include previous hepatitis B infection, heavy alcohol consumption, cirrhosis, and aflatoxin exposure. Alpha fetoprotein may be the best human cancer marker that appears in the serum, but levels of this marker are often not elevated until the tumor is beyond surgical treatment. No other serum or tissue marker is particularly useful. Screening of high-risk populations in China has detected previously undiagnosed HCC in 1,000 of 5 million individuals tested and has led to an increase in survival from 5.5 to 61.6% with surgical resection over those who are later diagnosed with HCC without screening. Elevations of AFP due to yolk sac tumors may be differentiated from those due to HCC on the basis of Concanavalin A reactivity. Immunodetection using radiolabeled anti-AFP and immunoscintigraphy have given inconsistent results that are not as sensitive as ultrasonography in detecting HCC in the liver. Various enzymes, isoenzymes, and other markers may be useful as adjuncts to diagnosis in selected cases, but are not generally as good as AFP alone. If a patient has an AFP-producing tumor, the serum levels of AFP provide an excellent means of monitoring its progression. If the serum AFP levels drop to normal and stay there, cure is almost certain. If, however, the serum AFP level does not fall at the normal catabolic rate after therapy, or subsequently rises, regrowth of metastases are indicated. Immunotherapy using anti-AFP has not been shown to induce remission, but experimental studies indicate that drug-conjugated anti-AFP is effective in inhibiting growth of AFP-producing tumors. Clinical trials using drug-conjugated anti-AFP are now underway. Monoclonal antibodies have not yet identified the "antigens" useful for the diagnosis or treatment of HCC, but epitopes identified by monoclonal antibodies have been studied experimentally in rats which indicate multiple cellular lineages to HCC in cases of experimental chemically induced hepatocarcinoma. PMID- 1713068 TI - Acute phase reactant proteins. PMID- 1713069 TI - Pancarcinoma T and Tn epitopes: autoimmunogens and diagnostic markers that reveal incipient carcinomas and help establish prognosis. PMID- 1713070 TI - Placental proteins as tumor markers. AB - Among the three placental proteins discussed, HCG is the only clinically useful tumor marker, and the value of HCG measurements is restricted to patients with gestational and nongestational trophoblastic disease. In patients with gestational trophoblastic disease, HCG levels may serve as an adjunct for the diagnosis, provide prognostic information, and be an objective parameter to evaluate the effects of therapy. Little or no additional information is obtained from HPL or SP-1 measurements. In patients with germ cell neoplasms of the testis, HCG measurements add useful information for clinical staging and monitoring of therapy, although discordance between tumor growth and HCG levels can be found in patients whose tumors contain several different elements. Therefore, AFP measurements must be made as well in these patients to monitor disease activity. Neither HPL nor SP-1 measurements are useful in these patients. None of the placental proteins are useful for screening, as prognostic indicators, or for evaluating the effects of therapy in groups of patients with nontrophoblastic neoplasms. In some patients with nontrophoblastic malignancies, each of the markers may accurately reflect changes in tumor burden during therapy. However, the problems with specificity and sensitivity of the tests and the fact that the majority of patients whose tumors produce the hormone have circulating concentrations that are at the limits of detection of the assays decrease the utility of these measurements and render them cost-ineffective for routine patient care. PMID- 1713071 TI - Development and evaluation of monoclonal antibody-based immunoassays: breast carcinoma-associated mucins as tumor markers. PMID- 1713072 TI - Strategies for heterogeneous enzyme immunoassays for tumor markers. PMID- 1713073 TI - Enhanced detection systems for enzyme-linked heterogeneous immunoassays: luminescence. AB - The use of luminescence detection as the endpoint for enzyme immunoassays offers the sensitivity and performance that had previously been obtained only with radioactive immunoassays. For systems that are optimized to give a continuous output of light, simple and convenient processing of tests can be achieved without the need for accurate timing of the signal generation. The applicability of these techniques for the analysis of a wide range of substances should lead to their establishment in cancer diagnosis as well as in other fields. PMID- 1713074 TI - The influence of properties of packing materials upon the recovery of biological substances isolated from urine by solid-phase extraction. AB - A series of packing materials with alkyl phase chemically bonded to silica gels of various porosity have been prepared. These packings have been used to isolate the test substances 5-hydroxyindole-3-acetic acid (5-HIAA) and serotonin (5-HT) from urine. The influence of the support porosity, structure of chemically bonded phase, length of alkyl chain, and coverage density on the recovery of isolated substances was studied. PMID- 1713075 TI - A method for assaying biofilm capacity on polyurethane-coated slides. AB - Isolates of coagulase-negative Staphylococci (CNS) were examined for their ability to form biofilms on polyurethane-coated slides. These slides provided a smooth plastic coating simulating polymeric plastic surfaces of medical grade catheter tubing. Slides were placed into plastic conical tubes containing tryptic soy broth inoculated with 10(6) bacteria per mL. The tubes were then incubated at 37 degrees for 48 hours. After incubation, 1 of the slides was stained with a fluorescent acridine orange stain and the other with a safranin stain. The incubation tubes were also stained with safranin. Forty-eight percent of the 65 CNS isolates were found to form a biofilm using acridine orange staining. Forty percent of the 65 CNS isolates were found to form a biofilm using the safranin stain on slides, whereas only 34% were found to adhere on sides of plastic tubes. Increased sensitivity of the fluorescent stain was probably due to enhanced visualization of smaller numbers of bacteria on the plastic. This method using fluorescent stained plastic-coated slides was easier to visualize and interpret than the tube method. PMID- 1713077 TI - [The structural characteristics of the phrenic nucleus in the cat and their functional significance]. AB - The structural elements of phrenic nucleus have been compared under the horseradish peroxidase introduction into the phrenic muscle and using a silver staining method. All the neurons of nucleus have been proposed to be motoneurons. The characteristic features of phrenic nucleus organisation are created by the groups of motoneurons and their dendritic bundles. Each nucleus contains about 800 motoneurons. Their sizes have been determined in three planes. Some functional characteristics of phrenic nucleus on the basis of its structural architecture are under discussion. PMID- 1713076 TI - [The role of plasmids in the cell cycle of Escherichia coli]. AB - The effect of R- and Hly-plasmid differing in phenotype, molecular size, conjugativity, stability and incompatibility properties on the cell cycle and nucleic acids content per cell has been studied in Escherichia coli. According to these properties the plasmids were divided into 3 categories. The possibilities of the autonomous plasmid replicon interactions with the host chromosome are discussed. PMID- 1713078 TI - [A quantitative evaluation of the reactive and dystrophic changes in the cerebellar cortex of mice during malnutrition and subsequent food rehabilitation]. AB - Neurones with chromatolysis, hyperchromatism and numerous nucleus have been counted of the paraffin cerebellar cortex sections coloured with kresil-violet and metil-green pironin. The alimentary deficiency has been shown to result in increase of cell number with chromatolysis and hyperchromatism and decrease of the number of numerous nucleus. The food rehabilitation doesn't take off completely damages caused by malnutrition. The use of carnitine leads to the complete rehabilitation that is due to the normalization of protein metabolism in cerebellum. PMID- 1713079 TI - Morphological study of the capsular organization around tissue expanders coated with N-carboxybutyl chitosan. AB - Expanders coated with N-carboxybutyl chitosan were inserted into surgical wounds in the dorsal skin of rabbits and the formation of capsular tissue was studied by scanning electron microscopy and transmission electron microscopy. N-carboxybutyl chitosan, in the course of the capsular organization, favours and potentiates the correct proliferation and organization of the tissue, rather than sustaining reactive processes leading to scar formation. N-carboxybutyl chitosan stimulates physiologically the tissue repair process and favours angiogenesis, whilst depressing fibrogenesis to a certain extent. Applications are envisaged in the treatment of wounds and in plastic surgery. PMID- 1713080 TI - [An electron microscopic radioautographic study of the viability of human cells after death. The postmortem activation of RNA synthesis in neutrophils]. AB - RNA synthesis in human neutrophils isolated from blood 2.5-67 hours after death of patients was studied by means of light and electron microscopy radioautography. The rate of RNA synthesis in neutrophils isolated from blood of cadavers was higher in comparison with the same parameter in neutrophils isolated from survivor's blood. The significance of this observation for resuscitation and transplantology is discussed. PMID- 1713081 TI - [Neurotrophic control of myosin synthesis in guinea pig slow muscle]. AB - After experimental cease of neurotrophic control of skeletal muscle by denervation no changes in myosin ATP-ase histochemistry and immunohistochemical profile in slow (m. soleus) muscle of guinea pig were found. All muscle fibers in intact muscle fibers). However after colchicine blockade of axoplasmic transport in this slow muscle some muscle fibers reacting with monoclonal antibodies against fast myosin heavy chain were found. At the same time no changes in histochemical ATP-ase profile were observed. Validity of myosin ATP-ase histochemistry for muscle fibers typing as well as possible influence of nerve activity and neurotrophic control itself were discussed. PMID- 1713082 TI - [The control of humoral transport of the eye tissues]. AB - Three series of investigations were carried out in experiments on rabbits with administration under the conjunctiva or by means of electrophoresis of lymphotrophic preparations of different mechanisms of actions with the use of a morphological marker: Gerot's mass and Indian ink jelly with subsequent histological study of the eyeball. Dalargin dilated structured liquorolymphatic drainage ducts of the eye. Terrylythin produced a selective effect on the pigment epithelium of the retina, and mannitol provided penetration of the marker into the retina neurons. Thus, it has been shown that it is possible to control selectively the humoral transport of some tissues of the eye by means of lymphotrophic agents. PMID- 1713083 TI - [The organizational characteristics of the APUD system in mammalian lungs at different stages of ontogeny]. AB - The organization peculiarities of APUD-system in the lungs of rabbits, rats and guinea pigs has been studied. The endocrine system in the lungs of rabbits in pre and postnatal ontogenesis is presented by the adipocytes and neuroepithelial bodies (NEB) containing a considerable number of monoamines. The number of argyrophil adipocytes and NEBs in the lungs of 21 and more day-old adult rats seem to be less than in fetuses and newborns. Monoamines are not revealed in the endocrine rat lung structures by means of the glyoxylic acid. In the lungs of guinea pigs the single argyrophil adipocytes and NEBs are determined in the gestation period. PMID- 1713084 TI - Identification of insulin-like growth factor binding proteins in breast cancer cells. AB - The insulin-like growth factors (IGFs) are potent mitogens for some breast cancer cell lines. Recent evidence suggests that IGF-induced mitogenesis may be influenced by specific IGF binding proteins (IGFBPs). In this study, breast cancer cell lines were examined for IGFBP protein and mRNA expression. Western ligand blot examination of conditioned media from breast cancer cell lines suggested that the IGFBP protein expression was heterogeneous. Although all breast cancer cell lines expressed a 24 kDa binding protein, MCF-7, an estrogen receptor positive (ER+) cell line, expressed a IGFBP compatible with reported sizes for IGFBP-2. Estrogen receptor negative (ER-) cells (MDA-MD-231, Hs578T) secreted IGFBPs consistent with sizes reported for IGFBP-1 and -3. Examination of mRNA expression supported these findings; IGFBP-2 was seen in all (4/4) ER+ cell lines while high levels of IGFBP-3 were found in ER- cell lines (3/5), although lower levels of IGFBP-3 mRNA could be found in some ER+ cell lines. In MCF-7 cells, steady state levels of IGFBP-3 mRNA were decreased by estradiol, while IGFBP-2 mRNA levels were slightly increased. These data suggest that IGFBP expression by breast cancer cells is heterogeneous, that the pattern of IGFBP expression is different between ER+ and ER- cell lines, and that in ER+ cells IGFBP mRNA may be regulated by estrogens. Thus, the IGFBPs may play an important role in mediating the mitogenic response of breast cancer cells to the IGFs. PMID- 1713085 TI - Interferon-alpha and gamma mediated gene responses in a human breast carcinoma cell line. AB - Interferons (IFNs) have been known to possess an antiproliferative effect on tumor cells besides their well characterized antiviral effect in cell cultures. The mechanism of action of the different IFNs is not fully understood, but in recent years a number of IFN-inducible genes, the presumed mediators of IFN action, have been identified. In the present study we examined the antiproliferative effect of IFN-alpha and IFN-gamma on human breast cancer cells (MCF-7) using (i) the MTT dye formation assay and (ii) anchorage-independent (AI) growth in soft agar. Both IFN-alpha and IFN-gamma were found to have an antiproliferative effect on the growth of MCF-7 cells. In addition, the kinetics of induction of a number of IFN-inducible genes was also examined. The expression of these genes was measured by mRNA analyses using specific [alpha-32P]-labeled cDNAs as probes. The induction of these genes by IFN-alpha and IFN-gamma is a primary effect of IFN, as de novo protein synthesis is not required for their induction. Our results on the kinetics of induction of these genes by IFN-alpha and IFN-gamma suggests a complex mechanism of ligand-dependent gene activation in this cell line with some similar and dissimilar pathways. PMID- 1713086 TI - Palliative laser therapy for tumours of the gastrointestinal tract. AB - The argon ion and Nd: YAG lasers were used initially in the mid 1970s to produce haemostasis in acutely bleeding peptic ulcers. With the evolution of treatment techniques, the main area of use of the Nd: YAG laser has now become the palliation of upper and lower GI malignancies. Thermal ablation of tumours may be achieved endoscopically by non-contact laser application at high power, or in the contact mode using artificial sapphire probes at much lower energy levels. Still lower powers can be employed therapeutically using interstitial hyperthermia, and this is best applied endoscopically to exophytic tumour nodules in the gut lumen or to tumours localized ultrasonically in solid organs, such as the liver or pancreas. PDT involves destruction of previously photosensitized tumours by the cytotoxic action of singlet oxygen released on exposure of the neoplastic tissue to light of an appropriate wavelength. Although the theory is attractive, the available experimental and clinical information suggests that treatment should, for the present, be confined to small or early malignancies whose depth of invasion can be verified by endoscopic ultrasound or other imaging techniques. PDT carries the biological advantage of healing by regeneration with preservation of connective tissue stroma, while the Nd: YAG laser causes destruction by thermal coagulation or vaporization and subsequent healing by fibrosis. Laser therapy of GI tumours expands the range of therapeutic endoscopic procedures in a relatively safe and readily repeatable manner which achieves high patient tolerance. By reducing morbidity, mortality and time spent in hospital, it offers significant advantages in the palliative treatment of conditions previously managed by conventional surgery, and also offers opportunities for treatment of previously inoperable disorders. Developments in laser technology and diagnostic imaging techniques are likely to promote laser therapy in the future as a primary treatment modality. PMID- 1713087 TI - Impact of variability among surgeons on postoperative morbidity and mortality and ultimate survival. AB - OBJECTIVE: To assess the differences among surgeons in postoperative complications, postoperative mortality, and survival in patients undergoing surgery for colorectal cancer. DESIGN: Prospective study of patients with colorectal cancer managed by one of 13 consultant surgeons, none of whom had a special interest in colorectal surgery. SETTING: Royal Infirmary, Glasgow. PATIENTS: 645 sequential patients with colorectal cancer presenting over the six years from 1974 to 1979. MAIN OUTCOME MEASURES: Postoperative complications, postoperative mortality (within 30 days), and survival (up to 10 years); predictive factors for postoperative mortality and survival; and relative hazard rate ratios for individual surgeons. RESULTS: The proportion of patients undergoing apparently curative resection varied among surgeons from 40% to 76%; overall postoperative mortality varied from 8% to 30%. After curative resection postoperative mortality varied from 0% to 20%, local recurrence from 0% to 21%, and the rate of anastomotic leak from 0% to 25%. Survival at 10 years in patients who underwent curative resection varied from 20% to 63%, two year survival in those who underwent palliative resection varied from 7% to 32%, and median survival in those who underwent palliative diversion varied from one to eight months. The hazard rate ratios among individual surgeons, taking into account the identified risk factors, varied from 0.56 to 2.03, from 0.17 to 1.92, and from 0.57 to 1.50 for curative resection, palliative resection, and palliative diversion, respectively. CONCLUSION: There were significant variations in patient outcome among surgeons after surgery for colorectal cancer; such differences compromise survival. A considerable improvement in overall survival might be achieved if such surgery were undertaken by surgeons with a special interest in colorectal surgery or surgical oncology. PMID- 1713088 TI - Megatherapy for soft tissue sarcomas. EBMT experience. PMID- 1713089 TI - Weaning in European and Latin American countries. PMID- 1713090 TI - Infantile nutrition--an update. The 4th International Symposium. Naples, May 10 12, 1990. PMID- 1713091 TI - Zinc therapy in children with cystic fibrosis. AB - The effect of oral zinc supplementation in patients with cystic fibrosis (CF) was investigated in a placebo-controlled, double-blind, crossover study with each treatment period covering 6 months. CF patients (n = 13, aged 2 years, 3 months to 19 years, 1 month) started with placebo and after 6 months, they received zinc therapy. Another 13 patients (aged 3 years, 5 months to 16 years, 10 months) started in the reverse order. Before zinc supplementation, all CF patients had low plasma levels of zinc which normalized during treatment. This effect was, however, transient. CF patients also had low concentrations of plasma selenium. A small decrease in the number of leukocytes was also noted during zinc therapy. In response to zinc treatment, no changes in the clinical status of the patients were observed either by the investigators or by the patients. Growth velocity was the same during the placebo and zinc treatment periods. No significant changes in lung function occurred in response to either placebo or zinc. It appears that the observed low plasma zinc concentration in CF patients was due to an impaired zinc absorption from the gut which was counteracted by an increased supply of oral zinc. No beneficial effect from zinc supplementation on clinical status, growth velocity, or lung function was found in this study. PMID- 1713092 TI - Zinc status in some chronic childhood diseases. PMID- 1713093 TI - Advances in prevention and treatment of metabolic diseases. PMID- 1713094 TI - Food allergy: diagnosis and strategies for prevention. PMID- 1713095 TI - Weaning as an approach to the 'non-self'. PMID- 1713096 TI - The child of today and the Mediterranean diet. PMID- 1713097 TI - Dietary treatment of liver glycogenosis. PMID- 1713098 TI - Development of the human gastrointestinal tract: implications for weaning. PMID- 1713099 TI - Role of proteins and fats in weaning. PMID- 1713100 TI - The diagnostic value of measurements for trace elements in biological material. PMID- 1713101 TI - Dietary trace elements in early infancy. PMID- 1713102 TI - Rubidium: a companion of potassium or an essential trace element of its own? PMID- 1713103 TI - Comparison of the effects of intra-arterial and aerosol administration of endothelin-1 (ET-1) in the guinea-pig isolated lung. AB - 1. Intra-arterial injection of endothelin-1 (ET-1, 400 pmol; 1 microgram) in guinea-pig isolated perfused lungs, induced increases in pulmonary inflation pressure (PIP) and perfusion pressure (PPP), associated with oedema formation and thromboxane B2 (TxB2) release but not with the generation of sulphidopeptide leukotrienes or release of histamine. In contrast, aerosol administration of ET-1 (3, 6, 10 micrograms ml-1, for 2 min) evoked a dose-dependent increase in PIP, without significant changes in PPP, oedema formation or TxB2 release. 2. Addition of indomethacin (5 microM) or BW 755C (10 or 100 microM), but not nordihydroguaiaretic acid (NDGA, 50 microM) or FPL 55712 (10 microM), to the perfusion medium led to a significant inhibition of the increases in PIP and PPP, TxB2 release and oedema formation evoked by intra-arterial injection of 400 pmol ET-1. In contrast, indomethacin (5 microM), BW 755C (100 microM) or FPL 55712 (10 microM), added to the perfusion medium 10 min prior to challenge, did not affect the increase in PIP induced by a 2-min aerosol of a solution of ET-1 10 micrograms ml-1. 3. In vivo aerosol administration of indomethacin (100 mg ml-1, for 20 min) to non-anaesthetized guinea-pigs, 15 min before lung removal, did not modify the bronchopulmonary response evoked in isolated perfused lungs by an aerosol of ET-1 10 micrograms ml-1. However, under the same experimental conditions, indomethacin significantly inhibited TxB2 release evoked by aerosolized arachidonic acid (2 mg ml-1). 4. In conclusion, the present study shows that when injected by the intra-arterial route, ET-1 effects are mediated primarily via the generation of cyclo-oxygenase metabolites of arachidonic acid, whereas when the aerosol route is used, the peptide appears to act on airway smooth muscle cells, through an indomethacin-insensitive process which may involve some other, as yet unidentified, mediator(s). PMID- 1713104 TI - Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro. AB - 1. Opioids have been shown to inhibit substance P (SP) release from primary afferent neurones (PAN). In addition, opioid receptors have been identified on PAN of the vagus nerves. Sodium cromoglycate (SCG) decreases the excitability of C-fibres in the lung of the dog in vivo. We have utilised a multi-superfusion system to investigate the effect of opioids and SCG on the release of SP from the rat trachea in vitro. 2. Pretreatment of newborn rats with capsaicin (50 mg kg-1 s.c. at day 1 and 2 of life) resulted in a 93.2 +/- 6.3% reduction in tracheal substance P-like immunoreactivity (SP-LI) content when determined by radioimmunoassay in the adult. 3. Exposure to isotonically elevated potassium concentrations (37-90 mM), capsaicin (100 nM-10 microM), and bradykinin (BK; 10nm 1 microM) but not des-Arg9-BK (1 microM) stimulated SP-LI release by a calcium dependent mechanism. 4. SCG (1 microM and 100 microM) did not affect spontaneous, potassium (60 mM)- or BK (1 microM)-stimulated SP-LI release. 5. Morphine (0.1 100 microM) caused dose-related inhibition of potassium (60 mM)-stimulated SP-LI release with the greatest inhibition of 60.4 +/- 13.7% at 100 microM. The effect of morphine was not mimicked by the kappa-opioid receptor agonist, U50,488H (10 microM) or the delta-opioid receptor agonist, Tyr-(D-Pen)-Gly-Phe-(D-Pen) (DPDPE). 6. The effect of morphine was totally abolished by prior and concomitant exposure to naloxone (100 nM) which had no effect on control release values. 7. We conclude that opioid receptors, predominantly of the MM-opioid receptor subtype, inhibit SP-LI release from PAN in the rat trachea and suggest that centrally inactive MM-opioid receptor agonists may have therapeutic potential in the treatment of asthma. PMID- 1713105 TI - GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro. AB - 1. The role of gamma-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system is well documented. Recently, GABAA and GABAB receptors have been identified in the peripheral nervous system, notably on primary afferent neurones (PAN). We have utilised a multi-superfusion system to investigate the effect of selective GABA receptor agonists and antagonists on the release of substance P (SP) from the rat trachea in vitro. 2. GABA (1-100 microM) did not affect spontaneous release of SP-like immunoreactivity (LI) but caused dose-related inhibition of calcium-dependent potassium (60 mM)-stimulated SP-LI release. The greatest inhibition of 77.7 +/- 18.8% was observed at 100 microM. 3. The inhibitory effect of GABA was mimicked by the GABAB receptor agonist, (+/-) baclofen (1-100 microM), but not the GABAA receptor agonist, 3-amino-1-propane sulphonic acid (3-APS, 1-100 microM). Baclofen (100 microM) had no effect on SP LI release stimulated by capsaicin (1 microM). 4. The inhibitory effect of baclofen (30 microM) was significantly reduced by prior and concomitant exposure to the GABAB receptor antagonist, phacolofen (100 microM) but not the GABAA receptor antagonist, bicuculline (10 microM). Neither antagonist, alone, affected spontaneous or potassium-stimulated SP-LI release. 5. We conclude that activation of pre-synaptic GABAB receptors on the peripheral termini of PANs in the rat trachea inhibits SP-LI release and suggest that GABAB receptor agonists may be of value in the therapeutic treatment of asthma. PMID- 1713106 TI - Comparison of endothelium-dependent responses of monkey cerebral and temporal arteries. AB - 1. Endothelium-dependency of vasodilator responses was compared in helical strips of monkey cerebral and superficial temporal arteries contracted with prostaglandin F2 alpha. Acetylcholine produced an endothelium-dependent relaxation in the temporal arteries, but did not consistently alter the tone of cerebral arteries. 2. Adenosine 5'-triphosphate (ATP) produced a transient contraction followed by a relaxation in the temporal and cerebral arteries; removal of the endothelium partially attenuated the relaxation of the cerebral arteries and markedly suppressed the relaxation in the temporal arteries. The dependency of adenosine 5'-diphosphate (ADP)-induced relaxations on the endothelium was also greater in temporal arteries than in cerebral arteries. 3. Histamine-induced relaxations in the temporal arteries were independent of the endothelium and were reversed to contractions by cimetidine. Cerebral arterial relaxations induced by histamine were partly dependent on the endothelium. Relaxations caused by substance P were reversed to contractions by removal of the endothelium in the temporal arteries, whereas the peptide did not consistently alter the tone of cerebral arteries. 4. The Ca2+ ionophore, A23187, relaxed the temporal and cerebral arteries to a similar extent; removal of the endothelium abolished these relaxations. Glyceryl trinitrate elicited similar relaxation of cerebral and temporal arteries, and these were independent of the endothelium. 5. These findings clearly indicate heterogeneity in the endothelium-dependency of several vasodilator responses in monkey intra- and extracranial arteries, although the ability of these arteries to respond to A23187 and glyceryl trinitrate does not appear to differ. The heterogeneous responses observed so far could therefore be due to different distributions of receptors or to variation in receptor-effector coupling in endothelial cells. PMID- 1713107 TI - Enhanced contractility of the rat stomach during suppression of angiotensin converting enzyme by captopril in vitro. AB - 1. Intragastric pressure (IGP) was used as an index, of the effect of serosal application of captopril (SQ 14,225; D-3-mercapto-2-methylpropanoyl-L-proline) on the contractility of rat stomach in vitro. 2. Captopril, at concentrations greater than 0.3 microM, enhanced the spontaneous gastric motility (GM) in a concentration-dependent manner whereas concentrations less than 0.3 microM selectively potentiated 4 nM bradykinin (BK)-evoked gastric contractions without significantly affecting the spontaneous GM. 3. The kallikrein inhibitor, aprotinin (100 u ml-1), markedly antagonized the enhanced GM to 1.4 microM captopril and BK (4 nM)-evoked contractions, without affecting the contractions evoked by angiotensin 1 (10 nM) and acetylcholine (0.4 microM). The angiotensin II antagonist, saralasin (50 microM) failed to mimic aprotinin. 4. The enhanced GM to captopril was markedly inhibited by tetrodotoxin (1 microM), and partially inhibited by atropine (1 microM). 5. These results indicate that in vitro, captopril (greater than 0.3 microM) enhances gastric contractility through kininase/ACE inhibitory action, presumably by increasing the concentration of undegraded tissue kinins and substance P. This motor response seems to be predominantly due to activation of the cholinergic neurones but non-cholinergic excitatory neurones are also involved. PMID- 1713108 TI - Effect of p-chlorophenylalanine on release of 5-hydroxytryptamine from the rat frontal cortex in vivo. AB - 1. Rats were given p-chlorophenylalanine (PCPA, 150 mg kg-1, i.p.) to inhibit partially 5-hydroxytryptamine (5-HT) synthesis so that its concentration in the frontal cortex fell by about half. The effects of this treatment on frontal cortex dialysate 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined before and after stimulation by increasing K+ concentration in the perfusion fluid by 100 mM for 20 min. Rates of 5-HT synthesis as indicated by the effects of 3-hydroxybenzylhydrazine (NSD 1015, 150 mg kg-1, i.p.) on frontal cortex tissue and dialysate 5-hydroxytryptophan (5-HTP) and dialysate 5-HIAA were also measured in rats that had not been stimulated with K+. 2. Dialysate 5-HT and 5-HIAA concentrations of both vehicle- and PCPA-treated rats fell into major (group 1) and minor (group 2) populations statistically distinguishable from each other by the high 5-HT and low 5-HIAA values of the latter group. 3. In group 1 animals, PCPA decreased both the dialysate 5-HT concentration and its rise following stimulation by K+ in proportion with the decrease of 5-HT in frontal cortex tissue. 5-HIAA fell more markedly than 5-HT and in similar proportion in both tissue and dialysate. The fall of dialysate 5-HIAA on stimulation by K+ was also attenuated to the same degree. The elevated 5-HT/5-HIAA ratios after PCPA treatment imply increased conservation of the depleted 5-HT stores. 4. PCPA decreased the above 5-HIAA values and the effects of NSD 1015 on tissue 5-HTP or dialysate 5-HIAA concentrations in similar proportion. However, PCPA had little effect on corresponding dialysate 5-HTP values. 5. The results are discussed with respect to relationships between synthesis, storage and release of 5-HT. They indicate that (under the conditions of the present study) the availability of 5 HT to receptors is directly proportional to total vesicular stores under both basal conditions and during neuronal firing. PMID- 1713109 TI - Evidence for facilitatory and inhibitory muscarinic receptors on postganglionic sympathetic nerves in mouse isolated atria. AB - 1. McNeil A 343 (10 microM-30 microM) enhanced the fractional stimulation-induced (S-I) outflow of radioactivity from mouse isolated atria which had been incubated with [3H]-noradrenaline. The enhancing effect of McNeil A 343 was not altered by hexamethonium (300 microM) suggesting that it was not due to an action at nicotinic receptors. It is also unlikely that McNeil A 343 enhanced the S-I outflow of radioactivity in mouse atria by blocking neuronal reuptake of noradrenaline since the effect persisted in the presence of cocaine (30 microM). 2. The facilitatory effect of McNeil A 343 on the S-I outflow of radioactivity was attenuated by atropine (0.3 microM), pirenzepine (0.2 microM or 1.0 microM), dicyclomine (1.0 microM) and methoctramine (1.0 microM) and was thus due to activation of muscarinic receptors. 3. In contrast to the effect of McNeil A 343, another muscarinic receptor agonist, carbachol (3.0 microM) significantly decreased the S-I outflow of radioactivity. The receptors through which McNeil A 343 acts to enhance the S-I outflow of radioactivity appear to be distinct from inhibitory prejunctional muscarinic receptors. The relatively M 1-selective antagonist, pirenzepine (0.2 microM), attenuated the facilitatory effect of McNeil A 343 whereas a higher concentration (1.0 microM) was required to block the inhibitory effect of carbachol. Conversely, the relatively M2-selective antagonist, methoctramine (0.1 microM), blocked the inhibitory effect of carbachol but a higher concentration of methoctramine (1.0 microM) was required to block the facilitatory effects of McNeil A 343. These results tentatively ascribe facilitatory muscarinic receptors as belonging to the Ml subtype and inhibitory muscarinic receptors as belonging to the M2 subtype. 4. The non selective muscarinic receptor antagonist, atropine, enhanced the S-I outflow of radioactivity, suggesting that there was tonic activation of inhibitory prejunctional muscarinic receptors by endogenous acetylcholine released from parasympathetic nerves. However, pirenzepine (0.03 pM-LO microM) did not decrease the S-I outflow of radioactivity, suggesting that under the conditions of the present study, facilitatory muscarinic receptors are not tonically activated by endogenous acetylcholine. PMID- 1713110 TI - A patch-clamp study of K(+)-channel activity in bovine isolated tracheal smooth muscle cells. AB - 1. Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K(+)-channels in these cells were studied by the patch-clamp recording technique. 2. Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 microM. Large unitary currents of both Ca(2+)-dependent and independent types were observed. Measured between + 20 and + 40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3. Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to much greater than + 40 mV, suggesting that both currents were carried by K(+)-channels. 4. The Ca(2+)-dependent and independent K(+)-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100 microM) applied to the cytosolic surface of the patches. 5. Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 microM. Ca(2+)-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS. 6. Activity of the Ca2+-dependent K+-channel detected in outside-out patches could be inhibited by application of TEA (1 mM), Cs+ (10mM), Ba2(+210mM) or quinidine (100 microM) to the external surface of the patch. 4-Aminopyridine (4-AP; 1 mM) was ineffective as an inhibitor. 7. The activity of the Ca2+-dependent K+-channel recorded from outside-out patches was reversibly inhibited by charybdotoxin (100 nM). 8. When whole-cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2 +] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nM-I microM) applied to the external surface of the cell.9. We conclude that bovine trachealis cells are richly endowed with charybdotoxin sensitive, large conductance, Ca2 +-dependent K+-channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2 + -independent K+ -channels remains unclear. PMID- 1713111 TI - Morphophysiology of synaptic transmission between type I hair cells and vestibular primary afferents. An intracellular study employing horseradish peroxidase in the lizard, Calotes versicolor. AB - Intracellular records with glass microelectrodes filled with horseradish peroxidase (HRP) were taken from primary afferents of the horizontal semicircular canal in the lizard, Calotes versicolor. A coefficient of variation (CV) of the interspike intervals of spontaneous action potentials (APs) was calculated and correlated with the terminal morphologies of afferents within the canal crista. Irregular fibers with CV greater than 0.4 always correlated with a nerve chalice or calyx afferent terminal expansion surrounding one or more type I hair cells; more regular fibers with CV less than 0.4 always correlated with a dimorphic or bouton only terminal expansion of afferents. Afferents with a CV greater than 0.4 demonstrated miniature excitatory postsynaptic potentials (mEPSPs) that summated to initiate APs. APs were blocked by tetrodotoxin and mEPSP frequency was modulated by caloric stimulation. Cobalt application reversibly blocked mEPSPs. Electron microscopic examination of physiologically studied afferents with CV greater than 0.4 revealed synaptic profiles consisting of typical synaptic bodies and synaptic vesicles in the type I hair cell presynaptic to the nerve chalice. Examples of the interspike baseline in regular and irregular afferents suggest differential modes of impulse initiation in these two fiber types. PMID- 1713112 TI - Acute and chronic effects of intrathecal galanin on behavioural and biochemical markers of spinal motor function in adult rats. AB - The spinal motor effects of galanin, which co-exists with 5-hydroxytryptamine (5 HT) and thyrotrophin-releasing hormone (TRH) in bulbospinal raphe neurones innervating spinal motoneurones, were examined by administering this neuropeptide through indwelling intrathecal cannulae to conscious adult Wistar rats. The acute effect of intrathecal galanin on spontaneous motor behaviour and the motor behaviours (back muscle contractions and wet-dog shakes) elicited by intrathecal injection of the non-selective 5-HT receptor agonist, 5-methoxy-N, N' dimethyltryptamine (5-MeODMT) or the TRH analogue, RX 77368 analogue, RX 77368 (pGlu-His-3,3'-dimethyl-ProNH2), respectively, and the chronic effect of galanin on neurochemical markers for bulbospinal raphe neurones and spinal motoneurones were determined. Intrathecal galanin (0.1 to 10 micrograms) did not produce any notable motor behaviours when given alone, but pretreatment with the neuropeptide (0.1 micrograms) significantly attenuated both the number of wet-dog shakes and the amount of forepaw-licking induced by RX 77368, without affecting 5-MeODMT induced back muscle contractions. Repeated intrathecal galanin administration (1 microgram, twice daily for 5 d) significantly elevated 5-HT (but not 5 hydroxyindoleacetic acid) and substance P-like immunoreactive (LI) levels and choline acetyltransferase (ChAT) activity in the dorsal, but not in the ventral, portion of the thoraco-lumbar spinal cord. In contrast, chronic intrathecal galanin did not alter the TRH- or calcitonin gene-related peptide (CGRP)-LI levels in either spinal cord region.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713113 TI - Localization of cardiac parasympathetic preganglionic neurons in the medulla oblongata of pigeon, Columba livia: a study using fragment C of tetanus toxin. AB - The binding fragment of tetanus toxin, fragment C, was injected into several different regions of the pigeon heart. Retrogradely and/or transneuronally labeled cardiomotor parasympathetic preganglionic neurons were found in two separate nuclei within the medulla oblongata. The majority of fragment C immunolabeled cells was confined to the caudal division of the nucleus ambiguus. This nuclear region is likely to be homologous to the ventrolateral nucleus of the external formation of the nucleus ambiguus in mammals. A smaller fraction (10 30%) of fragment C-positive cardiomotor preganglionic neurons were localized within a restricted portion of the ventrolateral subnucleus of the dorsal motor nucleus of the vagus nerve. This dual cardiac representation in an avian is very similar to the organization established in several mammalian species, and suggests that the brainstem organisation of cardiac parasympathetic efferents is evolutionarily stable across avians and mammals. PMID- 1713114 TI - Differential distribution of a neurofilament protein epitope in acetylcholinesterase-rich neurons of human cerebral neocortex. AB - The majority of acetylcholinesterase-rich pyramidal neurons in neocortical layers III and V of the human brain displayed intense immunostaining with SMI-32, a monoclonal antibody which recognizes a non-phosphorylated epitope of neurofilament proteins. In contrast, very few of the heteromorphic acetylcholinesterase-rich perikarya embedded in the white matter of the cerebral hemispheres are associated with this type of immunostaining. These two groups of acetylcholinesterase-rich cortical neurons can thus be differentiated not only on the basis of morphology and location but also on the basis of cytochemical signature. The concurrent visualization of SMI-32 immunoreactivity and acetylcholinesterase enzyme activity also showed that SMI-32 immunoreactive neurons can be subdivided into several subgroups on the basis of their perikaryal acetylcholinesterase activity. PMID- 1713115 TI - Parachloroamphetamine selectively alters regional cerebral metabolic responses to the serotonergic agonist metachlorophenylpiperazine in rats. AB - To determine if reported reductions of regional cerebral metabolic rates for glucose (rCMRglc) induced by the 5-HT agent metachlorophenylpiperazine (MCPP) (2.5 mg/kg) are due to a presynaptic action, 3-month old Fischer-344 rats were given parachloroamphetamine (PCA), a serotonin neurotoxin, and rCMRglc was measured 1 or 3 weeks later with the quantitative autoradiographic [14C]2 deoxyglucose procedure in 74 brain regions after administering saline, MCPP or other drugs. PCA alone increased rCMRglc significantly only in the raphe nuclei and in visual structures (visual cortex, lateral geniculate, superior colliculus). MCPP alone reduced rCMRglc in 75% of the regions studied. In PCA lesioned rats, metabolic responses to MCPP 2.5 mg/kg were virtually abolished and rCMRglc was increased in interanteromedial and centrolateral thalamic nuclei. rCMRglc responses to quipazine, a postsynaptic serotonin agonist, and to arecoline and bromocriptine, cholinergic and dopaminergic agonists, were unchanged by PCA-pretreatment. Selective abolition by PCA of the metabolic response to MCPP confirms that MCPP, at the dose studied, reduces rCMRglc in the forebrain via a presynaptic mechanism and that postsynaptic serotonergic function is not altered by PCA. PMID- 1713116 TI - Co-localization of enkephalin and TRH in perifornical neurons of the rat hypothalamus that project to the lateral septum. AB - Neurons of the lateral septal nucleus are surrounded by terminals immunoreactive for thyrotropin-releasing hormone (TRH) and enkephalin (Enk). Retrograde labeling from the lateral septum in combination with immunocytochemical analyses for Enk and TRH in colchicine-treated rats has revealed that nearly all Enk- (and TRH-) containing perifornical neurons project to the lateral septum. Immunostaining of adjacent, thin paraffin sections for either TRH or Enk and double staining of thick vibratome sections for the two peptides have shown that TRH and Enk immunoreactivities co-exist within the same neurons in the perifornical region of the hypothalamus. PMID- 1713117 TI - Prodynorphin- and substance P-containing neurons project to the medial preoptic area in the male Syrian hamster brain. AB - To determine if substance P- or prodynorphin-containing neurons of the medial nucleus of the amygdala and medial bed nucleus of the stria terminalis send projections to the medial preoptic area in the male Syrian hamster, we placed a fluorescent retrograde tract tracer (either Fluoro-gold, or rhodamine- or fluorescein-impregnated latex microspheres) into the medial preoptic area. Five to seven days later, the animals were treated with colchicine, allowed to survive for 48 h and the brains were processed for immunofluorescence histochemistry. Tissue sections were incubated in either rat anti-substance P or rabbit anti-C peptide (the C-terminal sequence of dynorphin B) antiserum followed by incubation in either fluorescein- or rhodamine-conjugated anti-rabbit or anti-rat antiserum. When the injection site of retrograde tracer was centered within the caudal one third of the medial preoptic area, labeled cell bodies were observed caudally in the medial part of the bed nucleus of the stria terminalis. Retrogradely labeled cell bodies were also observed in the posterodorsal subdivision of the medial nucleus of the amygdala. Both prodynorphin and substance P immunolabeling were observed in retrogradely labeled neurons in these two areas but fewer of these projection neurons were immunolabeled with substance P antiserum than with C peptide antiserum. These projections may play a role in the peptidergic modulation of reproductive behavior in this species. PMID- 1713118 TI - Immuno-electronmicroscopic visualization of cell adhesion molecule L1 in adult rat pyramidal tract: localization on neuronal and oligodendrocytic processes. AB - The immuno-electronmicroscopic localization of cell adhesion molecule L1 is investigated in adult rat pyramidal tract (PT) at the fifth/sixth cervical spinal cord segment, both by pre-embedding on vibratome sections and by immunogold labelling on ultra-cryosections. L1-immunoreactivity (L1-IR) can be noted not only on the surface of unmyelinated PT axons, the outer axonal membrane, but also within the axoplasm of myelinated PT axons as well as periaxonally between axolemma and compact myelin. Compact myelin is L1-negative. Interestingly, L1-IR is found in between the inner oligodendrocytic mesaxon and compact myelin. Hence, L1 is expressed by this type of glial cell in adult rat PT. In conclusion, L1 is suggested to be important in the adult rat PT, not only with respect to the adhesion between unmyelinated PT axons but also during stabilization of the mature neuron-oligodendrocyte interaction. PMID- 1713119 TI - Serotonin regulation of tachykinin biosynthesis in the rat neostriatum. AB - Serotonin (5-HT) neurotransmission was altered to determine its role in regulating the biosynthesis of tachykinins in the neostriatum (NS). Depletion of 5-HT with subchronic p-chlorophenylalanine (pCPA) treatment decreased preprotachykinin (PPT, the prohormone precursor to SP) mRNA levels in the NS. By contrast, raising extracellular 5-HT levels with zimelidine (a 5-HT uptake inhibitor) or clorgyline (a monoamine oxidase inhibitor) resulted in increased levels of PPT mRNA. To determine whether 5-HT receptors played a role in mediating the changes in PPT mRNA, animals were treated with the 5-HT2 agonist DOI. This drug significantly increased both PPT mRNA and SP-like immunoreactivity in the NS. These results together indicate that neostriatal tachykinin biosynthesis is sensitive to alterations in 5-HT neurotransmission. PMID- 1713120 TI - A subpopulation of displaced ganglion cells of the pigeon retina exhibits substance P-like immunoreactivity. AB - Immunohistochemical and retrograde tracing techniques were combined to demonstrate the occurrence of displaced ganglion cells (DGCs) exhibiting substance P-like immunoreactivity (SP-LI) in the pigeon retina. Following injections of rhodamine-labeled latex microspheres into the nucleus of the basal optic root (accessory optic system), about 5200 DGCs were observed to contain rhodamine fluorescence in the contralateral retina. Approximately 26% of the retrogradely labeled DGCs also contained SP-LI. The soma sizes of the doubly labeled DGCs ranged from 12 to 24 microns, and their distribution mirrored the overall distribution of DGCs projecting to the nucleus of the basal optic root. The density of doubly labeled DGCs ranged from 2 to 15 cells/mm2, with density peaks occurring in the superior-nasal and inferior-temporal retinal quadrants. Larger DGCs projecting to the nBOR (25-32 microns) were never seen to contain SP LI. Together with previous results of enucleation experiments, these data indicate the existence of a subpopulation of SP-LI DGCs which are connected with the accessory optic system in the pigeon. The present results also contribute information on the heterogeneity of retinal ganglion cells transmitters and modulators. PMID- 1713121 TI - Drug-related pulmonary toxicity in non-Hodgkin's lymphoma. Comparative results with three different treatment regimens. AB - Pulmonary toxicity may complicate the treatment of non-Hodgkin's lymphoma (NHL). The possible drug-related cause of pulmonary toxicity was investigated retrospectively in 207 NHL patients treated between 1981 and 1988 with three regimens containing cyclophosphamide with and without methotrexate or bleomycin: methotrexate, calcium, leucovorin, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m-BACOD) (n = 134); methotrexate, calcium, leucovorin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m ACOD) (n = 43); or cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) (n = 30) chemotherapy. These regimens contained the same drugs and were administered in the same schedule; the regimens differed primarily in the addition of bleomycin or methotrexate. Pulmonary toxicity occurred in 24 of 134 (18%) m-BACOD-treated and in six of 43 (14%) m-ACOD-treated patients (P = 0.65). Chest radiography revealed diffuse pulmonary infiltrates in 16 (67%) and six (100%) of the m-BACOD-treated and m-ACOD-treated patients with pulmonary toxicity, respectively. None of the CHOP-treated patients had pulmonary toxicity. The clinical features of pulmonary toxicity and the amount of chemotherapy administered before it occurred did not differ in patients treated with m-BACOD or m-ACOD, although the toxicity tended to be more severe in the m-BACOD group. Open lung or transbronchial biopsies done in six (38%) of the m-BACOD-treated and three (50%) of the m-ACOD-treated patients with pulmonary infiltrates revealed nonspecific pneumonitis compatible with drug-related toxicity. In summary, these results showed that pulmonary toxicity during m-BACOD and m-ACOD therapy occurred with similar frequency and clinicopathologic features. This suggested that bleomycin was not responsible uniquely for the pulmonary toxicity in m-BACOD treated patients. That pulmonary toxicity was not observed in patients treated with CHOP suggested that methotrexate may play an important role in the pathogenesis of the pulmonary toxicity. PMID- 1713122 TI - Secretion of gamma-glutamyl hydrolase in vitro. AB - gamma-Glutamyl hydrolase (also known as conjugase) is a ubiquitous enzyme that has the capacity to cleave folyl- and antifolylpolyglutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. H35 cells have lower cellular levels of gamma-glutamyl hydrolase than do hepatocytes but secrete a greater proportion of gamma-glutamyl hydrolase. More than 99% of the total enzyme from H35 cells accumulated in the medium after 48 h. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase, and lysosomal hydrolases other than gamma-glutamyl hydrolase were retained within the cell. Using the substrate 4-amino-10-methyl-pteroyldiglutamate (4-NH2-10-CH3-Pte-Glu2), the intracellular and secreted enzyme form(s) from H35 cells were found to have the following properties (a) Km values of 24.3 +/- 3.7 microM and 34.8 +/- 8.6 microM, respectively, and (b) maximal activity at pH 5 to 7 and apparent molecular weights of 120,000 by gel filtration. Both the cellular and secreted enzymes convert 4-NH2-10-CH3-PteGlu4 and pteroylpentaglutamate acid, to the corresponding monoglutamates with little or no appearance of intermediate chain length polyglutamates. This suggests that both act primarily as endopeptidases. Thus far, the cellular and secreted enzymes cannot be differentiated although the current studies do not establish this point unequivocally. Alterations in the cellular and secreted H35 cell gamma-glutamyl hydrolase levels in response to changes in culture conditions revealed that glutamine enhances activity while insulin diminishes it. Other transformed cells found to secrete this protein are Hep-G2 human hepatoma, JAR human choriocarcinoma, HeLa, and rat glioma. gamma Glutamyl hydrolase could not be detected in medium conditioned by human MCF-7 breast cancer cells, and relatively low activities were found in the medium from CCRF-CEM or K562 leukemia cells. These studies directly establish for the first time the secretion of gamma-glutamyl hydrolase in vitro. PMID- 1713123 TI - Abnormal expression of retinoic acid receptors and keratin 19 by human oral and epidermal squamous cell carcinoma cell lines. AB - We have analyzed the expression of the three retinoic acid receptor (RAR) (alpha, beta, gamma) mRNAs and the intermediate filament protein keratin 19 (K19) mRNA in cell lines cultured from oral and epidermal human squamous cell carcinoma (SCC) and from benign, hyperplastic, and hyperkeratotic (leukoplakia) lesions arising in various regions of the oral cavity. Seven of the SCC lines were derived from tumors arising in regions of the oral cavity in which the normal epithelial cells (keratinocytes) express RAR beta transcripts. Seven of the nine SCC lines tested did not exhibit detectable RAR beta mRNA levels, even in response to addition of retinoic acid (RA). The RAR beta gene did not appear to be rearranged or deleted in the five nonexpressing SCC lines examined by Southern analysis. The steady state RAR gamma mRNA levels were 2- to 4-fold lower in 6 of the 9 SCC lines than in their normal counterparts, whereas the RAR alpha message levels in SCC lines were similar to those of the normal cell strains. The expression of keratin 19 message, which is RA inducible in normal keratinocytes, was also abnormal in many of the SCC cell lines. Some SCC lines, e.g., those derived form tumors of the soft palate epithelium, did not express high levels of K19 message even though normal soft palate keratinocytes expressed high levels of K19 mRNA. Two of the nine SCC lines expressed higher than normal levels of K19 mRNA, and this expression was RA independent. Cells cultured from four oral leukoplakia lesions were also examined and found to express RAR beta mRNA at relatively normal levels, but they expressed RAR gamma message at half the level of epithelial cells cultured from normal tissue. These results show that the correlation between RAR beta gene expression and K19 gene expression that we have observed in the various normal keratinocyte subtypes of the oral cavity (D.L. Crowe et al., manuscript in preparation) is not present in transformed keratinocytes (SCC cells). The lack of apparent RA regulation of the K19 gene in SCC lines may be associated with other aberrations in differentiation which have been identified in SCC cells. Abnormally low expression of the RAR beta receptor may contribute to neoplastic progression in stratified squamous epithelia. It may also determine whether a tumor is responsive to RA as a chemotherapeutic agent. PMID- 1713124 TI - Immortalization by human papillomavirus type 16 alters retinoid regulation of human ectocervical epithelial cell differentiation. AB - Human cervical cells are a primary site of papillomavirus infection and 90% of all cervical tumors are positive for human papillomavirus (HPV) DNA. Over one half million cases of HPV-associated cervical, vulvar, and penile cancers are reported per year. Yet, in spite of the magnitude of this problem, the effects of HPV infection on cervical cell growth and differentiation are not well characterized. To study these effects we have developed a clonal cell line of HPV 16-immortalized ectocervical epithelial cells, ECE16-1. In the present study we demonstrate that under normal growth conditions the cytokeratin content of ECE16 1 cells is dramatically altered compared to normal cervical cells; the level of K5, K6, K14, K16, and K17 is reduced and the level of K7, K8, and K19 is increased. We demonstrate that this change is largely due to a difference in the response of the cells to retinoids, as growth in retinoid-free medium produces a complete normalization of cytokeratin levels. Upon addition of natural and synthetic retinoids, the levels of cytokeratins K5, K6, K14, K16, and K17 are reduced, while the levels of cytokeratins K19, K7, and K8 are increased. Cytokeratin K13 levels are only slightly altered. The level of involucrin, a precursor of the cervical cell envelope (superficial cell), is not changed by immortalization nor is it regulated by retinoids. Transglutaminase activity is also not appreciably altered by immortalization; however, ECE16-1 cells make fewer envelopes than normal ECE cells. Our results clearly indicate that natural and synthetic retinoids suppress the differentiation of HPV transformed cervical cells. In early, low grade, cervical intraepithelial neoplasia, transcription of the HPV16 E6/E7 oncogenes is confined to the suprabasal layers. Our results suggest that retinoids, because they inhibit the differentiation of HPV16 immortalized cervical cells, may reduce the extent of viral oncogene transcription and thus be useful in slowing the neoplastic process. PMID- 1713125 TI - Nucleolar organizer regions as a prognostic indicator for stage I non-small cell lung cancer. AB - When the number of silver-stained nucleolar organizer regions (Ag-NORs) was counted in 274 patients with non-small cell lung cancer, the mean number per nucleus in patients overall was 5.07 +/- 1.92 (SD). With the use of the tumor (T) nodes (N)-metastasis (M) classification, the mean Ag-NOR count for patients with T1 and T2 disease was statistically lower than that for those with T3 and T4 disease (P less than 0.01). The mean Ag-NOR counts were lower in patients with N0 disease than in those with N1 and N2 disease (P less than 0.01); lower in patients with stage I disease than in those with stage II, IIIA, IIIB, or IV disease (P less than 0.01); and lower in patients with adenocarcinoma than in those with squamous cell carcinoma (P less than 0.01) or large-cell carcinoma (P less than 0.05). In 131 patients with stage I disease, the mean Ag-NOR count was 3.80 +/- 1.32, and the 5-year survival rates of patients with Ag-NOR counts of less than 3.80 and greater than or equal to 3.80 were 78 and 44%, respectively, including 78% and 25% for adenocarcinoma, respectively (P less than 0.01). However, there was no statistically significant difference for those in stage II, IIIA, IIIB, or IV, and in stage I (without an adenocarcinoma). Because patients with stage I non-small cell lung cancer and a high number of Ag-NORs had a poor prognosis, Ag-NORs can serve as a pertinent marker of an early recurrence. PMID- 1713126 TI - Subtractive cloning of a hybrid human endogenous retrovirus and calbindin gene in the prostate cell line PC3. AB - A complementary DNA clone containing human endogenous retrovirus-like sequences spliced to sequences encoding human calbindin was discovered by complementary DNA subtraction analysis between two human prostate cell lines, PC3 and DU145. This gene is presumably activated by the long terminal repeat of the retrovirus-like sequence. It belongs to a member of the retrovirus-like H (tRNA(his) primer binding sequence) family. The level of this transcript is high in PC3, derived from a prostate bone metastasis, but not in DU145, derived from a prostate brain metastasis. PMID- 1713127 TI - The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. AB - Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis. PMID- 1713128 TI - Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. AB - We previously demonstrated that expression of an albumin-urokinase-type plasminogen activator (Alb-uPA) fusion construct in transgenic mice resulted in elevated plasma uPA concentration, hypofibrinogenemia, and neonatal hemorrhaging. Two lines of Alb-uPA mice were established in which only one half of the transgenic pups died at birth; surprisingly, plasma uPA concentrations in survivors gradually returned to normal by 2 months of age. The basis for this phenomenon is DNA rearrangement within hepatocytes that affects the transgene tandem array and abolishes transgene expression. Transgene-deficient cells selectively proliferate relative to surrounding liver, and this process culminates in replacement of the entire liver by clonal hepatic nodules derived from transgene-deficient progenitor cells. In some cases as few as two nodules can reconstitute over 90% of liver mass, highlighting the remarkable regenerative capacity of individual liver cells. PMID- 1713129 TI - Intracompartmental sorting of essential myosin light chains: molecular dissection and in vivo monitoring by epitope tagging. AB - The isoprotein-specific intracompartmental sorting of the three essential myosin light chains (LCs), the skeletal muscle LC-1f and LC-3f and the nonmuscle LC-3nm, was investigated. Epitope tagging was used to monitor the intracellular localization to different cytoskeletal structures of the exogenously introduced constructs in adult rat cardiomyocytes (ARCs), which exhibit both stress fibers and regenerating myofibrils. LC-1f and LC-3f bind almost exclusively to the sarcomeric myosin heavy chain (MHC) with high affinity, while the LC-3nm interacts with stress fibers and sarcomeres equally well. Sorting appears to be directed by a hierarchical order of different affinities. Domain mapping by deletion and by construction of a LC-1f/3nm chimera suggests that the LCs are composed of three functionally distinct domains: a basal MHC binding site in the C-terminus; the central part, modulating the preferential interaction with MHC isoforms; and the isoprotein-specific N-terminus of the essential LC, which is probably not involved in the sorting process. PMID- 1713130 TI - Epirubicin: a phase II study in recurrent small-cell lung cancer. AB - Epirubicin (4'-epidoxorubicin), an analogue of doxorubicin (Adriamycin), has established activity in the treatment of small-cell lung cancer (SCLC) when used at doses of 75 to 120 mg/m2 in previously untreated patients. We completed a phase II study of epirubicin (85 mg/m2 given intravenously at 3-week intervals) in 20 patients with recurrent SCLC, all of whom had received prior combination chemotherapy. Of 19 patients who were assessable for response, 2 achieved a complete response and 2 a partial response, for an overall response rate of 4/19 (21%); 95% confidence interval, 8%-43%). Myelosuppression and alopecia were the most frequent toxicities; epirubicin was otherwise well tolerated, with other toxicities such as nausea and vomiting being infrequent or mild. Epirubicin at a dose of 85 mg/m2 exhibits modest single-agent activity in previously treated SCLC and is generally well tolerated. Given as a single agent or in combination with other well-tolerated drugs, epirubicin would be suitable in cases in which palliation of symptoms without undue toxicity is required in the management of previously treated SCLC. PMID- 1713131 TI - Paracrine regulation of implantation and uterine function. PMID- 1713132 TI - Metformin causes a reduction in basal and post-venous occlusion plasminogen activator inhibitor-1 in type 2 diabetic patients. AB - The effects of metformin on the fibrinolytic system were studied pre- and post venous occlusion in 38 Type 2 diabetic patients in a double-blind, placebo controlled trial. After a 3-week run-in period, 21 patients received metformin and 17 placebo, for 6 weeks. In the metformin-treated patients basal plasminogen activator inhibitor-1 antigen (PAI-1Ag) fell from 57.4 micrograms l-1 before treatment to 36.1 (p less than 0.05) and 41.0 micrograms l-1 (p less than 0.01) after 3 and 6 weeks therapy. In this group post-venous occlusion PAI-1Ag also fell after 3 weeks (p less than 0.002) and 6 weeks (p less than 0.05) treatment. There were no changes in either basal or post-venous occlusion concentrations of PAI-1Ag in the placebo treated group. The fall in PAI-1Ag was not associated with an increase in basal plasminogen activator activity (PAA) which remained unchanged in both groups. Post-venous occlusion values for PAA in the metformin treated patients were increased at 3 weeks (p less than 0.05) although there was no difference at 6 weeks. PMID- 1713133 TI - Automated immunonephelometric assay for human alpha-1-microglobulin. PMID- 1713134 TI - Inhibition of aminoglycoside-induced nephrotoxicity in rats by polyanionic peptides. AB - In the present study, we compared poly-L-Asp with poly-L-Glu and poly-D-Glu in vitro and in vivo for their ability to inhibit the GM-induced nephrotoxicity. In vitro, all three polyanions (i) bound GM over a wide range of pH; (ii) displaced GM previously bound to negatively charged phospholipid bilayers at acid pH (i.e. under the conditions prevailing in lysosomes in vivo), and thereby (iii) decreased the inhibitory potency of GM towards lysosomal phospholipase A1. Thus, one was tempted to predict that all three polyanions would have the potential of protecting against AG-induced nephrotoxicity. However, when co-administered to rats with GM, poly-L-Asp and poly-D-Glu completely suppressed the development of lysosomal phospholipidosis, as assessed by biochemical criteria and increased drug accumulation, whereas poly-L-Glu did not offer such protection despite a relatively lower increase in drug accumulation levels. Histoautoradiography also confirmed that poly-L-Asp, but not poly-L-Glu, was a nephroprotectant against the tissue proliferative response induced by GM. Morphologically, poly-L-Asp almost completely and poly-D-Glu totally prevented the accumulation of myeloid bodies in lysosomes. In vitro incubation in the presence of purified lysosomal extracts revealed marked differences in the hydrolysis rate of these peptides (poly-D Glu:poly-L-Asp:poly-L-Glu = 1:1.2:16.9). Assuming that all three polyanionic peptides are transported and accumulated in lysosomes to the same extent, these results not only suggest that their stability in lysosomes is an essential requisite for protection against lysosomal phospholipidosis, but also strengthen our hypothesis that the site of action of poly-L-Asp is inside the lysosomes but not at the level of the renal membranes. In addition, poly-D-Glu alone or combined with GM induced another type of morphological lesion, not related to AG induced nephrotoxicity which, to our knowledge, has not yet been described. PMID- 1713135 TI - The discrepancy of serum alpha-1-microglobulin values obtained by different assay systems. PMID- 1713136 TI - Nephrotoxicity and tubular effects of contrast media. PMID- 1713137 TI - High and preferential accumulation in the kidney of anionic and cationic small proteins. PMID- 1713138 TI - Regulation of urinary excretion of alpha-2u-globulin during circadian rhythm. PMID- 1713139 TI - Non-small cell lung cancer. Part II: Treatment. AB - Squamous, large cell, and adenocarcinoma, collectively termed non-small cell lung cancer (NSCLC), are diagnosed in approximately 75% of patients with lung cancer in the United States. The treatment of these three tumor cell types is approached in virtually identical fashion because, in contrast to small cell carcinoma of the lung, NSCLC more frequently presents with localized disease at the time of diagnosis and is thus more often amenable to surgical resection but less frequently responds to chemotherapy and irradiation. Cigarette smoking is etiologically related to the development of NSCLC in the great majority of cases. Genetic mutations in dominant oncogenes such as K-ras, loss of genetic material on chromosomes 3p, 11p, and 17p, and deletions or mutations in tumor suppressor genes such as rb and p53 have been documented in NSCLC tumors and tumor cell lines. NSCLC is diagnosed because of symptoms related to the primary tumor or regional or distant metastases, as an incidental finding on chest radiograph, or rarely because of a paraneoplastic syndrome such as hypercalcemia or hypertrophic pulmonary osteoarthropathy. Screening smokers with periodic chest radiographs and sputum cytologic examination has not been shown to reduce mortality. The diagnosis of NSCLC is usually established by fiberoptic bronchoscopy or percutaneous fine-needle aspiration, by biopsy of a regional or distant metastatic site, or at the time of thoracotomy. Pathologically, NSCLC arises in a setting of bronchial mucosal metaplasia and dysplasia that progressively increase over time. Squamous carcinoma more often presents as a central endobronchial lesion, while large cell and adenocarcinoma have a tendency to arise in the lung periphery and invade the pleura. Once the diagnosis is made, the extent of tumor dissemination is determined. Since most NSCLC patients who survive 5 years or longer have undergone surgical resection of their cancers, the focus of the staging process is to determine whether the patient is a candidate for thoracotomy with curative intent. The dominant prognostic factors in NSCLC are extent of tumor dissemination, ambulatory or performance status, and degree of weight loss. Stages I and II NSCLC, which are confined within the pleural reflection, are managed by surgical resection whenever possible, with approximate 5-year survival of 45% and 25%, respectively. Patients with stage IIIa cancers, in which the primary tumor has extended through the pleura or metastasized to ipsilateral or subcarinal lymph nodes, can occasionally be surgically resected but are often managed with definitive thoracic irradiation and have 5-year survival of approximately 15%.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713140 TI - Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes. AB - The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions. PMID- 1713141 TI - Three epidermal and one simple epithelial type II keratin genes map to human chromosome 12. AB - We have localized the genes which encode the human type II epidermal keratins K5, K6a, and K6b and the simple epithelial keratin K7 (KRT5, KRT6A, KRT6B, and KRT7, respectively) to chromosome 12 using Southern blot analysis of somatic cell hybrids. In addition, we have sublocalized the genes for K6a and K7 to bands 12q12----q14 on the long arm of this chromosome by in situ hybridization of metaphase chromosomes. PMID- 1713142 TI - Lack of association between prostate-specific acid phosphatase RFLP genotypes and prostatic cancer or benign prostatic hyperplasia. AB - We have previously reported the identification and basic characterization of two biallelic TaqI RFLPs, A and B, of the 3' end of the human ACPP locus in an unselected Finnish population (Winqvist et al., 1989). In the present investigation, a similar allelic distribution was observed in patients with prostatic cancer or benign hyperplasia. In addition, it was found that the DNA sequences generating RFLP-B are located further downstream from the RFLP-A sequences. PMID- 1713143 TI - Intrathecal anti-myelin basic protein antibody production in Chinese patients with multiple sclerosis. AB - Cerebrospinal fluids and paired serums from 23 patients with multiple sclerosis (MS) were studied for intrathecal synthesis of oligoclonal band and antimyelin basic protein (MBP) antibody. Three parameters for anti-MBP antibody were designed to represent three different pathophysiological meanings, including value, index and ratio of anti-MBP antibody. Oligoclonal band was detected by agarose gel electrophoresis in 23 MS patients, and anti-MBP was assayed by enzyme linked immunosorbent assay in 11 MS patients to investigate these anti-MBP parameters in MS group and non-inflammatory control group. The anti-MBP assay revealed that intrathecal anti-MBP antibody production was increased during active stage of MS. However, anti-MBP antibody production correlated poorly with the IgG index, nor was it conspicuously higher than IgG production. This might imply that, except for MBP, still other protein components are involved in this immune process. PMID- 1713144 TI - Lectin staining of neoplastic and normal background colorectal mucosa in nonpolyposis and polyposis patients. AB - A lectin histochemistry approach was adopted for comparative assessment of a colon cancer risk. Binding of Ulex europaeus agglutinin-I (UEA-I), peanut agglutinin (PNA), Griffonia simplicifolia agglutinin-II (GSA-II), and Dolichos biflorus agglutinin (DBA) was investigated in tumor and background tissue from a total of 34 adenoma and 44 cancer patients and compared with reaction patterns in control and familial adenomatous polyposis (FAP) patients. Adenoma patients with UEA-I positive rectal mucosa were found to have a 33.3 percent familial history of large bowel cancer, which was significantly higher (P less than 0.05) than the respective 4.0 percent figure for patients with negative rectal mucosa. In the cancer patients, an even stronger correlation was noted, with a 63.2 percent UEA I positive family history association being recorded, as opposed to 4.0 percent in the negative rectal mucosa patients (P less than 0.01). Thus, the results suggest that, apparently, normal rectal background mucosa of individuals genetically at high risk for colon and rectal cancer demonstrates a specific lectin binding ability similar to that of FAP patients and that the simple method using UEA-I staining of rectal biopsy specimens can be of practical use in identification of high-risk colorectal cancer. PMID- 1713145 TI - [The bioenergetic properties of "dark" and "light" cells]. PMID- 1713146 TI - [Lennert's lymphoma and glomerulonephritis]. AB - A 49-year-old man noticed a swelling below the left ear. The histological diagnosis was chronic lymphadenitis with a small cell epithelioid cell reaction. A short time later he developed oedema of the legs, proteinuria and elevated serum creatinine levels (1.89 mg/dl). Renal biopsy showed mesangioproliferative glomerulonephritis. Over the next two years the tumour in the left side of the neck gradually increased in size. Computed tomography showed a space-occupying lesion 5 x 7 cm in the vicinity of the left parotid gland, with evidence of infiltrative growth. Histological examination of the tumour after removal revealed epithelioid cell tissue with numerous lymphocytes, and led to the diagnosis of a lymphoepithelial lymphoma (Lennert's lymphoma), a T-cell lymphoma of low malignancy. Complete remission was achieved after four chemotherapy cycles (COPP schedule) in reduced doses (creatinine concentration 2.09 mg/dl, creatinine clearance 36 ml/min); creatinine clearance subsequently improved to 63 ml/min. Later, however, the tumour recurred and the patient went into terminal renal failure, dying four years after the lymphoma first appeared. The glomerulonephritis may conceivably have been a paraneoplastic phenomenon. PMID- 1713147 TI - Effect of check size on the pattern reversal visual evoked potential. AB - Modifications of the components of pattern-reversal visual evoked potentials (PR VEP) with changes in check size of the stimulating pattern were studied in 11 healthy subjects. We made use of 8 different check sizes ranging between 10 and 90 min of arc. Changes in the check size modified in different manners the latencies and amplitudes of N75, P100 and N145. Two-step statistical analyses using the polynomial regression analysis method revealed significant modifications of latencies of the 3 components, but non-significant modifications of the amplitudes, except for N75. The latency and amplitude of N75 showed a significant inverse linear relationship with the logarithm of the check size, while the P100 and N145 latencies showed significant curvilinear relationships, with minimal latencies at check sizes around 35 min. These findings suggest different physiological properties of N75 from those of P100 and N145, and hence, the necessity to establish normal values for each check size of stimulation, for application in clinical studies. PMID- 1713148 TI - Mapped distribution of pattern reversal VEPs to central field and lateral half field stimuli of different spatial frequencies. AB - Visual evoked potentials (VEPs) to pattern reversal vertical bar stimuli of 3 different sizes (1, 2, 4 c/deg) were recorded from 19 scalp derivations in 50 controls. The stimuli were presented on a full-field (FF) screen of 24 degrees visual angle, and on left and right half-fields (HF) of 12 degrees radius. In 15 controls partial HF stimuli were presented on the central 3 and 6 degrees and as hemiannular stimuli of 12 degrees with occlusion of the central 3 and 6 degrees. An antero-posterior polarity reversal of the N1-P1-N2 sequence was observed for FF VEPs. A tangential polarity reversal was observed for HF VEPs. Also with central or hemiannular stimuli polarity reversals of all VEP components were observed within the scalp. Variants of VEP distribution, absence or prominence of some of the ipsi- or contralateral VEP components were observed in 8-40% of controls. The FF and HF VEP distribution, and the variant VEP asymmetries were partly dependent on the pattern spatial frequency. PMID- 1713149 TI - The extrastriate generators of the EP to checkerboard onset. A source localization approach. AB - The cortical origin of the pattern onset EP has been investigated over a time window which covers the entire positive-negative-positive complex of the pattern onset EP. On the basis of a dipole source localization approach, the position, orientation and strength of the underlying sources of the pattern onset EP were estimated. For large check stimuli, chosen to have a weak edge specific component in the response, still two components are needed to account for the variance of the responses. Each component corresponds to a single dipole source, and both originate in the extrastriate cortex. These components dominate, respectively, the initial and the late positive peaks of the pattern onset EP. The equivalent dipole sources of the two components show different behaviors with respect to the position of the stimulus in the visual field. The topography and behavior of the equivalent dipole source underlying the early positive component suggest an origin in area 18. The invariance with stimulus location of the dipole source underlying the late positive component suggests an origin beyond area 18. The different topographies of the components also account for the differences in surface distribution of the pattern onset EP to large check stimulation of the upper and lower sectors of the visual field. PMID- 1713150 TI - Optimal smoothing of coherence estimates. AB - We have previously described the usefulness of the magnitude-squared coherence function in analysis of auditory evoked potentials (AEPs) (Ear Hear., 1989, 20: 2 13). For each frequency of interest, the coherence value can be compared to a 'critical value' to determine whether a response is present. Coherence functions must be smoothed across either multiple subaverages or adjacent frequencies (or both) to be reliable, but there are trade-offs: increasing the degree of smoothing increases computational time and (in the case of frequency smoothing) reduces spectral resolution. Using AEPs to clicks and amplitude-modulated tones we investigated the effects of variable degrees of smoothing on threshold estimates in 10 normal human subjects. Thresholds were found to be lower for coherence estimates than for visual detection and are also lower for longer data collection periods. However, there appears to be little if any advantage to segment smoothing beyond 8-16 subaverages. The optimal degree of frequency smoothing is more difficult to specify, depending on the spectrum of the AEP being analyzed (especially the rate of change of phase), and the spectral resolution of the analysis system. PMID- 1713151 TI - Mapping study of somatosensory evoked potentials during selective spatial attention. AB - We have investigated the effects of selective spatial attention on early and middle-latency SEPs. Baseline control responses to electrical stimulation of 2 digits of the hand were recorded first in conditions of mental relaxation, in the absence of any cognitive task, to obtain truly 'neutral' responses uncontaminated by cognitive components. Then, during a 'task condition,' identical stimuli were applied to the same two fingers, but the subject's attention was driven towards the stimulated territory by the bias of mechanical taps delivered to the same digits. The earliest effect of directing attention towards the territory stimulated was a positive shift on contralateral somatosensory responses, with onset at 27.4 +/- 4 msec post stimulus. This SEP modification: (a) did not entail any change in the scalp distribution of components, as assessed by topographic mapping, and (b) was not present when attention was directed towards the hand contralateral to that receiving electrical stimuli. A second effect was represented by a parieto-central negativity in the 60-80 msec latency range; this feature could also be observed during contralaterally driven attention and was associated with topographical changes in SEP scalp distribution. Finally, a late centro-frontal negativity beginning at 90-100 msec (N140) appeared during ipsilateral attention, while P100 was not enhanced. Subcortical P14 and primary cortical N20 were not significantly affected by the tasks. We conclude that the 'early positive shift' is linked to the spatial aspects of selective attention and represents in part modulation of obligatory components (P25 through P45) existing in control SEPs; it probably corresponds with the deflections with similar polarity and time-course that have been described by others in response to somatosensory target stimuli. Conversely, 60-80 msec negative enhancement is less spatially selective and may represent non-specific arousal effects. The late negative component (N140) shares several features with the 'processing negativity' described in auditory paradigms and could represent the equivalent of this effect in the somatosensory system. PMID- 1713152 TI - Direct recording of somatosensory evoked potentials in the vicinity of the dorsal column nuclei in man: their generator mechanisms and contribution to the scalp far-field potentials. AB - Somatosensory evoked potentials (SEPs) in the vicinity of the dorsal column nuclei in response to electrical stimulation of the median nerve (MN) and posterior tibial nerve (PTN) were studied by analyzing the wave forms, topographical distribution, effects of higher rates of stimulation and correlation with components of the scalp-recorded SEPs. Recordings were done on 4 patients with spasmodic torticollis during neurosurgical operations for microvascular decompression of the eleventh nerve. The dorsal column SEPs to MN stimulation (MN-SEPs) were characterized by a major negative wave (N1; 13 msec in mean latency), preceded by a small positivity (P1) and followed by a large positive wave (P2). Similar wave forms (P1'-N1'-P2') were obtained with stimulation of PTN (PTN-SEPs), with a mean latency of N1' being 28 msec. Maximal potentials of MN-SEPs and PTN-SEPs were located in the vicinity of the ipsilateral cuneate and gracile nuclei, respectively, at a level slightly caudal to the nuclei. The latencies of P1 and N1 increased progressively at more rostral cervical cord segments and medulla, but that of P2 did not. A higher rate of stimulation (16 Hz) caused no effects on P1 and N1, while it markedly attenuated the P2 component. These findings suggest that P1 and N1 of MN-SEPs, as well as P1' and N1' of PTN-SEPs, are generated by the dorsal column fibers, and P2 and P2' are possibly of postsynaptic origin in the respective dorsal column nuclei.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713153 TI - Origin and distribution of brain-stem somatosensory evoked potentials in humans. AB - The distribution of somatosensory evoked potentials (SEPs) recorded from the brain-stem surface was studied to investigate their generator sources in 14 patients during surgical exploration of the posterior fossa. Two distinct SEPs of different morphologies and electrical orientation were obtained by median nerve stimulation. A small positive-large negative-late prolonged positive wave was recorded from the cuneate nucleus and its vicinity. There was a phase-reversal between the cuneate nucleus and the ventral surface of the medulla, depicting a dipole for dorso-ventral organization. From the pons and midbrain, triphasic waves with predominant negativity were obtained. This type of SEP had identical wave forms between the dorsal, lateral and ventral surface of the pons and midbrain. It showed an increase in negative peak latency as the recording sites moved rostrally, suggesting an ascending axial orientation. In a patient with pontine hemorrhage, the killed end potential, a large monophasic positive potential was obtained from the lesion. This potential occurs when an impulse approaches but never passes beyond the recording electrode. Therefore, the triphasic SEP from the pons and midbrain reflects an axonal potential generated in the medial lemniscal pathway. PMID- 1713154 TI - Bit-mapped imaging of somatosensory evoked potentials after stimulation of the posterior tibial nerves and dorsal nerve of the penis/clitoris. AB - Somatosensory evoked potentials (SEPs) to unilateral or bilateral posterior tibial nerve (PTN) stimulation and to stimulation of the dorsal nerve (DN) of the penis/clitoris were recorded on 32 channels in 10 volunteers. SEPs to unilateral PTN stimulation consisted of the classic 'W' complex P38-N45-P56-N75 maximal on the ipsilateral central and parietal leads, and two negative waves, N33 and N37, maximal on the contralateral post- and prerolandic areas, respectively. A lemniscal P30 was also recorded. Bilateral PTN stimulation caused, by algebraic summation, the disappearance of both N33 and N37; the W complex was symmetrical and the amplitude of P30 increased. The SEPs to DN stimulation were also symmetrical, and N33 and N37 were absent. These features can be explained by the bilateral character of DN stimulation. They also differed from bilateral PTN SEPs in 3 respects; the absence of P30, the small amplitude and the weaker gradients of field distribution of the 'W' complex, and the somewhat different distribution of penile from clitoral or bilateral PTN, N45 and P56. These differences can be explained both by physiological (the different fiber composition of the DN) and anatomical (the deeper localization of the DN cortical receiving area) mechanisms. PMID- 1713155 TI - Widespread N18 in median nerve SEP is preserved in a pontine lesion. AB - Widespread N18 potential to median nerve stimulation was preserved in a patient who had profound unilateral disturbance of deep sensation and a lesion of the pontine medial lemniscus confirmed by MRI. It was concluded from this result that at least a significant part of the N18 potential was generated caudal to the pontine level or at higher levels via extralemniscal pathways. Careful review of studies in man with intraoperative recordings seemed to support that the N18 potential already exists at the medullary level. We suggested that the potential generated at the cuneate nucleus which was described in cats may correspond to part of the N18 potential. PMID- 1713156 TI - Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines. AB - A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology. PMID- 1713157 TI - Effects of substance-P and neuropeptide-Y on in vitro steroid release by porcine granulosa and luteal cells. AB - The presence of substance-P (SP)- and neuropeptide-Y (NPY)-like immunoreactivity was recently shown in nerves that innervate the ovary. In the present in vitro study we demonstrate that both peptides have direct effects on ovarian steroidogenesis. In cultured porcine granulosa (G-) cells, neither peptide affected progesterone (P) production under basal conditions, but they both inhibited gonadotropin-stimulated P secretion. In luteal (L-) cell cultures, basal as well as hCG-stimulated P release were dose-dependently inhibited by NPY (ED50, 4 x 10(-9) M; identical for both, basal and stimulated release), while SP had only a moderate inhibitory effect (ED50, 6 x 10(-8) M). In the presence of AP13, a specific SP antagonist, the inhibitory effect of SP on P release was abolished, which suggests a receptor-mediated effect. In addition, we determined androstenedione (A) and estradiol (E2) release into G- and L-cell culture media. While E2 production in G-cell cultures was not influenced by SP and NPY, both peptides had a dose-dependent stimulatory effect on E2 secretion by L-cells. In contrast to E2 release, A secretion by G- as well as L-cell cultures was increased by gonadotropins. Both SP and NPY decreased gonadotropin-stimulated A secretion by G- and L-cells under basal as well as hCG-stimulated conditions. Furthermore, we demonstrate SP immunoreactivity in media of G- and L-cell cultures with a HPLC retention time identical to that of synthetic SP. This may suggest ovarian synthesis, in which case the peptide exerts auto- and/or paracrine effects on ovarian steroidogenesis. From these in vitro results we suggest that SP and NPY have a modulatory effect on ovarian function in pigs not only by their well known regulatory effects of blood supply, but also by a direct effect on ovarian steroidogenesis. PMID- 1713158 TI - Developmental expression of rat insulin-like growth factor binding protein-2 by astrocytic glial cells in culture. AB - Brain astrocytes were established in primary culture from postnatal and adult rats to characterize the developmental expression of secreted proteins. Astrocytes cultured from 21-day rat brain, but not 1-day rat brain, secreted a distinct group of proteins with Mr of 35,000 as determined by analysis of [35S]methionine-labeled proteins using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis of this protein group showed 100% identity to rat insulin-like growth factor binding protein-2 (rIGFBP-2), the BRL-3A IGFBP purified from a fetal rat liver cell line. An antiserum was generated against this astrocyte 35,000 Mr protein, and immunoblot analysis revealed a dramatic increase in rIGFBP-2 secretion in astrocytes cultured from 14-day, 21-day, and adult rat brain compared to astrocytes from 1-day and 7-day rat brain. Similar analysis of neonatal rat brain neurons in culture failed to show immunoreactive rIGFBP-2 in cell lysates or secreted protein. Ligand Western blot analysis demonstrated [125I]IGF-II binding to a single protein band which comigrated with a prominant rIGFBP-2 immunoreactive species in nonreduced conditioned medium from 21-day astrocytes. In comparison [125I]IGF-II binding proteins were detected only at low levels in medium from astrocytes cultured from 1-day rat brain and were undetectable in neuron-conditioned media. Northern blot analysis using a rIGFBP-2 complementary DNA revealed 5-fold greater messenger RNA levels in astrocytes from 21-day rat brain compared with astrocytes from 1-day brain, whereas neonate neurons showed no transcripts. Thus, rIGFBP-2 exhibits a pattern of developmental and cell specific expression in cultured rat brain cells. PMID- 1713159 TI - Leukotriene B4 increases intracellular calcium concentration and phosphoinositide metabolism in mouse osteoblasts via cyclic adenosine 3',5'-monophosphate independent pathways. AB - Leukotriene B4 is one of a number of agents which stimulate bone resorption by acting on osteoblasts. Some agonists, such as PTH or prostaglandins, are known to activate adenylate cyclase in osteoblasts, whereas others, such as vitamin D3, have no effect on adenylate cyclase. Recent evidence suggests that both classes of agonist may raise the intracellular calcium concentration, although the relative importance for bone resorption of calcium mobilization and adenylate cyclase activity in the osteoblast is not clear. Here it is shown 1) that leukotriene B4 does not activate but may be inhibitory toward adenylate cyclase in intact osteoblasts or membrane preparations, 2) that leukotriene B4 causes an elevation of intracellular calcium levels in osteoblast monolayers, 3) leukotriene B4 rapidly activates phosphatidylinositol bisphosphate breakdown in osteoblast membranes and intact osteoblasts, and 4) that leukotriene B4 stimulates phosphatidylinositol kinase activity concurrently with phosphoinositidase C in intact osteoblasts over a similar timescale. These results suggest that leukotriene B4 may increase the concentration of intracellular calcium in osteoblasts by stimulating phosphoinositide turnover, and support the proposal that calcium signaling rather than activation of adenylate cyclase in osteoblasts may be of overriding importance in the regulation of bone resorption. PMID- 1713160 TI - The direct in vitro effect of insulin-like growth factors (IGFs) on normal bovine mammary cell proliferation and production of IGF binding proteins. AB - Mammary epithelial cells isolated from pregnant, nonlactating heifers were grown in vitro using collagen substrates. Using these systems, the truncated form of insulin growth factor-1 (IGF-1) (des-3-IGF-1), IGF-1, and IGF-2 all stimulated a significant (0.5 to 1 fold) increase in cell proliferation (des-3-IGF-1 greater than IGF-1 greater than IGF-2). When grown in media containing serum plus IGF-1, normal bovine mammary cells also produced and secreted at least four species of IGF-binding protein (IGFBP) ranging from 21K to 48K (as demonstrated by ligand blot analysis). However, cells grown in serum free media secreted detectable quantities of only 2 major forms of IGFBP of 34K and 48K. Using immunoblot analysis, these proteins were identified as IGFBP-2 and IGFBP-3, respectively. Both proteins were inducible by the addition of IGF to the serum free media (relative potency; IGF-1 greater than des-3-IGF-1 greater than IGF-2). Using RIA analysis, bovine mammary cells cultured in the presence of IGF-1 produced 20-25 ng/ml IGFBP-2 compared to control cultures which secrete approximately 1.0 ng/ml. Cells exposed to des-3-IGF-1 produced 40-60% less IGFBP-2 whereas insulin and IGF 2 did not stimulate significant IGFBP-2 production. These data indicate that normal bovine mammary cells secret IGFBP-2 and IGFBP-3. This secretion is stimulated by IGF-1 and des-3-IGF-1 suggesting a mechanism for regulating local IGF activity. PMID- 1713161 TI - Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts. AB - Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels. PMID- 1713162 TI - Acute phosphate depletion dissociates hormonal stimulated second messengers in osteoblast-like cells. AB - The acute effect (24 h) of either phosphate depletion or phosphate surfeit on hormonal stimulated signal transduction systems was studied in the osteoblastic cell line UMR-106. Elevation of intracellular Ca2+ ([Ca2+]in), induced by different calciotropic hormones (PTH, prostaglandin E2, endothelin) was blunted by acute phosphate depletion, whereas at high inorganic phosphate (Pi) concentrations the rise in [Ca2+]in was augmented. Basal [Ca2+]in was not altered by either Pi depletion or Pi excess. The effect of acute phosphate depletion on hormonal mediated [Ca2+]in rise was not observed in the absence of extracellular Ca2+ suggesting that under these conditions, the release of Ca2+ from intracellular stores, is not affected. Also, nonhormonal calcium entry pathways such as depolarization-activated calcium channels or protein kinase C-activated Ca2+ channels were not affected by acute phosphate depletion. cAMP accumulation in the cells, either through receptor or nonreceptor-mediated mechanisms, increased under low Pi conditions and decreased as Pi concentration in the culture media was progressively increased from 0 to 2 mM during 24 h of incubation. Changes in Pi concentration had no effect on basal cAMP generation by the cells. The facilitative effect of acute Pi depletion on agonist-induced cAMP accumulation could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). PTH receptor binding assessed with [Nle8 Nle18 Tyr34] bovine PTH (1-34) NH2 was not altered by phosphate depletion. We conclude that exposure of osteoblasts to different Pi environments modulates the second messenger responses to hormones in a reciprocal fashion so that acute phosphate depletion down-regulates [Ca2+]in signals while augmenting cAMP generation and vice versa. Inasmuch as bone resorption processes can be modulated by Ca2+ and cAMP the data presented herein suggest that the altered bone resorptive response to calciotropic hormones (e.g. PTH), under surfeit or deficit of phosphate, is mediated by changes in [Ca2+]in and cAMP. PMID- 1713163 TI - Insulin-like growth factor (IGF) binding to cell monolayers is directly modulated by the addition of IGF-binding proteins. AB - Insulin-like growth factor-I (IGF-I) binds to specific receptors and IGF-binding proteins (IGFBPs) that are present on cell surfaces. The analysis of [125I]IGF-I binding to human fibroblasts is complicated by IGFBPs on the cell surface and their release into the medium during the binding assay. This release alters the distribution of [125I]IGF-I between type I IGF receptors and both soluble as well as cell surface-associated IGFBPs. In the present study we have determined the effects of three different forms of IGFBPs on [125I]IGF-I binding to cell surface binding sites of human fetal fibroblasts (GM10 cells) and porcine smooth muscle cells. Human 29,000 mol wt (Mr; IGFBP-1), bovine 34,000 Mr (IGFBP-2), and bovine 46,000 Mr (IGFBP-3) forms of IGFBP were compared. Each of the three IGFBPs inhibited [125I]IGF-I binding to the cell surface of both cell types. This effect was due to increased binding of [125I]IGF-I by the IGFBPs in the assay buffer. At equimolar concentrations, IGFBP-3 was more effective than either IGFBP-1 or IGFBP 2 in blocking cell surface binding. The addition of increasing concentrations of unlabeled IGF-I in the presence of each IGFBP showed that either IGFBP-1 or IGFBP 3, but not IGFBP-2, resulted in a paradoxical increase in [125I]IGF-I binding to the cell surface. The paradoxical increase occurred in the presence of excess insulin, indicating that unsaturated type I IGF receptors are not required to demonstrate this phenomenon. In a physiological salt solution, the order of affinity of the IGFBPs for IGF-I was IGFBP-3 greater than IGFBP-1 greater than IGFBP-2. These differences in affinity appear to account for the differences in IGF-I competition for binding that are seen when each of the three proteins is added. Thus, IGFBPs have the potential to alter the partitioning of IGF-I between cell surface-associated IGFBPs, membrane receptors, and the IGFBPs in extracellular fluids. The various forms of IGFBP affect IGF cell surface binding differently, and therefore, each may have distinct effects on IGF target cell actions. PMID- 1713164 TI - Seminal vesicle biopsies in the preoperative staging of prostatic cancer. AB - Twenty-five patients with localized prostate cancer underwent seminal vesicle biopsies before radical prostatectomy. A transrectal probe of 7 MHz, a 18-gauge needle and a biopsy gun were used. The preoperative biopsy established the absence of seminal vesicle invasion in 89% of cases. When the seminal vesicles are positive at biopsy, capsular penetration is observed in 100% of the cases and lymph node positivity in 50%. When seminal vesicles are negative at biopsy and the prostate-specific antigen level is less than 20 ng/ml (n less than 2.5), capsular penetration of greater than 1 cm is absent in 100% of cases and lymph nodes are positive in only 7% of cases. Biopsy of the seminal vesicle, as an outpatient procedure, improves the preoperative staging of prostate cancer before radical prostatectomy: negative biopsies are good predictors of the absence of lymph node invasion. PMID- 1713165 TI - 3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampus: role of Na+ entry on Ca2+ pools and of protein kinase C. AB - Slices of rat hippocampus, preincubated with [3H]noradrenaline [(3H]NA), were superfused continuously and stimulated by addition of 3,4-diaminopyridine (3,4 DAP; 100 microM) for 10 min to the superfusion medium. An overflow of 3H evoked by 3,4-DAP (representing [3H]NA release) was measurable not only in the presence but also in the absence of extracellular Ca2+. Both the protein kinase C (PKC) activator 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) and the PKC inhibitor polymyxin B, affected mainly the evoked release in the absence of extracellular Ca2+ in a facilitatory or inhibitory manner, respectively. Moreover, in the absence of extracellular Ca2+, both the 3,4-DAP-evoked [3H]NA release and the facilitatory effect of 4 beta-PDB were abolished in the presence of tetrodotoxin or in the absence of Na+ in the superfusion medium. Ruthenium red, a blocker of mitochondrial Ca2+ reuptake, potently increased 3,4-DAP-evoked [3H]NA release in Ca(2+)-free EGTA-containing medium. The facilitatory effects of ruthenium red and 4 beta-PDB were additive. From these and earlier observations we conclude (1) that the mechanism of 3,4-DAP-evoked [3H]NA release involves both Ca2+ influx into the nerve terminals and mobilization of intraneuronal Ca2+ pools. Most probably Ca2+ release from cytoplasmic Ca2+ stores (e.g. endoplasmic reticular pools or mitochondria) is induced by Na+ ions entering the nerve endings during 3,4-DAP-evoked repetitive action potentials. (2) The facilitatory effect of phorbol ester on 3,4-DAP-evoked NA release appears to be mediated not by changes in Ca2+ influx, but by enhancement of intraneuronal events distal to Na+ ion entry and increased intracellular Ca2+ availability. PMID- 1713166 TI - Determination of levels of cyclic AMP in the myenteric plexus of guinea-pig small intestine. AB - Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to investigate changes in levels of cyclic 3',5' adenosine monophosphate (cAMP) in response to stimulation of adenylate cyclase by forskolin and inhibition of phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX). A linear relation with a positive correlation coefficient greater than 0.98 was found between: (1) amount of cAMP and number of ganglia; (2) amount of protein and number of ganglia; (3) amount of DNA and amount of protein; (4) amount of DNA and number of ganglia. Basal levels of cAMP were 2.25 +/- 0.21 fmol per ganglion for 900 ganglia. Forskolin stimulated a dose-dependent increase in cAMP over a concentration range of 0.05 to 50 microM, with a level of 18.6 +/- 4.9 fmol/ganglion at 50 microM forskolin. The inactive forskolin analog 1,9 dideoxyforskolin did not elevate cAMP. Addition of IBMX to the incubation medium stimulated a dose-dependent increase in cAMP over a concentration range of 0.1 1000 microM, with a level of 17.58 +/- 3.38 fmol/ganglion at 1000 microM IBMX. Application of 1 mM IBMX strongly potentiated the stimulating action of forskolin on cAMP levels. Our results derived from direct determination of cAMP changes in small intestinal myenteric ganglia are consistent with existing electrophysiological evidence for second messenger function of cAMP in slow synaptic modulation of excitability in AH/Type 2 neurons of the enteric nervous system. PMID- 1713167 TI - Choleragenoid horseradish peroxidase used for studying projections of some hindlimb cutaneous nerves and plantar foot afferents to the dorsal horn and Clarke's column in the rat. AB - The aim of the present study has been to investigate the spinal projections of cutaneous hindlimb afferents particularly to the deep dorsal horn and to Clarke's column (CC), by using the B-subunit of cholera toxin conjugated to horseradish peroxidase. Injections into three different cutaneous hindlimb nerves in adult rats resulted in dense labeling in the dorsal horn laminae IIi-IV/V, moderate labeling in lamina I and modest labeling in dorsomedial parts of CC. Footpad injections gave similar results, except for a lack of labeling in CC and only weak labeling in laminae I and V. The results suggest that B-HRP should be a useful marker for studying cutaneous myelinated nerve fiber projections to the rat spinal cord. PMID- 1713168 TI - Topographic relationship between sagittal Purkinje cell bands revealed by a monoclonal antibody to zebrin I and spinocerebellar projections arising from the central cervical nucleus in the rat. AB - We have examined the topographic relationship between the sagittal bands of zebrin I immunoreactive Purkinje cells revealed by a monoclonal antibody, mabQ113, and the distribution of spinocerebellar fibers originating from the central cervical nucleus in the rat. The mossy fiber terminals were anterogradely labeled following injections of cholera toxin subunit B into the C1-C3 segments and visualized immunohistochemically. Zebrin I positive Purkinje cells appeared in seven sagittal bands (P1+ to P7+ bands). In lobules I-V of the anterior lobe, labeled mossy fiber terminals were distributed in the midline region, subjacent to the P1+ bands and at around 0.5 mm from the midline region, subjacent to the P2+ band in the lateral A1 to the medial A2 zones of Voogd et al. (1985). Labeled terminals were seen in the entire B zone and those distributed in its medial part were related to the P3+ band. In lobule VIII, labeled terminals were seen subjacent to the P1+, P2+ and P3+ bands, which were located in the lateral A1-A3 (or B) zones. In the copula pyramidis, labeled terminals appeared subjacent to the P4+, P5+ and the P6+ bands in the C1 and C2 zones (or the C1-C3 zones). Although the labeled terminals were seen beneath the zebrin I positive bands, the borders of terminal distribution were not well-delineated, and did not respect the borders of zebrin I positive bands. PMID- 1713170 TI - Ca2+ modulates an unspecific cation conductance in olfactory cilia of Xenopus laevis. AB - Olfactory neurones of Xenopus laevis were studied by the patch clamp technique under voltage-clamp conditions. Isolated receptor cells were obtained by dissociating the olfactory mucosa in a Ca(2+)-free solution. Usually some of the resulting isolated olfactory cells lost all of their cilia during the dissociation procedure. Comparing the currents of cells with cilia to those of cells without cilia, a marked difference was found. When all known voltage-gated currents except the Ca(2+)-current were blocked, cells without cilia showed the voltage-gated Ca(2+)-current alone whereas cells with cilia clearly had an additional conductance gc. It could be activated in two ways, either by Ca2+ entry through Ca(2+)-channels or by Ca2+ entry through the Na/Ca-exchanger working in the reversed mode at positive membrane potentials. This ciliar conductance gc had its reversal potential at 0 mV. Replacing extracellular Cl- by isethionate on the one hand, and Na+ by Cs+ or N-methyl-D-glucamine on the other showed that gc was permeable for cations but not for Cl-. In conclusion, there appears to be a Ca(2+)-dependent unselective cation conductance on the cilia of olfactory neurones. The probable role of gc as the last step an IP3/Ca mediated transduction pathway is suggested. PMID- 1713169 TI - Interactions between callosal, thalamic and associational projections to the visual cortex of the developing rat. AB - The patterns of callosal interconnections between the visual cortices of rats display considerable plasticity in response to various neonatal manipulations. In the present study, many neurones in the principal visual thalamic relay nuclei, the dorsal lateral geniculate nucleus (DLG) and to a lesser extent those in the lateral posterior nucleus (LP) were destroyed by injections of the neurotoxin - kainic acid - on the first day of postnatal life. Four weeks later, as demonstrated with the anterograde and retrograde transport of the enzyme horseradish peroxidase (HRP) injected into the occipital lobe of one hemisphere, callosally projecting neurones and terminals were distributed more widely in the retinotopically organized areas 17, 18a and 18b of the visual cortex ipsilateral to the lesioned visual thalamus than in unoperated control animals of the same age. By contrast, in the visual cortex contralateral to the lesioned visual thalamus the areal distribution of callosally projecting neurones and terminals was similar to that of the controls, that is, largely but not exclusively restricted to the common border of areas 17 and 18a. Both in unoperated and operated animals, cells in lamina V of several cytoarchitectonically defined areas that are not retinotopically organized (area 8 in the frontal lobe, area 29d in the retrosplenial limbic cortex and perirhinal areas 35/13 in the temporal lobe) also project to contralateral visual cortices. In areas 8 and 29d, the total numbers, laminar distributions and densities of labelled callosal cells both ipsilateral and contralateral to the kainate-injected visual thalamus were similar to those in the controls. However, in the temporal lobe, the areal distribution of the labelled callosal neurones was more extensive than that in the controls and labelled cells in areas 35/13 of the cortex contralateral to the kainate-lesioned visual thalamus merged with those in the neighbouring areas 20 and 36. By contrast, the areal distribution of associational neurones in area 18a and in nonretinotopically organized areas projecting to area 17 were very similar in controls and in operated animals (neonatal kainate lesion of the visual thalamus, neonatal section of the corpus callosum or both procedures combined). However, in operated animals, the labelled associational neurones projecting from the supragranular laminae (II/III) of area 18a to area 17 constituted a higher proportion of all cells than did those in the unoperated control animals. Thus, overall the number of associational neurones projecting from area 18a to area 17 was slightly increased by the experimental manipulations performed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713172 TI - Alveolar accumulation of hyaluronan and alveolar cellular response in bleomycin induced alveolitis. AB - Hyaluronan (HA) accumulating in the alveolar interstitial tissue of rats injured by a single intratracheal instillation of bleomycin has been visualized histologically and assayed. HA was present already by Day 1 after bleomycin treatment, increased to a maximum value on Days 3 and 7 and then declined. A time dependent relationship between this early connective tissue response and the invasion of inflammatory cells in the alveolar tissue was apparent. The dominating invading cells by Day 1 were granulocytes showing positive staining for the monoclonal antibody OX-42 reflecting the C3b receptor. The numbers of macrophages expressing class II antigens started to increase on Day 1, reaching a maximum on Days 3-7 and then declined. Macrophages were the dominating OX-42+ cells by Day 7. The appearance of W3/13+ cells ("pan-T-lymphocytes") showed a similar pattern to that for the class II expressing macrophages. The number of cells expressing CD4 antigen increased until Day 3 and levelled off on Day 30 whilst the largest number of cells expressing CD8 antigen was seen on Day 30. Few cells expressing B-cell phenotype outside lymph nodules were identified. Alveolar lining epithelial cells, probably epithelial type II cells, expressed class II antigens by Days 3-14. The time-related accumulation of HA and the appearance of T-cells, macrophages and granulocytes expressing signs of activation suggests that these cells may be involved in the early connective tissue response of the lung injured by bleomycin. PMID- 1713173 TI - Search for a retroviral cause for sarcoidosis: no evidence from peripheral blood studies. AB - Twenty six patients with sarcoidosis of recent onset or with severe progressive disease were studied for evidence of retroviral infection. Peripheral blood mononuclear cells (PBMC) were cultured in vitro and stimulated with phytohaemagglutinin and interleukin-2. Induction of syncytia (SI) and production of reverse transcriptase (RT) were sought as indicators of possible retroviral infection. PBMC from two patients showed syncytia formation and in one of these two there was associated production of low levels of reverse transcriptase. The remaining patients showed neither RT nor SI activity. The predominantly negative results of this study indicate that sarcoidosis is unlikely to be of retroviral aetiology; however, cell populations from sites of active disease should be studied before drawing this conclusion. PMID- 1713171 TI - Morphological characterization of rat entorhinal neurons in vivo: soma-dendritic structure and axonal domains. AB - We used in vivo intracellular labeling with horseradish peroxidase in order to study the soma-dendritic morphology and axonal projections of rat entorhinal neurons. The cells responded to hippocampal stimulation with inhibitory postsynaptic potentials, and thus likely received direct or indirect hippocampal input. All cells (n = 24) showed extensive dendritic domains that extended in some cases for more than 1 mm. The dendrites of layer II neurons were largely restricted to layers I and II or layers I-III, while the dendrites of deeper cells could extend through all cortical layers. Computed 3D rotations showed that the basilar dendrites of deep pyramids extended roughly parallel to the cortical layering, and that they were mostly confined to the layer containing the soma and layers immediately adjacent. Total dendritic lengths averaged 9.8 mm +/- 3.8 (SD), and ranged from 5 mm to more than 18 mm. Axonal processes could be visualized in 21 cells. Most of these showed axonal branching within the entorhinal cortex, sometimes extensive. Efferent axonal domains were reconstructed in detail in 3 layer II stellate cells. All 3 projected axons across the subicular complex to the dentate gyrus. One of these cells showed an extensive net-like axonal domain that also projected to several other structures, including the hippocampus proper, subicular complex, and the amygdalo-piriform transition area. The axons of layer III and IV cells projected to the angular bundle, where they continued in a rostral direction. In contrast to the layer II, III and IV cells, no efferent axonal branches leaving the entorhinal cortex could be visualized in 5 layer V neurons. The data indicate that entorhinal neurons can integrate input from a considerable volume of entorhinal cortex by virtue of their extensive dendritic domains, and provide a further basis for specifying the layers in which cells receive synaptic input. The extensive axonal branching pattern seen in most of the cells would support divergent propagation of their activity. PMID- 1713174 TI - The control of interferon-inducible gene expression. AB - The alpha beta interferons (IFNs) transiently induce genes through an IFN stimulable DNA response element (ISRE). IFN-cell surface receptor interaction triggers the cytoplasmic activation of the complex primary transcription factor E, which on translocation and interaction with the ISRE initiates transcription. Whether E is activated directly through the receptor(s) or through a more classical second message pathway(s) and the roles of additional factors in the alpha beta and gamma IFN responses remain to be established. Meanwhile analysis of mutants has revealed complexity and overlap in the alpha, beta and gamma IFN response pathways and the products of at least two viruses have been shown to inhibit IFN-inducible gene expression. PMID- 1713175 TI - Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1. AB - Plasmin is shown to specifically cleave vitronectin at the Arg361-Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-1 inhibitor of plasminogen activator (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI-1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event. PMID- 1713176 TI - [The role of histamine in neurogenic inflammations of the mouth mucosa in rats]. AB - The effect mechanism of neurogen inflammation incited by local capsaicin irritation was examined by the authors in the oral soft tissue of rats subsequent to antihistamin pretreatment. Furthermore, with the aid of muscarin blocking receptor also the parasympathetic component of the vasodilatation was examined. On basis of their results it was established that the vein-wall-permeability and microcircular changes observed in the course of the neurogen inflammation of the oral soft tissue partly expanded by the histamin through the H1-receptors. The H2 receptors as well as the parasympathetic fibers do not become activated in the process. PMID- 1713178 TI - [The effect of the experimental modulation of hypothalamic function on the development of the immune process]. AB - The organism immunological reactivity can be modulated through hypothalamic structures by means of patterns reproducing their activity during a developed immune response in rabbits. The partial modulation of the pattern specific for 4 5 days of developed immune response, was found to be the most efficient in changing the organism reactivity. PMID- 1713177 TI - [Structural effects in the sarcolemma of the cardiomyocytes under the action of modulators of the polyphosphoinositide system]. AB - The influence of activators (Ca(2+)-channels stimulator BAY K 8644 and phospholipase C stimulator fMLP) and inhibitors (Ca(2+)-channels blocker verapamil and alpha 1-adrenoceptors blocker prazosin) of the polyphosphoinositide system on cardiomyocytes sarcolemma, was studied. The structural effects of the PPI modifiers seem to be one of the mechanisms of the PPI physiological activity at the membrane level. PMID- 1713179 TI - Cystic adenomatoid malformation of the lung: an obstetric and ultrasound perspective. AB - Between 1986 and 1990, seven women were found to have fetuses with an abnormal antenatal ultrasound appearance of the chest and lungs suggestive of congenital cystic adenomatoid malformation of the lung (CCAM). All were confirmed by pathology. Cystic spaces were only visible in those women with macrocystic disease (Stocker types 1 and 11) whilst increased echogenicity of affected lung tissue and mediastinal deviation were seen in all types. Cystic adenomatoid malformation of the lung was also found in one woman with a raised maternal serum alpha-fetoprotein. The appearances are distinctive, and should be sought in all cases of polyhydramnios, fetal ascites and raised maternal serum alpha fetoprotein. PMID- 1713180 TI - The ethics of palliative care. PMID- 1713181 TI - Serological relationships of the O antigens of Klebsiella pneumoniae O5, Escherichia coli O8 and a new O serotype of Serratia marcescens. AB - Klebsiella pneumoniae O5, Escherichia coli O8 and Serratia marcescens 3255 were shown to cross-react in both ELISA and immunoblotting. The cross-reaction appeared to be due to the O antigen of their lipopolysaccharide (LPS). In addition, there was evidence that the reactions of these strains with their homologous antisera were due, in part, to determinants other than O polysaccharide. PMID- 1713182 TI - Treatment decisions in end-stage bladder cancer. Bilingual liaison rounds. AB - A bilingual conference was held in order to assist a Spanish-speaking patient and her physicians in planning for her care. The patient spoke only Spanish and her oncologists were primarily English-speaking. Her daughter, who was bilingual, did not attend the conference. One of the authors translated and served as interpreter and liaison between the patient and her other physicians. The patient was severely depressed and terminally ill with end-stage bladder cancer. Her depression and hopelessness complicated her care. PMID- 1713184 TI - Palliation of malignant obstructive jaundice--surgery or stent? PMID- 1713183 TI - Localisation of hyaluronan in the human intestinal wall. AB - By using biotin labelled proteoglycan core protein and an avidin enzyme system, hyaluronan (hyaluronic acid) was visualised in specimens of human jejunum. Intense staining for hyaluronan was seen in the loose connective tissue of the villi and of lamina propria while the epithelial layer was unstained. The muscularis mucosae showed only faint staining. The accumulation of hyaluronan in the subepithelial layer of the jejunal mucosa indicates that the previously reported high jejunal secretion of hyaluronan is due to passive diffusion from the subepithelial interstitium. The physicochemical characteristics conferred by hyaluronan may be important for the villi function. PMID- 1713185 TI - Isolation of tooth pulp cells for sex chromatin studies in experimental dehydrated and cremated remains. AB - In experiments designed to assess sex chromatin in artificially mummified and heated pulp tissue, a method was devised that successfully separates cells while minimizing nuclear damage. Sex chromatin (both Barr bodies and F-bodies) is shown to preserve in dehydrated human pulps up to one year. Human pulp tissue retains sex diagnostic characteristics when heated to 100 degrees C for up to 1 h. Parallel experiments on extracted teeth from young pigs reveals comparable tissue preservation. Heat penetration is retarded, however, in unextracted pig teeth in fleshed jaws such that temperatures could be raised to 300 degrees C for longer than 1 h. Heat penetration into fleshed material was further tested by the insertion of thermocouple probes to assess the temperature attained within the pulp chamber. At chamber temperatures up to 75 degrees C sex diagnosis in human pulps from extracted teeth was still possible. In outdoor incineration of fleshed pigs' heads in an open fire, 75 degrees C in the pulp chamber was reached at a fire temperature within the range 500-700 degrees C. The implications of these findings for forensic situations are described. PMID- 1713186 TI - [Surgical indications in ulcerative colitis and Crohn disease]. AB - Despite the progress made in recent years, treatment of chronic inflammatory bowel disease remains problematic. The highly individual spontaneous course of such conditions always makes an interdisciplinary management strategy mandatory. In view of the fact that continence-preserving procedures are possible in ulcerative colitis as well as bowel-preserving procedures in Crohn's disease, elective interventions in patients who fail to respond to what is accepted as suitable medication may be considered supplementary to the therapeutic spectrum. A consideration of all the factors involved suggests that too many reservations vis-a-vis surgery are no longer justified. PMID- 1713187 TI - Accelerated tumor growth and changes in images concomitant with vascularization in a patient with hepatocellular carcinoma. AB - We examined serial changes of tumor images in a patient with hepatocellular carcinoma. The hepatocellular carcinoma was initially detected as a homogeneous low-echo area with unclear margins, which was not enhanced by contrast media on computed tomograms, and did not reveal any vascular abnormalities on hepatic angiography. About 11 months later, the tumor growth accelerated, with a parallel increase in serum alpha-fetoprotein levels, and the ultrasonographic features of the tumor changed from a homogeneous low-echo area to a mixed low- and high-echo area with a peripheral low-echo zone. Hepatic angiography revealed a hypervascular tumor at this time. The present case indicates that tumor growth and imaging patterns of hepatocellular carcinoma are closely related to vascularization of the tumor. PMID- 1713188 TI - [Suppression of monocyte-macrophage activation by anti-CD4 therapy in patients with chronic polyarthritis]. AB - Monoclonal anti-CD4 antibodies have been introduced into the treatment of rheumatoid arthritis. A depletion of CD4+ T-cells down to 8% of the origin level (p less than 0.0001) followed the antibody application. Moreover, there was a significant reduction of blood monocytes to 30% (p less than 0.001). A reduced lymphocyte proliferation induced by antigens or mitogens was found in parallel. Prior to treatment, monocyte-macrophage activation in rheumatoid arthritis was signified by an increased expression of HLA-class II antigens and the CD14 antigen, and by an increased production of neopterin and interleukin-1. Anti-CD4 treatment resulted in a significant reduction of elevated levels of neopterin, beta 2-microglobulin in serum as well as IL-1 production in vitro. PMID- 1713189 TI - Do we need a pepton hypothesis? PMID- 1713190 TI - Positive correlation between oligonucleotide typing and T-cell recognition of HLA DP molecules. AB - The identification of 19 different HLA-DPB1 sequences implicates the existence of more DP specificities than can be typed for with cellular methods. How many of the DP beta sequences can be specifically recognized by T cells, and which of the polymorphic regions can contribute to the specificity of allorecognition, is not known. In order to investigate the distribution and the immunological relevance of recently described DPB1 alleles, we have typed a panel of 98 randomly selected Dutch Caucasoid donors for the HLA-DPB1 locus by oligonucleotide typing. Comparison of the typing results with primed lymphocyte typing (PLT) defined DP specificities shows an extremely good correlation. Moreover, additional alleles could be defined by oligonucleotide typing reducing the number of DP blanks in the panel. By selecting the appropriate responder stimulator combinations we were able to show that distinctive PLT reagents against oligonucleotide defined specificities DPB1*0401, DPB1*0402, DPB1*0901, and DPB1*1301 can be generated. To investigate in more detail which part of the DP molecule is responsible for the specificity of T-cell recognition, T-cell clones were generated against HLA-DPw3. The clones were tested for the recognition of stimulators carrying DPB1 alleles which had been defined by oligonucleotide typing and sequence analyses and which differed in a variable degree from DPB1*0301. The recognition patterns demonstrated that differences of one amino acid in polymorphic regions situated either in the beta sheets or alpha helix of the hypothetical model of the HLA class II molecule can eliminate T-cell recognition. Furthermore, sequence analyses revealed a new DPB1 allele designated DPB1*Oos. PMID- 1713193 TI - Macrophages produce nitrite, nitrate and nitrosamines after addition of catalase. AB - Mouse macrophages produced nitrite and N-nitrosomorpholine after incubation with catalase. A macrophage cell line, J774.1 (1 x 10(6) cells/ml), was incubated with catalase (500 U/ml) and morpholine (5 mM); after 48 h incubation at 37 degrees C, macrophages produced nitrite (100 microM) and N-nitrosomorpholine (1 microM). Stimulation of J774.1 cells with catalase enhanced interleukin-1 production and tumour-killing activity against mastocytoma P815 cells. Flow cytometric analysis showed that catalase was bound to the surface of the macrophages. PMID- 1713192 TI - Manufacture of a functional cDNA for the H-2Db molecule using a retroviral shuttle vector. PMID- 1713191 TI - Developmental and tissue-specific expression of the Q5k gene. AB - Expression of the Q5k gene was examined by northern blot analysis and polymerase chain reaction (PCR) in the AKR mouse and various cell lines, each of the H-2k haplotype. Our results show that Q5k mRNA is present during the whole postimplantational development of the AKR embryo/fetus (gestation day 6 to 15). In the juvenile mouse (week 2 to 4) transcription of the Q5k gene persisted in all organs examined. In contrast, in the adult animal expression of the Q5k gene was limited to the thymus and uterus of the pregnant mouse. Upon malignant transformation, the amount of Q5k-specific mRNA increased dramatically in thymus and could also be observed in the spleen of thymoma bearing animals. Expression of the Q5k gene was also detectable in several transformed mouse cell lines. Mitogen stimulation or treatment with cytokines induced Q5k expression in primary spleen cell cultures. A possible explanation for the tissue-restricted expression in the adult AKR mouse is discussed. PMID- 1713194 TI - Newly synthesized dithiocarbamates inhibit the metabolism and toxicity of N nitrosodimethylamine. AB - Several dithiocarbamates (DTC) of secondary amines and secondary amino acids were tested for their stability in aqueous solution and for their effect on nitrosamine metabolizing enzymes and on the acute toxicity of N nitrosodimethylamine (NDMA) in rats. The following results were found: (i) All DTC tested were stable in alkaline solution; in acidic milieu, only DTC derived from secondary amino acids were moderately stable. (ii) The activity of NDMA demethylase in rat liver microsomes was inhibited completely by all DTC tested. (iii) The excretion of unmetabolized NDMA in rat urine over 24 h increased from 0.1% without pretreatment to 3.6% of the given NDMA dose when combined with a single dose of DTC. (iv) The acute toxicity of NDMA was reduced by dihydroxyethyldithiocarbamate; when the sulfur compound was administered simultaneously and 24 h after the nitrosamine, lethality was almost completely inhibited. (v) The stability of a compound in aqueous solutions did not affect its activity in the enzyme tests. PMID- 1713195 TI - Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease. AB - The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease. PMID- 1713196 TI - Immunological characterization of recombinant antigens isolated from a Mycobacterium avium lambda gt11 expression library by using monoclonal antibody probes. AB - Nontuberculous mycobacteria, particularly Mycobacterium avium, have been isolated from a significant percentage of patients with AIDS. Early detection of M. avium infection is difficult, and treatment regimens are often ineffective. Much needs to be learned about antigens and factors responsible for immunity to and pathogenesis of the disease. Specific antigens and diagnostic procedures for infection need to be developed. To address some of these problems, we have generated 25 different monoclonal antibodies against a serovar 4 strain of M. avium isolated from a patient with AIDS. Protease sensitivity studies have demonstrated that each of these antibodies recognizes a protein-associated epitope. Immunoblot analyses suggest that seven of these monoclonal antibodies react specifically with M. avium and M. intracellular epitopes. Immunoreactive bacteriophages were identified from an M. avium lambda gt11 expression library with two of these monoclonal antibodies (3808 C3 and 3954 B12). Lambda lysogens, generated from the immunoreactive bacteriophages, overproduced beta-galactosidase fusion proteins which were reactive with the two monoclonal antibodies in immunoblot assays. The purified fusion proteins were shown to elicit skin test reactions in sensitized guinea pigs. PMID- 1713197 TI - Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms. AB - Infections caused by Haemophilus influenzae are a major worldwide health problem. In particular, nontypeable strains of H. influenzae are a common cause of otitis media in infants and children. A vaccine to prevent these infections would result in the prevention of substantial morbidity and cost savings. A problem in identifying an appropriate vaccine antigen has been the enormous antigenic heterogeneity among nontypeable strains of H. influenzae. The present study was undertaken to characterize the conservation of the P6 outer membrane protein (approximately 16,000 daltons) among strains of H. influenzae. A total of 20 type b strains and 20 nontypeable strains of diverse geographic and clinical origins was studied. Three approaches were taken. (i) Antigenic determinants recognized by monoclonal and polyclonal antibodies were present on P6 in all 40 strains tested. The molecular weight of P6 was identical in all strains. (ii) Comparison of the DNA sequences of the P6 genes from three epidemiologically and serologically unrelated strains demonstrated 100% homology at the amino acid level and 97 to 99% homology at the nucleotide level. (iii) Restriction fragment length polymorphism analysis demonstrated that the P6 gene and flanking sequences were highly conserved among all strains. These three independent series of experiments indicated that the P6 protein is highly conserved among strains of H. influenzae. P6 should receive serious consideration for inclusion in a vaccine to prevent infections caused by nontypeable H. influenzae. PMID- 1713198 TI - Human Mycobacterium tuberculosis-reactive CD4+ T-cell clones: heterogeneity in antigen recognition, cytokine production, and cytotoxicity for mononuclear phagocytes. AB - CD4+ T cells regulate the protective immune response which follows exposure to Mycobacterium tuberculosis by activating macrophages through the cytokines the CD4+ T cells secrete. In addition CD4+ T cells have been shown to be directly cytotoxic for antigen-pulsed mononuclear phagocytes (monocytes-macrophages). To explore the functional interaction between mycobacterial antigen-specific CD4+ T cells and mononuclear phagocytes further, CD4+ T-cell clones were derived from healthy purified protein derivative-positive individuals. Five T-cell clones were selected for detailed analysis. None responded to the purified recombinant or native mycobacterial antigens of 14, 19, 65, 71, and 30 (alpha-antigen/Ag6) kDa. However, the T-cell clones demonstrated heterogeneity in antigen recognition as measured by their Western blot (immunoblot) responses. Some T-cell clones made only interleukin 2, while others made only interleukin 4; all produced gamma interferon, although in differing amounts. Four of five T-cells clones were cytotoxic for purified protein derivative-pulsed monocytes at 1:1 and 10:1 effector-target cell ratios. When monocytes infected with live M. tuberculosis were used as targets, comparable levels of cytotoxicity were observed. The cytotoxicity was major histocompatibility complex class II restricted and inhibited by antibodies to ICAM-1 and LFA-1 and not by antibodies to tumor necrosis factor alpha, lymphotoxin, and gamma interferon. Cytotoxicity by CD4+ T cells for monocytes pulsed with mycobacterial antigens or infected with live M. tuberculosis is a common property of these cells and appears to be independent of the repertoire of lymphokines produced and not limited to recognition of defined mycobacterial heat shock proteins. Lysis of heavily infected mononuclear phagocytes may be one manner in which CD4+ T cells regulate host immune response to M. tuberculosis. PMID- 1713199 TI - Recognition of tachyzoite and bradyzoite antigens of Toxoplasma gondii by infected hosts. AB - Western immunoblots of tachyzoite and bradyzoite antigens of Toxoplasma gondii were probed with antisera from rabbits and mice at intervals between 2 and 8 weeks after infection and with human antisera with various titers of antibody. With rabbit and mouse antisera, two groups of antigens with molecular masses of 54 to 63 kDa (designated 58.5-kDa antigens) and 26 to 29 kDa (designated 27.5-kDa antigens) were demonstrated commonly for both stages, while those antisera reacted strongly with tachyzoite (but not bradyzoite) antigens with molecular masses of 29 to 54 kDa. Tachyzoite antigens of 21.5, 26.5, 31, 38, 40, 49, and 58 kDa reacted with antisera 2 to 4 weeks after infection, while bradyzoite antigens of 27, 51, 220, and 290 kDa reacted with antisera obtained 4 or more weeks after infection. The 58.5-kDa antigens of both stages reacted primarily with human antisera that had low titers of anti-T. gondii antibodies. Human (as well as rabbit and mouse) sera with high antibody titers reacted with the 27.5-kDa antigens as well as the 58.5-kDa antigens, but the reactivity of the 27.5- to 58.5-kDa antigens of tachyzoites was greater than that of bradyzoites. PMID- 1713200 TI - Potentiation of human natural killer cell cytotoxicity by Salmonella bacteria is an interferon- and interleukin-2-independent process that utilizes CD2 and CD18 structures in the effector phase. AB - Incubation of large granular lymphocytes (LGL) with glutaraldehyde-fixed bacteria stimulated in the supernatant the production of interferon (IFN), which proved to be mainly IFN-gamma. Even though IFN-gamma was produced upon exposure of LGL to bacteria, anti-IFN-gamma antibodies failed to interfere with induction of cytotoxicity by bacterial contact. Anti-IFN-gamma receptor antibodies had no effect on the induction of activated killing by bacterial contact either. We also tested the effect of anti-IFN-alpha antibody, but it failed to interfere with induction of cytotoxicity by bacterial contact. No interleukin-2 (IL-2) was detected in the culture supernatant of bacterially activated LGL by the mouse HT2 cell assay, nor did we detect any IL-2 mRNA in bacterially activated LGL by Northern RNA blot assay. Neutralizing anti-IL-2 antiserum had no effect on the induction of activated killing by bacterial contact, and recombinant IL-4 did not interfere with the induction of activated killing. We then studied the membrane structures involved in bacterially activated killing. Anti-CD18 monoclonal antibody did not interfere with the induction phase of bacterially activated killing. However, both anti-CD18 and anti-CD2 antibodies inhibited the effector phase of bacterially activated killing. The effector pathways utilized by activated LGL depended on the mode of activation in that even though bacterially activated LGL were sometimes blocked by anti-CD2 monoclonal antibody, recombinant IL-2-stimulated LGL were not. In conclusion, our present results suggest that there may be mediators other than exogenously secreted IFNs and IL-2 which are responsible for the induction of activated killing after bacterial contact. CD18 and CD2 structures were shown to be involved in the effector phase of bacterially activated killing. PMID- 1713201 TI - Analysis of immune responses of different hosts to Babesia divergens isolates from different geographic areas and capacity of culture-derived exoantigens to induce efficient cross-protection. AB - The immunoprecipitation of [35S]methionine-radiolabelled antigens from different Babesia divergens isolates by using bovine, gerbil, and human immune sera has shown that many B. divergens proteins contain epitopes shared between isolates. The cross-protective capacity of culture-derived soluble immunogens from the B. divergens Rouen 1987 isolate was tested against different B. divergens isolates. Results showed complete protection against the 7107b French isolate and substantial protection against the Weybridge 8843 English isolate (80% protection) and the Munich 87 German isolate (60% protection). In order to explain these vaccination results and to assess both the common and variable antigenicity of B. divergens, the antigenic patterns of the challenge isolates (Rouen 1987, 7107b, Weybridge 8843, and Munich 87) were compared by immunoprecipitation, using gerbil antisera raised against the Rouen 1987 vaccine isolate. Differences in the antigenic patterns and in the cross-protection of gerbils in these heterologous challenges were examined by studying the virulence and the antigenic status of each isolate. PMID- 1713202 TI - In vitro neutralization of Chlamydia trachomatis by monovalent Fab antibody specific to the major outer membrane protein. AB - Monovalent Fab antibodies to serovar- and subspecies-specific epitopes of the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized infectivity for hamster kidney cells by preventing chlamydial attachment. These findings exclude the aggregation of chlamydiae as a mechanism of anti-MOMP neutralization and provide additional evidence in support of the MOMP as a chlamydial adhesin. PMID- 1713203 TI - Heterogeneity in the immunolocalization of cytokeratin specific monoclonal antibodies in the rat eye: evaluation of unusual epithelial tissue entities. AB - The immunocytochemical localization of cytokeratin and vimentin in rat eye tissues was investigated using a panel of 39 monoclonal antibodies specific for single or multiple of cytokeratin polypeptides and one polyclonal anti CK20 antiserum. The retinal and the ciliary body pigment epithelial only expressed cytokeratins 8 and 18, whereas the fetal retinal pigment epithelium and focally the adult epithelium, in the transition zone of retina and ciliary body, exhibited a reactivity for cytokeratin 19. In contrast, the non-pigmented ciliary epithelium was positive for vimentin only. In the rat conjunctiva distributed goblet cell clusters were selectively stained with cytokeratin 7, 8, 18 and 19 specific monoclonal antibodies. Among them a group of cytokeratin 8 and 18 specific monoclonal antibodies which stained the goblet cells as well as cytokeratin 8 and 18 positive internal controls did not react with either the cytokeratin 8 and 18 positive neuroectodermal cells of the rat eye nor the rat choroid plexus epithelium. This indicates differences in the phenotype e.g. conformational epitope changes, of neuroectodermal derived and other cytokeratins. The corneal and conjunctival epithelium showed a more complex distribution of squamous epithelium type cytokeratins. The limbal region as a transient zone connecting both epithelia exhibited a changing cytokeratin pattern. In general, the study emphasized the necessity to work with an enlarged antibody panel to avoid misleading results in the immunolocalization of cytokeratins. PMID- 1713204 TI - HIV infection of human gastrointestinal submucosal cells: an in vitro model that mimics Kaposi's sarcoma. AB - Infection of human gastrointestinal submucosal mesenchymal cells with HIV-1 led to cell populations with abnormal growth properties, increased synthesis of endothelial cell and angioblast markers, and release of angiogenic factors. This system may be the first in vitro model for HIV-induced Kaposi's sarcoma. PMID- 1713205 TI - Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA. AB - The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serratia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants. PMID- 1713206 TI - Transcriptional attenuation control of ermK, a macrolide-lincosamide streptogramin B resistance determinant from Bacillus licheniformis. AB - ermK instructs bacteria to synthesize an erythromycin-inducible 23S rRNA methylase that confers resistance to the macrolide, lincosamide, and streptogramin B antibiotics. Expression of ermK is regulated by transcriptional attenuation, in contrast to other inducible erm genes, previously described, which are regulated translationally. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino-acid leader peptide together with its ribosome binding site. Additionally, the mRNA leader sequence can fold in either of two mutually exclusive conformations, one of which is postulated to form in the absence of induction and to contain two rho factor-independent terminators. Truncated transcription products ca. 210 and 333 nucleotides long were synthesized in the absence of induction, both in vivo and in vitro, as predicted by the transcriptional attenuation model; run-off transcription in vitro with rITP favored the synthesis of the full-length run-off transcript over that of the 210- and 333-nucleotide truncated products. Northern (RNA) blot analysis of transcripts synthesized in vivo in the absence of erythromycin indicated that transcription terminated at either of the two inverted complementary repeat sequences in the leader that were postulated to serve as rho factor-independent terminators; moreover, no full-length transcripts were detectable in the uninduced samples. In contrast, full-length (ca. 1,200 nucleotide) transcripts were only detected in RNA samples synthesized in vivo in the presence of erythromycin. Full-length transcripts formed in the absence of induction from transcriptional readthrough past the two proposed transcription terminators would fold in a way that would sequester the ribosome binding site together with the first two codons of the ErmK methylase, reducing its efficiency in translation. This feature could therefore provide additional control of expression in the absence of induction; however, such regulation, if operative, would act only secondarily, both in time and place, relative to transcriptional control. Analysis by reverse transcriptase mapping of in vivo transcripts from two primers that bracket the transcription terminator responsible for the 210 nucleotide truncated fragment supports the transcriptional attenuation model proposed and suggests further that the synthesis of the ermK message is initiated constitutively upstream of the proposed terminator but completed inductively downstream of this site. PMID- 1713207 TI - Structural analyses of a channel-forming fragment of colicin E1 incorporated into lipid vesicles. Fourier-transform infrared and tryptophan fluorescence studies. AB - Structural changes upon binding to the membrane of a COOH-terminal channel forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles. PMID- 1713208 TI - Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells. AB - A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-, beta-, gamma , and delta-subunit cDNAs plus the neor gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single approximately 9 S peak on sucrose gradients, and have the same ratio of alpha- to beta-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of alpha-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are alpha 2 beta gamma delta pentamers. PMID- 1713209 TI - Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl terminal nonapeptide. Evidence for similar post-translational processing in brain and gut. AB - An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK) 33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues. PMID- 1713210 TI - Epidermal growth factor, a modulator of luteal adenylate cyclase. Characterization of epidermal growth factor receptors and its interaction with adenylate cyclase system in bovine luteal cell membrane. AB - The epidermal growth factor (EGF) binding sites on bovine luteal cell membrane have been characterized in detail, and evidence has been obtained for a direct stimulatory effect of EGF on membrane-associated adenylate cyclase activity. The membrane fraction prepared showed the presence of high affinity (Ka = 1.2 +/- 0.7 x 10(-11) M-1), specific, and saturable EGF receptors of Mr = 170,000. The EGF receptors underwent rapid autophosphorylation and down-regulation following treatment of the cells with EGF. Treatment of the cells with 4 beta-phorbol 12 myristate 13-acetate resulted in a diminished binding of 125I-EGF to the receptors. When luteal cells were preincubated with EGF, both basal and forskolin stimulated adenylate cyclase activity was increased severalfold. This enhancement of the adenylate cyclase activity was dependent upon the duration of the exposure to EGF and on the concentration of the growth factor. An optimal enhancement was observed when the cells were preincubated with 10 ng/ml EGF for 10-15 min. Furthermore, when the membrane fraction prepared from luteal cells was preincubated in vitro with EGF, a similar dose-related and time-dependent increase in basal, as well as forskolin-stimulated, adenylate cyclase activity was observed. These results demonstrate that luteal cell adenylate cyclase activity is finely regulated by EGF. Such a direct interaction between EGF and membrane-associated adenylate cyclase has not been previously recognized. PMID- 1713211 TI - Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. AB - The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma. PMID- 1713212 TI - Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors. AB - The molecular basis for binding of alpha-macroglobulin-proteinase complexes to the human two-chain 500/85-kDa (alpha/beta) alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein was analyzed. Ligand blotting experiments showed that a 40-kDa protein, present in the affinity-purified alpha 2MR preparation, is bound to the alpha 2MR alpha chain and released by heparin. Removal of the 40-kDa protein resulted in a 3-5 fold increase in binding of alpha 2M-trypsin. Nitrocellulose-immobilized pure two chain alpha 2MR was incubated with human alpha 2M-trypsin, containing four identical subunits, and two monovalent ligands: rat alpha 1-inhibitor-3 chymotrypsin and the 18-kDa receptor binding fragment of the alpha 2M subunit. Binding of alpha 2M-trypsin to the alpha-chain of immobilized alpha 2MR was composed of a high (Kd = 40 pM at 4 degrees C) and a low (Kd = 2 nM) affinity component. alpha 1-Inhibitor-3-chymotrypsin bound to the same sites but with one component (Kd = 0.4 nM). Competition-inhibition experiments and dissociation experiments, using ligands with different valences, as well as experiments with alpha 2MR immobilized at different densities, led to the following model. The low (Kd = 2 nM) affinity of alpha 2M-proteinase is prevalent when only one of the four domains binds to alpha 2MR, i.e. when the receptor density is low or when neighboring receptors are occupied. The high (Kd = 40 pM) affinity is achieved by binding of at least two domains to adjacent receptors. PMID- 1713213 TI - Recombinant human pim-1 protein exhibits serine/threonine kinase activity. AB - The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity. PMID- 1713214 TI - Dissociation of platelet-derived growth factor (PDGF) receptor autophosphorylation from other PDGF-mediated second messenger events. AB - Activated p21ras alters the platelet-derived growth factor (PDGF) signal transduction pathway in fibroblasts by inhibiting autophosphorylation of the receptor as well as by inhibiting the induction of the growth-related genes c myc, c-fos, and JE. To elucidate the cause and effect relationships between receptor autophosphorylation and other second messenger events in the PDGF signaling pathway we created revertants of v-ras transformed cells by two methods: 1) the use of cAMP analogues, and 2) the introduction of a gene, Krev-1, which has been reported previously to revert ras transformed cells to normal morphology. Analysis of the revertants shows that the PDGF-mediated tyrosine phosphorylation of the 180-kDa PDGF receptor remains inhibited; however, the PDGF mediated activation of phospholipase C and the induction of the growth-related genes c-myc, c-fos, and JE have been restored. These data suggest the presence of parallel pathways for PDGF signal transduction which are not dependent on autophosphorylation of the PDGF receptor. PMID- 1713215 TI - Structural analysis of rod GTP-binding protein, Gt. Limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibody epitope. AB - The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489 496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23 and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t. PMID- 1713216 TI - Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. AB - Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template primer complex was determined from steady state kinetics and enzyme-template primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides. PMID- 1713217 TI - Parotid salivary secretion in diabetic autonomic neuropathy. AB - Parotid salivary flow rates and amylase concentrations were measured in three groups of eight subjects each (normal control, non-neuropathic diabetic, and neuropathic diabetic). Flow rates were significantly reduced in neuropathic diabetic patients as compared with normal controls (p less than 0.001) and non neuropathic diabetic patients (p less than 0.02). Amylase concentrations were similar. These data are consistent with parasympathetic denervation of the parotid gland in diabetic neuropathy and provide evidence for a widespread distribution of autonomic denervation in diabetes. PMID- 1713218 TI - Plasma free triiodothyronine response to thyrotropin-releasing hormone to predict the remission of Graves' disease treated with antithyroid drugs. AB - The responses of both plasma TSH and free T3 (FT3) to TRH were examined in 31 patients with Graves' disease who were euthyroid after treatment with antithyroid drugs, 6 patients with primary hypothyroidism, and 14 control subjects. TSH was measured 0, 15, 30, 60, 90, and 120 min and FT3 was measured 0, 30, 60, 90, 120, 150, and 180 min after TRH injection (500 microgram, iv). The increment in FT3 above the basal level (delta FT3) in normal controls ranged from 1.2-3.7 pmol/L, with a mean +/- SD of 2.2 +/- 0.8 pmol/L. The mean (+/- SD) delta FT3 in patients with primary hypothyroidism was 0.3 +/- 0.2 pmol/L. After the TRH test, antithyroid drugs were stopped in patients with Graves' disease. Nine of 31 Graves' patients relapsed within 6 months after the TRH test. The other 22 patients with Graves' disease were followed while in remission during the observation period of up to 48 months. The mean (+/- SD) delta FT3 were significantly lower in 9 Graves' patients who relapsed than in those who achieved remission (0.5 +/- 0.3 vs. 2.6 +/- 1.1 pmol/L; P less than 0.01). Eight of 9 Graves' patients who relapsed showed lower delta FT3 values than the lowest value (1.1 pmol/L) in 22 Graves' patients in remission. Although the mean increment of TSH above the basal level (delta TSH) was also significantly different between the Graves' patients who relapsed and those in remission (1.4 vs. 12.3 mU/L; P less than 0.01), there was considerable overlap between the 2 groups. These findings suggest that delta FT3 reflects the endocrinological recovery of the pituitary-thyroid axis and is a beneficial indicator for the termination of antithyroid drugs in Graves' disease. PMID- 1713219 TI - Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins in primary cultures of prostate epithelial cells. AB - Insulin-like growth factors (IGFs) are potent mitogens that bind with high affinity and specificity to IGF receptors and IGF-binding proteins (IGFBPs). We studied the roles of these three groups of proteins in prostate epithelial cells (PEC) in primary culture grown under serum-free conditions. Affinity cross linking of IGF-I and IGF-II to crude membranes prepared from PEC revealed an abundance of type 1 IGF receptors and no evidence of type 2 IGF receptors. Western ligand blots of conditioned media (CM) from PEC demonstrated the presence of two specific IGFBP bands similar to those previously demonstrated in seminal plasma, with approximate mol wt of 31 and 24 kDa. The 31-kDa band was immunoprecipitable with an antibody to IGFBP-2, and neither band could be deglycosylated with endoglycosidase-F. Northern blot analysis of poly(A)+ RNA prepared from PEC with cDNAs for hIGFBP-1, -2, and -3 documented the expression of mRNA for hIGFBP-2 only. Modifications of the serum-free conditions of PEC did not significantly alter the IGFBP profile of PEC CM. The ability of IGF-I, IGF II, and insulin to stimulate clonal growth of PEC was examined. IGF-I stimulated PEC growth with an ED50 of 0.1 ng/mL. IGF-II and insulin, respectively, were 1 and 3 orders of magnitude less effective than IGF-I in stimulating the growth of PEC. Radioimmunoassayable IGF-I and IGF-II levels in PEC CM were below the assay detection levels. In conclusion, we suggest that IGFs are important growth stimulators of PEC in culture, that their actions are mediated through the type 1 IGF receptor, and that PEC produce hIGFBP-2 and a 24-kDa IGFBP which may modulate IGF action in these cells. PMID- 1713220 TI - Octreotide stimulates insulin-like growth factor binding protein-1 (IGFBP-1) levels in acromegaly. AB - Insulin-like growth factors (IGFs) circulate in a complexed state with several binding proteins (BPs). Of these, IGFBP-1 is regulated by hormonal and nutritional factors. The somatostatin analogue, octreotide, has been used to effectively control hypersomaototropism in acromegaly. IGFBP-1 levels were measured by RIA in 17 acromegalic patients receiving octreotide. Serum hormone sampling was conducted hourly for 8 hr periods. Among 13 octreotide responders, mean pre-treatment basal GH, IGF-1, and IGFBP-1 levels were 19 +/- 5 micrograms/L, 1021 +/- 168 micrograms/L, and 36 +/- 8 micrograms/L respectively. One month following octreotide treatment, an acute subcutaneous injection (100 micrograms) maximally attenuated GH to 3 +/- 0.6 microgram/L (18% of control, P less than 0.03) and IGF-1 to 467 +/- 75 micrograms/L (46% of control, P less than 0.008) after 4 hrs. IGFBP-1 levels, however, were stimulated to 95 +/- 16 micrograms/L (297% of control, P less than 0.003) during the same time period. A significant increase in IGFBP-1 levels occurred within 2 hrs (158% of baseline, P less than 0.03), and was sustained until the 7th hr following injection. Insulin, a known suppressor of IGFBP-1, did not change during this time. Among the 4 octreotide non-responders, mean basal IGFBP-1 levels were 42 +/- 4 micrograms/L, and 4 hrs following octreotide administration IGFBP-1 was 40 +/- 7 micrograms/L. Octreotide induced a dynamic inverse relationship between circulating GH and IGFBP-1 levels (r = -0.73, P less than 0.001). The absence of IGFBP-1 changes in octreotide non-responders and the non-suppression of insulin in octreotide responsive patients, suggest a direct GH-mediated mechanism of IGFBP-1 regulation in octreotide treated patients with acromegaly. IGFBP-1 may be another useful marker in evaluating the response of acromegaly to octreotide treatment in patients who experience clinical benefit but equivocal GH and IGF-1 attenuation. PMID- 1713221 TI - Prealbumin in the diagnosis of bronchopulmonary carcinoid tumours. AB - The reliability of prealbumin as a diagnostic marker was studied in 60 cases of bronchopulmonary carcinoid tumours. There were differences in the incidence of positivity between typical and atypical carcinoids (well differentiated neuroendocrine carcinomas). Seventy five per cent of the carcinoid tumours were positive for prealbumin; (86.7% typical and 63.3% atypical carcinoids). In 15 cases, which were Grimelius negative, 10 were prealbumin positive. Only 8.3% carcinoids were negative with both prealbumin and Grimelius stains. Ten squamous, 10 adeno- and 10 small cell carcinomas showed only occasional scattered prealbumin positive cells. It is concluded that prealbumin is a useful marker for bronchopulmonary carcinoid tumours. It is cheap, readily available, and should be considered part of routine diagnostic procedures for the diagnosis of carcinoid tumours. PMID- 1713222 TI - Vessel associated adhesion molecules in normal skin and acute graft-versus-host disease. AB - Immunohistological staining of skin from normal donors and bone marrow transplant recipients was undertaken using antibodies to two vessel associated adhesion molecules, endothelial leucocyte adhesion molecule-1 (ELAM-1). In normal skin ELAM-1 staining was restricted to a variable but generally small number of endothelial cells which were significantly increased in graft-versus-host disease (GvHD), but only when the fully developed histological picture of epidermal basal damage and leucocytic infiltration was present. All other biopsy specimens from marrow recipients taken before or after transplantation were similar to those of normal controls even in the presence of a clinical rash consistent with early GvHD. Although VCAM-1 positivity was seen on a few endothelial cells in normal skin, staining was mainly observed on dermal dendritic cells surrounding blood vessels and adnexal structures. In specimens with histological evidence of GvHD, positive perivascular dendritic cells were increased and were accompanied by the appearance of large numbers of similar cells dispersed throughout the upper dermis. Biopsy specimens from marrow recipients before and after transplantation resembled those from normal donors except for the presence of a rash after transplantation when some specimens, which lacked the leucocytic infiltrate diagnostic of GvHD, showed an increase in VCAM-1 positive cells, particularly in the upper dermis. The identification of these cells may therefore be useful in diagnosing early GvHD. PMID- 1713223 TI - Use of leucocyte alkaline phosphatase (LAP) score in differentiating malignant from benign paraproteinaemias. AB - The leucocyte alkaline phosphatase (LAP) score of peripheral blood neutrophils was examined in 20 patients with multiple myeloma and compared with the score in 18 patients with monoclonal gammopathy of undetermined significance (MGUS). The mean (95% confidence limit) LAP score in those with multiple myeloma was 186 (169 218) compared with 92 (64-120) in the MGUS group. In the multiple myeloma group all but one patient had a high LAP score, irrespective of disease. No cause for raised LAP, such as infection, was present in any of the patients with multiple myeloma. In the MGUS group six patients had a raised LAP score; in two of them another cause for such a rise was present (autoimmune haemolytic anaemia and primary thrombocythaemia). In neither group did the LAP score correlate with duration of the disease, bone marrow plasma cell count, paraprotein concentration, haemoglobin, total white cell or neutrophil count. It is concluded that a normal LAP count in patients with paraproteinaemia suggests a benign condition, but a raised count does not indicate a malignant condition. PMID- 1713224 TI - Correlation analysis for clinical and gingival crevicular fluid parameters at anatomically related gingival sites. AB - This study was designed to evaluate the relationship of certain clinical and biochemical measures of periodontal pathology at anatomically related gingival sites. The maxillary first molar--second bicuspid region was studied in patients with gingivitis and periodontitis. The mesiobuccal site on the first molar was compared to the mesiopalatal and direct buccal sites on the molar and the distobuccal site on the second bicuspid. Probing depth, attachment level, gingival index, gingival crevicular fluid (GCF) volume, and GCF levels of the lysosomal enzyme B-glucuronidase (BG), the cytoplasmic enzyme lactate dehydrogenase, IgG and the protease-inhibitor alpha-2-macroglobulin were studied. For the 3 anatomical pairs that were analyzed, the correlation coefficients for the GCF constituents were generally higher than the correlations for the clinical parameters. The mean correlations for the GCF constituents were higher for the periodontitis patients as compared to the gingivitis patients. For the periodontitis patients, BG activity was correlated at adjacent proximal sites, approached significance at adjacent papillary sites, but was not significantly correlated at adjacent facial-proximal sites. This data suggests that sampling of BG activity from a mesiobuccal site provides information about the anterior papillary unit. In contrast, IgG in GCF collected from the mesiobuccal site on the first molar was significantly correlated with the total IgG in the 3 other sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713225 TI - Prenatal specification of callosal connections in rhesus monkey. AB - Anatomical tracing and quantitative techniques were used to examine the tempo and pattern of maturation for callosal projection neurons in the monkey prefrontal cortex (PFC) during fetal and postnatal development. Nineteen monkeys were injected with retrograde tracers (fluorescent dyes, horseradish peroxidase conjugated to wheat germ agglutinin [WGA-HRP] or HRP crystals) at various ages between embryonic day 82 (E82) and adulthood. The size of injection sites was varied in fetal, newborn, and adult cases. In adults, labeled neurons were found in greatest density in the homotopic cortex of the opposite hemisphere and considerable numbers were also observed in a constellation of heterotopic areas including the medial and lateral orbital cortex, the dorsomedial convexity, and the pregenual cortex. The majority of labeled neurons were consistently concentrated in the lower half of layer III in all areas. In cases with large injection sites, callosal neurons of layer III formed a continuous and uninterrupted band that extended over the entire lateral surface of the prefrontal cortex spanning both homotopic and heterotopic areas. In contrast, in cases with small injection sites, the labeling of layer III neurons exhibited discontinuities. Between embryonic ages E82 and E89, injections limited to the cortical layers labeled only a small number of neurons in the opposite hemisphere, indicating that few callosal axons have invaded the cortex by this age. However, by E111 comparable injections labeled a large number of callosal neurons and many features of their distribution were adult-like. The number and constellation of cytoarchitectonic areas that were labeled in the frontal cortex of the opposite hemisphere were the same as in adults and the majority of callosal neurons were found in supragranular layer III. Finally, in fetal animals beyond E111, labeled neurons extended as a nearly unbroken band over a wide expanse of the dorsolateral PFC, resembling the pattern seen in adult monkeys with large injections. The conclusion we draw from these results, together with our earlier findings (Schwartz and Goldman-Rakic: Nature 299:154, 1982), is that callosal neurons whose axons enter the cortical layers of the primate prefrontal cortex achieve their mature laminar and areal distribution prior to birth and do so largely by cumulative processes. PMID- 1713226 TI - Development of the laminar distribution of thalamocortical axons and corticothalamic cell bodies in the visual cortex of the wallaby. AB - The distribution of afferents from the dorsal lateral geniculate nucleus (LGNd) and the lateral posterior nucleus (LP) and of cell bodies projecting to these nuclei has been studied in the visual cortex of the wallaby (Macropus eugenii) throughout development to determine how the characteristic laminar distribution of afferents and efferents of the mature cortex is achieved. Young are born after 26-28 days of gestation and do not open their eyes until around 140 days after birth. Horseradish peroxidase conjugated to wheatgerm agglutinin was injected in the visual thalamus in adults and in pouch young aged from 22 days after birth, just after thalamic axons first reach the visual cortex, to 118 days, when cortical lamination resembles the adult. From 22 to 65 days, the developing visual cortex consists of a marginal zone (MZ), cortical plate (CP), and intermediate zone (IZ) including the superficial subplate (SP), subventricular zone, and ventricular zone. There is a thin compact cell zone (CCZ) at the top of the CP and below it a less densely packed region that increases in thickness with age. Retrogradely labelled cells in two bands were first seen at 40 days, one in the CCZ and the other at the base of the CP. Two bands of cells were seen at all subsequent times if the injection covered both LGNd and LP, and by 76 days, these cells were located within cytoarchitectonically recognizable layers V and VI. Anterograde label prior to 45 days was distributed densely and evenly throughout the IZ and the CP up to the CCZ. Label in MZ was first seen at 25 days and was substantial by 54 days. Anterograde label than became gradually reduced in the IZ, whereas in the CP it remained evenly and densely distributed until 82 days. At this age, coincident with the emergence of layer IV, label within the CP first showed variations in density and by 99 days was concentrated over layer IV and, to a lesser extent, over layer VI. By 118 days label resembled the adult after injections covering both LGNd and LP, with label concentrated in layer I, IV, and VI with a less dense projection to lower layer III and upper layer V. There is a relatively earlier initial ingrowth of axons into the visual cortex in the wallaby and throughout development thalamocortical axons appear to be more widely distributed in the depth of the visual cortex than has been demonstrated for placental mammals.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713227 TI - Volumetric and histological changes in the cochlear nuclei of visually deprived rats: a possible morphological basis for intermodal sensory compensation. AB - The cochlear nucleus of 90 day-old rats was studied to determine whether bilateral visual deafferentation at birth induced compensatory structural changes in the auditory system. Computer-assisted reconstructions of the cochlear nucleus were used to demonstrate the three-dimensional organization of the nucleus and for calculating volumes of the subdivisions. In enucleated rats the volume of the granular region of the nucleus (GCD) increased 22.6% relative to controls, and the volume of the anterior ventral nucleus (AVCN) also increased by 10.0%. The posterior ventral nucleus (PVCN) and dorsal nucleus (DCN) were not affected. Since the volume change in GCD was substantial, this region was subjected to further analysis. Two types of granular cells were identified in the GCD of both control and enucleated rats with Kluver-Barrera staining. Type 1 cells contained an ovoid dark nucleus with clumpy chromatin and possessed only a very thin rim of cytoplasm. Type 2 cells were larger with pale spheroidal nuclei and with a more prominent cytoplasmic rim. There was an obvious stratification of these two cell types in the superficial layers over the lateral aspect of the AVCN in both groups. In contrast to this, the subpeduncular corner was not distinctively laminated and contained predominantly Type 2 cells. In enucleated animals the nuclear size of Type 1 cells increased by 3.8% while the size of Type 2 nuclei was not affected. Granule cell numbers increased by 28% in the visually deafferented rats. The GCD seems to be more highly developed in species which are dependent on non-visual modalities for spatial location. The regional hypertrophy of the GCD and AVCN seen in the present study may indicate that intermodal sensory compensation is taking place in the auditory system of visually deafferented rats. PMID- 1713228 TI - Morphological organization of rat hippocampal slice cultures. AB - Using various histological methods, we investigated the cellular and morphological organization of rat hippocampal slice cultures. Many of the typical features of the hippocampus were retained in vitro over a long period of time. The principal cell types of the hippocampus and dentate gyrus, the pyramidal cells and granule cells, were well preserved and matured in vitro. Nonpyramidal cells and gamma-aminobutyric-acid (GABA) cells were also present in slice cultures and exhibited a strikingly similar dendritic appearance at the light microscopic level. Moreover, GABA-immunoreactive cell bodies and presynaptic terminals could be identified at the electron microscopic level; they expressed typical symmetric synaptic contacts with cell bodies and dendrites. The course of the intrinsic hippocampal fiber pathways--the mossy fibers, Schaffer collaterals, and alveus--was generally retained in vitro. Additional aberrant fiber projections could be identified. Finally, three types of nonneuronal cells could be distinguished on the basis of immunocytochemical methods. PMID- 1713229 TI - Topographic distribution of connections from the primary motor cortex to the corpus striatum in Aotus trivirgatus. AB - Connections between the primary motor cortex (MI) and the corpus striatum were studied in the owl monkey. Representations of specific body movements were elicited in MI by microstimulation techniques, and the efferent projections from these stimulation sites were labeled with wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP). Dense projections were found in the putamen ipsilateral to the injection site and sparser projections occupied comparable areas in the contralateral putamen. Representations of hindpaw regions projected to the dorsomedial portion of the putamen with only slight extensions across cell bridges into the caudate nucleus. Representations of the forepaw and head projected to progressively more ventrolateral zones of the dorsal two-thirds of the putamen. Projections from injections of cortex representing discrete body movements terminated in an irregular or patchy distribution over a relatively large portion of the putamen. Extensive projections from relatively discrete injections in MI are indicative of a large degree of divergence and provide evidence for possible overlap from representations of relatively disparate body parts. The results are consistent with observations reported in other species of primates, but different from those seen in carnivores. This difference is discussed with regard to possible general features of corticostriate organization. PMID- 1713230 TI - Activity-dependent regulation of glutamic acid decarboxylase in the rat barrel cortex: effects of neonatal versus adult sensory deprivation. AB - Immunocytochemical techniques were used to study the effects of tactual deprivation on glutamic acid decarboxylase (GAD) containing neurons in rat somatosensory barrel cortex. In normal rats GAD immunoreactive neurons and puncta are present in all laminae, with dense patches of GAD immunoreactive puncta centered on the barrels in lamina IV. Trimming whiskers of adult rats leads to a reversible decrease of GAD immunoreactivity in barrels corresponding to trimmed hairs. Intensity of GAD staining also is reversibly altered in supragranular laminae of nondeprived barrel columns flanked by deprived barrels. This indicates that GAD levels in the barrel cortex ordinarily fluctuate with changes in sensory input. By contrast, animals whose whiskers are trimmed from birth have normal GAD staining in both deprived and nondeprived barrels. Moreover, if trimmed whiskers of neonatally deprived animals are allowed to grow to normal lengths and are retrimmed later in adulthood GAD staining is not affected. Thus early tactual deprivation disrupts mechanisms that permit modulation of transmitter enzyme levels in cortical neurons following changes in sensory experience. PMID- 1713231 TI - Dopamine-immunoreactive neurones in the central nervous system of the pond snail Lymnaea stagnalis. AB - The distribution of dopamine and dopamine-immunoreactive neurones was studied in the central nervous system of the snail Lymnaea stagnalis. The results from immunocytochemical labelling were compared with those from the application of the glyoxylic acid fluorescence method and 6-hydroxydopamine-induced pigment labelling. Comparisons were also made between the number of dopamine immunoreactive neurones and the dopamine content of the ganglia, measured by high performance liquid chromatography. Dopamine immunocytochemistry proved to be superior to the other two histochemical techniques in terms of specificity and sensitivity. The 6-hydroxydopamine-induced pigment labelling failed to prove a useful tool for the in vivo identification of all dopamine-containing neurones. The distribution and number of dopamine-immunoreactive neurones and levels of biochemically measured dopamine in specific ganglia showed a close correspondence. By using the results of the dopamine immunocytochemistry and glyoxylic acid technique, a detailed map of dopamine-containing neurones was constructed. Dopamine-containing inter- and intra-ganglionic axon tracts were also demonstrated. The mapping of dopamine-containing neurones will facilitate further neurophysiological analysis of dopaminergic neural mechanisms in Lymnaea. PMID- 1713232 TI - Development and migration of avian sympathetic preganglionic neurons. AB - Modern neuronanatomical techniques were used to investigate the development of the avian sympathetic preganglionic cell column in the spinal cord of the chick embryo. [3H]thymidine autoradiography indicated that the majority of these preganglionic, or "Terni column" neurons are generated between stages 18 and 24 (days 2-4). This coincides with the genesis of the somatic motoneurons in the thoracic levels of the cord, and therefore differences in the time of origin cannot explain the divergent fates of these two neuronal populations. Data obtained from short-survival autoradiographic experiments indicated that many early born cells remain close to the ventral region of the ventricular epithelium until day 5 of incubation. Ventral root injections used to label retrogradely neurons projecting an axon into the ventral root (Terni cells and somatic motoneurons) have labeled neurons next to the ventricular epithelium at the same early stages. Thus, it seems likely that some Terni cells, if not all, maintain medial positions and do not migrate laterally to join a common motor column before initiating a dorsal migration. Analysis of a closely staged series of embryos, whose Terni column neurons were retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP), revealed that between days 5 and 8 of incubation, Terni column neurons migrated dorsally to attain their adult position adjacent to the central canal. These changes in position were reflected in the changing morphology of the Terni column neurons, visualized by the Golgi like HRP labeling. The positions of the migrating Terni cells differed from those of commissural cells, indicating that these fibers are not the substrate for the dorsal migration. The dorsal migration of Terni column cells was not disrupted by the surgical removal of the sympathetic ganglia, the synaptic targets of these neurons, nor by disruption of spinal afferents. Taken together, these results suggest that the migratory behavior of Terni cells in distinctive when compared to that of somatic motoneurons, and that local and/or intrinsic cues within the spinal cord guide the dorsal migration of Terni column cells. PMID- 1713233 TI - Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions. AB - Three neuroanatomical tracers have been employed to map the axonal projections formed between transplants of fetal spinal cord tissue and the surrounding host spinal cord in adult rats. Solid pieces of embryonic day 14 (E14) rat spinal cord were placed into hemisection aspiration cavities in the lumbar spinal cord. Injections of either (1) a mixture of horseradish peroxidase and wheat germ agglutinin- conjugated horseradish peroxidase, (2) Fluoro-Gold, or (3) Phaseolus vulgaris leucoagglutinin (PHA-L) were made into the transplants or the neighboring segments of the host spinal cord at 6 weeks to 14 months post transplantation. Injections of anterograde and retrograde tracers into the transplants revealed extensive intrinsic projections that often spanned the length of the grafts. Axons arising from the transplants extended into the host spinal cord as far as 5 mm from the host-graft interface, as best revealed by retrograde labeling with Fluoro-Gold. Consistent with these observations, iontophoretic injections of PHA-L into the transplants also produced labeled axonal profiles at comparable distances in the host spinal cord, and in some instances elaborate terminals fields were observed surrounding host neurons. The majority of these efferent fibers labeled with PHA-L, however, were confined to the immediate vicinity of the host-graft boundary, and no fibers were seen traversing cellular partitions between host and transplant tissues. Host afferents to the transplants were also revealed by these tracing methods. For example, the injection of Fluoro-Gold into the grafts resulted in labeling of host neurons within the spinal cord and nearby dorsal root ganglia. In most cases, retrogradely labeled neurons in spinal gray matter were located within 0.5 mm of the graft site, although some were seen as far as 4-6 mm away. The distance and relative density of ingrowth exhibited by host axons into the grafts, however, appeared modest based upon the results of HRP and Fluoro-Gold retrograde labeling. This was further confirmed with the PHA-L anterograde method. Whereas some host fibers were seen extending into the transplants, the majority of PHA-L containing axons formed terminal-like profiles at or within 0.5 mm of the host graft interface. The comprehensive view of intrinsic connectivity and host-graft projections obtained in these studies indicates that intraspinal grafts of fetal spinal cord tissue can establish a short-range intersegmental circuitry in the injured, adult spinal cord. These observations are consistent with the view that such grafts may contribute to the formation of a functional relay between separated segments of the spinal cord after injury.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713234 TI - The optic tectum of the dogfish Scyliorhinus canicula L.: a Golgi study. AB - The optic tectum of the dogfish Scyliorhinus canicula L. was studied by using the methods of Nissl, reduced silver nitrate, Golgi-aldehyde, and Golgi-Cox. Six layers and eight types of neurons were recognized. These cell types are not restricted to one layer; in fact some are found in all six tectal layers. The types of cells found are A) monopolar, B) triangular, C) radial bipolar, D) horizontal fusiform, E) large tectal, F) small tectal, G) pyriform, and H) stellate cells. In at least six of the cell types a series of dendritic specializations can be observed, such as spines in the form of "drumsticks" and thin varicose appendages, similar to those reported previously in the optic tecta of amphibians and teleosts. The optic tectum of the dogfish shows a degree of complexity comparable to that of amphibians and teleosts. PMID- 1713235 TI - Brain sites projecting to the spinal nucleus of the bulbocavernosus. AB - Motoneurons of the spinal nucleus of the bulbocavernosus (SNB) occupy a distinct dorsomedial position in the ventral horn of lumbar segments 5 and 6 and innervate sexually dimorphic striated muscles of the rat perineum, including the bulbocavernosus and levator ani. To begin the study of brain influences upon SNB function, we used retrograde tracers to identify brain regions that project to the area of SNB motoneurons. Our findings provide strong evidence that lateral vestibular and several reticular nuclei innervate the SNB. Additional possible afferents include the superior and medial vestibular nuclei, raphe nucleus, red nucleus, interstitial nucleus of the medial longitudinal fasciculus, and paraventricular nucleus. PMID- 1713236 TI - The calcium binding protein calbindin-D 28K reveals subpopulations of projection and interneurons in the cat superior colliculus. AB - The calcium binding protein calbindin-D 28K (CaBP) has been localized in the cat superior colliculus (SC). Four important features of SC organization have been revealed by using CaBP immunocytochemistry. 1) CaBP neurons formed three laminar tiers in SC, one within the upper one half of the superficial gray layer (SGL), the second bridging the deep optic (OL) and intermediate gray layers (IGL), and the third within the deep gray layer (DGL). 2) CaBP labeled several classes of interneuron in SC. In the upper CaBP tier, the labeled neurons were all small, but they varied in morphology and included horizontal, pyriform, and stellate neurons. A unique class of interneuron was labeled by anti-CaBP in the OL-IGL tier. This cell was stellate-like with highly varicose dendrites and broad dendritic trees. Other labeled neurons in the intermediate and deep tiers included nonvaricose stellate neurons and rare large neurons in the DGL. 3) A few anti-CaBP neurons were projection neurons. Virtually no CaBP neurons were retrogradely labeled after injections of HRP into the predorsal bundle and dorsolateral midbrain tegmentum or into the lateral posterior nucleus. However, 2.4% of anti-CaBP neurons were retrogradely labeled after HRP injections into the dorsal and ventral lateral geniculate nuclei. These represented 14.7% of all neurons projecting to the LGN complex. 4) A small percentage of CaBP neurons co localized GABA. A two-chromagen double-labeling technique showed that about 4.0% of labeled neurons were labeled by both antibodies. In summary, antibodies to CaBP densely labeled subpopulations of neurons in the cat SC, most of which were interneurons, some of which projected to the LGN, and a few of which co-localized GABA. PMID- 1713238 TI - Planar differences in nuclear area and orientation in the subventricular and intermediate zones of the rat embryonic neocortex. AB - Nuclear area and orientation in the subventricular and intermediate zones was studied quantitatively in coronal vs. sagittal sections of the dorsomedial neocortex. Nissl-stained methacrylate-embedded normal rat embryos were studied between embryonic days (E) 13 and E22. The area of nuclear profiles and the degrees their long axes (defined as a straight line through the two most distant points in the nuclear profile) deviated from the horizontal (defined as parallel to the pial membrane) were determined with a computer-graphics program. Because the nucleus is the most clearly outlined structure in embryonic cells, the area and orientation of the nucleus was taken to reflect the overall size and orientation of the cell body. Nuclear area is larger in the coronal plane than it is in the sagittal plane, especially between E17 and E20. Cell body orientation in the subventricular and lower intermediate zones is predominantly horizontal in the coronal plane and predominantly vertical in the sagittal plane. In the upper intermediate zone, cell body orientation is predominantly vertical in both planes, but more so in the sagittal plane. These data indicate that the majority of cell bodies in the subventricular and lower intermediate zones have a horizontally oriented, flattened elliptical shape with their larger diameters lying within the coronal plane and their smaller diameters in the sagittal plane. Because of the flattening, the cell bodies falsely appear to be vertically oriented in the sagittal plane. Qualitative observations in horizontal sections confirmed the quantitative computer analysis. These results are related to other findings with [3H]thymidine autoradiography concerning cell migration and the sojourn of cells in the subventricular and intermediate zones. PMID- 1713237 TI - Entorhinal cortex of the monkey: V. Projections to the dentate gyrus, hippocampus, and subicular complex. AB - The topographic and laminar organization of entorhinal projections to the dentate gyrus, hippocampus, and subicular complex was investigated in the Macaca fascicularis monkey. Injections of 3H-amino acids were placed at various positions within the entorhinal cortex and the distribution of anterogradely labeled fibers and terminals within the other fields of the hippocampal formation was determined. Injections of the retrograde tracers Fast blue, Diamidino yellow, and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were also placed into the dentate gyrus, hippocampus, and subicular complex, and the distribution of retrogradely labeled cells in the entorhinal cortex was plotted using a computer aided digitizing system. The entorhinal cortex gave rise to projections that terminated in the subiculum, in the CA1, CA2, and CA3 fields of the hippocampus, and in the dentate gyrus. Projections to the dentate gyrus, and fields CA3 and CA2 of the hippocampus, originated preferentially in layers II and VI of the entorhinal cortex whereas projections to CA1 and to the subiculum originated mainly in layers III and V. Anterograde tracing experiments demonstrated that all regions of the entorhinal cortex project to the outer two-thirds of the molecular layer of the dentate gyrus and to much of the radial extent of the stratum lacunosum-moleculare of CA3 and CA2. While the terminal distributions of entorhinal projections to the dentate gyrus, CA3, and CA2 were not as clearly laminated as in the rat, projections from rostral levels of the entorhinal cortex preferentially innervated the outer portion of the molecular layer and stratum lacunosum-moleculare, whereas more caudal levels of the entorhinal cortex projected relatively more heavily to the deeper portions of the entorhinal terminal zones. The entorhinal projection to the CA1 field of the hippocampus and to the subiculum followed a transverse rather than radial gradient of distribution. Rostral levels of the entorhinal cortex terminated most heavily at the border of CA1 and the subiculum. More caudal levels of the entorhinal cortex projected to progressively more distal portions of the subiculum (towards the presubiculum) and more proximal portions of CA1 (towards CA2). Lateral portions of the entorhinal cortex projected to caudal levels of the recipient fields and more medial parts of the entorhinal cortex projected to progressively more rostral portions of the fields. PMID- 1713239 TI - The surgeon's responsibility: operation and reoperation: the UCLA experience. PMID- 1713240 TI - Percutaneous transluminal balloon angioplasty of stenotic standard Blalock Taussig shunts: effect on choice of initial palliation in cyanotic congenital heart disease. AB - To date, attempted balloon dilation of stenotic standard Blalock-Taussig shunts has been largely disappointing. It has been suggested that this may be due to the use of balloons of insufficient diameter. Balloon dilation of stenotic Blalock Taussig shunts was attempted with use of relatively large balloons in five patients (11 to 67 months old) with cyanotic heart disease who were becoming progressively cyanotic and polycythemic (hemoglobin 17.9 +/- 1.1 g/dl) because of discrete shunt stenosis at the site of pulmonary anastomosis. Balloon diameters selected were equal to or within 1 mm of the unobstructed proximal shunt diameter. Before balloon dilation the diameter at the site of the stenosis was 2.8 +/- 0.8 mm (range 1.7 to 4); after balloon dilation it was 5.7 +/- 1.1 mm (range 4.5 to 7.5). The diameter increased in all patients (range 2.0 to 3.5 mm); the mean increase was 2.8 +/- 0.2 mm (p less than 0.005). Expressed as a percent, the increase in diameter at the stenosis ranged from 80% to 182.4% (mean 108.2 +/ 16.8%). Before balloon dilation the systemic oxygen saturation was 72.8 +/- 9.2% (range 55% to 80%) and after balloon dilation it was 83.6 +/- 2.9% (range 80% to 87%). A satisfactory increase (range 6% to 25%) in blood oxygen saturation was seen in all patients; the mean increase was 10.8 +/- 3.2% (p less than 0.01). At follow-up, the oxygen saturation by pulse oximetry was 85.8 +/- 2.9% (mean 5.8 +/ 1.7 months after balloon dilation) and the hemoglobin was 15.6 +/- 1.9 g/dl (mean 6.6 +/- 1.5 months after balloon dilation).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713241 TI - The gay voice in popular music: a social value model analysis of "Don't Leave Me This Way". AB - The gay voice in popular music and its potential to create positive social change regarding societal values about homosexuality is the focus of the present study. The historical development of the gay voice in popular music is reviewed as an introduction to a critical analysis of the Communards' music video "Don't Leave Me This Way." Using a modified version of the Social Value Model proposed by Rushing and Frentz, the video is analyzed on three levels: (a) narrative content, (b) use of symbols in the narrative, and (c) lyrical content. It is suggested that this video effected a dialectical synthesis of mainstream and homosexual values because it achieved mainstream commercial success while realistically expressing a gay perspective. PMID- 1713242 TI - [Progress in endocrinological therapy of basedow's and Hashimoto's diseases]. PMID- 1713243 TI - [Diagnosis and therapy of viral hepatitis]. PMID- 1713244 TI - Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay. AB - The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles. PMID- 1713245 TI - Characterization of a new apolipoprotein E5 variant detected in two French Canadian subjects. AB - We have found a novel apoE5 mutation, using isoelectric focusing (IEF), in two apparently unrelated French-Canadian subjects. Co-dominant inheritance was demonstrated in the family of the first proband, a healthy male subject. The presence of the apoE5 form was not associated with lipid abnormalities or cardiovascular disease in this family. The second proband was a hyperlipidemic female patient suffering from angina, with no informative relatives available for study. In both individuals, monoclonal antibody studies demonstrated that the mutation was associated with the loss of two overlapping epitopes at the amino terminus of the protein. Cysteamine treatment of the very low density lipoproteins indicated that the mutant apoE contained only one cysteine residue, suggesting that apoE3 was the parental form. Two-dimensional electrophoresis suggested that the mutated protein had a slightly lower molecular weight (by 1-2 kDa). However, DNA sequencing of the third exon of the apoE gene in both probands revealed a single G to A substitution at the 48th nucleotide, changing the amino acid at position 13 from glutamic acid to lysine. These results were confirmed by oligo melting experiments with allele-specific probes in relatives of the probands. The study of this apoE variant should provide additional insight into the structure-function relationship of apoE. PMID- 1713246 TI - Developmental disability. PMID- 1713247 TI - Professional attitudes toward the developmentally disabled patient. PMID- 1713248 TI - Expression and function of a variant T cell receptor complex lacking CD3-gamma. AB - A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma. PMID- 1713249 TI - Abnormal thymic development, impaired immune function and gamma delta T cell lymphomas in a TL transgenic mouse strain. AB - During derivation of transgenic mouse strains with various TL and TL/H-2 chimeric genes, one strain, Tg.Tlaa-3-1, introduced with a TL gene (Tlaa-3), was found to have an abnormal thymic T cell population and to develop a high incidence of T cell lymphomas. To investigate the etiology of the thymic abnormalities and of the lymphomas, the development of lymphoid organs in transgenic mice was studied. The thymus of these mice goes through three unusual successive events: perturbation of thymic development during embryogenesis, disappearance of thymocytes between day 14 and day 21 after birth, and subsequent proliferation of large blast-like cells. These events are associated with the abolishment of T cell receptor (TCR) alpha beta lineage of the T cell differentiation, leading to preponderance of cells belonging to the TCR gamma delta L3T4-Lyt-2- double negative (DN) lineage. Bone marrow transplantation and thymic graft experiments demonstrate that the abnormality resides in the bone marrow stem cells rather than in the thymic environment. The expression of TL antigen in the transgenic mice is greatly increased and TL is expressed in a wide range of T cells, including normally TL- DN cells and L3T4+ Lyt-2- and L3T4-Lyt-2+ single positive cells. These quantitative and qualitative abnormalities in TL expression most likely cause the abnormal T cell differentiation. The gamma delta DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, as T cell function is defective in antibody production to sheep red blood cells, in mixed lymphocyte culture reaction to allogenic spleen cells and also in stimulation with concanavalin A. These results indicate that the gamma delta cells are incapable of participating in these reactions. Molecular and serological analysis of T cell lymphomas reveal that they belong to the gamma delta lineage, suggesting that the gamma delta DN cells in this strain are susceptible to leukemic transformation. Based on cell surface phenotype and TCR expression of the DN thymocytes and T cell lymphomas, a map of the sequential steps involved in the differentiation of gamma delta DN cells is proposed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713250 TI - Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells. AB - Although thyroglobulin (Tg), the thyroid prohormone, is well known as a T cell dependent autoantigen in human and experimental autoimmune thyroid disease, very little is known about the molecular basis of Tg recognition by T cells. In this paper, we have characterized the epitopes recognized by two clonotypically distinct, murine Tg autoreactive T cell hybridomas, CH9 and ADA2. In vitro iodination of a Tg preparation which was deficient in in vivo organified iodine was first used to confirm our previous observation that these T cells recognize iodination-related epitopes in the Tg molecule. Affinity chromatography of tryptic peptides derived from normally iodinated human Tg revealed that these epitopes were exclusively located in thyroxine (T4) containing peptides. Through the use of synthetic T4-containing peptides, representing the four major hormonogenic sites in Tg, we demonstrated that both CH9 and ADA2 recognize an epitope containing the T4 at position 2553 in human Tg. Sets of overlapping 5mer to 12mer peptides around this T4 showed that the most potent peptide was a 9mer beginning at Asp 2551. The T4 was shown to be a critical residue, since its replacement with any of the 20 naturally occurring amino acids produced only nonstimulatory peptides. Since the T cell hybridomas could also be stimulated by major histocompatibility complex class II positive (interferon-gamma-treated) thyroid epithelial cells in vitro, and their parent T cell lines can induce thyroiditis on adoptive transfer, the T4-containing Tg sequence described here is implicated as a pathogenic epitope in murine thyroid autoimmunity. PMID- 1713251 TI - Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells. AB - Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells. PMID- 1713252 TI - Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding. AB - The human immunodeficiency virus (HIV) binds to the surface of T lymphocytes and other cells of the immune system via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gp120. By analogy with certain other enveloped viruses, receptor binding by HIV may be followed by exposure of the hydrophobic NH2 terminus of its transmembrane glycoprotein, gp41, and fusion of the virus and cell membranes. A similar sequence of events is thought to take place between HIV-infected and uninfected CD4+ cells, resulting in their fusion to form syncytia. In this study, we have used a soluble, recombinant form of CD4 (sCD4) to model events taking place after receptor binding by the HIV envelope glycoproteins. We demonstrate that the complexing of sCD4 with gp120 induces conformational changes within envelope glycoprotein oligomers. This was measured on HIV-1-infected cells by the increased binding of antibodies to the gp120/V3 loops, and on the surface of virions by increased cleavage of this loop by an exogenous proteinase. At 37 degrees C, these conformational changes are coordinate with the dissociation of gp120/sCD4 complexes from gp41, and the increased exposure of gp41 epitopes. At 4 degrees C, gp120 dissociation from the cell surface does not occur, but increased exposure of both gp120/V3 and gp41 epitopes is detected. We propose that these events occurring after CD4 binding are integral components of the membrane fusion reaction between HIV or HIV infected cells and CD4+ cells. PMID- 1713253 TI - Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast. AB - Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position. PMID- 1713254 TI - CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand). AB - CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF. PMID- 1713255 TI - Cytokine gene transcription in vascularised organ grafts: analysis using semiquantitative polymerase chain reaction. AB - Cytokine gene transcription has been analyzed by direct analysis of RNA obtained from mouse heterotopic cardiac transplants. The level of expression of the cytokine genes was assessed using semiquantitative polymerase chain reaction (PCR). Expression of the cytokines investigated fell into three groups. The first group included interleukin 1 beta (IL-1 beta), IL-5, IL-6, and interferon gamma (IFN gamma). These genes were expressed in normal heart tissue at low level and were upregulated following both syngeneic and allogeneic transplantation. Genes in the second group (IL-1 alpha, IL-3) were not expressed at detectable levels in normal heart but were induced following either syngeneic or allogeneic heart grafting. IL-2, IL-4, and tumor necrosis factor beta (IFN beta) comprised the third group and these cytokines were expressed only in allogeneic grafts after transplantation. PMID- 1713256 TI - Needs of handicapped infants, toddlers and families. Florida's response. AB - The State of Florida and its physicians are moving into a wonderful time of opportunity to serve the special needs of infants and toddlers at risk for developmental delays. If we meet our challenge we will no longer judge our success by a viable pregnancy or "graduation" from the nursery. We will begin to expect a community-wide response that prepares a child for education so successful that he or she will graduate prepared to work as a full member of the community. Not all our interventions will succeed at that level, but we have learned that good ones can place almost all our special needs children into the community. Physicians will have a major role in the success of this system of care for handicapped infants, toddlers and their families. PMID- 1713257 TI - Rapid laser nephelometric determination of amylase activity in serum and urine. AB - We describe herein a rapid and sensitive laser nephelometric method for the determination of serum and urinary amylase activities. Our data showed that the change in relative light scattering (RLS) of an amylopectin substrate measured by a laser nephelometer related directly with amylolytic activity of amylase from 50 to 600 IU/L. Within-run variations at 293 and 769 IU/L sera showed CV's of 5.0% and 3.1%, respectively. Day-to-day variation for the same sera showed CV's of 7.2% and 4.7%, respectively. Correlation studies using the manual Phadebas dye starch complex method and with the Roche amylochrome method showed correlation coefficients of 0.99 and 0.95, respectively. Using urine specimens, the correlation studies also showed a correlation coefficient of 0.98. These studies indicated that the proposed method was sensitive, fast, economical and easily adaptable to emergency and routine applications. PMID- 1713258 TI - Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A. AB - Spleen cells from mice immunized with a Bordetella pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0.22 and 2.2 micrograms mAb ml-1 (0.1 and 1 microgram per 450 microliter assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A. PMID- 1713259 TI - Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius. AB - The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194. PMID- 1713260 TI - Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. AB - Two canine isolates of simian virus 5 (SV5), termed CPI+ and CPI-, were examined for their ability to react with a bank of monoclonal antibodies (MAbs) that had been previously raised against a human isolate of SV5. CPI- virus was originally isolated from the brain of a gnotobiotic dog infected with CPI+ virus and establishes persistent infections more readily than CPI+ in vitro. Of more than 50 MAbs tested, only one (P-k) reacted with CPI+ but not CPI-, enabling distinction between the two canine isolates. It had been shown previously that MAb P-k reacts with an epitope common to both the P and V proteins. In order to characterize further the epitope binding site of this MAb the P/V genes of CPI+ and CPI- were sequenced. There were four nucleotide differences between CPI+ and CPI-, three of which resulted in predicted amino acid substitutions. Synthetic peptides corresponding to regions encompassing these changes were made and radioimmune competition assays were used to identify the epitope binding site of MAb P-k. Sequence comparison of the P/V gene of CPI+ with the published sequence of a monkey isolate of SV5 (W3) revealed 14 nucleotide differences with five amino acid substitutions. The only amino acid substitution observed between CPI+, CPI- and W3 which altered the predicted secondary structures of the P and V proteins was a leucine to proline change that induced a predicted beta-turn and resulted in the loss of binding of MAb P-k. PMID- 1713261 TI - Preliminary analysis of murine cytotoxic T cell responses to the proteins of the flavivirus Kunjin using vaccinia virus expression. AB - A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide. PMID- 1713262 TI - Localization of antigenic sites on the surface glycoprotein of mouse hepatitis virus. AB - A panel of murine hepatitis virus (MHV) surface (S) glycoprotein-specific monoclonal antibodies (MAbs), which recognize either continuous or discontinuous epitopes, were tested in competitive binding assays. The results indicate that the binding site of MAb 30B amino acids 395 to 406 in the amino-terminal S1 subunit, is involved in the discontinuous epitope designated antigenic site A. This site is a major determinant for the induction of neutralizing antibodies. These data define, for the first time, the location of a functionally important domain on the MHV S protein. PMID- 1713264 TI - Characterisation of a linear binding site for a monoclonal antibody to hepatitis B core antigen. AB - The complete amino acid (aa) sequence of the hepatitis B virus (HBV) core protein (HBcAg), ayw subtype, was synthesized as decapeptides with five overlapping aas. The peptides were tested for reactivity with monoclonal antibodies (mAbs) to HBcAg (35/312, 37/275, and 7/275). All the mAbs specifically inhibited human anti HBc by cross competition in assays for anti-HBc and anti-HBe. The mAb 35/312 recognised a peptide covering residues 76-85 of the HBcAg sequence. The other two mAbs did not react specifically with any linear peptide, suggesting discontinuous epitopes for these mAbs. The linear sequence EDPASR at residues 77-82 was found to constitute the epitope for mAb 35/312 when fine mapping the binding site. The most essential aas for mAb 35/312 were found to be the DP at residues 79-80, when peptides were synthesized where the aas at 77-83, were substituted by the other 19 aas. Since the mAb 35/312 inhibits the binding of human anti-HBc positive sera, which are known to recognise an SDS labile epitope, the sequence 77-82 might be a part of a larger discontinuous epitope. Alternatively the mAb 35/312 blocks the binding of human anti-HBc by steric hindrance. PMID- 1713263 TI - Coupling of killer virus transcription with translation in yeast cell-free extracts. AB - The cytoplasmically inherited killer virus of Saccharomyces cerevisiae expresses its dsRNA genome via apparently uncapped viral transcripts produced in the cytoplasm of infected cells. Virions of this naturally temperature-sensitive virus can be added to cell-free translational extracts of uninfected yeast cells resulting in a reaction in which viral transcription and translation are coupled at 15 degrees C in vitro. In this reaction nucleotides are incorporated into full length transcripts of the M and L-A dsRNA segments, with lower levels of incorporation into genomic RNA. In addition, incorporation of nucleotides is observed into a smaller RNA species showing no sequence relatedness to M or L-A. PMID- 1713265 TI - Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. AB - RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN 2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000 fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice. PMID- 1713266 TI - Detection of hantavirus RNA in tissues of experimentally infected mice using reverse transcriptase-directed polymerase chain reaction. AB - Detection of hantaviruses, the etiological agents of hemorrhagic fever with renal syndrome (HFRS), by virus isolation using experimental animals or cell culture is time-consuming. A more rapid but equally specific method is needed. We used a reverse transcriptase-directed polymerase chain reaction (RT-PCR) to detect hantavirus genomic sequences and compared its sensitivity with conventional virus isolation. RNA, extracted by the guanidinium isothiocyanate-cesium chloride method from hantavirus-infected Vero E6 cells and from tissues of infant mice inoculated intracerebrally with 100 LD50 of hantavirus, was initially reverse transcribed using avian myeloblastosis virus reverse transcriptase. The resulting complementary DNA (cDNA) was used as template to amplify the glycoprotein 2 encoding region of the hantavirus M segment. With this method, Vero E6 cell cultures infected with Hantaan virus strains 76-118 (prototype) and HV114 (an isolate from the urine of an HFRS patient in China) were positive, while control cultures were negative. Brain, lung, and heart tissues from hantavirus-infected mice were positive by RT-PCR at 5, 8, and 11 days after intracerebral inoculation. The specificity of the positive results was confirmed by restriction endonuclease digestion of the amplified fragments with AluI and HpaI. The sensitivity of the RT-PCR was equal to cell culture amplification but required less time. This method is being adapted for detection of hantavirus genomic sequences in clinical specimens and postmortem tissues from patients with HFRS. PMID- 1713267 TI - Rat paw oedema and mast cell degranulation caused by two phospholipase A2 enzymes isolated from Trimeresurus mucrosquamatus venom. AB - Two phospholipase A2 (PLA2) enzymes, TMVPLA2 I and TMVPLA2 II, isolated from Trimeresurus mucrosquamatus venom (TMV) induce rat hind-paw oedema in a dose dependent manner. This response is suppressed by pretreatment with diphenhydramine, methysergide or compound 48/80, which reduces tissue histamine content. In isolated mast cells, TMVPLA2 I and TMVPLA2 II cause concentration-, time- and calcium-dependent release of histamine and beta-glucuronidase. This effect is inhibited by disodium cromoglycate, mepacrine, nordihydroguaiaretic acid, piriprost and BW 755C, but not by aspirin or indomethacin. These observations indicate that the mast cell plays a predominant role in TMVPLA2 I- and TMVPLA2 II-induced paw oedema, and that venom PLA2 enzyme needs an intact lipoxygenase pathway to induce mast cell degranulation. PMID- 1713268 TI - Antigen presentation in the rheumatoid joint. PMID- 1713269 TI - Therapeutic use of monoclonal anti-CD4 antibody in rheumatoid arthritis. AB - Ten patients with severe rheumatoid arthritis were treated with a murine monoclonal anti-CD4 (B-F5) antibody in an open study (one with 10 mg/day, 2 with 15 mg/day, 7 with 20 mg/day) for 10 consecutive days. Tolerance was excellent. All patients improved during treatment clinically (Ritchie's index, morning stiffness, pain scale) (p = 0.005), as well as biologically C-reactive protein (p = 0.008) with an average 60% reduction of each of these variables at Day 15, and clinical benefit lasted over 6 months in some patients. No significative depletion was noted in total lymphocyte or CD3, CD4, CD8, CD20, positive cells after treatment. Evidence of murine immunization was found in only 2 patients. PMID- 1713270 TI - Pregnancy outcome in patients with high titer anti-RNP antibodies. A retrospective study of 40 pregnancies. AB - In a retrospective study the outcome of 40 pregnancies in 20 women with a high titer of anti-RNP antibodies was evaluated. In the 18 pregnancies that occurred after disease onset, transient proteinuria was noted in 3 and transient thrombocytopenia in 2. Deep venous thrombosis was observed in one patient. Preeclampsia in another woman necessitated cesarean sections in 2 pregnancies with successful outcome. The observed complications may all be seen in normal pregnancies. There was no evidence of exacerbation of maternal disease during pregnancy or in the postpartum period. Our study indicates that in women with high anti-RNP titer the risk of fetal loss or maternal worsening of disease seems slight. PMID- 1713271 TI - Supravital staining of cells in noninflammatory synovial fluids: analysis of the effect of crystals on cell populations. AB - Leukocyte differential counts are rarely performed on synovial fluids (SF) with low leukocyte (WBC) counts as they have been difficult to do with standard Wright stains. We performed SF analysis including supravital (SV) stains for differential counts on 100 consecutive relatively noninflammatory synovial fluids defined as any SF with less than 2,000 cells/mm3. SV stains gave clear cellular morphology. Monocytes were the most prominent cells in all noninflammatory synovial fluids. Thirty-eight fluids were found to have crystals (monosodium urate (MSU) in 15, calcium pyrophosphate dihydrate (CPPD) in 5, CPPD plus apatite like crystals in 9, apatite-like clumps alone in 8 and lipid liquid in 1). WBC counts, percentages of polymorphonuclear (PMN) and absolute PMN counts were greatest in fluids with MSU or CPPD crystals. Fluids with apatite-like clumps alone tended to have the lowest WBC counts. The presence of WBC over 500 cells/mm3, over 20% PMN and absolute PMN counts over 250 cells/mm3 should encourage a careful search for crystals, especially MSU, in noninflammatory synovial fluids. PMID- 1713272 TI - Genetic analysis in cystic fibrosis using the amplification refractory mutation system (ARMS): the J3.11 MspI polymorphism. AB - A new method of genetic analysis has been devised. The method, amplification refractory mutation system (ARMS), has been used to genotype the J3.11 MspI restriction fragment length polymorphism (RFLP) closely linked to cystic fibrosis (CF). The DNA sequence for both alleles of this dimorphism has been used to design ARMS primers. These allow genotyping of DNA isolated from blood, Guthrie cards, and buccal cells. PMID- 1713273 TI - Sigma subunit of Escherichia coli RNA polymerase loses contacts with the 3' end of the nascent RNA after synthesis of a tetranucleotide. AB - We have used photocrosslinking to analyze the contacts between the 3' end of the RNA and Escherichia coli RNA polymerase during the early steps of RNA synthesis using the nucleotide analog 8-azido-ATP (8-N3-ATP). The crosslinking group on 8 N3-ATP contacts the beta, beta' and sigma subunits when the analog is bound to the holoenzyme. We show here that 8-N3-ATP is a substrate for E. coli RNA polymerase and acts as an RNA chain terminator when incorporated into the 3' end of nascent RNA. 8-N3-AMP was incorporated uniquely at the 3' end of tri-, tetra- and pentanucleotides synthesized from a poly[d(A-T)] template and at the 3' end of pentanucleotides from two promoters (lambda PR' and E. coli rrnB P1). The oligonucleotides were covalently attached to the RNA polymerase by irradiation of transcription complexes with ultraviolet light. All RNAs labeled the beta and beta' subunits, but sigma was contacted only by the trinucleotide and tetranucleotide on poly[d(A-T)]. Sigma is still present in transcription complexes containing the pentanucleotide on poly[d(A-T)], despite the lack of labeling. Neither pentanucleotide from the authentic promoters contacted sigma. We conclude that as holoenzyme moves downstream, either two separate conformational changes occur, after synthesis of the trinucleotide and tetranucleotide, which result in movement of sigma away from the nucleotide binding site or, alternatively, sigma remains fixed relative to the DNA while the domain on core polymerase forming the nucleotide binding site moves downstream. PMID- 1713274 TI - Extracellular matrix of central nervous system white matter: demonstration of an hyaluronate-protein complex. AB - Monoclonal antibodies were raised against human glial hyaluronate-binding protein (GHAP), a major CNS-specific glycoprotein known to bind hyaluronate in vitro. Frozen sections of dog and human spinal cord were digested with Streptomyces hyaluronidase in order to ascertain whether GHAP is bound to hyaluronate in vivo. Digestion with hyaluronidase, prior to staining of the sections by conventional indirect immunofluorescence, led to a drastic reduction in the intensity of the staining reaction. Chondroitinase ABC (protease-free) was also effective in bringing about the release of GHAP from tissue sections. This enzyme also degrades hyaluronate. The effects of the chondroitinase were completely reversed by the addition of 1 mM Zn2+, a known inhibitor of this enzyme. The intact protein was released into the soluble fraction of human brain homogenates by testicular hyaluronidase. An immunoreactive species of 70 kD was released into the soluble fraction of dog spinal cord homogenates by Streptomyces hyaluronidase. Dog GHAP was isolated from spinal cord by means of ion exchange and affinity chromatography. This protein bound efficiently to hyaluronate in vitro. Dog and human GHAP had identical isoelectric points and similar peptide maps but different molecular weights. Dog GHAP (70 kD) was larger than its human counterpart (60 kD). These findings imply that GHAP exists in association with hyaluronate in CNS white matter. Immunoelectron microscopy revealed that GHAP fills the space between myelin sheaths in dog spinal cord white matter. One is led to conclude therefore that an hyaluronate based extracellular matrix exists in CNS white matter. PMID- 1713275 TI - Pulmonary hyalinizing granuloma: a rare cause of a solitary pulmonary nodule. AB - Pulmonary hyalinizing granulomata are unusual, noninfectious lesions of the lung of uncertain etiology that probably represent an exaggerated immune response. They present radiographically as noncalcified solitary or multiple pulmonary nodules, thereby mimicking primary or metastatic malignancy. The article discusses a case of this rare entity that presented as a solitary pulmonary nodule. PMID- 1713276 TI - A comparison of transurethral and transrectal microwave hyperthermia in poor surgical risk benign prostatic hyperplasia patients. AB - From 1987 to 1989, 36 poor surgical risk patients with benign prostatic hyperplasia (BPH) were treated with transrectal or transurethral microwave hyperthermia. Most of the 36 patients treated (85%) had severe signs and symptoms of urinary outflow obstruction. Of the patients 22 (61%) underwent transrectal and 14 (39%) underwent transurethral hyperthermia. Followup ranged from 10 to 28 months (mean 19 months) for the transrectal hyperthermia group and 7 to 16 months (mean 10 months) for the transurethral hyperthermia group. The patients were given 6 hyperthermia sessions of 30 minutes each with the temperature controlled on the rectal or urethral surface at 45C. Hyperthermia was well tolerated with mild acute toxicity and no late complications were observed. In the important subjective and objective parameters, major improvement was noted more frequently in the 14 transurethral than in the 22 transrectal hyperthermia treated patients (p less than 0.05). The Food and Drug Administration severity score, prostate volume, post-voiding residual volume and urethral flow showed substantial improvement in 79, 86, 79 and 79%, respectively, of the 14 transurethral hyperthermia treated patients compared to 41, 45, 82 and 82%, respectively, for the 22 transrectal hyperthermia treated patients. A prospective randomized trial comparing transrectal and transurethral hyperthermia is required to define the role of each treatment mode in patients with BPH. PMID- 1713277 TI - Small cell carcinoma of the genitourinary tract: an immunohistochemical, electron microscopic and clinicopathological study. AB - We examined 13 cases of small cell carcinoma of the genitourinary tract to evaluate and compare the immunocytochemical and ultrastructural features as well as the clinicopathological behavior. Immunohistochemical stains revealed that neuron specific enolase and chromogranin showed differences in staining between the bladder and prostate, as well as between the small cell and adenocarcinomatous components of the prostate. Also, synaptophysin was negative over-all in 12 of 13 cases. Epithelial membrane antigen, carcinoembryonic antigen and keratin showed strong focal positivity within the small cell component. Electron microscopy was performed in 4 cases, with 3 demonstrating neurosecretory granules. Clinically, 6 of the 7 patients with adenocarcinoma/small cell carcinoma of the prostate did poorly, all with a survival of 15 months or less. Of 5 patients with transitional cell/small cell carcinoma of the bladder 2 fared better (both had no evidence of disease at 12 months and 11 years, respectively). Based upon the immunostaining and electron microscopic findings, small cell carcinoma of the genitourinary tract is heterogeneous in appearance and, therefore, may arise from a multipotential cell of origin. This cell of origin may be organ-specific, as demonstrated by the variability in staining characteristics among the prostate, bladder and kidney, as well as by the differences in the clinical behavior of these malignancies. Small cell carcinoma of the prostate has a poor prognosis, while small cell carcinoma of the bladder may portend a better prognosis if diagnosed at an early stage. PMID- 1713278 TI - Voltage-gated calcium currents in isolated retinal ganglion cells of the cat. AB - Voltage-gated calcium current (ICa) was recorded from retinal ganglion cells dissociated from the adult cat under the voltage-clamp condition using a patch pipette in the whole cell configuration. ICa was isolated from the voltage dependent potassium and sodium currents by ion substitution and selective blockers. ICa was activated by depolarization of the cell from a holding potential (Vh) of -97 mV to more positive voltages than -57 mV. All recorded cells showed similar voltage dependence of ICa activation: 50% activation at about -23 mV. Current-voltage (I-V) relationship of ICa showed a symmetrical bell shape with a single peak at around -7 mV. The I-V curve recorded with Vh of -57 mV was nearly identical to that obtained with Vh of -97 mV. During a depolarizing command, the amplitude of ICa gradually decreased. Inactivation of ICa depended on Ca influx into the cell. ICa became more sustained either when the extracellular Ca was replaced by Ba, or when the cell was loaded with 30 mM EGTA. Nifedipine (10(-4) M) inhibited ICa reversibly. Effects of Bay K 8644 were bimodal: augmentation at a low concentration (10(-8) M) and suppression at a high concentration (10(-4) M). All these characteristics are identical to the previous findings for the high-threshold (L-type) ICa. The type of ICa recorded from the retinal ganglion cells in the adult cat is different from those in newborn rats. PMID- 1713279 TI - Lithium carbonate in the preoperative preparation of Graves' disease. AB - Lithium carbonate was given in the preoperative preparation of 12 patients with Graves' disease, the reasons for its use being side effects of thionamide in 9 patients, insufficient control by thionamide in 1 and psychic symptoms in 2. Lithium carbonate was often used in combination with other drugs, namely; thionamide in 4 patients, beta-adrenergic blockades in 5, reserpine in 5 and glucocorticoid in 1. This preoperative control significantly decreased the mean serum T2 and T4 levels from 656 +/- 55 ng/dl to 180 +/- 16 ng/dl and from 25.9 +/ 2.1 micrograms/dl to 9.7 +/- 1.5 micrograms/dl, respectively. The only adverse effect of lithium carbonate was pollakisuria observed in one patient. All patients underwent subtotal thyroidectomy uneventfully. It is concluded that the administration of lithium carbonate alone or in combination with other drugs is an effective method of preoperatively controlling hyperthyroidism when conventional antithyroid drugs show adverse effects. PMID- 1713280 TI - The effect of site and mode of expression of a heterologous antigenic determinant on the properties of poliovirus hybrids. AB - The antigenic site normally situated in the EF loop of VP2 (2EF) of poliovirus type 2 (Lansing) [PV2 (L)] was expressed in 2EF or in the BC loop of VP1 (1BC) of PV1 (Mahoney) [PV1 (M)]. A hybrid virus expressing the site in 2EF of PV1 (M) is known to be neutralizable by PV2 (L)-specific antisera and to induce neutralizing antibodies against PV-2 (L). In contrast, a hybrid expressing a related sequence in 1BC of PV1 (M), which maintained the length of the native BC loop, was not neutralizable by PV2 (L)-specific antisera and did not induce PV2 (L) neutralizing antibodies. However, when 1BC was extended, so that it was longer than the native 1BC, the resulting hybrids induced low titers of PV2 (L) neutralizing antibody although they were still not neutralizable by PV2 (L) specific antisera. Synthetic peptides copying the extended sequences raised neutralizing antibodies which were able to distinguish between the sequence expressed in the extended 1BC, in the native-length 1BC and in 2EF. The growth rates of the hybrids with modifications to 1BC depended upon the nature of the modifications. The hybrid with the native length 1BC grew poorly, but extending 1BC tended to improve the growth rate, and extending 1BC with PV1 (M)-specific sequences nearly restored wild-type growth rates. Thus, the size, precise sequence and location of a heterologous antigenic site expressed on poliovirus have significant effects on the properties of that determinant and of the hybrid expressing it. Antigenicity of hybrids can be modified and their growth rate can be enhanced by appropriate choices of these parameters. PMID- 1713281 TI - ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. AB - We describe a family of highly conserved, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences, 14 of which have been identified in Escherichia coli and Salmonella typhimurium and a further three in other enterobacterial species (Yersinia pseudotuberculosis, Klebsiella pneumoniae and Vibrio cholerae). ERIC sequences are 126 bp long and appear to be restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames. ERIC sequences are highly conserved at the nucleotide sequence level but their chromosomal locations differ between species. Several features of ERIC sequences resemble those of REP sequences (Stern et al., 1984) although the nucleotide sequence is entirely different. The question of whether ERICs have a specific function, or represent a form of 'selfish' DNA, is discussed. PMID- 1713282 TI - The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene. AB - A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical. PMID- 1713283 TI - Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same. AB - We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages. In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K. PMID- 1713284 TI - Structure and function of periplasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli. AB - The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunoblotting of subcellular fractions with an antiserum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone-like protein. Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilized this polypeptide and prevents its degradation by the cell envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed. PMID- 1713285 TI - Identification and assay of fibroblast growth factor receptors. PMID- 1713286 TI - Antiphosphotyrosine antibodies in oncogene and receptor research. PMID- 1713287 TI - The usefulness of the acridine-orange stain in identifying Helicobacter pylori in gastric biopsies. AB - The efficacy of the Acridine-orange stain (AOS) in identifying Helicobacter pylori (HP)-like organisms in biopsy smears from adults with gastroduodenal disease was studied. The results obtained indicate that AOS can replace Gram Stain in HP organism identification in gastroduodenal mucosa specimen. PMID- 1713288 TI - Human immunodeficiency virus type 1 infection of endothelial cells in vitro. AB - In order to establish whether endothelial cells are involved in immunodeficiency virus type 1 (HIV-1) infection, we performed a virological study on endothelial cells isolated from human adipose tissue and infected with HIV-1 in vitro. Supernatants from cultures showed a reverse transcriptase activity starting one day after HIV inoculation. Viral rescue was significantly impaired in cycloheximide treated cells confirming a de novo synthesis of viral products. PMID- 1713289 TI - Endocytosis constitute the infectious route of HIV-1 entry in human and rabbit monocytes lacking the CD4 receptor. AB - To obtain "functionally" CD4 negative human monocytes (0-5 CD4 +/1 x 10(6)/cells), 50 ng/5.10(5) cells of OKT4A were added daily after a pre incubation with OKT4A (100 ng/5.10(5) cells. In our experimental conditions the blocking the CD4 receptor of human monocytes with OKT4A monoclonal antibody did not prevent HIV-1 infection, although the level of virus replication appeared lower than that in cultures without OKT4A. "Naturally"CD4 negative rabbit monocytes infected with HIV-1 also released a detectable level of virus after 12 15 up 28-30 days. In "naturally" CD4 negative rabbit monocytes and "functionally" CD4 negative human monocytes, the virus particles entering via phagocytosis are not infectious because multiple well defined virions were observed in phagocytic vacuoles and the envelopes of these particles did not appear to interact with the vacuolar membrane. The infectious particles were represented by endocytic vesicles containing only the core of HIV after fusion between the viral envelope and endocytic membrane. Fusion between the viral envelope and plasma membrane on the cellular surface was never observed, in spite of examining greater than 1000 virions bound the surface of human and rabbit macrophage monocytes. The absence of cytopathic effect in the rabbit and human CD4 negative monocytes infected with HIV-1, and conversely the presence of specific sequences of HIV in the genomic DNA may indicate that the macrophages-monocytes serve as an important reservoir for the persistence of HIV in infected hosts, similar to the other related Lentiviruses. Our virological data have also demonstrated that virus infection can be transmitted from rabbit and human infected monocytes to uninfected H9 cells. This preliminary study may offer important evidence for the development and testing of vaccines and compounds that inhibit HIV penetration of susceptible cells. PMID- 1713290 TI - Maternal serum screening for neural tube defects and Down's syndrome. PMID- 1713291 TI - Differential regulation of myelin gene expression in SV40 T antigen-transfected rat glioma C6 cells. AB - Rat glioma C6 cells were stably transfected with a pSV3-neo plasmid containing SV40 T antigen gene, and geniticin-resistant transfectants (designated C6T cells) were cloned. The C6T cells grew as well-defined foci of cells showing squamous or irregular morphology. The doubling time for transfected cells was reduced by approximately 40% as compared to control C6 cells. The transfection with T antigen also affected the expression of genes coding for structural myelin proteins and for myelin-associated enzymes. The steady-state level of proteolipid protein (PLP)-specific mRNA in C6T cells was 44% lower than in parental C6 cells. On the other hand, the transfection upregulated the expression of myelin associated glycoprotein (MAG) by 153%. The activity of 2':3' cyclic AMP phosphodiesterase (CNP) was increased by approximately 80% in the C6T cells as compared to untransfected, control cells. The activity of calcium-activated neutral proteinase (CANP) was also significantly elevated in the transfectants by approximately 50% and 220% for millimolar and micromolar form respectively. The results indicate that T antigen affects the expression of myelin genes, although, individual genes appear to be differently regulated implying the existence of several independent regulatory mechanisms. PMID- 1713292 TI - Transcriptional effect of aflatoxin B1 on cytosine and/or hypoxanthine containing DNAs. AB - The effect of aflatoxin B1 (AFB1) on the template function for RNA synthesis of several single and double-stranded synthetic DNAs containing cytosine (C) and/or hypoxanthine (H) bases is studied in vitro. The results indicate that AFB1, after liver microsome activation, strongly inhibits the template function of poly[d(I C)] and has little, if any, effect on polydI.polydC, polydI, or polydC. This conclusion is reached whether rat liver nuclear free RNA polymerase or E. coli RNA polymerase is used for the transcription. The mechanism of this inhibition is believed mainly due to the inhibition of elongation of RNA synthesis, because autoradiography of the [alpha-32 P]GTP labeled RNAs after polyacrylamide gel electrophoresis clearly shows that the size of the RNA from AFB1 treated group is dramatically reduced. The evidence that the selective inhibition of poly[d(I-C)] template function is a direct reflection of the binding of AFB1 to poly[d(I-C)] is provided by the use of radioactive [3H]AFB1 for the binding and by spectrum analysis of the appearance of a broad AFB1-DNA adduct peak between 300 nm and 400 nm right after the typical DNA peak at 260 nm. These data, which are in direct support to our recent report (F.L. Yu, et al., Carcinogenesis, 11, 475-478, 1990), suggest that the binding of AFB1 prefers alternating, double-stranded DNA, and the binding affinity of AFB1 to DNA is greatly reduced when the bases are in either single- or double-stranded homopolymer forms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713293 TI - Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat. AB - Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10-30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p less than 0.05) and diaphragm (p less than 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p less than 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-I by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals. PMID- 1713294 TI - Epitope analysis of the oncofetal antigen alphafetoprotein using monoclonal antibodies. AB - Alphafetoprotein (AFP), an oncofetal antigen, plays very important roles in the early embryonic life and oncogenesis. Under various physiological and pathological conditions AFP exhibits microheterogeneity, probably as a result of differential expression of its epitopes. To analyse the epitopes we have developed a panel of monoclonal antibodies against human AFP purified by a new and efficient method using an immunoadsorbent consisting of polyclonal antibodies immobilized on cyanogen bromide activated Sepharose. Clones producing antibodies of various isotypes, e.g. IgG1, IgG2a, IgG2b, IgA and IgM have been subcloned and characterized. The antibodies showed high avidity for AFP (with half-maximal binding concentrations between 0.012 and 3.87 nM). Mutual inhibition efficiencies of a panel of 14 monoclonal antibodies were determined by RIA. Based on these inhibition data a computer program was used to group these antibodies with respect to their "epitope specificity distance". As a result of this grouping, clones have been identified which can recognize at least five different epitopes on AFP. This panel of antibodies may be very useful for analysis of the epitopic variation of AFP under various physiological and pathological conditions. PMID- 1713295 TI - Human and murine B-cells recognize the HBeAg/beta (or HBe2) epitope as a linear determinant. AB - The complete amino acid (aa) sequence of the hepatitis B virus (HBV) core protein (HBcAg), deduced from the genome of the HBV ayw subtype, was synthesized as decapeptides with five overlapping aas. The peptides were tested for reactivity with monoclonal antibodies (mAbs) to the beta (or HBe2) epitope of hepatitis B e antigen (HBe/b mAbs; 57/8, 78/3, 141/158 and 141/207). Cross-competition between the mAbs with a mAb to the HBe/alpha epitope (or HBe1) and an anti-HBc mAb showed that all the HBe/b mAbs specifically inhibited human anti-HBe/b binding. Screening the HBc/e peptides showed that all anti-HBe/b mAbs recognized a peptide covering the residues 126-135. Three of the mAbs, 78/3, 141/152 and 141/207, had a less restricted reactivity than the other two, suggesting the recognition of the HBe/b as a discontinuous determinant. Fine mapping of the region aa 126-135 was performed by synthesizing decapeptides with nine overlapping aas, covering residues aa 121-140. All mAbs, except 78/3, reacted with the linear sequence TPPAYR, at residues 128-133. An additional set of peptides was synthesized, where the six aas within the epitope 128-133 were substituted in turn by the other 19 possible aas. By this approach, the essential aas for mAb 57/8 were found to be the sequence of PPA at residues 129-131, and for mAb 141/158 the sequence PP-Y, at residues 129, 130 and 132, respectively. Human recognition of the linear HBe/b epitope was investigated by using a peptide covering residues 121-140 (p 33). Thirty-one sera from chronic carriers of HBsAg, of which seven were positive for HBeAg and the remaining 24 for anti-HBe, were investigated. Of the sera with HBeAg, two had low levels of anti/-HBe/b in the p 33 assay. Out of the sera with anti-HBe, eight were positive in the p 33 EIA. Thus, murine monoclonals and human sera may recognize the HBe/b epitope as a linear determinant residing around aa 130. PMID- 1713296 TI - Anti-idiotypic and anti-anti-idiotypic antibodies as highly specific tools in epitope and active site studies on human interferon-gamma. AB - Immunization of rabbits with F(ab')2 fragments of different monoclonal antibodies directed against human interferon-gamma yielded antisera with anti-idiotypic characteristics. Isolation of the anti-idiotypic fraction, resulting in a highly specific antiserum, allowed us to prove that out of six competing monoclonal antibodies directed against human interferon-gamma, only two really recognize the same epitope. The other monoclonal antibodies compete on the basis of steric hindrance, which is not surprising, because of the large difference in Mr between interferon-gamma and an immunoglobulin. The anti-idiotypes provided us also with a tool to study isolated epitopes on the human interferon-gamma molecule, a task which was previously not practicable. Exploration of the biological properties of these anti-idiotypes allows us to determine whether the investigated epitopes are involved in receptor binding. The production of an anti-anti-idiotypic antiserum not only proved that we generated real internal images, but also that these images conserved all of their properties, although with a decreased affinity in comparison with the original monoclonal antibody. As the former is a polyclonal antiserum, directed against a single epitope of the human interferon-gamma molecule, competition experiments yielded additional information on the relative position of three epitopes recognized by inhibiting monoclonal antibodies. These antisera will possibly open new ways for the affinity purification of interferon gamma and perhaps for the treatment of autoimmune diseases. PMID- 1713297 TI - [Polymerase and immunologic activity of reverse transcriptase of the HIV-1 virus, isolated from Escherichia coli]. AB - The recombinant reverse transcriptase of HIV-1 virus has been isolated from Escherichia coli cells transformed by the plasmid pRT40 DNA. The 103 Kd protein produced by these cells is shown to be processed to proteins with lower molecular masses by the reverse transcriptases own protease activity as well as Escherichia coli proteases. The resulting 103-66 Kd proteins possess the polymerase activity while 51 Kd and smaller proteins are lacking the activity. The 66 and 51 Kd reverse transcriptase fragments demonstrate the positive immunological reaction with the human blood serum from the people possessing antibodies to HIV-1 virus. The recombinant reverse transcriptase of HIV-1 produced by Escherichia coli cells is shown to be useful in AIDS diagnosis in humans. PMID- 1713298 TI - Expression site-associated genes transcribed independently of variant surface glycoprotein genes in Trypanosoma brucei. AB - Expression site-associated genes (ESAGs) of Trypanosoma brucei are found upstream of variant surface glycoprotein (VSG) genes in bloodstream expression sites. There are at least 6 different ESAGs in each of these expression sites, and each ESAG is repetitive in the genome. ESAGs are believed to reside only in VSG expression sites and to be co-transcribed with the VSG gene from a common alpha amanitin-insensitive promoter. Our results show that this is not always true. The transcriptionally active 1.22 metacyclic expression site contains no ESAGs, but ESAGs are highly transcribed in these cells. The level of transcription indicates that more than one copy of each of these genes is active. Furthermore, some of these genes are transcribed, to produce steady state RNA, in procyclic culture cells which do not express the VSG gene: there is differential expression of ESAGs between the bloodstream and procyclic phases of the trypanosome life cycle. Thus ESAGs can be transcribed outwith an active VSG gene expression site and in the absence of expression of the VSG. PMID- 1713299 TI - Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis. AB - BACKGROUND: Spirochetes are commonly associated with periodontal disease, but it is not known whether these treponemes are pathogenic or merely opportunistic. We sought to determine whether spirochetes present in periodontal disease share antigens thought to be unique to spirochetes that are known pathogens. METHODS: We examined dental plaque from 24 healthy subjects, from ulcerative sites in 17 patients with ulcerative gingivitis, and from areas of involvement in 19 patients with chronic periodontitis, using an immunocyto-chemical technique with monoclonal antibodies against pathogen-specific determinants on 47-kd and 37-kd molecules from Treponema pallidum subspecies pallidum. Serum was tested against T. pallidum by immunoblotting and by serologic assays for syphilis. RESULTS: Spirochetes with a pathogen-specific epitope on a 47-kd molecule were not found in plaque samples from any of the 24 healthy subjects, but they were identified in plaque samples from 11 of 17 patients with ulcerative gingivitis (P less than 0.001) and from 10 of 19 patients with periodontitis (P less than 0.01). Monoclonal antibodies directed against a 37-kd molecule reacted with spirochetes in plaque samples from 1 of 14 controls, from all 11 patients with gingivitis from whom samples could be obtained (P less than 0.001), and from 14 of 19 patients with periodontitis (P less than 0.001). Five of 18 normal subjects had IgG against 47-kd and 37-kd molecules, but none had IgG against 14-kd or 12-kd molecules from T. pallidum subspecies pallidum. Among 19 patients with ulcerative gingivitis, IgG was identified against 47-kd molecules in 15, against 37-kd molecules in 12, against 14-kd molecules in 4, and against 12-kd molecules in 15. CONCLUSIONS: The spirochetes found in dental plaque from patients with ulcerative gingivitis or chronic periodontitis have antigens that are thought to be unique to pathogenic treponemes. This close antigenic relation suggests that T. pallidum or a closely related organism may be involved in the pathogenesis of periodontal disease. PMID- 1713300 TI - Subcutaneous granulocyte colony-stimulating factor and acute anaphylaxis. PMID- 1713301 TI - Screening the keratinolytic activity of dermatophytes in vitro. AB - Sixteen strains out of 12 species dermatophytes were examined in respect to their ability of utilizing keratin substrates as the only sources of C and N. The employed keratin substrates included a solubilized preparation of feather keratin (KS) and native keratin, guinea pig hair and chicken feathers. It has been shown that the preparation KS constitutes a convenient model for a preliminary estimation of fungal keratinolytic activity and it can be a source of information about the localization of these enzymes. It has been found that, among the 16 fungal strains, 13 strains synthesize mainly intracellular keratinases while 3 strains of T. verrucosum release enzymes mainly to the medium. Native keratin from hair and feathers was degraded only by some of the examined strains which, under the experimental conditions, developed characteristic spore forms. Keratin of guinea pigs hair was attacked only by the T. mentagrophytes strains, T. verrucosum and K. ajelloi, and only T. gallinae grew on native keratin from chicken feathers. PMID- 1713302 TI - Cystic fibrosis. Channelling our thoughts. PMID- 1713303 TI - Serum antithrombin III and alpha-2-antiplasmin concentrations in patients with Hodgkin's disease in the course of chemotherapy. AB - Disturbances in hemostasis in cancer disease frequently occur. This clinical situation may contribute to the spread and metastasis of cancer. The determination of inhibitors controlling hemostasis especially during treatment may be of value for the evaluation of effectiveness of treatment and balancing of hemostasis. Twenty four patients diagnosed with Hodgkin's disease were introduced to our studies. The level of antithrombin III (AT-III) and alpha-2-antiplasmin (alpha-2-AP) in sera were determined before, during and after treatment with MOPP. We found decrease of AT-III concentration before treatment, and chemotherapy increased the level of this inhibitor but still below the level of control. Alpha-2-AP concentration was higher in patients and MOPP application decreased the level below the values found in controls. The determination of concentration of both inhibitors may be important for the evaluation of the effect of treatment in understanding how the disturbances of hemostasis may be equilibrated. PMID- 1713304 TI - Molecular and biological properties of hamster tumor cell lines transformed with B77 virus: expression of v-src does not correlate with metastatic potential. AB - Three hamster tumor cell lines (B77Hep, B77H1De and ML cl 3.1) were investigated with the aim to determine some biological and molecular properties of cells which are connected with invasive and metastatic ability. Except B77H1De all cell lines exhibited high metastatic capacity in syngeneic adult animals. Analysis of cell lines revealed a relationship between metastatic ability and growth properties (growth rate, saturation density and colony formation in soft agar). Southern blot analysis of genomic DNA samples from high metastatic cell line (B77Hep) and low metastatic (B77H1De) showed that the metastatic potential of these cell lines did not depend on the number of integrated proviral copies. Northern blot analysis was used to determine the level of mRNA encoded by v-src gene in B77Hep and B77H1De cell lines. We found a good correlation between the number of integrated proviral copies and the level of v-src gene expression in investigated cell lines, but not with their metastatic potential. No proviral sequences were found in genomic DNA isolated from ML cl 3.1 cell line. In cell lines used in this study we found differences in expression of endogenous proto-oncogenes c-myc and c-fos. PMID- 1713305 TI - [Neurologic aspects of clinical manifestations, pathophysiology and therapy of reflex sympathetic dystrophy (causalgia, Sudeck's disease)]. AB - The symptomatology of reflex sympathetic dystrophy (RSD), a diagnostic term which today includes causalgia and M. Sudeck, is characterized clinically by a triad of autonomic (sympathetic), motor and sensory disturbances. They develop following a noxious event--though independent of its nature and location--in a generalized distribution pattern at the distal site of the affected extremity. Pathophysiologically, a complex disturbance of the sympathetic vasoconstrictor system is involved, which mediates the dominant symptoms of RSD, namely the spontaneous pain and the swelling. This disturbance is thought to be initiated by nociceptive impulses, occurring in conjunction with the preceding noxious event, and to be maintained reflexly, in a form of a vicious circle, by means of the typical pain sensation accompanying the RSD-syndrome. From these ideas, an important part of the RSD therapy is deduced; i.e. the early interruption of the neuronal sympathetic activity by means of a sympathetic blockade. Such a blockade can interrupt the pain and at the same time also the vicious circle of RSD. Altogether, for the RSD syndrome there are relevant neurological aspects with respect to its clinical symptomatology, its pathophysiology and its therapy. PMID- 1713306 TI - Association of HLA-DQw4 with IgA nephropathy in the Japanese population. AB - We have studied HLA-A, B, DR and DQ phenotypes in 50 Japanese patients with IgA nephropathy. The antigen frequency of HLA-DQw4, which is known to be associated with DR4/Dw15 specificity, was increased in the patient group as well as in those of B35 and DR4. The increase of DQw4 (36.0%) was significantly different from that of national controls (corrected p less than 0.004). PMID- 1713307 TI - Heat shock protein expression in corpora amylacea in the central nervous system: clues to their origin. AB - Small bodies expressing epitopes of the 72 kD heat shock protein (HSP) have been identified in the brain and spinal cord in normal and neurologically abnormal individuals. These bodies resemble the 'pre-corpora amylacea' (pre-CA), thought to be the primary structure in the development of the mature body. Corpora amylacea are laminated hyaline bodies composed of polyglucosans. They are found in larger numbers with increasing age in the brain and spinal cord. Mature, histologically 'classical', corpora amylacea express epitopes of HSP chiefly at the periphery of the corpus, whilst smaller immature 'pre-corpora' stain intensely throughout the entire structure. A heat shock or stress response in neurons and glial cells may be part of the cellular reaction to accumulation of abnormal products. PMID- 1713308 TI - Cell proliferation in intracranial tumours: selective silver staining of nucleolar organizer regions (AgNORs). Application to surgical and experimental neuro-oncology. AB - A novel tool in diagnostic and experimental pathology, the AgNOR-technique, which consists of visualization of ribosomal gene activity by selective silver staining, was applied to 144 cytological specimens of human tumours of the nervous system. The number of silver-stained nucleolar organizer regions (AgNORs) was correlated with the biological behaviour of the tumours investigated; low AgNOR number were observed in benign neoplasms such as meningiomas and schwannomas and higher AgNOR numbers in glioblastomas and metastases. The mean AgNOR number per cell was 3.15 in astrocytomas, 4.5 in anaplastic astrocytomas and 5.86 in glioblastoma multiforme. Benign and malignant lesions showed different distribution patterns of AgNORs, with few but centrally located AgNORs in benign, and multiple but scattered AgNORs in malignant tumours. AgNOR number per cell and AgNOR area revealed an inverse relationship (correlation coefficient -0.15, linear regression). In addition to the human tumours, two N-nitroso-N ethyl-urea (NEU) induced tumors in BD-IX rats a mixed glioma (G-XIII) and a malignant schwannoma (N-XII), were investigated. Twelve G-XIII gliomas revealed homogenous AgNOR-counts (standard error of the mean less than 10%), with absolute values between the values obtained for human glioblastomas and metastases. Seven N-XIII subcutaneously transplanted schwannomas revealed higher AgNOR values than human schwannomas, but lower than experimental gliomas. It is concluded that the AgNOR method, as a technique for visualization of ribosomal gene activity, is valuable for assessing proliferative activity and malignancy in both diagnostic and experimental neuropathology. PMID- 1713309 TI - Granulomatous angiitis and cerebral amyloid angiopathy presenting as a mass lesion. AB - A woman, who presented with clinical and radiological signs of a right temporal mass suggestive of a brain tumour, was found to have granulomatous angiitis associated with cerebral amyloid angiopathy; the diagnosis was confirmed by biopsy. She is still well 13 years after excision of the lesion. The association of granulomatous angiitis and cerebral amyloid angiopathy constitutes a peculiar variety of central nervous system micro-angiopathy. Only a few similar cases have been described. PMID- 1713310 TI - Infective acute transverse myelopathy. Report of two cases. AB - Two children with acute transverse myelopathy following adenovirus and Borrelia Burgdorferi infections are presented. The diagnosis stems from the clinical presentation, the determination of specific antibodies in serum and the favorable response to penicillin treatment in the case of neuroborreliosis. Both children made a good recovery. The cerebrospinal fluid examination showed a highly increased myelin basic protein concentration, indicating demyelination. PMID- 1713311 TI - The psychomotor development during the first year of life of infants exposed to intrauterine alcohol of various duration. Fetal alcohol exposure and development. AB - The developmental abilities of 80 children exposed to alcohol of various duration in utero were assessed 1 to 3 times during their first year of life. The occurrence of developmental delay increased towards the end of the first year. The longer the exposure the more common and severe was the developmental delay. If alcohol consumption could be reduced by the second trimester only a slight abnormality of motor development was seen at the age of 12 months. If heavy maternal drinking continued throughout the second trimester cognitive development was also delayed. Psychomotor retardation by the age of one year occurred in 38% of children who had been exposed to continuous heavy alcohol consumption during pregnancy. The psychomotor retardation diagnosed at the age of one year could be found earlier in 80% of the children but pure motor or pure cognitive delay only in some cases. The beneficial effect of reducing maternal alcohol consumption before the last trimester of the child's development was clear. PMID- 1713312 TI - Localization of the lumbar pools of motoneurones which provide hindlimb muscles in the rabbit. AB - The localization of the pools of motoneurones (Mns) to the main hindlimb muscles was performed in the rabbit, using the retrograde transport of HRP from motor end plates. After 48 h survival time, the large alpha Mns were labeled. All the pools were met inside L6-S2 limits and a functional organization was observed: the pools to proximal muscles formed a ventral group of Mns and the pools to distal muscles a dorsal group, with flexor and extensor pools apart. The relative disposition of the different pools fits with that described in the cat and rat. PMID- 1713313 TI - Reduction of the amount of neurotensin retrogradely transported in dopaminergic neurons of senescent rats. AB - It is now clearly established that fast anterograde axonal transport can be altered during ageing, both in the central and the peripheral nervous systems, but no information is yet available concerning the modifications of fast retrograde axonal transport during senescence. In the present paper, we report the changes occurring in the retrograde axonal transport of neurotensin in dopaminergic neurons of old rats. When iodinated neurotensin was injected into the striatum, a diminution of approximately 50% in the amount of the labelling measured in the ipsilateral substantia nigra was observed in senescent rats by comparison with young adult rats. Nevertheless, the rate of neurotensin transport was not modified. Our results clearly indicate that less neurotensin is transported from the nerve terminals towards the cell bodies in senescent rats which may have possible consequences for dopaminergic neurons. PMID- 1713314 TI - Neuropeptide Y mRNA regulation in rat sympathetic ganglia: effect of reserpine. AB - To study the mechanism underlying trans-synaptic neuropeptide regulation, mRNA levels of neuropeptide Y were examined in the rat stellate and superior cervical ganglia using a specific neuropeptide Y cRNA probe. Basal levels of neuropeptide Y mRNA were detectable in total RNA extracts from single ganglia. Reserpine induced a large rise in ganglion neuropeptide Y mRNA. Decentralization prevented the increase of neuropeptide Y mRNA content in the ganglia. This suggests that the reserpine induced increase in neuropeptide Y mRNA was dependent on transsynaptic stimulation. Consequently, neuropeptide mRNA levels in sympathetic ganglia may be under the control of preganglionic impulse flow. PMID- 1713315 TI - Bacterial endotoxin-induced expression of metallothionein genes in rat brain, as revealed by in situ hybridization. AB - In order to clarify acute-phase response in brain, we investigated induction of metallothionein (MT) genes by administrating an endotoxin (lipopolysaccharide) in rat intraperitoneum. We performed in situ hybridization on the serial brain sections to identify the cells expressing the MT genes in acute-phase. After endotoxin administration, transcripts of MT genes were detected in the arachnoideal, ependymal cells and glial cells around the Purkinje cells of the cerebellum, while no significant induction of the MT genes by zinc ion was observed in brain. These results suggest that the acute-phase response occurs specifically in at least these 3 non-neuronal cells. PMID- 1713317 TI - Effect of neurokinin A, substance P and calcitonin gene related peptide in peripheral hyperalgesia in the rat paw. AB - The effect of neurokinin A (NKA), substance P (SP) and calcitonin gene-related peptide (CGRP) in peripheral hyperalgesia was studied in rats using a modification of the Randall-Selitto paw test. NKA was 10 times more potent than SP which was 500 times more potent than CGRP in inducing hyperalgesia in the rat paw, suggesting that NKA and SP but not CGRP could have an important role in acute hyperalgesic conditions. Furthermore, sensitization induced by several injections of subthreshold doses of NKA or CGRP suggest that these neuropeptides along with SP could participate as mediators or modulators of chronic pain. PMID- 1713316 TI - The acute convulsant effect of MPTP is dependent on intracerebral MPP+. AB - The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57 black mice causes an acute seizure syndrome the severity of which is dose dependent; there is also a good correlation between the seizure inducing potential of MPTP and the neostriatal dopamine (DA) depletion caused by MPTP. The simultaneous administration of MPTP and MAO B inhibitors attenuates both epileptiform phenomena and neostriatal DA depletion. On the contrary diethyldithiocarbamate (DDC) exacerbates both responses. All these pharmacological manipulations are known to affect the accumulation of 1-methyl-4 phenylpyridinium ion (MPP+) the main metabolite of MPTP. Thus the present data support the hypothesis of a strict dependence of the epileptiform phenomena on the presence of MPP+. Furthermore the tight correlation existing between the severity of epileptic events and DA depletion suggests that the acute excitotoxic syndrome may contribute to the long-term toxicity of MPTP. *On leave from the Department of Neurology, University of Pisa, Pisa, Italy. PMID- 1713318 TI - Astrocytes are responsive to endothelium-derived relaxing factor (EDRF). AB - The effects of endothelium-derived relaxing factor (EDRF) and sodium nitroprusside (SNP), which produces nitric oxide (NO) spontaneously, on cultured astrocytes prepared from cerebra of rat embryos were examined. EDRF produced by vascular endothelial cells in response to bradykinin stimulation, caused a significant rise in cyclic GMP levels of astrocytes. SNP also increased cyclic GMP levels of astrocytes in a dose-dependent way. These results revealed for the first time unambiguously that astrocytes are responsive to EDRF. PMID- 1713319 TI - The efferent innervation of the suboccipital muscles in the guinea pig: a study with retrograde transport of horseradish peroxidase. AB - The location and quantitative parameters of the motoneurons of three suboccipital muscles are studied by means of retrograde transport of horseradish peroxidase (HRP). Injections of HRP into the M. obliquus capitis superior and into the M. rectus capitis posterior maior result in labeling of somata within the first cervical spinal segment. Injections into the M. obliquus capitis inferior result in labeling within the second cervical spinal segment. All somata are situated within or close to the ventromedial nucleus and show average diameters of about 20-25 microns. Conclusions on the innervation of the suboccipital muscles are drawn. PMID- 1713320 TI - Patterns of corticothalamic terminations following injection of Phaseolus vulgaris leucoagglutinin (PHA-L) in the sensorimotor cortex of the rat. AB - The morphology and spatial distribution of terminals emitted by corticothalamic axons originating from the rat motor cortex (as defined by intracortical microstimulations) were studied using Phaseolus vulgaris leucoagglutinin (PHA-L) as an anterograde tracer. After PHA-L injection in the face, forelimb or hindlimb motor cortical areas, small and densely packed boutons (about 1 micron in diameter), en passant and terminaux, were seen in the ventrolateral nucleus of the thalamus and, more sparsely, in the reticular nucleus, the nucleus ventrobasalis and the posterior nucleus of the thalamus. A separate projection with giant boutons (5-10 microns in diameter), en passant and terminaux, terminated in the posterior nucleus of the thalamus exclusively. Giant boutons originated from corticothalamic axons distinct from those providing small boutons. The corticothalamic projection originating from the motor cortex has basic organizational properties comparable to previous data obtained in the auditory and somatosensory corticothalamic projection systems. PMID- 1713321 TI - [The histoarchitectonics of the uveal tract vessels in the rabbit eye in the dynamics of the burn process]. AB - By means of injecting soot into the vessels of the uveal tract of the rabbit eye the character of changes in the architectonics of the vascular bed in dynamics of the burn process could be detected. It was found that a burn trauma produces changes in practically the whole uveal tract, but to a greater extent, in its anterior segment. Reduction of blood filling of the vessels or their total segmentary "switching-off" is connected with the development of reversible (thrombosis, oedema of endotheliocytes and surrounding stroma, hemolysis) and irreversible (destruction of the vascular wall, obliteration of vessels) structural changes. The degree of disturbances in blood circulation and reversibility of these changes depend on the severity of the burn. In case of less severe burns, total structural restoration of the vascular network in the nearest time is recorded, while in severe burns--partial restoration with elements of vascular net reconstruction of adaptive character. PMID- 1713322 TI - Dentistry and ethics: a call for attention. PMID- 1713323 TI - Effects of ATP antagonists on purinoceptor-operated inward currents in rat phaeochromocytoma cells. AB - The effects of suramin, reactive blue 2 (RB2) and d-tubocurarine (d-TC) were investigated electrophysiologically to elucidate the mechanisms underlying their antagonism of P2 purinoceptor-mediated responses. All three compounds inhibited an adenosine triphosphate (ATP)-activated inward current in rat phaeochromocytoma PC12 cells in a concentration-dependent manner. The order of potency was RB2 greater than suramin greater than d-TC. The inhibition induced by suramin or RB2 was reversible, whereas that induced by d-TC was not reversed after a 5-min rinse. The inactivation of the ATP-activated current was accelerated by d-TC but not by suramin or RB2. RB2 administered simultaneously with ATP exerted much weaker inhibition compared to that induced by prior administration, suggesting that RB2 is a slowly acting antagonist. This was not observed for suramin or d TC. Suramin and RB2 caused a parallel shift in the concentration/response curve for the ATP-activated current. With d-TC the maximal response of ATP was decreased but the concentration producing half-maximal response was unchanged. The voltage dependency of the ATP-activated current showed less inward rectification in the presence of d-TC. Suramin or RB2 did not affect the voltage dependency. These results suggest that suramin and RB2 reversibly block binding of ATP to receptors, whereas d-TC blocks ion permeability through the ATP activated channel. PMID- 1713324 TI - Alterations of ionic currents after reoxygenation in isolated cardiocytes of guinea-pigs. AB - Single myocytes were isolated from ventricles of adult guinea-pig hearts. The patch-clamp technique in the whole-cell configuration was used to study ionic currents. Experiments were performed in an experimental chamber that allowed the cells to be exposed to a sufficiently low O2 pressure to cause metabolic inhibition after 4-35 min (mean 14.1 min, n = 20), which was indicated by the appearance of a large time-independent K current. Reoxygenation about 1 min after the first extra outward current was observed caused this current to vanish completely within 2-6 s if the calcium inside the pipette was buffered to negligible values with 20 mmol/l EGTA. With only 10 microM EGTA in the pipette, reoxygenation was followed by an arrhythmogenic period of 10-150 s duration, which was dominated by three types of event: (a) transient inward currents (Iti) developed during the first 5-10 s (26 cells); (b) the net current was increased by a factor of 1.9 +/- 0.4 (mean +/- SD, n = 17) yielding a reversal potential for the increased component of -77 +/- 4 mV (mean +/- SD, n = 4); and (c) the Ca current decreased by 20%-100% within the first 5-10 s. At the end of the arrhythmogenic period, Iti vanished, the net current recovered completely, and the Ca current recovered partially. At -45 mV, increasing preceding depolarization enlarged the amplitude of both the Iti and the net current, Iti being about four times more increased than the net current. The suppression of the Ca current was independent of the phase of the preceding Iti. We conclude that in isolated cardiocytes, after the induction of an anoxia-induced K current, reoxygenation causes a period of up to 150 s of cytosolic Ca overload, during which Iti is triggered, the net current is enhanced, and the Ca current is suppressed. PMID- 1713325 TI - Social support needs of family caregivers of psychiatric patients from three age groups. AB - Sixty family caregivers of child, adult, and elderly psychiatric patients were interviewed to determine their unique and common support needs. Content analysis of interview data was used to identify support categories and their properties. Support needs expressed by caregivers paralleled the general types of support described in the social support literature (emotional, feedback, informational, and instrumental); however, for many categories under each general type the specific meaning of the support was directly linked to the psychiatric caregiving role. Most of the differences in support needs among caregivers from the three age groups reflected the caregivers' stage of life and the length of time they had been caregiving. The group of caregivers of adult patients reported having the least support. Although many support needs were expressed, the needed support did not exist. PMID- 1713326 TI - [Monoclonal antibodies to insulin influencing its biological activity]. AB - Somatic hybridization was employed for obtaining 335 hybridomas producing monoclonal antibodies to insulin. Twelve hybridomas were cloned by a method of maximum dilutions, and specific immunoglobulins, secreted by them, were characterized by solid phase immunoenzyme assay. Monoclonal antibodies possessed different activity with relation to human, porcine and cattle insulins, indicating their specificity to different epitopes on the insulin molecule. Antibodies of IN-2 and IN-3 clones were capable of inhibiting insulin biological activity in vivo. In experiments on rabbits antibodies of IN-2 clone decreased a 3-fold insulin hypoglycemic action, and antibodies of IN-3 clone caused an increase in the life span of mice after administration of insulin at high doses to them. PMID- 1713327 TI - Characterization of His-X3-His sites in alpha-helices of synthetic metal-binding bovine somatotropin. AB - Variants of bovine somatotropin have been engineered to contain synthetic metal binding sites consisting of two solvent-exposed histidines separated by a single turn of an alpha-helix (His-X3-His variants). The affinities of these proteins for Cu(II) were characterized by measuring their partition coefficients in an aqueous two-phase polymer system. The partition coefficients were used to generate binding constants for formation of a complex between the engineered metal-binding site and Cu(II) chelated to an iminodiacetic acid derivative of polyethylene glycol. For three His-X3-His variants described here, these constants range from 2 x 10(4) to 1.6 x 10(6) M-1. The metal affinity of a His-X3 His site depends on the rigidity of the helix into which the site is engineered. The affinities of the His-X3-His sites for Cu(II) are large enough to dramatically increase not only the partitioning of these proteins in aqueous two phase systems, but also their retention times on a metal-affinity chromatography column. Both these features can greatly facilitate the purification of engineered proteins. Criteria for choosing positions for incorporating metal-binding sites are discussed. PMID- 1713328 TI - Inhibition of iloprost of the contractile effect of noradrenaline in mesenteric artery rings: evidence for a possible calcium-dependent mechanism. AB - Iloprost caused a concentration-dependent decrease in the response to noradrenaline in the rabbit isolated endothelium denuded rings from superior mesenteric artery but not thoracic aorta. Similar inhibition was obtained by verapamil using identical concentrations. In Ca(2+)-free EGTA containing medium noradrenaline both at lower and higher concentrations elicited a reduced contractile response and further addition of Ca2+ (2.5 mM) to the medium produced a second contraction in both mesenteric artery and aortic rings which was significantly and equally inhibited by iloprost and verapamil using identical concentrations in mesenteric artery but not in aortic rings. Prior addition of iloprost to the medium did not protect the inhibitory effect of phenoxybenzamine against noradrenaline-induced contraction. These results were taken as an evidence for the possible Ca2+ entry reducing effect of iloprost in mesenteric artery but not thoracic aorta. These results were also taken as an indirect evidence supporting the hypothesis that increased synthesis of prostacyclin by noradrenaline in the vascular wall may inhibit the contractile effect of the agonist by a (-) feedback mechanism mediated by Ca2+ entry into the vascular smooth muscle. PMID- 1713329 TI - Extracellular matrix molecules in development and regeneration of the leech CNS. AB - As neurons grow to their targets their processes elongate, branch and form specialized endings into which are inserted appropriate ion channels. Our aim has been to analyse the role of the extracellular matrix molecules laminin and tenascin in inducing growth and in determining the form and physiological properties of growing neurites. A preparation in which development and regeneration can be followed at the cellular and molecular level in the animal and in tissue culture is the central nervous system (CNS) of the leech. In leech extracellular matrix (ECM) both laminin and tenascin are present; the molecules are structurally similar but not identical to their vertebrate counterparts. Tenascin extracted from leech ECM shows a typical hexabrachial structure whereas laminin shows a typical cruciform structure in rotary shadowed preparations. Leech laminin purified by means of a monoclonal antibody is a molecule of about 1000 kDa, with a polypeptide composition of 340, 200, 180 and 160 kDa. Substrates that contain tenascin or laminin produce rapid and reliable outgrowth of neurites by identified cells. A remarkable finding is that the outgrowth pattern produced by an individual neuron depends in part on its identity, in part on the substrate upon which it is placed. For example, a Retzius cell grows in a quite different configuration and far more rapidly on laminin substrate than does another type of neuron containing the same transmitter (serotonin); and the pattern of outgrowth of the Retzius cell is different on laminin and on the plant lectin Con A (concanavalin A). Thus Con A induces the growth of processes that are shorter, thicker, more curved and contain fewer calcium channels than those grown on laminin. To determine whether laminin can also influence neurite outgrowth in the animal, immunocytological techniques have been used to follow its distribution in the extracellular matrix of normal, developing and regenerating leech CNS. In adult leeches neuronal processes in the CNS are not in contact with laminin which is confined to the surrounding extracellular matrix. In embryos however, laminin staining appears between ganglionic primordia along the pathways that neurons will follow. Similarly, after injury to the adult CNS, laminin accumulates at the very sites at which sprouting and regeneration begin. How the laminin becomes redistributed to appear in the region of injury has not yet been established. Together these findings suggest a key role for laminin and for other extracellular matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713330 TI - Survival of diploid yeast cells to bleomycin in combination with UV-light or hyperthermia. AB - In the present work we have investigated the possible interaction between Bleomycin (B) and UV light or hyperthermia (HT) in the induction of lethal events in diploid yeast populations in the stationary phase of growth. UV and B acting as single agents determine sigmoid survival curves. The combination of UV + B produces different degrees of sensitization depending on dose ranges. For [B] = 7.5 micrograms/ml combined with different UV fluences an exponential course is observed, suggesting overlapping lesion specificity of the involved repair pathways (excision and recombination). The hyperthermia plus Bleomycin treatment produces different degrees of inactivation depending on the sequences. Maximal inactivation effect was obtained for the sequence B + HT. In the case of HT + B ([B] greater than 7.5 micrograms/ml) the obtained sensitization is lower. PMID- 1713331 TI - Anatomical relationship between neuropeptide-containing fibers and efferent vagal neurons projecting to the rat corpus. AB - Injections of the retrograde tracers into the posterior surface of the stomach at the greater curvature resulted in labelling of the right half of the dorsal motor nucleus of the vagus. Whereas injections into the anterior and posterior surfaces of the corpus resulted in bilateral labelling in the medulla. Immunocytochemical staining of the labelled sections using antisera to substance P was confined to a dense network of fibers within the dorsal motor nucleus of the vagus and the nucleus tractus solitarius with no cell bodies being detected. Calcitonin gene related peptide-immunoreactivity was detected in nerve fibers in the nucleus tractus solitarius and cell bodies of the hypoglossal nucleus. Finally, neuropeptide Y-immunoreactivity was confined to nerve fibers within the vagal complex. Of the neurons labelled by the retrograde tracers injected into the corpus all were in close spatial contact with fibers containing substance P immunoreactivity. A smaller number were associated with neuropeptide Y-containing fibers with a few adjacent to calcitonin gene-related peptide-immunoreactive fibers. These results indicate that substance P and neuropeptide Y may directly regulate efferent neurons controlling gastric motility and acid secretion. Calcitonin gene-related peptide, however, is unlikely to directly modulate the cell bodies of the neurons in the dorsal motor nucleus but may modulate the dendrites from these neurons in the nucleus tractus solitarius. PMID- 1713332 TI - Increases in NPY in non-sympathetic nerve fibres supplying rat mesenteric vessels after immunosympathectomy. AB - The effect of nerve growth factor (NGF) deprivation on developing peripheral peptide-containing nerves has been examined in Wistar rats. Animals were treated from birth for 7 days with antibodies to NGF (10 microliters/g body weight) and killed at 4 or 8 weeks of age. The nerves of the mesenteric and femoral blood vessels, vas deferns and bladder were viewed with histochemical and immunohistochemical techniques. The effectiveness of anti-NGF treatment was monitored by viewing catecholamine (CA)-containing nerves, which were virtually absent from the blood vessels, but were little affected in the vas deferens and bladder in both age groups. Immunoreactivity for substance P and calcitonin gene related peptide was slightly reduced in the blood vessels. Immunoreactivity for neuropeptide Y (NPY) was reduced in the femoral blood vessels by 88% at both ages, but reductions in NPY immunoreactivity (NPY-IR) in the mesenteric vessels varied with age. In the mesenteric artery at 4 weeks, NPY-IR was reduced by 96% from control values, but at 8 weeks it was reduced by only 37%. Acute sympathectomy with 6-OHDA treatment reduced NPY-IR in the mesenteric artery by 98% at 4 weeks and 93% at 8 weeks. It is proposed that the increase in NPY-IR but not CA-containing nerves in the mesenteric artery between 4 and 8 weeks after immunosympathectomy is due to compensatory innervation from a non-sympathetic source (probably enteric neurons) that is available to mesenteric, but not to femoral blood vessels. PMID- 1713333 TI - [Importance of specific prostatic antigen to prostatic volume ratio in the selection of patients for ultrasonography-guided biopsy of the prostate]. AB - US-guided biopsy was performed in 94 patients with suspected lesions at transrectal US. Histology demonstrated carcinoma in 43 cases, benign hyperplasia in 44, and prostatitis in 7. In all cases the prostate specific antigen (PSA) was calculated, by means of US, together with prostatic volume (V). PSA was related to the corresponding gland volume, which resulted in PSA/V index. Subsequently, histology was correlated with both PSA value and PSA/V ratio. Our study showed PSA/V ratio to have higher sensitivity and specificity than absolute PSA value in the diagnosis of prostatic carcinoma. The authors believe prostate US-guided biopsy to be: a) necessary when the suspected area has PSA/V ratio greater than 0.15, and especially when PSA/V greater than 0.30; b) not indicated when echostructural alterations are associated with PSA/V less than 0.15, because they are most frequently due to benign lesions. The combined use of PSA/V ratio and US is therefore suggested to select the patients in whom biopsy is to be performed. PMID- 1713335 TI - Second international symposium on Supportive Care in Cancer Patients. March 1-3, 1990, St. Gallen, Switzerland. PMID- 1713334 TI - [Pulmonary hyalinizing granuloma. Apropos of 2 new cases]. AB - Pulmonary hyalinising granuloma are nodular or localised fibrosing lesions of the pulmonary parenchyma and are single or multiple. We report two new cases of this disorder which is rare, as only 62 cases have been published in the literature. It is a pathology with few symptoms, sometimes revealed by general signs. Radiologically there are nodules which are most often round or oval, intra parenchymal, well demarcated, single or multiple. The histological appearance is characteristic: the centre of the granuloma consists of a network of dense collagen fibres which are lamellar separated by clear spaces; the periphery is the seat of rich cellular infiltration of plasmocytes and lymphocytes in the peri vascular region. Most often there is a spontaneous benign outcome. The frequent association of pulmonary hyalinising granuloma in fibrotic disorders and the similarity in the histological appearance leads to the hypothesis of a common pathogenesis in these disorders. PMID- 1713336 TI - Optimizing palliative surgical support of cancer patients with visceral obstructions. PMID- 1713337 TI - The potential for the use of colony-stimulating factors in autologous bone marrow transplantation. PMID- 1713338 TI - News in managing cancer pain: an overview. PMID- 1713339 TI - Socioeconomic aspects of an implantable drug delivery device. PMID- 1713340 TI - Palliative care: a new reality in medicine. PMID- 1713341 TI - Cure and care: interaction between cancer centers and palliative care units. PMID- 1713342 TI - Optimizing palliative nursing skills by education. PMID- 1713343 TI - Education and palliative care: a different approach. PMID- 1713344 TI - Palliative care in German hospice: the medical and psychological concept of the Christophorus-Haus. PMID- 1713345 TI - Spiritual support and palliative cancer care. PMID- 1713346 TI - Correlation of serum interferon with some clinical and humoral signs of systemic lupus erythematosus. AB - The serum level of total interferon (IFN) was measured in 15 male patients with systemic lupus erythematosus (SLE) in the active phase and in remission, before and during corticotherapy. The values found were correlated with the clinical and humoral signs of disease. The IFN titer was high in the active phase of disease and was correlated with fever, extension of skin rash, polyarthritis, myositis, autoimmune hemolysis, cardiac and cerebral involvement as well as with ESR, reacting protein C, CIC, ANF and the percentage of LE cells. Isolated LE nephropathy without rapidly progressive or advanced renal failure was not associated with high IFN titer. PMID- 1713347 TI - [Therapy of immune thrombopenia]. AB - Idiopathic thrombocytopenic purpura (ITP) belongs to the family of autoimmune diseases. The term "idiopathic", however, is no longer correct as it is in fact an immunologically-related thrombocytopenia. This is why nowadays it is referred to as immune thrombopenia. Clinically the acute and chronic forms of ITP can be distinguished. We discuss the different forms of treatment based upon data provided by various studies of ITP. If treatment with prednisone or with gammaglobulins fails, or after unsuccessful splenectomy, then alternative experimental therapies may have to be used. Some of these treatments are described with reference to their therapeutic benefit and their function. PMID- 1713348 TI - [Homonymous lateral hemianopsia as a presenting sign of intracranial extracerebral cavernous hemangioma]. AB - A case of intracranial extracerebral cavernous hemangioma of the middle fossa in a woman who suffered from a multiple sclerosis, is reported. Appearance of a lateral hemianopia suggested an other disease than multiple sclerosis to explain this symptom. Intracranial extracerebral cavernous hemangioma is a rare tumor reported more often in the middle fossa than in other sites. It usually produces symptoms by compression of nearby structure. Diagnosis is based on neuroradiological examination and we describe the MRI aspect. It is difficult to remove because of massive hemorrhage. PMID- 1713349 TI - [Grief and suicidal behavior after losing a close person in endogenous and neurotic reactive depression in advanced age]. AB - Pathological reactions of mourning of 339 patients who are over 45 years old and who are being treated because of an endogenous, neurotic or reactive depression are investigated. In the case of 20 patients suicidal actions occurred in former years or before the present treatment after the death of their mother, father or husband or wife. Of essential importance are the reactions of the people closely about them, the setting-off of or the transition into an endogenous depression, a distinct dependence on the deceased and ambivalence of feelings, self-reproach and feelings of guilt, and the inability for or avoidance of conscious mourning. In the case of chronically suicidal patients the therapy turns out to be especially difficult, if a real work of mourning does not succeed. PMID- 1713350 TI - [Experiences with "Schedules for Clinical Assessment in Neuropsychiatry (SCAN)" within the scope of a multicenter field study]. AB - The "Schedules for Clinical Assessment in Neuropsychiatry" (SCAN), a comprehensive set of clinical assessment instruments were developed by the World Health Organization (WHO) and the London Institute of Psychiatry. A central aim is to provide clinicians and researchers with a standardized interview for ICD-10 diagnoses. Core instrument of SCAN is the 10th version of the "Present State Examination" (PSE) which has proved to be one of the most helpful psychopathological assessment instruments. PSE-10 covers a wide range of non psychotic as well as of psychotic symptoms and includes special modules for ICD 10 and DSM-IIIR diagnoses, e.g. alcohol and drug abuse, sleeping and eating disorders. A computer program (CATEGO-V) is also developed for standardized scoring and diagnostics. The paper introduces SCAN by describing its structure and applicability. Different rating periods and scales are explained, and PSE-10 is contrasted with PSE-9 in a whole. SCAN experiences within a reliability study are reported, completed by a critical evaluation of the most recent version of the SCAN system. PMID- 1713351 TI - [Psycho-educational family group for psychiatric patients of various diagnoses]. AB - The effectiveness of an eight-session psychoeducational group intervention with family members of psychiatric inpatients was evaluated in terms of benefits for the patients' clinical outcome, relatives' perceived burden and changes in qualitative aspects in family member-patients interactions. The study involved 12 inpatients and 16 immediate adult family members. At the nine-month follow-up the relapse rate was zero percent. Moreover, another effect was the decrease in family members' perceived burden. Additional findings also showed reductions in family members' facial expression of hostility in interaction with the patient. Results suggest that a psychoeducational intervention with family members of patients with different diagnoses may be effective as part of an inpatient treatment program. PMID- 1713352 TI - [Psychopathologic and psychophysiologic follow-up of inpatient depressed patients with standardized treatment with clomipramine and oxaprotiline]. AB - The aim of this study was to show psychopathological and especially psychophysiological data of depressed inpatients which were collected during a 28 day double-blind study with 68 depressed inpatients given clomipramine 150 mgr/die) or maprotiline/oxaprotiline 150 mgr/die). Using Hamilton-Depression Scale, Self-depression-scale (SDS by Zung), Beschwerdeliste (BL) and Befindlichkeitsskala (Bf-S) by v. Zerssen an impressive reduction of depression scores was observed in all rating scales with some superiority of clomipramine. But at the end of the study at treatment day 28 both groups - clomipramine and maprotiline/oxaprotilin - had similar results. During treatment each patient was part of a weekly habituation experiment using electrodermal activity in an orientation reaction as measure. Contrary to other studies (Lader 1975 or Heimann 1976, 1980) there was no difference in the course of psychophysiological data (EDA) between both treatment groups or between depressive agitation and psychomotor retardation. There was also no relationship between reactivity in EDA at study begin and treatment results. PMID- 1713353 TI - [Psychogenic reaction: course and prognostic factors]. AB - A follow-up was made of ninety patients with a diagnosed psychogenic reaction (adjustment disorder) fourteen years after the index hospitalisation. Contrary to expectation, at the follow-up only half of the patients showed no symptoms of the illness. Approximately a quarter of the patients suffered from a more serious illness (drug dependence, schizophrenia or organic mental disorder) than the index diagnosis. The other quarter showed symptoms of a psychogenic disorder (neurotic disorder, personality disorder, adjustment disorder). A number of factors which describe the course of the illness leading to the index hospitalisation permit a prediction of the outcome of the disease. PMID- 1713354 TI - [Molecular analyses of autoantigens]. PMID- 1713355 TI - Immunoscintigraphy of experimental transplantable tumours using monoclonal antibody against myelin basic protein. AB - Monoclonal antibody was prepared against myelin basic protein a so-called pancarcinoma antigen. After labelling with 131I the monoclonal antibody was injected into Lewis-lung cancer mice and rats with Walker breast cancer. Two, 24, 48, 72 and 96 hours after the labelled monoclonal antibody injection, radioimmunoimaging studies were carried out. After each gamma-camera study, organ distribution of the labelled monoclonal antibody was determined with radiobioassay technique which showed significantly higher activity in the tumour tissue than in healthy ones. Significant sample radioactivity could be recovered in the tumour masses 48 hours after injection, which persisted even after 96 hours. The later finding might enable diagnosing types of malignancy with isotope labelled monoclonal antibody against myelin basic protein. PMID- 1713356 TI - Induction of the protooncogene c-fos and recovery of cytosolic adenosine triphosphate in reperfused liver after transient warm ischemia: effect of nitrone free-radical spin-trap agents. AB - Ischemia and reperfusion stimulate several adenosine triphosphate (ATP)-dependent processes involving release of substances including free radicals. This cellular response is mediated through receptors responsive to transcriptional products of gene expression; c-fos acts as a transcriptional factor involved in the regulation of genes associated with cellular proliferation and differentiation. We hypothesized that nitrone free-radical spin traps promote restoration of cytosolic ATP during reperfusion and prevent c-fos induction. Four control rats had no ischemia. Global hepatic ischemia was induced in 19 rats in four groups: saline solution, phenyl-N-tert-butyl nitrone (PBN), alpha 1-pyridyl-N-oxide N tert-butyl nitrone (POBN), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). ATP and intracellular pH were measured at intervals before, during, and after ischemia. At 90 minutes of reperfusion, liver c-fos mRNA was measured. A fourfold elevation of c-fos occurred in the saline-treated group (p less than 0.001). PBN and POBN groups did not differ from the saline group. DMPO resulted in significantly less induction of c-fos than did NS. ATP depletion and recovery in all treatment groups was similar to that of the saline group. We conclude that (1) nitrone spin traps do not prevent c-fos induction or alter the pattern of ATP recovery after hepatic ischemia and reperfusion and (2) c-fos induction is not necessary for restoration of ATP, but the rate of ATP restoration is inversely related to c-fos induction. PMID- 1713357 TI - Vasoactive intestinal polypeptide inhibits c-myc expression and growth of human gastric carcinoma cells. AB - Vasoactive intestinal polypeptide (VIP) is a gut neuroendocrine polypeptide that increases cyclic adenosine monophosphate (cAMP) production in cells with VIP receptors. Some gastrointestinal cancer cells possess functional receptors for VIP; however, the role of VIP in regulation of growth of gastric cancer cells has not been determined. The purpose of this study was to determine whether VIP and other agents that increase cAMP regulate growth of a human gastric cancer cell line (AGS) and whether these agents regulate expression of c-myc proto-oncogene, which is required for cell proliferation. We measured levels of cAMP by radioimmunoassay, and we used Northern blot analysis to examine c-myc messenger RNA expression. Cell-growth studies were carried out in media supplemented with 3% serum, and cells were counted with a Coulter counter. We found that VIP significantly increased cAMP production of AGS cells in a dose-dependent manner, whereas secretin, glucagon, and peptide histidine methionine (PHM) did not stimulate cAMP production. Exogenous cAMP (8-bromo-cAMP) inhibited AGS cell growth in a dose-dependent manner. VIP acted synergistically with either isobutylmethyl-xanthine or forskolin to inhibit AGS cell proliferation. The increased c-myc expression, which was induced by serum, was inhibited by simultaneous treatment with VIP and isobutylmethyl-xanthine. We have found that AGS cells have specific, functional VIP receptors (activation of which are negatively correlated with cell growth) and that the mechanism by which VIP acts to inhibit cell growth appears to be due, in part, to cAMP-dependent regulation of c-myc proto-oncogene expression. PMID- 1713358 TI - FK 506 reverses acute graft-versus-host disease after allogeneic bone marrow transplantation in rats. AB - Severe graft-versus-host disease was induced by transplantation of ACI rat bone marrow and spleen cells into irradiated Lewis rat recipients. Treatment with FK 506 or cyclosporine A (CsA) was started after clinical and histologic evidence of acute GVHD was present. A 14-day course of FK 506 at 1.0 mg/kg/day could rescue 100% of the animals suffering from GVHD. In contrast only one half of the animals treated with CsA at a high dose of 25 mg/kg/day recovered. After cessation of immunosuppressive therapy, FK 506-treated animals displayed a marked prolonged disease-free interval as compared to CsA-treated bone marrow recipients. Recurrence of the disease in these animals could be prevented when FK 506 treatment was continued after the induction period with a low maintenance dose of 0.1 mg/kg/day every other day. PMID- 1713359 TI - RNA editing in mitochondrial mRNA of trypanosomatids. AB - The editing of mRNA coding sequences by the modification, removal or addition of nucleotides has recently been recognized as another form of RNA processing. Studies of the extensive editing of mitochondrial mRNAs in trypanosomatids have revealed the involvement of small guide RNAs (gRNAs) which are encoded by the minicircles of kinetoplast DNA. PMID- 1713360 TI - The effect of two new immunosuppressive agents, FK506 and didemnin B, in murine pregnancy. AB - The purpose of this study was to investigate two promising immunosuppressive agents, didemnin B (DB) and FK506 (FK), during pregnancy to assess potential adverse maternal or fetal effects. Pregnant C3H mice were randomized into control and high- and low-dose treatment groups for each drug. Animals received daily injections from day 1 to day 16, and on day 17 of gestation the maternal condition, litter size, fetal resorption rates, and fetal/placental unit weights were determined. Immunoglobulin (IgG) levels were obtained for DB treatment groups. Delayed type hypersensitivity was assessed in virgin females. Both DB and FK had dose-dependent immunosuppressive activity in the DTH assay, and DB caused elevated IgG concentrations. High doses of DB caused diarrhea and maternal wasting with no fetal survival; with low-dose DB, maternal weight gain was depressed, but pregnancy outcome was not different from control animals. High dose FK had no obvious detrimental effects on maternal health but caused resorption of all fetuses; administration of low-dose FK resulted in a higher number of resorptions, but fetuses that survived did not appear different from controls. We conclude that these immunosuppressive drugs can have adverse effects on pregnancy, but the maternal and fetal toxicity are dose-dependent. PMID- 1713361 TI - The immunosuppressive antagonism of low doses of FK506 and cyclosporine. AB - Clinical immunosuppression with potentially toxic agents may be optimized by combining drugs that act synergistically at low doses. The studies presented herein attempted to apply this strategy to the macrolide FK506 and the endecapeptide cyclosporine, which similarly inhibit T cell responses but display distinctive arrays of toxic side effects. The interaction between these agents was subjected to rigorous pharmacologic analysis using the median effect and combination index equations to determine synergistic, antagonistic, or additive drug interactions. FK506 and CsA showed pharmacologic antagonism in inhibiting in vitro proliferation upon phytohemagglutinin, anti-CD3 antibody, and mixed lymphocyte reaction (MLR) stimulation, and interleukin 2 generation by activated normal human peripheral blood lymphocytes. The antagonistic relationship was confirmed in vivo using low doses of FK506 in combination with CsA to treat Wistar-Furth recipients of heterotopic Buffalo rat cardiac allografts, a major plus minor histocompatibility barrier. This antagonistic relation suggests that FK506/CsA combination therapy does not permit dose reduction of the individual drugs to mitigate toxic complications. PMID- 1713362 TI - A protective effect of FK506 in ischemically injured rat livers. PMID- 1713363 TI - Preliminary experience with FK506 in thoracic transplantation. PMID- 1713364 TI - A comparison of in vivo responses to cyclosporine, FK506, and rapamycin following allogeneic immune challenge. PMID- 1713366 TI - Response of young rats to repeated oral administration of technical hexachlorocyclohexane. AB - Male and Female young rats given daily po doses of technical hexachlorocyclohexane (HCH) (5 or 25 mg/kg) for 90 d showed male rats more susceptible to HCH than female. Percent mortality and enzymatic changes were more pronounced in the male rat than in the female. The accumulation of residues in the vital organs of male rat was relatively more than that of the female rat. PMID- 1713365 TI - The effect of graft function on FK506 plasma levels, dosages, and renal function, with particular reference to the liver. AB - Plasma FK506 was studied in 49 liver, 13 heart, 3 double-lung or heart-lung, and 21 kidney recipients. The levels were correlated with the drug doses used, kidney function, and liver function. In all varieties of recipients, there was an early rise in the FK506 plasma levels that occurred at the time of intravenous administration of the drug. At the same time or shortly after, there were increases in serum creatinine that were transitory except in liver recipients with continuing suboptimal graft function. The quality of hepatic function dominated all aspects of FK506 management in the liver recipients. Those who received well-functioning grafts could be given about the same drug doses as recipients of kidneys and the thoracic organs. Liver recipients with defective grafts had astronomical rises in plasma FK506, a high incidence of renal failure, and probably increased neurotoxicity. In kidney transplant recipients, the FK506 plasma levels and doses were essentially the same in patients with prompt versus delayed renal function. These studies have highlighted the necessity, first, of close pharmacologic monitoring of patients who are given FK506 in the presence of abnormal liver function, and second, of using smaller intravenous induction doses than in past practice. PMID- 1713367 TI - Neurotoxicology of pyrethrin and the pyrethroid insecticides. AB - Natural pyrethrin and synthetic pyrethroid insecticides have been considered among the safest classes of insecticides available. Pyrethrins and pyrethroids are classified on the basis of their chemical structures and their toxicologic, neurophysiologic and pharmacologic effects. Cellular effects of pyrethrin and pyrethroid insecticides have been postulated to involve interactions with sodium channels, receptor-ionophore complexes, neurotransmitters, and ATPases. Toxicity is a function of chemical structure, metabolism, route of exposure, and the presence or absence of synergists. Pyrethroid insecticides are neurotoxic, and the development and severity of clinical signs is proportional to the nervous tissue pyrethroid concentration. Type I pyrethroid poisoning in mice and rats produces a syndrome characterized by tremors, prostration and altered startle reflexes. Type II pyrethroid poisoning in mice and rats causes ataxia, convulsions, hyperactivity, choreoathetosis and profuse salivation. A presumptive diagnosis of pyrethrin/pyrethroid poisoning is based upon history of exposure, development of appropriate clinical signs, and chemical analysis for insecticide residues. Treatment of pyrethrin and pyrethroid toxicosis involves basic life support, seizure control when needed, and the prevention of further insecticide absorption. PMID- 1713368 TI - [Methodological aspects of current research in the areas of biosynthesis and roles of intercellular substance proteins (review)]. PMID- 1713369 TI - [Determination of apoprotein A-1 by immunoenzyme analysis]. AB - Indirect solid-phase immunoenzymatic assay was improved and adapted for quantitative estimation of apo A-I in blood serum. Immunochemical identification of apo A-I was carried out and specific antibodies were obtained. Various procedures of blood serum pretreatment affected dissimilarly the detection of antigenic determinants in apo A-I. Presence of tetramethyl urea in blood serum increased by 30% the latent antigenic determinants accessibility. PMID- 1713370 TI - [The expression of a fragment of the HIV-1 Nef gene in Escherichia coli bacteria]. AB - The presence of antibodies to p27, the product of gene Nef, may be an important diagnostic sign since some sera from subjects of the risk groups negative to HIV 1 structural proteins may contain antibody to p27. The study resulted in construction of a hybrid plasmid determining in E coli bacteria the synthesis of a hybrid protein the N-terminus part of which is represented by full-size beta galactosidase and the C-terminus by a part of protein p27 with the main immunoreactive epitopes. The resulting polypeptide specifically interacts with sera of the infected subjects and may be used for detection of antibodies to the protein Nef in the blood of virus-carriers. PMID- 1713371 TI - [The differentiation of viruses of the tick-borne encephalitis complex by means of RNA-DNA hybridization]. AB - Nucleic acid spot hybridization with cloned cDNA of tick-borne encephalitis (TBE) virus, strain Sofjin, was used to differentiate strains of TBE and other flaviviruses. The cDNA probe reacted with strains of TBE and flaviviruses of TBE subgroup with the exception of Powassan virus. The probe did not react with viruses of Japanese encephalitis and Gendue subgroups. The viruses of TBE subgroup and some strains of TBE virus were differentiated from TBE strain Sofjin by thermal stability of RNA-DNA hybrids. Negishi and Louping ill viruses were found to be most closely related to TBE strain Sofjin among viruses of the TBE subgroup. PMID- 1713372 TI - [The antigenic structure of glycoprotein E2 in the Venezuelan equine encephalomyelitis virus studied using rat monoclonal antibodies]. AB - A collection of 21 rat hybridomas secreting high-affinity monoclonal antibodies to Venezuelan equine encephalomyelitis (VEE) virus was generated. Using a panel of 15 monoclonal antibodies to glycoprotein E2, the antigenic structure of this protein of VEE strains TC-83 and 230 was studied. A competitive radioimmunoassay suggested a new map of the antigenic structure of glycoprotein E2 in which 5 sites including 11 epitopes of monoclonal antibody binding were distinguished. Antibody to E2-2 site neutralized virus infectivity and blocked hemagglutination test and antibody to E2-3 site could only block hemagglutination. Antibodies to other E2 protein sites lacked any biological activity. PMID- 1713373 TI - [A surface membrane study of human cell lines with different interferon sensitivities]. AB - A comparative study of surface membranes of human cell J-96 and J-48 cultures with different sensitivity to alpha/beta interferon (IF). Reduced sensitivity of J-41 cells to IF-alpha/beta was found to be accompanied by a loss of highly specific receptors for IF-alpha, the lack of changes in the cell surface structures upon treatment with IF-alpha/beta, reduced intensity of cell fusion upon successive treatment with IF and polyethylene glycol. The results are discussed in connection with the observed changes in the activity of superoxide dismutase in J-96 and J-41 cell lines after treatment with IF. PMID- 1713374 TI - [The modulation of the interferon system by gamma-globulin preparations]. AB - Preparations of specific and commercial gamma-globulin against hepatitis A virus caused a marked inhibition of alpha- and gamma-links of the IF system 7 days after administration. In 80% of the subjects given commercial gamma-globulin 14 days after administration the above values returned to those observed before administration of the preparations. The recovery of the leukocyte capacity for alpha-IF production after administration of specific gamma-globulin was observed only in 50% of the subjects examined, while inhibition of the gamma-link of IF system persisted for 14 days (50%) or showed a poor trend to restoration not reaching the initial levels. PMID- 1713375 TI - [The biological properties of virus-induced and mitogen-induced interferons in dogs]. PMID- 1713376 TI - Media for the education of health professionals. AB - The benefits and pitfalls of applying media and communications techniques to the education of health professionals are considered in the context of their use in the classroom, for independent study and for distance education. The difficulties are emphasized for managing learning materials of this kind, and for keeping them up-to-date. PMID- 1713377 TI - Lindane toxicity in an infant. AB - A four-month-old infant with severe impetigo who had prolonged daily applications of lindane presented with two seizure episodes. His blood lindane level was 1.27 ppm. Caution should be exercised when using lindane in infants and children. Proper instruction and close supervision are essential. PMID- 1713378 TI - Double immunoenzyme staining. PMID- 1713379 TI - [An experimental study of the safety of a chemical monovalent tableted cholera vaccine in enteral administration]. AB - The safety of experimental chemical cholera monovalent vaccine in tablets, produced by the institute "Microbe" (Saratov, USSR), has been studied. The study has shown that the vaccine, administered to adult rabbits and germ-free suckling rabbits by the enteral route, retains residual toxicity, mainly due to the presence of O-antigen. One or two administrations of 1-2 human doses of this preparation to adult rabbits induce minimal structural changes admissible from the viewpoint of safety. After immunization made in two administrations immunobiological transformation develops more rapidly and is more pronounced than after immunization in a single administration. PMID- 1713380 TI - [The characterization of the spectrum of the antibody response to Mycobacterium tuberculosis antigenic determinants by immunoblotting]. AB - The spectrum of antibody response to M. tuberculosis antigenic determinants H37Rv and M. bovis antigenic determinants BCG was studied in serum samples from 33 healthy donors and 31 patients with infiltrative pulmonary tuberculosis by the method of immunoblotting. The study revealed that most frequently tuberculosis patients showed response to Ag-H37Rv with molecular weights of 52, 39, 35, 21, 31, 68 kD (44.4-22.2%) and Ag-BCG with molecular weights 60, 58, 50, 25, 54, 70 kD. (33.3-22.2%). By month 9 of effective chemotherapy binding predominantly with Ag-H37Rv determinants of 31, 62, 35, 75, 56, 28, 19, 5, 13 kD (75-37.5%) and Ag BCG determinants of 13, 34, 38, 44, 19, 36, 45, 52, 58, 60 70 kD (37.5-25%) were registered. Some differences in the spectra of antibody response to Ag-H37Rv and Ag-BCG determinants were noted. PMID- 1713382 TI - Removal of wrinkles and redundant skin in the region of the forehead. AB - On the basis of an analysis of recent results in 160 patients operated on for the purpose of removing wrinkles and redundant skin from the region of the forehead, and of an analysis of the author's own material in 80 patients operated on by means of a method elaborated by the author conclusions were drawn concerning the incision used in this operation. The incision is carried out depending on the height of the frontal region. Indications for the intervention on the frontal muscle were determined. Directions concerning the extent of the operation were revised. It has been found that a more extensive, wide mobilization of tissues including the bridge of the nose, the regions of the eyebrows and the temples along with the frontal region, yields more pronounced and lasting results. PMID- 1713381 TI - [The determination of antigen-specific immune complexes by an immunoenzyme method]. AB - The methodological approach permitting the detection of immune complexes containing specific antibodies to a definite antigen in the enzyme-linked immunosorbent assay (ELISA) is described. The basic conditions of the assay were optimized. Immune complexes were precipitated from blood serum with 3.5% polyethylene glycol 6000 for 4 hours. The precipitate thus obtained was dissolved and incubated in polystyrene plates with immobilized antigen at 37 degrees C for a long time (at least 6 hours) in a humid chamber. The amount of bound antibodies, determined by ELISA techniques, was conjectured from the level of antigen-specific immune complexes. The proposed approach can be used in the immunodiagnosis of infectious diseases. PMID- 1713383 TI - Musculocutaneous flaps for the treatment of chronic osteomyelitis of the heel. AB - Treatment for chronic osteomyelitis with musculocutaneous and muscular flaps is a highly effective modern method. It is effective even in such unfavourable localizations as the calcaneus. The scope of use provided by muscles in the plantar region is wide enough to permit the treatment of foci in the heel bone of diverse localizations. The range of possibilities includes use of m. abductor digiti quinti. A muscular or musculocutaneous flap elevated from the muscle is well suited for the treatment of defects on the external side of the heel. The group consisted of six patients with osteomyelitis of seven heel bones operated on as described above at the author's clinics between 1983 and 1987. One patient had both heel bones affected. The mean age was 47 years, ranging from 23 to 62 years. All of the patients showed clinical and X-ray signs of healing of the soft tissue defects and full remission of inflammatory complication. Control check-ups were made at 2-5 years postoperatively with an average postoperative period of 38 months. None of the patients developed any chronic osteomyelitis relapse at any point during that period. PMID- 1713384 TI - Use of the distraction method in hand surgery. AB - The author describes three prototypes of a miniature distractor for the bones of hand constructed according to his own design, and its uses in clinical practice. The distractors are used for lengthening bones of the hand after injuries involving loss of the thumb or fingers, and for the treatment of patients with congenital developmental defects of the hand. Over a period of three years, the distractors were used in thirteen cases and Ilizarov's original apparatus in three cases: for the lengthening of metacarpus I in seven cases, metacarpus II in two cases, metacarpi II-IV in one case, metacarpi II-V also in one case. Similarly, in one case each for the elongation of the proximal and medial phalanges of fingers. A lengthening of 3.5 to 3.7 was achieved. Ilizarov's apparatus was exploited in two cases to lengthen the bone of the forearm for congenital hypoplasia of the radius and ulna, and for metatarsal bone lengthening in a case of congenital aplasia of the toes and hypoplasia of the remaining bones of the foot. In the last three cases a lengthening of 10 cm was achieved. PMID- 1713385 TI - Proboscis lateralis type IV--a report from the Indian subcontinent. AB - Proboscis lateralis of type IV has not yet been reported from the Indian subcontinent. The authors gives a report on a case of this type and describes the technique of reconstruction not described before. A case of a right-sided proboscis lateralis associated with ipsilateral hemi-nasal aplasia and contralateral nostril and alar defect, a contralateral cleft lip and alveolus, hypertelorism and mild hydrocephalus is presented and documented on a boy coming from the Indian subcontinent. PMID- 1713386 TI - Primary reconstructive surgical treatment of patients with cancer of the mucous membrane of the floor of the oral cavity. AB - A new method of primarily reconstructive surgical treatment of patients with cancer of the mucous membrane of the floor of the oral cavity is described. By means of this method it is possible to preserve the natural ways of breathing and nutrition from the early postoperative period without neglecting the principles of radical treatment and considerably to reduce the number of postoperative complications. The proposed method of operation does not lead to disfigurement of the lower portions of the mandibulofacial region. PMID- 1713387 TI - Differences between facial configuration and development in complete and incomplete unilateral cleft lip and palate during the prepubertal period. AB - Mixed-longitudinal roentgencephalometric data were used for the determination of differences between the configuration and development of the face in complete and incomplete unilateral cleft lip and palate at the age of 8 to 12 years. As compared to incomplete clefts patients with complete clefts had a reduced height of the upper face and thus also of the face as a whole and an increased width of the nasal cavity. These findings were in agreement with the situation in adults, but contrary to adults we failed to disclose any difference in the depth of the maxilla and thus there were also no differences of the retrusion of the upper jaw, sagittal jaw relations, facial convexity, occlusion of incisors and the prominence of the upper lip. The thickness of the upper lip did not differ as well. The global results showed that the differences between facial configuration in these two types of clefts were much smaller up to the onset of puberty than in adults. Throughout the investigated period of time the growth and development of the investigated parameters proceeded identically in both forms of clefts. The reduction of upper face height in complete clefts confirmed an early, probably prenatal origin of this deviation from normal. PMID- 1713388 TI - Hypodontia in patients with isolated cleft palate, its relationship to etiopathogenesis. AB - The authors studied the incidence of hypodontia in 200 patients with isolated cleft palate. These patients were divided into 3 groups according to the degree of malformation (complete cleft palate, incomplete cleft palate, soft palate cleft). In the 3 groups, hypodontia was studied on permanent teeth, with the exception of the 3rd molars. Compared with the healthy population, hypodontia in patients with cleft palate appeared at a significantly higher rate (25.5%). No difference was found between boys and girls. Hypodontia in the mandible appeared in 18%, in the maxilla in 10%. The upper jaw showed maximum incidence in complete cleft palate (21%), while in the mandible hypodontia was observed most frequently in soft palate clefts (37% patients). In most cases the dental germs of the 2nd lower premolar failed to develop. Our results correlate with hypotheses based on experimental studies on the etiopathogenesis of facial clefts. PMID- 1713389 TI - The double folded pectoralis major musculocutaneous flap for pharyngostome closure. Case report. AB - A case is reported in which a double-folded pectoralis major musculocutaneous flap was successfully transferred for pharyngostome closure after partial horizontal laryngectomy. The double-folded flap was used for external and internal lining. PMID- 1713390 TI - Topographic distribution of scrapie amyloid-immunoreactive plaques in chronic wasting disease in captive mule deer (Odocoileus hemionus hemionus). AB - Chronic wasting disease (CWD), a progressive neurological disorder of captive mule deer, black-tailed deer, hybrids of mule deer and white-tailed deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation in neuronal perikarya and astrocytic hypertrophy and hyperplasia. We report the topographic distribution of amyloid plaques reactive to antibodies prepared against scrapie amyloid in CWD affected captive mule deer (Odocoileus hemionus hemionus). Scrapie amyloid immunoreactive plaques were found in the cerebral gray and white matter, in deep subcortical nuclei, in isolation or in clusters in areas of vacuolation, and perivascularly, in subpial and subependymal regions. In the cerebellum, immunoreactive amyloid plaques were observed in the molecular, pyramidal and granular layers. Scrapie amyloid-immunoreactive deposits were also seen in neuronal perikarya. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Our data corroborate that CWD in captive mule deer belongs to the subacute virus spongiform encephalopathies. PMID- 1713391 TI - Uptake of plasma proteins into damaged neurons. An experimental study on cryogenic lesions in rats. AB - Preliminary observations on human autopsy material have indicated that damaged neurons may take up plasma proteins early after the injury. These observations prompted an experimental study under controlled conditions. Focal brain lesions were produced in rats by extracranial application of dry ice for 90 s. This caused an immediate disruption of the blood-brain barrier with leakage of plasma components into the tissue and sharply circumscribed areas of necrosis of the underlying cortex. Five minutes after the lesion, uptake of albumin, fibrinogen and fibronectin into damaged neurons was demonstrated by immunostains. These proteins were retained in the injured neurons until they were phagocytized 2-4 days later. In addition, normal neurons whose axons or axon collaterals passed through or terminated in the lesion were labeled. This labeling was generally weaker than in damaged neurons and no labeling of neuronal nuclei was observed in these cells in contrast to those of damaged cells. Apart from nerve cells labeled through retrograde axonal transport, no staining of normal neurons was observed. Intravenous injections of Evans blue, which binds to plasma proteins, confirmed that albumin was taken up into damaged neurons almost immediately after the injury and showed that this uptake continued for at least 20 h. It is concluded that uptake of plasma proteins into damaged neurons may serve as early (and late) markers of neuronal injury. PMID- 1713392 TI - Regeneration of perivascular adrenergic innervation in rat tibial nerve after nerve crush. AB - Adrenergic innervation of blood vessels in the rat tibial nerve during degeneration and regeneration was studied using the formaldehyde-induced fluorescence method. The left sciatic nerve was crushed with suture threads to produce a 4-mm length of crushed nerve. At 1, 3, 7, 14, 28, 56 and 84 days after nerve crush, degenerative and regenerative changes in the nerve were verified using light microscopy. At each time point, adrenergic innervation was examined in epi-perineurial whole mount and nerve cross-section preparations. One day after nerve crush, fluorescence of adrenergic nerve fibers in the endoneurium was absent. Fluorescent adrenergic nerve fibers reappeared in the endoneurium at day 56 and reached the control density by 84 days. In the epi-perineurium, adrenergic innervation of small and medium-size arterioles was absent at 3 days, in large arterioles at 7 days. At 56 days, all epi-perineurial arterioles were reinnervated by a faint, sparse adrenergic network, which reached the control density at 84 days. The results suggest that adrenergic innervation in the rat peripheral nerve is lost during nerve degeneration, but recovers when the nerve has regenerated. PMID- 1713393 TI - Axons induce differentiation of neurofibroma Schwann-like cells. AB - Neurofibromatosis type 1 (NF-1, von Recklinghausen's disease) is characterized by the focal accumulation of Schwann-like cells (SLC) to form subcutaneous and plexiform neurofibromas and schwannomas. The aim of the present study was to determine whether NF-SLC are competent to differentiate in the presence of axons. Five dermal neurofibromas from five patients with NF-type 1 were enzymatically dissociated and the resultant cells were co-cultured with fetal rat dorsal root ganglion neurons. The cultures were studied by indirect immunofluorescence microscopy using antibodies against galactocerebroside (galC), P0 glycoprotein, human nerve growth factor receptor (NGFR) and human myelin-associated glycoprotein (MAG). SLC were strongly NGFR+ but galC- and MAG-SLC for the 2 weeks of coculture. After 3 weeks in vitro, SLC-NGFR was down-regulated but some of the spindle shaped cells had become galC+. MAG-SLC first appeared after 5 weeks in vitro but P0 glycoprotein was never detected when studied up to 6 weeks. Our data demonstrate that axons induce SLC to down-regulate surface NGFR and to express some myelin components in a qualitatively normal fashion. PMID- 1713394 TI - Modified tau is present in younger nondemented persons: a study of subcortical nuclei in Alzheimer's disease and progressive supranuclear palsy. AB - Tau-positive neurons relating to neurofibrillary tangles and diffuse cytoplasmic stainings were quantitatively examined in the brains of 61 nondemented persons including 24 age-matched controls, 10 patients with Alzheimer's disease (AD) and 5 with progressive supranuclear palsy (PSP). In nondemented persons, the locus ceruleus (LC) was found to contain tau-positive neurons initially in persons in their 30s, whereas the hippocampus contained such neurons initially in persons in their 40s. The LC had a higher incidence and density than the hippocampus in almost all age classes. As neuronal tau accumulation is considered a histological change occurring with normal aging, the LC might be involved in the earliest aging in the normal brain. In AD there was conspicuous tau accumulation in the same sites which were vulnerable to tau accumulation in the age-matched controls. In PSP tau accumulated heavily in a set of sites different from the age-matched controls and AD. Thus, subcortical tau accumulation in AD is increased far more than that under normal aging process, while that in PSP is not simply in an increased state of the normal aging process. PMID- 1713395 TI - Ultrastructural localization of argyrophilic substances in diffuse plaques of Alzheimer-type dementia demonstrated by methenamine silver staining. AB - The ultrastructure of argyrophilic substances in diffuse plaques of an Alzheimer type dementia brain was examined using methenamine silver (MS) electron microscopy with the pre-embedding method. Electron-dense substances, which were sparse aggregations of bundle-like structures with silver granules, were disseminated in the diffuse plaques. Comparison of serial MS and routine ultrathin sections revealed that diffuse plaques usually show scattered bundles of amyloid fibrils or amorphous materials, or both, between indistinct cell process membranes. Some of these processes were identified as astrocytic or dendritic in origin. A few degenerative neurites were frequently noted in large silver-positive areas but never in small areas. These findings suggest that the appearance of amyloid fibrils and amorphous materials between certain cell processes is a very early morphological change in the process of senile plaque formation. PMID- 1713396 TI - Spontaneous mineralization of the sciatic nerve of senescent rats. AB - A spontaneous mineralization of the sciatic nerve of senescent specific pathogen free-bred rats (aged 42 months) is reported. Deposits were found in the endoneurium of different branches of the nerve at mid-thigh level. They appeared as small discrete deposits or as large tubular-shaped concretions, probably formed by the growth and merger of the smaller deposits. Some of the concretions were found in close proximity to blood vessels. Deposits consisted of dense aggregations of randomly entangled spicules spreading within bundles of collagen fibrils. Calcium was detected by histochemistry and X-ray dispersion microanalysis. Phosphorus was also found, possibly associated with calcium to form hydroxyapatite. PMID- 1713397 TI - Increased reactivity of laminin in the basement membranes of capillary walls in AIDS brain cortex. AB - To verify how the components of the capillary wall are modified in the course of AIDS we studied the brain cortex from nine cases with AIDS. Cellular and extracellular components were delineated using antibodies for laminin and collagen IV for basement membranes and glial fibrillary acidic protein for astrocyte foot processes. We found a marked increase in reactivity for laminin in the basement membranes of capillary walls and hypertrophy and hyperplasia of astrocyte foot processes around vessels, when compared to control cortical tissue. We suggest that modifications of brain capillary wall may have a role in the pathogenesis of neurological disfunction in AIDS. PMID- 1713398 TI - Aberrant remyelination of axons after heat injury in the dorsal funiculus of rat spinal cord. AB - We studied the course of demyelination and subsequent remyelination of nerve fibers after heat injury in the dorsal funiculus of the rat spinal cord. Four weeks after heat treatment, we observed, in addition to normally remyelinated axons, a few aberrantly remyelinated axons which had both CNS- and PNS-type myelin sheaths: the CNS-type myelin sheaths were always situated inside the PNS type sheaths. This finding indicates that in some conditions Schwann cells can form myelin sheaths around those formed by oligodendrocytes. PMID- 1713399 TI - Compartment analysis of vascular effects of neuropeptides and capsaicin in the pig nasal mucosa. AB - The vascular effects of local infusion of capsaicin, substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were monitored in an experimental model on the pig nasal mucosa. Arterial, venous and superficial mucosal blood flow (laser-Doppler flowmetry) as well as mucosal volume, reflecting changes in capacitance vessels were studied in parallel. All substances induced concentration dependent increases in the parameters studied with the exception of the decrease in the superficial mucosal flow induced by vasoactive intestinal polypeptide. This latter finding was interpreted as a stealing phenomenon and suggests that vasoactive intestinal polypeptide mainly exerts its vasodilatory effect in the deeper glandular layers of the nasal mucosa. The vasodilatory effect of capsaicin, except the laser-Doppler signal, was markedly reduced by pretreatment with a combination of the ganglionic blocking agent chlorisondamine and atropine implying that capsaicin evokes a central reflex with a final parasympathetic pathway and release of agents like vasoactive intestinal polypeptide. The remaining capsaicin response may depend on a local effect with axon reflexes and the release of sensory neuropeptides with actions on superficial mucosal blood flow. PMID- 1713400 TI - Intertissular variations in osteonectin: a monoclonal antibody directed to bone osteonectin shows reduced affinity for platelet osteonectin. AB - Osteonectin, a major noncollagenous protein of bone, is also synthesized and secreted by various non-mineralized tissues and by platelets. To establish whether there are structural specificities of osteonectin according to its tissular origin, we raised 12 monoclonal antibodies against bovine bone osteonectin and screened them for their ability to recognize bone and platelet osteonectin. When hybridoma culture media were radioimmunoassayed all MAbs showed the same titer for [125I]human platelet osteonectin and for [125I]bovine bone osteonectin, except MAb 2, which poorly bound platelet osteonectin. Immunoprecipitation and immunoblotting experiments were performed on human bone protein extracts and on material secreted by human platelets upon thrombin stimulation; in these experiments MAb 2 recognized human bone osteonectin and only faintly human platelet osteonectin. A "sandwich" immunoradiometric assay was devised in which osteonectin bound to a solid phase by a first MAb was recognized by a 125I-labeled second MAb. In this assay MAb 2, used as a tracer, showed a 100 fold lower affinity for purified human platelet osteonectin than for purified human bone osteonectin. These results suggest the existence of structural variations in osteonectin obtained from bone and platelets. Whether these variations result from differences in sequence, post-translational processing, or postsecretional fate remains to be established. PMID- 1713401 TI - High-conductance anion channels in embryonic chick osteogenic cells. AB - Patch-clamp measurements done on excised membrane patches obtained from 1-5 day cultured embryonic chick osteoblasts, osteocytes, and periosteal fibroblasts revealed the existence of a high-conductance anion channel: 371 +/- 63 pS when measured under symmetrical 158 mM Cl- conditions. The channel frequently displayed subconductance levels. The ion selectivity of the channel expressed as the (an)ion to chloride permeability ratio was as follows: Cl- (1.0) greater than methylsulfate- (0.71) greater than gluconate- (0.25) greater than glutamate- (0.17) greater than Na+ = K+ (0.10). In addition, the channel had a significant permeability for inorganic phosphate ions. The channel was found in about 1% of the cell-attached patches, which indicates that the channel is under the control of as yet unknown intracellular factors. Once activated by patch excision, the channel was voltage dependent and active at potentials close to 0 mV. At potentials outside the range of +/- 10 mV channel activity decreased. This process proceeded faster at increasing membrane potentials of either polarity. Returning to potentials close to 0 mV caused reopening of the channels within seconds if the preceding voltage step led to complete closure of the channels. Channel activity did not depend noticeably on intracellular and extracellular CA2+ ions. The channel is not unique to (chick) osteogenic cells but has been demonstrated in excised patches obtained from excitable and other nonexcitable cells. Although its presence in a wide variety of cell types suggests that the channel plays a general role in as yet unknown cell physiologic processes, the channel may also have specific functions in osteogenic cells, for example providing a pathway for phosphate ions during mineralization. PMID- 1713402 TI - [Ciprofloxacin concentration in human prostatic tissue following 3 days' administration]. AB - The penetration of Ciprofloxacin (CPFX) into the prostatic tissue and the serum was examined. Forty-four patients with benign prostatic hypertrophy treated with transurethral resection of the prostate were entered in this study. CPFX was administered orally in a dose of 200 mg three times daily for three days preoperatively. The blood samples were taken simultaneously at the time of the tissue sampling. The patients were divided into groups 1 and 2. In group 1 (16 patients), the tissue sampling was done about 17 hours after the final drug administration. The mean concentration of CPFX was 0.61 +/- 0.40 microgram/g in the prostatic tissue and 0.27 +/- 0.19 micrograms/ml in the serum. In group 2 (28 patients), tissue sampling was done 5.5 hours after the final drug administration. The mean concentration of CPFX was 1.32 +/- 0.64 micrograms g in the prostatic tissue and 0.65 +/- 0.31 microgram/ml in the serum. PMID- 1713403 TI - Concealed rhythms by double ventricular parasystole. AB - Electrocardiograms taken from 11 patients in sinus rhythm with ventricular ectopic rhythms from two different foci were analyzed to find the number of sinus beats, S, between the ectopic rhythms (S values). Three out of 11 patients had the S values typical for concealed ectopic rhythms. One of them had concealed bigeminy of 2n-1 form that occasionally shifted to 2n form. Following the shift, S values of 2n-1 form were always achieved by the occurrence of double ventricular ectopic rhythms in succession. Concealed trigeminy of 3n and 3n-2 form was seen in the other two patients. Double ventricular ectopic rhythms had bizarre abnormal QRS complexes of two different morphologies and were inscribed in opposite directions. Ectopic rhythms in each case had parasystolic characteristics. These observations suggest bifocal automaticity as a mechanism for bidirectional ventricular tachycardia. PMID- 1713404 TI - Effect of reflex vagal activation on frequency of ventricular premature complexes. AB - To evaluate the antiarrhythmic effect of reflex-induced vagal activation, phenylephrine was infused in 17 patients with frequent ventricular premature complexes (VPCs). The role of heart rate reduction in suppressing VPCs was explored by pacing the atria at the preinfusion levels. Baroreceptor activation was considered effective when a greater than or equal to 20% decrease in heart rate was observed. Ten patients (59%) achieved the target heart rate decrease ( 29 +/- 5%), whereas in 7 (41%) the baroreceptor reflex was considered inadequate. In the former group ("responders"), heart rate decreased from 73 +/- 7 to 52 +/- 6 beats/min (p less than 0.0001). When heart rate was allowed to fluctuate, ectopic activity was completely abolished in 9 of 10 patients; mean number of VPCs decreased from 38 +/- 8 to 0.2 +/- 0.6/100 beats (p less than 0.0001). During pacing, VPCs reappeared but their mean number (22 +/- 10/100 beats) was still significantly reduced compared with control values (p = 0.003). In the "nonresponders," despite adequate blood pressure increases, VPC frequency was not affected. The QT interval lengthened during phenylephrine (392 +/- 17 ms) versus control conditions (372 +/- 18 ms, p = 0.0008) in the responders group, whereas no change was observed in the nonresponders. These results demonstrate that reflex vagal activation markedly reduces VPCs. This effect is only partially rate dependent; direct and indirect electrophysiologic changes secondary to baroreflex activation are also likely to be involved. PMID- 1713405 TI - Measurement of serum granulocyte colony-stimulating factor in a patient with congenital agranulocytosis (Kostmann's syndrome). AB - A 12-month-old boy with Kostmann's syndrome was admitted with cavitary pulmonary disease. He had also had bacterial conjunctivitis, periorbital cellulitis, pneumonitis, and otitis media since the age of 10 days. His umbilical cord had not fallen off until he was 3 weeks old. Neutropenia was diagnosed at 4 weeks of age. Antineutrophil antibody studies were negative. A bone marrow aspirate showed granulocytic hypoplasia and a maturation arrest at the promyelocyte stage. Hematopoietic cell culture showed normal numbers of colony-forming units granulocyte macrophage. Serum granulocyte-macrophage colony-stimulating factor level, was 0.24 ng/mL (normal, greater than 0.05 ng/mL). Serum granulocyte colony stimulating factor levels, measured by enzyme immunoassay, were undetectable. The patient was successfully treated with filgrastim (granulocyte colony-stimulating factor), with an increase in the absolute neutrophil count to 10.0 x 10(9)/L. Thus, our case of Kostmann's syndrome appears to represent a defect in regulation or production of granulocyte colony-stimulating factor. PMID- 1713406 TI - The effect of ERCP on circulating pancreatic enzymes and pancreatic protease inhibitors. AB - The pathogenesis of endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis is poorly understood. To elucidate a role for pancreatic enzymes in ERCP-induced pancreatitis, we measured serum amylase, lipase, trypsin, and elastase in 25 patients undergoing ERCP. Serum alpha 1-antitrypsin and alpha 2 macroglobulin, two major pancreatic protease inhibitors, also were measured. All pancreatic enzymes measured rose significantly after ERCP. Pancreatic duct cannulation was associated with a greater elevation in serum amylase and lipase. Circulating alpha 2-macroglobulin was reduced by 7% (p = 0.04) 6 h after ERCP, whereas circulating alpha 1-antitrypsin increased over the same time period. Papillotomy, stent placement, or underlying disease did not influence changes any further. Three patients developed ERCP-induced pancreatitis. All three patients had circulating alpha 2-macroglobulin levels below 243 mg/dl (p = 0.03). The ERCP induced alterations in circulating pancreatic enzymes and their inhibitors are similar to changes seen in clinical pancreatitis. Low circulating alpha 2 macroglobulin levels may predispose to ERCP-induced pancreatitis. PMID- 1713407 TI - Extracorporeal shock wave lithotripsy of pancreatic duct stones. AB - Chronic calcifying pancreatitis presents a major clinical problem, often requiring extensive surgery. Extracorporeal shock wave lithotripsy (ESWL) offers a new therapeutic option. We applied ESWL after endoscopic sphincterotomy of the pancreatic orifice in eight patients with impacted pancreatic duct stones. An electromagnetic lithotriptor (Siemens Lithostar, Erlangen, FRG) was used. Patients were treated in prone position under fluoroscopic control. A mean of 6,813 shock waves (range 1,500-10,000) was delivered in one or two sessions. Disintegration of stones was achieved in 6/8 patients, initial relief of pain in 7/8 patients, and total clearance of the pancreatic duct in 3/8 patients. One patient had an exacerbation of her pancreatitis one day after ESWL, which resolved rapidly with medical treatment. No other complications were observed. Four of five patients with fragmented stones had no abdominal complaints at follow-up (mean 17 months, range 3-27). Three patients in whom ESWL was not completely successful (two without and one with partial fragmentation) underwent an operation according to Puestow. Two of them still have abdominal complaints after surgery. From these data, we conclude that ESWL of pancreatic duct stones is a promising new alternative for surgery, when endoscopic stone extraction fails. PMID- 1713409 TI - Class I and class II HLA antigens in a homogeneous Argentinian population with Whipple's disease: lack of association with HLA-B 27. AB - The prevalence of class I and class II HLA antigen was analyzed in 14 patients (12 males, two females) with Whipple's disease, diagnosed an average of 9.7 yr (range 6 months to 25 yr) before the typing. They were compared with 174 healthy control subjects of the same geographic area in Argentina. Class I antigens (locus A, B, C) were studied by lymphocytotoxic test, and class II antigens (locus DR, DQ) were detected by the double immunofluorescence technique. HLA-B27 was positive in one patient (7.7%) and in 4% of the control population. No significant association was found with the antigens tested. We observed no difference in the clinical picture or in the frequency of arthralgias, compared with those reported in the literature. Our data suggest that there is no conclusive proof of an association between HLA-B27 and Whipple's disease. PMID- 1713408 TI - Intravenous heme-albumin in acute intermittent porphyria: evidence for repletion of hepatic hemoproteins and regulatory heme pools. AB - The purpose of this study was to assess effects of heme administered intravenously, complexed to human serum albumin, on activities of the hepatic hemoproteins, cytochrome(s) P-450, and tryptophan pyrrolase, and on the size of the heme pool that regulates activity of 5-aminolevulinate synthase. Effects were compared in six normal women and four women with acute intermittent porphyria. All porphyric subjects over-excreted heme precursors and had histories of acute neurovisceral porphyric attacks. All subjects were placed on a constant daily diet that included at least 3 g carbohydrate/kg body weight and sufficient total intake to provide 1.4 times the estimated resting energy expenditure. Urinary excretions of 5-aminolevulinate, porphobilinogen, porphyrins, and metabolites of tryptophan were measured daily before, during, and after infusions of heme albumin. In the porphyric subjects, intravenous heme [4 mg (6.1 mumol)/kg body weight (BWt) with equimolar albumin], given daily for 4 days, markedly reduced overexcretion of 5-aminolevulinate, porphobilinogen, and porphyrins, indicating repletion of the regulatory heme pool. The heme infusions also decreased mean urinary excretion of 5-hydroxyindoleacetic acid from 4.9 to 2.9 mg/g creatinine per day, suggesting increased activity of hepatic tryptophan pyrrolase, the rate controlling enzyme for metabolism of tryptophan to products not in the serotonin 5-hydroxyindoleacetic acid pathway. Heme-albumin infusions were without detectable effects on excretions of heme precursors or tryptophan metabolites in normal subjects. In contrast, in both normals and porphyrics, heme-albumin infusions significantly increased rates of antipyrine metabolism (by 159% and 330%, respectively), suggesting increased activities of cytochrome(s) P-450 were produced by the infusions. The infusions were well tolerated; no subject developed thrombophlebitis or bleeding. We conclude that such infusions are safe and effective in repleting deficient heme pools and hemoproteins in patients with acute porphyria, and that activities of cytochrome(s) P-450 in normal subjects may also be increased by heme administration. The therapeutic effect of heme in acute porphyria probably relates to its ability to decrease overproduction of precursors of heme or serotonin, as the result of its increasing critical cellular heme pools. PMID- 1713410 TI - CD5 positive immunoregulatory B cells in spleen populations from multiple myeloma patients. AB - CD19+CD5+ lymphocytes constitute a minority of peripheral blood B cells. In view of the importance of these cells in the pathogenesis of the immunoregulation of myeloma, their incidence in another lymphoid organ was determined. CD5+ B cells were studied in 9 spleens from patients with multiple myeloma and in 10 spleens from normal individuals removed secondary to trauma. The total number of CD19+ B cells were increased in myeloma spleens (44.4% +/- 12.6%) as compared to normal spleens (20.4% +/- 7.4%). Likewise, the percentage of CD19 cells which co expressed CD5 were increased in myeloma (25.3% +/- 12.4%) versus normal (4.4% +/- 2.3%) spleen. CD5+ B cells isolated from myeloma spleens, but not normal spleens, inhibit production of immunoglobulin in a pokeweed mitogen driven assay. Thus the spleen appears to be an important source of immunoregulatory B cells in multiple myeloma. PMID- 1713411 TI - The ligament mastocytosis with circulating mast cells. PMID- 1713412 TI - Colonic Cl channel blockade by three classes of compounds. AB - We compared the potency and inhibitory actions of three different classes of organic acids on a Cl channel derived from colonic enterocyte plasma membrane vesicles. Chloride channels were incorporated into planar lipid bilayer membranes to examine the effects of the anthranilic acids, diphenylamine 2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), the indanyl alkanoic acids, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H inden -5-yl)oxy] acetic acid (IAA-94) and its stereoenantiomer IAA-95, and the disulfonic stilbene, 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS). Except for DNDS, each of the blockers was equipotent from both the outer membrane and the cytoplasmic side of the channel protein. The potency order from the outmembrane side was DNDS greater than IAA-94 = IAA-95 greater than NPPB much greater than DPC. In contrast, the potency order from the cytoplasmic side was IAA-94 = IAA-95 greater than NPPB greater than DNDS much greater than DPC. DPC and NPPB caused a concentration-dependent decrease in the single-channel conductance (fast block). DNDS, IAA-94, and IAA-95 caused a flickery-type block and a concentration-dependent decrease in open-channel probability. Kinetic analysis revealed that blockade could be explained by a linear closed-opened blocked kinetic scheme. Similarities in the electrostatic potential maps of these open-channel blockers suggest they may bind to a single shared binding site within the channel protein. PMID- 1713413 TI - Release of galanin from isolated perfused porcine adrenal glands: role of splanchnic nerves. AB - We found a high concentration of galanin in extracts of porcine adrenal glands (114 pmol/g). By immunohistochemistry, galanin was localized to groups of medullary cells previously shown to produce norepinephrine. To study mechanisms for the release of galanin, we developed the following in vitro model: isolated perfused porcine adrenals with intact splanchnic nerve supply. When the nerves were electrically stimulated, epinephrine and norepinephrine secretion increased 276- and 291-fold, respectively, and galanin release increased up to 1,300-fold. Acetylcholine at 10(-6) M stimulated galanin release, and hexamethonium almost abolished the response to nerve stimulation. Galanin infusions had no effect on epinephrine and norepinephrine secretion in concentrations of 10(-8) and 10(-7) M, but increased both cortisol and aldosterone secretion (P less than 0.05). Splanchnic nerve stimulation in anesthetized pigs increased the concentration of galanin in the caval vein but not in arterial plasma. It is concluded that galanin, coreleased with catecholamines from the adrenal glands, may have endocrine functions but that galanin may also have local regulatory functions in the adrenals. PMID- 1713414 TI - Mechanism of secretagogue-induced HCO3- and Cl- loss from rat parotid acini. AB - The Cl(-)- and HCO3(-)-dependent components of muscarinic agonist (carbachol) induced K+ loss from a rat parotid mince were studied using 86Rb+ as a K+ marker. Both components of 86Rb+ loss were blunted by K+ and Cl- channel blockers and by removal of extracellular Ca2+, consistent with the hypothesis that 86Rb+ loss occurs via a Ca(2+)-activated K+ channel and that this cation loss serves to electrically balance a concomitant loss of the corresponding anion via one or more conductive pathways (channels). Two tissue "pools" of 86Rb+ were observed, a carbachol-sensitive pool and a carbachol-insensitive pool (approximately 70 and approximately 30% of the total 86Rb+ content, respectively). There was no evidence for a time-dependent desensitization of the muscarinic response of the carbachol-sensitive pool. Cl(-)-dependent 86Rb+ loss was not affected by HCO3- addition, suggesting that both Cl- and HCO3- secretion are accompanied by 86Rb+ loss from the same pool and thus occur from the same cells. HCO3(-)-dependent 86Rb+ loss was not enhanced by lowering the extracellular Na+ concentration, indicating that the HCO3- exit pathway is not a Na(+)-HCO3- symport. The data are consistent with the postulate that Cl- and HCO3- are secreted by rat parotid acinar cells via the same or very similar conductive transport pathways in response to muscarinic stimulation. PMID- 1713415 TI - Effects of calcium-modifying agents on integrity of rabbit isolated gastric mucosal cells. AB - The effects of Ca(2+)-modifying agents on the disruption of gastric isolated mucosal cells from the rabbit have been examined. Fundic mucosal cells were isolated and enriched by centrifugal elutriation, with cellular viability and disruption being assessed by trypan blue dye exclusion and by the release of lysosomal enzymes. The Ca2+ ionophore A23187 (3.125-50 microM) induced a concentration-dependent increase in enzyme marker release and decreased dye exclusion from the cells. Ionophore-induced enzyme release was reduced by removal of Ca2+ or by incubation with EDTA. Cells from the medium-sized fraction were found to be more sensitive to damage by A23187 than were larger-diameter cells. Enzyme release from these cells was also induced by the Ca(2+)-channel activator BAY K 8644 (1.5 microM). The Ca(2+)-channel antagonists, nifedipine, verapamil, and diltiazem (1 microM), abolished enzyme release in response to BAY K 8644 (1.5 microM) but did not affect A23187 (25 microM)-induced responses. Ethanol (5 and 8%) also induced a concentration-dependent increase in enzyme release and decrease in dye exclusion, but this effect was not dependent on the external Ca2+ concentration. However, threshold concentrations of A23187 (1.56 microM) substantially potentiated the cell damage induced by ethanol (5 or 8%), and this synergism was dependent on external Ca2+. These data suggest that agents that produce an inappropriate Ca2+ flux can disrupt or augment disruption of gastric mucosal cells and thus Ca2+ homeostasis is essential for maintenance of mucosal cell integrity. PMID- 1713416 TI - Cholinergic and noncholinergic submucosal neurons dilate arterioles in guinea pig colon. AB - Outside diameter of isolated submucosal arterioles in guinea pig colon were monitored to examine vasodilator innervation to these gastrointestinal microvessels. Electrical stimulation of intrinsic submucosal ganglia dilated submucosal arterioles that had been preconstricted with vasopressin or norepinephrine. In approximately 50% of arterioles examined, the muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 10 nM) abolished nerve-evoked vasodilations; 4-DAMP (500 nM) only partially inhibited the response in the other 50%. No significant differences in the nerve evoked vasodilations were observed after extrinsic denervation of sympathetic and sensory fibers and removal of myenteric neurons by surgical myectomy. Immunohistochemistry demonstrated both substance P (SP) and vasoactive intestinal polypeptide (VIP) fiber projections to arterioles after sensory denervation and myectomy. Superfusion with muscarine and SP, but not with VIP or calcitonin gene related peptide (CGRP), also dilated the arterioles; concentrations of muscarine and SP producing half-maximal responses were 35 and 6 nM, respectively. However, local pressure ejection of both CGRP and VIP dilated the arterioles. The SP receptor antagonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]SP reduced vasodilations in response to ganglionic stimulation as well as to pressure ejection of both SP and VIP. This study demonstrates that submucosal arterioles in the guinea pig distal colon receive a cholinergic as well as a noncholinergic vasodilator input from neurons in the submucosal plexus. SP and VIP are both likely candidates as the noncholinergic vasodilator transmitter to colonic gastrointestinal microvessels. PMID- 1713417 TI - Extent and modulation of junctional communication between pancreatic acinar cells in vivo. AB - To assess whether junctional communication may be of physiological relevance in the control of exocrine pancreas secretion, we have studied acinar cell coupling by microinjecting Lucifer Yellow CH in the intact pancreas of anesthetized rats. Reconstructions from serial sections showed that, under control conditions, pancreatic cells are extensively coupled within each acinus but do not communicate with centroacinar cells, duct cells, and cells of neighboring acini. Intravenous infusion of acetylcholine and caerulein, or electrical stimulation of the vagus nerve, increased pancreatic secretion (P less than 0.02-0.001). Under these stimulatory conditions, the extent of acinar cell communication was decreased (P less than 0.001) by 40%. The acetylcholine-induced uncoupling was prevented by treating rats with atropine. Thus, in the intact pancreas, acinar cells intercommunicate extensively within each acinus under resting conditions and reduce their coupling during stimulation. These data support the view that modulation of cell coupling is a physiologically relevant mechanism for the regulation of exocrine pancreas secretion in vivo. PMID- 1713418 TI - Low doses of ethanol have Ca2+ ionophore-like effects on apical membrane potential of in vitro Necturus antrum. AB - The effects of low doses of luminal ethanol on the amiloride-sensitive apical membrane potential of Necturus antral mucosa were studied using conventional microelectrode techniques. Luminal ethanol (0.250-4.0% vol/vol) caused a dose dependent hyperpolarization of the apical membrane potential (Vmc), an increase in transepithelial resistance (Rt) and resistance ratio (Ra/Rb), and a decrease in transepithelial potential (Vms). Luminal amiloride (100 microM) to 4% ethanol treated antra did not cause any additional hyperpolarization of Vmc. Compared with luminal 2% ethanol-Ringer, an equivalent osmotic mannitol solution depolarized Vmc and basolateral potential (Vcs), decreased Rt and Ra/Rb, and increased Vms. A single dose of 0.50% ethanol attenuated the effects of a second 2% ethanol exposure on Vmc. No change in periodic acid-Schiff (PAS)-positive mucous granule content could be found between control and 2% ethanol-treated antra. The Ca2+ ionophores A23187 or ionomycin (0.25-5.0 microM) dose dependently hyperpolarized the Vmc and Vcs, increased Rt and Ra/Rb, and decreased Vms. Luminal Ca(2+)-free Ringer had no effect on luminal 2.00% ethanol-induced changes in membrane potentials or resistances. Pretreatment with BAPTA blocked by approximately 70 and 55% the Vmc hyperpolarization of 2 and 4% ethanol, respectively. Pretreatment with ruthenium red (10-50 microM) also dose dependently reduced the 2% ethanol-induced changes in Vmc. The data indicate that 1) low doses of luminal ethanol and Ca2+ ionophores have similar effects on Necturus gastric antral membrane potentials and resistances, 2) ethanol-induced hyperpolarizations of the Vmc are partially mediated through an alteration in intracellular Ca2+, and 3) low doses of luminal ethanol do not cause the release of antral epithelial mucous granules at the time when significant changes are occurring in the Vmc. PMID- 1713419 TI - A Ca-activated K channel from rabbit renal brush-border membrane vesicles in planar lipid bilayers. AB - Rabbit renal brush-border membranes were fused to planar lipid bilayers to gain insight into the nature and properties of ion channels from the luminal membrane of the proximal tubule. Fusion was obtained using osmotic gradients. A large conductance channel was commonly observed. Measurements of reversal potentials indicated that the channel was selective for K over Rb, Na, and Cl. Channel open probability was increased by membrane depolarization and by increased Ca activity on the intracellular face of the channel. The channel was inhibited by charybdotoxin (CTX), a protein from leiurus venom, from the external side of the channel. The channel was also blocked by Ba and quinidine added to the intracellular bathing solution. Na added to the intracellular bathing solution reduced current amplitude in a voltage-dependent fashion. In addition, methylisobutyl amiloride, an analogue of the K-sparing diuretic amiloride, inhibited channel activity when added to the external solution. The possible physiological role of the channel is discussed. The usefulness to the study of renal ion channels of the technique of fusing membrane vesicles to planar lipid bilayers is evaluated. PMID- 1713420 TI - Colocalization of insulin-like growth factor-binding protein with insulin-like growth factor I. AB - We report the localization of insulin-like growth factor I (IGF-I) and a 25-kDa form of insulin-like growth factor-binding protein (IGF-BP-1) in adult rat kidney. The antigens were localized using a rabbit anti-human IGF-I antibody, and a rabbit anti-human IGF-BP-1 antibody raised against human 25-kDa IGF-BP-1 purified from amniotic fluid. Immunohistochemistry by the avidin-biotin peroxidase conjugate technique showed that both peptides are located in the same nephron segments, in the same cell types. The most intense staining was in papillary collecting ducts. There was moderate staining also in cortical collecting ducts and medullary thick ascending limbs of Henle's loop. In collecting ducts the antigens were shown to be present in principal cells but not in intercalated cells. In distal convoluted tubules, cortical thick ascending limbs, and in structures presumptively identified as thin limbs of Henle's loops there was only modest staining. The macula densa, however, lacked immunoreactivity. Colocalization of IGF-I and IGF-BP-1 in the same cells supports the notion, derived from studies on cultured cells, that the actions of IGF-I may be modified by IGF-BPs that are present in the same location. PMID- 1713421 TI - Platelet-activating factor alters glomerular barrier size selectivity for macromolecules in rats. AB - The effect of platelet-activating factor (PAF) on glomerular permeability to macromolecules was investigated in the isolated kidneys from normal male Sprague Dawley rats perfused at constant pressure. Compared with basal values, infusion of PAF (10 nM final concentration) into the isolated kidneys induced a progressive and significant increase in protein excretion (6.7 +/- 2.7 vs. 40.7 +/- 10.4 micrograms/min, P less than 0.01), completely reversible 20 min after PAF infusion was discontinued (8.5 +/- 1.3 micrograms/min). In additional experiments, during PAF infusion the fractional clearance of small neutral dextrans (radius 24-48 A), defined as the ratio of the clearance of neutral dextrans to the clearance of creatinine, was comparable to preinfusion values, whereas fractional clearance of large dextrans (greater than 50 A) was significantly elevated (P less than 0.005) above preinfusion values. The specific PAF receptor antagonist L 652731 completely prevented the increased fractional clearance of large dextrans induced by PAF. Finally, lowering Ca2+ concentration in the perfusion medium from 2.5 to less than 0.1 mM markedly reduced proteinuria in isolated kidneys exposed to PAF (80.0 +/- 10.3, 42.8 +/- 3.1, and 22.0 +/- 7.6 micrograms/min, respectively, for 2.5, 1.25, and less than 0.1 mM). These results indicate that in isolated perfused kidneys 1) PAF-induced proteinuria is a functional phenomenon reversible on discontinuing PAF infusion, 2) PAF modifies glomerular size-selective properties by increasing transmural passage of large dextran molecules, and 3) PAF-induced change in glomerular permselective properties is dependent on Ca2+ concentration in the extracellular medium. PMID- 1713422 TI - Sampling interstitial fluid from rat skeletal muscles by intermuscular wicks. AB - A modification of the implanted wick method (K. Aukland and H. O. Fadnes. Acta Physiol. Scand. 88: 350-358, 1973) was devised to sample interstitial fluid from rat muscles. Dry nylon wicks were inserted postmortem into intermuscular spaces between leg muscles by means of a plastic catheter, which was subsequently withdrawn. Inserting the wicks postmortem avoids contaminating wick fluid with proteins extravasated as a result of local inflammatory reactions; placing them intermuscularly avoids contamination by fluid and proteins from damaged muscle cells. Wick fluid protein concentrations (mg/ml) averaged 24.1 +/- 1.1 and 28.5 +/- 1.5 (means +/- SE) in medial and lateral hindlimbs muscles, respectively. The corresponding albumin concentrations were 13.0 +/- 0.7 and 13.9 +/- 0.7 mg/ml. Total protein and albumin concentrations in plasma were 54.1 +/- 0.8 and 22.5 +/- 0.3 mg/ml. Electrophoresis of wick fluid showed a pattern of peaks similar to that of plasma, with albumin relatively high and larger molecules relatively low. Proteins from muscle cells were not detected. Isotope studies (125I-labeled albumin, 51Cr-EDTA) showed that less than 2% of the albumin in wick fluid came directly from plasma and that wick fluid was not concentrated by cell swelling postmortem. Wick fluid from intermuscular wicks implanted in anesthetized rats in vivo had nearly the same total protein concentration as fluid from postmortem wicks, but albumin-to-globulin (A/G) ratios were slightly lower (1.22 +/- 0.07 vs. 1.53 +/- 0.21 measured by gel electrophoresis), and more significantly, nearly 50% of the albumin leaked to wick fluid from plasma as a result of wick implantation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713423 TI - Movement of cellular ions during stimulation with isoproterenol in simian eccrine clear cells. AB - The ionic mechanism of beta-adrenergic sweating is unknown. In isolated rhesus eccrine secretory coils, K efflux was determined by an extracellular K electrode and cellular monovalent ions by X-ray microanalysis. Isoproterenol (Iso) induced a small (dose-dependent propranolol-inhibitable) K efflux followed by net K reuptake. Similar K response was seen with forskolin, theophylline, or isobutyl methylxanthine (IBMX). The net K uptake often exceeded the net K efflux, causing a small net accumulation of K. Bumetanide (BT) and ouabain not only abolished the Iso (with or without IBMX) -induced net K uptake but increased the Iso-induced initial K efflux about threefold. BT and ouabain drastically decreased K and Cl concentrations in the clear cell (by X-ray microanalysis) only in the presence of Iso plus IBMX, suggesting that adenosine 3',5'-cyclic monophosphate (cAMP) may simultaneously stimulate both KCl efflux (by unknown mechanisms) and K reuptake (presumably by BT-sensitive cotransporters and ouabain-sensitive Na pumps). Thus the cAMP-mediated ion movement is different from the cholinergic mechanism that is characterized by the net KCl loss, cell shrinkage (Saga et al., J. Membr. Biol. 107: 13-24, 1989; Takemura et al., J. Membr. Biol. In press), and no augmentation of methacholine-induced K efflux by BT or ouabain. PMID- 1713424 TI - Conserved and variant epitopes of target antigens of transmission-blocking antibodies among isolates of Plasmodium falciparum from Malaysia. AB - Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms. PMID- 1713425 TI - Effect of vitamin A on the growth and differentiation of rat external auditory canal epithelium in organ culture. AB - Intact rat external ear canal explants were maintained either in retinoid deficient or retinoid-supplemented medium for 7, 10, 14, and 21 days. The morphology of the external ear canal epidermis was well maintained, including the presence of sebaceous glands even after 21 days in culture with retinoid deficient medium. However, after culturing in retinoid-supplemented medium, the external ear canal epithelium showed both a loss of keratohyalin granules and the formation of keratin. Extensive microvilli formation occurred, even though the desquamation process continued from the superficial layer after 7, 10, and 14 days in culture with retinoid-enriched medium. After 21 days in culture with retinoid-supplemented medium, the ear canal epithelium contained well-developed Golgi apparatus and secretory granules as well. It was concluded that the ear canal epithelium was transformed into a secretory-like mucosal epithelium by retinoid supplementation. PMID- 1713426 TI - [Comparative histological and light-microscopic cytomorphometric studies of the tissue repair processes in postoperative wound complications treated locally with helium-neon laser radiation or a proteolytic enzyme]. AB - Morphological studies were carried out on tissue material, removed from wound peripheral parts during application of secondary sutures on 69 complete perineal wound dehiscences of parturients prepared by helium-neon laser irradiation according to a method described by us, as of wound peripheral parts of 15 perineal wound dehiscences, prepared by alphahymotripsin dissolved in a solution with vitamin C. Histological preparations were stained by hematoxilin-eosin by van Gieson (for collagenous fibres), by Weigert (for elastic fibres) and by Gomori (for reticular fibres). Light microscopic cytomorphometric studies were made by a graduated ocular micrometer on stained with hematoxilin-eosin tissue sections with the help of standard optical system. The results of the performed histological examinations give foundation to accept the occurred more active stimulation of inflammatory and proliferative phase of wound reparative process after laser therapy with helium-neon laser irradiation. Light microscopic cytomorphometric studies indicate the presence of more advanced in its development granulation tissue after laser therapy by means of activation of local immunocompetent cells. PMID- 1713427 TI - Combined effects of volatile anesthetics and phosphodiesterase inhibitors on contractile performance in guinea pig hearts. AB - The cardiotonic effects of the phosphodiesterase inhibitors amrinone, milrinone, and 3-isobutyl-1-methylxanthine were studied in Langendorff-perfused guinea pig hearts exposed to the cardiodepressant concentrations of halothane or isoflurane. Left ventricular pressure, rate of change of pressure (dP/dt), heart rate, and perfusion pressure were measured in the presence of increasing drug concentrations. Under control conditions, both (dP/dt)max and heart rate were increased most with 3-isobutyl-1-methylxanthine and least with amrinone. In isoflurane-pretreated hearts (1.3%, vol/vol), the phosphodiesterase inhibitors increased (dP/dt)max to a larger degree, whereas the increase in heart rate remained similar to that in the control hearts. After exposure to halothane (0.8%, vol/vol), however, amrinone and milrinone were less effective with respect to enhancement of contractile performance. There was no difference in the 3 isobutyl-1-methylxanthine-induced increase of (dP/dt)max between hearts exposed to isoflurane and those exposed to halothane. Our results suggest that contractile performance of isolated hearts is more easily stimulated by milrinone and amrinone in the presence of isoflurane than in the presence of halothane. PMID- 1713428 TI - What is the mechanism of action of aprotinin? PMID- 1713429 TI - Effects of aprotinin on postoperative bleeding. PMID- 1713430 TI - Tachykinin receptors and noncholinergic bronchoconstriction in the guinea-pig isolated bronchi. AB - The aim of the study was to assess which type(s) of tachykinin receptor mediate the noncholinergic bronchoconstriction produced by activation (electrical field stimulation) of capsaicin-sensitive primary afferents in epithellum-denuded guinea-pig isolated bronchi. Experiments with natural and synthetic tachykinin agonists indicated the presence of both NK-1 and NK-2 receptors at this level. Experiments with the putative NK-1 (L668, 169) or NK-2 (MEN 10,207, MEN 10,376, L659,877, and R396) selective antagonists against NK-1 and NK-2 selective agonists further supported this conclusion. All the tachykinin antagonists tested reduced the noncholinergic bronchoconstriction to field stimulation with the order of potency MEN 10,207 = MEN 10,376 greater than L659,877 greater than L668,169 congruent to R396. In the presence of peptidase inhibitors, the activity of MEN 10,376 toward the noncholinergic bronchoconstriction was slightly reduced, whereas that of L668,169 was increased. These findings demonstrate that both NK-1 and NK-2 receptors mediate the noncholinergic constriction produced by endogenous tachykinins in guinea-pig bronchi and that the relative contribution of NK-2 receptors is greater than that of NK-1. These findings implicate a major role for neurokinin A rather than for substance P as an endogenous bronchoconstrictor in the guinea-pig isolated bronchi. In the presence of peptidase inhibitors, the relative contribution of NK-1 receptors is increased. PMID- 1713431 TI - Electroimmunology: membrane potential, ion-channel activities, and stimulatory signal transduction in human T lymphocytes from young and elderly. AB - There are conflicting data on the functional role and direction of the changes in membrane potential and ion currents accompanying lymphocyte stimulation. Recently, we discovered that a known sodium channel opener, bretylium tosylate (BT), may influence the stimulatory processes of lymphocyte activation at more than one site. Parallel flow cytometric and electrophysiological measurements with patch clamp techniques showed that BT quickly repolarizes previously slightly depolarized human peripheral blood as well as splenic murine lymphocytes. The repolarization occurred through opening ligand- and voltage gated, hitherto unknown sodium channels, and the sodium influx activated Na(+) K(+)-dependent, electrogenic ATP-ase activity. A comparison of the flexible responsiveness of the membrane potential was carried out between lymphocytes from young and elderly using the above mechanism and a number of combinations of channel blockers and ionophores in order to obtain information on the alleged changes in immunological behavior. A significant difference has been found between lymphocytes from human young and elderly volunteers in the readiness to respond to channel-activating perturbations. An explanation is offered, based upon known physicochemical changes in the plasma membrane during aging. PMID- 1713432 TI - Effects of different regimens of dietary restriction on the age-related decline in insulin secretory response of isolated rat pancreatic islets. PMID- 1713433 TI - The effect of aging on constitutive mRNA levels and lipopolysaccharide inducibility of acute phase genes. AB - Eukaryotic organisms possess natural defense processes associated with their response to injury, inflammation and pollutants. One of these, the acute phase (AP) host response, is characterized by a series of hepatic physiological reactions triggered by factors released as a result of bacterial infection, inflammation or tissue injury and is believed to be the mechanism by which cells and tissues are protected against further damage and injury. The capacity to respond to these physiological insults is known to be affected by aging. We propose that the AP response represents a series of intrinsic processes and interactions that may be affected by aging. Furthermore, we propose that this may be due to the progressive failure of the acute phase response. In this study we examine the relationship between aging and the expression of both positive and negative acute phase reactants, i.e., acute phase serum proteins whose levels are increased or decreased in response to systemic injury and infection. The mRNA levels of the positive acute phase reactants, alpha 1-acid glycoprotein (AGP), alpha 1-antitrypsin (AT), and the negative acute phase reactant, albumin were measured in both normal and inflammation-induced mice of ages 2, 7, 12, and 24 months. A significant decrease in the constitutive levels of AT and albumin mRNAs occurred as a function of increased age. Furthermore, aging decreased the ability of the AGP and albumin genes to respond to inflammation. Our studies indicate that aging may affect the transcription of these genes, processing of their mRNA or stability of the mRNA levels. PMID- 1713434 TI - Measurement of pancreatic amylase in macroamylasaemic sera. PMID- 1713435 TI - Serum alpha-fetoprotein: I. Evaluation of quantitative assays adapted to automated immunoassay systems. AB - Serum alpha-fetoprotein (s.AFP) has been established as a useful tool in monitoring of high-risk pregnancies, as an indicator of fetal neural tube defects, and has been used as an adjunct tumor marker and for monitoring therapeutic efficacy in the treatment of certain tumors. To date, the methods for measuring s.AFP are based upon the immunologic principle and are manual methods. The purpose here is to relate the evaluation of two automated systems for the assay of s.AFP. The automated systems are based upon the following immunoassay methods: a microparticle capture enzyme separation and final quantitation by reflectance fluorescence, and a solid phase 'sandwich' separation coupled with enzyme activity measurement (EIA). The reference method is a competitive binding radioimmunoassay. It has been found by us that the automated methods directly transfer analytically with the manual assay. All methods are referenced to the same standard (WHO 1st Intl. Std. for AFP 72/225). PMID- 1713436 TI - Serum alpha-fetoprotein: II. Clinical evaluation of two automated immunoassays for maternal serum screening. AB - A prospective clinical evaluation is reported for analysis of maternal serum alpha fetoprotein (s.AFP) testing by recently developed enzymeimmunoassay automated systems. The reference method for the study was a manual competitive binding radioimmunoassay which has been utilized by us in the Maternal Serum AFP Screening Program at the Medical University of South Carolina for the past five years. The patients were classified by weeks of gestation, 16 to 20. Mean concentrations of s.AFP increased with the increase in gestational week for each method; calculated multiples of median (MoM) values remained relatively constant regardless of the week of gestation. Mean and median concentrations of s.AFP measured by the radioimmunoassay and the coated tube enzymeimmunoassay methods showed close agreement, indicating the direct transferability of the methods for clinical purposes. The microparticle capture enzymeimmunoassay yielded higher results of s.AFP indicating the necessity for a correction if the method transfer is to be considered for sequential patient testing. (Multiples of median values for each method were uncorrected for maternal age, weight, or diabetes mellitus; lack of correction would not affect comparison of methods for clinical application. PMID- 1713437 TI - [A case of Whipple's disease with ankylosing spondylarthritis treated with trimethoprim-sulfamethoxazole]. AB - We report the case of a patient, suffering from Whipple's disease and HLA B27 positive ankylosing spondylitis with syndesmophytes and erosive discopathy. Since spinal radiographic aspects of spondylitis due to Whipple's disease are unusual, we are debating on their relation. We, then, took an interest in the treatment given to this patient: trimethoprim-sulfamethoxazole which would now appear to be the best antibiotic for Whipple's disease. PMID- 1713438 TI - Acetylcholine receptor-specific T-lymphocyte clones in the normal human immune repertoire: target epitopes, HLA restriction, and membrane phenotypes. AB - Potentially autoimmune T-lymphocyte lines specific for the nicotinic acetylcholine receptor of the neuromuscular junction have been isolated previously from patients with myasthenia gravis. We report on the isolation and expansion of T cells specific for the acetylcholine receptor of Torpedo californica or for a recombinant mammalian acetylcholine receptor alpha chain peptide (X4), from the peripheral blood of 11 healthy donors. Two major T-cell epitopes, located between amino acid positions 44-104 and 141-172, were identified using a panel of overlapping mammalian alpha chain fusion proteins. Most T lines recognized the acetylcholine receptor epitopes in the molecular context of HLA-DR molecules. Unexpectedly, all the T. californica acetylcholine receptor-specific T lines obtained from one DR4 (DRw53), DQw3 donor and two DR4, w8 (DRw53), DQw3 donors were restricted by DRw53 product(s). Using DR gene transfected L cells as antigen presenters, in 4 lines, a close relationship between the recognized epitope and the restricting DR element was revealed. The membrane phenotype of the T. californica acetylcholine receptor-and X4-specific T lines was predominantly CD4+CD8-, with some CD4+CD8+ components. It did not significantly differ from that of control, tuberculin purified protein derivate specific T lines raised from the same donors. These findings are in harmony with previous ones demonstrating the presence of potentially autoimmune T-lymphocyte clones within normal immune repertoires. PMID- 1713439 TI - Antiviral activity of sulfated polysaccharides against African swine fever virus. AB - The polyanionic substances lambda and kappa carrageenan, pentosan polysulfate, fucoidan, dextran sulfate and heparin were investigated for their inhibitory effect on the replication of African swine fever virus (ASFV) in vitro. Lambda carrageenan was the most efficacious with a selectivity index, as based on the ratio of the 50% cytotoxic concentration to the 50% antiviral effective concentration, of 120, followed by pentosan polysulfate with 30, kappa carrageenan 13.3 and fucoidan 10. Dextran sulfate and heparin were almost inactive. In general, the substances had low toxicity for Vero cells. The studies with radiolabeled ASF virions suggest that the sulfated polysaccharides inhibit virus adsorption. Inhibition of virus replication was found for all the polysaccharides only when the substances were present during virus adsorption, with the exception of lambda and kappa carrageenan, which were also inhibitory when added immediately after the adsorption period. PMID- 1713440 TI - Analysis of auditory comprehension performance in individuals with severe aphasia. AB - This research compared the performance of 20 individuals with global and mixed nonfluent aphasia across the four auditory comprehension subtests of the Boston Diagnostic Aphasia Examination (Word Discrimination, Body Part Identification, Commands, and Complex Ideational Material) and across the six subcomponents of the Word Discrimination Subtest (objects, actions, letters, colors, forms, and numbers). As expected, group means revealed severely reduced performance which was equally observable across all auditory comprehension tasks. To determine whether individual subjects demonstrated a pattern of consistently reduced performance for auditory comprehension tasks, z-scores and chi-square statistics were also calculated for each subject and task. The individuals with global aphasia demonstrated a greater number of statistically significant z-scores than was expected by chance. This was not true for the subjects with mixed nonfluent aphasia. Results of this research indicate that although as a group individuals with global aphasia may demonstrate consistently reduced auditory comprehension, when considered on an individual basis, this group may comprise a somewhat divergent population with respect to the configuration of preserved and impaired auditory comprehension skills. PMID- 1713441 TI - Palliation of malignant jaundice. AB - The great majority--perhaps 90%--of patients with malignant jaundice can only be treated by palliative means. The best method of palliation is yet to be defined. Some groups advocate routine surgical bypass, while others hold that all cases should be managed by the insertion of stents, either endoscopically or percutaneously. Recovery from surgery consumes a significant portion of residual lifespan, while stents produce long-term morbidity from stent blockage and cholangitis. The present study used a convenient and simple method to quantify the quality of life that follows surgical bypass and stent insertion. Six patients were followed for at least 6 months after open bypass, and nine after stent insertion. Four patients in each group were still alive at 12 months. The study suggests that there is no significant difference in the quality of life obtained by either method at 6 months, but that there is a clear-cut advantage in having surgical bypass by 12 months. The study points to the need to evolve better stents, to improve stent management and to define criteria which will identify patients who are likely to survive more than 6 months. PMID- 1713442 TI - Outpatient transurethral balloon urethroplasty. AB - Transurethral balloon dilatation was performed on 6 patients, who were assessed pre- and post-procedure on symptomatic and urodynamic criteria. Follow-up was on average 11.7 months. Only 1 patient had a successful result, despite early initial improvement in five. In view of our results and reports from other centres we cannot recommend transurethral balloon dilatation of the prostate as treatment in patients with bladder outlet obstruction due to benign prostatic hypertrophy, except in exceptional circumstances. PMID- 1713443 TI - A critical evaluation of peroxidase profiles in Parthenium argentatum. AB - Specific localization of peroxidases after electrophoresis on nondenaturing polyacrylamide gels is discussed. The use of a multifunctional analysis for the separation of isoperoxidases from polyphenoloxidases is suggested. PMID- 1713444 TI - Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues. AB - Mutant epidermal growth factor (EGF) receptors (obtained by substitution of one, two or three C-terminal autophosphorylable tyrosine residues with phenylalanine residues or by deletion of the C-terminal 19 amino acids, including the distal tyrosine) were expressed in mouse NIH-3T3 fibroblast clones at densities comparable (less than 25% difference) with those in control clones expressing the wild-type receptor. Total EGF-induced phosphorylation of the mutated receptors was not appreciably changed with respect to controls, whereas autophosphorylation at tyrosine residues was decreased, especially in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion mutants), intermediate in the dual mutants and almost complete in the triple mutants. Likewise, increases in intracellular Ca2+ concentrations [( Ca2+]i) induced by fibroblast growth factor were approximately the same in all of the clones, whereas those induced by EGF were decreased in the mutants, again in proportion to the loss of the phosphorylable C-terminal tyrosine residues. The same trend occurred with membrane hyperpolarization, an effect secondary to the increase in [Ca2+]i via the activation of Ca2(+) dependent K+ channels. We conclude that C-terminal autophosphorylable tyrosine residues play a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather in the introduction of strong negative charges at strategic sites of the receptor tail. As a consequence of autophosphorylation, the receptor could become competent for specific association with phospholipase C gamma, with ensuing activation by tyrosine phosphorylation followed by the chains of intracellular responses ultimately leading to DNA synthesis and cell duplication. PMID- 1713445 TI - Tissue-specific regulation of dipeptidyl peptidase IV expression during development. AB - The patterns of dipeptidyl peptidase (DPP) IV activity and protein amount in different rat organs during development were compared. In order to elucidate the molecular basis for these patterns, total RNA was isolated from lung and kidney at different stages of development and analysed by Northern-blot hybridization using an oligonucleotide derived from the DPP IV cDNA sequence. This oligonucleotide hybridized to two distinct mRNAs of approx. 3.2 and 4.8 kb respectively. During kidney development, the pattern for DPP IV mRNA paralleled that of DPP IV activity and protein amount, suggesting that, in kidney, the expression of DPP IV is primarily controlled at the transcriptional level. In contrast, the magnitude of DPP IV activity during lung development compared with that of DPP IV mRNA in lung suggests that post-transcriptional mechanisms are involved in regulating the expression of DPP IV in lung. Organ-specific regulation of DPP IV expression may provide a useful model for further comparative studies of transcriptional and post-transcriptional mechanisms of DPP IV expression within the same organism. PMID- 1713446 TI - Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts. AB - Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3 dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the 'fingerprint' region of the NAD(P)H binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns. PMID- 1713447 TI - Heterogeneous modulation of acute-phase-reactant mRNA levels by interleukin-1 beta and interleukin-6 in the human hepatoma cell line PLC/PRF/5. AB - The acute-phase response to tissue injury and inflammation is accompanied by a dramatic increase in the hepatic synthesis of plasma proteins known as acute phase reactants (APRs). This response is mediated by cytokines produced in part by activated macrophages at the site of inflammation; glucocorticoids have also been implicated as playing a regulatory role. The effects of the cytokines interleukin (IL)-1 beta and -6, alone or in combination, and in the absence or presence of the synthetic glucocorticoid dexamethasone, on the levels of APR mRNAs in the human hepatoma cell line PLC/PRF/5 were analysed. The accumulation of APR mRNAs [the complement components C3, factor B and Cl inhibitor; the major APRs C-reactive protein (CRP) and serum amyloid A protein and the CRP analogue serum amyloid P protein] was determined in dose-response and time-course studies. The APRs differed from each other in their responses to IL-1 beta alone, IL-6 alone, and IL-1 beta plus IL-6. Dexamethasone enhanced the cytokine-driven induction of a subset of APR mRNAs. These studies detail the heterogeneity of the 'in vitro' acute-phase response to defined mediators. PMID- 1713448 TI - Decreased expression of the myristoylated alanine-rich C kinase substrate in transformed BALB/C 3T3 mouse fibroblasts. AB - Expression of the myristoylated alanine-rich C kinase substrate was studied in the untransformed A31 and the transformed BPA31, DA31 and KA31 BALB/c 3T3 mouse fibroblast cell lines. The MARCKS transcript was markedly reduced in the three transformed cell lines. Southern analysis revealed similar gene copy number and no apparent DNA rearrangement. In the untransformed A31 cells, the MARCKS mRNA was constitutive in the cell cycle. PMID- 1713449 TI - Production of MUC1 and MUC2 mucins by human tumor cell lines. AB - A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29 SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins. PMID- 1713450 TI - Purification and characterization of human recombinant insulin-like growth factor binding protein 3 expressed in Chinese hamster ovary cells. AB - Recombinant human insulin-like growth factor binding protein 3 (hIGFBP-3) stably expressed in chinese hamster ovary cells (CHO cells) has been purified to homogeneity from serum-free culture media. The purified protein migrates as a doublet (45/43 kDa) upon SDS-PAGE. The purified recombinant hIGFBP-3 is fully active and binds one mole of IGF-I per mole of recombinant binding protein. When the transfected CHO cells are treated with tunicamycin a single 29 kDa hIGFBP-3 protein is observed. This expressed hIGFBP-3 protein maintains its ability to bind IGF-I. N-Glycanase treatment of the purified hIGFBP-3 protein results in a protein that migrates similar to E. coli-derived IGFBP-3 upon SDS-PAGE under reducing conditions (30 kDa). Carboxymethylation of hIGFBP-3 suggests that all 18 cysteines are involved in disulfide linkages. These results represent the first purification and characterization of recombinant hIGFBP-3 expressed in CHO cells. PMID- 1713451 TI - Glucose utilization by tumor cells: a post-translational modification of mitochondrial hexokinase may play a regulatory role. AB - In Northern blot analysis of a series of tumor cell lines a single hexokinase mRNA species of 4.3 Kb was detected. Detailed examination of one such line, the rat AS-30D hepatoma, revealed that two mitochondrial species of hexokinase are present with a molecular mass of 115 and 107 KDa. The smaller of the two species is 4-fold more active than the larger. Only the larger, less active species is detected in the well differentiated H-35 rat hepatoma cell line which exhibits a lower glucose catabolic rate. These results suggest that a post-translational proteolytic event may play a central role in regulating the glucose utilization capacity of tumor cells by modulating the relative levels of high and low activity forms of hexokinase. PMID- 1713452 TI - Cloning and functional expression of human cDNA for the ETB endothelin receptor. AB - We report the cloning of human cDNA encoding an ETB (non-isopeptide-selective) subtype of the endothelin receptor. The predicted amino acid sequence of the human ETB endothelin receptor was 87.8% and 62.9% identical with the previously cloned rat ETB and ETA receptors, respectively. COS cells transiently transfected with the cloned cDNA expressed specific, high-affinity binding sites for endothelin isopeptides and responded to the peptides with a transient increase of [Ca2+]i; endothelin-1 and endothelin-3 exhibited approximately equal potencies both in displacing 125I-labeled endothelin-1 binding and in eliciting [Ca2+]i transients. The ETB receptor mRNAs were expressed in various human tissues and also in the intact porcine aortic intimal cells ex vivo. PMID- 1713453 TI - Epitope mapping of the receptor-bound agonistic form of human chorionic gonadotropin (hCG) in comparison to the antagonistic form (deglycosylated hCG). AB - On the surface of free human chorionic gonadotropin (hCG), we can distinguish with our panel of monoclonal antibodies (MCA) 14 topographically distinct epitopes (designated alpha 1 - alpha 5, beta 1 - beta 5, alpha beta 1 - alpha beta 4, depending on the subunit they are attached to). Only 2, i.e. the adjacent beta 3 and beta 5 epitopes, of these 14 are accessible to 125I-labeled MCA binding, when hCG is first allowed to bind to the rat testis hCG receptor. This result indicates that the agonist hCG assumes a defined orientation in its receptor-bound state and that, except for that small area comprising the beta 3 and beta 5 epitopes, most of its surface is masked by the hCG receptor. We therefore asked whether the competitive antagonist deglycosylated hCG (degly hCG), which, when free, is antigenically (as to number and topography of epitopes) indistinguishable from native hCG, would interact with the receptor differently, that is, in a way that can be discerned by this epitope accessibility paradigm. Here we describe that on receptor-bound degly-hCG the beta 3 and beta 5 epitopes were concealed as were all other epitopes. This observation, together with finding the receptor affinity of degly-hCG to be 4 times higher than that of native hCG, suggests that degly-hCG assumes a signal transduction-incompetent ligand orientation and at the same time interacts with the receptor more intensively, i.e. establishes additional ("antagonist accessory") protein-protein contacts besides those involved in agonist binding. It thus appears that the carbohydrate moieties function to prevent formation of such accessory contacts. PMID- 1713454 TI - Isolation of a cDNA clone, encoding a human beta-galactoside binding protein, overexpressed during glucocorticoid-induced cell death. AB - In this report we describe the isolation and characterization of a cDNA clone overexpressed tenfold during the induction of apoptosis in the glucocorticoid sensitive human leukaemia cell line CEM C7. This clone was shown by DNA sequence analysis to represent the human HL14 gene, encoding a beta-galactoside binding protein, the mouse homologue of which has recently been reported to act as a cell growth inhibitory factor. PMID- 1713455 TI - Novel serine phosphorylation occurs in the fibroblast form of pp60c-src from Y79 retinoblastoma cells. AB - Two-dimensional tryptic peptide analysis showed that pp60c-src from the human retinoblastoma cell line Y79 gave a unique phosphopeptide, which was not found in human fibroblast RT59. There was no significant difference in the extent of phosphorylation of other peptides between the two cell lines. Only phosphoserine was detected in this phosphopeptide. Both the fibroblast form and the neuronal form of pp60c-src from Y79 cells had this unique peptide phosphorylated to the same extent. The phosphorylation site was inferred to be serine 97 by comparing the tryptic map and the arginyl-endopeptidase map. The specific protein kinase activity of pp60c-src from Y79 cells was nearly equal to that of RT59 pp60c-src. This unique serine phosphorylation in the fibroblast form was discussed in relation to the oncogenic change of Y79 cells. PMID- 1713456 TI - Toxic impact of lindane on aspartate and lindane aminotransferase activity levels of midgut gland of marine prawn, Penaeus indicus. AB - In vitro addition of lindane (organochlorine insecticide) causing significant changes in the protein and amino acid metabolic profiles of midgut gland by interfering with the aminotransferase system of Penaeus indicus. PMID- 1713457 TI - New agents to increase the permeability of the outer membrane of Escherichia coli. AB - Two diamines were prepared to investigate the structure-activity relationship required for an increase in the permeability of the outer membrane of Escherichia coli. It was found that diamine (a), bis[4-(2-methylaminoethoxy)phenyl]methane dihydrochloride, increased the permeability of the membrane, while diamine (b), 1,4-bis(2-methylaminoethoxy)benzene dihydrochloride, did not. The result indicated that the existence of bulky hydrophobic moiety is important to cause an increase in the permeability. PMID- 1713458 TI - Comparison of vasodilatory prostaglandins with respect to cAMP-mediated phosphorylation of a target substrate in intact human platelets. AB - The recent purification of a vasodilator-stimulated phosphoprotein (VASP) from human platelets and the development of a specific antiserum against VASP made it possible to study the quantitative effects of cAMP-elevating prostaglandins on cAMP-mediated phosphorylation of VASP in intact human platelets. Prostacyclin (PG I2), prostaglandin-E1 (PG-E1) and the stable prostacyclinanalog Iloprost, all agents used for the treatment of peripheral vascular disease, induced rapid, stoichiometric and reversible phosphorylation of VASP in human platelets mediated by the cAMP-dependent protein kinase. However, there were substantial differences between these three cAMP-elevating prostaglandins with respect to their effects on extent, duration and reversibility of VASP phosphorylation. Maximal VASP phosphorylation was induced both by PG-I2 and Iloprost, but the PG-I2 effect was only of short duration in comparison to that of Iloprost. The extent of PG-E1 induced VASP phosphorylation was less than that observed with PG-I2 and Iloprost. In endothelial cell-platelet coincubations, an endothelial cell-derived, indomethacin-sensitive factor caused a rapid elevation of platelet cAMP level and VASP phosphorylation. These results provided direct evidence that human endothelial cells are capable of producing biologically active quantities of cAMP elevating prostaglandins sufficient to induce stoichiometric cAMP-mediated protein phosphorylation in human platelets. VASP-phosphorylation induced by PG-I2 and PG-E1 was completely reversible after removal of the prostaglandins whereas this was only partially the case with Iloprost. In addition, evidence is presented that the prostaglandin-regulated adenylate cyclase system but not the cAMP-mediated protein phosphorylation desensitizes in human platelets after prolonged treatment with cAMP-elevating prostaglandins. VASP phosphorylation is proposed as a marker for quantitating aspects of vessel wall-platelet interaction. PMID- 1713459 TI - Sequence-dependent interaction of 5-fluorouracil and arabinosyl-5-azacytosine or 1-beta-D-arabinofuranosylcytosine. AB - We studied the cytotoxicity of arabinosyl-5-azacytosine (Ara-AC), a dCyd antagonist which inhibits DNA synthesis, in combination with 5-fluorouracil (FUra) in two human colon cancer cell lines, HCT 116 and SNU-C4. Clonogenic assays done following sequential or concurrent 24-hr exposures to Ara-AC and FUra showed that the sequence Ara-AC followed by FUra resulted in more than additive lethality in the HCT 116 cell lines and additive lethality in the SNU-C4 cells. In contrast, the reverse sequence, FUra followed by Ara-AC, was antagonistic in both cell lines. A similar interaction between FUra and 1-beta-D arabinofuranosylcytosine (Ara-C) was evident in HCT 116 cells; at concentrations which individually diminished viability by 34 and 62%, respectively, the sequence Ara-C followed by FUra decreased viability by 97%. Pulse-labeling with [3H]dUrd showed profound inhibition of DNA synthesis by the sequence Ara-AC followed by FUra, with over 90% inhibition lasting for up to 48 hr following Ara-AC exposure. When FUra preceded Ara-AC, however, earlier recovery from inhibition of DNA synthesis occurred. FUra pretreatment did not appreciably alter the quantity or distribution of [3H]Ara-AC or [3H]Ara-C nucleotides after a 4- to 6-hr exposure. Pre-exposure to FUra decreased Ara-AC incorporation into DNA by 37 and 73% at 6 hr in HCT 116 and SNU-C4, respectively. FUra pretreatment also inhibited Ara-C incorporation into DNA by over 50% at 6 and 24 hr. The antagonism of Ara-AC and Ara-C cytotoxicity by FUra pretreatment can thus be explained by diminished incorporation of the dCyd analogs into DNA resulting from inhibition of DNA synthesis by FUra-induced dTTP and dCTP depletion. In contrast, when Ara-AC or Ara-C preceded FUra, their incorporation into DNA was not disturbed, and prolonged inhibition of DNA synthesis was observed. PMID- 1713460 TI - Inhibition of cellular thymidylate synthesis by cytotoxic propenal derivatives of pyrimidine bases and deoxynucleosides. AB - A series of cytotoxic propenal (3-oxoprop-1-enyl) derivatives of pyrimidine bases and deoxynucleosides was evaluated for their ability to block thymidylate synthesis in intact and permeabilized murine leukemia L1210 cells. Several were potent inhibitors of this process, likely contributing to their cytotoxicity. The IC50 values of thymidine-3-propenal, the prototype of this series, in intact and permeabilized L1210, L-M and L-M(TK-) cells were 21, 7.5, and 75 microM and 1.5, 1.7, and 3.5 microM, respectively. The related base analogue, thymine-1-propenal, is a product of bleomycin-induced DNA strand-scission; the results of the present study bear on the mode of action of this antibiotic. PMID- 1713461 TI - Early effect of BCNU on rat astrocytes. Inhibition of S6 kinase activation by growth factors. AB - In primary cultures of astrocytes, methylmethane, 2-N-methyl 9-hydroxy ellepticinium acetate, ditercalinium, 1-(2-chloroethyl)-3-cyclohexyl-1 nitrosourea and 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) blocked to various extents the activation of S6 kinase by acidic fibroblast growth factor and insulin [or insulin-like growth factor 1 (IGF1)]. The effects of the most active agent, BCNU, were time and concentration dependent. Pretreatment of cells with 50 microM BCNU for 1 hr completely prevented S6 kinase activation by growth factors for at least 2 days. The S6 kinase activity of unstimulated cells was slightly affected. S6 kinase activation by 12-O-tetradecanoylphorbol 13 acetate was also strongly impaired by treating cells with BCNU whereas activation by 8-bromo cyclic AMP was slightly reduced. Cyclic AMP-dependent protein kinase and phospholipid and Ca(2+)-dependent protein kinase were unaffected. BCNU had no direct effect on IGF1 binding to cell surface receptors or on the S6 kinase activity of cell cytosols. PMID- 1713462 TI - High prostaglandin-E1 binding to serum protein in allergic subjects. AB - Protein binding to both salicylate and 3H-labelled prostaglandin-E1 ([3H]PGE1) was examined in the sera of 22 allergic and 16 normal individuals. Protein binding to salicylate (P less than 0.001) and to [3H]PGE1 (P less than 0.01) was significantly greater in the allergic than in the normal group. The nature of the binding sites of salicylate and PGE1 was investigated with two fluorescent probes, dansylamide and dansylsarcosine as specific marker ligands for established Sites I and II, found to be specific for anionic drugs. The serum protein of 11 allergic subjects showed a higher binding at Site I (P less than 0.05) and a lower binding at Site II (P less than 0.05) than that of seven normal subjects. Salicylate and [3H]PGE1 bound competitively at the two sites. It was concluded, when comparing allergic to normal subjects, that the high protein binding of allergic individuals to salicylate and PGE1 could be attributed to qualitative and/or quantitative differences in the lipophilic substances which are tightly bound to the albumin of normal sera, causing a reduction in binding ability at Site I. PMID- 1713463 TI - Hemodilution in cerebral infarcts. AB - To evaluate the effect of hypervolemic hemodilution on cerebral blood flow (CBF) two protocols have been performed: A) Ten randomly selected baboons have been treated with either low molecular dextrane (10 ml/kg) or normal saline (10 ml/kg). Regional cerebral ischemia was produced in all baboons. CBF increased selectively in the ischemic territory but not in the normally perfused tissue. B) Forty patients with acute cerebral ischemia were treated on day onset of symptoms with either dextrose, low molecular weight dextrane, hydroxyethyl starch or pentoxifylline. After intravenous infusion of the substances CBF increased only in the groups treated with dextrane- or starch-solution. Ischemic tissue benefitted more from hypervolemic hemodilution than normally perfused tissue. It was concluded that hypervolemic hemodilution leads to increase of cerebral blood flow, more in ischemic than in normally perfused brain tissue. PMID- 1713464 TI - Reduction of chromosomal damage by bleomycin in lymphocytes from subjects supplemented with carotenoids. Relevance in bleomycin tumour chemotherapy. Preliminary results. AB - In a one year-study, 9 healthy human donors were being supplemented with beta carotene (BC) plus canthaxanthin (CX), to determine the effect of carotenoids on chromosomal damage (micronuclei) induced in the donors' lymphocyte cell cultures by exposure to bleomycin (BLM), an antineoplastic drug that has been shown to produce chromosomal aberrations through the production of free radicals. The first four months monitoring data, including determination of carotenoid blood levels, are here reported. These data show that carotenoid supplementation significantly decrease (up to 50%) the formation of micronuclei induced by BLM in human lymphocyte cell cultures. This decrease is in correlation with carotenoid blood levels. PMID- 1713465 TI - Proliferative activity following induction chemotherapy in squamous cell carcinoma of the head and neck. A histopathological and immunohistochemical study using monoclonal antibodies. AB - Forty-six patients with advanced head and neck squamous cell carcinomas received two courses of chemotherapy with cisplatinum and bleomycin before undergoing cancer surgery. After surgery, histological serial sections of the resection specimens were examined. Biopsies from each resected specimen were shock-frozen for immunohistochemical examinations using the monoclonal antibodies Ki-67 and RPN-511, which are associated with cell proliferation. In no case did the morphologic analysis demonstrate complete tumor regression after chemotherapy. Thirty-seven patients showed partial tumor regressions histologically, as seen by tumor shrinkage of more than 50%. Nine of the specimens showed only minor regressions, with shrinkage less than 50%. The immunostaining of the frozen sections revealed in all cases the expressions of the Ki-67 nuclear antigen and the presence of specific transferrin (RPN-511) receptors in the proliferative compartments of the carcinomas. PMID- 1713466 TI - [Treatment of acute abscesses and gangrene of the lungs]. AB - Complex treatment of acute abscesses and gangrene of the lungs, including intensive therapy and resuscitation, nonoperative treatment with the use of methods for releasing microcirculation from blocking, inhibition of proteolysis in the focus, differentiated correction of the functional activity of phagocytes in the focus of affection, etc., is conducive to reduction of the frequency of the development of a chronic process and drop of mortality. PMID- 1713467 TI - Biological half-time and body burden of cadmium in dogs after a long-term oral administration of cadmium. AB - To investigate the kinetic behavior of cadmium, we conducted a long-term oral administration experiment, using beagle dogs. The experimental animals were given a commercial diet and pelleted food containing 1, 3, 10, 50, and 100 mg of cadmium per day in the form of cadmium chloride for 8 yr. A single injection of cadmium (as CdCl2) into dogs was also performed in order to obtain fundamental kinetic information for a dog. The kinetic behavior of cadmium in chronic experiment is described theoretically, using a two-compartment model. The model was selected based on the elimination pattern of cadmium from the blood in the single injection experiment. The parameters of the model were estimated from the acute and chronic experimental data. The theoretical value of the cumulative amount of cadmium excreted in urine agreed with the experimental one. This result suggests that the two-compartment model used in this study is useful to elucidate the kinetic behavior of cadmium after a long-term exposure to cadmium. The terminal biological half-time in the two-compartment model was estimated at about 1 to 2 yr for both male and female dogs given 1, 3, 10, and 50 mg of cadmium, and for the male dog given 100 mg of cadmium, but only 0.3 to 0.5 yr for the female dog given 100 mg of cadmium. The amount of cadmium in the central compartment and tissue compartment increased continuously and then gradually reached a steady state. The amount of tissue compartment was much higher than that of the central compartment for each beagle dog. PMID- 1713468 TI - Effects of three chelating agents, EDTA, NTA, and TPP, on the concentration of elements in rat tissues. AB - Ethylene diamine tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and tripolyphosphate (TPP) sodium salts were given orally to rats at the dose of 1 mmol/kg/d for 35 d. The concentrations of Na, K, Ca, Mg, P, S, Fe, Sr, Cu, and Zn were determined in blood, plasma, brain, heart, muscle, liver, kidney, duodenum, and bone of control rats and of the rats receiving EDTA, NTA, and TPP. The main effect induced by EDTA, NTA, and TPP was a decrease of the concentrations of several elements Ca, Mg, Fe, P in the duodenum. Otherwise, EDTA induced an increase of Zn in the kidney (+ 20%), NTA, an increase of Fe in liver (+ 29%), and particularly an increase of Zn in bone (+ 44%). TPP induced a slight decrease of Zn and Cu in liver. In conclusion, EDTA, NTA, and TPP taken orally at the dose of 1 mmol/kg/d for 35 d induced moderate changes of the concentrations of some elements in rat tissues, but without signs of toxicity. PMID- 1713469 TI - Hair chromium levels in patients with vascular diseases. AB - The chromium levels in the hair of patients with hyperlipemia and coronary heart disease were found to be similar to those of healthy controls (p greater than 0.2). In patients with cerebral hemorrhage and cerebral thrombosis, significantly higher hair chromium values were observed than in healthy subjects (p less than 0.001). The possible significance of these findings is discussed. PMID- 1713470 TI - Chromosomal aberrations induced by cobaltous chloride in mice in vivo. AB - The effects of cobaltous chloride in inducing chromosomal aberrations were observed on laboratory bred mice in vivo after single oral administration of different fractions (1/10, 1/20, 1/40) of the lethal toxic dose of the salt. Bone marrow cells were flushed out and processed for chromosome studies following colchicine, hypotonic, giemsa, air drying procedure. The parameters screened were chromosomal aberrations, with and without gaps and break per cell. Slides were screened after the expiry of 6, 12, 18, and 24 h. Statistical analysis indicated the clastogenic effects of the salt. The degree of chromosome damage was directly related to the concentration, and also to the period after administration. The different stages of the cell cycle were affected. PMID- 1713471 TI - Dietary fluoride prevents phosphorus-induced nephrocalcinosis in female rats. AB - The effect of dietary fluoride (F) on nephrocalcinosis was studied in young, female rats. Nephrocalcinosis was induced by a diet rich in phosphorus (P). F in the diet effectively counteracted P-induced nephrocalcinosis in a dose-dependent fashion. The feeding of increasing amounts of F caused decreasing calcium (Ca) and F concentrations in kidney. This suggests that the amount of Ca in kidney determines F accumulation in this organ, rather than F intake. Increasing amounts of F in the diet caused increasing rates of urinary and fecal excretion and whole body retention of F. Dietary F did not influence urinary and fecal excretion and plasma concentrations of Ca, magnesium (Mg), and P. The metabolic basis for the protective effect of F against the development of nephrocalcinosis remains to be established. PMID- 1713472 TI - Biochemical response to cadmium. Dose-time effect. AB - Dose- and time-related effects of Cd (II) (0.5 or 1.0 mg/kg, Cd as CdCl2.H2O, subcutaneously, daily for 48 h, 1, 3, or 6 wk) were investigated in rats. A dose related increase in the activity of plasma alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (GOT), and alanine aminotransferase (GPT) was evident only at 6 wk, whereas an early rise in ALP and LDH was seen at 3 wk in 1.0 mg Cd group only. The hepatic and renal metallothionein (MT) induction displayed a dose- as well as time-related increase with Cd accumulation. A significant increase in hepatic Zn and renal Cu, no change in hepatic Cu, and a slight increase in renal Zn was observed. Urinary ALP and leucine aminopeptidase (LAP) showed an initial increase at 48 h, thereafter returned to near normal. A second phase of enzymuria (ALP, LAP, GOT, GPT, gamma glutamyl transpeptidase), proteinuria, and aminoaciduria occurred at 6 wk in a dose-related manner. The urinary excretion of specific renal enzymes appeared closely related to the MT induction and organ Cd levels. PMID- 1713473 TI - Acute zinc deficiency and trabecular bone loss in rats with talc granulomatosis. AB - Subcutaneous inflammation induced by magnesium silicate (talc) leads to the suppression of bone elongation, osteoblast insufficiency, and subsequent bone loss in rats. Since bone and immunological changes in talc granulomatosis are similar to those observed in zinc deficiency, we investigated the kinetics of zinc tissue distribution and the effects of zinc supplementation on the development of bone loss in rats with talc-induced inflammation. Decrease in serum zinc concentration was observed between 5 and 15 h in rats with talc granulomatosis. It was paralleled by the accumulation of zinc in the liver and rapid disappearance of osteoblasts from the trabecular bone surfaces. However, talc-injected rats supplemented parenterally and orally with zinc sulfate exhibited a decrease in osteoblast trabecular surface comparable to that of unsupplemented rats bearing granulomas despite normalized serum zinc concentrations. Zinc supplementation slightly increased osteoblast trabecular surface in all supplemented groups, but this effect was not significant. We conclude that zinc is the earliest indicator of the acute-phase response in rats with talc granulomatosis. Although zinc appears to be important for the normal function of bone cells, there is no causative relationship between acute zinc deficiency and decreased osteoblast number and activity in rats with talc granulomatosis. PMID- 1713474 TI - Endogenous iron excretion. A quantitative means to control iron metabolism? AB - The effects of supplemental oral (0, 40, and 400 ppm) and parenteral iron (0 and 2.72 mg Fe iv given initially as a single dose) on iron absorption, excretion, and retention were determined in 30 rats. Endogenous fecal iron excretion was determined by the radioisotope dilution technique after im injection of 80 kBq Fe 59, using blood and certain body tissues as reference sources for the estimation of the specific activity (Bq Fe-59/micrograms Fe) of endogenous iron. The basal diet contained 3.6 ppm Fe. Fe(III)-hydroxide-polymaltose was used as the sole iron source in oral, iv, and im iron treatments. Iron balance as determined from day 14 to 20 of the experiment was not significantly affected by iv iron administration. Nevertheless, a temporarily reduced retention should have occurred, since differences in final body iron contents were lower than 2.72 mg, as compared to the respective untreated groups. Apparent iron absorption and iron retention increased with surplus oral iron, and the efficiency rates were highest with adequate iron supply (40 ppm). True absorption rates of iron were similar without any, and with 40 ppm Fe amounting 40 to 50% of the intake. In the iron deficient rats, half of the actually absorbed iron (about 16 micrograms/d) was lost by endogenous fecal re-excretion, and another 3 micrograms/d by the urinary route. Endogenous loss with feces and with urine increased with further oral iron supply, but at a considerably lower rate as total fecal excretion. Parenterally administered iron did not affect endogenous loss at all. The results indicate that endogenous excretion cannot be regarded as a means to eliminate excessive iron, and might actually be an inevitable loss. PMID- 1713475 TI - Detection of two Zn-finger proteins of Xenopus laevis, TFIIIA, and p43, by probing western blots of ovary cytosol with 65Zn2+, 63Ni2+, or 109Cd2+. AB - Two Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenile Xenopus laevis females by in vitro binding of radiolabeled divalent metals. Proteins fractionated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) were transferred by Western blotting onto nitrocellulose membranes, probed with 65Zn2+, 63Ni2+, or 109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 micrograms/well by 109Cd(2+)-probing, 0.13 micrograms/well by 65Zn(2+)-probing, and 0.26 mu/well by 63Ni(2+)-probing. Protein p43 was more clearly visualized by probing with 63Ni2+ than with 65Zn2+ or 109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide, 65Zn2+, 109Cd2+, and 63Ni2+ distinctly labeled the 22 kDa middle fragment; 65Zn2+ and 109Cd2+ also labeled the 11 kDa N-terminal fragment, but did not label the 13 kDa C-terminal fragment. These results are consistent with the notion that the radioligands were bound to finger-loop domains of TFIIIA, which occur in the middle and N-terminal fragments. Based on the abilities of nonradioactive metal ions to compete with 65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+ = Cu2+ greater than or equal to Hg2+ greater than Cd2+ greater than Co2+ greater than or equal to Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with 65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43. PMID- 1713476 TI - Distribution and effect of galanin on gallbladder and sphincter of Oddi motility in the pig. AB - This study was designed to determine the occurrence and topographical distribution of galanin-like immunoreactivity (GAL-LI) in the porcine gallbladder and sphincter of Oddi and to investigate the pharmacologic effect of GAL on gallbladder and sphincter of Oddi motility. By radioimmunoassay the concentration of GAL-LI in the gallbladder was 2.75 +/- 0.23, 9.73 +/- 1.33 in the common bile duct and 5.10 +/- 0.37 in the sphincter of Oddi (pmol/g +/- SE). By immunohistochemistry GAL-LI was found exclusively in ganglionic cells and in nerve fibers among the smooth muscle bundles. Gallbladder and sphincter of Oddi pressures were recorded before and during 5-minute local intraarterial infusion of 4, 8, 19, 39, 78 and 194 ng GAL - Kg-1 - min-1 in 12 anaesthetized pigs. GAL in doses greater than or equal to 39 ng.kg-1.min-1 significantly reduced sphincter of Oddi phasic wave frequency (4.8 +/- 0.4 vs. 2.1 +/- 0.5; p = 0.004) and sphincter of Oddi motility index (70.2 +/- 6.02 vs. 27.7 +/- 8.3; p = 0.002) but did not affect gallbladder pressure. We conclude that the distribution of GAL LI in the sphincter of Oddi and the effect that a pharmacologic dose of GAL has on sphincter of Oddi motor activity, suggests that GAL may be involved in the physiologic control of bile flow in the pig. PMID- 1713477 TI - Codon usage, transfer RNA availability and mistranslation in amino acid starved bacteria. AB - The fidelity of codon reading was examined in amino acid starved Escherichia coli. In one case the level of misincorporation of methionine was measured at an isoleucine residue encoded by either the commonly used AUU codon or the rarely used AUA codon. In this situation we found the frequency of methionine misincorporation to be very low and to be unaffected by the identity of the isoleucine codon. In other experiments histidine misincorporation for glutamine was measured in glutamine starved cells with normal levels of histidine-specific tRNA and cells overproducing this tRNA. Cells overproducing the tRNA had higher levels of misincorporation. PMID- 1713478 TI - Intracellular signaling of transcription and secretion of type IV collagen after angiotensin II-induced cellular hypertrophy in cultured proximal tubular cells. AB - Physiologic concentrations of angiotensin II (AII) can induce cellular hypertrophy in murine proximal tubular epithelium (MCT cells). This response is characterized by an increase in cell size, new protein synthesis, and by the secretion of new basement membrane type IV collagen in the absence of cellular proliferation. The present study was undertaken to evaluate the second messengers of these AII-induced cellular events with special reference to the increase in type IV collagen secretion. In initial experiments we observed that pretreatment of MCT cells with agents that increase concentrations of intracellular cAMP, like forskolin, dibutyryl cAMP, and isobutyl-methyl-xanthine abolish AII-induced amino acid incorporation, but have no effect on control cells or on their proliferation. In addition, 10(-8) M AII significantly decreased the concentration of intracellular cAMP. Phorbolesters were without significant effect on the hypertrophy or proliferation of AII-stimulated MCT cells or their rested controls. The transfection of MCT cells with reporter genes containing regulatory elements for type IV collagen revealed that the stimulatory effects of AII on collagen type IV depend, at least to some extent, on an increase in gene transcription. Agents increasing intracellular cAMP concentrations inhibited the AII-induced increase in transcription and secretion of collagen type IV, but had no effect on MCT cells grown in media without AII. Our findings provide evidence that AII-induced changes in tubular epithelium leading to the secretion of type IV collagen are mediated by a decrease in intracellular cAMP. PMID- 1713479 TI - Transgenic rice cell lines and plants: expression of transferred chimeric genes. AB - Cell suspension-derived rice (Oryza sativa L.) protoplasts were transformed by direct gene uptake. PEG-mediated transformation was more efficient than electroporation. Plasmid DNA containing a hygromycin phosphotransferase (HPT) gene (which confers hygromycin resistance) driven by the CaMV 35S promoter and a beta-D-glucuronidase (GUS) gene under control of the 1', 2' double promoter of the mannopine synthase (mas) locus of Agrobacterium tumefaciens was introduced into rice protoplasts. Southern analysis of DNA from transformed cell lines showed that the HPT and GUS genes were present intact. Both genes were expressed in transgenic cell suspensions. GUS activity was detected by histochemical staining of the cells and by enzyme assays. During a 12-day culture period the proportion of stained cells rose to a maximum and then decreased again. Considerably higher numbers of blue-stained cells were obtained when the transgenic cell lines were grown in the presence of 5-azacytidine. Transcripts of the GUS gene could not be detected, in contrast with the HPT gene. Plantlets were regenerated from one transgenic cell line. GUS activity was found in both leaf and root tissues of these plants, particularly, but not exclusively, in vascular bundles. A mouse dihydrofolate reductase coding sequence (DHFR), conferring methotrexate resistance, fused to the CaMV 35S promotor and the wild-type nopaline synthase (NOS) gene of A. tumefaciens were also introduced into rice protoplasts. Stable integration of both genes was confirmed by Southern analysis. Expression of the DHFR gene was demonstrated by high levels of resistance to methotrexate of the transgenic cell suspensions and by the presence of DHFR transcripts. Expression of the NOS gene at enzyme or RNA level was not detected. Southern analysis suggests that this gene was probably either methylated or scrambled in these lines. PMID- 1713480 TI - Photosynthetic electron transport controls degradation but not production of psbA transcripts in the cyanobacterium Synechocystis 6803. AB - Accumulation and stability of psbA and rbcL-S transcripts in Synechocystis 6803 was followed in the presence and absence of the photosynthesis inhibitors DCMU and methylviologen. Our results demonstrate that both transcript production and transcript stability are important regulatory elements for psbA gene expression in Synechocystis 6803. The production of psbA transcripts was stimulated by light in a process that operated independently of the photosynthetic electron transport. However, stability of the psbA transcript increased in the dark and was controlled by photosynthetic electron transport. The psbA transcript was remarkably stable in the dark, with a half-life of approximately 7 hours. By contrast, the regulatory pattern for the rbcL-S genes was quite different. The light-stimulated production of rbcL-S transcripts was dependent on an intact photosynthetic electron transport, and rbcL-S transcript stability was higher under illuminated conditions than in darkness. PMID- 1713481 TI - [Comparative biochemical systems]. PMID- 1713482 TI - Influence of gonadic hormones on the rat submaxillary gland. AB - The area of the granular tubules of the submaxillary gland was evaluated in male and female rats at 60 days of age. In males, this area was significantly greater than in females, corroborating the presence of sex dimorphism in this structure. Dimorphism disappeared after castration. Testosterone increased tubular area. Estrogen and progesterone as well as pregnancy reduced it. The tubular surface was significantly greater at term than at mid pregnancy. PMID- 1713483 TI - Liver fatty acid composition in the spontaneously diabetic BB rat. AB - The purpose of the present experiment was to investigate if the modulation by insulin of liver microsomal desaturase activities in the spontaneously diabetic adult male Bio-Breeding (BB) rat, with destructive insulitis resembling the lesions described in the human Type I (insulin-dependent) diabetes, corresponds to modifications in fatty acid composition, reflect of changes in fatty acid desaturation. We observed no significant differences between BB rats, during the hyper-(48 h), the normo-(17 h) and the hypo-glycemic (3 h) periods which followed the insulin injection, and control rats for the fatty acid composition of liver total lipids, phosphatidylethanolamines, phosphatidylcholines, triacylglycerols, cholesterol esters and non esterified fatty acids. On the other hand, linoleic acid of BB rats liver phospholipids increased, comparatively to control rats, while arachidonic acid decreased, in agreement with previously reported results on chemical diabetes and consistent with a defective delta 6 desaturation, particularly during the normo-and hyper-glycemic periods, and the fact that control of membrane lipid composition is multifactorial. PMID- 1713484 TI - Effect of arginine aspartate on the exercise-induced hyperammoniemia in humans: a two periods cross-over trial. AB - To investigate the effect of the ingestion of arginine aspartate (AA) in the decrease of the exercise-induced accumulation of ammonia in plasma, 11 voluntary subjects took part in a cross-over study where AA effect was tested against placebo. Both treatments were randomly administered in a double-blind procedure. To ensure the subjects would be able to present reproducible exercise-testing results during repetitive sessions, they were involved before the experiment in a cycle ergometer training program during 8 weeks. This training determined a significant 14% increase (P less than 0.001) in maximal oxygen uptake (VO2 max). The treatments were administered during 10 days and the two treatments were separated by a 10 day-wash-out period. A 45 min-cycle ergometer test was performed at 80% VO2 max during the 10th day of each treatment to measure plasma ammonia (p[NH4+]) and total blood lactate (b[lact]) concentrations at rest and at the 15th, 30th and 45th min of exercise (determinations of changes from rest; delta p[NH4+] and delta b[lact]). Both concentrations were unchanged between AA and placebo at rest but a significant lesser delta p[NH4+] was found under AA at the 15th min of exercise only (P less than 0.05). On the other hand, an order effect was found for delta p[NH4+] between the two periods of randomized treatment that was interpreted as a remaining training effect. This effect was highly significant at the 30th and 45th min of exercise (P less than 0.001). It was concluded that AA effect was minor with regard to the training effect. As it was not located at the same time of exercise, AA effect would not consequently have the same functional origin (postulated increase in the peripheral clearance of ammonia) than those of training (decrease in muscle production of ammonia). PMID- 1713485 TI - Enzymatic adaptations to treadmill training under the influence of naftidrofuryl acid in diaphragm and limb muscles of old rats. AB - The effects of training and naftidrofuryl treatment were observed in 21-month-old Long-Evans rats. Rats were injected intraperitoneally twice daily for 8 weeks with 7 mg.kg-1 of naftidrofuryl acid (SN, TN), or with 7 mg.kg-1 fumaric acid (SC and TC) or used as solvent. Training groups (TC, TN) started a progressive 8-week training programme of treadmill exercise. The activities of lactate dehydrogenase (LDH), hexokinase (HK), citrate synthase (CS) and 3-hydroxyacyl-Co-A dehydrogenase (HAD), were measured in Soleus (SOL), Extensor Digitorum Longus (EDL) and Diaphragm (DIA) muscles. The mean VO2max value was 65 ml.min-1.kg-1 for 21-month-old rats. The training protocol induced increases in the mean VO2max values in the TC and TN groups, 71.8 and 74.4 ml.min-1.kg-1. In sedentary groups (SN), naftidrofuryl increased enzymatic activities (HK, CS, HAD) in the three muscles examined. When the animals underwent 8 weeks of physical training, the enzymatic activities (HK, CS, HAD) increased in SOL, EDL and DIA. When training was combined with naftidrofuryl treatment the increases in enzymatic activities were greater than those induced by training alone. However, the total changes did not differ for the sum of the changes produced by each condition alone. PMID- 1713486 TI - Spontaneous activity of sodium loaded guinea-pig cardiac myocytes: contribution of Na+/Ca2+ exchange. AB - Guinea-pig cardiac myocytes loaded intracellularly with Na+ show spontaneous contractile activity and transient membrane depolarizations or inward currents. We have investigated whether both phenomena can be attributed to release of Ca2+ from overloaded cellular stores causing contractile activation and at the same time generating an inward current due to Ca2+ efflux by the electrogenic Na+/Ca2+ exchange. Variations in membrane potential by voltage clamp or in ionic composition of the superfusion medium changed the amplitude and frequency of spontaneous inward currents in the direction expected from computations of the Na+/Ca2+ exchange. Caffeine in concentrations that activate the Ca2+ release channel of the sarcoplasmic reticulum produced a vigorous contraction accompanied by a large current transient. After exposure to ryanodine both Isp and spontaneous contractions were abolished. Spontaneous inward currents were depressed by inhibitors of the Na+/Ca2+ exchange. The results with caffeine and ryanodine demonstrate that unimpaired sarcoplasmic reticular function is a prerequisite for both the contractile and the electrical event. From the characteristics of Isp it is proposed that this current is due to electrogenic Na+/Ca2+ exchange. PMID- 1713487 TI - Alteration of macrophage activity in experimental septic shock in the rabbit. AB - We studied alteration in macrophage activity during experimental septic shock and the effect of the protease inhibitor Trasylol on these alterations. Studies were carried out on three groups of 6 rabbits of each. One group (A) served as a control and in the other two groups (B,C) septic shock was induced using the cecal ligation technique. Group B received i.v. Trasylol prior to and following cecal ligation. The clearance and reticuloendothelial system (R.E.S.) distribution of 125I labelled polyvinyl pyrrolidone (PVP) was used to study macrophage function. PVP was injected into all animals 18 h prior to cecal ligation. For 48 h following the operation, PVP blood levels were repeatedly measured and clearance calculated. The animals were then sacrificed, and total radioactivity of the various organs was measured. In the early stages after cecal ligation a significantly higher PVP clearance rate was noted in groups B and C (P less than 0.01); In the later stages of the experiment, however, group C demonstrated the slowest clearance rate with intermediate values in group B. The highest PVP concentrations were found in the liver and spleen. A significantly higher PVP concentration was noted in the spleen of the animals in group A and B as compared to group C (P less than 0.01) while the difference between group A and B was not significant. Our results indicate that septic shock reduces macrophage function as measured by the changes in PVP clearance and distribution. Injections of Trasylol seem to ameliorate these changes. The model of 125I PVP clearance seems to offer a convenient, valid and informative model for measurement of macrophage activity in pathological conditions. PMID- 1713488 TI - Receptors mediating the contractile effect of serotonin (5-HT) in the isolated common bile duct of guinea-pig. AB - The contractile effect of 5-HT in the isolated common bile ducts of guinea-pigs was studied. 5-HT, administered non-cumulatively, evoked contractions which were concentration-dependent. Responses due to the low concentrations of 5-HT were antagonised significantly by ketanserin (10(-7) M and 10(-6) M) and methysergide (10(-6)M and 10(-5) M) whereas those induced by the higher concentrations of 5-HT remained unchanged. Atropine (3 x 10(-8) and 10(-7) M) and ICS 205-930 (10(-7) M and 10(-6) in contrast inhibited the contractions elicited by high concentrations of 5-HT without altering significantly the responses due to the lower ones. The results led us to conclude that 5-HT evoked contractions at low concentrations are predominantly mediated by 5-HT2 receptors whereas those at high concentrations are dependent on acetylcholine release via the stimulation of 5 HT3 receptors. PMID- 1713489 TI - Volume regulation in rat pheochromocytoma cultured cells submitted to hypoosmotic conditions. AB - The mechanisms at work in cell volume regulation have been studied in PC12 cultured cells. Results show, for the first time to our knowledge, that the volume readjustment process occurring after application of a hypoosmotic saline is sensitive to amiloride, IBMX and forskoline. The process is also inhibited by quinine hydrochloride and trifluoperazine. Volume readjustment is concomtant with a decrease in K+ and Cl- intracellular levels. The decrease in K+ level can be related to an assymetrical change in the fluxes in and out of the ion as shown by flux kinetics studies using Rb86. These results are interpreted considering that the control of the activity of the ion channel pathways associated with volume readjustment in PC12 cells may implicate the Ca(2+)-calmodulin - cAMP system. PMID- 1713490 TI - Oxygen-binding properties of bat hemoglobins. AB - The functional properties of hemolysates from the bats Rhinolophus ferrumequinum, Miniopterus schreibersi and Pipistrellus pipistrellus were studied at 25 degrees C and 37 degrees C over the pH range 7.0-7.4. The concentrations of 2,3-DPG and their effect on hemoglobin O2 affinity were also studied under the same conditions. At pH 7.4 and 37 degrees C hemoglobin O2 affinity was higher than in similarly-sized non-flying, normothermic mammals. The Bohr effect values in the three bat species were slightly lower than those reported for small non-flying mammals. The temperature sensitivities of the oxygenation reactions in bat hemoglobins were low, which may be a mechanism for avoiding the effects of abrupt body temperature changes on oxygen loading and unloading by hemoglobin. The levels of 2, 3-DPG high in red blood cells of active bats decrease when the bats are hibernating. Thus changes in hemoglobin O2 affinity are more probably due to changes in 2,3-DPG concentrations than to alterations of body temperature. PMID- 1713491 TI - Dose dependence of glutaraldehyde-induced changes in the electrical properties of the amphibian skin. AB - The effects of the protein cross-linker glutaraldehyde (GA) on the transepithelial short-circuit current (Isc), conductance (Gt) and impedance of the isolated frog skin were investigated at GA concentrations between 0.1 and 10 mM, i.e. up to three orders of magnitude less than used in fixative procedures. Below 0.5 mM GA increases Isc, with large variations among preparata. Millimolar GA concentrations induce fairly reproducible irreversible inhibitions of Isc, which proceed for about 3 h. The rate of Isc decrease and the amplitudes of the initial drop and subsequent recovery depend on GA concentration in a sigmoidal dose-effect way, reaching saturation at 10 mM. At this GA concentration, Gt is increased up to 5 times the control value. Transepithelial impedance measurements confirm the decreases in epithelial resistance (Rm) and show significant increases in epithelial capacitance (Cm). Rm is diminished by 20% at 0.5 mM GA and by 75% at 10 mM GA, while Cm is maximally augmented by 55% at 2.5 mM GA. It is concluded that protein cross-linking by mild GA treatment is a convenient procedure for changing the electrical characteristics of epithelia. PMID- 1713492 TI - An electromyographic study of an all-out exercise on a cycle ergometer. AB - The electromyograms of the vastus lateralis, the vastus medialis and the biceps femoris have been recorded with surface electrodes during an exhausting exercise on a Monark cycle ergometer, derived from the Wingate test (a 45 second all-out exercise against a braking force equal to 70 N) and during the velocity plateaus of submaximal exercises performed before this exhausting exercise on the same ergometer against the same resistance. This experiment has been carried out in order to study whether central fatigue occurs during an all-out anaerobic capacity test. One subject excepted (a former 800 m runner and road cyclist), there was a decrease in the electro expected integrated electromyogram (iEMG) during the exercise. The iEMG declines of the vastus medialis and the vastus lateralis were parallel and the revolution-to-revolution iEMG changes of the vastus medialis were positively correlated with those of the vastus medialis. These EMG changes suggest the occurrence of a central fatigue during this type of exercise. It was not possible to show a general trend for the effect of fatigue upon the EMG activity pattern of the biceps femoris because of large difference between subjects. PMID- 1713493 TI - The Na+/H+ exchanger role in the intracellular pH regulation of human platelets. AB - The intracellular pH of human platelets was measured with a fluorescent intracellular probe. When platelets were in basal conditions (pHo 7.4, [Na+]o 140 mM) the pHi was 6.98 +/- 0.04. Five minutes after the addition of EIPA 60 microM, an inhibitor of Na+/H+ exchange, the pHi fell in 0.075 +/- 0.022 pH units (P less than 0.05). Preincubation in a sodium free, acid medium (pHo 6.3) induced a cell acidification to pH 6.61 +/- 0.03 (P less than 0.01). Preacidified platelets showed a recovery in sodium-containing solution that is a function of [Na+]o. The initial rate of recovery depends on [Na+]o in a Michaelis-Menten fashion, showing a Km of 35.6 mM and a Vmax of 0.213 pH units/min. These results show that the pHi is maintained in human platelets by a Na+/H+ exchange that is active even in basal conditions. The properties of the Na+/H+ exchanger in human platelets and in the less accessible smooth vascular cells are similar; generalized pathological alterations, like hypertension, could be reflected by both tissues. PMID- 1713494 TI - Para-aminophenol and structurally related compounds as intermediates in lipofuscin formation and in renal and other tissue toxicities. AB - P-aminophenol is considered a minor nephrotoxic metabolite of phenacetin and acetaminophen (paracetamol) in man. Our experiments show that p-aminophenol readily undergoes oxidative polymerization during incubation in human blood or plasma, to form melanin, as a component of soluble lipofuscin. Haemolysis accompanies this process in whole blood. Unmetabolized phenacetin and acetaminophen do not form soluble lipofuscins. Long-term excessive use of phenacetin or acetaminophen has been associated with chronic renal disease, haemolytic anaemia, and increased solid lipofuscin deposition in tissues. Excessive use of phenacetin has also been associated with cancer of renal pelvis and bladder. It appears to us that p-aminophenol and other o- and p-aminophenol metabolites of these drugs are intermediates not only in the etiology of chronic renal disease, but in the other developments as well. P-aminophenol and other ex(end)ogenous aminohydroxyphenyl, aminopolyhydroxyphenyl, polyhydroxyphenyl and polyaminophenyl compounds with these groups in ortho and para positions (such as 3-hydroxyanthranilic acid, 6-aminodopamine, dopamine, p-phenylenediamine, etc.) can undergo autoxidations and metal-catalyzed and enzymatic oxidations in man to produce toxic (semi)quinones(imines), (semi)quinonediimines and reactive oxygen species. After depletion of antioxidants these very reactive (semi)quinones(imines) and (semi)quinonediimine intermediates, many of which are precursors of plasma soluble lipofuscins and melanoproteins, react with essential proteins, DNA, other macromolecules and can cause or contribute to renal and other tissue toxicity, haemolytic anaemia, neoplasia, and granular lipofuscin formation. The reactive oxygen species can also deplete antioxidants, damage essential proteins, DNA, and other macromolecules, and thereby injure cells and extracellular matrix. PMID- 1713495 TI - Adenohypophysis hormone gene products in 14 pituitary adenomas: analysis by immunohistochemistry and northern blotting. AB - We investigated 14 pituitary adenomas (10 silent adenomas; 3 prolactinomas and one GH-secreting tumor) for the presence of hormone gene transcripts (Northern blot) as well as for translation products (immunohistochemistry). The GH secreting tumor was shown to express the genes coding for GH and PRL and to synthesize the corresponding hormones. In the cases of prolactinomas, immunohistochemical data demonstrated the synthesis of prolactin only. In addition to the PRL gene, Northern blot analysis revealed the transcription of the alpha-subunit gene in one case. Hormone genes were found to be expressed in 7 out of the 10 silent tumors, whereas no hormone synthesis was detected in any of these tissues. LH-beta mRNA was found in 3 cases, FSH-beta mRNA in 5 cases and alpha-subunit gene was shown to be expressed in one case. Surprisingly, the level of expression of the FSH-beta gene was higher than in normal tissue. This study confirms that some so called "silent" adenomas are expressing alpha- and/or beta subunit glycoprotein hormone genes, even if no hormone is synthesized. The therapeutic action of bromocriptine described in some "silent" adenomas cases could be related to that hormone gene expression potentiality. PMID- 1713496 TI - Spontaneous activity in vitro of the uterine horns of unilaterally pregnant rats. Relations with glycogen and triglycerides levels. AB - Effects of the placental implantation on the in vitro spontaneous contractile activity of uterine strips incubated in a glucose-free KRB medium, and its relationship with the glycogen and triglycerides tissue levels, have been analysed using unilaterally pregnant rats. The spontaneous activity increased with pregnancy duration, both in the implantation zone (Impl) and the interembryonic segment (Inter) of the pregnant horn. It increased in the contralateral sterile horn (SH) also. Activity was significantly greater in SH and Inter than in Impl, at the same stage of pregnancy. Uterine glycogen, but not triglycerides, appeared to be the substrate used to sustain contractile activity, as its concentration was greater in Impl, the relatively quiescent zone. PMID- 1713497 TI - [Chemostimulation and catecholamine content of the rat adrenal medulla]. AB - Unanaesthetized rats whose arterial chemoreceptors were stimulated by an one hour acute exposition to hypoxic gaseous mixtures with various carbon dioxide concentrations, presented depletion of the catecholamines content of their adrenal glands only when hypocapnia or increased pH was present (non compensated hypoxia). Moreover, exposition to simultaneous hypoxia and hypercapnia increased the epinephrine stock of the adrenal glands. No changes were found in the myocardium amine content in the same conditions. When anaesthetized rats were treated by iv injection of almitrine bismesylate, a peripheral chemoreceptors stimulating drug, adrenal catecholamines content was insignificantly reduced. In the myocardium, the amines remained at control levels. The most powerful factor related to catecholamines depletion in the adrenals seems to be the hypocapnia or the alkalosis induced by the hyperventilation provoked by glomic stimulation. No indication has been found in favor of an effective adrenergic stimulation caused directly by chemoreceptors stimulation. PMID- 1713498 TI - Differential effects of caerulein and bombesin on the ethanol intake in the rat. AB - Male Wistar rats were allowed to drink either water or water mixed with ethyl alcohol (4%, 8% and 12%) as part of a water-deprived procedure. The decapeptide caerulein (1, 3 and 6 micrograms/kg i.p.), a cholecystokinin analog, decreased the intake of ethanol while the consumption of tap water remained unchanged in a choice paradigm. The addition of quinine (a bitter substance) in drinking bottles did not significantly modify the fluid consumption while the i.p. injection of caerulein produced a significant decrease in the consumption of the saccharin containing bottle. The i.p. administration of bombesin (10 and 20 micrograms/kg) failed to modify the intake of water or ethanol solution in water-deprived animals. Interpretations are given in terms of the action of the cholecystokinin analog on differences in the taste intensity induced by the beverage or in terms of a direct consequence of the caerulein-induced decreased gastric emptying effect leading to an accumulation of ethanol in the gut. PMID- 1713499 TI - Circadian cycles of central temperature in hot climate in man. AB - In order to appreciate eventual changes of the circadian rhythm of the body core temperature induced by hot climates, we measured for 24 h periods in summer (June) and in winter (December), in Senegal (latitude, 25 degrees north), the central temperature in two groups of 45 and 58 young soldiers living permanently in the Sahelian area. The subjects were free-running but without any strong activity during the experiments. Rectal temperatures were measured at rest each 3 h, in natural environment: in winter ambient temperature varied from 21 to 25 degrees C and in summer from 28 to 42 degrees C. We observed no change in the shape of the circadian cycle: minimal values were seen at 3 AM and maximal at 3 or 6 PM. But during the hot season, we observed increase of the mesor (36.85 and 37.10 degrees C, in december and june respectively) and of the amplitude of the nycthemeral variation (0.85 and 1.37 degrees C respectively). These results demonstrate a clear adaptation to high ambient temperatures in man, similar to that observed in other mammals. PMID- 1713500 TI - Tosyl-lysyl chloromethylketone inactivation of adenylate cyclase in separate regions of the rat brain. AB - Pretreatment of membranes from rat cerebral cortex, striatum and hippocampus with tosyl-lysyl chloromethylketone (TLCK) modified the adenylate cyclase activity. In the striatum preincubation with TLCK (0.5-6 mM), in the absence and presence of Gpp(NH)p and dopamine, dose dependently inactivated the basal activity of adenylate cyclase. In the cortex and hippocampus a biphasic action of TLCK on the basal activity of adenylate cyclase was observed. Low concentrations of TLCK (0.5 1 mM) enhanced the enzyme activity, while higher concentrations (3-6 mM) inhibited the activity. In the cortex and hippocampus this action of TLCK was found also in the presence of isoprenaline and 5-HT, respectively. In the three brain areas incubation with TLCK inactivated the basal and receptor agonist stimulated adenylate cyclase to equal degrees. In membranes pretreated with 1 mM TLCK the enzyme activity stimulated by forskolin, Gpp(NH)p, and receptor agonists was reduced in both the striatum and hippocampus. The present results indicate that TLCK affects the catalytic unit of adenylate cyclase. The distinct actions of TLCK in the striatum compared with those in the cortex and hippocampus may suggest region-specific differences in the regulation of the catalytic unit of adenylate cyclase in the brain. PMID- 1713501 TI - Effect of phenylpyruvate on mevalonate-activating enzymes from chick brain and liver. AB - Mevalonate-activating enzymes from chick brain and liver were stable when 105,000 x g supernatants were stored at -4 degrees C for 168 h. Mevalonate kinase and mevalonate 5-phosphate kinase retained their activities for 72 h at 4 degrees C while mevalonate 5-pyrophosphate decarboxylase activity significantly decreased after 24-48 h of storage at 4 degrees C. Direct addition of 2.5 mM phenylpyruvate to the reaction mixture produced a significant inhibition of decarboxylase activity in brain and liver. When enzyme preparations were preincubated with 2.5 mM phenylpyruvate for 20 min before the addition of substrate, an increased inhibition was observed. Mevalonate kinase and mevalonate 5-phosphate kinase from both tissues were not affected in the same conditions. The inhibition of brain and liver decarboxylase was progressive with increasing concentrations (2.5-10.0 mM) of phenylpyruvate. No significant difference was observed in the inhibition of decarboxylase after 10 or 20 min of preincubation. PMID- 1713502 TI - Cardiovascular and arginine vasopressin (AVP) responses to haemorrhage and nitroprusside infusion in conscious newborn calves. AB - The effects of normotensive and hypotensive hypovolaemia (haemorrhage) as well as isovolaemic hypotension (nitroprusside administration) on diastolic, systolic and mean arterial blood pressure, heart rate and plasma concentrations of arginine vasopressin were studied in two sets of experiments on 8-10 days old conscious newborn calves bearing an indwelling aortic catheter for continuous recording of arterial blood pressure. Removal of 20% of the estimated blood volume resulted in an average maximum decrease of systolic, diastolic and mean arterial blood pressure from 132 +/- 2 to 118 +/- 8 mm Hg (P less than 0.05), from 72 +/- 2 to 67 +/- 2 mmHg (P less than 0.05) and from 92 +/- 3 to 82 +/- 6 mmHg (P less than 0.05) respectively. In the same time heart rate increased from 124 +/- 3 to 143 +/- 5 beats.min-1. Plasma concentrations of arginine vasopressin increased before blood loss had induced any change in arterial blood pressure (from 5.7 +/- 0.7 pg/ml-1 at time 0 to 25.2 +/- 3 pg/ml-1 at time 20 min; P less than 0.01). The significant fall in blood pressure was accompanied by a further prompt increase in plasma concentration of arginine vasopressin which reached maximum values (90.1 +/- 9.7 pg/ml-1) at completion of haemorrhage. The conscious newborn calf responded to i.v. nitroprusside infusion (10 micrograms/kg-1/min-1 for 10 min) with a prompt fall in diastolic (-71%, P less than 0.01), systolic (-70%, P less than 0.01) and mean (-56%, P less than 0.01) arterial blood pressure within 3 min of the infusion. Time course changes in heart rate were opposite to those in arterial blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713503 TI - Ion transport in epithelial cells from the bovine trachea in culture: effects of dexamethasone and aldosterone. AB - The ion transport properties of epithelial cells from the bovine trachea grown in culture were investigated. The cellular uptake of 22Na+, 36Cl- and 86Rb+ was measured and the sensitivity of ion uptake to amiloride and bumetanide revealed the presence of the transport pathways that exist in the native tissue. The action of corticosteroids, dexamethasone and aldosterone (0.1 or 1.0 microM), was studied by measuring the short-circuit current and the uptake of Na+ and Cl- following a treatment of 18 h, 2 or 8 days. The results showed that, in each situation, dexamethasone enhanced markedly the amiloride-sensitive current and Na+ uptake. In contrast, aldosterone exerted no detectable effect on the ion transport and electrical properties. PMID- 1713504 TI - Excitatory responses of the dorsal root discharge to stimulation of cutaneous and muscle afferents in the cat. AB - Excitatory responses of the dorsal root discharge (DRD) consisting in transient increase in its frequency have been studied in non-anaesthetized low spinal cats. They were evoked by stimulation of cutaneous nerves (superficial peroneal and posterior tibial) and muscle nerves (gastrocnemius-soleus and posterior biceps semitendinosus), recorded from the central cut ends of single fibres of L7 dorsal roots. The excitatory responses were elicited by single volleys in low threshold cutaneous and group II muscle afferents. Group III afferents of gastrocnemius soleus nerve were also effective. Increase in strength of stimulation of posterior biceps-semitendinosus nerve from 4T to 40T which activated group III muscle afferents significantly decreased the excitatory effects of the DRD. Incidence of excitatory responses of the DRD to volleys both in low and high threshold cutaneous afferents, was 100%. Frequency of occurrence of excitatory effects to volleys in group II muscle afferents ranged from 23% to 43% (for responses to posterior biceps-semitendinosus and gastrocnemius-soleus volleys, respectively). It was increased to 47% and 72% when excitatory effects were elicited by group III afferent volleys. These findings indicate that only some types of afferent fibres evoke excitatory responses of the DRD. Variable incidence of the excitatory responses to stimulation of cutaneous and muscle afferents suggests important difference in effectiveness of connections between both types of fibres and interneurones generating the DRD. PMID- 1713505 TI - Characterization and pharmacological studies of an octopamine-sensitive adenylate cyclase from nerve cord of Locusta migratoria. AB - An octopamine-sensitive adenylate cyclase that is insensitive to stimulation by dopamine (DA) and 5-hydroxytryptamine (5-HT), was studied in a homogenized nerve cord preparation of the migratory locust, Locusta migratoria. The enzyme complex is similar to catecholamine-sensitive cyclases from mammals with respect to pH optimum, requirement for Mg++ and GTP, and sensitivity to forskolin. The octopamine-mediated elevation of adenylate cyclase activity is antagonized by a variety of drugs with the following order of potency: mianserin greater than phentolamine greater than promethazine greater than gramine greater than cyproheptadine greater than cis-flupenthixol greater than chlorpromazine greater than metoclopramide. PMID- 1713506 TI - Functional and metabolic effects of uridine and UTP on the skeletal muscle of the rat submitted to exercise. AB - Twenty millimolar and 2 mM uridine triphosphate and 2 mM uridine were injected intra-arterially into rat leg muscles during a 20 min period of intense exercise and during the recovery phase (20 min). Administration of 20 mM uridine triphosphate during exercise, provoked a complete depression of muscle contractility. On the contrary, supply of 2 mM uridine triphosphate or 20 mM uridine induced a reduction in the decrease of muscular developed tension during the exercise period of time and favoured functional recovery. This positive effect of pyrimidine compounds on muscular functional parameters did not seem to be correlated with metabolic effects. Indeed, 2 mM uridine supply did not modify the evolution of intramuscular glycogen and lactate concentrations and made worsened the adenine nucleotide degradation induced by exercise. PMID- 1713507 TI - [Relationship between oxygen deficit and VO2max in supramaximal running]. AB - The maximal accumulated oxygen deficit (DO2max, ml O2/kg) has been measured in 43 healthy male subjects with different VO2max (45-70 ml O2/kg.min) during the course of supramaximal runs performed until volitional exhaustion on an inclined treadmill. It was found that DO2max and VO2max were linearly related: DG2max = 39.7 + 0.31 VO2max (r = 0.701; P less than 0.001). Mean accumulated DO2max (58 ml O2/kg) corresponds to an amount of energy equal to 1212 J/kg. PMID- 1713508 TI - Differentiation in B-precursor acute lymphoblastic leukemia cell populations with CD34-positive subpopulations. AB - B-precursor acute lymphoblastic leukemia bone marrow specimens that contained subpopulations of cells with immunophenotypes corresponding to early (CD34) and late (CD20) and (CD22) stages of normal B-cell differentiation were studied. Subpopulations of cells were isolated according to immunophenotype and then analyzed by both a clonogenic assay and molecular genetic methods. Clonal equivalence of the early and late immunophenotypic subpopulations was confirmed for each case by the demonstration of identical lg gene rearrangements. The in vitro colony-forming assay consistently showed a growth advantage for the CD34+ subpopulations over the CD34- subpopulations. CD34 mRNA was detected readily in these isolated precursor cells. When two specimens in which virtually all of the leukemia cells were CD34+ and CD34+CD20+ and CD34+CD22+ subpopulations were also present the CD34 mRNA was limited to the cells without the late-stage differentiation antigens on their surface. Furthermore, the c-myb mRNA was found only in the subpopulations that also contained CD34 mRNA. Our results show that a limited program of differentiation reminiscent of normal B-cell development may be present in this leukemia. PMID- 1713509 TI - Potentiation of early hematopoiesis by tumor necrosis factor-alpha is followed by inhibition of granulopoietic differentiation and proliferation. AB - We have previously shown that tumor necrosis factor-alpha (TNF alpha) strongly potentiates interleukin-3 (IL-3)-induced short-term proliferation of human CD34+ hematopoietic progenitor cells (HPC). Using longer term cultures of CD34+ HPC, we demonstrate here that this initial potentiation ceases after 10 to 12 days; whereupon TNF alpha displays inhibitory effects. Thus, TNF alpha was found to inhibit cells of granulocytic affiliation while it potentiates the development of maturing cells of the monocytic lineage both in liquid and semi-solid (day 14 colony-forming unit) cultures. TNF alpha was demonstrated to reversibly block granulocytic differentiation at the level of uncommitted CD13-, CD15- blast cells that accumulate in IL-3 + TNF alpha cultures. Furthermore, growth of committed granulocytes (CD15+) from IL-3 cultures was also inhibited by TNF alpha through an arrest of cell cycle in G0/G1. Finally, the use of neutralizing anti-TNF alpha monoclonal antibody and limiting dilution studies indicate that the inhibitory effects of TNF alpha are direct. Taken together, our data demonstrate that, following a phase of potentiation of proliferation of early HPC, TNF alpha displays direct inhibitory effects due to negative interference with both granulocytic differentiation and proliferation of granulocytic cells. PMID- 1713510 TI - Hematologic effects of stem cell factor in vivo and in vitro in rodents. AB - Recombinant rat stem cell factor (rrSCF) administered to rats as a single intravenous injection causes a dose-dependent neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. Neutrophilia begins at 2 hours, peaks at 4 to 6 hours, and subsides between 12 and 24 hours. Lymphocytosis occurs at 0.5 hours and has subsided by 2 hours. rrSCF-induced neutrophilia and lymphocytosis are abrogated by boiling, demonstrating that endotoxin-contamination of the rrSCF preparation is not responsible for the observed hematologic effects. The bone marrow at 6 hours after injection of rrSCF shows a left-shifted myeloid and erythroid hyperplasia as evidenced by significant increases in the absolute numbers of morphologically recognizable early myeloid and erythroid precursors. A concurrent decrease in the absolute numbers of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, bone marrow smears demonstrate a remarkable mast cell hyperplasia accompanied by a decrease in total marrow cellularity and by a striking erythroid and lymphoid hypoplasia. rrSCF also causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type, but not mucosal-type, mast cells. In vitro long-term culture of lineage-depleted mouse bone marrow cells with rrSCF results in an almost pure outgrowth of mast cells. PMID- 1713511 TI - Differential transcriptional and posttranscriptional regulation of gene expression of the colony-stimulating factors by interleukin-1 and fetal bovine serum in murine fibroblasts. AB - Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run-on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS-induced increase in M CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL-1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level. PMID- 1713512 TI - Differential regulation of primitive human hematopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells. AB - Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems, but their role as microenvironmental regulators is poorly understood. To address this problem, we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2-10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early, intermediate, and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]), clonogenic cells, and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders, cells at all stages of hematopoiesis decreased to very low levels. In contrast, maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3 producing M2-10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However, this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand, in the presence of GM-CSF-producing feeders, the output of mature granulocytes and macrophages increased 20-fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long-term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications. PMID- 1713513 TI - Effect of single amino acid substitutions on the formation of the PlA and Bak alloantigenic epitopes. AB - The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2-associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)-specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes. PMID- 1713514 TI - Evidence of c-myc gene abnormalities in mediastinal large B-cell lymphoma of young adult age. AB - Six cases of mediastinal large B-cell lymphoma (MLCL) with sclerosis were analyzed for the presence and patterns of c-myc and bcl-2 loci rearrangements, and for the presence of Epstein-Barr virus DNA sequences by Southern blot hybridization, c-myc gene alterations were found in three of six cases. Two cases showed the presence of mutations or small rearrangements at the 3' end of the first exon. The c-myc gene abnormalities found in these two cases are similar to those observed in the translocation 8;14 of the endemic Burkitt's lymphomas or in its variants t(2;8) and t(8;22). A third case showed a major rearrangement of c myc gene, with truncation within its first intron, similar to those observed in sporadic Burkitt's and in acquired immunodeficiency-associated lymphomas. None of the cases displayed bcl-2 gene rearrangements or contained viral sequences. Our data suggest a possible role for a translocation-mediated c-myc activation in the pathogenesis of MLCL. Conversely, bcl-2 gene and Epstein-Barr virus do not appear to be involved in the pathogenesis of these peculiar lymphomas. The association between c-myc structural modifications and MLCL also seems to be of relevance in light of the peculiar tendency of this tumor to involve unusual extranodal site (eg, kidney), reminiscent of the spreading attitude of Burkitt's limphomas. PMID- 1713515 TI - Antibodies against human neutrophil LECAM-1 (LAM-1/Leu-8/DREG-56 antigen) and endothelial cell ELAM-1 inhibit a common CD18-independent adhesion pathway in vitro. AB - Neutrophil adhesion to interleukin-1 (IL-1)-stimulated human umbilical vein endothelial cells (HUVEC) involves the CD18 family of leukocyte integrins (lymphocyte function-associated antigen-1 [LFA-1], Mac-1, and p150,95) and LECAM 1 (DREG-56/LEU-8/LAM-1 antigen) on neutrophils and intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) on the endothelium. In this study, we compare CD18-independent adhesion pathways mediated by neutrophil LECAM-1 and endothelial ELAM-1 and find that these two pathways overlap in a variety of assays: (1) anti-LECAM-1 and anti-ELAM-1 monoclonal antibody (MoAb) inhibit neutrophil binding to HUVEC, and the inhibitory effect is not additive; (2) anti-LECAM-1 MoAb, like anti-ELAM-1 MoAb, inhibits neutrophil binding to HUVEC stimulated for 3 hours with IL-1, but not to HUVEC stimulated for 8 hours, by which time ELAM-1 expression is downregulated; (3) anti-ELAM-1 MoAb has no effect on transendothelial migration, a CD18 dependent, LECAM-1-independent neutrophil function. Interestingly, anti-ELAM MoAb has a reduced but significant inhibitory effect on the adhesion of activated neutrophils that have shed their cell-surface LECAM-1. We also show that neutrophil binding to ELAM-1-transfected L cells is inhibited not only by anti ELAM-1 but also by anti-LECAM-1 MoAb. These results suggest that LECAM-1 and ELAM 1 can operate in the same adhesion pathway, possibly as a receptor counterreceptor pair. LECAM-1 and ELAM-1 are likely to interact with other ligands as well, perhaps through carbohydrate determinants that modify more than one glycoprotein. PMID- 1713517 TI - Quantitative and functional studies on platelet glycoprotein IV. PMID- 1713516 TI - Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum. AB - When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC. PMID- 1713518 TI - [Determination of estradiol receptors by immunoenzyme technique and by radioligand binding in 2134 breast tumors]. AB - In order to test the qualities of the 2 assays, in the same laboratory and on the same tumors, a single-point dextran-coated charcoal radioligand binding assay (RLA-DCC) and Abbott enzyme immunoassay (EIA) were used for more than two years to perform estrogen receptor determinations on cytosols from 2,134 breast cancers. Statistical analysis of the data was performed according to the method of Passing-Bablock. The final regression curve between EIA (y) and RLA-DCC (x) was excellent y = 1.187 X fmol/mg of protein. However, from 1986 to 1988, a great variability was observed for this correlation. We report the study of this variability, which could be explained by several factors, especially calibration problems for the immunoassay kits and changes in our technical team. The binding assay appears to be more sensitive to the technicians' experience than the immunoassay. Technical points are discussed, particularly cytosol preparation and KCl presence or absence in the homogeneisation buffer. The conditions allowing for optimal correlation and routine determination fiability can therefore be defined. PMID- 1713519 TI - [Palliative care: definition and structure]. PMID- 1713520 TI - Acute myeloblastic leukemia and recombinant granulocyte colony stimulating factor. PMID- 1713521 TI - Effect of sex steroids on the circulating levels of alpha 2-macroglobulin in injured rats. AB - The effect of sex steroids on the plasma levels of alpha 2-macroglobulin (alpha 2 M) was investigated to explain the sexual dimorphism observed in response to the injury caused by ip administration of 5 micrograms endotoxin. Mean serum alpha 2 M concentrations were lower in female (46.82 +/- 8.10 arbitrary units) than in male injured rats (82.94 +/- 9.22 arbitrary units). A reduction of plasma alpha 2 M levels was observed after orchidectomy, and the administration of 1 mg testosterone to the same animals increased the response. The same response pattern was observed in ovariectomized rats under the same treatment. The response of androgenized rats (78.93 +/- 12.81 arbitrary units) to injury was similar to the male response. These results suggest the importance of testosterone as the major determinant of this sex-dependent response. PMID- 1713522 TI - MIP 28 forms channels in planar lipid bilayers. AB - Fiber cells, which constitute most of the lens tissue, have large amounts of a protein named the main intrinsic protein (MIP) in their plasma membrane. MIP seems to vary among species. On SDS-PAGE, MIP from bovine lens (MIP 26) migrates faster than MIP from chicken lens (MIP 28), which runs as a 28-kDa protein. Recently a number of laboratories have shown that MIP 26 forms channels in lipid bilayers. We have isolated membrane fractions highly enriched in MIP 28 from chicken lens and incorporated channel activity into planar bilayers from these membrane fractions before and after treatment with the detergent Triton X-100. Detergent treatment does not seem to affect channel properties. We have attempted to block channel activity with polyclonal antibodies against bovine and chicken MIP but failed to detect blockade using either detergent-free or detergent treated membranes. Single channel size in symmetric solutions of 300 mM K2SO4 (300-400 pS) agrees well with published results if one allows for corrections in ionic strength. Preliminary experiments indicate that the incorporated channels display voltage dependence. The channel activity recorded from MIP 28-enriched membrane fractions is qualitatively similar to that described for MIP 26 membrane fractions incorporated into bilayers. In contrast to previous reports, we do not find it necessary to add the membrane fractions to both sides of the bilayer to obtain channel incorporation. This may reflect the fact that MIP does not span two bilayers. PMID- 1713523 TI - Effect of turpentine on alpha 1-major acute phase concentration in normal and adrenalectomized rats. AB - Acute phase response of plasma alpha 1-major acute phase (alpha 1-MAP) was studied in normal and adrenalectomized rats. alpha 1-MAP basal levels were higher in female than in male rats. This protein, measured by radial immunodiffusion, increased significantly both in male and female rats 24 and 48 h after a turpentine stimulus, proving to be a positive acute phase protein for both sexes. In adrenalectomized male rats the increase of plasma alpha 1-MAP concentration was not different from that observed in sham-operated rats, suggesting that the acute phase response of this protein is not corticoid dependent. PMID- 1713524 TI - Distribution of extracellular tracers in perivascular spaces of the rat brain. AB - Large molecular weight tracers (india ink or albumin labeled with colloidal gold, Evans blue or rhodamine) were micro-injected into the perivascular space of an artery or vein on the brain surface, or within the cerebral cortex or the subarachnoid space of anesthetized rats. The subsequent distribution was followed both under intravital microscopy, in order to outline the pathways and direction of tracer movement, and in histological section, in order to describe the pathways of flow at the light and electron microscopic level. The tracers remained largely in the perivascular spaces and in the interconnecting network of extracellular channels, including the subpial space and the core of subarachnoid trabeculae. Tracer also leaked across the pia into subarachnoid CSF. Bulk flow of fluid within the perivascular space, around both arteries and veins, was suggested from video-densitometric measurements of fluorescently labeled albumin. However, this flow was slow, and its direction varied in an unpredictable way. These results confirm that perivascular spaces may serve as channels for fluid exchange between brain and CSF, but do not support the idea that CSF circulates rapidly through brain tissue via perivascular spaces. PMID- 1713525 TI - Selective reduction of glucocorticoid receptor immunoreactivity in the hippocampal formation and central amygdaloid nucleus of the aged rat. AB - Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713526 TI - Experience influences environmental modulation of function at the benzodiazepine (BZD)/GABA receptor chloride channel complex. AB - Function of the benzodiazepine (BZD)/GABA receptor chloride channel complex was selectively altered by specific aspects of an environmental challenge, i.e. encountering a stranger in a familiar versus an unfamiliar environment. Chloride (Cl-) enhancement of [3H]flunitrazepam [( 3H]flu) binding was facilitated in rats tested in an unfamiliar environment relative to that in rats tested in a familiar environment. Basal [3H]flu binding (binding in the absence of NaCl) also was greater in rats tested in the unfamiliar environment than in rats tested in the familiar environment, and Scatchard analysis of [3H]flu binding indicated that increased [3H]flu binding in the unfamiliar environment reflected an increase in both binding affinity and maximal binding capacity. In addition, both the sensitivity of [3H]flu binding to Cl- and the affinity of BZD recognition sites were decreased in handled control rats relative to non-handled control rats as well as to environmentally-challenged (prehandled) rats, suggesting that the experience of daily handling as well as familiarization with the environment modulates function at the BZD/GABA receptor complex. GABA-mediated 36Cl- uptake was facilitated by testing in either the familiar or unfamiliar environment relative to that measured in non-handled control rats. Thus, changes in GABA gated chloride channel function may reflect a more fundamental response of this complex to challenging situations. These findings suggest that components of the BZD/GABA receptor complex are differentially influenced by specific aspects of an environmental challenge. Furthermore, function at the BZD recognition site/chloride channel component of this receptor complex is influenced by both repeated and single exposure to specific environments. PMID- 1713527 TI - Existence of mutual synaptic relations between corticotropin-releasing factor containing and somatostatin-containing neurons in the rat hypothalamus. AB - Light microscopic studies of vibratome sections, which were double-immunostained for corticotropin-releasing factor (CRF) and for somatostatin (SS), suggested the presence of reciprocal synaptic relations between neurons containing immunoreactive (ir) CRF and those containing ir SS in the parvocellular paraventricular nucleus (parvo-PVN) and in the anterior periventricular area (APV) of the rat hypothalamus. In the sections the peptides included in neuronal fibers were labeled black with silver-gold particles, and the peptides included in neuronal cell bodies were labeled brown with diaminobenzidine (DAB). Thereby the brown cell bodies appeared to be surrounded by several black nerve terminals. In electron microscopic studies, the labeling was mostly performed in reverse fashion, because of the convenience for observing the ultrastructural details of the nerve terminals. The neuroplasm of the postsynaptic perikarya and dendrites was labeled with gold-coated silver grains, while the presynaptic axonal terminals were shown with scattered DAB particles. Granular structures in the perikarya or axonal terminals were labeled distinctively. The synaptic morphology appeared to be either symmetric or asymmetric connections. Then we found synaptic connections between presynaptic ir SS containing fiber terminals and postsynaptic ir CRF containing perikarya in the parvo-PVN, and those ir CRF containing fiber terminals and ir SS containing perikarya in the APV. The existence of such a reciprocal association between CRF and SS neurons may suggest that these neuronal systems intervene among different functional systems in the hypothalamus. PMID- 1713528 TI - Dual projections of gonadotropin releasing hormone containing neurons to the interpeduncular nucleus and to the vasculature in the female rat. AB - Immunofluorescence for gonadotropin releasing hormone (GnRH) in combination with retrograde labeling from the interpeduncular nucleus, as well as from the vasculature confirms that, in the rat, certain GnRH neurons project from the septum-diagonal band to the interpeduncular nucleus. However, about one half of these GnRH neurons also project to fenestrated capillaries as evidenced by uptake and retrograde transport of both peripherally injected Fluoro-Gold and centrally injected rhodamine labeled microspheres. The results indicate that the endocrine effects of GnRH are exerted in part by neurons which simultaneously project to neurohemal contact zones and to areas in the brain which are involved in the regulation of certain behaviors. It is suggested that certain GnRH neurons can directly couple endocrine events with other intracerebral events, such as regulation of lordosis behavior. PMID- 1713529 TI - Food restriction retards aging of the pineal gland. AB - The effects of chronic (40%) food restriction from 6 weeks of age were studied in aging male Fisher 344 rats. When compared with 3-month-old, ad libitum fed rats, pineal N-acetyltransferase (NAT) activity had declined to less than 30% and pineal and serum levels of melatonin to 40% after 28 months when feeding had been ad libitum. Food restriction significantly retarded this development (P less than 0.05) giving NAT and melatonin levels which were twice as high as in the ad libitum fed group. Nighttime levels of pineal serotonin (5-HT) were similar in food-restricted and ad libitum fed old rats but were nearly twice as high (P less than 0.05) as in young rats. There was also a tendency for increased production of 5-hydroxyindoleacetic acid (5-HIAA) in the pineal gland with higher levels of 5-HT. It is concluded that aging in the rat (Fisher 344) is accompanied by a reduction of pineal NAT activity, thereby reducing the production of melatonin and causing a buildup of 5-HT in the pineal gland. It is furthermore proposed that food restriction, which markedly increases the life span and reduces age related physiological deterioration and diseases in many animals, may mediate some of its effects through a sustained pineal activity in old age. PMID- 1713530 TI - Distribution of corticotropin-releasing factor mRNA and immunoreactivity in the central auditory system of the rat. AB - Hybridization histochemical and immunohistochemical methods were used to characterize the distribution of corticotropin-releasing factor (CRF) messenger RNA (mRNA) and peptide, respectively, in the central auditory system of the rat. Cell bodies expressing CRF mRNA and/or immunoreactivity (IR) were detected at each level of the system, including sparse or equivocal localizations in the dorsal cochlear nucleus, the medial nucleus of the trapezoid body, and the lateral superior olive. More prominent groups of cells expressing CRF mRNA and CRF-IR were found in the nuclei of the lateral lemniscus, the shell of the inferior colliculus, the medial division of the medial geniculate body and in primary auditory cortices. The latter showed the greatest density of CRF expressing interneurons, distributed primarily in layers II, III and V, of any neocortical area. Results obtained using the two staining methods were in good agreement, except in the cochlear and superior olivary nuclei, where cells displaying CRF-IR were apparent in far greater abundance than those expressing CRF mRNA. CRF-IR fibers and terminals were detected in regions generally consistent with the cellular localizations described above. These results provide evidence for a surprisingly widespread expression of CRF in the auditory system of the rat. This includes generally low levels of expression in components of the primary auditory path (cochlear nucleus, trapezoid body, superior olive, lemniscal nuclei). CRF appears to be more prominently expressed in so-called non primary components of the auditory system, including aspects of the medial geniculate body that constitute an interface between the auditory system and stress-related limbic system circuitry. PMID- 1713531 TI - Peripheral territory and neuropeptides of the trigeminal ganglion neurons centrally projecting through the oculomotor nerve demonstrated by fluorescent retrograde double-labeling combined with immunocytochemistry. AB - The peripheral territories of sheep trigeminal neurons which send their central process to the brainstem through the oculomotor nerve were investigated by the use of fluorescent tracers in double-labeling experiments. For this purpose Diamidino yellow (DY) injection into the oculomotor nerve was combined with Fast blue (FB) injection either into the extraocular muscles (EOMs), or the cornea, or the superior eyelid. Double-labeled DY + FB cells were found in the ophthalmic region of the trigeminal ganglion in addition to single-labeled DY or FB cells. The DY and DY + FB-labeled trigeminal cells were analysed immunocytochemically for their content of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and cholecystokinin-8 (CCK-8)-like. All single-labeled DY cells showed SP-, CGRP- or CCK-8-like immunoreactivity. Double-labeled DY + FB neurons innervating the EOMs were immunoreactive for each of the three peptides, whereas double-labeled neurons supplying the cornea were only CGRP-like positive. The findings suggest that, in the sheep, trigeminal neurons which send their process centrally through the oculomotor nerve supply the EOMs, the cornea, and the superior eyelid and contain neuropeptides which are usually associated with pain sensation. PMID- 1713532 TI - Manganese ion elicits a binding activity of placenta vitronectin receptor to fibronectin cell-binding domain. AB - Some members of the integrin family recognize the RGD sequence which is common to cell adhesive proteins in a divalent cation-dependent manner. In the presence of Ca2+ and Mg2+, the fibronectin receptor of placenta recognizes the RGD sequence of fibronectin, but not that of vitronectin, while the vitronectin receptor of placenta recognizes the RGD sequence of vitronectin, but not that of fibronectin, although both receptors recognize the same RGD sequence. We have found by performing an enzyme-linked immunosorbent assay (ELISA) using receptor-specific monoclonal antibodies and by electrophoretic analysis that in the presence of Mn2+ a vitronectin receptor of placenta binds to an affinity column coupled with the cell-binding domain of fibronectin. By replacing divalent cations from Mn2+ to Ca2+ and Mg2+, the vitronectin receptor was completely eluted from the column. When the synthetic peptides GRGDSP and GRGESP were applied to the column as competitors, the Mn(2+)-dependent binding was inhibited by both peptides. These results suggest that Mn2+ elicits a binding activity of the placenta vitronectin receptor to the RGD site of fibronectin. The modulation of ligand specificity by Mn2+ will provide an important clue in the elucidation of the cause of individual ligand specificity of RGD-recognizing integrins. PMID- 1713533 TI - Identification of a cloned sequence activated during multi-stage carcinogenesis in mouse skin. AB - Differential screening of cDNA libraries made from chemically induced malignant mouse skin squamous cell carcinomas (SCC) identified three sequences, including one called mal2, that were upregulated in their expression at both the benign papilloma and malignant SCC stages. The mal2 plasmid cDNA clone (containing a 350 bp insert) was used to screen lambda phage cDNA libraries made from chemically induced SCCs. Two of the largest mal2-related cDNA inserts obtained from the phage libraries were sequenced. In addition a mal2-related genomic clone was obtained by hybridization probing of a mouse spleen genomic DNA library. The sequence of the genomic clone overlapped and was identical with both the mal2 plasmid and lambda cDNA clones. Identity was found between the mal2 cDNAs, the mal2 genomic sequence and the cDNA sequence for a mouse hyperproliferative keratin called K6. A synthetic oligonucleotide specific for the 3' untranslated region of the mal2 or keratin K6 gene was used in Northern analyses to demonstrate elevated steady-state levels of K6 keratin transcripts in SCCs induced by various protocols involving both chemical and ionizing radiation initiation of tumors as well as complete chemical and radiation carcinogenesis protocols. Metastatic lung lesions derived from SCCs generated by repeated doses of benzo[a]pyrene showed moderate levels of K6 keratin transcripts, whereas normal lung showed very low levels of K6 transcripts. The overexpression of the mal2 or keratin K6 gene in malignant SCCs was independent of the protocol, either chemical or radiation, that was used to induce the tumors. PMID- 1713535 TI - Endothelin induces a nonselective cation current in vascular smooth muscle cells. AB - The potent vasoconstrictor endothelin leads to smooth muscle cell depolarization and increases in intracellular Ca2+. Although effects of endothelin on calcium channels have been described, it also has been speculated that endothelim may activate additional ion channels. The purpose of the present study was to identify an alternative ion current that could play a role in depolarizing cells in response to vasoconstrictors like endothelin and vasopressin. The effects of endothelin, vasopressin, sarafotoxin S6b, and phenylephrine were assessed using whole-cell patch-clamp recordings from primary dissociated rat aortic or mesenteric arterial smooth muscle cells cultured for 24-72 hours. From the usual resting potentials of these cells of -50 to -60 mV, endothelin (1-100 nM) induced a depolarization via an increase in membrane conductance. This depolarization was phasic, oscillating repeatedly from the resting potential to a relatively depolarized level and back to the resting potential. From a holding potential of 60 mV, endothelin-1, endothelin-3, vasopressin, or sarafotoxin S6b (but not phenylephrine) induced transient inward currents that also could be phasic. In external sodium, lithium, or cesium (but not Tris) and in internal potassium or cesium, these currents reversed near 0 mV. Although nifedipine-insensitive, the inward currents were absent in zero calcium, barium, or strontium, or in the presence of cobalt or nickel. These results represent the first report of a nonselective cation current in primary vascular smooth muscle cells that is calcium dependent and that could be responsible for the depolarizations induced from the resting potential by vasoconstrictors such as endothelin. PMID- 1713534 TI - Vascular response to basic fibroblast growth factor when infused onto the normal adventitia or into the injured media of the rat carotid artery. AB - Immunohistochemical techniques localize basic fibroblast growth factor (FGF) in endothelial and smooth muscle cells of the common carotid artery. Thus, we studied the effect in rats of basic FGF infused for 14 days onto the adventitia or into the media in vivo. In untreated rats, the adventitial layer is uniform, and few vessels are observed in cross sections (mean +/- SEM is 0.351 +/- 0.16 capillaries/field at a magnification of x 480). Whereas saline infusion increases the mean number of vasa vasorum to 2.73 +/- 0.011 capillaries/field (p less than 0.01), basic FGF (1 ng/microliter/hr) increases the capillary number to 13.4 +/- 0.67 capillaries/field. The effects are local and restricted to the site of delivery; no cell proliferation is observed even 2 mm from the site of infusion. There is also no evidence of the infiltration of macrophages and monocytes. In an effort to determine the effect of basic FGF in the media, a small longitudinal (1 mm) incision was made in the adventitia, and saline or basic FGF (1 ng/microliter/hr) was infused for 14 days into the arterial wall. Under these conditions, basic FGF is a potent inducer of smooth muscle cell proliferation in the vascular wall as well as of new capillaries. In these instances, however, the capillaries formed are thick-walled. The results support the hypothesis that basic FGF may be contributing to the growth and maintenance of the vasa vasorum and of vascular smooth muscle cells. PMID- 1713536 TI - Hypercholesterolemia enhances macrophage recruitment and dysfunction of regenerated endothelium after balloon injury of the rabbit iliac artery. AB - BACKGROUND: We studied the effects on and possible interaction of balloon denudation and hypercholesterolemia on large arteries in the rabbit with special regard to structure and vascular reactivity. METHODS AND RESULTS: New Zealand White rabbits fed a 1% cholesterol diet or a standard diet for 14 weeks underwent balloon denudation of the left iliac artery 4 weeks before death. Both the balloon-injured and the control iliac arteries were harvested for in vitro studies of vascular reactivity, for immunohistochemical staining with monoclonal antibodies directed at smooth muscle cells and macrophages, and for scanning electron microscopy. Balloon injury caused intimal smooth muscle proliferation with little macrophage infiltration and was followed by recovery of endothelium dependent vasodilator function within 4 weeks. Hypercholesterolemia caused macrophage-rich lesions confined to the intima with moderate impairment of endothelial vasodilator function. Balloon injury in the setting of hypercholesterolemia caused intimal smooth muscle cell proliferation and intense macrophage infiltration throughout the arterial wall and severe impairment of endothelial vasodilator function. Scanning electron microscopy confirmed regrowth of the endothelium in all balloon-injured vessels. In the balloon-injured arteries of hypercholesterolemic animals, the regenerated endothelium exhibited areas of atypical morphology not seen after balloon injury or hypercholesterolemia alone. CONCLUSIONS: The present study shows that balloon injury, hypercholesterolemia, and their combination cause distinct lesions and functional disturbances. An arterial balloon injury in the setting of hypercholesterolemia produces a diffuse inflammatory response that is accompanied by a sustained impairment of endothelial function and a marked proliferative response. PMID- 1713537 TI - Optimal right ventricular filling pressures and the role of pericardial constraint in right ventricular infarction in dogs. AB - BACKGROUND: Previous studies have reported an important role for right ventricular function in the pathophysiology of the low cardiac output state that can accompany right ventricular infarction. Some studies have suggested that right ventricular distensibility impairs right ventricular filling and stroke output; others have demonstrated that the pericardium can mediate depressed left ventricular filling and stroke output. METHODS AND RESULTS: To determine the role of pericardial constraint and optimal volume loading in an experimental model of right ventricular wall infarction, six mongrel dogs were studied before and after right ventricular wall infarction and after volume loading. The pericardium was then opened in two phases. In the first phase, the pericardium was opened partially to allow the atria to distend freely, and in the second phase, the pericardium was opened completely. The animals were preinstrumented with two sets of piezoelectric crystals attached to the right ventricular free wall, one in the infarct and the other in the noninfarct territory. Left ventricular size was estimated by left ventricular crystals on the anterior wall of the left ventricle. Right ventricular and left ventricular Millar catheters were used to assess intracavitary pressure, and a flat balloon was used to assess intrapericardial pressure. Right ventricular infarction reduced cardiac output by 23% and stroke volume by 30%. End-diastolic segment length and transmural pressure of the left ventricle decreased. Volume loading restored cardiac output to baseline values and was mediated by a significant increase in end-diastolic length in the noninfarct territory. This was achieved by increasing right ventricular end-diastolic pressure from 9 +/- 2 to 16 +/- 3 mm Hg (p less than 0.01). Partial opening of the pericardium mediated significant increases in both end-diastolic segment lengths of the left ventricle and the noninfarct territory. Left ventricular end-diastolic pressure decreased slightly by 3 mm Hg (p = NS). Complete opening of the pericardium increased cardiac output and stroke volume and mediated a significant decrease in right and left ventricular end-diastolic pressures. Left ventricular transmural pressure and end-diastolic segment lengths of the left ventricle and the noninfarct territory increased. Left ventricular diastolic pressure-segment length relations were shifted upward by right ventricular infarction. A partial opening of the pericardium shifted this relation downward in all animals, and complete opening of the pericardium shifted the relation rightward and further downward. CONCLUSIONS: Cardiac output is restored to baseline values by volume loading sufficient to increase the right ventricular diastolic pressure to 16 +/- 3 mm Hg. Evidence of pericardial constraint was observed and appears to be mediated by an atrioventricular interaction in addition to the direct ventricular interaction. PMID- 1713538 TI - Importance of accurate diagnosis in counseling for neural tube defects diagnosed prenatally. AB - In cases of fetal neural tube defects (NTD), termination of pregnancy without ascertainment of specific etiology may lead to provision of incorrect recurrence risks and erroneous diagnosis in future pregnancies. Four patients are presented who illustrate the etiologic diversity of neural tube defects. The patients were referred for prenatal diagnosis because of elevated maternal serum alphafetoprotein (AFP). All four chose pregnancy termination. Diagnostic methods included fetal ultrasound, amniocentesis for fetal karyotyping and amniotic fluid AFP/acetylcholinesterase (AChE) and/or fetal karyotyping after delivery, and dysmorphology evaluation of the fetus after intact delivery. These cases highlight the benefits of fetal karyotype analysis and of an intact delivery and thorough clinical examination of the fetus when patients choose to terminate pregnancies with fetal anomalies. PMID- 1713539 TI - Criteria for use of hetastarch. PMID- 1713540 TI - Fetal acidosis and hyperlacticaemia diagnosed by cordocentesis in pregnancies complicated by maternal diabetes mellitus. AB - Fetal blood samples were obtained by cordocentesis (ultrasound guided needle aspiration) from 28 pregnant Type 1 diabetic women between 20 and 40 weeks' gestation. Analysis of the deviations from normal values of blood pH and plasma lactate showed significant acidosis (p less than 0.001) and hyperlacticaemia (p less than 0.01) in the third trimester, but not in the second trimester. Blood PO2 and PCO2 levels did not differ significantly from normal values. The pH showed significant correlations with PO2 (r = 0.54; p less than 0.01) PCO2 (r = 0.70; p less than 0.001), lactate (r = -0.46; p less than 0.05), fetal glycosylated haemoglobin (r = -0.53; p less than 0.01), and maternal glycosylated haemoglobin (r = -0.57; p less than 0.01). Plasma lactate showed significant correlations with PO2 (r = -0.54; p less than 0.01), PCO2 (r = 0.50; p less than 0.05), and pH (r = -0.46; p less than 0.05). Neither pH nor lactate showed significant correlations with birthweight. These observations suggest that some fetuses of diabetic women are significantly acidotic and hyperlacticaemic in the third trimester. This may provide a possible explanation for the phenomenon of late intrauterine fetal death in pregnancies complicated by maternal diabetes mellitus. PMID- 1713541 TI - [Clinical significance of serum ferritin and acute phase reactant proteins levels in patients with cervical cancer]. AB - Serum ferritin (SF) levels from 162 patients with cervical cancer and their serum alpha 1-acid glycoprotein (alpha 1-AGP). alpha 1-antitrypsin (alpha 1-AT. Transferrin (Tf) in most patients were determined. The result showed that concentration of SF, alpha 1-AGP, alpha 1-AT were significantly higher, while TF significantly lower in cervical carcinoma patients during active period than from patients with benign tumors and normal persons. The levels of SF. alpha 1-AGP. alpha 1-AT and Tf were significantly increased during the remission period. The positive rate of SF, alpha 1-AGP and Tf in cervical cancer patients during active period was significantly higher than that of alpha 1-AT. Serial determinations of SF, alpha 1-AGP and. Tf may be helpful in the monitoring of disease development and early detection of recurrence and metastases. PMID- 1713542 TI - [Disturbance of immunoregulation in experimental allergic neuritis in rabbits: II. The relationship between the changes in CSF and serum serotonin concentrations and immune responses]. AB - The serotonin (5-HT) concentrations in the CSF and serum of the rabbits with experimental allergic neuritis (EAN) were determined. The relationships between the 5-HT concentrations and the rate of the specific lymphocyte transformation as well as that of the formation of the antimyelin antibody were investigated. The results indicated that after the rabbits were sensitized with myelin basic protein (MBP), the 5-HT levels in the CSF and serum of the rabbits with EAN increased. It was, therefore, considered that 5-HT might play a role in the inhibition of the lymphocyte transformation and the antibody production. PMID- 1713543 TI - Acquisition of immunity in cattle against the blue tick, Boophilus decoloratus. AB - It is well known that ixodid ticks have the ability to induce immunity in their host. We demonstrate, for the first time, that the tick Boophilus decoloratus induced immunity in its bovine host, since the mean weight of engorged females fed on naive animals dropped from 201.5 mg, to 173.7 mg and 155.3 mg, for females fed on calves previously exposed once and twice; respectively, to B. decoloratus infestations. Ticks which had been transferred from one individual host to another one were able to complete their feeding period on a sensitive host. Such ticks were significantly heavier (mean 245.2 mg) than those fed on a naive (mean 201.5 mg) host for the entire normal feeding period. A negative correlation between the mean weight of the engorged female ticks and the level of serum gamma globulins in the host was also demonstrated. PMID- 1713544 TI - Acute neonatal morbidity and long-term central nervous system sequelae of perinatal asphyxia in term infants. AB - Twenty-eight term neonates with severe perinatal asphyxia were referred to a tertiary neonatal intensive care unit (NICU). The morbidity of asphyxia included involvement of the pulmonary (n = 24 infants), central nervous system (n = 22), renal (n = 15), cardiac (n = 14), metabolic (n = 13) and hematologic (n = 10) systems. The majority of neonates had more than three organ systems involved. Twenty-four neonates survived the neonatal course and at NICU discharge all system effects other than the central nervous system had resolved. At 5 years (60 months), 14 children had a normal neurologic examination, 9 had spastic quadriplegia and one had hemiplegia. Nine children had a McCarthy General Cognitive Index (GCI) greater than or equal to 84, 3 had a GCI between 68 and 83 and 12 scored less than 67. Neonatal seizures, renal problems, microcephaly at 3 months, and post-neonatal seizures were associated with an abnormal neurologic outcome or a GCI less than 67. A neurologic examination during the first year of life may reveal whether children with birth asphyxia will be relatively normal at age 5 years or whether they will show considerable delay. PMID- 1713546 TI - American Association of Electrodiagnostic Medicine, 37th annual meeting. Chicago, IL, 7-8 September 1990. Abstracts. PMID- 1713545 TI - The neocortico to mesio-basal limbic propagation of focal epileptic activity during the spike-wave complex. AB - In order to localize epileptogenic electrophysiological sources, a multichannel MEG system was used in 3 patients with partial epilepsy during presurgical evaluation. MEG and EEG (including scalp, sphenoidal and intracranial foramen ovale electrodes) were recorded simultaneously during a period of intensive video EEG monitoring in order to observe single spontaneous spikes. In addition to MRI, SPECT and PET investigations were performed. Electrical activity subsequent to the activity of the epileptic focus could be localized by the MEG after noise reduction using a temporal correlation technique. Simultaneous registration of the magnetic field and the electrical field showed that the source of the primary focal epileptic activity (first period during the total spike wave complex where a dipolar magnetic field pattern is found) is localized in neocortical lateral regions, whereas another focal epileptic activity in a later phase of propagation occurs in temporal mesial regions. In 1 patient (case 1) the primary focal epileptic activity was localized in the surrounding neocortical tissue of an angioma and the middle and inferior temporal gyrus. The second phase of propagation is localized in temporo-basal-mesial regions, including para- and hippocampal structures. The latest center of activity occurred in posterior parts of the gyrus cinguli. In 2 other patients, the primary focal epileptogenic activity was localized at the insula and also spread into temporal basal mesial regions. A multi-modal approach to research of focal epilepsy, combining metabolic, electrical potential, magnetoencephalographic and morphological data, recorded by non-invasive techniques, offers new perspectives for the detection of involved brain regions. The 3-D and time-resolved localization of focal epileptic activity, correlated with the individual anatomy of the human brain, may improve the determination of neuronal populations involved in the individual epileptogenic process, especially in the interaction between temporal or extratemporal neocortex and limbic system. PMID- 1713547 TI - State-dependent spike detection: concepts and preliminary results. AB - In traditional methods of spike detection, spikes are defined in absolute terms (duration, amplitude) or relative to a few seconds of background. These methods result in many false positive detections during long-term epilepsy monitoring because of numerous artefacts and non-epileptic transients. To reduce significantly false detection, we propose to render spike detection sensitive to the state of the EEG. We thus defined 5 states (active wakefulness, quiet wakefulness, desynchronized EEG, phasic EEG and slow EEG) and designed a method for automatic state classification. We then designed procedures for identification of non-epileptic transients (eye blinks, EMG, alpha, spindles, vertex sharp waves). These procedures are to be applied only in the state in which they are likely to occur (e.g., eye blinks in wakefulness). We present preliminary results from 14 recordings each lasting 100 min, which indicate a state classification reliability of 85-90%, reduction in false detection of 65 90% if state classification were perfect; true spikes lost as a result of these procedures were under 5%. These results are encouraging and validate the concept of a spike detection system which analyses a wide temporal and spatial context before deciding the significance of a wave form. PMID- 1713548 TI - Reliability of topographic quantitative EEG amplitude in healthy late-middle-aged and elderly subjects. AB - Reliabilities of quantitative measures of absolute and relative EEG amplitudes were assessed in healthy older adults under the eyes closed (n = 46) and eyes opened (n = 45) conditions. For the theta, alpha, beta 1, and beta 2 bands, reliabilities of 28 scalp derivations were stable over the 4.5 month test interval. Reliabilities of delta were lower. When appropriate transformations were applied, the reliabilities of absolute EEG amplitude measures tended to exceed those of relative measures. There were not, however, striking differences in reliabilities under the eyes closed, as compared to eyes opened condition. We concluded that when coupled with the criterion of interpretability, the generally higher reliabilities of absolute, as opposed to relative, amplitude measures render them preferable in clinical research. PMID- 1713549 TI - Spatiotemporal modeling of cerebral evoked magnetic fields to median nerve stimulation. AB - We measured somatosensory evoked magnetic fields during median nerve stimulation in 6 normal subjects. We applied multiple dipole models to study the spatiotemporal structure of early somatosensory evoked magnetic fields (SEFs), as well as the number, 3-dimensional location and time activity of their underlying neuronal sources. Two dipole sources were necessary to model the first 40 msec of SEFs explaining 85% of the data variance. Source 1 was located deeper than source 2, showed primarily a tangential orientation, and accounted for a larger part of the variance; source 2 showed no consistent orientation across subjects. Both sources showed biphasic time activities corresponding to the previously described N20-P30 and P25-N35 components. Spatiotemporal modeling could identify sources which could not be modeled consistently above noise by single moving dipoles (P25 component), revealed small latency differences of the two sources in some subjects suggesting parallel activation of these sources, and allowed separation of sources overlapping considerably both in space and time. We conclude that spatiotemporal modeling of SEFs may be useful to study functional anatomy of human sensorimotor cortex non-invasively. PMID- 1713550 TI - Dipole models of eye movements and blinks. AB - Average EOGs were recorded from 4 subjects for vertical and horizontal eye movements of 15 degrees away from and back to a central fixation point, and for eyeblinks while looking at the fixation point. Using spatio-temporal dipole modelling, several alternative dipole models of the electrical activity of the eyes were compared. A reasonable fit was only obtained if the equivalent dipoles were allowed to take up different locations and orientations depending on the type of eye activity. It appears that (a) the equivalent ocular dipole is located away from the axis of rotation of the eyeball, (b) eyelid movements contribute to a change in location of the dipole in vertical eye movements and blinks, and (c) some of the apparent dipole movement is due to inadequacy of the 3-shell spherical head model near to the eyes. Consequences of the results for eye artifact correction are discussed. PMID- 1713551 TI - Human depth ERP in a visual threshold recognition task. AB - Event-related potentials (ERPs) were recorded from the globus pallidus, the N. ventro-lateralis thalami and adjacent areas in parkinsonian patients bearing gold electrodes for diagnosis and therapy. The patients participated in a recognition task in which visual stimuli (digits) were presented at threshold durations. The ERPs from each single recording site, as separate histograms and then in combination as composite histograms across all recording sites ('profiles of reactions' and 'profiles of reaction differences'), were computed for 3 response types (correct, non-recognition, and incorrect). Four groups of depth-ERP components (N100, P200, N300, P300) were observed. The N100 was not found to be affected by the quality of recognition, manifest in type of response, while the later components were, but each in its own way. A comparison of these data with those of multiunit activity recorded from these same sites (see Bechtereva et al. 1990) shows that the pattern of the depth N100 and P200 components with reference to the 3 types of response have no observable counterparts in the impulse activity manifestations of subcortical neuronal populations, while the depth N300 and P300 are indeed associated with the neuronal impulse activity. This in turn supports the hypothesis concerning multiple (including subcortical) generators of the P300. PMID- 1713552 TI - Automated staging of sleep in cats using neural networks. AB - Manual staging of sleep based on visual EEG criteria is a laborious and time consuming task. In an effort to automate sleep staging, we have developed a neural network that 'learns' to stage sleep on the basis of wave band count data alone, in the cat. Wave band count data are collected on a microcomputer, using period-amplitude analysis. Delta waves, spindle bursts, ponto-geniculo-occipital (PGO) waves, electro-oculogram (EOG), basal electromyogram (EMG) amplitude, and movement artifact amplitude are collected, and used to 'train' the network to score sleep. These wave count data serve as the input patterns to the net, and the corresponding manually scored sleep stages serve as a 'teacher.' We demonstrate that, when used to score the states of wake, slow wave sleep (SWS), desynchronized sleep (D), and the transition period from SWS to D (SP), these neural networks agree with manual scoring an average of 93.3% for all epochs scored. Neural network programs can learn both rules and exceptions, and since the nets teach themselves these rules automatically, a minimum of human effort is required. Because programming requirements are small for neural nets, this approach is readily adaptable to microcomputer-based systems and is widely applicable to both animal and human EEG analyses. The utility of this approach for the detection and classification of a variety of clinical neurophysiological disorders is discussed. PMID- 1713553 TI - Lesions of nucleus basalis alter ChAT activity and EEG in rat frontal neocortex. AB - The EEG was recorded from frontal, parietal and visual cortices of sham-operated control rats and rats having ibotenic acid lesions of the nucleus basalis. Recordings were made during a period of rest and during stimulus-evoked desynchronization. Spectral power was determined using a Fast Fourier Transform routine; 3 artifact-free 4 sec epochs of resting activity and two 4 sec epochs of activated EEG were analyzed. Choline acetyltransferase activity (ChAT) was measured in each cortical area and was reduced in lesioned animals an average of 25% in frontal cortex, 19% in the parietal region and 10% in visual cortex. The percent of low frequency activity (1-12 Hz) in the frontal EEG was significantly greater in lesioned animals than in the control group during quiet rest; a significant correlation was found between ChAT activity and power in this band. Desynchronized activity was largely unaffected except for a reduction in 25-31 Hz activity in the frontal cortex of lesioned animals. EEG activity in both the parietal and visual areas was unchanged from control values. PMID- 1713554 TI - 128-channel cable-telemetry EEG recording system for long-term invasive monitoring. AB - A 128-channel cable-telemetry EEG monitoring system has been developed for use on patients undergoing intensive neurodiagnostic monitoring with invasive intracranial electrodes. A unique head mounted preamplifier/multiplexor design allows continuous recording from any combination of intracerebral, subdural, epidural or surface electrodes. A computer is used to delay or buffer all 128 channels by 2 min or more. This computer is connected to a fileserver via a local area network and is used exclusively for data acquisition. Seizure files created by the activation of the seizure/event push button or detected by a second computer containing on-line seizure/spike detection algorithms are written directly on to the fileserver by the acquisition computer. The 128 channels of EEG recorded on the fileserver can then be remotely reviewed on a high resolution graphics terminal, printed out on a laser jet printer, archived or played out on paper, 16 channels at a time on any EEG machine. A time of day signal is also generated by the computer to permit time correlation with a continuous audio/video VCR unit which permits comparison of the EEG and clinical events. This system allows continuous EEG monitoring of extensive multichannel arrays not previously possible with conventional 16-, 32- or even 64-channel systems. PMID- 1713555 TI - High resolution EEG output using a laser printer. AB - With increasing computerization, digitization and complexity of 64- to 128 channel long-term monitoring (LTM), new problems have arisen with data presentation. We describe customized software that allows for high resolution EEG output on a standard laser printer. This system can display from 1 to 128 channels of EEG on standard or legal size paper with freedom to alter both time and amplitude scales as desired. This technique is inexpensive and provides a high resolution rectilinear paper record of selected EEG events that is convenient for review and storage. PMID- 1713556 TI - A new endoscopic method for treatment of malignant oesophagobronchial fistulas. AB - A short malignant oesophagobronchial fistula which could not be sealed using adhesive agents was successfully treated using a new endoscopic technique. The procedure provided good palliation and the results withstood the test of time in the patient. The method, which is described in detail, provides a useful modification to existing methods. PMID- 1713557 TI - Evaluation of pentisomide on stable ventricular premature beats. Comparison with placebo. AB - Pentisomide, a new class I anti-arrhythmic drug, was compared to placebo in 50 hospitalized patients with frequent (greater than 30 h-1) and stable ventricular premature beats (VPB) (variation less than 50% between two preliminary and one placebo 24-h Holter recordings). All patients underwent a single-dose acute oral testing followed by a short-term testing with 300 mg t.i.d. for 4 days and then by a 4-day placebo period. For the studied population, a 56.4% reduction of simple VPB and a 98.8% decrease of couplets and runs were the minimum required to define the drug efficacy and to exclude spontaneous variability, using the linear regression analysis. Pentisomide was found effective in 27 (54%) of the 50 patients after the acute test and in 23 (46%) after the short-term test. The drug induced a mild increase of PR and QRS intervals, while QTc, heart rate, blood pressure and ejection fraction showed no significant variations. Subjective tolerability was excellent. PMID- 1713558 TI - Familial bidirectional ventricular tachycardia. AB - The occurrence of incessant ventricular arrhythmia with bouts of polymorphous and bidirectional ventricular tachycardia is described in two totally asymptomatic sisters, with structurally normal hearts and no QT prolongation. Familial survey revealed the occurrence of increased ventricular ectopic activity in the father, brother and another sister. PMID- 1713559 TI - Non-Q-wave myocardial infarction associated with bleomycin and etoposide chemotherapy. AB - During chemotherapy with bleomycin and etoposide a 28-year-old male, suffering from germ-cell cancer, developed acute myocardial infarction. Under treatment with heparin and aspirin the patient revealed no Q-waves in ECG and recovery was without complications. Four weeks after onset of infarction, thallium-201 scintigraphy showed only a small irreversible, posteroseptal perfusion defect; coronary angiography was not performed. The chemotherapy regimen was continued and modified to etoposide as well as cisplatin and ifosfamide without recurrence of cardiac symptoms or ECG changes. PMID- 1713560 TI - [The toxic and immunomodulating properties of the somatic O-antigen polysaccharide of typhoid bacteria]. AB - In experiments on random bred mice and mice of various strains it was shown that when administered parenterally typhoid bacteria O-somatic antigen polysaccharide possesses the immunomodulatory properties. It stimulates the non-specific resistance of the organism to bacterial infection, produces the polyclonal activation of beta-lymphocytes, possesses the adjuvant properties, activates cells of the mononuclear phagocytic system. At administration in therapeutic doses the drug is not toxic, possesses no carcinogenic, mutagenic and allergenic properties. PMID- 1713561 TI - Retinal signs in sickle cell anaemia. PMID- 1713562 TI - Ion channels of glucose-responsive and -unresponsive beta-cells. AB - To assess whether different electrophysiological characteristics could account for the heterogeneous secretion of individual beta-cells in vitro, we used patch clamp configurations to study currents in plaque-forming (insulin-secreting) and non-plaque-forming rat pancreatic beta-cells that were distinguished in a reverse hemolytic plaque assay (RHPA) after a 30-min stimulation by 16.7 mM glucose. RHPA showed that the population of single beta-cells under study was stimulated (P less than 0.01-0.001) to secrete insulin by 16.7 mM glucose, 100 microM tolbutamide, 20 microM glyburide, or 30 mM KCl but, under these conditions, also comprised beta-cells that did not secrete detectable amounts of insulin. Under current clamp conditions, secreting and nonsecreting beta-cells showed analogous resting membrane potentials (approximately 60 mV) and were similarly depolarized by 30 mm KCl and 100 microM tolbutamide. Under voltage-clamp conditions, total membrane conductance (approximately 6 nS) was also similar in the glucose responsive and -unresponsive beta-cells, which, when monitored in the whole-cell configuration after RHPA, showed the following currents: a voltage-dependent Na+ current, a voltage-activated Ba2+ current, a voltage-dependent K+ delayed rectifier current, a voltage-dependent Ca(2+)-activated K+ current, and a voltage independent and tolbutamide-sensitive K+ current. In the cell-attached configuration and the presence of 2.8 mM glucose, secreting and nonsecreting beta cells displayed a similar single-channel activity that was abolished when glucose concentration was raised to 16.7 mM. We conclude that beta-cells studied after RHPA have an electrically normal membrane whether they release insulin in response to 16.7 mM glucose or not. PMID- 1713563 TI - Nutrition and somatomedin. XXVII. Total and free IGF-I and IGF binding proteins in rats with streptozocin-induced diabetes. AB - Impaired growth in diabetic humans occurs despite increased growth hormone and normal insulinlike growth factor I (IGF-I). Because IGF-I circulates complexed to binding proteins (BPs), we asked whether diabetes-related changes in IGF BPs could be associated with alterations in free unbound IGF-I--presumably the active form. Rats were given streptozocin (STZ) in increasing doses to produce graded severity of diabetes. IGF BP-1 and BP-3 were measured by ligand blotting, total IGF-I was determined by radioimmunoassay after separation from BPs by isocratic high-performance liquid chromatography (HPLC) at pH 3.9, and free IGF-I was estimated operationally as immunoreactivity with molecular weight equal to native IGF-I after HPLC at pH 7. Animals given 36 mg/kg STZ exhibited a glucose level of 9.74 mM and impaired weight gain, with little alteration in IGF BPs or total or free IGF-I. In contrast, animals given 72 mg/kg STZ (glucose level 24.64 mM and weight loss) had insignificant changes in total IGF-I and BP-3 but a 300% increase in BP-1 and a 50% fall in free IGF-I (both P less than 0.005). With 144 and 288 mg/kg STZ, animals had further metabolic decompensation and weight loss, with progressive fall in BP-3 and rise in BP-1; total and free IGF-I fell to 10 20% of control (both P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713564 TI - [Results of hormone therapy with aminoglutethimide (Rodazol) in postmenopausal metastatic breast cancer]. AB - 44 postmenopausal women with progressive soft tissue, osseous and/or pleuropulmonal metastases of breast cancer were treated with aminoglutethimide (Rodazol) and hydrocortisone. 40 of the 41 patients, evaluated for treatment results, were pretreated hormonally and 17 were additionally pretreated chemotherapeutically. Objective remissions were achieved in 8 of the total of 41 patients (20%) and in 7 of the women (35%), who had previously responded to tamoxifen. Pain reduction was achieved in 4 of 17 cases (23%) with skeletal pain as the main symptom. The response rate was lower than expected. Primary hormonal therapy with Rodazol cannot be recommended in the osseous type of metastatic spread. PMID- 1713565 TI - Hepatocellular expression of lymphocyte function-associated antigen 3 in chronic hepatitis. AB - T lymphocyte-mediated cytolytic immune reactions are considered a major cause of hepatocyte injury in chronic viral and autoimmune hepatitis. To further investigate local immune responses, we studied the expression of lymphocyte antigens and cell-cell interaction molecules known to be involved in effector target cell interactions by light and electron microscopy in liver biopsy specimens from patients with chronic viral and autoimmune hepatitis. CD8+ lymphocytes were found to be the predominant population of cells in the inflammatory infiltrate in chronic hepatitis B and non-A, non-B hepatitis. In contrast, CD4+ cells constituted a comparably higher proportion of cells and were more numerous than CD8+ cells in chronic autoimmune hepatitis. In both viral and autoimmune hepatitis, a substantial portion of lymphocytes expressed activation antigens such as T11/3 (CD2R) and IL-2-R (CD25). Lymphocyte function-associated antigen-3 (CD58), which mediates lymphocyte adhesion and activation and is the natural ligand of the CD2/T11 lymphocyte surface receptor, could be demonstrated on endothelial cells and hepatocytes. Hepatocellular lymphocyte function associated antigen-3 expression in chronic hepatitis showed membranous and cytoplasmic staining of hepatocytes and had a positive correlation with the degree of inflammatory activity. These results suggest that effector-target interactions between hepatocytes and lymphocytes mediated by the lymphocyte function-associated antigen-3/CD2 pathway play a role in chronic inflammatory liver disease. Possible functional consequences of this interaction include enhancement of antigen-specific immune reactions and antigen-independent mechanisms of T cell activation, which may contribute considerably to the degree of inflammatory activity and tissue damage in chronic hepatitis. PMID- 1713566 TI - Immunohistochemical detection of transforming growth factor-beta 1 in fibrotic liver diseases. AB - Transforming growth factor-beta 1 was localized by means of immunohistochemical reaction in liver biopsy specimens taken from patients having different chronic liver diseases with extending fibrosis. Two polyclonal antibodies that were produced in rabbits were directed against the amino terminal of transforming growth factor-beta 1. Staining by anti-CC(1-30) was primarily extracellular and located in the portal and periportal fibrotic areas of all seven cases with chronic active hepatitis. No staining was noted in the four chronic persistent cases studied. A strong reaction was seen with the antibody in nine of the ten cirrhotic samples, whereas it was negative in one inactive cirrhosis case and in all five cases with normal liver histological findings. No positive staining could be detected by the anti-LC(1-30) in any of the liver tissues. Detection of transforming growth factor-beta 1 in active liver diseases at the site of fibrosis suggests that transforming growth factor-beta 1 might have a role in the process and progression of fibrosis during the development of the disease. PMID- 1713567 TI - Inapparent transmission of hepatitis C: footprints in the sand. PMID- 1713568 TI - Interferon signalling through arachidonic acid-dependent pathways: a clue to adjuvant therapy for chronic viral hepatitis? PMID- 1713569 TI - Histamine release from cord blood basophils. AB - The histamine release (HR) after challenge with anti-IgE, concanavalin A, N formyl-met-leu-phe and the calcium ionophore A23187 from 97 cord blood samples was determined by a microfiber-based assay. Maximum HR with anti-IgE showed great inter-individual variation (median: 20.5; range: 1-104 ng/ml blood), but was not significantly different from the results obtained in identically treated blood samples from 50 adults (median: 23; range: 1-93 ng/ml blood). Both the maximum HR and the sensitivity to anti-IgE were dependent on total plasma IgE content. Blood samples with plasma IgE greater than or equal to 0.5 IU/ml (n = 15) had significantly higher maximum HR than those with plasma IgE less than 0.5 IU/ml (n = 82; median: 32 vs. 18 ng/ml blood; p less than 0.01). Passive sensitization with IgE-rich atopic plasma increased the maximum HR with anti-IgE only in samples with a plasma IgE content of less than 0.5 IU/ml, although sensitivity to anti-IgE was universally increased. Preincubation with pharmacologic agents modulating the IgE-mediated HR produced effects generally similar to previous findings in adult blood. However, the effects of inhibiting the cyclooxygenase pathway in cord blood differed from our observations in adult blood, and may represent a maturational phenomenon. The family history of allergy was obtained by a questionnaire, and clinical observations were gathered from patient records. None of these parameters were found to influence HR with any secretagogue. However, HR stimulated by the calcium ionophore A 23187 was found to be highly dependent on the storage time of the EDTA-anticoagulated blood samples, which should be carefully controlled. PMID- 1713570 TI - T-cell responsiveness towards various synthetic peptides of the P28 antigen in rat and mouse models during Schistosoma mansoni infection. AB - It has recently been demonstrated that the Schistosoma mansoni P28 antigen can induce a strong protective immunity after direct immunization in various experimental models. T lymphocytes from Fischer rats immunized with the recombinant P28 antigen were cultured in vitro in the presence of seven synthetic peptides derived from the amino acid sequence of the P28. The most significant and reproducible proliferation was obtained with the 24-43 and 115-131 synthetic peptides. In order to analyze whether these located determinants were also exposed to the host's immune system during the natural S. mansoni infection or after immunization with crude antigenic extracts of various development stages of the parasite, the T-cell responsiveness of infected or immunized Fischer rats and BALB/c mice was tested towards these synthetic peptides. The results showed that, in both permissive (mouse) and non-permissive (rat) hosts, 24-43 and 115-131 synthetic peptides are recognized during the course of infection and that there is a dynamic variation of this recognition. These peptides are also recognized by T cells educated against crude antigenic extracts of different developmental stages of the parasite which contained the native form of the P28 molecule. Taken together, the results indicated that these synthetic peptides derived from the recombinant P28 antigen can activate T lymphocytes educated against the native P28 molecule during the development and maturation of the parasite in their hosts. Therefore, they might be useful for the construction of synthetic vaccines against schistosomiasis. PMID- 1713571 TI - Differential effect of hypertonicity on FMLP-, anti-IgE- and Ca2+ ionophore A23187-induced histamine release from human basophil leukocytes. AB - Effects of different extracellular Na+ and K+ concentrations (respectively, 135, 155, 220, 260 mM NaCl, and 2.7, 20, 50, 100 mM KCl) on IgE-dependent and IgE independent histamine release from human basophils were examined. High extracellular Na+ and K+ concentrations were shown to reduce N-formyl-methionyl leucyl-phenyl-alanine- (FMLP), but not anti-IgE- or Ca2+ ionophore A23187-induced histamine release. A high extracellular Ca2+ (7.2 mM CaCl2) concentration increased basophil response to anti-IgE and FMLP. The enhancement of FMLP- but not of anti-IgE-induced histamine release was antagonized by high extracellular Na+ and K+ concentrations. When leukocytes were suspended in isotonic choline chloride solutions (choline is a nonpermeant monovalent cation), an enhancement of anti-IgE- and FMLP-induced histamine release was observed. This suggests that monovalent cations, namely Na+ ions, at physiological concentrations, downregulate histamine release from human basophils. At high choline chloride concentrations, FMLP-, but not anti-IgE-induced histamine release was inhibited. Thus, the reduction of FMLP-evoked histamine secretion from human basophils seems to be due to hypertonicity and not to the type of monovalent cation, either permeant or nonpermeant, contained in extracellular milieu. The different effects of a hypertonic solution on anti-IgE and FMLP-induced histamine release are probably related to the different cell activation pathways triggered by the two stimuli. PMID- 1713572 TI - Selective inhibition by magnosalin and magnoshinin, compounds from 'shin-i' (Flos magnoliae), of adjuvant-induced angiogenesis and granuloma formation in the mouse pouch. AB - Inhibitory effects of magnosalin and magnoshinin, compounds from the crude drug 'Shin-i' (Flos magnoliae), on angiogenesis and pouch granuloma formation induced by an adjuvant containing croton oil were investigated. Magnosalin inhibited angiogenesis 2.4-fold (intra-pouch) and 9.7-fold (intraperitoneal) more strongly than granuloma formation. The inhibition of angiogenesis by magnosalin was 5-fold (intra-pouch) and 21-fold (intraperitoneal) weaker than that by hydrocortisone. In contrast, intraperitoneal magnoshinin inhibited granuloma formation 2.5-fold more strongly than angiogenesis. The regression coefficients of anti-angiogenesis vs. the inhibition of granuloma formation were 1.79 for magnosalin, 1.11 for hydrocortisone, and 0.61 for magnoshinin. These results show that the anti chronic inflammatory effect of 'Shin-i' was caused by selective inhibition of angiogenesis by magnosalin and of granuloma formation by magnoshinin. PMID- 1713573 TI - Tumor IGF-II content in a patient with a colon adenocarcinoma correlates with abnormal expression of the gene. AB - We have recently reported abnormal insulin-like growth factor-II (IGF-II) mRNA levels in a number of human colorectal adenocarcinomas. Using an IGF-II radioimmunoassay, we have now detected high levels of both 10-kDa and 7.5-kDa IGF II species (2,370 ng/g) in a right colon tumor showing a 800-fold IGF-II gene over-expression in comparison to the normal adjacent tissue. The higher-molecular mass form represents 74% of the total immunoreactive IGF-II detected in the tumor. This form appears to be less reactive in the radioreceptor assay than in the radioimmunoassay. The insulin-like growth factor-I (IGF-I) concentration in the tumor is low. The patient's pre-operative serum IGF-II level is not increased and the proportion of the 10-kDa species is normal. In addition, the IGF-II/IGF-I ratio is 3 in the serum and 308 in the tumor. Our results show that the very high IGF-II level produced by the tumor does not influence the seric concentration of the growth factor. PMID- 1713574 TI - Relationships between epithelial basement membrane staining patterns in primary colorectal carcinomas and the extent of tumour spread. AB - In colorectal cancer an association has been found between lack of epithelial basement membrane (EMB) immunostaining in the tumour centre and more extensive malignant spread. Interestingly, ultrastructural investigations suggest that EBM loss at the tumour periphery may be part of an invasive mechanism. To further assess the significance of EBM deficiencies in different tumour areas, we carried out a detailed study of the basement membrane laminin immunostaining patterns in 130 cases of colorectal carcinoma. We find that discontinuous EBM staining in the tumour centre is associated with poor tumour differentiation (p less than 0.005), presence of lymph-node metastases (p less than 0.02), and more advanced Dukes stage (p less than 0.02). The latter association is strengthened by excluding cases in which numerous polymorphonuclear leukocytes (PMNs) are present adjacent to EBM breaks, suggesting that these inflammatory cells are a confounding factor. Discontinuous EBM staining is more frequently observed in tumour deep to muscularis propria than in submucosal tumour (p less than 0.02), indicating intra tumoral variation. At the tumour periphery, extensive EBM discontinuity shows no association with lymph-node involvement, but is linked with deeper local invasion (p less than 0.05). While EBM staining patterns around central and peripheral tumour glands are related (p less than 0.001), staining around peripheral glands is almost invariably more discontinuous. However, EBM lack at the tumour periphery is not as absolute as previously suggested, since in 18% of tumours fewer than 25% of peripheral tumour glands show EBM breaks. This appears consistent with the hypothesis that invasive changes at the tumour periphery are temporary and reversible. PMID- 1713575 TI - Modulation of cis-diamminedichloroplatinum(II) accumulation and sensitivity by forskolin and 3-isobutyl-1-methylxanthine in sensitive and resistant human ovarian carcinoma cells. AB - We have determined the effect of forskolin, an adenyl cyclase agonist, and 3 isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, on the accumulation and cytotoxicity of cisplatin (DDP) in 2008 human ovarian carcinoma cells. In DDP-sensitive 2008 cells, forskolin and IBMX caused 2.1-fold and 2.3 fold increases, respectively, in the short-term accumulation of DDP relative to untreated cells. The inactive analogue, 1,9-dideoxyforskolin, decreased DDP accumulation. Forskolin and IBMX also increased accumulation in A2780 cells. Neither forskolin nor IBMX had any effect on DDP accumulation in DDP-resistant 2008 cells. The effects were detectable as early as 1 min and persisted at 60 min. The concentrations for half-maximal stimulation of DDP accumulation were approximately 0.2 microM for forskolin and 0.2 mM for IBMX. Forskolin caused marked increases in cAMP levels in both sensitive and resistant 2008 cells within 1 min, although there were differences in the subsequent time-courses of the response. Both 2008 cell types had identical cAMP-dependent protein kinase (PKA) activity. These results suggest that there is a target downstream of PKA that is an important participant in DDP accumulation, and that this target is defective or missing in DDP-resistant cells. Following a 1-hr exposure to drugs, forskolin and IBMX at concentrations that were by themselves completely non-toxic increased the slopes of the clonogenic survival vs. DDP concentration curves in 2008 cells 1.9-fold and 3.3-fold, respectively. In DDP-resistant 2008 cells, however, forskolin and IBMX increased the slopes only 1.2 and 2.6-fold, respectively. These effects of forskolin and IBMX on DDP cytotoxicity did not directly correlate with the effects on the 1-hr DDP accumulation which suggested that, in addition to modulating DDP accumulation, these agents increase the cytotoxicity of the intracellular platinum. The results indicate that modulation of cAMP levels can have important effects on DDP accumulation and cytotoxicity in 2008 cells and that these effects are significantly diminished in DDP-resistant cells. PMID- 1713576 TI - Role of endoplasmic reticulum, an intracellular Ca2+ store, in histamine release from rat peritoneal mast cell. AB - By means of the Ca-antimonate precipitation technique, it was revealed that Ca2+ accumulates in the endoplasmic reticulum (ER) of rat peritoneal mast cells. The ER of rat mast cells was isolated using Percoll density gradient centrifugation, and its characteristics were studied. Although the uptake of 45Ca into the ER was enhanced by adenosine 5'-triphosphate (ATP) at concentrations lower than 2 mM, at higher concentrations ATP induced 45Ca release from the ER, suggesting that bidirectional translocation of Ca2+ takes place in the ER membrane. When apyrase was added to the reaction mixture to decompose all ATP molecules, the amount of 45Ca in the ER was decreased, indicating that ATP is necessary to retain Ca2+ in the ER. Not only inositol 1,4,5-trisphosphate (IP3) but also guanosine 5' triphosphate (GTP) was effective in releasing Ca2+ from the ER at concentrations higher than 2 microM, while guanosine 5'-[gamma-thio]-triphosphate (GTP-gamma S), a non-hydrolysable analogue of GTP, was not effective. This may indicate that a hydrolysis of GTP is necessary for Ca2+ release from the ER. Intracellular Ca2+ blockers such as 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and dantrolene sodium were effective in inhibiting Ca2+ release from ER induced by IP3. The Ca2+ release due to IP3 was also inhibited by flunarizine, a Ca2+ channel blocker, and by oxatomide, an antiallergic drug. Since these two compounds are diphenylpiperazine derivatives, it is suggested that a diphenylpiperazine moiety may be effective in inhibiting Ca2+ release from the ER. PMID- 1713577 TI - Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins. AB - The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification. PMID- 1713579 TI - Kinetic interaction of human immunodeficiency virus type 1 reverse transcriptase with the antiviral tetrahydroimidazo[4,5,1-jk]-[1,4]-benzodiazepine-2-(1H)-thione compound, R82150. AB - We examined the kinetic interaction of purified recombinant DNA-derived human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with R82150, a member of the tetrahydroimidazo[4,5,1-jk]-[1,4]-benzodiazepin-2(1H)-thione family of compounds (Pauwels, R., Andries, K., Desmyter, J., Schols, D., Kukla, M.J., Breslin, H.J., Raeymaeckers, A., Van Gelder, J., Woestenborghs, R., Heykants, J., Schellekens, K., Janssen, M.A.C., De Clercq, E., and Janssen, P.A.J. (1990) Nature 343, 470-474). R82150 inhibited noncompetitively the utilization of homopolymeric and heteropolymeric template-primers (KI range 280-300 nM). Inhibition of dNTP substrate incorporation was also noncompetitive (KI range 100 890 nM). In contrast, 100 microM R82150 did not inhibit human DNA polymerases alpha, beta, or gamma. Gel electrophoresis was used to analyze the effect of inhibitors on extension of heteropolymeric template-primers by HIV-1 reverse transcriptase. ddCTP induced accumulation of partially extended primers which had been terminated at sites requiring incorporation of deoxycytidylate. Competing template-primers reduced accumulation of both fully and partially extended primers. In contrast, R82150 induced accumulation of shortened primers that were terminated at various sites that did not correspond to any one particular deoxynucleotide species. Our results suggest that R82150 does not interact with HIV-1 reverse transcriptase as an analog of either template-primer or deoxynucleoside triphosphate substrate, but may bind allosterically at a site unique to this replicase. PMID- 1713578 TI - Interleukin-2 receptor regulates activation of phosphatidylinositol 3-kinase. AB - Interleukin-2 (IL-2) stimulates proliferation of T lymphocytes and is involved in the activation of both natural killer and lymphokine-activated killer precursor cells. The intracellular messengers which mediate IL-2-dependent events have not yet been identified. IL-2 receptor is not a protein-tyrosine kinase. Activation of a cellular protein-tyrosine kinase and direct association of a protein tyrosine kinase activity with the IL-2 receptor occurs within minutes of IL-2 stimulation. We investigated the activation of phosphatidylinositol 3-kinase (PI 3-kinase) in IL-2-mediated signal transduction using the IL-2-dependent murine T cell line, CTLL-2, and human phytohemagglutinin-stimulated peripheral blood lymphocytes (phytohemagglutinin blasts). Within a minute following stimulation of these cells with IL-2, PI 3-kinase activity could be detected in antiphosphotyrosine (anti-P-Tyr) antibody immunoprecipitates. IL-2 triggered a direct association of PI 3-kinase with the IL-2 receptor as detected in immunoprecipitates using anti-IL-2 receptor beta chain antibody. In vivo labeled CTLL-2 cells have a time-dependent increase in D-3-phosphorylated polyphosphoinositides following stimulation with IL-2. This is the first group of second messengers identified in IL-2-mediated signal transduction. PMID- 1713580 TI - Expression of genes related to the extracellular matrix in human endothelial cells. Differential modulation by elevated glucose concentrations, phorbol esters, and cAMP. AB - To identify agents and mechanisms responsible for the thickened basement membranes characteristic of diabetic angiopathy we examined the effects of high glucose (30 mM) on the expression of genes related to extracellular matrix composition and turnover and investigated whether the changes induced by high glucose were mimicked and sustained by activation of protein kinase C or A. In human umbilical vein endothelial cells high glucose increased fibronectin, collagen IV, tissue plasminogen activator (tPA), and plasminogen activator inhibitor 1 (PAI-1) mRNA levels 2-fold but did not affect type IV and interstitial collagenase expression. Acute treatment with phorbol esters resulted in increased collagen IV, tPA, PAI-1, and interstitial collagenase mRNAs; the type IV collagenase mRNA levels were instead suppressed to 50% of control. Upon longer exposure to phorbol esters (48 h) suppression of fibronectin and PAI-1 mRNAs also occurred. Intracellular elevation of cAMP led to over-expression of fibronectin and type IV collagenase and potentiated the effects of phorbol esters on collagen IV, tPA, and interstitial collagenase expression. The mRNA changes induced by high glucose occurred in the absence of protein kinase C activation or cAMP elevation. These studies indicate that events other than activation of protein kinase C or A bridge high ambient glucose to changes in endothelial cell gene expression that may contribute to diabetic angiopathy. PMID- 1713582 TI - Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit. AB - Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS 1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity. PMID- 1713581 TI - Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase. AB - In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA 10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP resistant phenotype. PMID- 1713583 TI - Early events in the synthesis of the multicopy single-stranded DNA-RNA branched copolymer of Myxococcus xanthus. AB - Myxobacteria and a variety of strains of Escherichia coli contain an unusual extrachromosomal element, a small single-stranded branched copolymer of DNA and RNA (msDNA). Interest in msDNA stems from the presence of a 2'-5' linkage between its DNA and RNA moieties and the possible involvement of reverse transcriptase in its synthesis. Two groups have proposed a model for the synthesis of msDNA that involves the following sequence of events: 1) synthesis of an RNA precursor; 2) addition of a dNTP in a 2'-5' linkage to the RNA precursor (branch priming); 3) synthesis by reverse transcriptase of complementary DNA on the primed RNA precursor; 4) concomitant processing of the RNA precursor by RNase H; and 5) further processing of the RNA precursor by RNase H; and 5) further processing of the branched polymer. The branch priming hypothesis (step 2) was originally based on pulse-chase experiments using a lengthy pulse of 30-min duration. Our experiments with shorter pulse durations demonstrate a variety of intermediate forms not predicted by this model. Specifically, we find that early in synthesis, intermediate msDNA forms appear that are apparently not branch-linked to corresponding RNA moieties. The concomitant RNase H hypothesis (step 4) was based in part on intermediate trapping experiments using dideoxy NTPs to interrupt msDNA synthesis. These experiments showed an apparent complementary relationship between DNA length and RNA length in the trapped forms. In contrast, our experiments show that DNA elongation and RNA processing follow different kinetics. These results suggest an alternate model for synthesis of msDNA in which a conventionally primed DNA moiety is joined with the RNA moiety in a branch ligation event taking place several minutes after the synthesis of both moieties. PMID- 1713584 TI - Structure and expression of TIS21, a primary response gene induced by growth factors and tumor promoters. AB - The TIS21 gene is a primary response gene that is induced rapidly and transiently in 3T3 cells by the tumor promoter and mitogen tetradecanoyl phorbol acetate. The predicted open reading frame of the TIS21 cDNA encodes a protein of 158 amino acids with no obvious similarity to any known protein. Antiserum prepared to TIS21 recombinant protein produced in Escherichia coli precipitates a 17-kDa protein from Swiss 3T3 cells. The 2040-nucleotide 3'-untranslated region of the cDNA includes an unusual T18 sequence. The TIS21 gene has a single 1.4-kilobase intron which interrupts the open reading frame and is otherwise identical to the cDNA sequence. The 5'-flanking sequence of the TIS21 gene contains TATA and CAAT box-type sequences, three potential Sp1 sites, two putative cyclic AMP response elements, two potential AP1 binding elements, and an AP2 element. A possible Z DNA structure of 29 AC repeats is present 660 nucleotides from the start of transcription. Expression from a luciferase reporter construct containing a 460 nucleotide fragment of the TIS21 promoter is induced by tetradecanoyl phorbol acetate, forskolin, epidermal growth factor, and serum, despite the absence of a consensus serum response element. PMID- 1713585 TI - Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule. AB - A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family. PMID- 1713586 TI - The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors. AB - The reverse transcription-polymerase chain reaction was used to examine alternative splicing at each of the three fibronectin exons known to undergo alternative splicing, i.e. extra domain A (ED-A), extra domain B (ED-B), and type III connecting sequence (IIICS). Ratios of fibronectin mRNAs with or without a given exon were determined in several rat tissues and human cell lines during aging in vivo and cellular senescence in vitro. We demonstrate that statistically significant shifts in the alternative splicing of fibronectin occur during aging in vivo and in vitro. Since all three alternatively spliced exons are spliced out at a higher frequency in aging tissues and cells, the fibronectin protein produced by old cells should be slightly smaller than that obtained from young cells. The reverse transcription-polymerase chain reaction demonstrates tissue specific patterns of alternative splicing in several tissues. Whereas fibronectin mRNAs from adult rat tissues were found to range from 0 to 25% ED-A+ and from 0 to 10% ED-B+, fibronectin mRNAs from cultured cell lines were found to be approximately 50-60% ED-A+ and 15-25% ED-B+. We observed similarity in splicing of fibronectin RNA by the different cultured cell lines obtained from many tissues and attribute this observation to the effect of growth factors. We demonstrate that serum deprivation; placement of cells into primary culture; and growth factors such as transforming growth factor beta 1, retinoic acid, and 1,25 dihydroxyvitamin D3 can all change the alternative splicing of fibronectin pre mRNA in the ED-A, ED-B, and type III connecting sequence exons. Possible mechanisms for the regulation of the alternative splicing of fibronectin RNA by growth factors are discussed. PMID- 1713587 TI - Characterization of the binding site for nevirapine (BI-RG-587), a nonnucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. AB - Nevirapine (BI-RG-587) is a potent and specific non-nucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. The compound is non competitive with respect to template, primer, and nucleoside triphosphates indicating that BI-RG-587 does not act directly at the catalytic site. The binding site for this inhibitor was investigated by employing an azido photoaffinity analogue, BI-RJ-70, to covalently label the enzyme. The resulting photoadduct was subjected to enzymatic digestion by trypsin and endoproteinase lys-C and a single, highly labeled peptide was identified as residues 174-199. Sequencing of this peptide identified Tyr-181 and Tyr-188 as labeled residues. PMID- 1713588 TI - Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. AB - The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain. PMID- 1713589 TI - Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme. AB - A system for the expression of recombinant human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in Escherichia coli has been developed, which allows purification of the heterodimeric form of the enzyme as well as the separate purification of the two subunits. It is shown that equilibrium formation between monomeric and homodimeric forms of the recombinant 66- and 51-kDa subunits is considerably more rapid than in the case of the corresponding homodimeric forms of HIV-1 RT. In accordance with our previously published studies on HIV-1 RT (Restle, T., Muller, B., and Goody, R.S. (1990) J. Biol. Chem. 265, 8986-8988) RNA-dependent DNA polymerase activity of the HIV-2 RT preparations can be exactly correlated to their dimer content. No significant heterodimer formation can be observed upon coexpression of the 66-kDa subunit of HIV-2 RT with the 51-kDa subunit of HIV-1 RT in the same cell, indicating differences in the dimerization domains of the two proteins. Recombinant HIV-2 RT is not recognized by a set of 23 monoclonal antibodies raised against HIV-1 RT, although it shows weak cross-reactivity with sera from HIV-1-infected patients. PMID- 1713590 TI - The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation. AB - Infection of cells with poliovirus results in a rapid inhibition of host RNA and protein synthesis. Concordant with this shutoff, the p220 subunit of the cap binding protein complex is cleaved, probably indirectly, by the poliovirus proteinase p2A (2Apro). To elucidate the mechanism of action of 2Apro in inhibiting protein synthesis in vivo, we studied the effect of transient expression of 2Apro in COS-1 monkey kidney cells. In cells transfected with a 2Apro expression plasmid, p220 was cleaved and the 2Apro mRNA was reduced 30-fold compared to an identical plasmid containing a translation termination codon within the 2Apro coding region. The reduced expression from the 2Apro vector results from a 4-fold reduction in DNA replication and 22-fold reduction in transcription by RNA polymerase II from the adenovirus major late promoter/SV40 enhancer utilized in this vector. In contrast, no decrease in transcription of the adenovirus virus-associated I RNA gene by RNA polymerase III was observed. The effect of 2Apro expression on cap-dependent mRNA translation was studied by producing a dicistronic beta-globin mRNA harboring the encephalomyocarditis virus leader and 2Apro coding region within the 3' end of the mRNA to mediate cap independent translation of 2Apro. Expression of this mRNA was also reduced 25 fold compared to an identical plasmid harboring a termination codon within the 2Apro coding region. Translation of the beta-globin marker gene from this mRNA was reduced 3-fold when corrected for mRNA level. These results suggest that p220 cleavage itself is not sufficient for complete inhibition of host translation and that an important effect of 2Apro expression on host protein synthesis is a reduction in RNA polymerase II transcription and to a lesser extent, DNA replication. This reduction could be a primary effect of 2Apro, or a secondary effect caused by the inhibition of translation. PMID- 1713591 TI - The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs. AB - Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell. PMID- 1713593 TI - Activation of signal transduction pathways protects quiescent Balb/c-3T3 fibroblasts against death due to serum deprivation. AB - Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin like growth factor-1 (IGF-1), and insulin protect density-inhibited murine Balb/c 3T3 fibroblasts against death by distinctive mechanisms. Determination of the cell survival-enhancing activity of growth factors by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep dose-response relationship in the 1-5 ng/ml range. The other factors display shallow log-linear relationships in the following ranges: EGF: 0.2-5 ng/ml; IGF 1: 2-80 ng/ml; and insulin: 57-4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate (Br-cAMP) and N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), markedly increase both short-term (5-h) and long-term (20-h) survival of cells. 2-Isobutyl-1-methylxanthine (IBMX) markedly enhances short-term survival, but its effect decays with time. The protein kinase C agonist 12-O tetradecanoyl phorbol-13-acetate (TPA) has a moderate protective effect at concentrations of 16-32 nM, and 64 nM TPA is highly effective. The synthetic diaclglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplementation of EGF with a protein kinase A or C agonist results in a varying additive increase in short-term (5-h) cell survival and supplementation of EGF + insulin or PDGF + EGF + insulin increases further the already high level of protection given by the growth factor combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of growth factors gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: 1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c-3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and 2) phosphorylation events catalyzed by protein kinase A largely serve to protect cells by a mechanism not requiring de novo RNA and protein biosynthesis. PMID- 1713592 TI - A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. AB - Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation. PMID- 1713594 TI - Factors influencing perpendicular elongation of embryonic frog muscle cells in a small applied electric field. AB - The mechanism controlling the perpendicular elongation of embryonic muscle cells exposed to a small applied electric field has been studied using a pharmacological approach. Inhibition of the inositol phosphate second messenger system, of calcium entry and of microfilament polymerisation all prevented perpendicular elongation. A model involving strengthened adhesion asymmetrically along the cathodal-facing side of round myoblasts and incorporating the above requirements is proposed to explain electric field-induced perpendicular differentiation. Some asymmetry of organelles is described also, with ribosomes, yolk granules and actin filaments all predominantly found on the anodal side of myoblasts. PMID- 1713595 TI - Neural crest cell locomotion induced by antibodies to beta 1 integrins. A tool for studying the roles of substratum molecular avidity and density in migration. AB - Migration of neural crest cells depends on direct, transient interactions between fibronectin molecules and their corresponding Arg-Gly-Asp integrin receptors. We have previously suggested that the moderate-activity interaction between integrin receptors and fibronectin may be critical for the transient association of the cells with their substratum. In order to test this hypothesis, we have examined the in vitro locomotory behavior of neural crest cells on substrata of differing apparent avidities for integrin receptors. As substrata, we used a variety of monoclonal and polyclonal antibodies to the integrin beta 1 subunit that were characterized for their respective relative apparent avidities for the receptor. Neural crest cells were able to migrate on these antibodies and exhibited an organization of substratum-adhesion sites and of cytoskeletal elements virtually identical to that observed on fibronectin, indicating that they can at least partially mimic the migration-promoting activity of fibronectin. However, the number of migrating cells as well as their morphology and their speed of locomotion varied significantly with both the concentration of the antibody substratum and its relative avidity for the receptor. Thus, on high-avidity monoclonal antibodies and on polyclonal divalent antibodies at high concentrations only a limited number of cells escaped from the neural tube, and the rate of their migration was reduced compared to that on fibronectin (23 +/- 5 microns h-1 versus 65 +/- 10 microns h-1). In addition, cells were unusually flattened and cohesive. Time-lapse videomicroscopy revealed that, on high-avidity substrata, neural crest cells were able to extend cell processes that adhered to the substratum, but showed a dramatically reduced capability of breaking pre existing substratum contacts. In contrast, the same antibodies at low concentrations produced neural crest cell migration at rates very similar to those on fibronectin at the same concentrations. Low-avidity monoclonal antibodies and polyclonal monovalent antibodies at all concentrations tested permitted extensive migration of neural crest cells, which exhibited the same morphology and locomotory behavior as on fibronectin. These results indicate that both the avidity of receptors for the substratum and the number of receptors bound to the substratum are critical in regulating the locomotory behavior of neural crest cells in vitro, and therefore might help to regulate the directionality of migration and final localization pattern of neural crest cells in vivo. PMID- 1713596 TI - Determination of 5-methoxyindoles in pineal gland and plasma samples by high performance liquid chromatography with electrochemical detection. AB - A liquid chromatographic analysis with electrochemical detection of 5 methoxytryptamine, 5-methoxytryptophol, 5-methoxyindoleacetic acid and melatonin is described. Optimal elution conditions were determined by studying several variables: pH, buffer salt, counter ion and organic modifier. Measurement of 5 methoxyindoles in the pineal gland and plasma of hamsters has been performed after extraction. This method is specific and sensitive, and enables detection of 5-methoxyindoles in a pool of two hamster pineal glands. This is also the first time that these three 5-methoxyindoles have been measured simultaneously in plasma. PMID- 1713597 TI - Simultaneous measurement of serotonin and 5-hydroxyindoleacetic acid in rat brain using a liquid chromatographic method with electrochemical detection. AB - A simple and sensitive method for the simultaneous measurement of 5 hydroxytryptamine and its main metabolite 5-hydroxyindoleacetic acid in rat brain is described. Brain tissue samples were only deproteinated and, without further extraction, were injected directly onto a high-performance liquid chromatography column and detected electrochemically. The detection limit for 5 hydroxytryptamine and 5-hydroxyindoleacetic acid was 16 and 8 g per injection, respectively. Within 8 min, the total separation of 5-hydroxytryptamine and 5 hydroxyindoleacetic acid is achieved. With this method over 100 analyses can be performed in a single working day. Brain samples from young and old, male and female Brown Norway rats were analysed for indoles by this method. The ratio 5 hydroxytryptamine 5-hydroxyindoleacetic acid, an index for serotonin turnover, showed significant differences between age groups and genders in the cortex, and a significant difference between genders in the hypothalamus. PMID- 1713598 TI - Marked improvement of a substance P radioimmunoassay by reduction of 125I labelled [Tyr8]-substance P prepared by the chloramine-T method with mercaptoethanol and subsequent purification by reversed-phase liquid chromatography. AB - [Tyr8]-substance P was radiolabelled with 125I by the application of the chloramine-T method. Due to the high oxidative potential of the 125I-chloramine-T system the purified reaction product was converted into a derivative, which presumably had been oxidized to the corresponding sulphoxide at Met11. This conversion was shown by reversed-phase high-performance liquid chromatography after consecutive reduction-oxidation experiments of the freshly prepared radiopeptide. The oxidized derivative exhibited only negligible binding to substance P receptors isolated from rat brain homogenates. However, in contrast, it showed marked cross-reaction to the antibody raised in rabbits against synthetic SP(1-11). The variance in the quantification of identical samples was marked, and the measurement of concentrations in the lower pg/ml range was not sensitive enough to determine levels of substance P-like immunoreactivity in human cerebrospinal fluid. Assay sensitivity could be substantially improved and variance significantly decreased by the use of a radiopeptide, which had been labelled by the chloramine-T method and which had been subsequently reduced with mercaptoethanol and purified by liquid chromatography. PMID- 1713599 TI - Relationship between bile tolerance and the presence of a ruthenium red staining layer on strains of Lactobacillus acidophilus. AB - Seventeen strains of Lactobacillus acidophilus were evaluated to determine the relationship between bile tolerance and the presence of an outer polysaccharide layer exterior to the cell wall when viewed by transmission electron microscopy. Bile tolerance is necessary for survival of lactobacilli in the intestinal tract, and the polysaccharide layer may be responsible for adherence to human intestinal tissue. These two factors may be the basis for use of L. acidophilus as a dietary adjunct. Ten strains exhibited a ruthenium red-stained outer polysaccharide layer. Three of the 10 strains had extremely dense layers, which may indicate stronger adherence properties. Seven strains did not contain a ruthenium red stained outer layer; however, six strains that did not have the stained layer were resistant to 1.0% bile concentration. Fourteen strains were tolerant to 1% bile, one strain was tolerant to 6% bile, and two strains were sensitive to bile. No relationship between bile tolerance and the presence of the ruthenium red stained outer polysaccharide layer was apparent. PMID- 1713600 TI - Liner collection cone and pH effects on postthaw motility, staining, and acrosomes of bovine spermatozoa. AB - Sixteen ejaculates were collected, four each from four bulls, using artificial vaginas with polyethylene or rubber liner collection cones in a crossover design experiment. The ejaculates were diluted with egg yolk-citrate extender at pH 6.4 or 7.2, cooled, glycerolated, equilibrated, packaged in .5-ml French straws, frozen in nitrogen vapor, and stored in liquid nitrogen. Thirty frozen straws from each ejaculate were thawed rapidly (46.5 degrees C for 12 s), pooled, and then incubated at 46.5 degrees C for periodic evaluation of progressive motility, differential staining, and acrosome morphology under thermal stress conditions. The postthaw motility of spermatozoa and percentage of unstained cells were higher both when collected in polyethylene than in rubber and when extended at pH 7.2 vs. 6.4, but no interaction was found between liner collection cone composition and pII for postthaw motility. Retention of spermatozoan motility during incubation under thermal stress was greater for cells collected in polyethylene, but not different due to pH. Neither pH nor composition of liner collection cone had an effect on postthaw acrosomal scores, but the time required for a 50% increase in severely damaged acrosomes was greater for spermatozoa collected in polyethylene than in rubber liner collection cones. PMID- 1713601 TI - Racial variation in reaction to physical stigma: a study of degree of disturbance by vitiligo among black and white patients. AB - The effect of race on reaction to impaired appearance is explored in a sample of 158 patients with vitiligo, a disfiguring skin disease. Blacks and Whites do not differ in degree of disturbance by the disorder. Psychological coping resources and variables related to negative labeling of the stigma are associated with variation in degree of disturbance. Self-esteem and perceived stigmatization are associated significantly with degree of disturbance among both Blacks and Whites. Gender, age, and visibility of the condition are not related to difference in degree of disturbance within either race, although there is some evidence that they may have an indirect relationship to degree of disturbance. Importance of appearance is associated with degree of disturbance for Whites only, because of the threat of the depigmentation induced by vitiligo to the racial identity of Blacks. The implications of these findings for theory and practice are discussed. PMID- 1713602 TI - Identification of T cell stimulatory peptides from the 38-kDa protein of Mycobacterium tuberculosis. AB - T cell specificity to individual antigenic epitopes could determine the distinction between protective and pathogenic host reactions in tuberculous infections. Therefore, T cell stimulatory epitopes of the Mycobacterium tuberculosis 38-kDa lipoprotein, of known structure and specificity and of prominent immunogenicity, have been examined. To identify potential T cell epitopes, eight peptides, seven of which were predicted to form amphiphatic helices, were used for immunization of various inbred mice and for elicitation of in vitro T cell proliferative responses. Three different response patterns were observed. 1) Lymph node cells from mice immunized with peptide, recombinant 38 kDa Ag, killed M. tuberculosis strain H37Ra, or live Mycobacterium bovis bacillus Calmette Guerin infection responded to peptide 38.G (residues 350 to 369). Responses were observed in mice of H-2b, H-2d, and H-2k haplotypes. 2) Peptide 38.C (residues 201 to 220) induced proliferation of lymph node cells from 38-kDa protein-, but not from peptide-immunized mice. 3) Peptide 38.F (residues 285 to 304) only elicited a response of the homologous peptide-primed cells. Analysis of CD4+ T cell lines confirmed the distinct specificities and stimulatory features of peptides 38.F and 38.G. The described attributes of peptide 38.C and 38.G could be of potential interest for diagnostic evaluation in tuberculous infections. PMID- 1713603 TI - CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. AB - CD28, initially detected on human T lymphocytes with the help of antibodies, and CTLA-4, obtained by reverse genetics through its preferential expression in mouse activated T cells, are both single-V domain members of the Ig superfamily. Early work showed a relationship between these two molecules, which we wished to further document, in particular because of the growing realization of the functional importance of CD28 in some T cell activation pathways. Isolation and analysis of the mouse CTLA-4 gene and further analysis of the human CTLA-4 gene showed that both of these and the human CD28 gene share the same overall intron/exon organization. The nucleic acid sequence homology of the exons was found to extend across both molecules and species, whereas the 5' and 3' flanking regions exhibited homology across species but not between molecules. Message expression of human CTLA-4 was only detected in activated T cells and, thus, shares with that of mouse CTLA-4 and of mouse and human CD28 a lymphoid tissue distribution, although apparently broader for the latter. Two main human CTLA-4 transcripts of about 1.8 and 0.8 kb were detected, the smaller of which may derive, as reported for human CD28, from the use of an alternate degenerated polyadenylation signal sequence. The nucleic acid sequence data allowed a direct comparison of the four putative complete protein sequences of CD28 and CTLA-4 in the mouse and the human, showing striking homologies, especially in some stretches (such as a MYPPPY hexamer in the hinge region) conserved across molecules and across species. The mouse CD28 gene was localized to chromosome 1 band C by in situ hybridization with three different radioactive probes, indicating, together with previous data, that the CD28 and CTLA-4 genes map to the same chromosomal region in both the mouse and the human. Thus, CD28 and CTLA 4 were found to be strikingly similar in most respects, in terms of structure, sequence, expression, and gene location, furthermore in two species, strongly suggesting that their genes are the direct products of a duplication event and raising the possibility of functional homologies between the corresponding proteins. PMID- 1713604 TI - Characterization of a new minor lymphocyte stimulatory system. I. Cluster of self antigens recognized by "I-E-reactive" V beta s, V beta 5, V beta 11, and V beta 12 T cell receptors for antigen. AB - In the mouse, two sets of V beta gene products have been shown to be associated with T cell recognition of endogenous self Ag. One of these is the set of V beta associated with T cell reactivities to stimulatory Mls gene products, Mlsa (V beta 6, V beta 8.1, V beta 9) or Mlsc (V beta 3); another is the set of V beta, such as V beta 5, V beta 11, V beta 12, or V beta 17a, which were originally found to be related to I-E recognition. Although the Mls system has been well characterized, little is known about the nature of the ligands for the second set of V beta. In this work, we describe the evidence that the natural ligand or ligands of V beta 5, V beta 11, and V beta 12 may be novel Mls determinants that are recognized by naive T cells at a high precursor frequency and function as the ligand for clonal deletion of self-reactive T cells by negative selection. However, surprisingly, unlike the conventional Mls system, in which all V beta associated with Mlsa recognition or Mlsc recognition are uniformly deleted in those animals expressing the relevant Mls type, expression of these three V beta segregates independently among strains. Based on these observations, the nature of T cell recognition for this new Mls gene product(s) is discussed. PMID- 1713605 TI - Presentation of autoantigen by human T cells. AB - Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag. PMID- 1713606 TI - Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells. AB - To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells. PMID- 1713608 TI - Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor). AB - IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA activated lymphoblasts and with cloned lines of human T cells indicated that IL 12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2 induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7. PMID- 1713609 TI - Function and evolutionary conservation of distinct epitopes on the leukocyte adhesion molecule-1 (TQ-1, Leu-8) that regulate leukocyte migration. AB - The leukocyte adhesion molecule-1 (LAM-1, TQ=1, Leu-8) in humans, like its murine homologue, MEL-14, is the principal receptor that mediates the binding of leukocytes to high endothelial venules (HEV) of peripheral lymph nodes. In this study, several regions of the protein which mediate receptor function were identified by using a large panel of murine mAb reactive with LAM-1. Individual mAb reacted with LAM-1+ cells with characteristic intensities of immunofluorescence staining, and each bound both lymphocytes and neutrophils. Lymphocyte attachment to HEV was significantly inhibited by the binding of five mAb. In contrast, only two of these mAb were able to completely block the binding of phosphomannan monoester core complex from the yeast Hansenula holstii cell wall (PPME), a phosphomannan monoester core polysaccharide that serves as a soluble model of the natural ligand of LAM-1. Interestingly, the binding of two anti-LAM-1 mAb to cells induced a significant increase in PPME binding, reminiscent of the increase in receptor affinity observed after leukocyte activation. Antibody cross-blocking studies indicated that many of the functionally important epitopes were spatially distinct, and domain mapping indicated that they recognized distinct domains of LAM-1. The expression and function of these epitopes were further assessed by using a variety of animal species to further characterize the functionally relevant epitopes defined in these studies. At least some anti-LAM-1 mAb reacted with leukocytes from monkey, cow, rabbit, sheep, dog, cat, pig, and goat, but not from chicken, rat, or mouse. The reactivity of anti-LAM-1 mAb in several animal species correlated with the ability of leukocytes to bind PPME, and mAb that inhibited lymphocyte binding to HEV in man could also inhibit this function in rhesus monkey and dog. Thus, several LAM-1 epitopes are structurally and functionally well conserved throughout recent mammalian evolution, emphasizing an important role for LAM-1 in the regulation of leukocyte traffic. PMID- 1713607 TI - Activation of AP-1 by IL-1 and phorbol esters in T cells. Role of protein kinase A and protein phosphatases. AB - We have examined the regulation of the AP-1 transcription complex in the IL-1 responsive murine T cell thymoma cell line EL-4 6.1 C10. Our results demonstrate that AP-1-mediated gene expression in T cells may be regulated by several signaling pathways and factors, including IL-1, protein kinase C, protein kinase A (PKA), and one or more serine/threonine-specific protein phosphatases. The activation of protein kinase C results in an increase in nuclear AP-1 DNA binding activity, as well as enhanced gene expression. IL-1 and agents that elevate intracellular cAMP levels do not, by themselves, induce AP-1 activation, but they synergize with phorbol esters. IL-1 and forskolin may enhance AP-1 function by different mechanisms, because forskolin enhanced gene expression without producing an increase in nuclear AP-1 DNA binding, whereas IL-1 increased AP-1 binding activity and gene expression. These observations, in conjunction with the lack of a demonstrable effect of IL-1 on cAMP production in EL-4 cells, are consistent with the view that IL-1 enhances AP-1 activation by a pathway that does not directly involve cAMP and PKA. However, the induction of AP-1 activity by IL-1 and phorbol esters is dependent upon the presence of PKA, as evidenced by the loss of AP-1 inducibility in cells transfected with a cDNA encoding protein kinase inhibitor, a specific inhibitor of PKA. The effect of protein kinase inhibitor on AP-1 activation in response to IL-1 and tetradecanoyl-phorbol-13 acetate was reversed in the presence of the serine/threonine protein phosphatase inhibitor okadaic acid. Thus, the level of AP-1 activity in T cells may be determined by the balance between the activities of several serine/threonine protein kinases and phosphatases. PMID- 1713610 TI - Human T cell recognition of influenza A nucleoprotein. Specificity and genetic restriction of immunodominant T helper cell epitopes. AB - The characterization of human T cell antigenic sites on influenza A nucleoprotein (NP) is important for subunit vaccine development for either antibody boosting during infection or to stimulate T cell-mediated immunity. To identify such sites, 31 synthetic peptides that cover more than 95% of the amino acid sequence from NP of influenza A/NT/60/68 virus were tested for their ability to stimulate PBL from 42 adult donors. The most frequently recognized region was covered by a peptide corresponding to residues 206-229 of NP, with 20/42 (48%) of responders. In many individuals this was also one of the peptides that stimulated the strongest T cell responses. Other regions that were also frequently recognized were 341-362 by 13/42 (30%), 297-318 by 10/42 (23%), and 182-205 by 9/42 (21%) of individuals. These peptides covered highly conserved regions in NP of influenza A viruses, suggesting that they could be useful in boosting cross-reactive immunity against the known type A virus strains. In order to define the class II restriction molecules used to present particular T cell epitopes, 22 persons from the donor panel were HLA-typed. The majority of persons who expressed DR2, and proliferated to NP also responded to the major immunodominant region 206-229. In addition, this peptide was also immunodominant in the one person expressing DRw13. The observation that recognition of this peptide is associated with DR2 was confirmed by using short term T cell lines and APC from a panel of typed donors. Further results with virus-specific T cell lines and clones and transfected L cells expressing DR molecules showed that DR1 could also present this peptide. Therefore the results suggest that recognition of 206-229 is associated with at least three different DR haplotypes and this may explain the high frequency with which this peptide is recognized in the population. The fine specificity of the response to peptide 206-229 was distinct when presented by DR1 or DR2-expressing APC. The DR1 response was dependent on the N terminus, and the DR2 response was directed to the C terminus of the peptide. PMID- 1713612 TI - Striatal restricted adenosine A2 receptor (RDC8) is expressed by enkephalin but not by substance P neurons: an in situ hybridization histochemistry study. AB - RDC8 has been recently cloned and characterized as an adenosine A2 receptor. This receptor is expressed exclusively by medium-sized neurons of the striatum as demonstrated by in situ hybridization. We have now studied the relationship of this receptor with three major components of the rat caudate-putamen: enkephalin, substance P, and choline acetyltransferase. Our results demonstrate that the adenosine A2 receptor is expressed exclusively by the enkephalinergic striatal subpopulation but not by the substance P-containing or cholinergic neurons. PMID- 1713611 TI - Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells. AB - Transforming growth factor-beta (TGF-beta) is a potent negative regulator of normal human B cell growth mediated by exogenous signals, including IL-2 and low m.w. B cell growth factor 12 kDa (BCGF-12 kDa). In the present study, we investigated the regulatory linkage between viral or nonviral transformation of human B cells and the growth inhibitory effects of TGF-beta 1. A panel of EBV+ and EBV- B cell lines, derived either by in vitro EBV B cell transformation, or from cases of lymphoma was used to quantitate the negative growth effects of TGF beta 1. The proliferative response of three EBV- B cell lines to rBCGF-12 kDa or serum was inhibited by low concentrations of TGF-beta 1 (0.2-0.5 ng/ml for 50% maximal effect), as measured by tritiated thymidine uptake and viable cellular recovery. In contrast, rBCGF-12 kDa or serum mediated proliferation of three EBV+ B cell lines was refractory to the growth inhibitory effects of TGF-beta 1. In an attempt to understand the mechanism(s) for this differential growth control in EBV+ and EBV- B cells, we studied the expression of TGF-beta 1, c-myc, and TGF beta 1 receptors. No correlation was observed between the expression of TGF-beta 1 or c-myc gene and growth inhibition by TGF-beta 1 in the cell lines studied. Our results indicate that sensitivity or resistance to TGF-beta 1 correlated with the presence or absence (loss) of high affinity receptors for TGF-beta 1. EBV- B cell lines expressed levels of high affinity receptors similar to those found on activated normal B or T cells. In contrast, EBV+ B cell lines showed no detectable high affinity receptors. Chemical cross-linking studies with a bifunctional reagent, dissuccinimidyl suberate revealed a normal expression of type I (65-70 kDa), type II (85-90 kDa), and type III (280-300 kDa) TGF-beta 1 high affinity receptors on EBV- B cell lines. In contrast, EBV+ B cell lines did not express type I and type II receptors, whereas type III receptors were expressed but could not be inhibited by unlabeled TGF-beta 1.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713613 TI - High expression of the HNK-1/L2 carbohydrate epitope in the major glycoproteins of shark myelin. AB - The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1 positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process. PMID- 1713614 TI - Lack of phospholipase D activity in chromaffin cells: bradykinin-stimulated phosphatidic acid formation involves phospholipase C in chromaffin cells but phospholipase D in PC12 cells. AB - The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin. PMID- 1713615 TI - Developmental expression of HNK-1-reactive antigens in rat cerebral cortex and molecular heterogeneity of sulfoglucuronylneolactotetraosylceramide in CNS versus PNS. AB - Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex. PMID- 1713616 TI - Effects of intranigral substance P and neurokinin A injections on extracellular dopamine levels measured with microdialysis in the striatum and frontoparietal cortex of rats. AB - Extracellular levels of dopamine (DA) and its metabolite, 3,4 dihydroxyphenylacetic acid (DOPAC), in the striatum and frontoparietal (sensorimotor) cortex in halothane-anesthetized rats were analyzed simultaneously using in vivo microdialysis. Basal DA levels, measured from the microdialysis perfusate, were 6.4 +/- 0.8 nM (n = 15) in the striatum and 0.9 +/- 0.1 nM (n = 15) in the frontoparietal cortex. Subcutaneous injections of d-amphetamine (2 mg/kg) increased DA levels 10-fold in the striatum and fivefold in the cortex. Injections of substance P (0.07 nmol/0.2 microliters) into the substantia nigra pars reticulata (SNR) increased DA and DOPAC levels approximately 30% in the ipsilateral striatum and approximately 50% in the ipsilateral frontoparietal cortex. Injections of neurokinin A (0.09 nmol/0.2 microliter) into the SNR increased DA and DOPAC levels approximately 30% in the ipsilateral striatum but did not significantly affect DA levels in the ipsilateral frontoparietal cortex, although DOPAC levels were increased by approximately 50%. It is suggested that striatal and cortical DA release is regulated differently by nigral substance P and neurokinin A terminals. PMID- 1713617 TI - Four decades of commitment. PMID- 1713618 TI - Leader with a cause. PMID- 1713619 TI - Endodontics. PMID- 1713620 TI - Oral & maxillofacial surgery. PMID- 1713621 TI - Periodontics. PMID- 1713622 TI - Public health dentistry. PMID- 1713623 TI - Metering dentistry. PMID- 1713624 TI - Specific decrease in liver insulin-like growth factor-I and brain insulin-like growth factor-II gene expression in energy-restricted rats. AB - Four-week-old male rats were maintained for 10 d on a series of diets containing a constant high level of dietary protein and total energy at 100, 70, 60 or 50% of the ad libitum intake rate. Under these conditions, growth rate varied as a function of dietary energy. Serum insulin-like growth factor (IGF)-I was decreased in the energy-restricted animals. Total hepatic IGF-I mRNA was decreased by approximately the same factor as circulating IGF-I protein. In contrast to previous results obtained with protein-restricted animals, serum albumin mRNA was not decreased in the energy-restricted animals. Brain IGF-II mRNA was slightly decreased in animals fed the 70 and 60% energy diets and was decreased by 50% in animals fed the 50% energy diet. Insulin-like growth factor binding protein-2 (IGFBP-2) gene expression was increased in the liver but not in the brain of the energy-restricted animals, indicating that dietary energy regulates IGFBP-2 gene expression differently in liver and brain. The results demonstrate specific changes in liver IGF-I and IGFBP-2 gene expression and brain IGF-II gene expression in animals that are growth-retarded because of a restriction of dietary energy. PMID- 1713625 TI - Identification of osteoclasts and their mononuclear precursors. A comparative histological and histochemical study in hamster periodontitis. AB - Quantification of osteoclast resorption, a good index of periodontitis destruction, is primarily based on osteoclast identification. As their identification is sometimes dubious, we compared osteoclastic counts in hamster specimens processed for either routine histology or tartrate-resistant acid phosphatase (TRAP) staining. No difference was found between the two approaches concerning the number of osteoclasts. However the mean bone-osteoclast interface was higher in the TRAP-stained specimens (+30%, p less than 0.02). As osteoclast precursors are also TRAP+ cells, they were quantified too. Compared with controls, there was a dramatic increase (p less than 0.0001) in periodontitis affected animals. Precursors were strongly correlated to active osteoclasts (r = 0.97). Our data suggest that precursors are recruited only when the disease is active in a given site. PMID- 1713626 TI - Pharmacokinetics of FK-506, a novel immunosuppressant, after intravenous and oral administrations to rats. AB - A pharmacokinetic study of FK-506 (FK), a novel immunosuppressant being about hundred times more potent than cyclosporin A (CyA) in the in vitro experiment, has been performed in rats after intravenous and oral administrations at two doses, 1.0 and 5.0 mg/kg. As compared with CyA, plasma, bile, urine and lymph FK levels were determined by a fluorescence high-performance liquid chromatographic method with chemiluminescence detection. Non-compartment pharmacokinetic parameters were calculated by area/moment analysis. After the i.v. injection of FK at 1.0 and 5.0 mg/kg, the total plasma clearance, CLtot, was 7.028 +/- 0.076 (mean +/- S.E.) and 7.651 +/- 0.755 ml/min, steady-state distribution volume, Vd.ss, was 4623 +/- 28 and 4201 +/- 529 ml/kg, elimination half-life at the terminal elimination phase, t1/2 beta, was 2.36 +/- 0.25 and 2.54 +/- 0.29 h, and the volume of the initial distribution space, V1, was 1336 +/- 150 and 1065 +/- 115 ml/kg, respectively. By comparing the pharmacokinetic parameter with CyA, the CLtot of FK is about three times greater than that of CyA. There is not a significant difference on t1/2 beta between CyA and FK. The V1 and Vd.ss of FK are 4-5 times greater than that of CyA. Therefore, higher clearance of FK is ascribed not only to the faster elimination from the rat body but also to the greater distribution space in the body. The mean percentage of FK transferred into the thoracic lymphatics over 6 h were 0.09 +/- 0.02% (1.0 mg/kg) and 0.16 +/ 0.02% (5.0 mg/kg), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713627 TI - [Prevention and treatment of decubitus ulcers. Up to date facts]. PMID- 1713628 TI - [Opening of a department for palliative care]. PMID- 1713629 TI - Molecular analysis of a human liver mitochondrial ornithine transcarbamylase deficiency. AB - The liver of a young girl which had been successfully transplanted was investigated at the ornithine transcarbamylase (OTC, EC 2.1.3.3) gene expression level. Northern blot hybridization using a human OTC cDNA probe showed a greater than 80% decrease in specific OTC mRNA although having the same molecular size as a normal control. OTC polypeptide was simultaneously synthesized with a normal molecular size but at a low level (20%) as shown by immunoblotting. The OTC enzyme from the deficient liver exhibited very little catalytic activity (7.2% as compared to the normal subject). These results may support several explanations of this disease such as mutation of the OTC gene promoter leading to a low transcriptional activity or mutation of the encoding sequence which results in a modified translation product but with a normal size. mRNA instability may also occur. PMID- 1713630 TI - Results of treatment and lessons learned from pathologically staged T4 non-small cell lung cancer. AB - Depending on the local extension of primary non-small cell lung cancer (NSCLC) and invaded T4 structure(s), 49 patients underwent complete (CR, n = 14) or palliative (PR, n = 13) resection of exploratory thoracotomy (ET, n = 22) between January 1982 and June 1988. Thoracic radiotherapy (TR) was given to all patients receiving PR (median dose, 43 Gy) and ET (median dose, 53 Gy). With a median follow-up of 44 months, overall 2- and 5-year survival was 25 and 5%, respectively. Patients undergoing ET plus TR had a significantly worse survival than those treated by CR (P = 0.041) and PR plus TR (P = 0.046). Only completely resected patients became long-term survivors (5-year survival, 29%) and significant predictors of their survival were previous weight loss, hemoglobin, and creatinine level, in univariate analysis, and previous weight loss in multivariate analysis. The site of initial treatment failure was mainly local for PR plus TR (85%) and systemic for CR (71%) and ET plus TR (86%). Presented results suggest that surgery might play a role for selected patients with T4 NSCLC, but advances in systemic and local therapy are necessary. PMID- 1713631 TI - The choice between surgical resection and radiation therapy for patients with cancer of the esophagus and cardia: a retrospective comparison between two treatments. AB - The choice of treatment for patients with cancer of the esophagus and cardia is controversial. Since overall survival is poor, the most important aim of treatment should be improvement of the main complaint: the inability to eat. In a retrospective analysis of 265 patients, referred to the University Hospital in Leiden, The Netherlands, comparisons were made between palliative effects of surgical resection (N = 92) and irradiation (N = 128). Several methods of comparing surgery with irradiation are possible: (1) all surgical patients vs. all irradiated patients; (2) only those surgical patients who survived the operation (N = 70) vs. all irradiated patients (N = 128); and (3) survivors after resection (N = 70) vs. only those irradiated patients treated with "curative" radiation (N = 62). Analysis of prognostic factors showed that in both surgical and irradiated patients, the only statistically significant factor was the (dis)ability to eat. Criteria to be considered to make individual recommendations for either treatment are presented. PMID- 1713632 TI - The investigation of antiepileptic action of qingyangshen (QYS)--effect of QYS on the concentrations of neuropeptides in rat brain. AB - The concentrations of central neuropeptides, somatostatin (SS) and substance-P (SP), were determined in the different brain regions of young-aged male rats after a long-term administration of anticonvulsants Qingyangshen (QYS), Diphenylhydantoin (DPH), and Carbamazepine (CBZ). The results were compared with Pentylenetetrazol (PTZ)-induced seizure model and normal saline-treated controls. No effects of QYS on the concentrations of SS and SP were found in the rats of four-week or eight-week group. Both of DPH and PTZ increased the SS levels in the midbrain of rats in four-week group. DPH, CBZ, and PTZ also increased the SP levels in the cerebral cortex, striatum, and brain stem of rats in eight-week group. Our present data indicated that the central neuropeptides SS and SP were involved in the processes of epilepsy and antiepilepsy. Since QYS did not influence the contents of SS and SP after a long-term administration, it suggested that the anticonvulsant mechanism of QYS may be different from those of DPH and CBZ, i.e. it may be not due to its effect on the central neuropeptide pathway. PMID- 1713633 TI - The effect of radix Salviae Miltiorrhizae (RSM) on substance P in cerebral ischemia--animal experiment. AB - The levels of substance P (SP) in rat brains were assayed in 64 rats. Bilateral common carotid artery ligation was done in 49 rats. Half an hour before ligation, 25 rats were given 10 g/kg of RSM; 24 rats were given the same volume of normal saline as controls. Sham operation was done in 15 rats. Half an hour and 3 hours after cerebral ischemia, the rats were quickly decapitated. SP concentration was assayed in the cerebral cortex, caudate nucleus and brain stem. In saline-treated animals, the SP level of caudate nucleus at 3-hour group was significantly decreased as compared with the 0.5-hour group and sham-operated group respectively. No significances were found among RSM-treated groups and sham operated groups. The SP levels were shown: brain stem greater than caudate nucleus greater than cerebral cortex. The preliminary results suggest that SP may be involved in the pathophysiologic procedures of cerebral ischemia and RSM may attenuate the dysfunction of SP during cerebral ischemia. PMID- 1713634 TI - D-factor: modulation of expression in fibroblasts. AB - Differentiation inducing factor (D-factor) is a recently described protein. The gene has been cloned, but little is known concerning regulation of expression of the gene. Our study showed that fibroblasts from a variety of tissues (lung, bone marrow, gingiva, foreskin) constitutively expressed D-factor RNA. Levels of expression of this gene increased in fibroblasts of each of the tissues after exposure to several stimuli including products of activated macrophages and lymphocytes (tumor necrosis factor (TNF), interleukin 1 and lymphotoxin). Other stimuli were those capable of activating either protein kinase C (12-O tetradecanoylphorbol-13-acetate (TPA) and teleocidin), G-binding proteins (NaF) or those inhibiting protein synthesis (cycloheximide). Accumulation of D-factor RNA by TNF may in part be explained by stabilization of D-factor transcripts; TPA and cycloheximide clearly stabilized D-factor transcripts. We and others have shown that these same signals similarly stimulated fibroblasts to express RNAs coding for a variety of cytokines including three colony-stimulating factors as well as interleukins 1 and 6. Taken together, D-factor probably is a participant in the cascade of cytokines that are produced in mesenchymal cells after various stimuli such as bacterial invasion. PMID- 1713635 TI - B cells of chronic lymphatic leukemia express V genes in unmutated form. AB - In order to investigate whether the leukemic B cells in B-CLL express immunoglobulin genes in mutated or unmutated form, independent cDNA clones of one patient with B-CLL expressing heavy and light chain V region genes were sequenced. The sequences of both the VH and V kappa clones were identical. The V genes could be assigned to known germline V genes. No somatic mutations were found. At the V kappa-J kappa border there is an insertion of N-sequences which are only rarely found in immunoglobulin light chain genes. Our study confirms other published data on V gene expression in B-CLL in that the surface immunoglobulins in these tumors are unmutated. PMID- 1713636 TI - Cell death in the human leukemia cell line, K-562, induced by antiserum and monoclonal antibodies. AB - Rabbit polyclonal antiserum, or derived gamma globulin, to K-562 cells induces decreased TdR uptake within hours and cell death without cytolysis in 2-4 days. A panel of nine mAb, reactive with K-562 cells, was grouped on the basis of no effect on growth or TdR uptake, increased uptake, or decreased uptake. Treatment of cells with antiserum, gamma globulin, or mAb of the last group caused single strand, but not double-strand, DNA fragmentation at a time when the cells were still viable. Cycloheximide did not inhibit the antibody effect suggesting that protein synthesis was not required. Aurintricarboxylic acid at certain concentrations markedly enhanced TdR uptake and protected the cells when antiserum was used but did not protect from mAb treatment. PMID- 1713637 TI - Expression of hematopoietic growth factor RNAs in human mesenchymal cells from various organs. AB - Experiments were undertaken to study expression of hematopoietic growth factor RNAs in mesenchymal cells from a variety of organs including bone marrow, foreskin, gingiva, and lung. Cells from each organ had negligible expression of RNAs coding for granulocyte (G), macrophage (M), and granulocyte-macrophage (GM) colony stimulating factor (CSF), interleukin 1 beta (IL-1 beta), and IL-6. Fibroblasts from each tissue had a comparable ability to express the same cytokine RNAs. Surprisingly, the stimuli for expression of G-CSF RNA was disparate from the stimuli for expression of the other cytokine RNAs. While IL-1 beta enhanced accumulation of G-CSF RNA, tumor necrosis factor alpha (TNF) and 12 O-tetradecanoylphorbol-13-acetate (TPA) did not. In contrast, IL-1 beta, TNF, and TPA equally stimulated increased levels of M-CSF, GM-CSF, IL-1 beta and IL-6 RNAs. PMID- 1713638 TI - Expression of hematopoietic progenitor cell associated antigen CD34 in chronic myeloid leukemia. AB - The expression of progenitor cell associated antigen CD34 was investigated in cells from 28 patients with chronic myeloid leukemia (CML). The CD34 positivity varied from 0-26% in patients with chronic phases CML (n = 17); from 6-64% in patients with accelerated phase CML (n = 4); and from 27-97% in the patients with blastic crisis of CML (n = 8). The difference in CD34 positivity between chronic (mean 10.1 +/- 2.3%), accelerated (37.7 +/- 13.3%) and blastic (58.0 +/- 7.3%) phases of CML is statistically significant (p less than 0.05), however, the number of patients studied, especially in accelerated and blastic phases is very small. There was no difference in the CD34 positivity of the cells in the peripheral blood and in the bone marrow. CD34 positivity was higher in patients with chronic phase CML at diagnosis (untreated patients) than in those who were studied during treatment. The possible importance of serially studying CD34 positivity in patients with CML is discussed in the paper. PMID- 1713639 TI - Increased risk of myelodysplasia and leukaemia after etoposide, cisplatin, and bleomycin for germ-cell tumours. AB - Among the cytostatic drugs only the alkylating agents have been firmly established as being leukaemogenic. This report describes 4 cases of acute myeloid leukaemia and 1 of myelodysplasia occurring in a cohort of 212 patients with germ-cell tumours treated with etoposide, cisplatin, and bleomycin. The mean cumulative risk of leukaemic complications was 4.7% (SE 2.3) 5.7 years after start of etoposide-containing chemotherapy, and, compared with the risk in the general population, the relative risk of overt leukaemia was 336 (95% CI 92-861). No leukaemias were detected in a previous cohort of 127 patients with germ-cell tumours treated with cisplatin, bleomycin, and vinblastine. The increased risk of leukaemia was most probably due to etoposide alone or in combination with cisplatin or bleomycin, since other published work has also not revealed an excess of leukaemias among patients with germ-cell tumours treated with only cisplatin, bleomycin, and vinblastine. The risk of leukaemia was dose related since all 5 patients with leukaemic complications were among the 82 who had received a cumulative dose of more than 2000 mg/m2 etoposide, whereas no leukaemias were observed among 130 patients who had received up to 2000 mg/m2 (p = 0.004). 3 of the leukaemic patients had balanced chromosome translocations affecting bands 11q23 and 21q22. These translocations, and perhaps also other balanced aberrations, seem to be characteristic of myelodysplasia and acute leukaemia occurring after therapy with cytostatic agents acting on DNA topoisomerase II. PMID- 1713640 TI - Treatment of chronic hepatitis C with inosine pranobex. PMID- 1713641 TI - Prediction of lymph node involvement in breast cancer. PMID- 1713642 TI - Drug interference on alpha-fetoprotein assays. PMID- 1713643 TI - Update: cholera--Western Hemisphere, and recommendations for treatment of cholera. AB - Epidemic cholera appeared in Peru in January 1991 and subsequently spread to Ecuador, Colombia, Chile, Brazil, Mexico, and Guatemala. Cholera can be a severe, life-threatening illness but is highly preventable and easily treated; however, few health-care practitioners in the United States have experience identifying and treating cholera. This report provides an update on cholera in the Western Hemisphere and provides recommendations on the clinical diagnosis and treatment of cholera in the United States. PMID- 1713644 TI - Synthesis and glycosylation of fibronectin in hepatocytes from vitamin A deficient rats. AB - Recent work from our laboratory (Kim and Wolf, J Biol Chem 262:365-371, 1987) has shown increased uptake of labeled amino acids into fibronectin (FN), increased net synthesis of FN and increased levels of FN-mRNA in primary cultures of hepatocytes from vitamin A-deficient rats compared to controls. We now find, surprisingly, decreased uptake of labeled sugars into the oligosaccharide chains of FN from vitamin A-deficient hepatocytes. This decrease could be reversed by added retinoic acid at physiological concentration. At the same time, FN from deficient hepatocytes (-A.FN) was more susceptible to proteolytic degradation. Decreased uptake of the core sugar mannose into -A.FN was similar to that of glucosamine, yet the percent of label in sialic acid was the same as in + A.FN, suggesting a smaller number of oligosaccharide chains per molecule of -A.FN. Upon enzymatic removal of oligosaccharide and labeling with sodium borotritide, it was found that both -A.FN and +A.FN had biantennary oligosaccharide structures. Selective enzymatic removal of sialic acid showed that +A.FN had both sialic acids in an alpha 2----3 linkage, whereas -A.FN apparently had one alpha 2----3 and one alpha 2----6-linked sialic acid. The borotritide experiments allowed us to calculate that +A.FN appeared to have 5 oligosaccharide chains per FN monomer, whereas the -A.FN showed only 4 chains. These results would account for the decreased glycosylation and increased susceptibility to proteolysis of the -A.FN. We conclude that vitamin A controls both the rate of synthesis of the polypeptide chain of FN via its mRNA, as well as the rate of its glycosylation. PMID- 1713645 TI - Single cell analysis of free cytosolic calcium changes in human lung mast cells- II. The relationship between desensitization and the cellular regulation of calcium changes. AB - Stimulation of mast cells results in two opposing reactions, activation events that cause degranulation and desensitization events that inhibit mediator release. Previous studies of human lung mast cells and murine mast cells have suggested that desensitization resulted from events that negatively regulated free cytosolic calcium ([Ca2+]i) levels; the current studies suggest otherwise. Stimulation of purified human lung mast cells with anti-IgE demonstrated that histamine release had reached a maximum at a time (5 mins) when [Ca2+]i levels were still near their maximum elevation. While there was a slow return of [Ca2+]i levels to baseline (T1/2 = 7.8 min), this rate of return could not clearly account for the cessation of histamine release. The heterogeneity in this decay parameter was also calculated to be insufficient to account for the heterogeneity in the peak calcium response while heterogeneity in the cell surface IgE density could adequately account for the heterogeneity in calcium responses. Preincubation of mast cells with anti-IgE antibody without extracellular calcium did lead to a progressive loss of the subsequent [Ca2+]i response when calcium was added back to the reaction, but the rate of desensitization determined by this measure, T1/2 of 8 min, was slower than the rate determined by measuring the progressive inhibition of histamine release (T1/2 of 4.5 min). In addition, no correlation existed for the rate of desensitization as measured by histamine release and that measured by the peak calcium response. These data suggested that the extent of histamine release was not strictly controlled by regulation of free cytosolic calcium and that desensitization events measured by the progressive loss in histamine release and calcium response were also not strictly related. PMID- 1713646 TI - A cluster of continuous antigenic structures in the transmembrane protein of HIV 1: individual patterns of reactivity in human sera. AB - We investigated the antigenicity of a highly conserved region in the transmembrane protein of the human immunodeficiency virus type 1 (HIV-1). In order to identify antigenically important residues, amino-acid sequences of synthetic peptides representing this region were varied systematically: single residues were omitted from the sequence of HIV-env 583-599; threonines were substituted for pairs of residues in HIV-env 581-599; the sequences of heptadeca peptides were shifted by single residues. The peptides were tested in an enzyme immuno-assay against fourteen HIV-1 antibody-positive human sera, which were previously found to react with HIV-env 583-599, and against rabbit antisera to the peptides HIV-env 583-599 and 586-606. Substitutions as well as deletions in the sequence 589-596 (AVERYLKD) aborgated the antigenicity of the peptides with most of the human sera. Changes outside this sequence affected the reactivities differentially. Six overlapping dodeca-peptides, shifted in the sequence by single residues, lacked antigenicity in a competition assay, suggesting antigenic dependence on an ordered peptide conformation, which the longer peptides may preferentially assume. 19- and 21-mers with overlapping sequences competed to different extents with each other for binding to the antibodies of 3 human sera, illustrating that more than one antigenic structure in this narrow region can be recognized by a single polyclonal serum. PMID- 1713647 TI - Monoclonal antibodies defining epitopes on human IgE. AB - Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C epsilon 1 or early C epsilon 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotypic marker. Another mAb reacted with heat-denatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C epsilon 1, C epsilon 2 and C epsilon 3-4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C epsilon 2 and C epsilon 3-4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C epsilon 2-4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc epsilon RI and Fc epsilon RII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C epsilon 1 and C epsilon 3-4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C epsilon 1-4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity. PMID- 1713648 TI - Psychological stress and susceptibility to the common cold. AB - BACKGROUND: It is not known whether psychological stress suppresses host resistance to infection. To investigate this issue, we prospectively studied the relation between psychological stress and the frequency of documented clinical colds among subjects intentionally exposed to respiratory viruses. METHODS: After completing questionnaires assessing degrees of psychological stress, 394 healthy subjects were given nasal drops containing one of five respiratory viruses (rhinovirus type 2, 9, or 14, respiratory syncytial virus, or coronavirus type 229E), and an additional 26 were given saline nasal drops. The subjects were then quarantined and monitored for the development of evidence of infection and symptoms. Clinical colds were defined as clinical symptoms in the presence of an infection verified by the isolation of virus or by an increase in the virus specific antibody titer. RESULTS: The rates of both respiratory infection (P less than 0.005) and clinical colds (P less than 0.02) increased in a dose-response manner with increases in the degree of psychological stress. Infection rates ranged from approximately 74 percent to approximately 90 percent, according to levels of psychological stress, and the incidence of clinical colds ranged from approximately 27 percent to 47 percent. These effects were not altered when we controlled for age, sex, education, allergic status, weight, the season, the number of subjects housed together, the infectious status of subjects sharing the same housing, and virus-specific antibody status at base line (before challenge). Moreover, the associations observed were similar for all five challenge viruses. Several potential stress-illness mediators, including smoking, alcohol consumption, exercise, diet, quality of sleep, white-cell counts, and total immunoglobulin levels, did not explain the association between stress and illness. Similarly, controls for personality variables (self-esteem, personal control, and introversion-extraversion) failed to alter our findings. CONCLUSIONS: Psychological stress was associated in a dose-response manner with an increased risk of acute infectious respiratory illness, and this risk was attributable to increased rates of infection rather than to an increased frequency of symptoms after infection. PMID- 1713649 TI - Administration of thyroxine in treated Graves' disease. PMID- 1713650 TI - Sodium channel density in hypomyelinated brain increased by myelin basic protein gene deletion. AB - Trophic control over the expression and membrane distribution of voltage dependent ion channels is one of the principal organizing events underlying the maturation of excitable cells. The myelin sheath is a major structural determinant of regional ion channel topography in central axons, but the exact molecular signals that mediate local interactions between the oligodendrocyte and axolemma are not known. We have found that large caliber fibre pathways in the brain of the mutant mouse shiverer (shi, gene on chromosome 18), whose developmental fate of myelination is averted by deletion of five exons in the myelin basic protein gene, have a striking excess of sodium channels. As cytoplasmic membranes of shiverer oligodendroglia still adhere to axons, the evidence indicates that myelin basic protein or a myelin basic protein-dependent glial transmembrane signal associated with compact myelin formation, rather than a simple glial-axon contact inhibition or an intrinsic genetic program of neuronal differentiation, could be critical in downregulating sodium channel density in axons. Here we use the shiverer mutant to show that mature central nervous system projection neurons with large caliber unmyelinated fibres sustain functional excitability by increasing sodium channel density. This axon plasticity, triggered by the absence of a single glial protein, contributes to the unexpectedly mild degree of neurological impairment in the mutant brain without myelin, and may be a potentially inducible mechanism determining the recovery of function from dysmyelinating disease. PMID- 1713651 TI - [The aphasic patient]. PMID- 1713652 TI - A GABA immunocytochemical study of rat motor thalamus: light and electron microscopic observations. AB - A light and electron microscopic study of GABA-immunoreactive neurons and profiles in the ventroanterior-ventrolateral and ventromedial nuclei of rat dorsal thalamus was conducted using antiserum raised against GABA. Less than 1% of the neurons in these motor-related nuclei exhibited GABA immunoreactivity, confirming previous reports that these nuclei are largely devoid of interneurons. Immunoreactive neurons in the ventral anterior-ventral lateral complex and ventromedial nucleus were bipolar or multipolar in shape, and tended to be smaller than non-immunoreactive neurons. GABA immunoreactivity in the neuropil consisted of labeled axon terminals and myelinated and unmyelinated axons, and was lower in the ventral anterior-ventral lateral complex and ventromedial nucleus than in neighboring thalamic nuclei. The density of neuropil immunolabeling was slightly higher in ventral anterior-ventral lateral complex than in ventromedial nucleus. GABA-immunoreactive axon terminals, collectively termed MP boutons for their medium size and pleomorphic vesicles (and corresponding to "F" profiles of some previous studies of thalamic ultrastructure), formed symmetric synapses and puncta adhaerentia contacts predominantly with large and medium-diameter (i.e. proximal) non-immunoreactive dendrites. Approximately 12 and 18% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, were GABA-immunopositive. Many of these immunoreactive profiles probably arose from GABAergic neurons in the thalamic reticular nucleus, substantia nigra pars reticulata and entopeduncular nucleus. Two types of non-immunoreactive axon terminals were distinguished based on differences in morphology and synaptic termination sites. Boutons with small ovoid profiles and round vesicles that formed prominent asymmetric synapses onto small-diameter dendrites were observed. Mitochondria were rarely observed within these boutons, which arose from thin unmyelinated axons. These boutons composed approximately 82 and 74% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, and were considered to arise predominantly from neurons in the cerebral cortex. In contrast, boutons with large terminals that contained round or plemorphic vesicles and formed multiple asymmetric synapses predominantly with large diameter dendrites were also observed. Puncta adhaerentia contacts were also common. Mitochondria were numerous within large boutons with round vesicles, which arose from myelinated axons. Many of the large boutons were likely to have originated from neurons in the cerebellar nuclei. Approximately 6% of the boutons in the ventral anterior-ventral lateral complex and 8% in ventromedial nucleus were of the large type.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713653 TI - Effects of neonatal gamma-ray irradiation on rat hippocampus--I. Postnatal maturation of hippocampal cells. AB - The axons of dentate granule cells, the mossy fibres, establish synaptic contacts with the thorny excrescences of the apical dendrite of CA3 pyramidal neurons. Dentate granule granule cells develop postnatally in rats, whereas the CA3 pyramidal cells are generated before birth. In the present studies, using unilateral neonatal gamma-ray irradiation to destroy the granule cells in one hemisphere, we have studied the effect of mossy fibre deprivation on the development of their targets. We show that such "degranulation" prevents the normal development of giant thorny excrescences, suggesting that the development of thorny excrescences in CA3 pyramidal neurons is under the control of mossy fibres. In contrast, irradiation of the hippocampus of the neonatal rat does not affect the development of the dendritic arborization of CA3 pyramidal cells and their non-mossy dendritic spines. PMID- 1713654 TI - Involvement of lipid peroxidation and inhibitory mechanisms on ischemic neuronal damage in gerbil hippocampus: quantitative autoradiographic studies on second messenger and neurotransmitter systems. AB - We investigated, to examine the involvement of lipid peroxidation and inhibitory mechanisms, a novel lipid peroxidation inhibitor (KB-5666) and a GABAA receptor effector (pentobarbital) on ischemic neuronal damage and the alterations in the second messenger and neurotransmitter systems in Mongolian gerbils by means of morphology and in vitro receptor autoradiography. Quantitative receptor autoradiography visualized binding sites for [3H]inositol 1,4,5-trisphosphate, [3H]forskolin, [3H]phorbol 12,13-dibutyrate, [3H]isradipine (PN200-110), [3H]N6 cyclohexyl-adenosine, and [3H]quinuclidinyl benzilate indicating binding sites for inositol 1,4,5-trisphosphate, forskolin, protein kinase C, L-type calcium channels (or dihydropyridine binding sites), adenosine A1, and muscarinic cholinergic receptors, respectively. In the morphological study, KB-5666, 10 and 50 mg/kg, i.v., 5 min before ischemia, protected against ischemic neuronal damage to the hippocampal CA1 subfield following 5 min of bilateral carotid artery occlusion in a dose-dependent manner. Pentobarbital, 30 mg/kg, i.v., 5 min before ischemia, also had a protective effect. In receptor autoradiographic studies, all receptor bindings decreased significantly in the CA1 subfield seven days after ischemia. In particular, [3H]inositol 1,4,5-trisphosphate binding in the CA1 subfield was completely lost after ischemia. [3H]Inositol 1,4,5-trisphosphate and [3H]forskolin binding decreased as early as 6 h after ischemia. In the CA3 subfield, [3H]inositol 1,4,5-trisphosphate, [3H]PN200-110, and [3H]N6 cyclohexyladenosine bindings decreased seven days after ischemia. In the dentate gyrus, [3H]inositol 1,4,5-trisphosphate binding decreased seven days after ischemia. KB-5666 and pentobarbital prevented reductions in these receptor bindings in the CA1 subfield at 6 h and seven days after ischemia. These results indicate that KB-5666 and pentobarbital protect the brain from both structural and functional damage after ischemia, and that lipid peroxidation and inhibitory mechanisms may play a pivotal role in the neuronal damage of the hippocampal CA1 subfield after ischemia. PMID- 1713655 TI - Collateralization of periaqueductal gray neurons to forebrain or diencephalon and to the medullary nucleus raphe magnus in the rat. AB - Antinociceptive effects elicited from the midbrain may involve both ascending and descending projections from the periaqueductal gray and dorsal raphe nucleus. To investigate the relationship between these different efferent pathways in the rat, we performed a double-labeling study using two retrograde tracers, colloidal gold-coupled wheatgerm agglutinin-apo horseradish peroxidase and a fluorescent dye. One tracer was microinjected in the medullary nucleus raphe magnus; the second was injected into one of several regions rostral to the periaqueductal gray that have been implicated in nociceptive and antinociceptive processes. The results can be grouped into two categories. First, injections into the ventrobasal thalamus, lateral hypothalamus, amygdala, and cerebral cortex labeled neurons in the dorsal raphe nucleus but not in the periaqueductal gray. Up to 90% of these projection neurons were serotonin immunoreactive, and up to 17% were also retrogradely labeled from the nucleus raphe magnus. Second, only injections into the ventrobasal hypothalamus (which included the beta-endorphin-containing arcuate neurons) or into the medial thalamus labeled neurons in the periaqueductal gray itself. Injections into the medial thalamus, but not into the ventrobasal hypothalamus, also labeled neurons in the dorsal raphe nucleus. Up to 20% of the neurons retrogradely labeled from these regions were also retrogradely labeled from nucleus raphe magnus. The presence of large populations of rostrally projecting periaqueductal gray neurons that collateralize to the nucleus raphe magnus implies that activity in ascending projections necessarily accompanies any activation of the periaqueductal gray-nucleus raphe magnus pathway. Possibly, projections from the medial thalamus and medial hypothalamus mediate antinociceptive effects that complement descending inhibition. Finally, possible antidromic activation of these pathways must be considered when interpreting the results of electrical brain stimulation studies. PMID- 1713656 TI - The kinetics and morphological characteristics of the macrophage-microglial response to kainic acid-induced neuronal degeneration. AB - Outside the nervous system myelomonocytic cells are known to play an important role in the inflammatory response and tissue repair after injury. In this study we have examined the myelomonocytic response to neuronal destruction following unilateral injection of the excitotoxin kainic acid into the mouse hippocampus. Intrahippocampal injection of kainate induces rapid, synchronous neuronal death. There is no neutrophil recruitment and a delay of at least 48 h before macrophage microglial cell numbers increase. The microglial reaction in the injected hippocampus consists of altered morphology, a 6-9-fold increase in mononuclear phagocyte cell numbers and enhanced expression of the macrophage-specific plasma membrane antigen, F4/80, assessed immunohistochemically and by Western blotting. Microglia also respond at distant sites related to the projection pathway and terminals of killed pyramidal cells but the reaction varies in cell numbers, kinetics and morphology. The absence of neutrophil recruitment and the delay in an increase in macrophage or microglial cells shows that the CNS differs from other sites in the body with regard to the kinetics and nature of the myelomonocytic cell inflammatory response. The role of mononuclear phagocytes in tissue repair in the CNS remains to be defined. PMID- 1713657 TI - Restricted cortical termination fields of the midline and intralaminar thalamic nuclei in the rat. AB - The projections from the midline and intralaminar thalamic nuclei to the cerebral cortex were studied in the rat by means of anterograde tracing with Phaseolus vulgaris-leucoagglutinin. The midline and intralaminar nuclear complex taken as a whole projects to widespread, predominantly frontal, cortical areas. Each of the constituent thalamic nuclei has a restricted cortical projection field that overlaps only slightly with the projection fields of adjacent midline and intralaminar nuclei. The projections of the intralaminar nuclei cover a larger cortical area than those of the midline nuclei. The laminar distributions of fibres from individual midline and intralaminar thalamic nuclei are different and include both deep and superficial cortical layers. The parataenial, paraventricular and intermediodorsal midline nuclei each project to circumscribed parts of the prefrontal cortex and the hippocampal and parahippocampal regions. In the prefrontal cortex, the projections are restricted to the medial orbital, infralimbic, ventral prelimbic and agranular insular fields, and the rostral part of the ventral anterior cingular cortex. In contrast to the other midline nuclei, the rhomboid nucleus projects to widespread cortical areas. The rostral intralaminar nuclei innervate dorsal parts of the prefrontal cortex, i.e. the dorsal parts of the prelimbic, anterior cingular and dorsal agranular insular cortical fields, the lateral and ventrolateral orbital areas, and the caudal part of the ventral anterior cingular cortex. Additional projections are aimed at the agranular fields of the motor cortex and the caudal part of the parietal cortex. The lateral part of the parafascicular nucleus sends fibres predominantly to the lateral agranular field of the motor cortex and the rostral part of the parietal cortex. The medial part of the parafascicular nucleus projects rather sparsely to the dorsal part of the prelimbic cortex, the anterior cingular cortex and the medial agranular field of the motor cortex. Individual midline and intralaminar thalamic nuclei are thus in a position to directly influence circumscribed areas of the cerebral cortex. In combination with previously reported data on the organization of the midline and intralaminar thalamostriatal projections and the prefrontal corticostriatal projections the present results suggest a high degree of differentiation in the convergence of thalamic and cortical afferent fibres in the striatum. Each of the recently described parallel basal ganglia thalamocortical circuits can thus be expanded to include projections at both the cortical and striatal levels from a specific part of the midline and intralaminar nuclear complex. The distinctive laminar distributions of the fibres originating from the different nuclei emphasize the specificity of the midline and intralaminar thalamocortical projections. PMID- 1713658 TI - Topographical gradient in expression of R2D5 antigen in superior olivary nuclei and hippocampal dentate gyrus of the cat. AB - An immunohistochemical analysis of the cat central nervous system revealed that a monoclonal antibody which recognizes a soluble cytosolic protein, R2D5, bound two regions in a prominent spatial gradient. In the medial and lateral superior olivary nuclei of the brainstem, R2D5 immunoreactivity appeared as a gradient across a population of topographically ordered principal neurons. The spatial gradient corresponded to the tonotopic organization in the superior olivary nuclei: i.e., R2D5 immunoreactivity tended to occur more frequently and intensely in low-frequency neurons than in high-frequency neurons. Granule cells in the hippocampal dentate gyrus also had a pronounced spatial gradient in R2D5 immunoreactivity expression, and this gradient corresponded to the septotemporal axis of the hippocampus. Granule cells of the temporal (ventral) portions of the hippocampus were labeled intensely with R2D5 antibody, while those located in progressively more septal (dorsal) portions had gradually less immunoreactivity. These results suggest that in both the superior olivary nuclei and the hippocampal dentate gyrus, neurons differ in intrinsic properties by their position along specific axes. They suggest also that the hippocampus has an intrinsic functional organization related to the spatial gradient along its septotemporal axis. PMID- 1713659 TI - Massage: positive strokes in palliative care. PMID- 1713660 TI - D-elg, a member of the Drosophila ets gene family: sequence, expression and evolutionary comparison. AB - We have cloned a cDNA from the Drosophila elg gene, a new member of the ets family of genes. The D-elg gene is located at 97D on chromosome 3R and is expressed as a 2.0kb RNA in the embryos, pupae and adults, with no detectable expression in third instar larvae. D-elg expression is observed in all cells of early stage embryos, prior to transcriptional activation of the zygotic genome, and is maintained throughout embryogenesis with no regional localization. The cDNA encodes a predicted protein of 15.4kD that has an 86 amino acid sequence with 72% similarity to the carboxy terminal region of the Drosophila ets-2 gene. Comparison with all known ets genes allows us to define a minimal region required for assignment to the ets gene family. PMID- 1713661 TI - Oncogenic potential of erbB-2 in human mammary epithelial cells. AB - Introduction of the normal erbB-2 gene into immortalized human mammary epithelial cells (184B5) by transfection conferred a growth advantage to these cells both in vitro and in vivo. The 184B5 cells overexpressing erbB-2 formed colonies in semi solid medium, frequently induced transient nodules in athymic mice and produced progressive tumors in vivo at a low frequency. Those tumors which did arise from erbB-2-transfected cells displayed substantially higher levels of normal gp185erb 2 protein when compared to the original transfectants, consistent with their selection for increased erbB-2 expression. Introduction of genes encoding genetically altered erbB-2 molecules into 184B5 cells increased their colony forming efficiency and converted the cells to a tumorigenic phenotype at a high frequency. When the biological and biochemical properties of human mammary carcinoma cell lines known to overexpress erbB-2 were compared to the transfected 184B5 lines, they behaved most like those overexpressing the normal erbB-2 protein. Results indicate that overexpression of normal erbB-2 may directly contribute to the transformation of human mammary epithelium if sufficient levels of erbB-2 protein are expressed or if the erbB-2 gene is genetically altered. PMID- 1713662 TI - Defective induction of Jun and Fos-related proteins in phorbol ester-resistant EL4 mouse thymoma cells. AB - Treatment of sensitive EL4 mouse thymoma cells with phorbol esters causes growth inhibition, adherence to substrate and production of several lymphokines including Interleukin 2. Resistant cells lack all of these responses. Since production of Interleukin 2 mRNA is dependent on protein synthesis, and the Interleukin 2 gene has a phorbol ester responsive element, we examined both cell lines for expression of the various Jun and Fos species which bind to this element. Phorbol ester induced c-fos, jun-B, and jun-D RNAs within 20 min in both cell lines. Fos-B was similarly induced in sensitive cells but induction was delayed and greatly enhanced in resistant cells. C-jun RNA induction was detected only in sensitive cells. Western analysis confirmed the induction of c-Jun and a Fos-related protein in sensitive cells only. Southern analysis indicated that both cell lines contain c-jun and fra-1 genes. These results suggest that defective induction of c-Jun and/or Fos-related proteins may contribute to the absence of phorbol ester-induced lymphokine production in resistant EL4 cells. PMID- 1713663 TI - CSF and blood pharmacokinetics of hydromorphone and morphine following lumbar epidural administration. AB - Sixteen consenting patients scheduled for elective thoracotomy were enrolled into a randomized trial of epidural morphine and hydromorphone. Each patient had a lumbar epidural catheter placed preoperatively for the purpose of post thoracotomy analgesia. Shortly before the end of the operative procedure each patient received 5 mg of morphine and 0.75 mg of hydromorphone via the epidural catheter. Blood was sampled at regular intervals following the opiate administration and patients were randomized to 1 of 7 cervical CSF sampling times. Blood and CSF samples were assayed for morphine and hydromorphone concentration to determine blood and CSF pharmacokinetic profiles. A maximum blood morphine concentration of 60 +/- 25 ng/ml (mean +/- S.D.) was obtained at 11 +/- 6 min (mean +/- S.D.). The blood hydromorphone peak of 14 +/- 13 ng/ml (mean +/- S.D.) occurred 8 +/- 6 min (mean +/- S.D.). The mean peak CSF opioid concentrations of 1581 ng/ml for morphine and 309 ng/ml for hydromorphone occurred 60 min after epidural administration. The blood and CSF pharmacokinetic profiles for morphine and hydromorphone are presented. These profiles are similar for the two drugs after lumbar epidural administration. PMID- 1713664 TI - A reappraisal of non-consensus mRNA splice sites. PMID- 1713665 TI - Targeted disruption of a human interferon-inducible gene detected by secretion of human growth hormone. AB - A new method is described for the sib-selection of 'targeted' mammalian cells that have undergone homologous recombination (HR) with a transfected DNA construct. This method has been used to disrupt the 6-16 gene, an interferon (IFN)-inducible gene of unknown function, in two different human cell lines. Disruption was caused by integration of a targeting construct containing a promoterless gene for human growth hormone (hGH) which was expressed after HR with the 6-16 gene. Homologous recombinants were detected in pools of non homologous recombinants by the appearance of hGH in the growth medium after the addition of IFN. Secondary and tertiary rounds of hGH assays were used to sib select 9 homologous recombinants that were shown to have 1, 2 or 3 copies of the targeting construct integrated at the 6-16 locus. The method, which should be applicable to other transcribed targets, provides an alternative to selection methods, and offers advantages over other screening methods in being simple, rapid, sensitive and reliable. PMID- 1713666 TI - Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome. AB - The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed, D. and Noller, H.F. (1987) Nature, 327, 389-394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A----G and 915A----G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)- and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin. PMID- 1713667 TI - PCR detection of the MspI polymorphism in the human IRBP gene (RPB3). PMID- 1713668 TI - [Palliative care: a need and not a fashion]. PMID- 1713669 TI - PMN-elastase in comparison with CRP, antiproteases, and LDH as indicators of necrosis in human acute pancreatitis. AB - We analyzed the role of polymorphonuclear granulocytes (PMN)-elastase in predicting the prognosis of patients with acute pancreatitis in comparison with C reactive protein (CRP), lactate dehydrogenase (LDH), and the two antiproteases alpha 1-antitrypsin (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M). Fifty-two patients with acute pancreatitis were subdivided according to morphological criteria into 29 patients with edematous pancreatitis and 23 patients with necrotizing pancreatitis. Within 5 days after the onset of acute pancreatitis, the accuracy rates for detecting necrotizing pancreatitis were 86%, 84%, 82%, 72%, and 69%, using cutoff levels of 120 mg/L for CRP, 120 micrograms/L for PMN elastase, 270 U/L for LDH, 1.5 g/L for alpha 2-M, and 3.5 g/L for alpha 1-AT, respectively. The median peak value of PMN-elastase was reached on day 1 of acute pancreatitis in contrast to the median peak of CRP, which was at its highest between days 3 and 4. PMN-elastase represents a reliable indicator, comparable with CRP, for the staging of acute pancreatitis. The advantage of PMN-elastase over CRP appears to be its earlier increase and the greater dynamism of its serum course. Finally, the results suggest that CT scanning for the evaluation of the extent of intra- and extrapancreatic necrosis could be restricted to those patients with increased values of PMN-elastase and CRP. PMID- 1713670 TI - Plasma concentrations of neurotensin and CCK in patients with chronic pancreatitis with and without enzyme substitution. AB - The peptide hormones neurotensin (NT) and cholecystokinin (CCK) are commonly attributed with a physiological role in the stimulation of exocrine pancreatic secretion. However, on the other hand, little is known about the effect of diminished exocrine pancreatic function and of the resulting maldigestion on postprandial plasma levels of these two gastrointestinal peptides. We investigated, therefore, the effect of enzyme substitution therapy on the magnitude and time course of plasma concentrations of both hormones in patients suffering from severe chronic pancreatitis. Pancreatic insufficiency led to elevated NT-concentrations, in response to a standard meal, which could be reduced by enzyme replacement therapy. Prior to enzyme therapy, the mean integrated postprandial release of NT amounted to 2800 +/- 250 pg/ml after 60 min in patients with severe chronic pancreatitis. This amount was significantly reduced to 1250 +/- 150 pg/ml after 60 min after enzyme therapy, compared to 810 +/- 90 pg/ml after 60 min in healthy volunteers after the standard meal. The integrated postprandial CCK level in patients investigated was significantly lower (35 +/- 4.8 pmol/L after 60 min) without any substitution therapy, compared to the integrated peptide amount in healthy volunteers (145 +/- 13.5 pmol/L after 60 min). Enzyme therapy in patients suffering from chronic pancreatitis led to an increased postprandial CCK-level (80 +/- 9.6 pmol/L after 60 min). Elevated CCK plasma concentrations have not been demonstrated in these patients with pancreatic insufficiency. We therefore suggest that CCK might not play a major role in feedback regulation in patients with chronic pancreatitis. However, in light of elevated NT plasma concentrations in patients with chronic pancreatitis, NT-mediated influence on the pancreas deserves further study. PMID- 1713671 TI - Synthetic CCK8 analogs with antagonist activity on pancreatic receptors: in vivo study in the rat, compared to non-peptidic antagonists. AB - The cholecystokinin octapeptide (CCK8)-derived synthetic peptides Boc-Tyr(SO3H) Nle-Gly-DTrp-Nle-Asp-O-CH2-CH2-C6H5 (JMV179) and Boc-Tyr(SO3H)-Nle-Gly-DTrp-Nle Asp-NH-CH2-CH2-C6H5 (JMV167) are antagonists of peripheral cholecystokinin (CCK) receptors in vitro. In the present study, antagonist activity of these peptides was studied on rat pancreatic secretion in vivo, and compared to those of other peptidic molecules and to the non-peptidic antagonists L364718, D-, L-, DL lorglumide, and proglumide. The decreasing order of antagonist potencies on amylase release in vitro was L364718 greater than JMV179 greater than lorglumide greater than JMV167 greater than proglumide; JMV179 was 25 times less potent than L364718 and 300 times more potent than JMV167. The decreasing order of antagonist potencies on protein output in pancreatic juice in vivo was L364718 greater than JMV167 greater than JMV179 greater than lorglumide greater than proglumide; JMV167 was two times more potent than JMV179 and only 8 times less potent than L364718. Increased potency of JMV167, relative to JMV179 under in vivo conditions, is probably due to the slower rate of catabolism of the phenylethylamide group, relative to the phenylethylester group, since the metabolite issued from hydrolysis of the ester bond was totally inactive. This study shows that it is possible to obtain peptidic CCK antagonists, which are active and potent in vivo, and provides a quantitative measurement of potency changes occurring in vivo for several peptidic and non-peptidic antagonists. PMID- 1713673 TI - The effect of the CCK receptor antagonist CR 1409 on bile reflux pancreatitis in the opossum. AB - Availability of specific cholecystokinin (CCK) receptor antagonists has the potential for contributing to delineation of the role of CCK in the development of pancreatitis and, perhaps, development of new therapeutic agents for treatment of the disorder. The purpose of this study was to evaluate the effect of a potent CCK receptor antagonist, CR 1409, on bile reflux pancreatitis. The opossum pancreatic duct enters the common duct in such a position that it is possible to ligate the common duct distal to the pancreatic duct, resulting in bile refluxing into the pancreatic duct and producing pancreatitis. CR 1409 was administered to opossums at the time of distal common duct ligation and at the time of cystic- and common ducts ligations. In a separate group, CR 1409 administration was begun 24 hours following onset of pancreatitis. Control experiments were performed, in which CR-1409 was not administered. Serum amylase, pancreas gland weights, inflammation, and systemic venous insulin, glucagon, and CCK concentrations were evaluated. Bile duct ligation resulted in significant hyperamylasemia, pancreas gland edema, inflammation, hyperglucagonemia, hypercholecystokinemia, and hypoinsulinemia. CR 1409, administered at the onset of pancreatitis, significantly decreased amylase concentrations, gland weight, and inflammation, when compared to control values. Hormonal changes associated with pancreatitis were also significantly altered by CR 1409 administration. When administered 24 hours following onset of pancreatitis, CR 1409 was not effective in altering the pancreatitis produced by bile duct ligation. The results suggest that CCK plays a permissive or contributory role in the inflammatory process and in associated hormonal changes during development of bile reflux pancreatitis in the opossum. PMID- 1713672 TI - Effects of the seleno-organic substance Ebselen in two different models of acute pancreatitis. AB - This study evaluated the effects of the seleno-organic substance Ebselen [2 phenyl-1,2-benzisoselenazol-3(2H)-one] in two models of acute hemorrhagic and acute edematous pancreatitis. Ebselen is known to catalyze glutathione peroxidase like reactions and to inhibit lipid peroxidation. Hemorrhagic pancreatitis was induced by feeding a choline-deficient, ethionine-supplemented (CDE) diet to mice for 66 h. Edematous pancreatitis was induced by 7-h subcutaneous injections of 50 micrograms/kg of cerulein in mice. Ebselen was given from the beginning of the CDE diet either as a subcutaneous injection of 100 mg/kg at 6-h intervals or was mixed in with the CDE diet to yield a daily dose of 100 mg/kg of Ebselen. In further experiments, Ebselen was given at various time intervals after the beginning of the CDE diet as subcutaneous injections of 100 mg/kg at 6-h intervals. In the cerulein model, Ebselen was given 5 min prior to each cerulein injection at doses from 10-500 mg/kg. Prophylactic administration of Ebselen given orally or subcutaneously significantly improved survival from 38.5% in the control group of saline-injected CDE-fed mice to 61.9 and 65.0%, respectively. Ebselen also reduced increases in serum amylase and pancreatic weight in the diet model. Therapeutic administration of Ebselen significantly increased survival only when injections were started 20 h after the beginning of the CDE diet (64%), but not when started after 40 h (44%). Similarly, increases in serum amylase and pancreatic weight due to the CDE diet were significantly reduced by Ebselen only when injections were started after 20 h but not when started after 40 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713674 TI - Involvement of tubular complexes in pancreatic regeneration after acute necrohemorrhagic pancreatitis. AB - Localized acute necrohemorrhagic pancreatitis was induced in rats by multiple trypsin injections. Morphological alterations were monitored by light and electron microscopy until complete recovery. In the acute phase, typical pictures of focal acute necrohemorrhagic pancreatitis were observed. In the postacute phase, fibrosis and tubular complexes are characteristic of damaged areas. Tubular complexes appear from the dedifferentiation of acinar cells. They are characterized by duct-like cells bordering wide, empty luminae. In the recovery phase, cellular proliferation was accompanied by differentiation, with progressive acquisition of the morphological characteristics of acinar cells at the periphery of the tubular complexes. In that instance, cellular proliferation was concomitant with the development of collagen septa in tubular complexes. In these structures both duct-like and acinar-like cells presented mitoses. Cell division persisted in the dedifferentiated cells until tubular complexes disappeared. A very similar process was observed in the embryonic pancreas, where organized parenchyma originated from proliferation and differentiation of protodifferentiated cells. We concluded that pancreatic repair following necrohemorrhagic pancreatitis involves proliferation of cells from intact acini and from tubular complexes, at variance with edematous pancreatitis, where regeneration is exclusively due to acinar cell proliferation. PMID- 1713675 TI - Immunocytochemical identification of monoclonal antibodies with binding activity to acinar cells but not islets. AB - The development of techniques for separating islets from acinar cells on a mass scale is a prerequisite to successful clinical attempts at islet transplantation. We have identified and partially characterized two blood group-reactive monoclonal antibodies (McAb), CAC1 and CAC2, with specific binding activity to acinar cells but not islets. These McAb are of the IgM subclass, and were found on immunocytochemical analysis to possess broad interspecies cross-reactivity, binding to antigens expressed in the acinar tissue of rats, dogs, and man. The antigens that these McAb recognize are glycolipid in nature and thus were not denatured by collagenase digestion of the pancreas. These properties, together with their ability to effect complement-mediated lysis, may make the McAb generally useful reagents in islet isolation and purification. PMID- 1713676 TI - ERCP- and endoscopic sphincterotomy-induced pancreatitis. AB - Acute pancreatitis may occur after the performance of endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy. During ERCP and endoscopic sphincterotomy, the pancreas is subjected to many types of potential injury--mechanical, chemical, hydrostatic, enzymatic, microbiological, allergic, and thermal. These factors may act independently or in concert to induce postprocedure pancreatitis. The potential role of each etiologic factor in the development of ERCP- and endoscopic sphincterotomy-induced pancreatitis is detailed. The management of this complication is reviewed. Patient factors that increase the risk for pancreatitis and techniques to prevent or limit this complication are described. A variety of agents have been shown to prevent or treat pancreatitis in animal models, but extrapolation to humans has been almost uniformly unsuccessful. Although postprocedure pancreatitis is unlikely to be completely eliminated, careful patient selection and attention to detail may reduce the incidence of this untoward event. PMID- 1713677 TI - Cytotactin expression in somites after dorsal neural tube and neural crest ablation in chicken embryos. AB - The spatiotemporal expression of the extracellular matrix protein cytotactin/tenascin during somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of neural crest (NC) development. In the present study, the spatial distribution and molecular forms of cytotactin in somites were examined using in situ hybridization, Western blotting, and immunohistochemistry during normal development and after injury. In situ hybridization showed that prior to NC cell invasion cytotactin mRNA was restricted to the caudal half of the newly formed epithelial somites. As each epithelial somite matured, giving rise to a sclerotome and dermamyotome, the mRNA was first restricted to the dermamyotome and later restricted to the rostral protion of the sclerotome, consistent with the previously reported protein distribution. Immunocytochemical analysis of the distribution of cytotactin and NC cells in embryos with ablations that removed NC cells, or with simple wounds that left NC cells in place, demonstrated that the presence of NC cells is neither necessary nor sufficient for the correct positioning of cytotactin. Immunoblotting analysis showed that cytotactin synthesized by sclerotomes in the absence of NC cells was of similar molecular mass to that produced in their presence. These findings are in accord with the notion that the abnormalities of cytotactin distribution are related to the wounding process. We conclude that, contrary to the suggestion of Stern et al. [Stern, C. D., Norris, W. E., Bronner Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319], there is no causal link between the presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin. PMID- 1713678 TI - Vascular cell adhesion molecule 1 induces T-cell antigen receptor-dependent activation of CD4+T lymphocytes. AB - Effective stimulation of CD4+ T cells in an immune response depends on activation signals transduced via not only the CD3-T-cell receptor (TCR) complex but also those generated by accessory cell-surface proteins, including some that mediate adhesion between T cells and antigen-presenting cells (APC). Three members of the Ig superfamily, CD54 [intercellular cell adhesion molecule 1 (ICAM-1)], CD58 [lymphocyte function-associated antigen 3 (LFA-3)], and B7, expressed on the surface of APC, have been shown to mediate both adhesion and signaling during T cell-APC interactions. Recently another member of the Ig superfamily, [vascular cell adhesion molecule 1 (VCAM-1; INCAM110)], has been identified. VCAM-1 mediates adhesion between endothelial cells and activated lymphocytes and certain tumor cells. Here, using a soluble VCAM-1 fusion protein with receptor globulin (Rg), we examined the role of VCAM-1 in T-cell activation. We observed that CD4+ T cells, which are inefficiently stimulated by immobilized anti-TCR-1 or anti-CD3 monoclonal antibody (mAb) alone, can be induced to proliferate when exposed to immobilized VCAM-1-Rg in conjunction with either immobilized anti-TCR-1 or immobilized anti-CD3 mAb. The costimulatory effects of VCAM-1-Rg on CD4+T cells is inhibited by mAb to either the CD29 (integrin beta 1)-CD49d [very late activation antigen 4 alpha (VLA-4 alpha)] complex on the surface of CD4+ T cells or to VCAM-1. Stimulation of CD4+ T cells with immobilized VCAM-1-Rg and anti-TCR or -CD3 mAb results in the synthesis of both interleukin 2 (IL-2) receptors and IL-2. In addition, anti-CD25 (anti-IL-2 receptor a) mAb significantly inhibited the VCAM-1-Rg/anti-TCR or -CD3 mAb-driven activation of CD4+ T cells, indicating that endogenously produced IL-2 is in part responsible for the observed T-cell proliferation. Collectively, these results suggest that VCAM-1 can play an important costimulatory role during the activation of CD4+ T cells. PMID- 1713679 TI - A molecular blueprint for the pore-forming structure of voltage-gated calcium channels. AB - A protein that imitates the sequence of a highly conserved segment predicted to line the pore of dihydropyridine-sensitive L-type calcium channels was designed and synthesized. Single-channel conductance properties were studied in planar lipid bilayers. The synthetic protein emulates the ionic conductance, ionic selectivity, and pharmacological properties of the authentic calcium channel, including the stereospecific action of agonist and antagonist enantiomers of the dihydropyridine BayK 8644. The identified sequence is identical in L-type calcium channels from skeletal muscle and isoforms of cardiac muscle, brain, and aorta. It is plausible that this structural motif represents the molecular blueprint for the pore-forming structure of voltage-gated calcium channels. PMID- 1713680 TI - Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription. AB - Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor alpha (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), we demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5' flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-kappa B and AP-1. Gel mobility shift assays demonstrate that TNF, IL-1, or LPS (but not IL-2, IL-4, IL-6, interferon gamma, histamine, or transforming growth factor beta) induces activation of NF-kappa B-like DNA binding activity in HUVEC. In contrast, neither TNF, IL-1, nor LPS activates proteins that bind to an AP-1 consensus sequence under these experimental conditions. Phorbol 12-myristate 13 acetate, a known activator of protein kinase C (PKC), weakly induces NF-kappa B like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-kappa B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kappa B-like proteins but does inhibit ELAM-1 gene transcription. We conclude that PKC-independent activation of NF-kappa B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription. PMID- 1713681 TI - Exclusive expression of Epstein-Barr virus nuclear antigen 1 in Burkitt lymphoma arises from a third promoter, distinct from the promoters used in latently infected lymphocytes. AB - Epstein-Barr virus transformation of human B lymphocytes in vitro results in the expression of six viral nuclear antigens (EBNAs) and three viral membrane proteins. However, examination of viral gene expression in fresh Burkitt lymphoma isolates has revealed expression of only one of the nuclear antigens, EBNA-1. Previous transcriptional analyses of the EBNA-encoding genes demonstrated that all these genes are driven from one of two distal promoters located near the left end of the viral genome, raising the question of how exclusive expression of EBNA 1 occurs in Burkitt lymphoma tumors. Although most established Burkitt lymphoma cell lines (group 3) exhibit the full-expression pattern of viral antigens seen in lymphoblastoid cell lines, a few cell lines have been established that retain the restricted pattern of viral gene expression (group 1). In this paper we characterize transcription of the EBNA-1 gene in a group 1 Burkitt lymphoma cell line and show that (i) neither Cp nor Wp, the promoters involved in driving EBNA gene expression in lymphoblastoid cell lines, are active in this cell line; (ii) treatment of this cell line with 5-azacytidine, previously shown to induce expression of all EBNA genes, induced Cp and Wp activity; (iii) sizes of the EBNA 1 transcripts detected in two group 1 Burkitt lymphoma cell lines correlated with each other and were distinct from the size of the EBNA-1 transcript seen in lymphoblastoid cell lines; (iv) the EBNA-1 transcripts in the group 1 Burkitt lymphoma cell lines do not hybridize to a probe containing the common 5' exons present in all the EBNA transcripts from lymphoblastoid cell lines; and (v) anchored-PCR cloning the 5' region of the EBNA-1 transcript from one of the group 1 cell lines identified two exons, FQ and U, upstream of the EBNA-1 coding exon. The FQ exon lies just downstream of a TATAA box, which may represent the promoter for transcription of EBNA-1 in these cells. It is particularly noteworthy that an incomplete EBNA-1 cDNA clone from a nasopharyngeal carcinoma tumor line that expresses EBNA-1, but not the other EBNAs, has been characterized; this EBNA-1 transcript also contains the FQ/U splice junction, suggesting that the organization of exons upstream of the EBNA-1 coding exon is the same and that this organization may reflect a viral program for exclusive EBNA-1 expression. PMID- 1713682 TI - Platelet-derived growth factor (PDGF) and PDGF receptor are induced in mesangial proliferative nephritis in the rat. AB - We investigated whether platelet-derived growth factor (PDGF), or its receptor (PDGF-R), was upregulated in a rat model of mesangial proliferative glomerulonephritis. A marked increase in both PDGF A- and B-chain mRNA could be demonstrated in glomerular RNA by Northern blot analysis 3 and 5 days after disease induction, corresponding to the time of mesangial cell proliferation. PDGF-R beta-subunit mRNA and protein were also increased in glomeruli in mesangial proliferative nephritis, being maximal at day 5. The principal cells expressing PDGF B-chain appeared by immunostaining to be a subpopulation of mesangial cells; in contrast, the majority of the mesangial cells expressed the PDGF-R beta-subunit protein. Both complement depletion and platelet depletion significantly reduced cell proliferation and expression of both PDGF and PDGF-R. Thus, in mesangial proliferative nephritis there is a platelet- and complement mediated induction of PDGF A and B chain and PDGF-R beta-subunit gene transcription and protein synthesis. The finding that the majority of PDGF is produced by the mesangial cell supports the role of PDGF as an autocrine growth factor in glomerulonephritis. PMID- 1713683 TI - Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis. AB - The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called "delta Phe508," a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, we evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-delta Phe508 heterozygotes, and delta Phe508 homozygotes to determine if the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium, to what extent they are expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and delta Phe508 CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells at approximately 1-2 copies per cell, more than 100-fold greater than in pharyngeal epithelium. Importantly, allele-specific hybridization studies demonstrated that the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium in similar amounts. PMID- 1713684 TI - TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells. AB - An anti-human immunodeficiency virus (anti-HIV) protein capable of inhibiting HIV 1 infection and replication has been isolated and purified to homogeneity from Trichosanthes kirilowii. This protein, TAP 29 (Trichosanthes anti-HIV protein, 29 kDa), is distinct from trichosanthin [also known as GLQ 223 (26 kDa)] in size, N terminal amino acid sequence, and cytotoxicity. In addition to three conservative substitutions--namely, Arg-29 to Lys, Ile-37 to Val, and Pro-42 to Ser--a total difference of residues 12-16 was found. TAP 29 yielded -Lys-Lys-Lys-Val-Tyr-, whereas trichosanthin has -Ser-Ser-Tyr-Gly-Val-. Although the two proteins exhibit similar anti-HIV activity, as measured by syncytium formation, p24 expression, and HIV reverse transcriptase activity, they differ significantly in cytotoxicity, as measured by their effects on cellular DNA and protein syntheses. At the dose level of the bioassays, 0.34-340 nM, trichosanthin demonstrates a dose-dependent toxic effect on host cells. TAP 29 displays no toxic effect, even at 100 X ID50, whereas trichosanthin demonstrates 38% and 44% inhibition on cellular DNA and protein synthesis, respectively. These results indicate that the therapeutic index of TAP 29 is at least two orders of magnitude higher than that of trichosanthin. Thus TAP 29 may offer a broader safe dose range in the treatment of AIDS. PMID- 1713685 TI - Molecular identification of a major palmitoylated erythrocyte membrane protein containing the src homology 3 motif. AB - The complete amino acid sequence of a 55-kDa erythrocyte membrane protein was deduced from cDNA clones isolated from a human reticulocyte library. This protein, p55, is copurified during the isolation of dematin, an actin-bundling protein of the erythrocyte membrane cytoskeleton. Fractions enriched in p55 also contain protein kinase activity that completely abolishes the actin-bundling property of purified dematin in vitro. The predicted amino acid sequence of p55 does not contain any consensus sequence corresponding to the catalytic domains of protein kinases but does contain a conserved sequence found in the noncatalytic domains of oncogene-encoded tyrosine kinases. This conserved src homology 3 (SH 3) motif appears to suppress the tyrosine kinase activity of various oncoproteins and has also been found in several plasma membrane associated proteins involved in signal transduction. Northern blot analysis indicated that p55 mRNA was constitutively expressed during erythropoiesis and underwent 2-fold amplification after induction of K562 erythroleukemia cells toward the erythropoietic lineage. The abundant expression of p55 mRNA, along with protein 4.1 mRNA, was evident in terminally differentiated human reticulocytes. Although p55 has many features consistent with known peripheral membrane proteins, its tight association with the plasma membrane is reminiscent of an integral membrane protein. This fact may be partly explained by the observation that p55 is the most extensively palmitoylated protein of the erythrocyte membrane. PMID- 1713686 TI - Inhibition of N- and L-type Ca2+ channels by the spider venom toxin omega-Aga IIIA. AB - omega-Aga-IIIA, an 8.5-kDa peptide toxin isolated from the venom of Agelenopsis aperta, was found to be a highly potent inhibitor of Ca channels in cardiac muscle and in peripheral and central neurons of rats and frogs. Cardiac L-type Ca channels were completely (Kd approximately 0.6 nM) blocked by omega-Aga-IIIA. In sensory neurons, the toxin inhibited most high-threshold Ca current but not T type Ca current. omega-Aga-IIIA blocked with similar potency (Kd approximately 1.5 nM) both omega-conotoxin GVIA-sensitive and dihydropyridine-sensitive current components but left a fraction (approximately 35%) of high-threshold current that was also resistant to omega-conotoxin and dihydropyridines. The toxin blocks N- and L-type channels with equal potency and therefore may identify a high-affinity binding site common to these two Ca channel types. PMID- 1713687 TI - Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13. AB - The 12-kDa FK506-binding protein (FKBP-12) is a cytosolic receptor for the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and subcellular localization of a 13-kDa FKBP (FKBP-13), which has a 21-amino acid signal peptide and appears to be membrane-associated. Although no internal hydrophobic region, and thus no transmembrane domain, is apparent within the 120 amino acids of mature FKBP-13, a potential endoplasmic reticulum retention sequence (Arg-Thr-Glu-Leu) is found at its C terminus. FKBP-13 has 51% nucleotide sequence identity and 43% amino acid sequence identity to FKBP-12; the N-terminal sequences are divergent, but the 92-amino acid C-terminal sequence of FKBP-13 has 46 identical and 20 related residues when compared with FKBP-12. The conserved residues that comprise the drug binding site and rotamase active site of FKBP-12 are completely conserved in FKBP-13. Therefore, the three-dimensional structures of FKBP-12 and the FKBP-12/FK506 complex are likely to be excellent models of the corresponding FKBP-13 structure. PMID- 1713689 TI - Oxathiin carboxanilide, a potent inhibitor of human immunodeficiency virus reproduction. AB - Oxathiin carboxanilide (OC), NSC 615985, a compound originally synthesized as a potential fungicide, was demonstrated to be highly active in preventing human immunodeficiency virus (HIV)-induced cell killing and in inhibiting HIV reproduction. Virus-infected CD4+ lymphocytes were completely protected by 0.5 microM OC, whereas no toxicity was observed at concentrations below 50 microM OC. Production of infectious virus, viral p24 antigen, and virion reverse transcriptase were reduced by OC at concentrations that prevented viral cell killing. A variety of CD4+ T-cell lines were protected by OC from HIV cytopathicity, and OC inhibited two distinct strains of HIV-1. However, HIV-2 infections were unaffected by OC. OC had no direct effect on virions of HIV or on the enzymatic activities of HIV reverse transcriptase or HIV protease. Time limited treatments of cells with OC before, during, or after exposure of cells to virus failed to protect cells from the eventual cytopathic effects of HIV, and OC failed to inhibit the production of virus from cells in which infection was established or from chronically infected cells. We conclude that the highly active OC has a reversible effect on some early stage of HIV-1 reproduction and cytopathicity. Pilot in vivo experiments showed that circulating concentrations of OC exceeding 1 microM could be achieved and sustained in hamsters for at least a week with no remarkable toxicological sequelae. OC represents a new class of anti-HIV agents that are promising candidates for drug development. PMID- 1713688 TI - Stable transformation of Leptomonas seymouri by circular extrachromosomal elements. AB - To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri. The vector is relatively small (6 kilobase pairs) and contains a portion of the L. seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418. This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid. In L. seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon. Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells. PMID- 1713690 TI - Biological transport processes and space dimension. AB - We discuss the generic time behavior of reaction-diffusion processes capable of modeling various types of biological transport processes, such as ligand migration in proteins and gating fluctuations in ion channel proteins. The main observable in these two cases, the fraction of unbound ligands and the probability of finding the channel in the closed state, respectively, exhibits an algebraic t-1/2 decay at intermediate times, followed by an exponential cutoff. We provide a simple framework for understanding these observations and explain their ubiquity by showing that these qualitative results are independent of space dimension. We also derive an experimental criterion to distinguish between a one dimensional process and one whose effective dimension is higher. PMID- 1713691 TI - Drosophila Rrp1 protein: an apurinic endonuclease with homologous recombination activities. AB - A protein previously purified from Drosophila embryo extracts by a DNA strand transfer assay, Rrp1 (recombination repair protein 1), has an N-terminal 427 amino acid region unrelated to known proteins, and a 252-amino acid C-terminal region with sequence homology to two DNA repair nucleases, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A, which are known to be active as apurinic endonucleases and as double-stranded DNA 3' exonucleases. We demonstrate here that purified Rrp1 has apurinic endonuclease and double-stranded DNA 3' exonuclease, activities and carries out single-stranded DNA renaturation in a Mg(2+)-dependent manner. Strand transfer, 3' exonuclease, and single stranded DNA renaturation activities comigrate during column chromatography. The properties of Rrp1 suggest that it could promote homologous recombination at sites of DNA damage. PMID- 1713692 TI - Mutations in the D strand of the human CD4 V1 domain affect CD4 interactions with the human immunodeficiency virus envelope glycoprotein gp120 and HLA class II antigens similarly. AB - CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4. PMID- 1713693 TI - Pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity. AB - Derivatives of pyridinones were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevent the spread of HIV 1 infection in cell culture without an appreciable effect on other retroviral or cellular polymerases. 3-[( (4,7-Dimethyl-1,3-benzoxazol-2-yl) methyl]amino ]-5 ethyl-6-methylpyridin-2(1H)-one (L-697,639) and 3-[[ (4,7-dichloro-1,3-benzoxazol 2-yl) methyl]amino]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,661), two compounds within this series, had HIV-1 RT IC50 values in the range of 20-800 nM, depending upon the template-primer used. The most potent inhibition was obtained with rC.dG and dA.dT as template--primers. With rC.dG, reversible slow-binding non competitive inhibition was observed. [3H]L-697,639 bound preferentially to enzyme template-primer complexes. This binding was magnesium-dependent and saturable with a stoichiometry of 1 mol of [3H]L-697,639 per mol of RT heterodimer. Displacement of [3H]L-697,639 was seen with phosphonoformate. In human T-lymphoid cell culture, L-697,639 and L-697,661 inhibited the spread of HIV-1 infection by at least 95% at concentrations of 12-200 nM. Synergism between 3'-azido-3' deoxythymidine or dideoxyinosine and either of these compounds was also demonstrated in cell culture. Based upon their specificity for HIV-1 RT activity, template-primer dependence on potency and ability to displace [3H]L-697,639; a tetrahydroimidazo [4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione derivative R82150 and the dipyridodiazepinone BI-RG-587 appear to inhibit RT activity by the same mechanism as the pyridinones. PMID- 1713694 TI - Axon collaterals indicate broad intraspinal role for sacral preganglionic neurons. AB - The classic view of preganglionic neurons in spinal autonomic nuclei is that they convey information exclusively from the central nervous system to autonomic neurons in peripheral ganglia. The present morphological study in the cat sacral spinal cord demonstrates that these neurons may also make abundant synaptic connections within the spinal cord. Neurons labeled intracellularly with neurobiotin or horseradish peroxidase exhibited an expansive distribution of axon collaterals in spinal cord laminae I, V, VII, VIII, IX, X, and the ventrolateral funiculi. This broad-ranging axon-collateral system, which has the potential for multiple neuronal contacts, indicates widespread integrative functions for sacral preganglionic neurons within the spinal cord, in addition to functions currently known in the periphery. PMID- 1713696 TI - The assay of angiogenesis. PMID- 1713695 TI - Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4. AB - Polyclonal antibodies to the amino- or carboxyl-terminated peptide sequences of the GLUT4 transporter protein were used in immunoelectron microscopic studies to demonstrate the location and insulin-induced translocation of GLUT4 in intact isolated rat adipocytes. Labeling of untreated adipocytes with the amino-terminal antibody revealed 95% of GLUT4 was intracellular, associated with plasma membrane invaginations or vesicles contiguous with or within 75 nm of the cell membrane. Insulin treatment increased plasma membrane labeling approximately 13-fold, to 52% of the total transporters, and decreased intracellular labeling proportionately. In contrast, labeling of untreated adipocytes with the carboxyl terminal antibody or with a monoclonal antibody (1F8) that binds to the carboxyl terminus of GLUT4 detected fewer transporters, only approximately 40% of which were intracellular. In insulin-treated cells, plasma membrane labeling increased approximately 20-fold, but the total number of labeled transporters also increased approximately 13-fold. The number of intracellular transporters was not changed. The insulin-induced increase in plasma membrane labeling was reversible. Thus, the vast majority of GLUT4 transporters in untreated adipocytes are intracellular in invaginations or vesicles attached or close to the plasma membrane. Insulin treatment causes translocation of transporters to the plasma membrane, which involves flow of transporters from invaginations to the cell surface and possible fusion of subplasma membrane vesicles with the plasma membrane. Differences in the labeling of intracellular transporters by peptide antibodies suggested the carboxyl-terminal epitope of intracellular transporters was masked. The unmasking of the carboxyl terminus during translocation to the plasma membrane may be part of the mechanism by which insulin stimulates glucose transport in rat adipocytes. PMID- 1713697 TI - Interactions of growth factors in tissue repair. PMID- 1713698 TI - Effect of subchronic antidepressants administration on serotonin-stimulated phosphoinositide hydrolysis in para-chlorophenylalanine-treated rat hippocampal slices. AB - 1. This study examines the effect of subchronic parachlorophenylalanine (PCPA) treatment upon serotonin (5-HT)-stimulated inositol monophosphate (IP-1) accumulation in rat hippocampal slices and also the effect of antidepressants upon this 5-HT response in the hippocampus from rats treated with or without concurrent administration of PCPA. 2. For high dose PCPA treatment, animals were injected intraperitoneally with 300 mg/kg daily for the first 5 days and then 100 mg/kg for 5 days, while for low dose PCPA treatment animals were injected for 10 days at a dose of 100 mg/kg. Imipramine or iprindole (15 mg/kg i.p.) was given once daily for 10 consecutive days. 3. 10-Day treatment with high dose of PCPA resulted in a significant increase in 5-HT-stimulated IP-1 accumulation, whereas low dose of PCPA had no significant effect upon the 5-HT response as compared to vehicle. 5-HT-stimulated IP-1 accumulation in rat hippocampus was not affected by subchronic treatment with imipramine or iprindole. The enhancement of the 5-HT response induced by high dose of PCPA was not attenuated by repeated antidepressants treatment. PMID- 1713699 TI - Current status of cardiac surgery in childhood. AB - In the 50 years since Gross (1938) obliterated a patent ductus arteriosus, congenital cardiac surgery has come of age, synchronized with the world explosion in microtechnology and space age materials. The late 1960s and early 1970s saw Barratt-Boyes pioneering complete intracardiac repairs on infants with congenital heart disease employing modifications of the Kyoto technique (Shirotani) for profound hypothermia and circulatory arrest. The past 10-15 years have been marked by the more widespread dissemination of increasingly safe techniques, and the application of progressive incremental refinement to the entire management package of complex congenital heart disease. Many innovative methods and concepts have been added to the therapeutic armamentarium of the congenital heart team. Currently, transplantation adds the prospect of "second chance", and in the future may constitute preferred primary management in certain complex forms of congenital heart disease. In the Western world the concept of "frequency sensitivity" and the value of rationalizing congenital heart surgery facilities, such that a single unit manages a population of 8-12 million, is established, though not necessarily widely accepted and acted upon. High-volume, low-risk units emerge such that operative mortality, despite the high acceptance rate of complex problems and high rates of neonatal and infant complex repairs, has dropped below 5%. Paradoxically, the so-called simple closed surgery (neonatal coarctation, shunts and other palliative procedures in complex congenital heart disease) retain relatively high risk and must be regarded as one of the areas of challenge over the next 5-10 years. PMID- 1713701 TI - Characterization of oral sustained release preparations of iloprost in a pig model by plasma level monitoring. AB - Iloprost (5-[(E)-1S,5S,6R,7R)-7-hydroxy-6-[(E)-3S,4RS)-3-hydroxy-4- methyl-octen 6-inyl]-bicyclo[3.3.0]-oct-3-ylidene)-pentanoic acid) is a chemically stable PGI2 mimetic with high pharmacological potency. Therapeutic efficacy in various disease stages (e.g. peripheral arterial occlusive disease, M. Raynaud and thromboangiitis obliterans) was shown after repeated once-a-day infusion treatment over several weeks. In order to facilitate drug therapy an oral dosage form is desirable. As a first step, a suitable animal model was needed to screen several formulation variants prior to characterization of promising candidates in man. After intravenous infusion treatment, the pig exhibited - similar to man - strictly dose-dependent steady state plasma levels and a total iloprost clearance of approximately 26 ml/min/kg (man approximately 20 ml/min/kg). Partial similarity of physiology and anatomy of the GI-tract and the possibility to administer intact capsule dosage forms led to a series of screen experiments with several sustained release preparations (pellets and matrix tablets) of iloprost exhibiting different in-vitro drug release profiles. A good correlation of in vitro dissolution and in vivo plasma level data was obtained for all preparations containing the pellet neutral polymer. For the other formulations slight differences between duration of liberation and plasma level or time of maximum dissolution rate and tmax of plasma levels was observed. In the case of ionized polymers or matrix tablets, in vitro dissolution profiles were slightly different from in vivo data. This might be due to different dissolution behaviour in the gastro-intestinal tract. The pig seems to be a model that is suitable for verifying drug liberation profile in-vivo. Based upon plasma levels obtained in animals, selection of a formulation for characterization in man can be made. PMID- 1713700 TI - The mechanisms of action of progesterone and the anti-progestin ZK 98734 on PGF2 alpha synthesis by early human decidua. AB - The inhibition of prostaglandin (PG) synthesis found in early human decidua is antagonized by the anti-progestin, ZK 98734. This action of ZK 98734 is abolished by actinomycin, an inhibitor of protein synthesis and by the calcium channel blocker, verapamil. Calmidazolium, an antagonist of the intracellular calcium binding protein calmodulin was less effective in inhibiting the stimulation of PG synthesis induced by the anti-progestin. Chronic stimulation of protein kinase C activity by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a slight reduction of PG release and was antagonized by polymixin. These findings suggest that inhibition of PG synthesis in early pregnancy is caused by progesterone and that increased release of PGs induced by anti-progestins is dependent on new protein formation and requires extracellular calcium. PMID- 1713702 TI - Fish oil fatty acids and experimental arthritis. AB - Maintenance of mice on dietary regimens containing fish oil decreases severity of collagen-induced arthritis. Macrophages from fish oil fed animals had decreased omega-6 and significant amounts of omega-3 polyunsaturated fatty acids in membrane phospholipids and produced significantly less prostaglandins than macrophages from corn oil fed animals. Gender differences in both prostaglandin production and susceptibility to arthritis were noted. PMID- 1713703 TI - [Management of ocular problems connected with diabetes]. PMID- 1713704 TI - [Surgical nursing is meeting]. PMID- 1713705 TI - [Continuing education. 60. Subject: medicine-surgery. Topic: the cardiac surgery patient. The care that is performed]. PMID- 1713706 TI - Characterization of a human trypsin resistant to Kunitz soybean trypsin inhibitor. Studies of duodenal juices after tube instillation of raw soybean extract. AB - Human duodenal juices collected during tube instillation of raw soybean extract into the duodenum contained free trypsin and free Kuntiz soybean trypsin inhibitor (KTI) in the simultaneous presence of trypsin-KTI complexes. It has previously been suggested that this KTI-non-inhibitable trypsin has a general resistance to serine protease inhibitors. Four different trypsin forms have been found and partly characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing followed by Western immunoblotting or enzyme staining. In addition, crossed immunoelectrophoresis and affinity chromatography with antibody-coupled gels have been used for identification of free and inhibitor-complexed trypsin. PMID- 1713707 TI - Plasma levels of gastrointestinal regulatory peptides in patients receiving maintenance hemodialysis. AB - The fasting plasma levels of 9 gastrointestinal regulatory peptides were measured by radioimmunoassay in 13 stable patients with chronic renal failure receiving hemodialysis treatment regularly and compared with those of 10 healthy controls. The plasma concentrations of gastrin-releasing peptide, motilin, neurotensin, pancreatic polypeptide, peptide YY, somatostatin, substance P, and vasoactive intestinal peptide were increased. The plasma level of gastrin was not statistically different from that of the controls (p = 0.077). We conclude that patients with chronic renal failure receiving hemodialysis treatment regularly have increased concentrations of eight of nine measured gastrointestinal regulatory peptides. The elevated levels of gastrointestinal peptides in patients with chronic renal failure may contribute to uremic gastrointestinal symptoms and dysfunctions. It is necessary to make a renal function evaluation before interpreting measured plasma levels of gastrointestinal regulatory peptides. PMID- 1713708 TI - Patient and physician treatment delay in patients with stomach cancer in Norway: is it important? The Norwegian Stomach Cancer Trial. AB - The effect and consequences of treatment delay were studied in 1165 patients with stomach cancer included in a Norwegian multicentre study. Median patient delay was 42 days; median doctor delay 37 days; and median total treatment delay 107 days. By Cox proportional hazards model analyses we found that an increase in weight loss was associated with an increase in total delay, whereas a more advanced stage of disease was related to a short total delay. Physician delay was more pronounced in women, increased with increasing Karnofsky performance index, but decreased in patients with stage-IV disease. By logistic regression analyses we found no association between delays and postoperative complication rate. The relationship between physician delay and postoperative mortality was statistical significant, with increasing number of deaths with decreasing delay. In conclusion, there is no evidence that long treatment delay is an important negative factor in relation to outcome of surgery. PMID- 1713710 TI - Recognition of a cell-surface oligosaccharide of pathogenic Salmonella by an antibody Fab fragment. AB - The 2.05 angstrom (A) resolution crystal structure of a dodecasaccharide-Fab complex revealed an unusual carbohydrate recognition site, defined by aromatic amino acids and a structured water molecule, rather than the carboxylic acid and amide side chains and a structured water molecule, rather than the carboxylic acid and amide side chains that are features of transport and other carbohydrate binding proteins. A trisaccharide epitope of a branched bacterial lipopolysaccharide fills this hydrophobic pocket (8 A deep by 7 A wide) in an entropy-assisted association (association constant = 2.05 x 10(5) liters per mole, enthalpy = -20.5 +/- 1.7 kilojoules per mole, and temperature times entropy = +10.0 +/- 2.9 kilojoules per mole). The requirement for the complementarity of van der Waals surfaces and the requirements of saccharide-saccharide and protein saccharide hydrogen-bonding networks determine the antigen conformation adopted in the bound state. PMID- 1713709 TI - Inhibition of cholecystokinin-stimulated pancreaticobiliary output in man by the cholecystokinin receptor antagonist MK-329. AB - MK-329 (formerly L-364,718) is a new nonpeptide antagonist for the peripheral (type-A) cholecystokinin (CCK) receptor, which has proved effective in blocking the actions of both exogenous and endogenous CCK in several species. To evaluate the effect of MK-329 on CCK-stimulated pancreaticobiliary output in man, six normal subjects received 10 mg MK-329 or placebo orally in a randomized, crossover fashion, before a background intravenous infusion of secretin (5 pmol/kg/h) and two doses of CCK-8 (approximately 15 and 40 pmol/kg/h, each for 1 h). Gastric and duodenal juice were aspirated separately via two double-lumen tubes, with 51Cr-ethylene-diaminetetraacetic acid as a duodenal marker. After placebo treatment the background infusion of secretin produced maximum plasma concentrations of secretin similar to postprandial values, averaging about 5 pM. After placebo treatment the low dose CCK-8 infusion (15 pmol/kg/h) increased circulating CCK concentrations from basal levels of 1.8 +/- 0.2 pM to levels similar to those observed postprandially, averaging 9.2 +/- 1.3 pM, and the high dose of CCK-8 (40 pmol/kg/h) induced supraphysiologic levels of CCK, averaging 23.4 +/- 3.2 pM. Plasma concentrations of secretin and CCK were not significantly different during MK-329 treatment. As expected, infusion of CCK-8 at both doses stimulated pancreatic exocrine secretion and gallbladder contraction in placebo controls, as indicated by increases in the output of trypsin, amylase, bicarbonate, and bilirubin. Whereas MK-329 did not significantly reduce basal pancreatic secretion, the integrated incremental output of trypsin, amylase, and bicarbonate in response to stimulation with the low (physiologic) CCK dose was inhibited by 74% (p less than 0.01), 89% (NS), and 75% (p less than 0.05), respectively. Basal bilirubin output was virtually abolished after treatment with MK-329, and the response to the low dose of CCK was reduced by 98% (p less than 0.01), indicating almost complete inhibition of gallbladder contraction at physiologic circulating concentrations of CCK. It is concluded that MK-329 is an orally active antagonist of CCK-stimulated pancreaticobiliary output in man and could thus be utilized to explore the physiologic regulation of the exocrine pancreas and gallbladder by CCK. PMID- 1713711 TI - Surgical treatment of distant metastases in renal cell carcinoma. AB - Twenty-six patients with metastatic renal cell carcinoma have been submitted to metastatectomy between 1975 and 1986. The actuarial 5-year survival of the 23 patients resected for cure is 31%. The medium disease-free survival was 11 months for the 11 synchronous metastases and 26 months for the 12 metachronous metastases. Three patients with disseminated disease had palliative surgery: 2 for pathologic fractures and one for tracheal compression. All improved after surgery, and survived 7, 16 and 27 months. It is confirmed that surgical resection of solitary or single organ site distant metastases from renal cell carcinoma is justified. PMID- 1713712 TI - Surgical considerations in patients with cancer of the colon and rectum. PMID- 1713713 TI - [The value of tumor markers in germ cell tumors]. AB - We present a patient with a germ cell tumour in the region of the pineal gland and discuss the symptomatology and diagnostic procedures of tumours in this region, emphasizing the value of tumourmarker determinations in serum and cerebrospinal fluid. PMID- 1713714 TI - Physiology of mutations affecting learning and memory in Drosophila--the missing link between gene product and behavior. PMID- 1713715 TI - The changing scene of neurotrophic factors. AB - The purification of brain-derived neurotrophic factor (BDNF), the elucidation of its primary structure, and the subsequent identification of neurotrophin-3 (NT-3) ended the monopoly of NGF as the only well-characterized, target-derived neurotrophic molecule. NGF, BDNF and NT-3 are members of a gene family called neurotrophins. They have strictly conserved domains that determine their basic structure. However, they also have distinctly variable domains that determine their different neuronal specificity mediated by different high affinity receptors, and that share a common low affinity subunit. These similarities and dissimilarities between the members of the neurotrophin gene family are also reflected by their regional distribution, cellular localization and developmental regulation. In this article the neurotrophins are compared with ciliary neurotrophic factor (CNTF), which is a representative of the category of neurotrophic molecules that, according to their regional distribution, developmental expression and cellular localization, do not fulfil the criteria of a target-derived neurotrophic molecule. The physiological and pathophysiological functions of neurotrophins and CNTF are discussed in the context of their potential use for the treatment of traumatic and degenerative diseases of the peripheral and central nervous systems. PMID- 1713716 TI - Neuroscience in Yugoslavia. AB - This survey is a personal account of the present status of neuroscience in Yugoslavia within the context of recent upheavals in Eastern Europe. The current situation in Yugoslavia, characterized by the absence of a Federal Ministry of Science and a poor scientific communication between federal states (republics), does not allow a comprehensive overview of neuroscience at the federal level. Even more difficult is to envisage the prospects of Yugoslav neuroscience in the light of European integration. Several problems serve to illustrate the present situation concerning Yugoslav neuroscience. First, the weakness of the self organization of science in Yugoslavia during the past 20 years is still the most important denominator in the current trend of neuroscience. Second, different Yugoslav republics have significantly different systems of science funding and evaluation, which reflect very plainly different levels of democratic (and socioeconomic) changes that were attained during 1990. Third, due to the different numbers of trained scientists, facilities and equipment, funds and levels of international scientific cooperation there are major differences between republics in the tempo of progress towards real achievements in science. Finally, the present explosive development of neuroscience and the proclamation of the 'Decade of the Brain' will hopefully stimulate Yugoslav neuroscientists to seek better programmes of neuroscience research and to improve the extent and quality of international cooperation. PMID- 1713717 TI - The pathogenesis of demyelinating disease: insights from cell biology. AB - Cellular and humoral immune mechanisms have been implicated in the pathogenesis of human and experimental demyelinating diseases of the CNS. How these interact in the complex sequence of events that culminates in phagocytosis of myelin by macrophages has yet to be resolved. The relationship between leakage of the blood brain barrier and demyelination, the reason why recurrent inflammatory demyelination occurs--seemingly in the absence of an antigen-specific immune response--and the lack of effective remyelination all require explanation if a coherent account of immunologically mediated demyelination is to be achieved. One approach to these problems is to study in vitro the developmental and cellular biology of oligodendrocytes--the glial cells responsible for the synthesis and maintenance of CNS myelin. This provides experimental opportunities not offered by more direct investigation of the intact nervous system, but carries the clear disadvantage that observations made in vitro cannot necessarily be extrapolated to humans. PMID- 1713718 TI - Synaptic homeostasis and Parkinson's disease. PMID- 1713719 TI - The path forward in Hungarian neuroscience. PMID- 1713720 TI - Long-term survival of anucleate axons and its implications for nerve regeneration. AB - Severed distal segments of nerve axons (anucleate axons) have now been reported to survive for weeks to years in representative organisms from most phyla, including the vertebrates. Among invertebrates (especially crustaceans), such long-term survival might involve transfer of proteins from adjacent intact cells to anucleate axons. In lower vertebrates and mammals, long-term survival of anucleate axons is more likely attributed to a slow turnover of axonal proteins and/or a lack of phagocytosis by macrophages or other cell types. Invertebrate anucleate axons that exhibit long-term survival are often reactivated by neurites that have grown from proximal nucleate segments. In mammals, induction of long term survival in anucleate axons might allow more time to use artificial mechanisms to repair nerve axons by fusing the two severed halves with polyethylene glycol, a technique recently developed to fuse severed halves of myelinated axons in earthworms. PMID- 1713721 TI - Molecular characterization of microtubule-associated proteins tau and MAP2. AB - Tau and MAP2 are two of the major microtubule-associated proteins in the vertebrate nervous system. They promote microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. In nerve cells immunohistochemistry shows complementary distributions, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites. Each protein consists of multiple isoforms that contain three or four homologous tandem repeats near the carboxy-terminus, which constitute microtubule binding domains. In humans, tau consists of at least six isoforms of related amino acid sequences that are produced from a single gene by alternative mRNA splicing and that are expressed in a stage- and cell type-specific manner. Tau is also a component of the paired helical filaments associated with Alzheimer's disease and other disorders of the CNS. Rat MAP2 consists of at least three isoforms produced from a single gene: high molecular mass MAP2a and MAP2b, and low molecular mass MAP2c. MAP2c is expressed only during early development and has so far been seen only in axons; MAP2a appears to replace MAP2c, whereas MAP2b is expressed throughout life. Messenger RNAs for MAP2 of high molecular mass are expressed both in cell bodies and in dendrites, consistent with the dendritic localization of the corresponding protein isoforms. PMID- 1713722 TI - Song-learning behavior: the interface with neuroethology. AB - Behavioral studies of song learning in birds continue to raise new problems for neuroethological investigation. Evidence is emerging for a new form of vocal plasticity, called 'action-based learning'. Motor patterns are overproduced during a particular phase of ontogeny, and are then subjected to attrition and selective reinforcement by various kinds of social stimulation as the young bird matures. This form of learning, analogous to operant conditioning, can occur at phases of development when the more traditional form of 'memory-based learning' is no longer possible. There is evidence that different physiological mechanisms are involved in the development and the maintenance of mature singing, with a transition occurring at the time of song crystallization. Neural events associated with the developmental stabilization of motor patterns are worthy of more study. PMID- 1713723 TI - Reassessing the mechanisms and origins of vocal learning in birds. AB - The most widely accepted hypothesis of vocal imitation in birds pre-dates many recent studies on the behavior, anatomy, physiology and cell biology of this phenomenon. It states that vocal learning involves two steps: (1) an auditory memory is laid down, and then (2) vocal output is modified until the auditory feedback it generates matches the model. This black-box model of vocal imitation disregards circuitry. We now know that the brain pathways for vocal learning in birds include a series of well-defined nuclei and projections. Some of these nuclei and projections develop late in ontogeny, at the time when auditory models are first acquired and imitated. We also know that the pathways involved in song production respond to sound, an observation that blurs the demarcation between what is an auditory and what is a motor circuit. These and other recent discoveries call for a reassessment of the mechanisms and origins of vocal learning in birds and mammals. PMID- 1713724 TI - [Mechanism of the antimutagenic effect of interferon in human fibroblasts based on induced protein analysis]. AB - Proteins induced in human fibroblasts after treatment of some antimutagens (interferon, p-aminobenzoic acid, heating and vaccinia virus infection) were identified by means of polyacrylamide gel electrophoresis (10-15%) followed by fluorography of the gel. Influenza virus proteins (A/WSN/33) were used as markers to determine the molecular weights of the new proteins. The results obtained suggest that interferon, p-aminobenzoic acid, heating and vaccinia virus infection induced proteins with mol. weights of 24 and 18 kD except the protein with 76 kD observed only after heating insult. PMID- 1713725 TI - [Polyfunctional inhibitors of proteolytic enzymes in the therapy of patients with chronic purulent maxillary sinusitis]. AB - Using clinical and biochemical data, the efficacy of the topical application of kontrikal, a polyfunctional inhibitor of proteinases, for the therapy of chronic purulent sinusitis is discussed. The drug was found to reduce the activity of proteolytic enzymes in the purulent exudate from the maxillary sinus. As compared with the controls (27 patients), the treated group (38 patients) showed a rapid disappearance of clinical manifestations, shortening of the therapeutic period, increase of proteinase inhibitors and decrease of protein in the exudate. PMID- 1713726 TI - [Signet-ring cell cancer of the large intestine in a child]. PMID- 1713727 TI - The 1989 revision of the U.S. Standard Certificates and Reports. AB - This report examines the procedures followed in the 1989 revision of the U.S. Standard Certificates of Live Birth and Death; License and Certificate of Marriage; Certificate of Divorce, Dissolution of Marriage, or Annulment; and Reports of Fetal Death and Induced Termination of Pregnancy. It outlines the history and basic principles of the standard certificates and reports and describes the principal additions, modifications, and deletions of items. In addition, it discusses changes in the format of the standard certificates and reports as well as the implementation of the new certificates and reporting forms. PMID- 1713728 TI - [Mechanisms of selective inhibition of pancreatic secretion after increasing the enzymatic activity of duodenal contents]. AB - In acute experiments on dogs it has been established that intraduodenal administration of trypsin, amylase and lipase induces selective inhibition of the introduced enzyme secretion by the pancreas. Atropine injection (0.2 mg/kg, intravenously, 4 times/h) removes the inhibitory effect of the introduced enzymes, the most pronounced atropine action being recorded in respect to trypsin. Intraduodenal trypsin administration decreases non-selective inhibitory effect of atropine on the stimulated secretion of the pancreas. It has been concluded that not only duodenal regulatory peptides but also the M-cholinergic mechanism participate in the selective inhibition. PMID- 1713729 TI - Iloprost and peripheral arterial disease in diabetic patients. PMID- 1713730 TI - [Current status of immunotherapy in oncology]. AB - Immunotherapy has evolved considerably since Ehrlich's concept of antigeneicity of tumour cells put forward at the beginning of the century. Whereas unspecific immunostimulatory interventions have yielded inconsistent and partly unreliable data, recently developed recombinant cytokines acting as biological response modifiers have delivered promising results. Such substances have been used as monotherapy, but also in combination with cytostatic drugs. An additional effort should be made to further define the standardization and the indication of the use of a specific cytokine for a specific malignant disorder. PMID- 1713731 TI - Tenascin in tissue perturbation repair. AB - Tenascin is an extracellular matrix glycoprotein consisting of six disulfide linked subunits with molecular masses of 190-250 kDa. The cDNAs of chicken, mouse and human tenascins were cloned and their amino acid sequences were determined. Molecular analysis of the tenascin gene revealed that it contains a region homologous to the fibrinogen gene, and repetitive sequences of the type III fibronectin and epidermal growth factor genes. Several isoforms of the tenascin gene as splicing variants have also been found. Culture studies have shown that tenascin has multiple functions including cell attachment and detachment, promotion and inhibition of neural crest cell migration, cell growth stimulation and hemagglutination. Immunohistochemistry of a variety of tissues, both normal and abnormal, from various animals has shown that the distribution of tenascin is characteristic, and spatially and chronologically restricted. Immunoreactive tenascin was demonstrated in the dense mesenchyme present around growing epithelia during embryogenesis and oncogenesis. Besides its oncofetal expression, tenascin was also found in many tissues with inflammation such as healing wounds, regenerating tissue and irritated tissue. These findings suggest that tenascin probably functions as a homeostatic factor in the repair of tissue perturbation. PMID- 1713732 TI - Malignant meningioma with cartilage and giant cells. AB - An autopsy case of recurrent and malignant meningioma is reported. This case was originally typical benign transitional meningioma of the falx, however, the histology of the tumor changed to show malignant features during successive recurrences. At autopsy, the tumor revealed findings consistent with malignant meningioma. One of the most interesting features was the presence of cartilage and giant cells in some parts. Immunohistochemistry showed positive immunoreactivity for S-100 protein in some cartilage and giant cells and for cytokeratin in some giant cells. Multidifferential potential of the meningioma cells was suggested in this case. PMID- 1713733 TI - Alpha-fetoprotein-producing urachal adenocarcinoma. AB - A 45-year-old Japanese male with a history of macroscopic hematuria for more than 6 months presented multiple metastatic lesions in the lungs. Cystoscopic examination demonstrated a large tumor mass protruding from the dome of the urinary bladder. Ultrasonography and CT highlighted a solid and cystic urachal tumor continuous from the vesical dome to the navel. Serum levels of alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) were elevated to 17,100 ng/ml and 17.7 ng/ml, respectively. He underwent palliative curettage of the vesical dome tumor twice, followed by chemotherapy with little effect. One year after admission, he died of progressive metastases to the lungs, left pleura, liver and brain. Final serum levels of AFP and CEA were 86,200 ng/ml and 60.9 ng/ml, respectively. The tumor was histologically classified as adenocarcinoma with a medullary growth pattern. Both papillotubular and solid (hepatoid) components were observed. The cancer cells were rich in glycogen and were immunoreactive diffusely for AFP and focally for CEA. CA15-3, CA19-9, epithelial membrane antigen and cytokeratin were also positive. In addition, argyrophilic cancer cells with immunoreactivities of neuron-specific enolase, chromagranin A and peptide YY were demonstrated. To our knowledge, this is the first reported case of AFP-producing adenocarcinoma of urachal origin. PMID- 1713734 TI - Effect of dantrolene on histamine release from rat peritoneal mast cells. AB - Dantrolene strongly and dose-dependently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE. Dantrolene inhibited Ca(2+) mobilization from intracellular Ca(2+)-store as well as histamine release in mast cells activated by anti-IgE, the effect on both these phenomena being closely correlated. These results suggested that the effect of dantrolene on histamine release from rat mast cell might be due to the inhibition of Ca(2+)-release from intracellular Ca(2+)-store. PMID- 1713735 TI - Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1). AB - Rapid intravenous administration of the glycopeptide antibiotic, vancomycin, may cause a hypotensive reaction which can usually be prevented by infusing vancomycin in dilute solutions. The release of histamine from circulating cells such as basophils and tissue mast cells has been implicated in hypotensive reactions since the effects can be prevented by antihistamine pretreatment. The direct effects of vancomycin on histamine release were therefore investigated in rat peritoneal mast cells and rat leukemic basophils (RBL-1 cells). Suspension cultures of mast cells or RBL-1 cells were exposed to vancomycin for 30-60 minutes at concentrations comparable to those infused clinically (2.28 or 4.56 mg/ml). Vancomycin induced a time- and dose-dependent release of histamine into the culture media from both cell types. The reference degranulating agent, Compound 48/80 (CP 48/80), was also shown to induce histamine release from mast cells and RBL-1 cells. Mast cells were significantly more sensitive to vancomycin and CP 48/80 than RBL-1 cells and, unlike RBL-1 cells, were responsive to the inhibitory effects of cromolyn sodium on histamine release. Cromolyn sodium did not inhibit vancomycin-induced histamine release in RBL-1 or mast cells. Morphologically, mast cells exposed to either vancomycin or CP 48/80 exhibited dose-related degranulation. On the other hand, treatment-related degranulation effects of either vancomycin or CP 48/80 on RBL-1 cells could not be reliably distinguished from controls by qualitative evaluation. Based upon these findings it is concluded that mast cells may represent a more useful model to evaluate the potential of investigational agents to release histamine and to study mechanisms of histamine release than RBL-1 cells. PMID- 1713736 TI - A toxic substance from the sea urchin Toxopneustes pileolus induces histamine release from rat peritoneal mast cells. AB - A toxic substance (P-II fraction), fractionated from the pedicellariae of the sea urchin Toxopneustes pileolus, dose-dependently caused the histamine release from rat peritoneal mast cells. The histamine release induced by P-II fraction increased with time, while compound 48/80 caused a more rapid histamine release. The dose-response curve for P-II fraction was studied with concentration 0.03-2.0 mg/ml. This reaction was dependent on Ca2+ and temperature. When glucose (5.5 mM) was omitted during the incubation step, the histamine release induced by P-II fraction was significantly reduced as compared to that of compound 48/80. Pyruvate reversed this reduction. On the other hand, the histamine release induced by P-II fraction was effectively potentiated by the addition of glucose (11.0 mM), but not that by compound 48/80. These results suggest that P-II fraction-induced histamine release differs from that of compound 48/80 disregards to the effects of glucose, because this histamine release appears to be more sensitive to the glycolytic pathway than compound 48/80-induced histamine release. PMID- 1713737 TI - Effect of the carboxylic ionophore monensin on histamine release from rat peritoneal mast cells. AB - The effect of the monovalent carboxylic ionophore monensin, which mediates a one for-one exchange of intracellular H+ for extracellular Na+, was investigated in purified rat peritoneal mast cells. Monensin inhibited histamine secretion induced by compound 48/80, adriamycin and the calcium ionophore A23187; the inhibitory effect was maximal when the compound was added at least 10 min before the secretagogues. Washing of cells before addition of the secretagogues did not abolish the inhibitory effect of monensin. On the contrary the carboxylic ionophore was completely ineffective in preventing concanavalin A-induced histamine release. When rat peritoneal mast cells were incubated in the presence of monensin for longer period (up to 5 hours), the substance induced a slow, progressive and dose dependent histamine release, which, at least for lower doses was noncytotoxic. The secretory effect of monensin was still present if the ionophore was washed away after 10 min of incubation, and the incubation continued in drug-free medium. Monensin stimulated histamine secretion was strictly dependent on extracellular Na+ concentrations, and independent on extracellular Ca++. PMID- 1713738 TI - Larynx preservation using induction chemotherapy plus radiation therapy as an alternative to laryngectomy in advanced head and neck cancer. A long-term follow up report. AB - Since 1977, we have used induction chemotherapy (CT) plus radiation therapy (RT) with curative intent in 35 advanced head and neck cancer (Ca) patients who otherwise would have required total laryngectomy. Fourteen patients had advanced Ca of the larynx or supraglottic larynx (SGL); 21 patients had Ca of the hypopharynx. In six patients the Ca was Stage III; in 26 patients it was Stage IV. Three patients had Stage II disease--2 with cancer of the pyriform sinus and one patient with Stage II SGL Ca who refused surgery. Chemotherapy consisted of platinum (P) + bleomycin in 18 patients until 1982, then P + fluorouracil in the next 17 patients. Total response rate was 77%--complete (CR) in 26% and partial (PR) in 51%. There were two toxic deaths. Surgery was limited to tracheostomy in 4 patients prior to CT and to radical neck dissection after CT in 4 others. Two patients required salvage laryngectomy at 11 and 31 months, respectively. One patient underwent partial laryngectomy with voice preservation. Thirty-two patients were evaluable for overall response after RT. Final disease-free status was achieved in 20/34. One long-term survivor was lost to follow-up (44 months) and 8 patients remained alive at 13+ to 109+ months. Median failure-free survival for all patients was no less than 24 months. Not counting 4 early deaths free of disease, 2-year local control using only chemotherapy plus radiation was 52% (16:31). Overall, 33 of 35 patients retained their voices. Sixteen patients (46%) have survived 2 years or longer. Survival of patients who achieved CR after induction chemotherapy was 48 months versus 14 months for those with less than a CR (p = 0.001). Patients with a hypopharyngeal primary had only a 33% 2-year local control rate with chemotherapy and radiation and a median survival of only 12 months versus 77% control and a minimum 39-month survival for those whose tumor arose in the larynx (p = 0.009). Induction chemotherapy plus radiation therapy is an effective strategy which can produce a high rate of larynx preservation, local control, and long-term survival in patients with advanced cancer of the larynx. Patients with hypopharyngeal primaries have a lesser rate of long-term survival and local control, despite similar overall response rates. PMID- 1713739 TI - The transient appearance of small blastoid cells in the marrow after bone marrow transplantation. AB - Of 14 patients who underwent allogeneic or syngeneic bone marrow transplantation, 6 had a transient appearance of small blastoid cells in the bone marrow after transplantation. Most of these patients (11) had leukemia, although 3 had severe aplastic anemia. The cells were 8-18 micron in diameter and had scant cytoplasm and dense nuclei with smooth, homogeneous chromatin. They often had distinct nuclear clefts. These cells constituted 4.0-21.3% of the total number of bone marrow cells. They were not reactive with peroxidase, alpha-naphtyl butylate esterase, naphthol AS-D chloroacetate esterase, or periodic acid-Schiff stains. Immunocytochemical analysis revealed that the small blastoid cells expressed terminal deoxynucleotidyl transferase, Ia-like, CD19, and CD10 antigens and cytoplasmic mu heavy chains, indicating a precursor B-cell phenotype. CD20 antigen was not expressed on these cells. The data suggest that cytoplasmic mu may be expressed earlier than CD20 antigen in the differentiation of B-cell lineage. The morphologic, cytochemical, and immunophenotypic characteristics did not distinguish these nonneoplastic cells distinctly from leukemic lymphoblastic cells. The increase of small blastoid cells was a transient and self-limited phenomenon, in contrast to that of neoplastic blasts. These cells should be recognized as a common component of the bone marrow of marrow transplant recipients. The significance and role of these cells in immune recovery and hematopoiesis remain uncertain. PMID- 1713740 TI - Potential use of monoclonal antibodies in the diagnostic distinction of gynecomastia from breast carcinoma in men. AB - Immunohistochemical (IHC) assays using the monoclonal antibodies (MoAbs) B72.3 and B6.2, recognizing two distinct and independently expressed breast tumor associated antigens (BTAAs), recently have been shown to significantly improve the accuracy of cytodiagnosis of breast nodules by fine-needle aspiration (FNA). To evaluate whether the same method may be useful diagnostically in distinguishing gynecomastia from breast cancer in men, a retrospective avidin biotin immunoperoxidase assay study was performed on 50 cases of gynecomastia and 30 cases of breast carcinoma in men, using a panel of five MoAbs known to recognize different BTAAs. The results of this study demonstrated that MoAbs B1.1, HMFG2, and MBr1 displayed a strong reactivity with gynecomastia and carcinoma, but MoAbs B72.3 and B6.2 separated benign and malignant lesions in a high percentage of cases. When used in combination, the latter two reagents reacted with 96% of the carcinomas that were analyzed but labeled only 67% of gynecomastia cases. Thus, the conjoint use of these two reagents may enhance the use of FNA biopsy as a valuable tool in the presurgical diagnosis of breast nodules in men. PMID- 1713741 TI - Expression of c-erbB-2 oncoprotein in mammary and extramammary Paget's disease. AB - Formalin-fixed, paraffin-embedded tissue sections from 45 patients with mammary and extramammary Paget's disease were stained immunohistochemically with the use of a polyclonal antiserum directed against a 14-amino acid segment of the c-erbB 2 oncoprotein. Positive membrane staining, which correlates with gene amplification, was found in 15 of 19 cases (79%) of mammary Paget's disease, 4 of 13 cases (31%) of vulvar Paget's disease, none of 8 cases of scrotal Paget's disease, and none of 5 cases of perianal Paget's disease. Of the 19 patients with mammary Paget's disease, specimens of underlying breast tissue were available from 14; all contained a concurrent ductal adenocarcinoma. Concordance of c-erbB 2 antigen staining between the underlying breast carcinoma and the pagetoid component was observed in 12 cases. Of the 13 patients with vulvar Paget's disease, 2 had superficial stromal invasion, and 3 had underlying, deeply invasive adenocarcinomas. One superficially invasive case was positive for c-erbB 2 expression. One additional case of vulvar Paget's disease had an associated primary pagetoid endocervical adenocarcinoma that spread into the endometrium; both the endocervical and vulvar components stained positively for the c-erbB-2 antigen. The results of this study indicate that the c-erbB-2 oncoprotein may play a role in the pathogenesis of extramammary Paget's disease. These results also suggest that the c-erbB-2 oncoprotein may function in vivo to promote intraepithelial spread of adenocarcinoma cells. PMID- 1713742 TI - Cell-surface ganglioside GD2 in the immunohistochemical detection and differential diagnosis of neuroblastoma. AB - The expression of the disialoganglioside GD2 was analyzed in 67 solid tumors and normal tissues from children by using the GD2-specific murine monoclonal antibody 3A7 and the indirect immunoperoxidase method. GD2 was expressed in all of 28 neuroblastomas and was most abundant in stroma-poor tumors. In differentiating stroma-rich neuroblastomas, neuroblastic clusters, neurofibrils, and most ganglion-like cells were positive, whereas Schwann's-cell stroma did not express GD2. In ganglioneuromas, only a few ganglion-like cells showed GD2, whereas all other structures were negative. Scattered foci of ganglioside GD2 also were found in some non-neuronal tumors, such as rhabdomyosarcomas and osteosarcomas, but not in lymphomas, Askin tumors, or most Wilms' tumors. The monoclonal antibody 3A7 is a useful aid in the immunohistochemical diagnosis of neuroblastoma. In addition, the intense cell surface staining of neuroblastoma cells by this reagent makes it potentially useful for detecting residual neuroblastoma in bone marrow samples and lymph node biopsies. PMID- 1713743 TI - Mycobacteria on Wright's-stained smears. PMID- 1713744 TI - Beneficial effects of isovolemic hemodilution using a perfluorocarbon emulsion in a stroke model. AB - In a clinically applicable cat stroke model, 16 purpose-bred adult animals were used to evaluate the beneficial effects of two treatment regimens: isovolemic hemodilution with either a perfluorocarbon emulsion or dextran 40 (a glucose polymer). Animals that received these treatment regimens were then compared with a control group of untreated animals. Focal cerebral infarctions were produced by transorbital ligation of the left middle cerebral artery. The randomly allocated treatment arms of the study were instituted 3 hours after ligation of the middle cerebral artery, thereby simulating a human clinical situation. In vivo mitochondrial metabolic activity of the peri-infarct cerebral tissue was continually assessed by means of a multiwavelength near-infrared spectrophotometer. This allowed measurement of cellular oxygenation at the cytochrome aa3 level, the terminal member of the cytochrome chain. Sequential proton-based magnetic resonance imaging was used to measure intracerebral water in vivo. Cardiac output, oxygen consumption/delivery, chemical, histologic, and rheologic parameters were also assessed. The data collected were analyzed by group means and standard statistical analyses, which revealed that the group treated with the perfluorocarbon emulsion had both less brain edema in the early post-infarct period (p less than 0.05), as well as a higher level of oxidation of cytochrome aa3 (p less than or equal to 0.025). This evidence supports the premise that isovolemic hemodilution with an oxygen-carrying hemodiluent may be beneficial in the treatment of ischemic strokes. PMID- 1713745 TI - Diagnostic and therapeutic strategies of white clot syndrome. AB - This study describes our experience with 12 patients with white clot syndrome encountered during a recent 36-month period. The diagnosis was based on the following criteria: (1) development of thrombocytopenia of less than 100,000/mm3 during administration of heparin therapy, (2) normalization of the platelet count after an interruption in heparin therapy, (3) exclusion of other causes of thrombocytopenia, (4) a positive heparin-induced platelet aggregation test, (5) detection of white clots on pathologic examination, and (6) the presence of thrombotic complications. Of 2,500 patients who received heparin therapy, 12 (0.48%) developed white clot syndrome. Various indications, routes of administration, and types of heparin were implicated. The mean platelet nadir was 26,900/mm3, and the mean time to onset of heparin-induced thrombocytopenia was 5 days. Thrombotic complications included arterial occlusions of the legs in 11 patients, deep vein thrombosis of the legs in 9 patients (4 had pulmonary embolism), and combined arterial and venous thrombosis in 8 patients. Treatment strategies included discontinuation of heparin in all patients and intravenous infusion of dextran, followed by arterial thrombectomy in four patients, urokinase therapy in two patients for arterial complications, and insertion of Greenfield filters in six patients. All patients were given warfarin. The mortality rate was 25% and the morbidity rate was 50%. An initial platelet count should be obtained on all patients prior to receiving heparin, followed by repeat platelet counts every 2 to 3 days. Once thrombocytopenia or thrombosis is diagnosed, heparin should be discontinued and other methods of therapy considered. PMID- 1713747 TI - [Value of early amniocentesis for prenatal diagnosis in the first trimester of pregnancy]. AB - For the prenatal diagnosis of the fetal status, amniocentesis was performed in 9 12-week pregnancy in 31 females at risk for birth of a baby with chromosomal abnormalities and congenital malformations of the central nervous system. There were no difficulties in carrying out the procedure. A balanced translocation bearing female was found to have a fetal chromosomal abnormality. Her pregnancy was interrupted at the 11th week; the prenatal diagnosis was evidenced by cytogenetic examination of the abortion specimen. The amniotic fluid alpha fetoprotein estimated by radioimmunoassay ranged from 15-18 to 550-620 ng/ml. The findings suggest that early amniocentesis may be useful in the prenatal diagnosis of the fetal status and further evidence should be accumulated. PMID- 1713746 TI - [An immunochemical analysis of the function of the hemato-encephalic barrier in acute fetal hypoxia and asphyxia neonatorum]. AB - The neurospecific protein alpha 1-globulin has been assayed in serum in order to evaluate the blood-brain barrier in newborns with acute intrapartum hypoxia. The study involved 35 term newborns with birth asphyxia of variable severity. The alpha 1-globulin levels correlated with severity of condition at birth, duration of intrauterine exposure to hypoxia and the presence of obstetric complications and clinical severity of cerebral circulatory disorders. A normal early adaptation and effective therapy reduced serum alpha 1-globulin levels 4-8-fold on the 3rd postnatal day and 6-16-fold on the 5th day. Deterioration of neurological symptoms was parallelled by a significant increase in protein levels (to 6400 ng/ml) at day 5. This evidence may confirm the fact that permeability of the blood-brain barrier is impaired by intrapartum hypoxia. PMID- 1713748 TI - Detecting proteins containing 3,4-dihydroxyphenylalanine by silver staining of polyacrylamide gels. AB - Proteins in which some or all of the tyrosine side chains are post translationally modified to dihydroxyphenylalanine have been found in several invertebrate phyla. In this paper we describe the unusual silver-staining properties of these 3,4-dihydroxyphenylalanine (Dopa)-proteins in silver-stained polyacrylamide gels. Our evidence suggests that the rapid silver staining of these proteins is due to the 3,4-dihydroxyphenol ring which is a highly effective reducing agent in the alkaline development conditions used in the final step of most silver-staining procedures. Normal proteins comprising the standard 20 amino acids and tyrosine on its own, do not reduce silver under these conditions. Pretreatment of the gels with acid-dichromate solutions abrogates the rapid staining of the Dopa-proteins. This rapid silver-staining technique will facilitate the rapid screening of many additional organisms for Dopa-proteins using sodium dodecyl sulfate gels and small amounts of tissue. PMID- 1713749 TI - Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol. AB - We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid. PMID- 1713750 TI - Simultaneous determination of intracellular calcium concentration and histamine secretion in rat basophilic leukemia cells (RBL-2H3). AB - In rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mast cells, the aggregation of IgE receptors initiates increase in the intracellular concentration of calcium ([Ca2+]i), monitored with the fluorescent Ca probe fura 2, and finally results in histamine secretion. In cell suspensions, however, the fluorescence gradually increases due to leakage and exocytosis of the dye. A superfusion system was developed to overcome these problems and [Ca2+]i was calculated from the ratio of fluorescence intensities at 505 nm of fura-2 excited at 340 and 380 nm. Histamine and beta-N-acetylglucosaminidase in granules are released during exocytosis, and both substances in the superfusates were determined simultaneously. This system is useful for studies on the relationships of cell stimulation, changes in second messengers, and final responses. PMID- 1713751 TI - Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus. AB - The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool. PMID- 1713752 TI - Reconstruction of the basement membrane in a cultured submandibular gland. AB - In a rat submandibular rudiment on day 16, both laminin (LM) and type IV collagen (Col-IV) were found in all cases to colocalize not only in the basement membrane, but also in the rough endoplasmic reticulum of the epithelial cells, indicating that the synthesis of the components of basement membrane is greatly enhanced at this particular stage of extensive branch formation. Using the submandibular gland from a 16-day embryo, the model system was developed to determine the structural organization of the basement membrane. The pre-existing basement membrane was digested with collagenase and dispase, causing its complete disappearance. The subsequent gradual reconstruction of an authentic basement membrane was confirmed by electron microscopy and immunohistochemistry of LM and Col-IV. In the model system, this recovery started at 4 h of culture, and formation was complete by 8 h. During the recovery, thick bundles of actin filaments appeared transitionally in the basal cytoplasm. Electron microscopic analysis indicated two precursor structures, aggregated fuzzy fibers (type 1 extracellular matrix (ECM)) and 10-nm-thick strand piles (type 2 ECM), and an authentic basement membrane structure appeared during the course of membrane reconstruction. LM and Col-IV were always located together in these three structures. These observations clearly indicate that the precursors, containing LM, Col-IV and most likely heparan sulfate proteoglycan, appeared to form immediately following their secretion into the extracellular space, and assembled into the rigid structure of basement membrane within 8 h. The ultrastructural and immunohistochemical process of basement membrane reconstruction appeared to coincide closely with that of the glomerular basement membrane in developing kidney.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713753 TI - A multicentre controlled clinical trial of high-volume fresh frozen plasma therapy in prognostically severe acute pancreatitis. AB - Fresh frozen plasma (FFP) has been proposed as a specific therapy for acute pancreatitis. It may replenish important circulating proteins, particularly the naturally occurring anti-protease system. To investigate this potential therapy, 72 patients with predicted severe disease were selected from 301 admissions with acute pancreatitis using the modified Glasgow prognostic scoring system. They were randomised within 6 h of diagnosis to receive FFP (8 units daily for 3 days) or a similar volume of colloid control as part of their intravenous fluid therapy. Clinical progress was monitored and specific blood proteins were measured on days 1, 3 and 7. FFP therapy significantly increased the day 3 concentrations of some of the acute phase proteins (C1-reactive protein P less than 0.02, D-dimer P less than 0.05 and fibrinogen P less than 0.05) as well as some proteins which showed a fall in circulating concentration during the early stages of the disease (alpha 2 macroglobulin P less than 0.001, antithrombin III P less than 0.01 and fibronectin P less than 0.001). However, there was no significant difference between the two groups in terms of clinical outcome. Mortality was 20% in patients who received FFP and 18% in the colloid control group. Despite the ability of FFP therapy to supplement circulating concentrations of several potentially useful proteins during acute pancreatitis, it does not appear to improve clinical outcome. PMID- 1713754 TI - Surgery offers the best palliation for carcinoma of the pancreas. AB - This debate discusses the palliative management of pancreatic cancer. The arguments in favour of surgical palliation are that this approach allows all symptoms to be treated or prevented, the diagnosis can be confirmed histologically and a final assessment of resectability can be made. The arguments against the use of surgery are that survival is short and that effective alternative therapies are available: endoscopic intubation, percutaneous coeliac plexus block and pancreatic enzyme supplements. The most appropriate policy, however, is to tailor the management plan to suit the individual patient. PMID- 1713755 TI - Improvement of outcome for infants of birth weight under 1000 g. The Victorian Infant Collaborative Study Group. AB - The two year outcome of extremely low birth-weight (ELBW) infants (birth weight 500 to 999 g), born in the state of Victoria over two distinct eras, 1979-80 and 1985-7, were compared. In the 1979-80 era, 25.4% of the ELBW infants survived to 2 years of age; only 12.5% of liveborn ELBW infants survived to 2 years with no neurological disabilities. In the 1979-80 era, ELBW infants born outside the level III centres in the state were significantly disadvantaged in both mortality and neurological morbidity. By 1985-7, the two year survival rate of ELBW infants rose significantly from 25.4% to 37.9%. By 1985-7, the proportion of ELBW infants who survived to 2 years free of neurological disabilities increased from 12.5% to 26.2%. Despite the improved survival, the absolute number of 2 year old children survivors with severe neurological disabilities remained constant at 8/year in both eras. By 1985-7, fewer ELBW infants were born outside the level III centres, their survival rate remained lower, but the severe neurological disability rate in survivors was no longer significantly higher. There has been a concomitant improvement in both survival and reduction in neurological morbidity. PMID- 1713756 TI - Role of basic fibroblast growth factor in revascularization of rabbit tracheal autografts. AB - Despite omentopexy of the bronchial anastomosis, donor airway ischemia remains a problem after lung transplantation. This study examined the hypothesis that surface abrasion and topical application of basic fibroblast growth factor (bFGF) would enhance omental revascularization of trachea in a rabbit heterotopic autograft model. Tracheal segments were excised, primary tracheal anastomoses performed, and the segments placed in the peritoneal cavity wrapped in omentum. Animals were randomized to one of six groups according to tracheal segment treatment: control, surgical abrasion, Surgicel wrap with topical bFGF, Surgicel wrap with bFGF vehicle, Gelfoam wrap with bFGF, and topical bFGF alone. One week later, animals were heparinized, perfused with Aquablak dye, and killed. Tracheal segments were excised and sectioned for light microscopic quantitative assessment of viability and dye perfusion. There was no significant improvement in viability or perfusion between abraded tracheal segments or segments treated with bFGF/Gelfoam or bFGF alone when compared with control segments. Airways wrapped in Surgicel had significantly greater ischemic injury compared with the control group, regardless of bFGF application. Neither surgical abrasion nor topical bFGF increased omental revascularization of transplanted tracheal segments after 7 days. PMID- 1713757 TI - Palliative reconstruction of right ventricular outflow tract in tetralogy. PMID- 1713758 TI - Relationship of factor XIIIa-positive dermal dendrocytes to Kaposi's sarcoma. AB - The histogenesis of Kaposi's sarcoma (KS) has been the subject of controversy, much of which has centered around whether the spindle cells of KS are derived from vascular endothelium or from lymphatics. Recently, some investigators have speculated that the spindle cells of KS are derived from dermal dendrocytes, a population of mononuclear dendritic cells normally present in the papillary and upper reticular dermis. These cells have been shown to proliferate in response to a variety of stimuli and have been reported to express the plasma proenzyme factor XIIIa. We examined immunohistochemically sections fixed in formaldehyde solution and embedded in paraffin from 20 tumor-stage, 15 patch-stage, and 15 plaque-stage lesions of KS with antibodies directed against factor XIIIa, factor VIII-related antigen, Ulex europaeus lectin, and LN3 (anti-HLA-DR) to investigate the relationship of dermal dendrocytes to KS in general and to try to clarify the histogenesis of this tumor. Our results revealed that the dermis of patch- and plaque-stage KS lesions contains an increased number of factor XIIIa-positive dermal dendrocytes compared with normal dermis and that some of these cells are spindle shaped. Many of the spindle cells in patch- and plaque-stage lesions of KS, however, are negative for factor XIIIa. The cells lining the slitlike spaces and some spindle-shaped cells in close proximity to the vascular spaces stain for factor VIII-related antigen and for Ulex europaeus lectin. LN3 labeled many cells resembling macrophages within the lesions and in papillary dermis. Less than 25% of the dendritic cells within the lesions and in the adjacent dermis expressed both factor XIIIa and LN3. Tumor-stage lesions showed focal but unequivocal staining of the spindle cells for factor VIII-related antigen and Ulex europaeus lectin. Tumor spindle cells were negative for factor XIIIa. Factor XIIIa-positive dendrocytes were plentiful in the uninvolved dermis and were aggregated around the periphery of the tumor nodules. The expression of factor VIII-related antigen and Ulex europaeus lectin by the spindle cells of nodular KS, and their lack of expression of factor XIIIa, suggests that the spindle-shaped tumor cells in all stages of KS are derived from endothelial cells and not from dermal dendrocytes. Dermal dendrocytes appear to undergo hyperplasia in response to KS of all stages. In patch- and early plaque-stage KS lesions, dermal dendrocytes are near factor VIII-related antigen-positive spindle cells and tumor vessels. The mechanism reactive dermal dendrocyte hyperplasia in KS remains obscure. PMID- 1713759 TI - Histologic observations and P-glycoprotein expression in gastric and esophageal adenocarcinomas treated with preoperative chemotherapy. AB - To describe histologic changes associated with chemotherapy response, we reviewed biopsy and resection specimens from 52 patients with locally advanced esophageal or gastric adenocarcinoma who were treated with preoperative chemotherapy, followed by resection. P-Glycoprotein expression in the adenocarcinomas was also determined with the use of antibody C219. Significant changes in morphologic appearance of the tumor between prechemotherapy and postchemotherapy samples was noted in 17 tumors (32.7%). The most frequent changes observed in these 17 tumors were a decrease in tumor cellularity and an increase in dense fibrosis in comparison with the prechemotherapy specimen. In signet-ring cell carcinomas, the intracytoplasmic mucin vacuoles were often smaller after chemotherapy, making tumor cells more difficult to identify. Another finding observed in tumors that showed histologic alteration after chemotherapy was the formation of large mucin pools that contained lymphocytes and macrophages. The remaining 35 tumors showed similar histologic features in prechemotherapy and postchemotherapy specimens. P Glycoprotein was identified in 15 (29.4%) of 51 specimens after chemotherapy. P Glycoprotein content of the residual tumors did not correlate with stage, degree of differentiation, or clinically determined chemotherapy response. We concluded that chemotherapy-induced changes in morphology were frequent in patients with upper gastrointestinal tract adenocarcinomas treated with preoperative chemotherapy. These changes should be recognized as they may cause difficulties in both gross and histologic evaluation of the extent of tumor in postchemotherapy resection specimens. The response of adenocarcinomas to this chemotherapy protocol does not appear to be linked to P-glycoprotein expression. PMID- 1713760 TI - HMB-45-positive malignant lymphoma. A case report with literature review of aberrant HMB-45 reactivity. AB - The monoclonal antibody HMB-45 is a highly specific and sensitive marker of malignant melanoma. Rarely, however, aberrant reactivity to nonmelanocytic tumors has been observed. We report an immunoblastic lymphoma of B-cell phenotype that reacted with HMB-45 in a 70-year-old white woman. To our knowledge, this is the first report of HMB-45-positive malignant lymphoma (excluding plasmacytoma). Of an additional 13 cases of benign and malignant lymphoid tissue, only benign plasma cells in one case reacted with HMB-45. PMID- 1713761 TI - Interferon detection in patients with neuroinfections: possibilities of clinical use. PMID- 1713762 TI - [Infiltrations in gonarthrosis, a therapeutic turning point: the use of a proteinase inhibitor]. AB - On the basis of evidence of the considerable damage caused by proteolytic enzymes in cases of degenerative arthritis, the authors propose the intra-articular introduction of an inhibitor of these enzymes (aprotinine). Both the immediate and long-term results of this therapy are very promising from a clinical standpoint. This drug not only acted on the symptoms of pain and stiffness, but also blocked the degeneration and consequent cell damage, restoring physiological homeostasis. Considering its high tolerability and absence of side effects, this drug represents a significant turning point in the treatment of degenerative joint disease, with results that are strikingly superior to those achieved with commonly used infiltrative drugs. PMID- 1713763 TI - Riboflavin carrier protein from carp (C. carpio) eggs: comparison with avian riboflavin carrier protein. AB - A protein exhibiting immunological cross-reactivity with the chicken egg-white riboflavin carrier protein was detected by radioimmunoassay in the eggs and serum of the fresh water fish Cyprinus carpio and subsequently purified to homogeneity by use of affinity chromatography. Fish riboflavin carrier protein resembled chicken riboflavin carrier protein with respect to most of its physicochemical characteristics. The major epitopes of chicken riboflavin carrier protein were shown to be conserved in the fish protein as probed with monoclonal antibodies to the avian vitamin carrier. PMID- 1713764 TI - High sensitivity to 5-azacytidine in LEC rats, a strain with a metabolic predisposition to hepatitis and hepatoma: possible involvement of DNA methylation in the expression of cytochrome P-450 and gamma-glutamyl transpeptidase. AB - The 5-methylcytosine (5-mCyt) content in hepatic DNA of LEC rats was measured in order to know the mechanism by which changes in the cytochrome P-450 content and gamma-glutamyl transpeptidase activity occur. At the age of 10 or 16 weeks, there was no difference in the extent of DNA methylation as compared with that of control strain (LEA) rats. However, in the hepatoma tissues that developed later in LEC animals, the percentage of 5-mCyt in the liver of LEC rats was markedly reduced. A single i.p. dose of 5-azacytidine brought about a significant reduction of 5-mCyt content with a concomitant decrease of cytochrome P-450 and an increase in gamma-glutamyl transpeptidase activity in LEC rats, whereas no such changes occurred in the control LEA rats. These results suggest that LEC rats are highly sensitive to 5-azacytidine and that a reduction in hepatic DNA methylation may play some role in the predisposition of the rats to hepatitis or hepatoma. PMID- 1713765 TI - [Phosphorylated derivitaves of All-Z and 3-demethyl-tri-TRANS, di-cis- hexaprenols as substrates for O-antigen polysaccharide biosynthesis in Salmonella anatum]. AB - Phosphates of all-Z- and 3-demethyl-tri-trans, di-cis-hexaprenols have been prepared and studied as substrates for enzymes of the Salmonella anatum O specific polysaccharide biosynthesis. Methyl group in alpha-isoprenic unit proved to be essential for the enzyme-substrate interaction, whereas the presence of E isoprenic units near the omega-end of the polyprenol is not significant. PMID- 1713766 TI - A murine monoclonal antibody that recognizes a genus-specific epitope in the Salmonella lipopolysaccharide outer core. AB - A murine monoclonal antibody 105 made from spleen cells of a mouse immunized with a mixture of common Salmonella serotypes reacted specifically with salmonellae from the most frequently encountered O serogroups of A (O:2) to E (O:3), and with strains from the less common O serogroups that represent the subspecies I, II, IIIb, IV, V and VI. Specificity for Salmonella was demonstrated by the lack of reactivity of monoclonal antibody 105 with any of the 30 other different species of Gram-positive and Gram-negative bacteria tested including 16 species in the family of Enterobacteriaceae. Studies to elucidate its binding epitope have shown that it reacts with the three distal sugar residues joined through specific anomeric linkages as present only in the Salmonella lipopolysaccharide outer core, which explains its specificity for the Salmonella. The failure of monoclonal antibody 105 to react with a subspecies IIIa Salmonella suggested a different outer core structure in this strain of Salmonella and also that monoclonal antibodies to the outer core of Salmonella lipopolysaccharide should be useful in the molecular analysis of their diversity. PMID- 1713767 TI - Binding of cloned S-fimbriated E. coli to human buccal epithelial cells- different inhibition of binding by neonatal saliva and adult saliva. AB - Investigations were carried out on the adhesion of cloned S-fimbriated E. coli, labelled with fluoresceinisothiocyanate (FITC) to human buccal epithelial cells. Fluorescence microscopic analysis revealed binding of bacteria to 75-95% of epithelial cells. Inhibition experiments with fetuin, alpha 1-acid glycoprotein and N-acetyl neuraminic acid confirmed the specificity of bacterial binding to sialoglycoproteins. Further studies using saliva as an inhibitor resulted in a 4 5 times stronger binding inhibition by newborn saliva in comparison to adult saliva coinciding with a 4-5 times higher content of total N-acetyl neuraminic acid in samples of newborn saliva. In Western blot analysis sialoglycoprotein bands with a molecular weight greater than 200 kD reacting with wheat germ agglutinin (WGA), were only identified in samples of newborn saliva. These bands are classified as mucins on account of molecular weight and staining. These data suggest that saliva mucins could represent a major defense mechanism against bacterial infections at a stage of ontogeny where the secretory IgA-system is not yet developed. PMID- 1713768 TI - Monoclonal antibodies to Legionella pneumophila serogroup 6: evidence of antigenic diversity. AB - Reactivity of monoclonal antibodies (Mabs) prepared against the type strain of Legionella pneumophila subsp. pneumophila serogroup 6 (Lp sg 6) was tested in the indirect immunofluorescence test using 16 environmental isolates of this sg and 58 strains of sg 1 to 5 and 7 to 14. Five out of 11 Mabs 5 were serogroup specific, i.e. there was a reaction with all sg 6 strains tested, but not with strains from other sg. Further 4 Mabs reacted with all sg 6 strains and a few strains of other sg. Two Mabs were only reactive with the type strain of Lp sg 6 and one sg 6 strain isolated in Bratislava, Czechoslovakia. This report shows further evidence that Lp sg 6 can be divided into antigenically distinct subtypes. PMID- 1713769 TI - [Androgen-like and anabolic action of Antheraea pernyi Guerin-Meneville Pas]. AB - It has been found that the ethyl acetate extract isolated from Antheraea pernyi Pas is able to increase the weight of prostate-semina and levator ani muscle bulbocavarnosus muscle of castrated mice. In addition, the extract also accelerates the growth of younger male mice and enhances the contents of RNA, DNA and protein in the liver tissue of mice. It has been determined that beta ecdysone is one of the effective constituents for the androgen-like and anabolic action. PMID- 1713770 TI - Correlation of DNA ploidy levels with altered cytokeratin patterns in rat bladder tumors. AB - Flow cytometry analysis of primary bladder carcinoma cells is appropriate for demonstrating the association between the presence of aneuploid cells and increased malignant potential for transitional cell carcinoma (TCC). Our laboratory previously has reported a specific alteration in the cytokeratin (CK) pattern of experimentally induced bladder carcinomas in rats. Unique immunohistochemically detectable changes in CK expression were the loss of CK 13 expression in invasive TCC cells and the loss of CK 19 expression in induced TCC. We now report the correlation of these changes in CK expression with cellular aneuploidy of the induced bladder carcinomas. Bladder carcinomas were induced in rats by direct instillation of N-methyl-N-nitrosourea. Immunohistochemistry was performed on the induced tumors using four commercially available monoclonal antibodies specific for CK 18 (TR1031), CK 13 (K8.12), CK 19 (K4.62), and the CKs 5, 7, and 8 (K8.13). Flow cytometry data from the induced bladder carcinomas was analyzed and the DNA index, proliferative index, and percent aneuploid cells were calculated for each time point. The percent aneuploid cells and CK 19 staining were tested statistically and were shown to be negatively correlated. We therefore hypothesize that the combination of the loss of CK 19 as detected by the antibody K4.62 coupled with the presence of aneuploid cells in histopathologically diagnosed invasive TCC is a significant factor in predicting the prognosis for any given diagnosis. PMID- 1713771 TI - cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues. AB - A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme. PMID- 1713772 TI - Structural and functional studies of full-length vascular cell adhesion molecule 1: internal duplication and homology to several adhesion proteins. AB - Full-length vascular cell adhesion molecule-1 (VCAM-1) cDNA cloned by polymerase chain reaction (PCR) of poly(A)+RNA from interleukin-1 (IL-1)-activated human umbilical vein endothelial cells (HUVEC) contained an insert of 276 nucleotides after position 1,034 of the previously published sequence. Synthetic oligomer probes, specific for each of the two possible species of VCAM-1 mRNA, detected only the longer form of VCAM-1 by Northern analysis of activated endothelial cell mRNA. This full-length VCAM-1 contains two internally repeated domains of approximately 273 amino acids with a high degree of homology. This new sequence information reveals homologies with additional members of the immunoglobulin superfamily and improves ALIGN scores for previously cited adhesion proteins. Removal of the transmembrane domain and the carboxy-terminal end of the full length VCAM-1 molecule allows the molecule to be secreted into the culture medium from cells transfected with an expression vector containing the corresponding VCAM-1 cDNA. PMID- 1713773 TI - Molecular cloning of the rhesus glycoprotein hormone alpha-subunit gene. AB - A rhesus monkey genomic library was screened with a cDNA for the glycoprotein hormone alpha-subunit. Genomic clones hybridizing with exon-specific probes were selected and the DNA sequences were determined for 1.6 kb of 5'-flanking DNA, all four exons, the second and third introns, all exon-intron junctions, and 357 bp of 3'-flanking DNA. Comparison with the 236 bp of 5'-flanking sequence data available for the human alpha gene indicates an overall homology of 95%. Primer extension analysis of rhesus placental and pituitary mRNA demonstrated that transcription initiation is identical to that in the human placenta. The rhesus gene contains an element nearly identical (21/22 bases) to the placental tissue specific element described for the human alpha gene. The rhesus gene has only one copy of the cAMP-response element (CRE), which is present as a direct repeat in the human gene. The rhesus CRE contains the consensus core sequence TGACG-TCA with the cytosine in the fourth position that is essential for placental expression of the human gene. The 5'-flanking region also has elements highly homologous to the consensus estrogen and progesterone/glucocorticoid response elements, as well as thyrotrope-specific and Pit-1-like binding sites described in rodent genes. The nucleotide sequence of four exons (predicted mRNA) have an aggregate homology of 92.7% with the human sequence. However, a 12-bp insertion to the second exon results in the addition of 4 amino acids to the amino-terminal end of the protein; these are homologous with the proteins of nonprimates but are lacking in the human alpha-subunit. The amino acid sequence of the deduced protein was slightly more homologous with the bovine than the human protein (91.6% vs. 89.6%). Thus, the rhesus glycoprotein alpha-subunit gene codes for a protein whose structure somewhat more closely resembles that of lower species, but the 5'-flanking DNA of the gene has gained the elements necessary for transcription in the placental syncytiotrophoblast which distinguishes the primate placenta from the other species examined. PMID- 1713774 TI - B and T cell epitopes of glycoprotein D of herpes simplex virus type 1. PMID- 1713775 TI - Detection of common polysaccharide antigen of Pseudomonas aeruginosa with O antiserum to Pseudomonas cerasi. AB - The identity of the structures of common polysaccharide antigen (CPA) of Pseudomonas aeruginosa and O-antigen of Pseudomonas cerasi was used for immunochemical study of polysaccharide antigens of seven immunotypes (IT) of P. aeruginosa. ELISA performed with O-antiserum to P. cerasi showed that CPA is present in all seven ITs in different amounts. In SDS-PAGE this antigen behaves as a lipopolysaccharide (LPS) and is detected by immunoblotting technique in five of seven ITs. PMID- 1713776 TI - The use of synthetic O-antigens of Salmonella for improvement of serological diagnosis of enteric infections. AB - Synthetic Salmonella O-antigens of a new copolymeric type, unlike natural antigens (lipopolysaccharides), exhibit monospecificity for groupspecific factors 0 : 3, 0 : 4, and 0 : 9 of Salmonella serogroups E, B, and D, and are, by 1-2 orders of magnitude, more sensitive in double immunodiffusion and passive hemagglutination inhibition tests. The use of the synthetic antigens for detection of antibodies in patients' sera by means of passive hemagglutination increased the specificity of this test considerably, thereby improving immunodiagnosis of salmonellosis. PMID- 1713777 TI - A peptide library expressed in yeast reveals new major epitopes from human immunodeficiency virus type 1. AB - In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA, providing a complete set of peptides derived from HIV-1. A library (4 x 10(4) clones) was first constructed in a new expression/secretion vector, using as inserts small fragments of HIV-1 DNA (50-150 bp) generated by random DNAse I cleavage. This peptide library, expressed in the yeast Saccharomyces cerevisiae, was screened with sera of HIV-1 infected individuals and human and murine anti HIV-1 monoclonal antibodies. Plasmids from immunoreactive colonies were recovered and the sequences of the HIV-1 derived inserts were determined. By using human sera, we have detected classical HIV-1 epitopes and identified two novel major epitopes, which may be used to improve diagnostic tests, localized in the p24 core protein and in the endonuclease. In addition, four minor epitopes were also defined by screening the library with monoclonal antibodies: in the protease, in the p17 core protein, in gp120 and near the C-terminal of gp41. This method is general and can be used for any protein from which a cloned cDNA is available. PMID- 1713778 TI - Grey platelet syndrome: evidence for alpha-granule localization of the platelet plasminogen activator inhibitor-1 pool. AB - The case of an 11-year-old boy with grey platelet syndrome is described. Platelets had the typical grey and ghostly appearance on May-Grunwald/Giemsa staining, caused by the absence of alpha granules confirmed by electron microscopy. Alpha granule protein content, i.e., beta-thromboglobulin and platelet factor 4, was less than 3% of normal and alpha granule secretion in response to thrombin was not detectable photometrically. The plasminogen activator inhibitor-1 pool in the patient's platelets was 5% of normal, confirming previous indirect evidence for the storage of this protein within the alpha-granule. Dense body secretion of adenosine triphosphate and 5 hydroxytryptamine was normal. Aggregation occurred normally in response to adenosine diphosphate and there was a slight delay in response to collagen. PMID- 1713779 TI - Use of photographs and audiovisual aids in office practice. AB - To insure complete and proper patient education, the use of audiovisual aids in every day practice is on the rise. It allows for a clear and concise convenience of information about the disease processes, use of medications, and treatment options. Transfer of information in this way improves compliance, clarifies expectations, and enhances treatment results. PMID- 1713780 TI - Teaching new acne patients with a customized slide sound program. AB - Automated teaching methods permit reliable dissemination of important information to patients. With the professional help of an audiovisual department, a physician can get information to patients in a bright, amusing, and reproducible manner. At the same time, the physician is free to perform other tasks. PMID- 1713781 TI - Standardization of the Feulgen reaction for absorption DNA image cytometry: a review. AB - The present paper gives a review of the current potentials and problems of a standardized Feulgen reaction for absorption DNA image cytometry. The cytochemical basis of the Feulgen reaction is described in the first part of this review. Subsequently, several preparatory factors which influence the performance of the Feulgen reaction, such as fixation, acid hydrolysis, composition of the Feulgen reagent and, in histology, embedding, are discussed in more detail. Some user-oriented recommendations for a standard Feulgen technique are given. PMID- 1713782 TI - Analysis of skin grafting techniques in the fetal rabbit. AB - The experimental model reported here was developed initially to examine the possibility of in utero coverage of congenital soft tissue defects using several types of reconstructive techniques. To pursue this, full-thickness skin grafts, pedicle flaps, and skin "islands" were fashioned on the backs of fetal rabbits; equivalent adult control wounds were also created. While all pedicle flaps and skin islands remained viable, none of the full-thickness grafts survived in the fetus. All adult control flaps, skin islands, and skin grafts were viable. Angiogenesis is crucial to full-thickness skin graft survival. These observations suggest that the death of full-thickness fetal skin grafts may be related to a failure of neovascularization in the graft bed. Further analysis using this model may help elucidate the factors involved in fetal angiogenesis. Additionally, this model may permit testing of putative angiogenic factors applied under a full thickness skin graft; graft survival offers an easy, objective, and quantifiable means of data analysis. PMID- 1713783 TI - Evaluation of three techniques to demonstrate retrograde transport of horseradish peroxidase. AB - Three methods of tracing neural connections by the use of horseradish peroxidase (HRP) were evaluated: (1) endoneurium injection, (2) injection followed by crushing at site of injection, and (3) nerve transection followed by capping of the proximal stump with a silicone cylinder containing HRP. The capping technique resulted in increased uptake and more uniform distribution of HRP in the nerves under study. PMID- 1713784 TI - The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids. AB - The exposure of [3H]arachidonate-radiolabelled murine peritoneal macrophages to alpha 2-macroglobulin-methylamine or alpha 2-macroglobulin-trypsin but not native alpha 2-macroglobulin (alpha 2M) results in the rapid secretion of [3H]eicosanoids. Resident peritoneal macrophages stimulated with 0.1 microM alpha 2M-methylamine exhibited an enhanced secretion within 10 min. The ability of alpha 2M 'fast' forms to stimulate secretion of [3H]eicosanoids was similar to that observed in the presence of the murine macrophage chemoattractant platelet activating factor. As observed for total [3H]eicosanoid secretion, alpha 2M 'fast' forms also rapidly enhanced the secretion of the cAMP-elevating prostanoid, prostaglandin E2, from resident peritoneal macrophages. Stimulated secretion of prostaglandin E2 in response to 0.1 microM alpha 2M-methylamine was less rapid than that observed using 0.1 microM platelet-activating factor. Similar amounts of secreted prostaglandin E2 were present in media of macrophage cultures after 1 h exposure to the two stimuli. In the presence of 0.1 microM alpha 2M-methylamine, secreted prostaglandin E2 remained elevated, compared to the appropriate buffer control, for at least 24 h. The present results indicate that receptor recognition of alpha 2M 'fast' forms by macrophages results in the rapid stimulation of eicosanoid secretion and suggest that secretion of prostaglandin E2 and other eicosanoids may be involved in the ability of alpha 2 M 'fast' forms to regulate various macrophage functional responses. PMID- 1713785 TI - Direct evidence for methylation of arginine residues in high molecular weight forms of basic fibroblast growth factor. AB - Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. Protein sequence analysis of bFGF isolated from tissue sources initially established that it is composed of 146 amino acids (apparent Mr 18,000). More recently larger apparent molecular weight forms have been identified and partially characterized. In addition, these high molecular weight forms (apparent Mr 22,000 and 25,000) have been shown to localize preferentially to nuclear fractions of transfected cells. In this report we demonstrate that the higher molecular weight, amino terminally extended forms of bFGF contain methylated arginine residues. The demonstration is based on 1) amino acid sequence analysis of a protein known to contain methylated arginine (myelin basic protein) and a comparison with amino acid sequence analysis of trypsin-derived fragments of the high molecular weight bFGF purified from guinea pig brain and 2) the ability to label in vivo the high molecular weight forms of bFGF with S-adenosyl-L-(methyl 3H)-methionine, the substrate of arginine-protein methylase I. These results are suggestive of a role of arginine methylation in directing nuclear localization of certain forms of bFGF. PMID- 1713786 TI - [Gram-negative bacilli in sputum]. PMID- 1713787 TI - [Comparative study of 3 methods for detecting bacteriuria]. PMID- 1713788 TI - Physico-chemical analysis of resorcin-fuchsin reagents and its relevance to staining quality of elastic fibres. AB - Three Weigert's resorcin-fuchsin reagents for elastic fibres were prepared by using different lots of basic fucsin and by preparing the staining solutions with ferric chloride or ferric perchlorate. The solutions were analyzed by spectrophotometry, thin layer chromatography (TLC) and spectrofluorimetry. Their ability to stain elastic fibres was tested on several human tissue. The strongest stain and faintest background were obtained with the fuchsin-chloride reagent, which showed the most significant reduction of the absorption at 575 nm by spectrofluorimetry, associated with an increased absorption at 510 nm with respect to the other stains. The results showed that physico-chemical analysis can represent a reliable, although empirical, way to predict the usefulness of a resorcin-fuchsin reagent for histological work. PMID- 1713789 TI - Negative staining and genesis of D-periodicity in native collagen fibrils. AB - An investigation was carried out on the mechanism which gives rise to the banding exhibited by collagen fibrils after negative staining. The negative staining (phosphotungstic acid) band patterns of native collagen fibrils (type I), isolated from calf reticular dermis, were compared with computer-drawn band patterns. The stimulations were based on the primary structure of bovine type I collagen, the "quarter stagger" molecular packing and different conformations of telopeptides. The results suggest that in negative staining, the stain exclusion effect depends on both spatial factors and water repelling factors being the "bulkiness" (molecular volume/length ratio) as well as the hydrophobicity of the amino acids of alpha 1 (I) and alpha 2 (I) chains directly involved. No final conclusion could be drawn about the contribution of positive staining to negative staining. Improvements in the simulations were achieved when the telopeptides were shaped according to particular conformational models. PMID- 1713790 TI - Copper tetrapyridino 'phthalocyanin' (Cuprolinic blue) differs in shape from the palladium and platinum analogues, and this affects staining of polynucleotides. AB - Palladium and platinum homologues of the copper-containing tetrapyridinotetraazaporphin cationic dye Cuprolinic blue were prepared. The affinities of all three dyes for a series of polyanions, with and without nucleotide bases, were assessed using the CEC (critical electrolyte concentration) approach, and expressed as molarities of MgCl2. Whereas Cuprolinic blue bound to RNA with much greater avidity than to DNA, the Pt and Pd analogues showed very high affinity for both. Patterns of affinities for non-nucleotide polyanions showed no differences. 1H and 13C NMR spectra showed that, although the Cu compound was planar, the Pt and Pd homologues must be non-planar. Taken together with previous findings, on e.g. methyl green and pyronin, these results suggest that DNA finds it easier to accommodate pairs of out-of-plane aromatic rings than RNA in stable complexes. CEC analyses can, as demonstrated here, uncover quite subtle differences between molecular recognition patterns of very closely related reagents. PMID- 1713791 TI - Electrophoretic studies on serum proteins in cows with traumatic pericarditis. AB - Diagnostic significance of electrophoretic findings of serum protein in cows with traumatic pericarditis was evaluated. Affected cows were classified into 3 groups according to autoptical findings: fibrinous, sero-fibrinous, and purulent types. Slight hypoprotenemia, moderate hypo-albuminemia, slight hyper-alpha globulinemia and tendency of hyper-beta globulinemia were commonly observed in the affected cows. The level of gamma globulin tended to be lower in the cows with fibrinous or sero-fibrinous, and higher in purulent pericarditis, than the level in healthy cows. In the serum protein electropherograms of the cows with fibrinous or sero fibrinous pericarditis, there was pathognostic pattern composed of slender albumin, acute shape of alpha globulin with a broad rising accompanied by double peaks and with main peak migrating toward the albumin side, tendency of rising beta globulin fraction, and large indentation between beta and gamma fractions. These findings except for the slender albumin fraction, however, was not or poorly observed in purulent pericarditis. Electrophoretic findings were subacute inflammatory pattern with non-selective serum protein losing in fibrinous or sero fibrinous, and chronic inflammatory pattern in purulent pericarditis. PMID- 1713792 TI - Analysis of protein compositions and surface protein epitopes of Anaplasma centrale and Anaplasma marginale. AB - Protein composition was compared and epitopes were analyzed among the isolates of Anaplasma centrale and A. marginale by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using bovine antisera and monoclonal antibodies, and enzyme-linked immunosorbent assay. Common and unique proteins were found among the isolates. All isolates tested had a major surface protein with an apparent molecular weight of 38 to 40 kilodalton which had slight molecular size variations between species. This protein was also a dominant immunogen to the host. At least two species-common epitopes, one of which might contain carbohydrate(s), were present on the major surface protein. One species-specific epitope was identified on the major surface protein of A. marginale isolates. PMID- 1713793 TI - Immunological cross-reactivity of the fragments of staphylococcal enterotoxins A and E generated by digestion of proteolytic enzymes. AB - Three major fragments were generated by limited digestion of staphylococcal enterotoxins A (SEA) and E (SEE) with papain, whereas five major fragments were generated by limited digestion with staphylococcal protease V8 (V8). All of these fragments were detected by immunoblotting with polyclonal anti-SEA and -SEE sera. Some of generated fragments were detected by monoclonal antibodies (MAbs) with specificities for SEA (A-111 and A-211), SEE (E-142), or both (AE-32, AE-37, and AE-53). This indicates that fragments of SEA and SEE containing the type-specific and cross-reacting epitopes may be generated by digestion of the toxins with either papain or V8. PMID- 1713794 TI - [Evolution of regulatory proteins]. AB - A concept of the evolution of signal molecules and their receptors is proposed. The formation of novel regulatory proteins by way of gene fusion and gene shuffling and the role of these processes in metabolic integration are considered. The different receptor variants may be due to alternative splicing which is regulated in a tissue-specific manner. PMID- 1713795 TI - [An active 2.5S RNA-polysaccharide complex]. AB - The information on the initial step of enzymatic reaction of 2.5S RNA isolated from muscle 1,4-alpha-glucane branching enzyme (EC 2.4.1.18), isomerase amylose, has been reported for the first time. The 2.5S RNA-polysaccharide (starch) complex was isolated and partly characterized. It was shown that the complex retains its integrity during precipitation with ethanol, gel-filtration on a Biogel P-150 column and electrophoresis but dissociates into RNA and starch upon fractionation on a DEAE-52 cellulose column. Treatment of the original preparation with RNAase fully reflects the complex formation: with a decrease in the RNA content in the original preparation that of the synthesized complex diminishes. The RNA-polysaccharide complex exhibits the properties of the branching enzyme; its enzymatic activity markedly exceeds that of the free RNA. PMID- 1713796 TI - A method for estimating the nearest neighbor base-pair content of RNAs using CD and absorption spectroscopy. AB - CD and absorption spectra are sensitive to the secondary structure of RNAs. By fitting the spectra contained in our basis set to the CD and absorption spectra of an RNA of known sequence, we could determine the fractions of base pairs, the fractions of each of the nearest neighbor base pairs, and the fractions of the single-stranded nucleotides in that RNA. The basis set included 58 CD and 58 absorption spectra. The fitting was done with a guided selection routine. The estimated error was about 0.05 for predicting the fractions of the nearest neighbor base pairs, 0.06 for predicting the fractions of A.U, G.C, and G.U base pairs, and 0.04 for predicting the fractions of the single-stranded nucleotides. PMID- 1713797 TI - The divalent cation-binding sites of gramicidin A transmembrane ion-channel. AB - The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites. It is inferred that Mn2+ ions are bound at the channel mouths at distances of 6.4, 8.6, and 8.8 A (+/- 2 A) from the oxygen atoms of exposed carbonyl groups of D-Leu 12, 14, and 10, respectively. The bounded Mn2+ retains its hydrate shell, the size of which (approximately 6 A) exceeds the inner pore diameter (approximately 4 A). That makes the gramicidin A channel impermeable for divalent cations. PMID- 1713798 TI - Canadian suicide mortality rates: first-generation immigrants versus Canadian born. AB - This article examines suicide mortality rates and trends in Canada for first generation immigrants and the Canadian-born population. Data are analyzed by age, sex and country of birth. Since 1950, suicide rates worldwide for both men and women have been increasing. In North America and most of Europe, suicide has been one of the major causes of death for many years. In Canada, suicide rates are also rising. However, this increase is due entirely to a rise in the rate for men; the rate for women has remained relatively stable. Several differences are apparent between the rates for the Canadian-born population and those for first generation immigrants. For example, three times as many Canadian-born men as women commit suicide. For first-generation immigrants, the ratio is two to one. Suicide mortality rates for the Canadian-born are higher than those for first generation immigrants in every age group except for the 65 and over groups. Canadian born males have higher ASMR than first generation immigrant males. The rates for women show that first-generation immigrant women have higher suicide mortality rates than their Canadian-born counterparts, and that the highest rate for all women is for immigrants born in Asia. PMID- 1713799 TI - The dexamethasone suppression test in Alzheimer's disease and major depression: relationship to dementia severity, depression, and CSF monoamines. AB - Patients with Alzheimer's disease (AD) have been reported to have a rate of nonsuppression on the dexamethasone suppression test (DST) comparable to that of patients with major depression. With symptoms of depression being increasingly recognized in patients with AD, studying their DST response may provide clues to the etiology of the abnormal response in both diagnostic groups. A correlation between dementia severity and post-dexamethasone cortisol was found within the group of male, but not female AD patients. Within the group of elderly depressives, a correlation between post-dexamethasone cortisol and ratings of depression was found. Serum dexamethasone levels were not significantly lower in the nonsuppressors as compared with suppressors in either diagnostic group. Within the AD group, dexamethasone levels themselves correlated significantly with ratings of dementia severity and with the Wechsler Memory Scale score. Cerebrospinal fluid (CSF) 3-methoxy-4-hydroxyphenylglycol (MHPG) correlated positively with 4:00 pm post-dexamethasone cortisol level and with ratings of dementia severity in the AD patients. Findings are discussed in light of the known clinical and other biological similarities between AD and major depression, followed by a review of theories regarding the etiology of the hypothalamic pituitary-adrenal abnormalities in these two illnesses. PMID- 1713800 TI - A tandem of alpha-tubulin genes preferentially expressed in radicular tissues from Zea mays. AB - The identification of a cDNA (MR19) corresponding to a maize alpha-tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two alpha-tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published alpha tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3' probes it appears that both genes are preferentially expressed in the radicular system of the plant. The alpha-tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members. PMID- 1713801 TI - Identification and characterization of highly conserved antigenic determinants in the laminin molecule. AB - 1. Fragments P1 and E8, the result of two different enzymatic digestions of the laminin molecule, represent interaction sites of laminin with specific cell receptors. By using negative and positive affinity purification of a rabbit antiserum against mouse laminin we have generated antibodies to these two fragments. 2. Antibodies against P1 were able to immunoprecipitate fragment E8 from elastase-digested laminin. By liquid phase competition experiments we demonstrated that the epitopes shared by P1 and E8 are a minor portion of the antigenic determinants of P1. When we checked for the presence of these shared epitopes in the human laminin molecule, they were the major fraction of the interspecies antigenic conservation. 3. A similar approach using polyclonal antibodies against human laminin has confirmed these results. 4. The shared epitopes present in both mouse and human laminin molecules seem to be spatially determined, because antibodies against these sites did not bind to fully denatured laminin. 5. Since human and mouse laminin bind to cell receptors and to other extracellular matrix proteins from both species, we conclude that these antigenic determinants may represent the actual sites for at least some of these interactions. PMID- 1713802 TI - Surgical collars: a survey of their prescription and use. AB - We report the results of a questionnaire survey of 124 orthopaedic, rheumatology, medical and accident and emergency outpatients prescribed a surgical collar during a 3-month period. Of these, 99 (80%) returned the questionnaire. Instructions received about when to wear the collar differed widely, especially between specialties. Most patients (76%) found benefit in wearing the collar. Pain was the symptom helped most (78%) whilst dizziness was helped least (40%). Problems with the collars were common, many being too hot (69%) and uncomfortable (48%). A third had difficulty putting the collar on. Despite these problems, compliance was good with 19 patients discontinuing collars for reasons other than recovery or medical advice. We believe this study highlights further areas for research and stimulates a re-evaluation of collar usage. PMID- 1713803 TI - Signal transduction. AB - Ordered cell proliferation relies on a complex interplay between diverse cell types, interstitial stroma and organ vasculature. At a cellular level the response to growth stimuli is dependent on the bidirectional exchange of information between the cell membrane and nucleus. Specific regulatory molecules enable the transfer of growth signals and constitute the signal transduction pathways. In malignant disease deregulation of growth regulatory pathways is believed to contribute to the characteristic features of neoplasia, unrestricted cell proliferation, direct invasion of surrounding tissues and the formation of distant metastases. Many proto-oncogenes and oncogenes encode proteins that are strongly suspected to function in aberrant signal transduction (Table 1). PMID- 1713804 TI - Elevation of circulating immune complexes and its relationship to alpha fetoprotein levels in patients with hepatitis B surface antigen-positive hepatocellular carcinoma. AB - In an attempt to evaluate the relationship between circulating immune complexes (CIC) and alpha-fetoprotein (AFP), CIC and AFP were detected in 93 hepatitis B surface antigen-positive (HBsAg+) patients with hepatocellular carcinoma (HCC) and 54 healthy controls. The median level of 3% PEG (polyethylene glycol)-CIC and Clq-CIC were higher in patients than in controls (p less than 0.001). In patients with HCC, the prevalence of elevated 3% PEG-CIC, Clq-CIC, and AFT was 27.9%, 55.9%, and 77.4%, respectively. There was association between AFP and 3% PEG-CIC positivity (p less than 0.01). The median level of 3% PEG-CIC and Clq-CIC increased as AFP levels elevated (p less than 0.05), but decreased as AFP exceeded 1599 ng/ml (p less than 0.05). For adjusting the effect of impaired liver function on the level of CIC, multivariate analysis with stepwise logistic regression revealed that 3% PEG-CIC was associated, in a dose-related fashion, with an increased risk for developing HCC (odds ratio = 1.003, p less than 0.001). These results imply that elevation of 3% PEG-CIC may be related to tumor mass. Additionally, 3% PEG-CIC is a useful marker to monitor therapy with transcatheter arterial embolization in patients with HBsAg+ HCC. PMID- 1713805 TI - The role of colony-stimulating factors in bone marrow transplantation. PMID- 1713806 TI - The use of hematopoietic growth factors in HIV infection and AIDS-related malignancies. AB - Human immunodeficiency virus (HIV) infection is associated with multiple defects in immune regulation and hematopoiesis. These defects include decreased proliferation of hematopoietic progenitor cells and increased destruction of mature cells. There are also disturbances of regulatory cytokines. As a result, hematopoietic cytopenias are common and the tolerance of myelosuppressive therapy is poor. One successful approach to the management of these clinical problems is the use of hematopoietic growth factors. To date, three agents have been studied in patients with HIV infection. In a Phase I trial, granulocyte macrophage-colony stimulating factor (GM-CSF) corrected leukopenia and pre-existing neutrophil defects in patients with HIV infection. In uncontrolled trials, GM-CSF also appears to reduce toxicity from zidovudine, ganciclovir, alpha-interferon, and antineoplastic therapy. In a placebo-controlled trial, erythropoietin (EPO) decreased transfusion requirements and corrected anemia in the majority of patients receiving zidovudine. In a Phase I/II trial, granulocyte colony stimulating factor (G-CSF) also corrected leukopenia and neutrophil defects in patients with AIDS without altering HIV expression. Combined G-CSF and EPO treatment corrected both anemia and leukopenia and reduced zidovudine toxicity. New combinations of hematopoietic stimulants are being used to decrease the toxicity from cytotoxic chemotherapy in the treatment of AIDS-related malignancies. Future treatments with other recombinant cytokines may result in both reduction in myelosuppression from drug therapy and, possibly, reconstitution of the immune and hematopoietic systems of HIV-infected patients. PMID- 1713807 TI - Toxic effects of colloids in the intensive care unit. AB - Colloid fluid solutions are frequently used as plasma volume expanders in the critically ill. As a group, these nonblood volume replacement solutions have in common a number of potential adverse effects. Intravascular volume overload, dilutional coagulopathy, extravascular extravasation across leaky capillary membranes, and anaphylactoid reactions may all occur with administration of any colloid. In addition, individual agents have unique toxic effects. Renal dysfunction has been associated with dextran 40, myocardial depression with albumin, hypotension with purified plasma protein, and hyperamylasemia with hetastarch. Because no ideal colloidal solution exists, knowledge of type, severity, and clinical significance of adverse effects is important in determining the appropriate plasma volume expander and monitoring its effects. PMID- 1713808 TI - [Current view on the anti-hepatitis B therapy]. PMID- 1713809 TI - [Activity of 5 alpha-reductase in human hyperplastic prostates]. AB - To Study the relationship between the activity of 5 alpha-reductase and benign hyperplastic prostate (BPH), we have measured the T and dihydrotestosterone (DHT) contents and the specific activity of this enzyme in mechanically separated stroma and epithelium and in both the nucleus and cytoplasm from 20 cases of BPH and 7 cases of normal human prostate. The results were as follows: (1) The content of DHT in the BPH group was about 3 times higher than that in the N group. (2) The Michaelis constant (Km) of 5 alpha-reductase in both the stroma and epithelium was similar. (3) The specific activity of the enzyme in the stroma was higher than that in the epithelium, and in the BPH group, increased activity was found in the tissue or stroma but not in the epithelium. The activity was predominant in the nucleus. So our experiment supports that the elevation of the activity of 5 alpha-reductase may be related to the pathogenesis of human BPH. PMID- 1713810 TI - [Isolation of human immunodeficiency virus (HIV) in epidemic area of HIV infection in Yunnan Province]. AB - Blood were collected from HIV infected persons in epidemic area of HIV infection in Yunnan province for isolation of HIV. The coculture method was used for cultivating the virus and reverse transcriptase assay (RT) was the main method for detection of HIV. Of 25 seropositive, 24 asymptomatic and one PGL, 10 showed positive RT activity (greater than 5,000 cpm/ml and with a steadily increase, some to more than 40,000 cpm/ml). The results were confirmed by the detection of HIV1 p24 Ag (ELISA) and HIV1 POL and GAG gene sequence (PCR]. In accordance with the reports from other labs, the viruses isolated from these group of persons infect only PMCs, grew slowly with gradual increase of RT activity and caused no CPE. Efforts are making, at present, to rise the virus titer with better culture system. The amplified gene sequence of the isolates are under investigating. PMID- 1713811 TI - Characterization of antibodies reacting with HIV gag proteins occasionally found in the serum of non-infected subjects. AB - The use of serological tests for the diagnosis of HIV infection has revealed that some non-infected persons have antibodies that react with HIV-1 gag proteins. Here, the sera of three non-infected subjects reacting with p17 and 11 non infected subjects reacting with p24 were investigated, using an enzyme immunoassay (EIA) with six recombinant gag antigens and Western blot analysis of proteolytic peptides of two of these gag antigens. The results indicate that whereas all p17-reactive sera could react with an unique epitope, individual p24 reactive sera recognize different epitopes. Investigations by EIA also demonstrated the role of sequences located far from the epitopes in making these epitopes accessible to the antibodies or in providing them with an antigenic conformation. In addition to the 14 subjects mentioned above, another subject was shown to have antibodies reacting with the p9 (NC) gag protein. Several proteins are known as having homology with HIV-1 gag proteins. Their possible role in eliciting cross-reactive antibodies is discussed. PMID- 1713812 TI - Polymorphism of insulin antibodies in six patients with insulin-immune hypoglycaemic syndrome. AB - Insulin antibodies in six patients with immune hypoglycaemic syndrome were studied. The antibodies displayed a higher affinity for bovine insulin in two patients, were specific for human insulin in one patient and non-species specific in the other three patients. The predominant IgG subclass of the insulin antibodies was IgG4 in two patients, IgG3 in two and IgG1 in two. In one of these, the other three subclasses were also detectable. Insulin autoantibodies of four patients were homogeneous with regard to light chains (kappa), and those of the other two contained both kappa and gamma light chains. Analysis of insulin immune complex size by fast protein liquid chromatography was possible in three patients and demonstrated immune complexes with elution profile close to that of IgG, although not exactly superimposable to the one obtained with a mouse monoclonal insulin antibody. In two patients, avidity was too low to permit chromatography of the immune complexes, and, moreover, in these two cases insulin antibodies were of the IgG3 isotype and spontaneously formed aggregates independently of insulin binding. We conclude that insulin antibodies of the insulin immune syndrome are polymorphic but different from those generated by insulin therapy. PMID- 1713813 TI - Serum CD14 levels in polytraumatized and severely burned patients. AB - Recently it has been demonstrated that the CD14 molecule which is expressed on monocytes and macrophages serves as a receptor for lipopolysaccharide (LPS) bound to LPS-binding protein (LBP) and thus mediates LPS-induced tumour necrosis factor (TNF) production. Here we report that CD14 is found as a soluble (s) molecule in serum. In healthy volunteers sCD14 levels (mean +/- s.e.m.) were 3.7 +/- 0.05 micrograms/ml (n = 30, 25-50 years of age) as determined by ELISA (detection limit 20 ng/ml serum) using two monoclonal antibodies in a sandwich technique. In polytraumatized patients (n = 16) significantly decreased levels (1.7 +/- 0.3) were detected immediately after the trauma, which increased to 4.9 +/- 0.3 micrograms/ml within the first 6 days post trauma. sCD14 remained elevated during the first 14 days post trauma in patients with the most severe injuries (injury severity score greater than 45 points), whereas a return to normal levels was observed in patients with an injury score of less than 45 points. In addition, the levels of the high-density lipoproteins that partially inactivate free endotoxin are significantly decreased post trauma. No correlation between parameters of inflammation (C3a and neopterin levels, leucocyte counts, amount of band cells), liver function and sCD14 levels was established. Comparable to polytraumatized patients, increased sCD14 serum levels were observed in five patients with burn trauma (burned area greater than 35%) within the second week post trauma when clinical signs of septicaemia were evident. PMID- 1713814 TI - In vivo treatment with interferon causes augmentation of IL-2 induced lymphokine activated killer cells in the organs of mice. AB - Interferon-alpha (IFN-alpha) has been shown to synergize with IL-2 in the regression of a variety of established murine tumours and studies are underway to explore this combination in patients with advanced cancers as well. To understand the mechanism of synergy we have studied lymphokine-activated killer (LAK) cell activity in various compartments of mice in response to IFN-alpha and IL-2 administration. The effects of IFN-gamma, TNF-alpha and IL-4 were also examined. C57BL/6 mice were injected intraperitoneally with HBSS, IL-2 alone, IFN-alpha alone or both, two times a day for 7 days. On days 4 and 8, LAK activity was tested in a 4-h chromium release in cells obtained from lungs, spleen, and liver using fresh MCA-102 tumour cells as targets. The cells from control mice failed to lyse the MCA-102 target. IL-2 caused the generation of LAK activity and an increase in total cell yield in all the organs after 3 days of injection. IFN alpha failed to generate LAK activity but when administered along with IL-2, caused synergistic enhancement of LAK lysis of MCA-102 target cells. Cell yield in this group was lower as compared with the IL-2-treated group. LAK activity tested after 7 days of IL-2 therapy was significantly decreased compared with that observed after 3 days. However, activity remained at as high a level after 7 days of therapy as after 3 days of therapy in animals treated with IFN-alpha and IL-2. FACS analysis revealed that asialo GM-1+ (ASGM-1) and NK1.1+ cells were increased in number in IL-2 and IL-2 plus IFN-alpha-treated spleen; however, the number of these cells was similar in both groups. In the liver, ASGM-1+ cells were higher in the IL-2 plus IFN-alpha group than in the group treated with IL-2 alone. By in vitro depletion utilizing antibody and Rbc' experiments, it was clear that both ASGM-1+ and NK1.1+ cells from the spleen mediated most of the cytotoxicity of MCA-102 targets. Pre-treatment irradiation (5 Gy) of mice completely abrogated the capability of IL-2 or IL-2 plus IFN-alpha to generate LAK activity. IFN-gamma also had a stimulatory effect on IL-2 induction of LAK activity. Tumour necrosis factor-alpha (TNF-alpha) and IL-4 failed to generate LAK activity and, in combination with IL-2, no additional stimulatory effect was observed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713815 TI - An approach to isolating T cell lines that react to antigens presented on the surface of dendritic cells. AB - We describe an approach that might be useful for identifying antigens on surfaces of antigen presenting cells. It is known that dendritic cells carry antigens in situ and are efficient at clustering antigen-specific T cells. Using the human mixed lymphocyte reaction (MLR) system, we have shown that alloreactive CD4+ T cells can be selected by their capacity to cluster with dendritic cells in the first 2 days of the MLR. Small numbers of clustered cells, 1-10/culture well, could then be expanded as antigen-specific lines in presence of either antigen or mitogen, sodium periodate. Few antigen-specific lines could be isolated from the nonclustered fraction. When T cell lines derived from the dendritic T cell clusters were maintained without antigen, i.e. using second party (syngeneic antigen-presenting cells (APC] or irrelevant antigen bearing APC, i.e. third party (HLA-mismatched) stimulator cells plus mitogen, the T cells retained their specificity for the original stimulating alloantigen over the time course tested, several weeks to months. These findings show that by using dendritic cells as immunoadsorbents one can prepare antigen-specific cell lines and maintain the specificity of the lines without the need for adding exogeneous antigen during either immunoselection or cloning. We discuss the possible use of dendritic cells as a means for raising T cell lines and clones that recognize antigens being carried by APC and which might be pertinent to protective immunity and autoimmunity. PMID- 1713816 TI - Inhibitory autoantibody to a conformational epitope of the pyruvate dehydrogenase complex, the major autoantigen in primary biliary cirrhosis. AB - The mitochondrial autoantibodies present in primary biliary cirrhosis (PBC) react with the 2-oxoacid dehydrogenase enzymes that include the pyruvate dehydrogenase complex (PDC). All epitopes so far demonstrable, including the inner lipoyl domain of PDC-E2, have been revealed by immunoblotting. To identify other epitopes, advantage was taken of the capacity of PBC sera to inhibit in vitro the catalytic function of the PDC enzyme. PBC sera were analyzed by affinity chromatography, using columns containing either recombinant PDC-E2 or intact PDC. Fractions that bound to the column (B) and nonbinding effluent fractions (NB) were tested by immunoblotting and ELISA and for their capacity to inhibit enzyme function. After separation on the PDC-E2 column the B fractions were reactive with PDC-E2 and intact PDC, whereas the NB fractions did not react by immunoblotting or ELISA with PDC-E2 but did react strongly by ELISA with PDC and did strongly inhibit the enzyme function. After separation of sera on the PDC column, the B fractions reacted more strongly with PDC than PDC-E2 by ELISA and strongly inhibited the enzyme function, whereas the NB fractions were nonreactive. Thus we describe a hitherto undetected population of autoantibodies in PBC sera that react only with intact PDC but not with the recombinant PDC-E2 subunit that contains the lipoyl epitope, are demonstrable by ELISA but not by immunoblotting, and notably, inhibit enzyme function. These nonblotting inhibitory autoantibodies in PBC are presumed to react with an exclusively conformational determinant perhaps presented by the tertiary structure of the entire enzyme complex. PMID- 1713817 TI - Histopathological effects of environmental pollutants beta-HCH and methyl mercury on reproductive organs in freshwater fish. AB - From various environmental pollutants studied so far, specific effects on the reproductive system of small fish species Poecilia reticulata (guppy) and Oryzias latipes (medaka) were noted in the case of beta-hexachlorocyclohexane (induction of vitellogenesis and hermaphroditism, both indicative of estrogenic activity; 32 micrograms/l) and methyl mercury (impaired spermatogenesis; 1.8 micrograms/l). The latter effect was attributed to a disturbance of mitosis. PMID- 1713818 TI - The effect of time on physiological changes in eel Anguilla anguilla, induced by lindane. AB - 1. Eel were exposed to a sublethal concentration of lindane (0.335 ppm) for 6, 12, 24, 48, 72 and 96 hr. 2. Concentrations of glycogen, glucose, lactate, pyruvate and lipids were determined in gill tissue after lindane exposure. 3. Gill glycogen decreased and glucose levels increased at 6 hr of treatment, lactate and pyruvate concentration increased between 6 and 48 hr. Total lipid values decreased between 6 and 24 hr; thereafter, the levels increased up to 72 hr of exposure. 4. Clear changes were found in all parameters tested in gill tissues. The observed effects of lindane on metabolism in fish are discussed in relation to acute stress syndrome. PMID- 1713819 TI - Sternohyoid muscle biopsy. A diagnostic technique in respiratory failure of neuromuscular origin. AB - Patients with neuromuscular disease may develop respiratory failure requiring mechanical ventilation. We describe a sternohyoid muscle biopsy technique as a diagnostic aid in such patients undergoing tracheostomy for prolonged ventilatory support. The biopsy procedure is quick and without added discomfort or morbidity for the patient. Our preliminary observations in three patients suggest that the sternohyoid muscle biopsy may be a useful diagnostic tool in this selected group of patients. PMID- 1713820 TI - Paradoxical relationship between substrates and agonist-induced contractions of opossum esophageal body and sphincter in vitro. AB - Possible differences in the abilities of esophageal body and lower esophageal sphincter muscles to utilize substrates to support agonist-induced contractions were studied. Strips of longitudinal, circular, and lower esophageal sphincter muscle from the opossum esophagus were first contracted to approximately 70% of the maximal contraction elicited by acetylcholine, histamine, or substance P. The tissues were then exhausted by exposure to 5 x 10(-4) M carbachol and a 15% O2-5% CO2-80% N2 gas mixture for 90 min. They were next reequilibrated with one of a number of alternative substrates and 95% O2-5% CO2 for 3 hr. Responses to the initial agonist doses were again noted and compared to controls. The alternative substrates were: 2-deoxyglucose, glucose, fructose 1-6 diphosphate, pyruvate, lactate, acetate, butyrate, caprylate, histidine, leucine, aspartate, alanine, succinate, acetoacetone, and beta-hydroxybutyrate. The results obtained show qualitative differences in the ability of the three muscle types to use these substrates. More importantly, however, the ability of any one substrate to support contractions was a function of the agonist used to stimulate the muscle. The evidence suggests, therefore, that not all pharmacologic receptors have equal access to intracellular energy sources. PMID- 1713821 TI - Evaluation of thiophosphoric acid alkaloid derivatives from Chelidonium majus L. ("Ukrain") as an immunostimulant in patients with various carcinomas. AB - This paper summarizes the preliminary results of two independent clinical trials conducted with the preparation "Ukrain", containing thiophosphoric acid alkaloid derivatives from the plant Chelidonium majus L. (greater celandine), in order to investigate whether it has immunopotentiating properties in cancer patients. A total of twenty-seven patients with various malignancies were treated with "Ukrain" given intravenously in a dose of 10 mg every three days. In all patients the cellular and humoral immune response was studied. There was an increase in both total T-cells and T-helper lymphocytes, a decrease in T-suppressor cells, and normalization of the helper/suppressor (HIS) ratio. A significant increase in erythrocyte-rosette-forming T-cells and NK cells was also demonstrated. Serum immunoglobulin levels, complement components (C3 and C4), and acute phase proteins were not significantly enhanced. Restoration of cellular immunity was accompanied by an improvement in the patients' performance status and in the clinical course of the disease. The treatment was generally well tolerated. The present study shows that some therapeutic benefit from the use of Chelidonium majus ("Ukrain") as an immunostimulant in cancer patients can be achieved. PMID- 1713822 TI - Predicting outcome in hypoxic-ischemic coma. A prospective clinical and electrophysiologic study. AB - A prospective analysis of 40 patients with hypoxic-ischemic coma lasting at least 6 h following sudden cardiac arrest was undertaken. The patients, all of whom had preserved brain-stem function, were studied electrophysiologically with electroencephalography (EEG), and median nerve somatosensory evoked potentials (SEPs) within 48 h to establish prognostic indices. Our results indicate that preserved brain-stem function does not necessarily predict favorable outcome following cardiac arrest as 26 of 40 (65%) patients died without awakening. The bilateral absence of cortical evoked potentials predicted death without awakening in 19 of 26 patients (73%) while malignant EEG change was similarly predictive in 11 patients (42%). Bilateral absence of cortical evoked potentials and/or malignant EEG change reliably predicted unfavorable outcome in 21/26 patients (81%). Patients with normal or delayed central conduction time (CCT) as well as 'benign' or 'uncertain' EEG findings had an uncertain prognosis as some entered a persistent vegetative state (PVS) or died without awakening. Fourteen patients (35%) awakened of whom 5 (13%) recovered completely while another 9 (23%) had varying degrees of motor or cognitive impairment. SEP and EEG findings did not distinguish between these outcomes. PMID- 1713823 TI - EEG and neuroimaging localization in partial epilepsy. AB - We have studied cortical localization provided by surface and sphenoidal electroencephalograms (EEGs) and that of computed tomography (CT), magnetic resonance imaging (MR) and single photon emission tomography (SPECT) in 58 patients with partial epilepsy. Each patient had EEG, MR and SPECT during a hospitalization period of 1-2 weeks. CT scans were obtained either during the same period or had been performed in the preceding year. EEG evaluation consisted of 3-5 days of continuous monitoring including video-telemetry and ambulatory recording as well as conventional EEGs with special electrode placements. Additionally 33 of 58 patients (55%) who were potential surgical candidates had sphenoidal recordings. All patients had an abnormal EEG which showed evidence of epileptic hyperexcitability. EEG abnormality was localized in 43 patients (74%). Neuroimaging studies were focally abnormal in 38 patients (66%); 12 CT (21%), 29 MR (50%) and 24 SPECT (41%). Thirty four of 43 patients with localized EEG had at least 1 focally abnormal neuroimaging study (79%), whereas 4 of 15 (27%) patients with non-localized EEG did so. Twenty-eight of 29 patients with focal MR (97%), 11 of 12 patients with focal CT (92%) and 20 of 24 patients with focal SPECT (83%) had a concordant focal EEG. EEG and neuroimaging localization agreed in all 15 patients in whom both MR and SPECT disclosed a concordant focal abnormality. This study demonstrates a significant (P less than 0.005) correlation between surface/sphenoid EEG and neuroimaging localization in partial epilepsy. PMID- 1713824 TI - Genetic determinants of EEG sleep: a study in twins living apart. AB - In order to investigate the genetic components of sleep and, in particular, of REM sleep, we performed 3 consecutive all-night EEG recordings in 26 pairs of normal male twins living apart (11 monozygotic and 15 dizygotic). Our results indicate that in man non-genetic rather than genetic influences substantially determine variance in stage REM, in contrast to stages 2, 4 and to delta sleep. In this sample of male twins, waking measures also showed a significant genetic component. PMID- 1713825 TI - Sphenoidal EEG recording using acupuncture needle electrode in complex partial seizure. AB - Sphenoidal EEG recording using an uninsulated acupuncture needle electrode were performed in 41 patients with or suspected of complex partial seizures of temporal lobe origin. The anterior temporal spikes were detected by the routine EEG in 17 patients (41%) and by the acupuncture sphenoidal needle in 29 patients (70%). The anterior temporal spikes recorded by the acupuncture needle were almost identical in configuration, amplitude and distribution to those recorded by conventional wire or insulated needle sphenoidal electrodes. The sequence in the frequency of spike detection by these 3 types of sphenoidal electrode were SP1-2, T1-2, F7-8 and A1-2 locations. The spikes of maximal amplitude were most frequently recorded by the SP electrode followed by the T1-2 electrode. The placement of the disposable acupuncture needle was simple and safe. Patients experienced minimal discomfort or pain that lasted at most 0.5 h. No complications occurred. The records were generally free of artifacts. It is concluded that the acupuncture needle can be used as sphenoidal electrode in outpatient EEG recording for the diagnosis of complex partial seizures of anterior temporal-origin. PMID- 1713826 TI - Physical exercise and voluntary hyperventilation in childhood absence epilepsy. AB - The aim of this study was to compare the effects of a physical exercise test and of voluntary hyperventilation between controls and children with absence epilepsy. Eighteen children (6 controls and 12 epileptics) were studied during rest (R), a maximal physical exercise test (15 min; PE), recovery (REC) and voluntary hyperventilation (3 min; VHPV). EEG and ECG were recorded during the experiment; respiratory parameters were measured to quantify PE; plasma levels of pH, lactate, pyruvate, glucose and antiepileptic drugs were determined. A decrease in the number of absences was observed during PE whereas an increase was observed during VHPV. We found significant positive correlations between the number of children with absences, the total number of absences for each state, frequency of absences per minute and the corresponding mean plasma pH, which demonstrate that the lower the pH is, the fewer absences occur. On the other hand, there was no relationship between the number of absences and the values of other parameters. Relations between variations of the plasma value of the pH, and thus the probable cerebral value of pH, and neuronal excitability are discussed. Our results indicate that children who suffer absence epilepsy should not be discouraged from sport practice. PMID- 1713827 TI - Unit activity in human thalamic reticularis nucleus. I. Spontaneous activity. AB - Microelectrode recording was carried out in the thalamic reticularis nucleus (Rt) during 51 stereotaxic operations performed in locally anesthetized dyskinetic patients. The spontaneous activity (SA) of 426 units was studied by means of computer processing techniques. Three types of unit (A, B, C) were shown to exist in Rt: with irregular low-frequency (0-10/sec) discharges (A type, 51%); bursting in short trains (10-30 msec) with unstable rhythmic pattern (2-5/sec; B type, 42%); presenting long duration (0.1-2 sec) high frequency bursts and relatively constant interburst silences (80-150 msec; C type, 7%). During short-term anesthesia A unit discharges disappeared; on the contrary the rhythmic bursts of B neurons were synchronized and presented a more stable frequency. The 3 types of cell were present in the whole Rt. However, a number of discharge characteristics (frequency, variation of rhythm) of A and B units changed significantly with the position of the cells in the Rt. No relationship was found between the frequencies of the rhythmic bursts and the parkinsonian tremor. With the use of a multiparametric statistical procedure, a relation was, however, found between the intensity of the peripheral tremor and the stability of the average frequency of the B type rhythmic bursts. The possible origins of rhythmic bursts of B and C neurons are discussed. PMID- 1713828 TI - Sleep and EEG disturbances in a rat neurological mutant (taiep) with immobility episodes: a model of narcolepsy-cataplexy. AB - The electroencephalographic sleep patterns recorded during short periods of time (3 h) of a neurological mutant rat (taiep) were studied. This rat exhibits, among other signs, immobility episodes that are similar to those observed in narcolepsy cataplexy. We describe findings of long term (6 months) electroencephalographic studies done in 9 mutant and 5 control rats. The mutant rats present electroencephalographic and behavioral disorders consisting of: (a) bursts of cortical waxing and waning waves occurring during the drowsy state; in some animals this activity represents up to 25% of the total drowsiness time; (b) shortened sleep time; (c) fragmented paradoxical sleep; (d) immobility episodes when the animals are subjected to an emotional excitement; and (e) electrographic activity of paradoxical sleep without atonia during the immobility episodes. These findings show that the taiep mutant shows several aspects of narcolepsy cataplexy and it may represent an experimental model for the study of this pathology. PMID- 1713829 TI - Drugs influencing the GABAergic neurotransmission have no effect on the non epileptic myoclonus of baboons. AB - In Papio papio baboons benzodiazepines can facilitate the appearance of a naturally occurring non-epileptic myoclonus, suggesting a possible role of GABAergic transmission in their physiopathology. Nevertheless, as this myoclonus is blocked by physostigmine, the effect of benzodiazepines is probably due to their indirect action on the cholinergic system. Therefore, in this study, we report the effects on the non-epileptic myoclonus of drugs influencing GABAergic transmission. Systemic injections of progabide (GABA precursor), baclofen (GABAB receptor agonist) and allylglycine (glutamic acid decarboxylase inhibitor) did not modify or induce the non-epileptic myoclonus. In the same way, localized chronic injections of GABA into various cerebral structures (prefrontal and motor cortical areas, reticular magnocellular nucleus and substantia nigra) had no effect. When the two types of myoclonus were present in the same photosensitive animal, the epileptic myoclonus induced by photic stimulation was blocked by benzodiazepines but was not influenced by physostigmine, thus differing from the non-epileptic myoclonus. This suggests that different neurochemical mechanisms are involved in the two types of myoclonus, the non-epileptic myoclonus not being directly influenced by the GABAergic transmission. PMID- 1713830 TI - Does interictal spiking change prior to seizures? AB - We studied 10 patients with intractable epilepsy being evaluated for epilepsy surgery for preictal changes in spiking. All patients were implanted with intracranial electrodes and underwent continuous EEG/audiovisual monitoring. Interictal spikes were detected and recorded continuously by a dedicated computerized system. Edited spikes were counted during 0-5, 5-10, and 0-60 min epochs before each seizure, during epochs of unvarying state of arousal (awake or sleep stage II). When comparing by repeated measures, 1-way ANOVA, total spiking (in all recording channels) did not differ among the different preictal epochs (0 5, 5-10, 0-60 min) in 45 seizures (F = 0.88, P = 0.40, using the Geisser Greenhouse adjustment--GGA). Likewise, no significant differences were obtained during those same epochs when comparing spiking originating from the channel of seizure onset in 5 patients with 28 seizures of localized onset (F = 1.19, P = 0.38 using the GGA). Our findings indicate that in patients with intractable epilepsy, no changes in spiking occur in the 5 min prior to seizures, when compared to more distant preictal epochs. PMID- 1713831 TI - Introduction: the first Lord Adrian Lecture. PMID- 1713832 TI - Neural mechanisms underlying brain waves: from neural membranes to networks. AB - In this review, a number of experimental findings and theoretical concepts that have led to new insights into the mechanisms underlying brain waves are presented. At the cellular level, the new evidence that certain types of neuron have intrinsic oscillatory properties that may underlie rhythmic EEG activities is discussed. In particular, the question of whether spindle oscillations are autonomous or input-dependent is addressed. At the neural network level, the main circuits of the thalamus and cortex that are responsible for the occurrence and modulation of spindles and alpha activity are described. In addition, the properties of rhythmic activities outside the alpha band are considered, particularly in relation to the prominent beta activity of the visual cortex. At the theoretical level, the possibility that neural networks may behave as complex dynamic systems with the properties of deterministic chaos is discussed. Finally, the fact that brain rhythms may have functional implications for the working of neural networks is examined in relation to 2 cases: the possibility that oscillations may subserve a gating function, and that oscillations may play a role in the formation of assemblies of neurons that represent given stimulus patterns. PMID- 1713833 TI - Long-term EEG monitoring in the early premature: developmental and chronobiological aspects. AB - Long-term cassette EEG monitoring in the neonatal intensive care unit has established prognostic criteria regarding the developmental outcome by quantifying seizure activity. The clinical significance of the organization of continuous and discontinuous EEG patterns in the early premature is still an open question. This report presents quantified EEG data from repeated 24 h records during the first week of life in premature infants (conceptional age less than 32 weeks) with and without ultrasound evidence of intracerebral hemorrhage. The repartition and evolution of EEG background activity is not a reliable parameter regarding pathology. The continuity index is rather a maturational variable and its ultradian fluctuation is an early expression of the "basic rest activity cycle" (BRAC) rhythm. PMID- 1713834 TI - The effect of the phase of prestimulus alpha activity on the averaged visual evoked response. AB - The relationship between the latencies and amplitudes of the N1 and P2 components of the averaged visual evoked potential (EP) and the phase of the alpha activity immediately preceding the time of the stimulus, has been investigated in 7 male subjects. Low intensity flashes, delivered randomly between 2 and 6 whole seconds, were used as the stimuli. The phase angle of the EEG at the moment of stimulation was computed for all trials containing more than 100 microV2 of prestimulus alpha power. The single trials were grouped into 8 classes on the basis of the phase angle value, and averaged EPs for each individual were computed from these groups. In addition, averaged EPs were computed in 3 ways: (1) a grand average consisting of all artifact-free trials, (2) an 'alpha average' consisting of all trials containing more than 100 microV2 of prestimulus alpha power, and (3) a 'non-alpha average' consisting of all trials with less than 100 microV2 of prestimulus alpha power. Each of these 3 averages were cross correlated with the phase-selective averages. It was found that the particular N1 component assessed in this experiment may possibly be entrained alpha activity, and that the measured P2 component is not an alpha process, yet it is influenced by the amount of prestimulus alpha activity. PMID- 1713835 TI - Alteration of SEP topography in Huntington's patients and their relatives at risk. AB - We studied the amplitude maps of median SEP parameters in patients with Huntington's disease (HDP) and their relatives at risk (HDF). Corresponding to the small amplitude of SEP in HDP, the power (microV2) was significantly smaller at all electrodes, and the maximum power was shifted anteriorly as a result of greater reduction of the power in the parietal than in the frontal region. In HDF, significant power reduction at the parietal region resulted in a similar anterior shift of the power to that noted in HDP. In addition to the overall reduction of SEP amplitude, the field distributions of parietal N20, frontal N29 and central N60 were significantly different in HDP, as compared to the normals. The typical relationship of the frontal positive and parietal negative fields normally present at N20 latency was lost in HDP due to the loss of the frontal P20. Frontal N29 was absent. Also N60 field shifted anteriorly. In HDF, the degree of deviation was in between those of HDP and normals. These alterations of SEP amplitude, wave form and field distribution in HDP and in some of HDF may be viewed as a result of aberrant modulatory effect exerted by the non-sensory system upon the somatosensory input. PMID- 1713836 TI - A prospective 1 year follow-up study with somatosensory potentials evoked by stimulation of the posterior tibial nerve in patients with supratentorial cerebral infarction. AB - Somatosensory potentials evoked by stimulation of the posterior tibial nerve (tibial nerve SEPs) were studied in 40 patients with supratentorial non haemorrhagic cerebral infarction and in 25 control subjects, SEPs were recorded twice in 39 patients and thrice in 35 patients. The first examination was carried out 4-19 days after the onset of the symptoms, the second examination 56-100 days after the stroke, and the third examination 348-393 days after the stroke. Increased side-to-side differences in the P57 and N75 peak latencies and absence of the P40 peak were the most frequent abnormal findings. The latency abnormalities were associated with involvement of the subcortical white matter of the rolandic region. The absence of the P40 peak was, in contrast, closely related to the extension of the infarcted area into the cortical gray matter of the rolandic region. When all SEP abnormalities were taken into account 55% of patients showed at least one abnormality in the tibial nerve SEP during the acute stage, 51% of patients had abnormal SEPs in the second examination and 43% of patients in the third examination. A nearly significant decrease was observed in the number of latency abnormalities, but the number of amplitude abnormalities, including absent responses, did not change during the 1 year follow-up period. PMID- 1713837 TI - Serial changes in somatosensory evoked potentials following cerebral infarction. AB - The relationship between somatosensory evoked potentials (SEPs) and recovery from stroke was investigated in 12 patients. All had suffered recent cerebral infarction. SEPs were performed within the first week, 6 weeks, 3 months and 6 months after stroke onset. Improvement of initially abnormal SEPs was maximal in the first 6 weeks and this correlated closely with the period of maximum clinical improvement. The results of this study suggest that the major effect of stroke on SEPs occurs acutely and is little affected by secondary degenerative processes. PMID- 1713838 TI - Laser-evoked brain potentials in patients with dissociated loss of pain and temperature sensibility. AB - Brief heat stimuli, elicited by a CO2 laser (10.6 microns wave length), activate the most superficial cutaneous nerve terminals of the thin myelinated A delta and unmyelinated C fibres which mediate heat and pain sensations. This paper investigates late cerebral potentials (SEPs) in response to laser pulses in comparison with those to conventional electrical stimulation in 18 patients with a dissociated sensory deficit (intact mechano-sensibility and disturbed temperature and pain sensation). Patients were stimulated in the most disturbed limb (affected area) and in a corresponding control area. In all 18 patients the SEPs elicited by laser stimuli were able to identify the body site with heaviest disturbances in pain and thermo-sensibility: the SEPs from the affected area were reduced or delayed, compared to the control area. In contrast, no alterations in SEPs could be observed after conventional electrical nerve stimulation, in agreement with the normal mechano-sensibility. However, the degree of SEP modulation in response to cutaneous heat stimuli did not correspond to the severity of the subjectively reported sensory deficit. Highest correlations between sensory deficits and abnormal SEPs were found in all those patients in whom computer tomography or MR imaging documented a localized destructive process in the CNS. All patients with the smallest SEP modulations despite a considerable sensory deficit had an inflammatory aetiology. Preliminary criteria to define a laser-evoked SEP as pathological are discussed. PMID- 1713839 TI - Clinical correlates of brain-stem auditory evoked potential variables in multiple sclerosis. Relation to click polarity. AB - The correlations between clinical signs and BAEP latency, amplitude and dispersion variables were investigated in 98 multiple sclerosis patients. A new dispersion variable, the wave IV-V "shape ratio" (SR IV-V), correlated most strongly with brain-stem signs (i.e., nystagmus). Severely reduced wave IV-V amplitude was frequently found in patients with vertical nystagmus or internuclear ophthalmoplegia, and interpeak latency (IPL) III-V correlated most strongly with cerebellar dysfunction (i.e., ataxia). The results may reflect different localizing ability among the various BAEP variables. The association between ataxia and increased IPL III-V was significantly stronger for BAEP to C clicks than to R clicks. Patients with abnormal BAEPs to one polarity (C or R) but not to the other, had significantly more clinical dysfunction than patients with normal BAEPs to both C and R clicks. Hence, C vs. R discordance may be interpreted to indicate possible brain-stem dysfunction. PMID- 1713840 TI - Wave III of brain-stem auditory evoked potentials analysed both with 3-channel Lissajous' trajectory and dipole localization method. AB - Wave III of the BAEP was analysed both with 3-channel Lissajous' trajectory (3 CLT) and a dipole localization method. The experiments were performed on 5 normally hearing subjects. The dipole analysis used an iterative algorithm assuming a spherical head model and homogeneous media. 3-CLT planar analysis was performed with a laboratory system. The parameters of plane C (azimuth and elevation) corresponding to wave III and those of the equivalent dipole showed a similar orientation of the plane and the dipole. This result is in agreement with previous investigations and confirms the interest of 3-CLT in far-field analysis and, at the same time, validates the dipole localization model used in this study, at least for BAEP analysis. PMID- 1713841 TI - Midlatency auditory evoked responses: differential effects of a cholinergic agonist and antagonist. AB - The effects of a cholinergic antagonist (scopolamine) and agonist (physostigmine) on the auditory middle latency evoked responses (MLRs) were studied in 7 normal male volunteers. Scalp recordings were made from a central (Cz) electrode referenced to linked ear lobes on one channel and to a non-cephalic, sternovertebral reference on a second channel. Three components were statistically analyzed for changes in latency and amplitude: Pa, with peak positivity in the 25-40 msec latency range, Nb, with peak negativity 40-50 msec, and P1, with peak positivity 50-65 msec. Control recordings included responses to click rates of 1, 5, 8 and 10/sec; as has been previously reported, P1 showed a marked decrease and disappeared at the faster rates of stimulation whereas Pa showed no change in amplitude. Intravenous injections of scopolamine resulted in a rapid and complete disappearance of P1 and a slight increase in Pa; concurrently, the subjects reported feeling drowsy but were awake with eyes open through the recordings. Subsequent injections of physostigmine resulted in a rapid reversal of the scopolamine effects so that the subjects became alert, Pa decreased, and P1 reappeared and increased to control amplitudes. Rapid click rates caused P1 to diminish, as in the control period, indicating a common P1 recovery cycle in both the control and physostigmine conditions. These data are discussed in terms of the hypothesis that the P1 generator system is comprised of a cholinergic brain-stem-thalamic component of the ascending reticular activating system. PMID- 1713842 TI - Laminar analysis of the origin of the various components of evoked potentials in slices of rat sensorimotor cortex. AB - In slices of rat sensorimotor cortex, extracellular field potentials evoked by electrical stimulation of the white matter were recorded at various cortical depths. In order to determine the nature of the various components, experiments were performed in 3 situations: in a control perfusion medium, in a solution in which calcium ions had been replaced by magnesium ions to block synaptic transmission, and in cortices in which the pyramidal neurons of layer V had been previously induced to degenerate. In the control situation, the response at or near the surface was a positive-negative wave. From a depth of about 150 microns downwards, the evoked response consisted usually of 6 successive components, 3 positive-going, P1, P3 and P6 and 3 negative-going, N2, N4 and N5. P1 and N4 were apparent in superficial layers only. The amplitude of the remaining waves was variable in the cortex but all diminished near the white matter. The early part of the surface positive wave arises from a non-synaptic activation of superficial elements, probably apical dendrites. The late part of the surface positive wave and the negative wave are due to the synaptic activation of neurons located probably in layer III. The large negative wave N2 represents principally the antidromic activation of cell bodies and possibly of proximal dendrites of neurons situated in layers III, IV and V, though the compound action potentials of afferent and efferent fibers may contribute to a reduced part to its generation. The late components N4 to P6 are post-synaptic responses. The negative component N5, the amplitude of which is largest in layers III and IV, represents excitatory responses of neurons located at various depths in the cortex. The nature of the positive component P6 is less clear, although the underlying mechanism might be inhibitory synaptic potentials. PMID- 1713843 TI - Dipole source localization in the study of EP generators: a critique. AB - (1) The decision to solve the inverse problem in terms of one or more localized generators implies the assumption of a particular generator model. This assumption permits carrying out inverse dipole estimation. However, it is perilous to justify the procedure on the basis of the result. (2) Goodness of fit is a necessary but not sufficient criterion of model adequacy. A particular quantitative inverse solution has meaning only if it is accompanied by consideration of sensitivity to small changes in all of the free modeling parameters and estimates of perturbing factors including noise and forward calculation uncertainties. PMID- 1713844 TI - Differences of cortical activation in spontaneous and reflex myoclonias. AB - Frontal and parietal components of somatosensory evoked potentials (SEPs) following median nerve stimulation and scalp potentials preceding myoclonic jerks (jerk-locked averaging, JLA) were compared in 6 patients with cortical reflex myoclonus. Giant potentials were found over the parietal cortex in both conditions. Prominent frontal activity was detected following median nerve stimulation which, however, was absent in jerk-locked averages. Therefore an identical generator of the giant SEP and the JLA is unlikely. As the frontal component is lacking in jerk-locked averaging, the spontaneous jerks produced in our experimental paradigm are believed to be due to spontaneous hyperactivity of the parietal cortex rather than to pathologically enhanced transcortical reflexes. PMID- 1713845 TI - In vivo somatic mutation in the lymphocytes of Hodgkin's disease patients. AB - While current medical therapies for Hodgkin's disease are usually quite effective, it has become increasingly clear that some of the therapies utilized carry an inherent risk for the induction of secondary malignancies. In order to examine the cellular and genetic responses to therapy for Hodgkin's disease among individuals, we have determined the mutant frequency of T-lymphocytes in 3 cohorts of patients (N = 86) and in controls (N = 71) using a T-cell cloning assay selecting for 6-thioguanine resistance. The Hodgkin's disease cohorts studied include 1) new and untreated, 2) radiotherapy, and 3) combined modality therapy patients. Additionally, two patients receiving chemotherapy alone were studied. In untreated patients, 3 of 18 (17%) mutant frequencies were above the upper 95% confidence limit for mutant frequency in controls (12.6 x 10(-6]. After therapy, 14 out of 45 (31%) of those treated with X-rays only and 10 of 23 (44%) patients treated with both X-rays and chemotherapy had mutant frequencies greater than 12.6 x 10(-6). Overall, the results indicated that the individual response to Hodgkin's disease therapy was a heterogeneous one with a sub-population of persons having elevated mutant frequencies even many years after their last treatment. The larger frequency of elevated MFs in those patients who received intensive therapy (chemotherapy and radiotherapy) is consistent with their increased risk for second cancer induction. PMID- 1713847 TI - n-3 fatty acids and acute-phase proteins. AB - The aim of this study was to determine the effects of n-3 fatty acids on acute phase proteins and response. Healthy male volunteers were submitted to standard bicycle ergometry once without supplementation and a second time after 3-weeks supplementation with highly purified n-3 fatty acids (1.75 g eicosapentaenoic acid and 1.05 g docosahexaenoic acid per day). Acute-phase proteins (immunoglobulin M, complement C4, haptoglobin, C-reactive protein, alpha 2 macroglobulin, coerulopasmin, fibrinogen, alpha 1-glycoprotein) were measured before, immediately after, 24 and 72 h after exercise. There were significantly lower values of immunoglobulin M (pre-exercise and at 72 h) and alpha 2 macroglobulin (pre-exercise) when cross-sectionally comparing the baseline data with and without n-3 supplementation. Longitudinal comparisons show that the ergometric test induced a discrete acute-phase reaction, which is evident with and without n-3 fatty acids. Yet the kinetics of the response seem to be altered by n-3 supplementation. The relative increase of most acute-phase proteins is numerically larger and the rise persists longer, which is particularly evident for fibrinogen and alpha 1-glycoprotein. The findings suggest that n-3 fatty acids lower acute-phase proteins at baseline and alter the pattern of change following acute exercise. PMID- 1713846 TI - The mode of action of quinolones: the paradox in activity of low and high concentrations and activity in the anaerobic environment. AB - All 4-quinolones that have been examined display rapid bactericidal activity which is biphasic. At concentrations above the MIC, the lethality of the drugs increases until a concentration known as the optimum bactericidal concentration (OBC) beyond which the bactericidal activity then declines. The biphasic response appears to be due to the inhibition of RNA synthesis at concentrations above the OBC, as RNA synthesis is required for the full bactericidal activity of the 4 quinolones. However, differences in the biphasic response are observed as some fluoroquinolones are still able to kill bacteria in the absence of bacterial protein or RNA synthesis, thus reducing the inhibition of bactericidal activity at concentrations above the OBC. It has been proposed that this ability to kill bacteria in the absence of protein or RNA synthesis is due to the possession of an additional bactericidal mechanism by these fluoroquinolones. Oxygen also appears to be essential for the lethality of the clinically available 4 quinolones although it is not required for the drugs to inhibit bacterial multiplication. Therefore these drugs are not bactericidal under anaerobic conditions. PMID- 1713848 TI - Antibodies against acetaldehyde-modified epitopes: an elevated IgA response in alcoholics. AB - Several recent reports have shown that antibodies reactive with acetaldehyde (AcH)-modified epitopes are present in alcoholics. However, similar antibodies have also been found in patients with non-alcoholic liver disease and control subjects. In each of these studies total immunoglobulin binding to the AcH modified proteins was measured, with no attempt being made to identify the classes of immunoglobulin involved. In the present study we employed an enzyme linked immunosorbent assay (ELISA) to assess the classes of immunoglobulin involved in this response, using plasma samples from 97 alcoholics with varying degrees of liver disease, 35 patients with non-alcoholic liver disease and 33 control subjects. All three groups exhibited a large IgM response and a negligible IgG response. However, the alcoholics exhibited a significantly higher IgA response than either of the other groups. This suggests that the measurement of the IgA response to AcH-modified epitopes may be a specific marker of ethanol abuse. PMID- 1713849 TI - Ca2+ channel modulation by dihydropyridines modifies sufentanil-induced respiratory depression in cats. AB - We analyzed the interaction between sufentanil, a selective mu agonist, and two dihydropyridines, the Ca2+ antagonist, nimodipine, and the Ca2+ agonist, Bay K 8644, on the respiratory actions induced in the brainstem of cats. Drugs were applied topically to the ventral medullary surface. Sufentanil (0.26-26 nmol) consistently induced an immediate and dose-dependent reduction in tidal volume. Respiratory frequency was only depressed by the higher doses of the opiate. Pretreatment with nimodipine (0.19 and 0.38 mumol) potentiated the respiratory depression induced by sufentanil (0.26 nmol). The potentiation included both frequency and tidal volume. On the other hand, under the influence of Bay K 8644 (0.28 nmol), the respiratory effect of the opiate (7.8 nmol) was partially antagonized. Our results indicate that modulation of the L-type Ca2+ channels by dihydropyridines modifies sufentanil-induced respiratory depression at the controlling medullary mechanisms of breathing. PMID- 1713851 TI - Qualitative changes of total cytoplasmic RNA size patterns in estrogen-induced adenoma in female rats. AB - Estradiol was administered 2.5 mg once a week for 4, 8 and 16 weeks to female Wistar rats. Hyperprolactinemia corresponding to the size of the pituitary developed along the pituitary enlargement after 4 weeks and macroscopically apparent pituitary adenomas after 8 and 16 weeks of treatment. The total pituitary cytoplasmic RNA was isolated from treated and control animals and the size distribution pattern of total RNA was obtained by sucrose gradient centrifugation. Estrogen treatment did not change that pattern in the first 4 weeks of treatment, but considerable RNA size distribution changes mainly in the medium size molecular range occurred after 8 and 16 weeks of estrogen administration. It can be hypothesized that an effect of estrogens on pituitary lactotroph hypersecretion and proliferation is not identical with events of irreversible adenomatous cell transformation which occurs later on during chronic estrogen administration. PMID- 1713850 TI - Decreased beta-adrenoceptor-mediated vasodilation in aorta from aged rats: possible involvement of a stimulatory GTP-binding protein. AB - KCl-contracted aortic rings from 18-month-old rats, in contrast with those from 2 month-old rats, showed a substantial reduction in the relaxant effects of the non selective beta-adrenoceptor agonist, isoproterenol, and of the selective beta 2 adrenoceptor agonist, clenbuterol, without changes in the relaxant actions of forskolin (an activator of the adenylate cyclase), 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) or acetylcholine (an endothelium- and cyclic GMP dependent vasodilator). The relaxant responses induced by adenosine and 2-Cl adenosine were also reduced in aged aortas. Isoproterenol and cholera toxin (an inhibitor of GTPase activity of the stimulatory GTP-binding protein) reduced cAMP production in aortas from 18-month-old rats. It is suggested that a decrease in the function of the stimulatory GTP-binding protein may contribute at least in part to the impairment in the vasodilation induced by activation of beta adrenoceptors in aortas from aged rats. PMID- 1713852 TI - A study of human calcitonin in an ovarian carcinoid and ovarian cancers. AB - High concentration of human calcitonin (hCT) was found in an ovarian carcinoid by radioimmunoassay. The hCT value was not affected by the presence of protease inhibitors. To confirm the presence of hCT in an ovarian carcinoid, hCT was isolated by the Baghdiantz method. The molecular weight of the ovarian hCT was determined using Sephadex G-75 gel filtration. Though the molecular weight of the ovarian hCT was variable, 90% corresponded to that of the authentic hCT. The carcinoid cells were examined by immunoperoxidase techniques. Those composed of strumal and trabecular structure were all argyrophilic, but hCT was only found in strumal structure. Significant concentrations of hCT were also found in ovarian cancers. PMID- 1713853 TI - Tectorecipient zone of cat lateral posterior nucleus: evidence that collicular afferents contain acetylcholinesterase. AB - The superficial layers of the cat's superior colliculus innervate the medial subdivision of the thalamic lateral posterior nucleus (LPm). LPm is set off from adjoining thalamic zones by its denser staining for acetyl-cholinesterase (AChE). We sought to learn whether the tectal afferents to LPm might themselves be the source of the enzyme staining by examining the effects of collicular lesions on the thalamic staining pattern. Large excitotoxin lesions of the colliculus largely eliminated AChE staining in the ipsilateral LPm. By contrast, fibersparing lesions of LPm itself left AChE staining nearly unchanged. Destruction of collicular neurons by excitotoxins dramatically reduced AChE staining in fibers of the brachium and superficial gray layer of the superior colliculus. The reduction was especially pronounced in the lower part of the superficial gray layer, in which LP-projecting collicular neurons are located. These results are consistent with the view that LP-projecting collicular neurons synthesize AChE and account for much of the histochemically detectable enzyme present both in the lower superficial gray layer and in LPm. In the colliculus, the excitotoxin lesions spared AChE staining in a thin sheet at the upper border of the superficial gray layer and in the enzyme-positive patches in the intermediate layers. This surviving tectal AChE thus is probably presynaptic and could be contained at least partly in cholinergic afferents from the parabigeminal nucleus and pontomesencephalic tegmentum. The collicular lesions had no obvious effect on AChE staining in the parabigeminal nucleus or in the C laminae or ventral division of the lateral geniculate nucleus. PMID- 1713854 TI - Delineation of the striate cortex, and the striate-peristriate projections in the guinea pig. AB - The size and position of the guinea pig area 17 were determined by transneuronal labeling after intraocular injections of 3H-proline or WGA-HRP. Area 17 occupies a large region of the occipital cortex located between two shallow fissures, the fissura sagittalis lateralis and the lateral groove. Area 17 extends for about 6 mm rostral from the occipital pole of the hemisphere, and encroaches occipitally for more than 1 mm upon the ventromedial surface of the hemisphere; the lateral width is up to 4.5 mm. Single injections of WGA-HRP into area 17 produced eight patches of transported tracer which formed the same general pattern in the peristriate cortex, regardless of the position of the injection within the visual field representation of area 17. Two of these patches were found in anteromedial peristriate cortex; three patches were distributed anterolateral and lateral of area 17; and three patches were located in posterolateral peristriate cortex. For several reasons, each of these patches was interpreted as representing a single striate projection onto a separate peristriate area. Comparison of these results with published findings indicates that the parcellation of the peristriate cortex into a variety of different areas, the pattern formed by these areas around area 17, and their reciprocal connections with area 17 follow a common plan in all hitherto studied terrestrial Old World and New World rodents. Lucifer Yellow injections into striate cells projecting to one of the recipient areas (AM) indicated that the pyramidal cells of this set of striate neurons are characterized by a short apical dendrite, and that the basal dendrites of the layer V pyramidal cells branch more profusely than those of the layer III pyramids. PMID- 1713856 TI - Autoregulatory control of actin synthesis in cultured rat hepatocytes. AB - ADP-ribosylation of actin by Clostridium botulinum C2 toxin resulted in a depolymerization of filamentous F-actin and an increase of monomeric G-actin in cultured hepatocytes. Simultaneously the de novo synthesis of actin was largely reduced, while the synthesis of albumin and of other proteins was not significantly impaired. The specific decrease of actin mRNA to 30% of the control indicates a down-regulation of actin synthesis at a pretranslational level. On the other hand, treatment with the mycotoxin phalloidin resulted in an increase of F-actin and a decrease of monomeric G-actin. Under this condition the de novo synthesis of actin was specifically enhanced and the level of actin mRNA was increased to 600% of the control. The data suggest an autoregulatory control of the actin synthesis. PMID- 1713857 TI - Identification and nucleotide sequence determination of the gene responsible for Ogawa serotype specificity of V. cholerae 01. AB - The gene encoding a protein of 27 kDa, which is specifically expressed in Vibrio cholerae of serotype Ogawa, was identified and its nucleotide sequence determined. The plasmid carrying this gene was found to convert serotype specificity from Inaba to Ogawa when introduced into the Escherichia coli DH5(pVCI112) cell which harbors a cloned 20-kilobase genomic DNA fragment of V. cholerae NIH35A3 and expresses the 01 antigen of Inaba serotype. PMID- 1713859 TI - Inhibition of HIV-reverse transcriptase activity by some phloroglucinol derivatives. AB - Four phloroglucinol derivatives, named mallotophenone (5-methylene-bis-2,6 dihydroxy-3-methyl-4-methoxyacetophenone), mallotochromene (8-acetyl-5,7 dihydroxy-6-(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-2,2 dimethylchromene), mallotojaponin (3-(3,3(dimethylallyl)5-(3(acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone) and mallotolerin (3-(3 methyl-2-hydroxybut-3-enyl)-5(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl) phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)-reverse transcriptase. Under the reaction conditions with (rA)n.(dT)12-18 as the template.primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 micrograms/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS-2 phage RNA as the template.primer. The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template.primer, (rA)n.(dT)12-18, and non-competitive with respect to the triphosphate substrate, dTTP. The Ki value of mallotojaponin for HIV-reverse transcriptase was determined to be 6.1 microM. PMID- 1713858 TI - Respiratory chain activity in tissues from patients (MELAS) with a point mutation of the mitochondrial genome [tRNA(Leu(UUR))]. AB - A heteroplasmic point mutation (transition A to G at position 3243 in the mitochondrial tRNA(Leu(UUR)) gene is indicative for myo-encephalopathy with lactic acidosis and stroke-like episodes (MELAS). Decreased respiratory chain complex activities measured in different tissues from four patients with MELAS syndrome do not correlate with the proportion of mutated mitochondrial genome. PMID- 1713860 TI - Keratinization is associated with the expression of a new protein related to the desmosomal cadherins DGII/III. AB - Amino acid sequencing of a 48/46 kDa glycoprotein from human plantar callus, recognised by antisera raised against the desmosomal cadherins DGII/III, has revealed N-terminal homology to the DNA-derived sequence of human and bovine DGII/III. However, a tryptic fragment has homology only with a bovine clone. We propose that there are two classes of DGII/III-like molecule, that represented by the bovine cDNA clone and the 48/46 kDa protein, a monoclonal antibody against which stains mainly the suprabasal layers of human epidermis, and that represented by the human cDNA clone, identified by a monoclonal antibody which stains uniformly the living layers of the epidermis. PMID- 1713861 TI - DNA damage by peplomycin and its repair in an in vitro system. AB - 1. DNA damage by peplomycin, an antitumor antibiotic, and its repair by cellular enzymes were studied using pUC18 plasmid DNA. The DNA damage and repair were measured by monitoring the conformational changes of pUC18 DNA. 2. Peplomycin induced DNA damage was enhanced by addition of ferrous ion and inhibited by deferoxamine, a specific iron chelator, suggesting iron-requirement for the DNA damage. 3. DNA damage by peplomycin was inhibited by superoxide dismutase in both native and heat-inactivated forms, possibly due to non-enzymatic interaction. 4. Peplomycin-induced, single-strand breaks in pUC18 DNA was repaired by incubating with a priming factor (an exonuclease purified from mouse ascites sarcoma cells), DNA polymerase beta, four deoxynucleoside triphosphates, T4 DNA ligase and ATP. The average repair patch size was estimated to be approximately four nucleotide length. PMID- 1713862 TI - Presence of an extracellular glycosyltransferase in human dental plaque. AB - 1. Glycosyltransferase activity incorporating 14C-radioactivity from [14C]sucrose into endogenous acceptor was demonstrated in human dental plaque. 2. The enzyme was localized in dental plaque into two forms: (a) associated form to bacteria (pellet 10,000 g) and (b) released as an extracellular form (supernatant 10,000 g). 3. The reaction product was insoluble in 95% ethanol, soluble in trichloroacetic acid, and it was a mixture of saccharides with different sizes, as was demonstrated by column chromatography. 4. Exogenous activity with Dextran T-10 as substrate was also demonstrated, and it represented 9% of the total endogenous activity. 5. Characterization of the extracellular glycosyltransferase, and comparative results with glycosyltransferase secreted by oral bacteria in cultures medium are discussed. PMID- 1713863 TI - Expression of c-kit encoded at the W locus of mice in developing embryonic germ cells and presumptive melanoblasts. AB - The W locus of mice encodes the c-kit tyrosine kinase receptor. In embryos homozygous for severe W mutations, the number of germ cells does not increase after 8 days of development, melanocytes do not appear, and production of erythrocytes and mast cells is deficient. To gain some insight into the role of the c-kit receptor, we have used in situ hybridization to explore the time period of expression of c-kit transcripts in early germ cells and melanoblasts. At 6 1/2 days of development, expression was not seen in the embryonic cylinder, but did appear in parietal endoderm. Germ cells displayed a low level of c-kit transcripts from their first appearance in the 7 1/2 -day embryo, continuing through early proliferation and migration to the gonad. During migration, surrounding tissues also expressed c-kit. Expression increased in gonia and then ceased as they became nonproliferative. Expression in presumptive melanoblasts was first seen in the cervical region of 10-day embryos and continued as they spread over the surface of the body, entered the epidermis, and differentiated in hair follicles after birth. The effects of mutations of c-kit on germ cells and melanoblasts can be interpreted as an absence of a proliferative signal shortly after their segregation from other cell types. This signal may be required throughout the proliferative phase of early germ cells [and also in postnatal stages of germ cell development (Manova et al. (1990). Development 110, 1057 1069]. In melanoblasts, c-kit may play a role during both proliferation and differentiation. PMID- 1713864 TI - Progressive modulation of endothelial phenotype during in vitro blood vessel formation. AB - "Sprouting" vascular endothelial cells were used as an in vitro model system to study the progressive morphologic and biosynthetic changes associated with the formation of tubular structures. In vitro, sprouting endothelial cells formed spontaneously without the addition of any exogenous factors from cultures of cloned endothelium exhibiting a polygonal/cobblestone phenotype. These phenotypically variant endothelial cells differentiated to form associated cell networks or nodules which gradually reorganized into tubular structures. Concomitant with these morphologic changes, the biosynthesis of extracellular matrix proteins was modulated, as determined by Northern blot analysis, metabolic labeling, and immunocytochemistry. The initial sprouting phase was characterized by the induction of type I collagen synthesis and the appearance of fibronectin containing the ED-A domain, in comparison to their absence in cloned cultures displaying a stable polygonal/cobblestone phenotype. The organizational stage, where the sprouting endothelial cells assembled into tubular structures, was additionally characterized by the expression of type IV collagen. These studies demonstrate that the progression from polygonal/cobblestone to sprouting cultures, and subsequent tubular organization, involves major alterations in extracellular matrix protein expression. This developmental phenomenon, although not completely analogous to blood vessel formation in vivo, nevertheless may be helpful in understanding the role of matrix macromolecules in the angiogenic process. PMID- 1713865 TI - Delay of testicular differentiation in the B6.YDOM ovotestis demonstrated by immunocytochemical staining for mullerian inhibiting substance. AB - It has been found that when the Y chromosome from Mus musculus domesticus (YDOM) is placed onto the C57BL/6J (B6) mouse background, the XY progeny (B6.YDOM) develop ovaries or ovotestes but not normal testes during fetal life. We examined the ontogeny of the abnormal testicular differentiation in the B6.YDOM ovotestis by immunocytochemical staining for Mullerian inhibiting substance (MIS). We found that the B6.YDOM ovotestis initiated testicular differentiation later in development than did the control B6 testis. When the YDOM was transferred onto the SJL J mouse background by crossing B6.YDOM males with SJL/J females, all XY progeny developed normal testes. The onset of testicular differentiation was at the same developmental stage as in the B6 male fetus. These results suggest that the delay of testicular differentiation is not due to the effect of the YDOM chromosome itself, but due to improper interaction of the testis-determining gene on the YDOM chromosome with autosomal genes of B6. In addition, we found a close correlation between the arrest of germ cells at the prespermatogonia stage and MIS production of adjacent somatic cells in the B6.YDOM ovotestis. This result may support the hypothesis that MIS is involved in the regulation of germ cell differentiation. PMID- 1713866 TI - Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro. AB - Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development. PMID- 1713867 TI - Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: effects of a cyclic AMP phosphodiesterase inhibitor. AB - The turnover of [32P]orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in [32P]phosphatidic acid (PA) and an increase in [32P] phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in [32P]PC and [32P]phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in [32P]PC and [32P]PE which accompanied GVBD. The increase in [32P]phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid. The third is associated with GVBD, and is cAMP-sensitive, and may represent stimulation of de novo synthesis of phospholipid, thereby permitting disruption of the nuclear membrane. PMID- 1713868 TI - 20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes. AB - Transcripts of ecdysone-dependent genes (EDGs) accumulate in isolated imaginal discs with 8 hr after exposure to a pulse of the steroid hormone 20 hydroxyecdysone (20-HE; 1 microgram/ml for 6 hr) but not in discs cultured in the continuous presence or absence of the hormone. Sequence analyses show that two of the EDGs are members of gene families encoding insect cuticle proteins. We conclude that a third EDG encodes a cuticle protein because the conceptual glycine-rich protein contains sequence motifs similar to those found in insect egg shell proteins and vertebrate cytokeratins and because expression of this gene is limited to tissues that deposit the pupal cuticle. Nuclear run-on assays show that the hormone-dependent expression of each of these EDGs is due to transcriptional regulation. Readdition of hormone to imaginal discs actively synthesizing the EDG messages causes rapid repression of EDG transcription. Thus, 20-HE acts as both a positive and a negative regulator of EDG transcription. Sequences in the promoter regions of two of the EDGs are similar to an ecdysone response element and may play a role in negative regulation. PMID- 1713869 TI - Binding and biological effects of insulin, insulin analogues and insulin-like growth factors in rat aortic smooth muscle cells. Comparison of maximal growth promoting activities. AB - Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of 125I-insulin was 0.9 +/- 0.2% of total 125I-insulin added, and the IC50-value was estimated to 8.9 pmol/l. The insulin analogue B10 Asp tended to be more potent than insulin in displacing 125I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of 125I-insulin-like growth factor-I after 4 h incubation at 10 degrees C was five times higher than the specific binding of insulin (4.4 +/- 0.4% of total 125I-insulin-like growth factor-I added), and the IC50-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing 125I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for 3H-thymidine incorporation into DNA: insulin-like growth factor-I greater than B10 Asp greater than insulin-like growth factor-II greater than insulin greater than or equal to B28 Asp greater than B9 Asp/B27 Glu greater than proinsulin. The maximal effect of insulin-like growth factor-I on 3H-thymidine incorporation was 71 +/- 16% higher than the maximal effect of insulin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713870 TI - Glucose regulates both glucose transport and the glucose transporter gene expression in a hamster-derived pancreatic beta-cell line (HIT). AB - We studied the effect of chronic exposure to high glucose on the glucose transport regulation in hamster pancreatic Beta cells in permanent culture (HIT). Cells were exposed to either 5.5 mmol/l or 16.7 mmol/l glucose for 48 h and then glucose transport was studied by measuring the (3H)-2-deoxyglucose uptake for 5 and 10 min at 37 degrees C. The 2-deoxyglucose uptake was lower in cells pre exposed to glucose 16.7 mmol/l for 48 h compared to cells pre-exposed to 5.5 (12.0 +/- 1.6 vs 19.1 +/- 1.2 nmol/0.1 mg after 5 min, and 22.2 +/- 2.6 vs 39.0 +/- 2.9 after 10 min respectively, mean +/- SEM, n = 5, p less than 0.01). In order to investigate the mechanism(s) for glucose impairment of glucose transport, we studied the glucose carrier gene expression in the same cells by Northern and slot-blot analysis. When total RNA was extracted from HIT cells cultured at either 5.5 or 16.7 mmol/l glucose and then hybridized to 32P-labelled cDNA probes for the glucose transporter 1, the glucose transporter 2 and beta actin, a significant reduction of both glucose transporter 1 (-63.9 +/- 4.1%, mean +/- SEM, n = 3) and glucose transporter 2 (-48.9 +/- 3.2%) mRNA was observed in HIT cells cultured with high glucose. In the same experiments no change of beta-actin mRNA was observed, suggesting that the effect of high glucose was specific on the glucose-transporter mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713871 TI - Inverse regulation of calcium channels and beta-adrenergic receptors in virus transformed human embryonal cells. AB - Monolayer cultures of human embryonal smooth muscle cells (HEC) were used to study the heterologous regulation of membrane beta-adrenergic receptors and Ca2+ channels. The relationships between the activation of membrane bound alpha-1 and beta-adrenergic receptors, the cyclic nucleotide response and Ca2+ channel binding were studied in a cellular model of latent virus infection (Herpes simplex, Type-2) in a human embryonal cell line. In the early stage of infection (72 h), there was a significant increase in the cell cAMP content, followed by a decrease in the binding capacity of the beta-adrenergic ligand with an increased total number of the 1,4-dihydropyridine Ca2+ channel agonist (-)-S-(3H)BAYK 8644 binding sites on the cell membrane of infected cells. The increased numbers of Ca2+ agonist binding sites were accompanied by an increased cAMP content in the cells and an increased membrane ATP-ase activity. Down-regulation of (3H)DHA binding, and an increased capacity of Ca2+ agonist binding were found after prolonged exposure of HEC to isoprenaline (10(-5) mol.l-1). Stimulation of alpha 1 adrenergic receptors with phenylephrine (10(-6) mol.l-1) was accompanied with only slight but significant increase in (3H)DHA binding and with a significant reduction in the total number of Ca2+ channel agonist binding sites. PMID- 1713872 TI - A comparison between two different procedures for bone marrow processing: a semiautomated procedure vs manual starch sedimentation. AB - After high-dose chemotherapy, autologous cryopreserved bone marrow infusion is employed to restore rapidly the compromised hematopoietic function. An efficient bone marrow processing reduces the infusion toxicity produced by hemolized red cells, granulocytes and platelets clumping and DMSO amount; moreover it increases freezing efficacy, a critical step in autologous bone marrow grafting techniques. Gravity sedimentation technique with 6% hydroxyethyl-starch (HES) or a semiautomated procedure using a blood cell processor were used in our center to manipulate ex-vivo the collected bone marrow. In our experience we compared these two different procedure and we evaluated their efficiency. PMID- 1713873 TI - CD34 or S313 positive cells selection by avidin-biotin immunoadsorption. AB - A column immunoadsorption method, based on the high affinity between the protein Avidin and the vitamin Biotin has been used to obtain positive CD34+ or S313+ marrow cells selection. Cell suspensions from marrow samples of six healthy subjects were incubated either the monoclonal antibody S313 or HPCA 1 (My 10 like) and a biotinilated goat anti-mouse Immunoglobulins antiserum, passed over a column containing an Avidin coated Polyacrylamide matrix, at the flow rate of 1 ml/min. The phenotype and the clonogenic efficiency of the recovered adherent cell fraction were studied by cytofluorimetric analysis and haemopoietic progenitors short term cultures. The results obtained show a mean CD34+ and S313+ cells recovery greater than 50% with a lower stem cells enrichment. Although these data could not considered optimal for clinical application in haematologic neoplasias, these preliminary studies demonstrate the possible use of the method for autologous bone marrow transplantation. PMID- 1713874 TI - Methodologies to estimate circulating hematopoietic progenitors for autologous transplantation in cancer patients. AB - Optimal criteria for harvesting circulating hematopoietic progenitors (CHP) for autologous transplantation to support myeloablative cancer therapy are still uncertain mostly because the CFU-GM assay, the commonly used indirect indicator of the hematopoietic recovery of the graft, is poorly standardized and provides information evaluable only retrospectively. Based on the knowledge that CHP express CD34 and CD33 differentiation antigens and facilitated by the availability of a very efficient fluorescein-conjugated CD34 antibody (8G12), we developed a direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. Recently, in a comparative study, both assays were applied to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide (7 g/m2) cancer therapy w/ or w/o rhGM-CSF. Results showed that: a) detectability of CD34+ CHP indicated an increase to greater than 500 CFU-GM/mL, a level clinically adequate for harvesting CHP; b) CD34+ cells correlated well with CFU-GM (R=0.89) and data fitted a linear regression line (y=388.3 + 64.0x; y=CFU-GM/mL and x=CD34+/uL); c) in a series of 8 patients treated with myeloablative chemoradiotherapy, early recovery of marrow functions was predicted more accurately by the number of transplanted blood CD34+/CD33+ cells than by nucleated cells, CFU-GM, CD34+/CD33-cells, or CD34-/CD33+ cells. As a guideline, provided platelets are greater than 70,000/uL, harvest of CHP by leukapheresis during recovery from chemotherapy induced pancytopenia should be started as soon as CD34+ cells appear in the circulation and continued until the threshold dose of 7.8x10(6) CD34+ cells/kg, equivalent to 50 x 10(4) CFU-GM/kg, is achieved.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713875 TI - Treatment of malignant lymphomas with very-high-dose CVB followed by transplantation of autologous blood stem cells collected after mobilizing chemotherapy. AB - In 8 patients (age 25 +/- 46 years, mean 34.5), 4 with Hodgkin's disease (HD) and 4 with non-Hodgkin's lymphoma (NHL), circulating stem cells (CSC) collected at the time of rapid leukocyte and platelet recovery after intense chemotherapy were employed for autologous hematological reconstitution after very-high-dose chemotherapy (CVB combination). At the time of graft 2 patients were in 1st remission and 2 in 2nd, while the remaining 4 had persistence or progression of disease. Autografted patients received 3.4 +/- 8.3 (mean 5.2) x 10e8/Kg mononuclear cells and 2.1 +/- 40.5 (mean 22.5) x 10e4/Kg CFU-GM. All patients had prompt and sustained engraftment, though in one case platelets never reached normal levels. Recovery time was respectively 10 +/- 17 days (mean 13.2) for granulocytes 0.5 x 10e9/L, and 10 +/- 49 days (mean 20.3) for platelets greater than 50.0 x 10e9/L, with an interval of less than 4 days from greater than 0.5 to greater than 1.0 granulocytes in 7 out of the 8 patients. All patients are currently alive at 57 +/- 645 (median 262) days, and 7 in remission at 61 +/- 645 (median 297) days from autologous blood stem cell transplantation (ABSCT). Our study demonstrates that CSC collected after mobilizing chemotherapy are able to promptly restore and sustain hemopoiesis after marrow ablative chemotherapy in patients with malignant lymphoma. CSC reduce toxicity and hospitalization so impressive, that in malignant lymphomas ABSCT could rapidly be considered for first line strategy. PMID- 1713876 TI - A technique for isolation of bone marrow cells using hydroxyethyl starch (HES) sedimentation agent. AB - Various methods have been investigated to concentrate hemopoietic stem cells before in vitro treatment and cryopreservation. Each procedure requires various degrees of cell manipulation; moreover, the extent of purification of the stem cell fraction depends on the further use of bone marrow. Fifty four bone marrows were treated with hydroxyethyl starch (HES) to isolate the mononuclear cell population. The manipulation with HES permitted to concentrate marrow stem cells in a small volume with removal of unwanted granulocytes, red blood cells and the donor isohemagglutinins in the major ABH incompatibility. We describe here a manual method which permits the transfusion of a very few number of red cells without depletion of progenitor cells and thus delay of engraftment. PMID- 1713877 TI - Plasmapheresis in the therapy of hyperthyroidism associated with leukopenia. AB - Surgery, the treatment of choice for hyperthyroidism due to nodular goiter, requires an euthyroid state, which is generally achieved with thionamides. Leukopenia is the most serious toxic effect of thionamides, and it causes controindication. We report a 50-year old woman with severe hyperthyroidism and leukopenia, in whom an euthyroid state before thyroidectomy was obtained with the use of therapeutic plasmapheresis. This procedure was carried out immediately before surgery using an intermittent flow separator; three sessions removed a total of 6,300 cc of plasma. Plasmapheresis caused a rapid reduction of both total and free thyroid hormone levels. Thyroidectomy was performed without any complications. Plasmapheresis can be considered a valid and safe method to prepare hyperthyroid patients for thyroidectomy when other therapies are ineffective or counterindicated. PMID- 1713879 TI - [4th Haemoblastosis Symposium of the Society of Haematology and Blood Transfusion of the DDR]. PMID- 1713878 TI - [Aggregative behavior and calcium content of platelets in thrombocythemia and thrombocytosis]. AB - Patients with thrombocythaemia due to myeloproliferative disorders (n = 21), with secondary thrombocytosis of various origin (n = 16), and a control group of healthy donors (n = 20) were investigated with respect to the aggregation behaviour and the total calcium content of blood platelets. The calcium content was significantly lower in both groups of patients as compared to controls (2 p less than 0.001). In 16 of 21 patients with myeloproliferative disorders platelet rich plasma did not respond to epinephrine (15 mumol/l), a concentration which induced at least weak aggregation in 14 of 16 patients with secondary thrombocytosis and also in healthy subjects. In patients with thrombocythaemia the mean extent of aggregation induced by epinephrine, collagen or adenosin diphosphate was significantly lower as compared to controls (2 p less than 0.001). PMID- 1713880 TI - Toxic effects of alkyl-lysophospholipids on human bone marrow and de novo leukaemias. A short overview. AB - Alkyl-lysophospholipids (ALPs) are reported to have an antineoplastic activity against leukaemic cells. We have tested some halogen-containing ALPs from the Central Institute of Molecular Biology (H. Brachwitz) in comparison with racemic 1-ostadecyl-2-methyl-glycero-3-phosphocholine (ET-18-OCH3) (P.G. Munder, Max Planck-Institut fur Immunobiologie, Freiburg, FRG). We found freshly dissolved ALPs to be very toxic both to human bone marrow and to leukaemic cells of patients. ALP-incubation before cryopreservation is more toxic to bone marrow (but not to AML blasts) than after cryopreservation. All experiments to test the selectivity and to establish a purging protocol should be done using 1, remission marrow including a cryopreservation step and 2, blasts of de novo leukaemias instead of cell as sensitive as HL 60 to ALP-incubation. We found direct toxicity of ALPs to be not suitable for purging lines of bone marrow from patients in remission. PMID- 1713881 TI - Prospective study on the influence of radiochemotherapy on pituitary function in children with acute leukaemia and NHL. AB - To assess effects of chemo- and radiotherapy on the endocrine system 31 children with acute leukaemia and NHL (3 AML, 24 ALL, 4 NHL) were investigated. Children were treated according to modified BFM protocols. 25 patients were before, 5 during and one after puberty (2 to 16 y.). Before treatment, during induction therapy, during cranial irradiation, 4-6 weeks later and during maintenance therapy the following hormone values were estimated: TSH and prolactin basal and 30 min. after TRH (5 micrograms/kg i.v.), LH and FSH basal. Final investigations included total T4 and T3. In conclusion, chemo- und radiotherapy lead to transient elevations of TSH and prolactin in a few patients, but without proof for permanent disorders. Due to the fact all 3 patients with hyperprolactinaemia showed high prolactin levels (700 to 770 mU/l) already before treatment it is unlikely therapy was the main cause of these observed alterations. Although basal LH and FSH values were in normal ranges for age the increasing values after cranial irradiation in prepubertal children may reflect a possible initiation of early maturation, reported by others. Furthermore a retrospective growth study was performed in children treated with 2 different protocols. Protocol LSA2L2 used in the past before 1981 resulted in a permanent reduction of the height. In contrast, the mean SDS for height in children treated with protocol VII declined only during the intensive period of treatment. A catch-up growth occured already during maintenance therapy. Prophylactic cranial irradiation with 18 Gy in our patients under protocol LSA2L2 did not affect growth during the first 5 years after diagnosis. PMID- 1713882 TI - Dose-reduced induction therapy for acute leukaemias decreases both the complete remission (CR) rate and the probability of leukaemia-free survival (LFS). AB - Especially in AML but also in ALL a dose reduction during the induction therapy effected distinctly both a diminution of the CR rate and a shortening of the LFS. For these reason reduced treated patients are to exclude from final analysis of study in order to obtain a objective comparison of the four postremission treatment modalities. There was no difference concerning treatment related mortality between "correct" and "reduced" induction therapy. PMID- 1713883 TI - Low dose cytosine arabinoside in the treatment of the primary myelodysplastic syndrome. AB - 23 patients with primary myelodysplastic syndrome was observed in 1982-1988. 8 patients with RAEB and RAEB-t according to FAB criterias were treated with low dose cytosine arabinoside. No complete response and only one partial response +10 months duration was achieved. Treatment had a minor influence on the natural course of disease, and doesn't protect patients from the transformation into acute leukaemias. PMID- 1713884 TI - T-cell depletion and haematological side effects of two cytotoxic monoclonal antibodies. AB - We investigated two cytotoxic monoclonal antibodies of BL-series (BL-IIIB4 and BL IIG2) according to T-lymphocyte depletion from bone marrow. Both antibodies work together with human complement similar Campath-1, which was tested parallelly. The extent of T-cell depletion is about 95% for all three antibodies. On the other hand the haemopoietic side effects tested by CFU-GM recovery and LTBMC are for the BL-antibodies not as strong as for Campath-1, especially in view of LTBMC. T-cell regeneration could be shown in long term cultures. Our results indicate a possible suitability of the two investigated antibodies for T-cell depletion of bone marrow. PMID- 1713885 TI - Bone marrow necrosis intravitally recognized in four cases of blastic leukaemia. AB - Bone marrow necrosis (BMN) is a rare intravitally recognized finding in acute leukaemia with an uncertain clinical significance. The clinical events in 4 patients with AML, ALL, AMoL and blastic transformation of CGL in whom bone marrow cytology and histology revealed BMN are reviewed. One patient with BMN at clinical presentation of AML entered complete, long lasting remission with marrow restoration after the standard DAT therapy. In the three remaining patients survival after BMN diagnosis was 6, 11, and 14 weeks. Clinical, haematological, histological and marrow scanning findings and their significance for early diagnosis and means to asses the extent and evaluation of BMN will be discussed. In contrast to the most earlier reports, BMN does not appear to confer a poor prognosis in all patients with blastic leukaemia. PMID- 1713886 TI - [Disturbances in iron utilization in acute leukemia and preleukemia]. AB - With Prussian blue reaction nonhaemoglobin iron in the erythroblasts is demonstrable. Three pathological sideroblast types are recorded separately: abnormal intermediate type I and II sideroblasts and ring sideroblasts, representing increasing levels of sideroachrestic disturbance. This permits the classification of sideroachrestic disturbances into four degrees of seriousness. The frequency of a sideroachrestic disturbance in 47 untreated patients with acute myeloid leukaemia was 87%. Among 11 patients with preleukaemic condition, 8 had a disturbance of iron utilisation. In both preleukaemia and leukaemia mainly intermediate sideroblasts were present. All patients with preleukaemia developed leukaemia within 1-20 months. In the course of preleukaemic condition a slight increase of iron misutilisation was obvious when terminating in overt leukaemia. This could be of prognostic importance. After treatment, pathological sideroblasts disappeared only in 2 out of 15 patients with complete remission. There was no correlation between effect of therapy and course of iron misutilisation. PMID- 1713887 TI - Cerebellar degenerative changes in patients with acute leukaemias and non Hodgkin's lymphomas. AB - In patients with neoplasms located outside the central nervous system can appear degenerative changes within nervous tissue. Neuropathological investigations have been done on 101 cases with acute Non-Lymphoblastic Leukaemias (ANLL) and Non Hodgkin's Lymphomas (NHL). Cerebellar degenerative changes especially granular layer rarefaction or atrophy appear more frequently in ANLL than in NHL. PMID- 1713888 TI - The activity of neutrophil myeloperoxidase in patients with Hodgkin's disease. AB - Neutrophil myeloperoxidase activity has been studied in twenty one patients diagnosed with Hodgkin's disease. The presented data indicate no differences in total MPO activity, whereas we observed some differences in the percentage of granulocytes with different degree of scores. Changes in the intensity of reaction may indicate the possibility of exocytosis as a mechanism releasing MPO from the cell to the surrounding area. In peripheral, circulating neutrophils, such a phenomenon seems to be of no avail and may disorganise anti-cancer defence. PMID- 1713889 TI - [Iron resorption and the effects of iron supplementation in blood donors]. AB - Topical values for some haematological factors such as Hb and Ery as well as transferrin and ferritin were determined in 7 female blood donors and 8 male blood donors during the years of their work as blood donors. Subsequently, an iron resorption test was carried out which unexpectedly resulted in low rates of resorption ranging from 5.6% in men to 3.7% in women. After supplementing with vitaferro for 3 months the ferritin values which initially lay around the lower limiting value of 20 or 10 mg/l in men or women had doubled. PMID- 1713890 TI - Transbilayer redistribution of phosphatidylserine in erythrocytes of a patient with autoerythrocyte sensitization syndrome (psychogenic purpura). AB - We have investigated a female patient with autoerythrocyte sensitization syndrome (AES syndrome), having a positive skin response to her own red blood cells (RBC) and to phosphatidylserine (PS). Using 2,4,6-trinitrobenzenesulfonic acid (TNBS), bee venom phospholipase A2 and merocyanine 540 binding, we have demonstrated that in RBC of patient more than 50% of PS is redistributed into the outer leaflet of the plasma membrane. Using homologous RBC from a healthy donor we were able to induce transbilayer PS redistribution by incubation with the patient plasma. The presence of immunoglobulin E against cardiolipin and PS was proved in patient's plasma. We elaborated a method for cytoskeleton visualization using indirect immunofluorescence technique. We found disorders in cytoskeleton organization in RBC of the patient. We recommend in vitro testing for AES syndrome diagnosis. The positive effect of chlorpromazine treatment is described. PMID- 1713891 TI - Laser nephelometer evaluation of factor IX. AB - Laser Nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetic of the reaction between antigen and antibody. In a few instances the method was also applied in the characterization of abnormal molecules. We developed assays for the measurement of Factor IX antigen and the results were compared with those obtained by conventional immunological methods such as rocket immunoelectrophoresis. Plasmas from patients with haemophilia B, on coumarin treatment, with liver cirrhosis were studied. A standard reference curve was obtained using pooled normal plasma. The factor IX levels obtained by laser nephelometer correlated fairly well with those obtained by electroimmunoassay. PMID- 1713892 TI - [Characterization of T lymphocyte defects in patients with Hodgkin's disease using enzyme chemical markers]. AB - Patients suffering from lymphogranulomatosis were studied with respect to cellular immune deficiencies. For this purpose, mononuclear cells from venous blood were separated and subjected to analysis of lymphocyte markers. T lymphocytes were enumerated by means of the sheep erythrocyte (SE) rosette test. T cell subpopulations were determined using enzyme cytochemical staining for dipeptidyl peptidase IV (DP IV) and unspecific acid alphanaphthylacetate esterase (ANAE). In 18 patients with M. Hodgkin a significant reduction in the T lymphocyte count in peripheral blood was found. This T cell defect is due to a selective decrease in the TM-subpopulation as identified by enzyme cytochemical markers DP IV and ANAE (focal reaction). From these results it is concluded that patients with lymphogranulomatosis have characteristic abnormalities in the immune system in the sense of a disturbed equilibrium of immune regulatory cells. PMID- 1713893 TI - Modification of exogenous haemopoietic spleen colony formation in irradiated mice by antisera against mouse thymocytes and mouse brain. AB - Treatment of donor bone marrow in vitro with both anti-thymocyte serum (ATS) and anti-mouse brain serum (RAMBS) inhibits the formation of haemopoietic spleen colonies in irradiated and reconstituted mice. This activity of the antisera may be completely (ATS) or partly (RAMBS) eliminated by absorption with thymocytes. The effect of the antisera is complement-independent. Most likely it depends on the existence and functionality of phagocytes (macrophages) in the recipients. PMID- 1713894 TI - [Routine hematologic parameters for thalassemia screening]. AB - There is an evaluation of routine examination of the blood film for the recognition of Thalassaemia (Th.). The results of 100 blood films from each group -Th. major, Th. minor, healthy persons (centre for Thalassaemia "Bishop Makarios"/Nikosia--Cypros)-were analyzed. MCV and MCH were most useful for the recognition of Th. minor. The constellation of increased red blood cells and normal hemoglobin seems to be typical for Th. minor. Haemoglobin and haematocrit are not suited because they were widely normal. Target cells also are not sufficient for screening--they were present only in 10% of Th. minor. To the contrary most values were clearly decreased in Th. major, and target cells were present here in almost 90%. PMID- 1713895 TI - Haemocompatibility of extracorporeal circulation technique with autooxygenation: influence on platelet function and homologous blood requirement. AB - Forty male patients: group A-autooxygenation and group B-bubble oxygenator used in extracorporeal circulation (ECC) were studied to evaluate the haemocompatibility of 2 types of ECC. The Plt count dropped significantly in group B patients: -73% of initial value vs only -27% in group A, (p less than 0.001). In both groups a rise in BTG was shown, but higher in group B, p less than 0.001. At the end of CPB aggregation decreased only slightly in group A after epinephrine and 4-ADP, and decreased hardly in group B with the significant difference between two groups (p less than 0.02 and p less than 0.001, respectively). In group A the mean blood loss was 278 +/- 49 ml/m2 and 483 +/- 67 ml/m2 in group B, p less than 0.001. The mean blood transfusion in group A and B was 198 +/- 82 ml/m2 and 427 +/- 85 ml/m2, respectively (p less than 0.001). We are positive that the elimination of artificial oxygenator from the ECC diminished markedly the decline in Plt count and Plt activation during CPB. PMID- 1713896 TI - [Review: heparin. 1. Methods of determination and inhibitory mechanisms of some coagulation factors]. AB - In a brief survey four groups of determining methods are reported for the purpose of monitoring the heparin therapy, with their advantages and disadvantages being assessed simultaneously. Emphasis is placed on the heterogeneity of heparin with approximately 120 fractions which are the cause of different manners of response and an impediment to the development of a rapid, reliable and economically advantageous method. Finally, all heparin factors in the plasma are enumerated and their mechanisms of action on individual coagulation factors are referred to. PMID- 1713897 TI - [Review: heparin. 2. Effect on thrombocytes, vascular walls and fibrinolysis]. AB - In a brief survey the impact on thrombocyte functions and thrombocyte values caused by heparin and its fractions is reported, with the acute and immuno allergic form of heparin-induced thrombopenia being widely dealt with. Furthermore, the relationship between heparin and vessel wall, particularly then its endothelial layer, is discussed and finally, its impact on the mechanisms of fibrinolysis is reported. PMID- 1713898 TI - Early prevention of childhood disability in developing countries. AB - The concept of prevention, while implicit in most early intervention efforts, has not been comprehensively articulated as a basis for conceptualizing early intervention services. The growing recognition of the importance of early identification and intervention for infants and young children, and involvement of the family, are factors which contribute to conceptualizations of services which are preventive in nature. This recognition parallels broader concerns for family support programmes which have a preventive focus and seek to enhance the development of children and families. The purpose of this paper is to present a comprehensive framework for the provision of child and family service by conceptualizing early intervention in terms of levels of prevention. Specifically, the concept of primary, secondary, and tertiary levels of prevention will be presented as a framework suitable to encompass the preventive function of community based rehabilitation. The relevance of early prevention is based on the premise that the condition of childhood disability can be prevented at primary, secondary, and tertiary levels. Viewed in this way, the problem or condition of developmental delay or disability in children can be addressed at each of the three levels to effect a reduction of its expression, its duration or extended impact. Primary, secondary, and tertiary prevention can be implemented in the context of community based rehabilitation to address these goals: (a) enhance development and minimize the potential for delay; (b) minimize the need for special education and related services; and (c) minimize the likelihood of institutional or other restrictive care outcomes. PMID- 1713899 TI - Applications of in situ hybridization. PMID- 1713900 TI - Interphase nucleolar organizer regions in cancer cells. PMID- 1713901 TI - Interactions between endothelial cells and the cells of the immune system. PMID- 1713902 TI - Prazosin concentration monitoring in the treatment of prostatic adenoma. AB - In a prospective study of 32 patients with BPH treated with Minipress (prazosin), determinations of drug plasma levels were carried out. The comparison of clinical results and serum plasma levels enabled optimalization of the dosage in long-term therapy. PMID- 1713903 TI - Zinc plasma levels in prostatic carcinoma and BPH. AB - Zinc in serum from patients with prostatic carcinoma and BPH before and after treatment was analyzed by atomic absorption spectrometry. There were significant differences between prostatic cancer and BPH. We also found distinct differences in the plasma content of zinc in patients with prostatic carcinoma before and after therapy. We conclude that the zinc concentration in serum may be a valuable index for the differential diagnosis and therapy of prostatic carcinoma. PMID- 1713904 TI - Blood loss during transurethral resection of the prostate injected with phenol solution. AB - Blood loss during transurethral resection of the prostate (TURP) still represents a major hazard. Several methods of reducing bleeding during TURP have been recommended [7, 8, 9, 13]. In 1986, Muzafer showed that blood loss during open prostatectomy of the prostate injected with 5% phenol in almond oil solution was significantly smaller than in the non-injected group [9]. PMID- 1713905 TI - Nucleotide sequence of the 3'-terminal region of carnation latent virus. AB - The sequence of 1,313 nucleotides of the 3'-terminal region of carnation latent virus RNA, determined from cDNA clones, was found to contain two major overlapping open-reading frames encoding proteins with relative molecular weights of 33.9 and 11.6 kD. The 33.9-kD protein, which is similar in size to the capsid protein, and the 3'-coterminal 11.6-kD protein show extensive homology with corresponding regions in other carlaviruses. Comparison of amino acid sequences of carnation latent virus coat protein and analogous regions in potexviruses showed large similarities. PMID- 1713906 TI - Capsaicin-induced release of tachykinins: effects of enzyme inhibitors. AB - We studied the effects of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) inhibition on the airway responses and the recovery of endogenously released substance P- and neurokinin A-like immunoreactivities (SP-LI and NKA-LI) after tracheal injection of capsaicin in isolated guinea pig lungs superfused through the trachea. Capsaicin in doses from 10(-10) to 10(-7) mol induced a dose dependent increase in airway opening pressure and release of SP-LI and NKA-LI. Airway opening pressure changes and the recovery of SP-LI and NKA-LI were significantly greater in lungs superfused with the NEP inhibitor SCH 32615 than in control lungs. ACE inhibition with captopril did not increase the mechanical response or the recovery of SP-LI compared with lungs not receiving captopril. In lungs from guinea pigs pretreated with high doses of capsaicin 7-10 days before study, a regimen designed to deplete endogenous tachykinins, there was a significant decrease in the content and release of NKA-LI and SP-LI. There were no detectable airway effects of acute capsaicin infusion even after doses of 10( 5) mol. Because NEP is important in modulating the airway effects of endogenously released tachykinins after tracheal infusion of capsaicin, but ACE is not, it seems likely that tracheal administration of capsaicin releases tachykinins from epithelial rather than endothelial loci. PMID- 1713907 TI - Neonatal capsaicin treatment alters basal pulmonary mechanics and response to methacholine in F344 rats. AB - Full methacholine dose-response curves were performed on anesthetized tracheostomized Fischer 344 adult rats treated neonatally with capsaicin (50 mg/kg) or with vehicle alone. Capsaicin, the hot extract of pepper, releases substance P (SP) from nonmyelinated sensory nerve endings and causes acute bronchoconstriction and airway microvascular leakiness. Chronic treatment with capsaicin leads to depletion of SP and other tachykinins from afferent C-fibers and can therefore be used as a tool to investigate the contribution of SP innervation to airway responses. The rats (9 controls and 6 treated with capsaicin) were paralyzed with succinylcholine and mechanically ventilated at a constant tidal volume and frequency. Airway resistance (RL) and dynamic compliance (Cdyn) were determined at each dose of methacholine from measurements of volume, flow, and transpulmonary pressure. Capsaicin-treated rats were found to have a significantly reduced baseline RL [0.150 +/- 0.039 (SD) vs. 0.225 +/- 0.050 cmH2O.ml-1.s, P = 0.009] and a correspondingly significantly elevated Cdyn (0.371 +/- 0.084 vs. 0.268 +/- 0.053 ml/cmH2O, P = 0.012). There was no significant difference in sensitivity to methacholine, but the maximal response to methacholine was significantly greater in the capsaicin-treated rats. In terms of RL, the maximal response for capsaicin-treated rats was 6.03 x baseline +/- 0.98 vs. 4.30 x baseline +/- 1.80 (P = 0.05) for controls, and for Cdyn changes the maximal decrease was 5.75 x baseline +/- 1.22 vs. 3.83 +/- 0.69 (P = 0.002). The observed differences in RL and Cdyn coupled with the differences in maximal responses can be attributed to the selective destruction of a subpopulation of pulmonary afferent C-fibers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1713908 TI - Molecular and cell biology of cystic fibrosis. AB - The questions emerged in better focus: we need to know, definitively, what CFTR is and what it does. We need to know how mutant CFTR expression leads to the relentless lung disease that takes the lives of the patients. We need to know how the different mutations in CFTR behave functionally. Much more information is needed on the pathways for ion transport in the airways in order for us to consider therapeutic alternatives. Better information on CFTR expression, particularly in the lung, would greatly facilitate consideration of pathophysiology as well as gene therapy. Many of these questions can be attacked by imaginative use of the tools already in hand. The need is urgent. The wondrous scientific advancements of the last five years and the additional money being spent on CF research have bought no dramatic increase in life expectancy for the patients. Every day, three more succumb. PMID- 1713909 TI - Delta beta thalassemia and hereditary persistence of fetal hemoglobin. AB - Delta beta Thalassemia and hereditary persistence of fetal hemoglobin (HPFH) constitute a heterogeneous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Coinheritance of these disorders with other beta chain hemoglobinopathies, such as beta thalassemia and the sickle cell (beta s) gene, can result in attenuation of the clinical severity of these hemoglobinopathies owing to the increased Hb F levels. The molecular basis of these disorders is quite heterogeneous and consists of both deletion and nondeletion types of mutations. The characterization of these molecular defects has provided new insights on the structure and function of important regulatory elements that are involved in the normal control of expression of the beta- and gamma-globin genes and in hemoglobin switching. PMID- 1713910 TI - Beta s-gene-cluster haplotypes in sickle cell anemia. Clinical and hematologic features. AB - Identification of the beta s-gene-cluster haplotype and alpha-gene status provides a useful tool for the detection of the high-risk SS patient. The DNA polymorphisms of the beta s-gene-cluster modulate the clinical course in sickle cell anemia, especially as it involves the risk of end stage organ failure of the kidney, lung, brain, eyes, bones, and leg ulcers. This is schematically represented in Figure 4. The disease severity is modified according to the beta s gene-cluster haplotypes and the co-inheritance of alpha-thalassemia-2. In both Africa and America, the CAR beta s haplotype increases the risk of developing an irreversible complication at an early age. The rate of progression of organ damage is regulated by the beta s-cluster haplotype from birth. The preservation of G gamma Hb F is haplotype dependent and correlates with the overall clinical course of the patient. Further modulation of the clinical course with the co inheritance of alpha-thalassemia-2 tends to decrease the risk of soft-tissue organ failure and increase the risk of osteonecrosis. Epidemiologic studies in Africa together with clinical correlative analysis in Southern California show that SS patients with a Ben haplotype have a less severe illness than those with a CAR and a more severe illness than those with a Sen. A single individual can be expected to fit into the overall pattern. Some sickle related illness will eventually occur in all. The variable clinical manifestations in sickle cell anemia are modified according to the interaction of alpha gene deletions and the beta s-gene-cluster haplotype, are distinct for each organ, and markedly influence the age of onset of end stage major organ failure. In the presence of a Senegal haplotype, the patient's health is better; with the CAR haplotype, it is always worse; severity is intermediate in the Benin haplotype. PMID- 1713911 TI - Hydroxyurea as treatment for sickle cell anemia. AB - Hydroxyurea may be the most promising drug suggested thus far as a treatment for patients with sickle cell anemia, but its safety and efficacy remain unproved, and it probably will not be evaluable for several years. It is not "the answer" for the disease, it seems likely that crises will not be eliminated by treatment, and at this time its use should be reserved for seriously affected adult patients who can participate in a controlled clinical trial. Every hematologist, internist, or pediatrician sees a few patients who are so severely afflicted by sickle cell anemia that their lives seem totally blighted. It is very tempting to consider treating them with hydroxyurea, because the drug is available in any pharmacy, but it is equally important to consider whether the treatment would accomplish any more than treating the physician's own sense of futility, or making the patient feel that "something was being done." If hydroxyurea were as safe as folic acid, treatment for these purposes would be reasonable. It is not, but its use may still be appropriate (although illegal unless used under an Investigational New Drug Agreement, because its use for this purpose is not approved by the FDA). Whether or not it is legal, prescription of hydroxyurea for patients with sickle cell anemia would be ethical or proper only if the potential risks, the variability of the Hb F response, and the lack of proof of clinical efficacy were clearly explained to potential recipients. PMID- 1713912 TI - Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA. AB - Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S 2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1. PMID- 1713913 TI - A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase. AB - A sensitive non-isotopic assay for specific detection of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is described using 5 bromo-2'-deoxyuridine triphosphate (BrdUTP) instead of tritiated thymidine triphosphate. After the RT reaction the template primer is degraded by alkaline hydrolysis. Single-stranded poly.(BrdU) is detected in an immunoenzymometric assay using monoclonal anti-BrdU antibodies. The specificity of the assay is demonstrated by the isolation of RT from virus lysate by an insolubilised monoclonal anti-HIV-1 RT antibody prior to the RT reaction. Immunological RT binding leads to a tenfold increase in analytical sensitivity since substances inhibiting the RT reaction can be removed. This non-isotopic assay is some 30 times more sensitive than the classical radioisotopic RT assay. In terms of RT determination, however, there is a good correlation between these tests (r = 0.96). Several filtrations are no longer necessary to remove non-incorporated nucleotides. The test can be adapted to microtitre plates and hence is easy to automate. PMID- 1713914 TI - Comparative evaluation of bovine immunodeficiency-like virus infection by reverse transcriptase and polymerase chain reaction. AB - Infection of embryonic bovine lung (EBL) cells by bovine immunodeficiency-like virus (BIV) were monitored by reverse transcriptase (RT), syncytia formation and polymerase chain reaction (PCR). Infection can be detected by PCR at 24 h while the presence of syncytia and RT were not detected until much later. The detection of BIV RT can be optimized by changing the pH and salt conditions. The enzyme is very sensitive to changes in pH but can tolerate a wider range of salt and MgCl2 concentrations. Infection of primary human cell cultures by BIV was monitored by both PCR and RT. No active infection of human cells were detectable. PMID- 1713915 TI - Influence of ELISA conditions on detection of serological relationships among luteoviruses. AB - Three variations in the ELISA procedure were used in an attempt to understand the basis for serological relationships among three isolates of barley yellow dwarf virus (BYDV-RPV-I from Illinois, BYDV-RPV-N from New York and BYDV-PAV from Illinois), beet western yellows virus (BWYV) and soybean dwarf virus (SDV). Detection of serological relationships was dependent on the state of the virus particle (e.g. dissociated or intact) and the method of detection (e.g. direct or indirect). In indirect ELISA, where virus particles were dissociated due to incubation in a high pH buffer, all five virus isolates were serologically related. In double antibody sandwich (DAS) ELISA, identification of serological relationships was based on detection of epitopes associated with intact virus particles, which resulted the detection of fewer serological relationships. Direct ELISA showed, depending on the Ig, that the state of the virus particle and/or the method of detection did effect the ability to detect some serological relationships. PMID- 1713916 TI - The polymerase chain reaction for the detection of HIV-1 genomic RNA in plasma from infected individuals. AB - HIV (human immunodeficiency virus) viraemia in serum or plasma of HIV-infected individuals was investigated by the polymerase chain reaction assay (PCR) in combination with reverse transcription to detect HIV-1 genomic RNA. Before PCR, plasma or serum was ultracentrifuged, precipitated virions were then treated with a RNase-free DNase, and a cDNA from the HIV-1 genomic RNA was synthesized. Thirty three fresh plasma and seven sera from either HIV-1 antibody-positive individuals or patients treated with AZT were tested. Plasma from three patients were assayed 3 or 6 months apart. Twelve sera from HIV-1 antibody-negative individuals were used as negative control. PCR was performed with primers in LTR, gag and env regions: 11 of 40 samples were positive with three primer pairs, 16 with two primer pairs and 11 with only one primer pair. PCR on HIV-1 genomic cDNA was positive in 38 out of the 40 plasma or serum samples (95%), regardless of the clinical stage of the infection: HIV-1 was detected in 14 of the 15 untreated subjects and in 24 of the 25 AZT-treated patients. HIV p24 antigen was detected in the serum of 38% of subjects (15 of 40). The results suggest that this method is suitable for the detection of viral particles in plasma or serum from HIV-1 infected individuals irrespective of antiviral treatment. PMID- 1713917 TI - Antigenic analysis of tick-borne encephalitis complex viruses by time-resolved fluoroimmunoassay with monoclonal antibodies. AB - The antigenic structure of 5 strains of tick-borne encephalitis (TBE) virus and 7 other viruses of the TBE complex was examined by the highly sensitive and specific technique of time-resolved fluoroimmunoassay (TR-FIA). A collection of 8 monoclonal antibodies to the Austrian strain. Neudorfl, was used in this study. The findings demonstrate the uniformity of the antigenic structure of TBE viruses from different geographic regions of the USSR. In addition, an epitope was detected which is characteristic of western variants of TBE virus, and another epitope was detected which permits the differentiation of the east-Siberian strain, Aina, from other TBE virus strains. The unique nature of Skalica virus was confirmed, and its similarity, but not identity, to Langat TP-21 virus was shown. Substantial variability in the antigenic structure of some TBE complex viruses was also demonstrated. PMID- 1713918 TI - Synthesis of fusion proteins of Epstein-Barr virus nuclear antigens in E. coli and their antigenicity. AB - Expression and yield in E. coli of a panel of fusion proteins containing various domains of Epstein-Barr virus nuclear antigens, EBNA-1, EBNA-2, EBNA-3, EBNA-4 and EBNA-6, were scrutinized. The antigenicity of the EBNA fusion proteins against human sera was examined. Monospecific antisera to the different EBNA domains were produced by immunizing guinea pigs and rabbits. An EBNA-6 fusion polypeptide was useful for separating anti-EBNA-6 antibody from human sera by immunoaffinity purification. The applications of the fusion proteins to clinical diagnosis are discussed. PMID- 1713919 TI - An ELISA detecting antibody to conserved pestivirus epitopes. AB - A monoclonal antibody based competition-ELISA is described for the detection of pestivirus antibodies directed against conserved epitopes on the p80 viral protein. The ELISA detected increases in serum antibody following experimentally induced infections of pigs, cattle and sheep with a wide range of pestiviruses, although the sensitivity of the test was not uniform for the different viruses studied. The ELISA was compared with virus neutralization tests for the assessment of porcine, bovine and ovine field sera. At a cut-off value of 50% inhibition, the ELISA showed a high specificity relative to virus neutralization tests, but appeared less sensitive for the detection of some weakly positive samples from pigs. Sera from both ruminants and pigs could be assessed without any modification of the test. PMID- 1713920 TI - Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells. AB - Insulin-like growth factor I (IGF-I) stimulates hematopoiesis. We examined whether bone marrow stromal cells synthesize IGF-I. Secretion of IGF-I immunoreactivity by cells from TC-1 murine bone marrow stromal cells was time dependent and inhibited by cycloheximide. Gel filtration chromatography under denaturing conditions of TC-1 conditioned medium demonstrated two major peaks of apparent IGF-I immunoreactivity with molecular weights of approximately 7.5-8.0 kD, the size of native IGF-I, and greater than 25 kD. Expression of IGF-I mRNA was identified by both RNase protection assay and reverse transcription/polymerase chain reaction. To determine whether the greater than 25 kD species identified by RIA possessed IGF-binding activity, a potential cause of artifactual IGF-I immunoreactivity, charcoal adsorption assay of these gel filtration fractions was performed. The peak of IGF-binding activity coeluted with apparent IGF-I immunoreactivity suggesting that TC-1 cells secrete IGF binding protein(s). Unfractionated conditioned medium exhibited linear dose dependent increase in specific binding of [125I]-IGF-I with a pattern of displacement (IGF-I and IGF-II much greater than insulin) characteristic of IGF binding proteins. Western ligand analysis of conditioned medium showed three IGF I binding species of approximately 31, 38, and 40 kD. These data indicate that TC 1 bone marrow stromal cells synthesize and secrete IGF-I and IGF-binding proteins and constitute a useful model system to study their regulation and role in hematopoiesis. PMID- 1713921 TI - Localization of the cystic fibrosis transmembrane conductance regulator in pancreas. AB - Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function. PMID- 1713922 TI - Immunohistochemical analysis of amylase isoenzymes in thyroid cancer. AB - The expression of amylase in various histological types of thyroid cancer was studied by an immunohistochemical technique, using a polyclonal antiamylase antiserum and two monoclonal antibodies specific for salivary and pancreatic-type amylases, respectively. Amylase was expressed in 21 of 24 (88%) thyroid cancers by polyclonal antiserum analysis. Analysis by monoclonal antibodies, however, showed that only 13 (54%) cases and three (13%) cases contained salivary-type and pancreatic-type amylases, respectively. Moreover, immunoreactivity for pancreatic type amylase was detected only in medullary carcinoma; other histological types were positive for salivary-type amylase. These results show that thyroid cancer frequently expresses amylase, and suggest that the differences between amylase isoenzymes in thyroid cancer may correlate with those found between cellular origin of tumour. PMID- 1713923 TI - Galanin-like immunoreactivity in the brain of teleosts: distribution and relation to substance P, vasotocin, and isotocin in the Atlantic salmon (Salmo salar). AB - The presence of galanin-like substances and their relation to substance P-, vasotocin-, and isotocin-immunoreactive neurons and fibers in the brain of teleosts was investigated with immunohistochemical methods. Two specific antisera against synthetic porcine galanin (GAL) revealed cell bodies and fibers in the brain of four different teleost species (Salmo salar, Carassius carassius, Gasterosteus aculeatus, and Anguilla anguilla). In all four species the main location of galanin immunoreactivity was in the hypothalamo-pituitary region. A detailed study of the distribution of galanin immunoreactivity in S. salar showed that galanin immunoreactive (GALir) perikarya were present in the nucleus preopticus periventricularis, an area that may be compared to the supraoptic nucleus in mammals, and in the nucleus lateralis tuberis, a nucleus involved in pituitary control in fishes that may be compared with the arcuate nucleus in mammals. GALir perikarya were found also in the nucleus recessus lateralis and in the nucleus recessus posterior. Numerous GALir fibers were present in the telencephalon and diencephalon, whereas only small numbers of fibers were found in the brainstem. In contrast to the situation in mammals, no GALir perikarya were observed in the brainstem areas corresponding to the noradrenergic locus coeruleus and serotonergic raphe nuclei in S. salar. We did not find any coexistence of GALir substances with arginine vasotocin or isotocin in neurosecretory neurons, as has been shown for galanin with the mammalian counterparts vasopressin and oxytocin. Also, the galanin-like substance(s) and their structurally closest related peptide family, the tachykinins, belong to separate neuronal systems in teleosts. The presence of GALir neurons in brain areas known to be involved in pituitary control, and a massive GALir innervation of the pituitary, strongly indicate a role for galanin-like substances in pituitary control also in teleosts. Furthermore, the presence of extrahypothalamic GALir fibers suggests involvement of galanin-like substances in other brain functions in teleosts. In conclusion, there are general similarities between teleosts and mammals concerning the distribution of galanin-like substances. However, there seem to be substantial differences in their distribution relative to functionally related peptides within the hypothalamo pituitary system. Whereas galanin appears to be colocalized and released together with vasopressin and oxytocin in mammals, in teleosts the homologous substances are contained within different sets of neurons that innervate the same target, the pituitary. PMID- 1713924 TI - The Edinger-Westphal nucleus: sources of input influencing accommodation, pupilloconstriction, and choroidal blood flow. AB - This study used neuroanatomical techniques to investigate sources of afferents to the Edinger-Westphal nucleus (EW) of the pigeon. The EW contains the parasympathetic preganglionic neurons that, by way of the oculomotor nerve, project to the ciliary ganglion (Narayanan and Narayanan, '76; Lyman and Mugnaini, '80). The ciliary ganglion, in turn, innervates the internal musculature of the eye; the ciliary body, the iris sphincter muscle, and the smooth muscle of choroidal blood vessels (Marwitt et al., '71; Pilar and Tuttle, '82). In the bird, the neurons in the ciliary ganglion that innervate the iris sphincter muscle and the ciliary body receive input specifically from cells in the lateral EW (EWl), whereas those that innervate choroidal blood vessels receive input from cells in the medial EW (EWm) (Reiner et al., '83). Thus neurons in the EWl mediate pupilloconstriction and accommodation, whereas neurons in the EWm modulate choroidal blood flow. To study the afferents to EW, injections of horseradish peroxidase (HRP) were placed in this nucleus. These injections resulted in labeled cells in the area pretectalis, a retinorecipient pretectal nucleus and the suprachiasmatic nucleus, a retinorecipient hypothalamic nucleus. We have previously identified both these areas as being sources of afferents to EW (Gamlin et al., '82, '84). In addition, these HRP injections into EW resulted in labeled cells in the medial mesencephalic reticular formation (MRF) lateral and ventral to the oculomotor nucleus and in a localized area of the rostral lateral mesencephalic reticular formation (LRF) dorsolateral to nucleus subpretectalis. Injections of tritiated amino acids into the MRF labeled the entire EW, while such injections into the LRF labeled only the lateral EW. Both of these projections were predominantly contralateral. This study has identified the sources of two previously undocumented inputs to the avian EW. Both sources of input, the MRF and rostral LRF, receive afferents from visuomotor areas of the telencephalon and visual structures in the midbrain. The MRF input to EW could have either direct or modulatory influences on pupil diameter, accommodation, and choroidal blood flow. The LRF input to EW could play a role in controlling accommodation and possibly the pupillary near response. PMID- 1713925 TI - Olfactory projections to the hypothalamus. AB - Electrophysiological recording, together with anterograde and retrograde axonal tracers, was used to provide a comprehensive description of the origin and distribution of the olfactory input to the lateral hypothalamus. This input was much more substantial to the caudal part of the hypothalamus than to the rostral part and originates from several different areas of the olfactory cortex. Positive responses to electrical stimulation of the olfactory bulb were found consistently in the postero-lateral hypothalamus, but only occasionally at more rostral levels. In agreement with this, injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in the posterior half of the lateral hypothalamus labeled cells in four cortical areas that receive input from the olfactory bulb: the anterior olfactory nucleus, the piriform cortex (in the deepest layer or ventral endopiriform nucleus), the olfactory tubercle (in the deep polymorphic layer), and the anterior cortical nucleus of the amygdala. Injections of WGA-HRP in the anterolateral hypothalamus labeled cells only in the anterior cortical nucleus of the amygdala. Anterograde axonal tracing confirmed these projections. Injections of 3H-leucine in the anterior olfactory nucleus, the piriform cortex, and the olfactory tubercle produced axonal label that was light and confined to the medial forebrain bundle in the rostral hypothalamus but was more substantial and extended throughout the lateral hypothalamic area caudally. Injections in the anterior cortical amygdaloid nucleus labeled axons in the anterior hypothalamus and in the premammillary nuclei as well as in the posterolateral hypothalamic area. In addition, a projection was demonstrated to the nuclei gemini from the polymorphic zone deep to the olfactory tubercle. Injections of two fluorescent retrograde tracers into the mediodorsal nucleus of the thalamus and the posterolateral hypothalamus showed that cells projecting to both diencephalic sites were intermingled in all of the olfactory cortical areas except the anterior olfactory nucleus, where cells were labeled only from the hypothalamus. In the deep layer of the piriform cortex and in the anterior cortical amygdaloid nucleus cells were also double labeled, indicating that they send collateral axons to both parts of the diencephalon. PMID- 1713926 TI - Efferent projections from the periventricular and medial parvicellular subnuclei of the hypothalamic paraventricular nucleus to circumventricular organs of the rat: a Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing study. AB - The heterogeneous hypothalamic paraventricular nucleus (PVN) is intimately involved in the regulation of several homeostatic functions. These regulations might, at least partly, be mediated via neuronal projections from the PVN to circumventricular organs outside the blood-brain barrier. To study the efferent projections of the medial and periventricular parvicellular subnuclei of the PVN with particular emphasis on the projections to the circumventricular organs, anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) was applied. Three major efferent pathways and one minor one coursed from the medial and periventricular parvicellular subnuclei to the circumventricular organs. The major fiber projections included a rostral, a lateral, and a dorsocaudal projection tract, whereas the minor projection coursed ventrally. Fibers of the rostral projection were followed to the preoptic area and along the fornix to the subfornical organ. Single fibers originating from this projection coursed further rostrally to the organum vasculosum laminae terminalis. The lateral projection equivalent to the hypothalamo-pituitary tract passed through the lateral hypothalamic area to the median eminence, and nerve terminals were observed throughout the rostrocaudal extent of this structure. A few fibers of this bundle continued into the infundibular stalk and some terminated in the posterior pituitary lobe. Few fibers of the lateral projection descended to caudal pontine levels, where they reached descending fibers of the dorsocaudal projection. The dorsocaudal projection was essentially restricted to midline structures. Along the midline, fibers were followed from the hypothalamus either dorsally through the thalamus to the dorsal part of the third ventricle or caudally alongside the ventricular wall to the mesencephalic periaqueductal grey. The density of fibers decreased along the caudal direction of the neuraxis. The dorsal part of this projection gave rise to terminals in the deep pineal gland and pineal stalk, whereas the caudal part of this projection sent terminating fibers into the area postrema. The minor ventrally directed projection could be followed through the periventricular region to the rostral part of the median eminence. The number of terminals in the circumventricular organs varied. Within the median eminence, a high density of afferents was observed in the entire rostrocaudal extent of the external zone, whereas a low density of fibers was seen in the internal zone. A medium density of afferents was observed in the organum vasculosum laminae terminalis, whereas a relative low density of nerve terminals was observed in the posterior pituitary, the deep pineal gland, the subfornical organ, and the area postrema.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713927 TI - Projections from the periaqueductal gray to the rostromedial pericoerulear region and nucleus locus coeruleus: anatomic and physiologic studies. AB - Previous studies showed that the nucleus locus coeruleus (LC) receives two major afferent inputs from 1) nucleus paragigantocellularis and 2) nucleus prepositus hypoglossi, both in the rostral medulla. Recent reports suggested that the midbrain periaqueductal gray (PAG) projects to the rostromedial pericoerulear area and LC. Since the PAG is a major site for control of central antinociception, and since descending noradrenergic fibers have been implicated in pain modulation, we have investigated in detail the functional anatomy of projections from PAG to the dorsolateral pontine tegmentum. A combined anatomical and electrophysiological approach was used to assess the organization and synaptic influence of PAG on neurons in the rostromedial pericoerulear region and in LC proper. Injections of the tracer wheatgerm agglutinin conjugated to horseradish peroxidase encompassing LC proper and the rostromedial pericoerulear area retrogradely labeled neurons in PAG located lateral and ventrolateral to the cerebral aqueduct; injections restricted to LC proper did not consistently label PAG neurons. Deposits of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into this same region of PAG labeled axons that robustly innervated the zone rostral and medial to LC. Only sparse fibers were observed in LC proper. Consistent with these results, focal electrical stimulation of LC antidromically activated only a few PAG neurons (6 of 100); all of these driven cells were located lateral and ventrolateral to the cerebral aqueduct. The majority of neurons in the rostromedial pericoerulear area were robustly activated by single pulse stimulation of PAG. In contrast, single pulse electrical stimulation of lateral PAG produced weak to moderate synaptic activation of some LC neurons; stimulation of ventrolateral PAG produced predominant inhibition of LC discharge, perhaps through recurrent collaterals subsequent to antidromic activation of neighboring LC cells. Taken together, these results indicate that PAG strongly innervates the region rostral and medial to LC, including Barrington's nucleus, but only weakly innervates LC proper. Although recent studies indicate that the dendrites of LC neurons ramify heavily and selectively in the rostromedial pericoerulear region, the results of the present physiological studies suggest that PAG preferentially targets rostromedial pericoerulear neurons rather than LC dendrites. PMID- 1713928 TI - Cortical connections of the caudal subdivision of the dorsolateral area (V4) in monkeys. AB - Evidence suggests that all primates have rostral and caudal subdivisions in the region of visual cortex identified as the dorsolateral area (DL) or V4. However, the connections of DL/V4 have not been examined in terms of these subdivisions. To determine the cortical connections of the caudal subdivision of DL (DLC) in squirrel monkeys, injections of the neuroanatomical tracers wheat germ agglutinin conjugated to horseradish peroxidase, Diamidino Yellow, and Fluoro-Gold were made in cortex rostral to V II. To aid in delineating the borders of DLC, cortex was also evaluated architectonically. Based on similar patterns of connections, DLC extends from dorsolateral to ventrolateral cortex. DLC receives strong, feedforward input from V II and projects in a feedforward fashion to the rostral subdivision of DL (DLR) and caudal inferior temporal (IT) cortex, including a separate location in the inferior temporal sulcus. DLC has weaker connections with V I, the middle temporal area (MT), cortex rostral to MT in the location of the fundal superior temporal area (FST), cortex dorsal to DLC, ventral cortex rostral to V II, and cortex in the frontal lobe, lateral to the inferior arcuate sulcus. Only lateral DLC has connections with V I, and only dorsolateral DLC has connections with cortex dorsal to DLC. The topographic organization of DLC was inferred from its connections with V II. Thus, dorsolateral DLC represents the lower field, lateral DLC represents central vision, and ventrolateral DLC represents the upper field. Limited observations were made on DLR. Confirming earlier observations (Cusick and Kaas: Visual Neurosci. 1:211, 1988), DLR is paler than DLC myeloarchitectonically. DLR receives only sparse feedforward input from V II, but stronger input from DLC. DLR has strong connections with cortex just rostral to dorsal V II, ventral posterior parietal cortex in the sylvian fissure, MT, the medial superior temporal area, FST, and the inferior temporal sulcus. DLR also shares connections with IT cortex. Thus, while both DLC and DLR are involved in the pathway relaying visual information to IT cortex, an area specialized for object vision, DLR also projects densely to areas such as MT involved in the pathway relaying to posterior parietal cortex, a region specialized for spatial localization and motion perception. PMID- 1713929 TI - Cortical connections of dorsal cortex rostral to V II in squirrel monkeys. AB - A region of dorsal cortex along the rostral border of V II has been described as comprising a visual area or areas separate from more lateral cortex in both New and Old World primates. To evaluate these possibilities in squirrel monkeys, we studied patterns of cortical connections by injecting Fast Blue, Fluoro-Gold, horseradish peroxidase, and wheat germ agglutinin conjugated to horseradish peroxidase into the dorsal region and related results to distinctions in myeloarchitecture. Our major conclusions are as follows. 1) The dorsal region (D) has distinctly different connections from the area found laterally, the caudal subdivision of the dorsolateral area (DLC). These include major connections with the rostral subdivision of the dorsolateral area (DLR), ventral posterior parietal cortex in the Sylvian fissure, the middle temporal area (MT), the medial superior temporal area (MST), ventral cortex just rostral to V II, and cortex in the inferior temporal sulcus. Weaker connections are with V I, V II, DLC, the fundal superior temporal area (FST), and the frontal lobe. In contrast, DLC has strong connections with V II and inferior temporal (IT) cortex, weaker connections with DLR, and lacks connections with ventral posterior parietal cortex (Steele et al: J Comp Neurol 306:495-520, 1991). 2) Caudal and rostral aspects of dorsal cortex differ in the magnitude of connections with V I, V II, DLR, and FST. These differences are consistent with the previous proposal that at least two visual areas, caudal and rostral, occupy the dorsal region in squirrel monkeys (Krubitzer and Kaas: Visual Neurosci 5:165, 1990), but they could also reflect regional differences in the connections of a single visual area. 3) The dorsal region is more densely myelinated than surrounding cortex; however, rostral aspects of dorsal cortex are less myelinated than caudal aspects, again suggesting the existence of at least two areas. 4) The distinctiveness of connections between dorsal cortex and rostral as compared to caudal dorsolateral cortex provides further evidence for dividing the region of DL into two visual areas, DLC and DLR (Cusick and Kaas: Visual Neurosci 1:211, 1988; Steele et al: J Comp Neurol 306:495-520, 1991). PMID- 1713930 TI - Plasma fibronectin and complement following infusion of colloidal solutions after spinal anaesthesia. AB - A randomized study of 30 patients undergoing uncomplicated surgery under spinal anesthesia was conducted to assess the influence of colloids on the kinetics of plasma fibronectin and complement. Both are opsonins of the reticuloendothelial system; moreover fibronectin is concerned with host resistance against septic complications following trauma and surgery. The patients were assigned to receive either Ringer's lactate (Group 1), gelatin (Group 2) or dextran 40 (Group 3). Blood samples were withdrawn before colloids or Ringer's infusion and during the 4 postoperative days. There was a reduction in plasma fibronectin throughout the study in groups 1 and 3, but an increase in group 2 by 24 h. The adhesion of plasma fibronectin to gelatin was maximal 1 h after infusion (44%) and remained significant up to day 2 in group 2. There was no relationship in groups 1 and 3. C3 and C4 components of complement exhibited a low value in the early post operative period, due to hemodilution. This study shows an in vivo fibronectin gelatin interaction, and suggests that gelatin infusion inhibits the increased shift of plasma fibronectin at the site of tissue injury after surgery. PMID- 1713931 TI - Transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride membranes. AB - We have developed a method to transfer proteins from a silver-stained polyacrylamide gel to a polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA). If the silver stained gels are rinsed in 2 x SDS Laemmli sample buffer prior to transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transfer, almost all proteins can be transferred comparably to non stained controls. Some proteins stained with silver can be directly transferred to a single sheet of Immobilon-P without a prior rinse in sample buffer. Most important in the Western blot the antigenicity of the transferred protein is retained in either way. The method described is simple, inexpensive and versatile. A slight modification of the technique permits one to extract minor proteins, or detect their antigenic activities, without contamination of contiguous proteins. PMID- 1713932 TI - Follicle stimulating hormone-secreting pituitary adenoma: inappropriate secretion and effect of pulsatile luteinizing hormone releasing hormone analogue (buserelin) administration. AB - A patient with an FSH secreting pituitary adenoma is reported. Elevated FSH and serum free alpha-subunit (SU) with low levels of LH and testosterone (T) were found. Immunostaining showed the presence of alpha-SU, FSH-beta and LH-beta subunits. LHRH analogue (buserelin) was administered in a pulsatile manner, by portable computerized infusion pump sc for ten days. During the first 24 h of treatment FSH, LH (p less than 0.001) and T (p less than 0.01) rose significantly. Ten days later, the expected desensitization phenomenon did not occur, but further increases of T (8.4 +/- 2.6, mean +/- SD, vs 17.4 +/- 4.1 nmol/l, p less than 0.001) and FSH (58.9 +/- 9.6 vs 70.7 +/- 3.8 mlU/ml, p less than 0.001) were registered. LH decreased (12.5 +/- 2.4 vs 7.1 +/- 0.6 mlU/ml, p less than 0.001) at day 10, but remained higher than basal level (5.0 +/- 0.6, p less than 0.001). Free alpha-SU also rose (2.8 +/- 0.4 vs 4.4 +/- 1.7 mlU/ml, p less than 0.001) after ten days of treatment. The chronic stimulatory effect of analogue on LH with a lack of desensitization suggests tumorous secretion despite a partially preserved negative feedback of testosterone. Low basal LH levels, in some patients with FSH secreting tumors may not be due to tumor mass effect, but rather may be the consequence of altered LH production and/or secretion by the tumor. Although buserelin may not have a therapeutic effect, it is of use in differential diagnosis of hypergonadotropinemia. PMID- 1713933 TI - Expanded ontogeny of neurotransmitters and their metabolites in the brains of fetal and newborn lambs. AB - To evaluate the ontogeny of the brain neurotransmitters norepinephrine, dopamine, serotonin and the metabolites hydroxyindoleacetic acid and homovanillic acid, we measured these neurotransmitters in 10 brain areas at three ages in fetal sheep and two ages in newborn lambs. Norepinephrine exhibited an increase only at 25-30 days after birth in the midbrain, lateral hypothalamus, dorsal medial hypothalamus and ventral medial hypothalamus. Dopamine concentration was very low and did not change over the ages examined. Homovanillic acid decreased after 125 days in the cerebellum, but this change is probably not biologically meaningful, since there were no statistically significant changes in homovanillic acid in other brain areas. Serotonin increased at 25-30 days after birth in the ventral medial hypothalamus, but changes in other brain areas were not significant. Hydroxyindoleacetic acid reached its greatest concentration at 1-5 days after birth in nine of the ten brain areas examined. Thus we conclude that the serotonin system is undergoing more change in the last third of gestation and first month of extrauterine life than the norepinephrine or dopamine systems. PMID- 1713934 TI - Insulin-like growth factor binding proteins: roles in regulating IGF physiology. AB - Insulin-like growth factor binding proteins (IGFBPs) are soluble proteins present in in extracellular fluids. They have high affinity for IGF-I and -II. Blood concentrations are controlled by nutrition and by hormones in a manner that in most, but not all, instances correlates with plasma concentrations of IGF-I or II. IGF binding proteins are secreted by a range of cell types in a manner that may serve to modulate the functions of the growth factors in a pericellular environment. IGF binding proteins cxan modify IGF interaction with the type I receptor and may thereby alter IGF signal transduction through this transmembrane signalling unit. Binding proteins may also act as inhibitors or potentiators of biological responsiveness and thereby directly cell type specific responses. PMID- 1713935 TI - Protease treatment of nitrocellulose-bound antigens enhances the sensitivity of western blots. AB - The sensitivity of Western blots is limited by the avidity of antibody-antigen interactions, and by problems of specificity in interactions between antibodies and antigens from different species. Using rat and human keratins as the antigens, and a set of antibodies against human and rat keratins, this study demonstrates that mild treatment of nitrocellulose blots with trypsin and/or pepsin enhances the sensitivity of the assay and permits the cross-species demonstration of antigens that are not otherwise detectable. PMID- 1713936 TI - Identification of epitopes recognized by a panel of six anti-human IgG2 monoclonal antibodies. AB - Human IgG2 contains several subclass specific amino acid residues or deletions in the CH1 and CH2 domains and also in the hinge region. These substituted residues are the structural correlates for IgG2 specific epitopes. Since human IgG2 has different biological properties from other subclasses, some IgG2 epitopes may be located in regions correlating with sites determining the biological functions. Previously, we produced three anti-IgG2 monoclonal antibodies (mAbs) with highly specific and interesting reactivities using improved immunization protocols. However, it has been almost impossible to identify epitopes conventionally, because human IgG2 is so resistant to proteolysis that various proteolytic fragments could not be isolated. In this study, we identified the epitopes recognized by anti-IgG2 mAbs by SDS-PAGE, Western blotting, amino acid sequence analysis and peptide/mAb binding ELISA, thus overcoming the need for fragment isolation. A panel of six anti-human IgG2 mAbs, including the current WHO/IUIS specificity standards (HP6002, HP6008, HP6014) and our own (HG2-6A, HG2-30F, HG2 56F), reacted with distinct epitopes. The residues essential to expression of the epitopes recognized by the mAbs were: Pro234, Val235 and Val309 for HG2-56F, HG2 30F and HP6008, respectively. HP6014 reacted with the epitope expressed by Thr214 and its neighboring residues. HG2-6A was reactive with the hinge region, and HP6002 was assumed to be directed against discontinuous epitopes requiring intact Fc for expression. Through these studies, the pepsin and papain cleavage sites of human IgG2 were also clarified. PMID- 1713937 TI - [Gene expression in the cells of the central nervous system]. PMID- 1713938 TI - Regulation of plasminogen activator production by endothelial cells: role in fibrinolysis and local proteolysis. PMID- 1713939 TI - The current status of targeting tumour vasculature as a means of cancer therapy: an overview. PMID- 1713940 TI - Ultrastructural studies of tumour angiogenesis in human xenotransplanted tumours. PMID- 1713941 TI - Tumour-associated hyaluronan: a potential regulator of tumour angiogenesis. PMID- 1713942 TI - Matrix integrity and the control of angiogenesis. PMID- 1713943 TI - Antitumour effects of indomethacin alone and in combination with radiotherapy: role of inhibition of tumour angiogenesis. PMID- 1713944 TI - Inhibition of angiogenesis with combination treatments of angiostatic steroids and suramin. PMID- 1713945 TI - The combination of a bacterial polysaccharide and tamoxifen inhibits angiogenesis and tumour growth. PMID- 1713946 TI - En bloc staining available for stereoscopic observation of epoxy resin Quetol 651 embedded thick sections under a high voltage transmission electron microscope. AB - This method has been devised for easy en block staining for stereoscopic observation of thick sections under a high voltage transmission electron microscope (HVTEM). It uses carbohydrazide as an osmium bridging agent and both osmium tetroxide and uranyl acetate as electron staining agents. Osmium tetroxide fixed and en bloc-stained tissue blocks are embedded in a Quetol 651 resin mixture. Thick sections (2-3 microns thick) without double staining are observed at an accelerating potential of 300 kV and a tilt angle of +/- 10 degrees by an H 9000 TEM with a side-entry goniometer. Stereoscopic electron micrographs can be obtained. PMID- 1713948 TI - Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium. AB - The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-. PMID- 1713947 TI - Interfering with calcium release suppresses I gamma, the "hump" component of intramembranous charge movement in skeletal muscle. AB - Four manifestations of excitation-contraction (E-C) coupling were derived from measurements in cut skeletal muscle fibers of the frog, voltage clamped in a Vaseline-gap chamber: intramembranous charge movement currents, myoplasmic [Ca2+] transients, flux of calcium release from the sarcoplasmic reticulum (SR), and the intrinsic optical transparency change that accompanies calcium release. In attempts to suppress Ca release by direct effects on the SR, three interventions were applied: (a) a conditioning pulse that causes calcium release and inhibits release in subsequent pulses by Ca-dependent inactivation; (b) a series of brief, large pulses, separated by long intervals (greater than 700 ms), which deplete Ca2+ in the SR; and (c) intracellular application of the release channel blocker ruthenium red. All these reduced calcium release flux. None was expected to affect directly the voltage sensor of the T-tubule; however, all of them reduced or eliminated a component of charge movement current with the following characteristics: (a) delayed onset, peaking 10-20 ms into the pulse; (b) current reversal during the pulse, with an inward phase after the outward peak; and (c) OFF transient of smaller magnitude than the ON, of variable polarity, and sometimes biphasic. When the total charge movement current had a visible hump, the positive phase of the current eliminated by the interventions agreed with the hump in timing and size. The component of charge movement current blocked by the interventions was greater and had a greater inward phase in slack fibers with high [EGTA] inside than in stretched fibers with no EGTA. Its amplitude at -40 mV was on average 0.26 A/F (SEM 0.03) in slack fibers. The waveform of release flux determined from the Ca transients measured simultaneously with the membrane currents had, as described previously (Melzer, W., E. Rios, and M. F. Schneider. 1984. Biophysical Journal. 45:637-641), an early peak followed by a descent to a steady level during the pulse. The time at which this peak occurred was highly correlated with the time to peak of the current suppressed, occurring on average 6.9 ms later (SEM 0.73 ms). The current suppressed by the above interventions in all cases had a time course similar to the time derivative of the release flux; specifically, the peak of the time derivative of release flux preceded the peak of the current suppressed by 0.7 ms (SEM 0.6 ms). The magnitude of the current blocked was highly correlated with the inhibitory effect of the interventions on Ca2+ release flux.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1713949 TI - Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species. AB - Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species. PMID- 1713950 TI - The use of 16S ribosomal RNA analyses to investigate the phylogeny of the family Legionellaceae. AB - The 16S ribosomal RNA sequences of Legionella pneumophila, L. erythra, L. hackeliae, L. spiritensis, L. longbeachae, L. bozemanii (Fluoribacter bozemanae) and L. micdadei (Tatlockia micdadei) were determined using reverse transcriptase. The sequences were compared with published sequences for Gram-negative bacteria and phylogenetic trees were constructed. The data confirm previous work which showed that the family Legionellaceae forms a monophyletic subgroup within the gamma subdivision of the Proteobacteria. The data show that all of the legionellae studied are highly related (greater than 95%) on the basis of 16S rRNA sequences and do not support the division of the family Legionellaceae into three genera. PMID- 1713951 TI - Immunoglobulin producing cells in bone marrow and blood of patients with multiple sclerosis and controls. AB - Multiple sclerosis (MS) is characterised by intrathecal synthesis of IgG, less frequently of IgA and IgM. Local production of antibodies to myelin basic protein (MBP) and other myelin components has also been reported, and autoimmune pathogenesis has been postulated. Whether MS is accompanied by a systemic B cell response is less clear. To elucidate this question, we examined bone marrow and peripheral blood from patients with MS and controls for cells secreting IgG, IgA and IgM, as well as anti-MBP antibodies of these three isotypes. Patients with MS without any signs of concurrent infections had higher numbers of IgG + IgA + IgM secreting cells both in bone marrow and peripheral blood compared with healthy controls. The same abnormalities were observed in patients with other inflammatory neurological diseases (OIND). When analysing individual isotypes, patients with MS and OIND had higher numbers of IgA secreting cells both in bone marrow and blood compared with healthy controls. Only one of 13 MS patients examined had anti-MBP antibody secreting cells in bone marrow and blood. The systemic B cell response registered in MS is also present in other inflammatory neurological diseases and its specificity and possible role in the pathogenesis of MS remains unknown. PMID- 1713952 TI - Are alpha-1-antichymotrypsin and inter-alpha-trypsin inhibitor peripheral markers of Alzheimer's disease? PMID- 1713953 TI - Purification of autoantibodies to myelin basic protein by antigen specific affinity chromatography from cerebrospinal fluid IgG of multiple sclerosis patients. Immunoreactivity studies with human myelin basic protein. AB - Immunoglobulin G (IgG) was purified by single-step protein A-Sepharose (Pharmacia) affinity chromatography from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and controls. Autoantibodies to myelin basic protein (anti-MBP) were isolated from the purified IgG fraction by two-step antigen specific affinity chromatography. Anti-MBP in the context of whole CSF or in purified form reacts equally to MBP prepared from non-MS or MS brain tissue. Kinetic studies of anti-MBP titers demonstrate that when anti-MBP is reacted with increasing amounts of non-MS or MS MBP, the autoantibody is immunoabsorbed by either antigen in vitro. Immunoabsorption of anti-MBP by MBP or its synthetic peptides may also be possible in vivo as a potential therapeutic tool. PMID- 1713954 TI - Effect of ceruletide on plasma monoamine metabolites in the rabbit. AB - Ceruletide, a cholecystokinin octapeptide-like substance, has been shown to have some effect on tardive dyskinesia. We, too, previously examined the effect of ceruletide on various types of involuntary movement, and found that responders tended to have high plasma homovanillic acid (HVA) levels. It is generally accepted that both central and peripheral sources make a contribution of plasma HVA. In this study, the response of plasma HVA in rabbits to ceruletide was investigated after pretreatment with debrisoquin sulfate, a drug which selectively blocks peripheral HVA production by inhibition of MAO. As a result, 8 and 50 micrograms/kg ceruletide treatment showed a tendency to decrease plasma HVA levels, but showed no significant differences; however, 140 and 200 micrograms/kg ceruletide showed a significant reduction of plasma HVA. These results are important to the understanding of the mechanism of ceruletide's effect on the brain, as well as to predict the effect of ceruletide on involuntary movements. PMID- 1713955 TI - Rethinking clinical endodontic diagnosis. PMID- 1713956 TI - Paradigm shifts in cleaning and shaping. PMID- 1713957 TI - Managing the obstructed root canal space: rationale and techniques. Ensuring the soundness of the remaining tooth structure. PMID- 1713958 TI - Surgical endodontic retreatment. PMID- 1713959 TI - An unconventional surgical technique: sealing a void in a root canal system. PMID- 1713960 TI - A plexiglass perfusion chamber for staining multiple electron microscope grids. PMID- 1713961 TI - Adaptation of a silver impregnation method for electron microscopic visualization of Alzheimer paired helical filaments. PMID- 1713962 TI - Albumin and a dialyzable serum factor stimulate feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum. AB - Previous studies demonstrated that third-stage, developmentally arrested larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to canine serum and hostlike temperature. Experiments to determine the identity of the serum stimulus are described. Serum from several nonhost species stimulated feeding, but to levels lower than canine serum. Heating the serum to 57 C had no effect on its stimulatory ability. Dialysis reduced serum stimulatory activity by 50%, and ultrafiltration through 10- and 30-kDa molecular weight cut-off membranes decreased activity in both the filtrates and retentates similarly. Recombination of the filtrates and retentates restored activity to whole serum control levels. Commercial canine and bovine albumin stimulated feeding to serum control levels at 10 and 50 mg/ml, respectively. These results suggest that albumin and an unidentified low molecular weight compound(s) are capable of inducing in vitro feeding by A. caninum L3. PMID- 1713963 TI - Effect of ecdysterone on histamine release from rat peritoneal mast cells. AB - Ecdysterone dose-dependently inhibited anti-IgE-induced histamine release from mast cells. Moreover, the rate and extent of histamine release from mast cells induced by Concanavalin A (Con A) are significantly diminished in samples incubated with ecdysterone. Ecdysterone inhibited both the initial and gradual rise in fluorescent response by anti-IgE and Con A. The effects of ecdysterone on the fluorescence response was correlated with the inhibition of histamine release. These results suggest the possibility that the inhibition of histamine release from rat mast cells by ecdysterone might be due to inhibition of Ca2+ mobilization from intracellular Ca2+ storage. PMID- 1713964 TI - Inhibitory effects of substance P and carbachol on the saturable sodium-dependent uptake process of myo-inositol in rat parotid gland. AB - myo-Inositol uptake in prisms of rat parotid glands was investigated by measuring both the accumulation of free myo-[3H] inositol into the cytosol and its incorporation into phospholipids. Total myo-[3H]inositol uptake involved two distinct processes, a prominent one which is saturable and sodium-dependent (Km, 95 microM; Vmax, 8 pmol/mg of protein per min) and a minor one, nonsaturable and sodium-independent. Phloretin and cytochalasin B, two inhibitors of hexose transport, and D-glucose, but only at high concentrations (greater than 10 mM), inhibited myo-[3H]inositol uptake. Dixon plots of the data indicated that D glucose inhibition was noncompetitive suggesting that myo-inositol and D-glucose are transported by different carriers. Electrogenic cotransport of sodium and myo inositol, rather than energy derived from mitochondrial oxidative metabolism, seems to be involved in the transport process. Thus, ouabain, monensin or veratridine, all of which increase intracellular sodium concentrations, reduced myo-[3H]inositol uptake, whereas dinitrophenol, potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone were without effect. Substance P affected only the sodium-dependent uptake process of myo-[3H]inositol, this inhibitory effect requiring extracellular calcium. Similar observations were made with the muscarinic agonist carbachol. From these results, an increase in intracellular sodium concentration linked to the activation of calcium-sensitive cation permeant channels appears to be responsible for the inhibitory effects of substance P and carbachol on myo-[3H]inositol uptake, these effects being mediated respectively by NK1 and muscarinic receptors coupled to a phospholipase C. PMID- 1713966 TI - Teaming up to cut costs. PMID- 1713965 TI - Binding of the dihydropyridine calcium channel blocker (+)-[3H] isopropyl-4 (2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-3 pyridinecarboxylate (PN200-110) to RINm5F membranes and cells: characterization and functional significance. AB - This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl 3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200 110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200 110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+) [3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites. PMID- 1713967 TI - The importance of communication between the anesthesiologist and the postanesthesia care unit nurse. PMID- 1713968 TI - Qualitative research methodologies: an overview, Part I. AB - In these days of budget constraints and recession, PACU nurses will have to demonstrate that their nursing interventions are economical and effective. Their nursing practice needs to be shaped by scientific knowledge. The purpose of this article is to present an overview of qualitative research methods that can be used by PACU nurses. The methods described and discussed in this article include phenomenology, ethnography, grounded theory, and case studies. Part II of this article, which will be published in a subsequent issue of this journal, will present research problems that are of interest and the ways in which PACU nurses can use the qualitative research designs discussed in part I of the article. PMID- 1713969 TI - White noise analysis of graded response in a wind-sensitive, nonspiking interneuron of the cockroach. AB - 1. A novel approach using a Gaussian white noise as stimulus is described which allowed quantitative analysis of neuronal responses in the cercal system of the cockroach, Periplaneta americana. Cerci were stimulated by air displacement which was modulated by a sinusoidal and a white noise signal. During the stimulation, intracellular recordings were made from a uniquely identifiable, nonspiking, local interneuron which locates within the terminal abdominal ganglion. The white noise stimulation was cross-correlated with the evoked response to compute first- and second-order kernels that could define the cell's response dynamics. 2. The interneuron, cell 101, has an exceptionally large transverse neurite that connects two asymmetrical dendritic arborizations located on both sides of the ganglion. 3. The first-order Wiener kernels in cell 101 were biphasic (differentiating). The waveforms of the kernels produced by the ipsilateral and contralateral stimulations were roughly mirror images of each other: the kernels produced by wind stimuli on the side ipsilateral to the cell body of the interneuron are initially depolarized and then hyperpolarized, whereas those on the other side are initially hyperpolarized. The polarity reversal occurred along the midline of the animal's body, and no well-defined kernel was produced by a stimulus directed head on or from the tail. 4. Mean square error (MSE) between the actual response and the model prediction suggests that the linear component in cell 101 comprises half of the cell's total response (MSEs for the linear models were about 50% at preferred directions), whereas the second-order, non linear component is insignificant. The linear component of the wind-evoked response was bandpass with the preferred frequency of 70-90 Hz. 5. Accounting for a noise, we reasonably assumed that at high frequencies the graded response in cell 101 is linearly related to a modulation of the air displacement and sensitive to the rate of change of the signal (i.e., wind velocity) and the direction of its source. It is suggested that the dynamics of the first-order kernel simply reflect the dynamics of sensory receptors that respond linearly to wind stimulation. PMID- 1713970 TI - Differential expression of complement regulatory proteins on subpopulations of human trophoblast cells. AB - Trophoblast cells forming the reactive interface between the mother and her semiallogeneic fetus risk attack by cellular and humoral elements of the maternal immune system. Biochemical, molecular, and immunohistologic studies have identified membrane cofactor protein (MCP) and decay accelerating factor (DAF) on trophoblast cells, which could assist in preventing lysis of the cells by complement-activating maternal antibodies. In this immunocytochemical study, differential expression of these two members of the family of complement regulatory proteins on subpopulations of human trophoblast cells and other types of cells in first and third trimester placentas was demonstrated. Staining with anti-MCP was particularly strong on villous cytotrophoblast cells and giant cells in first trimester tissues in comparison with other types of cells. In contrast, staining with anti-DAF was strong on proliferating cytotrophoblast in first trimester tissues, and on basal plate cytotrophoblast and decidual cells in term tissues. Placental villous mesenchymal cells but not trophoblast cells expressed a third regulatory protein, complement receptor 1. These observations support the postulate that complement regulatory proteins are critical to protection of the fetal allograft, and suggest specific requirements for trophoblast cells according to stage of differentiation and anatomic location. PMID- 1713971 TI - Does fetal seizure activity mean a poor outcome? A case report. AB - Fetal seizure activity is very rare: only three cases have been reported. A case of fetal seizure activity was detected with ultrasound. Such activity can be associated with a poor outcome. PMID- 1713972 TI - Adsorption with a soluble E. coli antigen fraction improves the specificity of ELISA tests for Lyme disease. AB - We reported that preadsorption of patient serum with heat killed E. coli increased the specificity of ELISA for antibodies to Borrelia burgdorferi. That procedure required extra specimen handling and a preincubation. We report the use of a soluble E. coli antigen fraction that is included in serum diluent, eliminating additional steps. Sera from 220 individuals were tested for antibodies to B. burgdorferi. Twenty sera were obtained from patients with Lyme disease and 200 sera were from a population that included healthy controls and patients with different inflammatory conditions (viral infections and various rheumatic disorders). Testing was performed using either a standard serum diluent or one containing soluble E. coli antigen fraction. Results demonstrate that inclusion of soluble E. coli antigen fraction in serum diluent increased assay specificity from 88% for the standard protocol to 98%, with no change in test sensitivity. PMID- 1713973 TI - The incidence of different cystic fibrosis mutations in the Scottish population: effects on prenatal diagnosis and genetic counselling. AB - We present an analysis of the frequency of 16 different cystic fibrosis (CF) mutant alleles in the Scottish population. Each allele was detected in DNA amplified by the polymerase chain reaction (PCR) either directly on polyacrylamide gels, on agarose gels after restriction enzyme digestion, or by using allele specific oligonucleotides. Among 506 CF chromosomes, of predominantly Scottish origin, the frequencies of the different mutations were delta F508 0.71, G551D 0.05, G542X 0.04, R117H 0.01, 1717-1G----A 0.01, A455E + delta I507 + R553X + R560T + W1282X + 621 + 1G----T combined 0.03, unpublished 0.01, and unknown 0.13. No examples of D110H, R347P, S549N, S549I, or 2566ins AT mutations were found. The relevance of this type of analysis for both prenatal diagnosis and heterozygote screening is discussed. PMID- 1713974 TI - Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel. AB - Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (Mr 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal by the endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope. 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983, Biochemistry 22:462-470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis. PMID- 1713975 TI - Ion channels in the plasma membrane of Amaranthus protoplasts: one cation and one anion channel dominate the conductance. AB - This report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons of Amaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier. The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K+) and relative permeability sequence K+ greater than Na+ much greater than Cl- (1:0.16:0.03): whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of subconductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (Vm) is depolarized; at millimolar levels of [Ca2+]cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2 mM) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO3- greater than Cl- greater than K+ greater than Aspartate (2.04:1:0.18 to 0.09:0.04). The relative permeability for K+ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clamp Vm at a preferred value in the face of events which would otherwise perturb Vm. PMID- 1713976 TI - Molecular cloning, expression and epitope mapping of autoantigens. PMID- 1713977 TI - A case of hepatocellular carcinoma with the sign of Leser-Trelat: a possible role of a cutaneous marker for internal malignancy. AB - A rare case of hepatocellular carcinoma who developed the complication of the sign of Leser-Trelat is reported. The patient, a 57-year-old male, visited our hospital with complaints of generalized malaise and anorexia. A diagnosis of hepatocellular carcinoma was made based on elevated alpha-fetoprotein measurement, ultrasonography, and hepatic arteriography findings. Chest x-ray film suggested pulmonary metastases of hepatocellular carcinoma. Thereafter, complications of the seborrheic keratosis developed in the trunk and the skin lesion was diagnosed as the sign of Leser-Trelat associated with hepatocellular carcinoma. The patient died of pneumonia 9 months after development of the sign of Leser-Trelat. PMID- 1713978 TI - Marked hypophosphatemia with decreased serum 1,25-dihydroxyvitamin D in a patient with hepatocellular carcinoma complicating liver cirrhosis. AB - A 61-year-old male with hepatocellular carcinoma (HCC) complicating liver cirrhosis presented hypophosphatemia progressing with HCC expansion and serum alpha-fetoprotein elevation. These changes were associated with an increased fractional excretion of phosphate and decreased theoretical phosphate threshold. There was increased nephrogenous cyclic adenosine monophosphate despite normal serum parathyroid hormone. Serum 1,25-dihydroxyvitamin D levels were markedly reduced with normal 25-hydroxyvitamin D levels. There were no symptoms of osteomalacia, however, a slightly increased osteoid seam was elicited on autopsy. The hypophosphatemia could be explained by presumed secretion from HCC of humoral factors which have a phosphaturic effect and also inhibit 25-hydroxyvitamin D-1 alpha-hydroxylase in renal tubular cells. PMID- 1713979 TI - [Various erythrocyte indices in patients with chronic hypoxia of different etiologies]. PMID- 1713980 TI - Inhibitory effects of somatostatin on rat hepatocyte proliferation are mediated by cyclic AMP. AB - Somatostatin (SS-14) is known as an antigrowth factor for a variety of cell types, including gastrointestinal mucosa, exocrine pancreas, lymphocytes, and some tumors. We have recently identified and biochemically characterized SS-14 binding protein on rat liver plasma membranes (S. E. Raper, P. C. Kothary, and J. DelValle, Gastroenterology 96: A408, 1989; P. C. Kothary et al., Digestion 46 (Suppl 1): 58, 1990). We hypothesized that SS-14 may affect liver growth as well and investigated cellular mechanisms of this phenomenon focusing on the second messenger cAMP. Freshly isolated rat hepatocytes were plated on tissue culture dishes coated with Matrigel (laminin, heparan sulfate, and type IV collagen). The medium was not supplemented with serum or hormones. Either dibutyryl-cAMP (1 mM) or isobutylmethylxanthine (IBMX, 0.1 mM) was added in the presence or absence of SS-14 (10 nM). DNA synthesis was estimated by the rate of [3H]thymidine incorporation into DNA and by the labeling index (an autoradiographic measurement of the number of labeled nuclei). SS-14 significantly inhibited both [3H]thymidine incorporation and labeling index of rat hepatocytes stimulated by dibutyryl-cAMP or IBMX. SS-14 also inhibited intracellular cAMP accumulation stimulated by IBMX. We conclude that SS-14 exerts at least part of its antiproliferative effects via the adenylate cyclase system. Further study using other signal transduction systems may yield more information about mechanisms of hepatocyte growth. PMID- 1713981 TI - Demographic factors and the antihypertensive effect of diltiazem. AB - The maximum blood pressure (BP) decrease obtained after dose titration with calcium antagonists is said to be greater in older patients. Because the dose necessary to achieve this maximum effect may also vary, it is not clear whether the sensitivity to treatment is actually increased in older patients. We evaluated the possible influence of pretreatment BP, age, and weight on the BP and heart rate (HR) response to 14-day treatment with a fixed dose of 120 mg diltiazem twice daily (b.i.d.) in 231 hypertensive patients aged 24-82 years (44 +/- 27). Diltiazem decreased BP from 171 +/- 1/103 +/- 7 to 156 +/- 1/91 +/- 1 mm Hg. Decreases in both systolic and diastolic BP (SBP, DBP) were related to their pretreatment values (p less than 0.0001 for both). Although pretreatment SBP was related to age (p less than 0.0001), its decrease with diltiazem was not. Neither pretreatment DBP nor its decrease with diltiazem was related to age; BP decrease was not superior in elderly patients (aged greater than 60 years) as compared with that in younger patients (SBP -16 +/- 2 vs. -15 +/- 1 mm Hg, NS; DBP -13 +/- 1 vs. -12 +/- 1 mm Hg, NS). In conclusion, the response to this average dose of diltiazem is related to pretreatment BP and is not affected by patient's age. Because this result is at variance with the concept that calcium antagonists are more effective in the elderly, this concept should not be used as a general therapeutic guideline. PMID- 1713982 TI - Effects of SR 44866, a potassium channel opener, on action potentials of rabbit, guinea pig, and human heart fibers. AB - The effects of various concentrations (3 x 10(-8) - 1 x 10(-5) M) of SR 44866, a K+ channel opener, on action potential (AP) characteristics were investigated in isolated rabbit sinoatrial node (SAN), rabbit Purkinje fibers, guinea pig ventricle, human atrium, and human papillary muscle. SR 44866 (up to 1 x 10(-5) M), like cromakalim and pinacidil, did not modify SAN AP and automaticity of the rabbit heart. In atrial, Purkinje and ventricular fibers of animal and human hearts, SR 44866 did not significantly change membrane resting potential, AP amplitude, or maximum rate of phase 0 (dV/dtmax). The main AP modifications induced by SR 44866 were concentration-dependent reductions in plateau amplitude and AP duration (APD): IC50 2 x 10(-7), 7 x 10(-7), 1.4 x 10(-6), 2.5 x 10(-6), and much greater than 10(-5) M for human atrium, human ventricle, guinea pig ventricle, rabbit Purkinje, and rabbit atrium, respectively. In isolated guinea pig heart, SR 44866 induced decreases in contractions (IC50 1.7 x 10(-6) M) and coronary perfusion pressure (CPP) (IC50 2.1 x 10(-8) M) with a very slight reduction (5% at 1 x 10(-6) M) in spontaneous heart rate (HR). Negative inotropic effect (guinea pig) and APD shortenings (guinea pigs and humans) of SR 44866 (1 x 10(-6) and 3.10(-6) M) were antagonized by glibenclamide (3 x 10(-7) to 3 x 10( 6) M), a specific blocker of cardiac K+ATP channels. The data support the hypothesis that SR 44866 activates ATP-sensitive K+ channels, which are present in the atria and ventricles of the human heart but not in pacemaker cells of rabbit SAN. PMID- 1713983 TI - Effects of bradykinin on inducible sustained ventricular tachycardia two weeks after myocardial infarction in pigs. AB - We studied the in vivo effect of bradykinin infusion on inducible sustained ventricular tachycardia (VT) 2 weeks after myocardial infarction in pigs, based on the assumption that the antiarrhythmic effect of angiotensin-converting enzyme (ACE) inhibitors may, apart from their angiotensin-II lowering effect, also be due to elevation of endogenous bradykinin levels. Of the six pigs with inducible VT in the control state, four were noninducible during subsequent bradykinin infusion (p less than 0.05). The ventricular effective refractory period (VERP) did not change during bradykinin infusion (from 237 +/- 37 to 239 +/- 42 ms), nor did intraventricular conduction change (filtered QRS duration was 45 +/- 17 ms before and 43 +/- 19 ms during infusion). Bradykinin caused both a significant systolic blood pressure (SBP) decrease (from 79 +/- 14 to 49 +/- 4 mm Hg, p less than 0.001) and diastolic BP (DBP) decrease (from 41 +/- 10 to 27 +/- 4 mm Hg, p less than 0.01). In conclusion, exogenous bradykinin reduced the inducibility of sustained VT 2 weeks after myocardial infarction. Because refractory periods or conduction velocity were not affected, the mechanism of action might be associated with the BP decrease, which can decrease wall stress. The previously reported antiarrhythmic effect of ACE inhibitors may be due in part to elevation of endogenous bradykinin levels. PMID- 1713984 TI - Effect of calcium channel blocking agents on infarct size after ischaemia reperfusion in anaesthetised pigs: relationship between cardioprotection and cardiodepression. AB - We compared the abilities of verapamil and nicardipine to protect the porcine myocardium from the consequences of ischaemia-reperfusion in vivo. Infusion of verapamil (50 micrograms/kg) into the left anterior descending coronary artery LAD (i.c.a.), in 15 min immediately before ligation depressed regional contractile function, reduced infarct size by 80%, and enabled contractile function to recover partially during reperfusion. Verapamil (10 micrograms/kg i.c.a.) did not depress contractile function before ligation or permit its recovery during reperfusion, despite reducing infarct size by 80%. Lower doses of verapamil were not cardioprotective. Nicardipine (10 and 30 micrograms/kg i.c.a.) depressed contractile function before ligation but did not permit its recovery during reperfusion. Nicardipine did not reduce infarct size development. Thus, drug-induced negative inotropic activity (which presumably reflects myocardial calcium channel blockade) and cardioprotection are not linked. Verapamil can markedly reduce infarct size development at a dose that exerts no detectable negative inotropic activity. This cardioprotective effect of verapamil was greatly reduced by intravenous (i.v) pretreatment with aspirin (30 mg/kg), which alone did not alter infarct size development. Thus, the cardioprotective effect of verapamil (10 micrograms/kg i.c.a.) appears to be mediated by a cyclooxygenase product, possibly prostacyclin. PMID- 1713985 TI - Converting enzyme inhibition in coronary artery disease: a randomized, placebo controlled trial with benazepril. AB - The antiischemic efficacy of the converting enzyme inhibitor (CEI) benazepril was investigated in a randomized, placebo-controlled double-blind study with intraindividual crossover in 11 normotensive patients with angiographically proven coronary artery disease. Bicycle ergometry and 24-h ambulatory ECG were performed before and after 2-week treatment with placebo and benazepril, respectively. Plasma concentrations of atrial natriuretic peptide (ANP) and plasma renin activity (PRA) were measured before each exercise test. Maximal exercise-induced ST-segment depression was not significantly influenced by benazepril therapy (placebo 2.09 +/- 1.22 mm, benazepril 1.91 +/- 1.00 mm). Systolic blood pressure/heart rate (SBP/HR) product at maximum workload remained almost constant with 253 +/- 43 with placebo and 253 +/- 39 with benazepril treatment. The number of anginal attacks and ischemic episodes detected by ambulatory ECG were not significantly reduced. PRA increased significantly from 2.18 +/- 3.76 to 9.62 +/- 8.49 ng/ml/h after benazepril (p less than 0.005), whereas plasma concentrations of ANP remained unchanged (28.04 +/- 12.39 vs. 26.73 +/- 11.09 pg/ml). Therefore, measurement of ST-segment depression with exercise in 11 normotensive patients with coronary artery disease produced no evidence of an antiischemic action for the CEI benazepril 10 mg twice daily (b.i.d.) for 2 weeks, but an improvement was observed in six patients. PMID- 1713986 TI - Role of blood pressure in the natriuretic response to acute calcium channel blockade in humans. AB - Glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and renal excretion of sodium and lithium were measured before and after acute oral administration of 20 mg nifedipine in 19 essential hypertensive patients. In 10 of them, with a diastolic pressure less than 105 mm Hg, nifedipine resulted in a decrease in mean blood pressure toward normal (109 +/- 2 to 97 +/- 2, p less than 0.001), a 27% increase in ERPF (p less than 0.001), no change in GFR, and an increase in fractional sodium excretion (28%, p less than 0.001). In nine subjects with a diastolic pressure greater than or equal to 105 mm Hg, nifedipine produced a decrease in mean blood pressure (133 +/- 6 to 117 +/- 4, p less than 0.001), which however remained higher than in mild hypertensives (p less than 0.001). ERPF rose by 29% (p less than 0.001), GFR remained unchanged, and fractional sodium excretion definitely increased more than in mild hypertensives (126%, p less than 0.001), as did fractional lithium excretion, used as an estimate of proximal tubular sodium handling. Acute nifedipine produces renal vasodilation in hypertensives, but with a greater natriuretic response in those subjects whose blood pressure remains elevated. Thus, acute natriuresis following nifedipine administration is largely dependent on the interaction between changes in arterial pressure and renal hemodynamics. PMID- 1713987 TI - Effects of nicardipine on pulmonary and systemic vascular reactivity to oxygen in patients with pulmonary hypertension secondary to chronic obstructive lung disease. AB - We compared the acute effects of nicardipine and a placebo on the response of pulmonary and systemic circulation to different inspiratory fractional concentrations of O2 (FiO2) in 10 patients with pulmonary hypertension secondary to chronic obstructive lung disease. After catheterization of the pulmonary and femoral arteries, gas mixtures containing 15, 21, and 30% O2 were randomly administered for 20 min each during infusion of saline and then nicardipine (0.06 mg/kg/min). Plasma nicardipine level was maintained at 30 ng/ml. During nicardipine infusion, cardiac index (CI) was significantly higher (+20%, p less than 0.05) than during placebo infusion, with no change in mean pulmonary artery pressure (MPAP). Pulmonary resistances also decreased significantly (-20%) during nicardipine. No change in arterial or mixed venous O2 contents was noted. Mean arterial pressure (MAP) and systemic resistances decreased significantly with nicardipine. Inhaling a hyperoxic mixture was followed by a significant decrease in arterial pressure during placebo infusion; this was not observed during nicardipine. In contrast with systemic circulation, the response of the pulmonary circulation to different FiO2 levels was unaffected by nicardipine. PMID- 1713988 TI - Thrombolytic properties of a novel modified human tissue-type plasminogen activator (E6010): a bolus injection of E6010 has equivalent potency of lysing young and aged canine coronary thrombi. AB - The thrombolytic properties of a novel modified human tissue plasminogen activator (E6010), in which cystein 84 in the epidermal growth factor domain is replaced by serine and that has a prolonged biological half-life, were examined. The thrombolytic efficacies of E6010 and recombinant human tissue plasminogen activator (rt-PA) on the duration of coronary artery thrombus were evaluated in a canine model (123 anesthetized dogs) with copper coil-induced left anterior descending coronary artery thrombus. Thrombi established for periods of 1, 3, or 6 h, as documented by coronary arteriography, were employed. A single bolus i.v. injection of E6010 or rt-PA and an i.v. infusion of rt-PA over 60 min were compared (n = 6). Thrombolytic efficacy was evaluated by three criteria: time to reperfusion (TR), reperfusion rate at 60 min (RR), and reocclusion rate at 60 min after reperfusion (OR). With a bolus i.v. injection of E6010 at a dose of 0.2 mg/kg or an i.v. infusion of rt-PA at a dose of 0.6 mg/kg/h, these parameters were as follows: TR, 30.0 +/- 15.3 and 27.5 +/- 4.8 min; RR, 100 and 100%; OR, 17 and 33% for 1-h aged thrombi; TR, 30.0 +/- 9.5 and 35.0 +/- 8.2 min; RR, 83 and 50%; OR, 20 and 67% for 6-h aged thrombi. These data indicate that a bolus injection of E6010 is almost equally efficacious in lysing thrombi aged both 1 and 6 h. On the other hand, in the case of rt-PA, the thrombi aged 6 h were lysed significantly less than the thrombi aged 1 h. Plasma half-lives of E6010 were t1/2 alpha, 4.8 +/- 0.95 (estimated by antigen level) and 3.0 +/- 0.78 min (estimated by activity), and t1/2 beta, 51 +/- 5.4 (antigen level) and 22 +/- 7.0 min (activity). The half-lives of rt-PA were t1/2 alpha, 3.6 +/- 0.23 (antigen level) and 2.1 +/- 0.61 min (activity), and t1/2 beta, 36 +/- 2.3 (antigen level) and 7.0 +/- 3.5 min (activity). We conclude that a bolus injection of E6010 may have a more potent and longer-lasting effect than i.v.-infused rt-PA in clot lysis therapy. PMID- 1713989 TI - An alpha-adrenergic coronary constriction during esophageal distention in the dog. AB - Previous work has shown an increase in sympathetic stimulation of the heart during chemical or mechanical irritation of visceral organs, but the involvement of the coronary circulation in such reflexes is not clear. In five preliminary experiments in anesthetized dogs, esophageal distention produced a sympathetic stimulation of the heart, as evidenced by an increase in heart rate, which was abolished by non-selective beta-adrenergic and muscarinic blockades. On the basis of these preliminary data, we further examined a sympathetic coronary constriction during acute esophageal distension in which any direct adrenergic coronary constriction was unmasked by muscarinic blockade with atropine (100 micrograms/kg, i.v.), and non-selective beta-adrenergic blockade with propranolol (1 mg/kg, i.v.). In seven dogs anesthetized with alpha-chloralose in an open chest procedure, the esophagus was rapidly distended to a pressure of 36 +/- 2 mm Hg, which was not significantly different from the distending pressure used in the preliminary experiments. During esophageal distention, the mean circumflex blood flow decreased to 77 +/- 10% (SEM) of the predistention value. This decrease was statistically significant (p less than 0.05). There was no change in left ventricular pressure, mean arterial pressure dP/dtmax, or heart rate. Intracoronary administration of the nonselective alpha-adrenergic antagonist phentolamine completely abolished the reduction in mean circumflex coronary blood flow caused by esophageal distention in the presence of beta-adrenergic and muscarinic blockades. These results demonstrate a direct sympathetic coronary vasoconstriction elicited by esophageal distention. This vasoconstriction was due to activation of coronary alpha-adrenergic receptors. PMID- 1713991 TI - Examination of two small-molecule antiperoxidative agents in a rabbit model of postischemic myocardial infarction. AB - Peroxidation of membrane phospholipid may be a causal contributor to the development of irreversible postischemic myocardial injury. In this study, two small-molecule antiperoxidative agents were tested for their ability to salvage reperfused rabbit myocardium and reduce infarct size as assessed by direct histological evaluation of hearts following a 30-min occlusion of a coronary arterial branch and a 72-h reperfusion period. The compounds tested are novel analogs of alpha-tocopherol (vitamin E), and share with the parent compound an ability to scavenge the peroxyl radicals that propagate and amplify lipid peroxidation initiated by partially reduced oxygen; however, the new analogs are significantly more potent peroxyl-radical scavengers that alpha-tocopherol. Each antiperoxidant (3 mg/kg) was administered by intravenous bolus injection in two stages: 20% of the total dose was given 15 min before occlusion, and the remaining 80% was given 5 min before reperfusion. The results revealed that neither antiperoxidant offered a sustained reduction in infarct size (expressed as a percentage of the region at risk) as compared to a nontreated vehicle control. The implications of the present study with respect to the purported cardioprotective effects of antiperoxidants in the setting of ischemia reperfusion and the pathogenic role of lipid peroxidation in the postischemic heart are discussed. PMID- 1713990 TI - Intracellular resistance in rat papillary muscle: interaction between cyclic AMP and calcium. AB - The influence of isoproterenol (10(-9)M) and high calcium solution (6 mM) on the intracellular longitudinal resistance (ri) on rat papillary muscle was investigated. The muscles were stimulated at 1 Hz. Isoproterenol (10(-5)M) reduced ri within 10 s while high calcium solution (6 mM) increased ri appreciably. In muscles previously exposed to high calcium solution, isoproterenol increased ri further. This increment of ri, which was suppressed by verapamil (10(-5) M), indicates that when the inward movement of calcium through surface cell membrane is appreciably enhanced, the increase in free (Ca)i counteracts the effect of cyclic AMP on ri. Forskolin (10(-5)M), an activator of adenyl cyclase, also reduced ri in muscles immersed in normal saline solution. The results indicate that cyclic AMP and calcium have opposite effects on the control of ri. PMID- 1713992 TI - A novel, orally active dopamine prodrug TA-870. V. Natriuretic and positive inotropic effects in rats: an assessment after chronic administration. AB - We investigated the acute natriuretic and positive inotropic effects of the dopamine prodrug TA-870 in rats before and after repeated administration for 2 weeks. Single intraduodenal (i.d.) administration of TA-870 (10-250 mg/kg) to saline-loaded anesthetized rats produced a dose-dependent increase in urinary flow and sodium excretion. It also produced a decrease in renal vascular resistance and an increase in renal blood flow. In another series of normal anesthetized rats, TA-870 caused dose-dependent increases in cardiac contractility [left ventricular dP/dtmax (LV dP/dtmax)] at i.d. doses of 10-250 mg/kg. Although the heart rate was also increased, this effect was much smaller than the effect on LV dP/dtmax. SCH-23390 (0.3 mg/kg i.v.), a selective DA1 dopamine receptor antagonist, strongly inhibited the above diuretic, natriuretic, and renal vasodilatory effects of TA-870. The positive inotropic effect of TA-870 was not inhibited by SCH-23390, but the latter effect was inhibited by pretreatment with propranolol (0.5 mg/kg i.v.). After repeated oral administration of TA-870 to rats (250 mg/kg twice a day for greater than 2 weeks), there was no significant differences in the natriuretic and positive inotropic responses to TA-870 between the TA-870-pretreated and control groups indicating a lack of pharmacological tolerance. In conclusion, TA-870, when administered enterally to rats, produced the natriuretic effect via the DA1 dopamine receptor and positive inotropic effects via the beta 1 adrenergic receptor stimulation, and these effects were not attenuated by chronic treatment with TA-870. PMID- 1713993 TI - Effect of nimodipine on subintimal hyperplasia of autologous vein bypass grafts in rats: a placebo-controlled study. AB - Autologous vein grafts are commonly used conduits for coronary bypass grafts. However, as many as 20% of the grafts may occlude in the first year to a subintimal hyperplasia. Although the initiating mechanism remains unclear, it has been demonstrated that subintimal hyperplasia is dependent upon smooth muscle cell proliferation and migration from the medial to the intimal layer. The present study focused on the prevention of smooth muscle cell proliferation using a calcium antagonist. A vein bypass graft from the jugular vein on the abdominal aorta was performed in 40 rats, divided into two groups of 20. Animals in the treated group received nimodipine (15 mg/kg of body weight), and those of the control group received a placebo. Nine months after grafting, the results showed that the group receiving nimodipine presented no or only slight subintimal hyperplasia as compared with the placebo group (p less than 0.001). The data presented in this study show that nimodipine can reduce subintimal hyperplasia in rats and strongly suggest that a calcium antagonist could be employed in the prevention of venous graft disease. PMID- 1713994 TI - Protein kinase C-mediated contraction in rabbit aorta is inhibited by CD-349, a dihydropyridine derivative. AB - We investigated the effects of nitroglycerin (NTG), atrial natriuretic peptide (ANP), and CD-349 on phorbol dibutyrate (PDBu)-induced contraction in rabbit aorta. In the presence of endothelium, both NTG (10(-8)-10(-5) M) and ANP (10( 10)-10(-7) M) inhibited the PDBu-induced contraction in a concentration-dependent manner. CD-349 (10(-8)-10(-5) M) also inhibited the PDBu-induced contraction in a concentration-dependent manner. This inhibitory effect of CD-349 was independent of the existence of endothelium. Inhibitory effects of both NTG and CD-349 but not ANP were antagonized by treatment with methylene blue. However, nifedipine (10(-5) M), nicardipine (10(-5) M), and diltiazem (10(-5) M) had little or no effect on PDBu-induced contraction. Furthermore, NTG, ANP, and CD-349 inhibited endothelin-induced contraction, which may be mediated through activation of protein kinase C. These results indicate that PDBu-induced and endothelin-induced contractions in rabbit aorta are inhibited by agents known to elevate vascular cyclic GMP content. PMID- 1713995 TI - Use-dependent block of atrial sodium current by ethylisopropylamiloride. AB - Ethylisopropylamiloride (EIPA) is a potent inhibitor of Na(+)-H+ exchange in many tissues and is frequently used to study cellular regulation of pH, but the electrophysiologic effects of EIPA on cardiac cells have not been studied previously. The use-dependent effects of EIPA on the sodium current (INa) of cultured embryonic chick atrial myocytes were investigated using standard whole cell patch-clamp techniques. With 150-ms depolarizations from -140 to 0 mV, applied at 1-3 Hz in the presence of 10 microM EIPA, a decrement in INa was observed. This use-dependent reduction equaled 31 +/- 6% of control INa at steady state during 1-Hz stimulation. Inhibition increased with stimulation rate and with depolarization of the holding potential to -100 mV, but there was no effect of pulse duration on the EIPA-induced inhibition over the range of 20-500 ms. Moreover, repetitive depolarizations to potentials that did not activate macroscopic current but that did yield pronounced channel inactivation did not result in a decrement in INa. The effect of EIPA increased over the concentration range of 1-30 microM so that with 3-Hz stimuli steady-state inhibition increased from 3 +/- 1 to 85 +/- 5%. Amiloride, which slows repolarization of the cardiac action potential, was at least 100-fold less potent than EIPA in reducing INa. We conclude that EIPA is an "open-channel" blocker of the cardiac sodium current at concentrations comparable to those of many type I antiarrhythmic agents. PMID- 1713996 TI - Effects of calcium channel blockers on stress protein synthesis in cardiac myocytes. AB - The detection of "stress proteins," certain protein groups of 70 or 30 kDa molecular weight synthesized de novo under stress conditions, serves as an assay for monitoring cellular toxicity. Typical toxins inducing stress protein formation in cardiac myocytes are CdCl2 and H2O2. The synthesis of 68, 71, and 30 kDa stress proteins is evoked by CdCl2, and H2O2 stimulates the formation of a 30 kDa protein. When fetal mouse myocardial cells are incubated first with CdCl2 and then with H2O2 or vice versa, an additive effect on stress protein synthesis can be documented. The calcium antagonists diltiazem, verapamil, and nifedipine, at concentrations above 0.05 mg/ml, stimulate the de novo synthesis of a 30 kDa stress protein. After preincubation of the cardiac myocytes with slow calcium channel blockers, the synthesis of the 70 kDa stress protein family evoked by CdCl2 is reduced. In contrast to the increased stress protein synthesis after heart cells are exposed to toxins, preincubation with calcium antagonists reduces the formation of certain stress proteins. These results indicate an interference of calcium channel blocking drugs with stress protein formation in cultured mouse myocardial cells. PMID- 1713997 TI - The role of endogenous angiotensin II in antidiuresis and norepinephrine overflow induced by stimulation of renal nerves in anesthetized dogs. AB - We examined the possible involvement of endogenous angiotensin II (ANG II) in norepinephrine (NE) overflow and antidiuresis induced by renal nerve stimulation (RNS). RNS at a frequency of 0.5-2.0 Hz, which did not influence renal hemodynamics, produced significant reductions in urine flow and urinary excretion of sodium, and elevations in NE secretion rate (NESR) and renin secretion rate (RSR). Intrarenal arterial (i.r.a.) infusion of phentolamine (10 micrograms/kg/min) abolished the RNS-induced antidiuresis. In dogs receiving captopril (15 micrograms/kg/min i.v.), RNS-induced antidiuresis and increase in NESR were significantly attenuated. The i.r.a. administration of propranolol at 5 micrograms/kg/min, a dose that inhibited completely the RNS-induced increase in RSR, did not influence the alterations in NESR and urine formation in response to RNS. During ANG II infusion (1 ng/kg/min i.r.a.), RNS produced a reduction in urine formation and an increase in NESR, at a magnitude similar to that seen without ANG II infusion. These results suggest that RNS at a low frequency increased the NESR and RSR without affecting renal hemodynamics and that the antidiuretic effect was probably produced via the activation of postsynaptic alpha-adrenoceptors, but not via the ANG II receptor, located on the renal tubules. The release of NE appears to be modulated by ANG II through the activation of a facilitatory prejunctional mechanism, which is maximally stimulated by endogenously and locally generated basal levels of ANG II. PMID- 1713998 TI - Effects of intracerebroventricular injected choline on cardiovascular functions and sympathoadrenal activity. AB - Intracerebroventricular (i.c.v.) injection of choline (50-150 micrograms) increased blood pressure (SP) and decreased heart rate (HR) in freely moving rats. Intracerebroventricular pretreatment of rats with mecamylamine (50 micrograms) blocked the reduction in HR and reduced the increase in SP induced by i.c.v. choline (150 micrograms). Central muscarinic blockade with atropine (10 micrograms, i.c.v.) reduced the pressor response to i.c.v. choline (150 micrograms) by about 70%, without influencing the decrease in HR. The decrease in HR induced by i.c.v. choline was prevented by intraarterial (i.a.) treatment of atropine methylnitrate (2 mg/kg). Intracerebroventricular choline (150 micrograms) produced a fivefold increase in catecholamine concentrations in adrenal venous plasma. Bilateral adrenalectomy reduced, but did not block, choline's effect on SP. Intracerebroventricular choline (150 micrograms) showed an ability to increase and restore SP in rats subjected to spinal cord transection or pretreatment with hexamethonium (15 mg/kg, i.a.) or with phentolamine (10 mg/kg, i.a.). Intracerebroventricular choline (150 micrograms) increased plasma vasopressin (VP) levels from 2.2 +/- 0.4 to 25.6 +/- 2.5 pg/ml. Pretreatment of rats with a VP antagonist reduced the pressor response to i.c.v. choline. It is concluded that (a) the reduction in HR results from a central nicotinic receptor-mediated increase in vagal tone, (b) the increase in SP appears to be due to activation of both nicotinic and muscarinic central cholinergic receptors, and that (c) the central activation of the adrenal medulla and the increase in plasma levels of VP are involved in the pressor response to i.c.v. choline. PMID- 1713999 TI - Effects of intracoronary gallopamil on coronary hemodynamics and myocardial oxygen consumption in humans. AB - The response of coronary hemodynamics to the intracoronary (i.c.) bolus administration of gallopamil, 1.5 and 3.0 micrograms/kg, was evaluated in 14 patients with normal coronary arteries. Gallopamil, 3.0 micrograms/kg, induced a small and transient decrease in systolic and mean arterial pressure and a small increase in the preejection period. Coronary sinus blood flow increased significantly at 30 s (p less than 0.01) and returned to baseline 10 min after gallopamil administration. Coronary vascular resistance was still reduced at 10 min and returned to baseline at 15 min. Myocardial O2 consumption and extraction decreased significantly (p less than 0.01) at 30 s. While myocardial O2 consumption returned to baseline 15 min after gallopamil administration, myocardial O2 extraction was still significantly reduced at this time. Milder and more transient changes were observed after i.c. administration of the lower dose (1.5 micrograms/kg), and no significant changes were found after i.c. administration of saline. These data show that i.c. gallopamil, in patients with normal coronary arteries, induces direct, transient, and dose-related peripheral coronary vasodilation. The reduction of myocardial O2 consumption and extraction suggests a direct negative inotropic and metabolic effect of gallopamil. PMID- 1714000 TI - Electrophysiologic effects of verapamil metabolites in the isolated heart. AB - The electrophysiologic effects of the metabolites of verapamil are unknown and may contribute to the observed differences between intravenous and oral verapamil. We examined the electrophysiologic effects of verapamil and its metabolites (norverapamil, N-dealkylverapamil (D617), and N-dealkylnorverapamil (D620)) at estimated, free therapeutic concentrations, in the retrogradely perfused, isolated rabbit heart. Verapamil at 5 and 10 ng/ml significantly prolonged anterograde (11 and 27%, respectively) and retrograde (10 and 25%, respectively) atrioventricular (AV) nodal block cycle lengths. Anterograde and retrograde AV nodal conduction times and refractory periods were also prolonged. Norverapamil at 100 ng/ml had qualitatively similar effects equivalent to 20-50% that observed with verapamil at 10 ng/ml. D620 had small but statistically significant effects on some AV nodal parameters. D617 had no effect. The combination of verapamil plus its principal metabolite, norverapamil, had additive effects. None of the compounds had any measurable effect on atrial conduction, His-Purkinje conduction, or atrial refractoriness. Ventricular refractoriness was significantly prolonged only by norverapamil. In conclusion, some of the metabolites of verapamil have important electrophysiologic AV nodal effects and may contribute to the clinical effects observed during chronic oral verapamil dosing. PMID- 1714001 TI - Effect of angiotensin II on plasma adenosine concentrations in the rat. AB - Our previous studies indicate that angiotensin II may stimulate release of adenosine from rat lungs, leading to an increase in plasma adenosine concentrations in renovascular hypertensive rats. Such an increase in plasma adenosine levels might be of physiological importance since this nucleoside is a known inhibitor of renin release and could therefore participate in a negative feedback loop whereby angiotensin II could limit its own biosynthesis. However, our previous studies examining angiotensin II-induced adenosine release were performed under nonphysiological conditions involving, in one case, perfusion of rat lungs with a salt solution and, in another case, collection of plasma adenosine samples from anesthetized, laparotomized rats. In the present study, we have addressed the hypothesis that angiotensin II stimulates an increase in plasma adenosine concentrations under more physiological conditions. Using microbore high-performance liquid chromatography, we determined the concentrations of adenosine in plasma samples collected from (a) the left ventricle and jugular vein of anesthetized rats during acute intravenous infusions of angiotensin II (1, 10, and 100 ng/min) and (b) the carotid artery of conscious, unrestrained rats exposed to chronic elevations of either exogenous angiotensin II (intraperitoneal infusion of angiotensin II, 125 ng/min) or endogenous angiotensin II (induction of renovascular hypertension by clipping the left renal artery or by ligating the suprarenal aorta). Both ventricular and venous plasma levels of adenosine were significantly elevated in anesthetized rats during acute infusions of angiotensin II at 10 ng/min but not at the higher and lower infusion rates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714002 TI - Myocardial adaptation to chronic propranolol therapy in diabetic rats. AB - The myocardial adaptation to chronic beta-adrenergic blockade was explored in diabetic and control female Wistar rats. Propranolol was administered by intraperitoneal injection, 30 mg/kg every 12 h. Treatment began 2 months after streptozotocin injection and at a comparable time in controls. Diabetes reduced the heart rate by 90 beats/min; propranolol decreased the heart rate by 30 beats/min further in diabetic rats. Propranolol reduced the heart rate by 100 beats/min in controls. Study of isolated ventricular papillary muscle showed qualitatively similar effects of propranolol in control and diabetic animals. Diabetes resulted in prolonged contraction duration and decreased shortening velocity; both developed tension and peak shortening were unchanged. In contrast, propranolol resulted in prolonged contraction duration but no change in shortening velocity; both developed tension and peak shortening were increased. The shortening of isometric relaxation in response to norepinephrine was exaggerated with propranolol therapy in control but not diabetic rats. These findings indicate that the myocardial adaptation to chronic beta-blockade results in increased developed tension and increased extent of muscle shortening in vitro, and differs qualitatively from the adaptation to diabetes. PMID- 1714003 TI - The diurnal rhythm of plasma potassium: relationship to diuretic therapy. AB - Plasma potassium levels have been implicated in the genesis of cardiac arrhythmias, particularly in patients receiving diuretic therapy. The present study was undertaken to evaluate the stability of plasma potassium levels throughout a 28-h period. Normal volunteers (n = 8) and subjects with essential hypertension (n = 10) were studied in a clinical research center while receiving controlled dietary intakes. Plasma potassium followed a diurnal rhythm in both groups, with a peak level at 12 h and a trough level at 24 h. The average peak-to trough difference was 0.62 +/- 0.05 mmol/L. Urinary potassium excretion also followed a diurnal rhythm, with the lowest excretory rate during the evening hours, when plasma potassium reached its nadir. Subjects with essential hypertension were restudied after 4 weeks of hydrochlorothiazide (50 mg/day) and then after an additional 4 weeks of hydrochlorothiazide (50 mg/day) and amiloride (5 mg/day). Hydrochlorothiazide alone reduced plasma potassium at all times of measurement without altering the diurnal rhythm. The combination of hydrochlorothiazide and amiloride resulted in higher plasma potassium levels in the morning, but did not significantly affect evening plasma potassium levels. The frequency of hypokalemia (K less than or equal to 3.0 mmol/L) was related to the time at which the plasma potassium was measured. We conclude that plasma potassium undergoes a diurnal rhythm and that diuretics shift this rhythm to uniformly lower values. This rhythm must be considered when defining the frequency of hypokalemia. PMID- 1714004 TI - Proarrhythmic activity of intracoronary endothelin in dogs: relation to the site of administration and to changes in regional flow. AB - The endothelium-derived peptide, endothelin, has been shown to exert powerful constrictor activity in both isolated and in situ coronary arteries. Recent in vitro data on isolated cardiac myocytes suggest that the substance might also possess electrophysiologic properties. We investigated the possibility that endothelin (ET-1) may exert proarrhythmic effects when infused selectively in the coronary circulation of open-chest-anesthetized dogs. Animals were instrumented for the measurement of left anterior descending (LAD) or left circumflex (LCX) coronary artery blood flow, left systolic ventricular pressure (LSVP), dP/dtmax, mean arterial pressure (MAP), and epicardial electrocardiogram (ECG; three leads). Data were recorded during infusion (2 min) of saline (n = 5) or increasing doses of endothelin (5-80 pmol/kg) given selectively in either the LCX (n = 10) or the LAD (n = 10). When infused into the LCX, endothelin produced a dose-dependent decrease in flow (40 +/- 23% at 80 pmol/kg, mean +/- SD, p less than 0.01) with a concomitant increase in coronary resistance and a decrease in dP/dtmax and MAP. ECG changes typical of myocardial ischemia paralleled the decrease in flow and culminated in ventricular fibrillation at the highest dose (80% of dogs). Endothelin caused similar hemodynamic effects when infused in the LAD, but fatal arrhythmias occurred for lower doses and for little or no change in coronary blood flow. Thirty percent of the animals died at 10 and 60% died at 20 pmol/kg, doses that induced only a moderate decrease (8 +/- 7 and 21 +/- 12%, respectively) in LAD total blood flow. Ventricular tachycardia always preceded ventricular fibrillation and death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714005 TI - Evidence for a central component in the cardiovascular effects of calcium channel blockers. AB - The possibility of a central component in the cardiovascular effects of peripherally administered calcium channel blockers has been explored through a comparison of the effects observed after intravenous (i.v.) and intracisternal (i.c.) administration of verapamil and diltiazem in chloralose-anesthetized and artificially ventilated cats. Both agents produced relatively greater effects after i.c. than after i.v. administration. The bradycardiac effect following i.c. as well as i.v. administration was totally abolished by bilateral cervical vagotomy, and the hypotensive effect was attenuated by this procedure. The results strongly suggest the existence of a central component in the cardiovascular effects of both agents. PMID- 1714006 TI - Adenosine attenuates the vasoconstrictor response to the cold pressor test in humans. AB - The forearm vasoconstrictor response to a standardized cold pressor test (CPT) was studied twice in eight healthy subjects, once during local intraarterial infusion of adenosine and once during infusion of equipotent dosages of the control vasodilator sodium nitroprusside (SNP). During local SNP infusion, the forearm vascular resistance (FVR) decreased from 70 +/- 14 to 30 +/- 7 arbitrary units (AU). Adenosine induced a comparable vasodilator response, with a decrease in FVR from 56 +/- 14 to 28 +/- 6 AU. Subsequent cold exposure induced a mean percentage increase in FVR of 62 +/- 17% during SNP, whereas the increase was only 27 +/- 12% during adenosine infusion (p = 0.014). There were no differences in the calculated cold-induced changes in forearm production of norepinephrine (NE) between the SNP and the adenosine tests. We conclude that adenosine attenuates forearm vasoconstrictor response to the CPT, probably by a postjunctional mechanism of action. PMID- 1714007 TI - Prostaglandin E2 and fetal oxygen tension synergistically inhibit response of isolated fetal rabbit ductus arteriosus to norepinephrine. AB - We wished to determine the effect of prostaglandin E2 (PGE2) on the response of the ductus arteriosus to norepinephrine (NE) and whether any effect of PGE2 was influenced by O2 tension. The vessel was isolated from fetal New Zealand White rabbits and studied in vitro. The response to NE was assessed in terms of both sensitivity (pEC50) and maximum contractile response (MCR) as determined from cumulative concentration-response curves. PGE2 caused a concentration-dependent inhibition of ductal sensitivity to NE. This was maximal in nanomolar concentrations of PGE2, in which ductal sensitivity to NE was decreased by approximately 50 times. This action was only minimally potentiated by 3% O2 (simulating fetal O2 tension, PO2). PGE2 could also inhibit the MCR to NE, but this was entirely dependent on PO2. In 95% O2, PGE2 had no effect on the MCR to NE, whereas in fetal PO2, PGE2 decreased the MCR. This decrease was maximal in 1 nM PGE2, in which the MCR in 3% O2 was about one fifth the size of the MCR in 95% O2. In 1 nM nM PGE2, 10% O2 also largely abolished the inhibitory effect of PGE2 on ductal MCR to NE, indicating that this effect of O2 is also observed in the range of the physiologic increase of arterial PO2, tension that occurs at birth. PGE2 also inhibited ductal sensitivity to 5-hydroxytryptamine, histamine, and potassium. We conclude that physiologic and therapeutic concentrations of PGE2 inhibit sensitivity of the ductus arteriosus to certain vasoconstrictors and can inhibit the maximum response to NE in fetal but not increased PO2. PMID- 1714008 TI - Reduced nitric oxide release causes nitrate tolerance in the intact coronary circulation. AB - We investigated the possible involvement of reduced nitric oxide (NO) formation in development of nitrate tolerance in an intact organ circulation. NO formation was measured spectrophotometrically on-line in the coronary effluent of Langendorff hearts of rabbits. Short-term (3 min) infusion of glyceryl trinitrate (GTN, 40 microM) or a sydnonimine (SIN-1, 2.3 microM), the active metabolite of molsidomine, into the coronary inflow tract resulted in a decrease in coronary vascular resistance and NO release into the coronary effluent. Pretreatment with 250 microM GTN for 30 min resulted in considerably reduced NO formation and coronary vasodilation, whereas NO release and coronary vasodilation subsequent to SIN-1 remained unchanged. In hearts pretreated with 250 microM SIN-1 for 30 min, there was no effect on GTN- or SIN-1-induced vasodilation and NO release. Studies of cyclic GMP formation in rat lung fibroblasts further indicated that GTN bioconversion rather than desensitization of the soluble guanylate cyclase is involved in GTN tolerance. These data suggest metabolic, endothelium-independent NO release from GTN during passage through the coronary circulation. This NO release is reduced in nitrate-tolerant cells and appears to be the major cause of nitrate tolerance in intact circulatory systems. PMID- 1714009 TI - Efficacy of a monoclonal antibody (MoAb 60.3) in reducing myocardial injury resulting from ischemia/reperfusion in the ferret. AB - The myocardial salvage efficacy of a monoclonal antibody (MoAb 60.3) directed at the CDw 18 membrane antigen complex essential for normal neutrophil function was evaluated in a ferret occlusion/reperfusion model. When infused i.v. over a 10 min interval beginning at the 45th minute of a 90-min occlusion at a fixed dose of 2 mg/kg, the antibody afforded 33 and 45% reductions in infarct size following reperfusion intervals of 6 and 24 h, respectively. Administration of that same dose via the left atrium over 1 h beginning at the 75th minute of occlusion and continuing until the 45th minute of reflow resulted in only a 14% reduction in infarct size. If MoAb 60.3 administration was withheld until the 5th-15th minute of reperfusion, the mean levels of salvage were 19 and 8%, respectively, following 6- and 24-h periods of reflow. Time-course hemodynamic data indicated that the monoclonal antibody caused no alterations in oxygen utilization that might be responsible for the levels of salvage observed. PMID- 1714010 TI - Antiarrhythmic activity of amiloride: mechanisms. AB - We have previously shown that amiloride suppresses the induction of sustained ventricular tachycardia both in dogs late following myocardial infarction and in patients. In those studies the only electrophysiologic correlate of amiloride's antiarrhythmic activity observed was prolongation of ventricular effective refractory period at the zone bordering the infarct. The purpose of this study was to assess the pharmacologic effects of amiloride associated with antiarrhythmic efficacy. However, amiloride has multiple pharmacologic effects, including inhibition of the slow inward calcium current (ICa), inhibition of the sodium-calcium and sodium-hydronium ion exchangers, acidification of intracellular pH resulting in partial inhibition of the inwardly rectifying potassium current (IK1), and increase in serum potassium and magnesium. The approach used in this study was to use selective pharmacologic probes to produce the known components of amiloride's pharmacologic effects. The selective agents consisted of verapamil (partial blockade of ICa), 3',4'-dichlorobenzamil (partial inhibition of the Na-Ca exchanger), 5-(N-ethyl-N-isopropyl) amiloride (partial inhibition of the Na-H exchanger), the combination of these congeners, KCl infusions to increase serum potassium, MgSO4 infusions to increase serum magnesium, the combination of KCl and MgSO4 infusions, barium (partial block of IK1), ryanodine (partial blockade of sarcoplasmic reticulum calcium release), and placebo. In this study only barium produced antiarrhythmic and electrophysiologic effects similar to those of amiloride. However, amiloride prolongs border zone refractoriness selectively, whereas barium prolongs refractoriness diffusely throughout the myocardium. Blockade of ICa by verapamil, increases in serum magnesium and potassium alone or in combination, and partial blockade of sarcoplasmic reticulum by ryanodine were not antiarrhythmic in this model. Monotherapies that produced partial blockade of the Na-Ca and Na-H exchangers separately did not produce antiarrhythmic activity. However, the combination of these amiloride congeners reproduced the antiarrhythmic activity of amiloride but did not prolong border zone refractoriness. From these studies we conclude that the antiarrhythmic activity of amiloride relates to (a) selective blockade of IK1 in the border zone and/or (b) combined inhibition of sodium-calcium and sodium hydronium ion exchangers. PMID- 1714011 TI - Continuous oral N-acetylcysteine treatment and development of nitrate tolerance in patients with stable angina pectoris. AB - The presence of sulfhydryl (SH) groups appears to be fundamental to nitrate induced vasodilation and N-acetylcysteine (NAC), a sulfhydryl (SH)-donor substance, potentiates hemodynamic responsiveness to nitrates. We investigated the effect of simultaneous administration of NAC and isosorbide dinitrate (ISDN) on development of nitrate tolerance. In a double-blind, randomized, placebo controlled cross-over study, seven patients with stable angina pectoris were treated for two 8-day periods with ISDN (40 mg four times daily, q.i.d.) together with NAC (controlled release 600 mg q.i.d.) or matching placebo. Bicycle exercise tests were performed before treatment was started, 1 h after treatment was started, and at day 8. After 8-day treatment with ISDN + placebo, responses determined by exercise testing were diminished as compared with responses obtained during acute therapy and did not differ from baseline values, suggesting development of tolerance to ISDN. During treatment with ISDN + NAC, time to 1-mm ST depression was significantly prolonged (441 +/- 44 vs. 381 +/- 40 s, mean +/- SEM) and total ST segment depression significantly reduced (1.9 +/- 0.7 vs. 3.5 +/- 0.8 mm) as compared with baseline values. The reduction in ST segment depression was significantly more pronounced during ISDN + NAC (46%) as compared with ISDN + placebo (23%). Although exercise time and time to angina pectoris were unaffected. NAC augmented the antiischemic effects of ISDN as assessed by ECG. This finding may suggest that development of nitrate tolerance is modified by chronic oral high-dose NAC administration. PMID- 1714012 TI - Voltage- and use-dependent modulation of calcium channel current in guinea pig ventricular cells by amiodarone and des-oxo-amiodarone. AB - Amiodarone is an effective antiarrhythmic drug handicapped by serious side effects. The mechanism of its antiarrhythmic activity is not known but is presumed to involve inhibition of current flowing through ion channels. Des-oxo amiodarone, a close structural analogue of amiodarone, was synthesized based on the hypothesis that the toxic and therapeutic properties reside in different parts of the molecule and that chemical modification could result in a less toxic agent that yet preserved amiodarone's antiarrhythmic efficacy. We compared the effects of amiodarone and des-oxo-amiodarone on Ca current in enzymatically dispersed guinea pig ventricular myocytes using the whole-cell patch-clamp method. Amiodarone caused both a tonic and a phasic (use-dependent) reduction of the Ca current. The relationship between membrane potential and the availability for channel opening upon depolarization (inactivation curve) was shifted toward more negative membrane potentials by amiodarone (delta - 10.6 +/- 2.2 mV, n = 7). The use-dependent reduction of the Ca current was also dependent on the frequency of the voltage clamp steps (0.5 Hz, 40.2 +/- 7.9%; 1.0 Hz, 50.0 +/- 6.7%). Dex oxo-amiodarone had a dual effect on the Ca current: After maintaining the membrane potential for several seconds at negative membrane potentials (less than -45 mV), the Ca current was increased by des-oxo-amiodarone. Des-oxo-amiodarone also shifted the Ca channel inactivation curve to more negative membrane potentials up to 16 mV. Consequently, Ca current could be increased or decreased depending on the experimental conditions. Enhancement of Ca current by des-oxo amiodarone was transient and was supplanted entirely by the antagonistic effects of the drug after approximately 5 min. The antagonistic effects of des-oxo amiodarone on Ca current were also use- and frequency-dependent. PMID- 1714013 TI - Nicorandil-induced vasorelaxation: functional evidence for K+ channel-dependent and cyclic GMP-dependent components in a single vascular preparation. AB - Using a series of functional criteria, we wished to evaluate the K+ conductance mechanism and the cyclic GMP mechanism implicated in the actions of nicorandil (NIC) as a vasodilator. In rabbit isolated superior mesenteric artery, NIC exhibited two relaxation dose-response curves (DRCs): one with a lower IC50 of 4.8 x 10(-6) M for norepinephrine (NE 5 microM) contraction, and another with a higher IC50 of 1.4 x 10(-4) M for 80 mM K+ contraction. K+ channel blockers (TEA 1-10 mM), Ba2+ (0.1-0.5 mM), glyburide (1 microM), and increased [K+]ex (20 mM), all caused significant attenuations in the ability of NIC to relax NE contraction, but did not influence the ability of NIC to relax high-K+ contraction. Pretreatment with 5 microM methylene blue, a guanylate cyclase inhibitor, produced a pronounced inhibition of nitroglycerine (NTG) relaxation, but only a marginal inhibitory effect on the NIC relaxation DRC for NE contraction. Functional studies demonstrated that the inhibitory effect of NIC on NE-sensitive intracellular Ca2+ release occurred in the same concentration range as that required for relaxation of 80 mM K+ contractions (10(-5)-10(-3) M). Furthermore, NIC also caused increases in cellular cyclic GMP levels at this higher concentration range. Finally, NIC relaxation of NE contraction was not prone either to self-tolerance (30 mM NIC preexposure) or cross-tolerance (0.55 mM NTG preexposure) development. In contrast, a modest but significant degree of self-tolerance to NIC could be demonstrated under high-K+ contraction condition. These studies thus show the existence of both cellular mechanisms for NIC in the same vascular preparation and further show that these two mechanistic components are separate and independent. The K+ channel-dependent component occurs at lower concentrations, is blocked by K+ channel blockers, is not inhibited by methylene blue, is not associated with increases in cyclic GMP, and is not prone to tolerance development. In this, NIC resembles other K+ channel openers. The cyclic GMP-dependent component is evident at relatively higher concentrations, is associated with inhibition of [Ca2+]i release, is associated with increases in cyclic GMP levels, and is prone to tolerance development. In this, NIC resembles other nitrovasodilators. A combination of these characteristics of the actions of NIC may contribute to the differences in the acute versus chronic hemodynamic profile of NIC. PMID- 1714014 TI - Protein kinase C and alpha 2-adrenoceptor-mediated inhibition of noradrenaline release from the rat tail artery. AB - In isolated rat tail arteries preincubated with [3H]noradrenaline, electrical field stimulation evoked the overflow of tritium. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activating phorbol ester, time-dependently increased the overflow at 1 mumol/L but not at 0.1 mumol/L. In contrast, the overflow was not altered by phorbol 13-acetate (PA, 1 mumol/L), which does not influence the activity of PKC. Polymyxin B (70 mumol/L), an inhibitor of PKC, depressed the overflow when given alone and, in addition, attenuated the effect of PMA, 1 mumol/L. The selective alpha 2-adrenoceptor agonist B-HT 933 depressed the overflow; PMA, 1 mumol/L, did not interfere with the effect of B-HT 933, 10 mumol/L. The results provide evidence for the participation of prejunctionally located PKC in the release of noradrenaline. However, PKC does not seem to be involved in the alpha 2-adrenoceptor-agonist-mediated inhibition of noradrenaline release. PMID- 1714015 TI - Cardiocirculatory responses to AII and AVP in conscious rats. AB - Cardiac and peripheral circulatory responses to changes in afterload with angiotensin II (AII) and vasopressin (AVP) were investigated in ganglion-blocked (hexamethonium) conscious rats. Cardiac output (CO) was measured by thermodilution. Both hormones were infused at a dose adjusted to increase mean arterial pressure 70% above baseline. AVP (11.4 +/- 2.2 ng/kg/min, n = 6) decreased CO from 43.4 +/- 2.3 to 34.1 +/- 2.9 ml/min/100 g (p less than 0.001), whereas AII (33.4 +/- 7.4 ng/kg/min, n = 7) increased CO from 38.7 +/- 2.6 to 44.9 +/- 3.4 ml/min/100 g (p less than 0.01). Heart rate did not change with the increase in afterload with either vasoconstrictor. To study whether the different effects of AII and AVP on CO may be explained by their different actions on the venous system, changes in venous tone were evaluated by measuring mean circulatory filling pressure (MCFP) and determining the pressure gradient for venous return (PGVR). AVP changed neither MCFP nor PGVR, whereas AII increased both these parameters, 20.7 +/- 2.8% (p less than 0.01) and 20.3 +/- 6.4% (p less than 0.01), respectively, above control. We also examined the effects of AII and AVP on ventricular dynamics: left ventricular systolic pressure and left ventricular dP/dtmax increased as aortic pressure was increased in a similar manner with both vasoconstrictors. However, AVP induced a greater increase in left ventricular end diastolic pressure than AII. Our results indicate that AII induces an increase in preload by its effect on venous tone, which is adequate to increase cardiac output. The decrease in cardiac output induced by increasing afterload with AVP can be explained by two mechanisms: an inadequate venous return and a failure of the left ventricle to overcome the increased afterload. PMID- 1714016 TI - Cigarette smoking alters sympathoadrenal regulation by decreasing the density of beta 2-adrenoceptors. A study of monitored smoking cessation. AB - We report the effects of monitored smoking cessation on adrenergic regulation in chronic smokers. The beta 2 adrenoceptor density of mononuclear leukocytes (MNLs) and plasma catecholamines was analyzed before cessation and 2, 3, and 8 weeks after cessation. We found a progressive increase in beta-adrenoceptor density after smoking cessation. During smoking the beta-adrenoceptor density was 1.456 +/- 83 (mean +/- SEM) binding sites per cell (n = 10), whereas 3 weeks after cessation the density was 1,774 +/- 157 sites per cell (n = 10; p less than 0.05), and at 8 weeks, 1,900 +/- 227 sites per cell (n = 8; p less than 0.05), representing an overall increase of 23%. Smoking cessation had no effect on binding affinity nor on lymphocyte subgroup distribution. The density of MNL cell beta-adrenoceptors in age-matched nonsmoking men was higher, at 1,896 +/- 271 sites per cell, than that of the chronic smokers before cessation, 1,419 +/- 117 sites per cell (n = 14; p less than 0.01). Plasma epinephrine decreased as a result of cessation from 0.36 pmol/ml (0.26-0.44, 95% confidence interval; baseline) to 0.26 pmol/ml (0.20-0.32) at 8 weeks (p less than 0.05), and norepinephrine decreased from 2.09 pmol/ml (1.38-2.80) to 1.69 pmol/ml (1.14 2.24; p = 0.06). We conclude that stopping smoking progressively increases beta 2 adrenoceptor density on MNL cells. Eight weeks after cessation the adrenoceptor density reaches the corresponding level of nonsmokers. These reversible changes in adrenergic regulation after smoking cessation may be associated with the relatively rapid reduction in cardiovascular disease risk among ex-smokers. PMID- 1714017 TI - Endothelium-dependent response of the rabbit aorta to femtomolar concentrations of angiotensin II. AB - Thirty-five percent of endothelium-intact rabbit aortic rings tested contracted biphasically to angiotensin II (AII). The threshold concentration in these rings varied, ranging from 10(-16) to 10(-12) M. The remaining 65% contracted monophasically with a range of threshold concentrations of 10(-11)-10(-8) M. All endothelium-denuded rings tested contracted monophasically with a threshold concentration of 10(-10)-10(-8) M. The contractions of both types of rings were inhibited by 1-sar-8-ala-angiotensin II, but not phentolamine or indomethacin, indicating that the contractions were not mediated by secondary agonists such as noradrenaline or arachidonic acid metabolites. Bioassay studies revealed that 10( 16) M AII released an endothelial factor that modulated the action of AII, converting the monophasic response of an endothelium-denuded ring to a biphasic response to AII (10(-16)-10(-7) M). The results demonstrate for the first time the endothelium-dependent response of an artery to femtomolar concentrations of AII and support the existence of arterial angiotensin receptors capable of being saturated by circulating levels of the hormone. PMID- 1714018 TI - Clonidine reduces blood pressure and heart rate oscillations in hypertensive patients. AB - Short-term fluctuations in blood pressure (BP) and heart rate (HR) were analyzed in a group of eight men with essential hypertension. Indirect finger BP was measured by a Finapres device. Analog-to-digital conversion of the BP was used to determine systolic (SAP), diastolic (DAP), and mean arterial pressure (MAP) and HR every second. The equidistant sampling allowed a direct spectral analysis using a fast Fourier transform algorithm. The effects of an oral dose of clonidine (150 micrograms) were studied in a double-blind, crossover, placebo controlled study. Clonidine markedly reduced the variability of BP and HR after 90 min as indicated by a reduction in the standard deviations of BP by 36.7% for SAP, 21.0% for DAP, 22.1% for MAP, and 26.0% for HR. At this time clonidine reduced the average BP by 19.7 mm Hg for SAP, 10.6 mm Hg for DAP, 16.0 mm Hg for MAP, and 1.0 beat/min for HR. Spectral profiles of BP and HR illustrated the alterations in the spontaneous oscillations underlying the standard deviation changes. Clonidine dramatically reduced the amplitude of BP and HR oscillations in the mid-frequency region 66-129 mHz, which depends on the activity of the autonomic nervous system. We suggest that an increased sensitivity of the baroreflex is responsible for the apparent better control of BP and HR with clonidine. PMID- 1714019 TI - Physiologic assessment of milrinone therapy in severe heart failure patients. AB - To assess the inotropic, vasodilator, and after-load-reducing effects of intravenous milrinone in patients with severe congestive heart failure, a simple noninvasive echocardiographic study coupled with a right catheterization was performed in 12 patients. Milrinone was administered intravenously as a 50 micrograms.kg-1 bolus followed by a 24-h milrinone infusion at a rate of 0.5 mg.kg-1.min-1 [corrected]. Echocardiographic left ventricular end-diastolic diameter (Ded), end-systolic diameter (Des), and wall thickness were measured at baseline and at 24 h of milrinone infusion, before and after a sublingual nitrate administration (0.8 mg of nitroglycerin) that induced load variations. Hemodynamic measurements were performed simultaneously. Left ventricular end systolic meridional wall stress (Ses) was then calculated. The slopes of percent fractional shortening (percent FS)/Ses and Ses/Des, obtained during sublingual nitrate administration, were traced. Both end-systolic relations are an index of the contractile state. Milrinone therapy improved hemodynamics in all patients, resulting in stabilized hemodynamic conditions between 12 and 24 h of continuous milrinone infusion. At these times, the cardiac index increased to 30% while the capillary pulmonary wedge pressure and systemic vascular resistance decreased to 26 and 24%, respectively (all p less than 0.01). The average slope of Ses/Des shifted upward from 47.5 +/- 30 to 69.25 +/- 34 (p less than 0.05) and the average slope of (percent FS)/Ses shifted from -0.032 +/- 0.025 to -0.082 +/- 0.061 (p less than 0.01), both variations attesting the inotropic effect of milrinone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714020 TI - Two weeks of intermittent dobutamine therapy restores cardiac performance and inotropic responsiveness in conscious rats with heart failure. AB - Dobutamine is frequently used for acute therapy in heart failure. In the present study, the hemodynamic effects of long-term intermittent dobutamine therapy were investigated in conscious rats with heart failure. Rats with healed myocardial infarctions received two i.p. injections of dobutamine per day for 2 weeks. Hemodynamic measurements were performed 90-180 min after the last injection. Two weeks of intermittent dobutamine significantly restored all hemodynamic changes induced by infarction. The maximal cardiac output during volume loading was depressed due to infarction and dose-dependently restored by 2 weeks of intermittent dobutamine. An increased stroke volume accounted for this improvement since the heart rate was not altered. In order to investigate changes in adrenergic responsiveness, the effects of acute dobutamine in nontreated and 2 weeks of dobutamine-treated infarcted rats were compared to those in control rats. Whereas chronotropic responses to acute dobutamine were comparable for all experimental groups, the inotropic response was reduced in nontreated infarcted rats but dose-dependently restored after 2 weeks of intermittent dobutamine therapy. From the data, we conclude that 2 weeks of intermittent dobutamine therapy in conscious rats with healed myocardial infarcts improved cardiac performance and restored the inotropic response to acute dobutamine administration. Data indicate that dobutamine has a long-term effect on cardiac function, which differs from the acute inotropic effect. PMID- 1714021 TI - Pharmacokinetic properties and antihypertensive efficacy of once-daily diltiazem. AB - The pharmacokinetic and clinical characteristics of a once-daily formulation of diltiazem are described. In a 20 subject, 5 day, steady-state pharmacokinetic study, 120 and 240 mg of once-daily diltiazem were bioequivalent on a dose adjusted basis and were bioequivalent to a conventional reference product administered four times daily. The conventional formulation showed marked diurnal variation in its pharmacokinetics. Plasma concentrations following its administration at 2100 and 0300 h were significantly lower than following administration at 0900 and 1500 h. One hundred forty-four hypertensive patients completed a 16 week placebo-controlled, dose-titrated study examining the effects of once-daily diltiazem at doses of 120, 240, and 360 mg. Blood pressure was measured manually and (in 121 patients) by ambulatory evaluation. Following dose titration, diltiazem given once daily reduced blood pressure with significant effects present at 24 h following drug administration. Ambulatory blood pressures were lower than those measured manually and data from the manual measurements demonstrated a placebo effect suggested to result primarily from investigator bias. The placebo-adjusted reduction in blood pressure 24 h following a dose of diltiazem was approximately 5 mm Hg and was comparable for manual (supine and standing) and ambulatory measurements. Diltiazem was well tolerated. The only significant findings were of tiredness/dizziness (9 patients of 144) or oedema (also 9 of 144). The incidence of headache was not different than placebo. On both pharmacokinetic and pharmacodynamic grounds, the results indicate that diltiazem can be formulated in a manner suitable for once-daily antihypertensive use. PMID- 1714022 TI - Nebivolol induces endothelium-dependent relaxations of canine coronary arteries. AB - Nebivolol is a new beta 1-antagonist that acutely reduces arterial blood pressure without depressing cardiac function. The present study was designed to determine the effect of nebivolol on coronary arteries. Rings of canine left anterior descending coronary (LAD) artery with or without endothelium were suspended in organ chambers and the isometric tension was recorded. In some experiments, the transmembrane potential of the smooth muscle cells was recorded by electrophysiological methods. During contractions to prostaglandin F2 alpha, nebivolol induced concentration-dependent relaxations of the coronary arteries. The enantiomer, l-nebivolol, also induced comparable relaxations; however, d nebivolol induced smaller relaxations. The relaxations induced by nebivolol and its enantiomer were significantly larger in tissues with than in those without endothelium. The differences between tissues with and without endothelium were abolished by nitro-L-arginine (3 x 10(-5) M) or methylene blue (10(-5) M). The nebivolol-induced relaxations were not affected by indomethacin (10(-5) M), phentolamine (5 x 10(-6) M), propranolol (5 x 10(-6) M), or methysergide (3 x 10( 6) M). Nebivolol at a subthreshold concentration for inducing relaxation (3 x 10( 7) M) did not significantly affect endothelium-dependent relaxations to acetylcholine but potentiated ADP-induced endothelium-dependent relaxations. The potentiation is stereoselective for l-nebivolol. Nebivolol induced a small hyperpolarization of the coronary smooth muscle with endothelium (1 mV).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714023 TI - Beta-adrenergic receptors and reflex tachycardia after single and repeated felodipine administration in essential hypertension. AB - To verify the possible contribution of beta-adrenergic receptor down-regulation to the reversal of reflex tachycardia during chronic treatment with a dihydropyridine calcium antagonist, 11 hypertensive patients were studied with noninvasive blood pressure (BP) and heart rate (HR) monitoring after a placebo period, and on the first and seventh day of felodipine administration, 5 mg twice daily. Plasma catecholamines and neutrophil beta-adrenergic receptors were measured on the first and seventh day of treatment, immediately before and 2 h after drug administration. The first administration of felodipine was followed by a significant drop in BP (peak reduction in mean BP 24 +/- 7 mm Hg), lasting 6 h and mirrored by reflex tachycardia (peak increase in HR 14 +/- 9 beats/min). On the morning of the seventh day, 12 h after the previous felodipine administration, mean BP (MBP) was 16 mm Hg lower than on the last placebo day, while HR was unchanged. The next administration of felodipine was followed by a smaller drop in BP (MBP - 15 +/- 7 mm Hg; NS vs. placebo), while reflex tachycardia was the same as after acute felodipine (HR 13 +/- 8 beats/min; p less than 0.05 vs. placebo, NS vs. acute administration). Plasma noradrenaline concentration increased after both acute and chronic administration (p less than 0.0001), and preadministration values were highest on day 7 (p less than 0.05). Neutrophil beta-adrenergic receptor density and affinity did not change either acutely or chronically. This study gives both indirect and direct evidence that beta-adrenoceptor down-regulation does not occur during repeated felodipine administration in hypertension. Reflex tachycardia is not abolished, but is reset to lower BP levels. PMID- 1714024 TI - Cardiovascular effects of elgodipine in conscious pigs with a normal coronary circulation and in conscious pigs with a healed myocardial infarction. AB - The cardiovascular effects of the phenyldihydropyridine derivative elgodipine (0.3, 1, 3, 10, and 30 microgram/kg/min) were studied in normal conscious pigs and in pigs with chronic left ventricular dysfunction (LVD, caused by coronary artery occlusion) without and after beta-adrenoceptor blockade with propranolol (0.5 mg/kg + 0.5 mg/kg/h). In normal pigs, elgodipine increased cardiac output from 2.57 +/- 0.09 to 5.21 +/- 0.24 L/min (p less than 0.05) as a result of a doubling of the heart rate. Mean arterial blood pressure decreased from 94 +/- 2 to 76 +/- 3 mm Hg (p less than 0.05) as a result of a decrease in systemic vascular resistance. Left ventricular (LV) dP/dtmax increased (by up to 78 +/- 9%), but left ventricular end-diastolic pressure (LVEDP) remained unchanged. After propranolol administration elgodipine did not increase LV dP/dtmax, and the increase in heart rate was attenuated, resulting in a smaller increase in cardiac output (from 2.11 +/- 0.13 to 3.09 +/- 0.23 L/min, p less than 0.05), but an unchanged vasodilator response. In pigs with LVD, elgodipine increased cardiac output and LV dP/dtmax less than in normal animals, but the vasodilator response was not affected. LVEDP decreased from 14.6 +/- 1.6 to 11.7 +/- 2.5 mm Hg (p less than 0.05). In animals with LVD, propranolol caused a more severe depression of systemic hemodynamics, but did not modify the cardiovascular responses to elgodipine. Its cardiovascular profile suggests that elgodipine may not only be useful in the treatment of cardiovascular disorders for which other dihydropyridines are already in use, but also in mild chronic heart failure. PMID- 1714025 TI - Effects of various calcium antagonists in isolated perfused hearts from diabetic and age-matched control rats. AB - The purpose of this study was to examine the effects of several calcium antagonistic drugs on left ventricular contraction (isovolumetric) and coronary flow in isolated perfused hearts from streptozotocin diabetic rats compared to age-matched controls, thereby hoping to throw further light on the changes in Ca2+ handling that occur in the diabetic heart. In the presence of Ca2+ (0.9-9.9 mM) left ventricular pressure (LVP) was not significantly different in either type of heart. From cumulative dose-response curves, pD2 values of various calcium antagonists for the negative inotropic activity were determined in diabetic and control hearts. The pD2 values for several calcium antagonists were significantly greater in diabetic hearts than in controls: 5.72 vs. 4.99 for diltiazem, 6.88 vs. 6.60 for verapamil, and 8.42 vs. 8.04 for (-)-devapamil. In hearts from diabetic rats, LVP was also more sensitive to TMB-8 compared with controls: a higher pEC20 value (instead of the pD2 value) was found (5.47 vs. 5.16). The pD2 values for (+)-devapamil, nifedipine, and ryanodine were not significantly different in either type of heart. Calcium-entry blockers increased the coronary flow in control hearts (range 60-80% of the initial flow). However, the increase in flow was significantly greater in diabetic hearts than in the controls (95-128%). TMB-8 and ryanodine had no effect on coronary flow in both types of hearts. In conclusion, the changes in sensitivity of LVP to several calcium antagonists may support a different Ca2+ handling in diabetic hearts compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714026 TI - Two types of voltage-dependent calcium channel currents and their modulation by parathyroid hormone in neonatal rat ventricular cells. AB - The positive inotropic and chronotropic actions of parathyroid hormone (PTH) in cardiac cells are considered to be related to the modulation of calcium influx. The underlying mechanisms, however, are unknown, and direct electrophysiological evidence at the single-cell level is required. In the present study, the whole cell configuration of the patch-clamp technique was used in neonatal rat ventricular cells to identify both transient (T) and long-lasting (L) voltage dependent calcium channels according to their electrophysiological and pharmacological characteristics. The active synthetic fragment of bovine PTH, bPTH-(1-34), at a concentration of 1 microM, significantly enhanced the magnitude of L-channel currents by 67.8% (n = 13, p less than 0.01). The steady-state activation curve of L-channel currents was shifted along the voltage axis toward more negative potentials by either bPTH-(1-34) or Bay K-8644. The suppression or amplification of PTH-induced enhancement of inward currents by nifedipine, 1 microM, or Bay K-8644, 5 microM, respectively, further indicated that the effect of PTH was specific for L-type calcium channels. However, bPTH-(1-34) failed to modulate the magnitude or kinetics of T-channel currents. This study directly demonstrates that PTH is a specific endogenous calcium-channel activator in neonatal rat ventricular cells. PMID- 1714027 TI - Differential effects of magnesium on basal and agonist-induced EDRF relaxation in canine coronary arteries. AB - In the present study the effects of alterations in extracellular magnesium concentration on spontaneous release of endothelium-derived relaxing factor (EDRF) and acetylcholine-, thrombin-, and calcium ionophore A23187-induced EDRF dependent relaxation in isolated canine coronary arteries were determined. Increasing the extracellular magnesium concentration from the standard 1.2 to 2.4 mM did not alter the coronary contractile responses, while complete removal of extracellular magnesium resulted in an EDRF-dependent relaxation. The averaged percent relaxation of coronary arteries previously constricted with either prostaglandin F2 alpha, norepinephrine, or barium chloride was -75.9 +/- 4.4 (mean +/- SEM of 27 vessel segments), -43.1 +/- 4.9% (20 segments), and -25.8 +/- 6.9% (15 segments), respectively. Complete removal of extracellular magnesium also resulted in a slight but significant rightward shift of the concentration response curves of acetylcholine- and thrombin-induced EDRF-dependent relaxation. The maximum relaxation was decreased to 86.5 +/- 1.7% and 69.5 +/- 4.6% of the control acetylcholine and thrombin relaxation, respectively. In contrast, the calcium ionophore A23187-induced EDRF-dependent relaxation was not altered following magnesium withdrawal, nor was the isoproterenol-induced endothelium independent relaxation. These results suggest that extracellular magnesium exerts a direct inhibitory effect on the spontaneous release of EDRF and is apparently important for the expression of endothelium-dependent relaxation induced by acetylcholine and thrombin, but not calcium ionophore A23187, in canine coronary arteries. PMID- 1714028 TI - Delayed visual maturation. PMID- 1714029 TI - Long-term continuous subcutaneous and intravenous opioid infusions. PMID- 1714030 TI - Decreases in repetitive extrasystole threshold in the conscious pig with myocardial infarct were reversed by tyrosine. AB - Reports indicate that the administration of tyrosine, the precursor amino acid for catecholaminergic neurotransmitters, may be beneficial under conditions of physiologic stress. We studied the effects of tyrosine on vulnerability to ventricular arrhythmia in conscious pigs with healing myocardial infarcts, and sham operated (intact) pigs. Mean arterial pressure and heart rate were measured via chronically implanted aortic catheters. The repetitive extrasystole threshold (defined as the energy in milliamperes (ma) needed to cause a spontaneous ventricular beat following a premature beat induced by an electrical impulse), was measured via a bipolar pacing catheter placed during instrumentation surgery in the apex of the right ventricle. One week after infarct, the myocardial infarct group was studied before and ninety minutes after the administration of tyrosine (8 mg/kg iv). Before tyrosine, the myocardial infarct group had a significantly lower repetitive extrasystole threshold (12 +/- 1 ma) compared to the intact group (19 +/- 2 ma). Ninety minutes after tyrosine, the repetitive extrasystole threshold in the myocardial infarct group was 17 +/- 1 ma. The availability of tyrosine did not alter the repetitive extrasystole threshold in the intact group. Thus, vulnerability to ventricular arrhythmia was enhanced in pigs with recent myocardial infarction. Tyrosine, which can be nutritionally manipulated, may reduce myocardial vulnerability to arrhythmia after infarct. PMID- 1714031 TI - Use of a conflict procedure in pigeons to characterize anxiolytic drug activity: evaluation of N-methyl-D-aspartate antagonists. AB - Because of its apparent effectiveness in detecting non-benzodiazepine anxiolytic agents, a recently introduced conflict procedure in pigeons was used to evaluate possible anti-punishment activity of various N-methyl-d-aspartate (NMDA) antagonists. Punished responding was significantly increased by competitive NMDA antagonists (CPP, CGS 19755), but not by noncompetitive NMDA antagonists acting at either the ion channel (PCP, ketamine, MK-801), the glycine site (kynurenic acid, 7-chlorokynurenic acid, ACPC), or the polyamine site (ifenprodil) of the NMDA receptor complex; the proposed glutamate antagonist, riluzole, was also ineffective. PMID- 1714032 TI - Eicosapentaenoic acid inhibits tube formation of vascular endothelial cells in vitro. AB - We previously have described a quantitative angiogenesis in vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20:5n-3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 micrograms/mL of arachidonic acid (20:4n-6), eicosapentaenoic acid or docosahexaenoic acid (22:6n-3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The context of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cell was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 micrograms/mL of eicosapentaenoic acid. At 5.0 micrograms/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition of in vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (less than 5.0 micrograms/mL) had no effect on the proliferation of endothelial cells cultured on plastic plates without collagen gel. However, when the cells were cultured between collagen gels, eicosapentaenoic acid inhibited cell growth in a dose-dependent manner with maximum inhibition being observed at 2.5 micrograms/mL. These data suggest that eicosapentaenoic acid suppresses tube formation of endothelial cells,at least in part, via its inhibitory effect on cellular proliferation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714033 TI - Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene. AB - The organization and expression of the mitochondrial (mt) genome of fertile, male sterile and restored lines of Helianthus annuus and of H. petiolaris were compared to identify alterations which might lead to cytoplasmic male sterility (CMS). The mtDNAs of fertile and male-sterile lines differ by an 11 kb inversion and a 5 kb insertion. The rearrangements seem to be the result of recombination events within an inverted repeat of 261 bp. Detectable alterations in the transcript pattern of the rearranged mtDNA regions are restricted to the atpA locus. The male-sterile line CMSBaso shows three additional transcripts of the atpA locus of about 2500, 1200 and 250 nucleotides which are not detectable in Baso. However, the coding sequences of the atpA gene are entirely identical in the fertile line Baso and the male-sterile line CMSBaso. But a new open reading frame (orfH522) of 522 nucleotides is co-transcribed with the atpA gene as an additional larger transcript of about 2500 nucleotides in CMSBaso. orfH522 is also included in a second additional transcript of about 1200 nucleotides. The predicted translation product of orfH522 might play a role in CMS in sunflower. PMID- 1714034 TI - Organization, promoter analysis and transcriptional regulation of the Staphylococcus xylosus xylose utilization operon. AB - The Staphylococcus xylosus xyl genes were cloned in Staphylococcus carnosus by complementation to xylose utilization. Xylose isomerase assays under inducing (xylose present) and non-inducing (xylose absent) conditions indicated the presence of a regulated xylA gene on the recombinant plasmid. The nucleotide sequence (4520 bases) revealed three open reading frames with the same polarity. They were identified by sequence homologies as xylR, encoding the Xyl repressor, xylA, encoding xylose isomerase and xylB, encoding xylulokinase. Primer extension analyses indicated constitutive transcription of xylR and xylose-inducible transcription of xylA. Promoter consensus sequences were found upstream of both transcriptional start sites. A transcriptional terminator between xylR and xylA separates the different transcriptional units. Potential regulatory elements were identified by sequence analysis and suggest a repressor-operator mechanism for the regulation of xylAB expression. PMID- 1714035 TI - Cross-talk between the virulence and phosphate regulons of Agrobacterium tumefaciens caused by an unusual interaction of the transcriptional activator with a regulatory DNA element. AB - Transcription of a virulence gene on the hairy-root-inducing plasmid A4, which is induced by plant factors in Agrobacterium tumefaciens, was also activated by phosphate limitation in both A. tumefaciens and Escherichia coli. The starting site of RNA synthesized under the two inducing conditions was the same, and an identical promoter was responsible for both inducible expressions. The response of the virulence gene to phosphate limitation did not require the positive regulator VirG for the virulence regulon, but depended entirely on the presence of PhoB protein, the positive regulator for the phosphate regulon. The DNA signal upstream of the virulence gene, which is targeted by the VirG protein, was recognized by the E. coli PhoB protein in vitro. These results indicate that cross-talk between the two regulons occurred during the recognition of a DNA signal by the regulatory protein. PMID- 1714036 TI - Isolation and characterization of nitrate reductase-deficient mutants in tomato (Lycopersicon esculentum Mill.). AB - Five nitrate reductase-deficient mutants of tomato were isolated from an M2 population after ethylmethanesulphonate (EMS) seed treatment by means of selection for chlorate resistance. All mutations were monogenic and recessive and complementation analysis revealed that they were non-allelic. Biochemical and molecular characterization of these mutants showed that four of them are cofactor mutants while one is an apoenzyme mutant. PMID- 1714037 TI - Transcriptional regulation by metals of structural genes for Azotobacter vinelandii nitrogenases. AB - Azotobacter vinelandii has three nitrogenases: a molybdenum (Mo) nitrogenase, a vanadium (V) nitrogenase, and a third nitrogenase (nitrogenase-3), which apparently lacks Mo and V. Mo represses synthesis of both V nitrogenase and nitrogenase-3, and in the absence of Mo, V represses synthesis of nitrogenase-3. We have investigated transcriptional regulation of the three nitrogenases by metals using Northern analysis and probes specific for transcripts of each of the three nitrogenases. Our results confirm that Mo is required for expression of the Mo nitrogenase structural genes (nifHDK), and substantiate the notion that Mo represses transcription of the structural genes for both V nitrogenase and nitrogenase-3. We show that repression by V of nitrogenase-3 is also effected at the level of transcription. Unexpectedly, V only represses transcription of the nitrogenase-3 structural genes (anfHDGK) if the V nitrogenase structural gene cluster vnfDGK is present. Further, deletion of nifHDK allows low expression of anfHDGK in the presence of Mo. Repression by Mo or V is independent of cofactor synthesis and therefore of enzyme activity. PMID- 1714038 TI - Cellular interactions. Out of equilibrium. PMID- 1714039 TI - Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. AB - A cyclic AMP-stimulated chloride conductance appears when the cystic fibrosis gene is expressed in non-epithelial cells by infection with recombinant viruses. Cyclic AMP-stimulated conductance in this system is mediated by the same ohmic, low-conductance Cl- channel as in human secretory epithelia, but control of this channel by phosphorylation has not been directly demonstrated. Here we report the appearance of the low-conductance Cl- channel in Chinese hamster ovary cells after stable transfection with the cystic fibrosis gene. The channel is regulated on-cell by membrane-permeant analogues of cAMP and off-cell by protein kinases A and C and by alkaline phosphatase. These results are further evidence that the cystic fibrosis transmembrane regulator is a Cl- channel which can be activated by specific phosphorylation events and inactivated by dephosphorylation; they reveal an unsuspected synergism between converging kinase regulatory pathways. PMID- 1714040 TI - Differential effects of clomipramine given locally or systemically on extracellular 5-hydroxytryptamine in raphe nuclei and frontal cortex. An in vivo brain microdialysis study. AB - The antidepressant drug clomipramine (CIM) blocks 5-hydroxytryptamine (5-HT) uptake in vitro. Electrophysiological studies have shown that CIM also reduces the firing of serotonergic neurons in the dorsal raphe nucleus. In order to assess the effects of CIM on serotonergic transmission in vivo, the technique of intracerebral microdialysis was used. CIM was administered either through the dialysis probe or i.p., and dialysate 5-HT and 5-hydroxyindoleacetic acid (5 HIAA) were determined in frontal cortex and/or raphe nuclei. In addition, the action of extracellular 5-HT in raphe nuclei on the release of 5-HT in frontal cortex was studied. The administration of CIM through the dialysis probe increased dialysate 5-HT in frontal cortex in a dose-dependent fashion. An actual ED50 of 3.15 microM CIM for the in vivo inhibition of 5-HT uptake can be calculated in this brain area. When given systemically (10 or 20 mg/kg i.p.), CIM did not increase dialysate 5-HT in the frontal cortex. The occurrence of extracellular 5-HT in the the raphe area was demonstrated. This pool of 5-HT increased markedly after local (10 or 40 microM) or systemic (20 mg/kg i.p.) administration of CIM. We also examined the effect of CIM applied locally in the raphe nuclei on extracellular 5-HT in the frontal cortex. The increased dialysate 5-HT in raphe after 10 or 40 microM CIM paralleled a decrease of dialysate 5-HT in the two areas correlated negatively. The administration of CIM through the dialysis probe slightly decreased dialysate 5-HIAA in the frontal cortex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714041 TI - Tachykinin antagonists inhibit the morphine withdrawal response in guinea-pigs. AB - This study was an investigation of the effects of the tachykinin antagonists, spantide and (D-Pro4, D-Trp7,9,10)substance P 4-11, injected intracerebroventricularly (ICV), on the locomotor and behavioural responses of guinea-pigs to substance P (SP) injected ICV and to naloxone-induced morphine withdrawal. SP, 50 nmol, produced increased locomotor activity and behaviour that mimicked the response induced by injection of naloxone hydrochloride, 15 mg/kg, in guinea-pigs treated 2 h previously with morphine sulphate, 15 mg/kg. Spantide or (D-Pro4, D-Trp7,9,10)SP4-11, 10 nmol, reduced the locomotor and behavioural responses to SP and to morphine withdrawal. The results support the suggestion that SP or a related tachykinin might be a mediator of the opioid withdrawal response in the central nervous system as has been proposed for the enteric nervous system. PMID- 1714042 TI - Limited proteolysis of single-chain tetanus toxin by tissue enzymes, in cultured brain tissue and during retrograde axonal to the spinal cord. AB - Single-chain toxin was investigated in vitro and in vivo for limited proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [3H]noradrenaline release in vitro. Cultured mouse brain generated fragments from 125I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i.v. into rats, 125I-single-chain-toxin disappeared from the blood with a half life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to limited proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems. PMID- 1714043 TI - Expression of the neuronal noradrenaline transporter in Xenopus laevis oocytes. AB - RNA isolated from both rat phaeochromocytoma (PC12) cells and bovine adrenal medulla was size-selected and injected into oocytes of Xenopus laevis. Expression of the neuronal noradrenaline transporter was assayed 3 days after injection by measuring the nisoxetine-sensitive noradrenaline uptake. A 2 kilobase fraction of either total RNA derived from both tissues or of polyadenylated mRNA obtained from bovine adrenal medulla caused Na(+)-dependent transport of 3H-noradrenaline in the injected oocytes. PMID- 1714044 TI - Plasma levels of 5-hydroxyindole-acetic acid in chronic renal insufficiency and their effect on platelet aggregation. PMID- 1714045 TI - Hemodynamic evaluation in patients with superficial temporal artery-middle cerebral artery anastomosis--stable xenon CT-CBF study and acetazolamide. AB - Sixteen patients with minor completed stroke in the chronic stage underwent superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis. The acetazolamide-activated regional cerebral blood flow (rCBF) was measured 20 minutes after the injection using inhalation of stable xenon and computed tomographic scanning (Xes CT-CBF study) pre- and postoperatively. Eleven patients (Group 1) showed immediate improvement in neurological state within a few days of the operation, while five (Group 2) showed no improvements. Preoperative rCBF in the ischemic areas without infarction was 30.8 +/- 3.0 ml/100 gm/min in Group 1 and 53.0 +/- 5.2 ml/100 gm/min in Group 2. Preoperative vasodilatory capacity with acetazolamide in Group 1 was 5.7 +/- 8.6 and significantly increased to 19.8 +/- 4.9 after surgery. In Group 2, pre- and postoperative vasodilatory capacity was 12.7 +/- 3.1 and 14.9 +/- 2.9, respectively, and there was no significant change. These results suggested that minor stroke patients with moderate decrease of affected side rCBF (less than 40 ml/100 gm/min) and with hemodynamic impairment may have the surgical indication for STA-MCA anastomosis. PMID- 1714046 TI - Contrast medium extravasation during cerebral angiography for ruptured intracranial aneurysm--clinical analysis of 26 cases. AB - Since 1976, the authors performed angiography on 837 patients with ruptured intracranial aneurysm, among which extravasation (EV) of contrast medium occurred in 26. Eleven patients underwent radical surgery within 3 hours after the EV, and four recovered well. The other 15 patients were conservatively treated, and all died. A large quantity of EV occurred when consciousness was severely disturbed and angiography was performed within 3 hours of the last attack. Most EV resulted in poor outcome. However, some patients with mildly disturbed consciousness before angiography, even with severe temporary disturbance of consciousness after the EV, recovered well after emergency radical surgery. PMID- 1714047 TI - Familial pituitary adenoma--report of four cases from two unrelated families. AB - The authors report four cases of familial pituitary adenomas from two unrelated families. No clinical or biochemical evidence of multiple endocrine neoplasia, type I (MEN-I) was demonstrated. Detailed study of the family trees disclosed no other family members affected by MEN-I. Familial occurrence of pituitary adenomas unassociated with MEN-I is rare. PMID- 1714048 TI - New method of MVD using a vascular tape for neurovascular compression involving the vertebrobasilar artery--report of two cases. AB - In the treatment of hemifacial spasm and trigeminal neuralgia by microvascular decompression (MVD), lack of improvement or recurrence may occur because of the difficulties in positioning prostheses and the involvement of the large vertebrobasilar arteries, even with use of fenestrated aneurysm clips or adhesives. We have developed a new method of MVD, in which a vascular tape is anchored to the dura mater to transpose the responsible large artery. This method achieved successful results in our two patients with nerve compression involving the vertebrobasilar arteries. PMID- 1714049 TI - Successful reconstruction of completely obstructed right CCA in Takayasu's disease: usefulness of rapid sequence scanning method--case report. AB - The authors describe a case of Takayasu's disease accompanied by complete occlusion of the bilateral subclavian, left vertebral (VA), and right common carotid arteries (CCA) and by severe stenosis of the right VA and left CCA. The patency of the right external and internal carotid arteries was demonstrated preoperatively by rapid sequence computed tomographic scanning. The right CCA was successfully reconstructed with an aorta-CCA bifurcation bypass. Simultaneously, the stenotic left CCA was also reconstructed with an aorta-CCA bypass using a Y shaped woven Dacron graft. PMID- 1714050 TI - Giant congenital capillary hemangioma of pericranium--case report. AB - The authors report a newborn male infant with a giant congenital capillary hemangioma of the pericranium. An elastic mass, measuring 6.5 x 6.9 x 3.9 cm, was located in the parieto-occipital region. Neurological examination revealed no abnormality. Angiographically, the tumor was fed symmetrically by the bilateral superficial temporal, occipital, and middle meningeal arteries. At surgery, the encapsulated tumor appeared to have arisen from the periosteum and was removed completely. Histological diagnosis was capillary hemangioma. Capillary hemangioma is a common benign tumor in infancy and usually present as a strawberry mark or port-wine stain. However, when the tumors seat relatively deeply as in the present case, they produce little or no discoloration in the overlying skin. Angiography is then useful to differentiate capillary hemangioma from other lesions and to choose an appropriate treatment. PMID- 1714051 TI - Ceruminoma with intracranial invasion--case report. AB - Ceruminous gland tumors (ceruminomas), which usually involve the external auditory canal, are rare. A case of ceruminoma invading the temporal bone and histologically proven to be papillary adenoma is presented. The tumor recurred and invaded intracranially after subtotal removal and was finally diagnosed as adenocarcinoma. The importance of early diagnosis and radical treatment is stressed. PMID- 1714052 TI - Multiple intracranial metastases following malignant evolution in recurrent pleomorphic adenoma of the lacrimal gland--case report. AB - A 54-year-old female presented with multiple intracranial metastases following malignant transformation in the third recurrence of pleomorphic adenoma of the left lacrimal gland, 25 years after the first surgical treatment. The preoperative computed tomography and magnetic resonance imaging demonstrated direct invasion of the orbital tumor into subdural and epidural spaces in the ipsilateral frontotemporal region and also an intracerebral metastasis in the ipsilateral parietal lobe. Histological examination of the surgical specimen revealed features of poorly differentiated adenocarcinoma, suggesting carcinomatous changes. The relevant literature is reviewed. PMID- 1714053 TI - Eosinophilic granuloma of the skull in identical twins--case report. AB - This is the first reported occurrence of identical twins with eosinophilic granuloma of the skull. These cases provide strong additional support that eosinophilic granuloma and Hand-Schuller-Christian disease are variants of the same basic disease process. PMID- 1714054 TI - Amino acid study of cerebral gliomas using positron emission tomography--analysis of (11C-methyl)-L-methionine uptake index. AB - Sixteen patients with gliomas (7 low grade, 9 high grade) were examined using positron emission tomography (PET) with intravenous administration of 22.2 MBq/kg (0.6 mCi/kg) of (11C-methyl)-L-methionine (C-11 Met). The tracer uptake in regions of interest was calculated on PET images taken 45 minutes after injection; the uptake index was represented as a percentage of the total count in the arterial blood summed over 45 minutes. C-11 Met uptake indices in the tumors ranged from 0.020 to 0.041% with a mean of 0.032% for the low-grade gliomas and from 0.013 to 0.044% with a mean of 0.036% for the high-grade gliomas. These indices significantly increased as compared with those in the contralateral gray matter (0.008-0.032% with a mean of 0.023%; p less than 0.01 vs. low-grade gliomas, p less than 0.001 vs. high-grade gliomas). In the low-grade gliomas, C 11 Met PET images clearly depicted the existence and even the extent of the tumors, although x-ray computed tomography (CT) did not always distinguish tumoral lesions. In the high-grade gliomas, the areas of tracer accumulation regionally extended to peritumoral low density on CT scans, where malignant tumor cell infiltration was proved by operative and follow-up CT findings. C-11 Met may be a useful radiopharmaceutical for differential diagnosis of gliomas, and the accuracy of tumor localization will give us a better rationale in therapeutic strategies for surgery and radiation therapy of gliomas. PMID- 1714055 TI - An attempt to localize the site of action of different agents within cholinergic motor neurones of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum by the triple bath method. AB - The site of action of cholinergic, adrenergic, peptidergic and opioid agents was studied in myenteric plexus-longitudinal muscle strips from the guinea pig ileum. A preparation in a special triple bath was drawn through two rubber membranes, dividing the strip into three segments. Neurogenic stimulation of the oral segment, set up nerve action potentials also in the neurones projecting axons up to the aboral segment. These axons, turning into varicose nerve terminals, conducted action potentials aborally across the middle segment, that was up to 10 mm wide. Finally, the nerve terminals, extending into the aboral segment, might be also invaded triggering twitches. Agents were added, either to the oral segment, to affect the genesis and spread of action potentials in the proximal parts of cholinergic neurones (cell bodies, axon hillocks, initial segments and axon preterminals) or they were added to the middle segment to affect propagation of action potentials in varicose nerve terminals. As a result, the amplitude of aboral twitches reflected their effects at each site, quantitatively. Noradrenaline and ethylketocyclazocine were more effective at the site of varicose nerve terminals, whereas substance P, acetylcholine and oxotremorine were more effective at the proximal parts; pilocarpine and nicotine were effective at both sites. Changes in membrane polarization might be the final common effect in the mechanism of action of all the stimulatory agents used. PMID- 1714056 TI - Rapidly progressive dementia in a patient with the Lewy body variant of Alzheimer's disease. AB - A 65-year-old woman presented with a mild memory impairment, spatial disorientation, and poor task initiation. Progression was rapid over 3 months. She developed severe apathy, delusions, extrapyramidal features, and psychometrically quantified cognitive deterioration. Her brain showed many neocortical neuritic plaques and neurofibrillary tangles along with neocortical and brainstem Lewy bodies and temporal lobe spongiform vacuolization. This case is the most rapid deterioration documented of a patient with Alzheimer's disease and Lewy bodies. PMID- 1714057 TI - Prognosis in AZT myopathy. AB - The myopathy caused by zidovudine (AZT) appears to be common but is incompletely characterized, particularly regarding prognosis. Twenty patients with HIV infection developed a necrotizing myopathy while taking AZT for 9 to 30 months. Ten presented with myalgia and 17 with proximal muscle weakness. Serum CK was elevated in all (two to 11 times normal), and EMG suggested active myopathy in all but two. There were scattered granular degenerating fibers, with scant or no inflammation, in a pattern consistent with a toxic myopathy in all 18 patients biopsied. Three patients with an HIV-related inflammatory myopathy were distinguished by histologic differences. After stopping AZT (n = 15), myalgia promptly resolved (10 of 10). Strength improved more slowly with 12 of 15 regaining normal or nearly normal strength, but three have persistent weakness. CK returned to normal in 12 of 15, and follow-up EMG (n = 11) documented reduced fibrillation density in all 11 patients. These findings underscore the need for early diagnosis of this reversible myopathy. PMID- 1714058 TI - Acetylcholine receptor-reactive T lymphocytes from healthy subjects and myasthenia gravis patients. AB - Peripheral blood lymphocytes from 23 of 114 (20%) myasthenia gravis (MG) patients showed positive T-cell proliferative responses to native acetylcholine receptor (AChR) purified from the electric fish Torpedo, compared with two of 25 (8%) healthy or other neurologic disease controls. Responsiveness appeared to fluctuate seasonally. Long-term T-cell lines and clones could be selected as readily from the two healthy responders as from the MG cases and showed similar culture behavior, CD4+ phenotype, and HLA class II restrictions. One clone from a control cross-reacted with recombinant human AChR alpha chain (r37-429A) and with the synthetic peptide 125-143(S-S) from its sequence. Both these human antigens stimulated primary proliferative responses at substantially higher frequencies (26 to 59%) than native xeno-AChR--in both patients and controls--demonstrating that truly autoreactive T cells are not inevitably deleted during normal T-cell development. PMID- 1714059 TI - Dystrophin deficiency in young girls with sporadic myopathy and normal karyotype. AB - We studied dystrophin in three young girls with a sporadic myopathy of early onset, manifested by mild to severe limb weakness, calf hypertrophy, high serum creatine kinase, normal karyotype, and morphologic features in muscle consistent with muscular dystrophy. DNA analysis did not reveal a deletion of the dystrophin gene. Immunohistochemical studies of dystrophin in muscle biopsies showed a mosaic of fibers with and without dystrophin, and immunoblot analysis showed partial dystrophin deficiency in all three patients, more severe in the patient with the highest proportion of dystrophin-deficient fibers. These observations suggest that the patients are Duchenne muscular dystrophy carriers. The data also support the concept that uneven lyonization in muscle is responsible for the clinical myopathy in these patients. We suggest that any girl with sporadic proximal limb weakness should be evaluated as a possible Duchenne carrier by dystrophin studies. PMID- 1714060 TI - Failure of copolymer I to inhibit the human T-cell response to myelin basic protein. AB - In clinical trials, copolymer I (Cop 1) appears to reduce the number of exacerbations in early relapsing-remitting multiple sclerosis. The mechanism of this effect is uncertain, but Cop 1 also reduces the severity of experimental allergic encephalomyelitis and inhibits the response of murine myelin basic protein (MBP)-specific T cells. We tested MBP-specific T-cell lines and clones from four subjects to determine whether Cop 1 also limits the human response to MBP. We found no inhibition by Cop 1 of the human T-cell response to MBP. PMID- 1714061 TI - Specific antibodies enhance alternative complement pathway activation by cuprophane. AB - Cuprophane activates the human alternative complement pathway. We have observed that the extent to which the alternative pathway was activated in the presence of fixed amounts of cuprophane in vitro greatly differed between individuals. Several lines of evidence indicated a role for anti-dextran antibodies in determining the extent of alternative pathway activation by cuprophane in whole serum: (1) alternative pathway activation by cuprophane was directly related to the extent to which it was activated by Sephadex in a given serum; alternative pathway activation by Sephadex was previously shown to be antibody-dependent; (2) alternative pathway activation by cuprophane was related to the serum titre of anti-dextran IgG antibodies; (3) adsorption of serum with Sephadex or cuprophane reduced the ability of the serum to be activated in the presence of fresh cuprophane. These results suggest that measuring serum values of anti-dextran antibodies may be potentially helpful for evaluating patients at increased risk of complement activation during haemodialysis with cuprophane membranes. PMID- 1714062 TI - Haemodialysis and platelet activation. PMID- 1714063 TI - Stimulation of biosynthesis of nerve growth factor by acidic fibroblast growth factor in cultured mouse astrocytes. AB - Bovine acidic and basic fibroblast growth factors (aFGF and bFGF) dose dependently stimulated the release of nerve growth factor (NGF) in a culture medium of mouse astrocytes. The NGF concentration in the medium started to increase compared to that of the control cultures 4 h later and was further sustained for 24 h when aFGF was contained. The content of NGF mRNA in the astrocytes treated with aFGF peaked at eightfold over the control level after 4 h. The astrocytes did not proliferate until after 72 h when treated with FGFs under the conditions employed. These results indicate that aFGF stimulates the biosynthesis of NGF in cultured astrocytes without promoting cell proliferation. PMID- 1714064 TI - Pertussis toxin-sensitive G-protein mediates galanin's inhibition of scopolamine evoked acetylcholine release in vivo and carbachol-stimulated phosphoinositide turnover in rat ventral hippocampus. AB - The effects of intracerebroventricular (i.c.v.) injections of pertussis toxin were investigated on the inhibitory action of galanin on acetylcholine release and phosphoinositide breakdown stimulated by muscarinic agents in rat ventral hippocampus. Pertussis toxin (0.6 micrograms, i.c.v., 96 h) counteracted the in vitro inhibitory effect of galanin (3.1 nmol) on phosphoinositide breakdown stimulated by carbachol without altering the stimulatory action of the cholinergic agonist on signal transduction, in miniprisms from rat ventral hippocampus. Pertussis toxin also abolished the in vivo effect of galanin on scopolamine-stimulated acetylcholine release in vivo but did not affect basal acetylcholine release. The results indicate that pertussis toxin-sensitive G protein(s) mediates the galanin receptor regulation of pre- and postsynaptic cholinergic functions in the ventral hippocampus. PMID- 1714065 TI - Lasting loss in substance P following administration of substance P antiserum to newborn rats. An immunohistochemical study. AB - Substance P (SP) antiserum (500 micrograms protein) was administered to rats on the second day of life and the animals were sacrificed 3 months later. This treatment produced a loss in SP immunoreactive fibers in the dorsal horn of the spinal cord, in the substantia nigra and in the periaqueductal gray matter, when compared to control animals receiving a neonatal treatment of non-specific immunoglobulins. In the dorsal horn, the observed depletion was greater in the superficial layers, lamina I and lamina IIo. Immunoreactivity for Met-enkephalin was apparently unchanged by SP antiserum. Results of this study provide cytochemical evidence for a specific and lasting deleterious effect of SP antiserum on different SP-containing neuronal systems. PMID- 1714066 TI - Cells of origin of avian postsynaptic dorsal column pathways. AB - Whereas in the cervical spinal cord of pigeons lamina IV and medial lamina V neurons are at the origin of postsynaptic pathways to the dorsal column nuclei, lumbar lamina IV neurons do not project substantially beyond the cervical enlargement. There is, however a distinct group of medially located lumbar lamina V neurons which projects ipsilaterally to the dorsal column nuclei. PMID- 1714067 TI - [Prevalence of hepatitis C virus antibodies in hospital personnel and the general population]. AB - The recent cloning of the genome of a parenterally transmitted non-A, non-B hepatitis virus, designated the hepatitis C virus (HCV), has been used for the development of an enzyme immunoassay for the detection of antibodies against HCV (anti-HCV). We have employed this assay to evaluate the prevalence of HCV antibodies in hospital personnel and voluntary blood donors. Twelve of 1018 sera (1.2 percent) from health care workers were repeatedly reactive in the enzyme immunoassay for anti-HCV. Specificity testing with a modification of the enzyme immunoassay (additional wash cycle with 8 mol/l urea) and a recombinant immunoblot assay demonstrated HCV antibodies in only 6 of the 12 sera. Thus, the true prevalence of anti-HCV in hospital personnel was 0.6 percent. Nine of 1046 sera (0.9 percent) from blood donors were reactive in the enzyme immunoassay for anti-HCV. In none of the 9 sera the presence of HCV antibodies could be confirmed by additional testing with the urea wash modification or the recombinant immunoblot assay. The true prevalence of anti-HCV in blood donors thus appears to be lower than 0.1 percent. Our results indicate that the risk of HCV transmission in the hospital setting appears to be low, but is significantly higher than that of the general population. PMID- 1714068 TI - [Developmental delay of school beginners with resulting partial performance limitations]. AB - There is an incomplete degree of school maturity in approximately ten per cent of our school beginners because of developmental delays which may have genetic or psychosocial causes or may be caused by an infantile cerebral dysfunction. A consequence of these developmental delays are the partial disturbances of performance frequently resulting in failure at school. An early registration of the partial disturbances of performance is important for the prognosis of these children in order to prevent the development of secondary unbalanced behaviour. Moreover an after maturation of the damaged cerebral centres can be reached by early support. PMID- 1714069 TI - Plasmodium falciparum (human malaria)-induced modifications in human erythrocyte band 3 protein. AB - A monoclonal antibody, 1C4, was produced which recognizes a 65 kDa protein that is localized to the plasma membrane of human erythrocytes infected with Plasmodium falciparum. By immunofluorescence the antigen was visualized as dots on the surface of the infected cell. The 65 kDa protein was present in 4 strains of diverse geographical origin, and in erythrocytes infected with a knobless strain. The 65 kDa protein was insoluble in non-ionic detergents, but was partly soluble in SDS and some high (1 M) salt solutions. The 65 kDa protein is recognized by antibodies specific for the cytoplasmic domain and the N-terminal side of the membrane-spanning region of human band 3, but was not recognized by an antibody specific to the C-terminal side of the membrane-spanning region. The results of treatment of the 65 kDa protein with trypsin and chymotrypsin are consistent with the 65 kDa protein being a truncated and covalently modified band 3 molecule which consists of the first 540 amino acids of human band 3. PMID- 1714070 TI - Detection of Chlamydia trachomatis cervical infection: a comparison of Papanicolaou and immunofluorescent staining in smears obtained by Ayre's spatula and cytobrush. AB - Two methods of staining (Papanicolaou versus direct immunofluorescence) and two methods of collection of the samples (Ayre's wooden spatula versus cervical Cytobrush) were compared in order to verify the efficiency in detecting Chlamydia trachomatis (CT) infections in the female genital tract. Out of 166 asymptomatic patients, 59 were positive for CT by means of direct immunofluorescence: 36 were detected in Cytobrush samples, 16 in Ayre's spatula samples, and 7 in the samples collected by both methods. Papanicolaou smears showed "moth-eaten" features suggestive of CT infection in a great number of metaplastic cells present in 35 cases: 24 collected by Cytobrush, 4 by Ayre's spatula and 7 by both methods. Our data show that Cytobrush is more efficient that Ayre's spatula in concentrating cellular material. It is thus possible to detect CT infection with more accuracy by means of direct immunofluorescence, and to suspect CT infection in smears collected by means of Cytobrush and stained by Papanicolaou's method. PMID- 1714071 TI - Antenatal predictors for Down's syndrome. PMID- 1714073 TI - [Development, structure and maturation of megakaryocytes. I]. PMID- 1714072 TI - [Endoscopic prosthesis of the biliary tract]. AB - In the years 1987--1989 endoscopic biliary tree stenting was performed in 64 patients. The straight prostheses type Amsterdam 10 or 12 F and double pigtail prostheses were applied. In 36 cases the indication for stenting was malignant stricture of the biliary tree (group I) caused by carcinoma of the ampulla of Vater (n = 12), carcinoma of the head of the pancreas (n = 15), common bile duct and bifurcation tumor (n = 5) and gallbladder cancer (n = 4). In 28 cases the indications were common bile duct stones (group II) where stone size made endoscopic removal impossible and surgery was contraindicated. Normalisation or improvement of elevated serum bilirubin level was observed quite quickly after stenting (it decreased to about 50% of initial value after 7 days). Mean duration of prosthesis patency in group I patients who avoided surgical treatment was 144 days and mean survival time was 220 days. In group II patients mean duration of prosthesis patency was 183 days. Endoscopic biliary tree stenting is an advantageous alternative to surgical palliative++ treatment of obstructive neoplastic jaundice and constitutes an efficient method of treating common bile duct stones in poor operative risk patients. PMID- 1714074 TI - Distribution of ICAM-1 within decidua and placenta and its gestational age associated changes. AB - Intercellular adhesion molecule 1 (ICAM-1), a ligand of leukocyte function associated antigen 1 (LFA-1), is present on many cells, including monocyte/macrophages. ICAM-1 is considered to play an important role in the induction and maintenance of inflammatory responses by permitting leukocyte adhesion. Its expression is inducible on endothelial and epithelial cells exposed to various inflammatory cytokines (preceding expression of HLA-DR) and is maturation dependent in certain cell lines. The distribution of ICAM-1 in decidua and placenta was evaluated using peroxidase-antiperoxidase immunohistochemistry. In decidua of first and third trimesters, scattered ICAM-1-staining cells were observed. In placentas of first and third trimesters, all types of trophoblasts stained negatively for ICAM-1. Prior to 10 weeks of gestation, the villous stroma was uniformly ICAM-1 and HLA-DR unreactive. Beginning in the chorionic plate at approximately 10 weeks, scattered ICAM-1-positive stromal cells were observed, whereas stromal cells of the terminal villi revealed no ICAM-1. By 14-16 weeks, approximately 40-50% of the terminal villous stromal cells were ICAM-1 staining. This parallels the 40-50% of the villous stromal cells that share other immunohistochemical markers, such as EB-11, with monocyte/macrophages. The lack of functional maturation of the villous stromal macrophage may explain the rarity of chronic villitis early in gestation. PMID- 1714075 TI - Sinus histiocytosis with massive lymphadenopathy: a spectrum of disease associated with immune dysfunction. AB - A detailed immunologic study of three cases of sinus histiocytosis with massive lymphadenopathy (SHML) was performed to better characterize this rare disorder. One patient had prominent cervical lymphadenopathy that regressed spontaneously, whereas the other two patients had persistent cervical lymphadenopathy and recurrent infections. The first patient was otherwise healthy and had normal immunologic studies. One of the latter patients had a relative increase in blood B cells, a decreased level of serum immunoglobulin A (IgA), decreased blood lymphocyte mitogenic responses to multiple mitogens (37-42% of controls), and cutaneous anergy. The other patient with persistent disease also had a relative increase in blood B cells, polyclonal hypergammaglobulinemia, and circulating immune complexes, as well as decreased blood T cells and markedly decreased blood lymphocyte responses to mitogens (12-37% of controls). Immunohistochemical stains of the lymph nodes of the three patients revealed a characteristic phenotype for the sinus histiocytes: S-100 protein, 3/3; CD14 (Leu M3) 3/3; CD11c (Leu M5), 1/1; CD71 (OKT9), 3/3; CD4 (Leu 3a), 2/3; CD1a (OKT6), 1/3; alpha-1-antitrypsin, 3/3; alpha-1-antichymotrypsin, 3/3; CD35 (C3b), 1/1; CD11b (Mo1), 0/3; CD15 (Leu M1), 0/3; HLA-DR, 0/3; and lysozyme, 0/3. This phenotype suggests that the cells of SHML have features of both the Langerhans/interdigitating cell and mononuclear phagocyte lineages. Emperipolesis by the histiocytes of B cells, T cells, and natural killer cells was demonstrated by a double-staining technique. Our findings indicate that patients with SHML may have a variably expressed immunodeficiency that predisposes them to recurrent infections. PMID- 1714076 TI - Zellweger syndrome: a histochemical diagnosis of two cases. AB - Until 17 years ago the diagnosis of the cerebrohepatorenal Zellweger syndrome (ZS) rested largely on clinical grounds confirmed by pathologic findings at autopsy. The observation that peroxisomes are not detectable morphologically or histochemically in liver or kidney of patients with this syndrome gave histopathologists the opportunity to make the diagnosis of this complex syndrome at biopsy. Catalase reaction of the peroxisomes can be used as a rapid and accurate method to differentiate between ZS and other clinical conditions in which the peroxisomes are present in normal or reduced number. We describe two patients in whom the diagnosis of ZS was made by the absence of histochemical staining for catalase in a liver biopsy. The findings were subsequently confirmed using standard biochemical tests. PMID- 1714077 TI - Hyperplasia of bombesin-immunoreactive pulmonary neuroendocrine cells and neuroepithelial bodies in sudden infant death syndrome. AB - The distribution and frequency of bombesin immunoreactive neuroendocrine (NE) cells including neuroepithelial bodies (NEB) was analyzed morphometrically in lung sections from 25 infants who died of sudden infant death syndrome (SIDS) and 25 control infants. The control group included infants age-matched to those with SIDS, as well as subjects ranging in age from early to late infancy, to define the postnatal development of pulmonary NE-cell system. Quantitative analysis was performed on lung sections immunostained with monoclonal antibody against bombesin and the contents of bombesin-like peptide in lung extracts were measured by a specific radioimmunoassay (RIA). In control infants, the frequency of NE cells was high at birth but decreased dramatically during the first year of life. In SIDS infants, the frequency of NE cells, the size of NEB, and the mean concentration of bombesin-like peptide detected by RIA were significantly increased compared to those values for age-matched controls. These findings suggest hyperplasia of bombesin-immunoreactive NE-cell system in the lungs of SIDS infants. Since NEB are thought to function as hypoxia-sensitive airway chemoreceptors and since these cells are prominent in the neonates but decline postnatally, we speculate that chronic hypoxia and/or developmental delay may be responsible for this alteration in the lungs of SIDS victims. Potential dysfunction of pulmonary NE-cell system, compounded by other abnormalities in the autonomic regulation of respiration may be of importance in the pathogenesis of SIDS. PMID- 1714078 TI - A co-twin fetus papyraceus as a cause of elevated AFP and acetylcholinesterase in the amniotic fluid of the normal co-twin. AB - An elevated amniotic fluid alpha-fetoprotein (AF-AFP) level together with a positive acetylcholinesterase (AChE) band is strongly predictive of neural tube defect (NTD) in the fetus. We report such results in a pregnancy in which the fetus was found to be normal after termination. Among the placental fragments was found a sac containing a prenatally undetected co-twin fetus papyraceus. We suggest that pregnant women with such laboratory results but lacking sonographic evidence of NTD should have a high-level untrasonographic investigation, as well as a thorough pathologic examination of both placenta and fetus in cases of termination. PMID- 1714079 TI - Anatomical location of phosphorylcholine and other antigens on encysted Trichinella using immunohistochemistry followed by Wheatley's trichrome stain. AB - This work investigated the location on the parasite of Trichinella antigens recognized by the mouse immune system and the question as to which of them bear the epitope phosphorylcholine (PC). Wheatley's trichrome stain (initially developed for faecal smears) proved to be excellent for visualization of Trichinella structures, enabling four types of stichocyte to be distinguished. By applying this stain on infected muscle sections after immunocytochemistry using (a) anti-PC BH8 monoclonal antibodies, (b) serum from mice that had been infected twice in the presence of 0.05% thiabendazole (to prevent reproduction by adult females) and then bled on day 7 post-reinfection, (c) serum from infected mice that were bled on day 14 postinfection, or (d) serum from infected mice that were bled on day 42 postinfection, we found (1) that PC is an abundant structural epitope on the hypodermis/muscle, genital primordium and intestinal tract but is absent from the cuticle and stichosome; (2) that the principle secretory cells of adult worms are delta- and beta-stichocytes, whereas those of migrating and encysted L1 larvae are alpha-stichocytes; and (3) that Trichinella antigens recognized in the encysted phase of the parasite's life cycle are present in parasitized myofibres in the sarcoplasmic matrix and in the nucleoplasm of hypertrophic nuclei. The significance of these findings is discussed. PMID- 1714080 TI - An identical epitope in Pneumocystis carinii and Toxoplasma gondii causing serological cross reactions. AB - A monoclonal antibody raised against membrane proteins of Toxoplasma gondii with molecular weights of 35 and 21 kDa also reacts strongly and "specifically" with surface antigens of Pneumocystis carinii with molecular weights of 394.2 and 69 kDa when used in a direct immunofluorescence antibody test, on the one hand, and in a immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), on the other. Whether or not this observation might have any phylogenetic relevance remains open. PMID- 1714081 TI - [The combined radio-chemotherapy of inoperable esophageal cancer]. AB - In a period of almost 6 years three various therapeutic methods of inoperable oesophagus carcinoma were applied. The best survival was attained by a combination of irradiation with 5-FU-infusions and bleomycin. The mean survival was 8 months, contrary to 4 months by a combination irradiation/5-FU-infusions and 5 months by unique radiotherapy. Further analyses to radio-chemotherapy of inoperable oesophagus carcinoma are desirable with that to improve the survival of these tumors further on. PMID- 1714082 TI - High-dose-rate intraluminal brachytherapy for esophageal cancer: 10 years experience in Hyogo College of Medicine. AB - From May 1980 through December 1989, 148 patients with thoracic esophageal cancer were treated with high-dose-rate intraluminal brachytherapy (HDRIBT) following external radiotherapy (ERT). The standard treatment protocol was 60 Gy/6 weeks of ERT and 12 Gy/1 week of HDRIBT. The patients were divided into two groups according to disease stage. Sixty-six patients had limited disease (LD), and 82 patients had extensive disease (ED). The 2-year survival rate was 37% in LD group, and 7% in ED group. The 5-year survival rate of LD group was 18%. The 1- and 2-year actuarial local control rate was 66% and 64% in LD group, and 49% and 45% in ED group, respectively. In the total patients, ulceration, stricture, and fistula were found in 42 (28%), 15 (10%) and 6 (4%) patients, respectively; however, major complication defined as one resulting in a second hospitalization or requiring surgical intervention was 3 of the 42 patients with ulceration, 1 of the 15 patients with stricture, and all of the six patients with fistula. As to cause of death, local failure, local failure with distant metastasis, distant metastasis, intercurrent disease, and unknown reason was 14, 6, 15, 16 and 0 in LD group, and 13, 24, 30, 9 and 1 in ED group, respectively. The technique and method of combined treatment are described in detail. PMID- 1714083 TI - Intraoperative brachytherapy alone for incomplete resected recurrent rectal cancer. AB - In order to determine the impact of intraoperative brachytherapy alone in patients with recurrent rectal cancer who, due to prior pelvic radiation therapy, were ineligible to receive further external beam pelvic radiation, we retrospectively reviewed the records of 36 patients with recurrent rectal cancer who had gross residual disease remaining in the pelvis following biopsy alone or subtotal resection. The median follow-up was 24 months (6-81 months). The median survival was 27 months and the 4 year actuarial survival was 25%. There was a suggestion of lower survival in patients who underwent biopsy alone compared with those who underwent a subtotal resection (21% vs. 34%). The local failure (LF) rate was 22% as the only site of failure and 44% as a component of failure. There was a lower but non-significant LF rate in patients who underwent subtotal resection vs. biopsy alone (33% vs. 66%) and those with an 125I implant volume of less than 40 cm3 vs. greater than or equal to 40 cm3 (39% vs. 100%). Four patients (11%) developed treatment-related severe complications (without evidence of LF). Our data suggest that, although it is not clear that intraoperative brachytherapy impacts on the ultimate survival rate in this group of patients, it does offer reasonable local control with acceptable morbidity. Since local control, in and of itself is an important endpoint in the treatment of rectal cancer, we continue to recommend brachytherapy as part of an overall aggressive approach in patients who are unable to receive pelvic radiation therapy. PMID- 1714084 TI - Effect of the specific cholecystokinin-receptor antagonist loxiglumide on bombesin stimulated pancreatic enzyme secretion in man. AB - We have investigated the effects of the specific cholecystokinin (CCK) receptor antagonist loxiglumide on basal and bombesin stimulated pancreatic enzyme secretion, bilirubin output and plasma CCK release in six healthy subjects. The data were compared with those obtained in control experiments where saline was infused instead of loxiglumide. Basal amylase output (4.7 +/- 0.8 kU/45 min), trypsin output (2.9 +/- 0.8 kU/45 min) and bilirubin output (7.7 +/- 2.8 mmol/45 min) gradually declined during infusion of loxiglumide to values of 1.3 +/- 0.3 kU/45 min, 0.5 +/- 0.1 kU/45 min and 0.4 +/- 0.0 mmol/45 min, respectively, reaching statistical significance (P less than 0.05) in the 30 to 45-min period after the start of the loxiglumide infusion. In the control experiments saline infusion failed to influence basal amylase, trypsin and bilirubin output, while bombesin stimulated amylase output from 4.7 +/- 0.8 kU/45 min to 25.1 +/- 5.1 kU/45 min (P less than 0.05), trypsin output from 2.9 +/- 0.8 kU/45 min to 11.6 +/- 2.0 kU/45 min (P less than 0.05) and bilirubin output from 7.7 +/- 2.8 mmol/45 min to 68.0 +/- 16.0 mmol/45 min (P less than 0.05). Loxiglumide failed to significantly influence bombesin stimulated amylase output (36.7 +/- 9.0 kU/45 min) and trypsin output (8.3 +/- 2.9 kU/45 min), but almost abolished bilirubin output (9.7 +/- 3.6 mmol/45 min) (P less than 0.05). Basal plasma CCK (2.4 +/- 0.1 pM) was not significantly influenced by loxiglumide (2.4 +/- 0.2 pM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714085 TI - Galanin induces contraction of isolated cells from circular muscle layer of pig ileum. AB - The effects of galanin and its interaction with cholecystokinin and acetylcholine on smooth muscle cells were studied in vitro on isolated cells obtained from pig ileum circular muscle layer. Galanin induced a concentration-dependent cell contraction with a maximal contraction (24.5% decrease in cell length from control) obtained at 1 nM. The concentration of galanin inducing a half-maximal contraction was 3 pM. Tetrodotoxin (10 microM) failed to inhibit cell contraction induced by galanin (1 nM), pentagastrin (10 nM) and acetylcholine (1 microM). Atropine abolished the contraction induced by acetylcholine (1 microM), but had no effect on galanin- and pentagastrin-induced contraction. L 364,718 inhibited the contraction induced by CCK8 but not the galanin-induced contraction. At the uneffective concentration of 10 fM, galanin had a synergistic effect with an uneffective concentration of CCK8 (1 pM). These results suggest that (i) galanin contracts smooth muscle cells from pig ileum by acting on a specific receptor; (ii) galanin and either CCK or acetylcholine may act in a synergistic way to induce cell contraction. PMID- 1714086 TI - Chagas' disease: CD5 B-cell-dependent Th2 pathology? PMID- 1714087 TI - Immunogenicity of Trypanosoma cruzi cysteine proteinase. PMID- 1714088 TI - Antigens of Trypanosoma cruzi that mimic mammalian nervous tissues: investigations of their role in the autoimmune pathophysiology of chronic Chagas' disease. PMID- 1714089 TI - Molecular mimicry and Chagas' heart disease: high anti-R-13 autoantibody levels are markers of severe Chagas heart complaint. PMID- 1714091 TI - Presentation of foreign antigenic determinants at the bacterial cell surface using the TraT lipoprotein. AB - The TraT protein is an oligomeric outer membrane lipoprotein, specified by plasmids of the IncF group, that is very highly exposed at the bacterial cell surface. We have investigated the feasibility of using the protein as a carrier of foreign antigenic determinants by genetic insertion of the C3 epitope of type 1 poliovirus into defined sites in the protein. Several of the hybrid proteins constructed had features characteristic of the native protein and one in particular retained the ability of function in surface exclusion and genetic suppression assays as well as to assemble into oligomers. Our results suggest that the TraT protein can be used to transport and present foreign antigenic determinants at the cell surface. PMID- 1714090 TI - Origin and significance of anti-heart and anti-skeletal muscle autoantibodies in Chagas' disease. PMID- 1714092 TI - Structure of viral B-cell epitopes. AB - Four categories of viral epitopes can be distinguished that have been designated cryptotopes, neotopes, metatopes and neutralization epitopes. Specific examples of each epitope type are presented and the methods used for locating their positions in viral proteins are described. The epitopes of four well characterized viruses, namely poliovirus, foot-and-mouth disease virus, influenza virus and tobacco mosaic virus are briefly described. PMID- 1714093 TI - Aromatic-dependent Salmonella as live vaccine presenters of foreign epitopes as inserts in flagellin. AB - Synthetic oligonucleotides specifying amino acid sequences identified as epitopes of various foreign antigens (cholera toxin subunit B, hepatitis B surface protein and others) have been inserted at an EcoRV-EcoRV deletion site in a cloned Salmonella flagellin gene; the resulting plasmids, when placed in flagellin negative Escherichia coli or Salmonella sp. strains, caused production of flagellin expressing the epitope. If the chimeric flagellin allowed formation of flagella, the epitope was exposed at the surface of the flagellar filaments. A delta aroA flagellin-negative S. dublin live vaccine strain given plasmids carrying various chimeric flagellin genes was administered to mice, etc. Serum antibody specific for the foreign epitope was in all cases evoked by parenteral administration; oral route administration was effective in the case of two epitopes of hepatitis B surface protein but not effective for several other epitopes. Several i.p. inocula of the live vaccine strain with an insert corresponding to the 15 N-terminal amino acids of the M protein of Streptococcus pyogenes type 5 evoked M-specific antibody with opsonic activity, and the mice were (incompletely) protected against a lethal challenge of S. pyogenes type 5. The non-virulence of Salmonella sp. strains with complete blocks in the aromatic biosynthesis pathway, even for animals with genetically determined or other defects in host defences, can be completely accounted for by their requirement for p-aminobenzoic acid, since non-leaky pabB mutations caused similar attenuation. Two transposon insertions at aroE caused little or no attenuation, presumably because they did not result in complete block of the relevant step in biosynthesis. The limited growth of delta aroA strains in mouse tissues parallels that which precedes the bacteriostasis caused by addition of a sulphonamide to a growing broth culture of a sulphonamide-sensitive strain; the final cessation of growth in each case presumably results from inability to initiate new protein chains with a formyl-methionine unit when the original folic acid content of the bacteria has been diluted out by residual growth. PMID- 1714094 TI - Immunogenicity of foreign peptide epitopes expressed in bacterial envelope proteins. AB - We have used two bacterial proteins from Escherichia coli to express heterologous peptides. Both proteins are situated in the E. coli cell envelope but have different properties: LamB is an integral outer membrane protein, and MalE a soluble periplasmic protein. The peptides were expressed as genetic inserts within "permissive sites" of these recipient proteins, i.e. sites which allow the insertion of foreign peptides without affecting the biological properties of the host protein. In this paper, we summarize preliminary rules governing the immunogenicity of resulting LamB and MalE hybrid proteins when expressed in E. coli. We focus on two model epitopes: either peptide 132-145 from the preS(2) region of hepatitis B virus or peptide 93-103 from poliovirus VP1 capsid protein. We also present first results obtained when the same hybrid proteins were expressed in attenuated Salmonella typhimurium. Plasmids encoding the hybrid proteins were transferred to aroA S.typhimurium by electroporation. In vitro, the hybrid proteins could be expressed at high levels by S. typhimurium. Mice were immunized by parenteral and oral routes. The effect of the carrier protein and the level of its expression on the in vivo behaviour of the immunizing bacteria and on the immune response induced will be discussed. PMID- 1714095 TI - Insertion of myoglobin T-cell epitopes into the Escherichia coli alkaline phosphatase. AB - We are interested in antigen processing mechanisms of antigen-presenting cells, and to what extent the susceptibility of protein antigens to degradation contributes to immunogenicity. Understanding the biochemistry of antigen processing may be essential for reliable prediction of T-cell epitopes and for the design of vaccines that are optimized for T-cell priming. To examine possible effects of protein structural context on antigen presentation, we used genetic engineering techniques to insert helper T-cell epitopes derived from sperm whale myoglobin into surface loops of the highly stable Escherichia coli alkaline phosphatase, with the expectation that presentation of the myoglobin guest epitopes might vary with their position in the carrier protein. Levels of recombinant protein expression in E. coli cells and residual enzyme activity depended on the location of the guest peptides in the alkaline phosphatase carrier. Mutants with insertions between residues 189-190 of the carrier were recovered with yields and activities similar to the wild type protein; however, insertion of the same peptides at a second site, between residues 165-166, led to low recoveries and diminished phosphatase activities. Subcutaneous injection of mice with one of the purified recombinant proteins in complete Freund's adjuvant induced T cells that responded to in vitro challenge with myoglobin. The potential use of this system to dissect processing mechanisms is discussed. PMID- 1714096 TI - [Transureteral thermotherapy (Prostatron) as treatment of prostatic adenomas. Viewpoint of the French-language urologist]. PMID- 1714097 TI - Evidence for the role of serotonin in the regulation of slow wave sleep in schizophrenia. AB - Nocturnal sleep data and cerebrospinal fluid (CSF) concentrations of the biogenic amine metabolites were measured in 20 male schizophrenics. Consistent with other reports of a stage 4 sleep deficit in schizophrenia, measures of stage 4 sleep were low relative to normal reference data. Measures of stage 4 sleep in absolute amounts and corrected for total sleep were positively correlated with CSF concentrations of the serotonin metabolite, 5-hydroxyindole acetic acid (5-HIAA). CSF 5-HIAA was also correlated with measures of stage 3 sleep and total sleep time suggesting that serotonin may modulate the amount of slow wave sleep broadly defined and possibly sleep duration. Total stage 4 time was also correlated with the dopamine metabolite HVA; consequently, the specificity of the finding might be limited. Also, in this study, schizophrenia was used as a particular model for stage 4 deficits; however, the association of measures of stage 4 sleep with CSF levels of 5-HIAA is not thought to be specific to schizophrenia. PMID- 1714098 TI - What roles have anti-interferon antibodies in physiology and pathology? AB - Until recently the generation of antibodies to interferons (IFNs) was considered an unlikely event, while it is now clear that natural interferons (except IFN beta) are practically nonimmunogenic, although recombinant interferons give rise to antibodies in about 30% of patients with occasional clinical complications. By realizing that normal individuals display spontaneously traces of IFN autoantibodies, in this review it is suggested that (if immunotherapy has to succeed) new generations of recombinant proteins should be the least antigenic as possible. PMID- 1714099 TI - In vitro infection with HIV of antigen-specific T cell clones derived from HIV seronegative individuals. Effects on cytokine production and helper function. AB - Three human T cell clones (TCC) specific for purified protein derivative of Mycobacterium tuberculosis were incubated in the presence of polybrene and phytohemagglutinin with irradiated mononuclear cells from one individual exhibiting seropositivity for human immunodeficiency virus (HIV) and high levels of circulating p24 antigen. After three weeks, TCC showed HIV integration in their DNA, as shown by polymerase chain reaction analysis and Southern blot technique. All the three HIV-infected TCC maintained their ability to recognize the specific antigen, even if their proliferative ability was reduced. The ability of the HIV-infected TCC to produce IL-2, IL-4 and IFN-gamma in response to phorbol myristate acetate plus anti-CD3 monoclonal antibody was decreased, whereas their ability to produce TNF-alpha was unaffected or even enhanced. Two out of the three HIV-infected TCC showed the ability to provide helper function for polyclonal immunoglobulin production when cocultured with autologous B cells in the absence of any stimulant. These data suggest that in vitro infection of normal human TCC may provide a useful model for the study of immunological alterations induced by HIV. PMID- 1714100 TI - Waves of B-lymphopoiesis in the establishment of the mouse B-cell compartment. PMID- 1714101 TI - Acute phase protein, serum amyloid A, inhibits IL-1- and TNF-induced fever and hypothalamic PGE2 in mice. AB - The effect of serum amyloid A (SAA) on fever induced by recombinant interleukin-1 beta (rIL-1 beta) or recombinant tumour necrosis factor alpha (rTNF alpha) was studied in mice. Serum amyloid A is an acute phase protein whose rise in pathological events is induced by the cytokines IL-1, IL-6 and TNF. Administration of human serum amyloid A to mice inhibited fever induced by rIL-1 beta or rTNF alpha in vivo, while the addition of human serum amyloid A to mice hypothalamic slices inhibited IL-1 beta- or TNF alpha-induced prostaglandin E2 (PGE2) production in vitro. Since serum amyloid A did not affect body temperature or hypothalamic PGE2 levels when administered alone, it may represent a specific servo-mechanism for fever regulation in acute events, and it suggests, for the first time, a possible feedback relationship between serum amyloid A and the immunoregulatory cytokines. PMID- 1714102 TI - [Prolonged erythrocytic T activation, depression of blood group A and increase of fetal hemoglobin in a myelodysplastic syndrome evolving into erythroleukemia]. AB - T cryptantigen can be exposed on the red cell membrane as a result of removal of terminal glycosides, either by bacterial enzymes or by incomplete synthesis of the cell membrane due to somatic mutation, usually caused by a neoplasm. T activated erythrocytes have been observed in different pathologies, but they have not been seen associated with other abnormalities of red blood cell proteins described in myelodysplastic syndromes or acute leukaemias. A patient with initial diagnosis of refractory anaemia that evolved into erythroleukaemia showed prolonged T-activation, a depressed A blood-group antigen and an increase of foetal haemoglobin, simultaneously. The evolutive pattern of T-activation suggests more an abnormal erythropoiesis than an enzymatic effect and a certain relationship with the haemolytic syndrome. PMID- 1714103 TI - The wound healing curve as a practical teaching device. AB - Fundamental concepts of clinical wound healing are commonly misunderstood. A hypothetical curve that describes the relationship between wound perfusion and risk of infection is constructed as a teaching device. Although many factors influence this curve, the most important are the presence of bacteria, dead space, necrotic tissue and motion. Subjecting the curve to clinical illustrations enhances its value as a tool for medical education. PMID- 1714104 TI - Long survival in rats after multivisceral versus isolated small-bowel allotransplantation under FK 506. AB - Abdominal multivisceral allotransplantation (MVTX) from Brown Norway donor rats to Lewis recipient rats was performed under a 14-day course of low (0.32 mg/kg) or high-dose (0.64 mg/kg) intramuscular FK 506 to which weekly further injections were added in some of the high-dose animals. With all three regimens, long survival was frequently achieved with good intestinal adsorption and weight gain, but histopathologic evidence of intestinal rejection existed in the most lightly treated animals. The liver, stomach, and pancreas had only minor abnormalities. Rejection of isolated intestinal grafts was more difficult to control based on histopathologic criteria, and satisfactory results were obtained only with the most aggressive treatment protocol, suggesting that the liver in the MVTX had provided an advantage to the companion organs of the graft, of which the intestine was most vulnerable. Histopathologically, the lymphoid elements of the intestine, including the Peyer's patches, appeared to be the most immunogenic component of the intestine. Epithelium near lymphoid areas was secondarily involved with villous atrophy, cryptitis, and abscess formation. Beginning within 12 days in successful MVTX experiments, the lymphoreticular components of the graft intestine, including the Peyer's patches, lamina propria, and mesenteric nodes, were shown with anti-Ia monoclonal antibodies to be repopulated with recipient cells. This finding in grafts that appeared to be permanently accepted was surprising and contrary to expectations from the literature on intestinal allotransplantation. PMID- 1714105 TI - Myoid and epithelial cell differentiation in myasthenic thymuses. AB - This study demonstrates that the stromal thymus elements of postcapillary venules are the source of desmin-positive mesenchyme from which both myoid and epithelial cells arise. The double staining revealed various degrees of desmin and keratin positivity in the same kind of cells in the medulla as well as in Hassall's corpuscles. Hassall's corpuscles seem to arise from several kinds of cells of which one appears to be monocytogenic and expressed S100 protein. PMID- 1714106 TI - [The autocrine regulation of proliferation in cell cultures. I. A study of the heparin-binding factors with growth-stimulating activity contained in media conditioned with transformed rat fibroblasts]. AB - A study was made of the medium conditioned by spontaneously transformed rat embryo fibroblasts of line Rec1-sf, which are capable of unlimited reproduction in medium free of serum and of other endogenous growth factors (c-medium). Addition of c-medium to stationary cultures of nontransformed rat embryo fibroblasts (REF), spontaneously transformed REF (line Rec1), and cells of Rec1 sf stimulated the incorporation of 3H-thymidine by 1.5-6 times. SDS polyacrilamide-gel electrophoresis of proteins, marked by 35S-metionine of c medium of the cell line Rec 1-sf, demonstrated that this medium had proteins with molecular mass from 10 to 110 kDa. The fractionating divisible by 100 ultra concentrates of c-medium with utilization of heparin-sepharose allowed to isolate two types of heparin-binding proteins. The proteins of the first type took about 5% of all the proteins of c-medium; they were eluted with 1.1 M NaCl and stimulated the incorporation of 3H-thymidine in REF, Rec1 and Rec1-sf cultures by 1.3-1.9 times. The second type proteins took about 1% of all the proteins of c medium and were eluted with 2M NaCl, and, like the main endogenic basic growth factor of fibroblasts, stimulated the incorporation of 3H-thymidine into REF and Rec1-sf, but not into the culture of Rec1 line cells. The results obtained are discussed in terms of a hypothesis of autocrine regulation of cell proliferation. PMID- 1714107 TI - Serum biomarkers in metastatic renal cell carcinoma. AB - To identify possible clinically valuable markers of metastatic renal cell carcinoma, we measured the serum concentrations of several commercially available biomarkers in 117 patients with this disease. The alpha-fetoprotein level was measured in 75 patients and was elevated in 8 (11%); elevation did not correlate with the presence of liver metastasis. Beta subunit of human chorionic gonadotropin levels increased in 8 of 83 patients tested (10%). C-terminal parathyroid hormone levels were measured in 79 patients and were elevated in 15 (19%); their serum creatinine level was normal. Thirteen of this group had normal serum calcium levels, whereas 7 patients with hypercalcemia and no clinically evident bone metastasis had normal parathyroid hormone levels. In only 2 of 72 patients, serum lactate dehydrogenase and its isoenzyme 1 were elevated. Only 1 of 85 patients had mildly elevated serum carcinoembryonic antigen, in contrast to 3 of 7 patients with metastatic transitional cell carcinoma of the renal pelvis who had moderately elevated carcinoembryonic antigen. Elevations in alpha fetoprotein, human chorionic gonadotropin, and parathyroid hormone correlated with the course of the disease in 13 patients for whom follow-up measurements were available; measurement of these markers, however, is only useful in a small proportion of patients with metastatic renal cell carcinoma. PMID- 1714108 TI - [Postextrasystolic potential in patients with a left ventricular postinfarct aneurysm]. AB - The author analyzes the effect of postextrasystolic potentiation of contraction of the asynergic regions of the myocardium in 18 of 34 patients with postinfarction aneurysm of the left ventricle evaluated by contrast ventriculography. There was a statistically valid increase of the extension of the zones of hypokinesia due to reduction of the relative length of akinetic regions as compared with data of control contractions, a significant rise of reduction of the sizes of hypokinetic zones, increase of the fraction of ejection of the contractile segment in the general fraction of ejection. It is recommended to use the effect of postextrasystolic potentiation of contraction on the function of asynergic regions of the myocardium with the purpose of assessment of their vitality, determination of resection borders and expediency of their revascularization in the course of aneurysmectomy. PMID- 1714109 TI - [Severe hyperemesis gravidarum--pathophysiologic observations and new therapeutic approach]. AB - It is reported on a 27 year old female patient who was hospitalized twice during her first pregnancy (16th and 28th week) because of severe hyperemesis gravidarum. Severe clinical symptoms associated with severe alterations in the clinical chemistry posed a series of differential diagnoses. Several diseases as potential causes for unappeasable vomiting were taken into account. All traditional therapeutic efforts to relieve hyperemesis gravidarum including H2 blockers in high dosages were not successful. Treatment with omeprazole proved to be effective by stopping the vomiting immediately. After the delivery of a healthy child in the 37th week of pregnancy, several investigations were performed to exclude organic diseases. Etiology and symptoms of hyperemesis gravidarum are discussed with regard to the gastrointestinal tract and thyroid gland function. PMID- 1714110 TI - Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli. AB - DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hallnas B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques. PMID- 1714111 TI - [Advances in gastroenterology 1990. 45th meeting of the German Society of Digestive and Metabolic Diseases with the Section for Gastroenterologic Endoscopy. Proceedings]. PMID- 1714112 TI - [Hepatic encephalopathy in fulminant liver failure. Clinical aspects and therapeutic approaches]. PMID- 1714113 TI - [Cholesterol absorption and metabolism in the small intestine]. PMID- 1714114 TI - The molecular basis of familial chylomicronemia. PMID- 1714115 TI - Lipoprotein(a): studies into the polymorphism and endothelial cell-binding. PMID- 1714116 TI - [Genetic variants of apo E: significance for triglyceride metabolism]. PMID- 1714117 TI - A new role for the low density lipoprotein receptor. AB - It is well established that the low density lipoprotein (LDL) pathway functions to maintain a constant concentration of cellular cholesterol, but LDL effects that are unrelated to cholesterol metabolism have not been studied in great detail. In the present investigation we demonstrate that the LDL receptor pathway regulates cellular levels of free arachidonic acid (AA) and hence prostaglandin (PG) synthesis. We used platelet-derived growth factor (PDGF)-stimulated fibroblasts as a model system to investigate mechanism of LDL-dependent PG synthesis. PDGF-stimulated but not quiescent cells formed radiolabelled prostacyclin (PGI2) and PGE2 upon incubation with LDL that had been reconstituted with cholesteryl-(1-14C)-arachidonate (rec-LDL), while fibroblasts from patients that are afflicted with the LDL receptor negative phenotype of familial hypercholesterolaemia (FH) failed to synthesize significant amounts of PGs. Furthermore cells that had been preincubated with chloroquine or an anti LDL receptor antibody, that prevents binding of LDL to its receptor, did not produce significant amounts of PGs upon incubation with rec-LDL. Moreover incubation of PDGF-stimulated cells with LDL or AA led to a time and concentration-dependent inactivation of PGH synthase, the rate limiting enzyme of PG synthesis. When taken together our results establish a new role of the classical LDL receptor pathway of Brown and Goldstein by demonstrating that LDL provides AA to fibroblasts for eicosanoid formation and that LDL has a profound inhibitory effect on the key enzyme of PG synthesis, the PGH synthase. PMID- 1714118 TI - [Practical procedure in the treatment of increased blood lipids--in whom, when and how?]. PMID- 1714119 TI - [Seminar I: Value of nuclear medicine methods in gastroenterology. introduction]. PMID- 1714120 TI - [Nuclear medicine studies of gastrointestinal transit; detection of reflux]. PMID- 1714121 TI - [Scintigraphic detection of gastrointestinal hemorrhage and ectopic mucosa]. PMID- 1714122 TI - [Nuclear medicine methods for diagnosis of abdominal inflammation]. PMID- 1714123 TI - [Liver circulation and biliary excretion]. PMID- 1714124 TI - [Value of nuclear medicine methods in hepato-gastroenterology--tumor detection]. PMID- 1714125 TI - [Resorption and excretion tests]. PMID- 1714126 TI - [Hepatorenal syndrome]. PMID- 1714127 TI - [Value of nuclear medicine methods in gastroenterology (summary and prospects)]. PMID- 1714128 TI - [Therapy of cholestatic liver diseases with ursodeoxycholic acid: a new treatment principle]. PMID- 1714129 TI - [New "diuretics" in therapy of ascites]. PMID- 1714130 TI - [Treatment of chronic hepatitis B and C with alpha interferon]. PMID- 1714131 TI - [Cyclosporin A: possible use in Crohn disease]. PMID- 1714132 TI - [New developments in therapy with 5-aminosalicylic acid preparations in ulcerative colitis and Crohn disease]. PMID- 1714133 TI - [Pro-kinetic agents. Developments and indications]. PMID- 1714134 TI - [New developments in drug therapy: omeprazole]. PMID- 1714135 TI - [Prostaglandin analogs in ulcer therapy]. PMID- 1714136 TI - [Somatostatin and octreotide in therapy of gastrointestinal diseases]. PMID- 1714137 TI - RUDER: interim evaluation of a 2-year, multicentre study of risk factors for duodenal ulcer relapse. PMID- 1714138 TI - [Peri-anal dermatopathies]. PMID- 1714139 TI - [Bowen's disease, Paget's disease and basalioma of the anal region]. PMID- 1714140 TI - [Acute variceal hemorrhage]. PMID- 1714141 TI - [Ulcerating, anal lesions--sexually transmissible diseases]. PMID- 1714142 TI - [Acute and chronic anal fissures]. PMID- 1714143 TI - [Anal Crohn lesions]. PMID- 1714144 TI - [Ulcerating, non-carcinomatous anorectal lesions--drug-induced lesions]. PMID- 1714145 TI - [Ulcus simplex recti]. PMID- 1714146 TI - [Limits of ESWL (extracorporeal shockwave lithotripsy)]. PMID- 1714147 TI - Direct contact dissolution of gallbladder stones with methyl tert-butyl ether: experience in 209 patients. PMID- 1714148 TI - [Non-surgical treatment of cholecystolithiasis: laser lithotripsy in the gallbladder]. PMID- 1714149 TI - [Laparoscopic cholecystectomy]. PMID- 1714150 TI - [Classical cholecystectomy]. PMID- 1714151 TI - [Combined therapy of inoperable esophageal cancer]. PMID- 1714152 TI - [Acute intestinal hemorrhage]. PMID- 1714153 TI - [Modern therapeutic strategies in stomach cancer]. PMID- 1714154 TI - [Therapy of inoperable cancers of the exocrine pancreas and liver]. PMID- 1714155 TI - [Possibilities of immunotherapy of colorectal cancers and pancreatic tumors]. PMID- 1714156 TI - [Interaction of gastrointestinal receptor tyrosine kinases with cytokines exemplified by pancreatic cancer]. PMID- 1714157 TI - [Role of the insulin receptor kinase in the effect of insulin and pathogenesis of type II diabetes]. PMID- 1714158 TI - Innervation and regulation of the pancreas by neurons in the gut. AB - Experiments were done in order to test the hypothesis that enteric neurons project to the pancreas and can modify pancreatic endocrine and exocrine activity. Injections of the retrograde tracer Fluoro-Gold (FG) into the rat pancreas labeled neurons in the myenteric plexus of the antrum of the stomach and in the first 6 cm of the duodenum. A subset of myenteric neurons were found in both the antrum and duodenum that were doubly labeled by retrograde transport of FG and anti-serotonin (5-HT) sera; therefore, some of the enteric neurons that innervate the pancreas are serotonergic. Within the pancreas, 5-HT immunoreactivity was not found in any neuronal cell bodies; however, 5-HT immunoreactive axons were observed. Varicose 5-HT-immunoreactive terminal axons were most commonly found in pancreatic ganglia. Anterograde tracers were microinjected into individual myenteric ganglia in order to determine the pancreatic targets of the enteric innervation. Following the microinjection of the B subunit of cholera toxin (B-CT) or 1,1", dioctadecyl-3,3,3',3' tetramethylcarbocyanine (Dil) into myenteric ganglia in the duodenum, labeled fibers were found in the pancreatic parenchyma. B-CT-immunoreactive terminals were most commonly observed in pancreatic ganglia, suggesting that pancreatic ganglia are the major targets in the pancreas of the enteric innervation. Experiments were also performed physiologically to determine whether enteric stimuli can influence pancreatic exocrine or endocrine activity via a neural pathway. For this purpose enteric neurons were stimulated in vitro by luminal application of veratridine (Ver), and the metabolic activity of neurons, islet, and acinar cells was determined in attached segments of pancreas by measuring their cytochrome oxidase (CO) activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714159 TI - [Neurophysiology of the enteric nervous system]. PMID- 1714160 TI - [Central nervous system regulation of gastric acid and duodenal bicarbonate secretion]. PMID- 1714161 TI - [Central nervous system regulation of exocrine pancreas secretion]. PMID- 1714162 TI - [Central nervous system regulation of the endocrine pancreas]. PMID- 1714163 TI - [The central nervous system, stress and motility of the gastrointestinal tract]. PMID- 1714164 TI - Neuropeptides and inflammatory bowel disease. AB - Pronounced changes in gut neuropeptide content and innervation patterns have been observed in the inflamed intestine of patients with inflammatory bowel disease. It is not known to date whether these changes in neuropeptides are due to altered synthesis and release from intrinsic and/or extrinsic neurons and nerve fibers. The changes in circular smooth muscle response associated with diminished VIP in the intestine of patients with Crohn's disease suggests that VIP may play an important role in the pathophysiology of motility in IBD. The pronounced increase in SP receptors at small vessels in all gut layers and at lymph nodules in the inflamed intestine of IBD patients supports the hypothesis that SP is a modulator of inflammation in IBD and possibly acts by release from extrinsic sensory nerves of the gut. Sensory nerve may play a role not only in enhancing an inflammatory response in the intestine, but also in tissue repair. An inflammatory response after tissue injury and subsequent wound healing presumably is the normal response in healthy tissue. In IBD however, this sequence may be deeply disturbed by an unrestricted immune response which does not lead to or delays intestinal tissue healing. Although it is intriguing to postulate that interactions between the immune system and nervous system exist and play a role in the pathophysiology of intestinal inflammation, in vivo studies blocking or mimicking neuropeptide action are needed to prove this bidirectional communication. PMID- 1714165 TI - [Modern concepts for the prevention of acute stress hemorrhage]. PMID- 1714166 TI - [Gastric balloon and gastroplasty: interventional therapy in morbid obesity]. PMID- 1714167 TI - [Stomach volume reducing balloon: its value in therapy of morbid obesity based on 7 years experience]. PMID- 1714168 TI - Studies on regulation of pancreatic gastrin gene to analyze the mechanisms of islet cell differentiation during fetal development. PMID- 1714169 TI - Trophic effects of gastrin and cholecystokinin. PMID- 1714170 TI - Molecular design of potent specific antagonists for the gastrin and cholecystokinin receptors. AB - Widespread distribution of receptors for the peptide hormones cholecystokinin (CCK) and gastrin in the gut and in the CNS suggests therapeutic potential for selective antagonists of these hormones. Discovery of the natural product Asperlicin provided a new class of non-peptidal CCK antagonists, but oral bioavailability in this class remained elusive. With Asperlicin as a guide, the new, selective, orally bioavailable, high affinity CCK-A antagonist, MK-329 (L 364,718; Devazepide) and CCK-B/gastrin antagonist, L-365,260 have been developed. Biological profiles of these compounds are presented and results of early clinical evaluation of MK-329 are described. The significance of these agents as models for development of non-peptidal ligands for other receptors are briefly summarized. PMID- 1714171 TI - [Characterization of receptors of the bombesin peptide family in gastrointestinal tissues]. PMID- 1714172 TI - [Second messenger systems in the gastrointestinal tract]. PMID- 1714173 TI - [Helicobacter pylori--a pathogen with many faces]. PMID- 1714174 TI - [Virulence factors of Helicobacter pylori and pathomechanisms of mucosal damage]. PMID- 1714175 TI - [Effect of Helicobacter pylori on stomach physiology]. PMID- 1714176 TI - [Helicobacter pylori infection: a facilitator of stomach cancer?]. PMID- 1714177 TI - [Epidemiology and diagnosis of Helicobacter pylori infection]. PMID- 1714178 TI - [Helicobacter pylori infection, gastritis and non-ulcer dyspepsia--is there a connection?]. PMID- 1714179 TI - [Prevention of stress hemorrhage in critical care patients]. PMID- 1714180 TI - [Relations between gastrointestinal interdigestive motility and secretion]. PMID- 1714181 TI - [Theses for determining and measuring characteristics of "quality of life"]. PMID- 1714182 TI - [The quality of life of patients with stomach and esophageal cancers]. PMID- 1714183 TI - Cholecystokinin as a regulator of rat pancreatic gene expression. AB - We examined the role of physiologic plasma concentrations of cholecystokinin (CCK) in the regulation of rat pancreatic gene expression. Postprandial plasma CCK concentrations, as determined by bioassay, were achieved by intraduodenal perfusion with soybean trypsin inhibitor (SBTI) or intravenous infusion of CCK-8. SBTI administration for 48h resulted in nonparallel regulation of digestive enzyme gene expression, as assessed by slot-blot analysis using cloned cDNA probes for trypsin, chymotrypsin, amylase and ribonuclease. As an indicator for pancretic growth stimulation, ornithine decarboxylase (ODC) gene expression was stimulated appr. 2-fold over the SBTI infusion period. Identical effects were seen with i.v. infusion of CCK-8. The CCK receptor antagonist L-364, 718 blocked the effects on pancreatic gene expression of both CCK infusion and SBTI administration. These data therefore indicate that postprandial plasma CCK concentrations regulate pancreatic digestive enzyme and ODC gene expression at a pretranslational level. PMID- 1714184 TI - [Ethics in a Leonardo world]. PMID- 1714185 TI - [Inhibition of pancreatic growth by exogenous and endogenous factors]. PMID- 1714186 TI - [Standards of laboratory diagnosis of acute pancreatitis]. PMID- 1714187 TI - [54-year-old patient with dysphagia]. PMID- 1714188 TI - [A 28-year-old patient with chronic vomiting and diarrhea]. PMID- 1714189 TI - [Jejunal diverticulosis in intestinal pseudo-obstruction]. PMID- 1714190 TI - [Spontaneous rupture of the esophagus in hypermotility disorder of the esophagus]. PMID- 1714191 TI - [Case report K.N.: Therapy refractory constipation following appendectomy]. PMID- 1714192 TI - [Toxic megacolon]. PMID- 1714193 TI - [Molecular mechanisms of signal transduction. Significance of signal transmission for immune regulation in the intestines with special reference to various differentiation stages of T-lymphocytes]. PMID- 1714194 TI - [Calcium and protein kinase C as second messengers]. PMID- 1714195 TI - [Specific and "half-specific" pathways of T-cell activation]. PMID- 1714196 TI - [Cytokines in intestinal mucosa]. PMID- 1714197 TI - [Integrins: cell receptors within the scope of regulation of differentiation]. PMID- 1714198 TI - [Hematopoietic growth factors and epithelial cell differentiation]. PMID- 1714199 TI - [Characterization and induction of phenotypic differentiation markers of the pancreatico-biliary system]. PMID- 1714200 TI - [Crohn disease--ulcerative colitis: therapy study]. PMID- 1714201 TI - [Emergency and critical care problems: acute intestinal ischemia]. PMID- 1714202 TI - [Nutrition of the intensive care patient]. PMID- 1714203 TI - [Classification of pancreatitis--comparison of the revised 1984 Marseille Classification and the 1983 Cambridge classification]. PMID- 1714204 TI - [Pathogenetic concepts of acute pancreatitis]. PMID- 1714205 TI - [Influence of gastrointestinal factors on adaptation of the pancreas--animal experiment studies]. PMID- 1714206 TI - [Acute necrotizing pancreatitis--clinical aspects, diagnosis and therapy]. PMID- 1714207 TI - [The natural course of chronic pancreatitis]. PMID- 1714208 TI - [Diagnostic standards in chronic pancreatitis. Exocrine pancreatic function tests]. PMID- 1714209 TI - [Diagnostic standards in chronic pancreatitis--imaging procedures]. PMID- 1714210 TI - [Enzyme therapy in the early stage of chronic pancreatitis?]. PMID- 1714211 TI - [Acute liver failure: definition, etiology, clinical picture and prognosis]. PMID- 1714212 TI - [Papillotomy in acute pancreatitis--never--selective--or always? Early ERCP and EST (endoscopic sphincterotomy)]. PMID- 1714213 TI - [Percutaneous therapy of pancreatic pseudocysts]. PMID- 1714214 TI - [Adenomatous polyps in the stomach-colon--when is polypectomy curative?]. PMID- 1714215 TI - [Early detection of hereditary hemochromatosis and Wilson's disease]. PMID- 1714216 TI - [Primary sclerosing cholangitis]. PMID- 1714217 TI - [Early diagnosis and therapy of primary biliary cirrhosis]. PMID- 1714218 TI - [Postoperative recurrence of Crohn disease]. PMID- 1714219 TI - [Pathophysiology of lipoprotein metabolism]. PMID- 1714220 TI - [The low-molecular protein of the cell wall in streptococci group A devoid of serological type specificity]. AB - The scheme for the isolation and purification of low-molecular cell-wall protein without type specificity, including the extraction of the cell walls of group A streptococci, type M 29, with 1% solution of Triton X-100, the separation of the extract by ion-exchange chromatography in DEAE-trisacryl M with the subsequent two-stage gel filtration in superfine Sephadex G-50, is described. The isolated protein had a molecular weight of 4,000 daltons and contained no admixtures of group-specific polysaccharide A, phosphorus, nucleic acids and Fc receptors and interacted with antisera to group A streptococcal cells of heterologous type M in the enzyme immunoassay (EIA). Purified protein was characterized by a high content of glycine. The antigenic determinants of immobilized protein, recognized by antibodies in EIA, were sensitive to the action of trypsin and resistant to the action of pepsin, papain, pronase E and sodium periodate. PMID- 1714221 TI - A new staining method for visualization of keratin filaments in hair fibre cross sections. AB - A new method of staining the keratin filament matrix allowing a visualization of the filaments in cross section of hair fibres has been developed. It differs from previously published methods in that the hair fibres are neither fixed with OsO4 nor treated with sulfur bond breaking agents. High contrast is still obtainable in sections thicker than 60 nm. PMID- 1714222 TI - Macrophages in developing mammalian skeletal muscle: evidence for muscle fibre death as a normal developmental event. AB - Macrophages in the diaphragm of fetal, growing and adult rats were investigated using electron microscopy. In addition, frozen sections of the diaphragm, muscles of the anterior abdominal wall and muscles of the calf were stained for acid phosphatase activity and examined with the light microscope. Macrophages were frequently observed in the muscles at 20 days of gestation and until the animals were 2 weeks old. They were less frequently found in the 4-week-old rats and very rarely found in the 8-week-old and adult rats. They were found in intimate contact with, sometimes apparently surrounding, certain muscle fibres which were very electron dense and considered to be degenerating. In addition, myofibrils were found as phagosomes within the macrophage cytoplasm. There was evidence to support the theory that macrophages develop from mesenchymal cells in embryonic tissue. Cells considered to be early macrophages and containing small lysosomes were found in the muscles at 16 and 18 days of gestation. In all other respects these cells resembled mesenchymal cells. It is proposed that cell death may be a normal developmental event in skeletal muscle, in which macrophages play an important role in the removal of the dead fibres. PMID- 1714223 TI - Immunophenotypic difference of keratin expression in normal mammary glandular cells from five different species. AB - The immunohistochemical reactivity of human, monkey, shrew, rat and mouse normal mammary glands was examined using methacarn-fixed paraffin-embedded specimens and acetone-fixed frozen sections using the avidinbiotin-peroxidase method for cell phenotype comparison. Actin was visualized using anti-smooth muscle actin antibody and keratin expression was determined by employing 12 different monoclonal antibodies. All these antibodies cross-reacted specifically with the species examined. Basal (myoepithelial) cells from all species showed muscle specific actin according to reactivity with HHF35 monoclonal antibody. Keratin expression showed significant phenotypic differences among species. In human and monkey, AEL-KS2, KL1, CK8.13, AE3 and 34BE12 stained luminal cells as well as basal cells. AE1, RPN1165, CK4.62, 35BE11, M20 and RPN1162 labeled only luminal cells whereas 312C8-1 preferentially bound to basal cells. In shrews, AEL-KS2, CK8.13 and AE3 reacted to both cell types, AE1 reacted only with luminal cells, and 35BE12 and 312C8-1 selectively stained basal cells. In rodents, AEL-KS2 reacted to both cell types, CK8.13, AE3, 34BE12 and 312C8-1 stained rat basal cells, and 34BE12 and 312C8-1 reacted to mouse basal cells. The data represents cytoskeletal differences among species. PMID- 1714224 TI - Repetitive monomorphic ventricular tachycardia in a 4-year-old boy with toxic multinodular goiter. AB - A case of toxic multinodular goiter associated with repetitive monomorphic ventricular tachycardia (VT) is reported. A 4-year-old boy was found to have asymptomatic VT. When treatment with antiarrhythmic agents turned out to be ineffective, thyrotoxicosis was suspected due to the rapid enlargement of the left thyroid gland and associated thyroid function studies. A diagnosis of toxic multinodular goiter was made on the basis of subsequent scintigraphy and ultrasonography. Treatment with antithyroid drugs and inorganic iodine restored the thyroid function to normal, and was accompanied by the disappearance of VT. A left thyroid lobectomy was performed, and the pathological findings were compatible with toxic multinodular goiter. After the operation, the patient was transiently hypothyroid and had no VT without medication. A review of the literature revealed no previously documented cases of VT with toxic multinodular goiter. PMID- 1714225 TI - Monocytoid B lymphocytes and epithelioid cell clusters in abscess-forming granulomatous lymphadenitis. With special reference to cat scratch disease. AB - In order to clarify the appearance of monocytoid B lymphocytes (MBLs) in abscess forming granulomatous lymphadenitis (AGL) and the relation between AGL and cat scratch disease (CSD), 48 cases of AGL were studied histologically. MBLs were present in about 50% of AGL cases. Warthin-Starry (WS) silver stain-positive bacteria, which are the causative agent of CSD, were present in 52.4% of AGL cases with MBLs and 59.2% of AGL cases without MBLs. The appearance of MBLs in AGL was not related to various clinical features, including disease interval from initial lymphadenopathy to lymph node biopsy. Histologically, epithelioid cell clusters appeared in about 70% of MBL-positive AGL cases, but were not observed in MBL-negative AGL. Therefore, a close interaction between MBLs and epithelioid cells in AGL is suggested, and we emphasize that the histological features of some AGL cases resemble those of toxoplasmic lymphadenitis. PMID- 1714226 TI - Multiple pulmonary hyalinizing granulomas associated with systemic idiopathic fibrosis. AB - A 41-year-old man with progressive nodular infiltration of the lung of about 2 years' duration died of cardiac and respiratory failure. Autopsy revealed bilateral multiple pulmonary hyalinizing granulomas (PHGs) diagnosed on the basis of the characteristic dense hyaline collagen bundles with nonspecific inflammatory infiltration. Constrictive pericarditis, retroperitoneal fibrosis, mediastinal fibrosis, fibrous thickening of the peritoneal and pleural surfaces, and fibrosis of soft tissue of the neck, flank, and hepatic hilar region were present, therefore, a diagnosis of systemic idiopathic fibrosis was made. The patient had anti-thyroglobulin and anti-thyroid microsomal antibodies and lymphocytic thyroiditis. The inflammatory process of PHG of the present case was active and the clinical course was progressive. PHG seems to be a lesion belonging to the systemic idiopathic fibrosis complex. Immunologic abnormalities may be related to PHG and systemic idiopathic fibrosis. PMID- 1714227 TI - Alpha-fetoprotein-producing papillary adenocarcinoma originating from a uterine body. A case report. AB - A rare case of alpha-fetoprotein (AFP)-producing papillary adenocarcinoma originating from the uterine body of a 55-year-old Japanese woman is reported. The histologic appearance of the tumor was reminiscent of a serous papillary carcinoma of the ovary, in clear contrast to that of common endometrial adenocarcinomas. No features of embryonal carcinoma or yolk sac tumor were evident. Immunohistochemical examination demonstrated the production of AFP by the tumor cells. To our knowledge there are six reported cases of AFP-producing malignant tumors of the uterine body in the literature, four of them being yolk sac tumor, one mixed mesodermal tumor, and one endometrial adenocarcinoma. This is therefore the first case of an AFP-producing papillary adenocarcinoma that developed in the uterine body. This report should contribute to a better understanding of the histogenesis of AFP-producing tumors in this organ. PMID- 1714228 TI - Antiexudative and capillaritonic effects of procyanidines isolated from grape seeds (V. Vinifera). AB - A purified and enriched fraction, containing procyanidines, was isolated from grape seeds of Bulgarian sorts of Vitis Vinifera. The effects of procyanidines on the local oedema, produced by subplantar injection of carrageenin and dextran in the hind rat paw, were studied. We also tested their effect on the capillary permeability using intravenous injection of Evans blue and local irritation by xylene. Procyanidines at a dose of 2 mg/kg applied orally three times daily for 6 days inhibited the carrageenin-induced hind paw oedema. On the same schedule of administration these compounds inhibited the dextran-induced oedema 4 hours after the development of the process. Procyanidines stabilized the capillary wall and prevented the increase of capillary permeability caused by local cutaneous application of xylene. PMID- 1714229 TI - Intracellular free magnesium concentration: relevance to cardiovascular medicine. AB - There is a growing awareness of the role hypomagnesemia plays in cardiovascular medicine. Recent experimental studies have also provided a new understanding og how Mg2+ ions influence various ion channels and transport mechanisms. Yet, the pathophysiological mechanisms that may be responsible for various phenomena associated with hypomagnesemia, have not been clarified. This is partly due to that there, until recently, has been a lack of convenient and reliable methods for measuring cytosolic free Mg2+ concentration. It is the hope that newly developed techniques for measuring the cytosolic free Mg2+ concentration will prove useful in this respect. PMID- 1714230 TI - Peplomycin-induced DNA repair synthesis in permeable mouse ascites sarcoma cells. AB - DNA repair synthesis induced in permeable mouse ascites sarcoma cells by peplomycin, an antitumor antibiotic, was studied. Mouse ascites sarcoma (SR C3H/He) cells were permeabilized with a low concentration of Triton X-100 in an isotonic condition. Permeable cells were treated with an appropriate concentration of peplomycin to introduce single-strand breaks in permeable cell DNA. DNA repair synthesis in peplomycin-treated permeable cells was measured by incubating the cells with four deoxynucleoside triphosphates in an appropriate buffer system. The DNA repair synthesis was enhanced by ATP and NaCl at near physiological concentrations. More than 90% of DNA synthesis in the present system depended on the peplomycin-treatment. The repair nature of the DNA synthesis was confirmed by a BrdUMP density shift technique. The repair patches were largely completed and ligated in the presence of ATP. Analyses using selective inhibitors for DNA polymerases showed that both DNA polymerase Beta and aphidicolin-sensitive DNA polymerases (DNA polymerase alpha and/or delta) were involved in the repair DNA synthesis. PMID- 1714231 TI - Antineutrophil cytoplasmic autoantibodies antigen specificity. AB - The results presented during the Third International ANCA Workshop, Washington, DC, 1990, allowed a better definition of the antigenic specificity of the antineutrophil cytoplasmic autoantibodies (ANCA). The large predominance of two major antigen specificities for proteinase 3 (PR3) and myeloperoxidase (MPO), in the group of vasculitic patients, was confirmed. PR3 and MPO are colocalized in the azurophilic granules of neutrophils and translocated to the cell surface during activation and thus are able to interact with ANCA after neutrophil preactivation. Furthermore, by comparison of amino acid and DNA sequences, the agreement was reached that PR3 was identical to AGP7, p29, and myeloblastin, described independently and involved in the control of growth and differentiation of leukemic cells. In addition to the two major ANCA antigens, a number of neutrophil cytoplasmic antigens recognized by ANCA have been previously identified (human leukocyte elastase [HLE], lactoferrin). It was established during the Third Workshop that these rare ANCA specificities, occurring in a limited number of patients, include a cationic antimicrobial protein (CAP57) and cathepsin G. However, the variety of ANCA antigen specificities contrasts with the fact that the vast majority of ANCA-positive sera are monospecific for a single ANCA antigen. Finally, the fine specificity of granulocyte-specific antinuclear antibodies (GS-ANA) occurring in rheumatoid arthritis and ulcerative colitis is still unknown, but clearly a substantial proportion of GS-ANA belongs to the ANCA family. PMID- 1714232 TI - Localization of the gene encoding the GABAA receptor beta 3 subunit to the Angelman/Prader-Willi region of human chromosome 15. AB - Deletions of the proximal long arm of chromosome 15 (bands 15q11q13) are found in the majority of patients with two distinct genetic disorders, Angelman syndrome (AS) and Prader-Willi syndrome (PWS). The deleted regions in the two syndromes, defined cytogenetically and by using cloned DNA probes, are similar. However, deletions in AS occur on the maternally inherited chromosome 15, and deletions in PWS occur on the paternally derived chromosome 15. This observation has led to the suggestion that one or more genes in this region show differential expression dependent on parental origin (genetic imprinting). No genes of known function have previously been mapped to this region. We show here that the gene encoding the GABAA (gamma-aminobutyric acid) receptor beta 3 subunit maps to the AS/PWS region. Deletion of this gene (GABRB3) was found in AS and PWS patients with interstitial cytogenetic deletions. Evidence of beta 3 gene deletion was also found in an AS patient with an unbalanced 13;15 translocation but not in a PWS patient with an unbalanced 9;15 translocation. The localization of this receptor gene to the AS/PWS region suggests a possible role of the inhibitory neurotransmitter GABA in the pathogenesis of one or both of these syndromes. PMID- 1714234 TI - Transmission probabilities are not correctly implemented in the computer program POINTER. PMID- 1714233 TI - Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM-negative subtype of the disease. AB - Four mutations of the porphobilinogen (PBG) deaminase gene that result in cross reacting immunological material (CRIM)-negative forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA from patients and by cloning of the amplified products in a bacterial expression vector. One mutation is a single base deletion which causes a frameshift and which is expected to result in the synthesis of a truncated protein. Two other mutations consist of single base substitutions and lead to amino acid changes. The fourth mutation is a single base substitution producing an aberrant splicing and resulting in an mRNA which would encode a protein missing three amino acids. DNAs from 16 unrelated CRIM-negative AIP patients were screened for the presence of these four mutations, by hybridization with oligonucleotides specific for each of the mutations, but none of the four mutations was identified in additional patients. The results indicate that mutations responsible for CRIM-negative AIP are highly heterogenous. PMID- 1714235 TI - Efficacy of intravenous gammaglobulin therapy in chronic refractory polymyositis and dermatomyositis: an open study with 20 adult patients. AB - PURPOSE: Polymyositis and dermatomyositis are inflammatory muscular diseases of unknown cause. Many interventions are available to treat patients with these conditions including corticosteroids, immunosuppressive drugs, plasmapheresis, and total body irradiation. However, these therapies are not always effective, and they may be associated with certain serious side effects. An attempt was made to evaluate the efficacy of polyvalent intravenous immunoglobulin (IVIG) in patients with polymyositis or dermatomyositis refractory to traditional treatment. PATIENTS AND METHODS: Twenty patients (16 women and 4 men; mean age 43 [16 SD] years), 14 with chronic refractory polymyositis and six with dermatomyositis, received high doses of IVIG because of the failure of traditional treatments (prednisone [19], methotrexate [10], azathioprine [6], cyclophosphamide [3], cyclosporine [3], chlorambucil [1], plasmapheresis [8], lymphopheresis [1], and total body irradiation [1]). In one patient with positive results on picornavirus serologic testing, IVIG was the first treatment choice. IVIG therapy was given with prednisone in 15 patients, with methotrexate in six patients, and with plasmapheresis in one patient. There were no changes in treatment in the 2 months before the introduction of IVIG therapy and no increases in dose during this treatment. Preparations of polyvalent human intravenous gammaglobulins with increased intact immunoglobulin G were used. Thirteen patients received 1 g/kg daily for 2 days each month, and seven patients received 0.4 g/kg daily for 5 days each month. The mean duration of treatment was 4 months. RESULTS: Clinical assessment, which consisted of the measurement of proximal muscle power, and biochemical studies were carried out before each treatment period. Significant clinical improvement was noted in 15 of the 20 patients. Mean muscle power estimated for the 20 patients before and after IVIG therapy was statistically significantly reduced (p less than 0.01). Eighteen patients showed biochemical improvement, and two patients with normal initial serum creatine kinase levels showed clinical improvement. Mean creatine kinase levels for the 20 patients during IVIG therapy showed a statistically significant decrease from the first IVIG perfusions (p less than 0.01). Side effects of IVIG therapy were noted in four patients; however, these effects were mild. During IVIG therapy, steroid doses were significantly reduced from the second or the third IVIG infusion (p less than 0.05). CONCLUSION: IVIG is an efficacious new therapy for polymyositis and dermatomyositis and should play a role in the treatment of these diseases, replacing or reducing steroid and immunosuppressive medications. PMID- 1714236 TI - Treatment of dermatomyositis with intravenous gammaglobulin. AB - PURPOSE: The mainstay of pharmacologic therapy in patients with dermatomyositis is corticosteroids. However, because patients sometimes become refractory to these drugs and because these drugs have potential short- and long-term toxicities, alternate therapy is highly desirable. Therefore, a pilot study was initiated using high-dose intravenous gammaglobulin (IVGG) in the treatment of dermatomyositis. PATIENTS AND METHODS: IVGG was administered to five patients with juvenile dermatomyositis. Prior to IVGG treatment, all patients had persistent muscle weakness despite daily corticosteroids and three patients had developed unacceptable steroid toxicity. Two of the patients had previously developed toxicity while receiving immunosuppressive therapy. RESULTS: IVGG therapy resulted in improved muscle strength and ameliorated skin rash in all patients. The percentage increase in muscle strength as measured by sphygmomanometry following the 9-month course of IVGG ranged from 56% to 606% in the proximal lower extremities and from 30% to 186% in the proximal upper extremities. Following IVGG therapy, prednisone could be discontinued or the dose reduced in all patients. CONCLUSION: This study suggests that IVGG may allow steroid sparing in dermatomyositis and may provide a safe alternative to cytotoxic therapy. PMID- 1714237 TI - Risks associated with an elevated amniotic fluid alpha-fetoprotein level. AB - Two and two-tenths percent of 85,000 consecutive amniotic fluid (AF) samples had alpha-fetoprotein (AFP) levels greater than or equal to 2.0 MoM. Half measured 2.0-2.4 MoM, and 93% had a normal outcome. Sixty-seven percent of those with higher levels had abnormalities. A positive acetylcholinesterase (AChE) increased the risk from 67% for levels between 2.0 and 2.4 MoM to 99% at greater than or equal to 5.0 MoMs. After a normal ultrasound and chromosome studies, the risk for a fetal abnormality was 1% for AF AFP measuring 2.0-2.4 MoM and 3% for higher levels. PMID- 1714238 TI - "Pseudomosaicism" for 4p- in amniotic fluid cell culture proven to be true mosaicism after birth. AB - Pseudomosaicism is noted in approximately 1% of amniotic fluid cell studies. Some represent numerical abnormalities, but pseudomosaicism for structural chromosomal abnormalities is also seen. Pseudomosaicism is not usually considered clinically significant. Recently, we evaluated a 13-month-old girl with developmental delay and minor anomalies suggestive of 4p- syndrome. In 5 of 100 peripheral lymphocytes, she had a deletion 46,XX,del(4)(p15). Review of a prenatal amniocentesis study performed on the mother of our patient disclosed that one colony of 18 examined from 2 in situ cultures had the same abnormality, whereas none of the 27 cells from a flask culture showed the abnormality. Results of this study had originally been reported as showing pseudomosaicism. To our knowledge, amniotic fluid pseudomosaicism of a structural abnormality has not previously been shown to reflect true mosaicism in fetal tissue or liveborn children. The actual incidence of this phenomenon is unknown, but it may be present in unexamined children with minimal clinical findings. Apparently only one previous case of mosaic 4p- in a liveborn individual has been reported. PMID- 1714239 TI - Risk of missing angle neovascularization by omitting screening gonioscopy in patients with diabetes mellitus. PMID- 1714240 TI - Chemosis associated with Whipple's disease. PMID- 1714241 TI - Low nm23 protein expression in infiltrating ductal breast carcinomas correlates with reduced patient survival. AB - Protein levels corresponding to nm23 were determined in normal and neoplastic breast tissues by immunoperoxidase staining. Nm23 protein levels were highest in normal breast epithelium, and lower in intraductal carcinomas. Based on nm23 staining, 39 infiltrating ductal carcinomas were separated into two groups: tumors with homogeneously high nm23 protein content, and tumors with low staining in either a homogeneous or heterogeneous pattern. Patients with low nm23 staining tumors, determined by three pathologists independently, had reduced survival times (alpha = 0.034, alpha = 0.012, alpha = 0.052 by the log rank test). Nm23 expression approached significance as an independent predictor of survival in Cox's proportional hazards model. The data provide the first correlation of low nm23 protein expression and reduced breast carcinoma patient survival. PMID- 1714242 TI - Immunohistochemical localization of glutathione-S-transferase and glutathione peroxidase in adult Syrian hamster tissues and during kidney development. AB - Tissues from adult Syrian hamsters were studied with immunoperoxidase techniques using polyclonal antibodies to glutathione-S-transferase (rat liver and human placental enzymes) and human erythrocyte glutathione peroxidase. Most tissues immunostained similarly with these antibodies. Most notable was the cytoplasmic staining of mesenchyme tissues, especially smooth muscle, by all three antibodies. Epithelial cells stained distinctively, but usually less intensely than mesenchyme. Epithelial cells from all levels of the gastrointestinal tract, respiratory epithelium, transitional epithelium, and epidermis all showed strong staining with these antibodies. Other epithelial cell types were usually positive but showed less dramatic staining. Most epithelial tissues showed both nuclear and cytoplasmic staining; some also showed cell-surface (eg, cilia) staining. The role of these enzymes in cell differentiation of a stable organ was studied by immunostaining the kidney during its development. Early stroma (13- and 15-day fetuses) of the kidney (metanephric mesenchyme) showed strong cell-surface staining for glutathione transferases and moderate staining for glutathione peroxidase; renal tubules (which are epithelial cells) at this stage were negative for these markers. As renal tubules differentiated, first cytoplasm and then nuclei stained moderately, suggesting that glutathione-S-transferases and glutathione peroxidase are markers of both mesenchymal cells, including embryonic mesenchyme, and terminal differentiation of at least some epithelial cells. PMID- 1714243 TI - Expression of endothelial leukocyte adhesion molecule-1 in septic but not traumatic/hypovolemic shock in the baboon. AB - Baboons were subjected to septic or traumatic/hypovolemic shock and their tissues were examined for the de novo expression of endothelial leukocyte adhesion molecule 1 (ELAM-1), using immunohistochemical techniques. In animals with septic shock induced with live Escherichia coli, there was widespread expression of ELAM 1, recognized by monoclonal antibodies H4/18 or ENA-1 in most tissues examined with strong staining in the lung, liver, and kidneys. Endothelial leukocyte adhesion molecule 1 expression was evident in capillaries, venules, small veins, arterioles, and arteries. In contrast, baboons with traumatic/hypovolemic shock had minimal levels of focal ELAM expression in all organs studied. Similarly evidence of neutrophil activation, measured by granulocyte elastase levels in the plasma was much more pronounced in animals with septic shock. The study documents that lipopolysaccharide (LPS)- and cytokine-induced endothelial activation occurs in vivo in septic shock. Much higher levels of ELAM-1 expression and plasma granulocyte-elastase titer in septic shock, as contrasted with traumatic/hypovolemic shock, are consistent with the higher levels of circulating tumor necrosis factor, other cytokines, and LPS in sepsis. PMID- 1714244 TI - Anterior cruciate ligament reconstruction using bone-patellar tendon-bone allografts. A biological and biomechanical evaluation in goats. AB - Twenty-eight goats underwent ACL reconstruction with freeze-dried bone-patellar tendon-bone allografts in one knee, the opposite knee serving as a control. One group of 16 knees was evaluated, in groups of four, at 6, 12, 26, and 52 weeks by histologic and vascular injection techniques. The other group of 12 knees was evaluated in two groups of six at 26 and 52 weeks by morphological and biomechanical techniques of analysis. Within the first 12 weeks these allografts were revascularized; in the first 26 weeks they had matured to resemble normal connective tissue. Graft stiffness was 29% of the control value and maximum force to failure was 43% of the control value. The results of this study indicated that freeze-dried bone-patellar tendon-bone allografts are biomechanically and biologically similar to patellar tendon autografts. PMID- 1714245 TI - Isotypic analysis, antigen specificity, and inhibitory function of maternally transmitted Plasmodium falciparum-specific antibodies in Gabonese newborns. AB - An analysis of Plasmodium falciparum-specific antibodies was performed in pairs of maternal and cord sera from Gabon, a region endemic for malaria. All paired sera (n = 59) had P. falciparum-specific antibodies. Immunofluorescence assays detected parasite-specific IgG1, IgG2, and IgG3 in 100% of the tested pairs (n = 26) and IgG4 in 42% of them. The titers of specific IgG2 and IgG3 were significantly lower in cord than in maternal sera. All maternal sera had specific IgM. Of the seven P. falciparum-IgM positive cord sera, six were associated with malaria-related histological placental changes (MRHPC). In addition, higher titers of specific IgG1 in maternal and cord sera and of specific IgG3 in cord sera were associated with MRHPC. Similar P. falciparum antigens were recognized by cord and corresponding maternal sera in radioimmunoprecipitation and Western blot assays (n = 40). Sixteen of 20 cord sera and 15 of 20 paired maternal sera significantly inhibited in vitro parasite growth. The extent of inhibition did not correlate with the titer of specific antibodies. These data confirm the very effective placental transfer of anti-malarial antibodies. The presence of IgM in some cord sera raise the question of intrauterine sensitization to malaria antigens. PMID- 1714246 TI - Follicular hybrid cysts. An expanded spectrum. AB - Currently it is well established that each of the three parts of the hair follicle (infundibulum, isthmus, and the inferior portion) originates different types of cutaneous cysts. Thus, follicular cysts include infundibular, trichilemmal, and matricial cysts. Brownstein in 1983 described a mixed type of cutaneous cyst combining epidermoid, infundibular, and trichilemmal types of keratinization. We review and illustrate the different combinations of follicular hybrid cysts reported to date: infundibular and trichilemmal cyst, infundibular and pilomatricoma cyst, trichilemmal and pilomatricoma cyst, eruptive vellus hair cyst and steatocystoma, and eruptive vellus hair cyst and trichilemmal cyst. Therefore, the concept of hybrid cyst should not be restricted to those composed of infundibular and trichilemmal cysts, because any cyst arising from the various parts of the pilosebaceous unit can combine with others to form a large series of follicular hybrid cysts. PMID- 1714247 TI - Occult herpesvirus folliculitis clinically simulating pseudolymphoma. AB - Two cases of cutaneous herpesvirus infection are described that clinically masqueraded as pseudolymphoma. Light microscopy demonstrated typical viral changes involving pilosebaceous complexes with sparing of the surface epithelium. Dermal changes consisted of a dense perivascular and perifollicular inflammatory infiltrate. Multinucleated lymphoid cells were found in the dermis in one case and viral inclusions in fibroblasts were present in the other case. Immunoperoxidase stains with antisera to herpes simplex virus types I and II were positive in one case and negative in the other case. Ultrastructural examination demonstrated viral particles consistent with herpesvirus in both cases. Recognition of typical histologicl features of herpesvirus folliculitis will lead to an accurate diagnosis in these types of clinically unsuspected cases. PMID- 1714248 TI - The ultrastructure of the transition zone between specialized cells ("Flugelzellen") of Huxley's layer of the inner root sheath and cells of the outer root sheath of the human hair follicle. AB - We studied biopsy specimens taken from the scalp of four normal volunteers by transmission electron microscopy (TEM). At the suprabulbous level of the follicle, where the cells of Henle's layer are fully keratinized but the cells of Huxley's layer are not, the cytoplasmic processes of Huxley's layer ("Flugelzellen") reach the cells of the outer root sheath through the keratinized cylinder of Henle's layer. This junction between the outer root sheath cells and the "Flugelzellen" has not been previously characterized ultrastructurally. It is peculiar in its total lack of desmosomes, and it may have the features of a gap junction. The function of this structure and its possible role in hair growth is unknown. It may be related to nutrition, differentiation, or both. PMID- 1714249 TI - Signet ring cell basal cell carcinoma. AB - A 63-year-old man presented with a signet ring cell basal cell carcinoma of the right infraorbital area. This is the third reported case of this rare variant of basal cell carcinoma characterized by tumor cells containing large, hyalinized, eccentric, intracytoplasmic inclusions that compress nuclei into crescent or ring shaped forms. Antibodies to both high and low molecular weight cytokeratins were strongly positive, staining the inclusions in a uniform fashion. Vimentin and actin antibodies did not stain the inclusions. These results support previous electron microscopic studies that show the inclusions to be aggregates of intermediate filaments blending into tonofilaments at their periphery. Although speculative, the formation of signet ring cells does not appear to be a degenerative or necrotic phenomenon, but probably a peculiar aberrant form of individual cell keratinization. PMID- 1714250 TI - Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase. AB - An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques. PMID- 1714251 TI - Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs. AB - Buoyant density of DNA in CsCl gradients with Hoechst 33258 (bisbenzimide) was investigated as a function of guanine plus cytosine content of the DNA (%GC; in mole percent). A formula for calculating %GC from the refractive index (nD) of the isopycnic CsCl/Hoechst 33258 solution over the range of 0-75 %GC was established: %GC = 351762.28 X nD - 123778.66 X nD2 - 249789.47 (the coefficients must not be rounded off). The shape of this curve indicates that under these conditions, in contrast to dilute buffers, Hoechst 33258 binds to single AT base pairs on DNA. Resolution of DNA bands in CsCl/Hoechst 33258 gradients is 1.6 to 2.1 times better than comparative CsCl gradients without the dye. Potential application to %GC determination is discussed. PMID- 1714252 TI - Introduction of unlabeled proteins into living cells by electroporation and isolation of viable protein-loaded cells using dextran-fluorescein isothiocyanate as a marker for protein uptake. AB - Commonly, microinjection has been the method of choice for introducing proteins into living cells. Viable cells containing an introduced protein can be then identified providing that the protein is fluorochrome conjugated. This approach is applicable only for adherent cells, and the number of cells that can be analyzed is small. In this study, we have established that electroporation can be used to load proteins into large numbers of cells with high efficiency. Furthermore, we have developed a method for the isolation of protein-loaded cells using fluorescein isothiocyanate-dextran (dextran-FITC) as a molecular marker for protein uptake. The essential features of this method are that dextran-FITC is included in the electroporation medium and, thus, is cointroduced with the protein of interest. Purification of cells containing dextran-FITC using fluorescence-activated cell sorting yields a population which is composed almost entirely of cells containing the protein of interest. PMID- 1714253 TI - Experimental and theoretical studies of rate constant evaluation for the solute matrix interaction in affinity chromatography. AB - In an investigation of the problem of determining kinetic parameters for the interaction of a solute with immobilized ligand sites on an affinity matrix, a combination of experimental studies and numerical simulations of frontal chromatography of methyl orange on Sephadex G-25 has yielded a simpler method than existing procedures for characterizing solute-matrix kinetics. A significant change in approach has entailed the direct evaluation of the kinetic contribution to boundary spreading from the flow-rate dependence of boundary variance under conditions of concentration-independent chromatographic migration (linear kinetics). This kinetic contribution is then interpreted in terms of an experimentally more appropriate form of a quantitative relationship for diffusion free chromatographic migration (H. W. Hethcote and C. DeLisi, 1982, J. Chromatogr. 240, 269-281). Finally, the results of numerical simulations of concentration-dependent chromatographic migration (Langmuir kinetics) have indicated that rate constants should also be determinable under these conditions by extrapolation of their apparent values obtained by the above procedure to infinite dilution. PMID- 1714254 TI - Immunolocalization of extracellular matrix components during organogenesis in the human small intestine. AB - The expression and distribution of several major extracellular matrix macromolecules were investigated at the epithelial-mesenchymal interface of the human fetal small intestine from 8 to 20 weeks of gestation. Localization of heparan sulfate proteoglycan, type-IV collagen and laminin, three basement membrane components, as well as fibronectin and tenascin, were assessed by indirect immunofluorescence staining on cryostat sections, and correlated to morphogenesis and epithelial cell differentiation. Basement membrane components and fibronectin were all detected as early as 8 weeks (a time when the epithelium is still stratified and does not express sucrase-isomaltase). Tenascin appeared only after short villi had developed (around 10 weeks) and was restricted to the connective tissue at the tip of villus rudiments. At 18 weeks, well-formed villi and crypts were apparent. The antibody against heparan sulfate proteoglycan stained exclusively the epithelial basement membrane. Anti-type-IV collagen and anti-laminin antibodies stained the epithelial basement membrane and also cellular and fibrillar structures in the lamina propria. Fibronectin was found uniformly distributed over the lamina propria except in the upper third position of the villus core. On the contrary tenascin was mainly localized in the stroma at the tip of the villi. Staining for tenascin was also detected at the epithelial-mesenchymal interface of the villus and in the mesenchyme immediately surrounding budding crypts. These results provide basic data concerning the development of the human gut, and suggest that extracellular matrix components could be involved in the remodelling process of the intestinal mucosa. PMID- 1714256 TI - Cytoskeleton in microridges of the oral mucosal epithelium in the carp, Cyprinus carpio. AB - Microridges produce a characteristic fingerprint-like pattern on the surface of fish oral mucosa. The cytoskeleton in these microridges was examined by immunofluorescence microscopy and transmission electron microscopy after detergent extraction and decoration with myosin subfragment 1. The effect of cytochalasin B on microridges was probed with scanning electron microscopy. Immunofluorescence microscopy revealed that actin filaments were present throughout the periphery of the epithelial cells and were especially localized beneath the free surface of the epithelium. In thin sections treated with Triton X-100, the majority of filaments in the microridges and their bases were found to be actin filaments and a plexus of keratin filaments that underlay the network of actin filaments. A part of the plexus of keratin filaments entered the microridges. After extraction with Triton X-100 and decoration with myosin subfragment 1, decorated actin filaments were found in the microridge cores, connected to the keratin filaments. The keratin filaments aggregated in the pattern of microridges and a few of them protruded into the microridges. Treatment with cytochalasin B caused microridges to disappear or to become thinner and lower or to change short or microvillus-like microridges. When most microridges disappeared, the surface of the superficial cells was prominently swollen, but the cell boundaries were fastened, and the microridges in the periphery were preserved. On the basis of these observations, the possible roles of actin and keratin filaments in the maintenance and the formation of microridges are discussed. PMID- 1714255 TI - Distribution of GABA and neuropeptides in the human cerebral cortex. A light and electron microscopic study. AB - Antibodies were used to identify neurons in human frontal and temporal cortex that were immuno-positive to gamma-aminobutyric acid (GABA) and the neuropeptides vasoactive intestinal polypeptide (VIP), substance P (SP) and somatostatin (SOM). Specimens were taken at surgical biopsy and fixed immediately after removal. The results described for both light and electron microscopy were obtained when relatively high concentrations of glutaraldehyde (2.5-3%) were present in the fixative. Specimens were examined from three adults and an infant aged 5 months. GABAergic neurons were present in all cortical layers, with fewest in layers I, deep III and V, and were mainly small, and round or oval. No labelled pyramidal neurons were detected. GABAergic puncta were common in the neuropil, probably representing axonal profiles. VIP-neurons were also found in all layers, including layer I, and were approximately twice as numerous as GABA-cells. SP positive cells were found throughout the layers, but were sparse in layers I and VI. They were about three times commoner than GABAergic neurons. SOM-reactivity was demonstrated in about the same number of cells as that for SP. Again, this involved all layers, but layer I least. Peptidergic neurons were larger, on the average, than GABAergic cells, and were frequently pyramidal in character. In the infant, the distribution, size and frequency of immunoreactive neurons were similar to those in the adult. However, GABAergic puncta were commoner. PMID- 1714257 TI - Cytoplasmic distribution of poly(A)-containing RNA in developing Necturus maculosus oocytes with reference to annulate lamellae. AB - The cytoplasmic distribution of poly(A)+ mRNA and its relationship to annulate lamellae were examined in developing Necturus maculosus oocytes by in situ hybridization with [3H]poly(U). The specificity of [3H]poly(U) binding was tested by incubating control ovarian sections with either KOH or RNase A before in situ hybridization. In both experiments, the silver grain densities were markedly reduced. Poly(A)+ RNA is uniformly distributed in the cytoplasm until the mid growth phase and then later in vitellogenesis becomes localized in the subcortical ooplasm. The silver grain density in the cytoplasm varied during oogenesis and was greatest in previtellogenic oocytes. Annulate lamellae commonly are observed with the light microscope in oocytes prior to vitellogenesis. In such oocytes, the labeled mRNA probe is observed over cytoplasmic regions of annulate lamellae. The results suggests that a differential localization of messenger RNA occurs during oogenesis in Necturus maculosus. Furthermore, poly(A)+ RNA is present in cytoplasmic regions of annulate lamellae. PMID- 1714258 TI - Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells. AB - The secretory granules of salivary glands often display complex internal substructures, yet little is known of the molecular organization of their contents or the mechanisms involved in packaging of the secretory proteins. We used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and alpha-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells. With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin, reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions of low and medium electron density, but not in the dense cores. Reactivity seen on the apical and basolateral membranes of acinar and duct cells was attributed to a shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in the dense cores and in areas of medium density, but not in the regions of low density. These results indicate that these two proteins are distributed differently within the secretory granules, and suggest that they follow separate pathways between the Golgi apparatus and forming secretory granules. Small vesicles and tubular structures that labeled only with the antibodies to the agglutinin were observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in transport of the putative membrane glycoprotein to the cell membrane. PMID- 1714259 TI - Influence of time of administration on allergic skin prick tests response. AB - This study evaluated influences of time of administration on the response to allergy skin prick tests. There was no significant difference between the mean +/ SEM wheal diameter of positive responses obtained in the morning, 2.98 +/- 0.19 nm, or in the evening, 3.14 +/- 0.18 mm (n = 175). PMID- 1714260 TI - [Inadequacy or breaks in the early child-mother interactions]. PMID- 1714261 TI - [Our experience with the 1st 100 cases of transurethral incision of the prostate performed for benign prostatic hypertrophy]. AB - Transurethral incision of the prostate (TUIP) or prostatotomy is a recently developed surgical technique whose favourable results have been reported elsewhere to range from 88% to 92%. It has a precise indication in small prostates and short urethras, and in well-selected cases its results are superior to that achieved by transurethral resection (TUR). Moreover, alterations of sexual function (impotence, retrograde ejaculation, etc.) are less and it affords the following advantages: the surgical technique is easy to perform, postoperative hospital stay is minimal, and patient comfort is enhanced. We have reviewed the first 100 prostatotomy procedures performed at the Urology Department of our hospital. Patient follow-up was 1 1/2 years. Good results were achieved in 65 (95.5%) of 68 patients with prostates less than 20 gms. In 20 patients with prostates between 20-30 gms., we achieved a success rate of 85% (3 poor results). The results were poor in 4 of 12 patients (60% success) with prostates more than 30 gms. The early complications were minimal and no late complications were observed. Thus, we believe this is a valid technique when performed in the appropriate cases. PMID- 1714262 TI - [Exophthalmos caused by orbital metastasis of prostatic carcinoma]. AB - A case of orbital metastasis from Whitmore stage D adenocarcinoma of the prostate is described. Clinically, it presented as rapidly progressing exophthalmos of the right eye with elevation (ptosis) and abduction paralysis. The associated clinical picture of a one-year history of prostatism prompted patient referral to our department. When a patient presents with an orbital tumor and a history of cancer localized to another site, the metastatic origin of the condition should be suspected and metastasis to other sites sought. A negative finding warrants performing orbital biopsy to confirm the diagnosis. Although excision of single metastatic tumors in this site has been described, coexisting metastasis to bone and lymph nodes, the hormone dependence that these present and prostatic cancer contraindicate resection of the orbital metastatic tumor. Following bilateral orchiectomy and hormone therapy with antiandrogens micturitional symptomatology improved, tumor size was reduced, and exophthalmos disappeared. The case described herein is not the first case of this type of metastatic lesion reported in the literature; 28 cases have been reported to date. This uncommon clinical presentation with extraurological manifestations gives us an idea of the broad clinical spectrum the biological behaviour of this tumor type can adopt. PMID- 1714263 TI - [Malignant priapism. Curettage of the corpora cavernosa as a palliative measure]. AB - A case of malignant priapism is described in a 58-year-old patient with metastatic carcinoma of the bladder. The diagnostic methods and the possibilities of palliative treatment are discussed. PMID- 1714264 TI - [Benign hypertrophy of the prostate: pathogenic aspects]. AB - In 106 male human cadavers the incidence of benign prostatic hyperplasia was found to be 63%. The present review confirmed age to be a determinant factor of the first order in the development of adenoma of the prostate. A delay in the presentation of BPH was observed in the cirrhotic alcoholic. Surprisingly, prostatic hyperplasia was not observed in those who had died from genitourinary, gastric or malignant hematologic diseases. No evidence of BPH was observed in 68.4% of patients who had died of cancer. PMID- 1714265 TI - [Transureteral resection of the prostate. Review of 20 consecutive operations (5 yr)]. AB - In the present study we reviewed our results in the treatment of prostatic obstruction of adenomatous origin over a period of 5 years and with a follow-up of 1 to 6 years. Of the 210 patients considered evaluable, good results were achieved in 84.28%, with mortality and morbidity rates of 0.45% and 22.82%, respectively. Infections were the most commonly observed complications in the early post-operative period. Urethral stenosis, the most common cause for reoperation, was observed in 9.04% in the late post-operative period. PMID- 1714266 TI - [Endourological treatment of benign prostatic hypertrophy. Preliminary report]. AB - We report on two patients with symptomatic Benign Prostatic Hyperplasia (BPH) who were submitted to balloon dilation of the prostate. The symptoms and signs of prostatism markedly improved in both patients; however, the observed improvement did not correlate with the transrectal ultrasound findings of the results of the urinary flow studies. This is a preliminary report of an ongoing prospective protocol. PMID- 1714267 TI - Intrahepatic recurrence after resection of hepatocellular carcinoma complicating cirrhosis. AB - To determine whether a careful evaluation of tumor extension by preoperative computed tomography scan after intra-arterial injection of ultrafluid lipiodol and by intraoperative ultrasound examination reduced the recurrence rate of hepatocellular carcinoma after resection, a series of 47 cirrhotic patients with a single tumor operated on from 1984 was studied. Alphafetoprotein level was less than 100 ng/mL in 26 patients (55%), size of the tumor was less than 5 cm in 28 patients (59%), and capsule was present in 30 patients (63%). The resection was performed with free margin measuring 1 cm or more. The overall cumulative survival rates at 3 and 5 years were 35% and 17%, respectively. Intrahepatic recurrence was observed in 28 patients (60%), located less than 2 cm from the resection margin in only four patients. The cumulative intrahepatic recurrence rate at 3 years was 81% and was significantly higher in patients with tumor greater than or equal to 5 cm and in patients with preoperative alphafetoprotein level of greater than or equal to 100 ng/mL. In this series the cumulative intrahepatic recurrence rate at 5 years was 100%. This high recurrence rate after resection, even with careful evaluation of tumor extension, indicates that liver transplantation might be envisaged for the treatment of cirrhotic patients with resectable hepatocellular carcinoma. PMID- 1714268 TI - Adverse effect of therapeutic vasoconstrictors in experimental acute pancreatitis. AB - Alpha-adrenergic drugs commonly are used to treat hypotension resulting from severe acute pancreatitis. It was shown previously that although systemic arterial pressure is increased by phenylephrine, pancreatic microcirculatory perfusion is decreased. Because inadequate tissue perfusion may be critical in the progression of edematous pancreatitis to parenchymal necrosis, it was hypothesized that vasoconstrictors might be harmful in pancreatitis. Therefore the effect of phenylephrine on cerulein-induced mild pancreatitis were studied. Sprague-Dawley rats (n = 54) were randomly allocated to 6 experimental groups and subjected to the following infusion regimens: (1) cerulein (cae) + phenylephrine (phe), (2) cae + saline (NS), (3) NS + phe, (4) cae + phenoxybenzamine (pbz) + phe, (5) NS + pbz + phe, and (6) NS. Initial and terminal hematocrit, serum amylase activity, and blood ionized calcium concentration were determined. The animals were killed 9 hours after starting the infusion. Macroscopic and histologic changes were scored by a 'blinded' pathologist. Phenylephrine increased the severity of cerulein-induced pancreatitis as manifested by statistically significant adverse changes in serum amylase, hematocrit, ionized calcium, peripancreatitic soap formation, and acinar cell vacuolization. These changes were antagonized by alpha-adrenergic receptor blockade with phenoxybenzamine. It is concluded that phenylephrine is deleterious in acute experimental pancreatitis, the first demonstration of such an effect by a pharmacologic vasoconstrictor, and suggested that microcirculatory changes may be important in the transition of mild to severe pancreatitis. Caution in the use of vasoconstrictor drugs in patients with acute pancreatitis is recommended. PMID- 1714269 TI - The local effects of cachectin/tumor necrosis factor on wound healing. AB - Previous experimental studies have suggested that tumor necrosis factor (TNF) may have either a beneficial or a detrimental role in wound healing. Control and doxorubicin-treated (6 mg/kg, intravenously) rats underwent paired dorsal 5-cm linear wounds and had either vehicle or recombinant (r)TNF (0.5, 5, or 50 micrograms) applied locally to the wound. Paired wounds were harvested at 7 and 14 days after wounding and analyzed for wound-bursting strength (WBS) and activity of the gene for type 1 collagen and TNF. Doxorubicin treatment decreased WBS at 14 days but not at 7 days after wounding. Local application of 50 micrograms of rTNF decreased WBS in saline-treated rats and concentrations of 5 and 50 micrograms decreased WBS in doxorubicin-treated rats when measured 7 days after wounding. These effects dissipated when WBS was measured 14 days after wounding. Doxorubicin decreased wound collagen gene expression and local TNF treatment decreased wound collagen gene expression in saline-treated rats and further decreased it in doxorubicin-treated rats. The decrement in collagen gene expression induced by rTNF increased as the local dose of rTNF increased. The gene for TNF was not detectable in wounds from normal or doxorubicin-treated rats at 3, 7, 10, or 14 days after wounding. These data suggest that the gene for TNF is not expressed in wounds and that the local application of TNF is detrimental to wound healing as it decreases WBS and activity of the gene for collagen. PMID- 1714270 TI - Argon laser photocoagulation for neovascular maculopathy. Five-year results from randomized clinical trials. Macular Photocoagulation Study Group. AB - With completion of follow-up of all patients enrolled in three randomized clinical trials of argon laser photocoagulation of extrafoveal choroidal neovascular membranes secondary to senile (age-related) macular degeneration, ocular histoplasmosis, or idiopathic causes, the Macular Photocoagulation Study Group has demonstrated that laser treatment of such lesions is beneficial in preventing or delaying large losses of visual acuity for at least 5 years. In eyes with senile (age-related) macular degeneration as the underlying cause, the relative risk of losing six or more lines of visual acuity from the baseline level among untreated eyes (n = 117) compared with laser-treated eyes (n = 119) was 1.5 from 6 months through 5 years after entry (P = .001). In addition, after 5 years, untreated eyes had lost a mean of 7.1 lines of visual acuity, while laser-treated eyes had lost 5.2 lines. Recurrent neovascularization had been observed in 54% of laser-treated eyes by the end of the 5-year follow-up period. Among eyes with ocular histoplasmosis, untreated eyes (n = 130) had 3.6 times the risk of laser-treated eyes (n = 132) of losing six or more lines of visual acuity (P less than .0001). Also, untreated eyes had lost a mean of 4.4 lines of visual acuity after 5 years, compared with only 0.9 lines lost by laser-treated eyes. Among laser-treated eyes, recurrent neovascularization had been observed in 26% by 5 years after enrollment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714271 TI - Characterization of infectious crystalline keratitis caused by a human isolate of Streptococcus mitis. AB - Streptococcus mitis isolated from a human with infectious crystalline keratitis was injected intrastromally into corneas of adult New Zealand white rabbits that were treated with tetracycline hydrochloride, methylprednisolone acetate, or a combination of tetracycline and methylprednisolone. Animals were followed up for up to 44 days; untreated corneas and those treated with tetracycline developed no disease or "fluffy" stromal infiltrates with overlying epithelial defects representing an abscess. Corneas treated with the combination of tetracycline and corticosteroid usually developed crystalline stromal opacities that on histopathologic examination were shown to be intrastromal aggregates of cocci. Transmission electron microscopy of crystalline lesions within 10 days of infection revealed typical cocci intermixed with a fibrillar material having periodicity characteristic of fibrinogen or fibrin, and immunoperoxidase staining for fibrinogen was positive. By 1 month, electron microscopy revealed aggregates of degenerated bacteria that were surrounded by cellular processes of activated keratocytes. Our studies demonstrate a model for crystalline keratitis in which organisms are seen to reside within the stroma for up to 44 days without an inflammatory response. Periocular corticosteroids appear to be necessary to create this model. It is possible that the organisms are isolated from the host response by fibrin or by keratocytes. PMID- 1714272 TI - Extrasystoles. PMID- 1714273 TI - Frequency of intermediate admissions and place of death of patients with advanced colorectal cancer. AB - An analysis was made of the place of death and the degree of institutional support required following surgery in patients with colorectal cancer (CRC) who had distant metastases. There was a high incidence of intermediate admissions to an acute hospital, and most patients died in an acute hospital bed. The number of readmissions and the place of death were not influenced by the patients' age, sex, site of tumour or their home situation at the time of diagnosis. In view of the high demand for acute surgical beds, there is a need to develop more appropriate facilities to care for patients in the terminal phase of this disease. PMID- 1714274 TI - Effects of tricyclic drug on induced circular dichroism spectra of dicumarol bound to alpha 1-acid glycoprotein. AB - Effects of both tricyclic and non-tricyclic drugs on the extrinsic Cotton effects of dicumarol bound to human alpha 1-acid glycoprotein (AGP) have been investigated. Basic tricyclic drugs caused the reversal of the signs of the induced Cotton effects of the circular dichroism (CD) spectra of the dicumarol AGP system while the basic drugs not possessing tricyclic rings and acidic drugs decreased the observed ellipticities without changing the signs of its CD spectra. There was no reversal of the CD signs of the drugs not containing two hydroxycoumarin rings bound to AGP by basic tricyclic drugs. Raising of pH and temperature, and the addition of guanidine hydrochloride decreased the observed ellipticities of the CD spectra of the dicumarol-AGP system without showing any change in the signs of the Cotton effects. The mutual displacement data showed that protriptyline increased its own binding and that of dicumarol with AGP. The results of CD titration and equilibrium dialysis experiments suggest that dicumarol-AGP and dicumarol-AGP-protriptyline form a 1:1 binary complex and a 1:1:1 ternary complex, respectively. PMID- 1714275 TI - "Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa--I. Characterization by means of agonists. AB - A previous study has suggested the presence of two distinct binding sites for gasrin and cholecystokinin (CCK) in isolated non-parietal cells from rabbit gastric mucosa: a receptor which binds CCK-8 and CCK-39 with a high affinity and a receptor which binds gastrin and CCK-8 with the same high affinity and CCK-39 with a lower affinity. To characterize these receptors, their ability to induce phosphoinositide breakdown was investigated. Gastrin (HG-17), CCK-39 and CCK-8 induced [3H]-inositol phosphate ([3H]InsP) accumulation from [3H]inositol prelabelled cells with a high potency (EC50: 0.3-2.7 nM) but CCK-8 exhibited a higher efficacy than HG-17 or CCK-39. HG-17, CCK-8 and CCK-39 induced a rapid accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2) and [3H]inositol trisphosphate ([3H]InsP3) but CCK-8 caused a two times higher accumulation than HG-17 or CCK-39. Histamine- and somatostatin containing cells appeared to be located in this non-parietal cells population. HG 17, CCK-8 and CCK-39 dose-dependently induced histamine release with the following order of potency: HG-17 = CCK-8 (EC50 approximately 0.2 nM) greater than CCK-39 (EC50 approximately 4 nM). In addition, HG-17 exhibited the highest efficacy. HG-17, CCK-8 and CCK-39 enhanced somatostatin-like immunoreactivity (SLI) release with the following order of potency: CCK-8 (EC50 approximately 0.1 nM) = CCK-39 greater than HG-17 (EC50 approximately 10 nM); CCK-8 and CCK-39 exhibited the highest efficacy. These results led us to the following conclusions: (i) existence of a "gastrin-type" and of a "CCK-type" receptor mediating phosphoinositide breakdown in these gastric non-parietal cells. CCK-8 interacts with both receptor-types with the same affinity; (ii) the release of histamine from histamine-containing cells could be induced following "gastrin type" receptors activation; (iii) somatostatin release from D-cells present in this non-parietal cells population could be induced following "CCK-type" receptors activation. PMID- 1714276 TI - "Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa-II. Characterization by means of selective antagonists (L-364,718 and L-365,260). AB - In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino) benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino) benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39 enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG 17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L 365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway. PMID- 1714277 TI - Comparison of pharmacological properties of optical isomers and a racemic mixture of epinastine. AB - Some pharmacological effects of d- and l-isomers of epinastine (WAL 801 CL, 3 amino-9,13b-dihydro-1H-dibenz [c,f]imidazo[1,5-a]azepine hydrochloride; CAS 80012 43-7) were studied in comparison with that of dl-epinastine. Although no significant difference was detected, d-epinastine is slightly more active than the l-isomer in the depressant actions of the central nervous system. However, in EEG power spectra recorded at the frontal cortex, occipital cortex, hippocampus and amygdala in conscious rats, no appreciable differences were recognized between two optical isomers and a racemic mixture. Furthermore, the extents of compound 48/80-induced lethality, antagonism on histamine-induced contraction of guinea pig ileum and histamine release from rat peritoneal mast cells were almost the same among the racemate and optical isomers of epinastine. In addition, two stereoisomers caused an almost equal degree of inhibition in antigen-induced histamine release from the lung pieces of sensitized guinea-pigs and the equivalent level of inhibition was exerted by the racemate. PMID- 1714278 TI - Mechanism of anti-inflammatory action of etodolac. AB - The effect of etodolac (CAS 41340-25-4) on the inflammatory reactions induced by histamine and bradykinin was compared with that of indomethacin and other nonsteroidal anti-inflammatory drugs. Etodolac (50 mg/kg p.o.), indomethacin (20 mg/kg p.o.), diclofenac Na (20 mg/kg p.o.) and acetylsalicylic acid (200 mg/kg p.o.) had no effect on the increase of vascular permeability induced by histamine or bradykinin and on passive cutaneous anaphylaxis in rats. Etodolac (5, 10 and 20 mg/kg p.o.) suppressed concanavalin A-induced paw edema in rats. Etodolac (10 mg/kg p.o.) and bromelain (10 mg/kg i.v.) significantly suppressed the heat induced elevation of bradykinin in perfusates of rat paws, but indomethacin (20 mg/kg p.o.) and diclofenac Na (20 mg/kg p.o.) did not. Etodolac inhibited bradykinin-forming enzyme activity in a concentration-dependent manner (IC50 = 1.5 x 10[-4) mol/l). These results suggest that etodolac is a unique nonsteroidal anti-inflammatory drug which can inhibit bradykinin formation, unlike indomethacin or diclofenac Na. PMID- 1714279 TI - Electrostatic energy and macromolecular function. PMID- 1714280 TI - Structure-function studies of voltage-gated ion channels. PMID- 1714281 TI - The Di Mascio Lecture. The allosteric modulation of GABAA receptors. Seventeen years of research. PMID- 1714282 TI - Stained colonies facilitate alignment in a nonradioactive colony hybridization. PMID- 1714283 TI - [Impact on the energetic-proteic nutritional status of total paracentesis combined with infusion of albumin or dextran-70 in the therapy of tension ascites in liver cirrhosis]. AB - The aim of this study was to investigate the effect of total paracentesis plus albumin or dextran-70 infusion on the nutritional status in cirrhotics with tense ascites. Seventeen patients were studied. Eight patients (group I) were treated with total paracentesis and albumin infusion, and in 9 cases (group II) dextran 70 infusion was associated to total paracentesis. The nutritional status was assessed before and two days after the procedure by measuring triceps skinfold thickness, mid-arm muscle circumference and serum albumin. No changes in anthropometric parameters were observed in either group. Patients in group I showed a significant increase in serum albumin levels (from 26.6 +/- 1.4 to 28.9 +/- 1.3 g/l; p = 0.007), whereas this parameter decreased in group II (from 25.5 +/- 1.3 to 23.1 +/- 1.4 g/l; p = 0.005). However, serum albumin levels returned to initial values in both groups one month after total paracentesis. There were no differences between both groups regarding the appearance of complications and mortality rate during admission. These results suggest that total paracentesis plus either i.v. albumin or dextran-70 has no long-term effect on the protein energy nutritional status. PMID- 1714284 TI - The role of NAD(P)H: quinone reductase (EC 1.6.99.2, DT-diaphorase) in the reductive bioactivation of the novel indoloquinone antitumor agent EO9. AB - EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2 electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in EO9 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of EO9 absorbance at 550 nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar Km of 1.4-3.1 microM, although the Vmax was 7 to 8-fold lower for the human preparation. EO9 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for menadione at around 15 microM and the Vmax was 6-fold lower at around 2.5 mumol of cytochrome c reduced mg-1 of protein. EO9 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100 microM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714285 TI - Isolation, identification, and assay of [3H]-porfiromycin adducts of EMT6 mouse mammary tumor cell DNA: effects of hypoxia and dicumarol on adduct patterns. AB - [3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed. PMID- 1714286 TI - [Carcinoid of the thymus]. AB - Thymic carcinoid is a rare lesion. Two cases are reported and diagnostic as well as therapeutic problems are discussed. PMID- 1714287 TI - [Internal biliary diversions in inoperable neoplasms of the biliary tract and pancreas]. AB - The authors report their experience with the treatment of mechanical jaundice in inoperable malignant neoplasms of the bile ducts and pancreas. They emphasize the importance of intrahepatic peripheral biliodigestive derivations (Soupault and Couinaud operation). After a review of the international literature, the authors consider the value of on accurate surgical strategy even in the presence of pathological and clinical features which may limit a radical choice. PMID- 1714288 TI - Prostate-specific antigen: a valuable clinical tool. AB - Because approximately 30% of patients with benign prostatic hyperplasia (BPH) only will have an elevated serum PSA concentration, it is unlikely that PSA by itself will become an effective screening tool for the early diagnosis of prostate cancer. However, if combined with digital rectal examination (DRE) and/or transrectal ultrasound, it may become a vital part of any early detection program. Although there is a direct correlation between the serum PSA concentration and clinical stage, PSA is not sufficiently reliable to determine the clinical stage on an individual basis. This finding also applies to pathologic stage. Low preoperative serum PSA concentrations in patients with previously untreated prostate cancers, however, are predictive of a negative radionuclide bone scan. With respect to monitoring patients after definitive therapy, PSA is an exquisitely sensitive tumor marker. Irrespective of the treatment modality, PSA reflects accurately the tumor status of the patient and is prognostic of eventual outcome. PMID- 1714289 TI - The use of intravenous gammaglobulin in the treatment of typical hemolytic uremic syndrome. AB - Nine children with acute typical post-diarrhea hemolytic uremic syndrome (HUS) were treated with intravenous gammaglobulin (IVIG). These children were compared to nine children with HUS who did not receive IVIG. The use of IVIG did not appear to have a beneficial effect on eight of the nine treated children. There were no significant differences found in the duration of hemorrhagic colitis, thrombocytopenia, elevation of the white blood count (WBC), anuria, dialysis, or hospitalization, or the presence of a central nervous system complication or pancreatitis. Although no significant difference was found in the duration of thrombocytopenia, there was a trend towards a longer duration of thrombocytopenia in children treated with IVIG (P = 0.13). One child demonstrated both an increase in her platelet count and a decrease in her WBC count within 24 h of receiving her first dose of IVIG. PMID- 1714290 TI - Immunodetection of the transforming growth factors beta 1 and beta 2 in the developing murine palate. AB - The expression of some members of the TGF beta family of genes in embryonic craniofacial tissue suggests a functional role for these molecules in orofacial development. In other systems, TGF beta 1 and TGF beta 2 have been shown to regulate cell proliferation and extracellular matrix metabolism, processes critical to normal development of the secondary palate. We have thus determined the amount and tissue distribution of both TGF beta 1 and TGF beta 2 in embryonic palatal tissue. Cellular extracts of murine embryonic palatal tissue from days 12, 13 and 14 of gestation were assayed for the presence of TGF beta 1 and TGF beta 2 by immunoprecipitation. Physiological levels, ranging from 0.05-20 ng/micrograms protein, of both growth factors were detected in all tissues examined. Immunostaining with antibodies directed against either TGF beta 1 or TGF beta 2 demonstrated the presence of these growth factors in palatal epithelium and mesenchyme early during palatal development (gestational day [GD] 12) a period characterized by tissue growth. On GDs 13 and 14, characterized by palate epithelial differentiation, immunostaining for both growth factors predominated in epithelial tissue. Immunostaining for TGF beta 1 and TGF beta 2 was also intense in mesenchyme surrounding tooth germs and in perichondrium. Chondrocytes and cartilage extracellular matrix did not stain for either TGF beta 1 or beta 2. Combined with existing evidence for the presence of functional receptors for the transforming growth factor-beta s in the developing palate, these results provide rationale for studies designed to clarify a specific role for these signalling molecules in palate epithelial differentiation and/or epithelial-mesenchymal interactions. PMID- 1714291 TI - In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs. AB - An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis. PMID- 1714292 TI - Stromal cells from murine developing hemopoietic organs: comparison of colony forming unit of fibroblasts and long-term cultures. AB - The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures. PMID- 1714293 TI - Treatment outcome in Hodgkin's disease in patients above the age of 60: a population-based study. AB - All persons in three Swedish counties afflicted with Hodgkin's disease between 1979 and 1988 were traced. The objective was to analyze, in unselected, population-based material, whether an assumed worse prognosis in the elderly could be due to differences in staging procedures, treatment intensity, decreased tolerance to therapy or to a more aggressive disease. After histopathological revision, 163 of 202 patients (autopsy cases excluded) were accepted as HD, 61 (37%) of them above the age of 60. Although staging procedures had been more intense in the young, the elderly patients had a more advanced stage at diagnosis, and tended more often to have B-symptoms. The intensity of staging procedures did not seem to influence survival. The 5-yr relative survival was 37% above and 85% below the age of 60. Radiotherapy was the primary treatment in 12 (20%) above and 41 (41%) below the age of 60 with 5-yr relative survival figures of 84% and 85%, respectively. Thirty-seven patients (61%) above and 61 (59%) below 60 were treated with combination chemotherapy (MOPP/ABVD, MOPP, ChlVPP/OPEC) with curative intent. The 5-yr relative survival was 33% and 86%, respectively. The majority of the elderly patients (54%) received less than 40% of the planned chemotherapy dose. The main reason for this pronounced reduction was intolerance to therapy, with 8 treatment-related deaths. We conclude that tolerance to combination chemotherapy in the elderly patients with HD is poor and could be the major reason for poor treatment outcome in this age group. PMID- 1714294 TI - Nail-fold capillary microscopy and chemotherapy-induced vascular toxicity. PMID- 1714295 TI - Disruption of keratin filaments in embryonic epithelial cell types. AB - The murine keratins Endo B and Endo A, which are homologs of the human keratins K18 and K8, constitute the intermediate filaments (IFs) that are found in all simple epithelia of the adult and in the first epithelial derivatives of the early embryo. The cellular role of simple epithelial keratins in development and differentiation was investigated by inducing filament collapse in HR9 endoderm and F9 embryonal carcinoma cells in which mutant Endo B protein was constitutively expressed. By immunolocalization techniques a perturbation of the keratin network was revealed as well as concomitant disruption of vimentin IFs and displacement of surface desmosomal proteins, demonstrating an intimate structural association of Endo B/A filaments with these cellular components. In aggregates of differentiating F9 cells displaying altered Endo A/B IFs, the formation of a compact, polarized visceral endoderm layer was significantly compromised. These results indicate that an intact keratin network influences the three-dimensional formation of cell-cell or cell-substratum contacts in embryonic visceral endoderm. PMID- 1714296 TI - Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. AB - A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2. Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full length products can be obtained. The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme. The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol). The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA. Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line. Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA. The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA. PMID- 1714297 TI - X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin. AB - A protease inhibitor from hagfish blood plasma, homologous to human alpha 2 macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods. PMID- 1714298 TI - Effect of salt and membrane fluidity on fluorophore motions of a gramicidin C derivative. AB - We have used fluorescence spectroscopy to investigate the effect of salt and membrane fluidity on the rotational motion of a 5-(dimethylamino)naphthalene-1 sulfonyl (dansyl) derivative of gramicidin C (dansyl-gC) in dimyristoylphosphatidylcholine bilayers, under conditions where the peptide is a formyl-NH to formyl-NH terminal dimer, and in octyl glucoside micelles, where the peptide is an intertwined helical dimer. Energy-transfer experiments showed no changes in either conformation or dimer aggregation under the conditions explored here (15-40 degrees C, 1-350 bar, 0-0.33 M Mg2+, and 0-1 M Na+). The addition of permeable (Na+) or nonpermeable (Mg2+) ions did not affect the temperature or pressure behavior of dansyl rotation. However, fluorescence lifetime measurements indicated an increase in solvent accessibility in the presence of sodium. In bilayers, the temperature dependence of the fluorescence polarization and lifetime shows strong interactions between the dansyl residue and the peptide, and at no time did the dansyl motions become solvent controlled as has been observed for aqueous solvent peptides [Scarlata, S. F., Rholam, M., & Weber, G. (1984) Biochemistry 23, 6789]. In micelles, the change in rotational motion with temperature followed solvent expansion, showing that in this case the dansyl residue does not associate extensively with the peptide. Our results indicate that because of the extensive coupling between the dansyl residue and the rest of the peptide, membrane fluidity does not play a major role in controlling side chain motions. PMID- 1714299 TI - A pancreatic exocrine cell factor and AP4 bind overlapping sites in the amylase 2A enhancer. AB - A factor found in pancreatic exocrine cell lines and pancreatic nuclei binds selectively to the alpha-amylase 2A transcriptional enhancer. Pancreatic exocrine cell extracts protect asymmetrically an unusually large, 35 base pair region from DNase I digestion in vitro, suggesting the involvement of a multimeric DNA binding complex. We show that this region of the enhancer contains a major affinity recognition sequence for the HeLa transcription factor AP4. A 4 base pair mutation in the enhancer sequence shown previously to abolish activity in vivo [Boulet, A. M., Erwin, C. R., & Rutter, W. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3599-3603] abolishes AP4 binding in vitro and weakens but does not eliminate the binding of adjacent enhancer factors. Further, sequences similar to the AP4 binding site are found within a consensus sequence of most pancreatic exocrine genes (Boulet et al., 1986). We have identified three AP4 binding sites in the pancreatic elastase gene: one occurs in the consensus sequence of the enhancer. Thus, protein(s) with the binding selectivity of AP4 may play a role in the expression of the pancreatic exocrine gene family. PMID- 1714300 TI - Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain. AB - Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action. PMID- 1714301 TI - Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs. AB - The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714302 TI - Analysis of an experimental cortical network: II). Connections of visual areas 17 and 18 after neonatal injections of ibotenic acid. AB - Lesions of cortical areas 17 and 18 were produced in newborn kittens by local injections of the excitotoxin ibotenic acid. In the adult this results in a microcortex which consists of superficial layers I, II and III, in the absence of granular and infragranular layers. Horseradish peroxidase, alone or wheat germ agglutinin conjugated, was injected in the microcortex or in the contralateral, intact areas 17 and 18. The microcortex maintains several connections characteristic of normal areas 17 and 18 of the cat. It receives afferents from the dLGN, and several visual areas of the ipsilateral and contralateral hemisphere. However, it has lost its projections to dLGN, superior colliculus, and, at least in part, those to contralateral visual areas. Thus some parts of the microcortex receive from, but do not project into, the corpus callosum. In addition, the microcortex maintains afferents from ipsilateral and contralateral auditory areas AI and AII which are normally eliminated in development. PMID- 1714303 TI - Diffusion model in ion channel gating. Extension to agonist-activated ion channels. AB - Previously, we described a model which treats ion channel gating as a discrete diffusion problem. In the case of agonist-activated channels at high agonist concentration, the model predicts that the closed lifetime probability density function from single channel recording approximates a power law with an exponent of -3/2 (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988a. Proc. Natl. Acad. Sci. USA. 85: 1503-1507). This prediction is consistent with distributions derived from a number of ligand-gated channels at high agonist concentration (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988b. Biophys. J. 54: 1165 1168.) but does not describe the behavior of ion channels at low activator concentrations. We examine here an extension of this model to include an agonist binding step. This extended model is consistent with the closed time distributions generated from the BC3H-1 nicotinic acetylcholine receptor for agonist concentrations varying over three orders of magnitude. PMID- 1714304 TI - Stochastic models for mechanical transduction. PMID- 1714305 TI - Ion transport in a model gramicidin channel. Structure and thermodynamics. AB - The potential of mean force for Na+ and K+ ions as a function of position in the interior of a periodic poly(L,D)-alanine model for the gramicidin beta-helix is calculated with a detailed atomic model and realistic interactions. The calculated free energy barriers are 4.5 kcal/mol for Na+ and 1.0 kcal/mol for K+. A decomposition of the free energy demonstrates that the water molecules make a significant contribution to the free energy of activation. There is an increase in entropy at the transition state associated with greater fluctuations. Analysis reveals that the free energy profile of ions in the periodic channel is controlled not by the large interaction energy involving the ion but rather by the weaker water-water, water-peptide and peptide-peptide hydrogen bond interactions. The interior of the channel retains much of the solvation properties of a liquid in its interactions with the cations. Of particular importance is the flexibility of the helix, which permits it to respond to the presence of an ion in a fluidlike manner. The distortion of the helix is local (limited to a few carbonyls) because the structure is too flexible to transmit a perturbation to large distances. The plasticity of the structure (i.e., the property to deform without generating a large energy stress) appears to be an essential factor in the transport of ions, suggesting that a rigid helix model would be inappropriate. PMID- 1714306 TI - Fluorescent studies directed towards the location of abnormal epithelial cells in cervical smears. AB - Cervical screening is concerned with the search for abnormal epithelial cells in smears prepared from scrapings from the uterine cervix. It is a highly skilled labour intensive operation and automated methods of detecting dyskariotic cells in cervical smears would be helpful. We report a fluorescence method of detecting abnormal cervical cells in smears and biopsies using a probe for guanidinobenzoatase. This approach has the potential for automation. PMID- 1714307 TI - Carcinoid tumour of male breast diagnosed by fine needle aspiration. AB - A primary carcinoid tumour of the breast in a 66-year-old man was diagnosed by fine-needle aspiration cytology. The nature of the lesion was proved by histochemical and immunocytochemical studies. The importance of a conclusive diagnosis is discussed and the value of immunocytochemical analysis as an aid to cytomorphologic diagnosis is demonstrated. PMID- 1714308 TI - Fine needle aspiration cytology of salivary gland lesions: advantages and pitfalls. AB - Fine needle aspiration cytology (FNAC) was performed on 52 patients with salivary gland lesions. A definitive cytodiagnosis was possible in 45 cases: a sensitivity of 89% and a specificity of 94% was achieved. The pitfalls of FNAC of salivary gland lesions are reflected by the false positive and false negative rates which were 4%. Errors of cytodiagnosis are due to the morphological variability of these tumours which make sampling and interpretation difficult. PMID- 1714309 TI - Nucleolar organizer regions in the fine needle aspirates of lung tumours. AB - Nucleolar organizer region associated proteins (AgNOR proteins) have been identified by means of an argyrophil technique in smears of 60 consecutive fine needle aspirates from lung tumours. AgNOR proteins were visualized as silver positive granules distributed in loose, intranuclear aggregates. Variations in the number as well as differences in the distribution pattern of AgNOR granules were found among different types of tumours. Except for small cell lung carcinoma, the count of AgNOR granules increased when the differentiation of tumours decreased. In particular well differentiated tumours had relatively few AgNOR granules, distributed in cohesive aggregates. Poorly differentiated tumours had many AgNOR granules organized in loose clusters and small cell lung carcinoma had relatively few granules dispersed throughout the nucleoplasm, showing characteristics unique among all lung neoplasms. The application of the AgNOR technique in cyto-preparations is useful in discriminating between small cell and non-small cell lung carcinomas. Moreover, the pattern of distribution of AgNOR proteins may be of diagnostic value in the assessment of tumours displaying overlap in AgNOR counts. PMID- 1714310 TI - Influence of lindane on the fluidity of the rat ventral prostate membranes. AB - The influence of lindane upon the dynamic properties of plasma membranes from rat ventral prostate has been investigated using a fluorescence polarization technique. Preincubation with lindane decreased the fluorescence polarization in a dose dependent manner. This effect, which is associated with an increased membrane fluidity, occurred in a very short period of time. Lindane also provoked a number of changes in lipid biosynthesis from acetate in the membrane. Less [1 14C]acetate was incorporated into cholesterol and more into phospholipids when this liposoluble toxicant was added to the preincubation medium. However, not all phospholipid classes were equally increased, because while the rate of acetate incorporation was greater into choline glycerophospholipids than into ethanolamine glycerophospholipids, both were higher than the rates of acetate incorporation into serine glycerophospholipids and sphingomyelin. PMID- 1714311 TI - Effects of propranolol on myocardial performance during acute normovolemic hemodilution. AB - The effects of propranolol and hemodilution on myocardial performance and oxygen delivery were evaluated in 36 anesthetized rats. Oral propranolol treatment consisted of 64 mg/kg/d for 6 weeks prior to the experiments, whereas intravenous (IV) propranolol treatment consisted of 5 micrograms/kg/min for 60 minutes after hemodilution. The hematocrit was reduced to 20% by a hetastarch-for-blood exchange. Animals were divided into six equal groups as follows: (1) no oral drug (water), no hemodilution, no IV drug (saline); (2) oral water, hemodilution, IV saline; (3) oral water, no hemodilution, IV propranolol; (4) oral water, hemodilution, IV propranolol; (5) oral propranolol, no hemodilution, IV saline; and (6) oral propranolol, hemodilution, IV saline. Left ventricular (LV) pressure, maximal dP/dt, ascending aortic blood flow, and response to preload (peak cardiac and stroke volume indices) and afterload (LV-developed pressure) stress were measured. In group 2, hemodilution significantly increased cardiac index, stroke volume index, and dP/dt, and decreased blood pressure, peripheral resistance, and oxygen delivery compared with group 1. Compared with group 2, IV propranolol after hemodilution in group 4 significantly decreased cardiac index, dP/dt, LV-developed pressure, and peak cardiac index, and increased peripheral resistance. Stroke volume index and peak stroke volume index after preload stress remained elevated in group 4, despite the negative inotropic effects of IV propranolol. Oral propranolol in group 6 did not prevent the hemodilution-induced increase in stroke volume index and peak stroke volume index in response to preload stress, although it did decrease cardiac index and dP/dt compared with group 2. Oxygen delivery was reduced in the hemodiluted animals in proportion to the decrease in hemoglobin, regardless of propranolol treatment. It is concluded that reduced myocardial contractility and cardiac performance by nonselective pharmacological beta-adrenoceptor blockade does not interfere with the compensatory increase in stroke volume index after hemodilution. PMID- 1714312 TI - Acute preoperative hemodilution in cardiac surgery: volume replacement with a hypertonic saline-hydroxyethyl starch solution. AB - Preoperative hemodilution (HD) is a recommended practice in cardiac surgery that conserves blood and reduces the complications of homologous blood transfusion. In 45 patients undergoing myocardial revascularization, HD was performed preoperatively. Withdrawn volume (10 mL/kg) was replaced either by a new hypertonic saline (HS) solution prepared in hydroxyethyl starch (HES) (2,400 mOsm/L, HS-HES group, n = 15) or by a standard low molecular weight hydroxyethyl starch solution (6% HES 200/0.5, HES group, n = 15) to maintain baseline PCWP (acute normovolemic hemodilution [ANH]). Fifteen comparable patients without HD served as controls. Significantly less HS-HES (210 +/- 20 mL) than HES 6% (890 +/ 90 mL) was necessary to sustain hemodynamics during HD. Stable cardiocirculatory conditions were obtained even after termination of bypass. Fluid balance during cardiopulmonary bypass as well as in the postoperative period was significantly lower in HS-HES-treated patients. With regard to hemodynamics, CI increased most in the HS-HES group (+36%), whereas systemic vascular resistance was lower in these patients. Right ventricular ejection fraction increased only in HS-HES patients (+15%). However, sodium concentration as well as osmolarity increased after volume replacement with HS-HES, without exceeding normal values. None of the patients suffered from organ failure. Pulmonary gas exchange (PaO2) was less compromised in the HS-HES patients. There were no renal function differences between the groups. In conclusion, HS solution prepared in HES is an attractive alternative for blood substitution in cardiac patients undergoing acute hemodilution for blood conservation. PMID- 1714313 TI - Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth. AB - The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s). PMID- 1714314 TI - An altered constitutive peptide in sym 5 mutants of Pisum sativum L. AB - Mutational analysis of Pisum sativum L. was used to search for constitutive proteins that might function in nodule formation. The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation. Comparison of two-dimensional polyacrylamide gels of in vitro translated root RNA showed a consistent difference in the migrational pattern of one peptide. In the nodulating parental cultivar 'Sparkle', a 66 kDa peptide had a pI of 5.9. In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8. This 66 kDa peptide is found in lateral root, tap root, and shoot. Its expression was independent of rhizobial inoculation, root temperature, or light. PMID- 1714315 TI - A gene coding for a basic pathogenesis-related (PR-like) protein from Zea mays. Molecular cloning and induction by a fungus (Fusarium moniliforme) in germinating maize seeds. AB - Pathogenesis-related proteins (PRs) are plant proteins produced in leaves in response to infection by pathogens including viruses, viroids, fungi and bacteria. Information on the presence and/or expression of PRs in monocotyledonous plants is scare. Here we report the identification of cDNA and genomic clones coding for a basic form of a protein from germinating maize seeds having a high homology with the group of PR-1 from tobacco. A cDNA library enriched in aleurone-specific sequences was prepared from maize seeds two days after germination. One clone was found to contain an open reading frame encoding a protein homologous to PR proteins from tomato (p14) and tobacco (PR-1 group). Sequence analysis of the corresponding genomic clone revealed that it was encoded by a single exon. Besides, DNA blot hybridization indicates that this PR-like protein is encoded by a single-copy gene in maize. The accumulation of its mRNA increases after rehydration of desiccated seeds. Furthermore, a relationship was found between its expression and infection by a natural pathogen of maize, the fungus Fusarium moniliforme. The possible role of this protein as a response mechanism following fungal infection in cereal seeds is discussed. PMID- 1714316 TI - Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding. AB - Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in lambda gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion. Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I-V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1-3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2-6-Tyr-Pro and (Gly)2-6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2-5-Arg repeats. PMID- 1714317 TI - Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Hevea brasiliensis. AB - Hevea brasiliensis is the major producer of natural rubber which is cis-1,4 polyisoprene. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is involved in the biosynthesis of rubber and other plant products. We have used a hamster HMGR cDNA clone as a heterologous hybridization probe to isolate and characterize cDNA and genomic clones of HMGR from H. brasiliensis. Sequence analysis revealed that these clones fall into two different classes, HMGR1 and HMGR2. Comparison of the two classes shows 86% nucleotide sequence homology and 95% amino acid homology. The carboxy-termini of Hevea HMGRs are highly homologous to those of hamster, yeast and Arabidopsis HMGR. The amino-terminus of Hevea HMGR contains two potential membrane-spanning domains as in Arabidopsis HMGR while seven such domains are found in the HMGRs of other organisms. The apparent molecular mass of Hevea HMGR was estimated in western blot analysis to be 59 kDa. Northern blot analysis indicated that the HMGR1 transcript of 2.4 kb is more highly-expressed in laticifer than in leaf. Genomic Southern analysis using 3' end cDNA probes indicates the presence of at least two HMGR genes in Hevea. PMID- 1714319 TI - Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana. AB - A cDNA encoding the NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) of Arabidopsis thaliana has been isolated and sequenced. The cDNA contains the complete reading frame for the precursor of the Pchlide reductase. The deduced amino acid sequence of the Arabidopsis enzyme closely resembles the corresponding sequences of barley and oat. The cDNA has been used as a template for the synthesis of the enzyme protein in Escherichia coli. An antiserum was raised against this enzyme protein and both the antiserum and the cDNA were used as experimental tools to study the effects of light on the Pchlide reductase in A. thaliana. When etiolated seedlings of Arabidopsis were exposed to light the enzyme activity and the concentration of the enzyme protein rapidly declined. Similar light effects have been described previously for other angiosperms. In contrast to most of these species, however, in Arabidopsis only minor changes in Pchlide reductase mRNA content could be observed when etiolated seedlings were exposed to light. PMID- 1714318 TI - Characterization of an alpha-amylase multigene cluster in rice. AB - Rice genomic clones containing eight different alpha-amylase genes have been previously classified into five groups based on DNA hybridization studies and restriction site mapping. This report describes the clustering of three Group 3 genes (RAmy3A, RAmy3B and RAmy3C) within 28 kb of genomic DNA. The genes are separated from each other by about 5 kb and transcribed in the same direction. At the protein level, RAmy3B and RAmy3C are 95% homologous while each is 78% homologous to RAmy3A. All three genes have relatively small introns in the first and third positions. RAmy3A; however, has an additional 409 bp intron in the second intron insertion site. Nucleotide sequence comparisons of the coding and 3' flanking regions suggest that clustering of the RAmy3 genes occurred by gene duplication resulting from unequal crossing-over at repetitive sequences. A comparison of the 5' flanking regions revealed several sequences that may be involved in transcription. Expression of RAmy3B/C first appears in the germinating seed after two days and at a higher level after four days. Quantitative primer extension analysis indicates that RAmy3B and RAmy3C contribute 25% and 75%, respectively, of the transcripts from this cluster at four days of germination. No primer extension band specific to RAmy3A transcripts could be detected at this time point. However, RAmy3A PCR products could be amplified from RNA isolated from embryo-derived callus tissue. PMID- 1714320 TI - A novel proline-rich protein from wheat. AB - A cDNA (WPRP1) encoding a wheat proline-rich protein has been isolated and sequenced. The amino acid composition shows 45% proline, with high levels of methionine, lysine and glutamic acid. The derived 378 residue amino acid sequence has a highly repetitive structure which is unlike those of other proline-rich proteins. The WPRP1 cDNA clone was used to determine the copy number and chromosomal location of the WPRP1 gene by restriction fragment length polymorphism analysis of wheat inbred lines. Although WPRP1 is encoded by a single-copy gene it is also a representative of a larger family of related sequences. RNA gel blot analysis showed that expression of WPRP1 is highest in rapidly growing tissue which together with its amino acid composition suggests a structural role for the encoded protein. PMID- 1714321 TI - Isolation and characterization of a soybean hsp70 gene. AB - Soybean, like many other organisms, responds to an increase in growth temperature by producing a set of new proteins, heat shock proteins. The heat shock proteins have been classified into several categories according to their molecular weight. Data are presented on the isolation, sequence characterization, and expression of a 70 kDa heat shock protein gene from soybean. A cDNA clone was isolated using a Drosophila hsp70 clone as a heterologous probe, and the cDNA was used for isolation of the soybean gene corresponding to the cDNA. The structure of this soybean is very similar to the hsp70 genes from other organisms. It has several sequences in the 5' untranscribed region that are similar to the well characterized heat shock consensus element found in other organisms. These heat shock consensus elements have the expected position relative to the start of transcription. Unlike hsp70-like genes previously isolated from other plants, this gene does not have an intron. This protein shows high amino acid sequence similarity to other hsp70 proteins from such diverse organisms as Drosophila, rat, and Xenopus. This soybean gene is only expressed during heat shock. In addition to the hsp70 gene isolated here, there is evidence for many other hsp70 like genes in soybean. PMID- 1714322 TI - Sequence, identification and characterization of cDNAs encoding two different members of the 18 kDa heat shock family of Zea mays L. AB - Heat-shocked maize seedlings (cv. Oh43) synthesize a characteristic set of heat shock proteins (hsps) which include an 18 kDa family containing at least six major isoelectric variants. A cDNA library was constructed from poly(A)+ RNAs isolated from the radicles of heat-shocked maize seedlings and screened with a DNA fragment from the theoretical open reading frame of a putative Black Mexican Sweet maize hsp18 genomic clone. Two clones, cMHSP18-3 and cMHSP18-9, were isolated, and the RNA transcripts generated from them were translated into proteins which immunoreact with antibodies directed against the maize 18 kDa hsps and exhibit the same electrophoretic characteristics as two different members of the 18 kDa hsp family. Nucleotide sequence analyses of the cDNAs in these clones reveal that their 5' and 3' untranslated regions exhibit 33-34% identity and that their protein encoding regions share 93% identity. The deduced amino acid sequences of these clones show 90% identity, and the apparent molecular masses and isoelectric points of these proteins agree with those established for two different 18 kDa hsps, numbered 3 and 6. This report substantiates that at least two of the 18 kDa hsps in maize are products of different but related genes. Moreover, it establishes that transcripts for these proteins accumulate during heat shock and that both their nucleotide and deduced amino acid sequences share extensive similarities with the class VI small hsps in soybean and with transcripts expressed during meiosis in Lilium. PMID- 1714323 TI - A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower. AB - Cytoplasmic male-sterile lines CMS89 and CMSBaso of sunflower (Helianthus annuus) differ from the fertile lines HA89 and Baso in a mitochondrial DNA sequence in the vicinity of the atpA gene. In addition, the transcriptional pattern of the atpA gene is changed in male-sterile lines compared to fertile ones. Besides one main transcript in the fertile lines, the male-sterile lines additionally show larger transcripts. Investigation of Baso and CMSBaso revealed that the two fertility-restored lines of CMS89 have the same transcripts as CMSBaso or a combination of CMSBaso and CMS89. Comparing the mitochondrial in organello translation products we observed a unique 16 kDa protein, which is expressed in male-sterile lines carrying the H. petiolaris cytoplasm but is not detectable in fertile lines with H. annuus cytoplasm. The 16 kDa protein can also be observed in restored lines but not in H. petiolaris. As the expression of the 16 kDa polypeptide seems to be linked to the interspecific cross between H. petiolaris and H. annuus it may play a role in CMS. By different criteria such as molecular mass, isoelectric point and peptide fingerprinting the alpha subunit of the F1 ATPase of male-sterile and fertile lines is very similar if not identical. PMID- 1714324 TI - Investigation of the mechanisms of monoclonal antibody-induced platelet activation. AB - Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation). PMID- 1714325 TI - Interferon-gamma enhances growth factor-dependent proliferation of clonogenic cells in acute myeloblastic leukemia. AB - Interferon-gamma (IFN-gamma) has been reported to antagonize the stimulatory effect of various conditioned media on the growth of normal hematopoietic progenitor cells and clonogenic blasts from patients with chronic myelogenous leukemia (CML) and acute myeloblastic leukemia (AML). In the present study, using purified recombinant cytokines and homogenous cell populations, we provide evidence for a synergistic or additive effect of IFN-gamma with recombinant human (rhu) hematopoietic growth factors in the stimulation of clonogenic blasts from most AML patients examined. Under conditions of limiting cell concentration, rhuIFN-gamma alone showed little effect on blast proliferation, whereas in conjunction with recombinant human interleukin-3 (rhuIL-3), IFN-gamma significantly enhanced colony formation in 13 of 15 AML cases. Maximal stimulation was obtained at low concentrations of IFN-gamma (2 to 20 pmol/L) in four cases and at higher concentrations (700 to 7,000 pmol/L) in the remainder. IFN-gamma also synergized with recombinant human granulocyte-macrophage colony stimulating factor (rhuGM-CSF) in 9 of 13 cases. Within 1 hour of exposure, IFN gamma induced a twofold to fourfold accumulation of tumor necrosis factor alpha (TNF alpha)-specific transcripts in AML blasts and several AML cell lines that include HL-60 and OCI-AML 1. Further, the synergy between IFN-gamma and IL-3 on AML blasts was partially or completely abrogated by a TNF alpha neutralizing antibody, suggesting that growth enhancement by IFN-gamma may be mediated through TNF alpha production in AML blast culture. Exposure of normal precursors (burst forming unit-erythroid [BFU-E] and colony-forming unit granulocyte-macrophage [CFU-GM]) to IFN-gamma also resulted in significant growth enhancement, suggesting that the proliferative response elicited by IFN-gamma was not limited to AML blasts. Finally, in M07-E, an IL-3-dependent human "megakaryoblastic" cell line, IFN-gamma also significantly enhanced IL-3-supported colony formation, much in the same way as in primary AML blasts. In contrast, IFN-gamma inhibited growth of all CSF-independent leukemic cell lines tested. This inhibition was partially alleviated by anti-TNF alpha antibody. In summary, our data indicate that IFN gamma can enhance or antagonize cell proliferation, depending on the cell type. Further, TNF alpha appears to mediate the biologic effect of IFN-gamma either in growth stimulation or growth inhibition. PMID- 1714326 TI - Anti-retroviral therapy of human immunodeficiency virus infection: current strategies and challenges for the future. PMID- 1714327 TI - Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony-stimulating factor. AB - Fc receptors are important effector molecules of neutrophilic granulocytes (polymorphonuclear neutrophils [PMN]), connecting phagocytic cells and the specific immune response. Neutrophils from healthy donors express the low affinity receptors for IgG Fc gamma RII (CD32) and Fc gamma RIII (CD16), but not the high-affinity receptor Fc gamma RI (CD64). The latter has been found on neutrophils from patients with certain bacterial infections and can be induced in vitro after incubation with interferon-gamma. We show here that neutrophils strongly express Fc gamma RI after in vivo application of recombinant human granulocyte colony-stimulating factor (rhG-CSF). PMN from patients receiving rhG CSF displayed higher cytotoxicity against Daudi lymphoma cells in vitro compared with control patients and with healthy donors. Fab fragments against Fc gamma RII (monoclonal antibody [MoAb] IV.3) inhibited neutrophil-mediated cytotoxicity of healthy donors but not of patients during rhG-CSF therapy. Therefore, expression of Fc receptors by PMN was investigated by flow cytometry and the mean fluorescence intensity (MFI) was compared. After staining with MoAb 32.2 against Fc gamma RL, the median MFI of neutrophils from G-CSF patients (median, 4.78; range, 2.40 to 8.50; n = 5) was significantly higher (P = .002 and P = .001, respectively) than the median MFI of patients not receiving G-CSF (median, 1.23; range, 1.01 to 1.58; n = 6) and the median MFI of healthy donors (median, 1.04; range, 0.67 to 1.12; n = 6). Fc gamma RI disappeared after the discontinuing of the G-CSF injections, but was reinduced during the next treatment cycle with rhG CSF. The high expression of Fc gamma RI during rhG-CSF therapy correlated with enhanced cytotoxicity. In vitro incubation with rhG-CSF also enhances cytotoxicity, but only minor increments in Fc gamma RI expression were observed. Thus, during in vivo application of rhG-CSF neutrophils acquire an additional potent receptor for mediating tumor cell killing in vitro by induction of the high-affinity receptor for IgG (Fc gamma RI, CD64). PMID- 1714328 TI - Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture. AB - We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL 6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G-CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU S number at the expense of stem cell function. PMID- 1714329 TI - The effects on hematopoiesis of recombinant stem cell factor (ligand for c-kit) administered in vivo to mice either alone or in combination with granulocyte colony-stimulating factor. AB - Stem cell factor (SCF) is the ligand for the receptor encoded by the c-kit proto oncogene. Mutations of either c-kit or the SCF gene are responsible for the defects of W and SI mutant mice, which both suffer a macrocytic anemia, the former associated with defective stem cells and the latter with a defective hematopoietic microenvironment. PEGylated recombinant rat SCF was administered to normal or splenectomized mice for up to 21 days. SCF was found to be a modest stimulator of peripheral blood neutrophil numbers in both groups of animals. The peak in neutrophil numbers was higher and occurred earlier in splenectomized mice. Bone marrow and spleen cellularity changed little during treatment but the content of interleukin-3-responsive progenitor cells and spleen colony-forming cells (CFU-S) reached very high levels, particularly in the spleen. Using recombinant human granulocyte colony-stimulating factor (rhG-CSF), we have shown that SCF induces a greater than additive increase in both blood neutrophils and blood-borne CFU-S. This synergy was seen throughout the dose range and may indicate a clinical role for SCF either alone or in augmenting the activity of G CSF upon blood neutrophils and transplantable stem cells. PMID- 1714330 TI - Purification of human marrow progenitor cells and demonstration of the direct action of macrophage colony-stimulating factor on colony-forming unit-macrophage. AB - To facilitate the investigation of the direct interaction between hematopoietic progenitors and colony-stimulating factors, we have developed a method to purify human marrow progenitor cells. Using density centrifugation, negative panning with concanavalin A coated plates, positive selection of CD34-positive cells with immunomagnetic microspheres, overnight adherence to a plastic dish, negative selection with a panel of monoclonal antibodies, and density centrifugation, human marrow progenitor cells were purified from 1.5% to 53.2%, a 42-fold purification, with a 4.8% yield. The purified cells consisted of 38% erythroid, 9% colony forming unit-granulocyte (CFU-G), 29% CFU-macrophage (CFU-M), 12% CFU eosinophil/basophil (CFU-Eo/Ba), and 4% CFU-mix. The purified cells cultured in serum-free fibrin clots with recombinant human macrophage colony-stimulating factor (rM-CSF) for 14 days developed a pure population of CFU-M colonies. An appearance of CFU-M colonies was present after the addition of 1 U/mL of rM-CSF and the maximum stimulation was found at 100 U/mL. When the purified cells were cultured in serum-free medium with rM-CSF in a limiting dilution assay and the percentage of nonresponder wells for CFU-M colonies was plotted against cell concentration, serum-free cultures yielded a straight line through the origin, indicating that CFU-M development did not depend on accessory cells and that rM CSF acted directly on the CFU-M. PMID- 1714331 TI - Indirect photocoagulation of subfoveal choroidal neovascularization in age related macular degeneration. AB - Forty consecutive patients with age-related macular degeneration and a choroidal neovascular membrane in the center of the macula were treated by dye laser photocoagulation. After a mean 24 month follow-up period, 21 patients (52.5%) remained stable or improved in vision. Of 21 patients with initial visual acuity of 20/100 or better, 11 patients (52%) remained 20/100 or better. In the control group only four patients (25%) remained stable or improved, and only two (15%) remained 20/100 or better. The theoretical and clinical advantages of the indirect laser treatment of subfoveal choroidal neovascular membranes in age related macular degeneration are discussed. PMID- 1714332 TI - Tangle-bearing neurons show more extensive dendritic trees than tangle-free neurons in area CA1 of the hippocampus in Alzheimer's disease. AB - To elucidate the pathogenetic significance of neurofibrillary tangles in Alzheimer's disease, the dendritic tree of tangle-bearing and unaffected pyramidal cells of area CA1 of the hippocampus was morphometrically examined. Golgi-stained neurons were assessed which were deimpregnated and counterstained with Congo red to visualize neurofibrillary tangles. The study revealed that tangle-bearing neurons have more extensive apical dendritic trees than tangle free neurons. It is concluded that metabolic processes associated with the formation of neurofibrillary tangles may increase neurotrophic activity on a single cell level and counteract the cellular degeneration process. PMID- 1714333 TI - Axonal transport of two major components of the ubiquitin system: free ubiquitin and ubiquitin carboxyl-terminal hydrolase PGP 9.5. AB - Ubiquitin (Ub), a stress protein thought to target abnormal proteins for degradation, is present in abnormal structures that occur in neuronal perikarya and axons of degenerative diseases including Alzheimer disease. To begin to assess the role of the Ub system in the axon, we studied expression and axonal transport of Ub and other stress proteins, as well as of Ub carboxyl-terminal hydrolase PGP 9.5, in the rat visual system in normal conditions and following heat-shock (HS). In the retina, both the constitutive and inducible forms of HSPs 70 were expressed under normal conditions, while in the superior colliculus the inducible form was detected only following HS. Ub, PGP 9.5 and HSPs 70 were transported in the axon exclusively with the slow component b (SCb), known to carry cytoskeletal and cytoplasmic proteins. The exceedingly long time needed for stress proteins to reach distant axonal locales at the rate of SCb (approximately 3 mm/day) makes it unlikely that they could contribute significantly to the stress response at those sites. PMID- 1714334 TI - Synaptic connections of a low-threshold mechanoreceptive primary neuron within the trigeminal subnucleus oralis. AB - The central axon of a primary afferent neuron that responded to indentation of the glabrous skin of the lower lip in a slowly adapting fashion was intra axonally injected with horseradish peroxidase. The labeled terminal within the subnucleus oralis was examined electron microscopically. The labeled ending had a pale axoplasm and contained clear spherical synaptic vesicles. The labeled ending formed a synaptic triad with a dendrite and an unlabeled axonal ending with pleomorphic vesicles (a mixture of oval, flattened and dense core vesicles). The labeled primary ending was presynaptic only to the dendrite, while the unlabeled ending was presynaptic to both the dendrite and the labeled primary ending. PMID- 1714335 TI - An ultrastructural study of neurotensin-like immunoreactive terminals in the mediodorsal thalamic nucleus of the rat. AB - Neurotensin-like immunoreactive (NTir) axon terminals in the mediodorsal nucleus of the thalamus (MD) in the adult rat were demonstrated by electron microscopic immunohistochemistry. Most NTir terminals were large (greater than 2 microns in diameter) with round synaptic vesicles and asymmetrical synaptic contacts although smaller (less than 1.5 microns in diameter) axon terminals were also labeled. Both types of terminals were found in the medial and central parts of MD with the greatest density in the medial part. These NTir boutons have similar ultrastructural features as anterogradely labeled terminals from the piriform cortex and the preoptic area, which have previously been identified as sources of NTir axons in MD. A few NTir boutons were also found in the medial part of MD with pleomorphic vesicles and symmetrical synaptic contacts. PMID- 1714336 TI - Selective decrease in extracellular DOPAC concentrations in rat striatum following in vivo dialysis with low concentrations of MPP+. AB - Using the technique of in vivo dialysis, 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was applied to the rat striatum and the effects of this treatment on the efflux of striatal dopamine (DA) and metabolites were monitored. The inclusion of low concentrations of MPP+ (1 and 10 microM) in the dialysis solution caused a progressive decrease in the efflux of dihydroxyphenylacetic acid (DOPAC), the major deamination product of DA, while homovanillic acid (HVA) and 5 hydroxyindoleacetic acid (5-HIAA) remained unchanged. Unlike the effects of dialysis with millimolar concentrations of MPP+, a large increase in the efflux of striatal DA was not observed. The effect of dialysis with 1 microM MPP+ was blocked if 1 microM GBR 12909, a specific DA reuptake blocker, was included in the dialysis fluid, suggesting uptake of MPP+ into striatal DA terminals mediated this effect. PMID- 1714337 TI - Distribution of substance P, GnRH, Met-enkephalin in the central nervous system of the pig. AB - The distribution of substance P (SP), gonadotropin releasing hormone (GnRH) and Met-enkephalin in the brain and spinal cord of the domestic pig is described for the first time. The levels of SP, GnRH and Met-enkephalin were measured by specific radioimmunoassays in various regions of the brain and spinal cord of the pig. Substance P and Met-enkephalin are widely distributed within the central nervous system of the pig. High levels of SP were found in the preoptic area (POA), suprachiasmatic area (SCA), medial basal hypothalamus (MBH) and brain stem while moderate amounts of SP were found in olfactory bulb (OB). High levels of Met-enkephalin were found in POA, SCA and MBH, and moderate levels of Met enkephalin in OB and brain stem. Both SP and Met-enkephalin levels were higher in the dorsal spinal cord in comparison with the levels of these peptides in the ventral spinal cord. This finding is in agreement with the predominant role played by these neural systems in primary afferent mediation of nociceptive impulses. The POA and SCA contained only low levels of GnRH while the MBH contained high levels of GnRH. Finally, some differences in the quantitative distribution of these peptides in the pig and rat are discussed. PMID- 1714338 TI - Anatomical distribution of substance P-immunoreactive neurons in human brainstem during the first postnatal year. AB - The localization of substance P (SP)-immunoreactive structures in the infant brainstem was investigated by immunohistochemistry using the peroxidase antiperoxidase technique. SP-immunoreactive structures are widely distributed throughout the brainstem region. SP-immunoreactive cell bodies are prominent in the superior colliculis, the central grey, the nucleus tractus solitarius and the reticular formation. A high density of SP-immunoreactive fibers is found in the nucleus tractus solitarius, the trigeminal nucleus and the dorsal horn of the spinal cord. Large SP-immunoreactive fibers are seen in the substantia nigra. In the present study, we also investigated the development of substance P immunoreactive fibers in the infant brainstem during the first postnatal year. We note a qualitative increase in the density of SP-immunoreactivity in some brainstem regions such as colliculus superior and substantia nigra with respect to age. PMID- 1714339 TI - Parathion (O,O-dimethyl-O-p-nitrophenyl phosphorothioate) induces pineal melatonin synthesis at night. AB - The effects of parathion on male rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels at the end of light period (2000 h) and at night (2300 h and 0100 h) were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5 HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Parathion was administered intragastrically at total doses (over 6 days) of either 6.5 or 13 mg/kg. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light:dark cycles of 14:10 with light off at 2100 h. Pineal NAT activity was increased at 0100 h following parathion administration at both doses, but HIOMT activity was unaffected. Pineal and serum melatonin levels were increased at night (2300 h and 0100 h) after the 13 mg/kg dose of parathion while the lower dose increased pineal melatonin only at 0100 h. Also, both doses decreased 5-HTP at 2000 h while the lower dose increased it at 2300; 5-HT was significantly decreased at 2300 h and 5-HIAA levels were lower but only significantly so for the 13 mg/kg dose at 2000 h. The results indicate that parathion has significant effects on pineal melatonin synthesis by mechanisms which remain unknown. PMID- 1714340 TI - DNA content and estrogen receptors in primary carcinoma of the breast. AB - DNA content and estrogen-receptor status were studied in 54 consecutive patients with primary breast carcinoma. Estrogen-receptor determinations were performed by immunohistochemical assay on frozen sections with a monoclonal antibody against the estrogen-receptor molecule and by biochemical analysis with a dextran-coated charcoal method. Nuclear DNA content was measured by flow cytometry performed on formalin-fixed, paraffin-embedded sections. Seventy-two percent of tumours were positive for estrogen receptors by immunohistochemical assay and 67% by biochemical assay. Comparison of the qualitative results of immunohistochemical and biochemical estrogen-receptor determinations revealed a strong correlation between the two assays, with agreement in 90% of the cases (p less than 0.001). Regression analysis showed only a weak relationship between the quantitative results of the two assays. DNA analysis was performed in 51 cases, and 54% demonstrated aneuploid stemlines by flow cytometry. An association was demonstrated between aneuploidy and low levels of estrogen receptor. The association was highly significant with the immunohistochemical assay but not with the biochemical assay. The authors' results suggest that immunohistologic determinations of estrogen receptor status may better reflect the biologic features of the tumour cells. However, improved standardization in reporting the results is necessary if the test is to have widespread use. PMID- 1714341 TI - Improved therapeutic efficacy of a monoclonal antibody radioiodinated using N succinimidyl-3-(tri-n-butylstannyl)benzoate. AB - Improvements in efficacy of radioimmunotherapy will require increased tumor uptake relative to normal tissue. We previously demonstrated that labeling the IgG2b glioma-reactive antitenascin monoclonal antibody 81C6 with 131I using N succinimidyl-3-(tri-n-butylstannyl)benzoate (ATE) increased tumor uptake and tumor-to-normal tissue ratios and decreased deiodination compared with labeling using Iodo-Gen. The present study was conducted to determine whether 131I 81C6 labeled using ATE (81C6 ATE) would demonstrate a therapeutic advantage over 131I 81C6 labeled using Iodo-Gen (81C6 IOD) in treating s.c. D-54 MG human glioma xenografts in athymic mice. The subclass IgG2b MAb 45.6 labeled with 131I using ATE (45.6 ATE) was used as a control. Animals were injected with saline or 500 microCi of 45.6 ATE (23 microCi/microgram), 81C6 ATE (26 microCi/microgram), or 81C6 IOD (24 microCi/microgram). With approximately 150 mm3 initial tumor volumes, growth delay for 81C6 ATE was significantly better by Wilcoxon rank sum analysis than saline (P = 0.0006 to 0.003), 45.6 ATE (P = 0.0006 to 0.002), and 81C6 IOD (P = 0.0008 to 0.007). Biodistribution data from similarly injected animals gave estimated radiation doses to tumor of 7723, 5200, and 1667 rad for 81C6 ATE, 81C6 IOD, and 45.6 ATE, respectively. In addition, 81C6 ATE administered at this dosage to animals with 50% larger initial tumors also improved tumor growth delay in comparison with 81C6 IOD given to animals with standard-size tumors. A similar experiment was conducted at 1000 microCi and, although radiation toxicity was noted in all labeled groups, two animals in the 81C6 ATE group had tumor regression for more than 240 days, and the other groups had no regressors. We conclude that the use of the ATE method may significantly improve the therapeutic efficacy of radioiodinated monoclonal antibodies. PMID- 1714343 TI - Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line. AB - Several tumorigenic (benign and malignant) clones have been raised from the human epidermal cell line HaCaT after transfection with the c-Ha-ras oncogene (val 12) (P. Boukamp et al., Cancer Res., 50: 2840-2847, 1990). In culture, these HaCaT ras clones expressed epidermal differentiation markers, such as keratins K1 and 10, at high density or upon depletion of retinoic acid. Accordingly, as HaCaT cells, the clones formed well-differentiated stratified epithelia synthesizing K1 and 10 in surface transplants, while simple and internal epithelial keratins seen in culture were suppressed (as upon retinoic acid depletion in vitro). In transplants of HaCaT cells, in contrast to those of normal keratinocytes, K1 appeared prematurely already in basal cells, while K10 localized rather normally in the suprabasal position. Keratins 1 and 10 were also synthesized in transplants of HaCaT-ras clones (again K1 preceding K10), but both generally shifted toward upper layers. This was particularly evident in thicker transplants of malignant clones. Staining for both keratins persisted "suprabasally" in invasive tissue masses, and this corresponded to their marked expression in solid carcinomas (after s.c. injection), seen by immunofluorescence and two-dimensional gel electrophoresis. Thus, notwithstanding some variations, differentiation potential was not significantly reduced in these clones disregarding levels of ras oncogene expression and malignant properties. PMID- 1714342 TI - Interaction of N,N',N''-triethylenethiophosphoramide and N,N',N'' triethylenephosphoramide with cellular DNA. AB - The antineoplastic agents N,N',N''-triethylenethiophosphoramide (thioTEPA) and N,N',N''-triethylenephosphoramide (TEPA) were studied for their interaction with the DNA of L1210 cells in the presence and absence of rat hepatic microsomes and NADPH. Alkaline elution was used to study 3 types of DNA lesions. When L1210 cells were incubated with thioTEPA alone, or with thioTEPA in the presence of microsomes and NADPH, no single-strand breaks were detected. However, incubation of L1210 cells for 2 h with thioTEPA, at concentrations greater than or equal to 100 microM, caused a dose-dependent increase in interstrand cross-linking that reached a maximum by 2 h after drug exposure. In the presence of rat hepatic microsomes and NADPH, this cross-linking was eliminated, but a different DNA lesion, alkali-labile sites, was produced. These alkali-labile sites were partially reparable with maximum repair achieved by 2 h after removal of drug. ThioTEPA was greater than 85% consumed by the microsomal incubation conditions employed, and TEPA was the only product of the microsomal metabolism of thioTEPA. Alkaline elution studies of L1210 cells that had been incubated with TEPA, alone or in the presence of microsomes and NADPH, demonstrated an elution pattern identical to that produced by thioTEPA in the presence of microsomes and NADPH. Lymphoblastoid cell lines derived from patients with Fanconi's anemia were far more sensitive to thioTEPA and mechlorethamine hydrochloride than were lymphoblasts derived from normal humans, but this hypersensitivity was not noted with TEPA or bleomycin. This is consistent with the known hypersensitivity of cells from patients with Fanconi's anemia to agents that produce interstrand cross-links and with the alkaline elution studies described above. In contrast, lymphoblastoid cell lines derived from patients with ataxia telangiectasia were no more sensitive to thioTEPA than were lymphoblasts derived from normal humans but were far more sensitive to bleomycin. One of these cell lines proved hypersensitive to TEPA, whereas the other was no more sensitive to TEPA than were lymphoblasts from normal humans. Our data imply that thioTEPA produces interstrand cross-links but that TEPA, the primary metabolite of thioTEPA, produces DNA lesions that are alkali labile. PMID- 1714344 TI - The hypersensitivity of the Chinese hamster ovary variant BL-10 to bleomycin killing is due to a lack of glutathione S-transferase-alpha activity. AB - As a means to understand the fundamental mechanisms of bleomycin cell killing, we previously isolated 19 bleomycin-sensitive mutants which represent at least six genetically distinct complementation groups (T.D. Stamato, B. Peters, P. Patil, N. Denko, R. Weinstein, and A. Giaccia. Cancer Res., 47: 1588-1592, 1987). One class of mutants represented by the cell line BL-10 displays only hypersensitivity to killing by bleomycin in both acute (16 h) and chronic treatments but no sensitivity to killing by other DNA-damaging agents. Complementation studies between this mutant and human fibroblasts suggested that the human gene which corrects the defect of BL-10 rested on human chromosome 6. It has been reported that the gene for human glutathione S-transferase (GST) alpha also resides on chromosome 6. Measurements of selenium-independent peroxidase (alpha-GST + glutathione peroxidase) activity in wild-type Chinese hamster ovary (CHO) cells, using cumene hydrogen peroxide as a substrate, gave a value of 112 nmol of glutathione oxidized/min/mg protein compared with 88.1 nmol of glutathione oxidized/min/mg protein for BL-10. Measurement of the selenium dependent peroxidase activity, using H2O2 as a substrate, resulted in 65.9 nmol of reduced glutathione oxidized/min/mg protein in CHO and 81.5 nmol of reduced glutathione oxidized/min/mg protein for BL-10. In other words, BL-10 cells did not exhibit a difference in their ability to metabolize both substrates in contrast to CHO cells. This indicates that BL-10 possesses little alpha-GST activity. Transfection of BL-10 cells with a mammalian expression vector containing the alpha-GST gene increases the survival of BL-10 to bleomycin and does not increase the bleomycin resistance of two other bleomycin mutants which lie in different genetic complementation groups. These data strongly implicate a role for alpha-GST in the resistance of cells to bleomycin. PMID- 1714345 TI - The improved effects of specific active immunotherapy on a rat fibrosarcoma by antitumor drugs. AB - We have tried to find out if the combination of a xenogenized tumor cell vaccine and antitumor drugs is able to induce a synergistic increase in the antitumor therapeutic effect. The degree of increase in the LTD50 (50% lethal tumor dose) is expressed numerically, as a quantitative index designed to compare degrees of transplantation resistance to tumor cell challenge. A LTD50 was achieved by an intradermal (i.d.) immunization with xenogenized tumor cells when challenged with tumor cells implanted intraperitoneally 2 weeks after the immunization: this LTD50 value was 527,000 times higher than that of the non-immunized group. When we combined this type of immunization with appropriate doses of bleomycin (BLM) or cyclophosphamide (CY), which are able to augment antitumor immunity, the LTD50 was 723,000-1,190,000 times higher than that of the non-immunized group. This increase in the LTD50 is definitely higher than that achieved by a single immunization with irradiated tumor cells (x 33,000) and combined with either BLM (x 93,000) or CY (x 140,000). We also studied the therapeutic effect of a tumor cell vaccine combined with antitumor drugs BLM or CY in tumor-bearing rats. We observed a synergistic effect caused by BLM or CY after i.d. immunization with xenogenized tumor cells: this showed a significant increase when compared with the therapeutic effects obtained by chemotherapy alone (P less than 0.01). Nevertheless, there was no evidence that the above antitumor effects is superior to the effect achieved by irradiated tumor cells. PMID- 1714346 TI - Electrophysiological studies of the gill ganglion in Aplysia californica. AB - 1. An electrophysiological analysis was made of gill ganglion neurons in Aplysia californica. 2. Gill ganglion neurons behave similarly to neurons in the abdominal ganglion (the central nervous systems; CNS) that are involved with gill withdrawal behaviors. 3. Some gill ganglion neurons are motor neurons much like those in the CNS. 4. Neurons in the gill ganglion are electronically and dye coupled. In addition, they receive common chemical synaptic inputs from the Int II network in the CNS. 5. Tactile stimulation of the gill or siphon evokes synaptic activity in gill ganglion neurons whether or not the CNS is present. 6. Pedal nerve stimulation results in synaptic activity in gill ganglion neurons and facilitates synaptic input evoked by tactile stimulation of the gill or siphon. 7. Antibody staining reveals serotonin-like fibers in the branchial nerve close to the gill ganglion but no cell bodies in the ganglion. 8. The gill ganglion may play a role in the mediation of adaptive gill reflex behaviors. It may be one of the loci where the CNS and peripheral nervous system (PNS) interact and form an integrated circuit to mediate gill withdrawal reflex (GWR) behaviors. PMID- 1714347 TI - Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes. AB - The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN. PMID- 1714348 TI - Erythrocyte membranes alteration in a shear stress measured by fluorescence anisotropy. AB - An experimental setup has been designed to allow fluorescence anisotropy measurements on labeled cell membranes under shear stress. An important change is observed when increasing the shear stress and varying the experimental parameters indicates that a decrease in membrane cohesion leads to a subsequent increase in the membrane alteration under shear stress. A model has been developed that shows, in agreement with experiment, that the effect observed is mainly the result of the alteration of the membrane, elongation, and orientation with respect to the fluid flow, which can be estimated. PMID- 1714349 TI - The relative merits of direct morphometry of reconstructions of whole cells, and statistical morphometry by stereology of random sections of cells. AB - Stereology, or the derivation of quantitative, three-dimensional (3-D) data about cells by statistical analysis of the structures of random sections, is widely used in cytology and pathology. However, there are situations where this approach is inadequate, and only an analysis of a homogeneous population of whole cells will give the required results. This involved 3-D reconstruction from physical or optical sections, or tomography or photogrammetry of whole-cell mounts. Use of stereo views of individual sections or projections adds considerably to the information available for both contouring and reconstruction. Recent image processing advances in clinical radiography have shown, for the first time, that rapid, high-resolution digitization and contrast enhancement enable nearly all structural details to be routinely extracted from the micrographs and adequately portrayed. Three-D whole-cell reconstructions provide the digital data for many kinds of morphometric measurements on both whole cells and their individual organelles and membranes. Rapid fixation or freezing allows improved quantitative structure/function correlations of organelles with disturbances in cell metabolism or gene expression. PMID- 1714350 TI - Localization of calcium and microfilament changes in mechanically stressed cells. AB - We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows: 1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium. 2. All micropipet aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips. 3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation. 4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA. It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood. PMID- 1714351 TI - Study of propidium iodide binding to DNA in intact cells by flow cytometry. AB - We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow cytometric technique for probing DNA structure in intact cells. PMID- 1714352 TI - The effect of free fatty acids on spectrin organization in lymphocytes. AB - Previous studies have shown that cis unsaturated free fatty acids (uFFAs) are able to cause alterations in the normal distribution pattern of certain cytoskeletal proteins in lymphocytes, including tubulin, actin, alpha-actinin, and myosin. The cytoskeletal protein spectrin naturally possesses a marked heterogeneity of distribution among resting T and B lymphocytes isolated from all murine lymphoid organs. In some cells, spectrin is observed in a ring-like staining pattern at the periphery of the cell, reflecting a likely association with the cell membrane; in other cells, spectrin is found within the cytoplasm as a large single aggregate or in several smaller aggregates. Addition of uFFA to freshly isolated murine lymphocytes causes disruption in the latter pattern of spectrin organization. Following short-term incubation (15 min) of tissue-derived lymphocytes (from spleen, thymus, and lymph node) and 1 microgram/mL uFFA (oleic [18:1 cis], linoleic [18:2 cis, cis], arachidonic [20:4], or elaidic [18:1 trans] acid) there is a loss of cytoplasmic aggregates of spectrin and a concomitant increase in cells in which spectrin is diffusely distributed. This effect is not seen when two saturated FFAs (sFFAs) were used. When using DO11.10 cells, a T cell hybridoma in which nearly all cells constitutively express a cytoplasmic aggregate of spectrin, a similar effect was observed, but greater concentrations (10-20 micrograms/mL) of FFA were needed to obtain the same effect. Addition of calcium to the incubation buffer substantially blocks spectrin reorganization. In several disease states, serum levels of FFA are observed to be excessively high; our data support the hypothesis that cytoskeletal reorganization in lymphocytes may be related to the altered immune function frequently observed in these conditions. PMID- 1714353 TI - Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. AB - In light of the possible role peripheral nerves may play in bone metabolism, the morphology of calcitonin gene-related peptide (CGRP)-, vasoactive intestinal peptide (VIP)-, substance P (SP)-, neuropeptide Y (NPY)-, and dopamine-beta hydroxylase (D beta H)-immunoreactive nerve fibers was examined in whole-mount preparations of periosteum of membranous bones (calvaria, mandible) and long bones (tibia) from the rat. Periosteum from animals treated to remove selectively either the sympathetic or fine-caliber primary afferent nerves was also examined to determine the origin of the nerve fibers. We found a consistent and often dense innervation of the periosteum. The innervation patterns of the calvaria and mandible were similar, with networks of nerves spread across the surface of the bone. Nerves in the tibial periosteum were oriented in the longitudinal axis and were more numerous at the epiphyses than in the mid-shaft region. CGRP immunoreactive fibers were widely and densely distributed. The presence of populations of CGRP-immunoreactive fibers of differing calibers and perivascular arrangements suggests that such nerves in bone tissues may serve different functions. SP-immunoreactivity was present in a fine network of varicose fibers in the superficial layers of the periosteum. CGRP- and SP-immunoreactive nerve fibers were dramatically reduced in periosteum of capsaicin-treated animals as compared to controls, indicating the sensory origin of these nerves. VIP immunoreactive nerve fibers were distributed in the periosteum of mandible and calvaria as small networks and individual fine varicose fibers. In tibial periosteum, larger networks of these fibers were visible. VIP-immunoreactive nerve fibers in the periosteum were associated with both vascular and nonvascular elements within the layers of cells closest to the bone, suggesting that VIP may serve more than one function in periosteal tissues. NPY-immunoreactive fibers were largely confined to vascular elements; occasional fibers were observed among the bone-lining cells. D beta H-immunoreactivity was associated only with blood vessels. VIP-, NPY-, and D beta H-immunoreactivities were dramatically reduced in the periosteum of guanethidine-treated animals, indicating the sympathetic origin of these nerves. PMID- 1714354 TI - Association between histamine-containing mast cells and sensory nerves in the skin and airways of control and capsaicin-treated pigs. AB - The association between mast cells (visualized by routine staining and immunohistochemistry for histamine) and capsaicin-sensitive nerves (containing calcitonin gene-related peptide (CGRP) and substance P (SP] was studied in the pig. In the 1-ethyl-3(3-diethylaminopropyl)carbodiimide (EDCDI)-fixed skin tissue, histamine-containing mast cells and CGRP/SP-positive nerves were found in close association around blood vessels. In the EDCDI-fixed airway mucosa, only single histamine-containing mast cells were detected. However, many alcian blue positive mast cells were found, sometimes close to the airway epithelium where CGRP SP-containing nerve were abundant. The CGRP/SP-containing nerve fibres were absent 2 days after systemic capsaicin pretreatment, but no changes in the number and distribution of tissue mast cells, granulocytes or lymphocytes, or the number of blood leukocytes were detected. Local injection of allergen, histamine and capsaicin into the skin of pigs actively sensitized with ascaris antigen caused a rapid light red-flare (vasodilation) reaction. Allergen and histamine, but not capsaicin, also produced plasma protein extravasation. In contrast to the absent flare, the protein extravasation response still occurred in capsaicin-treated pigs. The sensitivity to ascaris antigen was mediated by an IgE-like antibody. We conclude that a functional and morphological relationship exists between histamine-containing mast cells and capsaicin-sensitive sensory nerves in the pig skin. Mast cells and sensory nerves are also found in the airway mucosa and appear to be closely associated with the epithelium. PMID- 1714355 TI - RNA-mediated transfer of the gene coxII from the mitochondrion to the nucleus during flowering plant evolution. AB - The gene coxII, normally present in the mitochondrion, was functionally transferred to the nucleus during flowering plant evolution. coxII transfer is estimated to have occurred between 60 and 200 million years ago, whereas loss of coxII from the mitochondrion occurred much more recently, being restricted to a single genus of legumes. Most legumes have coxII in both the nucleus and the mitochondrion; however, no evidence is found for simultaneous coxII expression in both compartments. The nuclear coxII sequence more closely resembles edited mitochondrial coxII transcripts than the genes encoding these RNAs. Hence, gene transfer appears to have involved reverse transcription of an edited RNA intermediate. The nuclear gene contains an intron at the junction of the transit peptide sequence and the mature protein-coding sequence; exon shuffling may have played a role in assembling a functional coxII gene in the nucleus. PMID- 1714356 TI - Distribution of mitochondrial r1-type introns and the associated open reading frame in the yeast genus Kluyveromyces. AB - We have sequenced the intron in the large subunit ribosomal RNA gene from the mitochondrion of Kluyveromyces lactis. It is a typical group I intron but, unlike the corresponding intron (r1) in Saccharomyces cerevisiae, it does not contain an open reading frame. This intron is widespread in the genus Kluyveromyces although intron-less strains were also found in some species of this genus. Sequences homologous to the open reading frame of the S. cerevisiae ribosomal intron were detected in some strains of K. waltii, K. thermotolerans and K. africanus. PMID- 1714357 TI - Splicing of the Petunia cytochrome oxidase subunit II intron. AB - A comparative analysis of the plant intron-containing mitochondrial cytochrome oxidase subunit II (coxII) genes provides an indication that four conserved sequence motifs, present in exon 1 (intron-binding sequences; IBS), and complementary motifs (exon-binding sequences; EBS), present in domain I of the group II intron, may be involved in splicing of the intron. Two of these potential IBS motifs (IBS1 and IBS2) have been previously discussed. Two further potential IBS motifs (IBSa and IBSb), which occur twice within exon 1, could be involved in specification of the 5' splice site and of a 5' cryptic splice site. Nuclease-protection experiments and DNA sequence analysis of a spliced coxII cDNA have confirmed the predicted positions of the petunia coxII 5' and 3' splice sites. Evidence for the occurrence of splicing in vivo at the putative 5' cryptic splice site in petunia is provided by the detection of a nuclease-protected fragment corresponding to the size which is predicted if splicing at the proposed cryptic splice site occurs. The existence and location of a cryptic splice site, upstream of the normal coxII 5' splice site, is consistent with the proposed derivation of the cytoplasmic male sterility (CMS)-associated pcf gene from an abnormally spliced coxII transcript (Pruitt and Hanson 1989). PMID- 1714358 TI - Differential expression of the psbB and psbH genes encoding the 47 kDa chlorophyll a-protein and the 10 kDa phosphoprotein of photosystem II during chloroplast development in wheat. AB - The nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 kDa chlorophyll a-binding protein of photosystem II has been determined. The gene encodes a polypeptide of 508 amino acid residues which is predicted to contain six hydrophobic membrane-spanning regions. The psbB gene is located 562 bp upstream of the psbH gene for the 10 kDa phosphoprotein of photosystem II. A small open reading frame of 38 codons is located between psbB and psbH, and on the opposite strand the psbN gene, encoding a photosystem II polypeptide of 43 amino acid residues, is located between orf38 and psbH. S1 nuclease mapping indicated that the 5' ends of transcripts were located 371 and 183 bp upstream of the psbB translation initiation codon. Predominant transcripts of 2.1 kb and 1.8 kb for psbB and 0.4 kb for psbH were present in RNA isolated from etiolated and greening wheat seedlings. Immunodecoration of Western blots indicated that the 47 kDa polypeptide was absent, or present in very low amounts, in dark-grown tissue and accumulated on greening, whereas the 10 kDa polypeptide was present in similar amounts in both dark-grown and greening seedlings. The 10 kDa polypeptide was phosphorylated in vitro by incubating wheat etioplast membranes with [gamma 32P] ATP. PMID- 1714359 TI - Isopropylidine maltoheptosyl fructofuranoside, doubly blocked substrate for determination of endoamylase activity. AB - We synthesized o-(4,6-o-isopropylidene-alpha-D-glucopyranosyl)-(1----4)- [o-alpha D-glucopyranosyl-(1----4])5-o-alpha-D-glucopyranosyl-(1----2)- alpha-D fructofuranoside (IPG7F) and developed an assay for determining the activity of amylase in human serum and urine by using this substrate. Glucoamylase, alpha glucosidase, and mannitol dehydrogenase are used as coupling enzymes. The coupled reactions are monitored by continuously measuring the oxidation rate of NADH. In this procedure, various substances in the test specimens do not interfere with the detection of amylase activity. Exactly one molecule of NADH is oxidized by one attack of amylase on the substrate, although four products can be produced in the reaction. The within-assay coefficient of variation (CV) ranged from 1.0% to 4.1% and the between-assay CV ranged from 2.6% to 5.3%. The results of our new assay correlate well with those of the amylase assay involving p-nitrophenol maltoheptaoside as substrate (r = 0.978) and with those of the amylase assay involving maltopentaose (r = 0.987). PMID- 1714360 TI - Automated measurement of amylase isoenzymes by a double kinetic assay with "blocked" beta-2-chloro-4-nitrophenyl maltopentaoside as substrate and with wheat germ inhibitor. AB - We evaluated the enzymic mechanism by which 3-keto butylidene-beta-2-chloro-4 nitrophenyl maltopentaoside (3KB-G5-CNP) serves as a substrate for serum pancreatic (p-) and salivary (s-) amylases. In aliquots of the reaction mixture, three kinds of beta-2-chloro-4-nitrophenyl oligosaccharides (glucose, maltoside, and maltotrioside) were separated from the substrate by high-performance liquid chromatography. Both isoenzymes behaved nearly identically and produced almost the same products. We automated a double kinetic procedure for determining total (t-) and p-amylase with use of a selective inhibitor from wheat germ in a single channel on the Hitachi 7050 analyzer. Within- and between-run CVs were, respectively, 0.5% and 1.7% for t-amylase (240 U/L), and 0.7% and 2.3% for p amylase (230 U/L). The test results varied linearly with concentrations up to approximately 2000 U/L for t- and p-amylase activities. p/s ratios varied from 0.2 to 5.0. Results correlated well with those obtained by the monoclonal inhibition method (r = 0.992). PMID- 1714361 TI - Development and application of monoclonal antibodies to human cardiac myoglobin in a rapid fluorescence immunoassay. AB - Myoglobin (Mb) is considered a useful marker for early detection of myocardial infarction and for monitoring cardiac reperfusion after thrombolytic therapy. We developed eight monoclonal antibodies to human cardiac Mb, characterized their epitopic reactivity, and determined which combinations of the antibodies are useful in two-site immunoassays. We configured two of the monoclonal antibodies in a one-step, two-site particle concentration fluorescence immunoassay (PCFIA) for measurement of Mb. The PCFIA has rapid kinetics of reaction, being complete in 15 min, and has a linear analytical range of 20-675 micrograms/L for human Mb. Although the PCFIA has a high-dose "hook" effect, this is of no analytical importance at concentrations of Mb less than or equal to 148,000 micrograms/L. The assay is not subject to interference from icterus (bilirubin less than or equal to 360 mg/L), has no cross-reaction with hemoglobin (less than or equal to 42 g/L), and may be performed with either plasma or serum in approximately 1 h. The intra- and interassay imprecisions (CV) of the method are less than 10% for concentrations of Mb within the normal range and less than 4% at higher concentrations. A comparison of the PCFIA with a commercial radioimmunoassay showed that results of the two assays correlate well (PCFIA = 0.88 x RIA + 18, r = 0.990, n = 171). PMID- 1714362 TI - Interactions of the salivary and gastrointestinal systems. I. The role of saliva in digestion. AB - Considerable evidence now demonstrates that saliva and its components have multiple functions in the GI tract. Saliva aids in bolus formation; it lubricates, protects and cleanses the pharyngeal and esophageal mucosa. Salivary bicarbonate buffers esophageal acid in common reflux. Normal salivary flow decreases the duration of acid contact with esophageal mucosa, an important factor in the development of GERD. If salivary flow is depressed or if the esophagosalivary reflex is lost, a patient may be predisposed to develop GERD. Salivary EGF stimulates GI mucosal proliferation via a direct lumenal effect in the esophagus and stomach. The salivary enzymes LL and salivary amylase initiate fat and starch digestion. They are particularly significant in patients with pancreatic insufficiency such as neonates and patients with cystic fibrosis. PMID- 1714363 TI - The relative importance of transcriptional and post transcriptional regulation of Drosophila chorion gene expression during oogenesis. AB - To determine the relative roles of transcriptional and post-transcriptional events in establishing the temporal pattern of chorion gene expression in Drosophila, we have examined chorion gene transcription, RNA accumulation, and protein synthesis in follicles of selected pre-, early-, and late-choriogenic stages. Chorion gene transcription was assayed in follicle cell nuclei by nuclear run-on reactions. For the s15, s16, s18, s36, and s38 chorion genes, the periods of intense transcription are as predicted from the dynamics of RNA accumulation and protein synthesis, indicating that these genes are primarily regulated at the transcriptional level. In contrast, gene s19 appears subject to post transcriptional control at stage 14, when transcription rates are substantially higher than predicted from the observed RNA levels. Transcription of regions between the clustered and tandemly oriented chorion genes was also examined. In contrast to many RNA polymerase II transcribed genes, for the s18 and s36 chorion genes run-on transcription appears to terminate within about 100 base pairs downstream of the polyadenylation sites, corroborating previous reports based on electron microscopy of s36 [Osheim et al., EMBO J 5:3591-3596, 1986]. PMID- 1714364 TI - Histochemical determination of muscle fiber types in locomotor muscles of anuran amphibians. AB - 1. A histochemical study using myosin ATPase, succinate dehydrogenase and alpha glycerophosphate dehydrogenase reactions and a morphometric analysis with image analyser, was carried out in sartorius and gastrocnemius muscles of two anuran species, Rana perezi and Bufo calamita, that show different locomotor activities. 2. Four types of muscle fiber were found. There were interspecific variations in their proportions, with a predominance of oxidative muscle fibers in Bufo calamita. 3. These results agree with those obtained previously for the metabolic profile of several tissues from both species and point to a clear metabolic basis for the differences in locomotor activities between these two species. PMID- 1714365 TI - Organ and species specific differences in cytoskeletal protein profiles of cultured microvascular endothelial cells. AB - 1. Using two-dimensional gel electrophoresis and immunoblotting techniques we systematically document the structural diversity of cytoskeletal proteins in tight and leaky cultured microvascular endothelial cells (MEC). Bovine pulmonary and eel rete mirabile MEC primarily express cytokeratins 8 and 19. Cytokeratins 8 and 18 were found to be prominent in rat pulmonary MEC. Bovine retinal MEC contained cytokeratins 8, 18 and 19. Bovine adrenal MEC contain vimentin as their sole intermediate filament protein. 2. Four principal actin isoforms were resolved in micro/macrovascular endothelial cells as well as in vascular smooth muscle cells. Retinal pericytes expressed three principal actin isoforms. 3. These results indicate that MEC are diverse, highly differentiated cells displaying a large repertoire of cytoskeletal protein profiles suited for specific tissue functions. PMID- 1714366 TI - alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus. AB - 1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2 macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2 macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin. PMID- 1714367 TI - The role of alpha 2-macroglobulin in furunculosis: a comparison of rainbow trout and brook trout. AB - 1. The molecular basis for the high survival rate of rainbow trout, Oncorhynchus mykiss, infected with furunculosis was investigated. 2. Alpha 2-macroglobulin (alpha 2M), a major serum protease inhibitor, was partially purified from rainbow trout and brook trout, Salvelinus fontinalis, sera; the latter species shows marked disease susceptibility. 3. It is shown that a 10-fold species-based difference in alpha 2M inhibitory activity exists against a furunculosis associated bacterial protease. 4. A possible basis for the observed disparity is discussed. 5. Results suggest that the high mol. wt form of teleost (trout) albumin is a dimer composed of two 85,000 subunits. PMID- 1714368 TI - Serum amylase of non-parotid and non-pancreatic origin increases on feeding in rats and may originate from the liver. AB - 1. In order to evaluate possible non-salivary contributions to the content of salivary-type amylase in the circulation, parotid glands--the only salivary source of amylase in rats--have been totally removed and the effects on serum amylase have been assessed, after fasting and at different times after feeding. 2. Despite the parotidectomy the resting level of salivary-type amylase remained the same and an increase was still found to occur on feeding. 3. Isoelectric focusing has identified additional isoforms of amylase in serum and liver distinct from those occurring in parotid saliva. 4. The liver therefore may be contributing to the fasting levels of serum amylase and to the increases that occur on feeding, in rats. PMID- 1714369 TI - [Changes of monamine metabolites in the cerebral cortices and hippocampi following prolonged administration of diphenylhydantoin in the developing mice]. AB - It was previously reported by the authors that long-term diphenyl-hydantoin (DPH) administration impaired the learning behavior in the developing mice. The aim of the present study was to find out whether prolonged administration of DPH would affect the levels of monamine metabolites in the cerebral cortices and hippocampi of the developing mice. Each of the experimental animals was treated intraperitoneally with a dose of DPH of 25mg/kg q.d. for 50 days. The monamine metabolites were measured with high performance liquid chromatography. The results showed that chronic administration of DPH caused a significant elevation of the concentrations of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid (5 HIAA) and dihydroxy-phenyl acetic acid (DOPAC) in the cerebral cortices and hippocampi, whereas it did not raise the concentrations of dopamine (DA), homovanillic acid (HVA) and norepinephrine (NE) in these two brain regions. This suggested that DPH mainly enhanced the function of 5-HT in the brains. It was postulated that the elevation of the concentrations of 5-HT and 5-HIAA in the cerebral cortices and hippocampi might be responsible for the adverse effect of DPH on the learning behavior in the developing mice. PMID- 1714370 TI - Uptake and elimination of lindane by Lymnaea palustris (Mollusca: Gastropoda): a pharmacokinetic approach. AB - The uptake and elimination of lindane by adult Lymnaea palustris (Muller) were studied using a static contamination system. First-order one- and two-compartment models were used to quantitatively describe these phenomena. The accumulation of residues was triphasic and the observed steady-state bioconcentration factor lay between 36.8 and 56.4 but did not significantly depend on the initial lindane concentration (6, 60, and 600 micrograms liter-1). Accumulation was inferior to that observed for other aquatic organisms and this was attributed to the relatively low lipid content of L. palustris tissues (mean of 0.81 +/- 0.17% of fresh weight). The transfer of snails to lindane-free water after 72 hr of exposure was followed by a biphasic elimination of residues with a half-life of 0.7 hr in the central compartment and of 130.2 hr in the peripheral compartment. Additional experiments showed that the residues enter the snails through the foot and are afterward stocked within the visceral mass which contains approximately three times more lipids than the foot (1.03 +/- 0.13% of fresh weight vs 0.37 +/- 0.03%). PMID- 1714371 TI - Evaluation of the timing to reduce the initial dose of antithyroid drugs in patients with Graves' disease. AB - The present study was undertaken to evaluate whether the normalization of the serum TSH level in a supersensitive assay during the initial treatment with antithyroid drugs (ATD) is a useful indicator for the reduction of the initial dose of ATD in 50 patients with hyperthyroidism due to Graves' disease. The initial dose of ATD was continued until the achievement of the euthyroid state, and was then reduced either before the serum TSH level was in the normal range in 9 of 29 patients treated with methimazole (MMI) (group MMI-1) and 8 of 21 treated with propylthiouracil (PTU) (PTU-1), or after the serum TSH level was in/above the normal range in 20 of 29 treated with MMI (MMI-2) and 13 of 21 treated with PTU (PTU-2). Although there were no significant differences in age, sex, thyroid function, prevalence of autoantibodies, goiter size, duration of the disease or the initial and modified doses of ATD, the mean durations of the administration of the initial dose of ATD in MMI-2 and PTU-2 were significantly longer than those in MMI-1 and PTU-1, respectively. As a result, 4 (44%) in group MMI-1, 20 (100%) in MMI-2, 2 (25%) in PTU-1 and 7 (54%) in PTU-2 developed low free T4 levels, and 1 (11%) in MMI-1, 15 (75%) in MMI-2 and 3 (23%) in PTU-2 developed low free T3 levels. Serum TSH levels increased over the normal range in 3 (33%) in MMI-1, 18 (90%) in MMI-2 and 5 (39%) in PTU-2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714372 TI - Acquired aphasia, dementia, and behavior disorder with epilepsy and continuous spike and waves during sleep in a child. AB - Severe persistent neuropsychological disorders sometimes develop in the course of a focal epilepsy of unknown origin in previously normal children. Very frequent bilateral focal or generalized discharges are often noted on the sleep EEG records of these patients with no evidence of clinical seizures. The relation between these paroxysms and the observed deterioration remains unclear. We report a child with a partial complex epilepsy and severe disturbances of language, cognition, and behavior acquired in the early years of development who was followed for 15 years. A correlation between the evolution of the striking EEG abnormalities during sleep and the neuropsychological disorders could be established retrospectively. The observed sequence of onset and recovery of the aphasia, the dementia, and the "psychotic" behavior makes a direct causal relation between the deficits quite unlikely. Rather it suggests an association of independent symptoms with a specific language disorder becoming manifest in the course of the evolution. This child shows many of the main characteristics of the syndromes of "acquired aphasia with convulsive disorder" (Landau-Kleffner syndrome) and "epilepsy with continuous spike waves during sleep." Both syndromes describe probably different facets of a similar underlying, still unexplained cerebral dysfunction. PMID- 1714373 TI - The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding. AB - Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells. PMID- 1714374 TI - Characterization of the integrin alpha 8 subunit: a new integrin beta 1 associated subunit, which is prominently expressed on axons and on cells in contact with basal laminae in chick embryos. AB - In this article, we describe the primary structure, the biochemical characterization and the tissue distribution of a novel integrin alpha subunit, named alpha 8. This subunit associates with the integrin beta 1 subunit to form alpha 8 beta 1 heterodimers. By sequence analysis, alpha 8 is more closely related to the alpha 5 and alpha v subunits than to other characterized integrin alpha subunits, but is clearly distinct from each of these. The alpha 8 subunit is expressed at moderate levels in several epithelial cells where its localization adjacent to basal laminae suggests that alpha 8-containing heterodimers interact with at least one extracellular matrix constituent. In embryos, the highest levels of alpha 8 protein expression are seen in the nervous system where alpha 8 is strongly expressed by several classes of projection neurons. The alpha 8 subunit is concentrated in axon tracts, including major projections in the spinal cord, optic system and auditory system. This tissue specific expression and cellular localization suggest that alpha 8-containing integrin receptors might promote axon outgrowth in the embryonic nervous system. PMID- 1714375 TI - Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development. AB - Mutations at the dominant white spotting (W) and Steel (Sl) loci in mouse exert deleterious effects on three migratory cell lineages (primordial germ cells, melanocytes and hematopoietic stem cells) resulting in loss of pigmentation, reduced fertility and anemia. The W locus encodes the c-kit protein tyrosine kinase (TK) receptor. More recently, the Sl locus has been shown to encode a ligand for c-kit, which is variously known as mast cell growth factor (MGF), stem cell growth factor and c-kit ligand. Here we report an in situ hybridization analysis comparing the expression profiles of MGF and c-kit transcripts during mouse embryogenesis. The data are consistent with the c-kit receptor-ligand complex providing a homing mechanism during stem cell migration in early development and in stem cell proliferation, differentiation, or survival in late development. In the nervous system, an unexpected and complex pattern of expression is uncovered that suggests involvement of the W and Sl gene products in the organization of the neural tube and brain. PMID- 1714376 TI - A novel target cell for c-fos-induced oncogenesis: development of chondrogenic tumours in embryonic stem cell chimeras. AB - Embryonic stem (ES) cells were used to investigate the target cell specificity and consequences of c-fos when expressed ectopically during embryonic development. Chimeric mice generated with different ES cell clones selected for high exogenous c-fos expression were not affected during embryonic development; however, a high frequency of cartilage tumours developed as early as 3-4 weeks of age apparently independent of the extent of chimerism. The tumours originated from cartilagenous tissues and contained many chondrocytes. Expression of exogenous c-fos RNA and Fos protein was observed during development but was highest in tumour tissues, predominantly in differentiating chondrocytes. A number of primary and clonal tumour-derived cell lines were established which expressed high levels of c-fos, c-jun as well as the cartilage-specific gene type II collagen and which gave rise to cartilage tumours in vivo, some of which also contained bone. Interestingly, the levels of c-Fos and c-Jun appeared to be coordinately regulated in the cell lines as well as in chimeric tissues. Thus, we demonstrate that chondrogenic cells and earlier progenitors are specially transformed by Fos/Jun and therefore represent a novel mesenchymal target cell for c-fos overexpression. PMID- 1714377 TI - Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase. AB - Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c kit expression constructs abolished (W37) or reduced (W41) the Steel factor induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins. PMID- 1714378 TI - Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs. AB - The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective. PMID- 1714379 TI - Interference and synergism of glucocorticoid receptor and octamer factors. AB - We have analysed the interplay of glucocorticoid receptor (GR) and the lymphocyte specific factor Oct-2A with transient co-transfection assays. Our data confirm our previously described observation that GR and the apparently unrelated factors belonging to the Octamer family can synergize when permitted to bind in cis. However, when GR binding sites are not present in the reporter genes, we observe that the action of the cloned factor Oct-2A expressed in HeLa cells is strongly inhibited in the presence of active GR molecules. We can demonstrate that this GR mediated inhibition of Oct-2A action is neither due to competitive binding to DNA target sites nor to a reduction of DNA binding competent Oct-2A in the transfected cells. We observe that the phenomenon is not reciprocal, since co expression of Oct-2A does not inhibit GR-dependent transcription activation. Furthermore, we provide evidence that the observed GR-Oct-2A interference may be dependent on the type of cell line hosting the co-transfected molecules. We consider it likely that the GR-mediated inhibition is due to the exhaustion of some rate-limiting co-activators. PMID- 1714380 TI - Characterization of the mouse junD promoter--high basal level activity due to an octamer motif. AB - The product of the junD gene belongs to the Jun/Fos family of nuclear DNA binding transcription factors. This family regulates the expression of TPA responsive genes by binding to the TPA responsive element (TRE). Unlike its counterparts c jun and junB, junD expression is hardly inducible by growth factors and phorbol esters. In fact, junD is constitutively expressed at high levels in a wide variety of cells. To unravel the molecular mechanisms underlying constitutive junD expression, we have cloned and characterized the mouse junD promoter. We show that the high constitutive expression is caused by multiple cis-acting elements in its promoter, including an SP1 binding site, an octamer motif, a CAAT box, a Zif268 binding site and a TRE-like sequence. The octamer motif is the major determinant of junD promoter activity, while somewhat smaller contributions are made by the TRE and Zif268 binding site. The SP1 and CAAT box are shown to be of minor importance. The junD TRE is in its behavior indistinguishable from previously identified TREs. However, the junD promoter is not TPA inducible due to the presence of the octamer motif. PMID- 1714382 TI - Analysis of CpG methylation and genomic footprinting at the tyrosine aminotransferase gene: DNA methylation alone is not sufficient to prevent protein binding in vivo. AB - Specific DNA sequences from several DNase I hypersensitive sites located upstream of the tyrosine aminotransferase (TAT) gene are bound by ubiquitous nuclear factors in vitro. Genomic footprinting has shown, however, that proteins are excluded from their potential binding sites in cells where the gene is inactive and that the absence of in vivo footprints is correlated with CpG methylation and altered chromatin structures at these sites. In vitro, interactions of proteins with sequences of the TAT gene, including binding of the transcription factor CREB to the cAMP-responsive element (CRE), are prevented by a methylated CpG dinucleotide in the respective binding sites, suggesting that methylation of DNA might be sufficient to exclude proteins from their sites in vivo. To test directly whether the absence of in vivo footprints is the result of DNA methylation, we treated two different cell lines with 5-azacytidine to demethylate CpG dinucleotides. While genomic sequencing confirmed demethylation at two widely separated regions upstream of the TAT promoter, no footprints appeared in these cell lines, even though proteins capable of binding these sites in vitro were present in the nuclei. Thus, the simple model whereby protein exclusion in vivo is caused solely by DNA methylation is not appropriate in this case. The nucleosomal organization of the potential binding sites suggests that chromatin structure is a dominant determinant in maintaining the inactive state of these sites. PMID- 1714381 TI - Enhancer activation by a single type of transcription factor shows cell type dependence. AB - Promoter and enhancer elements contain multiple binding sites for ubiquitous and cell type-specific transcription factors which stimulate transcription synergistically. Here we show that cell type-dependent enhancer activity can be achieved by mechanisms other than interactions between cell type-specific transcription factors and DNA. The ability of the SV40 enhancer and the E2 transactivator from bovine papillomavirus (BPV-1) to stimulate transcription from a reporter gene with different minimal promoters was tested in four cell lines. In lymphoid BJA-B cells all minimal promoters that synergized with the SV40 enhancer did so also with the E2-dependent enhancer. In sharp contrast to these results, the E2 enhancer stimulated transcription only from a subset of promoters in fibroblasts and in epithelial cells. The results suggest that lymphoid cells, unlike fibroblasts and epithelial cells, contain auxiliary factor(s) which are necessary for the activation of certain promoters by the E2 enhancer and infer that the specificity of enhancers also is regulated at a co-activator level. PMID- 1714383 TI - The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene in. AB - Two chalcone synthase genes in maize have been cloned and molecularly characterized to be the C2 and the Whp (white pollen) locus. The two genes have highly homologous exon sequences but differ considerably in sequences 5' upstream and 3' downstream of the coding region, as well as in their introns. Northern and Western experiments of chalcone synthase expression in various tissues and in different genotypes indicated that C2 and Whp are differently regulated. The expression of Whp in maize aleurone is dependent on the presence of the recessive allele of the gene intensifier (in). The regulatory effect of in on Whp expression is not detectable at the transcriptional level, but seems to take place during translation. PMID- 1714384 TI - Cloning of a yeast U1 snRNP 70K protein homologue: functional conservation of an RNA-binding domain between humans and yeast. AB - We have cloned and sequenced a gene encoding a yeast homologue of the U1 snRNP 70K protein. The gene, SNP1, encodes a protein which has 30% amino acid identity with the human 70K protein and has a predicted molecular weight of 34 kDa. The yeast and human sequences are more closely related to each other than to other (non-U1) RNA-binding proteins, but diverge considerably in their C-terminal portions. In particular, SNP1 lacks the charged carboxy terminus of the human 70K protein. A yeast strain, a alpha 115, was constructed in which one allele of the SNP1 gene contained a 554 bp deletion. Tetrad analysis of a alpha 115 showed that the SNP1 gene is essential for the viability of yeast cells. The complete human 70K gene did not complement snp1, but the lethal snp1 mutation was rescued by plasmids bearing a chimera in which over half the yeast gene was replaced with the homologous region of the human 70K gene, including the RNA-binding domain. These results suggest that SNP1 encodes a functional homologue of the U1 snRNP 70K protein. PMID- 1714385 TI - An intact Box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. AB - The mammalian U3 snRNP is one member of a recently described family of nucleolar snRNPs which also includes U8, U13, U14, X and Y. All of these snRNPs are immunoprecipitable by anti-fibrillarin autoantibodies, suggesting the existence of a common binding site for the 34 kDa fibrillarin (Fb) protein. Two short nucleotide sequences, called Boxes C and D, present in each of these RNAs are the most likely sites for fibrillarin binding. We have developed a HeLa in vitro assembly system for binding of fibrillarin to human U3 snRNA. Reconstitution of the input RNA is specific in our assay since four of the other nucleolar small RNAs (U8, U13, X and Y) which have Boxes C and D become immunoprecipitable by anti-fibrillarin whereas two RNAs which lack these sequences (5S and 5.8S) do not. Deletion analyses of the U3 snRNA demonstrate that the presence of Box C but not Box D is required for fibrillarin binding. Moreover, seven single or double site-specific mutations in the U3 Box C abolish binding. The role of the Box C fibrillarin interaction in the biogenesis of the Fb snRNPs is discussed. PMID- 1714386 TI - A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. AB - The onset of S-phase and M-phase in both Schizosaccharomyces pombe and Saccharomyces cerevisiae requires the function of the cdc2/CDC28 gene product, p34, a serine-threonine protein kinase. A human homolog, p34cdc2, was identified by functional complementation of the S.pombe cdc2 mutation (Lee and Nurse, 1987). Using a human cDNA expression library to search for suppressors of cdc28 mutations in S. cerevisiae, we have identified a second functional p34 homolog, CDK2 cell division kinase). This gene is expressed as a 2.1 kb transcript encoding a polypeptide of 298 amino acids. This protein retains nearly all of the amino acids highly conserved among previously identified p34 homologs from other species, but is considerably divergent from all previous p34cdc2 homologs, approximately 65% identity. This gene encodes the human homolog of the Xenopus Eg1 gene, sharing 89% amino acid identity, and defines a second sub-family of CDC2 homologs. A second chromosomal mutation which arose spontaneously was required to allow complementation of the cdc28-4 mutation by CDK2. This mutation blocked the ability of this strain to mate. These results suggest that the machinery controlling the human cell cycle is more complex than that for fission and budding yeast. PMID- 1714387 TI - Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. AB - MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization. PMID- 1714388 TI - Purification of tobacco nuclear proteins binding to a CACGTG motif of the chalcone synthase promoter by DNA affinity chromatography. AB - The activity of various light-regulated and developmentally regulated plant gene promoters critically depends upon the presence of a conserved sequence with a central CACGTG motif. Using band-shift assays, we have identified nuclear factor(s) from Nicotiana tabacum, termed CG-1, that specifically recognize(s) this transcriptional element in the ultraviolet-light-regulated Antirrhinum majus chalcone synthase promoter. CG-1 activity is constitutively expressed in tobacco seedlings grown in the absence of ultraviolet light as well as in seedlings induced for chalcone synthase gene expression by ultraviolet light irradiation. CG-1 activity has also been detected in flower tissue. DNA-protein cross-linking studies identified three polypeptides with apparent molecular masses of 20, 32 and 42 kDa binding to the CACGTG motif. Proteins interacting with the CACGTG motif were purified from N. tabacum seedlings using differential sequence specific DNA affinity chromatography employing wild-type and mutated CG-1-binding sites. Denaturing polyacrylamide gel electrophoresis revealed major polypeptides of approximately 20, 30 and 40 kDa which are highly enriched in the affinity purified fractions binding specifically to the CACGTG motif. PMID- 1714389 TI - Comparative study of the structure/function relationship of wild-type and structurally modified maltopentaose-producing amylase. AB - Amylase A-180, which is secreted by a new alkaliphilic organism, isolate 163-26, consists of a single type of polypeptide chain of 186.5 kDa and hydrolyses starch by exo-attack releasing malto-pentaose as preferential product. The structure/function relationship of this unusual starch-degrading enzyme was analysed by introducing 3' deletions into the structural gene. It was found that removal of up to a 110-kDa portion from the C-terminus leaving 563 N-terminal amino acids still led to the formation of a fully active enzyme. The part of the structural gene coding for these 563 N-terminal amino acids was fused with the signal peptide-encoding segment of the cyclodextrin glucanotransferase gene from Klebsiella oxytoca and was cloned into an expression vector. The resulting truncated A-180 derivative, A-180/21, was efficiently transported through the cytoplasmic membrane and released into the medium by an Escherichia coli strain which 'leaks' periplasmatic components. A-180/21 was purified and its catalytic properties, i.e. specific activity and product specificity, proved to be identical to those of the wild-type enzyme; however, in contrast to the wild-type enzyme, it was unable to bind to raw starch and it displayed an altered temperature and pH dependence of activity. PMID- 1714390 TI - Localization on the mitochondrial F1 ATPase alpha subunit of an epitope masked in the membrane-bound enzyme using a monoclonal antibody and synthetic peptides. AB - The epitope of the monoclonal antibody 20D6 was localized by N-terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1 alpha subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven-amino-acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the alpha subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti-F1 antisera showed that this region is highly immunodominant. In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the alpha subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane. PMID- 1714391 TI - Alternative complement pathway-mediated myeloid cell cytotoxicity: repertoire of membrane factors participating in regulation of C3 deposition and cytolysis. AB - Most human nucleated cells and cell lines possess C3 step regulators, decay accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) and an inhibitor of membrane attack complex (MAC) formation (p18; CD59). Unless DAF and MCP were simultaneously blocked by their antibodies, Mg(2+)-EGTA-human serum treatment did not induce C3 deposition on most nucleated cells. Furthermore, less than 20% lysis occurred even after the block of all the three factors. In contrast, three myeloid cell lines, U-937, HL-60 and p39, were found to exhibit unusual C3 deposition or cytolysis. U-937 possessed DAF and MCP but lacked p18, and about 50% was lysed by treatment with anti-DAF and anti-MCP followed by Mg(2+)-EGTA-serum, which caused C3, C5 and C8 deposition. Anti-DAF evoked similar but less complement (C) deposition and cytolysis while anti-MCP alone did not, although it enhanced the anti-DAF-mediated C deposition and cytolysis. Thus, once the C3 step is overcome, U-937 is attacked by the late components leading to cytolysis because of the absence of p18. On the other hand, HL60 allowed the deposition of C3 by blocking of either DAF or MCP followed by the Mg(2+)-EGTA serum treatment. C5, C8 and C9 were subsequently deposited but resulted in no lysis. Lysis of 60% was attained by the additional blocking of p18. Thus, HL60 is poorly protected by C3 and C9 step regulation. Strikingly, extensive C3 deposition occurs on p39 without any antibody treatment, suggestive of the presence of unique alternative pathway activators. However, little cytolysis was induced on p39 even by blocking of all three inhibitors with antibodies. These results suggest that in activation of the alternative pathway on myeloid cells, C3 step is controlled by the inhibitors and alternative pathway activators, and C mediated cytolysis is blocked by p18 and additional regulatory mechanisms or factors which assist in protection of nucleated host cells from MAC attack. Susceptibility to homologous C of these cell lines, therefore, reflects relatively low potency of C regulation on their membrane. PMID- 1714392 TI - Comparative studies of decay-accelerating factor and HLA-DR within the CD8 brightly positive population. AB - Decay-accelerating factor (DAF) is a membrane protein that inhibits C3 convertase activity of autologous complement at the cell surface. We found that DAF+ cells and DAF- (DAFlow, if any) cells are clearly separated from each other among CD8 brightly positive (CD8bright) cells. Using three-color fluorescence flow cytometry, we found that whereas the CD8bright DAF- population express HLA-DR (class II major histocompatibility complex antigens, and an activated T cell marker), the CD8bright DAF+ population does not. Therefore, among the CD8bright T cells, DAF and HLA-DR are mutually exclusive. In addition, the CD8bright DAF+ population proliferates in the presence of recombinant interleukin 2 (rIL2) while the CD8bright DAF- population does not. After a 4-day cultivation of peripheral blood lymphocytes in the presence of rIL2, expression of HLA-DR increased in the CD8bright DAF- population and expression of IL 2Rp55 (alpha chain, the receptor for IL2, and a marker of T cell activation) occurred in the CD8bright DAF+ population. Furthermore, C3 deposition occurred in the CD8bright HLA-DR+ population which lacks DAF when lymphocytes, that had been cultured for 3 days in the presence of rIL2, were incubated with fresh autologous serum. This result suggests that the absence of DAF on CD8bright HLA-DR+ T cells might play a role in permitting complement reaction on the cells in vivo and may be related to the regulation of T cell activation. PMID- 1714393 TI - The sgp-60 molecule is linked to the plasma membrane via a glycosylphosphatidylinositol anchor. AB - We have recently identified a novel murine glycoprotein termed sgp-60, which is expressed on the cell surface of T and B lymphocytes. Because of the profound modulatory effects of sgp-60 on activation through the T cell receptor/CD3 complex, we have examined the membrane attachment domain of the molecule. sgp-60 is not expressed on the surface of variants of a T-T hybridoma cell line that are defective in glycosylphosphatidylinositol (GPI) anchor biosynthesis. In wild-type but not in mutant cells, sgp-60 can be labeled with palmitic acid. Furthermore, the molecule can be removed from the cell surface of both T and B lymphocytes by enzymatic digestion with a phosphatidylinositol-specific phospholipase C. We conclude that the sgp-60 molecule is linked to the plasma membrane via a GPI anchor. PMID- 1714394 TI - Inhibition of interleukin 4-promoted CD23 production in human B lymphocytes by transforming growth factor-beta, interferons or anti-CD19 antibody is overriden on engaging CD40. AB - Interleukin 4 (IL 4) is an essential component in the sequence of events directing IgE synthesis in uncommitted B lymphocytes. An early consequence of IL 4's interaction with the B cell is the induction of CD23, a low-affinity receptor for IgE (Fc epsilon RII). The present study was designed to explore the detailed regulation of this event. First, we report that transforming growth factor-beta (TGF-beta) is a potent inhibitor of IL 4-promoted CD23 production in human B lymphocytes. The level of inhibition achieved with TGF-beta was greater than that obtained with interferons, or with a monoclonal antibody (mAb) to CD19. Next, we identified three signals, each of which was capable of selectively counteracting the inhibitors of IL 4-promoted CD23 production: (a) the engagement of surface CD40 antigen with mAb was found to override the influence of all the inhibitors of CD23 expression; (b) mAb to surface IgM overcame the inhibitory actions of TGF beta and interferons but not that of CD19 ligation; (c) ligation of surface CD72 counteracted the inhibition mediated by TGF-beta but not that generated by interferons or anti-CD19 antibody. Inhibition of the IL 4 signal appeared to be selective for the pathway leading to CD23 induction: none of the inhibitors profoundly altered IL 4's ability to enhance surface IgM expression. The study has ramifications for the understanding of events leading to the promotion of IgE synthesis and consolidates the notion of a central role for CD40 in B cell regulation. PMID- 1714395 TI - Ca2+ channel agonists enhance thyrotropin-releasing hormone-induced inositol phosphates and prolactin secretion. AB - The dihydropyridine Ca2+ channel activator BAY K 8644 (1 microM) stimulated basal prolactin secretion from perifused primary cultures of anterior pituitary cells and potentiated the stimulation of prolactin secretion by 1 microM thyrotropin releasing hormone (TRH) 5-fold over 30 min. This potentiation was mimicked by other dihydropyridine agonists CGP 28392 and (+)-SDZ 202-791 and by (-)-BAY K 8644 (1 microM), but not by (+)-BAY K 8644. The Ca2+ channel antagonist nimodipine, at a concentration sufficient to block BAY K 8644-stimulated 45Ca2+ uptake in GH4C1 anterior pituitary tumor cells, decreased basal prolactin secretion and blocked the enhancement of basal and TRH-stimulated secretion by BAY K 8644. These results suggest that dihydropyridine agonists potentiate TRH induced secretion through interaction with known stereospecific sites on Ca2+ channels. In GH4C1 cells, BAY K 8644 alone did not affect inositol polyphosphate accumulation, but potentiated TRH-stimulated accumulation of inositol 1,3,4 trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Accumulation of the Ca(2+) mobilizing isomer inositol 1,4,5-trisphosphate was not potentiated, suggesting that potentiation of TRH-stimulated hormone secretion by BAY K 8644 does not result from synergistic stimulation of phospholipase C, but may correlate with enhanced inositol trisphosphate-3-kinase activity. PMID- 1714396 TI - Effects of phosphodiesterase inhibitors on vasoactive intestinal peptide-induced relaxation of isolated guinea-pig trachea. AB - The relaxant effect of vasoactive intestinal peptide (VIP) was investigated in isolated guinea-pig trachea in the presence of the phosphodiesterase (PDE) inhibitors, papaverine and 3-isobutyl-1-methylxanthine (IBMX), and the results were compared to those obtained with the cyclic AMP-dependent bronchodilators, isoproterenol and prostaglandin E2 (PGE2). The relaxant effect of VIP was greater when the magnitude of the leukotriene D4 (LTD4)-induced contraction was smaller. A similar effect was also observed for the relaxation induced by isoproterenol but not by PGE2. In the presence of papaverine (1 microM) and IBMX (3 microM), which reduced the 30 nM LTD4-induced contraction to the same extent, the relaxant effect of VIP was not changed, whereas the relaxant effects of isoproterenol and PGE2 were significantly potentiated. The potentiating effect of PDE inhibitors was also observed for the relaxation induced by the adenylate cyclase activator, forskolin, but not for the relaxation induced by the guanylate cyclase activator, sodium nitroprusside. These results suggest that the relaxation induced by VIP is different from that induced by cyclic AMP-dependent bronchodilator in the guinea pig trachea. PMID- 1714397 TI - Photoreceptor degeneration and loss of immunoreactive GABA in the Abyssinian cat retina. AB - GABA (gamma-amino butyric acid) and its synthesizing enzyme, GAD (glutamate decarboxylase; EC 4.1.1.15) were localized in the retina of Abyssinian cats homozygous for a recessively inherited retinal degenerative disorder which in several respects is similar to the human disease, retinitis pigmentosa. Clinically normal mongrel cats and heterozygous Abyssinian cats were studied for comparison. The GABA and GAD immunoreactive neurons of the heterozygous or young homozygous (clinically unaffected animals) had the same distribution and morphology as normal mongrel European type cats. The neuronal GABA immunoreactivity in both the inner and outer parts of the retina gradually disappeared in the course of the disease, with little or no loss of GAD immunoreactive neurons. Early in the disease, the changes were most severe in patches in the mid periphery of the eye and then spread both centrally and peripherally. Loss of photoreceptors was a prerequisite for the loss of GABA immunoreactivity. The observations show that retinal changes are not limited to the photoreceptors. The GABA loss is not likely to be due to a loss of neurons, because of the persistence of GAD immunoreactive neurons. PMID- 1714398 TI - The loss of fluorescein, fluorescein glucuronide and fluorescein isothiocyanate dextran from the vitreous by the anterior and retinal pathways. AB - The pathways by which fluorescein (F), fluorescein glucuronide (FG) and fluorescein dextran (FD) leave the vitreous body of the rabbit were examined by measuring the concentration distribution of the injected fluorophores in sections of the frozen eyes. The contours of F, as already known, show that it leaves the vitreous predominantly across the retinal surface. Mathematical analysis of the concentration gradient leads to an average outward permeability coefficient of 1.4 x 10(-3) cm min-1 for the retinal layers. The contours of FG and FD show that they leave predominantly by diffusion into the posterior chamber, encountering only a minor barrier at the anterior hyaloid membrane. The anterior contours indicate that there can be no substantial posteriorly directed fluid flow through the vitreous; if it occurs its velocity across the retinal surface must be less than 2 x 10(-5) cm min-1. The contours of FD near the posterior pole of the retina suggest that such a flow may be taking place. Some time after the systemic administration of F, an analysis of the rate of loss of fluorescence from the vitreous body shows that this corresponds to the movement of FG out through the anterior chamber. Its value bears little relationship to the condition of the blood-vitreal barrier. PMID- 1714399 TI - Gamma-aminobutyric acid (GABA) immunocytochemistry of laser coagulations in the rabbit retina. AB - Rabbit retinas were treated with laser coagulations of low intensity, using five different wavelengths. After coagulation (1, 4 and 28 days), the retinas were stained for GABA, using a direct biotin-avidin-horseradish peroxidase immunocytochemical method. These time intervals were chosen with regard to the existing knowledge of retinal repair. The staining for GABA was increased in the inner plexiform layer at the centre of the lesions at days 1 and 4 after coagulation. At the margins of the lesions, cells in the ganglion cell layer showed strong staining and cells in the amacrine cell layer showed cuffing, independent of the wavelength used. On day 28, a regression of the hyperstaining was noted. Some possible pathophysiological mechanisms are discussed. PMID- 1714400 TI - Initiation in DNA synthesis by colony-stimulating factors in subsets of human CD34+ marrow cells. AB - Initiation of DNA synthesis by recombinant colony-stimulating factors (CSFs) was assessed in normal human marrow blast cells isolated by expression of CD34 antigen (tritiated thymidine incorporation). Continuous exposure to CSF was required. A mild increase in DNA synthesis was initiated by granulocyte CSF (G CSF; greater than or equal to 1 ng/ml), to approximately 1.5 times control levels. A greater increase was initiated by granulocyte-macrophage CSF (GM-CSF), with a threshold of approximately 0.1 ng/ml and a plateau increment 2.5 times control levels. CD34+ cells were stimulated by interleukin 3 (IL-3) over a wide concentration range: two times control at 0.1/ml, three times control at 1 ng/ml, and four times control at 10 ng/ml. Overlap between responding populations was analyzed. G-CSF plus GM-CSF induced DNA synthesis greater than GM-CSF alone and supported the growth of much larger granulocyte-monocyte colonies. At saturating IL-3 concentrations, neither G-CSF nor GM-CSF induced additional DNA synthesis; at lower concentrations of IL-3, however, GM-CSF recruited additional cells into DNA synthesis. Using CD10 and CD19 antibodies to separate B-lineage cells, the CD34+ cells responding to CSF were observed to be in the non-B-lineage subset. Therefore 1) the response of CD34+ cell subsets CSFs is IL-3 greater than GM-CSF greater than G-CSF, and the IL-3-responsive population is heterogeneous for dose requirement; 2) a CD34+ subpopulation responding to concurrent G-CSF and GM-CSF includes increased proliferative potential cells; 3) IL-3-responsive cells include GM-CSF- and G-CSF-responsive cells, but cells responding to lower IL-3 concentration do not respond to GM-CSF; and 4) B-cell precursors do not respond to GM-CSF or IL-3 in this assay. PMID- 1714401 TI - Effect of recombinant human granulocyte colony-stimulating factor on hematopoiesis in normal cats. AB - The objective of this study was to determine how recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects hematopoiesis in normal cats. Recombinant human G-CSF was given at 3.0, 5.0, and 10.0 micrograms/kg to two cats each s.c. twice daily for 21 days. This resulted in significant (p less than 0.01) elevations of peripheral blood neutrophils from 3.0- to 9.2-fold above pretreatment levels and significantly (p less than 0.02) above levels of nontreated control cats (n = 4). A statistically significant dose-related response was not seen at these dosages in any parameter evaluated. The period of maximum neutrophilia occurred between days 10 and 14 of rhG-CSF treatment, with maximum neutrophil counts ranging from 20,370 cells/microliters to 61,400 cells/microliters (normal is less than 12,500). Lymphocytosis (greater than 7000 lymphocytes/microliters) and monocytosis (greater than 850 monocytes/microliters) were observed in 50% of the cats receiving rhG-CSF during the period of maximal neutrophil stimulation. Monocyte counts in treated cats were significantly (p less than 0.01) elevated over those of treatment controls on days 12-17. Lymphocyte numbers in rhG-CSF-treated cats were significantly elevated (p less than 0.05) over pretreatment controls on days 12 and 14 of rhG-CSF treatment. No significant changes were observed in reticulocyte counts, platelet counts, or hematocrit levels. By day 19, neutrophil levels had dropped significantly (p less than 0.01) from the maximum neutrophil levels, with one cat attaining a normal blood neutrophil count by day 21 of rhG-CSF treatment. Marrow aspirates revealed an overall increase in marrow cellularity through day 14 of treatment in rhG-CSF treated cats, with increased myeloid:erythroid ratios (two- to ninefold) over those of nontreated controls. The erythroid and lymphoid component of the marrow decreased from day 0 to day 14, whereas the early myeloid progenitors (myeloblasts, progranulocytes, and myelocytes) increased significantly (p less than 0.05). No significant differences in the percentage of later myeloid forms in the marrow were observed over the treatment period. In vitro colony-forming assays of marrow obtained from treated cats revealed increases in granulocyte macrophage colony-forming units (CFU-GM) through day 14, with subsequent decreases by day 21 of rhG-CSF treatment. Recombinant human G-CSF was also effective at in vitro stimulation of feline marrow cells from untreated cats in a dilution study, with maximal CFU-GM formation at 0.1 microgram rhG-CSF/ml assay.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714402 TI - Electron microscopic identification of the intracellular secretion pathway of human G-CSF in a human tumor cell line: a comparative study with a Chinese hamster ovary cell line (IA1-7) transfected with human G-CSF cDNA. AB - Using monoclonal antibodies specific for human granulocyte colony-stimulating factor (G-CSF), intracellular localization of G-CSF in a G-CSF-producing human tumor cell line (CHU-2) and its ultrastructural characters were described and compared with those of a Chinese hamster ovary cell line (IA1-7) transfected with human G-CSF cDNA. The CHU-2 line, which was derived from a poorly differentiated squamous cell carcinoma of the oral cavity, preserved the character of a poorly differentiated squamous cell carcinoma. In the CHU-2 cell line, there were few cells immunohistochemically positive for G-CSF under light microscopic analysis despite the high transcription level of G-CSF cDNA and secretion of G-CSF that were comparable with cDNA-transfected IA1-7 cells. Using electron microscopy, the reaction products were localized mainly in the perinuclear space (PNS) and rough endoplasmic reticula (RER) without dilation of the cisternae, but they were very rarely found in the Golgi complex and not at all in other intracellular organelles. In contrast, most cells were positive for G-CSF in the IA1-7 cell line. Reaction products in this cell line were also demonstrated in the PNS and RER without dilation of the cisternae. These immunohistochemical findings, in conjunction with the results of Western and Northern blot analysis, suggested that G-CSF was secreted via the PNS and RER without intracellular retention. PMID- 1714403 TI - Regulation of early hematopoiesis in serum-deprived cultures of mafosfamide treated and untreated CD34-enriched bone marrow cells. AB - Our experiments have addressed the regulation of early hematopoietic progenitor cell expansion by interleukin 3 (IL-3) and interleukin 1 beta (IL-1 beta) and its modulation by bone marrow fibroblasts in vitro. In a two-stage assay utilizing serum-deprived (SD) presuspension cultures of CD34-enriched bone marrow (BM) cells followed by clonal cultures, absolute numbers of granulocyte-macrophage progenitor cells (day-14 granulocyte-macrophage colony-forming units [CFU-GM]) increased progressively to 164% and 204% of input levels after 12 days of culture in the presence of IL-3 alone or in combination with IL-1 beta, respectively. Multilineage (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) and erythroid (erythroid burst-forming units, BFU-E) progenitor cell numbers increased above or were maintained at input levels after 4 and 7 days of liquid culture in the presence of IL-3 and IL-3 plus IL-1 beta, respectively, but in contrast to granulocyte-macrophage colony-forming units (CFU GM) they were essentially undetectable after 12 days of culture. Progenitors more primitive than colony-forming cells (pre-CFU) were assessed in SD-presuspension cultures of CD34-enriched BM cells purged with mafosfamide to eliminate base-line CFU-GM, CFU-GEMM, and BFU-E. Under these conditions and in the absence of stromal elements, CFU-GM but neither CFU-GEMM nor BFU-E developed in response to cytokines alone. In the additional presence of passaged bone marrow fibroblasts, however, IL-3 plus IL-1 beta and to a lesser degree IL-3 alone induced a pronounced amplification of BFU-E and CFU-GEMM, indicating that their development from a more primitive progenitor compartment requires growth activities in addition to IL-3 and IL-1 beta that are provided by marrow-derived stromal cells such as fibroblasts. PMID- 1714404 TI - Coexpression of the genes for interleukin 6 and its receptor without apparent involvement in the proliferation of acute myeloid leukemia cells. AB - Leukemic cells isolated from patients with either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) were screened for their capacity to express the interleukin 6 (IL-6) and IL-6 receptor genes, both at the RNA and protein levels. Variable levels (10 to greater than 600 U/ml) of an IL-6 activity, inhibited by neutralizing anti-IL-6 antibodies, were detected in AML cell supernatants using the B9 cell bioassay. High levels (greater than 100 U/ml) were observed in differentiated (M4 and M5 stages) AML, as well as in less mature (M1 and M2 stages) AML. Detection of the IL-6 transcript correlated with the biological activity. In addition, both IL-6 activity and IL-6 mRNA were detected in "fresh" leukemic cells, indicating that the glycoprotein was actually synthesized in vivo. In contrast, the IL-6 gene was less frequently expressed in ALL. The IL-6 receptor gene was transcribed in both AML and ALL; binding experiments showed that the protein was present at the cell surface. The spontaneous in vitro proliferation of leukemic cells coexpressing the transcripts for IL-6 and its receptor was not significantly inhibited by a neutralizing anti-IL-6 antibody, suggesting that IL-6 is not primarily implicated in the proliferation of the leukemic clone via an autocrine loop. Synthesis of IL-6 could, however, confer on leukemic cells a selective growth advantage through activation of the cytokine cascade. PMID- 1714405 TI - Peanut agglutinin in combination with CD19 monoclonal antibody has potential as a purging agent in myeloma. AB - Administration of high-dose chemotherapy to patients with myeloma, followed by rescue with autologous bone marrow transplantation (ABMT), sometimes induces complete disease remission but relapse is usual. We have attempted to reduce the risk of relapse by selective in vitro removal of myeloma cells from the autologous graft. A combination of the (gal-galNac)-binding lectin peanut agglutinin (PNA), which binds all plasma cells, and the pan-B monoclonal antibody CD19 was assessed for purging marrow of myeloma cells and their putative precursors using a magnetic bead method. Preliminary experiments performed on peripheral blood mononuclear cells spiked with fluorescent-labeled PNA+ Kirk tumor cells showed that a magnetic bead: target cell ratio of 40:1 resulted in a greater than 3-log reduction in PNA+ cells. This technique was then applied to 17 samples of myeloma bone marrow and to 18 samples of normal bone marrow spiked with PNA+ Kirk cells and CD19+ hairy cell leukemia cells. In each case all detectable plasma cells and CD19+ lymphocytes were effectively removed, and normal hemopoietic progenitor cell recovery was greater than 55%. This purging system deserves further study as a means of reducing relapse rates in myeloma patients treated by a combination of high-dose chemotherapy and ABMT. PMID- 1714406 TI - Distinct morphophenotypic features of chronic B-cell leukaemias identified with CD1c and CD23 antibodies. AB - Morphological criteria usually applied to diagnose various subtypes of B-cell chronic lymphoid leukaemia are largely subjective. Immunophenotyping of 61 relevant cases using a selected panel of monoclonal antibodies (mAb), showed that CD1c and CD23 mAb were able to separate B-cell chronic lymphocytic leukaemia (B CLL) from other chronic B-cell lymphoproliferative diseases. Lymphocytes of B-CLL were CD1c-, CD23+, whereas those of other types of chronic B-cell leukaemia were CD1c+/-, CD23-, and CD38/-. Non-B-CLL cases had a significantly higher amount of large peroxidase-negative (unstained) cells analyzed with an automated blood cell counter (Technicon H6000). This type of volumetric assessment allowed a separation between typical and "atypical" B-CLL, which otherwise were both CD1c-, and CD23+. These combinations of phenotypic markers corresponded to well-defined haematopathologic entities, conventionally diagnosed on peripheral blood (PB) and bone marrow smears, and on histologic sections of lymph nodes and spleen. PMID- 1714407 TI - Influence of cytostatic treatment on the coagulation system and fibrinolysis in patients with non-Hodgkin's lymphomas and acute leukemias. AB - Cytostatic therapy is known to aggravate tumor-induced coagulopathy. Therefore, we have studied the effect of different chemotherapeutic regimens on the activation of coagulation and fibrinolysis in patients with non-Hodgkin's lymphomas or acute leukemias. In non-Hodgkin's lymphoma patients treated with an aggressive protocol (COL-BLAM) and in leukemia patients (TAD-9) fibrinopeptide A, prothrombin fragment (F1 + 2) and thrombin antithrombin III complexes (TAT) increased (Tables 4 and 6), while D-dimer did not deviate significantly. The ratio D-dimer/TAT consequently showed a significant decrease, indicating increased formation of thrombin after release of procoagulant factors, which is not paralleled by an activation of fibrinolysis. Both these groups were also characterized by an increase in uric acid and in C-reactive protein and plasminogen-activator inhibitor, two acute-phase reactants. In contrast, patients with non-Hodgkin's lymphomas treated with a less aggressive protocol (COP) showed no significant changes in hemostatic variables, uric acid, or acute-phase reactants. The release of procoagulant factors relates to the cytostatic sensitivity of the tumor and to a high tumor-cell destruction. Our results further emphasize the need for large-scale studies on antithrombotic prophylaxis in patients undergoing cytostatic treatment. PMID- 1714408 TI - Antagonism of receptors for bombesin, gastrin and cholecystokinin in pancreatic secretion and growth. AB - The effects of bombesin, gastrin and cholecystokinin (CCK) on amylase secretion from the isolated rat pancreatic acini and on DNA synthesis (as biochemical indicator of trophic action) in the pancreas have been examined in 48-hour fasted and 16-hour refed rats with and without administration of specific receptor antagonists for bombesin, gastrin and CCK. Studies on the isolated rat acini revealed that bombesin, gastrin and CCK-8 all showed the same efficacy in their ability to stimulate amylase release. RC-3095, bombesin pseudo-peptide antagonizing bombesin receptors, was effective only in suppressing the amylase response to bombesin but not to gastrin or CCK. Benzodiazepine receptor antagonists for gastrin (L-365,260) and for CCK (L-364,718) showed higher efficacy in the inhibition of amylase release induced by pentagastrin and CCK, respectively, but failed to affect that induced by bombesin. These peptides administered 3 times daily for 48 h in fasted rats increased the rate of DNA synthesis as measured by the incorporation of [3H]thymidine into DNA. The blockade of bombesin receptors abolished the DNA synthesis induced only by bombesin but not by gastrin or CCK. The blockade of gastrin receptors by L 365,260 suppressed the DNA synthesis induced by gastrin while the antagonism of CCK receptors by L-364,718 was effective only against CCK. Refeeding of 48-hour fasting rats strongly enhanced DNA synthesis which was significantly reduced by blocking only the CCK receptors (with L-364,718), but not the bombesin (with RC 3095) or gastrin receptors (with L-365,260).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714409 TI - Elevated myocardial calcium and its role in sudden cardiac death. AB - The contribution of intracellular calcium to ventricular fibrillation (VF) was investigated using chronically instrumented dogs with healed myocardial infarctions. A 2-minute coronary occlusion was initiated during the last minute of exercise. Fourteen animals developed ventricular fibrillation (susceptible) whereas the remaining 12 did not (resistant) during this exercise plus ischemia test. The test was then repeated for the susceptible animals after pretreatment with the intracellular calcium chelator BAPTA-AM (1.0 mg/kg). BAPTA-AM significantly reduced left ventricular dp/dt max and prevented VF in 8 of 12 susceptible animals. Conversely, myocardial cytosolic calcium levels were increased in resistant animals using the calcium channel agonist Bay K 8644 (30 micrograms/kg) or phenylephrine (10 micrograms.kg-1.min-1 3-5 min before occlusion). Bay K 8644 induced VF in all 5 resistant animals tested whereas phenylephrine induced VF in 8 of 12 resistant animals. BAPTA-AM pretreatment attenuated the hemodynamic effects of Bay K 8644 or phenylephrine and prevented VF in five of five Bay K 8644- and four of seven phenylephrine-treated animals. Finally, the endogenous level of calcium/calmodulin (Ca-CaM)-dependent phosphorylation of 170- and 55-kDa substrate proteins was measured (as an index of intracellular free calcium concentration). In the susceptible dog heart, the endogenous level of Ca-CaM-dependent phosphorylation was estimated to be two- to threefold higher than that observed in resistant dog heart. Treatment of resistant dog tissue with the calcium ionophore A23187 increased the level of Ca CaM-dependent phosphorylation of these two proteins to the level observed in susceptible dog heart. These data suggest that elevated cytosolic calcium facilitates development of malignant arrhythmias and that elevated cytosolic calcium levels may be present in animals particularly susceptible to ventricular fibrillation. PMID- 1714410 TI - Mechanisms involved in the effects of phenidone, diclofenac and ethacrynic acid in rat uterus in vitro. AB - 1. The effects of phenidone (P, 10(-4)-10(-3) M), sodium diclofenac (D, 10(-5) 10(-4) M) and ethacrynic acid (E, 10(-5)-10(-4) M), proposed as inhibitors of eicosanoid synthesis, on the contractions of rat uterus induced by several agonists have been studied. 2. P, D and E inhibit the motility induced by oxytocin (4 mU/ml) (IC50: 4.62 x 10(-4), 2.55 x 10(-4) and 2.98 x 10(-5) M, respectively). 3. P (10(-3) M), D (10(-4) M) and E (10(-4) M) also inhibit the contraction induced by methacholine (10(-5) M), prostaglandin F2a (10(-6) M) and CaCl2 (6 mM), and relaxed, in a dose-dependent way, the tonic component of contraction to KCl (60 mM) (IC50: 5.81 x 10(-4), 6.67 x 10(-5) and 7.55 x 10(-5) M, respectively). 4. The CaCl2 (0.1-10 mM) reverted the relaxation of KCl contraction produced by P, but not by D or E. None of the inhibitions on CaCl2 (6 mM) are reverted by Bay K 8644. 5. D and E also relaxed the tonic contraction to vanadate (10(-4) M) in uterus incubated in calcium free solution P, enhances the vanadate-induced contractions. PMID- 1714411 TI - Inhibitory effects of cyclic AMP-elevating agents on norepinephrine-induced phosphatidylinositide hydrolysis and contraction in rat aorta. AB - 1. Exposure of rat aorta to forskolin, dibutyryl cyclic AMP, rolipram or isobutylmethylxanthine partially prevented the increased inositol monophosphate accumulation due to norepinephrine, while the contractile responses to norepinephrine were almost completely inhibited. 2. Inhibition of the increased phosphatidylinositide synthesis due to norepinephrine could not account for the decreased inositol monophosphate accumulation. 3. Although the increased inositol monophosphate accumulation due to norepinephrine was partially inhibited by the cyclooxygenase inhibitor, indomethacin, the inhibitory effects of the cyclic AMP elevating agents were still observed in the presence of indomethacin. 4. Inhibition of agonist-induced phosphatidylinositide hydrolysis may contribute, at least in part, to the vasodilatory effects of the cyclic AMP-elevating agents. PMID- 1714412 TI - Histamine release from rat mast cells induced by econazole. AB - 1. Econazole released histamine from rat mast cells in vitro. This response was not affected by the addition of calcium or by prior treatment of mast cells with EDTA or cromoglycate. 2. Rat mast cells treated with econazole were stained by the vital dye trypan blue. 3. The intradermal injection of econazole increased vascular permeability. This response was antagonized by chlorpheniramine and cyproheptadine. 4. Our results demonstrate that econazole releases histamine by the "nonselective" mechanism. It is suggested that econazole inflammatory effects may be due to histamine release from mast cells. PMID- 1714413 TI - Angiotensin II-induced formation of ionic channels in bilayer lipid membranes. AB - The interaction of angiotensin II (ANG II) with membrane was studied by measuring conductance and current-voltage characteristics (IVC) of bilayer lipid membranes (BLM) prepared of a mixture of egg lecithin with cholesterol, and of gramicidin D modified membranes of the same composition. Addition of physiological concentrations of ANG II (approx. 15 mumol/l) into the electrolyte (1 mol/l KCl, pH = 7) in contact with one side of BLM resulted in the appearance of discrete membrane conductance (symbol; see text) = (39.5 +/- 1.07) pS with a duration of the conductivity state tau = (52.15 +/- 6.44) s. Raising ANG II concentration to 75 mumol/l resulted in an additional conductance level of approx. 130 pS with a lifetime of approx. 1s. The electrolyte pH markedly influenced ANG II modified BLM conductance. A decrease of the electrolyte pH to 2.8 resulted in a reduction of the discrete conductance level to approx. 14 pS, whereas ANG did not induce any conductivity at pH = 11.5. The results obtained suggest that ion channels are formed consisting at least of two ANG II molecules. IVC of ANG II-modified BLM are superlinear within the range of electrolyte concentrations studied (between 0.01 and 3 mol/l KCl), i.e, the limiting stage of ion transport is the internal area of the conducting pore. ANG II affects in a cooperative manner the gramicidin D (GRD)-mediated transport, most likely by forming ANG II aggregates in the area of local inhomogeneities in the BLM structure of GRD channels. PMID- 1714414 TI - Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line. AB - E47 is a helix-loop-helix transcription factor that binds to sites in the immunoglobulin heavy-chain and kappa light-chain gene enhancers. Other proteins of this type are involved in cell-type determination. A possible role for E47 in B-cell development was tested by overexpressing a cDNA encoding E47 in the pre-T cell line 2017. We found a dramatic activation of a germ-line heavy-chain gene transcript in these stable transfectants and an equally large induction of immunoglobulin D-to-J rearrangement, the first recognized step in B-cell development. Germ-line kappa light-chain gene transcription and rearrangement were unaffected, but transcription of the recombination-activating genes RAG-1 and RAG-2 and the lymphoid-specific transcription factor Oct-2 was increased. These T cells did not transcribe their rearranged DJ alleles, however, and failed to progress to the next stage of heavy-chain gene assembly, V-to-DJ rearrangement. Because transcription factor E47 can induce pre-T cells to carry out events of B-cell differentiation, it may be a crucial determinant of the earliest stages of B-cell development. PMID- 1714415 TI - Importance of globin gene order for correct developmental expression. AB - We have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the genes. When the human beta-globin gene is linked as a single gene to the LCR it is activated prematurely in the embryonic yolk sac. We show that the correct timing of beta gene activation is restored when it is placed farther from the LCR than a competing human gamma- or alpha-globin gene. Correct timing is not restored when beta is the globin gene closest to the LCR. Similarly, the human gamma-globin gene is silenced earlier when present farthest from the LCR. On the basis of this result, we propose a model of developmental gene control based on stage-specific elements immediately flanking the genes and on polarity in the locus. We suggest that the difference in relative distance to the LCR, which is a consequence of the ordered arrangement of the genes, results in nonreciprocal competition between the genes for activation by the LCR. PMID- 1714416 TI - Inactivation of a sperm motility gene by insertion of an epidermal growth factor receptor transgene whose product is overexpressed and compartmentalized during spermatogenesis. AB - Transgenic mice were generated with a human epidermal growth factor (EGF) receptor cDNA driven by the chicken beta-actin gene promoter. One line (AE24) that exhibited a unique expression pattern in which dramatically elevated levels of EGF receptor RNA were found only in the testis was established, suggesting that the beta-actin promoter was being influenced by an adjacent testis-specific enhancer. EGF receptor RNA was detected in primary spermatocytes, whereas the synthesis of receptor protein was restricted to elongate spermatids, indicating that transgene expression was under translational control. At spermiation, the EGF receptor was sequestered in residual bodies and excluded from mature sperm by a compartmentalization mechanism. About half of AE24 homozygous males were sterile because of sperm paralysis, whereas heterozygous males and females of either genotype were completely fertile. Electron microscopic analysis of sperm flagella from sterile AE24 homozygotes revealed an aberrant axonemal structure in which outer doublet microtubules were missing from the middle piece, resembling changes observed in the sperm of some infertile humans. Flagellar axonemal disassembly was observed in the vas deferens and epididymis but not in the testis, suggesting that outer doublets were assembled in a grossly normal manner but possessed a latent instability. These results demonstrate that in the AE24 mouse line the EGF receptor transgene was integrated into and inactivated an endogenous autosomal gene, causing sperm flagellar axonemal disruption and male sterility. PMID- 1714417 TI - High-dose cisplatin and bleomycin neoadjuvant chemotherapy plus radical surgery in locally advanced cervical carcinoma: a preliminary report. AB - One course of chemotherapy containing cisplatin and bleomycin as a neoadjuvant treatment was given to 26 consecutive patients with previously untreated stage IB (bulky disease)-III cervical carcinoma and followed by radical surgery. After chemotherapy responses were detected in 23 patients (5 complete and 18 partial; overall, 88%) and permitted radical surgery in 21 cases (81%). Surgery consisted of type III-IV radical hysterectomy plus systematic para-aortic and pelvic lymphadenectomy. At histologic examination, complete responses were found in 5 (19%) and partial responses in 16 (62%) cases. The average number of lymph nodes removed was 61 (range, 38-118). A lower than expected incidence of lymph node metastases was detected (2/21, 9.5%). The chemotherapy-induced toxicity was mainly represented by nausea and vomiting. Chemotherapy did not seem to complicate surgery in these circumstances, even though moderate-degree postoperative complications occurred in 48% of cases. Eighteen months median follow-up time (range, 11-23) from hystological diagnosis has been reached in the operated patients, and no recurrences have been detected so far. PMID- 1714418 TI - Primary clear cell carcinoma of the peritoneum. AB - A case of primary clear cell carcinoma of the pelvic and abdominal peritoneum which occurred in a 67-year-old woman and with histological characteristics of Mullerian derivation is presented. To our knowledge, this is the first report of such a case. Although clear cell carcinomas have been previously described in peritoneal or retroperitoneal locations, these have been mass lesions thought to arise from endometriosis. All other cases of diffuse primary peritoneal adenocarcinomas have been of the serous type. PMID- 1714419 TI - An alternating chemotherapy combination (MACOBLE) for intermediate and high-grade non-Hodgkin's lymphoma. AB - From June 1983 to February 1986, 48 patients with intermediate or high-grade non Hodgkin's lymphomas (NHL) received a CHOP based combination with the addition of etoposide on days 1 and 2, bleomycin days 1 and 10 and methotrexate 1.5 g/m2 on day 10 (MACOBLE). Their median age was 59 years, 20 (42 per cent) had an ECOG performance status (PS) of 2 or 3, 24 (50 per cent) had stage IV disease, 25 (52 per cent) B symptoms and 21 (44 per cent) bulk (greater than 10 cm) disease. With a median follow-up of 62 months, 12 patients are alive, 10 of whom are disease free. Median overall survival was 13 months (95 per cent confidence interval 6-23 months) with actuarial 5-year survival of 25 per cent (95 per cent confidence interval 13-37 per cent). Factors associated with inferior survival were ECOG PS 2 or 3 (P = 0.004), B symptoms (P = 0.013) and bulk disease (P = 0.017). These data suggest that, when treating an unselected patient population, attempts to increase the intensity of first-line chemotherapy may not improve the outcome. PMID- 1714420 TI - Colony-stimulating factors: clinical status. PMID- 1714421 TI - Repeated dermal toxicity of technical HCH and methyl parathion (50EC) to female rats (Rattus norvigicus). AB - Repeated dermal application of hexachlorocyclohexane (HCH; 100 mg/kg/day) or methyl parathion (2 mg/kg/day) individually or in combination for 7, 15 and 30 days produced pathomorphological changes in skin, liver, kidney and brain of female rats along with significant enzymatic alterations in the activity of transaminase, alkaline phosphatase lactic dehydrogenase and acetylcholinesterase. The two insecticides in combination though produced severe toxicity on day 30 than at other periods, the changes were not suggestive of any additive or potentiation effect at the test doses. PMID- 1714423 TI - Synthesis and biological activity of substance P C-terminal hexapeptide and heptapeptide analogues. AB - The analogues [Glu(OBzl)11]SP6-11 and [Glu(OBzl)11]SP5-11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH3 group of Met11 is replaced by the COOCH2C6H5 group. 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 selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P. PMID- 1714422 TI - Allostimulatory analysis of a newly-defined and widely-distributed Mls superantigen. AB - We previously noted that Mlsa,c C58/J responder cells proliferated unexpectedly to H-2k-compatible Mlsa or Mlsc prototypic stimulator cells in a primary mixed lymphocyte reaction. The present investigation was performed to evaluate whether the response of C58/J T cells to these H-2- and Mls-compatible stimulator cells could functionally identify a newly-defined member of the Mls superantigen family through its allostimulatory ability. We observed that C58/J responder cells also proliferated when cultured with H-2-compatible prototypic Mls(null), Mlsb (nonstimulatory), or Mlsa,c splenic stimulator cells. The widely distributed nature of the non-MHC ligand recognized by C58/J T cells is indicated by the finding that 11 of 12 H-2k inbred mouse strains clearly expressed this specificity. A gradient of stimulatory capacity from low to high across this newly-defined non-MHC difference was detected with splenocytes from these different inbred mouse strains. The Mls(a,c) genetic composition of C58/J was confirmed by the observation that crossing C58/J with parental B10.BR (responsive to both Mlsa and Mlsc determinants) generated F1 progeny that were unresponsive to H-2k-compatible Mlsa, Mlsc, or Mls(a,c) stimulator cells. Like prototypic Mlsa and Mlsc, the non-MHC specificity recognized by C58/J responder cells, termed Mlsf, was particularly sensitive to radiation (versus mitomycin C) treatment of the stimulator cells, was greatly augmented after anti-IgD activation of splenic stimulator cells, was blocked with anti-MHC class II antibody, and was effectively presented by phenotypically normal female but not B cell-defective xid+ male CBA/N F1 stimulator cells. PMID- 1714424 TI - Radiotherapy-centered multimodal treatment of unresectable pancreatic carcinoma. AB - Multimodal treatment procedures, including intraoperative and external beam radiotherapy, chemotherapy and hyperthermotherapy, used for treatment of unresectable pancreatic carcinoma for the past two years have been described. Among the ten progressive cases where multimodal treatment was applied, marked reduction in tumor mass was observed in three cases. The cases receiving such treatment reported prolonged survival, the median survival period being 250 days as compared with 85.4 days in the non-multimodal group. The conclusion is that optimal palliative effects can be achieved by sustained application of both intraoperative and postoperative radiotherapy, hyperthermia and other techniques of multimodal therapy in cases of unresectable pancreatic carcinoma. PMID- 1714425 TI - [Chronic hepatitis--cause, pathogenesis, therapy]. PMID- 1714426 TI - [49-year-old patient with fever attacks, weight loss, arthralgia and abdominal tumor]. PMID- 1714427 TI - Neural spread of herpes simplex virus after anterior chamber inoculation. AB - A genetically engineered herpes simplex virus type 1 (HSV-1, strain RH116) that expresses beta-galactosidase (beta-gal) was used as a marker to trace the route of interocular spread of HSV-1 after anterior chamber (AC) inoculation into BALB/c mice. Because RH116 is thymidine kinase deficient (TK-), the wild-type TK+ KOS strain of HSV-1 was used as a helper virus to complement RH116 during in vivo infection. After coinfection of BALB/c mice with RH116 and KOS in the AC of one eye, beta-gal expression by RH116 was detected in both the eyes and in the central nervous system (CNS). Our results suggest that after AC inoculation into BALB/c mice: (1) virus spreads from the injected eye to the CNS through parasympathetic fibers of the oculomotor nerve that supply the iris and ciliary body; (2) virus spread in the CNS is limited primarily to nuclei of the visual system and the suprachiasmatic area of the hypothalamus; and (3) virus is transmitted from the CNS to the retina of the contralateral eye by retrograde axonal transport through the optic nerve along the endocrine-optic pathway between the retina and the suprachiasmatic nucleus of the hypothalamus. PMID- 1714428 TI - Characterization of a novel human corneal endothelial antigen. AB - The antigenic composition of the human corneal endothelium, a cellular layer essential for maintaining corneal function, has not been well characterized. A novel corneal endothelial antigen was identified by generating a monoclonal antibody (MAb) against normal human corneal endothelial cells. This MAb, designated 2B4.14.1, reacted strongly by immunoperoxidase staining with the endothelium of corneas from all human donors tested but not with other corneal components, including epithelium and stroma. Positive immunohistologic reactions of 2B4.14.1 with several other human tissues, including kidney (parietal epithelium of Bowman's capsule, proximal convoluted tubule, ascending limb of Henle's loop, and distal convoluted tubule), glandular epithelia of numerous organs, and mesothelial linings of several thoracic and abdominal viscera, also were observed. One of the renal antigens recognized by 2B4.14.1 was identified as Tamm-Horsfall glycoprotein (THGP), based on the ability of the antibody to recognize THGP in western immunoblots and the abrogation of immunohistologic reactivity of the antibody by preincubation with purified THGP. These findings raise the possibility that the human cornea expresses a molecule with homeostatic properties similar to those ascribed to THGP. However, it is unlikely that the corneal antigen recognized by 2B4.14.1 is conventional THGP; a MAb specific for THGP did not react with corneal endothelium. PMID- 1714429 TI - T6-positive Langerhans cells in diseased corneas. AB - Langerhans cells (LC) in normal human corneas (with the exception of newborns) lack thymocyte antigen T6, a highly specific marker for noncorneal LC. Because corneal LC could not be induced to express T6 antigen when cultured with various cytokines including interleukin-1 (shown to modulate T6 expression on gingival LC), some authors assume that corneal LC may represent a distinct LC subpopulation that is innately inactive. In this study, 62 corneas from patients with various corneal diseases were investigated for the presence of T6 and histocompatibility antigen HLA-DR on LC in the central and pericentral epithelium. Both T6- and HLA-DR-positive LC at a high density similar to that observed in normal epidermis could be detected in the epithelium of five corneas with epidermalization after alkali burns. Furthermore T6- and HLA-DR-positive LC at smaller densities also were detected in corneas from patients with chronic herpetic stromal keratitis, zoster keratitis, chronic allograft rejection, and bacterial corneal ulcers. Although the functional significance of T6 expression on corneal LC remains to be determined, the induction of T6 antigen on corneal LC may represent an important event for the antigen-presenting function of these cells in various corneal diseases including corneal allograft rejection. PMID- 1714430 TI - Immunohistochemical properties of human optic nerve glioma. Evidence of type 1 astrocyte origin. AB - The peroxidase-antiperoxidase method was used to study ten surgically obtained human optic nerve gliomas (pilocytic astrocytomas). All tissues were formalin fixed and paraffin embedded. Primary antisera included glial fibrillary acidic protein (GFAP), HNK-1 (type 1 astrocyte precursor marker), A2B5 (type 2 astrocyte precursor marker), S-100, vimentin, myelin basic protein (MBP), laminin, keratin, cytokeratin, epithelial membrane antigen (EMA), and neuron-specific enolase (NSE). Neoplastic astrocytes in optic nerve gliomas stained with GFAP, HNK-1, S 100, and vimentin. Oligodendrocytes and myelin sheaths stained for MBP, and NSE stained surviving axons in the tumors. Neoplastic astrocytes did not stain for A2B5, keratin, cytokeratin, EMA, or laminin. These results suggest that human optic nerve gliomas (pilocytic astrocytomas) arise from type 1 astrocytes. PMID- 1714431 TI - Treatment of experimental preretinal neovascularization using photodynamic thrombosis. AB - Retinal or preretinal neovascularization (NV) is the result of many ischemic conditions of the retina and is an important factor leading to severe visual loss in diabetic retinopathy. Panretinal photocoagulation does not always control its growth or bleeding sequelae. A potential new treatment modality, photodynamic therapy (PDT), was evaluated for limiting the progression of experimental NV in the rabbit eye. The NV was produced by injecting cultured dermal fibroblasts into the preretinal vitreous space after combined enzymatic and mechanical vitreolysis. This method results in traction retinal detachment with a rapid and consistent growth of NV. After administration of the photosensitizing dye rose bengal (20 mg/kg intravenously), PDT was done using a slit-lamp light source focused through a fundus contact lens (45 J/cm2). The NV was treated on two separate occasions during the active phase of growth (on days 13 and 21 after fibroblast injection). Control animals were exposed to light before injection of rose bengal. Eight randomly assigned animals in each group were followed between treatments and for 28 days after the second treatment. The appearance of NV was documented by frequent photography and fluorescein angiography. The PDT resulted in thrombosis of NV for at least 3 days. Reperfusion, however, was consistently noted at 7 days. Thrombosis was associated with a delay in the growth and maturation of NV fronds, which resumed after reperfusion. Twenty-eight days after the second treatment, NV in both experimental and control eyes had undergone atrophy. At that time (the conclusion of follow-up), however, the size of treated NV fronds (estimated from computerized image analysis of fluorescein angiograms) was significantly less than that of controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714432 TI - Age-dependent covalent changes in MP18 from bovine lens membrane. AB - Peptide antisera made against the putative MP18 sequence recognizes an 18 kilodalton protein on western blots of bovine lens membrane. These peptide antisera were used to investigate possible age-related changes in the MP18 molecule in membrane fractions from newborn and adult bovine lenses. Western blot analysis of membrane fractions with peptide antisera to sequences 45-50 (anti 45), 113-129 (anti-113), and C-terminus 169-173 (anti-Ct) showed similar patterns. All three antisera reacted more strongly with MP18 from membrane prepared from newborn lenses than with corresponding preparations from the adult lens. Binding ratios of anti-45 to anti-Ct showed a statistically significant difference between cortex and nucleus of newborn lenses (P less than 0.02) and between cortex of newborn and cortex of adult lenses (P less than 0.001). Differences in binding ratios of anti-113 to anti-Ct were the most striking, with anti-113 unable to detect MP18 in membrane preparations from adult lenses. Collectively, these results confirmed the putative sequence of MP18 and demonstrated covalent changes in this molecule during the process of aging in the normal bovine lens. PMID- 1714433 TI - Induction intra-arterial cisplatin and bleomycin in head and neck cancer. AB - Fifty-two consecutive patients, affected by large T2 (greater than 3 cm), T3, T4, N0, or N1 previously untreated squamous cell carcinoma of the head and neck, entered this phase I-II study. Treatment consisted of a continuous 8-day infusion on the following daily schedule: cisplatin 25 mg and bleomycin 15 mg administered for 4 and 20 hours, respectively. Technical-related toxicities were 1 case each of coagulation and displacement of the catheter and 1 case of reversible monoparesis of the contralateral arm. Drug-related relevant toxicities accounted for 4 cass of grade 3 or 4 leukopenia and 2 cases of peripheral palsy of the 7th and 12th cranial nerve, respectively. Forty-five of 50 evaluable patients obtained an objective response. In particular, 13 patients obtained a complete response, 22 a partial response greater than or equal to 75%, and 10 a partial response greater than or equal to 50%. Furthermore, 5 of 31 patients showed a complete pathologic disappearance of the tumor, whereas in 12 of 31 only a microscopic residue was found. PMID- 1714434 TI - Hb F-Charlotte, an A gamma variant with a threonine residue in position gamma 75 and a glycine residue in position gamma 136. AB - Structural analyses of an abnormal gamma chain, present in a relative amount of approximately 10% in a cord blood sample of a Black newborn baby, identified two substitutions (gamma 75 Ile----Thr and gamma 136 Ala----Gly) in an apparent variant of the A gamma chain. Gene mapping analysis of genomic DNA and hybridization with specific probes confirmed the presence of these two mutations and provided the evidence to indicate that the abnormal gamma chain was indeed a variant of A gamma with the two listed mutations. PMID- 1714435 TI - The human placenta: a model for tenascin expression. AB - Tenascin is a large glycoprotein of the extracellular matrix. Previous reports have demonstrated that it is associated with epithelial-mesenchymal interfaces and is expressed during embryonic and tumour development, wound healing, cell proliferation and it may be involved in immunomodulation. The human placenta shows numerous features related to these aspects. We have investigated the presence of tenascin in the human placenta throughout pregnancy by immunohistochemistry. We used monoclonal (mAb) and polyclonal (pAb) antibodies to tenascin, a mAb to fibrin, a pAb to fibrinogen, and the mAb Ki-67 as proliferation marker. Tenascin was highly expressed in the mesenchymal villi which are considered the basis of growth and differentiation of the villous trees. Moreover, fibrinoid deposits at the surfaces of the villous trees were always separated from the fetal stroma by tenascin. The stroma of villi encased in fibrinoid was also positive for tenascin. This glycoprotein was also expressed in the villous stroma directly apposed to cell islands and cell columns. In the proximal portions of both epithelial structures, cytotrophoblast was Ki-67 positive. These data show that tenascin is expressed during the development of the placenta, particularly in the mesenchymal villi, cell islands and cell columns. These structures are considered to be the proliferating units of the villous trees. Tenascin underlying fibrinoid deposits suggests that it also participates in repair mechanisms. Thus, in the human placenta tenascin expression can be correlated with villous growth, cell proliferation, and fibrinoid deposition. Its role in immunoprotection of fetal tissues in areas where syncytiotrophoblast as barrier is missing or damaged is discussed. PMID- 1714436 TI - Light microscopical detection of leukocyte cell surface antigens with a one nanometer gold probe. AB - The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grunwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens. PMID- 1714437 TI - Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head. AB - Four monoclonal antibodies that discriminate between structural domains of alpha (TU-01, TU-04) or beta-(TU-06, TU-12) tubulin and a polyclonal anti-tubulin antibody were used for immunostaining of human spermatozoa using immunofluorescence microscopy. Specificity of antibodies was confirmed by immunoblotting experiments. Antibodies TU-01 and TU-06 uniformly stained the whole tail and the neck, whereas antibodies TU-04, TU-12 showed differential distribution of corresponding epitopes in the stable arrays of flagellar microtubules. Of the monoclonal antibodies used, only TU-12 against the antigenic determinant on C-terminal domain of beta-tubulin showed strong reactivity with the equatorial segment of the head. The results document a differential exposure of tubulin epitopes at the single-cell level and suggest the existence of distinct tubulin populations in various structural compartments of the human spermatozoon. PMID- 1714438 TI - Progress in outgrowth culture from rabbit tracheal explants: balance between proliferation and maintenance of differentiated state in epithelial cells. AB - Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. PMID- 1714439 TI - Metabolic deviation of mouse liver by RhIL1-alpha or RhTNF/cachectin. AB - In this work deviation of liver metabolism by cytokines, especially recombinant human interleukin 1-alpha (rhIL1-alpha), was investigated. Administration of rhIL1-alpha or recombinant human tumor necrosis factor (rhTNF/cachectin) to normal mice resulted in rapid, dose-dependent induction of high liver ornithine decarboxylase (ODC) activity. The effects of these cytokines on liver ODC were not indirect effects mediated by eicosanoids. The induction of liver ODC by rhIL1 alpha was at least partly a direct effect on hepatocytes, and was due to increase in de novo synthesis of the enzyme protein after increase in ODC mRNA. No specific protein was required for increase in the level of ODC-mRNA. On IL1 treatment, actinomycin D caused superinduction of liver ODC, which was at least partly due to increased stability of the ODC enzyme, because actinomycin D doubled the apparent half-life (from 50 to 95 min). Daily administration of 2 x 10(3) U of rhIL1-alpha to mice for 3 days also caused decrease in the level of the differentiated type of pyruvate kinase isozyme (PK-L) and marked increase in that of the prototype isozyme (PK-M2) in the liver, but did not cause significant change in the isozyme patterns of the kidney, thymus, and spleen. RhIL1-alpha also induced hypertrophy of the spleen. These results indicate that rhIL1-alpha causes metabolic deviation of the liver similar to that in tumor-bearing hosts. PMID- 1714440 TI - Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme. AB - Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714441 TI - Monoclonal antibodies toward scallop (Patinopecten yessoensis) testis and wheat germ calmodulins. AB - A monoclonal antibody (IM7) toward scallop testis calmodulin and another one (PBE2) toward wheat germ calmodulin were produced. Ca2+ was required for IM7 to react with scallop calmodulin. IM7 reacted with the C-terminal region (Asp78 Lys148) of the calmodulin. As observed on competitive ELISA, IM7 reacted with chicken calmodulin, but not with Euglena gracilis or wheat calmodulin, troponin C, myosin light chains, or parvalbumin. It is assumed that the cluster of Thr143, Thr146, and Ser147 in the C-terminal region acts as the antigenic site. IM7 (and Fab of IM7) inhibited the activities of myosin light chain kinase and cAMP phosphodiesterase. PBE2 reacted with wheat germ calmodulin irrespective of the presence or absence of Ca2+, the antigenic site being in the N-terminal region (Ala1-Met37). It reacted with wheat and spinach calmodulins, but not with scallop, chicken, or Euglena calmodulin, troponin C, myosin light chains, or parvalbumin. PBE2 had no effect on the activities of myosin light chain kinase and cAMP-phosphodiesterase. PMID- 1714442 TI - Distribution and contents of free O-phosphoamino acids in animal tissues. AB - This study was carried out to investigate the distribution and contents of O phosphoserine (P-Ser), O-phosphothreonine (P-Thr), and O-phosphotyrosine (P-Tyr) as their free forms in animal tissues. After extraction of a tissue sample with trichloroacetic acid, these O-phosphoamino acids were converted into their N isobutoxycarbonyl trimethyl ester derivatives and then quantitated by GLC, with flame photometric detection, on a DB-1701 capillary column. Using this method, nanogram levels of O-phosphoamino acids in tissue samples could be accurately and precisely determined without any interference by coexisting substances. Free P Ser and P-Thr were widely found in animal tissues, pancreas, spleen, stomach, kidney, liver, and lung containing considerable amounts of these O-phosphoamino acids. On the other hand, free P-Tyr was not detected in any of the tissues investigated in this study. PMID- 1714443 TI - Identification of human galactoprotein b3, an oncogenic transformation-induced membrane glycoprotein, as VLA-3 alpha subunit: the primary structure of human integrin alpha 3. AB - Galactoprotein b3 is one of the cell membrane glycoproteins of fibroblasts showing enhanced expression in association with oncogenic transformation. Analysis of cDNA for this glycoprotein from hamster fibroblasts indicated that the glycoprotein is a member of the integrin superfamily [Tsuji, T., Yamamoto, F., Miura, Y., Takio, K., Titani, K., Pawar, S., Osawa, T., & hakomori, S. (1990) J. Biol. Chem. 265, 7016-7021]. In the present study, we examined the change in the amounts of mRNA for the mouse and human counterparts in fibroblasts after oncogenic transformation by Northern blot analysis using hamster galactoprotein b3 cDNA as a probe. In both human and murine fibroblasts transformed with SV-40, the homologous mRNA to galactoprotein b3 was also found to increase as compared with the progenitor cells. The human homologue of galactoprotein b3 cDNA was cloned from human bladder carcinoma cell line (T24) cDNA library. The cDNA codes for a single polypeptide of which the N-terminal sequence (21 amino acids) is identical with that of human VLA-3 alpha subunit. Based on this sequence identity and the structural similarities (i.e. the positions of most cysteine residues, the presence of a transmembrane domain near the C-terminus and the presence of metal binding sequences) with other integrins so far cloned, we conclude that human galactoprotein b3 is an integrin alpha 3 subunit. The mature integrin alpha 3 polypeptide was composed of 1,019 amino acid residues, and the overall structure was quite similar to the hamster counterpart.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714444 TI - Extracellular proteins that modulate cell-matrix interactions. SPARC, tenascin, and thrombospondin. PMID- 1714445 TI - Cyclosporin A inhibits an initial step in folding of transferrin within the endoplasmic reticulum. AB - As resolved by electrophoresis in non-reducing SDS gels, transferrin newly made in Hep G2 cells migrates as a very diffuse set of species. During a subsequent 1 h chase all transferrin polypeptides are converted to a single, rapidly migrating species. These changes in gel mobility are due to alterations in the pattern of disulfide bonding, are not caused by carbohydrate processing, and occur while the protein is in the rough endoplasmic reticulum. Cyclosporin A causes an approximately 10-min lag in transferrin folding, after which folding resumes at the normal rate. Cyclosporin A also retards transferrin maturation from the endoplasmic reticulum and its secretion, at concentrations that do not affect secretion of other hepatoma proteins. Neither FK506 nor rapamycin affect transferrin folding. We conclude that an initial stage in transferrin folding is accelerated by an endoplasmic reticulum peptidyl-proline isomerase that is inhibited by cyclosporin A. PMID- 1714446 TI - The interleukin-8 receptor is encoded by a neutrophil-specific cDNA clone, F3R. AB - Interleukin-8 (IL-8) is one of the most potent chemotactic agents for neutrophils and has been implicated as a major mediator of inflammation. The IL-8 receptor is expressed exclusively in neutrophils and belongs to the family of G-protein coupled receptors. In a recent paper we reported the characterization of a cDNA clone, F3R, isolated from a neutrophil cDNA library and showed that it encodes a G-protein-coupled receptor which is exclusively expressed in neutrophils. We also suggested, based on expression studies in Xenopus oocytes, that the F3R protein product is an isoform of the (fMLP) receptor (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20064). In this work, the F3R receptor cDNA is expressed in monkey kidney cells (COS-7) and is shown to encode the IL-8 receptor. F3R cDNA does not encode for a fMLP receptor isoform. We show conclusively that the F3R-transfected COS-7 cells express the IL-8 receptor at a density equivalent to that observed in neutrophils. The pharmacological profile of the F3R-transfected cells is the same as that of neutrophils. The apparent Kd values for binding of 125I-IL-8 to neutrophils and F3R-transfected COS-7 cell membranes were 1.2 and 1.4 nM, respectively. Antipeptide antibodies against a partial sequence of the F3R protein product specifically immunoprecipitate the IL 8 receptor from transfected cells as well as neutrophils. The molecular characterization of the IL-8 receptor should provide the basis for further studies on the identification of the binding domain of this inflammatory receptor. PMID- 1714447 TI - A carbohydrate domain common to both sialyl Le(a) and sialyl Le(X) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1. AB - The specificity of endothelial cell leukocyte adhesion molecule-1, ELAM-1, for binding to a panel of carbohydrate structures was determined by a sensitive cell binding assay with immobilized synthetic glycoconjugates. ELAM-1 cDNA transfectants were found to bind Sialyl Lea (sialylated lacto-N-fucopentaose II) or sialylated Lewis a antigen (NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc), as well as or slightly better than Sialyl Lex (sialylated lacto-N-fucopentaose III) or sialylated Lewis X antigen (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1 3)GlcNAc). A monoclonal antibody, HECA-452, which has been identified recently as recognizing ELAM-1 ligands in addition to those containing Sialyl Lex, was also found to bind both Sialyl Lex and Sialyl Lea. Hard sphere exo-anomeric (HSEA) calculations were performed on these two hexasaccharides. The conformations indicate that Sialyl Lea and Sialyl Lex show a high degree of similarity in both the nonreducing and reducing termini. As Lea and Lex show much weaker reactivity, the determinants recognized by ELAM-1 and HECA-452 probably involve neuraminic acid and fucose residues which on one face of both Sialyl Lex and Sialyl Lea can be similarly positioned. The finding that Sialyl Lea is a potent ligand for ELAM 1 is important, as circulating Sialyl Lea and Sialyl Lex containing mucins which are elevated in the serum of many cancer patients may block leukocyte interactions with ELAM-1 and may contribute to the pathological immunodepression observed in these patients. PMID- 1714448 TI - Asp383 in the second transmembrane domain of the lutropin receptor is important for high affinity hormone binding and cAMP production. AB - The lutropin (LH), follitropin, and thyrotropin receptors belong to the superfamily of G-protein coupled receptors and have some unique structural features. These glycoprotein hormone receptors comprise a C-terminal half and an N-terminal half of similar size. The C-terminal half is equivalent to the entire structure of other G-protein coupled receptors and has seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. In contrast, the hydrophilic N-terminal half is exoplasmic and unique to the glycoprotein hormone receptors. This large N-terminal half of the LH receptor has recently been shown to be capable of binding the hormone. Therefore, these glycoprotein hormone receptors are structurally and functionally different from other G-protein coupled receptors. In an attempt to define the role of the membrane-associated C-terminal half of the LH receptor, we have prepared several mutant receptors in which an Asp or Glu in the seven transmembrane domains has been converted to Asn or Gln, respectively. These include Asp383----Asn in the second transmembrane domain, Glu410----Gln in the third transmembrane domain, and Asp556----Asn in the sixth transmembrane domain. All these mutant receptors were successfully expressed in Cos 7A cells. The Glu410----Gln and Asp556----Asn mutants maintained normal affinities for hormone binding and cAMP production, but the Asp383----Asn mutant showed significantly lower affinities. Although Asp383 of the LH receptor is conserved in all G-protein coupled receptors cloned to date except the substance P receptor, which has Glu in the place of the Asp residue, this is the first observation of the critical role of the Asp in hormone binding and subsequent stimulation of cAMP production. PMID- 1714449 TI - Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites. AB - Of 14 identified epitopes on human GCase (acid beta-glucosidase), monoclonal antibodies (MCABs) recognizing 3 produced inhibition and 1 resulted in activation of GCase. MCABs F1 and F2 completely, and MCAB 61 partially (approximately 70%), inhibited GCase activity. Substrates and active site-directed inhibitors (specific sphingolipid and 5-amino-5-deoxyglucose derivatives) protected the enzyme from inhibition by MCAB F1 and F2, but not that by MCAB 61. Conduritol B epoxide did not protect GCase from the inhibition by these MCABs when covalently bound to the active site. These results indicated highly specific binding requirements of MCABs F1 and F2 for residues in a complex active site. In comparison, kinetic analyses using GCase transition state analogues, N-alkyl glucosylamines, and MCAB 61 demonstrated that this MCAB "freezes" the conformation of the enzyme and inhibits GCase by preventing formation of a conformer needed for maximal catalytic rates. The activating MCAB 122 mimicked the effects of saposin C and competed with this natural activator for residues on the enzyme. Interaction of saposin A and saposin C or MCAB 122 with GCase produced a synergistic effect leading to a marked sensitization of the enzyme to these activators. No such synergism or additivity was found for the maximal catalytic rate since it could be achieved by saturating amounts of any one or combinations of these activators. In the presence of MCAB 61, only 15 to 25% of the maximal activation of GCase was obtained by saposin C or MCAB 122, indicating that the major activation effects of these effectors derived from an induction of a GCase conformational change. These results demonstrate that saposins A and C mediate their activating effects by binding to distinct sites on GCase. Furthermore, major components of the mechanisms for catalysis and saposin C activation are due to conformational changes during the transition state. These findings have implications for understanding the perturbations of GCase function due to the missense mutations which cause Gaucher disease. PMID- 1714450 TI - Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14. AB - Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts coding for the entire gene. The genomic DNA was isolated and studied by restriction mapping, polymerase chain reaction analysis, and DNA sequencing. The gene was 11.5 kilobases in length and consisted of five exons separated by four introns. In addition, 0.8 kilobases of DNA from the 5'-flanking region were sequenced. The exon-intron boundaries all observed the "GT-AG" rule. The gene for protein C inhibitor was assigned to chromosome 14 by polymerase chain reaction analysis of human/hamster hybrid cell lines. The organization of the gene for protein C inhibitor is similar to the genes coding for alpha 1-antitrypsin and alpha 1-antichymotrypsin. The genes for these two proteins are also localized on chromosome 14 suggesting a recent evolution of the genes for these three proteins from a common ancestor. PMID- 1714451 TI - Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper responsive expression. AB - We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper repressible Cyt c6. A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C. reinhardtii polyadenylation signal. Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C. reinhardtii consensus intron/exon boundaries. Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine. Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes. Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells. These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels. Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo. PMID- 1714452 TI - Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism. AB - We present here the full-length cDNA sequence and genomic structure of the mouse homologue of the tumor-associated mucin, MUC1. This mucin (previously called polymorphic epithelial mucin) is present at the apical surface of most glandular epithelial cells. The mouse gene, Muc-1, encodes an integral membrane protein with 40% of its coding capacity made up of serine, threonine, and proline, a composition typical of a highly O-glycosylated protein. The mucin core protein consists of an amino-terminal signal sequence, a tandem repeat domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing transmembrane and cytoplasmic domains. Homology with the human protein is only 34% in the tandem repeat domain, mainly showing conservation of serines and threonines, presumed sites of O-linked carbohydrate attachment. Homology rises to 87% in the transmembrane and cytoplasmic domains, suggesting that these regions may be functionally important. The pattern of expression of the mouse mucin is very similar to that of its human counterpart and accordingly the two promoter regions share high homology, 74%, although previously identified potential hormone responsive elements are not conserved. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat polymorphism. We present evidence that suggests that the mouse gene was at one time polymorphic but has mutated away from this state. PMID- 1714453 TI - Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alpha-mannosidase. AB - We have isolated the gene from Saccharomyces cerevisiae encoding an alpha mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A. (1988) J. Biol. Chem. 263, 14757-14763). Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA. The labeled polymerase chain reaction products were used to screen a S. cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated. A 4.6-kilobase fragment which hybridized with the probes was sequenced. It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha mannosidase. The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases. The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain. It has three potential N-glycosylation sites and a calcium-binding consensus sequence. Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K. W., Schutzbach, J. S., Forsee, W. T., Neame, P., Bishoff, J., Lodish, H. F., and Robbins, P. W. (1990) Glycoconjugate J. 7, 401). Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity. Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth. These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability. PMID- 1714454 TI - Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL 60 cell clones. AB - In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA. PMID- 1714455 TI - The efficiency of the uncleaved secretion signal in the plasminogen activator inhibitor type 2 protein can be enhanced by point mutations that increase its hydrophobicity. AB - Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators that belongs to the serine protease inhibitor superfamily (SERPINS). PAI-2 exists in two molecular forms: an intracellular, non glycosylated form and a secreted, glycosylated form. Like ovalbumin, PAI-2 contains an uncleaved internal secretion signal. By deletion analysis, we have mapped the secretion signal to two mildly hydrophobic regions near the NH2 terminus. We also show that both of these regions become more efficient translocation signals when their hydrophobicities are increased. The PAI-2 secretion signal provides a unique example of a signal that, by virtue of its poor efficiency, allows the synthesis of both an extracellular and an intracellular form of the protein. PMID- 1714456 TI - Molecular structure of rat hepatic 3 alpha-hydroxysteroid dehydrogenase. A member of the oxidoreductase gene family. AB - 3-alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins. 3 alpha-HSD is also required for bile acid synthesis and has been suggested to play an important role in net bile acid transport across the hepatocyte (Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) Annu. Rev. Physiol. 51, 166-177). In order to characterize molecular forms and begin to determine its regulation, we now report the nucleotide sequence, tissue distribution, and homology to other members of the oxidoreductase superfamily. Rat hepatic 3 alpha-HSD cDNA encodes for a 322-amino acid protein with a predicted molecular weight of 37,022 expressed in a 2.4-kilobase (kb) message size. Northern blot analysis of total RNA revealed equivalent steady-state levels in liver and intestine in male rats with lower levels of expression in the colon and minimal expression in stomach, lung, and testis. Female liver contained approximately 2-3-fold greater steady state levels of mRNA as compared to the male liver with equivalent intestinal expression. Two hybridizing bands, 2.4 and 1.4 kb, were identified in total RNA from the ovary. 3 alpha-HSD exhibits 75% amino acid sequence homology with bovine lung prostaglandin F synthetase and 50% homology with human aldose reductases. Amino acid sequence analysis with short chain alcohol dehydrogenases identified a possible NADP(H) cofactor-binding site at the amino terminus. The significant homology of 3 alpha-HSD with both prostaglandin F synthetase and aldose reductases suggest a subdivision of monomeric, NADPH reductases within the larger oxidoreductases superfamily. PMID- 1714457 TI - Expression of the gene coding for a human mucin in mouse mammary tumor cells can affect their tumorigenicity. AB - The human epithelial mucin which is the product of the MUC1 gene is expressed by many carcinomas, including those of breast, ovary, colon, and lung. The core protein is aberrantly glycosylated in the tumors resulting in the exposure or appearance of novel epitopes. To examine the possibility of using the MUC1 gene and its products in active immunization against breast and other carcinomas, we have developed a syngeneic mouse model, by transfecting the gene into the mouse mammary epithelial tumor cell 410.4. An 8.3-kilobase EcoRI fragment of the gene was transfected using the expression vector pEMSV scribe alpha 2. Transcripts of the correct size, initiating from the transcriptional start site seen in human cells, were observed in the transfectants. The mucin was expressed in the cytoplasm and in the membrane, and the glycosylation pattern appeared to be similar to that seen in human tumor cells, since the core protein epitopes recognized by antibodies HMFG-1, HMFG-2, and SM-3 were exposed. The 410.4 transfectants expressing the human mucin showed a reduction in tumor incidence at low inocula and a delay in tumor growth at higher inocula. Pretreatment with 10(4) transfected cells could inhibit the development of tumors from a subsequent inoculum of 10(6) transfectants, but had no effect on the tumor development of the untransfected 410.4 cells. Our results suggest that the human mucin expressed by the 410.4 cells may mobilize an immune response which inhibits tumor development. They also indicate that the mouse model will be useful for evaluation of efficacy of immunogens based on the MUC1 gene and its product. PMID- 1714458 TI - Expression of S-antigen in retina, pineal gland, lens, and brain is directed by 5'-flanking sequences. AB - S-antigen (S-Ag) is an abundant protein of the retina and pineal gland that elicits experimental autoimmune uveitis and pinealocytis in several animal species. To study the elements regulating the expression of S-Ag, we generated transgenic mice expressing the chloramphenicol acetyl transferase (CAT) gene under the control of a 1.3-kilobase pair 5'-flanking segment of the mouse S-Ag gene. While all of the transgenic mice expressed CAT activity in the retina, in some animals CAT activity was also detected in the pineal gland, lens, and brain. Immunoblotting, polymerase chain reaction-mediated detection of RNA, and immunocyto-staining of transgenic tissues with antibodies to CAT and S-Ag established that the profile of expression of the transgene corresponded to that of S-Ag; both proteins were detectable in retinal photoreceptor cells, pinealocytes, lens fiber and epithelial cells, the cerebellum, and the cerebral cortex. These results indicate that S-Ag is expressed in a wider spectrum of the cell types than previously recognized and that a 1.3-kilobase pair S-Ag promoter segment contains sufficient information to direct appropriate tissue-specific gene expression in transgenic mice. PMID- 1714459 TI - Molecular characterization of transcription factors that bind to the cAMP responsive region of the substance P precursor gene. cDNA cloning of a novel C/EBP-related factor. AB - A cAMP response element (CRE) plays an important role in the cAMP-mediated gene regulation. Several factors that recognize a CRE have been characterized, and it has been shown that they need either covalent modification by protein kinase A or a cofactor such as the adenovirus Ela to function as an activator. In this study we show that the substance P precursor gene expression is regulated by protein kinase A and identify the CRE sequence in its promoter region. We find that a novel factor and ATF2 bind to the region containing the CRE of the substance P precursor gene. The sequence analysis indicates that the novel protein, designated CELF, has a significant homology to C/EBP gene family proteins in the carboxyl-terminal part containing the basic region and the leucine zipper motif. Ubiquitous expression of CELF suggests that this factor is utilized by various genes. Cell-free transcription analyses indicate that CELF is a constitutive transcriptional activator without apparent phosphorylation by protein kinase A. These results demonstrate that multiple factors are responsible for transcriptional control of the substance P precursor gene through the CRE region. PMID- 1714460 TI - Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins. AB - The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1. PMID- 1714461 TI - A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments. AB - To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness. PMID- 1714462 TI - Cytokeratin phosphorylation, cytokeratin filament severing and the solubilization of the maternal mRNA Vg1. AB - During meiotic maturation, the cortical cytokeratin filament system of the Xenopus oocyte disappears (Klymkowsky, M. W., and L. A. Maynell. 1989. Dev. Biol. 134:479). Here we demonstrate that this disappearance results from the severing of cytokeratin filaments into a heterogenous population of oligomers, with S- values ranging from 12S and greater. Cytokeratin filament severing correlates with the hyperphosphorylation of the type II cytokeratin of the oocyte. Both the severing of cytokeratin filaments and cytokeratin hyperphosphorylation are reversed by treatment with cycloheximide. These data suggest that fragmentation of cytokeratin filaments is controlled, at least in part, by the phosphorylation of the type II cytokeratin, and that the cytokeratin kinase activity responsible is biosynthetically labile. Cytokeratin filaments have been suggested to anchor the maternal mRNA Vg1 to the vegetal cortex of the oocyte (Pondel, M., and M. L. King. 1988. Proc. Natl. Acad. Sci. USA. 85:7216). By injecting fractions containing active maturation promoting factor or a purified, mutant cyclin protein, we find that the bulk of the Vg1 mRNA in the oocyte can be solubilized under conditions that block the fragmentation of cytokeratin filaments, and that the fragmentation of cytokeratin filaments itself leads to the solubilization of only a minor fraction of the Vg1 mRNA. Thus, at best, cytokeratin filaments directly anchor only a minor fraction of the Vg1 mRNA in the oocyte. Moreover, factors distinct from maturation promoting factor appear to be required for the complete solubilization of Vg1 mRNA during oocyte maturation. PMID- 1714463 TI - Inhibition of myogenesis by the H-ras oncogene: implication of a role for protein kinase C. AB - Expression of the oncogenic form of H-ras p21 in the mouse myogenic cell line, 23A2, blocks myogenesis and inhibits expression of the myogenic regulatory factor gene, MyoD1. Previous studies from a number of laboratories have demonstrated that the activation of ras p21 is associated with changes in phospholipid metabolism that directly, or indirectly, lead to elevated levels of intracellular diacylglycerol and the subsequent activation of protein kinase C (PKC). To assess the importance of PKC activity to the ras-induced inhibition of skeletal myogenesis, we examined the levels of PKC activity associated with the terminal differentiation of wild-type myoblasts and with the differentiation-defective phenotype of 23A2 ras cells. We demonstrate that there is a 50% reduction in PKC activity during normal myogenesis and that PKC activity is required for myoblast fusion, but not for the transcriptional activation of muscle-specific genes. In contrast, we found that the differentiation-defective 23A2 ras cells possess two- to threefold more PKC activity than wild-type myofibers and that reducing the PKC activity in these cultures does not reverse their non-myogenic phenotype. On the other hand, if PKC activity is downregulated in 23A2 cells before the expression of activated ras p21, myogenesis is not inhibited. These results suggest that activated ras p21 relies on a PKC-dependent signal transduction pathway to initiate, but not to sustain, its negative effects on 23A2 skeletal myogenesis and underscore the potential importance of PKC activity to the proper control of skeletal muscle differentiation. PMID- 1714464 TI - Alteration of proteoglycan metabolism during the differentiation of 3T3-L1 fibroblasts into adipocytes. AB - 3T3-L1 fibroblasts were induced to differentiate to 3T3-L1 adipocytes by dexamethasone, isobutyl-methylxanthine, and insulin. To study how differentiation affects extracellular matrix production, the accumulation of proteoglycans was studied by labeling the 3T3-L1 cells with [35S]sulphate for 24 h. The labeled proteoglycans were isolated from the medium and cell layer extracts by anion exchange chromatography. They were then taken to gel filtration chromatography on Superose 6 before or after chondroitin ABC lyase digestion. Hyaluronan was determined by radioimmunoassay. The rate of accumulation of proteoglycans and hyaluronan in the control 3T3-L1 fibroblasts increased with time whereas it decreased slightly in the age matched adipocytes where the differentiation had proceeded, as judged by the change of morphology and increase of the activity of the adipose conversion markers glycerol-3-phosphate dehydrogenase and hormone sensitive lipase. The main change noted was that the adipocytes accumulated 50 70% less amount of small proteoglycans (decorin) in the medium than the fibroblasts did. The amount of large chondroitin/dermatan sulphate proteoglycans was also decreased but to a considerably smaller extent (30%). In the cell layer, heparan sulphate proteoglycan decreased by 60% as compared with the control cells. Thus, the differentiation of 3T3-L1 fibroblasts into adipocytes, which changes the morphology and the function of the cells, is also accompanied by a decreased net production especially of proteoglycans typical of fibrous connective tissue. PMID- 1714465 TI - Trigeminal projections to the nucleus submedius of the thalamus in the rat. AB - Methods involving the anterograde and retrograde transport of wheat-germ agglutinin conjugated horseradish peroxidase and the retrograde transport of Fluoro-Gold were used in rats to examine the distribution within the spinal trigeminal nucleus of trigeminal neurons projecting to the nucleus submedius (Sm) of the thalamus, as well as the distribution of axon terminals within the Sm. Following injections into the trigeminal nucleus, axon terminals were seen in the dorsal part of the anterior Sm; the terminals occurred bilaterally but had an obvious contralateral dominance. To help determine the precise location of the Sm petal neurons, the border between trigeminal subnuclei interpolaris and caudalis was examined by the use of immunohistochemical procedures for calcitonin gene related peptide (CGRP). The Sm-petal neurons that were labeled retrogradely occurred only at the caudal interpolaris and rostral caudalis levels; the number of labeled neurons on the contralateral side was approximately six times that on the ipsilateral side. Most of these neurons were located in the ventral part of the caudal interpolaris and rostral caudalis and spinal trigeminal tract; in caudalis, the neurons were almost exclusively localized to its superficial layers. There were approximately three times more labeled neurons in interpolaris than in caudalis. In the experiments combined with immunohistochemistry for CGRP, many neurons (34%) were seen in proximity to CGRP-like immunopositive fibers. These results suggest that the Sm of the rat receives its orofacial afferent inputs from brainstem neurons that are localized to the caudal interpolaris and rostral caudalis. In view of previous studies that have implicated these three structures in somatosensory function, and in particular nociception, our data point to a role for this direct projection from interpolaris and caudalis to Sm in the central processing of pain. PMID- 1714466 TI - Neuroglial arrangements in the olfactory glomeruli of the hedgehog. AB - The olfactory glomeruli represent morphological and functional units in which olfactory information is processed in specialized synaptic arrangements established between the central processes of sensory neurons, whose cell bodies are located in the olfactory epithelium, and the terminal (intraglomerular) portions of the dendrites of periglomerular, tufted, and mitral cells. The olfactory glomeruli are surrounded by distinctive glial formations in which the peripheral glia interacts with the central glia. We have studied the morphology and organization of neuroglial cells in the layer of olfactory nerves and the glomerular layer of the olfactory bulb in the insectivorous hedgehog (Erinaceus europaeus) with the electron microscope, Golgi method, and immunohistochemistry by using antibodies to glial fibrillary acidic protein (GFAP) and "rip," a monoclonal antibody that stains oligodendrocytes and their processes in the rat (Friedman et al.: Glia 2:380-390, '89). The peripheral glia is represented by a special category of cells that are closely related to astrocytes and known as sheathing cells. They accompany olfactory axons to their entrance in the glomeruli where they interact with the central glia, represented by astrocytes and oligodendrocytes. The sheathing cells typically display indented nuclei and protoplasmic expansions forming laminar processes wrapping several axons together. Astrocytes surrounding the glomerular neuropil belong to the velate type. They display numerous sheet-like processes enveloping dendritic segments and periglomerular cell bodies. Oligodendrocytes were found surrounding the glomeruli and at the interstices separating different glomeruli. Myelinated dendritic segments and cell bodies were found surrounding the olfactory glomeruli. These myelin coverings probably derive from oligodendrocytes. Together with the astrocytic lamellar expansions, they provide a rigid structural support that contributes to the segregation of group of different cells while remaining relatively isolated from other influences at the periphery of the glomeruli. PMID- 1714467 TI - Idiopathic scrotal calcinosis is idiopathic. AB - The appearance of calcific masses within the dermis of scrotal skin is generally referred to as idiopathic scrotal calcinosis. There has been some debate about the pathogenesis of these calcium deposits. This debate centers on the question of whether the calcium deposition is truly idiopathic or whether it occurs as a result of preexisting epidermal cysts. We have performed immunohistochemical staining for keratin in nine patients with apparent idiopathic scrotal calcinosis and have found no evidence of keratin in the dermal tissue immediately adjacent to the calcium deposits. We conclude that idiopathic scrotal calcinosis is idiopathic. PMID- 1714468 TI - Intralesional bleomycin and Raynaud's phenomenon. PMID- 1714469 TI - The architecture of black and white facial skin. AB - This study yielded the following findings on the morphologic facial skin differences between black and white women: The epidermis of black skin has more and larger singly distributed melanosomes in the keratinocytes and corneocytes than that of white skin. The stratum lucidum in black skin is not altered by sunlight exposure. The epidermis of black skin rarely shows atrophied areas. The elaunin and oxytalan fibers in black skin are not disposed in candelabra-like formations. Black skin has minimal elastosis and elastic fibers stain pink or red with the hematoxylin and Lee procedure; none stain lilac or blue. The dermis of black skin contains many more fiber fragments composed of collagen fibrils and glycoproteins. Fibroblasts are more numerous, larger, have more biosynthetic organelles than white skin, and are often binucleated and multinucleated. The dermis of black skin has many binucleated and multinucleated macrophages and multinucleated giant cells. Black skin has many more mixed apocrine-eccrine sweat glands than does white skin and more blood and lymphatic vessels. PMID- 1714470 TI - Surgical management of severe aortic outflow obstruction in lesions other than the hypoplastic left heart syndrome: use of a pulmonary artery to aorta anastomosis. AB - Between December 1985 and April 1990, 50 infants with a variety of congenital cardiac lesions other than the hypoplastic left heart syndrome underwent surgical relief of aortic outflow obstruction by creation of a pulmonary artery to aorta anastomosis. The patients were grouped anatomically by ventriculoarterial alignment. Nineteen had normally aligned great arteries (group I); 25 had transposition of the great arteries, all with a univentricular heart of left ventricular morphology (group II); and 6 had a double-outlet right ventricle (group III). All patients had either aortic stenosis with atresia, subaortic stenosis or a restrictive ventricular septal defect. Sixteen had normal arch anatomy; 34 had arch anomalies consisting of arch hypoplasia (n = 17), coarctation (n = 11), interruption of the arch (n = 4) and complex arch anomalies (n = 2). Surgery was performed at a median age of 10 days (range 2 to 184). Of the 50 infants, 33 survived. No significant difference in early survival (30 days) was noted among the groups of varying ventriculoarterial alignment (68% group I, 72% group II, 83% group III) (p greater than 0.05). Overall actuarial survival was 63% at 18 months. Analysis of actuarial survival by arch anatomy, although not statistically significant, revealed a trend toward better survival at 18 months postoperatively in infants with normal arch anatomy (81%) than in infants with arch anomalies (54%). Of the 33 survivors, 26 have proceeded to the next surgical stage, including the Fontan procedure in 8, superior cavopulmonary anastomosis in 13 and biventricular repair in 5.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714471 TI - Nuclear magnetic resonance imaging of the palliative operation for hypoplastic left heart syndrome. AB - Electrocardiographic-gated nuclear magnetic resonance (NMR) imaging has been shown to be effective for the evaluation of congenital heart disease, particularly in supracardiac regions. This study evaluated the postoperative status after a stage I palliative operation (Norwood procedure) for hypoplastic left heart syndrome. The NMR images from three patients were compared with those of angiography and depicted all components of the reconstructed supracardiac and intracardiac anatomy after this operation. Nonobstructive anastomosis of the main pulmonary artery to the proximal aorta was clearly demonstrated in each patient. The caliber of the central or branch pulmonary artery, patency and caliber of the systemic to pulmonary artery shunt and the size of the atrial communication were also depicted in each patient and these findings corresponded with angiographic results. The results suggest that NMR imaging is effective for assessing the results of initial palliative surgery for hypoplastic left heart syndrome, which seems to be important for managing patients before subsequent definitive surgery. PMID- 1714472 TI - Origin of galanin-immunoreactive nerve fibers in the rat paracervical autonomic ganglia and uterine cervix. AB - Retrograde axonal tracing with fluorogold in conjunction with immunohistochemistry was used to examine the source of galanin-immunoreactive nerve fibers in the paracervical ganglia and uterine cervix of the female rat. Immunohistochemistry revealed galanin-immunoreactive neuron somata in lumbosacral dorsal root ganglia and around the central canal of the lumbosacral spinal cord (lamina X). Injection of fluorogold into the paracervical ganglia resulted in labelled cells in dorsal root ganglia and the sacral parasympathetic nucleus of the spinal cord; but fluorogold-labelled, galanin-immunoreactive cells were found only in dorsal root ganglia. Injection of the tracer in the cervix resulted in labelled cells in the paracervical ganglia and dorsal root ganglia; however, fluorogold-labelled, galanin-immunoreactive cells were again evident only in dorsal root ganglia. It is suggested that the galanin-immunoreactive nerve fibers and varicosities in the paracervical ganglia and uterine cervix are sensory fibers from spinal dorsal root ganglia. The galanin-immunoreactive varicosities in the ganglia could play a role in the modulation of pelvic visceral activity, while those in the musculature of the cervix could influence contractility. PMID- 1714473 TI - Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells. AB - We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation. PMID- 1714474 TI - IgE class switching is critically dependent upon the nature of the B cell activator, in addition to the presence of IL-4. AB - Cross-linkage of membrane IgD on resting murine B cells, by anti-IgD mAb conjugated to dextran (alpha delta-dex), induces high levels of proliferation, and in the presence of IL-2 or IL-5, Ig secretion in vitro. The structural and functional similarities between alpha delta-dex and TNP-Ficoll for B cell responses led us to propose that alpha delta-dex could provide a model system for studying B cell activation induced by T cell-independent, type II Ag. In this report, we study the effects of Ig class switch and differentiation factors on Ig isotype production by murine B cells activated by alpha delta-dex, and directly compare these to responses obtained after activation by LPS. We show that an IL-4 containing CD4+ T cell supernatant (Th2 SN) stimulates large increases in IgG1 and IgE production by LPS-activated B cells, but fails to stimulate detectable levels of IgE by alpha delta-dex-activated cells, despite inducing high levels of secreted IgM and IgG1. This is correlated with undetectable steady state levels of both germ-line and rearranged (productive) IgE-specific RNA in B cells stimulated with alpha delta-dex + Th2 SN. Alpha delta-dex is selective in its failure to costimulate IgE production in that IFN-gamma-containing T cell supernatant (Th1 SN) and transforming growth factor-beta-supplemented Th2 SN selectively stimulate a large IgG2a and IgA secretory response, respectively. Anti-IgD conjugated to Sepharose beads, in distinct contrast to dextran, costimulates a strong IgE response. These findings underscore the importance of the specific B cell activator, in addition to IL-4, in the regulation of IgE production. PMID- 1714475 TI - Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice. AB - Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice. PMID- 1714476 TI - Nonencephalitogenic CD4-CD8- V alpha 2V beta 8.2+ anti-myelin basic protein rat T lymphocytes inhibit disease induction. AB - Recently there has been a number of reports suggesting that CD4-CD8- T cells participate in the processes of inflammatory reaction. In an attempt to delineate the distinctive functions of double negative (DN) T lymphocytes in an autoimmune induced disease, we isolated and cloned such T cells, along with control CD4+ cells, from Lewis rats immunized with guinea-pig myelin basic protein in CFA. Both clones proliferated in response to the guinea-pig myelin basic protein and its synthetic encephalitogenic peptide, and expressed the same TCR V genes homologous to the mouse V alpha 2 and V beta 8.2 families that appear to be the defining entity of experimental autoimmune encephalomyeltis (EAE). Moreover, the TCR D and J region gene products of the DN cell were found to be similar to another encephalitogenic rat T cell clone. The two T clones did not differ markedly in their ability to produce TNF and IL-2 and to adhere to vascular wall derived extracellular matrix- and laminin-coated plates. Surprising, therefore, was the finding that, although the CD4+ T lymphocytes were capable of inducing EAE, the DN cells did not elicit disease but rather inhibited subsequent EAE induction. Thus, TCR V alpha 2V beta 8.2 and its junctional region gene products are not the only prerequisite segment for a T cell to become encephalitogenic. We suggest that the important determinants of the T cell ability to induce disease are features of the T cell, other than or in addition to, the T cell receptor. PMID- 1714477 TI - Lymphotoxin and an associated 33-kDa glycoprotein are expressed on the surface of an activated human T cell hybridoma. AB - A human T cell hybridoma, II-23.D7, was induced with phorbol ester to express a surface form of lymphotoxin (LT, TNF-beta) and an associated 33-kDa glycoprotein. The LT epitopes were detected by surface immunofluorescence staining and by immunoprecipitation from radioiodinated or biosynthetically labeled cells with the use of anti-rLT polyclonal and monoclonal antibodies. The epitopes detected by the antibody were related to LT because adsorption of the anti-rLT with PMA activated II-23.D7 cells resulted in the removal of the neutralizing titer of the anti-rLT antiserum. Immunoprecipitation of surface radioiodinated II-23.D7 cells revealed two bands of 25 kDa and 33 kDa that were specifically precipitated with anti-rLT, but not anti-rTNF antibodies. Enzymatic digestion with glycanases showed both proteins to have N-linked carbohydrate, with O-linked sugar limited to the 25-kDa protein. To determine the biochemical relationship between these proteins, the two LT-like forms were purified from detergent-solubilized II-23.D7 cells by immunoaffinity chromatography. Peptide mapping using CNBr cleavage showed the 25-kDa surface form to be identical to rLT, whereas the 33-kDa protein was different. Biosynthetic labeling studies showed that p33 contained both methionine and cysteine, whereas the p25 contained only methionine. Thus, the surface LT form lacks a leader peptide indicating an anchoring mechanism distinct from that described for membrane TNF. The nature of the attachment of this LT form to the membrane surface is not clear, however, neither TNF receptor binding nor lipid linkages appear to be involved. The accessory protein, p33, may anchor LT to the surface. These findings identify a new characteristic of LT and point toward an additional pathway by which T lymphocytes may mediate cytolytic activity and regulate inflammatory processes. PMID- 1714478 TI - Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1. AB - The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1 stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease. PMID- 1714479 TI - Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants. AB - The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5 positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain. PMID- 1714480 TI - Diversity in the fine specificity and idiotypic profile of mouse anti-HLA-DR monoclonal antibody elicited with the syngeneic anti-idiotypic monoclonal antibody F5-830. AB - Four hundred and sixty-six hybridomas were generated from a BALB/c mouse immunized with the syngeneic anti-idiotypic mAb F5-830 that recognizes an idiotope in the Ag-combining site of mAb AC1.59. At an appropriate concentration, the latter reacts with a determinant expressed by HLA-DR1, DRw8, and DRw9 Ag and subtypes of HLA-DR4 and DRw6 allospecificities. Serologic and immunochemical assays identified eight anti-HLA-DR anti-anti-idiotypic mAb. They are heterogeneous in their reactivity with a panel of HLA-typed B lymphoid cells: like mAb AC1.59, the anti-anti-idiotypic mAb MA1/38, MA1/40, MA1/47, and MA1/98 recognize the determinant shared by HLA-DR1, DRw8, and DRw9 Ag and subtypes of HLA-DR4 Ag. On the other hand, the anti-anti-idiotypic mAb MA1/52, MA1/157, MA1/281, and MA1/285 have a more restricted reactivity, inasmuch as the corresponding determinant(s) is detectable on only some of the allospecificities recognized by mAb AC1.59. Each anti-anti-idiotypic mAb varies in its extent of reactivity with HLA-DR allospecificities. These results suggest differences in the fine specificity of anti-HLA-DR anti-anti-idiotypic mAb and in the structural characteristics of the mAb AC1.59 defined determinant shared by HLA-DR1, DRw8, and DRw9 Ag and subtypes of DR4 allospecificities. Furthermore, the anti-anti idiotypic mAb are heterogeneous in terms of expression of idiotopes and of their spatial relationship with their Ag-combining site. The heterogeneity in the characteristics of anti-HLA-DR antibodies elicited with anti-idiotypic mAb F5-830 suggests that the Id cascade triggered by immunization with incompatible HLA allospecificities may account for the changes in the anti-HLA antibody specificity that have been observed in the course of an immune response to mismatched HLA alloantigens. PMID- 1714481 TI - Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation. AB - Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta pretreated EC. PMID- 1714482 TI - Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain. AB - The CD19 molecule is a 95,000 Mr cell-surface protein of human B lymphocytes with two extracellular Ig-like domains and a 240 amino acid cytoplasmic tail. cDNA encoding human CD19 and the cytoplasmic domain of the mouse CD19 Ag were previously isolated. In this report, those cDNA were used to isolate cDNA or genomic DNA encoding the complete mCD19 protein and a portion of CD19 from the guinea pig. Mouse pre-B and B cell lines expressed two CD19 mRNA species of 2.7 and 2.2 kb, whereas myeloma cell lines were negative as were T cell lines. Similarly, among mouse organs, only spleen contained detectable CD19 mRNA. These results suggest that only B cells express CD19 in mouse, as in man. Sequence determination revealed substantial conservation, with hCD19 and mCD19 being 66% and hCD19 and gpCD19 being 73% identical in amino acid sequence. The cytoplasmic region of CD19 was most highly conserved with human/mouse being 73% identical and human/guinea pig being 83% identical in amino acid sequence. Isolation of the hCD19 and mCD19 genes and determination of exon/intron boundaries revealed that both genes were structurally similar and were composed of at least 15 exons, 4 encoded extracellular domains, and 9 encoded cytoplasmic domains. Six of the exons that encoded cytoplasmic domains were essentially identical in sequence in all three species indicating that these regions have undergone considerable selective pressure to conserve sequences. Thus, CD19 appears to be well conserved in structure and expression through recent mammalian evolution and the highly conserved cytoplasmic domains may play a critical role in the transduction of CD19-mediated signals. PMID- 1714483 TI - In vivo and in vitro expression of Haemophilus influenzae type b lipooligosaccharide epitopes. AB - An indirect immunofluorescence system involving monoclonal antibodies (MAbs) directed against surface epitopes of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) was used to examine individual Hib cells in cerebrospinal fluid (CSF) from infants with Hib meningitis. In four of five CSF samples studied, 100% of the bacteria bound at least one LOS-directed MAb. When the bacteria from these CSF samples were grown in broth, most of these cells lost some or all of their ability to bind the MAb(s) that were bound by the same organisms present in human CSF. When in vitro-grown cells were used for intracisternal injection of rabbits, the populations of Hib cells observed in rabbit CSF after the development of meningitis generally resembled those of the respective broth-grown inocula in terms of their LOS antigenic characteristics. Hib cell populations recovered in infant rat CSF after intranasal challenge again had LOS antigenic characteristics similar to those of the in vitro-grown challenge inocula. These results indicate that a population of Hib cells growing in the infected human host may be quite different, with regard to its LOS antigenic characteristics, from the same Hib strain growing in vitro or in vivo in animal models. PMID- 1714484 TI - Respiratory viruses induce production of histamine-releasing factor by mononuclear leukocytes: a possible role in the mechanism of virus-induced asthma. AB - Histamine-releasing factor (HRF) is a cytokine produced by mononuclear leukocytes when stimulated with antigens or mitogens. HRF is capable of inducing degranulation of basophils and release of histamine. To determine if respiratory viruses can induce HRF production, mononuclear leukocytes from healthy adult donors were exposed to influenza or respiratory syncytial virus in vitro. HRF activity was tested by culturing the supernatants with fresh peripheral blood leukocytes and measuring the percentage of histamine released. Significant enhancement in histamine release was found in both virus groups compared with that of media controls. Thus, mononuclear leukocytes from normal individuals produce HRF in response to exposure to respiratory viruses, suggesting that this cytokine, which causes basophil degranulation, may play a role in the mechanism of virus-induced bronchospasm. PMID- 1714485 TI - Covariance of lowered capacity to induce interferon in human leukocytes and temperature sensitivity of type 3 poliovirus. AB - Attenuated poliovirus strains induce less interferon-alpha (IFN-alpha) in human leukocyte cultures than their parental wild-type strains or other neurovirulent strains. We have used a set of type 3 Leon/Sabin recombinant poliovirus strains to show that production of IFN in this system is closely associated with the genomic region that codes for capsid protein VP3; a mutation in this gene also causes the temperature sensitivity of the attenuated Sabin 3 strain and is partly responsible for the loss of neurovirulence of this vaccine strain. Sixteen independent poliovirus isolates, derived from the Sabin 3 virus but showing in vitro and in vivo markers of neurovirulence, were also tested. These gave IFN yields equivalent to or higher than those obtained with the neurovirulent Leon type 3 poliovirus. The relatively low IFN yields with the Sabin 3 virus seem not to be a direct consequence of its temperature sensitivity, but the two properties coincide with all the type 3 poliovirus strains tested. PMID- 1714486 TI - Regulation of laminin expression by interferon. AB - Regulation of laminin (LMN) expression by interferon (IFN) treatment has been studied in murine LB and 3T3 cells, human lung epithelial (A549) and foreskin fibroblast (FS4) cells, and bovine aortic endothelial cells. Using various morphological and biochemical techniques, our data show an increased expression of LMN following IFN treatment, with a corresponding increase in the mRNA levels. These observations may have considerable significance in various phases of wound healing, since exogenous LMN has been shown to increase the migration of epithelial cells and may lead to rapid healing of burn, traumatic, or infectious injuries. PMID- 1714487 TI - Transforming growth factor-beta inhibits the antiviral action of interferons in human embryonic fibroblasts. AB - Transforming growth factor-beta (TGF-beta) at concentration of 10 ng/ml inhibited the development of the interferon-alpha- (IFN-alpha) or IFN-gamma-induced antiviral state in quiescent human embryonic fibroblasts. The action of the cytokines was dose-related; TGF-beta had no direct effect on the replication of either vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) used as the challenge viruses in the IFN assays. We suggest that despite the fact that TGF-beta acts mainly as a "negative" growth factor, its interactions with IFNs in the antiviral assays resemble known effects of the typical "positive" peptide growth factors. PMID- 1714488 TI - Identification of epididymal proteins associated with hamster sperm. AB - The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm coating proteins. PMID- 1714489 TI - Weak-base amines inhibit the anterograde-to-retrograde conversion of axonally transported vesicles in nerve terminals. AB - Acidotropic weak-base amines were used to investigate the role of acidic compartments in the pathway of aterograde-to-retrograde conversion of axonally transported vesicles in axon terminals. A local concentrated population of nascent axon tips was produced by transecting the rat sciatic nerve in situ to allow local and direct exposure of the axon tips to test solutions. Immersion of the nascent axon tips in solutions containing 10 mM ammonium choloride or 10 mM propylamine caused the axon tips to become distended by an accumulation of elongated membranous tubules and occasional large vacuoles that were both distinct from retrograde organelles. To test whether this accumulation was the result of an impairment of anterograde-to-retrograde conversion, a radioactive pulse-labelling method was used together with a retrograde collection ligature, to quantify the proportion of anterogradely transported proteins that returned from the axon tips by retrograde transport. Exposure of the axon tips to 10 mM ammonium chloride caused the anterogradely transported membrane proteins to accumulate in the axon tips and reduced by about 50% the amount of protein that returned to the retrograde collection ligature. These observations implicate the involvement of acidic membranous compartments in the anterograde-to-retrograde conversion pathway that leads to the formation of retrograde organelles in axon tips. Exposure of nerves to Acridine Orange, which is a vital acidotropic fluorescent dye, confirmed the presence of acidic compartments in the axon tips. Based on these observations, we propose that the membranous tubules that accumulated in the axon tips in the presence of weak-base amines represent a transient intermediate in the pathway of anterograde-to-retrograde conversion of axonally transported vesicles in axon terminals, and that acidic membranous compartments within axon terminals are required for the conversion of these tubules into retrograde organelles. PMID- 1714490 TI - Expression of P0 mRNA in myelinating Schwann cells is related to fibre size. AB - This study examines whether there is a relationship between the abundance of expression for P0 mRNA in myelinated Schwann cells and fibre diameter. Individual teased sciatic nerve fibres from young adult rats were hybridized with radiolabelled probe for P0 mRNA which is expressed in the perinuclear cytoplasm of the mid-internode. Signal intensity was measured as optical density of the developed autoradiograms. A highly significant positive linear correlation was present between signal intensity and fibre diameter. In a companion study, individual fibres were mounted in Araldite resin and transversely serially sectioned at 4 microns for autoradiography. Grain densities were determined for fibres of different diameters. Again, larger diameter fibres were associated with higher grain densities. The results indicate that the abundance of P0 mRNA expressed by a myelin-producing Schwann cell is related to fibre diameter with axonal size probably being the critical determinant. Axons may regulate P0 expression through the number of signalling molecules exposed on or released from the axolemma. PMID- 1714491 TI - Distribution of P0 protein and the myelin-associated glycoprotein in peripheral nerves from Trembler mice. AB - The Trembler mouse has a dysymelination of peripheral nerves that includes hypomyelination, failure of myelin compaction, and demyelination/remyelination. We have localized the myelin proteins P0 and myelin associated glycoprotein in Trembler peripheral nerve and correlated their distributions with the ultrastructure of myelin internodes. Immunocytochemically, myelin-associated glycoprotein was localized in Schwann cell periaxonal membranes, Schmidt Lanterman incisures, paranodal loops, and internal and external mesaxons. P0 staining was located over compact myelin and regions of Schwann cell cytoplasm rich in Golgi membranes. An unusual abundance of small, P0-stained, Golgi-related vesicles was found in some Schwann cells. P0 protein was also detected in multiple spiral wraps of myelin-associated glycoprotein-positive mesaxon membranes. At some sites the periodicity of the myelin membranes was intermediate to that found in mesaxon membranes and compact myelin. The distance between apposing extracellular leaflets was similar to that found in mesaxon membranes, while the cytoplasmic leaflets were fused but twice as thick as normal major dense lines. These intermediate membranes were stained by P0 and myelin associated glycoprotein antiserum. These studies suggest that altered transport and/or translocation of P0 and myelin-associated glycoprotein results in defective myelin compaction in Trembler peripheral nerve. PMID- 1714492 TI - Responses of rod bipolar cells isolated from the rat retina to the glutamate agonist 2-amino-4-phosphonobutyric acid (APB). AB - Isolated rod bipolar cells were obtained by enzymatic (papain) and mechanical dissociation of the adult rat retina. Virtually all intact bipolar cells in the dissociates expressed protein kinase C (PKC) immunoreactivity, a selective marker for rod bipolar cells in the in vivo retina. Whole-cell recordings were performed using nystatin in the patch pipette to minimize washout of those cytoplasmic components necessary for the maintenance of ionic currents. At holding potentials of -33 mV, a tonic inward current was observed. The glutamate agonist 2-amino-4 phosphonobutyrate (APB) reduced this current by closing ion channels. Under normal conditions, Na+ appeared to be the main charge carrier. Both the internal and the external Ca2+ concentrations were found to exert a powerful influence on the APB-sensitive current. We conclude that the rod bipolar cell in situ is depolarized at light onset. PMID- 1714493 TI - Tau gene expression in rat sensory neurons during development and regeneration. AB - In the mature rat dorsal root ganglion (DRG), only one tau isoform is expressed, and this protein (110 kDa in apparent molecular weight) is considerably larger in size than the predominant tau isoforms found in brain. The size of the mRNA encoding the "big" tau mRNA in DRG [approximately 8 kilobases (kb)] is also much larger than that of the major rat brain tau mRNA species (approximately 6 kb). In this study, we examined the pattern of normal developmental changes in expression of this high-molecular-weight (HMW) tau and its encoding mRNA and also determined how axonal injury of adult DRG neurons effected the expression of this gene. RNA blotting experiments revealed that higher levels of HMW tau mRNA were present in the DRG at early postnatal times than in the adult. Immunoblotting of total DRG protein using a monoclonal tau antibody revealed that the immature DRG (7 d postnatal) contained a 62-kDa tau isoform in addition to the HMW tau isoform that was expressed in the adult DRG. Neither of the tau isoforms expressed in the immature DRG was present to any significant extent in either immature or adult rat brain. To examine how tau expression changed in adult DRG neurons during regeneration, the sciatic nerves of rats were unilaterally crushed, and the L4 and L5 DRG were harvested 1, 7, and 14 d later.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714494 TI - Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms. AB - Past studies revealed that NGF and fibroblast growth factor (FGF) prevent the death of PC 12 pheochromocytoma cells that otherwise occurs in serum-free medium. Additional agents were tested here for their abilities to promote long-term survival of naive and NGF-pretreated (primed) PC 12 cells in serum-free conditions. Forskolin and permeant cAMP analogs effectively prevented serum-free cell death, as did micromolar levels of insulin and 10-100-nM levels of insulin like growth factors I and II. In contrast to NGF and FGF, none of these agents caused neuronal differentiation of naive cells or neurite regeneration by primed cells. Each of the agents also prevented rapid cell death in a balanced salt solution, thus apparently ruling out a mechanism dependent on regulation of nutrient uptake. Epidermal growth factor and elevated K+ appeared to slow the rate of cell death, but did not promote long-term survival; phorbol ester, dexamethasone, or vanadate did not prevent cell death. Each of the survival promoting agents was effective even when macromolecular synthesis was blocked. Because the synthesis inhibitors themselves did not significantly prevent cell death, such findings indicate that survival was promoted by mechanisms that do not require synthesis of RNA or protein. In addition, various lines of experimental evidence (using the kinase inhibitor K-252a or PC 12 cell variants deficient either in protein kinase A activity or in responsiveness to NGF) further suggested that the effective agents maintain survival by independent initial pathways. Regulation of protein kinase activity appears to be a common feature of each pathway and may therefore play a key convergent role in mediating prevention of cell death. PMID- 1714495 TI - Sustained increase in intracellular calcium promotes neuronal survival. AB - Ciliary ganglion neurons, half of which normally suffer developmental death in the embryo, will survive in culture in medium supplemented with depolarizing concentrations of potassium. It is not known how elevated potassium acts inside the cell to promote survival. We report here that depolarizing concentrations of extracellular potassium promote neuronal survival by causing a sustained increase in intracellular calcium. Raising extracellular potassium from 5 to 40 mM, an optimal concentration for survival, caused a sustained increase in intracellular calcium from 250 nM to greater than 600 nM. By 26 hr, at which time greater than 90% of neurons in 5 mM potassium had died, the calcium concentration of neurons in 40 mM potassium was still above 400 nM. Reduction of extracellular potassium from 40 to 5 nM, which prevents the increase in survival, also reduced intracellular calcium back to rest levels. PN200-110, a dihydropyridine calcium channel blocker that inhibits the survival-promoting effect of elevated potassium, also prevented and reversed the potassium,-mediated increase in intracellular calcium. In addition, there was a strong, quantitative correlation between the percentage of neuronal survival and the intracellular calcium concentration over a wide range of extracellular potassium concentrations. These results suggest that elevated potassium opens dihydropyridine-sensitive calcium channels, causing a sustained increase in intracellular calcium that quantitatively determines the number of surviving neurons. PMID- 1714496 TI - Specialized vascularization of the primate visual cortex. AB - We have analyzed blood vessel distribution in the primary and secondary visual cortices of the squirrel monkey in relation to cortical modules, laminae, and cytoarchitectonic areas. Measurements of microvessel length in tangential sections through the primary visual cortex showed that blobs are more richly vascularized than intervening cortical regions. Thus, the mean total length of microvessel profiles per unit was 42% greater within these cortical modules than within adjacent (interblob) areas. Total microvessel length per unit area in another class of module, the stripes in the secondary visual cortex, was 27% greater than in interstripe regions. Microvessel distribution also varied systematically from layer to layer in the primary visual cortex, being greatest in lamina IVc. Finally, the overall microvessel length per unit area in sections of the primary visual cortex was 26% greater than that in the secondary visual cortex. These observations indicate that the modular, laminar, and regional organization of the primate visual cortex is reflected in the underlying distribution of cortical microvessels. These vascular patterns should be discernable in living animals with vascular contrast agents and appropriate imaging techniques. PMID- 1714497 TI - PET studies of fluorodeoxyglucose metabolism in patients with recurrent colorectal tumors receiving radiotherapy. AB - Forty-four patients with recurrent colorectal carcinoma were examined prior to a combination of conventional photon radiotherapy (40 Gy) and neutron therapy (10 Gy). Twenty-one of these underwent a PET examination after photon therapy and 12 also were studied after the end of combined therapy. CEA plasma levels were measured from blood samples taken immediately before the PET study. A significant decrease in FDG uptake despite good palliative results were observed in only 50% of the patients. This may be explained by inflammatory reactions caused by radiation injury. Inflammation and metabolically active residual tumor tissue cannot be distinguished. It is concluded that an observation interval longer than 6 mo may more effectively detect residual tumor activity. In 14 of 41 examinations, an increased FDG uptake was associated with a normal CEA value, and in only two cases were normal FDG uptake values and increased CEA levels found, suggesting that PET is more sensitive than the measurements of CEA plasma levels for tumor recurrence. PMID- 1714498 TI - A patient care travelling record for palliative care: a feasibility study. AB - The provision of palliative care can be a complex process. Patients are treated in a variety of settings, by multiple persons, thus risking loss of continuity of care. These patients take numerous medications and require many complex treatment decisions in the course of their illness, making the ready availability of current and accurate information a vital component of effective care. The use of a patient care travelling health record, while requiring time and commitment from all parties to be effective, has been shown to be both feasible and helpful to patients, families, and health professionals. Considerable education and commitment is necessary to ensure compliance by all involved parties. PMID- 1714499 TI - Can one die healed? PMID- 1714500 TI - Symptom prevalence and control during cancer patients' last days of life. PMID- 1714501 TI - Palliative care. PMID- 1714502 TI - The Edmonton Symptom Assessment System (ESAS): a simple method for the assessment of palliative care patients. AB - We describe a simple method for the assessment of symptoms twice a day in patients admitted to a palliative care unit. Eight visual analog scales (VAS) 0 100 mm are completed either by the patient alone, by the patient with nurse's assistance, or by the nurses or relatives at 10:00 and 18:00 hours, in order to indicate the levels of pain, activity, nausea, depression, anxiety, drowsiness, appetite, and sensation of well-being. The information is then transferred to a graph that contains the assessments of up to 21 days on each page. The sum of the scores for all symptoms is defined as the symptom distress score. The Edmonton Symptom Assessment System (ESAS) was carried out for 101 consecutive patients for the length of their admission to our unit. Of these, 84% were able to make their own assessment sometime during their admission. However, before death 83% of assessments were completed by a nurse or relative. Mean symptom distress score was 410 +/- 95 during day 1 of the admission, versus 362 +/- 83 during day 5 (p less than 0.01). Mean symptom distress scores throughout the hospitalization were 359 +/- 105, 374 +/- 93, 359 +/- 91 and 406 +/- 81 when the ESAS was completed by the patient alone, patient with nurse's assistance (p = N.S.), nurse alone (p = N.S.), or relative (p less than 0.01) respectively. We conclude that this is a simple and useful method for the regular assessment of symptom distress in the palliative care setting. PMID- 1714503 TI - The effect of platelet-activating factor on histamine release from guinea pig peritoneal mast cells. AB - The effect of platelet-activating factor (PAF) on histamine release from the peritoneal mast cells of male guinea pigs at 4 weeks of age and one week of age (weaning) was investigated. PAF as well as compound 48/80 and concanavalin A were not found to release histamine from the mast cells of either age of guinea pigs. On the other hand, Ca2+ ionophore A23187 showed a significant, concentration dependent histamine release from the mast cells obtained from guinea pig of either age group. PAF (3 x 10(-7) - 3 x 10(-6) g/ml) significantly inhibited the histamine release induced by Ca2+ ionophore A23187 from the mast cells of guinea pigs at one week of age, but not from those of the older ones. Such an inhibition was not seen with lyso-PAF in either age group. CV-3988, a PAF antagonist, neutralized the inhibitory effect of PAF on the A23187-induced histamine release from the mast cells of guinea pigs at one week of age. These results indicate that PAF does not have a histamine-liberating action on guinea pig peritoneal mast cells, and that PAF inhibits the effect of A23187 on histamine release from mast cells through activation of PAF receptor in guinea pigs at one week of age. PMID- 1714504 TI - Crystal structure of a Y35G mutant of bovine pancreatic trypsin inhibitor. AB - The structure of a Y35G mutant of bovine pancreatic trypsin inhibitor (BPTI) was solved by molecular replacement and was refined by both simulated annealing and restrained least-squares at 1.8 A resolution. The crystals belong to the space group P42212, with unit cell dimensions a = b = 46.75 A, c = 50.61 A. The final R factor is 0.159 and the deviation from ideality for bond distances is 0.02 A. The structure of the mutant differs from that of the native protein, showing an overall root-mean-square (r.m.s.) difference of 1.86 A for main-chain atoms. However, the change is mostly localized in the two loops (respective r.m.s. values of 2.04 A and 3.93 A) and the C terminus (r.m.s. 6.79 A), while the core of the protein is well conserved (r.m.s. 0.45 A). The change in the loop regions can be clearly attributed to the mutation while the difference in the C terminus might be only due to a different crystal packing. Seventy water molecules were included in the model but only seven of them are shared with the native structure. Thermal parameters are showing a good correlation with those for the wild-type of BPTI. PMID- 1714505 TI - Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. AB - The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3' 5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication. PMID- 1714506 TI - A sensitive fluorometric method for measurement of vascular permeability in spinal cord injury. AB - A sensitive fluorometric method was modified for the evaluation of drug action upon vascular permeability in rat spinal cord injury. Fluorescein isothiocyanate conjugated dextran (FITC-D MW 71,200), used as a macromolecular tracer, was injected iv 2 hours before sacrifice. The optimal pH for FITC-D fluorescence was 8.2. The recovery in spinal cord was 101.4 +/- 4.0% (mean +/- SD). The extent of FITC-D extravasation, expressed as the vascular injury index (VII), was increased in proportion to the trauma force. The peak of VII after trauma was at 2 hours. This fluorometric method is sensitive, simple, and reliable for evaluation of drug effects upon vascular permeability in CNS trauma. PMID- 1714507 TI - Phase I trial of fluorouracil modulation by N-phosphonacetyl-L-aspartate and 6 methylmercaptopurine riboside: optimization of 6-methylmercaptopurine riboside dose and schedule through biochemical analysis of sequential tumor biopsy specimens. AB - Preclinical and clinical studies demonstrate that the selective antitumor activity of fluorouracil (5-FU) is enhanced by agents which perturb certain intracellular nucleotide pools. We previously demonstrated that the combination of N-phosphonacetyl-L-aspartate (PALA), which depletes pyrimidine nucleotide pools, and 5-FU yielded a 43% response rate among 37 assessable patients with colorectal carcinoma. In preclinical tumor models, 6-methylmercaptopurine riboside (MMPR), an inhibitor of purine synthesis, elevates phosphoribosylpyrophosphate (PRPP) pools and promotes the anabolism of 5-FU to fluorinated nucleotides. In vivo, the addition of MMPR enhances the therapeutic efficacy of PALA-5-FU. In a phase I trial, we sought to determine the optimal dose and schedule of MMPR in combination with PALA (250 mg/m2 on day 1) and 5-FU (1300 mg/m2 by 24-hour infusion on day 2). MMPR (75-225 mg/m2) was given intravenously on day 1 to 27 patients with solid tumors (15 colorectal, seven breast, five other). Toxic effects were mild to moderate and included leukopenia, mucositis, nausea, or rash. Two of seven patients given MMPR at 225 mg/m2 had grade 3 diarrhea. PRPP was measured using a [14C]orotic acid 14CO2 release assay in tumor biopsy specimens obtained before and 12 hours and 24 hours after MMPR doses were given to 20 patients. The addition of MMPR elevated PRPP pools in human solid tumors. At 12 hours after treatment, two (50%) of four patients showed a twofold or greater elevation of PRPP at the MMPR dose level of 75 mg/m2; a similar elevation was observed in five (71%) of seven patients given 150 mg/m2 MMPR and in three (43%) of seven patients given 225 mg/m2 MMPR. At 24 hours after treatment, results for the respective dose levels of MMPR were two (33%) of six patients, one (20%) of five patients, and four (57%) of seven patients. Administration of the two highest MMPR dose levels appeared to result in a greater increase in tumor PRPP levels. However, toxicity was greater at the 225 mg/m2 dose level; therefore, the 150 mg/m2 dose level was favored. Tumor levels of PRPP decreased between 12 hours and 24 hours in nine (56%) of 16 patients. This time course indicates that MMPR should be administered at the beginning of the 24-hour infusion of 5-FU. PMID- 1714508 TI - Isolation and structural characterization of the murine tryptophan hydroxylase gene. AB - The mouse tryptophan hydroxylase gene was isolated and its intron/exon boundaries and putative regulatory sequences identified. To isolate the gene a mouse mastocytoma cDNA clone encoding tryptophan hydroxylase was used to identify and isolate ten overlapping DNA fragments from a mouse genomic library. Restriction mapping and sequence analysis of the clones revealed that the gene contains 11 exons and covers a region of DNA of approximately 21 kb. The transcription initiation site was mapped and the major site of initiation yields an untranslated leader sequence of 124 nucleotides. A minor initiation site is located 9 nucleotides 3' of the major site. The 5' untranslated sequence is interrupted by the first intron. Analysis of the sequence upstream of the initiation site showed the presence of several putative promoter and regulatory sequences. Nine of the ten intron/exon boundaries of tryptophan hydroxylase are conserved with tyrosine hydroxylase and phenylalanine hydroxylase, further delineating the evolutionary relationship of these three genes. PMID- 1714509 TI - Identification of mitochondrial and non-mitochondrial glutaminase within select neurons and glia of rat forebrain by electron microscopic immunocytochemistry. AB - Antibodies against the mitochondrial enzyme glutaminase (EC 3.5.1.2), have been used in previous immunocytochemical studies to help identify glutamate-releasing neurons among all glutamate-containing neurons. The studies were based on the idea that glutaminase is enriched within the releasable "transmitter" pools of glutamate. However, evidence is also available to suggest that the enzyme does not occur exclusively within glutamate-releasing neurons. Thus we sought to determine whether glutaminase was immunocytochemically detectable within presynaptic terminals forming asymmetric (putatively excitatory) synapses or, alternatively, occurs in association with mitochondria throughout the cell. For this purpose, we examined the cellular and subcellular distribution of glutaminase- immunoreactivity in neocortical (visual and somatosensory) areas known to contain glutamatergic perikarya. This localization was compared with the distribution in striatal (caudate-putamen and nucleus accumbens) regions recognized to contain high densities of glutamatergic terminals but fewer, if any, glutamatergic perikarya. Glutaminase-immunoreactive perikarya were numerous within the infragranular laminae of neocortex (approximately 1 per 1,000 microns 2 tissue area) but sparse within the caudate-putamen nuclei and accumbens nuclei (less than 1 per 20,000 microns 2.). In addition, heterogeneous distribution of small (less than 1 microns) punctate immunoreactive structures was notable. Relatively high densities of these punctate structures occurred within the supragranular laminae of neocortex, dorsolateral quadrant of the caudate-putamen nuclei, and surrounding certain groups of myelinated fiber bundles throughout the striatum. Electron microscopy revealed diffusely distributed peroxidase immunoreactivity in a select population of dendritic spines, glial processes, and axons. Eight percent of all synapses within the supra-granular laminae were formed by terminals labeled for glutaminase. These principally formed asymmetric junctions on spiny processes. When tissue was incubated with the antibody in the presence of a permeabilizing agent, Photo-flo, high levels of glutaminase immunoreactivity was detectable by electron microscopy within select mitochondria of neocortical (4%) and striatal (8%) perikarya and dendrites, while the diffuse distribution of immunoreactivity within axons and glia was greatly diminished. The differential ultrastructural conditions provide direct demonstration that glutaminase in brain occurs in at least two forms discriminable by their diffuse distribution within non-mitochondrial cytoplasm versus discrete localization within mitochondria. The morphological characteristics of synapses formed by axons exhibiting diffuse distributions of glutaminase immunoreactivity are consistent with the idea that glutaminase-enriched terminals mediate excitatory chemical transmission via the release of glutamate. Because glia containing glutaminase occur juxtaposed to the asymmetric junctions, the glia may utilize neuronally released glutamate for energy metabolism. PMID- 1714510 TI - Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons. AB - A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+) dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments. PMID- 1714511 TI - Expression of the beta-nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig. AB - Steady-state nerve growth factor (NGF) mRNA levels were estimated in male sex organs of the mouse, rat, and guinea pig by RNA blot hybridization analysis. The abundance of NGF mRNAs was in the order vas deferens greater than epididymis greater than or equal to seminal vesicles much greater than testis. NGF mRNA levels in these organs were compared with those estimated for other rat peripheral tissues and were found to correlate with the density of their sympathetic innervation, with the exception of guinea pig prostate. Castration had no significant effect on NGF mRNA levels in the guinea pig prostate, suggesting that NGF synthesis in this tissue is not under direct androgen control. NGF-like and proNGF-like immunoreactivities were localized by immunohistochemical techniques in the secretory cells of the glandular epithelium of the guinea pig prostate and in germ cells in the seminiferous tubules of the mouse testis. PMID- 1714512 TI - Sequelae of experimental tympanic and inferior canal wall perforations: the double meaning of epithelial migration. AB - The term "epithelial migration" has been used to describe both the normal surface movement of the tympanic epithelium and the movement of epithelial basal cells in repair processes. In an attempt to distinguish between these two processes and to determine their role in the repair of a wounded tympanic membrane, 20 guinea pig tympanic membranes were perforated inferiorly and tattooed with ink through an external incision. Sequential histology of whole temporal bones at intervals from the time of injury up to three weeks showed evidence of movement of the superficial layers of epidermis which was effective in the clearance of cellular debris but not in the closure of the perforation. Drum closure was effected more by the accumulation of exudate and epithelial hyperplasia. The migration of the epithelial basal cells was slow when compared to surface movement. It is suggested that tympanic epithelial surface movement is best described by a passive term such as "epithelial displacement" and that the term migration should be restricted to the activities of the living layers of epidermis. PMID- 1714513 TI - Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. AB - The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA primer bound near the 5' end of the genomic RNA at a position termed the primer-binding site (PBS). The PBS is an 18-nucleotide region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714514 TI - Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1. AB - Using antibodies directed against the TYB1 protein of the transpositionally competent retrotransposon Ty1-H3, we have identified three mature proteins of 23, 60, and 90 kDa and processing intermediates of 140 and 160 kDa that are derived from the 190-kDa TYA1-TYB1 polyprotein. Mature proteins and variable amounts of the precursors cofractionate with Ty viruslike particles. The map locations and precursor-product relationships of mature TYB1 polypeptides suggest that p23 is Ty1 protease, p90 is integrase, and p60 contains reverse transcriptase and RNase H. Immunoprecipitation and immunoblot analyses of Ty1 proteins show that p190 is cleaved to form p160. The p160 intermediate is cleaved to form p23 and p140, and p140 is cleaved to form p90 and p60. Processing of TYB1 proteins is dependent on Ty1 protease. Immunoblot analysis of TYB proteins from different Ty1 isolates reveal that correct processing of TYB1 proteins is a characteristic of functional Ty1 elements, whereas aberrant processing is a common defect found in transposition-incompetent elements. PMID- 1714515 TI - Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants. AB - To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization. PMID- 1714516 TI - Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16. AB - There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant. PMID- 1714517 TI - Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer. AB - Retroviruses mutate at a high rate during replication. We used a spleen necrosis virus-based vector system and helper cell line to characterize mutations occurring during a single round of retrovirus replication. The vector used, JD216HyNeo, codes for two drug resistance genes, hygromycin resistance (hygro) and neomycin resistance (neo). The downstream neo gene is expressed only when a mutation alleviates a block to splicing which is located in the upstream hygro gene. The mutations allowing splicing were large deletions, ranging in size from about 500 to about 2,000 bp. Most of the mutant proviruses lacked the encapsidation sequence, as shown by our inability to rescue the mutant proviruses with wild-type reticuloendotheliosis virus strain A and confirmed by Southern blotting and direct DNA sequence analysis. We therefore concluded that most of the deletions arose during reverse transcription in the target cell, rather than during transcription in the host cell. The sequence data also indicated that the deletions occurred by at least three different mechanisms: (i) misalignment of the growing point; (ii) incorrect synthesis and termination in the primer-binding sequence during synthesis of the plus-strand strong-stop DNA; and (iii) incorrect synthesis and termination before the primer-binding sequence during synthesis of the plus-strand strong-stop DNA. The second mechanism also led to the incorporation of cellular sequences into the proviral genome, pointing to a potential novel mechanism by which retroviruses can acquire cellular genes. PMID- 1714518 TI - Characterization of monoclonal antibodies against the human immunodeficiency virus matrix protein, p17gag: identification of epitopes exposed at the surfaces of infected cells. AB - Eight monoclonal antibodies reactive with the matrix protein of human immunodeficiency virus type 1 (HIV-1), p17gag, were isolated from rats which had been immunized with solubilized HIV-1 lysate. The epitope specificities of these antibodies were determined with a series of synthetic peptides representing overlapping regions of p17. Six of the antibodies were mapped to three distinct regions of p17, while two antibodies (G11g1 and G11h3) reacted only with intact recombinant p17, suggesting that they were directed against conformational or discontinuous epitopes. All the antibodies bound to HIV-infected cells which had been permeabilized with acetone, but only G11g1 and G11h3 reacted with live HIV infected cells. Specificity studies with diverse virus strains demonstrated that these two antibodies recognized distinct epitopes, one which was group specific for HIV-1, and one which was shared with HIV type 2 and simian immunodeficiency virus. Binding competition studies indicated that these epitopes were proximal in native p17. Despite their reactivity with intact cells, these two antibodies did not possess appreciable virus-neutralizing activity. These results indicate that a form of p17 is expressed on the surfaces of live HIV-infected cells which is accessible to some, but not all, antibodies against p17. These cell surface molecules may play a role in the generation of antibodies against p17gag that are characteristic of early stages of HIV infection, and they may act as natural targets for the immune system and as potential targets for immunotherapy of HIV infected cells. PMID- 1714519 TI - Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein. AB - Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1 specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans. PMID- 1714520 TI - Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies. AB - Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I. PMID- 1714521 TI - Endogenous interferon specifically regulates Newcastle disease virus-induced cytokine gene expression in mouse macrophages. AB - In macrophages from inbred mice, the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the If-1 locus, which carries the allele for either high (h) or low (l) IFN production. Here, we report that the activity of genes within the If-1 locus is influenced by macrophage-derived endogenous IFN. In addition to various other biological effects, we observed that endogenous IFN specifically downregulated NDV-induced IFN and interleukin 6 production. Preculture of bone marrow-derived macrophages (BMM) from BALB/c (If-1l) mice in macrophage colony-stimulating factor plus anti IFN-beta provoked a 30- to 50-fold increase in NDV-induced cytokine production compared with induced control cultures in macrophage colony-stimulating factor alone, whereas only a 4- to 6-fold increase was observed in anti-IFN-beta-treated BMM from C57BL/6 (If-1h) mice. This resulted in nearly complete abrogation of the genetically determined difference in the response to NDV. The increase was specific for NDV and was marked by strong additional activation of IFN-alpha genes. Studies using BMM from B6.C-H28c If-1l congenic mice gave results identical to those obtained with BALB/c BMM. Addition of 20 IU of recombinant IFN alpha 4 to anti IFN-beta-treated macrophages from B6.C-H28c mice 20 h prior to NDV infection strongly downregulated the IFN-alpha, IFN-beta, and interleukin 6 responses. The genetic difference between macrophages from If-1h and If-1l mice was thus reestablished, since the same treatment caused only weak reduction of NDV-induced cytokine gene expression in BMM from C57BL/6 mice. These data suggest that the If-1h and If-1l alleles harbor IFN-inducible genes that, following activation, specifically suppress subsequent cytokine gene expression in response to NDV. PMID- 1714523 TI - Several CD4 domains can play a role in human immunodeficiency virus infection in cells. AB - The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells. PMID- 1714522 TI - Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. AB - Human immunodeficiency virus type 1 (HIV-1)-specific pyridinone reverse transcriptase (RT) inhibitors prevent HIV-1 replication in cell culture (M. E. Goldman, J. H. Nunberg, J. A. O'Brien, J.C. Quintero, W. A. Schleif, K. F. Freund, S. L. Gaul, W. S. Saari, J. S. Wai, J. M. Hoffman, P. S. Anderson, D. J. Hupe, E. A. Emini, and A. M. Stern, Proc. Natl. Acad. Sci. USA 88:6863-6867, 1991). In contrast to nucleoside analog inhibitors, such as AZT, which need to be converted to triphosphates by host cells, these compounds act directly to inhibit RT via a mechanism which is noncompetitive with respect to deoxynucleoside triphosphates. As one approach to define the mechanism of action of pyridinone inhibitors, we isolated resistant mutants of HIV-1 in cell culture. Serial passage in the presence of inhibitor yielded virus which was 1,000-fold resistant to compounds of this class. Bacterially expressed RTs molecularly cloned from resistant viruses were also resistant. The resistant RT genes encoded two amino acid changes, K-103 to N and Y-181 to C, each of which contributed partial resistance. The mutation at amino acid 181 lies adjacent to the conserved YG/MDD motif found in most DNA and RNA polymerases. The mutation at amino acid 103 lies within a region of RT which may be involved in PPi binding. The resistant viruses, although sensitive to nucleoside analogs, were cross-resistant to the structurally unrelated RT inhibitors TIBO R82150 (R. Pauwels, K. Andries, J. Desmyter, D. Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykanti, K. Schellekens, M. A. C. Janssen, E. De Clercq, and P. A. J. Janssen, Nature [London] 343:470-474, 1990) and BI-RG-587 (V. J. Merluzzi, K. D. Hargrave, M. Labadia, K. Grozinger, M. Skoog, J. C. Wu, C.-K. Shih, K. Eckner, S. Hattox, J. Adams, A. S. Rosenthal, R. Faanes, R. J. Eckner, R. A. Koup, and J. L. Sullivan, Science 250:1411-1413, 1990). Thus, these nonnucleoside analog inhibitors may share a common binding site on RT and may all make up a single pharmacologic class of RT inhibitor. This observation may have important implications for the clinical development of these compounds. PMID- 1714525 TI - The vpx gene of simian immunodeficiency virus facilitates efficient viral replication in fresh lymphocytes and macrophage. AB - vpx is a unique open reading frame found in simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) but not in HIV-1. It encodes a 12 to 16-kDa virion-associated protein. Although vpx is dispensable for viral replication in several established human lymphocyte cell lines, there is no consensus regarding whether this gene is required for efficient viral replication in freshly isolated lymphocytes. We report here that the vpx mutant of SIVmac exhibits different degrees of impairment from wild-type SIVmac in freshly isolated lymphocytes. This defect is more pronounced in macrophages from the same donors. Our findings suggest that vpx is required for efficient viral replication in fresh lymphocytes and macrophages. PMID- 1714526 TI - International medical education. Common elements in divergent systems. PMID- 1714524 TI - Localization of immunogenic domains in the human immunodeficiency virus type 2 envelope. AB - Highly immunogenic domains have not yet been defined in the extracellular protein of the human immunodefiency virus type 2 (HIV-2) envelope. In this study, six contiguous segments covering the entire HIV-2ST envelope were amplified and cloned into a bacterial expression vector to localize the relative immunogenic reactivity of different regions of the molecule by Western blot (immunoblot) analysis. Our results demonstrate that the extracellular protein of the HIV-2 envelope contains highly immunogenic epitopes with potential value as type specific markers for HIV-2 infection. PMID- 1714527 TI - From the Centers for Disease Control. Update: cholera--Western Hemisphere, and recommendations for treatment of cholera. PMID- 1714528 TI - [Study of heterochromatin of metaphasic chromosomes in cultured lymphocytes of astronauts using C-stain]. PMID- 1714529 TI - High prevalence of benign prostatic hypertrophy in the community. AB - There is a strong suspicion among urologists that the prevalence of benign prostatic hyperplasia is higher than has been reported in clinical retrospective and necropsy studies. To find out the prevalence in one community all men aged 40 79 years registered with a group general practice were invited to complete a urinary symptom questionnaire and to undergo uroflowmetry. 705 men (77% of those eligible) participated. 214 men (84% of those invited) with signs and symptoms of prostatic dysfunction subsequently underwent transrectal ultrasonography (TRUS) for assessment of the volume (and by inference weight) of their prostates. The prevalence rate of benign prostatic hypertrophy (BPH), defined as enlargement of the prostate gland of equivalent weight greater than 20 g in the presence of symptoms of urinary dysfunction and/or a urinary peak flow rate less than 15 ml/s and without evidence of malignancy, was 253 (95% CI 221-285) per 1000 men in the community, rising from 138 per 1000 men aged 40-49 years to 430 per 1000 men aged 60-69 years. Thus apparently well men have a much higher frequency of BPH than was previously thought to be the case. PMID- 1714530 TI - Phylogeny of the Whipple's-disease-associated bacterium. AB - Efforts to culture and identify the intracellular bacteria associated with Whipple's disease have been unsuccessful. Nucleotide sequencing and amplification by the polymerase chain reaction was done on the bacterial 16 S ribosomal DNA present in a small-bowel biopsy specimen taken from a patient with Whipple's disease. A search by computer for similar rRNA sequences filed in databases showed the Whipple's-associated organism to be most similar to bacteria of the Rhodococcus, Streptomyces, and Arthrobacter genera, and more weakly related to mycobacteria. The biopsy specimen was estimated to contain around 10(7) cells of the organism. The probable aetiological agent for our patient's illness has not been identified previously in a patient with Whipple's disease. PMID- 1714531 TI - Ondansetron vs dexamethasone for chemotherapy-induced emesis. PMID- 1714532 TI - Comparison of ondansetron and ondansetron plus dexamethasone as antiemetic prophylaxis during cisplatin-containing chemotherapy. AB - Ondansetron, a serotonin antagonist, is effective in controlling the emesis associated with cancer chemotherapy; however, emesis in patients receiving high dose cisplatin is poorly controlled by ondansetron alone. Dexamethasone is an effective antiemetic with no known interaction with serotonin receptors and was thus chosen for study in combination with ondansetron. 31 patients (30 male, 1 female; median age 28.5 years, range 18-49) receiving a 4-day course of a chemotherapy regimen containing cisplatin (100-120 mg/m2) for metastatic germ cell tumours were entered in a randomised, double-blind, cross-over trial comparing oral ondansetron plus placebo with oral ondansetron plus dexamethasone as antiemetic prophylaxis. Ondansetron (8 mg every 8 h) was given to all patients for 8 days from the start of chemotherapy. Patients were given 8 mg of dexamethasone or placebo every 8 h starting 2 h before cisplatin (on day 4) and continuing for six doses (ie, for 2 days only). A second course of chemotherapy began 14 days after the start of the first, during which patients crossed over to the alternative antiemetic regimen. Results were available from 27 patients. In the 24-48 h after cisplatin 78% of patients taking ondansetron plus dexamethasone reported complete or major control of emesis compared with 30% of those taking ondansetron plus placebo (p = 0.001). Cross-over analysis showed a significant advantage for ondansetron plus dexamethasone in the control of nausea (p = 0.013) and emesis (p less than 0.001) over the 8-day study. 24 of 26 patients expressed a preference for the combination therapy (p less than 0.001). Ondansetron plus dexamethasone is effective antiemetic prophylaxis for high-dose cisplatin chemotherapy, has few side effects, and is active when given orally. PMID- 1714533 TI - [Revascularization of colonic anastomoses]. AB - The revascularization of colonic anastomosis after colon segmental resection in rabbits in 9 different end-on-end and inverting suture techniques was examined macroscopically, microangiographically, micropreparatorily and histologically. Independently of the suture technique revascularization started 4 days after surgery. 8 days postoperatively the suture line is mainly bridged, 14 days postoperatively the vasal connection to the opposite side is completed. The new vessels mainly originate from the subserosal and submucosal tissue. The adhesions participate in the revascularization, too. It (the revascularization) directly correlates with the development of granulation tissue. This is evident from the excess reactive revascularization of abscesses and ulcers in the anastomoses. There always results a vascular scar in the angioarchitecture of the colonic wall. Start and extent of the revascularization cannot--in contrast to former literature--be looked at as a guarantee of quality for the healing of anastomoses. PMID- 1714534 TI - The insulin-like growth factor binding protein-1 in low and high insulin responders before and during dexamethasone treatment. AB - To investigate the influence of normal insulin levels on levels of the insulin like growth factor binding protein-1 (IGFBP-1) we measured this peptide postabsorptively and during hyperglycemic clamp in 17 healthy subjects, nine with low insulin response (LIR) and eight with high insulin response (HIR). The study was performed before and after 60 hours of treatment with dexamethasone 6 mg/d. The fasting levels of IGFBP-1 were significantly higher in LIR, 36 +/- 2.5 micrograms/L, than in HIR, 22 +/- 2.6 micrograms/L (P less than .01), while no differences in glucose, insulin, and C-peptide concentrations were found. Dexamethasone induced an increase in basal concentrations of insulin, while IGFBP 1 levels decreased to 18.8 +/- 2 micrograms/L in LIR (P less than .01) and to 14.0 +/- 0.9 micrograms/L in HIR (P less than .05). There was no correlation between the individual basal IGFBP-1 concentrations and basal insulin levels. In contrast, basal levels of IGFBP-1 were inversely correlated to the integrated insulin or C-peptide concentrations during the hyperglycemic clamp both before (r = -.67, P less than .01) and during dexamethasone (r = -.79, P less than .001). Dexamethasone, which increased the insulin resistance, did not change the relationship between basal IGFBP-1 and the glucose-induced insulin release. In conclusion, the morning levels of IGFBP-1 in healthy subjects reflect the acute beta-cell responsiveness to glucose, which may correspond to integrated diurnal insulin levels. The inhibitory effects of dexamethasone on the morning levels of IGFBP-1 can be explained by attendant hyperinsulinemia. PMID- 1714535 TI - Serological studies on Leptospira strain Ictero No. I using monoclonal antibodies. AB - In order to elucidate the full serological characteristics of strain Ictero No. I, the first strain of Leptospira isolated by Inada and Ido in 1914, 17 monoclonal antibodies against the strain were produced by cell fusion technology. The antibody-producing hybridomas were designated IMAs 1 to 17. The reactivities of the monoclonal antibodies produced by the hybridomas were determined by the microscopic agglutination test. One of the 17 monoclonal antibodies, IMA 1, reacted with strains Ictero No. I, LT 1131 and Naam, but not with other strains of the serogroup Icterohaemorrhagiae including strain RGA used in the present study. Moreover, the reactivity of the antigenic determinant of IMA 1 was inactivated by treatment at 56 C for 30 min. The 16 other monoclonal antibodies, IMAs 2 to 17, showed different reaction patterns against Leptospira strains of the serogroup Icterohaemorrhagiae. All of the 16 antibodies reacted with both Ictero No. I and RGA strains. The antigens against antibodies IMAs 2 to 17 were thermostable. The present study thus clarified the presence of a thermolabile antigen in strain Ictero No. I, and allowed correct distinction between the serotype of strain Ictero No. I and that of strain RGA using IMA 1. Therefore, we propose that strain Ictero No. I represents serovar icterohaemorrhagiae, as originally designated by Inada and Ido. Moreover, strain Ictero No. I should be designated as the type strain of Leptospira interrogans. PMID- 1714536 TI - [Interferons. Basic principles and use in clinic and general practice]. PMID- 1714537 TI - Molecular cloning of the cDNA encoding human laminin A chain. AB - Laminin is a large basement membrane glycoprotein composed of three subunits designated the A, B1, and B2. We report here the isolation and nucleotide sequence of human laminin A chain cDNA. The nucleotide sequence spans 9505 bases and has an open reading frame encoding 3075-amino acids. The sequence covers a 77 nucleotide long 5' untranslated region and a 190-nucleotide long 3' sequence in front of the poly (A)+ tail. In analogy with the mouse A chain sequence, the deduced human amino acid sequence contains eight-distinct domains of four globular regions, three-cysteine-rich domains and an alpha-helical region, which is though to interact with the B chains of laminin. The deduced amino acid sequence is 14-amino acids shorter than the mouse A chain sequence. Seven of these amino acids are located in the putative signal sequence. The overall identity between the sequences from the two species is 78%. The carboxylterminal globular (G) domain contains five homologous subdomains characterized by a conserved seven-amino acid repeat within each subdomain. Both human and mouse A chain are about 39% identical to the G domain of merosin, a recently discovered A chain homologue. Unlike the mouse A chain, the human A chain contains a potential cell binding sequence (RGD) in this domain. The RGD sequence that is thought to be a cryptic cell attachment site in the amino-terminal domain IIIb of mouse laminin is not conserved in the human sequence. PMID- 1714538 TI - PCR technique as an alternative method for diagnosis and molecular epidemiology of rabies virus. AB - We have investigated the PCR amplification technique of viral nucleic acids as an alternative protocol for diagnosis and epidemiological studies of rabies virus. A primer set mapping in the nucleoprotein cistron allowed a specific and sensitive amplification of infected brain material, fulfilling the diagnosis requirements. One hundred samples checked by Southern or dot-blot analysis using both radioactive and non-radioactive probes showed identical results in parallel with routine techniques. For molecular epidemiological studies we selected another set of conserved primers flanking the highly evolutive pseudogene (psi gene) region. This set was found to be efficient for all tested fixed rabies virus strains or wild rabies virus isolates as well as the rabies-related Mokola virus. We describe a progressive characterization of the strain that could be extended from rapid typing by a limited panel of restriction enzymes, to the ultimate identification of the nucleotide sequence by an original direct sequencing technique of amplified segments. PMID- 1714539 TI - Mutagenic activity of 2-amino-N6-hydroxyadenine in the mouse spot test. AB - The base analogue 2-amino-N6-hydroxyadenine (AHA) was mutagenic in the spot test in (T x HT)F1 mouse embryos. Females were injected with single doses of 20 or 40 mg AHA per kg body weight on the 9th day of pregnancy. To rank the mutagenic potency of different compounds, the frequencies of genetically relevant spots induced by 1 mg/kg body weight were calculated. The observed somatic mutation frequency for 1 mg/kg AHA was lower (1.95 x 10(-3)) spots of genetic relevance) than that of mitomycin C (16 x 10(-3)), ethylnitrosourea (6.8 x 10(-3)) and cyclophosphamide (6.4 x 10(-3)) and therefore AHA was not classified as a very potent mutagen in this test system. The doubling dose to induce genetically relevant spots was calculated to be 20 mg/kg b.w. Based on these data, AHA is suggested to be a candidate to induce recessive specific-locus mutations in germ cells of mice. PMID- 1714540 TI - Genotoxicity of diesel-exhaust particles dispersed in simulated pulmonary surfactant. AB - Diesel-exhaust particles from two sources were dispersed in aqueous mixtures of dipalmitoyl phosphatidyl choline, a major component of pulmonary surfactant, and were tested for genotoxicity. Diesel samples from the same sources were extracted with dichloromethane and transferred into dimethyl sulfoxide and subjected to the same assays. Both types of extractions yielded similar results in both the Salmonella mutagenicity assay and the sister-chromatid exchange assay using V79 cells. After separation of the samples into supernatant and sediment fractions, the activity of both diesel samples was shown to reside exclusively in the supernatant fraction for the solvent-extracted samples, and exclusively in the sedimented fraction for surfactant dispersed samples. These findings indicate that genotoxic activity associated with diesel particles inhaled into the lung may be made bioavailable by virtue of the solubilization/dispersion properties of pulmonary surfactant components. PMID- 1714541 TI - Synergistic effect of CCNU and bleomycin on human lymphocytes exposed at late G1 and G2 states of the cell cycle. AB - The combined action of the antitumor antibiotic bleomycin and chloroethylnitrosourea (CCNU) was studied in human lymphocytes in vitro. All the experiments were carried out with 20 micrograms/ml bleomycin for a given treatment time. By adding 0.7 and 3.5 micrograms/ml CCNU at late G1-S phase we have demonstrated a considerable increase in both percent of aberrant cells and production of dicentrics and rings (5-fold, p less than 0.001). At late S-G2 the combined treatment led to a significant enhancement of breaks per cell (p less than 0.0001) and cells with more than 12 aberrations. A possible explanation could be the known repair-inhibitory potential of CCNU, but its pure clastogenic action still has to be considered. The results presented here point out the need for seeking chemotherapeutic regimens with reduced concentrations of the drugs in combination. PMID- 1714542 TI - Genetic toxicology testing and biomonitoring of environmental or occupational exposure. PMID- 1714543 TI - Temperature sensitivity of small-colony TFTres variants of L5178Y mouse lymphoma cells may be due to fastidious growth requirements of macromutations. PMID- 1714544 TI - Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay. PMID- 1714545 TI - The glucose-transporter protein and glucopenic brain injury. PMID- 1714546 TI - Enhance communication: avoid red and green in slides. PMID- 1714547 TI - Properties of kainate receptor/channels on cultured Bergmann glia. AB - Following the localization, at the electron microscope level, of the immunoreactivity towards a putative kainate receptor on Bergmann glial cells in the chick cerebellar cortex, cultures of Bergmann glia were used to establish the presence of functional kainate receptor/channels and study their properties. Bergmann glia were identified by their fusiform morphology and their ability to bind an anti-kainate binding protein monoclonal antibody, a kainate receptor high affinity ligand--kainyl-bovine serum albumin--and a glial marker--anti-vimentin monoclonal antibody. Membranes prepared from the culture cells displayed, using 25 nM [3H]kainate, the binding of 4.1 pmol of [3H]kainate/mg protein and showed the presence in Western blots of the two polypeptides of 49 and 93 kDa attributed to the kainate binding protein. Kainate, at concentrations above 0.1 mM, was found to increase the influx into cultured Bergmann glia of 22Na+, 86Rb+, 45Ca2+ and 36Cl- ions. The traffic of 22Na+, induced by kainate and glutamate, observed only in the presence of 1 mM ouabain, was blocked by kainate receptor antagonists and by 0.01 mM quisqualate. Analysis of the kinetics of incorporation of 22Na+ and 45Ca2+ ions showed an initial accumulation of 22Na+ and 45Ca2+ ions followed by their total dissipation. The results indicate that the kainate-induced influx of Na+ ions through the kainate receptor/channel causes the reverse transport of Na+ ions, by activation of the Na+/Ca2+ and Na+/H+ exchangers which remove intracellular Na+ ions. Pre-exposure of the cells to 0.5 mM dibutyryl cAMP was found to greatly enhance the kainate-induced 22Na+ ion influx. We propose that the Bergmann glia kainate receptors modulate the efficacy of the glutamatergic synapses between the parallel fibers and Purkinje cell spines and form part of a glial machinery responsible for plastic changes in synaptic transmission. PMID- 1714548 TI - Septal GABAergic neurons innervate inhibitory interneurons in the hippocampus of the macaque monkey. AB - The septohippocampal projection was visualized in three Macaca mulatta monkeys by anterograde transport of Phaseolus vulgaris leucoagglutinin. Following injections of the lectin into the medial septal nucleus, P. vulgaris leucoagglutinin labelled fibres were found in the hippocampal complex, mainly in stratum oriens of the CA1 subfield, throughout the CA3 subfield, and in the hilus and stratum moleculare of the dentate gyrus. The majority of labelled axons were varicose, and formed multiple contacts with cell bodies and dendrites of calbindin D28k- and parvalbumin-immunoreactive non-pyramidal cells. GABA immunoreactivity of P. vulgaris leucoagglutinin-labelled axons and their postsynaptic targets was investigated by sectioning varicose axon segments for correlated light and electron microscopy, and processing alternate ultrathin sections for postembedding immunogold staining for GABA. All P. vulgaris leucoagglutinin labelled boutons examined were GABA-immunoreactive and the majority of them formed symmetrical synapses with GABA-immunoreactive cell bodies and dendrites. The results demonstrate that a GABAergic septohippocampal pathway exists in the monkey, and, similar to the rat, terminates on different types of GABAergic neurons, including the parvalbumin- and calbindin D28k-containing non-pyramidal cells. PMID- 1714549 TI - Reconstruction of hippocampal granule cell electrophysiology by computer simulation. AB - A model of the hippocampal granule cells was created that closely approximated most of the measured intracellular responses from a neuron under a variety of stimulus conditions. This model suggests that: (1) A simple, four-conductance model can account for most of the intracellular behavior of these neurons. (2) The repolarization mechanism in granule cells may be different from that in squid axons. A weak potassium conductance may be present in hippocampal granule neurons, which simultaneously give rise to a small, passive depolarizing afterpotential. (3) The strength duration properties may assist in identifying the electronic and sodium channel properties with short stimulus pulse widths. (4) Repetitive firing responses are highly dependent on the cell's recent history of activation and the regulation of the slow potassium conductance and calcium dynamics. (5) The anodic break response is probably not a property of typical granule cells. Through thorough and precise comparison of experimental and model responses, computer simulations can help assembling channel information into verifiable models that accurately reproduce intracellular data. PMID- 1714550 TI - K+ channel and 5-hydroxytryptamine1A autoreceptor interactions in the rat dorsal raphe nucleus: an in vitro electrophysiological study. AB - Extracellular recordings were made from serotonergic neurons of the rat dorsal raphe nucleus in a slice preparation. In the presence of phenylephrine (3 microM) to restore the pacemaker activity of otherwise silent serotonergic neurons, superfusion with the 5-hydroxytryptamine1A agonist ipsapirone depressed the firing of these neurons with an IC50 of approximately 50 nM. Complete inhibition was achieved with 100-300 nM of the drug. Concomitant superfusion with the 5 hydroxytryptamine1A antagonists spiperone (100 nM) or propranolol (10 microM) markedly reduced the inhibitory effect of ipsapirone (100 nM). Superfusion with K+ channel blockers such as apamin (50-100 nM), charybdotoxin (100 nM) or Ba2+ (1 mM) did not induce any changes in the electrical activity of serotonergic neurons. However, 4-aminopyridine (0.1-1 mM) disrupted the regularity of their discharge without affecting the mean firing rate. The ipsapirone-induced inhibition was unchanged by apamin and charybdotoxin, but was markedly reduced by Ba2+ and 4-aminopyridine. Thus the IC50 of ipsapirone was shifted to approximately 150 nM in the presence of 1 mM of 4-aminopyridine. These results indicate that, in serotonergic neurons within the dorsal raphe nucleus, the K+ channel opened through the stimulation of 5-hydroxytryptamine1A autoreceptors is 4-aminopyridine-sensitive. PMID- 1714551 TI - Adrenergic innervation of the rat nucleus locus coeruleus arises predominantly from the C1 adrenergic cell group in the rostral medulla. AB - Focal iontophoretic injections of the retrograde tracer Fluoro-Gold into the locus coeruleus were combined with immunocytochemistry for phenylethanolamine N methyltransferase, the final enzyme in the synthesis of epinephrine. Retrograde labeling confirmed recent findings that the major afferents to the locus coeruleus are present in the ventrolateral (nucleus paragigantocellularis) and dorsomedial medulla (nucleus prepositus hypoglossi), areas containing the C1 and C3 adrenergic cell groups, respectively. The Fluoro-Gold label revealed morphologic details of locus coeruleus afferent cells. Labeled neurons in the prepositus hypoglossi region were typically round (10 microns diameter) or ellipsoidal and compressed against the ventricle wall (10 x 20 microns), while those in the paragigantocellularis were most often multipolar and ellipsoidal or triangular in shape (10 x 20-20 x 30 microns). Double labeling in the same tissue sections revealed that locus coeruleus afferent neurons are intercalated among phenylethanolamine N-methyltransferase-positive C1 and C3 neurons. Twenty-one per cent of locus coeruleus afferent neurons in paragigantocellularis stained for phenylethanolamine N-methyltransferase while only 4% of locus coeruleus afferents in the prepositus hypoglossi area exhibited phenylethanolamine N methyltransferase immunoreactivity. In paragigantocellularis, doubly labeled neurons were usually the smaller locus coeruleus afferents, while in the prepositus hypoglossi phenylethanolamine N-methyltransferase labeling was found in all cell types that project to the locus coeruleus. Phenylethanolamine N methyltransferase-positive fibers from the C1 and C3 cell groups form an adrenergic fiber bundle in the dorsomedial medulla; in the pons, these fibers appear to exit this bundle and innervate the locus coeruleus. Fibers from the neurons of the C3 cell group also appear to ascend on the dorsal surface of the medulla to innervate the locus coeruleus. The phenylethanolamine N methyltransferase fiber innervation in the locus coeruleus was dense and highly varicose. Phenylethanolamine N-methyltransferase innervation in the dorsal pons was not restricted to the locus coeruleus but was also prominent in neighboring areas such as Barrington's nucleus and the lateral dorsal tegmental nucleus of Gudden. PMID- 1714552 TI - Expression and localization in the developing cerebellum of the carbohydrate epitopes revealed by Elec-39, an IgM monoclonal antibody related to HNK-1. AB - The immunochemical and immunocytochemical reactivity of an anti-carbohydrate monoclonal antibody (Elec-39), obtained against acetylcholinesterase from Electrophorus electricus electric organ, was followed during the postnatal development of the rat cerebellum. The specificity of this antibody resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1 and NSP-4, as well as IgMs that occur in some human neuropathies. As revealed by immunoblotting techniques, the reactivity of Elec-39 is maximum around postnatal days 10-12. At this age, the antibody reveals eight major proteins of mol. wt ranging between 14 and 150 kDa. Some of them (with mol. wts of 14, 18, 28 and 31 kDa) are transiently expressed. They correspond to previously identified glycoproteins binding to the plant lectin concanavalin A and binding also to the endogenous mannose-binding lectin CSL and endogenous membrane-bound mannose binding lectin. In young animals, an important staining with the Elec-39 antibody can be observed on postmitotic precursors of granule cells, on astrocyte processes in the external granular layer, on newly formed parallel fibres and on unmyelinated axons of the white matter. In adult animals, the labelling is localized essentially in myelin and also in the cytoplasm of astrocytes. These results are discussed in relation to ontogenetic phenomena occurring during cerebellar development and the potential role of the carbohydrate epitope revealed with Elec-39 as a determinant in cell adhesion processes. PMID- 1714553 TI - Central projections of trigeminal primary afferents innervating the nasal mucosa: a horseradish peroxidase study in the rat. AB - The respiratory region of the nasal mucosa is innervated by the ethmoidal nerve. Chemical nociceptive stimulation of this area leads to upper airway reflexes that prevent access of noxious substances to the respiratory tract and the lungs. In the present study we examined the localization of the cell bodies of the respective primary afferent fibres within the trigeminal ganglion, as well as their central projections. In 25 rats a horseradish peroxidase-wheat germ agglutinin gel was applied to the right nasal cavity. The animals were killed after 48-72 h. For visualization of the tracer the tissue was processed according to the tetramethylbenzidine method. In the trigeminal ganglion almost all labelled cell bodies were localized in a medial band immediately caudal to the entrance of the ophthalmomaxillary branch. Transganglionic projections to the trigeminal brainstem nuclear complex were only localized in the superficial laminae of the subnucleus caudalis and in the subnucleus interpolaris, areas known to be involved in processing of nociceptive information. An additional labelled terminal field was observed in the interstitial subnucleus of the nucleus tractus solitarius, which is involved in respiratory control. These results are in favour of the hypothesis that the ethmoidal nerve in rat constitutes the afferent limb of protective upper airway reflexes since it transmits mainly nociceptive information. PMID- 1714554 TI - Neuropeptide Y and catecholamine synthesizing enzymes and their mRNAs in rat sympathetic neurons and adrenal glands: studies on expression, synthesis and axonal transport after pharmacological and experimental manipulations using hybridization techniques and radioimmunoassay. AB - The effects of reserpine treatment (10 mg/kg, i.p.) on the content of neuropeptide Y-like immunoreactivity and catecholamines were compared with the levels of mRNA coding for neuropeptide Y, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rat sympathetic neurons and adrenal gland. A reversible depletion of neuropeptide Y-like immunoreactivity was observed in the right atrium of the heart, kidney and masseter muscle, while the immunoreactive neuropeptide Y content in the stellate and lumbar sympathetic ganglia and its axonal transport in the sciatic nerve increased following reserpine. The increase in the stellate ganglion was maximal at 48 h and absent 9 days after reserpine treatment. The expression of neuropeptide Y mRNA and tyrosine hydroxylase mRNA in both the stellate and the superior cervical ganglion increased earlier than the neuropeptide Y content, with a clear cut two-fold elevation at 24 h after reserpine. The increase in both mRNAs in the superior cervical ganglion and the depletion of neuropeptide Y, but not of noradrenaline, in terminal areas was prevented after pretreatment both with a nicotinic receptor antagonist (chlorisondamine) and with surgical preganglionic denervation. A marked (75-90%) depletion of neuropeptide Y-like immunoreactivity and adrenaline in the adrenal gland, concomitant with 3-4-fold increases in neuropeptide Y mRNA and tyrosine hydroxylase mRNA expression, was present at 24 h after reserpine treatment. Also in the adrenal gland, there was a reversal of the reserpine induced increase in neuropeptide Y mRNA and tyrosine hydroxylase mRNA and depletion of neuropeptide Y and adrenaline following splanchnic denervation. Pharmacological, ganglionic blockade prevented the depletion of neuropeptide Y and the increased expression of neuropeptide Y mRNA, but not fully, the tyrosine hydroxylase mRNA elevation. In addition, a marked decrease in phenylethanolamine N-methyltransferase mRNA levels was noted after reserpine. This decrease was reversed by denervation and by ganglionic blockade. Denervation alone led to a small but significant decrease in all mRNAs examined both in the superior cervical ganglion and the adrenal medulla. The present data suggest that the depletion of neuropeptide Y-like immunoreactivity in sympathetic nerves and in the adrenal gland after reserpine is associated with a compensatory increase in neuropeptide Y synthesis and axonal transport, most likely due to increased nicotinic receptor stimulation. Whereas the reserpine depletion of neuropeptide Y in both sympathetic nerves and adrenal gland is related to neuronal activation, adrenal but not nerve terminal depletion of catecholamines can be prevented by the ganglionic blocker chlorisondamine.4+e difference in effect of pharmacological ganglionic PMID- 1714556 TI - [Rhabdomyosarcoma. Presentation of a case with abdominal localization]. AB - The Authors report a case of rhabdomyosarcoma observed in a 17-year-old boy. They emphasize that this rare form has an extremely rapid evolution, and in this case was also in an unusual site and of abnormal size. The difficulty of an early diagnosis and the impossibility of radical surgery are also underlined. PMID- 1714555 TI - Ion channels involved in the presynaptic hyperexcitability induced by herpes virus suis in rat superior cervical ganglion. AB - Rat superior cervical ganglia infected with herpes virus suis (pseudorabies virus) display a spontaneous bursting activity of still unknown origin. Previous intracellular recordings from the ganglionic neurons combined with pharmacological studies showed that the postganglionic action potentials are induced by acetylcholine release spontaneously from the preganglionic nerve. In this study we investigated whether the acetylcholine release is caused by mechanisms which are dependent on action potentials spontaneously generated on the preganglionic nerve or by mechanisms which occur without any changes in the excitability of presynaptic fibers. Simultaneous intra- and extracellular recordings from the ganglion cells and from the preganglionic nerve, respectively, were performed 32-38 h after the inoculation of herpes virus suis (strain Aujeszky) into the anterior chamber of one eye of the rat. Tetrodotoxin, well known to prevent the generation of action potentials by blocking the fast sodium channels, completely and reversibly abolished, whereas the potassium channel blockers 4-aminopyridine and apamin, enhanced the spontaneous, bursting activity at pre- and postsynaptic levels. The nicotinic receptor antagonist hexamethonium abolished the postsynaptic discharges and reduced the preganglionic activity by 50%. Pre- and postsynaptic electrical activities were suppressed in low calcium Krebs' solution, demonstrating that extracellular calcium is required not only for acetylcholine release but also for triggering the presynaptic action potentials. It is concluded that in the infected ganglia the spontaneous acetylcholine release is due to the generation of action potentials in the preganglionic nerve. Voltage-gated sodium and calcium channels contribute to the presynaptic electrogenesis, while the latter appears to be damped by the activation of voltage- and calcium-dependent potassium channels. Possible factors as well as mechanisms inducing such an increase in excitability are discussed. PMID- 1714558 TI - Fighting the fear of public speaking. PMID- 1714557 TI - [Clinical case of carcinoid of the gallbladder. Considerations on gallbladder cancer]. AB - Gallbladder cancer is the fifth of the digestive neoplasms. Diagnosis is often made very late sk that prognosis is really poor. We present 84 patients operated on for gallbladder cancer and 1 for carcinoid. Sixty-eight of them had a complete follow-up. PMID- 1714559 TI - An improved method of vital staining of the corneal endothelium. AB - We present an improved and simplified method for vital staining of the corneal endothelium, which allows high-quality images that are excellent for morphological and morphometric studies. This method is based upon the combination of trypan blue and alizarin red S. The main advantages of this technique are simplicity, rapidity and low cost. PMID- 1714560 TI - A method to quantify neovascularization in the mouse cornea. AB - A method for microscopical observation and quantification of corneal neovascularization in the living mouse is described. Measurements of the corneal vasculature were made on still-mode video-pictures of the cornea. The validity of the method was shown by the high correlation between the morphometric measurements and computerized values on the same vessel structures. The method was applied to describe the neovascular response of heat-killed tumour fragments implanted into corneal pockets. The neovascular response that was observed in some corneas was dependent on the distance from the edge of the pocket with heat killed material to the nearest vessel loop. The pocket area was vascularized only if this distance (measured the 3rd day after implantation) was less than 0.70 mm. Resorption of heat-killed tissue occurred in corneas without any neovascular response and it was not altered by penetration of vessel into the cornea. The described method proved to be suitable for studying the neovascularization induced by corneal pocket implants. PMID- 1714561 TI - Tumor angiogenesis. AB - Angiogenesis requires the migration, differentiation and proliferation of endothelial cells. Tumor growth is angiogenesis-dependent and angiogenesis is directly or indirectly induced by the tumor. Induction of angiogenesis is, therefore, an important stage in carcinogenesis and in the development of metastasis. Numerous angiogenic factors have been identified, most of which are mitogenic for endothelial cells and only some of which are responsible for tube formation. Pharmacologic compounds such as heparin, heparin fragments and corticosteroids have been shown to be anti angiogenic substances. Recently two new inhibitors of angiogenesis, a cartilage-derived inhibitor and platelet factor 4 have been described. PMID- 1714562 TI - HP 0.35, a cephalosporin degradation product is a specific inhibitor of lentiviral RNAses H. AB - Penicillins, cephalosporins and other betalactam antibiotics are widely used antibacterial drugs. Recently it was found that some of them also have effects on proliferating eukaryotic cells (Neftel, K.A. and Hubscher, U. (1987) Antimicrob. Agents Chemother. 31, 1657-1661), and one such effect was shown to be the inhibition of DNA polymerase alpha (Huynh Do,U., Neftel, K.A., Spadari, S. and Hubscher, U. (1987) Nucl. Acids Res. 15, 10495-10506). The data suggested that degradation products of betalactam antibiotics were responsible for the inhibitory effect on DNA polymerase alpha. There is some confirmation at the structural level, since we found that penicillin binding proteins, the natural target of the cephalosporins, share amino-acid homologies to DNA polymerases and also to reverse transcriptase from HIV1 (Hafkemeyer, P., Neftel, K.A. and Hubscher, U. Meth. Find. Exp. Clin. Pharmacol. 12, 43-46, 1990). We have purified and determined the structure of one product from the cephalosporin Ceftazidim and found one molecule (HP 0.35) that did not interfere with eukaryotic cell proliferation but rather had a specific inhibitory effect on the RNase H activity of human immunodeficiency virus 1 (HIV1) and feline immunodeficiency virus (FIV) reverse transcriptases, while the DNA polymerising activity of these enzymes was not affected. RNases H from HeLa cells, calf thymus and Escherichia coli on the other hand were much less affected by HP 0.35. The inhibitory concentration of 50% (IC50) was more than 10 times lower compared to those of all cellular RNases H. We therefore tested the effect of HP 0.35 on in vitro lentivirus infection as exemplified by FIV-infection of CD(4+)-cat lymphocytes in cell culture. Under conditions where cell proliferation was absolutely unaffected, HP 0.35 was able to inhibit FIV-infection in CD(4+)-cat lymphocytes. Moreover, preincubation of these lymphocytes with HP 0.35 rendered the cells completely unsusceptible to FIV infection. These data suggest that a degradation product of a clinically used betalactam antibiotic might represent an effective inhibitor class for lentiviral RNase H. PMID- 1714563 TI - 8-Methyladenosine-substituted analogues of 2-5A: synthesis and their biological activities. AB - 8-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me8A), p5'A2'p5'(me8A)2'p5'(me8A), p5'(me8A)2'p5'(me8A)2'p5'(me8A), and p5'(me8A) 2'p5'A2'p5'A, were prepared via a modification of a lead ion-catalyzed ligation reaction. These 2-5A monophosphates were converted into the corresponding 5' triphosphates. Substitution of an 8-methyladenosine residue at the third position (2'-terminus) of the oligonucleotides increased the stability to snake venom phosphodiesterase digestion. Both binding and activation of mouse liver 2-5A dependent ribonuclease (RNase L) by the various 8-methyladenosine-substituted 2 5A analogues were examined. Among the 8-methyladenosine-substituted trimer analogues, the analogues with 8-methyladenosine residing in the 2'-terminal position showed the strongest binding affinity and were several times more effective than 2-5A itself as an inhibitor of translation. PMID- 1714565 TI - Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207. AB - Mutations were constructed at three sites in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680. The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation. We constructed a deletion mutation at C967 (delta 967) and three substitution mutations at each of the following sites: G966, C967, and G1207. By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes. We found that delta 967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate. The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate. These results help to establish the importance of methyl-modified regions to ribosome function. PMID- 1714564 TI - Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron. AB - Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O (dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O (methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions. PMID- 1714566 TI - [The effective half-life of 131I during the treatment of autonomous thyroid disease with radioiodine]. AB - In order to compute effective half-life of 131I after application of therapeutic doses (Teff), the time course of whole-body radioactivity was evaluated retrospectively in 115 patients with benign thyroid diseases (multinodular autonomous adenoma, solitary autonomous adenoma or Graves' disease). Because of a large overlap of Teff in the various diseases analyzed, courses of all patients who did (group Ts, 24 cases) or did not (group kTs, 91 cases) receive antithyroid drugs during therapy were summarized. In group Ts a mean Teff of 5.0 +/- 0.9 d was found which was significantly (p less than 0.01) lower than the mean Teff of 6.3 +/- 0.9 d in group kTs. We believe that the mean Teff is a practical alternative in radioiodine dosimetry if an exact determination of Teff cannot be performed because of shortage of time. PMID- 1714567 TI - Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies. AB - The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies. PMID- 1714568 TI - Stability of 3' dangling ends on the core helix of AUGCAU at various Na+ concentrations. AB - Stability increments of 3' dangling ends on the core helices AUGCAU at various Na+ concentrations are reported. The results show that all 3' dangling ends except 3'U dangling at low Na+ concentrations can stabilize the helix and this stabilization is very sequence dependent. PMID- 1714569 TI - Possible evolutionary origin of primitive protein-encoding mRNAs as a virusoid like ribo-organism. AB - E. coli rnpA and rpmH genes encoding the protein portion of ribonuclease (RNase) P and L34 ribosomal protein were found to be homologous to the entire sequence of M1 RNA and virusoids. The resulting alignment strongly suggests that most primitive mRNAs must have emerged from virusoid-like ribo-organism. PMID- 1714570 TI - Ab initio investigations on the intermediates of RNA cleaving processes. AB - Ab initio molecular orbital calculations have been carried out on acyclic oxyphosphoranes in order to elucidate the origin of previously calculated energy differences of cyclic counterparts of RNA cleaving reactions. It appears that the orientation of the equatorial methoxyl group has pronounced effects in energy on the transition states than the metastable intermediates. PMID- 1714571 TI - In vivo RNA transcript-releasing plasmid possessing a universal pseudo-terminator by means of artificial ribozymes. AB - RNA transcript-releasing plasmid has been constructed by means of artificial hammerhead ribozymes. In this specific construct of pGENE8459v3 the ribozyme targeted for SFL1 gene (a yeast suppressor gene for flocculation) was fused between two other ribozymes called 5'-processing and 3'-processing ribozymes. Since the "Ribozyme for SFL1" portion (cassette) can be replaced by other RNA sequences, it is now possible to produce any RNAs with defined 5'- and 3'- ends. PMID- 1714572 TI - Base recognition mechanism of bleomycin: solution structure of d(GGGGAGCTCCCC)2 based on 1H-NMR and restrained molecular dynamics. AB - The self-complementary d(GGGGAGCTCCCC) dodecanucleotide cleavage by bleomycin occurs at G(4)pA(5) site rather than G(6)pC(7) site which is known as the preferential sequence. The molecular structure of this dodecamer is observed as the distorted B-DNA by the CD spectra and proton NMR measurements. Three dimensional structures in solution evaluated by molecular dynamics calculations with inter proton distance restraints have the distorted form of the minor groove structure at G(6)pC(7) site. These evidences offer the recognition mechanism model of the DNA base sequence by bleomycin. PMID- 1714573 TI - Facile cleavage of RNA via phosphodiester linkage fission by Co(III) complex. AB - Adenylyl (3'-5')adenosine (ApA) is effectively cleaved to two adenosine molecules by [Co(trien)(H2O)2]3+ complex (trien: triethylenetetramine). The complex (0.20 M) accelerates the cleavage by 10(5) fold, decreasing half-life of ApA from 4000 years to 9.3 days. The reaction involves general base catalysis by the hydroxide ion bound to the Co(III) ion for the formation of adenosine 2',3'-cyclic phosphate (A greater than p), followed by the prompt cleavage of the intermediate to adenosine. PMID- 1714574 TI - Synthesis of RNA having cap structure. AB - RNA consisting 43 nucleotides bearing cap structure was synthesized (Figure). In the first place, 9 mer of a leader sequence with the cap structure (F-1) was synthesized by the phosphotriester method and followed by the capping reaction. Next, 32 mer of a cistron was divided into two fragments and each was synthesized by the phosphoramidite method. The 3'-end nucleotide of the RNA, a modified guanosine 5'-phosphate, was introduced to F-3 by use of P1-2',3'-O methoxymethylene guanosine-5'-yl P2-adenosine-5'-yl diphosphate (A5' ppGmM) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated with T4 RNA ligase to afford the desired RNA. PMID- 1714575 TI - Stabilization of mRNA in the cell-free translation system from Escherichia coli. AB - In the RNA directed cell-free protein synthesizing system from E. coli, there is a problem of contaminating 3'-exonucleases which attack the mRNAs. Thus, we tried the following two methods to stabilize mRNAs against nucleases: (A) To use mRNAs having hairpin structures at their termini and (B) To hybridize mRNAs with small DNA fragments to the 3'-termini of mRNAs. It was found that degradation of a mRNA was inhibited by the method B rather than the method A in the translation system. PMID- 1714576 TI - Reinvestigation of phosphorylation of tRNA in Escherichia coli. AB - This report shows the results of the reinvestigation of tRNA phosphorylation in E. coli. The phosphorylation did not occur on suppressor seryl-tRNA but occurred on other tRNA species. The activity of tRNA phosphorylation was found in E. coli extracts and partially purified. On DEAE-Sephadex A50 and PAGE gel, the phosphorylated-tRNA showed a pattern different from that the natural suppressor serine tRNA. PMID- 1714577 TI - Something in the air. PMID- 1714578 TI - Induction of osteochondromas by periosteal resection. AB - Forty immature and 40 mature rabbits which received periosteal resection from the proximal metaphyses or diaphyses of tibiae were evaluated for the occurrence of osteochondromas at the sites of periosteal defects. Thirteen of 20 immature tibiae with the periosteal defects at their proximal metaphyses showed osteochondromas covered by hyaline cartilage caps. We assume that the occurrence of osteochondroma at the metaphyseal region of growing long bone, where the periosteal defects were made, is due primarily to a decrease in the power of the periosteum to control the circumferential growth of the metaphysis, and is due secondarily to an abnormal proliferation of cartilage progenitors located in Ranvier's groove. PMID- 1714579 TI - Hepatic disposition characteristics of electrically charged macromolecules in rat in vivo and in the perfused liver. AB - The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C-dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4-3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (Ess) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The Ess for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested. PMID- 1714580 TI - [Pathologic phenotypes of alpha 1-protease inhibitor in patients with pulmonary tuberculosis]. PMID- 1714581 TI - Intracellular Na+ modulates the cAMP-dependent regulation of ion channels in the heart. AB - The cAMP-dependent regulation of ion channels was studied by using the whole-cell configuration of the patch clamp technique. In isolated cardiac ventricular myocytes, the beta-adrenergically regulated Cl- current (ICl) exhibited an unusual dependence on Na+, such that replacement of extracellular Na+ with compounds such as tetramethylammonium, choline, Tris, or N-methyl-D-glucamine resulted in a reduction in current amplitude without changing the reversal potential. Replacement of extracellular Na+ with tetramethylammonium also reduced the magnitude of the beta-adrenergically enhanced Ca2+ current and delayed rectifier K+ current, suggesting that removal of Na+ was affecting the cAMP pathway that regulates all three currents. Replacement of extracellular Na+ also reduced ICl that was stimulated by (i) direct activation of adenylate cyclase with forskolin, (ii) inhibition of phosphodiesterase with 3-isobutyl-1 methylxanthine, (iii) exposure to the membrane-permeable cAMP derivative 8 bromoadenosine 3',5'-cyclic monophosphate, or (iv) direct phosphorylation of the channel with protein kinase A catalytic subunit. This suggests that the Na+ dependence is at a point beyond the activation of protein kinase A. The Na+ dependence of ICl regulation could not be explained by changes in intracellular Ca2+. However, the sensitivity of the ICl to changes in extracellular Na+ depended significantly on the intracellular Na+ concentration, suggesting that intracellular Na+ plays an important role in the cAMP-dependent regulation of ion channels. PMID- 1714582 TI - Differential accumulation of transcripts encoding protein kinase homologs in greening pea seedlings. AB - Degenerate oligonucleotides, corresponding to conserved regions within the catalytic domain of known protein-serine/threonine kinases, were used as primers for the polymerase chain reaction to amplify cDNA synthesized from poly(A)+ RNA purified from the apical buds of 7-day-old pea seedlings. Five partial cDNAs were obtained and designated PsPK1 through PsPK5 (for Pisum sativum protein kinase) in order of decreasing length. The deduced amino acid sequences show that each member of the PsPK series is different in length, and, although their sequences are quite similar overall, each has a unique sequence. Moreover, each member of the PsPK series has structural features typical of members of the protein serine/threonine kinase family of protein kinases. All are equally similar to cyclic nucleotide-dependent protein kinase and protein kinase C, suggesting that the pea homologs may be involved in signal transduction. DNA gel blots show that each PsPK cDNA is likely to be encoded by a single gene within the pea genome. RNA blot analyses show that the PsPK transcripts accumulate differentially during greening of etiolated seedlings. PsPK3 and PsPK5 transcripts show a large and rapid decline during deetiolation. In contrast, the level of PsPK4 RNA increases steadily during deetiolation whereas PsPK1 and PsPK2 transcripts show little change during the greening period. Thus light regulates changes in the levels of transcripts encoding putative protein kinases in plants. PMID- 1714583 TI - Integration and expression of a rabbit liver cytochrome P-450 gene in transgenic Nicotiana tabacum. AB - Cytochrome P-450 is involved in the oxidative metabolism of a broad range of substrates. We have made a chimeric construct, pSN002, containing the cDNA for rabbit liver cytochrome P-450 (IIC14) under the control of the TR2' promoter for mannopine synthase in the Agrobacterium Ti plasmid. Nicotiana tabacum was transformed with Agrobacterium tumefaciens harboring a cointegrated plasmid pSN002::pGV2260. The presence of mRNA and of the translated protein from the chimeric cytochrome P-450 gene in transgenic plants was confirmed by RNA blot hybridization and by Western blot and immunohistochemical analyses, respectively. The transformants in which the foreign cytochrome P-450 protein is expressed show marked phenotypic changes, notably a tendency rapidly to senesce. We detected 2 propenylpyrrolidine, a degradative metabolite of nicotine alkaloids, in transgenic tobacco showing this pronounced phenotypic change. Such metabolism is likely to be due to the effect of senescence and not directly to the presence of the cytochrome P-450. PMID- 1714584 TI - Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine. AB - Studies with purified nitric oxide synthase from rat cerebellum have confirmed previous reports that product formation is enhanced by tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin]. The effect of the natural isomer, (6R)-H4B, is observed at extremely low (less than 0.1 microM) concentrations and is remarkably selective. At these concentrations, only the diastereoisomer (6S)-H4B, the structural isomer 7-(L-erythro-1,2-dihydroxypropyl) 5,6,7,8-tetrahydropterin, and 7,8-dihydrobiopterin showed detectable effects. Our observations are inconsistent with a stoichiometric role for H4B in the oxygenation of arginine [e.g., Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, O. W., Feldman, P. L. & Wiseman, J. (1991) J. Biol. Chem. 266, 6259 6263]. Activity is initially independent of added H4B; enhanced product formation with H4B is observed only as incubation progresses. The effect of H4B is catalytic, with each mole of added H4B supporting the formation of greater than 15 mol of product. Recycling of H4B was excluded by direct measurement during nitric oxide synthesis and by the demonstration that nitric oxide synthase is not inhibited by methotrexate. These combined results exclude H4B as a stoichiometric reactant and suggest that H4B enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added H4B does not depend on catalytic turnover of the enzyme. The role of H4B may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity. PMID- 1714585 TI - Regulation of c-fos and c-jun protooncogene expression by the Ca(2+)-ATPase inhibitor thapsigargin. AB - Thapsigargin, a non-phorbol-ester-type tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca(2+)-ATPase. We used this drug to analyze the involvement of Ca2+ and Ca(2+)-ATPases in the control of growth- and transformation-related genes. Here we show that treatment of mouse NIH 3T3 fibroblasts with thapsigargin induced rapid expression of the c fos and c-jun protooncogenes. Inhibition or depletion of protein kinase C partially diminished the c-fos but not the c-jun response. Furthermore, thapsigargin could synergize with the tumor promoter phorbol 12-myristate 13 acetate to induce c-fos but not c-jun. However, thapsigargin had no effect on basal or phorbol ester-induced protein kinase C activity. Our results indicate that Ca2+ is a potent second messenger that controls expression of growth- and transformation-related genes. Since inhibition of the endoplasmic reticulum Ca(2+)-ATPase results in a strong induction of these genes, our data suggest that this Ca2+ pump may act as a negative regulator of cell growth. PMID- 1714586 TI - Regulation of skeletal muscle stiffness and elasticity by titin isoforms: a test of the segmental extension model of resting tension. AB - To explore the role of titin filaments in muscle elasticity, we measured the resting tension-sarcomere length curves of six rabbit skeletal muscles that express three size classes of titin isoform. The stress-strain curves of the split fibers of these muscles displayed a similar multiphasic shape, with an exponential increase in tension at low sarcomere strain followed by a leveling of tension and a decrease in stiffness at and beyond an elastic limit (yield point) at higher sarcomere strain. Significantly, positive correlations exist between the size of the expressed titin isoform, the sarcomere length at the onset of exponential resting tension, and the yield point of each muscle. Immunoelectron microscopic studies of an epitope in the extensible segment of titin revealed a transition in the elastic behavior of the titin filaments near the yield point sarcomere length of these muscles, providing direct evidence of titin's involvement in the genesis of resting tension. Our data led to the formulation of a segmental extension model of resting tension that recognizes the interplay of three major factors in shaping the stress-strain curves: the net contour length of an extensible segment of titin filaments (between the Z line and the ends of the thick filaments), the intrinsic molecular elasticity of titin, and the strength of titin thick filament anchorage. Our data further suggest that skeletal muscle cells may control and modulate stiffness and elastic limit coordinately by selective expression of specific titin isoforms. PMID- 1714587 TI - The low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation. AB - Protein tyrosine phosphorylation is a potential mechanism for initial signaling in PC12 cells during differentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of high-affinity NGF binding sites. In contrast to transfection of wild-type low-affinity p75 NGF receptors, transfection of p75NGFR with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NGFR is involved in initiating phosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosine kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation. PMID- 1714588 TI - Mutations in the su(s) gene affect RNA processing in Drosophila melanogaster. AB - We have studied the effect of mutations in the suppressor of sable [su(s)] gene on P element-induced yellow alleles. Two independent mutations tested, y76d28 and y1#7, contain a 1.1-kilobase (kb) P element inserted in the 5' transcribed untranslated portion of the yellow gene. Sequences responsible for the y1#7 mutation are inserted in the same transcriptional orientation as yellow and cannot be processed by splicing, and this mutation is not suppressed by su(s) mutations. P element sequences are located in a transcriptional orientation opposite to that of the yellow gene in y76d28; these sequences can be spliced from a composite P element-yellow mRNA, resulting in low accumulation of a functional 1.9-kb yellow transcript. The levels of both the putative precursor P element-yellow RNA and the 1.9-kb yellow transcript increase in y76d28 su(s) flies, suggesting that mutations in su(s) do not affect the efficiency of splicing of the P element sequences. Analysis of y76d28 cDNAs isolated from flies carrying a wild-type or mutant su(s) gene demonstrates that the choice of splice junctions to process P element sequences is unchanged in these different backgrounds, suggesting that mutations in su(s) do not affect the selection of donor and acceptor splice sites. We propose that the su(s) protein functions to control the stability of unprocessed RNA during the splicing reaction. PMID- 1714589 TI - Binding of the adenovirus VAI RNA to the interferon-induced 68-kDa protein kinase correlates with function. AB - In human cells infected with adenovirus, the virus-associated RNA VAI blocks the activation of the interferon-induced double-stranded-RNA-dependent 68-kDa protein kinase (p68) and maintains normal levels of protein synthesis at late times after infection. VAI antagonizes the kinase activity by binding to p68. The structure of VAI consists of two long, base-paired stems connected by a complex short stem loop structure. Previous work using a series of adenovirus mutants showed that the structural determinants of the VAI RNA that are essential for function reside in the central complex short stem-loop structure and adjacent base-paired regions (functional domain); the long duplex regions were found to be dispensable for function. To determine whether binding of VAI to p68 correlates with function and whether the structural determinants that are essential for function are also essential for binding, we studied the interaction of wild-type and several mutant VAI RNAs with p68 in whole cells. The p68-VAI complexes from mutant- and wild type-infected cells were immunoprecipitated by an anti-p68 monoclonal antibody. The mutant RNAs that functioned efficiently in the cells bound to p68 efficiently in the cells, whereas functionally impaired mutants failed to bind to p68, indicating that the binding of the VAI RNA to p68 correlates well with function. In vitro binding assays with immunopurified p68 confirmed these observations. Secondary-structure analysis of several mutant VAI RNAs suggests that the binding does not depend on the long duplex regions but requires all the elements of the functional domain. We propose that the functional domain and the p68-binding domain of the VAI RNA are identical. PMID- 1714590 TI - Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol. AB - Modification by covalent attachment of polyethylene glycol (PEG) can reduce the immunogenicity and prolong the circulating life of proteins, but the utility of this approach for any protein is restricted by the number and distribution of PEG attachment sites (e.g., epsilon-amino groups of lysine residues). We have developed a strategy for introducing additional sites for PEG attachment by using site-directed mutagenesis to selectively replace arginine with lysine codons and tested it with purine nucleoside phosphorylase (PNP) from Escherichia coli, an extremely stable but immunogenic enzyme, that could potentially be used to treat an inherited deficiency of PNP. A triple mutant, RK3, possessing three Arg----Lys substitutions was constructed that increased the number of lysines per PNP subunit from 14 to 17, providing an additional 18 potential PEG attachment sites per hexameric enzyme molecule. The wild-type and RK3 enzymes had similar catalytic activity, antigenicity, and immunogenicity. After PEG modification, both enzymes retained catalytic activity, the plasma half-life of both enzymes in mice increased from approximately 4 hr to 4 days, and the binding of both enzymes by antisera raised against each unmodified enzyme was markedly diminished. However, antibody raised against wild-type PEG-PNP did not bind the PEG-RK3 enzyme. PEG-RK3 PNP was also substantially less immunogenic than wild-type PEG PNP. Accelerated antibody-mediated clearance of PEG-PNP occurred in 2 of 12 mice treated with PEG-RK3 PNP, compared with 10 of 16 mice treated with the modified wild-type enzyme. This combined use of directed mutagenesis and PEG modification is aimed at permitting the widest choice of proteins, including products of genetic and chemical "engineering," to be used for therapy of inherited and acquired disorders. PMID- 1714591 TI - A myelin protein is encoded by the homologue of a growth arrest-specific gene. AB - Striking features of the cellular response to sciatic nerve injury are the proliferation of Schwann cells in the distal nerve stump and the downregulation of myelin-specific gene expression. Once the axons regrow, the Schwann cells differentiate again to reform the myelin sheaths. We have isolated a rat cDNA, SR13, which is strongly downregulated in the initial phase after sciatic nerve injury. This cDNA encodes a glycoprotein that shares striking amino acid similarity with a purified myelin protein and is specifically precipitated by a myelin-specific antiserum. Immunohistochemistry experiments using peptide specific polyclonal antibodies localize the SR13 protein to the myelin sheath of the sciatic nerve. Computer-aided sequence analysis identified a pronounced homology of SR13 to a growth arrest-specific mRNA (Gas-3) that is expressed in resting but not in proliferating 3T3 mouse fibroblasts. SR13 is similarly downregulated during Schwann cell proliferation in the rat sciatic nerve. The association of the SR13 as well as the Gas-3 mRNA with nonproliferating cells in two different experimental systems suggests a common role for these molecules in maintaining the quiescent cell state. PMID- 1714592 TI - Identification of a monocyte receptor on herpesvirus-infected endothelial cells. AB - The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation. PMID- 1714593 TI - Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster. AB - Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains. PMID- 1714594 TI - T-cell receptor peptide immunization leads to enhanced and chronic experimental allergic encephalomyelitis. AB - It has previously been reported that synthetic peptides corresponding to sequences derived from T-cell receptor variable regions identified as dominant in the T-cell-mediated autoimmune disease experimental allergic encephalomyelitis in both the mouse and the rat can down-regulate disease in Lewis rats. In contrast to these results, we have found that immunization of Lewis rats with such peptides in complete Freund's adjuvant prior to induction of experimental allergic encephalomyelitis with myelin basic protein leads to responses ranging from profound disease enhancement to lack of disease. In some cases, enhanced disease was followed by a prolonged neurologic deficit that resembles multiple sclerosis more closely than does acute experimental allergic encephalomyelitis. These findings, on the one hand, support previous results showing T-cell receptor peptide-induced modulation of the disease experimental allergic encephalomyelitis and, on the other, indicate that such immunization is not a reliable method for inducing suppression of encephalitogenic effector cells. PMID- 1714595 TI - Molecular characterization of a protein-tyrosine-phosphatase enriched in striatum. AB - A cDNA clone encoding a neural-specific putative protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been isolated from a rat striatal cDNA library. The deduced amino acid sequence predicts a protein of approximately 369 amino acids with a strong homology to other members of the family of protein-tyrosine-phosphatases. In vitro translation produces a protein with an apparent molecular mass of 46 kDa. A potential attachment mechanism to the cytoplasmic membrane is suggested by a myristoylation amino acid-consensus sequence at the N terminus of the protein. RNA analyses of various regions of rat brain reveal a 3-kilobase (kb) and a 4.4-kb mRNA. The 3-kb mRNA is highly enriched within the striatum relative to other brain areas and has been termed a "striatum enriched phosphatase" (STEP). In contrast, the 4.4-kb message is most abundant in the cerebral cortex and rare in the striatum. These two messages appear to be alternatively processed RNA transcripts of a single gene. PMID- 1714596 TI - An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P. AB - Deposition of the beta-amyloid protein in senile plaques is a pathologic hallmark of Alzheimer disease (AD). Focal deposition of beta amyloid in the adult rat cerebral cortex caused profound neurodegenerative changes, including neuronal loss and degenerating neurons and neurites. Chronic induction of the Alz-50 antigen appeared in neurons around focal cortical deposits of beta amyloid. Immunoblot analysis showed that beta amyloid induced Alz-50-immunoreactive proteins in rat cerebral cortex that were very similar to the proteins induced in human cerebral cortex from patients with AD. The neuropeptide substance P prevented beta-amyloid-induced neuronal loss and expression of Alz-50 proteins when coadministered into the cerebral cortex. Systemic administration of substance P also provided protection against the effects of intracerebral beta amyloid. Thus, beta amyloid is a potent neurotoxin in the adult brain in vivo, and its effects can be blocked by substance P. PMID- 1714597 TI - Morphology of a sensory neuron in Drosophila is abnormal in memory mutants and changes during aging. AB - Several mutations in Drosophila impair learning and the cAMP cascade. We report here that the fine morphology of an identified mechanosensory neuron is abnormal in two of these mutants, dunce (dnc) and rutabaga (rut). The neuron innervating the antero-notopleural bristle was filled with horseradish peroxidase and studied at the light- and electron-microscopy level. In the mutants dnc and rut, this neuron has an abnormally large number of side branches and varicosities in a defined segment of the axon. In wild-type flies, age tends to decrease the number of side branches and variacosities in the same axonal segment that is affected by the mutations. Ultrastructural studies are compatible with the interpretation that the varicosities are potential synaptic sites. The results suggest that the cAMP cascade plays a role in shaping neuronal connectivity. PMID- 1714598 TI - Localization of cholinergic differentiation factor/leukemia inhibitory factor mRNA in the rat brain and peripheral tissues. AB - Sympathetic neurons display considerable plasticity in the neurotransmitter and neuropeptide phenotypes they express in vitro and in vivo. The cholinergic differentiation factor (CDF, also known as leukemia inhibitory factor, LIF) induces cultured rat sympathetic neurons to become cholinergic, without affecting their survival or growth. To understand the role of this factor in normal development, it is essential to determine where it is produced in situ. To localize CDF/LIF mRNA, a semiquantitative, reverse transcription-polymerase chain reaction method was employed. Actin and tubulin mRNA were used as internal controls, and two different sets of CDF/LIF primers were compared. In postnatal rat peripheral tissues, CDF/LIF mRNA was selectively localized in the target area of developing, sympathetic cholinergic neurons; the mRNA was not detected in the targets of sympathetic noradrenergic neurons. This finding supports the hypothesis that CDF/LIF is a target-derived neuronal differentiation factor. In postnatal rat brain, CDF/LIF mRNA is localized selectively in two parts of the visual system, visual cortex and superior colliculus. Thus, CDF/LIF may play a role in this system as well. PMID- 1714599 TI - Planarian homeobox genes: cloning, sequence analysis, and expression. AB - Freshwater planarians (Platyhelminthes, Turbellaria, and Tricladida) are acoelomate, triploblastic, unsegmented, and bilaterally symmetrical organisms that are mainly known for their ample power to regenerate a complete organism from a small piece of their body. To identify potential pattern-control genes in planarian regeneration, we have isolated two homeobox-containing genes, Dth-1 and Dth-2 [Dugesia (Girardia) tigrina homeobox], by using degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain. Dth-1 and Dth-2 homeodomains are closely related (68% at the nucleotide level and 78% at the protein level) and show the conserved residues characteristic of the homeodomains identified to data. Similarity with most homeobox sequences is low (30-50%), except with Drosophila NK homeodomains (80 82% with NK-2) and the rodent TTF-1 homeodomain (77-87%). Some unusual amino acid residues specific to NK-2, TTF-1, Dth-1, and Dth-2 can be observed in the recognition helix (helix-3) and may define a family of homeodomains. The deduced amino acid sequences from the cDNAs contain, in addition to the homeodomain, other domains also present in various homeobox-containing genes. The expression of both genes, detected by Northern blot analysis, appear slightly higher in cephalic regions than in the rest of the intact organism, while a slight increase is detected in the central period (5 days) or regeneration. PMID- 1714600 TI - Critical role for the Val/Gly86 HLA-DR beta dimorphism in autoantigen presentation to human T cells. AB - Helper T lymphocytes recognize fragments of foreign (or self) antigens in the peptide-binding clefts of major histocompatibility complex class II molecules; their activation is a crucial step in the induction of many immune and autoimmune responses. While studying the latter, we raised a T-cell line from the thymus of a myasthenia gravis patient against recombinant alpha subunit of the human acetylcholine receptor, the target of this autoimmune disease. The line responds to the 144-156 region of the human sequence and not to the same region of the electric fish homolog, which differs by only three residues. These CD4+ T cells recognize this epitope only in the context of HLA-DR4 class II molecules, of which the variants with Gly86 are absolutely required. Thus the naturally occurring alternatives Dw14.2 (Gly86) and Dw14.1 (Val86)--which differ only at this one position in the entire antigen-binding region--show an all-or-nothing difference in presenting activity. This dimorphism at position 86 is widespread, occurring in subtypes of DR1, DR2, DR3, DR5, and DR6 alleles as well as DR4. Since other DR4 subtypes with substitutions at positions 70-74 also fail to present this peptide, and glycine residues can be uniquely flexible, we suggest that this replacement at position 86 acts locally or at a distance by altering the conformation of the peptide-binding cleft. Such profound functional consequences for T-cell recognition as we report here may explain this example of conserved major histocompatibility complex diversity. PMID- 1714601 TI - Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein tyrosine kinases. AB - Stimulation of resting B lymphocytes with antibodies to surface immunoglobulin (sIgD or sIgM) induces protein tyrosine phosphorylation, implicating one or more B-cell protein-tyrosine kinases (PTKs) in sIg signal transduction. We have evaluated whether members of the src family of PTKs are involved in this process. Our results show that addition of antibodies to IgD or to IgM can stimulate the PTK activity of the blk, fyn, and lyn gene products. Additionally, all three PTKs were found to coimmunoprecipitate with sIg in digitonin lysates from resting B cells. In all stimulatory conditions, whether initiated through sIgD or sIgM, the blk gene product p56blk displayed the strongest activation index. The kinetics of activation of these kinases, particularly that of p56blk, paralleled the early appearance of newly tyrosine-phosphorylated B-cell proteins, suggesting that this group of kinases may account for some portion of the tyrosine kinase activity in sIg-activated B cells. These observations demonstrate a functional and possible physical association between the members of the src family of PTKs and the B-cell antigen receptors. PMID- 1714602 TI - Autoantigen recognition by thyroid-infiltrating T cells in Graves disease. AB - Graves disease is a common form of human autoimmune thyroiditis. It shares many pathological features and HLA associations with other, less easily studied, organ specific autoimmune conditions such as insulin-dependent diabetes mellitus, and hence it is also a useful model for understanding these other diseases. We have previously shown that thyroid-infiltrating T cells in Graves disease that have been recently activated in vivo specifically recognize autologous thyroid epithelial cells. However, the autoantigens involved were not defined. In this study, we have made use of antigen-independent T-cell cloning techniques to show that at least three different thyroid antigens, three different epitopes on a single antigen, and two HLA class II elements are involved in this recognition process in a single individual. This demonstrates that T cells that are present and activated at the site of a human autoimmune disease may show considerable heterogeneity in their recognition of autoantigen on the target tissue. This contrasts with the limited heterogeneity recently reported in some animal models and has potentially important implications for both our understanding of the autoimmune process in humans and the design of immunotherapies to reverse it. PMID- 1714603 TI - Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic cells. AB - Recombinant rat stem cell factor (SCF) was studied for its ability to stimulate the growth of murine hematopoietic progenitor cells and to generate colony forming cells (CFC) from highly enriched populations of hematopoietic cells. In serum-deprived cultures, SCF alone stimulated few colonies but interacted with a number of other hematopoietic growth factors, particularly interleukin 3, to promote colony formation. The most marked effect was on the generation of mixed cell colonies. Hematopoietic cells were sorted into wheat-germ agglutinin negative, monocyte-depleted, rhodamine 123 (Rh123)-bright or Rh123-dull cells. Historically, Rh123-bright cells are capable of short-term (less than 1 mo) marrow engraftment, whereas among Rh123-dull cells are cells capable of long-term marrow engraftment. Enriched cells (2.5 x 10(3) were placed into serum-deprived liquid cultures with various hematopoietic growth factors. Initially, the Rh123 bright and Rh123-dull cells had few CFC but, in the presence of interleukin 3 and SCF, Rh123-bright cells gave rise to greater than 15,000 granulocyte/macrophage CFC, greater than 1500 erythroid burst-forming cells, and greater than 700 mixed cell CFC by day 5. In contrast, Rh123-dull cells proliferated only in the presence of interleukin 3 and SCF, but total cell numbers rose to a peak of 18,000 by day 21, and one-third of the cells were CFC. Thus, SCF, in combination with other growth factors, can generate large numbers of CFC from pre-CFC and appears to act earlier than hematopoietic growth factors described to date. PMID- 1714604 TI - Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1. AB - Adherence of human eosinophils to cytokine-stimulated endothelial cells, which was only partially due to CD18-dependent pathways, was also mediated by binding to endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Eosinophils bound specifically to both recombinant soluble ELAM-1 and recombinant soluble VCAM-1. Eosinophil binding to recombinant soluble VCAM-1 and to transfected CHO cells expressing VCAM-1 was inhibited with anti VCAM-1 (4B9) and anti-very late activation antigen 4 (anti-VLA-4; HP1/2 or HP2/1) monoclonal antibodies. Eosinophils, but not neutrophils, expressed VLA-4 detected by cytofluorography. Eosinophil adherence to tumor necrosis factor alpha stimulated human umbilical vein endothelial cells was partially blocked by monoclonal antibodies against ELAM-1 (BB11) and VCAM-1 (4B9) and against VLA-4 (HP2/1). Thus, while both eosinophils and neutrophils can bind to activated endothelial cells by adherence to ICAM-1 and ELAM-1, only eosinophils expressed VLA-4 and adhered to VCAM-1 on activated endothelial cells. Eosinophil adherence to VCAM-1 might provide a mechanism contributing to the selective recruitment of eosinophils into tissue sites of inflammation. PMID- 1714605 TI - Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast. AB - The ethylene-forming enzyme (EFE), which catalyzes the last step in the biosynthesis of the plant hormone ethylene, has never been purified and no molecular probes are available. Recently, a putative cDNA clone for tomato EFE (pTOM13) has been identified by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants. A direct test of its function has been made by expression of a pTOM13 gene in Saccharomyces cerevisiae. After cloning artefacts were discovered in the 5' region of the cDNA, a corrected cDNA (pRC13) was created by the fusion of the 5' end of a genomic clone to the 3' end of the cDNA and expressed in S. cerevisiae. Cultures of transformed yeast converted 1 aminocyclopropane-1-carboxylic acid (ACC) to ethylene, whereas control cells did not. This EFE activity displays similar characteristics to EFE found in plant tissue: it converts the trans isomer of the ACC analogue 1-amino-2 ethylcyclopropane-1-carboxylic acid to 1-butene in preference to the cis isomer, and it is strongly inhibited by cobaltous ions and 1,10-phenanthroline. Furthermore, information gained from the activity of effectors on yeast EFE activity supports the hypothesis that EFE is one of a group of hydroxylase enzymes. PMID- 1714606 TI - Modulation of peripheral leukocyte counts in mice by oral administration of interferons. AB - The ability of interferons (IFN) to exert a systemic effect following their oral administration was evaluated. One systemic effect of parenteral interferon administration has been shown to be a suppression of the number of peripheral white blood cells both in man and in mouse models. Using the mouse model of peripheral white blood cell suppression, the relative systemic effects of orally and subcutaneously administered interferons were determined. Murine IFN-beta, murine IFN-gamma and cross-reactive recombinant human IFN-alpha A/D were examined. The oral administrations of each of the three interferons were found to cause a dose-dependent suppression of the peripheral white blood cell counts. Significant levels of suppression were seen with as little as 5 units/day of murine IFN-beta and with 500 units/day of recombinant human IFN-alpha A/D and murine IFN-gamma. The dose-response curves obtained with orally administered interferons were much more shallow than those obtained with subcutaneously administered interferons. The results demonstrate that oral administration of interferons can provide a significant systemic effect. Further, the results support the possibility that the oral administration of interferons may have therapeutic potential. PMID- 1714607 TI - An angiogenic extract from skeletal muscle stimulates monocyte and endothelial cell chemotaxis in vitro. AB - The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 micrograms of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 micrograms of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-beta may be present in this acid/ethanol extract of skeletal muscle. PMID- 1714608 TI - Effects of forskolin and phosphodiesterase inhibitors on spinal antinociception by morphine. AB - The effect of intrathecal pretreatment with forskolin and the phosphodiesterase inhibitors Ro 20-1724, rolipram and 3-isobutyl-1-methylxanthine (IBMX) on the antinociceptive action of morphine administered intrathecally was examined using the rat tail-flick test to determine whether inhibition of adenylate cyclase contributed to spinal antinociception. Intrathecal pretreatment with forskolin (10 micrograms), Ro 20-1724 (15 micrograms) and IBMX (10 micrograms) inhibited the action of morphine in the tail-flick test. However, pretreatment with Ro 20 1724 (30 micrograms), rolipram (10 and 30 micrograms) and IBMX (30 micrograms) increased the action of morphine. These agents were devoid of intrinsic antinociceptive activity. Inhibition of spinal antinociception by morphine with agents which increase cyclic AMP levels in biochemical experiments is consistent with the hypothesis that some opiate actions are due to inhibition of adenylate cyclase. However, in view of the consistent increase in the effect of morphine with phosphodiesterase inhibitors at higher doses, this hypothesis may be insufficient to account for opiate interactions with the adenylate cyclase system in the spinal cord. Some effects on spinal antinociception also may be due to additional pharmacological actions of the agents used. PMID- 1714609 TI - The tachykinin NH2-senktide inhibits alcohol intake in alcohol-preferring rats. AB - The present study evaluated the effect of the intracerebroventricular injection of the tachykinins, substance P, neurokinin A and [Asp5.6,MePhe8]substance P(5 11) (also referred to as NH2-senktide), on the alcohol intake of genetically selected, alcohol-preferring rats. Animals were offered both water and 8% ethanol 2 h/day; tachykinins were administered just before access to fluids. Neurokinin A and substance P did not modify alcohol intake at doses up to 1000 and 2000 ng/rat, respectively. On the other hand, NH2-senktide potently suppressed alcohol intake at doses of 31.2-500 ng/rat. At the same doses, however, it did not significantly affect water intake. This finding suggests that its effect on alcohol intake might be rather selective and not due to general impairment of the behavior. Activation of tachykinin NK-3 receptors, for which NH2-senktide is a highly selective agonist, produces angiotensin II release in the brain; however, the effect of NH2-senktide on alcohol intake is probably not mediated by angiotensin II, as suggested by the fact that it is not modified by captopril pretreatment. PMID- 1714610 TI - Application of a CCD linear array camera in the quantification of tissue staining: NBT detection in ischaemic myocardium. AB - A method was developed to quantify the intensity of tissue staining using a CCD (charge-coupled device) camera. Reflection spectra of NBT-stained (nitro-blue tetrazolium-stained) and unstained myocardium were recorded via fibre optics coupled to a CCD camera, connected to a microcomputer. The calculation of the intensity of staining was based on evaluation of the NBT-related changes of the reflectance spectrum. In each of six anaesthetized sheep, global ischaemia was induced by cross-clamping of the aorta. The hearts were removed and incubated at 35 degrees C. At predetermined times two sections of ventricular myocardium were taken, one of which was then stained with NBT, the other being left unstained. Evaluation of the reflectance spectra from stained sections during the first phase of ischaemia showed a slight loss of NBT colour intensity followed by a more rapid loss of staining until the values of the unstained sections were reached. In contrast to the conventional visual evaluation, the method provides quantitative data on the intensity of staining, and allows use of the NBT technique for statistical evaluation of what happens over time, and the regional distribution of ischaemic injury of tissue. This method may also be applied to other staining techniques. PMID- 1714611 TI - Hepatocellular carcinomas with excessive copper accumulation: CT and MR findings. AB - Results of orcein staining and findings obtained with non-contrast-medium enhanced computed tomography (CT) and magnetic resonance (MR) imaging (T1- and T2 weighted images) were compared for 53 histologically confirmed hepatocellular carcinomas (HCCs). HCCs were 5 cm in the largest diameter. Three lesions with diffuse and strong orcein staining were highly attenuating on nonenhanced CT images. Electron x-ray microanalysis of one of them revealed copper. The high attenuation value at nonenhanced CT may have been due to the abundant copper binding protein in the cancer cells. The difference between orcein staining results and attenuation patterns at CT was statistically significant (chi 2, P less than .001). It could not be concluded that the paramagnetic effects of copper in HCC had an influence on signal intensity on MR images. The difference between orcein staining results and signal-intensity patterns on T1- and T2 weighted images was not statistically significant. PMID- 1714612 TI - Biomagnetic localization of ventricular arrhythmias. AB - The magnetic fields caused by electrical activity of the human heart can be coherently measured with a highly sensitive, multichannel, superconducting quantum interference-device system and can enable noninvasive localization of the underlying electrical activity. The magnetocardiograms (MCGs) of 10 patients with spontaneous premature ventricular complexes (PVCs), three patients with ventricular tachycardia (VT), and four healthy subjects with induced paced beats were recorded for 2-15 minutes. After correction for superimposed repolarization activity, the site of origin of the arrhythmias was localized from the magnetic field distribution at the onset of the ectopic beats. The localization results of paced beats showed an error of a few millimeters in relation to the position of the catheter tip. The results of spontaneous PVC and VT were confirmed with endocardial mapping or associated with ischemic lesions. The authors conclude that multichannel magnetocardiographic studies enable the completely noninvasive localization of ventricular arrhythmias. PMID- 1714613 TI - Tacrine: a pharmacological review. PMID- 1714614 TI - [Signal transduction by IGF-I receptor kinase]. PMID- 1714615 TI - [Hemopoietic growth factors and signal transduction]. PMID- 1714617 TI - [Growth factors of chondrocytes]. PMID- 1714616 TI - [Factors inducing differentiation of myeloid leukemic cells]. PMID- 1714618 TI - [Platelet-derived endothelial cell growth factor]. PMID- 1714619 TI - [Involvement of cellular responses to growth factors in angiogenesis and transformation malignant]. PMID- 1714620 TI - Interaction between prostacyclin and cortisol in fetal lung cells: effects on cAMP production. AB - Glucocorticoids secreted by the fetal adrenal, or administered for therapeutic reasons, stimulate fetal lung maturation in the human and other species. Prostacyclin, produced within the lung may be another agent with maturational effects. In this investigation we have demonstrated that glucocorticoids interact with lung cells and increase their response to a prostacyclin analogue (Iloprost, PGIp). This agent stimulates adenylate cyclase activity in fetal lung fibroblasts, fetal lung epithelial cells and in neonatal vascular smooth muscle cells. The cAMP response to PGIp in fibroblasts and epithelial cells occurred in the range 3nM-1 microM. When fibroblasts were pretreated with cortisol before PGIp, cAMP was increased 2-3 fold (p less than 0.01). There was a similar increase in cAMP after cortisol pretreatment in response to PGIp by fetal lung epithelial cells, but not with smooth muscle cells. The action of cortisol was blocked by an inhibitor of RNA synthesis (Actinomycin D) but not by an inhibitor of DNA synthesis (5-fluorodeoxy-uridine). Additional experiments with cholera and pertussis toxins, and with forskolin suggest that cortisol principally increases the quantity or activity of the adenylate cyclase sub-unit in fetal lung fibroblasts and, in doing so, increases the cAMP response to PGIp. PMID- 1714621 TI - [Effect of isoproterenol on the liberation of histamine from rat lung mast cells]. AB - Rat lung mast cells were stimulated with drugs with distinct mechanisms of action, namely concanavalin A, compound 48/80 ionophore A23187, in the presence of the beta adrenergic agonist (-)isoproterenol. Cells show a high response when they are stimulated with FNa-calcium. Isoproterenol does not inhibit histamine release induced by any stimuli, but enhances the response to concanavalin A and compound 48/80. Results point to the lack of beta activity on rat lung mast cells. PMID- 1714622 TI - [Inhibition of histamine liberation in mast cells from the lung and the intestine of the dog by isoproterenol]. AB - Enzymatically isolated dog lung and gut mast cells were stimulated with compound 48/80, ionophore A23187, concanavalin A and FNa-Ca. Cell response elicited by A23187, concanavalin A or 48/80 is almost completely inhibited by isoproterenol. Concanavalin A induced histamine release on gut mast cells is high, indicating an elevated degree of sensitization of these cells. Results point to the existence of beta adrenergic inhibitory activity on dog lung and gut mast cells. PMID- 1714623 TI - [Depression, nonverbal intellectual impairment and quality of life following left brain ischemic insult--results of a catamnestic study]. AB - The purpose of the present study was to establish the relationship between persisting aphasia and the extent of overall disability in the long-term outcome following left hemisphere ischaemic stroke. 55 right-handed patients who had sustained an initial left-sided cerebral infarction, verified by CT scan, were investigated after a mean observation period of six years. 39 patients were categorized as being non-aphasic, and 16 as being aphasic (3 Global, 6 Broca's, 1 conduction, 1 transcortical motor and 5 anomic aphasics) at the end of the follow up period. Regarding motor and sensory functions, a correlation between the presence of aphasia and the severity of deficits could be established at the end of the follow-up period. With respect to activities of daily living, a significantly larger number of aphasic stroke victims had to rely on help by others. Furthermore, the persistence of aphasia also negatively influenced the subsequent occupational capacity. With regard to social participation and leisure activities, a significant reduction was found in aphasic long-term stroke survivors as compared to non-aphasics. Concerning quality of life, both groups reported a marked decline at the end of the observation period; the presence of aphasia had an additional negative effect. However, as regards the long-term non verbal cognitive impairment, statistical analysis revealed no significant differences between both groups. In addition, aphasic stroke survivors did not demonstrate a higher incidence of depressive states than those without language deficit. On the basis of our results it is concluded that the presence of aphasia in left-hemispheric ischaemic stroke survivors indicates a more severe stroke, resulting in greater physical disability and social handicap in the long-term outcome.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714624 TI - The structure of the mycobacterial cell envelope is not yet understood. PMID- 1714626 TI - [Swollen joint, impaired kidney function and diarrhea in an elderly insurance professional]. PMID- 1714625 TI - [Chemical mediators and anti-mediator drugs]. PMID- 1714627 TI - Immunity to sexual stages of human malaria parasites: immune modulation during natural infections, antigenic determinants, and the induction of transmission blocking immunity. AB - Four antigens of Plasmodium falciparum have so far been identified as targets of transmission-blocking antibodies; three of them (Pfs 230, 48/45) are detectable in gametocytes and expressed on gametes, the fourth (Pfs 25) appears only after fertilization. Epitope analyses of each antigen were made with competitive immunoassays, and the extent of antigenic diversity determined amongst numerous isolates of P. falciparum. There was minimal variation within one of the two epitopes on Pfs 230 both of which induce transmission-blocking antibodies. The epitopes on Pfs 25 to which blocking monoclonal antibodies respond showed a variability amongst different isolates by immunofluorescence which was unexpected in view of sequence data on the molecule. Five epitope regions have been identified on Pfs 48/45 and antibodies to them interact in a complex manner. Antigenic diversity affecting these epitopes was minimal. In P. vivax malaria much greater polymorphism was seen amongst gamete surface antigens. Natural P. falciparum infections induce antibody responses to gametocyte/gamete surface antigens that will suppress infectivity to mosquitoes but these responses may involve reactivity with any of a series of different epitopes, interactions between antibodies, and may be sequential. In P. vivax infections antibody to the sexual stage antigens may suppress or enhance transmission depending on the antibody level. Cytokine production induced by sexual stage antigens may also modulate transmission, by rendering gametocytes non-infective. Experimental studies showed marked MHC-restriction of immune responses to gamete antigens (but not to the Pfs 25 zygote antigen); the evidence from studies in humans is less convincing. Antibody responses to the sexual stage antigens seem to be more frequent in persons who have experienced only one or a few attacks of malaria as opposed to those who have been exposed frequently. Some form of down-regulation may therefore be occurring. PMID- 1714628 TI - In situ expression of mRNA for proto-oncogenes in benign prostatic hyperplasia and in prostatic carcinoma. AB - Nine cases of prostatic carcinoma and 5 cases of benign prostatic hyperplasia (BPH) were examined for the in vivo expression of mRNA by an in situ hybridization technique for cellular proto-oncogenes, c-myc, c-fos, and c-sis. The latter of which encodes the B-chain of the platelet-derived growth factor (PDGF). High levels of c-myc and c-sis mRNAs were demonstrated in one highly differentiated prostatic carcinoma. Signals were localized in epithelial cells, both in packed cancer cells and in benign hyperplastic cells with glandular differentiation. A lower level of c-fos mRNA was also detected in the same areas. Four other carcinoma specimens showed c-sis mRNA, three of which also expressed c fos mRNA. One other carcinoma expressed only c-myc mRNA. Three BPH specimens also contained c-sis and c-fos mRNA with one also exhibiting c-myc mRNA in the glandular epithelial cells. The results support the possibility that local production of growth factors, i.e. PDGF, may play a role in proliferation of prostatic epithelial cells. PMID- 1714629 TI - [Palliative radiation therapy]. AB - Palliative oncological therapy attempts to maintain survival with a good quality of life in patients who cannot be cured. The aim of the treatment is in most cases the relief of agonizing or restricting symptoms. With metastases in particular, the shortest possible treatment period is desirable. The decision as to the best radiation treatment modality or technique depends on the individual needs and risks in each patient. Evaluation of the treatment results is difficult. It is worthwhile to use very specialized radiation techniques to improve the quality of life, but they should not replace supportive care and attention to the patients' psychological problems. PMID- 1714630 TI - [Laser therapy and death caused by arrhythmia in ischemic heart disease]. AB - The paper presents a new treatment of myocardial infarction which proved its high antiarrhythmic effectiveness in 300 MI patients followed up for 2 years. The method implies endogenous radiation of blood by He-Ne laser. Holter monitoring revealed that such radiation in the acute MI period promoted the arrest of high grade ventricular arrhythmia more efficiency than lidocaine++ and prevented primary ventricular fibrillation. The 2-year follow-up provides evidence for a significant reduction in the occurrence of high-grade and lethal outcomes which decreased by half. In view of high occurrence of high-grade extrasystole in most decreased control subjects, this may be attributed to less frequent arrhythmic deaths. Lethal outcomes in laser therapy were reported in persistent cardiac failure, repeat myocardial damage and in presenile and senile patients. PMID- 1714631 TI - Palliation of esophageal cancer--operative resection versus laser and afterloading therapy. AB - Since 1985, 71 patients with end-stage esophageal cancer have been treated either by surgical (n = 26) or endoscopic laser palliation (n = 45). In 16 of 45 patients treated by endoscopy, additional radiotherapy (extrenal and endoluminal irradiation) was performed. Surgery and Nd:YAG recanalization were initially effective in removing the malignant obstruction in 80% of cases. There were no significant differences in survival in either group. The stenosis-free interval was longer in patients who underwent surgery: 24 weeks; minimal stenosis-free interval: 20 weeks. Local recurrences occurred earlier in the endoscopic study group (mean survival: 36 weeks; minimal stenosis-free interval: 20 weeks). Most stenoses successfully underwent further laser treatment. Although only 35% of patients treated endoscopically underwent additional afterloading therapy, this treatment appears to prolong palliation (mean survival: 38 weeks; minimal stenosis-free interval: 36 weeks). A few patients bled after endoscopy and were treated conservatively. The most important complication in the afterloading group was esophagobronchial perforation, which caused one death in our series. Transient pulmonary problems were the most common complication (31%) in the surgical group with a hospital mortality of 19%. Overall, the improvement in the quality of life after surgery was better. However, our results show that Nd:YAG recanalization and afterloading therapy are effective therapeutic alternatives in patients unfit for surgery. PMID- 1714632 TI - Endoscopic placement of a prosthesis for benign anastomotic stenosis after oesophagectomy and colonic interposition. AB - After oesophagectomy for oesophageal carcinoma and retrosternal colonic interposition, a benign stenosis developed at the collar anastomosis. Bouginage was unsuccessful. Therefore, a prothesis was placed endoscopically, which enabled the patient to swallow without problems until his death as a result of diffuse metastasis 9 months later. PMID- 1714633 TI - Correlation of 125I-LSD autoradiographic labeling with serotonin voltage clamp responses in Aplysia neurons. AB - Autoradiographic receptor binding studies using 125I-LSD (2-[125I]lysergic acid diethyamide) revealed intense labelling on the soma of a symmetrically located pair of cells in the abdominal ganglion of Aplysia californica. This binding was blocked by micromolar concentrations of serotonin and lower concentrations of the serotonergic antagonists, cyproheptadine and mianserin (Kadan and Hartig, 1988). Electrophysiological investigation of responses to serotonin of neurons in the left upper quadrant, where one of the labeled neurons is located, revealed a range of serotonin responses. Cells L3 and L6 have a K+ conductance increase in response to serotonin that is not blocked by cyproheptadine or mianserin. Cells L2 and L4 have a biphasic response to serotonin: a Na+ conductance increase, which can be blocked by cyproheptadine and mianserin, followed by a voltage dependent Ca2+ conductance which is blocked by Co2+ but not the serotonergic antagonists. Cell L1, and its symmetrical pair, R1, have in addition to the Na+ and Ca2+ responses observed in L2 and L4, a Cl- conductance increase blocked by LSD, cyproheptadine and mianserin. LSD had little effect on the other responses. We conclude that the symmetrically located cells L1 and R1 have a Cl- channel linked to a cyproheptadine- and mianserin-sensitive serotonin receptor that is selectively labelled by 125I-LSD. This receptor has many properties in common with the mammalian serotonin 1C receptor. PMID- 1714634 TI - Effects of serotonergic denervation on the density and plasticity of brain muscarinic receptors in the rat. AB - This study investigated whether serotonergic lesion may affect density, sensitivity, and plasticity of muscarinic receptors in hippocampus and cerebral cortex. Intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) in rats produced a 90% reduction in cortical and hippocampal 5-hydroxytryptamine (5 HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents. In these brain areas, the 5,7-DHT lesion did not affect the overall density of muscarinic receptors or those of M1 and non-M1 muscarinic receptor subtypes as assayed using [3H]N methylscopolamine ([3H]NMS), [3H]pirenzepine, and [3H]NMS in the presence of pirenzepine, respectively. In addition, the binding of the muscarinic agonist [3H]oxotremorine-M (OXO-M), taken as an indirect index of coupling efficiency of non-M1 receptors with G-proteins, did not change significantly in cortex and hippocampus of 5,7-DHT-lesioned rats. Similarly, carbachol-induced accumulation of [3H]inositol phosphates (InPs) in hippocampal miniprisms showed no significant differences between tissues from 5,7-DHT-lesioned and sham-operated rats. In sham operated rats, an intraperitoneal (i.p.) injection of scopolamine (10 mg/kg once daily) during 21 days caused an increased density of [3H]NMS binding sites in cortex (+20%) and hippocampus (+26%). This up-regulation was restricted to non-M1 receptors subtypes. In 5,7-DHT-lesioned rats, chronic scopolamine failed to modify significantly the density of cortical or hippocampal M1 or non-M1 receptors. These results suggest 1) that 5-HT denervation did not affect the density and sensitivity of muscarinic receptors and 2) that the ability of cortical and hippocampal non-M1 receptors to up-regulate following repeated injection of scopolamine requires the integrity of 5-HT neurons terminating in these brain structures. PMID- 1714635 TI - Nomenclature for factors of the HLA system, 1990. PMID- 1714636 TI - Suppression of splenic accessory cell function in mice exposed to gallium arsenide. AB - Acute exposure of mice to a single intratracheal dose of gallium arsenide (50, 100, and 200 mg/kg) depresses the primary IgM antibody response to the T dependent antigen sheep red blood cells (SRBC) through alterations in the function of splenic accessory cells. To determine the mechanism by which GaAs exposure influences splenic accessory cells, the cells were isolated by adherence and their functional capability investigated 24 hr following GaAs exposure in the animal. Splenic adherent cells from GaAs-exposed mice were greatly impaired in their ability to process and present the particulate antigen SRBC to a SRBC primed T-cell population. However, GaAs exposure did not inhibit phagocytosis of fluorescent covaspheres by these cells, nor did it inhibit in vivo phagocytosis of 51Cr-labeled SRBC, indicating that the findings reported here were not due to decreased uptake of antigen by the accessory cells. Furthermore, production of IL 1 by these cells from exposed mice was not different from control and addition of exogenous IL-1 to cultures did not reverse GaAs-induced inhibition of the primary antibody response. GaAs exposure did not affect the percentage of Ia positive macrophages (F4/80 positive cells), but the amount of cell surface IAk molecules expressed was significantly decreased as measured by flow cytometry. In contrast to the SRBC response, GaAs did not suppress the ability of adherent splenocytes to process and present the antigen pigeon cytochrome c to the helper/inducer T cell clone F1.A.2 or the antigen KLH (keyhole limpet hemocyanin) to KLH-primed T cells. Therefore, GaAs exposure interferes with the capacity of splenic macrophages to process and/or present the particulate antigen SRBC, but not the soluble protein antigens pigeon cytochrome c or KLH. PMID- 1714637 TI - Chlordecone (Kepone) on the night of proestrus inhibits female sexual behavior in CDF-344 rats. AB - The effect of the estrogen-like chlorinated pesticide chlordecone (Kepone) on sexual behavior was examined in proestrous rats following treatment with 25, 50, or 75 mg/kg chlordecone. In most animals, sexual behavior, both receptivity and proceptivity, was reduced within 60 min following the higher dosage of chlordecone. Reduced sexual receptivity occurred more slowly with 50 mg/kg chlordecone (usually within 180 min) and no reduction was seen following 25 mg/kg chlordecone. The reduced sexual behavior after chlordecone treatment preceded the onset of marked chlordecone-induced tremor. A group of rats treated with 75 mg/kg chlordecone was euthanized at the time that behavioral inhibition began to develop. The content of serotonin, norepinephrine, and their principal metabolites was determined by high-performance liquid chromatography of extracts of brain tissue of these animals. In hypothalamus, increases in serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content, and a decrease in the level of norepinephrine (NE), were detected in chlordecone-treated rats relative to matched controls which received vehicle. The content of 5-HT was also increased in preoptic area of chlordecone-treated females. The content of the catecholamine metabolite, 3,4-dihydroxy-phenylacetic acid, was unaffected by chlordecone in either part of brain. These are the first observations of the parallel effects of chlordecone on receptive and proceptive behaviors, and on neurochemistry, in female rats; the results demonstrate short-latency effects of the pesticide treatment on the CNS events that mediate female reproductive behavior. Results of previous studies had led to the suggestion that chlordecone's inhibition of sexual behaviors resulted from its interaction with the intracellular estrogen receptor. However, the rapidity of the inhibition during the period of ongoing sexual behavior makes it unlikely that the inhibition is mediated by the pesticide's action at the intracellular estrogen receptor. Because of the importance of sexual behaviors to reproductive fitness, the current results indicate that nonsteroidal, behavioral mechanisms could contribute to chlordecone's neuroreproductive toxicity. PMID- 1714638 TI - Metabolic activation of the organophosphorus insecticides chlorpyrifos and fenitrothion by perfused rat liver. AB - The present study was undertaken to characterize the metabolic activation of the organophosphorus insecticides chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2 pyridyl) phosphorothionate] and fenitrothion [O,O-dimethyl O-(3-methyl-p nitrophenyl) phosphorothionate] by intact rat liver. Single-pass perfusions of rat livers with chlorpyrifos or fenitrothion to steady state conditions resulted in the appearance of their corresponding oxygen analogs in effluent. In addition, detoxification of chlorpyrifos oxon [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate] or fenitrooxon [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphate] by rat blood did not proceed at a rate rapid enough to prevent passage of at least some of these chemicals from liver to extrahepatic tissues, suggesting that hepatic biotransformation of chlorpyrifos and fenitrothion by rat liver results in their net activation. Although male rat livers produced more chlorpyrifos oxon and fenitrooxon from chlorpyrifos and fenitrothion, respectively, than did livers from female rats, the acute toxicities of chlorpyrifos and fenitrothion were greater in females than in males. Therefore, differences in hepatic activation of chlorpyrifos and fenitrothion in males and females cannot account for the sex differences in their acute toxicities in the rat. Finally, S-methyl glutathione and S-p-nitrophenyl glutathione were not detected in effluent or bile of livers perfused with fenitrothion, suggesting that glutathione-mediated biotransformation of this insecticide does not occur to any significant degree in intact liver. PMID- 1714639 TI - Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. AB - Developing mammals are more sensitive than adults to a variety of organophosphorothioate insecticides (OPs), compounds which act in vivo by inhibition of cholinesterase (ChE). Little is known, however, regarding age related differences in biochemical responses to these toxicants. The time course of ChE inhibition and recovery in whole brain was compared in neonatal (7 days of age) and adult (80-100 days of age) rats after treatment with maximal tolerated doses (MTDs) of either methyl parathion (MPS), parathion (PS) or chlorpyrifos (CPF). Neonatal rats were more sensitive than adults in all cases (MTDs for MPS, PS and CPF; neonates = 7.8, 2.1 and 45 mg/kg, s.c.; adults = 18, 18, and 279 mg/kg, s.c., respectively). In general, maximal brain ChE inhibition was similar (greater than 78%) in both age groups but ChE activity recovered faster in neonates. Plasma and erythrocyte ChE activities correlated relatively well (r = 0.794-0.943) with brain ChE activity in neonatal rats at all time points between 4 h and 7 days after treatment but similar correlations between circulating and brain ChE activities in adults were more variable (r = 0.211-0.917). The results indicate that neonatal rats are more sensitive to acute lethality from these compounds and that MTD exposures produce extensive brain ChE inhibition in both age groups. Significant inhibitor-related and age-related differences in the duration of ChE inhibition can ensue, however, following such OP exposures. PMID- 1714640 TI - Effect of manganese (II) bis(glycinate)dichloride on Ca2+ channel function in cultured chick atrial cells. AB - Manganese (II) bis(glycinate)dichloride (Mn(glycinate)2) is a coordination complex of manganese with application as a contrast enhancement agent for magnetic resonance imaging in the heart. To determine the cardioactivity of the manganese ion in this chelation cage, the effects of Mn(glycinate)2 on Ca channel function in the cultured chick atrial cell was studied. Mn(glycinate)2 decreased amplitude of contraction in chick atrial cells from embryos 14 days in ovo with complete inhibition of beating at 1 mM and half-maximal effect at 0.1 mM. Under control conditions, Bay K 8644, a Ca channel activator increased amplitude of contraction by 86% with a half maximal effect at 3.2 x 10(-7) M. In the presence of 0.025 mM Mn(glycinate)2, a concentration which had no effect on the amplitude of contraction, the maximum response to Bay K 8644 was decreased to 31%. Mn(glycinate)2 had no effect on the EC50 for the response to Bay K 8644, 1.7 +/- 0.1 x 10(-9) M (S.E.M., n = 4) in control cells compared to 2.2 +/- 0.4 x 10(-9) M (S.E.M., n = 4) in cells incubated with Mn(glycinate)2. 45Ca2+ uptake over 5 min in cultured chick atrial cells decreased from 2.0 nmol/mg protein in control cells to 1.5 nmol/mg protein in the presence of 10(-5) M PN200-110, a Ca2+ channel blocker, a decrease of 28%. 45Ca2+ uptake decreased to 0.94 nmol/mg protein (53%) in the presence of 1 nmol Mn(glycinate)2. Effects of Mn(glycinate)2 and PN200 were not additive. These data demonstrate that Mn(glycinate)2 exerts its negative inotropic effect, at least partially, by interfering with the function of the L-type Ca channels at high concentrations. PMID- 1714641 TI - Hepatitis C virus infection in patients undergoing allogeneic bone marrow transplantation. AB - Antibody to the recently identified hepatitis C virus was investigated in sera of 128 patients treated with allogeneic bone marrow transplantation, to determine the prevalence of HCV infection and its role in post-transplant liver complications. The overall prevalence of anti-HCV positivity was 28.6% (38/128 patients). The presence of pretransplant anti-HCV positivity (in 10/35 tested patients) did not seem to predict a more severe liver disease. In fact 8/10 anti HCV+ and 15/25 anti-HCV- patients had elevated transaminases at BMT, and posttransplant liver failure (due to VOD or subacute hepatitis), and post-BMT rises in transaminases occurred regardless of anti-HCV serology (P = 0.6 and 0.2, respectively). In patients tested for anti-HCV after BMT (n = 128), only two (one anti-HCV+ and one anti-HCV-) experienced VOD; the number of patients in whom liver failure contributed to death was comparable in anti-HCV-positive and anti HCV- negative patients (P = 0.4). Among 17 patients with documented posttransplant seroconversion (from anti-HCV- to anti-HCV+) the appearance of anti-HCV was concomitant with hepatitis exacerbation in 9 (53%). Histologic changes demonstrated a more severe liver damage in anti-HCV+ patients: a chronic hepatitis was diagnosed in 9/11 anti-HCV+ versus 1/7 anti-HCV- cases. Based on these observations, we conclude that hepatitis C virus has a role in liver disease in such patients, although its evaluation by the anti-HCV test is still of limited accuracy, due to low sensitivity and incomplete specificity. PMID- 1714642 TI - The effect of a chimeric mouse-human CD7 antibody on human T, natural killer, and lymphokine-activated killer cell activity in vitro. AB - A chimeric CD7 antibody has been constructed with mouse variable and human constant regions and is currently being assessed in the prophylaxis of renal graft rejection. In this study we have investigated if this antibody or its murine parental form inhibits the function of a number of immune effector mechanisms involved in host defense against infection and/or malignancy. Most memory T cells and all natural killer cells express the CD7 antigen and could therefore be affected by CD7 antibody. Murine and chimeric CD7 antibodies significantly inhibit the alloproliferation of naive (65 +/- 4% and 66 +/- 8%, respectively) but not memory T cells (86 +/- 2% and 98 +/- 4%, respectively) in a primary mixed lymphocyte reaction relative to the negative control CD10 antibody (P less than 0.001). The memory T cell proliferative response to recall antigen is also largely unaffected by murine and chimeric CD7 antibodies relative to the negative control antibody (91 +/- 12% and 103 +/- 10%, respectively). The CD7 antigen is almost completely modulated from the surface of NK cells after incubation for 24 hr with either the murine or chimeric CD7, but not the CD10, negative control. The modulation of CD7 antigen by antibody, however, does not affect the cytotoxic function of either the NK or lymphokine-activated killer cells significantly. Preincubation with the chimeric antibody however, consistently showed a small inhibition relative to the negative control of 75-80% in NK assays and to 80-90% in LAK assays. These data suggest that both murine and chimeric CD7 antibodies may have a selective effect on alloproliferation but may largely spare a major component of the host's innate immunity as well as memory T cell proliferation to previously encountered antigens. PMID- 1714643 TI - Phospholipase-C and Na-K ATPase activation by cyclosporine and FK506 in LLC-PK1, cells. Possible implications in blood pressure regulation. AB - Enhanced Na+ and water reabsorption by proximal tubular epithelial cells plays an important role in the development of systemic hypertension associated with cyclosporine immunosuppression. Since such Na+ reabsorption is subserved by sodium-potassium adenosine triphosphatase (Na-K ATPase), the current study compared the acute effects of hydrocortisone (H), cyclosporine, and FK506 on cultured LLC-PK1 cell viability and on Na-K ATPase activity. Phospholipase-C (PL C) activity was also investigated because of its possible regulatory effect on Na K ATPase activity. Culture medium containing low (5 nM, 4.1 ng/ml) or high (10 nM) concentrations of FK506 plus cyclosporine at 415 microM (500 ng/ml) resulted in cell death, whereas cyclosporine concentrations of 83 microM plus 5 nM or 10 nM FK506, or isolated use of the two drugs at high dosages, did not affect cell viability. As compared with controls, cyclosporine increased Na-K ATPase activity, particularly with addition of H (P less than 0.01). In contrast, FK506 reduced the specific activity of both PL-cyclosporine and Na-K ATPase (P less than 0.001-0.01); addition of H to FK506 resulted in an even greater fall in both the enzyme activities (P less than 0.001). Na-K ATPase activity increased in cell homogenates briefly incubated with cyclosporine in the ATPase reaction mixture (P less than 0.05) while FK506 reduced such enzyme activity (P less than 0.05), suggesting a direct effect of these agents on pump activity. These data in LLC PK1 cells pocessing proximal tubular epithelial cell characteristics indicate that the combined use of cyclosporine plus FK506 may be very deleterious to viability in such cells. The opposing effects of cyclosporine and FK506 on PL cyclosporine and Na-K ATPase activities and the possible potentiating effect of H on such responses are speculated to affect Na+ and water homeostasis in a manner that may explain differences in systemic blood pressure due to these agents. PMID- 1714645 TI - New horizons in immunosuppression. PMID- 1714644 TI - Anti-CD3 monoclonal antibody therapy. An approach toward optimization by in vitro analysis of new anti-CD3 antibodies. AB - Several problems remain associated with anti-CD3 monoclonal antibody therapy, including first-dose reactions, recurrent rejection, and the host humoral response to the xenogeneic mAb. One approach toward optimization of anti-CD3 mAb therapy involves the use of anti-CD3 mAbs of defined idiotype, isotype, and epitope specificity, so that the desired T cell activation and suppression properties are obtained and neutralization by antiidiotypic antibody is avoided. The purpose of the present study was to define the contribution of mAb isotype and epitope specificity in determining T cell activation and suppression potencies and to evaluate the idiotypic relationships among ten anti-CD3 mAbs and one anti-TCR alpha beta mAb selected for this study. Epitope mapping by flow cytofluorometry indicated that one mAb, OKT3D, possesses an epitope specificity distinct from that of other anti-CD3 mAbs. Analysis of early T cell activation, proliferation, and lymphokine production (TNF-alpha, gamma-IFN, and GM-CSF) indicated that mAb isotype exerted a profound effect on activation potency (IgG2a much greater than IgG1 greater than IgG2b), whereas epitope specificity exerted a minor effect. CTL inhibition studies demonstrated an epitope effect with highest inhibition potencies observed with OKT3D IgG1 and OKT3D IgG2b mAbs. Idiotypic analysis of nine mAbs indicated that all anti-CD3 mAbs except OKT3E possess idiotypes distinct from that of OKT3. Thus, two anti-CD3 mAbs, OKT3D IgG1 and OKT3D IgG2b, possess high immune suppression potency, low activation potency, and idiotypes distinct from OKT3. In conclusion, selection of anti-CD3 mAbs of defined idiotype and appropriate T cell activation and suppression properties may provide a means for mitigating problems associated with OKT3 therapy. PMID- 1714646 TI - Immunosuppression for organ grafting. PMID- 1714647 TI - Notes on FK 506. PMID- 1714648 TI - The influence of cyclosporine on the microvasculature of xenogeneic pancreatic islet grafts. PMID- 1714649 TI - Pathological and immunohistochemical examination in the rat treated with FK 506. PMID- 1714650 TI - Efficiency of FK 506 and CyA to prevent acute cellular rejection of pig liver allografts. PMID- 1714651 TI - The immunosuppressive effect of rapamycin on pancreaticoduodenal transplants in the rat. PMID- 1714652 TI - Prognostic relevance of silver-stained nucleolar proteins in sarcomatoid carcinomas of the breast. AB - Fourteen cases of sarcomatoid carcinomas of breast were evaluated by means of a silver technique that selectively stains proteins located in the nucleolar organizer regions (Ag-NORs). The mean area of Ag-NORs (MNORA) was in each case quantitatively analyzed by means of an automated image analyzer. Patients who died early of the disease had a higher MNORA than patients who survived longer than 3 years. The difference was statistically significant. Ag-NORs might be a novel parameter of prognostic relevance in specific cases. PMID- 1714653 TI - Risk factors for surgically treated benign prostatic hyperplasia in a prepaid health care plan. AB - The relationship of age, medical history, personal habits, and urologic symptoms to the incidence of surgically treated benign prostatic hyperplasia (BPH) was studied in a cohort of 16,219 men, aged forty years and over, who received multiphasic health checkups (MHCs) during 1971 and 1972 in Oakland or San Francisco while members of the Northern California Kaiser Permanente Medical Care Program, a large prepaid health care program. Follow-up was carried out for surgically treated BPH from the date of the MHC to the date of the earliest of the following: surgery for BPH (n = 1,027); incidence of prostate cancer (n = 329), bladder cancer (n = 119), or both (n = 10); other prostate surgery (n = 5); death (n = 2,525); membership termination (n = 4,235); or December 31, 1987 (n = 7,969). The mean length of follow-up was twelve years. In multivariate analysis utilizing the Cox proportional hazards model, the following characteristics were positively associated (p less than 0.05) with risk of surgically treated BPH: age, low body mass index, nonsmoking (vs. current smoking), urine pH greater than 5, history of kidney x-ray and of tuberculosis, and each of five urologic symptoms (dysuria, loss of bladder control, trouble starting urination, nocturia, slow urine stream). The risk of BPH associated with obstructive urologic symptoms decreased markedly with age. Some of these findings are consistent with those from other studies (age, nonsmoking), while others (high urine pH, history of tuberculosis) are new and should be examined in other study populations. PMID- 1714654 TI - Urinary symptom and quality of life questions indicative of obstructive benign prostatic hyperplasia. Results of a pilot study. AB - In a pilot study of a urinary symptom and health-related quality-of-life questionnaire for benign prostatic hyperplasia (BPH), responses from 64 Mayo Clinic patients with cystoscopic evidence of obstructive BPH were compared with those of 14 men with no cystoscopic evidence of BPH and a community sample of 64 comparably aged men with no medical history of prostate enlargement. Questions which best discriminated between the groups were those dealing with urinary symptom frequency, bother due to urinary symptoms, and worry and concern about urinary problems. The results suggest that urinary-symptom-bother and worry due to urinary symptoms may be important additions to the more usual questions asked about urinary frequency in the identification of men with BPH. These findings are preliminary, however, and will be verified in an ongoing natural history study of BPH. PMID- 1714655 TI - Mortality and reoperation following prostatectomy: outcomes in a Medicare population. AB - Data from a series of pilot projects undertaken by the Health Care Financing Administration and seven peer review organizations were used to evaluate the outcomes of prostatectomy. Outcomes in both the original random sample of 3,641 patients and subsample of 2,617 patients that had a diagnosis of benign prostatic hyperplasia and did not have a diagnosis of prostatic carcinoma were examined. Patients undergoing a transurethral resection had increased probabilities of reoperation and mortality. However, the increased risk associated with having a transurethral resection was not statistically significant after controlling for other variables associated with mortality. PMID- 1714656 TI - Incidence and outcome of surgery for benign prostatic hyperplasia among residents of Rochester, Minnesota: 1980-87. A population-based study. AB - The incidence and outcome of surgery for benign prostatic hyperplasia (BPH) was studied in Rochester, Minnesota, during the period 1980-1987. Three hundred thirty Rochester men without a diagnosis of prostate or bladder cancer underwent prostatectomy for BPH for the first time. Mean and median ages were both seventy (range: 46-95). The incidence of initial prostatectomy for BPH among men forty five years of age and older age-adjusted to the 1980 U.S. white male population was 642 cases per 100,000 persons per year (py). Among the 330 men undergoing initial prostatectomy for BPH, 14 (4.2%) had serious intraoperative complications, 32 (9.7%) were rehospitalized for urologic complications within thirty days of surgery, and 13 (3.9%) had other serious complications within thirty days after surgery, including 1 death (surgical mortality 0.3%). Forty five patients (14%) required blood transfusions within thirty days of surgery. The likelihood of reoperation within six years of the initial surgery was 15.1 percent (95% CI 9.7, 20.6). Short- and long-term postoperative mortality was not statistically significantly different than expected based on age- and sex specific mortality statistics for Rochester, Minnesota. PMID- 1714657 TI - Natural history of benign prostatic hyperplasia and risk of prostatectomy. The Baltimore Longitudinal Study of Aging. AB - The natural history of prostatism (clinically diagnosed benign prostatic hyperplasia) is examined based on symptom questionnaires and digital rectal examinations administered periodically to 1,057 men followed prospectively for up to thirty years in the Baltimore Longitudinal Study of Aging (BLSA). Benign prostatic hyperplasia (BPH) was clinically diagnosed in 527 men, 110 had a prostatectomy for BPH, and in 21 prostate cancer developed. Among men aged sixty or older with prostatic enlargement and obstructive symptoms, the twenty-year probability of surgery was 39 percent; for men aged fifty to fifty-nine years this probability was 24 percent; and for men aged forty to forty-nine years, the probability was 13 percent. The age-specific prevalence of clinically diagnosed BPH agreed closely at all ages with the age-specific autopsy prevalence of pathologically defined BPH from a published international compilation of 5 independent autopsy studies involving 1,075 prostates. PMID- 1714658 TI - Comorbidities and perioperative complications among patients with surgically treated benign prostatic hyperplasia. AB - We address the question of whether or not age and comorbidity are related to intra- and postoperative complications after a transurethral resection. The data are derived from a retrospective, population-based study conducted in Hagen, Germany, which included all patients with an initial prostatectomy for benign prostatic hyperplasia (N = 621) during the five-year period 1984-1988. Seventy seven percent of the patients had at least one of the following preoperative risk factors: heart disease, hypertension, smoking, chronic obstructive lung disease, and diabetes. There was no intraoperative death. The risk of intraoperative circulatory complications was found to be related to age only for patients without a history of heart diseases or hypertension. The incidence of major complications was 3.1 percent and was significantly higher in the oldest age group. Three patients (0.54%) died postoperatively in the hospital. Infections were the most frequent postoperative complications. The relationship of age and overall postoperative complications was not statistically significant either for patients with (p = 0.121) or without any comorbidity (p = 0.651). Based on this study it seems reasonable to conclude that age is not a clinically relevant risk factor for perioperative complications in patients who have a transurethral resection for benign prostatic hyperplasia. PMID- 1714659 TI - Age-related differences in risk factors for prostatectomy for benign prostatic hyperplasia: the VA Normative Aging Study. AB - The Veterans Administration Normative Aging Study (VANAS) is an ongoing study of a cohort of 2,280 men assembled between 1963-1968 and followed to the present time. Among this cohort of men, urinary symptoms were found to be strongly predictive of surgery for benign prostatic hyperplasia (BPH), and this risk varied by age. Nocturia (OR 1.8) and hesitancy (OR 4.3) were found to be predictive of surgery for younger men (age range 49-55), while only nocturia (OR 2.4) was predictive among older men (age range 62-68). This age-related difference in both identification of risk factors and magnitude of association with surgery was independent of assessing other commonly studied risk factors. Ultimately, the decision to operate is a combination of patient's need, access to care, and patient's preference. Further research will be required to assess which factor or factors are responsible for the age-related differences in surgical risk factors. PMID- 1714660 TI - Differentiation patterns in human testicular germ cell tumours. PMID- 1714661 TI - Immunohistochemical study of sarcoma-like mural nodules in a mucinous cystadenocarcinoma of the ovary. AB - We describe an ovarian mucinous cystadenocarcinoma with several sarcoma-like mural nodules (SLMN). The distinction between these lesions and foci of anaplastic carcinoma is important because of the poor prognosis of the latter. We have studied the potential value of immunohistochemistry in the differential diagnosis of these two lesions. In contrast to an anaplastic carcinoma, which was largely composed of keratin-positive cells, SLMN were negative or only focally positive. Therefore, in distinguishing SLMN from foci of anaplastic carcinoma, keratin strains may be added to other gross and microscopical differential features, such as size, demarcation, and presence or lack of obvious carcinomatous elements. PMID- 1714662 TI - Human immunodeficiency virus type 2 vpx protein augments viral infectivity. AB - The genomes of HIV and SIV are complex and contain several accessory genes which modulate viral replication and pathogenicity. One of these genes, vpx, is unique to the HIV-2/SIV group of viruses and encodes a virion-associated protein of unknown function. To examine the function of vpx, we constructed a vpx-deficient HIV-2 proviral clone and characterized its in vitro biological properties. Following transfection into immortalized T-cell lines, vpx-mutant HIV-2 was fully replication competent and exhibited growth kinetics and cytopathic properties equivalent to wild-type HIV-2. In addition, vpx-deficient virions were indistinguishable from wild-type HIV-2 in ultrastructure, composition of major structural proteins, and reverse transcriptase activity. In PHA-stimulated normal peripheral blood mononuclear cells (PBMCs), however, vpx-deficient virus replicated at substantially lower titers and required a 100- to 1000-fold higher inoculum to establish a productive infection. This defect was localized to early events in the viral life cycle since vpx-deficient virus exhibited a 5- to 10 fold reduction in initial (single cycle) viral DNA synthesis following acute infection of primary PBMCs. Paradoxically, in long-term (9-23 months) cultures of immortalized T-cells (SupT1) continuous high level replication of vpx-deficient, but not wild-type, virus was observed, indicating less efficient viral spread and cell killing and a more attenuated phenotype of vpx-deficient HIV-2. Taken together, these results demonstrate that vpx is required for the production of fully infectious and cytopathic HIV-2 virions and that it functions early in the viral life cycle by facilitating viral entry and/or reverse transcription. The pronounced replicative defect of vpx-deficient HIV-2 in primary PBMCs but not in short-term cultures of immortalized T-cell lines emphasizes the need to characterize the properties of nonessential HIV accessory gene products in natural target cells. PMID- 1714663 TI - Identification of residues in VP2 that contribute to poliovirus neutralization antigenic site 3B. AB - Amino acid substitutions were placed at residues 74, 243, and 246 in capsid protein VP2 of poliovirus serotype 1, using site-specific mutagenesis methods. The proximity of these residues to those previously identified in neutralization site 3B suggests that these residues may also contribute to neutralization site 3B and potentially be under antibody selective pressure to mutate. However, sequence analyses of independent serotype 1 isolates indicate high sequence conservation at these residues, suggesting selective pressures are present within the virus to maintain sequences within these loop regions. All viable mutants display partial or complete resistance to neutralization by the site 3B neutralizing monoclonal antibodies. Cross-neutralization data with the site specifically generated viral mutants confirm that these residues do indeed contribute to forming neutralization site 3B and also identify the participation of a new loop region within site 3B. However, many amino acid substitutions generate nonviable virus mutants and even conservative amino acid substitutions produce growth-compromised virus mutants. These data suggest that previous definition of neutralization antigenic sites by isolation of neutralization resistant mutants favors detection of viable mutant viruses with more normal growth characteristics and is inherently biased against detection of neutralization antigenic sites formed by residues critical for other stages of virus replication. PMID- 1714664 TI - Alleged common antigenic determinant of tobacco mosaic virus coat protein and the host protein ribulose-1,5-bisphosphate carboxylase is an artifact of indirect ELISA and western blotting. AB - We have reported the detection of an antigenic determinant shared by the tobacco mosaic virus coat protein and the large subunit of ribulose-1,5-bisphosphate carboxylase, a host protein (R.G. Dietzgen and M. Zaitlin Virology 155, 262-266, 1986). This conclusion was questioned by D. Zimmermann and M.H.V. Van Regenmortel (Arch. Virol. 106, 15-22, 1989). Thus we have reinvestigated this unexpected serological cross-reaction in Western immunoblotting and indirect ELISA. We found that when skimmed milk instead of bovine serum albumin was used as a blocking agent and as a diluent for antibodies and alkaline phosphatase conjugates, the alleged cross-reaction was abolished. In light of these findings, we retract our previous conclusions. PMID- 1714665 TI - ATP induces the assembly of polyoma large tumor antigen into hexamers. AB - Using zone velocity sedimentation and nondenaturing polyacrylamide gel electrophoresis, we have determined that purified polyoma large tumor antigen (Py T Ag) consists of discrete forms ranging from more abundant monomers and dimers to several higher but clearly distinguishable oligomeric species. Addition of ATP and MgCl2 to Py T Ag caused a dramatic increase in the appearance of Py T Ag hexamers, a form that, based on its SV40 T Ag counterpart, is likely to play a crucial role in its DNA replication functions. Other nucleotides in addition to ATP, as well as a nonhydrolyzable ATP derivative, were capable of inducing hexamer formation. This approach may further elucidate the role(s) of different forms of Py T Ag in viral regulatory processes. PMID- 1714666 TI - Expression of the F glycoprotein gene from human respiratory syncytial virus in Escherichia coli: mapping of a fusion inhibiting epitope. AB - A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus (strain A2) was inserted into a bacterial expression vector containing the lambda PR promoter. Upon heat induction, Escherichia coli cells harboring the vector produced a 45-kDa peptide which reacted with rabbit polyclonal antiserum to the native F protein. Expression of the F gene resulted in severe inhibition of bacterial growth, which was overcome by deletion of the DNA sequences encoding the F signal peptide. The region of the F protein which reacted with a virus-neutralizing and fusion inhibiting monoclonal antibody was probed by expressing cDNA fragments encoding different protein domains in E. coli and testing antibody reactivity by Western blot analysis. Analysis of six fragments yielded an overlapping antibody-reactive region between amino acids 253 and 298. Analysis of reactivity with a cassette of synthetic peptides confirmed that the virus-neutralizing epitope mapped between residues 289 and 298 defined by the amino acid sequence M-S-I-I-K-E-E-V-L-A. PMID- 1714667 TI - Type-specific and cross-reactive epitopes in human papillomavirus type 16 capsid proteins. AB - Genital human papillomavirus (HPV) 16 infection is frequently associated with cancer of the uterine cervix, as well as with precancerous lesions. In order to generate serologic reagents which might be useful in the diagnosis of HPV 16 infection, rabbit polyclonal and mouse monoclonal antisera were raised to carboxy terminal peptides from the HPV 16 L1 and L2 open reading frames (ORFs). Anti-L1 and -L2 peptide sera recognized HPV 16 L1 and L2 fusion proteins in Western blots and by immunoprecipitation. In Western blot analysis of L1 proteins from different HPV types, antisera to the L1 peptide reacted only with HPV 16, thus identifying an HPV 16 type-specific linear epitope. Anti-L2 peptide sera reacted with L2 fusion proteins from HPVs 6 and 16, but not from BPV, thus identifying a partially cross-reactive epitope in the HPV 16 L2. Computer analysis of carboxy terminal amino acid sequences of the L1 and L2 ORFs of multiple HPV types supported the Western blot findings. Despite the HPV 16 type specificity found in Western blots, anti-L1 peptide sera identified nuclear antigen by immunocytochemistry in cervical biopsies infected with HPV 16, as well as other genital HPV types. Anti-L2 peptide sera failed to recognize antigen in infected tissue. PMID- 1714668 TI - Host-associated selection of sequence variants from a satellite RNA of cucumber mosaic virus. AB - The sequence of lx-satRNA, a CMV-satRNA necrogenic for tomato that shows the unusual property of accumulating in cucumber and in squash to levels similar to those in tomato and in tobacco, was determined. When compared with the sequences of other necrogenic CMV-satRNAs that do not show this property, or with a sequence variant of lx-satRNA that retains it, the high accumulation of lx-satRNA in squash was correlated with the presence of two U/C transitions. The sequences of progeny lx-satRNA from passage experiments in tomato and in squash show that a sequence variant having a U at position 102 was selected in tomato and that the parent sequence with a C at 102 was consistently restored in squash when this U variant was used as inoculum. The results from these passage experiments may be explained by the heterogeneous nature of RNA populations, built from a number of variants of a master sequence, and illustrate the validity of this concept for CMV-satRNAs. The data also show that a minor sequence change, such as the transition C/U at 102, may have a major effect on the fitness of sequence variants of CMV-satRNAs in different hosts, and this may be relevant to the evolution in nature of these small RNAs. PMID- 1714669 TI - [Hemodynamic and clinical studies with Bonnecor--a new anti-arrhythmia drug]. AB - It is reported on clinical and haemodynamic results with Bonnecor, a new antiarrhythmic drug with class I and IV properties, on 68 test persons in parenteral and oral administration. The haemodynamic acute investigations (dosage 2 x 0.1 to 1 x 0.3 mg/kg body weight) which were performed on 10 test persons with compensated heart diseases did not show any negative haemodynamic effects of Bonnecor at rest and on exercition. The chronic effect on ventricular extrasystoles of the degrees of severity mostly II-IV were investigated in the open experiment on 17 patients as well as on 3 patients with supraventricular dysrhythmias and on 30 patients with ventricular extrasystoles Lown III-IV in the single blind cross-over experiments to verapamil retard. A clinically relevant result with regard to the reduction of the ventricular extrasystole about 75% was found in 23% of the patients (open study) and 33.3% of the patients (cross-over), respectively. The reduction of the ventricular extrasystole was significant compared with the empty value. Under the influence of verapamil a significant reduction of the ventricular extrasystole could not be verified, the differences between Verapamil and Bonnecor were, however, not to be ascertained statistically. The effect of Bonnecor on couplets and volleys was clinically relevant in 52 and 46%, respectively, of the patients (reduction greater than or equal to 93%) and approximately corresponds to the results with other antiarrhythmic drugs. The tolerability is to be estimated as relatively good.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714670 TI - Effect of CCK on food intake in man: physiological or pharmacological effect? AB - The present study was designed to determine in humans the dose of CCK which suppresses food intake. 18 male subjects received in randomized order either i.v. saline or Thr28 Nle31 CCK 25-33 (CCK-9) at 100 or 500 pmol/kgh, respectively. In addition, 7 subjects received CCK together with the opiate receptor antagonist naloxone to examine if activation of endogenous opioids might interfere with the potential satiating effect of CCK. Food intake during saline was 32 +/- 2 sandwiches (mean +/- SEM), during CCK-9 100 pmol/kgh 28 +/- 2 (n.s.) and only 12 +/- 3 during CCK-9 500 pmol/kgh (p less than 0.01). The respective water intake was 730 +/- 70 ml, 590 +/- 60 ml (n.s.) and 320 +/- 50 ml (p less than 0.01). Naloxone further reduced food and water intake during high but not low dose CCK or saline. During saline postprandial insulin levels rose by 49 +/- 6 microU/ml within 45 min which was attenuated during low dose (23 +/- 6 microU/ml; p less than 0.01) and high dose CCK-9 (1 +/- 1 microU/ml; p less than 0.001). Plasma glucagon did not change in control or CCK experiments. The postprandial rise of pancreatic polypeptide was attenuated during high dose CCK. Naloxone had no effect on the hormonal response except for a prolonged reduction of insulin and glucose levels following high dose CCK + naloxone. Plasma CCK levels rose by 5.4 pmol/l in controls but by 55 and 255 pmol/l during the low and high dose CCK infusion, respectively. These data demonstrate that suppression of food intake in man by i.v. CCK is a pharmacological rather than a physiological effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714671 TI - Comparison between the synthetic cholecystokinin analogues caerulein and Thr28NlE31CCK25-33(CCK9) with regards to plasma bioactivity, degradation rate and stimulation of pancreatic exocrine function. AB - A new synthetic analogue of cholecystokinin, Thr28Nle31CCK25-33(CCK9) is compared with caerulein with regards to plasma bioactivity, degradation rate, side effects, and stimulation of pancreatic secretion. 24 healthy male volunteers were intubated with a double lumen Lagerlof-tube. 30 min after correct positioning of the tube subjects received a continuous intravenous infusion of synthetic secretin (1 U/kg) together with either ceruletide (61.5 pM/kg) or CCK9 (30 pM/kg) both for 45 min. 30 pM/kg of CCK9 have been shown by others to cause maximal enzyme secretion (1). 61.5 pM/kg (= 100 ng) of caerulein are probably supramaximal but used by many centers for direct pancreatic function tests. Plasma CCK was measured by bioassay which compares amylase release of isolated rat pancreatic acini stimulated by plasma extracts with known standards of CCK8. Lipase, amylase, trypsin, chymotrypsin, and bicarbonate were measured in 15 min fractions after the onset of CCK infusion. Both drugs caused a similar stimulation of pancreatic enzyme secretion during infusion of the respective analogue which declined after termination. After the onset of CCK9 infusion plasma bioactivity reached a plateau around 20 pM at 15 min. Values declined after 30 min. After termination of infusion bioactivity rapidly declined within 3 min but still remained slightly elevated after further 15 min as compared to basal values (3.9 vs 0.6 pM). Plasma kinetics of caerulein were quite similar. However, despite a dose which was only twice as high as compared to CCK9, plasma bioactivity was six times higher with plateau values of about 120 pM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714672 TI - [The effect of electrical and acoustic stimulation in early ontogeny on the characteristics of higher nervous activity and on the nucleic acid content of the tissues in chum salmon fry]. AB - Stimulation of salmon larvae by electric current led further to unstable character of reactions in the open field. Acoustic stimulation both by tonal and musical signals favourably influenced the behaviour in the open field and the ability to elaborate conditioned reflexes. Changes of the content of nucleic acids were not found in the brain tissue but were found in the muscles, where they correlated with the growth speed and motor activity of the experimental fishes. The obtained data show the possibility of elaboration of applied methods of the control of CNS development and behaviour of young fishes at fishing plants. PMID- 1714673 TI - [The selective participation of brain-specific non-histone proteins of chromatin Np-3,5 during the reproduction of a defensive habit to food in edible snails]. AB - The role of brain-specific nonhistone proteins of chromatine Np-3.5 in the processes of reproduction of elaborated defensive habit to food was studied in previously learning snails. It was found, that gamma-globulines to Np-3.5 during tens of minutes inhibited behavioural and neuronal reactions elicited by a definite conditioned stimulus--carrot juice, without changing reactions to other conditioned stimulus--apple juice. gamma-globulines to other nonhistone proteins of chromatine did not influence the reproduction of food rejection habits. It was supposed that brain-specific nonhistone proteins of chromatine Np-3.5 were selectively involved in the molecular processes providing for neurophysiological mechanisms of information extraction from the long-term memory. PMID- 1714674 TI - [Optimization of the parameters that permit improvement in plate sorption activity for solid-phase immunoenzyme analysis]. AB - The possibility of increasing the sorption activity of polystyrene plates with an initially low capacity for sorption has been shown. The qualitative and quantitative parameters of the process of physical modification have been ascertained. The empirical formula for achieving the highest degree of the sorption activity of plates by their definite exposure to ultraviolet radiation has been obtained. PMID- 1714676 TI - Over the wall. PMID- 1714675 TI - Transurethral balloon dilatation of the prostatic urethra: effectiveness in highly selected patients with prostatism. AB - Transurethral balloon dilatation of the prostate has been shown to be a safe and potentially effective alternative to surgery in the treatment of benign prostatic hyperplasia, with a 66% success rate in relatively unselected patients. This study hypothesized that more careful patient selection might result in a significantly better rate of improvement. Ninety-one subjects with symptoms and signs of prostatism attributable to benign prostatic hyperplasia were studied. Group 1 comprised 42 patients with an initial mean symptom score of 16.8, residual urine of 249 ml, maximal flow rate of 7.9 ml/sec, and nomogram of maximal flow rate of -1.5. Group 2 comprised 49 less symptomatic patients with an initial mean symptom score of 14.5, residual urine of 105 ml, maximal flow rate of 10.7 ml/sec, and nomogram of maximal flow rate of -0.8. The difference in mean age and prostate size between groups was not statistically significant, but differences in baseline symptom score, residual urine, maximal flow rate, and nomogram of maximal flow rate were significant (p less than .04). Transurethral balloon dilatation of the prostate was performed under local anesthesia or IV sedation and analgesia with single-or double-balloon catheters with maximal diameters of 25-30 mm inflated to 2.5-4.0 atmospheres pressure for 10 min. Patients were followed up with repeat symptom scoring, uroflometry, and measurement of residual urine. After a mean follow-up of 22 months (range, 6-48 months), an improvement in symptom score was seen in 80% of group 2 patients compared with 43% in group 1. Improvement in symptom scores was statistically significant in both groups (p less than .04). We conclude that transurethral balloon dilatation of the prostate is more effective in patients with more moderate symptoms and with less marked signs of obstruction than in patients with more marked prostatism. PMID- 1714677 TI - Obstetric complications in a patient with Bernard-Soulier syndrome. AB - We describe the obstetric complications and management of a patient with Bernard Soulier syndrome. Severe bleeding at the time of delivery and delayed postpartum hemorrhage were prominent features of her pregnancies. Further complicating this woman's pregnancies was the development of antibodies to platelet glycoprotein IB/IX, leading to neonatal alloimmune thrombocytopenia. PMID- 1714679 TI - Prolonged exposure to wood preservatives induces endocrine and immunologic disorders in women. PMID- 1714678 TI - The mechanism responsible for diminished neutrophil production in neonates delivered of women with pregnancy-induced hypertension. AB - The neonatal neutropenia after pregnancy-induced hypertension is a function of diminished neutrophil production. These studies test the hypothesis that this diminution is due to decreased production of neutrophilic growth factors, reduced responsiveness of neutrophil progenitors to these factors, or the presence of an inhibitor. While the concentrations of placentally derived colony-stimulating factors were similar in normotensive and hypertensive gestations, bioassay demonstrated less colony-stimulating activity in placental conditioned media from hypertensive gestations. Evaluation of the responsiveness of progenitors to recombinant factors revealed no differences between those from normotensive and hypertensive gestations. However, neutrophilic colony formation in vitro was significantly inhibited after the addition of conditioned media or sera from hypertensive gestations, whereas the addition of these from normotensive gestations had no inhibitory effect. Thus this common maternal-fetal disorder is associated with an inhibitor of neutrophil production, which is elaborated by the placenta and present in cord blood serum. PMID- 1714680 TI - Potassium currents in freshly dispersed myometrial cells. AB - K+ currents in freshly dispersed cells from rat myometrium at estrus were studied with the patch-clamp technique (whole cell). Three types of K+ currents were identified: 1) a fast-activating current (IKf), 2) a slowly activating current (IKs), and 3) a transient current (IKt). IKf had a half-activation voltage of 12 mV and a time constant of activation (tau on) of approximately 3 ms at +50 mV. IKs had a tau on of approximately 9 ms at +50 mV and a half-activation potential of 33 mV. Both IKf and IKs were sustained and became potentiated by the entrance of Ca2+ from the patch pipette. These two Ca(2+)-activated K+ currents were inhibited by 100 nM external charybdotoxin and were blocked by external tetraethylammonium (TEA, 2-20 mM). The third current (IKt) was transient, had a faster tau on (approximately 1 ms), and a decay phase with a time constant of approximately 8 ms at +50 mV. This current had a half-activation potential of 22 mV. IKt was not potentiated by intracellular Ca2+, was sensitive to 4 aminopyridine (1 mM), was insensitive to external charybdotoxin (100 nM) and TEA (2 mM), and spontaneously decreased with time. PMID- 1714681 TI - Intestinal and renal Na+/glucose cotransporters share common structures. AB - Polyclonal antibodies were raised to peptides selected from three different regions of the cloned rabbit intestinal Na+/glucose cotransporter. Western blot analysis was used to identify the fully mature protein in intestinal and renal brush borders. Two of the antibodies specifically identified a approximately 70 kDa protein band in rabbit intestinal brush borders but did not specifically immunoreact with membranes that do not have Na(+)-dependent glucose transport activity. The immunoreactive proteins had an apparent isoelectric point between pH 4.7 and 6.8. The antibodies also specifically recognized a similar-sized protein in human and seven other mammalian intestinal brush borders. Similar protein bands were identified in four mammalian renal brush-border membranes, indicating shared epitopes between intestinal and renal cotransport proteins. In some species, e.g., lamb and pig, the epitope for one antibody was missing in both intestinal and renal brush borders, suggesting that this epitope is not essential for function. These results suggest that 1) the cloned intestinal Na+/glucose cotransporter is that identified in earlier biochemical studies, 2) there is close structural similarity between intestinal and renal cotransporters, and 3) the structure of these proteins has been conserved during evolution. PMID- 1714682 TI - Effects of secretagogues on cytosolic Ca2+ levels in rat submandibular granular ducts and acini. AB - Saliva is thought to be formed by a two-stage process, with the secretion of a "primary fluid" by the acinar cells followed by various ionic modifications in the salivary ducts. Both of these processes are under the control of autonomic stimuli. Although the role of the acini in salivary secretion has been studied in some detail, little is known about properties of ducts, particularly the intralobular ducts that make up the bulk of the ductal tissue. In the present study, microfluorometric methods were employed to examine the responses of intracellular Ca2+ concentration ([Ca2+]i) in individual male rat submandibular acini and intralobular (granular) ducts to various fluid secretory stimuli. We show that granular ducts respond to muscarinic (carbachol) and alpha-adrenergic (epinephrine) stimulation by increasing [Ca2+]i in a manner that is qualitatively similar to acini, but that in contrast to acini, these ducts do not respond to substance P. Because the transduction of a substance P peptidergic signal typically occurs via increased [Ca2+]i, this observation suggests that there are no substance P receptors on granular ducts. Ducts were also found to be somewhat more responsive to both carbachol and epinephrine than acini. Although muscarinic, alpha-adrenergic, and vasoactive intestinal peptide (VIP) stimulation are known to induce the secretion of epidermal growth factor from granular ducts, no significant increase in ductal [Ca2+]i in response to VIP (10(-9) to 10(-6) M) was observed. PMID- 1714683 TI - Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining. AB - In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white. PMID- 1714685 TI - Spontaneous anastomosis of transected vessels. AB - Experiments were performed on rats to establish the capacity for spontaneous repair of transected arteries and veins. The tests were done on the femoral vessels, with transection effected in a number of modifications. Veins proved capable of bridging even a triple transection within 2 weeks. Not a single artery, however, anastomosed. Vascular repair was on the basis of adventitial and perivascular networks. Explanations of arterial incapacity for spontaneous repair can presently be only speculative. The observations add to our knowledge concerning the revascularization of free grafts, and also indicate that in veins, spontaneous anastomosis can be induced by loosely approximating the stumps. PMID- 1714684 TI - Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography. AB - A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane. PMID- 1714686 TI - The effect of weight correction on risk calculations for Down's syndrome screening. AB - Recent advances in prenatal screening have led to the possibility that the risk of Down's syndrome associated pregnancy may be assessed by blood tests for maternal serum alphafetoprotein, human chorionic gonadotrophin (and possibly unconjugated oestriol) taken at 15-18 weeks of gestation. In neural tube defect screening correction of maternal serum alphafetoprotein for maternal weight has been recommended, although the precise method for weight correction is still under debate. We report an assessment of weight correction for maternal serum alphafetoprotein and human chorionic gonadotrophin based on 1408 singleton pregnancies and for unconjugated oestriol based on 197 singleton pregnancies. We demonstrate that weight correction of maternal serum alphafetoprotein and human chorionic gonadotrophin is statistically valid but that correction of unconjugated oestriol is not. PMID- 1714687 TI - Enzyme immunoassay of hyaluronic acid in serum and pleural fluid using sheep brain hyaluronectin. PMID- 1714688 TI - Mast cells in periodontal disease. AB - Since the roles of mast cells in periodontal tissues are not clear, this study aimed to count the number of mast cells in gingival tissues of healthy volunteers and patients with chronic periodontal disease before and three weeks after surgical treatment. The numbers of mast cells were found to be increased on postoperative healed tissues and inflamed tissues respectively, compared to healthy tissues. This increase is closely related to fibrosis in connective tissues. PMID- 1714689 TI - Neonatal meningitis in Harare, Zimbabwe: a 2-year review. AB - A 2-year review of neonatal meningitis was undertaken at a referral hospital in Zimbabwe to determine the pattern of bacterial isolates from the cerebrospinal fluid, the clinical presentation and the immediate outcome. During the study period, 94 cases were identified and the overall mortality was 41%. Group B Streptococcus was the predominant organism isolated (61%) and was associated with 42% mortality. Low-birthweight babies and babies with Gram-negative meningitis had mortality rates of 71 and 62%, respectively. PMID- 1714690 TI - Genetically determined neurodegenerative disorders: experiences in Saudi Arabia. AB - There is, to date, hardly any literature on genetic neurodegenerative disorders from developing countries. This paper reports a study of 98 Saudi children with genetic neurodegenerative disorders. The four most encountered diagnoses were: spinal muscular atrophy, storage (lysosomal) disorders, neurocutaneous syndromes and aminoacidopathies. Consanguinity has been noted in about 50% of the families. In view of the major advances made in recent years in the recognition and treatment of these disorders, the role of the physician is discussed. PMID- 1714691 TI - The pattern and prognosis of speech disorders among children in Enugu, Nigeria. AB - Of 965 children with neurological disorders seen in the Child Neurology Clinic of the University of Nigeria Teaching Hospital, Enugu over a 3-year period (1985 87), 80 (8.3%; 41 boys and 39 girls) had speech problems. The most common speech disorder was dyslalia. Twenty-six (32.5%) of the 80 children were mentally retarded. Besides speech problems, some of the children had other neurological disorders such as hyperactivity, recurrent seizures, microcephaly and deafness. Varying degrees of improvement in speech were observed in only eight girls and four boys, amounting to 24% of the 50 who were followed up for a period ranging from 3 months to 3 years (mean follow-up period 12.26 months). Eight of those who improved (66.6%) did so within the first 18 months of follow-up. There was a disturbingly high rate of default from follow-up, 30 of the patients (37.5%) failing to keep appointments at the clinic after the first assessment. Also, there was a long delay between onset of symptoms and presentation to hospital (mean delay 45.3 months). With the recent acquisition of the services of a speech therapist by the hospital, it is hoped that the prognosis for children with speech problems will be considerably improved. PMID- 1714692 TI - Monitoring the growth of the world's children. AB - Growth monitoring and promotion is a difficult operational strategy for communicating with mothers which is based on a simple technology of weighing and charting young children and an assumption that the undoubted early warning of future low nutritional status provided by monthly growth monitoring enables corrective action to be taken to avert death. Doubts have been expressed about the efficacy and cost-effectiveness of existing growth monitoring programmes. There is an urgent need for good demonstrations of programmes in which health professionals and communities cooperate in empowering mothers to promote their children's growth, while marshalling all the primary health care activities, and yielding valuable straightforward data on the growth of the world's children. PMID- 1714693 TI - Gross motor development of Nigerian children. AB - A prospective study of 320 Nigerian children was undertaken to determine their pattern of motor development by recording the age at attainment of 12 gross-motor milestones. The children were all born full term and were neurologically normal at birth. They were recruited in the first week of life and seen at regular intervals in a well-baby clinic, where their parents were questioned about the ages at attainment of milestones. Results show that most gross-motor milestones were attained at earlier ages by these children than by children studied to establish norms for the traditional tests of motor development that have long been in use. Our findings confirm several previous reports which emphasize the more rapid attainment of motor milestones such as 'sit without support', 'crawl', 'stand well alone' and 'walk well alone' by black as compared with white children. But, conversely, a number of transitional milestones such as 'roll over', 'pull self to stand' and 'stand holding on' were achieved later by children in this study than by their non-African counterparts. Girls in this study were slightly advanced, relative to boys, in their attainment of most milestones. The results of this study have been incorporated into a chart which can be used in well-baby clinics to detect children with motor delay. PMID- 1714694 TI - A survey of nasal carriage of Staphylococcus aureus in a neonatal ward in Ile Ife, Nigeria. AB - The nasal carriage rate of Staphylococcus aureus among maternal-infant pairs was 18% compared with 39% among hospital staff in Ile-Ife, Nigeria during a 12-week survey. Of the newborns, 46% tested positive compared with 26% of their mothers. The S. aureus phage types recovered were predominantly of the group III type (38%); however, 28% of the strains isolated were non-typable. All the S. aureus strains were resistant to penicillin, 84% to tetracycline, and 35 and 24% were resistant to streptomycin and chloramphenicol, respectively. Altogether 19% of the strains tested were resistant to methicillin. PMID- 1714695 TI - Glomerular filtration rate in healthy Nigerian children and in children with sickle cell anaemia in a steady state. AB - In a prospective study, the glomerular filtration rate (GFR) of 365 Nigerian children aged 4-14 years was determined at the University of Nigeria Teaching Hospital, Enugu. The subjects were made up of 305 healthy children and 60 children with sickle cell anaemia in a steady state. For the healthy children who served as controls, the mean (SD) GFR was 112 (35)ml/min/1.73m2, there being no sex difference. The peak GFR was recorded in boys at 9 years and girls at 13 years. In the children with sickle cell anaemia in a steady state, the mean (SD) GFR was 111 (36), a figure similar to that of normal controls. PMID- 1714696 TI - Prevalence of Mycoplasma pneumoniae in children with pneumonia in Zaria, Nigeria. AB - The prevalence of Mycoplasma pneumoniae in children suffering from pneumonia was investigated, using complement fixation and growth inhibition tests. From the sera of 104 children with pneumonia, 32 (31%) showed a CF titre greater or equal to 1:64, while all the 52 control children of the same age and sex had a CF titre less than 1:16. Children 6-10 years of age had the highest positive titre (41%) while the age groups 3-5 years and 0-2 years had positive titres of 30 and 28%, respectively. Both sexes were equally affected (33% male, 29% female). Mycoplasma pneumoniae was isolated in two children whose CF antibody titres were 1:16. It is concluded that M. pneumoniae plays an important role as an aetiological agent of pneumonia in children in Zaria, Nigeria, and could be included in the routine diagnostic protocol of pneumonia, especially during the dry harmattan months when cases of lobar pneumonia are prevalent. PMID- 1714698 TI - Prenatal diagnosis of alpha- and beta-thalassaemias in Singapore--current status. AB - Prenatal diagnosis was performed in 31 pregnancies where the fetuses were at risk for either homozygous alpha(0) - or beta-thalassaemia. First-trimester prenatal diagnosis by DNA analysis using chorionic villi was carried out for 17 pregnancies at risk for homozygous alpha (0)-thalassaemia. The alpha-globin genes in fetal DNA were detected by gene mapping using restriction endonuclease mapping and hybridization with cloned alpha-globin probe. Homozygous alpha (0) thalassaemia was detected in four fetuses and the results were subsequently confirmed by electrophoresis of the cord blood where only Hb Barts was detected. Prenatal diagnosis for beta-thalassaemia was carried out by globin chain biosynthesis using fetal blood at 18-20 weeks' gestation. Using carboxymethyl (CM) sepharose chromatography, homozygous beta-thalassaemia was predicted in six pregnancies, and one fetus carried Hb E-beta thalassaemia. The seven pregnancies were terminated and globin chain analysis using cord blood confirmed the prenatal diagnoses. The remaining seven fetuses were diagnosed as either normal or beta thalassaemia carriers. Using DNA analysis and globin chain biosynthesis for prenatal diagnosis of homozygous alpha(0)- and beta-thalassaemia, a 100% correlation was achieved with fetuses predicted to possess the homozygous condition. PMID- 1714697 TI - Iron fortification of infant milk formula: the effect on iron status and immune function. AB - We conducted a randomized double-blind trial of a cow's milk infant formula with increased iron fortification in order to confirm its safety and to measure its effects on iron status and immune function. A group of full-term, well nourished and healthy infants was followed from the age of 3 months to 1 year. A control group of 74 infants was given a commercially available infant formula containing 8.3 mg Fe/100g. The test group of 75 infants received a similar formula with 40 mg Fe/100 g. The formula with the extra iron proved to be safe and, when compared with the control group, the children in the test group had significantly improved iron status as reflected by the proportion of children classed as normal (25 of 61 cf. 44 of 65; p less than 0.003), and by the mean values of the haemoglobin concentration (11.5 cf. 11.9 g/dl; p = 0.04), red cell distribution width (15.5% cf. 14.4%; p = 0.0005), red cell zinc protoporphyrin (3.4 cf. 4.0 micrograms/g Hb; p = 0.04) and ferritin (29 cf. 17.3 micrograms/l; p = 0.004). The extra iron fortification depressed zinc concentration in plasma (90.6 cf. 83.5 micrograms/l; p = 0.05). There was no significant difference between the two groups for laboratory measures of immune function or for incidence of infection. No adverse effects such as infection could be attributed to the increased iron. We conclude that iron fortification of cow's milk infant formula may be safely increased to 40 mg/100 g (i.e. by a factor of 4.8 over the common concentration of 8.3 mg/100 g), but that this has less than the expected effect on iron status. Further studies are required to define (a) the long-term role of facilitators of iron absorption such as ascorbic acid, (b) the interaction of iron with absorption of divalent trace elements such as zinc, and (c) the effect of iron status on immune function and susceptibility to infection. PMID- 1714699 TI - Low birthweight in Riyadh, Saudi Arabia: incidence and risk factors. AB - In a multicentre prospective study, we have determined the incidence of low birthweight (LBW) and the main predisposing risk factors. Among 4651 consecutive births over a 5-month period in five hospitals in Riyadh, the overall incidence of LBW was 8.4%. When stillbirths were excluded the incidence of LBW was 7.4% of all live births. Statistical analysis was performed among 638 births (319 LBW infants, i.e. less than or equal to 2499 g and 319 babies weighing 2500 g or more). Of the 28 antenatal risk variables analysed, 13 were found to be significant when studied separately. Of these 13 variables, six were found to be significant predictors of LBW, using stepwise multiple logistic regression. These six variables together correctly predicted 72% and 88% of the LBW babies or normal birthweight babies, respectively. The risk factors thus identified were (i) short gestation, (ii) multiple gestation, (iii) low maternal body mass index, (iv) nulliparity, (v) availability of housework help, and (vi) absence of consanguinity. Measures for reducing these factors are also discussed. PMID- 1714700 TI - Tension teratothorax--a case report. AB - Respiratory distress caused by a giant mediastinal teratoma is hitherto unreported. This communication presents the case history of an 8-year-old boy who presented with this serious problem, along with perforation of the chest wall. PMID- 1714701 TI - Lipoid pneumonia in children following aspiration of animal fat (ghee). AB - Exogenous lipoid pneumonia induced by modified animal fat (ghee) in 10 children is described. The initial presentation was of an acute or chronic pneumonia which proved refractory to anti-microbial chemotherapy. The radiological presentation varied from mild perihilar consolidation to diffuse and extensive bilateral involvement, particularly of the posterior lung segments. A history of administration of ghee provided the initial clue to the diagnosis, which was confirmed by demonstration of fat by bronchoalveolar lavage or by open lung biopsy. Eight of the 10 patients improved with either steroid therapy alone or steroids with resection of the most involved lung segments. One patient, who had extensive superinfection with Mycobacterium fortuitum, died. Lipoid pneumonia should be considered in the differential diagnosis of 'non-resolving' pneumonias in communities where the cultural practice of infant feeding with ghee is prevalent. Public awareness through health education about the potential hazards of this practice to infants and children can contribute to reduce the incidence of the problem. PMID- 1714702 TI - Variegate (mixed) porphyria in a Nigerian girl. AB - A fatal case of variegate (mixed) porphyria in an 11-year-old Nigerian girl is reported. She presented with severe abdominal pain, vomiting, constipation, quadriplegia and cutaneous bullous dermatosis. Remission was temporarily achieved with chlorpromazine, high carbohydrate diet and physiotherapy. She finally died of respiratory paralysis 17 months after diagnosis. PMID- 1714703 TI - A prospective study of some diarrhoeagenic Escherichia coli in infants with diarrhoea in Mosul, Iraq. AB - The incidence of diarrhoeagenic Escherichia coli was investigated in 304 infants with diarrhoea in Mosul, Iraq by using standard biological assays and reversed passive latex agglutination (RPLA) procedures. Enterotoxigenic E. coli (ETEC) were found in 12.8% of the cases--27 (8.8%) strains produced heat-labile toxin (LT) only, 8 (2.6%) heat-stable toxin (ST) only and 4 (1.3%) produced both toxins (LT-ST)--whereas enteropathogenic E. coli (EPEC) were responsible for about 13.8% of the incidence of diarrhoea in the community. Detailed analysis revealed that 12 serotypes were involved. A greater number of cases of acute enteritis were seen in infants aged 0-18 months than in those aged between 19 and 36 months. The data indicate a highly significant level of resistance to most common antimicrobial drugs among diarrhoeagenic E. coli isolated, but most were highly sensitive to nalidixic acid, cephalothin and gentamicin. PMID- 1714704 TI - Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing. AB - The identification of the prokaryotic species which constitute marine bacterioplankton communities has been a long-standing problem in marine microbiology. To address this question, we used the polymerase chain reaction to construct and analyze a library of 51 small-subunit (16S) rRNA genes cloned from Sargasso Sea bacterioplankton genomic DNA. Oligonucleotides complementary to conserved regions in the 16S rDNAs of eubacteria were used to direct the synthesis of polymerase chain reaction products, which were then cloned by blunt end ligation into the phagemid vector pBluescript. Restriction fragment length polymorphisms and hybridizations to oligonucleotide probes for the SAR11 and marine Synechococcus phylogenetic groups indicated the presence of at least seven classes of genes. The sequences of five unique rDNAs were determined completely. In addition to 16S rRNA genes from the marine Synechococcus cluster and the previously identified but uncultivated microbial group, the SAR11 cluster [S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field. Nature (London) 345:60 63], two new gene classes were observed. Phylogenetic comparisons indicated that these belonged to unknown species of alpha- and gamma-proteobacteria. The data confirm the earlier conclusion that a majority of planktonic bacteria are new species previously unrecognized by bacteriologists. PMID- 1714705 TI - New version of the negative stain. AB - We have developed a new version of the negative stain which is very quick, reliable, and easy to perform and which uses a waterproof marking pen instead of nigrosin. It is ideal for teaching the negative staining technique to beginning student microbiologists. PMID- 1714706 TI - [Preoperative treatment of esophageal cancer with oral peplomycin jelly]. AB - Pepleo-jelly, peplomycin (PEP) compounded with sodium polyacrylate (PANa), was used as a local preoperative chemotherapy for esophageal cancer. Thirty milligrams of PEP emulsified in 10 ml of jelly was orally administered for 17 patients on consecutive bed time, and the average total dose was 470 mg. Concentrations of PEP in blood and tissue were measured with a bioassay. PEP was not detected in the blood but was detected in esophageal tissue and regional lymphnodes. A significant difference (p less than 0.05) of tissue concentrations between normal esophageal mucosa (0.11 microgram/g) and malignant region (0.34 microgram/g) was noted. Furthermore, PEP was detected in many regional lymphnodes. Histological antitumor effect (Ef2) was noted in three of the eight metastatic lymphnodes. A side effect of this treatment was not recognized. It is believed that this treatment is safe and useful in combination with other adjuvant cancer therapy. PMID- 1714707 TI - [Phase II study of YM881 (zinostatin stimalamer) suspension injected into the hepatic artery. Research Group for Intra-arterial Injection Therapy with YM881]. AB - A phase II study of YM 881 (zinostatin stimalamer) to determine the response and safety was conducted in patients with hepatocellular carcinoma by injecting a suspension of the drug into the hepatic artery. Repeated doses of 4 to 6 mg of the drug were given every 4 weeks so that the tumor tissues were filled with the suspension. Of the 195 registered patients, 15 were ineligible for the study, 8 dropped out, and data were missing for 5. A total of 167 patients completed the study. Response was assessed in the 167 patients who completed the study. CR was found in one, PR in 59, MR in 25, NC in 67, and PD in 15, with a response rate of 35.9. The safety of the drug was assessed in 177, excluding ineligible patients and 3 who dropped out because of the concurrent use of other drugs. Adverse reactions were found in 93.2% of the patients, and abnormal values in clinical laboratory tests in 60.5%. Major unwanted symptoms included fever, nausea, vomiting, and anorexia. Major abnormal changes in laboratory tests were elevated total bilirubin and LDH and abnormal hepatic function. About half the patients had malaise and pain related to the intra-arterial infusion therapy. The one year survival rate was 56.9%, and the duration of survival of 50% of the patients was 407 days. PMID- 1714708 TI - Changes in biochemical markers of hyperplastic and malignant prostatic tissues. AB - Some biochemical markers were evaluated in human prostatic tissue obtained from patients with benign hyperplasia. There was an almost threefold increase in the activity of acid phosphatase of these patients as compared to that of normal men. The changes in the acid phosphatase activity were directly correlated with citric acid concentration. Significant (p less than 0.001) increases in the content of citric acid, zinc, and calcium in benign hyperplastic prostates was found. There was no change in the activity of aminopeptidase. The activities of both enzymes were lower in malignant prostatic tissues. PMID- 1714709 TI - Low-molecular-weight heparin (enoxaparin) vs dextran 70. The prevention of postoperative deep vein thrombosis after total hip replacement. The Danish Enoxaparin Study Group. AB - A prospective randomized study compared the thromboprophylactic efficacy and safety of a low-molecular-weight heparin (LMWH), enoxaparin (40.6 mg subcutaneously once daily), with a standard regimen of dextran 70 in patients undergoing elective total hip replacement. Deep vein thrombosis was diagnosed by bilateral ascending phlebography 7 to 11 days after operation. Two hundred forty six patients were included and 219 were eligible for analysis. Deep vein thrombosis was diagnosed in seven of 108 patients in the LMWH group and in 24 of 111 patients in the dextran group. Clinical symptoms of pulmonary embolism did not develop in any patients during the study. In the postoperative period, patients receiving LMWH had a lower blood loss in drains and required fewer blood transfusions than patients receiving dextran, although no significant differences were noted between the groups with respect to the total number of blood transfusions required. Bleeding events and adverse events did not differ between the groups. None of the patients died in hospital during the study. One patient in the LMWH group died at home 15 days after the operation. Three patients receiving dextran had development of symptomatic deep vein thrombosis after hospital discharge. In conclusion, enoxaparin was a more effective thromboprophylactic than a standard regimen of dextran in patients undergoing total hip replacement. The two regimens were equally safe under the clinical conditions. PMID- 1714710 TI - The insulin-like growth factors: their putative role in atherogenesis. AB - Smooth muscle cell proliferation and leukocyte infiltration are characteristic features of all lesions of atherosclerosis. Although platelet derived growth factor (PDGF) is one of the major smooth muscle mitogens, other important mitogenic factors are found in plasma and in platelets. The insulin-like growth factors (IGF-I and IGF-II) are present in plasma complexed to one of a number of IGF-binding proteins (IGF-BP). They are also found at high concentrations within the alpha-granules of platelets. The IGFs are secreted by a number of cell types in-vitro and in-vivo, including smooth muscle cells and macrophages. The cellular effects of the IGFs are mediated by membrane bound high affinity receptors. IGF receptors are of two distinct types and are expressed by a wide variety of cells. The IGFs are potent smooth muscle cell mitogens and it is therefore possible that these polypeptides contribute to the formation of the atherosclerotic lesion by paracrine, autocrine or endocrine mechanisms. PMID- 1714711 TI - [Histogenesis of pleomorphic adenomas of the salivary glands]. AB - Histogenesis of pleomorphic adenomas was investigated by histological and electron microscopical methods. Histochemically alkaline phosphatase activity was revealed in transformed cells. Positive immunohistochemical reaction was shown in cells of pleomorphic adenomas with polyclonal antibodies against the smooth muscle myosin, human carbonic anhydrase III and monoclonal antibodies to cytokeratins N8, N17, N18, vimentin (clone NT-30) and to membrane epithelial antigen. It is concluded that these tumours are of the epithelial nature. PMID- 1714712 TI - [Patterns of keratin protein expression in basal cell, metatypical and squamous cell carcinoma of human skin]. AB - Comparative immunomorphological study of keratinous proteins was performed in 17 basaliomas, 4 metatypical (MT) and 1 squamous cell carcinomas by means of a spectrum of antibodies to the individual keratins. It is found that cells of MT carcinoma are distinguished from basalioma cells by the absence of keratins N8 and 17 and this may be used for the differential diagnosis of these two tumours. The spectrum of keratins expressed by basalioma cells coincides with that of early stages of hair follicles. Keratin N17 is locally induced in parabasal layers of the morphologically intact epidermis adjacent to the tumour. PMID- 1714713 TI - Response of rat parotid acini to barium. AB - The effect of barium on isolated acini was tested. Barium in the 0.1-10 mM concentration range non-competitively inhibited the efflux of 86Rb+ stimulated by carbamylcholine or substance P. This inhibition was independent of the presence of calcium in the extracellular medium. In the same preparation, barium did not affect the efflux of 45Ca2+ but, at a 10 mM concentration, it increased amylase release by 70%. Removal of extracellular calcium decreased basal amylase release and the response to carbamylcholine. Adding back calcium or barium to the incubation medium increased basal and carbamylcholine-stimulated amylase secretion, but calcium was more effective than barium. These results suggest that barium has two opposite effects on calcium-regulated processes in rat parotid gland: (1) it is an inhibitor of calcium-activated potassium channels; (2) it is a partial agonist of calcium-activated amylase secretion. PMID- 1714714 TI - The peripheral distribution of trigeminal nerve fibres in the rat temporomandibular joint studied by an anterograde axonal transport method with wheat germ agglutinin-horseradish peroxidase. AB - Wheat germ agglutinin-horseradish peroxidase was injected into the trigeminal ganglion to trace the peripheral distribution of the nerve fibres in the temporomandibular joint. It was transported anterogradely along trigeminal nerve fibres. Horseradish-peroxidase-labelled nerve fibres were found in the anterior and posterior bands of the articular disc, and terminated as nerve endings near the intermediate zone of the disc. However, the intermediate zone itself did not contain any nerve endings. Other nerve fibres penetrated from the subsynovial layer into the synovial membrane and also terminated as nerve endings close to the articular cavity. Thus, this method is suitable for tracing peripheral nerve fibres and nerve endings originating in the trigeminal ganglion. PMID- 1714715 TI - Light and electron microscopic demonstration of wheat germ agglutinin binding in the pericellular matrix of rat mandibular condylar cartilage. AB - Binding of wheat germ agglutinin to rat mandibular condylar cartilage was investigated with the avidin-biotin peroxidase complex. Binding sites were observed in the pericellular matrix of the hypertrophic cell zone. Two distinct patterns were identified: one showed binding to the pericellular matrix without apparent contour; in the other binding was confined to the matrix but with conspicuous condensation forming a pericellular rim. The first binding pattern was seen particularly in the upper part of the hypertrophic cell zone adjoining the mature cell zone; the second was localized in the lower part of this zone adjacent to the site of endochondral calcification. The binding sites in the pericellular matrix are assumed to be NANA and/or GlcNAc in view of the sugar specificity of this lectin. The presence of these binding sites in this region may be due to a structural alteration or modification of proteoglycans in the course of preparation for endochondral calcification. PMID- 1714716 TI - CO2 laser laparoscopic salpingotomy for treatment of tubal ectopic pregnancies: potential limitations. PMID- 1714717 TI - Effect of interleukin 6 on phenobarbital induction of cytochrome P-450IIB in cultured rat hepatocytes. AB - Human recombinant interleukin 6 (rhIL-6) caused a dose dependent decrease in the phenobarbital induction of benzyloxyresorufin O-deethylase activity in cultured rat hepatocytes. Decreased enzymatic activity was associated with a decrease in the amount of immunoreactive P-450IIB1/2. rhIL-6 also prevented the PB-induced increase in the steady state level of P-450IIB mRNA. These results suggest that altered P-450 levels observed in vivo during the acute phase reaction may be due to interleukin 6. PMID- 1714718 TI - Prostaglandins E2 and F2a mediate the increase in c-myc expression induced by EGF in primary rat hepatocyte cultures. AB - Epidermal Growth Factor (EGF) and prostaglandins (PGs) E2 and F2a, have been shown to stimulate primary hepatocyte proliferation. Verapamil (5-20 microM), a calcium channel inhibitor, inhibited hepatocyte DNA synthesis and c-myc expression, induced by EGF (50 ng/dish) and prostaglandins (1-12 micrograms/dish). Indomethacin (20-100 microM) decreased significantly the EGF induced hepatocyte DNA synthesis and c-myc expression. Addition of PGs (1-9 micrograms) in hepatocyte cultures treated with EGF+indomethacin (100 microM) restored the capacity of EGF to increase c-myc expression and DNA synthesis. We propose that arachidonic acid derivatives and calcium channel blockers modulate c myc expression in primary hepatocytes. PMID- 1714719 TI - Detection of allele-specific transcripts by the polymerase chain reaction (AST PCR). AB - We have applied the polymerase chain reaction to detect differences in relative amount of allele-specific transcripts of the low density lipoprotein receptor gene in individuals with heterozygous familial hypercholesterolemia (FH). This method is based on detection of loss of heterozygosity of exon-specific restriction fragment length polymorphisms present in amplified mRNA fragments as compared to amplified genomic DNA fragments. We detected loss of allele-specific mRNA in 20% of informative FH patients. In principle, this method enables the analysis of relative allele-specific transcript levels of any expressed gene and can thus be generally applied to study processes involving differential gene expression. PMID- 1714720 TI - Tumorigenesis of a v-Ha-ras-expressing pre-B cell line selects for c-myc activation. AB - Seven tumors independently derived from a v-Ha-ras-expressing pre-B cell line were examined to determine the oncogene activations cooperating with v-Ha-ras in in vivo tumor progression. The pre-B cell line was generated by infection with Moloney murine leukemia virus (MoMuLV) and a MoMuLV-derived recombinant expressing v-Ha-ras. Two of seven tumors possessed a MoMuLV integration immediately upstream and in reverse transcriptional orientation to c-myc. This correlated with a 3-fold increased level of c-myc mRNA. Two other tumors displayed elevated c-myc mRNA levels, although the mechanism of enhanced expression was unclear. Thus the tumor progression of a v-Ha-ras-expressing murine pre-B cell line selects for the activation of c-myc. PMID- 1714721 TI - Molecular cloning of the human histamine H2 receptor. AB - We utilized the technique of polymerase chain reaction with oligonucleotide primers based upon the nucleotide sequence of the canine H2 histamine receptor gene which we recently isolated to clone its human homologue. Transfection of a construct of this gene in Colo-320 DM cells led to the expression of a receptor that bound to [methyl-3H] tiotidine and was linked to 3',5'cyclic adenosine monophosphate (cAMP) generation in response to histamine. Both cAMP generation and [methyl-3H] tiotidine binding were inhibited with the H2 histamine receptor selective antagonist cimetidine but not diphenhydramine or thioperamide which are, respectively, H1 and H3 histamine receptor antagonists. These data confirm that we have successfully cloned a novel gene encoding the human H2 histamine receptor. PMID- 1714722 TI - Identification of tyrosine 67 in bovine brain myelin basic protein as a specific phosphorylation site for thymus p56lck. AB - The protein-tyrosine kinase p56lck exhibits a restricted substrate specificity in vitro but can efficiently phosphorylate bovine myelin basic protein (MBP). Results obtained from both peptide mapping and fast atom bombardment mass spectrometry indicate that tyrosine 67 in the sequence -Thr-Thr-His-Tyr67-Gly-Ser Leu-Pro-Gln-Lys- in bovine MBP is the specific phosphorylation site. p56lck does not phosphorylate the acidic, cytoplasmic domain of erythrocyte band 3. In contrast, p40, another protein-tyrosine kinase purified from bovine thymus that readily phosphorylates band 3, does not phosphorylate MBP. Therefore, MBP and band 3 may prove to be useful substrates for distinguishing between various tyrosine kinases on the basis of substrate specificity. In addition, identification of the recognition sequence in MBP for p56lck may contribute to an understanding of the structural features of physiological substrates for this kinase. PMID- 1714723 TI - Molecular cloning of human platelet thromboxane A synthase. AB - Complementary DNA coding for thromboxane A synthase was amplified by polymerase chain reaction using primers synthesized according to the partial amino acid sequences of human platelet thromboxane A synthase (Nusing, R., Schneider-Voss, S., and Ullrich, V. (1990) Arch. Biochem. Biophys. 280, 325-330) and cloned into pBluescript SK II(-). The primary structure of human platelet enzyme was deduced from the nucleotide sequence of the cDNA. The enzyme is composed of 533 amino acids with a molecular weight of 60,487. The primary structure of the enzyme exhibited a 34-36% homology to the amino acid sequences of cytochrome P450s classified in the P450 III gene family. The highly conserved cysteine-containing sequence involved in the heme-binding site of P450 was found near the carboxyl terminus (residues 472-492). The size of the major thromboxane A synthase mRNA from human platelets and human erythroleukemia cells was estimated to be approximately 2.2 kilobases by RNA blot analysis. PMID- 1714725 TI - Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1. AB - Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo. PMID- 1714724 TI - Elevated levels of c-jun and c-fos transcripts in the aged rat liver. AB - Age-associated increases of transcript levels of several genes were studied using aged rat liver. Northern hybridization showed marked increases of c-jun, c-fos, and glutathione S-transferase P (GST-P) in 24-month-old rats. These genes have the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-responsive element called TRE in their structures. Thus, TRE seems to be involved in important age-associated changes of gene expression. PMID- 1714726 TI - Potentiating effect of NH4Cl on vasoconstriction in rat aorta. AB - The effect on the vasocontractile response of pretreatment with NH4Cl at a concentration (10 mM) that made almost no change in the resting tension was investigated using aortic strips from rats. NH4Cl pretreatment for 10 min significantly potentiated strip contractions induced by KCl (less than or equal to 30 mM), BAY K 8644 (0.1 microM) and phenylephrine (0.01 microM). This potentiating action of NH4Cl was eliminated in presence of nifedipine (1 microM). KCl (14.7 mM)-stimulated 45Ca uptake in rat aorta was significantly potentiated by pretreatment with NH4Cl (10 mM) for 10 min, but this NH4Cl effect was also eliminated in the presence of nifedipine. These results suggest that NH4Cl potentiates contractions induced by KCl and agonists in rat aorta by facilitating calcium influx through the nifedipine-sensitive calcium channel. PMID- 1714727 TI - Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. AB - We have previously reported that cultured murine brain endothelial cells (MBE) produce large quantities of nitric oxide (NO) after activation with interferon gamma in combination with any of several immunoactivators including: bacterial endotoxin, tumor necrosis factor and interleukin-1. Since endothelial cells are the first example of a cell-type which may possess both a constitutive and an inducible type of NO synthase, it was of interest to compare the requirements of these two enzyme activities. Induction of NO synthesis in MBE by cytokines was abolished by the protein synthesis inhibitor, cycloheximide, and by 2,4-diamino-6 hydroxypyridine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate limiting enzyme for de novo synthesis of tetrahydrobiopterin (THB). In the presence of DAHP, NO synthesis was restored by sepiapterin (SEP), a substrate for the alternative pathway of THB synthesis occurring via pterin salvage. Moreover, SEP increased NO synthesis to greater than 150% of control values, suggesting that THB availability is rate-limiting for NO synthesis by cytokine-induced MBE. Methotrexate, an inhibitor of the pterin salvage pathway of THB synthesis, completely reversed the stimulation of NO synthesis by sepiapterin. Thus, cytokine-induced MBE NO synthase appears to have an absolute requirement for THB as cofactor. In additional studies we found that NO synthesis by cytokine activated MBE was inhibited by NG-monosubstituted arginine analogs with a rank order of potency NH2 greater than CH3 greater than NO2, in contrast with the rank order of NO2 greater than NH2 greater than CH3 previously described for inhibition of the constitutive endothelial cell enzyme. Using a kinetic assay for NO synthase activity, based on oxidation of myoglobin heme-iron, we have found that these rank orders of potency also apply to cytosol preparations of cytokine induced and untreated endothelial cells, respectively. Further differences between constitutive and cytokine-induced NO synthase were observed with regard to calmodulin requirements. Whereas constitutive NO synthase was potently inhibited by the calmodulin antagonists mellitin and trifluoperazine, cytokine induced NO synthase was unaffected. In summary, NO synthesis by cytokine activated MBE is THB-dependent, calmodulin-independent and inhibited by NG substituted arginine analogs with a rank-order profile distinct from that for untreated endothelial cells but identical to that for cytokine-activated macrophages. PMID- 1714728 TI - Separation of growth-promoting activity for human muscle cells from fetuin. AB - Whether the growth-promoting activity of Pedersen fetuin is due to fetuin itself or to a contaminant(s) has been a long-standing puzzle. The possibility that the growth-promoting activity of Pedersen fetuin for human muscle satellite cells (HMSC) could be caused by some other component of fetal bovine serum (FBS) that remained in the fetuin as a contaminant has been investigated. One liter of FBS was first precipitated with 50% saturated ammonium sulfate, which leaves the serum albumin in solution, and then with 25% polyethylene glycol, which leaves the fetuin in solution, to generate a fraction 50 PEG 2x that was enriched 11 fold in growth-promoting activity for HMSC, with 68% recovery of total activity. Further purification with FPLC anion exchange chromatography achieved 99-fold enrichment of the activity with 30% overall recovery. The activity is heat labile and pH sensitive, suggesting that it is of protein nature, and the size of the activity is above 70 kDa. SDS-PAGE of the most active fractions shows that they are virtually free of fetuin. Thus, although the active fractions are not homogeneous, these studies demonstrate that the growth-promoting activity for HMSC can be fully separated from fetuin. PMID- 1714729 TI - Tumor necrosis factor-induced interleukin-6 expression and cytotoxicity follow a common signal transduction pathway in L929 cells. AB - Interleukin (IL)-6 gene induction was studied in response to treatment with Tumor Necrosis Factor (TNF) in the sensitive murine L929 cell line. Under conditions where TNF-mediated cytotoxicity was either increased or decreased, depending on addition of activators or inhibitors, we found that the TNF-induced IL6 gene expression was likewise enhanced or repressed. We conclude that the signal (or part of the signals) going to the nucleus and responsible for gene activation is conducted along the reaction mechanism leading to cellular toxicity. PMID- 1714730 TI - Immunological properties of antibodies against Mg(2+)-ATPase from Acholeplasma laidlawii membranes. AB - In the present study, antibodies were raised against the Mg(2+)-ATPase and the immunological relationships between the enzyme and other ATPase from a variety of biological membranes were determined. The anti Mg(2+)-ATPase antiserum inhibited 85% of the enzyme activity from A. laidlawii membranes. We demonstrate a specific selectivity of Mg(2+)-ATPase antiserum for antigenic determinants of the A. laidlawii membranes. Immunoblot studies of A. laidlawii membrane peptides indicated labeling of five bands, 66KD, 49KD, 34KD, 26KD and 13KD, corresponding to five subunits of the ATPase in A. laidlawii membranes. PMID- 1714731 TI - N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) stimulation of K+ transport in a human salivary epithelial cell line. AB - Treatment of a human salivary epithelial cell line, HSG-PA, with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7; 20-70 microM) increased 86Rb (K+) influx and efflux in a manner similar to that resulting from muscarinic (carbachol; Cch) or calcium ionophore (A23187) stimulation. Unlike the Cch or A23187 responses, the W7 responses were not blocked by 0.1 mM atropine (muscarinic antagonist) or phorbol-12-myristate-13-acetate (0.1 microM). Like Cch or A23187-stimulated 86Rb fluxes, W7-stimulated 86Rb fluxes were substantially blocked by the K+ channel inhibitors quinine (0.25 mM) and scorpion venom containing charybdotoxin (33 micrograms/mL), while 5 mM tetraethylammonium chloride (K+ channel blocker), furosemide (0.1 mM; Na+,K+,2Cl- co-transport inhibitor) and ouabain (10 microM; Na+,K(+)-ATPase inhibitor) were ineffective. Purified charybdotoxin (10 nM) also blocked W7-stimulated 86Rb influx, as well as 86Rb influx stimulated by Cch or A23187. Although Quin 2 fluorescence measurements indicated that W7 increased free intracellular Ca2+ concentration ([Ca2+]i), the magnitude of the increase appeared to be insufficient to solely account for the W7-stimulated increases in 86Rb fluxes (i.e. K+ channel activity). Ca2+ was involved in the W7 response, however, as lack of Ca2+ in the incubation medium reduced the W7-stimulated increases in 86Rb influx and efflux. Taken together, our results suggest that W7 increased K+ fluxes in HSG-PA cells by interacting, directly or indirectly, with the K+ transport machinery (K+ channels) in a manner different from that observed during muscarinic stimulation, and also in a manner not accounted for solely by the formation of a typical muscarinic- or calcium ionophore-generated calcium signal. PMID- 1714732 TI - Oxidation of specific SH protein of mitochondria by photodynamic action of hematoporphyrin. Relevance to uncoupling of oxidative phosphorylation. AB - Photoexcited hematoporphyrin (Hp) induces the uncoupling of oxidative phosphorylation of mitochondria. The uncoupling was inhibited by pre-incubation of mitochondria with a fluorescent SH reagent, eosin-5-maleimide, which has been shown to react specifically with an essential SH group of the Pi/H+ symporter [Houstek and Pedersen, J Biol Chem 260: 6288-6295, 1985]. Eosin-5-maleimide labeled 33, 34.5 and 36 kDa proteins in untreated rat liver mitochondria. When eosin-5-maleimide was added after the treatment with Hp plus light, the proteins were not labeled. Singlet oxygen detection by the ESR spin trapping method during photoradiation of Hp was inhibited by amino acids. Cysteine inhibited it more efficiently than histidine, methionine, tryptophan, tyrosine or alanine under the conditions used. HPLC demonstrated that Hp plus light oxidizes cysteine to cystine together with a smaller amount of cysteinesulfinic acid. These results suggest that Hp plus light oxidizes the SH group of mitochondrial protein, probably the Pi/H+ symporter, with singlet oxygen as a mediator. The possibility of the uncoupling of oxidative phosphorylation through such a modification of the Pi/H+ symporter is discussed. PMID- 1714733 TI - Naloxone affects both pharmacokinetics and pharmacodynamics of morphine. Application of direct correlation analysis. AB - Direct correlation analyses between the distribution of morphine (pharmacokinetics) and the biochemical effects of the drug on monoamine metabolism (pharmacodynamics) are reported for dissected regions of the brain. Determinations of morphine and monoamine-related substances were carried out in the same sample by high performance liquid chromatography with electrochemical detection. Naloxone, an antagonist of morphine, significantly shortened the biological half lives of morphine in both the blood and brain tissue. Such pharmacokinetic behavior appeared to be related to the contractive effect of morphine on the bile duct, and naloxone facilitated the excretion of morphine via this route. In the striatum, significant correlations were observed between the concentrations of the metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and morphine with a shift to the right in the concentration-response curve on naloxone treatment indicating competitive antagonism. While significant correlations were also observed in this brain region for the metabolites of noradrenaline, 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG), and 5-hydroxytryptamine, 5-hydroxyindoleacetic acid (5-HIAA), a shift to the right did not occur. Significant correlations and shifts were noted for DOPAC, HVA and MOPEG in the hypothalamus. However, no correlation was found between the concentrations of 5-HIAA and morphine in this region. In other regions such as the hippocampus and medulla oblongata, similar correlations and shifts were not observed for MOPEG and 5-HIAA or for DOPAC and HVA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714734 TI - Amelioration of bleomycin-induced pulmonary fibrosis in hamsters by combined treatment with taurine and niacin. AB - Interstitial pulmonary fibrosis induced by bleomycin (BL) involves the production of reactive oxygen species and the impairment of repair of damaged epithelial cells. We have shown previously that taurine or niacin treatment partially attenuates BL-induced lung fibrosis in hamsters and that the two agents probably act through different mechanisms. In the present investigation, we have demonstrated that taurine and niacin in combination provide nearly complete protection against BL-induced pulmonary fibrosis. Based on the findings of this investigation, it is suggested that combined treatment with taurine and niacin offers the potential for a novel pharmacological approach in the prevention of lung fibrosis in humans. PMID- 1714735 TI - Low level hyperlipidemia impairs endothelium-dependent relaxation of porcine coronary arteries by two mechanisms. Functional change in endothelium and impairment of endothelium-dependent relaxation by two mediators. AB - We evaluated the effect of a low level of hyperlipidemia and the effects of in vitro exposure to atherogenic lipoproteins (LDL, VLDL) on the vascular responsiveness of isolated porcine coronary arteries. Firstly we studied the change in vascular responsiveness induced by feeding a cholesterol-rich diet to pigs for 4 and 9 weeks (C4 and C9 pigs). The serum cholesterol level in pigs fed a cholesterol-rich diet reached 218.5 +/- 32.9 mg/dl compared with 85.5 +/- 8.4 mg/dl in the controls. Segments of the left descending coronary artery were examined. The contraction induced by KCl or prostaglandin F2 alpha was not altered significantly by hypercholesterolemia nor was the relaxation induced by the Ca2+ ionophore, A23187, or by nitroglycerin. Endothelium-dependent relaxation (EDR) evoked by high, but not low, concentrations of bradykinin was reduced in the C4 pigs as compared with those in normal animals. EDRs evoked by bradykinin, substance P, and serotonin were significantly reduced in C9 pigs. Histologically, as observed by light and electron microscopy, fatty changes or intimal thickenings were not seen in the coronary arteries of the C4 pigs. Minimal changes (intimal thickening and fragmentation of internal elastic lamina) were observed only in parts of arteries of the C9 pigs. Secondly, the direct effects of LDL and VLDL on vascular responsiveness were studied. Although preincubation with LDL inhibited the EDR caused by exposure to bradykinin and A23187 in the coronary arteries of normal and cholesterol-fed pigs, preincubation with LDL inhibited the arterial relaxation induced by exposure to substance P or serotonin in both the C4 and the C9 pigs, but not in the control animals. The degree of inhibition was especially marked in the C9 pigs. The inhibitory effect of VLDL on EDR was weaker than that of LDL. Indomethacin (5 microM) did not alter this inhibitory effect of lipoproteins. Neither LDL nor VLDL had any effect on the vascular relaxation induced by nitroglycerin. These results are consistent with the idea that endothelium-dependent arterial relaxation is attenuated even at the very early stage of cholesterol-induced atherosclerosis. Atherogenic lipoproteins may further impair the decreased EDR in the arteries of hyperlipidemic pigs by two factors: one released on stimulation with bradykinin and the calcium ionophore A23187, the other released on stimulation with substance P and serotonin. PMID- 1714736 TI - Enhanced anti-cancer efficacy on lymph node metastasis using peplomycin adsorbed on small activated carbon particles. AB - A new dosage form (PEP-CH) of peplomycin was tested for therapeutic efficacy against lymph node metastasis in mice. PEP-CH is a suspension comprising 4 mg/ml of activated carbon, 2 mg/ml of peplomycin and 1.6 mg/ml of polyvinylpyrrolidone in saline. Mice were subcutaneously inoculated with 3 x 10(5) MH134 tumor cells into the left hind paw. Drugs were given on day 10 when cancer had been metastasized in the left popliteal lymph node. Mice were killed on day 17 and the left popliteal lymph node and the left deep inguinal lymph node were extirpated. Since the degree of the metastatic lesion and the lymph node weight correlated with a statistically high probability with each other, the degree of metastatic lesion was evaluated through comparison of lymph node weight. The left popliteal lymph node and the deep inguinal lymph node were 10.5 mg and 4.5 mg in average weight, respectively, in the mice given PEP-CH containing 0.1 mg of peplomycin subcutaneously into the left hind foot-pad. The weights were significantly smaller than those in the mice given an identical dose of peplomycin aqueous solution subcutaneously into the left hind foot-pad or intraperitoneally. PMID- 1714737 TI - The new potent immunosuppressant FK-506 reverses multidrug resistance in Chinese hamster ovary cells. AB - The macrolide FK-506, produced by Streptomyces tsukubaensis, is effective in sensitizing a multidrug resistant strain of Chinese hamster ovary cells to the cytotoxic action of vinblastine. FK-506 also promotes Adriamycin accumulation into both wild type and resistant cell lines. FK-506 may be a useful drug to enhance the effectiveness of anti-tumour cytotoxic drugs. PMID- 1714738 TI - Preferential methylation of the Ha-ras proto-oncogene by methylnitrosourea in rat mammary glands. AB - Activation of the Ha-ras proto-oncogene, but not the Ki-ras or N-ras genes, has been found in mammary gland carcinomas induced in female rats by a single dose of methylnitrosourea (MNU). Here we show that a 10-kb restriction fragment containing the Ha-ras gene was extensively methylated by MNU in DNA isolated from mammary glands of female rats 4 h after carcinogen treatment. Fragments of similar size containing either the Ki-ras or N-ras genes were methylated less extensively. The extent of methylation of the three ras genes by MNU correlated with their transcriptional activity. These results suggest that the extent of interaction of a carcinogen with an oncogene, which depends on its transcriptional activity, may be a factor in determining whether the gene is mutated during the initiation of carcinogenesis. PMID- 1714739 TI - Racial variation in the distribution of Ha-ras-1 alleles. AB - Restriction fragment length polymorphism analyses of the Ha-ras-1 proto-oncogene were undertaken in white and black populations residing in the Baltimore Washington metropolitan area to address whether specific rare alleles of the Ha ras-1 proto-oncogene locus vary in their distribution among different racial groups. High-molecular-weight genomic DNA samples from the lungs of 80 lung cancer patients and 92 accident victims were digested with appropriate restriction enzymes and subjected to Southern analysis using the 6.6-kb BamHI human Ha-ras-1 recombinant fragment from the plasmid pEC. Thirty allelomorphs of different sizes were detected among the 172 study subjects. An association was observed between race and specific alleles. Rare alleles were more frequent in black cancer patients and trauma victims than in whites. Within each racial category, lung cancer patients had an excess of rare alleles. These data indicate the importance of controlling for racial variation when designing studies to determine human cancer risk factors. PMID- 1714740 TI - Possible involvement of c-myc but not ras genes in hepatocellular carcinomas developing after spontaneous hepatitis in LEC rats. AB - LEC (Long-Evans with a cinnamon-like coat color) rats develop hepatocellular carcinomas (HCCs) spontaneously. We examined mutations of codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes in four HCCs by the polymerase chain reaction (PCR)-single-stranded DNA direct sequencing method. No ras gene mutations were observed, suggesting that ras activation is not involved in spontaneous hepatocarcinogenesis in LEC rats. The expression of mRNAs for c-myc, Ha-ras, c-raf, and the protein phosphatase 2A alpha gene (PP-2A alpha) was also examined in the four HCCs by northern blot analysis. Three of the four HCCs had c myc expression levels approximately 30-fold higher than that in the liver of control Long-Evans rats with an agouti coat color (LEA), a sibling line of LEC rats, while the remaining HCC had an expression level sevenfold higher than that of control. In contrast, the expression levels of the Ha-ras, c-raf, and PP-2A alpha genes were the same as those in the livers of control rats. Studies of c myc expression and mitotic index in five other HCCs, two hyperplastic nodules, and two nontumorous portions of livers of HCC-bearing LEC rats that had chronic phase hepatitis suggested that the high level of c-myc gene expression was not due only to increased cell proliferation but might possibly be more integrally involved in hepatocarcinogenesis. PMID- 1714741 TI - Isolation and partial characterization of a transformation-associated sequence from human nasopharyngeal carcinoma. AB - A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion sensitive mouse JB6(P+) C141 cells. To clone this transformation-associated sequence, we carried out three cycles of transfection, followed by cloning of anchorage-independent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in both soft-agar assay and nude-mice implantation, was used to make a genomic library in the vector lambda dash. Using the human repeated sequence Blur 8 to screen the library, we obtained 10 human Alu-positive clones. A cloned Alu positive insert of 16 kbp, CNE 323, was characterized in detail. CNE 323 transferred moderate transforming activity when introduced into JB6 P+ cells and showed no homology to Ha-, Ki-, or N-ras genes; human promotion sensitivity genes; src, myb, jun, myc, fos, raf, or int-2 oncogenes; or epidermal growth factor receptor. The isolated CNE 323 DNA sequence appeared to preserve the genomic structure of the original sequence found in CNE2 cells and in nude mouse tumors induced by CNE2 cells or by CNE/JB6 transfectant cells, indicating that the cloned NPC sequence was activated during NPC carcinogenesis and not during transfection or construction of the library, and that the cloned sequence or a larger sequence of which it was part played a role in tumor formation. Finally, we identified a 1.3-kb mRNA that hybridizes to a subclone of the 16-kb NPC sequence in CNE2 cell poly (A)+ RNA. PMID- 1714742 TI - Detection of proteins related to a salivary glycoprotein (EP-GP). Concentrations in human secretions (saliva, sweat, tears, nasal mucus, cerumen, seminal plasma). AB - With a highly specific monoclonal antibody against a previously isolated and characterized human salivary 19-20-kDa glycoprotein, designated as extra-parotid glycoprotein [Rathman et al. (1989) J. Biol. Buccale 17, 199-208], a common epitope was detected on proteins in several excretory human body fluids. With a quantitative ELISA the EP-GP epitope was measured in widely different concentrations in several secretory human body fluids in the descending order of seminal plasma much greater than tears approximately nasal mucus approximately sweat much greater than saliva. Crossreactivity was also observed in cerumen but not in milk, cerebrospinal fluid, blood plasma and urine. The relative amount of EP-GP in the positively reacting secretions was however, in the same order in each fluid per mg of protein on an average of 1% of the total protein amount. The EP-GP-epitope bearing proteins found in the various human secretions were further characterized by means of electrophoresis and immunoblotting. The molecular masses and the isoelectric points of the proteins in the different secretions display strong resemblance to values found for the salivary glycoprotein EP-GP (molecular masses 19 and 20 kDa; pI values between 4.8 and 5.4). All these findings point to the presence of proteins related to EP-GP in human secretions other than saliva. PMID- 1714743 TI - Ultrastructural study of cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band of broca in the basal forebrain of the rat. AB - The morphology, ultrastructure and synaptic relationships of the cholinergic and non-cholinergic neurons in the medial septal nucleus (MS) and vertical limb of the diagonal band of Broca (VDB) in the basal forebrain of the rat were studied at the light and electron microscopic levels. The cholinergic neurons were localized immunocytochemically using a monoclonal antibody against choline acetyltransferase (ChAT). Morphometric and statistical analyses showed that ChAT labelled cells presented a predominantly oval morphology in both nuclei. The sizes of the neurons were significantly larger in the VDB nucleus. Within the two nuclei, two populations of cholinergic neurons were differentiated. One of the large immunolabelled neurons presented deep indentations and prominent nucleoli in their non-immunoreactive nuclei. Their cytoplasm contained a well-organized endomembrane system composed of short cisternae of rough endoplasmic reticulum (RER). One or two lamellar bodies with a peculiar ultrastructure were frequently found intercalated in this system. The Golgi areas presented numerous coated vesicles, sequestration and multivesicular bodies, which was indicative of an intense metabolic activity in these cells. The second population of small immunolabelled neurons exhibited reduced cytoplasm with a poorly developed endomembrane system and apparent absence of lamellar bodies. The neighbouring non immunolabelled neurons presented a different type of organization of the endomembrane system which was composed of scattered and loosely arranged elongated cisternae of RER and infrequent lamellar bodies, with a structure different from that seen in the large cholinergic neurons. We propose that the structural differences in composition of the endomembrane system and lamellar bodies observed in the three types of neurons in this study indicate different metabolic activities. Symmetrical and asymmetrical synaptic contacts were observed on somata and dendrites of labelled neurons, the latter being more frequent. ChAT-labelled axon boutons were never seen. The absence of immunolabelled axon terminals and the presence of immunolabelled myelinated axons leads us to suggest that the majority of neurons in these areas are of the long projecting type. PMID- 1714744 TI - HNK-1 peripheral blood lymphocytes in HIV infected patients. AB - Peripheral blood cells with the HNK-1 phenotype were studied in HIV infected patients of which 28 with Asymptomatic Infection (AI), 64 with Persistent Generalized Lymphoadenopathy (PGL), 9 with AIDS Related Complex (ARC), and 12 with AIDS. Eight normal subjects served as controls. Two-color immunofluorescence by flow cytometry analysis showed in all of them a significant increase of the mean percentage of HNK+T3- lymphocytes (greater than 20%) as compared to controls (6%). Only in AI the mean absolute count was significantly higher (776/cmm) than control's one (152/cmm). Percentages and absolute counts of HNK+T3- lymphocytes were similar to normal ones. In AI and PGL HNK+T3+ cells correlated directly with T8 lymphocytes and inversely with T4 cells and T4/T8 ratio. These results indirectly suggests that HNK+T3+ cells represent a subset of suppressor/cytotoxic lymphocytes. The results in ARC and AIDS were somewhat equivocal and deserve further study in larger samples. No correlation was found between HNK+T3+ and HNK+T3- cells. The expansion of HNK+T3+ cells was parallel to that one of T8 lymphocytes expressing CD8 antigen at high surface density which were previously reported as having cytotoxic activity. Follow-up studies of the HNK cell peripheral pattern will clarify if it can be regarded as an early predictor of clinical outcome. PMID- 1714745 TI - Neuropoietic cytokines in the hematopoietic fold. AB - Among the molecules that determine the developmental fate of sympathetic neurons from noradrenergic to cholinergic function are two apparently unrelated proteins, cholinergic differentiation factor and ciliary neurotrophic factor (CDF and CNTF, respectively). The present work suggests a structural basis for their functional overlap: sequence pattern-matching and predictive structure analysis contends that CDF and CNTF are homologous and share a common helical framework. An integrated CDF/CNTF profile also reveals similar sequence/structure motifs in a group of hematopoietic cytokines composed of granulocyte colony-stimulating factor, interleukin-6, and a novel factor called oncostatin M; a more distant relationship is indicated with interleukin-3 and interferons-alpha/beta. Evolutionary ties between neuropoietic and hematopoietic cytokines predict a distinctive tertiary architecture for the uncharacterized CDF and CNTF receptors. The intertwined cytokine/receptor networks signal a closer relationship between the molecular mechanisms underlying neuro- and hematopoiesis. PMID- 1714746 TI - Immunohistochemical demonstration of peptidergic nerve fibers associated with the central lacteal lymphatics in the duodenal villi of dogs. AB - Immunohistochemical demonstration was made of the peptidergic nerves distributed in the central lacteal lymphatics of the canine duodenal villi. The central lacteal-associating nerve fibers were predominantly immunoreactive for both substance P (SP) and calcitonin gene-related peptide (CGRP). Observation of doubly immunostained sections evidenced that both peptides were located in one and the same nerve fibers. The SP/CGRP-immunoreactive fibers were concentrated in the intermediate portion of the villus height. Ultrastructurally, the SP/CGRP immunoreactive nerve fibers ran closely beneath the endothelial cells of the lacteal, some of them penetrating into the cytoplasm with knob-like swellings. The immunoreactive products were localized to large-cored vesicles in the sub- and intra-endothelial nerves. The occurrence of SP and CGRP in the nerve fibers distributed in the central lacteals which lack smooth muscles implies that these nerves may be sensory in nature. A mechanoreceptive function of the nerves is proposed on the basis of their peculiar knob-like projection into the lacteal endothelium. PMID- 1714747 TI - Shaking HIV-1 infected cells indicates novel behavior of MN strain. AB - The shaking method of harvesting human immunodeficiency virus type 1 (HIV-1) is a powerful method of obtaining high titer, highly infective virus solutions. In this method infected cells are suspended in a small volume of liquid and the mixture is shaken. Viral infectivity, measured by tissue culture infective dose (TCID50) studies, rises faster than virus titer, as measured by reverse transcriptase levels. It is postulated that this disproportionate increase in infectivity results from improved infectivity for the virus particles obtained from shaking the infected cells. Of the five strains of HIV-1 studied (IIIB, AL1212, 906, RJ4029, and MN), one strain, MN, behaved differently than the others. Upon shaking, its virus titer increased 18-fold, as opposed to the 5-10 fold increase demonstrated by the other strains. These results may indicate that MN virions are retained more on the surface of the infected cells, rather than budding off into the surrounding medium, than other HIV-1 strains. In support of this theory it was found that ratios of immunofluorescence assay scores to reverse transcriptase levels were higher for MN than for other strains. PMID- 1714748 TI - Immunization of chimpanzees with the HIV-1 glycoprotein gp160 induces long lasting T-cell memory. AB - The goal of the present study was to investigate the antigen-specific T-cell response to the recombinant HIV envelope glycoprotein (gp160) and to test the effect of various adjuvant formulations on the efficiency of T-cell priming as well as on magnitude and longevity of the gp160-specific T-cell response. Our studies revealed that, in combination with an appropriate adjuvant (lipid-based adjuvant or mineral carrier complex), immunization with recombinant gp160 led to the appearance of gp160-primed T cells. The T-cell response obtained was substantial (proliferative response of greater than 100,000 delta dpm after one primary and two booster immunizations), gp160-specific (proliferation only in response to gp160, no proliferation after addition of a mock gp160 preparation), and long-lasting (T cell responses of greater than 50,000 delta dpm were observed more than one year after the last booster). The results presented here differ from those of previous studies in that they show the presence of substantial and long-lasting T-cell memory toward the immunogen gp160. Therefore further investigations on the use of these preparations as HIV candidate vaccines appear to be justified. PMID- 1714749 TI - Zinc chelates bind human hemopexin. PMID- 1714750 TI - [Mechanical hepaticojejunostomy]. PMID- 1714751 TI - [Treatment of cancer of the esophagus: presentation of a case series]. PMID- 1714752 TI - [Radical and palliative treatment in carcinoma of the thoracic esophagus]. PMID- 1714753 TI - Macroamylasaemia--biochemical marker of polymyositis? PMID- 1714754 TI - Gastric volvulus: a rare cause of hyperamylasaemia. PMID- 1714755 TI - Pellagra in a patient with an eating disorder. AB - A case of pellagra is described that occurred in a patient with an eating disorder and who presented with marked photosensitivity and diarrhoea. We found urinary 5-hydroxy-indole-acetic acid to be low and suggest that this may be a useful screening test. To our knowledge this is only the second reported case of pellagra associated with an eating disorder. PMID- 1714756 TI - Effects of recombinant human granulocyte colony-stimulating factor on leucopenia in zidovudine-treated patients with AIDS and AIDS related complex, a phase I/II study. AB - Twelve male patients, eight with the acquired immunodeficiency syndrome (AIDS) and four with AIDS related complex (ARC), who had zidovudine associated neutropenia (less than 1 x 10(9) neutrophils/l) were treated with recombinant human granulocyte colony-stimulating factor (G-CSF) in a phase I/II study. Treatment consisted of daily subcutaneous injections with G-CSF in a weekly increasing dose of 0.4, 2, 5 or 10 micrograms/kg body weight until a neutrophil count of more than 3 x 10(9) neutrophils/l was observed. This effective dose was continued for up to 4 weeks, followed by 4 weeks observation period without G-CSF treatment. Two patients (both with ARC) reached target neutrophil counts at the lowest G-CSF dose, whereas nine patients needed 2 micrograms/kg. One patient discontinued treatment before he reached target neutrophil counts. Mean (+/- SD) neutrophil counts before and after 1 and 4 weeks of effective dose treatment were 0.65(+/- 0.188) x 10(9), 6.016(+/- 2.595) x 10(9) and 5.54(+/- 4.237) x 10(9)/l respectively (P less than 0.01). The number of monocytes increased from 0.171(+/- 0.113) to 0.501(+/- 0.274) and 0.474(+/- 0.374) x 10(9)/l after 1 and 4 weeks of treatment (P less than 0.01). Other haematologic parameters did not change significantly. Two weeks post-treatment the numbers of neutrophils and monocytes had returned to pre-treatment values. Mild side effects consisting of bone, joint or muscle pain were observed in three patients. Two patients (both with AIDS) did not complete the study. One patient stopped treatment because of fever and malaise, attributable to a generalized cytomegalovirus (CMV) infection and one patient had to stop zidovudine treatment because of severe thrombocytopenia. We conclude that G-CSF increases the number of circulating neutrophilic granulocytes in zidovudine-treated patients at relatively low doses and with few side-effects. PMID- 1714757 TI - Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in serum during induction treatment of acute leukaemia. AB - Granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) are increasingly used to stimulate granulopoiesis in neutropenic patients but in most cases without any knowledge of the endogenous CSF-levels. With the purpose to define serum levels of GM-CSF and G-CSF during induction chemotherapy and haematological reconstitution in patients with acute leukaemia we have used enzyme-linked immunosorbent assay (ELISA) techniques to measure these growth factors in 18 patients with acute myeloid leukaemia (AML) and eight patients with acute lymphoblastic or undifferentiated leukaemia (ALL/AUL). G-CSF above 0.05 ng/ml was detected in 54% of the analysed AML samples, median 0.29 (range 0.05-2.80) ng/ml; and in 40% of analysed ALL/AUL samples, median 0.09 (range 0.05-3.00) ng/ml. In patients with AML there was a clear correlation between an elevated serum concentration of G-CSF and documented infections. On the other hand, 15/18 of the patients with acute myeloid leukaemia and 8/8 patients with ALL/AUL had non-detectable levels of GM-CSF (less than 0.10 ng/ml). Two patients had measurable levels of GM-CSF in all samples, median 0.71 (range 0.26-1.18) ng/ml and in these patients the levels successively decreased during and after chemotherapy and did not increase in response to infections. In normals detectable levels of GM-CSF were found in 2/35 individuals and G-CSF in 0/10 individuals. PMID- 1714758 TI - Patterns of CD16 and CD56 expression in persistent expansions of CD3+NKa+ lymphocytes are predictive for clonal T-cell receptor gene rearrangements. The Yorkshire Leukaemia Group. AB - Phenotypic characteristics, and correlations between the expression of membrane NK-associated (NKa) determinants (CD11b, CD16, CD56 and CD57) and T cell receptor (TCR) genotypic patterns, were examined in 25 patients with persistent (greater than 6 months) expansions of CD3+WT31+NKa+ (CD8+ and CD8dim+) lymphocytes. These studies showed that distinct NKa phenotypic profiles were restricted to cases with rearranged TCR configurations and that clonal CD3+NKa+ components could be predicted in most cases by assessing relationships between membrane CD16 and CD56 expression. For all normal NKa subpopulations, there was a high correlation (P less than 0.0001; n = 31) between the expression of these two membrane determinants. Markedly increased CD16 expression by CD3+NKa+ cells, in relation to CD56 (i.e. a high CD16:CD56 ratio), was found exclusively in cases with rearranged TCR (13/16 cases); 2/3 of the remaining cases showing significantly reduced CD16:CD56 ratios and high (greater than 2.0) CD3+CD56+ absolute numbers. In contrast, 7/9 of the germline TCR cases had a normal CD16:CD56 ratio and 2/9 a decreased ratio with low (less than 1.0) CD3+CD56+ absolute numbers. A high ratio of CD16:CD56 expression by CD3+NKa+ lymphocytes was therefore informative for 82% of TCR rearrangements in this series; and analysis of CD16 and CD56 expression was predictive for germline and rearranged TCR configurations in 24/25 persistent CD3+NKa+ expansions. PMID- 1714759 TI - A phase II trial of recombinant human granulocyte colony-stimulating factor in the myelodysplastic syndromes. AB - We conducted a phase II study of the intravenous administration of a glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) for 7-14 d in 41 patients with the myelodysplastic syndromes (MDS). Administration of rhG-CSF elicited striking rises in both leucocyte and neutrophil counts in the majority of the patients irrespective of the FAB subtypes of MDS. The rises in neutrophil counts were dose dependent and 5 micrograms/kg/d of rhG-CSF yielded approximately an 8-fold increase in neutrophil counts. Leucocytes and neutrophil counts started to increase shortly after the first injection of 5 micrograms/kg, was maintained at significantly elevated levels during 14 d of treatment, and returned to the pretreatment levels within several days following discontinuation of rhG-CSF. The action of rhG-CSF was specific for neutrophils since leucocytosis was due exclusively to neutrophilic increase associated with an increased marrow myeloid maturation. There were no consistent changes in the monocyte, eosinophil, lymphocyte, platelet or reticulocyte counts. After treatment, the percentage of marrow blast cells was reduced in eight of 13 evaluable patients with refractory anaemia with an excess of blasts (RAEB) or RAEB in transformation (RAEB-t). No patients developed acute leukaemia during the treatment or in the immediate follow-up period. The treatment was well tolerated with only minimal toxicity. The results suggest that rhG-CSF is a safe and effective way to promptly improve neutropenia in MDS patients. PMID- 1714760 TI - Neovascular response in ischaemic central retinal vein occlusion after panretinal photocoagulation. AB - Twenty seven eyes treated for ischaemic central retinal vein occlusion with panretinal photocoagulation were reviewed. Prior to laser therapy anterior segment neovascularisation predominated (17 eyes) over posterior segment involvement (6 eyes). After photocoagulation anterior segment new vessels regressed or did not develop in the majority of cases. However, in five eyes previously absent posterior segment neovascularisation occurred. These results suggest that photocoagulation alters but does not eliminate retinal ischemia, thus modulating the neovascular response. PMID- 1714761 TI - A double blind clinical trial to assess the value of aprotinin in third molar surgery. AB - The bovine derived polypeptide, aprotinin, inhibits the activation of certain chemical mediators of acute inflammation. These mediators are responsible for causing pain and swelling in traumatised tissue. The properties of aprotinin were assessed in patients requiring surgical removal of third molars, a procedure which often results in considerable postoperative pain and swelling. A double blind clinical trial compared the effects of local infiltration of 1 ml of saline on one side of the mouth and aprotinin (10,000 international units) on the other, in patients requiring extraction of both mandibular third molars. Pain scores were assessed with a visual analogue scale, and swelling was subjectively assessed. The results, when analysed statistically, showed that aprotinin significantly reduced postoperative pain and swelling on the side of the mouth on which it was used, as compared to the control side (0.01% significance). PMID- 1714762 TI - Pointers on posters for the new presenter. PMID- 1714763 TI - Regional lymph node metastasis of early gastric cancer. AB - Of 122 patients hospitalized for early gastric cancer in 1974-1989, 15 were histologically found to have regional lymph node metastases. Tumor invasion was limited to the mucosa in 3 cases and to the submucosa in 12. An ulcer or ulcer scar was found in the tumor in 12 cases. Metastasis had occurred only to the perigastric nodes in 13 cases and also along the left gastric and/or common hepatic arteries in two. Preoperative endoscopic injection of Chinese ink into the submucosa revealed a high rate of uptake in regional lymph nodes along the left gastric, splenic and common hepatic arteries. Patients with early cancer should undergo gastrectomy with lymphadenectomy, particularly if the cancer contains an ulcer or has invaded the submucosa. If the patient's general health and life expectancy are poor, restricted surgery or endoscopic treatment should be considered. PMID- 1714764 TI - Acid ethanol fixation and polyester wax embedding combines preservation of antigenic determinants with good morphology and enables simultaneous bromodeoxyuridine (BrdU) labeling. AB - Lymphoid tissue, and/or isolated peripheral mononuclear blood cells were fixed in acid ethanol and embedded in polyester wax (melting point 37 C). The excellent cytomorphology obtained allowed distinguishing different types of individual lymphoid and nonlymphoid cells. Furthermore, this procedure was satisfactory in the immunophenotyping of histiocytes, endothelial, mesenchymal, epithelial cells, different (sub-) types of lymphocytes and also of lymphokine activated killer (LAK) cells. The staining patterns obtained with the different poly- and monoclonal antibodies on polyester wax sections were not only analogous to those obtained on frozen sections, but cells which had incorporated bromodeoxyuridine could be double labeled with specific antiserum. PMID- 1714765 TI - A method for the examination of the same cell using light, scanning and transmission electron microscopy. AB - A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis. PMID- 1714766 TI - Staining plant cells with silver. I. The salt-nylon technique. AB - A technique is described for selectively silver staining nucleoli, active nucleolus organizers, nucleolar material attached to chromosomes, kinetochores, synaptonemal complexes, and chromosome cores in plant cells. The technique, called salt-nylon silver staining, involves spreading cells on glass slides, treating the cells with a solution of saline sodium citrate, and incubating the cells in a silver nitrate solution covered with nylon screen. Selected variables important for achieving reliable silver staining are considered. PMID- 1714768 TI - A method for histological preparation of undecalcified bone sections containing acrylic bone cement. AB - An improved and time reducing method is presented for the histological evaluation of bone containing polymethylmethacrylate (PMMA) bone cement. The undecalcified bone was embedded in epoxy resin and section of 50-100 microns thickness were produced using a commercially available cutting grinding system. The sections were stained with Stevenel's blue and van Gieson picrofuchsin or a modified hematoxylineosin. PMMA bone cement was not dissolved and remained enabling examination in situ of an intact cement bone interface and tissue reaction without decalcification. PMID- 1714767 TI - Immunodetection of a tumor associated antigen (TAG-12): comparison of microwave accelerated and conventional method. AB - The microwave stimulated immunodetection of a tumor associated antigen (TAG-12) by monoclonal antibody 7A9 and an avidinbiotinylated alkaline phosphatase kit was compared with the conventional staining method. No difference in the staining pattern of antibody 7A9 was noticed in serial paraffin sections of 50 specimens including normal, benign and malignant breast tissues after microwave irradiated and conventional immunostaining. The results demonstrate that microwave stimulated immunostaining gives reliable results and can remarkably reduce the time of the staining procedure. PMID- 1714769 TI - Two methods for flat embedding sections in LR white cut from paraffin blocks. AB - Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistry. PMID- 1714770 TI - Histological preparation of the undecalcified rat otic bulla for mesoscopic observations. AB - Otic bullas of the rat, obtained by excision and formalin fixed, are successfully embedded in methylmethacrylate by dehydration and subsequent infiltration with plastic under vacuum. Sections 10 microns thick are obtained by cutting the trimmed and sandpapered acrylic blocks on an LKB multirange microtome. The sections are collected on adhesive tape and stained with a Trichrome stain (modified Weigert-van Gieson). Finally, the sections attached to the tape are mounted on microscope slides with glycerin-gelatin and sealed in the same medium. Serial sections are used for three-dimensional graphic reconstruction. PMID- 1714771 TI - A fresh mount method for cytochrome oxidase histochemistry. AB - A simple modification of cytochrome oxidase histochemistry was undertaken to prevent the artifactual condensation of reaction product. The quality and reliability of the histochemical method were greatly improved by using fresh mounted tissue [corrected]. PMID- 1714772 TI - Heat shock inhibits the cytotoxic action of TNF-alpha in tumor cells but does not alter its noncytotoxic actions in endothelial and adrenal cells. AB - We have previously demonstrated that a short heat treatment protects target cells from lysis by tumor necrosis factors (TNFs). Here we show that a similar heat treatment of human umbilical vein endothelial cells and human fetal adrenal cells does not alter noncytotoxic actions of TNF, suggesting that heat shock may specifically inhibit the cytotoxic action of TNF. To find clues to the mechanisms by which heat shock protects cells from TNF killing, its effects on TNF-alpha-TNF receptor interactions, on the metabolism of the ligand, and on the expression of mRNAs for possible protective proteins were studied. The affinity of binding and the internalization of the ligand were slightly reduced after heat shock. These effects were, however, very vague and seen both in heat-responsive tumor cells and in endothelial and adrenal cells. Thus, it is unlikely that they could explain the heat-induced TNF resistance. Heat shock increased the expression of mRNAs for heat shock proteins (hsps) 27 and 70 in all the cells studied, but did not alter the expression of manganous superoxide dismutase (MnSOD) mRNA, which has previously been shown to play a crucial role in TNF resistance. Based on these results, we suggest that cells have multiple mechanisms to escape TNF mediated lysis and that heat-induced protection from TNF killing may be mediated by hsps or other heat-inducible protective proteins, which act after receptor binding and protect cells from TNF-induced cellular damage without inhibiting the signal transduction mediating noncytotoxic effects of TNF. PMID- 1714773 TI - Effects of interleukin-6 and leukemia inhibitory factor on the acute phase response and DNA synthesis in cultured rat hepatocytes. AB - Human recombinant interleukin-6 (IL-6) and human recombinant leukemia inhibitory factor (LIF) similarly stimulate synthesis of typical acute-phase proteins in the primary rat hepatocyte cultures. LIF is, however, less effective in increasing uptake of alpha-aminoisobutyric acid than IL-6. Antiserum to human IL-6 abolishes induced protein synthesis and amino acid uptake elicited by hrIL-6 but has no effect on the acute-phase response of rat liver cells stimulated by LIF. Both IL 6 and LIF inhibit basal and epidermal growth factor-induced DNA synthesis in rat hepatocytes. PMID- 1714774 TI - Recombinant interleukin-1 (IL-1) stimulates prostaglandin E2 production by osteoblastic cells: role of calcium, calmodulin, and cAMP. AB - Interleukin-1 (IL-1) has been previously shown to stimulate prostaglandin E2 (PGE2) production by osteoblastic cells. This IL-1 effect has also been shown to be potentiated by parathyroid hormone (PTH), which activates both the calcium and the cAMP signal transduction pathways in osteoblastic cells. In the present study, the role of calcium, calmodulin, and cAMP in potentiating the IL-1 effect was examined. The calcium channel blocker verapamil (100 microM) completely inhibited the IL-1 effect. Similarly, the calmodulin antagonist W-7 (50 microM) inhibited the IL-1-induced stimulation. Conversely, the calcium ionophore A23187 (0.1 microM) potentiated the IL-1 effect. The phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX; 100 microM), which elevates cAMP levels in the cells, had a strong potentiating effect on the IL-1-induced PGE2 production. These results suggest that both the calcium and the cAMP second messenger systems can modulate the IL-1 effect on osteoblastic cells. PMID- 1714775 TI - Cathepsin B and D activities in intestinal mucosa during postnatal development in pigs. Relation to intestinal uptake and transmission of macromolecules. AB - The mucosal activities of cathepsin B and D were assayed in the small intestine of newborn pigs and of pigs 24 h, 6 days and 4-8 weeks old, respectively. Before sacrificing these animals, the intestinal capacity to internalise and further transmit macromolecules into the blood serum was evaluated by feeding a marker solution containing bovine IgG, bovine serum albumin and fluorescein isothiocyanate-conjugated dextran 70,000 (FITC-dextran). No significant differences in the activities of cathepsin B or D were observed in the mucosa from the various age groups of pigs. The transmission of the marker macromolecules into the blood was higher in the newborn pigs than in older postclosure animals, all of which only had low or undetectable serum marker concentrations. No apparent difference in the uptake of the markers into the intestinal epithelium could be detected between that of the newborn, preclosure pigs and pigs 24 h old. Both groups showed a similar pattern of mucosal fluorescence, indicating that there had been a high uptake of all markers into the epithelium. In the 6-day-old pigs, epithelial uptake was only visible in the distal small intestine, whereas no uptake at all could be found in the animals 4 8 weeks of age. The results suggest that intestinal closure in the pig, i.e. the dramatic decrease in the transfer of macromolecules from the intestinal epithelium in the blood at about 24 h of age, is not due to a decrease in the endocytotic ability of the enterocytes, nor to a higher degradation rate within these cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714776 TI - CSF monoamine metabolites and somatostatin in Alzheimer's disease and major depression. AB - Decreased cerebrospinal fluid (CSF), somatostatinlike immunoreactivity (SLI) and alterations in the CSF monamine metabolites 3-methoxy-4-hydroxyphenylethylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) have been reported in patients with probable Alzheimer's disease (AD) and in patients with major depression. In this study, we found CSF SLI to be significantly lower in a large group of AD patients (n = 60) and in a group of age-matched patients with major depression (n = 18) as compared with normal controls (n = 12). Mean CSF, MHPG, 5-HIAA, and HVA levels were not significantly different among diagnostic groups. Within a group of "depressed" AD patients, CSF levels of 5 HIAA showed a significant positive correlation (p = 0.03) with CSF SLI; a similar relationship was found within the group of patients with major depression. Further exploration of the relationship between the somatostatin and serotonin systems may provide clues as to how neuropeptides interact with monoamine neurotransmitters and what role they have in depression. PMID- 1714777 TI - Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463. AB - A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2 oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Brannstrom et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH. PMID- 1714778 TI - Open channel block by gadolinium ion of the stretch-inactivated ion channel in mdx myotubes. AB - Currents flowing through single stretch-inactivated ion channels were recorded from cell-attached patches on myotubes from mdx mice. Adding micromolar concentrations of gadolinium to patch electrodes containing normal saline produced rapid transitions in the single-channel current between the fully open and closed states. The kinetics of the current fluctuations followed the predictions of a simple model of open channel block in which the transitions in the current arise from the entry and exit of Gd from the channel pore: histograms of the open and closed times were well fit with single exponentials, the blocking rate depended linearly on the concentration of gadolinium in the patch electrode, and the unblocking rate was independent of the concentration of gadolinium. Hyperpolarizing the patch increased the rate of unblocking (approximately e-fold per 85 mV), suggesting the charged blocking particle can exit the channel into the cell under the influence of the applied membrane field. The rate of blocking was rapid and was independent of the patch potential, consistent with the rate of ion entry into the pore being determined by its rate of diffusion in solution. When channel open probability was reduced by applying suction to the electrode, the blocking kinetics were independent of the extent of inactivation, suggesting that mechanosensitive gating does not modify the structure of the channel pore. PMID- 1714779 TI - Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell free system. AB - Measurement of membrane transport in suspensions of isolated membrane vesicles provides averaged information over a potentially very heterogeneous vesicle population. To examine the regulatory mechanisms for ATP-dependent acidification, methodology was developed to measure pH in individual endocytic vesicles. Endocytic vesicles from proximal tubule apical membrane of rat kidney were labeled in vivo by intravenous infusion of FITC-dextran (9 kD); a microsomal fraction was obtained from dissected renal cortex by homogenization and differential centrifugation. Vesicles were immobilized on a polylysine coated coverglass and imaged at high magnification by a silicon intensified target camera. ATP-dependent acidification was not influenced by endosome immobilization. Endosome pH was determined from the integrated fluorescence intensity of individual labeled vesicles after background subtraction. Calibration studies with high K and nigericin showed nearly identical fluorescence vs. pH curves for different endosomes with a standard deviation for a single pH measurement in a single endosome of approximately 0.2 pH units. In response to addition of 1 mM MgATP in the presence of K and valinomycin, endosome pH decreased from 7.2 to a mean of 6.4 with a unimodal distribution with width at half-maximum of approximately 1 pH unit. The drop in endosome pH increased and the shape of the distribution changed when the time between FITC-dextran infusion and kidney removal was increased from 5 to 20 min. Differences in ATP-dependent acidification could not be attributed to heterogeneity in passive proton conductance. These results establish a direct method to measure pH in single endocytic vesicles and demonstrate remarkable heterogeneity in ATP-dependent acidification which was interpreted in terms of heterogeneity in the number and/or activity of proton pumps at serial stages of endocytosis. PMID- 1714780 TI - Characterization of ion channels on the surface membrane of adult rat skeletal muscle. AB - The channels present on the surface membrane of isolated rat flexor digitorum brevis muscle fibers were surveyed using the patch clamp technique. 85 out of 139 fibers had a novel channel which excluded the anions chloride, sulfate, and isethionate with a permeability ratio of chloride to sodium of less than 0.05. The selectivity sequence for cations was Na+ = K+ = Cs+ greater than Ca++ = Mg++ greater than N-Methyl-D-Glucamine. The channel remained closed for long periods, and had a large conductance of approximately 320 pS with several subconductance states at approximately 34 pS levels. Channel activity was not voltage dependent and the reversal potential for cations in muscle fibers of approximately 0 mV results in the channel's behaving as a physiological leakage conductance. Voltage activated potassium channels were present in 65 of the cell attached patches and had conductances of mostly 6, 12, and 25 pS. The voltage sensitivity of the potassium channels was consistent with that of the delayed rectifier current. Only three patches contained chloride channels. The scarcity of chloride channels despite the known high chloride conductance of skeletal muscle suggests that most of the chloride channels must be located in the transverse tubular system. PMID- 1714781 TI - Biochemical screening. PMID- 1714782 TI - Monoclonal antibodies in the cytodiagnosis of serous effusions. AB - In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1 glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1 glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy. PMID- 1714783 TI - Structure and biochemistry of mammalian hard keratin. AB - In this review, the structure and biological formation of hard alpha-keratin are drawn together. The hard keratins comprising wool, hairs, quills, hooves, horns, nails and baleen contain partly alpha-helical polypeptides which show homology with epidermal polypeptides only in the helical regions. These polypeptides (about 32 chains) are organized into intermediate filaments (IFs) of 7.5 nm diameter which are embedded in variable amounts of a matrix of non-helical cystine-rich proteins and glycine-tyrosine-rich proteins. The total number of proteins may exceed 100. In addition keratins contain a variety of lipid components. Wool and hair are produced in follicles in a multistep procedure. In the lower levels of the follicle, IFs without associated matrix are found. Subsequently matrix proteins are laid down between the IFs and further synthesis takes place concurrently. Finally the proteins are insolubilized by the oxidative formation of disulphide bonds. Keratinized fibres shows considerable complexity and diversity in the structural arrangement of IFs and matrix within cortical cells. Typically the IFs show hexagonal packing or give a whorl-like appearance in cross-section. PMID- 1714784 TI - Role of CD2 in regulation of CD3- LGL function. AB - CD2 is a differentiation marker present on T cells and NK cells. Cytotoxic T lymphocytes (CTL) can be activated by antibodies directed against the CD3/T-cell receptor complex and CD2 structures; however, the role of CD2 in regulation of CD3- large granular lymphocyte (LGL) functions has only recently been studied. Anti-CD2 monoclonal antibodies (mAbs) may be either augmenting or inhibitory and T-cell activation via the CD2 molecule occurs only when mAb binds defined combinations of the CD2 epitopes. Since LGL can be activated by a single stimulus (e.g., IL-2) to proliferate, produce IFN gamma, and increase their cytolytic potential, these functions were chosen to examine the effects of the anti-CD2 mAb and its combinations. Anti-CD2 mAb (D66, GT2, and X11-1) were incubated with LGL for various times in the absence or presence of IL2 and IFN gamma production was monitored. Single anti-CD2 mAb treatment demonstrated minimal augmentation of IFN gamma production. However, combinations of anti-CD2 (9.6) and the other anti-CD2 mAb resulted in a significant, synergistic enhancement of the IFN gamma production. Anti-CD2 mAb treatment appeared to inhibit production generated by optimal doses of IL-2 (1,000 U/ml). The effect of anti-CD2 mAb on IFN gamma production parallel their effects on LGL NK and LAK activity. These data suggested that mAb against the CD2 molecule were important in regulating LGL functions in the absence of a functional CD3 receptor in LGL.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714785 TI - Human monoclonal antibodies to cytomegalovirus recognize viral epitopes on the surface of virus-infected cells. AB - Four human monoclonal antibodies directed against human cytomegalovirus were produced by fusing Sp2/HPT heteromyeloma cells with peripheral blood lymphocytes, after stimulation in vitro for 6 days. The human hybridomas have been maintained in culture for one year and secrete, when cultured in serum-free medium, between 3.1 and 8.1 micrograms/ml of antibodies/10(6) cells/24 hours. HCV-1 and HCV-2 are IgG kappa, while HCV-3 and HCV-4 are IgG3 lambda. The four monoclonal antibodies immunoprecipitate a viral protein of 64 kD. Kinetic studies using indirect immunofluorescence indicate that this antigen appears late in the viral infectious cycle. All four monoclonal antibodies recognize human cytomegalovirus prototype strains AD-169, Davis and Towne, and 14 clinical isolates collected between 1984 and 1987. No reactivity was observed with other human herpesviruses. While no neutralizing activity could be observed with the human monoclonal antibodies, binding assays on unfixed infected cells showed that they recognized viral epitopes located on the cell surface membrane. These hybridomas may be useful for future therapy of immunocompromised patients. PMID- 1714786 TI - Evaluation of procedures for the fixation and processing of human tissue for immunohistochemical analysis of human monoclonal antibodies. AB - Preliminary investigations suggested the importance of an evaluation of different tissue preparation methods frequently used for immunohistochemical analysis of human or murine monoclonal antibodies on human tissue. Colon adenocarcinomas and adjacent morphologically normal colon epithelia were analyzed with an indirect immunoperoxidase technique. Duplicate tissue specimens were (1) snap frozen and fixed in acetone, (2) formalin fixed and paraffin embedded, with or (3) without ensuing treatment with pronase, or (4) alcohol fixed and paraffin embedded. Three different human monoclonal anti-colon cancer IgM antibodies, COU-1, D4213, and F10279, were used in the present study. Endogenous immunoglobulin and the secretory-component-mediated IgG binding were blocked on frozen sections with Fab' anti-IgM and anti-SC antibody. Bound monoclonal antibody was detected with horseradish peroxidase-anti-IgM. COU-1 was found to stain frozen sections of all 25 cancer and adjacent normal colon epithelia. In contrast, on formalin-fixed, paraffin-embedded tissue, only 80% (20/25) of the colon cancer and 44% (11/25) of the adjacent normal colon epithelia were positive. After treatment of the formalin-fixed sections with pronase, all cancers and normal adjacent epithelia were stained, but the cancer cells were more intensely stained than the normal colon epithelial cells. On alcohol-fixed tissues, intense staining was found in all the colon carcinomas analyzed, whereas no staining was found of the adjacent normal colon epithelia, except for a few cells in some of the sections investigated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714787 TI - Anesthetic management for myocardial revascularization following bleomycin chemotherapy. PMID- 1714788 TI - [The demonstration of a system of elastic fibers in a study of fibromatous lesions of the oral mucosa]. AB - The elastic fibers system, as visualised by orcein after potassium peroxymonosulfate oxidation in fibromatous lesions of the oral mucosa, is generally sparse. Rich orcein + fibers network was observed in only 5 of our 20 cases of reactive fibromas. Orcein + fibers are absent in the inflammatory areas. These observations rise the problem of the elastogenesis by the fibroblast in healing and neoplasia, and confirm the role played by the proteolytic enzymes during inflammation. PMID- 1714789 TI - Effect of fibrin glues on the mechanical properties of healing wounds. AB - Tissue glues are important in clinical practice. Fibrin based glues have many advantages over non-biological adhesives. The optimum formulation of these glues measured by their effect on mechanical properties of healing wounds has not been determined. Using a model involving standard dorsal skin incisions in adult male Wistar rats various glue formulations were compared by varying the fibrinogen, thrombin and factor XIII concentrations. Calcium (40 mumol/ml) and aprotinin (3000 kallidinogenase inactivator units, KIu/ml) concentrations were kept constant. Animals were killed at 8 days and wounds excised. Standard strips of these wounds were mechanically tested using an Instron tensiometer and the stress, strain, elasticity and work required to rupture wounds were calculated. Results indicate that a fibrin glue with a fibrinogen concentration of approximately 39 g/l and a thrombin concentration of 200-600 units/ml with no added factor XIII will result in wounds with significantly increased stress, energy absorption and elasticity values. PMID- 1714790 TI - Disposition of high-dose methotrexate in an obese cancer patient. AB - The disposition of many drugs in obesity is altered, although little is known of the effect of obesity on the pharmacokinetics of anti-cancer drugs. In this report the authors describe the pharmacokinetics of high-dose methotrexate in an obese woman (184% ideal body weight) with osteosarcoma. For this patient, both the volume of distribution at steady-state (0.398 l/kg) and systemic clearance (0.0956 l/h/kg) of methotrexate were increased compared with values observed in adult patients with osteosarcoma. The terminal elimination half-life (9.29 hours) of methotrexate was similar to other reported values in adult patients, thus indicating that increases in methotrexate volume of distribution and clearance may offset each other in obesity. Based on the experience gained from this patient, the authors suggest that renal function and methotrexate serum concentrations should be monitored in obese patients receiving high-dose methotrexate with leucovorin rescue. PMID- 1714791 TI - Secondary myelodysplastic syndrome complicating therapy for osteogenic sarcoma. AB - A 12-year-old girl with nonmetastatic osteogenic sarcoma received treatment with doxorubicin, methotrexate, cisplatin, cyclophosphamide, bleomycin, and dactinomycin. She developed unexplained persistent pancytopenia after completion of chemotherapy. Twenty-three months after the initial diagnosis of osteosarcoma an evaluation revealed a bone marrow pattern consistent with the diagnosis of refractory anemia with excess blasts, and karyotype analysis showed characteristic findings of therapy-related myelodysplasia (loss of chromosomes 5 and 7, as well as 12p and 17p deletions). Bone marrow transplantation from an human leukocyte antigen (HLA)-compatible sibling donor was performed 26 months after the diagnosis of the primary malignancy. Although it is unproven that the alkylating agents administered to this patient were responsible for the myelodysplastic syndrome, careful follow-up of osteosarcoma patients who receive alkylating agents is warranted. PMID- 1714792 TI - Multiple alpha-fetoprotein RNAs in adult rat liver: cell type-specific expression and differential regulation. AB - Multiple alpha-fetoprotein (AFP) RNAs are expressed in the rat liver and are differentially regulated during development. We examined the expression and cellular distribution of the full-length AFP RNA (major form, 2.1 kilobases highly expressed in fetal liver) and 3 variants of 1.7, 1.4, and 1.0 kilobases in normal rat liver, during fetal development, in regeneration, and in carcinogenesis. The 1.7-kilobase variant is expressed only in developing liver (by 15 days of gestation) and is much less abundant than the major form. In adult normal liver the 1.4- and 1.0-kilobase RNAs are the predominant forms. By cell separation studies we show that these variants are produced by parenchymal and nonparenchymal cells in normal rat liver, and that the full-length AFP mRNA is detectable in normal nonparenchymal cells. We demonstrate by in situ hybridization that the 2.1-kilobase mRNA is expressed by some ductular cells and a few nondividing hepatocytes (approximately 1 in 20,000). Further studies revealed that (a) the 2.1-kilobase AFP mRNA encodes translation products of molecular weight 68,000 and 70,000, and probably has multiple sites for translation initiation; (b) the 1.4-kilobase AFP RNA variant in adult rat liver encodes translation products of molecular weight 58,000, 54,000 and 44,000; (c) the 2.1-kilobase AFP RNA increases in liver nonparenchymal cells after CCl4 injury (20-30-fold) and in galactosamine-injured liver (60-100-fold), while the 1.4- and 1.0-kilobase variants change much less; and (d) after partial hepatectomy there are only small changes in any of the AFP RNAs, while during carcinogenesis oval cells contain large amounts of 2.1-kilobase AFP RNA and levels of the 1.4- and 1.0-kilobase species which are lower than those in normal liver. We suggest that after development synthesis of the full-length RNA is not shut off in a small proportion of rat liver cells and that ductular cells that express this RNA may constitute a facultative liver stem cell compartment. PMID- 1714793 TI - Growth hormone releasing hormone receptors on thymocytes and splenocytes from rats. AB - In the present study, we determined that rat mononuclear leukocytes possess specific receptors for growth hormone releasing hormone (GHRH). The results show that the binding of 125I-labeled GHRH to spleen and thymic cells was saturable and of a high affinity, approximately 3.5 and 2.5 nM for thymus and spleen cells, respectively. The Scatchard analysis revealed a binding capacity of approximately 54 and 35 fmol per 10(6) cells on thymus and spleen, respectively. The binding of GHRH was not competed by 10(-6) M growth hormone, corticotropin releasing factor, substance P or luteinizing hormone releasing hormone and vasointestinal peptide (VIP). Partial characterization of the receptor was accomplished by crosslinking 125I-labeled GHRH to thymus cells with disuccinimidyl suberate and polyacrylamide gel electrophoresis. Autoradiography of dried gels showed two major components in leukocytes and pituitary cells at approximately 42 and 27 kDa which could be diminished by unlabeled GHRH. The treatment of leukocytes with GHRH (10 nM) rapidly increased the intracellular free calcium concentration from a basal level of 70 +/- 20 nM to a plateau value of 150 +/- 20 nM in 6 min after stimulation. The functional activity of GHRH receptors was studied further by measuring lymphocyte proliferative responses and the increase in the level of cytoplasmic GH RNA. The presence of GHRH alone resulted in a dose-dependent increase in thymidine and uridine incorporation and a dose-dependent increase in the levels of GH RNA in the cytoplasm. Taken together, the results show that lymphocytes contain specific receptors for GHRH that are coupled to important biological responses and further support the concept of bidirectional communication between the immune and neuroendocrine tissues. PMID- 1714794 TI - Activation of human thymocytes by antibodies to the CD3/T-cell receptor complex: triggering of different epitopes results in different signals. AB - Monoclonal antibodies (mAb) against the CD3/T cell receptor (TcR) complex were analyzed for their ability to activate human thymocytes. In addition to mAb detecting epitopes on the CD3 complex (OKT3, BMA 030) the activation potential of recently developed mAb against common epitopes on the alpha/beta T-cell receptor (anti-TcR mAb: BMA 031, BMA 032) was evaluated. Several differences were observed between the two types of mAb: (a) Binding of the tested anti-CD3 mAb to thymocytes resulted in a rapid increase in the level of cytoplasmic free calcium ions [Ca2+]i, whereas no significant changes in [Ca2+]i were detected in thymocytes stimulated with BMA 031 or BMA 032. (b) Induction of effective proliferation induced by mAb OKT3 depended on exogenous IL-2 and in addition on the presence of accessory cells or phorbol-ester. Proliferation induced by BMA 031 only required exogenous IL-2. (c) OKT3 but not BMA 031 inhibited proliferation of thymocytes induced via the CD2 molecule. These studies indicate that anti-CD3 and anti-TcR mAb transduce different signals in thymocytes. Since the two types of mAb are directed to the same molecular complex the observed differences also support the idea that there are functionally different compartments in the CD3/TcR complex which may activate different signaling pathways. PMID- 1714795 TI - Comparative studies of CD3- and CD3+ CD56+ cells: examination of morphology, functions, T cell receptor rearrangement, and pore-forming protein expression. AB - Both CD3- and CD3+ CD56+ effector cells can mediate non-MHC-restricted lysis in the absence of activation. Previous studies have shown that both of these subsets can be augmented with IL-2. In the present study, we have examined further the phenotypic markers expressed on these cells as well as the functional capacities of these subsets, including LAK activity, cytokine expression, and pore-forming protein (PFP) production. In addition, these populations were analyzed for clonality by Southern blot analysis of the T cell receptor beta chain gene constant region. The CD3-, CD56+ and CD3+, CD56+ lymphocytes were quite similar in their phenotypic markers, although the CD3+, CD56+ lymphocytes lacked high levels of IL-2 receptor beta chain and did not express CD16. The CD3+, CD56+ lymphocytes mediated non-MHC-restricted lysis, but failed to express LAK activity or be induced by IL-2 to secrete IFN gamma, a characteristic of the CD3-, CD56+ lymphocytes. The T cell receptor beta chain gene pattern of the CD3+, CD56+ lymphocytes was characteristic of a polyclonal cell population. Of interest, both populations of cells appeared morphologically to be large granular lymphocytes that contain PFP in their cytoplasmic granules. Therefore these CD56+ subsets provide a new model to study several questions related to non-MHC-restricted target cell lysis, including the identification of novel receptors involved in target cell recognition and/or triggering as well as the biochemical pathways implicated in cellular lysis. PMID- 1714796 TI - Proliferation of resting lymphocytes is induced by triggering T cells through an epitope common to the three CD18/CD11 leukocyte adhesion molecules. AB - LFA-1, Mac-1, and p150,95 are a family of functionally important leucocyte integrins that share a common beta-subunit and participate in cellular adhesion. Monoclonal antibody to LFA-1 were described to block T-cell-mediated killing by inhibiting adhesion to target cells and to decrease T cell responses by preventing cell-cell contact. Recently it was demonstrated that LFA-1 molecule was involved in signal transduction. We report here that a monoclonal antibody termed 6.7 reacting with the three members of the leucocyte integrins is able in the presence of monocytes to directly induce the proliferation of resting peripheral blood T cells obtained from normal individuals. These results suggest the possibility that LFA-1 molecules could trigger T lymphocyte activation in addition to their role in homing, growth, and differentiation. PMID- 1714797 TI - [The effect of cholesterol on parameters of gramicidin D ion channels and on the macroscopic and microscopic characteristics of lipid bilayers]. PMID- 1714798 TI - [Inhibitory effect of bleomycin A6 on human colon cancer xenografts in nude mice]. AB - As evaluated by clonogenic assay, bleomycin A6 was found to be highly active against established human cancer cell lines derived from colon cancer (HT-29) and cecum cancer (Hce-8693). These human cancer cells have been serially transplanted in nude mice. At a tolerable dosage level, bleomycin A6 exerted remarkable growth inhibition on human colon cancer HT-29 and cecum cancer Hce-8693 xenografts (approximately 90% inhibition). No histopathological changes were found in the organs of treated animals. Compared on the basis of equitoxic doses (1/9 LD50), bleomycin A6 exerted much stronger growth inhibition against colon cancer HT-29 xenografts in nude mice than 5-fluorouracil and mitomycin C, with inhibition rates of 82%, 12% and 53%, respectively. More extensive necrosis was found in tumors treated with bleomycin A6 than in those treated with mitomycin C or 5 fluorouracil. The ratio values of non-necrotic tumor tissue to whole tumor tissue for bleomycin A6, mitomycin C and 5-fluorouracil were 0.33, 0.65 and 0.57, respectively. These observations indicate that bleomycin A6 is a potent antitumor agent against colon cancer xenografts and may be useful in human colon cancer chemotherapy. PMID- 1714799 TI - Effect of angiotensin converting enzyme inhibition on pressure-induced left ventricular hypertrophy in rats. AB - The influence of angiotensin converting enzyme inhibition on the development of left ventricular (LV) hypertrophy due to stenosis of the aortic arch was studied in female Sprague-Dawley rats. The aortic arch was banded to an outer diameter of 1.0 mm. After 14 days, LV and right ventricular functional parameters and transstenotic pressure gradient were measured in anesthetized rats. In addition, regional heart weights were determined, and myocytes of three different heart regions were isolated and subjected to morphometric analysis. To inhibit the angiotensin converting enzyme, ramipril was administered orally by gavage in a single daily dose of 1 mg/kg. Rats with aortic stenosis showed a marked increase in LV systolic pressure, mean prestenotic aortic pressure, and LV stroke work compared with sham-operated rats and demonstrated a systolic transstenotic pressure gradient of 82 mm Hg. This increase in LV hemodynamic load was paralleled by the development of LV hypertrophy as determined by a 37% increase in LV weight and by a 20% increase in cell volume of isolated LV myocytes. Concomitant ramipril treatment did not significantly affect LV functional parameters. The transstenotic pressure gradient was the same as in untreated rats with aortic stenosis. Likewise, the weight gain of the LV as well as the development of cellular hypertrophy of the LV were not influenced. Thus, in this model, angiotensin converting enzyme inhibition did not reduce the development of LV hypertrophy independent of the hemodynamic load. PMID- 1714800 TI - [Antigenic diversity of asexual schizont antigen GP195 of Plasmodium falciparum isolates from China]. AB - The antigenic diversity of asexual merozoite surface antigen precursor gp195 among 6 isolates of Plasmodium falciparum from Hainan and Jiangsu provinces of China was investigated with a panel of murine anti-gp195 McAbs by indirect immunofluorescence assay and Western blotting. The results show that all isolates express certain strain-common epitopes of gp195, and at the same time express or lack some strain-specific epitopes. The 6 isolates can be divided into two major gp195 serotypes according to the reactivity patterns with the panel of anti-gp195 McAbs. Remarkable molecular weight difference in gp195 was found among 4 isolates of P. falciparum in which the Jiangsu isolate possesses a large size of gp195 (210kDa) than the other 3 Hainan isolates (less than or equal to 200kDa). The polymorphism of gp195 and it's impact on the development of antimalarial vaccine is discussed. PMID- 1714801 TI - [Effect of electroacupuncture on the contents of substance P in the nucleus of the solitary tract in rats]. AB - Six pairs of rats were used in the experiment. The sciatic nerve was stimulated in one of every pair with the electric needle. Then the substance P (SP-like immunoreactive nerve fibers in the nucleus of the solitary tract (NST) were showed with immunohistochemical technique (ABC METHOD). The other of every pair was as the control. All experimental procedures were the same as the electroacupuncture group except that the sciatic nerve wasn't stimulated. The contents of SP of NST (at the area postrema segment) of both the electroacupuncture and the control were measured with the micro-spectroscopic image analyser. The results obtained were as follows: 1. The distribution positions of SP-like immunoreactive nerve fibers in the electroacupuncture group were the same as in the control group. That is, SP-like immunoreactive nerve fibers distributed from the caudal part to the rostral part in NST. 2. The contents and the area of SP-like immunoreactive nerve fibers of the electroacupuncture were more than the control. There was a significant difference between the two groups. The results mentioned above suggested that the role of NST is involved in the acupuncture analgesia. PMID- 1714802 TI - Isolation of an immunosuppressive metabolite of FK506 generated by human microsome preparations. AB - Metabolism of FK506, a 23 member macrolide under clinical investigation as immunosuppressant after transplantation, was studied using human liver microsomes. Two fractions isolated by semi-preparative HPLC were identified by negative fast atom bombardment mass spectrometry as FK506 metabolites with mass peaks at m/z = 790 indicating demethylation of the mother compound. The immunosuppressive activity of one metabolite was evaluated in a ConA-stimulated peripheral rat lymphocyte assay. FK506 had an IC50 of 0.186 nmol/L and the metabolite tested of 1.89 nmol/L. PMID- 1714803 TI - HIV-specific treatment. PMID- 1714804 TI - Purification and characterization of liver arylformamidase in rainbow trout and cattle. AB - 1. Arylformamidases were purified from the liver of rainbow trout and cattle. 2. Optimal pH's were 7.7 and 8.0 for the fish and cattle enzyme, respectively. Both enzymes showed optimum temperature at 40 degrees C. Thermal and pH stability ranges and Arrhenius plots of the enzymes differed. 3. Km and molecular weight of the fish enzyme were determined to be 0.28 mM and 32,700, respectively, while those for the cattle enzyme were 0.78 mM and 42,5000, respectively. PMID- 1714805 TI - Changes of glomerular fixed anionic charge sites in adriamycin nephrosis in rats. AB - A single intravenous injection of adriamycin (5 mg/kg) into rats induced nephrotic syndrome. In the various stages of the model, alterations of glomerular fixed anionic charge sites stained with colloidal iron were analyzed quantitatively by image analyzer, which is reported here for the first time. The results showed that there was a significant decrease in glomerular fixed anionic charge sites at 3 hours after administration of adriamycin as compared with the control animals (P less than 0.01). Marked loss of the anionic charge sites preceded the onset of proteinuria and steadily increased along with the progress of the disease. The results suggest that the loss of glomerular fixed anionic charge sites may play an important role in the development of proteinuria in nephrotic syndrome. PMID- 1714806 TI - [Experimental study on capacity of lung water regulation]. AB - The capacity of the regulation of lung water after fluid overloading or blood letting was measured in 70 rabbits. Body fluid in the whole blood was increased in both overloading and blood letting. The lung blood volume was increased after crystalloid overloading, but statistically insignificant (P greater than 0.05). On the contrary, a very significant (P less than 0.01) increase of lung blood volume was observed in all animals overloaded with colloid fluids. Blood letting caused an insignificant (P greater than 0.05) decrease of lung blood volume. Total lung water was increased in either groups, but, it was statistically insignificant in crystalloid group and highly significant in crystalloid group. Colloid fluid overloading caused a marked increase (P less than 0.01) of extravascular lung water (EVLW). Interesting to note is that animals received crystalloid fluids amounted up to 3-fold of its blood volume did not show any significant (P greater than 0.05) increase of EVLW. It is concluded that the capacity of lung water regulation was extraordinary intact even after the overloading with crystalloids in an amount equivalent to 2.5-fold of its blood volume. This phenomenon was not seen in the colloids group. PMID- 1714807 TI - Slowing of EEG in Parkinson's disease. AB - EEG studies of Parkinson's disease (PD) have shown that the incidence of EEG abnormalities is higher than in normal old individuals. The most common alteration in PD is generalized slowing of the EEG. We studied 18 patients with Parkinson dementia, 18 age-matched Parkinson patients without dementia and 20 controls. The absolute and relative amplitudes of delta, theta, alpha and beta bands and the peak and mean frequency were calculated from EEG spectra recorded from the T6-O2 derivation. All variables differed significantly in Parkinson dementia patients compared to controls. The most conspicuous finding was the increase of delta activity. Parkinsonian patients without dementia had more theta activity and the frequencies were slow compared to controls. We conclude that parkinsonian subgroups have distinct patterns of abnormality in EEG spectra: Parkinson patients with dementia have distinctly slower EEGs than patients without dementia. PMID- 1714808 TI - Sleep and cranial dystonia. AB - A nocturnal polygraphic study was performed on 10 patients with cranial dystonia (blepharospasm (BS) and oromandibular dystonia (OMD)). All patients showed impaired sleep efficiency and reduced slow and REM sleep, more marked in subjects with severe dystonia. Abnormal muscular activity decreased progressively with deeper sleep and during the first hours of the night, without disappearing. A disordered hypnic++ pattern and impaired motor control even during sleep are typical features in cranial dystonia. PMID- 1714809 TI - Modulation of early auditory processing during selective listening to rapidly presented tones. AB - Two dichotic listening experiments were performed in which stimulus and task conditions were optimized for the early selection of inputs. Subjects listened selectively to sequences of rapidly presented tone pips in one ear while ignoring tone pips of a different pitch in the opposite ear. In both experiments, an enhanced positivity between 20 and 50 msec (the 'P20-50') was observed over central and frontal sites in the ERPs to the attended-channel tone pips. At longer latencies, the effects of attention appeared to include an amplitude modulation of several exogenous ERPs, including subcomponents of the central N1 (60-150 msec) and P2 (170-230 msec) waves and the temporal T complex (80-150 msec). In contrast, the attention effect prefrontally consisted of a broad negativity that appeared to be largely endogenous. A signal processing technique (Adjar) was employed to correct for distortion of mutually overlapping ERPs elicited by successive stimuli presented at short interstimulus intervals (ISIs). It was confirmed that the P20-50 attention effect was not the result of differential overlap from previous ERPs. In addition, this technique allowed an analysis to be made of the effects of the preceding stimulus type and ISI on the attention-sensitive ERPs, which provided further support for the view that highly focused selective attention can directly modulate exogenous components of the auditory ERP. Moreover, these sequence-dependent ERP modulations were paralleled by variations in target discrimination performance. Taken together, these results provide strong support for the early selection hypothesis that attention can serve to selectively bias or gate stimulus processing before full perceptual analysis has occurred. PMID- 1714810 TI - Tonic changes in alpha power during immersion of the hand in cold water. AB - Phasic event-related desynchronization (ERD) of alpha activity briefly follows many types of stimulation. In order to define EEG changes resulting from longer stimulation. EEG records were made before and during hand immersion into cool and painfully cold water (cold pressor). Five minutes of 13-lead EEG records were obtained from 14 subjects for each condition. EEG frequency analysis was performed on artifact-free epochs from 60 to 240 sec following immersion. Following an initial phasic decrease in alpha power during cold water immersion, there was an augmentation of alpha power (8-12 Hz) in bilateral frontal and posterior electrodes. This augmentation was largely the result of an increase in the low alpha band (8-10 Hz). Alpha power at both central electrodes C3 and C4 changed little during cold water immersion. Cool water immersion produced less alpha power augmentation than cold water immersion. These observed changes were primarily in the high alpha band (10-12 Hz) and were larger in electrodes ipsilateral rather than contralateral to the stimulation. There was also an increase of beta bilaterally in frontal and posterior regions with cold water immersion. Our data demonstrate sustained topographic EEG responses during tonic stimulation from hand immersion in painfully cold water. These changes differ from those produced by stimulation with cool water immersion. PMID- 1714811 TI - Quantification of EEG irregularity by use of the entropy of the power spectrum. AB - A new method for quantifying irregularity of EEGs is proposed in this study. The entropy, an information measure, determines the uniformity of proportion distribution. The peakedness or flatness of the distribution of the EEG power spectrum, representing EEG rhythmicity, can be measured by the entropy, because the power spectrum consists of proportions of power at each frequency. The irregularity of the EEG was measured by the entropy of the power spectrum, called an irregularity index (II). The II was obtained from the power spectrum at F3, F4, C3, C4, P3, P4, O1 and O2 during rest and mental arithmetic in 10 normal subjects. Relative band powers of delta, theta, alpha and beta bands and alpha peak frequency were also obtained. EEGs during rest were significantly more irregular anteriorly than in the occipital areas. Alpha activity was also more irregular in the anterior region. A greater degree of EEG desynchronization during mental arithmetic was found over the left hemisphere and the right occipital area. The II was more sensitive to such desynchronization than alpha band power and alpha peak frequency. The differences in spectral structures between rest and mental arithmetic conditions, mainly over the left hemisphere, were also confirmed by the Kullback-Leibler information. PMID- 1714812 TI - On cortical folds and neuromagnetic fields. AB - A folded cortical source of neuromagnetic fields, similar in configuration to the visual cortex, was simulated. Cortical activity was modelled by different distributions of independent current dipoles. The map of the summed fields of the dipoles of this cruciform model changed, depending upon the statistical distribution of the electrical activity of the dipoles and its geometry. Arrays of dipoles of random orientations and strengths produced field patterns that could be interpreted as due to moving neural currents, although the geometry of the neural tissue remained unchanged and the average activity remained approximately constant. The field topography at any instant was apparently unrelated to the depth or orientation of the underlying structure, thus raising questions about how to interpret topographic MEG and EEG displays. Furthermore, asynchronous activity (defined as independent directions and magnitudes of activity of the dipoles) did not result in less field power than when the dipoles were synchronized, i.e., when the direction of current flow was correlated across all of the dipoles within the cruciform structure. Therefore, in this model 'alpha blockage' cannot be mimicked by desynchronization. More generally, for the cruciform or any other symmetrically folded and active cortical sheet, 'blockage' cannot be attributed to desynchronization. The same is true for the EEG except that smooth unfolded sheets of radially oriented dipoles would result in enhancement of voltage due to synchronization. Such radial dipoles do not contribute to the MEG. Blockage was simulated by reducing the amount of activity within different portions of the synchronized cruciform model. This resulted in a dramatic increase in the net field because attenuation broke the symmetry of the synchronized cruciform structure. With asynchronous dipoles populating the structure, the attenuation of the same portion of the structure had no easily discerned effect on the net field. However, maps of average field power were consistently related to the position of the region of attenuated activity. The locations of regions of attenuated activity were determined by taking the difference between the mean square field pattern obtained when all portions of the cruciform structure were active and the pattern obtained when a portion of the structure was relatively inactive. When activity of the same portions were incremented rather than attenuated, the resulting plot of average power was essentially the same as that of the attenuated portion derived by taking these differences between power distributions. The major conclusions are that the concepts of synchronization and desynchronization have no explanatory power unless the physical conditions under which they occur are specified precisely.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714813 TI - Methodological considerations for the evaluation of spatio-temporal source models. AB - The effects of physiological noise and modelling procedure on spatio-temporal source modelling (STSM) solutions were examined by adding EEG noise (3-4% of total energy) to synthetic signals from 3 dipoles producing topographies that partially cancelled at the surface of an homogeneous sphere. Three patterns of source activation profiles were each associated with two EEG records. The STSM solutions were subjected to statistical tests that detect signal left in the residuals. All models substantially accounted for noise. Essentially correct models left no consistent signal in the residuals, but other statistically acceptable models were also occasionally found. Unambiguous correct solutions were found for moderately correlated source activations. Statistically adequate 2 source and 3-source models were found for the 4 data sets incorporating nearly parallel activations of 2 sources. When all 3 sources had comparable amplitudes, the 2-source models represented compromises containing gross mislocalizations but correct 3-source models were found. When one of the parallel sources was attenuated by 75%, only 2-source models satisfactorily approximated the 2 main sources. The localization errors in the "correct" models ranged from 2.5% to 13% of the sphere radius. Thus, even with a perfect signal propagation model, STSM could at best claim approximate localizations from data containing structured noise. PMID- 1714814 TI - EKG artifacts suppression from the EEG. AB - A method is proposed to identify and eliminate EKG artifacts from a single channel of EEG recordings. The method identifies the EKG as outlying data in the EEG. The algorithm may be applied universally to all EEG recordings. PMID- 1714816 TI - Electromyography and motor control. PMID- 1714815 TI - Comments on article by Hufschmidt et al. PMID- 1714817 TI - 'Excitability changes of muscular responses to magnetic brain stimulation in patients with central motor disorders. AB - The 'excitability' and 'conductivity' of motor pathways during transcranial stimulation (TCS) have been investigated in 49 patients affected by multiple sclerosis (34), amyotrophic lateral sclerosis (7), spino-cerebellar ataxia (3), primary lateral sclerosis (4) and brain metastasis (1). Hyper-reflexia, spasticity and weakness were correlated with the central motor conduction time (CCT) and with the threshold intensity of TCS required to produce a motor evoked potential (MEP). MEPs to magnetic TCS were recorded from hand and foot muscles during relaxation, contraction and after tendon vibration. Thresholds and CCTs of the patients were compared with those of 30 healthy controls. Increased threshold was found in 37 out of 49 patients (75.5%). Prolongation of the CCT was found in 38 out of 63 clinically affected upper limbs (60.3%) and in 56 out of 77 clinically affected lower limbs (72.7%). Absent motor responses to maximal TCS were found in 20 out of 98 lower limbs (20.4%). Excluding ALS patients (in whom there was a lower threshold for MEP elicitation), a significant linear correlation was found between prolonged CCT and increased threshold. While MEPs with prolonged CCTs have elevated TCS threshold, it is important to note that an elevated threshold was found in 14 out of 49 patients (28.5%) despite unchanged CCT. Spasticity and/or hyper-reflexia were more frequently associated with increased threshold than with prolonged CCT, while weakness was correlated equally well with both these parameters. In this respect magnetic TCS proves to represent a new tool for the detection of abnormal 'excitability' of the central motor tracts. PMID- 1714818 TI - Motor potentials evoked by magnetic stimulation of the motor cortex in normal subjects and patients with motor disorders. AB - Motor evoked potentials (MEPs) elicited by magnetic coil stimulation of motor cortex were studied at rest and during maximum voluntary muscle contraction in 20 normal subjects and 42 patients with motor disorders. MEP parameters employed in this study included: onset latency, amplitude, MEP/M wave amplitude ratio and background EMG/MEP area ratio. Maximum voluntary contraction increased the amplitude of MEPs compared to the size of M waves elicited by peripheral nerve stimulation. A reduced MEP/M wave amplitude ratio had a higher correlation with pyramidal tract involvement than did a prolonged MEP onset latency. Analysis of MEP parameters may help in the differential diagnosis of cerebral infarction, ALS and cervical spondylotic radiculomyelopathy. The inhibitory period which follows MEPs during voluntary contraction was observed in all subjects; the mean duration in normal subjects was 126.6 +/- 29.5 msec. The mean duration of the inhibitory period in patients with cerebral infarction, ALS and cervical spondylotic radiculomyelopathy was 73.9 +/- 41.7 msec, 79.5 +/- 54.5 msec and 85.1 +/- 36.5 msec, respectively. These values were significantly shorter than in normal subjects. PMID- 1714819 TI - Spinal motor neuron excitability during the silent period after cortical stimulation. AB - During tonic voluntary muscle contraction, a period of electromyographic silence follows the motor evoked potential produced by transcranial stimulation of the contralateral motor cortex. We studied the silent period in the wrist flexors of 3 normal volunteers and a deafferented patient during 20% of maximal contraction. To test the excitability of the spinal motor neuron pool during the period of silence, the H-reflex was evoked in the normal subjects at different intervals after cortical stimulation. The amplitude of the H-reflex in the silent period was expressed as a percentage of the amplitude during complete muscle relaxation. The H-reflex was profoundly depressed at the beginning of the silent period (13.5 27% of the control measurement), but showed a clear tendency to recover toward the end of the silent period despite continued absence of muscle activation (71 84% of the control). Moreover, the silent period in the deafferented patient was of longer duration than can be accounted for by segmental mechanisms. These findings imply, at least in the late part of the silent period, that a reduction in the excitability of the spinal motor neuron pool plays only a minor role in determining the phenomenon and that it is probably caused by lack of cortical drive. PMID- 1714820 TI - Electrophysiological correlates of postural instability in Parkinson's disease. AB - Postural reflexes in response to sudden toe-up tilts of a supporting forceplate platform were studied in free-standing patients with stage III (N = 5) and stage IV (N = 5) Parkinson's disease and compared to 5 age- and sex-matched normals. Latencies of the short (SL), medium (ML) and long latency (LL) responses were normal in the patients. The normalized mean amplitudes of the ML responses were significantly increased only in the stage IV Parkinson patients (P less than 0.0005). The distal-proximal activation sequence of LL responses observed in all 5 controls was reversed in 1 of the stage III and 4 of the stage IV Parkinson patients. Both the enlargement of the ML response (P less than 0.01) and the inverted activation sequence of LL responses (P less than 0.01) were significantly correlated with severity of the disease. The data establish an association between abnormal modulation of postural reflexes in the lower extremity and clinically rated balance impairment in Parkinson's disease. PMID- 1714821 TI - Reaction times recording methods: reliability and EMG analysis of patterns of motor commands. AB - Different methods for estimating reaction times (RTs) from either finger flexion or finger extension responses have been evaluated. The onset of finger movement was recorded with a photoelectric method and the results are compared with RT measures based on microswitch closure or onset of electromyographic (EMG) activity in the prime move muscle. EMG analysis showed the voluntary motor commands to present a characteristic ballistic pattern in RTs. However, this was not true for a number of trials with unusually long RTs which involved ramp or double burst EMG patterns that were interpreted as reflecting errors in the force calibration of motor commands. RTs based on photoelectric recording of onset of finger extension were consistently related to the RTs estimated from EMG onset in the prime mover muscle. It is concluded that the EMG onset or the finger lift photoelectric method is best suited for reliable RT recording. PMID- 1714822 TI - Mechanical implications of paired motor unit discharges in pathological and voluntary tremor. AB - Paired motor unit discharges (PDs), i.e., two discharges of the same motor unit (MU) with short interval, and their relation to tremor amplitude were studied in the first dorsal interosseous muscle (FDI) of patients with parkinsonian or essential tremor and in normal subjects mimicking tremor. In both pathological and voluntary tremor, interdischarge intervals of PDs were inversely correlated with the amplitude of the subsequent tremor beat, i.e., PDs with shorter intervals were followed by higher tremor beats. To further assess their mechanical impact, PDs were simulated by applying paired stimuli to MUs of the FDI in normal subjects using intramuscular microstimulation. Following paired stimuli, summation of twitch responses was more than linear. This was also the case with the paired stimuli applied at a tremor-like repetition rate (5 Hz). These findings stress the importance of PDs for tremor expression. PMID- 1714823 TI - Neuromagnetic fields accompanying unilateral and bilateral voluntary movements: topography and analysis of cortical sources. AB - Movement-related magnetic fields (MRMFs) accompanying left and right unilateral and bilateral finger flexions were studied in 6 right-handed subjects. Six different MRMF components occurring prior to, and during both unilateral and bilateral movements are described: a slow pre-movement readiness field (RF, 1-0.5 sec prior to movement onset); a motor field (MF) starting shortly before EMG onset; 3 separate "movement-evoked" fields following EMG onset (MEFI at 100 msec; MEFII at 225 msec; and MEFIII at 320 msec); and a "post-movement" field (PMF) following the movement itself. The bilateral topography of the RF and MF for both unilateral and bilateral movements suggested bilateral generators for both conditions. Least-squares fitting of equivalent current dipole sources also indicated bilateral sources for MF prior to both unilateral and bilateral movements with significantly greater strength of contralateral sources in the case of unilateral movements. Differences in pre-movement field patterns for left versus right unilateral movements indicated possible cerebral dominance effects as well. A single current dipole in the contralateral sensorimotor cortex could account for the MEFI for unilateral movements and bilateral sensorimotor sources for bilateral movements. Other MRMF components following EMG onset indicated similar sources in sensorimotor cortex related to sensory feedback or internal monitoring of the movement. The results are discussed with respect to the possible generators active in sensorimotor cortex during unilateral and bilateral movement preparation and execution and their significance for the study of cortical organization of voluntary movement. PMID- 1714824 TI - Percutaneous cervical stimulation: effects on intraspinal structures. AB - Changes in the amplitude of motor evoked potentials (MEPs) from percutaneous cervical stimulation (PCS) obtained at rest in the thenar muscles, and smaller than 0.8 mV, were studied under 3 different experimental conditions. A significant enhancement was observed mainly with a conditioning subthreshold transcranial stimulus and when MEPs were obtained in coincidence with weak voluntary contraction of the target muscle. Subthreshold stimulation of Ia fibers of the median nerve seemed to have a smaller facilitatory effect. It is generally accepted that PCS excites the spinal motoneuron (SMN) axons at the spinal nerve. However, our results show that other SMNs, usually not recruited, may be triggered by PCS when they receive excitatory postsynaptic potentials from the pyramidal tract (PT) or Ia fibers. This behavior suggests that low intensity PCS also exerts subthreshold excitation of the PT fibers and, perhaps, of the incoming spindle afferents, which adds its effects to the conditioning stimuli. PMID- 1714825 TI - Is there an age-dependent continuous increase in the duration of the motor unit action potential? AB - The duration of the motor unit potential plays an important role in the diagnosis of neuromuscular diseases, but is also subject to physiological variations. In order to evaluate the age-related changes the mean motor unit potential duration was studied in 4 proximal and distal muscles of the upper and lower extremities in 111 healthy subjects between 20 and 80 years. Contrary to the results of previous researches no marked increase of mean duration could be found in subjects younger than 55 years. Subjects older than 55 showed a slight tendency towards increased duration of the motor unit potential. These findings are interpreted as the result of changes in fiber density and are in accordance with single fiber EMG records. PMID- 1714826 TI - An objective method for assessing graded electrically evoked afferent activity in humans. AB - A problem arises in human sensorimotor studies when attempts are made to equate the intensity of electrical stimulation of a peripheral nerve with the amount of afferent activity generated. Results presented here reveal that sural recruitment curves exhibit large session to session differences both within and among subjects. These differences hinder the prediction of afferent activation based solely upon stimulus intensity. An alternative method based upon measurement of the evoked potentials is described for improving on these predictions. Sural nerve stimulation is used to demonstrate this method, but the method should be applicable wherever a peripheral nerve is accessible for electrical stimulation and evoked recordings. PMID- 1714827 TI - Prime mover muscle in finger lift or finger flexion reaction times: identification with transcranial magnetic stimulation. AB - When recording the onset of the electromyographic (EMG) voluntary response in reaction time (RT) studies, the electrodes should be placed on the muscle which is first and foremost involved in executing the response. It is thus necessary to identify which is the prime mover muscle among active synergic muscles. This has been investigated for index finger lift or flexion RTs by delivering a magnetic stimulus to motor cortical areas prior to the subject's voluntary response. The EMG responses to the magnetic stimulus were selectively facilitated either in the extensor indicis proprius muscle (in index lift RTs) or in the first dorsal interosseous muscle (in index flexion RTs). These effects are robust and provide a method for identifying the prime mover muscle in voluntary movements. PMID- 1714828 TI - Expression and regulation of growth hormone (GH) receptor messenger ribonucleic acid (mRNA) in rat adipose tissue, adipocytes, and adipocyte precursor cells: GH regulation of GH receptor mRNA. AB - The effects of hypophysectomy and hormonal replacement therapy on GH receptor (GH R) gene expression was studied in rat adipose tissue with a cRNA probe corresponding to the amino-terminal of the hepatic GH-R. Male Sprague-Dawley rats, 50-65 days of age, were used. In all fat depots tested (epididymal, retroperitoneal, and sc), two transcripts with an estimated size of 4.0 and 1.2 kilobases (kb), respectively, were detected. An intermediate-size transcript (2.6 kb) was sometimes observed. Also, isolated adipocytes and adipocyte precursor cells from the epididymal fat pad expressed these GH-R transcripts. The pituitary dependance of GH-R gene expression was analyzed in epididymal fat. Hypophysectomies were performed at 50 days of age, and the rats were then given replacement therapy with L-T4 (10 micrograms/kg.day) and hydrocortisone (400 micrograms/kg.day). Hypophysectomy decreased the abundance of both the 4.0 and the 1.2-kb transcripts, an effect that in part was restored by GH treatment. A solution hybridization RNase protection assay was then used to further characterize the effect of GH treatment of hypophysectomized rats on GH-R gene expression. A single injection of human GH (100 micrograms/rat) increased GH-R mRNA levels within 1 h, and maximal levels were reached between 3-12 h after the injection. The increase in GH-R mRNA levels was dose dependent and was observed also after prolonged treatment (1 or 5 mg/kg.day for 6 days) with bovine GH. These results confirm that GH-R mRNAs are present in rat adipose tissue from different fat depots. GH-R transcripts of the same estimated size were detected in isolated adipocytes and adipocyte precursor cells. Furthermore, the results show that there is a rapid and GH-dependent regulation of GH-R mRNA levels in adipose tissue. PMID- 1714829 TI - Regulation of insulin-like growth factor (IGF) binding proteins in transgenic mice with altered expression of growth hormone and IGF-I. AB - The insulin-like growth factors (IGFs) are present in extracellular fluids bound to specific, high affinity IGF binding proteins (IGFBPs). IGFBPs are believed to mediate IGF transport to tissues and to modulate their actions on target cells. To determine whether IGF-I can modulate IGFBP concentrations in blood and to distinguish the effects of IGF-I from those of GH, we assessed serum IGFBP concentrations in four genotypically distinct groups of sibling transgenic (Tg) mice that differed in respect to their expression of IGF-I and GH. This unique physiological situation was created by crossing IGF-I Tg mice to GH-deficient, dwarf mice in whom somatotrophs were genetically ablated by the expression of a diphtheria toxin transgene in the somatotrophs. Because both Tg mouse lines are hemizygous for their respective transgene, progeny of the cross differ genotypically, according to whether or not they carry one or both transgenes, and phenotypically in regard to their relative expression of IGF-I and GH. GH deficient mice showed a 15.7-fold decrease in serum IGF-I and a 5.5-fold decrease in serum IGFBP-3, but no change in a serum doublet band of 29,000 to 34,000 Mr, as assessed by ligand blotting. When IGF-I was expressed in the GH-deficient mice, serum levels of IGF-I and IGFBP-3 were 69% and 64% of those in normal sera, respectively. The 29,000 to 34,000 Mr doublet bands also increased. The ternary 150 kilodalton IGF-IGFBP complex, however, was not restored, presumably because IGF-I has no influence on the expression of the acid-labile subunit in this complex. In mice with IGF-I overexpression, serum IGFBP-3 was increased 2.1-fold and the sum of the 29,000 to 34,000 doublet bands was increased 2.9-fold. Immunoblotting showed that the changes in the 29,000 to 34,000 Mr forms observed by ligand blotting appeared to be predominantly due to changes in IGFBP-2. The results show that IGF-I can induce IGFBP-3 and IGFBP-2 independently of GH and that IGF-I is a major controller of these binding proteins. PMID- 1714830 TI - Parathyroid hormone and calcium: interactions in the control of renin secretion in the isolated, nonfiltering rat kidney. AB - The present study was designed to characterize the interaction of calcium and PTH in the control of renin release in isolated rat kidneys perfused in a closed circuit at constant flow. Kidneys were rendered nonfiltering using low perfusion pressures (70 mm Hg) and a hyperoncotic perfusate (100 g/liter BSA). Under these conditions, differences in perfusion pressure were less than 9 mm Hg between control and PTH-treated kidneys over the 50 min of perfusion. In the absence of PTH, renin release was inversely correlated with ionized calcium (Ca2+) concentration, with the highest release of renin noted with 1 mM EGTA and no added calcium. Also, verapamil treatment markedly elevated renin release, even in the presence of 2 mM Ca2+. In contrast, renin secretion was strongly depressed by 20 nM BAY-K8644 in the perfusate. In medium containing normal calcium concentrations (1 mM Ca2+), rat PTH(1-34) induced a 2-fold greater renin accumulation than in the control, non-PTH-treated kidneys. Isoproterenol induced a 5-fold stimulation under the same conditions. In the 0 Ca2+/1 mM EGTA perfusion, PTH did not elevate renin secretion. Renin release in response to PTH in 2 mM Ca2+ was similar to that observed in the 1 mM Ca2+ perfusion. PTH also reversed the effects of BAY-K8644 to suppress renin release. In verapamil-treated kidneys, PTH failed to stimulate renin release. These results indicate that PTH stimulates renin release by a process independent of the baroreceptors and macula densa. The Ca2+ modulation of PTH-induced renin release is consistent with the reported ability of PTH to block calcium channels and relax vascular smooth muscle. PMID- 1714831 TI - Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line. AB - The Madin-Darby bovine kidney cell line was used to examine regulation of insulin like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1 62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth. PMID- 1714832 TI - Thyroid adenocarcinomas secondary to tissue-specific expression of simian virus 40 large T-antigen in transgenic mice. AB - A hybrid gene comprising the bovine thyroglobulin gene promoter and the coding region for the simian virus-40 large T- and small t-antigens was used to generate 30 transgenic mice by microinjection into the pronuclei of single cell embryos. All animals except three developed, as single primitive pathology, a dramatic enlargement of the thyroid gland. Compression of trachea and esophagus, accompanied by dyspnea, inspiratory stridor, and dysphagia, led to a progressive cachexia and premature death attributed to respiratory failure. Despite the large thyroid volume, T4 levels were abnormally low, and the progression of the syndrome could be delayed by a substitutive treatment with thyroid hormones. The rapid evolution of the disease, leading to the death of most founder transgenic animals before the breeding age, prevented transmission of the transgene to their offspring. Only two transgenic lines are presently surviving. Immunohistochemical analysis of the tissues revealed a specific expression of the simian virus-40 antigens in the thyroid cells. Hyperplasia was already obvious at birth. Older animals displayed moderately to poorly differentiated thyroid adenocarcinomas. Electron microscopy revealed, however, the persistence of cell polarity and the presence of microfollicles between the densely packed cells. Cell lines derived from these large T-expressing thyroids were shown to have lost expression of both thyroglobulin and thyroperoxidase, while expressing low levels of TSH receptors. These transgenic mice could constitute an interesting model of aggressive adenocarcinoma, sharing phenotypical similarities with the anaplastic type of human thyroid tumors. PMID- 1714833 TI - The modulation of the expression of IL-6 and its receptor in human osteoblasts in vitro. AB - Interleukin 6 (IL-6) probably plays a central role in the acute phase response and in haemopoiesis and may be involved in the control of bone turnover. We have studied the release of IL-6 from human trabecular bone cells treated with a variety of stimuli using a specific bioassay. In serum free medium, unstimulated human osteoblast-like cells produced IL-6 in the range of 1000-2050 pg/ml/24 h. Recombinant human interleukin 1 (IL-1 alpha) (10(-13)-10(-11) M), tumor necrosis factor alpha (TNF alpha) (10(-9)-10(-7) M) and lipopolysaccharide (5-500 ng/ml) all stimulated release of IL-6 from human bone cells. Maximal levels of 17,000 pg/ml were observed using the highest concentration of IL-1. 1,25(OH)2D3 and PTH did not stimulate IL-6 release. Using a specific sheep antihuman IL-6 antibody, all IL-6 activity could be neutralized. In parallel studies, ROS 17/2.8 rat osteosarcoma cells released around 50 pg/ml of IL-6 under basal conditions which were increased to a maximum of 900 pg/ml by treatment with PTH (10(-9) M). The cytokines were less effective and 1,25(OH)2D3 again had no effect. Modulation of expression of IL-6 mRNA in human osteoblast cells was examined using a human complementary deoxyribonucleic acid probe. The mRNA was constitutively expressed, and IL-1 (10(-11) M) and TNF (10(-7) M) induced further mRNA expression within 2 h, which was sustained over 24 h. 1,25(OH)2D3 (10(-7) M), IL-6 (2000 pg/ml), and PTH (10(-9) M) exerted no effects at any time point. Dexamethasone (10(-6) M) suppressed both basal and IL-1- and TNF-induced IL-6 mRNA expression. IL-6 receptor mRNA was constitutively expressed but was not regulated by any of the above agents. It is clear that rodent and human osteoblasts differ in their production of IL-6 and its modulation. These data support the hypothesis that IL 6 is produced locally in human bone by osteoblasts under the direction of other cytokines. This could have implications in bone remodeling, haemopoiesis, and systemic responses to local injury. PMID- 1714834 TI - Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3, and 4) in the rat ovary. AB - We investigated the possible presence of the mRNAs encoding four insulin-like growth factor-binding proteins (IGFBP-1, -2, -3, and -4) in the rat ovary. It has been demonstrated previously by Northern analysis that adult rat ovaries contain mRNA transcripts for IGFBP-2 and -3. Here we show by Northern analysis that adult rat ovaries contain mRNA for IGFBP-4, but the mRNA for IGFBP-1 was below the limit of detection. Using in situ hybridization, IGFBP-1 mRNA was not detected in any of the ovaries tested. The IGFBP-2 mRNA was localized specifically in thecal interstitial cells (TIC) and secondary interstitial cells of all ovaries. The IGFBP-2 signal was very strong in TIC of all Graafian follicles (healthy and atretic) and very strong in all secondary interstitial cells located in different regions of the ovary, but weak in TIC of preantral follicles. The IGFBP-3 hybridization signal was localized specifically to some corpora lutea. Here the hybridization single for IGFBP-3 was strong and distributed throughout the corpus luteum, strong but localized to only some luteal cells, or not detectable. The IGFBP-4 mRNA was localized almost exclusively to granulosa cells of atretic follicles. The intensity of the IGFBP-4 signals appeared to increase as the level of atresia increased. In some cases, IGFBP-4 signals were detected in the TIC, but they were weak and variable. These results show that the mRNAs for IGFBP-2, 3, and -4 are localized to the interstitial cells, corpora lutea, and atretic granulosa cells, respectively. The tissue-specific synthesis of IGFBP subtypes in specialized ovarian cells provides an excellent system to study the manner in which IGFBP synthesis is controlled and their potential role as an autocrine and paracrine factors. PMID- 1714835 TI - The heterogeneity of human chorionic gonadotropin (hCG). II. Characteristics and origins of nicks in hCG reference standards. AB - hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG. PMID- 1714836 TI - The heterogeneity of human chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nicked hCG. AB - Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714837 TI - Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons. AB - An immortalized hypothalamic neuronal cell line was recently developed by genetically targeting the expression of the simian virus-40 large T-antigen in LHRH neurons. These GT1 cells were subcloned to GT1-1, GT1-3, and GT1-7 cells, and they have been shown to express the mRNA for pro-LHRH and secrete LHRH-like immunoreactive (IR) materials into the media. The purpose of our study was to biochemically and immunologically characterize the IR materials within and secreted from these cells. Both LHRH- and GnRH-associated peptide (GAP)-like IR materials were present and were secreted from these four cell lines. Up to 3% of the total cellular protein was composed of LHRH and GAP materials. When materials from the cell lysate and media were separated according to mol wt (Mr), at least three different pro-LHRH species were detected. These precursors contained both LHRH- and GAP-like IR determinants, and they eluted in the void volume and at approximately 10,000-12,000 and 8,400-8,500 Mr. A material that contained GAP like IR eluted at approximately 6,500-6,800 Mr. This species is probably mouse GAP-(1-56) because it eluted on a reverse phase column in the approximate position of rat GAP-(1-56). Cell lysates contained a single LHRH-like IR form which coeluted on a size-exclusion column with synthetic LHRH. This material stimulated secretion of LH from anterior pituitary cells in a dose-response manner. By comparison, two different molecular forms of LHRH were detected in media at approximately 1,500 and 540 Mr. HPLC analyses revealed these peaks to be heterogeneous and to contain at least (Gln1)LHRH-(Gly11,Lys12,Arg13), (Gln1)LHRH (Gly1,Lys12), LHRH-(Gly11), and LHRH. These experiments demonstrate that the cells contain and secrete multiple molecular forms of the pro-LHRH and that processing of the prohormone must involve 1) cleavage by an endopeptidase to give GAP-(1-56) and a C-terminally extended LHRH, 2) removal of C-terminal basic amino acids by a carboxypeptidase, 3) amidation of LHRH-(Gly11) to LHRH, and 4) cyclization of glutamine to pyroglutamate at the N-terminal of LHRH. These results provide the first evidence for intermediates in the metabolic pathway of pro-LHRH to LHRH. PMID- 1714838 TI - Luteinizing hormone induces progesterone receptor gene expression in cultured porcine granulosa cells. AB - We have examined the effect of LH on the regulation of the progesterone receptor (PR) in cultured porcine granulosa cells. In this study we used the RNase protection assay to evaluate the PR mRNA levels with a porcine cDNA clone isolated by the polymerase chain reaction (PCR) method. This clone was regarded as part of the porcine PR cDNA because of its 98.3% and 95.7% homology to the hormone-binding domain of human PR cDNA in amino acid and nucleotide sequences, respectively. Treatment with LH (500 ng/ml) increased porcine PR mRNA to a maximum level of 8.6 +/- 1.1-fold (mean +/- SE) after 3-h exposure. This induction was mimicked by (Bu)2cAMP as well as by FSH and hCG, and the increased PR caused by LH and (Bu)2cAMP occurred in a dose-dependent manner. Basal and LH induced PR mRNA levels were not affected by progesterone (100 ng/ml), estrogen (100 ng/ml), and RU 486 (10 ng/ml) at 3 h. The mechanism of the increased PR mRNA levels was studied in the presence of actinomycin-D and cycloheximide. While inhibition of RNA synthesis with actinomycin-D blocked LH-induced PR mRNA expression, inhibition of protein synthesis with cycloheximide increased basal and LH-induced PR mRNA levels. These results indicate that the expression of PR mRNA is positively regulated by LH, and this induction does not require ongoing protein synthesis. There may be a cycloheximide-sensitive mechanism that modulates PR mRNA stability. From our results we suspect that progesterone modulates ovarian function through LH-induced PR in granulosa cells. PMID- 1714839 TI - Human galanin: molecular cloning reveals a unique structure. AB - Galanin, a novel neuropeptide/hypothalamic hormone originally identified and isolated by virtue of its carboxy-terminal amide group, has recently been shown to have a diverse range of biological activities, including potent effects on the secretion of insulin and growth hormone. The physiological role of galanin remains unclear, with different effects being observed when porcine and rat galanin have been used in various animal model systems and in human studies. Molecular cloning of cDNA encoding human galanin and galanin mRNA associated peptide (GMAP) from both pituitary and neuroblastoma sources has revealed a unique and unexpected structure. In contrast to porcine, bovine and rodent galanin, human galanin lacks a carboxy-terminal amide. By analogy to other neurohormones, the absence of carboxy-terminal amidation would be expected to have significant effects on functional properties such as affinity for different receptor subtypes and physiological half life, and may be responsible for the species specificity observed in the action of galanin. PMID- 1714840 TI - Predictive value of ventricular premature beats for subsequent ischaemic heart disease in apparently healthy subjects. AB - From 1978 to 1980, 260 healthy subjects, 40-79 years of age, underwent 24 h ambulatory electrocardiography in order to determine the prevalence and complexity of ventricular premature beats (VPBs) in adults without apparent heart disease. The number of types of VPBs seem in 5% or less were considered 'abnormal' and the present follow-up study undertaken in order to assess the significance of such 'abnormal' VPBs as predictors of subsequent ischaemic heart disease (IHD). Information concerning cardiac events within the follow-up period was available in 237 subjects. Nine were lost to follow-up and 24 refused clinical examination. IHD was documented in 13 (eight myocardial infarction, five angina pectoris). 'Abnormal' VPBs occurred in six out of 13 (46%) who later developed IHD compared to only 24 out of 213 (11%) without IHD (P less than 0.001). The presence of either more than 900 VPBs 24 h-1 or ventricular tachycardia of more than three beats, identified five out of 13 patients with IHD (sensitivity 38%), whereas 210 out of 213 with no evidence of IHD at follow-up were identified (specificity 98%). Four out of seven who initially had more than 900 VPBs 24 h-1 had IHD on follow-up. Our results have demonstrated a strong positive association between 'abnormal' VPBs observed in a random 24-h electrocardiographic recording of apparently healthy subjects 40-79 years of age and subsequent IHD. They also suggest that a 24-h ECG may be useful for the assessment of coronary risk even in asymptomatic subjects. PMID- 1714841 TI - Site of anti-inflammatory action of dexamethasone in rabbit skin. AB - Systemic pretreatment of rabbits with dexamethasone results in a time-dependent inhibition of oedema responses caused by intradermal injection of both 'direct acting' and 'neutrophil-dependent' stimuli. Local injection of actinomycin D, an inhibitor of RNA synthesis, inhibits this effect. Our studies suggest that a major action of dexamethasone in this model may be a local inhibition of increased permeability of the vascular endothelium. PMID- 1714842 TI - [Atmospheric air pollution and its prevention]. PMID- 1714843 TI - Ultrastructural localization of basic lysine-rich proteins during the nucleologenesis in preimplantation mouse embryos. AB - The paper describes nucleologenesis in preimplantation mouse embryos studied by electron microscopy after staining with ethanolic phosphotungstic acid (E-PTA), a substance which binds to basic lysine-rich proteins and makes it thus possible to follow their localization and distribution. A nucleolar precursor body (NPB) homogeneously stained with E-PTA first appears in pronuclei of a fertilized oocyte. In 2-cell stage embryos, the staining intensity of the central part of NPB is reduced, whereas the outer zone retains the PTA positivity. In the course of further development, reticulation takes place in the peripheral zone and all structures of a functional nucleolus are gradually formed. Tiny spherical bodies, called "remnant NPBs", can still be observed in reticular nucleoli of cells at the morula stage. The similarities in reaction and morphological appearance of the NPB and the corresponding structures in oocytes of the mouse and rat antral follicle are discussed. PMID- 1714844 TI - American Academy for Cerebral Palsy and Developmental Medicine, annual meeting 1991. Abstracts. PMID- 1714845 TI - The effects of ovine growth hormone on protein turnover in rainbow trout. AB - Ovine growth hormone (oGH) was administered to rainbow trout via an intraperitoneal cholesterol implant. After 21 days, plasma oGH levels were recorded as control group, less than 2 ng ml-1, i.e., not detectable, and oGH group, 19.2 +/- 2.8 ng ml-1. oGH-treated fish exhibited significantly increased whole-body growth rates, whole-body protein accretion rates, stimulated tissue protein synthesis, and tissue protein accretion rates. A dramatic decrease in white muscle protein concentration was also observed after oGH treatment. In some tissues (liver and stomach), elevated protein synthesis rates were the result of higher RNA/protein ratios. However, in other tissues (gill and ventricle), increased RNA activity accounted for the differences in rates of protein synthesis. The growth promoting effects of oGH on both whole-body and tissue protein turnover were generally accompanied with no change in the efficiency of deposition of newly synthesized protein. For the same ration size, the oGH group showed higher retentions of ingested nitrogen. It is concluded that oGH significantly enhances whole-body growth rates as a result of the stimulatory effect on protein synthesis rates with little effect on protein degradation. PMID- 1714846 TI - Partial characterization of an 89-kDa highly immunoreactive protein from Chlamydia psittaci A/22 causing ovine abortion. AB - An 89-kDa immunogen from Chlamydia psittaci A/22 causing ovine abortion was partially characterized. The 89-kDa protein, localized on the outer membrane complex of chlamydiae, was synthesized relatively early in the developmental cycle. The protein contained cysteine but was not extensively cross-linked by disulfide bonds. Treatment with proteases apparently did not cleave the protein. The infectivity of strain A/22 was partially (60%) reduced by treatment of chlamydial elementary bodies with monoclonal antibody BS/89 specifically reacting with the 89-kDa antigen. Species-specific as well as strain-specific antigenic determinants were present on the 89-kDa protein. PMID- 1714847 TI - [Homology between the Lpm system of allotypes in the American mink and the Gp system of allotypes in the domestic pig]. AB - The 5 alpha-macroglobulin allotypes alpha M1, alpha M2, alpha M3, alpha M4 and alpha M5 were identified in pig. The alpha M1 allotype was reported as a marker of pig alpha-macroglobulin, the latter being homologous to alpha 2-macroglobulins in human and in mink. The allotypes alpha M2-alpha M5 were specified as markers of the second isotypical variant of pig alpha-macroglobulins, which was homologous to mink Lpm macroglobulin (alpha 1M). As seen from data obtained by International Comparative Test ISABR 87-88, alpha M1 is a new allotype, while allotypes alpha M2--alpha M5 correspond to four allotypes in the Gp system (Janik et al.). Based on these data, a conclusion was made on the homology between the Lpm system in american mink and the Gp system in pig. Since the allotypes studied are the part of alpha-macroglobulins, a locus controlling them was designated the AM locus. We also find it more advantageous to apply the same name to the homologous locus in mink, instead of the Lpm used earlier. Genetic control of 5 allotypes was studied and the structure of the AM locus in pig analysed in detail. Comparative study of organization of the above locus and the homologous locus in mink was carried out. PMID- 1714849 TI - [4th Haemophilia symposium of the German Democratic Republic with international participation. Dresden, 22-25 November 1989]. PMID- 1714848 TI - An 'axis-like' material in the centromeric region of metaphase-I chromosomes from mouse spermatocytes. AB - This study reports the persistence of axis-like structures in the centromeric region of both homologues during the metaphase-I and anaphase-I stages of meiotic division of mouse spermatocytes. A novel type of silver 'argentaffin' technique (NH4-Ag) is employed. This technique includes the treatment of glutaraldehyde fixed tissues with dilute ammonium hydroxide followed by a reduction of aldehyde groups with sodium borohydride. Staining is accomplished with ammoniacal silver nitrate in darkness followed by sulfite washing. The lateral elements of synaptonemal complexes and the single chromosomal axes of diplotene spermatocytes show a prominent reactivity with this technique. The pattern of very small grains over condensed chromatin is uniform and gives only a light opacity to the electron beam. The presence of an axis-like structure is seen in every centromeric end of meiotic chromosomes at metaphase I and anaphase I. The chromatin (heterochromatin) that surrounds the centromeric filament and some material distributed in irregular linear arrays along some of the homologues also showed a higher electron opacity than the bulk of deoxyribonucleoprotein. While the former is related to C+ heterochromatin, the latter could represent dispersed material of diplotene axes. It is suggested that the disposal of axial material is differentially delayed at the centromeric regions. The present evidence supports the hypothesis that axial fragments or lateral-element segments persisting at these regions contribute to the cohesiveness of centromeres of sister chromatids during normal disjunction. PMID- 1714850 TI - [Joint status after prophylactic substitution. An investigation in hemophilic children and adolescents in the DDR]. AB - The joint condition in 98 children and adolescents with severe or moderate types of haemophilia participating in a summer camp between 1987 and 1989 had been analyzed. This was done by the centers' report and on the other hand with testing the patients by aid of the score system recommended by the World Federation of Hemophilia. According to the centers' reports more than 50% of all patients seem to be free of joint contractures. But according to the more sensitive score system more patients are affected by discrete symptoms of the chronic arthropathy. There had been discovered big differences between the centers and an increase of the patient's score by age. Not seldom an impairment of the findings within a single year was recognized. The joint problems in haemophiliacs are solved by the prophylactic treatment in an incomplete manner only. Lacks in the sophisticated evaluation of joints, with the management of prophylactic treatment as well as adjustment to prevention lead to avoidable damage crippling the patient on later age. PMID- 1714851 TI - Prevention of haemophilic arthropathy. AB - Early and adequate treatment of haemarthroses, will prevent or lessen future arthropathy. A prospective double blind controlled study of three different dose regimes of Factor VIII in the treatment of haemarthroses of the knees, elbows and ankles has shown that site and severity affect the initial response and should dictate the dose. High risk bleeds can be identified by the degree of movement loss and the presence of pain at rest, and tenderness. Reassessment of bleeds will indicate those not responding. Early recognition of delayed restoration of function requires an analysis of the normal processes. A study has revealed that the mean time for complete restoration of function was 3.6 +/- 3.3 days for elbows, 2.5 +/- 2.1 for knees and 1.1 +/- 0.9 for ankles. Departure from these norms should signal the need for early physiotherapeutic intervention. PMID- 1714852 TI - Synoviorthesis with 198Au colloid gold in haemophilia patients. A preliminary report. AB - In 1988-1989 fifteen patients with severe haemophilia A and recurrent bleedings into the knee joint, aged from 19 to 44 years were treated by an intraarticular injection of 198Au colloid gold. So far 10 of them were assessed after 6 months follow-up. In 6 cases cesseation and in 2 cases reduction in number and volume of bleedings were observed. Only in 2 patients the frequency of haemarthroses remained unchanged. No significant difference in tracer uptake was observed between pretreatment 99mTc-pertechnate gamma scans of the knee joints and controls completed 6 months after the radiogold injections. It is worthy to stress the lower costs of the 198Au synoviorthesis as compared with surgical synovectomy of the knee joint. The radioisotope method is also much less traumatic to the patient than the surgical one. PMID- 1714853 TI - [Arthrosonography--a modern method for morphologic and functional joint evaluation in hemophilia]. AB - The arthrosonography is a new depicting procedure for detection of destructing joint processes in haemophilia. A direct evaluation of the cartilage, of the border between cartilage and bone, of the synovial membrane with proliferative processes as well as the differentiation between soft-tissue bleeding and haemarthros are possible. An essential advantage is given by a combined morphological and functional joint evaluation. The diagnosis of joint instabilities can be recognized easier by sonography than by X-ray. Clinical findings are completed in an excellent manner and lesions of bone and cartilage are detected earlier by arthrosonography. PMID- 1714854 TI - [One-year analysis of bleeding events in 223 hemophiliacs in the DDR]. AB - The reports of 223 haemophiliacs living in the districts Halle, Leipzig, Karl Marx-Stadt, and Gera had been analyzed for bleeding events in 1986. There were suffering from haemophilia A 185, from haemophilia B 36 and 2 persons from a complex disorder. In each case 73 patients suffer from a severe and a moderate type of haemophilia. In 55 patients the diagnosis was mild form of haemophilia and in 22 subhaemophilia. Altogether 1802 bleeding episodes had been registered with the following locations: elbow joint 21.0%, knee joint 17.9%, ankle 14.2%, shoulder 4.3%. In 5% of all cases multiple joints had been affected at the same time. Soft tissue bleedings occurred in 16.7% of all cases. For substitution treatment of these 1802 bleeding events altogether 9020 transfusion units were needed, appropriate to 40.4 per year and 52.4 per year and bleeding patient respectively. Expense and duration of treatment depend on the bleeding location. Conclusions for the clinics and the transfusion service are possible. PMID- 1714855 TI - Genetics of plasma concentration of von Willebrand factor. AB - The plasma concentration of von Willebrand factor (vWf) shows a very wide range in individuals without bleeding disorders. In a twin study we found that 60% of the variance of the plasma concentration of vWf is due to genetic factors. Individuals with AB0 blood group 0 have a lower concentration of vWf than individuals with blood group A, B or AB. Thirty percent of the genetic variance was due to an effect of the AB0 locus. Since the Lewis substances show great structural similarity to the ABH blood group substances we compared the vWf concentration in individuals with and without the Lea antigen on the red cell surface. Individuals lacking the Lea antigen had a lower vWf concentration than individuals who had this antigen. Le(a+b-) people are nonsecretors and Le(a-b+) people are secretors of ABH substance. The lowest vWf concentration was found in blood group 0 secretors. Both the AB0 locus and the Secretor locus may be major loci for the determination of the plasma concentration of vWf. PMID- 1714856 TI - Disappearance of factor VIII antibodies upon frequent administration of factor VIII. AB - 20 haemophilia patients known to have antibodies against F VIII for at least more than three years were treated on a regular base with 25 U/kg b.w. F VIII every other day. All 5 patients with previous maximal anti F VIII antibody levels between 5 and 60 BU/ml showed a decrease of antibody level and normal F VIII recovery within 1-2 months. From 12 patients with previous antibody levels above 60 BU/ml, 8 showed a disappearance of antibodies within 2-26 months. In 3 patients in whom no previous highest inhibitor level was known, one was treated successfully. Another group of 6 young patients in whom an inhibitor against F VIII had just (less than 3 months) developed, was treated with F VIII as soon as an inhibitor was detected. The dose infused was 25 U/kg b.w. F VIII twice weekly. In 5 patients this regimen was successful within 1-7 months. In the 6th patient the dosage was increased to every other day. One year after the beginning of therapy no inhibitor was detectable. So our results show that regular administration of F VIII in intermediate or low dose can lead to rapid disappearance of anti F VIII antibodies especially in patients with moderate inhibitor levels. PMID- 1714857 TI - Anti idiotypic recognition of auto and allo antibodies to factor VIII by immunoglobulin preparations from single blood donors and large plasma pool. AB - Therapeutic immunoglobulins from commercial origin were administered to patients with allo and auto antibodies to factor VIII. The protocol used was similar to the prescription suggested for ITP, 0.4 g/kg body weight for five consecutive days. In patients with auto antibodies two kinds of effects were observed: A direct interaction between IVIg and the factor VIII inhibitor and long term interaction on antibody synthesis. The direct interaction observed in vivo was demonstrated to be mediated through Fab'2 fragments of both idiotypic interaction. This was also demonstrated using binding experiments and affinity chromatography experiments: Fab'2 fragments of IVIg were coupled to sepharose and Fab'2 fragments of patients IgG with allo and auto antibodies to Factor VIII were circulated through the column. The recovery of anti Factor VIII antibodies after elution was possible in 2 cases of allo antibodies out of 3 tested and 3 cases of auto antibodies out of 4. The question of anti idiotypes against factor VIII inhibitors in single individuals was investigated. IgG and Fab'2 fragments from single blood donors were isolated and tested for their inhibitory capacity against Fab'2 fragments of three different auto antibodies. The results did not show statistical differences between males and females however in the group of male blood donors, aging was a positive parameter for the presence of anti idiotypic antibodies against factor VIII inhibitors. Anti idiotypic antibodies against factor VIII inhibitors were present in 17.2% of the blood donor tested. PMID- 1714858 TI - Inhibitors to factor IX contain all IgG subclasses except IgG3. AB - Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development. PMID- 1714859 TI - [Antibodies to anticoagulants in rheumatic autoimmune diseases]. AB - The systemic lupus erythematosus (SLE) and the rheumatoid arthritis (RA) as the classic autoimmune diseases exhibit a great number of autoantibodies. Some of them are anticoagulants. Besides inactivating inhibitors against single coagulation factors interfering anticoagulants are known, belonging to the group of anti-phospholipid antibodies and detected as the lupus anticoagulants or anticardiolipin antibodies. Anti-phospholipid antibodies 184 patients with SLE or RA had been checked for. An enzyme immuno assay was used for detection of the anti-cardiolipin antibodies. The relations between occurrence of the anti cardiolipin antibodies and vascular processes as well as other immunologic parameters had been tested for clinical relevancy. PMID- 1714860 TI - Evaluation of wet pasteurization of a factor VIII concentrate produced by controlled-pore silica adsorption. AB - In the routine production of a factor VIII concentrate (produced by adsorption of contaminating proteins in cryoprecipitate to controlled-pore silica and concentration of the factor VIII effluent by ultrafiltration) the terminal dry heat treatment has been replaced by pasteurization in the liquid state. High effectivity of this procedure with respect to virus inactivation was demonstrated using a variety of both lipid- and protein-enveloped model viruses, including HIV. Pair-wise quality control of dry-heated and pasteurized product revealed no significant differences, except in the composition of the formulation buffer. In a clinical study in which 17 patients with haemophilia A participated the pasteurized product was well tolerated and in vivo recovery and half-life of factor VIII were in the same (normal) range as found for the dry-heated counterpart. PMID- 1714861 TI - Cryoprecipitate of intermediate purity produced in a closed thaw-siphon system from DDAVP stimulated blood donor plasma. AB - The effects of intravenous administration of DDAVP to blood donors and the use of DDAVP plasma for the production of cryoprecipitate in the closed thaw-siphon system were evaluated. DDAVP treatment produced on the average a 3.2-fold rise in plasma levels of factor VIII. Von Willebrand factor antigen increased to a lesser extent. Cryoprecipitate prepared from 220-280 ml aliquots of DDAVP stimulated donor plasma contained 472 +/- 210 units of factor VIII and 276 +/- 130 units of von Willebrand factor antigen. The average yield of factor VIII was 57% of that in the prefrozen plasma. The specific activity of factor VIII in cryoprecipitate was 0.77 +/- 0.44 U/mg protein, comparable to that for intermediate purity concentrates. Thus, by the use of DDAVP and the thaw-siphon technique it is possible to produce cryoprecipitate 4-7 times as potent as conventionally manufactured preparations. PMID- 1714862 TI - [Thrombogenicity of prothrombin complex concentrates]. AB - PCC's were isolated either by adsorption to DEAE-Sephadex A-50 (particle size 40 120 microns; Pharmacia Uppsala) or by adsorption to Molselect DEAE-50 (particle size 100-320 microns; Reanal Budapest) from human citrated plasma after separation of factor VIII by cryoprecipitation. The two products obtained (without heparin) were both compared by in vitro (NAPTT, TGt50) and in vivo (venous stasis) model in rats acc. to WESSLER) tests. The agent prepared by adsorption to DEAE-Sephadex A-50 showed a significantly higher thrombogenicity than that prepared by Molselect DEAE-50. ED50 values for thrombus formation to amounted 15 Factor IX U/kg for the first product and to 210 factor IX U/kg for the second. PMID- 1714863 TI - [Contribution to the mechanism of the "factor VIII bypassing activity" (FEIBA) reaction]. AB - The principle of the FEIBactivity hadn't been defined in a sufficient manner up to now. Probably the factor VII or factor VIIa are playing a key role. According to own experiments the factor VII activity increases considerably in activated prothrombincomplex concentrates together with the quotient factor VIIa/VII. Factor VII should be administered in his activated form for bypassing. Fractions with limitation of the activation process to factor VII are of a low thrombogenicity. Among activated coagulation factors factor VIIa persists longest in the circulation and a bypass efficacy can be provoked only by this. PMID- 1714864 TI - [Activated prothrombin complex preparations for the treatment of anticoagulant hemophilia. Preparation--method of operation--supply possibility]. AB - The mechanism of the bypass activity of prothrombin complex preparatives for treatment of haemophiliacs with circulating anticoagulants remains unclear up to now. The following parameters had been tested with preparatives produced in a different manner (absorption by DEAE-Sephadex A 50, Molselekt A 50, tricalcium phosphate or aluminium hydroxide): Coagulation factors II, VII, IX, X and XII, activated factors VIIa and Xa as well as the FEIB activity. The bypass activity is strongly correlated to the factor VIIa content of the preparatives, however. These results agree with the statements by HEDNER and KISIEL, which had used a factor VII a concentrate for treatment of haemophilia complicated by circulating anticoagulants with success. According to farther investigations factor VII is correlated with the lipid content of the starting plasma. This knowledge is important for optimizing the production procedure of prothrombin complex preparatives with bypassing activity. PMID- 1714865 TI - [Paradoxical bleeding as a complication of the treatment of hemophilia with factor VIII and factor IX preparations]. AB - Paradoxical bleedings are complications occurring under replacement therapy in haemophiliacs by disturbancies of the primary haemostasis. They have been observed during treatment with factor-VIII- and prothrombin-complex concentrates of long duration and in high dosage. Clinical complications, for example delayed wound healing as well as spontaneous bleedings into the skin and from the mucous membranes, have been observed in one quarter of haemophiliacs under substitution therapy. In one third of these patients pathological parameters of primary haemostasis (prolonged bleeding time, reduced retention, retraction, ADP- and collagen-induced aggregation and the platelet factor 3 release) were found out. The following mechanisms or substances may be the cause for these disturbancies: 1. fibrinogen and factor-VIII split products 2. high content of proteins predominantly fibrinogen and factor-VIII-related antigen 3. antigen-antibody reactions 4. development of inhibitors against the Willebrand factor. For treatment of the paradoxical bleedings freshly prepared cryoprecipitate, prednison and Etamsylatum have been used. PMID- 1714866 TI - Genomic diagnosis of haemophilia A and B in the German Democratic Republic. AB - Since 1986 the genomic diagnosis of haemophilia A and B in the GDR is realized as a national programme. Until Aug. 1989 56 families at risk of haemophilia A are analysed using RFLPs of different intragenic and intergenic probes (BclI/F8e 16 19, KpnI-XbaI/int 22, TaqI/St 14.1). 117 out of 162 females at risk being heterozygous were identified as carriers, in 40 cases the carrier state was excluded, and in 5 females the data were not informative. Prenatal diagnosis was offered to 93 carriers in reproductive age. Six genomic prenatal diagnoses in haemophilia A were performed. In four patients different partial deletions of factor VIII:C gene were found. 10 families of haemophilia B were analysed using intragenic and intergenic probes (P 1; pX58dIIIc). 14 females were identified as carriers, 11 were excluded. The application of direct and indirect gene diagnosis in haemophilia is discussed. PMID- 1714867 TI - Diagnosis of carriers of haemophilia A by RFLP analysis: Austrian results. AB - RFLP analysis with extragenic and intragenic probes was performed in 25 Austrian families with haemophilia A for the purpose of carrier diagnosis. Diagnosis could be established in 22 families, in 7 families with intragenic and extragenic probes, in 15 families with extragenic probes only. The remaining 3 families were not informative. During routine diagnosis we found three interesting cases which are described in the following paper. Firstly, we identified one deletion with Bcl I/p 114.12. Secondly, we observed an additional polymorphic band (5.2 kb) with Xba I/probe A. Thirdly, we saw one case where Bcl I/p 114.12 and Bgl I/1.8 were not in linkage disequilibrium. PMID- 1714868 TI - Genomic carrier detection and prenatal diagnosis of haemophilia A in families at risk using the polymerase chain reaction (PCR). AB - The polymerase chain reaction (PCR) was applied in genomic analysis of families at risk for haemophilia A using the intragenic Bel I and Hind III polymorphism in introns 18 and 19, respectively, of factor VIII gene. For the latter the primers derived from exon 19 and 20 sequences allowed to amplify the whole intron 19 resulting in a 730 bp fragment. Hind III restriction of this fragment provides polymorphic fragments of 250 bp or 160 bp and 90 bp respectively. An also occurring 480 bp fragment can be used as internal control to circumvent misdiagnosis due to incomplete or failure of restriction. The Hind III polymorphism was successfully used in prenatal diagnosis of an affected male in the first trimenon of pregnancy. Fetal sexing was also performed by PCR technique using Y specific primers. PMID- 1714869 TI - [Malondialdehyde formation by platelets of hemophiliacs]. AB - The platelets of haemophilic patients produce after stimulation with thrombin (5 NIH-units) a significantly reduced amount of malondialdehyde in comparison to the platelets of healthy children of the same age. There is a positive correlation between the platelet count in citrated whole blood and malondialdehyde production in the group of healthy children, however the same correlation is negative in adults and strongly negative in haemophiliacs. Because of the 24 hour-intervals between the last substitution and investigation in the majority of haemophilic patients, the reduced MDA-production of their platelets seems to be the result of side effects of the administration of plasma fractions. On the other hand, the reduced capacity for the MDA-production of the platelets of haemophiliacs can be explained as the result of the release reaction of platelets after haemostasis activation following bleedings. PMID- 1714870 TI - [Recommendations for a medical-meteorologic prognosis for hemophilia]. AB - A one year lasting study about the relation between bleedings in haemophiliacs and weather was performed. There had been included 1802 bleeding events. The influence of meteorologic factors to bleeding was proved. The influx of warm air proved inopportune. The decrease in atmospheric pressure as well as winter cold fronts lead to increase of bleeding tendency. Haemophiliacs should avoid physical and psychical stress in such unconvenient atmospheric situations. The replacement therapy should be adapted to the weather situation. PMID- 1714871 TI - Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication. AB - Serum and liver HBsAg bear an inverse relation to each other during the evolution of chronic hepatitis B virus infection and the quantity of HBsAg in tissue rises gradually with time. In this study, intracellular and extracellular levels of HBsAg were measured by radioimmunoassay in primary culture of hepatocytes from 30 patients with chronic hepatitis B virus infection to determine a possible relationship with hepatitis B virus replication. Serum levels of HBsAg correlated with markers of active viral replication (serum hepatitis B virus DNA, p less than 0.005, and tissue HBcAg, p less than 0.02) but inversely with tissue HBsAg (p less than 0.05). In similar fashion, in vitro export of HBsAg was also related to the presence of active viral replication markers (serum hepatitis B virus DNA, p less than 0.02, and tissue HBcAg, p less than 0.05) and negatively with tissue HBsAg (p less than 0.001). Export of HBeAg also correlated positively with markers of active viral replication (serum hepatitis B virus DNA, p less than 0.05 and tissue HBcAg, p less than 0.05). Further experiments indicated that intrahepatic pre-S1 and pre-S2 correlated closely with intrahepatic HBsAg, indicating that a failure to export HBsAg was unlikely to be attributable to deficient intracellular expression of pre-S1 or pre-S2. These data indicate that in vitro primary hepatocyte culture of hepatitis B virus-infected cells provides an accurate reflection of in vivo export of HBsAg and that this is closely related to the presence of active viral replication. PMID- 1714872 TI - Increased expression of intercellular adhesion molecule 1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis. AB - It has been suggested that immunological mechanisms involving lymphocyte-mediated damage are important in the characteristic bile-duct damage that occurs in primary biliary cirrhosis and primary sclerosing cholangitis. Because adhesion is necessary for the interaction of lymphocytes with their target structures, we have studied the expression of intercellular adhesion molecule 1, a ligand for the leukocyte adhesion receptor lymphocyte function-associated antigen 1 in the liver of patients with primary biliary cirrhosis and primary sclerosing cholangitis. Strong expression of intercellular adhesion molecule 1 was seen on interlobular bile ducts and proliferating bile ductules in both conditions. In primary biliary cirrhosis, medium-sized ducts, which are spared by the disease, were negative. Minimal bile-duct staining was seen in conditions in which bile duct damage is not a major feature, such as nonbiliary cirrhosis and acute liver diseases. In patients with cirrhosis from any cause, strong expression of intercellular adhesion molecule 1 was detected on the periseptal hepatocytes adjacent to new connective tissue. The intensity of immunohistochemical staining was recorded using a semiquantitative visual scoring system that was subsequently validated quantitatively by confocal laser scanning microscopy. The expression/induction of intercellular adhesion molecule 1 on bile ducts may be important in the pathogenesis of bile-duct damage in primary biliary cirrhosis and primary sclerosing cholangitis and is further evidence to support an immune pathogenesis in these two conditions. Furthermore, the induction of intercellular adhesion molecule 1 on hepatocytes may be an important factor in the liver-cell damage and fibrosis that occur during the development of cirrhosis. PMID- 1714873 TI - Distribution of dihydrolipoamide acetyltransferase (E2) in the liver and portal lymph nodes of patients with primary biliary cirrhosis: an immunohistochemical study. AB - The reason for the close association between primary biliary cirrhosis and the appearance of antibodies that recognize the E2 component of pyruvate dehydrogenase complex is not understood. The distribution of the three pyruvate dehydrogenase complex subunits was examined in the liver and lymph nodes of patients with primary biliary cirrhosis, patients with other liver diseases and normal subjects by immunohistochemistry using affinity-purified antibodies. Intensity of staining was assessed semiquantitatively and validated by scanning laser confocal microscopy. In primary biliary cirrhosis tissue, the E2 staining pattern did not parallel the reported distribution of mitochondria. E2 staining in biliary epithelial cells was consistently stronger than in hepatocytes. In primary biliary cirrhotic liver, staining of biliary epithelium was significantly stronger than in normal or other liver disease controls; many bile ducts in primary biliary cirrhotic liver demonstrated very high intensity, diffuse distribution of stain. No differences in staining intensity were seen between perivenular hepatocytes in primary biliary cirrhotic liver and those in controls; periportal hepatocytes in primary biliary cirrhotic liver were, however, more intensely stained than perivenular cells. In primary biliary cirrhotic portal lymph nodes, a subset of macrophages showed high-intensity, diffuse distribution of stain. By contrast, staining with antibodies to E1 and E3 (other components of pyruvate dehydrogenase complex) produced uniform-intensity, mitochondrial distribution both in primary biliary cirrhosis and control tissue. The increased intensity of E2 in primary biliary cirrhotic tissue could be explained in terms of abnormal metabolism of E2 by biliary epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714874 TI - Movement of IgM antibody from blood to bile in rats. AB - The movement from blood to bile of passively injected autologous IgM antibody against horse erythrocytes was studied in rats. Both native and neuraminidase treated antibody entered bile intact, with the peak titers for both measured between 60 and 90 min after injection. A small part (0.38%) of the injected dose of native antibody and 1.05% of the asialo-IgM antibody appeared in bile over 24 hr. These recoveries represented only a small fraction of the activity, which apparently disappeared from serum over the period. The pathways used by IgM antibody to enter bile were partially assessed. Prior injection of fetuin was found to abrogate the biliary secretion of native antibody and to significantly reduce the recovery of asialo-antibody in bile. In contrast, the presence of asialofetuin reduced (but not significantly) the secretion of native antibody and markedly changed the kinetics of appearance of asialo-IgM activity in bile. Some candidate routes to bile can be excluded as unlikely. The parahepatocellular pathway to bile canaliculi appears uninvolved because the secretion of antibody is slow and the inhibitory effects of fetuin argue against secretory component mediated transport. Instead, two secretory pathways appear to be present. The first appears to be available to both native and asialo-IgM antibody, and may involve the peribiliary capillary plexus. The second route to bile is available to asialo-IgM antibody alone and is probably initiated by binding to the hepatic asialoglycoprotein receptor. PMID- 1714875 TI - An in situ hybridization, molecular biological and immunohistochemical study of hepatitis delta virus in woodchucks. AB - The presence of hepatitis delta virus genomic RNA and hepatitis delta antigen was investigated in woodchuck liver and extrahepatic tissues by in situ hybridization using synthetic radiolabeled probes, Northern-blot analysis and immunohistochemical staining for hepatitis delta antigen. Hepatitis D virus RNA and hepatitis delta antigen were detected in the nuclei of infected hepatocytes but in none of the other tissues examined. Northern-blot analysis of total cell RNA confirmed these findings and revealed a series of hepatitis D virus transcripts, including full-length genomic RNA and dimers and trimers of hepatitis D virus RNA that may represent replicative intermediates. Use of single stranded probes showed genome-size monomers and dimers to be both of genomic and antigenomic polarity, although dimers were found to be predominantly antigenomic. These findings document the strict hepatotropism of hepatitis D virus and support the rolling-circle model of genome replication for this unique, defective RNA virus. PMID- 1714876 TI - Structure-function relationship of immunosuppressive drugs: a cautionary tale. PMID- 1714877 TI - Two monoclonal antibodies identify antigens preferentially expressed on normal human breast cells versus breast cancer cells. AB - In order to obtain antibodies with specificity toward normal mammary epithelial antigenic determinants, we immunized BALB/c mice with normal milk cells and screened the hybridomas against an undifferentiated breast cancer cell line H466B, peripheral blood lymphocytes and normal fibroblasts. Two hybridomas were generated, which produced BA6 (IgG1) and CA4 (IgM) monoclonal antibodies (MAbs). These MAbs did not react with 5 breast cancer cell lines. In cryostat sections of normal human breast tissue, BA6 was reactive with 6/6 and CA4 was reactive with 12/13 specimens both showing an apical staining of epithelial cells. Conversely staining of malignant cells in breast cancer biopsies was observed in 4/33 specimens with BA6 and in 4/19 specimens with CA4. Computerized image analysis (SAMBA) of immunostained sections showed homogeneous distribution of staining, with a high percentage of stained cell surfaces in normal breast (mean percentages of positive surfaces : BA6 : 75% and CA4 : 82%) while, in malignant samples, staining was heterogeneous, with a mean percentage of positive surface of 25% for BA6 and 12% for CA4. Both MAbs reacted strongly with human milk fat globule membranes (HMFGM) and skimmed milk. FPLC size exclusion chromatography of skimmed milk showed that CA4 and BA6 reactive materials eluted in distinct peaks in high molecular weight ranges. Electrophoretic separation of HMFGM followed by CA4 staining detected a high molecular weight reactive band (Mr 380-600 kDa). CA4 and BA6 reactivity was reduced by protease treatment of the antigen but was not affected by neuraminidase digestion, by methanol extraction or by Na metaperiodate oxidation. After perchloric acid treatment of HMFGM, BA6 activity was lost while the CA4 activity was found in the soluble fraction. The results reported suggest that the two MAbs identify two distinct novel epitopes of normal breast cells. PMID- 1714878 TI - Production and characterization of monoclonal antibodies to purified deglycosylated cystic fibrosis respiratory mucin: evidence for the presence of four immunologically distinct epitopes. AB - Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of three Cystic Fibrosis (CF) patients. The mucins were completely deglycosylated by treatment with trifluoromethanesulfonic acid and subsequent treatment with alpha-N-acetylgalactosaminidase. Over thirty hybrid clones secreting antibodies against the deglycosylated mucin (DGM) were obtained using standard hybridoma techniques. Hybrids with positive identification for CF-DGM were cloned twice using limiting dilution method to ensure the monoclonal nature of the antibodies. Eight stable clones (1a, 1b, 10a, 10c, 10d, 10e, 29d, and 30e) secreting monoclonal antibodies (MAbs) showing specificity of reaction to CF-DGM were obtained. Two clones, 29d and 30e, secreted antibodies of the IgM class while the other six clones secreted antibodies of the IgG1 subclass. Denaturation and reduction experiments suggested that MAbs 1b, 10e, 29d and 30e were directed against a given sequence of amino acids in the DGM while the other four MAbs, in addition to being sequence specific, were also conformation dependent. Further, competitive binding radioimmunoassays suggested that MAbs 1b, 10e, 29d and 30e recognize four distinct epitopes in the peptidic core of CF respiratory mucin. In summary, the MAbs may provide a promising approach to elucidate the structure of the polypeptide backbone of human respiratory mucins as well as for the screening of cDNA libraries for clones secreting mucin(s). PMID- 1714879 TI - Preparation of monoclonal antibodies to the beta A subunit of ovarian inhibin using a synthetic peptide immunogen. AB - Seven different synthetic peptides were prepared corresponding to regions of the beta A subunit of 32 KDa human ovarian inhibin predicted to contain possible continuous B cell epitopes. These were coupled to tuberculin as a carrier and used to immunize mice previously given a priming dose of human tuberculosis vaccine. Only one of these peptides, corresponding to sequence 82-114, consistently gave good titres of antibodies reactive with intact 32 KDa bovine inhibin by ELISA. From one of the mice immunized with this peptide, six stable hybridomas were prepared. Five of these secreted an IgM and the sixth (clone E4) secreted an IgG2b antibody. Immunoblotting experiments on follicular fluid concentrates, after treatment with sodium dodecyl sulphate and mercaptoethanol, showed strong reactivity of the antibody from clone E4 only with bands of about 13 kDa and 58 kDa corresponding to forms of the beta A subunit previously described. This monoclonal antibody, and an antibody to the alpha subunit previously made in this laboratory using synthetic peptides, should prove useful reagents for further study of inhibins and activins. PMID- 1714880 TI - Ultrastructural localization of gangliosides, calcium and a high-affinity Ca(2+) ATPase in nerve terminals: a contribution to the possible functional role of gangliosides. AB - By means of newly developed electron microscopical techniques (electron spectroscopic imaging, ESI; electron energy loss spectroscopy, EELS; immunogold labelling) a specific accumulation of endogenous calcium within the synaptic cleft and a distinct localization of a high-affinity Ca(2+)-ATPase at the inner sides of the pre- and postsynaptic membrane of nerve cells from fish brain have been demonstrated. Additionally, a differentiation-dependent expression of polysialoganglioside epitopes on the outer surface of nerve terminals in clustered arrangements was demonstrated using their ultracytochemical detection by means of the monoclonal antibody Q211. These results which are in agreement with parallel biochemical investigations on modulatory effects of exogenous gangliosides on a high-affinity Ca(2+)-ATPase in the CNS of vertebrates support our hypothesis that Ca(2+)-ganglioside complexes act as modulators for the processes of synaptic transmission and long-term neuronal adaptations. PMID- 1714881 TI - Mechanism of host cell membrane modification by tumour: a model for paraneoplastic syndromes. AB - A not well-appreciated but clinically important aspect of malignant tumours is their effects on distantly located host cells. The effects, termed paraneoplastic syndromes, also pose an intriguing mechanistic problem: how do malignant cells influence properties of host cells not in contact with them? Erythrocytes from the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma exhibit higher agglutinability with concanavalin A (Con A) than the cells from normal animals. Since the tumour and the red cells are not in contact, the enhanced agglutinability of the latter is a paraneoplastic effect. The mechanism by which the tumour brings about this effect is investigated as a model for paraneoplastic syndromes. The cell-free ascites fluid is able to impart high agglutinability on cells from normal animals in vitro. Also, when injected intraperitoneally in normal animals, the ascites fluid is able to enhance the agglutinability of erythrocytes in circulation. Apparently the tumour produces a substance(s) that appears in the ascites fluid and is able to diffuse into circulation, explaining the mechanism by which it can reach distant sites. From the cell-free ascites fluid three fractions have been isolated that are active in vitro. Of these, only one showed activity in vivo. From this fraction, a glycoprotein has been purified to homogeneity that confers maximal Con A-agglutinability on normal erythrocytes at 8 x 10(-7)M, at which concentration 6,400 molecules bind per cell. The protein has a molecular weight of 600 kDa in the native state and a pI of 5.35. It is made up of 4 identical subunits of Mr 170,000. It is detected in the plasma of tumour-bearing but not normal rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714882 TI - Production of antibodies to dextran using liposome as adjuvant. AB - The kinetics and nature of the immune response to dextran-BSA entrapped into liposomes was studied in rabbit. The antibody titer in the secondary immunization was found to be higher as compared to that in the primary response. In the primary response antibodies were of both IgG and IgM type but following secondary immunization the IgG level was increased. The conjugate entrapped into liposomes could establish an immunological memory for dextran. In order to eliminate the carrier effect of BSA, dextran was either entrapped or coupled to liposome and the immune response was studied in mice. The results revealed that dextran entrapped into liposomes or coupled to liposome surface induced IgM type immune response and antibody titer did not increase on secondary immunization. PMID- 1714883 TI - Report on the first international workshop on carcinoma-associated mucins. PMID- 1714884 TI - Characterization of the new bladder cancer cell line HOK-1: expression of transitional, squamous and glandular differentiation patterns. AB - The new continuous cell line HOK-1 derived from a grade-III transitional-cell bladder carcinoma with foci of squamous and glandular differentiation was shown to retain this phenotypical heterogeneity for more than 45 passages in vitro. Electron microscopy revealed transitional as well as a considerable proportion of squamous carcinoma and adenocarcinoma cells. PAS-positive mucus was detected in numerous cells. These features were principally maintained when grown as multicellular spheroids and in nude mice. More pronounced signs of differentiation (i.e., expression of cytokeratins 10 and 11, formation of glandular structures) were found in xenograft tumours. Independently, cytokeratins 13, 18 and 19 were detected in vitro and in vivo, reflecting the urothelial origin. The line forms distinct colonies in soft agar, expresses Lewis x and Lewis-y antigens and reacts with monoclonal antibodies (MAbs) against CEA, beta-HCG and URO-5. Cytogenetic analysis revealed several related clones with a rearrangement at chromosome 1 and loss of one X chromosome as common karyotypic changes in all clones. DNA content, as quantified by image analysis, showed a DNA stemline close to 2c. The new cell line HOK-1 can be used as an in vitro model to study the mechanisms of heterogeneous differentiation patterns in bladder cancer. PMID- 1714885 TI - A double-blind crossover comparison of flecainide and slow-release mexiletine in the treatment of stable premature ventricular complexes. AB - In 24 patients with stable premature ventricular contractions (PVCs) greater than or equal to 100/h, Lown class greater than or equal to 2 the relative anti arrhythmic efficacy of flecainide 150 mg twice daily and slow-release mexiletine 360 mg twice daily was evaluated in a double-blind placebo-controlled randomized crossover study. All the patients had normal ventricular function. Criteria of efficacy were: reduction greater than or equal to 70% of PVCs or reduction greater than or equal to 50% with abolition of Lown class greater than 2 arrhythmias or suppression of non-sustained ventricular tachycardias (nSVT). Twenty-two patients completed the study protocol. The placebo phases showed comparable results and no carry over effect. The criteria of efficacy were fulfilled in 20 of the 22 patients (91%) on flecainide and in 12 of the 22 (55%) on mexiletine. The absolute reductions of PVCs, couplets and nSVT obtained on flecainide and mexiletine, in comparison to the placebo, were statistically significant (p less than 0.01 for flecainide, p less than 0.05 for mexiletine). Flecainide was superior to mexiletine in overall PVC reduction (p less than 0.05). In the 17 patients with couplets the reduction obtained with flecainide was superior to mexiletine (p less than 0.05). Both drugs were highly effective on nSVT. At steady state, the mean plasma levels of both drugs were within the range of clinical efficacy. The drugs were well tolerated and no patient withdrew because of side-effects. It was concluded that at the dosages employed flecainide was superior to mexiletine in reducing premature ventricular contractions and in abolishing couplets. The efficacy of both drugs for non-sustained ventricular tachycardias was comparable. Both drugs were highly effective by comparison with the placebo. PMID- 1714886 TI - Toxicity trials of amsacrine (AMSA) and etoposide +/- azacitidine (AZ) in childhood acute non-lymphocytic leukemia (ANLL): a pilot study. AB - Recurrent or induction therapy-resistant ANLL carries a grave prognosis. The combination of AMSA at 100 mg/M2 daily for 5 days and etoposide at 200 mg/M2 daily for the first 3 days of therapy was given to 40 patients with refractory ANLL. An additional 17 patients received those two agents plus azacitidine at a dosage of 250 mg/M2 on days 4 and 5. All three drugs were given as one-hour infusions. All patients had normal electrolyte determinations daily and were on cardiac monitors during the period of drug administration. No arrhythmias were detected in 522 doses of AMSA. Toxicities observed were primarily related to myelosuppression. Forty-nine of the 57 patients required hospitalization for suspected or proven infection. Nausea/vomiting and mucositis were the next most commonly occurring toxicities. Responses were seen in 22 patients. PMID- 1714887 TI - Phase II evaluation of fludarabine phosphate in patients with central nervous system tumors. A Southwest Oncology Group trial. AB - Twenty-three patients with malignant central nervous system tumors were treated with fludarabine phosphate (2-FAMP) on a 5 day bolus schedule. One brief partial response was observed in 20 malignant astrocytoma patients. 2FAMP as given in this protocol is inactive in previously treated patients with recurrent malignant astrocytomas. PMID- 1714888 TI - [Congenital poikiloderma syndrome with early childhood blister formation (Brain syndrome) and unusual associated neurologic symptoms]. AB - We report on an 8-year-old girl with manifestations of congenital poikiloderma during her first year of life. Macroscopically, there was reticular teleangiectasia on cheeks and thighs, generalized de- and hyperpigmentation, dry skin with pityriasiform scaling and milia as a result of former blister formation. Histologically and ultrastructurally cytoid bodies, probably of keratinocyte origin, were observed. Associated findings were leucocytopenia, hyperlipoproteinaemia, spastic ataxia and lack of teeth. There is consanguinity in the family, but 3 sisters, the parents and the ancestors were completely healthy. Because of transient blister formation on the face, upper arms and elbows we would like to classify our case as Brain syndrome. PMID- 1714889 TI - An alternative method of teaching strain/counterstrain manipulation. AB - The range-of-motion box enables the operator to visualize how to carry a joint through extremes of motion range for range-of-motion evaluations and to locate the position of comfort in relaxation of tender points. It is helpful for teaching the novice counterstrain manipulation and for instructing fellow practitioners. Examples are illustrated for the cervicothoracic region, but the concept is applicable to other parts of the body as well. PMID- 1714890 TI - Application of acute phase reactants during antemortem and postmortem meat inspection. PMID- 1714891 TI - Premature ventricular contractions and apparent hypertension during anesthesia in an ostrich. AB - Premature ventricular contractions and apparent hypertension were seen in an adult ostrich anesthetized with isoflurane. The ostrich had septic joints and was anesthetized to allow joint lavage. The premature ventricular contractions occurred at a rate of 1 to 2/min, with a brief period of 12 to 15/min, and were not treated with any antiarrhythmic drugs. Normal blood pressures for awake or anesthetized adult ostriches are not readily available, but blood pressures in this bird were higher than in other ostriches measured with the same technique. Systolic pressures ranged from 199 to 249 mm of Hg, diastolic pressures from 107 to 177 mm of Hg, and mean pressures were from 165 to 220 mm of Hg during isoflurane anesthesia of approximately 45 minutes' duration. Recovery from anesthesia was complicated, although the ostrich died 12 days later from mycotic pneumonia. PMID- 1714892 TI - Visualization: contributions and disclaimers. PMID- 1714893 TI - A paradigm for the next millennium: health information science. AB - Although historically a major concern of both the artist and the scientist was the observation of nature, the two disciplines split when science became more wedded to mathematics and quantification. Today, with visualization, art and science can again together provide a view of the natural world. A prototype curriculum for a new multidisciplinary science--Health Information Science- incorporates aspects of computer science, cognitive psychology, bioengineering, biomedical visualization, medicine, dentistry, anthropology, mathematics, library science, and the visual arts. PMID- 1714894 TI - Evidence that a G-protein transduces signals initiated by the protein-tyrosine kinase v-Fps. AB - The protein-tyrosine kinase (PTK) v-Fps induces protein kinase C (PKC)-dependent expression of the transformation-related 9E3 gene in chicken embryo fibroblasts (Spangler, R., Joseph, C., Qureshi, S.A., Berg, K., and Foster, D.A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7017-7021). We present evidence here that a GTP binding protein (G-protein) is a component of this PKC-dependent signaling pathway. 1) A GTP analogue that stimulates G-protein-mediated signals induced 9E3 gene expression. 2) A GDP analogue that inhibits signaling through G-proteins inhibited expression of 9E3 and phosphorylation of a 67-kDa PKC substrate induced by v-Fps. The GDP analogue had no effect on phosphorylation of the PKC substrate or the expression of 9E3 induced by direct activation of PKC with phorbol ester. 3) Increased v-Fps PTK activity led to increased GTP binding to a 50-kDa protein. The molecular weight of this GTP-binding protein is consistent with the molecular weight of alpha-subunits of G-proteins of the heterotrimeric class. The data suggest that a G-protein functions upstream from PKC in a signaling pathway that connects v-Fps PTK activity to increased 9E3 gene expression. PMID- 1714895 TI - Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli. AB - The ubiquitin (Ub)-conjugating enzyme E2(25K) catalyzes the synthesis of multi-Ub chains in which successive Ub units are linked by an isopeptide bond involving the epsilon-amino group of Lys-48 of Ubn, and the COOH-terminal Gly residue of Ubn+1 (Chen, Z., and Pickart, C. M. (1990) J. Biol. Chem., 265, 21835-21842). We now describe the polymerase chain reaction (PCR)-based cloning of an E2(25K) encoding cDNA from a bovine thymus library, using degenerate oligonucleotide primers based on the sequences of two E2(25K) peptides. The cDNA encodes a 200 residue protein whose sequence bears similarities of 66 and 59%, respectively, to the sequences of the Ub-conjugating enzymes encoded by the UBC1 and UBC4/UBC5 genes of the yeast Saccharomyces cerevisiae. These three yeast E2s play key roles in Ub-dependent proteolysis (Seufert, W., McGrath, J. P., and Jentsch, S. (1990) EMBO J. 9, 4535-4541). Comparison of the amino acid sequence of E2(25K) with other known E2 sequences strongly suggests that Cys-92, one of two E2(25K) Cys residues, forms the Ub thiol ester adduct that is an intermediate in E2-catalyzed multiubiquitination. The E2(25K)-encoding cDNA was overexpressed in Escherichia coli, and the recombinant E2(25K) protein was purified to electrophoretic homogeneity; enzymatic assays showed that its multiubiquitinating activity was quantitatively identical with that of the native protein. The availability of a cloned cDNA will allow us to assess the physiological role of E2(25K). PMID- 1714896 TI - Interaction of pp60c-src, phospholipase C, inositol-lipid, and diacyglycerol kinases with the cytoskeletons of thrombin-stimulated platelets. AB - Phosphatidylinositol (PtdIns)-4- and -3-kinases, PtdIns(4)P-5-kinase, diacylglycerol (DAG) kinase, and PtdIns-phospholipase C were all detected in cytoskeletons of resting human platelets. The total cytoskeletal enzyme activities were greatly increased upon thrombin stimulation of the intact cells. Those reached a maximum after a 60-s stimulation for PtdIns(4)P-5-kinase and phospholipase C, while the other kinases appeared to be slightly delayed. Specific activities were stimulated from about 4-fold (PtdIns-3-kinase) to about 6-fold (PtdIns-4-kinase). Thrombin treatment also promoted a co-extraction of pp60c-src with the cytoskeletons and its disappearance from the Triton X-100 soluble fraction. These results suggest that stimulation of platelets by thrombin causes the association of enzymes responsible for lipid phosphorylation and hydrolysis with the cytoskeletons. This could occur at cytoskeleton anchoring points to the membranes. PMID- 1714898 TI - The protein HC chromophore is linked to the cysteine residue at position 34 of the polypeptide chain by a reduction-resistant bond and causes the charge heterogeneity of protein HC. AB - Protein HC, an extremely charge-heterogeneous lipocalin, carries a yellow-brown fluorescent chromophore of unknown structure covalently bound at an unidentified site of its polypeptide chain. Two chromophore-carrying peptides with 60% of the chromophore material (defined as material absorbing light at 330 nm) were isolated from pepsin-digested native human protein HC by reversed-phase high performance liquid chromatography. Sequence analysis of these peptides indicated that the chromophore was bound to the cysteine residue at position 34 of the protein HC polypeptide chain. Sequence analysis of a native chromophore tripeptide complex, isolated from pronase digests of the pepsin-produced peptides, identified the sequence Thr33-X34-Pro35, corroborating the position of the chromophore linkage. Quantitative amino acid analysis of the hydrolyzed, performic acid-oxidized, chromophore-tripeptide complex demonstrated approximately equal amounts of threonine, cysteic acid, and proline in the complex. Reduction and carboxymethylation of the native chromophore-tripeptide complex did not remove the chromophore from the peptide. The absorption spectrum of the chromophore-tripeptide complex was similar to that of native protein HC, implying that all of the heterogeneity of protein HC resides in its chromophore. PMID- 1714897 TI - Expression and cyclic AMP-dependent regulation of a high affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR). AB - The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5 fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation. PMID- 1714899 TI - A recombinant cDNA derived from human brain encodes a DNA binding protein that stimulates transcription of the human neurotropic virus JCV. AB - The human neurotropic virus JCV contains a 98-base pair repeat enhancer/promoter sequence that confers glial-specific transcription to the viral early and late promoters. The central region of this repeat, designated the B-domain, binds to a glial-derived nuclear protein that stimulates transcription of the viral promotor in vitro. We now report the isolation of a recombinant cDNA clone, termed glial factor-1 (GF1), from a brain expression library that encodes a novel protein which interacts with the JCV B-domain. Results from RNA studies indicate that the GF1 transcript is more abundant in brain than in other tissues and that the level of GF1 RNAs increases progressively during brain development. Cotransfection of the recombinant GF1 expressor plasmid with JCV promoters indicates that GF1 stimulates transcription of the JCV late promoter and to a lesser extent the JCV early promoter predominantly in cells of human glial origin. Thus, GF1 is a sequence-specific DNA binding protein that may play a role in determining the glial-specific expression of JCV. PMID- 1714900 TI - The alpha-adrenergic stimulation of atrial natriuretic factor expression in cardiac myocytes requires calcium influx, protein kinase C, and calmodulin regulated pathways. AB - It has been shown recently that alpha-adrenergic agonists can stimulate atrial natriuretic factor (ANF) expression in ventricular cardiac myocytes; however, little is known about the intracellular signals mediating this activation. The present study focused on the potential roles of calcium-regulated kinases and calcium influx in the alpha-adrenergic stimulation of ANF gene expression in ventricular myocardial cell cultures. Myocardial cells maintained for 48 h in serum-free medium supplemented with phenylephrine (PE) possessed up to 15-fold higher levels of ANF peptide and ANF mRNA than control cells. The removal of PE, or the addition of nifedipine, resulted in a rapid decline in ANF expression, suggesting that the sustained elevation of some intracellular messenger (e.g. calcium and/or phospholipid hydrolysis products) was required for the adrenergic response. The calcium channel agonist BAY K 8644 was capable of increasing ANF expression in a nifedipine-sensitive manner; however, unlike PE, it did not stimulate phosphoinositide hydrolysis. The protein kinase C inhibitor, H7, caused an approximate 75% reduction in PE-stimulated ANF expression, but had no effect on BAY K-stimulated expression. W7, a calcium/calmodulin inhibitor, completely blocked the effects of both PE and BAY K 8644. The addition of either H7 or W7 24 h after the PE addition resulted in a decline of ANF expression. These results indicate that alpha-adrenergic agonists augment ANF gene expression through at least two pathways, one that is H7-sensitive, perhaps involving the sustained activation of protein kinase C, and the other that is W7-sensitive, perhaps involving the sustained activation of calmodulin-regulated kinases. Further, it appears that BAY K 8644-mediated increases in ANF expression are independent of protein kinase C activation and dependent on calmodulin-regulated events. PMID- 1714901 TI - Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily. AB - gp49 is a Mr 49,000 glycoprotein expressed on the surface of mouse bone marrow derived mast cells, which are progenitors for the major in vivo mast cell subclasses, typified by intestinal mucosal mast cells and serosal mast cells. The amino-terminal amino acid sequence of gp49 was determined after isolation of the solubilized membrane protein by affinity chromatography with the B23.1 anti-gp49 monoclonal antibody. Redundant oligonucleotides were used to isolate a full length 1.3-kilobase cDNA from a mouse mast cell library. The predicted amino acid sequence contains a signal peptide of 23 residues, an extracellular domain of 215 residues with three potential sites of N-linked glycosylation, a transmembrane domain of 23 residues, and a cytoplasmic tail of 42 residues. Hybridization of the gp49 cDNA was limited to mRNA extracted from those cell types that also bound the B23.1 monoclonal antibody as assessed by cytofluorographic analyses. The predicted extracellular domain of gp49 contains two regions of 48 and 51 amino acids, each flanked by cysteine residues. Both regions meet criteria for being C2 type domains of the immunoglobulin superfamily based upon the alignment of consensus amino acids and their predicted secondary structure organization. Thus, gp49, a membrane glycoprotein preferentially expressed by the progenitor mast cell population, is a new member of the immunoglobulin superfamily. PMID- 1714902 TI - The high affinity Fc epsilon receptor gamma subunit (Fc epsilon RI gamma) facilitates T cell receptor expression and antigen/major histocompatibility complex-driven signaling in the absence of CD3 zeta and CD3 eta. AB - The T cell receptor (TCR) is a molecular complex formed by at least seven transmembrane proteins: the antigen/major histocompatibility complex recognition unit (Ti alpha-beta heterodimer) and the invariant CD3 chains (gamma, delta, epsilon, zeta, and eta). In addition to targeting partially assembled Ti alpha beta CD3 gamma delta epsilon TCR complexes to the cell surface, CD3 zeta appears to be essential for interleukin-2 production after TCR stimulation with antigen/major histocompatibility complex. The gamma chain of the high affinity Fc receptor for IgE (Fc epsilon RI gamma) has significant structural homology to CD3 zeta and the related CD3 eta subunit. To identify the functional significance of sequence homologies between CD3 zeta and Fc epsilon RI gamma in T cells, we have transfected a Fc epsilon RI gamma cDNA into a T cell hybridoma lacking CD3 zeta and CD3 eta proteins. Herein we show that a Fc epsilon RI gamma-gamma homodimer associates with TCR components to up-regulate TCR surface expression. A TCR composed of Ti alpha-beta CD3 gamma delta epsilon Fc epsilon RI gamma-gamma is sufficient to restore the coupling of TCR antigen recognition to the interleukin 2 induction pathway, demonstrating the functional significance of structural homology between the above receptor subunits. These results, in conjunction with the recent finding that CD3 zeta, CD3 eta, and Fc epsilon RI gamma are coexpressed in certain T cells as subunits of an unusual TCR isoform, suggest that Fc epsilon RI gamma is likely to play a role in T cell lineage function. PMID- 1714903 TI - Evidence for a molecular distinction between Golgi and cell surface forms of beta 1,4-galactosyltransferase. AB - beta 1,4-Galactosyltransferase (GalTase) is present on the plasma membrane of many cell types in addition to its traditional location within the Golgi compartment. Recently, the GalTase gene has been shown to encode two proteins that are identical throughout their length except that one has an additional 13 amino acid extension in its amino-terminal cytoplasmic domain. We present evidence here suggesting that the longer GalTase protein, containing this unique 13-amino acid peptide, is preferentially targeted to the plasma membrane, and the shorter GalTase protein resides primarily within the Golgi compartment. S1 nuclease protection assays of RNA from a variety of cells and tissues show that the relative abundance of the short and long GalTase mRNAs correlates with GalTase-specific activities in the Golgi and plasma membranes, respectively. Furthermore, transfection of cDNAs encoding either the long or short GalTase protein into F9 embryonal carcinoma cells suggests that the long GalTase protein is preferentially expressed on the cell surface. These results propose a molecular distinction between the Golgi and cell surface forms of GalTase as well as a novel mechanism for targeting glycoproteins to the cell surface. PMID- 1714904 TI - Missense mutation serine106----proline causes 17 alpha-hydroxylase deficiency. AB - Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities. We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam. We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast. Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities. Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities. An HhaI restriction site created by the mutation should permit screening of large populations. PMID- 1714905 TI - TIK, a novel serine/threonine kinase, is recognized by antibodies directed against phosphotyrosine. AB - We have isolated cDNAs encoding kinases from a murine pre-B cell line by screening a lambda gt11 cDNA expression library with anti-phosphotyrosine antibodies. One cDNA was identified to encode the previously isolated tyrosine kinase c-lyn. Among the remaining clones, we have characterized a cDNA encoding a novel kinase which we have designated TIK. Sequence analysis of this cDNA indicates that the TIK enzyme lacks the features thought to be conserved among protein tyrosine kinases. Although isolated on the basis of its reactivity with the anti-phosphotyrosine antibody, the TIK enzyme was found to have only serine and threonine kinase activity. The amino-terminal portion of the TIK protein contains a cdc2 phosphorylation consensus sequence. Three mRNA transcripts derived from the TIK gene are detected in a variety of adult murine tissues. PMID- 1714906 TI - Differentiation of lipid-associating helices by use of three-dimensional molecular hydrophobicity potential calculations. AB - Several types of lipid-associating helices exist: transmembrane helices such as in receptor proteins, pore-forming helices in ion channel proteins, fusion inducing peptides in viral proteins, and amphipathic helices such as in plasma apolipoproteins. In order to propose a classification of these helices according to their molecular properties, we introduce the concept of molecular hydrophobicity potential for such helical segments. The calculation of this parameter for alpha-helices enables the visualization of the hydrophobic and hydrophilic envelopes around the peptide and their three-dimensional representation by molecular graphics. We have used this parameter to differentiate between pore-forming helices with a hydrophobic envelope larger than the hydrophilic component, membrane-spanning helices surrounded almost entirely by an hydrophobic envelope, fusiogenic peptides with an hydrophobicity gradient both around the helix and along the axis, and finally, amphipathic helices with a predominantly hydrophilic envelope. The structure of the lipid protein complexes is determined by a number of different interactions: the hydrophobic interaction of the apolar faces of the helices with lipids, the polar interaction of the hydrophilic sides of different helices with each other, and the interaction of hydrophilic residues with the aqueous solvent. The relative magnitude of the hydrophobic and hydrophilic envelopes accounts for the differences in the structure of the lipid-protein complexes. Purely hydrophobic interactions stabilize transmembrane helical segments, while hydrophobic interactions with the lipid phase and with each other are involved in the stabilization of the pore-forming helices. In contrast, both hydrophobic interactions with the lipids and hydrophilic interactions with the aqueous phase contribute to the arrangement of amphipathic helices around the edges of the discoidal lipid-apoprotein complexes. PMID- 1714907 TI - The complete primary structure of a nematode alpha 2(IV) collagen and the partial structural organization of its gene. AB - We have isolated and characterized cDNA and genomic DNA clones which encode an alpha 2(IV) collagen chain from the parasitic nematode Ascaris suum. In addition we have determined, by nucleic acid sequence analysis, the structural organization of approximately two-thirds of the gene. This analysis has shown that the gene contains at least 15 introns, and those that have been characterized range in size from 141 to 854 base pairs. The derived protein sequence contains 1763 amino acids and includes a putative 26-amino acid signal sequence. The collagenous triple-helical region contains 17 interruptions, many of which occur in the same positions as those in the human alpha 1(IV) and alpha 2(IV) chains. Comparison of the genomic DNA sequence with the cDNA sequence has revealed the presence of a sequence within the gene which appears to be an intact and normal exon that is not represented in our cDNA sequence. The presence of this putative exon raises the possibility that the A. suum alpha 2(IV) collagen gene may undergo alternative splicing. PMID- 1714908 TI - In vitro regulation of human phagocyte cytochrome b heavy and light chain gene expression by bacterial lipopolysaccharide and recombinant human cytokines. AB - We examined the effects of bacterial lipopolysaccharide and several recombinant human cytokines (tumor necrosis factor alpha and granulocyte-, macrophage-, and granulocyte-macrophage colony-stimulating factors) on the expression of the genes for the phagocyte cytochrome b, an essential component of the superoxide generating oxidase. In vitro treatment with lipopolysaccharide, tumor necrosis factor alpha, or macrophage- or granulocyte-macrophage colony-stimulating factors increased the levels of transcripts for the cytochrome b heavy chain (gp91phox) 9 to 22-fold and transcripts for the light chain (p22phox) 2- to 5-fold in cultured human monocyte-derived macrophages. The same agents, except for macrophage colony-stimulating factor, induced the expression of the cytochrome b heavy chain gene 2- to 12-fold and light chain gene 2- to 6-fold in human granulocytes. The expression of the cytochrome b heavy and light chain genes was coordinated in both macrophages and neutrophils with regard to stimulus specificity and dose-response pattern. The time course for induction of the two genes was parallel in both cell types for all stimuli. The macrophage response to lipopolysaccharide occurred at least in part at the transcriptional level. These results show that a variety of physiological regulators modulate the coordinated expression of the cytochrome b genes. PMID- 1714909 TI - Cloning of the GATA-binding protein that regulates endothelin-1 gene expression in endothelial cells. AB - Previously, we have identified two regions (A and B) of the endothelin-1 promoter that are important for the expression of this gene in cultured vascular endothelial cells. The cis-acting sequence in one of these regions (Region A) includes the core binding motif GATA, raising the possibility that this region of DNA mediates binding of a member of the GATA-binding protein family. In this report, we describe the use of polymerase chain reaction in conjunction with cDNA cloning to characterize the GATA-binding protein expressed in endothelial cells. The nucleotide sequence of endothelial cell cDNA clones is highly homologous to that of the chicken GATA-2 (NF-E1b) gene, indicating that our clones encode the human GATA-2 gene transcript. By RNA blot analysis, this gene is expressed in cultured cell lines derived from a number of different tissues. Transactivation experiments utilizing human GATA-2 eukaryotic expression vectors indicate that the GATA-2 protein interacts with the endothelin-1 GATA sequence to increase transcription of reporter genes in both BAEC and HeLa cells. These data provide the first evidence for a non-erythroid target gene regulated by GATA-2 and indicate that GATA-2 may have a more broad role in transcriptional regulation than the erythroid-specific GATA-1 protein. PMID- 1714910 TI - A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa. AB - The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions. PMID- 1714911 TI - Osteoarthrotic changes after acute transarticular load. An animal model. AB - The canine patellofemoral joint was subjected to a standardized transarticular load of 2170 newtons for two milliseconds, and the gross and histological changes were examined at two, twelve, and twenty-four weeks after injury. Initially, the load creates fractures in the zone of calcified cartilage, with minimum damage to the articular cartilage surface. Surface fissures were visible in all patellae only after staining with India ink. Histologically, these surface clefts extended into the transitional or superficial radial zone, and they did not communicate with the subchondral bone except in two patellae. However, there were reproducible clefts in the region of the subchondral bone and the zone of calcified cartilage in all patellae. Six months after loading, there was a loss of safranin-O staining above the deep clefts, and there was new-bone formation in the subchondral region and fibrillation of the cartilaginous surface. Thus, the initial changes had progressed to osteoarthrotic-like conditions at six months. In this animal model, the joint is not invaded and the changes that result from loading are reproducible. The injury to the joint creates superficial disruption of the cartilage and subchondral changes that lead to arthritic-like degeneration of the cartilage within six months. PMID- 1714912 TI - Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs. AB - During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation. PMID- 1714913 TI - An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3-mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation. AB - The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required. PMID- 1714915 TI - Mechanism underlying protective effect of MK-801 against NMDA-induced neuronal injury in vivo. AB - The effects of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the dihydropyridine calcium antagonist nimodipine on NMDA-induced phenomena were investigated using an in vivo fluorometric technique with indo-1. Indo-1, a fluorescent cytosolic free calcium ([Ca2+]i) indicator, was loaded into the cat cortex approximately 500 microns in depth by superfusion with the membrane permeant indo-1 acetoxy-methyl ester (indo-1-AM). Changes in [Ca2+]i signals (400 and 506 nm) and reduced nicotinamide adenine dinucleotide (NADH) fluorescence (464 nm) were simultaneously measured directly from the cortex during ultraviolet excitation (340 nm). Superfusion of 100 microM NMDA over the exposed cortex induced an elevation of the [Ca2+]i signal ratio (400/506 nm), biphasic changes in NAD/NADH redox state (initial oxidation followed by progressive reduction), and characteristic changes in the EEG (abrupt depression in amplitude followed by an excitatory pattern of 18-22 Hz polyspikes or sharp waves). These changes were completely blocked by treatment with MK-801 and reduced by nimodipine. The mechanism underlying the protective effects of systemically administered MK-801 on the NMDA-induced neuronal injury was verified in vivo. PMID- 1714914 TI - Endoproteolytic cleavage in the extracellular domain of the integral plasma membrane protein CE9 precedes its redistribution from the posterior to the anterior tail of the rat spermatozoon during epididymal maturation. AB - Originally identified as a basolateral domain-specific integral plasma membrane protein of the rat hepatocyte, CE9 mRNA and protein were also detected at high levels in the testis of the rat by Northern and Western blotting and immunoprecipitation. CE9 proved to be a domain-specific integral plasma membrane protein of the rat spermatozoon: on testicular spermatozoa, it was concentrated within the posterior tail domain of the plasma membrane, whereas on vas deferens spermatozoa, CE9 was concentrated within the anterior tail domain. This change in the localization of CE9 was observed to take place in a offgressive fashion during the passage of the spermatozoa from the caput epididymidis to the cauda epididymidis and was preceded by the specific endoproteolytic cleavage of CE9 in the proximal portion of the caput epididymidis. Amino-terminal amino acid microsequencing of CE9 immunoaffinity purified from epididymis suggested that the cleavage occurred on the carboxy-terminal side of arginine-74 in the primary sequence of CE9, resulting in the loss of approximately 40% of the amino acids in the extra-cellular domain of this transmembrane glycoprotein. PMID- 1714916 TI - Two insulin-like growth factor (IGF)-binding proteins are responsible for the selective affinity for IGF-II of cerebrospinal fluid binding proteins. AB - We have studied the relationships between the structure and affinity of two insulin-like growth factor-binding proteins (IGFBPs) purified from human cerebrospinal fluid (CSF). Competitive binding studies were performed using preparations of human recombinant IGF (rhIGF-I, rhIGF-II, and their labeled homologs) and the truncated variant form of IGF-I, rh-Des-(1-3)-IGF-I. One of these BPs, which is the most consistently detected in CSF, corresponds to IGFBP 2. The other is a new form whose N-terminal sequence we reported earlier, which we call the 32-30K BP on the basis of its electrophoretic migration. Comparisons were made with an IGFBP-1 preparation purified from amniotic fluid and with two BPs purified from human serum, which are homologous to the CSF BPs. The CSF BPs have particularly strong affinities for IGF-II. The estimated affinity constants (Ka) were 2 X 10(10) M-1 for IGFBP-2 and 10(11) M-1 for the 32-30K BP. These affinities were 15-20 and 70 times stronger than the respective affinities for IGF-I. The affinity of the 32-30K BP is the strongest among the BPs identified to date. The two BPs isolated from serum, which correspond to the 32-30K CSF BP and IGFBP-2, had affinities for IGF-II and IGF-I similar to those of the CSF BPs. IGFBP-1 had nearly identical affinities for the two IGFs of approximately 10(10) M-1. Des-(1-3)-IGF-I failed to bind to the CSF BPs, but bound to IGFBP-1, although with a 40-fold weaker affinity than IGF-I. From our data it would seem that IGFBP-1 has two classes of IGF-binding site, one of high and one of low (less than 10(9) M-1) affinity for both IGFs. The other two BPs, by contrast, each possess a predominant class of high affinity binding site for IGF-II. A second class of lower affinity (greater than 10(9) M-1) sites bind both IGF-I and IGF-II. In the case of the 32-30K BP, these preferentially bind IGF-II; in the case of IGFBP-2, their binding of the two IGFs is similar. These different types of binding site may play an important role in controlling the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714917 TI - The shiverer mouse mutation shi/shi: rescue and preimplantation detection. AB - The shiverer mouse mutation has been used as a model in this series of experiments. Germ-line therapy of this disorder has been demonstrated by producing mice transgenic for the wild-type gene for myelin basic protein (MBP). The mutation has also been diagnosed in preimplantation mouse blastocysts. PMID- 1714919 TI - Substance P does not alter interleukin-1 expression by splenic or granuloma macrophages in murine schistosomiasis. AB - Substance P (SP) is an undecapeptide with neurotransmitter and immunoregulatory properties. In murine schistosomiasis, ova naturally induce liver and intestinal granulomas. These granulomas contain macrophages, and eosinophils that produce SP. A report showed that human blood monocytes isolated by adherence release interleukin-1 (IL-1) in response to SP (Lotz et al. (1989) Science 241, 1218). IL 1 is important for initiation of hypersensitivity granulomas. Therefore, it was determined whether SP modulates granuloma macrophage IL-1 production in murine schistosomiasis. Macrophages were obtained from lung and liver granulomas, and from spleens of infected mice. A thymocyte proliferation assay measured IL-1 activity in culture supernatants. Total RNA, extracted from macrophages, was assayed for IL-1 alpha and beta mRNA by Northern blotting using cDNA probes. In response to lipopolysaccharide (LPS), splenic macrophages and macrophages from young lung granulomas released appreciable IL-1. Macrophages from liver granulomas, that were lesions older than the lung granulomas, were unresponsive to LPS with regard to IL-1 secretion. Yet, granuloma macrophages spontaneously expressed IL-1 alpha and beta mRNA. LPS enhanced IL-1 mRNA expression in both splenic and granuloma macrophages. Exposure of macrophages from all sources to SP did not alter IL-1 secretion or gene expression. Similarly, the responsiveness of macrophages to LPS was not affected by concomitant exposure to SP. It is concluded that, in the murine system, SP does not directly influence splenic or granuloma macrophage IL-1 secretion or gene expression. Also, it appears that macrophage secretion of IL-1 is rapidly down-regulated following granuloma elicitation. PMID- 1714918 TI - Clonal diversity of basic protein specific T cells in Lewis rats recovered from experimental autoimmune encephalomyelitis. AB - T cell lines selected from Lewis rats recovered from experimental autoimmune encephalomyelitis (EAE) respond not only to the immunodominant 72-89 epitope of basic protein (BP), but also to secondary epitopes including the I-A restricted 43-67 region of guinea pig (Gp) BP and the I-E restricted 87-99 sequence of rat (Rt) BP. The current study demonstrates at the clonal level the diversity of T cell responses to Gp- and Rt-BP in EAE-recovered rats. As predicted from the response pattern of BP-selected T cell lines, T cell clones from the lines responded to both the dominant and secondary epitopes of BP. In addition, a new majority clonal type was identified that responded to whole BP but not to epitopes represented on enzymatic cleavage fragments or synthetic peptides spanning the BP molecule. Clones representative of each of the three types of Gp BP responses were characterized for phenotype, major histocompatibility complex restriction, and biologic activity in vivo. All of the clones were strongly CD4+ and co-expressed CD8 at modest levels as measured by both immunofluorescence and Northern blots. All three T cell specificities were I-A restricted. However, only the 72-89 responsive clone could transfer clinical EAE, due most likely to its unique ability to respond to Rt-BP. In contrast, the Gp-BP 43-67 reactive T cell clone transferred protection against EAE, whereas the whole Gp-BP reactive clone transferred delayed-type hypersensitivity response but was neither encephalitogenic nor protective. Thus, the recovery process from EAE is distinguished by an increased diversity of protective clones as well as innocuous clones that may be spawned as encephalitogenic T cells are regulated. PMID- 1714920 TI - Hypothesis: antigen-specific T cells prime central nervous system endothelium for recruitment of nonspecific inflammatory cells to effect autoimmune demyelination. PMID- 1714921 TI - Distribution of 125I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxytryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level. AB - The distribution of galanin-like immunoreactivity (GAL-LI) in the spinal cord of the cat was studied by use of indirect histochemistry and the peroxidase antiperoxidase (PAP) technique. In the ventral horn GAL-immunoreactive (IR) axonal fibers and terminals were most frequent in the ventral part of the motor nucleus. The GAL-IR axons also contained 5-hydroxytryptamine (5-HT)-LI, and they disappeared after spinal cord transection. It was concluded that these GAL-IR fibers belong to the serotoninergic bublospinal pathway. In the medulla oblongata from normal cats, scattered GAL-IR cell bodies were encountered within the nucleus raphe obscurus and nucleus raphe pallidus. Electron microscopic observations revealed that the fine structure of the GAL-IR axonal boutons in the motor nucleus was similar to that of 5-HT-IR boutons with a varying number of immunoreactive large dense core vesicles. The postsynaptic element in all cases studied was a dendrite. A dense GAL-IR axonal plexus was found in the superficial laminae I-II of the dorsal horn. Coexistence was found between the GAL- and substance P-LI in fibers within the dorsal horn plexus. Spinal cord transection did not alter the pattern of GAL-LI in the dorsal horn, while the vast majority of GAL-IR axonal swellings disappeared following dorsal root sectioning. Electron microscopic observations in lamina II (substantia gelatinosa) revealed that the GAL-IR axonal terminals could be divided into two main groups. One with small to medium-sized axonal boutons formed synaptic contacts with both dendritic and axonal profiles. The other formed the central axon terminals of glomeruli, suggesting that GAL-LI may be present in C-type primary afferents. Numerous small GAL-IR cell bodies were encountered in laminae II and III. GAL-IR cell bodies were also observed in lamina X. The dorsal root ganglia contained a low but consistent number of small to medium-sized GAL-IR cell bodies, which all contained immunoreactive calcitonin gene-related peptide (CGRP). Following peripheral sciatic nerve transection, the number and the labeling intensity of GAL-IR cell bodies in the corresponding dorsal root ganglia showed a moderate increase. Radioimmunoassay revealed that the concentration of GAL-LI increased along the rostrocaudal axis of the normal spinal cord, and was about three times higher in the dorsal than in the ventral regions. The concentration in the dorsal root ganglia was intermediate to those seen in the corresponding dorsal and ventral cord regions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714922 TI - Primary afferent projections from the upper respiratory tract in the muskrat. AB - The central projections of the ethmoidal, glossopharyngeal, and superior laryngeal nerves were determined in the muskrat by use of the transganglionic transport of a mixture of horseradish peroxidase (HRP) and wheat germ agglutinin (WGA)-HRP. The ethmoidal nerve projected to discrete areas in all subdivisions of the ipsilateral trigeminal sensory complex. Reaction product was focused in ventromedial portions of the principal nucleus, subnucleus oralis, and subnucleus interpolaris. The subnucleus oralis also contained sparse reaction product in its dorsomedial part. Projections were dense to ventrolateral parts of laminae I and II of the rostral medullary dorsal horn, with sparser projections to lamina V. Label in laminae I and V extended into the cervical dorsal horn. A few labeled fibers were followed to the contralateral dorsal horn. The interstitial neuropil of the ventral paratrigeminal nucleus was densely labeled. Extratrigeminal primary afferent projections in ethmoidal nerve cases involved the Kolliker-Fuse nucleus and ventrolateral part of the parabrachial nucleus, the reticular formation surrounding the rostral ambiguous complex, and the dorsal reticular formation of the closed medulla. Retrograde labeling in the brain was observed in only the mesencephalic trigeminal nucleus in these cases. The cervical trunk of the glossopharyngeal and superior laryngeal nerves also projected to the trigeminal sensory complex, but almost exclusively to its caudal parts. These nerves terminated in the dorsal and ventral paratrigeminal nuclei as well as lamina I of the medullary and cervical dorsal horns. Lamina V received sparse projections. The glossopharyngeal and superior laryngeal nerves projected to the ipsilateral solitary complex at all levels extending from the caudal facial nucleus to the cervical spinal cord. At the level of the obex, these nerves projected densely to ipsilateral areas ventral and ventromedial to the solitary tract. Additional ipsilateral projections were observed along the dorsolateral border of the solitary complex. Near the obex and caudally, the commissural area was labeled bilaterally. Labeled fibers from the solitary tract projected into the caudal reticular formation bilaterally, especially when the cervical trunk of the glossopharyngeal nerve received tracer. Labeled fibers descending further in the solitary tract gradually shifted toward the base of the cervical dorsal horn. The labeled fibers left the solitary tract and entered the spinal trigeminal tract at these levels. Retrogradely labeled cells were observed in the ambiguous complex, especially rostrally, and in the rostral dorsal vagal nucleus after application of HRP and WGA-HRP to either the glossopharyngeal or superior laryngeal nerves. In glossopharyngeal nerve cases, retrogradely labeled neurons also were seen in the inferior salivatory nucleus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1714923 TI - Dopaminergic innervation of substance P-containing striatal neurons by fetal nigral grafts: an ultrastructural double-labeling immunocytochemical study. AB - Evidence for survival and growth of fetal substantia nigra grafts in host striatum and partial reversal of behavioural and biochemical deficits in the host animal is well documented. Afferent synaptic connections arising from the graft and contacting host structures have also been reported; however, the properties of the neurons receiving this input is less clear. The purpose of this study was to determine if substance P-containing neostriatal neurons receive a dopaminergic input from nigral grafts. Fetal substantia nigra cell suspensions were stereotaxically implanted in the deafferented neostriatum of Wistar rats 2 weeks after a unilateral 6-hydroxydopamine (6-OHDA) lesion in the ipsilateral substantia nigra or medial forebrain bundle. The ultrastructural features of the graft-host synaptic interactions were analysed by employing an electron microscope immunocytochemical double-labeling technique. Tyrosine hydroxylase (TH) and substance P-immunoreactive structures were simultaneously demonstrated by means of the peroxidase-antiperoxidase method using two different chromogens with distinct reaction products easily differentiated at the light and electron microscope levels. TH-immunoreactive sites were first demonstrated using 3,3' diaminobenzidine tetrahydrochloride (DAB); then substance P immunoreactivity was localized using benzidine dihydrochloride (BDHC). TH-immunoreactive terminals of axons originating from the graft made synaptic contacts with substance P-positive cell bodies and dendrites from the host. These results indicate that at least partial restoration of the normal nigrostriatal circuitry can be achieved following nigral grafts. The demonstration of specific synaptic input on host substance P neurons provides an anatomical basis for direct functional modulation of the deafferented host neostriatum by the nigral graft. PMID- 1714924 TI - Prior optic nerve transection reduces capsaicin-induced degeneration in rat subcortical visual structures. AB - Capsaicin is a neurotoxin capable of causing degeneration in specific sites throughout the neuraxis, including the suprachiasmatic nucleus (SCh), the ventrolateral geniculate nucleus (VLG), the intergeniculate leaflet (IGL), and the olivary and medial pretectal nuclei (OPT and MPT). In this experiment, we tested the hypothesis that capsaicin-induced terminal degeneration in the SCh, VLG, IGL, OPT, and MPT results from destruction of retinal ganglion cells and their axonal projections to these sites. In the first experiment, silver stains were used to examine degeneration in the retina induced by systemic capsaicin treatment. Capsaicin caused degeneration of ganglion cells, bipolar cells, and nerve terminals in the retina, which could be observed between 2 and 24 hours after treatment. In the second experiment, 15-day-old rat pups were enucleated unilaterally. Five days or 2, 5, or 10 months later, they were injected systemically with capsaicin and killed 6 hours (pups) or 18 hours (adults) later for analysis with a cupric silver stain. In rats of all ages, prior monocular enucleation reduced or eliminated capsaicin-induced degeneration in the contralateral SCh, VLG, IGL, OPT, and MPT. In the third experiment, rat pups were treated systemically with capsaicin or vehicle solution at 12 days of age and given unilateral intravitreal injections of cholera toxin conjugated to horseradish peroxidase (CT-HRP) 3 days prior to sacrifice at 20 days of age. Transport of CT-HRP to the SCh, VLG, IGL, MPT, and OPT was attenuated but not abolished by capsaicin pretreatment. Results suggest that capsaicin causes degeneration in the SCh, VLG, IGL, MPT, and OPT by selective destruction of a subpopulation of retinal ganglion cells with axonal projections to these sites. PMID- 1714925 TI - Inhibition of contact sensitivity by interferon in mice infected with lactic dehydrogenase virus. AB - The effect of acute lactic dehydrogenase virus (LDV) infection was studied with respect to contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB). CS reaction was severely inhibited in the acute phase but not in the chronic phase of infection. The role of interferon (IFN) was studied to understand further the inhibition of CS during LDV infection. IFN in the blood was detected only in the acute phase, but not in the chronic phase of infection. When anti-IFN (alpha/beta) was administered to infected mice, no inhibition of CS was seen. CS was inhibited in uninfected mice treated with IFN (alpha/beta). These results suggest that IFN production in the blood may be responsible, at least in part, for inhibition of CS observed in the acute phase of LDV infection. PMID- 1714926 TI - High frequency of IL-4-producing CD4+ allergen-specific T lymphocytes in atopic dermatitis lesional skin. AB - In atopic dermatitis (AD) hypersensitivity reactions to allergens are commonly observed and are assumed to make a major contribution in the pathomechanism of the disease. It may be expected that allergen-reactive Th cells play a central role in these reactions. In the present study the occurrence and function of allergen-specific T lymphocytes in dermal inflammatory lesions were studied. To this aim panels of randomly cloned CD4+ T cells from lesional skin biopsies of two housedust mite Dermatophagoides pteronyssinus (Dp)-allergic AD patients were screened for reactivity with Dp allergens. The results were compared with similar tests for Dp reactivity of T-lymphocyte clones (TLC) from the peripheral blood of these patients. In the panels of TLC generated from lesional skin (S-TLC), a considerable number of TLC appeared to be Dp-specific, 47% (n = 17) and 10% (n = 29), respectively. In the panels from the peripheral blood, the percentages of Dp specific TLC were only 0% (n = 22) and 3% (n = 34), suggesting accumulation or expansion of these T cells in lesional skin. The function of these TLC was studied by assaying the secretion of IL-4 and IFN-gamma, which have been shown to be produced in aberrant ratios by Dp-specific TLC from the peripheral blood of AD patients (Wierenga et al: J Immunol 144:4651, 1990). All Dp-specific S-TLC produced IL-4 in combination with no or low levels of IFN-gamma, whereas many of the non-Dp-specific S-TLC and blood-derived TLC (B-TLC) were observed to produce high levels of IFN-gamma without significant amounts of IL-4. A functional consequence of these cytokine profiles was demonstrated by the finding that TLC producing substantial amounts of IL-4 enhanced expression of the low-affinity Fc receptor for IgE (CD23) on antigen-presenting cells to a greater extent than did IFN-gamma-producing TLC. PMID- 1714927 TI - Role of hair follicles in the repigmentation of vitiligo. AB - Vitiligo is a common pigment disease that is difficult to treat. The mechanism of repigmentation is not known. We combined Dopa-Toluidine Blue complex stain, hair follicle split-Dopa stain, and hair follicle split-scanning electron microscope (SEM) to observe the changes of melanocytes in 23 normal, 24 vitiliginous, and 36 repigmented skin specimens. We found that only active (Dopa-positive) melanocytes existed in the epidermis of normal skin. There were some inactive (Dopa-negative) melanocytes in the outer root sheaths of normal hair follicles, which form the melanocyte reservoir in human skin. In the patients with vitiligo the active melanocytes in the epidermis were totally missing, whereas the inactive melanocytes in the outer root sheaths of hair follicles were not affected. Treatment stimulated the inactive melanocytes in the middle and/or lower parts of the outer root sheaths of hair follicles to divide, proliferate, and migrate upward along the surface of the outer root sheath to the nearby epidermis, where the melanocytes continued to migrate radially to form the pigmented island visible clinically in repigmented vitiligo lesions. During the migration to the epidermis, the melanocytes matured gradually from an inactive phase to an active condition. In conclusion, the existence of these inactive melanocytes provided the melanocyte sources for repigmentation of vitiligo. PMID- 1714928 TI - Immune privilege in hair growth. AB - Immunostaining techniques were used to investigate the relationship between immune cells, proteoglycan, and class I MHC distribution in skin during the hair cycle in rats. The growth stage, anagen, was characterized by absence of class I MHC staining on most cells of the lower follicle and presence of chondroitin proteoglycan in the follicle sheath and dermal papilla. Immune cells were few in number and not associated with follicles. Dramatic changes were observed during regression in catagen; class I MHC was expressed on all follicle epithelium, large numbers of activated macrophages aggregated around the follicles, and the chondroitin proteoglycans disappeared from the follicle sheath and dermal papilla. During the resting stage, telogen, class I MHC remained on cells of the secondary germ, but macrophages and chondroitin proteoglycans were absent. These observations lead us to propose a hypothesis of immune privilege in hair growth. PMID- 1714929 TI - A potential role for CD1a molecules on human epidermal Langerhans cells in allogeneic T-cell activation. AB - The structural similarities of CD1a molecules to major histocompatibility complex (MHC) class I antigens, as well as their expression on epidermal antigen presenting cells suggest that CD1a molecules might be involved in the cutaneous immune response. In the present study, we investigated the effect of different anti-CD1a monoclonal antibodies (BL6, DMC1, and Na1/34) on T cell proliferation induced by allogeneic epidermal cells in vitro. A significant inhibition of the mixed skin cell-lymphocyte reaction was obtained with BL6 and DMC1 monoclonal antibodies (MoAb), which recognize the same epitope on CD1a molecule. The observed inhibition could not be related to a steric hindrance of MHC class II molecules, because Na1/34 MoAb, which reacts with another epitope on CD1a molecule, had no significant effect. BL6 and DMC1 MoAb interfered with an early event of T-cell activation, as shown by a time-course study. In the presence of these MoAb, the addition of exogenous interleukin 2 did not restore T-cell proliferation. Furthermore, the inhibitory effect of anti-CD1a MoAb was not mediated by a suppressor factor released by Langerhans cells (LC). These present data suggest that CD1a molecule may have an important function in self peptide presentation by human Langerhans cells. PMID- 1714930 TI - CD36(OKM5)+ dendritic cells in the oral mucosa of HIV- and HIV+ subjects. AB - In this study, we have investigated by light and electron microscopy the presence, distribution, and inner structure of CD36(OKM5)+ dendritic cells (DC) in the lamina propria and epithelium of the oral mucosa of HIV- and HIV+ subjects; in the latter, both clinically healthy areas and areas of hairy leukoplakia (HL) were studied. Perivascular CD36+ DC were present in the lamina propria of all the specimens studied. They were also found in small numbers in the epithelium of clinically healthy mucosa of HIV- and HIV+ subjects, but were practically absent from the epithelium of HL. CD36+ DC seemed to be regularly HLA DR+ in HIV-subjects; this positivity was recognized only in some cells in the clinically healthy mucosa of HIV+ subjects, and practically never in HL. Because the only perivascular cells observed in the clinically healthy areas of HIV+ subjects were CD36+, we investigated the ultrastructure of perivascular DC in these same areas. These cells were characterized by the presence of a prominent Golgi apparatus, many lysosomes, and focal adhesions to the extracellular matrix. It may be concluded that 1) CD36+ DC are physiologic components of the oral mucosa, 2) they share some ultrastructural features with macrophages, 3) no differences in numbers were found between HIV+ and HIV- subjects, and 4) these cells are affected in their expression of HLA-DR antigens during HIV infection, particularly in areas of HL. This may be a hint that the antigen-presenting function of these cells in the oral mucosa is negatively affected during HIV infection. PMID- 1714931 TI - Expression of beta 4 integrins in human skin: comparison of epidermal distribution with beta 1-integrin epitopes, and modulation by calcium and vitamin D3 in cultured keratinocytes. AB - The expression of beta 4 integrins in adult and fetal human skin as well as in cultured keratinocytes was studied by immunodetection with monoclonal antibodies, and compared with that of beta 1 integrins. The distribution of the beta 1 and beta 4 integrin epitopes was entirely different in the adult epidermis. As reported previously by us (J Clin Invest 84:1916, 1989), the beta 1 epitopes were present most prominently at lateral cell-cell contact points, whereas staining with the beta 4 antibody demonstrated a linear staining pattern polarizing to the basal surface juxtaposed to the dermal-epidermal basement membrane. In contrast, in fetal skin, the staining patterns both with beta 1 and beta 4 antibodies were similar and demonstrated the presence of epitopes surrounding the entire cell surface of both basal and suprabasal keratinocytes. Distinct polarization of beta 4 integrin epitopes was noted in cultured keratinocytes maintained in low-calcium (0.15 mM) medium, and the expression of these integrins was also noted in human papilloma virus-transformed keratinocytes. Switch of the cultures to high-calcium (1.2 mM) medium resulted in redistribution of the epitopes to a pattern suggesting their location at under-surface of the cells adjacent to the substratum. This Ca(++)-induced redistribution of beta 4 integrin epitopes could be counteracted by 10(-7) M vitamin D3. Finally, incubation with anti-beta 4 integrin antibody reduced the capacity of keratinocytes to attach to plastic substratum. The results provide further evidence for the role of beta 4 integrins in cell-matrix interactions. PMID- 1714932 TI - The role of endocavitary irradiation for limited lesions of the rectum. PMID- 1714933 TI - CTLA-4 is a second receptor for the B cell activation antigen B7. AB - Functional interactions between T and B lymphocytes are necessary for optimal activation of an immune response. Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation. CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28. It is not known, however, if CD28 and CTLA-4 also share functional properties. To investigate functional properties of CTLA-4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin C gamma chain. Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125I labeled extracts of these cells. The avidity of 125I-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd approximately 12 nM). Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes. These findings provide direct evidence that, like its structural homologue CD28, CTLA-4 is able to bind the B7 counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro. PMID- 1714934 TI - H-2Kd-restricted antigenic peptides share a simple binding motif. AB - We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule. PMID- 1714935 TI - Structure, expression, and T cell costimulatory activity of the murine homologue of the human B lymphocyte activation antigen B7. AB - Following occupancy of the T cell receptor by antigen, T cell proliferation and lymphokine production are determined by a second costimulatory signal delivered by a ligand expressed on antigen presenting cells. The human B cell activation antigen B7, which is expressed on antigen presenting cells including activated B cells and gamma interferon treated monocytes, has been shown to deliver such a costimulatory signal upon attachment to its ligand on T cells, CD28. We have cloned and sequenced the murine homologue of the human B7 gene. The predicted murine protein has 44% amino acid identity with human B7. The greatest similarity is in the Ig-V and Ig-C like domains. Murine B7 mRNA was detected in murine hematopoietic cells of B cell but not T cell origin. Cells transfected with murine B7 provided a costimulatory signal to human CD28+ T lymphocytes. These results demonstrate the costimulatory activity of murine B7 and provide evidence that the ligand attachment site is conserved between the two species. PMID- 1714936 TI - Fibrinolytic response to tumor necrosis factor in healthy subjects. AB - Tumor necrosis factor (TNF) may be involved in the disturbance of the procoagulant-fibrinolytic balance in septicemia, leading to microvascular thrombosis. To assess the dynamics of the fibrinolytic response to TNF in humans, we performed a crossover saline-controlled study in six healthy men, investigating the effects of a bolus intravenous injection of recombinant human TNF (50 micrograms/m2) on the stimulation and inhibition of plasminogen activation as well as on plasmin activity and inhibition. TNF induced a brief fourfold increase in the overall plasma plasminogen activator (PA) activity peaking after 1 h (p less than 0.0001), which was associated with rises in the antigenic levels of urokinase-type plasminogen activator (p less than 0.0001) and tissue-type plasminogen activator (p less than 0.0001). Plasminogen activator inhibitor type I antigen remained unchanged in the first hour, but showed a rapid eightfold increase thereafter (p less than 0.0001), which coincided with the decrease in PA activity. Generation of plasmin activity in the first hour was signified by an 11-fold rise in D-dimer levels (p less than 0.0001); inhibition of plasmin was reflected by a 36-fold rise in plasmin-alpha 2 antiplasmin complexes (p less than 0.0001), as well as by a transient 16% decrease in alpha 2 antiplasmin activity (p less than 0.01). In conclusion, TNF induced an early activation of the fibrinolytic system becoming maximal in 1 h, with a rapid inhibition thereafter. Earlier observations in the same subjects showed sustained coagulation activation for 6-12 h. The observed disbalance between the procoagulant and fibrinolytic mechanisms after TNF injection confirms the in vivo relevance of the effects of TNF on vascular endothelium in vitro and may explain the tendency towards microvascular thrombosis in septicemia. PMID- 1714937 TI - Maternal serum alphafetoprotein screening. University Medical Center. AB - University Medical Center in Jacksonville initiated a screening program for maternal serum alphafetoprotein (MSAFP) in October 1987. Women with both high and low values were recalled for testing. During the first two years, neural tube and other birth defects including cystic hygroma and gastroschisis were detected. No chromosomal abnormalities were found. The program meets the recommendation of the American College of Obstetrics and Gynecology to offer MSAFP testing to all pregnant women. Pregnant women in other areas of Florida could receive services through similar programs. PMID- 1714938 TI - On the interaction of bovine pancreatic trypsin inhibitor with maxi Ca(2+) activated K+ channels. A model system for analysis of peptide-induced subconductance states. AB - Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+) activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation. PMID- 1714939 TI - Analysis of the neutralization epitopes on human rotavirus VP7 recognized by monotype-specific monoclonal antibodies. AB - Three anti-VP7 monoclonal antibodies (MAbs) which neutralized only two strains (K8 and S12) of five serotype 1 human rotaviruses (HRVs) were obtained, and neutralization epitopes recognized by these 'monotype-specific' MAbs were analysed by epitope mapping and sequencing of the VP7 genes. Neutralization resistant mutants of K8 and S12 were selected by the monotype-specific MAbs and serotype 1-specific MAbs prepared previously. Cross-neutralization tests between MAbs and neutralization-resistant mutants of K8 and S12 indicated that epitopes of monotype-specific MAbs operationally overlap with those of serotype 1-specific and cross-reactive MAbs recognizing the S1 region. Sequence analyses of the VP7 genes indicated that VP7s of strains K8 and S12, which belong to a monotype of serotype 1 viruses, possessed amino acids at positions 42 and 87 different from other serotype 1 HRVs. Furthermore, amino acid substitution sites of representative mutants of K8, selected by the monotype-specific MAbs, were identified at positions 96, 97 and 100. These results imply that amino acids in variable region B (amino acids 87 to 101) are involved in the monotype-specific neutralization epitope as well as serotype-specific neutralization epitopes. PMID- 1714940 TI - Antigenic and functional characterization of rinderpest virus envelope proteins using monoclonal antibodies. AB - A total of 24 monoclonal antibodies (MAbs) against the haemagglutinin (H) and the fusion protein (F) of rinderpest virus (RPV) were used to characterize their antigenic structure and biological properties, and to analyse natural variation in the envelope proteins of morbilliviruses. The anti-H and anti-F MAbs defined seven and three distinct antigenic sites, respectively. The MAbs to six sites on H were able to neutralize the infectivity of RPV. The addition of guinea-pig complement or anti-mouse immunoglobulin increased the virus-neutralizing antibody titre of most of the anti-H MAbs, including those lacking neutralizing activity. One of the antigenic sites on H was conserved among morbilliviruses and the MAbs to this site had haemagglutination inhibition activity against measles virus (MV). The remaining sites were specific for RPV and varied antigenically between strains of RPV. The anti-F MAbs lacked neutralizing activity, but two of the five MAbs did show activity in the presence of complement or anti-mouse immunoglobulin. On the whole, the antigenic sites on F were conserved in some strains of MV, but not in canine distemper virus. All of the sites on the surface proteins were sensitive to SDS and, although those on F were not affected by 2 mercaptoethanol, five of the seven sites on H were destroyed by it. These results suggest that the epitopes on the envelope proteins are conformation-dependent. PMID- 1714941 TI - Characterization of Sendai virus-induced human placental trophoblast interferons. AB - Human placental trophoblast cultures produce a mixture of interferons (IFNs) when challenged with Sendai virus. High-performance dye-ligand and immunoaffinity chromatography of a trophoblast IFN (tro-IFN) preparation enabled the isolation of three antigenically distinct IFNs, alpha I, alpha II 1 and beta, with Mrs of 16K, 22K and 24K respectively, by reducing and non-reducing SDS-PAGE. The major IFN, responsible for 75% of the total antiviral activity, was tro-IFN-beta, with the remaining activity being due to tro-IFN-alpha I and tro-IFN-alpha II 1, as determined by an antiviral neutralization test using specific anti-human IFN antibodies. The antiviral activities of the tro-IFNs were stable at pH 2.0 for 24 h and tro-IFN-alpha II 1 and -beta were shown to be glycoproteins. The three tro IFNs showed different antiviral activities when assayed on human and bovine cell species; tro-IFN-alpha I and alpha II 1 protected both human and bovine (MDBK) cells from virus infection, whereas tro-IFN-beta showed a high degree of species specificity, protecting only the human cell types tested. PMID- 1714942 TI - Immunological characterization of the human immunodeficiency virus type 1 reverse transcriptase protein by the use of monoclonal antibodies. AB - Eighteen monoclonal antibodies (MAbs) directed against the purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) protein were produced. The antibodies were characterized by competitive ELISAs and Western blot experiments, and with nested, nine amino acid long peptides representing the whole 560 amino acid RT protein. By ELISA, the MAbs react with a minimum of seven epitopes of the protein. Four of the epitopes were located on the N-terminal 51K subunit and the remaining three epitopes were located at the C-terminal end of the protein. Using synthetic peptides, two epitopes at the N-terminal part were located at amino acids 294 to 302 and 350 to 354, respectively, from the N terminal start of the protein. One epitope was located at amino acids 442 to 450, just after the cleavage site between the N-terminal and C-terminal subunit at position 440. Antibodies located at amino acids 294 to 302 could inhibit the RT enzymic activity of the protein. Two other MAbs, directed at the N-terminal and C terminal parts of the protein, could also inhibit RT activity. PMID- 1714944 TI - Biochemical and immunological analysis of discontinuous epitopes in the family of human cytomegalovirus glycoprotein complexes designated gC-I. AB - The envelope of human cytomegalovirus contains a family of disulphide-linked glycoprotein complexes designated gC-I which contain two glycoproteins of 52,000 Mr (gp52) and 93,000 to 130,000 Mr (gp93-130). Epitopes recognized by several of our gC-I gp52-specific monoclonal antibodies (MAbs) were previously assigned to three domains based on reactivity with gC-I in a competitive binding assay. In this report, we have used additional gC-I MAbs to characterize three distinct discontinuous epitopes in the gC-I complexes. Two of these epitopes were in Domain I and one in Domain III. These epitopes were resistant to proteolysis, heat denaturation and SDS treatment. However, the discontinuous epitopes were lost after reduction of disulphide bonds. After digestion of gC-I complexes with chymotrypsin, two fragments of 43,000 (43K) Mr and 34,000 (34K) Mr were obtained which contained all discontinuous and continuous epitopes recognized by our gp52 MAbs. The Mr of these fragments could not be reduced further by longer digestion or by use of other proteases such as trypsin or pronase. The 43K fragment contained N-linked oligosaccharides not detected in the 34K fragment. These oligosaccharides may have prevented a complete proteolytic digestion so that the 34K fragment was not always obtained. It was established that 80 to 90% of the mass of these fragments was contributed by gp52. Thus the discontinuous epitopes were composed primarily of gp52 and not gp93-130. PMID- 1714943 TI - Selective induction of discrete epitopes of herpes simplex virus type 1-specified glycoprotein C by interference with terminal steps in glycosylation. AB - We have described two types of oligosaccharide modification influencing the antigenicity of the herpes simplex virus type 1 (HSV-1)-specified glycoprotein C (gC-1). First, the expression of several epitopes belonging to antigenic site II of gC-1 is dependent on the peripheral galactose of N-linked oligosaccharides. We have also shown that treatment of HSV-1-infected cells with 5-n-propyl-2' deoxyuridine (PdU) under certain circumstances results in other modifications of peripheral carbohydrate determinants, which are associated with increased antigenic activity of gC-1. In the present study we have mapped and characterized the epitopes susceptible to PdU induction by analysing the reactivity to a number of monoclonal antibodies defining several epitopes of antigenic sites I and II. The results indicate that the strict galactose dependence of epitopes and the PdU induced increase of antigenic activity are independent and unrelated phenomena. Thus, we identified galactose-dependent epitopes that were not PdU-inducible and vice versa, and some epitopes were both galactose-dependent and PdU-inducible. The results support a model where PdU treatment blocks synthesis of an antigen masking carbohydrate determinant. In addition, PdU treatment of HSV-1-infected cells seemed to increase the antigenic activity of other HSV-1 glycoproteins. PMID- 1714945 TI - Use of the polymerase chain reaction to analyse sequence variation within a major neutralizing epitope of glycoprotein B (gp58) in clinical isolates of human cytomegalovirus. AB - The heterogeneity of low passage human cytomegalovirus (HCMV) strains was determined by HindIII typing of 28 clinical isolates from transplant patients. These data have shown that, in general, each patient's strain has a unique restriction profile, usually comprising combinations of HindIII sites present in one or more of the tissue culture-adapted strains AD169, Towne and Davis. To map sequence changes in a more refined manner we performed detailed analyses of 33 low passage clinical isolates, including those aforementioned, analysing a sequence within glycoprotein B containing a major neutralizing epitope. A 149 bp sequence containing the epitope (amino acids 608 to 625) was amplified using the polymerase chain reaction, the products were cloned and their DNA sequence was determined. Comparison of the DNA and deduced amino acid sequences with those of HCMV strain AD169 revealed that there was a high degree of conservation of the epitope between the 33 clinical isolates. However 10 of the isolates possessed silent mutations and three isolates contained mutations producing amino acid changes within the neutralizing epitope. The possible functional significance of these changes is discussed. PMID- 1714946 TI - Immunofluorescence studies of biotype-specific expression of bovine viral diarrhoea virus epitopes in infected cells. AB - The expression of biotype-specific epitopes in cells infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea virus (BVDV) was analysed by immunofluorescence. Four monoclonal antibodies (MAbs) directed against different epitopes on the viral glycoprotein gp48 were used. With cells infected with cpBVDV strain NADL, the four MAbs yielded a strong and granular cytoplasmic fluorescence. The same pattern was observed when cells were infected with ncpBVDV 7443 with two of the MAbs (BVD/C12, BVD/C42). In contrast, reactivity with the other two MAbs (BVD/C38, BVD/C46) was restricted to a narrow perinuclear zone. These biotype-specific differences were not observed either with a gp53-specific MAb, or with an MAb specific for the non-structural protein p125/p80. Double immunofluorescence staining of living cells with a polyclonal BVDV-specific serum and with the MAbs revealed that expression of viral proteins on the surface of cells infected with cp- or ncpBVDV, respectively, was not detectable. PMID- 1714947 TI - The particle size of hepatitis C virus estimated by filtration through microporous regenerated cellulose fibre. AB - To estimate the particle size of hepatitis C virus (HCV), a major causative agent of post-transfusion non-A, non-B hepatitis, we filtered plasma or serum samples through microporous cellulose fibres with different pore sizes. The amount of HCV particles in samples before and after filtration was determined by a quantitative reverse transcriptase polymerase chain reaction (PCR) method. Since there is no quantitative biological assay for HCV, except for that in chimpanzees, the HCV titre obtained from the PCR method was used in an equation constructed previously for application to filtration experiments with a flavivirus which is distantly related to HCV. The particle was estimated to be between 30 and 38 nm in diameter, although the possibility remained that larger HCV particles or HCV aggregates with a diameter of more than 39 nm might exist. Double-step filtration through microporous cellulose fibres with a pore size of 35 nm reduced the HCV content to below levels detectable by our PCR method, indicating that it is possible to eliminate HCV particles by simple filtration techniques. PMID- 1714948 TI - Surveillance alone versus radiotherapy after orchiectomy for clinical stage I nonseminomatous testicular cancer. Danish Testicular Cancer Study Group. AB - From December 1980 to January 1984, all patients with stage I nonseminomatous testicular cancer in Denmark entered a randomized trial comparing surveillance only with radiotherapy after orchiectomy. One hundred fifty patients were assessable for the final analysis. Relapse occurred in 23 patients in the surveillance group and in 11 patients in the radiotherapy group. Radiotherapy completely prevented retroperitoneal relapse; 14 retroperitoneal relapses occurred in the surveillance-only group. All relapsing patients in the surveillance-only group are without evidence of disease with a median observation time after chemotherapy of 67 months. Two of the patients with relapse in the radiotherapy group died with disease; the others are alive without evidence of disease, with a median observation time after relapse treatment of 72 months. In the surveillance group, four relapses occurred later than 2 years after orchiectomy; only one such late relapse occurred in the radiotherapy group. Four of the retroperitoneal relapses occurred without concomitant increase in the serum marker levels (alpha-fetoprotein [AFP] and human chorionic gonadotropin [HCG]). It is concluded that surveillance only should replace radiotherapy after orchiectomy as standard treatment for clinical stage I nonseminomatous testicular cancer. Improved methods for control of retroperitoneal relapses, especially of embryonal carcinomas, are needed. PMID- 1714949 TI - A loading dose/continuous infusion schedule of fludarabine phosphate in chronic lymphocytic leukemia. AB - Using a loading dose/continuous infusion schedule, fludarabine phosphate was administered to 51 patients with previously treated chronic lymphocytic leukemia (CLL). All patients had evidence of active disease, and the majority had advanced Rai stages. Of the 42 patients assessable for response, 22 (52%) achieved a partial response, five (12%) had stable disease, and 15 (36%) progressed. Thirteen of the 22 responders improved their Rai stages with fludarabine therapy, including six patients who achieved stage 0. Response rates for pretreatment stages III and IV were 60% and 53%, respectively. Patients with final Rai stages 0 to II had better survival than those with stages III and IV. Patients who had undergone splenectomy before starting therapy were more likely to respond. Myelosuppression was the primary toxicity and did not appear to be cumulative. Severe leukopenia and thrombocytopenia, although infrequent, were associated with several deaths in the early cycles of treatment. Nonhematologic toxicity was mild with no serious neurotoxicity noted. Infections were common with 22 minor, 18 major, and 10 fatal episodes. Fludarabine phosphate by this alternative dosing schedule is effective in refractory advanced CLL and is well tolerated by the majority of patients. PMID- 1714950 TI - Intensive chemotherapy and low-dose radiotherapy for the treatment of advanced stage Hodgkin's disease in pediatric patients: a Pediatric Oncology Group study. AB - Sixty-two patients with advanced-stage Hodgkin's disease and a median age of 12 years (range, 3 to 22 years) were treated with four cycles of mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) alternating with four cycles of doxorubicin, vinblastine, bleomycin, and dacarbazine (ABVD) followed by low-dose radiotherapy (RT). We determined the feasibility, immediate safety, and rapidity of response of patients to this regimen, as well as the relationship between prognostic factors and the rate of complete remission (CR), event-free survival (EFS), and overall survival. Therapy was well tolerated, and the major toxicity was hematopoietic. At the end of chemotherapy, 54 of 62 patients (87%) were in CR by clinical restaging, with a biopsy of residual disease where necessary. The actuarial 3-year EFS is 77% (SE, 11%), with a median follow-up of 35 months, and the survival is 91% (SE, 7%). With respect to EFS, female patients and those with stage II or III disease fared statistically better than males and patients with stage IV disease, respectively. Six patients have died: three of progressive Hodgkin's disease, one of secondary acute myelocytic leukemia (AML), one of secondary non-Hodgkin's lymphoma (NHL), and one of overwhelming bacterial sepsis. The Pediatric Oncology Group (POG) is currently engaged in a randomized study of these eight cycles of chemotherapy with and without RT to assess the role of RT in achieving comparable results. PMID- 1714951 TI - Chemotherapy of metastatic and/or recurrent undifferentiated nasopharyngeal carcinoma with cisplatin, bleomycin, and fluorouracil. AB - Undifferentiated nasopharyngeal carcinoma (UCNT) is known to be radiosensitive and chemosensitive, but the latter has never been studied prospectively with phase II methodology. After an intensive work-up, 49 patients with recurrent (REC) and/or metastatic (MTS) UCNT were treated with three monthly cycles of cisplatin (CDDP) 100 mg/m2 day 1; bleomycin 15 mg intravenously (IV) day 1, and 16 mg/m2/d continuous infusion (CI) days 1 to 5; and fluorouracil (5FU) 650 mg/m2/d CI days 1 to 5 (PBF). Of the 49 patients, 33 were North African. The sex ratio was three males:one female, and the median World Health Organization (WHO) performance status was 1.6. In the 48 patients assessable for response, we observed nine (19%) complete responses (CRs) and 29 (60%) partial responses (PRs) (60%), for a 79% overall response rate (95% confidence interval, 68% to 90%) in the assessable group and a 78% global rate. There were eight CRs (24%) observed in the group without previous chemotherapy (33 patients) compared with one CR in the chemotherapy pretreated group (16 patients). Four patients are still alive without evidence of disease after 52+, 54+, 58+, and 58+ months, respectively. All of them had less than three bone MTS sites, and received radiation therapy in these sites. The results confirm the chemosensitivity of UCNT, and the observation of unmaintained long-term responders makes curability a possible consideration. PMID- 1714952 TI - Eye movements elicited by electrical stimulation of area PG in the monkey. AB - 1. Eye positions of monkeys were tracked while low-current electrical stimulation was delivered to area PG of the posterior parietal cortex. Stimulation was delivered while monkeys were in darkness, while they were in a dimly illuminated room, or while they actively fixated on small lamps to receive a liquid reward. 2. Resulting eye movements fell into one of three categories, depending roughly on the area stimulated. Stimulation of caudal regions generally resulted in saccades that were of approximately equivalent amplitudes and directions. When more rostral areas were stimulated, saccades were generally produced that directed the eyes toward roughly the same position in the head. Distributed throughout all regions were sites for which elicited saccades did not fall clearly into either of these coordinate bases. Stimulation of lateral areas produced low-velocity eye movements that were directed ipsilaterally from the stimulated hemisphere. 3. Stimulation made while monkeys fixated on target lamps produced saccades with more variability and less amplitude than those produced while monkeys were in darkness. Low-velocity eye movements could only be elicited while monkeys were in darkness. 4. Craniocentric saccades typically brought the eyes to within a 10-20 degrees area, and saccades could not be produced when the initial eye position was near this area. Craniocentric saccades were always greater than 5 degrees in amplitude. 5. It is concluded that area PG is organized into at least two zones that differ in the way by which they code saccades. A caudal region codes saccades in a way similar to that found in the frontal cortex and superior colliculus of primates. A rostral region codes saccades in a craniocentric manner, although it is restricted only to gross redirection of gaze without the accuracy monkeys are capable of using in directing their eyes. PMID- 1714953 TI - Immunohistochemical localization of collagenous components in healthy periodontal tissues of the rat and marmoset (Callithrix jacchus). II. Distribution of collagen types IV, V and VI. AB - The immunohistochemical distribution of collagen types IV, V and VI has been demonstrated in healthy periodontal tissues of rats and marmosets following decalcification of the maxillae and mandibulae in 0.2 N HCl. An intense fluorescence with anti-collagen type IV antibodies was demonstrated in the basement membranes of the epithelium and of the blood vessels and nerves. In the alveolar bone stroma and in the periodontal ligament (PL) collagen type IV was present only in the basal membranes of the blood vessels and nerves. In comparison, collagen type V was observed in a fibrillar pattern in the gingival connective tissue, as well as the PL. In the PL, type V collagenous fibers demonstrated a parallel distribution with stronger fluorescence near the cementum surface. Collagen type VI could be demonstrated in fine fibers present in the gingival connective tissue and the PL. Blood vessels and nerves were not stained in the marmoset, but were in the rat, where a localization of collagen type VI was demonstrated in these areas. Alveolar bone and cementum, as well as the Sharpey's fibers embedded in these tissues, were not stained with antibodies against collagen type V and type VI, but a pericellular localization of these collagenous components could be observed. Collectively, these results provide basic information on the relative distribution of different collagen types in normal tissues of rats and marmosets that will be required for future studies on the effects of pathological, reparative and regenerative processes. PMID- 1714954 TI - [Diarrhea and biogenic amines: regional changes in brain histamine concentration and serotonin metabolism following castor oil-induced diarrhea in rats]. AB - Regional changes in concentrations of histamine (HA), serotonin (5-HT) and 5 hydroxyindoleacetic acid (5-HIAA) in the rat brain were investigated after diarrhea induced by castor oil. Significant decreases in body weight were observed from the 3rd day after daily oral administration of castor oil (2.5 ml/kg). HA concentrations in most brain regions decreased in diarrhea induced by a single administration of castor oil. A significant decrease was recognized particularly in the case of the hippocampus. The influence has begun to appear in the thalamus and hypothalamus in consecutive (3 d) administration. HA concentration in the striatal and hypothalamic regions of the rat treated with castor oil for 9 d significantly decreased in comparison with the control group. On the other hand, an inhibition of 5-HT turnover was observed in the thalamus at 3 h after a single administration of castor oil. However, this inhibition was not found in rats treated with castor oil for 3 d. Moreover, 5-HT concentration in the midbrain significantly decreased in rats that acquired the adaptability for the occurrence of diarrhea. These data present a new finding that the occurrence of diarrhea or acquisition of adaptability for diarrheal occurrence affects the central histaminergic or serotonergic neuron system. PMID- 1714955 TI - Sperm immobilizing and fertilization-blocking monoclonal antibody 2C6 to human seminal plasma antigen and characterization of the antigen epitope corresponding to the monoclonal antibody. AB - A monoclonal antibody (Mab 2C6) with strong sperm immobilizing and agglutinating activities was generated by cell fusion between spleen cells from a mouse immunized with human seminal plasma (HSP) and mouse myeloma cells. It also showed a strong inhibitory effect on human sperm-egg interaction. The corresponding antigen was present on the whole surface of ejaculated spermatozoa. In male genital organs, immunostaining with Mab 2C6 was observed in epididymis and seminal vesicle but not in testis. By Western blotting, immunostaining with Mab 2C6 was detected around the 15-25 kDa region under both reducing and non-reducing conditions. The antigen corresponding to Mab 2C6 was susceptible to treatment with periodate or trifluoromethanesulfonic acid. The antigenic activities were slightly increased by treatment with neuraminidase but reduced by further treatment with glycosidases. Enzymatic digestions with pronase and papain also reduced the antigenic activities. The antigen molecules exhibited a strong binding affinity to RCA lectin. These results indicated that Mab 2C6 recognized one of the components which might be secreted from epididymis or seminal vesicle and bind to ejaculated spermatozoa as a sperm coating antigen. The corresponding antigen seems to be a glycoprotein and its carbohydrate moiety has an important role in the conformation of the antigen epitope. PMID- 1714956 TI - Novel gonadotropin-releasing hormone antagonists: peptides incorporating modified N omega-cyanoguanidino moieties. AB - In order to minimize the deleterious effects of histamine release resulting from the administration to rats and humans of some potent gonadotropin-releasing hormone (GnRH) antagonists, various arginine residues were replaced with the less basic N omega-cyano-N omega-alkyl- or -arylhomoarginine, -arginine, or -p aminophenylalanine and N omega-triazolyllysine, -ornithine or -p aminophenylalanine residues in active analogues. These novel analogues were synthesized on a solid-phase support via a two-step modification of the N omega NH2 of lysine, ornithine, or p-aminophenylalanine residues in otherwise protected resin bound peptides. Most analogues were tested in the rat antiovulatory assay (AOA) and three in vitro assays; a pituitary cell culture assay, a binding assay to pituitary cell membranes, and a histamine release assay. Introduction of the cyanoguanidino and N omega-triazolyl moieties into GnRH analogues yielded several water-soluble antagonists which showed a desirable therapeutic ratio (low histamine release activity to high in vivo potency). Among them, "Azaline" (10, [Ac-DNal1,DCpa2,DPal3,Lys5(atz), DLys6(atz),ILys8,DAla10]GnRH), inhibited ovulation in the rat by 90% at 2 micrograms/rat with an ED50 in the in vitro histamine release assay comparable to that of GnRH itself. PMID- 1714957 TI - Pseudopeptide analogues of substance P and leucine enkephalinamide containing the psi (CH2O) modification: synthesis and biological activity. AB - The isosteric methyleneoxy psi (CH2O) function was employed as a novel peptide bond surrogate and incorporated into sequences of two neuropeptides, substance P (SP) and enkephalin. A pseudopeptide analogue [pGlu6,Phe8 psi(CH2O)Gly9]SP6-11 (7) of SP related C-terminal hexapeptide [pGlu6]SP6-11 and two pseudopeptide analogues of [Leu5]enkephalinamide, [Tyr1 psi (CH2O)Gly2, Leu5] enkephalinamide (11) and [Gly2 psi (CH2O)-Gly3, Leu5]enkephalinamide (17), were synthesized. The N alpha-protected pseudodipeptidic units were incorporated in the appropriate peptide sequences by using conventional coupling methods in solution. Compound 7 was a potent agonist (EC50 = 4.8 nM) of substance P as compared to the parent peptide [pGlu6]SP6-11 (EC50 = 1.2 nM), in stimulating contraction of the isolated guinea pig ileum (GPI). Analogue 7 was more potent on the neuronal (NK-3) than on the muscular (NK-1) tachykinin receptors in the GPI as shown by the ratio of activities, EC50 (NK-1)/EC50 (NK-3) = 3.16, thus displaying an improved selectivity for the NK-3 tachykinin receptor subtype as compared to that of [pGlu6]SP6-11, EC50 (NK-1)/EC50 (NK-3) = 0.44. In the rat vas deferens (RVD) assay, a typical NK-2 system, the pseudopeptide analogue 7 was (EC50 = 2 microM) 10-fold more potent than the parent peptide and 20-fold less potent than eledoisin, an NK-2 selective tachykinin. The pseudopeptide enkephalin analogue 17 had low biological activity when tested in the electrically induced GPI (EC50 = 2.3 microM) and was inactive in the mouse vas deferens (MVD) assay. In the rat brain membrane (RBM) binding assay analogue 17 had low affinity (in the micromolar range) for both the mu and delta binding sites. In contrast, analogue 11 was a potent enkephalin agonist (EC50 = 30 nM), being equipotent to [D-Ala2, Leu5]enkephalinamide (DALE) in the GPI assay. In the MVD, analogue 11 showed a substantially reduced activity (EC50 = 92 nM), being about 10-fold less potent than DALE. In the RBM binding assay analogue 11 showed high affinity (in the nanomolar range) for both mu and delta binding sites with increased selectivity for the delta sites as shown by the ratio of the apparent affinities for both receptors, Ki (delta)/Ki (mu) = 2.1. The contribution of the modified peptide bonds in the mode of interaction of SP and enkephalin at their corresponding receptors is discussed. PMID- 1714958 TI - Solid-state and solution conformation of 3'-amino-3'-deoxythymidine, precursor to a noncompetitive inhibitor of HIV-1 reverse transcriptase. AB - The recent finding that 3'-amino-3'-deoxythymidine 5'-triphosphate is a noncompetitive inhibitor of the HIV-1 reverse transcriptase (Kedar, P.S.; et al. Biochemistry 1990, 29, 3603-3611), prompted an investigation of the conformation of 3'-amino-3'-deoxythymidine. An X-ray diffraction study has revealed that the glycosidic torsion angle of the nucleoside is in the less common syn region and this solid-state geometry is stabilized by a three-dimensional network of self associated hydrogen-bonded molecules. On the other hand, the aqueous solution conformation, as determined by 1H NMR, places the glycosidic torsion angle in the more usual anti region with the sugar in an equilibrium between C3'-endo and C2' endo puckering. The energy barrier between the solid-state and solution conformation is relatively low as was demonstrated by the MM2 calculations. PMID- 1714959 TI - Pertussis toxin stimulation of catecholamine release from adrenal medullary chromaffin cells: mechanism may be by direct activation of L-type and G-type calcium channels. AB - We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca2+]o-dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca(2+) channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca(2+)-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K+]o, nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba2+]o. All these data are consistent with the concept that PTX may act on Ca2+ channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca2+ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca2+ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX treated cells. Our measurements of whole cell inward Ca2+ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca2+ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca(2+)-channel conductance (6 nS/cell at 2.6 mM [Ca2+]o). PTX evoked the activation of a new class of Ca(2+)-selective channels (5 pS in 25 mM [Ca2+]pipet), which are rather insensitive to membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1714960 TI - Inner mitochondrial membrane anion channel is present in brown adipocytes but is not identical with the uncoupling protein. AB - Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8 +/- 8.7 pS (n = 16, 21 degrees C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 microM) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide binding, anion selective mitochondrial channel, that is not the UCP. PMID- 1714961 TI - Cl- channels in basolateral renal medullary membrane vesicles: IV. Analogous channel activation by Cl- or cAMP-dependent protein kinase. AB - We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl- concentrations in modulating the activity of Cl- channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, the cis and trans solutions face the extracellular and intracellular aspects of these Cl- channels, respectively. Raising the trans Cl- concentration from 2 to 50 mM increased the channel open time probability, raised the unit channel conductance, and affected the voltage independent determinant (delta G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990. J. Membrane Biol. 118:269 278). With 2 mM trans KCl, trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C PKA plus ATP) addition to cis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50 mM) trans Cl- concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl- channel activity by phosphorylation at or near the Cl(-)-sensitive site on the intracellular face of these channels. PMID- 1714962 TI - Evaluation of the sexual consequences of surgery: retrospective and prospective strategies. AB - To assess the impact of a stressor, it is desirable to evaluate affected individuals' status both prior to and following a stressful event. Because of the difficulties inherent in prospective designs, investigators often ask people who have experienced an aversive event to evaluate their prestressor adjustment retrospectively. Do such retrospective evaluations provide a reasonable alternative to prospective assessment? To answer this question we compared retrospective and prospective data gathering procedures in the evaluation of sexual adjustment after prostate surgery. One hundred fifty-two married males who had undergone prostatectomy for benign prostatic enlargement completed a battery of measures which evaluated pre- and postsurgical sexual adjustment either prospectively (i.e., before and after surgery) or retrospectively (i.e., ratings made after surgery of both pre- and postsurgical adjustment). Retrospective assessment indicated considerable sexual deterioration pre- to postsurgery. In subjects tested prospectively, however, the results showed that surgery had little impact on sexual adjustment. Moreover, direct comparisons of retrospective and prospective methodologies reveal that discrepancies are due to differences in evaluations of presurgery status, with retrospective evaluation yielding more favorable ratings than prospective assessment. The results highlight a variety of biases which may affect self-ratings of pre- and post-stressor adaptation and show that discrepancies associated with the two methodologies have important implications for understanding the impact of a stressor on adjustment. PMID- 1714963 TI - Conservation and potential role of RNA-instability motifs in urokinase gene 3' untranslated sequences. PMID- 1714964 TI - Is the Whipple procedure a better palliative option for pancreatic cancer? AB - In recent years, improved results with the Whipple operation have been reported because of improved case selection, thorough intraoperative assessment, aggressive nutritional management, and technical superiority. Ninety-four cases of pancreatic cancer using the Whipple procedure at the University of Oklahoma Health Sciences Center between 1980 and 1986 were reviewed. The median survival time for patients reviewed was 4.5 months; 1- and 2-year survival rates were 16% and 6%, respectively. No survivals at 5 years were observed. Those who underwent resection (Group A) survived 12.9 months with 1- and 2-year survival rates of 54% and 27%. Those undergoing bypass procedures (Group B) had a median survival time of 6 months, with 1- and 2-year survival rates of 16% and 4%. No statistical difference in survival distribution was observed between Groups A and B. The median survival time of patients receiving a staging laparotomy with no therapeutic intervention (Group C) was 2.3 months. Group D patients either refused abdominal exploration or demonstrated signs of inoperability. Surgical mortality in Groups A and B was 8% and 10%, respectively. We suggest that clinical Stage 1 and carefully selected Stage 2 cases of pancreatic cancer should be treated by pancreatoduodenectomy. Stage 3 and 4 patients warrant simultaneous duodenal-biliary by-passes. PMID- 1714965 TI - Serum amylase levels in homozygous sickle cell patients. AB - Serum amylase was evaluated in a group of 26 steady-state homozygous sickle cell (SS) patients and 11 age-matched HbAA controls. Half (50%) of the SS patients had values above normal while only two of the control group had slightly elevated values. The SS patients had a mean +/- SD value of 301.46 +/- 119.40 iu/L while the control group had 274.36 +/- 89.70 iu/L. The difference between these two values is statistically significant (P less than .05). Males in both groups had significantly higher mean serum amylase values. The values in the SS group did not show an association with age. Higher levels in SS patients suggest a predisposition to chronic pancreatitis. PMID- 1714966 TI - Peritoneal lavage enzyme determinations following blunt and penetrating abdominal trauma. AB - Diagnostic peritoneal lavage (DPL) provides a rapid and sensitive means of investigating the peritoneal cavity following blunt and penetrating trauma. However, its shortcomings include insensitivity in the early identification of isolated hollow viscus injuries. We have routinely assayed lavage amylase (LAM) and alkaline phosphatase (LAP) in acutely injured patients for more than 4 years to assess the contribution of lavage enzyme analysis to the overall accuracy of DPL. From 1,969 DPLs, LAM was analyzed in 1,881 (96%) and LAP in 1,734 (88%) of 1,536 blunt and 433 penetrating trauma cases. Of 28 patients with negative lavage by LRBC but LAM greater than or equal to 20 IU/L, 13 (46%) had clinically significant injury requiring laparotomy. Seventy-seven percent of these cases involved the small bowel. In this group, LAM greater than or equal to 20 IU/L had a sensitivity of 87%, specificity of 75%, and positive predictive value of 46% for significant intra-abdominal injury. Seven patients had LAM greater than or equal to 20 IU/L and LAP greater than or equal to 3 IU/L. These values had a sensitivity of 54%, specificity of 98%, and positive predictive value of 88% for significant abdominal injury. Elevations of LAM (greater than or equal to 20 IU/L) and LAP (greater than or equal to 3 IU/L) mandate laparotomy where the history is consistent with possible small bowel injury. Elevation of either enzyme alone should raise the suspicion of hollow visceral organ injury and warrant close observation. PMID- 1714967 TI - Choledochoscopic heat-probe therapy: an adjunctive palliative treatment for intrahepatic cholangiocarcinoma with hepatolithiasis. AB - From 1983 to 1988, 20 out of 440 patients undergoing surgery for hepatolithiasis were discovered to have proximal bile duct adenocarcinomas, an incidence of 4.54%. Pre- and intra-operative diagnosis in all patients was hepatolithiasis. Four patients had left lateral segmentectomies and were found to have intrahepatic cholangiocarcinoma on subsequent histopathologic studies. In 16 patients, choledocholithotomy and biliary drainage were performed requiring post operative choledochoscopy for removal of residual intrahepatic stones. Follow-up choledochoscopies revealed bile duct cancers in the biopsy specimens obtained. All patients refused any form of reoperation, eight patients accepted heat-probe therapy as an alternative, and were classified under group A. Group B patients (n = 8) refused any form of treatment whatsoever and agreed to periodic follow-ups and maintenance of external biliary drainage. At the half year follow-up, group A had a survival rate of 100%, whereas group B had a survival rate of 62.5%. At the end of one year, there was one survivor in group B, while group A has 2, 3 and 4 year survival rates of 50%, 37.5% and 12.5% respectively. This study has shown that gross and histological documentation of unsuspected proximal biliary cancers in hepatolithiasis patients are possible with the use of the flexible choledochoscope. Furthermore, we believe that choledochoscopic cauterization of tumors with the heat-probe is a reasonable palliative that can lead to appreciably longer survival rates. PMID- 1714968 TI - Somatostatin, substance P, prolactin and vasoactive intestinal peptide levels in serum and cerebrospinal fluid of children with seizure disorders. AB - Seventy patients aged from one month to 18 years with seizure disorders were classified into three groups: I. Patients who had hard control seizure attacks even under medication; II. those who had occasional seizure attacks (less than 6 times per year) and III. those who had no seizure attacks after receiving medication for at least one year. Blood samples were taken for somatostatin, substance P, prolactin and vasoactive intestinal peptide (VIP) assays. Lumbar puncture was made in 32 children and CSF samples were also assayed for neuropeptides. Somatostatin levels in serum were significantly elevated in group I and group II (P = 0.05, ANOVA) but not in group III and control group. Similar observations were made in substance P, prolactin and VIP studies. In CSF, the somatostation can better indicate the difference between epileptic and normal children (comparison with group I, P greater than 0.001; with group II, P less than 0.001; even with those who were seizure free after medication, P less than 0.05). In conclusion, the levels of several neuropeptides (somatostatin, substance P. prolactin, VIP) were elevated in children with seizure disorders both in serum and CSF. The present investigation provides a new category for the understanding of the pathogenesis, treatment as well as prognosis of seizure disorders. PMID- 1714969 TI - Effect of the addition of estrogen to medical castration on prostatic size, symptoms, histology and serum prostate specific antigen in 4 men with benign prostatic hypertrophy. AB - A total of 4 men with benign prostatic hypertrophy who underwent medical castration therapy with a long-acting gonadotropin-releasing hormone agonist (leuprolide) for more than 6 months elected to add an estrogen transdermal patch (0.05 mg. to the skin biweekly) to the leuprolide regimen. The average prostatic size (transrectal ultrasound), serum prostate specific antigen (PSA) levels and symptoms of prostatism were dramatically decreased with leuprolide alone. The addition of estrogen for 6 months did not result in any change in prostate size, symptoms or serum PSA levels over that seen with leuprolide alone. The development of squamous metaplasia was noted in 1 man with leuprolide alone and in 1 man after the addition of estrogen. Immunohistochemical staining with anticytokeratin 903 antibodies reveals that squamous metaplasia appears to arise from prostatic basal cells. We postulate that the target cell for estrogen action in the prostate is the prostatic basal cell. In the absence of androgen the only direct effect of estrogens is the induction of squamous metaplasia. PMID- 1714970 TI - Adenocarcinoma of the prostate involving 2 cell types (prostate specific antigen producing and carcinoembryonic antigen producing) with selective metastatic spread. AB - Of 3 patients with clinically localized adenocarcinoma of the prostate 2 were treated by radical prostatectomy and 1 was treated with radiation therapy. Serum prostate specific antigen (PSA) values were elevated before therapy. After treatment the PSA levels were decreased to zero. All 3 patients later had evidence of metastatic tumor spread to the liver with elevation of serum carcinoembryonic antigen but not PSA. Immunohistochemical staining of the 2 primary tumors from the prostatectomy specimens identified 2 cell clones, one immunoreactive to PSA and prostatic acid phosphatase (PAP) and nonimmunoreactive to carcinoembryonic antigen, and the other immunoreactive to carcinoembryonic antigen but not PSA or PAP. Biopsy of a hepatic metastasis in 2 patients confirmed anaplastic carcinoma of the carcinoembryonic antigen-producing cell type. Immunohistochemical staining of a lymph node metastasis identified the PSA producing cell type only. Such results suggest selective metastatic spread of each cell type to its own organ tropic site. Occasional carcinoembryonic antigen producing prostate cancers may metastasize to the liver. Serum carcinoembryonic antigen measurements occasionally may be useful in the management of certain prostate adenocarcinoma patients. PMID- 1714971 TI - Tumor induction in a rat model for ureterosigmoidostomy without evidence of nitrosamine formation. AB - Twenty rats were randomized into a vesicosigmoidostomy and an unoperated control group. In both groups the 24 hour excretion of secondary amines, nitrate, nitrite and nitrosamines was measured before and after gavage of proline and nitrate, piperazine and nitrate, N-nitrosoproline, mono-N-nitrosopiperazine. The urinary nitrosamine concentrations were not significantly different between both groups neither before nor after application of the several substances. Thirty rats were randomized into two vesicosigmoidostomy groups with and without antibiotic coverage and an unoperated control group. After ligation of distal rectum and mesosigmoid the rectosigmoids were removed. No significant concentrations of volatile nitrosamines could be measured in the rectosigmoid contents of the three groups. One hundred and twenty rats randomized into three groups following vesicosigmoidostomy received the potential nitrosamine antidotes sodium-2 mercaptoethane sulfonate or sodiumpentosan-polysulfate or acted as controls. 12/118 (10.2%) developed adenomas and 25/118 (21.2%) adenocarcinomas at the vesico-colonic anastomosis with no significant differences between the three groups concerning tumor incidence or mortality. The results show that colon carcinomas occur in a rat model for ureterosigmoidostomy without evidence for thus induced nitrosamine formation. This and the missing effect of nitrosamine antidotes suggest that other factors than nitrosation must be responsible for colon carcinogenesis following urinary diversion via intestine. PMID- 1714972 TI - Benign prostatic hyperplasia--video image analysis and its relationship to androgen and epidermal growth factor receptor expression. AB - Androgens are essential for the development and maintenance of the prostate. However, prostatic growth may be mediated by epidermal growth factor (EGF) and the expression of the EGF receptor (EGFR) may be influenced by the androgenic milieu. We have characterized the expression of cytosolic androgen receptor (ARc), nuclear salt extractable androgen receptor (ARn) and EGFR in 89 consecutive cases of benign prostatic hyperplasia, 84 of which were treated by transurethral prostatic resections. Image analysis morphometry was performed on the histological sections to determine the epithelial content of the gland. Our results indicate that there is a vast heterogeneity of receptor expression in benign prostatic hyperplasia. Expression of ARc ranged from zero to 1312 fmol/gm. tissue (mean +/- SD 265 +/- 290), ARn ranged from zero to 531 fmol/gm. tissue (mean +/- SD 145 +/- 98) and EGFR ranged from zero to 316 fmol/gm. tissue (mean +/- SD 121 +/- 76). A statistically significant association was found between expression of ARn and EGFR, and these were both significantly correlated with the epithelial content of the gland. PMID- 1714973 TI - [3H]bunazosin, a novel selective radioligand of alpha 1 adrenoceptors in human prostates. AB - The binding properties of a new radioligand, [3H]bunazosin, were studied in membranes of human prostates with benign prostatic hypertrophy (BPH). Specific binding of [3H]bunazosin was saturable, reversible, and of high affinity (Kd = 0.55 +/- 0.04 nM). The density of [3H]bunazosin binding sites (Bmax) was 676 +/- 33 fmol/mg. protein. [3H]Bunazosin rapidly associated with its binding sites in membranes of human prostates and reached steady state by 20 min. at 25C. The rate constants for association and dissociation of [3H]bunazosin binding were calculated to be 0.11 +/- 0.01/nM/min. and 0.05 +/- 0.02/min. (n = 4), respectively. Seven alpha 1 adrenoceptor antagonists competed with [3H]bunazosin for the binding sites in the rank order: R-(-)-YM-12617 greater than prazosin greater than SGB-1534 greater than bunazosin greater than terazosin greater than naftopidil greater than urapidil. In parallel studies with [3H]bunazosin, the Kd and Bmax values for [3H]prazosin binding in human prostates were slightly lower. There was a similarity in the potency and rank order of seven alpha 1, adrenoceptor antagonists for the inhibition of [3H] bunazosin and [3H]prazosin binding in human prostates. The new [3H]bunazosin binding assay in human prostates is remarkable for its low degree of nonspecific binding as compared to [3H]prazosin, especially at high ligand concentrations. Thus, [3H]bunazosin may become a useful radioligand for the further analysis of the alph 1 adrenoceptor binding sites in human prostates. PMID- 1714974 TI - Immortalization of human adult normal prostatic epithelial cells by liposomes containing large T-SV40 gene. AB - Simian virus SV40 has been widely used to immortalize epithelial cells of mammalian origin. We report here, for the first time to our knowledge, the immortalization of normal adult prostatic epithelial cells in culture by transfection of a plasmid containing SV40 genome with a defective replication origin (SV40 ori-) encapsulated into liposomes. These cells (PNT1) have now been cultured for more than 12 months, and shown to contain the SV40 genome. They express large T protein, present the phenotype of differentiated luminal prostatic cells (positive with antibodies to cytokeratin 18, 19, weakly positive for prostatic acid phosphatase and prostatic specific antigen, negative with anticytokeratin 14 and KL2 antibody). PNT1 cells contain high affinity receptors for dihydrotestosterone. These cells provide a useful tool to study the biology and the pathology of adult prostatic epithelial cells, specially to understand the steps leading to prostatic transformation. PMID- 1714975 TI - Purification and properties of gamma-glutamyl transpeptidase from the tissue of human benign prostatic hypertrophy. AB - The enzyme of gamma-glutamyl transpeptidase was purified to homogeneity from the tissue of human benign prostatic hypertrophy and its enzyme properties were studied. The enzyme activity was detected mainly in the luminal border of the epithelium lining ducts by histochemical staining. The enzyme was purified 759 fold that of the crude extract. The specific activity of the purified enzyme was 79,900 mU/mg protein. The following enzyme properties were obtained: Michaelis constant of the enzyme was 0.83 mmol/l. The molecular weight was 72 kDa, consisting of two subunits, 45 kDa and 27 kDa. The isoelectric point of the enzyme was 8.5. The optimum pH ranged from 8.2 to 8.5. By Concanavalin A sepharose affinity chromatography, more than 60% of the enzyme activity was eluted in the weakly bound fraction, suggesting biantennary complex sugar chain was the major type among the asparagine-linked sugar-chains of the enzyme. PMID- 1714976 TI - [Effect of lidocaine-epinephrine-dextran solution on the plasma epinephrine concentration during spinal surgery--how to avoid circulatory complications due to the use of epinephrine for hemostasis]. AB - Epinephrine infiltrated for hemostasis may cause adverse effect on the circulatory system and the effect can be potentiated by surgical stimulation. Local infiltration of lidocaine-epinephrine-dextran solution (LED) at the surgical site is advantageous for this purpose since nerve blocking action of lidocaine attenuates surgical stimulation and dextran suppresses the transfer of injected epinephrine to the blood stream, thereby reducing the adverse effect of epinephrine. LED, however, could be injected accidentally into vessels. This is one of the important causes of raising the plasma epinephrine concentration to a dangerous level. The author believes that the following maneuvers are helpful to avoid the rise of plasma epinephrine concentration to a dangerous level; 1) injecting not more than 0.05 ml.kg-1 at a time, 2) monitoring the arterial pressure waves and ECG during the injection by use of a continuous recording device, and 3) halting the injection immediately whenever sudden changes in the arterial pressure and in the height of the T wave in ECG were observed. More importantly, surgeon's cooperation is essential for anesthesiologists to execute these maneuvers. PMID- 1714978 TI - [Serum gamma-seminoprotein on patients suffering from urinary retention due to BPH]. AB - Serum gamma-Seminoprotein (gamma-Sm) of 33 patients suffering from urinary retention due to BPH was evaluated. In acute urinary retention gamma-Sm was 17.1 +/- 54.9 ng/ml (N = 33) and its positive rate was 72.7%. After indwelling urethral catherization for one week gamma-Sm fell to 3.7 +/- 3.6 ng/ml (N = 17) and its positive rate was 42.8%. gamma-Sm measured after one month was negative on all cases. It was showed that gamma-Sm in acute urinary retention increased and after releasing from urinary retention it was rapidly normalized. It is necessary to take into account the above mentioned findings on evaluation of gamma-Sm as a tumormarker of prostatic carcinoma. PMID- 1714977 TI - [Expression of Fc epsilon receptor 2 in minimal change nephrotic syndrome]. AB - In order to clarify the mechanism for elevation of serum IgE level in minimal change nephrotic syndrome (MCNS), we investigated Fc epsilon receptor 2 (Fc epsilon R2/CD23) expression, using fluorescein isothiocyanate (FITC) labeled anti CD20 monoclonal antibody, phycoerythrin (PE) labeled anti-CD23 monoclonal antibody and two-color flow cytometry. Moreover, serum IgE level was examined by radio-immunosorbent test. The subjects included 25 cases of MCNS, 17 cases of focal glomerular sclerosis (FGS), and 25 healthy volunteers as controls. The patients in the nephrotic stage of MCNS demonstrated significantly elevated levels of serum IgE, while those in the remission stage of MCNS showed no change in their serum IgE levels. In patients with nephrotics of MCNS, CD23+ cells, CD20+ CD23+ cells and CD23/CD20 ratio were significantly increased compared to normal controls. Furthermore, CD23/CD20 ratio in MCNS was significantly correlated with serum IgE level. Concerning FGS, there were no differences in serum IgE level and Fc epsilon R2 expression compared to those of normal controls. These results suggest that Fc epsilon R2 expression may be important in the mechanism of elevated serum IgE level in MCNS. PMID- 1714979 TI - [Hyperthermic treatment of benign prostatic hyperplasia by a prostathermer]. AB - Prostate thermotherapy consisting of 6 sessions of intrarectal 60 min irradiation of 915 MHz microwave generated by a prostathermer (Blodan, Isreal) was performed once or twice a week on 30 patients with benign prostatic hyperplasia. Excluding 2 cases who refused further therapy at the first session because of rectal discomfort, 28 cases were evaluated for the efficacy of the treatment. Good or fair improvement in subjective symptoms was observed in 24 (85.7%) of the cases. Nocturia was significantly relieved from 2.9 to 2.0 a night. Among the objective responses, no improvement was achieved as to residual urine or prostate size, while a significant increase of maximum or average flow rate on uroflowmetry was observed. Adverse effects were minor or moderate, including 2 cases of urethral bleeding and rectal discomfort, 2 cases of lower urinary tract infection and 1 case of hypotension, which occurred at the 5th session. The scoring criteria taking into account of both subjective and objective responses showed that the efficacy is good in 10 cases (33.3%) and fair or good in 20 cases (66.7%). These results indicated the usefulness of the prostathermer as one of non-surgical therapies for benign prostatic hyperplasia. PMID- 1714980 TI - [A case of retrovesical embryonal cell carcinoma]. AB - We report a case of an extragonadal germ cell tumor in the retrovesical region. The patient complained of a perineal and micturition pain. Urethrography, CT and MRI showed a retrovesical tumor protruding into the bladder. Alfa-fetoprotein was increased to 12,170 ng/ml. Bilateral testes did not contain any palpable mass by careful palpation. No tumor was detected by ultrasonography, either. Clinically, he was diagnosed as having a retrovesical extragonadal germ cell tumor associated with paraaortic lymph-nodes and bilateral pulmonary metastases. Although he was treated by combination chemotherapy (PVB and VAB-6 regimen) and irradiation, he died of carcinomatosis about 6 months after the admission. There was no evidence of tumor in bilateral testes on autopsy. This case was a second case of extragonadal germ cell tumor originating from the retrovesical lesion in the literature. PMID- 1714981 TI - [Treatment of ventricular arrhythmia resistant to sodium channel blockaders]. AB - The efficacy of antiarrhythmic agents of various groups (Na(+)- and Ca(2+) channel blockers, cordarone) was comparatively evaluated in the same patients with ventricular arrhythmias. Cordarone and Na(+)-channel blockers were equally effective, whereas verapamil was significantly less potent. Then the antiarrhythmic efficacy of verapamil and cordarone was assessed individually in rhythm disturbances responsive to Na(+)-channel blockers and resistent to their action. The efficacy of verapamil was significantly higher in patients resistant to Na+ blockers than in those responsive to them. The action of cordarone was equal in these two groups of patients, however, it occasionally abolished arrhythmias resistant to both Na(+)- and Ca(+)-current blockers. The findings suggest the pattern of choosing antiarrhythmic therapy in ventricular arrhythmias. PMID- 1714982 TI - [XI International Congress of Cardiologists (January 11-16, 1990, Manila, Philippines)]. PMID- 1714983 TI - [Multimodal treatment of pancreatic cancer]. AB - The experience with combined treatment of 122 patients with pancreatic cancer in the clinic of the Institute of Clinical & Experimental Surgery for the period of from 1988 to 1990 is summarized. Radical operations were performed in 54 patients, the palliative ones--in 68. After performance of a palliative operation, the patients underwent polychemotherapy, 5--in combination with UHF hyperthermia. To study the effect of chemotherapy on the immune system, the immunologic monitoring was carried out. It was established that under the influence of chemotherapy and hyperthermia, the indices of immunity in patients with pancreatic cancer improved. In cancer of the pancreatic head with involvement of pancreatic body, the performance of subtotal pancreatoduodenal resection with subsequent regional chemotherapy is expedient. Chemotherapy and hyperthermia in combination with creation of a shunt permit to improve the results of treatment of inoperable tumors. The use of regional chemotherapy in subtotal resection contributed to increase in 2-year survival of the patients. PMID- 1714984 TI - [Determination of carcinoembryonic antigen content as a diagnostic and prognostic test in colorectal cancer]. AB - Of the 120 patients with colorectal cancer, 24 had distant metastases of a tumor. In these patients, increase in the level of carcinoembryonic antigen (CEA) and ferritin in the peripheral blood serum was noted as compared with those in patients without tumor metastases (P less than 0.001). Measuring of CEA content in the peripheral and regional blood can be used in preoperative diagnosis of metastases of colorectal cancer. PMID- 1714985 TI - [The determination of the glycogen phosphorylase activity in blood serum]. AB - A method for measuring blood serum glycogen phosphorylase (GP) activity is described, informative at early stages of myocardial infarction. The method is sensitive and available for clinical biochemistry laboratories. It consists in preliminary purification of GP from serum proteins and metabolites by affinity chromatography in micro-columns and subsequent measurement of the activity in the eluate. The procedure involves selective GP sorption on starch, washing, and subsequent desorption with glycogen solution. GP activity is measured by the kinetic spectrophotometric technique, based on enzymic measurement of glucose-1 phosphate, the product of glycogen consumption reaction, at a wavelength of 340 nm. Conditions of serum GP chromatographic purification are modified in the suggested procedure, this improving the sensitivity of the enzyme measurement. Blood serum GP activities were measured in patients with various cardiac diseases -myocardial infarction (15 cases), angina of rest and effort (53), essential hypertension (30). Different methods of GP activity measurements are considered. Recommendations on the use of the described method, a sensitive test for the diagnosis of myocardial infarction, are given. PMID- 1714986 TI - [Determination of the esterase activity of serine proteinases using synthetic substrates]. AB - The authors suggest a highly sensitive rapid and simple method for measuring esterase activities of bovine pancreatic chymotrypsin, human neutrophilic cathepsin D, and elastase, and of human blood serum chymotrypsin-like esterase and elastase-like esterase activities, with fluorogenic synthetic ethers, amino acid derivatives, employed as substrates: N-benzoxycarbonylphenylalanine 4 methylumbelliferyl ester (Z-Phe-OMC) and tret-butyloxycarbonyl-1-alanine 4 methylumbelliferyl ester (BOC-Ala-OMC). PMID- 1714987 TI - [Immunochemical tests for the risk of inflammatory diseases]. AB - Immunochemical measurements of the blood serum acute-phase proteins (APP)--C reactive, pregnancy-related alpha 2-glycoprotein, lactoferrin, immunoglobulins A, M, and G, carried out in 153 healthy men and 46 children, has revealed that an elevated level of at least one, and, more so, of two or three APP evidences the presence of an inflammatory process in the examinee. In a number of cases enhanced APP production correlated with a marked reduction of IgA and IgG concentrations. Measurements of APP may be employed as available laboratory screening markers of nonspecific latent inflammation in adults and children. PMID- 1714988 TI - [Preparation of exact solutions of acids and bases]. AB - The procedure for the preparation of the necessary volumes of acid and base solutions in certain concentrations is described in brief, as is the determination of mass concentrations of the original acids and bases with the use of quantitative titrometric analysis. Mathematical formulae necessary for this work are presented. PMID- 1714989 TI - [A micromethod of determining the activity of factor XIII in whole blood]. AB - A simple, available, and sensitive micromodification of Sigg and Baluda's methods for measurements of Factor XIII activity is suggested with registration of the blood clot hemolysis degree in the urea. The method was tried in rabbits and guinea pigs during immunization against cholera. The results of Factor XIII activity measurements were found to be unrelated to blood fibrinogen level but they are in slight correlation with the blood fibrinolytic activity. PMID- 1714990 TI - [Complex forms of enzymes and prospects for their study (review of the literature)]. PMID- 1714991 TI - [A turbidimetric method of determining plasminogen]. AB - Plasminogen level in euglobulin plasma fraction was determined from the rate of the sample transparency changes in the course of fibrin clot formation and lysis. Natural plasmin substrate, fibrin is used for measuring plasminogen this improving the measurement specificity and accuracy. Errors due to high concentrations of fibrinogen and/or its degradation products in the sample influencing the amidolytic activity of streptokinase-plasminogen complex are ruled out. The method is available and does not involve the use of chromogenic substrate. PMID- 1714992 TI - [Characteristics of the cytogram in gastric mucosal epithelial dysplasia]. AB - Analysis of gastrocytograms of patients with chronic gastric diseases involving epithelial dysplasia of various gravity (as evidenced by histologic studies) helped detect signs of cellular atypia; the sum of these signs permits a cytologic diagnosis of epithelial dysplasia. Gastric carcinoma in the presence of severe epithelial dysplasia was diagnosed in 4.3 percent of patients with epithelial dysplasia followed up. Cytologic findings are recommended as an additional morphologic criterion when identifying subjects at risk of gastric carcinoma. PMID- 1714993 TI - [Cytologic diagnosis of papillomas and cancer of the bladder]. AB - Cystoscopy is the principal method in the diagnosis of bladder cancer and precancer, but though visual examination of the bladder cavity via a cystoscope is comparatively easy, early correct diagnosis is not always possible because of small volume of the bladder, urethral stricture, mucosal changes in the bladder. That is why so much attention is paid to the cytologic diagnosis of bladder conditions. To estimate the diagnostic value of the cytologic method for the recognition of bladder precancer states and cancer the authors have analyzed cytograms of 145 patients. Smears obtained by alcohol washing off were under study. Cytologic and cytochemical analysis of alcohol washings-off has proved to be a valuable extra method for the diagnosis of pretumor and tumor processes; along with cystoscopy and biopsy the cytologic method is recommended for wide practice, for its use will contribute to earlier detection of urologic conditions. PMID- 1714994 TI - [A new method of staining cytologic specimens for emergency studies]. AB - Staining of cytologic preparations with a new stain, heretofore not employed for this purpose, results in differentiated panchromatic high-quality staining of the preparations, achieved in few seconds. This method is used in emergency and planned cytologic investigations and in mass prophylactic check-ups. PMID- 1714995 TI - [Cytologic diagnosis of non-organ neoplasms in the small pelvis]. AB - Puncture biopsy specimens from 165 patients, 93 of these children, were examined to elucidate the potentialities and specific features of the diagnosis of inorganic neoplasms in the small pelvis. Cytologic diagnosis has proved to be a rational and sufficiently informative method, permitting a correct recognition of the process type in 83.6 percent of cases. Malignant tumors were correctly diagnosed in 94.1 percent of cases, benign and inflammatory processes in 45.6 percent. In the children teratoblastomas, neuroblastomas, and unclassified sarcomas predominated. PMID- 1714996 TI - [Cytologic diagnosis of malignant tumors of the cervix]. AB - Cytologic patterns of squamous-cell carcinoma, adenocarcinoma, malignant adenoma, and carcinoid of the cervix uteri are presented. Problems in the differential diagnosis of these conditions are discussed. Special attention is paid to the cytologic criteria for detection of intraepithelial and microinvasive squamous cell and glandular carcinomas. Cytologic investigation may essentially contribute to timely diagnosis of the condition. PMID- 1714997 TI - [Methods of isolating and identifying Campylobacter pylori (review of the literature)]. PMID- 1714998 TI - [Non-sporogenous anaerobes in the microflora of carious teeth]. AB - Microflora of carious cavities was examined in 38 children. The authors describe the methods for anaerobic microflora isolation and microflora composition. Obligate anaerobes make up 70 percent of the total number of microorganisms. Anaerobic microorganisms are isolated in associations in 84 percent of cases. Bacterial associations are analyzed in detail, and the ratio of obligate to facultative anaerobes is presented, which depends on the activity of the carious process. PMID- 1714999 TI - [Monospecific agglutinating sera for the identification of Yersinia strains]. AB - An original scheme of rabbit immunization with Yersinia O-antigen is suggested. Technology for preparation of monospecific Yersinia sera to be used in drop agglutination test on the glass has been developed. Field and laboratory trials evidence that the prepared sera are fit for serologic identification of Y. enterocolitica. PMID- 1715000 TI - [Control of microbial contamination of antiseptics and disinfectant solutions]. AB - Since various species of opportunistic and pathogenic bacteria and fungi are known to be present in working solutions of disinfectants and antiseptics, an original method for the detection of bacterial contamination of these solutions has been developed. This method has been introduced into practical activities of sanitary and epidemiologic stations and hospitals in Byelorussia as the principal element of sanitary inspection of antiseptic and disinfectant utilization. PMID- 1715001 TI - [Rapid determination of nitrate reductase in non-fermenting gram-negative bacteria]. AB - Experimental study of 22 types of nonfermenting gram-negative bacteria, carried out in 317 strains, has demonstrated a high accuracy and reproducibility of the rapid (taking but 3 hrs) measurement of nitrate reductase in these bacteria by a microtest in culture medium with 0.1 percent of NaNO3 in plates; 0.2 percent solution of rivanol and 12 percent hydrochloric solution are employed as the reagents for nitrates. PMID- 1715002 TI - [A rotational floating viscosimeter for hemorheological studies]. AB - The authors describe a modified Zimm-Krothers viscosimeter fit for hemorheological studies. The device is easy to make; it permits work within the range of shear rates from 1 to 100 sec-1 using but 1.5 ml of blood, plasma, or red cell suspension. The error of a single measurement is under 5 percent. PMID- 1715003 TI - [Cytomorphology of lymphoepitheliomas]. AB - Presents cytomorphologic criteria of lymphoepitheliomas of pharyngeal and thymic tonsils. Describes in detail the cytologic features of lymphoepithelioma peculiar epithelial cells and of lymphocytes that serve as the principal criteria in the cytologic diagnosis of the condition. PMID- 1715004 TI - [The diagnostic value of studying the N-acetyl-beta-D-glucosaminidase activity in urine (review of the literature)]. PMID- 1715006 TI - Effect of age and restricted feeding on polypeptide chain assembly kinetics in liver protein synthesis in vivo. AB - Polypeptide assembly rates during in vivo hepatic protein synthesis were studied as a function of age and restricted feeding in male rats. With ageing the time to assemble the average peptide in the liver of fully-fed rats significantly increased. In young rats maintained on a restricted feeding regime known to retard ageing, the time to assemble the average polypeptide was increased 2.5 times. With ageing the rate of peptide elongation increased so that at 2 years of age the underfed animals assembled peptides at a significantly faster rate than their age-matched controls. The rate of elongation of peptides during hepatic protein synthesis was shown to be directly dependent upon circulating T3 levels rather than the dietary status of the animal. On refeeding young diet restricted rats, polypeptide assembly kinetics did not immediately return to control values although the rate of protein synthesis was significantly increased. Total liver RNA content increased significantly in refed animals allowing for a greater rate of chain initiation to offset the slow rate of chain elongation. A period of 28 days of ad libitum feeding was required before assembly kinetics returned to control values and is probably indicative of a persistent impaired monodeiodination of T4 to T3. PMID- 1715005 TI - Changing pattern of CD5+ CD20+ double positive lymphocytes with ageing and cytotoxic chemotherapy. AB - In this cross-sectional clinical study, it was found that two subtypes of CD5+ B lymphocytes existed either with CD5-high and CD20-low or CD5-low and CD20-high expression, as determined by dual fluorescence analysis with fluorochrome-labeled monoclonal antibodies on a FACScan flowcytometer. In the normal healthy subjects (n = 20), the CD20 positive cells could be broken down into 3 subsets: CD5(2+) CD20+, 25.4 +/- 3.0% (mean +/- S.E.M.), CD5+ CD20(2+), 18.4 +/- 2.4% and CD5- CD20(2+), 56.2 +/- 2.7%. Similar values were observed in a group of patients (n = 29) suffering from a wide variety of benign or untreated malignant disorders. The CD5(2+) CD20+ subset was typically related to age (Spearman coefficient of correlation rho = 0.77, P less than 0.001 in healthy subjects and rho = 0.46, P = 0.02 in pathological cases). The CD5+ CD20(2+) subpopulation was a salient feature of newborns and little infants (n = 6, 75.4 +/- 2.4%, P less than 0.01). The CD5- CD20(2+) subset was characteristically depressed in patients treated with cytotoxics (n = 21, 41.2 +/- 3.6%, P = 0.001). As far as cytotoxic chemotherapy may represent a model of accelerated ageing, it is worth noting that, in patients treated with cytotoxics, the CD5 CD20 pattern was frequently disturbed in a hyperyoung or hyperaged picture. That age and cytotoxics can affect CD5 expression on CD20+ lymphocytes, suggests some specific B dysregulation and should be put together with the known emergence of autoantibodies, paraproteinemias and lympho-plasmocytic tumors with age and chemotherapy. PMID- 1715007 TI - [Palliative half-body irradiation in cancerous patients. Preliminary results]. PMID- 1715008 TI - [Whipple's disease manifested as a toxic syndrome and abdominal adenopathies]. PMID- 1715009 TI - Thiacetazone-induced hypersensitivity. PMID- 1715010 TI - Ruthenium red as a capsaicin antagonist. AB - Definition of the physiological and pharmacological properties of primary afferent neurons by the use of capsaicin and its analogues (e.g. resiniferatoxin) has represented one of the most active areas of research of the last decade (1-4 for reviews). In the past 3 years many important advancements have been made in this field, dealing with: a) discovery of the capsaicin (or 'vanilloid' receptor (5); b) discovery of capsazepine as a competitive receptor antagonist at the vanilloid receptor (6); c) definition of the cation channel coupled with the vanilloid receptor and the ionic basis for excitation and "desensitization" of primary afferents by capsaicin and related substances (7,8) and d) discovery of ruthenium red as a functional capsaicin antagonist. The aim of the present article is to briefly review the pharmacology of ruthenium red as a capsaicin antagonist and attempting to define the usefulness and the limits of this substance as a tool in sensory neuron research. PMID- 1715011 TI - Goals and rationale of cancer treatment. AB - OBJECTIVE: To provide an overview of the goals and rationale for cancer management. DATA SOURCES: Primarily the series "Changing concepts in the management of cancer" published in the Journal between December 1987 and September 1990 (see Box). STUDY SELECTION: Tumour types chosen for the series of articles were those with a high incidence, such as melanoma and tumours of the lung, breast and prostate, or curable tumours, such as acute leukaemias and lymphomas. DATA SYNTHESIS: Emphasis is placed on primary prevention, with lung cancer related to smoking as a model. Breast cancer serves as a model for secondary prevention and adjuvant therapy. The role of goal setting as the first step in cancer treatment is stressed. Some of the reasons why cure is now possible are discussed, as are criteria for the selection of tumours for adjuvant therapy and indications for starting palliative treatment. CONCLUSIONS: Cancer cure is no longer a myth but a reality. To overcome present barriers to the cure of some cancers, there is need for a better understanding of the biology of epithelial solid tumours including the mechanisms to overcome multiple drug resistance. The restoration of functional health after cure is attained is paramount. Quality of life as a measure of success should be included in all clinical trials. PMID- 1715012 TI - A patient's right to a good death. PMID- 1715013 TI - [Therapy of malignant pleural mesothelioma]. PMID- 1715014 TI - Effect of (+)-sparteine on nicotinic acetylcholine receptors in the neurons of rat superior cervical ganglion. AB - The effects of (+)-sparteine, a ganglionic blocking agent, on acetylcholine (ACh) induced membrane currents and on fast excitatory postsynaptic currents (EPSCs) were studied in the neurons of rat isolated superior cervical ganglion, with the whole-cell patch-clamp recording method and the two-electrode voltage-clamp method, respectively. (+)-Sparteine (2 microM) reduced the ACh-induced current caused by activation of nicotinic ACh receptors (AChRs) in a voltage-independent manner at membrane potentials of -50 mV to +30 mV, whereas its blocking effect increased at more negative membrane potentials. The dose-response relationship for ACh was modified by 2 microM (+)-sparteine at -50 mV and at -90 mV in a fashion typical for competitive rather than noncompetitive antagonists. The apparent mean open time of the AChR channel, as estimated from the power density spectrum of the ACh-induced current fluctuations at -90 mV, was not decreased by 2 microM (+)-sparteine, in contrast to what was observed with hexamethonium, the well known open-channel blocker for ganglionic AChRs. At higher concentrations, i.e., 5 microM and 10 microM (lower concentrations were not effective), (+) sparteine reduced the amplitude of the EPSC and the time constant of the EPSC decay. The former effect was voltage independent, whereas the latter effect was voltage independent at membrane potentials of -70 mV and more positive and increased at membrane potentials of -90 and -110 mV. These results suggest that (+)-sparteine produces in ganglionic AChRs a competitive blocking effect and, in addition, an open-channel blockade. The latter component probably provides a smaller contribution than does the former to the blockade by (+)-sparteine of the ACh-induced current. Conformational analysis of the (+)-sparteine molecule was performed, and the dimensions of the molecule were measured. Minimum dimensions of the space-filling profile for two conformers, high and low populated, were found to be 7.3 x 7.9 A and 6.8 x 7.5 A, respectively. Both profiles are larger than the channel profile at which the open-channel blockers have been suggested to bind, which may explain comparatively low open-channel-blocking activity of (+)-sparteine. PMID- 1715015 TI - Selective induction of cytochrome P450e by kepone (chlordecone) in primary cultures of adult rat hepatocytes. AB - Cytochromes P450b and P450e (IIB1 and IIB2, respectively) are two remarkably similar microsomal hemoproteins whose inductions in rat liver are generally believed to be coordinately controlled by such xenobiotics as phenobarbital. To critically examine this assumption, we used a new system of primary cultures of adult rat hepatocytes on Matrigel to evaluate whether organochlorine pesticides, as "phenobarbital-like" agents, directly induce these cytochromes in parallel in the liver parenchymal cell. For 14 of the pesticides we tested, as well as for phenobarbital, P450b and P450e mRNAs, measured on Northern blots, rose in concert as much as 58- and 6-fold, respectively, over the amounts in incubated control cultures. Kepone (chlordecone) treatment of the cultures increased P450e mRNA in a dose-dependent manner that disclosed a 10-fold greater potency, compared with cultures exposed to phenobarbital. Kepone also resembled phenobarbital in these experiments, in that there were dose-dependent increases in the amounts of hepatocellular P450p, P450pcn2, P450PB-1, P450f, and NADPH-cytochrome P450 oxidoreductase mRNAs. However, in the same kepone-treated cells, P450b mRNA or P450b immunoreactive protein was induced only slightly, if at all. In contrast, additions to the medium of mirex, a structural analog of kepone, effectively induced both P450b and P450e mRNAs and their proteins. Selective induction of P450e by kepone in the hepatocyte cultures, the first pharmacologic dissociation of the induction of P450b and P450e mRNAs and proteins, was not apparent in kepone-treated rats, where both P450b and P450e mRNAs were increased to equivalent extents. We conclude that the P450b and P450e genes may be expressed independently by process(es), possibly involving extrahepatic factors, that can be defined with the present hepatocyte culture system. PMID- 1715016 TI - Ethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in neuroblastoma cells and isolated adult mammalian neurons. AB - Recent studies indicate that ethanol (EtOH) potentiates ion current through the channel associated with the 5-hydroxytryptamine3 (5-HT3)-type serotonin receptor. The present study was designed to determine 1) whether such potentiation occurs in adult mammalian neurons expressing 5-HT3 receptors; 2) whether potentiation is selective for the 5-HT3 receptor, relative to other ligand-gated ion channels; and 3) possible mechanisms by which EtOH potentiates this response. EtOH potentiated 5-HT3 receptor-mediated ion current in freshly isolated nodose ganglion neurons at concentrations similar to those previously reported to be effective in neuroblastoma cells (25-100 mM). Current was blocked by the selective 5-HT3 antagonist ICS 205-930 even in the presence of EtOH, and current activated by a 5-HT3 agonist (2-methyl-5-HT) was potentiated by EtOH. Thus, EtOH appears to produce potentiation via an alteration in the function of 5-HT3 receptors and not through an independent effect. gamma-Aminobutyric acidA receptor-mediated Cl- current was not potentiated by EtOH in neurons in which potentiation of responses to 5-HT was observed. Methanol potentiated 5-HT3 receptor-mediated current with a potency lower than that of EtOH. Potentiation by EtOH decreased with increasing 5-HT concentration. In addition, EtOH increased the decay rate of current. EtOH did not alter the reversal potential of the 5-HT3 receptor-mediated current. These observations indicate that intoxicating concentrations of EtOH selectively potentiate 5-HT3 receptor-mediated responses by increasing the apparent potency of 5-HT for activating ion current. PMID- 1715017 TI - Ifenprodil blocks N-methyl-D-aspartate receptors by a two-component mechanism. AB - The inhibition of N-methyl-D-aspartate (NMDA) receptor channels by the vasodilatory and anti-ischemic agent ifenprodil was examined on cultured rat hippocampal neurons. Whole-cell and single-channel patch recordings were used. Ifenprodil inhibition of NMDA currents could be separated into two components, with IC50 values of 0.75 and 161 microM. The high and low affinity components were both voltage independent but could be separated by their kinetics and dependence on extracellular calcium and glycine. The maximal inhibition of inward current by ifenprodil (approximately 90%) was equally divided between the two components in 0.3 mM extracellular calcium and 500 nM glycine. The low affinity action of ifenprodil had rapid kinetics and appeared to result from allosteric inhibition of the glycine modulatory site on the NMDA receptor. The macroscopic kinetics of the high affinity component were slow. The rate of onset was concentration dependent, and complete recovery required 1-2 min. Unlike open channel blockers, ifenprodil block was not use dependent, and pre-exposure to ifenprodil also reduced subsequent NMDA responses. Low concentrations of ifenprodil were less effective after calcium-dependent inactivation of whole-cell currents, but the IC50 was unaffected, suggesting that calcium and ifenprodil act on a common set of channels. On outside-out membrane patches, ifenprodil reduced the frequency of channel opening without altering the single-channel conductance. Open time histograms of the large conductance events revealed two mean open times of approximately 2 and 8 msec, but only the duration of the long openings was decreased by ifenprodil. This effect was concentration dependent and revealed a blocking rate constant of 6 x 10(7) M-1sec-1. However, the proportion of current blocked by low concentrations of ifenprodil was larger in outside-out patches than in whole-cell recordings, suggesting that intracellular factors may influence ifenprodil efficacy. These results indicate that high affinity ifenprodil binding is extracellular and does not require agonist binding or channel opening. Because low concentrations of ifenprodil only partially inhibited the current and affected only the long openings, ifenprodil may promote a modal shift in channel gating. PMID- 1715018 TI - Glyburide-sensitive K+ channels in cultured rat hippocampal neurons: activation by cromakalim and energy-depleting conditions. AB - Previous studies in our laboratory have shown that cromakalim activates a tetraethylammonium-sensitive K+ current in cultured embryonic rat hippocampal neurons. This phenomenon was further characterized using whole-cell voltage-clamp and single-channel recording techniques. Glyburide (1-25 microM), an antagonist of ATP-sensitive K+ channels, produced a concentration-dependent depression of the cromakalim-activated current. In contrast, charybdotoxin (100 nM), an antagonist of some Ca(2+)-dependent and other K+ channels, not only failed to block the effect of cromakalim but actually produced a moderate enhancement of the cromakalim-activated K+ current. Neither glyburide nor charybdotoxin affected resting or voltage-activated K+ currents in the absence of cromakalim. Exposure of the cells to energy-depleting conditions (0.24 micrograms/ml oligomycin and 10 mM 2-deoxy-D-glucose) also activated an outward current. Single-channel recordings in the cell-attached configuration showed that cromakalim (100 microM) stimulated the opening of flickery single channels having a unitary conductance of approximately 26 pS and a prolonged burst duration (mean open time, approximately 131 msec); similar channel openings were observed in patches from cells exposed to energy-depleting conditions. In patches containing a single K+ channel, the open probability in the presence of cromakalim was approximately 0.6 and in the presence of energy-depleting conditions was approximately 0.8; in the absence of either of these treatments, channel openings were not observed. Glyburide produced a reversible inhibition of the channels activated by cromakalim and energy-depleting conditions. These data provide additional support for the existence of ATP-sensitive K+ channels in central neurons and indicate that the K+ channels whose opening is stimulated by cromakalim are likely to be of the ATP-sensitive type. PMID- 1715019 TI - Transactivation of pancreas-specific gene sequences in somatic cell hybrids. AB - Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT was expressed when transferred to pancreatic cell lines but not when transferred to GPT-fibroblast (L) cells or hepatoma cells. When the transformed L cells and hepatoma cells were fused with pancreatic cell lines, GPT was activated in the hybrid cells. Endogenous pancreas-specific genes from the L-cell and hepatoma parents were also activated in the hybrids. In addition, a pancreas-specific nuclear protein, PTF1, was produced in pancreatic and hybrid cells, correlating with GPT expression. The transformed L cells and hepatoma cells thus contained a nonexpressed construct which could be activated in trans by factors present in pancreatic cells. The hepatoma hybrid also continued to produce albumin, demonstrating the coexpression of liver and pancreas-specific genes in the hybrid-cell population. Cell lines carrying the amylase/elastase/GPT construct may be useful as a selection system for cloning of pancreatic transcription activators. PMID- 1715020 TI - Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation. AB - The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired. PMID- 1715021 TI - Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract. AB - Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc. PMID- 1715022 TI - Antifungal properties of the immunosuppressant FK-506: identification of an FK 506-responsive yeast gene distinct from FKB1. AB - FK-506 is a novel and potent antagonist of T-cell activation and an inhibitor of fungal growth. Its immunosuppressive activity can be antagonized by the structurally related antibiotic rapamycin, and both compounds interact with cytoplasmic FK-506-binding proteins (FKBPs) in T cells and yeast cells. In this paper, we show that FK-506 and two analogs inhibit vegetative growth of Saccharomyces cerevisiae in a fashion that parallels the immunosuppressive activity of these compounds. Yeast mutants resistant to FK-506 were isolated, and at least three complementation groups (fkr1, fkr2, and fkr3) were defined. These fkr mutants show no alteration in their levels of FK-506-binding activity. Likewise, strains carrying null alleles of FKB1 (the yeast gene coding for the FKBP) remain FK-506 sensitive, indicating that depletion of yeast FKBP is not sufficient to confer an FK-506 resistance phenotype, although fkb1 null mutants are resistant to rapamycin. FKB1 does not map to the three fkr loci defined here. These results suggest that yeast FKBP mediates the inhibitory effect of rapamycin but that at least one other protein is directly involved in mediating the activity of FK-506. Interestingly, the ability of FK-506 to rescue a temperature sensitive growth defect of the fkr3 mutant suggests that the FKR3 gene may define such a protein. PMID- 1715023 TI - Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly. AB - Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II. PMID- 1715024 TI - Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP. AB - The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN induced protein previously shown to be absent from mice of Gbp-1b genotype. PMID- 1715025 TI - Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. AB - The LINE-1 repeat family is interspersed throughout mammalian genomes and is thought to be the result of duplicative transposition of LINE-1 sequences via an RNA intermediate. This report describes a ribonucleoprotein particle with LINE-1 RNA in the mouse embryonal carcinoma cell line F9. This ribonucleoprotein particle is a potential intermediate in the transposition of LINE-1 in the mouse genome. PMID- 1715026 TI - Immunobiological properties of a recombinant simian retrovirus-1 envelope protein and a neutralizing monoclonal antibody directed against it. AB - We previously reported that an area encompassing amino acids 147-162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96-102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro. Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142-167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV 1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro. In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro. SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain specific and involves the recognition of envelope sequences unique to each strain. PMID- 1715027 TI - Monoclonal antibodies against human IgE. Identification of an epitope sharing properties with the high-affinity receptor binding site. AB - Three monoclonal antibodies to human IgE are described. One of them recognizes an epitope located within a region of 76 amino acids that has been shown to contain the Fc epsilon RI binding site. That epitope is shown to be susceptible to heating and to alkylation of cysteines involved in inter heavy chain bonds, but not to their reduction alone. In addition, this monoclonal antibody, although having a high affinity for free IgE, is unable to bind Fc epsilon RI-linked IgE. Based on these results, we discuss the possibility that the antibody recognizes the Fc epsilon RI binding site of the IgE molecule. PMID- 1715028 TI - Physical and molecular genetic analysis of Qa-2 antigen expression: multiple factors controlling cell surface levels. AB - We have examined the surface expression of Qa-2 on lymphocyte subpopulations and on splenocytes from inbred mouse strains by a radioactive binding assay using purified anti-Qa-2 antibodies and antibody fragments (Fab). Quantitative measurements by Scatchard analysis revealed that spleen cells from Qa-2high mice express (4-5) x 10(4) Qa-2 molecules/cell, whereas T lymphocytes have as high as (7-8) x 10(4) molecules/cell. In addition, it was determined that B lymphocytes express (5-6) x 10(3) molecules on their cell surface. The Qa-2 levels on anti CD3-activated T cells is 1.0 x 10(5) molecules/cell. Previous experiments have shown that the quantity of Qa-2 varies in a strain-specific fashion and may be classified into three groups: Qa-2high, Qa-2medium and Qa-2low. Our results indicated that Qa-2high strains express (4-5) x 10(4) Qa-2 molecules/cell, Qa 2medium strains (B6-K2, B10.A, A/J, BALB/c and DBA/2) express (1-1.7) x 10(4) molecules/cell, and Qa-2low strains (SWR/J and DBA/1) express no more than 6 x 10(3) molecules/cell. Detection of Q7 or Q9 mRNA by the polymerase chain reaction revealed that Qa-2high strains express two functional Qa-2 enconding genes, Q7 and Q9, whereas Qa-2medium and Qa-2low strains express either Q7 or Q9. These results strongly suggest that Qa-2 gene dosage contributes in part to the variation of Qa-2 levels on the cell surface. PMID- 1715029 TI - Mapping epitopic regions of cholera toxin B-subunit protein. AB - Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions. The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides. An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)--although in different order (Gln56-His57-Ile58)--in two parts of the B-subunit chain. Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein. Native holotoxin was more effective than native B-subunit. Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides- primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes. Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55-58, of the B-subunit amino acid sequence. Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein. Amino acid substitution revealed the essentiality of Gln and His residues to this epitope. Gly54 was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody. The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine. PMID- 1715030 TI - Sequence analysis of porcine immunoglobulin light chain cDNAs. AB - A porcine cDNA library was constructed using poly(A)+ RNA isolated from the spleen of an adult Minnesota miniature swine. Screening the library with antisera specific for porcine immunoglobulin light chains resulted in the selection and isolation of two recombinant clones, PLC18 and PLC3, which encode for kappa and lambda light chains, respectively. These cDNAs contain sequence information for a portion of the variable region and all of the constant region. The lengths of the constant regions are 105 amino acids for lambda and 108 amino acids for kappa. The deduced amino acid sequences of porcine immunoglobulin light chains share a high degree of homology with similar sequences from other species in both the fourth framework region and the constant region. PMID- 1715031 TI - Antigenic cross-reactivity potential of synthetic peptides immobilized on polyethylene rods. AB - A number of continuous epitopes of tobacco mosaic virus protein (TMVP) have been defined by the pepscan technique using polyclonal and monoclonal antibodies to TMVP as well as antisera raised against synthetic peptides. In general, the location of continuous epitopes agreed with the results of earlier studies with peptides synthesized by classical methods although there were some notable exceptions. Results obtained with the different types of antibodies used in this study indicated that a homology of three residues was sufficient to give rise to antigenic cross-reactions. In the case of antibodies raised against a peptide conjugated to ovalbumin, some unexpected cross-reactivities could be explained by assuming that antibodies to the carrier molecule recognized homologous tripeptide sequences in TMVP and ovalbumin. PMID- 1715032 TI - The immunological relationship of epitopes on major tree pollen allergens. AB - The major allergens of birch (Bet v I), alder (Aln g I), hazel (Cor a I) and hornbeam (Car b I) were investigated by means of high-resolution two-dimensional electrophoresis combined with immunoblotting. Eleven sera derived from patients allergic to birch pollen as well as mouse monoclonal antibodies BIP 1 and BIP 4, raised against Bet v I, were used as probes. Human IgE antibodies detected 10 spots in birch (Mr 17 kDa, pI 4.9-5.9); four spots in alder (Mr 18.5 kDa, pI 4.7 5.3); four spots in hazel (Mr 17 kDa, pI 5.0-5.8); and 12 + 7 spots in hornbeam (Mr 16.5 kDa, pI 4.9-6.6 and Mr 18 kDa, pI 5.2-6.7), respectively, representing major allergens. Each patient tested reacted in a similar fashion with the spot cluster(s) of a certain allergen. BIP 1 detected the same spot clusters as patients' IgE. BIP 4 reacted with the 17-, 18.5- and 18-kDa spots of birch, alder and hornbeam, but did not react with the 17-kDa spots of hazel and the 16.5-kDa spots of hornbeam. In inhibition experiments with birch pollen extract as inhibitor, IgE binding to Bet v I, as well as to Aln g I, Cor a I and Car b I was abolished, thus suggesting that IgE binding to major tree pollen allergens is confined to shared epitopes. These findings indicate that it might be sufficient to use only Bet v I for diagnostic procedures as well as for immunotherapy in patients with tree pollen allergy. PMID- 1715033 TI - Identity of BCA200 and c-erbB-2 indicated by reactivity of monoclonal antibodies with recombinant c-erbB-2. AB - BCA200 has been described as a 200,000 Mr monomeric cell surface glycoprotein associated with human breast cancer. Since the physical properties and cellular distribution of BCA200 resemble those of c-erbB-2, antibodies to BCA200 were tested for the ability to bind a recombinant protein containing the c-erbB-2 extracellular domain (erbB-2 ECD). Three antibodies to distinct epitopes of BCA200 reacted with erbB-2 ECD but not with a control protein expressed in a similar baculovirus lysate. Control myeloma proteins and antibodies to four other antigens did not react with erbB-2 ECD. A protein with the expected molecular weight for erbB-2 ECD was also immunoprecipitated by anti-BCA200 antibody 520C9. We conclude that BCA200 is another synonym for c-erbB-2. PMID- 1715034 TI - Lack of neurotoxicity after intra-raphe micro-injections of MDMA ("ecstasy"). PMID- 1715035 TI - The effects of buprenorphine in methadone-dependent volunteers. PMID- 1715036 TI - Cavernous angioma in the fourth ventricular floor--case report. AB - A rare case of cavernous angioma located in the fourth ventricular floor occurred in a 44-year-old female complaining of occipital headache, vomiting, diplopia, and dysarthria. Computed tomographic scans demonstrated a high-density area in the fourth ventricle and slight hydrocephalus. Magnetic resonance (MR) imaging showed a mixed intensity mass on T2-weighted images and high- or isointensity regions on T1-weighted images. The tumor was totally removed and histologically diagnosed as cavernous angioma. Postoperatively, ataxic gait, nausea, and vomiting disappeared gradually. MR imaging was useful to accurately evaluate the anatomic relationship between the lesion and the brainstem. PMID- 1715037 TI - Endarterectomy for persistent primitive hypoglossal artery--case report. AB - Persistent primitive hypoglossal artery, an anastomosis between the carotid artery and the vertebrobasilar system, is found in about 0.05% of cerebral angiograms. Though usually asymptomatic, it may occasionally cause ischemic disease. A 62-year-old male presented with left hemiparesis. Right carotid angiograms demonstrated a primitive hypoglossal artery originating from the internal carotid artery at the 2nd cervical spine. This artery supplied almost all blood to the basilar artery system. A marked stenosis extended from the origin of the internal carotid artery to the primitive hypoglossal artery. An endarterectomy of the internal carotid and primitive hypoglossal arteries was performed using a special internal shunt 46 days after the onset. Sudden arterial bleeding from the incised part of the internal carotid artery occurred 12 days after the operation. The carotid artery was resutured. The rupture of the carotid artery appeared to be caused by an infection of Pseudomonas aeruginosa, which was detected by culture of the chronic ear discharge. Rupture of the vessel wall due to infection is an important complication after endarterectomy. This is the second reported endarterectomy of the primitive hypoglossal artery. PMID- 1715038 TI - Arteriovenous malformation in the cerebellopontine angle presenting as hemifacial spasm--case report. AB - A 64-year-old female was admitted with a 6-year history of right hemifacial spasm. Neurological examination and precontrast computed tomographic (CT) scanning showed no abnormality. Vertebral angiography disclosed, however, a small arteriovenous malformation (AVM) in the right cerebellopontine angle. A postcontrast CT scan demonstrated a high-density area in the right cerebellomedullary junction which appeared as a flow-void signal on magnetic resonance images. A right retromastoid craniectomy was performed to separate an enlarged and tortuous loop of the right anterior inferior cerebellar artery from the right facial nerve using a Teflon-felt sheet. The AVM was not excised. Postoperatively, she was completely free of hemifacial spasm. PMID- 1715039 TI - Rotatostereoradiography: a new radiodiagnostic method--development of a new three dimensional radiodiagnostic device and evaluation in neurosurgical clinics. AB - The rotatostereoradiographic device uses an x-ray tube coupled with an image intensifier rotating through a 180 degree arc in 2.25 seconds. The rapidly rotating x-ray tube allows 180 degree-arc angiograms to be obtained with a single injection of contrast medium. Subtracted fluoroscopic angiograms can be viewed immediately after injection of the contrast medium with digital recording. These three-dimensional images are displayed on side-by-side monitors stereoscopically. The mortality and morbidity of subarachnoid hemorrhage can only be greatly reduced by surgical treatment of unruptured aneurysms and arteriovenous malformations detected by a wide survey of subarachnoid hemorrhage. Such a wide survey would be possible utilizing intra-arterial digital subtraction angiography via the ascending aorta and this new three-dimensional radiodiagnostic method. A fluoroscopic device must be used to allow easier manipulation of the catheter from the axillary or brachial artery. PMID- 1715040 TI - Cerebral blood flow measurements using stable xenon CT with very short inhalation times. AB - A noninvasive, simplified method using inhalation of stable xenon (Xe(s)) and computed tomographic (CT) scanning to estimate regional cerebral blood flow (rCBF) and regional partition coefficient (r lambda) is described. Twenty-four patients with cerebrovascular occlusive disease and six volunteer controls inhaled 30% Xe(s) and 70% oxygen for 180 seconds and exhaled for 144 seconds during serial CT scanning without denitrogenation. The end-tidal Xe(s) concentration was continuously monitored with a thermoconductivity analyzer to determine the build-up range (A value) and build-up rate constant (K value) for arteries with the curve fitting method. The time-CT number (Hounsfield unit) curve for cerebral tissue during the Xe(s) washin and washout phases was used to calculate r lambda and rCBF using least squares curve fitting analysis. The resultant r lambda and rCBF map demonstrated a reliable distribution between the gray and white matter, and infarcted areas. rCBF was high in gray matter, low in white matter, and much lower in infarcted areas than in white matter, r lambda was high in white matter, low in gray matter, and much lower in infarcted areas. Xe(s) CT-CBF studies with very short inhalation of 180 seconds is a clinically useful method for evaluation of rCBF in patients with cerebrovascular diseases. PMID- 1715041 TI - Subdural hematoma due to ruptured intracranial aneurysm. AB - Subdural hematoma (SDH) was observed in 15 of 484 cases of aneurysmal subarachnoid hemorrhage (SAH). There were four males and 11 females, with ages ranging from 39 to 75 years. The clinical grades (Hunt and Hess) on admission were 11 in three cases, III in two, IV in four, and V in six. The ruptured aneurysms were located in the middle cerebral artery (MCA) in six cases, anterior communicating artery in three, internal carotid artery in two, and distal anterior cerebral artery (ACA) in two, with two cases unconfirmed. A high proportion of aneurysms occurred in the MCA and distal ACA. Aneurysmal neck clipping and removal of SDH were performed in the acute stage of seven cases, without intraoperative rerupture. The outcomes 1 year after SAH of the seven patients undergoing surgery were good recovery in five, but in two, vegetative state due to preoperative rerupture or medical complications. All eight patients without surgical intervention died. A good prognosis for patients with ruptured intracranial aneurysms accompanied by SDH can be expected with direct surgical intervention in the acute stage, even if the clinical grade on admission is poor. PMID- 1715042 TI - Radical cranio-orbital reconstructive procedures for cloverleaf skull deformity in adult: operative technique for the longest survivor--case report. AB - Cosmetic cranio-orbital reconstructive surgery was carried out on a 22-year-old male, the longest surviving case of cloverleaf skull syndrome reported. He previously underwent classical linear suturectomy for synostotic sutures and temporal cranioplasty. Fortunately, hydrocephalus became arrested so did not require continuous cerebrospinal fluid drainage through shunt tube. His intelligence quotient was in the 40s. The present problem was mainly of cosmetic cranio-orbital corrections of shallow orbits with resultant exophthalmos, frontal dysgenesis, and marked temporal bossings. Bilateral orbital advance, lateral canthal/pterional reshaping, frontal remodeling, and temporal reduction cranioplasty were performed. The postoperative outcome was satisfactory. The cloverleaf skull deformity is etiologically and pathologically heterogeneous, so radical surgical reconstructive procedures should be planned and designed individually. PMID- 1715043 TI - Ciliated epithelial cell cluster in Rathke's cleft cyst--report of two cases. AB - The authors report two cases of symptomatic Rathke's cleft cyst. Cytological examination of the cyst contents identified clusters of ciliated epithelial cells. Rathke's cleft cyst is normally defined by mucoid fluid in the cyst cavity and cyst wall biopsy. However, intraoperative cytology was found useful in diagnosis and deciding operative strategy. PMID- 1715045 TI - Purine catabolites in cerebral interstitial fluid during progression of and recovery from ischemia. AB - The concentrations of purine catabolites in the cerebral interstitial fluid during progression of and recovery from ischemia were studied using brain microdialysis and high-performance liquid chromatography. Sealed 0.5-mm hollow dialysis fibers were stereotactically implanted into either the cerebral cortex or hippocampus of ketamine anesthetized gerbils and perfused with artificial cerebrospinal fluid at 2 microliters/min. Cerebral ischemia was induced by occlusion of the bilateral common carotid arteries. The reflectance spectra of oxy- and deoxyhemoglobin at the brain surface were monitored over the scalp to assess ischemia and confirm recirculation. Ischemia caused a rapid, 4.8-fold increase in the extracellular concentration of adenosine. The progressive increase in the concentration of adenosine, inosine, and hypoxanthine soon after recirculation is particularly interesting. The subsequent decrease in purine compound concentration was rapid for adenosine but more gradual for inosine and hypoxanthine. Calculated K values for adenosine deaminase and purine nucleoside phosphorylase were 0.045/min and 0.016/min, respectively. However, no xanthine, uric acid, or purine nucleotides were found in the perfusate. These observations indicated the presence of purine catabolites in the cerebral interstitial space as well as consecutive degradation and recycling involving the interconversion of purine compounds by biochemical metabolic pathways. PMID- 1715046 TI - Wrapping of intracranial aneurysms with gauze sponge. AB - A gauze sponge wrapping method to prevent recurrent subarachnoid hemorrhage (SAH) after incomplete obliteration of intracranial aneurysm or a residual neck after clipping was developed and tested. The authors performed the gauze wrapping for aneurysms with incomplete obliteration by clipping alone, abnormal changes of the parent artery wall which had the possibility of regrowth or rerupture, and surgical difficulties. The residual neck of the aneurysm or the abnormal arterial wall was tightly and completely wrapped, including the parent artery, and cemented with plastic adhesive. Seventy-eight (22.3%) of 349 surgically treated aneurysms were treated by gauze wrapping (26 wrapping only, 52 clipping and wrapping). The incidence of complications such as infection, angiospasm, and normal pressure hydrocephalus, and the clinical outcome were not significantly different for the wrapping and non-wrapping groups. No recurrent SAH was observed in the wrapping group during 3 months to 8.5 years follow-up. These results suggest that wrapping with gauze sponge is useful in the treatment for ruptured aneurysms which cannot be totally obliterated. PMID- 1715044 TI - Multiple brain tumors of different cell types with an unruptured cerebral aneurysm--case report. AB - A rare case of coexistent Burkitt-type lymphoma and meningioma associated with an unruptured cerebral aneurysm is presented. A 49-year-old male complaining of headache and right hemiparesis was admitted to our hospital. Neuroradiological examination revealed a multinodular mass in the left frontal convexity and an unruptured cerebral aneurysm at the M1 portion of the left middle cerebral artery. He underwent an operation for tumor removal and aneurysm clipping. Histological examination revealed the tumor to be a typical meningotheliomatous meningioma without malignancy. However, a second operation was necessary for another tumor invading into the left frontal lobe, which proved to be a Burkitt type lymphoma. The second tumor may have been resulted from irritative effect of the first tumor, but the aneurysm was considered purely coincidental. PMID- 1715047 TI - Multiple primary brain tumors of different histological types--report of two cases. AB - Simultaneous development of histologically different primary brain tumors aside from phacomatoses or previous irradiation is rare, and its preoperative diagnosis is still difficult. We report two such cases, a 49-year-old male with an acoustic neurinoma and a cerebellar hemangioblastoma and a 69-year-old female with a parietal convexity meningioma and an ipsilateral frontal lobe astrocytoma. The tumors in the first case developed closely, so some local stimulation probably acted as a developmental factor. However, the tumors in the second case developed in distant areas and were considered coincidental. PMID- 1715048 TI - Siblings with malignant glioma--report of two cases. AB - The authors report malignant glioma occurring in two sibling cases. The elder brother presented with right hemiparesis and hemihypesthesia at 14-year-old. Computed tomographic (CT) scanning demonstrated a low-density area in the left frontoparietal lobe. The tumor was partially removed. Histologic diagnosis was glioblastoma multiforme. Radiation therapy was given postoperatively, but he died due to tumor recurrence 15 months after onset. The younger sister was admitted comatose due to intratumoral bleeding at 19-year-old. CT scans showed a high density area in the right temporal lobe. The tumor was excised subtotally. Histological diagnosis was malignant astrocytoma (grade III). Radiation therapy, chemotherapy (ACNU), and immunotherapy (interferon) were given postoperatively, but she died due to recurrence 34 months after onset. PMID- 1715049 TI - Choroid plexus metastasis of lung carcinoma--case report. AB - Metastatic tumors in the choroid plexus are generally considered to be very rare. The authors present a case of lung large cell carcinoma with a single metastatic tumor in the choroid plexus of the lateral ventricle trigone. Precontrast computed tomographic (CT) scans showed an isodensity mass with extensive peritumoral edema, which was considerably enhanced on the postcontrast CT scans. Magnetic resonance (MR) images demonstrated the mass as a low-intensity area on the T1-weighted image and an iso-intensity area on the T2-weighted image. The tumor was clearly differentiated from the peritumoral edema by both CT and MR imaging. The diagnosis was confirmed by surgery. PMID- 1715050 TI - Isolated cerebral varix with magnetic resonance imaging findings--case report. AB - A rare case of isolated cerebral varix of the left deep sylvian vein was discovered incidentally in an 11-year-old boy by computed tomographic scanning, magnetic resonance (MR) imaging, and cerebral angiography. MR imaging was most useful in diagnosis of cerebral varix. Review of 21 similar reported cases shows no significant in age, sex, location, or size character. PMID- 1715051 TI - Multiple cerebral infarcts caused by emboli from carotid atheroma--a case confirmed by indium-111 platelet scintigraphy. AB - A 47-year-old male presented with frequent transient ischemic attacks (TIAs) of weakness of the left upper extremity. Computed tomography demonstrated multiple infarcts in the right cerebral hemisphere. Cerebral angiography showed marked atheromatous changes at the siphon of the right internal carotid artery, characterized by wall irregularities and heterogeneous filling. Indium-111 labeled platelet scintigraphy demonstrated abnormal tracer accumulation at the right carotid siphon. Following right carotid artery ligation, this abnormal platelet deposition was resolved and no further TIA was experienced. This suggests that the release of microthrombi from thrombogenic carotid atheroma contributes to embolic stroke. PMID- 1715052 TI - Extravasation from anterior choroidal artery in a child with moyamoya disease- case report. AB - A rare association of moyamoya disease with intra- and periventricular hemorrhage in an 8-year-old girl is presented. Angiography showed that the cause of hemorrhage was extravasation from the dilated anterior choroidal artery. Ventriculoperitoneal shunt and encephalomyosynangiosis have prevented clinical or neurological deficits for over 6 years. PMID- 1715053 TI - Delayed onset of intraventricular hemorrhage following removal of acute subdural hematoma--case report. AB - A 68-year-old male presented with neurological deterioration after a lucid interval following head trauma. Computed tomographic (CT) scans on admission demonstrated a subdural hematoma in the right frontotemporal region accompanied by subarachnoid hemorrhage in the right Sylvian and interhemispheric fissures. The subdural hematoma was removed via a right frontotemporoparietal craniectomy. However, immediate postoperative CT scans revealed hemorrhage in the third and both lateral ventricles, apparently separate from the primary hemorrhages. Decompressive rupture of damaged subependymal veins is suggested as the cause of the delayed traumatic intraventricular hemorrhage. PMID- 1715054 TI - Unusual posttraumatic porencephaly--case report. AB - An unusual case of posttraumatic porencephaly preceded by neither overt cerebral contusion nor hemorrhage is reported. The cerebral cortex just above the porencephalic cyst was found intra-operatively to be partially herniated into a fracture line, while the cortex elsewhere was completely intact. The porencephalic cyst communicated with the lateral ventricle. Apparently, brain herniation and the cyst-ventricle communication can be causative factors in the occurrence and growth of posttraumatic porencephaly. PMID- 1715055 TI - Human fetal adenohypophysis: morphologic and functional analysis in vitro. AB - Pituitaries from 24 human fetuses at 7-20 weeks of gestation were studied in culture to assess hormone release, response to adenohypophysiotropic hormones and cytodifferentiation by electron microscopy in cultures lasting 4-8 days and in some cases up to 28 days. At 7 weeks of gestation, ACTH was released by cultured cells which included recognizable corticotrophs. GH was released by cells cultured from 8- to 9-week fetuses and densely granulated somatotrophs were present in the cultures. alpha-Subunit of glycoprotein hormones was present in cultures from 10-week fetuses and TSH and LH were released from 12-week fetuses. FSH was found in cultures of a 13-week female fetus but not before 14 weeks in cultures from males. The levels of FSH and LH were higher in media from cultures of females than from those of males at all ages from detection to 20 weeks, whereas alpha-subunit was slightly higher in media from males. While cells with features of the glycoprotein hormone cell line were found in cultures from 10 week fetuses, no characteristic thyrotrophs or gonadotrophs were recognized. PRL was not measured in basal incubations before 14 weeks. The amounts of all hormones released were proportional to fetal age and decreased with duration of culture. Cortisol suppressed ACTH release in cultures from 7- to 8-week fetuses. Responsiveness of GH release to GRH/SRIH, of ACTH to CRH, and of FSH to GnRH, was found at 12 weeks; LH stimulation by GnRH and TSH response to TRH were documented at 14 weeks. Increments of gonadotropin release during incubation with GnRH were greater in cultures from females than in those from males. PRL release responded to GRH stimulation and to SRIH inhibition in parallel with GH; this behavior is consistent with production of both hormones by mammosomatotrophs. The onset of hormone release by cultured human fetal pituitaries correlates with the detection of hormones biochemically and immunohistochemically. Responsiveness of fetal adenohypophysial cells to hormonal influences indicates functional maturity early in gestation. PMID- 1715056 TI - Presence of substance P and angiotensin II in corticotropic cells of the lizard Gallotia galloti: immunochemical study in the adult and during ontogenesis. AB - The hypophysis of the lizard Gallotia galloti showed substance-P-like immunoreactivity in both the adenohypophysis (pars distalis, PD; pars intermedia, PI) and the neurohypophysis (median eminence and pars nervosa), whereas angiotensin-II-like immunoreactivity appeared only in PD and PI. The elution restaining procedure has allowed us to demonstrate the colocalization of both peptides with adrenocorticotropic hormone (ACTH) in PD and PI cells. Electron microscopic study revealed the presence of substance P immunoreactivity on ACTH secretory granules. The ontogeny of both peptides in corticotropic cells has been studied, revealing that the presence of substance P in ACTH-containing cells of the PI occurs from the embryonic stage 33 (S 33), whereas in the PD it occurs from S 34, coinciding with the appearance of ACTH within the same cells. In both median eminence and pars nervosa of the neurohypophysis, substance P appeared later in development, at S 38. Angiotensin II immunoreactivity in PI cells first appeared at S 38, while in PD it appeared from S 40. PMID- 1715057 TI - Ultrastructural changes in the juxtamembranous layer of ganglionar neurons with orthodromic pessimal stimulation. PMID- 1715058 TI - Comments on a paper by Peters and Large published in Pain, 41 (1990) 283-293. PMID- 1715059 TI - Diastolic potentials recorded by surface electrocardiographic signal averaging during sustained ventricular tachycardia: possible origin from the reentrant circuit. AB - ECG signal averaging can detect low amplitude diastolic potentials in sinus rhythm. We, therefore, recorded signal-averaged ECGs during eight episodes of inducible uniform sustained VT with coincident atrial pacing to look for continuous diastolic electrical activity. Simultaneous AV pacing in seven patients served as controls. The number of QRS complexes averaged (187 +/- 47 vs 183 +/- 63), the noise level (1.26 +/- 0.88 vs 1.39 +/- 0.47) and cycle length (385 +/- 52 vs 404 +/- 40) did not differ between VT and paced recordings. In each lead the difference in onset between the unfiltered surface recording and the filtered data (40 Hz bidirectional) was significantly greater in VT than the paced recordings (25 +/- 16 vs 11 +/- 8 msec, P = 0.0012). These late diastolic (pre-QRS) potentials were greater than 15 msec duration in 65% of the leads in VT versus 20% of paced recording (P = 0.021). The maximum value was greater than 20 msec in six VT (75%) versus one (14%) paced recording (P = 0.019). The earliest filtered onset in any lead preceeded the earliest surface activity by greater than 12 msec, in 6 VT versus one paced recording (P = 0.019). Early diastolic (post-QRS) potentials were also longer in VT than pacing (49 +/- 40 versus 5 +/- 20, P = 0.001) and exceeded 38 msec in seven of the VTs but none of the paced recordings (P = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715060 TI - The interaction of antiarrhythmic drugs and the energy for cardioversion of chronic atrial fibrillation. AB - Antiarrhythmic drugs may alter the energy for cardioversion of ventricular arrhythmias. This study compares the energy necessary for cardioverting chronic atrial fibrillation in 57 patients taking type Ia, Ic, or type III antiarrhythmic drugs. Patients taking Ia (n = 22) or III (n = 14) drugs had a median energy for cardioversion of 100 joules, while the patients taking Ic (n = 17) drugs had a median energy of 200 joules (P = 0.03). There were no differences in the frequency of unsuccessful cardioversion. There were no serious adverse events in any of the three groups, although three patients in the Ic group had greater than 3 second pauses after the shock. The data suggest that the use of Ic antiarrhythmic drugs results in a higher energy for cardioversion of atrial fibrillation. However with higher energies, conversion is as successful as for type Ia and type III. PMID- 1715061 TI - Catheter ablation of ventriculoatrial conduction in the treatment of pacemaker mediated tachycardia. AB - Pacemaker generator reprogramming does not always present an adequate solution to the problem of pacemaker-mediated tachycardia. A case is described where direct current shock catheter ablation of ventriculoatrial conduction prevented pacemaker-mediated tachycardia and allowed optimal dual chamber pacemaker programming. PMID- 1715062 TI - The pulmonary artery lasso: epicardial pacing lead causing right ventricular outflow obstruction. AB - Permanent pacing in small children may require placement of an epicardial pacing system. This report describes a young child who underwent pacemaker implantation with epicardial ventricular lead placement in infancy as an adjunct to antiarrhythmic therapy for congenital junctional ectopic tachycardia. At 5 years of age, a harsh systolic murmur was detected for the first time. Evaluation by catheterization and transluminal echocardiography showed right ventricular outflow obstruction (pressure gradient 40 mmHg) secondary to extrinsic compression by the epicardial lead. Surgical removal of the lead relieved the obstruction. PMID- 1715063 TI - Diagnosis of early cardiac transplant rejection by fall in evoked T wave amplitude measured using an externalized QT driven rate responsive pacemaker. AB - Reliable diagnosis of cardiac allograft rejection is at present only possible using endomyocardial biopsy. We have serially measured epicardial evoked T wave amplitude during ventricular pacing with an externalized QT driven rate responsive pacemaker telemetered to a TP2 analyzer in 13 patients (12 males) followed for 19 (14-26) days after transplantation. A total of 228 records were analyzed. Rejection was defined on endomyocardial biopsy. On 17 of the 31 occasions on which biopsy was performed during the study, specimens showed significant (moderate) rejection. In 11 patients the initial biopsy proven rejection episode was associated with a significant fall in the evoked T wave amplitude from 1.3 (0.7-2.3) mV to 0.6 (0.5-1.8) mV (P less than 0.005), which began 2 (1-4) days earlier. One patient with uncontrolled diabetes mellitus had no change in evoked T wave amplitude during rejection. The evoked T wave amplitude did not fall in the absence of histologic rejection. These results suggest a noninvasive method for detecting cardiac rejection, which appears both sensitive (92%) and specific (100%) in the first rejection episodes. PMID- 1715064 TI - Wenckebach periods in sinoatrial block: experimental and clinical evidence. AB - The reality of sinoatrial Wenckebach periods (WP) has been suggested, but not proven, in the literature. We report experimental and clinical data showing WP in sinoatrial blocks. Experimental sinoatrial blocks were induced by superfusion of bepridil (10(-5) M) in 15 preparations of isolated rabbit right atria. Different types of block were observed, including Blumberger I block, i.e., sinoatrial WP. The recordings showed that the typical pattern of Blumberger type IA block and sinoatrial WP may be due to transient acceleration of the sinus rate, without change in the increment. We also observed sinoatrial WP in a 72-year-old patient on direct recordings of the sinus node (SN) electrical activity. In this case, transient acceleration of the sinus rate also seemed to be involved in the genesis of sinoatrial WP. Analysis of these clinical and experimental data showed similarities that may explain the mechanism of the WP. Usually type IA Blumberger block is said to involve a decrease in the sinoatrial increment to explain the atrial sequence (decrease in PP interval followed by a pause shorter than twice the value of the preceding cycle, then a cycle longer than the one preceding the pause). In fact, this pattern can be observed when the acceleration of the higher structure, the SN, induces block within the lower structure, i.e., the atrium. PMID- 1715065 TI - Amplitude and direction of atrial depolarization using a multipolar floating catheter: principles for a single lead VDD pacing. AB - VDD stimulation using a single catheter for atrial sensing and ventricular sensing and pacing has become a reality. In order to compare the quality of the cavitary atrial electrogram (AEG) and to determine the intraatrial P wave direction and conduction time (CT), we compared, in an acute study, three different types of atrial electrode systems using four different leads, in 53 patients in sinus rhythm. The three electrode systems were: (1) one experimental system with quadripolar orthogonal electrodes using the Goldreyer concept; (2) one experimental system with quadripolar whole ring electrodes; (3) two systems with diagonally oriented half-ring electrodes, one experimental quadripolar and one bipolar CCS commercial (Polysafe A-Track lead). For the experimental systems, the four electrodes forming two independent bipolar pairs were situated on the intraatrial floating portion of a single lead and one supplemental electrode was distally positioned in the right ventricular apex. Bipolar AEGs were recorded at the high and at the low levels of the right atrium. For the CCS lead, the single bipolar AEG was recorded at the high level of the right atrium only. The highest AEG amplitude and the highest values for ventricular far-field rejection were provided by both diagonally oriented half-ring electrodes at the high atrial level and by the whole ring electrodes at the low atrial level. For both atrial levels, the orthogonal electrode system provided the smallest AEG amplitudes, the highest ventricular electrogram amplitudes, and therefore, the smallest values for ventricular far-field rejection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715066 TI - Monophasic action potential duration during programmed electrical stimulation. AB - To examine changes in monophasic action potential duration (APD) with a pacing protocol similar to that used during electrophysiological testing, action potentials were recorded in vivo from the left ventricular apical endocardium of 12 normal mongrel dogs. The atrioventricular node was ablated and the dogs paced from the anterior right ventricle at a baseline cycle length of 1000 ms between interventions. Mean steady-state APD (APDss) was 266 +/- 7 ms at a pacing cycle length (PCL) of 1000 ms. Two pacing protocols were used. The first consisted of a sudden acceleration in pacing from a cycle length of 1000 ms to one between 300 and 600 ms. The second consisted of an 8-beat train at a cycle length of 400 ms followed by a premature beat at a coupling interval of 280 ms followed by a pause. The inter-train pause varied between 1 second and 32 seconds. With a sudden acceleration in pacing rate, steady-state values for APD at the faster PCLs were significantly smaller than APDss at 1000 ms with a change to cycle lengths of 600 ms (247 +/- 29 ms), 500 ms (229 +/- 21 ms), 400 ms (220 +/- 17 ms), and 300 ms (203 +/- 31 ms; P less than 0.01 for all comparisons). The time constant of the change in APD was shorter at a PCL of 300 ms (14.9 +/- 0.8 s) than 600 ms (20.3 +/- 4.7 s; P less than 0.05). With drive train pacing and incorporating an inter-train pause, the percent drop in steady-state APD compared to APD for the first train ranged from 10.1% with a 1-second inter-train pause to 2.1% with a 32-second pause. The difference in APD between the first drive train and drive trains after at least 3 minutes of pacing when APD had stabilized was not significant for an inter-train pause exceeding 8 seconds. IN CONCLUSION: (1) with a sudden acceleration in pacing rate, endocardial APD in vivo decreases exponentially. The faster the new rate, the shorter the new steady-state APD and the shorter the time constant. (2) When pacing using an 8-beat drive train and an inter-train pause, there is a decremental shortening in APD for pause lengths shorter than 16 seconds. Thus, while performing programmed stimulation using a pause, a conditioning period of at least 2 minutes should be used prior to diastole scanning to allow APD to achieve a steady state. PMID- 1715067 TI - Successful catheter ablation of human ventricular tachycardia with radiofrequency current guided by an endocardial map of the area of slow conduction. AB - A case is presented of a 68-year-old male patient with a history of myocardial infarction and recurrent ventricular tachycardia who was successfully treated with a single 20-second transcatheter application of radiofrequency current. Prior to current application a complete endocardial map had been obtained of an area of slow conduction that extended caudo-cranially for approximately 2 cm along the lower left ventricular septum. Stimulation techniques yielded evidence that this area was critically related to tachycardia initiation and maintenance. Its central part was subsequently chosen as the site for current delivery. PMID- 1715068 TI - Phase image triangulation of accessory pathways in patients undergoing catheter ablation of posteroseptal pathways. AB - The outcome of posteroseptal accessory pathway ablation by direct current (DC) shocks delivered just outside the os of the coronary sinus was studied in 21 patients. Electrocardiographic and electrophysiological parameters as well as phase image patterns of equilibrium multiple-gated blood-pool scintigrams were studied to determine their usefulness in predicting the success of ablation. A second free-wall pathway was documented by electrophysiological or surgical findings in six patients, and the value of phase images in detecting this second pathway was studied as well. Ablation was successful in 57%. The cumulative mean energy of DC shocks amounted to 524 +/- 170 joules and was not predictive of ablation outcome, neither was the mean ventriculoatrial (VA) conduction time. The predictive value of the 12-lead maximally preexcited electrocardiogram was poor in the 15 patients with a single posteroseptal bypass tract. A new method to triangulate the site of the earliest phase angle on the atrioventricular (AV) valve plane successfully localized the bypass pathway in 14 of those patients. No specific phase pattern predicted successful ablation except for a symmetrical, concentric peripheral phase progression found to be predictive of ablation success in the four patients who showed this pattern. Phase analysis was able to localize the second, nonposteroseptal pathway in four of six patients. This study showed that a concentric peripheral phase progression in the gated blood-pool scintigrams is predictive for ablation success in patients with posteroseptal pathways. A free-wall localization of the earliest phase angle is suggestive of a second bypass tract in this area. PMID- 1715069 TI - Pacemaker malfunction is frequent during catheter ablation. PMID- 1715070 TI - Cardiac transplant rejection and evoked T wave amplitude. PMID- 1715071 TI - Implantable cardioverter defibrillators in cardiovascular practice: report of the Policy Conference of the North American Society of Pacing and Electrophysiology. NASPE Policy Conference Committee. PMID- 1715072 TI - An efficient technique for implantation of atrial leads from the left side. AB - Implantation of an endocardial lead into the right atrial appendage is itself a simple procedure but may offer complexity if the lead is introduced from the left side of the body. Experimentation on eight corpses allowed determination of an efficient method of rapid and reliable implantation of a lead using an S-shaped stylet. The method has been used successfully in 65 clinical instances. PMID- 1715073 TI - Implantable cardioverter defibrillator: possible hazards of electromagnetic interference. AB - We report on three patients with an automatic, implantable cardioverter defibrillator (AICD, CPI) in whom the device had been deactivated due to electromagnetic interference. In all cases, the source of the electromagnetic disturbances could be identified. PMID- 1715074 TI - Utility of adenosine administration during intraoperative mapping in a patient with the Wolff-Parkinson-White syndrome. AB - Adenosine is an endogenous nucleoside that is administered intravenously and has potent chronotropic and dromotropic effects. This drug is distinguished from verapamil by its very short half-life. This makes it an ideal agent for use in the operating room where long lasting electrophysiological effects may not be desirable. A 61-year-old man with preexcited atrial fibrillation was referred for surgical ablation of his accessory pathway. Preexcitation was minimal or absent on arrival in the operating room. Intravenous adenosine was given causing AV nodal block, and resulted in marked preexcitation, thus allowing computerized mapping to localize the site of the accessory pathway. Adenosine may be a useful agent for rapid and precise localization of accessory pathway(s) in a select group of patients with minimal preexcitation at the time of surgery. Its short half-life allows additional mapping without sustained electrophysiological effects on the AV node, or accessory pathway. PMID- 1715075 TI - Asymptomatic left ventricular malposition of a transvenous pacemaker lead through a sinus venosus defect: follow-up over 17 years. AB - The case of a woman with an asymptomatic transvenous left ventricular endocardial pacemaker lead is presented. The chest X ray and the electrocardiogram suggested pacemaker catheter malposition. By two-dimensional echocardiography, the pacemaker lead was shown to cross from the left atrium through the mitral valve and implant in the left ventricular endocardium. The underlying sinus venosus defect and the passage of the electrode through this interatrial communication were directly visualized by transesophageal echocardiography. No thrombotic material attached to the lead was detected corresponding to the patient's uneventful course for surprisingly more than 17 years without evidence of past or present neurological deficiencies or of peripheral embolic phenomena. Thus, no operative correction was performed. Warfarin sodium therapy, however, was initiated. PMID- 1715076 TI - Syncope secondary to paroxysmal high grade AV block in a heavily trained man. AB - J.B. is a well-trained male with syncope due to paroxysmal AV nodal heart block who ultimately required a permanent pacemaker despite an initial attempt at cessation of training only. Baseline sinus node function was normal, but AV nodal conduction remained abnormal even after autonomic blockade supporting intrinsic AV nodal dysfunction. This case illustrates that vigorous physical training may unmask previously unrecognized intrinsic dysfunction of AV nodal conduction or, as previously reported for physical training induced sinus node dysfunction, cause AV nodal dysfunction. Simple cessation of training to treat this problem is often recommended but may not be adequate for some patients who remain at risk for recurrent syncope during the deconditioning period. PMID- 1715077 TI - Controversies in pediatric emergency medicine: abdominal trauma cases. PMID- 1715078 TI - Left ventricular function in dogs 1 year after coarctectomy. AB - Juxtaductal aortic coarctation was surgically created in beagle puppies at the age of 2 months and resected at the age of 10 months, after the development of good collateral circulation. Control dogs undergoing sham operations on each occasion were studied in the same environment. Cardiac catheterization was performed 1 year after the coarctectomy to evaluate the recovery of the heart. Cardiac output, heart rate, end-diastolic pressure, and Vmax were similar in both groups of seven dogs, but the systolic pressure gradient (SPG) over the operated area during isoproterenol infusion was significantly higher in the coarctectomized group, with a mean of 8.9 +/- 6.3 (SD) vs. 0.1 +/- 0.3 mmHg (p less than 0.05). The preejection period [PEP, 58 +/- 8 vs. 47 +/- 5 ms (p less than 0.01)], electromechanical delay [EMD, 18 +/- 3 vs. 13 +/- 3 ms (p less than 0.05)], and isometric contraction time [ICT, 39 +/- 7 vs. 32 +/- 4 ms (p less than 0.05)], were all significantly longer in the coarctation group after isoproterenol infusion. The results demonstrate that, even though cardiac output increased adequately during loading and mechanical pumping efficiency was preserved, excitation-contraction coupling was still prolonged. Thus, an anatomical successful coarctectomy, even at the age of 10 months, does not fully restore left ventricular function in dogs after chronic experimental coarctation. PMID- 1715079 TI - Argyrophilic nucleolar organizer region counts and Ki-67 scores in human renal cell carcinoma. AB - The proliferative activity of 21 cases of renal cell carcinoma has been investigated by means of monoclonal antibody Ki-67 and Nucleolar Organizer Regions (AgNORs) analysis. The correlation between AgNOR counts and Ki-67 scores was only slightly significant (r = 0.53, r2 = 0.28) as determined by linear regression. Positive correlation was found between Ki-67 scores and tumour histologic grade. However, no correlation was observed between Ki-67 scores and tumour pathologic stage and between AgNOR counts and tumour histologic grade and/or pathologic stage. The results suggest that AgNOR counts cannot replace Ki 67 scores in evaluating the proliferative activity of renal cell carcinoma and that such activity and both histologic grade and pathologic stage seem to be independent parametres. PMID- 1715080 TI - Investigation on serum neurone-specific enolase in prostate cancer diagnosis and monitoring: comparative study of a multiple tumor marker assay. AB - A quadruple tumor marker serotest assay (neurone-specific enolase, NSE, prostate specific antigen, PSA, prostatic acid phosphatase, PAP, and carcino-embryonic antigen, CEA) was performed on sera from both 63 patients with untreated prostate cancer and 135 patients treated with orchiectomy, flutamide, diethylstilbestrol (DES), cyproterone acetate (CPA), and Estracyt. In untreated patients with local tumor elevated blood NSE concentrations were found more frequently (10/35, 28.6%) than in untreated subjects with disseminated disease (3/28, 10.7%). Elevated NSE values were measured more frequently in nonresponders to therapy 10/46 (21.7%), than in responders during prostate cancer partial remission (2/89, 2.2%). In none of NSE-positive neoplasms a small cell prostate cancer has been histologically detected. Many of NSE-positive tumors are also closely associated with elevated blood CEA values. The applied anticancer drugs were inefficient in the normalization of neither one from the pair of elevated NSE and CEA concentrations (regardless of the numerical values of the other two markers, PSA and PAP), but their values were found to decline occasionally only after surgical treatment. In patients with raised PSA, PAP, and CEA levels but with a normal NSE value, the application of the same treatment strategies was in the most of subjects sufficient to provoke either temporary or even lasting tumor response to therapy. Hence, it appears that the assessment of the NSE serotest, despite its minimal value in the overall tumor staging and monitoring, might furnish the decision making step related to the treatment of aggressive prostate cancer with an additional and powerful tool. PMID- 1715081 TI - Immunohistochemical study of metallothionein in normal and benign prostatic hyperplasia of human prostate. AB - Metallothionein (MT) in the human prostate gland was examined. Prostatic tissues were obtained from patients of urinary bladder cancer who had received radical cystoprostatectomy. MT content was 99.3 micrograms/g wet tissue (w.t.) in the peripheral zone (PZ), 12.0 micrograms/g w.t. in the preprostatic region (PR), 7.3 micrograms/g w.t. in the central zone (CZ), 6.8 micrograms/g w.t. in the anterior fibromuscular stroma, and 29.5 micrograms/g w.t. in benign nodular hyperplasia (adenoma) by radioimmunoassay. Immunohistochemical study demonstrated that MT was localized in the cytoplasm, nuclei of glandular epithelia, and secretory products. In general, a positive immunoreaction was strong in PZ and weak in CZ. It is considered that glandular epithelial cells in PZ, PR, CZ, and adenoma may react differently to heavy metals, e.g., zinc and cadmium. PMID- 1715082 TI - Adenosine: a natural modulator of L-type calcium channels in atrial myocardium? AB - The mechanism of action of adenosine at the level of atrial myocardium has been a matter of debate. Electrophysiological studies showed that adenosine increases K+ efflux which may reduce Ca2+ influx, indirectly, by shortening the myocardial action potential. Recently some authors proposed that adenosine also depresses Ca2+ influx by a direct action on the L calcium channel, but, this effect being lower than that on voltage-dependent K+ channels, it was considered of minor importance. The effect of adenosine and its stable analogues was studied in the presence of the dihydropyridine Bay K 8644, a highly specific L-type calcium channel agonist, on isolated guinea-pig atria. The inotropic effect of the calcium channel activator was found to be antagonized by adenosine A1-receptor agonists. Binding studies showed that the effect on Bay K 8644 was not due to the interaction between adenosine analogues and dihydropyridines at the level of a common receptor site on L-type Ca2+ channels. Inhibitors of K+ channels did not antagonize the effect of adenosine analogues against Bay K 8644. Experimental conditions aimed to unmask an effect on slow Ca2+ currents (i.e. K+ depolarized paced atria), further supported that adenosine analogues may act in atria as negative modulators on L-type Ca2+ channels. Finally, the use of 1,3-dipropyl-8 cyclopentylxanthine (DPCPX), a highly specific A1-receptor antagonist, demonstrated that the antagonism of Bay K 8644 by adenosine analogues is strictly dependent on A1 receptors. The above data support the possibility of a dual signal transduction pathway to ion channels (K+ and Ca2+) linked to A1 receptors in atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715084 TI - Hyperstimulation pancreatitis in mice induced by cholecystokinin octapeptide, caerulein, and novel analogues: effect of molecular structure on potency. AB - Acute pancreatic oedema with hyperamylasaemia was induced in mice by subcutaneous administration of cholecystokinin octapeptide (CCK8). Comparison with effect of caerulein showed that cholecystokinin is less potent in vivo. To investigate the observed difference in response, threonine3 CCK8 and methionine5 caerulein were synthesised and evaluated. Comparison of these peptides suggests that difference in bioactivity is related to possession of extra N-terminal residues rather than substitution of threonine for methionine. PMID- 1715083 TI - Organophosphate increases the sensitivity of human exocrine pancreas to acetylcholine. AB - Human pancreas contains two cholinesterase isoenzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In the present study, binding potency of two organophosphates for human cholinesterases were compared by the Ellman method. Echothiophate was found to have much greater potency than iso-OMPA for both cholinesterases. Using Karnovsky histochemical stains on human pancreatic tissue, the same results were confirmed. Dose-response studies with acetylcholine were done on viable pancreas fragments from nine human donors, without pancreatic disease (group I). Cold-preservation time was less than 30 h. Pancreas was minced into fragments, after the technique of Scheele and Palade, placed in Eagle's medium, and gassed with O2. Amylase release was measured by the Phadebas Method and corrected for basal release. There was a dose-dependent response to acetylcholine at 1 and 2 h, with a shift in peak amylase release to the left, when fragments were preincubated in 10(-4) M echothiophate. This indicated a 100 fold increase in sensitivity to acetylcholine. In three patients with chronic pancreatitis (Group II), there were variable patterns of response of amylase release to acetylcholine, and higher basal outputs. In Group III, prolonged storage conditions of over 40 h were tested for 4 pancreas donor tissues. There was no response to acetylcholine. These studies show that for up to 30 h cold storage, fragments of pancreas from human organ donors respond to acetylcholine in dose-dependent manner. An organophosphate, echothiophate (10(-4) M) which inhibits both cholinesterases, increases pancreatic sensitivity to acetylcholine, and these results are similar to findings from canine pancreas fragments, which also showed increased sensitivity. PMID- 1715085 TI - Aging and the trophic effects of cholecystokinin, bombesin and pentagastrin on the rat pancreas. AB - We examined the effect of age on the trophic response of the pancreas to chronic treatment with cholecystokinin (CCK), bombesin, or pentagastrin. Three age groups (3-, 12-, and 24-months) male F344 rats received saline; CCK-8 (5 ng/kg), bombesin (10 micrograms/kg), or pentagastrin (100 micrograms/kg) by intraperitoneal injection t.i.d. for 2 weeks. Rats were then killed and the pancreases excised, weighed, and assayed for DNA, RNA, protein, and polyamine (putrescine, spermidine, and spermine) concentrations and contents. We found that none of the treatments altered body weight at any age. All three hormones increased pancreas size and cell number in 3-month old rats, but by 12 months, all three had increased only pancreatic RNA content. Pancreatic spermidine concentration was decreased by all three hormone regimens in 3- but not in 12 month old rats, and pancreatic putrescine concentration and content were increased in 12-month old rats receiving all three hormones. There was no change in any parameter following any of the three hormones, tested at 24 months of age. We conclude that, at the dosages tested, the trophic response of pancreas to chronic administration of CCK, bombesin, and pentagastrin, which is normally present in young adult rats, is lost with aging. PMID- 1715086 TI - Essential role of cholecystokinin in pancreatic regeneration after 60% distal resection in rats. AB - The essential role of cholecystokinin (CCK) in pancreatic regeneration after pancreatitis or resection has been supposed, but not yet clearly demonstrated. In rats, 6-8 weeks after 60% distal resection of the pancreas a gradual increase in pancreatic weight and contents of DNA, protein, trypsin, chymotrypsin and amylase, occurred (there was no increase in lipase); Pancreatic regeneration stopped thereafter. Nonparallel increases in enzyme values were similar to those seen after CCK administration. Indeed, basal CCK levels increased significantly by the 6th week and declined thereafter. A one month s.c. administration of CCK octapeptide (CCK-8) (3 x 300 ng/kg/d) accelerated regeneration in the first month, but it had almost no effect during the second or third postoperative months. A two week s.c. administration of a specific CCK antagonist, CR 1409 (3 x 4 mg/kg/d) totally prevented regeneration by the fifth and sixth weeks, but did not diminish pancreatic weight or DNA and protein contents during the first two weeks. Alcohol administration (12 g/kg/d) reduced CCK release and prevented pancreatic regeneration during the three-month experimental period. These data indicate that CCK has an essential role in pancreatic regeneration and that the deleterious effect of alcohol on regeneration involves inhibition of CCK release. PMID- 1715087 TI - Inhibition of rat pancreatic exocrine secretion by neuropeptide Y: studies in vivo and in vitro. AB - Neuropeptide Y (NPY) is a unique 36 amino acid peptide with strong sequence homology to pancreatic polypeptide and peptide YY. In the rat pancreas, NPY positive fibers have been demonstrated in close association with exocrine structures, suggesting a regulatory role for the peptide. In conscious rats with pancreatic ductal cannulas, amylase output stimulated by cholecystokinin octapeptide (CCK-8: 0.2 microgram kg - 1 h -1) was dose-dependently inhibited by intravenous NPY infusion (20, 40, and 80 micrograms kg-1 h-1). Inhibitory effects were rapid in onset but reversed with cessation of NPY infusion. With continuous NPY infusion (40 microgram kg-1 h-1), prolonged inhibition of amylase output by the vagal stimulant 2-deoxyglucose (100 mg kg-1) was observed (greater than 50% inhibition in each of none consecutive 10-min periods). In contrast, NPY infusion in doses of 20, 40, or 80 micrograms kg-1 h-1 produced no alteration in immunoreactive somatostatin levels. In vitro, NPY incubation (10(-13)-10(-8) M) produced no change in basal amylase release from dispersed, purified acinar cells. In addition, co-incubation of NPY (10(-8)-10(-6) M) with CCK-8 (10(-13) 10(-8) M) produced no inhibition of CCK-stimulated amylase release from dispersed acini. In contrast, NPY (10(-6) M) produced significant inhibition of amylase release from pancreatic lobules that had been stimulated by 75 mM potassium (135 +/- 11% versus 177 +/- 18% of basal level) or by 25 microM veratridine (196 +/- 19% versus 398 +/- 152%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715088 TI - Aspiration of cystadenocarcinoma mimicking pancreatic pseudocyst. PMID- 1715089 TI - Increase in plasma corticosterone level does not participate in increase of amylase content induced by lorglumide in the newborn rat pancreas. PMID- 1715090 TI - Psychosocial factors and amine systems. PMID- 1715091 TI - Heartbeat and arrhythmia perception in diabetic autonomic neuropathy. AB - A comparative study of diabetics with autonomic neuropathy (N = 13) as against non-neuropathic diabetics (N = 16) and healthy control persons (N = 20) was carried out with respect to heart rate both at rest and under stress, frequency of cardiac arrhythmias in a 24-h ECG and accuracy of heartbeat and arrhythmia perception. In the subjects with diabetic autonomic neuropathy, the spontaneous variability and stress-induced reactivity of the heart rate as well as the number of tachycardic episodes were reduced, whereas the frequency of ventricular extrasystoles was somewhat increased. Impaired heartbeat perception and a complete loss of perception of arrhythmias as a consequence of neuropathic deafferentation could be demonstrated. Cardiac perception disorders also play a vital role in other clinical problems, e.g. silent myocardial infarction and lack of awareness of hypoglycaemia in diabetes mellitus. PMID- 1715092 TI - [Rheological and expander effect of hydroxyethyl-starch in intradural anesthesia]. AB - We have evaluated the effect of the infusion of hydroxyethylstarch (HES) on blood viscosity and its usefulness to prevent hypotension associated with intradural anesthetic blockade. The sample consisted of 20 healthy patients scheduled for elective surgery with intradural anesthesia (0.5% hyperbaric bupivacaine), in whom 500 ml of HES were infused in 20 minutes. Blood samples were taken before lumbar puncture and 20 minutes after it and once HES infusion had been finished. Blood viscosity, the erythrocyte and leukocyte mass parameters and biochemical values (total protein, BUN, creatinine, glucose) were measured. Blood pressure (systolic, diastolic, mean) and heart rate were monitored every 5 minutes. During the study time, systolic blood pressure did not show significant changes. Mean and diastolic blood pressure in the minutes 15 and 20 were reduced in less than 10 mmHg (p less than 0.01). Packed red cell volume diminished in 5.7% and the blood viscosity in 0.5-2.3 mPas. It was concluded that HES is a good option for intradural anesthesia because of its plasma volume expanding effect and the hemodilution it induces. PMID- 1715093 TI - New clue found to Alzheimer's. PMID- 1715094 TI - Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. AB - FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrested in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (i) strains that lack FKBP proline rotamase, encoded by FPR1, were viable and fully resistant to rapamycin and (ii) FK506 antagonized rapamycin toxicity in vivo. Mutations that conferred rapamycin resistance altered conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity were identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle. PMID- 1715095 TI - Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. AB - The N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate receptors, plays a key role in synaptic plasticity in the nervous system. After NMDA receptor activation, calcium entry into the postsynaptic neuron is a critical initial event. However, the subsequent mechanisms by which the NMDA receptor signal is processed are incompletely understood. Stimulation of cultured rat hippocampal cells with glutamate resulted in the rapid and transient tyrosine phosphorylation of a 39-kilodalton protein (p39). Tyrosine phosphorylation of p39 was triggered by the NMDA receptor and required an influx of Ca2+ from the extracellular medium. Because p39 was found to be highly related or identical to the microtubule-associated protein 2 kinase, the NMDA receptor signal may be processed by a sequential activation of protein kinases. PMID- 1715096 TI - The murine W/c-kit and Steel loci and the control of hematopoiesis. AB - The experiments summarized here indicate that germ-line mutations in either the c kit receptor tyrosine kinase (W mutants) or its ligand (Sl) lead to profound and pleiotropic developmental defects, including abnormalities within the hematopoietic system. These observations parallel findings in other developmental systems, notably the fruit fly Drosophila, that receptor tyrosine kinases play key roles in the determination of cell fate and the elaboration of developmental programs. Thus, it appears that the processes of hematopoiesis, melanogenesis, and gametogenesis in mammals involve a similar strategy to that used in other species for transmitting and receiving positional cues during development. Finally, it is interesting to note that what began as an attempt to understand the molecular basis of coat color mutations in the mouse has led to the identification of a key cell-signalling pathway in the development of at least three cell lineages in mammals. Further analysis of the W/Sl pathway should provide a more complete understanding of the early events in hematopoiesis, and may also lead to novel therapeutic applications involving the protein product of the Sl locus. PMID- 1715097 TI - [Nursing. Knowing a little about postoperative pain treatment]. PMID- 1715098 TI - [Nursing. None of that on principle. Pain treatment with the morphine pump]. PMID- 1715099 TI - Studies on synthesis of interleukin-6 and gammaglobulin in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. AB - Peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE) proliferated spontaneously and secreted an elevated level of IgG compared with that of normal controls. However, the levels of interleukin-6 (IL-6) produced by PBMC from patients with SLE with or without pokeweed mitogen (PWM) stimulation showed no significant difference from those of normal controls. The levels of IL-6 secreted spontaneously from PBMC of SLE patients correlated inversely with the percent and the absolute number of CD19 positive cells in PBMC, but not with the levels of IgG and IgM secreted spontaneously from PBMC. There was no significant difference in the levels of IgG produced by PBMC stimulated with IL-6 and also in the levels of IL-6 synthetized by T and B cells between SLE patients and normal controls. These data suggest that IL-6 may not play an important role in the hypergammaglobulinemia in SLE. PMID- 1715100 TI - Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein. AB - Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth. PMID- 1715101 TI - Effect of estrogen on human endometrial epithelial cell growth and differentiation in vitro. AB - Although physiologic parameters regulating endometrial proliferation and secretory maturation during the normal menstrual cycle are well characterized, cellular and molecular interactions directing these events under the influence of a changing hormonal milieu remain unclear. In the present study, the effect of estradiol on the growth and acquisition of differentiated function of purified endometrial epithelium was examined in an established model. Epithelial cells were isolated with high purity from human endometrial biopsies. When cultured on a biomatrix bed, cells established a polarized monolayer with gland-like invaginations. Cells isolated throughout the menstrual cycle were cultured under serum-free conditions, with or without estradiol, and secretion of tumor associated epitope TAG-72 was monitored by radioimmunoassay. Under steroid-free conditions, cells exhibited a distinct proliferative interval, followed by the acquisition of TAG-72 epitope secretory capability. Although estradiol had no apparent effect on proliferation or spatial organization of epithelial cells, a striking inhibition of TAG-72 epitope secretion was evident. This observation is believed to represent a direct effect of estradiol on endometrial epithelial cells in this model. PMID- 1715102 TI - Adenocarcinoma of the prostate: biopsy to whole mount. Denver VA experience. AB - Clinical understaging abounds in adenocarcinoma of the prostate. The preoperative prostate-specific antigen is not useful in preoperative staging, although enzymatic acid phosphatase elevation is associated with positive nodes in two thirds of patients. Whole mount evaluation of radical prostatectomy specimens reveals tumor multicentricity in more than half the patients and tumor extension beyond the prostatic capsule in the majority of patients. A significant number of patients have a final tumor grade higher than that initially assigned. Capsule penetration by tumor is a factor of tumor grade as is volume. PMID- 1715103 TI - Ratio of polyclonal-monoclonal prostate-specific antigen levels. Discrimination of nodal status in prostate tumors that produce low marker levels. AB - The preliminary data we present in this report suggest that the ratio of polyclonal-monoclonal serum prostate-specific antigen levels may add clinically useful information to that obtained using serum prostate-specific antigen levels alone. We hypothesize that the diversity of prostate-specific antigen ratios observed in our data reflects a diversity in the antigenic and structural attributes of prostate-specific antigen molecules found in the sera of patients with prostate cancer. Further, this heterogeneity of molecules is a reflection of the diverse and altered metabolic state of human prostate cancer and appears to be related to biologic behavior in individual patients. PMID- 1715104 TI - Significance of prostatic intraepithelial neoplasia on prostate needle biopsy. AB - Prostatic intraepithelial neoplasia (PIN) is a putative premalignant change in the human prostate. Previously, the spatial association of PIN with invasive carcinoma has been described in the study of total prostatectomies. PIN is frequently recognized in prostate needle biopsy specimens in which no carcinoma is apparent. To further define the potential significance of PIN, we performed repeat ultrasound-guided prostate needle biopsy in 21 men who had PIN identified on prostate biopsy performed because of an abnormal finding on digital rectal examination. Twelve patients (57%) had carcinoma identified on their second procedure including all who had intermediate- and high-grade PIN on the initial procedure. Prostate-specific antigen correlated with PIN grade and carcinoma on the secondary procedure, although this did not achieve statistical significance. Men with PIN on prostate needle biopsy should undergo repeat sampling to exclude missed carcinoma. PMID- 1715105 TI - Transurethral resection of prostate under sedation and local anesthesia (sedoanalgesia). Experience in 100 patients. AB - One hundred male patients (average age 68.2 yrs) with acute retention (n = 20) or with symptoms of outflow obstruction (n = 80) underwent transurethral resection of the prostate (TURP) under sedation (midazolam) and local anesthesia (lidocaine)--referred to as sedoanalgesia technique. Procedures lasted twenty four minutes on average (range 15-35 min), and the weight of prostatic tissue resected ranged from 2-35 g (average 11.1 g). There were no complications related to the use of midazolam or lidocaine. The technique of sedoanalgesia proved safe and acceptable to all patients regardless of their pre-existing medical condition. Where the weight of prostate to be resected is estimated to be less than 40 g, TURP under sedoanalgesia proves an effective alternative to general or regional anesthesia. PMID- 1715106 TI - Prostate-specific antigen and prostatic acid phosphatase: biomolecular and physiologic characteristics. AB - PSA is a 34-kd 240-amino acid glycoprotein produced by the prostatic epithelial cells. It is a member of the glandular kallikrein gene family and has a high sequence homology with human glandular kallikrein (hGK-1). PSA is a serine protease and has chymotrypsin-, trypsin-, and esterase-like activities. It is secreted into the seminal fluid where it degrades two seminal vesicle proteins that are important components of the semen coagulum, thus playing an important role in semen liquefaction. The production of PSA protein appears to be under the control of circulating androgens acting through the androgen receptor. Therefore, the significance of a low serum PSA value in a patient who has undergone previous antiandrogen therapy may not be the same as that for a patient who has not received endocrine treatment. PMID- 1715107 TI - Intravenous gamma-globulin for chronic inflammatory demyelinating polyneuropathy and myasthenia gravis. PMID- 1715108 TI - The axonal transport motor 'kinesin' is bound to anterogradely transported organelles: quantitative cytofluorimetric studies of fast axonal transport in the rat. AB - Monoclonal antibodies to the axonal transport ATPase kinesin were used in an immunofluorescent study on mammalian nerves. Following crushing of the sciatic nerve and the ventral roots of adult rats, immunoreactive material was found to accumulate rapidly, mainly proximal to a crush but also, to some degree, distal to a crush. The strongest immunofluorescence was observed after incubation with the H2 antibody against the heavy subunit of kinesin. Using the cytofluorimetric scanning (CFS) procedure, the accumulated amounts were quantified and it was found that the retrogradely accumulating kinesin-like immunoreactivity (IR) was about 4-12% of the anterogradely transported kinesin-IR. The results were compared to the vesicle marker p38 (synaptophysin), which was found to accumulate to a significant extent on both sides of the crush. Cytofluorimetric scanning measurements indicated that nearly 50% of the anterogradely accumulated p38-IR was recycling to the cell body. The results demonstrate that kinesin in the living axon is affiliated with anterogradely transported organelles. Retrogradely transported organelles appeared to carry very little kinesin-IR, suggesting that kinesin may be subject to turnover, distinct from that of p38, in the distal regions of the axon. PMID- 1715109 TI - The adenosine analogue R-PIA interacts with substance P binding in rat brain and spinal cord. PMID- 1715110 TI - Methylene blue attenuates vasodilation and enhances vasoconstriction in response to endothelin-1 in the pig nasal mucosa. AB - In thiopentone anaesthetized pigs (n = 10), simultaneous recordings of the vasomotor response to local intra-arterial infusion of endothelin-1 and -3 were performed in three different vascular compartments of the nasal mucosa. Sphenopalatine artery blood flow (reflecting nasal blood flow), nasal cavity volume (representing blood content in capacitance vessels) and laser Doppler flowmeter signal (reflecting superficial mucosal blood flow) increased in response to low doses (4 x 10(-9) to 4 x 10(-8) moles) of endothelin-1. In contrast, marked vasoconstriction (up to 50% of arterial blood flow reduction) occurred upon higher doses (4 x 10(-7) to 4 x 10(-6) moles). Endothelin-3 (4 x 10(-9) to 4 x 10(-6) moles) evoked at the highest dose a long-lasting increase of systemic arterial blood pressure resulting in a 22 +/- 4% increase in nasal vascular resistance. After local intra-arterial pretreatment with methylene blue (4 x 10(-4) mol), the vasodilatory responses to low doses of endothelin-1 were abolished for the three vascular parameters studied, whereas the vasoconstrictor responses in resistance and capacitance vessels evoked by higher doses of endothelin-1 were increased. In contrast, the laser Doppler flowmeter signal reduction in response to endothelin-1 was markedly attenuated. The vasodilatatory effect of substance P was abolished after methylene blue probably due to interference with effects of endothelium dependent relaxing factor (EDRF). These observations suggest that in the pig nasal mucosa in vivo, exogenous endothelin-1 induces a dose-dependent reduction of blood flow in resistance and capacitance vessels and in superficial mucosa.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715111 TI - Endothelium-dependent relaxation and effects of prostacyclin, endothelin and platelet-activating factor in human hand veins and arteries. AB - An altered release of endothelium-derived vasoactive factors has been implicated in several vasospastic conditions. Since the functional role of the endothelium in the hand vasculature is largely unknown, we examined the effects of 'endothelial removal' on vascular reactivity, and the effects of some 'endothelium-associated' substances in isolated human hand veins and arteries. Acetylcholine induced a large relaxation (Emax = 97 +/- 1%) in precontracted hand arteries. The relaxation was abolished by endothelial removal. In hand veins, acetylcholine induced a small relaxation (Emax = 13 +/- 4%), which was unaffected by endothelial removal. An endothelium-dependent relaxation was, however, obtained with high concentrations (greater than or equal to 10(-6) mol l-1) of the Ca2+ ionophore A23187. Contractile responses to noradrenaline, serotonin and prostaglandin F2 alpha did not differ between vein segments with and without endothelium. Endothelin was a potent constrictor of both veins and arteries. The potency and maximum response did not differ between the two types of vessel. Indomethacin pretreatment of veins did not influence the endothelin-induced contractions, suggesting that cyclo-oxygenase products are not involved in the response. In endothelin-contracted veins and arteries, the prostacyclin analogue iloprost elicited relaxation of similar potency and amplitude. The maximum relaxation in veins was, however, 3 times larger than that produced by prostacyclin itself. Platelet-activating factor was devoid of contractile and relaxant effects in both veins and arteries. The present study indicates differences between human hand veins and arteries regarding endothelial-dependent relaxation, and suggests that the modulatory role of endothelium-derived relaxing factor(s) is small in hand veins. The contractile and relaxant effects of endothelin and iloprost, however, did not differ between veins and arteries. PMID- 1715112 TI - Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells. AB - Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the 'state of equilibrium' at the cell surface caused by the superfusion (Uvnas et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 microM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 microM. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium. PMID- 1715113 TI - A calmodulin inhibitor with high specificity, compound 48/80, inhibits axonal transport in frog nerves without disruption of axonal microtubules. AB - The calmodulin inhibitor compound 48/80 has previously been shown to arrest axonal transport in vitro in the regenerating frog sciatic nerve. The inhibition was limited to the outgrowth region of nerves, which had been allowed to regenerate in vivo for 6 days after a crush lesion, before they were incubated with or without drugs in vitro overnight. The effects of compound 48/80 on the regenerating nerve were further investigated. A concentration of compound 48/80 (50 micrograms ml-1), which effectively inhibits axonal transport, did not cause observable changes of the microtubules of regenerating axons in the outgrowth region as judged by electron microscopy. Furthermore, it was shown that also a lower concentration (25 micrograms ml-1) inhibited axonal transport. As a measure of possible metabolic effects, the level of ATP was assessed in the regenerating nerve after exposure to compound 48/80. Compound 48/80 at 25 micrograms ml-1 did not change the level of ATP in the nerve. The assembly of bovine brain microtubule proteins in a cell-free system was unaffected by 25 micrograms ml-1 of compound 48/80 and slightly inhibited by 50 micrograms ml-1. At higher concentrations (greater than 100 micrograms ml-1) assembly of microtubules appeared stimulated, and microtubule spirals as well as closely aligned microtubules could be seen. These effects appeared to be unrelated to the transport effects. The present results indicate that compound 48/80 arrests axonal transport via mechanisms other than destruction of axonal microtubules or interference with the energy metabolism. It is possible that these mechanisms involve inhibition of calmodulin-regulated events essential to the transport. PMID- 1715114 TI - Selectivity of ruthenium red in inhibiting bronchoconstriction and CGRP release induced by afferent C-fibre activation in the guinea-pig lung. AB - In the present study we evaluated the effects of ruthenium red, a blocker of transmembrane Ca2+ fluxes, on bronchoconstriction and the release of calcitonin gene-related peptide-like immunoreactivity induced by different stimuli in the isolated perfused guinea-pig lung. Vagal stimulation (1 Hz, 1 min), capsaicin (10(-8) M, 10(-6) M), resiniferatoxin (3 x 10(-10) M), nicotine (10(-4) M), bradykinin (5 x 10(-6) M) and histamine (10(-5) M) evoked bronchoconstriction and calcitonin gene-related peptide-like immunoreactivity overflow. Ruthenium red (5 x 10(-6) M) almost completely inhibited the bronchoconstriction and calcitonin gene-related peptide-like immunoreactivity overflow induced by capsaicin and resiniferatoxin but did not influence the effects induced by vagal nerve stimulation, nicotine, bradykinin or histamine. The 20-deacetylated derivative of resiniferatoxin (ROPA), which lacks the homovanillyl ester group, did not evoke release or bronchoconstriction. Ruthenium red (3 x 10(-4) M) aerosol attenuated the cough induced by nebulized citric acid in conscious guinea-pigs. Citric acid induced coughing is mediated via capsaicin-sensitive neurons. However, cigarette smoke-induced coughing, which involves capsaicin-resistant mechanisms, was not affected by ruthenium red. In conclusion, ruthenium red selectively inhibits the capsaicin, resiniferatoxin and citric acid-induced excitation of the sensory nerves as revealed by calcitonin gene-related peptide-like immunoreactivity release, bronchoconstriction and coughing, suggesting that these agents share a common mechanism of action. PMID- 1715115 TI - Effects of centrally administered substance P on cyclic AMP levels in particular brain areas of rats. AB - Effects of centrally (intracerebro-ventricularly) administered synthetic substance P on the metabolism of cAMP in 19 brain areas were investigated. Substance P administration increased cAMP levels considerably in the frontal cortex, the central gray matter and in the nucleus of the solitary tract, and to a lesser extent in the bed nucleus of the stria terminalis and in the anterior hypothalamic nucleus. Cyclic AMP levels were decreased in the globus pallidus, the habenula, the posterior hypothalamic nucleus and in the locus coeruleus while other brain areas showed no changes in cAMP levels. PMID- 1715116 TI - Ultrastructure of pancreatic light and clear cells in normal and transplanted tissue fragments in the anterior eye-chamber of rats. AB - The ultrastructure of pancreatic light and clear endocrine cells in normal and transplanted tissue fragments in the anterior eye-chamber of rats was described by electron microscopy. Compared to the dark cells, the light cells contain a conspicuously poorly stained cytoplasmic ground substance with varying number of rough endoplasmic reticulum cisterns and secretory granules. The quantities of these two organelles are inversely proportional to each other. "Light" alpha, beta, delta and pancreatic polypeptide cell-types were identified in normal and transplanted tissue. Out of these, the light alpha cell is the most commonly occurring. The "clear" endocrine cells are much fewer in number than the light cells. They contain well stained cytoplasm with numerous low electron density vesicles and few secretory granules. There is no ultrastructural difference between the light and clear cells in normal and in transplanted tissue for 77 and 534 days. The occurrence of light and clear cells in the transplanted tissue shows that these transplants behave morphologically like normal tissue. These cell types might be related to the different stages of secretory granule synthesis and maturation. PMID- 1715117 TI - Interaction of 5-azacytidine and dexamethasone in the control of hepatic tyrosine aminotransferase. AB - The nucleoside analog 5-azacytidine is able to induce tyrosine aminotransferase several-fold in the liver of suckling rats. Bilateral adrenalectomy abolishes this inducing effect. The drug also decreases significantly the concentration of cytosolic glucocorticoid receptor accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. The antiglucocorticoid RU 486 which abolishes the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively is not able to inhibit either the induction of tyrosine aminotransferase or the translocation of the glucocorticoid receptor into the nuclei by 5-azacytidine. PMID- 1715118 TI - Synergistic combinations and peptides in the inhibition of human immunodeficiency virus. AB - Various combinations of inhibitors of HIV reverse transcriptase were tested for inhibition of HIV replication in order to reveal any potential synergism or antagonism. PFA, a pyrophosphate analogue, gave synergistic inhibition of HIV replication in combination with both of the thymidine analogues AZT and FLT. The combination of PFA and AZT-TP gave only additive or weakly synergistic inhibition in a reverse transcriptase enzyme assay. The combination of AZT and FLT also gave synergistic inhibition of HIV replication, whilst the combination of AZT-TP and FLT-TP gave only additive or weakly synergistic inhibition of reverse transcriptase. Thus, the synergy does not arise from effects on reverse transcriptase alone but must be owing to other, cellular factors, such as effects on nucleoside metabolism or metabolism of the analogues. The results are consistent with the hypothesis that AZT may have an alternative mechanism of inhibition other than inhibition of reverse transcriptase. The diminished cytotoxicity observed in addition to the synergistic inhibition makes these combinations attractive from the point of view of combination chemotherapy. The inhibition of HIV replication by peptides from various parts of the V3 region of gp120 whose sequences were homologous with the tryptase inhibitor trypstatin was tested. Inhibitory activity was displayed by two peptides containing cysteine in their sequence. Antibodies to two peptides containing the two conserved cysteine residues from opposite sides of the neutralizing loop of gp120 were previously associated with protection from vertical transmission of HIV. The V3 region thus seems to be important for the function of gp120 and the transmission of HIV. PMID- 1715119 TI - Multiple epitope specificity of monoclonal antibodies to a single synthetic peptide: use in the characterization of the GP IIb-IIIa binding domain of von Willebrand factor. AB - Monoclonal antibodies have been induced against the synthetic peptide with sequence Tyr-Glu-Val-Val-Thr-Gly-Ser-Pro-Arg-Gly-Asp-Ser-Gln-Ser-Ser. This peptide represents residues Glu1737-Ser1750 of the mature von Willebrand factor (vWF) subunit and contains the sequence Arg-Gly-Asp, thought to be important in mediating binding to the platelet receptor glycoprotein (GP) IIb-IIIa complex. Twelve antibodies were obtained, eight of which bound to native vWF as well as to the peptide immunogen insolubilized onto agarose beads. These antibodies defined at least three distinct epitopes, as demonstrated by antibody interaction with peptides having a single phenylalanine substitution at each position in the sequence. In particular, two antibodies bound to epitopes on vWF that included one or more of the three residues (arginine, glycine, aspartic acid) thought to be involved in binding to GP IIb-IIIa, whereas one antibody bound to an epitope that did not include any of those residues. Nevertheless, the three antibodies cross-reacted with each other, a finding explained by the fact that the corresponding epitopes had at least two residues in common, namely Gly1741 and Ser1742. In spite of the cross-reactivity for binding to vWF, only the two antibodies whose epitopes included residues in the Arg-Gly-Asp sequence inhibited vWF interaction with GP IIb-IIIa. The third antibody had no inhibitory effect even though it was bound to an epitope located at a distance of only few residues on the amino terminal side of Arg-Gly-Asp. These results demonstrate that monoclonal antibodies raised against a single synthetic peptide with sequence limited to fifteen residues may exhibit distinct epitope specificity and may be used to define functional domains in macromolecules with a high degree of resolution. PMID- 1715120 TI - Interactions between fibrin, collagen and endothelial cells in angiogenesis. AB - The role of fibrin in the generation of new blood vessels was examined in this study. Using a wound chamber model, we investigated the sequential interactions between endothelial cells and the extracellular matrix during angiogenesis. Silicone tubes 5 mm long and 1.4 mm in internal diameter were sutured to the cut ends of thigh muscles in the rats. The contents of the chamber were removed at intervals for histological, immunohistochemical and electron microscopic studies. We observed an initial phase of fluid accumulation in the wound chamber followed by formation of a fibrin/fibronectin clot. Migration of endothelial cells, macrophages and fibroblasts into the clot occurred after the 1st week. The subsequent phase of fibrinolysis was accompanied by deposition of collagen and organization of endothelial cells into capillary tubes. These findings support the view that angiogenesis is the product of interactions between endothelial cells and a changing extracellular matrix (ECM) and requires the participation of soluble and immobilized plasma proteins and local ECM factors. Our findings indicate that fibrin is intimately involved in both hemostasis and angiogenesis; these are sequential steps in the initial phase of wound healing. Thus, fibrin/fibrinogen occupies a central position and provides a vital link in the initiation of the cascade event of wound healing. PMID- 1715121 TI - Monoclonal antibodies for the detection of thrombosis. PMID- 1715122 TI - Immunochemical studies of A alpha chain crosslinking. PMID- 1715123 TI - Effects of laser-generated tissue debris on aggregation of human platelets. AB - This study examined the effects of laser-generated tissue debris from thrombus, atheroma, and normal aorta on platelet aggregation. Debris supernatant and suspension from lased thrombus induced dose-related aggregation, maximal at 48 +/ 12% and 65 +/- 2%, respectively. Debris suspension from normal aorta induced maximal aggregation of 35 +/- 12%, but the debris from atheromatous aorta surprisingly had no effect on platelet aggregation. The debris particle count was in the range of 10(10) to 10(12) per liter. Aspirin, 0.2 and 2.0 mmol/L, only weakly inhibited the debris-induced aggregation, and heparin up to 10 U/ml was ineffective. However, iloprost reduced aggregation to 40 +/- 11% of control at 0.3 ng/ml, and totally abolished it at 3 ng/ml. Soluble and particulate laser generated debris from vascular tissue and thrombus may cause platelet aggregation in vitro. This may have implications for laser coronary angioplasty. PMID- 1715124 TI - Effects of enflurane on inducibility of ventricular tachycardia. AB - The effects of enflurane on cardiac electrophysiologic parameters and on inducibility of ventricular tachycardia (VT) by programmed stimulation were studied in 12 patients (11 men, 1 woman, mean age +/- standard deviation 55 +/- 8 years) with drug refractory sustained monomorphic VT who underwent transcatheter ablation with high-energy direct-current shocks. One catheter ablation procedure was performed in 10 patients, whereas 2 ablation sessions were necessary in 2 patients. Programmed ventricular stimulation was performed on 2 separate days (mean interval 19). There were 2 baseline studies, 1 several days before ("baseline study I") and the second at the beginning of the ablation procedure ("baseline study II") while the patient was awake and nonsedated. The third programmed stimulation study was done 15 to 30 minutes after administration of anesthesia with enflurane, oxygen and nitrous oxide ("enflurane study"). Rate of sinus rhythm, QRS duration, PQ interval and ventricular effective refractory period were unaltered, whereas QTc interval increased significantly after initiation of anesthesia. Before and after induction of general anesthesia, clinical VT was inducible in all patients. However, in 1 patient, induction of VT was only possible by pacing in the left ventricle after enflurane administration. Based on these data, it is concluded that general anesthesia with enflurane, oxygen and nitrous oxide has no marked influence on inducibility of clinical VTs. Therefore, this type of anesthesia may be useful for nonpharmacologic, ablative procedures requiring general anesthesia. PMID- 1715125 TI - Effectiveness of imazodan for treatment of chronic congestive heart failure. The Imazodan Research Group. AB - A 12-week, multicenter, double-blind, randomized, placebo-controlled trial of imazodan, a type III phosphodiesterase inhibitor, was conducted in 147 patients with congestive heart failure to determine clinical efficacy and safety. Patients were randomized to placebo or 2, 5 or 10 mg of imazodan administered twice daily. Patients were maintained on their standard therapy including diuretics, digoxin and an angiotensin-converting enzyme inhibitor. The mean ejection fraction was 23 +/- 10%. Exercise time increased from baseline in all 4 groups. There was no significant difference observed between the placebo group and any of the treated groups with regard to exercise time, ejection fraction, frequency of ventricular premature complexes or ventricular tachycardia. When analyzed by intent to treat, the placebo mortality was 7% (3 of 44) and the imazodan mortality was 8% (8 of 103) (p = not significant). This study failed to demonstrate that imazodan provided any benefit in exercise performance when compared with placebo. PMID- 1715126 TI - Dipeptidyl aminopeptidase IV staining of cytologic preparations to distinguish benign from malignant thyroid diseases. AB - Staining for dipeptidyl aminopeptidase IV (DAP IV [EC: 3.4.14.5]) activity was applied to aspiration biopsy specimens or imprint preparations of surgical biopsy specimens from thyroid tumors. Material was obtained from 55 patients with histologically proven thyroid diseases: 9 with papillary carcinoma, 5 with follicular carcinoma, 11 with follicular adenoma, 13 with adenomatous goiter, 8 with Hashimoto's thyroiditis, and 9 with other benign conditions. Most tumor cells, follicular lumina in cell clusters, and intranuclear inclusions were strongly positive for DAP IV in all examples of papillary or follicular carcinoma. In contrast, only a few epithelial cells were labeled for DAP IV in follicular adenoma and adenomatous goiter. Some Hurthle cells in Hashimoto's thyroiditis also were positive for DAP IV. When a DAP IV scoring system based on the percentage of positive cells and staining intensity was used, all benign tissues except one (from a follicular adenoma) were found to have extremely low scores. These results indicate that staining for DAP IV activity is a simple but useful tool to aid in distinguishing benign from malignant thyroid neoplasms. PMID- 1715127 TI - Parathyroiditis associated with hyperparathyroidism and branchial cysts. AB - A 57-year-old man had renal stones, and biochemical investigation led to a diagnosis of primary hyperparathyroidism. Surgical exploration revealed bilateral inferior parathyroid enlargement. Both glands were removed; macroscopically, small cysts were seen on cut sections. Histologic examination showed broad bands of fibrosis, lymphoid follicles, and plasma cells that diffusely effaced the parathyroid architecture. Such features--if seen in the thyroid gland--would be reminiscent of an autoimmune process. The cysts were lined by respiratory and squamous epithelia and contained lymphoid follicles in their walls. Less affected areas of the parathyroid tissue were hyperplastic. It is believed that the inflammatory response in the parathyroid glands that is described in this article may be characteristic. It may result from the cysts or their contents or from an autoimmune reaction. PMID- 1715128 TI - Leukemia and lymphoma immunophenotyping in cell smears with immunogold-silver staining. AB - The potential of the immunogold-silver staining (IGSS) technique for immunophenotyping leukemia and lymphoma cells in cell smears was examined. Peripheral blood, bone marrow aspirates, lymph node biopsy specimens, fine-needle aspirates, and biologic fluids of 83 patients with acute or chronic leukemias, non-Hodgkin's lymphomas, or Hodgkin's disease were labeled. Cell smears, cytocentrifuge preparations, or imprints were fixed, incubated with the reagents, and counterstained with May-Grunwald-Giemsa. Stable immunostaining and good morphologic characteristics allowed accurate cell identification and rapid enumeration of the positive cells. The immunophenotypes obtained with the use of 35 monoclonal antibodies with different specificities were similar to those determined by flow cytometry or immunohistochemical studies on the same samples. This IGSS method was especially useful for the examination of poor samples or complex cell suspensions with rare malignant cells. It could be an alternative to the immunoenzyme methods that generally are used for this purpose. PMID- 1715129 TI - Difference in early development of presumed monozygotic twins with Rett syndrome. AB - Normal early development has generally been insisted on as an essential criterion for the diagnosis of Rett syndrome. A new set of monozygotic female twins is reported. Twin 1 was considered to be abnormal from birth while delay was not suspected in twin 2 until she was about one year old. Some regression occurred during the second year in both twins, who are now clinically indistinguishable from each other at age 4 years. Other than a slight difference in head circumference at birth, no environmental factor which could account for the clinical difference has been identified. PMID- 1715130 TI - Expression of renal Na(+)-Ca2+ exchange activity in Xenopus laevis oocytes. AB - The expression of a renal Na(+)-Ca2+ exchanger by Xenopus oocytes has been investigated. Each oocyte was injected with 50 ng of poly(A)+ RNA from either rat or rabbit kidney or with an equivalent volume of water. Na(+)-Ca2+ exchange was determined 3 days after injection, by measuring Ca2+ uptake by oocytes in the presence or absence of an outwardly directed Na+ concentration gradient. To manipulate Na+ concentration gradients, oocytes were first loaded with Na+ in Ca(2+)-free medium containing 90 mM Na+ and nystatin. They were then exposed to medium containing 45Ca and either 90 mM or 0 Na+. Na(+)-free media contained (in mM) either 90 K+, 90 choline or 85 choline plus 5 K+. Oocytes injected with rat kidney poly(A)+ RNA showed a Na+ gradient-dependent Ca2+ uptake of 6.6 +/- 0.8 (SE, n = 5) pmol.oocyte-1.30 min-1. This is significantly higher than the value of 3.4 +/- 0.5 (SE, n = 5) pmol.oocyte-1.30 min-1 obtained in water-injected oocytes (P less than 0.001). Similar results were obtained using poly(A)+ RNA from rabbit kidney cortex. Neither 10 microM nifedipine nor 0.5 mM D 600 significantly affected this Ca2+ uptake. However, 90% of the Ca2+ uptake was inhibited in the presence of 0.1 mM La3+. The poly(A)+ RNA-induced Na(+)-Ca2+ exchange activity was stimulated by the presence of 5 mM K+ in the extracellular choline solution compared with choline alone. Fractionation experiments indicate that the rat kidney Na(+)-Ca2+ exchanger was encoded by poly(A)+ RNA of 3-4 kb. PMID- 1715131 TI - Fibroblasts of rabbit kidney in culture. I. Characterization and identification of cell-specific markers. AB - There is currently no information available as to whether different renal fibroblast subpopulation can be identified and whether they show differences in functional properties. We therefore compared the growth characteristics of interstitial fibroblasts derived from the rabbit renal cortex and inner medulla (papilla) and sought cell-specific markers for the two populations of cells. Analyses of the population dynamics revealed that the mitotic lifespan of papillary fibroblasts (PF) is approximately 50% longer than that of cortical fibroblasts (CF), with the former going through 20 cumulative population doublings (CPD) before transition into terminally differentiated postmitotic cells compared with 9 CPD in CF. PF and CF populations contained three types of mitotically active cells (MFI, MFII, MFIII) and three types of postmitotic cells (PMFIV, PMFV, PMFVI) differentiating along a terminal cell lineage from MFI through PMFVI. In both PF and CF cultures the percent of MF-type cells decreased and the percent of postmitotic cells increased with successive doublings. Two dimensional polyacrylamide gel electrophoresis of uniform clonal populations of MFIII-type cells revealed two specific proteins for PF-MFIII-type cells, pf1 and pf2, and three specific proteins for CF-MFIII-type cells, cf1, cf2, and cf3. Additionally, a monoclonal antibody was raised that does not recognize CF in culture, but reacts strongly with PF. These studies demonstrate that rabbit renal PF have a pattern of growth in vitro that is distinct from that of CF and that they can be positively identified by specific immunological and protein markers in vitro. PMID- 1715132 TI - ATP-sensitive K+ channels from aortic smooth muscle incorporated into planar lipid bilayers. AB - Glibenclamide binding sites were identified in a membrane preparation from canine aortic smooth muscle. The dissociation constant for [3H]glibenclamide binding was 10 +/- 2 nM, with a density of 420 +/- 108 fmol/mg protein. The properties of ATP sensitive potassium (KATP) channels from the same membrane preparation incorporated into planar lipid bilayers were investigated. ATP was a potent inhibitor of the channels with half-maximal inhibition of channel activity by 41 microM ATP. Glibenclamide inhibited channel activity, and cromakalim activated the channel in the presence of ATP. Blockers of Ca(2+)-activated K+ (KCa) channels (charybdotoxin and tetraethylammonium ions) did not affect KATP channels in concentrations that caused significant block of KCa channels in bilayers. This membrane preparation should allow further biochemical and functional characterization of KATP channels and glibenclamide receptors in arterial smooth muscle. PMID- 1715133 TI - Trypanosoma cruzi: a carbohydrate epitope defined by a monoclonal antibody as a possible marker of the acute phase of human Chagas' disease. AB - An IgM monoclonal antibody (MAb) against a carbohydrate epitope present in Trypanosoma cruzi trypomastigote excretory-secretory antigens and expressed by different developmental stages of the parasite (epimastigote, trypomastigote and intracellular amastigote) was linked to a solid phase matrix and used as an antigen-capture antibody. Human serum complexes containing the epitope were then detected by using specific secondary antibodies against human immunoglobulin isotypes. Results of detection of IgM, IgG, and IgA serum complexes (SC) containing a T. cruzi polypeptide epitope showed that SC could be detected in 69% of the 13 Chagasic acute phase sera studied with IgG, in 84% with IgM, and in 75% with IgA. Only 16% (IgG-SC), 8% (IgM-SC), and 10% (IgA-SC) of chronic sera from 50 patients were positive. No patients with toxoplasmosis or rheumatoid factor were positive. Of the 11 leishmaniasis sera studied, four had IgG-SC, two had IgA SC, and five had IgM-SC. Of the eight Yanomamo Indians infected by Onchocerca volvulus, three were found to have IgG-SC, two had IgM-SC, and two had IgA-SC. Thirteen sera from healthy individuals living in an endemic area were also studied. One subject had IgG IgM and IgA-SC. The results presented in this study show for the first time, the specific detection of IgM, IgG, and IgA immune complexes using a MAb against T. cruzi. The presence of the epitope in association with IgM antibodies in sera from patients with the acute phase of the disease suggests that this antigen(s) carrying the epitope that reacts with the MAb could be a marker(s) of active infection. In addition, the specificity of the serum complex capture assay allowed the detection of Chagas' disease in two different endemic areas (Argentina and Venezuela). PMID- 1715134 TI - Neuroadaptive responses to chronic ethanol. AB - Cellular responses of neuronal tissue to chronic ethanol exposure are reviewed. Evidence for adaptive responses to the acute actions of ethanol is available for five systems: GABA-activated chloride channels, voltage-sensitive calcium channels, NMDA-activated cation channels, receptors coupled through stimulatory guanine nucleotide binding proteins, and membrane lipid order. We suggest that at least some of these adaptive responses occur because of ethanol actions at the level of gene expression. PMID- 1715135 TI - A combination histochemical stain for equine muscle. AB - The purpose of this study was to find a combination histochemical staining technique for the evaluation of equine skeletal muscle that is reliable and effective, while offering a substantial reduction in the labor and cost involved with currently used individual histochemical methods. Several combinations under varying conditions of pH were studied. The most uniform results were obtained using an acid preincubation step at an optimal pH of 4.2 followed by reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) and the remainder of the acid-ATPase procedure. PMID- 1715136 TI - Laser treatment of tumours in the gastrointestinal tract. PMID- 1715137 TI - [Radiotherapy of epidermoid cancers of the anal canal]. PMID- 1715138 TI - [Hepatoid adenocarcinoma of the stomach: an unusual anatomo-clinical entity?]. PMID- 1715139 TI - Cytotoxic effects of children's faeces: relation to diarrhoea due to Clostridium difficile and other enteric pathogens. AB - Cytotoxicity of faecal extracts was demonstrated in 47 of 88 children (54%) referred for microbiological investigation of stools. Cytotoxic Clostridium difficile and vertotoxigenic Escherichia coli (VTEC) were the pathogens identified most commonly but cytotoxicity was also found in association with Campylobacter jejuni, Salmonella spp, Shigella sonnei, Giardia lamblia, rotavirus, adenovirus and poliovirus type 1 which had been acquired by oral immunization. In two patients, one of whom had cystic fibrosis, cytotoxicity of faecal extracts was associated with isolation of Pseudomonas aeruginosa. In five of 13 patients with diarrhoea and cytotoxic C. difficile, other pathogens were also present, in agreement with the view that C. difficile are more readily recovered when the intestinal flora have been altered by colonization with other micro-organisms. There was no correlation between previous treatment with antibiotics and isolation of C. difficile. Cytotoxicity neutralized by antitoxin, usually to C. sordellii, is used to detect cytotoxic C. difficile. We suggest that cytotoxicity not neutralized in this way should be an indication for further investigation of stools for the presence of other pathogens such as VTEC or viruses. PMID- 1715140 TI - The serodiagnosis of tuberculosis in children: an evaluation of an ELISA test using IgG antibodies to M. tuberculosis, strain H37 RV. AB - An enzyme-linked immunosorbent assay (ELISA) for detecting serum IgG antibodies to an autoclaved suspension of Mycobacterium tuberculosis H37 RV was evaluated as a diagnostic tool in 132 clinical cases of childhood tuberculosis. The mean (SD) optical density value in these patients was 0.115 (0.122) compared with 0.022 (0.017) in the control group of patients who had non-tuberculous acute respiratory tract infections. Using as a cut-off level the mean of the control plus 2 standard deviations, the test sensitivity was 62% and the specificity was 98% in all the patients with a clinical diagnosis of tuberculosis. In the culture positive group (n = 35), the sensitivity was 69%. A positive correlation was shown between the optical density levels and increasing age and chronicity of infection but not with respect to tuberculin skin reactivity, nutritional status and the duration of prior therapy. In addition, BCG vaccination (presence of a scar) did not affect the ELISA result. We conclude that this ELISA test is a useful diagnostic test in children. PMID- 1715141 TI - Acute renal failure related to infectious disease in infancy and childhood. AB - In this prospective study done between 1983 and 1989, of 109 children with acute renal failure in Sivas, Turkey, infectious disease was found in 94. This was related mainly to preventable disease such as acute gastro-enteritis, sepsis and post-streptococcal acute glomerulonephritis. Within this infectious disease group, the highest incidence rate was found in sepsis. PMID- 1715142 TI - Childhood intussusception in Ile-Ife, Nigeria. AB - A retrospective review of 41 intussusceptions encountered in 39 children seen over an 8-year period in Ile-Ife, Nigeria is presented. Most cases (61.5%) occurred in infancy. This contrasts with previous reports from Nigeria where intussusception has been presented as being commoner in older children. Vomiting, abdominal pain, excessive crying and passage of bloodstained stools were the main presenting symptoms. An abdominal mass was palpable in only 28.2% of patients. Generally, patients presented late in hospital with only two (5.1%) arriving within 24 hours of the onset of illness. Hydrostatic reduction with barium enema was attempted in these two patients, and it successfully reduced the intussusception in one and caused partial reduction in the other. Nineteen patients (46.3%) required bowel resection. There were nine deaths, giving a mortality rate of 23.1%. The relatively high bowel resection and mortality rates were attributed to the delay in seeking medical treatment. PMID- 1715143 TI - Persistent or recurrent pneumonia in Saudi children seen at King Khalid University Hospital, Riyadh: clinical profile and some predisposing factors. AB - Children with persistent or recurrent pneumonia without an apparent cause constitute an important clinical category with much morbidity and mortality and can be perplexing and frustrating to the treating physician and the parents. To identify the clinical profile and predisposing factors in this group of patients, 18 Saudi children were studied. Their ages ranged from 3 months to 12 years (mean age 5.7 years) with male preponderance--12 boys and 6 girls (M:F ratio 2:1). About 44.4% had immune and inherited metabolic disorders. Anatomical abnormalities were found in four (22.2%). Two had measles as a predisposing factor. None had tuberculosis or pertussis. One child each had pulmonary candidiasis and laryngeal papilloma, probably contracted from their mothers as congenital infections. Though the pattern seems to follow that found in developed countries, it is noteworthy that cystic fibrosis was not identified in any of our patients. PMID- 1715144 TI - Aetiological profile of paediatric laryngeal stridor in an Indian hospital. AB - The case records of 180 infants and children with stridor were reviewed to note their clinical profile. We found that cases of acute stridor outnumbered cases of chronic stridor. The aetiology was congenital in 32.2% and acquired in 67.8%. Laryngomalacia (19.4%) was the commonest congenital cause. Laryngotracheobronchitis and diphtheria were the commonest infectious causes (35%). Tracheostomy was performed in 25% of cases, the most frequent indication being bilateral abductor paralysis. The problems peculiar to developing countries that influence the clinical status of a child in stridor are discussed. PMID- 1715145 TI - Scorpion sting: a management problem. AB - Admissions for scorpion sting in 1 year and deaths resulting from scorpion sting over 3 years were analysed. Features that indicated the severity of the clinical condition were identified. Pulmonary oedema and shock were the usual causes of death. Poor management of fluid therapy was responsible for the frequently unsatisfactory resolution of envenoming, especially when purified human plasma was used. The role of the scorpion antivenom used is questioned and controversy regarding the most appropriate sedative to use in the management of scorpion sting is still not resolved. An in-depth study of these management issues is urgently required. PMID- 1715146 TI - A review of accidental poisoning in Barbados--a new perspective (1981-1985). AB - A retrospective study is presented of accidental poisoning in 348 children who were admitted to the Queen Elizabeth Hospital during the 5-year period 1981-1985. These cases represented 4% of all admissions to the paediatric medical ward, with 56% of patients being boys and 43% girls. Most fell into the age group 13 months to 3 years, and all were aged between 6 months and 12 years. In the majority of cases, complications were mild or absent and no deaths were recorded. Their admission to hospital, however, did contribute greatly to the National Health Budget, costing more than BDS$130,000 over the 5-year period. Most of the children required only gastric decontamination and supportive treatment. Thirty four per cent of the poisonings were due to medications prescribed for friends or family. Common household and garden products were involved in a similar number (33%). Fortunately, kerosene poisoning has declined in importance, but still accounted for 20% of cases. These ratings contrast with previous studies where kerosene ingestion was the most frequent cause of poisoning. With the ever increasing variety of toxic drugs and chemicals now available, there is a greater need to continue to stress the prevention of accidental poisoning. This will decrease the likelihood of mortality and morbidity resulting from accidental ingestion and likewise decrease the cost of hospital admissions for a largely preventable condition. PMID- 1715147 TI - Accidental kerosene ingestion: a 3-year prospective study. AB - Accidental kerosene ingestion continues to cause morbidity and mortality in third world countries, where kerosene is still used for cooking, heating and cleaning. In this prospective study, 78 children aged from 10 months to 5 years were managed at Makassed Hospital in Jerusalem for kerosene ingestion. Respiratory distress developed in 60 (76.90%) children. Two who required mechanical ventilation died. Vomiting, which occurred in 49 cases, did not seem to increase the risk of respiratory complications, suggesting that aspiration occurs with the initial ingestion. Chest X-ray changes were noted in 60% of the children on admission. Pleural effusions occurred in three cases over 24 hours after the incident. CNS manifestations, most likely caused by anoxia, were seen in 27% of the children, but in only two were they severe in the form of convulsions, and both died. Fever occurred in about 50% of the children during their stay in hospital. Severe gastric dilatation developed in the four most severely ill children, two of whom died. The quantity of kerosene ingested by them was estimated to be large. PMID- 1715148 TI - Severe forms of status epilepticus. AB - Severe forms of status epilepticus needing ventilation with or without the need for a general anaesthetic were studied among the children admitted to the paediatric department at Assir Central Hospital, Saudi Arabia. From November 1988 to April 1990, 255 children were admitted because of fits, of whom 12 (4.7%) developed severe status epilepticus and required admission to the intensive care unit for ventilation. This group was studied in detail with regard to past history, course in the hospital and outcome. Five children recovered fully, four were left with neurological disability, and three died. The need to ventilate a patient or give a general anaesthetic for status epilepticus rarely arises in ordinary clinical practice. This study demonstrates the importance of these measures to reduce the risks of permanent neurological deficit and, possibly, death in severe status epilepticus. PMID- 1715149 TI - Should we resuscitate? Ethical dilemmas. AB - Over a 2-year period, there were 312 perinatal deaths. A total of 144 (46.2%) of these deaths were due to lack of or delayed resuscitation at birth. This group comprised 45 infants with multiple congenital abnormalities, 31 with severe birth asphyxia, 25 with meningomyelocele, 20 with infections and 15 with kernicterus. Taking the decision not to resuscitate may be difficult, perplexing and agonizing for the families and health professionals. The decision may be easier if one can prognosticate as to intact survival and development. The physician, though he is not God, has to be firm and decisive--if intact survival or satisfactory developmental outcome is very unlikely, then ther is probably no need to resuscitate. PMID- 1715150 TI - Plasma ferritin concentration in relation to vitamin A and E status of children with severe oedematous malnutrition. AB - The association between plasma ferritin concentration and vitamin A and E status was studied in 17 children aged 15-72 months with severe oedematous malnutrition. The controls were 10 children of similar age who were apparently well and with no obvious signs of clinical malnutrition. Plasma ferritin concentration in the patients was significantly higher than that in the control children. Conversely, the plasma concentrations of beta-carotene, alpha-tocopherol and retinol in patients were significantly lower than those in plasma of control children. The median (interquartile range) plasma alpha-tocopherol concentration of patients, 6.03 (5.29-9.50) mumol/l, is below the threshold of vitamin E deficiency (11.6 mumol/l). Fifteen of 17 (88%) malnourished patients were found to have plasma tocopherol concentrations below the normal threshold. However, all the patients had a tocopherol: cholesterol ratio greater than 2.22, indicating adequate vitamin E status for the level of cholesterol present in plasma. Twelve of 17 patients (70.5%) had plasma retinol concentration less than 0.70 mumol/l, indicative of marginal vitamin A status, while 3 patients had plasma retinol concentrations less than 0.35 mumol/l, indicating vitamin A deficiency. The median (interquartile range) plasma retinol concentration of patients, 0.51 (0.41 0.93) mumol/l, is significantly less than that of control children, 0.96 (0.74 1.09) mumol/l; p less than 0.01 Mann Whitney U test. Furthermore, anaemia (Hb less than 110 g/l) was widespread in the patients. The results also indicate no significant correlation between elevated ferritin concentration and the concentrations of beta-carotene, retinol and alpha-tocopherol in the patients' plasma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715151 TI - Immunization coverage survey in eight regions of Saudi Arabia. AB - A study of 1916 children in eight regions in the Kingdom of Saudi Arabia was conducted to find out the immunization coverage of the six expanded programme on immunization (EPI) target diseases. The eight regions surveyed were selected because they showed less than 80% coverage in a previous survey. The WHO cluster sampling technique was employed. The field workers were trained to conduct house to-house surveys, inspect the children's immunization cards or health registers and complete the data forms. The overall coverage in the eight regions was found to be 97.5% for BCG, 94.3% for DPT and polio and 86.0% for measles. Altogether, 84.8% of the children had completed their immunization schedule by the end of their 1st year of life. The situation improved further in the 2nd year of life to reach 91.9%. The immunization coverage varied from region to region and from one EPI target disease to another. The partially immunized children represented 6-18% while those who did not receive any immunization represented only 0-8%. The drop out rate between BCG and the third dose of DPT ranged from 1.9% to 9%. The factors behind the success are discussed and recommendations made for further improvement and maintenance of high immunization coverage. PMID- 1715152 TI - Co-sleeping and neonatal weight loss. AB - The degree of neonatal weight loss and the time required for a newborn to regain birth-weight vary between different populations. It has been suggested that co sleeping, the sharing of a single bed by mother and baby, might be an important factor in determining the neonatal weight change. In a rural setting in central Africa, 203 mother-baby pairs were studied. For 101 babies cared for in cribs and for 102 babies who shared their mothers' beds, sleeping location was associated with no significant difference either in the weight loss (6.3% for each group) or the time taken to regain birthweight (5.3 days for crib-cared babies and 5.4 days for co-sleepers). It is concluded that co-sleeping does not play a major role in determining the neonatal weight change. PMID- 1715153 TI - Risk factors for congenital syphilis. AB - A prospective study of newborns whose mothers had untreated or inadequately treated syphilis was undertaken. The infants were followed up for 3-4 months to ascertain whether they had congenital syphilis. A number of variables were analysed as possible predictive factors for the development of congenital syphilis. A maternal Venereal Disease Research Laboratory (VDRL) titre of 1:32 or above indicated which infants would develop congenital syphilis with a sensitivity of 93% and a specificity of 78%. The risk of a congenitally infected infant was significantly higher amongst the group of untreated mothers (p = 0.036). Low birthweight per se did not appear to be a predictor of the subsequent diagnosis of congenital syphilis. Using these simple predictive factors it may be possible to determine which at-risk infants would most benefit from careful supervision or a full 10-day course of therapy. PMID- 1715154 TI - [The changes in tumor marker levels after chemolipiodolization in early phase]. AB - Changes of the serum tumor marker levels were analyzed in 80 patients with hepatic malignancy treated with chemolipiodolization. In 52 cases, the levels of the tumor marker elevated on the 1st day after treatment with an exponential drop afterwards. The levels of the tumor marker on the first day compared with those before treatment were significantly higher in effectively treated cases. The exponentially declining curve dissociated about 10 days before reaching the lowest point, and it gradually rose again in 32 cases. The second treatment of chemolipiodolization should be applied before dissociation of the exponential curve. PMID- 1715155 TI - [Immunochemo-embolization therapy of hepatocellular carcinoma]. AB - To increase antitumor effects of transcatheter arterial embolization therapy (TAE) for hepatocellular carcinoma (HCC), immunochemo-embolization therapy via hepatic artery was performed with a mixture of doxorubicin and iodized oil (LPD) following a mixture of gamma-IFN, OK-432 and gelatin sponge, and then a mixture of actinomycin D and gelatin sponge. Three patients with HCC were treated by this procedure. One patient had tumor thrombus in inferior vena cava (IVC). The serum alpha fetoprotein (AFP) levels before this procedure ranged from 66 ng/ml to 8,360 ng/ml. Following this procedure, the serum AFP levels began to decrease for 1-3 months, then increased for 3-6 months, and again suddenly decreased under 10 ng/ml in two cases after initial procedure. The serum AFP levels of two cases revealed under 10 ng/ml for 9-18 months. CT after 2 weeks to 3 months of this procedure showed a low-density area around LPD-uptaking tumor and after 1-8 months decreased tumor in size with diminishing of the low-density area. Therapy for the main tumor of one case with tumor thrombus of IVC proved to be effective, but it was not effective for tumor thrombus of IVC. The reasons that the serum AFP level increased after decreasing for 1-3 months and then fell below 10 ng/ml following this procedure, may be some kinds of immunological antitumor effects produced by endogenous cytokines. PMID- 1715156 TI - [Problems with topical therapies for extensive squamous cell carcinoma of the buttocks]. AB - In patients in whom cancer originating from a bed-sore has advanced to a stage where radical treatment is impossible, topical treatment with ointment, or systemic anti-cancer chemotherapy, is usually applied. We recently attempted treatment of extensive squamous cell carcinoma of the buttocks, for which radical treatment was impossible, by chemotherapy using a reservoir for intraarterial injection. This therapy involved intraarterial injection of bleomycin and 5-FU, together with topical treatment with 5-FU ointment. We describe the problems encountered with this therapy, e.g., measures against fever after intraarterial injection, management of the affected area, evacuation, possibility and limits of radiation therapy, and assessment of its efficacy. PMID- 1715157 TI - Hepatic methotrexate content and progression of hepatic fibrosis: preliminary findings. AB - Liver tissue from 16 patients with rheumatoid arthritis was studied. The patients had received low dose methotrexate weekly for a minimum of 12 months between two liver biopsies. The progression of pericellular fibrosis was measured by computerised image analysis. Extracts of these liver biopsy specimens were pooled into five samples according to the progression of hepatic fibrosis and analysed by high performance liquid chromatography. The concentrations of methotrexate, 2,4 diamino-N(10)-methylpteroic acid, and methotrexate polyglutamate were markedly increased in the samples obtained from the three patients who recorded the greatest increase in fibrosis. These preliminary data suggest that progression of hepatic fibrosis is related to the retention of methotrexate and metabolites in the liver. PMID- 1715158 TI - Heterogeneity of the antigenic site responsible for the induction of neutralizing antibodies in infectious bursal disease virus. AB - Using neutralizing monoclonal antibodies, three categories of escape mutants were selected from a stock of wild-type infectious bursal disease virus (IBDV). Additional mutants were found, where alterations coexisted in two or three of these epitopes. Although each group of mutants had a distinct reaction pattern with neutralizing monoclonal antibodies, all types of mutants were neutralized by convalescent chicken sera to the same extent. In spite of the lack of homogeneity in these antigenic sites located on IBDV structural polypeptide VP2, all neutralizing monoclonal antibodies reacted with epitopes in extracts prepared from the bursa of Fabricius from animals that had died during recent outbreaks of infectious bursal disease in the F.R.G. and Africa. Since binding to VP2 of the escape mutants, demonstrable by immunoprecipitation, correlated with the neutralizing capacity of these antibodies, a combined immunoprecipitation immunoblotting technique was established as equivalent for a neutralization assay. The results of our experiments indicate that IBDV did not undergo a major antigenic variation in these two areas of Europe and Africa. The significance of protein conformation for the interaction of VP2 with neutralizing antibodies was underlined by the finding that renatured VP2 was capable of binding neutralizing antibodies; the antibodies induced in animals by immunization with this protein, however, were not neutralizing. PMID- 1715159 TI - Effects of prolonged parotid duct ligation, parotidectomy and acute hepatitis of rats on amylase activity. AB - Amylase activity in the parotid gland was extremely reduced (to 2.4 per cent) by prolonged duct ligation but that in the pancreas and serum as similar to controls. The serum amylase activity of parotidectomized rats was remarkably reduced within a week of surgery, but recovered to approximately the previous level in 4 weeks. The amylase activities in the pancreas and liver were not different between control and parotidectomized rats. The serum amylase activity of rats with acute hepatitis (1 day after administration of D-galactosamine) decreased to 18.5 per cent compared with controls; however, the activity recovered to its original level after 4-7 days. There was no pancreatic amylase in the serum either of parotidectomized rats or those with acute hepatitis. These observations show that the parotid glands may not be the only source of serum amylase. Serum amylase is mainly derived from the parotid gland, but liver may contribute to serum amylase in compensation for the parotid glands. PMID- 1715160 TI - Comparison of acute electrophysiological effects of amiodarone and its metabolite desethylamiodarone in Langendorff perfused guinea pig hearts. AB - During long-term treatment with amiodarone, slowing of conduction through the atrioventricular node, a prolongation of the QT-interval, and a prolongation of the atrial and ventricular myocardial refractoriness always developed. During short-term treatment, these effects were not found, except for depression of the AV-nodal conduction. This led to the suggestion that the electrophysiological effects of amiodarone during long-term treatment might be partly the result of the accumulation of its metabolite desethylamiodarone. Therefore, we examined the electrophysiological effects of amiodarone and desethylamiodarone on conduction and refractoriness in isolated spontaneously beating guinea pig hearts perfused by the method of Langendorff. Within 1 h of perfusion, desethylamiodarone caused a more pronounced prolongation of the AV-nodal, His-bundle, and intraventricular conduction intervals than did amiodarone. Desethylamiodarone, but not amiodarone led to a prolongation of the QT-interval. The refractoriness of sinoatrial-, AV nodal conduction, and of the atrial myocardium were significantly more prolonged by amiodarone than by desethylamiodarone. Both compounds showed a comparable strong rate-dependent effect on AV-nodal refractoriness. The ventricular refractoriness was similarily prolonged by either compound. These results show that for the class-III effects (i.e., prolongation of repolarization period) observed under chronic treatment of amiodarone the metabolite desethylamiodarone may be responsible. Desethylamiodarone also exerts more pronounced effects on the fast-channel-dependent parts of the conduction system than does amiodarone, a fact indicated by a higher prolongation of His-bundle and intraventricular conduction. PMID- 1715162 TI - Partition of serum lipoproteins in a polyethylene glycol/dextran two-phase system. AB - Aqueous two-phase systems containing polyethylene glycol (PEG) and dextran as phase forming polymers were used for the partition of unmodified and hypochlorite modified lipoproteins. Low density lipoprotein (LDL) was separated from high density lipoprotein (HDL) by sequential ultracentrifugation from human plasma. In agreement with the higher electrophoretic mobility, high density lipoprotein shows a higher value of the partition coefficient in contrast to low density lipoprotein. An increase in the concentration of chloride ions reduces the enrichment of lipoprotein in the top phase and favours the accumulation of aggregated material at the interface. The partition coefficient strongly depends on the age of the lipoprotein sample. Differences in the value of the partition coefficient could be obtained for the lipoprotein fractions HDL-2 and HDL-3. Hypochlorite modified LDL shows higher values of the partition coefficient due to the higher negative charge of the modified lipoprotein particle. PMID- 1715161 TI - On the mode of action of antiphlogistically active DL-omega-phenyl amino acid esters. AB - DL-omega-phenyl amino acid esters turned out to be inhibitors of the sheep vesicular gland prostaglandin H synthase in addition to their antiphlogistic action on the carrageenan-induced oedema of the rat paw and weak antihistaminic actions. The inhibition of the prostaglandin H synthase was dose-dependent, the inhibitory potencies were however much lower than that of indomethacin. Some but not all derivatives, such as DL-4-amino-4-phenylbutyric acid octyl ester, also caused inhibition of the pure lipoxygenase from rabbit reticulocytes and the conversion of arachidonic acid to leukotriene B4 and 5S-hydroxy-6E,8Z,11Z,14Z eicosatetraenoic acid by human polymorphonuclear leukocytes as well as inhibition of antigen-induced release of histamine from mast cells of ovalbumin-sensibilized rats. Since no clear relations between the data of the in vitro and in vivo models were obtained, further studies on the pharmacokinetics and possible biotransformations are required. PMID- 1715163 TI - Excessive dietary tryptophan enhances plasma lipid peroxidation in rats. AB - High plasma cholesterol levels and plasma lipid peroxidation are associated with atherosclerosis. The effect of excessive dietary tryptophan on plasma lipid peroxidation was studied in rats fed a diet containing soybean oil (control), as well as an atherogenic diet, containing coconut oil and cholesterol. Feeding the atherogenic diet resulted in a 5-fold increment in plasma cholesterol concentration with no significant effect of the tryptophan supplementation. The plasma obtained from the hypercholesterolemic rats exhibited a 67% increased lipid oxidation (measured as thiobarbituric acid reactive substances) in comparison to normocholesterolemic plasma. Dietary tryptophan supplementation increased plasma lipid peroxidation by 9 and 21% in the control and in the hypercholesterolemic animals, respectively. Similarly, the excessive dietary tryptophan enhanced macrophage cholesterol esterification rate by 40 and 38% following cell incubation with the plasma obtained from the control and from the hypercholesterolemic animals, respectively. Since tryptophan is the precursor of serotonin we have measured urine concentration of 5-hydroxyindoleacetic acid (5HIAA), the metabolite of serotonin, and found 22 and 118% elevation in 5HIAA in the tryptophan fed control and hypercholesterolemic rats, respectively. The direct effect of tryptophan and serotonin on in vitro lipid peroxidation was also studied. Low density lipoprotein (LDL) was peroxidized by incubation with copper ions in the presence of tryptophan or serotonin. Serotonin was shown to enhance LDL peroxidation whereas tryptophan had no effect on LDL peroxidation. We conclude that excessive dietary tryptophan may be atherogenic since it enhanced plasma lipid peroxidation in hypercholesterolemic rats and increased macrophage uptake of plasma cholesterol. These effects are probably associated with increased plasma concentration of serotonin following the consumption of a tryptophan supplemented diet. PMID- 1715164 TI - [Whipple's disease and tuberculosis. A previously unreported association]. AB - Whipple's disease and pulmonary and lymph node tuberculosis have been diagnosed in a 70 year-old man hospitalized for weight loss and fever. The tuberculous or Whipple's disease-related nature of associated non-necrotizing epithelioid hepatic granulomas could not be determined. The existence of cellular immune defects in Whipple's disease could explain the association with tuberculosis, which has not been previously reported in the literature. PMID- 1715165 TI - Rapamycin: FK506's fraternal twin or distant cousin? AB - Rapamycin (RPM) is the latest in a series of new bacterial-derived immunosuppressants. Here, Randall Morris compares and contrasts the mechanism of action and immune-modulating potential of RPM with the structurally-similar FK506. PMID- 1715166 TI - Natural autoantibodies: from 'horror autotoxicus' to 'gnothi seauton'. AB - The immune system of normal unimmunized animals is characterized by the presence of B cells synthesizing and secreting mainly polyreactive, but also monoreactive, IgM and IgG natural antibodies that can react with a variety of self constituents. These antibodies, like the autoantibodies appearing in several immunopathological states, use the same genetic elements as the antibodies directed against environmental antigens, and seem to be encoded by unmutated germ line genes. Accumulating evidence indicates that these natural auto-antibodies exert various biological roles, both related and unrelated to the immune system. In this article, Stratis Avrameas proposes that natural auto-antibodies, by interacting with the large number of self constituents present in an organism, establish an extensive dynamic network that contributes to the general homeostasis of the organism. PMID- 1715167 TI - The dominant self and the cryptic self: shaping the autoreactive T-cell repertoire. AB - Deletion of autoreactive T cells during the induction of self tolerance has been directly demonstrated. However, it is still relatively easy to detect self reactivity in normal healthy animals. In this article, Guy Gammon, Eli Sercarz and Gilles Benichou speculate on which T cells may escape tolerance induction and discuss how these cells could subsequently be involved in autoimmunity. PMID- 1715168 TI - In vitro diagnosis in asthma: the state-of-the-art. AB - "In vitro assays in asthmatic patients have been exploring the "allergic" component of asthma. Solid-phase IgE tests can supplement or replace skin tests. Histamine release and determination are performed with automated, or RIA/ELISA; however, these assays are still research oriented. More recently, tryptase has been investigated, and the serum levels of this enzyme correlate well with mast cell activation. But asthma is a multifactorial/facetted syndrome, and these assays provide minimal and specific informations which may just focus on limited etiological aspects of an elusive malady". PMID- 1715169 TI - Predicting common foldings of homologous RNAs. AB - A new approach is proposed for determining common RNA secondary structures within a set of homologous RNAs. The approach is a combination of phylogenetic and thermodynamic methods which is based on the prediction of optimal and suboptimal secondary structures, topological similarity searches and phylogenetic comparative analysis. The optimal and suboptimal RNA secondary structures are predicted by energy minimization. Structural comparison of the predicted RNA secondary structures is used to find conserved structures that are topologically similar in all these homologous RNAs. The validity of the conserved structural elements found is then checked by phylogenetic comparison of the sequences. This procedure is used to predict common structures of ribonuclease P (RNAase P) RNAs. PMID- 1715170 TI - Three-dimensional folding of Tetrahymena thermophila rRNA IVS sequence: a proposal. AB - We studied the Tetrahymena thermophila rRNA IVS sequence with the aim of obtaining a model of the structure characterized by the bases proximity of the self-reactions sites. The considered sequence kept up those fragments essential for its catalytic activity as demonstrated by deletion mutants. The first step was the theoretical analysis with a computer method previously proposed, to find optimal free energy secondary structures with the required features, under the suitable constrains. Then we tried folding the obtained secondary structures, in low resolution tertiary models, which kept up the proximity of the catalytic sites also in the space. The proposed tertiary folding seems to provide for a better explanation to the transesterification mechanisms and moreover it is in good agreement with the experimental data (activity of mutants, enzymatic cleavages, phylogenetically conserved regions). PMID- 1715171 TI - A method for the rapid detection of single base polymorphisms in genomic DNA. PMID- 1715172 TI - Assaying nanogram amounts of dilute protein. PMID- 1715173 TI - Simple modification of the single-step method of RNA isolation from rat liver. PMID- 1715174 TI - 3H-uridine incorporation in early porcine embryos. AB - The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages. PMID- 1715175 TI - Culture of human sebocytes and markers of sebocytic differentiation in vitro. AB - Human sebocytes obtained as explants after in vitro culture of isolated sebaceous glands were recently shown to maintain in part a sebocytic differentiation. The aim of this study was to further identify markers of sebocytic differentiation in vitro. Therefore, the morphology of cultured human sebocytes, and their differentiation with lipid storing and expression of cellular proteins were investigated by microscopy, electron microscopy, study of cell kinetics, cytochemistry and immunocytochemistry, and were compared to cultured human keratinocytes obtained from the same skin specimens. At first, sebocytes in all stages of sebocytic differentiation were detected in vitro. Abundant cytoplasmic lipids and the absence of desmosomes were identified as their ultrastructural characteristics. Secondly, an increasing number of sebocytes storing lipids was detected during cell proliferation. Sebocytes contained up to 4 times more lipids than keratinocytes in vitro. Squalene and increased quantities of wax/sterol esters could be extracted from secondary sebocyte cultures. Thirdly, the monoclonal antibodies 6B10 (keratin 4), RPN1162 (keratin 7), and OM-1 labeled only sebocytes in vitro. Furthermore, sebocytes presented a marked expression of keratin 19 in comparison to keratinocytes, as detected with CK 4.62, and a lack of RPN1161 (keratins 1 and 2) expression, which was typically found to be expressed in cultured keratinocytes. The culture of human sebocytes possessing several characteristics of sebocytic differentiation in vivo offers unique possibilities in investigating direct effects on sebaceous cell growth, differentiation and their regulation. PMID- 1715176 TI - Thrombin enhances lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis. AB - To clarify the role of thrombin in fibroblast growth and the development of pulmonary fibrosis in bleomycin-induced interstitial lung disease, we examined the relationship of thrombin activity to fibroblast growth-stimulating activity (FGA) in bronchoalveolar lavage (BAL) fluid from bleomycin-treated rats. Male Wistar rats were given a single intratracheal injection of bleomycin, BAL was performed 2, 6, and 15 days later, and the BAL fluid was assayed for thrombin activity and FGA. Higher FGA than the control value was detected in the BAL fluid from rats on day 6 after bleomycin administration. In bleomycin-treated rats, thrombin activity in the BAL fluid was significantly elevated on day 2 and maximal on day 6. The FGA of the BAL fluid from bleomycin-treated rats on day 6 was significantly decreased by its treatment with various thrombin inhibitors, such as alpha 1-protease inhibitor, antithrombin III, hirudin, and MD-805. In our assay, purified rat thrombin also showed FGA in vitro, and its FGA was inhibited by the same concentrations of these thrombin inhibitors as those inhibiting the activity in the BAL fluid. On ammonium sulfate fractionation, most of the thrombin activity was recovered in the fraction of 35 to 50% saturation in which most of the FGA was detected. These results suggest that the FGA of the BAL fluid from bleomycin-treated rats was at least partly due to thrombin is responsible, at least in part, for fibroblast growth and pulmonary fibrosis in bleomycin induced interstitial lung disease. PMID- 1715177 TI - Structure, secretion, and bacterial specificity of an endogenous lectin from cystic fibrosis lung. AB - Endogenous heparin-binding lectin purified from postmortem lung samples of two cystic fibrosis (CF) patients was compared to lectin derived from normal tissue with respect to structure, carbohydrate specificity, interaction with alginate derived from CF isolates of Pseudomonas aeruginosa, and secretion within the lung. Lectin was purified from extracts of lung tissue by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. Lectin purified from either CF lung or control tissue ran as two peptides of approximately 16,000 and 13,000 molecular weight on electrophoresis in sodium dodecyl sulfate. The lectins displayed similar carbohydrate specificity and interacted in much the same way with bacterial alginate. An increase in lectin secretion was seen in CF lungs affecting the bronchial epithelial cells and the mucosal glands. The data suggest that the major changes seen in endogenous heparin-binding lectin in CF are related to the quantity and distribution of lectin secretion. PMID- 1715178 TI - Isolation of a human T cell line specific for a streptococcal cell surface antigen. AB - A streptococcal cell surface antigen of Mr 185,000 (SAI/II) expressed by Streptococcus mutans has previously been well characterised. A T cell line specific for native SAI/II has been isolated from peripheral blood mononuclear cells (PBMC) of a naturally sensitised normal individual. This line has been maintained in culture for several months and was shown to be highly specific, not only for different preparations of native antigen but also for recombinant SAI/II protein. It did not respond to a homologous antigen SpaA (Mr 210,000), extracted from Strep. sobrinus. The phenotype of the line was CD3+ CD4+ CD8- TcR alpha beta +. HLA typing and inhibition studies showed that the response was restricted by both DR and DP encoded class II. PMID- 1715179 TI - Deproteination of nucleic acids by filtration through a hydrophobic membrane. AB - To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid. PMID- 1715180 TI - The ability of staurosporine to modulate pancreatic acinar cell desensitization by TPA, carbamylcholine and caerulein. AB - The implication of protein kinase C in the phenomenon of pancreatic acinar cell desensitization to carbamylcholine, caerulein and the phorbol ester 12-O tetradecanoylphorbol-13-acetate (TPA) was investigated using a potent PKC inhibitor, staurosporine. At a concentration of 1 microM, staurosporine caused a maximum 64% inhibition of amylase release from rat pancreatic acini stimulated by 100 nM TPA. At 100 nM, staurosporine reduced by 50 to 55% amylase secretion elicited by maximal concentrations of carbamylcholine or caerulein without affecting their potency. Staurosporine was also able to prevent completely desensitization by TPA of the subsequent secretory response to carbamylcholine and caerulein. Furthermore, staurosporine also totally prevented desensitization by caerulein of the subsequent secretory response to caerulein. In contrast, staurosporine only partially prevented desensitization by carbamylcholine of the subsequent secretory response to carbamylcholine. These results indicate that staurosporine is a potent inhibitor of protein kinase C as it inhibited the secretory response to carbamylcholine, caerulein and TPA. They also suggest that desensitization of the secretory response induced by TPA and caerulein used a common pathway involving protein kinase C activation. Finally, desensitization by carbamylcholine is more complex as it is only partially prevented at staurosporine; therefore, protein kinase C activation seems to be one of the factors involved. PMID- 1715181 TI - Inhibition of neutrophil superoxide production by adenosine released from vascular endothelial cells. AB - To investigate the inhibitory effect of adenosine released by endothelium on neutrophil superoxide (O2-) production, we treated confluent monolayers of cultured human umbilical vein endothelial cells with the enzyme adenosine deaminase, and then added human neutrophils. Superoxide (O2-) production by human neutrophils stimulated with 10(-6) M formyl-methionyl-leucyl-phenylalanine was inhibited by 49% in the presence of a confluent monolayer of human umbilical vein endothelial cells (5.1 +/- 0.1 versus 2.6 +/- 0.3 nmols O2-/10(6) neutrophils). Addition of 0.25 U/ml adenosine deaminase to neutrophils plus endothelial cells restored formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil superoxide production to the level seen with neutrophils alone. Deoxycoformycin (10(-4) M), an inhibitor of adenosine deaminase activity, prevented the increase in superoxide production associated with adenosine deaminase addition. The adenosine analogue 5'-(N-ethylcarboxamido)- adenosine (3 x 10(-4) M) caused increased inhibition of formyl-methionyl-leucylphenylalanine-stimulated superoxide release by neutrophils in the presence of endothelial cells and prevented neutrophil mediated endothelial cell damage, as measured by release of 3H-2-deoxy-D-glucose. Pairing 2-chloroadenosine (10(-5) M) or 5'-(N-ethylcarboxamido)-adenosine (3 x 10(-4) M) with a cyclic adenosine monophosphate phosphodiesterase inhibitor, 3 isobutyl-l-methyl-xanthine (10-4 M), produced greater inhibition of neutrophil superoxide production than occurred with either compound alone. The results support the hypothesis that vascular endothelial cells protect themselves from neutrophil attack by releasing adenosine to inhibit superoxide production. PMID- 1715182 TI - The E1b oncogene of adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody. AB - The cell lines KB8, 16, and 18 are KB cells which constitutively express adenovirus type 2 (Ad2) E1a, E1a plus E1b, and E1b genes, respectively. We show here that KB18 cells are completely resistant to cytolysis by tumor necrosis factor (TNF) or anti-Fas, although KB8 and KB or KB16 are highly and moderately sensitive, respectively. The levels of receptors for TNF and anti-Fas of KB18 were almost the same as compared with those of KB or other KB-cell lines. Expression of manganous superoxide dismutase (MnSOD) mRNA in KB18 was about 20 fold higher than that in KB or KB8 cells. KB, HT29, and A673 cells infected with dl337 (an Ad5 mutant defective in E1b function) are highly sensitive to TNF or anti-Fas, although wild-type Ad2-infected cells are resistant. Our results indicate that the E1b oncogene can confer cellular resistance to cytolysis by either TNF or anti-Fas in both KB cells and adenovirus-infected human cell lines through influencing intracellular events including regulation of MnSOD genes. Furthermore, we describe how anti-Fas mimics only the cytolytic activity of TNF, whereas TNF also has many other biological activities. PMID- 1715183 TI - IgG response is impaired in H2-c-fos transgenic mice. AB - Transgenic mice carrying the proto-oncogene c-fos under the control of H-2Kb promoter (c-fos mice) were generated from (C57BL/6 x SJL)F2 mice. One line was backcrossed with C57BL/6 mice for 10 generations. These semi-congenic c-fos mice express exogenous c-fos RNA in their hematopoietic tissues. The following immunological states are apparent. (i) Titers of serum IgM, IgG, and IgA of naive c-fos were within the control range. (ii) These mice could not produce primary IgG antibody specific for immunized antigen. (iii) Production of primary IgM and IgA antibody to the antigen was within the control range. (iv) There were no IgG memory B cells generated in the spleens of c-fos mice. (v) Activities of antigen presenting cells and carrier-specific helper T cells from c-fos mice were normal. These findings strongly suggest that the immune abnormality of c-fos mice is in limiting B cell function to the production of IgG to immunized antigens. PMID- 1715184 TI - Downregulation of CDC2 upon terminal differentiation of neurons. AB - The terminal differentiation of neurons occurs as precisely timed waves, with specific neuronal types differentiating in defined sequences. The precision of neuronal differentiation in the central nervous system offers an unusual opportunity to study terminal differentiation in vivo. The p34cdc2 kinase complex and the anti-oncogenes p53 and RB are central in the regulatory network that controls cell proliferation. We found high levels of expression of CDC2 mRNA and protein in proliferating neuronal precursor cells. The expression of both CDC2 and cyclin A was dramatically downregulated upon terminal differentiation of neurons in vivo and in a neuronal precursor cell line, ST15A. p53 mRNA expression was also downregulated but to a lesser extent; RB mRNA levels were unchanged during neuronal differentiation. Immunohistochemistry showed that p34cdc2 was expressed not only in the neuronal precursors of the cerebellar external granule layer but also in the early differentiating granule neurons. The expression of p34cdc2 in early neurons suggests a function for this enzyme in the events that occur soon after proliferation ceases. On the basis of the results reported here and other recent findings, we propose a model in which terminal differentiation is achieved by a switch in the neuronal precursors from p34cdc2-based proliferation to a differentiated state controlled by p34cdc2-related kinases. PMID- 1715185 TI - Cyclosporin A and FK-506 both affect DNA binding of regulatory nuclear proteins to the human interleukin-2 promoter. AB - The structurally unrelated immunosuppressive drugs cyclosporin A (Sandimmun) and FK-506 both interfere with the process of T-cell proliferation by blocking the transcription of the T-cell growth factor interleukin-2 (IL-2). Here we demonstrate that the transcriptional activation of this gene requires the binding of regulatory nuclear proteins to a promoter element with sequence similarity to the consensus binding site for NF-kappa B-related transcription factors. We present evidence that the binding by regulatory nuclear proteins to the kappa B element of the IL-2 promoter is affected negatively by cyclosporin A and FK-506 at concentrations paralleling their immunosuppressive activity in vivo. The decrease in DNA-protein complex formation induced by the immunosuppressive drugs correlates with a decrease in IL-2 production. FK-506 is 10 to 100 times more potent than cyclosporin A in its ability to inhibit sequence-specific DNA binding and IL-2 production. Our findings suggest that the actions of both drugs converge at the level of DNA-protein interaction. PMID- 1715186 TI - Recombination and modular exchange in the genesis of new lambdoid phages. AB - Comparison of the nucleotide sequence of the integrase genes of lambdoid phages 21 and 434 with the published sequences of phages HK022 and lambda shows that lambda and 434 are very closely related (98% base sequence identity), whereas HK022 and 21 respectively show 73% and 48% identity to lambda. It is likely that several homologous recombination events occurred in the int gene and flanking DNA among the progenitors of these phages. Sequence divergence to different alternative sequences at a common site (tL4) suggests that tL4 has been repeatedly used as a recombination site, despite the very limited homology it provides. A minor constitutive transcript that terminates at tL4 of lambda has been identified. We propose that the principal selective force acting to conserve tL4 is for terminator function, but that the use of tL4 as a recombination site has allowed the formation of selectively favored recombinants. By extension, we suggest that conservation of microhomologies at functional sites serves to keep lambdoid phages within a common gene pool despite extensive drift and divergence. PMID- 1715187 TI - The ionic channels formed by cholera toxin in planar bilayer lipid membranes are entirely attributable to its B-subunit. AB - The interaction of cholera toxin with planar bilayer lipid membranes (BLM) at low pH results in the formation of ionic channels, the conductance of which can be directly measured in voltage-clamp experiments. It is found that the B-subunit of cholera toxin (CT-B) also is able to induce ionic channels in BLM whereas the A subunit is not able to do it. The increase of pH inhibited the channel-forming activity of CT-B. The investigation of pH-dependences of both the conductance and the cation-anion selectivity of the CT-B channel allowed us to suggest that the water pore of this channel is confined to the B-subunit of cholera toxin. The effective diameter of the CT-B channels water pores was directly measured in BLM and is equal to 2.1 +/- 0.2 nm. The channels formed by whole toxin and its B subunit exhibit voltage-dependent activity. We believe these channels are relevant to the mode of action of cholera toxin and especially to the endosomal pathway of the A-subunit into cells. PMID- 1715188 TI - 2,2,4-Trimethylpentane induces Ca2+ release from the sarcoplasmic reticulum terminal cisterns. AB - Using quin2, the effects of aliphatic hydrocarbons on the system of Ca(2+) induced Ca2+ release in isolated membranes of rabbit skeletal muscle terminal cisterns have been studied. The hydrocarbons were inserted into the membranes by means of hydrocarbon-containing liposomes. 2,2,4-Trimethylpentane (isooctane) caused a rapid release of 70-75% of Ca2+ taken up by the terminal cistern vesicles during the Ca(2+)-pump operation. This effect was inhibited by the caffeine-induced Ca2+ release blockers--Mg2+, ruthenium red and tetracaine. The same was observed with a decrease in the concentration of ATP that is known to activate the terminal cistern Ca2+ channels. The effect of 2,2,4-trimethylpentane on the longitudinal cistern fractions practically devoid of Ca(2+)-channels was insignificant. Heptane, hexane and octane caused a slow release of 5-10% of the accumulated Ca2+ from the terminal cistern vesicles; no such effect was induced by decane. PMID- 1715189 TI - Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization. AB - A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation. PMID- 1715190 TI - Possible dual effect of synapses that are putatively purely excitatory or purely inhibitory: bases in stability theory and implications for neural network behavior. AB - Depolarization of an excitable membrane has a dual effect; excitatory in that it causes rapid opening of calcium and/or sodium channels but inhibitory in that it also causes those channels to inactivate. We considered whether apparently paradoxical or dual behavior might be exhibited by excitatory and inhibitory synaptic inputs. We used the classic Hodgkin-Huxley model for voltage-gated channels plus leakage channels of appropriate selectivity for ligand-gated postsynaptic channels. We summarize a model cell's behavior by calculating elicited firing frequency as a function of reversal potential and conductance of summed synaptic inputs, using stability theory and direct simulations. Dual behavior is elicited in the model with reasonable densities of ligand-gated channels. Thus a particular synaptic input to a neuron may be either excitatory or inhibitory depending on simultaneous activity of other synaptic inputs to the cell. This input-output map may give rise to biologically realistic and rich behaviors as an element of computed neural networks, and still be computationally tractable. PMID- 1715191 TI - Treatment of advanced chronic lymphocytic leukemia by fludarabine. Results of a clinical phase-II study. AB - In a clinical phase-II study fludarabine phosphate was given to 20 patients with advanced chronic lymphocytic leukemia who had failed on prior conventional therapy. Fludarabine was administered at a dose of 25 mg/m2/d for 5 days. Treatment cycles were repeated every 4 weeks until maximal response, followed by two cycles for consolidation. Four of the 20 patients achieved complete remission and seven patients partial remission, resulting in an overall response rate of 55% (11/20). Fludarabine therapy was well tolerated, with mild myelosuppression and secondary infections comprising the predominant side effects. These data warrant further confirmation and a randomized comparison of fludarabine with established regimens, which is currently underway. PMID- 1715192 TI - Pancreatitis in acute hemolysis. AB - Forty cases of hemolysis (drop of hematocrit greater than 12%/12 h) were retrospectively analyzed for hyperamylasemia and pancreatic complications. In 15 subjects the serum amylase level was greater than 360 U/l, i.e., three times the normal range, in ten the amylase level exceeded 900 U/l. Excluding patients in circulatory shock and/or hepatic coma, acute pancreatitis as defined by an elevation of serum amylase and clinical signs (epigastric pain) was present in four, with additional ultrasound findings (pancreatic swelling) and/or laparatomy/postmortem findings in a further six subjects (total ten patients = 25%) with various causes of hemolysis: autoimmune hemolysis 2, microangiopathic hemolytic anemia 2, toxicemia, G-6-PDH deficiency, septic abortion, malaria, Wilson's disease, and hypophosphatemia, one case each. In all subjects acute renal failure and in seven an activation of intravascular coagulation was seen. Three patients died (33% vs 47% of all hyperamylasemic patients and 46% of the whole group), but none of the deaths was attributed to pancreatitis. Pancreatic postmortem findings were diffuse edema and patchy parenchymal necrosis in two cases and petechial bleeding in one case. We conclude that acute pancreatitis is a complication of massive hemolysis, occurring at a prevalence of above 20%. It may progress from diffuse edema and inflammation to focal necrosis, rarely if ever to gross hemorrhage, and does not contribute to the high mortality of massive hemolysis. Back pain in hemolysis might originate from the pancreas rather than from the kidneys. PMID- 1715193 TI - Role of cyclic adenosine 3',5'-monophosphate in mediating the effect of prostaglandin E2 on decidualization in vitro. AB - When rat endometrial stromal cells from uteri sensitized for decidualization are cultured in vitro, there is an increase in alkaline phosphatase (ALP) activity paralleling that seen in vivo during decidualization. The addition of indomethacin to the culture medium decreases the endogenous production of prostaglandin E2 (PGE2) to below detectable levels and substantially reduces the increase in ALP activity. The addition of either PGE2 or its analog 16,16 dimethyl-PGE2, but not PGF2 alpha or its analog 15(S),15-methyl-PGF2 alpha, overrides this inhibitory effect, suggesting that PGE2 has a specific stimulatory effect upon ALP activity. This in vitro system was used to investigate the role of the cAMP pathway in mediating the stimulatory effect of PGE2 on ALP activity. The data indicate that PGE2 causes an increase in cAMP accumulation by the cells and that the addition of an analog of cAMP or substances which increase the level of cAMP in the cells (1-methyl-3-isobutyl xanthine, cholera toxin, forskolin) causes an increase in ALP activity. Collectively, the results suggest that the stimulatory effect of PGE2 is at least partially mediated by the cAMP pathway. PMID- 1715194 TI - Second messenger pathways mediating chicken luteinizing hormone secretion from dispersed pituitary cells. AB - A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2 acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715195 TI - Immunoneutralization of inhibin modifies hormone secretion and sperm production in bulls. AB - The objective of this study was to examine the role of endogenous inhibin in the regulation of FSH, LH, and testosterone secretion and sperm production in bulls. Bulls were actively immunized against bovine inhibin alpha 1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG) or HAG alone (controls) and emulsified in Freund's complete adjuvant. Primary immunization was at 14 wk of age, followed by booster immunizations in Freund's incomplete adjuvant at 28, 30, and 34 wk of age. Ten days after each booster immunization, scrotal circumferences and body weights were measured, and blood was sampled for determination of bINH antibody titer. Ten days after the third booster, blood was sampled at 1-h intervals for 8 h to quantify serum concentrations of FSH, LH, and testosterone. After this blood sampling period, bulls were castrated and testicular sperm production was determined. Serum diluted 1:4,000 from bINH-immunized bulls bound 36%, 52%, and 53% of radioiodinated bINH after the first, second, and third boosters, respectively. Serum from controls bound less than 1% radioiodinated bINH. After the third booster, serum concentrations of FSH and testosterone were increased (p less than 0.05) and LH concentrations were decreased (p less than 0.001) in bINH immunized bulls compared with controls. After the third booster, daily sperm production per gram of testicular parenchyma was increased (p less than 0.05) in bINH-immunized bulls compared with controls. Scrotal circumferences and body weights were similar between treatment groups throughout the experiment. We concluded that inhibin has a role in regulation of secretion of gonadotrophins and testosterone and testicular sperm production, but not testicular growth, in bulls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715196 TI - A ribosomal protein of Yersinia pseudotuberculosis having partial epitope identity with HLA-B27. AB - The phenotype HLA-B27 is common in patients who develop reactive arthritis after having an infection. One hypothesis concerning the pathogenesis of reactive arthritis is that molecular mimicry between HLA-B27 and certain bacterial components might be involved. It is known that an infection with Yersinia is commonly associated with reactive arthritis in B27 positive patients. Therefore, we were interested to investigate whether cross-reactivity between Yersinia and HLA-B27 exists. A gene library of Yersinia pseudotuberculosis was created in the plasmid vector pUC13. One of the resulting clones contained a gene encoding an intracytoplasmic protein that seems to have partial epitope identity with HLA B27. It reacted in western blot. ELISA and immunoprecipitation with three different HLA-B27 specific monoclonal and polyclonal antibodies of the IgG and IgM class. However DNA-sequencing of the cloned Yersinia gene and the predicted amino acid sequence revealed only a very remote similarity with HLA-B27 in the primary structure. Instead, an extremely high degree of similarity with the ribosomal protein L4 of the S10 operon of Escherichia coli was identified indicating that the protein encoded by the cloned Y. pseudotuberculosis gene is a corresponding ribosomal protein. PMID- 1715197 TI - Induction of interleukin-6 in murine bone marrow-derived macrophages stimulated by the Mycoplasma arthritidis mitogen MAS. AB - Cell-free supernatant of cultures from Mycoplasma arthritidis (MAS) functions as an extremely potent T-cell mitogen for human and murine lymphocytes. The T-cell response is dependent on the presence of accessory cells, presenting the intact E2 molecule on the cell surface. Until now, pure MAS protein has not been available. We developed a new multi-step method for MAS purification. The main steps in this protocol are ammonium sulfate precipitation, anion exchange and hydroxyapatite chromatography followed by gel filtration. With this efficient protocol we obtained fractions of extremely potent mitogenic properties, the purification rate was about 5 x 10(5). Although this protease-sensitive mitogenic activity was highly enriched, we failed to detect the protein by sensitive staining methods of SDS-PAGE. In previous studies, we showed that MAS induces the synthesis of interferon gamma in human and murine lymphocyte cultures. Here we demonstrate that MAS induces interleukin-6 (IL-6) in murine bone-marrow derived macrophage cultures. Since IL-6 is also induced by endotoxin, we used C3H/HeJ mice, which are known to be LPS-nonresponders, in all our studies. PMID- 1715198 TI - Respecrins--a new type of compound with targeted action. AB - A new technique for the creation of compounds with targeted action is described. In such compounds, termed respecrins (receptor-specific, screened toxin), a physiologically active component carries an epitope-containing fragment of the target antigen and is screened or masked by antibody specific to this antigen. Interaction of the respecrin with the target cell results in dissociation of this immunocomplex, and thereby activation of the physiologically active component. The kinetics of dissociation of a respecrin during interaction with a cell were investigated by flow cytometry. Possible applications of the principle are illustrated by investigation on the antigen-dependent activation of the mitogenic activity of staphylococcal enterotoxin A. PMID- 1715199 TI - Mitogenic effect of kallikrein from human urine on cultured human skin fibroblasts. Analysis of the combined action of kallikrein, insulin, and fibroblast growth factor on DNA and RNA synthesis. AB - Kallikrein isolated from human urine was capable of stimulating DNA and RNA synthesis in cultured human skin fibroblasts in media with a low serum content. The same concentration of kallikrein had a different effect on the DNA and RNA synthesis in different fibroblast lines, which was attributed to differences in the sensitivity of the cells to kallikrein. At a dose of 0.5 micrograms ml-1, kallikrein inactivated by heating at 100 degrees C caused an abrupt decrease in DNA synthesis in all the cell lines studied. When either active or inactivated kallikrein was added to the growth medium simultaneously with insulin there was a competitive effect on DNA and RNA synthesis. Preincubation of the cells with kallikrein prior to addition of insulin led to a reduction in the level of DNA synthesis compared to that seen upon simultaneous addition of kallikrein and insulin, suggesting that kallikrein and insulin competed for the same receptor. When kallikrein and fibroblast growth factor (FGF) were added simultaneously to the growth medium, there was a sharp decrease in both DNA and RNA synthesis in the cells compared to that seen on addition of FGF alone. Since heparin protected FGF from kallikrein inactivation, it is suggested that inactivation was caused by proteolytic degradation of part of the FGF molecule by kallikrein. It is concluded that kallikrein and insulin compete for the same receptor, possibly the insulin-like growth factor I (IGF-I), and that binding of kallikrein to this receptor is a prerequisite for mediation of the stimulatory effect of kallikrein on nucleic acid synthesis. PMID- 1715200 TI - The serine antiprotease aprotinin (Trasylol): a novel approach to reducing postoperative bleeding. AB - Aprotinin is a polypeptide from bovine lung which has been known since 1930, and which has previously been administered to humans as a relatively non-specific protease inhibitor in a number of disease states. In 1987, a remarkable use for this drug was realized, when by chance, it was discovered that by infusing large doses of aprotinin during cardiac surgery, it was possible to greatly reduce the bleeding which had hitherto been regarded as normal in such operations. The aim of this article is to briefly review the biochemistry and pharmacology of aprotinin, and to describe the background to its use in preventing postoperative bleeding. The use of aprotinin in reducing bleeding after cardiac surgery is then described in detail, and outlined for other types of major surgery. The possible modes of action of aprotinin in reducing bleeding are discussed, with particular reference to the bleeding which follows cardiac surgery. The profound effect of aprotinin, given in a novel dosage to reverse the postoperative haemostatic defect suggests that this drug may abolish the need for blood transfusions in most patients after major surgery. In addition, it is clear that aprotinin will provide a potent tool to probe some of the remaining mysteries of the haemostatic process. PMID- 1715201 TI - The influence of N-acetylcysteine on cardiac function and rhythm disorders during ischemia and reperfusion. AB - To assess the ability of N-acetylcysteine to reduce the vulnerability of the heart to arrhythmias and improve its mechanical function during ischemia and reperfusion, groups of Langendorff-perfused rat hearts (15 per group) were subjected to 20 minutes of aerobic perfusion, 10 minutes of regional ischemia and 10 minutes of reperfusion. The hearts were perfused with N-acetylcysteine throughout the study. The incidences of ventricular premature beats and ventricular tachycardia induced by ischemia fell from 93% and 67%, in the N acetylcysteine-free control group, to 40% and 27% with 8 microM N-acetylcysteine, to 33% and 27% with 80 microM N-acetylcysteine, and to 40% and 13% with 2000 microM N-acetylcysteine. The incidence of ventricular fibrillation induced by reperfusion was reduced from 93% to 60%, 67% and 47%, respectively. N acetylcysteine had no significant effect upon the coronary flow and heart rate. When the ischemic period was prolonged to 30 minutes, N-acetylcysteine (8 microM) was shown to delay the time of onset of arrhythmias during ischemia and reperfusion. Thus, the incidences of ventricular premature beats and ventricular tachycardia were greatest after 15 minutes of ischemia in controls but were maximal after 18 minutes in hearts treated with N-acetylcysteine. N acetylcysteine (8 microM) also improved the recovery of mechanical function after ischemia both in Langendorff preparations and in working preparations. In the working heart the post-ischemic recovery of aortic flow, coronary flow, cardiac output and aortic developed pressure after ischemia was improved from their control values of 30 +/- 5%, 82 +/- 2%, 43 +/- 2% and 63 +/- 4% to 59 +/- 7%, 98 +/- 5%, 71 +/- 3% and 80 +/- 6% respectively. Our results support the concept that agents which can alter redox state may exert protective effects against myocardial injury during both ischemia and reperfusion. PMID- 1715202 TI - Immunophenotyping of mesothelial cells and carcinoma cells with monoclonal antibodies to cytokeratins, vimentin, CEA and EMA improves the cytodiagnosis of serous effusions. AB - This paper presents an immunocytochemical study performed on cytocentrifuged deposits from 109 peritoneal and pleural effusions including 20 transudates, 43 malignant metastatic effusions and 46 effusions containing atypical cells, unidentifiable as reactive mesothelial or malignant epithelial cells on the classical morphological criteria. A panel of four monoclonal antibodies (MAb) was used, including KL1 directed to cytokeratins (KER), V9 to vimentin (VIM), NEO 723 to carcinoembryonic antigen (CEA) and E29 to epithelial membrane antigen (EMA). In most transudates the reactive mesothelial cells coexpressed VIM and KER with a ring-like pattern for the latter proteins. In contrast, they were unreactive to anti-CEA and weakly and inconsistently reactive to anti-EMA. In malignant effusions, most carcinoma cells coexpressed EMA, CEA and KER with a predominant diffuse cytoplasmic pattern for the latter. Only a few malignant epithelial cells from five metastatic adenocarcinomas weakly expressed VIM. When used on the 46 effusions with unidentifiable cells, the panel of MAb allowed reactive mesothelial cells and malignant epithelial cells to be distinguished from each other in 39 of 46 cases (85%). PMID- 1715203 TI - Channel protein engineering. An approach to the identification of molecular determinants of function in voltage-gated and ligand-regulated channel proteins. PMID- 1715204 TI - ATP-activated channels in excitable cells. AB - Extracellular ATP is an activator or modulator of ionic channels in a wide variety of excitable cells. There appears to be a class of related cation permeable ATP-activated channels in skeletal muscle, cardiac muscle, smooth muscle, and neurons; the channels in the different cell types appear to be similar, but not identical, in their ionic selectivity, receptor selectivity, and pharmacology. In all cases, these channels reverse near 0 mV and activation by ATP produces an excitatory effect. Much remains to be learned about these channels, their possible existence and roles in other cell types, and their relation to other types of ligand-gated channels. It will be especially important to develop more specific pharmacological blockers (and activators) in order to distinguish subtypes and to assess their physiological role. Another type of channel, so far described only in cardiac atrial cells, is identical to the channels in cardiac atrial cells activated by ACh receptors; it will be interesting to see if this type of receptor-channel complex is also found in neurons or other cells. In a variety of cells, ATP also acts as a modulator of voltage-dependent channels and of channels activated by other transmitters. It seems very likely that more instances of such modulation will be described in years to come. Possible second-messenger pathways mediating such modulation remain to be elucidated. PMID- 1715205 TI - Analytical diffusion models for membrane channels. PMID- 1715206 TI - Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes. AB - The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10-25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5' to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units. PMID- 1715207 TI - In vitro transcription from cauliflower mosaic virus promoters by a cell-free extract from tobacco cells. AB - We have studied transcription from the cauliflower mosaic virus 19S and 35S promoters in a cell-free system derived from tobacco cells in suspension culture. While a whole-cell extract is incapable of detectable transcription from these promoters, successive purification by column chromatography allows the preparation of two fractions which contain all factors necessary for transcription from the 19S promoter. In contrast, transcription from the 35S promoter leads to the accumulation of short RNAs. This accumulation can only be partially alleviated by modifying the conditions of transcription. PMID- 1715208 TI - Nucleotide sequence of a Hordeum vulgare gene encoding a glycine-rich protein with homology to vertebrate cytokeratins. PMID- 1715209 TI - Nucleotide sequence and secondary structure of apple scar skin viroid (ASSVd) from China. PMID- 1715211 TI - Glycine-rich RNA-binding proteins from Sorghum vulgare. PMID- 1715210 TI - Comparison of the expression of two highly homologous members of the soybean ribulose-1,5-bisphosphate carboxylase small subunit gene family. AB - Two soybean ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are highly homologous over a region that includes 4 kb of 5' and 1 kb of 3' flanking sequences. The expression of these genes was compared using synthetic oligonucleotide probes. Analysis of a soybean leaf cDNA library indicates that SRS1 and SRS4 are the most highly expressed members of the soybean SSU gene family. Similar changes were observed in the RNA levels for these genes in response to white light, far-red light and darkness, although SRS1 was expressed at a four-fold higher level in total RNA than SRS4 under all conditions. However, nuclear run-on assays indicate that SRS1 is transcribed at a lower rate than SRS4, which suggests that SRS1 RNA is more stable. S1 nuclease analysis and oligonucleotide directed RNase H cleavage indicate that transcripts from both genes are polyadenylated within two principle regions separated by 35 nt. Sequence analysis of 16 independent cDNA clones identified seven different polyadenylation sites, and six of these sites lie within these two regions. Although SRS1 RNA was poorly recovered during poly(A)+ fractionation, RNase H cleavage experiments showed that transcripts from SRS1 and SRS4 had similar poly (A) tail lengths ranging from 0 to 220 nt. In addition, and despite differences in the untranslated leader sequences, SRS1 and SRS4 RNAs are assembled into polysomes with equal efficiencies. The overall similarity in expression patterns for these two genes further illustrates the coordinate evolution of individual members of a SSU gene family and is consistent with the proposal that gene conversion homogenizes both the coding and regulatory regions of these genes. PMID- 1715212 TI - [Effects of dibutyryl derivatives of cyclic nucleotides on synthesis of total RNA and proteins in cultured fetal rat hepatocytes]. AB - During short-term (6 h) or long-term (24 h) incubation of fetal rat liver cells in primary cultures, 10(-3) M dibutyryl-derivative of cyclic AMP (Bt2cAMP) and sodium butyrate decreased total RNA and protein synthesis. In contrast, dibutyryl cyclic GMP (Bt2cGMP) at the same dose (10(-3) M) was without significant effect on RNA and protein biosynthesis. During short-term (4 h) incubation 10(-3) M Bt2cAMP and Bt2cGMP stimulated serum albumin production, while sodium butyrate was without effect. In long-term (22 h) incubation only 10(-3) M Bt2cAMP noticeably increased albumin production. The results obtained clearly show that Bt2cGMP, unlike Bt2cAMP, is not able to modify significantly total RNA and protein synthesis in cultured fetal rat liver cells. It is concluded also that the effects of dibutyryl-derivatives of cyclic nucleotides, at least on albumin production, are not mimiebet by butyrate. PMID- 1715213 TI - [Effects of antifeins with different memory effects on cAMP phosphodiesterase, lipid peroxidation and RNA synthesis in rat brain neurons]. AB - It has been shown that ethylnorantifein and its structural analogues with opposite effects on long term memory reduce the activity of membrane bound phosphodiesterase cAMP with high and low affinity and exert the same directed influence on lipids peroxidation in membranes. A positive correlation was observed only between the action of these substances on the long term memory and their influence on the RNA synthesis in the rat brain nuclei. Ethylnorantifein and its demethylated analogues increased RNA synthesizing activity while allyl- and propylnorantifeins decreased it. The molecular mechanisms of memory effects of neuroactive substances are discussed. PMID- 1715214 TI - [Study by the intracerebral microdialysis method of the effects of atypical neuroleptics and anxiolytics on striatal release and metabolism of dopamine in awake rats]. AB - Using brain microdialysis in awake rats effects of risperidone, ritanserin, buspirone, sulpiride and 5-methoxy-N,N-dimethyltryptamine (MeODMT) on striatal dopamine (DA) release and metabolism were studied. Risperidone, sulpiride and buspirone increased levels of DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Ritanserin failed to affect DA release, while increased DOPAC and HVA levels. MeODMT had no effect on striatal DA release and metabolism. Possible interaction between DA and serotonin systems is discussed. PMID- 1715215 TI - [Enzyme activity of interferon system in virus diseases]. AB - The enzymes activities of interferon system at viral infections of different etiology/influenza, parainfluenza, hepatitis B with delta infection and urticaria chronica with respiratory and herpes infection/has been studied and evaluation of the noted changes in the enzymes activity is discussed. It is shown that the same pathological changes in enzymes activities of interferon system were observed at different viral infections. PMID- 1715216 TI - [Proto-oncogene expression in the liver of male rats of different age]. AB - The expression of 10 protooncogenes has been quantitatively studied in liver of male rats L10 age of 1, 10.5, 22 and 37 months. It was shown that a number of specific mRNA transcripts and, therefore, the levels of expression of protooncogenes C-MYC, C-FOS, N-MYC, HA-RAS, KI-RAS, SIS, ABL, YES, MOS and MET in rat liver were constant during life span. These data are in accordance with resistance of the rat strain L10 to spontaneous hepatocarcinogenesis. PMID- 1715217 TI - [Immunoenzyme analysis of ovarian metastatic antigen-8]. AB - Immunoenzymatic method for the determination of new ovarian-metastatic antigen-8 in human blood serum was worked out. OMA-8 level was studied in blood serum of 40 healthy women, 40 healthy men, 16 neonates, 33 pregnant women, 103 patients with genital tumours. It was found out that OMA-8 level in blood serum of healthy women changed between 2 to 15.4 micrograms/l, in men between 5-17.0 microgramS/l. OMA-8 concentration higher than 15.4 micrograms/ was considered elevated. The elevated OMA-8 level was marked in neonates (100%) and in pregnant (39.3%). High level was discovered in the amniotic fluid. PMID- 1715218 TI - Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia. AB - A patient with primary plasma cell leukemia resistant to chemotherapy was treated for 2 months with daily intravenous injections of anti-interleukin-6 (IL-6) monoclonal antibodies (MoAbs). The patient's clinical status improved throughout the treatment and no major side effects were observed. Serial monitoring showed blockage of the myeloma cell proliferation in the bone marrow (from 4.5% to 0% myeloma cells in the S-phase in vivo) as well as reduction in the serum calcium, serum monoclonal IgG, and the serum C-reactive protein levels. The serum calcium and serum monoclonal IgG corrected by approximately 30%, whereas the C-reactive protein corrected to undetectable levels during treatment. No major side effects developed, although both platelet and circulating neutrophil counts decreased during anti-IL-6 therapy. A transient immunization was detected 15 days after the initiation of the treatment, which could explain the recovery of myeloma cell proliferation after 2 months of treatment (2% myeloma cells in the S phase). In conclusion, this first anti-IL-6 clinical trial demonstrated the feasibility of injecting anti-IL-6 MoAbs, and also a transient tumor cytostasis and a reduction in IL-6-related toxicities. It gave insight into the major biologic activities of IL-6 in vivo and may serve as a basis for further development of anti-IL-6 therapy in myeloma and other IL-6-related diseases. PMID- 1715219 TI - Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit. AB - Mice with W mutations characterized by hypopigmentation, sterility, anemia, and mast cell deficiency have abnormalities in c-kit, a receptor with tyrosine kinase activity. Recently, the ligand for c-kit was cloned by investigators in several laboratories. Zsebo et al identified and cloned a gene for a cytokine termed stem cell factor (SCF) in the medium conditioned by buffalo rat liver cells, and this cytokine proved to be c-kit ligand. We have examined the effects of recombinant rat SCF (rrSCF) on colony formation from primitive hematopoietic progenitors in culture. rrSCF and erythropoietin (Ep) supported formation of granulocyte/macrophage (GM) colonies as well as a small number of multilineage and blast cell colonies from marrow cells of normal mice. We then examined the effects of rrSCF using marrow and spleen cells of mice that had been treated with 150 mg/kg 5-fluorouracil (5-FU). Unlike single factors, combinations of factors such as rrSCF plus interleukin-3 (IL-3), rrSCF plus IL-6, and rrSCF plus granulocyte colony-stimulating factor (G-CSF) markedly stimulated the growth of multilineage colonies. In contrast to these factor combinations and a combination of IL-3 and IL-6, a combination of rrSCF and IL-4 did not support multilineage colony formation. Mapping studies of the development of multipotential blast cell colonies further indicated that rrSCF, like IL-6, G-CSF, and IL-11, shortens the dormant period in which the stem cells reside. When we tested the effects of rrSCF using pooled blast cells, which are highly enriched for progenitors and are devoid of stromal cells, rrSCF plus Ep supported formation of only a few multilineage colonies, indicating that rrSCF itself is ineffective in support of the proliferation of multipotential progenitors. However, rrSCF supported formation of a significant number of neutrophil and neutrophil/macrophage colonies from pooled blast cells, indicating that rrSCF is able to support directly the proliferation of progenitors in neutrophil/monocyte lineages. c-kit ligand may play important roles in adult hematopoiesis. PMID- 1715220 TI - On the target specificity of plasminogen activator inhibitor 1: the role of heparin, vitronectin, and the reactive site. AB - Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor XIIa (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3' residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2) Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3) Apart from the important role of the P1 residue, no consensus can be presented on the nature of other residues within the P3 to P3' region with regard to target protease specificity. PMID- 1715221 TI - Analysis at the clonal level of T-cell phenotype and functions in severe aplastic anemia patients. AB - The aim of this study was to analyze at the clonal level the phenotype and functions of T cells from patients with severe aplastic anemia (SAA). For this purpose we studied 175 T-cell clones obtained from peripheral blood (PB) and bone marrow (BM) of four SAA patients and 97 clones from two healthy controls. The percentage of CD8+ T-cell clones obtained from the patients' PB and BM was higher, but not significantly (P = .07 and P = .14, respectively), than that obtained in controls. A higher proportion of T-cell clones from SAA patients exhibited lectin-dependent cytolytic activity and especially natural killer-like activity when compared with controls (PB: P less than .01, P less than .05; BM: P less than .05, P less than .01, respectively). Lymphokine release was tested before and after mitogen stimulation. A number of patients' clones were able to release interferons (IFNs) spontaneously (PB: 28.6% v 0%, P less than .05; BM: 28.6% v 0%, P less than .10). After mitogen stimulation, patients' BM T-cell clones produced IFNs in greater proportions (90.9% v 46.7%, P less than .01) and in greater quantities (PB: 25.5 arbitrary units [AU]/mL v 5.7 AU/mL, P less than .03; BM: 26 AU/mL v 9.1 AU/mL, P = .011) as compared with controls. Tumor necrosis factor (TNF) activity was not found in supernatants of unprimed T-cell clones. After mitogen stimulation, PB T-cell microcultures produced TNF alpha in greater proportions (97.9% v 72.2%, P less than .01) and, also in this case, in greater quantities (PB: 7.2 AU/mL v 1.5 AU/mL, P = .007; BM: 9.9 AU/mL v 1.5 AU/mL, P = .003) than controls. In conclusion, T-cell clones from SAA patients exhibit predominantly a CD8+ phenotype, a greater cytotoxic activity, and can be shown to produce greater quantities of suppressor lymphokines when compared with controls. PMID- 1715222 TI - Solitary expression of CD7 among T-cell antigens in acute myeloid leukemia: identification of a group of patients with similar T-cell receptor beta and delta rearrangements and course of disease suggestive of poor prognosis. AB - In a series of 100 acute myeloid leukemia (AML) patients defined by cytochemistry and immunophenotyping, 20 expressed T-lymphocyte associated antigens on the surface of their blasts. While 15 expressed two or more T-cell antigens, five were found to express only CD7. All patients belonged to the French-American British type M4, and four were under the age of 40. Despite intensive chemotherapy, four never obtained a complete remission and the fifth died of relapse after an allogenic bone marrow transplantation. While 12 randomly selected T-cell antigen negative AML patients showed only few rearrangements in Ig- or T-cell receptor (TCR) genes, such genetic alterations were demonstrated in four of five patients for the TCR delta gene and in all patients for the TCR beta gene. Interestingly, DNA fragments of similar size were demonstrated in three of five patients for both the beta and delta genes. These data suggest that the solitary presence of CD7 among T-cell antigens in otherwise clearcut AML cases identifies a group of patients with similarities in antigen receptor gene configuration as well as outcome. PMID- 1715223 TI - Sexual abuse and learning disabilities. PMID- 1715224 TI - Prenatal screening for Down's syndrome. PMID- 1715225 TI - Perspectives in the prevention and treatment of acute graft versus host disease. PMID- 1715226 TI - Purification of aplastic anaemia (AA) marrow haemopoietic progenitors combined with long-term marrow cultures (LTMC) to assess haemopoiesis in AA. PMID- 1715227 TI - Contrasting effects of tachykinins and guanethidine on the acetylcholine output stimulated by nicotine from guinea-pig bladder [corrected]. AB - 1. Contractile responses and acetylcholine release evoked by nicotine in guinea pig detrusor strips were determined by isotonic transducer and radioimmunoassay, respectively. Nicotine stimulated acetylcholine release and a contractile response in guinea-pig detrusor strips treated with the cholinesterase inhibitor, methanesulphonyl fluoride (MSF). Both actions evoked by nicotine were antagonized by the nicotinic receptor antagonist, hexamethonium but were insensitive to tetrodotoxin. 2. A sympathetic nerve blocker, guanethidine and a tachykinin antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-substance P (rpwwL-SP) partially inhibited the acetylcholine release evoked by nicotine to much the same degree. The inhibitory effects of guanethidine and rpwwL-SP on acetylcholine release were significantly greater than corresponding effects on the contraction evoked by nicotine. 3. In preparations treated with rpwwL-SP to block the tachykinin receptors, guanethidine had no effect on the response to nicotine. Conversely, after treatment with guanethidine to block release of a mediator from sympathetic nerve endings, nicotine-induced responses were not affected by rpwwL-SP. 4. Nicotine-induced contraction was reduced to 30% by the muscarinic cholinoceptor antagonist, atropine and completely abolished after desensitization of P2 purinoceptors with alpha,beta-methylene ATP in the presence of atropine. 5. A concentration-contractile response curve to neurokinin A (NKA) was shifted to the left after cholinesterase inhibition with MSF. Atropine abolished the facilitatory effect of MSF and partially inhibited contractions induced by NKA at 100 nM to 1 microM. The contractile responses to substance P methyl ester (SPOMe) and Tyr0-neurokinin B (Tyr0-NKB) were not influenced by MSF or atropine. 6. After desensitization of NK, tachykinin receptors with SPOMe or preincubation with senktide, the cholinergic component of the nicotine-induced contraction was the same as the control value (100%). 7. Our findings give further support to our previous results: nicotine stimulates acetylcholine release in a tetrodotoxin resistant manner in guinea-pig bladder and acetylcholine release evoked by nicotine is increased by the coordinated action of sympathetic nerves and tachykinin(s). It is suggested that the tachykinin receptor subtype involved in acetylcholine release is NK,. PMID- 1715228 TI - Endothelium-dependent and endothelium-independent vasodilatation of the hepatic artery of the rabbit. AB - 1. The isolated hepatic artery of the rabbit contracted to exogenously applied noradrenaline (NA). There was no significant difference in the maximal contraction or the EC50 value in vessels where the endothelium was present and in endothelium-denuded preparations. 2. Acetylcholine (ACh) induced a vasodilatation of vessels preconstricted with NA which was entirely dependent on the endothelium. 3. Adenosine 5'-triphosphate (ATP), 2-methylthio ATP, adenosine and sodium nitroprusside induced concentration-dependent, sustained relaxations of vessels in which tone had been induced with NA. The relaxation responses were not reduced after removal of the endothelium. 8-Phenyltheophylline antagonized the relaxation response produced by adenosine, but not that due to ATP at lower concentrations. The maximum response to ATP was reduced in the presence of 8 phenyltheophylline. 4. alpha,beta-Methylene ATP produced further contraction of vessels preconstricted with NA in both endothelium-denuded preparations and in vessels where the endothelium remained intact. 5. Immunohistochemical analysis was used to show the presence of nerve fibres containing substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) in the hepatic artery. Application of SP induced a concentration-dependent relaxation which was entirely dependent on the presence of an intact endothelium. CGRP and VIP, however, elicited concentration-dependent relaxations which were independent of the endothelium. 7. It is concluded that in the rabbit hepatic artery, responses to ACh are dependent on the presence of intact endothelium. P1 , P2x- and P2y-purinoceptors, mediating relaxation to adenosine, vasoconstriction to ATP and vasodilatation to ATP respectively, are located on vascular smooth muscle. Furthermore, CGRP and VIP mediate a direct vasodilatation of smooth muscle both in the absence and the presence of the endothelium, whereas SP produces a relaxation via receptors located on the endothelium. PMID- 1715229 TI - Specific neurokinin receptors mediate plasma extravasation in the rat knee joint. AB - 1 Plasma extravasation in the rat knee joint was induced by intra-articular injection of neurokinins and specific neurokinin receptor agonists. 2 Pronounced plasma extravasation was produced by substance P (SP, 4-185 microM) and to a lesser extent by neurokinin-B (NKB, 83-413 microM), whereas neurokinin-A (NKA, 88 440 microM) and calcitonin gene-related peptide (CGRP, 26-130 microM) had no significant effect. 3 The specific neurokinin1 receptor agonist [Sar9, Met(O2)11] substance P (NK1 agonist) in doses of 0.4-70 microM appeared to be more potent than SP in eliciting plasma extravasation. The neurokinin2 receptor agonist [Nle10]-neurokinin A4-10 (NK2 agonist) was not effective at 70 microM but produced a small and significant effect at 330 microM, whereas the neurokinin3 receptor agonist [MePhe7]-neurokinin B (NK3 agonist) was without effect at 40 microM or 400 microM. 4 Injections of SP or NKA into the synovial cavity of the rat knee were equally effective in producing marked plasma extravasation in remote sites such as the forelimb and hindlimb paws. 5 Co-administration experiments showed that the effects of SP were synergistic with NKA or the NK1 receptor agonist, but not with CGRP or the NK2 receptor agonist. 6 The rank order of potency was NK1 agonist greater than or equal to SP greater than NKB greater than NK2 agonist suggesting that NK1 receptors mediate plasma extravasation in the rat knee joint. PMID- 1715230 TI - Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis. AB - Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen. PMID- 1715231 TI - Lollipop successful in providing analgesia to children before painful procedures. PMID- 1715232 TI - Elevated serum level of glycolipid sulfotransferase in patients with hepatocellular carcinoma. AB - Activity of glycolipid sulfotransferase (cerebroside sulfotransferase) in serum was elevated in 21 (33%) of 63 patients with hepatocellular carcinoma (HCC, mean +/- S.E., 349 +/- 32 pmol/ml per h, n = 63, P less than 0.001) compared to healthy subjects (172 +/- 12, n = 85). Ho significant elevation of the sulfotransferase level was observed in liver cirrhosis (219 +/- 28, n = 10) in which many of biochemical HCC markers increase concomitantly. The elevation of sulfotransferase was independent of the production of alpha-fetoprotein and of aminotransferase levels in HCC, providing complementary value for alpha fetoprotein-negative HCC cases. However, the sulfotransferase levels (234 +/- 21, n = 32, P less than 0.01) in sera from patients with renal cell carcinoma, in whose involved tissues the enzyme was demonstrated to increase markedly, were less than in HCC. PMID- 1715233 TI - A distinct kinase modulates the expression of IFN-inducible genes in human breast cancer cells. AB - The biological activity of interferons (IFNs) is presumed to be mediated through the induction of a number of IFN-inducible genes. IFN-mediated gene induction was examined in two human breast cancer cell lines, MCF-7 and BT-20. Both these cell lines were remarkably responsive to IFNs as a number of IFN inducible genes were rapidly induced. We examined the sensitivity of these genes towards 2-aminopurine (2-AP), a known inhibitor of double-stranded (ds) RNA dependent protein kinase. 2 AP has also been reported to inhibit the induction of IFN-beta 1 in response to dsRNA and the genes c-myc and c-fos in fibroblasts. In both MCF-7 and BT-20 cell lines, 2-AP selectively inhibited the IFN-induced gene responses. 2-AP did not affect levels of the oncogene, HER-2/neu. Tamoxifen (TAM), an antiestrogenic drug, which is known to inhibit the activity of protein kinase C at high concentrations, did not affect IFN-mediated gene induction. Our data is consistent with the concept that the 2-AP sensitive kinase is primarily associated with the IFN-induced gene systems and that positive and negative growth regulating stimuli in breast cancer may require the participation of distinct kinases. PMID- 1715234 TI - Angiogenic factor of a rat mammary tumor cell line (RMT-1) (I). Secretion of two distinct angiogenic factors into serum-free conditioned medium by RMT-1 cells. AB - Serum-free conditioned medium of a rat mammary tumor cell line RMT-1, established from a rat mammary carcinoma induced by 7,12-dimethylbenz[a]anthracene (DMBA), produced the complete angiogenic response in both rabbit cornea and chick embryo chorioallantoic membrane assays. The angiogenic activity in the RMT-1 conditioned medium was separated into two fractions on a column of heparin-Sepharose; one was eluted with 0.1 M NaCl and the other with 0.5 M NaCl, which are referred to hereafter as rAF-1 and rAF-2, respectively. These two angiogenic factors were further purified separately by FPLC on a Superose 12 column. The partially purified rAF-2 had an apparent Mr of 30,000-50,000 and seemed to exhibit mitogenic activity toward Balb/c 3T3 cells, while the partially purified rAF-1, with an apparent Mr of 10,000-30,000 did not have a mitogenic effect on these cells. Both rAF-1 and rAF-2 were resistant to heat and acid treatment, and exhibited trypsin sensitivity, suggesting that they are heat and acid stable peptides. The two angiogenic factors did not stimulate the proliferation of cultured vascular endothelial cells. These results suggest that RMT-1 secretes two distinct angiogenic factors into the medium and that these two secretable angiogenic factors participate cooperatively in the induction of the angiogenic response produced by a DMBA-induced rat mammary tumor in vivo. PMID- 1715235 TI - Increased c-Ki-ras expression in hamster lung exposed to N-nitrosodiethylamine and hyperoxia as detected by the polymerase chain reaction. AB - Neuroendocrine lung cancers can be induced in hamsters within 8-12 weeks by combined exposure to N-nitrosodiethylamine (DEN) and hyperoxia. The expression of the c-Ki-ras gene in this lung cancer model was studied using polymerase chain reaction analysis of mRNA (RNA/PCR). We used four different groups of hamsters, exposed for 6 weeks to DEN with hyperoxia (60% oxygen), DEN, hyperoxia, or ambient air, respectively. Total RNA was isolated from lung tissues and cDNA made prior to PCR amplification. A 234-bp product was amplified from c-Ki-ras cDNA and quantitated using scanning laser densitometry. The data obtained were normalized to the expression of the house keeping gene B-actin. The c-Ki-ras products were present after amplification of all hamster lung RNA samples. The hamster lungs exposed to DEN with hyperoxia displayed higher c-Ki-ras protooncogene expression than hamsters exposed to DEN, hyperoxia, or ambient air alone. Since the animals studied were sacrificed at 6 weeks, prior to the appearance of tumors, we conclude that this increased expression may indicate a role for c-Ki-ras in the initial steps in malignant transformation of neuroendocrine cells. PMID- 1715236 TI - Structure of the type-specific polysaccharide antigen of Streptococcus rattus. AB - The structure of the type-specific polysaccharide antigen of Streptococcus rattus was determined by methylation analysis, periodate oxidation, and by 2D-1H- and 13C-n.m.r.-spectroscopy. The polysaccharide was found to possess the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Galp-(1----3)] alpha-L-+ ++Rhap- (1----. PMID- 1715237 TI - Interleukin-6 does not support interleukin-2 induced generation of human lymphokine-activated killer cells. AB - The activity of lymphokine-activated killer (LAK) cells is supported by various cytokines. The objective of this study was to see if recombinant interleukin-6 (IL-6) either alone or in combination with interleukin-2 (IL-2) has any effect on the generation of LAK cells. Peripheral blood mononuclear cells of healthy donors were cultured for 4 or 6 days with both cytokines either alone or in combination. LAK activity against K562 and natural killer-resistant Daudi cells was assessed by a 4-h and an 18-h 51Cr-release assay at various effector to target ratios. IL 6 alone in increasing concentrations did not induce LAK cell activity. Neither additive nor synergistic effects of IL-6 with IL-2 were observed. Immunofluorescence analysis with phycoerythrin-conjugated anti-CD56 antibody demonstrated that IL-6 could not maintain or increase the number of CD56-positive cells over a 6-day culture period. These results suggest that IL-6 does not support LAK cell generation by itself or increase LAK cell activity in combination with IL-2. PMID- 1715238 TI - Calretinin immunoreactivity in cholinergic motor neurones, interneurones and vasomotor neurones in the guinea-pig small intestine. AB - Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26 +/- 1% of myenteric neurones and 12 +/- 3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones. PMID- 1715239 TI - Species differences in the coexistence of 5-hydroxytryptamine and substance P in presynaptic boutons in the cervical ventral horn. AB - Substance P (SP)- and 5-hydroxytryptamine (5-HT)-containing presynaptic boutons in the cervical ventral horn were studied in chicken, hamster, rat and monkey spinal cords, using PAP and protein A-gold double-labeling techniques in conjunction with monoclonal antibodies. In the chicken, the PAP method demonstrated that SP-immunoreactive boutons contained large spherical dense-cored vesicles (DCVs) whereas 5-HT-immunoreactive boutons displayed both elongated and spherical DCVs. Using the protein A-gold double-labeling technique, 10-nm gold particles for SP were localized over the spherical DCV-containing boutons whereas 15-nm gold particles for 5-HT were localized on elongated DCV-containing boutons. On the other hand, in the other species investigated, both SP- and 5-HT immunoreactive boutons had similar morphological features as shown by the PAP method; both contained elongated and spherical DCVs. The two different sized gold particles, each of which labeled either 5-HT or SP, were found together over DCVs in a single bouton. These results indicate that 5-HT and SP are contained in different presynaptic boutons in the chicken, although in the hamster, rat and Japanese macaque, the two neurotransmitters/modulators coexist in the same DCVs in a single bouton. Species differences have thus been demonstrated for the coexistence of 5-HT and SP in the spinal ventral horn. PMID- 1715240 TI - Presence of galanin in human pancreatic nerves and inhibition of insulin secretion from isolated human islets. AB - In several animal species, galanin occurs in pancreatic nerves and inhibits insulin secretion. However, the presence and action of galanin in the human pancreas have not been established. Therefore, we examined the presence and nature of human pancreatic galanin-like immunoreactive material (GLIR) and the effects of galanin on glucose-stimulated insulin secretion from isolated human islets. Immunofluorescent staining of human pancreas revealed GLIR in fine varicose fibers in both islets and exocrine parenchyma. Furthermore, acid extracts of pancreas (n = 3) and isolated islets (n = 3) contained 0.17 +/- 0.06 and 0.23 +/- 0.11 pmol GLIR/mg protein. Human pancreatic GLIR coeluted with synthetic porcine galanin from Sephadex G-50. Moreover, synthetic porcine galanin inhibited glucose-stimulated insulin secretion from collagenase-isolated human islets at dose rates greater than 10(-8) M. Thus, (1) human pancreas is innervated by galanin-containing nerves, (2) human pancreatic GLIR is of similar size as synthetic porcine galanin, and (3) porcine galanin inhibits glucose stimulated insulin secretion from human islets. Therefore, galanin could be an important local regulator of insulin secretion in man. PMID- 1715241 TI - Quantitative analysis of regional variability in the distribution of transverse tubules in rabbit myocardium. AB - The goal of the present investigation was to compare quantitatively the distribution of T-tubules between regions of the myocardium. The volume fraction and surface density of T-tubules in rabbit right atrial free wall, left atrial free wall, right ventricular free wall, left ventricular free wall, right ventricular papillary muscle, and left ventricular papillary muscle were measured using established, electron-microscopic, morphometric techniques. T-tubules were delineated using wheat-germ agglutinin conjugated to horseradish peroxidase as a tracer. No significant differences were found in the morphometric parameters between any two ventricular samples or between atrial samples. Furthermore, little difference between T-tubule volume fraction or surface density was found between individual animals for any given site. Both volume fraction and surface density of ventricular T-tubules were more than ten-times their values in atrial tissue (volume fraction: 3.43% +/- 0.35 vs. 0.20 +/- 0.09; surface density: 2.46 microns 2/microns 3 +/- 0.11 vs 0.10 +/- 0.04). Measurements show that there is greater variation of T-tubule volume fraction and surface density within atrial samples than within ventricular samples. This suggests greater inhomogeneity in T tubule distribution in atrial myocardium than in ventricular myocardium. Morphometric data also indicate that the mean diameter of atrial T-tubules is greater than that of ventricular T-tubules while qualitative observations show that atrial T-tubules are distributed less regularly and have a larger longitudinal component to their organization than those in the ventricular myocardium. PMID- 1715242 TI - Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments. AB - The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (less than 25 nm) gold particles into 40-50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (greater than 25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but no 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately. PMID- 1715243 TI - Galanin-immunoreactive nerves in the mouse and rat pancreas. AB - Galanin-containing nerve fibers have previously been observed in the human, dog, and pig pancreas. Whether the mouse and rat pancreas also contain galanin nerve fibers has been a matter of debate. Therefore, we examined the distribution of galanin in the mouse and the rat pancreas. Further, the possible localization of galanin to adrenergic nerves was studied using sequential immunostaining for galanin and tyrosine hydroxylase (TH). In the mouse pancreas, numerous galanin immunoreactive (GIR) nerve fibers occurred around blood vessels. They were less numerous in the exocrine parenchyma and in association with the islets. In contrast, in the rat pancreas, only a few GIR nerves were found. They were located around blood vessels and scattered in the exocrine parenchyma. Occasionally, GIR nerves were also observed in the islets. There was a dense distribution of TH-immunoreactive fibers in both the mouse and the rat pancreas. Sequential immunostaining revealed co-localization of galanin and TH immunoreactivity in nerve fibers in both the mouse and the rat pancreas. Following chemical sympathectomy using 6-hydroxydopamine (6-OHDA), not all GIR nerves disappeared. In the mouse pancreas a remaining population of galanin nerves was found around blood vessels, and occasionally in the islets. In the rat pancreas, a few GIR nerves were seen also after chemical sympathectomy. We conclude that intrapancreatic GIR nerves also occur in the mouse and the rat. These findings suggest that many of the GIR nerves are adrenergic but that non adrenergic, possibly intrinsic or sensory GIR nerves exist as well in both the mouse and the rat pancreas. PMID- 1715244 TI - Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. AB - Although the immediate receptors (immunophilins) of the immunosuppressants cyclosporin A (CsA) and FK506 are distinct, their similar mechanisms of inhibition of cell signaling suggest that their associated immunophilin complexes interact with a common target. We report here that the complexes cyclophilin-CsA and FKBP-FK506 (but not cyclophilin, FKBP, FKBP-rapamycin, or FKBP-506BD) competitively bind to and inhibit the Ca(2+)- and calmodulin-dependent phosphatase calcineurin, although the binding and inhibition of calcineurin do not require calmodulin. These results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and FK506, respectively, by forming drug-dependent complexes with and altering the activity of calcineurin calmodulin. PMID- 1715245 TI - [Studies on monoclonal antibody against recombinant human granulocyte colony stimulating factor]. AB - Three hybridomas producing monoclonal antibodies (McAb) against recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been established by fusing mouse myeloma SP 2/0 cells with spleen cells from a BALB/c mouse immunized with rhG-CSF. Ascites was produced from BALB/c mice by injecting substrain 2D4-B4 hybridoma cells intraperitoneally. The antibody was purified either by the caprylic acid-ammonium sulfate method or by euglobulin precipitation. The caprylic acid-ammonium sulfate method was superior to euglobulin precipitation in terms of purification and recovery of IgG. The 2D4-B4 McAb was determined to belong to the IgG3 subclass with a molecular weight of about 172,400 It was proved by Western Blot to react specifically with rhG-CSF. The stability and affinity of the McAb were analyzed as well. The potential clinical and experimental applications of this McAb are discussed. PMID- 1715246 TI - [Analysis of factors affecting the thermal stability of alpha 2-macroglobulin in solution]. AB - The factors affecting the thermal stability of alpha 2-macroglobulin (alpha 2M) in aqueous solution have been analyzed with an experiment of orthogonal design. The optimum conditions for heat treatment of alpha 2M have been established. Under these conditions, the trypsin-binding activity of alpha 2M can be fully retained after heat treatment at 60 degrees C for 10 hours. PMID- 1715247 TI - Inter-relations between growth hormone, insulin, insulin-like growth factor-I (IGF-I), IGF-binding protein-1 (IGFBP-1) and sex hormone-binding globulin in acromegaly. AB - Acromegaly is characterized by a hypersecretion of GH, which in turn results in an excess of IGF-I, an important mediator of its actions. IGF-I itself is intimately related to insulin both in structure and function. IGF-I circulates associated with specific binding proteins which appear to have important effects on its activity. We have examined the inter-relations between GH, prolactin, insulin, IGF-I and one of the binding proteins, IGFBP-1, in 62 patients with acromegaly of varying activity. Serum IGF-I levels were closely related to the logarithm of mean GH levels (r = 0.76; n = 62; P less than 0.001) but multiple regression analysis suggested that, after accounting for the variation due to GH, insulin accounted for some of the additional variation of IGF-I. IGF-I concentrations were independent of prolactin. Fasting insulin levels were high and unrelated to mean GH levels but correlated with those of IGF-I (r = 0.542; n = 57; P less than 0.001). This correlation coefficient was further improved by also accounting for variations in IGFBP-1 (r = 0.684; n = 57; P less than 0.001). Even in subjects whose acromegaly was well controlled or cured, as indicated by GH levels of less than 1 mU/l or IGF-I levels of less than 2 U/ml, fasting insulin levels remained significantly elevated in some individuals. The reason for this persistent abnormality is not clear. Fasting IGFBP-1 levels were low and unrelated to mean GH but were inversely related to fasting insulin levels (r = 0.593; n = 57; P less than 0.001). We propose that a cascade of events occurs in acromegaly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715248 TI - Epitope analysis of an HLA-B27-derived synthetic peptide: a possible approach to analyzing HLA class I antigens. AB - A monoclonal antibody, F3H7, was generated by immunizing mice with a synthetic peptide corresponding to residues 63-84 of the B*2705 allele of the HLA-B27 antigens. The reactive epitope and the contact residues on the peptide were localized by ELISA using a large panel of overlapping peptides as well as peptides with substituted amino acids. Residues corresponding to R75, D77 and L78 on the HLA-B27 protein appeared to be critical. The clarity of these results indicate that this is a potentially useful approach to the study of HLA class I epitopes. PMID- 1715249 TI - Hepatitis C virus in patients with polyarteritis nodosa. Prevalence in 38 patients. AB - In order to assess the prevalence of hepatitis C virus (HCV) in polyarteritis nodosa (PN), 38 patients with systemic necrotizing angiitis were retrospectively tested for the presence of anti-HCV antibodies (Ab). Twenty-one patients were hepatitis B virus (HBV) positive, comprising group A, and 17 were HBV negative, comprising group B. Two patients from group A had anti-HCV Ab (2/21: 9.5%). One was treated unsuccessfully with corticosteroids, then with vidarabine and plasma exchanges; HBe/anti-HBe seroconversion was not observed and anti-HCV Ab disappeared 8 months after the onset of PN. The second patient was successfully treated with corticosteroids, then vidarabine and plasma exchanges; he recovered from PN, HBV seroconversion occurred, and the anti HCV Ab remained detectable. These results show that: 1) the prevalence of anti HCV Ab in PN related to HBV is nearly the same (9.5%) as the prevalence of HCV Ab observed in patients with chronic hepatitis related to HBV infection; 2) the course of these two viral infections can be different and the role of HCV as an etiologic factor in PN has not been established. PMID- 1715250 TI - Update: gastroenterology and hepatology. PMID- 1715251 TI - Cutaneous trichilemmal cysts in three dogs. AB - Cutaneous trichilemmal cysts were recognized in 3 dogs. The lesions were multiple, asymptomatic, and occurred over the dorsal lumbar, lateral thoracic, dorsal neck, and cranial carpal regions. These cysts were not accompanied by other disease processes, and did not recur following surgical excision. Histologically, the trichilemmal cyst is characterized by trichilemmal differentiation of the entire cyst wall, and the formation of a predominantly amorphous and nonlaminated keratin in the cyst cavity. PMID- 1715252 TI - Somesthetic-visual matching disorders in right and left hemisphere-damaged patients. AB - Forty-nine patients with recent right (RHD) and left (LHD) hemispheric vascular lesions were compared on a task of somesthetic-visual matching of meaningful objects and of meaningless shapes. A selective impairment for shapes was found in RHD subjects, while LHD patients were impaired in object matching. This double dissociation conforms to the classical distinction between apperceptive and associative agnosia, and extends to the somesthetic modality the "double dissociation" between left and right hemispheric lesions and associative and apperceptive recognition disorders, which has been found in other modalities of agnosia. PMID- 1715253 TI - A morphological study of stromal microvasculature of nasopharyngeal precancerous lesions. AB - 24 specimens of nasopharyngeal precancerous lesions (NPP) were studied for stromal microvasculature by light microscope and image analysis for quantitation. FVIIIR: Ag was used as a vascular endothelium marker to show the blood vessels by immunohistochemistry. The microscopic and quantitative results showed that most cases had abnormal hyperplastic features similar to the stromal neovascularization of nasopharyngeal carcinoma (NPC). These findings suggest that abnormal hyperplastic vessels may be a prerequisite to cancerous transformation of nasopharyngeal epithelium. PMID- 1715254 TI - [The relation between ventricular late potential and ventricular arrhythmia in different stages of acute myocardial infarction]. AB - 50 cases of acute myocardial infarction (AMI) were examined for ventricular late potential (VLP) six times in total within 4 weeks. At the same time the patients clinical condition was observed. It is found that: (1) In our group the occurrence of VLP in AMI was 28% and VLP was unstable in both acute stage and recovery stage. (2) During acute stage, the difference of positive VLP rate is significant between the ventricular fibrillation group and normal rhythm group (P less than 0.05), but there is no significant difference between the ventricular ectopia group and normal rhythm group. During recovery stage, the difference of positive VLP rate is very significant between the ventricular ectopia and normal rhythm group (P = 0.001). As there was only 1 case of ventricular fibrillation occurring in the recovery stage, no comparison could be made. (3) There is no correlation between VLP and clinical conditions, such as age sex, location of the infarction, existence of old infarction and LVEF. However, the difference of the peak serum CPK level is significant between VLP positive and negative group (P less than 0.05). PMID- 1715255 TI - Usefulness of a near-total fine-needle aspiration biopsy retrieval method: a study of its use in 85 consecutive patients. AB - Fine-needle aspiration biopsy may provide miniscule material for diagnosis. A method was devised to ensure optimal retrieval of the specimen. If performed in the manner described, at least four to five different samples from each case may be obtained. This includes a smear stained with the Papanicolaou and hematoxylin and eosin methods, a cell block preparation, and at least two cytocentrifuge specimens. In 85 cases in which the method was applied and subsequently analyzed, we found the cell block and Papanicolaou-stained smears to be most effective for diagnosis, whereas the cytocentrifuge method was much less effective. The sensitivity and specificity were 93% and 100%, respectively. PMID- 1715256 TI - Modified EA staining solution using fast green in the Papanicolaou method. PMID- 1715257 TI - [The role of transcatheter ultra high frequency ablation of experimental arrhythmia at atrioventricular junctional area]. PMID- 1715258 TI - The mRNAs encoding acidic FGF, basic FGF and FGF receptor are coordinately downregulated during myogenic differentiation. AB - Acidic and basic fibroblast growth factors (FGFs) are members of a family of proteins that exert pleiotropic effects in a range of cell types including skeletal myocytes. Previous studies demonstrate that exogenously supplied FGFs stimulate proliferation of myoblasts and inhibit their differentiation in culture, but little information is available concerning endogenous expression of FGFs by skeletal myocytes. In this study acidic and basic FGF mRNAs were found to be expressed in murine and rat skeletal muscle, and expression was demonstrated to vary with the tissue and species examined. Myogenic cell lines were then analyzed to determine if FGFs are expressed in myoblasts, and if so, whether expression is regulated during myogenic differentiation. Murine Sol 8 and rat L6 myoblasts were found to express acidic and basic FGF mRNAs, and the expression of both growth factors was downregulated at the transcriptional level during myogenic differentiation. A decrease in expression of the mouse homologue of the human FGF receptor paralleled the decrease in acidic and basic FGF mRNAs in Sol 8 cells, indicating that the decrease in FGF receptor abundance previously observed during myogenic differentiation is regulated at the mRNA level. The results of this study suggest that a coordinate decrease in endogenously produced acidic and basic FGFs and their cognate receptor may participate in the regulation of myogenic differentiation. Furthermore, the observation that expression of a myogenic determination gene, myogenin, increases as FGF transcripts decline, together with previous data demonstrating suppression of myogenin expression by FGF, suggest a mechanism whereby endogenously produced FGFs may exert their effect on differentiation. PMID- 1715259 TI - Differentiating and mature neurons express the acidic fibroblast growth factor gene during chick neural development. AB - We have previously isolated and characterized acidic fibroblast growth factor (aFGF) from the embryonic chick brain. To analyze the expression of the gene encoding this growth factor a cDNA clone was isolated. The predicted amino acid sequence was found to be highly conserved (90%) between human and chick. Using single-stranded DNA probes, aFGF gene expression was detectable at day 3.5 in the embryonic chick brain. The mRNA level of the 1.7 kb transcript increased during embryonic development and reached the highest level in the adult brain. In situ hybridization results confirmed these developmental changes and revealed a localized expression in neurons. In the adult, Purkinje cells, deep cerebellar and brainstem neurons showed a high level of aFGF mRNA. In the embryonic brain, localized expression in neurons was detectable from day 6 onward. aFGF mRNA was also present in neurons of the peripheral nervous system. In dorsal root ganglia, aFGF was found to be expressed after embryonic day 6. Cells of blood vessels and the ependyma did not express detectable amounts of aFGF mRNA. These results suggest that aFGF may have a function as a differentiation or maintenance factor for postmitotic neurons or as a growth or differentiation factor for other cells in the nervous system mainly in later stages of development. PMID- 1715260 TI - Asymptomatic peptic ulcer disease. Is it worth looking for? PMID- 1715261 TI - Silent myocardial ischaemia. Implications for therapy. PMID- 1715262 TI - Current concepts in the pathogenesis of leprosy. Clinical, pathological, immunological and chemotherapeutic aspects. AB - In recent years there have been notable advances in the laboratory investigation and field management of leprosy. Progress, however, continues to be hindered by the lack of efficient methods for early diagnosis and implementation of control and treatment measures. Diagnosis is still made on the same principles as a century ago (clinical and histopathological findings), and only 1 in 3 known patients worldwide receives optimal chemotherapy. In 1988, nearly 1 in 10 newly diagnosed patients already had debilitating deformities. Contributing factors are operational, administrative and financial difficulties in implementing multidrug therapeutic regimens, inadequately trained personnel, and lack of priority and political commitment to leprosy control. The formulation and implementation of multidrug therapy is the most important development in leprosy in the past 10 years. Dapsone monotherapy was the mainstay for treatment and control for approximately 40 years, but secondary dapsone-resistant strains, first noted in 1964, now infect as many as 50% of all new patients. Multidrug regimens recommended by the WHO consist of various combinations of therapy using dapsone, rifampicin, clofazimine and a thionamide. Duration of therapy is limited to 6 months for paucibacillary and 2 years or more for multibacillary patients; relapse rates thus far are low. The average cost of treatment worldwide, including the cost of drugs, is estimated at $US150 per patient. The recent annual drop of nearly 8% in newly registered patients may be due to the implementation of these therapeutic regimens. Newer drugs that may be introduced into these regimens include fluoroquinolones, minocycline and clarithromycin. While knowledge of the microbiology of the leprosy bacillus and host response has advanced remarkably, there is little improvement in the understanding or amelioration of social aspects of leprosy. Better treatment and control reduces the stigma, but improvements in the attitudes of patients and society towards leprosy are as important as advances in medical science in achieving ultimate eradication of the disease. PMID- 1715263 TI - Haemodynamic monitoring. Problems, pitfalls and practical solutions. AB - The synthesis of adenosine triphosphate (ATP) depends on the coordinated interaction of oxygen delivery and glucose breakdown in the Krebs cycle. Cellular oxygen depots are non-existent, therefore the peripheral cells are totally dependent on the circulation for sufficient oxygen delivery. Shock is the clinical manifestation of cellular oxygen craving. The commonly measured variables--blood pressure, heart rate, urinary output, cardiac output and systemic vascular resistance--are not sensitive or accurate enough to warn of impending death in acutely ill patients nor are they appropriate for monitoring therapy. Calculated oxygen transport and oxygen consumption parameters provide the best available measures of functional adequacy of both circulation and metabolism. In order to optimise oxygen delivery (DO2), 4 interacting factors must be taken into account: cardiac output, blood haemoglobin content, haemoglobin oxygen saturation and avidity of oxygen binding to haemoglobin. For viscosity reasons, the optimal haemoglobin concentration is in the vicinity of 90 to 100 g/L, but for optimising the oxygen transport 100 to 115 g/L or a haematocrit of 30 to 35% seems better. The p50 (the pO2 at which haemoglobin is 50% saturated) describes the oxygen-haemoglobin dissociation curve; normally its value is +/- 27 mm Hg. It can be influenced by attaining normal body temperature, pH, pCO2 and serum phosphorous levels. In order to obtain an arterial blood saturation (SaO2) of more than 90% with acceptable haemodynamics, the ventilation mode and inspired oxygen fraction (FiO2) must be adapted; care must be taken not to stress the labile circulation with haemodynamic compromising ventilation techniques [e.g. high positive end expiratory pressure (PEEP) levels, inverse ratio ventilation, etc.]. The factor most amenable to manipulation is the cardiac output, with its 4 determinants--preload, afterload, contractility and heart rate. In daily clinical practice, heart rate should be between 80 and 120 beats/min; small variations are acceptable. Important deviations must be treated by chemically [isoprenaline (isoproterenol)] or electrically (pacing techniques) accelerating the heart, or with the different antiarrhythmic drugs. A wide variety of agents is available to decrease the preload: diuretics [especially furosemide (frusemide)], venodilators like nitroglycerin (glyceryl trinitrate), isosorbide dinitrate (sorbide nitrate) and sodium nitroprusside, ACE inhibitors, phlebotomy, and haemofiltration techniques (peritoneal or haemodialysis, continuous arteriovenous haemofiltration). To increase the preload, volume loading using a rigid protocol ('rule of 7 and 3'), preferably with colloids, or vasopressor agents [norepinephrine (noradrenaline), epinephrine (adrenaline), dopamine] are useful. Arterial vasopressors are needed to improve perfusion pressure of 'critical' (coronary and cerebral) arteries. Afterload can be decreased by arterial vasodilators. Predominantly arterial dilators are hydralazine and clonidine, while sodium nitroprusside, nitroglycerin and isosorbide dinitrate have combined arterial and venous dilating actions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1715264 TI - Current concepts in rational therapy for haemochromatosis. AB - Genetic haemochromatosis is characterised by an inappropriately high rate of iron absorption by the small intestine. The disease is transmitted as an autosomal recessive condition. The gene frequency in the Caucasian population is approximately 1 in 20 and the disease frequency is 1 in 400. Excessive iron deposition occurs in the liver, pancreas, heart, pituitary and joints and hepatic iron concentrations above approximately 400 mumol/g dry weight are always associated with fibrosis and usually with cirrhosis and progressive liver failure. Accurate diagnosis depends upon the demonstration of elevated hepatic iron stores. An hepatic iron index [hepatic iron concentration (in mumol/g dry weight) divided by patient age] of greater than 2.0 distinguishes homozygous subjects from the other conditions in which slight increases in hepatic iron concentration may occur, e.g. in a subject heterozygous for haemochromatosis or alcoholic liver disease. If cirrhosis is present, patients are at a high risk of developing hepatocellular carcinoma. Therefore, they should undergo regular abdominal ultrasound and alpha-fetoprotein estimation. In the absence of cirrhosis, phlebotomy restores life expectancy to normal. Venesection should be continued until all excess iron stores are removed as judged by failure of a rise in haemoglobin concentration on cessation of phlebotomy. Screening of first degree relatives should commence from a young age (e.g. 10 years). If serum ferritin or transferrin saturation are abnormal, liver biopsy should be undertaken. HLA typing of the family allows for the identification of those siblings who are most likely to develop the disease. Secondary iron overload is often multifactorial in origin. Iron chelation therapy with subcutaneous deferoxamine (desferrioxamine) should only commence after careful consideration of the potential benefits in each individual patient. PMID- 1715265 TI - Prophylactic treatment regimens for the prevention of hepatitis A. Current concepts. AB - Hepatitis A virus (HAV) occurs worldwide. In developing countries the virus is endemic, with the majority of the population being exposed to it in childhood, when the infection usually causes, at the most, a mild anicteric illness. In developed countries the majority of HAV infections occur at a later age, often in adults, especially those with a history of recent travel to developing countries. In adults, HAV infection usually causes a symptomatic icteric illness. In addition to community sanitation and hygiene measures, prophylactic prevention of hepatitis A infection can be achieved by 2 methods. The first is the established and widely used method of passive immunisation using human immune globulin from pooled serum. Indications for the use of human immune globulin are: (a) travellers who will be exposed to unhygienic conditions in high risk countries; and (b) contacts of patients with acute hepatitis A infection, in certain circumstances. The second method currently undergoing research, and trials, is active immunisation using either live-attenuated or killed vaccines, which have given encouraging results in a number of trials. Further vaccines, using molecular biology techniques, are currently being developed. PMID- 1715266 TI - Vigabatrin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in epilepsy and disorders of motor control. AB - Vigabatrin was specifically designed to enhance gamma-aminobutyric acid (GABA) function in the CNS. By increasing brain concentrations of this inhibitory neurotransmitter the drug appears to decrease propagation of abnormal hypersynchronous discharges, thereby reducing seizure activity. At this stage in its development, clinical experience with vigabatrin is limited primarily to patients with refractory seizure disorders. In this difficult-to-treat population, 'add-on' therapy with vigabatrin greater than or equal to 2 g/day has shown impressive efficacy, reducing seizure frequency by greater than or equal to 50% in approximately half of patients. Clinical efficacy does seem to vary with seizure type with the best response reported in adults with complex partial seizures with or without generalisation and in children with cryptogenic partial epilepsy or symptomatic infantile spasm. Vigabatrin appears to have a negative effect on absences and myoclonic seizures. Some disorders of motor control may also be amenable to enhanced GABAergic function. In the small number of patients with tardive dyskinesia treated to date, vigabatrin produced mild to moderate improvement in hyperkinetic symptom scores but Parkinsonism or schizophrenic symptoms occasionally worsened. The best response was reported in a study of patients who had been withdrawn from neuroleptic therapy. In a small but well controlled comparative trial, vigabatrin was as effective as baclofen in reducing spasm and improving some parameters of spasticity in patients with spinal cord lesions or multiple sclerosis. Most adverse reactions to vigabatrin are mild and transient with central nervous system (CNS) changes being reported most frequently. Of particular note, serial evoked potential studies and the few available histology reports have not found evidence of intramyelinic oedema during therapeutic use, as was reported in rats and dogs on chronic high-dose treatment. Thus, vigabatrin is a promising new anticonvulsant drug. Current evidence supports a trial of this agent as adjunctive therapy in patients with refractory seizure disorders, and future investigation of vigabatrin monotherapy and its efficacy relative to established agents is awaited with interest. Wider experience should help to clarify which patients - by seizure type and concurrent CNS pathology - are likely to benefit from vigabatrin and ongoing monitoring should further clarify the potential detrimental effects, if any, of long term use. In the meantime, it is a welcome addition in the difficult setting of resistant epilepsy. PMID- 1715267 TI - Acrivastine. A review of its pharmacological properties and therapeutic efficacy in allergic rhinitis, urticaria and related disorders. AB - Acrivastine is a short acting histamine H1-receptor antagonist with a rapid onset of action. Double-blind clinical trials have shown acrivastine (usually 8mg three times daily) to be an effective and well tolerated antihistamine in the treatment of chronic urticaria and allergic rhinitis. Acrivastine was more effective than placebo and similar in efficacy to clemastine or terfenadine in the treatment of seasonal allergic rhinitis. In the treatment of dermatoses in which histamine has a pathogenetic role, the efficacy of acrivastine was superior to that of placebo and similar to that of usual dosages of clemastine, hydroxyzine, chlorpheniramine, cyproheptadine or terfenadine. Acrivastine caused less drowsiness than clemastine, the incidence of adverse effects being indistinguishable from that with placebo or terfenadine. Thus, acrivastine is an effective addition to drugs currently available for the treatment of patients with allergic diseases in whom a histamine H1-receptor antagonist is indicated. Because of its rapid onset of action acrivastine will be particularly useful for 'on demand' therapy in patients with intermittent symptoms. PMID- 1715269 TI - Characterization of the adrenal 11 beta-hydroxylase in inbred salt-sensitive and resistant rats. AB - Rats have been bred for susceptibility and resistance to the hypertensive effect of dietary salt (S/JR & R/JR). S/JR have an abnormal adrenal steroid 11 beta,18 hydroxylase activity resulting in increased production of 18-OH-DOC. S/JR also produce increased quantities of 19-nor-DOC, which may be related, since the 11 beta,18-hydroxylase also catalyzes the 19-hydroxylation of DOC, a pivotal step in 19-nor-DOC biosynthesis. The purpose of the present studies was to further characterize the mutant S/JR adrenal steroid 11 beta,18-hydroxylase. Preliminary studies are also presented on assessing the renal 19-desmolase, the last step in 19-nor-DOC biosynthesis. Adrenal glands were harvested from R/JR and S/JR and prepared for incubation studies, protein immunoblotting, and RNA analysis. Kidneys from Sprague-Dawley rats were also used for isolated renal perfusion studies. Both S/JR and R/JR strains had a single immunostaining band for 11 beta,18-hydroxylase at 51,000 molecular weight which were equal in intensity. Both strains had a single RNA transcript at 4.3 kilobases which hybridized with equal intensity to the bovine cDNA (pB11-9). The Km for 11 beta- and 18 hydroxylation was identical within strains but was different between strains. The Km for 19-hydroxylation was different between S/JR and R/JR, and was much greater than 11 beta- and 18-hydroxylation in both strains. This suggests that the catalytic site for 19-hydroxylation is different than that for 11 beta- and 18 hydroxylation and that the S/JR enzyme binds the substrate with higher affinity than the R/JR enzyme. In the isolated perfusion studies the rat kidneys converted 80% of 19-oxo-DOC to either 19-oic-DOC or 19-nor-DOC. These data demonstrate that the difference in S/JR enzyme activity is probably due to a point mutation in the enzyme or to a change in a regulatory protein. PMID- 1715268 TI - Celiprolol. An updated review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in cardiovascular disease. AB - Celiprolol is a hydrophilic, beta 1-selective adrenoceptor antagonist with mild selective beta 2-agonist as well as weak vasodilator properties. Celiprolol 200 to 400mg once daily by mouth is similar in antihypertensive efficacy to usual doses of propranolol, atenolol, metoprolol and pindolol in patients with mild to moderate systemic hypertension. Similar doses of celiprolol are as efficacious as propranolol and atenolol in improving exercise tolerance and reducing the frequency of anginal attacks in patients with angina pectoris. Further clinical experience suggests that celiprolol does not produce bronchoconstriction and may have mild bronchodilating activity in asthmatic patients; it may also enhance the effects of bronchodilator drugs. Celiprolol has a slightly beneficial effect on serum lipid profiles, and does not appear to exert adverse effects on carbohydrate metabolism. If the apparent pharmacodynamic advantages of celiprolol translate into clinical benefits and are confirmed in well designed long term clinical trials, then celiprolol should represent a definite advance in beta blocker therapy. PMID- 1715270 TI - Analysis of bleomycin-induced mutagenic functions related to the PSO4 (= xs9) gene of Saccharomyces cerevisiae. AB - In the present work we analyse some properties of the pso4-1 (= xs9) mutant of the yeast Saccharomyces cerevisiae, which is blocked in both reverse mutation and recombination. We have applied the "apparent survival test" as a method to characterize putative inducible error-prone repair activities related to the PSO4 gene. As the mutagenic agent we have used bleomycin, which is an antitumour radiomimetic antibiotic. Survival, reverse mutation yield, and reverse mutation frequency were determined as a function of bleomycin concentrations (1-60 micrograms/ml) in the logarithmic phase of growth. It is shown that the PSO4 gene product is poorly involved in bleomycin sensitivity and that reverse mutation induced by this mutagen is dramatically reduced in the pso4-1 lys2 mutant strain, as compared with the isogenic SC7K (lys2) strain. M(x) functions exhibit linear quadratic courses for both the SC7K (lys2) and the pso4-1 lys2 strains. The apparent survival functions display "humps" in both strains, corresponding to the resistant components of the survival functions. These facts suggest that the mutagenicity of bleomycin depends on at least one inducible error-prone repair pathway and that the PSO4 gene product could act as a mutation triggering factor. PMID- 1715271 TI - Transcriptional regulation of interferon-stimulated genes. AB - Interferons (IFN) stimulate the expression of a number of genes following interaction with specific high-affinity plasma membrane receptors. The products of these genes either singly or coordinately mediate the antiviral, growth inhibitory or immunoregulatory activities attributed to IFN. While the gene products in some cases have been well characterized, other IFN-regulated genes encode proteins whose functions have yet to be elucidated. A feature common to all IFN-stimulated genes characterized thus far is the presence of a DNA element which constitutes an IFN-responsive enhancer, usually present in the 5' upstream region of the genes. This element, termed interferon-stimulated response element (ISRE) binds a nuclear factor(s) translocated from the cytoplasm to the nucleus following IFN-receptor-triggered signal transduction. The binding of these factors to the ISRE represents the initiating event in stimulating RNA-polymerase II-mediated transcription from IFN-responsive genes. Depending on the nature of the cells responding to IFN and the genes involved, induced transcription may be prolonged or rapidly terminated. The rapid termination of transcription is dependent in some cases on IFN-induced protein synthesis and also involves factor binding to the ISRE. Recent progress in detailing these events will be discussed including IFN-receptor interactions, signal-transduction pathways, comparing and contrasting IFN-regulated genes and elucidation of IFN-regulated factors. PMID- 1715272 TI - Binding of proteins of HeLa S3 cell extract to oligonucleotides containing the consensus interferon-response sequence (IRS) and to the IRS-containing fragment of the human c-myc gene. AB - The human c-myc proto-oncogene was recently found to contain a regulatory sequence similar to the consensus interferon-response sequence (IRS) of interferon-activating genes. Binding of regulatory protein(s) to this sequence of cloned fragment of c-myc, lacking the main part of 5'-nontranscribing region, regulates in vitro transcription from I1/I2 initiation sites located in the first intron of the gene. Here, we have shown that HeLa S3 nuclear extract contains different protein factors, at least two, that bind preferentially to the IRS sequence of either the c-myc gene or the interferon-dependent 6-16 gene. Moreover, each of these factors 'cross-binds' to the region of the other gene, although affinity of this interaction is lower. Binding constants of these proteins to oligonucleotide fragments of c-myc and 6-16 genes were determined. In vitro transcription of the human full-length c-myc gene (i.e. the gene containing the complete 5'-noncoding region) initiated from I1/I2 sites, that is controlled by the IRS region, was demonstrated to be blocked. A possible physiological role for the mechanisms described is discussed. PMID- 1715274 TI - Structural and immunochemical studies of O-specific polysaccharide of Proteus vulgaris 5/43 belonging to OX19 group (O-variants). AB - O-specific polysaccharide was obtained on mild acid degradation of lipopolysaccharide of Proteus vulgaris 5/43 belonging to OX19 (O-variants). It was found to contain D-glucose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido 2,6-dideoxy-L-glucose (QuiNAc, N-acetyl-L-quinovosamine) in the ratio of about 1:2:1, the last-named sugar being rather uncommon for bacterial antigens. A computer-assisted 13C-NMR-based analysis, methylation analysis, and selective cleavage with anhydrous hydrogen fluoride and dilute hydrochloric acid were applied for structural elucidation of the polysaccharide and the following structure was established:----2)-beta-D-Glcp-(1----6)-alpha-D-GlcpNAc-(1----3)- alpha-L-QuipNAc-(1----3)-beta-D-GlcpNAc-(1----. Serological studies of the O antigen and oligosaccharides derived therefrom revealed the importance of the trisaccharide fragment beta-D-Glcp-(1----6)-alpha-D-GlcpNAc-(1----3)-alpha-L- QuipNAc in manifesting the antigenic specificity. Serological cross-reactions were demonstrated between lipopolysaccharides of P. vulgaris 5/43, 8/44, and OX19 strains. PMID- 1715273 TI - Regulation of transcription of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase of Saccharomyces cerevisiae. AB - Transferring Saccharomyces cerevisiae cells from glucose- to oleate-containing growth media results in a significant increase in the number and volume of peroxisomes. To investigate this proliferation process we studied the transcriptional regulation of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase (EC 2.3.1.16) in response to the switch in carbon source. Expression was proved to be repressed during growth on glucose, derepressed during growth on glycerol, and induced during growth on oleate as the sole carbon source. By deletion and mutational analysis of sequences upstream of this gene, we have identified a region which is involved in the regulation of transcription. It is contained within a 52-base-pair sequence, UAST52 (upstream activation sequence thiolase 52), located between 203 and 151 nucleotides upstream of the translational initiation codon. This sequence proved to be required for repression, derepression and induction of transcription, and was able to activate transcription from the truncated version of the heterologous iso-1-cytochrome-c (CYC1) promoter in a similar way as in the wild-type promoter context. Sequence comparison revealed that the UAST52 contained a sequence motif ('beta-oxidation box') that is very similar to sequences located in the 5'-upstream regions of the genes coding for two other beta-oxidation enzymes of S. cerevisiae: the peroxisomal acyl-CoA oxidase and the peroxisomal trifunctional beta-oxidation enzyme of S. cerevisiae. Mutational analysis of the 'beta-oxidation box' indicates that this sequence motif acts as a UAS in vivo. Sequence comparison also revealed that just upstream of the 'beta-oxidation box', between positions 213 and -201, a potential binding site occurred for the yeast multifunctional autonomously replicating sequence binding factor ABF1. Gel-retardation competition experiments indicate that ABF1 binds specifically to this sequence. PMID- 1715275 TI - Guanine nucleotide sensitivity of [3H]iloprost binding to prostacyclin receptors. AB - A component of the displaceable binding of the stable prostacyclin analogue, [3H]iloprost, to membranes from human platelets and the somatic hybrid cell lines NG108-15 and NCB20, was inhibited by guanine nucleotides. The order of potency of a range of nucleotides for this effect was GTP gamma S greater than GppNHp greater than GTP greater than GDP = GMP; ATP, UTP and CTP were ineffective at concentrations up to 1 mM. In the presence of 100 microM GppNHp, iloprost binding curves were displaced to the right of curves obtained in the absence of guanine nucleotide, and their Hill slopes were greater. This was consistent with a conversion of a minor population of high affinity agonist binding sites to lower affinity sites in the presence of guanine nucleotides. These effects of guanine nucleotides on the binding of the agonist ligand [3H]iloprost were consistent with an interaction with a G protein coupled receptor. PMID- 1715276 TI - Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. AB - Mitochondrial nucleoids (mt-nucleoids) of the A2780 line of cultured human cells were stained with DAPI and observed using an epifluorescence microscope. The mt nucleoids appeared to be organized compactly in mitochondria. Numbers of mt nucleoids per mitochondrion ranged from 1 to more than 10, and 70% were "multinucleated" mitochondria. Intensities of fluorescence of mt-nucleoids in each mitochondrion were measured by a video-intensified microscope system (VIM system) and copy numbers of mitochondrial DNA (mtDNA) in each mitochondria were determined. The copy numbers of mtDNA per mitochondrion ranged from 1 to 15, and the average was 4.6. Because the cells had 107 mitochondria on average, the copy number of mtDNA per cell was estimated to be about 500. PMID- 1715277 TI - Cytoskeletal reorganizations in human umbilical vein endothelial cells as a result of cytokine exposure. AB - Treatment of HUVECs in culture with several cytokines and phorbol esters caused reorganizations of the actin and microtubule networks, as well as a redistribution of focal contract proteins. However, expression of the cytoskeletal proteins which link cells, via integrins, to the substrate, was not significantly affected. Indirect immunofluorescence microscopy of endothelial cells after treatment with interleukin-1 alpha and beta, gamma-interferon, tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, and phorbol 12,13 dibutyrate allowed us to observe reductions in the areas of cell-cell contact, redistribution of the stress fiber network, and concomitant changes in focal contacts. Microtubule arrays in TNF-treated cells became bundled. Phorbol esters induced formation of microtubule organizing centers not seen in resting or TNF treated HUVECs. Talin was distributed along stress fibers and not exclusively in focal contacts. Vitronectin receptor was observed in focal contacts, occasionally at cell-cell contacts, and in vesicular structures close to the lumenal surface, after both types of treatment. Although these morphological changes were easily observed by indirect immunofluorescence, no quantitative differences in specific cytoskeletal proteins were detected by immunoblots and [35S]cysteine metabolic labeling experiments. PMID- 1715278 TI - The small intestinal enterocytes of rats during lead poisoning: the application of the Timm sulphide silver method and an ultrastructural study. AB - Young male rats were divided into 3 groups which received approximately 100 mg of a lead acetate/kg b.w. per day in their drinking water during 2, 30 and 60 d, respectively. Samples from the jejunum were processed for transmission electron microscopy (TEM), and after 60 d of exposure blocks of tissue were also processed to evaluating the Timm sulphide silver reaction sites in the epithelial absorptive cells in TEM according to Dancher and Zimmer (5). The ultrastructure of enterocytes in poisoned rats at 2 days was similar to the controls. A marked feature of about one third of the rat enterocytes exposed to lead for 30 d was the presence of numerous, small rough-membraned vesicles and prominent, dilated Golgi complexes in their cytoplasm. Most of the enterocytes at the 60th d of lead exposed rats had a vacuolated cytoplasm associated with the prominent Golgi complexes and vacuoles of various size. They also had pleomorphic rough-membraned vesicles and dilated cisternae of the RER. The presence of the Timm reaction deposits has been observed on the microvillar surface of the brush border in connection with the enterocyte plasma membrane, and in the extracellular space between epithelial cells. Furthermore, Timm precipitates were found in extravascular spaces surrounding capillaries, between endothelial cells, and in the capillary lumen. PMID- 1715279 TI - How do viral reverse transcriptases recognize their RNA genome? AB - Reverse transcription is not solely a retroviral mechanism. Hepadnaviruses and caulimoviruses have RNA intermediates that are reverse transcribed into DNA. Moreover non-viral retroelements, retrotransposons, use reverse transcription in their transposition. All these retroelements encode reverse transcriptase but each group developed their own expression modes capable of assuring a specific and efficient replication of their genomes. PMID- 1715280 TI - Iron entry route in horse spleen apoferritin. Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4 maleimido-tempo. AB - Apoferritin has been selectively labeled with a maleimide nitroxide derivative at Cys-126, located in the hydrophilic 3-fold channels. Titration of this derivative with Fe(II), which gives rise to the initial Fe(III)-apoferritin complex, produces, at low metal-to-protein ratios, a decrease of the intensity of the label EPR signal due to the occurrence of a magnetic dipolar interaction. A label metal distance ranging between 8-12 A can be estimated from titrations performed with VO(IV), which is known to bind in the 3-fold channels, and likewise produces a decrease in the label EPR signal. The present findings indicate that iron binds in the hydrophilic channels in its higher oxidation state and that these channels represent the metal entry route at least at low metal-to-protein ratios. PMID- 1715281 TI - In vivo immediate early gene expression induced in intestinal and colonic mucosa by feeding. AB - Since the gut responds rapidly to food intake, the levels of expression of several immediate early genes were measured in mucosa from small and large intestine of rats starved for 3 days or refed. Within 1 h of refeeding, jejunal and ileal c-fos, jun B and zif/268 mRNA and colonic zif/268 dramatically increased. The zif/268 gene in jejunum corresponded in size to the full-length cDNA but, in ileum, an RNA band of about 1.2 kb in size increased greatly after feeding. This represents a physiologic in vivo model for the study of gene regulation associated with intestinal epithelial cellular responses to feeding. PMID- 1715282 TI - Light-induced D1 protein degradation is catalyzed by a serine-type protease. AB - Light-induced degradation of the D1 protein in isolated spinach photosystem II core preparations was studied after addition of various protease inhibitors. The degradation was selectively inhibited by several serine protease inhibitors in particular diisopropylfluorophosphate. The results demonstrate that the D1 protein is degraded by a serine-type of proteolytic activity that is an integral part of photosystem II. PMID- 1715283 TI - Fatty acid acylated Fab-fragments of antibodies to neurospecific proteins as carriers for neuroleptic targeted delivery in brain. AB - A method for targeted delivery of neuroleptics from blood in brain based on using Fab-fragments of antibodies to antigens of brain glia cells (acid gliofibrillar antigen and alpha 2-glycoprotein) is suggested. The essence of the technique is that the molecule of neuroleptic (trifluoperazine) is conjugated with Fab fragments of these antibodies. The conjugate thus obtained is modified by stearoylchloride in the system of Aerosol OT reversed micelles in octane. The study of the distribution of 125I-labelled conjugates in the rat organism after intracordial introduction is performed. On the contrary to the nonmodified conjugates and conjugate, containing fatty acylated Fab-fragments of antibodies, nonspecific to the rat brain, the conjugate of trifluoperazine with stearoylated Fab-fragments of antibodies to neurospecific antigens accumulate in brain tissues. The drastic increase of the neuroleptic activity of trifluoperazine resulting from its coupling with stearoylated Fab-fragments of antiglial antibodies is observed. PMID- 1715284 TI - Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C. AB - The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC. PMID- 1715285 TI - The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins. AB - A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis. PMID- 1715286 TI - Solubilization of rat kidney lysosomes in reversed micelles of aerosol OT in octane. AB - Intact lysosomes from rat kidneys were solubilized in the ternary system: surfactant (Aerosol OT)-buffer-organic solvent. According to data of laser light scattering analysis and kinetic experiments with the lysosomal marker enzyme, N acetyl-beta-D-hexosaminidase (EC 3.2.1.30), the solubilization of lysosomes in this system resulted in the destruction of the lysosomes and the entrapping of their components in reversed micelles. PMID- 1715287 TI - N-deglycosylation and immunological identification indicates the existence of beta-subunit isoforms of the rat GABAA receptor. AB - beta 2- and beta 3-subunits of GABAA receptors purified from the brains of 5-10 day-old rats were investigated in the intact or completely N-deglycosylated state using the beta-subunit-specific monoclonal antibody bd-17 and polyclonal antibodies directed against synthetic amino acid sequences specific for the GABAA receptor beta 2- or beta 3-subunits. The present results seem to indicate the existence of two different isoforms of the beta 3-subunit and several different isoforms of the beta 2-subunit of the GABAA receptor which probably are produced by alternative splicing. PMID- 1715288 TI - Extreme variations in the ratios of non-synonymous to synonymous nucleotide substitution rates in signal peptide evolution. AB - Nucleotide sequences encoding signal peptides from the precursors of alpha amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non-synonymous (KA) and synonymous (KS) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in KA/KS ratios has been observed; from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins, which are unrelated to those of the alpha-amylase/trypsin inhibitor family, and an average KA/KS of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints. PMID- 1715289 TI - Chemical detection of natural peptides by specific structures. Isolation of chicken galanin by monitoring for its N-terminal dipeptide, and determination of the amino acid sequence. AB - We have isolated galanin from chicken intestine by monitoring for the N-terminal glycyltryptophan, which constitutes a conserved part characteristic of the peptide. This monitoring method complements that previously used for C-terminal amide detection and proves chemical monitoring of specific structures to be useful. The isolation allowed determination of the structure, which was found to be unidentical to any of the known galanins. However, N-terminal pentadecapeptide parts are identical, showing this segment to be of special importance. In addition to common substitutions at positions 16, 18, 23, 26 and 29, chicken galanin has phenylalanine at position 28, where all known mammalian galanins have leucine. PMID- 1715290 TI - Brain nitric oxide synthase is a biopterin- and flavin-containing multi functional oxido-reductase. AB - Brain nitric oxide synthase is a Ca2+/calmodulin-regulated enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme-bound H4biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin-dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin. PMID- 1715291 TI - Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP). AB - The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN') and uridine and cytidine 2',3'-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the 'non secretory' liver-type enzymes rather than to the 'secretory' pancreatic-type RNases. PMID- 1715292 TI - Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor. AB - We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system. PMID- 1715293 TI - A 64 kDa protein from Mycobacterium bovis BCG shares the same antigenic determinants with line 10 hepatoma cells and has anti-line 10 tumor activity. AB - A 64 kilodalton (kDa) surface protein was isolated from the water-extracted materials from Mycobacterium bovis strain BCG, the determinants of which are antigenically shared by a 64 kDa major surface antigenic component of line 10 hepatoma cells. The 64 kDa protein showed anti-line 10 tumor activity in pre immunized guinea pigs, and this suggest that the BCG 64 kDa protein is probably identical with the tumor specific antigen. PMID- 1715294 TI - [The localization of neurons innervating the upper portion of the duodenum in the dorsal motor nucleus of the vagus nerve]. AB - In anesthetized cats, using method of the retrograde transport of the horseradish peroxidase and electrophysiological techniques, the dorsal motor nucleus neurons innervating upper portion of the duodenum were found. Maximal number of such neurons were found within 1.0 to 2.5 mm rostral to the obex. The effects of the neurons stimulation on the electrical activity of the duodenum smooth muscles were studied. Possible role of the dorsal motor nucleus neurons in realisation of gastro-duodenum motor reflexes is discussed. PMID- 1715295 TI - [The mechanisms of cellular complementarity in the erythroblast islands of the bone marrow]. AB - Phenylhydrazine was shown to reduce the content of erythroblast islands (EI) within 24 hrs in the rat bone marrow, the effect depending on the dose. Simultaneous strengthening of phagocytic activity, acidifying of lysosomes central macrophages EI and lowering of electrophoretic mobility, were observed. The proteinase inhibitor contrycal (50 and 500 i. u.) prevented the effect in the EI and intensified the regeneration of erythron. In vitro, trypsin and chymotrypsin decreased the EI content in the marrow cell suspension. Changing of functional condition of the central macrophages EI and, probably, products of its secretion, seems to affect the cell complementing in EI. PMID- 1715296 TI - [The effect of taurine on the electrically controlled ion channels of the somatic membrane of pond snail neurons]. AB - Isolated and internally dialysed neurons from the molluscs Lymnaea stagnalis were investigated under voltage-clamp conditions. The increasing of taurin concentration (from 1.10(-8) to 1.10(-2) mol/l) reduced the calcium inward--and delayed potassium outward currents, and exerted no effect on the fast potassium outward current. Sodium inward current was increased by taurin concentrations 1.10(-8)-1.10(-4) mol/l, and reduced by 1.10(-3)-1.10(-2) mol/l. Membrane leakage currents were reduced by small taurin concentrations and increased by large concentrations. PMID- 1715297 TI - Topical treatment with 13-cis-retinoic acid improves Darier's disease and induces the expression of a unique keratin pattern. AB - A patient with Darier's disease was treated topically with either 13-cis-retinoic acid 0.1%, all-trans-retinoic acid 0.05% or their cream base only. Both 13-cis retinoic acid and all-trans-retinoic acid were effective. However, all-trans retinoic acid had to be discontinued because of irritation. By contrast, 13-cis retinoic acid was well tolerated and resulted in a complete remission. Both retinoids caused a marked change in the expression of cytokeratins. The most remarkable observation was the expression of cytokeratins 4, 13 and 19 at retinoid-treated areas. These cytokeratins are absent in adult normal or diseased epidermis and hence provide a cell-biological tool to substantiate a retinoid effect. PMID- 1715298 TI - Sonographic features of an umbilical cord abnormality combining a cord pseudocyst and a small omphalocele; a case report. AB - The perinatal findings of a pregnancy complicated by an umbilical cord abnormality associated with a small omphalocele are presented. The lesion was first observed at 18 weeks of gestation and was associated with elevated maternal serum alpha-fetoprotein. Serial ultrasound examinations showed major changes in the cord appearances and an associated small omphalocele was identified during the third trimester. Colour flow imaging showed no blood flow within the lesion and no obstruction nor involvement of the main umbilical vessels. Postnatal investigations demonstrated focal myxoid degeneration of the cord, a small omphalocele and no other fetal abnormalities. PMID- 1715299 TI - Expression of the muscle regulatory factor MRF4 during somite and skeletal myofiber development. AB - The muscle regulatory factors MRF4, myogenin, myf-5, and MyoD constitute a family of proteins that can function as muscle-specific transcriptional activators. Although this gene family has been extensively studied, a specific role for each factor during myogenesis remains to be determined. Understanding how these factors function requires a detailed analysis of their expression patterns during development. Toward this goal, we examined the temporal pattern of expression of MRF4 and the other factors in the rat myogenic cell line L6J1-C, in newborn rat primary muscle cell cultures and in fetal and postnatal rat limb muscle. Our results demonstrate that MyoD, myogenin, and myf-5 transcripts accumulate maximally at various stages of myoblast differentiation and decline to low expression levels in adult muscle tissue. In contrast, MRF4 transcript accumulation is restricted to cell cultures containing multinucleate myofibers, and its expression in vivo increases sharply during late fetal muscle development. This level of MRF4 expression is maintained in the adult which, together with decreased expression of the other three muscle regulatory factors, makes MRF4 the predominant factor in adult muscle. In situ hybridization of mouse embryo tissue sections indicates that MRF4 transcripts accumulate in the limb beginning 13.5 days post coitum, which is 2 days later than the initial appearance of myogenin and MyoD transcripts. Hybridization to earlier stages of development reveals, however, that MRF4 mRNA initially is present in the myotomal compartment of the somites, just after myogenin but 2 days prior to MyoD expression. Unlike myogenin and MyoD, MRF4 expression declines in the myotomes at the time that multinucleate axial muscles begin to form in this region, although during later development MRF4 is expressed in the myofibers of axial muscles at levels comparable to those in the limb. Differences in the expression patterns for MRF4, myogenin, myf-5 and MyoD between myotomal and other skeletal muscle development suggest that the relative timing of expression for each muscle regulatory factor may control the distinct phenotypes associated with myotomal myocytes and multinucleate myofibers. PMID- 1715300 TI - The initial expression and patterned appearance of tenascin in scutate scales is absent from the dermis of the scaleless (sc/sc) chicken. AB - Morphogenesis of the anterior metatarsal skin (scutate scale region), from 9.5 to 12 days of development, results in the formation of orderly patterned scale ridges. It is after the initial formation of the Definitive Scale Ridge that the characteristic outer and inner epidermal surfaces differentiate. The hard, plate like beta stratum, with its unique beta keratins, characterizes the epidermis of the outer surface, while the epidermis of the inner surface elaborates an alpha stratum. The anterior metatarsal region of the scaleless mutant does not undergo scale morphogenesis. Therefore, scale ridges do not form nor do the outer and inner epidermal surfaces with their characteristic beta and alpha strata. We have found that the extracellular matrix molecule, tenascin, first appears in the scutate scale dermis at 12 days of development when the scale ridge is established. Tenascin is found in the dermis only under the scale ridge and is not associated with the dermal-epidermal junction. Tenascin is not found in scaleless anterior metatarsal dermis at this time. As outgrowth of the Definitive Scale Ridge takes place, tenascin distribution correlates closely with the formation of the outer epidermal surface of each scale ridge. By 16 days of development tenascin is also found in close association with the dermal-epidermal junction. Tenascin does not appear in scaleless anterior metatarsal dermis until 16 days of development and then it is randomly and sparsely distributed at the dermal-epidermal junction. Tenascin's initial appearance and pattern of distribution in the scutate scale dermis and its abnormal expression in the scaleless dermis suggest that morphogenesis plays a significant role in regulation of its expression. PMID- 1715301 TI - Developmental change in calcium-activated chloride current during the differentiation of Xenopus spinal neurons in culture. AB - The duration and ionic dependence of action potentials change during the differentiation of embryonic amphibian spinal neurons both in vivo and in culture. The development of sodium, calcium, and potassium currents has been characterized in these cells and the shortening of the action potential has been shown to depend to a great extent on developmental changes of potassium currents. Previous evidence suggests that a chloride current may also be present in these embryonic neurons. Chloride currents were investigated with intracellular current clamp and single-electrode and whole-cell voltage-clamp techniques. Most neurons exhibited a calcium-activated chloride current (ICl(Ca] that contributed to the postdepolarization following the action potential recorded in the absence of sodium and potassium currents. This current appeared to decrease in density and its deactivation rate increased during the first day in culture. Its incidence also declined during this period. A much larger Ca(2+)-dependent Cl- current was also observed in a subset of neurons after 24 hr, but was absent at earlier stages of development. The results suggest the presence of two Cl- currents with different developmental fates. The early current probably contributes to the repolarization of long calcium-dependent action potentials at initial stages of neuronal development, when potassium currents are small, and may serve to reduce the extent of repetitive firing. PMID- 1715302 TI - Effects of osmotic pressure and somatostatin on the cAMP messenger system of the osmosensitive prolactin cell of a teleost fish, the tilapia (Oreochromis mossambicus). AB - Altered osmotic pressure and somatostatin (SRIF) rapidly alter prolactin (PRL) release from the pituitary gland of the euryhaline teleost, the tilapia. The present studies were undertaken to determine whether altered osmotic pressure and SRIF influence cAMP metabolism in a manner that is correlated with the pattern of PRL release observed previously. Although PRL release is stimulated within 10-20 min when medium osmotic pressure is reduced, cAMP metabolism was not altered. However, following 1 hr of incubation in the presence of IBMX, cAMP accumulation was higher in PRL tissue exposed to medium of reduced osmotic pressure. This suggests that cAMP does not initiate an increase in PRL release in response to reduced osmotic pressure. By contrast, SRIF reduced the forskolin-stimulated increase in cAMP levels in a manner consistent with its rapid effects on PRL release. Moreover, the ability of SRIF to suppress the forskolin-stimulated increase in cAMP levels suggests that SRIF may act to render adenylate cyclase less responsive to direct stimulation by forskolin. PMID- 1715303 TI - Construction and characterization of an Escherichia coli mutant with a deletion of the metZ gene encoding tRNA (f1Met). AB - The Escherichia coli metZ gene encoding tRNA (f1Met) was replaced by the chloramphenicol-resistance-encoding gene. The resulting mutant exhibited slightly lower growth rates as compared to the wild type at 37 degrees C or 42 degrees C, but grew apparently slower than the latter at 30 degrees C, indicating a slight cold sensitivity of growth. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or trpA'::lac' Z fusion gene, in the metZ deletion mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB' ::lac'Z fusion gene, whose start codons are GUG, were also synthesised in the deletion mutant. These results provide evidence that tRNA (f1Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA (f1Met), a minor N-formyl methionine-specific tRNA, in the tRNA (f1Met)-depleted cells. PMID- 1715304 TI - Improving elderly recall with bimodal presentation: a natural experiment of discharge planning. AB - This study's goal was to better explain memory and its decline in a natural setting to improve social work practice among the elderly. The effect that bimodal (verbal and visual) presentation of information has on delayed incidental recall was compared with unimodal (verbal only) presentation. A significant increase occurred in overall recall and verbatim recall. Gist recall improved, but not significantly. PMID- 1715305 TI - [Sanitary and hygienic conditions and characteristics of work of female electric coil winders]. AB - The article contains a hygienic assessment of the working conditions of female coil winders engaged in high-powered electric engines' assembling. Relationships were established between the unfavourable labour conditions, anthropometric inadequacy of the equipment used and the female workers' labour capacity, their functional states and morbidity. Corresponding preventive measures were proposed to lower the labour intensity of female electric coil winders. PMID- 1715306 TI - Keratin expression in the normal breast and in breast carcinoma. AB - The immunohistochemical reactivities of 69 cases of breast carcinoma were examined on methacarn-fixed, paraffin-embedded sections using eight different monoclonal antibodies which recognize one or a few keratin polypeptides. In the normal breast, the monoclonal antibodies RPN1162, RPN1165 and AE1 stained almost all the luminal cells but not the basal (myoepithelial) cells. The monoclonal antibodies 35BH11, M20, CK5 and CK8.12 stained only a subset of the luminal cells. In contrast, 312C8-1 stained basal cells but not luminal cells. All the tumour specimens reacted with AE1, while over 80% of them also reacted with 35BH11 (57/69), CK5 (57/69) and RPN1165 (55/69); 30% reacted with CK8.12 (21/69) and 16% with RPN1162 (11/69). Basal cell-specific keratin, as defined by 312C8-1, was detected in only 1% of cases (1/69). Monoclonal antibodies to different keratin polypeptides may be of use in the characterization and subdivision of breast cancer. PMID- 1715307 TI - Fragile X expression and X inactivation. II. The fragile site at Xq27.3 has a basic function in the pathogenesis of fragile X-linked mental retardation. AB - The major concept of fragile X pathogenesis postulates that the fragile site at band Xq27.3 [fra(X)] represents the primary defect. The expression of fra(X) is predicted to be an intrinsic property of the mutated chromosome and, hence, should not be suppressed by X inactivation in females or induced by X-linked trans-acting factors. We made fibroblast clones of a fra(X)-positive female. Monoclonality was demonstrated using the DNA methylation assay at DXS255. The mutated X chromosomes and their states of genetic activity in the different clones were also defined by molecular methods. Five clones were selected to induce expression of fra(X) by 10(-7) M FUdR; two carried an active mutated X chromosome, in the other three the mutated X chromosome was inactivated. Fra(X) was found expressed in both types of clones. The percentages of positive cells were as high as 7-10%, regardless of the genetic activity of the mutated X chromosomes. DNA replicating patterns, obtained by BUdR labelling, demonstrated that expression occurred only on the mutated X chromosomes previously identified by molecular methods. The concept that the fragile site represents the primary mutation is now strongly supported by experimental evidence. The expression of fra (X) in females is independent of X inactivation and other trans-acting factors. PMID- 1715308 TI - Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene. AB - Three mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were discovered in a pancreas-insufficient patient with cystic fibrosis (CF) who displayed an uncommon combination of almost normal chloride concentration in sweat tests and typical symptoms of gastrointestinal and pulmonary disease. The R553Q mutation was found on the maternal delta F508-CFTR gene. Codon 553 is located within a consensus motif of the ATP-binding cassette transport proteins at a less conserved position. Other members of this protein superfamily contain a glutamine instead of arginine at the homologous position, suggesting a modulating rather than disease-causing role of the R553Q mutation in CFTR. The amplification refractory mutation system did not detect the R553Q mutation in a further 65 normal, 113 delta F508, and 91 non-delta F508 CF chromosomes. The index case carried the R553X nonsense mutation on the paternal chromosome. The R553X mutation was present on a further 9 out of 86 German non-delta F508 CF chromosomes linked with the XV2c-KM19-Mp6d9-J44-GATT haplotypes 2-2-2-1-1 and 1-1 2-1-2. The location of R553X on separate haplotypes including both alleles of the intragenic GATT repeat suggests an ancient and/or multiple origins of the R553X mutations. The association of the genotype of the CFTR mutation and the clinical phenotype was assessed for the patients carrying the related genotypes delta F508/delta F508 (n = 80), delta F508/R553X (n = 9) and delta F508-R553Q/R553X (n = 1). In compound heterozygotes, the median chloride concentration in pilocarpine iontophoresis sweat tests was significantly lower than in the delta F508 homozygotes (P less than 0.01). The patient groups were significantly different with respect to the distributions of the centiles for height (P less than 0.001) and weight (P less than 0.01) as the most sensitive predictors of the course and prognosis in CF. Growth retardation was more pronounced in the compound heterozygotes. PMID- 1715309 TI - Discrimination between recurrent mutation and identity by descent: application to point mutations in exon 11 of the cystic fibrosis (CFTR) gene. AB - A total of 75 non-delta F508 chromosomes from 59 German cystic fibrosis patients was screened for mutations in exon 11 of the cystic fibrosis (CFTR) gene. These Caucasian patients were found to possess an identical haplotype background for two common mutations (G551D, R553X) consistent with their being identical by descent. However, a different R553X associated haplotype found in American black patients was suggestive of recurrent mutation, a postulate supported by the location of the R553X alteration in a hypermutable CpG dinucleotide. Likelihood estimates for recurrent mutation and identity by descent were compared and strongly supported the hypothesis of recurrent R553X mutation. The ability to distinguish between these two alternatives provides an indication of whether or not the search for mutations should be restricted to chromosomes with similar haplotypes. PMID- 1715310 TI - The fragile X mutation does not have any major effect on the expression of the hypoxanthine phosphoribosyltransferase (HPRT) locus in human fibroblasts. AB - The possible influence of the fragile X mutation at Xq27 on the expression of the neighbouring gene (at Xq26) for hypoxanthine phosphoribosyl transferase (HPRT) was studied by determination of the levels of HPRT-RNA and HPRT enzyme activity in fibroblast cell cultures from 7 fragile X patients. These levels were lower (although not statistically significantly lower) than in normal fibroblast cultures. Hence, these data do not support the notion of a major effect of the fragile X mutation on the expression of the HPRT gene. PMID- 1715311 TI - Molecular analysis of cystic fibrosis in the Hungarian population. AB - Hungarian cystic fibrosis (CF) families (n = 33) including 114 family members have been analysed for the presence of the delta F508 mutation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and have been haplotyped with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CFTR gene. The delta F508 deletion was present in 64% of CF chromosomes. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV-2c: allele 1, KM-19: allele 2), which accounts for 95% of delta F508 CF chromosomes in our families. PMID- 1715312 TI - The delta F508 mutation and RFLP-linked loci in Spanish cystic fibrosis families. PMID- 1715313 TI - Effect of glycosylation of bacterial amylase on stability and active site conformation. AB - With a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. Glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. Several group specific inhibitors exhibited dose dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. The pH optima of native and glycosylated enzyme remained the same indicating that the ionization at the active site is not greatly influenced as a result of glycosylation. Although the native as well as the glycosylated enzyme bind to the substrate with the same affinity, the rate of reaction differed greatly at 90 and 100 degrees C. At 70 degrees C, the rate of reaction was similar for the conjugated as well as the unconjugated amylase. Thermostability at different temperatures clearly showed that the glycosylated enzyme had greater stability compared to the native enzyme. The divalent cation binding site in the amylase also appears to be unaltered upon glycosylation since EDTA inhibited both enzymes to the same extent and addition of calcium ion restored the activity to almost the same level. These studies showed that conjugating the amylase enzyme with a bulky molecule like dextran does not affect the conformation at the active site. PMID- 1715314 TI - Studies on the effect of various parameters on crotalarin-fetuin interaction. AB - With a view to optimise the interaction of crotalarin, a blood group A-specific lectin from the seeds of Crotalaria striata with fetuin the effect of various parameters on the reaction has been studied turbidimetrically. The formation of crotalarin-fetuin complex was dependent on time, temperature, pH and the ionic strength of the medium. The maximum turbidity appeared in 30 min at 20 degrees C and the pH optimum was 3.5. The binding constant (Ka) for crotalarin-fetuin interaction was 5.58 x 10(4) M-1 (pH 3.5) at 20 degrees C. Among the different inorganic salts tested, the cations with increasing concentrations had pronounced effect on binding. KCNS and KI, however, were noninhibitory. The turbidity slightly increased in presence of different sodium salts, whereas periodate and urea reduced the interaction. The different alcohols had no remarkable effect on the above reaction. PMID- 1715315 TI - Activation of human B cells through the CD19 surface antigen results in homotypic adhesion by LFA-1-dependent and -independent mechanisms. AB - Addition of CD19 monoclonal antibodies (mAb) to highly purified tonsillar B cells resulted in homotypic adhesion and the formation of cell clusters. This response was completely blocked by antibody to LFA-1, indicating an LFA-1-dependent adhesion mechanism. In contrast, aggregate formation by B cells activated with phorbol myristate acetate (PMA) was only partially inhibited by anti-LFA-1 antibody, and those formed in response to PMA plus CD19 antibody were not inhibited at all, suggesting aggregation of activated B cells stimulated with CD19 antibody was LFA-1 independent. This was confirmed with B-cell lines. The pre-B-cell line Nalm-6 formed aggregates in response to CD19 antibody which were not inhibited with anti-LFA-1. In addition, CD19 antibody induced aggregate formation by an Epstein-Barr virus (EBV)-transformed B-cell line derived from an LFA-1-deficient donor. These results suggest that different adhesion molecules may operate at different stages of B-cell activation, and that CD19 may be important in cell-cell interactions involved in regulation of antibody responses. PMID- 1715316 TI - Biochemical and developmental characterization of the murine cluster of differentiation 1 antigen. AB - The cluster of differentiation-1 (CD1) antigens are major histocompatibility complex (MHC) class I-like glycoproteins belonging to the immunoglobulin supergene family. Initially described in humans, more recently putative CD1 encoding genes have been identified in several other species, including the mouse where it has been clearly demonstrated that CD1 mRNA is expressed. However, in the mouse both its unusually wide tissue distribution and the prevalence of incompletely spliced RNA have raised the possibility that the mRNA did not encode a functional protein. We have utilized a rabbit polyclonal antiserum raised against an Escherichia coli-expressed recombinant murine CD1 fusion protein to characterize the murine CD1 protein. Here we demonstrate that the antiserum binds specifically to a set of glycoproteins (49,000-55,000 MW) which contain a common core protein with both a size (36,000 MW) and tissue distribution in accordance with those predicted. During thymic ontogeny, this protein is highly expressed by Day 14 of embryonic development and persists into adulthood, while its pattern of expression in other organs changes significantly during development. Thus, the mouse provides an amenable model system for the study of CD1 function. PMID- 1715317 TI - Comparison of the effects of FK-506, cyclosporin A and rapamycin on IL-2 production. AB - The immunosuppressive compounds FK-506, cyclosporin A (CsA) and rapamycin inhibit both the human and mouse mixed lymphocyte reactions (MLR) with IC50s of 2-5 x 10( 10) M for FK-506 and rapamycin and 10(-8) M for CsA. FK-506 and CsA were also potent inhibitors of A23187/PMA-stimulated IL-2 production by Jurkat and HuT-78 cells but had no effect on the response of mouse CTLL cells to IL-2. IC50 values for inhibition of IL-2 production closely matched those for inhibition of the MLR and both drugs were active only during the first 4-6 hr following stimulation. In contrast, rapamycin was a poor inhibitor of IL-2 production, although it inhibited cellular responses to IL-2. The IC50 values for these two activities indicated that neither alone accounted for rapamycin inhibition of the MLR. FK 506 and CsA affected IL-2 gene transcription in Jurkat cells by the same mechanism. Both inhibited the appearance of the transcription factor, NFAT, whereas rapamycin did not. The appearance of another transcription factor, NFK beta, was unaffected by all three drugs. The effects of FK-506 and CsA on IL-2 gene expression, therefore, are similar even though the two drugs act through distinct cytosolic receptors. PMID- 1715318 TI - Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription. AB - Attacins are antibacterial proteins synthesized by pupae of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. In this report we show that the previously described, attacin-induced alteration in the structure and the permeability of the outer membrane of Escherichia coli is associated with a specific inhibition of the synthesis of several outer membrane proteins, including OmpC, OmpF, OmpA, and LamB. The inhibition is expressed as a reduction in the steady-state mRNA levels and is at least in part the results of a block in transcription of the corresponding genes. Transcription directed by the promoter of ompR, the positive regulator of ompC and ompF expression in response to environmental conditions, is also affected by attacin. The effects on mutant strains show that the primary activity of attacin is not mediated by the ompR envZ regulatory system. Instead our data suggest the existence in E. coli of a previously unknown system for the transcriptional regulation of a large set of outer membrane proteins previously not known to be coordinately regulated. We propose that the activity of attacin is directed towards this system. PMID- 1715319 TI - Capsulelike surface material of Mycoplasma dispar induced by in vitro growth in culture with bovine cells is antigenically related to similar structures expressed in vivo. AB - Electron microscopy has been used to show that Mycoplasma dispar produces an external capsulelike material in vivo that has an affinity for both ruthenium red and polycationic ferritin. This extracellular material is lost upon passage in culture medium but can be regained with a single passage on bovine lung fibroblast (BLF) cells. To confirm that the extracellular material associated with cell-grown mycoplasmas was the same as that observed in infected calves, rabbit antibodies were produced to purified capsulelike material isolated by protease digestion of cell-grown organisms. These antibodies bound to capsulelike material on the surface of M. dispar cells colonizing the bronchial epithelium of infected calves and to capsulelike material from cell-grown mycoplasmas. Calves infected with M. dispar produced antibodies in lung secretions that were capable of binding to the purified capsulelike material. The Fab fragments of rabbit antibodies to in vitro-produced capsulelike material could block this binding, indicating that the capsulelike material was similar in both in vivo-grown and cell-grown organisms. The carbohydrate nature of the capsular material suggested by the ruthenium red and polycationic staining characteristics was confirmed by its binding to Ricinus communis agglutinin, a galactose-specific lectin. These studies confirm that capsule material produced during infections with M. dispar share antigenic determinants with the material produced under in vitro conditions and that association with mammalian cells induces production of this material. PMID- 1715320 TI - Antigenic cross-reactivity and functional inhibition by antibodies to Clostridium difficile toxin A, Streptococcus mutans glucan-binding protein, and a synthetic peptide. AB - A 10-amino-acid repeating sequence of the hemagglutinating portion of Clostridium difficile toxin A has been synthesized and used to produce antisera in rabbits. Antipeptide antibody inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. Immunoblot analysis with the antipeptide antibody revealed cross-reactivity with native toxin, a recombinant protein containing the toxin A repeats, and a glucan-binding protein from Streptococcus mutans whose primary structure has repeating amino acid motifs similar to those of the synthetic peptide. A polyclonal antibody against the glucan-binding protein, which cross reacted with purified toxin A, also inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. We recently identified toxin A and the glucan binding protein as members of a novel family of clostridial and streptococcal binding proteins based on conserved repeating amino acid motifs at the C-terminal region of the molecules. This study provides immunological and functional evidence of the predicted relationship between toxin A and the glucan-binding protein and further implicates the repeating subunits as ligand-binding domains in this family of proteins. PMID- 1715321 TI - Development and characterization of a monoclonal antibody specific for Listeria monocytogenes and Listeria innocua. AB - BALB/c mice were immunized with crude cell surface proteins of Listeria monocytogenes V7. Approximately 1,680 hybridomas were generated after two fusions, and the monoclone C11E9 was selected and used for further characterization. The monoclonal antibody (MAb) produced by C11E9 was immunoglobulin subclass G2b with kappa light chains. Dot and colony blot results indicated that MAb C11E9 was reactive to all the L. monocytogenes (34 of 34) and Listeria innocua (6 of 6) isolates without any cross-reaction to other organisms tested. Western blot (immunoblot) analysis of crude cell surface proteins in native polyacrylamide gel electrophoresis (PAGE) indicated that MAb C11E9 reacts with a single band in each species, with a molecular mass of 174 kDa for L. monocytogenes and 182 kDa for L. innocua. The MAb reacted with one major protein band in Western blot from acid-urea PAGE for both L. monocytogenes and L. innocua. Isoelectric focusing results indicated two immunoreactive protein bands with pIs of 8.1 and 7.4 for L. monocytogenes. Sodium dodecyl sulfate (SDS)-PAGE and Western blot analysis indicated several proteins with molecular masses of 76, 66, 56, and 52 kDa for L. monocytogenes and 66, 56, and 52 kDa for L. innocua. Reaction of MAb C11E9 to washed live cells indicated the possible binding of antibody to cell surface antigen. These cell surface antigens could be removed by 1 N HCl plus 9 M urea, 2% SDS-0.5% beta-mercaptoethanol, or 4 M guanidine-HCl. The epitope of MAb C11E9 binding site was shown to be protein in nature. Periodic acid-Schiff staining and glycoprotein immunoassay indicated that carbohydrate was absent in the epitope. The cellular locations of the MAb C11E9-reactive antigens were calculated to be 76 and 90% outside and 24 and 10% inside the cell membranes of L. monocytogenes and L. innocua, respectively, for 12- to 14-h cultures. PMID- 1715322 TI - The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene. AB - Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae. PMID- 1715323 TI - Induction of protective immunity by using Anaplasma marginale initial body membranes. AB - Anaplasma marginale initial bodies of the Norton Zimbabwe strain were disrupted and separated into two membrane fractions banding at 1.15 and 1.22 g/cm3 by sucrose density centrifugation. The membrane fractions differed in their morphology and polypeptide composition. Membranes banding at 1.22 g/cm3 shared epitopes with surface-exposed polypeptides of the Florida strain of A. marginale, confirming the outer membrane location of these polypeptides. Immunization of cattle with either membrane fraction induced protection against homologous challenge, as demonstrated by significantly less anemia and lower peak rickettsemia values compared with those of adjuvant-immunized and nonimmunized calves. Protection correlated with antibody titer to membrane polypeptides. Although both membrane fractions induced protection, a 31-kDa polypeptide was the only common antigen to both fractions, as shown by reactivity of immune sera. Identification of membrane antigens capable of inducing protective immunity should facilitate development of vaccines against anaplasmosis suitable for use in Zimbabwe. PMID- 1715324 TI - The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis. AB - A gene encoding the 33-kDa secreted protein of Mycobacterium tuberculosis (antigen 85-C) was isolated and sequenced. The corresponding DNA sequence contains a 1,020-bp coding region. The deduced amino acid sequence corresponds to a 340-residue protein consisting of a 46-amino-acid signal peptide and a 294 amino-acid mature protein. Comparison with previously described genes for the 30 kDa antigen (the alpha antigen of M. bovis BCG, also called antigen 85-B) and the 32-kDa antigens from M. bovis BCG and M. tuberculosis (antigens 85-A) indicates that the three genes share considerable sequence homology (70.8 to 77.5%) but may also code for distinctive epitopes. Strong differences among the three sequences are clearly visible upstream and downstream from the region coding for the mature proteins. The three genes have been detected in the genome of M. bovis BCG by Southern blot hybridization with three type-specific probes. Furthermore, hybridization of large DNA fragments (100 to 1,000 kbp) from M. tuberculosis separated by pulsed-field gel electrophoresis showed that the three genes coding for the antigen 85 complex are not clustered within the bacterial genome. PMID- 1715325 TI - Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope. AB - A genomic library of Pseudomonas aeruginosa DNA was screened with a monoclonal antibody (MAb 2528) specific for the P. aeruginosa 60-kDa heat shock protein. A positive clone, pAS-1, was isolated. The gene coding for P. aeruginosa chaperonin (hsp60) was localized to a 2-kb EcoRI fragment subcloned in pAS-2. A sequence analysis of pAS-2 and parts of pAS-1 identified two open reading frames that encoded proteins with calculated molecular masses of 10 and 57 kDa. In amino acid sequence comparison studies the sequences of these proteins, which were designated GroES and GroEL, exhibited up to 78% homology with known prokaryotic sequences of 10- and 60-kDa heat shock proteins (hsp10 and hsp60). In order to map the epitope recognized by MAb 2528, a series of GroEL nested carboxy-terminal deletion clones were tested with MAb 2528. We identified the clone with the shortest insertion that was still recognized by MAb 2528 and the clone with the largest insertion that was not recognized by MAb 2528. The 3' ends of the insertions were determined by sequencing and were found to delimit a region that encoded 25 amino acid residues. Synthetic oligonucleotides that coded for peptides possibly resembling the epitope within this region were ligated into expression vector pGEX-3X, and fusion proteins expressed by these clones were tested for reactivity with MAb 2528. By using this method we determined that the decapeptide QADIEARVLQ (positions 339 to 348 on GroEL) was responsible for the binding of P. aeruginosa-specific MAb 2528. PMID- 1715326 TI - Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting. AB - The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle. PMID- 1715327 TI - Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses. AB - Filamentous hemagglutinin (FHA), a 220-kDa protein that mediates the adhesion of Bordetella pertussis to eukaryotic cells, is a component of acellular vaccines against whooping cough. To identify the subregions of FHA that are immunogenic for T cells, 16 human T-cell clones were raised against purified FHA and tested for the recognition of recombinant and proteolytic fragments. The clones were found to map either in the carboxy-terminal or the amino-terminal part of the FHA molecule, but none of them recognized the central region, which contains a sequence that is homologous to that of the eukaryotic protein fibronectin. These data suggest that subregions of FHA that do not contain sequences that are potentially cross-reactive with self proteins may be sufficient to induce an immune response against the whole protein. PMID- 1715328 TI - Epitope mapping of the d flagellar antigen of Salmonella muenchen. AB - Contiguous exposed antibody-binding regions of the antigen d flagellin of Salmonella muenchen were identified by using octameric peptides synthesized on polyethylene pins. Identification was confirmed by the serological activity of immunoglobulins recovered from specified pin peptides. Peptides equivalent to four regions of the d flagellin reacted with three different sera tested. PMID- 1715329 TI - Strain variation of Babesia bovis merozoite surface-exposed epitopes. AB - Babesia bovis merozoites are exposed to antibodies during the extraerythrocytic phase, and surface polypeptides bearing exposed epitopes are possible immunogens. Monoclonal antibodies reactive with the merozoite surface bind either immunodominant epitopes expressed diffusely on the merozoite surface or, alternatively, epitopes expressed in a polar pattern. Epitopes expressed diffusely on the immunodominant 42- and 44-kDa merozoite polypeptides were not conserved among strains from geographically diverse regions. In contrast, epitopes expressed in a polar pattern on the merozoite surface were conserved among nine strains and clones. Identification of variables and conserved epitopes provides a basis for defining antigenic variation and cross-protective immunity. PMID- 1715330 TI - Comparison of neutralization of BPV-1 infection of C127 cells and bovine fetal skin xenografts. AB - BPV-1 induces focus formation in murine C127 cells and fibropapillomas in bovine fetal skin xenografts. In this study, we compared the specificity of neutralization of BPV-1 in both assay systems, using sera and monoclonal antibodies (MAbs) selected to define neutralizing epitopes. Sera from rabbits and cattle, inoculated with intact BPV-1 or PBV-2 virions, neutralize BPV-1 infectivity in both C127 cells and xenografts. Selected human sera and murine MAbs that react with intact BPV-1 particles, serum of a rabbit immunized with denatured BPV-1 particles, and sera from calves vaccinated with a recombinant LI fusion protein did not neutralize BPV-1 infection in either system. It was concluded that neutralization of BPV-1 infection of C127 cells and bovine fetal skin xenografts by hyperimmune sera is specific and concordant for both assay systems, and involves conformational BPV-1 capsid epitopes. PMID- 1715331 TI - Production and characterization of two immunotoxins specific for M5b ANLL leukaemia. AB - We describe the production and functional characterization of 2 monocytic-cell lineage-specific immunotoxins constructed with saporin emitoxin (SAP) from Saponaria officinalis. Interest in the production of these immunotoxins, of possible clinical relevance, has been raised by the availability of 2 MAbs of high specificity for circulating monocytes and M5b ANLL, thus envisaging their potential use in bone-marrow purging. SAP emitoxin was selected on the basis of the low cytotoxicity in unconjugated form, as opposed to highly specific cytotoxicity and favourable pharmacokinetical properties in the conjugated form. SPDP conjugation produced immunotoxins which retained serological specificity and protein-synthesis-inhibitory activity. The 2 immunotoxins did not interfere with bone-marrow progenitor-cell growth in a CFU-GM colony assay. On the contrary, they were capable of killing monocytic cells selectively, as demonstrated in phenotypical and functional assays. Thus these 2 novel immunotoxins appear to be promising reagents in purging autologous bone marrow prior to transplantation in patients suffering from monocytic leukaemia. PMID- 1715332 TI - Induction of airway hyperresponsiveness in allergic rats. AB - Brown-Norway rats (male) were sensitized with both dinitrophenylated-bovine serum albumin (DNP-BSA) and Bordetella pertussis simultaneously in order to induce airway hyperresponsiveness (AHR) as the first sensitization. At five days, DNP BSA was inhaled as a booster into the airways under thiopental anaesthesia. At eight days, inhalation of antigen markedly increased the tracheal pressure (TP) in sensitized rats (11.9 +/- 1.6 cmH2O) and slightly increased TP in non sensitized rats (1.1 +/- 0.4), the difference between the two groups being significant (p less than 0.001). Twenty-four hours after antigen challenge, the airway responsiveness to ACh in sensitized rats was markedly increased to about 4 fold as compared to that in non-sensitized rats. Inhalation of dinitrophenylated ovalbumin failed to increase the airway responsiveness to ACh in rats sensitized with DNP-BSA, although a marked increase in TP was induced immediately after antigen challenge. We thus succeeded in preparing a model of AHR by employing a new procedure of sensitization. PMID- 1715333 TI - Systemic associations of retinal vasculitis. PMID- 1715334 TI - [Surgical therapy of colorectal cancer]. PMID- 1715335 TI - Modulation of granuloma formation in vitro by endogenous mediators. AB - We have reported previously that in vitro granulomas are inducible by culturing murine spleen cells in the presence of artificial microparticles, dextran beads, and that macrophages and macrophage-derived cytokines (monokines) including interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) play a critical role in the initiation of bead-induced granulomas in vitro. To investigate regulatory mechanisms of granuloma formation, we examined the modulatory effects of various mediators such as IL-1, TNF-alpha, interferon-gamma (IFN-gamma), IL-4, IL-6, transforming growth factor-beta (TGF-beta), dexamethasone and prostaglandin E2 (PGE2) on the development of lesions, because these mediators are known to play a pivotal role in inflammatory responses. The lesions were suppressed by the addition of dexamethasone, PGE2 or certain T cell-derived lymphokines such as IL 4 and IFN-gamma. These results suggest that suppressive signals are different from granulomatogenic cytokines including IL-1 and TNF-alpha and that granulomas are regulated by multi-factor dependent mechanisms. PMID- 1715336 TI - Germinal center T cells are distinct helper-inducer T cells. AB - Germinal centers (GCs) contain a significant number of CD4+ T cells, but what role these T cells may play in the development of GC B cells has not been determined. To gain insight into their role, we studied the phenotype of GC T cells and the lymphokines secreted by GC T cells isolated from human tonsils obtained after tonsillectomies. In addition to confirming that a large fraction of GC T cells are Leu-7(CD57)+ and Leu-8-, we found that they have no binding sites for peanut agglutinin. Furthermore, we found that they are CD45RA- and CD45R0+, the phenotype of helper-inducer T cells. We also found that Leu-7(CD57)+ cells display CD69, a phenotypic marker of very early cell activation, but do not display three other markers of cell activation: CD25 [interleukin-2 (IL-2) receptor], CD71 (transferrin receptor), and DR. When isolated, Leu-7(CD57)+ cells were stimulated in vitro with a mitogen that can induce peripheral blood T cells with the helper-inducer phenotype to produce various cytokines, Leu-7(CD57)+ cells did not produce IL-2, interleukin-4 (IL-4), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) in significant amounts. Taken together, GC T cells from a distinct subpopulation of T cells with helper-inducer phenotype by their histologic location, by their surface phenotype, and by their ability to produce lymphokines. This finding is consistent with the possibility that GC T cells have been selectively recruited to actively help B cells develop in GCs. PMID- 1715337 TI - In vitro anti-human immunodeficiency virus type 1 activity of biliverdin, a bile pigment. AB - Biliverdin (BV) is a bile pigment having anti-allergic properties. We examined the effect of BV on human immunodeficiency virus type 1 (HIV-1) in vitro. BV completely inhibited the cytopathic effect of HIV-1 in MT-4 cells at concentrations of 22.2 micrograms/ml or more. This inhibitory effect was also observed when BV was present during the adsorption period of HIV-1. However, BV was cytotoxic to MT-4 cells at concentrations above 800 micrograms/ml. At a concentration of 66.7 micrograms/ml, BV completely inhibited syncytia formation by HIV-infected and uninfected MOLT-4 cells. Moreover, after exposure of HIV-1 particles to BV for 2 h, the infectivity of the virus was reduced in a dose dependent manner. It is speculated that the anti-HIV activity of BV is due to direct inactivation of virions and inhibition of virus binding to target cells. PMID- 1715338 TI - Relation between nucleolar size and growth characteristics in small cell lung cancer cell lines. AB - Relationships among cytological features, doubling time, S-phase percentage, expression of myc-family oncogenes, DNA ploidy and biochemical properties were studied in thirteen small cell lung cancer cell lines. Six cell lines that grew slowly (average doubling time 99 h) and had lower S-phase percentages (average 32%) showed inconspicuous nucleoli (average area of 1.5 microns 2), and the remaining seven cell lines that grew quickly (average doubling time 45 h) and had higher S-phase percentages (average 44%) showed large and prominent nucleoli (average area of 6.1 microns 2). DNA index value obtained from flow cytometric DNA histograms showed that all cell lines except for H-69 cell line displayed aneuploidy. Ribbon-like cell arrangements were observed in the 7 cell lines that grew quickly, and in 1 cell line that grew slowly. Biochemically, six slow growing cell lines and four fast-growing cell lines showed high levels of aromatic L-amino acid decarboxylase activity, while in the remaining three fast growing cell lines its level was low. A high level of c-myc or N-myc oncogene expression was observed in all 7 cell lines that grew quickly, but not in any of the 6 cell lines that grew slowly. It appears that small cell lung cancer cell lines that grow quickly can be expected to have large nucleoli and ribbon-like cell arrangements and to express high levels of myc-family oncogenes, and that nucleolar size is a good indicator for growth characteristics. PMID- 1715339 TI - A new CA125-like antigen (CA602) recognized by two monoclonal antibodies against a newly established ovarian clear cell carcinoma cell line (RMG-II). AB - A cell line designated RMG-II was established from the ascites of a patient with ovarian clear cell carcinoma. The chromosomal analysis revealed aneuploidy with a hypertetraploid modal number and 8 marker chromosomes. Radioimmunoassay and immunocytochemical staining showed that RMG-II cells produced some tumor markers such as CA125 and TPA. Two monoclonal antibodies, designated MA602-1 and MA602-6, were generated by immunization of mice with an extract prepared from the culture supernatant of RMG-II cells. The epitopes recognized by these two monoclonal antibodies were proved to differ from the CA125 epitope, but to exist on the molecule bearing CA125. We developed a double-determinant sandwich enzyme immunoassay using these two monoclonal antibodies, and the antigen defined by this assay was termed CA602. CA602 was frequently found in the sera of ovarian cancer patients; the positive rates were 92%, 38%, 60%, and 80% for serous, mucinous, clear cell, and endometrioid ovarian carcinomas, respectively, when the cut-off value was set at 60 U/ml (= mean + 3SD of healthy females). CA602 levels in serum were also high in endometriosis patients and in early pregnancy, as is the case for CA125, and the correlation coefficient between CA602 and CA125 was high (r = 0.88). Our preliminary evidence suggests that this CA602 assay system has higher sensitivity than the CA125 one. PMID- 1715340 TI - Detection of bone-type alkaline phosphatase by monoclonal antibodies reacting with human osteosarcoma-associated antigen. AB - The antigen detected by monoclonal antibodies reacting with human osteosarcoma associated antigen was shown to be a phosphatidyl-inositol (PI)-glycan-anchored protein, which can be released from the cell surface by PI-specific phospholipase C-treatment. The antigen detected by 2D3 and 2H10 antibodies exhibited alkaline phosphatase activity. Both antibodies strongly reacted with bone-type alkaline phosphatase. However, importantly, immunohistochemical analysis demonstrated that 2D3 and 2H10 did not react with alkaline phosphatase present in kidney or liver. In addition, neither placental nor intestinal alkaline phosphatase was recognized by 2D3 and 2H10 antibodies. These results indicated that two monoclonal antibodies, 2D3 and 2H10, are highly specific for bone-type alkaline phosphatase and can distinguish bone alkaline phosphatase from liver alkaline phosphatase in spite of the fact that liver and bone alkaline phosphatase are encoded by the same gene. PMID- 1715341 TI - Carbohydrates associated with the cell coat surrounding cells of the rainbow trout saccular macula as revealed by lectin probes. AB - The luminal surface of the saccular macula in the rainbow trout is covered with a glycoconjugate-rich cell coat. The aim of this study was to identify specific carbohydrate moieties present in this coat, using biotinylated lectins as probes. Saccular tissues were fixed in Karnovsky's fixative for 2 h at 1-2 degrees C, followed by incubation with biotinylated lectins for 12-16 h at 25 degrees C. Lectin binding was visualized by performing avidin-biotin-peroxidase reactions. As controls, specimens were reacted with solutions of lectins preincubated with their specific inhibitory sugars. Staining was observed that was consistent with the presence of glucose, galactose, fucose, mannose, N-acetylglucosamine, N acetylneuraminic acid, and N-acetylgalactosamine in the cell coat. The variability in the intensity of staining associated with the lectin-carbohydrate complexes suggests quantitative differences among the various carbohydrate moieties detected. The presence of these carbohydrates in the cell coat of the trout saccular macula also suggests biochemical similarities between cell coats in teleost and mammalian inner ear structures. PMID- 1715342 TI - Enhancement of keratin synthesis induced by lipokeratinogenoside, N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose, in association with alteration of the intracellular Ca(2+)-content and protein kinase in cultured keratinocytes (FRSK). AB - Lipokeratinogenoside [N-(O-linoleoyl)-omega-hydroxy fatty acyl sphingosyl beta glucose] is one of the epidermosides which were found to be glycosphingolipids characteristic of the epidermis of mammalian skin. On the addition of lipokeratinogenoside to cultured rat keratinocytes (FRSK), the amount of keratin in the cells increased, 48 and 144 h after cultivation, to 1.4 to 1.8 times higher than that without the addition of lipokeratinogenoside, and the number of cornified envelopes also significantly increased on cultivation of the cells with lipokeratinogenoside. Immunohistochemical staining with anti-keratin antibody revealed that the cells cultivated with lipokeratinogenoside were densely covered with keratin in distinct contrast to the control cells. The same enhanced syntheses of keratin and cornified envelopes were observed on cultivation in the presence of TPA, which has been shown to elevate the intracellular Ca(2+)-content and to translocate cytoplasmic protein kinase C to the plasma membrane in the initial stage of transmembrane signalling. Similarly, lipokeratinogenoside showed the ability to increase the intracellular Ca(2+)-content to the same extent as TPA did and to translocate protein kinase C to the membrane fraction. However, the above activities of lipokeratinogenoside decreased with removal of the linoleic acid moiety from lipokeratinogenoside with mild alkali, but linoleic acid alone did not show any activities, indicating that the lipokeratinogenoside molecule itself is required for expression of the activities. PMID- 1715343 TI - Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115. AB - An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)-like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC-3 cells in an autocrine mechanism. Since the androgen-induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC-3 cells. Heparin was found to exhibit significant inhibition of testosterone-induced growth in a concentration-dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 micrograms/ml. DNA synthesis of SC-3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC-3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen-induced growth factor in the conditioned medium from testosterone-treated cells. These results indicate that heparin inhibits autocrine loop in SC-3 cells induced by androgen. Thus, the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan. PMID- 1715344 TI - Secretion and biological actions of insulin-like growth factor binding proteins in two human tumor-derived cell lines in vitro. AB - The insulin-like growth factors (IGFs) I and II are present in extracellular fluids associated with specific binding proteins (IGFBPs) that can modify their biologic actions. These studies were undertaken to determine which forms of IGFBP are secreted by endometrial carcinoma (HEC-1B) and breast carcinoma (MDA-231) cells, to characterize variables that control IGFBP secretion, and to study the effect of IGFBP-1 and IGFBP-2 on IGF-I stimulated cell proliferation. Secreted IGFBPs were identified by ligand blotting and IGFBP-1 was quantified using a specific radioimmunoassay (RIA). MDA-231 cell conditioned media (CM) contained four (43,000, 39,000, 30,000 and 24,000 Mr) forms of IGFBP, and HEC-1B cell CM contained three forms (39,000, 34,000 and 30,000 Mr). Immunoblotting showed that the 30,000 Mr form secreted by both cell types was IGFBP-1. Likewise the 34,000 Mr band in HEC-1B media reacted with IGFBP-2 antiserum and the 39,000 and 43,000 Mr bands reacted with IGFBP-3 antiserum. IGF-I stimulated the secretion of IGFBP 3 from both cell types and IGFBP-2 from HEC-1B cells but either decreased or caused no change in secretion of IGFBP-1 and a 24,000 Mr form. In contrast, insulin inhibited the secretion of IGFBP-1 but increased the secretion of the 24,000 Mr form. Compounds that elevate intracellular cAMP levels increased the secretion of IGFBP-3, IGFBP-1, and the 24,000 Mr form from both MDA-231 and HEC 1B cells. When sparse cultures of MDA-231 cells were used, addition of IGF-I caused a 24% increase in cell number after 48 hr. This mitogenic response was enhanced by the presence of recombinant human IGFBP-1 (45% increase in cell number, P less than 0.001). Bovine IGFBP-2 did not potentiate IGF-I stimulated cell proliferation. These findings show that two tumor cell lines secrete distinct forms of IGFBPs and that there is differential regulation of IGFBP secretion. At least one form secreted by both tumors may act as a positive autocrine modulator of IGF-I's growth stimulating actions. PMID- 1715345 TI - Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin-like growth factor-1 and transforming growth factor-beta, and analysis of fibroblast growth factors in rat kidney. AB - Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose-dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot replace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin-like growth factor 1 (IGF-1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2-1.0 microgram/ml) of insulin required for cell growth can be replaced by a low concentration of IGF-1 (10-20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF-1 receptor. Transforming growth factor-beta 1 (TGF-beta 1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration-dependent manner. Northern blot analysis shows that mRNA for TGF-beta 1, IGF-1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF-like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF-beta, FGFs may also be important kidney-derived regulators of proximal tubule epithelial cell growth in vivo and in vitro. PMID- 1715346 TI - Allergenic fragments in Parietaria judaica pollen extract. AB - High-performance ion-exchange chromatography and immunoaffinity chromatography suggest that Par jI, the principal allergenic component of Parietaria judaica pollen, is a very unstable molecule, which tends to fragment in solution. Several fragments were obtained from Par jI and some of them show positivity toward the anti-Par jI monoclonal antibody, suggesting that they retain the entire structure of the allergenic determinant. These fragments could be the target for sequence and conformation studies. PMID- 1715347 TI - Partitioning of cells in dextran-poly(ethylene glycol) aqueous phase systems. A study of settling time, vessel geometry and sedimentation effects on the efficiency of separation. AB - The effect of prolonged settling times (up to 2 h), in high- and low-phase columns, on the cell partition ratios measured and on the separability of cell populations was examined. With closely related cell populations, modelled by rat erythrocytes in which subpopulations of red blood cells of distinct age were labeled isotopically, it was found that partitioning proceeds over the entire time period examined as evidenced by the continuous change in relative specific activity of cells in the top phase as the partition ratio falls. In control cell sedimentation experiments in top phase there was almost no change in the quantity of cells present when vertical settling (i.e., high-phase columns) was used and no separation of specific subpopulations was found. In the horizontal settling mode the initially higher cell partition ratio, as compared to vertical settling, decreased to a greater extent with longer time intervals; a given purity of cells only being obtained at a lower partition ratio than in the vertical settling mode. Cell sedimentation in top phase was appreciable with time in the horizontal settling mode but did not result in a separation of cell subpopulations. The effect of relative cell partition ratios and sizes in high- and low-phase columns on the efficiency of separation was examined by use of rat or sheep 51Cr-labeled red cells mixed with an excess of human unlabeled erythrocytes. Rat and sheep red cells are appreciably smaller than human erythrocytes. Rat red cells have higher, and sheep red cells lower partition ratios than human erythrocytes. With vertical settling, over a 2-h period, there is no appreciable contribution to the change in relative specific activities by cell sedimentation. However, the more rapid sedimentation of the larger human red cells has, with time, a measurable effect on the relative specific activities obtained during cell partitioning when run in the horizontal mode: enhancing the rat-human and diminishing the sheep-human cell separations. Partitioning cells in high-phase columns is of advantage with respect to increasing separation efficiency and virtually eliminating the influence of other physical parameters (e.g., cell size). Since the cell partitioning process continues for long periods of time, yielding ever-lower partition ratios with increasing proportions of cells with higher P values, a time may be selected which balances desired relative cell purity and yield. PMID- 1715348 TI - [2'-5' oligoadenylate synthetase activity in patients with acute viral hepatitis and chronic type B hepatitis, and its clinical significance]. AB - The levels of 2'-5' oligoadenylate synthetase activity (2-5AS) were measured in the lymphocytes and sera of 32 patients with acute viral hepatitis and 20 patients with HBeAg positive chronic type B hepatitis, and the relationship between the effectiveness of interferon (IFN) therapy and 2-5AS reactivity was then studied. The lymphocyte 2-5AS (L.2-5AS) and the serum 2-5AS (S.2-5AS) levels in patients with any type of acute viral hepatitis at the acute stage were significantly higher than in the healthy subjects. The 2-5AS level in cases of acute type A viral hepatitis was significantly higher than in type B and type non A non B hepatitis. However, no significant difference in the 2-5AS levels between type B hepatitis and type non A non B hepatitis was observed. After recovery, the L.2-5AS and S.2-5AS levels decreased in patients with any type of acute viral hepatitis. No significant difference between the L.2-5AS level in healthy subjects and patients with chronic type B hepatitis was observed. The S.2-5AS level was significantly higher in patients with chronic type B hepatitis than in healthy subjects. During IFN administration, the L.2-5AS and S.2-5AS levels increased, with the maximum level and maximum rate of increase obtained on the 3rd day with respect to the L.2-5AS, and after one week with respect to the S.2 5AS. Of the 20 patients with chronic type B hepatitis with positive HBe antigen, 11 showed normalization of DNA-P, 4 disappearance of HBe antigen and 2 seroconversion of HBe antigen. In cases with a higher maximum rate of increase of the L.2-5AS, the effect of IFN therapy was observed to be greater. PMID- 1715349 TI - A new experimental animal model of portal hypertension. Intrahepatic portal obstruction by injecting DEAE-cross-linked dextran microspheres into the portal vein in the rabbit. AB - We proposed a new experimental animal model for portal hypertension in the female Japanese white rabbit by an intraportal injection of DEAE-cross-linked dextran microspheres (100 +/- 25 microns in diameter). Histology of the liver revealed portal obstruction by the injected microspheres in almost all portal triads, resulting in a foreign body granuloma. A sustained elevation of the portal pressure by a mean of 36.7% as compared with the basal value was observed for at least eight weeks in association with the portal-systemic collateral circulation demonstrated by portography, the radioisotope labeled microsphere method and histological examination of the esophagus and the liver. The elevation in portal pressure in the eighth week was associated with an increase in the portal blood flow determined at the main portal trunk. This was in accordance with the forward theory of the pathogenesis of portal hypertension. Since this model appears to show the two main conditions characteristic of portal hypertension persistent elevation of portal pressure and both extra- and intrahepatic portal collaterals, mimicking those in humans, portal obstruction by injecting DEAE-cross-linked dextran microspheres into the portal vein of the rabbit could provide a versatile model for portal hypertension. PMID- 1715350 TI - Antigenic analysis of influenza viruses isolated in Thailand in the rainy season, 1989. PMID- 1715351 TI - Comparison of in vitro techniques to the in situ technique for estimation of ruminal degradation of protein. AB - The accuracy with which the Streptomyces griseus, ficin, and neutral protease with amylase in vitro methods could be used to predict in situ protein degradation of concentrate feeds was evaluated. Four types of soy products and two types of distillers grains were incubated using each of the four techniques for .5, 1, 2, 4, 8, 12, 24, and 48 h. The percentages of initial CP that remained undegraded over time were determined. Comparison of the degradation curves and contrast analysis of the data indicated that the in vitro methods generated degradability estimates in conflict with those obtained by the in situ method. However, the neutral protease with amylase method ranked the test feeds according to the extent of CP degradation at 24 h most similar to that in situ. Regression equations developed with the neutral protease with amylase degradability estimates explained 78, 76, and 74% of the variation in the in situ estimates obtained after 12, 18, and 24 h of incubation, respectively. At least 69% of the variation in the 18-h in situ estimates could be explained by the neutral protease with amylase estimates obtained after 1, 2, 4, 8, and 12 h of incubation. Little relationship was found between the ficin and S. griseus versus in situ results. Although none of the in vitro methods resulted in degradation curves consistently related to those generated by the in situ technique, relationships were found between protein degradability estimates obtained by the neutral protease with amylase method at specific time points and those obtained by the in situ technique. PMID- 1715352 TI - Methotrexate in bronchial asthma. PMID- 1715353 TI - Autoradiographic quantitation of radiolabeled proteoglycans. AB - Radiolabeled proteoglycans or glycosaminoglycans were precipitated with the cationic dye safranin O onto a sheet of nitrocellulose filter using a dot-blot apparatus. An autoradiography film was exposed against the nitrocellulose sheet. The developed film and the nitrocellulose sheet were separately digitized in a flat-bed-gray-scale scanner connected to a microcomputer. An image analysis program of the microcomputer was used to quantify the density of the radioactivity dots produced in the film, and the intensity of the dye spots on nitrocellulose. With this procedure, a single sample containing the minimum of about 20 ng uronic acid and 5 dpm of incorporated 35SO4 was quantified for both total glycosaminoglycan content and radioactivity. Unincorporated 35SO4 and low molecular mass radioactivity (e.g. products of glycosaminoglycan degrading enzymes) did not interfere since they were quantitatively washed through the membrane before the assay. PMID- 1715354 TI - Comparison of single-dose antithrombotic agents in the prevention of microvascular thrombosis. AB - A model of thrombosis was used to evaluate the efficacy of single-dose heparin, hemodilution, and 40,000 milliwatts dextran in the prevention of microvascular thrombosis. Forty Lewis rats (275 gm body weight) were divided into four groups (n = 10): hemodilution (6 ml NS), single-dose heparin (1 mu/gm), 40,000 mw dextran (0.3 gm/100 gm), and control (0.275 ml NS). After exposure of the superficial femoral arteries, a model of arterial crush with arteriotomy followed by an intimal abrasion was used. The animals randomly received one of the four solutions above. Experimental results included patency rates of 90% at 20 minutes and 10% at 24 hours in the hemodilution group, 100% at 20 minutes, and 70% at 24 hours in the single-dose heparin group, and 100% at both 20 minutes and 24 hours in the dextran group. A 100% thrombosis rate was observed in the control group at 20 minutes and 24 hours. Single-dose heparin and dextran significantly improved patency in comparison to both the control and hemodilution groups at 24 hours (p less than 0.01). Scanning electron microscopy of the vessels revealed thrombus deposition consistent with these findings. These results indicate that single dose heparin and single-dose dextran reduce thrombosis in this model of microvascular injury. PMID- 1715355 TI - Molecular cloning of the cDNA encoding an immunodominant antigen of Dirofilaria immitis. AB - An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 kDa antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a lambda gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consisted of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34,400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species. PMID- 1715356 TI - Spermatocyte chromosome analysis of Helicella virgata (Pulmonata: Helicidae): silver-stained and C-banded chromosomes. AB - Chromosome numbers of the snail Helicella virgata from the fields of Castellammare del Golfo (Sicily) are n = 26 and 2n = 52. Silver-staining analyses of testicular cells suggest that both mitotic and meiotic chromosomes are involved in nucleolus organization. A within-individual variability in NOR banding pattern is present in each of the 20 specimens analyzed. PMID- 1715357 TI - Lead and immunity: II. Suppression of humoral immune response to Hymenolepis nana in mice. AB - Serum protein changes in mice treated for varying durations with lead nitrate and subsequently infected with 1000 H. nana eggs were compared with their counterpart controls, only treated and only infected groups. Decreased values of beta and gamma globulins in all the experimental groups along with higher worm recoveries indicate suppression of humoral immune response by lead in association with higher worm recoveries indicate suppression of humoral immune response by lead in association with the cellular components since these globulins are known to contain antibodies. Lead treatment for a duration of 45 days proved to be most effective in suppressing the immune response. PMID- 1715358 TI - The role played the lipid fractions of Mycobacterium bovis BCG in the development of antituberculosis immunity in an animal experiment. AB - The authors investigated the antituberculosis and antitumour immunogenicity as well as tuberculin allergenicity of the lipid fractions from Mycobacterium bovis BCG strains of Danish, French, Japanese origin and of Czechoslovak 725. The fractions explored included phospholipids, Cord factor, ethanol-extractable lipids, waxes A, B, C + D and fats. The fractions were divided into three groups according to their effectiveness. 1. The Cord factor and phospholipids from all the studied strains were effective in the antituberculosis and antitumour models with the only exception of strain 725 phospholipids. Phospholipids from all strains were capable of inducing tuberculin allergy. 2. In the second group (waxes A, C + D and lipids extractable by ethanol) a variance was observed in the antigenic properties of identical fractions from different strains suggesting differing metabolism in the strains producing these fractions. A mixture of waxes C + D from the French and Danish strains showed a degree of suppression in its antituberculosis effectiveness. 3. Waxes B and fats were entirely ineffective in the antitumour model and, with the exception of waxes B from strain 725 and fats from the Japanese strain, in the antituberculosis model. The antituberculosis and antitumour effectiveness directly depended on the content of a mycolic acid complex in fractions. Tuberculin allergenicity was associated with the intensity of phospholipid production by mycobacteria. PMID- 1715359 TI - The 1A4 molecule (CD27) is involved in T cell activation. AB - We developed a new mAb, anti-1A4, which recognizes an epitope on the CD27 molecule distinct from those recognized by several known anti-CD27 mAb. Although it has been suggested that the CD27 molecule is a T cell activation Ag, there was little direct evidence that the structure was involved in the T cell activation process. In this study, we showed that anti-1A4 inhibited anti-CD2, anti-CD3, mitogens, or soluble Ag-induced T cell proliferation as well as PWM-driven B cell IgG synthesis. Interestingly, anti-1A4 inhibited IL-2 secretion without affecting IL-2R expression. In addition, pretreatment of T cells with anti-1A4 inhibited the normally sustained intracellular calcium mobilization seen after triggering of T cells via the CD2 or CD3 pathways. Thus, binding of anti-1A4 to the CD27 molecule appears to induce a negative effect on T cell activation. This may be due to either a direct signal to T cells or the blocking of an interaction between T cells and accessory cells or both. These findings support the notion that the CD27 molecule plays an integral role in the process of T cell activation. PMID- 1715360 TI - The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor. AB - Susceptibility to experimental autoimmune myasthenia gravis (EAMG), which is induced in mice by injection of purified Torpedo nicotinic acetylcholine receptor (TAChR), is influenced by the I-A locus products, which restrict presentation of AChR Th epitopes. The bm12 mutation of the I-Ab molecule in the C57BL/6 strain, which is highly susceptible to EAMG, yields the EAMG resistant mutant B6.C-H 2bm12 (bm12). We investigated here the consequences of the bm 12 mutation on the CD4+ response to the TAChR alpha subunit. Upon immunization with TAChR, CD4+ cells became sensitized to TAChR and anti-AChR antibodies were produced in both bm12 and C57BL/6 strains. Overlapping synthetic peptides, corresponding to the complete sequence of TAChR alpha subunit, were used to identify Th epitopes. CD4+ cells from C57BL/6 mice recognized peptides T alpha 150-169, T alpha 181-200, and T alpha 360-378. CD4+ cells from bm12 mice did not respond to any synthetic sequence. Upon injection of the three C57BL/6 Th epitope peptides, either individually or as a pool, CD4+ cells from C57BL/6 mice recognized each peptide and TAChR. Therefore they recognized epitopes similar or identical to those originated from TAChR processing. CD4+ cells from bm12 mice injected with the same peptides responded to T alpha 360-378 strongly, to a lesser extent to T alpha 181-200, never to peptide T alpha 150-169. Only CD4+ cells sensitized against the T epitope peptide T alpha 181-200 responded to TAChR. We tested if lack of response to T alpha 150-169, and the low response to T alpha 181-200, was due to inability of the I-Abm12 molecule to present the T epitope peptides. bm12 and C57BL/6 APC were used to present the T epitope peptides to specifically sensitized CD4+ cells from C57BL/6 mice. All T epitope peptides were presented by bm12 APC, although T alpha 150-169 was presented less efficiently than by C57BL/6 APC. Resistance to EAMG induced by the bm12 mutation may be due to the change in the epitope repertoire of AChR-specific Th cells, and lack of recognition of otherwise immunodominant Th epitopes. For at least one epitope this might be due to absence of potentially reactive, specific CD4+ clones. PMID- 1715361 TI - HIV-1 gag-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes. Fine specificity of the gag-specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells. AB - CTL directed at the highly conserved HIV-1 gag protein have been described in HIV 1 seropositive persons and may be an important host defense against this retrovirus. Presently only limited data are available regarding the specific epitopes recognized by these CTL. In this study, we have performed a detailed examination of the gag-specific CTL response in three HIV-1 seropositive subjects, using both unstimulated PBMC and cloned CTL. Lysis of gag-expressing targets was found to be mediated by CD3+CD8+ lymphocytes and restricted by class I Ag. Multiple class I Ag were found to restrict gag epitopes in each subject studied, with as many as three of these Ag involved in presenting gag CTL epitopes in a single subject. The majority of gag-specific CTL activity was found to be directed against epitopes in the p24 subunit of the gag protein, with at least seven different HLA class I-restricted CTL p24 epitopes identified in these three subjects. Less CTL activity was directed against p17 subunit of gag and two CTL epitopes were identified in this protein. Although as many as four different epitopes in gag were recognized using CTL from a single subject, none of the epitopes was recognized by CTL from more than one subject. Analysis of gag epitope recognition using cloned CTL demonstrated heterogeneity and specificity not appreciated using unstimulated PBMC. The identification of multiple relatively conserved epitopes in the HIV-1 gag protein and the heterogeneity of CTL responses to this protein may have important implications for vaccine development and our understanding of AIDS pathogenesis. PMID- 1715362 TI - IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts. AB - IL-12, formerly known as cytotoxic lymphocyte maturation factor, is a cytokine that stimulates proliferation of PHA-activated human peripheral blood lymphoblasts and synergizes with low concentrations of IL-2 in the induction of lymphokine-activated killer cells. IL-12 is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. mAb prepared against a partially purified preparation of natural IL-12 have been characterized by 1) immunoprecipitation of 125I-labeled IL-12, 2) immunodepletion of IL-12 bioactivity, 3) Western blotting of IL-12, 4) inhibition of [125I]IL-12 binding to its cellular receptor, and 5) neutralization of IL-12 bioactivity. Twenty antibodies immunoprecipitate 125I-labeled IL-12 and immunodeplete IL-12 bioactivity as assessed in the T cell proliferation and lymphokine-activated killer cell induction assays. Western blot analysis demonstrated that each antibody binds to the 75-kDa heterodimer and to the 40-kDa subunit. An IL-12R binding assay identified 12 individual antibodies that inhibited the binding of [125I]IL-12 to its cellular receptor. Two inhibitory antibodies, 4A1 and 7B2, were tested in the neutralization assay and found to block IL-12 bioactivity whereas one noninhibitory antibody, 8E3, was shown not to neutralize IL-12 bioactivity. Antibodies 4A1 and 8E3 can simultaneously bind to the 75-kDa heterodimer demonstrating that inhibitory and noninhibitory epitopes are spatially distinct on the 40-kDa protein. The ability of antibodies specific for the 40-kDa subunit of IL-12 to block receptor binding of [125I]IL-12 and to neutralize IL-12 bioactivity suggests that localized determinants on the 40-kDa subunit may be necessary for binding to the IL-12 cellular receptor. PMID- 1715363 TI - Hydrocortisone and IL-4 induce IgE isotype switching in human B cells. AB - Induction of IgE synthesis in human B cells requires two signals. The first signal is delivered by the cytokine IL-4. The second signal activates B cells and is delivered by T cells, EBV infection, or engagement of the B cell-specific Ag CD40. Hydrocortisone (HC) has recently been shown to synergize with IL-4 to induce IgE synthesis in CD5+ chronic lymphocytic leukemia B cells. We show herein that a combination of HC and rIL-4 induces IgE synthesis in highly purified normal peripheral blood B cells. HC and IL-4 acted directly on B cells, because T cells and monocytes were not required for IgE synthesis. IgE induction was shown to occur in surface IgE- B cells isolated by cell sorting. These results suggest that IgE synthesis results from isotype switching, rather than from expansion of a precommitted B cell population. Furthermore, IgE synthesis was induced in sorted CD5- B cells, indicating that the ability to produce IgE in response to HC and IL-4 is not constrained by CD5 expression. Endogenous IL-6 was critical for induction of IgE synthesis by HC and IL-4, because an anti-IL-6 antibody strongly inhibited IgE production. These data suggest that hormones may play an important role in the regulation of IgE synthesis. PMID- 1715364 TI - Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules. AB - mAb against human glycosyl-phosphatidylinositol-linked leucocyte surface Ag CD59 and CD55 immunoprecipitated from detergent lysates of HPB ALL cell line in addition to the respective Ag a common 80-kDa glycoprotein component and (glyco)lipids. The 80-kDa glycoprotein is different from otherwise similar CD44 Ag. The CD59 immunoprecipitate contained also a small amount of the CD55 glycoprotein and the CD55 immunoprecipitate minute amount of the CD59 Ag. These results are interpreted in terms of existence of noncovalent complexes resistant to dissociation by mild detergents and consisting of the 80-kDa glycoprotein, CD59 and CD55 glycoproteins, relatively tightly bound (glyco)lipids and possibly other so far unidentified components. These complexes contain probably also other glycosyl-phosphatidylinositol-linked Ag, as an anti-CD48 mAb immunoprecipitated also an apparently very similar complex. The complexes immunoprecipitated by mAb against the CD55, CD59, and CD48 Ag also contain a protein kinase activity. This type of complexes could not be demonstrated in several other cell types such as RBC, PBMC, and HeLa cells. However, a qualitatively very similar set of components was immunoprecipitated from the murine thymoma EL-4 cell line by an anti-Thy-1 mAb. PMID- 1715365 TI - Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology. AB - As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells. PMID- 1715366 TI - Selection of the same major T cell determinants of influenza nucleoprotein after vaccination or exposure to infectious virus. AB - In the present study, we have compared the T cell antigenic determinants on nucleoprotein (NP) of influenza A/NT/60/68 virus recognized by BALB/c mice (H-2d) after vaccination using several different vehicles with the determinants recognized after exposure to infectious virus. Mice were immunized s.c. with: 1) purified recombinant NP with three different adjuvants--alum, saponin, or CFA; 2) whole inactivated A/Okuda virus in PBS or saponin; or 3) live attenuated Salmonella typhimurium AroA- vector expressing NP. A series of overlapping synthetic peptides that cover more than 90% of the amino acid sequence of NP were used to map the Th cell epitopes. The results showed that the same limited number of major epitopes were recognized after each of the different immunization regimes. Secondary in vivo boosting using the same vehicles as for the primary immunization did not increase the number of different T cell sites recognized. The T cell responses after intranasal infection with infectious A/NT/60/68 or A/PR/8/34 virus also showed a similar pattern of recognition of the major CD4 positive T cell epitopes. The only exception was that the region corresponding to residues 401-419 was only recognized after exposure to NP from A/NT/60/68 but not A/PR/8/34. This is probably because the two viruses differ in amino acid sequence at positions 408 and 411 within this part of the NP molecule. In contrast to the results observed with CD4-positive T cell epitopes, the major determinant recognized by CD8-positive T cells was only presented after live viral infection. The results in this study have important implications for vaccine design, inasmuch as they indicate that the same dominant CD4 T cell determinants on NP presented by vaccination with NP are also recognized by T cells from mice exposed to infectious virus. PMID- 1715367 TI - Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-alpha promoter in primary macrophages. AB - In macrophages, the TNF-alpha promoter is specifically induced by bacterial endotoxin, and provides a good model for gene regulation during bacterial infections. We have analyzed the protein-binding characteristics and enhancer activity of four kappa B-like enhancers and of a MHC class II-like Y box found in the mouse TNF-alpha promoter. In addition to members of the NF-kappa B/rel transcription factor family, at least two of the kappa B sites also bound a nuclear protein identified as NF-GMa, a factor that binds to promoter sequences from many cytokines. When inserted upstream of an enhancer-less promoter, two of the kappa B sites were active as LPS-inducible enhancers in primary macrophages, whereas the other two were not. Mutations in nucleotides known to contact nuclear factors severely reduced affinity of the kappa B sites for NF-kappa B. Introduction of the same mutations into a construct containing 1059 bp of the TNF alpha promoter coupled to a CAT reporter gene resulted in a stepwise reduction in inducibility by LPS; mutation of all four sites (11 bp of 1059) reduced inducibility by 90%, providing compelling evidence for the role of transcription factors belonging to the NF-kappa B/rel family in the activation of the TNF-alpha promoter. The TNF-alpha Y box bound an abundant nuclear factor, but had no detectable activity in our assays, either as an enhancer or as a mutation sensitive controlling element. PMID- 1715368 TI - Selection of hybrid hybridomas by flow cytometry using a new combination of fluorescent vital stains. AB - A new combination of fluorescent dyes (rhodamine 123 and hydroethidine) was used to internally label hybridoma fusion partners. Murine hybridoma 520C9 (recognizing human c-erbB-2) was labeled with hydroethidine. Murine hybridoma 3G8 (recognizing human Fc gamma receptor III) was labeled with rhodamine 123, and verapamil was used to block rhodamine efflux via P-glycoprotein. Viability assays showed little cytotoxicity from these dyes at the concentrations used. The labeled cells were fused with polyethylene glycol, sorted for dual fluorescence on an Epics V cell sorter, and cloned. Hybrid hybridomas producing bispecific antibodies were selected for ability to promote lysis of SK-Br-3 breast cancer cells by human mononuclear cells. Several positive clones were obtained and shown to have a double content of DNA. Bispecific antibody produced by subclone 2B1 was purified by anion exchange chromatography and shown to bind both tumor cells and Fc gamma R III bearing cells. Using two parameter flow cytometric analysis, we were able to measure a 'bridging' effect of this bispecific antibody, which caused formation of complexes between PMNs and SK-Br-3 cells. Either parental antibody could compete with bispecific antibody to block such complexing. This fusion method provides several advantages over other techniques presently used (speed, convenience, low toxicity and automatic exclusion of dead cells) and can be applied to produce other hybrid hybridomas. PMID- 1715369 TI - Epitope mapping employing immobilized synthetic peptides. How specific is the reactivity of these peptides with antiserum raised against the parent protein? AB - Peptides synthesized onto polyethylene pins can be directly used to map the antigenic structure of proteins by ELISA (Geysen et al., 1987, J. Immunol. Methods 102, 259-274). The specificity of the reaction between anti-cytochrome c antibodies (IgG) and pin-bound hexapeptides of cytochrome c was tested using a competitive ELISA in which free and pin-bound peptides competed for the antibody. Competition was specific for many of the pin-bound peptides: only free peptides having the same sequence as the pin-bound peptides were able to compete for antibody-binding. However, non-specific reactivity was observed with pin-bound peptides whose sequence corresponded to the N terminal segment of cytochrome c. This segment is predicted to be particularly antigenic because of its high mobility and the nature of its amino acid sequence. In one case no competition by the free peptide could be observed even though the pin-bound peptide reacted strongly with anti-cytochrome c antibodies. PMID- 1715370 TI - The role of radiotherapy in palliative care. AB - Radiotherapy is an indispensable modality in the palliation of cancer. All palliative care programs should be acquainted with its indications and have a close working relationship with a radiation oncology department. The technical aspects of the subject may be intimidating to many staff and patients, and departments need to improve their outreach and education. The main indications are: pain relief (particularly bone pain), control of hemorrhage, fungation and ulceration, dyspnea, blockage of hollow viscera, and the shrinkage of any tumors causing problems by virtue of space occupancy. In addition, it has an important role in the palliation of three oncological emergencies: superior vena caval obstruction, spinal cord compression, and raised intracranial pressure due to cerebral metastases. More pragmatic fractionation schedules are being developed that are compatible with good results in terms of palliative end points, giving shorter courses with fewer hospital attendances for patient and family comfort and convenience. More clinical research and evaluation of palliative radiotherapy are required. PMID- 1715371 TI - Successful medical treatment of rhinocerebral mucormycosis complicating acute leukemia. PMID- 1715372 TI - Successful treatment of breakthrough bacteremia due to Pseudomonas aeruginosa with recombinant human granulocyte colony-stimulating factor in a patient with acute leukemia. PMID- 1715373 TI - Histopathology of prostatic tissue after transurethral hyperthermia. AB - Histopathological changes were studied in four patients who had moderate to severe urinary outflow obstruction due to benign prostatic hyperplasia (BPH) and were treated with transurethral microwave hyperthermia (TUMH). All these patients received TUMH with a helical antenna using a BSD 300 unit. The temperature was controlled on the urethral surface at 45 degrees C +/- 1 degree C. Each treatment session lasted 70 min at this temperature. Histological changes were periurethral, extending up to 6 mm radially and 4-5 cm longitudinally. They were symmetrical and most severe in the immediate periurethral zone. The severity and distribution of these histological changes correlated well with the thermal profile of the helical antenna. Acute changes, observed 24-48 h following administration of a single TUMH session, consisted of periurethral oedema, parenchymal haemorrhages and occasional, partial small-vessel thrombosis. Selective coagulation necrosis of the parenchymal smooth muscles with sparing of smooth muscle fibres in the vessel walls was noted. Histopathological changes found in patients 7 or 26 days following the administration of 10 TUMH treatments given twice-weekly for 5 weeks showed more severe and deeper lesions. They consisted of interstitial haemorrhages, complete obliteration of blood vessel lumina due to thrombosis and further evidence of coagulation and haemorrhagic necrosis. Evidence of a reparative process with ingrowth of granulation tissue in various stages of organization was clearly demonstrated. The effect of TUMH on BPH-obstructed urethra is probably expressed through selective shrinking and retraction of the periurethral prostatic parenchyma due to organizing localized tissue necrosis and cicatrization. Details of this complex process will be presented. PMID- 1715374 TI - Combined effects of hepatic arterial embolization using degradable starch microspheres (DSM) in hyperthermia for liver cancer. AB - Regional hyperthermia with a radiofrequency capacitive heating apparatus in combination with hepatic arterial embolization with degradable starch microspheres (DSM) was performed in 20 primary and six metastatic liver cancer patients. Efficacy was assessed primarily with regard to the improvement in heating efficiency. An angiocatheter was inserted into the hepatic artery in order to determine the DSM dosage adequate to arrest blood flow. The temperature rise in the tumours after heating alone and after heating combined with DSM embolization was compared. The maximum temperature and initial temperature rise within tumours were significantly improved by the combination therapy. Local tumour response could be evaluated in 10 primary and three metastatic liver cancer patients and tumour reduction over 50% was obtained in 40% and 33% respectively. Abdominal pain, nausea and vomiting, presumably due to reflux of the DSM, were experienced by several patients. In three patients heating could not be continued. However, all the symptoms were transient and responsive to symptomatic treatment, and no significant late complications were observed. Hepatic arterial embolization with DSM for liver tumours is considered effective and safe when combined with regional hyperthermia. PMID- 1715375 TI - T cell recognition in experimental autoimmune encephalomyelitis: prospects for immune intervention with synthetic peptides. AB - Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in mice of the H-2u haplotype. An important role for MHC class II molecules in "determinant selection" is revealed. Amino acids which determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides which bind MHC yet do not crossreact with the autoantigen. Two such peptides compete with the autoantigen for binding to the disease associated class II molecule and inhibit induction of experimental autoimmune encephalomyelitis in H-2u mice. Prospects for peptide mediated therapy are discussed. PMID- 1715376 TI - The presentation of self-peptides: tolerance and competition. PMID- 1715377 TI - Lipopolysaccharide/lipid A receptors on lymphocytes and macrophages. AB - Significant advances have been realized during the past five years in the understanding of the mechanism(s) by which endotoxic LPS interactions with mammalian lymphoreticular cells leads to characteristic cellular responses. There is now strong experimental evidence to support the concept that specific receptors for the lipid A component of LPS do, in fact, exist and are functional on these cells. While the available data do not rule out a potential contribution of nonspecific hydrophobic interactions of lipid A with the membrane bilayer in the cellular activation process, it would appear that interaction with the LPS receptor alone is sufficient to initiate triggering. Whether there exist more than one molecular entity which might function on mammalian cell membranes as a specific receptor for LPS, or whether different cell types may manifest different LPS receptors remains as an interesting area for future research. Further, the concept that molecular complexes of LPS with mammalian host proteins, such as the acute phase LPS binding protein, might trigger additional novel pathways for cell activation is both exciting and of potential importance. The precise mechanism or mechanisms by which LPS-receptor ligand interactions translate into appropriate transmembrane signalling events is currently uncertain. Clearly there exists evidence for contribution of many of the traditional second signals, although at present, the data are incomplete and not always consistent between laboratories. Of potential concern in this respect are the sometimes rather striking differences noted between lipid A and intact polysaccharide containing S-LPS. While such differences may be significant and important, it should be remembered that S-LPS itself is a potent stimulus for many lymphoreticular cell subpopulations, and any postulated pathways must encompass S-LPS as well as lipid A. In any case, it is likely that the further molecular-biochemical characterization of LPS receptors will yield crucial information for the eventual elucidation of the precise pathways for LPS transmembrane signalling. Such information will be invaluable in the future harnessing of the immunostimulatory potential of LPS as well as the abrogation of its profound deleterious pathophysiological effects in endotoxin shock. PMID- 1715378 TI - Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification. AB - Isolated sheep thyroid follicles release specific insulin-like growth factor binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissue may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by gamma-spectrometry. Iodine organification was assessed by incubation of follicles with 10(6) c.p.m. Na125I for 3 h before washing, solubilization in 0.1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A-Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol to OH or 3H medium significantly decreased the 125I labelled IGF-II associated with the 34- and 28-kDa IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P less than 0.01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function. PMID- 1715379 TI - Increased secretion of insulin-like growth factor-binding proteins and decreased secretion of insulin-like growth factor-II by muscle from growth-retarded neonatal rats. AB - Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) may be important factors in the control of neonatal growth. We have examined the production, in vitro, of IGFBPs and IGFs by hind-limb skeletal muscle from normal and small-for-gestational age (SGA) neonatal rats. Conditioned medium was collected from muscle strips after incubation at 37 degrees C for 2 h in Ham's F 12 medium. The conditioned medium was subjected to acid-gel permeation chromatography to separate IGFBPs from IGFs. The binding of 125I-labelled IGF-I to IGFBPs from both control and SGA muscle was displaced equipotently by IGF-I and IGF-II and not at all by insulin. IGFBPs from control and SGA muscles bound IGF-I with comparable affinities (Kd = 0.071 and 0.069 nmol/l respectively). When IGF-II was used as tracer, neither IGF-I nor insulin competed for binding. Western ligand blots of IGFBPs in conditioned media from both control and SGA muscles showed three bands of radioactivity at molecular masses equivalent to 24, 30 and 40 kDa. When the release of IGFBPs by muscle tissue in vitro was quantified by measuring the number of IGF-I binding sites in acid-fractionated medium it was apparent that the muscles from SGA pups secreted significantly more IGFBPs (39.3 +/- 7.5 fmol/mg muscle protein per 2 h) than the muscles from control pups (17.8 +/- 2.7 fmol/mg protein per 2 h; P less than 0.05). In contrast to the IGFBPs, more IGF activity was secreted by the muscles from the control pups (61.1 +/- 15.6 fmol/mg muscle protein per 2 h) than the muscles from the SGA pups (12.6 +/- 5.8 fmol/mg muscle protein per 2 h; P less than 0.05). Analysis of the IGF activity with assays specific for IGF-I and IGF-II showed that both SGA and control muscles secreted predominantly IGF-II with approximately 10% of the total IGF activity measurable as IGF-I. This differential secretion of IGFBPs and IGFs may be associated with the reduced growth potential of the SGA neonate. PMID- 1715380 TI - Substance P stimulation of polyphosphoinositide hydrolysis in rat anterior pituitary membranes involves a GTP-dependent mechanism. AB - Substance P (SP) stimulates polyphosphoinositide breakdown in the rat anterior pituitary through an NK-1 receptor. In the present study we present evidence that the coupling between the SP-NK1 receptor complex and polyphosphoinositide specific phospholipase C (PI-PLC) in rat anterior pituitary membranes may involve a mechanism consistent with a GTP-binding protein. The formation of inositol phosphates from [3H]myo-inositol-labelled anterior pituitary membranes induced by SP was potentiated by GTP and non-hydrolysable guanine nucleotides. The stimulatory effects of SP alone and SP plus GTP could be blocked by addition of GDP-beta-S (guanosine 5-O-(thiodiphosphate] in excess. Basal and SP plus guanine nucleotide-induced inositol phosphate formation were stimulated by fluoride, whereas the effect of SP alone was inhibited. Pretreatment of anterior pituitary membranes with sodium deoxycholate attenuated the inositol phosphate response elicited by GTP and GTP-gamma-S, whereas basal and SP-stimulated inositol phosphate production showed a peak at 1 mg sodium deoxycholate/ml. SP, fluoride and guanine nucleotide stimulatory effects on hydrolysis of polyphosphoinositide (PPI) were unaffected by pretreatment of anterior pituitary cells with cholera or pertussis toxin for 12h. Treatment of anterior pituitary membranes with cholera and pertussis toxin yielded [32P]ADP-ribosylation of two proteins with molecular masses of 45 and 41 kDa respectively. We conclude that SP coupling to PI-PLC through the NK1 receptor in the rat anterior pituitary involves a GTP-binding mechanism distinct from the G-proteins associated with adenylate cyclase, Gs and Gi. PMID- 1715381 TI - The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I, a potent IGF analogue, on growth hormone and IGF-binding protein secretion from cultured rat anterior pituitary cells. AB - The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I, a potent IGF-I analogue, on the secretion of GH and IGF-binding protein (IGFBP) from cultured rat anterior pituitary cells were measured. IGF-I and des(1-3)IGF-I stimulated GH secretion at low concentrations (maximally effective at 1 and 0.1 micrograms/l respectively) and inhibited GH secretion at higher concentrations. The half-maximal inhibitory concentrations (IC50) were approximately 20 micrograms/l and 1 microgram/l for IGF-I and des(1-3)IGF-I respectively. Thus des(1-3)IGF-I was more potent than IGF-I in these effects on GH secretion. We postulate that the increased potency of des(1-3)IGF-I in affecting GH secretion is due to decreased binding of this peptide by pituitary IGFBP compared with IGF I. In contrast with IGF-I and des(1-3)IGF-I, IGF-II did not stimulate GH secretion at low concentrations, but did inhibit GH secretion from pituitary cells with an IC50 of approximately 20 micrograms/l. Several IGFBPs ranging in molecular mass from 22,000 to 52,000 were detected in medium conditioned by cultured anterior pituitary cells. When measured by Western-ligand blotting and competitive ligand-binding techniques, these IGFBPs exhibited decreased binding of des(1-3)IGF-I compared with IGF-I and IGF-II. The production of IGFBP by anterior pituitary cells was stimulated by the addition of IGFs to the culture medium. PMID- 1715382 TI - The vascular architecture and innervation of the cerebral arteries in Leiothrix lutea. AB - The vascular architecture and innervation of the cerebral arteries in the robin billed leiothrix, Leiothrix lutea, were studied using catecholamine fluorescence, acetylcholinesterase active staining, and immunohistochemical techniques. The cerebral arteries in Leiothrix lutea consisted of the cerebral carotid and the basilar systems. The cerebral carotid artery can be divided into the anterior and posterior rami. Due to poor development of the posterior ramus, the posterior cerebral artery originated from the anterior ramus, and an anterior communicating artery between the cerebroethmoidal arteries formed the circle of Willis. The cerebral carotid system was supplied with aminergic nerve fibers (Amn), cholinergic nerve fibers (Chn) and peptides [substance P (SP), neurokinin A (NKA), calcitonin gene related peptide (CGRP) and vasoactive intestinal polypeptide (VIP)]-like immunoreactive (LI) nerve fibers in all regions. These nerve fibers were abundant in the cerebral carotid system, but were few and scattered in the basilar system. Only neuropeptide Y (NPY)-LI nerve fibers were recognized in moderate numbers in the cerebral carotid system, but were not found in the basilar system. Innervation of the small blood vessels of the cerebral parenchyma differed from that of the cerebral superficial arteries, SP-, NKA-, CGRP- and VIP-LI nerve fibers showed a dense distribution, but Amn and NPY-LI nerve fibers showed a sparse distribution, and almost no Chn was observed. Double staining in the cerebral arteries for SP-, NKA- and CGRP-LI nerve fibers demonstrated exactly the same distribution. This suggests that SP, NKA and CGRP co-exist in the same fiber.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715383 TI - Tumoricidal activity of interferon-r activated peripheral monocytes in colorectal cancer patients. AB - Peripheral blood monocytes obtained from 8 colorectal cancer patients and 6 normal controls were incubated in vitro with interferon-r (IFN-r) in the presence of bacterial lipopolysaccharide (LPS). The cytotoxic properties of the monocyte were determined subsequent to the interaction with radiolabeled autologous, allogeneic, as well as cultured colorectal cancer cells. Monocytes from normal controls and all colorectal cancer patients were activated in vitro to become tumoricidal; monocytes lysed tumorigenic cells but not nontumorigenic cells. Activators of protein kinase C (e.g. phorbol esters, PMA) and Ca2+ ionophores (A23187) when added alone did not effect the activation state of the monocyte. Whereas, PMA and A23187 cooperatively reproduced the ability of IFN-r to prime monocytes for tumoricidal activity. In the presence of PMA, A23187, and EGTA, the addition of excessive Ca2+ was sufficient for priming, whereas the addition of excessive Mg2+ was much less efficient. Priming by IFN-r, however, was not blocked by EGTA. An efflux of Ca2+ from preloaded monocytes was significantly increased by A23187 and by IFN-r. Quin-2/AM, an intracellular chelator of Ca2+, blocked priming by IFN-r. The results suggest that priming of monocytes for tumoricidal function by IFN-r may be involved in the activation of protein kinase C and mobilization of intracellular Ca2+. PMID- 1715384 TI - Prevalence of hepatitis C virus antibodies among patients infected with human immunodeficiency virus. AB - A study was undertaken to determine the prevalence and risk factors for serological evidence of hepatitis C virus (HCV) infection in patients infected with the human immunodeficiency virus (HIV). Tests for anti-HCV antibody were carried out by enzyme-linked immunoassay (EIA) on 101 HIV-infected patients from two university-based outpatient clinics. Anti-HCV antibody reactive samples were tested by using a recombinant immunoblot assay (RIBA) for HCV antibodies. Fourteen of 101 (13.9%) HIV-infected patients were anti-HCV reactive by EIA. Of these 14, only seven were reactive by RIBA: four were intravenous drug users as a sole risk factor for HIV infection; and the remaining three acquired HIV by blood transfusion, contaminated instrument exposure or IV drug use and sexual contact. Acquisition of HIV by sexual activity alone was not associated with HCV infection. It is concluded that HCV infection is found in approximately 7% of a university HIV clinic population. False-positive anti-HCV antibody serology may lead to overestimation of the prevalence of HCV infection. Female sex and intravenous drug use are significantly associated with HCV infection among HIV infected individuals. PMID- 1715385 TI - Immunohistochemical quantification of substance P in spinal dorsal horns of patients with multiple system atrophy. AB - Using a computer-assisted image analyser, an immunohistochemical quantification method of substance P-like immunoreactivity (SPLI) in laminae I + II of spinal dorsal horn was established and applied to 13 patients with multiple system atrophy (MSA) with no disturbance of pain sensation, including olivo-ponto cerebellar atrophy and striatonigral degeneration, and 13 neurologically normal controls. To investigate whether alteration of SPLI is related to an autonomic disorder, myelinated fibre counts of the fourth thoracic ventral roots were performed. Eleven of 13 MSA patients showed a significant decrease in small and large myelinated fibres, and were diagnosed with definite Shy-Drager syndrome (SDS), with the exception of two who had no apparent history of autonomic dysfunction. SPLIs in laminae I + II in 10 of these 11 patients, when adjusted for age, were significantly decreased at both levels of the fourth thoracic and third lumbar spinal segments. The results suggest the disorder of SP-containing synapses of primary afferent neurons and/or those of interneurons in SDS. PMID- 1715386 TI - Neuropathologic asymmetries in the brain of a patient with a unilateral status epilepticus. AB - Autopsy study of a patient who died after an episode of prolonged unilateral status epilepticus revealed neuronal loss in the hippocampus on the epileptic side, with gliosis confined to the CA1 and CA3 fields. There was loss of the parvalbumin-immunoreactive gamma-aminobutyric acid (GABA)-ergic interneurons in the hippocampus on that side. There was also loss of the normal laminar pattern of substance P staining with increased substance P immunoreactivity in the supragranular plexus on that side. Met-enkephalin immunoreactivity was also increased in the outer molecular layer of the dentate gyrus on the epileptic side. Mossy fibers on the epileptic side stained more strongly with the Hicks' silver stain and with antibodies against glutamate and taurine, but less intensely with antibodies against calbindin. In the contralateral cerebellum, there was Purkinje cell loss, injury to the remaining Purkinje cells, and increased prominence of the Bergmann glia. Our observations show that prolonged unilateral seizure activity can be associated with specific histochemical changes in the human hippocampus. PMID- 1715387 TI - Fos RNA accumulation in multiple sclerosis white matter tissue. AB - In order to better characterize the molecular events that accompany lesion development in multiple sclerosis (MS), we studied the accumulation of RNA specific to the nuclear proto-oncogenes c-fos and c-myb in post mortem white matter brain tissue. RNA was prepared from plaque and periplaque regions of 6 different MS brains, from "normal" white matter regions of 3 MS brains and from 6 normal control samples. Quantitation of specific RNA corresponding to each proto oncogene was performed by Northern blot hybridization and by scanning densitometry. Results indicate a 2-fold increase in c-fos RNA in MS white matter, compared to control tissue. No c-myb signal was identified in any sample. In situ hybridization studies confirmed the selective upregulation of c-fos RNA levels in MS tissue, and suggested that glial cells and not inflammatory cells were responsible for the enhanced c-fos signal. These results suggest that persistent glial cell activation is present within chronic MS lesions irrespective of whether the lesions are active (e.g., inflammatory) or inactive. PMID- 1715388 TI - The origin of thalamic inputs to the "hand" representation in the primary motor cortex. AB - We used retrograde transport of WGA-HRP to examine the origin of thalamic inputs to the "hand" representation in the primary motor cortex of macaques (Macaca nemestrina). Injections were placed in either the crest of the precentral gyrus or the rostral bank of the central sulcus. The sites for injection in the sulcus were determined by using intracortical stimulation to map the location of hand representation. We found that the precentral gyrus and central sulcus receive their predominant input from different subdivisions of the ventrolateral thalamus. Ventralis posterior lateralis pars oralis (VPLo) provides the most substantial input to a portion of the hand representation on the gyrus. In contrast, Ventralis lateralis pars oralis (VLo) provides the most substantial input to a portion of the hand representation in the sulcus. Prior studies have shown that VPLo is a major site of termination of cerebellar efferents and that VLo is a major site of termination of pallidal efferents. Thus, our results indicate that both the basal ganglia and the cerebellum "directly" influence the "hand" representation of the primary motor cortex. PMID- 1715389 TI - Amphetamine, cocaine, and fencamfamine: relationship between locomotor and stereotypy response profiles and caudate and accumbens dopamine dynamics. AB - Using in vivo microdialysis, the caudate and nucleus accumbens dopamine (DA) responses to the psychomotor stimulants amphetamine (AMPH), cocaine (COC), and fencamfamine (FCF) were evaluated in rats concurrent with characterization of their behavioral response profiles. Doses of each stimulant that produced either enhanced locomotion or a prolonged period of intense focused stereotypies were examined to evaluate the quantitative relationships between stimulant-induced behaviors and changes in DA dynamics and to test the hypothesis that a balance between mesostriatal and mesolimbic DA activity contributes to the appearance of specific stimulant-induced behaviors. Although 10 mg/kg COC and 1.7 mg/kg FCF promoted levels of locomotor activity substantially greater than 0.5 mg/kg AMPH, the magnitude of the DA increases in both caudate and accumbens were markedly less than was obtained following AMPH. Thus, stimulant-induced locomotion appears to be dissociated from the quantitative DA response in both brain regions. This behavioral/DA dissociation was also apparent at higher doses of AMPH (2.5 mg/kg), COC (40 mg/kg), and FCF (6 mg/kg), doses that promoted a behavioral pattern that included a prolonged period of intense stereotypy. Indeed, the regional DA responses to these high doses of COC and FCF were substantially less than the response to 0.5 mg/kg AMPH. Furthermore, there were no differences in the ratio of the caudate and accumbens DA responses as a function of dose for any of the three drugs. Thus, the balance between the regional DA activation does not appear to regulate the expression of the behavioral response. Additionally, the effects of these stimulants on regional DA metabolite concentrations were compared. The results indicate that AMPH promoted an identical pattern of effects on caudate and accumbens DA metabolites, suggesting that similar mechanisms govern the dynamics of DA in response to AMPH in both brain regions. In contrast, the DA uptake blockers promoted some region-specific effects on DA metabolites that may be due to regional differences in the DA metabolism and rates of impulse flow. PMID- 1715390 TI - Extracellular serotonin levels change with behavioral state but not with pyrogen induced hyperthermia. AB - Extracellular 5-HT in the anterior hypothalamus/preoptic area (AH/POA) and caudate nucleus of the freely moving cat was measured using in vivo brain microdialysis. Administration of 8-OH-DPAT, a 5-HT1A receptor agonist that decreases 5-HT neuronal activity, decreased extracellular 5-HT in both brain areas. Extracellular 5-HT levels were also examined in relationship to the sleep wake cycle, because previous data from our laboratory have indicated that behavioral state is the primary determinant of 5-HT neuronal discharge. As with 5 HT neuronal discharge, extracellular 5-HT was increased during active behavioral states and decreased during somnolent periods. These first two sets of findings confirm the ability of the microdialysis technique to measure physiological fluctuations in extracellular 5-HT levels and support the hypothesis that neuronal discharge is a major determinant of extracellular 5-HT levels. Levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA) in the AH/POA were also responsive to changes in behavioral state and administration of 8-OH-DPAT, though fluctuations in extracellular 5-HIAA were less robust and temporally delayed. Finally, extracellular 5-HT and 5-HIAA were examined in the AH/POA during fever induced by systemic injection of the synthetic pyrogen muramyl dipeptide. Previous data from our laboratory have indicated that 5-HT neuronal activity is unaffected by this manipulation, though 5-HT has been implicated specifically in thermoregulation. Pyrogen-induced hypothermia produced no specific change in 5-HT efflux, because any changes noted could be accounted for by behavioral state changes. These data are consistent with the hypothesis that the brain serotonergic system is closely linked to the sleep-wake-arousal cycle. However, extracellular 5-HT may be involved in thermoregulatory processes as part of a global role in modulating neuronal activity in coordination with the behavioral state of the animal. PMID- 1715391 TI - Rod bipolar cells in the macaque monkey retina: immunoreactivity and connectivity. AB - Rod bipolar cells in the macaque monkey retina were labeled by three antibodies: an antibody against the alpha- and beta-subspecies of protein kinase C (PKC), a polyclonal antiserum against the L7 protein from mouse cerebellum, and a monoclonal antibody against rabbit olfactory bulb (MAb 115A 10). The MAb 115A10 antibody also labeled some cone bipolar and some amacrine cells. The antibody against PKC was used to study the synaptic connectivity of rod bipolar cells. Reconstructions of 28 rod spherules showed that usually two and up to four rod bipolar processes invaginate each rod spherule. Six rod bipolar axons in the inner plexiform layer were reconstructed; they all showed the same pattern of connectivity. Synaptic output at rod bipolar dyads usually was onto two amacrine cell profiles: one that resembled the All amacrine cell and another that frequently made a reciprocal synapse. Rod bipolar cells did not contact ganglion cells. Synaptic input to rod bipolar cells came from reciprocal amacrine cells at dyads and other amacrine cells. In these respects, the rod pathway in the monkey is very similar to that described in cat and rabbit. The density of rod bipolar cells was determined and compared with the density of rods. There is a maximum of 15,000-20,000 rod bipolar cells/mm2 at 1-3 mm eccentricity, close to where rod density is maximum. Rod density is 10 times higher than rod bipolar cell density within 2 mm of the fovea, and 30 times higher at 15 mm eccentricity. This change in relative density is compensated by an increase in the number of rods contacted by individual rod bipolar cells (seen in Golgi-stained whole-mount retina) so that the number of rod bipolar terminal boutons in each rod photoreceptor remains relatively constant with changing eccentricity. We estimate that each rod bipolar cell is contacted by about 20 rods at 2-4 mm eccentricity and about 60 rods at 6 7 mm eccentricity. PMID- 1715392 TI - The remodeling of synaptic extracellular matrix and its dynamic relationship with nerve terminals at living frog neuromuscular junctions. AB - The question of whether the synaptic extracellular matrix undergoes remodeling and how this remodeling is related to nerve terminal plasticity was examined in living neuromuscular junctions of adult frogs. Sartorius muscles were double stained with a fluorescent nerve terminal dye 4-(4-diethylamino-styryl)-N methylpyridinium iodide (4-Di-2-Asp) and rhodamine-tagged peanut agglutinin (PNA) which recognizes synaptic extracellular matrix. Both nerve terminals and synaptic extracellular matrix in 200 identified normal junctions were visualized in vivo two or three times over a period of 2.6-6 months. The majority of neuromuscular junctions (NMJs) showed remodeling of both nerve terminals and synaptic extracellular matrix. Only 2.5% showed no changes in either synaptic element. The most commonly seen remodeling involved correlated changes in both nerve terminals and synaptic extracellular matrix. In this large group, while some junctions (20%) showed overall proportionate changes in all branches, most junctions (68%) showed disproportionate extension and/or retraction of some but not all individual branches. Another group of NMJs (9.5%) showed mismatched changes in the nerve terminal and synaptic extracellular matrix. In this group, some NMJs showed a decrease in the nerve terminal length without a corresponding reduction in synaptic extracellular matrix length. In other junctions that displayed extension of branches, the PNA-stained matrix was longer than the distal tip of the nerve terminal. Morphometric analysis indicated an average increase of 15.6% in total nerve terminal length and 13.6% in total synaptic extracellular matrix length. Although almost all NMJs displayed remodeling in at least one branch, about 50% of the 2201 individual branches examined did not show changes. The average change was 8.9% growth in the length of individual nerve terminal branches and 8.3% growth in the length of individual branches of synaptic extracellular matrix. There was no significant difference in the morphometry between the repeatedly observed junctions and the previously unobserved control junctions. Furthermore, junctions in which the synaptic extracellular matrix was longer than the nerve terminal also were seen in control as well as in experimental muscles. Cases where the nerve terminals were longer than the synaptic extracellular matrix were never observed in newly arising junctional branches. The present study has shown extensive remodeling in not only the nerve terminal but also the synaptic extracellular matrix in adult living frog NMJs. Results suggest that nerve terminals retract before the synaptic extracellular matrix. A hypothesis that extension of synaptic extracellular matrix precedes nerve terminal growth during synaptic remodeling is proposed. PMID- 1715393 TI - Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells. AB - Synapsin I is a neuron-specific phosphoprotein primarily localized at the presynaptic terminals, where it is thought to play an important role in the mechanisms involved in neurotransmitter release. Its interaction with cytoskeletal proteins and with small synaptic vesicles is regulated in vitro by phosphorylation by a calcium/calmodulin-dependent kinase. Here, we present the first evidence that, in the mouse retinal ganglion cells, synapsin I, moving along the axon with the slow component of axonal transport, is phosphorylated in vivo at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportion of differently phosphorylated molecules of synapsin I changes, and that these changes lead to a decrease of the overall content of phosphorus. The more basic forms, here collectively referred to as beta-forms, become predominant at the terminals after 7 d postlabeling, when the bulk of transported synapsin I has entered the superior colliculus. Along the axon, phosphorylation could be functional in preventing synapsin I from forming, with actin, a dense meshwork that would restrict organelle movement. On the other hand, at the terminals, the dephosphorylation-phosphorylation of synapsin I may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis. PMID- 1715394 TI - Utilization of bone scans in conjunction with prostate-specific antigen levels in the surveillance for recurrence of adenocarcinoma after radical prostatectomy. AB - Follow-up evaluation of patients who have undergone radical prostatectomy routinely consists of serial bone scintigraphy and, more recently, prostate specific antigen (PSA) levels. The utility of serial bone scans in combination with PSA levels is retrospectively reviewed in 118 men treated by radical prostatectomy for clinical Stage A or B disease who, at the time of surgery, had no evidence of metastatic disease. Of the 118 patients, 75.4% had no abnormality on either test (mean follow-up 32.4 mo), 9.3% demonstrated a detectable or rising PSA level with negative bone scan (mean follow-up 35 mo), and 8.5% exhibited a detectable and or rising PSA level and positive bone scan (mean follow-up 30.7 mo). Follow-up bone scans were read as either positive or indeterminate with undetectable PSA levels in 6.8% of patients (mean follow-up 27.3 mo). Critical review of the equivocal studies suggests that postoperative PSA levels more truly represent the clinical situation than bone scans. Following radical prostatectomy, routine bone scintigraphy provides little additional information when PSA levels are negative. If PSA becomes detectable or the patient develops symptoms, bone scintigraphy should then be performed. PMID- 1715395 TI - Negative staining-carbon film technique: new cellular and molecular applications. AB - Two techniques are presented which extend the original negative staining-carbon film technique into new areas of cellular and molecular application. These relate (1) to the production of negatively stained specimens of single-layer plasma membrane split from intact cells during the overall procedure that are negatively stained from the cytoplasmic face and (2) to the production of negatively stained specimens directly from glycerol-containing protein solutions, membrane or viral suspensions. In both cases in vacuo drying onto mica from glycerol is performed, prior to deposition of a carbon film. (For the cellular technique, freshly cleaved mica is firstly rendered positively charged by immersion in Alcian blue.) This is followed by release of the carbon film plus adsorbed membrane or protein by floating onto water, with subsequent negative staining. Selected preliminary applications using human erythrocyte membrane and the high molecular weight (native) human erythrocyte tripeptidyl peptidase-II complex are given and considered speculation as to the future application of the techniques is provided. PMID- 1715396 TI - Two different maturational stages of natural killer lymphocytes in human newborn infants. AB - The objective of this study was to assess the basis for the diminished natural killer (NK) lymphocyte activity of neonates. We found either severely reduced (63% of 68 neonates) or normal (similar to healthy adult) levels of NK activity. The percentages of cord blood mononuclear cells from the two groups of infants that expressed CD16, a differentiation antigen found in NK cells, were similar and within the range found in peripheral blood mononuclear cells of adults. However, infants with low NK activity had reduced numbers of cells in the CD16+56+ subpopulation, whereas the number of these effector cells present in cord blood mononuclear cells from infants with normal NK activity was within the range found in adults. Recombinant interleukin-2, but not recombinant interferon gamma, normalized the low NK activity of infants in a dose- and time-dependent manner. Analysis of the pattern of target cell susceptibility to lysis, together with the CD16+CD3- phenotype of the precursor and effector lymphocytes, demonstrated that the induced cytotoxicity was mediated by NK cells. In contrast, NK cells from infants with normal cytotoxic levels exhibited a functional response to interleukin-2 and interferon-gamma similar to that of adults. Our results indicate that NK cells in human neonates go through two different maturational stages. PMID- 1715397 TI - Indicators of the acute inflammatory and humoral immune responses in gingival crevicular fluid: relationship to active periodontal disease. PMID- 1715398 TI - Immunocytochemical characterization of cellular infiltrate, related endothelial changes and determination of GCF acute-phase proteins during human experimental gingivitis. PMID- 1715399 TI - Immunoglobulin A1 (IgA1) proteases from Prevotella (Bacteroides) and Capnocytophaga species in relation to periodontal diseases. PMID- 1715400 TI - Pineal gland "magnetosensitivity" to static magnetic fields is a consequence of induced electric currents (eddy currents). AB - During the past decade, a number of reports indicated that the mammalian pineal gland is magnetosensitive in terms of spatial orientation. This indication is based on observations that artificial alterations of the direction of the earth's magnetic field (MF) markedly decreased the gland's capability to synthesize melatonin. These findings, however, seem paradoxical since animals as well as humans experience such alterations whenever they turn their heads. Therefore, the potential of the pineal for sensing magnetic fields was re-investigated. During the dark phase, rats were exposed to repeatedly inverted MFs, generated by two identical pairs of Helmholz coils; one pair connected to a power supply automatically, the other pair manually using an integrated potentiometer. Only the pineals of animals exposed to the automatically activated field responded with a reduced activity of the rate-limiting enzyme serotonin-N acetyltransferase, lower melatonin levels and increases in serotonin and 5 hydroxyindole acetic acid. Hence, MF exposure itself did not affect the pineal. Rather, induced eddy currents in the animals, resulting from rapid On/Off transients of the artificially applied MF, are most likely the explanation. PMID- 1715401 TI - Characterization of polymorphs of tranilast anhydrate and tranilast monohydrate when crystallized by two solvent change spherical crystallization techniques. AB - Spherically agglomerated crystals of tranilast (oral antiallergic agent) with improved availability in vitro, as well as improved micromeritic properties such as flowability and packability, were prepared by a novel spherical crystallization technique. The agglomerates of tranilast were found to be composed of new monohydrate I, II, or III, depending on the crystallization solvent and the procedure employed. With dehydration by heating, monohydrate I transformed to the stable alpha form directly. On the other hand, monohydrates II and III converted to the amorphous and beta forms, respectively, followed by further transformation to the alpha form at 110 and 150 degrees C, respectively. The amorphous and beta forms of agglomerates were easily prepared by storing the monohydrates under 0% RH at 30-40 degrees C. Monohydrate II and the amorphous form of the agglomerate with high surface energy could enhance the solubility and the dissolution rate of tranilast. A phase diagram of polymorphs of agglomerated tranilast was constructed to exhibit their interconversions under various humidities and temperatures. PMID- 1715402 TI - Treatment options for the neonate with hypoplastic left heart syndrome. PMID- 1715403 TI - Liquid junction potentials and small cell effects in patch-clamp analysis. PMID- 1715404 TI - Dimensions of the ion channel in neuronal nicotinic acetylcholine receptor as estimated from analysis of conformation-activity relationships of open-channel blocking drugs. AB - Relationship between the size of the molecule in the series of organic ions Et3+N -(CH2)5--+NR1R2R3 (Ri--alkyl or cycloalkyl substituents) and their abilities to block nicotinic acetylcholine receptors (AChRs) due to their open-channel blockade in the neurons of autonomic ganglia and in frog end-plate was analyzed. All low-energy equilibrium conformations of the drugs were calculated by the molecular mechanics method. A unique rectangular channel profile 6.1 x 8.3 A, for which the best correlation between blocking activity of the drugs and total population of their conformations being able to penetrate into the channel, was deduced from all those tested. PMID- 1715405 TI - Ion channels in murine nuclei during early development and in fully differentiated adult cells. AB - The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores. PMID- 1715406 TI - Complexes formed by complementary RNA stem-loops. Their formations, structures and interaction with ColE1 Rom protein. AB - Regulation of replication of plasmid ColE1 involves interaction of two plasmid specified RNA transcripts. One of these RNAs (RNA II) serves as a primer for DNA synthesis, and the other (RNA I) is complementary to part of RNA II. The complementary regions of RNA I and RNA II form several stem-loop structures. Binding of these RNAs that regulates DNA replication begins by interaction at the loop regions. Plasmid-coded Rom protein stabilizes the product of the interaction. In this paper, the mechanism of the loop-to-loop interaction between pairs of RNA stem-loops having various nucleotide sequences is studied. Binding of two stem-loops containing six to eight nucleotides in their loops requires that the loop sequences be complementary, whereas the stem sequences need not be. The association rate constants for binding of complementary pairs with various sequences are relatively similar, around 1 x 10(6) M-1 S-1. On the other hand, the rates of dissociation of the complexes vary greatly depending on the loop sequence, even for complexes having the same base composition, suggesting a strong effect of base-stacking. All the complementary bases in the seven nucleotide loops participate in complex formation, and the resulting complex is bent a little at the interacting region. Rom binds and stabilizes any complex formed by pairs containing fully complementary loop sequences. Structures are proposed for the RNA complexes with and without Rom. PMID- 1715407 TI - Osmotic induction of gene osmC expression in Escherichia coli K12. AB - osmC, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned on multicopy plasmids, and its product, OsmC, was identified as a 14 kDa protein in maxicells. The DNA sequence of the gene and its upstream region were determined. The sequence of an osmC-phoA gene fusion confirmed the osmC reading frame. A deletion of osmC from the E. coli chromosome was constructed by gene replacement, demonstrating that it is not an essential gene. The osmCp promoter region was subcloned and a lac operon fusion transcribed under osmCp control was constructed. The expression of this operon fusion demonstrated that osmC regulation occurs at the transcriptional level. S1 nuclease protection experiments and deletion analysis identified two overlapping promoters with transcription start sites separated by ten nucleotides. All the sequences necessary for osmotic regulation of both promoters are located within a 137 base-pair DNA fragment extending from position -95 to +42 with respect to the putative osmC translation start. Two deletions were obtained that abolish the functioning of the upstream promoter. Yet, under our experimental conditions, the subsequent expression of the osmC-lacZ fusion was equivalent to that obtained from the tandem promoters. Mutations leading to constitutive expression of osmC were selected. Two independent mutations were obtained, both affected osmZ, the gene encoding the histone-like protein H1. PMID- 1715408 TI - Modulation of Ca currents in isolated frog atrial cells studied with photosensitive probes. Regulation by cAMP and Ca2+: a common pathway? AB - We have studied the regulation of cardiac Ca current by intracellular cyclic AMP (cAMP) and Ca2+, using photosensitive, caged compounds and the whole-cell, patch clamp technique in isolated frog atrial cells. Although both low voltage activated (LVA) and high voltage activated (HVA) Ca channels were found to be present in these cells, only the HVA Ca currents were sensitive to modulation by isoproterenol or dihydropyridines (DHPs). The application of extracellular isoproterenol, as well as the photorelease of intracellular cAMP or Ca2+ at micromolar and submicromolar concentrations, respectively, had no effect on LVA Ca currents. In contrast, these agents: (i) increased the amplitude of currents through HVA channels, carried by either Ca2+ or Ba2+ with a similar time-course, (ii) slowed the decay of the current when Ba2+ was the permeating ion, and (iii) modulated the agonist effect of the DHP Bay-K 8644. The strong similarities between the effects of cAMP and Ca2+ suggest that both of these intracellular messengers might eventually lead to the phosphorylation of HVA Ca channels. It is possible that Ca-dependent phosphorylation of the channels may account for the potentiation of Ca current induced by repetitive stimulation. PMID- 1715409 TI - The interferons. Mechanisms of action and clinical applications. AB - The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones. PMID- 1715410 TI - [The relationship between the anti-HCV titer and the state of chronic hepatitis C]. AB - By dilution of the serum, we quantitatively analyzed anti-HCV to examine the relation between the anti-HCV titer and the state of chronic hepatitis C, and determined the effectiveness of interferon therapy. The anti-HCV titer was related with the state of chronic hepatitis C, but not as closely as GPT. Cases with blood transfusion had a lower titer of anti-HCV, weaker histological finding and better response to interferon than those without blood transfusion. The anti HCV titer was not useful for determining the effectiveness of interferon therapy. PMID- 1715411 TI - [Demonstration of PIVKA-II and AFP production by gastric cancer by indirect immunoenzymatic staining of paraffin sections]. AB - The patient had elevated plasma PIVKA-II and serum AFP levels. However, no tumor was detected with an ultrasonography, angiography, abdominal computed tomography (CT), and magnetic resonance imaging (MR). Gastric endoscopic examination disclosed gastric cancer and then subtotal gastrectomy was done. Shortly after the surgery, both plasma PIVKA-II and serum AFP levels returned to each normal level. The extirpated tumor revealed histologically immature and mature cancer cells. It was pathologically diagnosed as IIc early cancer. The localization of PIVKA-II and AFP in gastric cancer cells was demonstrated by the immunostaining method using monoclonal antibody. Taken together, these results indicate that cancer cells may produce PIVKA-II or AFP in this patient. PMID- 1715412 TI - Changes in CNS levels of serotonin and its metabolite in SART-stressed (repeatedly cold-stressed) rats. AB - Central nervous system levels of serotonin (5-HT) and 5-hydroxy-indoleacetic acid (5-HIAA) in SART (specific alternation of rhythm in temperature)-stressed (repeatedly cold-stressed) rats were examined by HPLC-ECD. In SART-stressed rats, the levels of both 5-HT and 5-HIAA decreased in many brain areas. In the spinal cord, only the 5-HT level decreased. Therefore, the ratio of 5-HIAA to 5-HT increased only in the spinal cord. These results suggest that SART-stressed rats have some form of abnormality in the synthetic system of 5-HT. PMID- 1715413 TI - Cytosolic and membrane-bound nitric oxide synthase. AB - Cytotoxic activated macrophages were sonicated and centrifuged. The activity of nitric oxide (NO) synthase was present in the supernatant and independent of Ca2+. The pellets were washed three times and treated with buffer containing 0.1% Triton X-100 or buffer alone, followed by centrifugation. The supernatant containing Triton X-100 showed NO synthase activity that was dependent on Ca2+, whereas the supernatant without the detergent had little activity. These data suggest that there are two forms of NO synthase: cytosolic and membrane-bound enzymes. PMID- 1715414 TI - [Promotional effects of high fat diet on chemical carcinogenesis of the prostate]. AB - The effects of a high fat diet on progression of minimal cancerous lesions to manifest ones were investigated using a chemo-endocrine carcinogenesis model of rat prostate. Male Fischer 344 rats were alternatively given a diet containing 0.75 ppm of ethinyl estradiol (EE) for 3 weeks and the basal diet without EE for the following 2 weeks, and subcutaneously administered with 3,2'-dimethyl-4 aminobiphenyl (DMAB) at 50 mg/kg body weight 2 days after the diet with EE was changed to the basal one. This sequential treatment was repeated 10 times in 50 weeks, and the animals were fed with either normal fat diet (NF) or high fat diet (HF) during the following 30 weeks. At week 80, all the rats were sacrificed for histological examination of the prostate. Atypical hyperplasia and adenocarcinoma were induced in 15.4% (4/26) and 34.6% (9/26) in the rats fed NF and 44.8% (13/29) and 20.7% (6/29) in those group fed HF, respectively. The incidence of adenocarcinoma was significantly higher in the group fed NF than in the other. However, the number of rats with either atypical hyperplasia or adenocarcinoma was not significantly different between the two groups. These observations provided no supporting evidence that high fat content in diet has enhancing effects on prostatic carcinogenesis. Using different species or strain of rats, C3H/He mice and ACI/Seg rats, additional experiments were also conducted by a slightly modified protocol without changing on fat contents. Although ACI/Seg rats were known to spontaneously develop prostate cancer, the histopathological examinations revealed atypical hyperplasia at 25% (16/64) in mice and microadenocarcinoma at 8.1% (6/74) in rats. Apparently further studies are needed until a more useful and efficient model of prostate carcinogenesis is established. PMID- 1715415 TI - [Combination chemotherapy with methotrexate, vincristine, cisplatinum, cyclophosphamide, adriamycin, and bleomycin (MVP-CAB) for metastatic urothelial cancer]. AB - We studied 31 patients with bidimensionally measurable metastases of urothelial cancer who were treated with a planned regimen (20 mg/m2 methotrexate, 0.6 mg/m2 vincristine, 500 mg/m2 cyclophosphamide, 20 mg/m2 adriamycin, and 30 mg bleomycin on day 1, and 50 mg/m2 cisplatinum on day 2) in cycles given every 3 weeks. CR was achieved in 4 patients (13%) and PR in 17 patients (55%). The response rates according to the disease characteristics were 71% for TCC, 33% for SCC, 67% for renal pelvis tumors, 67% for bladder tumors, 73% for lung metastases, 67% for liver metastases, and 67% for lymph node metastases. The median response duration and the number of cycles of therapy were 32 months/3 cycles for patients with a CR and 6 months/6 cycles for those with a PR. The median duration of survival was 32 months (range: 3-46) in CR, 11 months (range: 1-37) in PR, and 6 months (range: 2-10) in non responders (NC + PD). A significant prolongation of survival was noted in patients with either CR (p less than 0.01) or PR (p less than 0.05). Then main toxic effects were pulmonary fibrosis and myelosuppression. Two patients aged 73 and 81 died of pulmonary fibrosis. However, it was possible to prevent pulmonary fibrosis by not administering bleomycin to patients over 70 years of age, or to patients with pulmonary dysfunction. WBC count nadirs of less than 2,000/m3 were noted in 22 patients (71%). Platelet count nadirs of greater than 5 x 10(4)/mm3 were noted in 7 patients (23%). However, there were no deaths due to myelosuppression. PMID- 1715416 TI - [A randomized trial of PVB, VAB-6, BVP regimen versus PEB chemotherapy in patients with disseminated testicular tumors]. AB - During 2 years and 7 months from June, 1985 to December, 1987, a randomized multi center trial of PVB, VAB-6, BVP regimen (group A) without etoposide versus PEB chemotherapy (bleomycin, etoposide and cisplatinum) (group B) was given to patients with disseminated testicular tumors. Of 34 patients registered, 10 patients were with minimal disease in stages IIA, IIIO and IIIA and 24 with extensive disease in IIB, IIIB2 and IIIC. Seminomas were found in 10 patients, while non-seminomatous tumors in 24. Among groups A and B, there was no statistical difference in clinicopathological profiles. A group patients were given either PVB, VAB-6 or BVP according to the physician's discretion. In groups A and B, 35% and 43% of the patients achieved complete response, and 45% and 50% achieved partial response, respectively. The difference in CR rates among both groups was not statistically significant even when calculated according to the stage or histologic grouping. Salvage treatments mainly with surgical resection of residual tumors after the chemotherapy, however, were more successful in group B (88%) than group A (61%). It appears likely that the higher response of induction chemotherapy in patients with extensive disease made the salvage surgery more successful in group B than in group A. The 3 year survival rate was 100% in group B, whereas it was 76% in group A. Although the incidence of myelosuppression and alopecia was significantly higher in group B, neuropathy was significantly more frequent in group A. From the above results, PEB seems to be a better induction chemotherapy than the conventional one for advanced testicular tumors. PMID- 1715417 TI - Angiogenic action of angiotensin II is important for the glomerular growth of maturing kidneys. PMID- 1715418 TI - [Acute experimental enzymatic cholecystitis (clinico-morphological comparisons)]. AB - In the experiment on 30 mongrel dogs, acute enzymatic cholecystitis (AEC) was modelled by means of intraduodenal connection with a fluoroplastic catheter of the common bile duct and pancreatic duct. The animals died at day 3-5 from transudative biliary peritonitis. A correlation between the severity of the clinical course, pronouncement of enzymologic shifts (increase in activity of amylase and phospholipase A2, content of biliary malonic dialdehyde) and morphological changes in the gallbladder and common bile duct in the form of necrosis and round-cell infiltration of the mucosa was revealed. Only decompression of the bile duct within the first 6 h from the moment of AEC development contributes to regression of clinical and enzymologic disorders and recovery of the animals. PMID- 1715419 TI - [Clinico-morphological studies of epididymal cysts]. AB - A clinico-morphologic study of epididymis cysts was made in 52 patients at mean age 39.4 years, observed over a period of 2 ys. 4 mos. In approximately half of the cases (49.2 per cent) the cysts were localized in the left epididymis. Four types of histologic structure were distinguished: 1. smooth outlines of the lumen, filled with spermatozoa, and wall built up of fibrous tissue and different proportion of smooth- muscle, elastic and reticular fibers, and lined with one row ciliary columnar or cubic epithelium; 2. similar to the former structure, but without spermatozoa in the lumen; 3. stellate-outlined lumen without spermatozoa in it, with a wall built up of connective tissue and lined with one-row ciliary columnar epithelium; 4. the wall is stellate-folded, built up of loose connective tissue and lined with one-row cubic epithelium, forming micropapillary structures, without presence of spermatozoa. Considerable difficulties are pointed out in determining the types of cysts according to the nature of their epithelial lining and the structure of their wall. Precise classification is possible when thorough clinico-morphologic parallel is used. PMID- 1715420 TI - [A micromodification of the turbidimetric method of determining lysozyme]. AB - A micromethod for lysozyme measurement with a Uniskan-1 spectrophotometer has been developed. The concentration of this enzyme was measured in 5-10 microliters of saliva and blood. Lysozyme levels were measured in miners of deep mines who had various dental conditions. PMID- 1715421 TI - [Determination of HBsAg using the method of competitive radioimmunologic analysis]. PMID- 1715422 TI - [An immunoenzyme method of controlling the concentration of theophylline in the blood of children with bronchial asthma during treatment with theophylline preparations]. PMID- 1715423 TI - [A commercial immunoenzyme reagent for the determination of ceruloplasmin]. AB - A diagnostic agent for enzyme immunoassay of ceruloplasmin has been developed. The method is highly sensitive and specific. The minimal detectable amount of ceruloplasmin is 25 ng/ml, variance coefficients with 1 lot being 2.6-7.3 percent and 4.2-10.8 percent within 3 lots. Blood serum ceruloplasmin concentration in health has made up 0.9 to 1.3 mg/ml. Ceruloplasmin levels were regularly shifted in Wilson's disease, cerebral lymphomas, sepsis, injuries, liver cirrhosis and other diseases. PMID- 1715424 TI - [A method of fractionating bone marrow cells in a density gradient using a mixture of Ficoll and Verografin]. AB - The authors suggest a method for hemopoietic tissue cell fractionation into populations enriched for various hemopoietic elements. Centrifugation in a three layer density gradient of mixtures of ficoll and verografin (d1 = 1.03 g/cm3, d2 = 1.09 g/cm3, d3 = 1.1 g/cm3) resulted in three fractions. The first contained lymphoid cells, the second erythroid cells (89.1 +/- 7.3%), and the third consisted of white blood cells (78.5 +/- 6.3%). PMID- 1715425 TI - [The migration capability of neutrophilic granulocytes in disseminated tuberculosis and sarcoidosis of the lungs]. AB - A method for estimation of neutrophilic granulocyte spontaneous migration in capillaries is suggested. Twenty-one patients with disseminated pulmonary tuberculosis, 19 ones with active pulmonary sarcoidosis, and 30 normal subjects were examined. The findings evidence a higher migration activity of neutrophilic granulocytes in the patients, 66.4 +/- 8.5 in tuberculosis and 65.1 +/- 7.7 in sarcoidosis as against that in health (32.4 +/- 4.0). Tuberculin was found to stimulate cell migration in tuberculosis patient; the migration index made up 1.3 +/- 0.07 in this case. This agent had no apparent effect on the phagocytes of sarcoidosis patients and normal subjects, and their migration index did not exceed 1.0. Neutrophilic granulocyte reaction to tuberculin may be used in the differential diagnosis between pulmonary tuberculosis and sarcoidosis. PMID- 1715426 TI - [Correlation of electrocoagulogram indices with coagulogram tests]. AB - New electrocoagulogram characteristics are suggested. Mathematical analysis has revealed a correlation between some acknowledged electrocoagulogram characteristics and the suggested tests, on the one hand, and plasma heparin tolerance, plasma recalcification time, prothrombin time, fibrinogen content, fibrinolytic activity, and fibrinase activity, on the other. The results of examinations of 76 patients with acute esophagogastroduodenal hemorrhages have confirmed a high informative value of the new characteristics and a possibility of determining the traditional coagulogram values from electrocoagulogram data. This recommends electrocoagulography as a rapid method for the diagnosis of blood coagulation disorders. PMID- 1715427 TI - [Coagulation and rheologic indices in the hyperviscosity syndrome]. AB - In vitro studies of blood coagulation by the macromethod and blood rheology measurements by rotation rheogoniometer helped assess blood aggregation status in normal subjects and patients with elevated viscosity syndrome associated with acute myocardial infarction. The findings also evidence relationships between stabilized index of fluidity limit and red cell deformability, fibrinogen concentration and fluidity limit, hematocrit and blood viscosity, clot density and total hemostatic index. PMID- 1715428 TI - [Functional activity of the neutrophils in the bronchoalveolar space in chronic bronchitis and bronchiectasis]. AB - Bronchoalveolar lavage fluid neutrophils were studied by the cytologic methods in 58 patients with chronic bronchitis, 63 ones with bronchiectasis, and 8 normal controls. The study included cytospectrophotometry of myeloperoxidase and alkaline phosphatase activity and estimation of active oxygen-producing cells in the NBT test. Neutrophilic functional activity was different in the patients with chronic bronchitis and bronchiectasis. Neutrophilic myeloperoxidase and alkaline phosphatase activities were lower in the patients with chronic bronchitis than in those with bronchiectasis, whereas the counts of cells active in the NBT test were low in both patient populations. PMID- 1715429 TI - [Dynamics of the cytologic and bacterioscopic changes in bronchoalveolar washings in acute focal pneumonia]. AB - The authors analyze cytologic and bacterioscopic changes in bronchoalveolar washings in 37 patients with acute focal pneumonia on days 4 through 44 of the disease. Bronchoalveolar washings obtained from 11 subjects without pulmonary conditions were examined for control. Immunofluorescent examination of brush biopsy specimens of the bronchial epithelium from 4 patients, taken at the peak of pneumonia, has revealed respiratory viruses antigens. The highest numbers of free bacterial colonies and intracellular bacteria were detectable also at the peak of pneumonia. In contrast to reference specimens, patients' bronchoalveolar washings were characterized by elevated absolute counts of the neutrophils, bronchoepitheliocytes, and destroyed cells during the peak and regress of the disease. During convalescence these cell counts were essentially reducing though persisting significantly elevated as against the control. Absolute counts of alveolar macrophages and lymphocytes in bronchoalveolar washings were virtually unchanged over the course of the disease. The authors claim that the cytologic and bacterioscopic characteristics of bronchoalveolar washings objectively and fully reflect the activity of the inflammatory reaction in the lungs. PMID- 1715431 TI - [Cytologic diagnosis of thyroid gland adenoma and goiter]. AB - Examinations of smear impressions of postoperative biopsy specimens of the thyroid from adenoma patients have revealed among polygonal structures 1 to 20 spherical complexes (per micropreparation) consisting of relatively monomorphic follicular cells with intensively blue cytoplasm. The 'ball' size was 200-900 micron or more. Cellular composition, specific features of cellular structure, and the presence of spherical structures permit differentiating adenoma from goiter. PMID- 1715430 TI - [The use of electron microscopy for diagnosing spongiform encephalopathies]. AB - The authors suggest an unsophisticated approach to laboratory diagnosis of spongiform encephalopathies induced by unconventional prion viruses, consisting in electron microscopic detection of abnormal helical Scrapie-associated fibrils. These fibrils are detectable in human and animal brain tissue samples and in cell cultures. PMID- 1715432 TI - [The determination of alpha-tocopherol in blood serum]. AB - A modified method for measuring vitamin E is described, making use of thin-layer chromatography with Silufol plates. Effects of various storage conditions of the blood serum on its vitamin E levels were examined. Vitamin E proved to be sufficiently stable at storage at -10 degrees C and defrosting under 20 degrees C. Comparison of this mode of storage with other ones has demonstrated its advantages. The method was tried in clinical practice in examinations of patients with pulmonary tuberculosis, chronic alcoholism, females suffering from infertility due to inflammations and healthy ones (controls) and found to be accurate, reproducible, and informative. PMID- 1715433 TI - [Haptoglobin phenotypes and serum enzyme activity]. AB - Haptoglobin (Hp) phenotypes were determined by thin-layer polyacrylamide gel electrophoresis in 2071 normal Russians, Tatars, and Bashkirs. Hp phenotype distribution was found similar in these populations, making up 13.4% for Hp 1-1, 48% for Hp 1-2, 38.6% for Hp 2-2. Hp1 gene incidence (0.37) was characteristic of the European populations. Serum alkaline phosphatase and gamma-glutamyl transpeptidase activities were similar in all the three Hp phenotypes. Alanine and aspartate aminotransferases and acid phosphatase activities were higher in Hp 1-1 phenotype by 25, 15, and 15 percent, respectively. PMID- 1715434 TI - [Laboratory diagnosis of Paecilomyces infections]. AB - Preincubation of blood, sputum, breast milk, and feces samples from patients in heparin-treated medium 199 for 18-24 h at ambient temperature to preserve viability of Paecilomyces tissue form when transferring the fungi from host to ambient temperature regimen results in transformation of the fungi into mycelium form in favorable conditions, which fact permits elevating the isolation rate of this fungus from 1.5 to 44-73 percent. PMID- 1715435 TI - [Specific antibodies to different mycobacterial antigens in patients with pulmonary tuberculosis]. AB - Antibodies to three mycobacterial antigens, Soviet tuberculin and ultrasound treated Mycobacterium tuberculosis (H37Rv) and M. bovis (BCG) were detected by enzyme immunoassay in 90 patients with pulmonary tuberculosis and 75 normal subjects. Antibody detection rates and levels were found related to the form of tuberculous process. The detection rate was rather low in focal tuberculosis and virtually the same for all the antigens (15.3 percent). In disseminated processes (infiltrative and particularly disseminated and fibrous-cavernous) antibodies are detected more often in higher titers than in local processes, and their levels are different with different antigens. The method was more sensitive with ultrasound-treated antigens of M. tuberculosis (H37RV) and M. bovis (BCG) than with PPD. Simultaneous enzyme immunoassay with three antigens detected up to 75 percent of antituberculous antibodies in infiltrative, up to 92 percent of antibodies in disseminated, and up to 100 percent of antibodies in fibrous cavernous tuberculosis. PMID- 1715436 TI - [Methods of testing the complement (review of the literature)]. PMID- 1715437 TI - [The etiologic structure of acute intestinal infections caused by opportunistic microorganisms (Irkutsk, 1984-1988)]. AB - The authors analyze the results of bacteriologic studies carried out in patients with intestinal infections at the laboratory of the Sanitary Epidemiologic Center of the town of Irkutsk over 1984-1988. The records of 24,490 case histories show infections induced by opportunistic microflora in 37 percent of cases. The most frequent etiologic agents were Staphylococcus aureus and Enterobacteriaceae, genera: Proteus, Enterobacter, Citrobacter, Klebsiella, Hafnia. Analysis of the incidence of acute intestinal infections induced by opportunistic bacteria in various age groups evidence that mostly infants aged under 1 suffer from them. In older children S. aureus is no longer etiologically significant and it is replaced by Enterobacter and Klebsiella. The authors find it interesting to compare the etiological patterns of enteritis induced by opportunistic microorganisms in various regions of our country. PMID- 1715438 TI - [The rapid determination of the hemagglutinating and the blood coagulating activity of staphylococci]. AB - A solid-phase preparation, designated by the authors "plasma pencil", has been developed for rapid measurements of plasma-agglutinating and plasma-coagulating activities of microorganisms. Plasma pencil employed instead of liquid plasma is economic, ready-to-use, may be stored for a long time, makes the procedure more rapid and simple. Measurements of the aforesaid activities in 388 staphylococcal clinical strains, carried out with the use of this pencil, have demonstrated a high efficacy of the preparation; the results coincided with those obtained by the routine methods. PMID- 1715439 TI - [A modification of the passive hemagglutination inhibition test to correct the results of the passive hemagglutination test in titrating tetanus antitoxin]. AB - The aim of this work was to accelerate passive hemagglutination inhibition test, simplify it, perform it without an incubator and antibody diagnostic agent, and confirm the results of this test the characteristics permitting not only specific, but quantitative assessment of the results of passive hemagglutination test. To achieve this, the authors suggest the following modifications: the working dose of purified tetanus antitoxin is titered in relation to the standard serum; antibody erythrocytic diagnostic agent is modified by adding the working dose of purified tetanus antitoxin; the test is performed at room temperature. PMID- 1715440 TI - [Evaluation of the work of bacteriological laboratories by their end results]. PMID- 1715441 TI - [Optimizing the hand washing of laboratory glassware for storing bacterial and blood preparations]. PMID- 1715442 TI - [Eosinophilic granuloma in a 9-month-old infant]. AB - Eosinophilic granuloma in an infant of 9 months is described; this condition is difficult to differentiate from Letterer-Siwe's disease. The final diagnosis of eosinophilic granuloma could be made only due to positive time course of the process eventuating in gradual normalization of the infant's health status. PMID- 1715443 TI - [A chromatographic method of determining verapamil and norverapamil in blood plasma]. PMID- 1715444 TI - [The fatty acid spectrum and free cholesterol level in exhaled air condensate]. AB - Gas chromatographic measurement of fatty acid spectrum and free cholesterol level in exhaled air condensate of children suffering from asthma has revealed a deficiency of polyunsaturated fatty acids and lipids at the expense of linoleic and arachidonic acids. PMID- 1715445 TI - [Detection of mites in domestic dust by measuring guanine]. PMID- 1715446 TI - Platelet-derived growth factor expression in mesangial proliferative glomerulonephritis. AB - Proliferation of mesangial cells and expansion of mesangial matrix are common histologic features of proliferative glomerular disease, a frequent cause of renal failure. Proliferation of glomerular mesangial cells occurs in response to platelet-derived growth factor (PDGF), and these cells release PDGF and express PDGF A and B chain mRNAs. However, all studies relating PDGF to potential changes in glomerular structure and function to date have been performed in vitro. To explore the role of PDGF in proliferative glomerulonephritides, we studied the expression of PDGF in vivo in two animal models of IgA nephropathy with different histologic patterns of glomerular injury: either predominant mesangial proliferation or expansion of mesangial matrix. Increased expression of PDGF and PDGF B-chain mRNA in whole kidneys from diseased mice was demonstrated by immunohistochemical techniques and by solution hybridization assay, respectively. Immunohistochemically, PDGF was localized primarily within the mesangial area of glomeruli and to a much lower extent in interstitium. The increased PDGF expression correlated with the degree of hypercellularity and clinical features of the disease. In addition, PDGF expression was increased in some forms of human glomerulonephritis, characterized by mesangial proliferation. These findings suggest that PDGF may be a major contributor to mesangial cell proliferation seen in proliferative glomerulonephritides. PMID- 1715448 TI - Cardiovascular hypertrophy. Its implication for pathophysiology, outcome and management of hypertension. Satellite symposium to the 13th scientific meeting of the International Society of Hypertension, Ottawa, June 29-30, 1990. PMID- 1715449 TI - Clinical significance of left ventricular hypertrophy: insights from the Framingham Study. AB - Left ventricular hypertrophy (LVH) is a cardiac end-organ response to increased pressure or volume load. For the past 40 years the electrocardiogram (ECG) has been used in the Framingham Heart Study for the detection of LVH. There is an increased risk of developing coronary heart disease following the appearance of LVH on the ECG. ECG LVH also carries a high risk for mortality. The overall risk of mortality in subjects with ECG LVH exceeds that following myocardial infarction. Over the past three decades there has been a significant decline in prevalence of ECG LVH concomitant with increased utilization of antihypertensive drug therapy. The recent introduction of echocardiography into the Framingham Heart Study has resulted in the development of new echocardiographic criteria for LVH. Echocardiographic LVH is more prevalent than ECG LVH. Corresponding prevalence rates for these two methods (per 1,000) are 174 vs. 24, respectively. Ambulatory ECG monitoring documents an association of echocardiographic LVH with increased risk for ventricular arrhythmias. Data from Framingham and elsewhere suggest that echocardiographically defined LVH is an important predictor of risk for cardiovascular disease morbidity and mortality. The extent to which prevention or regression of LVH, in response to antihypertensive drug therapy will alter the substantial risks associated with this condition, awaits additional investigation. PMID- 1715447 TI - Mitochondrial ultrastructural and molecular changes induced by zidovudine in rat hearts. AB - Zidovudine (azidothymidine (AZT)) inhibits human immunodeficiency virus replication, prolongs survival, and delays progression of acquired immune deficiency syndrome. We determined AZT-induced molecular and ultrastructural changes in the rat heart. Rats (3 per group) were given drinking water with or without AZT (0.2 to 1.0 mg/ml; 29 to 102 mg/kg/day). After 21, 35, or 49 days, hearts were glutaraldehyde-fixed by abdominal aortic perfusion, processed, and examined by transmission electron microscopy. In parallel, myocardial RNA was extracted from hearts (AZT dose: 1 mg/ml; 35 days) and subjected to Northern analysis using cDNA probes for: alpha c-actin, troponin C, mitochondrial creatine kinase and malate dehydrogenase, a portion of the mitochondrial genome containing cytochrome b coding region (pMM26), and glyceraldehyde-3-phosphate dehydrogenase. Results showed marked and widespread cardiac mitochondrial swelling with fractured and disrupted cristae after 35 days of 1 mg/ml AZT. After a 14-day recovery, these ultrastructural defects did not reverse. Changes were not present in myocardium after 21 days of AZT nor after 35 days of lower dose AZT (0.2 mg/ml). Mitochondrial cytochrome b mRNA expression was depressed in AZT-treated rat hearts (35 days; 1 mg/ml AZT). mRNAs encoding glyceraldehyde-3-phosphate dehydrogenase, alpha c-actin, troponin C, mitochondrial creatine kinase, malate dehydrogenase, and mitochondrial ribosomal RNAs remained unchanged. AZT disrupts cardiac mitochondrial ultrastructure and expression of mitochondrial cytochrome b mRNA in a dose- and time-dependent fashion. The mechanism of AZT cardiotoxicity may relate to inhibition of mitochondrial DNA replication (at the level of DNA polymerase gamma) as postulated by others. PMID- 1715450 TI - The effect of antihypertensive treatment on electrocardiogram voltages in the EWPHE trial. AB - Long-term effects of antihypertensive treatment on various electrocardiogram (ECG) voltages and the association between ECG findings at randomization and subsequent mortality were evaluated in the double-blind, placebo-controlled trial of elderly hypertensive patients, conducted by the European Working Party on High Blood Pressure in the Elderly (EWPHE). Patients were treated with a combination of hydrochlorothiazide and triamterene or matching placebo; methyldopa or placebo was added if blood pressures remained high. RaVL and SV1 + RV5 at randomization were related to systolic blood pressure (SBP) and RaVL was also related to diastolic blood pressure (DBP), after adjustment for age, gender, and body mass index (BMI). When adjusted for age and body mass index, the decreases in RaVL and SV1 + RV5 were not correlated with the changes in SBP after 1 year of active treatment but the decreases in SV1 + RV5 were positively related to the fall in DBP. After 4 years of active treatment, the fall in SBP was associated with decreases in RaVL and in SV1 + RV5, after adjustment for age and changes in BMI. PMID- 1715451 TI - Use of heterotopically isotransplanted rat heart to study the effect of load on cardiac mass. PMID- 1715452 TI - Cardiovascular response and cell membrane sodium transport systems in essential hypertension with cardiac hypertrophy. PMID- 1715453 TI - Role of intracellular cation abnormalities in development of left ventricular hypertrophy. AB - To elucidate factors determining the development of left ventricular hypertrophy, we performed measurements of intraerythrocyte sodium and intraplatelet calcium concentrations, measurements of plasma and urinary catecholamine levels and plasma renin activity, calculation of left ventricular mass using echocardiography, and 24-h ambulatory blood pressure (BP) monitoring. The results indicate that abnormalities of sodium and calcium mobilization may play an important role in the adaptation of left ventricular muscle to a persistent increase in BP. PMID- 1715454 TI - Stretch-activated channels in heart cells: relevance to cardiac hypertrophy. AB - Stretch-activated channels have been proposed as the transduction mechanism between load and protein synthesis in cardiac hypertrophy. Under this hypothesis, cardiac deformation is linked to an increased sodium (Na) influx, which, in turn, increases protein synthesis. We have tested whether stretch actually increases Na influx by applying patch-clamp techniques to cultured chick embryo cardiac myocytes and to freshly isolated adult guinea pig cardiomyocytes. Our experiments, in excised and cell-attached patches, revealed the existence of ionic channels that opened, or increased their frequency of opening, upon the application of negative pressures to the lumen of the patch-clamp pipettes. These stretch-sensitive channels allowed the passage of the major monovalent physiological cations, Na and potassium (K), and, to a much lesser extent, the major divalent cations calcium (Ca) and magnesium (Mg). Under normal conditions, the channels had a high open channel noise that prevented the customary, straightforward statistical analysis of single channel data. However, when one of the major monovalent cations was iso-osmotically replaced by sucrose, the open channel noise decreased significantly and permitted a good delineation of the open and closed channel states and, therefore, application of standard patch clamp, statistical analysis techniques. Under these "sucrose," "monoionic" conditions, the reversal potential was, as one should expect, close to the equilibrium potential for the major monovalent cation present. When high extracellular K solution was used to minimize the cell resting potential, the reversal potential for these stretch-activated currents was estimated to be around -40 mV. Therefore, under normal conditions, stretch should induce an inward, depolarizing current, carried mostly by Na ions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715455 TI - Interaction between sympathetic nervous system and adrenal medulla in the control of cardiovascular changes in hypertension. AB - The role of the sympathetic nervous system and adrenal medulla in the development of cardiovascular changes and hypertension was studied in spontaneously hypertensive rats (SHRs), and the results compared with age-matched normotensive Wistar-Kyoto (WKY) rats. Sympathectomy was initiated in newborn rats through daily injection with antiserum to nerve growth factor for 1 week, followed by daily injection with guanethidine for 3 weeks. Removal of the adrenal medulla was carried out in 4-week-old rats after the last guanethidine injection. Such a combination treatment was effective in permanently preventing the development of hypertension in the SHRs, and the blood pressure was maintained at the level of WKY rats. The heart rate of the SHRs and WKY rats was not affected by such treatment. Hypertrophy of the heart and of the vessel wall in the mesenteric arteries was also prevented by such treatment. We conclude that in the SHR, the sympathetic nervous system and the adrenal medulla are essential for the development of cardiovascular changes and hypertension. PMID- 1715456 TI - Interleukin-6 stimulates proliferation of cultured vascular smooth muscle cells independently of interleukin-1 beta. AB - The effects of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on proliferation of cultured vascular smooth muscle cells (VSMCs) were investigated. Treatment with IL-6 caused a rapid increase in the c-myc mRNA level, and resulted in increases in DNA synthesis and cell number. IL-1 beta stimulated the DNA synthesis of the cells. EGF showed synergistic and PDGF or IL-1 beta showed additive effects with IL-6 on the DNA synthesis. These results suggest that IL-6, independently of IL-1 beta, may be important in the proliferation of VSMCs. PMID- 1715457 TI - Changes in contractile and noncontractile protein metabolisms in both ventricles in monocrotaline-treated rats. AB - To clarify the metabolism of contractile and noncontractile proteins of both ventricles (BVs) during the development of right ventricular hypertrophy (RVH) induced by pressure overload, monocrotaline (M) was injected subcutaneously into Sprague-Dawley (SD) rats. Myosin isoenzymes (MIEs) were analyzed by pyrophosphate gel electrophoresis. Acid-soluble collagens were analyzed using improved noninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue collagen concentrations were also measured. M induced RVH, but not left ventricular hypertrophy, at 2 weeks, and severe RVH at 4 weeks. In right ventricles (RVs) of M-treated rats, MIE significantly shifted from V1 to V3, and the proportions of type III and V collagens increased compared to control at 2 and 4 weeks. In the left ventricles (LVs) of M-treated rats, similar but less remarkable MIE shifts were found without remodeling of collagen types at 2 and 4 weeks. Collagen concentrations of BVs treated with M did not show any significant changes compared to control at 2 and 4 weeks. Our results show remodelings of contractile and noncontractile proteins in RVs during the development of RVH, and also provide evidence for the changes in protein metabolism of the counterpart of RVs (i.e., LVs) during the development of cardiac hypertrophy. PMID- 1715458 TI - Left ventricular hypertrophy is more marked in salt-sensitive than in salt resistant hypertensive patients. AB - Besides the duration and severity of hypertension, several other factors have been shown to be related to left ventricular hypertrophy (LVH) in essential hypertension. The present study was conducted to examine the influence of salt sensitivity on LVH. Fifteen essential hypertensive ambulatory patients were submitted to a low-salt (30 mEq of Na/day for 7 days) and a high-salt (200 mEq of Na/day for 7 days) diet after 12 weeks on placebo. Daily urine collection was obtained during the whole study. After the placebo period, all patients were submitted to a complete clinical and laboratory investigation that included an echocardiogram (M-mode and two-dimensional). Five patients were salt-sensitive (mean blood pressure (BP) increase from the seventh day of the low- to the seventh day of the high-salt diet greater than 10%). No differences in weight, sex ratio, and duration of hypertension were obtained between salt-sensitive and resistant patients. The initial BP was higher in the salt-sensitive patients. However, the difference was small and without statistical significance. The left ventricular weight was higher in the salt-sensitive than in salt-resistant patients (148 +/- 51 vs. 109 +/- 32 g/m2, p less than 0.05). The left ventricular end-diastolic diameter was also higher in the salt-sensitive patients (50 +/- 10 vs. 43 +/- 6 mm, p less than 0.05). The interventricular septum and posterior wall thicknesses were higher in salt-sensitive patients, although they did not reach statistical significance. In conclusion, salt-sensitive essential hypertensive patients are at a higher risk to develop LVH. PMID- 1715459 TI - Neither awake nor sleep blood pressures better predict target-organ effect. AB - We sought to assess the strengths of commonly used ambulatory blood pressure (ABP) parameters as predictors of transmitral flow/velocity ratio (E/A) and the dimensions that govern the left ventricular (LV) mass index. ABP, E/A, and LV dimensions were assessed in 47 subjects, with inclusion of 45 in the final data analysis. The results of four weekly (96 h total) ABP studies were averaged for each subject. No single ABP parameter immerged as a "best predictor." Furthermore, casual blood pressures taken at the beginning of the echo examination had predictive strength for LV mass which was similar to that of the ambulatory data. In the present study, neither awake nor sleep ABPs differed significantly with respect to correlations with indices of LV structure. PMID- 1715460 TI - Cardiac performance in elderly hypertensive patients with left ventricular hypertrophy: responses to isometric exercise and beta-agonists. AB - To assess the cardiac functional reserve of elderly hypertensive patients with left ventricular hypertrophy (LVH) we studied cardiac function after isometric exercise and beta-adrenergic stimulation. Forty-five elderly hypertensive and 16 normotensive patients (NC group) were recruited for the study. The hypertensive patients were divided into two groups: those with LVH (n = 17) mass index (LV mass index greater than 130 g/m2; H1 group) and those without LVH (n = 28) (H2 group). Echocardiographic studies were performed before and after isometric exercise (handgrip) and isoproterenol (ISP) administration. We measured the LV mass index, fractional shortening (FS), isovolumic relaxation time (IRT), and the ratio of late and early diastolic transmitral flow velocity (A/E). The FS at rest in the H1 group was significantly higher than those in the H2 and NC groups. In the H1 group, the IRT was elongated and A/E was greater than in the NC group, which indicated the impaired diastolic function in the H1 group. After the HG stress, the FS in the H1 group significantly decreased whereas it did not change in the H2 or NC groups. The FS increased in all three groups after the infusion of ISP, although the increment of FS was smaller in the H1 group. In conclusion, (a) diastolic function was impaired whereas systolic function was supranormal at rest in the hypertrophied heart of the elderly hypertensive patients and (b) when exercise or pharmacological stress was loaded, the systolic function deteriorated, suggesting the impairment of cardiac reserve in those patients. PMID- 1715461 TI - Impaired left ventricular function during exercise in hypertensive patients with normal coronary arteriograms. AB - Patients with left ventricular hypertrophy (LVH) often exhibit manifestations of myocardial ischemia. In 17 hypertensive patients (group 1, mean age of 56 +/- 4 years, 10 females, 7 males) with ST-segment depression during the exercise electrocardiogram (ECG) and effort angina and normal coronary arteriograms, the left ventricular function at rest and during exercise was studied by heart catheterization. The results were compared with 17 hypertensive patients (group 2, mean age of 56 +/- 6 years, 6 females, 11 males) with coronary artery disease (CAD). The normal pulmonary wedge pressure at rest (group 1, 8.9 +/- 3 mm Hg; group 2, 8.9 +/- 3 mm Hg) was pathologically increased (p less than 0.001) in both groups (group 1, 27.1 +/- 5 mm Hg; group 2, 28.8 +/- 7 mm Hg) even at a work load of 50 W with a further increase at 75 W to 31 +/- 4 and 29.7 +/- 4 mm Hg, respectively. Cardiac output was normal. There was no significant correlation between ST-segment depression, pulmonary wedge pressure, LVH, and Holter ECG. Hypertensive patients without CAD may reveal a disturbed pump function due to ischemia even at low work loads, which does not differ significantly from patients with CAD. This may provoke subendocardial fibrosis and thereby contribute to the development of heart failure. PMID- 1715462 TI - Left ventricular hypertrophy as a risk factor for arrhythmias in hemodialysis patients. AB - The contribution of left ventricular hypertrophy in determining ventricular arrhythmias (VAS) was studied in 81 chronic renal failure patients in chronic hemodialysis using two-dimensional echocardiographic and electrocardiogram Holter monitoring. The prevalence of LVH was 93% (96% in hypertensive and 87% in normotensive patients). The prevalence of VA was 48%. These arrhythmias were associated with increased cardiac mass, lack of potassium supplementation to the hemodialysis bath, and low K+ and PaO2 during dialysis. Severe forms of VA occurred in 19 of 78 patients, and the risk factors for this occurrence were (a) largely increased cardiac mass indices (exceeding in more than 40% the upper limit of normal for each sex) and (b) prolonged periods of time in hemodialysis treatment (34 +/- 5.5 vs. 17 +/- 2.7 months, p less than 0.05). Changes in potassium or oxygen content of the blood were not significantly associated with the occurrence of severe forms of VA. PMID- 1715463 TI - Dipyridamole-thallium tests are predictive of severe cardiac arrhythmias in patients with left ventricular hypertrophy. AB - In a population of patients with chronic renal failure (CRF) and a high prevalence of left ventricular hypertrophy (LVH) undergoing chronic hemodialysis, we investigated the association between the results of dipyridamole-thallium tests (DTTs) and the occurrence of ventricular arrhythmias. We observed a positive significant association between positive DTTs and the occurrence of severe forms of ventricular arrhythmias. A significant association was also observed between the presence of severe LVH and the occurrence of severe ventricular arrhythmias. However, no association was found between the presence of LVH and the positivity of the DTT. As most of our patients with positive DTTs had unimpaired coronary circulations, we conclude that positive DTTs, although falsely indicative of impaired myocardial blood supply, does have an important clinical relevance, indicating increased risk of morbidity (and, possibly, mortality) due to ventricular arrhythmias in a population of CRF patients submitted to chronic renal function replacement program. PMID- 1715464 TI - Signals for the remodeling of the cardiac interstitium in systemic hypertension. AB - Cardiac myocyte growth is the common denominator in myocardial hypertrophy irrespective of the hypertrophic stimulus. The hypertrophic remodeling of the myocardium may or may not also include the growth of nonmyocyte cells, thereby creating the potential for heterogeneity in tissue growth. Hypertrophy, therefore, need not be a uniform process, especially if trophic factors responsible for myocyte and nonmyocyte growth are independent of one another. To examine this hypothesis further, we determined the relative importance of hemodynamic and hormonal factors in augmenting ventricular mass and cardiac fibroblast-induced collagen accumulation in several rat (Sprague-Dawley) models of arterial hypertension: renovascular hypertension (RHT), infrarenal aorta banding (IRB), and chronic aldosterone (ALDO) administration. Elevations in arterial pressure were comparable in each, whereas circulating angiotension II (Ang II) and ALDO were dissimilar: in RHT, each was increased; with IRB they were normal; and with chronic ALDO, Ang II was suppressed whereas ALDO was increased. We reasoned that because of the in-series arrangement of the ventricles, where only the left ventricle (LV) experienced an elevation in systolic pressure, the right ventricle (RV) served as a negative control regarding hemodynamic factors. Relative to the in-parallel arrangement of the ventricles, provided by the coronary circulation, the RV served as a positive control for circulating hormones.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715465 TI - Electrophysiological responses of hypertrophied rat myocardium to combined hypoxia, hyperkalemia, and acidosis. AB - We studied whether electrophysiological response to ischemia could be different in hypertrophied left ventricle of spontaneously hypertensive rats (SHRs) and in normal left ventricle of normotensive Wistar-Kyoto (WKY) rats. For that purpose, we used a two-compartment tissue bath in which one-half of a left ventricular strip was exposed to normal conditions and the other part to ischemic conditions (low pH, hypoxia, and hyperkalemia). Electrical activity was recorded using standard microelectrodes in normal and hypertrophied preparations. Under basal conditions, the action potential duration (APD) and effective refractory period (ERP) were longer in SHRs than in WKY rats (118 +/- 9 ms vs 81 +/- 3 ms, p less than 0.05 and 90 +/- 9 ms vs. 74 +/- 4 ms, p less than 0.05, respectively). During ischemia, the APD and ERP changed in opposite ways in both groups and we observed the development of postrepolarization refractoriness that was greater in hypertrophied than in normal hearts. Maximum upstroke velocity (Vmax) values were 229 +/- 12 and 227 +/- 10 V/s in the SHR and the WKY preparations, respectively. Three minutes after the induction of ischemia, Vmax values decreased to 46 +/- 7 V/s in SHRs and to 106 +/- 12 V/s in WKY rats. A significant increase in subendocardial collagen density was measured in SHR left ventricle compared to WKY rats (4.39 +/- 0.34% vs. 1.66 +/- 0.15%, p less than 0.05), which might partly explain the impaired conduction observed in hypertrophied preparations during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715466 TI - Prevalence of late ventricular potentials in hypertensive patients. AB - Late ventricular potentials detected by signal averaging are used for predicting ventricular arrhythmias and sudden cardiac death in patients with coronary artery disease. We studied the prevalence of signal-averaged late ventricular potentials in 37 male patients (mean age of 56 years) with angiographically normal coronary arteries. Seventeen patients were hypertensive (group A) and 20 patients were normotensive (group B). In group A, 5 of 17 patients (29%) had late ventricular potentials. In group B, only 1 of 20 patients (5%) had late potentials (p less than 0.05). Late ventricular potentials detected by signal averaging are more common in hypertensive patients than in healthy controls. Whether late ventricular potentials can be used to predict malignant ventricular arrhythmias or sudden cardiac death in hypertensive patients will have to be investigated in further studies. PMID- 1715467 TI - Scintigraphic assessment of cardiac adrenergic innervation in patients with essential hypertension. AB - To assess the regional cardiac adrenergic innervation in patients with essential hypertension (EHT), simultaneous iodine-123 metaiodobenzylguanidine ([123I]MIBG) and thallium-201 (201Tl) myocardial imagings were performed in five patients with EHT, seven patients with hypertrophic cardiomyopathy (HCM), and seven normal subjects. Short axial images at rest were divided into five segments: anterior, septal, posterior, lateral, and apical segments. Percent regional uptake (%RU) of 201Tl except the septal segment in patients with EHT showed no significant difference. However, the %RU of [123I]MIBG at posterior, lateral, and apical segments was significantly lower than that at anterior and septal segments in EHT. This intraimage heterogeneity of [123I]MIBG was also observed in HCM. These results suggest that there is a difference in regional adrenergic innervation of the left ventricle with myocardial hypertrophy. PMID- 1715468 TI - Capillarization of the hypertrophic heart: discrepancy of the results obtained by the triangulation and domain methods. AB - The geometry of capillary networks was examined in pressure-overloaded hypertrophic rat myocardium. All hearts were exposed to a staining protocol that distinguished the arteriolar (AC) and venular (VC) regions of capillaries. In control hearts, the triangulation method (one-dimensional distance between neighboring capillaries) and the domain method (two-dimensional area surrounding capillaries) exhibited similar results, showing increased capillarization for the venular region of capillaries networks. In hypertrophic hearts, the triangulation method did not demonstrate differences between arteriolar and venular capillaries (AC = 27.2 +/- 0.4 microns, VC = 27.0 +/- 0.2 microns, p = 0.56). However, the domain method still detected smaller tissue supply regions for venular capillaries (AC = 547 +/- 6 microns 2, VC = 464 +/- 5 microns 2, p less than 0.01). To resolve this discrepancy in hypertrophic heart, we have measured capillary spacing from longitudinal sections at discrete intervals along the A-V path length. These results indicate a stepwise decrease in intercapillary distance from arteriole to venule for both groups, which would support results obtained by the domain method. A possible explanation for the discrepancy between these two methods, specific to hypertrophic heart, may be changes in the clustering patterns of arteriolar and venular capillaries. PMID- 1715469 TI - Antiproliferative effects of the serotonin type 2 receptor antagonist, ketanserin, on smooth muscle cell growth in rats. AB - We defined the role of a serotonin type 2 receptor antagonist, ketanserin, in the growth of aortic vascular smooth muscle cells (VSMCs) from Wistar rats, using cell culture and cell synchrony methods. Deoxyribonucleic acid (DNA) replication in the G0/G1- or G1/S-synchronized VSMCs was assessed by [3H]thymidine uptake into DNA. Ketanserin at 2 x 10(-5) M significantly decreased the thymidine uptake by 48% in the proliferating VSMCs, whereas methysergide, a nonspecific serotonin inhibitor, unaffected the thymidine uptake. Ketanserin at 10(-5) M did not influence the duration of the G1 resting period. However, this dose of ketanserin significantly lowered DNA replication in the DNA synthetic (S) period in a dose dependent manner. Neither methysergide nor the alpha 1-adrenoceptor antagonist, prazosin, affected DNA synthesis in the S period. Ketanserin exhibits antiproliferative effects on rat VSMC growth probably through the suppression of DNA replication in the S phase. This property would also contribute to the vascular protective effects of ketanserin with its well-documented antihypertensive action. PMID- 1715470 TI - Effects of long-term blood pressure control on left and right ventricular structure and function in hypertensive patients. AB - The functional and anatomical abnormalities of the right ventricle may occur in hypertensive patients with left ventricular hypertrophy (LVH). The present study was designed to assess the functional and structural changes in both left and right ventricles induced by chronic antihypertensive therapy. Doppler and standard echocardiography were performed in 10 hypertensive patients with LVH before and after 1 year of treatment with captopril alone (5 patients) or captopril plus nifedipine (5 patients). We found that the left ventricular mass index and right ventricular thickness were significantly reduced in all patients when compared with pretreatment values. Furthermore, the Doppler-derived diastolic filling indexes show a significant improvement of both ventricular chambers. Our data suggest that anatomical and functional changes induced by therapy in hypertensive patients are not limited to the left ventricle but also involve the right ventricle. PMID- 1715471 TI - Improvement of diastolic filling in hypertensive patients treated with cilazapril. AB - The aim of this study was to assess the behavior of the diastolic left ventricular (LV) filling pattern and cardiac hypertrophy after treatment with cilazapril. Twelve patients (9 male and 3 female) with mild to moderate essential hypertension, aged 46 +/- 14.1 years, were treated with cilazapril for 1 year. They underwent Doppler echocardiography at the beginning (I), after 6 months (II), and after 1 year (III) of treatment. The following parameters were evaluated: mean arterial pressure (MAP) automatically recorded for 24 h, interventricular septal and posterior wall thickness, LV and end-diastolic diameter, LV mass index, early (E) and late (A) diastolic filling flow velocities, and the ratio E/A. A significant reduction in LV hypertrophy and an improved diastolic filling pattern of the left ventricle was shown after 6 months of therapy with cilazapril; this improvement still remained after 1 year of therapy. PMID- 1715472 TI - Effects of antihypertensive treatment on cardiac hypertrophy and cardiac function in elderly hypertensive patients. AB - The effects of antihypertensive treatment with calcium antagonists or angiotensin converting enzyme (ACE) inhibitors on the reversal of left ventricular hypertrophy and the left ventricular function in elderly hypertensive patients were examined. Twenty-four elderly hypertensive patients with cardiac hypertrophy, aged from 65 to 79 years (mean +/- SEM of 71 +/- 1 years), were treated with a calcium antagonist (nifedipine or nicardipine) or ACE inhibitor (captopril or enalapril) for 3 months. Thirteen patients had essential hypertension [EH: systolic blood pressure (SBP) greater than or equal to 160 mm Hg and diastolic blood pressure (DBP) greater than or equal to 90 mm Hg, aged 70 +/- 1 years] and 11 had isolated systolic hypertension (ISH:SBP greater than or equal to 160 mm Hg and DBP less than 90 mm Hg, aged 74 +/- 2 years). All patients underwent M-mode echocardiography to assess left ventricular mass index (LVMI) and left ventricular function (ejection fraction, EF) before and after 3 months of treatment. BP significantly decreased from 174 +/- 3/97 +/- 1 to 144 +/- 5/84 +/- 2 mm Hg in EH and from 167 +/- 3/82 +/- 2 to 144 +/- 4/74 +/- 2 mm Hg in ISH. The LVMI was also significantly reduced from 204 +/- 14 to 174 +/- 16 g/m2 in EH and from 179 +/- 14 to 156 +/- 12 g/m2 in ISH. EF showed no significant changes with treatment in either group. In elderly hypertensive patients, the antihypertensive treatment with calcium antagonist or ACE inhibitor reduced cardiac hypertrophy without any deterioration of left ventricular function in both essential hypertension and isolated systolic hypertension. PMID- 1715473 TI - Contrasting effects of calcium antagonists vs. arterial vasodilators on cardiac anatomy and sympathetic activity in spontaneously hypertensive rats. AB - In spontaneously hypertensive rats (SHRs) we evaluated the effects of 35 and 70 days of treatment with nisoldipine (2 mg/g of food) vs. minoxidil (120 mg/L of drinking water) on cardiac anatomy [i.e., left ventricular (LV) and right ventricular (RV) weights and LV internal diameter and wall thickness] and cardiac sympathetic activity assessed by the norepinephrine turnover rate. The minoxidil induced antihypertensive response was associated with a marked increase in cardiac sympathetic activity, potentiation of RV hypertrophy (RVH), and the development of eccentric LV hypertrophy (LVH). Nisoldipine decreased both blood pressure (BP) and cardiac sympathetic activity, but caused only small decreases in LV weight and LV wall thickness and no change in RV weight. With regard to minoxidil, the increase in sympathetic activity may contribute to the minoxidil induced potentiation of cardiac mass. Nisoldipine, despite decreasing BP as well as cardiac sympathetic activity, unexpectedly resulted in only a small decrease in cardiac mass, suggesting that additional mechanisms may play a role in the effects of calcium antagonists on cardiac mass. PMID- 1715474 TI - Opposite effects of arterial vasodilators on cardiac vs. arterial hypertrophy and sympathetic activity in spontaneously hypertensive rats. AB - To investigate whether cardiac and arterial structure and sympathetic activity changes in a similar fashion during chronic arterial vasodilation, we evaluated the morphology and sympathetic activity of the mesenteric arterial bed and the left (LV) and right (RV) ventricles of 16-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs) after 35 and 70 days of treatment with the arterial vasodilator minoxidil. The minoxidil-induced antihypertensive response was associated with an increase in ventricular sympathetic activity, potentiation of RV hypertrophy (RVH), and the development of eccentric LV hypertrophy (LVH). In the mesenteric arterial bed, minoxidil decreased the sympathetic activity, increased the lumen of the superior mesenteric artery, and decreased the medial area of the large and small mesenteric arteries. We conclude that the contrasting effects of minoxidil on cardiac vs. arterial structure may--in part--relate to selective effects on regional sympathetic activity. PMID- 1715475 TI - Effect of nifedipine GITS on left ventricular mass and diastolic function in severe hypertension. AB - Treatment of severe hypertension is beneficial, but reversibility of target-organ damage has not been characterized. Serial studies were performed in 15 patients with severe essential hypertension (age of 56 +/- 3 years, mean +/- SEM) treated for 1 year with 60 to 150 mg/day of continuous-release nifedipine; 3 patients required 50 mg of chlorthalidone/day to lower diastolic blood pressure (BP) to less than 95 mm Hg. Left ventricular (LV) structure and function was evaluated with two-dimensional-directed M-mode echocardiography, digitized from videotape and analyzed blindly. BP was markedly reduced from 194 +/- 8/115 +/- 4 to 146 +/- 4/88 +/- 14 mm Hg (p less than 0.0001) and maintained at this level for 1 year. Posterior wall and septal LV thickness, elevated at entry (12.9 +/- 0.1 and 13.4 +/- 0.1 mm), dropped steadily over 1 year into the normal range (10.0 +/- 0.03 and 11.2 +/- 0.1 mm, p less than 0.001). LV mass index, above 95% for normals at entry, decreased by 19% at 6 months (129 +/- 10 to 104 +/- 7 g/m2, p less than 0.01), and remained at this level at 1 year. LV fractional shortening rose steadily over 1 year from 34 to 42% (p less than 0.02). Atrial natriuretic peptide, which reflects LV filling pressures, was markedly elevated at entry, but was significantly reduced by 6 months (76 +/- 22 vs. 45 +/- 14 pg/ml, p less than 0.05). Sustained reduction of arterial BP with continuous-release nifedipine for 1 year normalizes LV mass, improves LV systolic function, and reduces circulating levels of atrial natriuretic peptide. PMID- 1715476 TI - Improvement of cardiac contractile response to beta-adrenergic stimulation in normal and two-kidney, one-clip hypertensive rats treated with nitrendipine. AB - To evaluate the effect of nitrendipine on cardiac hypertrophy and inotropic response to isoproterenol in two-kidney, one-clip (2K,1C) renovascular hypertension, male Wistar rats (n = 56) were divided into a clipped group (K) (n = 28) and a sham group (S) (n = 28). Twenty-one days after surgery, the rats were placed in metabolic cages where they received either a normal diet (S and K rats) or a similar one containing N (18 mg/day) (SN and KN rats) during 3 weeks. Arterial pressure and body weight were measured twice a week. At the end of the experimental period, the hearts were excised, the ventricles were weighed, and cardiac proteins were measured by the Lowry method in seven hearts of each group. In the remaining hearts, the left ventricular papillary muscle was excised and mounted in a bath where the developed tension (dt) and the maximal rate of rise of developed tension (+T) were recorded in basal conditions, and after cumulative doses of isoproterenol (10(-11) to 10(-4) M). The arterial pressure, ventricular weight, and cardiac proteins were similar in S, SN, and KN groups and were significantly higher in the K group (p less than 0.05). No difference in the basal values of +T and dt were observed among the four groups. The increment in +T after isoproterenol was higher in treated (KN and SN) than in nontreated (K and S) groups (p less than 0.05). In conclusion, nitrendipine enhances the contractile response to isoproterenol and reverses the cardiac hypertrophy in the 2K, 1C model. PMID- 1715477 TI - Improved left ventricular systolic and diastolic function after regression of cardiac hypertrophy, treatment withdrawal, and redevelopment of hypertension. AB - The aim of this study was to differentiate the effects of changes of arterial pressure from those of regression of left ventricular hypertrophy (LVH) on systolic and diastolic function at rest and during a rapid increase in afterload, induced by handgrip and cold pressor tests. An additional purpose was to assess the hemodynamic mechanisms responsible for the increase in arterial pressure after treatment withdrawal. Therefore, we evaluated the cardiac anatomy and function (TM echo) at rest and during handgrip and cold pressor tests in 23 hypertensive patients, before therapy and 20-30 days after withdrawal of treatment, which had lasted 6-12 months, when left ventricular mass index (LVMI) was significantly reduced (from 140 +/- 44 to 113 +/- 13 g/m2, p less than 0.001) but the arterial pressure had increased again to pretreatment values (166 +/- 19/105 +/- 7 mm Hg before treatment vs. 162 +/- 15/100 +/- 12 mm Hg). The LVMI reduction was due to a decrease in LV wall thickness, whereas the LV internal dimensions did not change. Systolic function was evaluated by the relationship between LV end-systolic stress (ESS) and fractional shortening (FS): at rest as well as at the peak of handgrip and cold pressor tests, highly significant negative correlations between ESS and FS (range of -0.71 to -0.96) were found. Considering each point of relation between ESS and FS in hypertensive patients, in comparison with 95% prediction limits of the correlation obtained in normal subjects, a "supernormal" systolic function at rest was observed in 10 of 23 patients and a reduced systolic function was not present after treatment withdrawal and redevelopment of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715478 TI - Once- vs. twice-daily nitrendipine in the treatment of mild to moderate hypertension, Canadian Nitrendipine Study Group. AB - Improved measurement of the plasma concentration of nitrendipine demonstrated a plasma half-life of 17-21 h, allowing once-daily (o.d.) instead of currently twice-daily (b.i.d.) dosing. To determine the effectiveness of nitrendipine given o.d. vs. b.i.d., 78 hypertensive patients, [supine diastolic blood pressure (DBP) of 95-114 mm Hg] were randomized in a double-blind fashion to 12 weeks of treatment with either nitrendipine 10 mg b.i.d. (n = 39) or nitrendipine 20 mg o.d. (n = 39) after a 2-week placebo baseline period. Blood pressures (BPs) were measured in the morning at the end of the dosing interval. Mean +/- SD reduction in supine systolic BP (SBP) and DBP in patients evaluable for efficacy (greater than or equal to 14 days treatment) were 7.2 +/- 16.5 and 7.7 +/- 10.3 mm Hg, respectively, after nitrendipine b.i.d. (n = 38) and 9.4 +/- 15.1 and 9.5 +/- 7.0 mm Hg, respectively, after nitrendipine o.d. (n = 36). Similar falls in BP were found for both regimens in patients completing the full 12 weeks of treatment period (n: o.d. = 28, b.i.d. = 32). Discontinuation due to adverse experiences (AEs) occurred in three patients on b.i.d. and eight patients on o.d., the latter mostly in the first 2 weeks of therapy. Overall, AEs were higher in the o.d. group (% AEs at least possibly related to study medication: o.d. = 44%, b.i.d. = 33%). Most frequent AEs were headache and flushing.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715479 TI - Sympathetic activity: modulator of myocardial hypertrophy. AB - The mechanisms regulating myocardial hypertrophy are largely unknown. Furthermore, the hypertrophic phenotype can be associated with either normal or abnormal function. To study the molecular mechanisms involved in myocardial hypertrophy, we have established a cell culture system in which stimulation of the alpha 1-adrenergic receptor leads to the development of myocardial cell hypertrophy. In addition to producing a generalized twofold increase in both cell size, total protein, and total RNA, activation of the alpha 1-receptor produces specific alterations in gene expression that are reflected by changes at both the mRNA and protein levels. In particular, alpha 1 stimulation leads to an increase in the expression of the c-myc oncogene as well as a selective increase in skeletal alpha-actin and beta-myosin heavy-chain isogene expression, isoforms normally found only in fetal/neonatal hearts. Similar changes in gene expression are seen in pressure-load hypertrophy in vivo. Skeletal alpha-actin gene expression is induced preferentially to that of the cardiac actin isogene resulting from a specific preferential increase in gene transcription. Work with subtype-specific inhibitors indicates that it is a particular alpha 1-receptor subtype that is responsible for the development of hypertrophy in culture. The finding that alpha 1 stimulation leads to an increase in protein kinase C activity is suggestive of a potential second messenger involving the phosphorylation of a transcriptional factor or factors. PMID- 1715480 TI - Significance of cardiovascular hypertrophy in the development and maintenance of hypertension. AB - The amplifier properties associated with the structural changes in the heart and resistance vessels in chronic hypertension together play a major role in maintaining the elevated blood pressure (BP) in chronic hypertension, which is greater than that of the initiating cause. In patients with primary hypertension, long-term antihypertensive drug therapy causes substantial regression of the structural changes, as assessed by normalization of the total peripheral resistance (including the nonautonomic component) and of the left ventricular (LV) mass. Reversal of LV hypertrophy (LVH) took considerably longer than reversal of the vascular changes and the more complete the reversal of LVH, the slower the rate of redevelopment of hypertension upon cessation of treatment. We observed no change in sympathetic activity or in renin-angiotensin-aldosterone levels during the redevelopment phase and we hypothesized that the cardiovascular amplifiers played a role in pathogenesis. This hypothesis was examined in spontaneously hypertensive rats (SHRs), where the vascular amplifier properties were developed substantially by 4 weeks of age, i.e., before the development of hypertension, whereas LVH occurred pari passu the rise in BP. When young SHRs were treated with enalapril for brief periods, there was almost complete long term regression of vascular amplifier properties, but attenuation of LVH and hypertension were both smaller and more transient. In 4-week-old SHRs, complete abolition of sympathetic activity had a minimal effect on vascular amplifier properties, but affected LVH. Our findings suggest that LVH is as important in both the development and maintenance of hypertension as the structurally determined amplifier properties of the resistance vessels. PMID- 1715481 TI - Left ventricular mass as an indicator of hemodynamic load in hypertension. PMID- 1715482 TI - Does hypertension potentiate atherosclerosis via vascular hypertrophy? PMID- 1715483 TI - Coronary circulation in arterial hypertension. AB - Arterial hypertension is the most common cause of congestive heart failure and an important risk factor in coronary artery disease (CAD). However, even in the absence of CAD, coronary reserve is frequently impaired in hypertensive patients. To study whether the reduced coronary reserve is due to the degree of left ventricular hypertrophy (LVH) or is a consequence of primary vascular alterations, coronary reserve was determined in 31 hypertensive patients (age of 56 +/- 10 years; systolic/diastolic blood pressure of 167 +/- 18/98 +/- 9 mm Hg) with angina pectoris and normal coronary angiogram. Coronary reserve was determined by measuring coronary resistance before and after dipyridamole (0.5 mg/kg of body weight i.v.). Coronary blood flow was measured quantitatively by the gas chromatographic argon method. LV muscle mass was measured by ventriculography. Twelve normotensive patients (age of 52 +/- 8 years) were studied for comparison. Coronary resistance was 20% higher in hypertensive than in normotensive patients, whereas coronary blood flow at rest was not significantly different. The maximal coronary blood flow after dipyridamole was 40% lower in hypertensive than in normotensive patients; accordingly, minimal coronary resistance was significantly increased by 112% in hypertensive patients (p less than 0.0005). Coronary reserve was reduced by 37% (p less than 0.001) in hypertensive patients compared with normotensive patients. Between the impairment in coronary reserve and left ventricular muscle mass, no significant correlation (r = 0.024, n.s.) was found. The impaired coronary vasodilator reserve is reflected by episodes of transient myocardial ischemia during ST-segment monitoring. PMID- 1715484 TI - Effects of coronary artery occlusion in animals with hypertension and left ventricular hypertrophy. AB - Chronic arterial hypertension (HT) and left ventricular hypertrophy (LVH) increase the morbidity and mortality of acute myocardial infarction in patients. In this article, we discuss earlier studies from Koyanagi et al. in our laboratory that showed that when animals with chronic HT and LVH (HT-LVH) were subjected to acute coronary artery occlusion (CAO), there was a 3.5-fold increase in mortality and a 35% increase in infarct size expressed as a percent of the area at risk. We subsequently determined the effect of HT-LVH on the wavefront of myocardial infarction. Dogs were made hypertensive using a single-kidney, single clip model of renovascular hypertension that produced mean arterial blood pressure (BP) = 141 +/- 3 mm Hg and left ventricular:body weight = 5.8 +/- 0.1 g/kg (p less than 0.05 vs. control animals). Conscious animals with HT-LVH and control animals were subjected to 1 or 3 h of CAO. Infarct and risk areas were measured using triphenyltetrazolium chloride (TTC) stain and barium angiography, respectively. The results suggested that the wavefront of infarction was accelerated in animals with HT-LVH. Further studies suggested that the wavefront of myocardial infarction could be markedly retarded by normalizing blood pressure (nitroprusside) 1 h following CAO. Recent studies in an animal model of HT-LVH suggested that electrophysiological abnormalities occur when these animals were subjected to CAO. Sixty-five percent of animals with HT-LVH had sudden death during CAO compared to 27% of the control group. We studied whether chronic beta adrenergic blockade would reduce mortality associated with CAO in animals with HT LVH.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715485 TI - Determinants of ventricular arrhythmias in cardiac hypertrophy. AB - Ventricular arrhythmias occur with increased frequency in both experimental and human cardiac hypertrophy. Although the process of hypertrophy itself may be arrhythmogenic, other factors may contribute to the high prevalence of arrhythmias in hypertensive patients with left ventricular hypertrophy (LVH). Disease of the large epicardial coronary arteries or of the small intramyocardial vessels (coronary microangiopathy) may lead to myocardial ischemia and thus predispose to arrhythmia. Myocardial fibrosis, a common sequelae of cardiac hypertrophy, has also been shown to be associated with ventricular arrhythmias in experimental models. Other possible determinants of ventricular arrhythmias in this group of patients include metabolic abnormalities; studies relating to the importance of hypokalemia in particular have yielded conflicting results. Thus a number of factors may combine to explain the high prevalence of ventricular arrhythmias in hypertensive patients with LVH. PMID- 1715486 TI - Antihypertensive drugs and cardiac trophic mechanisms. AB - In hypertension, increased systolic blood pressure (BP) and the resulting increase in systolic wall stress are major determinants of the degree of left ventricular hypertrophy (LVH). Antihypertensive drugs all decrease BP, but different classes of these drugs may activate other trophic mechanisms and therefore may have different effects on LVH. Angiotensin-converting enzyme (ACE) inhibitors decrease the major cardiac growth-promoting factors such as systolic wall stress, diastolic wall stress, and cardiac sympathetic and renin activity, and consistently cause regression of LVH. On the other end of the drug spectrum, arterial vasodilators may decrease systolic wall stress, but increase diastolic wall stress and cardiac sympathetic and renin activity, resulting in either the absence of regression or even progression of LVH. Other classes of antihypertensive drugs nonuniformly change neural, humoral, or mechanical stimuli, so that the net effect ranges from full regression, partial regression, to none. Age and reactivity of sympathetic and/or renin activity may play a major role in determining the response of cardiac mass to BP lowering. PMID- 1715487 TI - Resistance vessel structure: effects of treatment. AB - The purpose of this article is to review the evidence that the resistance vasculature is altered in hypertension, the role that it may play in the pathogenesis of the disease, and the effect of antihypertensive treatment on the abnormalities. In some models of hypertension, functional changes (i.e., increased vascular smooth muscle sensitivity) have been found, but in human essential hypertension, it appears that structural changes in the resistance vasculature predominate. The structural changes result in an increased media:lumen ratio of the resistance vessels, but it is not clear if these changes are also associated with an increased synthesis of vascular wall material, or whether they can alone be due to a remodeling of the vascular wall (i.e., redistribution of existing material). In both essential hypertension and in the spontaneously hypertensive rat, vascular smooth muscle volume appears to be normal, but is increased in renal hypertensive rats, suggesting that hypertrophy may be a response to imposed increases in load. Although there seems to be a close correlation between altered media:lumen ration and blood pressure in all forms of hypertension investigated, it is generally found to be difficult to obtain full regression of vascular structure. The reason for this remains obscure. PMID- 1715488 TI - Effects of antihypertensive drug classes on regression of connective tissue components of hypertension. AB - Hypertension leads to structural and functional adaptations which, although initially protective for the cardiovascular system, ultimately work to sustain and reinforce the hypertensive state. Although blood pressure (BP) may be effectively lowered by a variety of treatments, it is becoming clear that these structural adaptation, and the risks and consequences of hypertension, may persist long after BP has been restored to normal levels. Cardiovascular tissues appear to be highly sensitive to increased BP, responding quickly with large and proportional increases in collagen and elastin. The sensitivity of this response to increased pressure, together with the slow turnover of these connective tissue proteins, suggests a mechanism by which transient or intermittent episodes of hypertension may lead to cumulative and persistent structural changes in cardiovascular tissues. With some exceptions, studies directly investigating the reversibility of these connective tissue changes have generally confirmed that increased cardiovascular collagen and elastin persist for long periods of time after BP lowering, independent of the treatment method used to achieve that lowering. Because maintenance of elevated levels of collagen and elastin is principally caused by the slow turnover of the proteins rather than by their continued production, rapid and effective reversal of cardiovascular connective tissue changes may require the development of pharmacological agents that promote or accelerate the turnover of these proteins. PMID- 1715489 TI - Signals for cardiac muscle hypertrophy in hypertension. AB - Hypertension is associated with a rise in arterial pressure and a compensatory increase in cardiac mass, which if not treated effectively, progresses to decompensated congestive heart failure. This decompensation of an initially compensatory hypertrophy has intensified interest in the factors that initiate and maintain the development of cardiac hypertrophy. The potential signals that induce the development of cardiac hypertrophy are grouped as hemodynamic, growth promoting hormonal, vasoconstriction-promoting hormonal, and genetic factors. Growth-promoting hormones such as insulin and thyroxine appear to play a permissive, but essential, role in the development and maintenance of cardiac hypertrophy. However, changes in cardiac load, both above and below normal, result in parallel changes in cardiac mass, which will return to normal when a normal load is restored. This adaptive response of the myocardium in direct response to elevated and depressed loads demonstrates that cardiac structure, composition, and function are not fixed postneonatal cardiac properties, but instead are regulated dynamically by the cardiocyte loading environment. This adaptive response is subject to modulation by vasoconstriction-promoting hormones and genetic factors. The current thrust in this research area is to elucidate the cellular signals that transduce the physical stimulus for hypertrophy into biochemical events underlying hypertrophic cardiac growth. To remove complex systemic interactions in vivo from the experimental paradigm, several in vitro models have been used to examine three general, but distinct, cellular pathways involving protein kinase C activation, cyclic AMP formation, and increased ion fluxes. Each pathway demonstrated a stimulatory effect on general protein synthesis, which is necessary for growth in all cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715490 TI - Effects of exercise and other nonpharmacological measures on blood pressure and cardiac hypertrophy. AB - Reversal of left ventricular hypertrophy (LVH) is an important target of antihypertensive therapy. Nonpharmacological approaches such as weight reduction and exercise training have favorable effects on other risk factors. However, there are few data on their effects on LVH. Athletes have eccentric rather than concentric LVH. A 12-month exercise program in 13 unmedicated hypertensive subjects altered LV geometry, reducing LV wall thickness and increasing LV internal diameters (LVID). LV mass was unchanged, and the thickness/radius fell by 9%. Shorter-term studies have shown that the cardiac structural changes with a moderate exercise program occur rapidly and their onset lags only about 2 weeks behind blood pressure (BP) effects. Assessment of weight loss effects on LVH is complicated by the strong relationship between body weight and ventricular wall thickness. LVID, and LV mass. To some extent, this can be overcome by arbitrarily indexing to body surface area or height. The wall thickness/radius ratio is not related to body size. Weight reduction reduces BP and thickness/radius by 10% in controlled trials. Small studies have also reported reduction in LV mass after sodium restriction in hypertensive subjects. Studies with other nonpharmacological measures could make a substantial contribution to knowledge of their efficacy. PMID- 1715491 TI - Cardiac mass and aortic distensibility following calcium blockade in hypertension. AB - Calcium-entry blockade produced by verapamil was studied in 13 subjects with sustained essential hypertension. Noninvasive echo-Doppler methods were used to evaluate cardiac structure and function, maximum aortic acceleration (used as an indirect index of cardiac performance), and carotid-femoral pulse-wave velocity. Measurements were performed in baseline conditions, following 12 weeks of active treatment, and 4 weeks of placebo. Following verapamil, blood pressure (BP) significantly decreased (p less than 0.001) with a slight reduction in cardiac output (p less than 0.05) and a more substantial decrease in heart rate (p less than 0.002). No significant change occurred in maximum acceleration and in the curve relating percent fractional shortening to end-systolic stress, suggesting that verapamil decreased BP with minor changes in cardiac performance. Cardiac mass decreased by approximately 6.3% whereas pulse-wave velocity did not change, suggesting that the small decrease in cardiac mass could be related to the lack of improvement in the capacitative component of vascular impedance, as judged from the determination of pulse-wave velocity. The study provides evidence that calcium blockade due to verapamil results in a dissociation between the antihypertensive effect and the absence of significant changes in cardiac and arterial structure and function. PMID- 1715492 TI - Regression of cardiac hypertrophy and left ventricular pumping ability postregression. AB - As a result of the increasing afterload imposed upon the left ventricle in hypertension, the heart adapts structurally and functionally. Structural changes involve an increased muscle mass achieved by left ventricular hypertrophy (LVH). Unless therapy is interdicted, left ventricular failure will ensure as the major cardiac hemodynamic consequence. LVH is also associated with a risk that is independent of the pressure. Although antihypertensive therapy reduces risk from the hemodynamic alterations, the independent risk of LVH has not been demonstrated. Moreover, should risk be demonstrated with therapy, before it can be ascribed to any specific agent(s), it must be dissociated from its effects on pressure, coronary blood flow (and flow reserve), or electrophysiological effects and be directly related to its action on reducing mass and reversing hypertrophy. Current investigative areas, involving new concepts of molecular biology of the cardiac myocyte, may provide great promise to the quest of unraveling these heretofore unexplainable and newly postulated questions. PMID- 1715493 TI - Long-term studies on regression of left ventricular hypertrophy. AB - The long-term effects of antihypertensive therapy on echocardiographically proven left ventricular hypertrophy (LVH) were investigated in 117 previously untreated hypertensive patients (mean age of 46 +/- 9 years; 15 women, 102 men). Twenty-two patients (group 1) received daily 100 mg of gallopamil, 25 (group 2) received 200 mg of metoprolol, 35 (group 3) received both 50 mg of atenolol and 20 mg of nifedipine (follow-up of 5 years), 14 (group 4) received 200 mg of acebutolol and 20 mg of nifedipine, and 21 (group 5) daily received 50 mg of atenolol and 10 mg of enalapril (follow-up of 4 years). For the entire population, there was a significant (p less than 0.001) decrease in left ventricular mass index (LVMI) of 24.5% after 1 year, which increased further to 44.1% after 5 years of treatment. In addition, fractional fiber shortening increased by 16% (p less than 0.001). In 82% of the patients, almost complete regression of LVH was achieved. However, the time course of regression of LVMI differed significantly between the five treatment groups, despite similar blood pressure reduction under resting conditions. PMID- 1715494 TI - Effects of different antihypertensive drug classes on survival in animal models. AB - Data on the influence of antihypertensive drug treatment on mortality of hypertensive rats are reviewed. Dihydropyridine calcium antagonists, verapamil, the angiotensin-converting enzyme (ACE) inhibitor captopril, and a triple combination of reserpine, hydralazine, and chlorothiazide normalized or markedly prolonged survival. Captopril was less effective in sodium chloride-induced, low renin Dahl rat hypertension. Dihydralazine prolonged but did not nearly normalize survival. The K(+)-channel activator minoxidil was relatively ineffective. Data on diuretics or beta-blockers are insufficient or unavailable. Calcium antagonists nitrendipine and nimodipine and the ACE inhibitor captopril improved survival and prevented vascular lesions and calcinosis even at doses that failed to achieve normotension. All drugs that normalized survival also reduced heart weights. Minoxidil invariably increased heart weights and failed to improve survival. (Di)hydralazine assumed an intermediate position. PMID- 1715495 TI - Ultrastructural localization of blood-brain barrier-specific antibodies using immunogold-silver enhancement techniques. AB - The blood-brain barrier in vivo is situated at the level of the endothelia in brain microvessels and functions to regulate the composition of the extracellular milieu of brain. In order to study the expression of barrier-specific proteins (BSPs) within the microvessels, an antiserum was prepared that reacted specifically with microvasculature in bovine brain tissue. Attempts at immunocytochemical localization of the BSP antigens on endothelia using pre embedding and post-embedding methods with sections of bovine brain were hampered by either the poor penetration of immune reagents and/or the poor ultrastructure in preparations where fixation was light enough to preserve antigenicity. These problems were addressed by applying a method whereby the primary and secondary antisera were reacted with microvessels isolated by enzymatic digestion of bovine brain; the secondary antibody was labelled with 1 nm gold particles and, after fixation with glutaraldehyde and osmium, the size of the gold probe was amplified by a one-step silver enhancement. The microvessel pellets were then processed as for routine electron microscopy. The BSP antiserum localized to the luminal and the abluminal membranes of the endothelial cells, and there was also immunolabelling of the endothelial tight junctional area. Comparison of these preparations made with the 1 nm gold conjugates with those made with standard 5 10 nm immunogold probes demonstrates that this immunogold-silver enhancement of the 1 nm probe is a simple and more sensitive method of immunolocalization of brain capillary antigens. PMID- 1715496 TI - An enzyme-linked immunosorbent assay for myelin basic protein-specific protein methylase I. AB - A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed to determine myelin basic protein (MBP)-specific protein methylase I. Rabbit immunoglobulin anti-bovine MBP-specific protein methylase I, purified by Sepharose-A affinity chromatography, was utilized as the primary antibodies, while the same antibodies which had been conjugated to peroxidase were employed as the indicator antibodies. This assay method was about 280 times more sensitive than the conventional trichloracetic acid (TCA) precipitation method. Employing the ELISA, the level of MBP-specific protein methylase I during mouse brain development was examined; the peak level of the methylase was shown to be at 16th postnatal day, indicating temporal correlation with myelination. Among several species of brains examined, human showed the highest and carp the least amount of MBP-specific protein methylase I; 6.33 micrograms and 0.33 micrograms per mg of brain cytosol protein, respectively. Dysmyelinating jimpy hemizygous mouse brain showed the immunoreactive MBP-specific protein methylase only 60% that of the control at 20 days of age. The high sensitivity of the method together with the fact that MBP-specific protein methylase is present in human cerebrospinal fluid suggests a possible clinical application of this method for evaluating demyelinating disorders. PMID- 1715497 TI - A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin. AB - The hydrochloride salt of a new, small molecular weight (M.W. = 286) biotin containing compound referred to as biotinamide (N-(2-aminoethyl)biotinamide) was compared with biocytin (M.W. = 372) for its use in intracellular labeling of neurons and in neuronal tracing experiments using avidin conjugates for histochemical detection. The DC resistance and current passing ability of electrodes filled with 1-2 M potassium chloride, potassium acetate or potassium methylsulfate and containing 1-4% of these compounds were compared. Although differences were observed due to the electrolyte, with KCl electrodes being the least resistant, no electrode differences could be attributed to the concentration or type of tracer. However, whereas biocytin could be electrophoresed with either positive or negative current with roughly similar facility, biotinamide was selectively ejected with positive current. This would be beneficial to electrophysiologists using hyperpolarizing current to stabilize the membrane potential of neurons prior to recording. In addition, biotinamide HCl could be dissolved at concentrations of 2-4% in either 1 or 2 M salt without precipitation, whereas biocytin precipitated in some of these solutions. Both compounds were equally useful for neuronal tracing experiments with survival times of 2 days, but labeling was much weaker with longer survival times. There was also little difference in the ability to histochemically localize these compounds using avidin conjugates, including avidin-biotin-horseradish peroxidase complex. In conclusion, biotinamide shares many of the useful features of biocytin, but can be selectively electrophoresed with positive current and can be dissolved at higher concentrations with little detriment in the electrical properties of the recording electrode. PMID- 1715498 TI - Neuropeptides can interfere with the recording properties of voltammetric carbon fibre electrodes. AB - The effects of neuropeptides on the recording properties of carbon fibre micro electrodes used with differential pulse voltammetry were examined both in vitro and in vivo. The in vitro voltammetric signal recorded in a solution containing 3,4-dihydroxyphenylacetic acid (10(-4) M) was attenuated after the addition of thyrotropin releasing hormone, neuromedin N and neurotensin (10(-5) M). The administration of neurotensin (1 or 3 micrograms/microliters) into the nucleus accumbens adjacent to the carbon fibre electrode produced a decrease in the extracellular 3,4-dihydroxyphenylacetic acid peak. However, evidence was obtained that peptides alter the recording properties of the microelectrodes when present at sufficient concentrations and such an effect may explain the result obtained in vivo. In contrast, neurotensin administered into the ventral tegmental area increased the 3,4-dihydroxyphenylacetic acid peak in the nucleus accumbens as previously reported. Thus, attenuation of the voltammetric signal was caused by an effect of the peptide on the recording properties of the electrode rather than real diminution of the free oxidisable amine around the electrode. Caution should be taken when investigating the effects of peptides on extracellular amines or their metabolites using in vivo voltammetry with carbon fibre electrodes if the peptides are administered in high concentrations close to the electrode. PMID- 1715499 TI - A simple method for organotypic cultures of nervous tissue. AB - Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture. PMID- 1715500 TI - Comparative study of the adverse effects of various radiographic contrast media, including ioversol, a new low-osmolarity medium. I. Histamine release. AB - Histamine release induced by some radiographic contrast media (RCM) such as iohexol, iopamidol and meglumine sodium amidotrizoate (MSA) was studied in comparison with ioversol, a new low-osmolarity contrast medium. Although all of the RCM caused a dose-related histamine release from rat peritoneal mast cells, that induced by ioversol was the least. MSA induced more remarkable histamine release than did sodium chloride at each of the corresponding osmolarities, except under hypoosmolar conditions. Moreover, in guinea pig lung perfusion, RCM, especially ioversol, iohexal and iopamidol, induced only a weak histamine release in both non-sensitized and sensitized animals. However, when MSA was perfused in sensitized animals, a large amount of histamine was detected in the lung effluents. These results suggest that nonionic RCM, including ioversol, can be used more safely than MSA for diagnostic examination. PMID- 1715501 TI - Placebo-controlled, double-blind, cross-over trial of growth hormone treatment in prepubertal children with chronic renal failure. AB - Stunted growth is a serious problem for children with chronic renal failure (CRF) despite normal endogenous growth hormone secretion and normal or elevated plasma concentrations of insulin-like growth factors (IGF) I and II. Biosynthetic growth hormone (GH) was given to 20 prepubertal children (eleven boys, nine girls; mean age 9.5 years, range 4-16) with CRF and severe growth retardation in a placebo controlled, double-blind, cross-over trial. 6 months of subcutaneous injection of GH (4 IU/m2 per day) was either preceded or followed by 6 months of placebo injection. The patients had a full examination every 3 months. Sixteen children completed the study. Height velocity improved significantly with GH therapy (p less than 0.0001) and placebo (p less than 0.04), but the GH-induced height velocity increase exceeded that of placebo by 2.9 cm per 6 months. There was a positive relationship between prestudy height velocity and height-velocity increase. Bone maturation was not affected. GH caused a significant increase in IGF-I and a moderate increase in IGF-II plasma concentrations. The pretreatment elevation of IGF-binding protein-1 decreased by almost 50% during GH therapy, while IGF-binding protein-3 increased significantly in concentration, although this increase was significantly smaller than the GH-induced increase in IGF-I. Fructosamine, lipid, and parathyroid concentrations remained constant. Renal function deterioration did not accelerate. Impressive height-velocity increase can be achieved with GH therapy in children with CRF and growth retardation without changes in renal function. Bone maturation appears unaffected suggesting improved final height. Treatment is best started before growth retardation becomes considerable. PMID- 1715502 TI - Solving the weanling's dilemma: power-flour to fuel the gruel? PMID- 1715503 TI - Pilot study of screening for prostate cancer in general practice. AB - The success of a screening programme for cancer depends on the sensitivity of the tests used and on the proportion of the target population that comes forward for screening. To assess the value of digital rectal screening and prostate-specific antigen (PSA) measurement as screening measures, the 814 men in a city general practice aged between 55 and 70 were recruited in one of five different ways. Men with a palpably suspicious prostate or a serum PSA greater than 4 ng/ml were referred for transrectal ultrasonography and, if indicated, biopsy. 472 men (58%) were screened; of these 68 underwent transrectal ultrasonography and 29 biopsy. In 7 the biopsy specimen showed carcinoma. Serum PSA was better than digital examination as a screening test--all men with prostate cancer had raised concentrations of serum PSA, whereas only 1 had a palpably abnormal prostate. All 7 had localised disease, and 5 underwent radical prostatectomy. The best methods of patient recruitment were to send an appointment for screening and to "tag" the patient's notes. PMID- 1715504 TI - Magnesium and chronic fatigue syndrome. PMID- 1715505 TI - In vitro proton spectroscopy of normal and abnormal prostate. AB - Previous biochemical and 13C NMR spectroscopic data have suggested that the metabolism of citrate, a secretory product of normal prostate, may be interrupted in prostate cancer. In the present study in vitro 1H NMR spectroscopy was used to see if cell strains derived from prostate cancers could be reliably distinguished from those of normal prostate epithelium. High-resolution one-dimensional and two dimensional J-resolved 1H NMR spectra as well as gas chromatography coupled with mass spectroscopy were used to study extracts of highly defined cell strains from normal peripheral zone, normal central zone, adenocarcinoma, and benign prostatic hyperplasia. Resonances assigned to citric acid and related metabolites were identified. Cell strains derived from prostate cancers tended to have smaller amounts of citrate than those from normal prostate epithelium. However, the differences were small and not statistically significant. The lack of statistically significant differences may reflect the variability present in both normal and abnormal cell strains and thus underscore the well-known difficulty in differentiating normal and cancerous tissues. PMID- 1715506 TI - Comparison of three forward mutation systems in Saccharomyces cerevisiae for sensitivity to polycyclic and heterocyclic compounds. AB - Forward mutation to cycloheximide resistance, L-canavanine resistance and DL alpha-aminoadipic acid resistance in Saccharomyces cerevisiae wild-type strain S7a was tested for sensitivity to nine mutagens in treat-and-plate assays. Eight of these agents, 2-aminofluorene, 2-acetylaminofluorene, benzo[a]pyrene, benzidine, cyclophosphamide, acriflavine, 7,12-dimethylbenz[a]anthracene and 2 aminoanthracene were known or suspected to be difficult to detect whilst one, methyl methanesulphonate, was known to be very active in yeast. Forward mutation to cycloheximide resistance was, overall, the most sensitive system, detecting all nine agents under optimal conditions, although neither benzidine nor benzo[a]pyrene were consistently positive. Mutation to adipic acid resistance occasionally gave responses superior to those at the cycloheximide loci, but mutation to canavanine resistance was never more sensitive than the cycloheximide resistance system. We conclude that forward mutation in strain S7a using both cycloheximide and adipic acid resistance loci is capable of detecting the genetic effects of a range of polycyclic and heterocyclic compounds with greater sensitivity than is seen in other published gene mutation assays with yeast. Although sensitivity is much lower than in bacterial assays, such yeast assays provide a reasonable alternative to bacterial genotoxicity screening for agents such as potent bactericides. PMID- 1715508 TI - [Conformational analysis of protein side chains using two-dimensional Overhauser nuclear effect spectroscopy]. AB - The method has been proposed to determine the conformations of protein side chains (dihedral angles chi 1) using two-dimensional nuclear Overhauser effect spectroscopy data. This method is grounded of the algorithm prepared on the basis of joint consideration of proton-proton distance dependences in dipeptide units of L-amino acid residues on the dihedral angles phi, psi and chi 1 with the accounting of the local sterical conditions of the polypeptide chain. The obtained results gave the possibility to bring the different regions of space (phi, psi) of amino acid residues into the line with the specific sets of nuclear Overhauser effect spectral parameters which unambiguously characterize in most cases the conformational states of their side-chains. The method efficiency was displayed on the test calculation with the utilization as the experimental data of the "model" nuclear Overhauser effect contacts derived from the X-ray atomic coordinates of the bovine pancreatic trypsin inhibitor molecule. PMID- 1715507 TI - Characterization of monoclonal antibodies identifying type and strain-specific epitopes of human immunodeficiency virus type 1. AB - Several hybridoma cell lines were raised against the highly cytopathic Zairian isolate of Human Immunodeficiency Virus (HIV), HIV1-NDK. The specificity of the secreted monoclonal antibodies (mAb) was demonstrated by immunoblotting, radioimmunoprecipitation and immunofluorescence. Two hybridoma cell lines secreted mAb reacting with independent epitopes of the NDK p17 capsid protein and its precursors. One, RL16.24.5, is specific for the NDK isolate whereas the other, RL16.45.1, along with anti-p25 RL16.30.1 mAb, bind all HIV1 isolates but not HIV2. Together with the previously described mAb RL4.72.1 those reagents define lentivirus subfamily (HIV1, HIV2, SIV) type/subtype (HIV1) and strain (HIV1-NDK) specific epitopes expressed on HIV1-NDK core proteins. The last mAb RL16.76.1 binds the env gene products gp160 and gp120. PMID- 1715509 TI - [Exposure of the major immunodominant epitope of the gp51 envelope protein of bovine leukosis virus on the surface of the hepatitis B core antigen capsid]. AB - Insertion of 48 amino acid long sequence of envelope protein gp51 of bovine leukemia virus (BLV), located from position 56 till 103 of mature protein, into Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids. These capsids preserve morphology of intact HBcAg but expose on their outer surface BLV epitopes which are localised in the inserted gp51 fragment and responsible for the recognition of chimeras by monoclonal anti-gp51 antibodies MAK14. The anti-genicity of gp51 epitopes within chimeric capsids is not disturbed after shortening of C terminal part of inserted gp51 fragment by deletion of amino acids 73-103. The resulting chimeras show the same capsid forming ability as well as HBcAg and gp51 antigenic properties. PMID- 1715510 TI - [A new recipient cell line for transfection of biologically active oncogenes]. AB - The properties of the new immortalized rat cell line (REF-1) were analyzed. These cells can be used as recipient ones in transfection assays. REF-1 cells never convert spontaneously to transformed phenotype during long-term passages in vitro unlike NIH3T3 cells. This peculiarity allowed to use REF-1 cells for identification of oncogenes, which induce slow-growing tumors. The following oncogenes were used in gene transfection experiments in order to test their effects: activated human EJ-ras; N-ras; v-myc; v-mos; activated tpr-met and c-hu met. REF-1 cells, transfected with the members of ras family; v-mos and tpr-met were found to be transformed in vitro and induce tumors in nude mice, on the contrary of c-hu-met- and v-myc-transfected cells, which are non-tumorogenic. A number of clonal cell lines carrying different oncogenes were obtained. The detailed analyses of integration and expression of exogenous sequences of different oncogenes has been presented in 18 clonal cell lines. PMID- 1715512 TI - Endogenous xenobiotic enzyme levels in mammalian cells. AB - The response of mammalian cell lines to chemicals depends, in part, on the exogenous activation system used for the induction of a biological response. This could be attributed to differences in the expression of enzymes involved in xenobiotic metabolism. We have measured the activities of benzo[a]pyrene hydroxylase, dimethylaminoazobenzene N-demethylase, catalase, superoxide dismutase, peroxidase and glutathione-S-transferase in human lymphoblast TK6, mouse lymphoma L5178Y, Chinese hamster ovary (CHO) and lung (V79) and mouse C3H10T1/2 cell lines as well as in primary hepatocytes and S9 preparations of liver from male F344 rats. Nitroreductase was also measured in some of these preparations. Human lymphoblast TK6 and mouse C3H10T1/2 cells had the capacity to metabolize dimethylaminoazobenzene and the latter cell line also metabolized benzo[a]pyrene, indicating the presence of constitutive mono-oxygenase activity. Cytochrome P450 could not be detected spectrophotometrically in the cell lines. Western blot analysis indicated that P450 from the P450IIA family is expressed in C3H10T1/2 cells. Reactivity was also observed with an antibody to P450IA2; however, the identity of this protein remains uncertain. Superoxide dismutase, catalase and peroxidase, which protect cells against oxygen radical damage, were found in all the cell lines and in rat hepatocytes and S9. The human lymphoblast TK6 cell line, however, had the least of each of these three enzymes. Glutathione S-transferase activity was detected at varying levels in all cell types. Nitroreductase activity was high in S9 and Chinese hamster ovary cells and lower in mouse lymphoma and Chinese hamster V79 cells. PMID- 1715511 TI - Biological low-dose radiation effects. AB - It is theorized that biological responses to ionizing radiation in the low dose range are determined according to a doubly dichotomous pattern. Energy depositions fall into 2 categories: events at thermal energy levels where they may be experienced by cells as rates even at background exposure conditions, and events at energy levels of the order of 10-100 eV where damage to DNA may be caused. Variations in background exposure intensity may or may not lead preemptively to changes in the cell's capacity for response to radiation damage. High-level energy depositions lead post hoc to an initial stabilizing reaction largely leading to the fixation of the initial DNA damage, and to a subsequent restorative or palliative repair process. This model entails reinterpretation of some experimental results. The model has implications for the relationship between scientific analysis of low-dose effects and the regulatory needs for simplicity and homogeneity in risk evaluation. This represents a new challenge for the acceptability of radiation protection norms. PMID- 1715513 TI - Structure-activity relationships of a series of antitumoral 5,8-quinazolinediones in mutagenicity studies. AB - Four antitumoral 5,8-quinazolinediones were examined for their ability to induce mutation in Salmonella typhimurium. Each compound was tested at several concentrations in 4 strains. Relationships were established between the structure of the quinones and their mutagenic activities. The mutagenicity was influenced by (i) the nature of the substituent(s) of the quinonic moiety: the methoxyquinone had no mutagenic properties and the aziridinylquinones were mutagenic in the 4 strains with or without activation by S9 mix; (ii) the presence or the absence of a diaminopolymethylenic chain in the 4 position; (iii) the monomeric or the dimeric structure of the tested compound. Interestingly, the data indicated that the aziridinylquinazolinedione bearing the dimethylaminopropylamino chain in the 4 position was less mutagenic and had greater antitumor activity than the dimeric quinone. PMID- 1715514 TI - Measurement of prostate-specific antigen as a screening test for prostate cancer. PMID- 1715515 TI - Signal transduction. Immunosuppressants at work. PMID- 1715516 TI - Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. AB - Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit. PMID- 1715517 TI - Effects of the steel gene product on mouse primordial germ cells in culture. AB - Mutations at the steel (sl) and dominant white spotting (W) loci in the mouse affect primordial germ cells (PGC), melanoblasts and haemopoietic stem cells. The W gene encodes a cell-surface receptor of the tyrosine kinase family, the proto oncogene c-kit. In situ analysis has shown c-kit messenger RNA expression in PGC in the early genital ridges. The Sl gene encodes the ligand for this receptor, a peptide growth factor, called here stem cell factor (SCF). SCF mRNA is expressed in many regions of the early mouse embryo, including the areas of migration of these cell types. It is important now to identify the role of the Sl-W interaction in the development of these migratory embryonic stem cell populations. Using an in vitro assay system, we show that SCF increases both the overall numbers and colony sizes of migratory PGC isolated from wild-type mouse embryos, and cultured on irradiated feeder layers of STO cells (a mouse embryonic fibroblast line). In the absence of feeder cells, SCF causes a large increase in the initial survival and apparent motility of PGC in culture. But labelling with bromodeoxyuridine shows that SCF is not, by itself, a mitogen for PGC. SCF does not exert a chemotropic effect on PGC in in vitro assays. These results suggest that SCF in vivo is an essential requirement for PGC survival. This demonstrates the control of the early germ-line population by a specific trophic factor. PMID- 1715518 TI - Requirement for mast cell growth factor for primordial germ cell survival in culture. AB - Mast-cell growth factor (MGF) is encoded by the murine steel (Sl) locus and is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c kit at the murine dominant white spotting (W) locus. Mutations at both these loci affect mast cells, primordial germ cells (PGCs), haemopoietic stem cells and melanocytes. In many Sl and W mutants, the rapid proliferation of PGC that normally occurs between day 7 and 13.5 of embryonic development fails to occur. As c-kit is expressed in PGCs while MGF is expressed in the surrounding mesenchyme, MGF might promote the proliferation of PGCs. Here we report that MGF is essential for PGC survival in culture, but does not stimulate PGC proliferation. Moreover, whereas both the transmembrane and soluble proteolytic cleavage forms of MGF stimulate mast-cell proliferation, soluble MGF has a relatively limited ability to support survival of PGCs in culture, thus explaining the sterility in mice carrying the steel-dickie (Sld) mutation, which encodes only a soluble form of MGF, and providing a functional role for a transmembrane growth factor. PMID- 1715519 TI - Specific interaction between HPV-16 E1-E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network. AB - The human papillomaviruses (HPV) are associated specifically with epithelial lesions, ranging from benign warts to invasive carcinoma. The virus encodes three late proteins, which are produced only in terminally differentiating keratinocytes, two of which are structural components of the virion. The third, E1-E4, is derived primarily from the E4 open reading frame, which represents a region of maximal divergence between different HPV types. E1-E4 does not seem to be a component of the virus particle or to be needed for transformation in vitro, but accumulates in the cytoplasm, where in certain benign lesions it can comprise 20-30% of total cell protein. We show here that expression of the HPV-16 E1-E4 protein in human keratinocytes (the natural host cell for HPV infection) results in the total collapse of the cytokeratin matrix. Tubulin and actin networks are unaffected by E1-E4, as are the nuclear lamins. PMID- 1715520 TI - The war of the whorls: genetic interactions controlling flower development. AB - The analysis of mutations affecting flower structure has led to the identification of some of the genes that direct flower development. Cloning of these genes has allowed the formulation of molecular models of how floral meristem and organ identity may be specified, and has shown that the distantly related flowering plants Arabidopsis thaliana and Antirrhinum majus use homologous mechanisms in floral pattern formation. PMID- 1715521 TI - Direct access to serum macromolecules by intraerythrocytic malaria parasites. AB - Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents. PMID- 1715522 TI - Excitatory responses to scyliorhinins I and II in smooth muscle strips isolated from the carp intestinal bulb (Cyprinus carpio). AB - 1. The effects of scyliorhinins I (SCY I) and II (SCY II) on longitudinal (LM) and circular muscle (CM) strips isolated from the carp intestinal bulb were investigated in vitro and compared with that of substance P (SP). 2. SP (0.3 nM-1 microM), SCY I (0.3-300 nM) and SCY II (0.3 nM-1 microM) caused transient concentration-dependent contractions of LM strips. The EC50 values for SP, SCY I and SCY II were 16 nM, 15 nM and 39 nM, respectively. Tetrodotoxin and atropine partly decreased the contractile responses to SP, neurokinin A and neurokinin B, but did not change those to SCY I and SCY II. Spantide, methysergide, pyrilamine and naloxone did not decrease the contractile responses to SP, SCY I and SCY II. SP-induced desensitization selectively decreased the responsiveness of LM strips to SCY I and SCY II, and in addition, SCY I- or SCY II-induced desensitization decreased that to SP, SCY I and SCY II. 3. SP, SCY I and SCY II (1 nM-1 microM) caused concentration-dependent contraction of CM strips. The time course of the contractile response of CM strips was different from that of LM strips. Neither tetrodotoxin, atropine, methysergide nor spantide decreased the contractile responses to these tachykinins. 4. These results indicate that SCY I and SCY II act directly on tachykinin receptors located on smooth muscle cells and thus cause the excitatory response in the carp intestinal bulb. PMID- 1715523 TI - Pyrogenicity of polyadenylic.polyuridylic acid in rabbits. AB - Polyadenylic.polyuridylic acid injected intravenously into rabbits produced a rapid-onset, monophasic fever. Pyrogenic tolerance occurred in rabbits following daily injections of polyadenylic.polyuridylic acid. However, direct injection of the agent into the preoptic anterior hypothalamic region of rabbit's brain produced a markedly different fever. After an intrahypothalamic injection of polyadenylic.polyuridylic acid, fever was delayed in onset and persisted for a longer period. At room temperature, the fever was due to both increased metabolism and cutaneous vasoconstriction. In a colder atmosphere the fever was due solely to increased metabolism, whereas in the heat the fever was due to reduction in cutaneous blood flow and respiratory evaporative heat loss. In addition, the fever induced by intravenous polyadenylic.polyuridylic acid injection was reversed by a cyclooxygenase inhibitor, but not by a protein synthesis inhibitor. Polyadenylic.polyuridylic acid was shown to stimulate PGE2 production from rabbit's hypothalamus in vitro. The results reveal that this agent is a prostaglandin-dependent pyrogen. PMID- 1715524 TI - [The effect of intracellular calcium on an increase in the cAMP-mediated calcium current]. AB - Intracellularly perfused neurons of Helix pomatia in which serotonin-induced increase of calcium current (Ica) is mediated by cAMP were studied by the voltage clamp technique. It was established that an increase of the calcium intracellular concentration ([Ca2+]i) caused inhibition of the serotonin (5-HT) effect. The 5 HT effect value at 10(-6) and 10(-7) mol/l [Ca2+]i was 60 and 32% of the control one, respectively. Addition of the calmodulin antagonist (5.10(-5) M trifluoperasine) and phosphodiesterase blockers (5 mM theophylline or 10(-4) mol/l IBMX) sharply reduced the Ca2+ ability to inhibit the 5-HT effect. It is concluded that the Ca(2+)-calmodulin-dependent phosphodiesterase is a key link in the interaction between two signal transduction pathways--Ca2+ and cAMP in modulation of the calcium channel activity. PMID- 1715525 TI - [Voltage-gated influx ion channels expressed in Xenopus oocytes after the administration of brain mRNA]. AB - Expression of "fast", TTX-sensitive sodium and high-threshold calcium channels in the membrane of Xenopus oocytes following mRNA injection from the rat brain has been detected using two microelectrode voltage clamp technique. Barium current through expressed calcium channels was blocked by 200 mumol/l Cd2+ and was insensitive to D-600 (20 mumol/l) and nitrendipine (50 mumol/l). Expressed barium current was inhibited within 20-40 min by omega-conotoxin, a peptide neurotoxin known to block high-threshold calcium channels of the neuronal membrane, in 1 mumol/l concentration. A steady-state inactivation curve for this current could be fitted by the Boltzmann relation with V1/2 = -50 mV and k = 14 mV. Voltage dependent and pharmacological properties of calcium channels which appeared in the oocyte membrane following mRNA injection from the mammalian brain resembled most of all those of high-threshold inactivating (HTI- or N-type) calcium channels of neurons in spite they did not demonstrate prominent time-dependent inactivation. Evidences in favour of expressed calcium channels heterogeneity were not obtained. PMID- 1715526 TI - [The spatial organization of the vestibulospinal neurons in the guinea pig]. AB - Quantitative investigation of the spatial organization of vestibulospinal neurons has been performed on a guinea pig using the horseradish peroxidase retrograde transport method. After unilateral injection in the upper cervical and low thoracic spinal cord the labelled neurons were found unilaterally in the lateral vestibular nucleus and bilaterally in descending and medial vestibular nuclei. Distribution of vestibulospinal neurons within the brainstem has been analyzed. It was revealed that projections of some neurons of medial and descending vestibular nuclei coincided with spinal projections of cortical and rubral descending pathways. Functional significance of the vestibulospinal system in the supraspinal motor control was discussed. PMID- 1715527 TI - Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures. AB - Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10(-5)M and 10(-8)M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [3H] and [35S] methionine and [3H]ethanolamine as precursors showed an increased methylation of [3H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [3H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [3H]- and [35S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased 3H- and 35S labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [3H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-3H] label after incubation of neurons with [3H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms. PMID- 1715528 TI - Physiological and pharmacological properties of 5-HT3 receptors--a patch clamp study. AB - Whole cell and patch clamp techniques were used to investigate the properties of 5-HT3 receptors of a murine neuroblastoma cell line (N1E-115) and adult rabbit nodose ganglion neurones. In addition, some preliminary results from guinea-pig nodose ganglion neurones are presented. In such cells, voltage-clamped at -60 mV, 5-HT (10 microM) induced an inward current associated with a conductance increase. The results of ion substitution experiments suggest that the 5-HT activated ion channel is permeable to both Na+ and K+ ions with a permeability ratio (PNa/PK) of 0.94 and 0.92 for rabbit nodose ganglion cells and N1E-115 cells respectively. On outside out membrane patches excised from rabbit nodose ganglion neurones, 5-HT (1 microM) activated clearly discernible single channel currents with a conductance of 16.6 +/- 0.7 pS (n = 4). In contrast, fluctuation analysis of 5-HT induced whole cell currents suggests that the single channel conductance of N1E-115 cells is only 0.3 pS, a value some 50 fold lower. The 5-HT induced whole cell currents recorded from all three preparations were antagonised by the selective 5-HT3 receptor antagonist ondansetron (GR38032F) and by the less selective agents metoclopramide, cocaine and (+)-tubocurarine. However, these preparations demonstrate a differential sensitivity to some antagonists. In particular, (+)-tubocurarine was a potent antagonist in N1E-115 cells (IC50 = 0.85 nM) but was approximately 200 fold (IC50 = 156 nM) and 1200 fold (IC50 = 10 microM) less potent in rabbit and guinea-pig nodose ganglion neurones respectively. Additionally, a novel effect of ketamine (10 microM) to potentiate the 5-HT-induced current of rabbit nodose ganglion neurones is described. PMID- 1715529 TI - Studies on the effect of systemic PD134308 (CAM 958) in spinal reflex and pain models with special reference to interaction with morphine and intrathecal galanin. AB - The effect of intravenous (i.v.) PD134308, which is a CCK-B antagonist, morphine and intrathecal (i.t.) galanin (GAL) on the excitability of the spinal nociceptive flexor reflex and in the hot plate test was examined in rats. PD134308 (1 mg/kg, i.v.) caused a weak, naloxone-reversible depression of the flexor reflex and moderate antinociception in the hot plate test. PD134308 significantly potentiated the antinociceptive effect of morphone, as well as its depressive effect on the flexor reflex. PD134308 and i.t. GAL synergistically depressed the flexor reflex, which was reversed by naloxone. Finally, the magnitude and duration of the depression of the flexor reflex by morphine was synergistically increased by coadministering i.v. PD134308 and i.t. GAL. The results demonstrate that a CCK antagonist directed to the central CCK-B receptor potentiates the analgesic effects of opioid and non-opioid drugs at spinal level in the rat, thus supporting the notion that CCK in the CNS may be an endogenous, physiological opioid antagonist. PMID- 1715530 TI - Retinofugal projections in the house musk shrew, Suncus murinus. AB - Retinofugal projections in the house musk shrew (Suncus murinus) were studied with the WGA-HRP method. After WGA-HRP injection into the vitreous cavity of one eye, terminal labeling was seen in the suprachiasmatic nucleus, dorsal and ventral lateral geniculate nuclei, pretectum and superficial layer of the superior colliculus. The terminal labeling in the suprachiasmatic nucleus was more marked on the side ipsilateral to the injection than on the contralateral side, whereas that in other regions was seen mainly on the contralateral side. A retino-intergeniculate leaflet projection was observed. No unequivocal terminal labeling was found in the lateroposterior thalamic nucleus. PMID- 1715531 TI - The noradrenergic innervation of the spinal cord: differences between two substrains of Sprague-Dawley rats determined using retrograde tracers combined with immunocytochemistry. AB - We have recently described the spinal cord terminations of noradrenergic neurons located in the A5, A6 and A7 cell groups. However, recent reports from another laboratory, using similar experimental methods, have described results that are profoundly different. The present experiments were designed to determine whether these discrepant results are due to fundamental differences between the substrains of rats used in the conflicting experiments. To this end, retrograde tract tracing experiments were done using Sprague-Dawley rats from either Sasco, Inc. or Harlan Sprague-Dawley, Inc. The results indicate that noradrenergic neurons in the pontine catecholamine cell groups exhibit remarkably different spinal cord projections in these two substrains of Sprague-Dawley derived rats. PMID- 1715532 TI - Many diverse types of retinal neurons show tracer coupling when injected with biocytin or Neurobiotin. AB - This study demonstrates that the junctional connections between rod-signal interneurons in mammalian retina can be visualized by tracer coupling, following intracellular injection of the biotinylated compounds, biocytin and Neurobiotin. In addition, many other types of retinal neurons -including B-type horizontal cells and several types of retinal ganglion cells-show specific patterns of tracer coupling, usually to cells of the same neuronal type but occasionally to cells of other neuronal classes. These findings suggest that electronic transmission occurs commonly throughout the retina and, consequently, diverse types of retinal neurons may form functional networks of coupled cells. PMID- 1715533 TI - Inward currents elicited by stream of fluid during transmitter-induced current oscillations in RNA-injected oocytes of Xenopus laevis. AB - A stream of fluid elicited an inward current in RNA-injected oocytes of Xenopus laevis during transmitter-induced current oscillations (stream evoked inward current, Ii,st). The Ii,st showed the following characteristics: (1) amplitude and duration (half width time) ranged between 10 and 300 nA and 1 and 3 s, respectively. (2) The Ii,st could be evoked only during transmitter-induced current oscillations; with blockade of the oscillations the Ii,st disappeared. (3) The induction of the Ii,st was independent of the composition of the washing fluid. PMID- 1715534 TI - An antibody to beta amyloid and the amyloid precursor protein inhibits cell substratum adhesion in many mammalian cell types. AB - An epitope-specific antibody directed against the first 16 amino acids of the beta amyloid protein (anti-BP16) immunoprecipitated the secreted form of the amyloid precursor protein (APP) from the conditioned medium of PC12 cells. This antibody caused neurite retraction in differentiated PC12 cells and inhibited cell-substratum adhesion in many neuronal and non-neuronal cell types. The inhibitory effect of anti-BP16 was abolished by preabsorption of the antibody with BP16 peptide. Antibodies directed against other domains of APP did not inhibit cell adhesion. The secreted form of APP may be important for cell adhesion in many different mammalian cell types. PMID- 1715535 TI - Immunohistochemical localization of the alpha 1, alpha 2 and alpha 3 subunit of the GABAA receptor in the rat brain. AB - The immunohistochemical distribution of the alpha 1, alpha 2 and alpha 3 subunit of the gamma-aminobutyric acid-A (GABAA) receptor was investigated in the rat brain using affinity-purified antibodies against unique parts of the amino acid sequence of the respective subunits. The distribution of the 3 subunits differed markedly from each other indicating heterogeneity of the GABAA-receptor composition in different brain regions and at various receptive compartments (dendrites or somata) of neuronal cells. PMID- 1715536 TI - Postnatal development of neurogenic inflammation in the rat. AB - We have studied the development in the rat of neurogenic inflammatory mechanisms that mediate cutaneous plasma extravasation. At birth and at postnatal day 10, intradermal injection of substance P, histamine, and bradykinin produced no significant plasma extravasation. At day 13 through adulthood (days 42-49), all test agents produced significant plasma extravasation which increased with increasing age. In the adult rat, pretreatment with 6-hydroxydopamine, to eliminate sympathetic postganglionic nerve terminals, attenuated the plasma extravasation elicited by substance P, histamine and bradykinin. The possible role of the sympathetic postganglionic neuron in the age-dependent changes in neurogenic inflammation is discussed. PMID- 1715537 TI - Appearance of nerve growth factor and acidic fibroblast growth factor with different time courses in the cavity-lesioned cortex of the rat brain. AB - Time-dependent changes in both nerve growth factor (NGF) and acidic fibroblast growth factor (aFGF) levels in the rat brain after cortical cavity lesioning were examined, by using sensitive enzyme immunoassays (EIA) specific for each factor. In the cavity fluid, the NGF level increased rapidly and temporarily with a sharp peak 16 h after lesioning. A relatively high level was sustained during the next 3-6 days. Contrary to NGF, aFGF was first detectable only 10 days after lesioning, and its level increased gradually until 30 days. These results suggest that NGF and aFGF would play some roles for neuronal repair in different ways. PMID- 1715538 TI - Presence of a cholinergic projection from ventral striatum to amygdala that is not immunoreactive for NGF receptor. AB - By combining the retrograde axonal transport of a fluorescent dye with nerve growth factor (NGF) receptor or choline acetyltransferase (ChAT) immunocytochemistry, we show that the cholinergic neurons that project most strongly to the basolateral nucleus of the amygdala in the ferret do not possess NGF receptor immunoreactivity in their soma and are situated in the ventral striatum, an area known to receive a massive reciprocal projection from the basolateral nucleus of the amygdala. PMID- 1715539 TI - GABA and non-GABA immunostained neurones in the nucleus prepositus and the periparabigeminal area projecting to the guinea pig superior colliculus. AB - We have investigated the possibility that GABAergic neurones may be involved in two ascending projections to the superior colliculus, originating in the nucleus prepositus hypoglossi and in the periparabigeminal area of the mesencephalon, respectively. The projecting neurones of both structures were identified using gold-WGA-apoHRP, a retrogradely transported tracer, injected unilaterally into the superior colliculus. GABA was detected in these neurones by means of immunocytochemical staining. The results show that 25% of the projecting neurones in the prepositus hypoglossi are indeed GABA-immunoreactive. They could exert a direct inhibitory influence on the colliculus. By contrast, only a few (7%) gold filled-GABAergic cells were detected in the periparabigeminal area, which suggests that this region cannot participate in an important inhibitory afferent system to the colliculus. PMID- 1715540 TI - Serotonergic involvement in the cardiovascular stimulation by thyrotropin releasing hormone (TRH) in anesthetized rats. AB - The cardiovascular effects of intracerebroventricularly administered thyrotropin releasing hormone (TRH) were studied in anesthetized rats in the presence of serotonin (5-HT) depletion induced by pretreatments with p-chloroamphetamine (PCA) or p-chlorophenylalanine (PCPA). After PCA the reduction of the whole brain 5-HT and 5-HIAA (5-hydroxy-indoleacetic acid) was 53% and 32% of control, respectively. PCPA reduced the brain 5-HT and 5-HIAA levels even to a greater extent, corresponding levels were 9% and 17% of control. TRH 1-100 nmol/kg increased dose dependently blood pressure and heart rate. PCPA pretreatment significantly attenuated the pressor effect and the tachycardia induced by TRH, whereas PCA did not modify the effects of TRH, which may be related to its weaker capacity to deplete 5-HT in TRH sensitive brain areas. These results suggest the involvement of the central serotonergic system in the TRH-induced cardiovascular stimulation. PMID- 1715541 TI - Nerve growth factor: increased angiogenesis without improved nerve regeneration. AB - Nerve growth factor (NGF) and laminin are important factors for neural development and regeneration. We examined the effects of increasing the local concentration and duration of action of NGF and laminin on peripheral nerve regeneration in the adult mouse sciatic nerve. A Silastic (silicone rubber) channel with intraluminal NGF solution was secured between transected nerve ends. The second channel tested, formed from a polysaccharide called chitosan, was prepared with NGF and laminin in the channel walls and provided a sustained release of NGF. At six weeks post-implantation, no improvement in nerve regeneration was identified in those channels prepared with NGF when comparing electromyographic thresholds (microA), maximum potentials (mV), nerve diameter, myelin sheath thickness, myelinated axon counts, or diameter. However, increased angiogenesis was demonstrated within the chitosan and Silastic channels prepared with NGF compared to those channels without NGF. Silastic exhibited minimal inflammation. Chitosan was associated with inflammation in many nerve channels. PMID- 1715542 TI - Immunomodulation of the induction phase of lymphokine-activated killer activity by acute phase proteins. AB - Effective treatment of head and neck cancer with biologic response modifiers may be benefitted by an understanding of in vivo factors capable of modulating the lymphokine-activated killer (LAK) cell phenomenon. Eighteen patients with squamous cell carcinoma of the head and neck were studied. Killer cells from each patient, activated by recombinant interleukin-2 (10 U/ml), were induced in either complete medium alone or complete medium plus 10% autologous serum solution and analyzed. Cytotoxicity against both K562 and squamous cell carcinoma (MDA686-Ln) cell lines was determined by use of standard chromium-release assays. The immunomodulatory capacity of serum was correlated with levels of various acute phase proteins. Autologous serum significantly inhibited the induction phase of the LAK phenomenon in 61% of patients and stimulated it in 22%. No patients with early stage I or II disease had significant inhibition of induction. No direct correlation between inhibition and serum acute phase protein levels were seen. An inverse relationship was seen between the C3 component of complement and induction inhibition (r = -0.6). These findings suggest that advances of in vivo immunomodulatory therapy will require elucidation of mechanisms of serologic inhibition of the induction phase of the LAK phenomenon. Such studies may lead to serologic modification to enhance treatment efficacy of biologic response modifiers. PMID- 1715543 TI - Sensors in implantable cardiac devices. PMID- 1715544 TI - The subpectoral pocket: the preferred implant site for pediatric pacemakers. AB - Implantation technique for pediatric pacemaking has evolved from predominantly epicardial to predominantly endocardial. One of the potential problems with endocardial pacing in children is their very thin subcutaneous tissue, which can result in an unpleasant cosmetic result. The superficialness of the pacemaker pulse generator also may render it more susceptible to erosion and infection. A series is presented of pediatric patients who underwent implantation of a transvenous bipolar pacing system in the pediatric catheterization laboratory. The pacemaker was implanted under the pectoralis major muscle after a muscle spreading incision was made. The lead was also introduced into the subclavian vein under the pectoralis muscle. No complications resulted, and the cosmetic result was judged to be good to excellent by the parents and physicians. No infections or erosions occurred. The subpectoral pocket is recommended as the preferred site for implantation of transvenous pacemakers in pediatric patients. PMID- 1715545 TI - Right-sided infective endocarditis with acquired tricuspid valve stenosis associated with transvenous pacemaker: a case report. AB - Five years prior to presentation, a 29-year-old woman received a transvenous pacemaker (DDD) for sick sinus syndrome and nodo-hisian pathology. After pacemaker insertion, she complained of recurrent febrile episodes. Her pacemaker related endocarditis was quite unusual for the infecting organism (a micrococcus) and for an acquired tricuspid valve stenosis. The suspected cause was confirmed at surgery. PMID- 1715546 TI - A prospective study of changes in right ventricular dP/dt during ventricular tachycardia. AB - The automatic implantable cardioverter defibrillator (AICD) has significantly decreased mortality in high risk ventricular tachycardia (VT) patients. The AICD provides treatment based on ventricular rate, sometimes leading to high energy shocks in conscious patients with stable VT, or patients with sinus or supraventricular tachycardia. Other physiological parameters, such as maximal positive and negative systolic right ventricular (RV) dP/dt (RV + dP/dtmax, RV - dP/dtmax, respectively), may be included in detection algorithms for future implantable defibrillators. We studied frequency band limited positive and negative RV dP/dtmax before, during, and after 13 episodes of VT lasting at least 40 beats in duration in nine male patients. The mean (+/- SEM) RV + dP/dtmax, dropped by 120 +/- 28 mmHg/sec (P less than 0.001) during the first five beats of VT. RV + dP/dtmax then slowly rose toward baseline levels until a significant overshoot occurred during the first ten beats following VT termination (delta = 234 +/- 58 mmHg/second, P less than 0.002). RV + dP/dtmax correlated poorly with mean arterial pressure (r = 0.32, P greater than 0.1), systolic blood pressure (r = 0.19, P greater than 0.1), and VT cycle length (r = 0.34, P greater than 0.1). Conversely, RV - dP/dtmax rose during the first ten beats of VT (74 +/- 27 mmHg/sec, P greater than 0.05) and then slowly drifted back toward baseline levels. Like RV + dP/dtmax, RV - dP/dtmax overshot baseline levels during the recovery phase (-108 +/- 48 mmHg/sec, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715547 TI - Bradycardia-dependent early afterdepolarizations in a patient with QTU prolongation and torsade de pointes in association with marked bradycardia and hypokalemia. AB - Endocardial monophasic action potentials (MAPs) were recorded at the right ventricular apex in a patient with QTU prolongation and torsade de pointes (TdP) in association with marked bradycardia and hypokalemia. There was a distinct hump on phase 3 repolarization of the MAPs characteristic of early afterdepolarizations (EADs), which was associated with marked prolongation of the QTU interval on the surface electrocardiogram. EAD amplitude was bradycardia dependent, and there was a strong correlation (r = 0.91) between the preceding RR interval and the amplitude of the EAD (percent of MAP amplitude). Intravenous administration of lidocaine or right ventricular pacing suppressed the ventricular premature complexes and TdP in association with the suppression of the EADs on the MAPs. Furthermore, these EADs were not recorded on the MAPs 1 month later when the QTU prolongation and TdP had disappeared. These findings suggest that the TU abnormality and QTU prolongation responsible for TdP were due to bradycardia-dependent EADs. PMID- 1715548 TI - Complete atrioventricular block following mediastinal irradiation: a report of six cases. AB - Complete atrioventricular block (AVB) following radiotherapy has been reported rarely, usually after high dose mediastinal irradiation for Hodgkin's disease or lung or breast carcinoma. We report six new cases of episodic complete infranodal AVB, requiring permanent pacemaker implantation. The mean age was 48-years old (ranging from 25-60) at the first Adams Stokes attack, mean delay was 12 years after irradiation (10-18), and mean radiation dose was 5,200 rads (4,000-6,500). All patients had abnormal interval electrocardiograms (right bundle branch block in two, left bundle branch block in three, alternating left and right bundle branch block in one). Electrocardiograms during the episode of AVB or Holter recordings were consistent with infranodal block in all patients; electrophysiological study performed in five patients confirmed infranodal AVB in four, and one was normal. Pericardial disease was constant, which included pericardial constriction in four patients. Two patients died after failure of pericardiectomy to improve congestive heart failure, due to epicardial, myocardial, and endocardial involvement. Noncardiac mediastinal lesions were present in four cases. Since this delayed complication may occur in patients of such age that the relation between the AVB and the chest irradiation is questionable, we propose the following etiologic criteria; high radiation dose (over 4,000 rads); delay of 10 years or more; abnormal interval tracings; pericardial involvement; and associated cardiac or mediastinal radiation-induced lesions. PMID- 1715549 TI - The initial clinical experience with an implantable cardioverter defibrillator/antitachycardia pacemaker. AB - Guardian antitachycardia pacing (ATP) 4210 is a third generation, multi programmable cardioverter defibrillator undergoing Phase I clinical trials. The tiered response includes ATP, low energy cardioversion or defibrillation, and bradycardia support. Extensive telemetry is available, including an episode log and details of all episode events. Five patients underwent the implantation of Guardian ATP 4210 as part of a Phase I trial at the University of Louisville. Two of the five patients had multiple VT episodes that were reverted successfully using ATP pacing (slow VT) and defibrillation (fast VT) and VF episodes, which resulted in defibrillation therapy over a follow-up period of 6 to 8 months. Four of the five patients required bradycardia support for bradyarrhythmias unassociated with ATP therapy or defibrillation and one patient required bradycardia support postdefibrillation therapy. The device design is microprocessor based and requires continuous interrogation of the microprocessor memory and checks of the validity of programmed parameters to continue its operation. When the safety check fails, the device is designed to shut down its antitachycardia and defibrillator functions. This design feature has a potential for leaving the patient unprotected if the device shuts down. Modification of this feature is required to ensure the device's long-term safety. PMID- 1715550 TI - Clinical electrophysiological effects of intravenous recainam: an antiarrhythmic drug under investigation for the treatment of ventricular and supraventricular arrhythmias. AB - This open-label, multicenter study was designed to assess the electrophysiological properties of intravenous recainam, an investigational Class I antiarrhythmic agent. In 25 patients undergoing electrophysiological studies for the evaluation of arrhythmias, recainam was administered intravenously in a loading infusion (0.1 mg/kg/min) for 40 minutes, followed by a maintenance infusion (0.02 mg/kg/min) until the completion of the study. Electrophysiological measurements were obtained at baseline, 30 minutes after initiation of the loading infusion, and 30 minutes after termination of the infusion during washout. Conduction intervals, refractory periods, and sinus node recovery times were measured during sinus rhythm and during atrial or ventricular pacing. Vital signs were obtained and recorded before, during, and after recainam infusion. The results showed no change in mean arterial pressure, but heart rate increased slightly by 4 beats/min following recainam infusion. Recainam produced a generalized slowing of intracardiac conduction. The mean intraatrial conduction time, measured at an atrial paced cycle length of 600 msec, increased during recainam loading infusion by 44%, from 38.8% +/- 2.8 to 53.0 +/- 5.4 msec; intranodal conduction time increased by 10%, from 102.0 +/- 5.5 to 112.1 +/- 5.2 msec; and infranodal conduction time increased by 31% from 53.1 +/- 3.0 to 70.7 +/- 3.8 msec. Slowed conduction persisted during washout. The mean right atrial effective refractory period was significantly prolonged (+7% at 600 msec cycle length and +8% at 450 msec cycle length, P less than 0.05 and P less than 0.01, respectively) during recainam loading and remained so during washout.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715551 TI - Retrospective analysis of patients undergoing one- or two-stage strategies for myocardial revascularization and implantable cardioverter defibrillator implantation. AB - Internal defibrillation leads were placed at time of coronary revascularization in 79 patients. In 34, an implantable cardioverter defibrillator (ICD) was placed simultaneously (group I). A two-stage strategy (selective implantation of the ICD in patients with postoperative spontaneous or inducible ventricular tachycardia [VT]) was followed in 45 patients (group II). Group I patients had failed more antiarrhythmic drug trials (2.9 +/- 1.6 vs 1.5 +/- 1.6; P = 0.02), including amiodarone (62% vs 20%; P less than 0.001). There were four operative deaths in each group. Postoperatively, VT was present in 27 group II patients (60%), 25 of whom received an ICD (two refused device implantation). Patients with postoperative VT had a lower left ventricular ejection fraction than those without VT (33 +/- 9 vs 47 +/- 16; P = 0.01). Actuarial survival at 1, 2, and 3 years was 88 +/- 6, 88 +/- 7, and 88 +/- 10 in group I; and 83 +/- 6, 76 +/- 7, and 76 +/- 11 in group II (NS). No patient without an ICD (based on the postoperative electrophysiological study [EPS]) died suddenly. Five patients (6%) had ICD system infection. Sudden death was largely prevented by either strategy, but relatively high rates of operative mortality and ICD system infection were observed. Prospective studies should identify patients more likely to benefit from one or another strategy. PMID- 1715552 TI - Dispersion of repolarization induced by a nonuniform shock field. AB - Dispersion of repolarization may contribute to arrhythmias. To determine whether an electrical field stimulus (S2) with a nonuniform potential gradient can induce a dispersion of repolarization, we applied 5 ms rectangular S2 that had a nonuniform or uniform potential gradient during the action potential (AP) of bathed frog ventricular strips. One group had a partitioned bath to produce a nonuniform S2 of 39 +/- 11 V/cm (mean +/- SD) in one half of the 1 x 6 mm strip (H) and 0.3 +/- 0.2 V/cm in the other half (L), and simultaneous intracellular AP recordings in H and L with glass microelectrodes positioned 1.4 +/- 0.4 mm apart. Another group had uniform S2 and an AP recorded near the center of the strip. S1 pacing at 0.5 Hz was performed at one end of the strip and conduction along the strip was monitored. In each experiment, the S2 trials had an S1-S2 interval of 300 ms so that S2 was given during an AP (shocked AP). In both H and L, nonuniform S2 produced cumulative shortening of paced APs and lengthening of each shock AP compared with the paced AP preceding it. Uniform S2 of 1 V/cm did not shorten the paced APs or lengthen the shocked APs indicating that the AP changes in L were not due to the small potential gradient in L. Before beginning nonuniform S2 trials, the AP duration determined at the maximum repolarization rate was 601 +/- 72 ms in H and 602 +/- 71 ms in L (P = ns). During 13-20 nonuniform S2 trials over a 60-80 minute period, paced APs were shortened to 490 +/- 51 ms in H and 515 +/- 39 ms in L while each shocked AP was lengthened, compared with the paced AP preceding it, to 636 +/- 40 ms in H and 561 +/- 21 ms in L (P less than 0.05). Therefore, paced APs after shocks repolarized 25 ms earlier in H than in L and shocked APs repolarized 75 ms later in H than in L. The results show that during the shortened AP in H, the AP in L is shortened, which is consistent with intracellular current from L to H during repolarization. During the prolonged AP in H, the AP in L is prolonged compared with the paced AP preceding it, consistent with intracellular current from H to L during repolarization. Thus, nonuniform shocks can induce a dispersion of repolarization and may induce cell-to-cell interactions during repolarization. PMID- 1715553 TI - Physical and dynamic characteristics of DC ablation in relation to the type of energy delivery and catheter design. AB - We evaluated and compared the in vitro characteristics of direct current ablation using high energy ablation (Hewlett-Packard defibrillator) and a new form of low energy ablation (low energy ablation power supply, Cardiac Recorders, UK). Two new catheters with a large distal electrode have been recently introduced for catheter ablation: a low energy 7F bipolar catheter (Bard) with a contoured distal electrode, and a 7F deflectable catheter with a 4-mm tip (Mansfield). In vitro studies were carried out in a large tank filled with physiological saline while recording voltage, current, and pressure. High speed cinematography at 32,000 frames per second (Cordin, Utah) was done to assess the dynamic behavior of the vapor globe with both systems of energy delivery. We evaluated shocks of 50, 100, 150, 200, and 300 joules with the conventional system, and shocks of 10, 15, 20, 30, and 40 joules with the new system, and also compared the effects of varying catheter design with both systems of energy delivery. The conventional system using high energy showed significant arcing and increases in pressure. Low energy direct current ablation produces nonarcing shocks with 20 joules or less, and significantly less vapor globe and gas formation during arcing shocks, with a shorter duration of increase in pressure. This new system using low energy direct current may reduce the risk and complications reported with high energy ablations. PMID- 1715554 TI - Inability of the signal-averaged electrocardiogram to determine risk of arrhythmia recurrence in patients with implantable cardioverter defibrillators. AB - Signal-averaged electrocardiography has been used to identify patients at risk for arrhythmic death after myocardial infarction. Since patients with implantable cardioverter defibrillators (ICDs) are at high risk for arrhythmic events, they should also be expected to have a high incidence of abnormal signal-averaged electrocardiograms (SAECGs). However, whether the SAECG can discriminate patients who will have arrhythmia recurrence and receive appropriate ICD shocks from those who will have no recurrence and no shocks is unknown. This study examines the usefulness of the SAECG to separate appropriate users from non-users of the ICD. Fifty patients with ICDs participated in this study. Those who received a shock preceded by symptoms, a shock without preceding symptoms but with electrocardiographic documentation of ventricular fibrillation or ventricular tachycardia, or a shock while asleep were classified as ICD users. All other patients were classified as nonusers. The SAECG was classified as normal if the QRS duration on the standard electrocardiogram was less than or equal to 110 msec and if the total filtered QRS duration was less than 120 msec, the root-mean square voltage of the terminal 40 msec was greater than 25 muV, and the terminal low amplitude signal duration measured less than 38 msec. The SAECG was classified as abnormal if the QRS duration on the standard electrocardiogram was less than or equal to 110 msec and any one of these three criteria were outside the "normal range." The SAECG was classified as indeterminate if the QRS duration on the standard 12-lead electrocardiogram was greater than 110 msec. For the entire group of 50 patients, 8 (16%), 12 (24%), and 30 (60%) had normal, abnormal, and indeterminate SAECGs, respectively. Of the 22 ICD users, 1 (5%), 5 (23%), and 16 (73%) patients had normal, abnormal, and indeterminate SAECGs, respectively. Of the 28 ICD nonusers, 7 (25%), 7 (25%), and 14 (50%) patients had normal, abnormal, and indeterminate SAECGs, respectively. ICD users had lower left ventricular ejection fractions (P = 0.0002), a higher incidence of ventricular tachycardia (P = 0.04), prior exposure to a greater number of antiarrhythmic drugs (P = 0.04), and a lower likelihood for survival (P = 0.02) compared to the ICD nonusers. There was no statistically significant difference between the ICD users and nonusers as stratified by SAECG classification regardless of whether or not the interminate studies were included or excluded from the analysis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1715556 TI - Late complication of radiotherapy to the chest. PMID- 1715555 TI - Two phase radiofrequency catheter ablation of isolated ventricular endomyocardium. AB - This report describes a two phase radiofrequency (TPRF) energy source producing two radiofrequency sinusoidal voltages of similar frequency but different phase angles between three points of wire. When delivered through an orthogonal electrode catheter array (OECA) TPRF energy produces a square-shaped lesion of the area covered by the five electrodes (0.8 cm2). The purposes of the study were: to create square-shaped lesions using TPRF energy; to compare the size of lesions created by single phase radiofrequency (SPRF) to that of TPRF energy; and to study the depth of such lesions and to create lesions of desired size by adjacent placement of the OECA using TPRF energy. Ablations were created in nine isolated bovine hearts using three power settings (10, 20, and 40 watts) and three pulse durations (5, 10, and 20 seconds). Pathological examination was performed to document the length, width, depth, and the microscopic changes of ablations. TPRF energy increases the size of lesion (P less than 0.001) and utilizes less power (P less than 0.008) at the same power setting and pulse duration compared to SPRF energy. This is possibly related to earlier rise in impedance with TPRF compared to SPRF ablations. The largest lesion for both SPRF (0.51 +/- 0.08 cm2) and TPRF (1.03 +/- 0.18 cm2) ablations were observed at 20 watts for 20 seconds. By adjacent placement of the OECA and TPRF energy desired size (6 cm2) lesions were created. There was no significant difference between the depth of SPRF versus TPRF ablations at comparable power setting and pulse duration. Pathological examination revealed the shape of lesions were elliptical or cross-shaped for SPRF and square for TPRF ablations. Microscopic examination revealed coagulation necrosis, edema, and few necrotic cardiac muscle strands. CONCLUSIONS: TPRF energy can cause 1.2 cm2 lesions. TPRF compared to SPRF energy causes larger lesions but depth of lesions are not different than SPRF energy at the same power setting and pulse duration. By adjacent placement of OECA and TPRF energy desired size lesion can be created (6 cm2). PMID- 1715557 TI - Performance of implantable cardiac rhythm management devices. PMID- 1715558 TI - [Refsum's disease with 25 years' follow-up. A long term prognosis transformed by diet therapy]. PMID- 1715559 TI - [Transurethral or transvesical prostatectomies. Do they apply to the same patients?]. PMID- 1715560 TI - Structural and functional domains in human tumour necrosis factors. AB - Human tumour necrosis factors (hTNFs) alpha and beta are related pleiotropic cytokines which share many activities and compete with each other for binding to two receptor components on many cell types. Although structural and biological data indicate that the active form of hTNF-alpha may be a symmetrical trimer, the manner in which hTNFs interact with their receptors to trigger a myriad of cell type-dependent responses is not clear. A combination of chemical modification, epitope mapping and site-directed mutagenesis approaches suggest that at least four distinct peptide sequences are important for the biological activity of hTNF alpha. In particular, certain peptide sequences between amino acid positions 11 and 35 in hTNF-alpha appear to be critical for receptor binding and triggering biological responses. The recent cloning of the two hTNF-alpha/beta receptors opens the way for precise mapping of the functional domains in hTNFs. PMID- 1715561 TI - Molecular modeling of antibody-antigen complexes between the Brucella abortus O chain polysaccharide and a specific monoclonal antibody. AB - A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement. PMID- 1715562 TI - Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo. AB - The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids. Two methods for improving the efficiency of translation have been tested. First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS. The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS. The expression levels are described by an approximately Gaussian histogram. The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor. Under the expression conditions, the protein is partially proteolysed. The proteolysis is significantly decreased by adding salt to the growth medium. After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions. An efficient purification protocol is described. PMID- 1715563 TI - On the multiple-minima problem in the conformational analysis of polypeptides. V. Application of the self-consistent electrostatic field and the electrostatically driven Monte Carlo methods to bovine pancreatic trypsin inhibitor. AB - In connection with the accompanying paper to test various models for the hydration of polypeptides, we have explored a limited portion of the conformational energy hyperspace of the small protein bovine pancreatic trypsin inhibitor (BPTI) with the aid of two search methods developed in this laboratory. A series of low-energy conformations was obtained as a result of this study. These conformations constitute a set of local minima in the conformational energy space of the molecule as described by the ECEPP/2 (Empirical Conformational Energy Program for Peptides) potential energy function, without the inclusion of hydration. Five different initial conformations were used in this exploration: the first corresponds to an energy-refined structure based on the crystallographic coordinates (4PTI) provided by Deisenhofer and Steigemann and reported previously by Meirovitch and Scheraga. The remaining four initial conformations were obtained by using a Variable-Target-Function procedure, applied to the experimental Cartesian coordinates (5PTI) reported by Wlodawer et al. The self-consistent electrostatic field (SCEF) and the electrostatically driven Monte Carlo (EDMC) methods were used to search the conformational space. The SCEF and EDMC methodologies assume that a polypeptide or protein molecule is driven toward the native structure mainly by the action of the electrostatic interactions. Application of these methodologies led to a set of conformations (up to 50 kcal/mol lower than the starting ones) with ECEPP/2 energies lower than any of those that we had previously found. Application of both methods to the initial conformation generated from 4PTI led to a series of low-energy conformations exhibiting similar rms deviations with respect to the experimental data (4PTI) as did the starting conformation. However, statistical analysis of the runs that had started from the conformations generated by using the variable target-function procedure (and applying the EDMC method) indicated that the rms deviations of the atomic positions of the new low-energy conformations tended to increase as the energy improved, when compared with the X-ray data from which the starting conformations had been generated. The structures with the lowest energies also had radii of gyration smaller than the experimentally observed one. These results indicated a need to include hydration in the potential function, and provided the conformations used in the accompanying paper to test various hydration models. PMID- 1715564 TI - Empirical solvation models can be used to differentiate native from near-native conformations of bovine pancreatic trypsin inhibitor. AB - Several hydration models for peptides and proteins based on solvent accessible surface area have been proposed previously. We have evaluated some of these models as well as four new ones in the context of near-native conformations of a protein. In addition, we propose an empirical site-site distance-dependent correction that can be used in conjunction with any of these models. The set of near-native structures consisted of 39 conformations of bovine pancreatic trypsin inhibitor (BPTI) each of which was a local minimum of an empirical energy function (ECEPP) in the absence of solvent. Root-mean-square (rms) deviations from the crystallographically determined structure were in the following ranges: 1.06-1.94 A for all heavy atoms, 0.77-1.36 A for all backbone heavy atoms, 0.68 1.33 A for all alpha-carbon atoms, and 1.41-2.72 A for all side-chain heavy atoms. We have found that there is considerable variation among the solvent models when evaluated in terms of concordance between the solvation free energy and the rms deviations from the crystallographically determined conformation. The solvation model for which the best concordance (0.939) with the rms deviations of the C alpha atoms was found was derived from NMR coupling constants of peptides in water combined with an exponential site-site distance dependence of the potential of mean force. Our results indicate that solvation free energy parameters derived from nonpeptide free energies of hydration may not be transferrable to peptides. Parameters derived from peptide and protein data may be more applicable to conformational analysis of proteins. A general approach to derive parameters for free energy of hydration from ensemble-averaged properties of peptides in solution is described. PMID- 1715565 TI - Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I. AB - The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human IGFBP-1 cDNA construct) and human hepatoma (HepG2) cells secrete 32P-labeled IGFBP-1 following incubation with [32P]orthophosphate. Phospho amino acid analysis of 32P-labeled IGFBP-1 revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms. Using this technique we demonstrated that human amniotic fluid and human fetal serum contain a large proportion of nonphosphorylated IGFBP-1, as well as phosphorylated forms. In contrast, HepG2 cells and human decidual cells secrete predominantly the phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. We conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification. PMID- 1715566 TI - Expression of two forms of carp gonadotropin alpha subunit in insect cells by recombinant baculovirus. AB - There are two types of cDNA clones (designated alpha 1 and alpha 2) encoding the alpha subunit of carp gonadotropin. These two cDNAs are derived from different genes and encode proteins that differ by seven amino acid residues (three in the signal peptide and four in the mature polypeptide). Expression of these two cDNAs in insect cells by recombinant baculovirus revealed that the alpha 1 subunit, after noncovalent association with the beta subunit, has the same potency as the native alpha subunit purified from the pituitary. In contrast, the alpha 2 subunit can associate with the beta subunit, but only to form an inactive gonadotropin. Competition of the alpha 2 subunit with the alpha 1 subunit for association with the beta subunit decreases the gonadotropin activity of the alpha/beta complex. In addition, both alpha 1 and alpha 2 subunits are secreted into the culture medium by insect cells and have an apparent molecular mass approximately 5 kDa higher than that of the native alpha subunit. These results indicate that the insect cell-derived alpha 1 subunit is biologically active and that those four amino acid changes in the mature of alpha 2 protein affect the biological activity and thus provide valuable clues for the study of the structure-function relationship of the alpha subunit of glycoprotein hormones. PMID- 1715567 TI - cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. AB - A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl( )-indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell current-voltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl- flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (delta F508), the most common mutation causing cystic fibrosis. These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl- channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basic mechanisms underlying this CFTR-associated ion permeability and to study the structure and function of the CFTR. PMID- 1715569 TI - Mouse macrophage clones immortalized by retroviruses are functionally heterogeneous. AB - Murine macrophage clones were generated from thymus, spleen, brain, and bone marrow by in vitro immortalization with recombinant retroviruses carrying an avian v-myc oncogene. The cloned cell lines express F4/80 molecules, exert phagocytosis, have nonspecific esterase activity, and express class II molecules after interferon gamma activation. The macrophage clones are diploid and their karyotypes have remained stable for greater than 3 years in culture. After the macrophage clones were activated, their pattern of cytokine production was investigated. Functional heterogeneity in cytokine transcription was demonstrated: one of six liposaccharide-activated macrophages was unable to transcribe interleukin 1 alpha, whereas all of the liposaccharide-activated clones were able to transcribe tumor necrosis factor alpha. Interleukin 6 production was detected in three of six clones. The production of nitrite and tumor necrosis factor alpha as effector molecules of cytotoxicity was detected in all clones, thus showing that a single macrophage can exert more than one cytotoxic mechanism. The results indicate that immortalized and cloned macrophages have a differentially regulated expression of cytokine genes, adding further evidence for the existence of functional heterogeneity among cloned macrophages. This heterogeneity seems to derive from differentiation-related mechanisms rather than from external constraints. PMID- 1715568 TI - Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo. AB - The lectin homing receptor LECAM-1 (LAM-1, Leu8) and the beta 2 integrins, particularly Mac-1 (CD11b/CD18), participate in leukocyte-endothelial cell interactions in inflammation. LECAM-1 is rapidly shed while Mac-1 expression is dramatically increased upon neutrophil activation, suggesting functionally distinct roles for these molecules. Using intravital video microscopy, we have compared the effect of antibodies against LECAM-1 and CD18 on leukocyte interactions with rabbit mesenteric venules. Anti-LECAM-1 monoclonal antibody and its Fab fragments inhibited initial reversible leukocyte rolling along the vascular wall. Anti-CD18 monoclonal antibody had no effect on rolling but prevented subsequent firm attachment of leukocytes to venular endothelium. These results support a two-step model of leukocyte-endothelial cell interactions: reversible rolling mediated in part by LECAM-1 facilitates leukocyte recruitment by the local microenvironment and precedes activation-dependent firm attachment involving beta 2 integrins. PMID- 1715570 TI - Pelizaeus-Merzbacher disease: a valine to phenylalanine point mutation in a putative extracellular loop of myelin proteolipid. AB - In the central nervous system, myelin proteolipid protein isoforms (PLP and DM20) play an essential structural role in myelination. It has been shown in several species that myelination is impaired by molecular defects resulting from single base mutations in the PLP gene. We have used DNA amplification by polymerase chain reaction to study the PLP gene coding regions from 17 patients in 15 unrelated families with similar Pelizaeus-Merzbacher phenotype. In one case amplification of peripheral nerve PLP/DM20 cDNAs revealed that a silent T----C transition was unrelated to the disease. In one family a nonsilent mutation was identified that leads to a phenylalanine substitution for valine-218 in PLP/DM20 proteins. We investigated the inheritance of the mutant allele in 19 subjects of this four-generation family and found a strict cosegregation of the Phe218 substitution with transmission and expression of the disease. The effect of the Val218----Phe mutation is discussed in the frame of a recently suggested topological model of PLP/DM20, according to which Val218 is part of an extracellular loop that connects the last two of four membrane-spanning alpha helices. PMID- 1715571 TI - Depolarization-induced changes in cellular energy production. AB - Addition of high concentrations of KC1 to preparations of rat brain synaptosomes incubated with either glucose or pyruvate caused a transient stimulation of oxygen uptake. This increased respiration was insensitive to 1 mM ouabain and 10 microM ruthenium red but was dependent upon the presence of calcium. With 40 mM KCl in the incubation medium, the levels of high-energy phosphate compounds in the synaptosomes were unaltered, whereas pyridine nucleotides underwent a rapid, albeit small and temporary, oxidation. It is postulated that there is a calcium dependent mechanism in synaptosomes through which the function of the mitochondrial respiratory chain or of oxidative phosphorylation is stimulated directly without the involvement of either adenine nucleotides or mitochondrial dehydrogenases. PMID- 1715572 TI - Expression of two different forms of fibroblast growth factor receptor 1 in different mouse tissues and cell lines. AB - The fibroblast growth factor receptors (FGFRs) form a multigene family of at least four members, all having extracellular regions consisting of either two or three immunoglobin-like (Ig-like) domains. By RNase protection analysis we have analyzed the expression of FGFR-1 mRNA in various tissues and cell lines and demonstrated that all of the cell lines studied expressed at least two different forms of the FGFR-1 at similar levels. Although muscle and heart express forms having either two [FGFR-1 short (FGFR-1S)] or three [FGFR-1 long (FGFR-1L)] Ig like domains, the developing brain and adult brain express only mRNA encoding the longer form. The two forms of the receptor were characterized further by stably introducing expression vectors expressing them into Rat-2 fibroblasts and FDC-P1 myeloid cells. Treatment of the transfected Rat-2 cells with acidic FGF (aFGF) or basic FGF (bFGF) resulted in focus formation. The transformed phenotype was observed even without addition of ligand after growth in culture for greater than 2 months. Cross-linking of 125I-labeled bFGF (125I-bFGF) to Rat-2 cells expressing either FGFR-1L or FGFR-1S yielded two similar complexes of 150 and 110 kDa. Although Rat-2 cells expressing FGFR-1L yielded similar complexes with 125I labeled aFGF (125I-aFGF), only the 150-kDa complex was formed with cells expressing FGFR-1S. The 150-kDa complex was also observed when 125I-aFGF or 125I bFGF was cross-linked to FDC-P1 cells expressing FGFR-1L. Significantly, these complexes were only observed when heparin was present in the cross-linking reaction. FDC-P1 cells expressing FGFR-1 bound aFGF and bFGF with high affinity but only in the presence of heparin. The factor dependence of these cells could be switched from interleukin 3 to FGF in the presence of heparin. PMID- 1715573 TI - A prion-like protein from chicken brain copurifies with an acetylcholine receptor inducing activity. AB - The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well. PMID- 1715574 TI - Two nonallelic insulin genes in Xenopus laevis are expressed differentially during neurulation in prepancreatic embryos. AB - Insulin, traditionally regarded as a metabolic hormone, also can potently stimulate growth and differentiation in many cell types. To study further the potential role of insulin during early embryogenesis, we have used the amphibian Xenopus laevis, a versatile model of vertebrate development. Using (i) nucleotide sequences of two previously cloned cDNAs that correspond to two different nonallelic Xenopus insulin genes (both of which are expressed in the adult pancreas) and (ii) a modification of the highly sensitive reverse transcription polymerase chain reaction (RT-PCR) method developed in our laboratory, designated RNA template-specific PCR (RS-PCR), we now find that mRNAs for both Xenopus insulins I and II are present in mature (stage VI) oocytes but not in less-mature oocytes (stages I and IV) or in unfertilized eggs. The Xenopus insulin II gene is differentially expressed during early neurulation (stage 13), while only the insulin I gene is expressed at stage 21, when the neural tube is closing and cephalization is beginning. During later stages (i.e., stage 26) there is a region in the head that appears to be transcribing only the insulin I gene, while mRNAs for both insulins I and II are present in the body region. These findings show that the two nonallelic insulin genes are expressed differentially in Xenopus embryos in a stage- and region-specific manner; because appropriate receptors are also present, we suggest a role for insulin during early nervous system development well before the emergence of pancreatic beta cells. PMID- 1715575 TI - Expression cloning of a rat B2 bradykinin receptor. AB - A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl- current oocytes expressing the receptor with the rank order of potency: bradykinin approximately Lys-bradykinin greater than [Tyr8] bradykinin much greater than [Phe6]bradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 microM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [Thi5,8,DPhe7]bradykinin, where Thi is beta-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle, rather than a neuronal, B2 receptor subtype. Receptor message has a distribution consistent with previous reports of bradykinin function and/or binding in several tissues and is found in rat uterus, vas deferens, kidney, lung, heart, ileum, testis, and brain. Receptor subtypes are a possibility because several tissues contain two or three message species (4.0, 5.7, and 6.5 kilobases). Southern blot high-stringency analysis demonstrated that the rat, guinea pig, and human genomes contain a single gene. As bradykinin is a key mediator of pain, knowledge of the primary structure of this receptor will allow a molecular understanding of the receptor and aid the design of antagonists for pain relief. PMID- 1715576 TI - Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif. AB - The Rev protein of human immunodeficiency virus type 1 is a sequence-specific RNA binding protein that is essential for viral replication. Here we present evidence that Rev is a stable oligomer both in vitro and in vivo. Analysis of Rev mutants indicates that oligomerization is essential for RNA binding and hence Rev function. The oligomerization and RNA binding domains overlap over 47 amino acids. Within this region is a short arginine-rich motif found in a large class of RNA binding proteins. Substitution of multiple residues within the arginine rich motif abolishes oligomerization, whereas several single-amino-acid substitution mutants oligomerize but do not bind RNA. Thus, Rev's arginine-rich motif participates in two distinct functions: oligomerization and RNA binding. PMID- 1715577 TI - A functional isoform of the human granulocyte/macrophage colony-stimulating factor receptor has an unusual cytoplasmic domain. AB - The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) transduces a signal that results in the proliferation, differentiation, and functional activation of hematopoietic cells. This study sought to determine whether functional isoforms of the receptor exist that may be important in generating this diversity of cellular response. We have isolated a cDNA encoding an isoform of the low-affinity human GMR that is a product of alternative splicing of the GMR gene and results in a predicted 410-amino acid protein with a cytoplasmic domain that is rich in serine residues, a feature of regions critical in signal transduction for other receptors of the hematopoietin receptor superfamily. This receptor bound ligand and was functionally active when introduced into a murine factor-dependent cell line; mRNA transcripts representative of this isoform were coexpressed with those for a previously isolated 400-amino acid isoform of the GMR in normal hematopoietic and leukemic cells. In view of the recent isolation of a cDNA, designated GM-CSF R beta, that confers high-affinity binding of GM-CSF in cotransfection experiments with the low-affinity receptor, we suggest that the previously isolated low-affinity receptor be designated GM-CSF R alpha 1 and the one described in this report be designated GM-CSF R alpha 2. PMID- 1715578 TI - Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells. AB - We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability. PMID- 1715579 TI - Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. AB - A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN. PMID- 1715580 TI - Differential expression of epidermal growth factor-related proteins in human colorectal tumors. AB - Amphiregulin (AR) and cripto are proteins that are structurally related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is also functionally related to this family of growth regulatory molecules and is able to bind and activate the 170-kDa EGF receptor (EGFR). Human EGFR-3 (HER3)/ERBB3 is a recently identified protein related to the EGFR that is widely expressed in breast carcinomas and is a candidate receptor for EGF-like growth factors. Differential expression of these putative ligands and receptors in transformed cells suggests that they may function in an autocrine manner to regulate tumor cell growth. Specific mRNA transcripts for TGF-alpha [4.8 kilobases (kb)], AR (1.4 kb), cripto (2.2 kb), and HER3 (6.2 kb) were expressed in a majority of human colon cancer cell lines. HER3 mRNA was detected in 55% of primary or metastatic human colorectal carcinomas but in only 22% of normal colon mucosa and 32% of normal liver samples. In contrast, cripto and AR mRNA were expressed in 60-70% of primary or metastatic human colorectal cancers but in only 2-7% of normal human colonic mucosa. Immunostaining also detected AR protein in primary and metastatic colorectal tumors but not in normal colon or uninvolved liver. These findings suggest that cripto and AR may be useful markers to discriminate between normal and malignant colonic epithelium and may provide a selective growth advantage for colorectal carcinomas. PMID- 1715581 TI - Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. AB - NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity. PMID- 1715582 TI - Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets. AB - Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation. PMID- 1715583 TI - Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene. AB - Vascular cell adhesion molecule 1 (VCAM-1) is a cell surface glycoprotein adhesive for certain blood leukocytes and tumor cells, which is expressed by activated endothelium in a variety of pathologic conditions including atherosclerosis. Genomic clones encoding the VCAM1 gene were isolated and the organization of the gene was determined. The gene, which is present in a single copy in the human genome, contains 9 exons spanning approximately 25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5. A consensus TATAA element is located upstream of the transcriptional start site. The VCAM1 promoter contains consensus binding sites for NF-kappa B, the GATA family of transcription factors, as well as an AP1 site. The VCAM1 gene was assigned to the 1p31-32 region of chromosome 1 based on the analysis of human-mouse hybrid cell lines and in situ hybridization. Structural analysis of the human VCAM1 gene provides the basis for alternative mRNA splicing and an initial approach to elucidating the regulation of VCAM-1 expression. PMID- 1715584 TI - Cloning and expression of a rat cardiac delayed rectifier potassium channel. AB - We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2. PMID- 1715585 TI - Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver. AB - Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p less than 0.05 to p less than 0.001) lower binding capacity and binding affinity for 3H-PGE1, 3H-PGE2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 +/- 7% for 3H-PGE2, 75.3 +/- 9% for 3H-PGE1 and 78.9 +/- 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p less than 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver. PMID- 1715586 TI - [The behavior of substance P concentration in rat plasma in the acute single- and combined-actions of trichloroethylene and ethanol]. AB - In experimental studies in male Wistar-rats exerted for determination of trichloroethylene, ethanol and combined action influences on the substance P plasma concentrations. These concentrations were determined by a radioimmunological method. The acute exposure of trichloroethylene and ethanol lead to a significant rise of substance P. We found high substance P concentration peaks, in principle, 4 h after acute exposure and the combined action shows the greatest effect. PMID- 1715587 TI - Structural elements in RNA. PMID- 1715588 TI - Nuclear RNA-binding proteins. PMID- 1715589 TI - [Studies on impaired angiogenesis activity of the leucocytes in patients with oral candidiasis]. AB - The preliminary characteristics is reported of the cells causing active suppression leading to a decrease of the angiogenesis activity of leucocytes isolated from patients with oral candidiasis. The effect of three immunomodulators on this suppression was studied. PMID- 1715590 TI - Transcriptional regulation by cholecystokinin-pancreozymin in rat pancreas. AB - Cholecystokinin-pancreozymin (CCK-PZ) is involved in the regulation of pancreatic protein synthesis and secretion. We demonstrate here that CCK-PZ also stimulates RNA synthesis. Rats were killed 0, 30, 60, 120, 240 or 480 min after intraperitoneal injection of CCK-PZ (8 U/kg). Nuclei were prepared from pancreata and used for in vitro RNA synthesis ('run-on' experiments) in the presence of [alpha-32P]UTP. Total RNA synthesis increased after CCK-PZ with maximum UTP incorporation at 60 min. Contributions of RNA polymerase II, responsible for mRNA synthesis, and RNA polymerases I and III could be separately estimated by using alpha-amanitin. RNA polymerase I and III activities increased by 68% after CCK PZ, whereas RNA polymerase II activity increased by 113%. Rates of synthesis of amylase, chymotrypsinogen B, trypsinogen I and actin mRNAs were estimated by quantitative hybridization of newly synthesized transcripts to specific cDNA clones. Synthesis of the four RNAs increased with a maximum between 60 and 120 min after CCK-PZ, stimulation being more important for the serine proteinases than for amylase. It was concluded that, in rat pancreas, CCK-PZ controls gene expression at the transcriptional level. PMID- 1715591 TI - Neuropeptides of the primary sensory neurones in rat skin: an ontogenic study. AB - Cutaneous primary sensory neurones contain a number of biologically-active peptides, including substance P (SP), neurokinin A (NKA) and calcitonin gene related peptide (CGRP). However, little information is available on ontogenic changes in the tissue concentrations of these neuropeptides. In this study, the concentrations of these neuropeptides have been assessed in dorsal and ventral abdominal rat skin at various stages of development from foetal, early neonatal, late neonatal, weaner to adult, using sensitive and specific radioimmunoassays. In addition, the levels of peptide histidine isoleucine (PHI), a peptide found in non-sensory cutaneous nerves, were assessed to control the study. The levels of PHI and NKA immunoreactivity did not change significantly at any stage of development. However, the levels of SP and CGRP immunoreactivity were significantly elevated in the early neonate with CGRP remaining elevated in the late neonate. The levels of both SP and CGRP were not significantly different between other developmental groups. Significant elevations in cutaneous SP and CGRP concentrations in early neonatal life in the rat, at a time when the pups are blind and naked, may be related to control of cutaneous sensitivity, which during this period of development, has positive survival value for the pups. PMID- 1715592 TI - Substance P, neurokinin A and calcitonin gene-related peptide during development of the rat gastrointestinal tract. AB - Substance P, neurokinin A and calcitonin gene-related peptide (CGRP) were determined in the stomach and small intestine of rats during late foetal development and up to 35 days postnatal life. Concentrations of substance P in stomach and intestine increased from 14 gestational days to 3 days postpartum, and declined thereafter. Concentrations of neurokinin A in stomach declined from 14 days gestation over the period 3-35 postnatal days. In the intestine, concentrations of neurokinin A increased steadily from 14 days gestation to 21-35 postnatal days. Concentrations of CGRP in stomach and intestine declined from 14 days gestation to 7 postnatal days. Thereafter, concentrations of CGRP increased in both stomach and intestine. Total contents of each of the three peptides increased progressively with gestational and postnatal age in parallel with increasing stomach and intestinal weights. The results demonstrate different patterns of change in the concentrations of substance P, neurokinin A and CGRP during the dynamic phases of growth and maturation of the gastrointestinal tract in the foetal and postnatal rat. PMID- 1715593 TI - Role of norfluoxetine in the inhibition of desipramine metabolism and in the inhibition of serotonin uptake after fluoxetine administration to rats. AB - Fluoxetine, a serotonin uptake inhibitor, is known to inhibit the metabolism of some drugs including desipramine, resulting in increased brain and blood levels of desipramine when the drugs are co-administered to rats. Norfluoxetine, the N desmethyl metabolite of fluoxetine, was found to be less potent than fluoxetine in increasing brain and blood levels of desipramine in rats. Norfluoxetine was essentially equipotent to fluoxetine in decreasing brain concentrations of 5 hydroxyindoleacetic acid (5-HIAA) as a consequence of serotonin uptake inhibition. After the injection of fluoxetine into rats, brain levels of fluoxetine predominated over those of norfluoxetine at 1 hour, but at longer times (out to 24 hours), norfluoxetine levels were higher in brain (and in liver) than fluoxetine levels. Brain levels of 5-HIAA were decreased for at least 24 hours after fluoxetine injection, due apparently to the persistence of and inhibition of serotonin uptake by norfluoxetine. When desipramine was injected 16 hrs after fluoxetine injection, brain levels of desipramine were no longer elevated. The results suggest that norfluoxetine contributes in a major way to the inhibition of serotonin uptake after fluoxetine administration but contributes less, if at all, to the inhibition of desipramine metabolism. PMID- 1715594 TI - Acute phase proteins in grass sickness (equine dysautonomia). AB - Four acute phase proteins were assayed in the serum of normal horses and those with acute, subacute and chronic grass sickness, colic and inflammatory conditions, in order to investigate their diagnostic value in grass sickness. The grass sickness and inflammation group had a significantly increased haptoglobin concentration (P less than 0.01-P less than 0.001). Orosomucoid was elevated in acute, subacute and chronic grass sickness and inflammation (P less than 0.001, P less than 0.001, P less than 0.05 and P less than 0.05, respectively). Highest concentrations of haptoglobin and orosomucoid were recorded in subacute grass sickness. Ceruloplasmin was significantly higher in acute grass sickness cases than all other groups except the colic group (P less than 0.05-P less than 0.01). alpha 2-macroglobulin was significantly higher in acute grass sickness than normal, colic and chronic grass sickness cases (P less than 0.01, P less than 0.05 and P less than 0.05). The time scale of changes suggests that the stimulus to haptoglobin and orosomucoid synthesis occurs at the onset of clinical signs whereas the increase in ceruloplasmin and alpha 2-macroglobulin is more likely to reflect haemoconcentration. PMID- 1715595 TI - Echinococcus granulosus: antigenic proteins in oncospheres and on the surface of protoscoleces identified by serum antibodies from infected dogs. AB - Proteins present in oncospheres and on the surface of living protoscoleces of Echinococcus granulosus were radioiodinated by the lodogen technique and immunoprecipitated with sera from dogs with E granulosus infection and several categories of control sera. Analysis of immunoprecipitates was performed using sodium dodecyl-sulphate polyacrylamide gel electrophoresis to identify antigenic protein components specific for E granulosus. Sera from dogs with E granulosus infection identified antigenic proteins of around Mr 37,000, 30,000 or 22,000 in oncospheres, and proteins of around Mr 70,000, 43,000, 36,000, 27,000 (triplet), 20,000 or 14,000 on the surface of protoscoleces. These antigens appear to be both species- and stage-specific and may be useful for serological discrimination between 'current' and 'recent past' prepatent and patent E granulosus infections in dogs. PMID- 1715596 TI - Primary culture of chicken bursal plical epithelium. AB - Plical epithelial cells were obtained by trypsin-EDTA treatment of chicken bursa of Fabricius and cultured in the presence of type IV collagen. The culture became confluent six to seven days after seeding. The grown cells showed a positive reaction for cytokeratin by immunostaining and had ultrastructural characteristics of the epithelial cells in vivo. The cell culture will be useful for parasitological and virological studies. PMID- 1715597 TI - Evidence of phenotypic dichotomy within an individual Pasteurella multocida type strain and among some haemorrhagic septicaemia-causing field isolates. AB - Haemorrhagic septicaemia-causing strains of Pasteurella multocida were identified by a disease-specific ELISA. Some strains, however, were of the same serotype as those which cause haemorrhagic septicaemia (HS) but were negative when tested in the disease specific ELISA. The suspect false negative isolates were passaged in mice and retested in the HS ELISA with the same result. Immunoelectron microscopy was used to examine further these suspect HS-causing strains. Monoclonal antibodies and protein A-gold showed that the suspect negative organisms were a mixture of phenotypes with less than 10 per cent, and usually less than 2 per cent, of the population expressing HS-associated epitopes. The degree of staining on the organisms expressing the HS-epitopes was of the same intensity as the positive control organism. Expression of the HS-associated epitopes is presumably too low to allow detection in the current HS ELISA. PMID- 1715598 TI - Establishing a cancer patient education system for ambulatory patients. AB - Communication, coordination, and education are fundamental components of ambulatory care nursing. Yet the level of activity in most ambulatory care settings prohibits focusing exclusively on patient education. Rather, patient teaching procedures, tools, and programs must be incorporated with other activities. Several considerations for effective outpatient education include learning needs, barriers to learning, assessment of available resources (eg, environmental, human, financial), delineating roles for providing information, and selecting teaching tools and methods. PMID- 1715599 TI - [Infant mortality and health and social conditions in the Americas. A correlation study]. AB - The relation between the infant mortality rate, as a health indicator, and various demographic, social and health care development indexes is explored by means of an ecological, study based on correlation theory. Results show that the variables of greatest influence are maternal education and birth rate. It seems apparent that, once a minimal level is achieved, an increase of resources devoted to medical care, does not by itself, improve the infant mortality rate in these countries. PMID- 1715600 TI - Alpha-2-macroglobulin decreases parallel to albumin and haemoglobin after elective surgery. AB - Plasma levels of the plasma protease inhibitor alpha-2-macroglobulin (alpha 2-M) were followed for 7 days in 90 patients subjected to various surgical procedures. Alpha 2-M was found to decrease strictly in parallel with the decrease seen for haemoglobin and albumin levels in all patients. Changes were most pronounced after extensive operations; total hip replacement (n = 7), pulmonary resection (n = 11), extensive colo-rectal resection (n = 15), and less pronounced after 'minor' operations; mastectomy (n = 23) proximal gastric vagotomy (n = 5) and moderate colo-rectal resection (n = 29). Levels were lowest on the second to third postoperative day, whereafter they slowly returned to normal, preoperative levels during the 7-day study period. Functional and quantitative alpha 2-M levels almost paralleled each other throughout the 7 days studied. Chromogenic peptide substrate assays indicated circulating plasmin-alpha 2-M complexes, while no protease-alpha 2-M complexes could be demonstrated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF) analyses. Local accumulation and consumption of proteins within wounded tissues, together with haemodilution, were probably the major factors responsible for the decreased plasma levels seen. It is concluded that the plasma levels of alpha 2-M decrease after major elective surgery strictly in parallel with the decrease seen in haemoglobin and albumin levels, and that circulating plasmin alpha 2-M complexes are probable. The decrease seems to be graded, that is, proportional to the extent of the operative trauma, similar to the postoperative increase seen in positive acute-phase proteins. Thus, alpha 2-M cannot be used as an internal, unchanged plasma protein standard for other protein changes seen after trauma. PMID- 1715601 TI - Tumour marker CA 125 in patients with digestive tract malignancies. AB - The serum levels of tumour marker CA 125 were measured in 162 patients with various digestive tract malignancies and in 155 patients with benign digestive tract diseases. The highest frequency of elevated CA 125 values (greater than 35 U ml-1) was found in patients with liver cancer (78%), but the level was equally often elevated in liver cirrhosis (78%). Two-thirds of the patients with biliary tract cancer had an increased CA 125 concentration, while four patients with benign biliary diseases had an elevated value. The serum level of CA 125 was elevated in only 20% of 60 patients with primary colorectal cancer, and in none of those with local disease (Dukes A or B). The CA 125 concentration seldom increased in patients with recurrent colorectal carcinoma. Twenty-three per cent of 44 patients with gastric cancer had an elevated CA 125 value. Two of 33 patients with benign colorectal and one of 68 patients with benign gastric diseases had an increased CA 125 concentration. The serum values of CA 125 showed no correlation with those of tumour markers alphafetoprotein (AFP), carcinoembryonic antigen (CEA) or CA 19-9. AFP was superior to the other markers in the diagnosis of liver diseases, while CA 19-9 showed the greatest accuracy in gastric diseases. In colorectal diseases, CEA had a higher sensitivity, but a lower specificity than CA 125 and CA 19-9. CA 125 and CA 19-9 had similar sensitivities for biliary tract cancer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715602 TI - The role of CD45RA on human B-cell function: anti-CD45RA antibody (anti-2H4) inhibits the activation of resting B cells and antibody production of activated B cells independently in humans. AB - Anti-CD45RA antibody defined by anti-2H4 monoclonal antibody has been reported to split CD4+T cells into two distinct subpopulations. CD45RA antigen is present on the surface of virtually more than 95% B lymphocytes in the purified tonsillar B cell preparations. We examined the role of CD45RA antigen on human B-cell function using this antibody. The addition to anti-2H4 to tonsillar B cells inhibited the proliferative response induced by Staphylococcus aureus Cowan strain I(SAC) in a dose-dependent manner. Kinetic analysis indicated that anti 2H4 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72-h culture period. Anti-2H4 inhibited the transferrin receptor expression without interfering with the expression of the IL-2 receptor on SAC-stimulated B cells in a short-term culture. Anti-2H4 blocked the progress of SAC-stimulated B cells from the G1 to S phase of the cell cycle. These events suggested that anti-CD45RA MoAb inhibited the proliferative response by directly acting on B cells in the G1 phase. In addition, anti-CD45RA antibody also had a suppressive effect on early phase of B-cell differentiation. This effect appeared to be independent of its suppressive effect on proliferation, because anti-CD45RA did not inhibit the proliferative response of preactivated B cells with lymphokines. These studies suggested that the restricted epitope recognized by anti-2H4 antibody may be directly involved in regulatory function on B cells. PMID- 1715603 TI - T-cell regulation of CD5+ B-cell activity in normal mice. AB - The differentiation of plaque-forming cell (PFC) precursors against bromelain treated syngeneic erythrocytes (Br MRBC) into PFC induced in vitro by LPS is down regulated by nylon non-adherent (nylon-passed--NP) T cells and by nylon adherent (NA) T cells. NA T cells are more potent inhibitors than NP T cells. This regulatory activity of NA and NP T cells results from an interaction between CD4+ radioresistant and CD8+ radiosensitive T cells. Furthermore CD4+ T cells from the NA fraction but not from the NP fraction are activated cells: their inhibitory activity is abrogated after preincubation with cycloheximide. These results are discussed within the overall framework of T-cell regulation of autoimmune anti-Br MRBC B-cell subsets. PMID- 1715604 TI - Fine molecular specificity of linear and assembled antibody binding sites in HIV 1 p24. AB - A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11-kDa 104-mer peptide covering the C-terminal residues 270-373 of the p24 gag protein (HIV-1BRU strain). All monoclonal antibodies recognized HIV 1IIIB infected MOLT3 cells by fluorescence and gave positive Western blot signals with viral gag peptides (p55 and/or p24). Oligopeptide binding regions were located with competitive enzyme-linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104-mer region. The antibodies bound to p24 peptide sequences located within the 275-293 and 351-368 regions. One antibody (LH104-B) which reacted with residues 357-362 bound to p55 alone. In contrast, another antibody (LH104-I), which recognized the residues 358-363, i.e. with five out of six residues in common with antibody LH104-B for its epitope region, reacted exclusively with p24. At least two of the antibodies (LH104-C and -A) which bound to p24 alone, apparently recognized conformational epitopes. They gave positive reactions with the regions 288-293/351-356 and 284-289/351-356, respectively. This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins. PMID- 1715605 TI - Expression of the CD2 activation epitope T11-3 (CD2R) on T cells in rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and Lyme disease: phenotypic and functional analysis. AB - CD2R is an activation-associated epitope unmasked by a conformational change of the CD2 cell-surface glycoprotein. In spite of elaborate studies on the role of CD2 and CD2R in adhesion and stimulation of T cells in vitro, no instances of CD2R expression in vivo were known to date. We report high levels of CD2R observed on blood and synovial fluid T cells in rheumatoid arthritis and on peripheral blood T cells in juvenile rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and Lyme disease. In vivo, expression of CD2R was restricted to T cells, not limited to a particular T-cell subset and not correlated with the expression of p55 interleukin 2R (IL-2R) (CD25) or major histocompatibility complex (MHC) class II molecules. When stimulated to proliferation via CD2 or CD3, ex vivo CD2R+ T cells showed the same basic activation requirements as CD2R-T cells. PMID- 1715606 TI - MPB 64 possesses 'tuberculosis-complex'-specific B- and T-cell epitopes. AB - We have developed monoclonal antibodies (MoAb) reactive with a protein from Mycobacterium tuberculosis of apparent molecular mass 24 kDa. This protein was shown to be identical with MPB 64 (Harboe et al.,) MoAb bound to four different epitopes of which two were restricted to the 'tuberculosis complex' and two were also found in mycobacteria not belonging to the 'tuberculosis complex'. The cross reactive MoAb demonstrate that MPB 64 is present in more mycobacterial species than previously assumed. MPB 64 was shown to induce strong delayed type hypersensitivity (Dth) reactions in outbred guinea pigs immunized with M. tuberculosis and M. bovis bacille Calmette-Guerin (BCG). No reaction was observed in animals immunized with mycobacteria not belonging to the 'tuberculosis complex'. The Dth-inducing capacity of MPB64 was compared with that of another 24 kDa protein purified from M. tuberculosis and of the previously described 38 kDa protein. The Dth responses to these three antigens were further analysed in four inbred guinea pig strains. A genetic restriction of the ability of the animals to respond to MPB 64 as well as to the 38 kDa protein was observed. PMID- 1715607 TI - Hepatitis C virus antibody in Poland. AB - To assess the epidemiology of hepatitis C virus (HCV) in Poland, anti-HCV was studied in patients with acute and chronic non-A, non-B (NANB) hepatitis, in healthy adults, and in subjects at risk. Anti-HCV prevalence was 2% in 152 blood donors, 78% in 95 parenteral drug addicts, 21% in 112 alcoholics, and 86% in 42 patients with chronic NANB hepatitis. Among 34 prospectively followed patients with acute NANB hepatitis 17 (50%) developed anti-HCV. It seems that HCV infection is responsible for the majority of NANB hepatitis in Poland and is common in parenteral drug abusers and alcoholics. PMID- 1715608 TI - New antipsychotics: classification, efficacy, and adverse effects. AB - Compared to traditional neuroleptics, most of the new antipsychotics are characterized by a low extrapyramidal side effect (EPS) liability and varying antipsychotic efficacy. This topic is reviewed for four principal classes of new, established, and potential antipsychotics: (1) Antipsychotics such as sulpiride and remoxipride that block a subgroup of dopamine (DA) D2/D3 receptors produce a relatively low level of side effects, including EPS, and have an antipsychotic effect equal to or slightly weaker than traditional neuroleptics. D1 antagonists demonstrate a low level of EPS in primates and may prove to be a valuable new type of antipsychotic drug. (2) Theoretically, partial D2 agonists have the advantage of producing few or no EPS and a specific beneficial effect in negative symptoms, but as yet the expectations have not been fulfilled. (3) Nondopamine drugs such as serotonin (5HT1) agonists, 5HT2 antagonists, 5HT3 antagonists, and gamma-amino-butyric-acid-A (GABA-A) benzodiazepine agonists have anxiolytic, antidepressant, antiaggressive, and maybe antiparkinsonian effects and may play an adjunctive role in the treatment of schizophrenia. 5HT3 antagonists (e.g., ondansetron), partial benzodiazepine agonists, and partial glutamate agonists may prove to be effective antipsychotics. (4) Antipsychotics such as clozapine and risperidone, which affect D2/D3 receptors as well as 5HT, alpha 1, and/or D1 receptors appear to have the most pronounced antipsychotic effect. PMID- 1715609 TI - [Treatment of suppurative-necrotic decubitus ulcers in patients with spinal injuries by using alginate coating teralgim and algimaf]. AB - Alginate coating teralgim and algimaf were applied to 24 decubitus ulcers of various sites in 19 spinal patients. Stage--specific choice of applications produced a good cleansing effect and ulcer healing in 62.5% of the lesions within 48.5 days on the average. Indications for surgery were defined in 37.5% of the cases (repair of the ulcer defect) which were also treated with alginate coating with a good preoperative effect. PMID- 1715610 TI - [A case of successful complex treatment of locally disseminated cancer of the thoracic esophagus]. PMID- 1715611 TI - MK-801 inhibition of nicotinic acetylcholine receptor channels. AB - MK-801 is a potent inhibitor of the NMDA subtype of glutamate receptors. Single channel and macroscopic currents indicate that MK-801 also inhibits nicotinic acetylcholine receptors (nAChRs). MK-801 does not significantly increase desensitization of the nAChRs or compete for the ACh binding site. Although there is a slight inhibition of the closed nAChR, the main action of MK-801 is to enter and block the open channel. The voltage dependence for block is consistent with a single binding site within the channel that is 50% of the way through the membrane field. The IC50 for block is 3 microM at -70 mV for currents induced by 0.5 microM ACh. The data from both single-channel and macroscopic currents can be used to estimate a Kd (0) of 7 microM, which is about 40 times higher than the Kd (0) for MK-801 binding to the NMDA receptor. The relative potency of tricyclic compounds like MK-801 for various neurotransmitter systems points out that the pharmacologic action of these drugs could involve complicated interactions in vivo. PMID- 1715612 TI - Organization of mitochondria in olfactory bulb granule cell dendritic spines. AB - In contrast to dendritic spines with only postsynaptic functions, the spines of olfactory bulb granule cells subserve both pre- and postsynaptic roles. In single sections these spines were previously seen to contain mitochondria, most likely needed to provide energy for presynaptic functions, but their frequency and distribution were unknown. In order to understand the organization of mitochondria in these specialized dendritic appendages, we have studied the geometry and cytoplasmic organization of granule cell spines with computer assisted reconstructions of serial electron micrographs. The spine heads were seen to be elliptical in shape with a single pair of reciprocal synapses on the concave face apposed to the mitral/tufted cell dendrite. Mitochondria were found localized in the spine neck as well as the spine head and often extended between the two compartments. Based on their variable distribution it seems reasonable to suggest that these mitochondria are motile and move in and out of spine compartments from the parent dendrite. Spine apparatus was apparent in most of the spines as membrane bound cisterns of smooth endoplasmic reticulum located close to mitochondria. The possible role of spine apparatus in facilitating the movement of mitochondria in the necks and heads of granule cell spines in the absence of microtubules is discussed. PMID- 1715613 TI - Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S. AB - A convenient method for staining cartilage with several basic stains after alizarin red S staining of bone was investigated in rat fetuses. It was found that bromophenol blue was useful and effective for staining of the margin and center areas of cartilage, even in specimens stored in glycerin for over 10 years. The specimens were washed in running tap water for 1 hr, and subsequently were immersed in water or in 70% ethanol at pH 4 for 1 hr or longer. The specimens were then stained with 0.005% bromophenol blue in 40% ethanol adjusted to pH 4 for 2 hr, or with 0.001% bromophenol blue in 40% ethanol adjusted to pH 4 for 24 hr. Furthermore, the bromophenol blue stain color actually faded when the specimens were immersed in water or in 70% ethanol at pH 8. Descending order of the stain-effective action on fetal rat cartilage for the basic stains tested was bromophenol blue, aniline blue, Evans blue, methyl violet, trypan blue, and water blue. PMID- 1715614 TI - Late results following closed mitral valvotomy in isolated mitral valve stenosis: analysis of thirty-five years of follow-up in 240 patients using Cox regression. AB - Between 1950 and 1985, 240 patients (male/female ratio: 1/3.9, mean age 47.2 +/- 10.4 years [11-71]) with isolated mitral valve stenosis underwent closed mitral valvotomy (CMV). Ten hospital deaths (4.2%) were excluded from further analysis. Follow-up totaled 3,572 patient-years. Pre- and intraoperative predictability of long-term event-free (i.e. no reoperation) survival was examined using the Cox regression analysis. Six preoperative- and one intraoperative variables were found to have independent predictive value: mean pulmonary capillary wedge pressure (25 mmHg), age, congestive heart failure, sex, mean systemic blood pressure (116 mmHg), mitral opening snap, and postoperative regurgitation (judged intraoperatively). The Kaplan-Meier estimate of survival at 1-, 5-, 10-, 15-, 20 , 25-, 30 and 34 years +/- SE were 95 +/- 1%, 85 +/- 2%, 72 +/- 3%, 51 +/- 3%, 32 +/- 4%, 15 +/- 4%, 14 +/- 4%, and 9 +/- 5% respectively. NYHA functional class improved from a mean of 2.8 preoperatively to 1.7 at one year and 1.9 at 5-years. Long-term complications: thromboembolism of 0.67 per 100 patient-years; endocarditis of 0.47 per 100 patient-years. Reoperation was done on 28% with a hospital mortality of 6.3%. The time span from first CMV was mean 14.6 years (0.2 27.8). At reoperation 92% had stenosis, 81% regurgitation, and 8% endocarditis. In conclusion CMV offers good long-term palliation of symptoms from isolated mitral stenosis in patients without signs of irreversible organ damage and with pliable valves. The frequency of endocarditis and thromboembolic complications is low.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715615 TI - Cytogenetic analysis of human bile for mutagenicity and co-mutagenicity. AB - Cytogenetic analysis was used to test whether or not human bile induced chromosome abnormalities in lymphocytes grown in culture. Bile was obtained from gallbladders resected for various reasons such as cholecystitis, cholelithiasis, polypus and cancers of the biliary tract, stomach and pancreas. After adding human bile to a final concentration of 25 microliters/ml or 12.5 microliters/ml, the culture medium was incubated at 37 degrees C for 72 hr. Air-dried slides were stained with conventional Giemsa and the numerical and structural chromosome abnormalities were scored. Positive and negative controls in terms of chromosome abnormalities were established by using 0.03 micrograms/ml mitomycin C (MMC) and 0.9% normal saline, respectively. Cytogenetic analysis was successfully performed in 6 out of 10 bile samples (60.0%). Bile alone did not induce numerical or structural chromosome abnormalities. Structural abnormalities increased significantly in the 25 microliters/ml bile + 0.03 micrograms/ml MMC group, compared with the 0.03 micrograms/ml MMC group: 36.0% vs. 20.7% in the chromatid type gaps and breaks, 27.8% vs. 22.7% in the chromosome-type gaps and breaks, and 8.3% vs. 3.2% in the exchange-type abnormalities. It is likely that the interaction between bile and MMC is synergistic rather than additive. PMID- 1715616 TI - Correlations between IL-2 enhancing activity and clinical parameters in patients with rheumatoid arthritis and systemic lupus erythematosus. AB - In a previous paper (Tomura, K. et al. Tohoku J. Exp. Med., 1989, 159, 171-183), we discovered IL-2 enhancing factor(s) designated B cell derived-growth enhancing factor-2 (BGEF-2), which enhanced IL-2 dependent cell proliferation, and reported that BGEF-2 was produced by B cells of the patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) only when they were in the active stage of the disease. In this paper, we studied relationship between each IL-2 enhancing activity from B cell supernatant of the patients with these diseases and clinical parameters. IL-2 enhancing activities did not correlate with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), but correlated with plasma concentrations of gamma-globulin from the patients with RA and SLE in the active stages. IL-2 enhancing activities correlated with hypocomplementemia and leukocytopenia in the patients with SLE, and also correlated with RAHA titer in the patients with RA. Moreover, on several patients with RA or SLE in the active stages, diminution of IL-2 enhancing activity was found when they were in the remission stage after treatments. These findings suggested that IL-2 enhancing activity (i.e., BGEF-2 activity) correlated with activity of these diseases and supported the hypothesis that BGEF-2 played an important role in the polyclonal B cell activation and autoantibody production in patients with these diseases. PMID- 1715617 TI - Tandem sequence repeats in transmembrane channel proteins. AB - The flow of ions and small molecules out of and between cells is mediated by various classes of transmembrane proteins. One group of putative channel proteins, including the abundant lens protein MIP, is widely distributed from prokaryotes to vertebrates. This article suggests that these proteins contain a structural twofold repeat and may have arisen by gene duplication. Such a model has implications for the tertiary structures of these important proteins. PMID- 1715618 TI - [Conformational characteristics and packing of endogenous lipid fractions of transcriptionally active and repressed chromatin]. AB - The fluorescence and polarography methods are used to establish certain differences in conformation characteristics of transcriptionally active and repressed forms of rat liver chromatin. The data obtained prove the nucleo-soma ability to aggregation in the repressed chromatin and confirm a decondensed character of packing in transcriptionally active chromatin. It is supposed that these peculiarities are connected with different content of calcium and iron ions in the studied fractions of chromatin. Lipids, being the components of chromatin fractions, are packed with proteins in a form of bilayer structure. The packing character is similar in the both chromatin fractions. But microviscosity of the lipid bilayer in transcriptionally active chromatin is significantly lower than that in the repressed one and this may be one of reasons of different transcriptional activity and ability to peroxidation of the studied chromatin forms. PMID- 1715619 TI - Biological applications of photoelectron imaging: a practical perspective. AB - Photoelectron imaging is finding a promising niche in the study of biological specimens. The features of photoelectron imaging that contribute to its uniqueness for this application are described. Image formation and the major contrast mechanisms of photoelectron microscopy, material contrast and topographical contrast are reviewed and illustrated with examples of photoelectron images of cultured cells and of DNA. General considerations in sample choice and preparation are also presented. Strategies for photoelectron labeling are discussed including the use of immunogold labeling, silver enhancement and cesium-based photocathodes. PMID- 1715620 TI - Homotypic and heterotypic serum and milk antibody to rotavirus in normal, infected and vaccinated horses. AB - The homotypic and heterotypic antibody response to rotavirus was determined in three pony mares and their foals. The normal concentrations of anti-rotavirus antibodies in mares' milk and mares' and foals' serum over the first 10 weeks post-partum were measured using IgA, IgG and rotavirus serotype-specific enzyme linked immunosorbent assays. Experimental infection of the foals with serotype 3 equine rotavirus produced a rapid, serotype-specific response which peaked 10 days after infection and a slower heterotypic response which peaked 32 days later. In contrast, vaccination of the mares with an inactivated, adjuvanted serotype 6 bovine rotavirus produced a heterotypic response similar to that of the homotypic response in both serum and milk, although the predominant response in serum was IgG, while in milk it was IgA. These results suggest that non serotype-restricted passive protection of foals against rotavirus may be achieved by parenteral vaccination of mares. PMID- 1715621 TI - Production, characterization and protective effect of monoclonal antibodies to Haemophilus paragallinarum serotype A. AB - Monoclonal antibodies (mAbs) to Haemophilus paragallinarum serotype A were obtained by fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with whole cells of strain 221. Enzyme linked immunosorbent assay with whole cells was used to show that the monoclonal antibodies are specific for serotype A of H. paragallinarum. Four monoclonal antibodies indicated hemagglutination-inhibition (HAI) activity against serotype A; their titers were 10(4)-10(5). By western blotting, two of these monoclonal antibodies reacted with a protein of molecular weight 39,000. Chickens treated with mAbs possessing HAI activity survived without clinical signs of infection. No challenge strain was isolated from these chickens, indicating that four mAbs with HAI activity suppressed growth of the challenge strain in the nasal cavity, whereas mAbs without HAI activity showed no passive protective effect. These results demonstrated that HI antibodies contributed to protection, and strongly suggest that hemagglutinin (HA) antigen, especially the epitopes which were recognized by these mAbs are important for protective immunity in chickens. PMID- 1715622 TI - Erythrocyte maturation in neonatal dwarf and landrace kids. AB - The morphology of the erythrocyte cell series was investigated in external jugular vein blood samples from Dwarf and Danish Landrace goats aged from one day to 12 months. Three erythrocytic cell types were observed in neonates after supravital staining with new methylene blue. The first type were macrocytes which were stained uniformly dark to muddy blue. They formed the majority of the erythrocytic cells at birth and were categorized as diffusely basophilic chromatophilic erythrocytes. The second type were punctate and aggregated reticulocytes, and the third type were mature erythrocytes. The size ranges of the three erythrocytic cells were 4.2-5.6, 4.9-6.3 and 2.8-3.5 microns diameter respectively in the Dwarf kids and 5.6-9.7, 4.2-9.0 and 3.8-4.2 microns respectively in the Landrace kids during the first week of life. Romanowsky stained blood smears from neonatal kids were characterized by anisocytosis and poikilocytosis in which polychromatophilic macrocytes were numerous, but reticulocytes were not clearly identifiable. The ranges of erythrocyte diameters in Romanowsky-stained neonatal blood films were 4.4-5.8 (5.2 +/- 0.39) and 4.1 6.7 (5.1 +/- 0.67) microns in Dwarf and Landrace kids respectively, decreasing to 3.0 +/- 0.15 and 3.3 +/- 0.13 microns in the two broods respectively by 12 months of age. The numbers of diffusely basophilic polychromatophilic erythrocytes and punctate/aggregated reticulocytes diminished with age and they were not observed in 1-2-month-old kids. PMID- 1715623 TI - Tenascin gene expression in rat liver and in rat liver cells. In vivo and in vitro studies. AB - Tenascin is a major glycoprotein constituent of the extracellular matrix with a strong affinity to fibronectin; its distribution is believed to be temporarily and spatially limited. Tenascin gene expression is increased during wound healing processes. As repair mechanisms in chronic liver diseases resemble wound healing we studied tenascin gene expression in rat liver and in isolated rat liver cells. In normal rat liver a tenascin specific antiserum stains sinusoidal cells with fiber-like prolongations, which at the same time are desmin-positive (ITO-cells). In the CCl4-acutely-damaged liver a strong tenascin staining is detected in cells located among the mononuclear cells of the inflammatory infiltrates in the areas of necrosis and in cells of the sinusoids. In CCl4-chronically-damaged liver a strong tenascin staining is demonstrable in the connective tissue septa. In both cases, many of the tenascin-positive cells can be identified as desmin-positive by means of the double-staining fluorescence technique. The wall of larger vessels is always tensacin-negative. The staining pattern obtained with a fibronectin-specific antiserum is somewhat comparable with that of tenascin but the vessel wall was positive. hepatocytes, Kupffer cells, ITO-cells and endothelial cells were isolated from rat liver and studied for their capacity to express the tenascin gene. Biosynthetically labeled tenascin was immunoprecipated from supernatants and cell lysates obtained from cultured ITO-cells and to a much lesser extent from intracellular lysates obtained from endothelial cells; its synthesis in ITO-cells increased during the time in culture. Tenascin was also identified immuno-cytochemically in increasing amount in ITO-cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715624 TI - Silver-stained nucleolar organizer proteins in chondrosarcoma. AB - Silver-stained nucleolar proteins (AgNORs) were counted in primary chondrosarcomas of three histologic grades and in metastatic chondrosarcomatous lesions in the lung. The AgNOR numbers of neoplastic cells in primary tumors increased stepwise from grade 1 (4.42 +/- 1.11) through grade 2 (4.94 +/- 1.31) to grade 3 (6.97 +/- 1.10). There was a significant difference in AgNOR numbers between grade 3 and both grades 1 and 2 (p less than 0.001). Furthermore, the mean number of AgNORs in metastatic lesions (9.75 +/- 0.83) was significantly higher than that in primary sites (p less than 0.001). The number of AgNORs therefore reflects the grade of the chondrosarcoma. The results in the present study indicate that silver colloid staining is a useful technique for determining the histologic grade and evaluating the proliferative activity of chondrosarcomas. PMID- 1715625 TI - [The characteristics of the evolutionary variability of influenza A (H1N1) viruses]. AB - Studies of the antigenic structure of hemagglutinins of influenza A (H1N1) viruses isolated in 1978-1988 using monospecific and monoclonal antibodies demonstrated the strains of the H1N1 subtype to be highly apt to antigenic drift. The evolutional variability of that period was peculiar and characterized by antigenic drift in various directions. In those years, the variants were regularly isolated which had retained the determinants of viruses of 1933-1957 circulation period in their hemagglutinin structure. The variants containing in their hemagglutinin 2 antigenic sites common with A/USSR/090/77 virus and antigenic groupings characterizing the strain specificity of each isolate, were epidemically active. At the same time, epidemically important variants were dominant whose properties were markedly different from those of previously known viruses. Their hemagglutinin contained 2 basically new antigenic determinants. This direction of evolutional development of influenza A (H1N1) virus is the most prospective epidemically. PMID- 1715626 TI - [The humoral immunity system of mice with experimental slow influenzal infection]. AB - The status of the interferon system and level of immunoglobulins were studied in C57BL6 mice with slow influenza infection. These mice showed signs of immunosuppression: low endogenous interferon production, synthesis of alpha- and gamma-interferon by splenocytes of these mice in vitro 4-8 times lower than by those of the controls, lower levels of IgG in the blood serum. These data indicate general suppression of humoral immunity. PMID- 1715627 TI - [The heterogeneity of an influenza virus A population due to differences at individual hemagglutinin H3 sites]. AB - Using monoclonal antibodies to hemagglutinin and nucleoprotein of various influenza virus strains, the populations of long-passaged strain A/Hong Kong/1/68 and of recently isolated strain A/sparrow/Ukraine/83 belonging to the H3N2 serovariant were shown to have subpopulations of virions differing in the structure of antigenic sites of hemagglutinin and in nucleoprotein domain which correlated with the degree of electrostatic interaction of virions with an ion exchanger. The results of the study indicate a possibility of separation in the course of stepwise ion-exchange chromatography on DEAE-Sephadex A-50 of antigenic variants of influenza virus strains which is very important for the understanding of the mechanisms of population variability as well as for investigation of individual epitopes of hemagglutinin and nucleoprotein domains of influenza virus in evaluation of antigenic relationships of virions belonging to the same strain of a certain serotype. PMID- 1715629 TI - [The persistence of the distemper virus in continuous human cells]. AB - Two cultures chronically infected with distemper virus (HEP-2 and L-41) were obtained. The cultures produced a small-plaque cell-associated virus and a virus specific antigen which was demonstrated by the fluorescence antibody technique in 40%-60% of the cells. The chronically infected cells produced interferon as judged by their resistance to superinfections with heterologous viruses. The virus-carrier state was characterized by temperature sensitivity. PMID- 1715628 TI - [The design and trial of conjugates for performing lanthanide immunofluorescence analysis]. AB - The possibility of using a number of complexons for labeling of antibodies to Venezuelan equine encephalomyelitis virus and to adenovirus with europium ions was studied. The resultant conjugates, irrespective of the type of complexon, were shown to retain their immunochemical activity and could be used for lanthanide immunofluorescence analysis of virus-specific antigens. PMID- 1715630 TI - [The interferon system in HIV infection]. PMID- 1715631 TI - [The action of tomicide on macromolecular synthesis in bacterial cells]. AB - The study of the rate of incorporation of labeled precursors for nucleic acids and protein into Staphylococcus aureus 209 P cell fraction, insoluble in trichloroacetic acid, has revealed that in the presence of tomicide in the medium in a dose of 1 MCI (600 micrograms/ml) the synthesis of DNA in inhibited rapidly and almost completely (by 90%). The inhibition of the rate of incorporation of 3H thymidine into the cells of staphylococcal culture by tomicide directly correlates with the concentration of the preparation within the range 100-600 micrograms/ml, the inhibition of the synthesis of RNA and protein being less pronounced than the inhibition of the synthesis of DNA. PMID- 1715632 TI - [Antibodies to the rhamnose determinants of the polysaccharide of streptococci group A in the sera of rheumatism patients and donors]. AB - In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens. PMID- 1715633 TI - Nerve growth factor receptor immunostaining in the spinal cord and peripheral nerves in amyotrophic lateral sclerosis. AB - In animal experiments, nerve transection is followed by expression of nerve growth factor receptors (NGFR) on Schwann cells of both motor and sensory nerve fibres distally to the site of the lesion. To determine whether denervated Schwann cells in amyotrophic lateral sclerosis (ALS) similarly express NGFR, a study was made of post-mortem material of peripheral nerves and ventral roots from ALS cases and age-matched controls, using immunolabelling methods. Dorsal roots and spinal cords were also examined for the presence of NGFR. In all the ALS cases and controls, NGFR immunostaining was seen in the outer layer of vessel walls, perineurial sheaths, connective tissue surrounding fascicles in nerve roots and in the substantia gelatinosa of the spinal cord. In ALS, NGFR staining was also present in the Schwann cells of degenerated nerve fibres in mixed peripheral nerves, in ventral roots and, to a lesser extent, in dorsal roots. NGFR immunoreactivity was also seen in elongated cells extending from the perifascicular connective tissue into the nerve fascicles. It is concluded that denervated Schwann cells in ALS express NGFR and that NGFR immunostaining on Schwann cells may be used as an indicator of axonal degeneration. The NGFR labelling in the dorsal roots supports the notion that ALS is not a pure motor syndrome. PMID- 1715634 TI - Interferon inhibitor in the blood of patients with systemic lupus erythematosus. AB - Systemic lupus erythematosus (SLE) patients at advanced stages of the disease have an interferon inhibitor in the blood circulation. This inhibitor can block antiviral activity of all three types of human interferons and can significantly reduce the synthesis of interferon alpha by the treated lymphocytes obtained from normal healthy individuals. Available evidence suggests that inhibitor activity is neither because of the antibody to interferon nor due to high level of protease-like activity in the plasma. The inhibitor has also been shown to be effective in eliminating the interferon-mediated enhancement of natural killer cell activity. Interferon inhibitory activity was not detected in any of the sera taken from normal healthy individuals. Identification and characterization of interferon inhibitor has direct bearing upon effective utilization of interferons in the clinic. PMID- 1715635 TI - Long-term culture growth of CD4-CD8- lymphocytes exhibiting elevated non-MHC restricted cytotoxic activity. AB - We have developed a culture system for "long-term" growth of human lymphokine activated killer (LAK) cells exhibiting an elevated, wide-spectrum anti-tumor cytotoxicity. The system allows the exponential growth of monocyte- and B lymphocyte-depleted CD4-CD8- lymphocytes in the presence of human AB serum and recombinant human interleukin-2 (IL-2) (2 x 10(2) U/ml) combined with interleukin (IL-1) beta (50 ng/ml). After 21 days in culture, these cells undergo massive amplification (i.e., the cell yield rises up to 30-120 times the starting values), and exhibit a marked anti-tumor cytotoxic activity against a panel of natural killer (NK)-resistant tumor cell lines. Interestingly, this activity correlates with the high level of perforin RNA. The membrane phenotypes of the final cell population, assessed by a panel of monoclonal antibodies (MoAbs) indicate a mixed population comprising two cell types in variable proportions (i) NKH-1+, T cell receptor (TCR) alpha/beta-, TCR gamma/delta-, CD3-, Leu 23+; (ii) NKH-(+), TCR alpha/beta-, TCR gamma/delta+, CD3+, Leu 23+. This culture system may provide a tool for cellular and molecular studies on the mechanisms of anti tumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced cancers. PMID- 1715636 TI - Construction and characterization of hybrids containing genes coding for proteins of the central part of bacteriophage T4 baseplate. AB - The central part (hub or plug) of bacteriophage T4 baseplate consist of several proteins which are present in only few copies per phage particle. The presence of these minor baseplate components was inferred from the genetic data but only some of them were identified as distinct proteins species by biochemical analysis. We have constructed a number of plasmids containing segments of bacteriophage T4 genome coding for baseplate proteins. The following genes were cloned into expression vectors: 54, 48, 29, 28, 27, 51, 26 and 25. The presence of a particular gene product was confirmed by in vivo complementation test. On the basis of these results we could more precisely localize the position of a particular gene on T4 phage genetic map. The hybrids contain sets of genes which make aggregation impossible, so bacteria harbouring these plasmids are convenient starting point for the purification of baseplate proteins. PMID- 1715637 TI - Sensitivity of Shigella flexneri and Escherichia coli bacteria to bacteriophages and to colicins, lost or established by the acquisition of R plasmids. AB - Lac+ recombinant of Shigella flexneri 2a carrying R-factors of the standard set have been constructed. The R+ transconjugants obtained have been compared with the original strain with respect to their susceptibility to a set of bacteriophages recommended for phagetyping of Shigella strains and to a set of colicins. It have been found that as a result of acquiring some R-factors the susceptibility to bacteriophages changes; presence of certain R-factors causes susceptibility, that of other R-factors--resistance. Similar changes have been observed in the case of susceptibility to colicins. Propable mechanisms of the changes of susceptibility to these factors are discussed. PMID- 1715638 TI - Screening of microorganisms for improvement of beta-galactosidase production. AB - The strain of Penicillium notatum 1 most effective for producing beta galactosidase (see lactase 3.2.1.23), was selected out of 110 moulds belonging to 15 different species, by the test-tube microculture method. The dynamics of beta galactosidase synthesis was investigated in P. notatum 1 during its culture by submerged method. PMID- 1715639 TI - Functions coded by plasmids in chemolithotrophic bacteria. PMID- 1715640 TI - Studies on the adaptation of influenza virus replicated at low temperature. III. Biochemical studies. AB - Five strains of influenza viruses A(H3N2) replicated at low temperature passaged in cotton rats were reisolated. The properties of these strains replicated at low temperature were compared before and after passage in susceptible animals to check the stability of some its markers. At the same time original viruses replicated at 37 degrees C--which are different in epidemiological potency--were compared. The following parameters being tested: NA activity, HA titers, heat inactivation NA and Ha, Michaelis constants and optimum pH. We observed some differences between strains both replicated at low temperature after passage in the susceptible animal organism and original viruses from 37 degrees C. Viruses replicated at low temperature from original epidemiostrain are really cold adapted and remained stable after passage in the animals when the others derived from no epidemic strain are not stable. PMID- 1715641 TI - Restriction map of Thiobacillus versutus plasmid pTAV1. AB - A restriction map of Thiobacillus versutus plasmid pTAV1 was constructed using EcoRI, BamHI and SalI restriction enzymes. Knowledge of the restriction map is an obligatory starting point for genetic and molecular studies of this, so far cryptic, plasmid. PMID- 1715642 TI - An extracellular material observed in Clostridium difficile strains. AB - Some extracellular material-- "capsule" has been observed among Clostridium difficile strains. C. difficile strains (44 examined) contain different proportions of cells with "capsule" and without "capsules". Toxigenic strains contained fewer capsulated cells than non-toxigenic strains. PMID- 1715643 TI - Relationship of bacteriological status of the mammary gland to lactose level in milk yield. AB - The analysis of variance showed a significant (P less than or equal to 0.01) effect of the presence of pathogenioc bacteria in the udder on lactose content in milk. The relationship between lactose level and milk yield was significant (P less than or equal to 0.05). PMID- 1715644 TI - Sensitivity of Shigella strains to bactericidal activity of human serum. I. Participation of two independently active mechanisms in bactericidal action against Shigella sonnei phase II. AB - Normal human serum is strongly bactericidal for all studied Shigella sonnei phase II (10 strains). The studied bacteria were sensitive to two alternative mechanisms of the bactericidal activity of serum factors. The first mechanism involves the action of serum in which complement (C) is activated by the studied bacteria via the classical pathway. Lysozyme did not participate in this reaction. The second mechanism involves the combined action of two factors: C activated via the alternative pathway and lysozyme. PMID- 1715645 TI - Sensitivity of Shigella strains to the bactericidal activity of human serum. II. Shigella flexneri 3 a is killed by complement activated via the classical pathway, with participation of lysozyme. AB - Normal human serum is bactericidal for all studied strains (15) of Shigella flexneri serotype 3a. The activity of the serum was similar irrespective of the invasiveness of the bacteria or its lack. The studied bacteria were susceptible to a single mechanism of bactericidal activity involving complement activated via the classical pathway, accompanied by the action of lysozyme. PMID- 1715646 TI - Sensitivity of Shigella strains to the bactericidal activity of human serum. III. The diversity of activity variants of bactericidal serum factors against Shigella flexneri serotypes. AB - Normal human serum is bactericidal for all studied Shigella flexneri strains (38) belonging to nine serotypes. Six variants of bactericidal activity of serum factors for these bacteria were determined. PMID- 1715647 TI - Role of Staphylococcus saprophyticus in human infection. AB - Biological properties of Staphylococcus saprophyticus strains isolated from urinary tract infection and respiratory tract secretions were investigated. The majority of S. saprophyticus strains exhibit moderate surface hydrophobic properties, as measured by Hydrophobic Interaction Chromatography. There was no significant difference between two groups of isolates in respect to electrostatic charge. It was found that the ability to form a diffuse type of growth is characteristic for most of S. saprophyticus strains despite of source of isolation. Five carbohydrates on the surface of 25% of S. saprophyticus strains were shown by means of specific lectin agglutination. Some of the tested strains were capable to bind labelled fibrinogen. PMID- 1715648 TI - Studies on adaptation of influenza virus replicated at low temperature.IV. Sensitivity of neuraminidase and hemagglutinin to some proteolytic enzymes, detergents and chemical agents. AB - Resistance against proteolysis enzymes, detergents and chemical group-specific reagents has been compared for the neuraminidase and hemagglutinin of influenza virus replicated at low temperature and original strain replicated at 37 degrees C before and after passage in the susceptible animal organism - cotton rat. Our study indicated great differences between strains and temperature conditions to resistance of the neuraminidase and hemaglutinin to proteolytic enzymes, detergens and chemical group specific reagents. No differences was found for sensitivity of influenza virus replicated at low temperature before and after passages for 3 strains -A/Pol/L/71, A/Phil/2/82, A/Pol/79/85 which are really cold adapted viruses. On the other hand neuraminidase of this strains was more resistance to these treatments. PMID- 1715649 TI - Kinetics of haemolytic activity in Proteus strains. AB - Proteus species produces toxins and constitutes a causative agent of some chronic and recurrent infections. For the study of haemolytic activity and the production and inhibition kinetics, a total of 140 local isolates were diagnosed and examined by the general biochemical methods, and their ability of haemolysis were tested by both direct and indirect methods utilizing the enrichment procedure for all strains. Two antibiotics, erythromycin and keflex (cephalexin), were tested for the study of haemolysis inhibition and its kinetisc. Rof further study, examples of Proteus species were selected; the new approach was based on mixing procedure between P. aeruginosa (also pyocyanine) and Proteus species for inhibition of haemolytic activity. Spectrophotometric analysis were used parallel to these studies to support quantitatively the observed results as all samples show an absorption centre at 542 +/- 1 nm. Results of such analysis of haemolytic activity and inhibition kinetics are presented. PMID- 1715650 TI - Amino acid auxotrophy increases sensitivity of Saccharomyces cerevisiae to a quaternary ammonium salt IM. AB - Relationships between amino acid auxotrophy and N-dodecyloxy-carboxy-methyl-N-N-N trimethyl ammonium chloride (IM) sensitivity have been investigated in isogenic yeast strains Saccharomyces cerevisiae and their meiotic segregants. It has been found, that auxotrophy increases the level of sensitivity to this salt markedly. A gene conferring resistance to that drug cancels the auxotrophy-dependent sensitivity. PMID- 1715651 TI - Uranium and lead accumulation in cells of Streptomyces sp. AB - Lead and uranium were accumulated equally well both in the viable and dry biomass of Streptomyces sp. The process occurred in less than 5 min. Uranium was accumulated selectively from a polymetallic solution containing U, Pb, Cu, Zn, Ni, Co. The optimum pH for the process was 5.0, and the concentration of each metal in the solution was 10(-3) M. Under these conditions, the dry biomass of Streptomyces amounting to 1 mg/cm3 accumulated over 60% of the uranium in the solution. With the same amount of cell wall preparation it was possible to remove from the solution ca. 90% of U. In this case, the accumulated uranium reached 21% of the sorbent dry mass. Electron micrographs show that lead accumulated in Streptomyces cells is mainly concentrated in the cell wall structures although in the case of uranium this is not so clear. PMID- 1715652 TI - Production of B--vitamins by heterotrophic planktonic bacteria isolated from littoral zone of lake Jeziorak. AB - Different genera and groups of heterotrophic planktonic bacteria of the littoral zone of the lake Jeziorak produced different B-group vitamins. Most numerous among the planktonic bacteria of this zone of the lake were biotin producers and least numerous were organisms synthesizing riboflavin and nicotinic acid. Most bacteria produced one or two vitamins. Three or more vitamins were produced by only a few strains. In autumn no strain produced all five vitamins studied in this work. PMID- 1715653 TI - The toxicity of Bacteroides thetaiotaomicron cell-surface antigens to chicken embryos. AB - The toxicity of B. thetaiotaomicron lipopolysaccharides and capsular antigen to 11-day-old chicken embryos was examined. CELD50 (chicken embryo LD50) of these preparations were determined. All examined preparations showed the lethal activity for chicken embryos. The toxicity of lipopolysaccharides was much higher than the toxicity of capsular antigen. PMID- 1715654 TI - The influence of morphine on development of HSV-1 and M-MSV virus infection in mice. PMID- 1715655 TI - Background paper. Mapping epitopes on coronavirus glycoproteins. PMID- 1715656 TI - Binding of antibodies that strongly neutralise infectious bronchitis virus is dependent on the glycosylation of the viral peplomer protein. PMID- 1715657 TI - Enteric coronavirus TGEV: mapping of four major antigenic determinants in the amino half of peplomer protein E 2. PMID- 1715658 TI - Location of antigenic sites of the S-glycoprotein of transmissible gastroenteritis virus and their conservation in coronaviruses. PMID- 1715659 TI - Topological and functional analysis of epitopes on the S(E2) and HE(E3) glycoproteins of bovine enteric coronavirus. AB - Monoclonal antibodies (Mabs) were selected which reacted with bovine enteric coronavirus S and HE. Mabs to S were used to identify 2 cleavage products of S, S/gp105 and S/gp90. Monoclonals to S/gp105 and HE neutralised the virus; only Mabs to the latter inhibited haemagglutination and acetyl-esterase activity. Topological distribution of epitopes was studied on these 3 glycoproteins by means of competition binding experiments. Two independent epitopes were characterised on HE, 4 on S/gp105, and 2 on S/gp90. Neutralising Mabs defined one major site on both S/gp105 and HE; however a minor neutralisation epitope was also delineated on S/gp105. Functional mapping using neutralisation-resistant mutants confirmed the topological distribution of epitopes on S/gp105. PMID- 1715660 TI - Linear neutralizing epitopes on the peplomer protein of coronaviruses. PMID- 1715661 TI - The nucleocapsid protein of IBV comprises immunodominant determinants recognized by T-cells. PMID- 1715662 TI - Protection of mice from lethal coronavirus MHV-A59 infection by monoclonal affinity-purified spike glycoprotein. AB - Numerous studies have provided indirect evidence that the spike glycoprotein of coronaviruses (E2 or S) bears determinants for pathogenesis and the induction of protective immunity. In order to directly evaluate its immunogenicity, the E2 glycoprotein of the murine hepatitis virus, strain A59, was purified by immunoaffinity chromatography. High titers of neutralizing and fusion inhibiting antibodies were induced in mice vaccinated with purified E2/S in Freund's adjuvant, which were protected from an intracerebral challenge with 10 LD50 of MHV-A59. This study provides a direct demonstration of the importance of the coronavirus spike glycoprotein in the induction of a protective immune response. PMID- 1715663 TI - Background paper. Functions of the coronavirus nucleocapsid protein. PMID- 1715664 TI - Characterization and location of the structural polypeptides of turkey enteric coronavirus using monoclonal antibodies and enzymatic treatments. PMID- 1715665 TI - Sequence analysis of the 3' end (8740 nucleotides) of BECV genome; comparison with homologous MHV nucleotide sequence. PMID- 1715666 TI - Is Perls stain specific for hemosiderin? PMID- 1715667 TI - Small cell carcinoma of the stomach: a clinicopathologic study of 17 cases. AB - Of 17 cases of small cell carcinoma of the stomach, three were early and 14 were advanced. Grossly, the tumors were mostly polypoid at the early stage, and as they advanced, deep ulcerations developed. Histologically, only one tumor was "oat cell type," and the other 16 were "intermediate cell type." With regard to tumor components, five were "pure" tumor, and 12 were "composite" admixing glandular and/or squamous differentiation. Argyrophil cells were seen in eight tumors. Immunohistochemically positive cells for chromogranin, neuron-specific enolase, and keratin were seen in 12, 10, and 7 tumors, respectively. Carcinoembryonic antigen was negative in the small cell component of most tumors as opposed to strong positivity in the glandular component. Electron-dense core granules were evident in seven of nine tumors examined. These findings suggest that histologic variety is quite characteristic of the small cell carcinomas of the stomach, and this type of carcinoma takes an aggressive clinical course like its counterparts in other organs. PMID- 1715668 TI - Respiration-deficient cells are caused by a single point mutation in the mitochondrial tRNA-Leu (UUR) gene in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). AB - MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) is a major subgroup of heterogeneous mitochondrial diseases. For identifying a mutation in the mitochondrial gene, we isolated, from the same muscle tissue from a patient with MELAS, cell lines with distinctly different phenotypes: one was respiration-deficient, and the other was apparently normal. Compared with the normal cells, only one A-to-G nucleotide transition at nucleotide 3243 in the tRNA-Leu (UUR) gene was found in whole mtDNA of the respiration-deficient cells. This mutation was also found in eight patients, from unrelated families, who had MELAS in a heteroplasmic manner but was not found in control individuals. Therefore, the single point mutation causes the functional abnormality in the respiratory chain of mitochondria. PMID- 1715670 TI - Role of vascular factors, including angiogenesis, in the mechanisms of action of sucralfate. AB - This brief overview of our recent results implicates vascular factors in the mechanisms of acute and chronic actions of sucralfate. Pretreatment of rats with sucralfate and its components, such as SOS and sodium sulfate, prevented the ethanol-induced microvascular injury and maintained blood flow in the gastric mucosa. Thus, preservation of microvascular integrity seems to be one of the mechanisms of acute gastroprotection by sucralfate. Chronic experiments with subcutaneously implanted sponges containing sucralfate or SOS revealed that both compounds stimulated angiogenesis, whereas only sucralfate enhanced the area of granulation tissue. These processes may have a role in the ulcer-healing action of sucralfate. PMID- 1715669 TI - The neurofibroma in von Recklinghausen neurofibromatosis has a unicellular origin. AB - von Recklinghausen neurofibromatosis (NF1) is the most common hereditary syndrome predisposing to neoplasia. NF1 is an autosomal dominant disease caused by a single gene which maps to chromosome 17q11.2. The most common symptomatic manifestation of NF1 is the benign neurofibroma. Our previous studies of tumors in NF1, studies which detected a loss of heterozygosity for DNA markers from the NF1 region of chromosome 17 in malignant tumors, did not detect a loss in neurofibromas. We report here that a more extensive study, including the analysis of neurofibromas from 19 unrelated NF1 patients by using seven probes, failed to detect a single instance of loss of heterozygosity. This finding suggests that neurofibromas are either polyclonal or monoclonal in origin but arise by a mechanism different from that of NF1 malignancies. In order to investigate the first possibility, we analyzed neurofibromas from female NF1 patients by using an X chromosome-specific probe, from the phosphoglycerokinase (PGK) gene, which detects an RFLP. The detected alleles carry additional recognition sites for the methylation-sensitive enzyme HpaII, so that the allele derived from the active X chromosome is digested by HpaII while the one from the hypermethylated, inactive X chromosome is not. We analyzed neurofibromas from 30 unrelated females with NF1. Eight patients were heterozygous for the PGK RFLP. By this assay, neurofibromas from all eight appeared monoclonal in origin. These results suggest that benign neurofibromas in NF1 arise by a mechanism that is different from that of malignant tumors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715671 TI - Enhancement of the protective qualities of gastric mucus by sucralfate: role of phosphoinositides. AB - The effects of intragastric administration of sucralfate on the physicochemical properties of gastric mucus, and the mechanism of its protective action against alcohol-induced mucosal injury were investigated using in vivo and in vitro models. The experiments in vivo were conducted with groups of rats receiving a dose of 100 mg sucralfate twice daily for 5 consecutive days. The animals were sacrificed 16 hours after the last dose, their stomachs dissected, and the mucosa subjected to physicochemical measurements. In the in vitro studies, gastric mucosa was cultured in the presence of sucralfate, ethanol, or both. The in vivo results revealed that sucralfate elicited an 8% increase in mucus gel dimension, while its sulfo- and sialomucin content increased by 63% and 81%, respectively. The changes in mucus gel mucin content with sucralfate were accompanied by a 9.5% increase in mucus hydrogen ion (H+) retardation capacity, 1.9-fold increase in viscosity, and a 60% increase in the gel hydrophobicity. The mucus elaborated in the presence of sucralfate exhibited 14% lower protein content and 62% higher content of carbohydrate than that of control, and contained more neutral lipids. Furthermore, the gastric mucus of the sucralfate group showed a marked increase in mucus glycoprotein polymeric form. The data obtained with gastric mucosal culture demonstrated that sucralfate elicited a significant increase in mucin synthesis, which was reflected in the enhanced metabolism of mucosal phosphoinositides. In contrast, ethanol, which exhibited detrimental effects on mucin synthesis, also caused alterations in the phosphoinositide signal pathway. The changes in mucin and phosphoinositide distribution patterns evoked by ethanol were prevented by sucralfate. Our results suggest that the mucosal strengthening action of sucralfate occurs through the stimulation of the metabolism of phosphoinositide-derived messenger molecules. PMID- 1715672 TI - Administration of sucralfate prolongs survival of animals with experimental peptic ulceration. AB - Ligation of the pig bile duct (BDL) results in 100% incidence of pars esophageal ulceration within 48 hours of the procedure. Usually such ulceration is uniformly fatal unless a highly selective vagotomy is performed simultaneously with the BDL. The administration of sucralfate to pigs with BDL prolonged their survival for up to 7 days, with evidence of healing of the ulcer on macroscopic and histologic observations. An increase in cell proliferation in the squamous epithelium of the ulcerated area was also seen in this sucralfate group. These features were not seen in controls, pigs with BDL only, or pigs with BDL and with magaldrate (Riopone), colloidal bismuth subcitrate (DeNol), or carbenoxolone. Analysis by Sepharose 2B gel filtration showed that there was no significant difference in the amounts of polymeric mucin in any group, with a wide scatter of the data seen especially for pigs in the untreated BDL-only group. This study suggests that sucralfate may enhance healing in this experimental pig ulcer model via a mechanism independent of the stimulation of mucus secretion. We propose that coating the mucosa with sucralfate provides a temporary substitute barrier that creates a microenvironment conducive to wound repair by mucosal proliferation. PMID- 1715673 TI - Mechanisms of gastroduodenal protection by sucralfate. AB - Over the past 5-10 years, a number of studies have shown that topical sucralfate enhances a number of gastric and duodenal mechanisms, e.g., the "mucus bicarbonate barrier," mucosal hydrophobicity, mucosal blood flow, cell viability, and local production of prostaglandins, as well as endogenous mediators of tissue injury and repair. It seems likely that the complex actions of sucralfate are in part related to direct interaction between the drug or its components (aluminum, sucrose, and sulfate) and gastric mucosal tissues, and in part related to effects of the drug on the various mucosal mediators of tissue injury and repair. Local actions may play a role in accelerating healing of ulcer-damaged mucosa, but this does not explain the protective actions of sucralfate on normal mucosa. Thus sucralfate appears to enhance the protective function of the "mucus-bicarbonate" barrier by actions on both components. This may depend in part on an interaction with the unstirred layer overlying gastric epithelium. Sucralfate has also been shown to increase the hydrophobicity of mucus gel. There is little doubt that sucralfate increases local production and release of protective prostaglandins (PGs), but the precise role played by these agents in mediating mucosal protection and in chronic ulcer healing remains uncertain. Currently, the mechanism of action of sucralfate on vascular integrity remains unknown and the role of PGs in this protective function is unclear. There is little evidence that epidermal growth factor plays any role in mediating mucosal protection by sucralfate, but it may be important in its ulcer-healing action. Sucralfate has been shown to be truly "cytoprotective" in that it protects isolated epithelial cells from damage by noxious agents. In animals treated with sucralfate, the surface epithelial cells were disrupted, but necrotic lesions in the deep proliferative zone were virtually absent. It seems likely that investigations of the actions of sucralfate and its components will move ever closer to defining the target cells, the intracellular events, and the mediators that bring about its protective and ulcer-healing activity. PMID- 1715674 TI - Full-field electroretinograms in patients with the carbohydrate-deficient glycoprotein syndrome. AB - We examined five patients who had carbohydrate-deficient glycoprotein syndrome with full-field electroretinograms. Only two of the patients showed fundus changes typical for retinitis pigmentosa, whereas abnormal electroretinograms were seen in all patients. There was no recordable rod response; however, a delay in the cone b-wave implicit time was noted. All patients had nyctalopia. These observations suggest that patients with the carbohydrate-deficient glycoprotein syndrome have a progressive tapetoretinal degenerative disorder of the retinitis pigmentosa type with defined alterations in the electroretinogram. PMID- 1715675 TI - Diagnostic and management considerations of acquired epileptic aphasia or Landau Kleffner syndrome. AB - A small number of children have been identified as having an interruption in their communicative progress known as Landau-Kleffner syndrome, acquired epileptic aphasia, or aphasia with convulsive disorder. Although presenting symptoms have differed among the cases reported, a progressive or acute language loss and inattentiveness to auditory stimuli are the most common manifestations. Typically, these children begin developing language normally and then, for no apparent reason, language progress is disrupted. This disruption is accompanied by the onset of seizure activity and/or abnormal electroencephalographic (EEG) findings. While this disorder appears to be relatively uncommon, its frequency is questionable due to its unfamiliarity among the audiology and otology communities and, thus, it is subject to the likelihood of misdiagnosis. A case of acquired epileptic aphasia is described herein. A team diagnostic and management approach, which can include audiology, otology, psychology, neurology, and speech-language pathology is recommended for such cases. Earlier identification of this debilitating disorder is needed in order to secure appropriate intervention and reestablish communication systems for these children. PMID- 1715676 TI - What is the role of an ATP stain to detect facial muscle atrophy? PMID- 1715677 TI - Lateral ventricle-brain ratio and balance between CSF HVA and 5-HIAA in schizophrenia. AB - OBJECTIVE: Lateral ventricle enlargement in schizophrenia has been positively correlated with poor premorbid competence, negative symptoms, and poor treatment response and negatively correlated with concentrations of homovanillic acid (HVA), a dopaminergic metabolite. The authors provide further evidence of a reciprocal relationship between lateral ventricle size and dopaminergic activity in schizophrenia. METHOD: They assessed the relationship between lateral ventricle enlargement (ventricle-brain ratio, VBR) and CSF neurotransmitter metabolite concentrations (HVA and 5-hydroxyindoleacetic acid [5-HIAA]) in 45 patients with schizophrenia, 28 with affective disorders (19 patients with major depression and nine with bipolar disorder), and 91 normal comparison subjects. RESULTS: No group mean differences were significant. Although individual correlations of VBR with HVA and 5-HIAA were not statistically significant, the ratio of HVA to 5-HIAA was significantly correlated with VBR in the patients with schizophrenia, a finding consistent with dopaminergic-serotonergic balance hypotheses. CONCLUSIONS: These data suggest that it is the balance between HVA and 5-HIAA rather than their absolute levels which is associated with brain morphology and that this relationship between brain chemistry and morphology may be characteristic of the normal range of functioning for these systems. In other words, independent of whether brain morphology and chemistry differentiate psychopathological from nonpsychopathological states, there may be an orderly relationship between lateral ventricle size and the balance between HVA and 5 HIAA balance that is especially prominent in schizophrenia. PMID- 1715678 TI - Striatal c-fos induction in neonatally dopamine-depleted rats given transplants. PMID- 1715679 TI - Biotechnology: its impact on medicine and oncology. AB - Biotechnology has been fundamental to exploration and manipulation of life structures and processes at the molecular level. Regulation of expression of specific genes has impacted on understanding of cancer development and immunopathologic diseases. An even greater impact has been the production of new therapeutic molecules. Cytokines have been produced by recombinant DNA technology and are established as effective therapeutic modalities for cancer and infectious diseases. High quality pharmaceutical molecules have been produced which can substantially regulate cell function. Interferons typify this progress and are now licensed for both viral and neoplastic disease in more than 43 countries around the world. It seems certain that by the year 2000, the impact of biotechnology on human medicine will even be wider than that of today. Findings of biotechnology will have implications not only for advancement of human health but also will create debate regarding appropriate use. PMID- 1715680 TI - Antisense RNA. PMID- 1715681 TI - The epidemiological evolution of HIV infection. PMID- 1715683 TI - [Effect of phospholipase A2 inhibitor on substance P-induced histamine release from rat peritoneal mast cells]. AB - Rat peritoneal mast cells purified on a Percoll gradient were challenged with substance P and effect of phospholipase A2 inhibitor ONO-RS-082 on substance P induced histamine release from the cells were investigated. Substance P at the concentration of 10(-5) M caused a significant histamine release and the amount of histamine release reached to its submaximum at 1 min after the challenge and then slowly increased. ONO-RS-082 inhibited the substance P-induced histamine release in a concentration-dependent manner at the concentration from 10(-6) to 10(-4) M, suggesting that phospholipase A2 may play some roles in the process of substance P-induced histamine release from rat peritoneal mast cells. PMID- 1715682 TI - Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface. AB - PhoE protein is an abundant outer membrane protein of the Escherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foregin antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein of Mycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuated Salmonella strains, which can be used as live oral vaccines, was shown. PMID- 1715684 TI - Association of the HNK-1 epitope with 5'-nucleotidase from Torpedo marmorata (electric ray) electric organ. AB - 5'-Nucleotidase isolated from the electric organ of the electric ray (Torpedo marmorata) has a molecular mass of 62 kDa and, on two-dimensional electrophoresis, separates into up to 13 isoforms within a pI range of 5.9-6.7. The N-terminal sequence data show a 71% identity over 17 amino acids with that previously published for the rat liver enzyme. All forms of 5'-nucleotidase are recognized by the HNK-1 monoclonal antibody. HNK-1 immunoreactivity is found at the surface of the Schwann-cell processes covering the synaptic terminals and in this respect corresponds to that of 5'-nucleotidase in the same tissue. Since a number of glycoproteins involved in cell recognition and cell adhesion carry the HNK-1 epitope, 5'-nucleotidase may play a role in cell-cell or cell-extracellular matrix interaction in addition to its activity as an enzyme. PMID- 1715685 TI - Altered affinity of insulin-like growth factor II (IGF-II) for receptors and IGF binding proteins, resulting from limited modifications of the IGF-II molecule. AB - The binding affinities of seven analogues of recombinant human insulin-like growth factor II (hIGF-II) were characterized for the IGF type-I and type-II receptors and insulin receptors, as well as for IGF-binding protein (IGFBP)-1, IGFBP-2, IGFPB-3 and human serum IGFBPs. A switch of two of the three cysteine bridges in hIGF-II, 9-47 and 46-51 to 9-46 and 47-51, severely impaired the binding of this analogue to all receptors and to the IGFBPs. The affinities for the IGF type-I receptor and the IGFBPs were decreased over 100-fold, while the binding to the insulin receptor and the IGF type-II receptor was less affected, with a 6-10-fold decrease in affinity. Slight modifications of the N-terminus had only minor effects upon the binding of hIGF-II to the IGFBPs or to the receptors. Deletion of both the N-terminal amino acid and the two C-terminal amino acids resulted in moderate decreases in affinity, with a 60% decrease in affinity for IGFBP-1 and the IGF type-I receptor. Acetylation of the N-terminus of Ala1 and the epsilon-nitrogen of Lys65 decreased the affinity, by 60-90%, of hIGF-II for all of the IGFBPs and receptors. The experiments involving acetylation of IGF-II or switching of its cysteine bridges indicated that these modifications (no substitution, deletion or addition of any of the 67 amino acids of hIGF-II) may lead to a severe impairment of the binding affinity of IGF-II for both the IGFBPs and the receptors. Acetylation of the epsilon-nitrogen of Lys65, which causes a charge change, or alteration of the three-dimensional structure, as shown by the cysteine bridge switch, lead to a severe impairment of the binding affinity for the binding proteins and for the receptors. In general, care should be taken with the synthesis of analogues and the interpretation of resulting binding data, since affinity alterations ascribed to amino acid changes may instead be caused by alterations of the charge or the three-dimensional structure of the protein. PMID- 1715686 TI - Dephosphorylation of autophosphorylated insulin and epidermal-growth-factor receptors by two major subtypes of protein-tyrosine-phosphatase from human placenta. AB - The identity of protein-tyrosine-phosphatases (PTPases) active against autophosphorylated insulin receptor was probed by using an insulin-receptor related peptide phosphorylated on tyrosine (peptide 1142-1153). Two major peaks of PTPase activity were resolved from the particulate (Triton X-100-soluble) fraction of human placenta by chromatography on DEAE-cellulose. The two peaks were purified 1300-2300-fold; other peaks of PTPase activity (greater than 15%) were not detected. Properties of the PTPases indicated that they corresponded to subtypes 1A and 1B. Both subtypes appeared capable of catalysing dephosphorylation of all autophosphorylation sites in three domains of the insulin receptor, with no appreciable difference in the pattern of dephosphorylation detected by two-dimensional tryptic-peptide mapping. The tyrosine-1150 domain of the insulin receptor in triply phosphorylated form was found to be highly sensitive to the action of both PTPases, and was dephosphorylated at least 4 times faster than the doubly and singly phosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains by either PTPase. This is significant, as the level of the triphosphotyrosine-1150 species has been shown to correlate well with the capacity of the insulin-receptor tyrosine kinase to phosphorylate other proteins. Both subtypes also dephosphorylated autophosphorylated epidermal-growth-factor (EGF) receptor by greater than 95%. Placental particulate (and cytosolic) PTPase activity against either receptor distributed approximately 2:1 between subtypes 1A and 1B as assayed in the presence of EDTA. In summary, PTPases within two major subtypes have been identified as phosphotyrosyl-insulin and -EGF-receptor phosphatases in vitro. The PTPases identified exhibit high affinities for substrates and high activities in cells, which is commensurate with the PTPases being important in vivo in controlling or reversing autophosphorylation-induced regulatory or signalling events. PMID- 1715687 TI - Murine osteogenic protein (OP-1): high levels of mRNA in kidney. AB - The murine OP-1 gene (EMBL accession No. X56906) encoding the homolog of human osteogenic protein-1 was isolated from cDNA and genomic libraries using human OP 1 cDNA as probe. The deduced murine OP-1 amino acid sequence revealed 11 amino acids changes, three of them in the mature protein. Murine OP-1 probes were used for analysis of OP-1 mRNA in mouse embryo and organ tissues. Northern blot hybridization revealed multiple mRNA species: the major species of 2.2 kb, minor species of 1.8 and 2.4 kb and a large 4 kb species, which may represent alternative splices. Tissue specific expression was studied in brain, lung, heart, liver, spleen, kidney, adrenal and bladder tissue. Maximal levels of OP-1 mRNA were found in kidney which may explain the phenomenon of epithelial osteogenesis, first described by Huggins in 1931 using epithelium from the urinary tract. Moreover, our data suggest that kidneys may be the main site of OP 1 synthesis, even though it is distant from its physiological site of action, skeletal bone. PMID- 1715688 TI - Evidence for a structural mutation (347Ala to Thr) in a German family with 3 ketothiolase deficiency. AB - The molecular basis of 3-ketothiolase deficiency (3KTD) was examined in a 3KTD family. Immunochemical analyses showed that mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts (GK06) was unstable and that the parents and brother were obligatory carriers of 3KTD. When sequencing the PCR-amplified patient's T2 cDNA, we noted a G to A replacement which caused 347Ala to Thr substitution of the mature T2 subunit. Transfection analysis revealed that this substitution resulted in an instability of the T2 protein. Analyses of the T2 cDNA and gene of the family indicated that the patient was a compound heterozygote; the allele that derived from the mother had a point mutation (347Ala to Thr) and the other allele from the father has a mutation which would abolish the T2 gene expression. This report is apparently the first definition of a mutant allele for 3KTD, at the gene level. PMID- 1715689 TI - The inhibitory effect of [D-Arg1,D-Phe,D-Try7,9,Leu11] substance P on endothelin 1 binding sites in rat cardiac membranes. AB - The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments. PMID- 1715690 TI - Nerve growth factor binds to the 140 kd trk proto-oncogene product and stimulates its association with the src homology domain of phospholipase C gamma 1. AB - The cellular actions of nerve growth factor (NGF) involve regulation of protein phosphorylation. In PC-12 pheochromocytoma cells, exposure of [125I]NGF followed by crosslinking indicates that the ligand binds to two discreet receptors, the previously described 75 kd protein, as well as the trk proto-oncogene product pp140c-trk. Competition experiments reveal that of the two, pp 140c-trk binds to NGF with higher affinity. Following exposure to NGF, pp140c-trk undergoes a rapid autophosphorylation on tyrosine residues, and concomitantly phosphorylates and associates with phospholipase C gamma 1 (PLC gamma 1), through interaction with its src homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent activation of its tyrosine kinase activity and the specific association with the effector molecule, PLC gamma 1, suggests that this is the biologically relevant signaling receptor for NGF. PMID- 1715691 TI - Inhibition of human immunodeficiency virus type-1-induced syncytium formation and cytopathicity by complestatin. AB - Complestatin, an anti-complement agent, was shown to be a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) infection in vitro. It inhibited HIV 1-induced cytopathicity and HIV-1 antigen expression in MT-4 cells; the 50% effective doses for these effects were 2.2 and 1.5 micrograms/ml, respectively. No toxicity for MT-4 cells was observed at concentrations up to 400 micrograms/ml. In addition, the agent inhibited the focus formation in HT4-6C cells (CD4-positive HeLa cells); the concentration for 50% focus reduction was 0.9 microgram/ml. HIV-1-induced cell fusion in cocultures of MOLT-4 cells and MOLT-4/HTLV-IIIB were also blocked by complestatin (the concentration for 50% cell fusion inhibition, 0.9 microgram/ml). Complestatin had no ability to inhibit HIV-1 reverse transcriptase activity. When MT-4 cells were pretreated with complestatin for 2 hrs prior to the exposure to HIV-1, the HIV-1-induced cytopathicity was markedly inhibited, while pretreatment of HIV-1 with the agent did not affect the infection. These results suggest that complestatin primarily interacts with cells and inhibits viral adsorption to the cell surface as well as adsorption of infected cells to adjacent cells. PMID- 1715692 TI - Retinoic acid receptor transcripts in human umbilical vein endothelial cells. AB - Human umbilical vein endothelial cells contain high levels of mRNA for the beta retinoic acid receptor, and very low levels of alpha-retinoic acid receptor transcripts. The cells responded to retinoic acid with a significant induction of tissue transglutaminase expression but no alterations in the expression of beta retinoic acid receptor transcripts. The physiological implications of the constitutive expression of this receptor in endothelial cells is discussed. PMID- 1715693 TI - Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues. AB - Rat brain plasma membranes were solubilized in detergent and a glycoprotein enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro. PMID- 1715694 TI - Glucuronosyl transfer to galactose residues in the biosynthesis of HNK-1 antigens and xylose-containing glycosaminoglycans: one or two transferases? AB - An early step in the assembly of the xylose----serine-linked proteoglycans is the transfer of glucuronic acid to the C-3 position of a galactose residue in the carbohydrate-protein linkage region. Since a similar reaction occurs in the biosynthesis of NHK-1 antigens, the question arose whether these processes are catalyzed by the same enzyme. In the present study, the proteoglycan-related glucuronosyltransferase activity in embryonic chick brain was found to be firmly membrane-associated, while the majority of the activity towards N acetyllactosamine - a model substrate for HNK-1 antigen biosynthesis - was readily solubilized. No activity towards N-acetyllactosamine was found in embryonic chick cartilage, which is a rich source of the proteoglycan-related enzyme. Together with the results of mixed substrate experiments, these findings strongly indicate the existence of two separate glucuronosyltransferases catalyzing transfer to galactose residues. PMID- 1715695 TI - Sequence of a human brain adenylyl cyclase partial cDNA: evidence for a consensus cyclase specific domain. AB - A cDNA coding for a human brain adenylyl cyclase was isolated and sequenced. The deduced partial 675 amino-acid sequence was compared with those of other known adenylyl and guanylyl cyclases. Comparison of this predicted amino-acid sequence with that of bovine brain (type I) and rat olfactory (type III) adenylyl cyclase indicated a significant homology with the carboxyl-terminal halves of both enzymes. The homology between the human adenylyl cyclase and the other two mammalian adenylyl cyclase also appears at the topographic level. Indeed, the human enzyme includes a extremely hydrophobic region containing six potential membrane-spanning segments followed by a large hydrophilic domain. At the beginning of the hydrophilic domain, there is a 250 amino-acid region which shows not only a striking homology with the bovine and rat adenylyl cyclase (86% of similarity and 57% of identity), but also a significant homology with non mammalian adenylyl cyclase and guanylyl cyclases. We found that this 250 amino acid domain contains a sequence of about 165 amino-acids which is highly conserved in most of the known nucleotide cyclases suggesting that it includes residues that are critical for the function of the enzymes. PMID- 1715696 TI - 2,3,7,8-Tetrachlorodibenzo-P-dioxin as a possible activator of HIV infection. AB - The effects of 10-150 nM 2,3,7,8-TCDD on the HIV infection in the various lymphoid cell cultures have been studied. The level of reproduction of HIV was examined by determination of virus antigen as well as by measurement of reverse trascriptase activity. 2,3,7,8-TCDD does not exert any toxic influence in this concentration on the MT-4 cells and causes the induction of cytochrome P-450 containing monooxygenase. Furthermore, in MT-4 cell culture infected by HIV-1 virus an increase of virus production after a treatment of cells with 2,3,7,8 TCDD has been revealed. PMID- 1715697 TI - Presence of IDF45 (mlGFBP-3) binding sites on chick embryo fibroblasts. AB - IDF45 (inhibitory diffusible factor) a mouse insulin-like growth factor binding protein (mlGFBP-3) has been shown to 100 percent inhibit DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF). Our previous results suggested that this large inhibition by IDF45 of serum stimulation was not just the result of its inhibitory activity toward IGF present in serum. The addition of Mn2+ (10( 3)M) in the incubation medium enables us to show the presence of numerous binding sites per cells (about 60,000) of mlGFBP-3. However the dissociation constant (10(-8)M) indicated that this mouse IGFBP-3 bound to the membrane with low affinity. These findings lend new support to the assumption of the bifunctional property of IGFBP-3, which would have an effect outside the cell (binding of IGF in the medium) and another effect within cells or on the surface. PMID- 1715698 TI - Intact but not truncated insulin-like growth factor binding protein-3 (IGFBP-3) blocks IGF I-induced stimulation of osteoblasts: control of IGF signalling to bone cells by IGFBP-3-specific proteolysis? AB - IGFBP-3 is the predominant IGFBP in serum and the major IGFBP secreted by osteoblasts. Native and recombinant IGFBP-3 and a truncated form lacking the carboxyterminal third were tested for their effects on 2 osteoblastic cell lines. Intact but not truncated IGFBP-3 blocked IGF I-stimulated DNA and glycogen synthesis. Inhibition was dose-dependent and found whenever the concentration of intact IGFBP-3 exceeded the concentration of IGF I. Truncated IGFBP-3 appears to result from proteolytic cleavage and does occur in vivo. The loss of inhibition by IGFBP-3 may be regulated at the site of IGF target cells and thus be essential for IGF I-induced osteoblast growth. PMID- 1715699 TI - Interleukin 8-inhibitor and inducer of histamine and leukotriene release in human basophils. AB - We have shown previously that interleukin 8 (IL-8) induces histamine and leukotriene release in human basophils exposed to interleukin 3 (IL-3). We now found that pretreatment with low concentrations of IL-8 selectively inhibits this response. Inhibition was significant at 0.01 nM IL-8 and virtually complete at 1 nM, which is about 100-fold lower than the concentration required for induction of mediator release. IL-8 dependent responses were also inhibited, albeit to a lesser extent, by preincubation with neutrophil-activating peptide 2 (NAP-2), but not with connective tissue-activating peptide III (CTAP-III) or platelet factor 4 (PF4). Release induced by C5a, fMet-Leu-Phe, or anti-IgE antibody, by contrast, was not affected. PMID- 1715700 TI - Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes. AB - Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5 trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration. PMID- 1715701 TI - Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin. AB - We previously observed that Ser378 in the heparin-binding domain of vitronectin becomes phosphorylated by a protein kinase in plasma upon addition of ATP and divalent cations. We now report that purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein and that vitronectin becomes phosphorylated during biosynthesis in human hepatoma (HepG2) cells. In vitro, rabbit muscle cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain (75 kDa) vitronectin but does not phosphorylate the two chain (65/10 kDa) form cleaved at Arg379. Heparin affects neither the time course nor the extent of phosphorylation of Ser378 at neutral pH. The extent of phosphorylation of Ser378 achieved with cAMP-dependent protein kinase (greater than or equal to 0.3 mol phosphate per mol vitronectin) is greater than that obtainable in plasma and should enable comparisons to be made of the activities of the native and phosphorylated forms. PMID- 1715702 TI - Phosphorylase kinase activity in I/strain neonatal skeletal muscle with a deficiency in alpha/alpha' subunit mRNAs. AB - In the adult I/LnJ mouse skeletal muscle, phosphorylase kinase activity is 0.2% of that in normal. This deficiency results from a paucity of mRNA's for the phosphorylase kinase regulatory subunit- alpha and its isoform alpha'. However, in the I/LnJ neonatal skeletal muscle phosphorylase kinase activity is 20-25% of that in normal. During the first two months of development this activity decreases while in normal tissue it increases. The developmental differences in the magnitude of the I/LnJ deficiency indicate the possibility of stage specific mechanisms regulating the accumulation of alpha/alpha' mRNAs. To investigate this possibility, the abundance of alpha/alpha' mRNAs and of the catalytic subunit, gamma, mRNAs were compared by Northern Blot analysis. The results demonstrate that neonatal and adult I/LnJ skeletal muscle have a similar paucity of alpha/alpha' mRNAs whereas accumulation of gamma mRNAs is not significantly different from normal. PMID- 1715703 TI - A transgenic mouse line developed to express human amyloidogenic transthyretin cDNA in the brain. AB - To elucidate the role of amyloid deposits in the pathogenesis of degenerative disorders of brain, we attempted to develop transgenic mice producing amyloidogenic human variant transthyretin (TTR) in the brain. A minigene, in which the promoter region of the mouse myelin basic protein (MBP) gene was ligated to the human mutant TTR cDNA, was used for microinjection. A transgenic mouse line expressing the human TTR mRNA specifically in the brain was thus derived. However, human TTR was not detected in the brain or in the serum of these mice. These data suggest that the human mutant TTR mRNA is not efficiently translated in the mouse brain. PMID- 1715704 TI - Spectrofluorometric determination of lipase activity. AB - A fluorescent triglyceride, 1(3)-pyrenylbutanoyl-2,3(1,2)-dipalmitoyl-sn glycerol, was synthesised, characterised by NMR and mass spectrometry, incorporated into a lipid emulsion and used as a fluorescent substrate for pancreatic lipase. It is shown that the product of the reaction, pyrene butyric acid, diffuses into the aqueous phase resulting in a decrease in the excimer fluorescence of the pyrene fluorophore in the emulsion and an increase in its monomer fluorescence. The phenomenon can be used to assay the enzyme thereby cirumventing the need to extract the fatty acid product. PMID- 1715705 TI - Purification and properties of adenosine 5'-phosphosulfate deaminating enzyme from marine macroalga Gloiopeltis furcata. AB - An enzyme which catalyzed the hydrolytic removal of the 6-amino group of adenosine 5'-phosphosulfate (APS) into inosine 5'-phosphosulfate was purified from the marine red macroalga Gloiopeltis furcata by means of salt fractionation, affinity, anion-exchange, and hydrophobic interaction chromatographies. The native enzyme had a Mr of about 285,000. Dissociation yielded a form with a Mr of about 70,000. The enzyme catalyzed the irreversible deamination of adenosine and its 5'-substituted compounds in addition to APS. Thus the enzyme seemed to be a nonspecific adenine nucleotide deaminase. Some properties were determined and compared with those of other nonspecific adenine nucleotide deaminases. PMID- 1715706 TI - Isoelectrofocusing of rat muscle adenylosuccinase. AB - Adenylosuccinase catalyses the conversion of adenylosuccinic acid to AMP and fumarate. We have developed a coupled enzyme staining procedure applicable to nitrocellulose blots after agarose gel isoelectrofocusing of rat muscle adenylosuccinase. The coupling enzymes, fumarase (fumarate to L-malate) and malic enzyme (L-malate to pyruvate and NADPH), are adsorbed to nitrocellulose prior to blotting. The NADPH, mediated by phenazine methosulfate, converts a tetrazolium salt to its blue formazan. This procedure demonstrated that rat muscle adenylosuccinase consists of three isomeric forms present in similar amounts. PMID- 1715707 TI - Molecular heterogeneity of angiotensin converting enzyme in human cerebrospinal fluid. AB - Three different molecular forms of angiotensin converting enzyme (ACE) (approximately Mr 150,000, 80,000 and 40,000, respectively), have been recovered from human cerebrospinal fluid. All three enzymes were inhibited by captopril and enalapril and their activity was potentiated by chloride ions. They were capable of degrading Leu-enkephalin-Arg6 and substance -P, but gave no conversion of neurokinin A. In all these aspects, the CSF enzymes were identical with the human pulmonary enzyme. The Mr 40,000 form of ACE is the smallest active form of the enzyme hitherto reported and is likely to represent a fragment of the C-terminal part of native ACE, where its active center is located. PMID- 1715708 TI - Identification of mRNA for epidermal growth factor and transforming growth factor alpha present in low copy number in human endometrium and decidua using reverse transcriptase-polymerase chain reaction. AB - The presence of mRNA for epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) was demonstrated in small fragments of human endometrium and decidua by use of the technique of reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers. The presence of mRNA encoding EGF and TGF alpha has not been shown in human endometrium previously. Other studies using conventional techniques, such as Northern blot or in-situ hybridization, showed the presence in low copy number of EGF but not TGF alpha in murine endometrium. Messenger RNA for EGF was not present in peripheral leukocytes or platelets, suggesting an endometrial source for the message. Messenger RNA for TGF alpha was found in these blood components, thus preventing confirmation of the source of TGF alpha mRNA. PMID- 1715709 TI - Signal transduction alterations in GH(1)2C1 rat pituitary tumour cells following treatment with 5-azacytidine. AB - In GH(1)2C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl 5'-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of G alpha s RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of G alpha i and/or G alpha o. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH(1)2C1 rat pituitary tumour cells with 5 azacytidine therefore causes a marked but temporary increase in the ratio of G alpha i/G alpha s protein levels. PMID- 1715710 TI - Differentiation of human monocytes into CD14 negative accessory cells: do dendritic cells derive from the monocytic lineage? AB - Human peripheral-blood monocytes, when cultured in the absence of serum, are prevented to differentiate to macrophages (M phi). Instead, they develop into accessory cells which by various properties resemble dendritic cells. Signals that control development either into M phi or monocyte-derived accessory cells (m AC) have been investigated by us. By applying such triggers, m-AC phenotypes and functions approached those known from lymphoid dendritic cells. Only the monocyte marker CD14, which is absent from dendritic cells, remained positive on m-AC as a last indicator of the monocytic origin of the cells. We now report that this most stable marker of the monocyte/M phi lineage can completely be down-regulated by combining tissue culture techniques with the inductive property of interleukin-4. Evidence has also been obtained by us that the conversion of monocytes into both m-AC and M phi represents a true differentiation, as demonstrated by the expression of the nuclear marker lamin A/C. PMID- 1715711 TI - Small (CD14+/CD16+) monocytes and regular monocytes in human blood. AB - Compared to the obvious phenotypic and functional heterogeneity of tissue macrophages, little information is available on subsets of blood monocytes. We have employed two-color immunofluorescence and flow cytometry for the definition of regular and small monocytes, the latter characterized by the low-density expression of CD14 and the strong expression of the CD16 (Fcy-RIII) antigen. These cells comprise 15% of the blood monocytes and they appear to be similar in phenotype to the alveolar macrophage. The CD14+/CD16+ small monocytes can perform phagocytosis and they produce reactive oxygen, while their capacity for cytokine production is strongly reduced when compared to regular monocytes. At this point it is still unclear as to whether the CD14+/CD16+ small monocytes comprise a specific level of activation or differentiation or a distinct sublineage of human blood monocytes. PMID- 1715712 TI - Cytokine regulation of the myeloid glycoprotein CD14. AB - Monocyte membrane CD14 (mCD14) antigen expression was measured in normal human monocytes and blood monocyte-derived macrophages. Seven-day culture of monocytes in serum-containing medium lead to an increase in mCD14. Addition of interferon gamma (IFN gamma) or interleukin-4 (IL-4) to monocytes caused a dose-dependent reduction in mCD14 within 3 or 45 h respectively. These effects were strong in monocytes, weak in macrophages, and they were blocked by anti-IFN gamma and anti IL-4 antibodies, respectively. Interleukin-2 and interferon-alpha produced a decrease in mCD14 in mononuclear leukocyte cultures but not in purified monocytes. Their effect was abolished in the presence of anti-IFN gamma. Other cytokines (TFN alpha, Ill beta, IL-6, IL-3, IL-5, GM-CSF, TGF beta 1) did not change mCD14. In conclusion, IFN gamma and IL-4 are revealed to be the only cytokines which directly affect monocyte CD14 expression. PMID- 1715713 TI - Activation of monocytes during inflammatory bowel disease. AB - Inflammatory bowel diseases lead to a systemic acute-phase response. Monocyte activation plays a central role during systemic acute-phase response via secretion of inflammatory cytokines. We determined the activation of peripheral blood monocytes in patients with inflammatory bowel diseases by measuring their interleukin-6 (IL-6) secretion. Blood was obtained from patients with active Crohn's disease before treatment [mean Crohn's disease activity index (CDAI) = 332 +/- 34] and from patients after treatment with prednisolone (mean CDAI index = 139 +/- 20). The mean serum IL-6 levels measured by a hybridoma growth assay (B9) were 23 +/- 4 U/ml before therapy and fell to 16 +/- 3 U/ml after treatment with prednisolone. Healthy persons and patients with inactive Crohn's disease usually had serum IL-6 levels below the detection limit of 4 U/ml. An ex vivo whole-blood system was used to measure IL-6 secretion by peripheral-blood monocytes with and without stimulation. Spontaneous IL-6 secretion in this system was about 9 U/ml in patients with Crohn's disease and below the detection limit of 4 U/ml in healthy controls. Moderate stimulation of blood cells [100 pg/ml lipopolysaccharide (LPS)] from patients with active Crohn's disease before and after treatment led to mean IL-6 concentrations of 1,160 +/- 514 and 131 +/- 54 U/ml, respectively. Maximal stimulation of peripheral blood before and after therapy by LPS (100 ng/ml) led to mean IL-6 concentrations of 5,570 +/- 1,660 and 6,220 +/- 1,630 U/ml, respectively. Thus, administration of glucocorticoids led to a rapid down-regulation of IL-6 synthesis by peripheral-blood monocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715714 TI - Regulation by endogenous interferon of virus-induced cytokine gene expression in mouse macrophages. AB - In macrophages from inbred mice the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the locus If 1, with C57BL/6 carrying the 'high-producer' allele If-1h whereas BALB/c have the 'low-producer' allele If-1l. The IFN produced consists of 90% IFN-beta and there are 10-fold differences between macrophages from If-1h and If-1l mice. Recently, we observed that interleukin-6 (IL-6) is coinduced by NDV in macrophages and seems to be under the same genetic control. Noninduced macrophages have been shown to secrete low amounts of antiviral activity endogenously when cultured in the presence of the macrophage-colony-stimulating factor (M-CSF). Here, we report that the amount of this endogenous IFN varies between macrophages from different mouse strains. Macrophages from BALB/c were found to secrete 5-10 times more endogenous IFN compared to C57BL/6. The antiviral activity could be identified as IFN-beta. Interestingly, we observed that endogenous IFN specifically down regulates NDV-induced IFN and IL-6 production. Preculture of BALB/c macrophages in M-CSF plus anti-IFN-beta to neutralize the biological effects of the endogenous IFN provoked a 30- to 50-fold increase in NDV-induced cytokine production, resulting in a nearly complete abrogation of the genetically determined difference since the same treatment only caused a 6-fold increase in C57BL/6 macrophages following NDV infection. This increase in cytokine gene expression was specific for NDV and marked by a strong additional activation of IFN-alpha genes. Addition of mouse recombinant IFN-alpha 4 to anti-IFN-beta treated macrophages for 18 h prior to NDV infection down-regulated again IFN gene expression and reestablished the genetic differences between macrophages from If 1h and If-1l mice.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715715 TI - Treatment of cancer patients with endotoxin induces release of endogenous cytokines. AB - This study sought to determine whether endogenous tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G CSF) were detectable in sera of lipopolysaccharide (LPS)-treated cancer patients. Twenty patients received an intravenous bolus of purified LPS from Salmonella abortus-equi (4.0 ng/kg). Patients were pretreated with ibuprofen (1,600 mg) to prevent constitutional side effects like fever and chills. Serum TNF-alpha levels increased from less than 0.01 ng/ml before treatment up to maximal levels of 21 ng/ml, peaking 1.5 h after LPS injection. Similarly, serum IL-6 concentrations increased from less than 0.01 to 11 ng/ml, but peak levels were obtained 30 min later than TNF-alpha. Circulating G-CSF appeared still later than TNF-alpha and IL-6. It was detectable within 3 h and peaked 6 h after LPS injection. Parallel to the release of the above cytokines a marked increase in granulocyte counts was observed. In all patients administration of LPS led to an acute-phase response as measured by C-reactive protein. PMID- 1715716 TI - Further characterization of carboxymethylated aspartic acid agarose. Purification of human alpha 2-macroglobulin and hemopexin. AB - alpha 2-Macroglobulin and hemopexin were purified by affinity chromatography on a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with cobalt(II). Adsorption was performed at neutral pH and the proteins were eluted by lowering the pH to 5.0. An alternative method for desorption as well as comparison with iminodiacetic acid agarose coupled with cobalt(II) is also described. PMID- 1715717 TI - Detection, quantification and sequencing of HIV-1 from the plasma of seropositive individuals and from factor VIII concentrates. AB - A highly sensitive and reliable RNA polymerase chain reaction method has been developed which has been used to detect, quantify and sequence cell-free HIV RNA directly from the plasma of seropositive individuals. Plasma from 10 out of 12 haemophiliacs tested was found to contain detectable levels of HIV-1 RNA [log mean value: 1.2 x 10(3) copies for Centers for Disease Control (CDC) group II patients, 5.5 x 10(3) copies for CDC group IV patients]. The presence of cell free circulating virus in both symptomatic and asymptomatic individuals suggests that viral replication continues throughout the course of infection. The same procedure has been used to detect and sequence HIV-1 RNA in two batches of unheated commercial factor VIII concentrate distributed in 1981 and 1983. The sequences obtained revealed a closer relationship to North American than to African strains of HIV-1. PMID- 1715718 TI - Identification of T-cell epitopes without B-cell activity in the first and second conserved regions of the HIV Env protein. AB - We have previously hypothesized that an effective vaccine against HIV should elicit cell-mediated immunity without antiviral antibody production. As a first step towards this goal we have identified potential T-cell epitopes, without B cell activity against the native protein, from the first and second conserved sequences, and from three functionally important regions of the HIV-1 envelope protein gp160. For this approach, short peptide sequences selected by established computer programs were synthesized and chemically modified to generate either polymers with disulfide bonds, or micelles with two palmitic acid residues attached to the amino-terminal lysine. In both configurations several peptides were immunogenic without the need for coupling to carrier molecules. Of the 19 peptides we tested in our present studies, seven induced good T-cell proliferative response in mice representing four major histocompatibility complex haplotypes. None of these seven peptides produced antibodies that could recognize the envelope protein gp160. PMID- 1715719 TI - Calcium channel drugs affect nocturnal serotonin N-acetyltransferase (NAT) activity in rat pineal gland. AB - The effects of different organic compounds that block and increase Ca2+ influx through the voltage-sensitive calcium channels (VSCC) on the nocturnal serotonin N-acetyltransferase (NAT) activity was investigated in vivo in rats. Systemic administration of VSCC antagonists, i.e. nimodipine, nifedipine, verapamil and diltiazem, resulted in a marked suppression of the nighttime pineal NAT activity. Bay K 8644, a VSCC agonist, injected to rats before the time of the light offset of the light-dark cycle significantly enhanced the nocturnal increase of the pineal NAT activity. Although Bay K 8644 given during the dark phase of an imposed illumination cycle had little effect on the nocturnal pineal NAT activity, it antagonized the nimodipine- and verapamil-induced decrease in the enzyme activity. These results support the role of Ca2+ influx through the VSCC in the nocturnal increase of NAT activity in the pineal gland of rat. PMID- 1715720 TI - [Social resources of the aged: isolation and family stress as health risk factors. Results of the Rome Elderly Study (RES)]. PMID- 1715721 TI - [Cardiovascular risk factors: their associations, their correction]. PMID- 1715722 TI - [First isolation of Chromobacterium violaceum strains, also achromogenic, in drinking water]. PMID- 1715723 TI - [Environmental spread of barium: health effects and and hygienic relevance of contamination of drinking water]. PMID- 1715724 TI - [Air pollution and zonation of epiphytic lichens in the city of Isernia]. PMID- 1715725 TI - [Patulin]. PMID- 1715726 TI - [Serological study on hepatitis C (HCV) in 4371 blood donors]. PMID- 1715727 TI - [Prevalence of hepatitis virus (HBV and HCV) and HIV-1 infections in a prison community]. PMID- 1715728 TI - The cellular measurement of time. AB - This review describes three biological processes in which there is evidence for single cells being able to measure elapsed time. We describe the work that has led to this view, and review more recent work that has provided new insights into possible mechanisms for the measurement of time. PMID- 1715729 TI - Lymphocyte trafficking in inflamed liver. AB - Cell-mediated immune reactions occurring in liver diseases involve infiltration of inflammatory cells into the hepatic parenchyma. Almost nothing is known about cellular and molecular mechanisms regulating this leukocyte traffic during the afferent and efferent arms of the hepatic immune response. This overview emphasizes that various liver parenchymal cells appear to participate actively in the development of the inflammatory response. PMID- 1715730 TI - Immunocytochemistry of cytokeratins in primary human liver tumors. AB - Human liver parenchymal cells have a very simple cytokeratin composition and express only one cytokeratin pair: cytokeratin 8 (a type II cytokeratin, molecular weight 52 kD) and cytokeratin 18 (a type I cytokeratin, molecular weight 45 kD). Intrahepatic bile duct cells contain in addition to cytokeratins 8 and 18 also cytokeratins 7 (a type II cytokeratin, molecular weight 54 kD) and cytokeratin 19 (a type I cytokeratin, molecular weight 40 kD). The paper deals with the cytokeratin expression in various types of benign and malignant primary liver tumors as assessed by immunohistochemical methods or by the use of gel electrophoresis and immunoblotting of cytoskeletal extracts. PMID- 1715731 TI - The value of immunohistochemistry in the differential diagnosis of endometrial carcinomas. AB - Endometrial carcinomas may originate from endometrial glandular epithelium and show endometrial differentiation, or from various types of metaplasias developing in the endometrium from pluripotent Mullerian epithelium. They then show endocervical or serous papillary differentiation. Because of their differences in spread, speed of growth and survival rates, it is important to subclassify these endometrial carcinomas. Immunohistochemically, adenocarcinoma with endometrial differentiation including adenoacanthomas and adenosquamous carcinomas can be recognized by their coexpression of cytokeratin 8 and vimentin, and by their negative reaction for CEA. Distinction from adenocarcinomas with mucinous differentiation, including mucoepidermoid adenocarcinomas, is possible by their negative reaction for vimentin and by their positive reaction for CEA. On the other hand, carcinomas with mucinous differentiation primarily located in the endometrium can not be distinguished from those primarily located in the endocervix by immunohistochemistry; that distinction must be made topographically. The same holds true for clear cell carcinomas of both locations. Over the past decade, mucinous adenocarcinomas and clear cell carcinomas originating from the endometrium have increased, whereas adenocarcinomas with endometrial differentiation have become less frequent. This shift is closely related to the altered postmenopausal hormone substitution with the addition of the synthetic gestagens. These apparently stimulate proliferation of endocervical epithelium not only in the endocervix, but also that arising in endocervical metaplasias of the endometrium. PMID- 1715732 TI - Immunotherapy and other novel therapies. AB - Although no true breakthroughs occurred, publications during the 12-month period of this review added substantial definition to certain novel immunotherapies potentially applicable to the treatment of rheumatoid arthritis. Overall, this period witnessed maturation in the field of biologic interventions. Clinical trials provided further data needed to assess the efficacy of high-dose intravenous gamma-globulin therapy in patients with systemic juvenile rheumatoid arthritis, and extended uncontrolled experience with interferon-gamma in adult rheumatoid arthritis was obtained. An intriguing immunostimulant and antiviral drug, isoprinosine (inosine pranobex), failed in a scientifically rigorous trial in rheumatoid arthritis. Provocative insights into totally new approaches surfaced in additional reports from a variety of immunologic areas. Although seemingly distal to rheumatoid arthritis, these papers are cited because their further development or adaptations could reach a stage where clinical trials in rheumatoid arthritis are warranted. PMID- 1715733 TI - Increased calcitonin gene-related peptide (CGRP), substance P, and enkephalin immunoreactivities in dorsal spinal cord and loss of CGRP-immunoreactive motoneurons in arthritic rats depend on intact peripheral nerve supply. AB - The distribution of peptides thought to be involved in pain modulation--substance P, calcitonin gene-related peptide (CGRP), and enkephalin--were studied in the spinal cord and dorsal root ganglia of polyarthritic rats and in rats with one sciatic nerve sectioned prior to induction of arthritis. In arthritic rats there was a bilateral increase of CGRP- and substance P-immunoreactive fibers and appearance of enkephalin-immunoreactive cell bodies in the dorsal horn of the lumbar (L4) spinal cord when compared to controls. In the corresponding dorsal root ganglia there were significant increases of CGRP- (P less than 0.02) and substance P- (P less than 0.001) immunoreactive cell bodies compared to controls. In the ventral horn of the control rats CGRP-immunoreactive motoneurons were abundant but were significantly (P less than 0.001) reduced in the arthritic spinal cord. Less pronounced changes were seen in the contralateral L4 spinal cord of arthritic rats with unilateral sciatic nerve section. In the ipsilateral dorsal horn, however, CGRP- and substance P-immunoreactive fibers were markedly depleted, and no enkephalin cell bodies were present. Furthermore, a number of CGRP-immunoreactive motoneurons were observed. In the ipsilateral L4 ganglia CGRP (P less than 0.02) and substance P- (P less than 0.02) immunoreactive cells were significantly decreased compared to the contralateral side. The data suggest that pain perception is linked to complex interactions between CGRP, substance P, and enkephalin in sensory pathways and an intact peripheral input. The loss of CGRP immunoreactive motoneurons may reflect muscular dysfunction associated with the arthritic condition. PMID- 1715734 TI - Current status of quality of life measurement in lung cancer patients. AB - Quality of life in patients with inoperable lung cancer receiving palliative treatment has become an important issue. As in other types of cancer, lack of standardization of quality of life data makes comparison between trials difficult, sometimes even impossible. Much effort is being given to the development of suitable instruments to measure quality of life and strategies to incorporate these measurements into clinical trials. The authors review studies in palliative lung cancer treatment where two crucial questions are being addressed: Do patients improve in performance status and symptom relief and does treatment toxicity outweigh disease palliation? PMID- 1715735 TI - New approaches to the chemotherapy of melanoma. AB - Several new investigational agents have shown some promise in the treatment of patients with disseminated melanoma. While earlier studies of combination chemotherapy led to increased toxicity without improved anti-tumor effect, more recent combination regimens have produced responses up to 50%. One of these is a dose-intensification regimen using high-dose cisplatin and the chemoprotective drug WR-2721. Other treatments under study include autologous bone marrow transplantation plus thiotepa, a combination regimen of chemotherapy and interferon, and newer agents such as taxol, tauromustine, and detrorubicin. PMID- 1715736 TI - Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. AB - The primary structures of the 16S rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (greater than 99%) that were consistent with the close relationships shown by previous DNA hybridization studies. The sequences of B. anthracis Sterne and B. cereus emetic strain NCTC 11143 were found to be identical for a continuous stretch of 1,446 bases and differed from the sequence of B. cereus NCDO 1771T (T = type strain) by only a single nucleotide. The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by four to nine nucleotides. PMID- 1715737 TI - Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA. AB - The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings. PMID- 1715738 TI - Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees. AB - A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper. PMID- 1715739 TI - Computer-aided quantification of RNA levels detected by in situ hybridization of tissue sections. AB - A semi-automatic program, designed for non-computer scientists, was developed for quantification of RNA levels detected by in situ hybridization in heterogeneous tissues. A video camera was used to acquire microscopic images of autoradiographed tissue sections which are then digitized on a video monitor for semi-automated quantification of silver grains. We describe a data entry and analysis procedure for systematic quantification of RNA levels in which about 300 cells per tissue sample can be analysed within 10 min. When compared with visual counting, computer-aided quantification was found to be more objective and reliable, with the highest variation coefficient between individuals being 7.5% using computer-aided quantification, compared to 24% with manual counting of the same section areas. A comparative study of c-myc oncogene expression in 11 mammary adenocarcinomas from 3 independent experiments showed the good reproducibility of results using the computer-aided method, with an 18% maximum variation between experiments. The program, with its simple user-interface, reliability and rapidity, is convenient for measuring specific genetic expression levels in clinical studies requiring large numbers of specimens. PMID- 1715740 TI - Overview of FK506 in transplantation. AB - FK506 is a potent immunosuppressive agent which is undergoing clinical testing in liver, kidney, heart, and bone marrow transplantation. It has been shown to effectively prevent and reverse ongoing rejection in these models. From the outset, FK506 was used with low-dose steroids to treat 110 primary liver, 30 heart, and 66 kidney graft recipients. FK506 was also used in the setting of complications related to CsA or to ongoing chronic or acute rejection. One hundred seventy-three liver, 21 kidney, 10 heart, and 11 bone marrow recipients were converted to FK506 and low-dose steroids, from a combination of CsA, steroids, and/or Aza. A randomized, prospective trial comparing FK506 with CsA in primary liver transplantation has verified the lower incidence of rejection and greater ease in treating rejection episodes, with fewer adverse effects. In summary, FK506 has proven to be an effective baseline immunosuppressive agent, as well as a dose-adjustable agent for the treatment of rejection. PMID- 1715741 TI - HLA epitopes detected by serology. AB - There is evidence that many of the so-called multispecific cytotoxic antisera react to the amino acid-defined epitopes of the HLA-A and B loci molecules. This evidence is based on the finding that a group of sera produced allelic reactions which corresponded to amino acid substitutions. Antisera for 29 amino acid variants were identified at 67 variable residues on the A locus and for 17 of the 51 variable sites on the B locus. The results were validated by showing that the reactions produced by testing cells from 111 individuals on an epitope typing tray fit the Hardy Weinberg equilibrium. This means that the alleles defined by the antisera behaved as alleles in the random population. Tests of homozygous cells on the epitope typing trays produced completely mutually exclusive reaction patterns as expected on the basis of the amino acid substitutions. Two examples are cited in which immunization across a single HLA "antigen" difference existed between donor and recipient, resulting in a "multispecific" antiserum. The specificities contained in the serum exactly fit an amino acid difference between the donor and recipient. PMID- 1715742 TI - HLA class II epitope detection by serology. AB - The amino acid sequence for the Class II specificities on 70 homozygous cell lines was compared with the cytotoxic reactions produced by 13,000 allogenic antisera against these lines. Many of the cytotoxic reaction patterns correlated completely with unique amino acids at a given residue. Therefore, we inferred that the antibodies were reacting directly with these amino acid-defined residues. Among 131 variable DRB1 residues, several well-correlated antisera were found to 64 (49%). Among 94 DQB1 residues, antibodies were found to 59 (63%). The location of these serologically defined epitopes on the molecule is shown in the table. We conclude that much of the serologic reactions of Class II can be explained by reactivity to the amino acid-defined epitopes. It can be shown that many of the sera that were previously considered multispecific are directed against these epitopes. These serologically defined epitopes are clearly immunogenic and are probably important in transplant matching. PMID- 1715743 TI - Similar properties of cGMP-activated channels between cones and rods in the carp retina. AB - Using patch-clamp techniques, properties of cGMP-activated channel were studied at a single-channel level in order to examine (1) whether any differences are recognized between the cGMP-activated channels of rods and cones in the same animal species, and (2) whether the channel properties of the same photoreceptor class differ in different animal species. Experiments were performed on inside out membrane patches excised from outer segments of rods and morphological subtypes of cones in the carp retina. Single-channel activities could be recorded when the patches were perfused with low concentrations of cGMP (less than 10 microM). Throughout five morphological subtypes of cones and rod, single-channel currents showed no significant rectification at membrane hyperpolarization in a low divalent cation solution, and single-channel conductances were almost the same: 13.8 +/- 0.2 pS (mean +/- S.E.M., n = 23) in cones and 12.7 +/- 0.8 pS (n = 3) in rods. These values were significantly smaller than that reported in catfish cones (about 50 pS), and that in rods of the toad and the tiger salamander (about 25 pS). In rods and all subtypes of cones of the carp, open durations of cGMP activated channels were brief. In addition, kinetic parameters of channel openings and closings showed no differences throughout all subtypes of cones and rod. PMID- 1715744 TI - Provocation of thyroid neoplasms in female Wistar rats fed n-3 or n-6 fatty acids in the feed. PMID- 1715745 TI - Lack of effect of verapamil on diurnal and thyrotropin releasing hormone stimulated thyrotropin levels in man. AB - We have evaluated the effects of oral administration of the calcium blocker verapamil on the pituitary secretion of TSH. Verapamil was administered 120 mg tid to 6 healthy volunteers. Plasma TSH was measured with an ultrasensitive immunoradiometric assay. TSH secretion was assessed by a 24-hour profile of unstimulated plasma concentrations and by the effect of TRH stimulation before and after 7 days of verapamil administration. No effects of verapamil was found on either basal or stimulated secretion of TSH. PMID- 1715746 TI - Identification of serum proteins, thyroglobulin and antithyroid antibodies in the fluid of thyroid cysts. AB - Fluid from fourteen thyroid cysts has been submitted to electrophoretic and immunochemical procedures in order to describe the biochemical structure of the fluid. Two patterns have been identified: a pattern showing a composition similar to that of normal serum ("serum-like"); thyroglobulin concentration is low, IgG are sometimes above normal and hTg-Abs are frequently detectable. A second pattern shows instead, a picture in which serum protein fractions are largely denatured and not identifiable ("denatured"); thyroglobulin concentration has been found markedly increased. It is suggested that in "serum like" the formation of the cysts occurs in the interstitium, whereas in the "denatured" type cyst the hemorrhagic necrosis appears to be the consequence of a massive damage of the follicular tissue. PMID- 1715747 TI - Massage therapy on neck: a contributing factor for destructive thyrotoxicosis? AB - A 57-year-old woman with Hashimoto's disease is described who developed transient destructive thyrotoxicosis subsequently after she underwent physical massage therapy over her shoulder to neck, together with the goiter, for muscle stiffness in that part. This case supports the concept that physical vigorous manipulation might be a contributing factor for thyrotoxicosis in cases predisposed with autoimmune thyroiditis. PMID- 1715748 TI - Hemostasis of the thyroid carcinoma invading the skin and trachea. Conservative treatment. AB - In most instances, the prognosis of well-differentiated thyroid carcinoma is generally favorable and adequately controlled by surgery alone. In some cases, however, in which the tumor has remained or in recurrent cases, infiltrates surrounding the skin or trachea can result in uncontrollable hemorrhage which cannot be treated with conventional medical or surgical therapy. We present our attempts at hemostatic procedures in such cases. When a thyroid cancer for which radical surgery is not indicated invades the skin with hemorrhage, we remove the ulcerated skin area with as much of underlying cancerous tissue as possible, and cover the defect skin area with a Bakamjian's deltopectoral flap or a musculocutaneous flap. In the 4 cases we have experienced, there had been no ulceration of the flap skin or recurrent. When a thyroid cancer invades the tracheal wall resulting in an intratracheal tumor accompanied by hemoptysis, we attempt to perform bronchoscopic cauterization using Nd-YAG Laser. Cauterization is repeatedly performed weekly, up to five times in one series. In all cases, radioiodine therapy is used before or after treatment. In all 4 cases we have experienced, hemostasis and shrinkage of the tumor was achieved. No complication such as ulcer formation or perforation was noted. PMID- 1715749 TI - Ouham-Pende: a new endemic goiter area in Centro-African Republic (C.A.R.). Preliminary observations on school children. AB - In this work, the Authors make several epidemiological observations and report a series of biohumoral data relative to a new endemic goiter area in the north western region of the Centro-African Republic (CAR). In this province whose chief town is Bocaranga, living conditions are very poor and primitive, manioca being the staple food. The survey was carried out in 11 rural schools and in 2 schools in the chief-town; 4009 subjects were examined; 2839 were males, 1170 females. The goiter prevalence observed in the whole sample population was 79.75%. The prevalence for each step of the grading were: 34.6% for 1a; 32.9% for 1b; and 11.8% for 2. In the schools of the chief town the goiter prevalence was found to be significantly lower than in the rural schools (71.9% versus 83.6% with P less than 0.01). Being the goitrous F/M ratio equal to 1.041, the goiter prevalences and gradings in females were higher and more severe than in males. After calculating these differences by means of the cumulated frequencies and the Kolmogorov Smirnov test, they were found to be significant. In 45 goitrous subjects, high TSH (7.19 microU/ml +/- 5.1), T3 (1.65 ng/ml +/- 0.56), and TG serum levels (410 ng/ml +/- 280) were found; however, low normal levels were observed for rT3 (8.97 ng/dl +/- 7.5) and fT4 (0.50 ng/ml +/- 0.51). In 42 casual urine samples, iodine excretion was 23.2 micrograms/l +/- 12.8; thyocianate excretion was 9.72 mg/l +/- 6 with a I/SCN ratio = 2.38.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715750 TI - Observer variation in the assessment of scintigraphy of the thyroid gland. AB - In order to determine observer variation in the assessment of thyroid scintigrams two specialists in nuclear medicine and two specialists in endocrinology independently evaluated 240 thyroid pertechnetate scintigrams twice, and assessed a number of variables concerning size and isotope uptake. The observed agreement between pairs of observers for the variables ranged from 0.70 to 0.98. By the use of the kappa coefficient the observed agreement was adjusted for change agreement. Kappa can variate from -1 (total disagreement) to +1 (perfect agreement). Kappa values between 0.29 and 0.86 were found. In the intraobserver study the observed agreement ranged from 0.83 to 0.99 resulting in kappa coefficients between 0.53 and 0.96. Thus the level of agreement in the present study was "fair to substantial" for agreement in the interobserver part and "substantial to almost perfect" for agreement in the intraobserver part. No difference was found in the level of agreement between the nuclear specialists and the endocrinologists. Although the treatment of patients is based on knowledge of the case histories and clinical and laboratory findings the high degree of observer variation may lead to misclassification of a number of patients with thyroid disease and subsequently a less optimal choice of treatment. PMID- 1715751 TI - Clinical correlation is required to avoid erroneous thyroid image interpretations. PMID- 1715752 TI - Multicompartmental analysis of triiodothyronine (T3) distribution and metabolism in clinically euthyroid obese individuals. AB - Triiodothyronine (T3) kinetic studies were performed on ten clinically non fasting euthyroid obese individuals and on eight normal subjects. Kinetic analysis was carried out according to a three-pool model whose basic approach involved the acquisition of data from both vascular and extravascular sources. The former were represented by the plasma disappearance curves of iv injected radiolabeled T3. The latter included fecal and urinary losses and liver, kidney and thigh uptake. A detailed comparison of the T3 kinetics of obese and normal individuals did not uncover many differences between these two groups in the way the hormone is distributed, metabolized and excreted. The mean value for the plasma equivalent distribution volume of T3(VD3) in obese individuals (27.05 L) was not significantly different from that in controls (24.60 L) nor were significant differences observed between the mean value for the plasma appearance rate of the hormone (PAR3) in obese subjects (29.80 micrograms/day) and that in controls (30.05 micrograms/day). The mean value for the size of the slow pool (Qc), including fatty tissue as well as skeletal muscle, was unchanged in obese individuals when compared with controls, although in the obese subjects the mean value for the mass of fatty tissue was about 5 times greater. In addition, in obese individuals, the mean value for the fractional rate transfer from plasma to the slow pool (Kca), which was 9.06 day-1, did not significantly differ from that in controls (9.22 day-1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715753 TI - 4.5S RNA: does form predict function? AB - 4.5S RNA is a stable RNA of Escherichia coli, and functional homologs of the molecule apparently exist in all prokaryotes: eubacteria, archebacteria, and mycoplasma. Genetic and physiological measurements of the function of 4.5S RNA in E. coli indicate a role for this RNA in protein synthesis. A conserved domain of 4.5S RNA displays structural similarity with the eukaryotic 7S RNA that functions in protein secretion. Although complementation by eukaryotic 7S RNAs remains to be demonstrated, a number of archaebacterial 7S RNAs are able to replace 4.5S RNA for growth of E. coli, and 4.5S RNA is able to mediate a number of 7S RNA functions in vitro. Surprisingly, no effects on protein secretion in E. coli have been directly attributed to 4.5S RNA. These observations raise the question of whether molecules of similar structure necessarily perform the same function. PMID- 1715754 TI - Specificity for molecules of the major histocompatibility complex mediated by a hybrid immunoglobulin-T cell receptor. AB - A chimeric T-cell receptor (TCR) alpha-chain gene was produced by shuffling the immunoglobulin VDJH from a 40-140 digoxin-specific hybridoma onto alpha-chain constant region (C alpha) exons. This hybrid immunoglobulin-TCR gene was used to produce transgenic mice. Previous results indicated that this chimeric gene encoded a polypeptide that associated with endogenously encoded beta chains to form a hybrid TCR. T cells expressing this receptor could be stimulated with antibodies specific for CD3 or the 40-140 idiotype (Id40-140), and also with digoxin coupled to bovine serum albumin (digoxin-BSA). We were interested in determining whether a hybrid receptor such as this could also recognize the natural ligand of T cells, namely allelic variants of major histocompatibility complex (MHC) molecules. A T-cell hybridoma was produced that expressed a hybrid receptor with specificity for an IAk-encoded determinant, digoxin-BSA, or staphyloccocal enterotoxin B. Transfection experiments showed that the specificity for MHC determinants was dependent on both the hybrid alpha chain and a particular beta chain. These results indicate that a V beta domain combined with a VH domain can produce a receptor capable of reacting with MHC molecules, and at the same time retain specificities mediated by the beta chain and alpha chain alone. A conclusion is that the pervasive MHC specificity of the TCR is not unique to the family of TCR heterodimers, but is selected, and can be mediated by immunoglobulin domains. PMID- 1715755 TI - Tissue- and development-specific expression of HBGF-1 mRNA. AB - The gene for heparin-binding growth factor-1 (HBGF-1) encodes a 15.5-18 kDa polypeptide that affects the proliferation and differentiation of a broad range of mammalian cells and is widely distributed among normal adult tissues. In this study, we show that normal tissues of the adult rat express HBGF-1 transcripts in one of three patterns: a 4.4 kb mRNA was the predominant HBGF-1 transcript in brain, heart and lung; a 1.4 kb mRNA was the predominant transcript in the liver; approximately equal levels of the 1.4 and 4.4 kb mRNAs were found in the kidney. HBGF-1 expression was localized in two tissues: central nervous system expression of HBGF-1 was significantly higher in the brain stem compared to the cerebrum and cerebellum; renal expression of HBGF-1 was significantly higher in the medulla compared to the cortex. Analysis of the postnatal changes in HBGF-1 expression using the newborn rat kidney revealed that the level of HBGF-1 mRNA is low at birth and does not rise to adult levels until the seventh postnatal day. These findings demonstrate that HBGF-1 expression is specific for tissue type and stage of development. PMID- 1715756 TI - Functional expression of urea channels in amphibian oocytes injected with frog urinary bladder mRNA. AB - In amphibian urinary bladder epithelium, vasopressin increases passive urea permeability, concomitant with the appearance of a facilitated urea transport. Amphibian oocytes from Xenopus laevis and Rana esculenta were microinjected with total or fractionated poly(A+) RNA isolated from frog urinary bladder epithelial cells. After several (3-5) days at 18 degrees C, the urea flux was assayed by measuring the uptake and efflux of [14C]urea in water-injected and mRNA-injected oocytes. A 2 to 3-fold increase of urea transport was detected in oocytes injected either with total mRNA or with a 6-10 kilobase mRNA fraction, when compared with water-injected oocytes. This expression of urea channels was inhibited by 0.1 mM phloretin (50% inhibition) and 0.1 mM nitrophenylthiourea (up to 70% inhibition). On the contrary, no expression was detected in brain mRNA injected oocytes. These results show the specific functional expression of the phloretin- and NPTU-sensitive urea channel (or carrier) from frog urinary bladder epithelial cells, providing an approach for the expression cloning of these urea channels. PMID- 1715757 TI - The characterization of synthetic by-products by B/E linked scanning and high resolution thermospray mass spectrometry. AB - Characterization of unknown synthetic by-products from a test synthesis of the experimental inhibitor of HIV-1 reverse transcriptase, BI-RG-587, is presented. Seven impurities were separated and characterized by thermospray liquid chromatography/mass spectrometry using low-resolution, high-resolution, and B/E linked scanning methods. The high-resolution mass measurements which were obtained were accurate to within 10 ppm of structures proposed for synthetic by products present at the 0.1% level. Linked scans produced daughter ion spectra for each impurity to further aid in the characterization. PMID- 1715758 TI - Monoclonal antibodies and mechanistic studies on recA protein. AB - A protein has various epitopes, and a monoclonal antibody specifically binds to the protein by recognizing 1 of the epitopes. This characteristic of the monoclonal antibody has opened various new approaches in a wide variety of research works. In studies about recA protein and its promoted various reactions relating to genetic recombination, anti-recA protein-monoclonal antibodies are very useful to analyse reaction mechanisms and to detect transition in the higher order-structure of the protein, as well as to measure the amounts of recA protein in vitro or in vivo and to identify the related proteins. In this article, we will review studies on recA protein in which monoclonal antibodies were used as major tools. By using anti-recA protein-monoclonal IgGs as specific inhibitors, the partial reactions of the homologous pairing and strand exchange promoted by recA protein were separated, and by use of a set of anti-recA protein IgGs the stages of activation of recA protein in the above reactions were discriminated. PMID- 1715759 TI - Identification of a mouse cDNA fragment whose expressed polypeptide reacts with anti-recA antibodies. AB - We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein. Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies. The cDNA fragment is 601 nucleotide long and was called KIN17(601). It contains an open reading frame coding for a 200 amino acid polypeptide. In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein. Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins. PMID- 1715760 TI - Characterization of complement-independent neutralizing epitopes on pseudorabies virus glycoprotein gp50. PMID- 1715761 TI - Negative regulation of interleukin-2 production in primary lymphocytes by 12-O tetradecanoylphorbol-13-acetate. AB - Stimulation of quiescent T lymphocytes to proliferate involves a complex series of events both between and within cells. At least 70 genes are known to be induced or activated from the time of the initial stimulation until DNA synthesis. While some of these gene products, e.g., interleukin-2 (IL-2) and IL-2 receptors, are required for proliferation, others, e.g., gamma-interferon and colony-stimulating factor, are ancillary to activated T cell function. Several biochemical signal transductions are among the early events. One of the earliest is phospholipase C-mediated hydrolysis of phosphatidylinositol leading to release of diacylglycerols and inositol phosphates, which in turn activate protein kinase C and elevate intracellular free calcium levels. The discovery that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) both enhances proliferation and activates protein kinase C strengthens the evidence for a general role of protein kinase C in proliferation. Yet, the exact consequences of stimulation of protein kinase C in regard to specific proliferation proteins is still not clear. In this study, we present evidence that protein kinase C activation is directed to production of IL-2 but not to IL-2 receptors. Under conditions of TPA treatment in which protein kinase C was chronically reduced in T lymphocytes, IL-2 production was greatly depressed as were the level of IL-2 mRNA and [3H]thymidine incorporation. In contrast, these cells still expressed high affinity IL-2 receptors and proliferated when endogenous IL-2 was added. Because neither phosphatidylinositol metabolism nor Ca2+ flux was affected, the block appeared to be mediated directly or indirectly through protein kinase C. PMID- 1715762 TI - Role of calcium on interleukin-1 production by monocytes: its relevance during T cell proliferation. AB - T cell proliferation, in the presence of monocytes, triggered either by an anti CD3 monoclonal antibody (mAb) or by a mitogenic pair of anti-CD2 mAbs was inhibited either by the calcium chelator EGTA or the calcium channel blocker nifedipin. Antibodies against interleukin-1 (IL-1) inhibited T cell proliferation in both mitogenic systems. However inhibition achieved by anti-IL-1 beta Ab was greater than by anti-IL-1 alpha Ab while the combination of both anti-rIL-1 alpha Ab + anti-rIL-1 beta could completely inhibit the CD3-triggered T cell proliferation. On the other hand, IL-1 production by LPS-stimulated monocytes was strongly decreased both by EGTA and nifedipin. Northern blot analysis showed that this inhibition paralleled a decrease of IL-1 alpha and beta messenger RNA (mRNA) expression only in the presence of EGTA but not in the presence of nifedipin. These results indicate that EGTA acted at the transcriptional level while nifedipin acted at a yet undefined posttranscriptional level. Thus, it is suggested that the impairment of T cell proliferation by calcium inhibitors could result not only from an effect on Ca2+ influx in T cells but also from interfering with the function of accessory cells, such as the production of IL-1. PMID- 1715763 TI - Neutrophil proteases in plasminogen activation. AB - Leukocyte elastase and leukocyte cathepsin G degrade porcine plasminogen primarily by hydrolysis of the A447-I448 bond between kringle4 and kringle5. The rate of formation of des-kringle1-4-plasminogen is faster with elastase (k"obs greater than 10(5) mol-1 s-1) than with cathepsin G (kobs less than 300 mol-1 s 1). In contrast to elastase, leukocyte cathepsin G does not inactivate alpha 2 antiplasmin. Consequently, plasminogen activation by urokinase in the presence of alpha 2-antiplasmin is elastase-dependent, but cathepsin G does not overcome the action of alpha 2-antiplasmin. The rate-enhancing effect of fibrin(ogen) fragments in plasminogen activation by tissue-type plasminogen activator is also disabled efficiently by these proteases. It is concluded that enhancement of plasmin expression by neutrophil proteases is accounted for primarily by the action of elastase. PMID- 1715764 TI - Proton conductance by the gramicidin water wire. Model for proton conductance in the F1F0 ATPases? AB - The gramicidin channel contains a single strand of water molecules associated through hydrogen bonds. Previous work has shown that channels of similar size are formed by association of transmembrane alpha helices of synthetic leucine-serine peptides. Both types of channels translocate protons with considerable selectivity relative to other cations, and it has been proposed that the selectivity arises by proton "hopping" along hydrogen-bonded chains of water, whereas other cations must cross by ordinary diffusion processes. It is possible that a similar mechanism underlies proton transport in the Fo subunit of the F1F0 ATP synthase. Using the gramicidin channel as a model, we have tested whether a single strand of water is kinetically competent to translocate protons at a rate sufficient to support known rates of ATP synthesis. We found that the gramicidin channel saturates at approximately 530 pS of protonic current in 4 M HCl, more than sufficient for typical ATP synthesis rates. It follows that proton diffusion to a putative channel in Fo, rather than the channel itself, may limit ATP synthesis rates. PMID- 1715765 TI - Microscopic model for selective permeation in ion channels. AB - Ionic permeation in the selectivity filter of ion channels is analyzed by a microscopic model based on molecular kinetic theory. The energy and flux equations are derived by assuming that: (a) the selectivity filter is formed by a symmetrical array of carbonyl groups; (b) ion movement is near the axis of the channel; (c) a fraction of water molecules is separated from the ion while it moves across the selectivity filter; (d) the applied voltage drops linearly across the selectivity filter; (e) ions move independently. Energy profiles, single channel conductances, and the degree of hydration of K+ in a hypothetical K+ channel are examined by varying the following microscopic parameters: ion radius and mass, channel radius, number of effective water dipoles, and number of carbonyl groups. The i-V curve is linear up to +/- 170 mV. If the positions of energy maxima and minima are fixed, this linear range is reduced to +/- 50 mV. Channel radius and ion-water interactions are found to be two major channel structural determinants for selectivity sequences. Both radius and mass of an ion are important in selectivity mediated by these interactions. The theory predicts a total of 15 possible kinetic selectivity sequences for alkali cations in ion channels with a single selectivity filter. PMID- 1715766 TI - Time-correlation analysis of simulated water motion in flexible and rigid gramicidin channels. AB - Molecular dynamics simulations have been done on a system consisting of the polypeptide membrane channel former gramicidin, plus water molecules in the channel and caps of waters at the two ends of the channel. In the absence of explicit simulation of the surrounding membrane, the helical form of the channel was maintained by artificial restraints on the peptide motion. The characteristic time constant of the artificial restraint was varied to assess the effect of the restraints on the channel structure and water motions. Time-correlation analysis was done on the motions of individual channel waters and on the motions of the center of mass of the channel waters. It is found that individual water molecules confined in the channel execute higher frequency motions than bulk water, for all degrees of channel peptide restraint. The center-of-mass motion of the chain of channel waters (which is the motion that is critical for transmembrane transport, due to the mandatory single filing of water in the channel) does not exhibit these higher frequency motions. The mobility of the water chain is dramatically reduced by holding the channel rigid. Thus permeation through the channel is not like flow through a rigid pipe; rather permeation is facilitated by peptide motion. For the looser restraints we used, the mobility of the water chain was not very much affected by the degree of restraint. Depending on which set of experiments is considered, the computed mobility of our water chain in the flexible channel is four to twenty times too high to account for the experimentally measured resistance of the gramicidin channel to water flow. From this result it appears likely that the peptide motions of an actual gramicidin channel embedded in a lipid membrane may be more restrained than in our flexible channel model, and that these restraints may be a significant modulator of channel permeability. For the completely rigid channel model the "trapping" of the water molecules in preferred positions throughout the molecular dynamics run precludes a reasonable assessment of mobility, but it seems to be quite low. PMID- 1715767 TI - Characterization of S-[2-(N1-adenyl)ethyl]glutathione as an adduct formed in RNA and DNA from 1,2-dibromoethane. AB - The major DNA adduct derived from 1,2-dibromoethane is known to be S-[2-(N7 guanyl)-ethyl]glutathione; minor nucleic acid DNA adducts were characterized in view of the possibility that some might be unusually persistent or biologically active. RNA was modified in vitro by treatment with 1,2-dibromoethane and glutathione in the presence of rat liver cytosol, and bases were released by mild acid hydrolysis, which liberated greater than 99% of the bound radioactivity. One of the minor adducts was identified as S-[2-(N1-adenyl)ethyl]glutathione on the basis of its UV, mass, and NMR spectra. This adduct could be synthesized by reaction of S-(2-chloroethyl)-glutathione with adenosine. The material was desulfurized by treatment with Raney Ni to give N1-ethyladenine in low yield. The Raney Ni reaction was accompanied by considerable formation of the corresponding N6-adenine derivative via Dimroth rearrangement. Another adduct was identified as S-[2-(N7-guanyl)ethyl]cysteinylglycine by its UV, mass, and NMR spectra, but the material was demonstrated to be formed from the major DNA adduct, S-[2-(N7 guanyl)-ethyl]glutathione under conditions of mild acid hydrolysis. The imidazole ring opened derivative of S-[2-(N7-guanyl)ethyl]glutathione was synthesized and found not to be formed in DNA in vitro or in vivo. The two remaining minor adducts account for 1-2% of the total binding, but insufficient quantities were recovered to allow for structure determination; however, neither of these (uncharacterized) minor products are seen after the reaction of S-(2 chloroethyl)glutathione with guanosine or adenosine. S-[2-(N1 Adenyl)ethyl]glutathione was formed in rat liver RNA and DNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715768 TI - Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration. AB - Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration. PMID- 1715769 TI - Biochemical and biological analysis of human interleukin 6 expressed in rodent and primate cells. AB - The cDNA for human interleukin 6 (IL 6) was stably expressed at high levels in the three mammalian cell lines COS-7, PA317, and GH3 to yield IL 6 proteins of 25 to 27, 26, 22 to 24, and 23 kDa molecular mass. Both size and relative amounts of the recombinant IL 6 (rIL 6) species produced correspond to those of natural IL 6 secreted by LPS-stimulated monocytes. Oligosaccharide analysis of recombinant IL 6 utilizing tunicamycin and endoglycosidases revealed O- and N-linked glycosylation that is comparable to that of natural IL 6 derived from human monocytes and fibroblasts. IL 6 expressed in each of the three cell lines was phosphorylated similarly to the IL 6 produced in human monocytes and fibroblasts. IL 6 secreted by the three different cell lines have marked differences in specific biological activities. COS-7 IL 6 appeared to be 12-fold more active in its hybridoma growth factor activity than that made in PA317 or GH3 cells. In contrast, PA317 and GH3 IL 6 were 230 and 6.7 times more effective than COS-7 IL 6 in inducing Ig production in CESS cells. Also, PA317 and GH3 IL 6 were more effective than COS-7 IL 6 in inducing the acute-phase protein fibrinogen in human hepatocytes. The rIL 6 species exhibited no antiviral activity. PMID- 1715770 TI - Modification of biological responses to interleukin-1 by agents that perturb signal transduction pathways. AB - In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-1). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular cAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-1 induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E2. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-1, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate. PMID- 1715771 TI - Identification of structural differences between different forms of interleukin-2 (IL-2) using anti-(human recombinant) IL-2 monoclonal antibodies. AB - We have generated a panel of murine monoclonal antibodies (mAbs) directed against recombinant human interleukin-2 (rh IL-2). All mAbs have similar affinities (Kaff approximately equal to 10(-8) M) and are of the IgG1 isotype. Specificity of the mAbs has been established using an ELISA method and immunoprecipitation of native human interleukin-2 (nh IL-2) present in supernatants from PHA-stimulated human mononuclear cells (PBMNC). Four of the nine mAbs inhibit the rh IL-2-dependent proliferation of a murine cytotoxic T-lymphocyte line (CTLL-2). The estimated ID50, in the presence of 0.75 IU rh IL-2/ml, ranges from 2.0 micrograms/ml to 30 micrograms/ml final concentrations of the antibodies. Using different forms of IL 2 we found that the mAbs give different patterns of inhibition of the IL-2 dependent proliferation. One mAb (AMCIB 27) is able to discriminate between rh and nh IL-2. Findings with another mAb (AMCIB 24) indicate that possible functional differences between human IL-2 and recombinant murine (rm) IL-2 are caused by differences in the active sites. The results of this investigation show that it is possible to obtain mAbs, after immunization with rh IL-2, that differ in their ability to inhibit the biological action of different forms of IL-2. PMID- 1715772 TI - Biology of the RANTES/SIS cytokine family. PMID- 1715773 TI - The collagen fibril--a model system for studying the staining and fixation of a protein. AB - A collagen fibril is made up of long rod-like molecules regularly D-staggered with respect to one another. This means that (i) its axially projected fine structure, resolvable to approximately 2 nm in electron micrographs, repeats D periodically (D = 67 nm), and (ii) the amino acid residues contributing to each element of the fine structure can be inferred from sequence data. Electron optical data from a fibril D-period can can therefore be correlated directly with chemical data. Such correlations confirm the electrostatic nature of the staining reaction when a fibril is positively stained. After negative staining, the principal factor determining the small-scale distribution of stain is local exclusion by 'bulky' amino acid side-chains. ('Bulkiness' is the average cross sectional area, or 'plumpness', of a side-chain.) A small superimposed positive staining contribution can also be detected. Fixation of collagen by aldehydes and diimidoesters occurs via an initial reaction with lysyl (and hydroxylsyl) side chains and alpha-amino groups, followed by secondary cross-linking reactions that differ from fixative to fixative. These secondary reactions determine the nature and abundance of the cross-links and the extent to which they influence subsequent staining behaviour. PMID- 1715774 TI - Negative staining of proteins. AB - Negative staining, some closely related alternative preparation techniques and radiation stability are considered. An attempt is made to clarify the mechanism of action and ultimate resolution limit of negative staining. The results of electron diffraction investigation of thermitase microcrystals embedded in glucose and glucose + stains are presented. It is shown that at doses not exceeding 10 electrons/nm2 electron diffraction from thermitase crystals demonstrate diffraction fields up to 0.2 nm. When adding heavy-atom salts to glucose or using negative staining, the relative intensities of reflections change and electron diffraction patterns for every type of heavy-atom additive (or negative stain) have their specific features. Such characteristic changes of reflection intensities indicate specific interaction of these additives (or stains) with the object. In the case of electron diffraction from the crystals stained using the routine negative staining technique the ordering was preserved down to 0.4-0.5 nm. Increasing the dose up to the normal value results in fading of distant reflections. Thus, negative staining with radiation doses less than the critical one could yield resolution down to 0.4 nm. Yet, the structure may change due to interaction with the stain. Nevertheless, the possibility that such resolution could be obtained for a limited number of objects should not be excluded. Some examples of the application of negative staining for investigation of quaternary and domain structure of proteins (nitrogenase, glutamine synthetase, mitochondrial ATP-synthase, membrane monooxygenase enzymes), tubular and two-dimensional protein crystals (catalase, phosphorylase, HWV protein, hydrogenase), as well as ribosomes and bacteriophages are given in the review. PMID- 1715775 TI - Immortalization of magnetically separated human lymphocytes by electrofusion. AB - Human lymphocytes from peripheral blood (MNC) were separated on magnetic beads for the presence of different surface markers. Cells from positive and negative fractions were successfully immortalized by electrofusion with the heteromyeloma line CB-Fu2. B cells, which were separated on anti-CD 19 coated beads, could be immortalized at a rate between 10(-5) and 10(-4) even if the fusion was conducted with just a few hundred thousand cells. Comparison of the frequency of Ig positive hybridomas in B cell, T cell, and unseparated MNC fusions indicated that also non-B cells may give rise to HAT resistant hybridoma clones, although the fusion frequency was low. PMID- 1715776 TI - A human monoclonal autoantibody specific for human platelet glycoprotein IIb (integrin alpha IIb) heavy chain. AB - Splenocytes from a patient with chronic, immune-mediated thrombocytopenic purpura (ITP) were transformed with Epstein-Barr virus. A stable lymphoblastoid cell line (LCL) derived from this transformation (2A3) produces IgM antibody reactive with platelet glycoprotein IIb. 2A3 was fused to the 6-thioguanine-resistant ouabain resistant, murine-human heteromyeloma cell line, F6. The resultant heterohybridomas were selected by growth in medium containing hypoxanthine/aminopterin/thymidine and ouabain. One hybridoma line, 2E7, produces high levels of IgM antibody (2 to 4 micrograms IgM/ml/24 hr/10(5) cells) reactive with glycoprotein IIb. 2E7 has been repeatedly subcloned by limiting dilution and has been maintained in continuous culture for 26 months. 2E7 binds to human platelets but not endothelial cells, as determined by flow cytometry, and does not react with platelets of patients with Glanzmann's thrombasthenia that lack IIb-IIIa. The epitope recognized by 2E7 is likely to be a contiguous peptide sequence since the antibody binds to the IIb heavy chain in immunoblot assays of denatured, reduced platelet protein. Treatment of intact platelets or purified IIb-IIIa with papain or chymotrypsin, but not SV8 protease, destroys the epitope. Thus, the 2E7 epitope may be at or very close to a site on IIb that is cleaved by these proteases. The expression of the 2E7 epitope is significantly affected by the presence of divalent cations. Treatment of intact platelets with EDTA at 37 degrees C results in a three-to four-fold increase in the number of 2E7 molecules bound per platelet and an eight-fold increase in the affinity of the antibody. The binding of 2E7 to normal platelets does not inhibit any of the functions attributed to IIb-IIIa, such as fibrinogen-dependent platelet aggregation or clot retraction. 2E7 represents the first human monoclonal antibody reported to recognize an epitope on platelet glycoprotein IIb. The epitope is unique to IIb and not shared by other integrin alpha subunits. PMID- 1715777 TI - Effect of total parenteral nutrition on histamine in the rat brain and other tissues. AB - The effects of a constant infusion of total parenteral nutrition on the histamine content of the hypothalamus was studied in adult male rats. Histamine content of the stomach, kidney, liver, and heart was also determined. The relationship between tissue histamine levels and duration of the infusion was examined. Rats receiving intravenous infusions of total parenteral nutrition had significantly higher (p less than 0.001) histamine levels in the hypothalamus than did orally fed control rats. No difference was observed in the histamine level in the remainder of the brain. Animals receiving parenterally infused nutrient solutions had a higher histamine content in the kidneys but lower histamine levels in the stomach than did the control rats. No changes in the histamine content of liver or heart were observed. It appears that increases in histamine levels in the kidney and hypothalamus of rats given total parenteral nutrition persist up to 5 days and 9 days, respectively. The mechanisms responsible for and the consequences of the alterations of histamine content of these tissues remain to be established. PMID- 1715778 TI - Hepatocellular carcinoma in Karachi. AB - The present study defines the clinical presentation and examines possible aetiological factors in the occurrence of hepatocellular carcinoma in Karachi, Pakistan. Histologically proven cases of hepatocellular carcinoma (n = 366) were seen over 16 years. The maximum frequency occurred in the age range 51-60 years (range: 8-98 years), and the male to female ratio was 2.5:1. The place of birth, place of longest stay and various addictions did not have any association with the occurrence of liver carcinoma. Only three cases had a history of liver cancer in their immediate relatives. The main presenting features were right hypochondrial mass (85%) and pain (79%). The liver size did not correlate with the duration of illness. alpha-Fetoprotein titres were more than 200 ng/mL in 62% of cases. Using a reverse passive haemagglutination assay method, HBsAg and anti HBc positivity were 32% and 60%, respectively. Antigen figures rose to 60% when radio-immunoassay was used; 41% of cases were anti-delta positive (EIA). Aflatoxin contamination varied between 10% and 17% in various localities of Karachi, suggesting an association of liver cancer with HBsAg and aflatoxins. Histopathologically 73% were trabecular, and 2.5% were mixed hepatocholangiocarcinomas. Follow-up was available in 45% of cases. All except two (1.2%) died within 6 months. PMID- 1715779 TI - Management of malignant bile duct obstruction. PMID- 1715780 TI - Proteolipid DM-20 predominates over PLP in peripheral nervous system. AB - The major central nervous system (CNS) myelin proteolipid (PLP) is also expressed in the peripheral nervous system (PNS). This paper gives evidence that DM-20, an isoform of PLP, also occurs in rat sciatic nerves, where, in contrast to what is seen in CNS myelin, it predominates over PLP. This conclusion was reached on the basis of results obtained by immunoblot analysis of a crude proteolipid extract from adult peripheral nerve with two site-specific anti-proteolipid (PLP and DM 20) antibodies. This finding was further corroborated by characterization of the products obtained by Polymerase Chain Reaction (PCR) amplification of cDNAs synthesized from total RNA of 14-day-old sciatic nerves. The significance of the occurrence of these proteolipids in PNS remains obscure. PMID- 1715781 TI - Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. AB - Reduction of turgor in pea shoots caused the accumulation of several poly(A) RNAs. cDNA clones derived from three different poly(A) RNAs which accumulate in wilted pea shoots were isolated, sequenced and expression of the corresponding genes examined. Clone 7a encoded a 289 amino acid protein. The C-terminal 180 amino acids of this protein were homologous to soybean nodulin-26. RNA hybridizing to cDNA 7a was abundant in roots, and induced in shoots by dehydration, heat shock and to a small extent by ABA. Hydropathic plots indicate that the protein encoded by cDNA 7a contains six potential membrane spanning domains similar to proteins which form ion channels. Clone 15a encoded a 363 amino acid protein with high homology to cysteine proteases. RNA hybridizing to cDNA 15a was more abundant in roots than shoots of control plants. Dehydration of pea shoots induced cDNA 15a mRNA levels whereas heat shock or ABA treatment did not. Clone 26g encoded a 508 amino acid protein with 30% residue identity to several aldehyde dehydrogenases. RNA hybridizing to cDNA 26g was induced by dehydration of shoots but not roots and heat shock and ABA did not modulate RNA levels. Levels of the three poly(A) RNAs increased 4-6-fold by 4 h after wilting and this increase was not altered by pretreatment of shoots with cycloheximide. When wilted shoots were rehydrated, RNA hybridizing to cDNA 26g declined to pre stress levels within 2 h. Run-on transcription experiments using nuclei from pea shoots showed that transcription of the genes which encode the three poly(A) RNAs was induced within 30 min following reduction of shoot turgor. One of the genes showed a further increase in transcription by 4 h after dehydration whereas transcription of the other 2 genes declined. These results indicate that plant cells respond to changes in cell turgor by rapidly increasing transcription of several genes. Furthermore, the expression of the turgor-responsive genes varies with respect to the time course of induction and reversibility of the wilting induced changes. PMID- 1715782 TI - A primary transcript in spinach chloroplasts that completely lacks a 5' untranslated leader region. AB - A fifth, previously undetected transcript for the plastid gene encoding the beta subunit of spinach chloroplast ATPase (atpB) has recently been identified in vitro. In this report we show that this transcript is present in vivo and its 5' end is at the translation start codon. When synthesized in vitro using spinach chloroplast RNA polymerase, the 5' end of this novel transcript is located at the beginning of the second codon of the atpB coding sequence. Although this atpB transcript lacks an untranslated leader region, it is an abundant RNA in vivo and is associated with crude polysomal fractions, as are the four larger atpB transcripts. In vitro synthesis of the leaderless transcript does not occur for a plasmid DNA in which the "-35' region of the promoter is deleted. PMID- 1715783 TI - Two genes encoding the soybean translation elongation factor eEF-1 alpha are transcribed in seedling leaves. AB - A cDNA and a genomic DNA library from soybean (Glycine max L.) were used to identify and sequence two genes coding for the alpha-subunit of the translation elongation factor eEF-1. Within the coding part, the two genes (tefS1 and tefS2) diverge in 80 wobble positions thus yielding an identical protein composed of 447 amino acids. The soybean protein has about 95% similarity with eEF-1 alpha proteins of Arabidopsis thaliana and tomato. Both genes S1 and S2 contain, within the coding part at a site seemingly unique to higher plants, a single short intron of 86 and 116 nucleotides, respectively. The untranslated leader part of both genes is interrupted by a large intron (partially sequenced). Genes S1 and S2 are transcribed in young leaves. cDNA and gene-specific oligonucleotide probes interact with a unique transcript of close to 1.9 kb. Northern hybridization studies using RNAs from dark- and light-grown seedlings show that light sharply increases the level of stable transcripts (1.9 kb). A peak value is measured after about 3 h of illumination, afterwards the transcript concentration drops to about 10% of the peak value. Genes S1 and S2 follow a similar transcription pattern in developing seedling leaves, which is distinct from that of the rbcS genes measured in parallel experiments. According to northern results, S1 transcripts are more abundant in leaves at all measured stages of development than S2 transcripts. PMID- 1715784 TI - Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. AB - A highly efficient and synchronous in vitro tuberization system is described. One node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for both in vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz- and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud. These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development. PMID- 1715785 TI - Differential regulation in tobacco cell suspensions of genes involved in plant bacteria interactions by pathogen-related signals. AB - Six cDNA clones whose corresponding mRNAs accumulate early during the hypersensitive reaction in tobacco leaves have been classified into 2 groups according to their maximum levels of accumulation in an incompatible versus a compatible interaction with Pseudomonas solanacearum. We present evidence that, at least in the first stages of the interaction, tobacco cell suspensions retain the ability to respond differentially to compatible and incompatible isolates of P. solanacearum. In addition, studies on the effect of a fungal elicitor on the accumulation of the mRNAs corresponding to the cDNA clones in cell suspensions indicate that only one group of genes responds to this treatment. PMID- 1715786 TI - Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants. AB - An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation. PMID- 1715788 TI - Use of keratin antibodies in tumor diagnosis. AB - The tissue specific expression shown by intermediate filament proteins, their ease of extraction and their antigenicity has led to the use intermediate filament antibodies in diagnostic pathology, particularly antibodies to keratin intermediate filaments because of the predominant involvement of epithelia in cancers. We review the principles of differentiation-specific keratin expression and detail 100 of the best-known monoclonal antibodies to keratins which are currently in use: in many circumstances antibodies can now be reliably used as in situ markers of keratin expression. We also suggest a database scheme for better dissemination of information on available monoclonal antibodies to keratins in the future. PMID- 1715787 TI - Wound-regulated accumulation of specific transcripts in tomato fruit: interactions with fruit development, ethylene and light. AB - Regulation of three cDNA clones (pT52, pT53, and pT58) was analyzed in terms of wounding alone and wounding in conjunction with developmental and environmental cues (ripening, ethylene, and light) in tomato fruit tissue. The pT52-specific transcript level is induced by wounding in early-red and red stage fruit and by ethylene. The pT58-specific transcript level is also induced by wounding and ethylene in early-red stage fruit but is not induced by wounding in red fruit. The pT53-specific transcript level is repressed by wounding in early-red and red stage fruit. Like the pT52- and pT58-specific transcripts, the pT53-specific transcript is induced by ethylene. Furthermore, the level of the pT52-specific transcript is regulated by light. Analysis of unwounded tissue showed that the abundance of each cDNA-specific transcript changes during fruit ripening and that each of the transcripts is present in other plant organs as well. This analysis provides information about the interactions between developmental and environmental factors affecting these genes. PMID- 1715789 TI - Isolation of c-kit receptor-expressing cells from bone marrow, peripheral blood, and fetal liver: functional properties and composite antigenic profile. AB - To define the cellular targets for c-kit ligand (KL) and to study their functional properties and composite antigenic profile, we isolated cells expressing c-kit receptor (KR) from bone marrow (BM), peripheral blood, and fetal liver (FL) using immunoadherence to a recently obtained antibody (SR-1) against the human KR. Cells isolated by this approach (designated SR-1Ad) have the morphology of blasts and represent 1% to 4% of the original BM or FL populations. SR-1Ad cells from either source are highly enriched in progenitors (12% to 73%) and respond to KL in distinct patterns. In SR-1Ad cells from BM, the greatest impact of KL stimulation is on burst-forming units-erythroid (BFU-E), whereas in SR1-Ad cells from FL, the most significant KL effect is on a mixed erythroid/nonerythroid progenitor (erythroid/macrophage, colony-forming unit-mix [CFU-Mix]). When antibody SR-1 is continually present in culture, it neutralizes the effects of added KL. Furthermore, in the absence of added KL, it greatly diminishes the erythropoietin- and interleukin-3-dependent BFU-E growth in BM; whereas in FL, a wider spectrum of inhibition is observed, with CFU-Mix most severely curtailed. SR-1Ad cells coexpress other progenitor-associated antigens in a combination reflecting the dominant presence of erythroid progenitors (high expression of CD34, DR, CD38, and Ep-1; low expression of CD33). Several cytoadhesion molecules, ie, alpha L/beta 2 and alpha 4/beta 1 integrins, and intercellular adhesion molecule 1 and homing cell adhesion molecule 1, are also coexpressed. Our data provide new information on the isolation and characterization of KR expressing cells from normal, adult, and fetal hematopoietic tissues. On these biologically relevant target cells, the impact of ligand-induced stimulation or antibody-mediated ablation of KR function has been gauged. PMID- 1715790 TI - Identification of the c-kit ligand: end of the road for understanding aplastic anemia in steel mutant mice? AB - We here report the initiation of hematopoietic recovery in congenitally hypoplastic S1/S1d mice by the cytotoxic ablation of cells bearing the natural killer (NK) phenotype (NK 1.1+). The most striking finding was the early several fold increase in the cycling fraction of stem and progenitor cells (with the exception of progenitors committed to megakaryocytopoiesis) in the anti-NK 1.1+ antibody-treated group. This increase resulted in an early, complete restoration of total marrow cellularity to the normal (+/+) littermate level. Our data suggest that NK 1.1+ cells exert functions critical to the negative control of hematopoietic cell proliferation in S1/S1d mice. PMID- 1715791 TI - Suppression of chronic myelogenous leukemia colony growth by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activity. AB - In this study, we investigated the role of interleukin-1 beta (IL-1 beta) in the malignant evolution of chronic myelogenous leukemia (CML) and the functional activity of IL-1 inhibitors. Bone marrow (BM) and peripheral blood (PB) low density cells from 38 CML patients were studied in the colony-forming unit granulocyte, erythrocyte, monocyte, megakaryocyte colony culture assay. Samples from patients with early stage, interferon-alpha (IFN)-sensitive disease formed hematopoietic colonies in the presence of fetal calf serum (FCS), erythropoietin (Epo), and one of the following: granulocyte-macrophage colony-stimulating factor (10 ng/mL), IL-3 (15 ng/mL), both, or phytohemagglutinin-conditioned medium. The addition of IL-1 beta augmented IFN-sensitive CML colony growth in a dose dependent manner at concentrations of 10 to 100 U/mL. In sharp contrast, addition of the above growth factors did not augment the colony growth-promoting effect of FCS and Epo in samples from IFN-resistant patients; further, adherent cell fractionation or T-lymphocyte depletion attenuated the "autonomous" colony growth. Lysates of 2.5 x 10(7) low-density cells from each of six IFN-resistant and six IFN-sensitive CML patients and three normal volunteers were tested for intrinsic IL-1 beta content in an enzyme-linked immunosorbent assay and yielded a mean of 610 pg, 54.6 pg, and 49.4 pg of IL-1 beta, respectively (P less than .045). Interestingly, both soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) at concentrations of 5 to 100 ng/mL (sIL-1R) and 10 to 500 ng/mL (IL-1RA) inhibited CML colony growth in a dose-dependent fashion, with maximal inhibition of 64% and 65%, respectively. A similar effect was noted with the use of anti-IL-1 beta neutralizing antibodies. These data implicate IL-1 beta in CML disease progression and suggest that the inhibitory effects of molecules such as sIL-1R and IL-1RA could conceivably be the basis of a novel therapeutic strategy against this disorder. PMID- 1715792 TI - Autoantibody-associated cross-reactive idiotype-bearing human B lymphocytes: distribution and characterization, including Ig VH gene and CD5 antigen expression. AB - Monoclonal antibodies (MoAbs) specific for autoantibody associated cross-reactive idiotypes (CRIs) frequently recognize the Igs of neoplastic B cells in patients with chronic lymphocytic leukemia (CLL) and/or Waldenstrom's macroglobulinemia. Very little is known regarding the normal B cells expressing CRIs (CRI-positive B cells). Using a variety of MoAbs against CRIs we investigated the distribution and topographic localization of CRI-positive B cells in normal adult human lymphoid tissues. We found that CRI-positive B cells represent a significant B cell subpopulation expressing surface IgM (greater than 90%), IgG (approximately 5%), or IgA (approximately 2%). CRI-positive B cells are homogeneously distributed throughout all lymphoid tissues, accounting for 10% to 15% of all B lymphocytes, with the exception of the thymus, in which they represent the predominant B cell population. Immunophenotypic studies showed (1) that a small subpopulation (3.7% +/- 0.8%) of CRI-positive B cells are activated in vivo, based on CD25 and CD38 antigen expression; and (2) that approximately 50% of CRI positive B cells express the 67-Kd pan-T-lymphocyte CD5 antigen, suggesting that the CRI-positive B-cell subset and the recently described CD5-positive B-cell subset are closely related. This hypothesis is supported by the fact that CRI positive B cells produce oligo or polyreactive Igs, which are a characteristic feature of CD5-positive B cells, and also by the fact that both B-cell subpopulations appear to use similar and restricted Ig VH gene family members. PMID- 1715793 TI - Alpha-thalassemia resulting from deletion of regulatory sequences far upstream of the alpha-globin structural genes. AB - We describe an alpha-thalassemia determinant in which alpha-globin expression is silenced by a deletion located 27 kb 5' to the transcription start site of the alpha 2-globin gene. This alpha-thalassemic determinant, (alpha alpha)MM, is a member of a newly described group of thalassemic mutations resulting from deletion of locus-controlling sequences critical to globin gene expression. PMID- 1715794 TI - Inhibition by galanin and by high K+ of human basophil histamine release triggered by calcium ionophores but not responses induced by anti-IgE, chemotactic peptide or phorbol ester. AB - 1. Galanin concentration-dependently blocked human leukocyte histamine release triggered by the calcium ionophores A23187 and ionomycin. Almost complete inhibition of release was recorded at 410 nM whereas 41 nM galanin mediated close to 50% inhibition of responses. 2. Pretreatment of the cells with pertussis toxin did not influence the inhibitory effects of galanin. 3. Leukocyte responses triggered by anti-IgE, the chemotactic peptide formyl-methionyl-leucyl phenylalanine (FMLP) or 4 beta-phorbol 12-myristate 13-acetate (PMA) were not affected by galanin. 4. Ionophore-induced basophil histamine release, but not responses triggered by anti-IgE, FMLP or PMA, was inhibited when cells were challenged in medium containing high potassium (120mM). 5. We conclude that galanin and a depolarizing medium selectively inhibit ionophore-induced basophil histamine release. PMID- 1715795 TI - Naloxone-induced cardiovascular depression in rats that had received chronic morphine-treatment. AB - 1. Cardiovascular changes in response to intravenous injection of naloxone were studied in pentobarbitone-anaesthetized rats which had been given morphine in their drinking water for 1-21 days. The mechanisms of the observed changes were investigated in intact animals and in isolated hearts and tail arteries. 2. In rats that had received chronic morphine-treatment, intravenous administration of naloxone caused immediate decreases in blood pressure, heart rate, left ventricular pressure and dLVP/dtmax which were followed by the occurrence of atrial or ventricular extrasystoles and other signs of opiate withdrawal such as faecal passage and muscle twitching. 3. The intensities of the naloxone precipitated cardiovascular changes were directly related to the duration of chronic morphine pretreatment, reaching statistically significant levels on day 2 or 3 and maximal levels on day 7 or 14. This phenomenon disappeared on days 3 to 14 following opiate withdrawal in animals which had been treated previously with morphine for 21 days. 4. Either atropine or clonidine pretreatment significantly prevented the occurrence of faecal passage or muscle twitching during naloxone precipitated opiate withdrawal. However, clonidine, but not atropine or yohimbine, abolished the decreases in various haemodynamic parameters. The occurrence of cardiac extrasystoles was not affected. 5. In isolated heart or tail artery preparations from chronically morphine-treated rats, naloxone administration did not elicit reactions which differed from those of the preparations from naive animals. These findings suggest that under pentobarbitone anaesthesia, the cardiovascular systems of rats that had received chronic morphine treatment exhibit inhibitory, instead of excitatory, reactions to naloxone-precipitated opiate withdrawal. PMID- 1715796 TI - A comparison of the effects of angiotensin II and Bay K 8644 on responses to noradrenaline mediated via postjunctional alpha 1-and alpha 2-adrenoceptors in rabbit isolated blood vessels. AB - 1. The effects of angiotensin II (AII) and Bay K 8644 on responses to noradrenaline (NA) mediated via postjunctional alpha 1- and/or alpha 2 adrenoceptors have been compared in three isolated venous preparations from the rabbit, the lateral saphenous vein, the left renal vein and the ear vein. 2. A similar action of AII and Bay K 8644 was observed only in the lateral saphenous vein; each potentiated responses to NA after isolation of a homogeneous population of postjunctional alpha 2- adrenoceptors. However, even in this preparation the mechanism of action for these agents was not identical. The sensitivity of KCl-induced contraction to changes in extracellular calcium ions (reflecting activation of voltage-dependent Ca2+ channels) was enhanced by Bay K 8644 but reduced by AII. 3. All produced a selective facilitation of responses mediated via postjunctional alpha 2-adrenoceptors. In the lateral saphenous vein it reduced the effectiveness of prazosin and facilitated responses after isolation of alpha 2-adrenoceptors with phenoxybenzamine and rauwolscine. It directly enhanced responses to NA in the ear vein, where only alpha 2 adrenoceptors are involved. In contrast, AII did not influence responses mediated via postjunctional alpha 1-adrenoceptors in the left renal vein (even after the receptor reserve had been removed with phenoxybenzamine) nor the 'rauwolscine resistant' component of responses to NA in the saphenous vein. 4. Bay K 8644 enhanced contractile responses to NA mediated both via alpha 2-adrenoceptors, in the lateral saphenous vein, and via alpha 1-adrenoceptors in the left renal vein. Thus, unlike angiotensin II, no preferential effect was apparent. 5. Bay K 8644 was inactive against responses to NA in the rabbit isolated ear vein. Since the sustained component of responses to NA in this preparation is dependent upon the influx of extracellular Ca2 , these observations suggest that the influx of Ca2+ stimulated by NA is mediated via receptor-operated (1,4-dihydropyridine resistant) Ca2 + channels. PMID- 1715797 TI - Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission. AB - 1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L 659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA. The response to bombesin was not affected by spantide, L-659,877 or MEN 10,376. 7 P2. purinoceptor desensitization by repeated administration of alpha,betal methylene ATP depressed the twitch response to electrical stimulation of postganglionic nerves but did not affect the peak or the late response to capsaicin. 8. We conclude that multiple TKs are coreleased by capsaicin in the rat bladder and mediate the capsaicin-induced contraction by activating both NKI and NK2 receptors. Endogenous TK with preferential affinity for the NK, receptor (putatively SP) are selectively involved in the peak response to capsaicin while endogenous TK with preferential affinity for the NK2 receptor (putatively NKA) are selectively involved in the late response to capsaicin and partly contribute to the peak response. These findings provide pharmacological evidence for tachykinin-mediated cotransmission in the rat urinary bladder. ATP is unlikely to be involved in the efferent function of capsaicin-sensitive sensory nerves in the rat bladder. PMID- 1715798 TI - Stage I seminoma of the testis. Adjuvant radiotherapy or surveillance? AB - Lately the role of radiotherapy in stage I seminoma of the testis has been questioned by some authors who reported on a "surveillance" strategy for these patients. Since 1980, 124 patients with seminoma of the testis have been referred to this institution; 97 of 116 patients analysed presented with stage I disease and 10 of these had elevated levels of beta HCG. A total of 64 patients were given radiotherapy after orchiectomy and 33 entered a surveillance protocol. After a median follow-up of 48 months, 3 patients in the surveillance group relapsed after 5, 13 and 49 months and 2 of the irradiated patients did so after 25 and 33 months. Elevation of beta HCG was not significant because none of these patients showed progression. A low rate of progression and excellent survival are associated with standard treatment (orchiectomy and radiotherapy) and good results have been achieved with chemotherapy in cases of relapse. A surveillance policy is not recommended in stage I seminoma because of its slower growth compared with non-seminomatous germ cell tumours (NSGCT), the absence of a specific tumour marker, the 10% risk of occult metastases and the 3-fold higher progression rate compared with irradiated patients. We suggest the use of a reduced dosage and radiation field. PMID- 1715799 TI - Data, statistics, and theory: a comment on Bates, McDonald, MacWhinney, and Applebaum's "A maximum likelihood procedure for the analysis of group and individual data in aphasia research". PMID- 1715800 TI - Nature of spelling errors in a Thai conduction aphasic. AB - A Thai conduction aphasic's performance on a written confrontation naming task is reported. Analysis of his spelling errors indicated that errors rarely violated Thai phonotactic constraints; consonant substitutions were phonologically similar to the target stimuli; longer stimuli were more likely to be in error; distribution of errors was the same across consonants, vowels, and tones; and distribution of error types varied between segmentals (consonants, vowels) and suprasegmentals (tones). Error patterns were similar to those observed in oral reading and repetition. The pattern of impaired writing performance is discussed in relation to a functional model of the spelling process, and it is hypothesized to reflect primarily a functional lesion to the phonological buffer. PMID- 1715801 TI - Elemental composition and water content of myelinated axons and glial cells in rat central nervous system. AB - The distribution of elements (e.g. Na, Cl, K) and water in CNS cells is unknown. Therefore, electron probe X-ray microanalysis (EPMA) was used to measure water content and concentrations (mmol/kg dry or wet weight) of Na, Mg, P, S, Cl, K and Ca in morphological compartments of myelinated axons and glial cells from rat optic nerve and cervical spinal cord white matter. Axons in both CNS regions exhibited similar water content (approximately 90%), and relatively high concentrations (wet and dry weight) of K with low Na and Ca levels. The K content of axons was related to diameter, i.e. small axons in spinal cord and optic nerve had significantly less (25-50%) K than larger diameter axons from the same CNS region. The elemental composition of spinal cord mitochondria was similar to corresponding axoplasm, whereas the water content (75%) of these organelles was substantially lower than that of axoplasm. In glial cell cytoplasm of both CNS areas, P and K (wet and dry weight) were the most abundant elements and water content was approximately 75%. CNS myelin had predominantly high P levels and the lowest water content (33-55%) of any compartment measured. The results of this study demonstrate that each morphological compartment of CNS axons and glia exhibits a characteristic elemental composition and water content which might be related to the structure and function of that neuronal region. PMID- 1715802 TI - Is nerve growth factor required for the survival of retinal ganglion cells during development? AB - Staining for nerve growth factor receptor was observed in the ferret's retinal ganglion cell layer, optic nerve and tract, and in the lateral geniculate nucleus and superficial layers of the superior colliculus in the prenatal period, but had disappeared by birth. Thus the incidence of this transient staining does not correspond with the ganglion cell death that is known to occur in the ferret retina during the first postnatal week. PMID- 1715803 TI - Interactions between serotonin and substance P in the spinal regulation of nociception. AB - Interactions between 5-hydroxytryptamine (5-HT) and substance P (SP) in the mouse spinal cord were investigated using the tail-flick test and the behavioral response evoked by intrathecal (i.th.) SP or i.th. 5-HT. I.th. injection of 5-HT (20 micrograms) or the 5-HT1 receptor agonists (+)-8-hydroxy-2-(di-n propylamino)tetralin ((+)-8-OH-DPAT) (20 micrograms) or 5-methoxy-3(1,2,3,6 tetrahydropyridine-4-yl)-1H-indole (RU 24969) (20 micrograms) markedly inhibited the tail-flick reflex. The effect of these compounds was reduced when SP (5 micrograms) was given i.th. 55 min, or 55 and 45 min before the agonists. The tail-flick latencies recorded 5 min before injection of a 5-HT receptor agonist were similar in animals treated with SP or vehicle. The changes in the tail-flick test were not due to changes in tail skin temperature since only minimal differences in the skin temperature were recorded between the groups injected with SP or vehicle. I.th. injection of SP (10 ng) or 5-HT (2 micrograms) produced a similar behavioral response consisting of biting, licking and scratching of the caudal part of the body, indicative of nociceptive stimulation. The responses both to i.th. SP and 5-HT were reduced after i.th. application of the SP receptor antagonist [D-Arg1,D-Trp7.9,Leu11]-SP (Spantide) (5 micrograms), as well as 5 min after i.th. injection of the 5-HT receptor antagonist metergoline (4 micrograms). The data may indicate functional interactions between SP and 5-HT in the mouse spinal cord, which may take place in neurons involved in the processing of nociception. PMID- 1715804 TI - Dopaminergic and serotonergic activity in the hypothalamus during early and late pregnancy. AB - Push-pull cannulae were implanted into the arcuate nucleus of pregnant or ovariectomized (OVX) rats, and the perfusate samples were analyzed for biogenic amines and their metabolites. Injection of pargyline, a monoamine oxidase inhibitor, resulted in a decrease in 3,4-dihydroxyphenylacetic acid (DOPAC) and 5 hydroxyindoleacetic acid (5-HIAA) levels in the samples, and detectable amounts of dopamine (DA) and serotonin (5-HT), which were usually not measurable prior to injection. Administration of 5-hydroxytryptophan resulted in a sharp increase in 5-HIAA and 5-HT levels in the perfusates, and no change in DOPAC levels. Push pull perfusion was done between midnight and 06.00 h on day 8 and 16 of pregnancy. In those rats which showed a nocturnal prolactin (PRL) surge on day 8, 5-HIAA levels were very high compared to those that did not, or compared to those on day 16, which had chronic low PRL levels. DOPAC levels were not significantly different in the 3 groups. Perfusion of medial basal hypothalamic (MBH) fragments taken from mother rats on day 8 of pregnancy during the PRL surge spontaneously released more DA or 5-HT than did fragments taken on day 16 at the same time of day. These results suggest that serotonergic activity in the MBH is higher on day 8 of pregnancy, in parallel with the occurrence of PRL surges, than on day 16 when no surges are present. Dopaminergic activity, as measured by DOPAC levels in push-pull samples, does not appear to be different between the two days. PMID- 1715805 TI - Oleic acid reversibly opens the blood-brain barrier. AB - This study examined the effect of intracarotid oleic acid infusion on blood-brain barrier permeability. Oleic acid was infused for 30 s at a rate of 6 ml/min into the right internal carotid artery at concentrations of 10(-6), 10(-5), 2 x 10(-5) and 5 10(-5) M. Extensive Evans blue-albumin extravasation was observed 15 min after the administration of 2 x 10(-5) M oleic acid. The permeability surface area product for alpha-aminoisobutyric acid (AIB), determined 1-11 min following the infusion of oleic acid was increased 10-fold following infusion of 10(-5) M oleic acid and 20-fold following the administration of 5 x 10(-5) M oleate. The blood-brain barrier opening to AIB proved to be reversible 80-90 min after the infusion of 2 x 10(-5) M oleic acid. The possible mechanisms of the oleic acid effect are discussed. PMID- 1715806 TI - Relationship of Met-enkephalin-like immunoreactivity to vagal afferents and motor dendrites in the nucleus of the solitary tract: a light and electron microscopic dual labeling study. AB - Methionine (Met5)-enkephalin has been implicated in autonomic functions involving vagal reflexes within the nucleus of the solitary tract (NTS). We examined the light and electron microscopic relationships between neurons containing methionine (Met5)-enkephalin-like immunoreactivity (MELI) and vagal afferents and motor dendrites in the rat NTS. A polyclonal antibody raised against Met5 enkephalin and showing maximal cross-reactivity with this peptide was localized by immunoautoradiography. In the same sections, vagal afferents and motor neurons were identified by histochemical detection of anterogradely and retrogradely transported horseradish peroxidase (HRP). By light microscopy, the MELI was detected in perikarya distributed principally in the dorsomedial, intermediate and parasolitary subdivisions of the NTS. These subnuclei as well as medial and commissural divisions of the NTS also showed: (1) aggregates of silver grains thought to overlie terminals containing MELI, and (2) anterogradely transported HRP in varicose processes. Electron microscopic analysis of the dorsomedial NTS at the level of the area postrema established that MELI was detectable in perikarya, dendrites, and axon terminals. Most of the MELI was associated with large dense core vesicles (dcvs). These opioid terminals formed primarily symmetric synapses on proximal and asymmetric synapses on distal dendrites. Analysis of the dendritic targets of terminals containing MELI revealed that 13/222 were in synaptic contact with dendrites also containing MELI. The remainder of the terminals containing MELI either lacked recognized junctions or formed synapses with unlabeled dendrites. In comparison to the terminals containing MELI in the same series of sections, anterogradely labeled vagal terminals extensively formed asymmetric junctions with distal dendrites and spines. Of the observed anterogradely labeled terminals 6/84 formed synapses with dendrites containing MELI and 3/84 with dendrites containing retrogradely transported HRP. The remainder of the junctions were with dendrites lacking detectable immunoautoradiographic or HRP-labeling. The majority of the recognized synapses on labeled dendrites were at more proximal sites possibly reflecting more limited detection of both MELI and retrogradely transported HRP in smaller dendrites. However, the presence of even a few junctions at proximal sites on dendrites where synaptic transmission is known to be more effective suggests a potentially strong modulation of both opioid and vagal motor neurons by visceral afferents in the NTS. In addition to forming synapses on dendrites, both vagal afferents and terminals containing MELI showed frequent synaptic associations with unlabeled terminals, but not with each other. This finding suggests that the previously demonstrated opiate binding sites on vagal afferents is most likely attributed to other endogenous opiates. PMID- 1715807 TI - Survival and regeneration of the adult rat vagus nerve in culture. AB - Here I report that the vagus nerve from the adult rat survives in culture for several days. The cultured preparation retains its ability to propagate action potentials and transport proteins within its sensory axons. Regeneration could be induced by a crush lesion and nerve fibers regenerated at a rate of 1.4 mm/day. The production of proteins, later subjected to retrograde axonal transport, and outgrowth of neurites could be prevented by inhibition of protein synthesis at the site of the crush lesion. PMID- 1715808 TI - [The Wolf-Parkinson-White syndrome and peroperative arrhythmias]. AB - The influence of anaesthetic drugs or techniques on the development of arrhythmias in patients with the Wolff-Parkinson-White syndrome still remains controversial. Most of the literature is based on single case reports from which no clear-cut attitude can be defined. In this retrospective study we tried to evaluate if a particular drug or technique could be recommended. We also discuss on the basis of electrophysiology of the Wolff-Parkinson-White syndrome situations in which the potential for arrhythmias is enhanced. PMID- 1715809 TI - Chemotherapy of head louse (Pediculus humanus capitis) infestation gamma benzene hexachloride (gamma-BHC) among school children in Szu-Hu District, Yunlin County, Central West Taiwan. AB - In order to evaluate the effectiveness of 1% gamma-BHC emulsion against head louse infestation, 1,527 school children were examined using observation by ocular and combing methods were used and infested children were treated with three regimes of 1% gamma-BHC emulsion. An overall infestation rate of 40% was found. The infestation rate was highest in Tung-Kuang Primary School (59%) and lowest in Chien-Hua Primary Schools (7%). The rate was highest among school children grade 2 (45%) and lowest in grade 3 (35%). The rate of girls (65%) was much higher than that of boys (9%). A total of 443 lice were collected from 78 infested school girls: 56 males, 59 females, and 328 nymph. The average number of head lice in each infested girl was 5.7. Follow-up examination was conducted one week after treatment. The cure rates for dosages of 10.0 ml, 5.0 ml, and 2.5 ml 1% gamma-BHC emulsion were 96%, 88%, 68% for girls and 100%, 92%, and 33% for boys, respectively. Only mild and transient itching and burn sensation of scalp were reported by a few children. The overall infestation rate 5 months (April September 1981) after treatment was 23% (286/1,245). The rate of girls decreased from 65% to 40% and that of boys from 9% to 3%. Results of the present study indicates that 1% gamma-BHC emulsion is an effective pediculicide at a dosage of 5 ml or 10 ml. However, the overall infestation rate remained high (23%) 5 months after treatment. These findings suggest that treatment of head louse infestation must be conducted continuously. PMID- 1715810 TI - Monoclonal antibodies against pili of serologically distinct Bacteroides nodosus. AB - Several monoclonal antibodies (McAbs) against pili of Bacteroides nodosus were examined to determine their reactivity with 11 different serotypes. One McAb was identified by enzyme-linked immunosorbent assay (ELISA) analysis that bound to nine of the 11 serotypes and another that bound to the remaining two serotypes tested. In addition, some McAbs demonstrated specificity for a single serotype, while others displayed specificities for up to five other serotypes. Comparison of immunoblot analysis with the ELISA revealed that the former method was not as sensitive in that all McAbs positive by the ELISA, were not positive by immunoblot. Possible explanations of these findings are discussed. There appear to be several antigenic determinants on B. nodosus pili and considerable sharing of these determinants between pili types. The 11 serotypes analyzed by the McAbs in this report are representative of all 20 US serotypes as well as the A-set and D-set categories of Australia. Therefore, the two epitopes recognized by two of the McAbs reported herein encompass all of the currently characterized B. nodosus serotypes and may provide a basis for bivalent vaccines efficacious for all types of B. nodosus induced footrot in sheep. PMID- 1715811 TI - Basic aspects of cystic fibrosis. PMID- 1715812 TI - Long-term treatment with hepatic tumor promoters inhibits mitogenic responses of hepatocytes to acidic fibroblast growth factor and hepatocyte growth factor. AB - Studies with hepatocyte cultures have defined four hepatocyte mitogens which can transmit a complete mitogenic signal in cultures kept in completely defined conditions. These four mitogens are epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), hepatopoietin A/hepatocyte growth factor (HPTA/HGF) and hepatopoietin B (HPTB). In this study, we investigated the effect of aFGF, HGF and the mito-inhibitor transforming growth factor beta (TGF-beta) on cultured hepatocytes isolated from livers of rats treated with the xenobiotic hepatic tumor promoters phenobarbital (PB) and alpha-hexachlorocyclohexane (alpha HCH). Male F344 rats were treated with each of these two xenobiotics to stimulate hepatic DNA synthesis and augmentative hepatomegaly. At different times on the regimens with tumor promoters, hepatocytes were isolated and placed in primary culture. DNA synthesis of hepatocytes in culture stimulated by these two growth factors and the suppression of DNA synthesis affected by TGF-beta were examined as a function of time of treatment in vivo with these two promoters. Following day 10, hepatocytes from both promoter regimens became unresponsive to these two growth factors for the rest of the duration of the treatment (day 90). TGF-beta suppressed DNA synthesis stimulated by growth factors but did not affect the high background DNA synthesis stimulated by xenobiotics themselves. PMID- 1715813 TI - Postischaemic reperfusion injury in the isolated rat heart: effect of ruthenium red. AB - STUDY OBJECTIVE: The aim was to investigate the effect of attenuating mitochondrial calcium uptake with ruthenium red on myocardial function and the resultant necrosis following prolonged ischaemia and reperfusion in isolated rat hearts. Mitochondrial dysfunction, secondary to increased calcium uptake, has been implicated as an important mediator of reperfusion injury in the heart. DESIGN: To examine the role of mitochondrial calcium uptake in mediating ischaemic and reperfusion injury, isolated rat hearts were perfused with ruthenium red (n = 6), a polysaccharide dye which inhibits calcium uptake by mitochondria, and were compared to control perfused hearts (n = 7). After stabilisation, hearts were subjected to 60 min no flow ischaemia, immediately followed by 40 min reperfusion. EXPERIMENTAL MATERIAL: Hearts were used from male Wistar rats weighing 300-350 g. MEASUREMENTS AND MAIN RESULTS: Cardiac high energy phosphates (ATP, phosphocreatine, inorganic phosphate) and pH were continuously monitored during ischaemia and reperfusion using phosphorus magnetic resonance spectroscopy. Contractility (dP/dT), coronary flow, creatine kinase release, and the time to the onset of ischaemic contracture were also measured. No differences in metabolic abnormalities or time to peak contraction during ischaemia were found between groups, suggesting that ruthenium red does not alter the metabolic consequences of ischaemia. However, upon reperfusion, the following differences in the ruthenium red perfused hearts were observed when compared to control hearts (p less than 0.05): ATP and phosphocreatine recovery were more complete, myocardial contractility was greater, coronary flow was greater, and myocyte necrosis was attenuated. CONCLUSIONS: Combined with the known inhibitory effect of ruthenium red on mitochondrial calcium uptake, these data suggest that an important component of myocardial injury following ischaemia and reperfusion in the isolated rat heart is the result of mitochondrial calcium accumulation. PMID- 1715814 TI - Spectra of intracellular Fura-2. AB - In the theory of measurement of calcium ion activity by determination of Fura-2 fluorescence at two excitation wavelengths, the accuracy of the result depends upon the accuracy both of the sample measurements and of the calibration measurements which are made on calcium-bound and free dye. Two factors underlie adequate calibration and accuracy. The first is the elimination of systematic error due to spectral shifts arising from the intracellular environment felt by the dye. To this end, detailed comparisons between complete spectra of both calcium-bound and calcium-free Fura-2 can be used to help separate spectral effects due to light absorption by cellular constituents versus polarity and viscosity of the intracellular milieu. The second major factor which determines accuracy is the experimental uncertainty (in both sample and calibration measurements). For samples in which the ratio of bound to free dye is large, the uncertainty in the ratio is also large, even when it is expressed as a percentage of the ratio itself. The errors in calibration measurements impact on the accuracy of the method primarily through the measurements made at wavelengths which are off the spectral peaks of the bound or free dye, since these are the least accurate. In order to obtain a guide to the choice of wavelengths and estimation of the reliability of results, a mathematical expression is derived for the dependence of the accuracy of the method on the accuracy of both sample and calibration measurements. PMID- 1715815 TI - Diphenylamine-2-carboxylate (DPC) reduces calcium influx in a mouse mandibular cell line (ST885). AB - Non-selective cation channels are found in many diverse cell types and have been proposed as a potential entry path for Ca2+. ST885 cells contain large numbers of these channels which are active in the resting cell. We have used Fura-2 to monitor changes in intracellular free Ca2+ ([Ca2+]i) in response to step changes in extracellular Ca2+ ([Ca2+]o). We found that DPC, a blocker of the non selective cation channel in these cells, caused a reduction of approximately 50% in the rate of rise in [Ca2+]i following a step increase in [Ca2+]o. Since our experiments demonstrate that this phenomenon is not due to DPC blockade of Cl- channels, the Na+/Ca2+ exchanger or cyclooxygenase, we conclude that it is attributable to a direct effect of DPC on the non-selective cation channel. It thus appears that the non-selective cation channel is a significant pathway for basal Ca2+ entry in these cells. PMID- 1715816 TI - Cyclic AMP-mediated modulation of immunoglobulin production in B cells by prostaglandin E1. AB - We had previously demonstrated in a transformed human B cell line, LA350, the existence of an inverse relationship between cyclic adenosine monophosphate (cAMP) content and immunoglobulin secretion using the cAMP-elevating agents such as cholera toxin and forskolin. In this paper we report that cAMP acting as a second messenger for prostaglandin exerts a similar effect on the antibody response of B lymphocytes. Incubation of the cells with PGE1 in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) produced a concentration- and time-dependent elevation of intracellular cAMP. Significant increases of cAMP production were observed at physiologically relevant levels of PGE1 (10(-7) and 10(-8) M). Immunoglobulin production, whether measured as the total number of immunoglobulin-secreting cells by a reverse hemolytic plaque assay or as specific immunoglobulin production (IgM) by an enzyme-linked immunoadsorbent assay, was suppressed in a dose-dependent fashion by the presence of IBMX. This suppression of immunoglobulin production was significantly enhanced by the presence of PGE1. Phorbol myristate acetate-induced IgM production was also inhibited by the presence of PGE1. These results imply that prostaglandins regulate B cell activation and immunoglobulin production by signal transduction mechanisms involving cyclic nucleotides. PMID- 1715817 TI - Resuscitation of hypovolemic sheep with hypertonic saline/Dextran: the role of Dextran. AB - We evaluated the role Dextran-70 plays in small volume resuscitation of hemorrhage using hypertonic saline dextran solutions. Sheep were hemorrhaged (1.5 to 2 liters) over 2 hr to an arterial pressure of 50 mm Hg and were then resuscitated with 100 ml of either 7.5% NaCl (HS/0% Dex) alone, 7.5% NaCl/6% Dextran-70 (HS/6% Dex), or 7.5% NaCl/24% Dextran-70 (HS/24% Dex). During hemorrhage cardiac output was reduced to 40-50% of baseline levels. The major effect of the added dextran was a greater initial increase and more sustained normalization of plasma volume and cardiac output. At 15 min post-infusion, plasma volume was expanded 17 +/- 2% with HS/0% Dex; 27 +/- 2% with HS/6% Dex; and 56 +/- 8% with HS/24% Dex. A dose response effect of the added dextran was also apparent in the post-resuscitation increases in arterial pressure and cardiac output. At 3 min post-infusion, both variables improved with all solutions, but baseline levels were reached and exceeded only in the HS/24% Dex. The increased cardiac output correlated significantly with the degree of vascular expansion. Regression and extrapolation of the post-resuscitation data of plasma volume expansion and increased cardiac output to no volume expansion suggests that the hypertonic saline also augments cardiac output by an additional mechanism independent of volume. Our data show that the addition of dextran to hypertonic saline can play an important role in small volume hypertonic resuscitation by improving both the initial cardiovascular response and the sustainment of that response. Higher concentrations of dextran than currently used in hypertonic formulations may make small volume resuscitation more efficacious. PMID- 1715818 TI - Reentrant excitation around a fixed obstacle in uniform anisotropic ventricular myocardium. AB - BACKGROUND: The purpose of this study was to investigate the role of tissue anisotropy and dispersion of refractoriness on initiation of reentrant ventricular tachycardia (VT). METHODS AND RESULTS: A ring of perfused uniform anisotropic ventricular epicardium in Langendorff-perfused rabbit hearts was created by an endocardial freezing technique. High-resolution mapping (248 channels) was used to analyze epicardial activation of the left ventricle. One to three premature beats were induced at a total of 272 points in 17 experiments (16 different points around the ring in each experiment). Reentrant VT could be initiated at 43 of the 272 points tested. The cycle length of VT was stable and ranged from 128 to 198 msec (mean, 161 +/- 19 msec). Correlation between conduction velocity and the angle between the circulating activation wave and epicardial fiber orientation showed that in segments of the ring where conduction was perpendicular to the fiber axis, mean conduction velocity was 25 +/- 5 cm/sec compared with 60 +/- 7 cm/sec when conduction was parallel to the fiber orientation. Analysis of the site of unidirectional conduction block showed that in 41 of 43 cases, block occurred while the impulse was propagating parallel to the fiber orientation. Measurement of the refractory periods at either side of the line of unidirectional block showed that only in 12 of 43 cases did block occur while the impulse was propagating into an area with a longer (more than 10 msec) refractory period. CONCLUSIONS: In uniform anisotropic ventricular myocardium, reentrant VT is initiated because lowering the stimulating efficacy of the depolarization wave by premature beats leads to preferential conduction block parallel to the fiber orientation. PMID- 1715819 TI - Stage IIB osteogenic sarcoma. AB - Two hundred seventy-one consecutive patients treated from 1976 through 1986 were reviewed to estimate long-term survival. Disease-free survival for the entire cohort was 77% at five years and 74% at ten years. Humeral lesions had the best probability of survival (84% at ten years), followed by tibial lesions (81%) and femoral lesions (67%). Histologic response to preoperative chemotherapy was the strongest predictor of outcome. Those with little response had a survival estimate of 54% at ten years as compared to 68% for partial responders and approximately 90% for complete responders. Local recurrence was seen in 6.6% and was associated with an adverse effect on survival. Only two of the 18 patients with local recurrence have been rendered long-term disease-free survivors. PMID- 1715820 TI - Local control and survival from the Cooperative Osteosarcoma Study Group studies of the German Society of Pediatric Oncology and the Vienna Bone Tumor Registry. AB - The use of aggressive chemotherapy undoubtedly has brought about a dramatic increase in the cure rate of osteosarcoma. The authors' investigations have increased the authors' knowledge of chemotherapy for osteosarcoma, the differential efficacy of currently used agents, and the pronounced schedule dependency and relative route independency of their efficiency. The authors were able to confirm the prognostic significance of tumor response after preoperative chemotherapy. Preoperative chemotherapy in itself has facilitated and promoted limb-salvage surgery. Also, more patients can be cured today by use of aggressive thoracic surgery in case of primary or secondary pulmonary metastases. The authors' efforts to steadily increase metastasis-free survival rates by intensifying chemotherapy in this series of studies, however, have been only moderately successful. Still, chemotherapy-related acute toxicity is considerable and increases with aggressiveness of treatment, and the manifestations of late toxicity may continue to increase with follow-up time. Future trials should be targeted toward exploration of the minimum indispensable amount of toxic treatment yielding comparable or even better results than those currently attainable. PMID- 1715821 TI - [The expression of the protein product of the c-src proto-oncogene in human tumors]. PMID- 1715822 TI - [Single calcium channels from the membranes of the adrenal medullary substance]. PMID- 1715823 TI - Localization of dopamine and its relation to the growth hormone producing cells in the central nervous system of the snail Lymnaea stagnalis. AB - The distribution of dopamine in the central nervous system of the pond snail Lymnaea stagnalis was investigated by using immunocytochemistry and HPLC measurements. With both methods it was demonstrated that dopamine is predominantly present in the cerebral and pedal ganglia. The dopamine immunoreactivity was mainly observed in nerve-fibers in the neuropile of the ganglia. Relatively few dopamine-immunopositive cell bodies (diameters 10-30 microns) were found. A large cell in the right pedal ganglion (the so-called RPeD1) stained positively with the dopamine antibody. It has previously been demonstrated that the growth hormone producing cells (GHCs) possess dopamine receptors on their cell bodies. However, dopamine-immunopositive fibers were observed only in the vicinity of the GHC nerve-endings and not close to the GHC cell bodies. PMID- 1715824 TI - Detection of the release of 5-hydroxyindole compounds in the hypothalamus and the n. raphe dorsalis throughout the sleep-waking cycle and during stressful situations in the rat: a polygraphic and voltammetric approach. AB - In the present work, voltammetric method combined with polygraphic recordings were used in animals under long-term chronic conditions; the extracellular concentrations of 5-hydroxyindole compounds (5-OHles) and in particular 5 hydroxyindoleacetic acid (5-HIAA) were measured in the hypothalamus and in the nucleus Raphe Dorsalis (n.RD). The hypothesis that extracellular detection of 5 HIAA, in animals under physiological conditions, might reflect serotonin (5-HT) release is suggested by the following observations: serotoninergic neurons are reported to contain only monoamine oxidase type B (MAO-B);--an inhibitor of such an enzyme, MDL 72145 (1 mg/kg), fails to decrease the extracellular 5-HIAA peak 3 height:--MAO type A is contained in non-5-HT cells or neurons;--only the inhibitor of this last type of enzyme (Clorgyline 2.5 mg/kg) induces a complete disappearance of the voltammetric signal. The 5-HIAA measured in the extracellular space thus comes from the 5-HT released and metabolized outside the 5-HT neurons. Throughout the sleep-waking cycle, 5-OHles release occurs following two different modes: 1--during sleep, in the vicinity of the 5-HT cellular bodies in the n.RD; this release might come from dendrites and be responsible for the 5 HT neuronal inhibition occurring during sleep; 2--during waking, at the level of the axonal nerve endings impinging on the hypothalamus; this release might be related to the synthesis of "hypnogenic factors". Finally, we have observed that in the hypothalamus, 30 min. of immobilization-stress (IS) induces a larger increase of the voltammetric signal (+80%) than a painful stimulation of the same duration (+30%); the possible link between the 5-OHles release occurring in this area during an IS and the subsequent paradoxical sleep rebound is discussed. PMID- 1715825 TI - Location of saccade-related neurons in the macaque superior colliculus. AB - The locations of saccade-related neurons were studied in the superior colliculi of two adult rhesus monkeys (Macaca mulatta) by placing marking lesions at the sites of physiologically characterized cells and comparing these histologically identified sites with the collicular laminae and acetylcholinesterase (AChE)-rich patches. Three major conclusions were drawn on the basis of 39 histologically identified sites at which saccade-related neurons were recorded. First, saccade related neurons were distributed from the ventral half of the optic layer through the deep gray layer, and were most concentrated in the intermediate gray and white layers. Second, there was a clear relationship between the discharge characteristics of these saccade-related neurons and the depths at which they were found. Neurons having presaccadic bursts, defined as clipped and partially clipped, tended to be encountered more dorsally, and neurons that did not have bursts (unclipped) were encountered more ventrally. Although cells having different discharge characteristics seemed to be organized along a dorsoventral axis, there was no compelling evidence that these properties were specified by their laminar locations. Third, there was no clear correlation between the locations of saccade-related neurons and the distribution of individual AChE-rich patches. Saccade-related cells were found both in the caudal superior colliculus where patches were located and in the rostral superior colliculus where patches were not found; both within and between the two tiers of AChE-rich patches in the caudal superior colliculus; and both within and between individual AChE-rich patches. However, the depth-level at which saccade-related neurons occurred generally matched the region bounded by the two tiers of AChE-rich patches in the intermediate and deep layers, and the dorsal and ventral extent of saccade related neurons was the same as that of the AChE-rich patches. PMID- 1715826 TI - The contribution of GABA-ergic neurons to horizontal intrinsic connections in upper layers of the cat's striate cortex. AB - The contribution of neurons containing gamma-aminobutyric acid (GABA) to horizontal intrinsic projections in layers I-III of cat's striate cortex was investigated by combining GABA-immunohistochemistry with axonal tracing. After intracortical injections of Rhodamine-labelled latex microspheres Rhodamine labelled neurons form patch- or bandlike aggregations (clusters) separated from each other by regions containing fewer, evenly distributed or no labelled neurons. Of the Rhodamine-labelled neurons about 5% display GABA-immunoreactive material (double labelled = DL-neurons). Approximately 70% of the DL-neurons occur at distances of less than 1 mm, and the remaining 30% at distances between 1 mm and 2.5 mm from the injection. About 60% of the DL-neurons reside within clusters and 40% are located in regions between clusters; the respective percentages of the Rhodamine labelled GABA-negative neurons are about 85 and 15. Considering their small number and their spatial distribution inhibitory interneurons seem to make only minor contributions to the clustered pattern of intrinsic connections. Our results demonstrate that the topographical organization of neurons giving origin to lateral inhibitory interactions in upper layers of cat's striate cortex is different from that of neurons mediating excitatory functions. PMID- 1715827 TI - Topographical projections from the cerebral cortex to the nucleus of the solitary tract in the cat. AB - The distribution of cerebral cortical neurons sending projection fibers to the nucleus of the solitary tract (NST), and the topographical distribution of axon terminals of cortico-NST fibers within the NST were examined in the cat by two sets of experiments with horseradish peroxidase (HRP) and HRP conjugated with wheat germ agglutinin (WGA-HRP). First, HRP was injected into the NST. In the cerebral cortex of these cats, neuronal cell bodies were labeled retrogradely in the deep pyramidal cell layer (layer V): After HRP injection centered on the rostral or middle part of the NST, HRP-labeled neuronal cell bodies were distributed mainly in the orbital gyrus and caudal part of the infralimbic cortex, and additionally in the rostral part of the anterior sylvian gyrus. After HRP injection centered on the caudal part of the NST, labeled neuronal cell bodies were seen mainly in the caudoventral part of the infralimbic cortex, and additionally in the orbital gyrus, posterior sigmoid gyrus and rostral part of the anterior sylvian gyrus. The labeling in the infralimbic cortex, orbital gyrus and anterior sylvian gyrus was bilateral with a predominantly ipsilateral distribution, while that in the posterior sigmoid gyrus was bilateral with a clear-cut contralateral dominance. In the second set of experiments, WGA-HRP was injected into the cerebral cortical regions where neuronal cell bodies had been retrogradely labeled with HRP injected into the NST: After WGA-HRP injection into the orbital gyrus, presumed axon terminals in the NST were labeled in the rostral two thirds of the nucleus bilaterally with an ipsilateral predominance. After WGA HRP injection into the rostral part of the anterior sylvian gyrus, a moderate number of presumed axon terminals were labeled throughout the whole rostrocaudal extent of the NST bilaterally with a slight ipsilateral dominance. After WGA-HRP injection into the middle and caudal parts of the anterior sylvian gyrus, no labeling was found in the NST. After WGA-HRP injection into the caudal part of the infralimbic cortex, presumed terminal labeling in the NST was seen throughout the whole rostrocaudal extent of the nucleus bilaterally with a dominant ipsilateral distribution. After WGA-HRP injection into the posterior sigmoid gyrus, however, no terminal labeling was found in the NST. The results indicate that cortico-NST fibers from the orbital gyrus terminate in the rostral two thirds of the NST, while those from the infralimbic cortex and the rostral part of the anterior sylvian gyrus project to the whole rostrocaudal extent of the NST. PMID- 1715828 TI - The immunogold-silver staining procedure in the study of freshly suspended Langerhans cells at the transmission electron microscopic level. AB - The potential of an immunogold-silver staining for the study of human suspended Langerhans cells at the transmission electron microscopic level was evaluated. Cells were labeled, by using a preembedding technique, with 5-nm colloidal gold particles followed by silver enhancement. The use of small colloidal gold particles permits a detection of small quantities of antigen; the metallic silver deposition around gold granules gives rise to a large electron-dense marker which can be easily detected even at low magnification. Ultrastructural details were well preserved, and the background was not significant. The major advantage of the present immunogold-silver staining is that it enables to detect labeled cells easily, even when limited amounts of antigenic moieties are present on a low percentage of cells. Therefore, a rapid and simultaneous evaluation of both immunophenotype and ultrastructural details of investigated cells is allowed. PMID- 1715829 TI - Glycation of rat sciatic nerve tubulin in experimental diabetes mellitus. AB - Diabetic neuropathy is associated with some early defects of axonal transport in experimental animals. Axonal transport is dependent on intact microtubules, and unsubstituted lysine residues of tubulin are essential for microtubule polymerization. As lysine residues are the major target for the non-enzymatic attachment of glucose, the effect of diabetes on the extent of glycation of tubulin was investigated. There was a more than four-fold increase in the extent of glycation of tubulin in the sciatic nerve of rats with streptozotocin-induced diabetes of 2 weeks duration compared with control rats. In contrast, no such increase in glycation was observed in brain microtubule protein from diabetic rats at that stage of diabetes. Incubation of brain microtubule protein with glucose prior to in vitro polymerization showed that the early stages of glycation were not associated with inhibition of microtubule assembly. The observed glycation of peripheral nerve tubulin in early experimental diabetes may nevertheless contribute to axonal transport abnormalities through an as yet undetermined impairment of microtubule function. PMID- 1715830 TI - NCI-Black-Reiter (NBR) male rats fail to develop renal disease following exposure to agents that induce alpha-2u-globulin (alpha 2u) nephropathy. AB - The NCI-Black-Reiter (NBR) rat is the only strain of male rat known not to synthesize the hepatic form of the low molecular weight protein, alpha 2u globulin. In previous studies, NBR rats were shown not to develop renal disease when exposed to decalin, a compound known to induce alpha 2u-globulin nephropathy in other rat strains. The objective of this study was to show that the presence of alpha 2u-globulin (alpha 2u) is essential for the development of this syndrome in rats exposed to 2,2,4-trimethylpentane (TMP), 1,4-dichlorobenzene (DCB), isophorone (IP), PS-6 unleaded gasoline (UG), and d-limonene (d-L). The induction of alpha 2u-nephropathy in F344 male rats with lindane was used as a positive control and this response was contrasted to male NBR and female F344 rats treated with lindane. Five to seven 11-week-old male NBR rats were exposed to TMP (500 mg/kg/day), DCB (500 mg/kg/day), IP (1000 mg/kg/day), UG (500 mg/kg/day), d-L (1650 mg/kg/day), or lindane (10 mg/kg/day) and five 11-week-old male and female F344 rats were exposed to lindane (10 mg/kg/day) by oral gavage on 4 consecutive days. NBR male and F344 male and female rats gavaged with corn oil were incorporated in the study as vehicle controls. The presence of hyaline droplets was assessed in perfusion-fixed kidneys by staining paraffin sections with Mallory-Heidenhein stain and in GMA sections with Lee's methylene basic blue fuchsin stain. Paraffin sections were also analyzed immunohistochemically for the presence of alpha 2u. Under exposure conditions that clearly induce alpha 2u nephropathy in male F344 rats, no lesions, hyaline droplets, or alpha 2u were detectable in treated or control male NBR and female F344 rats. It is thus concluded that the presence of alpha 2u is causal to the development of renal disease in rats exposed to TMP, DCB, IP, UG, d-L, and lindane. PMID- 1715831 TI - Effects of subchronic exposure of rats to 2-methoxyethanol or 2-butoxyethanol: thymic atrophy and immunotoxicity. AB - Male Sprague-Dawley rats were exposed to either 2000 or 6000 ppm of 2 methoxyethanol (ME) or 2-butoxyethanol (BE) and females were exposed to either 1600 or 4800 ppm of these compounds in the drinking water for 21 days. Body weights were decreased in male rats exposed to the high doses of both chemicals, while body weights of females exposed to either dose of BE were decreased. Male and female rats exposed to either concentration of ME had a dose-related reduction in thymus weights. Testis weight was significantly lower in male rats exposed to the high dose of ME. Dose-related increases in natural killer (NK) cell cytotoxic activities and decreases in specific antibody production were observed in all rats treated with ME. Rats exposed to the low dose of BE also had enhanced NK cell activity. Splenocyte production of interferon-gamma was decreased in male rats exposed to either dose of ME and in females treated with the high dose of ME. Spleen cell numbers were reduced in males exposed to the high dose of ME and females given either dose of ME. It appears that the immune system is a sensitive target of ME but not BE. The effects of ME on immune function differ depending on the immune parameter assessed. Enhanced NK cell activity may partially explain the observations of others that certain glycol ethers have antitumor effects in vivo. PMID- 1715832 TI - Hepatitis C virus infection and antiviral treatment. PMID- 1715833 TI - Therapeutic efficacy of interferon on HCV-RNA in chronic hepatitis C. AB - The efficacy of interferon therapy for chronic hepatitis C were evaluated by the changes of serum RNA of the hepatitis C virus (HCV) by the polymerase chain reaction. Before the treatment, HCV-RNA was detected in all of the 17 patients. 5 patients given IFN for 4 weeks, 12 cases given IFN by the 2 weeks/on 2 weeks off schedule (4 cases given 4 cycles and 8 given 6 cycles). Immediately after the treatment ended, the HCV-RNA was not detected in 12 (71%) cases. Five cases were persistently positive for HCV-RNA and showed continuously abnormal level of ALT. In 7 of 12 cases who became negative for HCV-RNA just after the treatment ended, HCV-RNA was detected again at first and 12th month after therapy. The ALT level were continuously abnormal in 4 of these 7 cases during and after the treatment, and became normal transiently in the other 3. In the 5 cases who became persistently negative (at least 1 year), ALT level decreased to normal or near normal during follow up for more than one year. According to our results, we classified the therapeutic efficacy of IFN therapy for chronic hepatitis C. Type I response is complete response. In this type, HCV-RNA became negative persistently and ALT became normal or near-normal for at least 12 months after therapy. Type II is partial response. In this type, HCV-RNA became negative transiently and ALT level was transiently normal or poor. Type III is ineffective. In this type, HCV-RNA did not become negative and the effect for ALT was poor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715834 TI - A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis. AB - Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base substitutions that could possibly influence gene expression. Mutations were made such that the DNA sequence upstream of the ATG start codon was not changed. Moreover, care was taken that the substitutions, which were all within the first six codons, neither affected the amino acid sequence of the gene product nor introduced codons rarely used in L. lactis. The results suggest that mRNA secondary structure contributes to the efficiency of translation initiation in L. lactis. PMID- 1715835 TI - [Various forms of the anti-nicotine propaganda (work experience of the Mozyr health center)]. PMID- 1715836 TI - [Features of the immune status in patients with pulmonary tuberculosis living in areas of intensive use of pesticides]. PMID- 1715837 TI - Palliation of malignant dysphagia: surgery, radiotherapy, laser, intubation alone or in combination? PMID- 1715838 TI - Characteristic behavior of serum elastase 1 in pancreatic cancer. AB - Diagnostic significance of serum immunoreactive elastase 1 (IRE) in pancreatic cancer was evaluated in 53 patients with pancreatic cancer. Frequency of abnormally high serum IRE levels in pancreatic cancer was 66.0%; 87.0% in head cancer (N = 23), 55.0% in body & tail cancer (N = 20) and 40% in diffuse cancer (N = 10). Serum IRE level in resectable cancer was significantly higher than that in unresectable cancer. Comparative studies of serum pancreatic enzymes revealed that serum IRE was the most sensitive marker for diagnosis of pancreatic cancer. The characteristic behavior of serum IRE throughout the course of pancreatic cancer was that abnormally high levels of IRE are maintained for a longer period of time when compared to that of pancreatitis. In the comparative study of serum IRE and CA19-9, of the 9 cases of pancreatic cancer with CA19-9 levels less than 100U/ml, 7 showed abnormally high values of IRE, and of these 4 were resectable. These results indicate that in order to detect early pancreatic cancer, any elevation of serum IRE before CA19-9 increase should be noted with care, and patients who particularly show elevated IRE values for more than one month should be subjected to more extensive morphological examinations. PMID- 1715839 TI - Primary intracranial sarcomas: histopathological features of 19 cases. AB - Nineteen primary intracranial sarcomas out of a total of about 25,000 brain tumour biopsies are reported. Subtypes included malignant fibrous histiocytoma (6 cases), leiomyosarcoma (3), rhabdomyosarcoma (2), angiosarcoma (2), and one case each of fibrosarcoma, low-grade fibromyxoid sarcoma, malignant ectomesenchymoma, mesenchymal chondrosarcoma, differentiated chondrosarcoma and Ewing's sarcoma. Histological and immunohistochemical features corresponded to those of extracranial sarcomas. Nests of pleomorphic astrocytes mimicking glioma were detected in the five storiform-pleomorphic malignant fibrous histiocytomas. Our results indicate that intracranial sarcomas can be classified like their extracranial counterparts. The low incidence compared with earlier series is related to changes in classification and progress in histogenetic clarification. PMID- 1715840 TI - An immunohistological study of granulomatous prostatitis. AB - Granulomatous prostatitis may result from tuberculosis and fungal infection and has been described following prostatic surgery. In most cases, however, the aetiology is unknown, although it may be due to a reaction to extravasated or altered prostatic secretions. We have investigated cells (macrophages, lymphocytes), serum proteins (fibrinogen, alpha 1-antitrypsin) and prostatic epithelial products (prostatic-specific antigen and prostatic acid phosphatase) in diffuse granulomatous prostatitis (3 cases), focal periacinar prostatic granulomas (9) and focal prostatic infarcts (5), using an immunohistological technique. T-lymphocytes and macrophages are present in diffuse and focal granulomatous prostatitis, but few B-lymphocytes occur. Fibrinogen-related antigen is absent from granulomas, but a small amount is present within infarcts, whereas plentiful alpha 1-antitrypsin was detected both in granulomas and infarcts. Significant reduction in prostatic-specific antigen and acid phosphatase reactivity occurs in granulomatous prostatitis. This suggests that cytokines derived from activated macrophages and T-lymphocytes may be exerting a cell regulatory effect and altering cell secretions, as well as causing destruction of the prostatic epithelium. PMID- 1715841 TI - A blue naevus of the prostate: a light microscopic study including an investigation of S-100 protein positive cells in the normal and in the diseased gland. AB - A blue naevus of the prostate in an 80-year-old patient is presented, including electronmicroscopical examination and the demonstration of S-100 protein positivity. Cells expressing S-100 protein were sought in prostates of infants, young adults and elderly patients with hyperplasia and carcinoma. Positive staining was found in stromal cells, but the number diminished with age and disease. These findings could explain the rarity of blue naevus and the virtual absence of primary malignant melanoma in the prostate. PMID- 1715842 TI - Nucleolar organizer regions in benign and malignant prostatic disease. AB - A modified silver stain technique for visualizing nucleolar organizer regions (AgNOR counting) was applied to 24 benign and 23 malignant prostatic biopsies. Marked inter-observer variation was found, particularly in sections with high AgNOR counts. After averaging the AgNOR counts of both observers, there was no significant difference in counts between the benign and the malignant biopsies. The AgNOR count appeared to be increased in tumours up to Gleason histological grade 6, but not in tumours of Gleason histological grade 7 or more. PMID- 1715843 TI - Langerhans cells in nasal mucosa of patients with grass pollen allergy. AB - Langerhans cells (LC) are known to be present in squamous epithelia of the human body. They are dendritic cells (DC) and characterized by the presence of Birbeck granules (BG). In previous studies, DC positive for CD1a and HLA-DR were found in the cylindrical epithelium and the lamina propria of the nasal mucosa. In our study, more CD1a cells occurred in the allergic patients than in the non-allergic controls. In a combined light microscopy (LM) and electron microscopy (EM) study, biopsies of nasal mucosa in allergic patients were studied. We used monoclonal antibodies against CD1a and HLA-DR, to identify DC in LM cryostat sections. The presence of BG identified most of the intra-epithelial DC as LC on the EM level, whereas a minority of DC in the lamina propria also contained BG. The ultrastructure of LC and DC in the ciliated cylindrical epithelium and the lamina propria is compared. PMID- 1715844 TI - Kinetics of MHC class II and transferrin receptor expression by colostral cells stimulated in vitro with concanavalin A and myelin basic protein. AB - The colostral cells, regarded generally as being protective, have been shown to differ in a number of membrane properties (rosetting, adherence, mobility) from the corresponding peripheral blood mononuclear cells. After in vitro stimulation with Con A or MBP 50% to 70% of the human colostral cells appeared HLA-DR positive at the first 24 h of culturing. The CD71 expression reached a maximum on culture days 2-3 coinciding with the maximal proliferative response. With regard to the phenotypic characteristics and their kinetics, the human colostral cells did not show significant differences from the peripheral blood mononuclear cells. PMID- 1715845 TI - Neutral endopeptidase (EC 3.4.24.11) does not hydrolyze recombinant human interleukin-1 beta. AB - We have previously shown that neutral endopeptidase (NEP; EC 3.4.24.11) regulates neuropeptide-induced responses. Recently, Pierart et al. reported that NEP degraded purified interleukin-1 (IL-1) using thymocyte proliferation assay. Since IL-1 is an important cytokine in the immune response and inflammation, we have assessed whether NEP hydrolyzes recombinant human IL-1 beta using three assay systems (bioassay, immunoassay, and HPLC analysis). NEP on the NALM-6 cells (both intact cells and the solubilized plasma membrane fraction) efficiently hydrolyzed Met5-enkephalin and substance P. However, NEP did not significantly decrease the amount of rhIL-1 beta assessed by the growth inhibitory activity of a human melanoma, by the immunoassay, or by the direct analysis on HPLC. Therefore, we conclude that NEP does not significantly hydrolyze rhIL-1 beta. Our results suggest that, in contrast to the regulatory role of NEP in neuropeptide-induced responses, NEP is not a regulatory enzyme for IL-1-induced responses. PMID- 1715846 TI - Probing the idiotype/anti-idiotype antibody interaction with a set of synthetic peptide homologues. AB - Anti-idiotypic (anti-Id) antibodies were raised against two murine monoclonal antibodies (mAb 1/1 and mAb 2/1) which recognise two distinct and well characterised epitopes on a 24-residue synthetic peptide representing part of the haemagglutinin (HA) of influenza virus. A monoclonal anti-Id antibody, specific for mAb 2/1, could bind to mAb 2/1 when the paratope of the latter was occupied with peptide, indicating that this anti-Id antibody is directed to a framework idiotope. In contrast, an anti-Id mAb derived from mAb 1/1-immunised mice was inhibited in its binding to Id by the parent peptide and also by the heptapeptide NVPEKQT which constitutes the epitope recognised by mAb 1/1. The small size of this synthetic peptide eliminates the possibility of significant steric inhibition in the system, and establishes that this mAb is a true paratope directed anti-Id antibody. The interaction of this anti-Id mAb with the paratope of mAb 1/1 in the presence of a set of peptide homologues of the epitope was also examined. A peptide as short as 5 residues, which contains two of the three irreplaceable residues of the epitope, could inhibit binding between the two mAbs. PMID- 1715847 TI - Generation of recombinant, carbohydrate-free intercellular adhesion molecule-1 (ICAM-1) and ICAM-1 fragments in Escherichia coli and mapping of epitopes recognized by anti-ICAM-1 monoclonal antibodies. AB - Intercellular adhesion molecule-1 (ICAM-1) has been shown to interact with the integrin leukocyte function associated antigen-1 (LFA-1) in a variety of cell cell adhesion phenomena. Furthermore, it serves as a receptor for the majority of the Rhinoviruses and for Plasmodium falciparum-infected human erythrocytes. We generated recombinant, carbohydrate-free ICAM-1 and several ICAM-1 fragments by expression in Escherichia coli using the fusion protein expression system pUEX1 3. In Western blot and dot blot analyses we tested mAbs (7F7, 8B9, P3.58-BA3, BA11, -BA14, -BA19, -BA21, -BA23, -BA24, -BA26, CL203.4 and 84H10) and a polyclonal antiserum directed against native ICAM-1 for their reactivity with these constructs. We were able to localize the binding site for the mAbs P3.58 BA3, -BA11, -BA14, -BA19, -BA21, -BA23, -BA24 and -BA26 at domain 5, whereas the mAbs 7F7, 8B9, CL203.4 and 84H10 did not recognize the recombinant, carbohydrate free ICAM-1. Our findings suggest the presence of an immunodominant epitope on domain 5 of ICAM-1. PMID- 1715848 TI - Influence of time of injection of recombinant porcine somatotropin (rpST) relative to time of feeding on growth performance, hormone and metabolite status, and muscle RNA, DNA, and protein in pigs. AB - Thirty-six barrows were used in a 2 X 3 factorial treatment array to determine the effects of time of injection (0800 [AM] or 1800 [PM]) of recombinant porcine somatotropin (rpST) (0, 50, or 100 micrograms.kg BW.d-1 adjusted weekly) relative to time of feeding on growth performance, carcass composition, serum hormones and metabolites, and muscle RNA, DNA, and protein. Pigs were fed at 85% of ad libitum and allowed access to feed between 0800 and 1200. Treatments were initiated at 38 kg and continued until each pig consumed an average of 7.5 Mcal of DE/d. There was no significant effect of injection time for any measure of growth performance or composition of gain even though rpST treatment improved most criteria evaluated. Treatment with rpST increased ADG by 30%, improved feed:gain by 23%, reduced lipid accretion by 46%, increased protein accretion by 69%, and increased loin eye area (LEA) by 26%. Time of injection also had no effect on serum hormones or metabolites, except for nonesterified fatty acids (NEFA), which were 20% higher in the AM-injected animals. Treatment with rpST increased (P less than .05) RNA concentration (21%) and RNA/DNA (17%) in longissimus dorsi (LD) muscle. In semimembranosus (SM) muscle, however, rpST administration resulted in an increased (P less than .05) DNA concentration (10%) and a decreased DM content (4%). These results suggest that rpST influences growth differently in these two muscles. We conclude that time of rpST administration (AM vs PM) has no effect on its metabolic properties and, therefore, no effect on growth performance in the pig. PMID- 1715849 TI - Insulin-like growth factor (IGF)-I and -II and IGF binding proteins in serum and mammary secretions during the dry period and early lactation in dairy cows. AB - Concentrations of IGF-I and IGF-II, and IGF binding proteins (IGFBP) in serum and mammary gland secretions were surveyed during the dry period and early lactation of 30 Holstein cows. Although there was a threefold drop in the concentration of IGF-I in serum from the last week of the dry period to parturition (81 +/- 7 to 24 +/- 3 ng/ml, P less than .01), there was no significant change in serum IGF-II concentration during this period (150 +/- 17 vs 173 +/- 13 ng/ml, P greater than .05). Furthermore, a 57% increase in serum IGF-I was observed from the last week of lactation to the second week of drying off (100 +/- 5 to 157 +/- 8 ng/ml, P less than .05). Changes in serum IGF-II were not observed (126 +/- 11 vs 150 +/- 10 ng/ml, respectively; P greater than .05). Although IGF-I, IGF-II, and IGFBP concentrations in mammary secretions peaked 2 wk before parturition (2.95 +/- 1.1, 1.83 +/- .6, and 7.27 +/- .76 micrograms/ml, respectively), total output/quarter was highest in colostrum (394 +/- 119, 295 +/- 132, and 2,680 +/- 1,967 micrograms/quarter, respectively). Weekly milking of two individual quarters during the dry period did not affect (P greater than .05) IGF-I or IGF II concentration (ng/ml) or total output (microgram/quarter) and milk yield in colostrum and milk (2 wk and 7 wk) compared with the ipsilateral quarter. The data support the hypothesis that IGF-I may be transported by the mammary gland epithelium. Furthermore, the secretion mechanisms of IGF-I, IGF-II, and IGFBP by the gland may be related to each other. PMID- 1715850 TI - Transferrin- and albumin-directed expression of growth-related peptides in transgenic sheep. AB - Chimeric genes containing either the mouse transferrin (Trf) enhancer/promoter fused to the structural sequences encoding bovine growth hormone (GH) or the mouse albumin (Alb) enhancer/promoter fused to the gene for human growth hormone releasing factor (GRF) were microinjected into sheep zygotes. A low percentage of resulting transgenic sheep chronically expressed the respective genes, resulting in elevated plasma concentrations of circulating GH or GRF, respectively. Growth hormone-releasing factor expression induced elevated plasma levels of endogenous GH production. In addition, elevated levels of circulating insulin-like growth factor-I were observed in the bovine GH-expressing Trf transgenic sheep. Growth of these founder transgenic sheep relative to controls were not enhanced. In part, this may be due to the development of the diabetic condition exhibited by both transgenic groups. These results demonstrate that the mouse Trf and Alb enhancer/promoters are active in sheep and suggest that alternate strategies for expressing growth-related genes may be required to modulate growth in sheep. PMID- 1715851 TI - Intrathoracic accumulation of 111In-labeled neutrophils in guinea pigs in response to PAF. AB - The intrathoracic content of neutrophils, labeled with 111In-oxine has been measured in the anesthetized guinea pig by using an automated isotope-monitoring system. Intravenous infusion of platelet-activating factor (PAF; 5.6, 10, or 18 ng.kg-1.min-1 over 5 min) caused a dose-related abrupt intrathoracic accumulation of neutrophils, which dispersed from the thorax within 20 min. Repetition of this procedure after 1 h gave responses of comparable magnitude and duration. Anti platelet antiserum pretreatment did not influence the response of neutrophils to PAF. Iloprost infusion (10 ng.kg-1.min-1 over 15 min) did not affect the response of neutrophils to PAF, whereas accumulation of radiolabeled platelets in the lung was totally suppressed by this dose. Intrathoracic accumulation of neutrophils in response to PAF can be considered to be independent of platelet activation. PMID- 1715852 TI - The effects of malnutrition on the motor, perceptual, and cognitive functions of Filipino children. AB - The motor, perceptual, and cognitive abilities of 99 Filipino children, aged 4-6 years with a documented history of malnutrition from a nutritionally depressed area of Manila were determined using the Revised Manila Motor-Perceptual Screening Test. They were classified into four groups of: (1) normal; (2) acutely malnourished; (3) stunted but not malnourished; and (4) chronically malnourished using the Waterlow classification. Thirty-one normal children of comparable ages and background from a nationwide pool were similarly tested and served as the control group. Motor (p = 0.001) and perceptual skill (p less than 0.03 to less than 0.001) scores were significantly lower than in their normal counterparts, especially in the chronically malnourished children. Cognitive abilities were not evidently affected by malnutrition. PMID- 1715853 TI - Physical map locations of the genes that encode small stable RNAs in Escherichia coli. PMID- 1715854 TI - Survey of multicopy single-stranded DNAs and reverse transcriptase genes among natural isolates of Myxococcus xanthus. AB - Twenty different isolates of the soil bacterium Myxococcus xanthus were examined for the presence of multicopy single-stranded DNA (msDNA)-producing retroelements, or retrons. Each strain was analyzed by ethidium bromide staining for msDNA, 32P labeling of the msDNA molecule by the reverse transcriptase (RT) extension method, and DNA hybridization experiments with probes derived from two retrons, Mx162 and Mx65, previously cloned from M. xanthus DZF1. These analyses revealed that all M. xanthus strains contain an msDNA very similar to Mx162 msDNA, and 13 strains also contain a second smaller msDNA very similar to Mx65 msDNA. In addition, the strains contained retron-encoded genes msr and msd, which code for msDNA, and a gene for RT responsible for the synthesis of msDNA. These genes show greater than 80% nucleotide sequence similarity to retrons Mx162 or Mx65. The near-ubiquitous occurrence of msDNA retrons among M. xanthus strains and their homogeneous nature are in marked contrast to the highly diverse but rarely occurring msDNA-producing elements of Escherichia coli. The possible origin and evolution of RT and retron elements is discussed in view of these findings. PMID- 1715855 TI - The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12. AB - The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2. PMID- 1715856 TI - Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis. AB - srfA is an operon required for the production of the lipopeptide antibiotic surfactin, competence development, and efficient sporulation in Bacillus subtilis. The expression of srfA is induced after the end of exponential growth and is dependent on the products of late-growth regulatory genes comP, comA, and spo0K. To begin to understand the mechanism of srfA regulation, the srfA promoter region was identified and characterized. To examine srfA promoter activity, the srfA promoter was fused to lacZ and inserted into the B. subtilis chromosome as a single copy at the SP beta prophage. The location of the transcription start site of srfA was determined by primer extension analysis and shown to be preceded by a sequence that resembles the consensus promoter recognized by the sigma A form of RNA polymerase. The srfA operon was found to have a sequence corresponding to a long, untranslated leader region of the srfA mRNA (300 bp). A nucleotide sequence and mutational analysis of the promoter identified a region of dyad symmetry required for srfA-lacZ expression. A similar sequence is found in the region upstream of the degQ promoter, transcription from which is also regulated by ComA. This region of dyad symmetry found upstream of these promoters may be the target for ComA-dependent transcriptional activation. PMID- 1715857 TI - marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli. AB - Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K 12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance. PMID- 1715858 TI - Regulation of ompF porin expression by salicylate in Escherichia coli. AB - The expression of ompF, the gene encoding a major outer membrane protein of Escherichia coli, is regulated by various environmental factors. The mechanism by which salicylate (SAL) drastically reduces ompF expression was studied here by means of lacZ fusions to ompF, ompC, and micF, by sodium dodecyl sulfate-gel electrophoresis of outer membrane proteins, and by measurements of outer membrane permeability. Growth of E. coli in LB broth containing SAL strongly reduced ompF specific translation of an ompF-lacZ fusion. The extent of this reduction varied with the SAL concentration from 64% at 0.5 mM to 95% at 2 mM and greater than 99% at 10 mM. ompF-lacZ transcription was not affected by SAL, whereas ompC-lacZ transcription was elevated by 70%. Since the micF transcript is antisense to a portion of the ompF transcript and is capable of decreasing the translation of ompF, the effect of SAL on micF transcription was measured in a micF-lacZ fusion strain. SAL-grown cells contained three- to fourfold more micF transcript during the logarithmic phase of growth than did the control cultures. However, micF was not absolutely required for the response to SAL. In micF-deleted strains, the effects of SAL on ompF translation, on OmpF in the outer membrane, and on outer membrane permeability were diminished but still evident. The effect of SAL on ompF expression was independent of the osmolarity of the medium and was epistatic to certain ompB regulatory mutations: the high levels of ompF expression found in envZ3 and ompR472 strains were greatly reduced by growth in SAL. Unexpectedly, the OmpC- phenotypes of these mutants were suppressed by SAL. Thus, growth in SAL severely decreases the translation of ompF while enhancing the transcription of micF and ompC. In this respect, SAL-grown cells resemble certain marA and tolC mutants that have high levels of micF and ompC transcripts and low levels of OmpF. PMID- 1715859 TI - Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis. AB - The sequence and properties of the comQ gene are described. comQ was predicted to encode a 34,209-Da protein, and the product of comQ was shown to be required for the development of genetic competence. The apparent transcriptional initiation and termination sites of comQ were mapped, and the location of a likely E sigma A promoter was inferred. The expression of comQ was maximal early in growth and declined as the cells approached the stationary phase. This expression was not dependent on any of the competence regulatory genes tested (comA, comP, sin, abrB, degU, and spo0A). Disruption of comQ in the chromosome prevented the development of competence as well as the transcription of comG, a late competence operon. This disruption also decreased the expression of srfA, a regulatory operon needed for the expression of competence. These and other results suggest a role for ComQ early in the hierarchy of competence regulatory genes, probably as a component of a signal transduction system. PMID- 1715860 TI - Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli. AB - We report on the cloning and characterization of the rfb gene cluster of the O75 lipopolysaccharide from a urinary tract isolate of Escherichia coli. Deletion cloning defined the minimum region of DNA that expressed the O75 antigen in E. coli host strains to be on a 12.4-kb insert. However, the E. coli strain expressing this region did not produce a polymerized O chain as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. A slightly larger DNA clone of 13.4 kb produced a polymerized O chain in E. coli S phi 874 but was found to be abnormal in its distribution over the surface membrane. Normal wild-type E. coli, as with Salmonella spp., has a bimodal distribution of the lipopolysaccharide on the surface which is seen as an abundance of long and short O chains attached to the lipid A-core structure. We found in a region adjacent to the cloned rfb region, and on the opposite side from where the putative polymerase (rfc) is encoded, a novel protein of 35.5 kDa expressed from a 1.75-kb DNA fragment. This protein was shown to complement in trans the E. coli strains carrying plasmids that expressed abnormal, unregulated lipopolysaccharides. The expression of these complemented strains was bimodal in distribution. Mutation of the gene encoding this protein destroyed its ability to regulate O-chain distribution. We propose to call this regulator gene rol, for regulator of O length. PMID- 1715861 TI - Construction, characterization, and complementation of Rhodospirillum rubrum puf region mutants. AB - Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 light-harvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the puf region. Southern blot analysis demonstrated that the defective copies of puf sequences had replaced their normal chromosomal counterparts through homologous recombination. The phenotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semi aerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination. PMID- 1715862 TI - Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. AB - High-frequency persistence to the lethal effects of inhibition of either DNA or peptidoglycan synthesis, the Hip phenotype, results from mutations at the hip locus of Escherichia coli K-12. The nucleotide sequence of DNA fragments which complement these mutations revealed an operon consisting of a possible regulatory region, including sequences with modest homology to an E. coli promoter, and two open reading frames which are translated both in vitro and in vivo. The stop codon of a 264-bp open reading frame, hipB, and the start codon of a 1,320-bp open reading frame, hipA, share an adenine residue. Assays of promoter strength, the location of the probable promoter with respect to the start of transcription, and codon usage all indicate that hipB and hipA are weakly expressed genes. The activity of the promoter is impaired by an adjacent downstream sequence which includes the coding region of hipB. The impairment is partially relieved by insertion of a premature translation termination signal within the coding region of hipB, suggesting involvement of the HipB protein in the regulation of this promoter. The arrangement of hipB and hipA within the operon and the toxicity of hipA for strains defective in or lacking hipB suggest an important interaction between the products of these genes. PMID- 1715863 TI - Constitutive and UV-mediated activation of RecA protein: combined effects of recA441 and recF143 mutations and of addition of nucleosides and adenine. AB - The recF143 mutant of Escherichia coli is deficient in certain functions that also require the RecA protein: cell survival after DNA damage, some pathways of genetic recombination, and induction of SOS genes and temperate bacteriophage through cleavage of the LexA and phage repressors. To characterize the role of RecF in SOS induction and RecA activation, we determined the effects of the recF143 mutation on the rate of RecA-promoted cleavage of LexA, the repressor of the SOS genes. We show that RecA activation following UV irradiation is delayed by recF143 and that RecF is specifically involved in the SOS induction pathway that requires DNA replication. At 32 degrees C, the recA441 mutation partially suppresses the defect of recF mutants in inducing the SOS system in response to UV irradiation (A. Thomas and R. G. Lloyd, J. Gen. Microbiol. 129:681-686, 1983; M. R. Volkert, L. J. Margossian, and A. J. Clark, J. Bacteriol. 160:702-705, 1984); we find that this suppression occurs at the earliest detectable phase of LexA cleavage and does not require protein synthesis. Our results support the idea that following UV irradiation, RecF enhances the activation of RecA into a form that promotes LexA cleavage (A. Thomas and R. G. Lloyd, J. Gen. Microbiol. 129:681-686, 1983; M. V. V. S. Madiraju, A. Templin, and A. J. Clark, Proc. Natl. Acad. Sci. USA 85:6592-6596, 1988). In contrast to the constitutive activation phenotype of the recA441 mutant, the recA441-mediated suppression of recF is not affected by adenine and nucleosides. We also find that wild-type RecA protein is somewhat activated by adenine in the absence of DNA damage. PMID- 1715864 TI - Targeting of lysosomal integral membrane protein LIMP II. The tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes. AB - Time course experiments of the localization of rat LIMP II expressed in COS cells show that the protein is transported directly from the Golgi complex to lysosomes. Substitution of the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II for the native cytoplasmic tails of the plasma membrane proteins CD36 and CD8 resulted in straight transport of both proteins to lysosomes. The synthetic tyrosine-containing heptapeptide, RGTGVYG, did not replace the natural carboxyl cytoplasmic tail of LIMP II in its ability to transport both CD36 and CD8 to lysosomes, and the two constructs were transported to and expressed at the plasma membrane. Substitution of the cytoplasmic tails of either CD36 or CD8 for the carboxyl cytoplasmic tail of LIMP II resulted in transport of the mutants to the plasma membrane where they underwent endocytosis before accumulating into lysosomes. The results indicate that a motif contained in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient to target proteins directly from the Golgi complex to lysosomes. PMID- 1715865 TI - Dihydropyridine-sensitive calcium channels from skeletal muscle. I. Roles of subunits in channel activity. AB - Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are hetero oligomeric proteins. Little is known about the functional roles of the various subunits, except that the alpha 1 subunit is the essential channel unit. We have reconstituted both partially purified holomeric channels and the separated subunits into liposomes and measured their properties using an assay based on the Ca2+ indicator dye fluo-3. The holomeric channels exhibited Ca2+ influx that was sensitive to membrane potential achieved by the addition of valinomycin in the presence of a K+ gradient. Dissipation of the K+ gradient resulted in the loss of the valinomycin-sensitive Ca2+ flux. In addition, the reconstituted channels were: 1) activated by the dihydropyridine Ca2+ channel activator Bay K 8644 in a dose-dependent manner with a Kd of 20 nM; 2) inhibited by various types of Ca2+ channel inhibitors including the dihydropyridine (+)-PN 200-110, the phenylalkylamine verapamil, and the benzothiazepine d-cis-diltiazem; and 3) modulated in a stereoselective manner by the enantiomers of the dihydropyridine S 202-791. The purified channels used in this work possessed an alpha 1 subunit of 165 kDa and did not appear to contain a larger alpha 1 subunit of approximately 210 kDa, suggesting that channel activity with properties similar to those observed in intact cells can be supported with an alpha 1 subunit of 165 kDa. Reconstituted channels that were 85% depleted in the alpha 2/delta subunits showed a significant decrease in the initial rate of Ca2+ influx induced by valinomycin, but retained responsiveness to Bay K 8644 and (+)-PN 200-110. When the separated alpha 2 and delta subunits were added back to the alpha 1 subunit containing preparation, the channels exhibited their normal rate of Ca2+ influx. These results demonstrated that the dihydropyridine-sensitive Ca2+ channels from skeletal muscle require the presence of the alpha 2.gamma complex in stoichiometric amounts to exhibit full activity. PMID- 1715866 TI - Covalently cyclized agonist and antagonist analogues of bombesin and related peptides. AB - During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures. PMID- 1715867 TI - Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina. AB - Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function. PMID- 1715868 TI - Channel permeant cations compete selectively with noncompetitive inhibitors of the nicotinic acetylcholine receptor. AB - Previous work suggests that noncompetitive inhibitor (NCI) ligands and channel permeant cations bind to sites within the nicotinic acetylcholine receptor ion channel. We have used ethidium as a fluorescent probe of the NCI site to investigate interactions between NCI ligands and channel permeant cations. We found that ethidium can be completely displaced from the receptor by a variety of inorganic monovalent and divalent cations. The rank order of monovalent cation affinities was found to be Tl+ greater than Rb+ greater than or equal to K+ greater than Cs+ greater than Na+ greater than Li+. The monovalent cation Kd values vary markedly over a 40-fold range, from 3 to 121 mM. The Kd values and rank order correspond to values determined previously from electrophysiological data. Hill plots of the back titrations yield slopes of 1.0 for all monovalent cations, indicating a single class of independent sites, as shown previously for NCI ligands. Scatchard analysis of ethidium binding in the presence of Tl+ reveals a reduction in affinity and no changes in the maximal number of sites. In the presence of agonist the kinetics of ethidium dissociation induced by the addition of phencyclidine or cations alone or the simultaneous addition of both are nearly identical. The ethidium dissociation rate induced by either phencyclidine or cations is regulated by the occupation of the agonist sites in a similar manner. These results indicate that the effect of cations on NCI ligand binding occurs by mutually exclusive competition. We suggest that NCIs can regulate cation binding at a physiological cation recognition site that is likely part of the cation permeation path through the receptor channel. PMID- 1715869 TI - The plasma cell membrane glycoprotein, PC-1, is a threonine-specific protein kinase stimulated by acidic fibroblast growth factor. AB - A 32P-labeled protein that co-purified with acidic fibroblast growth factor (aFGF) receptor from bovine liver proved to be a distinct membrane protein, which itself has kinase activity that is stimulated by aFGF. The protein was designated MAFP for major aFGF-stimulated phosphoprotein. MAFP was purified from bovine liver using immunoaffinity chromatography with monoclonal antibody to MAFP following Triton X-100 extraction of plasma membranes and wheat germ lectin Sepharose 4B column chromatography. The purified MAFP showed molecular masses of 130 kDa and 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. Purified MAFP elicited aFGF-stimulated Thr-specific autophosphorylation activity and phosphorylation activity toward protein substrates (myelin basic protein and histone). Amino acid sequence analyses of 16 peptide fragments of MAFP, produced by endoproteinase Lys C digestion followed by reduction and S-pyridylethylation, showed approximately 80-100% homology with the cDNA-deduced amino acid sequences of human and mouse plasma cell membrane glycoprotein, PC-1 (Buckley, M. F., Loveland, K. A., McKinstry, W. J., Garson, O. M., and Goding, J. W. (1990) J. Biol. Chem. 265, 17506-17511), suggesting that MAFP is the bovine version of PC-1. The amino acid sequences of bovine MAFP, human and mouse PC-1 reveal a putative ATP binding site in their extracellular domains. These results suggest that MAFP(PC-1) is an ectoprotein kinase. In addition to the kinase activity, MAFP(PC-1) was also found to possess alkaline nucleotide phosphodiesterase activity. It is now clear that several of the unique properties previously attributed to the aFGF receptor kinase are actually properties of this novel Thr-specific ectoprotein kinase, which co-purifies with the aFGF receptor and is responsive to stimulation by aFGF. PMID- 1715870 TI - Amphipathic beta structure of a leucine-rich repeat peptide. AB - Long tandem arrays of a characteristic leucine-rich repeat motif on the order of 24 amino acids in length have been found in the primary structure of an increasing number of proteins. The most striking feature of these repeats is an amphipathic sequence, with leucine as the predominant hydrophobic residue. Based on this amphipathic sequence and the function of the proteins in which they have been found, the repeats have been proposed to be involved in protein-protein and protein-lipid interactions. As a step toward elucidating the structure and biochemical properties of the leucine-rich repeat motif, we have studied a synthetic leucine-rich repeat peptide (LRP32) representing one of the repeats found in Drosophila chaoptin. We have shown that: (i) LRP32 is soluble in aqueous solution but will bind quantitatively to phospholipid vesicles; (ii) LRP32 has a partial beta structure in aqueous solution and is predominantly a beta structure in the presence of phospholipid; (iii) LRP32 integrates into lipid bilayers to form 60-A intramembrane particles as seen using freeze-fracture electron microscopy (these putative oligomeric structures appear to contain a central aqueous core as indicated by their ability to generate conductances in planar lipid bilayers); and (iv) LRP32-lipid complexes generate 2H NMR spectra characteristic of integral membrane proteins. This study is consistent with LRP32 forming an amphipathic beta sheet. We propose that protein segments containing tandem arrays of leucine-rich repeats also may form amphipathic beta sheets. PMID- 1715871 TI - Cloning, sequencing, and expression of a cDNA encoding rat LIMP II, a novel 74 kDa lysosomal membrane protein related to the surface adhesion protein CD36. AB - LIMP II is a glycoprotein expressed in the membrane of lysosomes and secretory granules with lysosomal properties. Sequence analysis of a CNBr-cleaved peptide allowed the synthesis of a 47-mer oligonucleotide that was used to screen a rat liver cDNA library in lambda gt11. This resulted in isolation of a 2-kilobase cDNA containing 1,434 bases encoding the entire protein. The deduced amino acid sequence indicates that LIMP II consists of 478 amino acid residues. The segment spanning residues 4-6 to 26 constitute an uncleavable signal peptide. LIMP II possesses a hydrophobic amino acid segment near the carboxyl end, that together with the uncleaved signal peptide may anchor the protein to the membrane through two distant segments. The major portion of the protein resides on the luminal side and displays 11 potential N-glycosylation sites and 5 cysteine residues. Two short cytoplasmic tails, 2-4 and 20-21 amino acids long, correspond to the NH2- and COOH-terminal ends of the protein, respectively. Transfection of COS cells with the cDNA of LIMP II resulted in expression of the protein and its transport to lysosomes. Comparison of the entire sequence to various data bases of known proteins revealed extensive homology between LIMP II and the cell surface protein CD36 involved in cell adhesion. No significant homology was detected with the two families of lysosomal membrane proteins A and B, recently described. PMID- 1715872 TI - The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy. AB - Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule. PMID- 1715873 TI - Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix. AB - In this study, we have examined the capacity of various cell types, which express cell surface hyaluronan receptors, to organize a chondrocyte-like pericellular matrix when given chondrocyte-derived extracellular matrix macromolecules exogenously. The assembly of a pericellular matrix was visualized by a particle exclusion assay. Without the addition of exogenous macromolecular components, none of the cell types studied exhibited significant pericellular matrices extending from their plasma membranes. However, upon the addition of high molecular weight hyaluronan in combination with aggregating cartilage proteoglycan monomers, large pericellular matrices were formed within two hours of incubation. No pericellular matrices were formed if these macromolecular components were added separately at equivalent concentrations or if the components were added in the presence of hyaluronan hexasaccharide, a competitive inhibitor of hyaluronan interaction with cell surface hyaluronan receptors. Fully assembled pericellular matrices could also be displaced by the subsequent addition of hyaluronan hexasaccharides. Nonliving, glutaraldehyde-fixed cells, which retained functional hyaluronan receptors, maintained the capacity to assembly pericellular matrices with exogenous components, in serum-containing or serum-free medium. Cells that were incubated with exogenous matrix macromolecules for 24 h, followed by a chase incubation in medium minus the exogenous macromolecules, continued to maintain the matrix for up to 6 h on live cells and more than 24 h on glutaraldehyde-fixed cells. Cell types that did not express hyaluronan receptors were not capable of organizing such pericellular matrices when incubated with these exogenous components. These findings suggest that cells expressing hyaluronan receptors have a significant capacity to organize their immediate extracellular environment via hyaluronan-hyaluronan receptor interactions. Possible physiological functions for this type of matrix organizing capacity are discussed. PMID- 1715874 TI - A common epitope is shared by ciliary rootlets and cell-cell adherens junctions in ciliated ependymal cells. AB - Using immunoblot, light and electron immunocytochemistry, we investigated the presence and the localization of polypeptides cross-reacting with the monoclonal antibody CC.310 (mAb CC.310), which is mainly directed against a 175K (K = 10(3) Mr) ciliary rootlet protein. In hypothalamic ependymal cultures, the unique antigen recognized by mAb CC.310 was associated with the Triton X-100-insoluble fraction in these cultures and electrophoretically migrated to these cultures and electrophoretically migrated to 94K. mAb CC.310, which appears to be a very suitable marker for ciliated ependymocytes, allowed us to observe ciliogenesis during the growth of the ependymal cultures, from a single spot in each undifferentiated ependymal cell to a massive labeling in ciliated ependymal cells. In fully differentiated ciliated ependymocytes, mAb CC.310 strongly reacted with fibrous structures corresponding to ciliary rootlets, as confirmed by ultrastructural observations. In addition, a weaker immunostaining was also found along the intercellular junctions, and showed that proteins sharing a common epitope are located in ependymal ciliary rootlets and near adherens-type junctional complexes. Immunofluorescence studies confirmed the presence of positive labeling at the level of junctional complexes between cells in two epithelial lines, HeLa and PtK2, in which mAb CC.310 mainly reacted with one polypeptide of 85K. PMID- 1715875 TI - Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain. AB - All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly. PMID- 1715876 TI - Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes. AB - During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715877 TI - Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins. AB - Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate. PMID- 1715878 TI - Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies. AB - A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described. Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed. The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories. It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test. Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous. PMID- 1715879 TI - Molecular characterization and proposal of a neotype strain for Bartonella bacilliformis. AB - Bartonella bacilliformis, the etiologic agent of bartonellosis, was characterized biochemically and by DNA hybridization, guanine-plus-cytosine content, genome size, and 16S rRNA sequencing. DNAs from the two strains in our collection exhibited 97% relatedness in hydroxyapatite reactions done at 55 degrees C (optimal reassociation criterion) and 100% relatedness in reactions done at 70 degrees C (stringent reassociation criterion). There was no evidence of divergence within the related sequences. B. bacilliformis DNA showed no relatedness to the cat scratch disease bacillus or to a strain of a second species in the same genus as the cat scratch disease bacillus in hybridization reactions done at 65 degrees C. The guanine-plus-cytosine contents of DNAs from the two B. bacilliformis strains were 39 and 40 mol%. Time course reassociation, done by determining spectrophotometrically the time required for one-half of the denatured DNA to form duplexes, indicated that B. bacilliformis has a genome size of approximately 4 x 10(8). The 16S rRNA sequence analysis indicated that B. bacilliformis is in the alpha-2 subgroup of the purple bacteria, class Proteobacteria, and that its closest relatives are Rochalimaea quintana and Brucella abortus. Strain KC583 (= Herrer 020/F12,63 = ATCC 35685) is proposed as the type strain of B. bacilliformis. PMID- 1715880 TI - Comparison of ribotyping with conventional methods for the type identification of Enterobacter cloacae. AB - An Escherichia coli rRNA probe was compared with a combination of O serotyping, phage susceptibility, and biotype pattern for the type identification of strains of Enterobacter cloacae. Forty-five isolates of E. cloacae from 36 patients in nine hospitals were examined. By conventional typing, only 26 (57.7%) could be assigned to a specific serotype and 6 (13.3%) were autoagglutinating owing to rough lipopolysaccharide antigens. All isolates could be assigned to one of three biotypes, but many phage sensitivity patterns were evident. Twenty-nine distinct strains were identified by combined typing. Probing of EcoRI and BamHI digests of chromosomal DNA with a cDNA copy of E. coli rRNA proved to be highly discriminating between strains. Thirty different ribotypes based on 28 bands were recorded. Overall, agreement between the ribotyping and combined typing methods was good (84.4%), and discrepancies were generally confined to serologically unclassifiable strains and variability in biotype codes. Ribotyping was reproducible, and five of six pairs of isolates from the same and different patients gave identical hybridization profiles on separate occasions. We conclude that ribotyping is a highly discriminatory and reproducible method for the typing of E. cloacae, but in most outbreaks it offers little increase in discrimination over traditional methods. PMID- 1715881 TI - Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. AB - To determine the minimum number of Cryptosporidium oocysts that can be detected in stool specimens by diagnostic procedures, stool samples seeded with known numbers of Cryptosporidium parvum oocysts were processed by the modified Formalin ethyl acetate (FEA) stool concentration method. FEA concentrates were subsequently examined by both the modified cold Kinyoun acid-fast (AF) staining and fluorescein-tagged monoclonal antibody (immunofluorescence [IF]) techniques. Oocysts were more easily detected in watery diarrheal stool specimens than they were in formed stool specimens. For watery stool specimens, a 100% detection rate was accomplished at a concentration of 10,000 oocysts per g of stool by both the AF staining and IF techniques. In formed stool specimens, 100% of specimens seeded with 50,000 oocysts per gram of stool were detected by the IF technique, whereas 500,000 oocysts per g of stool were needed for a 100% detection rate by AF staining. Counting of all oocysts on IF slides indicated a mean oocyst loss ranging from 51.2 to 99.6%, depending on the stool consistency as determined by the FEA concentration procedure. Our findings suggest that the most commonly used coprodiagnostic techniques may fail to detect cryptosporidiosis in many immunocompromised and immunocompetent individuals. PMID- 1715882 TI - DNA polymorphism in strains of Listeria monocytogenes. AB - DNA polymorphism in 35 Listeria monocytogenes strains belonging to serovars 1/2a, 1/2b, 1/2c, and 4b was studied by genomic DNA digestion. The restriction endonucleases ApaI and NotI, which cleave DNA at rare sequences, were used, and DNA fragments were analyzed by pulsed-field gel electrophoresis. Restriction fragment length polymorphism varied among different serovars and was used for epidemiological studies, but serovar 1/2c isolates could not be analyzed because their restriction patterns were indistinguishable. The genome sizes were calculated by addition of the sizes of the ApaI fragments and were found to be about 2,660 kb for serovar 1/2a strains, 2,640 kb for serovar 1/2b strains, and 2,710 kb for serovar 4b strains but only 2,340 kb for serovar 1/2c strains. This last group therefore appears to differ from the other serovar strains by the absence of restriction fragment length polymorphism and a chromosome that is 15% shorter, suggesting that strains of serovar 1/2c have quite recently emerged. PMID- 1715883 TI - Biochemical properties of the outer membrane of Treponema denticola. AB - The outer membranes (OMs) from serovars a, b, and c of Treponema denticola, originally isolated from periodontal patients, were prepared. Dialysis of the OMs against 20 mM MgCl2 yielded the aggregable (A) and the nonaggregable (NA) moieties of the OMs. The absence of muramic acid, adenosine triphosphatase, hexokinase, and nucleic acid as well as electron microscopy indicated that the OM preparations were homogeneous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the A and NA moieties of the OMs showed approximately 25 Coomassie brilliant blue R-250 stain-positive bands or 47 silver-stained polypeptides. The relative molecular masses ranged between 14 and 97 kDa. The electrophoretic polypeptide profiles of the A and NA moieties shared many similarities among serovars a, b, and c. However, they exhibited variation in the overall pattern, intensity, or location of the polypeptide stained zones. This was especially true for serovar b. Two-dimensional electrophoretic studies showed an excess of 100 silver-stained spots with isoelectric points of 4.6 to 7.0 and relative molecular masses in the 14- to 97-kDa range. The OMs contained simple proteins, glycoproteins, and lipoproteins. The NA moieties of the OMs contained 4 to 6, 10 to 12, and 4 to 6 glycopeptides as well as two, seven, and two lipoprotein bands for serovars a, b, and c, respectively. The A moieties of the OMs showed 7 to 9, 11 to 13 and 5 to 6 glycopeptides as well as four, five, and three lipoprotein bands for serovars a, b, and c, respectively. Lipopolysaccharide was detected in the OMs of the three serovars following removal of proteins with proteinase K, pronase and silver staining of sodium dodecyl sulfate-polyacrylamide gels, or removal of lipopolysaccharide from the OMs by hot phenol extraction. The 66- and 53-kDa bands were present in serovars b and c, while a band with a relative molecular mass of 45 kDa was present only in serovar c. Endotoxin-like activity was also shown in the OMs of the three serovars by the Limulus amebocyte clotting assay and the chick embryo lethality test. This is the first report on selected biochemical properties of the OM macromolecules of three known serovars of T. denticola. PMID- 1715884 TI - Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates. AB - Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats. PMID- 1715885 TI - A Cl- channel activated by parathyroid hormone in rabbit renal proximal tubule cells. AB - Previous data suggested an active Cl- conductance in the renal proximal convoluted tubule, although single channel conductance and regulation were not found. We have investigated the presence and regulation of the Cl- channel in proximal convoluted tubules by patch clamp analysis. The current-voltage relationship of whole cells with 130 mM NaCl in the pipette was nonlinear. The addition of 1-34 PTH (10(-8) M), forskolin, or cAMP significantly increased whole cell Cl- conductance. We found a single Cl- channel in excised apical membranes possessing conductance of 33 picosiemens (pS) at positive and 22.5 pS at negative potential, which was blocked by 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid (10(-4) M) and was selective to Cl- (Cl/Na = 10). The channel was activated by prolonged membrane depolarization, by a catalytic subunit of protein kinase A (PKA), or by purified kinase C (PKC), but not by Ca2+ (1 microM) inside the membrane. During cell-attached patch clamping, the channel was similarly activated by PTH, phorbol ester, or dibutyryl cAMP in a dose-dependent manner. To investigate second messenger contributions to the PTH-action, the PTH-evoked channels were modified further by the subsequent addition of several blockers of the second messengers. This suggested that PKA and PKC were involved in Cl- channel activation. We therefore conclude that renal proximal convoluted tubule cells possess an apical Cl- channel activated by PTH via the PKA and PKC pathways. PMID- 1715886 TI - Acidic polyamino acids inhibit human eosinophil granule major basic protein toxicity. Evidence of a functional role for ProMBP. AB - Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells in vitro, is a single polypeptide chain rich in arginine. MBP has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma. The MBP cDNA indicates that MBP is translated as a slightly acidic preproprotein with an acidic propart. To test the hypothesis that the acidic pro-part of proMBP inhibits the toxicity of mature MBP, acidic polyamino acids (aa) were used as antagonists of MBP toxicity to K562 cells and guinea pig tracheal epithelium and used as antagonists of MBP airway hyperresponsiveness in primates. The acidic poly aa inhibited MBP toxicity and MBP airway hyperresposiveness. The acidic poly aa inhibited MBP toxicity in a charge-dependent manner similar to that proposed for proMBP, suggesting that the acidic pro-part of proMBP functions to mask mature MBP toxicity. This inhibition was not limited to MBP, but also applied to polyarginine and eosinophil cationic protein. These acidic poly aa may be useful to inhibit the actions of a number of cationic toxins released by the eosinophil in numerous hypersensitivity diseases. PMID- 1715887 TI - Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa. AB - The object of this study was to further localize autoantigenic structures on IIb IIIa and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of IIIa that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to IIIa on intact platelets as well as in an immunoblot assay under nonreduced conditions. Using an immunoblot assay, a carboxy-terminal region of this fragment (33 kD) that contains the cysteine-rich domains of IIIa was found to carry the epitope(s) recognized by the prototype autoantibody RA. The amino-terminal amino acid sequence of the reduced 33-kD fragment, the smallest fragment that retains the RA epitope, is XPSQQDEXSP, and that of the reduced 50-kD fragment is IVQVTFD. This indicates that the 33-kD fragment consists of approximately 175 amino acids beginning at residue 479 and extending at least through residues 636-654, while the 50-kD fragment spans the same region but begins at residue 427. It is apparent that the 33-kD fragment is generated from the 50-kD fragment by additional chymotryptic hydrolysis but remains associated because of the multiple disulfide bonds that are characteristic of this cysteine-rich domain. Sera from 48% of patients with chronic ITP and 2 of 8 patients with acute ITP contain antibodies that bind to the 50-kD fragment in an ELISA. Antibodies of the same specificity are also found in one-third of patients with either secondary immune thrombocytopenia or apparent non-immune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of IIIa is a frequent target of autoantibodies in ITP, but that such antibodies may also be present in cases of thrombocytopenia that cannot be linked to an apparent autoimmune process. PMID- 1715888 TI - Construction of peptides encompassing multideterminant clusters of human immunodeficiency virus envelope to induce in vitro T cell responses in mice and humans of multiple MHC types. AB - To make synthetic peptide vaccines effective in a broad population of outbred humans, one would have to incorporate enough antigenic determinants to elicit recognition by T cells of most HLA types. We have previously defined multideterminant regions of the human immunodeficiency virus (HIV) envelope that include overlapping determinants seen by proliferating T cells of three or four haplotypes of mice. We have now tested the hypothesis that synthetic peptides encompassing such multideterminant regions will be recognized by T cells of multiple murine MHC types as well as by human T cells representing multiple HLA types. Six such peptides of 20-33 residues in length were prepared, and tested for their ability to stimulate T cells from mice of four distinct MHC types immunized with recombinant envelope protein rgp 160, as well as from 42 HIV infected humans of different HLA types. Results identify several such peptides that are broadly recognized by mice of four H-2 types and by 52-73% of infected humans who still retain IL-2 productive responses to control recall antigens such as influenza A virus or tetanus toxoid. 86% of such infected donors tested against at least three peptides respond to at least one of the six peptides, and 77% of an additional group of seropositives respond to a mixture of the peptides. Moreover, the peptides can be used to immunize mice to elicit T cells reactive with the intact HIV envelope protein. These peptides therefore may be useful for both vaccine development in the broad human population, and diagnostic or prognostic use. PMID- 1715889 TI - Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells. AB - Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment. PMID- 1715890 TI - The barrelettes--architectonic vibrissal representations in the brainstem trigeminal complex of the mouse. I. Normal structural organization. AB - The organization of the brainstem trigeminal complex (BTC) of the mouse is described, with emphasis on the normal organization of the vibrissal representations. Thionin staining for Nissal substance was employed to reveal the cytoarchitecture. Cytochrome oxidase histochemistry was used to reveal the chemoarchitecture. Golgi impregnation methods, in combination with thionin staining, were used to examine the neuronal dendritic morphology within a defined cytoarchitectonic context. An in vitro horseradish peroxidase labelling method was used to study the distribution and morphology of primary trigeminal afferent terminals within the BTC. The BTC consists of four distinct subnuclei: principalis (nVp), oralis (nVo), interpolaris (nVi), and caudalis (nVc). The present study shows that these sub-nuclei can be distinguished from each other on the basis of several anatomical criteria, including the distribution and density of neuronal size classes, histochemical staining intensity, morphology and orientation of neuronal dendrites, and size and texture of primary afferent terminal arbors. Anatomical manifestation of vibrissal representations within the BTC can be described in nVp, nVi, and nVc, but not in nVo. Within the three subnuclei where they are found, anatomical vibrissal representations are composed to architectural subunits that form an overall pattern homeomorphic to the pattern of vibrissae on the face of the animal. Each sub-unit forms a cylindrical tube running in a rostrocaudal orientation within the BTC. These sub-units will be called barrelettes. Cytologically, each barrelette consists of cell-dense "sides," surrounding a practically cell-free "hollow." Individual sub-units are separated by narrow, cell-free "septa." Histochemically, each subunit is manifested as a discrete patch of positive-staining reaction products. Differential interference contrast optics shows that these patches correspond precisely to the barrelette hollows. Evidence is presented to show that the barrelettes are the functional units for the processing of vibrissal sensory information. Terminal arborizations of individual primary afferents seem to be confined to the hollow of single barrelettes. The majority of neurons that form the sides of a barrelette have bitufted dendritic arbors, which project predominantly into the barrelette hollow, although a minority of neurons, particularly in nVi and nVc, also extend part of their dendritic arbors into adjacent barrelette hollows. The barrelette hollows are thus the principal neuropil region in which primary afferents and their target neurons interact. Contacts are made mainly between en passant varicosities and terminal boutons on primary afferent collaterals and dendritic spines and shafts of second order neurons.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1715891 TI - Glutamate-like immunoreactivity in retinal terminals in the nucleus of the optic tract in rabbits. AB - The ultrastructural organization of retinal terminals within the nucleus of the optic tract of rabbits was investigated with a combination of anterograde tracing and immunocytochemistry. The anterogradely transported WGA-HRP injected in the vitreous of the eye was visualized with the sensitive gold-substituted silver peroxidase (GSSP) method. Glutamate and GABA immunoreactivity were identified with postembedding colloidal gold particles. Retinal ganglion cell terminals (R terminals) in the nucleus of the optic tract formed asymmetric synapses and contained spherical vesicles and electron lucent mitochondria. R-terminals were observed in large clusters in the neuropil and in synaptic contact with large initial dendrites and somata. Within the clusters of neuropil the R-terminals formed two types of glomeruluslike arrangements: (1) an R-terminal centrally located and surrounded by small dendritic and axonal profiles and (2) several R terminals surrounding a single dendrite or a group of dendritic profiles, presumably of interneuronal origin. All R-terminals identified with WGA-HRP as well as those exhibiting similar ultrastructural characteristics showed high levels of glutamate immunoreactivity, but no GABA immunoreactivity. The presence of glutamate and the absence of GABA in R-terminals suggest that glutamate is involved in neurotransmission in the pathway from retina to the nucleus of the optic tract of rabbits. PMID- 1715892 TI - Rat tooth pulp projections to spinal trigeminal subnucleus caudalis are glutamate like immunoreactive. AB - It has been shown that glutamate-like immunoreactive axon terminals are present within the spinal trigeminal nucleus, including subnucleus caudalis. The morphology of many of these terminations is consistent with their identification as primary afferents. To establish whether primary afferent projections to subnucleus caudalis are glutamate-like immunoreactive, we injected an anterograde tract tracer into rat incisor tooth pulp, histochemically visualized this tracer within subnucleus caudalis, and then used an immunocytochemical technique to label glutamate-like immunoreactive profiles within these same sections. The anterograde tract tracer used, the B subunit of cholera toxin conjugated to horseradish peroxidase (B-HRP), is transported transganglionically and can be used to localize tooth pulp projection fibers in the spinal trigeminal nucleus. A majority of B-HRP projection fibers from rat lower incisors terminated ipsilaterally in axon terminals in the dorsal region of subnucleus caudalis. Labeled axon terminals were both scallop-shaped and smooth in profile. Small numbers of fibers containing B-HRP extended into laminae I-III caudally and were present in both the border zone between laminae IV and V and the most lateral region of lamina V rostrally. Approximately 75% of the B-HRP-labeled projection fibers were glutamate-like immunoreactive, providing evidence that the excitatory amino acid glutamate functions as a neurotransmitter in a subpopulation of these fibers. Terminals reactive for both B-HRP and glutamate-like immunoreactivity contained small, spherical round vesicles, formed asymmetric synapses, and participated in axoaxonic and axodendritic synaptic junctions. These results support the hypothesis that glutamate may be a transmitter of A delta and C fibers involved in relaying nociceptive information from the tooth pulp. PMID- 1715893 TI - Chlorpyrifos resistance in German cockroaches (Dictyoptera: Blatellidae) from restaurants. AB - Visual inspections and trapping revealed the presence of German cockroaches, Blattella germanica (L.) in 62 of 100 randomly selected commercial food-handling establishments. From live cockroaches trapped in 55 restaurants, 35 strains were established and tested for chlorpyrifos resistance. Poor sanitation and the likelihood of trapping cockroaches were associated. Twenty strains had resistance ratios (RR) as LD50 greater than 10-fold, a value exceeding the pest management threshold level for this insecticide. Resistance ratios were not associated with numbers of cockroaches trapped, the level of sanitation, numbers of different insecticide products used, or the number of treatments per month. Significantly more categories of insecticides (e.g., carbamate, organophosphate, inorganic) were used in restaurants with RR greater than 10 than in those with RR less than 10. PMID- 1715894 TI - Abnormal distribution of epidermal protein antigens in psoriatic epidermis. AB - The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis. PMID- 1715896 TI - Comparisons of various acidic treatments of bovine serum on insulin-like growth factor-I immunoreactivity and binding activity. AB - Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I) binding protein complexes during gel chromatography: one of Mr 150,000 and the other of Mr 40,000-45,000. The majority of the immunoreactive IGF-I was associated with the Mr 150,000 complex. Following acid-ethanol extraction of serum, the binding activity at Mr 150,000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose-response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment. PMID- 1715895 TI - Intracellular mechanisms involved in the stimulation of growth hormone release from rat anterior pituitary cells by Russell's viper venom. AB - We have previously shown that a heat-stable component of Russell's viper venom (RVV) releases GH in a dose-dependent manner from cultured rat anterior pituitary cells. We have now investigated the intracellular mechanisms involved in RVV stimulated GH release by concomitant administration of RVV with known intracellular mediators in rat pituitary cells. 3-Isobutyl-1-methylxanthine (IBMX; 0.5 mmol/l), added to cultured rat anterior pituitary cells simultaneously with RVV, at concentrations up to a maximally effective dose of 10 micrograms/ml, increased GH release (3.7-fold, 4.0-fold and 2.0-fold; P less than 0.001) compared with the effect of venom alone. These effects were additive, indicating that RVV and IBMX stimulate through different intracellular messengers. RVV failed to increase the formation of basal or IBMX-stimulated intracellular cyclic AMP (cAMP), confirming that RVV affects GH release through a cAMP-independent pathway. 12-0-Tetradecanoylphorbol-13-acetate (TPA; 0.1 mumol/l), added simultaneously with various doses of RVV (0.1-10 micrograms/ml), did not increase GH release beyond the maximal effect of RVV. This result indicates that RVV might be stimulating GH release through a similar mechanism to that of TPA (by activating protein kinase C). When pituitary cells were perifused with Ca(2+) free medium or verapamil (50 mumol/l), RVV-stimulated GH release was inhibited by 65 and 42% respectively. This reflects the recognized requirement of Ca2+ for secretory processes. However, RVV (10 micrograms/ml) had no significant effect on intracellular free Ca2+ concentrations as measured using the fluorescent Ca2+ probe quin-2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715897 TI - Inhibition of human hepatocellular carcinoma and hepatoblastoma cell lines by deferoxamine. AB - Inhibition of human hepatocellular carcinoma (PLC/PRF/5 and Hep3B) or hepatoblastoma (Hep G2) cell lines by inclusion of deferoxamine mesylate (desferrioxamine) (DFX) in the culture medium was evaluated. When PLC/PRF/5 cells were maintained for 7 days in 30 or 60 microM DFX, the cell number was decreased by 30-60%, little or no alpha-fetoprotein (AFP) was produced, and supernatant endpoint dilution titers of hepatitis B surface antigen (HBsAg) were reduced 1-2 logs. PLC/PRF/5 cells maintained for 7 days without DFX (simultaneous controls) grew to confluence, produced AFP that reached 10-60 ng/ml in the supernate, and the HBsAg titer remained constant or increased 1 log. Similar effects were observed in Hep3B and Hep G2 cells maintained in DFX (except that Hep G2 cells do not produce HBsAg), compared to simultaneous control cells grown in the absence of DFX. The growth of a human embryonic lung fibroblast cell line (Wl 38) was not significantly inhibited by DFX, although it grew at a slower rate than simultaneous control cells grown without DFX. Subsequent growth in FeSO4 of PLC/PRF/5, Hep3B, and Hep G2 cells that previously had been maintained in DFX did not reverse the effects of DFX. PLC/PRF/5 cells were also inhibited when maintained in medium containing equimolar concentrations of DFX and FeCl3 and in medium containing equimolar concentrations of DFX and FeSO4. PLC/PRF/5 cells were not inhibited by maintenance in up to 60 microM of another chelating agent that has a similar affinity for iron, calcium disodium versenate (EDTA). These studies show that DFX inhibits the growth of human hepatocellular carcinoma and hepatoblastoma cell lines regardless of the presence (PLC/PRF/5, Hep3B) or absence (Hep G2) of integrated hepatitis B virus DNA. The findings also suggest that the inhibition may have been due to mechanisms other than iron chelation. PMID- 1715898 TI - HIV reverse transcriptase inhibiting antibodies detected by a new technique: relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables. AB - A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection. PMID- 1715899 TI - Orthognathic surgery. PMID- 1715900 TI - The decade of the consumer. PMID- 1715901 TI - Polaroid photography: an important tool for esthetic dentistry. PMID- 1715902 TI - Intra-oral video photography: questions and answers. PMID- 1715903 TI - Computer imaging: an aid to treatment planning. PMID- 1715904 TI - Emergence of lasers in dentistry. PMID- 1715905 TI - Forensic dentistry: investigation of the Los Angeles Airport disaster. PMID- 1715906 TI - Oral health and aging: dental implications of a grayer America. PMID- 1715907 TI - The status and practice of complete dentures--a personal view. PMID- 1715908 TI - A case history: intra-alveolar root fracture. PMID- 1715909 TI - Immunoalkaline phosphatase with cerium-based cytochemistry for double staining at the transmission electron microscopic level. AB - Immunoelectron microscopic localization of surface antigens on lymphocytes is possible using alkaline phosphatase combined with cerium-based cytochemical methods. Distinctive electron-dense deposits are easily identified at sites of antibody binding. Mouse splenocytes showing surface immunoglobulin localization and human peripheral blood lymphocytes stained for the MHC-Class II antigen HLA DR illustrate the results. Double staining for murine Ia antigen by the alkaline phosphatase procedure, combined with immunogold labeling of antigens identified on dendritic cells, i.e., NLDC-145, demonstrates the utility of the cerium cytochemical method. PMID- 1715910 TI - Circular harmonic averaging of rotary-shadowed and negatively stained creatine kinase macromolecules. AB - The structure of mitochondrial creatine kinase is investigated by high-resolution shadowing at very low temperature and conventional negative staining. The electron microscopic images are analyzed with circular harmonic averaging, a method suited for the processing of single molecules. The rotational alignment and averaging is performed with the circular harmonic components, which allows data compression and several steps of noise reduction to be carried out within the averaging procedure. In addition, the symmetry can be deduced. For the mitochondrial creatine kinase, a fourfold symmetry is found that is compatible with the biochemical and biophysical characterization of the molecule. PMID- 1715911 TI - Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: the utility of methylamine tungstate stain and Butvar support film in the study of macromolecules by transmission electron microscopy. AB - The structure of ornithine decarboxylase (Mr approximately 1.04 x 10(6] from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted +/- 45 degrees. The two edge views were related by a 30 degrees rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-A resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10 degrees with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy. PMID- 1715912 TI - A method for high-resolution scanning electron microscopy of organic cartilaginous components. PMID- 1715913 TI - [Results of chemo-radiotherapy for nasopharyngeal carcinoma]. AB - We reported the data on 25 patients with nasopharyngeal carcinoma from 1984 to 1989, who received chemo-radiotherapy for initial treatment and follow-up by additional maintenance of chemotherapy for 2 years. The result of this study is as follows. 1. The 20 males and 5 females were included. 2. A median age of the patients was 62 years. 3. Stage grouping study showed 3 cases of stage II, 6 of stage III, and 16 of stage IV. 4. The local control rate was 92% and the rate of control in the cervical region was 96%. 5. However, the overall 4-year survival rate obtained by the Kaplan-Meiers method was 50%. 6. The cause of patients' death was mainly distant metastasis. PMID- 1715914 TI - [Motor innervation of the canine arytenoid muscle]. AB - Dual motor innervation by the bilateral recurrent laryngeal nerves (RLNs) has been demonstrated in the human arytenoid muscle (AR). Whether AR of the dog receives dual motor innervation remains to be cleared yet, although the canine larynx is frequently used in experimental studies. To answer this question, the author observed the muscular structure in detail and anastomotic nerve branch between the bilateral RLNs, and then carried out glycogen depletion experiments on AR of dog compared with typical unpaired ARs of monkey and of guinea pig. 1) Muscular structure AR of the dog consisted of three parts of muscle bundles: the transverse arytenoid muscle (TVA), ventricular muscle (VT) and anonymous small bundle provisionally named the smaller interarytenoid muscle (IAm). While TVA and VT were paired type, IAm was unpaired type and lay horizontally on the dorsal aspect of the sesamoid cartilage around the midline. So the canine AR displayed a trigastric muscle as a whole. 2) Anastomotic nerve branch By the vital staining with methylene blue, the arytenoid branch of canine RLN ramified in three directions: anteriorly to the bellies of TVA and VT, medially to the anastomotic branch and superomedially to IAm. By the silver impregnation method of Barker and Ip, the bilateral IAm ramuli were found to form collateral anastomoses and terminate disorderly on the individual fibers. 3) Glycogen depletion experiments When an electrical stimulation was applied to the unilateral RLN in the monkey and the guinea pig, about one half of AR fibers were unstained with PAS staining and, in turn, these unstained fibers were known to be innervated by the ipsilateral RLN. While these unpaired ARs receive dual motor innervation as a whole muscle, every individual fiber is innervated by the unilateral RLN. In the canine VT and TVA, almost 90% of fibers were depleted of glycogen on the belly of the stimulated side, while the reverse was on the nonstimulated side. This finding suggests that most fibers of canine VT and TVA are ipsilaterally and the remaining fibers are contralaterally innervated. About one half of fibers of IAm were unstained and the others were stained. This pattern was similar to that observed in the monkey and the guinea pig. Therefore, IAm receives dual motor innervation from both RLNs as a whole muscle. PMID- 1715915 TI - [The role of stromal components and Ca++ ATPase in pleomorphic adenoma and adenoid cystic carcinoma]. AB - In order to clarify the neoplastic-mesenchymal cell interaction, tumor structures were histologically classified into duct, solid and scattered types, and stromal changes were observed in each type with histochemical techniques. The quantitative and qualitative differences of the stromal components as proteoglycan and collagen were histochemically differed in these morphological features. Ca++ ATPase was ultrastructurally localized on the plasma membrane of myoepithelial cell in salivary glands. The activity of Ca++ ATPase changes in tumor cell differentiation of pleomorphic adenoma and adenoid cystic carcinoma. These results suggest that the stromal components and Ca++ ATPase play an important role on the tumor cell proliferation and differentiation in these tumors. PMID- 1715916 TI - Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells. AB - 1. Single-channel currents evoked in outside-out membrane patches from rat cerebellar granule cells by glutamate, aspartate, N-methyl-D-aspartate (NMDA), kainate and quisqualate were studied. Each agonist produced openings to five discrete amplitude levels. At a membrane potential of -70 mV, these levels correspond to single-channel conductances of about 8, 17, 30, 40 and 50 pS. NMDA, aspartate and glutamate evoked mainly 50 pS openings and also substantial numbers of 40 pS events. Kainate evoked primarily 8 and 17 pS openings. 2. The relative proportion of openings to each conductance level showed no dependence on membrane potential. At membrane potentials negative to -100 mV, current-voltage plots for 30, 40 and 50 pS openings showed substantial inward rectification. 3. With NMDA, aspartate and glutamate, the most common type of direct transition was between the 50 pS open level and the shut level. Transitions between the 30, 40 and 50 pS levels were also relatively common. With few exceptions, 8 and 17 pS openings appeared to arise directly from, and return directly to, the shut level. The differences between granule cells and certain other central neurones, in the types of transitions associated with NMDA receptor channels, provide evidence for the existence of more than one type of NMDA receptor. 4. Four exponential components were identified consistently in the shut-time distributions that were obtained with NMDA, aspartate and glutamate. Mean time constants for the briefest two components were 30 to 65 microseconds and 0.65 to 1.00 ms. The mean duration of these 'gaps within bursts' differed for different agonists, but did not vary with membrane potential. 5. Two exponential components were distinguished in most open-time distributions for the 50 pS level (time constants, 0.9-1.2 and 3.2-3.9 ms at -100 mV), whereas open-time distributions for 30 and 40 pS events were described adequately by single exponentials with time constants below 1.0 ms. The duration of 50 pS openings decreased with hyperpolarization. 6. Mean open times for 8 and 17 pS events produced by NMDA, aspartate and glutamate were 0.3 to 0.7 ms. The longest such openings were observed with quisqualate. 7. Three exponential components were present in distributions of burst length, and of total open time per burst, that were obtained with NMDA, aspartate and glutamate. The slowest two burst-length components had mean time constants of 1.7-2.4 and 10.6-13.0 ms and originated from the kinetic behaviour of the 50 pS state.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1715917 TI - Single channels activated by high concentrations of GABA in superior cervical ganglion neurones of the rat. AB - 1. Single-channel currents evoked by high concentrations of GABA (10-2000 microM) have been analysed to investigate the characteristics of GABAA receptor channels in outside-out patches from rat sympathetic neurones. When high concentrations of GABA were applied to a patch, channel openings occurred in prolonged clusters (3.8 +/- 3.7 s (mean +/- S.D.) at 50 microM-GABA) consisting, on average, of 350 apparent openings per cluster. Individual clusters were separated by long silent intervals. 2. Channel openings were to many (often ill-defined) conductance states (range 7-36 pS), but the most frequently observed conductance level was approximately 30 pS, (29.6 +/- 0.34 pS). Only these clusters during which the channel was open to this main state conductance for at least 95% of the cluster open time were used in the analysis of probability of being open. Other less frequently observed conductance levels were 15-18 and 22-23 pS, while levels of 33-36 and 7-9 pS were occasionally, but reliably, observed. 3. Bursts within clusters were defined as a series of openings separated by closed intervals shorter than some critical value, tc. At 50 microM-GABA the mean burst length was 439 +/- 434 ms (+/- S.D., tc = 50 ms). 4. The probability of being open, po, during bursts within clusters has been analysed as a function of GABA concentration. As expected, increasing the concentration of GABA resulted in an overall increase in po. However, for a given agonist concentration there was a wide spread in po, far greater than that predicted for a population of identical and independent receptor channels (demonstrated by comparison with stimulated channel activity). 5. The wide range of po values at a particular concentration of GABA was not due to inappropriate selection of tc. At 50 microM-GABA the range of po values was similar for tc of 20-1000 ms, although the overall mean po became lower (0.64 rather than 0.81). 6. On the basis of simulated channel activity, it appears that most clusters which do not contain multiple openings, arise from the activity of one individual channel. Furthermore, there was no detectable tendency for gaps between bursts to be shorter in the middle of a cluster than at its ends. Therefore it is unlikely that variability in po arose from overlapping activity of two or more channels. 7. The values for po, mean open time, and mean shut time for bursts within the same cluster, and between different clusters, were compared by a randomization test.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1715918 TI - Two types of Ca2+ currents are found in bovine chromaffin cells: facilitation is due to the recruitment of one type. AB - 1. Whole-cell Ca2+ currents in cultured bovine chromaffin cells were studied using patch-clamp electrophysiology. With Ba2+ or Ca2+ as the current carriers, two separate components of whole-cell current could be distinguished by biophysical and pharmacological criteria. These components of Ca2+ current were different from T- or N-type Ca2+ channels previously described, as they were not inactivated at a holding potential of -60 mV. 2. Depolarization of the cells past -20 mV in 10 mM-Ba2+ activated a single component of Ca2+ current, called the 'standard' current. This current showed no detectable voltage-dependent inactivation, but did show marked current-dependent inactivation as steady-state inactivation (H-infinity) plots obtained in the presence of Ba2+ were quite different from those obtained from Ca2+. 3. In most chromaffin cells large pre depolarizations or repetitive depolarizations in the physiological range activated a second component of Ca2+ current called 'facilitation'. Facilitation was observed with either Ca2+ or Ba2+ as the charge carrier. Recruiting facilitation increased whole-cell currents by an average of 60%. 4. Pre-pulses to +120 mV lasting 200 ms completely activated facilitation. Pre-pulses longer than 800 ms started to inactivate facilitation, while pre-pulses longer than 2500 ms completely inactivated this component of Ca2+ current. Because only outward currents were recorded at +120 mV, it is likely that facilitation inactivated in a voltage-dependent manner. 5. When the extracellular Ba2+ concentration was increased in the range from 2 to 90 mM activation of both facilitation and standard Ca2+ currents shifted in the depolarizing direction. In 2 mM-Ba2+ facilitation activated at potentials 10 mV more negative than the standard component, while in 90 mM-Ba2+, facilitation activated at a potential about 10 mV more depolarized than the standard component. Thus, the voltage sensor for the facilitation Ca2+ current appeared to sense more surface charge than did the standard Ca2+ current. 6. Tail currents measured at -20 and -30 mV in the absence of facilitation (without pre-pulses) showed one time constant for current deactivation. Tail currents measured with both facilitation and standard currents activated showed a significantly slower deactivation rate than that seen with the standard current alone. 7. The dihydropyridine antagonist nisoldipine (1 microM) completely suppressed the facilitation Ca2+ current even when cells were held at negative holding potentials (-80 mV). In contrast, the standard current was unaffected by 1 microM-nisoldipine, even at depolarized holding potentials (-20 mV).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1715919 TI - Structure-activity relationships of vasopressin analogues on release of factor VIII in dogs. AB - In order to study structure-activity relationships as to Factor VIII release conscious dogs were injected with analogues of vasopressin. The peptides used were chemically modified either in the hexapeptide ring structure of the vasopressin molecule or in the C-terminal tripeptide or in both. The results showed that an intact C-terminal appears to be of importance for retaining Factor VIII releasing activity of the analogues, whereas at least some modifications of the ring structure are tolerated without loss of activity. Decreased activity was also observed when the disulphide bridge was substituted with a monocarba bond. PMID- 1715920 TI - Relationship of orientation with affinity and activity of receptor-bound glycosylation variants of human chorionic gonadotropin (hCG), as visualized by monoclonal antibodies (MCA). AB - When hCG was receptor-bound, only 2 epitopes (i.e. beta 3 and beta 5) out of its previously mapped total of 14 surface epitopes (10, 11, 12) could be detected by the respective 125I-MCA. Clearly, this indicates a non-random orientation of hCG in this particular state (1). Now we report that on receptor-bound desialylated (asialo-hCG) as well as on receptor-bound deglycosylated hCG (degly-hCG), the beta 3 and beta 5 epitopes were inaccessible for 125I-MCA as were the remaining epitopes, although both variants, when not receptor-bound, were indistinguishable from native hCG with respect to number and topography of epitopes. Thus, the carbohydrate (CHO) units of hCG neither seem to be part of these 14 antigenic sites nor to contribute to the affinity of receptor binding: both variants had even higher affinities than native hCG. However, since the CHO are known to be obligatory for triggering the postreceptor responses of hCG-stimulated target cells, as seen by the 50% reduced or totally abolished biological potency of asialo-hCG and degly-hCG, respectively, the here demonstrated clear-cut differences in epitope accessibility can be related to differences in receptor bound orientations which reflect signal transduction-competent and incompetent modes of interaction with the receptor. We believe that the CHO, while not contributing to receptor binding per se, function to assure correct positioning of hCG in the ligand recognition domain to allow for proper protein-protein interactions between ligand and receptor and thus optimal activation and hence transduction of the external signal to the cell's interior. In addition, the fact that most of the surface epitopes were masked on receptor-bound hCG, represents the first experimental support for the sofar unproven hypothesis that this unusually long (i.e. 341 amino acid residues) extracellular N' terminal domain of the recently cloned hCG receptor (hCG-R) (23,24), is indeed involved in ligand recognition. PMID- 1715921 TI - The use of PCR to probe calcium channel diversity. AB - Voltage-dependent calcium channels are a diverse set of proteins that can be classified into at least 3 classes based on their electrophysiological and pharmacological behavior. Our studies have focused on the dihydropyridine sensitive L-type class, which has two isoforms that have been cloned and expressed. In this report we describe the development of a polymerase chain reaction (PCR, Cetus) to probe for the expression of L-type calcium channel isoforms. We describe the optimization of the PCR reaction in terms of the following: methods for producing the template cDNA, concentration of primers, and magnesium concentration. In addition, we discuss our efforts to understand the factors involved in the design of oligonucleotides for PCR primers. These studies led to the following conclusions: 1) that primers should be less than 30 base pairs in length, 2) that the addition of extraneous polylinker sequences on the 5' end of the primer has no effect, 3) that the primer should not be located in regions where secondary structure may exist, and 4) that non-degenerate primers can be used to amplify homologous gene family members. We also present methods for subcloning PCR fragments, which allow the product of a single reaction to be subcloned and sequenced. We illustrate the use of all these techniques with RNA from mouse ovary, where we have discovered the expression of the cardiac isoform of the dihydropyridine-sensitive L-type calcium channel, and the expression of a novel sequence that we postulate to be an isoform of L-type calcium channels. PMID- 1715922 TI - Functional properties of the nicotinic and glutamatergic receptors. AB - Several important physiological processes such as plasticity, memory, cell death, and rhythmic firing involve the N-methyl-D-aspartate (NMDA)-type of glutamatergic receptor. Nicotinic acetylcholine receptors (AChR), recently demonstrated in the central nervous system (CNS), are also of great interest. We have used several ligands to study the physiology and pharmacology of the agonist recognition sites of these receptors and kinetic properties of associated ion channels using whole cell, cell-attached or outside-out variants of the patch-clamp technique. Enzymatically dissociated frog interosseal muscles were used to study peripheral AChRs, and tissue cultured or acutely dissociated hippocampal neurons and retinal ganglion cells (RGCs) for CNS receptors. For reproducible and fast solution changes when recording in the whole-cell configuration, we modified the "U" shaped tube system to obtain different outputs from the same outflow port. We used fluorescent rhodamine-labeled latex microspheres to identify RGCs. Our studies provide important information regarding the molecular mechanisms of several clinically used agents. Additionally, similar actions of noncompetitive agents on the ion channels of the nicotinic ACh and NMDA receptors support the concept of a receptor ion channel superfamily. PMID- 1715923 TI - Endoscopic insertion of Celestin tubes in carcinoma of the oesophagus. PMID- 1715924 TI - Tumor cell adhesion to endothelial cells: endothelial leukocyte adhesion molecule 1 as an inducible adhesive receptor specific for colon carcinoma cells. AB - Activation of endothelial cells by the two inflammatory mediators interleukin-1 (IL-1) and tumor necrosis factor strongly increases tumor cell adhesion. We describe antibody inhibition studies showing that the endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell-surface glycoprotein selectively expressed by cytokine-activated endothelial cells and responsible for neutrophil adhesion, is the major, if not the only, mediator of colon carcinoma cell adhesion to activated endothelial cells. Among the different tumor cell lines tested, seven colon carcinoma cell lines were sensitive to ELAM-1 antibodies. Adhesion of melanoma, osteosarcoma, and lung, cervix, or kidney carcinoma cell lines to IL-1 treated endothelial cells was not affected by the ELAM-1 antibody. This result suggests that ELAM-1 is selectively recognized by colon carcinoma cells and that adhesion of tumor cells to activated endothelial cells could be mediated by different and specific mechanisms. PMID- 1715925 TI - Monoclonal antibody Br4 recognizes specific neuronal cell types. AB - A monoclonal antibody termed as Br4 was prepared by a fusion of the myeloma P3 x 63-AG8-653 with spleen cells from BALB/c mice immunized with chick embryonic brain cells. Immunocytochemically, it reacts strongly with certain neurons in cerebral cortex, hippocampus, substantia nigra, and granular layer of cerebellum from rat brain but does not react with the white matter. A monoclonal antibody Br4 also reacts with primary cultured chick neurons, but not with cultured astrocytes. Western blots show that Br4 recognizes three proteins of 145,000, 108,000, and 97,000 molecular weight from the rat brain. The protein with molecular weight 145,000 (p145) and the protein with 97,000 molecular weight (p97) are essentially soluble; p145 is especially enriched in the synaptosomal soluble fraction. The protein with 108,000 molecular weight (p108) is found in both membrane-bound and soluble forms. Western blots show that among the tissues examined the three proteins are found most abundant in brain and especially enriched in the cerebral cortex and hippocampus. The Br4 antigens may be useful in identifying neuronal cell subpopulations in the central nervous system. PMID- 1715926 TI - [Immunosuppressive agents--advances of the developmental studies and the mode of action]. PMID- 1715927 TI - Pharmacological studies on lappaconitine: antinociception and inhibition of the spinal action of substance P and somatostatin. AB - The pain response of mice to an injection of 0.5% formalin into the dorsal surface of a hindpaw is biphasic, with a first phase lasting for 5 min and a second phase lasting from 10 to 30 min post-injection. Intrathecal (i.t.) injection of [D-Pro2, D-Trp7,9]-substance P inhibited the first phase, and i.t. cysteamine inhibited the second phase. Lappaconitine (LA) and morphine (MOR) inhibited both phases equally in a dose-dependent manner. Diclofenac inhibited both phases, but the second phase was inhibited by lower doses. An i.t. injection of substance P (SP) or somatostatin (SOM) produced a characteristic behavioral response (scratching, biting, and licking). This behavioral response to SP and SOM was inhibited by s.c., intracerebroventricular (i.c.v.), or i.t. injection of MOR. In contrast, LA inhibited the SP- and SOM-induced response when injected s.c. or i.c.v., but had no effect when injected intrathecally. These results indicate that LA may act supraspinally to inhibit the transmission of nociceptive information by SP and/or SOM. PMID- 1715928 TI - [Surgical management of congenitally corrected transposition of the great arteries]. AB - From 1962 to 1990, we have experienced 12 patients with congenitally corrected transposition of the great arteries (CTGA). Associated cardiac defects were present in all cases, most frequently ventricular septal defect (100%), and pulmonary stenosis (67%), Palliative procedures were done in 5, corrective operations in 6. One patient underwent corrective procedure 27 years after palliation. The hospital mortality rate was 8% (1/12), and 1 late death (8%) was seen in this series. In most cases, we approached the defect through the mitral valve, and the deLeval method was very useful in placing stitches along the trabecular septum. AV conduction disturbance could be avoided in 5 of 6 patients who underwent the closure of ventricular septal defect. Pulmonary stenosis was relieved by valvotomy and/or infundibulectomy, or implantation of an extracardiac conduit according to their anatomy. Cardiac function of systemic ventricle (morphologic right ventricle) were well preserved in the case undergoing intracardiac repair and also in the palliative cases. Careful observation is needed in these cases. PMID- 1715929 TI - [Regulation of ATP-dependent potassium channels]. PMID- 1715930 TI - [Separation of bone marrow cells in a Percoll discontinuous density gradient]. AB - Human bone marrow cells were separated by centrifugation in percoll discontinuous density gradient with 1.020-1.080 g/ml densities. CFU-GM test and EPICS analysis were used to characterize cell compositions of the resultant fractions. The employed mode of separation permits obtaining cell fractions rich for monocytes, macrophages, T and B lymphocytes. PMID- 1715931 TI - [An automated scintillation counter for poorly contrasting microparticles]. AB - A cell scintillator intended for analysis of the disperse composition and concentration of poor contrast biologic objects suspended in transparent liquid media has been designed at the Analitpribor Research and Production Amalgamation in Tbilisi for the Cytology Institute of the USSR Academy of Sciences in Leningrad. The apparatus is based on light blocking and hydro-focussing physical principles. Automated system of the apparatus calibration permits measurements of the particles 3 to 150 microns in size at the level of 10%. The mean error of cell concentration estimation within the range of 10000-150000 cell/ml is 3%. PMID- 1715932 TI - [The cytochemical determination of lysosomal cationic proteins in the blood neutrophils in hypertrophic cardiomyopathy]. AB - Examinations of 33 patients with hypertrophic cardiomyopathy have revealed Stages I-II circulation insufficiency in 15. The findings were compared with those in 20 untrained normal subjects and 8 athletes. Lysosomal cationic protein levels were measured in polymorphonuclear leukocytes by the cytochemical method at rest, at the peak of dosed exercise (permanently increasing bicycle spiroergometry) and over the course of the recovery period--in 3, 12, and 24 h after exercise. The neutrophil distribution was also estimated by the staining intensity. Lysosomal cationic protein levels in hypertrophic cardiomyopathy patients with and without circulation insufficiency were changed to a different degree; these characteristics essentially varied in the patients, normal untrained subjects, and athletes. Changed levels of lysosomal cationic proteins appear to reflect the microcirculation disorders and the augmenting hypoxemia in the patients with circulation insufficiency, and thus may be used as a criterion to assess the clinical course of hypertrophic cardiomyopathy. PMID- 1715934 TI - [Lactoferrin and fibrinogen degradation products in saliva]. AB - The authors suggest a simple and convenient procedure for measuring acute-phase proteins, lactoferrin, fibrinogen and its degradation products, in mixed human saliva by the agar immunodiffusion test with appropriate monospecific antisera. Examinations of 1205 salivary samples have revealed that salivary fibrinogen degradation products and lactoferrin detection rates do not depend on the subject's sex but are growing with age and intensity of the aggressive admixture effects in inhaled air; they are also related to the intensity of the inflammatory and proliferative processes in the oral cavity and bronchopulmonary system, the values varying from 4-16 percent in health to 68.8-71.8 percent in disease. In monitoring salivary acute-phase proteins detection rates make up 100 percent in the patients and 0-4 percent in normal subjects. The authors come to a conclusion that measurements of salivary acute-phase proteins may be used in screening of the population to detect subjects at risk for further diagnostic examinations. PMID- 1715933 TI - [Leukocyte indices in infections of premature infants]. AB - The problem of search for objective criteria for the prediction of infectious inflammatory conditions in preterm newborns is still a pressing one. The authors demonstrate the informative value of unsophisticated diagnostic tests--leukocytic indexes--in infections of the preterm newborns born to mothers excreting M. hominis and Ur. urealyticus during delivery. PMID- 1715935 TI - [A micromethod of determining the Willebrand factor in blood]. AB - The suggested method for Willebrand's factor detection differs from the known methods by a 2-3 times higher sensitivity and takes 10 times less blood. The technique is based on estimation of washed formalin-treated donor platelet ristomycin aggregation index by scintillating these cells in Laborskel automated scintillator in supernatants of 2 samples with 0.1% ristomycin solution after the tested platelet-free plasma is added to one of these samples. Willebrand's factor level is found on the calibration curve of standard dilutions of a mixture of platelet-free donor plasma. The method is sufficiently simple, operative, and sensitive; it permits the diagnosis of not only Willebrand's disease, but of the hyperaggregation syndromes associated with vascular endothelium involvement that occur in various conditions, as well; it helps monitoring the efficacy of substitute and antiaggregation therapy. PMID- 1715936 TI - [A comparative evaluation of methods of determining melanogen in the urine of patients with steroid-dependent bronchial asthma]. AB - Fifty-four patients with steroid-dependent asthma of varying severity during exacerbation were examined to compare the efficacies of urinary melanogen measurement techniques. Jaksch's, Tormelen's methods, and the technique with 2M sulfuric acid were used. The tests were positive in but 3 (15%) patients with a medium severe course of the process and in 23 (68%) ones with a grave condition. Jaksch's and Tormelen's tests were similar in sensitivity, and the hydrochloric acid test was less demonstrative. Melanuria manifestation may be regarded as an early preclinical sign of adrenal insufficiency. Pigment metabolism studies in patients treated with steroids for a long time may be used as an extra method for the assessment of adrenocortical function and for monitoring the course of glucocorticosteroid therapy. PMID- 1715937 TI - [A method of determining the adhesive properties of sputum]. AB - Adhesive characteristics of the sputum are assessed from adhesion to glass. The force and time of preliminary pressure on the tested material are not taken into consideration, nor is the rate of augmentation of the force directed to disruption of the adhesion contact. The strength of the adhesion contact was found directly depending on the force and time of preliminary pressure and inversely related to disrupting force augmentation rate. A conclusion is made on the necessity of stabilizing work schedules (in reference to the above parameters) for the standardization of the method. PMID- 1715938 TI - [The determination of amylo-1,6-glucosidase in biopsy specimens from human chorion]. AB - This method has been developed for the prenatal diagnosis of a grave hereditary disease, a deficiency of amylo-1,6-glucosidase (A-1,6-G) activity. The methods suggested earlier did not rule out the effects of other glucosidases, that could result in false-positive data if the fetus was involved. The new method for measuring A-1,6-G activity includes sedimentation of acid alpha-glucosidase with rabbit blood antiserum to human placental acid alpha-glucosidase. A-1,6-G activity is estimated from the difference in glucose accrement with limit dextrin and glycogen used as substrates. A-1,6-G activities were measured in 15 chorion samples obtained in the first trimester of pregnancy by the developed method, and normal values of this parameter established, making up 39.77 +/- 8.1 nmol/h per g of protein. The method may be useful in the prenatal diagnosis of type III glycogenosis. PMID- 1715939 TI - [Characteristics of the distribution and reference values of 12 biochemical indices of the blood serum]. AB - Twelve blood serum parameters: sodium, potassium, chlorine, total protein, cholesterol, glucose, urea nitrogen, creatinine, bilirubin, alkaline phosphatase, alanine and aspartate aminotransferases activities were measured in 488 normal subjects (male), residents of Leningrad, by the Technicon (USA) microanalyzer. Relationships between the estimated reference values and the distribution patterns of the referent blood parameters (variability, symmetric pattern) were analyzed. The authors compare the reference values of the Leningrad population with those obtained by this microanalyzer in the USA. PMID- 1715940 TI - [A method of measuring the triboluminescence of blood using the TRA-2 rapid analyzer]. AB - The procedure of triboluminescent analysis of blood lipid peroxidation processes using TRA-2 express analyzer is described. 0.02 ml of the blood is sufficient for analysis that takes 40 seconds; all the measurements are automated, no laboratory assistants participate here; no special chemical reagents are necessary. Results of laboratory clinical trials evidence good prospects of employing this method for the differential diagnosis between oncologic and inflammatory diseases. PMID- 1715941 TI - [Neutrophil adhesion: pathogenetic and methodologic aspects (review of the literature)]. PMID- 1715942 TI - [A modification of the fluorescent stain method for identifying a deficit of erythrocyte phosphoglycerate kinase in mass screening]. AB - The developed modification may be used in clinical practice as a diagnostic test and in mass screenings for phosphoglycerate kinase deficiency: more than a hundred tests may be made in a day. If blood samples have to be sent by mail, their quality is unchanged for up to 7 days. PMID- 1715943 TI - [Commercial preparations for the immunologic diagnosis of infections in pediatric hospitals]. AB - The authors analyze the shortcomings of Soviet commercial agents intended for the diagnosis of infectious diseases at pediatric hospitals. Prospects for the possible solution of these problems and for commercial manufacture of new agents are outlined. Possible schemes of multicomponent procedures applicable at pediatric hospitals of various profiles are presented, that may be used in the production of multicomponent purposeful test systems. PMID- 1715945 TI - [The prognostic significance of cellular immunity indices in patients with peptic ulcers]. PMID- 1715944 TI - [Stable erythrocyte reagents for detecting antigen-binding lymphocytes]. AB - Fixation of native chicken red cells with a solution containing 14.4 percent of acetate aldehyde and 0.85 percent of NaCl (pH 7.0-7.2) permitted preparation on the basis of these red cells of stable immunoreagents for the detection of antigen-fixing lymphocytes using any (optimal for the antigen used) procedure. Experiments have demonstrated a high efficacy of detecting staphylococcal, proteus, or Ps. aeruginosa etiology of pyoinflammatory and septic conditions in indirect rosette formation test with the developed immunoreagents. The levels of the detected antigen-fixing lymphocytes varied in the patients from 0.7 to 8.0 percent, the mean value being 2.1 +/- 0.1 percent. PMID- 1715946 TI - [The use of semi-starvation nutrient media for phage typing of Staphylococcus aureus]. AB - A total of 161 Staphylococcus aureus strains grown in Hottinger's broth and semi starvation medium (1% peptone water with 1% glycerol) were phage-typed. 107 (66.4 +/- 3.1%) of the 161 cultures grown in the semi-starvation medium could be typed and only 77 (48.1 +/- 3.63%) of the 161 grown in Hottinger's broth, which fact speaks in favor of semi-starvation nutrient media employment for phage-typing of S. aureus. PMID- 1715947 TI - [Evaluation of the efficacy of collection media used in hospital inspections]. AB - A total of 2303 parallel studies of washings off hospital environmental objects were carried out with 1% sugar broth and 1% peptone water used as enrichment media. Sugar broth was found preferable for the isolation of Staphylococci and gram-negative nonfermenting bacteria. Direct inoculations of washings off the environmental objects collected at treatment and prophylactic institutions for Staphylococci were found ineffective. PMID- 1715948 TI - [A method of determining glucose-6-phosphate dehydrogenase in bacteria of the genus Yersinia]. AB - A method for qualitative assessment of glucose-6-phosphate dehydrogenase in Yersinia is suggested. The reaction mixture was prepared by mixing the ingredients in the ratios as follows: 10 microliters of 0.01 M glucose-6 phosphate, 10 microliters of 0.0075 M NADP, 30 microliters of 0.75 M tris HCl pH 7.8, 10 microliters of 0.2 M MgCl2, 20 microliters of distilled water. The reaction was triggered by adding 20 microliters of cell-free bacterial extract. After 110-130 min incubation 10-20 microliters aliquots w re collected on filter paper (Whatman No 1) strips. The stains were dried at ambient temperature and examined in UV light at 365 nm with the use of a routine UV lamp. This method shows intensive fluorescence of the stain in M. tuberculosis and very poor fluorescence in Y. pestis. This technique is convenient when the scope of identification tests and differentiation between these two agents is high. PMID- 1715949 TI - [The use of micromethods for the identification of Vibrios]. AB - The authors recommend micromethods for laboratory studies of Vibrio; such methods may be widely used at bacteriologic laboratories for examinations of biochemical characteristics of these microorganisms, for rapid identification of V. cholerae 01, and for serologic identification (typing) of V. cholerae non 01, since they accelerate the diagnosis and are much simpler than macromethods. PMID- 1715950 TI - [The effect of the growth period of a culture of L-929 cells on its sensitivity to the action of Vibrio cholerae cytolysin]. AB - The activity of purified V. cholerae cytolysin was estimated from its cytotoxic effect on L-929 cells in various growth phases. The period of target cell development was supposed to influence the sensitivity of the test. Cytolysin preparation is thermolabile, and its effect is neutralized with homologous antiserum. PMID- 1715951 TI - [An immunoenzyme method of detecting Vibrio cholerae cytolysin]. AB - Enzyme immunoassay was used in detection of V. cholerae cytolysin. Conjugate of immunoglobulins to purified cytolysin with horseradish peroxidase was used, obtained by the periodate technique. The method sensitivity is 2 ng/ml of purified cytolysin. The results of hemolytic activity measurements in supernatants of 40 V. cholerae strains and enzyme immunoassay findings were in high correlation. PMID- 1715952 TI - [A false positive reaction to lactose in polycarbohydrate media]. AB - In combined media for primary identification Salmonella typhi are detectable by the lactose test only within a certain range of proteolytic activities, which fact is explained by specific features of these media. Reduced proteolytic activity and thiosulfate reductase activity in S. typhi cultures resulted in false-positive lactose test and false-positive hydrogen sulfide production test, this leading to identification of these cultures as Escherichia in accordance with the universally acknowledged classification scheme. Taking this feature into consideration, the author has additionally isolated 20 typical S. typhi strains of the 22 cultures isolated in the laboratory. PMID- 1715953 TI - [Qualification requirements for physician-bacteriologists]. AB - The authors present the qualification requirements to laboratory physicians specialized in bacteriology. The knowledge and skills of a bacteriologist, necessary for his organizations, methodologic, prophylactic, and diagnostic activities, are listed. PMID- 1715954 TI - [Current problems of personnel policy in clinical biochemistry]. PMID- 1715955 TI - [Methodology of calculating the net cost of biochemical clinical studies]. AB - A method for estimating the net cost of biochemical investigations has been developed as exemplified by the clinical laboratory of the N. N. Petrov Research Institute of Oncology of the USSR Ministry of Health. A data bank including information on the wages fund, cost of material used, equipment wear, and other expenditures was created for these estimations. Labor consumption for each of the 27 types of biochemical investigations comprised the production time of physicians (79.3 percent) and laboratory assistants (87.3 percent). Net cost of physicians' productive minute has made up 0.04 roubles, that of laboratory assistants' minute, 0.02 roubles. Wastes unrelated to the time of investigation were also taken into consideration. PMID- 1715957 TI - [Calculation of the expenditure of time on laboratory studies (methodological recommendations)]. PMID- 1715956 TI - [Provision of quality control in hemoglobin studies]. AB - Hemolysate with 30% of ethylene glycol was used for plotting calibration graph and for calibration of micropipets employed in blood collection and hemoglobin determination. The hemolysate was treated for 20 days; statistical characteristics were determined (mean value, root-mean-square deviation, coefficient of variations). A control chart was plotted for every laboratory assistant engaged in blood collection and measurement of hemoglobin concentration. Errors possible in estimation of hemoglobin concentration at clinical diagnostic laboratories are analyzed. PMID- 1715958 TI - [Evaluation of the morphofunctional status of peripheral blood cells using the GTsMK-3 hemocytometer]. AB - The suggested complex method for assessment of red blood cell and leukocyte counts and membranous function helps assess the time course of cellular membrane and peripheral blood status from the minimal volumes of the blood by precise estimation of cell loss using conductometry. PMID- 1715959 TI - Acute myeloid leukemias with chromosomal abnormalities involving the 21q22 region identified by their in vitro responsiveness to interleukin-5. AB - Among 52 patients diagnosed as acute myeloid leukemia (AML), nine cases were found in which interleukin-5 (IL-5) induced a proliferative response in the leukemic cells, as measured by the stimulation of DNA synthesis or colony formation in vitro. All cases (n = 7) with the cytogenetic abnormality t(8;21)(q22;q22) belonged to this group of IL-5 responders. Of the additional two cases, one had an apparently normal karyotype, but the other expressed a dicentric chromosome 21, an abnormality also involving the breakpoint region 21q22. The leukemic cells of the IL-5 responsive patients could also be stimulated to proliferate by IL-3, GM-CSF and G-CSF, and in some cases by IL-6 or M-CSF. Immunophenotypic analysis revealed the presence of the immature hematopoietic cell antigen CD34, the myelomonocytic maturation antigens CD13 and CD33, in association with the B-cell related surface marker CD19 on the leukemic cells. Immunoglobulin mu and T-cell receptor beta-genes in the leukemic cells were in germline configuration. Upon incubation in colony culture, clonogenic cells were capable of producing progeny showing eosinophilic or neutrophilic maturation following stimulation with IL-5 or G-CSF, respectively. It is concluded that IL-5 responsive AML represents a subgroup of leukemia with distinct immunotypic and cytogenetic features. PMID- 1715960 TI - Deficiency of a phosphatidylinositol-anchored cell adhesion molecule influences haemopoietic progenitor binding to marrow stroma in chronic myeloid leukaemia. AB - The interactions between haemopoietic progenitor cells and marrow stromal cells that are essential for the regulation of normal haemopoiesis are defective in chronic phase chronic myeloid leukaemia (CML). The presence of primitive progenitor cells (blast colony-forming cells, Bl-CFC) in the blood of patients with CML is reflected by their reduced capacity to bind to marrow derived stromal layers in vitro. Whereas normal bone marrow Bl-CFC bind irreversibly to cultured stromal layers (and none are found in normal blood), the Bl-CFC in CML bind transiently and then detach. The normal cell adhesion mechanism is partially sensitive to treatment with phosphatidylinositol-specific phospholipase C (Pl PLC), indicating the participation of a phosphatidylinositol (Pl)-linked structure; however, when CML cells were treated with Pl-PLC it had no effect on progenitor binding. Two other Pl-linked structures, decay-accelerating factor (DAF) and lymphocyte function associated antigen-3 (LFA-3) were normally expressed on CD34 positive CML cells and normally susceptible to Pl-PLC treatment. The treatment of normal cells with Pl-PLC, to mimic the situation in CML, resulted in the indiscriminate and inefficient binding of Bl-CFC to stroma. Moreover, treatment of the normal cells with 5637 conditioned medium (CM), which contains haemopoietic growth factors, also reduced the binding capacity of normal Bl-CFC; 5637CM treatment did not alter the expression of DAF. It is proposed that a Pl-linked cell adhesion molecule (CAM) is deficient in CML as a consequence of the constitutive activation of ABL kinase whilst, in normal cells, CAMs attached in this manner are responsible for efficient adhesion to stroma and are regulated by growth factors. PMID- 1715961 TI - OCI/AML-4 an acute myeloblastic leukemia cell line: regulation and response to cytosine arabinoside. AB - This paper describes the properties of a continuous cell line derived from the blast cells of a patient with acute myeloblastic leukemia (AML), secondary to the treatment of Hodgkin's disease. The line grows slowly without stimulation but responds to interleukin-3 (IL-3), GM-CSF and mast cell growth factor (MGF), a ligand for the receptor encoded by the c-kit oncogene. When OCI/AML-4 cells are exposed to MGF with IL-3 or GM-CSF, additive or synergistic effects are seen. Combinations of MGF and G-CSF, IL-6 or CSF-1 give less growth than MGF alone. OCI/AML-4 cells are sensitive to retinoic acid; a dose related decrease in clonogenic cells is observed when OCI/AML-4 cells are exposed to retinoic acid in suspension culture. OCI/AML-4 cells are sensitive to cytosine arabinoside (ara C), but the ara-C dose-response curve can be changed by altering the regulatory milieu in suspension culture. The cells are more ara-C sensitive in MGF or G-CSF than in IL-3 or GM-CSF. Following a 24 h exposure to retinoic acid, the ara-C sensitivity increases; in contrast, after a similar exposure to hydrocortisone, the cells become less ara-C sensitive. These changes in ara-C sensitivity occur in cells that are actively making DNA, as indicated by the reduction in colony formation after exposure to tritiated thymidine. Since OCI/AML-4 cells respond to many of the regulators that affect the growth of freshly obtained AML blast cells, it is proposed that this cell line may be useful for the study of regulation on AML in general and the interaction between different regulators in particular. PMID- 1715962 TI - Euthanasia and other medical decisions concerning the end of life. AB - This article presents the first results of the Dutch nationwide study on euthanasia and other medical decisions concerning the end of life (MDEL). The study was done at the request of the Dutch government in preparation for a discussion about legislation on euthanasia. Three studies were undertaken: detailed interviews with 405 physicians, the mailing of questionnaires to the physicians of a sample of 7000 deceased persons, and the collecting of information about 2250 deaths by a prospective survey among the respondents to the interviews. The alleviation of pain and symptoms with such high dosages of opioids that the patient's life might be shortened was the most important MDEL in 17.5% of all deaths. In another 17.5% a non-treatment decision was the most important MDEL. Euthanasia by administering lethal drugs at the patient's request seems to have been done in 1.8% of all deaths. Since MDEL were taken in 38% of all deaths (and in 54% of all non-acute deaths) we conclude that these decisions are common medical practice and should get more attention in research, teaching, and public debate. PMID- 1715963 TI - Prevalence of antibody to hepatitis C virus in prospectively followed acute non A, non-B hepatitis, from different epidemiological categories. AB - We have studied the prevalence of antibody against hepatitis C virus (anti-HCV) and its relation to the time of onset of the symptoms in 57 patients with acute non-A, non-B hepatitis: 16 post-transfusion, 25 drug addicts and 16 sporadic cases. In the 1st month after the onset of illness, anti-HCV was positive in 25% of patients with post-transfusion hepatitis, 44% of drug addicts and 25% of sporadic hepatitis. In the 3rd month this antibody was detected in 75%, 88% and 31.2%, and in the 6th month in 87.5%, 96% and 31.2%, respectively. The prevalence in the 3rd and 6th months was significantly higher in post-transfusion patients and drug addicts than in sporadic cases. In the 6th month the prevalence of anti HCV in patients who progressed towards chronicity was also significantly higher than in those with acute resolving non-A, non-B hepatitis (94% vs 50%, p less than 0.001). These results show that HCV is probably the main agent in acute post transfusion non-A, non-B hepatitis and in those occurring in drug addicts, and that in a high proportion of these patients the anti-HCV can be detected in the 3rd month after the beginning of the symptoms. On the other hand, the relation of hepatitis C virus with sporadic acute non-A, non-B hepatitis may be doubtful. PMID- 1715964 TI - Long-term follow-up of chronic hepatitis non-A, non-B--with special reference to hepatitis C. AB - One hundred and twenty-seven patients with histologically verified chronic non-A, non-B hepatitis were followed for up to 23 years (mean 6.3 years). Thirty-nine were infected by blood transfusion, 58 were drug-addicts and 30 had no obvious source of infection. Chronic persistent hepatitis (CPH) was diagnosed in 84 (66%), while 43 patients (34%) had chronic active hepatitis (CAH) with or without cirrhosis. Patients with CPH were significantly younger (29.7 years vs 46.8 years; p less than 0.01), irrespective of the type of virus exposure. Antibodies to hepatitis C virus (anti-HCV) were detectable in 91 patients (72%) and 36 (28%) were anti-HCV negative. Fifteen patients with acute onset, and negative for anti HCV at the start, became positive during follow-up; 12 of them within 4.5 months. We found no differences among anti-HCV positive and anti-HCV negative patients in liver function tests, resolving rate, morphological progression in serial biopsies or mortality rate. Five previously anti-HCV positive patients became negative during follow-up and in two of them this was accompanied by decreasing hepatic inflammation. PMID- 1715965 TI - Myosin in hepatocytes is essential for bile canalicular contraction. AB - Active dynamic contraction of bile canaliculi has been observed in cultured doublet hepatocytes using time-lapse cinephotomicrography. This contractile movement plays an important role in normal bile formation. The mechanism of bile canalicular contraction has been proved to involve the Ca(2+)-calmodulin system and pericanalicular actin filaments. However, the role of myosin in this system is still unknown. In this study, using the newly synthesized myosin light-chain kinase inhibitor ML-9, we found that the treatment of cultured doublet hepatocytes with ML-9 inhibited canalicular contraction. This inhibitory effect suggests that myosin is involved in this complex cellular function and that the integrity of the actin-myosin system, as well as the Ca(2+)-calmodulin system is essential for normal bile canalicular contraction. PMID- 1715966 TI - Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes. AB - This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits. PMID- 1715967 TI - Spontaneous electrical activity regulates vasoactive intestinal peptide expression in dissociated spinal cord cell cultures. AB - Activity-dependent expression of vasoactive intestinal peptide (VIP) was investigated in spinal cord/dorsal root ganglia cultures derived from embryonic mice. Since all spinal cord neurons appear to exhibit spontaneous action potentials after one week in vitro, activity-dependent regulation of VIP transcripts (mRNAVIP) could be studied with or without electrical blockade induced by tetrodotoxin (TTX). In 10-day-old cultures, a 50% decrease in mRNAVIP was observed after 3 days of treatment with TTX. The decrease in mRNAVIP was reversed upon removal of the TTX and was dependent on the age of the cultures: no decreases from control were observed in 5-day-old cultures and much smaller decrements were produced in one month old cultures treated with TTX. A variety of neuroactive substances were tested for effects on mRNAVIP in electrically active and electrically blocked cultures. Application of 8-bromo-cAMP (cAMP), N-methyl-D aspartate (NMDA), substance P, muscimol, A23187 and VIP to electrically active cultures resulted in a 2- to 3-fold increase in mRNAVIP, while phorbol myristate 13-acetate (PMA) and 8-bromo-cGMP (cGMP) had no effect. In contrast, electrically inactive cultures exhibited a 3 to 4-fold increase in mRNAVIP after treatment with PMA, cAMP and VIP, while NMDA, substance P, muscimol, A23187 and cGMP produced no increases. In summary, the regulation of VIP gene expression in embryonic spinal cord neurons shows a temporal sensitivity to TTX-induced electrical blockade and may be mediated by multiple neurotransmitter inputs which converge on cAMP- and calcium-related processes in an activity-dependent manner. PMID- 1715968 TI - Effects of unilateral cortex lesions on gene expression of rat cortical cholecystokinin neurons. AB - In rat neocortex, the gene encoding preprocholecystokinin is expressed in interneurons which also synthetize gamma-aminobutyric acid. An injury to the meninges and the underlying cortex increased the concentration of mRNA coding for preprocholecystokinin in all ipsilateral cortical areas. Simultaneous treatment of the rats with the anti-inflammatory agent diclofenac did not affect the injury induced change in gene expression indicating that inflammatory processes were not involved. The injury also enhanced the expression of the immediate early gene c fos in the ipsilateral cortex in a time-dependent manner. There was an increase in c-fos mRNA 1 h after the operation, which was no longer observed 3 h later. Twenty-four hours after the operation, cells containing c-fos mRNA were found in cortical layers II, III, V and VI. The neurons which showed an increased expression of preprocholecystokinin were also in these layers. The N-methyl-D aspartate (NMDA) receptor antagonist MK-801 prevented the injury-induced increases in both preprocholecystokinin and c-fos gene expression, indicating that stimulation of this glutamate receptor subtype may initiate the changes in expression of both genes. It is hypothetized that the immediate early gene c-fos is activated first and this then leads to the increase in preprocholecystokinin mRNA. PMID- 1715969 TI - Human astrocytoma U251 RNA and genomic brain glycogen phosphorylase sequences. AB - There are two versions of human brain glycogen phosphorylase (B-GP) cDNA in the literature that differ significantly in their C-terminal coding and 3' untranslated regions; one isolated from human fetal brain, and the other from human brain astrocytoma cell line U251. A 280 bp absence in the cDNA sequence isolated from human brain astrocytoma cell line U251 changes the predicted protein length from 842 aa (estimated from fetal brain cDNA) to 862 aa. RNA and genomic DNA were isolated from U251 cells and the 280 bp region of interest was amplified by the polymerase chain reaction (PCR). Sequence analysis of the amplified region has unexpectedly confirmed the presence of the 280 bp in U251 RNA and genomic DNA. Thus, the predicted protein length of 842 aa, as reported for fetal brain glycogen phosphorylase, is most likely the correct one. PMID- 1715970 TI - Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue. AB - The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissue-specific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715971 TI - The transposable element Tam1 from Antirrhinum majus shows structural homology to the maize transposon En/Spm and has no sequence specificity of insertion. AB - We present the genomic structure of Tam1, a transposable element from Antirrhinum majus. The Tam1 element is 15.2 kb long and includes two genes that are transcribed to produce a 2.4 kb (tnp1) and a 5 kb mRNA (tnp2). These transcripts partially overlap and the exons are scattered over the whole element. Tnp1 encodes a 53 kDa protein as deduced from the cDNA sequence. The 5 kb transcript of tnp2 contains an open reading frame that shares 45% homology with part of the tnpD gene of En/Spm from maize and 48% homology with an open reading frame of the Tgm element from Glycine max. We discuss the possible functions of these genes by analogy with En/Spm. Additionally, a number of flanking sequences of Tam1 insertions were analysed to investigate the sequence specificity of insertion. From these studies we conclude that Tam1 transposes predominantly into AT-rich regions that can be unique as well as repetitive. No specific target sequence of insertion could be found. PMID- 1715972 TI - A novel ras-related rgp1 gene encoding a GTP-binding protein has reduced expression in 5-azacytidine-induced dwarf rice. AB - Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Schizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development. PMID- 1715973 TI - Effects of Tnt1 tobacco retrotransposon insertion on target gene transcription. AB - The effects of Tnt1 retrotransposon insertion on nitrate reductase (NR) gene transcription have been analyzed in three NR-deficient insertional, mutants of Nicotiana tabacum. In the three mutants, named h9-Nia4, h9-Nia5 and h9-Nia6, Tnt1 was inserted into exon 3, exon 2 and exon 1 of the nia2 NR alloallelle, respectively. The mutants h9-Nia4 and h9-Nia6, which contained Tnt1 insertions that were oriented opposite to the direction of nia2 gene transcription, expressed chimaeric nia2-Tnt1 RNAs, respectively 12 kb and 10 kb long. The size observed in h9-Nia6 was close to the expected size for a full-length hybrid transcript starting and ending under the control of nia2 signals (about 9 kb). The larger transcript found in h9-Nia4 was shown to be due to a failure to splice the nia2 intron 2. The mutant h9-Nia5, which contained a Tnt1 insertion oriented in parallel with the direction of nia2 transcription expressed two truncated nia2 Tnt1 RNAs, 2 kb and 6.7 kb long. These transcripts arose from termination in the long terminal repeats (LTRs) of Tnt1. Since no full-length hybrid RNA was detected, we suggest that Tnt1 carries efficient termination signals, which are more efficiently recognized in the 3' LTR than in the 5' LTR. PMID- 1715974 TI - Regulation of metallocarboxypeptidase inhibitor gene expression in tomato. AB - Tomato fruits contain a metallocarboxypeptidase inhibitor (MCPI) the sequence of which has already been determined. Here we report the isolation of a tomato cDNA clone that encodes the mature MCPI protein as well as an N-terminal signal peptide for entry into the secretory system and an eight amino acid carboxyterminal extension. MCPI RNA is present at very high levels in anthesis stage ovaries and decreases quite rapidly during fruit development. MCPI protein accumulation reflects the pattern of MCPI RNA accumulation in fruit, consistent with a transcriptional control of MCPI gene activity. In leaves, the levels of MCPI RNA and protein are very low. Wounding of the leaves causes a dramatic (100 fold) increase in steady-state level of MCPI RNA without a concomitant increase in MCPI protein level suggesting a control at the post-transcriptional or translational level of gene expression. Genomic DNA blot hybridization data indicate that MCPI in tomato may be encoded by a single gene. PMID- 1715976 TI - Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny. AB - In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1-2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carrying the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species. PMID- 1715975 TI - CYP1 (HAP1) is a determinant effector of alternative expression of heme-dependent transcribed genes in yeast [corrected]. AB - The CYP1 (HAP1) gene of Saccharomyces cerevisiae is known to activate a number of target genes in response to the presence of heme. Several features of the protein, deduced from the sequence of the gene, suggest that CYP1 is a general sensor of the redox state of the cell. To investigate further the function of CYP1, we analysed its effects on the transcription of two genes, HEM13 and 14DM, which are preferentially expressed in anaerobiosis. HEM13 encodes coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway and 14DM encodes lanosterol-14-demethylase which is involved in sterol biosynthesis and is a member of the cytochrome P450 family. Isogenic CYP1+ and cyp1 degree deleted strains, either heme-sufficient or heme-deficient (HEM1 disrupted), were grown in aerobic or anaerobic conditions, and transcripts of HEM13 and 14DM were analysed on Northern blots. The results show that in anaerobic and in heme-deficient cells, CYP1 activates the transcription of HEM13 and inhibits that of 14DM. Opposite effects of CYP1 are observed in aerobic, heme sufficient cells. We concluded that: (i) CYP1 is an efficient activator especially in heme-depleted cells; (ii) CYP1 exerts both positive and negative regulatory effects; (iii) the nature of the regulatory function of CYP1 depends on the target gene; and (iv) for a given gene, the presence or absence of heme or oxygen reverses the sense of CYP1-dependent regulation. PMID- 1715977 TI - The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli. AB - Using T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E. Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity. We also found that T7 RNA polymerase terminates within the 5S rRNA gene. PMID- 1715979 TI - Nucleolar organizer regions in vascular and neoplastic cells of human gliomas. AB - The number of nucleolar organizer regions in vascular cells and neoplastic cells of human gliomas was counted by a combined staining technique: one-step silver colloid method for nucleolar organizer region-associated argyrophilic protein (AgNOR) and periodic acid-Schiff staining for basement membrane of vascular components. The number of AgNORs (mean +/- SD) in the vascular and neoplastic cells of various tumors tested was as follows: benign astrocytoma (Grade 2, n = 4), 1.52 +/- 0.07 and 1.80 +/- 0.13, respectively; anaplastic astrocytoma (Grade 3, n = 7), 1.98 +/- 0.23 and 2.87 +/- 0.50; and glioblastoma multiforme (Grade 4, n = 11), 2.05 +/- 0.29 and 3.13 +/- 1.13. AgNOR scores in vascular cells of benign astrocytomas, anaplastic astrocytomas, and glioblastomas were significantly higher than those of vascular cells in normal brain tissue without neoplastic alteration (1.26 +/- 0.05; n = 3; P less than 0.01, P less than 0.001, and P less than 0.001, respectively). Moreover, the AgNOR scores of vascular cells in high-grade gliomas (Grades 3 and 4) were significantly higher than those in low-grade gliomas (Grade 2) (P less than 0.01). These results indicate that the proliferative activity of both vascular and neoplastic cells in gliomas increased as histological malignancy advanced, and that the quantification of AgNORs was useful in evaluating proliferative activity in vascular cells as well as in assessing the malignancy of neoplastic tissues. PMID- 1715978 TI - RNA-protein interactions at transcript 3' ends and evidence for trnK-psbA cotranscription in mustard chloroplasts. AB - In vitro transcripts from the 3' flanking regions of mustard chloroplast genes were tested for protein binding in a chloroplast extract. Efficient and sequence specific RNA-protein interaction was detected with transcripts of the genes trnK, rps16 and trnH, but not with the 3' terminal region of trnQ RNA. The transacting component required for specific complex formation is probably a single 54 kDa polypeptide. The protein-binding region of the rps16 3' terminal region was mapped and compared with that of the trnK transcript determined previously. Both regions reveal a conserved 7-mer UUUAUCU followed by a stretch of U residues. Deletion of the trnK 3' U cluster resulted in more than 80% reduction in the binding activity, and after deletion of both the U stretch and the 7-mer motif no binding at all was detectable. RNase protection experiments indicate that the protein-binding regions of both the rps16 and trnK transcripts correlate with the positions of in vivo 3' ends, suggesting an essential role for the 54 kDa binding protein in RNA 3' end formation. In the case of the trnK gene, evidence was obtained for read-through transcripts that extend into the psbA coding region, thus pointing to the possibility of trnK-psbA cotranscription. PMID- 1715980 TI - Influence of computer-modulated profile haemodialysis on cardiac arrhythmias. AB - To reduce hamodialysis-induced ventricular arrhythmias, each of 15 patients (age 66.9 +/- 6.2 years) with end-stage renal disease and cardiac irregularities was treated subsequently with four different computer-modulated bicarbonate haemodialysis profiles (A-D) for 2 weeks respectively: (A) constant UF, dialysate Na (138 mmol/l) and K (2 mmol/l); (B) decreasing UF, otherwise as (A); (C) decreasing UF and Na (starting with 10% higher than serum Na), otherwise as (A); (D) decreasing UF and Na, adapted K to achieve a maximal reduction of serum K of only 15%/h. Cardiac monitoring was done by 11 h ECG. Only in haemodialysis profile D a distinct reduction of ventricular extrasystoles during and after haemodialysis was obtained. It was accompanied by an improvement in the Lown classification. In addition, a weak but highly predictive correlation between the number of ventricular extrasystoles in the last hour of dialysis and the difference between pre- and post-dialysis potassium concentration in the serum could be established (r = 0.37; P less than 0.004). Computer-modulated potassium profile haemodialysis is a useful tool to reduce the number and severity of ventricular extrasystoles. PMID- 1715982 TI - Molecular mechanism of negative autoregulation of Escherichia coli crp gene. AB - Transcription of the Escherichia coli crp gene encoding cAMP receptor protein (CRP) is negatively regulated by CRP-cAMP complex that binds to a specific site located downstream from the transcription start site. The binding of CRP-cAMP to this site activates transcription from a second divergent overlapping promoter. The mechanism of this negative autoregulation of the crp gene has been investigated by in vitro transcription, gel shift, DNase I footprinting, and exonuclease III protection assays. We demonstrated that the crp and divergent promoters are reciprocally and coordinately regulated by CRP-cAMP. The abortive initiation assay revealed that the divergent RNA itself is not required for the inhibition of crp transcription. Detailed binding studies revealed that CRP-cAMP stimulates the binding of RNA polymerase to the divergent promoter and thus blocks the occupation of the crp promoter by RNA polymerase. PMID- 1715981 TI - A comparative study of the ribosomal RNA operons of Streptomyces coelicolor A3(2) and sequence analysis of rrnA. AB - S. coelicolor A3(2) contains six ribosomal RNA operons. Here we describe the cloning of rrnA, rrnC and rrnE, thereby completing the cloning of all operons. Southern hybridisation of genomic DNA with a heterologous probe from the E.coli rrnB 16S rRNA gene showed differences in hybridisation among the six rRNA operon containing bands. The nucleotide sequence of the 16S rRNA gene and the upstream region of rrnA was determined and compared with the corresponding sequence of rrnD, showing that the 16S rRNA genes are 99% identical. Substantial differences were found, however, in the upstream regions corresponding to the P1 and P2 promoters of rrnD. Southern analysis showed that some of the other rRNA operons of S.coelicolor A3(2) also differed in this part of the upstream region. PMID- 1715983 TI - A sequence tagged site (STS) detects EcoRI polymorphisms in the human acidic fibroblast growth factor gene. PMID- 1715984 TI - A new polymorphic probe on chromosome 3p: lambda LIB17-11 (D3S1092). PMID- 1715985 TI - A new polymorphic probe on chromosome 3p: lambda LIB13-67 (D3S591). PMID- 1715986 TI - A new polymorphic probe on chromosome 3p: lambda LIB37-96' (D3S1192). PMID- 1715987 TI - A new polymorphic probe on chromosome 3p: lambda LIB4-59 (D3S575). PMID- 1715988 TI - A new polymorphic probe on chromosome 3p: lambda LIB18-88 (D3S1093). PMID- 1715989 TI - The identification of a novel NCA-related pancreatic tumour-associated antigen, DD9-antigen: a comparison with the expression of other tumour antigens by the pancreatic tumour cell line GER. AB - Pancreatic tumour-associated monoclonal antibody DD9E7, raised against the GER pancreatic adenocarcinoma cell line, recognises a protein epitope on a novel family of membrane-bound cell surface glycoproteins (Mr 80-115,000). Western blot analysis of SDS/PAGE gels of tumour biopsies and of normal adult pancreas has shown that these glycoproteins are highly expressed in most pancreatic tumours but cannot be detected in normal adult pancreas. Using monoclonal antibodies directed against other antigens that have been associated with pancreatic adenocarcinoma (Du-Pan-2, Ca 19-9, CEA, NCA-95/55, EMA, and FAP), we have been able to show that although some of the antigens are also expressed by the GER pancreatic tumour cell line, the glycoproteins identified by monoclonal antibody DD9E7 are distinct from those other antigens in both molecular weight and antibody binding characteristics. Neuraminidase, periodic acid, and tunicamycin treatment of cultured cells has shown that monoclonal antibody DD9E7 recognises an epitope on the protein core of the antigen. This epitope is also present in NCA-1, but not in CEA, which suggest that there may be an association between DD9 antigen and other members of the NCA/CEA supergene family. PMID- 1715990 TI - Bile exclusion from the gut exacerbates acute pancreatitis caused by pancreatic duct obstruction in rats. AB - Acute pancreatic duct obstruction causes hyperamylasemia and mild pancreatic inflammation. We hypothesized that bile exclusion from the gut, which stimulates pancreatic secretion, exacerbates acute pancreatitis caused by pancreatic duct obstruction. Rats were surgically prepared with gastric, duodenal, bile, and pancreatic fistula catheters and a jugular vein catheter. After a 4-day recovery, groups of rats (a) served as controls, (b) had complete pancreatic duct obstruction for 6 h, or (c) had bile excluded from the gut for 24 h and then, during the final 6 h, complete pancreatic duct obstruction. Plasma amylase was measured, and all rats were euthanized at the end of experiments. Each pancreas was excised and weighed, and portions were fixed in formalin and glutaraldehyde. In blind fashion, each pancreas was examined under light microscopy and assigned a pancreatitis score based on presence of edema and severity of acinar cell changes and inflammation. Acute pancreatic duct obstruction was associated with increased pancreas weight, hyperamylasemia, and elevated pancreatitis score; moderate acinar cell vacuoles, which were observed in the cytoplasm near the basolateral membrane, and loss of microvilli were noted with electron microscopy. Bile exclusion from the gut exacerbated the acute pancreatitis caused by pancreatic duct obstruction; acinar cell distortion and destruction, as well as marked inflammation, were seen microscopically. These observations suggest that the absence of intestinal bile contributes to the pathogenesis of acute pancreatitis associated with pancreatic duct obstruction. PMID- 1715991 TI - Lack of effect of cerulein on pancreatic growth of rats fed a low-protein diet. AB - The effect of dietary protein deficiency and protein concentration of the diet on the pancreatic trophic response to a CCK analogue (cerulein) were studied. Rats were fed for 14 days with semipurified diets containing 5, 30, or 60% casein. During the final 4 days, they received 2 micrograms/kg cerulein or gelatin vehicle subcutaneously three times/day, and the effects on pancreatic weight and pancreatic content of protein, RNA, DNA, amylase, and chymotrypsin were determined. Cerulein failed to increase significantly any pancreatic parameter in rats fed 5% casein, while stimulating significant increases in almost all parameters in rats fed 30 and 60% casein diets. In the absence of cerulein treatment, increases in dietary protein levels caused progressive increases in all pancreatic growth parameters with the exception of amylase. In the presence of cerulein, increases in dietary protein concentrations caused progressive increases in pancreatic growth parameters (except amylase), which were maximal at 30% casein concentration of the diet for most parameters. The results confirm that pancreatic growth is stimulated by increasing protein concentration of the diet and indicate that a low protein diet, acting through a deficiency of dietary nitrogen and essential amino acids, limits the pancreatic trophic response to CCK or analogues. These results explain the failure of trypsin inhibitors to stimulate pancreatic growth in rats fed low levels of dietary protein. PMID- 1715992 TI - The site of action of neurotensin in the rat pancreas. AB - The tridecapeptide neurotensin, present in endocrine cells of the ileal mucosa and in nerve bodies of cerebral nuclei, may play a role in the physiology of exocrine pancreatic regulation. This assumption is based on two observations: Neurotensin appears in the blood stream after a fatty meal and neurotensin stimulates exocrine pancreatic secretion. There has, however, been controversy on the site of action of this peptide since some investigators did not observe an effect on the pancreas in vitro and suggested, therefore, an indirect, perhaps neural or even central site of action. In the present investigation, we compared the action of neurotensin on the exocrine pancreas in vivo in the anesthetized rat after intracerebroventricular (i.c.v.) injection and after i.v. infusion and in vitro after incubation of the pancreas in three different preparations: isolated dispersed acini, isolated lobuli, and isolated total pancreas. We found that i.c.v. application of neurotensin stimulated exocrine pancreatic secretion only when doses were applied that led to elevated peripheral plasma neurotensin levels. In vitro, the action of neurotensin was very weak and the optimal dose was integrity dependent; e.g., a concentration of 10(-4) and 10(-5) M neurotensin was necessary to stimulate amylase release from isolated acini, 10(-8) M neurotensin induced amylase release from isolates lobuli, and 10(-9) M neurotensin released amylase from the intact pancreas. Intravenously, neurotensin resulted in a greater amylase release over basal than in any of the in vitro experiments. We conclude that neurotensin acts directly on the pancreatic acini but that the sensitivity of the pancreas is greatly enhanced when the organ is intact and has normal neural and vascular communications.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1715993 TI - Galanin fragments and analogues: effects on glucose-stimulated insulin secretion from isolated rat islets. AB - Galanin occurs in intrapancreatic nerves and inhibits insulin secretion both in vivo and in vitro. To investigate which part of the galanin molecule accounts for this inhibition, we studied the effect of porcine galanin, four galanin fragments and three galanin analogues with substitutions of the 2nd amino acid in galanin, on glucose-stimulated insulin secretion from isolated rat islets cultured overnight. The islets were incubated for 1 h at 8.3 mM glucose. Porcine galanin1 29 inhibited insulin secretion at dose levels from 10(-8) M to 10(-6) M (p less than 0.001). Also, the galanin fragments GAL1-15, GAL2-29, and GAL3-29 significantly inhibited insulin secretion at and above concentrations of 10(-7) M (p less than 0.001), 10(-8) M (p less than 0.001), and 10(-7) M (p less than 0.001), respectively. Galanin analogues where the 2nd N-terminal amino acid had been changed from tryptophan to tyrosine (GAL-TYR2) or isoleucine (GAL-ILE2) inhibited insulin secretion, as did porcine galanin1-29, whereas after substitution with phenylalanine (GAL-PHE2) no effect was observed. Furthermore, the C-terminal fragment GAL10-29 did not influence insulin secretion. We conclude that the inhibitory action by galanin on glucose-stimulated insulin secretion from normal islets resides in the N-terminal part of the molecule. In contrast, the C-terminal part of galanin apparently does not influence insulin secretion. PMID- 1715994 TI - CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA Aw68.1. AB - The crystal structure of the HLA-Aw68.1 antigen binding site revealed a negatively charged pocket centred on aspartic acid 74 (Garrett et al. 1989). Access to this '74 pocket' is blocked in HLA-Aw68.2 and HLA-Aw69 by two substitutions at positions 97 and 116. This key feature suggests that the Aw68.1 peptide-specific interactions may involve salt bridges between oppositely charged residues. In this paper, the influenza epitope recognized by virus-specific HLA Aw68.1-restricted cytotoxic T lymphocytes (CTL) has been defined in vitro with a synthetic peptide corresponding to residues 89-101 of the nucleoprotein (NP). Amino acid substitutions of the peptide NP 89-101 showed that the arginine at position 99 is an anchor point of the peptide within the Aw68.1 antigen binding site. Consistent with this we find that neither HLA-Aw68.2 nor HLA-Aw69 positive cells can present peptide NP 89-101 to Aw68.1-restricted CTL. Our results therefore suggest a model in which presentation of NP 89-101 by HLA-Aw68.1 is dependent upon interaction of the positively charged arginine residue at position 99 of the peptide, with the negatively charged aspartic acid in the '74 pocket' of HLA-Aw68.1. We also show that influenza-virus-specific HLA-Aw68.1-restricted CTL are CD8 independent. This result is consistent with the low affinity of HLA Aw68.1 for CD8 (Salter et al. 1989) and reveals a unique example of CD8 independent priming of CTL by natural infection with a common pathogen in humans. PMID- 1715995 TI - The protective effects of Iloprost and thromboxane synthetase inhibitor, UK 38485, against glycerol--induced acute renal failure in rats. AB - Tissue protective activities of Iloprost, a stable analogue of PGI2, and of UK 38485, an inhibitor of thromboxane synthetase, were investigated in rats, in which acute renal failure was elicited by the injection of glycerol. The effects of these compounds on PGE2- and LTC4-like activities in the kidney tissue were also studied. Glycerol injection caused acute kidney damage as evidenced by light microscopic examination and abundant hematuria. Glycerol injection also caused an increase in tissue PGE2- and LTC4-like activities. Although both metabolites were increased, the ratio of PGE2/LTC4 was found to be decreased when compared with the control value. Both Iloprost and UK 38485 partially prevented tissue damage due to glycerol and caused an increase in the ratio of PGE2/LTC4. The preventive effects of the drugs were more pronounced when both drugs were used in combination. The participation of arachidonic acid metabolites in the mechanism of the production of kidney damage due to glycerol and possible preventive effects of the compounds are discussed. PMID- 1715996 TI - [Biodegradable gentamicin-depot implants made of beta-tricalcium phosphate ceramics. 3. In vivo studies on drug release, tissue tolerance, and biodegradation]. AB - In vivo drug release properties and biocompatibility of gentamicin-loaded controlled release implants made of beta-tricalcium phosphate ceramics designed for the local antibiotic treatment of bone infections were investigated. Controlled release pellets containing 0.4 and 0.8 mg of gentamicin were implanted into the femoral bone of rats. Drug release was measured from renal excretion over a time period of 3 weeks. The excretion pattern can be described by an initial phase of increased drug release was faster at higher drug loading. Drug release from glyceride-containing controlled release pellets occurs at a significantly slower rate than from drug-loaded pellets without glycerides. Histological studies after implantation of the pure ceramic pellets and the controlled release pellets into the bone tissue of rats and rabbits are showing a high tissue tolerance and the biodegradability of the implants. However, the glyceride-containing pellets are degraded at a slower rate than the pure ceramic pellets. PMID- 1715997 TI - Taste of glutamate salts in young and elderly subjects: role of inosine 5' monophosphate and ions. AB - Taste sensitivity to five glutamate salts (sodium glutamate, potassium glutamate, ammonium glutamate, calcium diglutamate, and magnesium diglutamate) were determined in sixteen young (mean age 25.58 years) and eighteen elderly (mean age 86.89 years) subjects. The effect of inosine 5'-monophosphate (IMP) and ions on taste perception of glutamate compounds was also investigated. The detection thresholds for glutamate salts were 5.04 times higher in elderly subjects than in young subjects; the recognition thresholds were 3.84 times higher. For young subjects, 0.1 mM IMP lowered detection and recognition thresholds for all 5 salts. A stronger concentration of IMP (1 mM) had this effect in both young and elderly groups. Elderly subjects perceived suprathreshold concentrations as less intense than young subjects. Chloride and acetate salts of sodium, potassium, and calcium reduced the detection and recognition thresholds of L-glutamic acid but had no effect sodium glutamate thresholds. PMID- 1715998 TI - Bleomycin induces similar survival variations through the cell cycle as do X rays with and without hypertonic shock. AB - In an earlier report [H. Utsumi and M. M. Elkind, Radiat. Res. 119, 534-541 (1989)], it was shown that the survival of V79 Chinese hamster cells treated with bleomycin was significantly reduced by a posttreatment with anisotonic phosphate buffered saline in a manner that was qualitatively similar to what had been observed with X rays [H. Utsumi and M. M. Elkind, Radiat. Res. 77, 346-360 (1979)]. This similarity suggested that similarities might exist in the cyclic variation in the suppression of the repair of potentially lethal damage following treatment with bleomycin or X rays. Accordingly, the age-response variations of survival, with or without a posttreatment challenge with hypertonic buffer, were compared in the same experiment when cells were treated with either agent. Although a significant difference was observed near the G1/S-phase border, in general the damage induced by the two agents showed a similar dependence on cell age, and posttreatment with hypertonic buffer enhanced cell killing appreciably following either treatment. The results support the inference that bleomycin is a radiomimetic agent. PMID- 1715999 TI - The biophysics of peptide models of ion channels. PMID- 1716000 TI - When the diagnosis is pancreatic cancer. PMID- 1716001 TI - Enhancement of the glutamate response by cAMP-dependent protein kinase in hippocampal neurons. AB - Receptor channels activated by glutamate, an excitatory neurotransmitter in the mammalian brain, are involved in processes such as long-term potentiation and excitotoxicity. Studies of glutamate receptor channels expressed in cultured hippocampal pyramidal neurons reveal that these channels are subject to neuromodulatory regulation through the adenylate cyclase cascade. The whole-cell current response to glutamate and kainate [a non-NMDA (N-methyl-D-aspartate) receptor agonist] was enhanced by forskolin, an activator of adenylate cyclase. Single-channel analysis revealed that an adenosine 3',5'-monophosphate-dependent protein kinase (PKA) increases the opening frequency and the mean open time of the non-NMDA-type glutamate receptor channels. Analysis of synaptic events indicated that forskolin, acting through PKA, increased the amplitude and decay time of spontaneous excitatory postsynaptic currents. PMID- 1716002 TI - Severity, pathobiology, epistatic effects, and genetic markers in sickle cell anemia. PMID- 1716003 TI - The impact of marrow transplant preparative regimens on subsequent growth and development. The Seattle Marrow Transplant Team. AB - These data demonstrate that endocrine function abnormalities influencing subsequent growth and development in children after marrow transplantation rarely occur after preparation with CY alone. However, after administration of regimens containing TBI, multiple endocrine function abnormalities can occur. Although longer follow-up is needed, it appears that the addition of BU to CY does not result in thyroid function or growth abnormalities. Pubertal development and gonadal function, however, may be adversely influenced by BU and patients may require supplementation with appropriate gonadal hormones. PMID- 1716004 TI - Prevention and therapy of viral hepatitis. PMID- 1716005 TI - Anemia in the newborn: a diagnostic approach and challenge. PMID- 1716006 TI - [Immobilized granulated drugs with magnetic properties based on nDNA, RNA, cardiolipin in the diagnosis and treatment of systemic lupus erythematosus]. AB - To advance diagnosis and treatment of systemic lupus erythematosus (SLE), the authors have developed a number of specific diagnostic preparations basing on nDNA, RNA, cardiolipin immobilized into the three-dimensional lattice of polyacrylamide gel. Using such granulated antigenic preparations with magnetic properties, a correlation has been derived between SLE activity, visceral pathology and a level of specific antibodies. In view of a successful experimental use of the preparations as selective sorbents, they may be introduced into extracorporeal treatment. Immobilized granulated preparations can be used in immunofluorescent tests and enzyme immunoassays. PMID- 1716007 TI - [Effects of beta blockers on the parameters of acute phase response and level of circulating immune complexes in myocardial infarct]. PMID- 1716008 TI - [Antigenic spectrum of circulating immune complexes and its diagnostic significance in salmonellosis]. PMID- 1716009 TI - Intestinal parasitoses and the modern description of diseases of poverty. PMID- 1716010 TI - Mapping movements within a moving motor map. PMID- 1716011 TI - Alzheimer plaques and tangles: advances on both fronts. PMID- 1716012 TI - Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence. AB - The discovery in the late 1970s that cholinergic neurons in the basal forebrain degenerate in Alzheimer's disease (AD) greatly accelerated research on the role of cholinergic mechanisms in learning and memory. As is often the case in science, the early enthusiasm for the cholinergic hypothesis has been tempered by the results of subsequent research. Although there is substantial pharmacological evidence that unspecified cholinergic systems in the brain play important roles in some forms of learning and memory, recent findings in humans indicate that antimuscarinic drugs do not model the deficits seen in AD. In addition, the goal of elucidating the functions of these basal forebrain neurons in animals has proved to be difficult and is yet to be achieved. Despite substantial effort, therefore, the cognitive and behavioral consequences of cholinergic pathology in AD remain unknown. Under these circumstances, attempts to develop cholinergic pharmacotherapies for these deficits in AD are based on questionable assumptions. PMID- 1716013 TI - How do retinal axons find their targets in the developing brain? AB - Tissue culture studies show that cell survival and process outgrowth from retinal ganglion cells depend on the molecular composition of the substrates over which the neurites grow, and on diffusible factors present in the medium. Recent work has begun to show that at least some of these components might be interactive. Since the conditions in a culture dish, as well as the patterns of antigen expression on cells in vitro, can differ considerably from those encountered in vivo, it is important to design experiments in vivo that examine how growing neurites relate to their natural microenvironment. By the use of transplantation techniques, it has been possible to provide evidence for a comparable duality of substrate-dependent and target-derived controls of optic axon growth, which might provide insight into the normal developmental process. PMID- 1716014 TI - A brief sketch of Soviet neuroscience. AB - Social and cultural philosophy in the guise of various forms of materialism has long been the basis on which Soviet science (including neuroscience) is founded. Neuroscientific research developed within a number of 'schools', each associated with the doctrine of their founders and concentrated in a few large cities. Currently, central governmental funding covers research within state neuroscience programmes. PMID- 1716015 TI - Hereditary cerebral hemorrhage with amyloidosis--Dutch type: its importance for Alzheimer research. AB - Alzheimer's disease is now commonly regarded as a form of 'amyloid encephalopathy'. Amyloid deposits in the cerebral blood vessels and parenchyma consist mainly of a unique protein called amyloid beta protein (A beta P), which has a molecular weight of 4 kDa and is 42 amino acids long. These deposits are thought to be of pathogenetic importance in Alzheimer's disease. Recently, therefore, attention has been focused on the process of turnover of the precursor of A beta P to amyloid fibrils, and the deposition and persistence of A beta P in this disease. The study of several other diseases with cerebral A beta P deposition can be informative in this respect, because they allow the comparison of different pathogenetic mechanisms that lead to this type of deposition. One of these diseases is hereditary cerebral hemorrhage with amyloidosis- Dutch type (HCHWA-D), which is the subject of this review. PMID- 1716016 TI - The neurobiology of narcolepsy. AB - Narcolepsy is characterized by excessive sleepiness and abnormal manifestations of rapid eye movement (REM) sleep. Neurochemical studies of human and canine narcolepsy have demonstrated disturbed monoaminergic and cholinergic function and suggest that deficits of noradrenaline availability in specific brain regions may account for much of its disordered pathophysiology. Genetic susceptibility to narcolepsy is closely linked to a specific region of the major histocompatibility complex on chromosome 6 and an important direction for future research will be to unravel the relationship between this gene region and the neurochemical abnormalities of narcolepsy. PMID- 1716017 TI - Not such a unifying hypothesis? PMID- 1716018 TI - Regulation of receptor expression by agonists: transcriptional and post transcriptional controls. AB - The molecular cloning of certain members of several distinct classes of receptors has opened up new avenues by which the regulation of signal-transducing proteins can be investigated. The information derived from molecular cloning permits not only studies of functional domains via mutagenesis, but also the study of gene regulation and the cell biology of these receptors by the use of molecular probes. All receptors are bifunctional, possessing domains for ligand binding as well as for signal propagation through binding to other protein(s), DNA or ion channels. Regulation of receptor expression and function in response to agonist stimulation is a central feature of receptor biology. In this article, Drs Hadcock and Malbon focus on the regulation of receptor expression by agonists at the transcriptional and post-transcriptional levels. Regulation of the expression of three classes of receptors central to neurobiology is highlighted: namely, steroid hormone receptors (estrogen receptor), G protein-coupled receptors (beta2 adrenergic receptor) and tyrosine kinase receptors (epidermal growth factor receptor). The emerging idea of cross regulation between receptors is also discussed in order to demonstrate the complexities of studying receptor expression. PMID- 1716019 TI - Stimulus classification by ensembles of climbing fiber receptive fields. AB - Although the local structure of the cerebellum is fairly uniform and its inputs are often widely shared, outputs from different regions of the cerebellar cortex reach different parts of the cerebellar and vestibular nuclei, which can affect the rest of the nervous system in different ways. In this review, we explain how different ensembles of climbing fiber responses in the anterior lobe and paramedian lobule can be generated by a tactile stimulus to the distal hindpaw. Apart from differing in degree of activation, the cortical regions differ also in the detailed pattern of the activation transmitted. The anterior lobe can distinguish a greater diversity of stimuli to various skin surfaces than can the paramedian median lobule. This differential classification of particular stimulus arrays by the two cerebellar regions could produce distinct patterns of neuronal activity in various corticonuclear compartments. PMID- 1716020 TI - From behavior to molecules: an integrated approach to the study of neuropeptides. AB - Despite extensive information on many aspects of peptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory peptides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes in target sensitivity and the distribution of the CAP containing neurons, including a set of peptidergic neurons that alter their transmitter phenotype postembryonically. Studies show that inositol 1, 4, 5 trisphosphate (IP3), linked to intracellular Ca2+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides. PMID- 1716021 TI - Preoperative prostate-specific antigen in predicting pathologic stage and grade after radical prostatectomy. AB - Preoperative serum prostate-specific antigen (PSA) was measured in 63 men who had clinically localized, previously untreated adenocarcinoma of the prostate and underwent subsequent radical prostatectomy and bilateral pelvic lymph node dissection. Pathologic stage and grade were correlated to the serum PSA value. Patients with organ-confined (P1, P2) and extracapsular (P3, P3N +) prostate cancer had elevated preoperative serum PSA levels (greater than 4 ng/mL) in 61 and 90 percent of cases, respectively. Patients with low-grade and high-grade tumor histology had elevated preoperative PSA levels in 62 and 80 percent of cases, respectively. In distinguishing between organ-confined and extracapsular disease with a preoperative serum PSA of 10 ng/mL as a cutoff value, the sensitivity was 68 percent, the specificity was 66 percent, the positive predictive value was 46 percent, and the negative predictive value was 83 percent. Although there was a trend of increasing preoperative serum PSA levels with higher pathologic stage or grade, there was no significant difference in preoperative serum PSA values with pathologic stage and/or grade considered as a group or in determining stage and/or grade preoperatively on an individual basis. PMID- 1716022 TI - Prostate cancer screening in younger men: prostate-specific antigen and public awareness. AB - The American Cancer Society recommends annual digital rectal examination for men over forty years of age. We evaluated 414 men between forty and fifty-nine years of age with a questionnaire, digital rectal examination (DRE), and prostate specific antigen (PSA) determination. One hundred ninety were forty to forty-nine years old, and 224 were fifty to fifty-nine years old. Four patients in the forty to forty-nine age group had elevated PSA determinations, and 7 had abnormal findings on DRE. Using prostate ultrasound and biopsy, no cases of prostate cancer were diagnosed. Ten patients in the fifty to fifty-nine age group had elevated PSA determinations, and 5 were diagnosed to have prostate cancer. These data suggest that PSA may have utility in detecting cancer in younger men. PMID- 1716023 TI - Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain. AB - We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV 1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV 1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents. PMID- 1716024 TI - HTLV-1 envelope sequences from Brazil, the Caribbean, and Romania: clustering of sequences according to geographic origin and variability in an antibody epitope. AB - We sequenced the envelope genes of Human T-cell leukemia type I viruses (HTLV-I) derived from five Brazilian, two Caribbean, and one Romanian case of adult T-cell leukemia after amplification of the complete env gene by PCR. A comparison with previously reported HTLV-I sequences revealed that, although highly homologous, no two env sequences were identical. All envelope sequences differed from each other by 0.3-2.1% nucleotide differences. The five Brazilian sequences clustered together and were about as different from each other (0.5-0.75% nucleotide difference) as were three previously reported Japanese sequences (0.7-0.95%). In contrast, sequences of Caribbean origin were less homogeneous (0.5-1.9% nucleotide differences within this group). The Romanian sequence was not significantly more divergent than any of the others and was closest to our two Caribbean sequences. We observed two changes in a region (aa 176-209) which has previously been shown to contain a linear antibody epitope recognized by most human sera from seropositive individuals. One of these changes affects the binding of monoclonal antibodies to this epitope demonstrating the variability of an antibody epitope in the HTLV-I envelope. PMID- 1716025 TI - Compatibility of rev gene activity in the four groups of primate lentiviruses. AB - The compatibility of rev genes derived from various primate immunodeficiency viruses of all distinct subgroups identified was assessed in three experimental systems: complementation experiments between proviral rev and gag mutants, evaluation of the ability of the rev gene products to activate proviral reporters, and examination of the capacity of various viruses to augment marker gene expression in the infected reporter cell lines. In all systems, human immunodeficiency virus type 1 (HIV-1) rev was not substantially substituted or was extremely poorly substituted by the rev of the other viruses. The rev of simian immunodeficiency virus (SIV) from a mandrill could be exchanged by HIV-1 rev. In contrast, the rev gene products of all viruses efficiently activate HIV-2 and SIV from an African green monkey. PMID- 1716026 TI - The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA stimulated NTPase. AB - Recently it has been reported that a membrane fraction can be isolated from West Nile virus-infected BHK cells which contains the viral nonstructural (NS) proteins as major constituents (Wengler et al., 1990). In this report we show that treatment of these membranes with subtilisin releases the carboxy-terminal segment of the NS 3 protein as a soluble protein of about 50 kDa apparent molecular weight. This molecule, which is called the p50-S protein, can be purified by standard chromatographic procedures. The p50-S protein binds to poly(A) and apparently represents a nucleoside triphosphatase which is stimulated in the presence of ssRNA molecules. The data represent experimental support for the predicted role of this segment of the NS 3 protein as an RNA helicase. Some properties of the p50-S protein are described and a possible function of this protein segment during RNA synthesis is discussed. PMID- 1716027 TI - Polymorphic human gene(s) determines differential susceptibility of CD4 lymphocytes to infection by certain HIV-1 isolates. AB - Independent isolates of HIV-1 differ widely in their tropisms for CD4-positive T cell lines. This study demonstrates that tropisms of 10 different HIV-1 isolates also differ widely, as much as 1000-fold, for normal peripheral blood lymphocytes (PBLs) cultured from any given donor. This could only be reproducibly demonstrated by end point titrations. In addition, the degree of susceptibility of PBLs from 12 random donors to productive infection by any given HIV-1 isolate also varied in a reproducible pattern from donor to donor, with some donors relatively resistant to one or a few isolates. The HIV susceptibility pattern of each donor was manifested at the level of the CD4 lymphocyte and it segregated within a family, conclusively demonstrating that it was genetically determined. PMID- 1716028 TI - Polyadenylated RNA sequences produced in vaccinia virus-infected cells under aberrant conditions inhibit protein synthesis in vitro. AB - We have previously demonstrated that small nontranslated polyadenylated RNAs (POLADS) produced in vaccinia virus (VV)-infected cells inhibit the translation of cellular mRNAs, but minimally affect the translation of VV mRNAs in a cell free protein synthesizing system. Infection of HeLa cells with ultraviolet irradiated vaccinia virus or infection in the presence of actinomycin D (ACD) amplifies the synthesis of POLADS compared to the amount produced in cells infected under normal conditions. The effect of these POLADS on translation was studied in the reticulocyte lysate system. Polyadenylated RNAs isolated from cells infected with wild-type virus (V-POLADS) had a greater inhibitory effect on HeLa cell protein synthesis than on VV protein synthesis. Polyadenylated sequences obtained from cells infected with ultraviolet-irradiated virus (UV POLADS) or from cells infected in the presence of ACD (ACD-POLADS), however, inhibited translation of both HeLa and viral mRNAs. Ultraviolet-POLADS and ACD POLADS were found to possess, on average, longer poly(A) tails than V-POLADS. The inhibition of translation of both host and viral mRNAs effected by V-POLADS, UV POLADS, and ACD-POLADS was reversed by poly(A) binding protein. PMID- 1716029 TI - Recombination between satellite and genomic RNAs of turnip crinkle virus. AB - New recombinant molecules formed from satellite and genomic RNAs of turnip crinkle virus (TCV) have been characterized. Known collectively as sat-RNA CX, these molecules are composed of a nearly full-length segment of a previously characterized TCV satellite RNA (sat-RNA D) at the 5' end joined to variable lengths of TCV genomic RNA 3' terminal sequence. Sat-RNA CX molecules fall into two classes: molecules of 420 to 435 bases and larger species of 501 to 506 bases. The TCV sequence at the junction of the larger molecules is purine-rich and is similar to a motif found at the 5' ends of the TCV satellite RNAs and at the junctions of some TCV defective interfering RNAs. The TCV sequence at the junction of the smaller sat-RNA CX molecules is pyrimidine-rich and is similar to the sequence at the right side of a junction of one TCV defective interfering RNA as well as sequence immediately downstream of the internal initiation site of the 1.45-kb TCV subgenomic RNA. We propose that the latter motif is another putative signal recognized by the viral replicase during the generation of defective interfering and recombinant RNAs in the TCV system. PMID- 1716030 TI - Localization of a VP3 epitope of infectious bursal disease virus. AB - An immunodominant region of VP3, one of the two structural proteins of infectious bursal disease virus (IBDV strain 002-73), has been mapped by restriction site specific deletion analysis and subcloning in Escherichia coli, followed by immunoblot analysis of the synthesized products. The epitope located within 58 amino acids reacted very strongly with a mouse monoclonal antibody (MAb 17/80) raised against IBDV 002-73. This immunodominant region may be useful in serodiagnosis of IBDV infection in poultry. PMID- 1716031 TI - [Serotonin and kinins in the cerebrospinal fluid in brain tumors]. AB - Concentrations of serotonin, 5-hydroxyindoleacetic acid, melatonin and parameters of the kinin system in the cerebrospinal fluid were monitored in 109 cases of brain tumor. All the levels were increased prior to surgery but returned to normal postoperatively unless disease followed an unfavorable course. It was suggested that the above-mentioned physiologically active substances play a certain role in compensatory mechanisms assuring recovery of CNS function. PMID- 1716032 TI - [Survival of patients after combined treatment for rectal cancer]. AB - The analysis of end results of treatment of rectal cancer showed combined therapy to assure long survival in the whole group of radically operated cases, viz. two third of them survived 10 years. Conversely, mean survival of non-operated or surgically palliated patients was as low as 10.1 and 26.7 months, respectively. Survival of radically operated cases was found to be related to various clinico anatomic factors, the most close relationship being observed for extent, histology and growth pattern of tumor and regional nodal status. The influence of site and surgical procedure on patient survival proved far less significant. PMID- 1716033 TI - [Reactions of the cardiovascular system to volume load and strophanthin administration in the early period after radical thoraco-abdominal surgical interventions in cancer patients]. AB - Cardiovascular reactions were assessed in 65 cancer patients who had been given volume load to the heart with sequential strophanthin in early postoperative period following thoraco-abdominal surgery. Positive hemodynamic reaction to the volume load was observed in 60% of cases whereas negative--in 35.4% while after strophanthin treatment the figures were 52.3 and 43.1%, respectively. Combination of values observed in the two tests proved indicative of the pathogenetic mechanism underlying postoperative circulatory disorders: cardiac failure, hypovolemia or syndrome of peripheral vasoconstriction. These data should be considered in choosing infusion and other medicinal treatment in early postoperative period. PMID- 1716034 TI - [Fetomaternal transfusions in early pregnancy]. AB - Fetomaternal bleeding is the major cause for rhesus immunization in pregnancy. This study evaluates the amount of fetomaternal bleeding in 345 pregnancies before 28th week of gestation. Fetomaternal bleeding was of clinical relevance (HbF greater than 0.015%) in 2.2% of all uncomplicated pregnancies. In case of abortion or vaginal bleeding in early pregnancy we observed a statistical significant increase of transplacental hemorrhage. The need for anti-D prophylaxis in this patients is obvious. PMID- 1716035 TI - Delineation of protective epitopes on the E2-envelope glycoprotein of Semliki Forest virus. AB - Two short linear peptides, 17 and 14 amino acids long, on the Semliki Forest virus (SFV) E2-envelope polypeptide are shown to be involved in the protection of mice against lethal challenge with SFV. Peptides corresponding to these two regions, designated H and L, were selected for study on the basis of our model for prediction of protective epitopes on E2 polypeptide of alphaviruses. These peptides were produced in Escherichia coli as recombinant proteins fused to the amino terminus of beta-galactosidase. Both the H epitope (amino acid positions 227-243 on E2) and L epitope (amino acid positions 297-310) are recognized by antibodies raised against SFV, and both trigger antibodies that interact with native SFV-E2. Vaccination of mice with the H-beta-galactosidase polypeptide confers 64-87% protection against a lethal viral challenge (250 LD50), and immunization with L-beta-galactosidase leads to 23-66% protection of challenged mice. The efficacy of the L-based synthetic vaccine could be improved further (up to 100% protection) by presentation of this epitope as a dimer fused to beta galactosidase. These results provide evidence that the algorithm and the methodology proposed by us previously are effective tools for identification of linear protective epitopes on E2-envelope of SFV. PMID- 1716036 TI - [The range of antigenic specificity of Bifidobacterium peptidoglycan]. AB - The antigenic relationships of Bifidobacterium bifidum 1 peptidoglycans with different strains of this species (LVA-3, 791, GO-4), bifidobacteria of other species (B. adolescentis GO-13, B. breve 79-38, B. lactentis 79-41, B. longum GO 3) and bacteria of remote taxonomic groups (Streptococcus faecalis 6-3. Staphylococcus aureus COM 885, S. epidermidis COM 2124. Lactobacillus plantarum 1, Escherichia coli M-17) were studied on the basis of a highly sensitive test system permitting the registration of normal human antibodies to peptidoglycans. The level of cross reactions with staphylococci and streptococci correspond to intraspecific and antigenic affinity to L. plantarum and E. coli was considerably less pronounced. Copying a number of epitopes of bifidobacteria, S. aureus peptidoglycan seems to possess additional antigenic determinants which participate in the formation of immunological responsiveness in man. PMID- 1716037 TI - [The development of a C1q-solid-phase immunoenzyme method for determining circulating immune complexes]. AB - The method of quantitative enzyme immunoassay (EIA) for the determination of circulating immune complexes (CIC) was developed on the basis of solid-phase human C1q. The calibration curve was plotted with the use of aggregated human gamma-globulin (AHGG), the optimum range of concentration being 15-500 microg/ml. In the process of approbation on clinical material the method revealed an elevated level of CIC in the sera of patients in comparison with their level in the sera of healthy donors. Out of 40 studied serum samples from patients with Yersinia infection, in 3 serum samples the levels of CIC was 26, 65 and 94 microg of AHGG equivalents per ml. In 4 out of 46 studied serum samples obtained from patients with diagnosed Yersinia arthritis the level of CIC was 12, 27, 46 and 186 microg of AHGG per ml, and in serum samples from healthy donors this level was 8.6 microg/ml [corrected]. PMID- 1716038 TI - Plasma C3c in immune-mediated neurological diseases: a preliminary report. AB - Plasma C3c levels were examined in 56 patients with immune (27) and non-immune (29) mediated neurological diseases by crossed immunoelectrophoresis. Plasma samples were collected during the active phase of illness in both groups, usually within 7 days of admission. 11 patients (4 Guillain-Barre Syndrome-GBS, 3 chronic inflammatory demyelinating polyneuropathy-CIDP, 4 myasthenia gravis-MG) had their plasma saved sequentially during the active and the recovery phase. Plasma C3c levels were elevated in the group with immune mediated diseases when compared with those of non-immune mediated diseases. The sensitivity and specificity of C3c as a diagnostic test for immune mediated neurological diseases were 61.4 and 100% respectively with a positive and negative predictive value of 100 and 41%. the C3c levels in plasma correlated well with disease severity in MG and GBS patients. Such a correlation was also evident in all CIDP patients except one that had persistent elevation in the presence of clinical improvement. Results suggest that the plasma C3c level may be useful for differentiating immune from non-immune mediated neurological diseases. Plasma C3c may also be used for monitoring disease severity, particularly in myasthenia gravis. PMID- 1716039 TI - [Indications and results of surgical treatment of carcinoma of the hypopharynx and cervical esophagus]. PMID- 1716040 TI - [Mechanism of enhancement of bleomycin A5 antitumor activity by verapamil]. AB - The mechanism of enhancement of Bleomycin A5 antitumor activity by verapamil was explored by flow cytometry and tracing the radiolabelled bleomycin A5 in vivo. Verapamil was found to increase the G2 blocking effect of bleomycin A5 prominently in mouse S-180 and human HEP-2 cell lines. The distribution of 57Co bleomycin A5 in mice bearing S-180 sarcoma was changed by verapamil and accumulation of the drug in tumor was increased. In contrast, the labelled drug in the lung was decreased. It seems that the effects of verapamil in enhancing the antitumor activity of bleomycin A5 are to increase the accumulation of the drug in tumor cells and enhance the G2 blocking effect of the drug in cell cycle. PMID- 1716041 TI - Low AFP and congenital diaphragmatic defects. PMID- 1716042 TI - Coexpression of CD15 and CD20 by Reed-Sternberg cells in Hodgkin's disease. AB - The immunophenotype of the Reed-Sternberg cells in Hodgkin's disease is heterogeneous among different cases; this heterogeneity has contributed to the continuing uncertainty regarding the normal counterpart of the Reed-Sternberg cell. In this study, the authors demonstrate coexpression of the B-cell marker, CD20, and the granulocyte associated antigen, CD15, by Reed-Sternberg cells in three of 20 cases of nodular sclerosis and mixed cellularity Hodgkin's disease using a double-labelling technique in one case and staining of serial sections in three cases. Additionally, the authors found that expression of CD20 occurred more often in tumors with a monomorphous proliferation of mononuclear and binucleate Hodgkin's and Reed-Sternberg cells, without numerous eosinophils or polymorphonuclear neutrophils. In contrast, expression of CD15 by Reed-Sternberg cells was associated with a greater granulocyte infiltrate. The presence or absence of fibrosis, plasma cells, and histiocytes did not correlate with antigen expression. These results suggest that there may be a continuum of antigen expression by Reed-Sternberg cells, with some cells expressing CD20, some CD15, and others expressing both antigens; cells coexpressing both CD15 and CD20 may represent an unstable intermediate in the process of antigen switching. The possibility that antigen expression by the neoplastic cells in a given case may modulate depending on the background infiltrate could explain the heterogeneity of immunophenotype among cases of Hodgkin's disease. PMID- 1716043 TI - Amyloid precursor protein and ubiquitin immunoreactivity in dystrophic axons is not unique to Alzheimer's disease. AB - A distinctive feature of Alzheimer's disease (AD) is the presence of dystrophic neurites that immunoreact with antibodies to amyloid precursor protein (APP) and ubiquitin (Ub). The authors examined dystrophic axons (DA) present in other chronic conditions such as familial infantile neuroaxonal dystrophy (INAD), aging, cystic fibrosis, and biliary obstruction as well as in conditions of shorter duration such as human immunodeficiency virus (HIV) leucoencephalopathy, infarction and radiation therapy to determine whether APP and Ub immunoreactivity was unique to the DA of AD. A large number of DA immunoreacted with antibodies to the A4, C- and N-terminal regions of APP as well as to Ub. Ub and APP immunoreactivities often, but not always, colocalized. "Acute" DA generally reacted more intensely and in larger number with antibodies to APP than to Ub, whereas the reverse was true for "chronic" DA. Structureless DA immunostained diffusely. In DA with cores or granules, the Ub immunoreaction was occasionally limited to these structures, whereas reaction with antibodies to APP was more diffuse. In view of the contention that impairment of proteolysis is the common pathogenetic step in the formation of DA, Ub immunoreactivity in all DA may indicate a vicarious attempt to degrade accumulated components through an activation of the Ub system. The role of APP in the formation of DA remains to be determined. PMID- 1716044 TI - Immunohistochemical colocalization of amyloid precursor protein with cerebrovascular amyloid of Alzheimer's disease. AB - Molecular cloning and cDNA sequencing have indicated that the fibril-forming, amyloidogenic beta/A4 peptide of cerebrovasculature and plaque core in AD is encoded as part of a larger precursor, amyloid precursor protein (APP). A panel of antibodies directed against synthetic peptides, which correspond to distinct domains of this putative APP molecule (i.e., amino acid residues 45-62, 587-596, 597-606, 597-638 [beta/A4 peptide], 638-658 and 653-661), were used to probe immunohistochemically serial sections of formalin-fixed, paraffin-embedded Alzheimer's disease (AD) brains for the presence of APP and/or its derivatives. Histochemical staining of adjacent sections with Bielschowsky's silver impregnation and with Congo red or thioflavin S-staining techniques was also done to identify the structures with amyloid deposition. All these antibodies exhibited intense immunoreactivity with amyloidotic cerebral vessels, including meningeal and parenchymal. This observation indicates that the amyloidotic vasculature of AD brain contains, in addition to the fibril-forming beta/A4 protein, nonamyloidogenic APP and/or its derivatives. More importantly, this APP immunoreactivity colocalized with angiopathic amyloid, which is characterized by phenol-resistant, birefringent congophilia. Parallel analyses with a dual SABC/silver impregnation procedure further confirmed that APP and/or its derivatives, including the amyloidogenic beta/A4, colocalized with argentophilic amyloid in the cerebrovasculature of AD. PMID- 1716045 TI - Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine. AB - Oval cells may function as facultative liver stem cells and tumor progenitors in liver carcinogenesis. The authors determined whether oval cells proliferate and if small hepatocytes might be generated from epithelial cell progenitors in noncarcinogenic liver injury. The authors found that oval cells similar to those detected in early carcinogenesis proliferate in response to D-galactosamine (GaIN). Oval cells expressed gamma-glutamyl transpeptidase activity, bile duct type cytokeratins and peanut agglutinin binding. Two unusual types of hepatocytes also appeared after injury: small hepatocytes (less than or equal to 16 microns in diameter) and hepatocytes lining atypical ductlike structures. In situ hybridization studies showed that the fetal form of alphafetoprotein mRNA was expressed by many oval cells, some bile duct cells, and occasional hepatocytes. By following the fate of epithelial cells labeled early after GaIN administration, the authors conclude that duct cells can generate both oval cells and small hepatocytes in response to GaIN. PMID- 1716046 TI - Image analysis microspectroscopy shows that neurons participate in the genesis of a subset of early primitive (diffuse) senile plaques. AB - Amyloid is a component of the senile plaques that characterize one of the major neuropathologic changes in patients with Alzheimer's disease (AD). The sequence of events leading to the accumulation of amyloid precursors in senile plaques is unknown. In previous studies, the authors have shown that congophilic deposits in a subset of mature amyloid plaques are angiocentric. In this study, the authors used image analysis microspectroscopy and an antibody directed against a synthetic beta-protein (beta) or A4 sequence to examine the distribution patterns of this protein in serial sections from brains of patients with AD and in normal aged brains after quantitative immunohistochemistry. Image analysis of early primitive plaques disclosed two main patterns of early beta/A4 deposition, which consisted of neurocentric and angiocentric decreasing concentration gradients. In most instances, these gradients were not recognizable by the naked eye but appeared strikingly conspicuous after image subtraction and pseudocoloring. The described neurocentric gradients suggest that deposition of this protein, in at least some early primitive plaques, is related to neurons and possibly originates from these cells. The opposite viewpoint, i.e., that peripherally synthesized beta/A4 protein would 'sink in' toward neurons, is not supported because in very early plaques the highest immunoreactivity within the gradient was the neuronal body itself. A hypothesis is offered to reconcile the presence of both neurocentric and angiocentric depositions of these substances. PMID- 1716047 TI - Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys. AB - Simian immunodeficiency virus (SIV), like the human immunodeficiency virus (HIV), is a lentivirus that is both immunosuppressive and neurovirulent. Rhesus macaques (Macaca mulatta) inoculated with SIV often develop a giant cell encephalitis similar to that seen in humans infected with HIV. The authors examined SIV expression by immunohistochemistry and RNA in situ hybridization in the cerebrum, cerebellum, choroid plexus, and spinal cord from five macaques with and two macaques without giant cell encephalitis. Selected portions of the central nervous system (CNS) also were examined by electron microscopy. Simian immunodeficiency virus was detected in the CNS of all seven monkeys whether or not they had giant cell encephalitis. Both SIV antigen and RNA were present in all levels of the CNS examined. Macrophage/giant cell lesions always contained viral RNA and antigen and were the only sites where viral particles were detected by electron microscopy. However, SIV antigen and RNA also were commonly associated with small vessels, the choroid plexus, and meninges; these were the only locations where virus was detected in animals without giant cell encephalitis. Immunophenotyping showed that the cellular infiltrates consisted primarily of monocyte/macrophages and occasional CD8-positive T cells. Macrophages and T cells also were present in the stroma of the choroid plexus and were intimately associated with vessels in the CNS of SIV-infected but not uninfected macaques. Simian immunodeficiency virus infection of the macaque CNS provides an excellent model for studying the pathogenesis, treatment, and prevention of HIV-1-encephalitis. PMID- 1716048 TI - Induction of tenascin in rat arterial injury. Relationship to altered smooth muscle cell phenotype. AB - Arterial smooth muscle cells produce large amounts of extracellular matrix molecules during repair processes and in primary culture. This occurs after a transition of the cells from a contractile to a synthetic phenotype and together with the acquisition of proliferative capacity. Here, the deposition of the glycoprotein tenascin in the extracellular matrix of rat arterial smooth muscle cells in vivo and in vitro was studied by immunofluorescence microscopy and immunoblotting. Tenascin was found in the intimal layer of the adult rat aorta and carotid artery, but not in the media. Tenascin also appeared in the neointima formed by proliferating smooth muscle cells 2 weeks after balloon catheter injury of the carotid artery. To determine if the deposition of tenascin in the neointima after arterial injury was related to changes in smooth muscle function, freshly isolated rat aortic smooth muscle cells were seeded on a substrate of plasma fibronectin and allowed to modulate from a contractile to a synthetic state in a serum-free medium. In these quiescent cultures, a tenascin-containing extracellular matrix was formed after 3 to 4 days. Taken together, the results show that tenascin production is induced concomitantly with changes in smooth muscle phenotype both in vivo and in vitro. PMID- 1716049 TI - Chloride conductance pathways in heart. AB - Nonelectrogenic movement of Cl- is believed to be responsible for the active accumulation of intracellular Cl- in cardiac muscle. The electro-neutral pathways underlying this nonpassive distribution of Cl- are believed to include Cl(-)-HCO3 exchange, Na(+)-dependent cotransport (operating as Na(+)-Cl- and Na(+)-K(+)-2Cl cotransport), and K(+)-Cl- cotransport. The electrogenic movement of Cl- in cardiac muscle is particularly interesting from a historical perspective. Until recently, there was some doubt as to whether Cl- carried any current in the heart. Early microelectrode experiments indicated that a Cl- conductance probably played an important role in regulating action potential duration and resting membrane potential. Subsequent voltage-clamp experiments identified a repolarizing, transient outward current that was believed to be conducted by Cl-, yet further investigation suggested that this transient outward current was more likely a K+ current, not a Cl- current. This left some doubt as to whether Cl- played any role in regulating membrane potential in cardiac muscle. More recent studies, however, have identified a highly selective Cl- conductance that is regulated by intracellular adenosine 3',5'-cyclic monophosphate, and it appears that this Cl- current may play an important role in the regulation of action potential duration and resting membrane potential. PMID- 1716050 TI - Regulation of Na-K-2Cl cotransport in osteoblasts. AB - Uptake of 86Rb was used to follow the activity of Na-K-2Cl cotransport in the osteosarcoma cell line UMR-106-01. The ouabain-resistant fraction of 86Rb uptake was sensitive to bumetanide and furosemide. Furosemide-sensitive 86Rb uptake required the presence of Na+, K+, and Cl- in the incubation medium. These observations indicate the presence of a Na-K-2Cl cotransport system in osteoblasts. Cotransporter activity was stimulated by agonists which increase adenosine 3',5'-cyclic monophosphate (cAMP), cytosolic free Ca2+ ([Ca2+]i), and protein kinase C (PKC) activity such as parathyroid hormone (PTH) and prostaglandin E2 (PGE2). However, endothelin, which increases [Ca2+]i and PKC activity without affecting cellular levels of cAMP, was ineffective in stimulating the cotransporter. Accordingly, increasing cellular cAMP with forskolin was as effective as PTH and PGE2 in stimulating the cotransporter. Stimulation of PKC with TPA inhibited the cotransporter in a time- and concentration-dependent manner. No stimulation of cotransport could be demonstrated at any 12-O-tetradecanoyl-phorbol-13-acetate (TPA) concentration or incubation time. The Na-K-2Cl cotransporter was stimulated by cell shrinkage. Maximal stimulation was observed after swelling the cells in hypotonic medium and subsequent shrinkage in isotonic medium. Stimulation by cell shrinkage can be demonstrated in control, agonist-, cAMP-, and TPA-treated cells. These observations suggest that 1) the osteoblastic Na-K-2Cl cotransporter is activated by calciotropic hormones predominantly through an increase in cellular cAMP, and 2) in osteoblasts, the cotransporter is independently regulated by different biochemical pathways. PMID- 1716051 TI - Regulation of cell volume by the osteosarcoma cell line UMR-106-01. AB - Determination of cell volume by an electronic cell-sizing technique was used to study the role of ion transporters in cell volume regulation by the osteosarcoma cell line UMR-106-01. Swelling the cells in hypotonic medium was followed by regulatory volume decrease (RVD). The rate of RVD was strongly dependent on the subpassage used and increased with increasing subpassages. Swelling-evoked changes in cytosolic free Ca2+ ([Ca2+]i) did not account for this behavior, since it was similar in cells from all subpassages. Increasing plasma membrane K+ permeability with valinomycin resulted in a similar rate of RVD in cells from different subpassages, suggesting increased K+ channel activity or other electrogenic transporter with increased subpassages. In contrast, the mechanisms responsible for regulatory volume increase (RVI) were fully active in cells from all subpassages. Increasing medium osmolarity of cells bathed in isotonic medium induced slow and incomplete RVI. In addition, shrinking cells exposed to hypotonic medium before completion of RVD resulted in impaired RVI. Effective RVI could be observed only after completion of RVD of cells exposed to hypotonic medium. Removal of extracellular Na+ or K+ completely blocked RVI, whereas removal of external Cl- partially blocked RVI. The effect of K+ removal probably reflects in part inhibition of Na-K-2Cl cotransport and in part inhibition of the Na+ pump.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716052 TI - Isolation and characterization of rat submandibular intralobular ducts. AB - Intralobular (granular) salivary ducts were purified by isopycnic centrifugation after collagenase/hyaluronidase digestion of the rat submandibular gland. The resulting ductal fraction (density, 1.056 +/- 0.003) was highly enriched in kallikrein (a ductal cell marker) and contained little amylase activity (an acinar cell marker). The resting intracellular calcium level in the ductal preparation was 103 +/- 4 nM. Increased intracellular calcium concentrations (2-3 times resting levels) were observed in response to muscarinic (carbachol) and alpha-adrenergic (epinephrine) agonists, but little response was observed to substance P, suggesting the absence of substance P peptidergic receptors on rat submandibular ducts. Intracellular adenosine 3',5'-cyclic monophosphate levels were increased 35-fold in response to beta-adrenergic stimulation (isoproterenol) and forskolin. The ducts secreted kallikrein in response to epinephrine, carbachol, and isoproterenol but not in response to substance P. Epinephrine was the most potent inducer of kallikrein release with a K0.5 of approximately 3 microM and a maximal secretory rate approximately nine times unstimulated levels. Taken together, these results provide strong evidence for the functional integrity of the ductal preparation. This preparation should prove useful for the further elucidation of the properties of intralobular salivary ducts structures which heretofore have only been studied indirectly. PMID- 1716053 TI - Acetylcholine Ca2+ stores refilling directly involves a dihydropyridine-sensitive channel in dog trachea. AB - Repetitive stimulation of the smooth muscle with acetylcholine (ACh) in the continuous presence of nifedipine resulted in a progressive decrease in the developed tension. This was associated with a decrease in the content of the agonist-sensitive intracellular Ca2+ stores. Agonist-sensitive internal Ca2+ stores appeared to be readily depleted by successive or prolonged agonist stimulation in Ca(2+)-free medium. The refilling of the empty stores when the muscle is at rest required extracellular Ca2+, was decreased by nifedipine, and was increased by BAY K 8644 and by increased external Ca2+ concentration. Refilling of stores during ACh stimulation in Ca(2+)-containing medium was decreased by nifedipine and by cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, and was potentiated by BAY K 8644. BAY K 8644 reversed the inhibitory effect of CPA on stores Ca2+ refilling. Ryanodine in normal Krebs increased muscle resting tension, an effect not observed in Ca(2+) free medium, blocked by nifedipine and enhanced by BAY K 8644. We propose that the refilling of ACh-sensitive internal Ca2+ stores involves two distinct pathways, one dependent on the uptake of cytosolic Ca2+ via a CPA-sensitive SR Ca(2+)-adenosinetriphosphatase, and the other pathway dependent on extracellular Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel and is CPA insensitive. The refilling pathway between plasmalemma and SR may involve a plasmalemma L-type Ca2+ channel (dihydropyridine sensitive) and the SR Ca2+ release channel (ryanodine sensitive). PMID- 1716054 TI - Hypertrophy of isolated adult feline heart cells following beta-adrenergic induced beating. AB - Catecholamine-induced beating and myocardial hypertrophy were evaluated in isolated adult feline cardiomyocytes maintained in culture for up to 30 days. Adult feline cardiomyocytes were used in this study because they displayed several unique characteristics that facilitated assessment of factors regulating cardiomyocyte hypertrophy in vitro. These characteristics included the following. 1) A single heart provides a high yield of 20-40 x 10(6) calcium-tolerant rod shaped myocytes. 2) In culture, isolated adult feline cardiomyocytes maintain a stable population of differentiated myocytes that could be maintained without the dramatic loss of cell number, DNA content, or cell structure seen in adult rat cardiomyocyte cultures. 3) Cultured feline cardiomyocytes remained quiescent in culture unless appropriately stimulated to begin beating. 4) Sustained regular beating activity could be readily initiated up to 3 wk in culture by addition of 1 x 10(-5) M isoproterenol, other beta-adrenergic agonists, or agents known to elevate adenosine 3',5'-cyclic monophosphate. Beating could be maintained indefinitely in the presence of isoproterenol, but ceased upon removal of isoproterenol from the medium. Initiation of beating in 7-day-old cultures resulted in a profound restructuring of cardiomyocyte morphology compared with quiescent cultures. Beating heart cells were 66% larger with increased protein content, and they had significantly greater development of striated myofibrillar structure than quiescent myocytes at the same age in culture. We conclude that maintenance of an organized myofibrillar structure in cultured adult cardiac myocytes requires activation of intrinsic beating. Cardiomyocyte hypertrophy also develops following beta-adrenergic activation of beating, but it is unclear whether beating per se is required for inducing hypertrophy in isolated adult cardiomyocytes in vitro. PMID- 1716055 TI - Integration of embryonic nephrogenic cells carrying a reporter gene into functioning nephrons. AB - We developed a procedure to introduce and stably express foreign genes into the kidney. The Lac Z reporter gene encoding the bacterial protein beta-galactosidase was introduced by retrovirus-mediated gene transfer into rat nephrogenic mesenchymal cells, which were induced for 24 h with embryonic spinal cord in vitro. The Lac Z-tagged mesenchymal cells were subsequently transplanted underneath the capsule of the neonatal kidney. Two weeks after transplantation, the Lac Z-tagged cells derived from transplants were identified by their beta galactosidase expression. Well-differentiated Lac Z positive cells were observed in glomerulus and proximal and distal nephron segments. To determine if the tagged mesenchymal cells developed into functional nephrons, fluorescein isothiocyanate-labeled dextran was infused into transplanted animals before death. We observed that fluorescent apical vesicles were colocalized to beta galactosidase positive proximal tubular cells, indicating that the transplanted mesenchymal cells were integrated into reabsorbing nephrons. These results show the feasibility of introducing foreign genes into epithelia of functioning nephron segments. PMID- 1716056 TI - Atrial natriuretic polypeptide secretion via selective activation of kappa-opioid receptor: role of dynorphin. AB - The present study was designed to investigate the direct effect of dynorphin on atrial natriuretic polypeptide (ANP) secretion in cultured rat atrial cardiocytes via a kappa-opioid receptor activation as well as the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) system in the secretion of ANP from cardiocytes. Dynorphin stimulated ANP secretion dose and time dependently from 2 day cultured atrial cardiocytes. The dynorphin-induced ANP secretion was partially antagonized by MR2266, a selective kappa-opioid receptor antagonist. U 62066E, a selective kappa-opioid receptor agonist, stimulated ANP secretion. This stimulation was also antagonized by MR2266. However, no stimulation of ANP secretion was seen with [D-Ala2,D-Leu5]enkephalin, methionine (Met)-enkephalin, or [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin. Dynorphin at 10(-6) M significantly decreased the production of cAMP in the cultured cardiocytes. However, 10(-6) M Met-enkephalin had no effect on cAMP at all. The decrease in cAMP production by the addition of dynorphin was partially antagonized with a simultaneous addition of MR2266. The dynorphin-induced ANP secretion, as well as the basal secretion, were significantly decreased by the addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, as compared with the respective controls. Dibutyryl cAMP at 10(-3) M significantly decreased the basal secretion of ANP as compared with the control. Therefore, the present studies show that dynorphin selectively stimulates ANP secretion, at least in part, via the activation of a specific kappa-opioid receptor. PMID- 1716057 TI - Functional subtyping of muscarinic receptors on canine esophageal mucosa. AB - Serosal addition of muscarinic agonists elicited rapid changes in electrical parameters across the isolated canine esophageal epithelium set up in vitro. Both carbachol and the M1-selective agonist, McNeil A343 (McN), increased transmucosal potential differences (PDs), decreased transmucosal resistances (R), and increased short-circuit currents (Isc). Carbachol was more potent and more effective than McN. Muscarinic antagonists were used to define the muscarinic receptor involved. The pA2 values obtained with Schild plots were as follows: atropine 9.14, 4-DAMP 8.98, AFDX-116 6.71, and pirenzepine 7.12. Low concentrations of pirenzepine (10(-8) M), produced a rightward shift in the dose response curve to McN, without inhibiting responses to carbachol. Thus the receptor subtype is clearly not an M2. As in other glandular systems, M3 receptors are present. Whether M1 receptors also exist requires better definition of receptor densities-reserves in this tissue. Carbachol induced net secretion of Na and Cl and converted a predominantly absorptive tissue to a secretory one. PMID- 1716058 TI - Pancreatic gene expression is altered during acute experimental pancreatitis in the rat. AB - We investigated pancreatic gene expression in the rat in response to taurocholate induced acute pancreatitis. Concentrations of transcripts encoding pancreatic protein showed noncoordinated alterations. Contents in amylase, trypsinogen I, chymotrypsinogen B, elastase 1, and procarboxypeptidase A mRNAs decreased by greater than 50% during the acute phase (days 0-2), whereas actin and lithostathine mRNAs increased 5 and 0.6 times, respectively, and pancreatitis associated protein (PAP) mRNA increased greater than 200 times, indicating redirection of the pattern of gene expression. Synthesis of pancreatic proteins was also altered in a noncoordinated manner. During the acute phase, it decreased more for trypsinogen I and chymotrypsinogen B than for amylase and lipase, whereas synthesis of the PAP increased dramatically. For amylase and chymotrypsinogen B, we compared the patterns of changes in mRNA concentrations, rates of synthesis, and pancreatic contents. Changes in enzyme contents and synthetic rates were temporally correlated during the acute phase. On the contrary, changes in mRNA concentrations and enzyme synthesis were not coordinated, suggesting that control of synthesis partly occurred at the posttranscriptional level. It was concluded that induction of pancreatitis is accompanied by transcriptional and posttranscriptional modifications resulting in rapid and massive rearrangement of the pattern of pancreatic protein gene expression. PMID- 1716059 TI - Stimulation of proximal small intestinal mucosal growth by luminal polyamines. AB - The purpose of this study was to determine whether luminal polyamines stimulate intestinal mucosal growth in vivo. Rats received 2% alpha-difluoromethylornithine (DFMO) added to their drinking water throughout the experiment. The polyamines spermidine and spermine (3 mg each/100 g body wt) were given intragastrically in combined doses once at 9:30 A.M. and again at 5:30 P.M. Duodenal and jejunal mucosal ornithine decarboxylase (ODC) activity in the DFMO-treated rats was inhibited significantly for the duration of the study. DFMO also markedly decreased the rate of [3H]thymidine incorporation into DNA of duodenal and jejunal mucosa. The decrease in [3H]thymidine incorporation was significant 4 days and maximal 6 and 8 days after beginning treatment with DFMO. Decreased ODC activity and DNA synthesis were paralleled by decreases in total mucosal DNA, RNA, and protein content. Administration of the polyamines significantly reversed the effects of DFMO except the inhibition of ODC. In fact, there were no significant differences in mucosal growth parameters between the controls (without DFMO) and those treated with DFMO plus polyamines. Oral administration of spermidine and spermine at a dose of 4.5 mg each/100 g body wt for 6 days to rats not treated with DFMO increased the normal rate of mucosal growth in the duodenum and jejunum as well. Polyamine accumulation in IEC-6 cells was measured to determine whether it was altered by DFMO. IEC-6 cells took up [3H]putrescine and [3H]spermidine from their surrounding environment and the uptake was stimulated by serum. DFMO (5 mM) totally inhibited the increase in ODC activity but had no effect on the cellular uptake of polyamines in the presence of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716060 TI - Regulation of Na(+)-Ca2+ exchange in cultured human mesangial cells. AB - Na(+)-Ca2+ exchange contributes to regulation of cytosolic free Ca2+ levels ([Ca2+]i) of cultured human mesangial cells following phospholipase C stimulation, as shown by larger responses to vasoconstrictors such as angiotensin II (ANG II) or endothelin 1 in Na(+)-free media. In turn, previous activation of phospholipase C by vasoconstrictors significantly enhances the amplitude of the [Ca2+]i elevation induced by Na+ withdrawal. We studied the mechanisms of upregulation in monolayer cultures loaded with the fluorescent Ca(2+)-sensitive probe fura-2. The exchanger was stimulated by insulin and inhibited by chronic exposure to serum. A rise of [Ca2+]i was not sufficient per se to enhance exchange activity, as prior elevation of [Ca2+]i with the ionophores ionomycin or 8-bromo-A23187 failed to augment the response to Na+ withdrawal. Protein kinase C (PKC) activation by phorbol 12-myristate-13-acetate (PMA), alone or in combination with a rise of [Ca2+]i, potently inhibited basal and vasoconstrictor enhanced Na(+)-Ca2+ exchange. Suppression of the effects of ANG II was not due to frustrated phospholipase C activation by PMA, because addition of PMA after ANG II also inhibited Na(+)-Ca2+ exchange. PKC downregulation by 24-h pretreatment with PMA or inhibition with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or staurosporine did not prevent activation by ANG II. The exchanger was markedly potentiated by Na+ loading the cells with gramicidin D or reducing extracellular K+. ANG II failed to stimulate Na(+)-Ca2+ exchange when added in the absence of extracellular Na+. Therefore vasoconstrictors promote Na(+)-Ca2+ exchange by a mechanism independent of [Ca2+]i and PKC while presumably linked to Na+ influx. PMID- 1716061 TI - Cellular mechanism of lithium-induced nephrogenic diabetes insipidus in rats. AB - One of the mechanisms by which Li evokes polyuria is thought to be impairment of arginine vasopressin (AVP)-sensitive adenylate cyclase (AdC) in cells of the renal collecting duct. To investigate how AdC is influenced by chronic administration of Li, we created nephrogenic diabetes insipidus (NDI) in rats and microdissected the medullary collecting tubule from both control and NDI rats. In the NDI group, the 10(-6) M AVP-stimulated cAMP contents failed to increase completely, and the levels were significantly lower than that of the control group (10.4 +/- 1.4 vs. 48.4 +/- 4.7 fmol/mm, P less than 0.001). Pretreatment with pertussis toxin (PT), an inhibitor of inhibitory G protein (Gi), did not affect the basal cAMP levels in both groups, although it increased AVP-stimulated cAMP production in the NDI group in a dose- and time-dependent manner. AVP stimulated cAMP production with over 100 ng/ml PT in the NDI group reached the levels observed in the control group. Incubation with cholera toxin, an agonist of stimulatory G protein (Gs), increased the cAMP content in the two groups to almost equal levels. To exclude the possibility that prostaglandin E2 (PGE2) is involved in the cellular mechanism of Li-induced NDI, the effect of indomethacin (Indo) on PT action was examined. However, Indo (10(-5) M) did not influence either the basal or AVP-dependent cAMP contents. From these results it is suggested that Li impairs AVP-sensitive AdC not through inhibition of Gs but through activation of Gi and that PGE2 may not be involved in the cellular pathogenesis of NDI at least in the rat at the step of cAMP formation. PMID- 1716062 TI - Hormone responses of proximal Na(+)-H+ exchanger in spontaneously hypertensive rats. AB - Na(+)-H+ exchange activity is increased in hypertensive rat strains and could be a predisposing factor in the pathogenesis of essential hypertension. Previously we demonstrated that proximal nephron Na(+)-H+ exchange is stimulated by alpha adrenergic agonists and angiotensin II (ANG II) and inhibited by parathyroid hormone (PTH) and dopamine (DA). To test the hypothesis that hormonal regulation of proximal nephron Na(+)-H+ exchange could differ with hypertension, alterations in Na(+)-H+ exchange were determined by 1) amiloride analogue-suppressible 22Na+ uptake and 2) change in intracellular pH (pHi) as monitored with the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)carboxyfluorscein acetoxymethyl ester. Spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats had similar tail cuff pressures at 4 wk, but SHR blood pressure was significantly elevated at 8 and 16 wk compared with WKY. No significant differences were observed between SHR and WKY basal ethylisopropyl amiloride-suppressible 22Na+ uptakes or rates of pHi change. alpha-Adrenergic agents and ANG II significantly increased (P less than 0.05) Na(+)-H+ exchange, but, in contrast, 8- and 16-wk-old SHR tubules lacked responsiveness to PTH (10(-8) M) and DA (10(-6) M) observed in WKY. A significant reduction (57-79%, P less than 0.05) in norepinephrine and ANG II stimulation was observed with 8- and 16-wk-old WKY tubules incubated in combination with PTH or DA, but only a 3-33% reduction was produced in 8- and 16-wk-old SHR tubules. PTH- and DA-stimulated adenosine 3',5'-cyclic monophosphate accumulation was significantly reduced in SHR compared with WKY tubules at 4 and 8 wk. It appears that proximal nephron Na(+)-H+ exchange activity is a balance between ANG II and NE activation and PTH and DA inhibition. The data suggest SHR proximal hormone responses are different from WKY and may alter the balance of net Na(+)-H+ exchange activity, possibly contributing to the development or maintenance of hypertension in the SHR. PMID- 1716063 TI - Effects of PGE2 and a thromboxane A2 analogue on uptake of IgG complexes and LDL by mesangial cells. AB - Vasoactive agents such as angiotensin (ANG) and eicosanoids can influence macromolecular deposition in the glomerular mesangium. We studied whether prostaglandin E2 (PGE2) and U-46619 (a stable analogue of thromboxane A2) could directly affect the uptake of immunoglobulin G (IgG) complexes or low-density lipoprotein (LDL) by cultured rat mesangial cells (MC). Preincubation of MC with PGE2 (10(-6) M) resulted in decreased uptake (in counts.min-1.well-1) of IgG complexes (PGE2, 2,589 +/- 72; control, 3,840 +/- 114; P less than 0.001) and LDL particles (PGE2, 23,176 +/- 1,145; control, 37,216 +/- 4,520; P less than 0.05). MC preincubated with forskolin (10(-5) M) also showed decreased uptake of IgG complexes (forskolin, 2,896 +/- 196; control, 3,840 +/- 114; P less than 0.005) and LDL particles (forskolin, 23,176 +/- 1,145; control, 37,216 +/- 4,520; P less than 0.05). In contrast, preincubation with ANG II (5 x 10(-7) M) showed significantly higher uptake of IgG complexes (ANG II, 4,475 +/- 282; control, 3,787 +/- 277; P less than 0.05) and LDL (ANG II, 48,573 +/- 1,079; control, 44,697 +/- 770; P less than 0.05). Similarly, preincubation with U-46619 (10(-6) M) resulted in significantly higher uptake of IgG complexes (U-46619, 5,370 +/- 300; control, 3,659 +/- 307; P less than 0.02) and LDL (U-46619, 48,298 +/- 1,418; control, 44,697 +/- 770; P less than 0.05). After preincubation with cyclooxygenase inhibitor meclofenamate (10(-6) M), ANG II (5 x 10(-7) M) resulted in a significantly higher uptake of IgG complexes compared with uptake by MC treated with ANG II alone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716064 TI - Leukotrienes, thromboxane A2, and prostaglandins during systemic anaphylaxis in sheep. AB - We investigated the roles of eicosanoid mediators in acute systemic anaphylaxis in anesthetized sheep. Sheep were sensitized with dinitrophenylated Ascaris suum extract and were challenged with an intravenous injection of dinitrophenylated bovine serum albumin. During anaphylaxis, cyclooxygenase inhibitors eliminated the elevation of arterial plasma levels of thromboxane B2 and 6-ketoprostaglandin F 1 alpha but markedly elevated the levels of leukotriene E4 in lung lymph without significantly eliminating elevation of plasma levels of histamine. Most of the measured physiological abnormalities accompanying anaphylaxis were aggravated by cyclooxygenase blockade. Enhancement of this anaphylactic mediator response was associated with an accentuated and prolonged increase of airway pressure (P less than 0.05, compared with sensitized, antigen-challenged but otherwise untreated sheep), a more intense hypoxemia (P less than 0.0001), and leukopenia (P less than 0.001), changes that were largely eliminated by pretreating with the sulfidopeptide leukotriene (SPLT) antagonist FPL 55712, suggesting that the SPLTs were important mediators of these responses. In contrast, the prolonged, but less severe, systemic vascular collapse and the reduced pulmonary hypertension induced by cyclooxygenase inhibitors were not influenced by the SPLT antagonist. These results demonstrate that in sheep cyclooxygenase metabolites are mainly involved in the acute, but transient, systemic and pulmonary vascular response of systemic anaphylaxis, whereas SPLTs are primarily implicated in the airway and secondary cardiovascular response. SPLT may act either directly or by potentiating the release of and reactivity to histamine and other mediators. Our data therefore suggest that a combination of cyclooxygenase and lipoxygenase inhibition will be necessary to more effectively protect against the consequences of an anaphylactic reaction. PMID- 1716065 TI - Skeletal muscle amino acid and myofibrillar protein mRNA response to thermal injury and infection. AB - Skeletal muscle changes associated with severe injury were investigated in male Wistar rats subjected to 30% full thickness scald injury (burn) and thermal injury followed by immediate colonization with 10(8) colony-forming units of Pseudomonas aeruginosa (BI). Freely fed animals (FF) and animals pair fed to the BI animals (PF) served as controls. Thermal injury in conjunction with infection produced a rapid and sustained muscle cellular membrane depolarization (transmembrane potential difference at 12 h after injury: FF 92.1 +/- 0.3 and BI 85.2 +/- 2.3 mV; P less than 0.05). This was followed by body weight loss and skeletal muscle protein wasting (gastrocnemius protein at 7 days: FF 0.35 +/- 0.01 and BI 0.16 +/- 0.03 g; P less than 0.05) and intracellular high-energy phosphate depletion (ATP at 10 days: FF 6.6 +/- 0.4 and BI 4.5 +/- 0.4 mumol/g tissue; P less than 0.05). These body and cellular changes were not accounted for by the anorexia alone. Marked alterations in intracellular free amino acids were also noted in the BI group characterized by increases in levels of all amino acids (total intracellular free amino acids at 7 days: FF 51 +/- 7 and BI 91 +/- 12 mM; P less than 0.05) except intracellular glutamine (at 7 days: FF 6.0 +/- 0.2 and BI 2.4 +/- 0.6 mM; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716066 TI - Neuropeptide regulation of feeding in dogs. AB - Norepinephrine and four families of neuropeptides, namely, neuropeptide Y (NPY), opioid peptides, galanin, and growth hormone-releasing factor (GRH), have been shown to stimulate feeding after central administration. Because these studies were mainly done on laboratory rats, the present study was designed to ascertain the central stimulators of feeding in dogs. We have shown that porcine and human pancreatic polypeptides (PPs), when administered into the third cerebral ventricle (intracerebroventricularly), increased food and water intake of satiated animals but that the COOH-terminal fragments [hPP-(18-36) and hPP-(23 36)] did not do so at the same molar dose (11.9 nmol). The kappa-opioid receptor agonist dynorphin (A-(1-17) also stimulated food and water intake, whereas alpha neoendorphin and Met-enkephalin did not. These results suggest the structural specificity of PPs and dynorphin peptides for stimulating feeding. Surprisingly, neither intracerebroventricular injections of NPY and peptide YY nor intracerebroventricular pretreatment with anti-hNPY gamma-globulin modulated feeding, stressing the species differences in the feeding response to exogenous substances and the underlying physiology. Intracerebroventricular injections of norepinephrine, GRH, galanin, and pancreastatin also failed to increase food intake, although most substances tended to or did increase water intake. These results suggest that neuropeptides play a role in a species-specific way in modulating appetite regulation. PMID- 1716067 TI - The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye. AB - N,N-dipentadecylaminostyrylpyridinium iodide is a dye that is approximately 100 fold more intensely fluorescent in a lipid than aqueous environment. This observation suggests its potential as a fluorescence stain for lipoproteins. This work reports the staining of LDL with this dye for use in studies of cellular binding. The staining procedure is simple, resulting in stable attachment of the dye as determined by transfer experiments, physical properties essentially identical to native LDL as demonstrated by virtually identical electrophoretic mobility, and consistent results in studies of cellular binding using flow cytometry. Increased signal to noise ratio over other dyes used for lipoprotein staining including the widely used Dil (3,3'-dioctadecylindocarbocyanine iodide) allows determinations of greater sensitivity and precision to be made. This is demonstrated by the flow cytometric determination of the 4 degrees C binding curve of LDL with freshly isolated human peripheral blood lymphocytes (i.e., cells not LDL receptor upregulated). Mediation of binding by the LDL receptor is demonstrated by correspondence between the LDL receptor dissociation constant derived from this work and literature values; increased specific binding in lymphocytes cultured in lipoprotein-deficient media to up-regulate the LDL receptor; and decreased specific binding in lymphocytes cultured in the presence of 25-hydroxy cholesterol for 48 h to suppress the LDL receptor. PMID- 1716068 TI - A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels. AB - A method is described in which individual proteins labeled with weak beta emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum. PMID- 1716069 TI - A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge coupled device after staining with silver or labeling with fluorophore. AB - A multiple mini two-dimensional electrophoretic method which results in three two dimensional protein spot patterns being positioned side by side in an individual gel has been developed. Preparation time has been minimized by employing disposable capillary tubes for the isoelectric focusing gels and reducing the number of second-dimensional gels required. Commercially available vertical slab units were used for the second-dimensional electrophoresis. The protein spot patterns were visualized either by staining the second-dimensional gel with silver or fluorescently labeling the focused proteins while present in the isoelectric focusing gel and subsequently electrophoresing them into the second dimensional gel. The fluorescently labeled second-dimensional gel was imaged while still present in the glass mold immediately following electrophoresis. Two fluorophores were compared: 2-methoxy-2,4-diphenyl-3(2H)-furanone and 5-(4,6 dichlorotriazin-2-yl)aminofluorescein hydrochloride. A rapid imaging system based on a cooled charge-coupled device was used to view both the silver-stained and fluorescently labeled two-dimensional spot patterns. The sensitivity of detection of protein spots in the mini two-dimensional gels was similar for the two types of fluorescently labeled gels and the silver-stained gels. PMID- 1716070 TI - Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay. AB - A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716071 TI - Isolation of nucleic acids from plants by differential solvent precipitation. AB - The purification of nucleic acids from plant tissue is often made difficult by the presence of contaminating carbohydrate polymers and polyphenols. A procedure for the simultaneous isolation of DNA and translatable RNA from plants is described. The method removes most of the polysaccharides and polyphenols extracted with nucleic acids in a single step by taking advantage of differences in solubility of these compounds in the solvent 2-butoxyethanol. Stepwise addition of 2-butoxyethanol to phenol extracts of specific ionic strength precipitates nucleic acids largely free of contaminants. Subsequent separation of RNA from DNA by precipitation with LiCl was optimised to give a high recovery of translationally active RNA. Successful isolation of nucleic acids from strawberry (Fragaria X ananassa) receptacle, a particularly recalcitrant tissue, and from a range of tissues of other plant species demonstrates the general applicability of the method. PMID- 1716072 TI - Quantitation of mRNA by the kinetic polymerase chain reaction assay: a tool for monitoring P-glycoprotein gene expression. AB - The overexpression of P-glycoprotein (mdr1) induces the phenotype of multidrug resistance to many chemically unrelated drugs. Absolute mdr1 mRNA levels in tissues, neoplasms and cell lines from various rodents and man were estimated by primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labeled nucleotide. Aliquots taken during PCR were analyzed for product formation by two-dimensional densitometry of autoradiograms of separated cDNA on dried agarose gels. The kinetic RT/PCR assay was calibrated for the range of 10(-3) to 10(3) amol of specific mRNA by utilizing mdr1 RNA transcribed from a full-length cDNA as the external standard and aldolase mRNA as the internal standard in order to assess RNA degradation of the specimen. For various cDNA fragments differing between 200 and 600 bp in size, the yield of cDNA during logarithmic growth increased by 1.78 +/- 0.02 per cycle. Hence, the sensitive and reproducible quantitation of transcript is feasible by monitoring RT/PCR kinetics without the need for any competing cRNA or cDNA fragment provided that a high-performance thermal cycler is employed for PCR. PMID- 1716073 TI - Determination of urinary 5-hydroxyindole-3-acetic acid by high-performance liquid chromatography with electrochemical detection and direct sample injection. AB - A method for the routine quantitative determination of the major serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is described. 5-HIAA was analyzed without prior sample cleanup, using an automated high-performance liquid chromatography system with isocratic elution and electrochemical detection (+0.60 V versus a Ag/AgCl reference electrode). The urine samples were mixed with a solution of the internal standard (5-hydroxyindole-3-propionic acid) and centrifuged. The supernatant was transferred to sealed glass vials, and a 2 microliters aliquot was injected directly onto a C18 reversed-phase analytical column, using an automatic sample injector. Samples of urine could be stored for several months at -80 or at +7 degrees C for 2 days without loss of 5-HIAA. However, a gradual decline with time occurred in crude samples stored at room temperature or above, as well as in urine samples diluted with the mobile phase. The detector response was linear in the range of 0-65 mumol/l 5-HIAA, and the intra- and interassay coefficients of variation were about 5 and 7%, respectively (n = 10). PMID- 1716074 TI - Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay: application in determining epitope accessibility on ribosomes. AB - A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined. We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay. Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes. This method of detecting antibody migration is more sensitive than the conventional means of using A260nm to monitor the antibody-mediated dimerization of ribosomes. Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies. Thus, the relationship of different accessible epitopes in situ can be readily examined. Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems. PMID- 1716075 TI - Quantitative determination of the intracellular concentration of the various forms of HPr, a phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system in growing cells of oral streptococci. AB - A simple procedure for quantitative estimation of the different phosphorylated forms of the phosphocarrier protein HPr in growing cells of oral streptococci is described. The growth of the cells was rapidly stopped by acidification of the medium and concomitant addition of the ionophore Gramicidin D. This procedure inactivated Enzyme I, HPr(Ser) kinase, HPr(Ser-P) phosphatase, and the enzymes involved in the metabolism of the allosteric effectors as well as the substrates of HPr phosphorylation. The cellular concentrations of HPr (His approximately P), HPr (Ser-P), HPr (His approximately P) (Ser-P), and free HPr were then determined by crossed immunoelectrophoresis. PMID- 1716076 TI - Fast and sensitive silver staining of DNA in polyacrylamide gels. AB - The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF). PMID- 1716077 TI - Platelet activation and aggregation during cardiopulmonary bypass. AB - Increases in plasma concentrations of platelet granule products such as platelet factor 4 and beta-thromboglobulin during cardiopulmonary bypass suggest that platelets are activated during extracorporeal circulation. Subsequent circulation of these activated platelets may be responsible for the ubiquitous platelet dysfunction associated with cardiopulmonary bypass. Using flow cytometry and a monoclonal antibody directed against an alpha-granule membrane protein, granule membrane protein 140 (GMP-140), which is expressed on the platelet surface membrane after activation, we directly measured the percentage of circulating activated platelets in 41 patients before, during, and after cardiopulmonary bypass. In addition, we compared the GMP-140 expression with platelet aggregation in response to adenosine diphosphate (ADP). Cardiopulmonary bypass produced a significant increase in the percentage of GMP-140-positive platelets persisting in the circulation; the percentage peaked at a mean of 29% (range 10-58%) before separation from extracorporeal circulation. A significant percentage of these activated platelets continued to circulate in the early postoperative period. Simultaneous measurement of platelet aggregation in response to ADP demonstrated an aggregation defect that had a time course distinct from platelet activation and whose magnitude did not correlate with the degree of platelet activation in individual patients. We conclude that cardiopulmonary bypass causes a complex constellation of platelet defects, which include alpha-granule release, prolonged circulation of activated, "spent" platelets, and impaired platelet aggregation. PMID- 1716078 TI - Effects of calcium-free solution, calcium antagonists, and the calcium agonist BAY K 8644 on mechanical responses of skeletal muscle from patients susceptible to malignant hyperthermia. AB - The purpose of this investigation was to determine if alteration in the function of the dihydropyridine receptor may in turn modify halothane-induced contractures in muscle bundles from patients susceptible to malignant hyperthermia (MH). The effects of Ca(2+)-free Krebs Ringer (KR) solution, 5 microM verapamil, 5 microM nifedipine, and 10 microM of the Ca2+ agonist BAY K 8644 on halothane-induced contracture were therefore investigated. The halothane-induced contracture was prevented in the absence of extracellular Ca2+ and significantly reduced in the presence of verapamil or nifedipine. BAY K 8644 significantly enhanced the 0.5-, 1.0-, and 1.5-vol % halothane-induced contracture in MH-susceptible muscle bundles. When BAY K 8644 was dissolved in Ca(2+)-free KR solution, no contracture was observed in MH-susceptible muscle bundles. These results on cut MH susceptible human muscle bundles support the hypothesis that halothane-induced contracture in MH can be modified by the binding of Ca2+ agonists or antagonists to the dihydropyridine receptor. The role of Ca2+ entry phenomena remains unclear, but the results suggest that extracellular Ca2+ is required to reprime or to bind to some sites of the dihydropyridine receptors. PMID- 1716079 TI - Blood conservation techniques and platelet function in cardiac surgery. AB - Postoperative alterations in platelet function induced by cardiopulmonary bypass (CPB) are of importance. The effect on platelet aggregation of three different techniques for reducing blood consumption was studied in 30 patients undergoing elective aortocoronary bypass grafting from the beginning of anesthesia until the 1st postoperative day. The patients were randomly divided into three groups, in which 1) a cell separator was used during and after CPB; 2) a hemofiltration device was used; and 3) high-dose aprotinin was used in order to reduce the need of homologous blood. A fourth group undergoing neurosurgery procedures served as a control. Platelet aggregation induced by adenosine diphosphate (concentration 0.25, 0.50, 1.0, and 2.0 microM), collagen (4 microliters/ml), and epinephrine (25 microM) was determined by the turbidimetric method. Platelet aggregation was not significantly changed in the control group, indicating that the operation itself did not impair platelet function. At the end of the operation (after retransfusion of the salvaged pump blood), the maximum aggregation and maximum gradient of aggregation induced by all three inductors were most reduced (significantly) in the cell-separator patients. On the 1st postoperative day, platelet aggregation in the hemofiltration patients and the patients treated with aprotinin had normalized. Aggregation of patients pretreated with high-dose aprotinin was not different from that of the hemofiltration patients throughout the investigation. Blood loss was significantly highest in the cell-separator group (770 +/- 400 ml on the 1st postoperative day) but was not different between the hemofiltration (390 +/- 230 ml) and the aprotinin-treated patients (260 +/- 160 ml).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716080 TI - Isoflurane and an alpha 2-adrenoceptor agonist suppress nociceptive neurotransmission in neonatal rat spinal cord. AB - Analgesia is an important component of general anesthesia. alpha 2-adrenoceptor agonists such as clonidine and dexmedetomidine are effective analgesics at the spinal level, and furthermore, they reduce the volatile anesthetic requirement. In order to probe a possible spinal-level contribution to general anesthetic induced analgesia, the effects of dexmedetomidine were tested in an isolated spinal cord preparation. The effects of dexmedetomidine were compared with those of isoflurane, and dexmedetomidine-isoflurane interactions were explored. The test response was a nociceptive-related slow ventral root potential (slow VRP) recorded from the isolated neonatal rat spinal cord in response to electrical stimulation of a dorsal root. At 0.2-1.28 vol%, isoflurane reversibly depressed the slow VRP. At a lower concentration (0.14 vol%), isoflurane increased the slow VRP in three of five preparations. At 1.0-1.28 vol%, isoflurane also depressed the monosynaptic reflex. Recovery on washout usually was to a level greater than control. The N-methyl-D-aspartate (NMDA) receptor antagonist (DL)-2-amino 5 phosphonovalerate (10 microM) prevented the rebound to levels above control on isoflurane washout. The earlier components of the slow VRP were more sensitive to isoflurane than were the later. Dexmedetomidine (0.5-10 nM) depressed the slow VRP and had no effect on the monosynaptic reflex. The slow VRP depends on both substance P and glutamate NMDA-receptor-mediated neurotransmission; isoflurance and dexmedetomidine depressed responses to both substance P and NMDA. Although the two agents depress responses to the same neurotransmitters, there is no evidence that they act at the same cellular site(s). There was no significant interaction between dexmedetomidine and isoflurane. The results suggest that isoflurane exerts marked inhibitory effects on spinal neurotransmission, depressing both substance P and glutamate-mediated pathways. There is a possible biphasic effect on the NMDA receptor. To the extent that nociception depends on these neurotransmitters, isoflurane may be expected to exert profound analgesic effects at the spinal level. By blocking responses to strongly arousing stimuli, these effects may contribute to general anesthesia. Suppression of nociceptive neurotransmission at the spinal level may contribute to dexmedetomidine's anesthetic-sparing properties as well as to analgesia by this agent. PMID- 1716081 TI - Anesthetic considerations in patients receiving colony-stimulating factors (G-CSF and GM-CSF). PMID- 1716082 TI - Phenotyping of human tumor-infiltrating lymphocytes before and after exposure to different in vitro stimulation conditions. AB - Tumor-infiltrating lymphocytes (TiL) and autologous peripheral blood lymphocytes (PBL), mainly from breast and kidney tumor patients were cultivated with high- and low-dose rIL-2 under addition of autologous tumor cells or nonmalignant epithelial cells. Tumor cells added to TIL-cultures induced an additional proliferative response. Before cultivation, CD8+ and CD25+ lymphocytes were more frequent in TIL when compared to PBL, while CD16+, CD19+ and CD56+ cells were rare. After culture, lymphocytes of both origins showed an increase of CD2+, CD25+, CD56+ and HLA-DR+ cells and a relative decrease of CD3+ cells. The CD4+/CD8+ ratios and a number of CD56+ cells were rIL-2 dose dependent. PMID- 1716083 TI - Effects of antimicrobial drugs on the cytotoxicity of epirubicin, bleomycin, estramustine and cisplatin. AB - The effect of concomitant treatment with different antibiotics on the cytotoxicity of epirubicin, bleomycin, estramustine and cisplatin was studied in vitro on fibroblasts (V79) and two cancer cell lines (colon cancer HT29 and lung cancer P31). The cell lines were propagated under standard tissue culture conditions and evaluated as the number of surviving cell clones in comparison to untreated controls. Fifteen commonly used antibiotics were tested and thirteen of these were found to modify the cytotoxic effect in one or several of the combinations tested. One antibiotic agent could affect the toxicity of different cytostatics in opposite directions and there were marked differences between the cell lines tested. Only in one of the situations, the combination of bleomycin and ceftazidim, did the antibiotic cause opposite effects on the toxicity of a cytostatic when comparing fibroblasts and carcinoma cells. A most impressive observation was the pronounced increase in cisplatin-induced cytotoxicity by amphotericin B. In conclusion, the results suggest that antibiotics can interact with the cytotoxicity of antitumoral drugs but that the feature of this interaction is seemingly an erratic phenomenon. Further studies are certainly justified, especially regarding the effects of amphotericin B and its mechanisms in enhancing the cytotoxicity of cisplatin. PMID- 1716084 TI - Ethyl alcohol as a cocarcinogen with special reference to the aerodigestive tract: a cytogenetic study. AB - Although prior to its metabolic conversion alcohol has no mutagenic activity, an interaction of alcohol with tobacco in carcinogenesis of the head and neck region is well characterized. In a recent ecogenetic case control study of these cancers we documented an interaction between alcohol use and in vitro mutagen-induced chromosome damage in cancer risk. The present report evaluates the in vitro genotoxicity of five mutagens (including cigarette smoke condensate) tested in conjunction with 2% and 4% ethanol in human lymphoid cell lines. Ethanol alone did not exert a demonstrable clastogenic effect, as measured by the frequency of chromatid breaks per cell. However, the clastogenic potential of all mutagens increased when ethanol was added concurrently with the mutagens, and there was a dose-dependent potentiation of clastogenicity by ethanol, with a threshold dose between 0.5% and 1.0%. Preliminary experimental results strongly indicate that ethanol, at relatively high doses, inhibits DNA and chromosome repair systems. These data support the epidemiologic evidence of an interaction between alcohol and mutagens in carcinogenesis and suggest that alcohol may have co-carcinogenic properties. PMID- 1716085 TI - A simple fluorescent technique for the location of tumour cells in frozen sections of the head and neck region. AB - Tumour cells possess cell surface proteases referred to as guanidinobenzoatase (GB) which are closely similar to plasminogen activator. Previous studies have demonstrated different isoenzymic forms of GB on tumour cells and normal cells which can be recognised by cytoplasmic protein inhibitors extracted from frozen sections of appropriate tissues. We now show that normal human serum possesses inhibitors which selectively recognise the isoenzymic forms of GB associated with normal cell surfaces but do not recognise the GB on tumour cell surfaces in frozen sections of tissue obtained from the head and neck regions. PMID- 1716086 TI - Antigenic expression changes occurring in adriamycin resistant MCF-7 mammary carcinoma cells. AB - The antigenic phenotypic repertoire of MCF-7 human breast carcinoma cell line variants that display different sensitivity to adriamycin (Adr) was analyzed using monoclonal antibodies (MoAbs) recognizing five different tumor associated antigens (TAAs) and the external domain of the epidermal growth factor receptor (EGF-R). ELISA and cytofluorimetry determinations were used and results indicate a diminished expression of one antigenic determinant of the carcinoembryonic antigen (CEA) molecule, the disappearance of all the other TAAs and the de novo expression of the EGF-R in the MCF-7 AdrR (IC50/Adr:10 uM). Treatment with recombinant alfa-Interferon (alfa-IFN) did not enhance antigenic expression in MCF-7 AdrR cells. PMID- 1716087 TI - Interaction of hyperthermia with bleomycin and liblomycin: effects on CHO cells in vitro. AB - The relative ability of hyperthermia to potentiate the cytotoxicity of bleomycin A2 (BLM) and the lipophilic bleomycin analog liblomycin (LBM) was examined in chinese hamster ovary (CHO) cells. A 60-min exposure to either BLM or LBM inhibited CHO clonogenic survival in a concentration-dependent manner (IC50 values: 11 microM and 2 microM, respectively). Hyperthermia alone also inhibited CHO cell survival in a temperature- and time-dependent manner. In combination with hyperthermia, the cytotoxicity of both drugs was enhanced, however, combination of LBM with hyperthermia exhibited a greater synergistic effect than that observed with the BLM + heat regimen. The greatest inhibitory effects of combined drug + heat treatments occurred when hyperthermia was applied simultaneously with drug exposure. Hyperthermic potentiation of drug cytotoxicity could not be explained by increased uptake of radiolabeled BLM or LBM by CHO cells, or heat-mediated potentiation of the ability of these drugs to cleave plasmid pBR322 DNA in vitro. Our data demonstrate the greater efficacy of LBM, as well as the greater synergy between LBM and hyperthermia against the BLM sensitive CHO cell line. PMID- 1716088 TI - Antiretroviral activities of anthraquinones and their inhibitory effects on reverse transcriptase. AB - Alizarin complexone (AC), alizarin Red S (ARS) and various other anthraquinones were evaluated for their inhibitory effects on Rous-associated virus 2 reverse transcriptase (RAV-2 RT). Some 1,2-dihydroxyanthraquinones were active against this enzyme and AC was the most potent inhibitor among these compounds [50% inhibitory concentration (IC50): 3.8 micrograms/ml]. AC slightly inhibited Rous sarcoma virus RT (RSV RT) and human immunodeficiency virus type 1 RT (HIV-1 RT) (IC50: 100 micrograms/ml and 45 micrograms/ml, respectively). However, AC efficiently inhibited focus formation by Rous sarcoma virus (RSV) and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1). Simultaneous administration of AC with RSV to newborn chickens also delayed tumor induction by RSV. PMID- 1716089 TI - A cell culture assay for compounds which inhibit hepatitis B virus replication. PMID- 1716090 TI - Prophylactic and therapeutic activities of 7-thia-8-oxoguanosine against Punta Toro virus infections in mice. AB - The biological response modifier 7-thia-8-oxoguanosine was evaluated in mice against the hepatotropic Adames strain of Punta Toro virus. When administered intraperitoneally in divided doses, significant protection from death was conferred at doses of 50 and 100 mg/kg/day given 24 and 17 h pre-virus inoculation, 25-100 mg/kg/day administered 4 h pre- and 3 h post-virus challenge, and 12.5 to 100 mg/kg/day administered 24 and 31 h after virus inoculation. These doses preventing death reduced liver icterus scores, serum alanine aminotransferase and aspartate aminotransferase levels, and liver and serum virus titers relative to placebo controls. Full daily doses administered at 24 h were somewhat less protective to mice than divided daily doses starting at the same time. The initiation of treatment could be delayed as late as 36 h after virus inoculation, resulting in complete protection from mortality at 100 mg/kg/day. This prevention of death occurred despite the acute nature of the infection which resulted in deaths by 96 h in the placebo-treated controls. These results show that 7-thia-8-oxoguanosine has both prophylactic and therapeutic potential as an anti-Phlebovirus agent. Interferon induction appears to be the reason for antiviral activity in this model, since up to 10,000 units of interferon/ml were induced in mice 1 h after treatment with 100 mg 7-thia-8-oxoguanosine per kg, and antibody to interferon alpha/beta administered shortly after treatment with the nucleoside negated the antiviral effect. PMID- 1716091 TI - [Neoadjuvant chemotherapy of head and neck cancer]. AB - Chemotherapy for head and neck cancer has made great progress after CDDP was introduced at the clinical level. Chemotherapy prior to regional treatment (neoadjuvant chemotherapy) has become a popular approach for incorporating chemotherapy into multimodality treatment. Numerous studies support the general observation that response rates, especially the rates for complete response, are higher in patients who have not had prior regional treatment. The goals of this treatment are 1) to increase the effectiveness of local treatment (surgery and radiation therapy) while obtaining superior disease-free survival, and 2) to prevent metastases by eradicating micrometastatic lesions. Despite the excellent response rates after reported for regimens with CDDP, randomized studies have failed to prove statistically significant superior disease-free survival. The reason for these negative results is considered to be the patient's compliance and difficult of obtaining sufficient numbers of patients in each kind of primary sites, because this type of cancer is relatively infrequent. More study of neoadjuvant chemotherapy with high quality control is needed. Some trials with neoadjuvant chemotherapy to preserve the function of larynx or hypopharynx are promising new strategies. Concurrent chemo-radiotherapy using CDDP was proved to potentiate radiotherapy and showed a good response rate. Adjuvant chemotherapy after surgery or radiotherapy is also expected to prolong disease-free survival when it is incorporated into multimodality treatment. PMID- 1716092 TI - [Clinical evaluation of hypopharyngeal cancer]. AB - Clinical results of cases of hypopharyngeal carcinoma treated from 1966 to 1990 at Kyushu University Hospital were investigated. In 1966-1972, all cases were treated with only radical operation. We used FAR therapy since 1972, and preoperative chemotherapy (CDDP and Peplomycin) in addition to FAR therapy since 1984. The crude five-year survival rate of the operative therapy-group (25 cases in 1966-1972) was 24%, and that of the FAR therapy-group with or without operation (47 cases in 1972-1980) was 38%. Although it was cumulative survival rate by Kaplan-Meier's method, the five-year survival rate of the group (16 cases in 1984-1990) treated with a combination of FAR therapy, preoperative chemotherapy, and operation was 76%. The improvement in the survival rate and the role of preoperative treatment (FAR therapy and chemotherapy) are discussed. PMID- 1716093 TI - [A case of RAEB-t treated by G-CSF showing complete remission after MST-16 treatment for subsequent reversible leukemia]. AB - A seventy-five-year-old female with general fatigue, high fever and anemia was admitted. Her chest X-ray film revealed pneumonia. She was diagnosed as RAEB-t with the normal karyotype by peripheral blood film and bone marrow aspiration; 125 micrograms/ml of G-CSF was administered s.c. daily in order to increase neutrophil count because of the prolongation of pneumonia. Her blast cells in both peripheral blood and bone marrow showed a remarkable increase by G-CSF. After the cessation of G-CSF administration, blast cells decreased rapidly, and neutrophil count in the peripheral blood increased. Her pneumonia was then cured. After 5 months of stable hematological state, 60% of her bone marrow cells became occupied by blast cells again. So 2 consecutive courses of 14 days p.o. administration of 1,200 mg MST-16/day were tried. Three months after the first MST-16 trial, her bone marrow showed complete remission (CR) which lasted about 4 months. But she died of sepsis after the first relapse. Her bone marrow in CR still revealed several features of dyspoiesis. PMID- 1716094 TI - [Successful treatment of recurrent kidney pelvic squamous cell cancer with chemotherapy and radiotherapy: a case report]. AB - We report a case of recurrent squamous cell carcinoma of the renal pelvis. A 61 year-old woman was readmitted to our hospital 4 months after left nephrectomy. The medical imaging method revealed a left retroperitoneal tumor and squamous cell carcinoma related antigen (SCC-Ag) elevated (82 ng/ml). We suspected a recurrent tumor from renal pelvic cancer. She received 2 courses of systemic chemotherapy with 5-FU and CDDP, but the tumor did not change. As a second treatment, combined radiotherapy with PEP was given. The tumor was reduced and SCC-Ag returned to the normal level. The patient is alive with no recurrence or metastasis at one year following these therapies. PMID- 1716095 TI - [Tumor markers--personal experience. A case of advanced extragonadal germ-cell tumor showing complete remission by high-dose combination chemotherapy with autologous bone marrow transplantation]. AB - A twenty-four-year-old male patient with stage III advanced extragonadal germ cell tumor obtained complete remission after comprehensive treatment including high-dose combination chemotherapy with autologous bone marrow transplantation. He had massive tumors in cervical, mediastinal, abdominal and inguinal lymph nodes, bilateral lungs and liver. Ascites, plural effusion and pericardial effusion were also noted. Cancer cells were demonstrated from his bloody sputum, pericardial drainage and an aspirate from supraclavicular tumor. His tumor produced hCG, AFP, placental ALP and LDH, and hCG was the best marker for the diagnosis and monitoring. The initial serum hCG level was high at 460,000 mIU/ml, but fell to 13 mIU/ml after 3 courses of PVP therapy. However, it rose ten-fold in a week. After ultra-high dose combination chemotherapy with autologous BMT, the patient's hCG fell to 1.8 mIU/ml and remained at that level thereafter. He has remained well with no sign of recurrence after 25 months. PMID- 1716096 TI - Influence of virginiamycin on the digestive physiology in precaecal re-entrant cannulated pigs. AB - The present experiment was set up to study the effect of Virginiamycin, a nutritional growth promoting antibiotic, on the digestive physiology using precaecal cannulated pigs. The semipurified diet used provided 21% protein and 60% of nitrogen free extract (NFE), 2/3 as starch and 1/3 as lactose. Due the older age of the pigs, the lactose induced some degree of malabsorption as precaecal flow rate of digesta was markedly higher (3.2 kg/d) compared with published data obtained with purified diets without lactose. This was also reflected in the rather low ileal digestibility of the nutrients (%): dry matter 68.6, protein 79.6, fat 78.4 and NFE 75.0. Virginiamycin markedly lowered flow rate (2.2 kg/d) and significantly improved apparent precaecal digestibilities (%): dry matter 74.0, protein 81.4, fat 81.9 and NFE 78.7. The faecal apparent digestibility was comparable with published data. There were no treatment differences indicating that precaecal digestibilities are much more sensitive then faecal ones. In order to explain the differences obtained the mean retention time in the upper intestine was measured. Although significant differences were noted (control: 5 h, Virginiamycin: 6 h) a direct cause-effect relationship was not evident. Also the activity of selected pancreatic enzymes in ileal contents was compared. There were no consistent differences between the two treatments, except for a lower lipase activity during Virginiamycin treatment. PMID- 1716097 TI - Role of hepatocellular regeneration and hepatolobular healing in the final outcome of liver injury. A two-stage model of toxicity. PMID- 1716098 TI - FK-506 and cyclosporin A: selective inhibition of calcium ionophore-induced polymorphonuclear leukocyte degranulation. AB - This paper investigates the abilities of FK-506 and cyclosporin A (CsA) to inhibit human polymorphonuclear leukocyte (PMNL) degranulation. PMNLs, purified from human blood, were stimulated in vitro with A23187, ionomycin, the complement derived peptide C5a, formylmethionylleucinylphenylalanine (FMLP) or phorbol myristate acetate (PMA). Degranulation was assessed by measuring the release of either lactoferrin or N-acetyl-beta-D-glucosaminidase (NAG). Both FK-506 and CsA produced a concentration-related inhibition of degranulation induced by either A23187 or ionomycin but did not affect C5a-, FMLP- or PMA-induced degranulation. The IC50 values for inhibition of degranulation (approximately 0.7 nM for FK-506 and 33.7 nM for CsA) are very close to the published values for inhibition of human T-cell proliferation. Removal of calcium from the incubation medium with ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) totally inhibited calcium ionophore-induced degranulation but had no effect against C5a-, FMLP- or PMA induced degranulation. Preincubation of PMNLs with actinomycin D or cycloheximide did not affect either A23187- or PMA-induced degranulation. Non-immunosuppressive analogs of CsA were ineffective at inhibiting degranulation. Rapamycin, a macrolide structurally related to FK-506, did not inhibit degranulation but it did antagonize the inhibition produced by FK-506. Given the similar profiles of activity of FK-506 and CsA in neutrophils and T cells, we conclude that similar activation or signal transduction pathways may be present in both T cells and neutrophils. Because A23187-induced PMNL degranulation was not sensitive to either actinomycin D or cycloheximide, it is apparent that the signal transduction pathways ultimately control different cellular functions. PMID- 1716099 TI - Identification of the novel rat liver IBMX-insensitive phosphodiesterase as a non specific phosphodiesterase capable of hydrolysing cCMP. PMID- 1716100 TI - [Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22]. AB - Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131 152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV. PMID- 1716101 TI - [Complementary addressed alkylation of 16S rRNA of Escherichia coli by 2',3'-O-[4 N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligodeoxyribonucleotides. V. Study of the factors affecting selectivity of modification]. AB - We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribonucleotide) stabilizing duplex RNA.reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2 chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16s rRNA was determined. The temperature rise from 20 to 40 degrees C brought about a 1.5-fold increase in the relative extent of modification at the target site 771-781. In the presence of oligonucleotide-effector, which is a full complementary copy of the 782-789 fragment of 16S rRNA (reagent concentration is 1 x 10(-6) M), the selectivity of the RNA modification at the target site is doubled and a high level of the modification is retained. When the reagent concentration in the reaction mixture was decreased down to 1 x 10(-7) M, the same level of selectivity was achieved without the oligonucleotide-effector. Under these conditions, however, a drastic (20-fold) drop of the level of the 16S rRNA alkylation was observed. PMID- 1716102 TI - [Peptide synthesis--protein fragments from Plasmodium falciparum and their conjugates with protein and synthetic carriers]. AB - To study coat proteins of Plasmodium falciparum, seven putative antigenic determinants of PMMSA, SHARP, GBP and four known determinants of CSP, CRA and RESA were synthesized. Computerized methods for predicting protein antigenic determinants were employed to select the peptides. For immunochemical studies the peptides were conjugated to proteins and synthetic carriers by means of nonspecific and regiospecific heterobifunctional reagents. PMID- 1716103 TI - [Immunochemical study of antigenic determinants of surface protein s of the tropical malaria pathogen Plasmodium falciparum using synthetic peptides]. AB - To develop new approaches to diagnostics and therapy of malaria, we carried out immunochemical study of the surface proteins of the tropical malaria parasite Plasmodium falciparum with the use of synthetic peptides corresponding to the suggested antigenic determinants of the parasite proteins. Rabbit antisera raised against the synthetic peptides bound to parasite proteins as shown by ELISA and immunoblotting. Affinity purified anti-peptide antibodies inhibited, in some cases, the parasite growth in the human erythrocytes culture. PMID- 1716104 TI - [3'-Mercapto-3'-deoxythymidine-5'-triphosphate as a terminator of DNA synthesis catalyzed by RNA-dependent DNA-polymerases]. AB - 3'-Mercapto-3'-deoxy-TTP was synthesized and tested as DNA chain terminator nucleotide for calf thymus alpha DNA polymerase, E. coli DNA polymerase I (Klenow fragment), terminal deoxyribonucleotidyltransferase (Bollum enzyme) and reverse transcriptase from AMV- and HIV-I-viruses. It was shown that the compound terminates DNA chain elongation by reverse transcriptases selectively and irreversibly. Other tested DNA polymerases do not use this nucleotide analogue as a substrate. 3'-Mercapto-3'-deoxythymidine was tested on lymphoblastoid T-cell line MT-4 with HIV-viruses and shown to suppress viruses as efficiently as 3' azido-3'-deoxythymidine. PMID- 1716105 TI - [Identification of a protective epitope--a fragment of Lassa virus nucleoprotein]. AB - Several peptides from Lassa virus glyco- and nucleoproteins were predicted as probable T-cell epitopes. Their synthesis was performed by solid phase method. The study of possible protective effect in vivo with Lassa-sensitive CBA mice revealed protective epitope within the 277-303 nucleoprotein region. Further studies reduced the protective epitope structure to the 287-300 nucleoprotein fragment. PMID- 1716106 TI - [Beta-endorphin and substance P in the perioperative period]. AB - The modifying impact of anaesthesia on the stress reaction related to surgical trauma was investigated on the basis of the neuropeptidergic parameters of 66 patients who had to undergo a gynaecological radical operation. Anaesthesia was either performed as neuroleptanaesthesia or as epidural analgesia by using bupivacaine in combination with general anaesthesia. The plasma concentrations of substance P and beta-endorphin were taken as neuropeptidergic parameters. Both regulatory peptides show numerous corresponding synergisms. An acceleration of these neuropeptide systems is assumed to be present in severe disturbance of homeostasis. Plasma concentrations of substance P and beta-endorphin were examined at 11 measuring points in the perioperative and intraoperative periods. The plasma concentration of substance P significantly declines in the preoperative period while the concentration of beta-endorphin in the plasma remains at a relatively constant level. In the dynamics of beta-endorphin in the plasma significant differences between the two anaesthetic techniques become apparent in the intraoperative period. Those patients given epidural analgesia have a significantly higher maximum concentration at a later date. This difference is attributed to the possible loss of the adrenal medullary function due to partial sympathetic blocking. Single observations in patients pregnant in the last trimester testify to an extraordinary adaptability at the end of pregnancy. PMID- 1716107 TI - Epitope mapping. AB - The immune response to extrinsic and intrinsic antigens involves specific antigen receptors on T and B cells. The precise antigenic determinants, or epitopes, recognized by these receptors are discrete sequences within the native antigen. The ability to identify and manufacture key epitopes in the immune response has important implications for disease diagnosis and immunointervention. Consequently, increasingly sophisticated technologies are being applied to epitope mapping. This report from a recent workshop gives a balanced view of progress to date and the challenges ahead. PMID- 1716108 TI - Interactions between cytokines and alpha 2-macroglobulin. PMID- 1716109 TI - [Tubular structures revealed using tannic acid on the surface of the epithelium of the tympanic cavity of the chicken]. AB - We have studied the ultrastructure of the epithelium lining the tympanic cavity of chicken, using two embedding techniques. In certain cases, the epithelium was minced in Karnovsky fixative, post-fixed in osmium, dehydrated and embedded in Epon, following the usual methods. No morphologically detectable structures were seen at the level of the epithelium surface. In other cases, the epithelium was immersed in Karnovsky fixative and subsequently in glutaraldehyde (1%) to which, however, tannic acid (1%) was added; the specimens were then osmicated, dehydrated with Epon and embedded in polar Epon mix. This method was sometimes used to study the alveolar surfactant, since this makes it possible to preserve its phospholipid fraction. The epithelium, in this way, is seen to be covered by an electron-dense material made up of thin, intertwined tubules. The hypothesis formulated is that the tubules are related to the presence of surfactant substances. PMID- 1716110 TI - [Lingual innervation in the buffalo]. AB - The lingual and papillary mucosa innervation has been studied in the buffalo by histochemical and immunohistochemical techniques and by gold impregnation method of Ruffini. In lingual mucosa the presence of ganglionic structures has been noticed in which there are numerous cellular bodies and bundles of fibres variously directed. Particularly in the vallate papillae, the papillary stroma contains myelinic and unmyelinated fibres and terminal nerve corpuscles. Along stromal myelinic fibres the presence of the proteins S-100 and NF has been demonstrated. PMID- 1716111 TI - Oral and pharyngeal adenosquamous carcinoma. A report of four cases with immunohistochemical studies. AB - Four cases of adenosquamous carcinoma from the oral and pharyngeal cavities were analyzed by light microscopy and immunohistochemistry. Lymph node metastases were present in three cases. One patient died 2 years after treatment. All four carcinomas presented a mixture of squamous and glandular mucus-secreting neoplastic elements. Immunostaining for high-molecular-weight cytokeratins (KL1) was constantly positive in both squamous and glandular tumor cells. Antibodies against low-molecular-weight cytokeratins (K19) and carcinoembryonic antigen were positive only in the glandular component. The histological aspect and the immunohistochemical phenotype of these tumors is similar to the ordinary squamous cell carcinoma and adenocarcinoma, respectively. PMID- 1716112 TI - Cytokeratin expression in the epithelia of the adult human cochlea. AB - Epithelia can be characterized by the specific expression pattern of their cytokeratin components. Therefore, we investigated the immunohistochemical expression of different cytokeratin subunits in frozen sections of chemically fixed, non-decalcified, adult human cochleas. The organ of Corti and the marginal cells of the stria vascularis showed reactivity for cytokeratin subunits 8, 18 and 19, whereas the other cochlear epithelia in addition expressed cytokeratin 7. The expression of cytokeratins 7, 8, 18 and 19 by the epithelia of the adult human cochlea is typical of "simple" epithelia. The deviant cytokeratin pattern of the organ of Corti and marginal cells of the stria vascularis may well reflect their differences in functional state and/or differentiation as compared to the other cochlear epithelia. PMID- 1716113 TI - Immunohistochemical observations of dividing cells in olfactory epithelium using anti-BrdU antibody. AB - To examine the dividing cells in the olfactory epithelium, an experiment using a novel immunohistochemical technique with bromodeoxyuridine (BrdU) was performed. Tissue specimens were obtained from the olfactory epithelium of guinea pigs at days 7 and 21 after olfactory nerve axotomy. BrdU uptake was detected in the epithelial cell layer directly above the basal cell layer rather than in the basal cells per se. The BrdU-immunoreactive cells were found more numerously at 7 days than at 21 days after axotomy. The basal cells showed no immunoreaction to the anti-BrdU antibody on either day. The cells reacting with the anti-BrdU antibody also showed no reaction to the anti-cytokeratin antibody used to identify the basal cells. These findings indicate that the cells showing mitotic activity have characteristics different from those of basal cells, which has been considered previously to be the precursors of regenerating olfactory receptor cells. PMID- 1716114 TI - Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis. AB - To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures. PMID- 1716115 TI - Distribution of lectin receptors sites in the zona pellucida of follicular and ovulated rat oocytes. AB - Carbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm-egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus-oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR-Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin tagged lectins as probes, and avidin-biotin-peroxidase complex as visualant. For EM examination, thin LR-Gold sections were labeled with RCA-I colloidal gold complex (RCA/G) and stained with uranyl acetate. LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S-WGA), Griffonia simplisifolia agglutinin-I (GS-I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA-I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA-I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S-WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716116 TI - [An electron microscopic study on the RNA component of synaptonemal complexes in spermatocytes of Mus musculus]. AB - The ultrastructural and cytochemical features of synaptonemal complexes (SC) in sections of spermatocytes of Mus musculus were studied under electron microscope. In specimens stained with uranyl acetate and lead citrate the SC was found consisting of three main elements. the lateral element (LE), the central element (CE) and the transverse filament (L-C filament). When stained with the Bernhard's technique, the SC was recognized as a contrasted, tripartite structure which was usually located in the bleached area occupied by the condensed chromatin and composed of highly electron-dense LEs and medium electron-dense CE and L-C filaments. The SC and the LE, stained either by uranyl acetate-lead citrate or by the Bernhard's technique, always showed diameters of about 210 nm and 60 nm, respectively. The results suggest that RNA may be an important component of the SC. PMID- 1716117 TI - [Localization of substance P in middle ear mucosa and peripheral auditory pathways in guinea pigs]. AB - The distribution and intracellular localization of substance P (SP) in middle ear mucosa (MEM), cochlea and spiral ganglion (SG) were studied by immunohistochemical technique and immunoelectron microscopy. There was a widespread distribution of SP positive nerve fibers (NF) along the median and small vessels of MEM. SP-immunoreactivity (SP-IR) positive cells could be seen in the MEM near the promontorium tympani. In the Corti's organ, SP-IR positive products were located at the base of inner hair cells. The majority of positive NF emerged like strings of beads and were radially distributed from osseous spiral laminal to the Corti's organ. About 50% of the SG cells were SP-IR positive. Two types of SP-IR positive NF were found in the VIII cranial nerve by light microscopy. Small clear vesicles with a diameter of 50-70nm were localized in the cytoplasm of the type-I SG cells by immunoelectron microscopy. In the outer membrane and inside the mitochondria, SP-IR positive substances could be distinguished as an electron dense matter. The possibility of SP as an afferent neurotransmitter or modulator in cochlea and the significance of its presence in the MEM were discussed. PMID- 1716118 TI - Pharmacological management of perennial rhinitis. PMID- 1716119 TI - [Age changes in concentrations of monoamines and related substances in the cerebrospinal fluid]. AB - We studied age changes of concentrations of thirteen substances including monoamines, their precursors and metabolites in the cerebrospinal fluid, using high performance liquid chromatography. Cerebrospinal fluid was obtained from 106 subjects without neurological diseases (44.2 +/- 17.3 years) who underwent minor operations under lumbar anesthesia. Concentrations of dopamine, norepinephrine, tyrosine and 3-methoxy-4-hydroxy-phenylalanine were significantly increased with age, while concentrations of other monoamine precursors and metabolites were unchanged. There was a significant positive correlation between concentrations of the following substances: dopamine and norepinephrine; tyrosine and tryptophan; tyrosine and 3-methoxy-4-hydroxy-phenylalanine; tyrosine and 5-hydroxytryptophan; homovanillic acid and 5-hydroxyindoleacetic acid. Norepinephrine concentrations were positively correlated with concentrations of 3-methoxy-4-hydroxy phenylglycol, whereas dopamine concentrations were not with homovanillic acid concentrations. The significance of these results was discussed with regard to age changes of transmitter secretion and metabolism, the binding capacity of receptors and cerebrospinal fluid kinetics of the measured substances. PMID- 1716121 TI - [Neuritis and neuroma of the sensory branches of the radial nerve. Apropos of 44 cases]. AB - The authors present a series of 44 cases of sensory disorders in the radial nerve territory. Eighteen cases of pure painful neuromata gave, excellent results according to Herndon criteria in 11 cases with various techniques. Wartenberg neuritis (22 cases) was found to respond favorably to conservative treatment in the majority of cases (19 cases). A legal problem can arise from association with De Quervain disease if the condition has not been recognized before surgical release of the first compartment. Finally 9 cases are presented with intricate pathology of neuroma plus neuritis. Treatment addressing both conditions provided good results. PMID- 1716120 TI - [The axial orientation of the phalanges following the curling up of the fingers]. AB - This study follows and modestly completes those by Dubousset and Kapandji concerning the phenomena of axial (or longitudinal) rotation of the phalanges combined with flexion-extension of the fingers. Our analysis of the orientation of axial rotation of the skeletal elements of the digital chain, in relation to each other and during digito-palmar flexion was supported by simple observation, impressions in silicone paste, study of anatomical preparations, three dimensional computed tomography. The index finger and middle finger tend to supinate. The ring finger undergoes virtually no rotation and the distal end of the little finger tends to pronate. These phenomena, essential for good adaptation to grip and fine manipulation must be taken into account by rehabilitation physicians when the amplitude of these movements are limited by immobilisation or disease. PMID- 1716123 TI - [Fractures of the proximal pole of the scaphoid bone. Anatomo-clinical and therapeutic entity]. AB - The authors present their anatomical and clinical findings concerning fractures and nonunions of the carpal scaphoid. They stress the proximal pole type I et II of the Schernberg (type C of Herbert) classification, and they suggest isolation of an anatomical entity, by the study of blood vessels, the ligament connections, and especially the pathomechanics as well as a clinical entity, by the mechanism of fracture and the surgical treatment, emphasizing the proximo-distal screwing, with excellent results. They present a series of 29 patients, with type I or II proximal fractures, either fresh or non-unions. PMID- 1716122 TI - [Improvement of muscular re-innervation by using an enriched biological glue in the rat]. AB - The use of biological glue for the repair of nerve lesions offers the possibility of acting on axonal regeneration by means of trophic substances included in the glue. A comparative study conducted in the sciatic nerve of the rat shows that incorporation of the liquid exuded by sectioned nerve extremities into the glue to repair a section of the sciatic nerve trunk of other animals significantly increases the muscular tension of the force jerk and the of tetanic contraction of the reinnervated extensor digitorum longus 21 and 28 days after repair. It therefore appears possible to influence the regeneration or the maturation of the regenerating axons by local application of exogenous substances. PMID- 1716124 TI - [A new test for the functional evaluation of the hand and its contribution to the study of toe transfers: the 5 matches test called "Take Five"]. AB - After studying a series of thumbs reconstructed by 2nd toe transfer, we devised a new dexterity test: the "five matches test" or "Take Five Test" (R.S.). This test consists of the standardised timed and comparative pick-up of 5 identical fine objects (matches), permitting a simple and quick evaluation which objectively quantifies dexterity in fine pinches. The specificity of our test is that it is bilateral and comparative providing a narrow range of normality. In fact, a marked variability of time-scores is found between different individuals of a normal population, making it difficult to determine a "normal dexterity" criterion. But in the same person, there is little variability between two successive experiments or between the dominant and non-dominant hands. Examining a reconstructed hand, we have chosen as the "normal dexterity" criterion the time score of the contralateral spared hand, timed first. The final score (from 0 to 4 points) is based on the difference between time-scores of the 2 hands (delay tested hand/spared hand). The "Take Five Test" (five matches pick-up test) has largely proven its efficiency in studying a series of thumbs reconstructed by 2nd toe transfer (operated on by V.M.). Fine pinch dexterity is satisfactory in 45% of cases (65% when long digits are spared), scoring at least 2 points (delay less than 4 seconds). The average score is 1.6 pts (range: from 0 to 4). This test could prove the functional value of parameters such as phalangeal mobility and a short delay between injury and reconstruction. PMID- 1716125 TI - ["Reduction-effect" ARUM-type intra-focal pins in the osteosynthesis of fractures of the lower end of the radius]. AB - Used for 13 years, the "Intra-Focal Pinning" procedure for the fixation of the Colles fractures, has been criticized on certain points: pin expulsion, risks of injuring the tendons by the cut end of the pin. A new type of pin in proposed, the "ARUM" pin, that is composed of a 20/10 mm screwed pin and a special nut, which has a curved conical form, with a convex base bearing a cruciform groove. Its axial canal has a little space at the base. There are two ancillary tools: a special wire-cutter with asymmetric cutting blades, that can cut the pin close to the nut, and a compound screwdriver and pin-holder. With this tool, it is possible to put the pin in its right place and to screw the "ARUM" nut very precisely: the concave conical form of the nut makes it possible to slide between the tendons without injuring them and widening the fracture site: this is the "reduction effect". In practice, the pins are introduced in the same way as the previously described procedure, by they must protrude beyond the opposite wall by 6-8 mm; the difference is in the screwing of the "ARUM" nut: first it is screwed as the reduction stress is increased so its conical part penetrates between the fracture edges; then the pin is cut; and the nut is unscrewed so the cut end of the pin will be included in the space of its base. Unscrewing is continued until the sharp end of the pin just extends beyond the cortical wall, but without losing the reduction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716126 TI - [Osteoid osteoma of the distal end of the radius. Review of the literature in light of a case]. AB - A case of osteoid osteoma in the distal extremity of the radius is reported. Clinical examination, treatment and clinical course are described and previous publications are reviewed. PMID- 1716127 TI - [Isolated metastasis to the capitate bone]. PMID- 1716128 TI - [Tuberculoid tenosynovitis of the hand. Apropos of 3 cases of unusual and various causes]. PMID- 1716129 TI - [Acrometastases. Apropos of 2 cases. Review of the literature]. AB - Bone metastases in the hand and foot bone (acrometastases) are rarely observed. The authors report two new cases. The first case is chiefly interesting because of its multifocal appearance and its particularly rare origin (oesophagus). The second case is remarkable as it is the initial expression of a lung neoplasm. They propose a review of the literature on this subject and emphasize the origin, the mechanisms of spread the prognosis and treatment of this disease. The difficulty of the diagnosis is also stressed. PMID- 1716130 TI - [The combined luxation of the trapezoid and 2nd metacarpal bones. Apropos of a case]. AB - The authors report a case of posterior dislocation of the trapezoid bone together with the second metacarpal. Among the 25 cases of trapezoid dislocations reported in the literature, no similar case has been found. The patient was seen 4 months after the injury and there was also a scaphoid instability. Open reduction and a scapho-trapezio-trapezoid arthrodesis were performed. The diagnosis, the mechanism and the treatment of this very unusual injury are discussed. PMID- 1716131 TI - [Massive osteolysis of the metacarpal bones. Apropos of a case of an osteolytic form of psoriatic rheumatism]. AB - The authors report a case of massive osteolysis of all of the metacarpal bones of the right hand in a 26 year old man. The disease commenced 9 years previously with painful symptoms associated with inflammation and the initial X-rays showed periosteal reaction of the 2nd and 3rd metacarpals. Massive osteolysis of all of the metacarpal bones, including the 1st metacarpal, developed progressively over a period of one year, although no signs of osteolysis were observed in the carpal bones or phalanges. A surgical operation, performed to realign the ring and little fingers, revealed a 4 mm thick periosteal sheath explaining the maintenance of a certain degree of stability of the metacarpals despite the massive osteolysis. Histological examination eliminated any neoplastic or infectious aetiology and confirmed the inflammatory origin with vascular and lymphocytic proliferation. The development of palmo-plantar psoriasis several months after the onset of the painful symptoms suggested the diagnosis of psoriatic rheumatism. This is a rare site with an unusual clinical course with massive osteolysis of all of the metacarpal bones, but it appears to be the most likely hypothesis. The clinical course was stabilised by non-steroidal anti inflammatory agents but there was no bony reconstruction. PMID- 1716132 TI - [Abrikossoff's tumor of the hand. Apropos of a case. Review of the literature]. AB - This mucous tumor (tongue) is uncommon. The cutaneous site is even rare. We report a recurrent granular cell tumor of the thumb and the clinical, diagnostic and prognostic aspects of this usually non malignant tumor. PMID- 1716133 TI - [Compression of the median nerve in the carpal tunnel due to an intra-canal palmar muscle]. AB - Five cases of palmaris longus muscle in the carpal tunnel responsible for median nerve compression are reported. This represents less than 0.5% of all carpal tunnel syndromes operated over a ten year period. A review of the literature reports this abnormality in about 0.5% of tendons examined. It is important to recognise these forms for carpal tunnel and tendon transfer surgery. PMID- 1716134 TI - [Osteoid osteoma of the trapezoid bone]. AB - The present report describes a case of osteoid osteoma of the trapezoid. It'is an unusual localisation. Synovitis was the first clinical symptom. Tomograms allowed diagnosis and guided the treatment. Radical excision prevented recurrence. PMID- 1716135 TI - [An unusual displacement fracture of the 1st metacarpal bone. Apropos of a case reviewed after 3 years]. AB - An unusual fracture of the base of the first metacarpal with in contrast with Bennett's fracture displacement of the thumb and avulsion of the Abductor Pollicis Longus tendon is described after a "Rugby" trauma. Immediate repair and stabilization of the injured structures followed by specific rehabilitation, produced a satisfactory result with normal function of the thumb three years after the injury. PMID- 1716136 TI - [Achromic ungual melanoma. Apropos of a case]. AB - A 55-year-old woman presented with suppurative paronychia that became chronic. In fact, this was an exceptional case of amelanotic subungual melanoma. This case is compared with a general review of subungual melanomas. Diagnosis is often delayed, due to the difficulty caused by the misleading clinical symptoms. PMID- 1716137 TI - [Metacarpal bone chondrosarcoma. Apropos of a case]. AB - On the basis of a personal case of non-symptomatic chondrosarcoma of the metacarpal bone and a review of the literature, the authors advise the biopsy of any lesion which resembles a chondroma of the hand. Only histological examination can provide evidence for diagnosis and guide effective treatment. PMID- 1716138 TI - [Polydactyly]. AB - The authors reports the experience of the plastic surgery unit in the Kassab's Institute of Orthopedics in Tunis. This series consists of 44 cases of polydactyly dominated by duplication of the little finger and the thumb. A slight male predominance was observed and the mean age at surgery was 8 years. Two types of polydactyly were observed: simple, such as rudimentary digits, complex, with associated bone malformation or syndactyly. In ulnar polydactyly, associated duplication of the toes in frequently noted. The most frequent surgical approach was amputation of the more dystrophic finger with reconstruction and correction of the other congenital malformation observed. Some cases of digital redistribution were also performed. A review of the literature is presented together with an objective analyse of esthetic and functional results. PMID- 1716139 TI - Direct correlation between methylation status and expression of the human O-6 methylguanine DNA methyltransferase gene. AB - To assess the role of DNA cytosine methylation in the expression of the O-6 methylguanine DNA methyltransferase (MGMT) gene, the methylation status of selected CpG-containing dinucleotides in and surrounding the coding regions of the gene were examined and correlated with steady state expression of MGMT mRNA in 13 human cell lines. Additionally, tumor cells which exhibited very high levels of MGMT expression were chronically exposed to 5-azacytidine to assess the effects of changes in gene methylation on MGMT expression. Results of these studies demonstrate that the degree of methylation of multiple MGMT gene regions correlates with gene expression, but in a direct rather than an inverse fashion, and that 5-azacytidine-induced demethylation of the MGMT gene correlates with a significant reduction, rather than induction, of MGMT steady-state mRNA expression. These results suggest a unique, potentially alterable methylation related regulatory mechanism for the MGMT gene. PMID- 1716140 TI - The influence of Org 10172, a low molecular weight heparinoid, on antipyrine metabolism and the effect of enzyme induction on the response to Org 10172. AB - 1. We have investigated the effect of repeated s.c. Org 10172 (a low molecular weight heparinoid; Lomoparan) treatment (1000 anti-Xa units twice daily for 5 days) on antipyrine (500 mg orally) metabolism, and the effect of enzyme induction by pentobarbitone (100 mg for 12 days) on the pharmacokinetics and pharmacodynamics of Org 10172 following an intravenous bolus injection of 3250 anti-Xa units. 2. Org 10172 treatment caused a small increase in the formation rates of all antipyrine metabolites (P less than 0.05), while the overall kinetics of antipyrine did not change significantly. 3. Oxidative enzyme induction by pentobarbitone, as demonstrated by an increased clearance of antipyrine, was associated with an increase in the area under the anti-thrombin activity vs time curve (P less than 0.05). No influence was seen on the kinetics of plasma anti-Xa and thrombin generation inhibiting (TGI) activity. 4. The pharmacodynamics of Org 10172, as determined by clotting tests, was not influenced by enzyme induction. 5. The clinical relevance of these observations is likely to be limited. PMID- 1716141 TI - Nursing care of pituitary surgery: an example of advanced clinical practice. AB - Because we have one of the leading pituitary surgeons in Canada, Dr. Harley Smyth, on our neurosurgical service we have nursed over 53f cases of pituitary tumor resection at the Wellesley Hospital. This series of patients represents a major ongoing international research project in Endocrinology and Neurosurgery and involves close teamwork between these two services and the nursing service. The paper gives a brief overview of pituitary surgery from its beginnings with Sir Victor Horsley in 1904 and details major breakthroughs since the 1960's. The three principal clinical groups of pituitary problems treated at the Wellesley are then outlined. The preoperative work-up on the endocrinology ward delineating clinical symptoms and precise hormonal values, the operation with the new anatomical approach developed by Dr. Smyth, and postoperative care with precise monitoring of fluid and electrolyte balance, methods of nasal packing, treatment of cerebrospinal fluid leaks and tracking of hormone levels are all examined in detail. We explain new developments such as the move to less steroid coverage and the better understanding of the ultrastructure and functional microbiology of pituitary tumors. We conclude by pointing out the excellent management of these patients, with a short hospital stay and much of the preoperative work-up done on an outpatient basis. As well these patients are an extremely satisfying group to nurse because of the high correction rate achieved. Finally, as nursing protocols constantly change and as we continually learn more from this research project, we emphasize the need for the flexibility and knowledge of the advanced nursing practitioner. PMID- 1716142 TI - Role of polyamine in the regulation of RNA synthesis in uterine nucleoli. AB - Administration of estradiol (E2) to ovariectomized mature rats has been shown to result in synthesis of uterine polyamines in the same temporal manner as E2 regulation of nucleolar transcription. Data is presented on the in vivo and in vitro effects of polyamines on uterine nucleolar RNA synthesis. Transcervical intrauterine administration of putrescine (100 micrograms), spermidine (100 micrograms), or spermine (100 micrograms) resulted in an increased transcriptional activity of 93 and 82% in uterine nucleoli isolated from putrescine and spermidine treated animals, respectively. Spermine administration was without effect on uterine nucleolar transcription. The polyamine-induced increase in transcription was totally accounted for by an increased rate of elongation of previously initiated RNA chains. No effect on the number of nucleolar RNA chains in the act of synthesis was observed. Preincubation of uterine nucleoli, isolated from control animals (no E2) with putrescine, spermidine, or spermine in the presence, but not in the absence of ATP, resulted in 44, 83 and 31% increased nucleolar RNA synthesis, respectively. In vitro polyamine-induced nucleolar RNA synthesis was correlated with a polyamine activated phosphorylation of nucleolar proteins of 110,000 24,000, 18,000 and 14,000 Da. Results suggest that early E2 action may result in activation of the polyamine pathway which modulates nucleolar protein kinase activity; initiating an increase in nucleolar transcription. PMID- 1716143 TI - Analysis of functional sites on a peptide antigen, p43-58, in I-A or I-E restricted T cell responses. AB - It has been shown that two different sites (an agretope and an epitope) on a peptide antigen function independently in T cell responses to the antigen. By virtue of these sites, antigens, MHC molecules, and TCRs constitute trimolecule complexes which eventually result in T cell activation. In our previous reports, we have defined that residues 46 and 54 on synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58, AEGFSYTDANKNKGIT) and its analogs function as an agretope and residue 50 as an epitope in both I-Ab and I-Ak-carrying mice. In the present study, to extend our method to the other MHC class II molecules (I E), we used two peptide antigens, 46D50V54R and 50V54R, which had been prepared by substitution of amino acids at positions, 46, 50 and 54 or 50 and 54 of p43-58 D, V, R or V, R, respectively, and compared the immunogenicity with those of other peptide analogs. The 46D50V54R was shown to be non-immunogenic in I-Ab carrying mice and the 50V54R was non-immunogenic in I-Ak-carrying mice. In contrast, the 46D50V54R or 50V54R could induce I-E-restricted proliferative responses of T lymphocytes in I-Eb/k- or I-Ek/k-carrying mice, respectively. Furthermore, residues 46 and 54 were shown to function as agretopes and residue 50 as an epitope in the I-E-restricted responses as they did in the I-A restricted responses, even though some differences were seen between peptide-I-E interaction and peptide-I-A interaction. These agretopes and epitope functioned independently. PMID- 1716144 TI - Location of human cytotoxic T cell epitopes within a polymorphic domain of the Plasmodium falciparum circumsporozoite protein. AB - Studies in mice have shown that cytotoxic T lymphocytes (CTL) specific for epitopes within the circumsporozoite (CS) protein of malaria sporozoites can prevent malaria probably by destroying infected hepatocytes. This has provided a model for the development of a sporozoite vaccine. It has not been shown whether humans can mount a CTL response to this protein nor what determinants on the protein could be considered as target epitopes for such cells and thus merit inclusion in a sporozoite vaccine. We have used a novel technique to study a caucasian population which would benefit from a sporozoite vaccine and have been able to demonstrate that some individuals with a history of sporozoite exposure do contain peripheral blood CTL specific for the Plasmodium falciparum CS protein. The prevalence of CTL among different individuals is low and there is evidence that recent malaria exposure may be a prerequisite for finding such CTL. In three individuals, CTL could be repeatedly found and in all cases the epitopes mapped to one of the two polymorphic C-terminal domains. Using a CTL line, we mapped a recognition site to residues 351-395 of the CS protein, overlapping the region of the protein recognized by murine CTL. PMID- 1716145 TI - Evolution of a VH gene family in low vertebrates. AB - Antibody genes represent one of the most complex receptor gene systems which have evolved in the vertebrate lineage. One of the central questions is how antibody variable region genes diversify the complementarity determining region (CDR) in evolution while they are subject to forces for sequence homogenization operating in multigene families. Information on germ line antibody genes in lower vertebrates is still fragmentary and it would be fruitful to gain insight into this question. We have studied the evolutionary stability of a rainbow trout VH family (RTVH 431) by Southern blot hybridization in various fish species. Comparison of our results with paleontological/zoological evidence suggests that this VH family has been preserved in the genomes of many fish species over a time span of 150 million years. We also show that there exist species-specific residues common to rainbow trout VH genes, which strongly supports the presence of genetic processes for homogenization. These results are compatible with the view that antibody V genes evolve at a fairly constant rate and that the homogenization process (e.g. gene duplication) may be slow enough to allow diversification of CDR by mutation, selection and drift. PMID- 1716146 TI - CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria. AB - Immunization of BALB/c mice with radiation-attenuated Plasmodium yoelii sporozoites induces cytotoxic T lymphocytes (CTL) specific for an epitope located within the amino acid sequence 277-288 of the P. yoelii circumsporozoite (CS) protein. Several CD8+ CTL clones were derived from the spleen cells of sporozoite immunized mice, all displaying an apparently identical epitope specificity. All the clones induced high levels of cytolysis in vitro upon exposure to peptide incubated MHC-compatible target cells. The adoptive transfer of two of these clones conferred complete protection against sporozoite challenge to naive mice. This protection is species and stage specific. Using P. yoelii specific ribosomal RNA probes to monitor the in vivo effects of the CTL clones, we found that their target was the intrahepatocytic stage of the parasite. The protective clones completely inhibited the development of the liver stages of P. yoelii. Some CTL clones were only partially inhibitory in vivo, while others failed completely to alter liver stage development and to confer any detectable degree of protection. The elucidation of the effector mechanism of this CTL mediated protection against rodent malaria should facilitate the design of an effective malaria vaccine. From a broader perspective this model may provide further insight into the mechanism(s) of CTL mediated killing of intracellular non-viral pathogens in general. PMID- 1716147 TI - Interactions of intermediate filament proteins from wool. AB - Filaments of wool are heteropolymers formed by interaction of type I and type II intermediate filament (IF) proteins. There are four proteins in each of these two classes. Interaction of the reduced wool IF proteins was studied by two dimensional electrophoresis which showed that complexes between type I and type II proteins were formed in solution at urea concentrations below 6 M. Complex formation between the carboxymethyl derivatives of wool IF proteins was studied using a filter binding assay in which radio-labelled individual components were allowed to react under various conditions with SDS-PAGE separated components after transfer to nitrocellulose. The results suggested that (i) absolute type specificity of interaction was maintained, (ii) fine specificity, i.e. preferential reaction between specific components is observed, (iii) wool IF proteins (hard keratins) also react, with the same type specificity, with soft keratins isolated from cow snout, (iv) the initial step in the polymerization sequence that leads to filament formation yields heterodimers. PMID- 1716148 TI - Biological activity of 3-deazauridine nucleoside analogue: a theoretical study. AB - The incorporation model of Sanyal et al. has been used to understand the biological activity of the cytostatic compound 3-deazauridine. The interaction energies of various types of binding pattern of the enterant molecule with nucleic acid fragments have been computed. The energy values and the sites of association of the analogous base, obtained by optimization of energy values as well as the sites of association of nucleic acid bases during the transcription process have been compared. The specificity of the binding of the interacting molecule has been discussed, along with the inhibitory effect of 3-deazauridine. They are in agreement with the experimentally observed evidence. PMID- 1716149 TI - Exon organization of the human FKBP-12 gene: correlation with structural and functional protein domains. AB - FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper we report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting from the topology of the antiparallel beta-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel beta-sheet and alpha-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene also provided insight into the genetic evolution of the immunophilin family. Knowledge of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes. PMID- 1716150 TI - Effect of base, pentose, and phosphodiester backbone structures on binding and repair of pyrimidine dimers by Escherichia coli DNA photolyase. AB - Photolyases reverse the effects of UV light on cells by converting cyclobutane dipyrimidine photoproducts (pyrimidine dimers, Pyr mean value of Pyr) into pyrimidine monomers in a light-dependent reaction. Previous work has suggested that, based on substrate preference, there are two classes of photolyase: DNA photolyase as exemplified by the Escherichia coli enzyme, and RNA photolyases found in plants such as Nicotiana tabacum and Phaseolus vulgaris. In experiments aimed at identifying substrate determinants, including the pentose ring, for binding and catalysis by E. coli DNA photolyase we tested several Pyr mean value of Pyr. We found that the enzyme has relative affinities for photodimers of T mean value of T greater than or equal to U mean value of T greater than U mean value of U much greater than C mean value of C and that the E-FADH2 form of the enzyme repairs these dimers at 366 nm with absolute quantum yields of 0.9 (T mean value of T), 0.8 (U mean value of T), 0.6 (U mean value of U), and 0.05 (C mean value of C). The enzyme also repairs an isolated thymine dimer and the synthetic substrate, 1,1'-trimethylene-bis (thymine) cyclobutane dimer. Unexpectedly, we found that this enzyme, previously thought to be specific for DNA, repairs uracil cyclobutane dimers in poly(rU). The affinity of photolyase for a uracil dimer in RNA is about 10(4)-fold lower than that for a U mean value of U in DNA; however, once bound, the enzyme repairs the photodimer with the same quantum yield whether the dimer is in ribonucleoside or deoxyribonucleoside form. PMID- 1716151 TI - Enzymatic release of 5'-terminal deoxyribose phosphate residues from damaged DNA in human cells. AB - Activities that catalyze or promote the release of 5'-terminal deoxyribose phosphate residues from DNA abasic sites previously incised by an AP endonuclease have been identified in soluble extracts of several human cell lines and calf thymus. Such excision of base-free sugar phosphate residues from apurinic/apyrimidinic sites is expected to be obligatory prior to repair by gap filling and ligation. The most efficient excision function is due to a DNA deoxyribophosphodiesterase similar to the protein found in Escherichia coli. The human enzyme has been partially purified and freed from detectable exonuclease activity. This DNA deoxyribophosphodiesterase is a Mg(2+)-requiring hydrolytic enzyme with an apparent molecular mass of approximately 47 kDa and is located in the cell nucleus. By comparison, the major nuclear 5'----3' exonuclease, DNase IV, is unable to catalyze the release of 5'-terminal deoxyribose phosphate residues as free sugar phosphates but can liberate them at a slow rate as part of small oligonucleotides. Nonenzymatic removal of 5'-terminal deoxyribose phosphate from DNA by beta-elimination promoted by polyamines and basic proteins is a very slow mechanism of release compared to enzymatic hydrolysis. We conclude that a DNA deoxyribophosphodiesterase acts at an intermediate stage between an AP endonuclease and a DNA polymerase during DNA repair at apurinic/apyrimidinc sites in mammalian cells, but several alternative routes also exist for the excision of deoxyribose phosphate residues. PMID- 1716152 TI - Amino acid sequence modulation of gramicidin channel function: effects of tryptophan-to-phenylalanine substitutions on the single-channel conductance and duration. AB - Linear gramicidins with one, two, or three Trp----Phe substitutions in the gramicidin A sequence form beta 6.3-helical channels that have widely varying conductances and average durations. The variations in single-channel conductance and average duration are uncoupled. The single-channel conductance decreases as a monotonic function of the number of Trp----Phe substitutions, and the relative conductance decrease induced by a given Trp----Phe substitution is only weakly affected by substitutions at other positions. These results suggest that each Trp influences the conductance independently, most likely through electrostatic interactions between the Trp dipole(s) and the permeant ion (as was deduced previously for aromatic side-chain substitutions at position one [Koeppe, R. E., Mazet, J.-L., & Andersen, O. S. (1990) Biochemistry 29 (2), 512-520]). Trp----Phe substitutions exert a complex, nonadditive influence on average duration as well as the energetics of heterodimer formation. These changes are presumably due to sequence-specific differences in the channel's surface chemistry--which may be related to ability of the Trp indole NH moieties to form hydrogen bonds with the lipid backbone oxygens and/or interfacial H2O. PMID- 1716153 TI - A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization. AB - A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G 100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined. PMID- 1716154 TI - Phagocytosis of protein coated colloidal-gold-agarose-gelatin microbeads by cultured uterine glandular epithelial and stromal cells. AB - A procedure for fixing and immunostaining whole cells from primary cultures of ovine and bovine uterine gland fragments was used to identify keratin in intermediate filaments of epithelial cells to distinguish them from stromal cells. Colloidal gold encapsulated agarose-gelatin microbeads were coated with different proteins and used to investigate uptake by epithelial and stromal cells in culture. Microbeads were taken up by stromal cells and by epithelial cells on the outskirts of colonies. These cells formed ridges where they contacted and grew above stromal cells. Electron microscopy demonstrated that the microbeads had been internalized and appeared to be nontoxic. Individual cells could harbor more than 90 microbeads within their cytoplasm for at least seven to ten days with no apparent harm. Some cells with microbeads were seen to divide. PMID- 1716155 TI - Motor nerve terminal staining combined with catecholamine histofluorescence or immunocytochemistry. AB - A number of excellent techniques are available to stain and characterize different types of neurons and nerve terminals. However, because these different techniques are frequently not compatible, their usefulness in determining the relationships between specific axons and neuromuscular junctions is often limited. The goal was to develop specific procedures for simultaneous visualization of different types of unmyelinated axons and motor nerve terminals in the same preparation. First we modified the formaldehyde/glutaraldehyde staining solutions of the aqueous aldehyde fluorescence technique (Faglu) to observe catecholamine containing axons in whole mount amphibian skeletal muscle. The compatibility of this modified staining solution with other histological procedures made it possible to stain both motor nerve terminals with tetrazolium salts and, in the same preparation, to observe unmyelinated axons with aldehyde induced catecholamine histofluorescence. This same general formaldehyde/glutaraldehyde staining procedure was also used with immunocytochemical techniques to visualize fluorescent antibody stained nerves and motor nerve terminals in the same whole mount preparation. PMID- 1716156 TI - The reaction of reducing sugars with histones. AB - The epsilon amino groups of histone proteins were eliminated by condensation with glucose, fructose or mannose. The trichloroacetic acid extracted, DNA negative nuclei treated with reducing sugars, stained easily with basic dyes. PMID- 1716157 TI - A nuclear marker for mammalian cells and its use with intracerebral transplants. AB - The Hoechst dye staining method has been successfully applied to the central nervous system in mammals and its use has been demonstrated in intracerebral transplantation. The technique is rapid, simple and based on intrinsic nuclear properties. It was found to be permanent and valid whatever the animal strains or ages, allowing the distinction of rat cells from those of mouse, studied either separately or in a cross-transplantation model. It permitted the detection of grafted cells in the area of transplantation and the observation of early dispersion around the implantation site. Moreover, it can be combined with immunohistochemistry as demonstrated by a myelin marker in a relevant model. Immunodetection can thus help to directly observe grafted cells, at distance from the locus of transplantation, confirming their presence in the graft-type myelin patches. Because of its rapid performance, this technique can be used systematically after transplantation to check for the presence of grafted cells in the host. PMID- 1716158 TI - A multichromatic stain for Lowicryl K4M embedded tissues. AB - A staining procedure using celestine blue and chromotrope 2R for Lowicryl K4M embedded tissues is presented. The stain produces a reliable multichromatic stain for light microscopy on Lowicryl embedded semithin sections. PMID- 1716159 TI - C-banding with specific fluorescent DNA-ligands: a new approach to constitutive heterochromatin heterogeneity. AB - The employment of certain DNA-specific fluorescent stains on unbanded and C banded chromosomes of two species of grasshoppers shows remarkable differences among C-heterochromatic regions supposed to be similar in their base pair composition, according to their response to the standard fluorescence techniques. The possible interspersion of the opposite DNA base pairs in these regions as well as the role played by proteins in chromosome banding are discussed. PMID- 1716160 TI - Nuclear staining of Colletotrichum gloeosporioides f. sp. malvae conidia with fluorescent and nonfluorescent stains. AB - Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores. PMID- 1716161 TI - Efficient lipid staining in plant material with sudan red 7B or fluorol [correction of fluoral] yellow 088 in polyethylene glycol-glycerol. AB - Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general. PMID- 1716162 TI - A method to prevent wrinkles in autoradiographic emulsion when staining plastic embedded sections with hematoxylin and eosin-phloxine. AB - A procedure was developed which prevents wrinkles in autoradiographic emulsion when sections, embedded in glycol methacrylate, are stained with hematoxylin and eosin-phloxine. Craniofacial tissues labeled with tritiated thymidine were collected and mounted on slides. Slides were dipped in emulsion, stored for one month and developed. The slides were immersed in liquefied celloidin and subsequently stained with a modified hematoxylin and eosin-phloxine procedure. Results showed that the emulsion did not wrinkle and the procedure did not effect the occurrence of labeled cells. PMID- 1716163 TI - Demonstration of monophenoloxidase activity of tyrosinase after electrophoresis. AB - The improved method presented here for localizing monophenoloxidase activity of tyrosinase (E.C. 1.14.18.1) after electrophoresis is based on the transfer of electrons from the monophenolic substrate, tyrosine methyl ester, to an artificial acceptor, phenazine methosulfate, and subsequent reduction of nitro blue tetrazolium into a violet formazan. This method is rapid, sensitive and versatile compared to the standard method. The electron transferred from monophenol can be accepted directly by nitro blue tetrazolium; although the background of the gel is clear, the sensitivity is decreased. The monophenol-PMS NBT method is suitable for both plant and animal samples. This method can also be used for histochemical demonstration of monophenoloxidase activity. PMID- 1716164 TI - Modification of the silver proteinate impregnation technique for protozoa and cultured nerve cells. AB - Silver impregnation with silver-protein compounds is widely used for staining tissue sections and cell cultures. Some authors report that the results obtained with these methods have not always been reproducible because the reagent's composition varies according to the manufacturer. To avoid this problem in the method described in this paper, a silver proteinate, produced in our own laboratory is used. Although our method is based on Bodian's, the modifications we have made allows its use for both free-living cells (protozoa) and cells grown in culture (nerve cells). The significant modifications are 1) different fixation, 2) postfixation with Cajal's formol-bromide, 3) changes in the duration of the impregnation steps technique and 4) elimination of metallic copper. The method reported here enables us to use silver proteinate whenever we require it and to control the composition of the silver proteinate. This technique can be used for cells cultured in either plastic or glass. PMID- 1716165 TI - Differential staining of mucin granules from epoxy resin sections by a phosphotungstic acid-methyl green procedure. AB - After treatment of epoxy resin semithin sections from glutaraldehyde fixed rat large intestine with 5% aqueous phosphotungstic acid (PTA), staining with unpurified 0.2% solutions of methyl green at 60 C for 5 min produces a color differentiation between mucin granules of goblet cells. Some mucin granules and the glycocalyx appear deep green while the remaining granules, luminal mucin and collagen fibers are pink. The known contamination of unpurified methyl green with crystal violet seems to be responsible for the pink staining reaction of the latter structures, which also present an orange-red fluorescence under green exciting light. Electron microscopic observations show selective contrast of mucin granules which appear with a different amount of PTA deposits. This procedure is useful to reveal the heterogeneity of mucin granules in light and electron microscopy. PMID- 1716166 TI - A modified silver impregnation technique for the observation of thick sections of lymph nodes perfused with colloidal carbon. AB - For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network. To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 microns thick sections from rat nodes, fixed in a solution of Bouin Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions. PMID- 1716167 TI - Silver enhancement of nickel-diaminobenzidine as applied to single and double immunoperoxidase staining. AB - A reliable and practical method is proposed for increasing sensitivity and detection efficiency of immunocytochemical techniques, based on silver enhancement of the nickel-diaminobenzidine product of the peroxidase reaction. The procedure produces a strong signal at the site of the end product of the peroxidase reaction which is visible as black grains at the light microscopic level. The method has been used to detect peroxidase labeled probes in immunocytochemical tissue preparations and blotting assays and is ideal for the purposes of double staining and photographic documentation. PMID- 1716168 TI - Morphometric studies in duodenal biopsies from patients with coeliac disease: the effect of the steroid fluticasone propionate. AB - Morphometric measurements have been performed on small intestinal biopsy specimens from patients with untreated coeliac disease before and after six weeks oral treatment with a steroid of low systemic bioavailability (fluticasone propionate). Measurements were obtained by point counting and also by a computer aided measuring system with reference to a constant area of the muscularis mucosa. Fluticasone propionate led to a parallel reduction in the intraepithelial lymphocyte count within the surface (P less than 0.001) and crypt epithelium (P less than 0.01). The intra-epithelial lymphocyte count assessed by reference to constant areas of the muscularis mucosa and surface epithelium were decreased two fold (P less than 0.01) and seven-fold (P less than 0.001) respectively. Fluticasone propionate treatment also led to significant increases in the absorptive surface epithelium as shown by an increase in the villus:crypt ratio (P less than 0.01), the epithelial cell height (P less than 0.01) and two- to three-fold increases in the area and length of the surface epithelium (P less than 0.001). Short-term fluticasone propionate treatment appears to exert a powerful beneficial effect upon duodenal morphology in patients with coeliac disease. Whether the alterations seen are comparable to a similar period of gluten withdrawal is not yet known. PMID- 1716169 TI - Lack of correlation between the coupling interval of ventricular premature beats and the preceding cycle length. AB - The accepted variation of 80-120 ms in the coupling interval of non-parasystolic ventricular premature beats has been linked to the duration of the preceding cycle length, but a lack of correlation between these factors has often been found. As a result, there is no explanation for variations in the coupling interval of such ventricular premature beats. To analyze the influence of the preceding cycle length on the coupling interval of isolated ventricular premature beats throughout an entire day, 10 otherwise healthy patients with frequent monofocal non-parasystolic isolated ventricular premature beats were studied with Holter recordings. A sample of the electrocardiogram was obtained at the beginning of each quarter of an hour and coupling intervals as well as preceding cycle lengths of ventricular premature beats not belonging to a bigeminy or trigeminy sequence were measured. Preceding cycle lengths changed in a way similar to the prevailing heart rate, with a marked prolongation during sleep. Coupling intervals showed an irregular pattern during the waking period but increased consistently from the first to the last hour of sleep. The average coupling interval during sleep was significantly longer than during waking (439 +/- 74 vs 466 +/- 80, p less than 0.01). In each patient a similar preceding cycle length within a narrow range of 40 ms was searched for during waking as well as during sleep and the corresponding coupling intervals were significantly longer during sleep (446 +/- 80 vs 466 +/- 85, p = 0.009).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716170 TI - Human T cell receptor gamma delta + T cells. AB - TCR gamma delta + T cells represent a minority of CD3+ T cells in many species including man. The molecular structure of the TCR gamma and delta loci in man is well understood. The gamma and delta loci contain V, D, J and C gene segments. These segments do not rearrange randomly but in a coordinated, ordered fashion during thymic development. Therefore, the structure of gamma and delta genes of early fetal TCR gamma delta + thymocytes differ drastically from those in postnatal TCR gamma delta + thymocytes. In contrast to postnatal TCR gamma delta + thymocytes, early fetal-TCR gamma delta + produce substantial levels of IL-4 and IL-5 and the possibility is discussed that the early fetal TCR gamma delta + cells are involved in development of TCR gamma delta + cells. In man, unlike in mouse, no preferential homing of early fetal TCR gamma delta + cells has been observed so far. Mature human peripheral TCR gamma delta + cells can recognize a great variety of cell surface antigens including 'classical' and 'non-classical' MHC antigens, immunoglobulins and other undefined antigens. In addition, TCR gamma delta + can recognize bacterial products. So far, no class of antigens has been defined that is preferentially recognized by TCR gamma delta + T cells and the function of these cells remains elusive. PMID- 1716171 TI - Specificity of gamma delta receptor bearing T cells. AB - T cells expressing the gamma delta receptor heterodimer can recognize a broad array of different antigens, including classical and non-classical major histocompatibility complex (MHC) proteins, MHC-like CD1 antigens, and bacterial antigens such as the mycobacterial heat shock proteins and staphylococcal enterotoxins. Reactivity to self antigens including autologous stress proteins implicates TCR gamma delta T cells in autoimmune disease. It is as yet unclear whether the responses of gamma delta T cells specific for soluble proteins are restricted by conventional or non-classical MHC molecules. Correlations of TCR gamma delta usage with specificity suggest that, like TCR alpha beta, sequences encoded within both the V regions and the V(D)J junctions are important in determining receptor specificity. PMID- 1716172 TI - Murine T cells with invariant gamma delta antigen receptors: origin, repertoire, and specificity. AB - T cells bearing invariant gamma delta T cell antigen receptors localize to distinct epithelial sites in the adult mouse. These gamma delta T cells differ from the lymphoid alpha beta and gamma delta T cells in several ways. The epithelial gamma delta T cells appear to be the product of the earliest waves of TCR expressing cells in the fetal thymus. Additionally, the rearranged TCR junctions exhibit a distinct lack of diversity, possibly a result of the fetal origin and a specialized selection process. The use of a single invariant TCR and strict tissue localization suggest that these gamma delta T cells may provide a specialized function in the epithelial tissues distinct from that of the circulating alpha beta and gamma delta T cells. PMID- 1716173 TI - The co-receptor function of CD4. AB - CD4 is a critical component of the T cell receptor complex that recognizes peptides bound to MHC class II molecules. This can be observed at all stages of T cell development, activation, and function. CD4 has been termed a co-receptor to indicate that its most important activity is to bind the same peptide: self class II MHC complex as the T cell receptor and to transduce positive activating signals in conjunction with the T cell receptor. This behavior has been shown by several independent experimental systems: direct cross-linking of the T cell receptor to CD4, the inhibition of T cell activation by anti-CD4, the transfection of CD4 into CD4- T cells, and by the phenomenon of epitope interference, as described in this review. All of these approaches suggest that the participation of CD4 as a co-receptor in antigen: self class II MHC recognition potentiates activation by 100-fold. Given the complex nature of the ligand recognized by the T cell receptor, the co-receptor function of CD4 virtually eliminates the possibility of CD4 T cells recognizing peptides presented by class I MHC molecules, in keeping with many in vivo observations. PMID- 1716174 TI - Projection from the external pallidum to the reticular thalamic nucleus in the squirrel monkey. AB - Small injections of biocytin in the external segment of the pallidum (GPe) of the squirrel monkey (Saimiri sciureus) led to anterograde labeling of fibers in the thalamic reticular nucleus (NRT). These fibers reached NRT by coursing along the ventral tip of the internal capsule or by directly piercing the internal capsule more dorsally. They arborized profusely within the entire rostrocaudal extent of the nucleus. Within NRT, biocytin-labeled fibers were long, slightly varicosed, and emitted numerous short collaterals whose terminal portions consisted of clusters of large varicosities. Some of these varicosities were closely apposed to cell bodies and proximal dendrites of NRT neurons. Small injections of wheat germ-agglutinated horseradish peroxidase in the rostral pole of NRT led to retrograde cell labeling within the entire rostrocaudal extent of GPe. These retrogradely-labeled cells did not display immunoreactivity for choline acetyltransferase. Hence, beside the well-established projection from the internal pallidum to the thalamus, our findings support the existence of another pallidothalamic projection whereby GPe neurons could exert a powerful influence upon the thalamocortical neurons via a relay in NRT. PMID- 1716175 TI - Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain. AB - Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells. When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria. The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells. Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency. Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures. PMID- 1716176 TI - Modulation of DNA synthesis in aortic smooth muscle cells by dinitrotoluenes. AB - Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced 3H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1-100 microM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1-100 microM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions. PMID- 1716177 TI - Study of the biological effects of theophylline on V79 cells: viability, membrane permeability, and metabolic cooperation. AB - The effect of theophylline, a specific inhibitor of phosphodiesterase, on gap junction-mediated intercellular communication between Chinese hamster V79 cells was examined. It was found that addition of theophylline to coculture of 6 thioguanine-resistant (TGr) and 6-thioguanine-sensitive (TGs) V79 cells significantly increased the recovery of TGr cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations less than 0.3 mg/ml, which were neither cytotoxic (after short or continuous exposure) nor inhibited the synthesis of DNA, RNA, and proteins. At the tested concentrations, no change was found in the membrane permeability of cells. Theophylline did not increase the incorporation of glucose into the cells. PMID- 1716178 TI - Cytokine activity in bovine mammary gland secretions during the periparturient period. AB - The presence of cytokine activity in periparturient bovine mammary secretions was evaluated. Mammary secretions were modified for use in biological assays for interleukin-2 (IL-2) like and antiviral activity. The level of IL-2 like activity in mammary gland secretions was lower during the last week of gestation when compared to levels detected approximately two weeks prepartum. Antiviral titers gradually increased as parturition approached. Results from Western blots indicated that the antiviral activity observed in prepartum secretions may be due to tumor necrosis factor (TNF). Interferons (IFN) were not detected in the colostrum samples. PMID- 1716179 TI - Intravascular stents in the management of superior vena cava syndrome. AB - Superior vena cava syndrome can be effectively palliated with the use of intravascular stents. Adjunctive modalities which may be utilized prior to stent placement are thrombolytic therapy and balloon angioplasty. Six patients with an underlying malignancy were treated with these modalities. Complete resolution of symptoms occurred in five patients, and partial resolution occurred in the sixth. Two of the patients who had initial, complete resolution of symptoms had recurrences. One involved rethrombosis of the superior vena cava which occurred twice and required percutaneous thrombectomy, and the second involved restenosis requiring a percutaneous transluminal angioplasty of the SVC just distal to the stent. Both of these patients with second procedures, again, had complete resolution of symptoms. Intravascular stents are a valuable additional treatment of superior vena cava syndrome. PMID- 1716180 TI - Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. AB - CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity. PMID- 1716181 TI - When worlds collide: immunosuppressants meet protein phosphatases. PMID- 1716182 TI - The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140. AB - LECAM-1 (leukocyte-endothelial cell adhesion molecule 1), the lymphocyte lectin ("selectin") homing receptor for peripheral lymph nodes (PLNs), participates in the earliest interactions of polymorphonuclear neutrophilic leukocytes (PMNs) with inflamed venules. Here, we present evidence that LECAM-1 mediates this function through a novel mechanism--presentation of oligosaccharide ligands to the inducible vascular selectins endothelial-leukocyte adhesion molecule (ELAM-1) and granule membrane protein 140 (GMP-140). PMN, but not lymphocyte, LECAM-1 is modified with the vascular selectin ligand sialyl Lewis x (sLex) and specifically binds ELAM-1-transfected cells. Although only a small fraction of total cell surface sLex, LECAM-1-associated sLex appears to play a prominent role in PMN interactions with cell-associated ELAM-1 and GMP-140, as anti-LECAM-1 monoclonal antibodies or selective removal of cell surface LECAM-1 inhibits PMN binding to vascular selectin transfectants by up to 70%. The enhanced function of LECAM-1 associated sLex may reflect the striking concentration, shown here, of LECAM-1 on PMN surface microvilli, the site of initial cellular contact. PMID- 1716183 TI - [Thrombosis test in vitro of 107 cases with arteriosclerosis obliterans]. AB - Thrombosis test in vitro of 107 cases with arteriosclerosis obliterans were measured and controlled by 36 normal persons. Of 107 cases, 13 cases belonged to ischemic phase, 47 cases nutritional disturbance phase, 47 necrosis (24 cases were first degree, 9 cases second degree, 14 third degree). Types distribution were: simple blood stasis 65 cases, blood and damp stasis 34, and 8 others. 46 patients were treated systematically by TCM-WM. The result proved that the indexes of thrombosis test in vitro of patients were much higher than that of normal persons (P less than 0.001). The relevant indexes were different in varied periods, degrees and types (P less than 0.05-0.01) and between before and after treatment (P less than 0.001). Of treated 46 cases, total effective rate was 86.96%, amputation rate was 13.04%. Formation of thrombus in vitro lowered 88.03%. The authors suggested that measurement of thrombosis test might be a accurate way to reflect the course of the disease and a basis for treatment and differentiation of symptoms. PMID- 1716184 TI - An enzyme-linked lectin assay for sialidase. AB - A procedure for the detection of low activities of sialidase (= neuraminidase) is described. Natural substrates for sialidase (human erythrocytes, fetuin or gangliosides) were coated onto the wells of microplates and incubated at 37 degrees C with the enzyme. Sialidase-induced desialylation of these natural substrates unmasks saccharides that are specifically recognized by the peanut agglutinin lectin (PNA). The use of a peroxidase-conjugated PNA (Po-PNA) allowed the binding of the lectin to the desialylated substrate to be quantified. The amount of bound Po-PNA correlated directly with the amount of sialic acid released from the substrate, and therefore with the sialidase activity. With this method, it was possible to detect sialidase activity associated with bacteria, myxoviruses and cells from higher organisms. This method may have important clinical implications as the use of ELISA allows automation and concurrent analysis of numerous samples. PMID- 1716185 TI - Detection of the common Hb F Sardinia [A gamma (E19)Ile----Thr] variant by isoelectric focusing in normal newborns and in adults affected by elevated fetal hemoglobin syndromes. AB - A simple and rapid conventional isoelectric focusing technique for the detection of the silent Hb F Sardinia variant, containing the mutated A gamma T chain, is described. The method is based on thin-layer gels of shallow pH gradient (pH 6.7 7.7) and allows the direct detection of this rather common and widespread Hb variant at a screening level. 15-30 hemolysates from newborns and adults affected by elevated Hb F syndromes, both in the heterozygous and homozygous condition, could be examined simultaneously. The frequency of the A gamma T gene in Sardinian newborn (f = 0.175), in beta 0-thalassemia (f = 0.722), in beta (+) thalassemia (f = 0.346), and in the non-deletional type of A gamma-HPFH (f = 0), as evaluated with this method, is in accordance with that previously reported by means of other methodologies. PMID- 1716186 TI - Effects of gonadotrophin releasing hormone antagonist and agonist on the pulsatile release of gonadotrophins and alpha-subunit in postmenopausal women. AB - OBJECTIVE: The present study was designed to further assess the mechanism of action of GnRH and GnRH analogues. DESIGN AND PATIENTS: Both the Nal-Glu GnRH antagonist and the D-Trp6 GnRH agonist were administered sequentially to nine normal, post-menopausal women. MEASUREMENTS: A baseline study of pulsatile LH, FSH and free alpha-subunit secretion was performed, with sampling every 10 min for 8 h, and then repeated 8 h after a single subcutaneous injection of Nal-Glu GnRH antagonist (5 mg). Sampling was repeated 21 days after the intramuscular injection of a depot preparation of D-Trp6 GnRH (3.75 mg) in the same women. RESULTS: The baseline sampling period showed synchronous pulses of LH and free alpha-subunit. The antagonist Nal-Glu decreased plasma LH (71%) and free alpha subunit (43%). However, with the single dose of 5 mg, pulsatile LH and free alpha subunit release were not completely suppressed and remained temporally correlated. The GnRH agonist had a potent inhibitory action on plasma immunoreactive LH (IRMA) (93%). In contrast, it increased the mean plasma levels of free alpha-subunit from 1.66 +/- 0.01 to 5.06 +/- 0.02 micrograms/l (205%). The pulsatile secretory patterns of both LH and free alpha-subunit were abolished by the agonist. Immunoreactive FSH levels were decreased by the antagonist (24%) and suppressed by the agonist (93%). CONCLUSIONS: The pulsatile study confirms the different mechanism of action of GnRH analogues. Following antagonist administration, low amplitude free alpha-subunit pulses persist and are synchronous with residual LH pulses. In contrast, LH and free alpha-subunit are not maintained under agonist treatment. These data provide evidence for the differential regulation of LH and free alpha-subunit by GnRH. PMID- 1716187 TI - The presence of cation-dependent proteases for insulin-like growth factor binding proteins does not alter the size distribution of insulin-like growth factors in pregnancy. AB - OBJECTIVE: The aim was to investigate the sera of pregnant women for the presence of specific proteases for insulin-like growth factor binding proteins (IGFBPs) and to determine the effect of these on the distribution of IGFs in the circulation. DESIGN: The method used was the chromatographic and electrophoretic analysis of patients' serum. PATIENTS: Sera were examined from normal women during pregnancy: first trimester (n = 4), second trimester (n = 4) and third trimester (n = 10). Eight women with Type I diabetes in the third trimester were also studied along with sera from ten normal adult volunteers. MEASUREMENTS: Circulating IGF-I and IGF-II levels were measured by RIA and their distribution examined by gel filtration. The pattern and stability of the IGFBPs was investigated by Western ligand blotting. RESULTS: A marked reduction in the serum levels of IGFBP-2, IGFBP-3 and IGFBP-4 on Western ligand blotting, which was associated with the presence of three independent, cation-dependent proteases that were specific for different IGFBPs, was found in late pregnancy. Gel filtration of third trimester serum revealed most of the IGF-I to be present in a complex larger than 130 kDa, with a similar distribution to that found in serum of non-pregnant women. The enzymatic modification of the binding proteins made apparent by the decrease in binding protein bands on Western ligand blotting of preincubated samples had no effect on the distribution of IGF-I following size fractionation. CONCLUSIONS: There appear to be at least three independent enzymes that are induced or activated during pregnancy to modify IGFBP-2, IGFBP-3 and IGFBP-4 sufficiently to prevent their detection by ligand blotting. However, this enzymatic processing does not alter the distribution of IGFs, suggesting that the altered binding proteins are still able to carry IGFs but with reduced affinity. Such an alteration in the carrying mechanism of IGFs may have profound effects upon the bioavailability of the IGFs to the maternal tissues and contribute to the altered metabolic demands of pregnancy. PMID- 1716188 TI - Serum and tissue alpha-L-fucosidase activity in the pre-clinical and clinical stages of hepatocellular carcinoma. AB - 1. To assess the value of serum alpha-L-fucosidase (EC 3.2.1.51) as a marker for hepatocellular carcinoma, the activity was measured in patients with hepatocellular carcinoma and in control subjects. 2. Mean serum alpha-L fucosidase activity was significantly greater in 35 patients with hepatocellular carcinoma (225 +/- 69 nkat/l) than in 35 patients with cirrhosis and 20 normal subjects (134 +/- 30 and 93 +/- 28 nkat/l, respectively). The overlap between hepatocellular carcinoma and cirrhosis, however, was such as to severely limit any value of the enzyme as a diagnostic test. 3. In four cirrhotic patients with hepatocellular carcinoma, an increased enzyme activity was detectable in samples taken up to 66 months before the tumours were diagnosed clinically. 4. The serum activity of alpha-L-fucosidase fell to within the reference range after liver transplantation for hepatocellular carcinoma in three patients and in one of these a subsequent rise was associated with tumour recurrence which was diagnosed at 8 months after the rise in activity. Ineffective cytotoxic chemotherapy was also associated with a progressive rise in serum alpha-L-fucosidase activity. 5. alpha-L-fucosidase activity in tumour tissue was significantly lower than that seen in non-tumour tissue from cirrhotic patients. These reductions may represent increased transport from the tissue and may partly account for the increased serum activity found in some patients with hepatocellular carcinoma. PMID- 1716189 TI - Muscle composition in relation to age and sex. AB - 1. A method is described enabling the determination of fat, water, electrolytes, protein, DNA, RNA and total creatine in a single sample of human muscle obtained by the percutaneous needle-biopsy technique. The amino acid content can also be analysed in the same muscle sample. 2. Fifty healthy subjects were studied: 29 between 19 and 40 years of age, 11 between 41 and 60 years of age, and 10 between 61 and 85 years of age. The two groups aged less than 60 years showed only marginal differences in muscle composition, whereas the highest age group showed increases in muscle fat content in relation to tissue weight and decreases in alkali-soluble protein content in relation to both tissue weight and tissue DNA content. Also, potassium, magnesium, total creatine and RNA contents were decreased in this age group when related to tissue DNA content. When alkali soluble protein was used as a reference base, only magnesium content was decreased. 3. A comparison was also made between female (n = 23) and male (n = 18) subjects in the age groups below 60 years. Differences observed included a higher fat content in female muscle, and an increase in total creatine content in relation to tissue weight. The alkalisoluble protein content was lower per muscle cell in the females when calculated on the basis of DNA content. 4. The results show that in the assessment of muscle constituents, age and sex must be taken into account. PMID- 1716190 TI - Differentiation of Enterobacteriaceae, Pseudomonas aeruginosa, and Bacteroides and Haemophilus species in gram-stained direct smears. AB - The accuracy of examination of the Gram-stained direct smear to classify presumptively Gram-negative rods into three morphotype groups, that is, (a) Enteric bacteria, (b) Pseudomonas, and (c) Bacteroides or Haemophilus, was evaluated. Randomly selected clinical strains (4-9) each of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Ps. aeruginosa, B. fragilis, and H. influenzae were used to produce peritonitis or subcutaneous abscesses in mice. A Gram-stained direct smear was prepared from exudate collected from each animal. The direct smears were examined to classify bacteria observed into one of the three morphotype groups. The percent accuracy was 82, 56, and 95, respectively, and 76 overall. The assumption was made that classification was based primarily on differences in length and width of the organisms. To test this hypothesis, we prepared scanning electron photomicrographs from each specimen of exudate and measured the lengths and widths of bacteria. Examination of the Gram-stained direct smear was more accurate for classification of enteric bacteria, H. influenzae, or B. fragilis. Electron microscopy was more accurate for classification of Ps. aeruginosa. The higher length-width radio should be helpful in recognizing Ps. aeruginosa in direct smears. PMID- 1716191 TI - Use of lactophenol cotton blue mounts of corneal scrapings as an aid to the diagnosis of mycotic keratitis. AB - Lactophenol cotton blue (LPCB) mounts of corneal ulcer scrapings were assessed as a diagnostic tool in cases of mycotic keratitis over a period of 15 months. We investigated 568 cases of ulcerative keratitis by microbiological techniques consisting of direct microscopic examination of LPCB mounts and Gram-stained smears as well as culture of material scraped from the ulcer. Fungi were isolated in large numbers on multiple media from the corneal scrapings of 179 patients (culture-proven mycotic keratitis). Direct microscopic examination of LPCB mounts of corneal scrapings yielded positive results in 140 (78%) of 179 culture positive patients and negative results in 371 (95%) of 389 culture-negative patients. There was a significant difference between the percentage of positive results obtained by LPCB mounts and by Gram-stained smears in the culture positive cases. The LPCB mount was positive in greater than 80% of cases of keratitis due to Fusarium spp. and Aspergillus spp. The LPCB mount is strongly recommended as a simple, rapid, inexpensive, and sensitive diagnostic technique in cases of mycotic keratitis. PMID- 1716192 TI - Diagnosis of cytomegalovirus hepatitis by histopathology and in situ hybridization in liver transplantation. AB - Hematoxylin-eosin staining (H&E) of cytomegalovirus (CMV) inclusion bodies was compared with in situ hybridization using a biotinylated DNA probe for the detection of CMV. Of 29 biopsy specimens selected from 23 patients with clinical CMV hepatitis and typical CMV inclusions on histopathologic examination, 23 (79%) were positive by DNA probe and 17 (59%) were detected in cell cultures. The mean number of CMV foci per tissue section was higher by DNA probe (12) compared with H&E (5). We do not recommend in situ hybridization in microbiology laboratories as a replacement for histopathology for the diagnosis of CMV in tissue specimens. PMID- 1716193 TI - Hard contact lens-induced corneal neovascularization treated by oxygenation. AB - A 39-year-old man suffered from corneal neovascularization, through to result from hypoxia caused by improper use of polymethylmethacrylate (PMMA) hard contact lenses. The condition was successfully treated by oxygenation of the corneas under swimming goggles. PMID- 1716194 TI - Primary, adjuvant, and palliative radiation therapy. PMID- 1716195 TI - [The changes of monoamine metabolites in CSF of patients with cerebral stroke]. AB - For understanding of the role of monoamines in cerebral ischemia, 3-methoxy-4 hydroxyphenylglycol (MHPG), hydroxyindoleacetic acid (5HIAA) homovanillic acid (HVA) the three major monoamine metabolites in CSF of 33 patients and 18 controls were measured with high-performance liquid chromatography. Results showed MHPG was more sensitive to cerebral ischemia than the two others. All three metabolites were elevated in patients with severe ischemia but only MHPG and 5 HIAA were significantly elevated. A positive correlation between any two of metabolites was found in controls and in patients in the first week after stroke but altered at the end of the second week. Computer assisted multivariate analysis indicated 5-HIAA might contribute more to the state of illness in the acute stage while HVA the least. Clinically, MHPG appeared to be the most significant element on reflecting the degree of the damage and the prognosis of the disease among the metabolites. PMID- 1716196 TI - [Alpha-fetoprotein in non-hepatocellular malignant tumors]. AB - Alpha-fetoprotein (AFP), a very useful tumor marker for hepatocellular carcinoma and yolk sac tumor, is rarely elevated in other malignant tumors. However, since 1978, 9 cases of non-hepatocellular malignant tumor with elevated AFP (greater than 400 ng/ml), 3/206 gastric carcinoma, 1/112 esophageal carcinoma, 1/87 colorectal carcinoma, 2/83 pancreatic carcinoma and 2/192 lung cancer had been collected. Seven of the 9 cases had liver involvement. AFP might be originated from the tumor cells of the primary cancer and, in cases with liver metastases, from the regenerative hepatocytes surrounding the metastatic foci. It is of the opinion that elevated AFP is not specific for hepatocellular carcinoma, as it may also be seen in the other malignant tumors. PMID- 1716197 TI - On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis. AB - The quantification of proteins in silver-stained electrophoresis gels has been limited by the differences in "stainability" of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to differences in di- or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed. PMID- 1716198 TI - Counterflow affinity isotachophoresis on cellulose acetate membranes. AB - Counterflow isotachophoresis on cellulose acetate membranes of human alpha fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method. PMID- 1716199 TI - Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels. AB - The effect of the cationic dye Alcian Blue on the silver staining of bacterial lipopolysaccharide and its polysaccharide and lipid A portions in polyacrylamide gels was investigated. The polysaccharide was only stained when the gel was previously treated with the dye. The polysaccharide moiety was found to be responsible for the lipopolysaccharide staining with silver, whereas the lipid A seemed unimportant. Treatment with Alcian Blue may prove useful to detect hydrophilic components of lipopolysaccharide samples that could not be stained by the usual silver staining procedures. PMID- 1716200 TI - Quantification of proteins in sample buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis using colloidal silver. AB - A rapid and simple assay for quantification of proteins in sodium dodecyl sulfate sample buffer is described. Proteins are bound to a nitrocellulose membrane, stained with colloidal silver, and quantified by transmission densitometry. The staining requires 1 microL of protein sample and takes less than 20 min. Good linearity between the staining intensity and amount of proteins is in the range of 5-100 ng. PMID- 1716201 TI - O-specific polysaccharide of Hafnia alvei lipopolysaccharide isolated from strain 1211. Structural study using chemical methods, gas-liquid chromatography/mass spectrometry and NMR spectroscopy. AB - The Hafnia alvei strain 1211 O-specific polysaccharide is composed of 3-amino-N (D-3'-hydroxybutyryl)-3,6-dideoxy-D-galactose, N-acetyl-D-galactosamine, N-acetyl D-glucosamine and D-glucose (1:1:2:2). On the basis of sugar and methylation analyses, Smith degradation, and one- and two-dimensional 1H- and 13C-NMR spectroscopy, the polysaccharide was shown to be an O-acetylated polymer of the repeating hexasaccharide unit, ----2D(4-OAc)Fucp3NAcyl beta 1----6DGlcpNAc alpha 1---- (DGlcp beta 1----3)4DGalpNAc alpha 1----3DGlcpNAc beta 1----2DGlcp beta 1-- -, where DFucp3NAcyl = 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D- galactopyranose. The O-specific polysaccharide showed some microheterogeneity due to incomplete substitution by terminal glucose. PMID- 1716202 TI - A novel mechanism of Sarcophaga lectin gene expression. Possible involvement of oxidation of the sulfhydryl group of a fat-body protein. AB - The Sarcophaga lectin gene, an acute-phase-responsive protein of Sarcophaga, was found to be activated when the fat body was incubated in phosphate-buffered insect saline (10 mM NaH2PO4/Na2HPO4, pH 6.2, 2 mM NaHCO3, 1 mM MgCl2, 5 mM KCl, 1 mM CaCl2 and 120 mM NaCl). This activation was selectively suppressed by addition of 2-mercaptoethanol. 2-Mercaptoethanol did not affect the transcription directly, suggesting that the oxidation of the sulfhydryl group of a fat-body protein is required for activation of the Sarcophaga lectin gene. This oxidation reaction seemed to occur immediately when the fat body was incubated in vitro, and once this reaction was initiated, 2-mercaptoethanol no longer inhibited transcription of the Sarcophaga lectin gene. This gene is known to be activated when the larval-body wall of Sarcophaga is injured. It is suggested that the Sarcophaga lectin gene is activated via this novel mechanism on its acute-phase expression in vivo. PMID- 1716203 TI - Conformational and epitope mapping of herpes-simplex-virus type-1 thymidine kinase using synthetic peptide segments. AB - Adjacent peptide segments covering the complete sequence of herpes-simplex-virus type-1 thymidine kinase (HSV1-TK) of 376 amino acids were synthesized in order to experimentally verify the three-dimensional structure of the HSV1-TK active site, which was previously determined by molecular modeling. 26 peptides have been prepared by multiple solid-phase synthesis using the 9-fluorenylmethoxycarbonyl strategy. The purified peptides were linked covalently to bovine serum albumin. The peptide/ELISA of the synthesized bovine-serum-albumin conjugates using polyclonal rabbit anti(HSV1-TK)serum resulted in ten epitopes, which correlate excellently with the computer-proposed active site of HSV1-TK. CD spectra of the HSV1-TK peptides were recorded in trifluoroethanol/water (9:1 by vol.) An eigenvalue method based on CD spectra of 15 well known protein structures was used to calculate the relative percentage of secondary structures from the CD data. The computer model of the HSV1-TK showed full conformity with the folding pattern determined by CD of the synthetic peptide segments. Therefore, conformational peptide mapping with CD-based secondary structures combined with epitope mapping from the peptide/ELISA is an efficient and reliable method to support computer-aided protein design. PMID- 1716204 TI - The role of magnetic resonance imaging and computed tomography in the treatment evaluation of retroperitoneal lymph-node metastases of non-seminomatous testicular tumors. AB - The role of Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) in the treatment evaluation of retroperitoneal lymph-node metastases of non-seminomatous testicular tumors (NSTT) was prospectively studied in 10 consecutive patients before, during and after chemotherapy. The results thus obtained were compared with laparotomy findings before and after chemotherapy, the histology of the primary testicular tumor, and that of retroperitoneal residual lesions after chemotherapy. MRI and CT proved to be equivalent in detection and in determining the anatomical localization and size of the retroperitoneal lymph node metastases. Unlike CT, MRI revealed unmistakable changes in the structure of the retroperitoneal lymph-node metastases during chemotherapy, for which no histological cause was found except in mature teratoma. In mature teratoma a high T2 signal was found within the metastases, corresponding with a high water content. On the basis of tumor consistency and signal intensity in the T1- and T2 weighted images, MRI cannot yet warrant any conclusion about the ultimate effect of chemotherapy. PMID- 1716205 TI - Glucocorticoid-dependent and -independent mechanisms involved in lipopolysaccharide tolerance. AB - Injection of bacterial lipopolysaccharide (LPS) into animals results in a transient increase in serum tumor necrosis factor (TNF). Maximal increases in TNF were detected by 1 h and 3-4 h serum TNF was no longer apparent. These animals were LPS tolerant and a repetitive LPS stimulus did not result in an additional peak in TNF. Regulation of TNF expression in LPS-tolerant animals was at the transcriptional level as TNF mRNA was not apparent in spleen or peritoneal macrophages following a second LPS stimulus. Adrenalectomized (adrex) mice, in contrast, did not become LPS tolerant and sera from these animals demonstrated an additional peak in TNF 1 h following a second LPS stimulus. Concomitant with the secondary rise in serum TNF in adrex mice was an increase in splenic TNF mRNA. The ability of adrex mice to become LPS tolerant was restored by exogenous glucocorticoids. LPS tolerance was also investigated in the galactosamine LPS model which like the adrex model is characterized by a thousandfold increase in the sensitivity of these animals to the lethal effects of LPS. Consistent with the absence of LPS tolerance in adrex mice, galactosamine-sensitized mice were also responsive to a second LPS stimulus and did not become LPS tolerant. While LPS-treated adrex mice had no significant increases in serum corticosterone, corticosterone levels in LPS-treated galactosamine-sensitized mice were comparable to LPS-stimulated normals suggesting that LPS tolerance involves both glucocorticoid-dependent and -independent components. Finally, prophylactic administration of a monoclonal antibody against murine TNF protected normal and galactosamine-sensitized mice from a lethal dose of LPS and yet had no protective effect in adrex animals. PMID- 1716206 TI - Antigen processing by endosomal proteases determines which sites of sperm-whale myoglobin are eventually recognized by T cells. AB - This study reports an identification of the major processing products of an exogenous protein antigen, viz, sperm-whale myoglobin, as obtained after cell free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by high performance liquid chromatography analysis from those generated after uptake of myoglobin inside live macrophages. The concerted action of the endosomal proteases cathepsin D and cathepsin B can account for nearly all cleavages observed. Cathepsin D appears to be mainly responsible for the initial cleavage of myoglobin, while cathepsin B catalyzes the C-terminal trimming of initially released fragments. The fragments released by cathepsin D contain most, if not all, major epitopes for murine myoglobin-specific helper T cells. Interestingly, each known T cell epitope of myoglobin is located at the very N terminus of a different myoglobin fragment released upon processing. In order to explain this correspondence, noted also in several other protein antigens, a structural relationship is proposed between antigen processing by cathepsin D and antigen recognition by major histocompatibility complex (MHC) class II products. As is demonstrated here, this relationship may be used as a predictive tool for the identification of MHC-binding sequences as well as of T cell epitopes in their naturally occurring form. PMID- 1716207 TI - Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line. AB - Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post-implantation-stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well-defined class I product, we introduced the H-2Dd gene under control of the human beta-actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H-2Dd heavy chains and beta 2-microglobulin protein, but only small amounts of H-2Dd surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H-2Dd-transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly-defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post-implantation-stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post-translationally at the level of MHC class I assembly. PMID- 1716208 TI - Diabetes-prone BB rats express the RT6 alloantigen on intestinal intraepithelial lymphocytes. AB - The RT6 alloantigen of the rat is expressed on most peripheral T cells but not on thymocytes and thus represents a marker for postthymic T lymphocyte maturation in this species. Diabetes-prone (DP) BB rats exhibit a genetically determined T cell lymphopenia associated with a deficiency of RT6+ T cells. In this study we have analyzed the expression of RT6 on lymph node (LN) cells and intestinal intraepithelial lymphocytes (IEL) in two DP BB strains (BB/OK and BB/Mol) and two control strains (non-lymphopenic BB/PhiK and LEW) by flow cytometry. In the DP BB rats the number of LN T cells was substantially reduced (less than 25% TcR2+ cells) and completely lacked RT6 expression. The IEL population was also reduced in number and in marked contrast to normal rats consisted predominantly of CD4+ cells. The majority of IEL, however clearly expressed RT6. Treatment with a phosphatidylinositol (PI)-specific phospholipase C markedly reduced the RT6 density showing that PI-mediated anchoring of RT6 in the cell membrane also applies to IEL of DP BB rats. The results demonstrate that the DP BB strains possess a functional RT6 gene and are also able to generate the PI anchor. The defect in RT6 expression is thus unlikely to be the primary cause of the T cell lymphopenia. PMID- 1716209 TI - Biased VH gene expression in murine CD5 B cells results from age-dependent cellular selection. AB - Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single-stranded DNA, trimethyl ammonium and bovine gamma-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms. PMID- 1716210 TI - Genetic differences in the T cell receptor alleles of LEW rats and their encephalomyelitis-resistant derivative, LER, and their impact on the inheritance of EAE resistance. AB - Experimental allergic encephalomyelitis (EAE) is an animal model for the human disease, multiple sclerosis. The LEW rat strain is very susceptible to induction of EAE, whereas the closely related, major histocompatibility complex (MHC) identical, inbred strain LER is resistant. In this report, the two rat strains have been compared for differences at a number of immunologically relevant loci by restriction fragment length analysis and by nucleotide sequencing. A major difference between the two strains was discovered at the T cell receptor beta chain locus (TcR beta). Both variable (V beta 8) and constant (C beta 1) region elements of TcR beta showed allelic variation between LEW and LER. The known genetic influences in rat models of autoimmunity are currently limited to those encoded by the rat MHC, RT-1. In this study we report our characterization of the allelic differences in TcR beta chains between two rats which differ in their susceptibility to induced EAE, with the goal of understanding the role played by these allelic forms of TcR in the pathogenesis of EAE. The importance of the TcR beta allelic difference in resistance or susceptibility to EAE was assessed in a study of backcross rats scored for both EAE and for the novel LER TcR beta allele. We found that the TcR beta allele from the susceptible strain was present in three out of four susceptible rats, suggesting that it is an important, but not the only, genetic factor in EAE. Supporting this conclusion were the observations that 12 of 13 rats with homozygous LER-derived TCR beta alleles were resistant to EAE. PMID- 1716211 TI - Structure-function studies on human C4b-binding protein using monoclonal antibodies. AB - Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked beta chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I-labeled C4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated. PMID- 1716212 TI - Allospecific recognition of hemic cells in vitro by natural killer cells from athymic rats: evidence that allodeterminants coded for by single major histocompatibility complex haplotypes are recognized. AB - We have previously shown that large granular lymphocyte (LGL)-enriched cell populations have the capacity to spontaneously recognize and kill allogeneic small lymphocytes and bone marrow cells (BMC) in vitro in certain strain combinations of rats. Here, we have studied the alloreactivity of natural killer (NK) cells from PVG nude (RT1c) rats against a panel of major histocompatibility complex (MHC) incompatible hemic cells. Both lymphocytes and BMC from the AO (RT1u), DA (RT1a), BN (RT1n) as well as the MHC-congenic PVG-RT1u (RT1u) rat strains were efficiently killed in vitro, whereas cells from syngeneic PVG rats were spared. The structures recognized on lymphocytes and BMC were probably similar since the two cell populations inhibited each other in cross-competition experiments. A number of features aligned the alloreactive effector cells with NK cells and not T cells. (a) Only about 5% of the effector cells from nude spleens expressed the T cell antigens CD3, CD5 or T cell receptor (TcR) alpha/beta whereas greater than 50% of the cells expressed markers present on NK cells (CD2, CD8, OX52 and the rat NK cell-specific marker NKR-P1 recognized by the monoclonal antibody 3.2.3). (b) The alloreactive cells were granular since pretreatment of nude spleen cells with the lysosomotropic agent L-leucine methyl ester which eliminated LGL, simultaneously abolished the cytolysis of both allogeneic lymphocytes and YAC-1 tumor cells. (c) Nude spleen cells stimulated with human recombinant interleukin 2 for 1 week in vitro generated large granular proliferating cells which were CD3-, CD5-, TcR alpha/beta-, but greater than 95% 3.2.3+. These cells efficiently killed allogeneic hemic cells from the same rat strains as did freshly isolated effector cells. (d) The cytolysis of allogeneic hemic cells could effectively be inhibited with unlabelled NK-sensitive (YAC-1 and K-562), but not NK-resistant (Roser leukemia) tumor cells. Cross-competition studies showed that PVG nude NK cells discriminated between AO, BN and DA BMC, suggesting that different alloantigens were positively recognized by subsets of NK cells. The mode of inheritance of the allodeterminant specifically recognized on AO BMC was investigated in crosses and backcrosses between AO and BN or DA rats. A gene dosage effect was observed in that this determinant was expressed at a slightly reduced level in F1 hybrids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716213 TI - Involvement of both T cell receptor V alpha and V beta variable region domains and alpha chain junctional region in viral antigen recognition. AB - We have studied the lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T cell response in transgenic mice expressing either the T cell receptor (TcR) alpha (V alpha 2/J alpha TA31) or the corresponding TcR beta (V beta 8.1/D beta/J beta 2.4) chain originally isolated from the LCMV glycoprotein specific (residues 32-42), H-2Db-restricted T cell clone P14. The expression of single transgenic TcR chains did not influence the corresponding endogenous TcR V gene usage in unstimulated T cells indicating that one particular TcR alpha or beta chain can randomly pair with different V beta or V alpha chains without any obvious bias. However, upon infection with LCMV, reactive cytotoxic T lymphocytes (CTL) from P14 beta-transgenic mice were predominantly V alpha 2+ whereas CTL from P14 alpha transgenic mice preferentially expressed V beta 8.1 and unexpectedly also V beta 8.3 (but not V beta 8.2). Correspondingly, the LCMV-specific CTL response in both alpha and beta TcR-transgenic mice was strongly biased to the original P14 T cell epitope (LCMV glycoprotein residues 32-42). Sequence analysis of a large panel of LCMV-reactive "half-transgenic" TcR from P14 single receptor chain-transgenic mice revealed a highly conserved VJ alpha and a more diverse VDJ beta junctional region. This report demonstrates that the antigen specificity of the studied TcR depends on the specific combination of both TcR alpha and beta chains which implies that amino acids located in the TcR V alpha and V beta segments as well as in the junctional region are involved in binding of the viral antigenic fragment. PMID- 1716214 TI - Distinction of naive and memory BoCD4 lymphocytes in calves with a monoclonal antibody, CC76, to a restricted determinant of the bovine leukocyte-common antigen, CD45. AB - A murine IgG1 monoclonal antibody (mAb), CC76, has been produced that, based on findings of the relative molecular mass of polypeptides that it recognized, staining of leukocytes in blood and tissues, and the biological properties of the T lymphocyte subpopulations with which it reacts, is considered to identify an isoform of the leukocyte common antigen (LCA) family of molecules in cattle. The mAb is more similar to human CD45R which detect products requiring the presence of the B exon within the LCA gene and to the anti-rat mAb MRC-OX22, than to CD45RA or CD45R0. mAb CC76 reacts with an antigen expressed by subpopulations of cells in bovine blood that express BoCD2 and either the BoCD4 or BoCD8 antigens. T cells that express the gamma/delta T cell receptor identified with mAb to BoWC1 antigen did not react with CC76. The molecule detected is expressed on B cells but not on monocytes or granulocytes. Only 2% of cells in thymic suspensions stained with mAb CC76. Immature cortical thymocytes that were BoCD1+ did not react with CC76 and 90% of the cells in thymic suspensions that were CC76+ had the phenotype of mature thymocytes. These cells were primarily in the medulla. The LCA isoform detected thus appears to be acquired by mature cells shortly before emigration from the thymic medulla into the periphery. Expression of the molecule detected by mAb CC76 on cells from lymph nodes was similar to that in blood, but expression on cells from the gut mucosa was quite different. Almost all, 95% and 93% respectively, of the BoCD4+ cells in the gut mucosa or discrete Peyer's patches were CC76-. A greater proportion of BoCD8+ cells from these sites, 35% and 26%, expressed the antigen. Lymphocytes from animals that had been immunized with Trypanosoma brucei were sorted into BoCD4+, CC76+ and BoCD4+, CC76 populations and cultured in vitro with the variable surface glycoprotein antigen from the parasite. Lymphocyte transformation responses were entirely within the CC76- population indicating that the mAb distinguished naive from memory BoCD4+ T cells in cattle. Major histocompatibility complex (MHC) class I-restricted cytotoxic precursor cells that expressed the BoCD8 antigen sorted from cattle that were immune to Theileria parva were both CC76+ and CC76- indicating that different isoforms of the LCA may be expressed on MHC class I- and class II restricted memory cells and that BoCD8 memory cells are heterogeneous with respect to the LCA isoform that they express. PMID- 1716215 TI - Resistance of leukemic blasts to lymphokine activated killer (LAK)-mediated cytotoxicity is not related to their adhesion properties. AB - The expression of adhesion molecules on blasts from 14 patients with acute myeloid leukemia (AML) was investigated by immunofluorescence and flow cytofluorometry. All tested blast populations expressed CD18/CD11a complex [leukocyte function antigen-1 (LFA-1)] and CD29 (very-late antigen (VLA)) and the majority were positive for CD54 [intercellular adhesion molecule-1 (ICAM-1), 78.6%] and CD56 [neural cell adhesion molecule (NCAM), 64.3%]. The expression of two other alpha chains of CD18/CD11b and CD11c varied considerably (64.3% and 42.8% of positive cases, respectively). Only one case (AML-M4) showed a weak expression of the activated platelet antigen CD41b. None of the tested blasts expressed the vitronectin receptor (CD61/CD51). No significant correlation between the expression of adhesion molecules and the FAB type of leukemia could be found. All tested blast populations were completely resistant to NK-mediated cytotoxicity and relatively resistant to LAK-mediated cytotoxicity in the standard 51Cr release assay. While no statistically significant correlation of the results in cytotoxicity assays with the expression of adhesion molecules or the expression of HLA-DR antigen could be observed, 2 out of 3 completely resistant cases lacked ICAM-1. These results show that even leukemic blasts which express all of the tested adhesion molecules can still be resistant to LAK mediated cytotoxicity. PMID- 1716216 TI - Constitutive production of granulocyte colony-stimulating factor and interleukin 6 by a human lung cancer cell line, KSNY: gene amplification and increased mRNA stability. AB - A human lung squamous cell carcinoma cell line designated KSNY was established from a patient suffering from marked neutrophilia and polyclonal hyper-gamma globulinemia. In our previous report, we demonstrated colony-stimulating activities in the culture supernatant of this cell line. To determine the exact molecules for the activities, we investigated the gene expression of various cytokines in KSNY cells and showed the mRNA expression of both granulocyte colony stimulating factor (G-CSF) and interleukin-6 (IL-6). We also detected substantial amounts of G-CSF and IL-6 in the culture supernatant with sensitive enzyme-linked immunosorbent assays (ELISA). The amplification of the gene locus for G-CSF, but not for IL-6 was shown by Southern blot analysis. Furthermore, we also showed that the mRNAs for G-CSF and IL-6 were relatively stable in KSNY cells. These findings are thought to be related to the constitutive production of the cytokines in KSNY cells. PMID- 1716217 TI - Substance P immunoreactive nerves in airways from asthmatics and nonasthmatics. AB - Substance P has been localized to nerves supplying smooth muscle, blood vessels and glands in the human lung and may play a major role in the pathophysiology of asthma. We performed a morphological study, using the avidin biotin peroxidase immunostaining technique, to examine sections of airway wall from subjects with and without asthma for the presence of substance P immunoreactive nerve fibres. Airways of 200 microns-12 mm were obtained from autopsy, lobectomy and bronchoscopy. Quantitative morphological analysis was performed on 3 mm diameter airways from three asthmatic and three nonasthmatic subjects collected at autopsy, and on biopsies of 10 mm diameter airways from eight asthmatic and thirteen nonasthematic subjects. There was an increase in both the number and the length of substance P immunoreactive nerve fibres, in airways from subjects with asthma when compared with airways from subjects without asthma. Fibres were found in the lamina propria and surrounding vessels and glands. The fibres were commonly seen as bundles rather than as single fibres. There was no difference in the number of substance P nerves between normal subjects and subjects with chronic airflow limitation (CAL). The difference in the number, length and morphological characteristics of the substance P immunoreactive nerves between asthmatic and nonasthmatic subjects were striking. PMID- 1716218 TI - Site-specific antibodies as probes of the structure and function of the brain D2 dopamine receptor. PMID- 1716219 TI - Protein synthesis in the lungs of young rats in vivo and its nutritional modification. PMID- 1716220 TI - Biochemical subfractionation of retrogradely transported proteins from rat sciatic nerve. PMID- 1716221 TI - Fluo-3 an ideal calcium indicator for measuring calcium fluxes in SR and ER. PMID- 1716222 TI - Phosphoproteins of dentine extracellular matrix: silver colloid staining. PMID- 1716223 TI - Detection by PCR of thyroid hormone receptor transcripts in Xenopus oocytes and eggs. PMID- 1716224 TI - Regulation of Ca2+ mobilization by Ins (1,4,5)P3 and intraluminal Ca2+ in permeabilized hepatocytes. PMID- 1716225 TI - The acute phase protein response in Crohn's colitis. PMID- 1716226 TI - Recombinant human granulocyte colony stimulating factor (rhG-CSF) and superoxide production in a haematopoietic cell line. PMID- 1716227 TI - Role of oncogenes in the regulation of MHC antigen expression. AB - Class I and class II major histocompatibility complex (MHC) antigens are required for CD8+ cytotoxic T cells and CD4+ helper T-cells, respectively, to recognize foreign antigen. Regulating the levels of expression of these MHC antigens regulates the T-cell responses [1]. This regulation is mainly carried out by the interferons (IFN), which are produced in the disease state. Type I IFN (IFN alpha or IFN beta; collectively 'IFN alpha beta) up-regulates class I MHC and IFN gamma up-regulates class I and class II MHC. We and others [1-3] have shown that transfection of cells with a variety of oncogenes including ras and myc affects the level of MHC antigen expression. This and other data provide evidence for a scheme in which the signal transduction mechanisms whereby IFN up-regulates MHC antigens involve several (proto) oncogenes. PMID- 1716228 TI - Control of proliferation by myeloid growth factors. AB - At present the molecular events which regulate the proliferation and developmental fates of haemopoietic cells are poorly understood. Until recently, the only receptor for the myeloid growth factors which had been characterized extensively was that for M-CSF (c-fms). The molecular cloning of receptors for IL 3, GM-CSF and G-CSF should now permit rapid progress in the analysis of receptor mediated haemopoietic cell differentiation and development, and should also reveal how the process of leukaemic transformation effects these events. PMID- 1716229 TI - Stretch and force generation induce rapid hypertrophy and myosin isoform gene switching in adult skeletal muscle. AB - Using electrical stimulation to control force generation and limb immobilization to alter the degree of stretch, we have studied the role of mechanical activity in inducing hypertrophy and in determining fast and slow muscle fibre phenotype. Changes in gene expression were detected by analysing the RNA in hybridization studies employing cDNA probes specific for fast and slow myosin heavy chains and other genes. As a result of overload in the stretched position, the fast contracting tibialis anterior muscle in an adult rabbit is induced to synthesize much new protein and to grow by as much as 30% within a period as short as 4 days. This very rapid hypertrophy was found to be associated with an increase of up to 250% in the RNA content of the muscles and an abrupt change in the species of RNA produced. Both stretch alone and electrical stimulation alone caused repression of the fast-type genes and activation of the slow-type genes. it appears that the fast-type IIB genes are the default genes, but that the skeletal slow genes are expressed as a response to overload and stretch. These findings have implications as far as athletic training and rehabilitation are concerned. PMID- 1716230 TI - Regulation of myelin basic protein gene transcription in glial cells. PMID- 1716231 TI - Immunohistochemical study of colorectal adenocarcinomas and adenomas with antibodies against carcinoembryonic antigen (CEA), CA19-9, keratin, alpha-tubulin and secretory component (SC). AB - The immunohistochemical localization of five antibodies against carcinoembryonic antigen (CEA), CA19-9, keratin, alpha-tubulin and secretory component (SC) was investigated in 14 lesions of adenocarcinoma (AC), 22 of adenoma with high-grade atypia (AH), 50 of adenoma with low-grade atypia (AL), and 15 of non-neoplastic mucosa (NNM) of the large intestine. The positive patterns for each staining were divided into three categories (patterns 1, 2, and 3). All neoplastic lesions (AC, AH and AL) were positive for CEA, while 85.7% of AC, 36.4% of AH and 6.0% of AL showed strongly positive staining (pattern 3). 78.6% of AC and 54.5% of AH were positive for CA19-9 in comparison to 20.0% of AL. For keratin, more than 95% of the neoplastic lesions were positive, while 78.6% of AC, 27.3% of AH and 22.0% of AL showed strongly positive staining (pattern 3). For alpha-tubulin, more than 85% of neoplastic lesions were positive, while 50.0% of AC, 36.3% of AH and 26.0% of AL showed strongly positive staining (pattern 3). For SC, in contrast, 42.9% of AC, 27.3% of AH and 8.0% of AL were negative, but 93.3% of NNM were positive. It was concluded that the positive staining rate, especially the rate of pattern 3 for each antibody correlated with the degree of atypia of the colorectal neoplastic lesions (AC, AH and AL). PMID- 1716232 TI - Serological and immunohistochemical studies on sialylated carbohydrate antigens in colorectal carcinoma. AB - Sialylated carbohydrate antigens, such as CA19-9 (sialyl Lea), CA-50 (sialyl Le4), CSLEX1 (sialyl Lex) and SLX (sialyl Lex-i), were assayed in the same preoperative serum samples of 63 patients with colorectal cancer, and compared with CEA. In addition immunohistochemical expressions of sialyl Lea, sialyl lex and sialyl Lex-i antigens were studied in 62 colorectal carcinomas and 42 normal mucosal sites remote from the malignant lesion using monoclonal antibodies CSLEA1, CSLEX1 and FH-6, respectively, in order to elucidate their tumor specificity and clinical usefulness as a tumor-associated antigen. Serologically, the percent positive rates of CA19-9, CA-50, CSLEX1, SLX and CEA were 30.2%, 17.7%, 23.8%, 16.1% and 44.4%, respectively. In dukes' A and B, these sialylated carbohydrate antigens, especially CSLEX1 and SLX, showed low positive rates, but the percent positive rates of CSLEX1 and SLX correlated with operative radicality. The positive spectrum of CSLEX1 differed from that of CA19-9 in sera, and CEA had no correlation with these two antigens. The immunohistochemical expression rates of sialyl Lea, sialyl Lex and sialyl Lex-i were 88.1%, 17.0% and 9.5% in normal mucosa, but were 77.8%, 90.5% and 71.4% in carcinoma, respectively. These data suggested that the type 2 chain antigens CSLEX1 and SLX, which have high tumor-specificity compared with CA19-9, may be useful in preoperative diagnosis for extension of carcinoma and operative radicality, although early diagnosis using these sialylated carbohydrate antigens may be difficult, while the combined use of CA19-9, CSLEX1 and CEA should make it possible to detect a wide range of colorectal cancer patients. PMID- 1716233 TI - Histomorphological study on the minor duodenal papilla. AB - The morphological characteristics of the minor duodenal papilla were studied histologically using surgically resected specimens according to the presence or absence of an opening of the accessory pancreatic duct in 30 patients (22 with a ductal opening and 8 without such an opening) who had undergone pancreatoduodenectomy within the past 4 years. The following results were found: 1) The minor duodenal papilla was composed fundamentally of the accessory pancreatic duct, pancreatic tissue of the dorsal pancreas which penetrated the duodenal proper muscular tunics and the surrounding fibrous connective tissue. 2) In those specimens with a ductal opening, smooth muscle fiber bundles derived from the duodenal proper muscular tunics surrounded the accessory pancreatic duct and seemed to possess sphincter action similar to that of Oddi sphincter muscles. 3) In those specimens without a ductal opening, the accessory pancreatic duct terminated blindly in the minor duodenal papilla which was comprised mostly of pancreatic tissue and seemed to have no sphincter muscles. 4) Islet cells in the pancreatic tissue of the minor duodenal papilla were rich in B-cells which were round to ovoid in shape and sharply outlined. PMID- 1716234 TI - Effects of omeprazole on gastric mucus glycoprotein biosynthesis. PMID- 1716235 TI - A modified argyrophilic nucleolar organiser region (AgNOR) staining method for nucleolar staining in formalin-fixed, paraffin-embedded materials. PMID- 1716236 TI - A new technique for thin sections of the gallstones; microscopic detection of mucopolysaccharides in gallstone. PMID- 1716237 TI - Sporadic vs. posttransfusion hepatitis C: why the differences? PMID- 1716238 TI - Bidirectional communication between mast cells and nerves controls intestinal secretion. PMID- 1716239 TI - [Fetomaternal transfusions in complicated and uncomplicated pregnancy]. AB - Fetomaternal bleeding in pregnancy is the most common cause of rhesus immunization. In this study we evaluated the amount of fetomaternal bleeding during pregnancies with and without complications. The data from 1204 patients are analyzed. Fetomaternal bleeding was of clinical relevance (HbF greater than 0.01%) in 6% of all uncomplicated pregnancies. There was no increased fetomaternal bleeding in pregnancies complicated by gestosis, preliminary labour, placenta praevia, trauma, and diabetes in pregnancy. In cases with premature rupture of the amnion or vaginal bleeding in pregnancy we observed an increased percentage of fetomaternal bleeding into the mother's circulation. Nearly 25% of all patients with hydrops fetalis had clinical relevant fetomaternal bleeding (HbF greater than 0.01%). PMID- 1716240 TI - [Systemic therapy in recurrent and primary advanced cervix cancer]. AB - Therapeutic results in advanced cervical cancer have not been showing any progress for more than 30 years. Only by means of systemic therapy does it seem possible, that further treatment results may be obtained similar to those in squamous cell carcinoma of the head and neck. Therefore, between 1985 and 1990 we tried to apply an aggressive polychemotherapy regimen for 5 days with cisplatinum and 5-fluorouracil with supportive treatment in 47 patients with either primary advanced inoperable (n = 25) or recurrent (n = 22) cervix carcinomas. Complete (n = 12) and partial remissions (n = 24) could be obtained in 36 out of 47 cases (= 77%). However, this high response rate conceals the fact, that this aggressive palliative treatment in case of recurrence and/or distant metastases means a considerable deterioration of quality of life with a median survival time of only 9.5 months. Although primary therapy with combined chemoradiotherapy shows good results, it is, however, only a real advantage for patients with complete response. Taking into account the short period of observation (median 25 months), the overall survival of all primary advanced cervix carcinomas is 13 + months, in case of complete remission 20 + months. It remains to be seen, whether sequential or simultaneous chemoradiotherapy will prove superior. Due to severe haematoxicity (WHO grade IV) after the first 3 treated patients, a dose reduction of 20% of chemotherapy was necessary. No further severe toxicities occurred. By means of supportive care, other side effects could also be well controlled, so that this effective polychemotherapy regimen is not only efficacious but also practicable. Prospective randomised studies are required to determine the importance of exclusive or combined chemoradiotherapy in advanced as well as in operable cancer of the cervix. PMID- 1716241 TI - [Final comment on discussion comments by H. Alfthan and U. H. Stenman, "Beta-HCG in serum of females with normal pregnancy is not an artefact"]. PMID- 1716242 TI - Identification of an unusual structure in the Drosophila melanogaster transposable element copia: evidence for copia transposition through an RNA intermediate. AB - The Drosophila melanogaster transposable element copia is usually 5 kb long with long terminal repeats (LTRs), and its major transcripts are a full-length 5-kb RNA and a 2-kb RNA. We have previously shown that the 2-kb RNA is generated through splicing. Here, we have cloned a genomic intronless copia using an oligodeoxyribonucleotide probe which is specific for the junction of the two exons. The unusual copia is bounded by two LTRs and lacks precisely the intron of the 2-kb copia RNA. Identification of genomic intronless copia strongly suggests that copia transposes through an RNA intermediate. Moreover, we have found that copia virus-like particles (VLPs), in which reverse transcription of copia RNA seems likely to occur, packages the spliced copia RNA much less efficiently than the full-length copia RNA. This result leads to the suggestion that much lower copy number of genomic intronless copia, as compared with that of 'normal' copia, may be responsible for the inefficient packaging of the spliced copia RNA into the VLP. PMID- 1716243 TI - Molecular cloning and sequence analysis of the murine cDNA for the cystic fibrosis transmembrane conductance regulator. AB - We have cloned the mouse homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR) using clones isolated from a mouse lung cDNA library and using amplification of cDNA to isolate specific regions. The cDNA was 6304 bp in length and encoded a polypeptide of 1476 amino acids. Comparison of the deduced amino acid sequence showed that the mouse protein has high homology to the human protein; overall identity was 78.3%. The amino acid identity was high for both transmembrane domains (first transmembrane domain, 86.7%; second transmembrane domain, 81.1%) and for both ATP-binding folds (first ATP-binding fold, 80.5%; second ATP-binding fold, 83.9%), suggesting the functional importance of these regions. On the other hand, the R domain was less well conserved (68.9% identity). All of the published missense mutation sites and the site of the common delta F508 mutation were conserved between human and mouse. PMID- 1716244 TI - CA/GT microsatellite alleles within the cystic fibrosis transmembrane conductance regulator (CFTR) gene are not generated by unequal crossingover. AB - The gene responsible for cystic fibrosis (CF) has recently been identified, and a three-nucleotide deletion (delta F508 mutation) that results in the loss of a phenylalanine residue in the first putative ATP-binding domain of the predicted protein (CF transmembrane conductance regulator, CFTR) has been found to be the major CF mutation. Although several other mutations have been identified in the CFTR gene, most of them are very rare, making their application to genetic diagnosis difficult. While characterizing the genomic region encompassing the CF locus, we have identified three CA/GT blocks that flank exon 9 of the CF gene. One of the CA/GT blocks exhibits a highly informative variable number of dinucleotide repeats (VNDR) polymorphism. This intragenic VNDR microsatellite should, by itself, provide full information for genetic analysis in approximately 80% of CF families and will help elucidate the associations between DNA polymorphism haplotypes and specific gene mutations. Haplotype analyses of CF chromosomes with and without the delta F508 mutation suggest that the different alleles are generated by slipped-strand mispairing within the dinucleotide repeat during DNA replication, rather than by unequal crossingover within a recombination hot spot. PMID- 1716245 TI - Effect of ricin on acute phase response in rats. AB - Ricin, a highly toxic protein from castor beans was administered (ip) to rats in a dose of 1.25 micrograms/100 g to selectively deplete at least 60-70% of Kupffer cells. This dose spared hepatocytes. This rat model was used to study acute phase protein synthesis and the role of Kupffer cells in acute phase response (APR). Ricin itself induced an APR, similar in pattern but of lower magnitude, than that induced by turpentine. However, the effect of combination of ricin and turpentine on APR was not additive. Kupffer cells appear to play permissive role in APR through mediators like hepatocytes stimulating factors. PMID- 1716246 TI - Transurethral resection of very large prostates. A retrospective study. AB - Twenty-one patients with benign prostatic hypertrophy (BPH), and a weight of transurethrally resected tissue exceeding 80 g (Group 1), were compared to a control group of 30 patients with a weight of resected tissue less than 80 g (Group 2) with regard to the peri- and postoperative course and the symptomatic and urodynamic results of surgery. All patients were followed 12 months postoperatively. In both groups more than 90% of the patients were satisfied with the results of the operation. However, the obstructive symptoms were better relieved than the irritative symptoms. The group who had large resections performed had a longer operating time and a greater perioperative blood loss than the group of minor resections. No differences were found with regard to other peri- or postoperative complications or subjective results. Transurethral resection is safe and efficient in treating BPH, also with very large prostates. PMID- 1716247 TI - Gross haematuria in a Negro population: an analysis of 100 adult cases. AB - One hundred consecutive cases of gross haematuria seen at a Nigerian Urologic Clinic are analysed. The causes were identified in 95 cases. Benign prostatic hypertrophy was the commonest cause accounting for 27% of the cases, followed by trauma and infection. Schistosomiasis was an uncommon cause. The pattern of the distribution of aetiological factors and valuable investigations are discussed. PMID- 1716248 TI - Why palliative medicine? PMID- 1716249 TI - Palliative care in the 1990s: special issues. PMID- 1716250 TI - Immunobiologic aspects of head and neck cancer. Clinical and laboratory correlates. AB - The role of the immune system in the pathogenesis and treatment of cancer remains to be determined. Impairment of humoral and cellular immunity has been associated with various factors implicated in head and neck cancer--tobacco, alcohol, and malnutrition. Whether these immune dysfunctions are causative or a secondary effect is not known. Patients with head and neck cancer have well-documented defects in immune function. In general, depression in cellular immunity has been noted, which can be correlated with stage of disease and prognosis. Conversely, increased humoral immune responses are seen, especially salivary IgA and circulating immune complexes. It may be that IgA or immune complexes are capable of suppressing the cellular immune response. Clinical trials utilizing biologic response modifiers in head and neck cancer have begun. Preliminary studies with heavily pretreated patients have demonstrated antitumor activity, suggesting that immunotherapy may provide a treatment alternative. In conclusion, the weight of evidence suggests that the immune response to head and neck cancer does play a role in the pathogenesis of the disease. A better understanding of the host-tumor interaction will allow for improved utilization of biologic response modifiers in the treatment of head and neck cancer. PMID- 1716251 TI - Heparin-binding (fibroblast) growth factors are potential autocrine regulators of esophageal epithelial cell proliferation. AB - A serum-free culture system supplemented with neural tissue extract for normal and tumor human esophagi was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life span but retained morphology and other characteristics of normal epithelial cells. The cells formed a small cyst consisting of keratinized squamous epithelium in syngenic hosts. A screen for growth factors that stimulated growth of the nonmalignant MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed large invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 and proliferating nonmalignant MEE cells had properties similar to HBGF-1 (acidic fibroblast growth factor). These results constitute a cultured mouse esophageal epithelial cell model for study of conversion of immortalized premalignant cells to malignant cells, and suggest that conversion from a state of cell cycle dependent autocrine expression of one or more members of the HBGF family to a state of constitutive expression correlates with and may contribute to malignancy. PMID- 1716252 TI - Close similarity between cultured human omental mesothelial cells and endothelial cells in cytochemical markers and plasminogen activator production. AB - The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments. The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor 1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-Pa may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells. PMID- 1716253 TI - Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa. AB - Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factor beta-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13 acetate or an elevated Ca2+ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. PMID- 1716254 TI - Comparative growth of normal and malignant mouse mammary epithelium cultured serum-free on a biomatrix from preadipocytes. AB - Growth of normal and malignant mouse mammary epithelial cells (MMEC) on a biomatrix of substrate-attached material from 3T3-L1 preadipocytes was evaluated to devise culture conditions that are suitable for transformation studies but do not involve embedding cells in a gel. The biomatrix was prepared as described by Levine and Stockdale, and serum-free medium contained bovine serum albumin, insulin, progesterone, prolactin, and linoleic acid. Each cell type produced a distinctive pattern of colony architecture in this culture system. Cells from virgin mice (vMMEC) usually formed elaborate, three-dimensional structures resembling ducts and alveoli; cells from pregnant mice (pMMEC) grew as flat monolayers; and tumor cells grew in multilayered clusters. Cell growth was monitored by an assay for succinate dehydrogenase. Similar growth rates were found through Day 8 in cultures of vMMEC and D2 carcinoma cells. Growth of vMMEC slowed thereafter, whereas tumor cells typically continued growing through Day 14 to 18. Increase in cell number during 18 days in culture was 3-, 7-, 9-, and 11 fold for cells from pregnant and virgin mice, BALB/cfC3H and D2 carcinomas, respectively. The percent cells in S phase on Day 2 of culture was 9% for pMMEC, 4 to 11% for BALB/cfC3H tumor cells, 20% for vMMEC, and 24% for D2 tumor cells. Thus, this culture system promotes extended growth of MMEC and offers several advantages over embedding cells in a collagen gel. It may therefore be applicable to in vitro transformation studies with MMEC. PMID- 1716255 TI - Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium. AB - We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells. PMID- 1716256 TI - Induction of specific T-cell responsiveness to allogeneic bone. AB - The capacity of fresh murine allogeneic bone to induce a specific immune response in vitro was studied. T-cells stimulated by allogeneic bone in vitro were collected and were characterized for state of activation, cell-surface phenotype, and antigen specificity. The stimulating antigens were determined by genetic mapping with use of recombinant inbred strains of mice and by blocking of mixed lymphocyte cultures with use of neutralizing antibodies. Purified T-cells were cultured alone or with allogeneic or syngeneic bone. In some experiments, the bone marrow was removed before in vitro culture. Responding cells were recovered after a secondary exposure to the stimulating bone. Primed cells were used immediately or cell-lines were developed. The data demonstrated that (1) allogeneic bone activated T-cells and induced their proliferation; (2) bone induced proliferation of T-cells was specific for antigens that map to the major histocompatibility complex of the bone donor; (3) within the major histocompatibility complex, the antigens responsible for proliferation of T-cells were apparently class-I and class-II determinants; (4) removal of bone-marrow cells had no effect on the ability of that bone to stimulate alloreactivity; and (5) all of the alloreactive T-cells had the cell-surface phenotype Thy-+ CD8+ CD4 . PMID- 1716257 TI - Three-dimensional observations on microvascular growth in rat glioma using a vascular casting method. AB - The microvascular growth of ethylnitrosourea-induced rat glioma was observed using vascular casting and scanning electron microscopy (SEM). Light microscopy showed central necrosis and marginal invasive tumor cell growth with increased vascularity, and suggested that adopted pre-existing circulation was dominant in the inoculated brain tumors. In SEM, numerous buds or nodular protrusions and a few large and tortuous vessels along the tumor margin were seen at the early stage. In the intermediate stage, microaneurysms, buds with septum formation and anastomotic arches appeared, and these tumor vessels became more tortuous and larger, and extended as the tumor grew. Several "potato-shaped" huge vessels and linear nodular large vessels also appeared. In the late stage, glomeruli appeared and potato-shaped huge vessels increased in number. The neovascularization and microvascular growth of the tumors was characterized by three patterns: (a) growth of the parent vessels forming buds, (b) vascular growth in a meshwork formation producing glomeruli, and (c) vascular enlargement without a definite pattern creating potato-shaped huge vessels. The tumor vessels gradually lost their natural three-dimensional structure. PMID- 1716258 TI - A research for the relationship between human papillomavirus and human uterine cervical carcinoma. II. Molecular genetic and ultrastructural study on the transforming activity of recombinant retrovirus containing human papillomavirus type 16 subgenomic sequences. AB - In order to elucidate the role of HPV-16 in the development of genital cancer, NIH3T3 cells were transfected by HPV-16 whole genome and its two early genes, E6 E7. Besides ordinary calcium phosphate/DNA coprecipitation technique, a newly designed recombinant retrovirus containing the HPV-16 genome or subgenomes was used to infect cells for transfer of the target genes. The transforming activities have been demonstrated to be most efficient when a bioengineering technique of this kind is used. HPV-16 DNA was proved to have transforming potential for NIH3T3 cells, and the DNA of HPV-16 was proved to undergo multisite integration into transformed cells and nude mice tumour cells. The E6-E7 open reading frames are sufficient for transforming NIH3T3 cells independently in vitro, which implies that E6-E7 open reading frames are transforming genes or even viral oncogenes of HPV-16. The RNA transcribed by the E6-E7 of HPV-16 was expressed in transformed cells and in tumour cells of nude mice. The use of a recombinant retrovirus for gene transfer in this study is much more efficient than that of calcium phosphate/DNA coprecipitation. The lack of a tissue-culture system suitable for HPV replication in vitro makes HPV gene recombination into a specially engineered retrovirus for viral-mediated gene transfer of particular significance for the possible application of viral carcinogenesis, both in vitro and in vivo, for basic and clinical research. PMID- 1716259 TI - Impaired formation of the ternary insulin-like growth factor-binding protein complex in patients with hypoglycemia due to nonislet cell tumors. AB - In some subjects with hypoglycemia associated with tumors of mesenchymal origin, high insulin-like growth factor-II (IGF-II) levels have been described in serum and in the tumors. Tumor IGF-II of 10-15 kDa circulates in a 60-kDa complex, in contrast to the ternary 150-kDa complex in which serum IGFs normally circulate together with the IGF-binding subunit (IGFBP-3) and the acid-labile subunit (alpha-subunit). This study examines the molecular distribution and complex forming activity of the components of the ternary complex in the serum of subjects with mesenchymal tumor hypoglycemia. Total serum IGFBP-3 levels were 60% of normal in tumor patients and appeared at 60 kDa on gel chromatography, shifting after tumor removal to 150 kDa. Total alpha-subunit levels were 40% of normal in patients with tumors, increasing after tumor removal to 70% of normal and changing in elution profile from a peak typical of uncomplexed alpha-subunit to the normal broad peak representing both complexed and uncomplexed alpha subunit. Although low by RIA, alpha-subunit activity in a ternary complex formation assay was normal, indicating that the ability of free alpha-subunit in the patients' circulation to combine with exogenous IGFBP-3 plus IGF-I was not impaired. In contrast, in an assay that tested the ability of IGF-IGFBP complexes in the patients' circulation to combine with pure alpha-subunit, complex formation activity was 75-85% below normal in preoperative sera, despite low normal IGFBP-3 levels. Therefore, the cause of hypoglycemia in these patients may be the inability of complexes between the abnormal tumor IGF-II and IGFBP-3 to be sequestered in the biologically inactive ternary complex. PMID- 1716260 TI - Variables controlling the secretion of insulin-like growth factor binding protein 2 in normal human subjects. AB - Insulin-like growth factor binding protein-2 (IGFBP-2) is one of a family of IGFBPs that are present in extracellular fluids, and binds both IGF-II and IGF-I with high affinity. These studies were conducted to determine the nutritional and hormonal variables that regulate plasma IGFBP-2 concentrations in humans. The mean plasma IGFBP-2 concentration for 38 normal adult subjects was 150 +/- 61 micrograms/L and was 4.7-fold greater than their mean fasting IGFBP-1 value. Mean IGFBP-2 values in cord sera of 26 normal term infants was 3.8-fold greater than the normal adult mean value. Likewise, the mean value for 44 hypopituitary adults was increased 2-fold compared to normal. There was no suppression of IGFBP-2 values in acromegaly. Normal adult subjects showed minimal fluctuations (less than 2-fold changes) in plasma IGFBP-2 concentrations during a 48-h sampling period. These changes were significantly less than the changes that occurred in plasma IGFBP-1 during the same interval. Plasma IGFBP-2 did not change significantly post prandially or after a glucose infusion. Extreme insulin deficiency, after 9 days of fasting, was associated with a 1.7-fold increase in plasma IGFBP-2. Administration of GH, which is known to cause a major decrease in plasma IGFBP-1 and in IGFBP-2 in hypophysectomized animals, did not result in a change in calorically restricted normal adult subjects, suggesting that a normal caloric intake is required for GH to suppress IGFBP-2. In summary, these results show that plasma IGFBP-2 is regulated differently than IGFBP-1. Acute stimulation of insulin secretion does not suppress IGFBP-2, and there is much less daily fluctuation compared to IGFBP-1. These findings suggest that plasma IGFBP-2 levels are more stable than IGFBP-1, and therefore IGFBP-2 may serve as a larger reservior that is available for IGF transport. PMID- 1716261 TI - Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector. AB - The sequential epitopes on the human thyroperoxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease were investigated using a recombinant DNA technique. Previous studies led to the isolation of two overlapping cDNA clones that encode polypeptides of TPO (85 residues, C2; 100 residues, C21) recognized by sera from several patients with autoimmune disease that contained antimicrosomal autoantibodies. In this report the vector pUEX1 was used to clone and express small random fragments of TPO cDNA in Escherichia coli as a beta-galactosidase fusion protein. Colonies were screened with a serum from a patient with Hashimoto's thyroiditis, and immunoreactive peptides were identified by sequencing the corresponding DNA inserts. Two linear epitopes of human TPO (amino acids 590-622 and 710-722) were recognized by the autoantibodies. This confirmed our previous results and provide a more precise localization of the antigenic determinants involved. The same approach has been applied in an attempt to identify the binding site(s) for autoantibodies on the human TSH receptor. In contrast to the data obtained with TPO, sera from patients with blocking (from idiopathic myxoedema) or stimulating (from Graves' disease) activity did not recognize the linear TSH receptor peptide fragments generated in our libraries. PMID- 1716262 TI - Determination at the molecular level of a B-cell epitope on thyroid peroxidase likely to be associated with autoimmune thyroid disease. AB - In a panel of 13 mouse monoclonal antibodies generated against native (nondenatured) human thyroid peroxidase (TPO), only 1 (monoclonal antibody 47) recognized TPO protein fragments expressed in a human TPO cDNA sublibrary. Determination of the nucleotide sequences of 18 clones recognized by monoclonal antibody 47 localized its epitope to 9 amino acids (residues 713-721) in the human TPO protein. On Western blot analysis, only TPO monoclonal antibody 47 recognized the 933-amino acid TPO molecule after denaturation and reduction of the latter, supporting the concept that the major part of the epitope is represented by a continuous portion of the TPO sequence. The binding of TPO monoclonal antibody 47 to native TPO is inhibited by immunoglobulin G in the serum of patients with autoimmune thyroid disease. The epitope for monoclonal antibody 47 defined in the present study is, therefore, part of or in the vicinity of an epitope for autoimmune thyroid disease-associated TPO antibodies. PMID- 1716263 TI - Intravenous gamma-globulin in myasthenia gravis: interaction with anti acetylcholine receptor autoantibodies. AB - Clinical improvement has been observed in myasthenia gravis patients treated by intravenous immunoglobulin (IVIg). In order to investigate the mechanism of action of these IVIg, we looked for an in vitro interaction between IVIg and the anti-acetylcholine receptor autoantibodies. Significant inhibition by IVIg of anti-acetylcholine receptor autoantibody activity from 30 MG sera was observed and binding of anti-acetylcholine receptor autoantibodies on IVIg was found for four of five myasthenia gravis sera. These observations suggest that IVIg contains Ig directly binding to and inhibiting pathogenic autoantibodies. PMID- 1716264 TI - Morphology of Borrelia burgdorferi: structural patterns of cultured borreliae in relation to staining methods. AB - The microscopic recognition of Borrelia burgdorferi in biologic fluids and tissues is difficult and challenging because of low numbers of organisms occurring as single isolated spirochetes, the apparent lack of colony formation in tissues, and differing lengths and structural morphologies. To identify the most common morphologic forms, we studied numerous cultures by a variety of microscopic techniques. Culture suspensions of B. burgdorferi were stained by several different histochemical procedures (Gram, Wright, Wright-Giemsa, Giemsa, and polychromes), fluorochromes (thioflavin-T, acridine orange, and rhodamine), silver impregnation techniques (Warthin-Starry, modified Dieterle, modified microwave Dieterle, and Bosma-Steiner) and immunocytochemical methods with different polyclonal and monoclonal antibodies. Additionally, borrelia culture suspensions were embedded in agar and also injected into skin biopsies and processed for histologic study. The different staining properties were found to be influenced by fixation methods and the type of antibody in immunocytochemical stains. This study provides evidence for marked cytomorphic variations in individual spirochetes. Variations detected by these staining procedures provide a basis for future study of tissue sections and for how borreliae can be expected to appear in tissue sections. PMID- 1716265 TI - Acridine orange stain for determining intracellular enteropathogens in HeLa cells. AB - Green-fluorescent intracellular enteropathogenic bacteria were observed after infected HeLa cell monolayers were stained with acridine orange and counterstained with crystal violet at least 3 h after infection. PMID- 1716266 TI - Comparison of commercially available cytokeratin antibodies in normal and neoplastic adult epithelial and non-epithelial tissues. AB - Five commercially available cytokeratin antibodies (lu-5, AE1/AE3, CAM 5.2, MFN116 and anti-cytokeratin 18) were used to stain a wide range of normal and neoplastic epithelial and non-epithelial tissues to assess their potential value in diagnostic histopathology. All five showed good specificity, with some cross reactivity in smooth muscle cells. The wider reactivity of AE1/AE3, lu-5, and MFN 116, which includes cytokeratins 8,18 (Moll's catalogue) expressed in simple epithelia and their tumours, as well as cytokeratins expressed in complex stratified squamous epithelia, permits identification of a wider range of epithelial derived tumours. This wider spectrum of reactivity may allow these antibodies to be used in a diagnostic panel for the identification of poorly differentiated tumours. PMID- 1716267 TI - Phrenic motoneuron morphology in the neonatal rat. AB - The morphology of neonatal rat phrenic motoneurons was studied following retrograde labeling with horseradish peroxidase, which resulted in Golgi-like fills of phrenic motoneuron somata and dendrites. At birth, these neurons have well-developed dendritic trees with many characteristics described for phrenic motoneurons in the adult rat. The dendrites form tightly fasciculated bundles that emerge from the phrenic nucleus primarily along four axes: ventromedial, ventrolateral, dorsolateral, and rostral/caudal, with smaller and more variable projections directly lateral and ventral. Although sparse, some dendritic appendages were also present, and in a few animals, somata clustering was apparent. The most significant difference between adult and neonatal rat phrenic motoneurons is in the extent to which medially and laterally projecting dendrites extend beyond the borders of the ipsilateral gray matter. In the neonate, unlike the adult, these dendrites project extensively past the gray/white border to the edge of the hemicord. Ventromedial dendrites occasionally cross to the contralateral ventral horn in the ventral white commissure and laterally projecting dendrites could be seen reaching the edge of the cord, turning and traveling rostrally or caudally for up to 100 microns. Phrenic motoneurons are not unique in having long dendrites at birth. A brief comparative study showed that neonatal cervical, thoracic, and lumbar motoneurons also have long dendrites that project to the medial and lateral borders of the hemicord. PMID- 1716268 TI - Unmyelinated axons of the auditory nerve in cats. AB - This paper describes some central terminations of type II spiral ganglion neurons as labeled by extracellular injections of horseradish peroxidase (HRP) into the auditory nerve of cats. After histological processing with diaminobenzidine, both thick (2-4 microns) and thin (0.5 microns) fibers of the auditory nerve were stained. Whenever traced, thick fibers always originated from type I spiral ganglion neurons and thin fibers always from type II ganglion neurons. Because the labeling of type II axons faded as fibers projected into the cochlear nucleus, this report is limited to regions of the ventral cochlear nucleus near the auditory nerve root. The central axons of type II neurons are unmyelinated, have simple yet variable branching patterns in the cochlear nucleus, and form both en passant and terminal swellings. Under the light microscope, most swellings are located in the neuropil but they are also found in the vicinity of cell bodies, nodes of Ranvier of type I axons, and blood vessels. Eighteen en passant swellings in the neuropil were located by light microscopy and resectioned for electron microscopy; two of these swellings exhibited ultrastructural features characteristic of chemical synapses. The data indicate that inputs from outer hair cells might be able to influence auditory processing in the cochlear nucleus through type II primary neurons. PMID- 1716269 TI - Projections to the basis pontis from the superior temporal sulcus and superior temporal region in the rhesus monkey. AB - The present investigation was designed to determine the origins in the temporal lobe, and terminations in the pons, of the temporopontine pathway. Injections of tritiated amino acids were placed in multimodal regions in the upper bank of the superior temporal sulcus (STS), and in unimodal visual, somatosensory, and auditory areas in different sectors of the lower bank of the STS, the superior temporal gyrus (STG), and the supratemporal plane (STP). The distribution of terminal label in the nuclei of the basis pontis was studied using the autoradiographic technique. Following injections of isotope into the multimodal areas (TPO and PGa) in the upper bank of the STS, intense aggregations of label were observed in the extreme dorsolateral, dorsolateral, and lateral nuclei of the pons, and modest amounts of label were seen in the peripeduncular nucleus. The caudalmost area TPO projected in addition to the ventral and intrapeduncular pontine nuclei. The second auditory area, AII, and the adjacent auditory association areas of the STG and STP contributed modest projections to the dorsolateral, lateral, and peripedunuclar nuclei, but generally spared the extreme dorsolateral nucleus. The lower bank of the STS, which subserves central vision, the somatosensory associated region at the fundus of the rostral STS, and the primary auditory area did not project to the pons. The higher order, multimodal STS contribution to the corticopontocerebellar circuit may provide a partial anatomical substrate for the hypothesis that the cerebellum contributes to the modulation of nonmotor functions. PMID- 1716270 TI - Efferent projections of the infralimbic cortex of the rat. AB - On the basis of stimulation studies, it has been proposed that the infralimbic cortex (ILC), Brodmann area 25, may serve as an autonomic motor cortex. To explore this hypothesis, we have combined anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde tracing with wheat germ aggutinin conjugated to horseradish peroxidase (WGA-HRP) to determine the efferent projections from the ILC. Axons exit the ILC in one of three efferent pathways. The dorsal pathway ascends through layers III and V to innervate the prelimbic and anterior cingulate cortices. The lateral pathway courses through the nucleus accumbens to innervate the insular cortex, the perirhinal cortex, and parts of the piriform cortex. In addition, some fibers from the lateral pathway enter the corticospinal tract. The ventral pathway is by far the largest and innervates the thalamus (including the paraventricular nucleus of the thalamus, the border zone between the paraventricular and medial dorsal nuclei, and the paratenial, reuniens, ventromedial, parafasicular, and subparafasicular nuclei), the hypothalamus (including the lateral hypothalamic and medial preoptic areas, and the suprachiasmatic, dorsomedial, and supramammillary nuclei), the amygdala (including the central, medial, and basomedial nuclei, and the periamygdaloid cortex) and the bed nucleus of the stria terminalis. The ventral efferent pathway also provides descending projections to autonomic cell groups of the brainstem and spinal cord including the periaqueductal gray matter, the parabrachial nucleus, the nucleus of the solitary tract, the dorsal motor vagal nucleus, the nucleus ambiguus, and the ventrolateral medulla, as well as lamina I and the intermediolateral column of the spinal cord. The ILC has extensive projections to central autonomic nuclei that may subserve a role in modulating visceral responses to emotional stimuli, such as stress. PMID- 1716271 TI - Blueprint for proficient practice: an adult medical-surgical nursing orientation program. AB - Medical-surgical nursing is a specialty that is long overdue for acknowledgment. Our hospital orientation program provides an integration of theory and practical experience using the principles of adult learning. Faculty maintain "facilitator" rather than "instructional" roles and develop supportive and collaborative relationships with the new nurse. Its concise plan and design is intended to have a favorable impact on professional nursing practice and patient care outcomes. In addition, the program has promoted a recruitment advantage in today's arena of educational competitiveness. PMID- 1716272 TI - Reduced responsiveness of adenylate cyclase in alveolar macrophages from patients with asthma. AB - Alveolar macrophages from patients with asthma accumulated less cyclic adenosine monophosphate when these macrophages were exposed to isobutyl methylxanthine, salbutamol, or prostaglandin E2, compared to cells from control subjects without asthma, and the degree of the hyporesponsiveness was related to the severity of asthma. In addition, a significantly lower adenylate cyclase activity was observed in crude membrane fractions of macrophages from the group with asthma in the presence of salbutamol and prostaglandin E2. The refractoriness observed in patients with asthma is thus not accounted for by a specific beta-adrenergic desensitization at the adenylate cyclase receptor level but should rather be explained by a cyclic adenosine monophosphate-dependent postreceptor mechanism. PMID- 1716273 TI - Monitoring human basophil activation via CD63 monoclonal antibody 435. AB - On activation of human basophilic granulocytes with anti-IgE or with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, the expression of the CD63 antigen on the cell surface, detected by monoclonal antibody (MAb) 435, increased up to 100-fold. The kinetics of CD63 up regulation and histamine release were identical, and a strong correlation was found between percentage of MAb 435-binding basophils and extent of histamine release. Immunoelectronmicroscopy demonstrated that the epitope for MAb 435 in resting basophils is located on the basophilic granule membrane. After basophil activation, MAb 435 bound to the exterior of the plasma membrane. Experiments with various doses of anti-IgE demonstrated that the binding of MAb 435 to basophilic granulocytes follows an all-or-nothing-like response per cell. Basophils either do not bind the MAb at all, or they bind a maximal amount of the MAb. We also measured the up regulation of the CD11/CD18 leukocyte adhesion complex. Here, too, we noted an increase in cell-surface exposure of all subunits after activation. This increase was not as strong as increase found with MAb 435. Thus, MAb 435 is an interesting new tool for investigating the activation of human basophils, in addition to the measurement of mediator release. This MAb may be useful for the detection of basophil activation in vivo. PMID- 1716274 TI - Sesame seed oil anaphylaxis. PMID- 1716275 TI - Influence of age and long-term dietary restriction on plasma insulin-like growth factor-1 (IGF-1), IGF-1 gene expression, and IGF-1 binding proteins. AB - The purpose of these studies was to determine more accurately the relationship between IGF-1 and life span, and to determine whether moderate dietary caloric restriction alters the age-related changes in IGF-1. Studies included an assessment of plasma IGF-1, hepatic IGF-1 mRNA, and plasma IGF-1 binding proteins (IGF-1-BP). Rats (6, 12, 22, and 28 months of age) were fed ad libitum or maintained on a diet 60% of ad libitum. In ad libitum fed animals, plasma IGF-1 decreased by 20% between 6 and 28 months of age. Similar age-related declines were observed in dietary restricted animals but levels were generally 14-25% lower at each age group. IGF-1 mRNA levels demonstrated similar decreases with age in ad libitum fed animals, but in dietary restricted animals, levels plateaued at 22 and 28 months. IGF-1 binding protein analysis revealed 3 bands at approximate molecular weights of 40k, 30k, and 24k. All bands demonstrated a decrease in relative IGF-1-BP concentration with age, as well as a decrease in the 40k and 30k binding proteins after dietary restriction. These results indicate that (a) aging in ad libitum fed animals is associated with decreases in plasma IGF-1, IGF-1-BP, and IGF-1 mRNA levels; and (b) long-term dietary restriction decreases plasma IGF-1 and IGF-1-BP levels in each age group although the age-associated decreases in IGF-1 mRNA levels are prevented by dietary restriction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716276 TI - New operative technique to reduce surgeons' risk of HIV infection. AB - The surgical team is potentially at risk of acquiring human immunodeficiency virus (HIV) from the patient. Assuming that the probability of an accidental injury during surgery is 0.01 (P2), the prevalence of HIV is 0.01 (P3) and the seroconversion rate is 0.01 (P1), we have estimated the risk (actuarial model) for a surgeon as 0.2% per year, and 5.82% for 30 years of surgery. In view of this we have made changes in surgical technique to reduce the risk to the surgical team from splash or injury. The surgeon must handle tissue with instruments only and minimize the use of fingers. Whenever possible, sharp instruments should be replaced by a blunt type. The surgical nurse loads needles to the needle carrier using forceps. Sharp instruments are placed in a neutral zone on the nurse's stand so that the surgeon and the nurse never touch the same sharp instrument at the same time. Movements should be controlled, and instrument handling accompanied by eye contact. We consider that these changes will reduce the risk of accidental injuries and thereby the transmission of HIV during operations to a greater degree than knowledge of the patient's HIV status. PMID- 1716277 TI - Therapy of chronic viral hepatitis. AB - Chronic infection with hepatitis B virus (HBV), the delta agent (HDV) or hepatitis C virus (HCV) carries high risks of chronic liver disease which can result in cirrhosis and hepatocellular carcinoma. Many antiviral agents have been tried to inhibit viral replication and thereby limit infectivity and the risks of eventual serious liver disease. Interferon offers a 30-40% chance of viral clearance to the hepatitis B carrier, offers a good chance of clinical response in parenterally acquired chronic non-A non-B hepatitis and may be of benefit for some patients with chronic delta infection. PMID- 1716278 TI - Phenotypic complexity of intraepithelial lymphocytes of the small intestine. AB - A detailed phenotypic analysis of intraepithelial lymphocytes (IEL) of the small intestine was performed using multicolor fluorescence flow cytometry. CD4+8+ IEL (double positives; DP) could be detected in significant numbers in preparations from several mouse strains. DP IEL expressed Tcr alpha, beta and Thy1. Comparison of Tcr alpha, beta levels of thymocytes and IEL revealed that whereas the majority of DP thymocytes expressed low Tcr levels, DP IEL expressed high, mature T cell levels of Tcr. In addition, DP IEL were generally Ly3- (CD8 beta), unlike their thymic counterparts, which are Ly3+. Ly3 was not present on Tcr gamma, delta IEL, whereas CD4-8+ Tcr alpha, beta IEL contained Ly3- and Ly3+ subsets. The Ly3- population in either Tcr-bearing subset could be further subdivided by Thy1 expression. Ly1 (CD5) expression was also examined, and none of the Tcr gamma, delta IEL were Ly1+. Based on Thy1, Ly3, and Ly1 expression, four CD4-8+ Tcr alpha, beta IEL subsets were detected. The results indicate the cellular complexity of the IEL compartment rivals that found in the thymus. These findings are discussed in light of recent data suggesting an extrathymic origin of some IEL. PMID- 1716279 TI - Successful treatment of experimental allergic encephalomyelitis with transforming growth factor-beta 1. AB - Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine with immunosuppressive effects on T cells in vitro. Experimental allergic encephalomyelitis is an archetypal T cell-mediated autoimmune demyelinating disease of the central nervous system that often serves as a model for multiple sclerosis. In vivo administration of TGF-beta 1 into SJL mice was successful in reducing the incidence of clinical disease and the histologic severity of inflammation and demyelination in the brain and spinal cord. Immunohistochemical studies performed on control animals showed that TGF-beta-1, -2, and -3 were present in inflammatory perivascular lesions in the brain. The use of a naturally occurring cytokine with immunoregulatory functions in the treatment of an autoimmune disease is novel. However, potential long term complications of such therapy must be addressed before its use in human autoimmune disease such as multiple sclerosis. PMID- 1716280 TI - Successful treatment of paralytic relapses in adoptive experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance. AB - We have previously demonstrated that Ag-specific tolerance induced by the i.v. administration of splenocytes coupled with neuroantigens, such as mouse spinal cord homogenate, myelin basic protein (MBP), and proteolipid protein, and their encephalitogenic peptides, results in dramatic inhibition of clinical and histologic signs of both actively induced and adoptively transferred relapsing experimental autoimmune encephalomyelitis (R-EAE). We report here that the administration of splenocytes coupled with mouse spinal cord homogenate (i.e., a mixture of neuroantigens), after the first paralytic episode of adoptive R-EAE triggered by MBP-specific T cells but before the appearance of the first relapse, effectively reduced the onset and severity of all subsequent relapses, as determined by both clinical and pathologic criteria. In contrast, the i.v. administration of splenocytes coupled with MBP (i.e., the specificity of the initiating T cell response), under similar conditions, effectively inhibited the initial clinical relapse, but subsequent relapses occurred with the same incidence rate and severity as those in control animals. Collectively, these results demonstrate that neuroantigen-specific tolerance is effective at specifically down-regulating an ongoing autoimmune response. This may have potential clinical applicability for treatment of autoimmune diseases. The results also support the hypothesis that the neuroantigen specificity of later relapses of R-EAE may be due to effector T cells with specificities different from those that triggered the initial clinical episode. Thus, potential therapy for the advanced stages of R-EAE, and perhaps other autoimmune diseases, may have to be directed not simply against the effector cells initiating the disease but also against effector cells with differing specificities recruited as a result of tissue damage occurring in the initial acute disease. PMID- 1716281 TI - Sequential appearance of IL-1 and IL-6 activities in rat carrageenin-induced pleurisy. AB - IL-1 and IL-6 activities were measured in the pleural exudate of rats during carrageenin-induced pleurisy to examine the relationship of the local production of cytokines to the inflammatory reaction. Time courses of appearance of the cytokines and inflammatory parameters in the exudate were compared. IL-1 activity and exudate volume started to increase at 1 h after the carrageenin injection, and then slightly later IL-6 activity and leukocyte number began to increase. IL 1 showed peak activity of approximately 700 U at 3 h and IL-6, of 6000 U at 5 h in the exudate, whereas exudate volume and number of polymorphonuclear leukocytes continued to increase thereafter. Furthermore, IL-6 level in the plasma of the carrageenin-injected rats showed a peak at 4 h (30 U/ml), and when rhIL-1 alpha (100,000 U) was intrapleurally injected, the more rapid increase in plasma IL-6 level was demonstrated at 1 h (30 U/ml). This latter rise was neutralized with simultaneous injection of anti-rhIL-1 alpha antibody. These facts indicate the possibility that IL-1 produced in the exudate or injected could rapidly propagate a signal to induce IL-6 production in the circulation. It took several hours to transmit an inflammatory signal that stimulated the liver to synthesize the acute phase protein, T-kininogen. The time lag from the peak induction of IL-1 to the T kininogen-increase in the pleurisy corresponded well to the interval for T kininogen-increase by exogenous rhIL-1 alpha injection. These results strongly suggest that the initial inflammatory stimulus induces sequentially IL-1, IL-6, and T-kininogen production in this carrageenin inflammation. PMID- 1716282 TI - Thymus-dependent network responses to a monoclonal cross-reactive antiidiotypic antibody. AB - BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3 nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3 nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced. PMID- 1716283 TI - Wheat germ agglutinin and other selected lectins increase synthesis of decay accelerating factor in human endothelial cells. AB - Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium. PMID- 1716284 TI - Binding sites for endotoxins (lipopolysaccharides) on human monocytes. AB - The nature of the binding sites for LPS on human monocytes was investigated using [3H] labeled intact LPS from Neisseria meningitidis and from Salmonella minnesota R7, and the [3H] labeled purified inner core region (PS-OMe) of S.m. R7 LPS. In the presence of serum, intact LPS from enterobacterial and nonenterobacterial strains bound to monocytes in a dose-dependent, saturable, and displaceable fashion. N.m. LPS and LPS from the enterobacterial strain of Escherichia coli 0111-B4 bound to the same sites on monocytes as assessed in competitive binding experiments. Specific binding of intact LPS to monocytes occurred through the CD14 molecule as shown by the ability of mAb and of F(ab')2 fragments of mAb directed against specific epitopes of CD14 to inhibit the binding of [3H]-LPS to cells and by the lack of binding of intact LPS to CD14-deficient cells from patients with paroxysmal nocturnal hemoglobinuria. Specific binding of LPS to monocytes was not mediated by the CD11/CD18 complex because mAb to the alpha and beta chains of the Leu-CAM molecules did not alter the binding of LPS to cells and because LPS did not inhibit the binding of labeled mAb to monocytes. [3H]-PS OMe also bound in a dose-dependent and displaceable fashion to monocytes involving an unidentified, non-CD14, binding site on the cells. Binding of LPS to monocytes also involved nonsaturable binding sites for hydrophobic structures of LPS as evidenced in binding experiments performed in the absence of serum. These observations indicate that intact LPS may interact with the monocyte membrane in at least three ways including serum-dependent binding to CD14 and to a lectin like receptor, and serum-independent hydrophobic interactions. PMID- 1716285 TI - IL-8 inhibits histamine release from human basophils induced by histamine releasing factors, connective tissue activating peptide III, and IL-3. AB - We have previously purified and partially characterized histamine releasing factors (HRF), which were derived from a mixture of human mononuclear cells and platelets. We now report the effect of IL-8 upon HRF-, connective tissue activating peptide III (CTAP III)-, and IL-3-induced histamine release from human basophils. We determined that IL-8 itself, at concentrations between 10(-7) to 10(-11) M, does not release histamine from basophils, although positive results are observed in two of 26 subjects at 10(-7) M. Unfractionated (crude) HRF released histamine in 25 of 26 donors, in the range of 6.7% to 100% of total basophil histamine stores. When basophils were preincubated with IL-8 (10(-7) to 10(-11) M) for 5 min, followed by a 40-min incubation with HRF, histamine release was significantly inhibited in 20 of 25 donors. Inhibition was observed at as little as 10(-11) M IL-8, with maximal inhibition being attained at 10(-9) M. HRF containing supernatants contain a mixture of different histamine-releasing moieties. To better define which factor(s) may be inhibited by IL-8, fractionated supernatants, purified CTAP III, and IL-3 were studied. Histamine release produced by two different HRF-containing chromatographic fractions (HRFvoid and HRFpeak 2) and purified CTAP-III (5 micrograms/ml) was inhibited by IL-8 in 10 of 12 donors, three of three donors, and seven of 10 donors, respectively. IL-3 (5000 U/ml)-dependent histamine release was inhibited by IL-8 in all subjects tested. In contrast, histamine release by anti-IgE and FMLP was not affected by IL-8. Thus, IL-8 appears to be an inhibitor of cytokine-like molecules that induce histamine release and may represent the previously described 8-kDa histamine release inhibitory factor present in mononuclear cell supernatants. PMID- 1716286 TI - Specific endotoxic lipopolysaccharide-binding receptors on murine splenocytes. III. Binding specificity and characterization. AB - In previously published studies, we employed a photoreactive radioiodinated derivative of LPS from Escherichia coli 0111:B4 to identify and characterize a membrane-localized specific LPS binding protein of approximately 80-kDa molecular mass. Our more recent studies demonstrating that mAb with specificity for this 80 kDa protein will act as an agonist in mediating macrophage activation have established that this protein serves as a specific receptor for LPS. In the experiments reported here, we have more accurately determined the apparent molecular mass of this protein to be 73 kDa (p73). We have also extended the sources of LPS-derivatized photo-cross-linking preparations (including Re-LPS) to determine generality of LPS binding to this receptor. Binding to the p73 LPS receptor is demonstrated with all of the LPS derivatives synthesized in our laboratory, as well as probes synthesized by other investigators. Binding of S LPS is readily inhibited by Re chemotype LPS, and we have shown that this competitive inhibition is most likely not the result of formation of LPS aggregates. These results confirm and extend our earlier studies suggesting that the binding of LPS to the p73 receptor is lipid A specific. We further demonstrate that, in contrast to results published in a recent report, the p73 LPS receptor has no significant binding specificity for a variety peptidoglycan polymer preparations. Finally, we show that this LPS receptor can be detected on murine fibroblast, macrophage, and mastocytoma cell lines. Differences have been observed in the level of expression of LPS receptors on the various cell lines studied. PMID- 1716287 TI - Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL. AB - Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact. PMID- 1716288 TI - The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level. AB - The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes. PMID- 1716289 TI - Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli. AB - Protective immunity against Toxoplasma gondii is recognized to be cell mediated and IFN-gamma is considered to be the major mediator of resistance. Thus, protective Ag of the parasite must induce IFN-gamma-producing T cells. In order to identify such Ag, we have constructed a T. gondii cDNA library in the cloning/expression vector lambda gt11, screened this library with a pool of sera of immune donors, and further screened the set of selected recombinant Ag using, as probe, a T. gondii-reactive T-cell clone (TCC) derived from an infected/immune individual and producing a high level of IFN-gamma. One recombinant Ag was shown to induce TCC proliferation and was characterized. The corresponding mature T. gondii Ag has an apparent molecular mass of 54 kDa and the sequence of the cDNA clone suggests that it is membrane associated. The epitope defined by the TCC on this Ag was found to be present in three Toxoplasma strains independently of their phenotype (virulent or cyst forming). Recognition of this Ag by the TCC was shown to be restricted by HLA-DPw4, the most frequent allele in the Caucasian population (approximately 40%). The use of this Ag as a vaccine component is proposed. PMID- 1716290 TI - Molecular mapping of Osp-A mediated immunity against Borrelia burgdorferi, the agent of Lyme disease. AB - It is paradoxical that although antibodies to the outer surface protein (Osp) A of Borrelia burgdorferi protect mice against infection and that immunization of uninfected mice with Osp-A is protective, antibodies to Osp-A induced early in natural infection of mice are not curative. A region recognized by a neutralizing mAb is also recognized by sera from chronically infected or immunized mice but is not bound by sera from mice infected for 15 days. Infection in mice, despite the presence of early Osp-A antibody, may therefore be explained in part by the lack of response to this epitope. Sera from infected humans recognizes this region, although in this case the immune response to Osp-A occurs only late in infection. Nonetheless, the fact that both human sera and sera from mice immunized with Osp A bind a conformationally dependent epitope that localizes to the same region tentatively suggests that humans are able to respond to a protective epitope and that an Osp-A-based vaccine may elicit protective immunity in humans. PMID- 1716291 TI - T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements. AB - No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products. PMID- 1716292 TI - Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence. AB - A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1 domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1 domain with the alpha-chain is more important than the presence of the alpha chain of the parental species. PMID- 1716293 TI - Complementation of expression of carcinoembryonic antigen and tumor associated glycoprotein-72 (TAG-72) in human colon adenocarcinomas. AB - Enzyme-labeled monoclonal antibodies (MAbs) were used in an immunohistochemical, dual-staining study of 10 colon adenocarcinomas. MAbs B72.3 and COL-4, reactive with the high molecular weight tumor-associated glycoprotein-72 (TAG-72) antigen and carcinoembryonic antigen (CEA), respectively, were labeled with horseradish peroxidase or alkaline phosphatase. Dual staining using the two MAbs on a single tissue section (formalin-fixed, paraffin-embedded) showed that greater numbers of carcinoma cells could be detected by using the combination of the two MAbs than could be detected by use of either MAb alone. In many tumors, some carcinoma cells reacted with MAb B72.3, some reacted with MAb COL-4, and some cells reacted with both MAbs. Only 1 of 10 carcinomas showed greater than 75% reactive cells when stained with each MAb individually. In 9 of 10 cases, however, greater than 75% of cells reacted when the combination of MAbs was used. Cell surface and cytoplasmic patterns of reactivity were observed with both MAbs while some pools of extracellular mucin were composed of both TAG-72 and CEA. This study supports the rationale for the use of a combination of anti-TAG-72 and anti-CEA MAbs for in vitro immunologic detection and potential in vivo immunodiagnostic and immunotherapeutic applications for these MAbs in colon adenocarcinoma patients. PMID- 1716294 TI - [A case of fetal triploidy associated with low maternal serum alpha-fetoprotein]. PMID- 1716295 TI - Androscopy for anogenital HPV. AB - BACKGROUND: Strong evidence now links anogenital intraepithelial neoplasia to the transmission of the human papillomavirus (HPV) through sexual intercourse. While there is increasing research on women with this disease, less is known about their male sexual partners. METHODS: Male patients whose female sexual partners had been diagnosed as having anogenital intraepithelial neoplasia were recruited for the study. The genital regions of the male patients were examined and biopsied with the aid of a colposcope after application of a 5% acetic acid solution. Treatment was based on the specific findings in each patient. RESULTS: Genital lesions were found on 65% of the male patients examined, even though no disease had been detected by the individual. Seventy-nine percent of patients who were compliant with the prescribed treatment protocol had no detectable HPV related lesions at the time of their last androscopic examination. CONCLUSIONS: Magnified examination of the male genitalia using an androscope following the application of 5% acetic acid solution is an effective method by which the primary care physician can detect and treat male HPV-related anogenital lesions. PMID- 1716296 TI - Effect of alpha-interferon therapy on hepatitis C viraemia in community-acquired chronic non-A, non-B hepatitis: a quantitative polymerase chain reaction study. AB - Sera from 30 patients with community-acquired, biopsy-proven chronic non-a,non-B hepatitis (NANBH) were tested for antibodies to the C100 protein of hepatitis C virus (HCV). The 20 patients who showed reactivity in this assay were followed prospectively for 6 months, during which time seven were treated with recombinant alpha-interferon. HCV RNA was detected by "nested" polymerase chain reaction (PCR) in 19 of the 20 anti-C100-positive sera taken at the onset of the study and also in five of the ten anti-C100-negative sera. Pretreatment viraemia levels ranged from 2 x 10(3) to 2 x 10(8) HCV genomes/ml. After 6 months of interferon, elevated serum alanine aminotransferase (ALT) levels had fallen to normal in four of the seven treated patients. In each case the response to interferon was accompanied by either a disappearance of or a decline (1 log to 8 log reduction) in viraemia. HCV genome titres in the three nonresponders and in the 13 untreated anti-C100-positive patients did not change significantly over this 6 month period. These findings confirm the aetiological role of HCV in community-acquired NANBH and suggest that quantitative PCR will become a valuable technique for monitoring the antiviral effect of interferon and other experimental treatments. PMID- 1716297 TI - Detection of human immunodeficiency virus type 1 genomic RNA in plasma samples by reverse-transcription polymerase chain reaction. AB - An application of the polymerase chain reaction (PCR) to the direct detection of human immunodeficiency virus type 1 (HIV-1) viremia is described. The amplification of specific HIV-1 sequences of gag and env viral genes was carried out after the reverse-transcription of plasma samples (plasma RT-PCR) from seropositive subjects. The assay is faster and cheaper than detection of specific HIV-1 transcripts from peripheral blood mononuclear cells by RT-PCR. The data suggest that HIV-1 viremia is detectable by plasma RT-PCR in a large proportion of seropositive individuals. PMID- 1716298 TI - Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations. AB - Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-(1+)-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1+ cells compared to the remaining HNK-1+ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1+ subpopulations were grown together with HNK-1- cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1+ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1- cells. In the second part of this study, we investigate the specificity of differentiation of HNK-(1+)- and HNK-(1-)-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-(1+)-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-(1-)-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-(1+)- and the HNK-(1-)-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives. PMID- 1716299 TI - Ionic currents of the nodal membrane underlying the fastest saltatory conduction in myelinated giant nerve fibers of the shrimp Penaeus japonicus. AB - The myelinated giant nerve fiber of the shrimp, Penaeus japonicus, is known to have the fastest velocity of saltatory impulse conduction among all nerve fibers so far studied, owing to its long distances between nodal regions and large diameter. For a better understanding of the basis of this fast conduction, a medial giant fiber of the ventral nerve cord of the shrimp was isolated, and ionic currents of its presynaptic membrane (a functional node) were examined using the sucrose-gap voltage-clamp method. Inward currents induced by depolarizing voltage pulses had a maximum value of 0.5 microA and a reversal potential of 120 mV. These currents were completely suppressed by tetrodotoxin and greatly prolonged by scorpion toxin, suggesting that they are the Na current. Both activation and inactivation kinetics of the Na current were unusually rapid in comparison with those of vertebrate nodes. According to a rough estimation of the excitable area, the density of Na current reached 500 mA/cm2. In many cases, the late outward currents were induced only by depolarizing pulses larger than 50 mV in amplitude. The slope conductance measured from late currents were mostly smaller than that measured from the Na current, suggesting a low density of K channels in the synaptic membrane. These characteristics are in good harmony with the fact that the presynaptic membrane plays a role as functional node in the fastest impulse conduction of this nerve fiber. PMID- 1716300 TI - Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. AB - The embryonic lethal abnormal visual system (elav) gene of Drosophila melanogaster is required for the development and maintenance of the nervous system. Transcripts from this locus are distributed ubiquitously throughout the nervous system at all developmental stages. A product of this gene, the ELAV protein, has homology to known RNA binding proteins. The localization of the ELAV protein was studied in all developmental stages using antibodies that were generated against a hybrid protein made in Escherichia coli. In general, these data are consistent with previous results and demonstrate that (1) the ELAV protein is detected in the developing embryonic nervous system at a time coincident with the birth of the first neurons, (2) the ELAV protein is first detected in the majority of neurons of the central and peripheral nervous systems of embryos, larvae, pupae, and adults, (3) the ELAV protein appears to be localized to the nucleus, and (4) the ELAV protein is not detected in neuroblasts or identifiable glia. These data also provide new information concerning elav expression and show that (1) ELAV is not expressed in the ganglion mother cells (GMCs), (2) while the ELAV protein is localized to the nucleus, it is not uniformly distributed throughout this structure, and (3) other Drosophila species do express an ELAV-like antigen. We propose that the elav gene provides a neuronal-housekeeping function that is required for the successful posttranscriptional processing of transcripts from a set of genes the function of which is required for proper neuronal development and maintenance. PMID- 1716301 TI - A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons. AB - During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1' dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6 fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons. PMID- 1716302 TI - Testosterone and the incidence of hormone target cells in song-control nuclei of adult canaries. AB - Adult male canaries learn to produce high-amplitude complex courtship songs each breeding season, whereas females do not, and brain nuclei involved with the production of song behavior are much larger in breeding males than in nonbreeding males or females (Nottebohm, 1980, 1981). However, treatment of adult females with testosterone (T) causes them to produce male-like song and stimulates pronounced growth of some song-control brain nuclei such as the caudal nucleus of the ventral hyperstriatum (HVc). We reexamined the effects of T on song-control nuclei in deafened birds. In order to examine whether the pattern of hormone accumulation varies as a function of circulating testosterone levels we described the distribution of testosterone-concentrating cells in HVc and the magnocellular nucleus of the anterior neostriatum (MAN) in hearing adult male, female, and T treated female canaries, as well as in deaf T-treated and untreated females. In contrast to our previous findings (Bottjer, Schoonmaker, and Arnold, 1986a), we observed no tendency in this study for testosterone-induced growth of HVc to be attenuated in deafened birds. There was no difference between deaf and hearing birds in the incidence of labeled cells within HVc. We also observed no sex or hormone-induced differences in the percentage of hormone-concentrating cells in HVc: normal females have approximately the same proportion of hormone target cells as do males and T-treated females. However, males normally have many more neurons in HVc than do control females, and systemic exposure to testosterone induces a pronounced increase in the number of HVc neurons of adult females. Therefore, the absolute number of hormone target cells in HVc is likely to be much greater in males and T-treated females than in normal females. As in HVc, there were no differences among groups in the proportion of labeled cells within lateral MAN (IMAN), a nucleus that has been implicated in song learning (Bottjer, Miesner and Arnold, 1984). In contrast, the incidence of hormone target cells in medial MAN (mMAN) did vary as a function of hormonal condition: although mMAN of normal females is rarely visible in Nissl-stained sections and cells in this region are not hormone labeled, mMAN is clearly visible in Nissl-stained sections of males and T-treated females and contains many hormone-labeled cells. This testosterone-induced change in the phenotype of mMAN cells suggests a possible role for mMAN in learned song behavior. PMID- 1716303 TI - Cell lines derived from mouse neural crest are representative of cells at various stages of differentiation. AB - In order to study mammalian neural crest differentiation in vitro, a series of clonal neural crest (NC) cell lines have been generated by infection of migrating mouse neural crest cells with two recombinant retroviruses containing either the c-myc or N-myc proto-oncogenes. Many cell lines were generated which could be subdivided into three groups based on their appearance in culture. Eleven of these cell lines representative of each of the morphological groups were characterized for the expression of six antigenic markers expressed by neural cells. In addition, mRNA was prepared from these cell lines and analyzed for the expression of a number of neural specific genes. These analyses show that the cell lines are representative of the following cell types: (1) neural crest-like cell lines that do not differentiate in 10% serum; (2) progenitor cell lines, some of which can partially differentiate in culture; and (3) mature neuronal cell lines or bipotential cell lines. Southern blot analysis of DNA from these lines indicated that they have multiple integration sites for the provirus and suggest that phenotypically different cell types have arisen from a single cell. None of the cell lines showed any proliferative or morphological response to nerve growth factor (NGF), whereas over two-thirds of the lines showed both marked proliferative and morphological responses to fibroblast growth factor (FGF). These data indicate that we have generated a range of cell lines representative of a spectrum of mouse neural crest derivatives. PMID- 1716304 TI - An asymptomatic radiolucency of the posterior maxilla. PMID- 1716305 TI - A series of 14 new monoclonal antibodies to keratins: characterization and value in diagnostic histopathology. AB - A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C 35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis. PMID- 1716306 TI - Validity of a screening test for school learning problems in a pediatric clinical setting. AB - Children brought to pediatric outpatient clinics with the primary complaint of developmental difficulty often arrive with little or no information as to the nature of their problem. This study sought to determine if a brief screening test could facilitate the referral process by predicting the primary diagnosis subsequently given to the child based on a comprehensive interdisciplinary evaluation. Subjects for the study were 176 English-speaking children between the ages of 5 and 11 who had been evaluated at the child development center of a large urban medical school. Of the children later diagnosed as learning handicapped, 89% failed the routing test, while 78% with other primary diagnoses passed it. The results of this preliminary study suggest that a brief screening test can be used effectively to form initial hypotheses about the problems (learning vs. nonlearning) experienced by students referred to pediatric clinics for developmental difficulties. Suggestions for further research are offered. PMID- 1716307 TI - Variable colonial phenotypic expression and comparison to nuclei number in Blastomyces dermatitidis. AB - We examined colonial phenotypes of five isolates of Blastomyces dermatitidis at 33, 35 and 37 degrees C on four growth media. Three different colony types were identified: yeast, mycelial, and a mixed type consisting of both yeast and mycelia. Each isolate varied in its ability to grow on the different media and at different temperatures, and in the types of colonies it produced in the various temperature-media combinations. Quantification of the number of nuclei per yeast cell by fluorescent staining revealed no correlation between the number of nuclei per cell and the colonial phenotype. These results indicate that the colonial phenotype of B. dermatitidis varies with the isolate as well as with temperature and culture medium, but is not correlated with the number of nuclei per yeast. These findings could provide a start towards typing B. dermatitidis isolates. PMID- 1716308 TI - Induction of experimental Candida arthritis in rats. AB - Experimental arthritis, caused by intravenously (IV) introduced Candida albicans, has been induced for the first time in rats. Four-week-old Sprague-Dawley rats were inoculated IV with three different strains of C. albicans and observed for 4 weeks. Each of the three strains tested was able to produce arthritis. The incidence of Candida arthritis increased in a dose-dependent manner and was more than 90% at sublethal doses. Joints of the limbs were affected predominantly, and at higher doses arthritis was produced in multiple (four or five) joints in individual animals, showing it to be polyarthritis. C. albicans was recovered from all cultures of affected limb joints, which were excised 12, 19 and 28 days after inoculation and showed different stages and degrees of joint swelling. Results of histopathology and radiography showed that the Candida arthritis involved not only periarticular inflammation but also changes in joint bones. In particular, metaphyseal enlargement, punched-out lesions at the diaphysis and the appearance of osteoclasts were the most prominent changes in affected bones. These pathological features are compared with those of Candida arthritis in humans. PMID- 1716309 TI - Expression of EGF-receptors on epithelial and stromal cells of normal and inflamed gingiva. AB - Immunolocalization techniques were used to examine the expression of the cell surface receptors of EGF in normal and inflamed gingival tissue. Detectable levels of receptor were not observed in any (0/6) of the normal tissue biopsies examined; in contrast, the EGF-receptor was expressed by both epithelial and stromal cells in 7/9 of the inflamed tissue biopsies. Receptor expression by epithelial cells in inflamed tissues exhibited a variable distribution pattern. In the majority of sections, staining was confined to cells in the spinous, granular and cornified cell layers, with little in the basal layer. Occasionally, isolated islands of stained epithelial cells were present, suggesting their clonal origin. Staining for the EGF receptor was also observed in fibroblasts and endothelial cells throughout the lamina propria of inflamed tissue. Positive staining for the receptor ligand (EGF) was observed in both normal and inflamed tissue. These data suggest that an up-regulation of cell surface receptors for EGF occurs during the inflammatory response, this resulting in an increased cellular responsiveness to EGF. PMID- 1716310 TI - Inhibition of human platelet aggregation by endothelium-derived relaxing factor, sodium nitroprusside or iloprost is potentiated by captopril and reduced thiols. AB - We have examined whether inhibitors of angiotensin-converting enzyme-containing sulfhydryl groups such as, captopril (CPT) or SQ 14,534, the nonsulfhydryl containing angiotensin-converting enzyme inhibitors, teprotide (TPR) or enalaprilat (ENA) and other structurally unrelated sulfhydryl-containing compounds, N-2-mercaptopropionylglycine (MPG) or N-acetyl-L-cysteine (NAC), could influence platelet aggregation. Incubation of human washed platelets with CPT, SQ 14,534, TPR, ENA, MPG or NAC (0.1-0.5 mM) did not modify their aggregatory responses to thrombin. However, the antiaggregatory properties of endothelial cells cultured from bovine aorta were potentiated by CPT, SQ 14,534, MPG or NAC but not by TPR or ENA (40-100 microM). CPT (100-500 microM) or NAC (50-200 microM) but not ENA (100 and 500 microM) also potentiated the antiaggregatory effects of sodium nitroprusside (1.0 microM) or iloprost (0.2 nM). The ability of the thiol-containing compounds (CPT or NAC) to potentiate the antiaggregatory effects of sodium nitroprusside or iloprost was not associated with an elevation of platelet cyclic GMP or cyclic AMP levels, respectively. Thus, CPT and other sulfhydryl-containing compounds can synergize with antiplatelet compounds, thereby enhancing the ability of endothelial-derived autocoids to inhibit platelet aggregation. The mechanism responsible for this potentiating effect of thiols on platelet aggregation is not known, but may relate to the ability of thiol-containing compounds to act as intracellular scavengers of oxygen-derived free radicals. PMID- 1716311 TI - Protein kinase C modulates immunoglobulin E-mediated activation of human mast cells from lung and skin. I. Pharmacologic inhibition. AB - It is widely appreciated that protein kinase C (PKC) is activated in a variety of secretory cells after stimulation. By using two complementary pharmacologic approaches, we have investigated the role of PKC in immunoglobulin E (IgE) mediated secretion from human lung, bronchoalveolar lavage and skin mast cells. We examined the effect of the PKC inhibitor, staurosporine, on goat anti-human IgE-induced mediator release. In lung mast cells, the IC50 for staurosporine on histamine release was 2.8 +/- 1.4 nM (n = 9). The drug was slightly more potent in skin mast cells where the IC50 was 0.64 +/- 2.0 nM (n = 6), but staurosporine had no effect on the IgE-mediated release of histamine from bronchoalveolar lavage mast cells. Mast cells were also incubated overnight with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to down regulate PKC and the response to goat anti-human IgE was measured. Prolonged exposure to 100 ng/ml of TPA reduced IgE-mediated histamine release from 28 +/- 6 to 6 +/- 1% in the skin mast cells whereas similar treatment only reduced the response of lung mast cells from 36 +/- 5 to 26 +/- 6%. Despite using agents which act by different mechanisms, short-term inhibition with staurosporine and long-term treatment with TPA, we obtained consistent results which suggest that PKC is playing a prorelease role in IgE-mediated signaling. Our results also suggest that skin and lung mast cells are more sensitive to PKC down-regulation than bronchoalveolar mast cells. This heterogeneity between human mast cells at the level of PKC regulation of cell activation is previously undescribed. PMID- 1716312 TI - Pharmacological characterization of the vascular muscarinic receptors mediating relaxation and contraction in rabbit aorta. AB - Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3 chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1 piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells. PMID- 1716313 TI - Methylxanthines block antigen-induced responses in RBL-2H3 cells independently of adenosine receptors or cyclic AMP: evidence for inhibition of antigen binding to IgE. AB - Activation of a novel adenosine receptor in a rat tumor mast cell line (RBL-2H3 cells) elicits a transient generation of inositol 1,4,5-trisphosphate and an equally transient increase in the level of free cytosol Ca++: Such responses promote little exocytosis, but markedly enhance the secretory response to antigen. A variety of xanthine adenosine receptor antagonists did not suppress the responses to the adenosine analog 5-N-ethylcarboxamidoadenosine. However, 3 isobutyl-1-methylxanthine (IBMX) and certain related xanthines inhibited antigen (dinitrophenylated bovine serum albumin, DNP-BSA)-induced generation of inositol phosphates, the increase in level of free cytosolic Ca++ and exocytosis in RBL 2H3 cells that were primed with a monoclonal DNP-specific immunoglobulin E (from hybridoma H1 DNP-epsilon-26.82). The same compounds inhibited the binding of antigen to cell attached DNP-specific IgE in a highly selective manner. Incorporation of an aromatic or cycloalkyl group in the 8-position of IBMX or theophylline, for example, resulted in compounds that were more potent inhibitors than the parent compounds. Conversely, substituents in the 7- or 9-position of IBMX resulted in inactive compounds. 1,3-Diethylxanthine and 1,3-dipropylxanthine had no activity, suggesting that substituents as large as ethyl or propyl are not tolerated at the 1-position. Inhibition by IBMX was not observed when cells were activated by nonimmunological stimulants or when cells were primed with certain other monoclonal preparations of DNP-specific IgE and stimulated by DNP BSA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716314 TI - Large conductance calcium-activated non-selective cation channel in smooth muscle cells isolated from rat portal vein. AB - 1. Membrane currents of isolated smooth muscle cells from rat portal vein were studied using the whole-cell patch-clamp technique. 2. In addition to the Ca(2+) dependent Cl- current, single-channel activities were recorded in response to external applications of 10 mM-caffeine, in the absence of EGTA in the pipette solution. The conductance of this novel type of channel was around 200 pS for membrane potentials ranging from -100 to +60 mV. 3. The single-channel activities were also induced by external application of noradrenaline (10(-5)M) or acetylcholine (10(-5)M), by Ca2+ entry through voltage-dependent Ca2+ channels, and by intracellular application of ryanodine (10(-5)M). The caffeine-activated single-channel currents disappeared when 10 mM-EGTA was added to the pipette solution or after replacement of external Ca2+ with Ba2+. These results show that these channels are Ca2+ dependent. 4. Alterations of the Cl- equilibrium potential did not produce any change in the reversal potential of the caffeine activated single-channel current indicating that it was not carried by Cl- ions. The value of the reversal potential was about +10 mV, irrespective of the CsCl-, KCl- or NaCl-containing solutions used to fill the pipette. This observation indicates that the channel was equally permeable to these monovalent cations. 5. Caffeine activated single-channel currents when cells were bathed in 90 mM-Ba2+ plus 1 mM-Ca(2+)- or 91 mM-Ca(2+)-containing solutions, showing that divalent cations permeate the channels. The permeability for Ca2+ over Na+ was high as the ratio PCa/PNa was estimated to be 21. 6. Single-channel activities induced by caffeine were not modified by Ca2+ channel blockers such as dihydropyridines and phenylalkylamines. 7. It is concluded that portal vein smooth muscle cells possess Ca(2+)-activated nonspecific channels which are highly permeable to Ca2+ ions. PMID- 1716315 TI - Inactivation characteristics reveal two calcium currents in adult bovine chromaffin cells. AB - 1. Two calcium currents were identified by differences in their inactivation characteristics in adult chromaffin cells maintained in short-term primary culture (3-5 days). Calcium currents were recorded by means of the whole-cell configuration using an intracellular medium highly buffered for pH and pCa. 2. Calcium current evoked from a holding potential of -90 mV inactivated along two components: an initial transient with a time constant of 250 ms followed by a plateau. 3. Steady-state inactivation followed two processes which developed at two distinct membrane potentials. One process was half-inactivated at low voltages around -55 mV and affected mainly the initial transient component. The other process, which affected mainly the sustained component of the calcium current, was half-inactivated at voltages around -10 mV. The proportions of these two processes varied greatly from cell to cell. 4. The dihydropyridine antagonists (nicardipine and nifedipine applied at 10(-5) M) and the phenylalkylamine D600 (5 x 10(-6) M) shifted the half-inactivation value towards 55 mV, indicating the suppression of the sustained component. The snail toxin, omega-conotoxin, had the opposite effect; it shifted the half-activation value towards -10 mV. 5. The calcium channel agonist Bay K 8644 (10(-5) M) either had no effect or induced only a slight increase of the response, as did its (-) enantiomer (10(-6) M). To interpret the present results, we suggest that the L component was maximally activated in our recording conditions. 6. In chromaffin cells, the calcium current recorded in whole-cell conditions is composed of two components with properties close to those of N- and L-type currents described in sympathetic neurons. PMID- 1716317 TI - Clinical classification of Swedish snuff dippers' lesions supported by histology. AB - From a total material of 184 Swedish users of loose packed moist snuff and 68 users of portion-bag packed moist snuff, cases were selected from subgroups based on a four-point clinical grading scale. The selected material for the study comprised 70 cases (ten from each clinical grade group, no Degree 4 lesion was found among portion-bag users). Features recognized in biopsies from these cases together with findings in previous studies correlated well with the use of a four point scale for the grading of clinical changes, especially in the context of discriminating lesions for which special efforts should be undertaken to make the patient stop or change the snuff dipping habit and for selecting patients in whom regular clinical follow-up including a biopsy should be carried out. In this article is also discussed the labeling of the clinical oral mucosal changes seen at the site where a quid of snuff is regularly placed. The conceptual use of "snuff dippers' lesions" is recommended instead of e.g. snuff-induced leukoplakia. PMID- 1716316 TI - Two types of calcium channels are expressed in adult bovine chromaffin cells. AB - 1. Calcium channel activity was recorded in chromaffin cells in the cell-attached condition, using 110 mM-Ba2+ as the permeant ion. 2. One type of calcium channel had a conductance of 16 pS, was completely inactivated at a holding potential of 20 mV and was insensitive to dihydropyridine agonists and antagonists. These characteristics correspond to a calcium channel of the N-type. 3. A second type of calcium channel was active at holding potentials of -30 mV and above, had a channel conductance of 31 pS, and was sensitive to the dihydropyridine agonist, Bay K 8644. The channel opened along two dominant modes with characteristic time constants of 0.5 and 5 ms. The main effect of Bay K 8644 was to increase the probability of both short and long openings with no change in their relative proportions (6 to 1 respectively). These characteristics correspond to a calcium channel of the L-type. 4. omega-Conotoxin affected the activity of both N- and L type channels. It drastically reduced the number of N-type channel openings and produced complex changes in L-type channel activity. Long openings were less frequent and the conductance during short openings was slightly smaller than that measured in the presence of Bay K 8644. 5. The discussion focuses on modulation of L-type channel activity. Openings of L-type channels are rarely recorded in the cell-attached configuration under control conditions. Addition of Bay K 8644 is needed to reveal the presence of L-type channels. By contrast, L-type currents recorded in the whole-cell configuration are always observed and are insensitive to Bay K 8644. These results indicate that L-type channels are normally inoperable in chromaffin cells. PMID- 1716318 TI - Architectural organization of human oral epithelium as visualized by keratin staining pattern in tobacco-associated leukoplakias. AB - The keratin staining pattern in clinical normal buccal mucosa in smokers and non smokers and in tobacco-associated leukoplakias histologically characterized by the chevron type of keratinization were studied. No differences in keratin staining pattern were found in normal buccal mucosa in smokers and non-smokers. In the tobacco-associated leukoplakias we found distinct differences in staining pattern between areas overlying connective tissue papillae and epithelial ridge areas indicating a lateral organization of oral epithelium related to the ridge system. It seems possible to explain this pattern in terms of current hypotheses of the proliferative conditions in squamous epithelia as previously studied in detail in the epidermis and tongue filiform papillae. We conclude, that our results confirm the presence of subpopulations of suprabasal and basal cells and support the presence of proliferative units in oral epithelium. Furthermore, smoking does not seem to influence the keratin staining pattern in clinically normal buccal mucosa. PMID- 1716319 TI - Application of molecular techniques in the rapid diagnosis of EBV-associated oral hairy leukoplakia. AB - A rapid method for the detection of EBV-DNA in paraffin sections of lesions of oral hairy leukoplakia (OHL) is described. The method makes use of advances in molecular technology, including the use of synthetic oligonucleotides with digoxigenin labelling in an in situ hybridisation (ISH) reaction, which can be completed in 24 h. Using this method, sections from 15 of 17 patients clinically diagnosed as having OHL contained readily detectable EBV-DNA in small foci along the upper layers of the stratum spinosum. The sections examined from the two remaining patients appeared to be EBV-DNA negative but both patients were on AZT therapy and one was in addition receiving acyclovir. PMID- 1716320 TI - Evaluation of the nucleolar organizer region associated proteins in minor salivary gland tumors. AB - Forty-three intraoral salivary gland tumors were studied to determine the value of the AgNOR technique in the assessment of these neoplasms. Well defined black dots were visible in the nucleii of all the specimens studied. The mean AgNOR count per nucleus for each tumor was calculated as follows: pleomorphic adenoma (n = 15) 1.52; Polymorphous low-grade adenocarcinoma (n = 12) 1.90; adenoid cystic carcinoma (n = 6) 2.92; mucoepidermoid carcinoma (n = 4) 1.93; carcinoma ex mixed tumor (n = 4) 2.05; undifferentiated carcinoma (n = 1) 3.13 and epithelial-myoepithelial carcinoma (n = 1) 2.23. The difference between the means of benign and malignant tumors (P less than 0.01) and polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (P less than 0.01) were highly significant. The overlapping of the AgNOR count between various tumors prohibited the use of this technique as an absolute criterion in establishing a final diagnosis. It could however be used as a diagnostic aid in differentiating between salivary gland neoplasms. PMID- 1716321 TI - Cough and cold treatment with OTC medicines. PMID- 1716322 TI - Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression. AB - The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute and chronic CdCl2 exposures resulted in the induction of the MT-1 gene in lymphocytes as measured by standard RNA Northern blot analysis. Twenty-four hours following an ip injection of 3.4 mg/kg CdCl2, a ninefold increase in MT gene expression was observed in lymphocytes, as well as five- and sevenfold increases in liver and kidney, respectively. Oral exposure of rats to 1-100 ppm CdCl2 via drinking water resulted in an approximate twofold enhanced MT signal in lymphocytes after 6 wk, and a threefold increase after 13 wk of exposure to 100 ppm Cd. No increases in lymphocyte MT gene expression were observed after 3 wk of Cd exposure. Liver MT gene expression was substantially induced following chronic Cd exposure, while kidney was not, although this organ had a higher basal expression of the MT-1 gene. Analysis of tissue Cd burdens demonstrated a dose-response Cd accumulation in liver and kidney, but only kidney burdens increased substantially with prolonged Cd exposure. These results demonstrate the utility of lymphocyte gene expression assays to detect in vivo toxicant exposure, and thus their applicability as molecular biomarker assays for human exposure assessment. PMID- 1716323 TI - Differential expression of MAG isoforms during development. AB - The myelin-associated glycoproteins (MAG) mediate the cell interactions of oligodendrocytes and Schwann cells with axons that are myelinated. MAG exists in two developmentally regulated isoforms: large MAG (L-MAG) and small MAG (S-MAG). In this paper, we have studied the tissue-specific and developmentally regulated alternative splicing of these isoforms using monospecific antibodies that recognize epitopes common to both isoforms or that are present only on L-MAG. In the central nervous system (CNS), L-MAG is the major form synthesized early in development, and it persists as a significant proportion of the MAG present in the adult. In the peripheral nervous system (PNS), L-MAG is expressed at modest levels during development; it is virtually absent in the adult. Thus, the expression of L-MAG is not limited to the CNS, as was formerly believed, suggesting that it plays a common role during the early stages of myelin formation by both oligodendrocytes and Schwann cells. In both the CNS and PNS, S MAG is the predominant isoform in the adult. A higher-molecular-weight form of MAG is present in the PNS at low abundance, that is developmentally regulated, and appears to be a glycosylation variant. An analysis of the carbohydrate residues on MAG demonstrates that it contains both N-linked and O-linked sugars that could be modulated during development. These results suggest a possible mechanism for the regulation of MAG function during myelinogenesis via the expression of alternative isoforms and carbohydrate modifications. PMID- 1716324 TI - Expression of protein kinase C isozymes in primary neuronal cultures of the rat cerebellum. AB - Protein kinase C (PKC), a family of closely related enzymes, has been implicated in molecular processes involved in differentiation in a variety of cells, including neuronal cells. We studied the presence and distribution of four PKC isozymes immunocytochemically in primary neuronal cultures of the rat cerebellum. We employed four anti-PKC antisera raised against synthetic peptides predicted from the cDNA sequence of the C-terminal portion of four PKC isozymes, alpha, beta I, beta II, and gamma. The majority of neurons were PKC(beta II) immunoreactive both in the early and late (14 days) stage of culture, whereas PKC(alpha)-, (beta I)-, and (gamma)-immunoreactive neurons were most abundant in the late stage of culture. Immunoreactivity of each PKC was high in the cytoplasm, processes, and growth cones. Prominent nuclear staining was observed with anti-PKC(gamma) antibody. These results are in contrast with in vivo results where each PKC isozyme is localized in a distinct population of neurons and subcellular compartment, suggesting the presence of regulatory mechanisms for PKC expression and compartmentalization in vivo. PMID- 1716325 TI - [Paroxysmal nocturnal hemoglobinuria (PNH) deficiency of major complement regulatory membrane proteins on erythrocytes]. AB - The significance of the deficiency of the major complement-regulatory membrane proteins, decay-accelerating factor (DAF) and CD59, to the lysis of paroxysmal nocturnal hemoglobinuria (PNH) red blood cells was investigated. DAF and CD59 were demonstrated to be deficient simultaneously on affected PNH red blood cells (PNH-III) by two-color FACS analysis. At least in some patients with PNH, PNH-I was also revealed to be deficient partially in DAF. Purified DAF and CD59 ameliorated the complement sensitivity of PNH red blood cells, partially and completely, respectively. Functional blocking of these molecules on nomrla human red cells by monoclonal antibodies to DAF and CD59 rendered A or AB type blood cells complement-sensitive but not O or B blood type blood cells. The differences of complement-sensitivity among blood types were revealed to reside on the step of binding of C9 to C5b-8, i. e. C9 can bind to C5b-8 more on A type blood cells than on O type blood cells. We conclude that the deficiency of DAF and CD59 play a major role for the complement sensitivity of PNH red blood cells and that other factors reported to be deficient in PNH do less than these two proteins. PMID- 1716326 TI - [Ectopic salivary amylase-producing IgA- lambda-type multiple myeloma with expression of MDR-1/P-glycoprotein]. AB - We report a case of ectopic salivary amylase-producing IgA- lambda-type multiple myeloma. A 70-year-old man was admitted because of anemia and renal failure. After chemotherapy for eight months, the serum amylase markedly increased. Amylase activity in the supernatant of cultured myeloma cells, which were obtained from the bone marrow, also increased. The myeloma cells expressed MDR 1/P-glycoprotein). The case implies the association of drug resistance and the ectopic amylase production in a case of multiple myeloma. PMID- 1716327 TI - [Cytogenetic analyses of diesel engine exhaust by sister chromatid exchange and micronucleus tests with mouse fetal liver cells]. AB - The genotoxic effect of maternal exposure to diesel engine exhaust was studied by analyses of sister chromatid exchanges (SCEs) and formation of micronuclei with fetal liver cells. Fetal liver cells were obtained from pregnant ICR/JCl mice at the 16th day of gestation. Diesel engine exhaust was generated by running a small engine (YANMAR NSA-40CE, used as an electric generator, displacement: 269 cc). The concentrations of pollutants in the inhalation chamber were as follows; CO: 40-100 ppm, NO2: 2-4 ppm, and particulates: 2-4 mg/m3. Maternal mice were divided into three groups: the long-term exposure group, the short-term exposure group and the non-exposed control. Mice of the long-term group were exposed to the exhaust 4 hours daily from day 0 to the 16th day of gestation. Mice of the short term group were similarly exposed but only on the 15th and 16th days of gestation. The number of SCEs of fetal liver cells was significantly increased in both the exposed groups. The long-term exposure group showed a significant increase in the number of SCEs in comparison with that of the short-term exposure group. In the micronucleus test with fetal liver cells, no significant change in the frequency of micronucleated cells was induced by exposure to diesel exhaust. PMID- 1716328 TI - [Consistency in viabilities of mycobacteria detected by FDA/EB staining and colony forming units on the medium]. AB - We have previously reported that the staining of mycobacteria with fluorescein diacetate (FDA) and ethidium bromide (EB) detects viable bacteria and distinguish them from heat-killed bacteria. Whether this method can be applied to clinical specimens has been an important question. In the present experiment, we treated mycobacteria with either UV-irradiation, 70% ethanol, 0.2% benzalconium chloride, 5% saponated cresol solution, or 5% phenol for 24 hours, and stained with FDA/EB to evaluate the effect of sterilization. We also stained samples of sputum from a tuberculosis patient with FDA/EB after treatment with 4% NaOH for 30 min. and neutralized with 1N H2SO4. All the samples were determined for the colony forming units on 1% Ogawa egg media. A good correlation was observed between the results of FDA/EB staining and colony formation. We believe that FDA/EB staining is useful method to detect viable mycobacteria in clinical samples. PMID- 1716329 TI - Ion channel regulation in the thick ascending limb of the loop of Henle. PMID- 1716330 TI - First steps of tumor-related angiogenesis. AB - We present morphological data of the early steps of tumor-induced angiogenesis and show the distribution of the three main components of the basal lamina (BL), laminin, collagen IV, and fibronectin during these early processes. Tumor cells of a line of BSp73 AS a nonmetastasizing tumor isolated from a pancreatic adenocarcinoma were injected subcutaneously into the back of BDX rats. Two days after tumor inoculation, the BL of the dilated mother vessels around the whole circumference of the vessel has either disappeared, become fragmented, or developed several successive layers. By immunoelectron microscopy, we demonstrate that the fragmented and multilayered BL is strongly stained for laminin and collagen IV but less strongly for fibronectin. Around the surface of the dilated mother vessels which are free of any detectable BL material (by electron microscopy standards), we can see accumulation of all three components in the connective tissue. Simultaneously with the alteration of the BL, the proliferation of the endothelial cells (EC) and the pericytes and the migration of the EC from the wall of the mother vessel have started. EC migration begins in two different ways. Either one EC migrates from the wall of the mother vessel into the surrounding connective tissue, or two or more EC form nearly parallel processes toward the connective tissue. The tips of these processes are connected by intracellular junctions. Around the cellular protrusions of these cells material of the BL deposited into the nearby connective tissue can be observed neither by conventional nor by immunoelectron microscopy. During the outgrowth and migration, the EC remain in contact via junctions with the EC of the original vessel. When migration during which the EC retain their polarization continues, a slit-like lumen forms immediately between the migrating EC. This lumen always remains in direct connection with the lumen of the mother vessel. It is sealed at its border by intercellular junctions. Such junctional complexes can develop a length (in sections) of several hundred micrometers. A BL detectable in the electron microscope can neither be found around the tip of the migrating EC nor around young capillaries not yet surrounded by pericytes. By immunoelectron microscopy, however, only the cellular protrusions at the tip of migrating EC are free of deposited material of the BL. The basal surface of longer (new) capillaries is covered by a continuous layer of amorphous material.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716331 TI - Proliferation index in various stages of breast cancer determined by Ki-67 immunostaining. AB - To investigate factors involved in progression of breast cancer, we estimated the growth fraction of malignant cell populations in various stages of mammary cancer growth. Frozen sections were immunostained with the Ki-67 monoclonal antibody and the proliferation index determined using static image analysis. Pure intraductal carcinoma, intraductal carcinoma coexisting with invasive disease, and metastatic sites coexisting with primary tumors were studied. The proliferation index of pure intraductal carcinomas (mean 4.5%, median 1.8%) was not significantly different from invasive mammary cancers (mean 5.1%, median 2.2%). The proliferation index determined for the in situ component of primary cancers (mean 3.8%, median 1.5%) was not significantly different from values obtained from the invasive component of growth (mean 4.2%, median 2.1%). Variability between in situ and invasive components for individual cases was minimal in tumors whose proliferation index was less than 3.0%; for tumors with higher proliferation indices, the differences were greater. However, there was no trend toward a decrease or increase in growth fraction for the two components of primary tumor growth. The mean proliferative index for primary tumors (mean 4.9%, median 4.0%) was not significantly different from the mean proliferative score from a matched group of metastatic sites in the same patients (mean 5.7%, median 5.5%). Comparison of individual cases uncovered differences in some tumors; again no consistent trends in either direction were noted. An increase (or decrease) in growth activity does not accompany the transition from intraductal (in situ) disease to invasive mammary cancer, nor does a change in growth fraction necessarily accompany progression of mammary cancer from the primary to regional metastatic site. PMID- 1716332 TI - Adenocarcinoma of the pancreas: a multimodality approach--a single surgeon's experience (1979-1988). AB - A retrospective review of a single surgeon's experience with adenocarcinoma of the pancreas was performed. One hundred-one patients were treated over a 10-year period from 1979 to 1988. Seven patients underwent potentially curative resections and 28 patients presented with metastatic (stage IV) disease. Sixty four patients had locally advanced and unresectable primary lesions. A total of 51 patients received I-125 seed implantation. There was no statistically significant difference in morbidity (33% vs. 30%) or mortality (6% vs. 8%) between patients receiving I-125 implantation and those undergoing palliative surgical procedures without implantation. Operative mortality was highest in patients presenting with stage IV lesions (11%). In those patients with locally advanced and unresectable carcinomas, there was a nonsignificant increase in survival (12.8 mo vs. 10.7 mo) in those receiving intraoperative I-125 implants when compared to those who did not when both groups received postoperative adjuvant chemotherapy and external beam radiotherapy. Based on these encouraging results, it is concluded that I-125 implantation can be performed safely and shows a trend toward improving long-term survivorship in patients with locally advanced pancreatic carcinoma when used in conjunction with chemotherapy and external beam radiation. PMID- 1716333 TI - The immune system as a neural network: a multi-epitope approach. AB - The term "neural network" has been applied to arrays of simple activation units linked by weighted connections. If the connections are modified according to a defined learning algorithm, such networks can be trained to store and retrieve patterned information. Memories are distributed throughout the network, allowing the network to recall complete patterns from incomplete input (pattern completion). The major biological application of neural network theory to date has been in the neurosciences, but the immune system may represent an alternative organ system in which to search for neural network architecture. Previous applications of parallel distributed processing to idiotype network theory have focused upon the recognition of individual epitopes. We argue here that this approach may be too restrictive, underestimating the power of neural network architecture. We propose that the network stores and retrieves large, complex patterns consisting of multiple epitopes separated in time and space. Such a network would be capable of perceiving an entire bacterium, and of storing the time course of a viral infection. While recognition of solitary epitopes occurs at the cellular level in this model, recognition of structures larger than the width of an antibody binding site takes place at the organ level, via network architecture integration of, i.e. individual epitope responses. The Oudin Cazenave enigma, the sharing of idiotypic determinants by antibodies directed against distinct regions of the same antigen, suggests that some network level of integration of the individual clonal responses to large antigens does occur. The role of cytokines in prior neural network models of the immune system is unclear. We speculate that cytokines may influence the temperature of the network, such that changes in the cytokine milieu serve to "anneal" the network, allowing it to achieve the optimum steady-state in the shortest period of time. PMID- 1716335 TI - Circulating hemopoietic progenitors mobilized by cancer chemotherapy and by rhGM CSF in the treatment of high-grade non-Hodgkin's lymphoma. PMID- 1716334 TI - ProMACE-MOPP vs MACOP-B in high grade non-Hodgkin's lymphomas: a randomised study in a multicenter cooperative study group (NHLCSG). AB - From January '85 to April '87, 81 patients (pts) with diffuse intermediate and high-grade non-Hodgkin's lymphomas were treated with the ProMace/MOPP protocol in a large Italian Cooperative Study Group (NHLCSG). Criteria for entry into the study included: no prior therapy, stage III-IV or stage II with bulky disease and/or B-symptoms, age below 65. 79 pts were evaluable for response. Almost all pts received six courses of chemotherapy, plus radiotherapy on bulky disease. 53 pts (67%) achieved complete remission (CR), 7 (9%) partial remission (PR), 4 (5%) were considered stable disease (SD) and 15 (19%) progression disease (PD) with 5 of them died early during treatment. The actuarial overall survival (OS) and disease free survival (DFS) are respectively 54% at 61 mos and 62% at 41 mos. The median follow-up from the end of therapy is 56 mos (range 40-68). Until now 20 pts (38%) relapsed on a median time of 8 mos (range 2-21) from CR. These data allowed to us to consider this regimen as effective as the third generation protocols also taking into account the multicenter basis of this study. With the aim to evaluate the impact of the third generation regimen on the outcome of these pts, a randomized study has been performed comparing ProMACE-MOPP with the third generation regimens MACOP-B. Therefore, from 1988 up to now, 206 pts with similar clinical and histological characteristics, have been enrolled in the two arms. No differences in terms of CR and DFS have been registered between the two treatments, with roughly the same toxicity. An analysis of prognostic factors in the larger series of pts treated with ProMACE-MOPP in the first and in the second study (167 pts) was performed. On these basis it seems reasonable that our next step would be to candidate these poor prognosis pts to a new therapeutic strategy which included the use of ABMT and/or PBSC transplantation as first line. PMID- 1716336 TI - Non-Hodgkin's lymphomas of primary follicle/mantle zone origin. AB - Recent immunopathologic studies have demonstrated that primary follicles and the mantle zones of secondary follicles are composed largely of virgin B lymphocytes which migrate from the bone marrow to these areas, and are the precursor cells of germinal centers. Non-Hodgkin's lymphomas corresponding to these immature B cells include mantle zone lymphoma (MZL), a primary follicle variant of MZL without reactive germinal centers, and diffuse intermediate lymphocytic (centrocytic) lymphoma. Diffuse intermediate lymphocytic lymphoma (DILL) is considered a late stage in the progression of MZL. Cytologically, these lymphomas usually resemble their normal cellular counterparts and consist predominantly of atypical small lymphoid cells with slightly-irregular and indented nuclei, moderately-coarse chromatin, inconspicuous nucleoli, and scant cytoplasm. Small lymphocytic, cerebriform and blastic variants have also been described. In smears and touch preparations, the neoplastic cells are usually prolymphocytes. Immunologically, the cells have features of virgin B cells, bearing pan-B cell antigens along with monoclonal surface IgM, with or without surface IgD, and CD5 (Leu 1) antigen, and lacking common acute lymphocytic leukemia associated (CALLA) antigen. Cytogenetically, the t(11;14)(q13;q32) has been associated with this group of lymphomas, and expression of the putative cellular oncogene bcl-1 (11q13) has been demonstrated in 30-50% of cases. Clinically, the patients have a median age of 60 years and usually present with advanced stage disease. Splenomegaly, often massive, is present in 80% of those with MZL. Patients with MZL have a significantly longer median survival (74-77 months) than those with DILL (30-33 months), and survival in both groups is significantly prolonged if a complete clinical remission is attained. Based on clinical studies, MZL should be considered a low grade lymphoma and DILL should be considered an intermediate grade lymphoma by Working Formulation criteria. The lymphomas of primary follicle/mantle zone origin are a distinct clinicopathologic entity biologically analogous to the follicular and diffuse lymphomas of germinal center origin, from which they should be distinguished in current and future classifications of non Hodgkin's lymphoma. PMID- 1716337 TI - The management of mycosis fungoides at Stanford--standard and innovative treatment programmes. PMID- 1716338 TI - MACOP-B vs F-MACHOP in the treatment of high-grade non-Hodgkin's lymphomas. AB - From September 1988 two hundred-sixty-seven (267) untreated patients (pts) with stage II to IV high grade non Hodgkin's lymphoma (NHL) have been enrolled in a multicenter, randomized, still ongoing study, comparing two third-generation combination chemotherapy regimens, MACOP-B versus F-MACHOP. At the present time, 177 pts have completed the treatment program and are evaluable, with a median follow-up of 13 months. Clinical, histologic and laboratory characteristics are equally distributed in both groups. Among the 92 pts treated with MACOP-B, 58 (63%) achieved a complete remission (CR), 17 complete responders have relapsed (29%), and 21 have died (23%), including 3 treatment-related deaths. Among the 85 pts who received F-MACHOP, 65 (76%) achieved a CR, 9 complete responders have relapsed (14%), and 11 pts have died (13%), including 3 treatment related deaths. 30 months-projected survival is 64% for MACOP-B treated pts compared to 84% for F MACHOP treated pts; 30 months-projected relapse- free survival is 80% and 84%, respectively. F-MACHOP seems to be superior in immunoblastic lymphoma (overall survival, OS, 82% vs. 54%) and in Burkitt-type lymphoblastic lymphoma (OS 100% vs, 42%). The degree of hematological and non-hematological toxicity was similar in both regimens. More reliable conclusions will be drawn after a longer follow up. PMID- 1716339 TI - MACOP-B for advanced stage large cell lymphoma (DLCL). More is better? Italian Multiregional Cooperative Study Group (IMCSGL). AB - A study of the predictive value for CR, DFS and OS of the presenting features was carried out on 180 patients with advanced stage DLCL treated with MACOP-B between June 1986 and March 1989. A multivariate regression analysis identified LDH level, bone marrow involvement and tumor burden as independent risk factors with a 4 year survival rate of 79%, 58% and 28% respectively. Therefore MACOP-B proved to be an adequate treatment for the first two groups of patients but not for the third which requires a more aggressive treatment. A sequential single drug high dose chemotherapy with collection of peripheral blood stem cell program followed by bone marrow harvesting, super-intensive radio-chemotherapy and bone marrow transplant has been activated. Seven patients have been so far enrolled: preliminary results demonstrated the feasibility of the program. A larger number of cases and a longer follow-up is required for assessing the efficacy of this approach. PMID- 1716340 TI - ProMACE-cytaBOM versus MACOP-B in intermediate and high grade NHL. Preliminary results of a prospective randomized trial. AB - One hundred seventy-nine patients with intermediate or high-grade non-Hodgkin's lymphoma were randomized to receive either ProMACE-CytaBOM (P-C) or MACOP-B (M B). At last follow-up 71 patients in the P-C arm and 78 in the M-B arm were assessable for response. Forty-one patients treated with P-C (58%) and 49 patients treated with M-B (63%) achieved a CR. Moreover 18 and 22 patients achieved PR with P-C and M-B, respectively. Twenty-five patients relapsed, 12 in the P-C arm and 13 in the M-B arm. Thirty-nine patients died, 32 from disease progression, 5 from treatment related causes, and 2 from other causes. No differences between the two treatment groups were observed as regard to relapse or death-rate. At 27 months the survival rate was of 71.9% for patients treated with P-C and 70.7% for those treated with M-B. At 2 years the RFD rate was 64% and 60% for patients in P-C and M-B arm, respectively. Patients treated with M-B experienced an high rate of methotrexate-related toxicity. ProMACE-CytaBOM and M B seem provided with similar activity. However P-C seem less toxic and more manageable in an outpatient setting. PMID- 1716341 TI - Wood preservatives, solvents, and thrombocytopenic purpura. PMID- 1716342 TI - Analysis of LPS released from Salmonella abortus equi in human serum. AB - We investigated in which form lipopolysaccharide (LPS) is released from live bacteria incubated with human serum and whether the released LPS can interact with high density lipoprotein (HDL), the main transport protein for purified LPS in circulation. Live biotinylated Salmonella abortus equi bacteria were incubated with fresh serum (37 degrees C; 2 h). The released LPS was isolated by immunoprecipitation or immunoabsorption using specific anti-O antibodies. It was analysed and compared with purified LPS, also incubated with serum under identical conditions. Immunoprecipitation led to a 35% recovery and immunoabsorption to quantitative recovery of released or purified LPS. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblot analysis revealed that all molecular species present in the purified LPS were present in the released LPS. The rough fraction, which was co-isolated from serum together with the true smooth (O-polysaccharide-containing) molecules, exhibited S. minnesota rough mutant Rb antigenic specificity. In the immunoprecipitated material two forms of released LPS were identified. One represented LPS associated with a biotinylated bacterial component with an apparent molecular mass of 35-36 kDa, which was identified as OmpA, a major outer membrane protein. The OmpA-associated LPS was free of HDL. Another part of the released LPS was free of biotinylated bacterial components. This portion of LPS was associated with HDL, indicating that the interaction with HDL may also proceed with a part of LPS released from bacteria. PMID- 1716343 TI - The prostate plasma membrane as an androgen receptor. AB - The pivotal role of the cell nucleus in androgenic control of target organs, such as the prostate, has become increasingly suspect. Equally qualified receptor activities have been found in the cytosol, endoplasmic reticulum, and plasma membrane. It is presently difficult to explain how a sex steroid can manage proliferation, metabolism, biosynthesis and secretion, all through chromatin directed signals. In my search for a more satisfactory mediator of androgen action, I discovered that the sodium-potassium-dependent ATPase of the prostate plasma membrane binds androgen, and is activated by the hormone's presence to serve as a metabolic pacemaker. This paper is my terminal status report on one aspect of this hypothesis; namely, the nature and site of androgen binding, with clues as to the mode of action. SDS-PAGE indicates that androgen can be bound to the beta-subunit of prostatic Na,K-ATPase. Selective enrichment of the enzyme by reversible coupling to either concanavalin A or a DHT-affinity column support this conclusion. Several studies show the dynamic effect of androgen binding: increased ouabain binding; enhancement of this binding by facilitated phosphorylation; spectroscopic evidence of conformational shifts, possibly consequences of these suggested activities for regulation, especially of metabolism, are examined. PMID- 1716344 TI - New trends in microencapsulation of physiologically-active substances- homogenization of liquid diphase systems. Theory and applications. PMID- 1716345 TI - Cloning of a recombinant complementary DNA to a baboon (Papio anubis) estradiol dependent oviduct-specific glycoprotein. AB - The estradiol-dominated baboon oviduct synthesizes and secretes a glycoprotein (mol wt, 120,000) that binds to oocyte zona pellucida in vivo. This glycoprotein separates into two major isoelectric variants (pl 8.0 and 4.5) on two-dimensional electrophoretic gels. This study was undertaken to further characterize the steroid-controlled gene expression of this glycoprotein. A recombinant cDNA library was prepared to poly(A)+ RNA isolated from oviducts obtained from estradiol-treated baboons. The library was screened with a polyclonal antibody prepared against the acidic component (pl 4.5) of the glycoprotein. A single positive plaque was purified. Digestion of the recombinant plasmid with EcoRI revealed fragments of 230 and 1000 basepairs. The antibody used to screen the library was epitope selected against the fusion protein produced by the purified recombinant phage. The epitope-selected antibody, when incubated with Western blots of oviductal explant culture medium obtained from estradiol-treated baboons, recognized both the acidic and the basic variants of the glycoprotein. Northern blot hybridization with a 32P-labeled insert indicated that a single message of 2.8 kilobases was present in oviducts obtained from estradiol-treated baboons; no hybridization was detectable to RNA from oviducts obtained from progesterone-treated baboons. A mRNA of comparable size was also found in human, hamster, rabbit, and mouse oviduct tissue. In vitro translation of oviductal poly(A)+ RNA from an estradiol-treated baboon followed by immunoprecipitation with the polyclonal antibody against the acidic component indicated that the protein component of the oviductal glycoprotein had a mol wt of 66,000.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716346 TI - Cytopathology in pediatrics. PMID- 1716347 TI - Altered Na+ channel activity and reduced Cl- conductance cause hyperexcitability in recessive generalized myotonia (Becker). AB - Intact muscle fibers or resealed fiber segments from 7 patients with recessive generalized myotonia were studied in vitro. All fibers had normal resting membrane potentials and normal resting [Ca2+]i several hours after removal. Contractions were characterized by slowed relaxation which was due to electrical after-activity. Often spontaneous depolarizations were recorded intracellularly. In all fibers, the steady state voltage-current relationship was abnormal, due to a reduced Cl- conductance. However, this conductance ranged from 0% to 66% of the total membrane conductance, whereas, in normal muscle, it was 80%. Theoretically, myotonic after-discharges would not appear until the Cl- conductance is below 20%. Thus, the membrane hyperexcitability must be due to another defect, at least in the preparations in which the Cl- conductance was only slightly reduced. In all patches from all patients investigated with the patch clamp technique, we observed reopenings of the Na+ channels throughout depolarizing pulses (such behavior was absent in normal muscle). If a patch was polarized to potentials less negative than the resting potential, the duration of the reopenings increased. We conclude that a combination of reduced Cl- conductance and the reopenings of Na+ channels underlie the electrical after-activity in recessive generalized myotonia. PMID- 1716348 TI - Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. AB - Mitogen activated protein (MAP) kinases (MAPKs) are a family of protein serine/threonine kinases activated as an early intracellular response to a variety of hormones and growth factors. They are unique in requiring both serine/threonine and tyrosine phosphorylation to become active and are the only examples of protein-serine/threonine kinases activated by tyrosine phosphorylation. Nerve growth factor (NGF) promotes differentiation of phaeochromocytoma (PC12) cells, which respond by conversion within hours from a chromaffin-like to a sympathetic neuron-like phenotype. NGF stimulation of PC12 cells increases the activity of two protein kinases by greater than 20-fold within minutes, both strikingly similar to MAPKs. They are inactivated by either protein-tyrosine phosphatases or the protein-serine/threonine phosphatase termed protein phosphatase 2A (ref. 8), they activate protein S6 kinase-II (refs 9, 10), and they phosphorylate identical threonine residues on myelin basic protein (our unpublished results) to those phosphorylated by other MAPKs. Immunological data indicate that these protein kinases, termed peak-I and peak-II (Fig. 1a) are probably ERK2 and ERK1, respectively, two widely expressed MAPK isoforms. Here we identify the 'MAP kinase kinases' (MAPKKs) in PC12 cells which are activated by NGF and report that MAPKKs are dependent on serine/threonine phosphorylation for activity and promote phosphorylation of serine/threonine and tyrosine residues on MAPKs. PMID- 1716349 TI - Comparison of the presence and actions of substance P and neurokinin A in guinea pig taenia coli. AB - The presence and sites of action of two closely related tachykinins, substance P (SP) and neurokinin A (NKA), were examined in the taenia coli of the guinea-pig. SP- and NKA-like immunoreactivity (LI) were demonstrated histochemically in nerve fibres supplying the taenia. Chromatographic characterization of aqueous acetic acid extracts of taenia showed only one peak of SP-LI, corresponding in retention time to authentic SP, whereas there were multiple peaks of NKA-LI, the major one of which corresponded to authentic NKA. SP-LI and NKA-LI, determined by radioimmunoassay, were in a molar ratio of SP equivalents to NKA equivalents of 8.5:1 in taenia extracts. Extrinsic denervation of the caecum had no significant effect on the concentration of either SP-LI or NKA-LI or on their immunohistochemical distributions. Both SP and NKA (10(-10) to 10(-5) M) caused contractions of the taenia that were unaffected by hyoscine (10(-6) M), mepyramine (10(-6) M) or tetrodotoxin (5 x 10(-7) M), indicating that both peptides act directly on the smooth muscle of the taenia. Contractions to SP occurred after a short, but concentration-dependent, delay, reached a peak quickly, and then decayed. In contrast, NKA caused contractions after longer latencies, the peak was reached more slowly, and the response was maintained for up to 10 min. (D-Pro2, D-Trp7,9)-SP (10(-5) M) antagonised responses to SP and NKA to a similar degree. It is concluded that both NKA and SP should be considered as transmitter candidates for non-cholinergic nerve-mediated excitation in the taenia. PMID- 1716350 TI - Functional properties of strychnine-sensitive glycine receptors expressed in Xenopus oocytes injected with a single mRNA. AB - Mature rat spinal cord cDNA libraries constructed in lambda gt10 and lambda ZAPII were screened with an oligonucleotide probe (39 mer), and 4 clones that possess DNA-inserts encoding a glycine receptor subunit were obtained. The cloned cDNAs were used to reconstruct the nucleotide sequence of the full-length open reading frame consisting of 1350 base pairs (bp) as well as the 5'-(184 bp) and 3'-(591 bp) non-coding regions. Synthetic RNA transcribed in vitro from the glycine receptor cDNA induced Xenopus oocytes to synthesize functional glycine receptor that generated large Cl- currents. The electrophysiological properties of the wild-type receptor and some mutant receptors produced by site-directed mutagenesis were analyzed. PMID- 1716351 TI - Divergent axon collaterals from fastigial oculomotor region to mesodiencephalic junction and paramedian pontine reticular formation in macaques. AB - Collateralization of efferent fibers from the fastigial oculomotor region (FOR) to the paramedian pontine reticular formation (PPRF) and the mesodiencephalic junction (MDJ) was studied in macaque monkeys using a fluorescent double-labeling technique. Retrogradely-labeled neurons in the contralateral FOR were examined following injections of fast blue (FB) into the MDJ and diamidino yellow (DY) into the PPRF, or vice versa. Some FOR neurons were labeled with FB, while some other FOR neurons were labeled with DY and intermingled within the FOR. While single-labeled cells in the FOR projected either to the PPRF or to the MDJ, the presence of double-labeled cells indicated that the FOR contains neurons whose axons collateralize to project to both the MDJ and PPRF. These are regarded as the preoculomotor nuclei responsible for vertical and horizontal saccades, respectively. PMID- 1716352 TI - Application of bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody for the in vivo analysis of proliferative characteristics of human leukemia cells in bone marrows. AB - To investigate the proliferative characteristics of human bone marrow leukemic cells in vivo, we developed a new method using bromodeoxyuridine (BrdU) and its monoclonal antibody. Thirty minutes after a bolus BrdU infusion, a lst bone marrow aspiration (BMP) was performed to calculate the BrdU labeling index (BrdU LI). Then, a 150-min continuous infusion of BrdU was started. Immediately after the infusion, a 2nd BMP was done to analyze the increasing rate of the BrdU-LI for 3 h. Each smear was stained by an immunohistochemical method. The BrdU-LI, the duration of S phase (Ts) and the cell cycle time (Tc) of leukemic cells from 13 previously untreated patients with acute leukemias were estimated. A mean value of 7.2% for BrdU-LI was obtained. Furthermore, mean values of 9.7 h and 152.5 h for Ts and Tc respectively were calculated. This method may prove to be useful in the in vivo cell cycle analysis of leukemias and other malignancies. PMID- 1716353 TI - Corneal staining after sequential instillations of fluorescein over 30 days. AB - The corneal staining patterns of 6 healthy female subjects were monitored Monday through Friday for 4 consecutive weeks using daily sequential instillations of fluorescein. The patterns showed that in these subjects there was no predictable pattern of staining, that each subject had different staining characteristics, and that corneal staining varied day to day. PMID- 1716354 TI - Calcifying odontogenic cyst. A review of ninety-two cases with reevaluation of their nature as cysts or neoplasms, the nature of ghost cells, and subclassification. AB - Ninety-two cases of calcifying odontogenic cyst (COC) were reviewed with special consideration of their nature as cysts or neoplasms, the nature of ghost cells, and classification on the basis of clinicopathologic features. The cases were divided into 79 (85.9%) cysts and 13 (14.1%) neoplasms. The cysts occurred as four variants: (1) nonproliferative COC (35 cases), characterized by a simple unicystic structure; (2) proliferative COC (17 cases), characterized by a cystic structure with multiple daughter cysts, extensive ghost cell formations, and marked tendency for calcification; (3) ameloblastomatous COC (11 cases), characterized by ameloblastoma-like, cyst-lining epithelium with ghost cells and calcifications; and (4) COC associated with odontoma (16 cases), which combined features of COC and odontoma. The neoplasms occurred as three variants: (1) ameloblastoma ex COC (two cases), which showed unifocal and multifocal intraluminal and intramural ameloblastoma proliferating from the COC-lining epithelium; (2) peripheral epithelial odontogenic ghost cell tumor (eight cases), which occurred in the gingiva and resembled peripheral ameloblastoma except for clustered ghost cells in the central portion of epithelial islands and the presence of juxtaepithelial dentinoid; and (3) central epithelial odontogenic ghost cell tumor (three cases). The latter showed ameloblastomatous or adenomatoid odontogenic tumor-like epithelial clusters with ghost cell formation and juxtaepithelial dentinoid. The clinical features of cystic and neoplastic variants were tabulated and described. On the basis of histopathologic features and their immunohistochemical reaction to polyclonal antikeratin antibody, it is suggested that ghost cells might be the result of coagulative necrosis. PMID- 1716355 TI - Central granular cell tumor of the jaw. An electron microscopic and immunohistochemical study. AB - Central granular cell tumor of the jaw was formerly known as granular cell ameloblastic fibroma; recently, the term central granular cell odontogenic fibroma has been proposed. This study attempts to determine the ultrastructural features and selected immunohistochemical properties of the tumor cells. Four formalin-fixed specimens were processed for electron microscopy, and for immunohistochemical staining with antiactin, anti-glial fibrillary acidic protein, and OKT6 (CD1) with the avidin-biotin-peroxidase complex method. Tumor cells contained many primary lysosomes, autophagic vacuoles, and phagocytic vacuoles. The phagocytic vacuoles appeared to contain collagen fibrils. Tumor cells stained positive with antiactin and OKT6 (CD1), and negative with anti glial fibrillary acidic protein. The results indicate that tumor cells are actively phagocytic and suggest that tumor cells might arise from Langerhans' cells. PMID- 1716356 TI - Immunolocalization of 15-kDa membrane proteins in the kidneys of normal and acidotic rats. AB - Proteins with apparent molecular masses between 15 kDa and 17 kDa were enriched from rat renal brush-border membranes by preparative gel electrophoresis and used for immunization of rabbits. The serum of one of the rabbits reacted in Western blots of separated renal brush-border proteins with a single 15-kDa band. A comparably strong reaction is seen with a 15-kDa band of renal endosomal proteins. Basolateral membranes show a much weaker reaction. In light- and electron-microscopic studies the serum stains brush-border membranes and endosomes in rat proximal tubule cells, but not mitochondria and basolateral membranes. In cortical collecting ducts, principal cells are not stained with the antiserum. alpha-type (H(+)-secreting) intercalated cells bind the antibodies at apical tubulovesicles. The luminal membrane is scarcely labelled. Conversely, beta-type (HCO3(-)-secreting) intercalated cells exhibit antibody binding to their basolateral membrane. Thus, the antiserum detects 15-kDa proteins differently sorted in alpha- and beta-intercalated cells. After induction of an acute (6 h) metabolic acidosis, the antibody-binding pattern changes only in intercalated cells, type alpha, and occurs at the markedly enlarged luminal plasma membrane. The amount of alpha-type intercalated cells with enlarged luminal membrane ("secreting cell") increases at the expense of alpha cells with apical tubulovesicles ("resting cell"). Taken together, the antiserum detects 15 kDa proteins, the localization and adaptive changes to metabolic acidosis of which are similar to H(+)-ATPases. The functional role of the 15-kDa proteins needs to be established in further studies. PMID- 1716357 TI - The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression. AB - We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA. PMID- 1716358 TI - The muscle specific domain of mouse N-CAM: structure and alternative splicing patterns. AB - The neural cell adhesion molecule (N-CAM) is an important mediator of calcium independent cell-cell interactions. Variations in the primary structure of the protein are due to alternative splicing of pre-mRNA in the region encoding the extracellular, trans-membrane and cytoplasmic domains. In order to identify the patterns of exon usage during development of skeletal muscle and brain of the mouse, a coupled reverse-transcriptase/polymerase chain reaction was used to identify the murine homologues of the muscle-specific domain (MSD), located between exons 12 and 13 in human N-CAM mRNA. The cDNAs produced have been cloned and sequenced, or analysed directly. The amplification reactions were shown to maintain the concentration ratios of the initial cDNAs. The results indicate that the mouse homologue to exon MSD1a is under tissue and developmental regulation that is independent of exons MSD1b and MSD1c. The inclusion of the triplet exon AAG is also regulated in a cell- and stage-specific manner, which is independent of the other alternatively spliced exons of this domain. PMID- 1716359 TI - Differential expression of novel Gs alpha signal transduction protein cDNA species. AB - The Gs alpha guanine nucleotide-binding signal transduction protein is part of a heterotrimeric complex that is involved in the stimulation of adenylate cyclase upon activation of membrane receptors. We report the characterization of 16 Gs alpha cDNA clones isolated from the human adult retina and fetal eye libraries. Molecular heterogeneity in the 5'-region defines four novel Gs alpha cDNA species which are generated either by alternate splicing or by using alternative promoter. The novel exons upstream of exon 2 interrupt the highly conserved 'region A' in the Gs alpha polypeptide. Non-AUG codons in the novel 5'-exon can initiate translation of these Gs alpha species in vitro. Reverse transcription of total RNA coupled with polymerase chain reaction (RTPCR) using specific primers and in situ hybridization to mRNA in baboon tissue sections with a specific oligonucleotide probe show a high level of expression of these species in retina and brain but not in liver. Differential expression of alternatively spliced Gs alpha species suggests novel signal transducing pathways. PMID- 1716360 TI - Sequence analysis of Saccharomyces exiguus mitochondrial DNA reveals an RNase P RNA gene flanked by two tRNA genes. PMID- 1716361 TI - Sequenase should be used instead of the Klenow fragment for the synthesis of oligonucleotides labeled to a high specific activity. PMID- 1716362 TI - The MspI polymorphism in intron 6 of p53 (TP53) detected by digestion of PCR products. PMID- 1716363 TI - A new RFLP identified at the D3S48 locus. PMID- 1716364 TI - Unraveling the mystery of pain. PMID- 1716365 TI - Ibuprofen for urinary symptoms. PMID- 1716366 TI - Effect of in vivo chromate, acetone and combined treatment on rat liver in vitro microsomal chromium(VI) reductive activity and on cytochrome P450 expression. AB - Cytochrome P450 (P450IIE1) in rat has previously been shown to exhibit high chromate [Cr(VI)] reductase activity (Mikalsen et al. 1991). The present study reports on the effect of chromate treatment in vivo in rats and on the modulating effect of acetone + fasting on chromium(VI) toxicity. No effect of intraperitoneal injection with 5 mg chromate/kg was observed, whereas 15 mg chromate/kg decreased the liver microsomal Cr(VI) reductase activity by about 30% in in vitro microsomal incubations. In addition, the P450 and cytochrome b5 contents were decreased by about 30% and 25% respectively. Acetone + fasting caused increases of total microsomal P450 and cytochrome b5 contents, associated with similar increases in apoproteins P450IIE1 and P450IIB1 + 2, and their corresponding mRNA, and apoprotein NADPH-P450 reductase, as well as NADPH-P450 reductase and microsomal Cr(VI) reductive activities. Related to acetone + fasting alone, when given in combination with chromate (15 mg/kg) the Cr(VI) reductive activity was decreased by about 30%, associated with decreases in the P450 and cytochrome b5 contents, 65% and 35% respectively. This further reduced the apoprotein levels of P450IIB1 + 2, P450IIE1, and NADPH-P450 reductase to 90%, 60%, and 40%, respectively, and the mRNA levels of P450IIB2 and P450IIE1. No effect was observed on NADPH-P450 reductase activity. This dose also caused some macroscopic alterations in the liver. In contrast, the P450IIE1 apoprotein level in the lung was apparently stabilized or even increased by chromate in rats treated with acetone + fasting.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716368 TI - New information relating to the prognosis of cardiac arrhythmias. PMID- 1716367 TI - Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin. AB - The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy. PMID- 1716369 TI - Newly recognized lipid carrier proteins in fetal life. AB - An attempt is made in this review to summarize new findings that propose the direct involvement of two fetal proteins (i.e, fetuin and alpha-fetoprotein) in fatty acid transport and delivery to cells. The presence of these proteins at a high plasma concentration in fetal life and the critical need for fatty acids during development strongly support this hypothesis. Tissue and cell specificities involved in the uptake of these fetal proteins and the mechanism of fatty acid delivery awaits further research. PMID- 1716370 TI - The peptide Glu-His-Ile-Pro-Ala binds fibrinogen and inhibits platelet aggregation and adhesion to fibrinogen and vitronectin. AB - Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-Ile-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-Ile-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site. PMID- 1716371 TI - Nortrilobolide, a new potent guaianolide secretagogue from Thapsia garganica. PMID- 1716372 TI - Money meanings and madness: a psychoanalytic perspective. PMID- 1716373 TI - [Alpha fetoprotein and hepatic metastasis]. PMID- 1716375 TI - The combination of symbolic and numerical computation for three-dimensional modeling of RNA. AB - Three-dimensional (3-D) structural models of RNA are essential for understanding of the cellular roles played by RNA. Such models have been obtained by a technique based on a constraint satisfaction algorithm that allows for the facile incorporation of secondary and other structural information. The program generates 3-D structures of RNA with atomic-level resolution that can be refined by numerical techniques such as energy minimization. The precision of this technique was evaluated by comparing predicted transfer RNA loop and RNA pseudoknot structures with known or consensus structures. The root-mean-square deviation (2.0 to 3.0 angstroms before minimization) between predicted and control structures reveal this system to be an effective method in modeling RNA. PMID- 1716374 TI - The sources of chemical substances in allergic nasal fluid. AB - The sources of different chemical substances in the NF of allergic patient, such as albumin, secretory IgA, histamine, leukotriene, kinin and substance P were investigated. To accomplish this, we challenged the inferior turbinate on one side, but separately collected NF from both sides in patients with nasal allergy to house-dust. Provocation was done with paper disc containing dried allergen extract. Collection was done by suction for the first five minutes immediately after the onset of a positive response to nasal provocation. The total amount of the chemical substances on each side was analyzed separately and compared. Significant differences were seen between both sides only for histamine and leukotriene. In consideration with the previous reports, it is suggested that in nasal allergen challenge the major sources are glandular secretion for secretory IgA, and albumin, and secretion for migrating cells for histamine and leukotriene. The major sources responsible for kinin and substance P, however, are not defined. PMID- 1716376 TI - Some aspects of the regulation of erythropoietin gene expression. PMID- 1716377 TI - Regulation of erythropoietin gene expression. PMID- 1716378 TI - Enhancement of the thrombolytic efficacy of prourokinase by lys-plasminogen in a dog model of arterial thrombosis. AB - Current findings suggest that the efficacy of thrombolytic therapy may be limited by the availability of active forms of plasminogen at the thrombus site. The purpose of this study was to determine if the systemic administration of 0.5 mg kg-1 glu-plasminogen (glu-plg) or 0.5 mg kg-1 lys-plasminogen (lys-plg) could safely increase the efficacy of a single intravenous bolus injection of 50,000 U kg-1 prourokinase (proUK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by direct visual examination. Forty dogs (mean wt 10.3 +/- 2 kg) were randomly sorted into 4 groups of 10 each. The dogs in each group were given either saline plus saline, saline plus proUK, glu plg plus proUK, or lys-plg plus proUK 60 minutes after formation of an occlusive arterial thrombus. Ninety minutes after drug administration the dogs receiving saline plus proUK, glu-plg plus proUK, and the lys-plg plus proUK showed greater thrombolysis (41%, 43%, and 66%, respectively) than the control (saline plus saline) group (15%, P less than 0.01). The lys-plg plus proUK treatment caused greater lysis than the saline plus proUK or the glu-plg plus proUK treatment (P less than 0.05). All of the dogs (10/10) receiving lys-plg plus proUK had patent vessels at the end of the 90 minute monitoring period, whereas only 4/10 and 5/10 vessels were patent in the saline plus proUK and glu-plg plus proUK groups, respectively. None of the dogs in the saline plus saline group had patent vessels. No significant changes were observed in the various coagulation parameters tested for any of the 4 treatment groups. The results show that lys plg can safely increase the thrombolytic efficacy of proUK. PMID- 1716379 TI - Quantitative isolation of RNA from human platelets. AB - We have adapted the acid-guanidinium-phenol-chloroform extraction procedure of Chomczinsky and Sacchi to achieve efficient rapid recovery of total RNA from human platelets. Sufficient platelet RNA (20 micrograms of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot analysis on individual donors and detect the mRNAs for glycoproteins IIb1(GP IIb) and IIIa1(GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our technique allows simultaneous Northern blotting and PCR, and therefore should be of great help to the characterization of inherited platelet disorders such as Glanzmann's thrombasthenia. PMID- 1716380 TI - Nonradical excited oxygen species induce selective thrombolysis in vivo. AB - All the thrombolytic agents currently in clinical use act as plasminogen activators. In this study evidence is presented that also oxidants of the phagocyte type are of fibrinolytic efficiency in vivo. Activated phagocytes participate in physiologic fibrinolysis. The cells generate plasminogen activators and reactive oxidants of the nitrogen-chlorine type. Experimental mimicry of this oxidative inflammatory response induces selective thrombolysis in a rabbit jugular vein model. Intravenous bolus administration of sub-millimolar blood concentrations of chloramine-T resulted in thrombolysis after about 30 min without notable systemic toxicity; the coagulation parameters activated partial thromboplastin time (aPTT), thrombin time, fibrinogen, and alpha-2-antiplasmin were not influenced. Control experiments with 2000 IU of urokinase/kg induced thrombolysis after about 90 min with systemic changes of the hemostatic system. The fibrinolysis promoting effect of the oxidants of the phagocyte type could be inhibited by quenchers of singlet molecular oxygen and was not affected at all by inhibitors of oxygen radicals. The data gives evidence that nonradical excited oxygen species (NEOS) act as powerful pro-fibrinolytic and anti-coagulant agents in vivo. It might be suggested that NEOS could represent a novel class of regulators of the fibrinolytic system. The long lived and hydrophilic chloramine derivatives can either accumulate or diffuse far from their site of generation. Therefore, on the one hand oxidants in high (local) concentrations might be considered as direct pro-fibrinolytic agents due to their powerful protein modulating efficacy. On the other hand, oxidants at low concentrations may act as indirect pro-fibrinolytic compounds, i.e. as chemoattractants to concentrate phagocytes to the site of a thrombus. In this case the oxidants would play the role of signal elements faraway from the thrombus, a self amplifying mechanism possibly mediated by oxidation of blood arachidonat/lipid metabolites. PMID- 1716381 TI - Platelet membrane glycoproteins and platelet functions during storage in the presence of a proteinase inhibitor. AB - The effect of the proteinase inhibitor aprotinin on membrane glycoproteins and functions of platelets stored for 5 days in platelet-rich plasma was tested. Platelet membrane glycoprotein content was determined by flow cytometry or immunoblot techniques using different monoclonal antibodies. ADP- and ristocetin induced platelet aggregation and adhesion to collagen were tested in parallel. Using the flow-cytometry technique i) a progressive decrease in the percentage of platelets reacting with the different monoclonal antibodies was observed during storage ii) a 30% reduction of the GPIb mean fluorescence intensity (MFI) was observed after 5 days storage while the MFI of the GP IIb-IIIa complex was not modified. Using the immunoblot technique, a decrease in the amount of both the GPIb alpha and the component of Mr 100,000 was observed, while a 50,000 Mr fragment appeared progressively. Platelet adhesion and aggregation were reduced after 24 hours of storage. Aprotinin prevented neither the GPIb alpha reduction nor the modifications of the functions of human platelets stored in their autologous plasma. PMID- 1716382 TI - Potentiation of BrCCl3 hepatotoxicity by chlordecone: biochemical and ultrastructural study. AB - Previous work has established that chlordecone (CD) potentiates the hepatotoxicity of BrCCl3. This interaction occurs at nontoxic levels of CD and BrCCl3. The present research was designed to investigate the mechanism governing the pathogenesis of potentiated hepatic injury and lethality induced by a low dose of BrCCl3 after dietary pretreatment with 10 ppm of CD for 15 days. On Day 16, a single dose of BrCCl3 (30 microliters/kg) was administered ip to rats maintained either on normal diet (ND) or on a diet contaminated with 10 ppm CD. Blood and liver samples were collected at 0, 3, 6, 12, 24, 36, and 48 hr after the halomethane administration for biochemical (ATP, bilirubin, glycogen) and for ultrastructural studies. A continuous increase in serum bilirubin and decrease in hepatic ATP and glycogen were observed in CD + BrCCl3 combination, indicating progressive injury, but not in other treatment groups. In ND + BrCCl3 combination, all biochemical indices were either normal or close to normal after 36 hr, suggesting complete recovery from hepatotoxicity. The most extensive ultrastructural changes characteristic of halomethane hepatotoxicity (necrosis, ballooned cells, and dilation of rough endoplasmic reticulum) were observed after the CD + BrCCl3 combination treatment. The progressive and early depletion of hepatic ATP and glycogen, and the progressive increase in toxicity along with decreased cell division in CD + BrCCl3-treated rats, indicate the association of compromised energy status and suppression of cell division and tissue repair in CD-potentiated BrCCl3 toxicity. These findings suggest that the suppression of stimulated hepatocellular regeneration results in the loss of the essential mechanism of tissue repair leading to continuation of the toxic liver injury associated with the CD + BrCCl3 combination treatment. PMID- 1716383 TI - Male rat specific nephrotoxicity resulting from subchronic administration of hexachlorobenzene. AB - Male rats are more sensitive to the nephrocarcinogenic effect of hexachlorobenzene (HCB) than are female rats. The purpose of this study was to shed light on this phenomenon by investigating mechanisms of subchronic nephrotoxicity of HCB. Groups of rats were administered HCB in corn oil (po) at 100 mg/kg, 5 days per week for 15 days or at 50 mg/kg, 5 days per week for 50 days. Urine was collected on Days 1, 8, and 15 for the 15-day treatment and on Day 50 for the 50-day treatment. Glucosuria, proteinuria, and enzymuria (gamma glutamyl transpeptidase) were measured to assess renal function. Twenty-four hours after the last HCB treatment, the animals were killed and kidneys were removed for histopathological evaluation. Urine analyses showed no indication of renal dysfunction in treated animals compared to controls during the 15-day treatment. However, histology of male rat kidneys revealed degenerative and regenerative cellular foci accompanied by an increased accumulation of protein droplets in epithelial cells of the proximal tubules. The same histological observations were also made in male rats after a 50-day HCB treatment but this time they were accompanied by renal function alterations. In female rats, no such renal functional or histological alterations were observed. The histopathological observations in male rats correspond well with the protein droplet nephropathy; the latter is characteristic of the accumulation in kidney cells of alpha 2u globulin probably caused by the reversible binding of a chemical to alpha 2u that renders the protein indigestible to kidney proteases. alpha 2u-Globulin was measured in the cytosol of male rats and was found to be increased 11-fold compared to controls. Also, HCB was found to be bound reversibly to alpha 2u. These results suggest that HCB induces a male rat specific nephropathy that could explain the higher incidence of kidney tumors in male rats compared to female rats. PMID- 1716384 TI - Sia-b1 and I antigens recognized by Mycoplasma pneumoniae-induced human cold agglutinins. AB - Postinfection cold agglutinins (CAs) with anti-Sia-b1 (i.e., anti-Sialo-branched) specificity frequently occurring together with anti-I CAs recognize antigenic determinants that are present on Oh red cells and are partially destroyed by endo beta-galactosidase on the red cell surface. They differ markedly from the monoclonal anti-Sia-b1 FI CA, which recognizes an epitope not expressed on Oh cells, and resistant to the enzyme. On the other side, Sia-b1 determinants reacting with postinfection CAs share the characteristics of I determinants, with the exception that Sia-b1 determinants require sialyl groups as an immunodominant component. Five sera containing coexisting anti-Sia-b1 and anti-I CAs were tested against Oh and enzyme-treated group O red cells. The results are consistent with a postinfection autoimmune response against a sialo-type 2 structure common for Sia-b1 and I determinants, which could serve as a receptor for Mycoplasma pneumoniae. PMID- 1716385 TI - Preliminary studies of photoinactivation of human immunodeficiency virus in blood. AB - The transmission of human immunodeficiency virus (HIV) by blood or blood components is a major concern in blood banking. A photodynamic flow cell system was designed to inactivate cell-free HIV mixed with blood from a healthy donor. Blood containing 4 x 10(3) infectious units of HIV was treated with 10 and 20 micrograms per mL of commercially available dihematoporphyrin ether (DHE) per mL. Aliquots of this mixture were then held in the dark or irradiated in a flow cell illuminated at a light energy density of 5 J per cm2 provided by a xenon light source equipped with a 630 +/- 5 nm band-pass interference filter; the aliquots were subsequently placed in A.301 cells. All infected cultures were assessed for reverse transcriptase (RT) activity for 17 days. RT activity for either concentration of dye was significantly reduced in irradiated samples as compared to that in samples held in the dark. Blood samples from volunteers also were assessed for the effects of the inactivation process on red cells at concentrations of DHE up to 200 micrograms per mL. No effects were observed on red cell 2,3 DPG or ATP, whole blood potassium concentrations, red cell osmotic fragility, or blood cell antigens. PMID- 1716386 TI - RNA recognition: towards identifying determinants of specificity. AB - Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition. PMID- 1716387 TI - Light-induced, GTP-binding protein mediated membrane currents of Xenopus oocytes injected with rhodopsin of cephalopods. AB - Xenopus oocytes that were injected with rhabdomeric membranes of squid and octopus photoreceptors acquired light sensitivity. The injected oocytes showed a light-induced current having characteristics similar to other G-protein-mediated Cl- currents induced by the activation of other membrane receptors. Pretreatment of the oocytes with pertussis toxin before the injection suppressed the generation of the light-induced current, indicating an ability of cephalopod rhodopsin to cross-react with an endogenous G-protein of Xenopus oocytes. PMID- 1716388 TI - Effects of IBMX on the rod ERG of the isolated perfused cat eye: antagonism with light, calcium or L-cis-diltiazem. AB - Full-field electroretinograms (ERGs) were recorded from isolated cat eyes perfused through the ophthalmociliary artery with the cGMP-PDE inhibitor, 3 isobutylmethylxanthine (IBMX). Under dark-adapted conditions perfusion with IBMX resulted in reduced ERG b-wave amplitudes at low stimulus luminances and supernormal b-wave amplitudes at high stimulus luminances with reduced b-wave sensitivity; b-wave implicit times were more delayed at low than at high stimulus luminances. Presentation of a steady white background or high calcium fully reversed the supernormal amplitudes and partially reversed the delayed implicit times produced by IBMX. Rod ERG b-wave sensitivity, reduced with IBMX alone, was partially reversed with calcium but further reduced with background light. Perfusion with the cation channel blocker, L-cis-diltiazem, also reversed the supernormal amplitudes produced by IBMX but had no effect on implicit times or b wave sensitivity. Possible mechanisms of action of these antagonists and clinical implications of these findings are considered. PMID- 1716389 TI - [Gamma-globulins in the prevention and therapy of herpesvirus infections]. PMID- 1716390 TI - [The characteristics of influenza A/H1N1 viruses related to A/PR/8/34 isolated in the Mongolian People's Republic]. AB - Investigations of the antigenic structure and genome of influenza A (H1N1) viruses isolated in the Mongolian People's Republic in 1982, 1983, 1986 and 1987 from children with acute respiratory diseases using monoclonal antibodies and nucleotide sequencing revealed 4 strains identical to the prototype strain A/PR/8/34 (H1N1), variant M. Sinai, and 8 strains closely similar to the epidemic strain A/USSR/90/77 (H1N1) in the antigenic structure and A/Leningrad/54 (H1N1) in the primary structure of HA. PMID- 1716392 TI - [The detection of the human immunodeficiency virus antigen in the lymphoid cells of seropositive subjects]. AB - Cells from anti-HIV-positive persons were used in experiments for virus isolation. The RT-activity, viral antigen, nucleic acids, electron microscopic morphology, and infectivity were studied. The data presented allow a conclusion that the virus was isolated. These data confirmed the previous diagnosis of HIV infection. PMID- 1716391 TI - [2-site competitive immunoenzyme analysis based on monoclonal antibodies for the detection and titration of antihemagglutinating measles antibodies]. AB - Using monoclonal antibodies (MCA) to measles virus (strain Leningrad-16) hemagglutinin, a competitive enzyme immunoassay (cEIA) was developed for the detection and titration of antihemagglutinating antibodies in human sera. Serum samples from children, collected in measles foci, were tested in cEIA, SP-EIA, HI, and PHA tests. The results evidence a high correlation of antihemagglutinin titres in cEIA and HI tests, the correlation coefficient of cEIA and PHA being 97.7%. The cEIA based on MCA to measles virus hemagglutinin is highly specific for the detection and titration of measles antihemagglutinins, it does not involve the use of simian erythrocytes. A significant advantage of this test system consists in the possibility of using unpurified antigen prepared from the infected cell lysate. PMID- 1716393 TI - [The differentiation of viruses in the Venezuelan equine encephalomyelitis complex by using monoclonal antibodies and lanthanide immunofluorescence analysis]. AB - Potentialities of differentiation between Venezuelan equine encephalomyelitis (VEE) complex viruses by time-resolved fluoroimmunoassay and enzyme immunoassay were studied. For this, 4 test systems were used based on different combinations of native and labeled polyclonal antibodies to VEE virus, strain Trinidad, and monoclonal (MCA) antibody MAK 14-7 to protein EL of this virus. The maximal sensitivity and specificity was achieved in the test system formed from native MCA MAK 14-7 for sensitization of the solid phase and labeled polyclonal immunoglobulins for demonstration of the test results. This combination of antibodies allowed to differentiate the epidemic variant of VEF/Trinidad (IA) from epizootic variants of Mucambo (III), Pixuna (IV) and attenuated strain No. 230. PMID- 1716394 TI - [The interferon status in recurrent genital herpes]. PMID- 1716395 TI - [The protease inhibitor treatment of the disseminated intravascular coagulation syndrome in patients with liver cirrhosis]. AB - The results of the treatment of the syndrome of disseminated intravascular coagulopathy (DIC) in 41 patients with liver cirrhosis are reported. A relation between the severity of the liver impairment and the degree of the increase of the fibrin split products (FSP) was established. FSP above 100 micrograms/ml were found in patients with III class liver failure according to Child-Turcotte-Pugh and rating above 6.5 according to Z. Krustev. The administration of 30,000-60,000 U of the protease inhibitor contrykal led to mastering of the hemorrhagic diathesis in 19 of 21 patients (group A). The increased FSP in the serum fell (chi 2 = 5.79; p less than 0.001). In contrast, the administration of PAMBA and styptanon did not affect the hemorrhagic diathesis and DIC syndrome in 16 of 20 other patients (group B--chi 3 = 20.54; p less than 0.001). The authors recommend protease inhibitors as the treatment of choice of DIC syndrome in liver cirrhosis. Only in case of lack of effect should heparin be added. PMID- 1716396 TI - Localization and developmental change of indoleamine 2,3-dioxygenase activity in the human placenta. AB - Previously, we pointed out the importance of the kynurenine metabolism in fetuses and neonates. We examined localization and developmental change of indoleamine 2,3-dioxygenase activity in human placenta. The indoleamine 2,3-dioxygenase was found localized in syncytiotrophoblast in the placenta. The indoleamine 2,3 dioxygenase activity was not detected in placenta in the early stage of gestation. It was first detected at around 14 weeks of gestation, increased rapidly thereafter and was maintained at high levels till near term. The indoleamine 2,3-dioxygenase activity was significantly lower in placenta with retarded intrauterine development. These results suggest the importance of placental indoleamine 2,3-dioxygenase during fetal development. PMID- 1716397 TI - Purification of lipid-associated basic protein from guinea pig spinal-cord myelin. AB - Myelin basic protein (MBP) was purified from guinea pig spinal-cord in a native like form retaining the binding to all the myelin lipids. Since the guinea pig MBP was found to be much more labile than the corresponding MBP from bovine brain, the original procedure based on the use of hydroxyapatite and detergents was slightly modified as reported here. The product of this purification, lipid bound MBP, may represent an alternative to lipid-free MBP for the induction, the study and the treatment of experimental allergic encephalomyelitis. PMID- 1716398 TI - Myelin basic protein in lipid-bound form induces experimental allergic encephalomyelitis and demyelination in Lewis rat. AB - Myelin basic protein (MBP) was isolated from guinea-pig spinal cord in a form retaining the binding to all the myelin lipids. This new, lipid-bound and native like preparation was used to immunize Lewis rats in complete Freund's adjuvant (CFA) in order to produce experimental allergic encephalomyelitis (EAE). The clinical features were compared with those of Lewis rats immunized with lipid free MBP (LF-MBP), myelin, LF-MBP + octyl-POE (the non-ionic detergent used for the purification of LB-MBP) and octyl-POE alone. The clinical observation indicate that LB-MBP exerts an encephalitogenic activity on Lewis rats which is more intense than LF-MBP and includes demyelinating lesions in the central nervous system (CNS). The data suggest that LB-MBP is a new encephalitogenic antigen, which may induce more intensive immunization in rats and may be relevant in humans for autoimmune demyelinating diseases of the CNS. PMID- 1716399 TI - Spontaneous adhesiveness of lipid-free myelin basic protein to immune cells as detected by a double labelling technique. AB - We have investigated whether immunocompetent cells have the capacity to interact directly with the myelin basic protein (MBP) of the central nervous system. To this end we have applied a double tagging system in order to study whether purified lipid-free MBP is able to bind to normal peripheral blood mononuclear cells (PBMC) without the need to purify the cells. Evaluation of MBP binding to PBMC was determined with biotinylated MBP and fluoresceinated avidin, and lymphocytes population was identified by the corresponding phycoerythrinated monoclonal antibody (MoAb). The contemporary use of MoAbs and avidin unambiguously showed that MBP is able to bind to both B and T lymphocytes. The biological significance of MBP adherence to immune cells still needs clarification. PMID- 1716400 TI - Restricted endogenous proteolysis of myelin basic protein of zinc-treated myelin. AB - Isolated myelin of bovine spinal cord was saturated with 500 mu ZnCl2 to immobilize myelin basic protein (MBP) and to assess the degradation of endogenous or exogenous MBP by neutral proteinase activity of myelin. While the initial degradation of endogenous MBP was not affected by Zn2+ a single fragment of approximately 17kDa accumulated in zinc-treated myelin as compared to several fragments in the control. In contrast exogenous MBP was fully degraded even by zinc-saturated myelin. In immobilizing MBP zinc appears to limit the access of endogenous proteinases to a terminal portion of the primary structure of myelin associated MBP. PMID- 1716401 TI - Zinc as an inhibitor of myelin basic protein proteolytic breakdown in the central nervous system. AB - It is known that central nervous system myelin contains proteolytic enzymes which degrade the myelin basic protein (MBP). We have found that zinc acetate is able to inhibit MBP cleavage at concentrations of 1 mM or higher. Furthermore, the Zn inhibitable MBP-degrading activity was found to be water-soluble and able to recognize MBP also if this protein was protected by its lipidic environment in the native-like, lipid-bound form. Data presented here suggest a Zn-dependent metallo-protease which recognize MBP as a substrate probably even in the myelin sheath, when the protein is not yet released from the membrane. PMID- 1716402 TI - Interaction of cations with lipid-free myelin basic protein. A spectroscopy study. AB - The interaction of some divalent cations with myelin basic protein (MBP) in buffer and in model membranes was studied by using the static fluorescence of the intrinsic tryptophan residue of the protein. Results were indicative of Zn++ ability to bind to MBP. The observed binding could facilitate the interaction of MBP with lipids and have a role in stabilizing the myelin sheath. PMID- 1716403 TI - Myelin basic protein and its free and bound antibodies in cerebrospinal fluid. All three must be determined on each specimen. AB - Myelin basic protein (BP) in the cerebrospinal fluid (CSF) is an important marker of brain damage, especially of white matter, but low or "normal" values can be misinterpreted if the CSF is not also examined for free and bound antibodies to BP. BP has many epitopes (antigenic determinants) and is very susceptible to fragmentation by proteolytic enzymes that are frequently very active in CSF, especially in patients with neurological diseases. This combination of factors permits BP to be degraded as it is released from myelin and to allow antibodies to those epitopes destroyed by enzymatic action to persist free in the CSF. Other free anti-BP antibodies may simply represent antibody excess. The relatively frequent formation of soluble antigen-antibody complexes with other epitopes on BP permits the existence of anti-BP antibodies bound to BP also in CSF. In addition to these factors that contribute to low values of free BP in CSF, if the analyses are not performed promptly on CSF collected in plastic tubes (polystyrene but preferably polypropylene), the "natural" adhesiveness of BP (immediately to glass, slowly to plastic) can remove it from the CSF, resulting in artifactually low values which can also be misinterpreted as "normal". PMID- 1716404 TI - Serum gamma-seminoprotein determination in prostatic cancer. AB - Serum gamma-seminoprotein (gamma-Sm) was evaluated as a new marker for prostatic cancer in comparison with prostatic acid phosphatase (PAP). The sensitivity of gamma-Sm and PAP for untreated prostatic cancer was 81% and 67%, respectively. gamma-Sm showed a higher positive rate over all stages than in benign prostatic hypertrophy (BPH). There was no correlation between gamma-Sm and PAP in prostatic cancer. Improved sensitivity was obtained by simultaneous measurement of gamma-Sm and PAP. Specificity of gamma-Sm and PAP for BPH was 87% and 90%, respectively. gamma-Sm normalized after endocrine therapy for stage D2 more often than did PAP. These results indicate that gamma-Sm is another useful marker to evaluate prostatic cancer. PMID- 1716405 TI - [Significance of serum ferritin level in testicular tumors]. AB - The clinical value of serum ferritin level in patients with testicular cancer was studied. Seven cases of seminoma and nine cases of non-seminoma from 1983 to 1989 were evaluated. The serum levels of ferritin, human chorionic gonadotropin (beta HCG), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) were estimated before and after treatment. Abnormally high values of serum ferritin before treatment were noted in 4/7 (57%) in seminoma, 3/9 (33%) in non-seminoma and 7/16 (44%) in total. The total rate showing abnormally high values of serum ferritin was lower than that of beta-HCG and LDH. Meanwhile it was the same as that of AFP and higher than that of CEA. Changes in the serum ferritin level did not always correspond with the clinical course. In 3 out of 6 tumor free patients, higher levels of serum ferritin before treatment became normal after treatment. In one patient with a high level of serum ferritin before treatment, the level of serum ferritin remained higher and retroperitoneal lymph node metastasis developed after treatment. In 9 cases with normal serum ferritin level, 7 showed the normal range of ferritin level throughout the treatment course. These findings suggests that in some patients with testicular cancer, the serum ferritin level might serve as a tumor marker indicating the efficacy of the treatment and the tumor recurrence. PMID- 1716406 TI - [Analysis of ferritin immunostaining in testicular tumors]. AB - Localization of ferritin in testicular tumors was studied by the immunohistochemical method and the usefulness of ferritin was evaluated compared with the clinical course. Seven cases of seminoma and 9 cases of non-seminoma were used for the study. Formalin-fixed, paraffin-embedded tissue sections were stained by the avidin-biotin complex method. Commercial rabbit anti-human ferritin polyclonal antibody in 1/100 dilution was allowed to react at room temperature for one hour. In normal testicular tissues, the epithelium in germinal cells was not stained for ferritin. In seminomas, some tumor nests were stained for ferritin. Interstitial cells, especially histiocytes, were also stained for ferritin. In stained tumor cells, cytoplasm was stained uniformly. Necrotic cells were not stained. The same findings were obtained in non seminomas. In metastatic lesions and tumor thrombi in the vessels, some tumor cells were stained as intensely as in the origin. A case was calculated positive if more than 5% of the tumor cells in the specimen were stained. The positive rate in ferritin immunostaining was significantly higher than that of human chorionic gonadotropin (beta-HCG), alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) immunostaining with the same materials. The specimens from cases with abnormally high serum ferritin level, were stained more intensely than those from cases with normal serum ferritin level. The result suggests that ferritin might be a useful tumor marker in some of testicular tumors. PMID- 1716407 TI - [Clinical efficacy and safety of long-term administration of YM617 for urinary obstruction of the lower urinary tract]. AB - The efficacy and safety of YM617, a new alpha 1-blocker, were evaluated in 121 patients with urinary obstruction of the lower urinary tract in the long-term administration study for 1 year. A total of 121 patients, i.e., 111 with benign prostatic hypertrophy (BPH), 6 with bladder neck sclerosis (BNS), 3 with BPH with BNS and 1 with female urinary obstruction, were orally administered 0.1 to 0.4 mg of YM617 once daily. Patients showing improvement of patient's impression and of subjective symptoms judged by the physician were increased until the 16th to 20th week, and it had been maintained there after. Objective findings were improved to a constant level after the 4th week. Patient's impression and subjective symptoms were improved in a certain dose-response relationship Side effects were observed in 3 patients but none were serious. YM617 proved to be useful in the treatment of patients with urinary obstruction of the lower urinary tract like BPH because it maintained the efficacy and safety in the long-term administration. PMID- 1716408 TI - [The development of continuous crystallizer and observation of formation and growth of calcium oxalate crystals]. AB - A continuous flow crystallizer system of calcium oxalate has been developed. Crystal nucleation rate (B0) and growth rate (G) were calculated by the formula by Randolph and Larson. Nucleation rate, growth rate and crystal mass concentration were decreased with increase of residence time. A linear relationship was observed between growth rate and nucleation rate (B0 = 1.58 x 10(4)G0.924, r = 0.981). Crystals formed in the solution were composed of calcium oxalate dihydrate by infrared spectroscopic analysis. Growth rate and crystal mass did not change between pH 5.0 and 6.5 but increased above pH 7.0. CG-120 and sodium pentosan polysulfate inhibited the nucleation rate, crystal mass concentration but did not influence the growth rate of the crystals. PMID- 1716409 TI - [Clinical and pathological study of tumor marker in benign prostatic hypertrophy and incidental prostatic cancer]. AB - To determine the value of prostatic markers for prostate cancer, serum prostatic acid phosphatase (PAP), prostate specific antigen (PSA) and gamma-Seminoprotein (gamma-Sm) were measured in 81 patients with benign prostatic hypertrophy and in 12 patients with incidental prostatic cancer. gamma-Sm was the most sensitive but the least specific of the three markers. Large prostate glands, especially hyper glandular type tended to be associated with high gamma-Sm levels in our study. Patients with acute urinary retention, acute prostatitis and necrosis also showed positive markers. Out of 12 patients with incidental cancer, 5 patients had more than 2 elevated markers. Four patients with poorly differentiated adenocarcinoma failed to show increased markers. PMID- 1716410 TI - [Ceftazidime concentration in human prostatic tissue and serum following intravenous injection]. AB - The present study was undertaken to evaluate the penetration of Ceftazidime (CAZ) into prostatic tissue (P) and serum (S). Thirty-seven patients with benign prostatic hypertrophy took part in this study. CAZ was administered intravenously at a dose of 1g preoperatively. Blood samples were taken simultaneously at the time of tissue sampling by transurethral resection of the prostate (TUR-P). The patients were divided into 3 groups. In group 1 (twelve patients), intravenous injection of the drug was given 60 minutes before tissue sampling. The mean concentrations of CAZ were 23.4 +/- 7.4 micrograms/g in the prostatic tissue and 45.0 +/- 15.9 micrograms/ml in the serum. In group 2 (thirteen patients), the injections were given 120 minutes before tissue sampling. The mean concentrations of CAZ were 18.0 +/- 8.2 micrograms/g in the prostatic tissue and 39.8 +/- 21.3 micrograms/ml in the serum. In group 3 (twelve patients), the injections were given 240 minutes before tissue sampling. The mean concentrations of CAZ were 11.3 +/- 3.2 micrograms/g in the prostatic tissue and 22.3 +/- 9.6 micrograms/ml in the serum. These findings indicate that CAZ is useful for the treatment of prostatitis and preventive medication before TUR-P. PMID- 1716411 TI - Late intravenous gamma globulin treatment in infants and children with Kawasaki disease and coronary artery abnormalities. PMID- 1716412 TI - Ki-67 staining as a means to simplify analysis of tumor cell proliferation. PMID- 1716413 TI - The slide centrifuge gram stain as a urine screening method. AB - A slide centrifuge Gram stain procedure was performed to screen for bacteriuria 4161 urine specimens submitted in urine preservative tubes for routine culture. For slide centrifuge Gram staining, each urine sample was mixed well. Thereafter, 0.2 mL of each sample was placed, using a pipette, into a slide centrifuge chamber and centrifuged at 2,000 rpm for 5 minutes. The slides were heat fixed, Gram stained, and read by laboratory personnel who scanned 12 consecutive oil immersion fields using a set pattern. The presence of the same organism in six or more fields was defined as a positive urine screen. Urine samples were cultured using a 0.001-mL loop and a comparison of culture growth with slide centrifuge screening was made. When growth of 100,000 or more colony-forming units per milliliter (CFU/mL) was the reference for comparison, the screen had a sensitivity rate of 98%, a specificity rate of 90%, a negative predictive value of 99%, and a positive predictive value of 65%. When a lower colony count of 10,000 or more CFU/mL was the reference for comparison, the screen had a sensitivity rate of 88%, a specificity rate of 95%, a negative predictive value of 96%, and a positive predictive value of 84%. The slide centrifuge Gram stain is a very sensitive screening method to detect bacteriuria in an adult male population. PMID- 1716414 TI - Tumor proliferative fraction in solid malignant neoplasms. A comparative study of Ki-67 immunostaining and flow cytometric determinations. AB - Tumor proliferative fraction (TPF) has been shown to correlate with prognosis in some malignancies. A method for its determination that is practical, accurate, and reproducible is still being sought. In this comparative study of techniques, TPF values were determined in mirror-image samples of 126 consecutive solid malignant neoplasms using flow cytometry and immunostaining with Ki-67, a monoclonal antibody that recognizes an unknown nuclear antigen expressed during the entire cell proliferation cycle but not in resting cells. The mean TPF values for all cases were 19.5 +/- 15.6% (percentage of tumor cells stained) by Ki-67 (range, 1-86%) and 15.7 +/- 9.6% (S + G2M) by flow cytometry (range, 3-60%), which correlated significantly at r = 0.53 and P = 0.005. The correlation was less strong in tumors with low S-phase values (less than or equal to 10%, r = 0.28) than in tumors with intermediate and high S-phase values (r = 0.66). Ki-67 staining percentages did not correlate with patient age, sex, or tissue origin of the tumor. Ki-67 staining appears comparable to flow cytometry determination of TPF in solid malignancies with intermediate and high S-phase values. In tumors with low S-phase values, Ki-67 immunostaining shows higher TPF values, which perhaps reflect an increase in the proportion of G1-phase cells or dilutional effect of nonneoplastic cells in the tumors with low proliferative fraction. PMID- 1716415 TI - Glassy cell features in adenosquamous carcinoma of the uterine cervix. Histologic, ultrastructural, immunohistochemical, and clinical findings. AB - Glassy cell features (GCF) were identified as composing a predominant pattern (more than 85% of histology) in six cases and a focal pattern (33-85% of histology) in 10 cases of a series of 53 adenocarcinomas (AC) and adenosquamous carcinomas (ADSQ) of the uterine cervix. In three cases examined ultrastructurally, GCFs correlated with many cytoplasmic polyribosomes and abundant rough endoplasmic reticulum, but Golgi complexes and tonofilaments were scant and intracytoplasmic lumina were absent. Intracellular mucin was present in the areas showing GCFs of four ADSQs with predominant GCFs and six ADSQs with focal GCFs. Two of three cases examined ultrastructurally showed intracellular electron-dense material that corresponded to mucin secretory material. Immunohistochemical studies of the six ADSQs with predominant GCF cases showed the following pattern of reactivity: monoclonal carcinoembryonic antigen (CEA), 2 of 6 cases; polyclonal CEA, 3 of 6; CA 125, 0 of 6; CA 19-9, 0 of 6; placental alkaline phosphatase, 0 of 6; and vimentin, 1 of 6. Focal GCF areas showed monoclonal CEA, 4 of 9 cases; polyclonal CEA, 3 of 9; vimentin, 4 of 9; while CA 125, CA 19-9, and placental alkaline phosphatase were negative in areas of GCFs. One of three patients with ADSQ with predominant GCFs and five of nine patients with ADSQ with focal GCFs with at least 1 year of follow-up were disease free. No association between GCFs (combined focal and predominant) and recurrent disease was present when compared to the other 29 AC and ADSQ patients with follow-up. Recurrent disease in our series of AC and ADSQ was only associated with stage III or IV disease at presentation (P less than 0.001). There was no association with adenosquamous histology, histologic grade, lymphatic invasion, or age. There were insufficient cases of ADSQ with predominant GCFs with follow-up to evaluate fully prognostic significance of this subgroup. Our study suggests that GCFs are part of the spectrum of differentiation in ADSQ of the cervix rather than a distinct histologic type of carcinoma with unique clinical significance. PMID- 1716416 TI - Estrogen and progesterone receptors in lymphangioleiomyomatosis, epithelioid hemangioendothelioma, and sclerosing hemangioma of the lung. AB - The therapeutic options in the treatment of lung neoplasia usually have not included hormonal therapy, unlike those for primary tumors of other sites (e.g., breast). However, two mesenchymal proliferations of lung, lymphangioleiomyomatosis and epithelioid hemangioendothelioma (EHE), and one epithelial tumor, sclerosing hemangioma (SH), have a significant female predilection and may benefit from such hormonal therapy. The authors investigated five cases each of EHE and lymphangioleiomyomatosis and four cases of SH for expression of estrogen and progesterone receptors and 17-beta estradiol in paraffin-embedded tissue. Only one case each of lymphangioleiomyomatosis and EHE expressed 17-beta estradiol. All of the other cases were negative. These findings are contrary to the viewpoint held in published literature, especially in case reports of lymphangioleiomyomatosis, describing patients with positive estrogen and progesterone receptor results. Consequently, a number of issues must be considered in the clinical and immunohistochemical evaluation of the estrogen and progesterone receptor status of these rare pulmonary neoplasms. PMID- 1716417 TI - Desmoplastic small cell tumors of the peritoneum coexpressing mesenchymal and epithelial markers. AB - Desmoplastic small cell tumors arising diffusely within the abdomen and lacking an apparent organ of origin are rare. Most previously reported cases occurred in children, but young adult patients also have been described. Light microscopic examination shows the tumors to be composed of nests of small cells surrounded by an abundant desmoplastic stroma. Immunohistochemical findings reveal multidirectional differentiation with coexpression of cytokeratin, milk fat globule, neuron-specific enolase, Leu-7, desmin, and vimentin. Electron microscopic examination demonstrates paranuclear condensations of intermediate filaments. The authors describe two patients who died of their disease, despite aggressive chemotherapy and surgical intervention. PMID- 1716418 TI - Aprotinin blocks the binding of pro atrial natriuretic peptides 1 to 30, 31 to 67, and 99 to 126 to human placental membranes. AB - Two peptides with natriuretic and diuretic properties consisting of amino acids 1 to 30 and 31 to 67 of the 98 amino acid N-terminal end of the prohormone of atrial natriuretic factor, which normally circulates in humans, were investigated to determine if they have specific binding sites of placental membranes. Competitive binding experiments revealed that atrial natriuretic peptides 1 to 30, 31 to 67, and 99 to 126 (i.e., C-terminus) each had specific and separate binding sites. The dissociation constants for atrial natriuretic peptides 1 to 30, 31 to 67, and 99 to 126 binding to human placental membranes were similar at 4.3 +/- 0.6 nmol/L, 3.1 +/- 0.4 nmol/L, and 2.9 +/- 0.5 nmol/L, respectively. Each peptide bound to the placental membranes between 10(-8) and 10(-11) mol/L but could bind to the other peptides' receptors only at supraphysiologic concentrations of 10(-6) and 10(-7) mol/L. The protease inhibitor aprotinin (50 micrograms/ml) blocked the binding of the atrial natriuretic peptides 1 to 30, 31 to 67, and 99 to 126 to their respective receptors. These results suggest that atrial natriuretic peptides 1 to 30 and 31 to 67 do not work through the atrial natriuretic factor receptor but rather have their own separate and distinct receptors on placental membranes and that the protease inhibitor aprotinin antagonizes their respective binding to placental membranes, suggesting that integral membrane proteases may be modulators of atrial natriuretic peptides receptor function. PMID- 1716419 TI - Risks associated with an elevated maternal serum alpha-fetoprotein level. AB - Among 58,187 women tested, 1002 had a maternal serum alpha-fetoprotein measuring greater than or equal to 2.5 multiples of the median after correction for race, weight, and insulin-dependent diabetes. They were stratified into three groups: group 1, 2.5 to 2.9; group 2, 3.0 to 5.0; group 3, greater than or equal to 5.0 multiples of the median. The initial risk of a serious abnormality detected by ultrasonography or amniocentesis was 17% (5%, 12% and 65% in groups 1, 2, and 3, respectively). After correction for twins and dates, this risk became 23% (7%, 18%, and 71% in groups, 1, 2, and 3, respectively). Among the women with high maternal serum alpha-fetoprotein levels, 556 (77%) had normal ultrasonographic and amniocentesis studies, and the risk of adverse pregnancy outcome ws 27% (19%, 29%, and 70% in groups 1, 2, and 3, respectively). There was a statistically significant increase in late fetal and perinatal death, prematurity and growth retardation, oligohydramnios, abruptio placentae, preeclampsia, and congenital abnormalities. The overall risk for abnormality or adverse outcome was 24% in group 1, 41% in group 2, and 91% in group 3. PMID- 1716420 TI - Prospective evaluation of maternal serum human chorionic gonadotropin levels in 3428 pregnancies. AB - As part of a multicenter prospective study, second-trimester human chorionic gonadotropin and alpha-fetoprotein concentrations were evaluated. Data included maternal age, human chorionic gonadotropin level, alpha-fetoprotein level, weight, race, and pregnancy outcome of 3428 pregnancies at between 15 and 20 weeks' gestation. The results of the study indicate that human chorionic gonadotropin levels decrease as maternal weight increases, that weight-adjusted human chorionic gonadotropin levels for Oriental and black women are higher than for white or Hispanic women, and that twin pregnancies have higher human chorionic gonadotropin levels than singleton pregnancies. Of 255 pregnancies that did not have normal outcomes, 54 (21.2%) had human chorionic gonadotropin levels greater than 2.0 multiples of the median and 26 (10.2%) had alpha-fetoprotein levels greater than 2.5 multiples of the median. Of 11 pregnancies with fetal aneuploidy, 6 (54.5%) had human chorionic gonadotropin levels greater than 2.0 multiples of the median. It is concluded that in human chorionic gonadotropin screening programs for fetal Down syndrome, weight and race adjustments are necessary for accurate risk assessment. PMID- 1716421 TI - Expression of cytokine receptors and markers of differentiation in human papillomavirus-infected cervical tissues. AB - Human papillomavirus infection of the uterine cervix is associated with a spectrum of benign, premalignant, and malignant epithelial lesions, a process that appears to require the coordinated effects of secondary cellular and environmental events. We have used flow cytometry and immunohistochemistry to examine the expression of the cellular markers for proliferation (interleukin-1, epidermal growth factor receptor, and transferrin receptor) and the markers of cellular differentiation (filaggrin and low-molecular-weight cytokeratin) in normal and human papillomavirus--infected human cervical tissues representing the natural range of human papillomavirus--induced disease. The results were correlated with the histologic grade of disease, human papillomavirus type, cellular deoxyribonucleic acid content, and cell cycle status. Interleukin-1 and transferrin receptor were slightly increased in high-grade dysplasias and in squamous cell carcinomas. Filaggrin expression was found to be inversely related and cytokeratin and epidermal growth factor receptor expression directly related to the degree of neoplasia. These findings indicate that cytokeratin and epidermal growth factor receptor are useful markers of cell proliferation in human papillomavirus--infected tissues and that their expression may directly increase as a result of infection. PMID- 1716422 TI - Free beta-protein studies need confirmation. PMID- 1716423 TI - Value of high and low maternal serum alpha-fetoprotein levels in screening singleton pregnancies. PMID- 1716424 TI - Effects of drugs on cholesterol esterification in normal and Niemann-Pick type C fibroblasts: AY-9944, other cationic amphiphilic drugs and DMSO. AB - AY-9944 and other cationic amphiphilic drugs (CADs) have been reported to cause a reduction of acid sphingomyelinase activity in fibroblasts and various tissues in the rat, and DMSO has been known to correct a partial deficiency of acid sphingomyelinase activity in Niemann-Pick type C (NPC) fibroblasts. Furthermore, in the present study we demonstrated that AY-9944 and other CADs caused a marked reduction of cholesterol esterification in control fibroblasts and an excessive intracellular accumulation of unesterified cholesterol as in NPC cells, and that this reduction could partially be corrected by the addition of 2% DMSO to the medium. These characteristics of the drug-treated cells mimic the phenomena seen in NPC fibroblasts. Therefore, fibroblasts treated with CADs may be used as a model of drug-induced lipidosis of NPC. The effect of DMSO suggests the possibility of its usefulness in the treatment of NPC patients. PMID- 1716425 TI - Quantitation of early myocardial ischemia using acridine orange fluorescence--an experimental study. AB - It has been observed in the present investigation that ischemic myocardium consistently produces bright green fluorescence after Acridine Orange (AO) staining. The area of ischemia in the left ventricular myocardium at different time intervals after onset of experimental ischemia has been calculated by use of this AO fluorescence technique. Zonal distribution of ischemia in the epicardial, middle, and endocardial zones has also been evaluated quantitatively from ten minutes to six hours after ligation of the anterior descending branch of the left coronary artery in the Wistar strain of albino rats. No similar study was available for comparison from the literature reviewed. The total area of left ventricular ischemia showed an increase with the prolongation of duration of coronary ligation from 13.39 +/- 2.69 mm2 at ten to twenty-five minutes to 32.99 +/- 5.69 mm2 at six hours after ligation. Statistical analysis of the zonal area of ischemia has shown that ischemia in the middle and endocardial zones was greater than that in the epicardial zone at all time intervals recorded. Middle zone ischemia extended over a larger area than that over the endocardial zone at all intervals except at intervals II (30-45 min), IV (2-2 1/2 hrs) and V (3-4 hrs). The results of this experimental investigation are significant for these point to the value of Acridine Orange fluorescence in detecting early myocardial ischemia and in demarcating zonal differences in ischemia. The authors have successfully utilized the method in a few human cases of clinically suspected myocardial infarction and recommend the technique for routine use to detect early human myocardial ischemia. PMID- 1716426 TI - A MspI restriction fragment length polymorphism at the ovine locus for 2',3' cyclic-nucleotide 3'-phosphohydrolase. PMID- 1716427 TI - A MspI restriction fragment length polymorphism at the ovine locus for neurotensin. PMID- 1716428 TI - Restriction fragment length polymorphisms at the ovine locus for the alpha subunit of pituitary glycoprotein hormones. PMID- 1716429 TI - Clinical and inflammatory responses to exogenous tachykinins in allergic rhinitis. AB - Our purpose was to characterize the tachykinin receptor type involved in nasal obstruction to exogenous substance P in rhinitic patients. We also attempted to assess biochemical and cellular events associated with this response. Nasal challenges were performed in seven patients with allergic rhinitis. They received increasing doses (10 to 80 nmol) of substance P, of neurokinin A, of the N terminal fragment of substance P, substance P(1-9), and of saline on 4 different days separated by 14 days. Nasal airway resistance (NAR) increased in a dose dependent manner on substance P. Maximal increase reached 4.5-fold basal NAR. Response to neurokinin A was significantly lower (less than 2-fold basal NAR). No effect was observed on substance P(1-9) and saline. This order of activity [substance P much greater than neurokinin A greater than substance P(1-9) = saline] indicates an NK1 receptor-mediated mechanism inducing local vasodilation. No histamine release was found after any of the four challenges. Proteins significantly increased in nasal lavage fluid on both substance P and neurokinin A, whereas substance P(1-9) and saline had no effect. The percentage of albumin increased in nasal lavage fluid from 30 to 50% of total proteins on substance P and neurokinin A, indicating microvascular leakage. Polymorphonuclear cells significantly increased from 9 to 36% on substance P, from 13 to 49% on neurokinin A, and from 13 to 55% on substance P(1-9). Eosinophils increased in five patients on substance P (from 0.1 to 5% for the group), in three patients after neurokinin A, and in two after substance P(1-9).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716430 TI - Clinical applications of serum tumor markers. AB - The pursuit of the ideal tumor marker has generated many tests for use in the diagnosis and management of cancer, several of which are now widely available. Tumor markers have five potential uses in patient care: They can be used for screening, for diagnosis, for establishing prognosis, for monitoring treatment, and for detecting relapse. The value of a marker in a given setting depends on two marker-related characteristics--sensitivity and specificity. The value of a marker in a particular malignancy also depends on the effectiveness of therapy for the malignancy. Tumor markers have been used to screen for occult cancer but have proved to be valuable only in selected cancers. As diagnostic tools, tumor markers have limitations: Nearly all markers can be elevated in benign disorders, and most markers are not elevated in the early stages of malignancy. Extreme marker elevation often indicates a poor prognosis and in some malignancies can indicate the need for more aggressive treatment. Tumor markers have their greatest value when used to monitor therapy in patients with widespread cancer. Nearly all markers show some correlation with the clinical course of disease, with marker elevation in any stage declining to normal after a curative intervention. Recurrent disease can be accompanied by increased marker levels, but markers can detect an occult recurrence in only a few diseases, thereby facilitating a second attempt at cure. Although it seems unlikely that an ideal tumor marker will be identified for every malignancy, several workable markers are already available. Increasing our knowledge about the capabilities and limitations of existing markers will enable us to use them judiciously in the treatment of cancer. PMID- 1716431 TI - Fetal hemoglobin alters hemoglobin A1c measurements. PMID- 1716432 TI - Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin antigens: IV. Suppression of chronic relapsing disease in the Lewis rat and strain 13 guinea pig. AB - Oral administration of proteins is a long-recognized method of inducing antigen specific peripheral immune tolerance. We previously showed that oral administration of myelin basic protein suppresses monophasic experimental autoimmune encephalomyelitis in the Lewis rat when it is given in association with immunization and prior to disease onset. As a potential therapy for human autoimmune disease, it is crucial to determine whether oral tolerance can ameliorate an ongoing immune response. We therefore asked whether oral administration of myelin antigens, after sensitization and disease expression has occurred, could affect immunological, clinical, or pathological features of experimental autoimmune encephalomyelitis. Chronic relapsing experimental autoimmune encephalomyelitis was induced in the Lewis rat and strain 13 guinea pig by immunization with whole guinea pig cord homogenate, complete Freund's adjuvant, and Mycobacterium tuberculosis. Following recovery from the first attack, animals were orally given bovine myelin, guinea pig myelin, or guinea pig myelin basic protein three times per week for up to 3 months. Animals receiving myelin products orally had decreased severity and frequency of clinical relapses, decreased delayed-type hypersensitivity responses to myelin antigens, diminished inflammation in the central nervous system (CNS), and decreased areas of CNS demyelination. In the rat, guinea pig myelin basic protein was as effective as guinea pig myelin in ameliorating the disease and also resulted in decreased serum anti-myelin basic protein antibody levels. No exacerbation of disease or worsening of pathological findings occurred in the animals given myelin products. These results demonstrate that oral administration of myelin antigens can suppress chronic relapsing experimental autoimmune encephalomyelitis and have direct relevance to therapy of human demyelinating disorders such as multiple sclerosis. PMID- 1716433 TI - Lichen sclerosus et atrophicus, morphea, and coexistence of both diseases. Histological studies using lectins. AB - Histological studies using three lectins, lens culinaris agglutinin, soybean agglutinin, and Ulex europaeus agglutinin-I, were carried out in a case of coexistent lichen sclerosus et atrophicus and morphea, five cases of morphea, and two cases of lichen sclerosus et atrophicus. The lectin staining patterns of the formaldehyde-fixed epidermis of patients with morphea were not different from those of normal epidermis, but epidermis of patients with lichen sclerosus et atrophicus showed different staining patterns. Lens culinaris agglutinin stained the basal and the spinous layers of the normal epidermis and that of patients with morphea but stained only the basal cells of the epidermis from patients with lichen sclerosus et atrophicus; epidermal Ulex europaeus agglutinin binding was observed only in the cases of lichen sclerosus et atrophicus. Moreover, in the patient with coexistent diseases, the morphea lesion showed the staining profiles of morphea and the lichen sclerosus et atrophicus lesion showed the staining patterns of lichen sclerosus et atrophicus, respectively. PMID- 1716434 TI - [Fusion of protoplasts of inactive variants of 2 producers of actinomycin C and the biosynthesis of an antibiotic of non- actinomycin nature]. AB - Fusion of protoplasts of double auxotrophic mutants of spontaneous inactive variants of two cultures producing actinomycin C, i.e. Streptomyces chrysomalus 305 and Streptomyces sp. 26-115 induced by PEG-600 yielded a number of stable recombinants. One of the recombinants requiring proline for its growth was designated as recPro. Unlike its parent strains, it synthesized an antibiotic substance active against gram-positive bacteria and Saccharomyces cerevisiae. The nature of the substance is under investigation. PMID- 1716435 TI - T cell defined HLA epitopes and T cell receptor polymorphism in insulin dependent diabetes mellitus. AB - T cell defined epitopes on class II HLA molecules (epitopes distinguishable by T cells but not by antibodies) seem to be important determinants of IDDM susceptibility/resistance. Although HLA-DR4 is associated with IDDM in many populations, DR4-positive HLA haplotypes vary greatly (relative risk from greater than 10 to less than 1). This variation seems to depend on both the DQ allele and T cell defined subtypes of the DR4 allele. These IDDM associated alleles at the two loci (DQB1 and DRB1) are not correlated with each other in the healthy population, so they clearly are independent risk factors. HLA-DR2 has universally been associated with lack of IDDM, and seems to be protective. However, not all DR2 haplotypes protect, and the protection or lack of protection correlates with T cell defined subtypes of DR2. In this case, however, the DR2 subtypes do correlate with DQ alleles, so it is unclear which locus (loci) is (are) actually affecting the disease process. It may be significant that, for both DR2 and DR4, only the more protective subtypes have arginine at amino acid position 71. Other portions of the DR beta chain are clearly important, however. Although TCR alpha and beta seemed to be promising candidates for additional IDDM susceptibility genes, in fact the various TCR alpha and beta haplotypes are equal, or nearly equal, with regard to IDDM susceptibility. The importance of HLA alleles in IDDM susceptibility, and the lack of importance of TCR alpha and beta alleles, may be due to the different means by which the HLA and TCR molecules achieve antigen binding diversity: HLA molecules by multiple loci and allelic diversity, and TCR molecules by the tremendous diversity that can be generated from a single TCR allele during T cell maturation. PMID- 1716436 TI - Structural and functional constraints on HLA class II dimers implicated in susceptibility to insulin dependent diabetes mellitus. PMID- 1716437 TI - The gene for the common alpha subunit of porcine pituitary glycoprotein hormone. AB - The gene for the common alpha subunit of the porcine anterior pituitary glycoprotein hormones was cloned from a genomic library constructed in EMBL3. The nucleotide sequence of the entire coding sequence of the porcine common alpha subunit gene was determined in addition to one intron and 1059 and 160 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the common alpha-subunit gene is present as a single copy. The transcriptional unit of the porcine common alpha subunit spanned about 14 kb and contained four exons interrupted by three introns of about 11.5, 1.2 and 0.4 kb. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues +9/+10 and +71/+72 were highly conserved among the rat, human and bovine common alpha-subunit genes. In the proximal portion of the 5'-flanking region, one TATA box and one CCAAT box were present. A steroid-responsive element was not found up to 1059 bases upstream from the transcription start site. The potential AP-1 and AP-2 factor-responsive elements were present at three and one positions respectively in the 5'-flanking region. This feature suggests that hypothalamic gonadotrophin-releasing hormone stimulates the expression of the common alpha-subunit gene predominantly by a signal-transduction system, with the protein kinase C cascade and factors AP-1 and AP-2 as mediators. The cyclic AMP-responsive element was also present at two positions, but a single base substitution was found in each sequence compared with the consensus sequence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716438 TI - Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion. AB - While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716439 TI - Molecular analysis of microtubule-associated protein-2 kinase cDNA from mouse and rat brain. AB - We have isolated and characterized brain cDNA clones encoding microtubule associated protein-2 (MAP-2) kinase for rat (rMNK1) and mouse (mMNK1). The nucleotide sequences diverged by only 5% whereas the amino acid sequences were identical except for one conservative residue change. Conservation of the expressed sequence extended into other mammalian species. These findings constitute the first demonstration of a strict evolutionary conservation of MAP-2 kinase. Genomic restriction patterns revealed a single MAP-2 kinase gene that shares homology with other genomic sequences. The 3' terminal half of the gene appears to be encoded by four exons. rMNK1 and mMNK1 differed from a recently reported MAP-2 kinase cDNA, termed ERK1, because of a nonconservative change in position 82, from Gly in ERK1 to Arg in rMNK1. The rMNK1 gene was found to be expressed mainly as a 1.8-kb transcript that was highest in brain and in lung. In contrast to ERK1, rMNK1 showed two equally prominent mRNA species in liver, at 1.8 kb and 5 kb, which imply differential processing of the primary transcript. Results derived from the immunological screening of an expression library showed that MAP-2 kinase might share epitopes with two prominent protein kinase C substrates, MARCKS (an 80-kD protein kinase C substrate) and GAP-43, suggesting the possibility that MAP-2 kinase could interact with kinase C. PMID- 1716440 TI - Enhancer, repressor, and promoter specificities combine to regulate the rat alpha fetoprotein gene. AB - The upstream transcription control region of the rat alpha-fetoprotein (AFP) gene was analyzed using transient expression of CAT genes in HepG2 cells which express the gene; H4C3 cells which repress the AFP gene but express the albumin gene; and four nonexpressing cell lines. Deletion analysis based on the DNA sequence resolved three upstream enhancers corresponding to the mouse AFP enhancers, but showed additional weak effects from flanking sequences. Quantitative experiments demonstrated that the three enhancers were additive when acting through a single promoter and did not confirm the presence of a distal upstream repressor. All three enhancers stimulated the AFP, albumin, or thymidine kinase (tk) promoter in HepG2, but only the tk and albumin promoters in H4C3. Deletion of a proximal repressor region near the AFP promoter allowed expression in H4C3 cells with the AFP promoter. Thus, the liver-specific developmental repressor is near the AFP promoter, and H4C3 cells provide an in vitro system for analysis of this repressor in transfection assays. The repressor region also blocked expression of the SV40 enhancer through the AFP promoter in hepatic and nonhepatic cell lines, but when this enhancer was combined with an AFP promoter from which the repressor region was deleted, the combination showed expression in all six cell lines studied. AFP expression results from a combination of enhancer, promoter, and repressor activities, and the repressor is functional with a heterologous enhancer in a variety of cells. PMID- 1716441 TI - Modeling of nucleic acid complexes with cationic ligands: a specialized molecular mechanics force field and its application. AB - A potential energy force field designed for modeling nucleic acids and particularly their complexes with cationic ligands is presented. The force field is a modified version of that developed by Weiner, S.J., Kollman, P.A., Nguyen, D.T. and Case, D.A.,J. Comp. Chem. 7,230-252 (1986) and is based upon the use of a distance dependent dielectric constant, epsilon = 4rij, and partially neutralized phosphates to represent solvent and counterion. Changes from the Weiner et al. force field include additional atom types and modifications to van der Waals, electrostatic, hydrogen bonding and torsional parameters. Molecular modeling test cases of the force field are presented for a number of simple small molecules, as well as uracil and benzene dimerization, thymine-adenine and cytosine-guanine base pair formation, and adenosine/deoxyadenosine pseudorotation. Several DNA and RNA oligomers and DNA/RNA intercalation complexes with ethidium are also modeled with the force field. In all cases, the modeling results compare favorably with available experimental results. Additionally, conformational trends observed experimentally for nucleic acids by NMR and X-ray crystallographic techniques are reproduced. The modeling results for ethidium intercalation indicate a complex in which the favorable interactions are primarily van der Waals contacts, and in which electrostatic interactions are a relatively minor component. We feel the force field is particularly useful for molecular mechanics aided drug design, and an analysis of modeling results with respect to design of drugs which bind selectively to RNA is presented. PMID- 1716443 TI - Long distance image transfer: first results of its use in histopathological diagnosis. AB - Histopathological images were transferred by use of normal telephone lines between three pathology institutes located in three different cities in the FRG. Images were digitized using a colour TV camera and stored in a special computerized image transmission system. The stored image was transferred and visualized on a (receiver) colour TV screen after dialing the telephone number connected to the receiver image transmission system. An additional telephone dialogue was activated by use of a normal acoustic telephone, and the diagnostic difficulties of the underlying image were discussed. Diagnostic assistance was possible in all transferred cases as well as histopathological diagnosis. Resolution of the images was set at 512 x 512 pixel x 8 bit. Image transfer time was 3.2 min on average. The differences between the original and transferred image were measured by "retransfer" of the original image and by subtracting the two images from each other. No major transfer errors could be measured. PMID- 1716442 TI - Production and of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins. AB - Eighteen monoclonal antibodies (MAb) to simian immunodeficiency virus (SIV) envelope have been characterized. All MAb were shown to bind to viral antigens on the surface of unfixed SIV-infected cells and to precipitate surface glycoproteins of SIVmac251. In Western blot 11 MAb bound to gp160 and gp120, five bound to gp160 and the transmembrane protein gp41 and two MAb did not react with denatured antigen. Preliminary competition assays identified the existence of six competition groups; two groups were within gp41 and four were within gp120. Of the latter four groups, three contained MAb with neutralizing activity. Two of the neutralizing MAb (KK5 and KK9) did not react with denatured antigen in Western blot suggesting that they may recognize conformational epitopes. Enzyme linked immunosorbent-assay titres of MAb against SIVmac251 ranged from 10(2.4) to 10(5.6) and although similar titres were obtained with some MAb against other SIV and HIV antigens, the presence of isolate specific and shared group epitopes was demonstrated. PMID- 1716444 TI - Intermediate and fine cytofilaments in cutaneous and subcutaneous leiomyosarcomas. AB - The expression of fine and intermediate cytofilaments in 10 cutaneous and seven subcutaneous leiomyosarcomas was studied immunohistochemically. All the tumors contained tumor cells which showed a positive immunoreactivity for desmin in formaldehyde-fixed and paraffin-embedded sections, but none of the seven anti desmin antibodies used alone produced a distinct positive staining in all the tumors. A lack of correspondence in terms of immunoreactivity between tumor cells and the supposed muscle of origin was observed, especially in the subcutaneous leiomyosarcomas. In all cases, antibodies to muscle-specific and smooth muscle specific actin were found to produce a positive staining in both the tumors and the supposed muscle of origin. Vimentin was detected in 8/10 cutaneous and 4/7 subcutaneous leiomyosarcomas, while the supposed muscle of origin was positive in 3/10 and 7/7 cases, respectively. Four of the cutaneous and three of the subcutaneous leiomyosarcomas contained tumor cells which stained positively for cytokeratins, while the supposed muscle of origin showed no positivity. It thus appears that a phenotypic shift in terms of vimentin and cytokeratin expression occurs in the tumor cells of cutaneous and subcutaneous leiomyosarcomas compared with the supposed muscle of origin. It is recommended that more than one monoclonal anti-desmin antibody is used to characterize these tumor entities. It is also concluded that the immunoreactivity for muscle-specific actins in superficial leiomyosarcomas is more constant, although less specific, than that of desmin and that the demonstration of the simultaneous expression of muscle specific actins and desmin is helpful. PMID- 1716445 TI - Activated alpha 2-macroglobulin is a principal defensin-binding protein. AB - Defensins are highly abundant and variably cationic peptides that possess antimicrobial, cytotoxic, and chemoattractant properties and equip mammalian phagocytes for participation in host defense and inflammatory processes. We studied the binding of the human defensin HNP-1 by proteins in plasma and serum and identified activated (F-form) alpha 2-macroglobulin (alpha 2M) as a principal binding protein for HNP-1. In contrast, native (S-form) alpha 2M bound little HNP 1. The binding of HNP-1 by F-form alpha 2M was resistant to salt and boiling in 2% sodium dodecyl sulfate but was ablated by dithiothreitol. Pretreatment of methylamine-activated serum or plasma by iodoacetamide substantially decreased the binding of HNP-1 to alpha 2M, suggesting that thiol groups in activated alpha 2M play a role in defensin binding, possibly by covalently trapping defensins via thiol-disulfide exchange. Western blots of conventionally collected sera showed endogenous defensins complexed with the F-form of alpha 2M, indicating that the generation of defensin-alpha 2M complexes was not limited to the in vitro model of methylamine-activated serum or plasma and radiolabeled HNP-1. Previous studies indicated that native alpha 2M can be converted to its F-form by many proteases, including those released by neutrophils and platelets, and that the F-form is recognized and internalized by specific receptors on macrophages and hepatocytes. Our findings suggest that the alpha 2M system may function as a scavenger of defensins and other peptide mediators in inflamed tissues and may constitute an important mechanism for the regulation and containment of inflammation. PMID- 1716446 TI - Molecular diversity of basement membrane collagen: elucidation of the Goodpasture's epitope. PMID- 1716447 TI - Nuclear RNA processing. AB - Continued progress has been made during the past year in understanding the basic biochemical mechanisms involved in nuclear RNA processing. Of particular importance have been the advances made in purifying and characterizing protein factors involved in splicing and polyadenylation of pre-mRNAs. PMID- 1716448 TI - Cisplatin and 5-FU combined with radiotherapy and surgery in the treatment of squamous cell carcinoma of the esophagus. Palliative effects and tumor response. AB - The combination of cisplatin (90-120 mg/m2) and 5-fluorouracil (5-FU) (1,000 mg/m2/day in continuous infusion for five days) was given for 2-3 cycles, prior to combined radiotherapy and surgery, to 73 patients with esophageal squamous cell carcinoma, 60 with limited disease (LD), and 13 with extensive disease (ED) (i.e. with metastasis) of whom 3 had recurrent disease. Before preoperative radiotherapy among 60 LD patients, 12 (20%) had complete response, 21 (35%) partial response, 25 (42%) had stable disease, and 2 (3%) progressive disease. Swallowing was improved in 35/73 (48%) of the cases. In the resected specimens, no tumor was found in 8/53 (15%) of the cases, microscopic tumor in 18/53 (34%) and macroscopic tumor in 27/53 (51%). In the ED group, complete response of distant metastases was obtained in 6/13 (48%) of the patients, one of whom is still alive with no evidence of disease 62 months after the start of treatment. PMID- 1716449 TI - Nutritional regulation of IGF-I and IGF binding proteins. AB - The secretion of both IGF-I and IGF-II, peptides that are structurally related to insulin, is linked to nutrient intake. These growth factors and their binding proteins appear to be major links between nutrient intake and cellular anabolic responses. Understanding the mechanisms by which nutrients control IGF synthesis and regulate transport to target tissues should permit the formulation of strategies for treatment of catabolic disorders. Understanding the alterations of such processes in catabolic states should enable development of better methods for nutritional repletion and wiser uses of anabolic agents in the treatment of these conditions. PMID- 1716450 TI - [Hematopoietic tissue growth factors: clinical factors]. AB - Hematopoietic growth factors (HGF) are glycoproteins controlling proliferation, differentiation and function of bone marrow derived cells. Results of recent studies, both in vitro and in vivo, in animal models, indicate that these molecules might have a therapeutical value in several different clinical settings. Thanks to advances in the expression of recombinant genes. HGF have recently become available in sufficient quantities to be tested in clinical investigation. During the last few years, a number of phase I/II trials have been performed with the use of recombinant colony stimulating factors and erythropoietin, suggesting the efficacy of these substances in treating diseases associated with hematopoietic failure or impaired cell function. This review article takes a critical look at the most important recent preclinical and clinical experience on HGF, and also examines some of the future directions in which clinical medicine will probably benefit from these molecules. PMID- 1716451 TI - Regulation of 1,4-dihydropyridine and beta-adrenergic receptor sites in coronary artery smooth muscle membranes. AB - The receptor sites for 1,4-dihydropyridine (DHP) calcium channel ligands were identified and pharmacologically characterized in partially purified canine coronary artery smooth muscle (CSM) membranes (purification factor for 1,4-DHPs 2.8 and 2.2 respectively) using Ca2+ channel agonist (-)-S-[3H]BAYK 8644 and antagonist (+)-[3H]PN 200-110 as radioligands. The beta-adrenergic receptors were identified with (-)-3-[125I]iodocyanopindolol (ICYP). Specific binding of 1,4 DHPs and ICYP to membrane fraction was saturable, reversible and of both high and low affinity. The Kd for 1,4-DHP Ca2+ channel agonist was 0.59 +/- 0.05 and for antagonist 0.35 +/- 0.06 nmol/l and for low affinity binding sites Kd = 9.0 +/- 0.18 and 18.0 +/- 1.1 nmol/l. The high affinity 1,4-DHP binding (Bmax = 265 +/- 21 and 492 +/- 12 fmol/mg protein), showed stereoselectivity, temperature dependence as well as pharmacological specificity: isoprenaline- and GTP sensitivity, positive modulation with dilthiazem and negative modulation with verapamil, that is, properties characteristic of 1,4-DHP receptor sites on L-type Ca2+ channels. The low affinity binding sites were characterized as nonselective, temperature independent, dipyridamol-sensitive and represented a nucleoside transporter. The proportion of high affinity binding sites identified in the CSM membranes was 1.85 : 1.0 in favour of the antagonist. Results obtained with [125I]omega Conotoxin GVI A demonstrated that CSM membrane fractions isolated from median layers of coronary artery were devoid of substantial contamination with fragments of neuronal cells. PMID- 1716452 TI - Temperature and benzyl alcohol as probes of the antilipolytic mechanism of insulin action in adipocytes. AB - The temperature dependence of cAMP accumulation and glycerol release in response to epinephrine and insulin in adipocytes is examined. (1) Glycerol release in the presence of epinephrine demonstrated linear Arrhenius kinetics to 41 degrees C, and above 45 degrees C glycerol release was progressively inhibited. (2) In contrast, incubation of the cells with both epinephrine and insulin resulted in glycerol release rates that were relatively temperature insensitive. (3) Calculation of the efficacy of insulin to inhibit epinephrine-stimulated glycerol release as a function of temperature yielded a biphasic response, with a distinct optimum around 41 degrees C, in a similar manner to the effects of insulin on hexose transport activation determined previously. (4) A saturating dose of insulin (40 ng/ml) was found to have no significant effect on epinephrine stimulated intracellular cAMP over the temperature range studied. (5) Addition of benzyl alcohol (to 40 mM) resulted in substantial inhibition of basal, epinephrine stimulated, and insulin inhibited glycerol release, without affecting the magnitude of insulin inhibition. We conclude from these studies that (a) insulin inhibition of glycerol release can not be mediated directly by intracellular cAMP modulation, (b) as in the case of hexose transport activation, the signalling mechanism by the occupied insulin receptor appears to be relatively independent of the membrane lipid environment. PMID- 1716453 TI - Characteristics and performance of a bispecific F (ab'gamma)2 antibody for delivering saporin to a CD7+ human acute T-cell leukaemia cell line. AB - We have investigated the efficacy of a F(ab'gamma)2 bispecific antibody (BsAb) with dual specificity for the CD7 molecule in one Fab arm and for the ribosome inactivating protein (rip) saporin in the other arm, for delivering saporin to the acute T-cell leukaemia cell line HSB-2. Saporin titration experiments revealed that BsAb increased the toxicity of saporin 435-fold for HSB-2 cells, reducing the IC50 for saporin alone from 0.1 mumol to 0.23 nmol when BsAb was included. The rate of protein synthesis inactivation brought about by BsAb mediated toxin delivery to HSB-2 cells was very similar to that described for conventional immunotoxins (IT's) with a t10 (time taken for a one log inhibition of protein synthesis compared with controls) of 46 h obtained at a saporin concentration of 1 nmol and 226 h at 0.1 nmol. BsAb titration studies demonstrated a clear dose response effect of BsAb concentration on target cell protein synthesis inhibition and cell proliferation. The absolute specificity of toxin delivery was unequivocally demonstrated by a failure of BsAb to deliver an effective dose of saporin to the CD7- cell line HL60 and by the blocking of BsAb mediated delivery of saporin to HSB-2 cells with an excess of F(ab)2 fragments of the anti-CD7 antibody, HB2. These studies have clearly demonstrated the effectiveness of this BsAb for delivering saporin to a T-ALL cell line utilising CD7 as the target molecule on the cell surface. BsAb's would therefore appear to offer a realistic alternative to IT's for toxin delivery to tumour cells and may even offer certain advantages over conventional IT's for clinical use. PMID- 1716454 TI - Antibody BNH9 detects red blood cell-related antigens on anaplastic large cell (CD30+) lymphomas. AB - Two new monoclonal antibodies--BNH9 and BNF13--were generated by using a human lung adenocarcinoma cell line and standard hybridoma techniques. Both were found to react with epithelial and endothelial cells in routinely fixed and embedded tissues. Unexpected membrane labelling of some large cell lymphomas while non reacting with normal lymphoid cells, prompted further characterisation. The antibodies were found to recognise red blood cell-related oligosaccharide antigens. The specificities were directed towards H and Y determinants. A distinctive pattern of reactivity was found for BNH9 in studying 480 cases of various lymphoid neoplasms. Strong expression of H and/or Y antigens was observed in 65/127 (51%) cases of anaplastic large cell(ALC) (CD30+) lymphomas, which are also known to co-express epithelial membrane antigen (EMA) frequently. Only a minority (less than 6%) of other non-Hodgkin's lymphomas (NHL) (CD30-,EMA-; 208 cases) and Hodgkin's disease (HD) (CD30+, EMA-; 126 cases) were positive. Expression of H and Y antigens was inducible on normal lymphocytes by mitogenic stimulation and by Epstein-Barr virus infection. The data suggest remarkable biological differences of ALC lymphomas within NHL and from HD. PMID- 1716455 TI - Argyrophilic nucleolar organiser region counts and prognosis in pharyngeal carcinoma. AB - The prognostic significance of argyrophilic nucleolar organiser regions (AgNORs) has been evaluated in biopsy specimens from 61 primary squamous and undifferentiated carcinomas of the pharynx prior to therapy. The univariate Kaplan-Meyer survival analysis showed a significant correlation between 3- and 5 year survival rates and the mean AgNOR number per tumour cell (P less than 0.001). No significant correlation was found between prognosis and patients age and sex, tumour location, clinical stage, histologic grade, extent of lymphocytic infiltration, HMFG-2 positivity of tumour cells and UCHL1, LN2, MB2 positivity of infiltrating lymphocytes. There was no significant association between AgNOR counts and tumour histologic grade or clinical stage. Multivariate survival analysis showed that only two variables were significantly correlated with prognosis: AgNOR counts (P less than 0.001) and the extent of lymphocytic infiltration (P less than 0.027). Our results indicate the prognostic value of AgNOR counts and suggest the use of this method as a significant parameter in the pretherapeutic assessment of the aggressiveness of pharyngeal carcinomas. PMID- 1716456 TI - Acute-phase proteins in stroke: influences of its cause (cerebral hemorrhage or infarction), of the cerebral site of infarction, and of the sex of patients. AB - In most of the 129 patients with a recent stroke by cerebral hemorrhage or infarction a note-worthy acute-phase response was found, as demonstrated by important quantitative alterations of blood levels of several acute-phase proteins (APP). These alterations were different in patients with cerebral hemorrhage as compared to those with cerebral infarction. The alterations due to cerebral infarction were not different according to the site of the infarction in brain, i.e. in the brain territories irrigated by the carotid artery system or by the basilar artery system. The APP alterations do not depend on the sex of patients or on the time elapsed from stroke-onset to blood collection. PMID- 1716457 TI - Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase. AB - We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716458 TI - Cell-binding domain of endothelial cell thrombospondin: localization to the 70 kDa core fragment and determination of binding characteristics. AB - Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70 kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716459 TI - Role of divalent metal ions in the hammerhead RNA cleavage reaction. AB - A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716460 TI - Cholecystokinin receptor occupation and cholecystokinin-induced calcium mobilization in the early phase in rat pancreatic acini. AB - We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini. PMID- 1716461 TI - [Structural-membrane regulation of intercellular contacts of low-molecular weight peptides]. AB - It has been shown by microelectrode techniques and microcytofluorometry that fragments of the substance P and L-vasopressin--low molecular physiologically active peptides of wide regulatory action--bind to the receptors on the surface of plasma membranes outside the region of high permeable gap contact in unimolar concentrations and cause an increase in the rate of intracellular diffusion exchange of inorganic ions and fluorescein molecules in the salivary gland cells of Drosophila larva. The growth of intracellular contact permeability induced by low peptide concentrations is blocked by carbodiimide--a reagent cross-linking the membrane proteins. On the basis of the previous data it is suggested that the functions of intracellular contacts are regulated by the distant action mechanism involving the generalized structural rearrangements of plasma membranes. PMID- 1716462 TI - [Decoding common mechanisms of cellular genetic-epigenetic control in eukaryotes]. AB - The mechanism of genetic epigenetic operation at genomic and chromosomic levels within the limits of imitation model of eucaryotic cellular compartment is postulated, this compartment including left and right operators. Probable pattern of interactions during reproduction, determination and expression of genes as a manifestation of genetic, epigenetic memory and memory of water is shown. A specific character and rate of transformations of nucleotides and proteins are realized through different operation mechanisms over hierarchic processes directed on the preservation of DNA in the line of cellular generations and also determining dynamics of the genome with DNA variations. The mechanism of programmed provision of genetic-epigenetic interaction lies in the ways of control, regulation, adaptation and modulation of nucleotides and proteins transformations which occur on the basis of specific (complementary, kinetic and tunnel effects) choice of directions, place, time and aim of nucleotide nucleotide, nucleotide-protein, protein-nucleotide and protein-protein interactions. PMID- 1716463 TI - Noncontiguous regions in the extracellular domain of EGF receptor define ligand binding specificity. AB - Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high affinity binding site typical of human EGFR. PMID- 1716464 TI - Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM 110/VCAM-1. AB - Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM 1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis. PMID- 1716465 TI - Renal cell carcinoma. AB - Renal cell carcinoma represents a significant challenge to surgeons and oncologists treating urologic malignancies. Diagnostically, it is critically important to identify the precise extent of the tumor prior to therapeutic intervention. Therapeutically, a number of controversies continue to be debated, including the role of renal-conserving surgery and the role of surgery in patients with metastatic disease. New research is beginning to identify factors involved in the multidrug-resistant properties of these tumors that may allow us, in the future, to treat these tumors more effectively with systemic chemotherapy. Utilizing immunotherapy in the form of autolymphocytes, interferon, interleukin 2, or combinations of these regimens, a number of exciting advances have been made in the treatment of metastatic renal cell carcinoma. This review examines the most recent literature on each of the above-mentioned aspects in the treatment of this difficult and challenging tumor. PMID- 1716466 TI - Prognostic factors and therapy for superficial and invasive bladder cancer. AB - Although most of the transitional cell carcinomas are superficial at presentation and pose little problem as far as survival is concerned, predicting the ones that will recur and, more importantly, recur as invasive tumors, is crucial. Determination of serum and urine human chorionic gonadotropin-beta levels and epidermal growth factor receptor analysis are promising in this respect. Intravesical bacillus Calmette-Guerin, which is widely used to prevent further recurrences and to treat carcinoma in situ, augments the host's immune status, probably by increasing interleukin-2 and tumor necrosis factor levels. Intravesical interferon seems to be a promising agent, especially against carcinoma in situ, and has exceptionally few side effects. Multidrug chemotherapy regimens such as methotrexate, vinblastine, doxorubicin, and cisplatin, or cisplatin, methotrexate, and vinblastine have achieved 30% to 40% objective complete remission rates in patients with metastatic bladder cancer. These regimens are also used in a neoadjuvant setting with or without irradiation in the hope that more patients will be spared cystectomy and enjoy similar if not better survival. At the same time, continent diversions and nerve-sparing cystoprostatectomy open a new era in the treatment of invasive bladder cancer by improving the quality of life after cystectomy. Although important progress has been made, there is still room for research and clinical controlled studies to achieve higher rates of response, prolonged disease-free survival, and perhaps even improved rates of cure. PMID- 1716468 TI - Rapid diagnosis of bovine cryptosporidiosis with a modified commercial acid-fast staining procedure. PMID- 1716467 TI - A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus. AB - A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave greater than 20% inhibition) that ranged from 32 to greater than 1.024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by cELISA. The sensitivity and specificity of the cELISA test was further confirmed by analysis of a panel of bovine test sera supplied by the National Veterinary Services Laboratories, indicating that the cELISA is a superior test for detection of BTV group-specific antibodies in sera from ruminants in the United States. PMID- 1716469 TI - Comparison of four anti-HIV screening assays which belong to different test generations. AB - There are three different test generations of enzyme-linked immunosorbent assays (ELISA) for the detection of human immunodeficiency virus (HIV) infection, depending on whether virus lysate, recombinant proteins or synthetic peptides are used as solid phase antigen. Four different assays, i.e., three sandwich ELISAs and one competitive test, were used to demonstrate differences between the three systems with regard to the content of different diagnostically relevant virus proteins. The sensitivities and specificities of these assays were compared by using 312 anti-HIV positive sera and 500 sera of healthy blood donors. The highest sensitivity and specificity were achieved by the competitive ELISA based on recombinant proteins, and by the sandwich ELISA based on synthetic peptides. PMID- 1716470 TI - Reduced cerebrospinal fluid dynorphin A1-8 in Alzheimer's disease. AB - Cerebrospinal fluid (CSF) measures of dynorphin A were compared in three groups. Alzheimer patients (n = 9), elderly depressives (n = 9), and age-matched normal controls (n = 9). The Alzheimer patients revealed a 40% decrease in CSF dynorphin compared with controls (36 +/- 15 versus 60 +/- 21 pg/ml, p less than 0.05). In contrast, other peptide measures [Neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and galanin] remained unchanged across groups. This finding was further supported when an additional 20 Alzheimer patients with similar clinical backgrounds also showed reduced CSF dynorphin (37 +/- 13 pg/ml). CSF dynorphin did not correlate significantly with clinical variables or other CSF measures of monoamine metabolites [i.e., 3-methoxy-4-hydroxyphenylglycol (MHPG), 5 hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA)]. Given the previous report of increased kappa binding of Alzheimer brains at autopsy, the authors speculate about a possible up-regulation of opiate receptors in Alzheimer's disease and suggest ways to test this hypothesis in vivo. PMID- 1716471 TI - [Does aprotinin lessen intraoperative blood loss?]. PMID- 1716472 TI - [The manipulation of the hematocrit--where are the limits? The view of the experimental surgeon]. PMID- 1716473 TI - [Ventricular arrhythmias]. PMID- 1716474 TI - [Severe hypertension secondary to nasal decongestants]. PMID- 1716475 TI - Mapping of autoantigenic epitopes on recombinant thyroid peroxidase fragments using the polymerase chain reaction. AB - Cloned cDNA templates of thyroid peroxidase (TPO) have been used in conjunction with the polymerase chain reaction (PCR) to express selected segments of the thyroid microsomal/peroxidase antigen (TMA/TPO) as recombinant protein in E. coli. Six small, different recombinant fragments averaging 120 amino acid residues and one large fragment (269 amino acids) of TPO which together encompass 80% of the extracellular region of the molecule have been produced and autoantibody (aAb) binding sites analysed by immunoblotting. A minimum of six independent, sequential antigenic determinants have been localized on the recombinant proteins and these map to the amino terminal, the central core region and the carboxyl terminal of the TPO molecule. More accurately, the six antigenic sites reside on overlapping recombinant TPO preparations termed R1a + R1b (residues 1 to 160) R1c (residues 145 to 250), R2b (residues 457 to 589), R3a (residues 577-677), R3b (residues 657-767) and R3c (residues 737-845). The large fragment of TPO termed R3 (residues 577-845) encompassing R3a, R3b and R3c also reacts with the aAbs. Different sera from patients with autoimmune thyroid disease contain antibodies to TMA/TPO which differ in their fine specificity. The use of recombinant molecular biological techniques together with PCR to prepare small segments of a large autoantigen as recombinant protein will now allow studies to progress on autoepitope mapping of the precise amino acid sequences of the TPO molecule with the use of synthetic peptides. PMID- 1716476 TI - Intrathyroidal accumulation of T cell phenotypes in autoimmune thyroid disease. AB - In this study we have correlated peripheral T cell subset phenotypes with intrathyroidal lymphocyte accumulation in patients with autoimmune thyroid disease (Graves' and Hashimoto's disease). Our study utilized euthyroid family members for one of our control groups (n = 48) thus significantly limiting familial, but not disease-specific, influences on these T cell phenotypes. Our principal new observations were found only in patients with Graves' disease. As previously reported, there was a decrease in CD8+ (suppressor/cytotoxic) T cells in the peripheral blood of patients with untreated hyperthyroid Graves' disease (n = 27) (mean +/- SEM, 19 +/- 1.1% in patients compared with 25 +/- 1.2% in controls, p = 0.03), a finding not observed in treated, euthyroid Graves' disease patients or their relatives. However, the relative number of CD8+ T cells, assessed by CD4:CD8 ratios, was increased in the intrathyroidal T cell populations (n = 10), when compared to normal and patient peripheral blood. There were no consistent changes in total CD4+ (helper) T cells in the peripheral blood of patients with treated and untreated Graves' disease but a reduction in CD4+2H4+ (suppressor-inducer) T cells was seen in patients undergoing surgery for Graves' disease (13 +/- 6.9% compared with 39 +/- 3.4%). Again, however, this T cell subset was increased within the target organ of the same patients (41 +/- 5.9%). These data point to either a selective accumulation, or a specific "homing", of certain T cell subsets within the thyroid gland of patients with Graves' disease where T cell differentiation may be strongly influenced by antithyroid drug treatment and the local immune environment. PMID- 1716477 TI - The evolution of red blood cell and lymphocyte Ro/SSA. AB - Recent studies have demonstrated that the Ro/SSA autoantigen is heterogeneous as is the corresponding autoimmune response. In addition the autoimmune responses is highly species specific and preferentially reactive with the human antigen. Quantitative ELISA study shows that red blood cell Ro/SSA evolves much more rapidly than lymphocyte Ro/SSA and Western Blot analysis shows that the quantitative ELISA results are mirrored by changes in the 60 kD Ro/SSA molecules but not the 52 kD and 54 kD Ro/SSA molecules. The 52 kD and 54 kD Ro/SSA molecules seem to be relatively conserved as indicated by the Western immunoblotting experiments. These studies add weight to the concept that the antigenic epitopes of these related proteins are under the control of separate genes which have undergone different rates of evolution. PMID- 1716478 TI - The effect of dithiotreitol on thyroid peroxidase and microsomal antigen epitopes recognized by auto and monoclonal antibodies. AB - The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD). PMID- 1716479 TI - Mutagenesis and expression of putative class II susceptibility genes: a "reverse immunogenetics" approach to analysis of HLA and disease. AB - Immune activation events regulated by the human MHC are triggered by interactions between HLA class II molecules, antigenic peptides, and reactive T cells. In most autoimmune diseases, little is known about the latter two elements of this trimolecular complex, while the HLA class II contribution is being deciphered in increasingly sophisticated detail. In two cases in particular, type I diabetes and rheumatoid arthritis, susceptibility is tied to structural elements within specific HLA class II genes. Site-directed mutagenesis and gene expression studies provide a means to directly test the contribution of specific residues within individual candidate disease susceptibility genes to peptide and T cell interactions. PMID- 1716480 TI - The molecular genetics of three thyroid autoantigens: thyroglobulin, thyroid peroxidase and the thyrotropin receptor. PMID- 1716481 TI - Prevention of in vitro fibrinogenolysis during laboratory monitoring of thrombolytic therapy with streptokinase or APSAC. AB - In the present study, we systematically investigated aprotinin, epsilon aminocaproic acid (EACA) and tranexamic acid as inhibitors of fibrinogen breakdown and of the generation of fibrinogen degradation products (FgDP). The experimental setting very closely imitated the conditions in practice when collecting blood from patients receiving thrombolytic therapy with streptokinase or APSAC. The minimal concentration of aprotinin required to completely inhibit fibrinogen breakdown and FgDP generation was 200 KIU/ml blood. This was sufficient even at the highest concentrations of streptokinase and APSAC expected to occur in patients (300 U/ml and 46 nM, respectively). However, 200 KIU/ml aprotinin heavily interfered in the determinations of plasminogen and alpha 2 antiplasmin. Relatively low concentrations of EACA (200 mM) and tranexamic acid (35 mM) were sufficient to prevent FgDP generation, but they interfered in the Clauss assay of fibrinogen. A non-interfering concentration of EACA (7 mM) allowed the inhibition of lower concentrations of APSAC (20 nM) and streptokinase. We conclude that at least 200 KIU aprotinin per ml blood is necessary to effectively inhibit in vitro fibrinogenolysis under circumstances likely to be met in clinical practice during thrombolytic therapy. PMID- 1716482 TI - Platelet activation during preparation and storage of concentrates: detection by flow cytometry. AB - This paper describes a rapid, whole blood micro-method for assessing the activation status of platelets that can be used to monitor changes occurring during the preparation and storage of platelet concentrates. Platelets are examined for the expression of markers of early activation (fibrinogen binding) and of degranulation (GP53 and GMP-140 expression), using flow cytometry. Fibrinogen binding can be detected in a number of blood packs even before processing. Platelet concentrates prepared by the washed platelet method become further activated during centrifugation and separation. More than 90% of platelets in freshly prepared concentrates carried bound fibrinogen (90.9 +/- 7.7%) whilst the GP53 and GMP-140 antigens were expressed on 11.3 +/- 3.3% and 13.7 +/- 5.1 of these cells respectively (mean +/- SD). Fibrinogen binding fell on storage of the platelets for four days, attributed to shedding of the fibrinogen molecule. PMID- 1716483 TI - Synthesis of a recombinant DNA-derived HBV e antigen and its application in diagnosis. AB - The gene coding for HBV e antigen (HBeAg) and core antigen (HBeAg) are located in the C region of the HBV genome. In this paper 1.8 kb BamHI-EcoRI DNA fragment carrying HBc gene from adw2 HBV DNA was digested with Bal31 and HapII, a series of fragment with different length were produced and inserted into pUR222 and pUC18 vectors to construct various recombinant plasmids. Two new recombinants able to direct high level synthesis of HBeAg in E. coli were obtained. Recombinant HBeAg was then purified by DEAE chromatography and affinity chromatography. The preparation obtained were identified by electrophoresis and immunochemical analysis. Molecular weight of the recombinant HBeAg determined by SDS-PAGE was about 38 kd. It is considered to be a dimer of two fused antigen molecules in size of 19 kd. Bacterial extracts prepared from cells harboring one of the constructed plasmids have been used successfully as a diagnostic reagent for the detection of HBeAg and anti-HBe in human sera by ELISA. PMID- 1716484 TI - Cytokines and transcription factors. AB - Transcriptional induction is mediated by transcription factors, which influence gene expression by interacting with specific elements in their regulatory regions. Here we discuss transcription factors involved in the regulation of cytokines and their receptors (interleukin-2, interleukin-2 receptor alpha chain, and interferon-beta), as well as transcription factors that are activated by cytokines (interleukin-1, tumor necrosis factor, interferons, and transforming growth factor beta). PMID- 1716485 TI - Induction of in vitro human lymphocyte migration by interleukin 3, interleukin 4, and interleukin 6. AB - The effects of interleukin 3 (IL 3), IL 4, IL 6, and interferon-gamma (IFN-gamma) on lymphocyte migration have been investigated and compared with those of transforming growth factor-beta 1 (TGF-beta 1), granulocyte colony stimulating factor (GCSF), and macrophage colony stimulating factor (MCSF). Potent, temperature-dependent stimulation of lymphocyte migration was obtained in response to IL 3 and IL 4 (ED50 less than 10(-11) M and less than 10(-13) M, respectively) and this migration was abolished in the presence of 3 micrograms ml 1 cytochalasin B. IL 6 and IFN-gamma were less active (ED50 greater than or equal to 10(-9) M and greater than or equal to 10(-8) M, respectively), maximal migration in response to IFN-gamma being only 30% above background as compared with approximately 250% for IL 3 and IL 4. TGF-beta 1, GCSF, and MCSF failed to stimulate lymphocyte migration in doses similar to those used for IL 3, IL 4, and IL 6. The presence of antisera to IL 3, IL 4, and IL 6 specifically inhibited lymphocyte migration induced by the corresponding cytokines (IC50 values being 1/10,000, greater than 1/30,000, and greater than 1/30,000 dilution of antibody, respectively). Cross-desensitization experiments using IL 3 and IL 4 demonstrated that neither IL 3 nor IL 4 were able to stimulate dose-related lymphocyte migration in cells preincubated with IL 3. Cells preincubated with IL 4 were only stimulated by a supraoptimal concentration of IL 4 (10(-11) M). The induction of lymphocyte migration by IL 3, IL 4, and IL 6 therefore appears to be a specific and potentially important effect of these cytokines. Cross-desensitization of lymphocytes by IL 3 and IL 4 raises the possibility that the induction of lymphocyte migration by these cytokines may occur through a common postreceptor signal transduction mechanism. PMID- 1716486 TI - Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application. AB - Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL. PMID- 1716487 TI - Differential regulation of IL 6, IL 1 A, IL 1 beta and TNF alpha production in LPS-stimulated human monocytes: role of cyclic AMP. AB - Interleukin 6 (IL 6), IL 1 alpha, IL beta and tumor necrosis factor (TNF) alpha are four cytokines induced in monocytes by lipopolysaccharide (LPS); however, it is unclear whether the mechanisms which control their production are similar. In this study, we report the effects of prostaglandin E2 (PGE2), and two other cAMP elevating agents, dibutyryl cAMP and 3-isobutyl-1-methyl-xanthine, on the in vitro LPS-induced production of IL 6, IL 1 alpha, IL 1 beta and TNF alpha by human monocytes. The production of these four cytokines was found to be selectively regulated in monocytes, by increases in intracellular cAMP levels. In effect, such agents enhanced, in a dose-dependent manner, both extracellular and cell-associated IL 6 production by LPS-stimulated monocytes. In contrast, it was confirmed, using the same samples, that these cAMP-elevating agents inhibit both extracellular and cell-associated TNF alpha production in a dose-dependent manner. IL 1 alpha and IL 1 beta production, measured by means of specific immunoreactive assays, were not significantly modified. Kinetic analysis showed that the potentiating effect of cAMP on IL 6 production, along with its inhibiting effect on TNF alpha production, could be seen as early as 1 hr after LPS stimulation. These results demonstrate that IL 6, TNF alpha, IL 1 alpha and IL 1 beta production can be differently modulated by an agent, PGE2, which is produced simultaneously by LPS-stimulated monocytes. Such differential autocrine modulation may play an important role in the regulation of the production of cytokines participating in immune and inflammatory responses. PMID- 1716488 TI - The effects of cytokines, platelet activating factor, and arachidonate metabolites on C3b receptor (CR1, CD35) expression and phagocytosis by neutrophils. AB - The cytokines tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G CSF), and interleukin 1 (IL 1) all caused an upregulation of C3b receptors (CR1) on neutrophils that ranged from around 76% (G-CSF and IL 1) to 93% (TNF alpha and GM-CSF) of the upregulation obtained by pretreatment of the neutrophils with the chemotactic peptide FMLP. However, only TNF alpha and G-CSF caused a significant increase in phagocytosis of opsonized microspheres. Platelet derived growth factor, interleukin 2, and transforming growth factor beta had no effect on either of these parameters. The mediators platelet activating factor (PAF) and leukotriene B4 (LTB4) both caused a large upregulation of CR1 (93% and 80%, respectively, of the FMLP-mediated value); however, only PAF caused a significant enhancement of phagocytosis by the neutrophils. Prostaglandin E2 and thromboxane B2 had no effect on these parameters. Considerable individual variation was observed among some of the untreated and mediator-treated neutrophil preparations regarding CR1 expression and phagocytosis. The upregulation of CR1 and associated increase in phagocytic capacity of neutrophils caused by certain cytokines and other mediators may be important in host defense. Also the lack of enhancement of phagocytosis accompanying an upregulation of CR1 is unusual and may have important implications regarding the cellular mechanisms of phagocytosis by neutrophils. PMID- 1716489 TI - Lymphokines and the acute-phase response in clinical bone marrow transplantation. AB - This paper reviews the role of the acute phase response and of cytokines in clinical bone marrow transplantation. Data are discussed from the literature and from the authors experience which show that measurement of C-reactive protein is a rather non-specific marker of tissue injury, but that it is elevated in graft versus-host disease, and especially in infection. Cytokines are clearly implicated in several aspects of transplantation, and tumour necrosis factor in particular may be important. Although there are some data which associate high TNF levels with severe graft-versus-host disease, this association may not hold true in individual patients. PMID- 1716490 TI - Is "tumor necrosis factor" the major effector of pulmonary fibrosis? AB - A major role of TNF in pulmonary fibrosis is supported by the following evidences obtained from pulmonary fibrosis induced in mice by bleomycin or silica particles; 1) these diseases are associated with a marked and lasting increase of the TNF mRNA within the lung, 2) they can be prevented by a treatment with anti TNF antibody or aggravated by a perfusion of mouse recombinant TNF, 3) an infusion of TNF-alpha can reproduce the alterations observed during pulmonary fibrosis such as growth of fibroblasts, collagen deposition, cell necrosis. PMID- 1716491 TI - Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM CSF): two cytokines increasing histamine synthesis by hematopoietic cells. PMID- 1716492 TI - In vivo administration of recombinant human interleukin-3 elicits an acute phase response involving endogenous synthesis of interleukin-6. AB - Interleukin-6 has been shown to stimulate in vitro synthesis of C-reactive protein (CCRP) in hepatocytes by enhancing the transcriptional rate of the CRP gene. It has also been demonstrated that IL-6 spurs the terminal differentiation of B-cells into immunoglobulin secreting cells when synergizing with IL-3. IL-6 may therefore act as a prime molecule in the acute phase and immune response. We have administered rh IL-3 to cancer patients in a phase I/II clinical trial. Endogenous IL-6 levels increased in a dose-dependent fashion upon i.v. bolus injection of rh IL-3 and continued to be significantly elevated above pretreatment levels when IL-3 was further administered by the s.c. route. Increases of IL-6 levels were associated with enhanced production of CRP in vivo detected after 24 h of injection. At day 14 of rh IL-3 treatment plasma immunoglobulin concentrations were measured and IgM was found to be increased by greater than 2.5 fold above starting levels. These results indicate that rh IL-3 also augments the acute phase response in vivo and contributes to increased synthesis of IgM and that induction of endogenous IL-6 is involved in these events. PMID- 1716493 TI - Interleukin-6 (IL-6) and acute-phase proteins in rats with biliary sepsis. AB - We measured serum interleukin-6 (IL-6) and acute-phase proteins, alpha 1-acid glycoprotein (AGP) and alpha 2-macroglobulin (alpha 2M), after a retrograde intrabiliary bacterial infection in rats with biliary obstruction. Maximum serum IL-6 was obtained at 6 h in rats following inoculation of bacteria (10(6) CFU/ml E. Coli) in the bile duct and it was higher than that observed in rats undergoing a bile duct ligation or a laparotomy. There was a strict relationship between the level of IL-6 at 6 h and the modified levels of AGP and alpha 2M at 48 h. AGP and alpha 2M levels were the highest in sera of rats with bile duct infection as compared with those found in sera of rats with bile duct ligation or laparotomy. After inoculation of E. Coli or E. Fecalis, blood IL-6 level was always higher at 6 h in inferior vena cava as compared with that found in the supra hepatic vein. These results indicate that IL-6 is synthesized after a biliary sepsis and that its blood level is higher in the systemic circulation than in the local circulation. PMID- 1716494 TI - Thymic lymphocytes and thymic epithelial cells in 4 cases of congenital immunodeficiency. AB - Congenital defects of predominantly cell-mediated immunity without other major defects or recognizable enzyme deficiency are classified "severe combined immunodeficiency" (SCID). Affected thymuses are severely reduced in size, microscopically they show only few lymphocytic cells and a cortico-medullary boundary is missing as well as Hassal's corpuscules. The primary defect is not yet clear. In this study 4 such cases were examined. The intrathymic epithelial cells showed expression of different cytokeratins and the typical strong positivity for the major histocompatibility complex. Lymphoid cells were phenotyped immunohistochemically and appeared as a very small population of cortical thymocytes, which was especially clear in a prenatally diagnosed and aborted fetus. His thymus, not altered by stress of infection or bone marrow transplantation, showed those few lymphocytes clustered around epithelial cells. The finding suggests an arrest in maturation and cell amplification rather than the absence of stem cells in the pathogenesis of SCID. Alterations in the thymic microenvironment could not be revealed by our methods. PMID- 1716495 TI - Landau-Kleffner syndrome: epileptic activity in the auditory cortex. AB - The Landau-Kleffner syndrome (LKS) is characterized by electroencephalographic spike discharges and verbal auditory agnosia in previously healthy children. We recorded magnetoencephalographic (MEG) spikes in a patient with LKS, and compared their sources with anatomical information from magnetic resonance imaging. All spikes originated close to the left auditory cortex. The evoked responses were contaminated by spikes in the left auditory area and suppressed in the right--the latter responses recovered when the spikes disappeared. We suggest that unilateral discharges at or near the auditory cortex disrupt auditory discrimination in the affected hemisphere, and lead to suppression of auditory information from the opposite hemisphere, thereby accounting for the two main criteria of LKS. PMID- 1716496 TI - A novel glycine-rich cell wall protein gene in rice. AB - A genomic clone isolated from rice (Oryza sativa var. IR 36) contains a gene that encodes a glycine-rich cell wall protein. This gene is a member of a multi-gene family. Two transcripts with different 5' termini are encoded by this gene and the expression of these two transcripts is differentially regulated. The amino acid sequence derived from the DNA sequence of this gene has a putative amino terminal signal peptide, and a highly repetitive structure in the putative mature protein. A model is proposed which describes the potential monomeric structure of this glycine-rich protein and how these monomers may be covalently linked to produce a network-like structure. PMID- 1716497 TI - Limited expression of a diverged beta-tubulin gene during soybean (Glycine max [L.] Merr.) development. AB - We examined the developmental expression of a diverged soybean beta-tubulin gene (designated sb-1), which had been cloned and sequenced previously. A probe specific for the sb-1 gene was constructed from the 3' transcribed untranslated sequence. As a control, a more general probe for beta-tubulin genes and their transcripts was constructed from a highly conserved region of the third exon of another soybean beta-tubulin gene, sb-2. Poly(A)+ RNA, extracted from various soybean tissues and organs, was probed alternatively with the sb-1 gene-specific probe and with the generic beta-tubulin probe. Levels of beta-tubulin transcripts recognized by the generic probe differed by a factor of approximately 3 in the different tissues and organs and varied with the state of organ development. Highest levels were found in young, unexpanded leaves and they decreased as leaf maturation occurred. In contrast, transcripts of sb-1 were nearly undetectable in young leaves, and they increased as leaf maturation occurred. Levels of sb-1 transcript were low in all organs of the light-grown plant examined, except the hypocotyl, where they were approximately 10-fold higher. However, the highest levels of sb-1 transcripts were observed in elongating hypocotyls of etiolated seedlings. Exposure of six-day-old etiolated seedlings to light for 12 hours halted further hypocotyl elongation and brought about a dramatic, nearly 100 fold, decrease in the steady-state level of sb-1 transcripts. PMID- 1716498 TI - Low water potentials affect expression of genes encoding vegetative storage proteins and plasma membrane proton ATPase in soybean. AB - We have examined growth, water status and gene expression in dark-grown soybean (Glycine max L. Merr.) seedlings in response to water deficit (low water potentials) during the first days following germination. The genes encoded the plasma membrane proton ATPase and two proteins of 28 kDa and 31 kDa putatively involved in vegetative storage. Water potentials of stems and roots decreased when 2-day-old seedlings were transferred to water-saturated air. Stem growth was inhibited immediately. Root growth continued at control rates for one day and then was totally inhibited when the normal root-stem water potential gradient was reversed. Expression of mRNA for the 28 kDa and 31 kDa proteins, measured independently using specific 3'-end probes, occurred about equally in stems. However, only the mRNA for the 31 kDa protein was detected in roots and at a lower abundance than in stems. Low water potentials increased the mRNA only for the 28 kDa protein in stems and the 31 kDa protein in roots. This differential expression followed the inhibition of stem growth but preceded the inhibition of root growth. The expression of the message for the ATPase, measured using a probe synthesized from a partial oat ATPase clone, was low in stems and roots but there was a 6-fold increase at low water potentials in roots. The increase followed the inhibition of root growth. This appears to be the first instance of regulation of ATPase gene expression in plants and the first demonstration of differential expression of the 28 kDa, 31 kDa, and ATPase messages. The correlation with the differential growth responses of the stems and roots raises the possibility that the differential gene expression could be involved in the growth response to low water potentials. PMID- 1716499 TI - Anther-specific, developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower. AB - We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower. PMID- 1716500 TI - Blue light is required for survival of the tomato phytochrome-deficient aurea mutant and the expression of four nuclear genes coding for plastidic proteins. AB - When dark-grown aurea mutant tomato seedlings which lack more than 95% of the phytochrome present in isogenic wild-type seedlings are kept in white or blue light, four nuclear-encoded transcripts coding for plastidic proteins (the light harvesting chlorophyll a/b-binding protein of photosystem I and II [cab-PSII], plastocyanin and subunit 2 of photosystem I) are present in comparable amounts. These transcript levels in red light are strongly reduced in aurea seedlings when compared with those of wild type. Thus, blue light is required for normal expression of these genes in the mutant, while red light alone is not sufficient. Red light-grown aurea seedlings are very sensitive to blue light, even 10 minutes of blue light every day suffices to cause a measurable increase in cab-PSII transcript level. The action of blue light on the expression of cab-PSII in the mutant is under phytochrome control. After 8 days of blue light, phytochrome is almost as effective in inducing cab-PSII mRNA as in the isogenic wild type, whereas after 8 days of red light, only a small phytochrome response was observed in the mutant. It is concluded that blue light sensitizes the mutant to the residual phytochrome which allows normal gene expression and survival of the mutant under daylight conditions. PMID- 1716501 TI - Angiogenic growth factors. AB - Angiogenesis, the formation of new blood vessels, plays a central role in a variety of physiological and pathological processes such as embryonic development, wound healing and tumor growth. It is a complex, multi-step process that involves the migration and proliferation of capillary endothelial cells. Several factors that stimulate the proliferation of endothelial cells in vitro have been shown to induce angiogenesis in vivo. Among these angiogenic growth factors are wide-spectrum multifunctional mitogens (e.g. the fibroblast growth factors) and the recently identified factors with distinct specificity for vascular endothelial cells (e.g. the platelet-derived endothelial cell growth factor). Another group of factors apparently induce angiogenesis indirectly (e.g. transforming growth factor-beta) by stimulating target cells to release angiogenic factors or by other mechanisms. The differential expression, release and activation of these factors might regulate angiogenesis under various physiological and pathological conditions. PMID- 1716502 TI - Heat shock proteins and autoimmunity. AB - Heat shock proteins are major antigens of the T cell response to many pathogens. Because of their marked conservation of sequence in pro- and eukaryotes, they have also been postulated as target antigens in autoimmune disease. Despite substantial in vitro evidence to suggest T cell recognition of self hsp, it has proved more difficult to implicate such recognition in autoimmune disease. Nevertheless, their dominance in T cell responses generally and the ability to manipulate certain autoimmune diseases by altering responses to hsp suggests that T cell recognition of hsp is important in these diseases; however at present the mechanisms involved remain obscure. PMID- 1716503 TI - Heat shock proteins implicated in antigen processing and presentation. AB - The recognition of antigen by helper T lymphocytes requires that the antigen be processed and presented by a cell expressing the Major Histocompatibility Complex (MHC) class II molecules. Antigen is taken into an acidic intracellular compartment where it is proteolytically degraded, releasing peptide fragments which become displayed on the cell surface in association with the MHC class II molecules. At present, little is known of the discrete steps in this process or the molecular mechanisms underlying the assembly of the antigenic peptide-MHC class II complex. Although antigenic peptides have been shown to bind directly to the MHC class II molecules, the unusual characteristics of this binding, namely extraordinarily slow association and dissociation rates, make it likely that the binding of peptide to MHC is facilitated within the cell by unknown mechanisms. The functions recently ascribed to several members of the heat shock proteins, in particular their binding to newly synthesized, denatured or inappropriately folded proteins, make them attractive candidates to play a role in antigen processing. In searching for proteins which might facilitate the binding of peptides to the MHC class II molecules, we isolated a peptide binding protein of 72/74 kDa Mr (PBP72/74). Antibodies raised to PBP72/74 block antigen processing and/or presentation, indicating a role for PBP72/74 in this process. Recent studies show that PBP72/74 is serologically related to the heat shock protein (hsp) family and that PBP72/74 shares a second characteristic of this family, namely ATP binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716504 TI - [Effects of various proteins on erythrocyte aggregation effected by proteoglycans]. PMID- 1716505 TI - [Electron-radioautographic study of cell viability of the human heart after death of the body]. AB - Human heart cells RNA-synthesis was investigated by electron-microscopic radioautography in the 3rd, 4th, 8th and 13th hour after death. In many blood vessels cells and in some myocytes via creation of adequate regimen a capacity to restore RNA-synthesis persevered for 8-13 hours after death. PMID- 1716506 TI - Effects of tachykinins on [3H]taurine release from human astrocytoma cells (U-373 MG). AB - Substance P (SP) stimulated [3H]taurine release from human astrocytoma cells (U 373 MG). This effect was concentration dependent and the EC50 was 0.3 nM. This stimulatory effect of SP can be inhibited by spantide, a SP receptor antagonist. The rank order of potencies of related tachykinins and their analogues in stimulating the release of [3H]taurine was SP much greater than neurokinin A much greater than neurokinin B and [Glp6,L-Pro9]SP (6-11) much greater than [Glp6, D Pro9]SP (6-11) which conformed to that reported for the tachykinin NK-1 receptor. In addition to SP, isoproterenol, a beta-adrenergic agonist, can also increase the release of [3H]taurine from these cells and the effects of SP and isoproterenol were additive. PMID- 1716507 TI - Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. AB - 1. Body weight and the weight of the digestive organs and activities of some digestive enzymes were determined from hatching to 23 d of age. 2. Relative daily growth rate peaked at 11 d of age (22% gain/d) and then decreased gradually. 3. The vitelline residue was decreased rapidly from 4.6 g at hatching to negligible values from 4 d of age. 4. Maximal allometric growth of the pancreas and small intestine was 4-fold and that of liver 2-fold greater than that of the body. 5. Activities (units/kg body weight) of the digestive enzymes measured in the pancreas and intestinal contents increased with age. In the pancreas maximal values were attained on day 8 for amylase and lipase and 11 for trypsin and chymotrypsin. In the small intestine maxima were attained on day 4 for lipase, 11 for trypsin and chymotrypsin and 17 for amylase. 6. The development of secretion of digestive enzymes in the post-hatched chick could be a limiting factor in digestion and subsequently in food intake and growth. PMID- 1716508 TI - Monoclonal antibody based sandwich enzyme-linked immunosorbent assay for chicken growth hormone. AB - 1. Monoclonal antibodies which bind to different epitopes of chicken growth hormone (cGH) were used to develop a homologous sandwich enzyme-linked immunosorbent assay (ELISA). 2. The first antibody, which is species specific, was immobilised on microtitre plates and concentrations of cGH in biological fluids were estimated by revealing bound hormone using a second, biotinylated monoclonal antibody. 3. The sensitivity was 0.024 ng/ml, which is at least ten fold greater than current radioimmunoassays (RIA) and there was no cross reactivity to other chicken pituitary hormones or to growth hormone from other species. 4. The accuracy and precision of the assay were similar to RIA, and the growth hormone concentrations measured in plasma samples by both RIA and this new ELISA showed a high degree of correlation. 5. The assay takes only 4 h using pre coated plates which can be stored at 4 degrees C in sucrose. The advantages of being rapid and non-isotopic make this method attractive to both research and industrial laboratories. PMID- 1716509 TI - Ethics of euthanasia. PMID- 1716510 TI - Prostate-specific antigen levels from completely sectioned, clinically benign, whole prostates. AB - Clinically benign whole, untrimmed prostates and pelvic lymph nodes were obtained from 105 patients at autopsy. All 105 patients had premortem serum from which prostate-specific antigen (PSA) levels were obtained. Sixty-eight did not have carcinoma of the prostate (CAP), 28 had CAP less than 1 ml and 9 had CAP larger than 1 ml. Eleven untrimmed prostates weighed 80 g or more and eight had elevated PSA levels (more than 4.0 ng/ml): five of eight without CAP, two of two with CAP less than 1 ml, and one of one with CAP larger than 1 ml. Ninety-four whole untrimmed prostates weighed less than 80 g and 20 had elevated PSA levels: ten of 60 without CAP, two of 26 with CAP less than 1 ml, and eight of eight with CAP larger than 1 ml. This study suggests that PSA levels from patients with untrimmed prostates weighing 80 g or more (equivalent to a 60-g trimmed prostate) are usually elevated regardless whether CAP is present. However, CAP less than 1 ml, in untrimmed prostates less than 80 g, usually does not elevate PSA levels whereas CAP larger than 1 ml usually does (P less than 0.0001). The likelihood that elevated PSA levels, from patients with untrimmed prostates less than 80 g, are due to CAP larger than 1 ml increases as the PSA level increases. PMID- 1716511 TI - Prospective quality-of-life analysis after palliative photoablation for the treatment of malignant dysphagia. AB - Forty patients were treated for the relief of malignant dysphagia by using laser photoablation. Their quality of life was assessed before the start of treatment and at monthly intervals until death. Two methods were used, a physician's assessment (QL index) and a patient's self-assessment, the linear analogue self assessment (LASA). There was significant correlation between assessments done at different times by different physicians (QL index rs, 0.786; P less than 0.001; LASA rs, 0.865; P less than 0.001). The correlation coefficient of the QL index and the LASA score with the patient's dysphagia grade was 0.459 and 0.336, respectively. The patient's swallowing ability, QL index, and LASA all were improved significantly at some time after laser therapy. The mean survival was 16 weeks with 58% of patients dying at home, 28% in the hospital, and 18% in a hospice. It was concluded that laser photoablation improves the overall quality of life in patients with malignant dysphagia. PMID- 1716512 TI - Photoimmunotherapy of human ovarian carcinoma cells ex vivo. AB - Photodynamic therapy is a relatively new and potentially selective experimental approach to the treatment of malignant neoplasms. Its inherent dual selectivity is reinforced by the use of photosensitizer-monoclonal antibody conjugates. The goal of this study was to evaluate the phototoxicity and selectivity of an immunoconjugate (IC) synthesized from a chlorin derivative chlorin e6 monoethylenediamine monoamide (CMA) as the photosensitizer and an anti-ovarian carcinoma monoclonal antibody OC125. Binding efficiency and specificity of the IC were determined by enzyme-linked immunosorbent assay, and specific covalent linkage of the monoclonal antibody to the photosensitizer was demonstrated by fluorescence and electrophoresis. Phototoxicity was tested against ascites or pleural fluid cells from 15 patients with ovarian and nonovarian cancers. Tumor cells from the fluid were treated with the IC at 3 microM equivalent CMA concentration and irradiated at 654 nm (lambda max CMA in IC) at 25 J/cm2 from an argon ion-pumped dye laser. Phototoxic efficacy was assayed by [3H]thymidine incorporation. Ovarian cancer cells exhibited high cytotoxicity with [3H]thymidine incorporation of 2.4 +/- 2.2%, while nonovarian cancer cells under identical conditions exhibited none to reduced cytotoxicity with [3H]thymidine incorporation of 70 +/- 54%. Using a Wilcoxon test, there was a statistically significant difference between these two groups (P less than 0.001). Dose response curves revealed reciprocity in photosensitizer concentration and fluence. These results demonstrate that photoimmunoconjugates retain significant antigen binding specificity and affinity, are effective in the selective photochemical eradication of target cells, and merit further evaluation as photochemotherapeutic agents. PMID- 1716513 TI - Human high-molecular-weight melanoma-associated antigen mimicry by mouse antiidiotypic monoclonal antibody TK7-371. AB - Since the human high-molecular-weight melanoma-associated antigen (HMW-MAA) represents a useful target to implement active specific immunotherapy with mouse antiidiotypic monoclonal antibody (mAb), the present study is aimed at developing and characterizing mouse antiidiotypic mAbs which bear the mirror image of the determinant defined by the anti-HMW-MAA mAb TP61.5. To this end, a BALB/c mouse was immunized with the syngeneic mAb TP61.5. Screening of the 703 generated hybridomas showed that 13 of them secrete antiidiotypic mAbs which inhibit the binding of the immunizing mAb TP61.5 to melanoma cells by at least 75%. The dose of antiidiotypic mAb required to inhibit by 50% the binding of mAb TP61.5 to melanoma cells ranged between 4 and 200 ng. The 13 antiidiotypic mAbs recognize conformational idiotypes, since they did not react in Western blotting with the heavy and light chain of mAb TP61.5 isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Furthermore, the 13 antiidiotypic mAbs did not react with 23 mAbs which recognize 13 determinants of the HMW-MAA distinct from that defined by mAb TP61.5. Analysis of the 13 antiidiotypic mAbs for their ability to elicit cellular and humoral anti-HMW-MAA immunity showed that only mAb TK7-371 elicited a delayed-type hypersensitivity reaction to HMW-MAA-bearing cells in syngeneic hosts and anti-HMW-MAA antibodies in BALB/c mice and in rabbits. Since rabbits express the HMW-MAA, the present results indicate that the antiidiotypic mAb TK7-371 can induce humoral immunity to self HMW-MAA. Therefore, the antiidiotypic mAb TK7-371 may be an efficacious immunogen to implement active specific immunotherapy in patients with melanoma. PMID- 1716514 TI - Induction of growth factor RNA expression in human malignant melanoma: markers of transformation. AB - Alteration in the expression of growth factors is widely accepted as being one of several critical defects in the generation of the malignant cell. In the present study, 19 human metastatic melanoma cell lines were compared to 14 normal human foreskin melanocyte cell lines for the production of RNA transcripts specific for 11 different growth factors. Using the extremely sensitive technique of polymerase chain reaction to amplify growth factor-specific complementary DNAs, we analyzed the following: transforming growth factor (TGF) types alpha, beta 1, beta 2, and beta 3, acidic (a) fibroblast growth factor (FGF), basic (b) FGF, FGF 5, keratinocyte growth factor (KGF), HST, and platelet-derived growth factor (PDGF) types A and B. There were clear distinctions among the patterns of growth factor RNA expression by normal melanocytes and malignant melanoma cells. The prototypic melanocyte pattern of expression included TGF beta 1, TGF beta 3, and KGF. A subset of melanocyte cell lines also expressed PDGFA transcripts. In contrast, melanoma cells characteristically expressed RNA transcripts of TGF beta 1, TGF beta 2, TGF beta 3, TGF alpha, bFGF, KGF, and PDGFA. Subsets of melanoma cell lines also expressed aFGF, FGF-5, and PDGFB. The results presented indicated that TGF beta 2, TGF alpha, and bFGF may be particularly important in melanomagenesis and that these, as well as FGF-5, aFGF, and PDGFB, can be used as markers of transformation in this tumor type. PMID- 1716515 TI - Characterization of tenascin secreted by human melanoma cells. AB - Tenascin is a large glycoprotein of the extracellular matrix. It shows a site restricted expression during embryogenesis and can be found in adult tissues during wound healing and tumorigenesis. Because of the potential involvement of tenascin in adhesion and invasion during metastasis, the study of the interactions of tumor cells with tenascin is of considerable interest. Using five anti-melanoma monoclonal antibodies to four different epitopes of human tenascin, we found that most melanoma cells secrete tenascin in vitro constitutively. Transforming growth factor beta 1 in the medium increased secretion in tenascin producing cells. Tenascin was present in sera of melanoma patients, with significantly elevated levels in patients with advanced melanomas as compared to patients with low tumor burden or to normal donors. Normal and malignant melanocytes did not attach to tenascin as substrate within 1 to 2 h and tenascin could also inhibit fibronectin-dependent adhesion. These results indicate that tenascin may play a critical role in cell-substrate interactions of melanoma cells. PMID- 1716516 TI - Qualitative and quantitative changes of human tenascin expression in transformed lung fibroblast and lung tumor tissues: comparison with fibronectin. AB - Qualitative and quantitative alterations of human tenascin (TN) expression in virally transformed lung fibroblasts and in lung tumor tissues were investigated using S1 nuclease protection analysis in comparison with those of fibronectin (FN). Transformed fibroblasts and fetal lung tissues expressed more TN mRNA with an extra sequence encoding the sixth FN type III repeat than did normal cells and adult tissues. The splicing pattern of TN mRNA was also altered in many lung cancer tissues, showing increased or sometimes decreased expression of the TN mRNA with the extra sequence when compared with their surrounding normal tissues. These results provide additional evidence for the oncodevelopmental regulation of alternative RNA splicing in human lung tissues, first observed with FN mRNA (F. Oyama, et al., Cancer Res., 50: 1075-1078, 1990). Quantitative analysis of the levels of TN and FN mRNAs showed that the ratio of TN mRNA to FN mRNA was significantly increased in transformed fibroblasts and in some lung tumor tissues, when compared with their normal counterparts. Among different types of lung tumors, a significant increase of the TN/FN ratio was observed with most squamous cell carcinoma but with only a small fraction of adenocarcinoma. Since TN has been shown to inhibit cell adhesion to FN, the altered ratio of TN mRNA to FN mRNA may well affect the adhesive and migratory properties of tumor cells in lung cancer tissues. PMID- 1716517 TI - Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development. AB - Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6 ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development. PMID- 1716518 TI - Induction of interleukin-5 responsiveness in resting B cells by engagement of the antigen receptor and perception of a second polyclonal activation signal. AB - Experiments were performed to examine the nature of agents which could induce IL 5 responsiveness in small, resting splenic B lymphocytes. First, IL-5 increased plaque forming cell responses to the TI-1 antigen TNP-LPS. A second set of experiments using anti-IgM + LPS which allowed limiting dilution analysis showed induction of IL-5 responsiveness in about 20% of the resting B cell population. In the same system, IL-4 increased the percentage of proliferating cells by about 40%. A third system using the TI-2 analog conjugate anti-IgD-dextran (anti-delta dextran) also rendered small, resting B cells responsive to IL-5. An additional system employing anti-IgM plus dextran sulfate, which also allowed limiting dilution analysis, induced IL-5 responsiveness in at least 10% of resting B cells. The features common to all four systems inducing B cell IL-5 responsiveness are at least twofold. Each system directly accesses the B cell antigen receptor and causes crosslinking. Second, each system also provides an additional polyclonal activating moiety, some of which may be similar to those in thymus independent antigens. These results suggest that some resting B cells may become IL-5 receptive after perception of at least two kinds of signals one of which perturbs sIg and the second being nonspecific and polyclonally activating. PMID- 1716519 TI - HgCl2-induced perturbation of the T cell network in experimental allergic encephalomyelitis. I. In vitro characterization of T cells involved. AB - Mercuric chloride (HgCl2) induces in Lewis (LEW) rats a non-antigen-specific immunosuppression and is able to down-modulate experimental allergic encephalomyelitis in about 70% of the rats. The aim of the present study was to determine the frequencies of lymph node cells involved in the proliferative response to myelin basic protein in rats injected with HgCl2 and immunized with myelin by using limiting dilution analysis (LDA). Highly frequent CD8+ T suppressor cells and at least 10-fold less frequent protein basic-specific T helper cells were detected in these rats. A third cell type allowing the proliferative response of Th cells in spite of Ts cells was also demonstrated. These cells, which could act as contrasuppressor cells, were CD4+ and adhered to Vicia villosa lectin; their frequency was in the same range as that of T helper cells. These data illustrate the potential role of different levels of T cell immunoregulatory activity in autoimmunity and the major interest of LDA in their analysis. PMID- 1716520 TI - CD45 isoforms associated with distinct functions of CD4 cells derived from unusual healthy donors lacking CD45RA- T lymphocytes. AB - We now report two healthy individuals whose T lymphocytes were over 95% positive for CD45RA antigen expression. However, these donors normally expressed both the CD29 high (CD29+) and CD45RO high (CD45RO+) antigens on approximately 40 and 50% of their CD4 cells, respectively. Despite the strong CD45RA expression on the surface of almost all CD4 cells, the CD29 marker allowed T cells from these donors to be divided phenotypically into subsets having distinct in vitro function. CD4+CD29+ cells from these donors responded maximally to recall antigens such as TT and provided strong helper function for B cell Ig synthesis. In contrast, CD4+CD29- cells responded poorly to recall antigens and had poor helper function for B cell Ig synthesis, but had strong suppressor activity. Thus, CD29 antigen expression was still predictive of the in vitro functional activity as previously described for normal donors. Furthermore, biochemical analysis of the distribution of individual CD45 isoforms on the surface of these subsets of CD4 cells revealed distinct differences. The CD4+CD29 high (CD4+CD29+) subset of cells primarily expressed the 180-, 190-, and 205-kDa CD45 isoforms, while the CD4+CD29 low (CD4+CD29-) cells primarily expressed the 190-, 205-, and 220-kDa CD45 isoforms. These results suggest that despite the superficial phenotypic similarity of CD4 cells in these donors, distinctions in the distribution of both CD29 and the 180- and 220-kDa CD45 isoforms exist and might play a role in the different functions of freshly isolated CD4 lymphocytes. PMID- 1716521 TI - Selective induction of B7/BB-1 on interferon-gamma stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway. AB - The B cell activation antigen B7/BB-1 is the natural ligand for the T cell antigen CD28 and these two molecules are capable of mediating T-B cell adhesion. Engagement of the CD28 pathway provides a costimulatory signal to T cells leading to enhanced lymphokine production. We report that interferon-gamma (INF-gamma) induces the expression of B7/BB-1 on monocytes. This induction was very specific since other cytokines and stimuli which activate monocytes including M-CSF, GM CSF, IL3, TNF-alpha, and LPS were unable to induce B7/BB-1. Following culture of monocytes with INF-gamma, maximal mRNA and cell surface B7/BB-1 expression was detected at 12 and 24 hr, respectively. In addition to antigen presentation, optimal T cell activation and lymphokine synthesis require an additional cell to cell contact signal provided by the antigen presenting cell. The induction of B7/BB-1 on monocytes and subsequent heterophilic interaction of B7/BB-1 with CD28 may provide a mechanism for the amplification of T cell proliferation and lymphokine production by INF-gamma activated monocytes. PMID- 1716522 TI - IL-4 down-regulates the expression of J11d on murine B cells. AB - T cell-derived IL-4 has many effects on murine B cells, including the up regulation of class II antigens and the induction of isotype switching. The development of memory B cells and the decreased expression of J11d antigens on these cells are influenced by T cells. In this report, we determined whether the decreased expression of J11d can also be mediated by T cell-derived lymphokines and, in particular, IL-4. We found that IL-4 can down-regulate the expression of J11d on both large and small B cells, that this effect becomes significant after 48 hr of culture and occurs at doses of IL-4 that are similar to those required to up-regulate murine class II MHC antigens encoded by I-region alpha genes (IA). Anti-IL-4 antibody completely blocks this effect but IFN-gamma does not. Other lymphokines (IL-1, IL-2, IL-3, IL-5, IL-6) neither induce a decrease in J11d nor alter the ability of IL-4 to down-regulate J11d expression. The decrease in J11d expression on B cells is not due to a preferential survival of cells expressing lower levels of J11d, although IL-4 has a more pronounced effect on these B cells. Finally, the down-regulation of J11d and up-regulation of IA by IL-4 occurs on all inducible B cells. PMID- 1716523 TI - Analysis of the human CD36 leucocyte differentiation antigen by means of the monoclonal antibody NL07. AB - The murine monoclonal antibody (MoAb) NL07 was generated by immunization with human platelet extracts. NL07 MoAb recognized a molecule expressed by human platelets, monocytes, and endothelial cells, as well as by the myelomonocytic line U937 and by some melanoma cells or lines. Normal endothelial cells and the melanoma cells express the NL07 epitope only while adhering to a substrate. SDS polyacrylamide gel electrophoresis and two-dimensional gel analysis indicate that the molecule recognized by NL07 MoAb on platelets is a single chain structure featuring a molecular weight of 85 kDa under reducing conditions, with an acidic isoelectric point ranging from 5.2 to 5.5. The specific phenotype distribution and the biochemical structure indicate that NL07 MoAb recognizes the platelet GPIV (CD36) molecule, a surface glycoprotein with a functional role of thrombospondin receptor. The results of competition tests with OKM5 MoAb (specific for the CD36 molecule) confirm the molecular specificity and epitope coincidence. Furthermore, upon binding to the platelets, NL07 MoAb is able to transmit via CD36 an activation signal which is followed by a potent aggregation. On the contrary, there is lack of evidence concerning the ability of the CD36 molecule of transmitting signal(s) on the U937 cells. PMID- 1716524 TI - [Serum levels of trophoblast-specific beta-1-globulin (SP1) and alpha-1 fetoprotein (AFP) in pregnant women with rheumatoid arthritis]. AB - In 19 pregnant women treated on account of rheumatic disease (17 women with rheumatoid arthritis or juvenile rheumatoid arthritis, one woman with non specified arthralgia, one woman with rheumatic fever in the case-history) were examined at least once during pregnancy for SP1 and AFP serum levels. For the assessment of SP1 simple radial immunodiffusion was used, for the assessment of AFP Sevatest ELISA AFP Micro I. The SP1 and AFP levels were compared with the physiological range in the pertaining week of gestation. In nine women of 19 patients raised SP1 levels were found, in seven elevated AFP levels. In all patients with elevated SP1 levels the disease improved, while in four of ten women with normal or reduced SP1 levels the disease deteriorated or developed during pregnancy. This relationship was not found in AFP. PMID- 1716525 TI - [Acute phase proteins during labor and surgical stress in women with normal pregnancy and late gestoses]. AB - The authors investigated within the framework of non-specific immunity reactions proteins of the acute phase during spontaneous delivery and Caesarean section up to the 5th day after delivery and on the 1st, 5th and 10th day after delivery in forty women, half of them healthy and half suffering from late gestoses. Half the women had spontaneous deliveries, half had deliveries by Caesarean section. The proteins of the acute phase reacted sensitively to the stress of childbirth during spontaneous delivery and their activation was inhibited by general anaesthesia in deliveries by Caesarean section. After spontaneous delivery the elevated proteins of the acute phase returned to baseline values by the 5th day, after operation their levels rose further up to the 5th day, orosomucoid to the 10th day after operation. In late gestoses with spontaneous delivery or by Caesarean section there were significantly elevated alpha-1-antitrypsin and alpha 2-macroglobulin and orosomucoid levels, whole in patients with late gestoses in the prealbumin levels were reduced. The elevated levels of proteinase inhibitors in late gestoses support the view on a possibly impaired coagulation equilibrium in late gestoses, and the high ceruloplasmin levels suggest an enhanced catecholamine breakdown during the stress alarm reaction. PMID- 1716526 TI - [Metabolites of neurotransmitters in the cerebrospinal fluid in children]. AB - Metabolites of neurotransmitters, homovanillic acid (HVA) and 5 hydroxyindolacetic acid (5-HIAA) were assessed in the cerebrospinal fluid of 149 children of different age from neonatal age to 13 years by the method of high pressure liquid chromatography with electrochemical detection. The mean concentration of the two metabolites in cerebrospinal fluid correlated indirectly with the childrens age--the highest mean concentrations of HVA and 5-HIAA were found in the group of neonates, lower values in infants and the lowest ones in the group of toddlers and older children. In the group of toddlers and older children with severe mental disorders and repeated tonic-clonic or myoclonic seizures a lower mean 5-HIAA was found in the cerebrospinal fluid than in children of equal age, similarly mentally affected but without seizures in the case-history. PMID- 1716527 TI - Interferon in haematological disorders. PMID- 1716528 TI - Inhibition of avian myeloblastosis virus reverse transcriptase by heterocyclic quinones: structure-activity correlation. AB - Synthetic heterocyclic quinones (107 samples) consisting of o- and p-quinoline quinones, o-isoquinoline quinones and p-quinoxaline quinones as well as o- and p naphthoquinones (3 samples) were tested for their inhibitory activities against avian myeloblastosis virus reverse transcriptase (AMV-RT) and cytotoxic activities against mouse lymphoblastoma L5178Y cells. In general, o-quinoline quinones (i.e., the 5,6-quinolinedione derivatives) are more potent inhibitors of AMV-RT than p-quinoline quinones (the 5,8-quinolinedione derivatives). Furthermore, the growth of L5178/Y cells were significantly refractory to the 8 methoxy-5,6-quinolinedione derivatives whose suppressive effects on AMV-RT function were fairly comparable to those of the other o-quinoline quinones. The longer the chain length of 7-alkyl substituent in o- or p-quinoline quinones, the lower the biological activities. PMID- 1716529 TI - Targeting of mitomycin C to the liver by the use of asialofetuin as a carrier. AB - Desialylated fetuin (asialofetuin) was adopted as a carrier for introducing drugs in parenchymal liver cells. Mitomycin C, as a model guest-compound, was covalently attached to asialofetuin through a spacer of the succinyl group. The asialofetuin-mitomycin C conjugate contained 4.4 w/w% of mitomycin C and liberated it gradually at physiological conditions (t1/2 = 37 h). The survival time of the conjugate in rat blood circulation was significantly smaller than that of the non-desialylated fetuin conjugate; the elimination half-life was 7.3 min after intravenous injection. At 30 min after injection of the conjugate in rats, 40% of the dose was present in the liver. Parenchymal cells in the liver selectively took up the conjugate, which was highly distributed to the lysosomal fraction in the cell. The greater uptake of the conjugate by hepatocytes reflected the increased excretion in the bile; totally 10.4% of the dose was recovered. PMID- 1716530 TI - The effect of Yoshida sarcoma cell transplantation to rats on the rate of acute phase protein synthesis in the liver. AB - The effect of transplantation of Yoshida sarcoma cells into rats on the concentrations of both the acute-phase proteins (APPs) in the serum and their mRNAs in the liver has been investigated. In rats that responded to transplantation by forming solid tumors, the serum APP levels increased with tumor size. Some of the APP was found to be infiltrated in the area of the tumor cells. Concentrations of the APP mRNAs in the liver were enhanced to an extent comparable to that observed for the encoded proteins, indicating that hepatocytes were the major site of their synthesis. In rats in which no formation of solid tumors occurred, the serum APP concentrations expressed a tendency to decrease below the control values. An inverse relationship between the rates of APP synthesis and the capability of rats to prevent proliferation of transplanted ascites tumor cells in the solid tumor form was discussed. PMID- 1716531 TI - Protein synthesis is required for the initiation of dendritic growth in embryonic rat sympathetic neurons in vitro. AB - We have utilized an experimental paradigm which allows the manipulation of dendritic growth in sympathetic neurons in culture to examine the effects of inhibitors of protein synthesis and RNA synthesis on the development of dendrites. Embryonic rat sympathetic neurons extend only axons when they are grown in serum-free medium on a polylysine substrate. The addition of an extract of basement membrane proteins (BME) to this culture system elicits dendritic growth within 48 h. Both cycloheximide and actinomycin-D inhibited BME-induced dendritic growth in greater than 80% of the neuronal population and reduced the number of dendrites extended by greater than or equal to 97%. In contrast, cycloheximide was found to have minimal effects on axonal growth in short-term (less than or equal to 18 h) cultures as measured with respect to the percentage of the population with axons and the number of axons per neuron. However, this inhibitor did significantly reduce (84%) the length of the axonal plexus extended. These results indicate that dendritic and axonal growth in sympathetic neurons are differentially dependent on protein synthesis such that the formation of dendrites requires protein synthesis whereas the initiation, but not the elongation, of axons is relatively independent of protein synthesis. PMID- 1716532 TI - Development of the cerebellar cortical efferent projection: an in-vitro anterograde tracing study in rat brain slices. AB - This study reports the results of an anterograde HRP analysis of the development of cerebellar cortical efferents studied in-vitro with cerebellum-brainstem slices from embryonic (E-18) through newborn rats. From E-18, Purkinje cells in the medial region of the cerebellum are seen to project to the medial cerebellar and vestibular nuclei, their appropriate targets in the adult animal. Thus, the cortical efferent system is forming during late embryogenesis and may establish synaptic contacts with target cells as early as E-20. PMID- 1716533 TI - Multiple labeling and time-resolvable fluorophores. AB - A new time-resolved fluorescence immunoassay system involving use of the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label is reviewed. This stable chelate by itself is not very fluorescent but, used in multiple labeling strategies, improves the achievable detection limits. By using multiple labeling, streptavidin tailing, and Eu3+ activation, one can create a very stable, easy-to-use reagent that is suitable for devising highly sensitive immunoassays and other biotechnological assays. This reagent, a streptavidin-based macromolecular complex, is able to detect approximately 300,000 molecules (approximately 0.5 amol) of alpha-fetoprotein in a model noncompetitive immunoassay. PMID- 1716534 TI - Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. AB - Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene. PMID- 1716535 TI - Time course of changes in pancreatic enzymes, isoenzymes and, isoforms in serum after endoscopic retrograde cholangiopancreatography. AB - Kinetics of the catalytic activities of total amylase (AMY; EC 3.2.1.1), pancreatic (P)-AMY isoenzyme, P2 and P3 isoforms, and pancreatic lipase (LPS; EC 3.1.1.3), and of the mass concentration of LPS in serum were studied in 10 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) and showed a distinct pancreatic injury. The temporal characteristics of enzyme changes described were (a) the maximal rate (Ka) at which enzymes are released into blood, (b) the time lag from ERCP until maximum concentration value, (c) the peak value of each serum enzyme, and (d) the rate (Kd) at which each enzyme is cleared from serum. LPS activity and mass concentrations increased and decreased faster than AMY and isoamylases, and the time of the LPS peak tended to be earlier than that of the other enzymes, but not significantly. The average peak increase of LPS values was higher than that of total AMY, P-AMY, and P2 isoform (P less than 0.001). The P-AMY time-activity curve was a composite of curves attributable to its isoforms; the isoforms increased and peaked sequentially, with P3 returning to normal more slowly than did P2. LPS mass and activity concentrations showed excellent parallelism, with no important differences. At 50 h after ERCP, only LPS values still exceeded the upper reference limit, returning to normal 70 h after the examination. PMID- 1716536 TI - Prostate-specific antigen in serum occurs predominantly in complex with alpha 1 antichymotrypsin. AB - Immunologic measurements of the serum concentration of prostate-specific antigen (PSA), an abundant prostatic-secreted serine proteinase, are frequently used to monitor patients with prostate cancer, though it has not been ascertained whether this immunoreactivity represents a PSA zymogen, the active proteinase, or PSA complexed to extracellular proteinase inhibitors. To characterize the PSA immunoreactivity in serum, we used monoclonal antibodies produced against PSA and a polyclonal rabbit IgG against alpha 1-antichymotrypsin in the design of three noncompetitive PSA assays: assay T, which detected PSA both when present as the active proteinase and when complexed to alpha 1-antichymotrypsin; assay F, which recognized the active proteinase but most poorly detected PSA complexed to alpha 1-antichymotrypsin; and assay C, which was specific for PSA complexed to alpha 1 antichymotrypsin. We used the three assays to measure PSA immunoreactivity in 64 patients' sera and in the effluent after gel chromatography of sera from four patients. This identified an 80- to 90-kDa complex between PSA and alpha 1 antichymotrypsin as the predominant fraction of the PSA immunoreactivity in blood plasma; an immunoreactive 25- to 40-kDa compound was the minor fraction. PMID- 1716537 TI - Absorption spectra of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin, and methemoglobin. AB - We determined the millimolar absorptivities of the four clinically relevant derivatives of fetal and adult human hemoglobin in the visible and near-infrared spectral range (450-1000 nm). As expected, spectral absorption curves of similar shape were found, but the small differences between fetal and adult hemoglobin absorptivity were important enough that they should be taken into account in multicomponent analysis of hemoglobin derivatives. Common pulse oximeters, however, involving light of 660 and 940 nm, are so insensitive to the presence of fetal hemoglobin that they can be used safely in neonates. The error in pulse oximetry caused by the presence of carboxyhemoglobin is insubstantial, but methemoglobin gives either an understimation or an overestimation at high or low oxygen saturation, respectively, the turning point being near 70% saturation. PMID- 1716538 TI - Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers. AB - We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes. PMID- 1716539 TI - Interference of serum mercury with the Abbott TDx. PMID- 1716540 TI - Estimation of amounts of anti-La(SS-B) antibody directed against immunodominant epitopes of the La(SS-B) autoantigen. AB - The contribution of circulating anti-La(SS-B) antibody to the hypergammaglobulinaemia seen in primary Sjogren's syndrome is unknown. In this study levels of anti-La(SS-B) antibody directed against three immunodominant epitopes of the anti-La(SS-B) autoantigen were measured by ELISA in 84 anti-La(SS B)+ sera using purified recombinant protein and antibody affinity-purified against the three anti-La(SS-B) fusion proteins. There was marked variation in the amounts of IgG anti-La(SS-B) antibody detected, with levels ranging from 0.02 mg/ml to 11 mg/ml. The anti-La(SS-B) levels were greater than 1 mg/ml in 61% of patients; in 18% of sera the anti-La(SS-B) level constituted 10% or more of the total serum IgG. However, other patients were seen with marked hypergammaglobulinaemia and low anti-La(SS-B) concentrations. These results support an antigen-driven mechanism for the anti-La(SS-B) response and suggest that anti-La(SS-B) antibody production is regulated independently of other immunoglobulins. PMID- 1716541 TI - CD5+ B cells and naturally occurring autoantibodies in cancer patients. AB - We have determined the percentage of CD5+ B lymphocytes in the peripheral blood of cancer patients and healthy controls, using antibodies directed at the CD5 and CD19 (pan-B) markers. The frequencies of CD5+ B cells, expressed as a percentage of total B cells, ranged from 14.3 to 57.5% in the controls and from 14.8 to 82.8% in the patient population. One-third of the cancer patients had frequencies greater than 2 s.d. above the mean of the control population. The CD5+ B cell fraction expressed as a percentage of total lymphocytes was also significantly elevated in this group of cancer patients. These results suggest that the CD5+ B cell compartment may be affected by the malignant process or by the therapy modality employed. The plasma levels of several naturally occurring autoantibodies, the products of the CD5+ B cells, were also assessed in cancer patients and controls. No significant differences were observed when reactivity to several autoantigens was measured. These included nuclear components and phospholipids. PMID- 1716542 TI - Serial study of CD5+ and CD5- B cell subpopulations in 335 HIV seropositive patients. AB - B cell subpopulations, as defined by double-labelling techniques with CD5 and CD19 monoclonal antibodies (MoAbs), were serially studied in 335 HIV-1 seropositive patients. At the time of the first consultation, no important modifications in either CD5+ or CD5- subpopulations were observed, whatever the stage of the disease. However, in 18 out of the 335 patients (5.37%), a sharp increase in B cells exceeding 20% and 300/mm3 was observed. This increase in B cells was mainly accounted for CD5-CD19+ B cell subpopulations and was associated with: (i) evolution of the disease, since only four patients presented it at their first consultation (one lymphadenopathy-associated syndrome (LAS) and three AIDS); (ii) advanced stages of disease since, at the time of B cell augmentation, two patients were staged as LAS, four as ARC and 12 as AIDS; (iii) a high incidence of non-Hodgkin's lymphomas (NHL) since three out of the 18 patients presented a histologically confirmed NHL and three others a clinical pattern compatible with this diagnosis. However, in three patients with B hyperlymphocytosis, polymerase chain reaction (PCR) studies of immunoglobulin gene rearrangement revealed the existence of a polyclonal expansion of B cells. These results justify inclusion of a pan-B cell marker in routine phenotypic studies of HIV-infected individuals, as well as the search for NHL among patients presenting this abnormality. PMID- 1716544 TI - Transcriptional control of hepadnavirus gene expression. PMID- 1716543 TI - Monoclonal antibodies VIB-E3, IB5 and HB9 to the leucocyte/epithelial antigen CD24 resemble BA-1 in recognizing sialic acid-dependent epitope(s). Evidence that VIB-E3 recognizes NeuAc alpha 2-6GalNAc and NeuAc alpha 2-6Gal sequences. AB - The specificities of seven monoclonal antibodies to the human B cell differentiation marker CD24 have been investigated with respect to sialic acid containing carbohydrates. These are antibodies HB8, HB9, VIB-C5, VIB-E3, AL1a, LC66 and IB5, which are known to bind to polydisperse sialoglycoprotein(s) on Nalm-6 B lymphoblastoid cells. Three of the antibodies, HB9, VIB-E3 and IB5, have been found to resemble the first described antibody in this series, BA-1, in binding also to bovine submaxillary mucin. As with BA-1, the binding of the antibodies is abolished or reduced markedly after desialylation of the epithelial glycoprotein, and the binding to neuraminidase-treated Nalm-6 cells is also reduced. There is evidence for the involvement of of non-O-acetylated sialic acid in the determinants recognized by these antibodies, since there is a substantially enhanced binding following mild-alkali treatment of the epithelial mucin which removes O-acetyl groups. One of the antibodies, VIB-E3, is deduced to recognize the oligosaccharide sequences NeuAc alpha 2-6GalNAc and NeuAc alpha 2 6Gal as part of larger antigenic structures. This conclusion has been reached from the results of inhibition-of-binding experiments using a series of structurally defined sialo-oligosaccharides and direct binding experiments using oligosaccharides chemically linked to lipid (neoglycolipids). PMID- 1716545 TI - [Assessment of the preparation Carietest, Septodont]. AB - The usefulness for staining of plaques on teeth was assessed using the preparation Carietest manufactured by Septodont. Carietest is a ready for use green fluid prepared, according to the producer, of blue sulphane and yellow tartrazin. It is applied painting the teeth in any area in which poor oral hygiene is suspected. The preparation was tried out using as reference standard Plakk-festo erythrosin tablets of Hungarian origin. The studied group comprised 25 patients. The range of staining of dental plaques by both preparations was determined using a simplified fuchsin index. The study showed a good suitability of the preparation Carietest for staining of bacterial dental plaques. PMID- 1716546 TI - A double-blinded, randomized trial of hydrocortisone in acute hepatic failure. The Acute Hepatic Failure Study Group. AB - The Acute Hepatic Failure Study Group (AHFSG) has conducted a double-blinded, randomized evaluation of hydrocortisone in patients with acute hepatic failure. From July 1975 through August 1978, a 38-month period, 18 medical centers in the United States and one in Canada participated in this trial. A total of 64 patients were accessed and found eligible to participate in the study; two of them were subsequently eliminated from our analysis. Eighteen patients received placebo; 23 received 400 mg hydrocortisone per day, and 21 patients were administered 800 mg hydrocortisone per day. We did not observe any therapeutic effect of hydrocortisone, and the survival rates for placebo versus 400 mg and versus 800 mg hydrocortisone per day were 22%, 9%, and 24%, respectively. Fulminant hepatitis associated with drug hepatotoxicity or non-A, non-B hepatitis seemed to have a worse prognosis than fulminant B, although these differences were not significant. Serum alpha-fetoprotein had a modest prognostic value of survival and seemed to be limited to fulminant B. The AHFSG recommends, therefore, that corticosteroid use in acute hepatic failure with hepatic encephalopathy be discontinued. PMID- 1716547 TI - [The use of monoclonal antibodies for research on fibrin polymerization processes]. PMID- 1716548 TI - [The characteristics of the functioning of the protein-synthesizing apparatus in hibernating Citellus undulatus]. PMID- 1716549 TI - [The activation of the transposition cycle Ty element by the reverse transcriptase of the human immunodeficiency virus in Saccharomyces cerevisiae yeasts]. PMID- 1716550 TI - [Nuclear matrix DNA binding sites in the vicinity of the alpha-globulin gene domain replication region in chickens]. PMID- 1716551 TI - [Resting cells preincubated with actinomycin D do not suppress DNA synthesis in heterokaryons with stimulated cells]. PMID- 1716552 TI - [The role of hemagglutinin during the adaptation of the influenza virus to a new host and its acquisition of virulent properties]. PMID- 1716553 TI - Interactions of the Drosophila gap gene giant with maternal and zygotic pattern forming genes. AB - The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development. PMID- 1716554 TI - A newt type II keratin restricted to normal and regenerating limbs and tails is responsive to retinoic acid. AB - In order to understand the molecular mechanisms underlying the regenerative ability of the urodele limb, it is important to identify regeneration-associated proteins and to study their regulation. We have recently shown that the anti cytokeratin monoclonal antibody LP1K reacts strongly with newt blastemal cells, while its reactivity is restricted in normal limbs. By screening a cDNA expression library from the newt blastema with LP1K, we have identified cDNA clones coding for a type II keratin (NvKII) expressed both in the mesenchyme and the specialized wound epithelium of the blastema. While the rod domain of the protein is highly conserved, the homology between NvKII and mammalian type II keratins drops markedly at the N- and C-terminal regions. The expression of this keratin was analysed by Northern blotting and RNAase protection analysis of various newt tissues, and appears to be organ specific, since it is restricted to normal and regenerating limbs and tails. In particular, we have investigated the expression of this keratin mRNA in normal and regenerating limbs. The transcript is barely detectable in the proximal portion of the normal limb, but its level is high in the distal one. After amputation, NvKII mRNA is expressed both in proximal and distal blastemas, although at higher levels distally, indicating that this keratin is regeneration associated. The NvKII transcript is detectable both in mesenchyme and in the wound epithelium of the regenerate, while no transcript is detectable in normal epidermis. The level of NvKII mRNA is markedly down-regulated both in normal and regenerating limbs following intraperitoneal injection with retinoic acid, a putative endogenous morphogen in limb regeneration. PMID- 1716555 TI - Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos. AB - We have cloned a Xenopus cDNA that encodes a homologue of the human myogenic factor, Myf-5. Xenopus Myf-5 (XMyf5) transcripts first accumulate in the prospective somite region of early gastrulae. The pattern of XMyf5 expression is similar to that of the Xenopus MyoD (XMyoD) gene, except that XMyf5 transcripts are largely restricted to posterior somitic mesoderm even before any somites have formed. Transient ectopic expression of XMyf5 activates cardiac actin and XMyoD genes in animal cap cells, but does not cause full myogenesis, even in combination with XMyoD. These results suggest that XMyf5 acts together with XMyoD as one of the set of genes regulating the earliest events of myogenesis, additional factors being required for complete muscle differentiation. PMID- 1716556 TI - Three-dimensional human pattern visual evoked potentials. I. Normal subjects. AB - Pattern visual evoked potentials (PVEPs) were obtained from 30 normal adult volunteers, recording from both a conventional horizontal occipital array and three orthogonal bipolar antipodal channels approximating the three dimensions of space. Central and eccentric fixation of 60' checks and central fixation of 30' checks under binocular and monocular viewing conditions was employed. The three antipodal wave forms were displayed as a single 3-D Lissajous trajectory which contained four apices, corresponding to P40 (apex A), N70 (apex B), P100 (apex C) and N125 (apex D). The 3-D evoked potentials depicted the dynamic nature of the human PVEP in terms of changes in the 3-D voltage-voltage-voltage plots of the recordings. The orientation of the A-B, B-C and C-D curvilinear segments reflected the stimulating condition (central fixation vs. right vs. left hemi field stimulation) for all subjects with more accuracy than did the wave forms from the conventional array. Spherical statistical methods are described for quantifying and evaluating 3-D evoked potential recordings. PMID- 1716557 TI - Three-dimensional human pattern visual evoked potentials. II. Multiple sclerosis patients. AB - Pattern visual evoked potentials were obtained from 46 patients with definite relapsing/remitting multiple sclerosis, using both a conventional 5-channel occipital array and a 3-D recording technique consisting of three bipolar derivations approximating the three dimensions of space. These three orthogonal wave forms were displayed as a 3-D Lissajous trajectory for each subject. Two of the 15 patients with completely normal conventional pattern VEPs had abnormalities of the orientation of the B-C curvilinear segment of the 3-D pattern VEPs. Delays in the first major occipital positive component (P100) were evident using both techniques; the correlation between P100 latency and the latency of the corresponding trajectory apex was r = 0.99 (P less than 0.01). Post-chiasmal MRI abnormalities were associated with 3-D VEP orientation abnormalities. Three-dimensional pattern VEPs are moderately more sensitive than conventional pattern VEPs at detecting dysfunction posterior to the optic chiasm in demyelinating disease and do not require the use of eccentric fixation to do so. PMID- 1716558 TI - Effects of scopolamine on visual evoked potentials in aging and dementia. AB - The unusual combination of a normal pattern reversal VEP and a delayed flash VEP has been reported in patients with dementia of Alzheimer's type (DAT). Hyoscine hydrobromide has been reported to produce a similar VEP abnormality in young, healthy subjects. In the present study, we assessed the relative sensitivity of DAT patients and healthy young, middle-aged and elderly subjects to temporary cholinergic blockade. We report VEP latency values following 3 doses of scopolamine and after a peripheral anticholinergic agent. Flash P2 latency was not significantly slower in DAT patients than in the healthy elderly. Scopolamine increased P2 latency in the young controls but did not affect any other group. The pattern reversal P100 was normal in DAT, and a significant increase in latency occurred following scopolamine administration in both the control and patient groups. PMID- 1716559 TI - SEP testing in deeply comatose and brain dead patients: the role of nasopharyngeal, scalp and earlobe derivations in recording the P14 potential. AB - Median nerve somatosensory evoked potentials (SEPs) were tested in 50 patients (20 brain dead, 18 comatose and in 12 progressing from coma to brain death, i.e., 32 cases with brain death and 30 cases with coma were recorded). Derivations were taken from nasopharynx, earlobes, scalp, and neck using cephalic and non-cephalic references. Cortical and subcortical SEP components were evaluated, focussing on the P14 potential. There is evidence that rostral and caudal parts of the P14 generator (lemniscus medialis) are differently affected in brain death, resulting in an abolition of the rostral part, while occasionally leaving intact for some time the caudal part. Non-cephalic referenced scalp records pick up the whole P14 dipole, whereas nasopharyngeal and earlobe derivations pick up different parts of P14, depending on the reference used. Scalp-to-nasopharynx records derive the most rostral part of P14; this "rostral P14" was bilaterally lost in all brain dead patients, but preserved in all deeply comatose patients with diffuse brain stem injuries. Scalp-to-earlobe records, in contrast, picked up a P14 dipole segment reaching more caudally, resulting in a P14 potential also in brain dead patients. It is concluded that midfrontal scalp-to-nasopharynx derivations give the most valuable contribution to the electrophysiological assessment of brain death versus deep coma. PMID- 1716560 TI - Middle-latency somatosensory evoked potentials following median and posterior tibial nerve stimulation in Down's syndrome. AB - Middle-latency somatosensory evoked potentials (SEPs) following median and posterior tibial nerve stimulation were studied in 40 patients with Down's syndrome and in age- and gender-matched healthy controls as well as in middle aged and aged healthy subjects. In median nerve SEPs, latencies of the initial cortical potentials, N18 and P18, showed no significant difference, but the following potentials N22, P25, N32, P41 and P46 were relatively or significantly shorter in latency in Down's patients than in the controls. Amplitudes of all components in Down's patients were significantly larger than those of age- and gender-matched controls as well as of those of middle-aged healthy subjects, but there was only a small difference in their amplitudes from aged healthy subjects. Results of posterior tibial nerve SEPs were generally consistent with those of median nerve SEPs. Therefore, 'short latency with large amplitude' is the main characteristic of middle-latency SEPs in Down's syndrome, possibly related to accelerated physiological aging of the central nervous system. PMID- 1716561 TI - Nerve, spinal cord and brain somatosensory evoked responses: a comparative study during electrical and magnetic peripheral nerve stimulation. AB - Somatosensory evoked potentials (SEPs) and compound nerve action potentials (cNAPs) have been recorded in 15 subjects during electrical and magnetic nerve stimulation. Peripheral records were gathered at Erb's point and on nerve trunks at the elbow during median and ulnar nerve stimulation at the wrist. Erb responses to electrical stimulation were larger in amplitude and shorter in duration than the magnetic ones when 'electrical' and 'magnetic' compound muscle action potentials (cMAPs) of comparable amplitudes were elicited. SEPs were recorded respectively at Cv7 and on the somatosensory scalp areas contra- and ipsilateral to the stimulated side. SEPs showed a statistically significant difference in amplitude only for the brachial plexus response and for the 'cortical' N20-P25 complex; differences were not found between the magnetic and electrical central conduction times (CCTs) or for the peripheral nerve response latencies. Magnetic stimulation preferentially excited the motor and proprioceptive fibres when the nerve trunks were stimulated at motor threshold intensities. PMID- 1716562 TI - Comparison of somatosensory evoked potentials recorded from the scalp and dorsal column nuclei to upper and lower limb stimulation in the rat. AB - Responses from the dorsal column nuclei (DCN) in the rat to stimulation of the upper limbs (median nerve) and lower limbs (sciatic nerve) showed a difference in the wave forms of the two responses. These results support results of earlier studies in the cat, monkey, and man that showed that only slow-conducting cutaneous afferents from the lower limbs travel in the dorsal column, while all afferents from the upper limbs travel in the dorsal column and synapse in the DCN. A comparison between the response from the DCN and that from the vertex to stimulation of the upper limbs showed correspondence between short-latency peaks, while no clear earlier waves could be discerned in the response from the vertex to stimulation of the lower limbs. Even when the dorsal column was transected on one side, the correspondence between the early peaks in the scalp and the DCN responses to stimulation of the upper limbs was maintained. The effect of the dorsal column lesion on the response recorded from the surface of the DCN to stimulation of the sciatic nerve was mainly a reduction in the number of peaks. Transection at the midbrain level resulted in elimination of the long-latency response in the scalp recording, but the initial negative peak was maintained, which corresponded to the initial negative peak of the DCN response to stimulation of the upper limbs. PMID- 1716563 TI - Effect of hyperthermia on somatosensory evoked potentials in the anaesthetized rat. AB - In the rat heat stroke model, established by heating to a climatic chamber temperature of 42 degrees C, the brain temperature was found to be consistently lower than the rectal temperature, suggesting efficient brain cooling mechanisms in the rat. In response to heating, with increasing brain temperature, the latencies of the somatosensory evoked potentials (SEPs) showed an initial decrease followed by an increase (inflection point). Studies were done on rats heated up to, before, or after the inflection point and then cooled. Reversibility with cooling of functional and structural changes induced by heat was evaluated by analysis of SEPs, survival time, brain blood perfusion and histopathology. The evidence from these studies demonstrated that the brain temperature at which the inflection in wave P2 latency occurred was critical, beyond which hyperthermia produced irreversible changes in the SEP, shorter survival time, relative reduction in brain blood perfusion and evidence of brain histopathological damage. The suggestion that endorphins may mediate brain dysfunction in hyperthermia was investigated. In rats heated and then cooled after wave P2 latency inflection naloxone, the endorphin antagonist, was injected (10 mg/kg, intravenously) just prior to the inflection. It produced reversibility of SEP changes as well as longer survival time (P less than 0.001) compared to saline-treated rats. PMID- 1716564 TI - Brain-stem auditory evoked potentials and brain death. AB - BAEP records were obtained from 30 brain-dead patients. Three BAEP patterns were observed: (1) no identifiable waves (73.34%), (2) an isolated bilateral wave I (16.66%), and (3) an isolated unilateral wave I (10%). When wave I was present, it was always significantly delayed. Significant augmentation of wave I amplitude was present bilaterally in one case and unilaterally in another. On the other hand, in serial records from 3 cases wave I latency tended to increase progressively until this component disappeared. During the same period, wave I amplitude fluctuations were observed. A significant negative correlation was found for wave I latency with heart rate and body temperature in 1 case. Two facts might explain the progressive delay and disappearance of wave I in brain dead patients: a progressive hypoxic-ischaemic dysfunction of the cochlea and the eighth nerve plus hypothermia, often present in brain-dead patients. Then the incidence of wave I preservation reported by different authors in single BAEP records from brain-dead patients might depend on the moment at which the evoked potential study was done in relation to the onset of the clinical state. It is suggested that, although BAEPs provide an objective electrophysiological assessment of brain-stem function, essential for BD diagnosis, this technique could be of no value for this purpose when used in isolation. PMID- 1716565 TI - Memory-dependent auditory evoked potentials to change in the binaural interaction of noise signals. AB - When non-identical binaural noise signals suddenly become coherent in the two ears, or coherent noise suddenly becomes incoherent, long latency binaurally evoked potentials (BINEP) are elicited which consist of P70, N130 and P220 components. Responses of similar morphology and latency were recorded to a change in the frequency of monaural click trains. The responses to onset or offset of the click trains were 20-50 msec shorter in latency. BINEP are also evoked when the sound image suddenly shifts due to the introduction of a short inter-aural delay in coherent noise signals. Responses to "isolated" shifts occurring once every 7 sec were 2-3 times (N130) or 3-5 times (P220) larger than responses to "frequent" shifts (6/7 sec) of the same magnitude in the same direction. Responses to "infrequent" shifts (1/7 sec) interspersed with frequent shifts in the opposite direction were of intermediate size, significantly larger than frequent responses. The BINEP could reflect the activity of location-specific neurones in the auditory cortex, but it seems more likely that they are due to a common neuronal pool responsive to any shift in the location of the sound image. Similar neuronal pools may be concerned with the detection of change in other auditory dimensions such as pitch. The difference between isolated, infrequent and frequent responses suggests that the BINEP amplitude is dependent on a memory of the shifts which have occurred in the preceding few seconds. The underlying process may be similar or identical to that which governs generation of the "mismatch negativity." PMID- 1716566 TI - Neuromagnetic responses elicited by auditory stimuli in dichotic listening. AB - We measured N1m and P2m components of the magnetic field responses that were elicited by random series of a tone burst given to the left ear and a monosyllabic speech sound given to the right ear. The magnetic responses had smaller amplitudes and/or longer peak latencies of the N1m and the P2m when the stimulus was preceded by a stimulus at the same ear than when preceded by a stimulus at the different ear. This reduction of the response by preceding stimulation of the same ear was significant over the hemisphere contralateral, but not ipsilateral, to the ear stimulated. The peak latencies of N1m and P2m were significantly longer in the response over the hemisphere contralateral than ipsilateral to the stimulated ear. PMID- 1716567 TI - Differential impact of hypothermia and pentobarbital on brain-stem auditory evoked responses. AB - Two experiments were conducted to determine the effects of hypothermia and pentobarbital anesthesia, alone and in combination, on the brain-stem auditory evoked responses (BAERs) of rats. In experiment I, unanesthetized rats were cooled to colonic temperatures 0.5 and 1.0 degrees C below normal. In experiment II, 2 groups of rats were cooled and tested at 37.5, 36.0, 34.5 and 31.5 degrees C. One group was anesthetized during testing and the other group was awake. The rat BAER was sensitive to cooling of 1 degree C or less. Peak latencies were prolonged and peak-to-peak amplitudes were increased by hypothermia alone. The effect on amplitude may be related to the time course of temperature change or to stimulus level. Pentobarbital significantly affected both latencies and amplitudes over and above the effects of cooling. The specific effects of pentobarbital differed by BAER peak and by temperature. The findings point up the importance of the potential confound of anesthetic drugs in most of the evoked potential literature on hypothermia and, for the first time, quantify the complex interactions between pentobarbital and temperature which affect the BAER wave form. PMID- 1716568 TI - Auditory brain-stem evoked potentials in cat after kainic acid induced neuronal loss. I. Superior olivary complex. AB - Auditory brain-stem potentials (ABRs) were studied in cats for up to 45 days after kainic acid had been injected unilaterally or bilaterally into the superior olivary complex (SOC) to produce neuronal destruction while sparing fibers of passage and the terminals of axons of extrinsic origin connecting to SOC neurons. The components of the ABR in cat were labeled by their polarity at the vertex (P, for positive) and their order of appearance (the arabic numerals 1, 2, etc.). Component P1 can be further subdivided into 2 subcomponents labeled P1a and P1b. The correspondences we have assumed between the ABR components in cat and man are indicated by providing a Roman numeral designation for the human component in parentheses following the feline notation, e.g., P4 (V). With bilateral SOC destruction, there was a significant and marked attenuation of waves P2 (III), P3 (IV), P4 (V), P5 (VI), and the sustained potential shift (SPS) amounting to as much as 80% of preoperative values. Following unilateral SOC destruction the attenuation of many of these same ABR components, in response to stimulation of either ear, was up to 50%. No component of the ABR was totally abolished even when the SOC was lesioned 100% bilaterally. In unilaterally lesioned cats with extensive neuronal loss (greater than 75%) the latencies of the components beginning at P3 (IV) were delayed to stimulation of the ear ipsilateral to the injection site but not to stimulation of the ear contralateral to the injection. Binaural interaction components of the ABR were affected in proportion to the attenuation of the ABR. These results are compatible with multiple brain regions contributing to the generation of the components of the ABR beginning with P2 (III) and that components P3 (IV), P4 (V), and P5 (VI) and the sustained potential shift depend particularly on the integrity of the neurons of the SOC bilaterally. The neurons of the lateral subdivision (LSO) and the medial nucleus of the trapezoid body (MNTB) of the SOC have a major role in generating waves P3 (IV) and P4 (V). PMID- 1716569 TI - Auditory brain-stem evoked potentials in cat after kainic acid induced neuronal loss. II. Cochlear nucleus. AB - Auditory brain-stem potentials (ABRs) were studied in cats for up to 6 weeks after kainic acid had been injected unilaterally into the cochlear nucleus (CN) producing extensive neuronal destruction. The ABR components were labeled by the polarity at the vertex (P, for positive) and their order of appearance (the arabic numerals 1, 2, etc.). Component P1 can be further subdivided into 2 subcomponents, P1a and P1b. The assumed correspondence between the ABR components in cat and man is indicated by providing human Roman numeral designations in parentheses following the feline notation, e.g., P2 (III). To stimulation of the ear ipsilateral to the injection, the ABR changes consisted of a loss of components P2 (III) and P3 (IV), and an attenuation and prolongation of latency of components P4 (V) and P5 (VI). The sustained potential shift from which the components arose was not affected. Wave P1a (I) was also slightly but significantly attenuated compatible with changes of excitability of nerve VIII in the cochlea secondary to cochlear nucleus destruction. Unexpectedly, to stimulation of the ear contralateral to the injection side, waves P2 (III), P3 (IV), and P4 (V) were also attenuated and delayed in latency but to a lesser degree than to stimulation of the ear ipsilateral to the injection. Changes in binaural interaction of the ABR following cochlear nucleus lesions were similar to those produced in normal animals by introducing a temporal delay of the input to one ear. The results of the present set of studies using kainic acid to induce neuronal loss in auditory pathway when combined with prior lesion and recording experiments suggest that each of the components of the ABR requires the integrity of an anatomically diffuse system comprising a set of neurons, their axons, and the neurons on which they terminate. Disruption of any portion of the system will alter the amplitude and/or the latency of that component. PMID- 1716570 TI - P300 variations in parkinsonian patients before and during dopaminergic monotherapy: a suggested dopamine component in P300. AB - P300 was recorded, using an 'odd ball' paradigm, in 18 parkinsonian patients before and during dopaminergic monotherapy. The data were compared with those of a homogeneous standard group of 20 subjects. The main finding was an increase in the P300 latency of parkinsonian patients before therapy, which recovered during dopaminergic monotherapy. In 11 voluntary healthy subjects the same therapy did not produce a reduction of the P300 latency. The data are discussed in relation to a possible dopaminergic component in P300 origin. PMID- 1716571 TI - Auditory event-related potentials in obstructive sleep apnea: effects of treatment with nasal continuous positive airway pressure. AB - Event-related potentials (ERPs) were recorded in 47 patients with obstructive sleep apnea (OSA) syndrome prior to and after 6 weeks of treatment with continuous positive airway pressure (CPAP). Compared with a control group, the OSA patients showed ERP abnormalities: lengthened P3 latencies and decreased N2 P3 amplitudes. After 6 weeks of CPAP treatment, there was a highly significant improvement in the abnormal ERPs: the P3 and N2 latencies were shortened, but remained longer than in controls, and the N2-P3 and N1-P2 amplitudes were increased. No correlations could be established with various sleep variables. ERPs may be used as an electrophysiological marker of brain dysfunction; treatment of OSA with CPAP is probably responsible for functional brain modifications. On the other hand, possible relationships between the ERP abnormalities and the neuropsychological disorders observed in OSA remain to be established. PMID- 1716572 TI - Ruthenium red as a selective capsaicin antagonist of the motor response to capsaicin in the human isolated ileum. AB - We have investigated the effect of ruthenium red, omega-conotoxin fraction GVIA (CTX) and tetrodotoxin (TTX) on the relaxation produced in the circular muscle of the human isolated ileum by capsaicin, electrical field stimulation (EFS) or vasoactive intestinal polypeptide (VIP). Ruthenium red (10 microM) selectively blocked the capsaicin-evoked relaxation while leaving the response to EFS or VIP unaffected. CTX had no significant effect on the various stimuli. TTX blocked the relaxation due to EFS but not that due to capsaicin or VIP. It is concluded that capsaicin excitation of primary afferents in the human ileum, leading to VIP release and muscle relaxation, occurs with mechanisms similar to those operating in animal tissues and that ruthenium red acts as a selective capsaicin antagonist in the human ileum. PMID- 1716573 TI - Activity of spantide I and II at various tachykinin receptors and NK-2 tachykinin receptor subtypes. AB - We compared the ability of spantide I and II to antagonize tachykinins in monoreceptor bioassays. Both peptides antagonized the response to substance P methylester in the guinea-pig ileum (NK-1 receptor-mediated) with greater affinity than the responses mediated by NK-2 or NK-3 receptors in other bioassays. Spantide II was about 10 times more potent than spantide I as an NK-1 antagonist and also possessed some selectivity for the NK-2 receptor subtype present in the hamster trachea. Spantide II is a suitable tool to assess the role of NK-1 receptors in the central and peripheral nervous system. PMID- 1716574 TI - Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation. AB - Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25-2 microM) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents. PMID- 1716575 TI - Neurokinin receptors in the rabbit iris sphincter characterised by novel agonist ligands. AB - We have used novel selective agonist ligands to examine neurokinin receptors mediating the contractile response to tachykinins in the rabbit iris sphincter preparation in vitro. The selective NK-1 receptor agonist delta-amino valeryl-[L Pro9,N-Me Leu10]SP-(7-11) (GR73632) and the NK-3 receptor-selective agonist succ [Asp6,N-Me-Phe8] SP-(6-11) (senktide) were both very active (concentration range 0.032 pM-10 nM and 0.1 pM-32 nM respectively), and were 933 and 16.6 times more potent than substance P, respectively, in contracting the iris. In contrast, the NK-2 selective agonist [Lys3,Gly8-R-gamma-lactam,Leu9]NKA-(3-10) (GR64349) was active only at the highest concentrations tested (3.2 nM-32 microM), and had 0.054 the activity of substance P. The presence of several peptidase inhibitors was without effect on the concentration-response relationship to substance P, GR73632, GR64349 or senktide. Tachykinins differed in their offset kinetics. Responses to GR73632, GR64349 and senktide were rapid in offset (times to reach half maximal responses were 1.5, 1.1 and 5.1 min, respectively), whereas responses to substance P were very much more prolonged in duration (time to reach half maximal response was 35.3 min). These results suggest the presence of both NK-1 and NK-3 receptors mediating contraction of the rabbit iris sphincter preparation. In addition, differences in response offset kinetics seem not to be due to differences in peptide metabolism, and suggest a property of substance P not shared by the other tachykinins used in this study. PMID- 1716576 TI - Anti-inflammatory effect of gangliosides in the rat hindpaw edema test. AB - The influence of total brain gangliosides on acute inflammation was investigated using the rat hind paw edema test. Total gangliosides (10 micrograms/paw) inhibited the edema produced by the injection of bee venom phospholipase A2 (5 micrograms/paw) when the lipids were co-injected or injected 15 min before the phospholipase A2. Sulphatide (10 micrograms/paw) did not inhibit the edema but potentiated it. Gangliosides (40 micrograms/paw) inhibited the edema induced by carrageenin 1% when they were injected 1 h before the agent. However, gangliosides (up to 200 micrograms/paw) failed to inhibit the dextran-induced edema. The edema test was also used to investigate the effect of gangliosides on the production of mediators of inflammation by peritoneal adherent macrophages. Gangliosides inhibited the production of mediators of inflammation only when they were incubated with these cells before the stimulation with phospholipase A2 or carrageenin. Gangliosides did not inhibit the production of mediators of inflammation when arachidonic acid was added to the cells. These results suggest that the anti-inflammatory effect observed with gangliosides is mediated by inhibition at or before endogenous phospholipase activity. PMID- 1716577 TI - Protein kinase C-dependent diacylglycerol formation is mediated via Ca2+/calmodulin in parotid cells. AB - The kinetics of carbachol-induced sn-1,2-diacylglycerol (DAG) formation and the underlying mechanism(s) involved in parotid acinar cells were investigated. Supramaximal concentrations of carbachol for amylase secretion (10 microM) caused a transient rise in DAG levels at 10 s. In contrast, this rapid rise was not elicited by 1 microM carbachol, which is the maximally effective concentration for amylase secretion. Carbachol (10 microM) also increased DAG levels linearly up to 20 min, which were sustained for up to a further 10 min. DAG formation stimulated by 1 microM carbachol was biphasic; the first peak was observed after 5 min and the second after 20 min. DAG formation induced by 0.01-0.1 microM carbachol was concentration-dependent and monophasic, peaking at 5 min. The second peak evoked by carbachol was partly inhibited by Ca2+ deprivation from the extracellular space, whereas the first peak was not. Similar results were obtained in experiments using Ca2+ antagonists such as verapamil and LaCl3. The protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine, and a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1 naphthalenesulfonamide (W-7), significantly inhibited the second DAG peak produced by 1 microM carbachol, but did not alter the first peak. The degree of inhibition of the second peak by these antagonists was comparable. Furthermore, the inhibitory effect of staurosporine and W-7 was concentration-dependent. The A23187-induced accumulation of DAG also was abolished by both staurosporine and W 7. These data indicate that a protein kinase C-dependent mechanism(s) is involved in mediating the second DAG accumulation peak induced by 1 microM carbachol and is mainly regulated by the Ca(2+)-calmodulin complex. PMID- 1716578 TI - Norepinephrine inhibits a Ca2+ current in rat sympathetic neurons via a G protein. AB - The effects of norepinephrine on a Ca2+ current from acutely isolated and short term (24 h) cultured adult rat superior cervical ganglion neurons were studied using the whole-cell variant of the patch-clamp technique. Norepinephrine produced a rapid, reversible and concentration-dependent reduction of the Ca2+ current. Accurately timed applications of norepinephrine (3 microM) showed that the development of Ca2+ current inhibition was delayed by up to 11 s after application of norepinephrine. Internal 500 microM guanylyl-imidodiphosphate (Gpp(NH)p) or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) decreased the Ca2+ current amplitude and induced a biphasic rising phase of the Ca2+ current. Under these conditions, the reduction of Ca2+ current amplitude by 3 microM norepinephrine was virtually abolished when compared with cells dialysed with GTP containing internal solutions. Internal dialysis with solutions containing 2 mM guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) increased the Ca2+ current amplitude and reduced the inhibition produced by 3 microM norepinephrine compared to cells dialysed with control internal solution. Treatment with 200 ng/ml pertussis toxin for 12-16 h greatly reduced the norepinephrine-induced Ca2+ current inhibition. Internal dialysis with solutions containing 500 microM cyclic adenosine 3',5'-monophosphate (cyclic AMP) and 3-isobutyl-1-methylxanthine had no significant effect on either the Ca2+ current inhibition by norepinephrine or the Ca2+ current amplitude. These results suggest that norepinephrine blocks a Ca2+ current in adult rat superior cervical ganglion neurons via a pertussis toxin sensitive G-protein which is independent of intracellular cyclic AMP. PMID- 1716579 TI - Beta-adrenoceptor induced inhibition of muscarinic receptor-stimulated phosphoinositide metabolism is agonist specific in bovine tracheal smooth muscle. AB - The ability of the beta-adrenoceptor agonist isoprenaline to inhibit agonist stimulated phosphoinositide metabolism was examined in bovine tracheal smooth muscle slices prelabelled with [3H]inositol. Accumulation of [3H]inositol phosphates was enhanced by the muscarinic agonists carbachol, oxotremorine and pilocarpine although the latter were only partial agonists for this response. Histamine stimulation of [3H]inositol phosphates was sensitive to mepyramine but maximal responses were only comparable to those of pilocarpine. Preincubation of tracheal slices with isoprenaline reduced the maximal phosphoinositide response to histamine and pilocarpine but the responses to carbachol and oxotremorine were unaffected. The inhibitory effect of isoprenaline (IC50 = 0.04 microM) was reversed competitively by 1 microM propranolol. The non-selective phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (1 mM) resulted in a more severe suppression of the histamine and pilocarpine responses and also produced a significant suppression of the maximal response to oxotremorine and a small shift in the carbachol dose-response curve. The different susceptibility of agonist-stimulated phosphoinositide hydrolysis to isoprenaline and IBMX are discussed in relation to the relative intrinsic activity of the agonists and/or the role of different muscarinic receptor subtypes. PMID- 1716580 TI - Evidence for the interaction of mast cell-degranulating peptide with pertussis toxin-sensitive G proteins in mast cells. AB - K(+)-channel blocker properties have been reported for mast cell-degranulating peptide (MCD) in the central nervous system, but its action mechanism in mast cells remains unknown. We studied the effect of MCD on the membrane potential of rat peritoneal mast cells using the fluorescent probe bis-oxonol. Unexpectedly, MCD induced a decrease in bis-oxonol fluorescence, in a rapid and then a slower phase, suggesting hyperpolarization of mast cells. Other K(+)-channel blockers, tetraethylammonium and 4-aminopyridine, did not significantly modify the bis oxonol fluorescence and did not alter the effect of MCD. The late phase of bis oxonol fluorescence decrease was inhibited by ouabain and by potassium deprivation, whereas histamine release was not affected. The first phase of putative hyperpolarization induced by MCD coincided with histamine release and with the generation of inositol polyphosphates. Prior treatment of the cells with pertussis toxin inhibited these effects of MCD. MCD stimulated the GTPase activity of purified G proteins (G0/Gi) in a concentration-dependent manner. These results indicate that the effect of MCD on mast cells is unrelated to K+ channels but that it is relevant to the activation of pertussis toxin-sensitive G proteins leading to the activation of phospholipase C. A direct interaction of MCD with G proteins is proposed, which, unlike mastoparan, does not require positive cooperativity. PMID- 1716581 TI - Complex allosteric interaction of heparin with neurokinin-1 receptors. AB - The effects of several heparin preparations on rat striatal neurokinin-1 receptor labelling were investigated. A low concentration of heparin dose-dependently stimulated [125I]Bolton-Hunter substance P ([125I]BH-SP) binding to rat striatal membranes. This effect was mainly due to an increase of the association velocity reflected by a decrease of the equilibrium dissociation rate constant. A higher heparin concentration inhibited specific [125I]BH-SP labelling by a negative allosteric mechanism as a result of an increase of the dissociation velocity. PMID- 1716582 TI - Rat chromosome 5 (q22-23) contains elements that control cell morphology and interactions with the extracellular matrix: a study of normal fibroblast x malignant hepatoma cell hybrids. AB - Cell interactions with the extracellular matrix are consistently modified in neoplasia. Malignant transformation has been correlated with modifications in the synthesis and distribution of matrix components and with alterations of cell adhesive properties to these components. A particular class of genes, able to suppress the transformed phenotype in normal cells, may be involved in those phenotypic changes. By studying somatic cell hybrids between mouse hepatoma (BWTG3) cells and normal rat skin fibroblasts (RSF), Islam and co-workers were able to localize a gene or a group of genes controlling anchorage dependence and cell growth in vitro. This (or these) gene(s) was (were) assigned to the q22-23 fragment of rat chromosome 5. In the present study, we compare the morphology and the interactions with the extracellular matrix proteins (laminin, fibronectin, and collagen IV) and the synthesis of these proteins by RSF X BWTG3 hybrid cells that had either retained (BS181p10) or lost (BS181a5) the q22-23 region of rat chromosome 5. Our results suggest that the rat 5q22-23 fragment controls a part of the cell differentiation program including morphology, attachment to extracellular matrix, and synthesis of some matrix proteins, particularly alpha 1 and alpha 2 chains of collagen IV. PMID- 1716583 TI - SV40T-immortalized lung alveolar epithelial cells display post-transcriptional regulation of proliferation-related genes. AB - To study the regulation of proliferation of lung alveolar epithelial type 2 cells, we have established a cell line derived from neonatal type 2 cells by transfection with the SV40 large T antigen gene. We find that this cell line, designated SV40-T2, displays the same post-transcriptional control of expression of proliferation-related genes, including c-myc, ornithine decarboxylase, thymidine kinase, and histone, that we have previously described in primary isolates of type 2 cells (Clement et al., Proc. Natl. Acad. Sci. USA 87, 318-322, 1990). Both proliferating and nonproliferating SV40-T2 cells express these genes at high levels, but their translation products are only detected in proliferating cells. Using the histone gene as an example, we have found that regulation of expression occurs at the level of transcription and of mRNA turnover, as previously described in other mammalian systems. However, in addition, regulation of expression also occurs at the level of translation of the histone mRNA, because its protein product is not detectable in nonproliferating SV40-T2 cells. We have analyzed the steps which are potentially involved in this translational regulation of histone gene expression in SV40-T2 cells. In both proliferating and nonproliferating cells, histone mRNA was found to be efficiently transported from the nucleus to the cytoplasm and to associate with the translationally active heavy polysomal fractions. These results indicate that control of histone gene expression (and perhaps that of other proliferation-related genes) in lung epithelial cells may involve either rapid and selective degradation of histone protein or binding factor(s) which modulate translational efficiency of histone mRNA. PMID- 1716584 TI - Heat-shock induction and cytoplasmic localization of transcripts from telomeric associated sequences in Chironomus thummi. AB - Transcription of telomeric-associated sequences has been detected in the salivary gland cells of the larvae Chironomus thummi. In this species, a heat shock induces puffing at some telomeres, especially at one of the telomeres of chromosome III. We found that this process was concomitant with an increase in the overall telomeric transcript levels. Transcription was also observed in all the telomeres under control conditions, by in situ hybridization, even when these telomeres appeared to be in a nonpuffed state. The telomeric transcripts were found in both, the nuclei and, at higher levels, in the cytoplasmic extracts of salivary gland cells. The heat-shock activation, however, appeared to be restricted to the nuclear level. Telomeric transcription and the peculiar behavior of C. thummi telomeres after a heat shock are discussed. PMID- 1716585 TI - MPF is activated in growing immature Xenopus oocytes in the absence of detectable tyrosine dephosphorylation of P34cdc2. AB - Tyrosine-phosphorylated p34cdc2 and cyclin B2 are present and physically associated in small growing stage IV oocytes (800 microns in diameter) of Xenopus laevis. Microinjection of M-phase promoting factor (MPF) into stage IV oocytes induces germinal vesicle breakdown and the activation of the kinase activity of the p34cdc2/cyclin B2 complex measured on p13suc1 beads. During the in vivo activation of MPF in stage IV oocytes, p34cdc2 tyrosine dephosphorylation is not detectable, in contrast to stage VI oocytes. Addition of cycloheximide in MPF injected stage IV oocytes induces neither the inhibition of histone H1 kinase activity nor the cyclin B2 degradation. Therefore, the activation mechanism of histone H1 kinase in stage IV oocytes does not require detectable tyrosine dephosphorylation of p34cdc2. It is suggested rather that the tyrosine phosphorylation of p34cdc2 plays a role in inhibiting cyclin B2 degradation. PMID- 1716586 TI - Expression of the HSP 70 gene family in rat hepatoma cell lines of different growth rates. AB - We have studied the expression of different members of the HSP 70 gene family in MH1C1, FAO, and 3924A hepatoma cell lines, which possess different growth rates and show different levels of histone H3 gene expression. The cells have been subjected to mild (42 degrees C/1 h) or severe (45 degrees C/25 min) heat shock that causes a decrease in cell proliferation and histone H3 gene expression correlated to the severity of stress: previous mild heat shock protects against the effects of the subsequent severe exposure. All cell lines, irrespective of their growth rate, show a high constitutive expression of the HSC 73 gene, which is barely detectable in normal liver, and a good induction of the heat-inducible HSP 70 gene, which, however, seems to be induced less than in the normal tissue. The relative amount of grp 78 mRNA is high in all hepatoma cells lines, but only FAO cells maintain a significant expression of the albumin gene. The basic diversity in HSP 70 family gene expression between normal and tumors is still maintained in hepatoma cell lines, but the growth-related, quantitative differences among the transplantable hepatomas that we previously found in the animal (Bardella et al., Br. J. Cancer 55, 642-645, 1987; Cairo et al., Hepatology 9, 740-746, 1989), seem to be lost, or at least strongly blunted, in vitro. PMID- 1716587 TI - Effects on spleen colony-forming unit self-renewal after retroviral-mediated gene transfer of multi-colony-stimulating factor, granulocyte-macrophage colony stimulating factor, or granulocyte colony-stimulating factor. AB - C57BL/6 mice were established to constitutively produce multi-colony-stimulating factor (multi-CSF), granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor by transplantation with post-5-fluorouracil treated syngeneic marrow cells infected with retroviral vectors bearing the corresponding growth factor complementary DNAs. At 2-4 weeks after transplantation, these mice had a 2-7-fold increase in spleen colony-forming unit (CFU-S) numbers when compared to control animals transplanted with MPZipNeo infected marrow cells. Most of the CFU-S in the former animals were located in the spleen, whereas those of control mice were found in the marrow. The increase in total CFU-S content in recipients of CSF-infected mice was not due to an increase in self-renewal ability but rather to the recruitment of more primitive cells, as there were no differences in CFU-S content of spleen colonies generated from marrow cells of either group. Neither were there any differences in the cellular composition of these spleen colonies or in the size or distribution of cell types in secondary spleen colonies generated from these spleen colonies, suggesting the inability of any of the three CSFs to alter the differentiation pattern of CFU-S. PMID- 1716588 TI - The clearance of a single i.v. bolus of recombinant human erythropoietin from the serum of patients with myelodysplastic syndromes and its effects on erythropoiesis. AB - The serum erythropoietin (EPO) concentration in patients with myelodysplasia (MDS) varies widely at similar hemoglobin concentrations, although the reasons for this variation are unclear. We have studied the pharmacokinetics of an i.v. bolus of recombinant human EPO in ten subjects with myelodysplasia. Basal serum EPO concentration varied from 210 to 5984 mU/ml. Plasma half-time clearance (t1/2) varied from 3.9 to 20.0 h. A significant positive correlation was found between t1/2 and basal EPO concentration. An increase in immature peripheral blood reticulocytes was found on days 1 and 2 after EPO treatment; this may represent either an effect on hemopoiesis or on reticulocyte release from the bone marrow. PMID- 1716589 TI - Derivation of mixed phenotype cell lines with ras-myc- and raf-myc-containing retroviral vectors after infection of murine long-term bone marrow cultures. AB - Murine long-term bone marrow cultures were infected with retroviral vectors expressing the viral ras, raf, and myc oncogenes, alone and in combination. Stably transformed clonal cell lines were obtained after infection with ras-myc and raf-myc retroviruses but not by vectors expressing ras, myc, or raf alone. Northern blot analysis demonstrated that the two clonal cell lines expressed high levels of vector-specific transcripts. Phenotypic analysis of the cell lines by flow microfluorimetry and histochemical staining suggested that both cell lines expressed markers associated with cells of the megakaryocyte differentiation pathway. Histochemical staining demonstrated that these cell lines also expressed cytoplasmic enzymes associated with granulocytes and/or monocytes/macrophages. These cell lines, despite their clonal origin, are therefore of a mixed phenotype. PMID- 1716590 TI - Blast colony-forming cells and precursors of colony-forming cells detectable in long-term marrow culture express the same phenotype (CD33- CD34+). AB - We have previously shown that CD33- CD34+ human marrow cells are capable of giving rise to colony-forming cells (CFC) in long-term marrow culture (LTMC) but are mainly depleted of progenitors that directly form colonies in semi-solid media. In contrast, the CD33+ CD34+ population contains most of the CFC but not the precursors of these cells detectable in LTMC. The purpose of the present study was to determine if a form of CFC with self-renewal potential, the blast CFC, is contained within the CD33+ CD34+ or CD33- CD34+ population. The results demonstrate that blast CFC segregate within the CD33- population, representing a distinct progenitor population that may be analogous to progenitors for CFC detectable in LTMC. PMID- 1716591 TI - Effect of exogenous recombinant human granulocyte and granulocyte-macrophage colony-stimulating factor on neutrophil function following allogeneic bone marrow transplantation. AB - Functional activity of peripheral blood granulocytes was assessed in seven patients and in their normal donors following allogeneic bone marrow transplantation (BMT). Functions studied included superoxide generation (O2-), intracellular killing of Staphylococcus aureus, phagocytosis, and killing of Candida albicans. Neutrophils were tested following preincubation with 300 pM granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.2 nM granulocyte colony-stimulating factor (G-CSF), or buffered solution (diluent) as control. Our data indicate that following BMT, both recipients and their normal donors show GM CSF- and G-CSF-induced increases in: 1) O2- production in response to fMet-Leu Phe (fMLP), 2) killing of S. aureus, and 3) phagocytosis of C. albicans. In two patients that showed low candidacidal activity, GM-CSF and G-CSF markedly enhanced the cytotoxic activity of the cells. Our studies indicate that GM-CSF and G-CSF increase "oxygen-dependent" oxidative activities in neutrophils from BMT recipients and their normal donors and enhance the antimicrobial activity of the cells. PMID- 1716592 TI - Regulation of lymphokine-activated killer activity in T-replete and T-cell depleted human bone marrow by interleukin 4. AB - Donor-derived lymphokine-activated killer (LAK) cells appear to play a role in mediating an antileukemia effect in recipients of both T-replete and T-cell depleted (TCD) bone marrow transplants. LAK activity, however, is subject to regulation by cytokines other than interleukin 2 (IL-2). The purpose of this study was to examine the effect of interleukin 4 (IL-4) on the induction of LAK activity in both T-replete and TCD bone marrow. IL-4 inhibited the induction of LAK activity in a time- and dose-dependent manner in both T-replete and TCD bone marrow cultures, although there appeared to be a differential effect, suggesting that T and non-T LAK precursors have different thresholds of sensitivity to IL-4. Single-cell cytotoxicity assays indicated that IL-4 did not inhibit binding of LAK effectors to targets but did reduce the frequency of lytic conjugates. Kinetic analysis techniques demonstrated that IL-4 decreased the maximal rate of target cell lysis by IL-2-activated LAK precursors and inhibited the rate of lytic programming. These data indicate that IL-4 is able to regulate the induction of LAK activity in both T-replete and TCD bone marrow and may play a role in modulating the generation of effector cells with potential antileukemia reactivity in vivo. PMID- 1716593 TI - A rush on "stem cell factor": is it or isn't it? PMID- 1716594 TI - Prosencephalic connections of striate and extrastriate areas of rat visual cortex. AB - Afferent connections of rat primary visual cortex (area 17 or V1 area) and the rostral and caudal parts of areas 18a and 18b were studied, by placing in each of the areas, small electrophoretic injections of enzyme horseradish peroxidase (HRP) or wheat germ agglutinated-HRP. The results indicate that: 1) each of the areas has a distinct pattern of distribution of afferent neurons in the ipsilateral visual thalamus - area 17 receives its principal thalamic input from the dorsal lateral geniculate nucleus, the caudal parts of areas 18a and 18b receive a major thalamic input from the lateral posterior nucleus and a minor input from the posterior nucleus, while the rostral parts of areas 18a and 18b receive a major input from the posterior nucleus, and a minor projection from the lateral posterior nucleus; 2) the rostral and caudal parts of areas 18a and 18b each receive an associational input from area 17; 3) the rostral parts of areas 18a and 18b each receive associational input from three different extrastriate regions, the caudal part of the same extrastriate area, and the rostral and caudal parts of the other extrastriate area, whereas the caudal parts of areas 18a and 18b receive associational inputs only from one or two extrastriate regions; 4) area 17, area 18b and rostral area 18a each receive a substantial associational input from lamina V of the caudal part of the frontal eye field (FEF) in the motor cortex; however the input from the FEF to caudal area 18a (if present) is very small; 5) The extrastriate areas studied receive associational input from the restrosplenial cingulate area 29d; however, the input from area 29d to area 17 appears to be very small. The distinct patterns of distribution of prosencephalic afferents suggest to us that multiple retinotopically organized areas described previously in the rat cortex (cf Montero 1981; Espinoza and Thomas 1983) represent functionally distinct areas. PMID- 1716595 TI - Thermoreceptive lamina I trigeminothalamic neurons project to the nucleus submedius in the cat. AB - The technique of antidromic mapping with a roving array of electrodes was used to demonstrate that lamina I trigeminothalamic cells responsive specifically to skin temperature project to the n. submedius (Sm) in the medial thalamus of the cat. This finding indicates that Sm receives thermoreceptive in addition to nociceptive information. PMID- 1716597 TI - Human granulosa contain messenger ribonucleic acids encoding insulin-like growth factor-binding proteins (IGFBPs) and secrete IGFBPs in culture. AB - OBJECTIVE: To determine if luteinizing human granulosa cells contain messenger ribonucleic acid (mRNAs) encoding insulin-like growth factor-binding protein (IGFBPs) and if cultured granulosa secrete IGFBPs into conditioned medium (CM). DESIGN: Northern analysis, using IGFBP-specific complementary deoxyribonucleic acid probes, was used to detect granulosa-derived IGFBP mRNAs. Western ligand blot analysis of CM was used to detect IGFBPs secreted by granulosa cultures with and without human chorionic gonadotropin (hCG). SETTING: Granulosa cells were obtained from the In Vitro Fertilization (IVF) Program at Stanford University, a private teaching institution. PATIENTS, PARTICIPANTS: Patients undergoing IVF for tubal disease. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Transcripts of IGFBP mRNA and IGFBPs secreted into CM were detected by autoradiography of Northern and Western ligand blots, respectively. RESULTS: Transcripts of IGFBP-3, IGFBP-2, and IGFBP-1 mRNA were detected in human luteinizing granulosa. Cultured granulosa secreted IGFBPs with molecular weights corresponding to IGFBP-3, IGFBP-2, and IGFBP-1, and the latter two IGFBPs increased with 10 ng/mL hCG. A 24 kd IGFBP was noted, which may be newly characterized IGFBP-4. CONCLUSIONS: These data show that luteinizing human granulosa cells express mRNAs encoding three IGFBPs, secrete IGFBPs into culture medium, and that production of at least two of the IGFBPs is hCG-dependent, further supporting a role for the IGF system in human folliculogenesis. PMID- 1716596 TI - Endocrine responses to long-term administration of the antiprogesterone RU486 in patients with pelvic endometriosis. AB - OBJECTIVE: To examine endocrine and clinical responses to long-term administration of RU486 in patients with endometriosis. DESIGN: Prospective open trial. SETTING: Faculty practice of the authors. PATIENTS, PARTICIPANTS: Six normally cycling women with endometriosis were recruited. INTERVENTIONS: Subjects received RU486 100 mg/d for 3 months. MAIN OUTCOME MEASURE(S): Hormonal changes during RU486 were compared with control data obtained in the preceding cycle during the early follicular phase. Clinical responses were determined by patient assessment and second-look laparoscopy. RESULTS: All women became amenorrheic, and daily urinary levels of ovarian steroid metabolites remained acyclic. Mean luteinizing hormone (LH) (P less than 0.02) and LH pulse amplitude (P less than 0.05) were increased without changes in LH pulse frequency. An antiglucocorticoid effect was demonstrated by an increase in serum cortisol (P less than 0.01) and adrenocorticotropic hormone (P less than 0.05) levels. Treatment resulted in an improvement in pelvic pain in all subjects without significant change in the extent of disease as evaluated by follow-up laparoscopy. CONCLUSIONS: Daily administration of RU486 results in acyclic ovarian function and improvement in the subjective painful symptoms of endometriosis. PMID- 1716598 TI - Fertilologists and surgeons. PMID- 1716599 TI - [Effect of Bacillus mucilaginosus biomass on the exocrine function of the pancreas in piglets]. AB - The effect of mucous bacilli on the excretory activity of pancreas in pigs has been investigated. The use of biomass at doses of 0.1, 0.2 and 0.3 g/kg stimulates to a different extent the pancreatic exocrine function in pigs: the pancreatic juice volume increases, on the average, by 17-20% and the secretion of amylolytic, proteolytic and lipolytic enzymes of the pancreatic juice becomes 1.6 2.2, 1.5-1.8 and 1.4-1.7 times higher, respectively. The increased functional activity of pancreas indicates the high reserve secretory potential of the pancreatic secretory apparatus. When brought into action, this apparatus favours more complete segregation and absorption of food nutrients. PMID- 1716600 TI - Oral cavity metastasis from carcinoma of the cervix. AB - A case report of a patient with carcinoma of the cervix with metastasis to the oral cavity clinically mimicking a primary oral cavity neoplasm is described. The patient presented one year after completing radiotherapy followed by radical hysterectomy and pelvic lymphadenectomy for carcinoma of the cervix. Excellent palliation was produced with radiotherapy. We are not aware of any reported case of oral cavity metastasis from carcinoma of the cervix. PMID- 1716601 TI - Significance of soluble Fc epsilon receptor II (sFc epsilon RII/CD23) in serum and possible application of sFc epsilon RII for the prevention of allergic reactions. AB - The significance of sFc epsilon RII in IgE-mediated allergic disease was examined. sFc epsilon RII in serum was found to decrease following clinical improvement, suggesting sFc epsilon RII in serum may be an indicator of allergic diseases. Significant proportions of sFc epsilon RII in serum were present as complexes with IgE in normals as well as in atopic patients, and these complexes were more prominent in the former than in the latter group. From these observations, attempts were made to inhibit IgE-mediated allergic reactions in vitro employing recombinant sFc epsilon RII. sFc epsilon RII inhibited IgE binding as well as IgE-mediated release of chemical mediators from Fc epsilon RI and Fc epsilon RII expressing cells. These results show the functional significance of sFc epsilon RII in the negative regulation of IgE-mediated allergic reactions. PMID- 1716602 TI - Update on middle ear disease. PMID- 1716603 TI - Pharmacokinetics, effect on clotting tests and assessment of the immunogenic potential of hirudin after a single subcutaneous or intravenous bolus administration in man. AB - The pharmacokinetics and the anticoagulant effects of hirudin were investigated in 12 healthy volunteers after single subcutaneous or intravenous bolus administrations. Hirudin concentrations in citrated plasma and urine were determined with a radioimmunobioassay, which detects the inhibitor by its thrombin-binding capacity. Plasma profiles could be adequately described by the equation for an open two-compartment model (intravenous route) and by the Bateman equation (subcutaneous route), respectively. Within 24 h half of the administered hirudin dose was recovered in the urine in biologically active form. The prolongation of clotting times (activated partial thromboplastin and thrombin time) was dependent on the hirudin plasma concentration. All test subjects tolerated the hirudin injection without visible or measurable side effects. No hirudin-specific antibodies were found after single parenteral administrations. PMID- 1716604 TI - A fast photometric assay for the determination of hirudin. AB - Recombinant hirudin, a potent and specific thrombin inhibitor, is considered for anticoagulant therapy. Therefore we developed a fast and sensitive chromogenic assay for its determination in plasma. Samples can be assayed directly if aprotinin, Polybrene and urea are added to the reagent mixture. The influence of progressive inhibitors is excluded by a short incubation time. The samples (20 microliters) are diluted with 1 ml reagent mixture (0.2 M Tris, 0.025 M NaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitory units/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH units/ml bovine thrombin). After an incubation time of 1 min, 100 microliters Chromoyzm TH (Tos-Gly-Pro-Arg-pNA, 1.9 mM) is added. delta absorbance/min is linear at least up to 3 min. The calibration curve is linear up to at least 800 ng hirudin/ml plasma. The inter- and intraassay coefficient of variation in hirudin spiked normal plasma is below 5%. The detection limit is at 25 ng hirudin/ml. In plasma samples obtained from healthy subjects under hirudin therapy, a good correlation was found to the activated partial thromboplastin time (r = 0.89). In conclusion, we describe a fast and simple chromogenic substrate test to assay hirudin in plasma. Under assay conditions, the influence of endogenous thrombin inhibitors can be neglected. PMID- 1716605 TI - Antithrombotic effects of recombinant hirudin in different animal models. AB - The effect of recombinant hirudin (r-hirudin; HBW 023) was studied in four different models of thrombosis as well as on bleeding time. Arteriolar thrombus formation was induced in rats either by argon laser injury or by photochemical reaction. r-Hirudin, given by intravenous infusion, significantly inhibited arteriolar thrombus formation and arterial bleeding time at a dose of 40 micrograms/kg.min. In rabbit jugular veins and carotid arteries, occluding thrombi were produced by stenosis and endothelial damage. r-Hirudin reduced the incidence of thrombosis dose dependently (ED50 0.7 and 1.0 mg/kg i.v. in arteries and veins, respectively). Caval vein thrombosis was initiated in rats by insertion of a stainless steel coil. This thrombosis was dose dependently reduced by the injection of r-hirudin (ED50 0.16 mg/kg i.v.). The duration of the antithrombotic effect of 0.25 mg/kg of r-hirudin in caval veins was about 60 min, whereas the arterial bleeding time was significantly prolonged in these rats for only 20 min. The plasma level of r-hirudin had dropped to 0.12 micrograms/ml 60 min after injection. These results suggest that the duration of the effect on bleeding time may be shorter than the antithrombotic action of r-hirudin. PMID- 1716606 TI - Oxidative damage and changes in the glutathione redox system in erythrocytes from rats treated with hexachlorocyclohexane. AB - The concentration of reduced glutathione in the erythrocytes of rats was significantly decreased 24-72 hr after the rats were treated with 300 mg commercial hexachlorocyclohexane/kg body weight (one-third of the LD50), given ip. The activities of glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase were also significantly decreased 24 hr after treatment but there was no change in glutathione peroxidase activity. The results suggest that hexachlorocyclohexane produces significant changes in the glutathione redox system of rat erythrocytes leading to oxidative membrane damage. PMID- 1716607 TI - Single amino acid substitution in the V3 domain of CD4 is responsible for OKT4 epitope deficiency. PMID- 1716608 TI - Characterization of glutamate uptake systems in astrocyte primary cultures from rat brain. AB - The dependence of 3[H]-L-glutamate uptake on the presence of sodium, chloride, or calcium ions or on a combination of the three was investigated in astrocyte primary cultures. A stimulating effect on glutamate uptake by each of the ions tested was found. In addition to the comparably small effect by calcium alone, calcium exhibits a synergistic effect on the sodium- and chloride-dependent uptake. The sodium-dependent transport accumulates the glutamate analogue D aspartate as well as L-glutamate. L-aspartate is taken up by about 50% of the values observed for L-glutamate transport. Sodium-dependent glutamate uptake is strongly inhibited by aspartate-beta-hydroxamate (A beta H) and threo-beta hydroxyaspartate (T beta H). Quisqualate is less potent in inhibiting this uptake. In contrast, the chloride- and the calcium-dependent uptake systems do not handle D- and L-aspartate as substrates. A beta H and T beta H are only poor inhibitors of these transporters while quisqualate reduces glutamate uptake almost completely. Kinetic data of all uptake systems were estimated. High and low affinity components of each individual system are demonstrated by Eadie Hofstee analysis. Hill plots indicate that high and low affinity uptake may be due either to two respective uptake sites for Na(+)-, Cl(-)-, and Ca(++) dependent glutamate transport, or to two glutamate binding sites for each single transport system with negative cooperativity. PMID- 1716609 TI - Mapping and topographic localization of epitopes of the Yersinia pseudotuberculosis invasin protein. AB - The Yersinia pseudotuberculosis invasin protein is a 986-amino-acid outer membrane protein that promotes bacterial penetration into mammalian cells by binding to beta 1-chain integrin receptors. We previously showed that the integrin binding domain is encoded by the carboxyl-terminal 192 amino acids. To further investigate the structure of this protein, we characterized a set of 32 monoclonal antibodies (MAbs) directed against invasin. Invasin deletion derivatives and fusion proteins carrying different segments of invasin were used to map the epitopes of this set of MAbs into 10 overlapping but distinct intervals. Indirect immunofluorescence of intact bacteria expressing invasin demonstrated that two large regions of invasin contain epitopes exposed on the bacterial surface. To assess the role of these surface-exposed regions in the binding and invasion of mammalian cells, each of the MAbs was tested for its ability to inhibit these processes. All of the MAbs that recognized bacterial surface-exposed epitopes in the cell binding domain of invasin inhibited both cell attachment and cell penetration, and no other MAbs inhibited either activity. PMID- 1716610 TI - The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide. AB - Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS. PMID- 1716611 TI - Sucrose-promoted accumulation of growing glucosyltransferase variants of Streptococcus gordonii on hydroxyapatite surfaces. AB - Streptococcus gordonii exhibits a phase variation involving expression of high (Spp+) or low (Spp-) glucosyltransferase activity. The related bacterial accumulation on hydroxyapatite (HA) and saliva-coated HA surfaces was examined and found to be significant. Spp+ cells growing anaerobically in a defined medium utilize about 30% of the glucose available from sucrose to make insoluble glucans. These glucans formed cohesive masses on HA beads, which contained 80 to 90% of the total bacteria. The bacterial polymer mass had a volume of about 40 microns3 and contained more than 5 x 10(10) viable cells per cm3. In the absence of sucrose, the beads were saturated by 1 x 10(8) to 2 x 10(8) Spp+ cells. Spp- bacteria, which make 30-fold less glucan than do Spp+ bacteria, did not accumulate on surfaces in numbers significantly above the saturation level of 1 x 10(8) to 2 x 10(8) cells in the presence or absence of sucrose. Insoluble glucan synthesized by Spp+ cells from sucrose also enabled these bacteria to accumulate on saliva-coated HA seven times more effectively than the Spp- cells and 10 times more effectively than the Spp+ cells grown in medium without sucrose. PMID- 1716612 TI - Protective ribosomal preparation from Shigella sonnei as a parenteral candidate vaccine. AB - A parenteral Shigella ribosomal vaccine (SRV) was investigated in animals for safety, antibody-inducing capacity, and protective activity. Ribosomal preparations from a Shigella sonnei phase I avirulent strain were obtained and shown to possess chemical, sedimentation, and other properties typical of bacterial ribosomes. No endotoxin contamination was revealed by a ketodeoxyoctonate assay, although the presence of some kind of O antigen was evidenced by serological findings and the high activity of SRV in inducing the O antibody response and immunological memory in animals. SRV was nontoxic in mice, guinea pigs, and monkeys and induced no local reactions when injected subcutaneously in reasonable doses. Significant protection against a local Shigella infection (Sereny test) was seen in guinea pigs injected with SRV (efficiency index, about 60%) and the specificity of the protection was evident from cross-challenge experiments. The protective efficiency of SRV was especially high in rhesus monkeys challenged orally with virulent Shigella cells (89%, as calculated from the summarized data of several experiments in 71 animals). Protection in monkeys was long lasting and could be demonstrated several months after injection of SRV. An inexpensive technique can be used for the production of SRV on a large scale. The high immunogenicity of SRV is discussed in terms of the amplifying effect of the ribosome, which serves as a delivery system for polysaccharide O antigen. Further study of SRV as a candidate vaccine for humans seems justified by the data obtained. PMID- 1716613 TI - Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity. AB - We synthesized Cryptococcus neoformans serotype A glucuronoxylomannan (GXM) conjugate vaccines under conditions suitable for human use to prevent disseminated cryptococcosis. The purified, sonicated GXM was derivatized with adipic acid dihydrazide through either hydroxyl or carboxyl groups and then covalently bound to tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA). The immunogenicity of these conjugates was evaluated in BALB/c and general purpose mice by subcutaneous injection in saline. The conjugates elicited higher GXM antibody responses than GXM alone. Booster immunoglobulin G (IgG) and IgM responses were elicited by all conjugates in BALB/c mice. The conjugates prepared through hydroxyl activation (GXM-TT2 and GXM-rEPA) were more immunogenic than the one prepared through carboxyl activation (GXM-TT1). GXM antibody response was enhanced by the administration of monophosphoryl lipid A 2 days following the injection of GXM-TT2 (P less than 0.03). The conjugates also elicited IgG antibodies to the carrier proteins. Gel diffusion tests using conjugate-induced hyperimmune sera and chemically modified GXMs suggested that the specificity of GXM-TT1-induced antibodies was conferred by the O-acetyl groups. Hyperimmune sera generated by GXM-TT2 precipitated with the chemically unmodified and the de-O-acetylated GXMs but not with the carboxyl-reduced and de O-acetylated GXM. GXM-TT2-induced hyperimmune serum also precipitated with the capsular polysaccharides of C. neoformans serotypes D, B, and C. The conjugate vaccines prepared through hydroxyl activation of the GXM are sufficiently immunogenic and appear to be suitable for clinical evaluation. PMID- 1716614 TI - Lymphokine secretion and cytotoxic activity of human CD4+ T-cell clones against Bordetella pertussis. AB - Human CD4+ T-cell clones specific for pertussis toxin and other Bordetella pertussis antigens have been tested for their cytotoxic activity, lymphokine production, and capacity to induce immunoglobulin synthesis. Clones specific for the S1 subunit of pertussis toxin were cytotoxic for autologous Epstein-Barr virus-transformed B cells, which had been pulsed with the native antigen, the recombinant S1 subunit of pertussis toxin, or synthetic peptides derived from the S1 amino acid sequence. The killing of antigen-pulsed target cells was class II restricted. All of the T-cell clones produced mostly interleukin-2 and gamma interferon and assisted allogeneic B cells in the production of immunoglobulins M and G but not immunoglobulin E. The potential in vivo role of the cytotoxic activity of these clones is discussed. PMID- 1716615 TI - Direct expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli and Salmonella typhimurium aroA. AB - Nonfused (i.e., nonhybrid) filamentous hemagglutinin (FHA) of Bordetella pertussis was efficiently expressed in Escherichia coli K-12 and Salmonella typhimurium aroA at levels higher than those found in wild-type B. pertussis when the upstream signals of the gene were replaced and the translation initiation region was engineered to optimize translational efficiency. Inclusion of part of the C-terminal FHA open reading frame, whose translation product does not appear to be part of the major secreted species of FHA, was shown to be important in achieving protein expression in both E. coli and S. typhimurium aroA; removal of the downstream gene sequence abolished recombinant FHA production. The levels of expression observed varied widely according to the construct and host bacterium used. PMID- 1716616 TI - Prevention of murine cerebral malaria by a stable prostacyclin analog. AB - Iloprost, a synthetic prostacyclin analog, successfully prevents the development of cerebral malaria in mice. Malaria antigen-induced tumor necrosis factor (TNF) production could be inhibited by iloprost in vitro and in vivo. Northern analysis of TNF mRNA revealed that malaria antigen-induced TNF expression was suppressed at the transcription level. PMID- 1716617 TI - Effect of a short maximal physical exercise on coagulation, fibrinolysis, and complement system. AB - In 11 healthy young subjects, the plasma concentrations of the thrombin antithrombin III complex, fibrinopeptide A, tissue-plasminogen activator, complement fragments C3a and C4a, and histamine were measured before and after a graded maximal bicycle exercise test. The analyses were carried out 30 min before and immediately before exercise, immediately after exercise, and 30 and 60 min later. All post-exercise values were corrected for plasma volume changes, which were calculated from hematocrit and hemoglobin values. Immediately post-exercise, thrombin-antithrombin III, tissue-plasminogen activator, complement fragments C3a and C4a, and histamine were all significantly elevated (p less than 0.01), compared with the pre-exercise values; 30 and 60 min later the values normalized and significant differences from the pre-exercise values could no longer be measured. Fibrinopeptide A did not change significantly after exercise. The present results provide evidence for a simultaneous activation of coagulation, fibrinolysis, and complement system as well as for a release of histamine after a short maximal exercise. PMID- 1716618 TI - Prevention of herpes keratitis by monoclonal antibodies specific for discontinuous and continuous epitopes on glycoprotein D. AB - Seven monoclonal antibodies (mAb) specific for defined discontinuous and continuous epitopes on glycoprotein D of herpes simplex virus type 1 (HSV-1) were surveyed for their capacity to protect against virus-induced corneal disease in a murine ocular infection model. A known amount of purified mAb was transferred passively to BALB/c mice 24 hr after topical infection with HSV-1 on their scarified corneas. At high doses (50-136 micrograms), all seven mAbs protected against the development of persistent necrotizing stromal keratitis. Significant protection was also observed at low doses (20 micrograms) with two mAbs to discontinuous epitopes and two mAbs to continuous epitopes. Selected high-dose mAbs also were able to reduce the severity of blepharitis. These results indicated that at least seven different antigenic sites on glycoprotein D can serve as targets for effective antibody therapy in the murine model of HSV-1 ocular infection. PMID- 1716620 TI - Hepatitis C--screening of blood donations. PMID- 1716619 TI - Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells. AB - The authors tried to determine whether human corneal endothelial cells in primary culture synthesize messenger RNA (mRNA) coding for epidermal growth factor (EGF), EGF receptor, basic fibroblast growth factor (FGFb), transforming growth factor beta-1 (TGFb1), and interleukin-1 alpha (IL-1 alpha). Oligodeoxythymidine-primed complementary DNA (cDNA) was generated from total cellular RNA extracted from eight independent primary corneal endothelial cell cultures. Four of these cultures, maintained 18-51 days, had obvious increases in cell numbers and mass over the 2 weeks before RNA extraction and were populated primarily with cells that were small, uniform, and mononuclear (proliferative cultures). The morphology of the cells in other four cultures, maintained 47-78 days, was predominantly large, irregular, vacuolated, and occasionally multinucleated. These cells were identical to senescent cells found in previous studies, and the cell number did not increase in these cultures over the 2 weeks preceding RNA extraction (senescent cultures). The polymerase chain reaction (PCR) was used to amplify the growth factors (EGF, FGFb, TGFb1, and IL-1 alpha), EGF receptor, and beta actin sequences from each of the cDNA samples. The EGF receptor, FGFb, and beta actin mRNAs were present in all eight cDNA samples. The EGF mRNAs were detected by PCR alone in four of the samples from proliferative cultures, TGFb1 mRNAs in three, and IL-1 alpha mRNAs in three. In the samples from senescent cultures, 0, 1, and 0 mRNAs were detected, respectively. Southern blots of the PCR products were probed with oligonucleotides complementary to sequences in each of the amplified products. This technique showed that the appropriately sized amplification products were specific.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716621 TI - Ventricular extrasystoles masquerading as aberrantly conducted atrial extrasystoles because of postectopic T wave change. AB - This presentation reflects a case where broad and bizarre premature QRS complexes are preceded by sinus beats whose T wave is "abnormal," and seems to contain a premature P wave. A diagnosis of atrial extrasystoles with aberrancy thus could be entertained. The extrasystoles, however, are ventricular in origin. The pattern is explained on the basis of postectopic T wave change, that is, the change in configuration of the T wave that occurs in the sinus beat after an extrasystole. PMID- 1716622 TI - Detection of hepatitis C virus infection by enzyme-linked immunosorbent assay system using core protein expressed in Escherichia coli. AB - Enzyme-linked immunosorbent assay of the core protein of hepatitis C virus (HCV) expressed in E. coli led to detection of the antibody against this virus in patients with chronic hepatitis. Some of the negative results obtained using a different viral protein became positive with this E. coli-expressed viral protein, and were also positive for the viral RNA. Thus, use of the core protein of HCV facilitates accurate detection of HCV infection. PMID- 1716623 TI - Establishment of an alpha-fetoprotein-producing gastric cancer cell line in serum free media. AB - A new cell line, designated ISt-1, was established in serum-free medium from a 63 year-old gastric cancer patient with an extremely high serum level of alpha fetoprotein (AFP). ISt-1 exhibited a typical morphology of epithelial cells in culture. The population doubling time was 31 hours. Production of AFP was demonstrated by immunohistochemical study and high levels of AFP were detected in the conditioned medium of ISt-1 cells. ISt-1 is considered to be an AFP-producing gastric cancer cell line and will provide a useful tool for the study of production and secretion of AFP from cells. PMID- 1716624 TI - Encapsulation of coagulase-negative staphylococci of bovine origin. AB - Capsule expression was assessed in six coagulase-negative staphylococcal strains in serum-soft agar and by india ink and electron microscopy. Classification of strains as encapsulated by serum-soft agar and india ink methods differed. Staphylococcus chromogenes, Staph. hyicus, and Staph. simulans grew as diffuse colonies in serum-soft agar and unstained halos were detected in india ink preparations. Staphylococcus hominis and Staph. simulans grew as diffuse colonies in serum-soft agar but no unstained halo was seen in india ink preparations. Staphylococcus hyicus was the only strain that gave negative results with serum soft agar and india ink assays. Conventional electron microscopy revealed the presence of capsular polysaccharides on the cell surface of Staph. chromogenes, Staph. hominis and Staph. hyicus. Conventional electron microscopic technique used to examine the surface of cells was detrimental to capsule structure. During dehydration the capsule collapsed and appeared as electron dense aggregates at the surface of cells. To confirm results of conventional electron microscopy and to visualize clearly the cell surface, encapsulated Staph. hyicus and unencapsulated Staph. simulans were observed after freeze-fracture and etching by scanning electron microscopy. The fibrous nature of capsular polysaccharides surrounding cells of Staph. hyicus were distinct and confirmed observation by conventional electron microscopy. A rapid transmission electron microscopic technique is described also for observation of capsule. Results of the rapid TEM method agreed with conventional TEM and SEM. The finding that coagulase-negative staphylococci isolated from bovine milk are capable of capsule production may be important when investigating pathogenicity of these micro-organisms. PMID- 1716625 TI - The differential fluorescence of bacteria stained with acridine orange and the effects of heat. AB - Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence. PMID- 1716626 TI - Chloroplast translational initiation factor 3. Purification and characterization of multiple forms from Euglena gracilis. AB - The chloroplast translational initiation factor 3 (IF-3chl) has been purified by a combination of gravity and high pressure liquid chromatographic steps. IF-3chl activity has been resolved into three forms designated alpha, beta, and gamma. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the alpha form corresponds to a single polypeptide with a molecular mass of approximately 34 kDa. The beta and gamma forms have been purified to near homogeneity, and both forms appear to function as monomers with molecular masses of about 39-42 kDa. All three forms are heat stable. All the forms of IF-3chl detected enhance the poly (A,U,G)-dependent binding of the initiator tRNA to chloroplast 30 S ribosomal subunits in the presence of Escherichia coli IF-1 and IF-2. The chloroplast factor, unlike the corresponding bacterial factor, does not have a strong RNA binding activity. PMID- 1716627 TI - Triorganotins inhibit the mitochondrial inner membrane anion channel. AB - The inner membrane of liver and heart mitochondria possesses an anion uniport pathway, known as the inner membrane anion channel (IMAC). IMAC is inhibited by matrix Mg2+, matrix H+, N,N'-dicyclohexycarbodiimide, mercurials and amphiphilic amines such as propranolol. Most of these agents react with a number of different mitochondrial proteins and, therefore, more selective inhibitors have been sought. In this paper, we report the discovery of a new class of inhibitors, triorganotin compounds, which block IMAC completely. One of the most potent, tributyltin (TBT) inhibits malonate uniport via IMAC 95% at 0.9 nmol/mg. The only other mitochondrial protein reported to react with triorganotins, the F1F0ATPase, is inhibited by about 0.75 nmol/mg. The potency of inhibition of IMAC increases with hydrophobicity in the sequence trimethyltin much less than triethyltin much less than tripropyltin less than triphenyltin less than tributyltin; which suggests that the binding site is accessible from the lipid bilayer. It has long been established that triorganotins are anionophores able to catalyze Cl-/OH- exchange; however, TBT is able to inhibit Cl- and NO3- transport via IMAC at doses below those required to catalyze rapid rates of Cl-/OH- exchange. Consistent with previous reports, the data indicate that about 0.8 nmol of TBT per mg of mitochondrial protein is tightly bound and not available to mediate Cl /OH- exchange. We have also shown that the mercurials, p-chloromercuribenzene sulfonate and mersalyl, which only partially inhibit Cl- and NO3- transport can increase the IC50 for TBT 10-fold. This effect appears to result from a reaction at a previously unidentified mercurial reactive site. The inhibitory dose is also increased by raising the pH and inhibition by TBT can be reversed by S2- and dithiols but not by monothiols. PMID- 1716629 TI - Undulin is a novel member of the fibronectin-tenascin family of extracellular matrix glycoproteins. AB - We characterized cDNA clones specific for the extracellular matrix glycoprotein undulin. Two sets of cDNA clones were isolated from a human placental lambda gt11 expression library and from a rhabdomyosarcoma cell line encoding two partially identical carboxyl-terminal polypeptides of 843 (Un1) and 443 (Un2) amino acids suggesting differential splicing of a single gene transcript. Northern blot analysis of human rhabdomyosarcoma cell poly (A) RNA with cDNA specific for Un1 identified transcripts of approximately 4.2, 6.5, and 8.5 kilobases, whereas a probe specific for Un2 detected a single mRNA of approximately 5 kilobases. Since a monoclonal antibody that is reactive with a sequence encoded by Un1 and not by Un2 detects the bands considered characteristic for undulin in Western blots, the mRNAs related to Un1 may code for the major part of the undulin molecule. The protein sequences deduced from Un1 and Un2 reveal an amino-terminal differentially spliced von Willebrand factor A domain, characteristic of proteins that interact with interstitial collagens, which is linked to fibronectin-like type III homology units by a unique sequence of 57 amino acids. Whereas Un2 encodes two complete and one incomplete type III homologies followed by a unique acidic carboxyl-terminal domain of 118 amino acids, Un1 codes for seven complete and one truncated type III homologies, followed by a short proline-rich carboxyl terminal segment of 23 amino acids. Considering the 298 amino acids occurring in identical segments, the 989 different amino acid positions deduced from clones Un1 and Un2 represent an estimated 40% of the overall undulin sequence. In the context of 1) rotary shadowing electron microscopy data showing undulin as a structure composed of nodules that are interconnected by flexible rods of varying size, 2) the presence of three major bands of Mr 270,000, 190,000, and 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 3) common antigenic epitopes and similar peptide maps (Schuppan, D., Cantaluppi, M.C., Becker, J., Veit, A., Bunte, T., Troyer, D., Schuppan, F., Schmid, M., Ackermann, R., and Hahn, E.G. (1990) J. Biol. Chem. 265, 8823-8832), our finding of differentially spliced type III homology units, as found in tenascin and fibronectin, suggests that undulin is another member of the fibronectin-tenascin family of extracellular matrix glycoproteins. Furthermore, as in fibronectin and tenascin, undulin bears an additional subset of interactive domains tailored to specific structural and functional roles in development and differentiation. PMID- 1716628 TI - Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover. AB - Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding. PMID- 1716630 TI - Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion. AB - We have used the human Lewis blood group fucosyltransferase cDNA and cross hybridization procedures to isolate a human gene that encodes a distinct fucosyltransferase. Its DNA sequence predicts a type II transmembrane protein whose sequence is identical to 133 of 231 amino acids at corresponding positions within the catalytic domain of the Lewis fucosyltransferase. When expressed by transfection in cultured cell lines, this gene determines expression of a fucosyltransferase capable of efficiently utilizing N-acetyllactosamine to form the Lewis x determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc). By contrast, biochemical and flow cytometry analyses suggest that the enzyme cannot efficiently utilize the type II acceptor NeuNAc alpha 2----3Gal beta 1--- 4GlcNAc, to form the sialyl Lewis x determinant. In Chinese hamster ovary cells, however, the enzyme can determine expression of the alpha 2----3-sialylated, alpha 1----3-fucosylated structure known as VIM-2, a putative oligosaccharide ligand for ELAM-1. Cell adhesion assays using VIM-2-positive, sialyl Lewis x negative transfected Chinese hamster ovary cells indicate that surface expression of the VIM-2 determinant is not sufficient to confer ELAM-1-dependent adhesive properties upon the cells. These results demonstrate that substantial structural similarities can exist between mammalian glycosyltransferases with closely related enzymatic properties, thus facilitating isolation of their cognate genes by cross-hybridization methods. The results further suggest that cell surface expression of the VIM-2 determinant is not necessarily sufficient to mediate ELAM 1-dependent cell adhesion. PMID- 1716631 TI - Stimulation of protein tyrosine phosphorylation, phosphoinositide turnover, and multiple previously unidentified serine/threonine-specific protein kinases by the Pan-B-cell receptor CD40/Bp50 at discrete developmental stages of human B-cell ontogeny. AB - CD40/Bp50 B-cell receptor has been implicated as having an important function for the regulation of human B-cell growth and maturation as well as antigen-driven selection of tonsillar B-cells in germinal centers. The purpose of the present study was to examine the biochemical events triggered by the engagement of the CD40 receptor in human B-lineage lymphoid cells corresponding to discrete developmental stages of human B-cell ontogeny. The engagement of the CD40 receptor on pro-B-, pre-pre-B-, pre-B-, or activated mature B-cells but not on resting mature B-cells with the agonistic anti-CD40 monoclonal antibody G28-5 resulted in enhanced tyrosine phosphorylation of four distinct phosphoproteins with molecular masses of 67, 72, 96, and 113 kDa and induced a rapid increase in the production of inositol 1,4,5-trisphosphate. Further, we have identified five electrophoretically distinct renaturable CD40-regulated serine/threonine-specific protein kinases (PK120, PK93, PK76, PK55, and PK48) that showed markedly increased in vitro activity after CD40 stimulation. Protein kinase C inhibitor 1 (5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) abrogated the stimulation of the in vitro activity of PK120, PK93, PK55, and PK48 and attenuated the stimulation of the in vitro activity of PK76 in response to the engagement of the CD40 receptor but did not influence the enhanced tyrosine phosphorylation of cellular substrates after CD40 stimulation. Notably, genistein and herbimycin A, two potent inhibitors of tyrosine-specific protein kinases, not only abrogated the CD40-induced enhanced tyrosine phosphorylation on the 67-, 72-, 96-, and 113 kDa substrates, but they also inhibited the CD40-induced stimulation of phosphoinositide turnover as well as the CD40-induced increase of the in vitro activity of renaturable serine/threonine-specific protein kinases.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716632 TI - Identification of divalent metal ion-dependent inhibition of activated protein C by alpha 2-macroglobulin and alpha 2-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin. AB - The half-life of activated protein C (APC) was 31 min in citrated blood and 18 min in whole blood. Immunoblotting analysis of citrated blood identified APC protein C inhibitor (APC-PCI) and APC-alpha 1-antitrypsin complexes. Whole blood contained two additional APC-inhibitor complexes, one stimulated by Ca2+ and another by Mg2+. The former was identified as APC-alpha 2-macroglobulin (APC alpha 2M) while the latter was not identified. APC-alpha 2-antiplasmin complexes (APC-alpha 2AP) were identified, comigrating with APC-PCI complexes. Purified alpha 2M and alpha 2AP inhibited APC in the presence of Ca2+ (k2 = 99 and 100 M-1 S-1, respectively. Inhibition of APC and Factor Xa by alpha 2M and inhibition of APC by alpha 2AP was stimulated by Ca2+, Mn2+, and Mg2+. Inhibition of thrombin by alpha 2M and of plasmin by alpha 2AP was not altered by EDTA or Ca2+, suggesting divalent metal ions affect APC and Factor Xa rather than the inhibitors. k2 values for the APC inhibitors and their plasma concentrations suggest that PCI and alpha 1-antitrypsin are the more important APC inhibitors and that alpha 2M and alpha 2AP are metal ion-dependent auxiliary inhibitors. Inhibitors can account for the in vivo half-life of APC. PMID- 1716633 TI - Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP. AB - The microfilament lattice, composed primarily of filamentous (F)-actin, determines in large part the mechanical (deformability) properties of neutrophils, and thus may regulate the ability of neutrophils to transit a microvascular bed. Circulating factors may stimulate the neutrophil to become rigid and therefore be retained in the capillaries. We hypothesized that cell stiffening might be attenuated by an increase in intracellular cAMP. A combination of cell filtration and cell poking (mechanical indentation) was used to measure cell deformability. Neutrophils pretreated with dibutyryl cAMP (db cAMP) or the combination of prostaglandin E2 (PGE2, a stimulator of adenylate cyclase) and isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) demonstrated significant inhibition of the n-formyl-methionyl-leucyl phenylalanine (fMLP)-inducing stiffening. The inhibition of cell stiffening was associated with an increase in intracellular cAMP as measured by enzyme-linked immunoassay (EIA) and an increase in the activity of the cAMP-dependent kinase (A kinase). Treatment with PGE2 and IBMX also resulted in a decrease in the F-actin content of stimulated neutrophils as assayed by NBD-phallacidin staining and flow cytometry or by changes in right angle light scattering. Direct addition of cAMP to electropermeabilized neutrophils resulted in attenuation of fMLP-induced actin assembly. Neutrophils stimulated with fMLP demonstrated a rapid redistribution of F-actin from a diffuse cortical location to a peripheral ring as assessed by conventional and scanning confocal fluorescence microscopy. Pretreatment of neutrophils with the combination of IBMX and PGE2 resulted in incomplete development and fragmentation of the cortical ring. We conclude that assembly and redistribution of F-actin may be responsible for cell stiffening after exposure to stimulants and that this response was attenuated by agents that increase intracellular cAMP, by altering the amount and spatial organization of the microfilament component of the cytoskeleton. PMID- 1716634 TI - Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice. AB - Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear. PMID- 1716635 TI - Acidic fibroblast growth factor in the developing rat embryo. AB - Compared to basic fibroblast growth factor (bFGF), a widely distributed, broad spectrum mitogen and mesoderm inducer, acidic fibroblast growth factor (aFGF) is reported to have an essentially neural distribution and to be undetectable in the early embryo. In the present investigation, we used immunoblotting and immunochemistry to assess the cellular and tissue distributions of aFGF and bFGF in 11-20-d rat embryos. Immunoblotting of crude and heparin-bound embryo extracts revealed faint bands at the expected 17-18-kD and predominant bands at an apparent molecular mass of 26 to 28-kD (despite reducing conditions) using multiple specific antibodies for aFGF and bFGF. Pretreatment with 8 M urea yielded 18-20-kD aFGF and bFGF and some 24-26-kD bFGF. Immunoreactivity for both aFGF and bFGF was positive and similar in the cytoplasm, nuclei, and extracellular matrix of cells of neuroectodermal and mesodermal origin, while it was negative in endoderm-derived cells. The distribution of immunoreactive aFGF and bFGF also showed changes during development that were associated with the process of cellular and tissue differentiation. For example, intensity and extent of immunoreactivity for both peptides progressively increased in the middle layer of the spinal cord with increasing differentiation of the neural cells. The immunostaining patterns were very similar for aFGF and bFGF for each organ and at each stage. In conclusion, high molecular mass forms of immunoreactive aFGF and bFGF are present in the rat embryo. Acidic FGF and bFGF are both widely distributed in tissues of neuroectodermal and mesodermal origin, and their distribution was very similar. PMID- 1716637 TI - Two-parameter mobile phase optimization for the simultaneous high-performance liquid chromatographic determination of dopamine, serotonin and related compounds in microdissected rat brain nuclei. AB - A new high-pressure liquid chromatography method with electrochemical detection is described that allows the simultaneous determination of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, 5 hydroxytryptophan and 5-hydroxyindoleacetic acid in microdissected nuclei from individual rat brains. No sample pre-treatment steps are required. Resolution and analysis time were optimized by a simple limited optimization procedure, involving two-parameter factorial design. PMID- 1716636 TI - Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly. AB - Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected. PMID- 1716638 TI - Subfractions of membranes from calf brain synaptosomes obtained and studied by liquid-liquid partitioning. AB - Synaptosomes isolated from calf brain cortex were lysed and fragmented by Yeda press treatment. The obtained membranes have previously been fractionated in a counter-current distribution process using a liquid-liquid two-phase system consisting of water, dextran, Ficoll and poly(ethylene glycol) [J. Chromatogr., 358 (1986) 147]. Using the fact that there are discrete membrane populations, a rapid preparative method for isolation of the two main fractions is presented in the present work, as well as a subfractionation of one of them using liquid liquid extraction with dextran-bound Procion yellow HE-3G. The content of several membrane constituents, i.e. protein, acetylcholinesterase, succinate dehydrogenase and ATPase, as well as opiate binding, were determined for the three fractions. Counter-current distribution of the fractions elucidates their heterogeneity and the effectiveness of the purification. PMID- 1716639 TI - Competitive dissociation of encephalitogenic complexes between antigen presenting cells and myelin basic protein. AB - Splenic T cells from myelin basic protein (MBP)-immunised Lewis rats were activated to transfer experimental autoimmune encephalomyelitis (EAE) by co culture with MBP-pulsed lymphoid dendritic cells (DC). MBP-pulsed DC could be kept for at least 24 h at 37 degrees C in antigen-free medium without affecting their ability subsequently to activate encephalitogenic T cells. However, MBP pulsed DC were rendered much less stimulatory after a 6 h, but not 2 h, secondary incubation with ovalbumin. Thus, although encephalitogenic complexes between MBP and DC appear very stable in the absence of competing antigens, in their presence, antigen exchange can take place over a period of a few hours; this has positive implications for therapy of EAE by antigen competition. PMID- 1716640 TI - Enhancement of antigen-specific T-cell reactivity on the affected side in stroke patients. AB - We have analyzed the impact of stroke with subsequent hemiparesis and sensory loss on in vivo mediated immune functions. The delayed-type hypersensitivity (DTH) reaction to purified protein derivate (PPD, tuberculin) was used as a measure of antigen-specific T-cell reactivity, and subcutaneous immunization with influenza vaccine was employed to evaluate T-cell-dependent B-cell function. Thirty-two of the 50 stroke patients tested displayed positive DTH reaction to PPD. All but two showed equal or stronger DTH reaction on the paretic arm compared to the contralateral side (p less than 0.0001). This stroke-induced enhancement of DTH reactivity was evident in patients with combined motor and sensory deficit as well as in subjects with hemiparesis alone. In contrast, immunization of stroke patients with influenza vaccine, a T-cell-dependent B-cell antigen, raised equal antigen-specific serum IgG, IgA and IgM antibody responses irrespective of side (paretic or not paretic). We conclude that stroke enhances antigen-specific T-cell reactivity on the affected side of the body, and that motor but not sensory deficit seems to be required for this enhancement. Antigen specific B-cell reactivity was not significantly influenced by the hemiparesis. PMID- 1716641 TI - Identification of glycoconjugates which are targets for anti-Gal(beta 1-3)GalNAc autoantibodies in spinal motor neurons. AB - Human IgM anti-Gal(beta 1-3)GalNAc antibodies which bind to GM1 and GD1b, are implicated in the pathogenesis of predominantly motor neuropathy or motor neuron disease. By immunofluorescence microscopy, the human antibodies immunostain the surface of motor neurons from bovine spinal cord. The motor neurons are also immunostained by cholera toxin (CT), which is specific for GM1. Glycolipid analysis using thin-layer chromatography (TLC) and immunostaining reveals that the relative concentration of GM1 and GD1b in motor neurons is greatly reduced in comparison to whole spinal cord, and that other motor neuron gangliosides are unreactive with the anti-Gal(beta 1-3)GalNAc antibodies. By Western blot analysis, the antibodies react with several protein bands in motor neuron extracts, and many of the same proteins are also recognized by PNA. These data suggest that both glycoproteins and glycolipids might be targets for anti Gal(beta 1-3)GalNAc antibodies in spinal motor neurons. PMID- 1716642 TI - Cerebrospinal fluid levels of myelin basic protein-like material and soluble interleukin-2 receptor in multiple sclerosis. AB - The presence, level and disease activity relationships of soluble interleukin-2 receptor (sIL-2R) in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients are unresolved. We measured CSF immunoreactive myelin basic protein (MBP), a marker of acute myelin damage, and sIL-2R levels in the CSF from 11 patients with active relapsing remitting (RR) MS, five with stable RR MS, eight with chronic progressive (CP) MS, five with other neurologic diseases, and three normal controls. No measurable (less than 100 units/ml) sIL-2R was present in any of the samples. Conversely, MBP levels were elevated in the active RR group compared to the other four groups. These results indicate that, at the sensitivity of assays currently available, levels of CSF sIL-2R do not correlate with the diagnosis or disease activity of MS. PMID- 1716643 TI - Comparison of immunocytochemical staining of astrocytes, oligodendrocytes, and myelinated fibers in the brains of carbonic anhydrase II-deficient mice and normal littermates. AB - Immunostaining for carbonic anhydrase (CA) was performed in paraffin sections from the brains of CA II-deficient mutant mice and their normal littermates. Double immunofluorescence staining showed CA in myelinated tracts and oligodendrocytes in the cerebellum of the normal but not the CA II-deficient mice, and also in astrocytes in the cerebral cortex of the normal mice but not the mutants. The data show that the CA in normal oligodendrocytes, astrocytes, and myelin is the II isoenzyme, because these structures in the mutants would be positively stained if the staining normally were due to a contaminant in the antiserum or an antibody against a different isoenzyme. The findings in normal gray matter also suggest that many neuronal cell bodies are surrounded by a network of fine, CA-positive astrocytic processes. PMID- 1716644 TI - Influence of chlorthalidone on the pharmacokinetics and pharmacodynamics of Org 10172 (Lomoparan), a low molecular weight heparinoid, in healthy volunteers. AB - The influence of chlorthalidone (100 mg PO) on the pharmacokinetics and pharmacodynamics of Org 10172 (IV bolus injection of 3250 anti-Xa units), a low molecular weight heparinoid, was studied in six healthy male volunteers using an open randomized two-way crossover design. Chlorthalidone produced a slight decrease in clearance of anti-Xa activity from 7.1 +/- 1.0 to 6.6 +/- 0.8 mL/min and a decrease of the volume of distribution from 0.20 +/- 0.05 to 0.16 +/- 0.04 L/kg, whereas the volume of distribution of antithrombin activity increased from 0.14 +/- 0.05 to 0.26 +/- 0.10 L/kg (all differences P less than .05). During the entire study period no adverse events occurred. In summary, chlorthalidone showed separate effects on different fractions of Org 10172. The clinical implication of the slight change observed in plasma anti-Xa activity is likely to be limited, whereas the 80% increase in distribution volume of plasma antithrombin activity can not be defined as yet in terms of clinical relevance. PMID- 1716645 TI - Central projections of primary sensory neurons innervating different parts of the vibrissae follicles and intervibrissal skin on the mystacial pad of the rat. AB - The cell bodies and central projections of neurons innervating the vibrissae follicles and adjacent skin in the rat were investigated by retrograde and transganglionic transport of HRP. The cell bodies of neurons innervating the vibrissa follicle via the deep vibrissa nerve (DVN) were the largest, followed by those innervating the follicle via the superficial vibrissa nerve (SVN). The smallest cell bodies were those innervating the intervibrissal skin. The DVN neurons terminated centrally as an almost uninterrupted column through the trigeminal sensory nuclear complex. The DVN projections to nucleus caudalis and C1 dorsal horn were entirely restricted to laminae III, IV, and V. Besides the projections to lamina V, the DVN projections were strictly localized somatotopically at all levels replicating the peripheral organization of the vibrissae. The SVNs projected sparsely to midlevels of the main sensory nucleus but not to nuclei oralis and interpolaris. The main SVN projections appeared in laminae I-III of nucleus caudalis. In addition, a small projection to lamina V was observed. The projections to laminae II and III were organized mediolaterally in a similar way as the DVN projections; those to laminae I and V were less restricted. The intervibrissal skin neurons projected sparsely to the caudal main sensory nucleus and to the border between nuclei oralis and interpolaris. The projections to nucleus caudalis were restricted to laminae I-III and V and were organized in a similar way as the SVN projections. PMID- 1716646 TI - Epitopes located in spatially separate domains of each neurofilament subunit are present in Parkinson's disease Lewy bodies. AB - Subcortical Lewy bodies are the pathological hallmark of idiopathic Parkinson's disease. This study sought to determine the extent to which each neurofilament subunit [low (NF-L), mid (NF-M), or high (NF-H)] was present in Lewy bodies by using light, confocal, and electron microscopy. A battery of 37 antineurofilament antibodies, characterized as to subunit specificity, epitope domain, and phosphorylation status, was employed to probe substantia nigra Lewy bodies from 15 Parkinson's disease cases. All 37 antibodies labelled Lewy bodies. The epitopes recognized by these antibodies included those in the NF-L rod and tail domains; the NF-M head, rod, and tail domains, as well as epitopes within, and flanking, the multiphosphorylation repeat site; and the NF-H rod domain and multiphosphorylation repeat sites. With these probes, nearly the entire length of each subunit could be demonstrated in Lewy bodies. However, the staining pattern of the Lewy bodies suggested that the tail domains of NF-M and NF-H were present in the periphery of the Lewy body core and in the Lewy body corona, but they appeared to be altered or missing in the center of the Lewy body core. In contrast, the head domain of NF-M, the tail domain of NF-L, and the rod domains of all three subunits are present throughout the Lewy body. These results strongly suggest that the entire extent of each neurofilament subunit is found in Lewy bodies but that the neurofilament subunits may be altered during the processing of these filaments into Lewy bodies. PMID- 1716647 TI - A Golgi study of dendritic development in the dorsal lateral geniculate nucleus of normal ferrets. AB - The development of neurons in the dorsal lateral geniculate nucleus (dLGN) of pigmented ferrets was studied by using the Golgi-Hortega technique. In adult ferrets, four dLGN cell classes were defined on the basis of somatic and dendritic morphology. Classes 1 and 2 were divided into stellate and oriented subtypes. Class 1 and 4 cells are characterized by filiform appendages, class 2 cells by club-like appendages, and class 3 cells by stalked appendages. At birth, dLGN neurons have simple dendritic arbors. During the first postnatal week, dendritic length and proximal branching density increase markedly. By postnatal day 21 (P21), dendritic morphology begins to take on mature characteristics and by the time of eye opening (P30-P35), most neurons can be classified. Also by that time, dLGN cells are covered with abundant filiform appendages. Developmental changes in appendage density were quantified for class 1 stellate cells. These data reveal that appendage density reaches a peak at P56, decreases sharply until P90, and then gradually declines to mature levels by P180. Elaboration and elimination of transient appendages occurs centrifugally; at maturity appendage density remains greater distally. PMID- 1716648 TI - Effect of maternal dietary hexachlorocyclohexane exposure on pup survival and growth in albino rats. AB - In adult albino rats, maternal dietary beta- and gamma-hexachlorocyclohexane (HCH) intake during gestation upto 400 ppm level did not affect the number of litters produced. But about 50 and 100% pup mortality was found in 200 and 400 ppm beta-HCH group within 5 days of birth. Maternal mortality was observed in 800 ppm beta-HCH group during third week of gestation. The effect of maternal dietary intake of HCH isomers at 50 and 250 ppm level during gestation and/or lactation on perinatal development was also studied. The body weights and sizes of the newborn litters of mother rats exposed to dietary HCH isomers did not differ from controls. Similarly, the growth and development of the litters of HCH exposed mother rats that survived 28 day lactation period were found to be comparable to controls as evidenced by the body weight and weight of vital organs. However, liver weight increases were found in the 28 days weaned litters wherever the mothers had been exposed to HCH isomers during lactation. Lowered kidney weight was seen in litters of mother rats fed 250 ppm gamma-HCH during gestation and lactation. The brain and testis weights were not affected in the litters of any experimental groups. PMID- 1716649 TI - Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro. AB - Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of anti-HIV-1 agents. Zidovudine (AZT, 3'-azido-3' deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-alpha A (rIFN-alpha A) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZT-resistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-1 were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFN-alpha A in two-drug regimens against AZT resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT containing regimens may involve inhibition of AZT-sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro. PMID- 1716650 TI - Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies. AB - A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in Mr. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion. PMID- 1716651 TI - Antigenic variation of European haemorrhagic fever with renal syndrome virus strains characterized using bank vole monoclonal antibodies. AB - Monoclonal antibodies (MAbs) against Puumala (PUU) virus, the aetiological agent of nephropathia epidemica, were produced by fusing activated spleen cells from a bank vole (Clethrionomys glareolus) with the mouse myeloma cell line SP2/0. This novel approach, utilizing the natural vector of PUU virus for hybridoma production, proved to be highly efficient, and eight stable PUU virus-specific heterohybridomas were isolated and characterized. The bank vole MAbs were all specific for the nucleocapsid protein (N) of PUU virus, as determined by immunoprecipitation. When evaluated by additivity immunoassays, the MAbs were found to recognize several different, distinct or overlapping, epitopes on N. The MAbs were used in immunofluorescence assays to compare eight PUU-related virus isolates, and the prototype Hantaan, Urban rat and Prospect Hill viruses. The reactivity varied among the different MAbs and could be classified into five groups. One MAb reacted exclusively with PUU-related viruses; two MAbs reacted with all PUU-related virus strains tested, as well as Prospect Hill virus, but did not react with Urban rat virus and Hantaan virus; one MAb reacted with all PUU-related virus strains tested and weakly with Hantaan virus, but not with Urban rat and Prospect Hill viruses; two MAbs reacted with all the virus strains tested. Two virus strains, K-27 and CG-1820, isolated in the western U.S.S.R., were distinguished from the other PUU-related virus strains by two MAbs, suggesting that the large group of independently isolated PUU-related viruses may be more heterogeneous than previously believed. PMID- 1716652 TI - Typing of hepatitis C virus genomes by restriction fragment length polymorphism. AB - Recently, we reported that hepatitis C virus (HCV) can be classified genetically into two types, HCV-K1 and HCV-K2, which show 67% and 71% identity at the nucleotide and amino acid sequence levels in a 340 bp region which encodes the NS5 gene Gly-Asp-Asp motif. To develop a rapid method to classify the genomes of HCV isolates, we identified restriction fragment length polymorphisms (RFLPs) in reverse transcriptase-polymerase chain reaction products encoding a portion of the NS5 gene. AluI and AccII enabled HCV to be classified into the K1 and K2 types, and Sau96I enabled classification into the K1 type, and the K2a and K2b subtypes. These RFLPs also generally allow Japanese isolates to be distinguished from the prototype (PT, an isolate from the U.S.A.), which is a K1 type. Sequence analysis of the 5'-untranslated regions of Japanese isolates revealed near identity between the K1 type and PT, and 93 to 94% identity between the K1 and K2 types, indicating that there are type K1- and K2-specific RFLPs in this region. Our results suggest that the nucleotide sequences of the K1 and K2 types are different throughout the HCV genome. The incidence of HCV types K1, K2a and K2b, and PT in 50 samples was 74%, 16%, 8% and 2%, respectively. PMID- 1716653 TI - The trans-activating C-type retroviruses share a distinct epitope(s) that induces antibodies in certain infected hosts. AB - Using sera from hosts infected with bovine leukaemia virus (BLV), human T cell lymphoma virus types I and II (HTLV-I and -II), or simian T cell lymphoma virus type I (STLV-I), we found that the major gag proteins of these viruses cross react immunologically. The specificity of this cross-reactivity was demonstrated by absorption using purified viral proteins, virus lysates and extracts of infected cells. The data strongly suggested that the cross-reacting epitope(s), referred to as CE, differs from those responsible for cross-reactions between the major gag proteins of HTLV-I, HTLV-II and STLV-I, and between those of BLV and HTLV-I reported previously. The prevalence of antibodies to CE was low, even amongst infected hosts with high titres to other epitopes present in the major gag proteins of the homologous viruses. CE was not detected in any of the other C or D-type retroviruses, or lentiviruses examined. Therefore, it is likely that CE can be used to define serologically a subgroup of C-type retroviruses, the genomes of which display unique features and functional activities. PMID- 1716654 TI - A comparison of WIN 51711 and R 78206 as stabilizers of poliovirus virions and procapsids. AB - The thermal denaturation of poliovirus virions and procapsids in the 42 to 48 degrees C range was studied using N- and H-specific monoclonal antibodies. The half-life of the N antigen of Mahoney and Sabin 1 virions was extended 50- to 250 fold by either 10 microM WIN 51711 or R 78206. The minimum concentrations required for full stabilization at 46 degrees C (1.0 microM for WIN 51711, 0.2 microM for R 78206) were independent of the strain or serotype of the virus; 30 to 60 molecules of stabilizer per virion were required for full protection. R 78206 was the most efficient stabilizer of Mahoney procapsids; the half-life of the N-specific epitopes of these particles at 44 degrees C was extended from less than 1 min to 1 day. PMID- 1716655 TI - Influence of the C terminus of the small protein subunit of bean pod mottle virus on the antigenicity of the virus determined using monoclonal antibodies and anti peptide antiserum. AB - Middle component particles of bean pod mottle virus (BPMV) containing small protein subunits with a cleaved C terminus were used to produce monoclonal antibodies (MAbs). All MAbs were specific for cryptotopes, i.e. epitopes present only on dissociated BPMV protein. The MAbs reacted more strongly with virus protein preparations containing the cleaved form of the small subunit than with preparations containing only the uncleaved form. It seems that the presence of additional residues at the C terminus of the intact small subunit interferes with antibody binding. Antibodies raised against synthetic peptides corresponding to the C terminus of the uncleaved small subunit reacted with both intact virions and dissociated subunits. This C-terminal region seems to play a dominant role in the antigenicity of the virus. PMID- 1716656 TI - The structural polypeptide VP3 of infectious bursal disease virus carries group- and serotype-specific epitopes. AB - Two independent non-overlapping epitopes could be demonstrated on the structural protein VP3 of infectious bursal disease virus by non-neutralizing monoclonal antibodies produced against serotypes I and II. Both serotypes have one epitope in common, whereas the second epitope is distinct for serotype I and serotype II. PMID- 1716658 TI - Measuring symbolic play style in infancy: a methodological approach. AB - Researchers have examined symbolic play style as a qualitative variable distinguishing two groups of children: patterners and dramatists. We have conceptualized style not as a typology of children but as reflecting dimensions of difference between children which are amenable to quantitative analysis. For the construct of style to be invoked, it must be measurable by many variables, it must be observable over time, and it ought to be demonstrably independent of general developmental level. Fifteen boys and 15 girls were each seen twice, once at 21 months of age and again 6 weeks later. The Session 2 pattern of correlations among hypothesized dramatist measures was not entirely consistent with that observed at Session 1; however, three measures, narrative vocalizations, spontaneous play, and elicited-appropriate play were primary dramatist measures at both sessions. PMID- 1716657 TI - A synthetic peptide elicits antibodies reactive with the external glycoprotein of human T cell lymphotropic virus type I. AB - A synthetic peptide derived from the external glycoprotein of human T cell lymphotropic virus type I (HTLV-I) (Env-5; amino acids 242 to 256) reacted with IgG antibodies in serum specimens from HTLV-I-infected individuals. C-terminal residues of Env-5 were crucial for the antibody reactivity. Polyclonal rabbit antibodies to Env-5 did not inhibit syncytium formation but such antibodies reacted specifically with gp68env and gp46env glycoproteins of HTLV-I in an immunoblot analysis. Immunoprecipitation of the surface-labelled MT-2 (HTLV-I infected) cell line with anti-Env-5 precipitated the gp68env precursor protein. It was concluded that peptide Env-5 mimics a surface-exposed epitope on the HTLV I external glycoprotein. PMID- 1716659 TI - Acute toxicity of mosquitocidal compounds to young mosquitofish, Gambusia affinis. AB - Toxicity of Florida mosquito larvicides and adulticides to 3-5 day old Gambusia affinis was determined in the laboratory. After 24-h exposure, the larvicides, temephos, fenoxycarb and petroleum distillates had LC50 values of 5.60, 1.05 and 593.4 ppm, respectively. After 24 h the adulticides resmethrin, fenthion, naled and malathion had LC50 values of 0.007, 2.94, 3.50 and 12.68 ppm, respectively. The only compound toxic to young mosquitofish at maximum field application rates was resmethrin. However, in the light of earlier tests, aerially applied adulticides generally reach the water surface at reduced concentrations and thus probably pose little or no risk to mosquitofish populations. PMID- 1716660 TI - Residual concentration and efficacy of three temephos formulations for control of larval Aedes aegypti. AB - The residual concentration and efficacy of Abate 5CG (impregnated in celatom granules) and plaster pellets impregnated with either Abate 4E or technical temephos were compared against late 3rd-instar Aedes aegypti larvae. Both plaster pellet formulations resulted in 100% larval mortality during the 6-wk test, compared with 2 wk for a similar level of mortality for the celatom formulation. The maximum temephos concentration in water treated with the celatom formulation occurred 30 min after treatment at 0.071 ppm. Temephos concentration of water treated with the 4E plaster formulation peaked at 12 h at 0.148 ppm while the concentration of the technical plaster formulation peaked at 48 h at 0.30 ppm. PMID- 1716661 TI - A monoclonal antibody that reacts immunohistochemically with amyloid deposits in the brain tissue of Alzheimer patients binds to an epitope present on complement factor 4. AB - The mouse monoclonal antibody SMP has previously been demonstrated to react immunohistochemically with neurofibrillary tangles, argyrophilic plaques, and leptomeningeal vascular amyloid deposits in the brain tissue of individuals dying from pathologically diagnosed Alzheimer's disease. In preliminary studies the antibody was shown, by size exclusion chromatography, to bind to a protein with an apparent molecular mass of 260 kDa present in the CSF and serum of demented individuals. Chromatographic separation of a 40% ammonium sulphate precipitate of CSF and serum yielded immunoreactive fractions that were subjected to 9% sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by western blotting. Probing the nitrocellulose blot with the antibody revealed that the antibody selectively binds to a protein chain with an apparent molecular mass of 100 kDa. By using a combination of affinity chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis, coupled with western blotting, the serum component with which the antibody reacts has been identified as complement factor 4. In addition, the antibody has been shown to react specifically with an epitope on the alpha-chain of this protein. PMID- 1716662 TI - Effect of tetrahydrobiopterin on serotonin synthesis, release, and metabolism in superfused hippocampal slices. AB - The effects of 6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R-BH4 did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KCl. Under the same incubation conditions, 6R-BH4 altered neither the synthesis (basal or tryptophan-stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6 methyl-5,6,7,8-tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R-BH4, did not. The 6S isomer of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R-BH4 in stimulating serotonin release. 6R-BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R-BH4, like that produced by p-chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KCl-induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R-BH4 increases the depolarization induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R-BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase. PMID- 1716663 TI - Astroglial-induced in vitro angiogenesis: requirements for RNA and protein synthesis. AB - Astrocytes are believed to affect microvascular endothelial cell differentiation in brain and retina. Bovine retinal microvessel endothelial cells formed capillary-like structures when cocultured with C6 astroglial cells or in the absence of C6 cells in response to the reconstituted basement membrane protein Matrigel. Using quantitative computer-assisted image analysis, the requirements for RNA and protein synthesis in these two complementary models of in vitro microvessel morphogenesis were examined. Astroglial-dependent capillary-like structure formation was inhibited by up to 87% in a dose-dependent fashion by cycloheximide (0.01-0.1 micrograms/ml), puromycin (0.1-0.25 micrograms/ml), and actinomycin D (0.01-0.025 micrograms/ml). In contrast, the astroglial-independent process in response to Matrigel was not affected by these metabolic inhibitors. These findings suggest that capillary-like structures form in response to astroglial cells in two distinct sequential stages. The first consists of inductive astroglial-endothelial interactions requiring both RNA and protein synthesis. This initiates endogenous endothelial morphogenic events that do not appear to require RNA or protein synthesis, consistent with posttranslational regulatory mechanisms. The first astroglial-dependent step is relevant to the regulation of microvessel formation in brain and retina, whereas the second may represent a morphogenic pathway common to microvessel formation in many tissues. PMID- 1716664 TI - An ion channel locus for the protein kinase C potentiation of transmitter glutamate release from guinea pig cerebrocortical synaptosomes. AB - The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+) dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels. PMID- 1716665 TI - Plasma exchange in the treatment of Refsum's disease (heredopathia atactica polyneuritiformis). AB - Five cases of heredopathia atactica polyneuritiformis (HAP--Refsum's disease) were treated by serial plasma exchanges. In all patients a reduction in calorie intake and body weight had been associated with a rise in plasma phytanic acid, followed by an exacerbation of the ataxia and neuropathy. Lowering the plasma phytanic acid by plasma exchange produced a rapid clinical improvement. The main indication for plasma exchange in HAP is a severe or rapidly worsening clinical condition. A lesser indication is failure of dietary management to reduce a high plasma phytanic acid level. PMID- 1716666 TI - Experimental superficial siderosis of the central nervous system. I. Morphological observations. AB - Autologous washed red blood cells were injected weekly over a period of three to six months into the cisterna magna of adult New Zealand white rabbits. After three months, the surface of the brain stem, cerebellum, and piriform cortex showed a distinct brown color, and staining of the gross specimens for iron produced an intense blue color which extended for a distance of 1-2 mm into the brain parenchyma. Enhanced iron stains of vibratome sections revealed the accumulation of reaction product in microglia and Bergmann glia of the cerebellar cortex, and in microglia and astrocytes of the piriform cortex. Ferritin immunocytochemistry revealed reaction product in cerebellar microglia and Bergmann glia which strongly resembled that obtained by the enhanced iron stain. In the piriform cortex, only microglia were reactive with anti-ferritin. Electron microscopy confirmed the accumulation of electron-dense ferritin granules only in the cytoplasm of microglia. Bergmann glia in the cerebellum and astrocytic processes in the piriform cortex were replete with intermediate filaments and contained an excess of glycogen. After six months, small granules of hemosiderin began to appear in cerebellar and piriform cortices. The observations support that the sequence of conversion of hemoglobin to ferritin and hemosiderin occurs in brain as in other organs. PMID- 1716667 TI - Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. I. Production following colchicine application to the sciatic nerve. AB - Neurofilament (NF) triplet proteins are normally poorly phosphorylated in neuronal perikarya, the two high molecular weight polypeptides becoming extensively phosphorylated once the NF enters the axon. Abnormal expression of phosphorylated NF (pNF) epitopes in neuronal perikarya has been revealed using monoclonal antibodies in a variety of human and experimental conditions. In the present study, we asked whether pNF epitopes are expressed in sensory neurons in the L4 and L5 dorsal root ganglia (DRG) following blockade of fast axonal transport in a model producing few (less than 1%) degenerating fibers. Colchicine (5 mM) was briefly (45 minutes) applied to the sciatic nerve at mid-thigh twice (once weekly) and the animals studied two weeks following the first colchicine application; contralateral nerves were either treated with saline or crushed. Modest to intense immunoreactivity was found with antibody 07-05 (directed against pNF epitopes on the two high molecular weight NF polypeptides) in 30.4% and 45.1% of DRG neurons from colchicine-treated and crushed nerves, respectively; only a rare cell body demonstrated immunostaining from the contralateral saline-treated nerves. Immunoreactivity was not observed with antibody 07-05 at two and five days following single colchicine application. In a separate study, colchicine or saline was applied (as above) 1-2 cm proximal to a nerve crush. Colchicine application did not influence the extent of DRG neurons expressing pNF epitopes; immunostaining with antibody 07-05 was present in 44.7% and 43.8% of DRG neurons from saline-treated and colchicine-treated crushed nerves, respectively. The results indicate that structural interruption of nerve target contact is not necessary to induce aberrant NF phosphorylation in neuronal perikarya. It is suggested that loss of a retrogradely transported "trophic" signal(s) triggers this response. PMID- 1716668 TI - Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. II. Correlation with continued axonal elongation following axotomy. AB - Neurofilaments (NF) are normally poorly phosphorylated in neuronal perikarya and highly phosphorylated in axons. Aberrant NF phosphorylation in the neuronal perikaryon has been demonstrated in a number of human and experimental disorders. In this study, we have asked whether expression of these phosphorylated NF (pNF) epitopes is dependent upon continued axonal regeneration following nerve transection (axotomy). This hypothesis was tested using the neurotoxic chemical acrylamide (AC) which is known to inhibit axonal regeneration following systemic administration. First, we examined whether AC acts at the level of the neuronal perikaryon to inhibit axonal elongation. Systemic, high dose intraperitoneal (IP) AC administration totalling 150 mg/kg (75 mg/kg x 2) did not impair the axotomy induced reordering of slow axonal transport in the neuronal perikaryon. Next, we studied the ability of AC to directly prevent nerve outgrowth at the growing tips of axons. Subperineurial injection of AC (0.1 M), which in preliminary studies was found not to produce nerve fiber damage, markedly reduced the extent of nerve outgrowth when injected proximal to a nerve crush; this was shown by a reduction in the extent of radiolabeling and number of axonal sprouts in the distal stump seven days following nerve crush. Using this protocol, a 67% decrease in the number of neuronal perikarya in the L4 and L5 dorsal root ganglia demonstrating immunoreactivity to antibody 07-05 (directed against pNF epitopes) was observed in AC-injected compared to contralateral saline-injected crushed nerves. Taken together, the results indicate that inhibition of axonal regeneration in the distal stump by AC reduces aberrant NF phosphorylation in the neuronal perikaryon following axotomy. PMID- 1716669 TI - Comparative histology of experimental allergic neuritis induced with minimum length neuritogenic peptides by adoptive transfer with sensitized cells or direct sensitization. AB - Using synthetic peptides representing specific regions of the bovine myelin protein P2, the minimum peptide length requirement for the T-cell epitope necessary for successful production of experimental allergic neuritis (EAN) has been shown to involve residues 61-70 of the P2 protein. In this study, morphometric analysis was used to compare the histologic changes in sciatic nerves of Lewis rats after disease was induced by P2 specific neuritogenic T-cell lines (P(2)3) or, alternatively, by direct inoculation of synthetic peptides representing residues 60-70 or 61-70 of the P2 protein sequence. Inoculation with cell line P(2)3 stimulated with residue 61-70 failed to elicit demyelination in sciatic nerves. However, cells stimulated with residue 60-70 produced inflammation, endoneurial edema, mild demyelination and axonal degeneration within seven days. In contrast, disease induced with either peptide by direct sensitization was more severe. Morphometric analysis revealed that inflammation was most severe in animals sensitized to the decapeptide. In the sciatic nerve, axonal degeneration was proportional in frequency to the extent of inflammation. Inflammation was especially intense in spinal roots with extensive destruction of axons including unmyelinated fibers. Spinal root changes were associated with Wallerian degeneration in the posterior white matter tracts of the spinal cord and were apparently secondary to endoneurial inflammation. Disruption of the blood-nerve-barrier (BNB), evident as physical separation of endothelial cells in association with severe perivascular inflammation, was observed. PMID- 1716670 TI - Longterm remission of multiple brain metastases with tamoxifen. AB - A case of multiple brain metastases from breast carcinoma treated with tamoxifen is described. She remained in remission for 58 months and is still alive after 82+ months of hormone therapy. The case indicates that hormonal treatment may be very effective in the management of brain metastases from breast carcinoma. PMID- 1716671 TI - Acute effects of human recombinant tumor necrosis factor-alpha on the cerebral vasculature of the rat in both normal brain and in an experimental glioma model. AB - The effects of intravenous (IV) infusion of human recombinant tumor necrosis factor-alpha (rTNF-alpha, Cetus) on normal brain and malignant glioma in rats were examined. Twelve Fischer 344 rats were given either a single injection of 10(6) U rTNF-alpha or injections of 5 x 10(5) U rTNF-alpha for three days. One day post-rTNF-alpha injection(s), rats were injected IV with horseradish peroxidase (HRP) to determine blood-brain barrier (BBB) breakdown and, one hour later, were perfused with an aldehyde fixative and processed for histologic examination. Treatment of normal rats with rTNF-alpha by either dosage or schedule caused no remarkable histopathologic changes in the brain and no alteration in BBB integrity. Human glioma models were produced by intracerebal inoculation of 10(4) syngeneic RT-2 glioma cells into the right parietal lobe of 30 rats. Animals received single IV injections of 10(6) U human rTNF-alpha or its excipient (TNF-E) as above on day 3, 7, or 10 post-tumor inoculation or multiple injections of 5 x 10(5) U rTNF-alpha beginning on day 7, 10, or 12 post-tumor inoculation. With a single IV injection of either rTNF-alpha or its excipient, 3 day models showed a similar pattern of HRP extravasation limited to the extracellular space of the tumor inoculation site. In 7-day models treated with a single IV injection of rTNF-alpha or TNF-E, HRP extravasated throughout the tumor, but did not exceed peritumoral margins. In 10-day models treated with a single injection of TNF-E, HRP was found only in the tumor and immediate peritumoral regions while rTNF-alpha-treated rats showed more extensive areas of BBB breakdown with HRP evident throughout the entire right hemisphere and extending via the corpus callosum into the contralateral hemisphere. Pericapillary halos were also evident around the small blood vessels within the edematous areas of the corpus callosum. Within tumors, hemorrhagic necrosis and adherence of neutrophils to vessels was observed only in animals treated with rTNF-alpha at 10 days post-tumor inoculation. Multiple IV injections of rTNF alpha in 7 and 10-day models triggered widespread hemorrhagic necrosis, neutrophil adherence and infiltration in the tumor. There was also extravasation and diffusion of HRP from the site of glioma into the contralateral hemisphere. Twelve-day models treated with multiple rTNF-alpha injections, in addition, showed irregular luminal surfaces and gaps between adjacent endothelial cells of tumor vasculature.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716672 TI - Development of subrenal capsule tumours following transplantation of rat RN6 multicell spheroids. AB - We describe here the growth rate, morphology and ultrastructure of the RN6 neurinoma spheroids in culture and after syngeneic transplantation under the renal capsule of adult BDIX rats, where sequential growth and tumour angiogenesis studies were performed. The tumour cells in solid avascular multicell spheroids (MTS) and in subrenal capsule tumours (SCT) showed similar morphology and ultrastructure. The vascularization of spheroids started 2 to 3 days after implantation under the renal capsule from proliferating venule-like vessels of the kidney parenchyma. The MTS were soon densely penetrated by hairpin loops of newly formed capillaries. Such vascularized spheroids grew rapidly. The morphology, ultrastructure and permeability characteristics of newly formed tumour vessels and their relationship to the development of tumour necrosis are described. This syngeneic model of subrenal capsule transplantation of neurogenic tumour spheroids appears to be useful for studies of the mechanisms of tumour angiogenesis, tumour necroses and the host response to malignant tumours of the nervous system. PMID- 1716673 TI - [Estimation of left ventricular diastolic characteristics in patients with myocardial ischemia by pulsed Doppler echocardiography: transmitral blood flow velocity in postextrasystolic beats]. AB - To investigate diastolic dynamics of the left ventricle in postextrasystolic (PES) beats during myocardial ischemia induced by rapid atrial pacing (RAP), transmitral blood flow velocity was evaluated using pulsed Doppler echocardiography in 13 subjects. The subjects consisted of eight patients with ischemic heart disease and five healthy subjects. An atrial extrasystole was artificially induced before and immediately after RAP, while the transmitral flow velocity was recorded continuously. The ratio of the early peak diastolic velocity to the peak atrial velocity (A/E) on PES beat was less than that in a basic beat without an extrasystole. The A/E increased in the PES beat in four patients who experienced myocardial ischemia; whereas, other patients and the normals showed a decrease in the A/E in the PES beat even after RAP. These findings indicated that left ventricular dynamics coupled with left atrial contraction are quite different in myocardial ischemic and nonischemic situations, and that postextrasystolic potentiation may be a useful means of detecting the ventricular diastolic dysfunction. PMID- 1716674 TI - Factors released from endocardium of the ferret and pig modulate myocardial contraction. AB - 1. In isolated heart muscle preparations, selective removal of the endocardium results in a characteristic and unusual negative inotropic effect. Possible mechanisms for this effect were investigated in this study. 2. In endocardium intact preparations of ferret papillary muscle, 8-bromo-cyclic GMP, sodium nitroprusside, atrial natriuretic peptide (ANP) and substance P each induced changes in contractile behaviour similar to selective endocardial removal, and each significantly elevated myocardial cyclic GMP levels. Substance P failed to elevate myocardial cyclic GMP levels following removal of endocardium or in the presence of haemoglobin, suggesting that it may act by releasing endothelium derived relaxing factor (EDRF) from endocardium. However, there was no change in myocardial cyclic GMP levels following endocardium removal alone. 3. In cascade bioassay experiments, it was confirmed that porcine cultured endocardial cells released an unstable humoral agent whose effects on an endothelium-denuded pig coronary artery were indistinguishable from EDRF. 4. The negative inotropic effects of endocardium removal were reversed in bioassay experiments where an endocardium-denuded papillary muscle was exposed to the effluent from a column of porcine cultured endocardial cells on microcarrier beads. This demonstrates for the first time the release of a 'contraction prolonging factor' from endocardium, the tonic release of which would explain the negative inotropic effect of endocardium removal. 5. It is concluded that elevation of ferret papillary muscle cyclic GMP (as for example with EDRF) produces changes in contractile performance similar to those induced by endocardium removal. We also demonstrate that superfused porcine cultured endocardial cells release a humoral agent (provisionally named 'endocardin') which causes reversal of the changes in mechanical properties seen after endocardial removal. PMID- 1716676 TI - Monovalent cation conductance in the ryanodine receptor-channel of sheep cardiac muscle sarcoplasmic reticulum. AB - 1. The ryanodine receptor protein of sheep cardiac muscle sarcoplasmic reticulum membranes functions as a ligand-regulated ion channel following solubilization with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1- propane sulphonate); purification by density gradient centrifugation, reconstitution into proteo-liposomes and incorporation into planar phospholipid bilayers. 2. In the absence of divalent cations, measurable conductance is observed with the group 1a cations and with some larger organic cations. In symmetric 210 mM solutions the following conductance sequence was determined: K+ greater than Rb+ = NH4+ greater than Na+ = Cs+ greater than Li+ much greater than Tris+. 3. Other organic cations, e.g. TEA+, do not produce measurable current under these conditions. 4. Single-channel conductance saturates with increasing ionic activities of K+, Na+ and Li+. Saturation curves are described by Michaelis Menten kinetic schemes with the following values of maximal conductance and apparent dissociation constant: K+ 900 pS, 19.9 mM; Na+ 516 pS, 17.8 mM; Li+ 248 pS, 9.1 mM. 5. The channel displays only minor differences in permeability amongst the group 1a cations. Relative permeability, monitored under bi-ionic conditions, yields the following sequence: Na+, 1.15 greater than K+, 1.00 = Li+, 0.99 greater than Rb+, 0.87 greater than Cs+, 0.61. Under similar conditions the permeability ratio of NH4+ to K+ was found to be 1.32 and that for Tris+ to K+ was 0.22. 6. The K+ conductance is reduced by low concentrations of the impermeant cation TEA+. Block appears as a smooth reduction in single-channel current amplitude and the degree of block is dependent upon applied voltage. These observations are consistent with a single-site blocking scheme in which TEA+ has access to a site within the voltage drop of the channel from only the cytosolic face of the channel protein and interacts with a site located approximately 90% of the electrical distance across the channel. The zero-voltage dissociation constant for TEA+ block is 50 mM. 7. Single-channel conductance measurements in mixtures of K(+)-Na+ and K(+)-Li+ reveal no anomalous behaviour as the mole fraction of the ions is varied. 8. With monovalent cations as permeant species, the sheep cardiac sarcoplasmic reticulum ryanodine receptor protein functions as a poorly selective, ligand-regulated channel. Under the conditions described here the channel functions as a single-ion pore. It is proposed that discrimination is largely dependent upon the strength of interaction of the permeant ion with a binding site in the conduction pathway. PMID- 1716675 TI - Dopamine actions on calcium currents, potassium currents and hormone release in rat melanotrophs. AB - 1. Intracellular and whole-cell recordings were made from primary cultures of rat intermediate pituitary cells; beta-endorphin secretion was also measured by radioimmunoassay. The effects of dopamine receptor activation on hormone secretion, calcium currents and resting potassium conductance were compared. 2. Spontaneous sodium-dependent action potentials occurred in 82% of cells recorded with intracellular microelectrodes and 64% of cells recorded with whole-cell patch electrodes; the same proportion of cells showed spontaneous calcium dependent depolarizations in the presence of tetrodotoxin. 3. Calcium currents recorded from holding potentials of -90 or -70 mV showed transient and sustained components, both of which activated at -40 mV and had similar current-voltage relations. Bay K 8644 (1 microM) increased both components by about 130% while nifedipine (1-10 microM) decreased them by a maximum of 30%. Nickel (500 microM) inhibited transient and sustained components by 68 and 50%; cadmium (100 microM) abolished the current. omega-Conotoxin (1 microM) reversibly inhibited the transient component by 26%. 4. The dopamine D2 receptor agonist, quinpirole (0.1 10 microM) inhibited transient and sustained components in all cells by a maximum of 40 and 25% respectively. Quinpirole did not alter the time course of the current. 5. Quinpirole (1-100 nM) hyperpolarized 90% of cells from which intracellular recordings were made and 55% of cells recorded from with whole-cell patch pipettes. Maximum hyperpolarization of 16 +/- 4 mV from a resting potential of -44 +/- 5 mV was observed with 100 nM-quinpirole; concentration producing half maximal effect was 3 nM. The hyperpolarization resulted from an increase in potassium conductance. 6. Quinpirole (1-100 nM) decreased basal beta-endorphin secretion by 55% and abolished secretion stimulated by Bay K 8644 or isoprenaline; concentrations producing half-maximal inhibitions were 5-10 nM. Tetrodotoxin (1 microM), nifedipine (1 microM), nickel (500 microM) and cadmium (100 microM) did not alter basal or stimulated secretion although higher concentrations of cadmium did inhibit stimulated hormone release. 7. Pertussis toxin pre-treatment prevented all actions of quinpirole. 8. Thus, concentrations of quinpirole that abolished stimulated hormone secretion did not alter calcium currents; conversely, concentrations of calcium channel blockers that partially or completely inhibited calcium currents did not alter basal or stimulated secretion. These results may indicate that calcium influx through the voltage dependent calcium channels measured in these experiments does not contribute significantly to hormone release from melanotrophs.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716677 TI - Modulation of noradrenaline-induced membrane currents by papaverine in rabbit vascular smooth muscle cells. AB - 1. The modulation of noradrenaline-induced membrane currents by papaverine was studied with the nystatin whole-cell technique in freshly isolated rabbit ear artery and portal vein cells. 2. At a holding potential of -50 mV (in potassium free conditions), papaverine inhibited the Ca(2+)-activated inward chloride current ICl(Ca) evoked by ionophoretic application of noradrenaline in rabbit portal vein cells in a concentration-dependent (10(-5)-10(-4) M) manner. 3. With potassium-containing solutions (at 0 mV) 10(-4) M-papaverine reversibly abolished the Ca(2+)-activated outward potassium current IK(Ca) induced by noradrenaline in both protal vein and ear artery cells. Occasionally, 10(-4) M-papaverine itself activated IK(Ca) before it abolished the response to noradrenaline. 4. Higher concentrations of papaverine (up to 10(-3) M) always evoked both ICl(Ca) and IK(Ca) at -50 and 0 mV respectively, before it abolished these responses to noradrenaline in both ear artery and portal vein cells. In the presence of 10(-3) M-papaverine higher doses of noradrenaline evoked the non-selective cation current in some portal vein cells. 5. In the presence of 10(-2) M-caffeine, which elicited both IK(Ca) and ICl(Ca), papaverine (10(-3) M) failed to activate either current and 10(-3) M-papaverine reduced the caffeine-induced IK(Ca) by 94%. In contrast, IK(Ca) induced by the calcium ionophore ionomycin was unaffected by papaverine. In Ca(2+)-free, 1 mM-EGTA external solutions, 10(-3) M-papaverine still induced IK(Ca) and blocked the response to noradrenaline. 6. The alpha 1 adrenoceptor antagonist prazosin (5 x 10(-8) M) completely blocked the noradrenaline-induced IK(Ca) but currents evoked by papaverine (10(-3) M) were unaffected by prazosin. 7. It was concluded that papaverine modulated noradrenaline-induced membrane currents in vascular smooth muscle cells by releasing and depleting caffeine-sensitive intracellular Ca2+ stores. PMID- 1716678 TI - Ca(2+)-activated K+ channels in rat thymic lymphocytes: activation by concanavalin A. AB - 1. The role of ion channels in the mitogenic response of rat thymic lymphocytes to concanavalin A (ConA) was studied using single-channel patch-clamp recordings and measurements of membrane potential with the fluorescent probe bis-oxonol. 2. ConA (20 micrograms ml-1) evoked a rapid membrane hyperpolarization; Indo-1 measurements indicated a concurrent increase in [Ca2+]i. The hyperpolarization was blocked by cytoplasmic loading with the Ca2+ buffer BAPTA (bis(O-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid), or charybdotoxin, a component of scorpion venom known to block K+ channels in lymphocytes. 3. Cell-attached patch clamp recordings showed that both ConA and the Ca2+ ionophore ionomycin activated channels with high selectivity for K+. Two conductance levels were observed -6-7 pS and 17-18 pS-measured as inward chord conductance at 60 mV from reversal potential (Erev) with 140 mM-KCl in the pipette. The current-voltage relationship for the larger channel displayed inward rectification and channel open probability was weakly dependent upon membrane potential. 4. These experiments provide the first direct evidence for mitogen-activated Ca(2+)-gated K+ channels (IK(Ca)) in lymphocytes. This conductance is relatively inactive in unstimulated rat thymocytes but following the intracellular Ca2+ rises induced by ConA, IK(Ca) channels are activated and produce a significant hyperpolarization of the cell potential. PMID- 1716679 TI - Multiple effects of tetraethylammonium on N-methyl-D-aspartate receptor-channels in mouse brain neurons in cell culture. AB - 1. The mechanisms of tetraethylammonium (TEA) antagonism of N-methyl-D-aspartate (NMDA) responses were investigated in cultured mouse cortical neurons by analysing single-channel and whole-cell currents from patch clamp recordings. TEA (1-5 mM) decreased whole-cell NMDA responses. Kainate and quisqualate receptor mediated responses were unaffected at these TEA concentrations. 2. In whole-cell recordings, increasing the NMDA concentration while keeping the TEA concentration constant resulted in greater inhibition by TEA. Thus, TEA-mediated inhibition of NMDA responses was not due to competitive antagonism, and the greater inhibition by a single dose of TEA as NMDA concentration was elevated indicated some form of non-competitive inhibition. In single-channel recordings, two inhibitory effects were seen in 1-5 mM-extracellular TEA: single-channel conductance (gamma) was decreased, and the frequency of channel events was decreased. These effects were not accompanied by any change in average channel open time. 3. Single-channel current-voltage (I-V) curves obtained in 2, 5, 10 and 30 mM-TEA indicated the decrease in NMDA channel conductance was voltage dependent with larger reduction occurring as patches were hyperpolarized. The data were well fitted by the Woodhull model with the dissociation constant (KD) showing an e-fold increase in inhibition for a 43-45 mV change in membrane potential. The 0 mV KD was 45 mM-TEA decreasing to about 11 mM at -60 mV. The TEA block site appeared to sense approximately 60% of the transmembrane potential field (delta = 0.6) for extracellular application of TEA. 4. The decrease in channel opening frequency seen in TEA was concentration dependent and generally more sensitive to extracellular TEA than the channel block effect. There was a 50% reduction in the number of NMDA channel openings observed in 5 mM-TEA. Increasing either NMDA or glycine concentrations in constant TEA concentration caused an additional decrease in the frequency of NMDA channel opening. In contrast to extracellular TEA, intracellular TEA had no noticeable effect on open-state probability. 5. NMDA single-channel currents were observed at positive potentials after completely replacing pipette Cs+ by 140 mM-TEA-Cl indicating TEA could serve as a current carrier through NMDA channels. Single channel I-V curves obtained with pipettes containing 70 or 140 nM-TEA in place of equivalent amounts of Cs+ were fitted by the Goldman-Hodgkin-Katz (GHK) equation over the range of -80 to +70 mV assuming a permeability of 0.45 compared with a Cs+ permeability of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716681 TI - Spontaneous and clastogen induced chromosomal breakage in scleroderma. AB - Spontaneous and clastogen induced chromosomal damage was evaluated in lymphocytes of 10 patients with scleroderma and 21 controls. Peripheral blood lymphocytes from patients with scleroderma not only had elevated rates of spontaneous chromosomal damage (p = 0.002), but also bleomycin induced (p = 0.041), streptonigrin induced (p = 0.035), and 4-nitroquinoline-1-oxide induced (p = 0.032) chromosomal breakage. Our findings suggest that scleroderma lymphocytes may have a generalized susceptibility to DNA damage caused by free radicals. PMID- 1716680 TI - Conductance and kinetic properties of single nicotinic acetylcholine receptor channels in rat sympathetic neurones. AB - 1. The unitary conductance of nicotinic acetylcholine (ACh) receptor channels in rat sympathetic neurones has been studied. Conductance estimates varied from 26 48 pS with a mean of 36.8 pS in 1 mM-Ca2+. The main conductance level varied from patch to patch and the presence (or absence) of additional conductance levels also varied. 2. The channels showed large open channel noise and experiments with 300 mM-NaCl in the patch pipette substantially increased the open channel noise. The appearance of detectable step-like transitions within this noise strongly suggested the existence of closely spaced discrete levels. 3. Removal of divalent cations from the external solution increased the unitary channel conductance. Altering the main permeant ion in divalent-free solutions gave the following conductance sequence: K+ (93 pS) greater than Cs+ (61 pS) greater than Na+ (51 pS) greater than Li+(23 pS). 4. Replacement of Na+ by Cs+ in the external solution considerably reduced the current evoked by ACh in whole-cell recordings and the channel-opening frequency in outside-out patches. 5. The kinetic properties of channels activated by ACh and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) were also studied. At low concentrations of ACh and DMPP the gap distributions were complex and best fitted by the sum of four exponential components. Individual activations (bursts) were interrupted by the two shortest closed periods the briefer of which had time constants of 36 microseconds for ACh and 67 microseconds for DMPP. 6. The distribution of burst lengths had two components for each agonist, each component making up about 50% of the total area under the distribution. For ACh, the time constant of the longer component (12.2 ms) was similar to the decay time constant of excitatory postsynaptic potentials (EPSCs) at similar temperature and potential. For DMPP the time constant of the longer component was 17.6 ms. 7. The relative number of brief gaps per long burst was much larger for ACh than for DMPP. Therefore the corrected mean open time for ACh (0.86 ms) was much shorter than that for DMPP (2.3 ms). 8. In terms of receptor mechanism, the values of the channel opening equilibrium constant (beta/alpha) estimated from these numbers (ACh, 23; DMPP, 25) suggest that both agonists are efficaceous. 9. DMPP is a potent blocker of the channel with an equilibrium dissociation constant (KB) of around 50 microM and blockage gaps of around 1 ms duration. ACh also blocks the channel but with a higher KB of around 470 microM.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716682 TI - Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists. AB - Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950 2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini. PMID- 1716683 TI - 2-Pyridinone derivatives: a new class of nonnucleoside, HIV-1-specific reverse transcriptase inhibitors. PMID- 1716684 TI - NMR studies of an FK-506 analogue, [U-13C]ascomycin, bound to FKBP: conformation and regions of ascomycin involved in binding. PMID- 1716685 TI - A comparison of probes for restriction fragment length polymorphism (RFLP) typing of Legionella pneumophila serogroup 1 strains. AB - The use of probes derived from rRNA sequences to detect restriction fragment length polymorphisms (RFLPs) associated with the ribosomal RNA genes for epidemiological typing (ribotyping) is a powerful and readily applicable tool. Different probes and enzymes for ribotyping were compared for a series of 73 unrelated Legionella pneumophila serogroup 1 strains. The probes compared were cDNAs, transcribed from L. pneumophila or Escherichia coli rRNA subunits, and a cloned L. pneumophila rRNA gene. The cloned rRNA gene probe gave the best discrimination and this probe was further compared with cloned probes comprised of randomly selected (non-rRNA) parts of the L. pneumophila chromosome. In this instance the greatest discrimination was achieved when one of the non-ribosomal RNA gene probes was employed. The overall discrimination of RFLP typing was enhanced by combining the data obtained with both rRNA and non-rRNA probes. PMID- 1716686 TI - Comparison of calcium release from sarcoplasmic reticulum of slow and fast twitch muscles. AB - The mechanism of Ca2+ release from the sarcoplasmic reticulum (SR) of slow and fast twitch muscle was compared by examining biochemical characteristics, ryanodine binding, Ca2+ efflux, and single Ca2+ channel properties of SR vesicles. Although many features of the Ca2+ release channel were comparable, two functional assays revealed remarkable differences. The comparable properties include: a high molecular weight protein from both types of muscle was immunologically equivalent, and Scatchard analysis of [3H]ryanodine binding to SR showed that the Kd was similar for slow and fast SR. In the flux assay the sensitivity to the agonists caffeine, doxorubicin, and Ca2+ and the antagonists Mg2+, ruthenium red, and tetracaine differed only slightly. When SR vesicles were incorporated into lipid bilayers, the single-channel conductances of the Ca2+ release channels were indistinguishable. The distinguishing properties are: When Ca2+ release from passively 45Ca(2+)-loaded SR were monitored by rapid filtration, the initial rates of Ca2+ release induced by Ca2+ and caffeine were three times lower in slow SR than in fast SR. Similarly, when Ca2+ release channels were incorporated into lipid bilayers, the open probability of the slow SR channel was markedly less, mainly due to a longer mean closed time. Our results indicate that slow and fast muscle have ryanodine receptors that are biochemically analogous, yet functional differences in the Ca2+ release channel may contribute to the different time to peak contraction observed in intact slow and fast muscles. PMID- 1716687 TI - An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing. AB - Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus. PMID- 1716689 TI - Zidovudine-resistant human immunodeficiency virus selected by passage in cell culture. AB - Variants of human immunodeficiency virus (HIV) with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine) have been selected by passage of virus in cell culture in the presence of drug. Wild-type, sensitive virus became partially resistant to zidovudine by passage 12 (50% inhibitory dose values measured in HeLa CD4+ cells increased from 0.014 to 0.2 microM), and genetic analysis using the polymerase chain reaction revealed that mutations in the reverse transcriptase coding region identical to those seen in clinical isolates from treated individuals had occurred. The order of appearance of these resistance mutations in passaged virus was also similar to that in clinical isolates. The partially resistant strain, HIVRTMC/F, became highly zidovudine resistant by passage 12 (50% inhibitory dose values increased from 0.4 to 2.5 microM during passages 7 to 11). Nucleotide sequence analysis of the reverse transcriptase from this variant revealed a novel amino acid substitution (Lys----Glu) at codon 219. A different substitution at this codon (Lys----Gln) had been seen previously in clinical isolates. When this mutation was created in HIVRTMC/F by site-directed mutagenesis, the resulting partially resistant virus became highly resistant, thus confirming the significance of this change. In view of the possibility that this mutation might occur in HIV isolates during treatment of patients, we adapted our selective polymerase chain reaction procedure to enable screening for this change in clinical samples. The virus passage procedure described here may be useful for gaining further insight into the mutational events occurring during the development of resistance to zidovudine and other HIV inhibitors. PMID- 1716688 TI - Characterization of the bovine herpesvirus 4 major immediate-early transcript. AB - The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain. PMID- 1716690 TI - Mutational analysis of the simian immunodeficiency virus SIVmac nef gene. AB - We are using site-directed mutagenesis of single viral genes to identify and analyze the genetic determinants of human and simian immunodeficiency virus pathogenicity. In a first approach, we have constructed a series of simian immunodeficiency virus SIVmac nef mutants by partial deletion and insertions in the nef gene, as this gene is a candidate gene for the establishment and maintenance of latency. nef insertion mutants replicated faster than wild-type SIVmac, suggesting that the nef gene product acts as a negative factor for replication. Surface phenotyping revealed that cultures permanently infected with nef mutants exhibit an enhanced expression of viral proteins on the outer cell surface. We have analyzed the properties of the mutant viruses in cell culture and intend to use rapidly replicating mutants (putatively unable to undergo latency) as model vaccine viruses in the rhesus monkey. PMID- 1716691 TI - Characterization of two distinct major histocompatibility complex class I Kk restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin. AB - Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes. PMID- 1716692 TI - Nucleotide sequence and expression of the capsid protein gene of feline calicivirus. AB - The sequence of the 3'-terminal 2,486 bases of the feline calicivirus (FCV) genome was determined. This region of the FCV genome, from which the 2.4-kb subgenomic RNA is derived, contained two open reading frames. The larger open reading frame, found in the 5' end of the subgenomic mRNA, contained 2,004 bases encoding a polypeptide of 73,467 Da. The smaller open reading frame, encoded in the 3' end of the mRNA, was composed of 318 bases, encoding a polypeptide of 12,185 Da. The AUG initiation codon of the second open reading frame overlapped the UGA termination codon of the first, with the sequence AUGA. The nucleotide sequence of the region containing this overlap resembles the -1 frameshift sequences of the retroviruses. The 5' end of the 2.4-kb subgenomic RNA was mapped by primer extension analysis. There were two apparent transcription initiation points, both of which were 5' to the AUG initiation codon of the large open reading frame. Transcription from these sites yielded RNA transcripts with 5' nontranslated leader regions of 17 and 18 bases. The total length of the 2.4-kb subgenomic RNA was 2,375 bases (from the 5'-most start site) excluding the poly(A) tail. Edman degradation of the purified capsid protein of FCV showed that the capsid protein was encoded by the large open reading frame. Western immunoblot analysis of FCV-infected cells using a feline anti-FCV antiserum demonstrated that translation of the capsid protein was detectable at 3 h postinfection and continued to accumulate until 8 h postinfection, the last time examined. PMID- 1716693 TI - Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies. AB - We identified and mapped the regions responsible for neutralization in the human parvovirus B19 structural protein by using region-specific human antibodies derived from seropositive blood donors. The region-specific antibodies were purified by using affinity columns coupled with synthetic peptides of the hydrophilic regions including the beta-turn structure deduced by the predicted secondary structure of VP2. Fifteen highly specific antibodies against the synthetic peptides were obtained. Ten of them were able to precipitate the radiolabeled virus. Six of them proved to be able to protect the colony-forming unit erythroid cells in human bone marrow cell cultures from injury by the virus. The sequences recognized by the six neutralizing antibodies were sites corresponding to amino acids 253 to 272, 309 to 330, 325 to 346, 359 to 382, 449 to 468, and 491 to 515 from the amino-terminal portion of VP2. These observations suggest that the neutralizing epitopes were distributed in the region from amino acid 253 in the amino-terminal portion of VP2 to the carboxyl terminus of VP2. PMID- 1716694 TI - Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180. AB - The human cervical carcinoma cell line ME180 was examined for human papillomavirus (HPV) DNA and RNA. The integrated DNA of a presumably new HPV type showing a relationship closer to HPV39 than to HPV18 was cloned and sequenced. HPV sequences from the E6-E7-E1 region are expressed as poly(A)+ RNAs. PMID- 1716695 TI - The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization. AB - By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis. PMID- 1716696 TI - Prostate specific antigen in the management of patients with localized adenocarcinoma of the prostate treated with primary radiation therapy. AB - The records of 143 patients treated at 5 institutions with external beam megavoltage irradiation for localized prostatic cancer were reviewed to evaluate post-treatment changes in prostate specific antigen (PSA) in the context of subsequent events. Complete responders were defined as patients clinically well with normal PSA, clinical failures were patients with documented local tumor recurrence or distant metastases and chemical failures were patients clinically well but with a PSA level above the upper limits of normal. Correlations with pre treatment PSA values were also made for the 50 of 143 patients for whom pre treatment PSA data were available. Median patient followup was 27 months (range 18 to 91 months). The data were analyzed with parametric and nonparametric univariate and multivariate statistical procedures. Pre-treatment PSA levels increased with increasing tumor stage (p = 0.004) but not with increasing summed Gleason pattern scores (p = 0.15). The probability of remaining a complete responder decreased with increasing stage (p = 0.008) but not with increasing Gleason score (p = 0.14). Increasing pre-treatment PSA correlated with clinical failure (p = 0.01) and chemical failure (p = 0.006). Of the patients with a pre treatment PSA level of less than 4 times the upper limits of normal 83% remained as complete responders compared to 30% of those with a higher pre-treatment PSA (p = 0.0002). The return of PSA levels to the normal range within 6 months after treatment was strongly correlated with a favorable outcome when analyzed by multivariate logistic regression. The status at last followup of patients who had a normal PSA level at 6 months versus those with an elevated PSA level 6 months after treatment is 94% versus 8% for complete responders (p = 0.0001), 0% versus 60% for clinical failures (p = 0.002) and 6% versus 32% for chemical failures (p = 0.14). Similar results occurred when analyzing outcomes in relationship to PSA normalization within 12 months after treatment (p = 0.001 for clinical failures, p = 0.02 for chemical failures and p = 0.001 for complete responders). We conclude that the pre-treatment level of PSA is an independent prognostic factor for prostate cancer patients treated with primary radiation therapy, and that the failure of PSA to return to the normal range within 1 year after completion of treatment identifies a group of patients at high risk for tumor recurrence. PMID- 1716697 TI - Prostate specific antigen and prostatic acid phosphatase for monitoring therapy of carcinoma of the prostate. AB - Serial serum prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) measurements were performed in 871 patients treated with hormonal combination therapy for stage C (95 patients) or stage D2 (776) prostate cancer for an average followup of 26 months. The relative efficacy of serum PAP and PSA for predicting recurrence of the disease was evaluated by 2 statistical methods at the time of progression as well as 6 and 12 months before clinical relapse of disease using optimized cut-off values of 2.0 and 4.0 micrograms/l. for serum PAP and PSA, respectively. At the time of progression the sensitivity (plus or minus standard deviation) of the 2 tests was estimated at 61.1 +/- 3.2% and 86.7 +/- 3.1% for PAP and PSA, respectively, while the specificity (plus or minus standard deviation) was calculated at respective values of 79.6 +/- 1.3% and 92.4 +/- 4.1%. Receiver operating characteristic analysis disclosed a greater accuracy for PSA at 89.2 +/- 1.7% versus 78.7 +/- 1.6% (plus or minus standard deviation) for PAP. The somewhat lower positive predictive value of the PSA test (81.4% versus 89.6%) is more than compensated by its superior negative predictive value (92.4% versus 79.6%). The present data also show that serum PSA measurements are superior to those of serum PAP for predicting disease recurrence in stages C and D prostate cancer patients treated by combination endocrine therapy and they indicate that measurement of serum PAP does not add significantly to single measurement of serum PSA alone. PMID- 1716698 TI - This month in investigative urology: transurethral laser treatment of benign prostatic hyperplasia. PMID- 1716699 TI - Transurethral ultrasound-guided laser-induced prostatectomy (TULIP procedure): a canine prostate feasibility study. AB - We describe the TULIP procedure, a new system to relieve bladder outlet obstruction caused by benign prostatic hyperplasia. This device is composed of a real-time 7.5 MHz ultrasound transducer coupled to a Nd:YAG laser with a 1.064 microns wavelength that fires through an intact intraprostatic balloon. A series of feasibility studies in 21 canine prostate glands was performed with a follow up time to 3 months. Results indicate that the Nd:YAG laser in the 20 to 40 W range at a pull rate of approximately 1 mm. per second is an effective means of removing substantial amounts of canine benign prostatic hyperplasia. Transurethral ultrasonography was a reliable means of identifying essential landmarks and of controlling the laser. Prostatectomy by laser coagulation necrosis resulted in no bleeding or postoperative obstruction. Intraoperative irrigation fluids were not required, eliminating systemic volume related problems. PMID- 1716700 TI - Expression of the insulin like growth factor-binding protein 3 (IGFBP-3) gene is increased in human renal carcinomas. AB - After we had established that the IGFBP-3 gene is expressed in normal human kidney we examined renal adenocarcinoma tissue for alterations of the expression of this gene. For this purpose we prepared poly(A)+ RNA from normal kidney tissue and adjacent renal adenocarcinoma of 18 adult patients and compared the levels of IGFBP-3 mRNA by Northern analysis in both samples. The mean content by densitometry was markedly increased in the carcinoma tissues; in 17 of 18 patients the carcinoma contained significantly more IGFBP-3 mRNA than the normal kidney sample. The highest mRNA levels were found in patients with N2 and N3 lymph node extensions. Comparative Southern analysis of paired samples of four of these patients did not reveal amplification of the gene as the cause of these increased mRNA levels. In one patient, however, we identified a restriction fragment length polymorphism (RFLP) present in normal and malignant cellular DNA. This suggests a participation of the IGFBP-3 gene in the development of human renal cell cancer. PMID- 1716701 TI - Transureteroureterostomy and terminal loop cutaneous ureterostomy in advanced pelvic malignancies. AB - Transureteroureterostomy was combined with terminal loop cutaneous ureterostomy, without complications, in 8 patients with advanced pelvic malignancy and a poor prognosis. Urinary diversion was palliative in all patients and followed pelvic exenteration in 4, debulking of pelvic tumor in 2 and radical cystectomy in 1, while 1 had inoperable bladder cancer. All patients had at least unilateral hydroureteronephrosis preoperatively. In each case a postoperative excretory urogram revealed significant improvement of the hydroureteronephrosis and the serum creatinine improved or stabilized. No patient had ureteral stomal stenosis or retraction. Mean survival was 5 months, with the longest survival being 1 year. Transureteroureterostomy in conjunction with terminal loop cutaneous ureterostomy is an effective technique of urinary diversion in selected patients with a poor prognosis and advanced pelvic malignancy, decreasing operative time while avoiding the morbidity associated with a ureterointestinal operation or nephrostomy. PMID- 1716702 TI - Stimulation of protein, RNA, and nucleotide synthesis in lymphocytes after abdominal surgery is not affected by postoperative amino acid supply. AB - The effects of abdominal surgery on protein, RNA, and de novo purine nucleotide synthesis in lymphocytes, and modification of these changes by postoperative amino acid supply, were investigated in 24 patients undergoing cholecystectomy (n = 12) or removal of gastric cancer (n = 12). Mono-nuclear cells were isolated from the peripheral venous blood and incubated with radioactive tracers in vitro. Protein and RNA synthesis, as measured using [14C] glycine and [3H]uridine, respectively, increased postoperatively. Nucleotide synthesis determined by the incorporation of radioactivity from [14C] glycine into nucleotides increased simultaneously. The concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP) estimated by the incorporation of [14C]adenine into nucleotides also increased. These changes were greater and of longer duration in patients with cancer operation than in those with cholecystectomy. In neither case were they affected by the amount of amino acid intake, or increases in energy intake. These results suggest that abdominal surgery stimulates protein and ribonucleic acid (RNA) synthesis in lymphocytes. Increased RNA synthesis may be ensured by increased synthesis of nucleotides, and increased PRPP concentrations appear to regulate the rate of nucleotide synthesis. The responses are apparently dependent upon the severity of surgery, but unrelated to the amount of amino acid supplied postoperatively. PMID- 1716703 TI - [Progress in diagnosis and therapy of pancreatic diseases]. PMID- 1716704 TI - [Problems in laboratory diagnosis of pancreatic diseases--reevaluation of screening strategies from laboratory medicine]. PMID- 1716705 TI - [Exocrine pancreatic function tests]. PMID- 1716706 TI - [Abnormalities in cough reflex]. AB - Enzymes degrading peptides may participate in the regulation of the cough reflex. Thus, decreases in enzyme activities may enhance cough reflex and a decrease in cough reflex may lead to aspiration pneumonia. Down- and up-regulation of cough reflex is important for the understanding of cough reflex. PMID- 1716707 TI - Macular lesions predisposing to senile disciform macular degeneration. AB - Senile disciform macular degeneration (SMD) is a neovascular/exudative form of age-related macular degeneration (AMD). In this study 340 eyes were followed up to assess the progression of SMD. The 340 eyes consisted of two groups. Group 1 was composed of 157 eyes with age-related macular changes other than choroidal neovascular membrane. Group 2 was made up of the contralateral eyes of 183 unilateral SMD eyes. Average ages were 61 and 64 in groups 1 and 2, respectively, and respective follow-up periods were 44 and 52 months. Choroidal neovascular membrane developed in 12 eyes in group 1 (7.6%) and in 19 eyes in group 2 (10.4%), a total of 31 eyes (9.1%). Retinal pigment epithelium (RPE) detachment was found as a predisposing lesion in 25 of these 31 eyes. Choroidal neovascular membrane developed in 12 of the 24 eyes with large RPE detachments. In 3 eyes neovascular membrane developed from an RPE detachment which had evolved from soft drusen. There were 3 eyes among the 62 eyes with soft drusen in which neovascular membrane developed directly from soft drusen. Based on these results, we classified AMD into 3 types; 1) atrophic, 2) predisciform, which includes RPE detachment and soft drusen, and 3) SMD. PMID- 1716708 TI - Release of digestive enzymes from isolated rat pancreatic acini following muscarinic stimulation: a comparative study with enzyme release and receptor binding. AB - Effect of cholinergic stimulation on the release of digestive enzymes from isolated rat pancreatic acini prepared by collagenase digestion was investigated. The release of enzymes (amylase, chymotrypsinogen, lipase) increased linearly with the time of incubation in the absence of secretagogues. Carbachol, a muscarinic agonist, induced a remarkable increase in the release of these enzymes. This carbachol-stimulated amylase release showed a biphasic curve, and its maximal response was observed at 10(-5) M. The release patterns of chymotrypsinogen and lipase were similar to that of amylase. This carbachol stimulated amylase release was inhibited by atropine in a dose-dependent manner, with an ED50 value of 1.17 X 10(-8) M. Other cholinergic agonists such as methacholine also stimulated the release of these enzymes, and these increases in the release were inhibited by atropine. Scatchard analysis for [3H]quinuclidinyl benzilate ([3H]QNB) binding to isolated pancreatic acini revealed that the binding site of [3H]QNB was a single component with a Kd value of 0.09 nM and a Bmax value of 89.3 fmol/mg protein, respectively. The effect of other cholinergic antagonists, pirenzepine and AF-DX 116 (11-[[2-[(diethylamino) methyl]1 piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one), on pancreatic acini was also investigated. Based on these results, it has been concluded that isolated rat pancreatic acini have muscarinic receptors and are useful for analyzing the mechanism of pancreatic enzyme secretion. PMID- 1716709 TI - [Qualitative analysis of the immunoreactive hCG beta-like substance produced by transitional cell carcinoma of the bladder]. AB - The hCG beta-like immunoreactivity (IR-hCG beta) in the sera and urine of three patients with transitional cell carcinoma of the bladder was characterized by enzyme-immunoassay which specifically recognized the hCG beta-related molecular species (whole hCG, free hCG beta and beta-core fragment). There was a qualitative difference in IR-hCG beta between the serum and the urine in all of the patients. Free hCG beta was exclusively detected in the serum, whereas both free hCG beta and beta-core fragment (beta-CF) could be detected in the urine. Whole hCG could not be virtually observed in the sera and the urine from all the patients examined. The concentrations of urinary beta-CF were consistently much higher than those of urinary and serum free hCG beta in all the patients. The immunohistochemical study was performed by using two kinds of the antibody against the core and the carboxyterminal portions of the hCG beta molecule. Immunoperoxidase staining was positive not only in the syncytiotrophoblastic giant cells but also in the transitional cell carcinoma cells for both antibodies. The present results suggest that free hCG beta is secreted from the tumor cells and metabolized into beta-CF may provide a new index to detect a transitional cell carcinoma of the bladder. PMID- 1716710 TI - [Objective evaluation of various extracardiac factors of cerebral origin in extrasystole]. AB - Partial integral rheography was used to study the volume velocity of brain blood supply (BBS) in 39 flying operators aged 20-39 years during continuously increasing bicycle ergometer testing. In good exercise tolerance, the dynamics of cerebral blood flow was found to be biphasic: 1) a rapid increase in BBS volume velocity at the first stages of exercise and 2) its subsequent stabilization. Stabilization of cerebral blood flow at 25% levels of blood minute volume was achieved by tonic tension autoregulation of the intracerebral arteries and adequate venous return. During the bicycle ergometer testing, the examinees with transient premature contractions showed impaired autoregulation, i.e. a considerable decrease in regional artery tone, cerebrovascular hyperperfusion and circulatory disorder resulting in the ++cerebro-cardiac syndrome with extracardiac premature beats. With drug therapy of extracardiac premature contractions, the tone of cerebral arteries and the level of intracranial hemodynamics should be borne in mind and corrected. PMID- 1716711 TI - [Lipid peroxidation in vasopressin-induced pulmonary edema and hyperbaric oxygenation therapy]. AB - Experiments in 150 albino rats have revealed that hyperbaric oxygenation (303.98 kPa) causes no decrease in pulmonary tissue hyperhydration and its blood filling induced by pituitrin administration. Hyperbaric oxygen therapy enhances pulmonary tissue peroxidation of lipids, without modifying them in blood. Application of lipid peroxidation inhibitors to prevention of pulmonary edema fails to affect pulmonary hydration and blood filling, which does not rule out the possibility of using them as protective agents against the toxic effects of oxygen in myocardial infarction which may be complicated by acute pulmonary edema. PMID- 1716712 TI - Significance of anti-phospholipid antibodies in patients with lupus nephritis. AB - Anti-phospholipid antibodies (APA) as markers or mediators of thrombosis in lupus could be of pathogenetic significance in nephritis, since glomerular capillary thrombi are an indicator of subsequent renal dysfunction. Isotype specific APA antibodies were measured, using cardiolipin as the antigen, in 76 patients with lupus nephritis. Twenty-nine percent of the patients had elevated IgG APA. Overall, 43% of patients showed raised levels of at least one isotype. In general, APA had specificity for anionic phospholipids. In vitro lupus anticoagulant activity was associated with all three isotypes of APA, but only the IgM isotype correlated with the biological false positive test for syphilis. APA were not associated with thromboses or neurological involvement, and only the IgA isotype correlated with thrombocytopenia. We confirmed an association between the presence of intraglomerular thrombi and serum IgG APA. However, we found no association between APA and renal histological pattern, or long-term renal function. Our data, therefore, do not support a major pathogenetic role for APA in the nephritis of lupus in treated patients. PMID- 1716713 TI - Angiotensin converting enzyme inhibition improves glomerular size-selectivity in IgA nephropathy. AB - Clearances of uncharged dextrans of broad size distribution were used to evaluate the effects of a 30 day course of enalapril on glomerular barrier function in 10 patients with IgA nephropathy and proteinuria (from 1.4 to 5.6 g/day). Dextran clearance experiments were repeated three times: before enalapril therapy, after 30 days of enalapril and again 30 days after enalapril withdrawal. GFR, but not RPF, was significantly reduced by enalapril (basal 38.3 +/- 11.9, enalapril 30.2 +/- 12.6 ml/min/1.73 m2) and returned to basal values after enalapril withdrawal. Urinary protein excretion and fractional clearance of albumin were both significantly reduced by enalapril (basal 2.3 +/- 1.1 g/day and 102 +/- 90 x 10( 5), enalapril 1.2 +/- 0.6 g/day and 51 +/- 23 x 10(-5), respectively) and returned to basal values after enalapril withdrawal. Transglomerular passage of large dextrans (radii 54 to 62 A), but not of lower size (26 to 42 A) were significantly lowered by enalapril. When enalapril was withdrawn the dextran sieving profile returned comparable to the baseline levels. A theoretical analysis of dextran-sieving profiles indicated that enalapril lowered the radius of largest membrane pores. This effect was independent from glomerular hemodynamic changes. We conclude that angiotensin converting enzyme inhibitors (CEI) in humans with IgA nephropathy reduces urinary protein excretion by a primary action on the intrinsic glomerular membrane properties enhancing barrier size-selective function. The hypofiltration associated with enalapril therapy in these patients, which was eliminated by its withdrawal, has to be taken into account as a possible undesired effect of CEI in long-term treatment. PMID- 1716714 TI - [The treatment of wounds from burns and mechanical trauma with deskhizan]. PMID- 1716715 TI - [Carcinoma of the pancreas--the palliative therapeutic possibilities outside of a radical surgical operation]. PMID- 1716716 TI - [Ion channels--the molecular background of neural transmission]. AB - Methodological developments in recent years, particularly patch clamp technology and the techniques of molecular biology, have advanced our knowledge of the molecular basis of synaptic transmission. The review is a brief summary of some findings concerning the ion channels that respond to hormones transmitters, or change in cell membrane potential, and of advances in our understanding of transmitter storage and release. Ion channels have been found to be targets for several drugs in clinical use, including anaesthetics, bensodiazopines, so-called calcium antagonists and the sulphonylureas used in the treatment of diabetes. The latter drugs have been shown to interact specifically with a potassium channel regulated by ATP (adenosine triphosphate). In pancreatic beta-cells, this channel controls membrane potential and insulin release under physiological conditions; in several other cells, it is activated under hypoxic or ischaemic conditions. PMID- 1716717 TI - Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex. AB - To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+) evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes. PMID- 1716718 TI - Effect of lindane upon the beta-adrenergic stimulation of cyclic AMP accumulation in rat renal cortical tubules caused by alterations in membrane fluidity. AB - The influence of lindane (gamma-hexachlorocyclohexane) on fluidity and lipid composition in rat renal cortical tubules has been investigated. Lindane increased membrane fluidity as measured by a fluorescence polarization technique using the probe diphenylhexatriene. This effect was dose-dependent and was accompanied by a 70% inhibition of the beta-adrenergic stimulatory activity upon cyclic AMP accumulation after 30 min of preincubation with lindane at 25 degrees C. Experiments with increasing concentrations of isoproterenol indicated that the efficacy, but not the potency, of the beta-adrenergic effect upon cyclic AMP accumulation was affected by lindane. Lindane toxicity could also be associated with variations in the incorporation of acetate into various lipid classes. Lindane increased acetate incorporation into phospholipids and decreased that into cholesterol. PMID- 1716719 TI - Neisseria gonorrhoeae LPS variation, serum resistance and its induction by cytidine 5'-monophospho-N-acetylneuraminic acid. AB - Inherent serum resistance and the effect of the serum resistance inducing factor cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were studied on Neisseria gonorrhoeae with different lipopolysaccharides (LPS). Strain M01 and LPS variants of strain Gc40 (variants D1, D2, D4 and D5) were examined after incubation in the presence or absence of CMP-NANA by bactericidal assays using normal human or immune sera and by SDS-PAGE followed by silver staining or Western blotting. The blots were probed with monoclonal antibody CC1, specific to epitope C of the LPS. Variants D1 and D5 were inherently serum resistant, variants D2 and D4 and strain M01 were susceptible. CMP-NANA induced marked changes in the LPS of all gonococci. However, only some gonococci were converted to serum resistance. Gonococci which were converted to serum resistance had LPS with components of relatively large molecular mass, expressing epitope C. Variants which did not convert to serum resistance had LPS with low molecular mass components only, without epitope C. Conversion to serum resistance increased the size of the large LPS components without affecting the expression of epitope C. The results indicate that conversion to serum resistance by CMP-NANA is not a general occurrence but depends on the quality of the LPS. PMID- 1716720 TI - Evaluation of the effect of pharmacologic agents on crush-avulsion arterial injuries: a scanning electron microscopy study. AB - This study was designed to investigate the short-term formation and composition of thrombus occurring at sites of arterial intimal injury in rabbits. Anti coagulants, platelet anti-aggregation agents, and fibrinolytic agents were evaluated for their influence upon the developing thrombus, utilizing scanning electron microscopy to determine thrombus surface composition. These agents included: heparin, aspirin, dextran 40, streptokinase, and prostacyclin. The results are consistent with the known nature of each of these agents. When two agents were given simultaneously, each acting on different aspects of thrombogenesis (platelets vs. fibrin), the outcome appeared synergistic, with substantial reduction in thrombus accumulation; clear inhibition of both platelet aggregation and fibrin polymerization was noted. These findings support the clinical use of two agents used simultaneously, one inhibiting fibrin strand formation and the other inhibiting platelet adherence aggregation, for the prevention of micro-arterial thrombosis. PMID- 1716721 TI - Palliative care in the 1990s? PMID- 1716722 TI - FK 506--an investigational immunosuppressant. PMID- 1716723 TI - [A proposal of a new histological method for the dynamic diagnosis of the disease stage in oral lichen planus]. AB - The paper describes a histological fixing and staining technique capable of identifying the stage of disease in oral lichen planus. This is based on staining the nuclei on the basal stratum blue instead of red-orange as normally occurs using Mallory's technique. Under particular conditions of increased cellular protein synthesis these become a typical blue colour. PMID- 1716724 TI - Characterization of transcription initiation sites on the soybean mitochondrial genome allows identification of a transcription-associated sequence motif. AB - Transcription initiation sites on the soybean mitochondrial genome have been characterized by sequence analysis of in vitro-capped soybean mtRNAs and corresponding mtDNA regions. The most abundant, discrete soybean mtRNA species labeled by guanylyltransferase and [alpha-32P]GTP are shown to correspond to the major transcript of the atp9 gene and to a group of small RNAs consisting of a discrete 80 nucleotide (nt) species plus heterogeneous species ranging in size from 133 to 148 nt. The 133-148 nt RNAs represent a set of transcripts with a common 5' terminus and ragged 3' ends, while the 80 nt RNA corresponds to positions 53-133 of the 133 nt species. The major, discrete in vitro-capped RNA species thus correspond to primary transcripts originating at three sites located in two regions of the soybean mitochondrial genome. The sequences extending from 13 nucleotides upstream to 8 nucleotides downstream of the initiation sites for the atp9 and 133-148 nt transcripts are identical at 18 of 21 positions. Sequences closely resembling this motif are located at some other 5' transcript termini of dicot plant mitochondria. Less closely related sequences are found at transcription initiation sites of wheat and maize mitochondria. PMID- 1716725 TI - Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture. AB - Three open reading frames (ORFs) in the actII region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2), which are involved in the export of the antibiotic are carried on two divergent transcripts. A monocistronic transcript carries actII-ORF1, encoding a putative repressor protein, and a bicistronic transcript codes for actII-ORF2 and -ORF3, whose products have been postulated to form an antibiotic export complex. The actII ORF1 and actII-ORF2/3 transcripts each have a single promoter and the promoters for the two transcripts overlap. Both promoters are most active in cultures that have developed to the stage of actinorhodin production. The promoters resemble consensus promoters of the vegetative class in Escherichia coli and Streptomyces. We also demonstrate that these promoters are expressed in E. coli and use this finding to reveal a regulatory role for the repressor, using the xylE reporter gene on promoter-probe shuttle vectors and regulated expression of the actII-ORF1 gene under control of Plac. The actII-ORF2/3 promoter is strongly repressed by the ORF1 product and the ORF1 product also represses its own promoter. The finding that the operator/promoter arrangement, and regulatory interconnection, of an antibiotic export/repressor gene pair in Streptomyces strikingly resemble those for tetracycline resistance in bacteria of clinical importance supports the hypothesis of an evolutionary origin of such genes in an ancestral actinomycete. PMID- 1716726 TI - Molecular analysis of the Bacillus subtilis recF function. AB - recF resides between the dnaN and gyrB genes of Bacillus subtilis. The recF15 mutation results in replacement of a glutamate residue in the wild type with a lysine residue in the mutant RecF protein. We investigated the in vivo regulation of recF using a transcriptional fusion to the xylE gene and assaying mRNA production. We found that novobiocin leads to a four-fold induction in recF gene expression, but this is not observed in a gyrB mutant strain. Enhancement of expression of the recF gene in the presence of novobiocin is unrelated to the SOS response. The RecF protein, which has a predicted molecular mass of 42.2 kDa, does not seem to be involved in its own regulation. PMID- 1716727 TI - 10Sa RNA, a small stable RNA of Escherichia coli, is functional. AB - The ssrA gene, coding for the metabolically stable 10Sa RNA, affects cell growth. A mutant in which the chromosomal 10Sa RNA gene is interrupted by a cat insert does not produce detectable levels of 10Sa RNA, and it grows more slowly than the parental strain. PMID- 1716728 TI - Desensitization of N-methyl-D-aspartate receptors in neurons dissociated from adult rat hippocampus. AB - Desensitization of the N-methyl-D-aspartate (NMDA) receptor-channel complex was studied in isolated rat hippocampal neurons using a fast drug application system. 1) Desensitization rate was slower at more negative membrane potentials and when external [Ca2+] was lowered. 2) In the presence of 10 microM glycine, 2-amino-5 phosphonovalerate neither induced desensitization nor prevented recovery from it. 3) Preincubation in 500 microM aspartate or 10 microM glycine alone elicited desensitization only weakly or not at all. 4) Aspartate appeared to bind at its receptor site in the absence of glycine, and vice versa. It is proposed that, for the NMDA receptor, channel opening is necessary for the occurrence of desensitization and, thus, that desensitization involves structural changes in the channel-lining section of the protein rather than the glycine or NMDA binding sites. PMID- 1716729 TI - Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins. AB - The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins. PMID- 1716730 TI - Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes. AB - Avermectins are a family of potent broad-spectrum anthelmintic compounds, which bind with high affinity to membranes isolated from the free-living nematode Caenorhabditis elegans. Binding of avermectins is thought to modulate chloride channel activity, but the exact mechanism for anthelmintic activity remains to be determined. In this report, the properties of an avermectin-sensitive membrane current were evaluated in Xenopus laevis oocytes that were injected with poly(A)+ RNA from C. elegans. In such oocytes, avermectins increased inward membrane current at a holding potential of -80 mV. An avermectin analog without anthelmintic activity had no effect. Half-maximal activation of current was observed with 90 nM avermectin. The reversal potential for avermectin-sensitive current was -19.3 +/- 1.9 mV, and it shifted with external chloride, as expected for a chloride current. Avermectin increased membrane current in C. elegans injected oocytes that were also injected with the Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The response to avermectin was greatest in the 1.0-2.5-kilobase class of size-fractionated C. elegans poly(A)+ RNA. Oocytes that responded to avermectin were insensitive to gamma aminobutyric acid and the avermectin-induced current was blocked by picrotoxin. PMID- 1716731 TI - Differentiation of erythrocyte-(GLUT1), liver-(GLUT2), and adipocyte-type (GLUT4) glucose transporters by binding of the inhibitory ligands cytochalasin B, forskolin, dipyridamole, and isobutylmethylxanthine. AB - The binding affinities of the glucose transporter isoforms GLUT1, GLUT2, and GLUT4 for the inhibitory ligands cytochalasin B, forskolin, dipyridamole, and isobutylmethylxanthine (IBMX) were compared in membranes from human erythrocytes and rat brain containing the erythrocyte-type glucose transporter (GLUT1), in membranes from rat liver containing the liver-type glucose transporter (GLUT2), and in membranes from adipocytes and heart containing predominantly the adipose/muscle-type glucose transporter (GLUT4). The binding affinities of cytochalasin B for GLUT1 and GLUT4 were virtually identical (KD) in membranes from erythrocytes, 190 nM; in brain, 130 nM; in adipocytes, 160 nM; and in heart, 170 nM). In contrast, no specific glucose-inhibitable binding of cytochalasin B was detected in liver membranes. The binding affinity for forskolin of GLUT1 was significantly lower than that of GLUT4 (KD in erythrocytes, 2360 nM; Kl in brain, 4360 nM; and KD in adipocytes, 200 nM; and in heart, 210 nM); specific glucose inhibitable binding to GLUT2 was not detectable. Like forskolin, the glucose transport inhibitors dipyridamole (Kl in adipocyte membranes, 1.2 microM; in erythrocytes, greater than 40 microM) and IMBX (Kl in adipocyte membranes, 60 microM; and in erythrocytes, greater than 500 microM) bound with higher affinity to GLUT4 than to GLUT1. These data demonstrate striking differences of GLUT1, GLUT2, and GLUT4 with respect to their binding affinity for the inhibitory ligands cytochalasin B, forskolin, dipyridamole, and IBMX. It is suggested that the complex differences result from interaction of more than one heterogeneous binding site at the glucose transporters with the inhibitory ligand. PMID- 1716732 TI - Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides. AB - A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52 kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716733 TI - [The c-fos proto-oncogene promotor is not regulated by serum, epidermal growth factor, and phorbol ester in embryonal fibroblasts transformed by E1Aad5+cHa-ras oncogenes]. AB - Regulation of c-fos protooncogene activity in rat embryonal fibroblasts (REF), E1Aad5-immortalized REF cells, and E1Aad5 + cHa-ras transformed REF cells has been investigated. The analysis of regulation of fos-promoter activity was done by means of transient and stable transfection of fos-CAT plasmid into immortalized and transformed cells. In parallel, the regulation of cellular c-fos as well as c-jun and c-myc genes expression has been studied. It has been found that in E1Aad5 + cHa-ras-transformed cells the expression of c-fos promoter has a constitutive, non-inducible character while in REF cells and cells immortalized by E1Aad5 the fos-promoter can be regulated by serum growth factors, EGF, and TPA. PMID- 1716734 TI - [Features of the structure and evolution of complex tandemly organized Bsp repeats in the fox genome. II. Tissue-specific and recombinant BamHI-dimer sites]. AB - The complex structure of the clustered Bsp-repeats in fox genome seems to have evolved throughout a long period of time as a result of multiplication, recombination and divergence events. The sequence of the subrepeat (SR) approximately 245 b.p long is the basic substructure for the hierarchically arranged Bam HI-repeat 1468 b.p. long. The monomer consists of 3 SRs with a 43 59% homology. A dimer is composed of 2 monomers with a 93% homology. Amplification of the Bsp-repeats during evolution seems to have occurred at least twice: first--on the SR ancestral form level, second--on the monomer level. Despite profound divergence, there are still conservative regions in SRs with sequences homologous to known functional sites in eukaryotes. However qualitative and quantitative composition of most functional motifs is stringently individual in every SR. The performed analysis revealed that throughout evolution SRs acquired significant amount of motifs homologous to promoter and enhancer regions in tissue-specific genes and virus regulatory regions. Functional motifs in separate SRs are being differently grouped. Most inducible motifs are located in the III and II subrepeats, putative promoters--in the II one; elements participating both in transcriptional and replicational processes--mainly in the I subrepeat. A few ensembles of functional motifs remotely resemble extended regulatory regions of some tissue-specific genes. The monomers are potentially capable of ensuring diverse aspects of transcriptional regulation. As a whole, motifs of the 3 SRs are potentially capable of regulating the RNA synthesis periodicity with respect to the cellular cycle, activation and repression of genetical material in response to signals from the environment (AP-1, AP-2, AP-4, T-antigen, etc) and temporal ("octamers") etc. Apart from the BamHI-dimer, a few homologues fragments were isolated from fox genome and sequenced. Some of them were rearranged with respect to the BamHI-dimer. Inversion locally alters the composition of motifs and the sequence acquires new functional potential. Thus, the analysis of the emergence and development of Bsp-repeat structural variations allows us to consider repetitive DNA sequences as an ideal material in constructing multiprofile regulatory sequences. PMID- 1716735 TI - [Localization of the site of human transferrin interacting with cellular receptors using monoclonal antibodies]. AB - Antigenic determinants of the human transferrin molecule on the sublobe and lobe levels were localized for 7 monoclonal antibodies. Antibodies used have different effects on the interaction of the transferrin with its receptor. It was concluded that transferrin-receptor recognition was determined by NH2-lobe, the N2-sublobe playing major part. Dimerization of the transferrin molecules in solution was detected. Using the panel of monoclonal antibodies it was shown that dimerization accomplished by means of the COOH-lobes of transferrin molecules, the sites of interaction of the NH2-lobe with receptor being exposed. A model of the transferrin - receptor complex is proposed. PMID- 1716736 TI - [RNA and DNA synthesis catalyzed by a DNA-polymerase alpha complex with primase from silkworm cells on single-stranded DNA]. AB - Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation. PMID- 1716737 TI - [Organization of promotor regions in Drosophila retrotransposons]. AB - The absence of TATA-box-like sequences and the presence of TCAGT sequences in the region of transcription initiation is the common feature of a number of Drosophila retrotransposons (e.g. mdg1, mdg3, mdg4). To reveal the LTR sequences, responsible for the transcription, a series of deletion mutants, containing a bacterial gene of chloramphenicholacetyltranspherase (CAT) under the control of different LTR fragments of these mdgs, was constructed. Such constructions were transfected into Drosophila Schneider2 cultured cells and the CAT-activity was analysed. The transcription of these constructions was also investigated by Northern blot hybridization and primer extention methods. The analysis of mdg1 promoter region showed, that it has two different elements. The distal one is responsible for the RNA synthesis level, while the proximal one is localized in the site of transcription origin and is responsible for the precision of transcription initiation. Therefore, a new type of RNA-polymerase II promoter elements, responsible for the accuracy of transcription initiation was revealed. The analysis of mdg3 and mdg4 promoters showed, that these retrotransposons also have such elements, but in contrast to mdg1, they have no promoter sequences analogous to the distal one in mdg1. PMID- 1716738 TI - Recovery of spleen cell natural killer activity by thyroid hormone treatment in old mice. AB - The age-dependent changes in thyroid-hormone blood levels and the effects of in vivo and in vitro thyroid-hormone administration on both basal and lymphokine induced spleen cell natural killer (NK) activities have been investigated in young and old Balb/c mice. Both thyroxine (T4) and triiodothyronine (T3) plasma levels decline progressively with increasing age of the mice, displaying in 25 month-old mice only 50 and 60% of the T4 and T3 blood levels, respectively, found in young mice. In vivo T4 administration to old mice causes a significant increment in endogenous NK activity (2.2-fold increase), which approaches the values observed in young animals, while it does not modify NK activity in young mice. The T4 injection in old mice does not induce changes in the lymphocyte sub populations. When T4 is administered in vitro alone or in combination with interferon (IFN) and/or interleukin 2 (IL-2), no effect is observed either on basal activity or IL-2-induced cytotoxicity, whereas the IFN sensitivity of spleen cells from old mice is significantly recovered (4-fold increase). T4 is able to increase IFN-induced cytotoxicity even when administered in vitro simultaneously with IFN to the cytotoxic assay (1.5- and 2.7-fold increases in young and old mice, respectively). Under these conditions, IFN alone is not able to exert any boosting effect even at a young age. In vivo propylthiouracil (PTU) administration completely abrogates the IFN responsiveness of spleen cells in young mice. The interruption of the PTU treatment results in a recovery of IFN inducible NK cytotoxicity. Taken together, our findings point out the important role of thyroid hormones in the modulation of NK cell activity and provide a new insight into the mechanisms by which the endocrine system is able to influence the expression of natural immunity. PMID- 1716739 TI - In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator. AB - Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The principal manifestations of CF include increased concentration of Cl- in exocrine gland secretions, pancreatic insufficiency, chronic lung disease, intestinal blockage and malabsorption of fat, and male and female infertility. Insight into the function of CFTR can be gained by correlating its cell-specific expression with the physiology of those cells and with CF pathology. Determination of CFTR messenger RNA in rat tissues by in situ hybridization shows that it is specifically expressed in the ductal cells of the pancreas and the salivary glands. In the intestine, decreasing gradients of expression of the CFTR gene are observed on both the crypt-villus and the proximal-distal axes. This expression is consistent with CFTR being responsible for bidirectional Cl- transport, secretion in the intestinal crypts and reabsorption in the silivary gland ducts, and suggests that in these tissues CFTR functions as a regulated Cl- channel. In the lung, a broad band of hybridization includes the mucosa and submucosa of the bronchi and bronchioles. In the testis, CFTR expression is regulated during the cycle of the seminiferous epithelium. Postmeiotic expression is maximal in the round spermatids of stages VII and VIII, suggesting that CFTR plays a critical role in spermatogenesis and that deficiency of this function contributes to CF male infertility. PMID- 1716740 TI - [Intraarterial injection of low molecular weight dextran and urokinase for acute cerebral infarction]. AB - Strategic recanalization of the occluded cerebral vessels has shown promise as a therapy for embolism and thrombosis in the acute stage. A single-dose of urokinase (UK) administered by intravenous and intraarterial routes was usually designed to restore patency of the infarct-related arteries and reperfuse the area of infarction. However, thrombolytic agents which have been available to date may lack resoluvability, limiting the amount of doses, because overdosage may induce hemorrhagic complication. This newly-designed therapy, intraarterial injection of UK-low molecular dextran (LMWD) complex was introduced in order to overcome the danger shown in the previous study. A high-resolvent allows cut-down of urkinase doses. Patients with acute cerebral infarction were selected for treatment with the resolvent if they satisfied the following conditions: 1) up to 79 years old without serious systemic diseases, 2) less than 12 hours from the onset, 3) better than the score of 8 in GCS, 4) no abnormality in CT scan, 5) apparent neurological deficit and 6) responsible pathology in angiography. LMWD of 15ml, UK of 240000IU and 15ml saline-complex was injected as one course at 2.0 2.5ml/min for 11 cases. Recanalization was observed in seven cases of embolism, and lack of reperfusion in five cases of thrombosis. The minimum effective dose was determined as 480000IU of UK in two courses. In terms of time lag from the onset, six hours may be the inferior limit, within which five of six cases (83%) succeeded in the recanalization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716741 TI - Capsaicin-induced release of neurokinin A from muscle and mucosa of gastric corpus: correlation with capsaicin-evoked release of calcitonin gene-related peptide. AB - It has been proposed that capsaicin-sensitive nerves might participate in a gastric defence mechanism, possibly via a local release of sensory neuropeptides. In this study, it was examined whether capsaicin might induce the release of neurokinin A (NKA), substance P (SP) and calcitonin gene-related peptide (CGRP) from different regions of rat stomach. Firstly, the tissue content of NKA-, SP- and CGRP-like immunoreactivity (LI) was measured in the fundus, in the corpus, in the muscle layer and in the mucosa of corpus of control rats and rats pre-treated with systemic capsaicin, s.c. (50 mg/kg as newborn). A large depletion (about 80%) of CGRP-LI following capsaicin treatment was observed in all regions examined, while no difference was observed for NKA-LI and SP-LI. NKA-LI, SP-LI and CGRP-LI release induced by capsaicin was measured in different regions of the rat stomach. Both in the gastric fundus and in the corpus, capsaicin (10 microM) produced a remarkable release of of CGRP-LI and NKA-LI, but not of SP-LI. A second administration of the drug had no longer effect, indicating desensitization. In the gastric corpus, the capsaicin-induced NKA-LI and CGRP-LI release was larger from the muscle layer than from the mucosa. The present findings provide neurochemical evidence that both NKA-LI and CGRP-LI are released from different regions of the rat stomach and both peptides should therefore be taken into account when considering the efferent function of capsaicin-sensitive primary afferents at gastric level. PMID- 1716742 TI - Effect of tachykinins in small human airways. AB - We have compared the contractile responses of substance P (SP) and neurokinin A (NKA) to that of the non degradable muscarinic agonist, carbachol, in small and large human airways in vitro. We have also investigated the effects of the neutral endopeptidase (NEP) inhibitor, thiorphan (100 microM) on these responses. NKA contracted large and small airways to a different extent (56% vs 92% of carbachol maximal contraction, respectively). NKA was significantly less potent in large vs small bronchi (EC50 = 150 +/- 15 vs 12 +/- 5 nM respectively, p less than 0.05). SP had a lower contractile effect in large (26% carbachol maximum) and small airways (59%) with EC50 values higher than 0.5 microM. The enkephalinase inhibitor thiorphan shifted the concentration-response curve to NKA to the left in large (EC50 = 35.2 +/- 8.2 nM) and small bronchi (EC50 = 2.8 +/- 1.3 nM, p less than 0.02). This shift was associated with an increase in the maximal contraction to NKA (75% in large vs 123% in small bronchi). The amplitude of contraction to SP was also potentiated in large (45%) and in smaller bronchi (101%). In conclusion, we have demonstrated that NKA has a significantly greater constrictor effect than a cholinergic agent in more peripheral human airways in vitro. This suggests that non cholinergic constrictor pathways are more likely to be important in more peripheral airways. PMID- 1716743 TI - Immunoautoradiographic localisation of enkephalinase (EC 3.4.24.11) in rat gastrointestinal tract. AB - Enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) is a zinc peptidase expressed by neurons and a variety of epithelial cells, and responsible for the inactivation of enkephalins in brain. Its functions in the gastrointestinal (GI) tract are less well understood although enkephalinase inhibitors were reported to induce a constellation of antisecretory and motor responses. Its localisation in various segments of the rat GI tract was established autoradiographically using a 125I-labelled monoclonal antibody. All along the GI tract, the highest immunoreactivity was found in mucosal layers e.g., in intestinal villi, basal epithelial layers of the oesophagus or gastric cardia, muscularis mucosae of the stomach and large intestine. The immunoreactivity was also high in the stomach submucosae and moderate in the muscularis propria of the caecum. A faint patchy immunoreactivity was also observed in several other layers. This distribution suggests that the membrane peptidase is expressed by enterocytes and a variety of other cells. Its high expression in mucosal layers is consistent with its participation in protein digestion and also in the inactivation of endogenous peptides, particularly the enkephalins, acting at this level to control secretory mechanisms and hydroelectrolytic fluxes. Its presence in submucosal layers may account for some naloxone-reversible motor responses elicited by enkephalinase inhibitors. PMID- 1716744 TI - Synaptic reorganization by mossy fibers in human epileptic fascia dentata. AB - This study was designed to identify whether synaptic reorganizations occur in epileptic human hippocampus which might contribute to feedback excitation. In epileptic hippocampi, (n = 21) reactive synaptogenesis of mossy fibers into the inner molecular layer of the granule cell dendrites was demonstrated at the light microscopic and electron microscopic levels. There was no inner molecular layer staining for mossy fibers in autopsy controls (n = 4) or in controls with neocortex epilepsy having no hippocampal sclerosis (n = 2). Comparing epileptics to controls, there were statistically significant correlations between Timm stain density and hilar cell loss. Since hilar neurons are the origin of ipsilateral projections to the inner molecular layer, this suggests that hilar deafferentation of this dendritic zone precedes mossy fiber reafferentation. Quantitative Timm-stained electron microscopy revealed large, zinc-labelled vesicles in terminals with asymmetric synapses on dendrites in the inner molecular and granule cell layers. Terminals in the middle and outer molecular layers did not contain zinc, were smaller and had smaller vesicles. These histochemical and ultrastructural data suggest that in damaged human epileptic hippocampus, mossy fiber reactive synaptogenesis may result in monosynaptic recurrent excitation of granule cells that could contribute to focal seizure onsets. PMID- 1716745 TI - Morphological, neurochemical, and behavioral studies on serotonergic denervation and graft-induced reinnervation of the rat hippocampus. AB - A procedure was developed to conduct simultaneously immunocytochemical and neurochemical studies on the serotonergic system in adjacent 300-micron-thick slices of rat hippocampus. This procedure was applied to correlate morphological (innervation pattern and density), neurochemical (5-hydroxytryptamine and 5 hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake and release) and behavioral (spatial learning) effects of neurotoxin-induced denervation and reinnervation by grafting fetal mesencephalic raphe cells. Intracerebroventricular injections of a low dose of 5,7-dihydroxytryptamine caused a discrete serotonergic denervation of the hippocampus. Eleven months after lesioning, 5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake capacity were decreased by 50-60%. By this time, the residual fibers displayed an enhanced vulnerability towards K(+)-induced depolarization. Grafting of a fetal raphe cell suspension resulted in a reinnervation of the host hippocampus. The pattern of reinnervation was comparable to control innervation and the density was supranormal at the level of the graft. As observed semiquantitatively, the innervation density decreased with distance from the core of the graft. Neurochemical studies showed that the fibers were capable of synthesizing, metabolizing and releasing 5-hydroxytryptamine. The turnover of 5-hydroxytryptamine in both the denervated and the reinnervated hippocampus was comparable to that in control tissue. Previous behavioral testing of the denervated and of the denervated and implanted animals did not reveal any effect on spatial learning, either in an individual or in a social test paradigm. The latter data substantiate the notion that interference with the hippocampal serotonergic innervation does not hamper adequate spatial learning. PMID- 1716746 TI - Development of intrastriatal striatal grafts and their afferent innervation from the host. AB - The morphological maturation of cell suspension grafts of fetal striatal tissue (obtained from 14-15-day-old rat fetuses) was followed from two days to eight weeks after implantation into intact and ibotenic acid-lesioned striata of adult rats. The development of host afferent innervation of the grafts from the substantia nigra (tyrosine hydroxylase immunoreactive), mesencephalic raphe (serotonin immunoreactive), and the frontal cortex (anterogradely labelled with Phaseolus vulgaris leucoagglutinin) were revealed by immunohistochemistry. During the first weeks post-grafting, the striatal implants consisted of a mixture of mature- and immature-looking cell clusters. Grafts implanted into ibotenic acid lesioned striatum grew rapidly (about five-fold) in volume over the first week. The areas of immature (probably proliferating) cells gradually disappeared, and by six to eight weeks the grafts had a fully mature appearance with patches of neurons which stained densely for DARPP-32 (i.e. were striatum-like) embedded within areas of essentially DARPP-32-negative (i.e. non-striatum-like) tissue. Peripheral clusters of grafted cells gradually intermingled with nearby areas of the surrounding lesioned host, and already by two to four days after implantation, coarse and densely immunoreactive host fibres from the substantia nigra, mesencephalic raphe and frontal cortex were present within the grafts. By four to five days the first DARPP-32-immunoreactive neurons appeared in patches within the mature portions of the grafts, and one to two days later the tyrosine hydroxylase-positive fibres began to sprout thin axons selectively within the DARPP-32-positive patches. Similarly, the serotonergic and cortical fibres in the grafts increased in number over the next two weeks, but they showed no preference for the DARPP-32-positive regions. Rich terminal networks were established by two to three weeks post-grafting, and by six to eight weeks the nigral, raphe and cortical afferents had reached terminal densities similar to those seen previously in long-term surviving grafts. Grafts implanted into dopamine denervated hosts showed a normal morphological maturation of both DARPP-32 positive and -negative areas, although no tyrosine hydroxylase-positive innervation appeared within the grafts. Grafts implanted into non-lesioned striata did not grow beyond their initial size. The implanted cells showed less intermingling with the surrounding host striatum, thus resulting in sharply delineated graft-host borders. DARPP-32-positive patches developed, but they were smaller in size and generally present only in the most peripheral graft portions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716747 TI - Ultrastructural localization of adrenocorticotrophic hormone and the phosphoprotein B-50/growth-associated protein 43 in freeze-substituted, Lowicryl HM20-embedded mesencephalic central gray substance of the rat. AB - Previous studies have shown that the endogenous phosphorylation of the neuron specific protein B-50 in isolated synaptic plasma membranes is inhibited by adrenocorticotrophic hormone(1-24). The aim of this study is to examine if there is a specific neuroanatomical interaction of adrenocorticotrophic hormone and B 50 in the mesencephalic central gray substance of the rat. With light microscopy, high B-50 immunoreactivity was detected throughout the mesencephalic central gray substance, overlapping with those areas where adrenocorticotrophic hormone immunoreactive fibres were present. To study the ultrastructural localization of B-50 and adrenocorticotrophic hormone, we employed a method of immunogold labelling on ultrathin sections of freeze-substituted and Lowicryl HM20-embedded fixed brain tissue. This offered optimal morphological preservation together with high retention of antigenicity. At the electron microscopic level, adrenocorticotrophic hormone immunoreactivity was detected in dense-core secretory granules present in non-junctional regions of axoinal varicosities. This suggests a non-synaptic release of adrenocorticotrophic hormone from the axons. Using double immunolabelling techniques we showed that in adrenocorticotrophic hormone-innervated areas of the mesencephalic central gray substance B-50 immunoreactivity was present at plasma membranes of all unmyelinated axons and axonal varicosities and virtually absent in dendrites. The result on B-50 localization agrees well with previous studies in the hippocampus [Van Lookeren Campagne et al. 1990 J. Neurocytol. 19, 948-961] and in the pyramidal tract [Gorgels et al. 1989 J. Neurosci. 9, 3861-3869] of the rat and suggests that in the mature rat central nervous system, B-50 expression in axons is a general phenomenon. For the adrenocorticotrophic hormone-innervated areas, we discuss the proposal that non-synaptically released adrenocorticotrophic hormone modulates B-50 phosphorylation in axons and axon terminals. PMID- 1716748 TI - Colchicine enhances mRNAs encoding the precursor of calcitonin gene-related peptide in brainstem motoneurons. AB - Hybridization signals indicating mRNAs encoding the precursor of calcitonin gene related peptide (CGRP) and CGRP immunoreactivity were detected on parallel sections containing brainstem motor nuclei using in situ hybridization histochemistry and immunohistochemistry. In untreated and saline-injected rats the motoneurons in the hypoglossal, facial motor nuclei and in the ambiguus nucleus showed weak to moderate hybridization signals. In these motoneurons CGRP immunoreactivity was restricted to the Nissl bodies of the perikarya. Twenty-four and 42 hours after intracerebroventricular colchicine injection the intensity of both the hybridization signal and the immunoreaction product increased. The distribution of CGRP immunoreactivity changed from discrete perikaryal localization to diffuse reaction in the perikarya and along the proximal dendritic tree. Motoneurons in the rest of the brainstem motor nuclei (VIth, Vth, IVth and IIIrd) of untreated and saline-injected rats showed neither hybridization signal nor CGRP immunoreactivity. After intracerebroventricular injection of colchicine these motoneurons showed both hybridization signal and CGRP immunoreactivity. In all nuclei the size of motoneurons decreased and their Nissl structure changed to an amorphous basophilic mass following colchicine treatment. PMID- 1716749 TI - Trophic functions of primary sensory neurons: are they really local? AB - The results of the present study, in the rat and cat, indicate that not only a lesion of peripheral nerve or capsaicin pretreatment but also pharmacological deafferentation with local anaesthetic or disruption of the connections between primary sensory neurons and the central nervous system are effective in producing dystrophic changes in tissues. These effects of deafferentation do not seem to depend on the sympathetic or parasympathetic efferents. Dystrophic changes are connected with microcirculation disturbances: slow down of local blood flow, elevation of the vascular permeability, oedema and leucocyte infiltration. The findings indicate that capsaicin-sensitive primary sensory neurons are the afferent part of some reflex arrangement which participates in the regulation of microcirculation and the maintenance of trophic processes in peripheral tissues. The efferent part of this arrangement is unknown. PMID- 1716750 TI - Effects of basic fibroblast growth factor on the development of GABAergic neurons in culture. AB - Six-day-old neuronal cultures derived from 14-day-old embryonic rat cerebral hemispheres were highly enriched in GABAergic neurons, as was demonstrated by immunocytochemistry using an anti-glutamate decarboxylase antiserum. They contained about 64% glutamate decarboxylase-positive neurons. About 8% of these neurons proliferated, as shown by a combination of glutamate decarboxylase immunocytochemistry and [3H]thymidine incorporation into cell nuclei. The proliferative activity of GABAergic precursor cells and changes in the cellular concentrations of the non-essential amino acids, including GABA under the effect of basic fibroblast growth factor were studied. When basic fibroblast growth factor was added to the cultures 4 h after seeding, the proliferation of the GABAergic neurons was stimulated about threefold. Under this culture condition, the concentration per cell of all amino acids increased, except those of GABA and beta-alanine. When basic fibroblast growth factor was added to cultures only on day four, the proliferation of the neuronal cells was no more enhanced. Under this condition of treatment, the concentrations of all non-essential amino acids, including those of GABA and beta-alanine were enhanced. Under both basic fibroblast growth factor treatments the concentration of GABA per GABAergic cell was increased. In contrast, the specific activity of glutamate decarboxylase was not stimulated under these conditions. We hypothesize that under the effect of basic fibroblast growth factor the capabilities of the cells to store GABA are improved. PMID- 1716751 TI - Quantitative sensory testing before and after regional guanethidine block in patients with neuralgia in the hand. AB - Using reference values from healthy volunteers, thermal and vibration-induced pain thresholds and the sensibility for warm and cold were studied in 18 patients with neuralgia in one hand following a traumatic injury or surgery. All patients had spontaneous pain and allodynia to vibration. They were treated with intravenous regional guanethidine block (RGB). Quantitative sensory testing was performed on both hands before and 1-3 days after treatment. Eleven patients benefitted considerably from the block, with pain relief for 2 weeks or more. Ten of these 11 patients had mild nerve injuries caused by compressive trauma to the nerve. Before RGB they showed a moderate loss in temperature discrimination capacity; their heat pain thresholds were reduced and they exhibited allodynia to cold and vibration on the injured side. After RGB, the pain thresholds were normalised both to thermal and vibratory stimuli. These patients were classified as having sympathetically maintained pain (SMP). Seven patients reported no or only minor pain-relieving effect of RGB lasting 1-5 days. Severe nerve injuries were most frequent in this group of patients. On the injured side, before RGB, their ability to discriminate between warm and cold was markedly impaired, thermal pain thresholds were normal, and they showed allodynia to vibration. After RGB, there was no change in thermal pain thresholds and the allodynia to vibration persisted. These patients were classified as having sympathetically independent pain (SIP). The results indicate that quantitative thermal sensory tests, together with clinical evaluation of the nerve trauma, can help to predict which patients will have long-lasting pain alleviation after RGB treatment. PMID- 1716752 TI - Pain characterization in cancer patients and the analgetic response to epidural morphine. AB - In 48 patients with pain related to malignancy, a pain characterization was performed during oral opioid therapy. After an optimal epidural morphine regimen had been established, the alteration in pain relief was evaluated by means of a visual analogue scale. The CSF and plasma morphine concentrations at minimum steady state were then analysed in 28 patients and related to the degree of pain relief. The efficacy of the spinal treatment ranked in the following order: somatic greater than visceral greater than radiating = 0, but the difference was only significant between the somatic and radiating pain groups. There was a tendency for continuous pain to be better relieved than intermittent pain. No correlations were found between the CSF or plasma morphine concentrations and the degree of pain relief, suggesting that not all pain impulses are modulated in a dose-dependent manner by morphine at the spinal level. Pain characterization may be instrumental in providing an optimal spinal opioid analgesia in malignancy. Moreover, there is a need for better defined diagnostic criteria for clinical pain characterization. PMID- 1716753 TI - Chronic pain: a PET study of the central effects of percutaneous high cervical cordotomy. AB - We have studied 5 patients with unilateral, severe chronic pain due to cancer before and after percutaneous, ventrolateral cervical cordotomy to investigate the central effects of the procedure. The aim was to identify the functional anatomical correlates of abolishing unilateral nociceptive input to the brain. Patients were investigated by positron emission tomography using C15O2 to evaluate cerebral blood flow. Comparisons were made between the patients with unilateral pain before cordotomy and normal volunteers. These demonstrated significantly less blood flow in 3 out of 4 of the individual quadrants of the hemithalamus contralateral to the side of pain (P less than 0.01-0.05). These differences were abolished by cordotomy. Comparison of the patients before and after cordotomy showed a significant decrease in blood flow in the dorsal anterior quadrant of the thalamus contralateral to the side of pain (P less than 0.05) which was normalised after cordotomy. There were no significant changes in the prefrontal or primary somatosensory cortex. We conclude that chronic pain results in a decrease of synaptic activity at thalamic level either from decreased activity in neurones projecting to that region and/or attenuated local neuronal firing. We have demonstrated no secondary remote effects in cortex, indicating the importance of subcortical mechanisms in central responses to chronic pain. PMID- 1716754 TI - Failure to confirm the presence of a retrovirus in cultured lymphocytes from patients with Kawasaki syndrome. AB - We and others previously reported DNA polymerase activity in culture supernatants of peripheral blood mononuclear cells from patients with acute Kawasaki syndrome (KS). In the present study, we further characterized the previously detected polymerase activity and attempted to confirm its presence in cultured peripheral blood mononuclear cells from additional patients with KS. Characterization experiments indicated that the polymerase activity was typical of a DNA-dependent DNA polymerase rather than viral reverse transcriptase. Peripheral blood mononuclear cell cultures from 17 additional KS patients were negative for reverse transcriptase activity in three laboratories. Our findings do not provide support for a retroviral etiology of KS. Further studies should continue to focus on infectious agents in efforts to elucidate the etiology of KS. PMID- 1716755 TI - Identification of a secretory IgA receptor on breast-milk macrophages: evidence for specific activation via these receptors. AB - Binding of secretory IgA (sIgA) to human milk macrophages was identified by rosette formation with sIgA-sensitized indicator cells and competitive inhibition binding studies with [125I]-sIgA. Macrophages formed rosettes with sIgA sensitized sheep red blood cells that were inhibited by sIgA (87%) but not IgG. Both IgA1 and IgA2 inhibited sIgA-sensitized sheep red blood cell rosette formation. Rosette formation was completely inhibited by galactose, partially inhibited by asialofetuin, and unaffected by mannose. [125I]-labeled sIgA binding to macrophages reached a plateau after 60 min, was dependent on the number of macrophages in culture, and was specifically inhibited by unlabeled sIgA. Incubation of macrophage monolayers with increasing concentrations of sIgA-anti IgA immune complexes resulted in a progressive increase in the oxidative burst and increased secretion of prostaglandins. These studies indicate that human milk macrophages have a receptor for sIgA and that activation of these macrophages may occur via these receptors. PMID- 1716756 TI - Subcutaneous infusions for cancer pain. PMID- 1716757 TI - [Recombinant human erythropoietin stimulates synthesis of fetal hemoglobin in patients with renal anemia treated by repeated hemodialysis]. AB - Effect of intravenously administered recombinant human erythropoietin (rHu EPO) on haemoglobin (Hb) level, haematocrit (Ht), reticulocyte count and foetal haemoglobin (HbF) concentration was assessed in 10 patients with anaemia, treated by repeated haemodialysis due to end-stage kidney. As compared to the initial values, erythropoietin treatment brought about a significant increase in all the parameters examined. During the subsequent therapy with lower, supporting doses of erythropoietin, the elevated HbF values fell back to normal, whereas the higher level of total Hb and Ht were maintained. PMID- 1716758 TI - [Arrhythmia in patients with mitral valve prolapse]. AB - An incidence of cardiac arrhythmias was evaluated in 119 patients with mitral valve prolapse. The disease was made basing on the results of clinical symptoms, echo-, angio- and phonocardiography. Electrocardiograms were recorded from the standard 12 lead and Holter technique for 24 hours in each patient to assess present arrhythmias. It was found that the most frequent cardiac arrhythmias accompanying mitral valve prolapse are ventricular extrasystolic contractions of Lown's class 1a and 1b. Only examination of strictly selected groups of patients (age groups with or without co-existing mitral valve insufficiency for adequate period of time) will facilitate precise evaluation of an incidence of different cardiac arrhythmias accompanying the underlying disease. PMID- 1716759 TI - [Intraoperative staining of tissues]. AB - Possibilities of vital dyeing to determine blood supplied from non blood supplied tissues are subject of many years' investigations. They may play an important role in traumatology, reconstructive and septic surgery. In acute traumatic changes one of the methods known is the so called immediate surgical treatment with delayed operation. In chronic and septic pathological changes there exists a great difficulty distinguishing necrotic parts of bone or soft tissues, which are the cause of long lasting inflammation and external fistulas. This process can last for months or even years if not treated by a proper and precise surgical procedure. Very helpful may be vital dying of tissues which was introduced by K. Klemm in 1970 for the surgical treatment of septic bone necrosis using the Disulphine Blue, produced by Imperial Chemical Industries Limited, Pharmaceuticals, Division, Macclesfield Cheshire, G. B. The tissue dyes are used widely for subcutaneous injections in lymphography, i.e. in about 30,000 patients in USA yearly. Disulphine Blue is a water soluble and partly spirit soluble powder giving a blue solution, which can be sterilized in autoclave. After an intravenous application of 0.25-0.5 ml/kg body of 6.2% solution a green colour of skin and conjunctivas appears in 5 minutes. The dye can appear in bile, bronchial tree, feces and in intestine secretions. It is evacuated with urine. Green colour of the skin disappear after 36 hours in children and after 48 hours in adults. This dye has been used as a direct visual test to investigate normal blood supplied and necrotic soft and bone tissues. This phenomenon can have a special significance for defining the vitality in inflammation of bone tissue, in burns and in necrosis of soft tissues a.a. of the Achilles tendon. A demonstrative case report. Patient K. J., 32 years old, a worker and sportsman was hospitalized in our clinic because of chronic inflammation and purulent fistulas 10 weeks after two operations in one of hospitals because of traumatic right Achilles tendon rupture. During the first operation the tendon was sutured and the leg immobilized for a period of 6 weeks. After that time the plaster dressing was removed and during the rehabilitation was stated a secondary Achilles tendon rupture. The tendon was resutured. The course after the operation was complicated with haematoma and purulent fistulas. Antibiotics were used but without effect. In our Clinic 10 weeks after the primary injury and unsuccessful local treatment we performed a subsequent operation after slow intravenous injection of 10 ml Disulphine Blue.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716760 TI - Prenatal diagnosis of recurrent Larsen syndrome: further definition of a lethal variant. AB - Larsen syndrome is characterized by multiple congenital joint dislocations and flattened facies. Some cases have been familial, with both autosomal dominant and recessive patterns of inheritance. Reports of a form of Larsen syndrome, lethal in the neonatal period, are reviewed. We present a family in which recurrence of the syndrome was diagnosed prenatally, but a lethal outcome again resulted despite preparation for anticipated perinatal complications. Because of the wide clinical variation and the lack of a known metabolic defect, delineation between the various forms of Larsen syndrome is difficult. While the lethal variant appears to be a combination of the Larsen phenotype and pulmonary hypoplasia, other features noted in the lethal cases, such as abnormal palmar creases and laryngotracheomalacia, are also seen in patients with Larsen syndrome who survive. PMID- 1716761 TI - Screening for fetal Down's syndrome with maternal serum markers--an experience in Italy. AB - The effectiveness of maternal serum alpha-fetoprotein, unconjugated oestriol, and human chorionic gonadotrophin in screening for Down's syndrome (DS) was evaluated on 840 women who underwent amniocentesis for fetal karyotype on account of their age. The risk of a DS pregnancy was established using the method of Wald et al. (1988b), which combines the age-specific risk with that indicated by the levels of the three serum markers. In women over 35, at cut-off risk levels of 1:250 and 1:380, the false-positive rate was 24 and 34 per cent, respectively. In all nine cases of DS, the estimated risk was higher than 1:250. The best screening strategy with the lowest false-positive rate was obtained by combining the three serum markers. The results suggest that this kind of screening can be proposed during genetic counselling for women under 35 and older women wishing to avoid the risk of miscarriage induced by amniocentesis. PMID- 1716762 TI - Cu/Zn superoxide dismutase quantification from fetal erythrocytes--an efficient confirmatory test for Down's syndrome after maternal serum screening and sonographic investigations. AB - An enzyme immunoassay especially designed for the quantification of Cu/Zn superoxide dismutase (SOD) in erythrocytes has been applied to measure the SOD of outcomes with high risk for Down's syndrome. From 148 fetuses SOD was quantified from erythrocytes of umbilical vein blood and related to the number of cells, the content of haemoglobin (Hb), and to the haematocrit (Hc). Comparative studies between the SOD content of erythrocytes from the fetuses and their mothers resulted in similar SOD levels (14.09 +/- 1.20 for fetal and 14.48 +/- 1.63 for maternal cells) with a 1.84-fold smaller variance for fetal cells. The best differentiation between normal fetuses and fetuses with Down's syndrome resulted from the SOD/cell ratio followed by the SOD/Hb ratio. Fixing a cut-off value from the probability density functions that the method results in a specificity of 99.99 per cent, the sensitivity to detect cases of Down's syndrome was 99.71 per cent for the SOD/cell ratio, 70.92 per cent for the SOD/Hb ratio, and 60.21 per cent for the SOD/He ratio. Nine cases with Down's syndrome were correctly diagnosed by the SOD/cell ratio determination. Eight of these were confirmed as free trisomy 21 by karyotype analysis and one was found to be a triploidy. The latter was not detected by the SOD/Hb and SOD/Hc ratios because of the one-third higher content of haemoglobin and the larger volume of the erythrocytes which resulted in ratios within the normal range. PMID- 1716763 TI - [Immunologic cytopenia associated with chronic lymphoid leukemia. Role of high dose intravenous gammaglobulins]. PMID- 1716764 TI - Association of p21ras with phosphatidylinositol 3-kinase. AB - In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3 kinase activity. PMID- 1716765 TI - Light-dependent channels from excised patches of Limulus ventral photoreceptors are opened by cGMP. AB - The identity of the second messenger that directly activates the light-dependent conductance in invertebrate photoreceptors remains unclear; the available evidence provides some support for cGMP and Ca2+. To resolve this issue we have applied these second messengers to membrane patches excised from the light sensitive lobe of Limulus ventral photoreceptors. Our results show that these patches contain channels that can be opened by cGMP, but not by Ca2+. These cGMP activated channels closely resemble the channels activated by light in cell attached patches. This evidence suggests that cGMP is the messenger that opens the light-dependent channel in invertebrate photoreceptors. PMID- 1716766 TI - Transcription factor AP2 and its role in epidermal-specific gene expression. AB - The epidermis is a stratified squamous epithelium whose major differentiation specific products are keratins. To elucidate factors controlling keratinocyte specific gene expression, we previously identified proximal and distal regulatory elements that act synergistically to drive keratinocyte-specific expression of the gene encoding human epidermal keratin K14. Control by the proximal element is mediated by a transcription factor, KER1, which is more abundant in nuclear extracts of keratinocytes than in extracts of other cell types, including fibroblasts, lymphocytes, and simple epithelial cells. In this report, we identify this factor as transcription factor AP2, shown to be transcribed in cells of epidermal and neural crest lineages. Furthermore, we demonstrate functional AP2 binding sites upstream from three additional epidermal genes, suggesting that AP2 may be generally involved in epidermal gene regulation. Finally, although AP2 is necessary, it is not sufficient for epidermal gene expression: a distal element contributes to tissue-specific expression of the human keratin K14 gene as judged by its ability to enhance expression of a heterologous promoter in keratinocytes but not in hepatoma cells. These results imply that a combination of factors is likely to contribute to epidermal-specific expression of the human keratin K14 gene. PMID- 1716767 TI - Antigen mobility in the combining site of an anti-peptide antibody. AB - The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 57-84), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, Trp-62, Trp-63, and Ile-78) do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity. PMID- 1716768 TI - Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription. AB - Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3' terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5' phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA. PMID- 1716769 TI - Identification of monoclonal antibody epitopes and critical residues for rhinovirus binding in domain 1 of intercellular adhesion molecule 1. AB - Intercellular adhesion molecule 1 (ICAM-1) is the cellular receptor for the major group of human rhinoviruses (HRVs) and the adhesion ligand of lymphocyte function associated antigen 1. Analysis of a series of chimeric exchanges between human and murine ICAM-1 shows that two distinct epitopes recognized by monoclonal antibodies that block rhinovirus attachment and cell adhesion map to the N terminal first domain of ICAM-1. Furthermore the specificity for HRV binding is entirely contained within the first 88 amino acids. Mutagenesis of the four sites of N-linked glycosylation within the second domain shows that carbohydrate is not involved in virus recognition. Homologue replacement mutagenesis localizes the epitopes for virus-blocking antibodies to two regions of domain 1 predicted to form beta strand D and the loop between the F and G strands of an immunoglobulin fold structure. Analysis of virus binding to the mutants predicts a large surface of contact between HRV and ICAM-1 domain 1 but shows that the regions most important for virus binding are coincident with the monoclonal antibody epitopes. PMID- 1716771 TI - Solid phase extraction of 5-hydroxyindole-3-acetic acid from urine and its application to HPLC and spectrophotometric methods. PMID- 1716770 TI - Engineered secreted T-cell receptor alpha beta heterodimers. AB - We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent heterodimers of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa dimers and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR heterodimers and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha 2 (V delta 8) and V beta 2 gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface. PMID- 1716772 TI - Skin recycling following neovascularization using the rat musculocutaneous flap model. AB - The recycling of a skin territory as part of a musculocutaneous flap despite prior division of existing musculocutaneous perforators or vice versa within an axial cutaneous flap using skin from a previous musculocutaneous flap may sometimes be done safely if an adequate time period has been allotted to permit sufficient neovascularization from adjacent tissues. In order to test this clinically observed phenomenon, a musculocutaneous flap model based on perforators from the rat rectus abdominis muscle was developed and observed to have complete reliability. Groups of five Sprague-Dawley rats each were sequentially utilized to prove that by a single week following creation of a rectus abdominis musculocutaneous flap adequate peripheral neovascularization would evolve to permit total viability of secondary axial epigastric cutaneous flaps incorporating the same skin that initially was the cutaneous portion of the muscle flap. The converse was also confirmed possible, again using sequential groups of five rats each, in that by 2 weeks the skin of an initial abdominal cutaneous flap could instead be safely transposed and nourished as part of a rectus abdominis musculocutaneous flap. The proposition concerning the reliable reuse of identical skin territories as part of disparate metachronous flap configurations appears to be valid. PMID- 1716773 TI - Enhanced neovascularization of pedicle flaps with low perfusion. PMID- 1716774 TI - Use of an ELISA for differential diagnosis of Mycoplasma agalactiae and M mycoides subspecies mycoides (LC) in naturally infected goat herds. AB - Contagious agalactia is an ovine and caprine mycoplasmosis which manifests as mastitis, arthritis and keratoconjunctivitis. Mycoplasma agalactiae is recognised as a causal agent but M mycoides subspecies mycoides (LC), and M capricolum may also be responsible for this syndrome in goats. The clinical signs are not pathognomonic; diagnostic procedures are based on isolation of the organism from diseased animals or by detection of seroconversion. An ELISA specific for M agalactiae and M m mycoides (LC) is described. The specificity of the antigens was demonstrated by immunoblotting and by ELISA using monospecific hyperimmune rabbit sera. A correlation of ELISA activity with other serological tests and isolation of mycoplasmas was carried out in two goat herds under field conditions. Results indicate the ability to detect subclinical mycoplasma infection and individual carrier goats on the basis of ELISA, a finding which will assist control procedures. PMID- 1716775 TI - Effect of pancreatectomy on plasma activities of amylase, isoamylase, lipase and trypsin-like immunoreactivity in dogs. AB - The contribution of the pancreas to the plasma activities of amylase, isoamylase, lipase and the concentration of trypsin-like immunoreactivity (TLI) in the dog was examined by measuring the activities of these enzymes before and after total pancreatectomy. Pancreatectomy was followed by a decrease in the concentration of TLI (from 6.2 +/- 0.3 micrograms litre-1 to 1.2 +/- 0.3 micrograms litre-1; P less than 0.001) and activity of isoamylase peak 4 (from 1257 +/- 105 iu litre-1 to 894 +/- 171 iu litre-1; P less than 0.05). Though significantly reduced, the activities of peak 4 isoamylase were still within the normal range for control dogs. Pancreatectomy did not significantly alter the activities of amylase, lipase or isoamylase peaks 1, 2 and 3. These findings provide strong evidence that the pancreas is not the sole source of circulating amylase, isoamylase and lipase activities. In contrast the marked reductions in TLI to values close to the limits of assay sensitivity suggest that TLI is derived from the pancreas alone. The results indicate that assay of circulating TLI provides a more sensitive and specific indicator of pancreatic exocrine mass than plasma amylase, lipase or isoamylase activities. PMID- 1716776 TI - Effect of cytokines and lipopolysaccharides on HIV infection of human macrophages. AB - Human blood-borne monocytes (MO) differentiating into mature macrophages (MAC) were cultured on hydrophobic Teflon membranes. The cells were infected with two different monocytotropic HIV isolates: HIV1D117III obtained from a perinatally infected child, and HIV2D194 obtained from an AIDS patient who suffered exclusively from neurological symptoms. Virus production monitored by reverse transcriptase activity and HIV-antigen ELISA in cell-free supernatant was of a high level and continued for several weeks. To investigate possible modulatory pathways interfering with HIV infection in MAC we tested various recombinant cytokines as well as bacterial lipopolysaccharides (LPS) in our culture system. Whereas interleukin-1 (IL1) accelerated and increased HIV replication in MO/MAC, the interferons (IFN) alpha, beta and gamma effectively suppressed or delayed infection depending on the concentration used. Suppression was seen at concentrations as low as 0.3 U/ml and was most effective when the IFN were given prior to infection. No effect was observed with IL6 up to 2,000 U/ml. LPS affected virus infection in a complex manner: at 1-100 ng/ml virus replication was inhibited, but it was enhanced at subnanogram concentrations (25-100 pg/ml). PMID- 1716777 TI - In vitro infection of macrophages by HIV: correlation with cellular activation, synthesis of tumour necrosis factor alpha and proteolytic activity. AB - Macrophages were obtained after differentiation of healthy donor monocytes. Seven to 9 days after isolation, cells were infected with HIV1. Tumour necrosis factor alpha (TNF alpha) biological activity, TNF alpha- and 1-6-fructose-diphosphatase gene expression and gelatinase activity were sequentially determined and correlated with viral infection and replication. TNF alpha was only detectable when mature viral particles were isolated in cell culture supernatants; 1-6 fructose diphosphatase mRNA was hyperexpressed in infected cells and its proteolytic activity was tremendously decreased during the early days postinfection. These results would seem to indicate that in human macrophage activation, cytokine secretion and microbicidal proteolytic activity are strongly modified by HIV infection. PMID- 1716778 TI - Interferon-regulated viral replication in chronically HIV1-infected promonocytic U937 cells. AB - To further document the role of interferons in the restriction of HIV1 replication in cells of the monocyte/macrophage lineage, the antiviral effects of alpha-interferon (IFN alpha) were studied in chronically HIV 1-infected promonocytic cells U937, in which IFN alpha was endogenously produced or to which recombinant IFN alpha was added. Protein analysis performed after immunoprecipitation of culture media revealed that the addition of anti-IFN alpha antibody led to an increase in the production of viral particles, whereas addition of IFN alpha caused its decrease in a dose-dependent manner, indicating that IFN alpha mainly affects viral assembly and virion release. However, the treatment of HIV 1-infected cells with IFN alpha did not cause an increase in the amount of intracellular viral proteins, suggesting that this cytokine might act on other steps in the viral life cycle. No decrease in the level of the 3 main types of viral RNA was observed by Northern blot analysis, indicating that proviral transcription was not restricted by IFN alpha. Furthermore, cotransfection experiments with TAR(trans-activation responsive)-element containing expression plasmids demonstrated that viral replication appears to be restricted at either a post-transcriptional and/or a translational level. These experiments suggest that the double-stranded (ds) inverted repeat TAR sequence present in HIV1 leader RNA may inhibit viral protein synthesis by phosphorylating the dsRNA-activated protein kinase induced by IFN. These results provide an impetus for achieving antiretroviral therapy based on the constitutive expression of IFN in monocytes and macrophages. PMID- 1716779 TI - Interferon induction by cell-cell interaction of HIV-infected monocytes/macrophages with lymphocytes. AB - An in vitro model of interferon (IFN) induction in HIV infection was established. IFN are induced by a cooperative mechanism, involving monocytes/macrophages (M/M) as well as lymphocytes. M/M non-lytically infected with HTLV-IIIB did not produce IFN, but were able to induce high titres of IFN activity when cocultured with peripheral blood lymphocytes, cell-cell contact being required. IFN alpha as well as IFN gamma were induced, as shown by neutralization with specific antisera. The antiviral activity of HIV-induced IFN was shown by infectivity reduction assays, and the immune-modulating capacity was demonstrated by activation of M/M leading to neopterin release. PMID- 1716780 TI - Antigen detection ELISAs: pretreatment of serum to reduce interference by specific host antibodies. AB - The pretreatment of serum to reduce interference by specific host antibodies was investigated as a means of improving the sensitivity of antigen detection ELISAs whilst screening serum samples. Four antigen detection assays based on monoclonal antibodies directed against antigens of the bovine filariid Onchocerca gibsoni were used in this study and, of these, three assays suffered a dramatic drop in sensitivity when detecting male O. gibsoni antigen in the presence of bovine serum as compared with antigen in buffer. A number of methods for pretreating serum to eliminate the problem of antibody interference with antigen detection were attempted, including heat and alkali treatments, detergent treatment of heat treated samples and the use of a reducing agent. The pretreatment of serum by boiling for 5 minutes in the presence of an equal volume of 0.1 M Na2EDTA pH 4.0 and recovery of the supernatant fluid following centrifugation at 16000 g was the most effective method of restoring the sensitivity of each of these three assays whilst screening bovine serum. Pretreatment of serum using this method produced up to a 512-fold increase in sensitivity compared with results obtained in assays with non-treated serum. PMID- 1716781 TI - [A morphometric study of 74 cases of oral lichen planus]. AB - We have carried out a morphometric study of the lesions of 74 patients with oral lichen planus. We have found as important data that the parameters differentiating the reticulated clinical lesions from the atrophic or erosive lesions are the epithelial thickness and the length of the dermic papillae. On the other hand, we have seen no difference between both types of lesions as regards the quantity of infiltrate in the connective tissue. PMID- 1716782 TI - [An unusual form of primary epithelioma of the mandible: odontogenic clear cell carcinoma. Clinical and morphologic study]. AB - One case of aggressive intramandibular epithelial tumor is presented. This tumor demonstrated an unusual pattern with areas of follicular ameloblastoma together with undifferentiated trabeculae or lobules composed of basophilic cells and of clear glycogenic-rich cells. The odontogenic nature of this tumour and its analogies with common ameloblastoma were demonstrated by electron microscopy, immunohistoenzymology and immunohistochemistry. The signification of such a neoplasm is discussed. PMID- 1716783 TI - Reexamination of the folding of BPTI: predominance of native intermediates. AB - Bovine pancreatic trypsin inhibitor (BPTI) continues to be the only protein for which a detailed pathway of folding has been described. Previous studies led to the conclusion that nonnative states are well populated in the oxidative folding of BPTI. This conclusion has broadly influenced efforts to understand protein folding. The population of intermediates present during the folding of BPTI has been reexamined by modern separation techniques. It was found that all well populated folding intermediates contain only native disulfide bonds. These data emphasize the importance of native protein structure for understanding protein folding. PMID- 1716784 TI - A combinatorial approach toward DNA recognition. AB - A combinatorial approach has been used to identify individual RNA molecules from a large population of sequences that bind a 16-base pair homopurine homopyrimidine DNA sequence through triple-helix formation. Fourteen of the seventeen clones selected contained stretches of pyrimidines highly homologous to the target DNA sequence (T.AT and C+.GC). In addition, these RNA molecules contained hairpin loops, interior loops, and nonstandard base triplets [C+(or C).AT, U.GC, G.GC, and A.AT] at various positions. Affinity cleavage experiments confirmed the ability of selected sequences to bind specifically to the target DNA. Systematic variation in both the target DNA sequence and buffer components should provide increased insight into the molecular interactions required for triple-helix-mediated recognition of natural DNA. PMID- 1716785 TI - Oligoclonal expansion and CD1 recognition by human intestinal intraepithelial lymphocytes. AB - A human intestinal intraepithelial lymphocyte (IEL) T cell line was established from jejunum to characterize the structure and function of the alpha beta T cell antigen receptors (TCRs) expressed by this population. Single-sided polymerase chain reaction (PCR) amplification cloning and quantitative PCR amplification of the TCR chains from the cell line and from fresh IELs demonstrated that IELs were oligoclonal. The IEL T cell line exhibited CD1-specific cytotoxicity and a dominant IEL T cell clone was CD1c-specific. Thus, human jejunal intraepithelial lymphocytes are oligoclonal and recognize members of the CD1 gene family. PMID- 1716786 TI - A mechanosensitive channel in whole cells and in membrane patches of the fungus Uromyces. AB - Bean leaf stomata provide a topographical signal that induces germlings of the phytopathogen Uromyces appendiculatus to develop specialized infection structures. Protoplasts from germ tubes of this fungus, when examined with patch clamp electrodes, displayed the activities of a 600-picosiemen mechanosensitive ion channel. This channel passes a variety of cations, including Ca2+, and is blocked by Gd3+ at 50 micromolar. This channel could transduce the membrane stress induced by the leaf topography into an influx of ions, including Ca2+, that may trigger differentiation. PMID- 1716788 TI - Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. AB - Serial human immunodeficiency virus type-1 (HIV-1) isolates were obtained from five individuals with acquired immunodeficiency syndrome (AIDS) who changed therapy to 2',3'-dideoxyinosine (ddI) after at least 12 months of treatment with 3'-azido-3'-deoxythymidine (zidovudine, AZT). The in vitro sensitivity to ddI decreased during the 12 months following ddI initiation, whereas AZT sensitivity increased. Analysis of the reverse transcriptase coding region revealed a mutation associated with reduced sensitivity to ddI. When this mutation was present in the same genome as a mutation known to confer AZT resistance, the isolates showed increased sensitivity to AZT. Analysis of HIV-1 variants confirmed that the ddI resistance mutation alone conferred ddI and 2',3' dideoxycytidine resistance, and suppressed the effect of the AZT resistance mutation. The use of combination therapy for HIV-1 disease may prevent drug resistant isolates from emerging. PMID- 1716787 TI - The roles of the subunits in the function of the calcium channel. AB - Dihydropyridine-sensitive voltage-dependent L-type calcium channels are critical to excitation-secretion and excitation-contraction coupling. The channel molecule is a complex of the main, pore-forming subunit alpha 1 and four additional subunits: alpha 2, delta, beta, and gamma (alpha 2 and delta are encoded by a single messenger RNA). The alpha 1 subunit messenger RNA alone directs expression of functional calcium channels in Xenopus oocytes, and coexpression of the alpha 2/delta and beta subunits enhances the amplitude of the current. The alpha 2, delta, and gamma subunits also have pronounced effects on its macroscopic characteristics, such as kinetics, voltage dependence of activation and inactivation, and enhancement by a dihydropyridine agonist. In some cases, specific modulatory functions can be assigned to individual subunits, whereas in other cases the different subunits appear to act in concert to modulate the properties of the channel. PMID- 1716789 TI - Serving rural families of developmentally disabled children: a case management model. AB - Family-centered, community-based case management is a key feature of new federal legislation mandating educational and preschool services for all developmentally disabled children. Social work skills, including counseling, planning, coordination, and knowledge of family and community dynamics, are germane to this task. The goal of family-centered case management is the empowerment of parents as caretakers and planners for their children. Highly skilled case managers will be particularly needed in rural areas where families must deal with scarce resources and other environmental constraints. The authors describe a program employing master's-level social work case managers who serve rural Appalachian families with developmentally disabled children. Included is a case illustration that delineates both case management activities and the contextual elements important in working in an Appalachian rural community. PMID- 1716790 TI - Effects of desmopressin and dextran on coagulation and fibrinolysis in healthy volunteers. AB - The effects of desmopressin and dextran on haemostasis and fibrinolysis were studied in four healthy volunteers. Both drugs were compared to placebo, each volunteer being subject to four experiments. Dextran 70 (30 g i.v.) moderately decreased VIII:C and vWF:Ag and slightly increased antithrombin III, also when haemodilution and diurnal variation were considered. Desmopressin (0.3 micrograms/kg BW i.v.), alone as well as in combination with dextran, increased VIII: C, vWF:Ag, protein C and tPA and decreased PAI-1. The combination of desmopressin and dextran stimulated coagulation and fibrinolysis and might be of relevance to surgical blood loss as well as to postoperative thromboembolism. PMID- 1716791 TI - The effect of alpha 2-macroglobulin on human polymorphonuclear cathepsin G. PMID- 1716792 TI - Absence of side effects of hydroxyethylstarch 200 in a porcine model of experimental arterial thrombosis. PMID- 1716793 TI - Uric acid as an inhibitor of biochemical changes induced by cyclophosphamide in mice. AB - The biochemical changes induced by uric acid, cyclophosphamide and uric acid plus cyclophosphamide were evaluated in Swiss albino male mice. Uric acid dissolved in water was administered orally in different doses for 7 days. Some mice from each group were injected intraperitoneally with cyclophosphamide (25 mg/kg) and sacrificed after 30 h. The blood of all animals was analyzed for uric acid levels. Uric acid was found not to affect the biosynthesis of nucleic acids and proteins in liver, testes and brain at 10-100 mg/kg/d. Pretreatment with uric acid provided significant protection against cyclophosphamide-induced impairment of DNA, RNA and protein biosynthesis. PMID- 1716794 TI - Effects of a secondary and a tertiary amine tricyclic antidepressant on cerebral biogenic amines as a function of mouse strain: a comparative neurotoxicological evaluation. AB - The effect of equal-dose regimens of amitriptyline and nortriptyline on the concentrations of serotonin, dopamine and major acidic metabolites was compared in 5 distinct brain regions as a function of inbred mouse strain. Amitriptyline increased to a greater extent the regional brain serotonin levels in the albino BALB/c mouse than did nortriptyline. Both drugs increased serotonin levels but decreased cerebral 5-hydroxyindoleacetic acid levels in some distinct brain regions of the black C57BL/6 mouse strain. The results suggest a strain-dependent differential increase in brain serotonin turnover in specific mouse strain brain regions which may account for the greater incidence of amitriptyline-induced sedation and seizures. The BALB/c mouse was also found to be more sensitive than the C57BL/6 strain to the action of both drugs on dopamine and major acidic metabolites with amitriptyline showing more regional brain potency than nortriptyline. The data suggest an increase in dopamine turnover particularly in brain areas associated with motor function and posture which may account for tricyclic-antidepressant-induced extrapyramidal disorders. The results also indicate that the C57BL/6 mouse strain may be of experimental value for studying the mechanism underlying tricyclic-induced adverse reactions relevant to sedation and movement disorders as a function of genetic predisposition. PMID- 1716795 TI - In vitro staining of islets of Langerhans for fluorescence-activated cell sorting. AB - A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets. PMID- 1716796 TI - The effect of octreotide acetate on meal-stimulated exocrine secretion in canine pancreatic autografts. AB - Octreotide acetate (Sandostatin), a long-acting somatostatin analogue, has been demonstrated to have an inhibitory effect on exocrine secretion in the neurally intact pancreas. This study was designed to evaluate the effect of this agent on exocrine secretion in the denervated canine pancreas, utilizing animals with pancreatic autografts and functioning pancreaticocystostomies. The rates of secretion of urinary (autograft) amylase (units/min) and bicarbonate (mM/min), over a five-hr interval, were determined in the basal state (group A, n = 10), after a bolus injection of 400 micrograms of Sandostatin (group B, n = 5), after a standard meal (group C, n = 5), or a meal preceded by 400 micrograms of Sandostatin (group D, n = 5). Basal secretion of amylase was decreased for 4 hr following Sandostatin, although this decrease was not significant. Conversely, basal bicarbonate secretion was not inhibited by Sandostatin. When compared with group C (22.4 +/- 3.2), a significant inhibition of meal-stimulated amylase release was demonstrated in group D (5.4 +/- 0.21, P = 0.0006) during the first hour after Sandostatin was given. This inhibition remained significant at 2 hr (group C = 38.5 +/- 5.2 versus group D = 9.4 +/- 0.8; P = 0.0006) and 3 hr (group C = 38.6 +/- 6.3 versus group D = 17.5 +/- 0.9; P = 0.0108) after Sandostatin was given. In addition, meal-stimulated bicarbonate secretion was significantly inhibited for 2 hr following Sandostatin (group C = 0.19 +/- 0.03 versus group D = 0.07 +/- 0.02, P = 0.0096; and group C = 0.23 +/- 0.03 versus group D = 0.10 +/ 0.01, P = 0.0018, respectively). These studies demonstrate that Sandostatin has a profound inhibitory effect on meal-stimulated enzyme and bicarbonate release in a denervated canine autograft model. Although the site of action of this agent remains to be defined, Sandostatin may have therapeutic potential in clinical pancreas transplantation. PMID- 1716797 TI - In vivo effect of FK506 on human pancreatic islets. AB - The purpose of this study was to evaluate the in vivo effect of FK506 on human pancreatic islets. Twenty-five nude mice were made diabetic by one intravenous injection of streptozotocin. Approximately 600 islets were administered in the renal subcapsular space 3-5 days following streptozotocin administration. One week after transplantation, the mice were divided in four groups. In group 1, the animals received 1 injection of 0.5 ml of diluent i.p. daily for one week. In groups 2, 3, and 4 the treatments were daily i.p. injection of 0.3, 1, and 3 mg/kg FK506, respectively. After treatment, the functional integrity of the transplanted human islets was tested by measuring the plasma glucose and human C peptide response to intraperitoneal glucose injection in groups 1 and 4. IPGTT alone was assessed in groups 2 and 3. The results indicate that i.p. administration of FK506 for one week at a dose 0.3 mg/kg/day did not result in any significant alteration of glucose disappearance and C-peptide response to IPGTT. Higher doses of FK506 produced a significant delay in glucose disappearance in groups 3 and 4, and a significant inhibition of glucose-mediated C-peptide response in group 4. PMID- 1716798 TI - Inhibition of complement-mediated endothelial cell cytotoxicity by decay accelerating factor. Potential for prevention of xenograft hyperacute rejection. AB - Complement plays a major role in hyperacute rejection of discordant xenografts. In immediately vascularized xenografts, such as porcine organs to humans, C activation contributes to triggering of endothelial cell activation and adhesion of leukocytes and platelets to the endothelial cells, which is followed by thrombosis and tissue necrosis. We investigated the potential utility of the membrane-associated inhibitor of C, decay accelerating factor (DAF), in the prevention of C-mediated tissue injury. We used an in vitro model of xenotransplantation consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and C. Because C inhibitors such as DAF may be relatively species-specific, we tested whether human DAF would incorporate into porcine endothelial cells and function to inhibit cytotoxicity of such cells by human C. We found that purified radiolabeled human DAF incorporated into porcine endothelial cells in a dose dependent manner and that human DAF very significantly protected the endothelial cells from the cytotoxic effect of human C. PMID- 1716799 TI - Prolongation of renal allograft survival in the rat treated with amniotic fluid. AB - To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy. PMID- 1716800 TI - Enhancement of allograft survival by donor-specific transfusion one day prior to transplant. Importance of timing and specificity when DST is given with cyclosporine. AB - Donor-specific transfusion effects were studied in the ACI-to-Lewis rat heterotopic heart allograft model using cyclosporine immunosuppression. Low-dose CsA for 1 week plus a single fresh or stored DST given 1 day before allografting significantly prolonged graft survival over CsA therapy alone (median survival time 23.5 days vs. 10 days, P less than 0.01), but third-party transfusion did not (11.5 vs. 10 days, NS). When CsA was started at the time of DST and continued for 2 weeks, maximal graft enhancement was achieved after just one DST. DST/CsA was equally efficacious if given on any day before transplantation, provided CsA was started on the same day as the transfusion. However, pretransplant DST given without CsA shortened subsequent graft survival of day -1 DST/CsA treatment (14.5 days, n = 6, vs. 60 days for controls, n = 10; P less than 0.01). The addition of methylprednisolone to the DST/CsA protocol had no effect on graft survival (51 vs. 53 days, P = NS), but extending the period of postoperative CsA therapy for 4 weeks at reduced dose (2.5 mg/kg/day) significantly prolonged median survival (111 days, n = 11) and resulted in 45% permanent engraftment (greater than 120 days survival). CsA permits graft enhancement with a single DST as early as 1 day before grafting. This avoids the risk of sensitization from DSTs and can extend DST use to cadaveric graft recipients. PMID- 1716801 TI - Comparison of the effects of FK506 and cyclosporine on bone mineral metabolism in the rat. A pilot study. PMID- 1716802 TI - Induction of antibodies to canine herpesvirus in mice by immunization with anti idiotypic antibodies. AB - Anti-idiotypic antibodies (anti-Id Abs) were produced in rabbits after inoculation with two mouse monoclonal antibodies (mAbs) directed against canine herpesvirus (CHV) glycoproteins (gps). One of the mAbs, 12H11, was directed against an epitope on gp 145/112 of CHV which induced virus neutralizing (VN) antibodies and against a cross-reacting epitope on the gp 143/108 of feline herpes-virus type 1 (FHV-1). The other mAb, 11F7, was directed against epitopes on CHV gp47 which induce VN and hemagglutination-inhibition (HAI) antibodies. Using VN-inhibition and HAI-inhibition assays with CHV and FHV-1, the anti-Id Abs obviously inhibited the activities of autologous mAbs, suggesting that anti-Id Abs mimic the epitopes of CHV gp 145/112 or FHV-1 gp 143/108 and CHV gp47 by binding the anti-combining site of the mAbs. These anti-Id Abs, when injected into mice, elicited specific CHV-neutralizing and HAI antibody responses, and one of them also elicited a specific FHV-1-neutralizing antibody response. These data supported the idea that immunization with anti-Id Ab can induce specific VN antibody response, as has been theorized by other workers. PMID- 1716803 TI - Family structure and children's health: United States, 1988. AB - This report describes the family arrangements of children 17 years of age and under and the association between family structure and various demographic and socioeconomic characteristics of the children and their families. The focus of the report is on the relationship between family structure and children's health and well-being. Physical health, educational attainment, and emotional health are compared for children in the four most common types of family. Data are from the 1988 National Health Interview Survey on Child Health. PMID- 1716804 TI - [Programmed ventricular stimulation in the evaluation of the clinical significance of premature ventricular contractions]. AB - The authors subjected 34 patients (27 men and 7 women) to invasive electrophysiological examination. All patients had early ventricular contractions class 3-5 according to Lown which were recorded during ECG monitoring in bed or by Holter's system. A pathological excitability of the ventricular myocardium was found during programmed stimulation in 29% of the subjects. The excitability of the heart muscle was substantially enhanced by the incidence of ischaemic heart disease and reduced left ventricular function. The decreased frequency and severity of early ventricular contractions during bicycle ergometry, the absence of palpitations and syncopes increases the probability of normal excitability of the ventricular myocardium. PMID- 1716806 TI - [Direct positive DDP 111 as a possible means of documentation for electrophoresis]. AB - The positivepaper DDP 111 manufactured in the Fotopapierwerk Dresden is presented. It allows to make copies (1:1) of electrophoreses. Independently of the used staining procedure it is a simple, quickly and cameraless possibly to obtain reproductions with a high quality. The DDP 111 allows to resolve the problem of documentation of electropherograms (especially of wet flat gels) and to build up collections and data files respectively. PMID- 1716805 TI - [Septic shock: pathophysiology, therapeutic approaches, perspectives]. PMID- 1716807 TI - Overcoming inhibition of antibody responses to a malaria recombinant vaccinia virus caused by prior exposure to wild type virus. AB - Pre-injection of mice with vaccinia virus inhibited the subsequent antibody response to a recombinant polypeptide expressed by vaccinia virus. The inhibition was overcome following additional challenges with recombinant vaccinia virus. This suggests that a potential disadvantage in vaccinia-immune individuals can be circumvented and may be outweighed by the advantages of the vector. PMID- 1716808 TI - Epitopes of the human malaria parasite P. falciparum carried on the surface of HBsAg particles elicit an immune response against the parasite. AB - The development of recombinant subunit vaccines against pathogenic organisms requires not only the identification of epitopes eliciting a protective immune response but also suitable carriers with adjuvant function. B- and T-cell epitopes of the malaria vaccine candidate gp190 were selected on the basis of a systematic search along the gp190 molecule and by computer prediction based on the amino acid sequence. Using some of the epitopes identified, we have redesigned the surface of the hepatitis B surface antigen lipoprotein particles by replacing the major antigenic determinants with malaria-specific sequences of up to 61 amino acids in length. Upon expression via vaccinia virus the hybrid particles elicit an anti-gp190 immune response in animals. Monoclonal antibodies derived from such infections recognize the native parasite. PMID- 1716809 TI - Model using a peptide with carrier function for vaccination against different pathogens. AB - It has been shown that pre-immunization with a protein can inhibit the antibody response to a new B-cell sequence which is coupled to the protein (epitope specific suppression). By utilizing a peptide with carrier function from the protein, rather than the entire protein, the antibody response to the new B-cell sequence is enhanced in protein-primed mice. The present results extend this observation by showing that mice which have been pre-immunized with a protein followed by a B-cell sequence linked to a carrier peptide from the protein produce an enhanced antibody response to a new B-cell sequence linked to the same carrier peptide sequence. This indicates that it would be possible to reuse a carrier peptide sequence coupled with newly defined protective B-cell epitopes in a given individual to achieve immunity against new pathogens. PMID- 1716810 TI - In vivo evidence of immunological masking of the Vibrio cholerae O antigen of a hybrid Salmonella typhi Ty21a-Vibrio cholerae oral vaccine in humans. AB - The immunogenicity of the live oral hybrid vaccine organism Salmonella typhi Ty21a/V. cholerae Inaba (EX210) following its growth in media containing variable concentrations of supplemental galactose was examined in human volunteer subjects. The local intestinal IgA-specific antibody responses to both typhoid and cholera lipopolysaccharide (LPS) preparations were determined. It was observed that the immunogenicity of the galactose-independent Vibrio cholerae O antigen in vivo was dependent upon the variation in galactose-dependent long chain S. typhi O antigen production which was directly proportional to the media galactose concentration. It is likely that this observation was a result of steric hindrance of the presentation of the V. cholerae O antigen by S. typhi Ty21a in the presence of the longer, immunodominant S. typhi Ty21a O antigen. This observation may have relevance to the use of S. typhi vectors in vaccine development involving the presentation of LPS-associated heterologous antigens. PMID- 1716811 TI - [Characterization of collagen of canine intervertebral disks using the N-methyl benzothiazol-2-on-hydrazone reaction]. AB - Collagen types were studied in the lumbar intervertebral discs of non chondrodystrophic dogs, 1 to 3 years old. Parts of intervertebral discs were frozen and sections were subjected to the 3-methyl-benzothiazol-2-on-hydrazone (MBTH) method, which permits differentiation of genetic types and maturation stages of collagen. The annulus fibrosus showed a blue (collagen I) and a violet colour (collagen II) at the external zone. The internal zone was more homogeneous and contained a mixture of both colours (collagen I and II). The nucleus pulposus showed only the violet colour, indicative of collagen II. Discs were also dissected at three different zones and submitted to an extraction technique to obtain collagen, then subjected to SDS-polyacrylamide gel electrophoresis and determination of spectrophotometric reactions of MBTH. Both studies demonstrated type I and type II collagens in the external and internal zones of the annulus fibrosus, but only type II in the nucleus pulposus. Collagen characterization, using this technique, would allow for evaluation in pathologic events of the intervertebral discs as well as for establishing the degree of alteration. PMID- 1716812 TI - Effects of galanin on electrical activity of pancreatic islet cells. AB - Intracellular microelectrode technique was used to investigate the effect of galanin on the electrical activity of beta cells of Langerhans islets of mice. Galanin 0.15 and 0.3 mumol/L in the perfusion medium inhibited the basic electrical activity induced by glucose 5.5, 11.1 and 20 mmol/L in decreasing the frequency and amplitude of the spikes. In some cells, during galanin inhibition there was a hyperpolarization after the spike. Verapamil (30, 60 and 90 mumol/L), a blocker of voltage-dependent Ca channels, blocked dose-dependently the electrical activity induced by glucose and attenuated the depolarization induced by KCl 50 mmol/L. Galanin 0.3 mumol/L also attenuated the depolarization induced by KCl 50 mmol/L, similar to the effect of verapamil. The results suggest that the effect of galanin on inhibition of the electrical activity of beta cell might be due to blocking of voltage-dependent Ca2+ channels. PMID- 1716813 TI - Mercaptodextran--a new copper chelator and scavenger of oxygen radicals. AB - The therapy of copper poisoning with mercaptodextran inhibits the copper-induced haemolysis, whereas 2,3- dimercaptopropanesulfonic acid (DMPS) may accelerate such haemolysis. Some aspects of the mechanisms of these effects were investigated. The possible generation of activated oxygen species during the interaction of Cu++ and chelating thiols was studied using a chemoluminiscent method detecting oxygen radicals. It was found that incubation of DMPS with copper ions or erythrocyte membranes was accompanied by generation of oxygen radicals. Mercaptodextran added to similar suspensions did not lead to oxygen radical production. And unlike DMPS, mercaptodextran acted as a scavenger of radicals generated by the xanthine oxidase/acetaldehyde system. The different ability of the chelating thiols to cope with free radicals may explain their different potentials to protect against copper-induced haemolysis. Our results also indicate that mercaptodextran may be a useful therapeutic agent in cases of haemolytic crisis in Wilson's disease. PMID- 1716814 TI - [Effects of 7 drugs on cutaneous blood flow evoked by electric stimulation of rat sciatic nerve]. AB - The non-invasive technique of laser-Doppler flowmeter (LDF) was used to measure the change of cutaneous blood flow evoked by electric stimulation of rat sciatic nerve. The sciatic nerve was cut centrally and placed on bipolar electrodes. Drug were infused in a carotid artery. Electric stimulation of the sciatic nerve containing sensory afferent fibers caused an increase in cutaneous blood flow. This increase was not modified by the ia infusion of adrenergic blocking agents (phentolamine, 0.1 mg/kg and propranolol, 0.5 mg/kg), anti-muscarinic agent (atropine 0.5 mg/kg), anti-histamines (mepyramine, 10 mg/kg and cimetidine, 10 mg/kg) and 5-HT antagonist (methysergide, 0.5 mg/kg). Pretreatment with capsaicin (50 mg/kg, sc) in the newborn rat or the ia infusion of spantide (1, 2 mumol/kg) significantly inhibited the the stimulation-induced increase of the blood flow. These results suggest that substance P released from the peripheral endings of sensory nerve may be involved in vasodilation following electric stimulation of the sciatic nerve in rat. PMID- 1716815 TI - [Enhancement of antitumor activity of bleomycin A5 in mouse sarcoma 180 cells in vitro and in vivo by verapamil]. AB - Verapamil (Ver), a calcium influx blocker, enhanced the cytotoxicity of bleomycin A5 (BLM) in cultured S-180 cells. IC50 of BLM to the cells was about 2.6 micrograms/ml when the cells were treated with BLM alone, but when the cells were treated with BLM plus Ver 1, 5, 10 micrograms/ml, the IC50 were 1/1.2, 1/5.2, 1/7.4 of the cells treated with BLM alone, respectively. In colony test, the survival fractions of the cells treated with BLM 20, 60, 100 micrograms/ml plus Ver 60 micrograms/ml were 1/7, 1/8, 1/11 of the cells treated with BLM alone, respectively. Ver enhanced the growth inhibitory actions of BLM in mice bearing S 180 sarcoma by 60% or over. The mean survival the growth inhibitory actions of BLM in mice bearing S-180 sarcoma by 60% or over. The mean survival time of the mice bearing S-180 ascites sarcoma treated with BLM plus Ver was prolonged 21% from that of the mice treated with BLM alone. The results suggest that Ver may be used clinically as an antitumor enhancer of BLM. PMID- 1716816 TI - Modulatory effect of oxytocin and arginine-vasopressin on functional properties of three types of acetylcholine receptors in molluscan neurons. AB - It was found that 10(-7)-10(-8) mol/l oxytocin (OT) or arginine-vasopressin (AVP) applications produced effects on functional properties of three types of acetylcholine (ACh) receptors on various neurons identified in the ganglia of Helix pomatia under voltage clamp conditions. OT and AVP depressed ACh-induced sodium-potassium-calcium current in neuron RBc3 without shift of reversal potential. Our data show that there are two types (subtypes) of ACh receptors which are connected with chloride current in neurons of Helix pomatia. OT decreased ACh-induced chloride current in neuron D4 and enhanced ACh-induced chloride current in neuron D5. These effects of OT were mimicked by the intracellular injection of cyclic AMP or application of isobutylmethylxanthine and an active cyclic AMP analog. AVP as a rule mimicked the effects of OT on functional properties of ACh receptors, but in neuron F8 effects of OT and AVP were independent. The present results suggest that cyclic AMP may be the second messenger mediating the OT- and AVP-induced modulations of functional properties of three types of ACh-receptors. PMID- 1716817 TI - Interferon inhibition of neoplastic phenotype in cell lines harbouring human papillomavirus sequences. AB - The transformed phenotype is markedly affected by Interferon (IFN) long term treatment in HeLa and Siha cell lines. Two months of continuous exposure to 100 and 1000 International Units (IU)/ml of human recombinant alpha IFN causes a drastic reduction of anchorage independent growth in soft-agar. No effect on in vitro growing potential was observed at the same concentrations. This antiproliferative effect in soft-agar was more pronounced in Siha than in Hela cells and was concentration and time dependent. The Human Papillomavirus (HPV) sequences inserted in the cell genome apparently were unaffected as their RNA expression, indicating that IFN may modulate aspects of transformed phenotype regulated by genes other than HPV sequences. PMID- 1716818 TI - ELISA analysis of soybean trypsin inhibitors in processed foods. AB - Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed. PMID- 1716819 TI - Nutritional value of processed rapeseed meal. AB - Supplementation of iodine at the level of 3.5 ppm reduced weight gain of the rats fed rapeseed oil meal (ROM) diets. Treatment of ROM with ammonia at the level of 2 or 4% tended to increase metabolizable energy value and availability of dry matter, crude protein and crude ash of ROM in the chicken. Potential goitrin level of ROM was reduced by ammoniation at 6% level. On the other hand, level of potential isothiocyanantes increased by ammoniation. Treatment of ROM with ammonia at the level of 3% and above reduced weight gain of the chickens fed treated ROMs. Weight of thyroid glands of the birds increased as the level of ammoniation of ROM increased. Supplementation of Avoparcin to the diets containing ROM improved weight gain and dressing percentage of the broiler chickens. PMID- 1716820 TI - Central (third) fat pad of the upper eyelid. AB - A clinical study on the surgical anatomy of the upper-eyelid fat pads was performed on 55 consecutive patients who underwent a blepharoplasty. It was confirmed that the periorbital fat is encapsulated in compartments and that the number of fat pads varies. In 56% of the cases there were two fat pads and in the 44% three fat pads in the upper eyelid. The third fat pad is anatomically and histologically an accessory medial extension of the lateral fat pad. However, for the sake of clarity, the term central fat pad of the upper eyelid is proposed as a denominator of this structure. The purpose of this article is to make the less experienced surgeons aware of variations in the configuration of the periorbital fat and to remind them that after two fat pads are removed from the upper eyelid there might still be a third. PMID- 1716821 TI - Patterns of histamine release in the brain. AB - The pattern of histamine release has been investigated in various brain areas of anaesthetized cats and conscious, freely moving rats by the push-pull technique. In the hypothalamus, medial amygdaloid nucleus and mamillary body of the anaesthetized cat, histamine was found to be released according to an ultradian rhythm with a frequency of 1 cycle per 1-2 h. Additionally, oscillations have been observed in the medial amygdaloid nucleus and mamillary body with a frequency of 1 oscillation per 10 min. In the posterior hypothalamus of the conscious rat, histamine is also released rhythmically with a frequency of 1 cycle per 1.5 h. Moreover, the release rate of histamine is increased in the night. PMID- 1716822 TI - Influence of catecholamines on the in vivo release of histamine in the hypothalamus. AB - The hypothalamus of conscious, freely moving rats was superfused with artificial CSF through push-pull cannulae and the release of endogenous histamine was determined radioenzymatically in the superfusate. Superfusion with potassium chloride enhanced the release rate of histamine. The effect of potassium chloride was abolished by alpha-fluoromethylhistidine. Noradrenaline (alpha 1- and alpha 2 agonist) and clonidine (alpha 2-agonist) decreased the release rate of histamine and inhibited the potassium-induced histamine release. In preliminary experiments, yohimbine (alpha 2-antagonist) seemed to increase the release rate of histamine in the superfusate. beta-Agonists and antagonists (isoprenaline, salbutamol, propranolol) did not influence the release of histamine. PMID- 1716823 TI - Mast cells from human gastric mucosa: a comparative study with lung and colonic mast cells. AB - Mast cells isolated from human gastric mucosa released histamine on challenge with IgE-directed ligands and calcium ionophores but were essentially unresponsive to a variety of non-immunological stimuli. Moreover, immunologically induced histamine secretion from these cells was inhibited by a number of anti allergic agents including anti-asthmatic chromones, beta-adrenoceptor agonists and phosphodiesterase inhibitors. In total, these data indicate that mast cells from the human gastric mucosa are in many respects functionally similar to their lung and colonic counterparts. PMID- 1716824 TI - Properties of tuberomammillary histamine neurones and their response to galanin. AB - Histaminergic neurones in the tuberomammillary nucleus possess electrophysiological properties which distinguish them from other neurones in their neighborhood. Their resting potential is -50 mV and they are spontaneously active at about 2 Hz in a slice preparation. They display a transient outward rectification and an anomalous inward rectification. Bath application of galanin (0.1 microM) reduced their firing rate significantly and hyperpolarized them slightly. PMID- 1716825 TI - The human skin mast cell: a comparison with the human lung cell and a novel mast cell type, the uterine mast cell. AB - The functional and histochemical properties of isolated mast cells from human skin, uterine myometrium and lung parenchyma were compared. The skin cell showed a marked difference in responsiveness to both secretagogues and anti-allergic compounds when compared to mast cells from the uterus and lung. The latter cell types responded more strongly to immunologically-directed ligands and calcium ionophores than the skin cell. However, the skin cell released histamine in response to a wide variety of polycationic compounds whilst the lung and uterine cells were essentially refractory to these agents. The antiallergic drugs, disodium cromoglycate (DSCG) and nedocromil sodium, were weakly inhibitory against the skin cell but were in contrast more effective against the lung and uterine cells. PMID- 1716826 TI - Acid blockade by omeprazole or ICI 162846 in a chronic duodenal ulcer model. AB - Oral treatment with the H2-antagonist ICI 162,846 or omeprazole for five days inhibited both basal and pentagastrin stimulated acid secretion by 50% or more in mice. Either treatment increased the luminal secretion of histamine in the basal (12-fold) and stimulated (9-fold) states. Mice treated with the H2-antagonist had a 27% reduction (p less than 0.05) in mural histamine in the acid-producing area of the stomach. Mice were treated so as to induce duodenal ulcers (abscopal model) and were then treated with the H2-antagonist ICI 162,846, omeprazole or vehicle, orally for one week. Fewer duodenal ulcers were found in animals receiving drug treatments than in the oral vehicle group. Both the H2-receptor antagonist and the proton pump blocker inhibit acid production; acid blockade by either drug is accompanied by a massive increase in secretion of histamine. This rise was associated with depletion of the gastric histamine store only with H2 receptor blocker. Both means of acid inhibition reduce the formation of ulcers in this model. PMID- 1716827 TI - Mast cell regulatory effect on lymphoid cell proliferation. AB - It has been shown rat mast cells (MC) can modulate lymphocyte proliferation in vitro. Depending on concentrations tested both serosal MC and their supernatants enhanced the spontaneous and T-mitogen-induced proliferation of spleen and lymph node cells. In addition T-mitogen-induced thymocyte proliferation was also increased. The enhancing effect of MC on lymphoid cell proliferation appeared after MC and lymphocytes were cocultured for 24, 48 or 72 h. The highest enhancing action of MC was observed when MC and lymphocytes were plated simultaneously. In contrast, when MC were added 24 or 48 h after the start of lymphocyte culture, the enhancing action of MC decreased or was abolished, respectively. No dependence was found between histamine concentration in MC supernatants and the enhancing activity of supernatants. After chromatographic separation of MC supernatants the fractions with molecular weights between 1-6 KDa augmented lymphoid cell proliferation. PMID- 1716828 TI - Vascular responses to histamine at low temperatures in normal digital skin and Raynaud's phenomenon. AB - A defective histaminergic dilating system in the digital vasculature has been proposed for the pathophysiology of Raynaud's phenomenon but this is not supported by studies of digital intradermal responses to histamine or agents which cause histamine release. The vascular responses (measured by planimetry and laser Doppler flowmetry) of digital skin over the middle phalanx to intradermal histamine, compound 48/80 and Substance P have now been studied at low temperatures (because it is in the cold that Raynaud's phenomenon occurs) in normal controls and patients with primary Raynaud's phenomenon. A cold-related attenuation of mast cell histamine release by compound 48/80 was observed in both normal and Raynaud's subjects. These results do not support a major histaminergic defect in the pathogenesis of Raynaud's phenomenon. PMID- 1716829 TI - Histamine release induced by opioid analgesics: a comparative study using porcine mast cells. AB - Studies in vitro have demonstrated histamine release induced by opioid analgesics from rat peritoneal mast cells and human skin mast cells. In humans, elevated plasma histamine levels have also been found following intravenous or oral administration. This study compared the reactivity of five opioid analgesics on mast cell suspensions obtained from porcine heart, kidneys, liver and lungs. The five compounds investigated were hydromorphone, levomethadone, morphine, pethidine and oxycodone. Both morphine and hydromorphone produced little histamine release from all cell types tested. The other drugs demonstrated clear differences between the different cell populations. Thus, the cardiac cells responded most strongly to oxycodone but were the least responsive to pethidine. These differences, if confirmed in vivo, could indicate which drugs may be more suitable for patients with different underlying disease states. PMID- 1716830 TI - Histamine release during paediatric cardiopulmonary bypass. AB - Histamine release occurs during paediatric cardiopulmonary bypass at the time of removal of the aortic cross-clamp. Left atrial histamine levels are significantly (p less than 0.02) higher than right atrial levels at the time of reventilation of the lungs. These results suggest that histamine is released from the pulmonary vasculature following reperfusion. PMID- 1716831 TI - In vivo and in vitro tests in anaphylactic reactions to anaesthetic agents. AB - The majority of patients with anaphylactoid reactions (AR) under general anaesthesia showed histamine release from basophil leucocytes (HRL) by neuromuscular blockers in vitro (none in normals) and high levels of serum IgE antibody (detected by the 'new' paper radioallergosorbent test, RAST; little or none in normals) which reacted with the quaternary ammonium group of choline, and less frequently of alcuronium, strongly suggesting that IgE antibody, HRL and AR are related. Skin tests on their own are of limited value. PMID- 1716832 TI - Comparison of histamine release from peritoneal mast cells derived from diabetic and control rats. AB - Clinical and experimental diabetes are associated with an increased number of mast cells and elevated tissue histamine concentrations. This study compared histamine release from peritoneal mast cells derived from diabetic and control rats. Experimental diabetes was induced by a single i.v. injection of streptozotocin (50 mg/kg body weight). Measurement of plasma glucose levels confirmed the diabetic state. Peritoneal mast cells were stimulated for 10 min with the lectin concanavalin A (0.5-100 micrograms/ml) in the presence or absence of phosphatidylserine, clinical dextran (0.6-1200 micrograms/ml) in the presence of phosphatidylserine, the calcium ionophore A23187 (0.1-1 microM) or the basic releasing agent compound 48/80 (0.1-10 micrograms/ml). Histamine release induced by these agents was similar in both populations. Further studies will compare the differences in histamine release from mast cells isolated from different tissues, e.g. heart and lung. In addition, physiological stimuli which are altered in the diabetic state (e.g. hyperosmolalar solutions and free radical generating systems) are under investigation. PMID- 1716833 TI - In vitro and in vivo studies of radiographic contrast media-induced histamine release in pigs. AB - Routine clinical use of radiographic contrast media (RCM) causes adverse reactions in some patients. To elucidate the mechanisms of these reactions both in vitro and in vivo studies are necessary. In this study, RCM-induced histamine release from isolated mast cells was compared with the in vivo release of histamine and cardiovascular symptoms using a porcine model. The 2 non-ionic preparations examined (Solutrast and Ultravist) released little or no histamine from the 4 cell types tested (porcine pulmonary, cardiac, hepatic, and renal mast cells). The 4 ionic preparations (Angiographin, Hexabrix Rayvist, and Telebrix) caused histamine release from most of the cell suspensions. In almost all cases, the cardiac mast cells were the most sensitive followed by the hepatic mast cells. All 4 RCM tested in vivo produced elevated plasma histamine levels in some animals. The highest incidence was observed using the ionic, high osmolal Rayvist (6 of 12 animals), followed by the non-ionic RCM with the lowest osmolality Ultravist (4 of 12 animals). In vivo, mechanisms in addition to direct histamine release may also be involved in RCM-induced adverse reactions, since low osmolal, non-ionic RCM can cause elevated plasma histamine levels without in vitro release. The susceptibility of cardiac mast cells to RCM-induced histamine release suggests that patients undergoing e.g. coronary angiography may be especially at risk for an adverse reaction. PMID- 1716834 TI - Characteristics of histamine and serotonin release from rat mast cells induced by thymic peptides. AB - Thymopoietin peptides release histamine and serotonin from rat mast cells. The release has the characteristics of other basic secretagogues in that it occurs rapidly, does not require extracellular Ca2+ ions, and is inhibited by benzalkonium chloride (BAC). These similarities indicate that chemically different basic compounds have common mechanisms for histamine and serotonin release from rat mast cells. PMID- 1716835 TI - Effect of chloroquine on isolated mast cells. AB - Chloroquine liberated a relatively low amount of histamine from isolated rat mast cells. In a dose-dependent way, this drug inhibited histamine liberation from mast cells stimulated with compound 48/80, A23187, concanavalin A plus phosphatidylserine (Con A + PS) and abolished histamine liberation induced by exaprolol. The degranulation was decreased in cells stimulated with 48/80, Con A + PS and exaprolol. Chloroquine significantly inhibited the formation of thromboxane B2 in mast cells stimulated with 48/80, Con A + PS and A23187. We assume that chloroquine interferes with mast cells at a plasmic membrane site as well as intracellularly. PMID- 1716836 TI - The effect of nitric oxide generators on ischemia reperfusion injury and histamine release in isolated perfused guinea-pig heart. AB - Experiments were carried out to provide evidence of the effect of L-arginine (L Arg), its analogue NG-monomethyl-L-arginine (MeArg) and of some nitrovasodilators (sodium nitroprusside, NaNP; 3-morpholino-sydnonimine, SIN-1) which spontaneously release nitric oxide (NO) on ischemia-reperfusion injury, histamine release and mast cell degranulation, occurring after multiple ligature and release of the left anterior descending (LAD) coronary artery in isolated perfused guinea-pig hearts. The reopening of the LAD coronary artery leads to a release of histamine related to a decrease in microdensitometry of cardiac mast cells and to calcium overload. The perfusion of the heart with NO-donors significantly reduces either the release of histamine, the loss of mast cell metachromasia and the overload of calcium. These effects were potentiated by SOD. The results suggest that the endogenous formation of NO and molecules able to generate NO have a role in the prevention of post-ischemic tissue injury. PMID- 1716837 TI - A place for free radicals in platelet-derived histamine releasing factor (PDHRF) and evidence that histaminergic receptors modulate platelet aggregation. AB - The release of histamine from rat serosal mast cells induced by coincubation with resting and activated human platelets, or by exposure of mast cells to supernatants obtained from activated platelets, is significantly reduced by anti free radical interventions, and is coupled with the generation of membrane lipid peroxidation products. These results suggest free radical participation in the activity of PDHRF. Human platelets possess specific binding sites for an H1 receptor antagonist, suggesting that H1-receptors could modulate the intracellular calcium levels in a pro-aggregatory fashion. PMID- 1716838 TI - Rat mast cells synthesize a nitric oxide like-factor which modulates the release of histamine. AB - Rat serosal mast cells were evaluated for their capacity to generate a nitric oxide-like factor by two bioassays: inhibition of platelet aggregation and stimulation of mast cell guanylate cyclase. Incubation of mast cells with human washed platelets, both treated with indomethacin, inhibited thrombin-induced platelet aggregation which was potentiated by superoxide dismutase and reversed by oxyhaemoglobin. When mast cells alone were stirred at 1000 rpm, a time dependent increase in the levels of their cGMP but not cAMP was observed. Preincubation of mast cells with NG-monomethyl-L-arginine significantly enhanced E. coli lipopolysaccharide-evoked histamine release. Our results show that mast cell histamine release can be modulated by an intrinsically generated nitric oxide-like factor. PMID- 1716839 TI - Comparison of histamine release in cord blood and adult blood. AB - Histamine release (HR) after stimulation with anti-IgE, concanavalin A (ConA) and Formyl-Met-Leu-Phe (FMLP) from 97 cord blood samples was compared to results obtained in identically treated blood samples from adults. The maximal HR obtained with anti-IgE did not differ significantly from the values obtained in adult blood, although a ten times higher concentration of anti-IgE was required for maximum release. Passive sensitization with IgE-rich plasma caused a significant increase in maximal anti-IgE-induced HR in the majority of cord blood samples, and the dose-response curve was similar to that obtained in adult blood. Challenge with ConA and FMLP caused a HR similar to that seen in adult blood, but the close correlation between anti-IgE- and concanavalin A-induced HR seen in adult blood was absent in cord blood. PMID- 1716840 TI - Effect of picumast on histamine release from rat cardiac and peritoneal mast cells. AB - Compound 48/80-induced histamine release (HR) from the isolated perfused rat heart was markedly and significantly inhibited by picumast (PIC), possibly by acting as a calmodulin antagonist (CMA) or membrane stabilizer. Trifluoperazine (TFP, another CMA in clinical use) had a similar effect. However, an action as CMA being the basis of inhibition of HR could not be confirmed in another 'allergy' model, namely HR from rat peritoneal mast cells (RPMC). PIC, TFP and two other CMA, W7 and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide) failed consistently to inhibit 48/80-induced HR from RPMC, and when used on their own at high concentration these compounds caused HR. PIC and TFP also potentiated the heat-induced haemolysis of rat erythrocytes, i.e. lacked membrane stabilizing effect in this model. PMID- 1716841 TI - Characteristics of deoxycholic acid-induced histamine release from mast cells of guinea-pig rectocolonic mucosa and rat peritoneal cavity. AB - Deoxycholic acid (DA) caused a dose-related release of histamine (HR) from mast cells of rat peritoneum (RPMC) and mucosal cells of guinea pig rectocolon (RCMC). In both cell populations, DA-induced HR was: (1) accompared by a parallel release of lactate dehydrogenase (LDH), (2) not affected by metabolic inhibitors, (3) dependent on time of incubation, temperature and pH, and affected by Ca++ concentration in RPMC but not in RCMC. DA-induced HR from RCMC may be involved in certain functional disorders of the colon. PMID- 1716842 TI - A novel source of mast cells: the human placenta. AB - The presence of moderate amounts of histamine in the human placenta was confirmed (0.72 +/- 0.10 microgram/g wet weight), and the hitherto unknown storage site of this biogenic amine was elucidated. Mast cells were identified by their characteristic morphology, staining reactions and secretory activity measured in terms of histamine release. Human placental tissue contains 7.6 x 10(5) mast cells/g wet weight, identified by staining with toluidine blue or alcian blue, and these cells were positive for chloro-acetate-esterase. Light microscope studies of placental tissue stained with HRP-conjugated anti-human IgE demonstrated cells with a typical 'halo' effect indicating cell-bound IgE, and electron microscopy revealed cells containing membrane-bound electron dense granules. A single mast cell was calculated to contain approximately 1 pg of histamine. Enzymatic digestion of placental tissue with collagenase (1.5 mg/ml) yielded viable cell suspension. containing mast cells in a purity of 0.6% which exhibited a low spontaneous output of histamine (12%). Placental mast cells released histamine in a concentration dependent manner upon challenge with anti human IgE and the calcium ionophore A23187. Also, unlike other human mast cells so far studied. with the exception of skin, those dispersed from human placenta were responsive to the polybasic secretagogue compound 48/80. These findings represent a novel source of human mast cells and, since placentas are readily available in quantity, such tissue is proposed as an ideal source of mast cells for biochemical and pharmacological use. PMID- 1716843 TI - G-proteins as targets for non-immunological histamine releasers. AB - The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction. PMID- 1716844 TI - Molecular basis for cellular effects of naturally occurring polyamines. AB - The naturally occurring polyamines, putrescine, spermidine and spermine, and the analogue cadaverine, induce a dose-dependent histamine release from rat peritoneal mast cells. Spermine was the most active among these polycationic metabolites, followed by spermidine and putrescine. The histamine release was inhibited by a 2 h pretreatment of the cells with pertussis toxin (100 ng/ml), demonstrating the involvement of a pertussis toxin-sensitive GTP-binding regulatory protein during the exocytotic process. Experiments performed with purified Go/Gi proteins reconstituted into phospholipid vesicles showed a direct stimulation of GTPase activity by the polyamines. This direct stimulation of G proteins and the consequent activation of the coupled effectors may represent a new mechanism of action for natural polyamines controlling receptor-dependent processes. PMID- 1716845 TI - Changes in rat mast cell responses in sodium-free media. Lack of demonstrable sodium channel activity. AB - Exposure of rat mast cells to isotonic sucrose (employed as a sodium free medium) increased several-fold the sensitivity to calcium, which itself became a stimulus for exocytosis. Concentrations of the cation as low as 25 microM permitted maximal histamine release. Preincubation of cells in sucrose to allow sodium efflux before adding the ionophore A23187 led to a slower release of histamine. We postulate that sodium efflux can generate a membrane potential that causes the increased sensitivity to calcium and the delay in response after preincubation. The response to A23187 is somewhat unspecific since the ionophore can release histamine from internal calcium reservoirs. Saxitoxin or veratridine did not affect cell responses, so that sodium activity is not mediated through defined sodium channels. PMID- 1716846 TI - Enalaprilat versus cilazaprilat: a comparison of allergic skin reactions in the guinea pig. AB - ACE-inhibitors have for some time been used in the treatment of hypertension. Apart from inhibiting the conversion of angiotensin I to II, the drugs also affect the metabolism of some inflammatory agents, like bradykinin and substance P. Egg albumin (EA)-sensitized guinea pigs were pretreated with the ACE inhibitors. Measurement of flare and wheal areas induced by an intradermal injection of EA, showed that enalaprilat significantly increased, whereas cilazaprilat slightly decreased, the reaction area. Enalaprilat also showed an enhancement in histamine and substance P (SP) contents in the skin. In vitro incubation of guinea pig biopsies with enalaprilat potentiated EA- but not SP induced histamine release. The EA-induced effect was abolished if the animals were pretreated with capsaicin. The conclusion is that cilazaprilat, in contrast to enalaprilat, does not potentiate inflammatory reactions in the guinea pig. PMID- 1716848 TI - Study of the activity of differentiation-induction in a Chinese herb-viscum alniformosanae. Part 1: In vitro induction of differentiation in HL-60 leukemic cell line by conditioned medium secreted from viscum alniformosanae-stimulated mononuclear cells. AB - A conditioned medium(CM), designated as 572-CMF-, was a Chinese herb viscum alniformosanae (V.A.) stimulated mononuclear cells. This CM has the capacity to induce the promyelocytic cell line HL-60 to differentiate into morphologically and functionally mature monocytoid cells. However, our results on the effect of a combination of 572 conditioned medium and IFN-r, TNF and IL-2 were neither synergistic nor additive. Further investigation of the nature of this conditioned medium remains to be performed. PMID- 1716847 TI - Supplementation of patients with homozygous sickle cell disease with zinc, alpha tocopherol, vitamin C, soybean oil, and fish oil. AB - Thirteen patients (aged 0.7-17.9 y) with homozygous sickle cell disease were supplemented with alpha-tocopherol, vitamin C, zinc, and soybean oil (suppl 1; for 8 mo) and alpha-tocopherol, vitamin C, and fish oil (suppl 2; for 7 mo). Urinary zinc (suppl 1), plasma vitamin C, plasma cholesterol ester and erythrocyte (RBC) omega 3 fatty acids (suppl 2), and plasma and RBC alpha tocopherol (suppl 1 and 2) increased. Suppl 1 decreased irreversibly sickled cells by 37.5%, decreased RBC protoporphyrin and urinary porphyrins, and increased the RBC total fatty acid-cholesterol ratio. Suppl 2 decreased plasma triglycerides, further increased the RBC alpha-tocopherol, moderately increased the RBC double-bond index, but decreased the RBC total fatty acid-cholesterol ratio. Zinc, copper, and porphyrins showed prolonged changes. The supplements did not change hemoglobin concentrations, RBC age (reticulocytes, polyamines), or number of aplastic and vasoocclusive crises. Zinc reduces irreversibly sickled cells. Augmentation of RBC antioxidant status by alpha-tocopherol and vitamin C and incorporation of omega 3 fatty acids into RBCs do not affect hemolytic component. Effects on vasoocclusive component are unclear. PMID- 1716849 TI - Intravenous gamma globulin decreases platelet-associated IgG and improves transfusion responses in platelet refractory states. AB - In an attempt to improve platelet transfusion responses, intravenous immunoglobulin (IV-IgG) was administered to 19 patients who were refractory to random and best available HLA-matched platelets. A response to IV-IgG was defined as two or more successive transfusions of HLA-matched products that provided recoveries greater than 30%. Thirteen of 19 (68%) patients responded to therapy at a median time of 7 days after initiation of IV-IgG (range = 2-17). Baseline platelet associated IgG levels (PalgG) were elevated in both the responders (61.6 +/- 76.2) (mean +/- SD) and the non-responders (47.0 +/- 46.3 fg/plt). Post therapy, PalgG levels remained unchanged in the nonresponders but were decreased significantly (p = 0.05) to 11.1 +/- 6.2 fg/plt in the responders. The latter levels were similar to those (11.6 +/- 8.2 fg/plt) measured in a series of 36 transfusion responsive patients. This apparent decline in PalgG was not explained by differences in lymphocytotoxic antibodies (LCT-Ab) after therapy. Moreover, a high degree of alloimmunization was associated with a poorer response to IgG. Only two of eight patients with LCT panel-reactive antibody (PRA) of greater than 85% were responders. By contrast, improved transfusion outcomes were seen uniformly in patients with PRA greater than or equal to 85%. Improved recoveries were obtained using LCT-Ab compatible but not incompatible platelets. The median increment (% predicted) with compatible platelets before therapy was 6.0 +/- 9.9 (SD). Post-IgG, median recoveries were 37.0 +/- 31.2 percent, P less than 0.001. These findings suggest that IV-IgG may alter destructive mechanisms that affect the survival of compatible platelets in refractory patients. PMID- 1716850 TI - Heterogeneity of peripheral blood reticulocytes: a flow cytometric analysis with monoclonal antibody HAE9 and thiazole orange. AB - The expression of a human erythroid cell surface antigen recognized by monoclonal antibody (mAB) HAE9 has been studied on peripheral blood reticulocytes by one- and two-color flow cytometry. Total reticulocyte count was determined using Thiazole Orange (TO) and flow cytometry. In normal individuals, 4.56% of reticulocytes were stained by FITC-labeled mAB HAE9. The correlation between reticulocyte percentage by TO and HAE9 staining was 0.828 (P less than 0.0001) in patients with hematocrits less than 0.25. A HAE9-positive reticulocyte percentage of 6-44% was observed when analyzed by two-color flow cytometry with TO and mAB HAE9. These findings, in conjunction with previous studies, suggest that mAB HAE9 recognizes an early, less differentiated population of peripheral blood reticulocytes. Enumeration of immature reticulocytes may be of clinical utility. PMID- 1716851 TI - Ultrastructural evidence for the origin of mast cells in normal human bone marrow. AB - Whether the origin of the progenitor of mast cells occurs in the basophil/mast cell system or the myeloid cell system of neutrophils and monocytes/macrophages remains controversial. Lactoferrin or lysozyme is known to be present in the myeloid cell system. By electron microscopy, antibodies against lactoferrin and lysozyme were observed to stain the granules of normal bone marrow mast cells. This supports the proposal that the precursor cell of bone marrow mast cells is nearer to the myeloid cell system than the basophil/mast cell system. PMID- 1716852 TI - CD7 false-positive acute myelogenous leukemia and promyelocytic leukemia cell line HL-60: characterization of CD7 epitopes by four monoclonal antibodies. AB - Phenotypes of cells from 12 patients with acute myelogenous leukemia (AML) were analysed by means of a fluorescence-activated cell sorter utilizing a panel of monoclonal antibodies (MAbs). A majority of the cells from peripheral blood coexpressed the antigens against MAbs CD11, CD13, and CD33 but did not express the antigens against CD1, CD3, CD4, CD5, CD8, CD19, CD20, CD21, CD41 and 42, and glycophorin A. Three out of the 12 cases expressed CD7 antigen. However, one of them showed no reaction with Tp40 MAb, whereas the others showed reaction with Leu9 and T55. The discrepancy of reactivities between Leu9 and Tp40 MAbs prompted us to study the promyelocytic leukemia cell line HL-60, which showed similar reactions against Leu9 and Tp40 MAbs. Leu9, OKT16, and T55 MAbs reacted strongly with HL60 cells, whereas Tp40 MAb, which reacted strongly with T-cell leukemia cell line Jurkat, showed no reaction. The reactivity of Leu9, OKT16, and T55 MAbs with HL-60 cells was completely inhibited after preincubation with aggregated human immunoglobulin G (AHIG), which clearly shows the existence of nonspecific binding between these 3 MAbs and HL-60 cells via Fc gamma R. On the basis of our experiments, we conclude that HL-60 cells bind nonspecifically with Leu9, OKT16, and T55 MAbs via FcRI, and this is suggestive that de novo AML cells probably behave in the same fashion. Hence, we recommend that the utilization of murine IgG2a and IgG3 MAbs should be avoided especially in cell surface analysis of myeloid leukemic cells. PMID- 1716853 TI - Unexpectedly high toxicity of MACOP-B in young patients with low grade lymphoma. AB - We report the results of treatment with MACOP-B in 11 young patients with low grade lymphoma (LGL). Complete remission was obtained in 4 (57%) and partial remission in 3 (42.8%) of 7 evaluable patients. However, this aggressive chemotherapy has not offered any advantage because of an unacceptably high treatment-related morbidity and mortality (17%). Serious infection during neutropenia was the most common complication. PMID- 1716855 TI - Compatibility of hydromorphone hydrochloride and tetracaine hydrochloride. PMID- 1716854 TI - Molecular characterization of the human delta-aminolevulinate dehydratase 2 (ALAD2) allele: implications for molecular screening of individuals for genetic susceptibility to lead poisoning. AB - The second enzyme in the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALAD), is a homooctameric protein encoded by a gene localized to human chromosome 9q34. Expression of the two common alleles, ALAD1 (p = .9) and ALAD2 (q = .1), results in a polymorphic enzyme system with three distinct charge isozymes, designated 1-1, 1-2, and 2-2. Individuals heterozygous (2pq = .18) or homozygous (q2 = .01) for the ALAD2 allele have significantly higher blood lead levels than do ALAD1 homozygotes, when exposed to low or high levels of lead in the environment. To investigate the molecular nature of this common polymorphism, total RNA from an ALAD2 homozygote was oligo-dT primed and reverse transcribed, and then the ALAD2 cDNA was amplified, subcloned, and sequenced. Compared with the ALAD1 sequence, the only difference in the ALAD2 cDNA was a G-to-C transversion of nucleotide 177 in the coding region, which created an MspI restriction site. This base substitution predicted the replacement of a positively charged lysine by a neutral asparagine (K59N), an amino acid change consistent with the more electronegative charge of the ALAD-2 subunit. The ALAD1 and ALAD2 alleles were easily detected by amplification of a 916-bp region of genomic DNA and MspI digestion which results in 582- and 511-bp products, respectively. Molecular analysis of 85 ALAD1/ALAD2 heterozygotes and of eight ALAD2 homozygotes revealed no discrepancy between the predicted genotype and the erythrocyte isozyme phenotype, indicating that all the ALAD2 alleles analyzed had the G-to-C transversion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716856 TI - How to prepare and deliver pharmacy presentations. AB - Advice to pharmacists on the preparation and delivery of oral presentations is given. First, a topic must be selected that is appropriate for the speaker, the audience, and the situation or occasion. The audience's educational level, prior knowledge of the topic, and beliefs or attitudes toward the topic must be considered, as well as the time allotted. A title should be chosen to succinctly describe the presentation and to arouse interest. Next, the information should be organized into an outline. Preparing learning objectives helps to identify concepts that the speaker should emphasize; the objectives can also be used to assess the audience's knowledge after the presentation. Visual aids should be prepared in draft form before the text is written, so that the oral presentation will expand upon material presented through the visual aids. Guidelines are given for preparing and presenting overhead transparencies and slides. Although no more than 25% of the speaker's allotted time should be spent on the introduction and conclusion, the introduction is crucial because it must tell the audience the purpose and the topics to be covered, as well as stimulate the listeners' interest. The conclusion should summarize and clarify the information and answer any questions posed in the introduction. Delivery involves effective use of eye contact and body language. The presentation should be spoken in a voice that is intelligible and uses appropriate stress, variation, quality, and pace. Tips for polishing and rehearsing the presentation are given. Pharmacists can enhance communication with an audience by paying close attention to topic selection, organization, visual aids, preparation of the text, and methods for effective delivery. PMID- 1716857 TI - Transplantation's newest weapon FK 506. PMID- 1716858 TI - [Comparative study of immunosuppressive properties of pregnancy- associated protein A, alpha 2-glucoprotein and alpha 2-macroglobulin]. AB - Effects of high-molecular proteinase inhibitors on spontaneous and mitogen induced lymphocyte proliferation were examined in males and pregnant and non pregnant females. All three proteins, A protein, alpha 2-glycoprotein, and alpha 2-macroglobulin were found to be potent immunosuppressors and their activity was determined by the stage of pregnancy, the dose and mode of mitogenic lymphocyte stimulation. PMID- 1716859 TI - Treatment for facial neuronitis: a new approach to Bell's palsy. AB - The presence of sensitive symptoms, symptoms of parasympathetic ganglion's affection, as well as signs of affection of other cranial nerves accompanying to Bell's palsy, led us to consider and treat this disorder as a symptom of motor lesion of a possible primary neuron inflammation. Although we have not found valid statistical correlations between the lesion and location patterns, we have found, however, that continues electromyographic studies, and the early treatment carried out, may be of great interest to avoid sequels. The treatment was focussed to eliminate both the possible cause and the inflammatory process, as to improve neuron regeneration. From 60 patients selected to enter this study, 91,66 percent recovered the motor function. PMID- 1716860 TI - Collection of nonpolar organic compounds from ambient air using polyurethane foam granular adsorbent sandwich cartridges. AB - Glass cartridges containing 6-10 g of Tenax-GC or 15 g of XAD-2 resin packed between two slices of polyurethane foam (PUF) were used in a General Metal Works PS-1 high-volume sampler to collect chlorobenzenes (CBs) containing three to six chlorine atoms, hexachlorocyclohexanes (HCHs), and two-ring aromatic hydrocarbons from 35-385 m3 of air. Laboratory experiments were run by vaporizing known quantities of analytes into a clean airstream for sampling by the PS-1 system at 20 degrees C. Collection efficiencies, determined from mass balance of the quantities introduced and recovered, ranged from 70 to 120% for individual compounds and averaged 93% overall. Penetration of analytes to backup adsorbent traps showed an inverse correlation to vapor pressure. The method was used to collect the above compounds from urban and rural air. PMID- 1716861 TI - [Understanding of ichthyosis]. PMID- 1716862 TI - Treatment of subacute sclerosing panencephalitis with alpha interferon. PMID- 1716863 TI - Comparison of insulin receptor tyrosine phosphorylation under in vitro and in situ conditions: assessment of specific protein tyrosine phosphorylation without the use of 32P-phosphate-labeled substrates. AB - An assay which permitted a comparison of insulin receptor tyrosine phosphorylation induced under in vivo and in situ conditions was developed. It was demonstrated that the level of tyrosine phosphorylation of the receptor induced by insulin under in situ conditions exceeded by 2.3-fold the level of tyrosine phosphorylation induced under in vitro conditions. In addition, chronically treated, down-regulated cells demonstrated a significant decrease in the level of insulin receptor tyrosine phosphorylation compared to cells acutely treated with insulin. The procedure utilized an antibody specific for the insulin receptor which allowed separation of the receptor from other cellular proteins which are potential substrates for tyrosine phosphorylation. A second iodinated antibody specifically directed against phosphotyrosine residues allowed quantitation of the phosphorylated residues. Since the use of 32P-labeled substrates for phosphorylation was not required, this procedure allowed a comparison of protein phosphorylation induced under in vitro and in situ conditions. This procedure should permit investigations into the roles of other cellular factors, such as phosphatases and serine kinases, in modulating protein tyrosine phosphorylation. PMID- 1716864 TI - A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response. AB - A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8] vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation. PMID- 1716865 TI - Zn(2+)-dependent tyrosine phosphorylation of a 68-kDa protein and its differentiation from Mg(2+)-dependent tyrosine phosphorylation in sheep platelets. AB - A 68-kDa protein that was tyrosine phosphorylated in the presence of Zn2+ and two proteins of 52 and 46 kDa that were tyrosine phosphorylated in the presence of Mg2+ were separated by column chromatography of a sheep platelet high speed supernatant on poly(Glu, Tyr)4:1 copolymer-Sepharose or tyrosine-Sepharose. Phosphorylation of the 68-kDa protein occurred maximally in the presence of Zn2+ while Mg2+ was ineffective. The kinases responsible for the Zn(2+)- and Mg(2+) dependent tyrosine phosphorylation could also tyrosine phosphorylate poly(Glu, Tyr)4:1, histone, and angiotensin II with the same metal ion specificity. The two tyrosine kinase activities could be also distinguished by their differential response to polyamines and quercetin. Zn2+ stimulation did not appear to be due to the inhibition of a protein tyrosine phosphatase. Sephadex G-100 gel filtration of the fraction showing Zn(2+)-dependent tyrosine phosphorylation of the 68-kDa protein showed that the tyrosine kinase activity corresponded to a molecular mass of 68,000 and it showed a protein band of 68 kDa as detected by silver staining on sodium dodecyl sulfate-polyacrylamide gel. PMID- 1716866 TI - Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylhydrazine, and diamide: evidence for oxidative stress involvement. AB - The induction of heme oxygenase in rat liver by cobaltous chloride (CoCl2) and Co protoporphyrin IX is entirely prevented by the administration of alpha-tocopherol and allopurinol. CoCl2 was converted in the liver into Co-protoporphyrin IX before it induced heme oxygenase activity. Actinomycin and cycloheximide affected to a similar degree the induction of heme oxygenase by both CoCl2 and Co protoporphyrin IX. Administration of either CoCl2 or Co-protoporphyrin strongly decreased the intrahepatic GSH pool, a decrease which was completely prevented by the administration of either alpha-tocopherol or allopurinol. The latter compounds prevented heme oxygenase induction as well as the decrease in hepatic GSH when administered 2 h before, together with, or 2 h after CoCl2. However, when given 5 h after administration of CoCl2, alpha-tocopherol and allopurinol showed no preventive effect. Similar results were obtained when Co-protoporphyrin IX was used, with the difference that when alpha-tocopherol and allopurinol were given 2 h after administration of the inducer, they showed no protective effect. Phenylhydrazine and diamide also induced heme oxygenase activity in rat liver. This inductive effect was preceded by a decrease in the intrahepatic GSH pool, which took place several hours before induction of the oxygenase. Administration of alpha-tocopherol and allopurinol prevented induction of the oxygenase but had no effect on the decrease in GSH levels. These results suggest that the induction of heme oxygenase by phenylhydrazine and the diamide is preceded by an oxidative stress which very likely originates in the depletion of GSH. The induction of heme oxygenase by hemin was not prevented by administration of alpha-tocopherol or allopurinol. Coprotoporphyrin IX did not affect the pattern of the molecular forms of hepatic biliverdin reductase, at variance with CoCl2, which is known to convert molecular form 1 of the enzyme into molecular form 3. PMID- 1716867 TI - Disposition of mitochondrially bound hexokinase at the membrane surface, deduced from reactivity with monoclonal antibodies recognizing epitopes of defined location. AB - A panel of monoclonal antibodies against rat brain hexokinase (ATP: D-hexose 6 phosphotransferase, EC 2.7.1.1) has been employed to investigate the orientation of the mitochondrially bound enzyme on the mitochondrial surface. Based on their ability to immunoprecipitate truncated forms of the protein, obtained by in vitro translation of truncated versions of the mRNA, the epitopes for seven monoclonal antibodies were mapped to regions consisting of 20-50 amino acid residues within the sequence of the N-terminal half of the enzyme. There is extensive sequence similarity between the N- and C-terminal halves of this enzyme, which is thought to have evolved by a process of gene duplication and fusion. However, these antibodies react selectively with epitopes in the N-terminal half, and thus epitopic regions for several of these antibodies could be further defined by eliminating from consideration regions showing substantial sequence similarity with the C-terminal half. The epitope for one of the monoclonal antibodies, designated 4D4, was shown to involve the extreme N-terminus of the enzyme; selective proteolytic modification of this region resulted in loss of immunoreactivity. Relative location of epitopes for three other antibodies, designated 2B, 1C5, and 4C5, within a 20-residue segment was deduced from effects of modifying sulfhydryl residues within this segment on immunoreactivity. Thus, by a combination of sequence analysis and experimental methods, the epitopes for these seven antibodies could be localized to defined regions within the overall sequence. The ability of these antibodies to prevent binding of hexokinase to mitochondria, and their ability to recognize the mitochondrially bound enzyme, provided a basis for assessing the relative proximity of the corresponding epitopes to the mitochondrial surface when the enzyme was bound. The disposition of the bound enzyme on the mitochondrial surface was deduced by relating these results to the proposed structure for brain hexokinase. PMID- 1716868 TI - Modulation of proteoglycan synthesis by bovine vascular smooth muscle cells during cellular proliferation and treatment with heparin. AB - Proliferating cultures of bovine vascular smooth muscle cells synthesized a variety of proteoglycans corresponding closely to those reported previously for monkey smooth muscle cells. These included a chondroitin sulfate proteoglycan (CSPG) (47%), a dermatan sulfate proteoglycan (DSPG) (22%), and a heparan sulfate proteoglycan (HSPG) (6%) which were secreted into the medium. Heparan sulfate proteoglycan (6%) and a second dermatan sulfate proteoglycan (14%) were also present in the cell layer. Confluent cultures synthesized a similar spectrum of proteoglycans although the medium CSPG and DSPG were of smaller hydrodynamic size. The cell layer HSPG was much reduced relative to DSPG in early proliferating cultures. Previous reports have shown that heparin inhibits vascular smooth muscle cell proliferation. Heparin had two effects on proteoglycan synthesis. In control cultures, 35S-Labeled proteoglycan synthesis doubled during the first 12 h after releasing cells from growth arrest, decreasing during the following 12 h during which time cell division occurred. Treatment with heparin delayed the onset of proliferation by 24 h and this was accompanied by a corresponding delay in the increase in 35S-labeled proteoglycan synthesis associated with the early phase of the cell cycle. Secondly, heparin treatment resulted in an increase in the anionic properties of heparan sulfate proteoglycan synthesized by the cells. This was independent of the proliferative state of the cultures. Pentosan polysulfate, semi-synthetic heparin, and a highly sulfated heparan sulfate modulated both cell proliferation and heparan sulfate proteoglycan synthesis in the same way as heparin. PMID- 1716869 TI - Inhibition of dicarboxylic anion transport by fluorescein isothiocyanate in skeletal sarcoplasmic reticulum. AB - It has been demonstrated previously that dicarboxylic anions are cotransported during ATP-dependent Ca2+ transport by skeletal muscle sarcoplasmic reticulum (SR) membranes, and that anion cotransport stimulates Ca2+ transport. In the current study, we present evidence that dicarboxylic anion cotransport and Ca2+ transport are kinetically distinct in SR, but both functions are mediated by the CaATPase protein. Preincubation of SR with 40 microM fluorescein isothiocyanate (FITC) (pH 7.0) inhibited essentially all of the Ca2+ ATPase activity, as well as active oxalate-supported and oxalate-independent 45Ca2+ accumulation. The addition of 1 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the preincubation media fully protected the dicarboxylic anion-independent Ca2+ ATPase activity and the oxalate-independent active 45Ca2+ accumulation from the inhibitory effects of FITC; however, the ATP-associated [14C]oxalate accumulation, the oxalate-dependent 45Ca2+ accumulation, and the oxalate- and maleate-dependent stimulation of Ca2+ ATPase activity were not protected by AMP PCP. Thus, the dicarboxylic anion accumulation and the stimulation of Ca2+ uptake by dicarboxylic anions could be functionally separated from the ATP-dependent, anion-independent Ca2+ translocation. FITC bound exclusively to the 100-kDa (CaATPase) and 92-kDa (phosphorylase) proteins in the SR membranes and to purified CaATPase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 1 mM AMP-PCP inhibited 50-55% of the FITC fluorescence on the 100-kDa protein, but did not significantly alter fluorescence on the 92-kDa protein. Two-dimensional gel analysis demonstrated a single 100-kDa protein in longitudinal SR membranes. FITC appears to inhibit ATP-dependent Ca2+ transport, and dicarboxylic anion translocation through interaction at separate domains of the CaATPase protein. PMID- 1716870 TI - Isolation and fine-structure characterization of four monoclonal antibodies reactive with glycoconjugates containing terminal GlcNAc residues: application to aspects of lacto-series tumor antigen biosynthesis. AB - A series of murine monoclonal antibodies, each reactive with terminal GlcNAc residues expressed on glycolipids, have been isolated after immunization with the glycolipid nLc5 (GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1--- 4Glc beta 1----1Cer). The derived antibodies, designated TE-4, TE-5, TE-6, and TE-7, were tested for binding specificity with a variety of terminal GlcNAc containing oligosaccharides expressed on glycolipids and glycoproteins. Antibody TE-4 was found to be reactive only with linear and branched terminal GlcNAc beta 1----3Gal containing structures present in lacto-series carbohydrates irrespective of core chain length. The binding specificity of TE-7 was similar except that no reactivity was observed with the short chain structure Lc3 and was weakly reactive with branched agalacto-I structures, suggesting a longer recognition epitope than for the TE-4 antibody. Antibodies TE-5 and TE-6 reacted with terminal GlcNAc beta 1----3Gal structures and as well GlcNAc beta 1--- 2(6)Man structures present on BSA-oligosaccharide conjugates. Weak binding was also observed with GlcNAc beta 1----6Gal structures with these antibodies. TE-5 was found to be particularly sensitive to low amounts of terminal GlcNAc containing glycolipids in both solid phase assays and in TLC-immunostaining studies of neutral glycolipids extracted from colonic adenocarcinoma cell lines and tumors. No reactivity was observed with internal GlcNAc residues with any antibody tested. The panel of antibodies was applied to studies of binding to Triton X-100-solubilized fractions from normal mucosal and adenocarcinoma cell lines after desialylation and Smith degradation to expose terminal GlcNAc residues on glycoproteins and glycolipids. Binding of antibodies TE-4 and TE-7 was restricted to adenocarcinoma-derived cell fractions. Application of these antibodies in studies of lacto-series core chain synthesis and in immunodiagnostic procedures after initial treatments to concentrate lacto-series antigens into terminal GlcNAc-containing structures is discussed. PMID- 1716871 TI - Transforming growth factor-beta in calf articular cartilage organ cultures: synthesis and distribution. AB - Transforming growth factor beta 1 (TGF-beta 1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828-12,831, 1988). In this study, we compare two closely related TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-beta 1 and TGF-beta 2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at approximately 5 ng/ml. Synthesis of TGF-beta by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-beta, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-beta from tissue extracts and conditioned culture medium. The net amount of extractable TGF-beta 1 and TGF-beta 2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-beta 1 (60-85%) and the total TGF-beta 1 + TGF-beta 2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (approximately two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF beta which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand. PMID- 1716872 TI - Estradiol induces class I alcohol dehydrogenase activity and mRNA in kidney of female rats. AB - Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be similar or identical to the class I ADH expressed in liver. Both tissues contain a 1.6-kb transcript which hybridizes with an ADH cDNA under stringent conditions. Kidney ADH activity is responsive to estradiol. The enzyme activity in the kidneys of sham-operated and ovariectomized animals was the same. Treatment of either group of animals by intramuscular injection of estradiol (1 mg/kg body wt/day) for 10 days induced ADH activity in kidney two- to threefold, whether the activity was expressed as U/g tissue, U/g protein, or U/mg DNA. Estradiol induced kidney ADH mRNA in both ovariectomized and sham-operated rats approximately twofold. Thus, induction of ADH mRNA accounts for the increase in ADH activity. In situ hybridization indicated that the ADH mRNA was present in the inner cortex and medulla of the kidney. Methylation patterns of the ADH gene were examined. The gene resides in a methylated region of chromatin without any of the typical features of a HpaII tiny fragment (HTF) island. Two MspI sites flanking the transcription start site are undermethylated in liver compared with kidney and spleen. This suggests that methylation of this gene may play a role in the tissue specific expression of ADH. PMID- 1716873 TI - Collagenase synthesis by cloned rabbit pulp cells--molecular and immunological identity of pulp cell and fibroblast enzyme. AB - Cultures of cloned rabbit pulp (RP) cells without stimulation produced collagenase of a concentration as high as reference rabbit skin fibroblast cultures which were stimulated with phorbol myristate acetate (PMA, 100 ng/ml). The RP cell collagenase was compared with reference fibroblast collagenase in Western blot analysis using monoclonal antibodies prepared against RP cell collagenase and a polyclonal antibody prepared against rabbit fibroblast collagenase. Both enzyme preparations revealed, with either antibody, identical bands of approximate molecular masses 57,000, 52,500, and 45,000. These antibody preparations variously inhibited RP cell collagenase activity. Intracellular collagenase in RP cells in culture was demonstrated by the indirect immunofluorescence antibody technique using polyclonal anti-fibroblast collagenase antibody. RNA samples from RP cells hybridized with rabbit fibroblast collagenase cDNA (clone H9) and showed a distinct band at 2.7 kb. Both control and PMA-stimulated RP cells and PMA-stimulated reference skin fibroblasts demonstrated strong cytoplasmic hybridization between H9 and collagenase mRNA. The results indicate that RP cell collagenase is identical to rabbit fibroblast collagenase, and that the RP cell line provides a useful in vitro reference system for the study of collagenolysis in the rabbit model. PMID- 1716874 TI - Allosteric regulation of purine nucleoside phosphorylase. AB - Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM). PMID- 1716875 TI - Carbohydrate-binding specificity of calcyclin and its expression in human tissues and leukemic cells. AB - Binding of biotinylated fetuin in a solid-phase assay served as activity assay for purification of calcyclin, the product of a cell growth-related cDNA with homologies to Ca(2+)-binding proteins. Asialofetuin failed to bind to calcyclin, emphasizing the importance of sialic acids. Binding of fetuin was most effectively reduced by N-glycolylneuraminic acid within a panel of mostly negatively charged sugars. Bovine submaxillary mucin and the ganglioside GM1, but not asialo-GM1, proved more effective than neoglycoproteins, carrying negatively charged carbohydrate moieties. Extension of N-acetyl-neuraminic acid to its lactosyl derivative increased its inhibitory potency. Among charge-free carbohydrate residues, only N-acetylglucosamine, lactose, and mannose, but not fucose, melibiose, or N-acetylgalactosamine affected fetuin binding, substantiating the inherent selectivity. Chemical modification with group specific reagents revealed that lysine and arginine residues appear to be involved in ligand binding that is optimal in the presence of Ca2+, but not Zn2+ and stable up to 1 m NaCl. Biotinylation of calcyclin by modification of carboxyl groups facilitated performance of solid-phase assays with calcyclin in solution, yielding similar results with (neo)glycoproteins in relation to assays with immobilized calcyclin, thereby excluding an impact of binding to nitrocellulose on calcyclin's specificity. Subcellular fractionation disclosed the presence of fetuin-binding activity in all fractions, the specific activity decreasing from the nuclear to the particulate cytoplasmic fraction and the cytoplasmic supernatant. Affinity-purified antibodies were employed to detect high levels of calcyclin expression in acute lymphoblastic, myelogenous, and monocytic leukemia cell lines, but not in myeloma or lymphoblastoid cells. In comparison, most cells were nearly devoid of an O-acetylsialic acid-specific protein that is more abundant in various tissue types than calcyclin. PMID- 1716876 TI - The structure of human acidic fibroblast growth factor and its interaction with heparin. AB - The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo. PMID- 1716877 TI - The use of monoclonal antibodies to study the structure and function of cytochrome f. AB - Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b6/f complex but MAb-ED4 did not. These results indicate that MAb JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b6/f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were L-D-Q-P-L-T-S-N. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were N-I-L-V-I-G-P-V. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154. PMID- 1716878 TI - Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin. AB - Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons. PMID- 1716879 TI - Sialagogue-stimulated protein phosphorylation related to ornithine decarboxylase induction in cultured rat parotid explants. AB - Both beta-adrenergic (isoproterenol) and cholinergic (carbachol) sialagogues increase amylase secretion, ornithine decarboxylase activity and DNA synthesis in murine parotid gland in vivo and in vitro. These agonists enhanced the incorporation of labelled inorganic orthophosphate into parotid proteins in rat parotid explants cultured on siliconized lens paper floating on serum-free 199 medium. Analysis of the labelled proteins by SDS-PAGE and autoradiography revealed that isoproterenol enhanced the phosphorylation of four proteins with apparent molecular weights of 17, 20, 31 and 32 kDa and carbachol stimulated the phosphorylation of 31 and 32 K proteins. Isoproterenol-dependent ornithine decarboxylase induction and phosphorylation of the proteins were selectively suppressed by monensin but not by polymyxin B, whereas carbachol-dependent ornithine decarboxylase induction and protein phosphorylation were inhibited by polymyxin B but not by monensin. Neither monensin nor polymyxin B suppressed isoproterenol- or carbachol-stimulated amylase secretion. Time course experiments showed that sialagogue-stimulated protein phosphorylation preceded the increase of ornithine decarboxylase activity and had almost disappeared when it was maximal. Propranolol and atropine, antagonists of isoproterenol and carbachol, respectively, completely inhibited not only amylase secretion and ornithine decarboxylase induction but also protein phosphorylation stimulated by the corresponding agonists. These findings suggest that increased phosphorylation of specific proteins is associated with sialagogue-stimulated ornithine decarboxylase induction but not amylase secretion. PMID- 1716880 TI - Kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin as measured by small-angle neutron scattering. AB - The kinetics of the urea-induced dissociation of human plasma alpha 2 macroglobulin into two half-molecular fragments was investigated at 21.0 degrees C by using small-angle neutron scattering. The relative change in molecular mass that occurs upon dissociation was monitored by recording the forward scattering of neutrons as a function of time. All these kinetic data can be explained by a reaction that is first-order with respect to the concentration of undissociated alpha 2-macroglobulin. The velocity constant is a function of urea concentration and it varies within wide limits. For instance, the half-life of the reaction at the lowest concentration of [2H]urea studied (2.70 M) is 328 h, whereas the same value at the highest concentration of [2H]urea (6.24 M) is only 8 min. Measurements were made both with [1H]urea in 1H2O and with [2H]urea in 99% 2H2O, and it was found that there is a pronounced kinetic isotope effect, i.e. the dissociation is 4 times faster in the 1H-containing medium as compared with the 2H-containing medium at the same molar concentration of urea. From the angular dependence of the neutron scattering it can be concluded that the dissociation is associated with a drastic change in structure. This is directly shown by the radius of gyration, which increases from about 7.4 nm immediately after the addition of urea up to about 9.4 nm when the protein is fully dissociated. A structural analysis shows that the scattering curve of urea-dissociated alpha 2 macroglobulin can best be explained by that of a Gaussian coil with a radius of gyration equal to 9.44 nm. These data indicate that the so-called non-covalent interaction of alpha 2-macroglobulin probably is more complicated than just a pure hydrophobic interaction. Finally, it is also shown that the dissociation is accompanied by a loss in trypsin-binding activity, which is directly related to the fraction of dissociated protein. PMID- 1716881 TI - Protein kinase C regulates the stimulated accumulation of 3-phosphorylated phosphoinositides in platelets. AB - We have shown that platelets stimulated with thrombin or guanosine 5'-[gamma thio]triphosphate (GTP[S]), both of which activate phospholipase C and protein kinase C (PKC), show enhancement of 3-phosphorylated phosphoinositide accumulation (3-PPI). We now report the following. (1) Inhibition of thrombin- or GTP[S]-stimulated PKC by pseudo-substrate peptide (RFARK) added to permeabilized platelets markedly inhibits 3-PPI, whereas the serine/threonine phosphatase inhibitor, okadaic acid, promotes 3-PPI. PKC activity, insufficient in itself for fully activating 3-PPI, appears crucial to receptor and post-receptor stimulation of 3-PPI, even when tyrosine phosphorylation is unimpaired. (2) Alteration of Gi by ADP-ribosylation only slightly affects the stimulation of 3-PPI by thrombin, and activation of the G-protein Gi by adrenaline has no effect on 3-PPI. (3) Inhibition of PKC blocks activated secretion of platelet-derived growth factor (PDGF). However, PDGF cannot promote platelet 3-PPI, and thus cannot account for the inhibitory effects of RFARK on 3-PPI. PMID- 1716882 TI - Hepatocanalicular organic-anion transport is regulated by protein kinase C. AB - In order to investigate the regulation of canalicular organic-anion transport, we used a hepatocyte transport assay in which canalicular secretion of a model organic anion, dinitrophenyl-glutathione (GS-DNP), was measured in the presence of stimulators and inhibitors of the Ca2+/protein kinase C (PKC) second-messenger system and of the cyclic AMP (cAMP) second-messenger system. Vasopressin (24 nM) and the phorbol ester phorbol 12-myristate 13-acetate (1 microgram/ml), both stimulators of PKC, stimulated GS-DNP efflux by 65 +/- 36% and 55 +/- 28% respectively, whereas staurosporine (10 microM), an inhibitor of PKC, inhibited efflux by 53 +/- 13%. Glucagon and forskolin, both stimulators of the cAMP second messenger system, as well as the cAMP analogue dibutyryl cAMP and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, did not significantly influence the GS-DNP efflux. It can be concluded that canalicular organic-anion transport in hepatocytes is either directly or indirectly regulated by PKC. PMID- 1716883 TI - Antibody mapping of tissue factor implicates two different exon-encoded regions in function. AB - Tissue Factor (TF), a small transmembrane glycoprotein, is the cellular receptor for the zymogen Factor VII and the serine protease Factor VIIa (VIIa). TF provides cofactor function for VIIa in the catalytically active (TF: VIIa) binary complex. To explore the structural loci of TF that are responsible for binding of VII and VIIa, monoclonal antibodies (MAbs) and sequence-specific polyclonal antibodies to the native TF protein were analysed for inhibition of VII binding. Two independent epitopes of MAbs were localized by reciprocal competition and by binding of the MAbs to different proteolytic fragments of TF. The epitopes were also characterized in part by progressive C-terminal deletional mutation of the TF protein. Reactivity of the anti-(locus II) MAb TF9-6G4 is consistent with epitope localization in residues Thr40-Val83, encoded by exon 3. In contrast, the anti-(locus I) MAb TF9-5G9 was reactive with fragments encompassing exon 4 (Thr106-Lys165). Antibodies to linear sequences encoded by the same two exons also inhibited VII binding. These data suggest a minimum requirement for two of the four exon-encoded regions of TF for the functional integrity of this receptor cofactor with respect to ligand recognition and high-affinity binding. PMID- 1716885 TI - Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. AB - Recently the lectin-like domain on ELAM-1 (endothelial leukocyte adhesion molecule-1) was shown to recognize a carbohydrate antigen, sialyl Lewis x. In this paper we demonstrate, by a series of inhibition experiments utilizing specific monoclonal antibodies and pure glycolipid preparations, that the sialyl Lewis a antigen serves as a specific ligand for ELAM-1 as well as sialyl Lewis x and plays a significant role in the ELAM-1-mediated binding of human cancer cells to activated endothelial cells. PMID- 1716884 TI - Identification of a functionally conserved surface region of rat cytochromes P450IA. AB - A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450. PMID- 1716886 TI - Stabilization of the FK506 binding protein by ligand binding. AB - Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed. PMID- 1716887 TI - Enhancement by IL-1 beta and IFN-gamma of platelet activation: adhesion to leukocytes via GMP-140/PADGEM protein (CD62). AB - We have examined the effect of inflammatory cytokines on the platelet activation. IL-1 beta and IFN-gamma were found to enhance the adhesion of thrombin-treated platelets to monocytic leukemia cells (U937), when the adhesion was assayed by platelet-mediated cell agglutination. The agglutination was inhibited by a monoclonal anti-GMP140 antibody or EDTA, suggesting that the enhanced platelet adhesion to the leukemic cells was mediated by GMP140. In addition, these cytokines also increased the release of 5-HT from platelets in the presence of a low concentration of thrombin. These data suggest that platelet functions are regulated by the cytokines and that activated platelets participate in inflammatory process. PMID- 1716888 TI - Expression of the Tn antigen on T-lymphoid cell line Jurkat. AB - Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr). PMID- 1716889 TI - Inhibitors of poly (ADP-ribose) polymerase suppress lipopolysaccharide-induced nitrite formation in macrophages. AB - Stimulating bone marrow derived macrophages with LPS results in the induction of NO-synthase as measured by NO2- formation. Inhibitors of poly(ADP ribose)polymerase, namely nicotinamide, 3-aminobenzamide and 3-methoxybenzamide, prevented NO2- formation in a dose dependent manner. Inhibition was most effective if the inhibitors were added at the same time as LPS. When added 10 h after exposure to LPS, a time at which expression of the enzyme had reached its maximum, no inhibition was observed. The inhibitors also blocked early events in activation such as protein and RNA-synthesis as well as DNA-synthesis. Thus prevention of NO2- formation may be related to inhibition of these events. Activation of macrophages by LPS was not accompanied by an increase but rather by a small decrease in ADP-ribosyltransferase activity. Whether this decrease plays a physiological role in activation needs further exploration. PMID- 1716890 TI - Thrombospondin binds normally to glycoprotein IIIb deficient platelets. AB - Glycoprotein IIIb (GPIV, CD36) has been proposed as the platelet receptor for thrombospondin (TSP). We found two healthy blood donors, whose platelets were shown to be GPIIIb deficient. These platelets expressed endogeneous TSP as control platelets and their binding capacity for exogeneous TSP was the same. These results indicate that GPIIIb is not the major TSP receptor on platelets. However, it is not yet possible to exclude that in GPIIIb-deficient platelets other proteins may substitute for GPIIIb in TSP binding. PMID- 1716891 TI - Effects of the triazolopyrimidine trapidil on force of contraction, beating frequency and phosphodiesterase I--IV activity in guinea-pig hearts. AB - The effects of the triazolopyrimidine trapidil (5-methyl-7-diethylamino-s triazolo [1,5-alpha]pyrimidine, CAS 15421-84-8) on force of contraction, beating frequency and phosphodiesterase (PDE) activity were investigated in isolated preparations from guinea-pig hearts. The effects of 3-isobutyl-1-methylxanthine (IBMX), theophylline and milrinone were studied for comparison. Trapidil exerted a concentration-dependent (1000-3000 mumol(s)/l) positive inotropic effect (EC50 562.4 mumol(s)/l) in guinea-pig papillary muscles. The positive inotropic effect was accompanied by a shortening of the duration of contraction as described for IBMX, or isoprenaline. The efficacy of trapidil was lower than that of IBMX or milrinone. Both agents maximally enhanced force of contraction to a 3fold (milrinone) or even 6fold greater amount (IBMX). The potency of trapidil was almost in the same order of magnitude as that of milrinone. The positive inotropic effect of trapidil is at least partially due to a cyclic adenosine monophosphate (cAMP)-dependent mechanism because carbachol antagonized the increase in force of contraction. Trapidil concentration-dependently but nonselectively inhibited the activities of cAMP PDE isoenzymes I-IV as did theophylline or IBMX. Based on IC50 values (275 mumol(s)/l on the average) trapidil had a potency similar to that of theophylline while IBMX was about one order of magnitude more potent. Regarding the inhibition of PDE III, IBMX was 49fold and milrinone 114fold more potent than trapidil. Trapidil revealed only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles was increased by 13% at most. Trapidil did not produce any tachyarrhythmias or contractures. It is concluded that the positive inotropic effect of trapidil is mainly due to PDE inhibition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716892 TI - Efficacy of the glyceryl trinitrate transdermal therapeutic system in a dog model of heart failure. AB - The efficacy of a controlled-release topical dosage form of glyceryl trinitrate (Nitroglycerin Transdermal Therapeutic System, Nitroderm TTS, NTG-TTS; CAS 55-63 0) was studied in the experimental model of congestive heart failure in beagles. NTG-TTS suppressed the increase in left ventricular end-diastolic and central venous pressure, and total peripheral resistance resulting from propranolol, dextran and l-phenylephrine infusion. NTG-TTS antagonized the decrease in cardiac output in this model. These effects of NTG-TTS on congestive heart failure were presumably attributable to the reduction of pre-load, together with direct vasodilation of the peripheral arteries. PMID- 1716893 TI - [Pharmacokinetic parameters as criteria for clinical use of hydroxyethyl starch preparations]. AB - Pharmacokinetic Parameters as Criteria for Clinical Use of Hydroxyethyl Starch Preparations In a study with volunteers (n = 2 x 6) pharmacokinetic data of two only marginally differing starch preparations were investigated. We were able to demonstrate that there exist significant differences in raw materials used which determined the pharmacokinetic data in humans. Newly implemented analyzing methods (LALLS) were used. In addition to the degree of substitution, further differences concerning the position of hydroxyethylization at the anhydroglucose molecule could be documented. The C2/C6 positions of hydroxyethylization at the molecule seem to be most essential. To classify and to differentiate starch preparations we propose to include these data in general informations for clinicians because these differences might determine clinical usage and efficacy. PMID- 1716894 TI - Podoscyphic acid, a new inhibitor of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase from a Podoscypha species. AB - A novel enzyme inhibitor of RNA-directed DNA-polymerases of avian myeloblastosis and murine leukemia virus was isolated from fermentations of an tasmanian Podoscypha species. Its structure was elucidated by spectroscopic methods and oxidative degradation as (E)-4,5-dioxo-2-hexadecenoic acid (1). The enzyme inhibitor, which was named podoscyphic acid, did not inhibit DNA and RNA synthesis in permeabilized L 1210 cells nor did it affect RNA synthesis in isolated nuclei of L 1210 cells. 1 inhibits protein synthesis in whole L 1210 cells and rabbit reticulocyte lysate and shows very weak antimicrobial and cytotoxic properties. The testing of ethyl (E)-4,5-dioxo-2-hexadecenoate (2) and (E)-4-oxo-2-tetradecenoic acid (11) revealed the importance of the free gamma oxoacrylic acid unit for the biological activities of 1. PMID- 1716895 TI - A method to quantitate the relative nuclear area of colorectal polyps by image analysis. AB - Image analysis of the nuclear area was performed on Feulgen-stained sections from 94 colorectal polyps: 57 colorectal adenomas with or without invasive growth, 28 hyperplastic polyps and 9 Peutz-Jeghers hamartomas (without dysplasia or carcinoma). The lowest mean values of nuclear area were recorded in control cases: in hyperplastic polyps (27.3%), followed by hamartomatous polyps (34.7%). The nuclear area was significantly increased in tubular adenomas with low-grade dysplasia (37.9%), in villous adenomas with low-grade dysplasia (41.16%) and in tubular and villous adenomas with high-grade dysplasia (47.0% and 46.0%, respectively). The nuclear area in villous adenomas with invasive growth was significantly higher than in all other adenomas without invasive growth (56.0%). The present data suggest that the nuclear area in colorectal polyps (as deduced by image analysis studies) correlates well with the degree of nuclear atypia. Thus, measurements of nuclear area appear to be an important parameter in studies aiming to elucidate the malignant potential of these lesions. PMID- 1716896 TI - Karyometric features in nuclei near colonic adenocarcinoma. Statistical analysis. AB - The normal mucosa adjacent to colonic adenocarcinoma (marginal or transitional mucosa) has been shown to have subtle alterations of architecture, surface glycoproteins and proliferative activity. To evaluate possible changes in nuclear configurations in this marginal mucosa, a large set of cytometric features was evaluated using a computer-assisted video analysis system. Preliminary statistical analysis of the measurements identified six nuclear features useful for discriminating marginal mucosa nuclei from normal (control) mucosa nuclei: total optical density (OD), nuclear area, chromatin texture (from gray value cooccurrence matrix), chromatin coarseness, average OD of nuclear staining and peripheral tendency of the chromatin in the nucleus. An analysis of variance revealed that both patient-to-patient and gland-to-gland variation would limit the usefulness of any one feature as a screening tool. As a group, however, these six features should be investigated further as markers of preneoplastic changes in histologically normal-appearing mucosa. PMID- 1716897 TI - Human monoclonal autoantibody 2E7 is specific for a peptide sequence of platelet glycoprotein IIb. Localization of the epitope to IIb231-238 with an immunodominant Trp235. AB - 2E7 is a human monoclonal IgM autoantibody that binds to a site on the heavy chain of the human platelet integrin alpha subunit glycoprotein IIb. The epitope recognized by 2E7 is stable to denaturation with sodium dodecyl sulfate and reduction of disulfide bonds but is destroyed by proteolysis with papain, chymotrypsin or elastase. By evaluating the reaction of 2E7 with a number of protein sequences from the IIb heavy chain, we have determined that the epitope is located in the octapeptide Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser (FDGYWGYS), corresponding to residues 231-238, and that substitution of the Trp at position 235 completely destroys the epitope. This represents the first precise localization of an epitope on the human platelet integrin IIb-IIIa or on any platelet membrane glycoprotein that is recognized by a human autoantibody. PMID- 1716898 TI - Nucleotide sequence of the human autoantibody 2E7 specific for the platelet integrin IIb heavy chain. AB - The platelet glycoprotein (GP) IIb-IIIa complex figures prominently as an immunogen in autoimmune (idiopathic) thrombocytopenic purpura (ITP). 2E7 is a human monoclonal IgM autoantibody, derived from splenocytes of a patient with ITP, that recognizes a specific octapeptide amino acid sequence, Phe-Asp-Gly-Tyr Trp-Gly-Tyr-Ser, on the heavy chain of GPIIb. This represents the first precise identification of an epitope on GPIIb-IIIa recognized by a human antibody. In this study, we have isolated total mRNA from 2E7, synthesized the corresponding cDNA using reverse transcriptase, and amplified the immunoglobulin mu and kappa chain cDNA by the Taq 1 polymerase chain reaction (PCR) using specific primers. The 2E7 mu chain variable region is encoded by a VH3 gene segment that is 98% homologous to the germline gene VH1.9III, a D-gene that is not homologous to any of the germline D-genes reported to date, and a JH6 gene segment that is essentially germline. The heavy-chain sequence, save for the unique D-gene, is similar to that of a number of human autoantibodies. The 2E7 kappa variable region is encoded by a Vk1 gene segment linked to a Jk1 gene segment. The Vk1 sequence of 2E7, with the exception of one nucleotide, is identical to that of autoantibody HF2-1/17, a prototype of SLE-associated anti-DNA autoantibodies bearing the 16/6 idiotype. The single base substitution results in a relatively conservative exchange of Asp for Glu at position 70 of the protein sequence. Despite this near identity in sequence, 2E7 does not bind to either single stranded or double-stranded DNA. From these results, we conclude that specificity of 2E7 is likely to reside in either or both the D-JH region (CDR3) of the mu chain and the Jk region (CDR3) of the kappa chain. In addition to the identification of a novel D-gene, we also provide evidence that the 2E7 VHIII gene is probably a prototype of a VHIII subfamily, that the germline Vk1 gene shared by 2E7 and autoantibodies of the 16/6 idiotype probably represents a separate Vk family, and that this Vk gene cannot itself attribute specificity for DNA. PMID- 1716899 TI - Natural IgG to epidermal cytokeratins vs IgG to the stratum corneum of the rat oesophagus epithelium, so-called 'antikeratin antibodies', in rheumatoid arthritis and other rheumatic diseases. AB - In order to study the relationships between the circulating IgG autoantibodies to epidermal cytokeratins (AECK), which were described in normal human sera as well as in sera from patients with various diseases, and the so-called 'antikeratin' IgG antibodies ('AKA'), which are highly specific for rheumatoid arthritis (RA), we simultaneously investigated AECK by a specific ELISA using cytokeratins from human stratum corneum (SC) and 'AKA' by semiquantitative indirect immunofluorescence assay on rat oesophagus epithelium, in a large series of 595 rheumatic sera including 229 RA. AECK were found to be present in all the 595 sera, with large inter-individual variations in titre. Whatever the titre chosen as threshold, the autoantibodies (auto-Ab) were never found to be specific for any rheumatic disease. Moreover, in RA, they were found to vary independently of IgM rheumatoid factor (IgM-RF), erythrocyte sedimentation rate (ESR) and C reactive protein (CRP), while they were found to vary in parallel with the total serum IgG concentration. In contrast, although 568 of the 595 rheumatic sera contained antibodies that labelled the rat oesophagus SC, the highest titre-like values were obtained with RA sera. At a convenient threshold, 95 (41.5%) of the 229 RA were detected while only three false positives (0.08%) remained among the 366 non-RA sera. Moreover, in RA, 'AKA' were found to be related to IgM-RF, ESR and CRP, while their titre was found to be independent of the total serum IgG concentration. Lastly, no statistical correlation was found between the antibodies, either in the whole sample of 595 sera or in any diagnostic group. In conclusion, the simultaneous investigation of AECK and 'AKA' showed that they differ from each other in all the aspects explored. AECK belong to the widely explored family of natural auto-Ab against cytoskeleton components and do not constitute a diagnostic marker while, on the other hand, 'AKA' confirmed their high diagnostic specificity for RA. It can also be asserted that, in spite of their name, 'AKA' do not recognize human epidermal cytokeratins, at least in the denatured form they present in ELISA. Therefore, they recognize either conformational epitope(s) appearing on cytokeratins during the late stages of the cornification process, or epitope(s) borne by rat cytokeratins but absent on human cytokeratins, or lastly a non-cytokeratin SC antigen. PMID- 1716900 TI - Immunoblotting for the detection of TSH receptor autoantibodies. AB - Immunoblotting was optimized to detect autoantibodies to TSH receptors from human and porcine thyroid tissue and to determine their epitope specificity. Autoantibodies to putative TSH receptor proteins in thyroid particulate membranes were detected in approximately 35% of sera from patients with Graves' disease. However, despite modifications to increase immunoblotting sensitivity and specificity, only a minority (less than 15%) of Graves' disease sera contained autoantibodies that identified epitopes within TSH affinity-purified human or porcine receptor proteins. In these sera there was no correlation between the TSH receptor antibody titre, determined by radioreceptor assay, and receptor epitope reactivity. The sensitivity of immunoblotting was limited by reduced transfer of purified receptor from the gel. However, in addition, the inability to immunoblot the purified receptor with a majority of Graves' sera, under conditions designed to enhance receptor renaturation, appears to reflect a strict conformational requirement for immunoreactivity. Immunoblotting of purified receptors therefore has a limited application in detecting, and defining the epitope reactivity of, TSH receptor autoantibodies. PMID- 1716901 TI - Isoamylase levels in bone marrow transplant patients are affected by total body irradiation and not by graft-versus-host disease. AB - The mean total serum amylase levels in patients was 3.2 +/- 0.5 mukat/l (+/-SE) before total body irradiation (TBI) prior to bone marrow transplantation of which 50% was due to pancreatic isoamylase and 50% salivary isoamylase. Total serum amylase increased to a maximum of 100.3 +/- 12.3 mukat/l on the first day after TBI and most of this increase was due to an increase in salivary isoamylase (90.0 +/- 12.1 mukat/l). In association with this, all patients had clinical symptoms of parotitis. An increase in pancreatic isoamylase was found in 27% of the patients; however, none of them had clinical symptoms of pancreatitis. Serum amylase levels returned to normal within 5 days after TBI but then decreased to subnormal values, remaining below the normal range for 3 weeks. Pancreatic isoamylase returned to pre-irradiation levels 1.5 months after TBI, while salivary isoamylase remained low for the rest of the observation time. TBI of 7.5 Gy at 26 cGy/min gave significantly lower salivary amylase at 2 days after TBI compared with 10 Gy at 4 cGy/min: 32 +/- 4 versus 76 +/- 13 mukat/l (P less than 0.05). At 2.5 and 6 months after TBI significantly higher total amylase levels were recorded for patients treated with 7.5 Gy of TBI compared with 10 Gy: 2.5 +/ 0.4 and 2.7 +/- 0.3 versus 2.0 +/- 0.5 and 0.8 +/- 0.3 mukat/l, respectively (P less than 0.01, P less than 0.05, respectively). Acute or chronic GVHD did not affect acinar cells in this investigation. PMID- 1716902 TI - Conjugate of N4-(4-carboxybutyryl)-ara-C and ethylenediamine-introduced dextran. Drug release profiles and further in vivo study of its antitumor effects. AB - In vitro and further in vivo work with a conjugate formed from the cytotoxic drug 1-beta-arabinofuranosylcytosine (ara-C) and dextran 2000 are described. In the preparation of this conjugate, functionalisation of ara-C was via N4-(4 carboxybutyryl)-ara-C (glu-ara-C), permitting conjugation with amino groups introduced by prior reaction of the oxidised dextran with ethylenediamine; by varying the proportions of the reaction components, 5.4 to 7.7% w/w loadings of ara-C were obtained. At physiological pH, in vitro, drug release from a 5.4% loaded conjugate was gradual and was dominantly ara-C; at lysosomal pH (pH 5) the release rate was much slower and more ara-U was formed. Antitumour effects were evaluated in L1210 leukaemic mice following single (1 day after inoculation) or double (2 and 6 days after inoculation) intraperitoneal injection of ara-C, glu ara-C, or a 7.7% loaded conjugate at three dose levels. In all cases, the increase in lifespan was greatest following use of the conjugate, but the differences in the effects of ara-C and the conjugate were only significant at the lowest dose level. Glu-ara-C was virtually inactive under all conditions. PMID- 1716903 TI - T-cell recognition of myelin basic protein. AB - Multiple sclerosis is a chronic inflammatory disease of the central nervous system which has been hypothesized to be autoimmune in nature. To test whether this is the case, Kai Wucherpfennig and colleagues have developed a set of criteria that must be met to satisfy the hypothesis. Here, they present these criteria and assess the extent to which studies to date satisfy them. PMID- 1716904 TI - A novel family of leucocyte surface antigens. PMID- 1716905 TI - Biogenic amines and metabolites in spinal cord of patients with Parkinson's disease and amyotrophic lateral sclerosis. AB - In four human controls, four cases of Parkinson's disease and three cases of amyotrophic lateral sclerosis analysis of dopamine, noradrenaline, serotonin and the metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5 hydroxyindoleacetic acid was performed in various segments of postmortem spinal cord. In controls the concentrations of dopamine are about 1/3 to 1/4 that of noradrenaline; the significantly highest content of noradrenaline was found in the lumbar, and dopamine in thoracic, lumbar and sacral segments of the spinal cord. Intersegmental distribution of monoamines was only present in spinal cord of controls, while in the spinal cord of parkinsonian patients such a difference was not found. Otherwise, biogenic amine and metabolite concentrations in spinal cord segments of parkinsonian patients did not differ significantly from those in the control subjects. However, it cannot be excluded that these segments are sensitive to drugs including neuroleptics and combined L-DOPA treatment. In subjects with amyotrophic lateral sclerosis significantly lower concentrations of noradrenaline in the cervical and thoracic, and of dopamine and homovanillic acid in the thoracic and lumbar segments were found in comparison with controls. The concentrations of serotonin and 5-hydroxyindoleacetic acid in the thoracic segments of amyotrophic lateral sclerosis were significantly lower than that of controls. Differences in the inter-segmental distribution of noradrenaline in lumbar, lumbar-sacral, and serotonin in lumbar segments of spinal cord were found in this group. PMID- 1716906 TI - Effect of yohimbine on brain monoamines: an in vivo study. AB - Following the administration of yohimbine, an alpha 2-adrenoreceptor antagonist, the levels of norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5HIAA) increased significantly in the lateral ventricular fluid of rats. These increases were abolished when animals were pretreated with alpha-methyl-para-tyrosine or reserpine. Dopamine (DA) was not detected in ventricular fluid either before or after yohimbine administration. Yohimbine administration did, however, increase intracellular DA levels in the corpus striatum. These findings indicate that yohimbine promotes NE and DA release in the brain and suggest that it also modifies the activity of the serotonin system. PMID- 1716907 TI - Neuropeptide levels in the basal ganglia of aged common marmosets following prolonged treatment with MPTP. AB - Aged common marmosets were treated with 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP; 0.5-2.0 mg/kg/week i.p.) for 16 or 24 weeks, observed for a total of 30 weeks and then killed for measurement of biochemical parameters in basal ganglia. The MPTP treatment induced a marked depletion in dopamine, 3,4 dihydroxyphenylacetic acid and homovanillic acid levels in the caudate nucleus and putamen. In contrast, the concentrations of five neuropeptides: [Met5] enkephalin, [Leu5]-enkephalin, cholecystokinin, substance P and neurotensin as measured by a combined HPLC/RIA method, remained unaltered in all basal ganglia regions examined. Enkephalin precursor levels, as reflected by cryptic [Met5] enkephalin content, were increased in the putamen, but not in the caudate nucleus, as a consequence of MPTP administration. Cryptic [Leu5]-enkephalin content remained unchanged in the striatum of MPTP treated marmosets. Overall, these results suggest an increase in striatal [Met5]-enkephalin release following chronic MPTP treatment of aged marmosets. However, the chronic treatment of aged marmosets with MPTP does not reproduce the neuropeptide alterations characteristic of Parkinson's disease. PMID- 1716908 TI - Induction of experimental allergic encephalomyelitis by myelin proteolipid protein-specific T cell clones and synthetic peptides. AB - Proteolipid protein (PLP) is the major protein of central nervous system (CNS) myelin. SJL(H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 (HSLGKWLGHPDKF) develop acute experimental allergic encephalomyelitis (EAE). In the present study a T cell line and 4 clones were derived from SJL/J mice following immunization with this synthetic peptide. Severe clinical and histological EAE could be induced by adoptive transfer of the peptide-specific T cell line and 3 of 4 T cell clones. The T cell line/clones all responded strongly to PLP peptide 139-151 in in vitro proliferative assays. However, two different reactivity patterns emerged when truncated PLP peptides 141-150 and 141-149 were tested, suggesting that more than 1 epitope may be present within the PLP 139-151 determinant. To evaluate the encephalitogenic potential of the truncated peptides, we compared the ability of 2 truncated PLP peptides to induce EAE in vivo and proliferative responses in vitro. Immunization with PLP peptide 141-150 induced acute EAE in about 70% of mice tested, but PLP peptide 141-149 induced a comparatively mild form of EAE in 4 out of 9 mice tested. Lymph node cells from mice immunized with these peptides showed in vitro proliferative responses to each of the peptides, but the response to peptide 139 151 was always strongest. These combined in vivo and in vitro data further define the epitopes involved in PLP-induced EAE in SJL mice. Furthermore, the availability of multiple PLP-specific T cell clones will enable us to study the diversity of the T cell repertoire to PLP. PMID- 1716909 TI - Density-dependent regulation of epidermal growth factor receptor expression. AB - Previous studies have demonstrated that binding of peptide growth factors such as epidermal growth factor (EGF) decreases as cell density increases. We now report that binding of EGF to MDA 468 breast carcinoma cells is reduced as cells increase in density with time in culture. Cells at low density bound more EGF per cell than cells at higher density. The reduction of EGF binding was due to a reduction in receptor number. Metabolic labeling of MDA 468 cells with [35S] methionine followed by immunoprecipitation of the EGF receptor (EGFR) indicated that the amount of total receptor protein was decreased. Using RNA blot hybridization, we found that high-density cells contained decreased amounts of EGFR transcripts. Northern analysis revealed that both the 10- and 5.6-kilobase mRNA transcripts encoding the EGFR were decreased. These data suggest that increasing cell density with time in culture results in modulation of EGFR content, with changes at the level of both protein and mRNA expression. PMID- 1716910 TI - Bioavailability of ketoprofen from orally administered ketoprofen-dextran ester prodrugs in the pig. AB - The bioavailability of ketoprofen after oral administration of aqueous solutions of various ketoprofen-dextran ester prodrugs in pigs was assessed. Conjugates derived from dextran fractions in the molecular weight range 10,000-500,000 were employed. Compared to the administration of an oral solution of an equivalent dose of parent ketoprofen, the average absorption fractions for the different prodrugs ranged from 100 to 67%. Relatively small inter-individual variation of ketoprofen bioavailability was observed. Apparently, the molecular size of the employed dextran transport group only has a minor influence on the pharmacokinetic parameters. The ketoprofen plasma profiles for all the administered prodrugs exhibited a characteristic lag time of ketoprofen appearance in the blood (2-3 h). Quite similar results were obtained from identical experiments carried out in the pig, employing naproxen-dextran esters. Thus, the present study adds support to a more versatile application of the dextran ester prodrug approach to providing selective colon delivery of drugs possessing a carboxylic acid functional group. PMID- 1716911 TI - [Kawasaki disease]. PMID- 1716912 TI - Characterization of a gene encoding a basic protein of the spermatid nucleus, TNP2, and its close linkage to the protamine genes in the bull. AB - During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome. PMID- 1716913 TI - Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain. AB - A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT). The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC. Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein. The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site. The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated. The chimeric gene product from a crude E. coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E. coli proteins were eliminated after elution with 20-35 mM imidazole. The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel. The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein. The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed. PMID- 1716914 TI - T-cell-mediated activation of B cells. AB - Antibody responses to protein antigens are dependent on helper T lymphocytes, which provide necessary growth and differentiation, inducing stimuli to antigen specific B cells. Recent investigations have focused on the mechanisms of T-B interactions, and the nature of the T-cell-derived signals that are needed for activation and expansion of B cells. PMID- 1716915 TI - Retention modeling of diesel exhaust particles in rats and humans. AB - The objective of this study was to predict the lung burden in rats and humans of diesel exhaust particles from automobile emissions by means of a mathematical model. We previously developed a model to predict the deposition of diesel exhaust particles in the lungs of these species. In this study, the clearance and retention of diesel exhaust particles deposited in the lung are examined. A diesel particle is composed of a carbonaceous core (soot) and adsorbed organics. These materials can be removed from the lung after deposition by two mechanisms: (1) mechanical clearance, provided by mucociliary transport in the ciliated airways as well as macrophage phagocytosis and migration in the nonciliated airways, and (2) clearance by dissolution. To study the clearance of diesel exhaust particles from the lung, we used a compartmental model consisting of four anatomical compartments: nasopharyngeal, tracheobronchial, alveolar, and the lung associated lymph node compartments. We also assumed a particle model made up of material components according to the characteristics of clearance: (1) a carbonaceous core of about 80 percent of particle mass, (2) slowly cleared organics of about 10 percent of particle mass, and (3) fast-cleared organics accounting for the remaining 10 percent of particle mass. The kinetic equations of the retention model were first developed for Fischer-344 rats. The transport rates of each material component of diesel exhaust particles (soot, slowly cleared organics, and fast-cleared organics) were derived using available experimental data and several mathematical approximations. The lung burden results calculated from the model showed that although the organics were cleared at nearly constant rates, the alveolar clearance rate of diesel soot decreased with increasing lung burden. This is consistent with existing experimental observations. At low lung burdens, the alveolar clearance rate of diesel soot was a constant, equal to the normal clearance rate controlled by macrophage migration to the mucociliary escalator, whereas at high lung burdens, the clearance rate was determined principally by transport to the lymphatic system. The retention model of diesel exhaust particles for rats was extrapolated to humans of different age groups, from birth to adulthood. To derive the transport rates for the human model, the mechanical clearance from the alveolar region of the lung was assumed to be dependent on the specific particulate burden on the alveolar surface. The reduction in the mechanical clearance in adult humans caused by exposure to high concentrations of diesel exhaust was found to be much less than that observed in rats. The reduction in children was greater than that in adults.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1716916 TI - Effects of 4 percent and 6 percent carboxyhemoglobin on arrhythmia production in patients with coronary artery disease. AB - In this study, we assessed the effects of exposure to 4 percent and 6 percent carboxyhemoglobin on ventricular arrhythmias in 41 subjects (nonsmokers) with documented coronary artery disease. We used a randomized, double-blind, crossover design. On day 1, a training session with no exposure, the baseline carboxyhemoglobin level was measured, and a supine bicycle exercise test was done. On days 2 through 4, subjects were exposed to room air, 100 parts per million (ppm)2 carbon monoxide (target, 4 percent blood carboxyhemoglobin), or 200 ppm carbon monoxide (target, 6 percent blood carboxyhemoglobin), and they then did a supine bicycle exercise test. Radionuclide ventriculography was performed at rest and during exercise. Ambulatory electrocardiogram recordings were made during the four consecutive days to determine the frequency of premature ventricular contractions at various intervals. The frequency of single premature ventricular contractions per hour during exercise was significantly greater on the 6 percent carboxyhemoglobin day than on the room air day (167.72 +/- 37.99 for 6 percent carboxyhemoglobin compared with 127.32 +/- 28.22 for room air, p = 0.03). The frequency of multiple premature ventricular contractions per hour was also significantly greater during exercise on the 6 percent carboxyhemoglobin day compared with the room air day (9.59 +/- 3.70 for the 6 percent carboxyhemoglobin day compared with 3.18 +/- 1.67 for the room air day, p = 0.02). Patients who developed increased arrhythmias during exercise on the 6 percent carboxyhemoglobin day were significantly older than those who had no increased arrhythmia, and, in addition, exercised longer and had a higher peak workload during exercise. No effect of carbon monoxide exposure was seen on the 4 percent carboxyhemoglobin day. PMID- 1716917 TI - Regression of atherosclerosis. A role for calcium antagonists. AB - The key processes responsible for plaque in atherosclerosis are cholesterol accumulation, caused by increased net uptake of atherogenic lipoproteins; cell proliferation and recruitment, caused by mitogenic and chemotactic activity; extracellular matrix expansion and collagen accumulation, caused by increased intimal cell synthesis; and high rates of cell death, caused by increased metabolic needs of tissue in an unfavorable milieu. Cell death is associated with neovascularization, dense fibrosis, and calcification, with the subsequent plaque complications of hemorrhage and thrombosis. An additional functional feature of atherosclerosis, even in prestenotic stages, is severe vasomotor abnormalities, caused in part by endothelial dysfunction. Reversal of atherosclerosis involves interrupting these processes. Calcium antagonists may block specific stages of atherogenesis. Anticalcifying agents (diphosphonates, thiophenes, and metallic ions) inhibit accumulation of connective tissue. Calcium channel blockers reduce endothelial injury and intimal permeability, block proliferation of intimal cells, inhibit lipid accumulation by a number of mechanisms, and reduce exaggerated vasoconstrictor responses. Thus, calcium antagonists may promote regression of lesions beyond what can be achieved by reducing risk factors alone. PMID- 1716918 TI - Modulation of a gated ion channel admittance in lipid bilayer membranes. AB - A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication. PMID- 1716919 TI - Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. AB - Lymphocytes adhere to other cells and extracellular matrix in the process of immunological recognition and lymphocyte recirculation. This review focuses on regulation of lymphocyte adhesion and the use of adhesion mechanisms by lymphocytes to obtain information about their immediate environment. The CD2 and LFA-1 adhesion receptors appear to have distinct roles in the regulation of adhesion and modulation of T lymphocyte activation. Adhesion mediated by interaction of CD2 with LFA-3 is dramatically altered by surface charge and adhesion receptor density in such a way that this pathway is latent in resting T lymphocytes but becomes active over a period of hours following T-cell activation. CD2 ligation can mediate or enhance T-cell activation, suggesting that signals from CD2/LFA-3 adhesive interactions are integrated with signals from the T-cell antigen receptor during immunological recognition. A model for the role of LFA-3 lateral diffusion in adhesion is presented, based on the lateral diffusion of different LFA-3 forms in glass supported planar membranes. Interaction of LFA-1 with ICAMs is also regulated by cell activation but in a different way than in interaction of CD2 with LFA-3. LFA-1 avidity for ICAMs is transiently increased by T-cell activation over a period of minutes. Cycles of avidity change are also observed for other T lymphocyte integrins which bind to extracellular matrix components. We propose that integrin avidity cycles may have an important role in the interconnected phenomena of locomotion, initial cell cell adhesion, and cell-cell deadhesion. Recent observations on recirculation of T lymphocyte subpopulations are discussed in the context of general lessons learned from study of the CD2/LFA-3 and LFA-1/ICAM adhesion mechanisms. PMID- 1716920 TI - The specificity and regulation of T-cell responsiveness to allergens. AB - CD4+ T lymphocytes initiate and regulate both the specific and nonspecific effector mechanisms of allergic immune responses. The definition of immunogenic components within allergens has allowed the specificity of the T-cell repertoire to be examined, and the refinement of their molecular structure is now facilitating the mapping of functional epitopes. Nevertheless, the immunological mechanisms that distinguish between atopic and nonatopic states remain unclear; as knowledge of the complex network of cytokines in the regulation of immune responses unfolds, this problem may be resolved. Population genetics indicates that susceptibility to allergic disease is partly determined by products of the major histocompatibility gene complex. The use of molecular genetic probes and monoclonal T cells demonstrates the functional importance of both the HLA-DRAB1 and -DRAB3 gene products in T-cell recognition of allergen. Collectively, this information has made it possible to develop in vitro models examining the modulation of responsiveness to allergens. PMID- 1716921 TI - Slide presentation graphics using a personal computer. A comparative evaluation of available software. AB - Most of the available methods for preparing professional slides to be used in oral presentations can be cumbersome, time-consuming, and expensive. Creating high-quality 35-mm slides may require the use of expensive medical illustrators and photographic equipment not available to all physicians. More recently, technology has emerged that permits professional in-office generation of quality slides using commercially available computer graphics software and film recorders. Such a system is simple to establish and maintain when compared with commercial slide production facilities. We will review the hardware requirements of such a system, as well as the advantages and disadvantages of several commercially available software packages. Using a personal computer-based slide making system, any physician communicating with small or large groups of people can now produce professional-looking slides easily, rapidly, and inexpensively. PMID- 1716922 TI - Further characterization of a novel phospholipase B (phospholipase A2- lysophospholipase) from intestinal brush-border membranes. AB - The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species. PMID- 1716923 TI - Isolation and some properties of 11- and 9-Gla prothrombins from normal plasma. AB - The relationship of prothrombin structure to function with respect to gamma carboxyglutamic acid (Gla) residues can be effectively evaluated by characterizing the behavior of prothrombin isomers differing in Gla content. In addition to the isolation of a whole spectrum of Gla-deficient, 0- to 9-Gla isomers from dicoumarol-treated plasma, prothrombin isomers containing 11 (10.90) and 9 (8.85) Gla residues have now been isolated from normal bovine plasma. The isomers were isolated by barium citrate adsorption, elution, and finally by heparin-agarose, DEAE-cellulose, and immuno-affinity chromatographies. Each of the purified isomers showed a single component by agar gel and sodium dodecyl sulfate - polyacrylamide gel electrophoresis. By agar gel electrophoresis, the 11 Gla prothrombin isomer moved the fastest, followed by the 10-, and lastly the 9 Gla isomer, independent of Ca2+. The corresponding 9-, 10-, and 11-Gla prothrombin fragments 1 exhibited similar migration tendencies. By gel electrofocusing, 11- and 9-Gla fragments 1, respectively, focused anodal and cathodal to 10-Gla fragment 1. The Ca2+-induced decrease in the intrinsic fluorescence in 11-, 10-, and 9-Gla fragments 1 was 48, 40, and 45%, respectively. This metal-induced structural change did not correlate with the functional, thrombin-generating property of the isomers, as the 9-Gla variant exhibited 75%, and the 11-Gla 110-115%, of normal coagulant activity. PMID- 1716924 TI - Studies on the ionic selectivity of the GABA-operated chloride channel on the somatic muscle bag cells of the parasitic nematode Ascaris suum. AB - The reversal potential for the GABA-elicited hyperpolarization (EGABA) of the muscle bag cells of the parasite nematode Ascaris suum is near to the equilibrium potential for chloride ions (ECl) indicating that this is a chloride-mediated event. The value of EGABA in the presence of different anions indicates the selectivity sequence of the GABA-operated chloride channel as SCN- greater than Br-, I- greater than ClO3-, NO3-, Cl- greater than C2H5COO- greater than CH3COO-, HCOO-, OHCH2CH2SO3H-, CH3SO4-, BrO3-, SO4(2-). The cut-off point in terms of relative hydrated size for permeation is between ClO3- and BrO3- suggesting that the diameter of the channel is between 0.29 and 0.33 nm. The selectivity sequence of the resting membrane chloride channels was indicated by the degree of muscle cell hyperpolarization upon addition of the anion to the bathing medium and was SCN- greater than NO3- greater than I-, Br-, greater than ClO3- greater than Cl-. These sequences are consistent with the anions interacting with a cationic site of low field strength at the selectivity determining portion of the channel for both the GABA-operated and the resting chloride channel. PMID- 1716925 TI - Conductance rise induced by the calcium ionophore A23187 in unfertilized eggs of tilapia. AB - Current-clamp measurements on unfertilized tilapia eggs gave linear current voltage relations in the range 0 to -120 mV. Eggs had low resting potentials (-22 mV) and high specific membrane resistances (500 k omega cm2) and specific membrane capacitances (0.9 microF cm-2) indicating no marked folding of their plasma membrane. Application of A23187 induced a small depolarization and a conductance rise, the response having a reversal potential of about -16 mV. The results suggest that tilapia eggs have a calcium-gated conductance probably activated at fertilization. PMID- 1716926 TI - Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor. AB - The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield. PMID- 1716927 TI - Time-resolved immunofluorometric detection of antigens separated by high performance liquid chromatography and coated to polystyrene. AB - We use high-performance liquid chromatography with fraction collection to separate an antigen of interest. The antigen is then immobilized on polystyrene microtiter wells and detected with a specific antibody, followed by a second biotinylated antibody and streptavidin labeled with a fluorescent europium chelate. Fluorescence can be quantified with the use of time-resolved fluorescence. Antigen detectability down to 2-3 x 10(-17) mol was achieved. This method could be used as an alternative to Western blot in certain applications. PMID- 1716928 TI - Prominent expression of acidic fibroblast growth factor in motor and sensory neurons. AB - Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor. PMID- 1716929 TI - Localization of the hair cell's transduction channels at the hair bundle's top by iontophoretic application of a channel blocker. AB - In order to understand how the hair cell's mechanoelectrical transduction channels are gated during mechanical stimulation, it is essential to determine their location with respect to the hair bundle's constituent stereocilia. We localized the transduction channels by focally blocking receptor currents with iontophoretically ejected gentamicin, an aminoglycoside antibiotic that acts as a reversible channel blocker. The drug was most effective when directed at the top of a hair bundle, whereas application at the bundle's bottom or at the cuticular plate had little or no effect. Computer simulations of blocking agreed with experimental data only when the transduction channels were hypothesized to occur near the bundle's top. These results confirm that the hair cell's transduction channels are located near the stereociliary tips. PMID- 1716930 TI - Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells. AB - Activation of kainate receptors causes Co2+ influx into neurons, type-2 astrocytes, and O-2A progenitor cells. Agonist-activated Co2+ uptake can be performed using cultured cells or fresh tissue slices. Based on the pattern of response to kainate, glutamate, and quisqualate, three functionally different kainate-activated ion channels (K1, K2, and K3) can be discriminated. Co2+ uptake through the K1 receptor was only activated by kainate. Both kainate and glutamate activated Co2+ uptake through the K2 receptor. Co2+ uptake through the K3 receptor was activated by all three ligands: kainate, glutamate, and quisqualate. Co2+ uptake occurred through a nonselective cation entry pathway permeable to Co2+, Ca2+, and Mn2+. The agonist-dependent activation of divalent cation influx through different kainate receptors could be correlated with expression of certain kainate receptor subunit combinations. These results are indicative of kainate receptors that may contribute to excitatory amino acid-mediated neurotoxicity. PMID- 1716931 TI - Pulmonary edema and coagulopathy following intrauterine instillation of 32% dextran-70 (Hyskon). AB - Hysteroscopy using 32% Dextran-70 in 10% dextrose (Hyskon) is a common outpatient procedure. We report a case of pulmonary edema and coagulopathy in a 38-year-old, 48 kg female who had hysteroscopy and removal of an endocervical polyp using 700 ml of Hyskon over a 2-hour period. Low percutaneous oxygen saturation (SpO2) and vaginal bleeding developed postoperatively. A vaginal hysterectomy was performed. The coagulation profile showed coagulopathy, which was treated with transfusions of fresh frozen plasma and platelets. The patient developed overt pulmonary edema following the hysterectomy and was treated with positive pressure ventilation. The pathophysiology for the development of pulmonary edema and coagulopathy is discussed. Recommended limits for Hyskon volumes and instillation times are emphasized, as is careful recording of these parameters. PMID- 1716932 TI - Low dose aprotinin as blood saver in open heart surgery. AB - Bleeding after open heart surgery is still a great concern for the surgeon, especially when the surgical field has been revised accurately and hemostatic stitches and electrical cauterization have been used extensively. Among non surgical adjuncts, aprotinin has been reported as very effective in reducing complications. At the time we started using this drug, we intended to test two different dosages lower than those reported in the literature. We evaluated three groups of 18 patients: the first (A) received about 350 mg of aprotinin from the start of anesthesia up to the end of operation (140 mg in the priming of cardio pulmonary bypass and 70 mg/h i.v. during the procedure; the second (A/2) received half that dose (i.e. 70 mg and 35 mg, respectively), and the third (C) did not receive aprotinin. We compared in these groups: postoperative bleeding, blood transfusions, red blood cells, hemoglobin, hematocrit, platelets. The results were good only in the A group: bleeding was reduced and few transfusions were required. The patients in the A/2 and C groups did not show significant differences. From our observations we conclude that aprotinin is a useful adjunct, but has to be given in the proper dose. PMID- 1716933 TI - [Disorders of cardiac rhythm and conduction after radical correction of tetralogy of Fallot]. AB - The work is based on the results of examination of 78 patients conducted before, in the immediate, and in the late-term periods after the operation (6.5 +/- 5.0 years on the average). Holter's monitoring and bicycle ergometry conducted before the operation revealed rhythm disorders in 55% of patients: complete block of the right limb of the bundle of His in 30, I-III degree atrioventricular block in 9%, supraventricular arrhythmias in 2.5%, ventricular arrhythmias in 5%, and combined arrhythmias in 7.5% of patients. Complete block of the right limb of the bundle of His was discovered in all patients in the late-term postoperative periods, and other types of rhythm disorders were found in 62% of patients: I degree atrioventricular block in 2.5%, bifascicular block in 2.5%, ++tri-fascicular block in 1%, ventricular arrhythmias in 26%, and combined arrhythmias in 30% of patients. The results of the examination showed that: (1) the presence of stable block of the right limb of the bundle of His, bifascicular block, as well as ventricular arrhythmia of I-II gradation after Laun-Wolf does not lead to decrease of myocardial working capacity and contractile function. In contrast, III-IV gradient ventricular arrhythmia is attended by significant diminution of myocardial contractility; (2) the incidence of ventricular arrhythmias grows with increase of the patients' age at the time of the operation and intensification of the degree of initial arterial hypoxemia and the anatomical severity of the anomaly; (3) correction of the anomaly contributes to the disappearance of the preoperative arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716934 TI - Palliation of bone metastases. PMID- 1716935 TI - A prospective, randomised double-blind crossover study to examine the efficacy of strontium-89 in pain palliation in patients with advanced prostate cancer metastatic to bone. AB - The palliative efficacy of strontium-89 chloride has been evaluated in a prospective double-blind crossover study comparing it with stable strontium as placebo in 32 patients with prostate cancer metastatic to bone. Response was assessed 5 weeks after each treatment. 26 patients were evaluable. Complete pain relief was only reported following strontium-89 injection. Statistical comparison between placebo and strontium-89 showed clear evidence of a therapeutic response to strontium-89 compared with only a limited placebo effect (P less than 0.01). PMID- 1716936 TI - [New in situ hybridization technique with non-radio-labeled probe--detection of choline-acetyltransferase gene expression in rat spinal cord]. AB - We have established a new in situ hybridization method utilizing non-radiolabeled probes. Using this technique, we have attempted to detect the choline acetyltransferase (ChAT) gene expression in rat spinal cord. It was revealed that the ChAT gene was expressed mainly in the cytoplasm of motor neurons and para central cells. On the other hand, ChAT protein has already been reported to exhibit a diffused distribution in the cholinergic fibers. Comparing the localization of the ChAT gene with that of the ChAT protein, the ChAT gene was shown to exist only in the cytoplasm surrounding the nuclei. However, the ChAT gene was not expressed in axon terminals where ChAT protein synthesized acetylcholine. This result indicates that the ChAT gene is translated into protein around the nuclei and is thereafter transported toward the action site. We now think that there are two different patterns of neurotransmitter gene distribution. After mRNA is translated into protein, this protein is carried to the action site. On the other hand, mRNA itself is delivered to the action site and translated into protein. After the translation, this protein form exerts its own function. The ChAT gene is suspected as belonging to the first category of gene distribution. In Alzheimer disease, not only the acetylcholine system but also its biosynthetic enzyme, ChAT, system are supposedly destroyed by an unknown factor. If we can clarify the regulatory mechanism of the ChAT gene, this will lead us to the molecular pathogenesis of Alzheimer disease. Additionally, this new in situ hybridization technique should shed some light on the complex brain networks. PMID- 1716937 TI - [The effect of a new immunosuppressive agent, FK-506, on xenogeneic neural transplantation in rodents--comparison with cyclosporin A]. AB - The present study was performed to compare the immunosuppressive effect of FK-506 (FK) with cyclosporin A (CyA) in xenogeneic neural transplantation in rodents. The solid grafts of embryonic ventral mesencephalic tissue from 14-day rat embryos were transplanted into the right lateral ventricle of 20 mature male mice using a stereotactic approach, and the mice were sacrificed 14 days after transplantation. The removed brains, which were sectioned coronally, were stained with antibodies against glial fibrillary acidic protein (GFAP), Lyt-2 or tyrosine hydroxylase (TH) in addition to hematoxylin-eosin and Nissl stainings. The subcutaneous (s.c.) administration of FK at a dose of 10.0 mg/kg daily for 14 days enabled the neural xenografts to survive and grow (n = 5), although all of the neural xenografts treated with CyA at a dose of 10.0 mg/kg s.c. (n = 5) or FK at a dose of 1.0 mg/kg s.c. (n = 5) daily for 14 days and ones treated without such an immunosuppressive agent (n = 5) were rejected by immunological reactions. We conclude that FK is a more powerful immunosuppressive agent than CyA in xenogeneic neural transplantation as well as in cardiac and renal allograftings. PMID- 1716938 TI - [Distribution of glycoconjugates in gerbil hippocampal neurons--histochemical study with lectins]. AB - Paraffin-embedded sections of gerbil hippocampus were made and stained by use of lectins with different sugar specificities: Concanavalin A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), Phaseolus vulgaris agglutinin (PHA-E), peanut agglutinin (PNA), soybean agglutinin (SBA), and Vicia villosa agglutinin (VVA) to investigate the distribution of glycoconjugates in this brain region. Nuclear membranes of all the neurons in the hippocampus were positively stained with Con A and PHA-E, whereas WGA revealed definite staining of cell membranes. Endothelial cells of blood vessels were intensely positive to RCA, suggesting its usefulness as a marker of endothelial cells. With SBA and VVA, a few neurons in Ammon's horn were positively stained, while no positive cells were observed in the dentate gyrus. CA2 and the medial part of the CA1 were also positive bilaterally with SBA and VVA. Some neurons in Ammon's horn and dentate gyrus were selectively stained on the surface of their cell bodies with PNA. The present results show that lectins used distinguish different subpopulations of hippocampal neurons, indicating a new possible classification of hippocampal neurons based on their differences in glycoconjugates. PMID- 1716939 TI - Laser therapy and insertion of Wallstents for palliative treatment of esophageal carcinoma. AB - A combination of endoscopic laser therapy (ELT) and insertion of Wallstents is a good alternative therapy for palliation of esophageal carcinoma and was performed in 12 patients. The method allows repeated laser therapy and, if necessary, supplementary insertion of stents to maintain the patients' ability to swallow during their remaining lifespan. PMID- 1716940 TI - Inhibition of 5-fluorouracil activation by oxipurinol in preparations of normal rat tissues and Jensen sarcoma: tumor-selective stimulation by inosine. AB - The effects of inosine on oxipurinol-induced inhibition of 5-fluorouracil (FUra) activation were investigated in a transplantable rat tumor and three normal rat tissues in vitro. FUra activation directed toward RNA was assessed in preparations of small intestine and Jensen sarcoma by determining the uptake of 14C-labeled FUra into purified total RNA of each tissue under identical conditions of incubation. Oxipurinol reduced the incorporation of FUra into intestinal RNA by as much as 93% but inhibited FUra uptake into tumor RNA by only about 45%. In the presence of sufficient oxipurinol to induce inhibition at approximately these levels, inosine failed to stimulate FUra uptake into intestinal RNA, whereas 0.1 mM inosine restored FUra uptake into Jensen sarcoma RNA to the level found in oxipurinol-free controls, and 0.5 mM inosine increased FUra uptake 29% above the controls. Both tissues had a nearly equivalent enzymatic capacity to cleave inosine by phosphorolysis. In view of what is currently known about the mechanism of action of oxipurinol and the metabolism of inosine, these results suggested that under our in vitro conditions rat small intestine was incapable of utilizing ribose-1-phosphate derived from inosine to stimulate the conversion of FUra to 5-fluorouridylate (FUMP), whereas Jensen sarcoma efficiently used this source of ribose-1-phosphate to increase the production of FUMP from FUra. In the absence of oxipurinol, inosine increased FUra uptake into total RNA of Jensen sarcoma by as much as 57%, compared to inosine-free controls, but markedly inhibited FUra uptake into total RNA of the small intestine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716941 TI - Whipple's disease, familial Mediterranean fever, and adult-onset Still's disease. AB - Whipple's disease is a multisystem disorder thought to be caused by infection by rod-shaped bacilli. Early diagnosis remains difficult, because initial clinical features are nonspecific. Ultrasonography and computed tomographic scanning were used to demonstrate distinctive lymphadenopathy in Whipple's disease. Magnetic resonance imaging showed central nervous system lesions that were reversible with antibiotic therapy. Familial Mediterranean fever, or recurrent polyserositis, is an autosomal recessive disorder common among patients of Mediterranean heritage. Erysipelas-like skin lesions are commonly described. Other skin lesions, including Schonlein-Henoch purpura, nonspecific purpura, diffuse erythema, and angioneurotic edema are now reported. Renal complications, thought previously to be due primarily to amyloid, are also caused by immunoglobulin deposition resulting in mesangial proliferative glomerulonephritis. Adult-onset Still's disease is a systemic illness characterized by quotidian fever and a fleeting, salmon-colored rash. The long-term evolutions of juvenile-onset and adult-onset Still's disease were compared and found to be similar, except for the occurrence of amyloidosis in the latter group of patients. Prognosis of patients with articular features was worse than that of patients with extra-articular features. A multicenter survey of Japanese patients found few significant differences between Japanese and non-Japanese cases. Less well-recognized features of adult onset Still's disease, including neurologic complications, uveitis, and peritonitis, are reported. PMID- 1716942 TI - Cholera situation in the Americas. An update. PMID- 1716943 TI - AIDS surveillance in the Americas. PMID- 1716944 TI - [Occult hepatic metastases from colorectal carcinoma: diagnostic and prognostic considerations]. AB - Occult hepatic metastases (OHM) from colorectal cancer are those not evident to the surgeon at laparotomy. In this retrospective and "deductive" study the Authors evaluated the accuracy of hepatic CT scan and ultrasonography (US) to detect hepatic metastases. The CT and US accuracy rate was 78.4% and 79.8% respectively, and proved to be correlated to the intraoperative dimensions of the lesions. Sensitivity of these examinations, in the light of OHM identification, decreased to 69% and 69.2% respectively. This study shows that hepatic US and CT scan are not sufficient to identify OHM; the attempt to reduce the frequency of OHM by means of intraoperative ultrasonography could allow to obtain a more careful stadiation and prognosis of these neoplasms with possible therapeutic advantages. PMID- 1716945 TI - The effect of sodium repletion on growth and protein turnover in sodium-depleted rats. AB - This study examines the consequences of sodium chloride supplementation to young rats previously made salt deficient by feeding them a sodium-deficient, chloride replete diet. Salt-deficient rats received the test diet and distilled water for 10 days. As in our previous studies, rats cared for in this manner grew more slowly than rats fed the identical diet but allowed to drink 37 mM sodium chloride. On day 11, half of the salt-depleted animals received 37 mM sodium chloride in their drinking water. Sodium-deficient and supplemented rats were studied 1,2,5-6 and 11-12 days later. Urinary sodium rapidly rose from undetectable of 46 mEq/l urine within 1 day of supplementation and there was no further increase the next day, suggesting that extracellular fluid volumes were rapidly repleted. Food intake increased in the supplemented rats compared with the deficient animals but the difference in food intake equalled only 2.25 g/day for the first 2 days of supplementation. Over the last 12 days of the study, the slopes of both weight and length gains were equal in both the supplemented and the control group and significantly higher than those in the deficient rats. Over the course of the study, full catchup was not obtained in either length or weight. In addition to total weight and length gains, liver and kidney weights increased proportionately and by 5-6 days of supplementation were equivalent to the weights seen in the control group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716946 TI - Growth hormone resistance and inhibition of somatomedin activity by excess of insulin-like growth factor binding protein in uraemia. AB - Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) were studied in children with end-stage renal failure (ESRF, n = 31) and chronic renal failure (n = 11) with residual glomerular filtration. Somatomedin bioactivity in patient sera was found to be decreased while IGF-I and IGF-II levels by radio immunoassay (RIA) were normal. In contrast, IGFBP-1 and IGFBP-3 levels (measured by RIA) were markedly increased in uraemia. Excess IGFBP was shown to be able to bind IGF by determination of the free IGF binding capacity. Using high performance liquid chromatography a shift of the IGFBP-3 profile to low molecular weight components could be demonstrated in ESRF. Affinity cross-linking experiments showed that these low molecular weight IGFBP-3 immunoreactive forms are biologically active. In normal urine only IGFBP-3 forms smaller than 60 kDa were detected with a major peak at 12-20 kDa. Removal of excessive IGFBP from patient sera by affinity chromatography on an IGF-II Sepharose column resulted in a significant increase in somatomedin bioactivity. Model calculations on the interaction of IGF and IGFBP using empirical data suggested a reduction of IGF secretion in uraemia by an order of magnitude. It is concluded: (1) that renal failure causes an accumulation of low molecular weight IGFBP, (2) that the resulting excess of IGFBP acts as a somatomedin inhibitor, and (3) that in uraemia there is a relative growth hormone resistance with respect to IGF production. PMID- 1716947 TI - Serum alpha 1-microglobulin and beta 2-microglobulin for the estimation of fetal glomerular renal function. AB - As proteins cannot cross the placenta levels of the microproteins alpha 1 microglobulin (alpha 1MG) and beta 2-microglobulin (beta 2 MG) can be used to assess fetal glomerular renal function. alpha 1MG, beta 2MG and creatinine were routinely determined in cord and maternal blood of 133 newborns [gestational age (GA) 25-42 weeks]. Twenty-nine patients with suspected impaired maternal or fetal renal function were studied separately and two fetuses were studied in utero. The mean fetal beta 2MG concentration fell from 3.87 +/- 0.56 mg/l in the 25-31 weeks GA group to 2.60 +/- 0.50 mg/l in the mature newborn group. alpha 1MG concentration fell from 3.10 +/- 0.51 to 2.25 +/- 0.49 mg/dl. In contrast, the mean maternal beta 1MG concentration rose from 1.73 +/- 0.69 mg/l in the 25-31 weeks GA group to a mean of 1.83 +/- 0.48 mg/l in the mature newborn group; alpha 1MG rose from 3.96 +/- 0.58 to 4.33 +/- 1.6 mg/dl. Maternal and fetal creatinine levels were identical. Fetal microprotein levels fall during intra-uterine development as glomerular filtration rate (GFR) rises. There is no correlation between cord blood and maternal alpha 1MG or beta 2MG concentrations. In 13 children with urological anomalies only 1 had elevated microprotein levels and he later developed renal insufficiency. Determination of microprotein levels in fetal serum can be used to detect severe renal function disturbances and to estimate GFR independently of maternal renal function. PMID- 1716948 TI - Chlamydia-specific lymphocytes in conjunctiva during ocular infection: limiting dilution analysis. AB - The immunopathogenesis of ocular infection with Chlamydia trachomatis can be investigated in the cynomolgus monkey model of trachoma. During ocular infection, both follicles (germinal centers) containing primarily B lymphocytes and a heavy perifollicular T cell infiltrate are observed. Limiting dilution analyses were performed on conjunctival and peripheral blood lymphocytes to quantify the proportion of antigen-specific cells elicited by ocular C. trachomatis infection. The frequencies of chlamydia-specific cells were 10-100 times higher in conjunctiva than in peripheral blood. Similar frequencies were observed during active infection and during the ocular delayed hypersensitivity response elicited in sensitized monkeys by topical challenge with a triton X-100 extract containing the 57 kD chlamydial stress protein. Given the antigen-specific proliferative response, these cells are most likely T lymphocytes. The finding of large numbers of antigen-specific conjunctival lymphocytes at the site of chlamydial infection suggests that they play an important role in the pathogenesis of this potentially blinding disorder. PMID- 1716949 TI - Cyclosporin A inhibits DNA synthesis by epidermal Langerhans cells. AB - Cyclosporin A, a potent immunosuppressive drug currently used in organ transplant recipients, has been shown to exert in vitro a direct antiproliferative effect on a number of cell types present in the skin, including keratinocytes, fibroblasts, and endothelial cells. Although in vitro studies suggest that cyclosporin A may interfere with the functional capacities of epidermal Langerhans cells, there is no evidence that the treatment influences the distribution or number of Langerhans cells in vivo. We used a model of normal human skin graft to "nude" mice, which is free of the human systemic control mechanisms, for studies on the DNA synthesis of human Langerhans cells under the influence of cyclosporin A. The grafted animals were given daily subcutaneous (50 mg/kg) or intraperitoneal (5, 12.5, and 25 mg/kg) drug injections during three weeks, which resulted in mean blood levels comparable to those observed in treated patients with organ transplants or psoriasis, respectively. BrdU administered during the last week of the experiment was incorporated by all cells synthesizing DNA, including those passing through S-phase. Langerhans cells were detected on deparaffinized or frozen tissue sections of xenografts with anti-CD1a and anti-HLA DR monoclonal antibodies, and the number of BrdU-positive cells was determined by double labeling. Our results indicate that the Langerhans cell DNA synthesis is impaired by therapeutic levels of cyclosporin A. PMID- 1716950 TI - Cytokine interactions in the central nervous system. PMID- 1716951 TI - Palliative medicine education: bridging the gap between acute care and hospice. PMID- 1716952 TI - c-src structure in human cancers with elevated pp60c-src activity. AB - We used RNAase protection and restriction fragment length polymorphism assays to detect activating mutations of c-src in a spectrum of human tumours. No mutations were detected at codons 98, 381, 444, and 530. We conclude that mutational activation is not the mechanism of enhancement of pp60c-src-specific kinase activity found in a number of human cancer types. PMID- 1716953 TI - An assessment of combined tumour markers in patients with seminoma: placental alkaline phosphatase (PLAP), lactate dehydrogenase (LD) and beta human chorionic gonadotrophin (beta HCG). AB - We have assessed the tumour markers placental alkaline phosphatase (PLAP), lactate dehydrogenase (LD), and human chorionic gonadotrophin (beta HCG) using 2,000 serum samples from 286 patients with seminoma. The ROC curves show that no one marker performs adequately for the detection of disease either at initial staging or during follow-up. We used a Markov model heuristically to devise strategies, in which marker results were assessed in combination, which might be useful in clinical practice. We found that the best strategy was to consider a test result abnormal only if either the beta HCG was greater than 6 Ul-1 or the LD was greater than 400 U l-1 and the PLAP level was greater than 60 U l-1. This will detect about 50% of patients with disease and the false-positive rate is 2%. In practical terms this means that PLAP need only be estimated in patients whose beta HCG is less than 6 IU l-1 and whose LD is greater than 400 U l-1. PMID- 1716954 TI - The differentiation and proliferation of newly formed epidermis on wounds treated with cultured epithelial allografts. AB - Fifteen patients, eight with burn or scald wounds and seven with split-thickness donor sites, were treated with cultured epithelial allografts. Skin was obtained from HIV-negative donors undergoing circumcision and sheets of epithelium were cultured using the 3T3 feeder method. Multiple post-operative biopsies were performed at various time intervals and stained with a panel of monoclonal antibodies against cytokeratins, involucrin, transferrin receptor and epidermal growth factor receptor. Fresh cultured epithelial sheets, normal skin, standard treated donor sites and burns treated with autografts were also studied. Cytokeratin-10 expression was not observed at treated sites until 4 weeks post grafting, when normal suprabasal levels were observed. Cytokeratins 13 and 16, usually observed in highly proliferative states such as psoriasis, were observed in epithelial-treated sites for up to 6 months. Other proliferation markers such as Ki67 and transferrin receptor were only expressed 2-3 weeks post-operatively. Involucrin, a marker of keratinocyte terminal differentiation, was expressed throughout newly formed epidermis until 15 weeks, when the normal pattern of granular expression was observed. These results indicate that although the cultured 'allograft' does not survive, it may modulate the proliferation and differentiation of spontaneously regenerating epithelium. PMID- 1716955 TI - B cells expressing CD5 antigen are markedly increased in peripheral blood and spleen lymphocytes from patients with immune thrombocytopenic purpura. AB - By two-colour flow cytometric analysis, we examined the proportion of B lymphocytes bearing CD5 cell surface antigen (CD 5+ B cells), which are capable of producing autoantibodies, both in peripheral blood and spleen from patients with chronic immune thrombocytopenic purpura (ITP). The percentage of CD5+ B cells in peripheral blood lymphocytes (PBLs) was significantly increased (P less than 0.005) in patients with ITP (3.7 +/- 2.2%, n = 30) as compared with normal controls (1.7 +/- 0.7%, n = 28). However, there was no correlation between the percentages of circulating CD5+ B cells and platelet counts. The percentage of splenic CD5+ B cells in ITP patients was much more increased (9.0 +/- 4.5%, n = 9), P less than 0.005) compared with that of other disorders (3.2 +/- 0.5%, n = 5). Furthermore, isolated splenic CD5+ B cells from two out of five ITP patients produced high levels of IgM-type, platelet-bindable antibodies (PBIgM) after stimulation with Staphylococcus aureus Cowan I (SAC), while CD5- B cells isolated from the same spleen or splenic CD5+ B cells from other non-autoimmune disorders failed to produce significant amount of PBIgM. In three ITP patients, no increase in PBIgM was detected despite SAC stimulation. The increased proportion of CD5+ B cells in peripheral blood and spleen, and their ability to produce anti-platelet antibodies indicate that they are directly involved in the autoimmune pathogenesis in ITP. PMID- 1716956 TI - Antigenic determinants responsible for the reactions of drug-dependent antibodies with blood cells. AB - In an attempt to determine the antigenic combining sites of drug-dependent antibodies in patients with drug-related immune haemolysis, we have assessed the reactivity of 35 nomifensine-induced antibodies against human red blood cells (RBC) in the presence of 11 closely related compounds: nomifensine, its three main metabolites including their methoxylated analogues and diclofensine with its three main metabolites. Three types of antibody reactivity patterns could be differentiated: Firstly, antibodies most strongly reactive with nomifensine (n = 12); secondly, antibodies primarily directed against one of its main metabolites (n = 7), and thirdly, antibodies optimally reactive with its unknown metabolites (n = 16). The antibodies preferentially directed against nomifensine showed varying cross reactions with nomifensine-related compounds and in almost all cases also with diclofensine and/or its metabolites. Those antibodies which were optimally reactive with metabolites reacted only with nomifensine-derived compounds and only one of them was non-crossreactive. The third group of antibodies showed no (n = 12) or only weak (n = 4) cross reactions with nomifensine and/or its metabolites. Although none of the substances used bound firmly to RBC, certain blood groups have been identified in previous studies to be defined. RBC antigens were required for the reaction in approximately 40% of all antibodies, independent of their reactivity with the compounds. Thus, even when the specificity of some antibodies appeared to be predominantly controlled by certain structural features of the compounds, the actual antigenic combining site of each antibody was different and seemed to comprise parts of the drug related determinants as well as different constituents on RBC membranes. These findings indicate firstly that RBCs function as 'carrier-like' macromolecules, since they are directly involved in the reaction; secondly, that the drugs and their metabolites act as 'pseudohaptens', in as much as they do not bind tightly to the cells; and, thirdly, that the determinants which govern the immune response appear to result from an accidental attachment rather than from a predetermined selection of antigenic membrane structures, since each antibody shows a unique reaction pattern. PMID- 1716957 TI - Heterogenous expression of decay accelerating factor and CD59/membrane attack complex inhibition factor on paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes. AB - In order to clarify the characterization of phenotypes of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes (E), we analysed the expression of decay accelerating factor (DAF) and CD59/membrane attack complex inhibition factor (MACIF) on the membrane surface of PNH-E by means of the flow cytometric method using anti-DAF and/or CD59/MACIF monoclonal antibodies in nine PNH-patients. In two-colour analysis, this expression of PNH-E was classified into three fractions; negative, intermediate and positive according to intensity. The negative fraction was classified into two groups; one group an exclusively negative population, and the other a negative population having slightly DAF positive E. The intermediate fraction was recognized on PNH-E of cases with PNH II-E and extremely heterogenous. In the positive fraction, this expression was almost the same as on normal E except for case 8. In single-colour analysis for DAF or CD59/MACIF, three fractions were classified as well as the definition in two-colour analysis. In single-colour analysis, the expression on PNH-E was also heterogenous in each fraction and among PNH-patients. However, the intermediate fraction for CD59/MACIF was not found on PNH-E of cases without PNH II-E, although intermediate fraction for DAF was recognized on PNH-E of some cases with PNH III-E in addition to those with PNH II-E. The results suggest that expression of CD59/MACIF and DAF on the membrane surface of PNH-E phenotypes is heterogenous and varies among PNH patients. PMID- 1716958 TI - Probable in vivo induction of differentiation by recombinant human granulocyte colony stimulating factor (rhG-CSF) in acute promyelocytic leukaemia (APL). PMID- 1716959 TI - Polyclonal CD5 + B-lymphocytosis resembling chronic lymphocytic leukaemia. PMID- 1716960 TI - Emerging of DAF-negative erythrocyte clone in aplastic anaemia before and during haematologic recovery. PMID- 1716961 TI - Induction of granulocyte and granulocyte-macrophage colony-stimulating factors from human monocytes stimulated by Fc fragments of human IgG. AB - The effect of human IgG on human haemopoiesis has been studied in vitro. Dialysed purified IgG stimulated haemopoietic colony growth by bone marrow mononuclear cells (MNC) but not by monocyte-depleted MNC. Culture media, conditioned by IgG stimulated peripheral blood MNC, augmented formation of neutrophil-macrophage, eosinophil, and megakaryocyte colonies by monocyte-depleted marrow MNC. Serum free IgG-conditioned media also contained colony-stimulating activity (CSA). IgG Fc fragments and heat-aggregated IgG promoted the secretion of CSA, but F(ab')2 fragments, Fab fragments or ultracentrifuged IgG did not. In the cell-selection studies, CSA was produced by highly enriched monocytes following stimulation with Fc fragments. The antiserum against human granulocyte colony-stimulating factor (G-CSF) and/or granulocyte-macrophage CSF (GM-CSF) neutralized the CSA produced by Fc fragment-activated monocytes. Enzyme immunoassays showed G-CSF and GM-CSF in media conditioned by monocytes stimulated with the Fc fragments, heat aggregated IgG and anti-D-sensitized red blood cells (RBC). Northern hybridization analysis showed mRNA encoding G-CSF and GM-CSF in RNA extracted from MNC and monocytes cultured with the Fc fragments, but not in the RNA from unstimulated cells or monocyte-depleted MNC. These results indicate that IgG Fc fragments, aggregated IgG and antigen-antibody complexes induce monocytes to produce G-CSF and GM-CSF in vitro. The CSFs release induced by IgG may be involved in the in vivo regulatory network in haemopoiesis. PMID- 1716962 TI - Interleukin-4 prevents the induction of G-CSF mRNA in human adherent monocytes in response to endotoxin and IL-1 stimulation. AB - Human recombinant interleukin-4 (IL-4) was studied for its effects on the expression of granulocyte-colony stimulating factor (G-CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL 1). IL-4 (15 ng/ml) did not induce G-CSF transcripts in monocytes but suppressed the endotoxin-induced G-CSF expression when added simultaneously. Sequential treatment of monocytes with IL-4 followed by endotoxin suppressed G-CSF mRNA induction totally. This effect was independent of the presence of fetal bovine serum but dependent of the IL-4 dose. Comparable results were obtained with IL-1. IL-1 (50 U/ml) induced G-CSF expression in human adherent monocytes which could be counteracted by IL-4 pretreatment. In addition, it was shown that the induction of G-CSF mRNA by the calcium-ionophore A23187 or by c-AMP elevating agents could be blocked by IL-4. These suppressive effects of IL-4 were not related to changes in the half-life of G-CSF mRNA and were independent of protein synthesis. Finally it was demonstrated that IL-4 had comparable effects on the G CSF secretion of endotoxin and IL-1 stimulated human monocytes by using a murine bone marrow assay. These results indicate that IL-4 down-regulates the expression of G-CSF gene and secretion of proteins in human activated monocytes. PMID- 1716963 TI - Two different forms of homozygous sickle cell disease occur in Saudi Arabia. AB - Haematological, clinical and some molecular genetic features of homozygous sickle cell (SS) disease in Saudi Arabia have been compared in 33 patients from the Eastern Province (Eastern) and 30 from the South Western Province (Western). Eastern patients all had the Asian beta globin haplotype whereas Western patients were more variable but predominantly of the Benin haplotype. Eastern patients had more deletional alpha thalassaemia, higher total haemoglobin and fetal haemoglobin levels, and lower HbA2, mean cell volume, reticulocytes, and platelet counts. Clinically, Eastern patients had a greater persistence of splenomegaly, a more normal body build and greater subscapular skin fold thickness, and Western patients had more dactylitis and acute chest syndrome. Painful crises and avascular necrosis of the femoral head were common and occurred equally in both groups. The disease in the Eastern province has many mild features consistent with the higher HbF levels and more frequent alpha thalassaemia but bone pathology (painful crises, avascular necrosis of the femoral head, osteomyelitis) remains common. The disease in the West is more severe consistent with the Benin haplotype suggesting an African origin. PMID- 1716964 TI - The erythrocytes in paroxysmal nocturnal haemoglobinuria of intermediate sensitivity to complement lysis. AB - The sensitivity to lysis by complement of the erythrocytes of 56 patients with paroxysmal nocturnal haemoglobinuria (PNH) was compared to the membrane expression of decay accelerating factor (DAF, CD55), membrane inhibitor of reactive lysis (MIRL, CD59) and acetylcholinesterase (AChE). Most patients (36/50 72% in whom the analysis could be made) appeared to have erythrocytes of intermediate sensitivity to complement in the blood. These cells appeared as a discrete population of cells (PNH II cells), as a 'tail' of cells slightly less sensitive than the predominant PNH III cells (previously called PNH IIIb cells), or as a continuous spectrum of cells sensitive to complement. The PNH III cells totally lacked all three proteins (DAF, MIRL, AChE) by flow cytometric analysis whereas PNH I cells appeared to have normal or nearly normal amounts of each. The cells of intermediate sensitivity (PNH II) had coordinately decreased expression of all three proteins; the level of expression of DAF and MIRL paralleled the sensitivity of the cells to the haemolytic action of complement. PMID- 1716965 TI - Sex-specific transcriptional regulation of the C. elegans sex-determining gene her-1. AB - Expression of the sex-determining gene her-1 is required in C. elegans for the normal male development of XO animals. Abnormal expression in XX animals, which normally develop as hermaphrodites, results in aberrant male development. We have isolated a molecular clone of the her-1 gene and have identified two transcripts that are present in XO animals at all stages of development: an abundant 0.8 kb transcript and a less abundant 1.2 kb transcript. In preparations of XX animals, the 0.8 kb transcript was observed only at very low levels in embryos or L1 larvae and the 1.2 kb transcript was not detected. Two gain-of-function her-1 mutations result in high levels of the 1.2 and 0.8 kb transcripts in XX animals. The levels of these transcripts are also elevated in XX animals carrying a loss of-function mutation in either sdc-1 or sdc-2, consistent with the proposed roles of these genes as negative regulators of her-1. These results demonstrate that expression of the her-1 gene in males and hermaphrodites is controlled at the level of transcript synthesis or accumulation. This mode of regulation contrasts with that found for the Drosophila sex-determining genes, whose sex-specific expression is controlled by differential splicing in males and females. PMID- 1716966 TI - Genetic polymorphism of human glutamate oxaloacetate transaminase (GOT1) detected using isoelectric focusing and a sensitive and positive staining method. AB - Genetic polymorphism of glutamate oxaloacetate transaminase (GOT1) was demonstrated in human erythrocytes by isoelectric focusing in thin layer polyacrylamide gels and a sensitive and positive detection method. Using this technique, five phenotypes, GOT1 1, 2, 2-1, 3-1 and 3-2 were determined and the estimated gene frequencies of GOT1*1, GOT1*2 and GOT1*3 in the Japanese population were 0.9740, 0.0173 and 0.0087, respectively. PMID- 1716967 TI - Identification of fetal hemoglobin and simultaneous estimation of bloodstain age by high-performance liquid chromatography. AB - A method using reverse-phase high-performance liquid chromatography (HPLC) for the identification of fetal hemoglobin (Hb F) and the simultaneous estimation of bloodstain age is described. Umbilical cord and neonatal bloodstains can be differentiated from adult stains by the presence of gamma-globin chains which are characteristic of Hb F. With this method, cord and neonatal blood could be distinguished from adult blood in stains up to 32 weeks old. The age of the stain was estimated from the ratio of the peak area of the alpha-globin chain to that of heme on the same chromatogram. The ratio decreased gradually with an increase in the age of the stain up to 20 weeks old. Studies performed at each time period revealed no significant difference in the ratios of cord and neonatal bloodstains or in the ratios of cord and adult stains. The regression equation calculated from the ratios (y) and the ages of stains in weeks (x) expressed logarithmically is y = 2.5758-0.2497 In (x) and the coefficient of correlation is -0.7491 (n = 252, P less than 0.001). The present method, having the advantages of simplicity, speed and sensitivity, should be of great value to forensic science. PMID- 1716968 TI - Immunohistochemical study on the localization of the epitope defined by a human saliva-specific mouse monoclonal antibody (P4-5C). AB - A novel mouse monoclonal antibody (P4-5C) has been developed which recognizes the core portion of the protein carrying ABO(H) blood group antigens in human saliva. This proved to be specific for human saliva using immunochemical investigations such as enzyme-linked immunosorbent assay, Ouchterlony method and counter immunoelectrophoresis. By light and electron microscope studies with immunohistochemical techniques using this human saliva-specific P4-5C as primary antibody, it was shown that P4-5C reacted specifically and exclusively with mucus from the mucous gland cells of human salivary glands. P4-5C reacted neither with the mucous gland cells of other primates (hamadryas baboon, Japanese monkey and Rhesus monkey) and four mammals (dog, cat, rabbit and mouse) nor with other human tissues. The epitope on the core portion of the ABO(H)-carrying protein was defined by P4-5C and could be discriminated from the epitope of ABO(H) blood group antigens using immunoelectronmicroscopy, although these 2 epitopes were localized relatively close to each other. The P4-5C monoclonal antibody can be also used for morphological species identification of tissue specimens from submandibular glands. PMID- 1716969 TI - Time-dependent RNA synthesis in different skin layers after wounding. Experimental investigations in vital and postmortem biopsies. AB - Incision wounds were made on the outer ear of rats and two biopsies were taken for examination after different survival times. In each case a biopsy was made of vital tissue and a second of postmortem tissue after refrigeration for 24 h. The biopsies were exposed to a solution containing the RNA precursor 3H-cytidine for 1 h, washed and fixed in formalin. Sections 5 microns thick were then autoradiographically prepared and automatically evaluated using Quantimet 920. The intravital specimens showed a significant increase in 3H-cytidine incorporation in the basal cell layer after survival times of 10-24 h. No increase was seen in the stratum corneum, corium or cartilage tissue. The investigated distance from the wound margin did not have any significant bearing on the results. The 3H-cytidine incorporation rate in postmortem tissue was practically identical with that of vital tissue, but no increase was observed in the rate of RNA synthesis in the basal cells as a function of the age of the wound. It may therefore be assumed that this method provides no additional information as to the age of wounds in postmortem examination. PMID- 1716970 TI - New site-directed polyclonal antibody maps N-terminus of occluded region of the non-transformed glucocorticoid receptor oligomer to within BUGR epitope. AB - Using a 17-mer synthetic peptide for immunization, a polyclonal antibody (WS933) directed against amino acid residues 395-411 of the mouse glucocorticoid receptor (GCR) has been raised and used to probe the significance of this region in forming the receptor oligomer and to localize the truncation site of the mutant GCR of the P1798 lymphosarcoma. This region of the receptor, which encompasses the BUGR epitope, is amino-terminal of and immediately adjacent to the DNA binding domain. The polyclonal antibody WS933 reacted with both native and denatured forms of the wild-type mouse GCR as judged by its ability to shift the transformed receptor peak on Sephacryl S300 columns, to immunoadsorb the receptor to protein A Sepharose, and by immunoblot analysis where it identified the 98 kDa receptor protein in the cortisol-sensitive line of the P1798 mouse lymphosarcoma. WS933 also reacted with rat and rabbit GCR, but not human GCR. These characteristics were shared by the BUGR-2 monoclonal antibody. Unexpectedly, there were two highly significant differences between WS933 and BUGR-2. The first was the ability of WS933 to bind to the mutant 45 kDa GCR of the cortisol resistant P1798 lymphosarcoma as judged by its capability of shifting the receptor peak on Sephacryl S300 columns. BUGR-2, in contrast, was unable to shift this mutant receptor peak. Secondly, WS933 was unable to react with the non-DNA binding form of the wild-type (or mutant) GCR, whereas BUGR-2 could react with the non-DNA-binding form of the wild-type GCR. The first observation suggests that the truncation site of the mutant receptor may lie within a portion of the BUGR domain. Additionally, the second observation implies that at least part of the region lying within amino acid residues 395-411 of the mouse GCR is occluded in the receptor oligomer and that this site only becomes available upon transformation of the GCR to the DNA-binding form. This data provides the first mapping of the amino-terminus of the occluded region of the non-transformed receptor, and suggests that WS933 will be a useful probe for characterizing mutant as well as wild type glucocorticoid receptors. PMID- 1716971 TI - Immunoassay using the depolarized and forward scattered light intensity fluctuations from latex spheres. AB - A new optical method is presented here for detecting immunoreaction by means of the forward and depolarized light scattering by coated carrier particles. By this approach, a short-time measurement of antigen-antibody reactions was concisely achieved. The method covered in this article is based on using double-scattered light. While light that is single-scattered by microspheres is polarized parallel to the incident light polarization, double-scattered light contains depolarized components. The normalized fractional variance in the single-scattered field is inversely proportional to the particle concentration, whereas that in the double scattered field is inversely proportional to the square of the particle concentration. Due to this difference, the decrease of the particle concentration during the agglutination reaction is more sensitively detected through the measurement of the fractional variance in the double-scattered field. In addition, undesirable light scattered from non-aggregated microspheres is reduced by using the method summarized here. This study is based on the assumption that the particle concentration of the double-scattered field is 4.5 x 10(11) particles/cm3. The latex spheres coated with antibody molecules aggregated after adding the antigen and, as a result, the fractional variance rapidly increased before leveling off after 5-10 min. The antigens measured were alpha-fetoprotein (AFP) and immunoglobulin E (IgE). PMID- 1716972 TI - Treatment of murine CD5- B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways. AB - Anti-Ig stimulated murine B cells express high levels of surface CD5 (ly-1) and increased CD44 while maintaining surface IgD, CD23 and J11d. Sorting of CD5- and CD5+ cells demonstrates that anti-Ig induces CD5 expression rather than the selective expansion of CD5+ cells. Anti Ig plus interleukin-6 (IL-6) induces the CD23, IgD, low ly-5 (B220) (CD45low), J11dhigh phenotype of typical CD5+ peritoneal B cells. In contrast, lipopolysaccharide (LPS)-stimulated B cells have high levels of CD44 but decreased surface IgD, CD23 and J11d and no CD5. Thus LPS and anti-Ig generate activated cells with differing phenotypes. Induced CD5+ cells have increased viability, even in the absence of added exogenous factors, while the viability of CD5- B cells is dependent on factors such as IL-4. We conclude that conventional CD5- B cells can be activated by either of two pathways: one generating CD5+ B cells; the other yielding conventional activated cells. We hypothesize that the first path requires slg cross-linking and corresponds to T-independent (type 2) stimulation, while cognate interaction with helper T cells in the absence of slg cross-linking induces B cells to enter the second path. PMID- 1716973 TI - CD22 antigen: biosynthesis, glycosylation and surface expression of a B lymphocyte protein involved in B cell activation and adhesion. AB - The CD22 antigen, although present in the cytoplasm of early and immature B cells, first appears on the cell surface of mature B lymphocytes. The phenotypic patterns and some functional properties of the surface-expressed CD22 antigen are well described, but little is known about its molecular structure. We have therefore investigated the relationship of the two CD22 glycoproteins (140/130 kd), and the influence of their complex N-glycosylation on surface expression and antibody recognition. Comparative peptide mapping of the 100 and 80 kd protein cores, obtained by endoglycosidase F treatment, revealed a common structure shared by both protein cores. In pulse-chase experiments the mature glycoproteins originated from two separate precursor molecules, indicating that the two proteins may be generated by different RNA processing. In cell lysates a CD22 specific polyclonal anti-serum recognized both molecules the size of the protein cores and glycosylated CD22 molecules, whereas in membrane preparations only the glycosylated forms were detected. The CD22 mAb HD39 reacted exclusively with the glycoprotein froms in either cellular preparation. The influence of glycosylation on surface expression and epitope recognition was investigated in more detail by applying various inhibitors of the glycosylation pathway. 1-Deoxymannojirimycin and Swainsonine, which block glycosylation at the high-mannose and hybrid-type stages respectively, modulated the CD22 antigen but did not alter its surface expression. Tunicamycin blocked de novo glycosylation and led to reduced surface recognition of the CD22 antigen. Together, these results suggested that comparatively simple oligosaccharide structures of high-mannose type are sufficient for surface expression of the CD22 antigen and for epitope recognition by mAb HD39. It is most likely that glycosylation is required to stabilize epitopes in the protein moiety recognized by CD22 mAb. Finally, we demonstrated the presence of glycosylated, cytoplasmic CD22 antigen in CD22 surface negative B lineage ALL cells. This finding led us to conclude that complex glycosylation does not provide the determining signal for the switch from cytoplasmic to surface expression of the CD22 antigen. PMID- 1716974 TI - Accumulation of CD16-CD56+ natural killer cells with high affinity interleukin 2 receptors in human early pregnancy decidua. AB - Most human peripheral blood natural killer (NK) cells express the phenotype CD16+CD56+. However, a very minor subset of NK cells express CD16-CD56+, and these NK cells bear both interleukin 2 receptor (IL-2R)alpha (p55) and IL-2R beta (p75) (high affinity IL-2 receptors). In this report, we demonstrate that in human early pregnancy decidua--an interface between maternal immunocompetent cells and fetus (placenta)--abundant (approximately 83%) CD16-CD56+ NK cells with high affinity IL-2 receptors were present, and these cells responded to low amounts of IL-2 (4.5 pM). These CD16-CD56+ NK cells significantly expressed an early activation antigen, CD69, in vivo, whereas peripheral CD16-CD56+ NK cells did not express CD69. These findings suggest that CD16-CD56+ NK cells in early pregnancy decidua may be activated in vivo, and may play an important role in immunoregulation during early pregnancy. Also, decidual lymphocytes may be useful materials to study the mechanism of MHC-unrestricted cytotoxicity of this type of NK cells. PMID- 1716975 TI - Selective expansion of idiotype sharing T and B cells in cyclosporin A-mediated autoimmunity. AB - CBA/N mice submitted to autologous bone marrow reconstitution after lethal irradiation and simultaneous Cyclosporin A (CsA) treatment develop a chronic graft-versus-host disease with autoimmune characteristics. When compared to normal controls, diseased mice show an overrepresentation of V beta 8-expressing T cells (65-80% of all CD3+ lymphocytes), together with a marked increase in the titres of serum Ig that specifically bind to F(ab')2 fragments of anti-V beta 8 F23.1 antibodies. Such 'V beta 8-like' Ig V regions are abundantly represented among the IgG2b and mAbs of an unselected collection of hybridomas derived from these mice. These mAbs are not multireactive Ig as they fail to bind to a panel of various antigens and antibodies, but often show simultaneous reactivity with anti-idiotypic mAbs to F23.1 and auto-binding. These molecules may provide the structural basis of V-region specific complementarities, driving the expansion of restricted T and B cell repertoires associated with pathological autoimmunity. PMID- 1716976 TI - Glycine loops in proteins: their occurrence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins. AB - Quasi-repetitive, glycine-rich peptide sequences are widespread in at least three distinct families of proteins: the keratins and other intermediate filament proteins, including nuclear lamins; loricrins, which are major envelope components of terminally differentiated epithelial cells; and single-stranded RNA binding proteins. We propose that such sequences comprise a new structural motif termed the 'glycine loop'. The defining characteristics of glycine loop sequences are: (1) they have the form x(y)n, where x is usually an aromatic or occasionally a long-chain aliphatic residue; y is usually glycine but may include polar residues such as serine, asparagine, arginine, cysteine, and rarely other residues; and the value of n is highly variable, ranging from 1 to 35 in examples identified to date. (2) Glycine-loop-containing domains are thought to form when at least two and to date, as many as 18, such quasi-repeats are configured in tandem, so that the entire domain in a protein may be 50-150 residues long. (3) The average value of n, the pattern of residues found in the x position and the non-glycine substitutions in the y position appear to be characteristic of a given glycine loop containing domain, whereas the actual number of repeats is less constrained. (4) Glycine loop sequences display a high degree of evolutionary sequence variability and even allelic variations among different individuals of the same vertebrate species. (5) Glycine loop sequences are expected to be highly flexible, but possess little other regular secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716977 TI - Conformational epitopes on interstitial collagens. AB - The antigenic response to the helical domain of collagens is normally very low, with the nature of the epitopes recognized by antibodies being dependent on the species of origin. Thus, in certain species, for example rabbit, sequential determinants on single alpha-chains are found, whereas in other species such as mouse, conformational epitopes are predominant. A variety of techniques for identification of epitopes, including rotary shadowing, examination of specific fragments and chemical modification reactions are discussed. The application of these techniques is illustrated using a range of monoclonal antibodies to interstitial collagens. These antibodies show that epitopes are distributed over the length of the collagen molecule. PMID- 1716978 TI - Decreased expression of decay-accelerating factor on endothelial cells of immune complex-mediated vasculitic skin lesions. AB - Endothelial cells may be damaged directly by the membrane attack complex of complement in immune complex vasculitis of the skin. However, for endothelial cell membrane injury to occur, normal regulatory mechanisms must fail. One of the main complement regulatory proteins of endothelial cells is decay-accelerating factor, a surface protein which interferes with either the classical or alternative pathway C3 and C5 convertases. We have investigated the expression of decay-accelerating factor in 4 patients with histologically proven cutaneous immune complex vasculitis, using an immuno-electronmicroscopic technique. We demonstrated that endothelial cells of upper dermal vessels in vasculitic lesions were almost completely devoid of decay-accelerating factor. By contrast, the expression of this protein on endothelial cells in uninvolved skin of the patients was the same as in skin of healthy volunteers. As yet, the mechanism responsible for depletion of decay-accelerating factor is not clear. Absence of decay-accelerating factor may follow enzymatic release from the phosphatidylinositol anchor, proteolytic stripping from the cell membrane or a down-regulation of decay-accelerating factor synthesis. Regardless of mechanism, endothelial cell injury or death could serve a phlogistic function to facilitate complement-mediated destruction of endothelial cells for removal and repair. PMID- 1716979 TI - Cotinine-assisted intervention in pregnancy to reduce smoking and low birthweight delivery. AB - OBJECTIVE: To investigate the feasibility and impact of integrating a cotinine assisted smoking intervention programme with an existing antenatal maternal serum alpha-fetoprotein (AFP) screening service for open neural tube defects. DESIGN: A multisite randomized controlled trial. SETTING: 139 physician offices and clinic sites in Maine providing antenatal care. SUBJECTS: 2848 pregnant women who smoked 10 or more cigarettes daily, enrolled at between 15 and 20 weeks gestation, from a population base of approximately 18,000 pregnancies. INTERVENTIONS: The women were individually allocated at random to intervention or control groups within each centre at the time the serum sample was received for AFP measurement. The intervention group received an interpreted measurement of the serum cotinine, reported through the physician to the woman, along with a self-help smoking cessation booklet and a repeat serum cotinine measurement one month later, again interpreted and reported through the physician to the woman. Women in the control group received the usual anti-smoking advice provided by the antenatal care site and were not told of the study. MAIN OUTCOME MEASURES: Birthweight, physician cooperation with study protocol (as measured by effectiveness in obtaining repeat serum samples for cotinine measurements). RESULTS: Pregnancy outcome data were available for 97% of the study population, including birthweight for 2700 singleton viable pregnancies. The smoking intervention programme led to a significant 66 g increase in mean birthweight (P = 0.03; 95% CI+9 to +123 g) and to a 30% reduction in the rate of low birthweight in pregnancies managed by the 70 physicians who secured the highest rate of obtaining repeat serum samples for cotinine measurements in their intervention group. Among the remaining 69 physicians, intervention had no detectable effect on birthweight. CONCLUSION: A cotinine-assisted smoking intervention programme managed from a central location as an adjunct to a maternal serum AFP screening service can, with the cooperation of physicians responsible for antenatal care, lead to a significant and cost effective reduction in the number of low birthweight babies. This programme is inexpensive, requires little extra effort, and does not need specially trained personnel. PMID- 1716980 TI - Early amniocentesis: alphafetoprotein levels in amniotic fluid, extraembryonic coelomic fluid and maternal serum between 8 and 13 weeks. AB - OBJECTIVE: The aim was to establish a normal range of alphafetoprotein (AFP) concentrations in amniotic fluid from 8 to 12 weeks gestation, and to determine any difference between AFP levels in amniotic fluid and extraembryonic coelomic fluid. DESIGN AND SUBJECTS: 150 women had a transvaginal ultrasound guided amniocentesis before termination of an apparently normal first trimester pregnancy. Separately identified samples of amniotic fluid and extraembryonic coelomic fluid were obtained and assayed by radioimmunoassay for AFP. RESULTS: In amniotic fluid, very high levels of AFP were present at 8 weeks, levels falling rapidly up to 10 weeks after which there was a slight rise. Thus over the period 8 to 10 weeks, there was a significant inverse correlation between amniotic fluid AFP and gestational age (r = 0.67; P less than 0.001). In extraembryonic coelomic fluid, by contrast there was no trend in AFP relative to gestational age. CONCLUSIONS: The rapidly changing levels of AFP from 8 to 10 weeks as well as the small volume of the amniotic cavity makes the use of amniocentesis impracticable before 11 weeks gestation. The lack of any relation between AFP levels in amniotic fluid and extraembryonic coelomic fluid emphasises the importance of identifying the site of amniocentesis in the first trimester. PMID- 1716981 TI - Maternal serum unconjugated oestriol and human chorionic gonadotrophin levels in twin pregnancies: implications for screening for Down's syndrome. AB - OBJECTIVE: To investigate maternal serum unconjugated oestriol (uE3) and human chorionic gonadotrophin (hCG) levels in twin pregnancies and to consider the implications of the results for antenatal screening for Down's syndrome. DESIGN: Measurement of maternal serum uE3 and hCG levels from 15-22 weeks of gestation in twin and singleton pregnancies. Previously available maternal serum alpha fetoprotein (AFP) levels were also presented. SETTING: Stored serum samples collected from women receiving routine antenatal care in Oxford were used. SUBJECTS: 200 women with a twin pregnancy and, for each, three singleton control pregnancies matched for gestational age (same completed week of pregnancy) and duration of storage of the serum sample (same calendar quarter). RESULTS: The median uE3, hCG and AFP levels in the twin pregnancies were respectively, 1.67 (95% CI 1.56-1.79), 1.84 (95% CI 1.64-2.07) and 2.13 (95% CI 1.97-2.31) multiples of the median (MoM) for singleton pregnancies at the same gestational age. The variance of values for the three serum markers (expressed in logarithms), and the correlation coefficients between any two, were similar in the twin and singleton pregnancies. CONCLUSION: In maternal serum screening programmes for Down's syndrome dividing uE3, hCG and AFP MoM values in twin pregnancies by the corresponding medians for twin pregnancies will, in expectation, yield a similar false-positive rate in twin pregnancies as in singleton pregnancies. PMID- 1716982 TI - Cell-substratum interactions and the cytoskeleton in cell shape-mediated growth regulation of lens epithelial cells. AB - Cell attachment to a suitable substratum is a precondition for the mitotic growth of nontransformed lens epithelial cells. Cultering of cells in suspension results in a strong decline of the DNA synthetic rate, whereas reattachment induces the reentrance into the cell cycle. Further studies revealed that not anchorage itself but cell flattening is prerequisite for the entrance of cells into the cycle. Flattened cells exert tension to the substratum via numerous filopodia. If the rigidity of the substratum is reduced by loosening of the collagen gel from the bottom of the petri dish, the gel becomes contracted by the traction forces of the cells and the cell shape becomes transformed from a flattened shape into a more spheroidal or longstretched one. This cell shape transition is connected with a decrease in RNA- and protein synthesis and a stop of DNA synthesis. During further experiments it was demonstrated that microfilaments are involved in gel contraction and cell shape alteration, respectively. Furthermore, intact microfilaments are needed for G0-G1-S-transition. Desintegration of microfilaments by cytochalasin is without influence on ongoing DNA synthesis but hinders strongly the entrance of cells into the S-phase. The survey gives some recent results on the molecular basis of cell substratum interactions as well as the structure and function of the cytoskeleton. The role of the cytoskeleton in cell shape-mediated growth regulation is discussed. PMID- 1716983 TI - RNA today. Molecular Evolution of Introns and Other RNA Elements: a Keystone Symposium, Taos, NM, USA, February 2-8, 1991. AB - RNA research is alive and well. The joyride for those studying the biochemistry and molecular biology of RNA continues, although perhaps not at the thrill-a month pace of recent years. The Keystone Symposium provided an opportunity to gain deeper insight into RNA-based biological phenomena by attempting to place current research in an evolutionary context. In this sense the meeting was an unqualified success. The meeting participants, having been warmed by the New Mexico sun and the chile-laden cuisine, now return to their laboratories determined to pursue not only the details of RNA biochemistry and molecular biology, but also the evolutionary implications of their work. PMID- 1716984 TI - Effect of increased chain packing on gramicidin-lipid interactions. AB - To study the effect of lipid packing on the dynamics of membrane proteins, the changes in the rotational motion of gramicidin tryptophans with increased packing brought about by high hydrostatic pressure through fluorescence spectroscopy were determined. In fluid phase dimyristoylphosphatidylcholine, the rotational motion of the residues decreased slightly with increased packing, but in the gel phase a significant reversible increase was observed. The magnitude of this increase was temperature dependent and much greater at lower temperatures. Quenching studies show that the increase in rotational motion is not due to a change in the location of the peptide in the membrane under pressure. Aromatic ring stacking between residues 9 and 15 appears to be stabilized under pressure, and there is no evidence of pressure-induced changes in peptide aggregation. The increase in rotational motion could be caused by a destabilization of hydrogen bonds between the indole hydrogens and the lipid head group oxygens due to an increase in the thickness of the compressible lipid bilayer with pressure without a concomitant lengthening of the peptide. These results indicate that specific interactions between lipids and proteins may play a major role of regulating the dynamics of membrane proteins. PMID- 1716985 TI - Transient induction of the mitochondrial permeability transition by uncoupler plus a Ca(2+)-specific chelator. AB - Determinations of aqueous space volumes, swelling and Mg2+ release experiments demonstrate that EGTA plus uncoupler causes the permeability transition in Ca(2+) loaded mitochondria. Extramitochondrial Mg2+ is required to obtain this effect. Changes in transition-dependent parameters are smaller and more varied when induced by EGTA plus uncoupler than when induced by Ruthenium red plus uncoupler, although inhibitor-sensitive experiments show that the same basic mechanism is involved in both cases. Measurements of sucrose trapping and sucrose or inulin accessible space, after changes in transition-dependent parameters are complete, indicate that rapid reversal occurs when the transition is induced by EGTA plus uncoupler, explaining why limited responses are obtained. Data support the hypothesis that an external divalent cation binding site regulates activity of the mitochondrial Ca2+ uniporter. PMID- 1716986 TI - Why is inorganic phosphate necessary for uncoupling of oxidative phosphorylation by Cd2+ in rat liver mitochondria? AB - The phosphate (Pi)-dependent uncoupling action of Cd2+ in oxidative phosphorylation in rat liver mitochondria was studied mainly in terms of Pi transport. Cd2+ at 2 microM caused full uncoupling in the presence of 10 mM Pi, but no uncoupling in the absence of Pi. Cd2+ released state 4 respiration after a certain lag-time, and then the respiration increased progressively with time. After its addition, Cd2+ was taken up by mitochondria in a similar period to the lag time before respiratory release. KIH-201, a potent and specific inhibitor of Pi transport via the Pi/H+ symporter, abolished the uncoupling completely. Cd2+ caused dissipation of the electric transmembrane potential (delta psi) and swelling of mitochondria in a Pi-dependent manner. Uncoupling by Cd2+ was found to take place in parallel with the uptake of Pi into mitochondria via the Pi/H+ symporter, suggesting that the uncoupling was due to acceleration of H+ influx through the Pi/H+ symporter activated by Cd2+. PMID- 1716987 TI - A zein signal sequence functions as a signal-anchor when fused to maize alcohol dehydrogenase. AB - A chimeric gene, preZad, was constructed encoding a zein signal sequence fused precisely to the amino terminus of maize alcohol dehydrogenase 1. Translocation and processing of this chimeric preZad protein were assayed in vitro using a rabbit reticulocyte lysate translation system supplemented with canine pancreatic microsomes. PreZad was cotranslationally translocated across the vesicular membranes. Unexpectedly, the signal sequence was not removed although a suitable cleavage site was preserved and presented within the vesicle lumen. Failure to cleave the signal sequence was apparently not due to the lack of a beta-turn near the processing site. When a beta-turn was introduced near the cleavage site through site-directed mutagenesis, no processing was observed. PreZad was not solubilized by alkaline treatment of the microsomes, indicating an integral membrane association. Resistance to proteolysis, in the absence of detergent, indicates that preZad is associated with the membranes in a type II orientation (C-terminus in and N-terminus outside the vesicles). Analysis of truncated versions of preZad showed that it is the uncleaved signal sequence that functions as a signal-anchor. Changing the ratio of net charge flanking the signal sequence to less than 1 (N-terminal:C-terminal) did not alter the type II membrane orientation, as would have been predicted by the 'positive-in rule'. Our results provide additional insight into the role of the passenger protein and signal sequence-flanking regions in recognition of a signal peptidase processing site, and the orientation of insertion of a signal-anchor sequence into the endoplasmic reticulum membrane. PMID- 1716988 TI - Regulation of glucose transport in Clone 9 cells by thyroid hormone. AB - Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1 2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT 1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre existing membrane transporter sites. PMID- 1716989 TI - Potentiation of the activity of mouse liver-derived HBGF-1-like growth factor by heparin and dithiothreitol: evidence for the involvement of a plasma factor. AB - The potentiation of mouse liver derived heparin binding growth factors 1 and 2 (HBGF-1, HBGF-2) activity has been investigated. It was found that both heparin and various sulfhydryl reagents (such as dithiothreitol, DTT) markedly potentiated HBGF-1 activity, but not HBGF-2 activity. Further studies with HBGF-1 indicated that the growth factor would interact with a plasma factor, in a temperature-dependent manner, to become inactive, and that sulfhydryl reagents would reverse this inactivation. Inactivation would not occur either in the presence of heparin or DTT, indicating that heparin and DTT can protect the growth factor from plasma inactivation. When assayed in the absence of plasma, both heparin and DTT were required to reactivate plasma inactivated HBGF-1-ML. A model is presented to explain these data. This model predicts that either DTT or heparin can block the plasma induced inactivation process, but that once inactivation has occurred only sulfhydryl reagents can restore activity. Furthermore, heparin is thought to activate growth factor activity in the absence of plasma by blocking non-productive growth factor binding to the extracellular matrix. The identification of a plasma inactivating factor for mouse liver derived HBGF-1 has important implications for understanding the regulation of extracellular growth factor activity. PMID- 1716991 TI - A fast reliable method for the measurement of intraperitoneal dextran 70, used to calculate lymphatic absorption. AB - The use of intraperitoneally administered dextran 70 was investigated for measurement of lymphatic absorption in CAPD patients. For this purpose a fast, highly accurate HPLC method was developed, that was not influenced by the high glucose concentration in peritoneal dialysate, nor by the inulin that was added to the dialysate for the measurement of residual volume. Pretreatment of the samples consisted of deproteinization with trichloroacetic acid, followed by incubation at 45 degrees C to hydrolyze inulin. This was followed by a further rinsing step using a Sephadex G-25 PD-10 column. The HPLC was performed on a Bio Gel XL guard column with refractive index detection. Because of the short guard column the chromatographic analysis was only 5 minutes for one sample. With the method described lymphatic absorption rate was measured in 30 CAPD patients and was found to range from 0.1 to 3.5 mL/min, median 1.0 mL/min. PMID- 1716992 TI - Individual characterization of the peritoneal restriction barrier to macromolecules. AB - The transport of macromolecules such as proteins and dextrans during CAPD is restricted by the permeability of the peritoneum. When its degree is determined by diffusion characteristics of macromolecules, the intrinsic permeability of the peritoneal membrane can be expressed as the relation between clearance of macromolecules and parameters of diffusion velocity. In order to characterize the peritoneal restriction barrier in individual patients, such a relationship has to meet the following conditions: (a) a good fit to linearity, (b) a low inter- and intra individual variability and (c) similarity for proteins and equal sized dextran fractions. In the present study a comparison is made between the reciprocal plot (RP): C-1 = s (esr) + A and the diffusion plot (DP): C = A'. Ds20,w. In these equations C is clearance, esr is Einstein Stokes radius, D20,w is the free diffusion coefficient in water, A and A' are constants, while s is the slope of the regression line and therefore represents intrinsic peritoneal permeability to macromolecules. The 95% confidence interval of the correlation coefficient r was above 0.95 in all studies for the RP and the DP. In general it was somewhat higher for the DP. The inter-individual coefficient of variation was lower in the DP than in the RP. This was also the case for the intra-individual coefficient of variation of the slope for proteins (DP 4% vs RP 27%, p less than 0.01) and for dextrans (DP 18% vs RP 47%, p less than 0.05). The correlation coefficient between dextran slopes and protein slopes was higher for DP (r = 0.68) than for RP (r = 0.50).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1716990 TI - Phytoestrogens: new ligands for rat and human alpha-fetoprotein. AB - The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat alpha fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA greater than equol greater than enterolactone greater than enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants (Kd) for phytoestrogens binding to AFP were: Kd NDGA = 5 +/- 1.2.10(-7) M, Kd equol = 6.7 +/- 0.8.10(-6) M, Kd enterolactone = 1.7 +/- 0.4.10(-5) M and Kd enterodiol = 2.2 +/- 0.6.10(-5) M. The Kd for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of esterone binding sites remained unchanged. This, plus the results of double reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased Kd), but high concentrations induced conformational changes in rat AFP, as both Kd and the number of binding sites (n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development. PMID- 1716993 TI - [Common and peculiar features of the interaction of hemoglobins with hydrated dipalmitoyllecithin]. AB - It was demonstrated that human and horse hemoglobin variants having quantitative difference in the interaction with dipalmitoyllecithin exhibit features of generality. The latter is manifested in the established hydrophobic contacts between protein and lipid in hydrated films discovered by IR spectroscopy. The arrangement of hydrophobicity profiles of hemoproteins demonstrated in amino acid sequences of chains the existence of intermittent hydrophobic and hydrophilic regions. Such a composition of hemoglobins could underlie their property to participate in hydrophobic interactions with lipids. PMID- 1716994 TI - [Nonlinear modelling of the propagation of action potentials]. AB - The role of nerve fibre membrane inductance in the action potential spreading was studied. We received a nonlinear differential equation for the action potential. This equation has soliton solutions. On the basis of the suggested model the numeral calculation results were given in our paper. PMID- 1716995 TI - RecBC promoted repair of bleomycin damage in Escherichia coli. AB - The repair response of Escherichia coli K-12 to bleomycin was examined in Rec- mutants showing differential sensitivity to this agent. Sedimentation analysis of the cellular DNA showed incision after bleomycin treatment. The subsequent reformation of the DNA, found in the wild-type and the recD mutant, was abolished in the recB and delayed in the recF and recBC sbcB mutants. The bleomycin-induced SOS response was reduced in strains containing recB or recBC sbsB mutations. It is suggested that the RecBCD pathway has the main role in the efficient repair of bleomycin-induced DNA damage. PMID- 1716996 TI - Day-to-day variability of protein transport used as a method for analyzing peritoneal permeability in CAPD. AB - The transperitoneal transport of macromolecules is dependent on both effective peritoneal surface area and intrinsic permeability of the peritoneum. For passage of small solutes, the effective surface area is the main determinant. We hypothesized that day-to-day variations in peritoneal clearances are caused by changes in the effective surface area and not in the intrinsic permeability. Four CAPD (continuous ambulatory peritoneal dialysis) patients without peritonitis were investigated on 28 consecutive days. Concentrations of beta-2-microglobulin, albumin, IgG, and alpha-2-macroglobulin were determined daily in dialysate (night bags) and weekly in serum. Clearances and their coefficients of variation were calculated. Mean coefficients of the intraindividual variation of protein clearances increased, the higher the molecular weight: they ranged from 12% for beta-2-microglobulin clearance to 22% for alpha-2-macroglobulin clearance. Correlations were present between the clearances of albumin, IgG, and alpha-2 macroglobulin, but not between any of these and beta-2-microglobulin clearance. In all patients, protein clearance (C) was a power function of the free diffusion coefficient in water (D) according to the equation: C = a. Db in which b represents the restriction coefficient of the peritoneum, and thus intrinsic permeability. The coefficient of variation of the restriction coefficient was low (range 4-6%). This supports our assumption that the intrinsic permeability is fairly constant on the short term. Day-to-day variations in protein clearances are thus mainly caused by alterations in the effective peritoneal surface area. Long-term follow-up of the restriction coefficient in individual patients might identify those at risk for the development of structural changes in the peritoneal membrane. PMID- 1716997 TI - Compound heterozygosity for a beta zero-thalassemia (frameshift codons 38/39; -C) and a nondeletional Swiss type of HPFH (A----C at NT -110, G gamma) in a Czechoslovakian family. AB - We have analyzed the levels and composition of the fetal hemoglobin (Hb F) in several members of a Czechoslovakian family with a heterozygosity for a newly discovered beta zero-thalassemia (codons 38/39; -C), or for a newly detected nondeletional hereditary persistence of fetal hemoglobin (a form of Swiss-HPFH with an A----C mutation at nucleotide -100 5' to the Cap site of G gamma), or with a compound heterozygosity for these two conditions. The Hb F level in the beta zero-thalassemia heterozygotes averaged approximately 0.3% with low G gamma values (approximately 28%) and relatively high A gamma T values (approximately 50%), that in the two Swiss-HPFH heterozygotes averaged 0.8% with approximately 95% G gamma, while that of the compound heterozygote was 3.1% with approximately 95% G gamma. The low Hb F levels were determined with a recently published cation exchange high-performance liquid chromatography (HPLC) procedure that is accurate at the 0.1%-0.2% Hb F level. This method, together with a reversed-phase HPLC procedure, made it possible to detect this unusual type of nondeletional G gamma HPFH and provided the data indicating that the increased Hb F in the compound heterozygote was derived mainly from the chromosome with the HPFH determinant. PMID- 1716998 TI - [Hybridase cleavage of RNA. III. Use of UV-spectroscopy for studying the kinetic properties of E. coli RNAase properties]. AB - A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml. PMID- 1716999 TI - Application of stains-all for demarcation of cement lines in methacrylate embedded bone. AB - Cement lines provide a record of sites of past remodeling buried in the matrix of bone. A method is reported for application of Stains-all, a cationic carbocyanine dye, for demarcation of cement lines in bone. The method, which is simple, works well for both glycol methacrylate and methyl methacrylate undemineralized embedments and produces good concomitant staining of cytoplasm and nuclei of osteoblasts, osteoclasts and marrow cells. PMID- 1717000 TI - Short Nissl staining for incubated cryostat sections of the brain. AB - Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poorly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods. PMID- 1717001 TI - A method to stain decalcified bone without loss of structural detail. AB - A modification of Bodian's protargol-S technique is done on 7 microns sections of decalcified bone. Light microscopic results are greatly improved when compared to either ground bone sections or decalcified bone stained routinely with hematoxylin and eosin. PMID- 1717002 TI - Potentiated 5-hydroxytryptophan response suppression following 5,7 dihydroxytryptamine raphe lesions in an animal model of depression. PMID- 1717003 TI - New approaches and concepts in the study of differentiation of oral epithelia. AB - Epithelial structural proteins, the keratins and keratin-associated proteins, are useful as markers of differentiation because their expression is both region specific and differentiation-specific. In general, basal cells in all stratified oral epithelia express similar keratins, while the suprabasal cells express a specific set of markers indicating commitment to a distinct program of differentiation. Critical factors in the regulation of epithelial protein expression are now under investigation. The promoter regions of keratin genes are being characterized to determine what sequences within the genes are responsible for differential expression. One important extracellular factor that influences epithelial protein expression is retinol (vitamin A), which exerts its effects via a group of nuclear receptor proteins that may also be expressed in a region specific manner. These molecular biological approaches enhance our understanding of the mechanisms regulating differentiation of oral epithelia and its regional complexity. PMID- 1717004 TI - Disease specificity of antibodies to poly (ADP-ribose); their relationships to anti-DNA antibodies and to disease activity in lupus. AB - In this study we have measured the level of anti poly (ADP-ribose) antibodies in the sera of a number of patients with SLE and their relatives, patients with a wide variety of other autoimmune and infectious diseases, and a group of normal healthy controls. It was found that these antibodies were not disease specific but were present in nine out of thirteen groups tested in significant numbers. The levels of anti poly (ADP-ribose) antibodies and anti DNA antibodies in SLE patients bled serially were also measured. The level of these antibodies fluctuated in parallel in many of these patients, although the anti poly (ADP ribose) antibodies reflected disease activity more accurately in some. PMID- 1717005 TI - Predicting antigenic determinants of autoantigens. AB - Predicting antigenic determinants of foreign proteins from their amino acid sequence and/or conformation is of growing importance in the production of synthetic vaccines and antigens. Unlike foreign antigenic proteins, little is known of the suitability of predictive techniques for defining antigenic regions of self proteins recognised by autoantibodies. In this study we describe our use of two computer programmes (HYDRO 3 and ACROPHILICITY [ACRO], Hopp, 1986) for the prediction of antigenic determinants of autoantigens of the cell nucleus. Using the amino acid sequence of the protein, HYDRO 3 and ACRO respectively, provide information on the hydrophilic and surface regions of the protein. Both methods were used to predict the antigenic determinants of known autoantigens, including histones, the A, B", E and 70 kD proteins of snRNPs, SS-B/La, proliferating cell nuclear antigen (PCNA) and others. Our analysis of the antigenic determinants of histones agreed with other studies which have used antihistone antibodies and fragments of histones to show that autoantibody reactive sites reside in the terminal portions of these proteins, particularly the amino terminus. A detailed study of histone 2B correctly identified most regions recognised by antibodies, particularly autoantibodies. In addition the recently described epitope of the autoantigen ribosomal protein P2 was predicted by this analysis. From these observations we hypothesize that linear antigenic sites of self proteins can be predicted. Our hypothesis can be proven experimentally by demonstrating specific interaction between autoantibodies and synthetic peptides homologous with the predicted determinant. PMID- 1717006 TI - Binding of bovine and porcine pituitary glycoprotein hormone alpha-subunit to TSH antibody in serum of patients with Graves' disease. AB - The characteristics of autoantibodies reactive with bovine (b) TSH were examined in the sera of six patients with Graves' disease selected on the basis of highly negative values in the TSH receptor assay. Test sera were incubated with other 125I-labeled pituitary glycoprotein hormones and their isolated subunits (alpha and beta) [human (h) TSH, bTSH, porcine (p) TSH, pFSH, bFSH, bLH and equine (e) chorionic gonadotropin (CG)] (purity was confirmed by gel-filtration on Sephadex G-100 and SDS-PAGE), and the antibody bound fraction was precipitated by the addition of anti-human gamma-globulin (goat). Almost all sera showed detectable binding to bTSH, pTSH, pFSH, pTSH-alpha, bFSH-alpha, bLH-alpha, but not to hTSH, hTSH-alpha, hTSH-beta, hFSH, hLH, hCG, pTSH-beta, bLH-beta, eCG-alpha. Exceptions were very low binding to bLH-beta by one serum and to pTSH-beta, by two sera. The level of binding (B/T%) of the patients' sera to pTSH-alpha, bFSH-alpha and bLH alpha was 3.0-27.7%, 2.6-45.3% and 2.2-39.0%, respectively; that of sera from normal healthy adults was 1.9 +/- 0.3%, 0.8 +/- 0.2% and 0.9 +/- 0.2% (mean +/- SD), respectively. These results indicate that the TSH antibodies recognize mainly an epitope in the alpha subunit of bovine and porcine pituitary glycoprotein hormones (TSH, FSH, LH). PMID- 1717007 TI - Effects of FK506, 15-deoxyspergualin, and cyclosporine on experimental autoimmune uveoretinitis in the rat. AB - The effects of two novel immunosuppressants, FK506 and 15-deoxyspergualin (15 DSG), on experimental autoimmune uveoretinitis (EAU) were evaluated in the rat. Rats were immunized with retinal soluble antigen (S-antigen) and treated with FK506 or 15-DSG, and the disease induction as well as the immune responses to the antigen were examined. The results were compared with animals treated with cyclosporine (CsA) which has been widely used to treat refractory uveitis in humans. A dose-response study showed that FK506 suppressed EAU induction at doses 10-30 times lower CsA when given on days 0-14 postimmunization, while 15-DSG suppressed the disease development at doses very similar to CsA. As with CsA, FK506 suppressed EAU induction when given only in the induction phase (days 0-5 postimmunization) or in the effector phase (days 7-12), but at doses much lower than CsA. On the other hand, 15-DSG suppressed EAU only at toxic doses with these schedules. The antigen specific mitotic response of lymphocytes was markedly suppressed by the three agents, while the antigen specific antibodies in sera were suppressed by 15-DSG and FK506 but not by CsA at doses effective in suppressing the disease induction. More significant findings were uniquely prolonged immunosuppressive effects of FK506: EAU induction as well as the immune responses to S-antigen were suppressed long after the cessation of drug treatment. PMID- 1717008 TI - FR900506 (FK506) and 15-deoxyspergualin (15-DSG) modulate the kinetics of infiltrating cells in eyes with experimental autoimmune uveoretinitis. AB - In order to evaluate two new immunosuppressive agents, FR900506 (FK 506) and 15 Deoxyspergualine (15-DSG), the kinetics of infiltrating cells in the eyes of Lewis rats with experimental autoimmune uveoretinitis (EAU) were studied. Rats were immunized with retinal S-antigen and treated with different doses of either FK 506 or 15-DSG. The inflammatory ocular tissues obtained at various intervals during the process of EAU were examined using an immunoperoxidase technique. The results were compared with those eyes developing EAU without treatment or with suboptimal doses or a suboptimal dose of Cyclosporine (CsA). Both FK 506 and 15 DSG, like CsA, delayed the cellular kinetics during the course of EAU. However, FK 506 had the greatest effect on the kinetics of T lymphocyte subsets by causing the greatest increase in the recruitment time of the T suppressor/cytotoxic population. FK506 treatment resulted not only in the highest inhibition of expression of IL-2 receptors on T cells, but also in the prevention of the expression of MHC class II antigens on ocular resident cells. Treatment with 15 DSG resulted in general immunosuppression on various infiltrating inflammatory cells. PMID- 1717009 TI - Demonstration of IgE antibodies to nucleic acid antigens in patients with SLE. AB - Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target tissues. PMID- 1717010 TI - Studies with recombinant autoepitopes of thyroid peroxidase: evidence suggesting an epitope shared between the thyroid and the gastric parietal cell. AB - In a previous work we characterized a major epitope of thyroid peroxidase (TPO), which was recognised by 66% of 157 patients with autoimmune thyroid disease (AITD) and 9 out of 50 patients with non-thyroidal autoimmune disease (NTAID) 6 of whom had antibodies to the gastric parietal cell antigen (PCA). In the present study we have affinity purified C2 antibodies and demonstrated that they bind to the native TPO enzyme in a radioimmunoassay (RIA). We have measured antibodies to the C2 peptide and a second TPO peptide, C21, in enzyme-linked immunosorbent assays (ELISA) in 30 patients with NTAID all of whom have antibodies to the gastric PCA, having first determined the incidence of antibodies to C21 in 98 patients with AITD who do not have antibodies to the gastric PCA. 58% of patients with AITD have antibodies to C21, a peptide of TPO which overlaps C2 by 21 amino acids in the region containing an 11 residue fragment which is very similar to a fragment of the H+ K+ ATPase, which has recently been shown to be a major component of the gastric PCA. In patients having NTAID and antibodies to the gastric PCA, 60% are positive for C2 Ab and 100% for C21 Ab, which is suggestive of an epitope shared by TPO and H+ K+ ATPase, corresponding to TPO 659----669 and H+ K+ ATPase 177----187. We conclude that C2 antibodies are heterogenous and comprise activities which bind to the intact enzyme and activities binding to an epitope which may be common to TPO and H+ K+ ATPase. PMID- 1717011 TI - Recombinant reverse transcriptase of Rous sarcoma virus: characterization of DNA polymerase and RNAase H activities. AB - Enzyme preparations of Rous sarcoma virus (RSV) reverse transcriptase have been isolated from a culture of E. coli HB101(pMF14). The enzyme has been purified to homogeneity and been shown to consist of two subunits, of molecular mass 97.4 and 61.3 kDa, respectively. The optimum conditions for the DNA polymerase and RNAase H activities, fidelity of DNA synthesis on a homogeneous RNA template, and the inhibitory effect of azidothymidine triphosphate have been determined. Data on the use of RSV recombinant reverse transcriptase for cDNA synthesis are given. PMID- 1717012 TI - Local activation of lymphoid cells in rheumatoid synovium. AB - Lymphoid cell phenotype was investigated in the peripheral blood, synovial fluid, and synovial tissue of sixteen patients with rheumatoid arthritis (RA). In the peripheral blood of RA patients the proportion of cells expressing HLA-DR and beta 2-microglobulin receptors was higher than in normal controls, whereas the proportion of cells that were CD5+ (i.e. were T lymphocytes) was lower. Expression of the other cell surface antigens studied remained in the normal range. In the synovial tissue and synovial fluid of RA patients there was an increased percentage of cells expressing HLA-DR, beta 2-microglobulin receptors, CD25, CD5, CD4, and Thy-1, but the proportion of CD8+ cells was significantly decreased compared with that seen in peripheral blood. The CD4+/CD8+ ratio in RA joints was therefore significantly higher than that in peripheral blood. The proportion of cells expressing HLA-DR correlated with disease activity. PMID- 1717013 TI - The effect of chronic alcoholism on neuroendocrine and immune parameters. AB - Parameters of cellular and humoral immunity, and the levels of neurotransmitters and hormones both before and after the administration of various stimulators and blocking agents that affect the immune and neuroendocrine systems were investigated in 100 patients suffering from chronic alcoholism. The results direct attention to the significance of the impairment of interactions between these biological systems during chronic alcoholism. PMID- 1717014 TI - Two-colour flow cytometry study of lymphocyte subpopulations in patients with primary immunodeficiencies. AB - Immunofluorescent flow cytometric examination of one hundred and eighty-five children with different primary immunodeficiency syndromes and sixty-nine control patients revealed twenty-six cases with a bimodal distribution of antigens CD5 and CD7. Such abnormalities were most frequently found in patients with total antibody deficiency, namely those with common variable hypogammaglobulinaemia (10/24 patients) and congenital agammaglobulinaemia with lack of B cells (10/40), but were never seen in normal controls. Two-colour flow immunofluorescence demonstrated that antigen CD4 was expressed only on intensely fluorescent CD5+ cells, irrespective of the immunodeficiency state. Antigen CD4 was detected on cells with both high and low expression of antigen CD7, but a small percentage (2%-5%) of CD4+ lymphocytes did not belong to the CD7+ population. Antigen CD8 was found equally on intensely and weakly fluorescent CD5+ and CD7+ cells. In some immunodeficient patients suffering from ataxia-telangiectasia (12/36) and in some with Wiskott-Aldrich syndrome (2/6) there was a significant excess (greater than 20%) of CD7+ over CD5+ cells. In these patients a considerable number of the CD8+ cells were not part of the CD5+ population, but were always part of the CD7+ population. Cell populations with the phenotype CD5-, CD7+ consisted mainly of lymphocytes showing weak expression of antigen CD8. PMID- 1717015 TI - Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating. AB - The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity. PMID- 1717016 TI - Dynamics and aggregation of the peptide ion channel alamethicin. Measurements using spin-labeled peptides. AB - Two spin-labeled derivatives of the ion conductive peptide alamethicin were synthesized and used to examine its binding and state of aggregation. One derivative was spin labeled at the C-terminus and the other, a leucine analogue, was spin labeled at the N-terminus. In methanol, both the C and N terminal labeled peptides were monomeric. In aqueous solution, the C-terminal derivative was monomeric at low concentrations, but aggregated at higher concentrations with a critical concentration of 23 microM. In the membrane, the C-terminal label was localized to the membrane-aqueous interface using 13C-NMR, and could assume more than one orientation. The membrane binding of the C-terminal derivative was examined using EPR, and it exhibited a cooperativity seen previously for native alamethicin. However, this cooperativity was not the result of an aggregation of the peptide in the membrane. When the spectra of either the C or N-terminal labeled peptide were examined over a wide range of membrane lipid to peptide ratios, no evidence for aggregation could be found and the peptides remained monomeric under all conditions examined. Because electrical measurements on this peptide provide strong evidence for an ion-conductive aggregate, the ion conductive form of alamethicin likely represents a minor fraction of the total membrane bound peptide. PMID- 1717017 TI - Opening rate of acetylcholine receptor channels. AB - The nicotinic acetylcholine (ACh) receptor is responsible for rapid conversion of chemical signals to electrical signals at the neuromuscular junction. Because the receptor and its ion channel are components of a single transmembrane protein, the time between ACh binding and channel opening can be minimized. To determine just how quickly the channel opens, we made rapid (100-400 microseconds) applications of 0.1-10 mM ACh to outside-out, multichannel membrane patches from BC3H-1 cells, while measuring the onset of current flow through the channels at 11 degrees C. Onset time is steeply dependent upon ACh concentration when channel activation is limited by binding of ACh (0.1-1 mM). At +50 mV, the 20-80% onset time reaches a plateau near 110 microseconds above 5 mM ACh as channel opening becomes rate limiting. Thus, we calculate the opening rate, beta = 12/ms, without reference to specific channel activation schemes. At -50 mV, the combination of a rapid, voltage-dependent block of channels by ACh with a finite solution exchange time distorts onset. To determine opening rate at -50 mV, we determine the kinetic parameters of block from "steady-state" current and noise analyses, assume a sequential model of channel activation/block, and numerically simulate current responses to rapid perfusion of ACh. Using this approach, we find beta = 15/ms. In contrast to the channel closing rate, the opening rate is relatively insensitive to voltage. PMID- 1717019 TI - Development-specific genes and their expression at the RNA level. AB - A total of seven clones, which are specific for the development of carrot somatic embryo cells, were constructed and screened from the cDNA expression library of phage lambda gt11. The purification of plaques, isolation of DNA from the phages, digestion of the clone's DNA with EcoRI restriction enzyme, identification of the inserts on the agarose gel by electrophoresis and subcloning of cDNA inserts from phage lambda gt11 vector into plasmid pIBI or pUC18 were performed respectively. The data of cross hybridization by Southern blot analysis indicated that clones 16,22,50 and 60 are new cDNA genes. In addition, the new cDNA genes tissue specific expression on variety organs in the carrot plant, such as flower, petiole, leaf and root, as well as time-course expression in callus and embryo cells of different culture periods were determined through Northern blot analysis. The results suggested that the corresponding gene for clone 22 is a specific gene which is regulated by development and closely associated with carrot somatic embryogenesis. PMID- 1717018 TI - Time course of receptor-channel coupling in frog sympathetic neurons. AB - The M-type potassium current and the N-type calcium current are inhibited by several different neurotransmitters in frog sympathetic neurons. These effects seem to be mediated via G proteins, but it is not clear whether diffusible second messengers are involved. Using a rapid (approximately 100 ms) flow tube perfusion system to apply agonists, the inhibition of calcium current develops and recovers rapidly but not instantaneously (t1/2 = 1-2 s). M-current inhibition is considerably slower, with t1/2 approximately 30 s for recovery from inhibition. At least for M-current inhibition, there appears to be sufficient time for involvement of an enzymatic cascade in receptor-channel coupling. PMID- 1717020 TI - Alpha-fetoprotein. AB - The past year has seen major challenges to existing maternal serum alpha fetoprotein testing protocols for both neural tube defects and chromosomal anomalies. These challenges are reviewed along with the physiology of alpha fetoprotein; the use of amniocentesis, ultrasonography, and additional serum markers in women with elevated alpha-fetoprotein levels; and epidemiologic implications of maternal serum alpha-fetoprotein screening. PMID- 1717021 TI - Prenatal diagnosis of structural abnormalities. AB - In the past year, improved ultrasonographic quality has allowed the imaging of more and more subtle anomalies. A major focus of recent discussion, ie, whether the combination of ultrasonographic and maternal blood test results (eg, serum alpha-fetoprotein level) is a satisfactory screening method for commonplace anomalies such as Down's syndrome or spina bifida, is reviewed. Whether these noninvasive tests need to be supplemented by amniocentesis is also discussed, as are ultrasonographic markers for various trisomy syndromes and the association between renal abnormalities and chromosomal defects. PMID- 1717022 TI - Primary oesophageal rhabdomyosarcoma. AB - A case is presented of a rhabdomyosarcoma of the oesophagus with a description of the cytology, light microscopy, and immunocytochemical findings and a discussion of spindle cell tumours occurring at this site. Cytologically, large bizarre shaped pleomorphic cells were seen in which desmin was demonstrated in order to confirm the diagnosis after destaining a Papanicolaou stained slide and restaining it with antibody to desmin. PMID- 1717023 TI - Examination of induced sputum in the diagnosis of Pneumocystis carinii pneumonia. AB - The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced. PMID- 1717024 TI - Evaluation of immunocytochemical staining as a method of improving diagnostic accuracy in a routine cytopathology laboratory. AB - Immunocytochemical stains in a routine cytopathology laboratory can be used to distinguish between benign and malignant cells, and to identify tumour type. In our laboratory 30 problematic cases were selected for immunocytochemical stains and the results analysed in this paper. The following markers were used: cytokeratin (CAM5.2), carcinoembryonic antigen (CEA), kappa and lambda light chains, leucocytic common antigen (LCA), chorionic gonadotrophin (hCG), prostate specific antigen (PSA), L26, UCHL1, S100-protein and vimentin. Twelve FNA (four lymph nodes, one parotid swelling, two from lungs, two from pleura and chest wall, one from lumbar region, two from soft tissue masses), and 18 effusions (12 pleural effusions, five ascitic fluids, one pericardial effusion) were investigated. We found immunocytochemical stains of value in formulating the cytological diagnosis in 11/12 of FNA and 15/18 of effusions. PMID- 1717025 TI - Medullary carcinoma of thyroid: a re-evaluation of the cytological criteria of diagnosis. AB - We have reviewed the fine needle aspiration cytology appearances of a series of 31 consecutive and histologically confirmed medullary carcinomas of the thyroid. Despite the absence of a totally specific diagnostic feature, this retrospective re-evaluation indicates that a preoperative diagnosis of medullary carcinoma of the thyroid is possible in almost every case. The features occurring most commonly include a dispersed cell pattern in which round or spindle shaped cells with eccentric speckled nuclei were seen showing slight pleomorphism with inconspicuous nucleoli. In a third of cases fine red granularity was present in the cytoplasm in slides stained with Giemsa and by the Papanicolaou technique. PMID- 1717026 TI - Intralymphnode injection of human monocyte macrophage colony stimulating factor (CSF-1) as a method of obtaining high titer anti-CSF-1 antibodies. AB - We describe a rabbit intralymphnode immunization technique for obtaining a high titer antihuman CSF-1 antiserum with small amounts of antigen. This procedure provided a rapid (42 days after primo-injection), stable maximum immune response with a high titer antiserum precipitating 30% of 125I CSF-1 at a 1:25.000 dilution. The specificity of the immune serum was assessed by competitive binding experiments in RIA and neutralization of the CSF-1 biological activity in culture. The antiserum was also tested for its ability to detect CSF-1 in Western blotting, immunocytochemistry and immunohisto-chemistry. The results show that the immune serum specifically recognizes the biological active domain of human CSF-1 molecules from different origins and only detects dimeric forms. The potential uses of this anti CSF-1 antiserum are discussed. PMID- 1717027 TI - Modulation of bone marrow cell functions in vitro by bestatin (ubenimex). AB - Bestatin (ubenimex), the microbial leucil-aminopeptidase B inhibitor, has been shown previously to stimulate both interleukin-1 (IL-1) and IL-2 production and to enhance T-cell, as well as macrophage mediated immunoreaction when administered in vivo in mice. Here we show that although Bestatin has no direct growth stimulatory activity, it enhances the growth of GM-CFU populations in semisolide culture and stimulates the cell production in liquide organotypic Hematon cultures in synergy with recombinant human GM-CSF. In long term human bone marrow culture Bestatin accelerated the adipocytic differentiation among colony forming stroma cells (F-CFU). Our data provide further evidences that Bestatin may interact with the hemopoietic cell renewal system at different levels of biological organisation. PMID- 1717028 TI - Immunohistochemical evaluation of reverse transcriptase in breast carcinoma with polyclonal antibodies raised in rabbit. AB - In this study, the presence of reverse transcriptase in breast tumours was examined with immunoperoxidase staining using antibodies raised in rabbit against reverse transcriptase of Moloney murine leukemia virus and against reverse transcriptase of avian myeloblastosis virus. The specificity of such antibodies was investigated with ELISA and Western blotting techniques. Five cases of infiltrating ductal carcinomas were found positive with the immune serum anti reverse transcriptase of Moloney murine leukemia virus on 28 studied infiltrating ductal carcinomas, 2 infiltrating lobular carcinomas and 2 fibroadenomas. PMID- 1717029 TI - Epitopes of cartilage core proteins and GAG pattern in human non-Hodgkin lymphoma xenografts. AB - Glycosaminoglycan and core protein components of proteoglycans (PGs) have been studied in three human non-Hodgkin lymphoma xenografts of B cell origin. Lymphomas showed similar GAG content, but different composition of GAG subtypes. This variability was accompanied by an individual capacity to adhere to extracellular matrix elements. The core proteins identified by monoclonal antibodies raised against human cartilage chondroitin sulfate PG were also distinctly expressed and released. These proteins shared by different cell types may have biological significance. PMID- 1717030 TI - Peritoneal absorption of pancreatic enzymes in bile-induced acute pancreatitis in dogs. AB - To clarify the contribution of peritoneal absorption of enzyme-rich exudate to the persistent elevation of serum amylase in bile-induced pancreatitis in dogs, serum amylase, lipase and immunoreactive trypsin (IRT) levels were measured during 24 h after induction of pancreatitis with and without peritoneal lavage. The basal level of serum amylase activity (m +/- s.e. = 1291 +/- 111 U/L) reached a plateau at 30 min (2688 +/- 185) after induction of pancreatitis and continued to rise until 24 h (7201 +/- 424). This persistent amylase elevation could be reduced significantly by peritoneal lavage. Serum IRT rose to a peak (378 +/- 103 ng/mL) at 30 min from the basal (20 +/- 5), then decreased until 3 h (211 +/- 34) and maintained a consistent level thereafter. Serum lipase elevation took an intermediate course between the levels of serum amylase and IRT. Intraperitoneal injection of 5 mL pancreatic juice could reproduce similar elevations to those of the respective enzymes, except lipase, seen in pancreatitis. These results suggest that transperitoneal absorption of pancreatic enzymes contributes to the elevation in serum enzymes levels and that rates of peritoneal absorption and serum disappearance differ from enzyme to enzyme. PMID- 1717031 TI - Bacterial biofilm formation on nasogastric tubes. AB - We examined the external surfaces of the gastric portion of nasogastric tubes recovered from hospitalized patients. Most of these surfaces were covered with a thick amorphous biofilm which was shown to be largely composed of microbial microcolonies in which individual cells were enclosed in their exopolysaccharide glycocalyces. Bacteria of many different morphotypes and some yeast cells were found in a fibrillar matrix that appeared to mediate their attachment to the silastic tubing. Within these mature biofilms some microcolonies were structurally intact and apparently vigorous, while others were composed largely of dead cells and empty cell walls. We conclude that nasogastric tubes that remain in patients for as short a time as one day are heavily colonized by a rich variety of biofilm-forming bacteria and yeasts. PMID- 1717032 TI - Rapid diagnosis of Campylobacter pylori infection by urea test paper. AB - Antral biopsy specimens from 106 patients were examined by culture, Gram stain and silver stain for Campylobacter pylori. Biopsies were also examined by a urea test paper test (UTPT). Of 106 patients studied C. pylori was detected in 68 (64.2%) by Gram stain, silver stain and culture. The UTPT was positive in 63 (59.4%) specimens. Five had false negative results using the UTPT with no false positive subjects. Thus, UTPT has a sensitivity of 92.6% (63/68) and a specificity of 100%. Of the 63 specimens that were UTPT positive, 45 were positive within 1 min, 58 were positive within 5 min. The remainder became positive between 5 and 15 min. There was a negative correlation between the time required for positive UTPT and the number of C. pylori per pit as seen on silver stained sections (P less than 0.05). UTPT is a rapid and sensitive method for detecting C. pylori in gastric mucosa. This enables early therapy, if indicated, before discharge from hospital. Moreover, the urea test paper method is cheap and easily stored. PMID- 1717033 TI - Protection by gabexate mesilate (FOY) of the exocrine pancreas in rats with acute pancreatitis induced by a supramaximal dose of caerulein. AB - Earlier studies have reported that interstitial oedematous pancreatitis characterized by hyperamylasaemia can be seen during the early stage of stimulation of supramaximal dose of caerulein. The present study investigated the changes in both cellular and lysosomal fragility and the protective effects of a synthetic protease inhibitor gabexate mesilate (FOY) on this non-invasive model of experimental pancreatitis. The infusion of FOY (50 mg/kg/h) prevented the caerulein-induced increase in serum amylase and pancreatic oedema formation and reduced the elevated amylase content significantly. The administration of FOY with caerulein also reduced the increased lactic dehydrogenase (LDH) discharge significantly and inhibited the cathepsin B leakage from lysosomes in an in vitro incubation system. These results indicate that FOY plays its protective role at the subcellular level--that is, in lysosomes by inhibiting some proteases such as phospholipase A2. The importance of esterases in the pathogenesis of acute pancreatitis is demonstrated. This type of esterase inhibitor may be valuable clinically in the treatment of acute pancreatitis and these results also suggest the role of lysosomal fragility in the pathogenesis of progression of acute pancreatitis. PMID- 1717034 TI - Review of hepatitis C in Japan. PMID- 1717035 TI - Oligodendrocyte cell surface recognized by a novel monoclonal antibody specific to sulfatide. AB - A rat monoclonal antibody (OL-1) was obtained by in-vitro immunization of rat spleenocytes with paraformaldehyde-fixed primary cultured glial cells derived from newborn rat brain and subsequent fusion with a rat myeloma cell line. The antibody secreted by the hybridoma immunostains live rat and mouse oligodendrocytes in primary and secondary cultures. The antibody binds specifically to oligodendrocytes and myelin structures in-situ. Radioimmunolabelling assays with a number of purified glycolipids offer thin layer chromatography separation show that OL-1 antibody binds strongly to sulfatide and to a lesser extent to galactosyl diglyceride. PMID- 1717036 TI - Carbamazepine blocks NMDA-activated currents in cultured spinal cord neurons. AB - The antiepileptic agents, carbamazepine and phenytoin, suppress seizures in man and convulsant-induced hyperactivity in spinal cord nerve cell cultures. In the present study, we have shown by whole cell recording that carbamazepine, in contrast to phenytoin, blocks N-methyl-D-aspartate (NMDA)-activated membrane currents in cultured neurons in a dose-dependent fashion. The NMDA receptor activated channel, which is blocked at physiological concentrations of Mg2+ at resting membrane potential, can be activated by glutamate in depolarized neurons and thus be involved in epileptogenesis. Therefore, the block of NMDA-evoked membrane currents in cultured neurons may contribute to the clinical effectiveness of carbamazepine. PMID- 1717037 TI - 5-HT3 receptor antagonists inhibit sensory neuropeptide release from the rat spinal cord. AB - 5-HT3 receptors may be present on primary afferent neurons containing substance P (SP), neurokinin A (NKA) or calcitonin gene-related peptide (CGRP). We investigated the release of SP-, NKA- and CGRP-immunoreactivities (IR) from rat spinal cord slices. Thirty mM potassium chloride caused an increased outflow of all three peptides, i.e. 140-190% of spontaneous release. This release was slightly enhanced in the presence of 3 x 10(-5) M 5-hydroxytryptamine (5-HT). In contrast, a significant inhibition of potassium-evoked, but not of basal NKA-IR and CGRP-IR release was observed when 10(-7) M BRL 43694 or ICS 205-930, two specific 5-HT3 receptor antagonists, were superfused together with 5-HT. In conclusion, 5-HT may facilitate the evoked release of peptides from central terminals of primary sensory neurons via 5-HT3 receptors. PMID- 1717038 TI - Different sensitivities to dihydropyridines of catecholamine release from cat and ox adrenals. AB - Catecholamine release evoked by quick pulses of Ca2+ (2.5 mM) given sequentially at 30 min intervals to cat and ox adrenal glands perfused continuously with Ca2+ free Krebs-Tris solutions containing 35 or 118 mM K+, was studied. In the feline, the secretory response was highly sensitive to various dihydropyridine (DHP) derivatives. For instance, secretion was completely blocked by nM concentrations of (+)isradipine (IC50 between 3 and 4 nM) and markedly potentiated by (-)Bay-K 8644. In contrast, the bovine secretory responses were resistant to blockade by nitrendipine or (+)isradipine, as well as to potentiation by Bay-K-8644, even at microM concentrations. From these experiments, it seems clear that distinct subtypes of Ca2+ channels might mediate a similar secretory response in ox and cat adrenal chromaffin cells, at least in the present experimental conditions. PMID- 1717039 TI - Induction of basic fibroblast growth factor in Alzheimer's disease pathology. AB - Alzheimer's disease (AD) and its hallmark, plaques, may be due to an imbalance of trophic support. It has been suggested that plaque biogenesis may involve a growth factor which induces sprouting of neurites to form plaques. Thus, we studied the distribution of basic fibroblast growth factor (bFGF), in the hippocampus from AD brain and in rodent brain after entorhinal ablation. Both cases have a partial deafferentation of the hippocampus. The strongest bFGF immunoreactivity in AD was shown in plaques of the dentate gyrus. Rodent brains showed a lesion-induced increase of bFGF in the dentate gyrus, primarily localized to astrocytes. Our results indicate that bFGF may serve an important biological function in plaques and possibly attract neurites. PMID- 1717040 TI - ADP-ribosylation of myelin basic proteins isolated from normal and mutant mouse brains. AB - Myelin basic proteins (MBPs) have been shown to be ADP-ribosylated in-vitro by cholera toxin in the presence of NAD. Since acid-soluble extracts of brain contain other proteins in the 14-32 kD range (such as histones) in addition to MBP's, the identification of the ADP-ribosylated proteins was uncertain. To determine that only the MBP's were ADP-ribosylated, the acid-soluble fractions from several murine mutants were prepared. Thus, in the Shiverer mutants, none of the proteins in the 14-32 kD range were ADP-ribosylated; in the Myelin-deficient mutant, some of ADP-ribosylation was observed but none in the Jimpy mutant, consistent with our demonstration that the least cationic isomer cannot be ADP ribosylated. PMID- 1717041 TI - Glutamate-like immunoreactivity in medulla oblongata catecholamine/substance P neurons. AB - Immunofluorescence histochemistry was performed on sections of the rat medulla oblongata with a well characterized antibody to the amino acid glutamate (GLU) combined with antisera to catecholamine synthesizing enzymes and substance P. GLU like immunoreactive (LIR) neurons were seen in many areas of the medulla, and were particularly intensely stained in the rostral ventrolateral medulla. In this area, nearly all adrenaline neurons were GLU-LIR. This immunoreactivity was also seen in catecholamine neurons of the C2, C3, A1 and A2 cell groups. Many adrenaline neurons, especially of the C1 group, contained substance P-LI in addition to GLU-like immunoreactivity (LI). PMID- 1717042 TI - Protein loss from axonal transport occurs without diminution of vesicle traffic. AB - Protein loss from the rapid anterograde axonal transport system of amphibian sensory nerve fibers was compared with the numbers and sizes of anterogradely transported vesicles in the axons. Protein was found to be lost at a rate of approximately 2% per millimeter of nerve traversed. However, no changes were observed in either the numbers or sizes of vesicles in the nerve at two locations separated by 60-75 mm. The results show that protein loss is not explained as a loss of vesicles from the transport system nor by a reduction in vesicle size. PMID- 1717043 TI - Steroids inhibit nicotinic acetylcholine receptors. AB - Application of progesterone to Xenopus oocytes expressing a cloned neuronal nicotinic acetylcholine (nAChR) revealed two effects. The first effect was a fully reversible reduction of the current induced by acetylcholine (ACh), its onset being nearly instantaneous. The second effect, which developed in a few hours, was an irreversible suppression of ACh-evoked currents. The transient inhibition had an apparent Ki of 7 microM when tested with 50 nM ACh, but the percentage of inhibition was positively correlated to the ACh concentration. A reduction of ACh-induced currents which appeared immediately upon progesterone application was also observed with muscle nAChR expressed in oocytes and with nAChR on membrane patches isolated from ciliary ganglion neurons. Thus nAChRs are modulated by progesterone and steroids may play an important role in nicotinic cholinoception. PMID- 1717044 TI - Antibodies to the alpha v beta 3 integrin label a protein concentrated in brain synaptosomal membranes. AB - Immunochemical methods were used to test whether vitronectin receptors exist in synaptosomal membranes (SPMs) and hence are positioned to play a role in synaptic adhesion. Antibodies against the alpha v beta 3 integrin detected proteins in brain homogenates that correspond to conventional integrin subunits. Conversely, these antigens were not found in SPMs prepared from the same brain tissue. The antibodies did, however, express strong immunoreactivity towards a 27 kDa polypeptide that was greatly concentrated in SPMs from major brain regions and that was not found in tissues other than brain. This is an example of an integrin epitope contained in a synaptic polypeptide that is too small to be a conventional matrix receptor, thus, suggesting the possibility that synaptic adhesion involves unusual proteins. PMID- 1717045 TI - Excitation of substantia nigra pars compacta neurones by 5-hydroxy-tryptamine in vitro. AB - Incoming serotonergic fibres are known to make direct synaptic contact with dopamine-containing neurones in the substantia nigra pars compacta (SNc). However, the effects of 5-HT (5-hydroxytryptamine) on these cells have not been thoroughly investigated. In the present study we show that application of 10-50 microM 5-HT increases the firing frequency of SNc neurones in-vitro, and produces inward rectification in a voltage region negative to -50mV. This effect is sensitive to extracellular Cs+, but not to Ba2+, and has similar properties as the intrinsic inward rectifier current, Ih. Antagonists of the 5-HT1A and 5-HT2 receptors were inefficacious. It is concluded that 5-HT excites SNc neurones via an enhancement of the conductance underlying Ih. PMID- 1717046 TI - Modulation of glycine-activated membrane current by adamantane derivatives. AB - Pressure application of the inhibitory neurotransmitters glycine and GABA, respectively, evoked a Cl- current in spinal cord neurons of fetal mice in culture. Memantine, in low concentrations (0.3-5 microM) enhanced the glycine mediated current at both negative and positive holding potentials. At concentrations of more than 5 microM, memantine increased the glycine-evoked current only when the membrane potential was positive. At negative potentials the current was inhibited. 3-isopropylamantadine and 3,5-diethylamantadine augmented the glycine-activated membrane current irrespective of the concentrations applied, whereas amantadine was ineffective. The GABA-activated Cl- current was not altered by the adamantanes. These data are evidence of a specific glycinergic interaction with putative antispastic agents and can explain some conflicting effects of memantine in experimentally induced seizures. PMID- 1717047 TI - Patchy intrinsic connections of the ferret primary auditory cortex. AB - Small iontophoretic injections, of the leucoagglutinin extracted from Phaseolus vulgaris (PHA-L), were made at a depth of 600 microns, into the ferret primary auditory cortex (AI). The lectin anterogradely labelled the axons of pyramidal cells located in the upper layers. The axons had collaterals which terminated in 5-8 columns within AI. The columns were 0.25-0.8 mm in diameter and, based on their position, appeared to be in areas of cortex where the cells would have higher and lower as well as equivalent characteristic frequencies to those in the injection site. PMID- 1717049 TI - Distribution of (fucogalactosyl-)epitope expressing glycoconjugates in rat brain. AB - Glycoconjugates are known to be concentrated in plasma membranes, especially in synaptic junctions, where they subserve various functions in neural connectivity. Here we report the cellular distribution of a new monoclonal antibody recognizing (fucogalactosyl) sequences in carbohydrate structures. The most pronounced immunoreactivity was found in fibrous astrocytes, in many parts of the brain and with lower density in various neuronal elements. This points to the expression of identical carbohydrate sequences on molecules within certain glial and neuronal elements. Previous intracerebral injections of the antibody interfered with long term memory formation. Therefore, functions mediated by corresponding glycoproteins in neurons and glia cells or even neuron-glial interactions, might be relevant for information-processing. PMID- 1717048 TI - Dark-induced supersensitivity of dopamine D-1 and D-2 receptors in rat retina. AB - Light deprivation of rat retina leads to a rapid (within 6 h) development of a state of supersensitivity (upregulation) of dopamine D-1 receptors (positively coupled to adenylate cyclase), which are essentially involved in the modulation by light of the electrical activity and communication between horizontal cells. In contrast, the supersensitivity of D-2 receptors (negatively coupled to cAMP generating system) appears to develop only after 2 days (better after 4 days) of dark adaptation, although these receptors are linked to multiple light-dependent retinal functions. These results suggest the existence of different mechanisms of sensory adaptation for these two subtypes of dopamine receptors. PMID- 1717050 TI - 'Phytoantibodies': a general vector for the expression of immunoglobulin domains in transgenic plants. AB - Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development. PMID- 1717051 TI - Phenotypic characterization of murine thymic microenvironments. AB - The thymus provides the necessary microenvironments for the differentiation of T lymphocytes. Thymic non-lymphoid cells, such as epithelial cells, macrophages and interdigitating cells are thought to promote sequential stages in T cell differentiation. However, their specific role in each step of T cell differentiation remains to be established. With the development of new monoclonal antibodies it has now become possible to characterize the different thymic stromal cell types. In this review, various aspects of thymic stromal cells and their functions in T cell differentiation are discussed, such as: (1) phenotypic analysis of stromal cells in situ; (2) the application of new "chimeric' monoclonal antibodies which "link' developing thymocytes and stromal cells; (3) perturbation of thymic microenvironments after cyclosporin-A treatment; (4) perturbation of thymic microenvironments in new transgenic mouse lines; (5) phenotypic analysis of in vitro growing stromal cell lines. PMID- 1717052 TI - Functional roles of ion channels in lymphocytes. AB - The application of patch-clamp and video-imaging techniques has enabled responses of lymphocytes to be examined at the level of individual cells. Eight distinct types of ion channel activity have been revealed in T lymphocytes. A variety of external stimuli shifts the pattern of channel activity from the resting state, which is dominated by voltage-gated K+ channels. Channel regulation is achieved both by acute modulation and by altered expression in the membrane. During mitogen stimulation, Ca2+ channels and Ca(2+)-activated K+ channels become active and appear to underlie Ca2+ oscillations. These acute changes are followed by increased expression of voltage-gated K+ channels. In response to osmotic challenge in hypotonic media, cell swelling initiates activation of Cl- channels, which may, in turn, indirectly activate K+ channels and trigger a regulatory decrease in cell volume. PMID- 1717053 TI - Cell lineages in haemopoiesis: comments on their regulation. AB - The regulation of cell production and the determination of cell lineage in haemopoiesis are the result of multiple processes involving cell-cell interactions and the action of specific haemopoietic growth factors as well as other cytokines. Additive and synergistic interactions between these factors, and also with those involved in the regulation of lymphopoiesis have been described, which involve modulation of the growth and differentiation of haematopoietic precursors. However, the mechanisms which determine the self-renewal and the differentiation of the pluripotent lymphohaemopoietic stem cell remain largely undefined. PMID- 1717054 TI - Development of murine B cell subpopulations. AB - The generation of diverse B cell subpopulations in the mouse is the subject of considerable current interest. In this review, I first describe a general framework for antigen-independent B cell differentiation in the adult bone marrow including results from in vitro culture systems. In following sections, effects on B cell development in several immunodeficient strains and transgenic lines are presented. Next, the CD5+ B cell subpopulation is covered in some depth, including data on development, physiology and repertoire selection. Finally, another selected population, memory B cells, together with their likely origins in the germinal center are discussed. PMID- 1717055 TI - [The intermediate lobe of the pituitary, model of neuroendocrine communication]. AB - The intermediate lobe of the pituitary is composed of a homogeneous population of endocrine cells, the melanotrophs, which secrete several bioactive peptides including alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. In contrast to most endocrine glands which are richly vascularized, the intermediate lobe of the pituitary contains very few blood vessels; in some species, the pars intermedia is virtually totally avascular. In contrast, pituitary melanotrophs are richly supplied by nerve fibers originating from the hypothalamus. The pars intermedia thus appears as a pure model of neuroendocrine communication, i.e. it is an archetype of the mode of transducing interface between the central nervous system and endocrine effectors. In mammalian species, different types of nerve terminals containing dopamine, norepinephrine, gamma-aminobutyric acid (GABA) and serotonin have been identified. In lower vertebrates, particularly in fish and amphibians, the pars intermedia is also innervated by peptidergic fibers which are though to take part in regulation of the secretory activity of the melanotroph. In these animals, the pars intermedia is regarded as a major center of neuroendocrine integration and an exceptional model to investigate the process of communication between the brain and the endocrine glands. The purpose of the present review is to summarize our current knowledge on the synthesis, processing and release of peptide hormones from pars intermedia cells and to survey the multiple regulatory mechanisms which are involved in the control of the activity of pituitary melanotrophs. Proopiomelanocortin, a multifunctional precursor. Pituitary melanotrophs synthetise a major precursor protein called proopiomelanocortin (POMC) which generates through proteolytic cleavage several biologically active peptides including adrenocorticotropic hormone (ACTH), endorphins and MSHs. In lower vertebrates, alpha-MSH is generally considered as the major hormone secreted by melanotrophs, in that it is involved in the process of skin colour adaptation. The post-translational processing of POMC, which yields to the mature hormones released by melanotrophs, includes a number of steps: glycosylation, phosphorylation, tissue-specific proteolytic cleavage, amidation and acetylation. Some of these posttranslational modifications can be regulated by neuroendocrine factors. For instance, in frogs, it has been shown that dopamine inhibits acetylation of alpha-MSH and thus reduces the secretion of the biologically active form of the peptide. The intermediate lobe of the pituitary: a model of neuroendocrine integration. In most vertebrate species, the intermediate lobe of the pituitary is innervated by catecholamine-containing fibers. In particular, the presence of dopaminergic nerve fibers has been observed in the pars intermedia of mammals and poikilotherms.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717056 TI - [Osmoregulatory function and ecophysiology of anticipation in migratory amphihaline fish]. PMID- 1717057 TI - Inward movement of Ca2+ in K(+)-depolarized rat vascular smooth muscle. AB - The kinetics of the Ca2+ (45Ca2+) uptake in segments of rat aorta and its relationship with tension development in the presence of K+ concentrations ([K+]o) ranging from 3.5 to 100 mM was investigated. It was found that: 1) The curve relating 45Ca2+ uptake and time was virtually linear during the first 3 min. Therefore, the influx calculated as uptake in 3 min/3 min was not significantly different from that calculated as the slope of the uptake curve. The actual uptake of the cation during the loading period would be underestimated by about 30 percent if the loss of Ca2+ from that compartment during a 2 h clearing of the extracellular space in La3+ medium, is ignored. 3) 45Ca2+ activity remaining in the tissue after a 3 min labelling period followed by a 2 h washout can reliably be corrected to calculate Ca2+ influx. 4) The K(+)-induced tension development showed no significant correlation with the total exchangeable Ca2+ while it appeared as a steep function of Ca2+ influx between 15 and 80 mM [K+]o. 5) In [K+]o = 100 mM Ca2+ influx was 5 to 6 times larger than in normal [K+]o (5.3 mM). The estimated Ca2+ permeability (PCa) increased linearly as a function of [K+]o (5.3 to 100 mM). In [K+]o = 100 mM, PCa was estimated to be about 17-fold that in [K+]o = 5.3 mM. Similarly, the increased in Ca2+ conductance (GCa) for the same change in [K+]o was estimated to be 14-fold. PMID- 1717058 TI - Metabolic adaptation of renal carbohydrate metabolism. IV. The use of site specific liver gluconeogenesis inhibitors to ascertain the role of renal gluconeogenesis. AB - The in vitro and in vivo effects of several different inhibitors of carbohydrate metabolism have been studied. The in vitro addition of 5-methoxyindole-2 carboxylic acid (MICA), pent-4-enoic acid, and quinolinic acid to the perfusion medium significantly inhibited liver gluconeogenesis in 48-hour-starved rats (100% inhibition when MICA and quinolinic acid were added at 0.8 and 2.4 mM, respectively). In vivo the level of inhibition varied greatly depending upon whether MICA was administered by intragastric tube or intraperitoneal injection. In all cases the inhibitory capacity of MICA on liver gluconeogenesis was significantly higher when injected intraperitoneally. On the other hand, the administration of MICA produced a significant, dose-dependent, increase in renal gluconeogenesis in both fed and 48-hour-starved rats, more so when the inhibitor was administered by intraperitoneal injection. PMID- 1717059 TI - Effects of bilateral hysterectomy on the hepatic lipid reserves in rats. AB - Wistar adult female rats have been hysterectomized. Compared to controls, these hysterectomized animals presented significant increase in body weight, due to lipid accumulation. Their liver had significantly higher tissue somatic index due to identical deposition of lipid fractions. Development of these lipid reserves can be related to ovary inactivation, as shown by estradiol low levels in blood. PMID- 1717060 TI - Effects of cefroxadine on L-leucine absorption in rat jejunum. AB - The effect of cefroxadine, an aminocephalosporin (beta lactam antibiotic) on rat intestinal L-leucine transport has been studied. Cefroxadine inhibited the L leucine uptake by the intestinal mucosa in a dose-dependent fashion. In vivo studies showed that cefroxadine reduced L-leucine absorption. This effect was irreversible. Only the active transport component of the absorption was inhibited. Oxygen consumption of the mucosa was reduced by cefroxadine which inhibited the activity of the basolateral (Na(+)-K+) ATPase also. PMID- 1717061 TI - Lysosomal glycosidase activities in rat testis during sexual maturation. AB - Concentrations of testosterone and its metabolite dihydrotestosterone were determined in whole rat testis during the transition from the prepuberal to the mature status. The activities of five glycosidases (beta-N-acetylhexosaminidase, alpha-L-fucosidase, beta-D-galactosidase, alpha-D-glucosidase and alpha-D mannosidase) were also investigated in the seminiferous tubules and interstitial tissues. The same androgens and enzyme activities were measured in testis of rats treated with 17-beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5-alpha-androstan-3-on e, a potent inhibitor of the 5-alpha-reductase-mediated conversion of testosterone into dihydrotestosterone. Lysosomal glycosidic activities in seminiferous tubules were found to vary slightly with the age of the animals, and thus seemed not to depend on the hormonal status of the tested animals. In contrast, the enzyme activities were low in the immarure interstitium but increased sharply during the onset of sexual maturity. The activity of each glycosidase reached a maximum between the 45th and the 65th day, except for beta N-acetylhexosaminidase which did not decrease significantly following the 55th day. The five lysosomal enzyme activities decreased in the interstitial compartment of rat testis treated with DMAA, suggesting a relationship between these glycosidic enzyme activities and dihydrotestosterone. PMID- 1717062 TI - Effect of theophylline on the electrolyte permeability of the isolated skin of the toad Bufo arenarum. AB - 1. Short-circuit current (SCC, an index of active sodium transport) increased in response to theophylline (Theo) in the isolated skin of Bufo arenarum, regardless of the presence of chloride in the bath. 2. Tissue conductance, G, and the transepithelial potential difference, PD, also increased moderately in skins bathed in chloride-free solutions (main anion: sulfate or iodide); the increases in SCC and G were fully blocked by further addition of amiloride to the epidermal bath. 3. The increase in G following theophylline was greater in the presence of chloride, whereas PD decreased; the increased G was not modified by amiloride at a concentration that completely abolished the SCC. 4. Establishment of a chloride gradient across the theophylline-treated skin whose SCC was completely abolished by amiloride or by removal of sodium from the bath, induced a diffusion current (SCCg) in a direction consistent with the transepithelial flow of chloride following its gradient. The intensity of the SCCg was directly related to the magnitude of the gradient. 5. For a given gradient, both the intensity of the SCCg and the increase in G were greater when the gradient was directed inward (i.e., when the chloride concentration was higher in the epidermal than in the dermal bath), as compared to the opposite case. 6. Exposure of the skin to an epidermal bath made hyperosmotic by addition of urea (epidermal hyperosmolarity, EH, which reversibly increases the permeability of the paracellular pathway by "opening" the tight junctions) induced similar increases in G, whereas SCC and PD decreased irrespective of the anion present in the solution.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717063 TI - Effects of the stress of sampling and of anaesthesia on the metabolic status of suckling rabbits. AB - The stress of anaesthesia and of sampling on the concentrations of blood metabolites and tissue glycogen content was studied in awake, anaesthetized and decapitated young rabbits 1-2 days old ("young") and 17-18 days old ("old"), 24 h after the last controlled suckling. Stress of sampling and anaesthesia was particularly evidenced by high blood lactate observed for the three protocols in young rabbits and to a lesser extent in old animals. Decapitation appeared as the less aggressive procedure for blood lactate assessment in "young" and "old" rabbits. In "young" rabbits, blood glucose was not significantly modified by the mode of blood sampling while in "old" rabbits blood glucose was significantly lower in awake than in anaesthetized animals. Muscle and liver glycogen content data were not significantly different between the three protocols in old and young rabbits. From a comparison of these results with those found in adult animals in various species, it appears that blood puncture in awake "young" and "old" rabbits is a suitable procedure for determining the majority of blood metabolites except lactate in "young" animals. PMID- 1717064 TI - [In vitro effects of 24R,25-dihydroxyvitamin D3 on alkaline phosphatase and gamma glutamyltransferase in the kidney of hypophysectomized rats]. AB - The effects of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3 on alkaline phosphatase (PAL), gamma-glutamyltransferase (GGT) and acid phosphatase (PAC) activities were investigated on renal cortex slices of hypophysectomized rats. Indeed after hypophysectomy renal 24,25-(OH)2D3 production was increased and renal PAL and GGT activities were decreased. After 5h incubation with physiological concentrations (0.1-10 nM) of 24,25-(OH)2D3 significant increases of PAL and GGT activities were produced. The maximum stimulation obtained with 1 nM was +23% for PAL and +26% for GGT as compared to controls. PAC was not modified. The time course of these effects was studied from 45 min to 8 h. In the presence of 24,25-(OH)2D3 (1 nM), delayed (3h) stimulation of PAL and GGT appeared. It reached the maximal value after 6h, +37% for PAL and +30% for GGT and persisted again at 8h. Cycloheximide added to incubation medium with steroid inhibited the stimulating effect on PAL only. Actinomycin D suppressed the induction of both enzymes, indicating that the observed actions of 24,25-(OH)2D3 depend on protein synthesis whose responsible mechanisms were different. These protein synthesis inhibitors did not modified enzymatic activities. Physiological significance of these renal effects is to be clarified. PMID- 1717065 TI - Electrophysiological and microiontophoretic analysis of the habenulo-hippocampal circuit. AB - In the cat, the effects of lateral habenula stimulation, at different ranges of frequency, on hippocampal units were studied. Habenular stimulation at low frequency excited, while at high frequency inhibited the greater part of hippocampal units. Moreover, in order to clarify the possible pathway involved in the habenulo-hippocampal circuit, the effects of iontophoretic acetylcholine and serotonin on hippocampal units were compared with those of habenular stimulation. Iontophoretic acetylcholine induced both excitatory and inhibitory responses while serotonin induced only inhibitory responses. Iontophoretic atropine blocked the effects of acetylcholine ejection but did not antagonize stimulation effects; ion-tophoretic methysergide induced an increase of basal firing of hippocampal units and antagonized both serotonin and habenular stimulation inhibition. The results suggest an influence of lateral habenula to the hippocampus which does not appear to be cholinergically-mediated. A possible involvement of the raphe as a relay station in the habenulo-hippocampal pathway is discussed. PMID- 1717066 TI - Neonatal red blood cells as a model for the study of ouabain-furosemide interaction on active K+ transport. A theoretical approach. AB - In a former paper by Serrani et al. (1990), an overlapping action of ouabain and furosemide on potassium influx was observed. In the present paper we demonstrated that Na, K-ATPase from neonatal red cells ghosts is a target of both drugs. Furthermore the combined action of ouabain and furosemide on potassium (86Rb) influx (in its greatest fraction mediated by primary active transport) was studied by generalized equations for the analysis of inhibitors. The isobologram and the combination index performed from the data obtained point to the existence of synergism or antagonism between both inhibitors according to the fraction affected. Clinical implications of these findings could be postulated. PMID- 1717067 TI - [The implantation of self-expanding metal prostheses (stents) into the bronchial system in central neoplasms]. AB - This paper reports the use of an endobronchial metallic prosthesis (wall stent) for palliative management of 6 patients with central neoplasms and life threatening airway obstruction. In five of six patients implantation of the stent resulted in improvement of their dyspnoea. However, functional data showed only a slight amelioration. In case of endobronchial growth, tumour may protrude easily through the meshwork of the stent. Therefore, use of the stent in these patients only seems appropriate if combination with other forms of therapy (radiation, laser) is possible. The easy feasibility of stent implantation and the lack of severe side effects and migration indicate that the stent may be an effective means in patients suffering from peribronchial tumour compression. PMID- 1717068 TI - Enrichment and characterization of murine hematopoietic stem cells that express c kit molecule. AB - The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK-2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac 1(macrophage)+ or Gr-1(granulocyte)+ cells were c-kit-low positive. Colony forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c-kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis. PMID- 1717069 TI - Dynamics of leukocyte-platelet adhesion in whole blood. AB - The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes. PMID- 1717070 TI - Activated and unactivated platelet adhesion to monocytes and neutrophils. AB - To examine the possible receptor-ligand pairs mediating adhesion of activated and "unactivated" platelets to leukocytes and the kinetics of leukocyte-platelet binding, we developed a flow cytometric assay using isolated cell fractions to accurately measure heterotypic cell adhesion, including both total leukocyte platelet conjugate formation as well as the number of platelets bound per leukocyte. We have shown that (1) activated platelet binding to both polymorphonuclear leukocytes (PMN) and monocytes is dependent on both a specific epitope (blocked by monoclonal antibody G1) of granule membrane protein-140 (GMP 140) and the presence of divalent cations; (2) unactivated platelets bind to 87% of viable, resting monocytes but to only 34% of PMN; (3) the receptor(s) on unactivated platelets that mediate adhesion to PMN and monocytes do not require divalent cations and become nonfunctional after thrombin activation; and (4) the kinetics of platelet adhesion to monocytes and PMN indicate that monocyte adhesion is favored over neutrophil adhesion. We conclude that platelet heterotypic cell adhesion is a dynamic process reflecting the activation status of the platelet and differential binding abilities of leukocytes. PMID- 1717071 TI - Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia. AB - Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly. PMID- 1717072 TI - CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation. AB - The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, "enkephalinase"). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met enkephalin, formyl-met-leu-phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides. PMID- 1717073 TI - Involvement of tyrosine kinases in the activation of human peripheral blood neutrophils by granulocyte-macrophage colony-stimulating factor. AB - The aim of the present study is to evaluate the involvement of human neutrophil tyrosine kinase(s) in the signal transduction mechanism of granulocyte-macrophage colony-stimulating factor (GM-CSF). Stimulation of neutrophils with GM-CSF resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 40, 55, 74, 97, 118, and 155 Kd, detected by immunoblot using a monoclonal antibody directed against phosphotyrosine. GM-CSF-induced tyrosine phosphorylation was inhibited in a dose- and time-dependent manner by the tyrosine kinase inhibitor erbstatin. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of GM-CSF on human neutrophils. Pretreatment of neutrophils with erbstatin before incubation with GM-CSF completely inhibited the GM-CSF induced intracellular alkalinization, downregulation of the leukotriene B4 receptor, enhancement of fMet-Leu-Phe-induced intracellular calcium mobilization, as well as the accumulation of mRNA for the proto-oncogene c-fos. Taken together, these data suggest that tyrosine kinase activation in human neutrophils plays a critical regulatory role in both the stimulation and priming of neutrophil function by GM-CSF. PMID- 1717074 TI - Inactivation of human immunodeficiency virus type 1 by ozone in vitro. AB - A device was designed to deliver a constant source of given concentrations of ozone to fluids containing human immunodeficiency virus type 1 (HIV-1). Ozone was found to inactivate HIV-1 virions in a dose-dependent manner. Greater than 11 log inactivation was achieved within 2 hours at a concentration of 1,200 ppm ozone. Similar concentrations of ozone had minimal effect on factor VIII activity in both plasma and immunoaffinity-purified preparations of factor VIII treated for the same time period. The data indicate that the antiviral effects of ozone include viral particle disruption, reverse transcriptase inactivation, and/or a perturbation of the ability of the virus to bind to its receptor on target cells. Ozone treatment offers promise as a means to inactivate human retroviruses in human body fluids and blood product preparations. PMID- 1717075 TI - Synergistic effects of interleukin-1 beta and interleukin-3 on the expansion of human hematopoietic progenitor cells in liquid cultures. AB - In the present study, we show that recombinant human interleukin-1 beta (rhIL-1 beta), which has no effect on the proliferation of human progenitor cells, has synergistic effects on the expansion of human progenitor cells induced by rhIL-3 in liquid cultures. The synergistic effects of rhIL-1 beta with rhIL-3 were observed in liquid cultures using not only fresh bone marrow mononuclear cells, but also selected populations of nonadherent cells, non-T nonadherent cells, and CD34-positive cells. Anti-granulocyte-macrophage colony-stimulating factor (anti GM-CSF) antibody partially blocked the synergistic effects of rhIL-1 beta on the proliferation of colony-forming unit (CFU)-GM burst-forming unit-erythroid (BFU E), and CFU-Mix in liquid cultures in the presence of rhIL-1 beta plus rhIL-3, suggesting that the synergistic effects of rhIL-1 beta plus rhIL-3 are explained in part by the secondary production of GM-CSF. Limiting dilution assays and liquid culture assays using CD34-positive cells indicate that rhIL-1 beta directly increases the numbers of colony-forming cells in liquid cultures. These results suggest that rhIL-1 beta has unique direct and indirect effects on the expansion of hematopoietic progenitor cells in liquid cultures. PMID- 1717076 TI - Stem cell factor in combination with granulocyte colony-stimulating factor (CSF) or granulocyte-macrophage CSF synergistically increases granulopoiesis in vivo. AB - Recombinant rat stem cell factor (rrSCF) and recombinant human granulocyte colony stimulating factor (G-CSF) coinjected for 1 week in rats cause a synergistic increase in mature marrow neutrophils accompanied by a striking decrease in erythroid and lymphoid marrow elements. The spleens of the same rats show increased granulopoiesis as well as increased erythropoiesis as compared with the spleens of rats treated with either growth factor alone. Splenic extramedullary erythropoiesis may act to compensate for the decrease in marrow erythropoiesis. The coinjection of rrSCF and G-CSF causes an increase in marrow mast cells at the end of 1 week, but the increase is much less than in rrSCF-alone-treated rats. The combination of rrSCF and G-CSF increases the rate of release of marrow neutrophils into the circulation and causes a dramatic synergistic peripheral neutrophilia, beginning especially after 4 days of treatment. Colony-forming assays of all experimental groups showed a synergistic increase in colony-forming unit granulocyte-macrophage (CFU-GM) in the marrow, but not in peripheral blood, after coincubation with SCF plus granulocyte-macrophage CSF (GM-CSF) as opposed to GM-CSF alone, showing anatomic compartmentalization between a more primitive marrow CFU-GM subset and a more mature peripheral blood CFU-GM subset. In vivo daily administration of SCF plus GM-CSF results in a synergistic increase in marrow neutrophils, but not the striking synergistic increase in circulating neutrophils that is observed with SCF plus G-CSF. PMID- 1717077 TI - Role of AUUU sequences in stabilization of granulocyte-macrophage colony stimulating factor RNA in stimulated cells. AB - RNAs for transiently expressed genes such as oncogenes and cytokines, including granulocyte-monocyte colony-stimulating factor (GM-CSF), have a short half-life (T1/2). A cluster of AUUU sequences identified in the 3' untranslated (UT) region of these RNAs has been implicated in controlling stability of these transcripts. We examined the role of AUUU sequences in mRNA stability of GM-CSF after stimulation of cells. Human fibroblasts (W138) were stably transfected with chimeric constructs containing the beta-globin gene linked to a 52-bp tail of GM CSF containing either eight ATTTT (pNEOR beta G-AT) or eight repeats in which the AT sequences have been changed to GC sequences (pNEOR beta G-GC). Data confirmed that AUUU sequences in 3'UT region of GM-CSF play a major role in GM-CSF RNA instability. Stimulators of protein kinase C (PKC), cycloheximide (CHX), sodium fluoride (NaF), and, to a more limited extent, interleukin-1 beta (IL-1 beta), appear to stabilize GM-CSF RNA through these AUUU sequences, but tumor necrosis factor-alpha (TNF-alpha) induces stabilization of GM-CSF RNA through a mechanism independent of their AUUU sequences. PMID- 1717078 TI - Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions. AB - Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A 1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen-induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions. PMID- 1717079 TI - Refractory period phenomenon in the induction of tissue factor expression on endothelial cells. AB - Tissue factor (TF) is the first factor of the extrinsic pathway of coagulation. Normally, TF is not expressed on the surface of endothelial cells. However, expression of TF can be induced in these cells in response to stimulation by diverse inflammatory mediators such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and phorbol 12 myristate 13-acetate (PMA). We have studied the effect of these mediators on the kinetics of the induction of TF-related procoagulant activity (PCA) on human umbilical vein endothelial cells (HUVECs). PCA is transiently induced on HUVECs, attaining a peak some 4 to 8 hours after addition of inflammatory agents, with maximal accumulation of TF messenger RNA (mRNA) occurring 3 to 5 hours earlier. Because the expression of PCA by treated HUVECs returns to basal levels by 20 to 30 hours, we examined the response of these cells to a second inflammatory stimulus. Continuous incubation of cells with a single inflammatory agent for 24 to 48 hours induces a hyporesponsive state with respect to the reinduction of TF expression by the same agent (14% of the initial stimulation for IL-1 beta, 39% for TNF-alpha 30% for LPS, and 7% for PMA). Such a diminution in PCA was also observed in the levels of TF mRNA. By contrast, pretreatment of HUVECs with one agent did not dramatically affect the reinduction of TF by any of the three other factors. We subsequently focused our attention on the induction of the autologous refractory period by IL-1 beta. De novo protein synthesis was not required during the preincubation of ECs for hyporesponsiveness to be observed. The establishment of the refractory state did not depend on the downmodulation of IL-1 beta receptor affinity or expression. Moreover, pretreatment of HUVECs with IL-1 beta increased prostacyclin (PGI2) production in response to a second stimulation by IL-1 beta, although such cells were unable to reexpress TF under the same conditions. This result suggests that distinct secondary messenger pathways are involved in TF induction and PGI2 synthesis by IL-1 beta in HUVECs. PMID- 1717081 TI - Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self renewal, multilineage differentiation, and long-term in vitro hematopoiesis. AB - Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages. PMID- 1717080 TI - Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: effective prophylaxis for graft-versus-host disease in patients with hematologic malignancies. AB - Seventy-one patients with hematologic malignancies received bone marrow from a histocompatible sibling (n = 48) or a partially matched relative (n = 23) that had been depleted of CD5+ T cells with either an anti-CD5 mooclonal antibody (MoAb) plus complement (anti-Leu1 + C) or an anti-CD5 MoAb conjugated to ricin A chain (ST1 immunotoxin [ST1-IT]). These patients received intensive chemoradiotherapy consisting of cytosine arabinoside, cyclophosphamide, and fractionated total body irradiation. Both anti-Leu1 + C and ST1-IT ex vivo treatments effectively depleted bone marrow of T cells (97% and 95%, respectively). Overall, primary and late graft failure each occurred in 4% of evaluable patients. The diagnosis of myelodysplasia was a significant risk factor for graft failure (P less than .001), and if myelodysplastic patients were excluded, there were no graft failures in major histocompatibility complex (MHC) matched patients and 2 of 23 (8.7%) in MHC-mismatched patients. The actuarial risk of grade 2 to 4 acute graft-versus-host disease (GVHD) was 23% in MHC matched patients and 50% in MHC-mismatched patients. In MHC-matched patients, acute GVHD tended to be mild and treatable with corticosteroids. Chronic GVHD was observed in 6 of 36 (17%) MHC-matched patients and none of 11 MHC-mismatched patients. There were no deaths attributable to GVHD in the MHC-matched group. Epstein-Barr virus-associated lymphoproliferative disorders were observed in 3 of 23 MHC-mismatched patients. The actuarial event-free survival was 38% in the MHC matched patients versus 21% in the MHC-mismatched patients. However, if outcome is analyzed by risk of relapse, low-risk patients had a 62% actuarial survival compared with 11% in high-risk patients. These data indicate that the use of anti CD5 MoAbs can effectively control GVHD in histocompatible patients, and that additional strategies are required in MHC-mismatched and high-risk patients. PMID- 1717082 TI - In search of the self-renewing hematopoietic stem cell. PMID- 1717083 TI - Applicator exposure to airborne concentrations of a termiticide formulation of chlorpyrifos. PMID- 1717084 TI - Cadmium stimulates osteoclast-like multinucleated cell formation in mouse bone marrow cell cultures. PMID- 1717085 TI - Automated differential counting. PMID- 1717086 TI - Parathyroid hormone-related protein, chromogranin A, and calcitonin gene products in the neuroendocrine skin carcinoma cell lines MKL1 and MKL2. AB - We studied the production of parathyroid-hormone-related protein, chromogranin A, calcitonin and calcitonin-gene-related peptide in the neuroendocrine skin cell line, MKL1, and a subsequently derived cell line designated MKL2. Both cell lines had cytological, histological and electron-microscopic features typical of neuroendocrine differentiation. Immunohistology and radioimmunoassay studies demonstrated the presence of parathyroid-hormone-related protein, chromogranin A, calcitonin-gene-related peptide, and calcitonin in the MKL2 cell line and the last three substances in both cell lines. The secretion of each of the first three substances was regulated by phorbol in the MKL2 cells. Additional immunohistochemical studies demonstrated the variable expression of bombesin, substance P, and vasoactive intestinal polypeptide in MKL2 cells, and the expression of synaptophysin in both MKL1 and MKL2 cells. These studies demonstrate the neuroendocrine characteristics of the MKL cell lines and provide a novel model for studies of the production and interactions of several neuroendocrine proteins and peptides by human skin cells. PMID- 1717088 TI - Aprotinin and cardiac surgery. PMID- 1717087 TI - Screening for Down's syndrome based on individual risk. AB - OBJECTIVE: To evaluate the effectiveness of biochemical screening of individual pregnancies for Down's syndrome risk. DESIGN: Retrospective determination of risk. SETTING: Obstetric and cytogenetic services in Tayside, Scotland. SUBJECTS: 3436 pregnant women who had screening for neural tube defects in the second trimester during November 1988 to March 1990 and whose pregnancies were dated by ultrasonography. Three women with pregnancies associated with Down's syndrome reported later in 1990. MAIN OUTCOME MEASURES: Individual risk calculated from age at estimated date of delivery; chorionic gonadotrophin and alpha fetoprotein concentrations in serum samples obtained at precisely determined gestational ages in second trimester. Results of karyotype determination and outcome of pregnancy. RESULTS: During November 1988 to March 1990 karyotypes were determined for 5% of pregnancies for reasons of maternal age and genetic history and one of the eight affected fetuses was detected. Individual risk could not be calculated for 347 pregnancies, but screening on this basis would have detected five of the cases and required screening in 194 out of 3089 (6.3%) pregnancies; all three affected pregnancies reported later in 1990 would also have been detected, giving a success rate of 73% (95% confidence interval 39% to 94%). The age distribution of women according to individual risk suggests that women over 35 would be screened effectively. CONCLUSION: Screening based on individual risk would use resources more effectively than screening based on maternal age and genetic history without affecting detection rates in older women. PMID- 1717089 TI - Evolutionary conservation of an epitope associated with striated rootlets in different epithelial ciliated cells. AB - In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells, to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct. PMID- 1717090 TI - Microtubule diversity in ciliated cells: evidence for its generation by post translational modification in the axonemes of Paramecium and quail oviduct cells. AB - The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre- and post-embedding EM immunocytochemistry with a variety of anti-tubulin antibodies. The 6-11B-1 monoclonal antibody, specific for the post-translational acetylation of Lys 40 of alpha-tubulin, and a polyclonal antibody raised against Paramecium axonemal tubulin (anti-PA tubulin antibody) both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti-PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti-tubulin antibody and the anti-PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti-PA tubulin one. This provided a strong indication that the anti-PA tubulin antibody is directed against a post translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti-PA tubulin antibody however, suggesting that in Metazoa the post-translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post-translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes. PMID- 1717091 TI - Substance P and calbindin D-28k-immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co-localization. AB - During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co localized. PMID- 1717092 TI - Failure of purged autologous bone marrow transplantation in high risk acute lymphoblastic leukaemia in first complete remission. AB - Patients in first remission of acute lymphoblastic leukaemia (ALL) considered to be at high risk of relapse were offered autologous bone marrow transplantation (ABMT) using purged marrow as a therapeutic alternative to cranial irradiation and maintenance chemotherapy. Twenty-seven bone marrows taken in remission, were purged using monoclonal antibodies (anti CD7 for T lineage and anti CD10 and/or anti CD19 for B lineage leukaemias) plus rabbit complement. Retrospective analysis of 19 purged marrows by immunophenotyping or immunoglobulin gene rearrangement studies demonstrated no evidence of disease. Engraftment was seen in 26 of the patients. No correlation was found between the numbers of infused nucleated cells or colony forming units-granulocyte-macrophage (CFU-GM) and subsequent engraftment kinetics. The actuarial disease-free survival (DFS) is 32% at 7 years (median follow-up 3.4 years). There were two transplant related deaths (actuarial risk 8%); the main cause of treatment failure has been disease recurrence with an overall actuarial risk of 67%; 76% for T-ALL (five of nine), 62% for common ALL (five of 10), two of five pre B and none of three patients with B-ALL. In these 27 high risk patients in vitro purging of remission marrow as part of ABMT appears not to improve patient outcome, although confirmation of this would require a randomized trial. PMID- 1717093 TI - Modulation of gastric contractions in response to tachykinins and bethanechol by extrinsic nerves. AB - 1. Extrinsic reflexes elicited by changes in gastric wall tension play an important role in regulating gastric tone. The present study investigated whether such reflexes modulate gastric contractions induced by close arterially administered neurokinin A (NKA), substance P (SP), SP-methylester and bethancehol in anaesthetized rats. 2. Reflex pathways were acutely interrupted by either subdiaphragmatic vagotomy or prevertebral ganglionectomy. C-fibre afferent nerve activity was abolished by pretreating rats with capsaicin 10 to 16 days before the experiments. 3. The order of potency in inducing gastric contractions was NKA greater than SP greater than bethanechol. SP-methylester was markedly less effective than SP and its effects did not fit sigmoid dose-response curves (DRCs). The maximal responses to NKA, SP, and bethanechol were similar, whilst the DRC for SP was significantly flatter than those for NKA or bethanechol. Pretreatment of the rats with the peptidase inhibitors phosphoramidon or captopril did not increase the contractile response to SP. 4. Prevertebral ganglionectomy had no significant effect on the DRCs for SP and NKA, whereas vagotomy shifted the DRCs for all three test substances to the left. 5. Capsaicin pretreatment did not change the DRC for NKA in rats with intact vagus but shifted that for bethanechol to the left. The leftward of the DRC for NKA caused by vagotomy was prevented in capsaicin-pretreated rats whereas the vagotomy-induced shift of the DRC for bethanechol remained unaltered. The shift of the DRC for SP seen in response to vagotomy was only slightly reduced by capsaicin pretreatment. 6. These data may be interpreted as demonstrating two neuronal mechanisms for modulating drug-induced gastric contractions. First, the contractions themselves activate a vago-vagal negative feedback involving capsaicin-sensitive afferents. Second, NKA, and to a lesser degree SP, seem to induce a nonvagal non-splanchnic mechanism which via capsaicin-sensitive afferent neurones reinforces tachykinininduced gastric contractions. PMID- 1717094 TI - Strontium-89 chloride for pain palliation in prostatic skeletal malignancy. AB - In a multi-centre study strontium-89 was shown to be effective in relieving bone pain from prostatic carcinoma in patients who had failed conventional therapies. Of 83 patients assessed at 3 months, following the administration of a dose of at least 1.5 MBq/kg, 75% derived benefit and 22% became pain free. Symptomatic improvement usually occurred within 6 weeks and continued for between 4 and 15 months (mean 6 months). Based on the dose estimation part of this study the recommended dose of strontium-89 is 150 MBq. Toxicity was low, provided platelet levels were above 100 x 10(9) l-1 at the time of treatment. Repeat treatments with strontium-89 may be given at intervals of not less than 3 months. Strontium 89 is administered intravenously on an out-patient basis with no special radiological protection precautions. PMID- 1717095 TI - Cytokeratin shedding in urine as a biological marker for bladder cancer: monoclonal antibody-based evaluation. AB - Cytokeratin shedding into urine was measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) in 282 individuals. Samples included urine from normal controls, patients with urogenital conditions and bladder cancer patients. A monoclonal antibody prepared against cytokeratins extracted from a hyperkeratotic low grade squamous cell carcinoma (UNME/K1) was used in the assay. The results indicated reasonable levels of sensitivity (83%), specificity (67%) and overall accuracy (70%) in the detection of bladder cancer. The levels of sensitivity in detecting squamous and transitional cell carcinoma patients were 87 and 73% respectively. The low level of specificity was due to a high frequency of false positive results (55%) within the urogenital controls; this suggests that further immunochemical and immunohistopathological analyses of associated urothelial cytokeratins are required. PMID- 1717096 TI - Screening for prostate cancer. Comparison of transrectal ultrasound, prostate specific antigen and rectal examination. AB - In seeking to define the relative value of transrectal ultrasound (TRUS), prostate specific antigen (PSA) and digital rectal examination (DRE) in the diagnosis of prostatic cancer, 863 patients were studied and the findings compared. DRE detected malignancy in 0.3% of the population of asymptomatic "normal" men undergoing routine health screening, and in 1.6% of patients who consulted their General Practitioner for one reason or another. In patients who attended our out-patient department with a variety of urological symptoms (not necessarily prostatic), TRUS suggested malignancy in 2% of those glands which were pronounced normal on DRE. Significantly elevated PSA detected malignancy in 0.3% of the patients undergoing routine health screening. (Although this figure equals the pick-up rate by DRE in this group, they were not necessarily the same patients). When these 3 investigations are summated, the pick-up rate is twice as high as when a single parameter is used. PMID- 1717097 TI - The intraurethral catheter: long-term follow-up in patients with urinary retention due to infravesical obstruction. AB - Insertion of 43 intraurethral catheters (IUC) was performed on 31 patients in order to replace an indwelling catheter for urinary retention due to benign prostatic hyperplasia. The insertion of the IUC was done under local anaesthesia with lignocaine jelly in the out-patient clinic. The device has been in place for 3 to 14 months (mean 7), in 14 cases for greater than 3 months and in 29 cases for greater than 6 months. In 7 of the 43 inserted IUCs the device had to be removed earlier than intended (after 4.2-8 months) for reasons related to the device. Of the remaining 36 devices which were functioning well, 6 were removed at prostatectomy 3.5 to 8 months after insertion and an additional 6 IUCs had to be removed for reasons not related to the device; 24 IUCs are still in place and functioning satisfactorily. The mean maximal flow rate in these patients is 13.9 ml/s. Of the 31 patients, 27 had a positive urine culture before IUC insertion. None of the patients with the IUC had a clinically evident urinary tract infection, although in 7 cases the urine culture was positive during the follow up period. All patients were in a better psychological state, and those who were able to perform sexually before they had the indwelling catheter were also able to perform sexually with the device in place. After long-term follow-up, the IUC has proved to be a cost-effective, successful alternative to an indwelling catheter and can be used as an alternative to prostatectomy in patients who are at high risk for surgery. PMID- 1717098 TI - Low dose bleomycin with etoposide and cisplatin for metastatic testicular teratoma. AB - Thirty-nine men with metastatic testicular teratoma were treated with a combination of bleomycin, etoposide and cisplatin (BEP). Unlike the usual regimen of these 3 agents, bleomycin and cisplatin were given on day 1 only of the cycle, with etoposide for 3 days. Thirty patients (77%) are alive and disease-free after a median follow-up of 31 months--24/25 (96%) with disease confined to lymph nodes but only 6/14 (43%) patients with lung involvement. Modified BEP chemotherapy is a well tolerated alternative to standard BEP chemotherapy for small volume nodal disease; it minimises in-patient time, hospital visits and the risk of bleomycin lung toxicity. However, omission of the weekly doses of bleomycin and shortening of the administration schedule of cisplatin and etoposide may be detrimental in patients with more extensive disease, for whom more intensive therapy may be necessary. PMID- 1717099 TI - Re: Gamma-seminoprotein--a new tumour marker in prostatic cancer? PMID- 1717100 TI - Tropic role of enteroglucagon in pancreatic adaptation to subtotal enterectomy. AB - Proximal small bowel resection causes pancreatic hyperplasia, presumably via a humoral mechanism. Although cholecystokinin can stimulate pancreatic growth, its proximal distribution in the gut makes it an unlikely intermediary after proximal small bowel resection. The potential roles of neurotensin and enteroglucagon were studied, since these hormones are mainly secreted from the ileum and proximal colon. Male Wistar rats (n = 50) weighing 200-250 g were randomized to receive 90 per cent proximal small bowel resection or jejunal transection and resuture (control). Rats were killed at 1 week or 1 month, when plasma was obtained for hormone assay and the pancreas was excised for protein and nucleic acid measurement. Proximal small bowel resection increased circulating enteroglucagon levels by 150 per cent at 1 week (P less than 0.002) and by 83 per cent at 1 month (P less than 0.005); neurotensin levels were unchanged. Pancreatic wet weight was 21 per cent greater 1 month after proximal small bowel resection (P less than 0.001). Proximal small bowel resection increased protein, RNA and DNA contents of the pancreas both at 1 week and at 1 month. Since plasma enteroglucagon correlated with these indices of pancreatic mass, enteroglucagon may have a pancreatotropic role (in addition to its enterotropic role). PMID- 1717101 TI - Accurate intraoperative transhepatic U tube placement. AB - Cholangiocarcinoma of the main hepatic duct junction is best treated by curative resection, although this is not often possible as most lesions are associated with local infiltration, portal vein invasion or metastases. For unresectable lesions, we advocate the use of a wide bore U tube followed by radiotherapy. Placement of the U tube must be accurate and meticulous. This paper describes a simplified method allowing accurate intraoperative transhepatic U tube placement. PMID- 1717102 TI - An ELISA for the detection of serum antibodies to both transmissible gastroenteritis virus and porcine respiratory coronavirus. AB - A competition ELISA utilizing a mAb directed towards a peplomer protein epitope common to TGEV, PRCV and related feline and canine coronaviruses is described. PMID- 1717103 TI - Low-dose intrathecal morphine facilitates the spinal flexor reflex by releasing different neuropeptides in rats with intact and sectioned peripheral nerves. AB - The facilitatory effect of intrathecal (i.t.) morphine on the excitability of the nociceptive flexor reflex was examined in decerebrate, spinalized, unanesthetized rats with intact or sectioned sciatic nerves. Low doses of i.t. morphine (10 ng in rats with intact nerves and 10 or 100 ng in rats with sectioned nerves) facilitated the flexor reflex. Higher doses of morphine caused facilitation followed by reflex depression. Facilitation of the flexor reflex induced by 10 or 100 ng morphine was prevented by i.t. naloxone (1 microgram). In rats with intact sciatic nerves the facilitation was partially antagonized by the tachykinin antagonist spantide II (D-NicLys1,3-Pal3,D-Cl2Phe5,Asn6,D-Trp7,9,Nle 11) substance P (SP), indicating that the reflex facilitation evoked by low doses of morphine may be due to the release of SP and perhaps other neuropeptides. In axotomized animals, 14-20 days after unilateral sciatic nerve section, spantide II failed to antagonize morphine-induced facilitation, suggesting that SP or other tachykinins, no longer played a role in this effect. In contrast, the vasoactive intestinal peptide (VIP) antagonist (N-Ac-Tyr1,D-Phe2)-GRF (1-29)-NH2 blocked morphine-induced reflex facilitation in axotomized rats, but not in rats with intact nerves. The present study provides evidence that low doses of morphine may induce the release of excitatory neuropeptides, thereby facilitating spinal nociceptive transmission. The identity of the neuropeptides depends on whether or not peripheral axons are intact, tachykinins in rats with intact nerves and VIP in axotomized rats. PMID- 1717104 TI - Human cochlear nucleus: comparison of Nissl-stained neurons from deaf and hearing patients. AB - In this study of the effects of deafness on the morphology of the human cochlear nuclei, non-parametric statistical analysis is used to quantify differences in sizes and shapes of neuron somata. Data on 81,007 neuron somata from 11 patients are presented, as well as the total volume and surface area of the cochlear nuclei. Soma size of deaf patients, especially postlinguistically deaf ones, was smaller than that of controls, but not significantly so; the same was true for total cochlear nucleus volume. The data also indicate a greater soma size on the right side (as well as a greater ventral cochlear nucleus volume), a caudal-to rostral decrease in soma size, and a correlation between soma size and shape. The data base is being continually extended and in future will allow comparisons with measurements from patients suffering from various forms of hearing loss. PMID- 1717105 TI - Neuron regeneration reverses 3-acetylpyridine-induced cell loss in the cerebral cortex of adult lizards. AB - Systemic administration of the neurotoxin 3-acetylpyridine to adult lizards results in extensive loss of neurons in the medial cerebral cortex, other brain areas remaining largely unaffected. After the neurotoxic trauma, new cells are produced by mitotic division of cells in the ventricular wall. The new cells migrate along radial glial fibers and replace lost neurons in the medial cortex. Electron microscopic examination of cells labeled with [3H]thymidine confirms that the newly generated cells are neurons. Thus, neuron regeneration can occur in the cerebral cortex of adult lizards. PMID- 1717106 TI - Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons. AB - The effects of mercuric chloride and methylmercury chloride on the rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch clamp technique. gamma-Aminobutyric acid-induced chloride currents were augmented by mercuric chloride in a potent and efficacious manner; at concentrations of 1 and 10 microM, the current amplitude was increased to 130% and 200% of the control. Methylmercury even at 100 microM did not augment but rather decreased the GABA-induced chloride current. Both mercuric chloride and methylmercury generated slow inward currents by themselves. These currents are not mediated by the GABA-activated chloride channels or by voltage-activated sodium, potassium or calcium channels, and are likely to be due to non-specific cation channels. PMID- 1717107 TI - Galanin and NADPH-diaphorase coexistence in cholinergic neurons of the rat basal forebrain. AB - The distribution of the enzyme NADPH-diaphorase in the rat basal forebrain was examined in relation to the neuropeptide galanin and the neurotransmitter synthetic enzyme choline acetyltransferase. Immunoperoxidase staining permitted camera lucida mapping of galanin and choline acetyltransferase distributions in serial sections through the basal forebrain for comparison with adjacent sections prepared for NADPH-diaphorase histochemistry. Photographs of sections subjected to indirect immunofluorescence for galanin and choline acetyltransferase were compared to photographs of the same sections taken after NADPH-diaphorase histochemistry. This permitted the direct investigation of co-localization within the cholinergic basal forebrain. The distributions of choline acetyltransferase- and galanin-immunoreactive neurons in the basal forebrain agreed with previous descriptions. NADPH-diaphorase histochemistry selectively stained a population of magnocellular basal forebrain neurons with a distribution similar to that observed with galanin immunohistochemistry. Double and triple staining experiments indicated that NADPH-diaphorase labels a majority of the magnocellular cholinergic neurons in the medial septum and diagonal band nuclei. Most of these neurons also contain galanin immunoreactivity. However, small populations of galanin-positive/diaphorase-negative or diaphorase positive/galanin-negative cholinergic neurons were also observed. In the more caudal portions of the cholinergic basal forebrain, very few galanin or NADPH diaphorase-positive neurons were observed. Thus, galanin and NADPH-diaphorase coexist in the majority of cholinergic basal forebrain neurons in the regions innervating limbic structures. The neocortically projecting cholinergic cells in the caudal basal forebrain appear to lack these other neurochemical markers. PMID- 1717108 TI - Ruthenium red antagonism of the effects of capsaicin mediated by extrinsic sensory nerves on myenteric plexus neurons of the isolated guinea-pig ileum. AB - The effects of Ruthenium red and its antagonism of capsaicin-induced action on the electrophysiological behavior of myenteric neurons were investigated with intracellular recording techniques in the isolated guinea-pig ileum. Ruthenium red antagonized dose-dependently (1-10 microM) a capsaicin-induced marked long lasting slow depolarizing action associated with increased input resistance, during which the cells spiked repeatedly or displayed anodal break excitation. This action of capsaicin has been found to be mediated via a release of substance P from sensory nerve endings. The slow depolarizing response to exogenous substance P applied by pressure microejection, which mimicked the capsaicin induced action, was not affected by Ruthenium red. Therefore, present results indicate that Ruthenium red antagonizes the specific effect of capsaicin on myenteric neurons by acting on the presynaptically located peripheral nerve terminals of sensory neurons and inhibiting the release of substance P. Electron microscopic examination showed that the neurotoxic action of capsaicin towards extrinsic sensory nerve fibers was also dose-dependently (1-10 microM) protected by pretreatment of ruthenium red. Present results suggest that Ruthenium red inhibits a capsaicin-induced activation of cation channels at the cell membrane of sensory nerves. PMID- 1717109 TI - Levodopa replacement therapy alters enzyme activities in striatum and neuropeptide content in striatal output regions of 6-hydroxydopamine lesioned rats. AB - The effects of striatal dopamine denervation and levodopa replacement therapy on neuronal populations in the rat striatum were assessed by measurement of glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) activities in the striatum, dynorphin and substance P concentrations in the substantia nigra, and enkephalin concentration in the globus pallidus. Rats with a unilateral 6 hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway were treated for 21 days with levodopa (100 mg/kg/day, i.p., with 25 mg/kg benserazide) on either an intermittent (b.i.d.) or continuous (osmotic pump infusion) regimen and sacrificed following a three day drug washout. In saline-treated control rats, striatal GAD activity and globus pallidus enkephalin content were elevated and nigral substance P content was reduced ipsilateral to the 6-OHDA lesion. Intermittent levodopa treatment further increased GAD activity, decreased CAT activity, restored substance P to control levels, markedly increased dynorphin content, and had no effect on enkephalin. In contrast, continuous levodopa elevated globus pallidus enkephalin beyond the levels occurring with denervation, but had no effect on any of the other neurochemical measures. These results indicate that striatal neuronal populations are differentially affected by chronic levodopa therapy and by the continuous or intermittent nature of the treatment regimen. With the exception of substance P, levodopa did not reverse the effects of the 6-OHDA lesion but, rather, either exacerbated the lesion induced changes (e.g. GAD and enkephalin) or altered neurochemical markers which had been unaffected by the lesion (e.g. CAT and dynorphin).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717110 TI - Suppression of noradrenergic innervation compensates for behavioral deficits induced by lesion of dopaminergic terminals in the lateral septum. AB - Spontaneous alternation which is disrupted by lesion of septal dopaminergic (DA) afferents was chosen as a behavioral marker for the study of functional interactions between DA and noradrenergic (NA) innervation of the lateral septum. Three groups of rats were studied: a solvent group which received only vehicle injection, and two lesioned groups, one with DA lesion and the second with simultaneous DA + NA lesion of the septal innervation. DA lesion was produced by infusing 6-hydroxydopamine (6-OHDA) into the lateral septum after pretreatment with desmethylimipramine (DMI) injected intraperitoneally. The DA + NA lesion was produced by infusing 6-OHDA without DMI pretreatment. The lesion of DA innervation alone led to a disturbance of alternation behavior in a Y-maze, but performance was not affected by the combined DA + NA lesion. The group with septal DA lesion was then injected with 6-OHDA into the pedunculus cerebellaris superior (PCS) in order to destroy NA efferents from the locus coeruleus. The two other groups were sham-operated. After post-operative recovery, the rats were retested for spontaneous alternation. The rats with the PCS NA lesion subsequent to the DA septal lesion displayed normal alternation behavior. Their performance was not different from that of animals with both NA and DA lesions in the septum. Thus the NA lesion appears to prevent the alternation deficits induced by the DA septal lesion, and also abolishes the deficits induced by the prior DA lesions. These results may have therapeutic implications. PMID- 1717111 TI - Compartments in rat dorsal and ventral striatum revealed following injection of 6 hydroxydopamine into the ventral mesencephalon. AB - The catecholamine selective neurotoxin, 6-hydroxydopamine, was injected into the ventral mesencephalon of rats and the distribution of tyrosine hydroxylase immunoreactivity in the striatum was compared to that of substance P and calbindin immunoreactivities, recognized histochemical markers of striatal compartments. Two components of the TH-IR mesostriatal innervation were identified. A more vulnerable component, present in the core of the nucleus accumbens and matrix of the caudate-putamen, excepting its ventrolateral part, was eliminated rapidly, unmasking a less vulnerable component which was present primarily in the shell of the nucleus accumbens and patch(striosome) compartment of the caudate-putamen. The TH-IR innervation in the ventrolateral caudate putamen also was patchy following these lesions but the patches corresponded consistently to neither patch nor matrix compartments. PMID- 1717112 TI - Neuronal-specific expression of human copper-zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effects in Down's syndrome. AB - It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Down's syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammon's horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Down's syndrome patients. PMID- 1717113 TI - Reversal of rapid axonal transport at a lesion: leupeptin inhibits reversed protein transport, but does not inhibit reversed organelle transport. AB - The hypothesis that the reversal of rapid axonal transport requires a proteolytic conversion of anterograde transport vesicles was examined using sciatic nerve preparations from Xenopus laevis and leupeptin as an inhibitor of proteolysis. The transport of newly synthesized 35S-labeled proteins was studied with a position-sensitive detector of radiation. Organelle transport in isolated myelinated axons was studied by video microscopy. Leupeptin (0.1-0.4 mM) reduced the anterograde-to-retrograde reversal of protein transport adjacent to an axonal lesion. In experiments in which organelle transport was observed close to lesions in axons maintained in a medium compatible with intracellular function, 1.0 mM leupeptin inhibited neither the anterograde-to-retrograde nor the retrograde-to anterograde reversal of organelle transport. In addition, in experiments in which conditions approximated those used to study protein transport, organelle transport away from the lesion was not inhibited by 1.0 mM leupeptin. A comparison of the morphology of rapidly transported organelles that underwent anterograde transport to the morphology of those that returned from a lesion (with or without the presence of leupeptin) provided no evidence that a morphological conversion was a necessary step in transport reversal. PMID- 1717114 TI - Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study. AB - Although abundant evidence suggests a major role for substance P (SP) and other neurokinins (NK) in the transmission of nociceptive information, it is not known whether the various NK receptor classes are differentially located in the substantia gelatinosa of the spinal cord where primary afferent fibres mostly terminate. In order to investigate this issue, we studied the effects of unilateral dorsal rhizotomy on binding of 125I-Bolton-Hunter-SP, (2 [125I]iodohistidyl1)-neurokinin A, and 125I-Bolton-Hunter-eledoisin as respective radioligands for the NK-1, NK-2 and NK-3 receptor sub-types. Seven, 14, 21 and 28 days following unilateral lumbosacral dorsal horn deafferentiation, NK receptor binding parameters were evaluated using quantitative receptor autoradiography. Rhizotomy produced an increase in the densities of NK-1, NK-2 and NK-3 binding sites in the superficial laminae of the dorsal horn. Increases were maximal at 14 days, post-operatively, for both NK-1 and NK-2 sites; slight recovery being observed thereafter. For NK-3 sites, unilateral rhizotomy induced a progressive increase in binding without evidence of recovery over time, at least up to 28 days post-lesion. NK-1 receptor binding parameters around the central canal and in the ventral horn were not affected by the dorsal rhizotomy. These data suggest that all 3 NK receptor classes are located post-synaptically to afferent fiber terminals in laminae I, II and X of the dorsal horn of the spinal cord. PMID- 1717115 TI - Do neuropeptides in the dorsal horn change if the dorsal root ganglion cell death that normally accompanies peripheral nerve transection is prevented? AB - Peripheral nerve section causes the death of dorsal root ganglion cells and changes in neuroactive peptides in the dorsal horn of the spinal cord. The relationship between these 2 events has not been previously studied, however. One approach would be to prevent sensory cell death and then determine changes in peptide immunoreactivity. To do this, transected rat sciatic nerve stumps were placed in an impermeable silicone tube for one month. The tube was then removed and after 30 additional days the cells were counted. The data indicate that no cell death occurred. We conclude that the sensory cells are first saved due to some factor present in the tube, and then after 30 days, the cells become independent of the tube and its contents. In these same animals, all of the peptides we examined were significantly changed. Four of the peptides, calcitonin gene-related peptide (CGRP), substance P (SP), cholecystokinin octapeptide (CCK) and galanin (GAL) were significantly depleted in the medial L4-L5 superficial dorsal horn, and vasoactive intestinal polypeptide (VIP) was significantly increased. We conclude that there are major changes in spinal peptide systems following peripheral nerve transection even if there is no accompanying death of sensory neurons. Thus we suggest that dramatic central changes in peptide immunoreactivity following peripheral nerve transection are independent of the sensory cell death that usually occurs in response to this injury. PMID- 1717116 TI - Monte Carlo simulations of ionic channel selectivity. AB - Monte Carlo simulations have been developed to study the selectivity of ionic channels in biological membranes. The channel is considered to be in either of two possible states: (i) densely packed with ions, the ions moving in single file in one direction, or alternatively, (ii) sparsely packed, where holes could appear at any particular time thereby allowing bidirectional movement of ions. The two models enable us to envisage a quantitative flux of permeable ions in the presence of smaller sized ions, taking into consideration their concentrations in the bulk solutions, the ion-channel interactions and probability with which they fill up the channel. The programs are written in FORTRAN-77 (MS-FORTRAN) for an IBM-compatible personal computer. From the simulation results we observe an enzymatic function of the channel and also note that the smaller sized ions tend to block the movement of permeable ions. The simulations represent a technique for visualization of the factors that decide ionic permeability and help in manipulating their effects with ease and speed which would otherwise involve intricate experimental setups. PMID- 1717117 TI - Phylogenetic comparative analysis of RNA structure on Macintosh computers. AB - A Macintosh Hypertalk program (Hypercard 'stack') for use in phylogenetic comparative analysis of RNA structure is described. The program identifies covariations and compensatory changes in RNA sequence alignments, for use in the construction of secondary structure models or the identification of tertiary interactions. The results of an analysis are presented either as a list of positions in the alignment which covary, or as a 2-dimensional matrix in which potential helices in the secondary structure appear as diagonal patterns. PMID- 1717118 TI - A unique human IgE-binding epitope on the Bermuda grass pollen recognized by mouse lambda-type monoclonal antibodies. AB - A group of six mouse monoclonal antibodies (MoAbs) with the unusual lambda-type light chain were generated by fusion of NS-1 cells with splenic cells derived from BALB/c mice immunized with crude extracts of Bermuda grass pollen (BGP). Four of them were IgG1, one was IgG2b, and one was IgG3. Binding inhibition assay showed that they recognized the same (or very similar) epitope. Using sera from BGP-allergic patients, it was found that the specific binding between the IgE antibodies and the MoAb 26-11-fixed antigen could be blocked by MoAb 26-11 itself and another MoAb 9-13 in a dose-dependent manner. It appears that the epitope recognized by the lambda-type MoAbs is a human IgE-binding antigenic determinant. Further physico-chemical analyses showed that this epitope was stable under heat but sensitive to treatments of sodium periodate and proteinase K. Results from these studies indicate that this unique epitope which leads to the generation of lambda-type MoAbs is part of a glycoprotein. PMID- 1717119 TI - The correlation between the increase in slow outward current and in contraction induced by caffeine, ryanodine, and rapid cooling in voltage-clamped frog muscle fibers. AB - The effects of caffeine, ryanodine, and rapid cooling were tested on the depolarization-induced contraction and the apamin-insensitive slow outward current (Iso) of voltage-clamped (double mannitol gap) single frog muscle fibers. Subthreshold caffeine concentrations (0.5-2 mM) induced a monotonic increase in contractile and Iso amplitude. Whatever the concentration, the increase in contraction was roughly twice the one in current. Similar results were obtained upon rapid cooling (20-4 degrees C) in the presence of 0.5 mM caffeine. In the absence of external Na+ (choline-substituted) 10(-5) M ryanodine induced a delayed increase (approximately 30 min) in contraction and in current, shortly before the development of a drastic and irreversible contracture. Here again, the increase in contraction was twice that in current. In the presence of 5 mM tetraethylammonium (TEA) and (or) 25 nM charybdotoxin, 2 mM caffeine still induced a strong facilitating effect on contraction but the parallel increase in current was strongly reduced. The linear relationship between the increase in current and contractile amplitude has a slope approximately 0.5 (whatever the drug used to increase contractility); it is approximately 0.1 in the presence of TEA and (or) charybdotoxin. In conclusion, provided the changes in contractile amplitude are caused by parallel changes in depolarization-induced sarcoplasmic reticulum Ca2+ release, about 50% of the apamin-insensitive Iso is controlled by internal Ca2+ release. The main part of this current corresponds to the TEA- and charybdotoxin-sensitive component of Iso. PMID- 1717120 TI - Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine evoked release of adenosine from spinal cord synaptosomes. AB - The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20 1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717121 TI - Neoadjuvant chemotherapy consisting of cisplatin and continuous infusions of bleomycin and 5-fluorouracil for advanced head and neck cancer. The need for a new stratification for stage IV (M0) disease. AB - A Phase II study of cisplatin (100 mg/m2 on day 1) and bleomycin (15 mg intravenous push day 1) followed by 5 days of continuous intravenous infusions of 5-fluorouracil (5-FU) (650 mg/m2/d) and bleomycin (16 mg/m2/d) repeated at 21-day intervals was performed in 54 previously untreated patients with nonmetastatic (M0), locoregionally advanced head and neck squamous cell carcinoma (SCC). The aim of this study was to increase the complete response rate to chemotherapy and to identify prognostic factors that may influence local control and disease-free survival. From April 1986 until August 1988, 5 patients with Stage III and 49 with Stage IV (International Union Against Cancer-American Joint Committee on Cancer 1986 [UICC-AJCC]) disease received this regimen. Thirty (61%) patients with Stage IV disease had bulky nodal disease (9 N2c and 21 N3) and 29 (53%) had T4 primary lesions. The response rate was 59% (95% confidence interval, 47% to 71%) and the complete response rate to chemotherapy was 13% (95% confidence interval, 0% to 26%). The response rate was greatly influenced by tumoral volume and performance status (PS). The complete response rate to chemotherapy was 40% for patients with Stage III disease (2 of 5 patients) versus 10% for patients with Stage IV disease (5 of 49 patients; P = 0.02). The response rate for patients with Stage III disease was 100% (5 of 5 patients) versus 55% for patients with Stage IV disease (27 of 49 patients; P = 0.14). For patients with Stage IV bulky nodal disease (N2c-N3), the response rate was 43% (13 of 30 patients) and the complete response rate to chemotherapy was 3% (1 of 30 patients) versus 68% (13 of 19 patients; P = 0.13) and 21% (4 of 19 patients; P = 0.07), respectively, for patients with Stage IV less than N2b disease. The local control rate after definitive therapy was 100% for patients with Stage III disease, 70% (17 of 24 patients) for patients with Stage IV less than N2b disease, and 17% (5 of 30 patients) for patients with bulky nodal disease (P = 0.0005). As of February 1991, with a median follow-up time of 38 months (range, 30 to 53 months), 4 of 5 patients with Stage III disease and 7 of 19 patients with Stage IV less than N2b disease were alive with no evidence of disease (37%) versus 0 of 30 patients with bulky nodal disease (P = 0.001).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717122 TI - Clinical significance of CD7-positive stem cell leukemia. A distinct subtype of mixed lineage leukemia. AB - Ten leukemia cases with mixed phenotype were investigated in terms of clinical characteristics and cellular origin. Three patients were infants and six patients were older children. Six of them had a high leukocyte count and a mediastinal mass was found in three cases. All but one showed hepatosplenomegaly and/or lymphoadenopathy. In spite of intensive chemotherapy, most of them responded poorly. Cytochemical analysis of their leukemic cells revealed a low percentage of positivity for myeloperoxidase reactivity (less than 25%) in two cases and electron microscopic platelet peroxidase reactivity was found in one of three analyzed cases. Phenotypically, these cells all expressed CD7, and other T lineage-associated, B-lineage-associated, and/or myeloid-associated antigens were also detected to some extent. In addition, three cases expressed CD41 and one case expressed CD56. The T-cell receptor (TCR) genes and immunoglobulin gene were in the germline configuration in seven cases. In three rearranged cases, two showed only the TCR-delta gene rearrangement, and one had both TCR-gamma and delta gene rearrangements. Cell culture studies with 12-0-tetradecanoyl-phorbol 13-acetate (TPA) revealed differentiation to the T-lineage in two cases and to a myeloid lineage in one case. Megakaryocytic differentiation was detected in two cases in culture without TPA. These results suggest that the cells from these cases arose from stem cells capable of both lymphoid and nonlymphoid differentiation. Although the cells were heterogeneous with regard to their potency of differentiation, they have similar clinical characteristics. Because of poor prognosis, it is important to identify this type of leukemia, and allogenic or autologous bone marrow transplantation should be considered. PMID- 1717123 TI - Soluble interleukin-2 receptors and other markers in primary lung cancer. AB - Serum levels of soluble interleukin-2 receptors (sIL-2R), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), beta-chorionic gonadotropin (beta-HCG), pregnancy-specific glycoprotein (SP1), and beta 2-microglobulin (beta 2M) were taken in 92 patients with primary lung cancer and 43 controls. The mean value of sIL-2R in the cancer group was twice as high as that of the controls (P less than 0.001) and the highest values were observed in those with small cell carcinoma (SCC) (P less than 0.0001). Of the cancer patients, 51.1% had CEA values higher than the cutoff level of 5 ng/ml. Extended-disease patients had a higher percentage of increased CEA values than those with limited disease. Adenocarcinoma (ADCC) and SCC groups had the highest percentages of increased CEA levels. There was no significant difference between the groups for beta-HCG, AFP, SP1, and beta 2M, and intermarker correlation was not seen. The results suggest that sIL-2R and CEA may be useful in monitoring the extent of disease and possibly indicate the histologic subtype, thus having a bearing on treatment and prognosis. PMID- 1717124 TI - Elevated prostate markers in metastatic small cell carcinoma of unknown primary. AB - Numerous ectopic hormones and markers have been described in small cell carcinoma of the lung as well as in extrapulmonary small cell carcinomas. The authors report a case of a patient with metastatic small cell carcinoma of unknown primary who had very high prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) levels. Results of multiple prostate examinations, as well as blind biopsies, were normal. His course was significantly longer than that of the usual patient with extensive small cell carcinoma. At autopsy the prostate showed only mild benign prostatic hypertrophy. There are no previous reports in the literature of abnormal prostate markers in small cell carcinomas. Physicians should be aware of the increasing complexity and the unusual biologic markers associated with neuroendocrine carcinomas. In some of these cases, the tumors ability to produce an ectopic product may portend an improved prognosis. PMID- 1717125 TI - A phase II trial of interleukin-2 by continuous infusion and interferon by intramuscular injection in patients with renal cell carcinoma. AB - Fifteen patients with advanced, measurable renal cell carcinoma entered a Phase II clinical trial of interleukin-2 (IL-2) (Teceleukin, Hoffmann-La Roche Inc., Nutley, NJ) and interferon (IFN) (Roferon A, Hoffmann-La Roche Inc.). IL-2 was administered by continuous infusion daily for 4 days and IFN was administered by intramuscular injection daily for 4 days; therapy continued for 4 weeks. Eight men and seven women were treated in this trial (median age, 61 years). Toxicity was moderate to severe with fatigue, nausea, vomiting, hypotension, and elevated blood urea nitrogen bunion and creatinine levels seen in all patients. Two patients achieved a complete remission and two patients achieved a partial remission. The median duration of response was 18 months. IL-2 and IFN is an active combination in the treatment of renal cell carcinoma and warrants further investigation. PMID- 1717126 TI - Serum CA 19-9 and alpha-fetoprotein levels in primary hepatocellular carcinoma and liver cirrhosis. AB - Serum CA 19-9 and alpha-fetoprotein (AFP) levels were determined in 211 patients with liver cirrhosis and 27 with primary hepatocellular carcinoma (HCC) associated with liver cirrhosis. This was done to determine the usefulness of CA 19-9 level with respect to AFP level in distinguishing between these two illnesses, and to assess the influence of some clinical and biochemical variables on these tests in patients with liver cirrhosis with or without primary HCC. Pathologic AFP values were found in 23 of 27 (sensitivity, 85%) patients with HCC; CA 19-9 levels increased in only 12 of 27 (sensitivity, 44%) HCC patients, the values being comparable with those of patients with liver cirrhosis. In liver cirrhosis a substantial number of false-positive values was found for both markers, although they were higher for CA 19-9 (50 of 211 versus 39 of 211). In liver cirrhosis correlations were found between AFP level and alanine amino transferase level; and between CA 19-9 level and (1) total bilirubin value, (2) alkaline phosphatase level, and (3) pseudocholinesterase level. The authors conclude that CA 19-9 level is a poor biochemical marker, inferior to AFP level, in the detection of a carcinomatous transformation of liver cirrhosis. The finding of false-positive AFP values in liver cirrhosis seems mainly attributable to cellular proliferation and necrosis. Cholestasis seems to greatly affect serum CA 19-9 level variations, probably by reducing its liver metabolism. PMID- 1717127 TI - Endometrial carcinoma with trophoblastic differentiation. An aggressive form of uterine cancer. AB - Three cases of poorly differentiated endometrial adenocarcinoma showing trophoblast-like differentiation are reported. The multinucleated, syncytiotrophoblast-like cells were strongly positive for beta-human chorionic gonadotropin (beta-HCG) by immunohistochemical study. High levels of beta-HCG were also present in the patients' serum, but dropped significantly after treatment. The patients had an unusually rapid and progressive clinical course with widespread dissemination and death by tumor. PMID- 1717128 TI - Total gastrectomy for advanced cancer. A worthwhile palliative procedure. AB - Subtotal gastric resection usually provides the best palliation in advanced gastric cancer; however, if total gastrectomy (TG) is required there is doubt about its benefit. The authors reviewed 53 consecutive patients undergoing TG for advanced gastric adenocarcinoma between 1980 and 1989. Indications for TG were tumor location in 30% and extent of tumor in 70%, including nine patients (17%) with linitis plastica. Four patients (8%) died postoperatively, and six patients required reoperation for postoperative complications. The median postoperative hospital stay was 13 days. The median survival was 19 months, and 13 patients (24%) lived for more than 2 years. The quality of life was graded in survivors as good in 59%, satisfactory in 28%, and poor in 13% of patients. It was concluded that TG is a worthwhile procedure for selected patients, even in the presence of advanced disease, providing prolongation of good quality of life with low morbidity and mortality. PMID- 1717129 TI - Nodular hepatocellular carcinoma. Treatment with subsegmental intraarterial injection of iodine 131-labeled iodized oil. AB - Internal radiation therapy with subsegmental arterial injection of iodine 131(131I)-labeled iodized oil (Lipiodol; Laboratorie, Guerbet, France) was evaluated in 24 patients with nodular hepatocellular carcinoma (HCC) ranging from 2.5 to 8.0 cm in size. 131I Lipiodol (555 to 2220 MBq in 3 to 8 ml) was injected depending on the tumor size. Tumor reduction was seen in 88.9% of tumors smaller than 4.0 cm in diameter, 65.5% of tumors between 4.1 to 6.0 cm, and 25.0% of tumors larger than 5.1 cm. The tumor size reduction corresponded to the gradual drop of serum alpha-fetoprotein (AFP) levels and devascularization on follow-up angiography. Adverse reactions from treatment included fever, mild abdominal pain, nausea, and elevation of transaminases. These were mild and well tolerated by patients. This method provided long-term local control without complications related to the thyroid, lung, gastrointestinal tract, and bone marrow. PMID- 1717130 TI - A comparison of 5-fluorouracil metabolism in human colorectal cancer and colon mucosa. AB - The metabolism of 5-fluorouracil (5-FU) was studied in biopsy specimens of primary colorectal cancer and healthy colonic mucosa obtained from previously untreated patients immediately after surgical removal. The conversion of 5-FU to anabolites was measured under saturating substrate (5-FU) and cosubstrate concentrations. For all enzymes, the activity was about threefold higher in tumor tissue compared with healthy mucosa of the same patient. The activity of pyrimidine nucleoside phosphorylase with deoxyribose-1-phosphate (dRib-1-P) was about tenfold higher (about 130 and 1200 nmol/hr/mg protein in tumors) than with ribose-1-phosphate (Rib-1-P), both in tumor and mucosa. Synthesis of the active nucleotides (5-fluoro-uridine-5'-monophosphate [FUMP] and 5-fluoro-2' deoxyuridine-5'-monophosphate [FdUMP]) was studied by adding physiologic concentrations of adenosine triphosphate (ATP) to the reaction mixture; the rate of FdUMP synthesis was 50% of that of FUMP (about 4 and 7 nmol/hr/mg protein in tumors). Direct synthesis of FUMP from 5-FU in the presence of 5-phosphoribosyl-1 pyrophosphate (PRPP) was about 2 nmol/hr/mg protein. With the natural substrate for this reaction, orotic acid, the activity was about 14-fold higher. To obtain insight into the recruitment of precursors for these cosubstrates, the authors also tested the enzyme activity of pyrimidine nucleoside phosphorylase with inosine and ribose-5-phosphate (Rib-5-P, as precursors for Rib-1-P) and deoxyinosine (as a precursor for dRib-1-P); enzyme activities were approximately 7%, 7%, and 3%, respectively, of that with the normal substrates, both in tumors and mucosa. However, when ATP and Rib-5-P were combined, the synthesis of FUMP was about 70% of that with PRPP, but only in tumors. In normal tissues no activity was detectable. These data suggest a preference of colon tumor over colon mucosa for the conversion of 5-FU to active nucleotides by a direct pathway; a selective antitumor effect of 5-FU may be related to this difference. PMID- 1717131 TI - Characterization of conservatively resected renal tumors using automated image analysis DNA cytometry. AB - The DNA histograms of 57 conservatively resected renal tumors were studied using automated image analysis DNA cytometry (Leytas II). Forty-nine of the analyzed tumors were renal cell carcinomas, six were oncocytomas, one was an angiomyolipoma, and one was a renal cell adenoma. On the basis of their DNA histograms, diploid, tetraploid, and aneuploid tumors could be distinguished. Aneuploid tumors could be subtyped further according to the DNA content of the stem cell line as hyperdiploid, hypertriploid, or hypertetraploid. Eight of the tumors were characterized by a combination of diploid and hypertriploid stem cell lines. During a mean follow-up of 5 years, only the two patients with a pure hypertriploid tumor died of distant metastases. These results indicate that automated DNA image analysis cytometry is able to differentiate among several types of renal tumors with obviously different prognoses. PMID- 1717132 TI - Functional evaluation of tumor-infiltrating mononuclear cells. Detection of endogenous interferon-gamma and tumor necrosis factor-alpha in human colorectal adenocarcinomas. AB - Quantitative evaluation of the levels of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the extracts of tumors and their corresponding normal tissues resected from 43 patients with colorectal adenocarcinoma was done using solid-phase, sandwich radioimmunoassay. The levels of both IFN-gamma and TNF-alpha detected in the tumor tissues were higher than those in the corresponding normal colorectal tissues obtained from each patient. A significant negative correlation was observed between the level of IFN-gamma and TNF-alpha in each tumor extract. The decrease of the level of IFN-gamma in the tumor correlated with the advance of clinical stage, and the levels of IFN gamma of the patients with distant metastases were significantly lower than those of the patients without distant metastases. However, an increase in the level of TNF-alpha correlated not only with an enlarged diameter but also with the extent of the primary tumor. Immunohistochemical staining of IFN-gamma and TNF-alpha producing cells in tumor tissues showed that IFN-gamma was mainly produced by CD4+ CD8- T-lymphocytes and TNF-alpha was mainly produced by CD11c+ cells with macrophage-like morphology. These results suggest that CD4+ T-lymphocytes that produce IFN-gamma might play an important role in the antitumor response against cancer progression in human colorectal adenocarcinomas. PMID- 1717133 TI - Etiologic and clinical characteristics of peripheral and hilar cholangiocarcinoma. AB - A 20-year experience with 112 patients with cholangiocarcinoma was reviewed with reference to the demographic, etiologic, and clinical features and prognosis in the following two types: peripheral (originating from the intrahepatic small duct radicles) and hilar (originating from the major hepatic ducts at or near the junction of the right and left hepatic ducts). Seventy of the 112 patients were in the hilar group, and 42 were in the peripheral group. Prolonged high alcohol consumption was a prominent feature in both categories (45% and 37%, respectively). Among the women, 35% of those with the peripheral tumor had used oral contraceptive preparations. The major identifiable etiologic factor among the hilar tumors was ulcerative colitis, with or without sclerosing cholangitis, which was documented in 20 of 70 cases (28.6%), with an additional 4 patients having Crohn's disease. The hilar group mainly had obstructive jaundice initially, whereas abdominal pain and weight loss were the predominant symptoms in the peripheral type. Tumor recurrence was frequent in those undergoing resection or transplantation, and none of those undergoing chemotherapy or radiation therapy showed any objective evidence of response. Overall median survival time was poor in both groups at 12 months. PMID- 1717134 TI - The role of prostate-specific antigen as part of the diagnostic triad and as a guide when to perform a biopsy. AB - The authors reviewed the results and relationship of prebiopsy prostate-specific antigen (PSA) assay, digital rectal examination (DRE), and transrectal ultrasound (TRUS) in 124 consecutive patients who underwent a prostate biopsy because of abnormal results of either DRE or TRUS. Results of the three tests (PSA, DRE, and TRUS) showed abnormalities in 54%, 75%, and 84.6% of patients, respectively; biopsy results were positive for cancer in 45.2%. Cancer detection rate increased as the PSA value increased from less than or equal to 4 ng/ml (17.5%) to more than 4 ng/ml (68.7%) to more than 20 ng/ml (83.3%), and as the number of positive tests increased (6.9% for one, 32.7% for two, and 82.6% for three). The PSA assay was the most important parameter of the diagnostic triad. These results suggested that regardless of DRE and TRUS findings, PSA less than or equal to 4 ng/ml confers a low prostate cancer risk, PSA more than 4 ng/ml but less than or equal to 10 ng/ml confers an intermediate prostate cancer risk, and PSA more than 10 ng/ml confers a high prostate cancer risk. Regardless of other findings, all patients with a PSA value more than 10 ng/ml require biopsy because of the high likelihood of cancer. All patients with abnormal DRE or TRUS results still require biopsy despite a low index of suspicion of prostate cancer when the PSA value is less than or equal to 4 ng/ml. PMID- 1717135 TI - Enhanced expression of low molecular weight keratins during progressive diethylnitrosamine-induced hepatocarcinogenesis in rats is associated with the presence of high levels of gamma-glutamyl transpeptidase and glycogen-deficient islands. AB - The sequential expression of keratin proteins as a function of tumour progression was studied in the rat liver and compared with several tumour markers like histochemical gamma-glutamyl transpeptidase (GGT)-positive foci, quantitative GGT activity and glycogen deficient islands at corresponding stages using diethylnitrosamine (DEN) as a carcinogen. The enhanced expression of low molecular weight keratins indicating undifferentiated nature of the tumour is associated with the increased levels of tumour markers. The findings are discussed in relation to cytoskeletal alterations during progressive hepatocarcinogenesis in rats. PMID- 1717136 TI - Novobiocin modulates cytokeratin assembly and differentiation of human hepatoma cells induced by butyrate and teleocidin. AB - A differentiation inducer butyrate and a tumor promoter teleocidin had inhibitory effects on the proliferation of PLC/PRF/5 hepatoma. Both of these reagents stimulated the production of procollagen type III peptide, enhanced the cytokeratin assembly and altered the morphological appearance. Novobiocin, a topoisomerase II inhibitor, enhanced the cytokeratin assembly induced by butyrate but antagonized that induced by teleocidin without changing the expression and the phosphorylation state of cytokeratin proteins. In addition, novobiocin acted synergistically with butyrate but not with teleocidin in stimulating the procollagen production and the acetate uptake. These results suggest that butyrate and teleocidin induce cell differentiation via distinct signaling pathway and that novobiocin and butyrate can be used as subsidiary drugs in preventing the growth of hepatoma. PMID- 1717137 TI - Muscle-specific gene expression in rhabdomyosarcomas and stages of human fetal skeletal muscle development. AB - Rhabdomyosarcomas (RMS) bear a morphological resemblance to developing striated muscle. It has been reported that two histologically distinct subtypes of RMS, embryonal and alveolar, behave differently in many clinical aspects, such as age distribution, primary site, and prognosis. We have investigated the expression of various genes, which are preferentially expressed in normal muscle tissue or cell culture (actins, myosins, and creatine kinases, and myogenic regulatory genes MyoD, myogenin, MRF4, and Myf5), in embryonal and alveolar subtypes and compared the results to the stages of developing human fetal limb muscle. The data showed that each of the RMS tumors tested, regardless of histological features, expressed MyoD1 and MRF4 transcripts. Expression of the myogenin gene was detectable in all alveolar RMS (n = 8), whereas only 5 of 8 embryonal RMS expressed myogenin transcripts. Trace levels of Myf5 transcripts were visible in all alveolar RMS and 7 of 8 embryonal RMS. The alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoplasmic actin transcripts were detectable in all alveolar RMS. While the beta- and gamma-cytoplasmic actin transcripts were evident in all embryonal RMS, only 3 of 8 and 6 of 8 embryonal RMS expressed detectable levels of alpha-skeletal and alpha-cardiac actin transcripts, respectively. The embryonic form of myosin heavy chain was detectable in 1 of 8 of each type of tumor. Myosin light chain-1/3 transcripts were detectable in 4 of 8 alveolar RMS and 5 of 8 embryonal RMS. Brain creatine kinase transcripts were detectable in all alveolar RMS and 4 of 8 embryonal RMS, whereas none of the RMS samples contained detectable levels of the muscle form of creatine kinase. A comparison of the expression profiles with those of normal developing human fetal limb muscle (from 7.5 to 24 weeks' gestation) suggested that RMS resembled a relatively restricted segment of fetal muscle development. Furthermore, the data also showed a great deal of overlap in the differentiation state achieved by the embryonal and alveolar subtypes of RMS, suggesting that the clinicopathological difference between these two may not be due to malignant transformation of the cells from different positions in the normal pathway of myogenesis. PMID- 1717138 TI - Expression of insulin-like growth factor I, its binding proteins, and its receptor in ovarian cancer. AB - Insulin-like growth factor (IGF) I is a polypeptide hormone important in normal growth and development. Although IGF-I is a mitogen for many cancer cell lines, previous work has suggested that autocrine production of IGF-I is uncommon in cancers of epithelial origin. In this study, expression of IGF-I, its binding proteins, and its receptor were examined in ovarian cancer cell lines and tissues. Of 10 ovarian cancer cell lines, 3 (OVCAR-3, OVCAR-7, and PEO4) expressed IGF-I mRNA. RNase protection assays using probes derived from IGF-IA, IGF-IB, and alternate exon 1 IGF-I complementary DNAs demonstrated that these cells contained a predominant IGF-I transcript with an alternate first exon. RNA extracted from primary and metastatic ovarian cancer tissues also expressed IGF-I mRNAs (7 of 7) with the alternate first exon. IGF-I protein was detected in OVCAR 3-conditioned media; this activity was secreted in conjunction with several IGF binding proteins (IGFBPs). IGFBP-2, IGFBP-3, an Mr 24,000 species, and an Mr 30,000 species could also be demonstrated in OVCAR-3. Type I IGF receptor mRNA was found in all 10 of the ovarian cancer cell lines and all 7 of the primary or metastatic ovarian cancer tissues. IGF-I was a mitogen for OVCAR-3, demonstrating the presence of a functional type I IGF receptor. These data show that all the necessary components for an IGF-I-mediated autocrine loop are expressed by ovarian cancer cells. PMID- 1717139 TI - Differential expression of the normal and mutated K-ras alleles in chemically induced thymic lymphomas. AB - The presence of point mutations in the K-ras gene was examined in murine thymic lymphomas induced by a single dose of N-methylnitrosourea by the RNase A mismatch cleavage method and by allelic-specific oligonucleotide hybridization of in vitro amplified DNA by polymerase chain reaction. The results show that the frequency of mutations is lower than that of tumors induced by multiple N-methylnitrosourea treatments. Four mutations identified were the aspartic acid at codon 12, a G:C to A:T transition in its second position. A G:C to T:A transversion in codon 146 was also found in one thymic lymphoma, changing the amino acid alanine to serine. The use of the RNase A assay allowed an estimation of the relative expression levels of both normal and mutant K-ras alleles. The results show that in approximately one half of the tumors the mutant allele is predominantly expressed, suggesting that the normal allele has been lost or that the mutant allele has been amplified relative to the normal. Altogether, these findings are consistent with ras mutations occurring in some instances during tumor development and with a ras effect being not strictly dominant but favoring selection for increasing levels of expression from the oncogenic allele. PMID- 1717140 TI - Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin. AB - The cyanomorpholino derivative of doxorubicin (MRA-CN) is a DNA intercalator and alkylator that is a highly potent cytotoxin, non-cross-resistant in multidrug resistant cells, and noncardiotoxic in comparison with doxorubicin. To further examine mechanisms of action and resistance to MRA-CN, a cell line resistant to MRA-CN, ES-2R, was established by growing a human ovarian carcinoma cell line, ES 2, in increasing concentrations of the drug. The resistant subline was 4-fold resistant to MRA-CN and cross-resistant to other DNA cross-linking agents, cisplatin (7-fold) and carmustine (3-fold), as well as to the DNA strand-breaking agents etoposide (6-fold), doxorubicin (2-fold), bleomycin (5-fold), and ionizing radiation (2-fold). In contrast, ES-2R cells were not cross-resistant to vinblastine. Several months of additional growth of ES-2R cells in MRA-CN did not yield higher, stable levels of drug resistance. A low level of P-glycoprotein was detectable in the ES-2R cells. However, the extent of intracellular accumulation of [3H]MRA-CN by this resistant cell line was identical to that of the sensitive line. The number of DNA cross-links formed by cisplatin in ES-2R was only 50% of that of the ES-2 cells and was associated with a 50% increase in the rate of repair of these cross-links in the resistant cells. Ionizing radiation induced similar amounts of single- and double-strand breaks in the ES-2 line as well as in the ES-2R cells. There was no apparent difference between the two cell lines in the rate and extent of repair of these DNA breaks. Thus, enhanced DNA repair cannot explain the phenomenon of cross-resistance to radiation. Comparisons of glutathione (GSH) content and the enzymes involved in GSH homeostasis showed significant differences. Resistant cells contained 1.5-fold more GSH, a 2.2-fold increase in gamma-glutamyltranspeptidase activity, and a 2.4-fold increase in GSH reductase compared with ES-2 cells (all P less than 0.05). Total glutathione-S transferase (GST) activity was 2.6-fold higher (P less than 0.01) in the ES-2R line. The pi-class GST subunit by Western blotting and GST activity toward ethacrynic acid were increased 2-fold in the resistant cells. Depletion of GSH levels in ES-2R cells by buthionine sulfoximine restored the sensitivity of ES-2R to MRA-CN. These findings implicate a role for GSH metabolism in the resistance phenotype of ES-2R cells. We have previously reported that these cells have an increased generation time and decreased topoisomerase II content. Thus, the ES-2R cell line exhibits a complex phenotype of broad cross-resistance, which is likely to involve multiple mechanisms, and includes enhanced DNA repair and increased GSH content and GST activity. PMID- 1717141 TI - Production of neuromedin B and neuromedin B gene expression in human lung tumor cell lines. AB - Gastrin-releasing peptide (GRP), a mammalian bombesin-like peptide, has been shown to be an important autocrine growth factor for small cell lung cancer (SCLC). However, not all SCLC cell lines express the GRP gene or respond mitogenically to GRP stimulation, suggesting the existence of other autocrine pathways in this tumor. Neuromedin B (NMB), the mammalian counterpart of amphibian ranatensin, has been shown to be a mitogen for SCLC cell lines in vitro. To determine whether NMB is a potential autocrine growth factor for lung tumors, NMB gene expression, peptide synthesis, and secretion have been investigated in a panel of SCLC and non-SCLC (NSCLC) cell lines. Northern blot analysis and enzymatic amplification from mRNA by polymerase chain reaction showed that the NMB gene was expressed in all SCLC and NSCLC cell lines examined. In contrast, the GRP gene was expressed in four of six classic SCLC cell lines but not in variant SCLC or NSCLC cell lines. Immunoreactive NMB was detected by radioimmunoassay in the majority of classic SCLC, in one of three variant SCLC and in one of three NSCLC cell lines, and secreted NMB was detected in medium conditioned by a SCLC and a NSCLC cell line. The present study also demonstrated the presence of immunoreactive GRP in the absence of detectable GRP gene expression. The antiserum used in the GRP radioimmunoassay failed to cross-react with NMB but showed some cross-reactivity with amphibian phyllolitorin raising the possibility that certain SCLC cell lines may produce a phyllolitorin-like peptide. PMID- 1717142 TI - Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins. AB - To study the mechanism of p53 involvement in malignant transformation, we compared the tumor development patterns induced by a parental p53 nonproducer pre B cell line with those by cell lines generated from this parental cell line following transfection of either wild type or mutant p53. It was found that whereas mutant p53 facilitated tumor development, expression of wild type p53 restrained tumor development. Cell lines expressing the wild type p53 induced the development of faster regressing tumors than the parental cell line. The parental p53 nonproducer and the wild type p53 producer regressor tumors underwent in vivo cell differentiation, manifested as IgG production. Mutant p53, producer cell lines, on the other hand, failed to show any immunoglobulin synthesis and gave rise to highly proliferative lethal tumors. Our results support the conclusion that these pre-B cells develop regressor tumors because they have undergone differentiation. Whereas the wild type p53 facilitates this differentiation, mutant p53 cells block it. We suggest that, in addition to inactivating the growth-suppressive activity of wild type p53, the expression of mutant p53 facilitates malignant transformation. PMID- 1717143 TI - Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells. AB - Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5 fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range. PMID- 1717144 TI - Mechanisms of resistance to etoposide and teniposide in acquired resistant human colon and lung carcinoma cell lines. AB - Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9 , 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5 , 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines. PMID- 1717145 TI - CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines. AB - One of several splice variants of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to the non-metastatic variant of a rat pancreatic carcinoma line (U. Gunthert et al., Cell, 65: 13-24, 1991). The variant-specific rat CD44 sequences were used to detect RNA expression in human cell lines: in carcinoma lines from lung, breast and colon; and in keratinocyte lines. By polymerase chain reaction amplification, complementary DNAs encoding human homologues were isolated and sequenced. The largest splice variant has been found in a large cell lung carcinoma line and in keratinocyte cell lines. It carries at least 5 additional domains (exons) encoding a total of 338 amino acids in the membrane-proximal extracellular region of the standard CD44. Various alternative splice products have been detected in other human tumor cell lines. The distribution of CD44 splice variants is consistent with the speculation that they fulfill functions in only a few restricted differentiation pathways and that in tumor cells these pathways have been reactivated. PMID- 1717146 TI - Evidence for the involvement of transforming growth factor alpha and epidermal growth factor receptor autocrine growth mechanism in primary human ovarian cancers in vitro. AB - We examined 35 primary human ovarian adenocarcinomas for the presence of epidermal growth factor (EGF) receptor (EGFR) in plasma membranes from cancer tissues by using 125I-EGF as a ligand. Specific 125I-EGF bindings were observed in 20 (57%) of these 35 cases. Scatchard analysis showed a class of high affinity EGF receptor: Kd 5.0 +/- 1.0 x 10(-10) M; Bmax, 83.3 +/- 12.1 fmol/mg protein (mean +/- SE, n = 20). Northern analysis in polyadenylated RNA from 15 EGFR(+) cancers using pretransforming growth factor alpha (pre-TGF alpha), prepro-EGF complementary DNA, and pE7, a complementary DNA clone of human EGFR, as probes revealed that pre-TGF alpha and EGFR mRNAs but not prepro-EGF mRNA were expressed in all cases examined. Immunocytochemical studies using monoclonal antibodies (mAbs) against TGF alpha, EGF, and EGFR showed that TGF alpha and EGFR but not EGF proteins were present on ovarian cancer cells in all cases. These data suggested a possible TGF alpha/EGFR autocrine mechanism in EGFR(+) ovarian cancers. We, therefore, examined the biological significance of this autocrine mechanism by using primary monolayer cell cultures. In primary cultures from EGFR (+) cancers, TGF alpha added to the culture medium stimulated the [3H]thymidine incorporation dose dependently. Moreover, the addition of mAbs against TGF alpha and EGFR but not EGF inhibited [3H]thymidine incorporation dose dependently in EGFR(+) cancer cells. On the other hand, in primary cultures from EGFR(-) cancers, TGF alpha and anti-TGF alpha, -EGFR, and -EGF mAbs did not show any effects on [3H]thymidine incorporation. All these results suggested the possible crucial role of a TGF alpha/EGFR autocrine growth mechanism in primary human ovarian cancers which express EGFR. PMID- 1717147 TI - Folate-binding protein is a marker for ovarian cancer. AB - We describe the isolation of a complementary DNA (cDNA) sequence encoding the ovarian cancer-associated antigen recognized by monoclonal antibody MOv18 and its identification as a high-affinity folate-binding protein (FBP). Functional cDNA clones were isolated using mRNA from the ovarian carcinoma cell line SKOV3 and colon carcinoma cell line HT29, by transient expression in WOP cells and selection of expressing cells by adhesion to antibody-coated magnetic beads. The cDNAs differed in the lengths of 5'- and 3'-noncoding regions, but they encoded identical peptides. A database search clearly showed them to be adult high affinity FBPs with amino acid sequences identical with those isolated from normal placenta and several carcinoma cell lines. Reactivity of cell lines with MOv18 was quantitatively consistent with the expression of FBP mRNA. Southern hybridizations show evidence of a family of related genes and/or pseudogenes and were mapped to chromosome 11q13.3-14.1 by fluorescent in situ hybridization using cosmid clones containing part of this region. Also identified were two PstI polymorphisms of four and three alleles, respectively, and a two-allele MspI polymorphism. The folate-binding protein locus was not amplified in any of the 16 carcinoma cell lines tested and in only 1 of 10 serous adenocarcinomas, indicating that overexpression of FBP in ovarian cancer cannot, in general, be due to gene amplification. PMID- 1717148 TI - Increased number of accessible sugar epitopes defined with monoclonal antibody AM 3 on colonic mucins is associated with malignant transformation of colonic mucosa. AB - Monoclonal antibody AM-3 detects a mucin sugar epitope (AM-3 epitope) the expression of which increases in the course of human colon carcinogenesis parallel to the gradual morphological alterations (so called adenoma-carcinoma sequence). In the present report the AM-3-positive mucin has been purified from human normal and carcinomatous colonic tissue. About 300-fold enrichment of the epitope per protein from both sources was achieved after ultracentrifugation, gel filtration on Sepharose CL-6B and isopyknic gradient centrifugation. Slot-blot and enzyme-linked immunosorbent assays of the purified preparation indicated not only different amounts of the mucins but also a consistent qualitative difference between the molecules from both sources. The qualitative difference could be obliterated by a partial removal of the AM-3 epitope from the tumor-derived mucin with neuraminidase. The visualization of the molecules by rotary shadowing indicated that the mucins from both sources have similar length distribution, 80% of the molecules being 100-600 nm long. The reaction with AM-3 antibody followed by rotary shadowing showed that in the purified preparations more than 95% of the tumor-derived molecules and 74% of the normal colon tissue-derived molecules carried the epitope. The tumor-derived mucins bound, on the average, 34 +/- 15 (SD) antibodies/1000 nm of the protein core while the mucin from normal colon tissue carried 12 +/- 11 antibodies/1000 nm of the protein core. These data indicate that the increased expression of AM-3 epitopes during malignant transformation of the human colon is due to accumulation of AM-3-positive mucin as well as a higher number of accessible AM-3 epitopes on this mucin. PMID- 1717149 TI - Immortalization of normal human bronchial epithelial cells by human papillomaviruses 16 or 18. AB - Human papillomaviruses (HPV) are associated with papillomatosis of the larynx, trachea, and bronchi in decreasing order of frequency, and these papillomatosis lesions may become malignant. When the patients are not selected for a history of papillomatosis, the frequency of HPV in bronchogenic carcinoma tissue is 1-5%. In order to develop a model for investigating the role of HPV in human bronchogenic carcinogenesis, normal human bronchial epithelial cells were transfected with cloned full-length HPV16 or HPV18. Two HPV18-transformed cell lines (BEP1 and BEP2) and one HPV16-transformed cell line (BEP3) were established. These nontumorigenic epithelial cell lines have: (a) attained over 100 population doublings in vitro; (b) mutually exclusive human marker chromosomes; (c) HPV DNA in forms that are consistent with chromosomal integration by Southern analysis; (d) HPV E6, E7, and E6* mRNA transcripts by Northern and reverse transcriptase polymerase chain reaction analysis; and (e) diminished confluence-induced squamous differentiation. These cell lines should be useful for studying mechanisms involved in proliferation, differentiation, and neoplastic transformation of human bronchial epithelial cells. PMID- 1717150 TI - Regional heterogeneity and complementation in the expression of the tumor associated glycoprotein 72 epitopes in colorectal cancer. AB - We analyzed the immunohistochemical expression of three epitopes of the tumor associated glycoprotein 72 (TAG-72) in whole cross-sections of primary colorectal carcinomas and in regional lymph node metastases using monoclonal antibodies (MAbs) B72.3, CC-49, and CC-83, which recognize distinct carbohydrate antigenic determinants. B72.3, CC-49, and CC-83 reacted with 13 of 27 (48%), 25 of 27 (92%), and 21 of 27 (77%) carcinomas, respectively. The immunoreactivity with lymph node metastases followed a similar pattern; MAb CC-49 was again the most reactive of the three antibodies, since it labeled 13 of 15 metastatic lesions. Positive reactions of the MAbs with the primary tumors were not always predictive of the immunorecognition of their metastases. Distinct areas within whole cross sections of TAG-72-positive primary carcinomas demonstrated marked differences in the expression of the three epitopes. CC-49 tended to react with the highest number of areas and with the highest percentages of carcinoma cells within each area. In no instances did B72.3 demonstrate reactivity superior to that of either CC-49 or CC-83. Tumors negative for the CC-49 epitope in any area also did not express the other two TAG-72 epitopes. However, the comparison of the immunostaining obtained with each MAb in TAG-72-positive primary lesions revealed areas where CC-83 was clearly more reactive than CC-49. Moreover, one lymph node metastasis, negative for CC-49, was recognized by CC-83. Thus, the combined use of MAbs CC-49 and CC-83 resulted in additive immunostaining of primary and metastatic colorectal carcinoma cells. The study provides evidence of intratumoral heterogeneity in the glycosylation pattern of the TAG-72 antigen in colorectal cancer and emphasizes the advantages of cocktails of anti-tumor associated antigen MAbs in the immunodetection of colorectal tumor cells. PMID- 1717151 TI - Evaluation of nontumorous tissue damage by transcatheter arterial embolization for hepatocellular carcinoma. AB - The serial changes in serum hepatic enzyme activities by transcatheter arterial embolization (TAE) were analyzed in 17 patients with hepatocellular carcinoma to estimate the contribution to the value by the damage of tumor or nontumorous hepatic cells. The serum levels of relatively tumor-specific fructose 1,6 diphosphate (FDP) aldolase were elevated after TAE in the cases of both superselective and nonsuperselective TAE that were performed from the segmental and the nonsegmental hepatic artery, respectively, but we found the marked elevation of FDP aldolase in the cases of the superselective TAE. In contrast, the non-tumor-specific fructose 1-phosphate (F1P) aldolase was markedly elevated only in the cases of nonsuperselective TAE. The total amount of FDP aldolase released by TAE correlated significantly with the integrated tumor tissue volume (P less than 0.005), whereas the total amount of F1P aldolase output correlated significantly with the integrated nontumorous tissue volume (P less than 0.005) as defined by lipiodol accumulation on computerized tomography scan. The consequent changes in the total nontumorous liver volumes after TAE were also analyzed by the follow-up computerized tomography scan. The nonsuperselective TAE caused the significant total nontumorous liver atrophy when compared with the superselective TAE. The progression of the total nontumorous liver atrophy correlated significantly with F1P aldolase output by TAE (P less than 0.001) but not with FDP aldolase output. These results suggest that the outputs of FDP and F1P aldolase are useful to estimate the degree of the tumorous and nontumorous tissue damage by TAE, respectively, and F1P aldolase output can be used to predict the progression of liver atrophy caused by TAE. PMID- 1717152 TI - Carcinoembryonic antigen has a different molecular weight in normal colon and in cancer cells due to N-glycosylation differences. AB - Carcinoembryonic antigen, an apical membrane glycoprotein expressed in normal human colonic epithelial cells, colonic polyps, tumor, and tissue culture cell lines originating from colonic adenocarcinomas, is generally considered to have a molecular weight of 180,000. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis associated with immunoprecipitation or immunoblotting with both monoclonal (Mab 517 and Mab 601) and polyclonal antibodies, we observed that carcinoembryonic antigen was actually expressed as two discrete apparent molecular weight forms in normal tissues: a broad band averaging at Mr 200,000 and a sharp band at Mr 130,000. This constituted the phenotype of the normal colon. In cancer cells we detected a single band at Mr 170,000 or lower. This variation was mainly the consequence of a modification of the glycosylation pattern of the molecule since deglycosylation by N-glycanase or biosynthesis in the presence of tunicamycin always produced a single molecular weight form, whether or not the source of tissue was normal or cancerous. By close inspection of benign, moderately transformed, and carcinomatous human colonic polyps we noticed that this shift in the molecular weight of carcinoembryonic antigen preceded the detection of other cancer markers such as nonspecific cross-reacting antigen at Mr 95,000 or the histological modifications leading to malignant diagnosis. Carcinoembryonic antigen constitutes, therefore, an important model with which to study the modifications of the glycosylation pattern induced during cancer biogenesis. PMID- 1717153 TI - Acidic and basic fibroblast growth factors are present in glioblastoma multiforme. AB - Immunohistochemistry was performed on paraffin sections from human glioblastoma multiforme and normal brain tissue. Acidic fibroblast growth factor (FGF) was abundantly present in astrocytes from all glioblastomas studied. Basic FGF was found in the matrix surrounding proliferating blood vessels in most of the glioblastomas. In contrast, astrocytes from normal brain did not contain acidic FGF, and perivascular matrix staining was not demonstrated for basic FGF in the normal brain. Both growth factors could be demonstrated in neurons, Purkinje cells, capillary endothelium, and arterial walls in the normal brain. This study implicates both growth factors in the pathogenesis of malignant glioma. Both may be significant mediators of angiogenesis in glioblastoma. PMID- 1717154 TI - Neurofilament protein triplet immunoreactivity in the dorsal root ganglia of the guinea-pig. AB - Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding. PMID- 1717155 TI - Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. AB - In a transgenic mouse model, dermal fibrosarcomas develop in a pathway comprised of at least three stages: mild fibromatosis, aggressive fibromatosis, and fibrosarcoma. The latter two stages are highly vascularized when compared with both the normal dermis and the initial mild lesion. Analysis of cell cultures derived from biopsies of these lesions has revealed that basic fibroblast growth factor (bFGF) is synthesized in all three stages and in normal dermal fibroblasts derived from the same mice. Unexpectedly, there is a change in the localization of bFGF from its normal cell-associated state to extracellular release in the latter two stages, which is concomitant both with the neovascularization seen in vivo and with the tumorigenicity of these cell lines. Thus, in this multistep tumorigenesis pathway there appears to be a discrete switch to the angiogenic phenotype that correlates with the export of bFGF, a known angiogenic factor. PMID- 1717156 TI - The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and alpha 2-6 sialyltransferase, CD75, on B cells. AB - Functional maturation of B lymphocytes correlates with expression of the B lineage-specific cell surface glycoprotein CD22. Two CD22 polypeptides have been characterized and suggested to play a role in B cell-B cell interaction as well as in B cell adhesion to monocytes. In this work we provide evidence that CD22 is directly involved in the cognate interaction between B and T cells. One of the two CD22 polypeptides, CD22 beta, interacts with a specific ligand on a subpopulation of CD4+ T cells. Our results suggest that the T cell ligand of CD22 is CD45RO, an isoform of the leukocyte common antigen class of phosphotyrosine phosphatases associated with the helper T cell phenotype. We further demonstrate that CD22 recognizes a second ligand, CD75, expressed predominantly on activated B cells and shown to be a cell surface alpha 2-6 sialyltransferase. PMID- 1717157 TI - Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. AB - Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation. PMID- 1717158 TI - RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. AB - L-glutamate, the principal excitatory transmitter in the brain, gates ion channels mediating fast neurotransmission. Subunit components of two related classes of glutamate receptor channels have been characterized by cDNA cloning and shown to carry either an arginine or a glutamine residue in a defined position of their putative channel-forming segment. The arginine residue in this segment profoundly alters, and dominates, the properties of ion flow, as demonstrated for one channel class. We now show that the genomic DNA sequences encoding the particular channel segment of all subunits harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in mRNAs of three subunits. Multiple genes and alternative exons were excluded as sources for the arginine codon; hence, we propose that transcripts for three subunits are altered by RNA editing. This process apparently edits subunit transcripts of the two glutamate receptor classes with different efficiency and selectivity. PMID- 1717159 TI - CD62/P-selectin recognition of myeloid and tumor cell sulfatides. AB - CD62, also called PADGEM protein, GMP-140, or P-selectin, is a granule membrane protein of endothelial cells and platelets that is mobilized to the plasma membrane following exposure to mediators such as thrombin, histamine, complement components, or peroxides. Data presented to date suggest that one ligand of CD62 includes CD15 (Lewis x determinant) and sialic acid. We show here that sulfatides, heterogeneous 3-sulfated galactosyl ceramides, are an apparently unrelated ligand of CD62. Sulfatides are expressed on the plasma membrane of, and are excreted by, granulocytes, and constitute the principal ligand for CD62 on the plasma membrane of some tumor cells. CD62 binds to sulfatides adsorbed to plastic as avidly as it binds to myeloid or tumor cells. We find that granulocytes excrete sulfatides at a rate predicted to allow them to be rapidly released from CD62 once they have exited the bloodstream. PMID- 1717160 TI - Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. AB - The Brachyury (T) gene is required for mesoderm formation in the mouse. In this paper we describe the cloning and expression of a Xenopus homolog of Brachyury, Xbra. As with Brachyury in the mouse, Xbra is expressed in presumptive mesodermal cells around the blastopore, and then in the notochord. We show that expression of Xbra occurs as a result of mesoderm induction in Xenopus, both in response to the natural signal and in response to the mesoderm-inducing factors activin A and basic FGF. Expression of Xbra in response to these factors is rapid, and will occur in dispersed cells and in the presence of a protein synthesis inhibitor, indicating that this is an "immediate-early" response to mesoderm induction. PMID- 1717161 TI - Selectins: a family of adhesion receptors. PMID- 1717162 TI - Cationization of protein antigens. VI. Effects of cationization on the immunoregulatory properties of a bovine serum albumin peptide, a.a. 506-589. AB - Cationization of bovine serum albumin (BSA) causes a profound increase in its immunogenicity. To establish if immunoregulatory properties of an immunosuppressive peptide are affected by cationization, a BSA peptide, a.a. 506 583, was cationized and tested for its immunogenic properties. A greatly reduced amount of cationized peptide compared to native peptide was required to stimulate BSA-primed T cells to proliferate in vitro. Mice primed with the cationized peptide administered with an adjuvant responded with a significantly greater anti BSA response than mice immunized with the native form of the peptide. In the absence of an adjuvant i.v. or i.p. administration of the native peptide was immunosuppressive, while the cationized form was immunoenhancing. Both forms of the peptide stimulated in vivo induction of L3T4+ (CD4), and Lyt-2+ (CD8) T cells. Removal of Lyt-2+ T cells from lymph node cultures following immunization with the native peptide caused a significant increase in the proliferation of the remaining T cells. This increase was not observed when the mice were immunized with the cationized peptide. No major BSA B cell determinants were present within the peptide sequence. Mice immunized with the peptide exhibited a negligible anti BSA antibody response compared to those immunized with the whole BSA molecule. Furthermore, the peptide did not inhibit anti-BSA antibody binding to BSA. We demonstrated that cationization modifies immunoregulatory properties of an immunosuppressive BSA-derived peptide. PMID- 1717163 TI - Antibodies to different members of the beta 1 (CD29) integrins induce homotypic and heterotypic cellular aggregation. AB - Members of the beta 1 (CD29) integrin family are involved in cellular adhesion to extracellular matrix. However, there have been several reports of CD29 integrin participation in intercellular adhesion. For example, the treatment of the human T cell line Jurkat with antibodies to alpha 4 (CD49d) causes homotypic aggregation. The present report describes the induction of aggregation of Jurkat cells by antibodies to alpha 5 (CD49e) and to a lesser extent by antibodies to the common beta 1/CD29 chain of these integrins. The metabolic requirements for these aggregations are compared with that of the CD49d-induced process. The possible involvement of fibronectin in the cytoadhesion appears to be unlikely as (1) the aggregates form in the absence of plasma fibronectin; (2) antibodies to fibronectin do not inhibit the cell adhesion; and (3) exogenous fibronectin does not influence the process. One of the cognate partners involved in the CD49e induced aggregation appears to be present on non-antibody-treated Jurkat cells as the cells are coincorporated into aggregates of anti-CD49e-stimulated cells. The adhesion does not appear to involve members of the CD2, CD3, CD4, or CD18 receptor groups. These results indicate that interaction with alpha 4, alpha 5 chain or the beta 1 chain of the CD29 integrins leads to the induction of intercellular adhesion. The possible biological significance of this process is discussed. PMID- 1717165 TI - Regulation of murine IgE responses: induction of long-lived inhibition of allergen-specific responses is genetically restricted. AB - We previously reported that administration of high Mr glutaraldehyde-polymerized ovalbumin (OA-POL) 10 days prior to OA Al(OH)3 immunization results in 85 to 99% inhibition of primary and secondary anti-OA IgE responses and 10(2)- to 10(4) fold increases in OA-specific IgG2a production in each of 14 inbred and 1 outbred murine strains tested. Administration of unmodified OA under the same conditions fails to inhibit IgE synthesis and yields only minor increases in IgG2a production. In the present report, the genetic restrictions placed on the capacity of this modified allergen to elicit long-lived reciprocal regulation of specific IgE and IgG2a responses were examined. Virtually permanent, antigen specific inhibition of IgE production (greater than 90% for greater than 22 months) was elicited in C57B1/6 mice following administration of a single course of OA-POL. This inhibition was paralleled by substantial (250-fold) increases in specific IgG2a production and was dependent on the activity of extremely long lived regulatory CD4 T cells. In contrast, BALB/c mice failed to maintain an IgE unresponsive state beyond 10-12 weeks and exhibited transient and less intense increases in IgG2a production. Examination of MHC and Igh congenic strains revealed that the induction of long-term split tolerance by these modified allergens is under multigenic control and is not solely attributable to MHC, Igh, or background genes. PMID- 1717164 TI - Monocyte-induced down-modulation of CD16 and CD56 antigens on human natural killer cells and its regulation by histamine H2-receptors. AB - Human natural killer (NK) cells carry CD16/FcR and CD56 cell-surface Ag but lack the T-cell marker CD3. Here we show that incubation of resting human NK cells with CD3-/16+/56+ phenotype with autologous monocytes induced the disappearance of CD16 and CD56 cell-surface Ag on NK-cells but did not affect CD2 or CD3 Ag expression on T-cells. Monocyte-induced down-modulation of NK-cell-surface Ag was cell-contact dependent and induced only by freshly isolated monocytes, recovered from peripheral blood by counter-current centrifugal elutriation. Adherence of monocytes abrogated the capacity to induce down-modulation of NK-cell-surface Ag. The biogenic amine histamine dose-dependently reversed the monocyte-induced down modulation of CD16 and CD56 on CD3- NK-cells. The effect of histamine was mediated by H2-type receptors on monocytes. The data presented are suggestive of a cell-cell-mediated interaction between monocytes and NK-cells which modulates surface expression of NK-cell Ag and its histaminergic regulation. PMID- 1717166 TI - Multiple cytokine interactions regulate Ly-6E antigen expression: cooperative Ly 6E induction by IFNs, TNF, and IL-1 in a T cell lymphoma and in its induction deficient variants. AB - The cell surface Ly-6E antigen, known to play a role in T cell activation, is up regulated by IFNs. In the present study, we investigated the possible interactions between IFNs and other cytokines in this regulation. As a model system, we used the YAC T cell lymphoma, in which Ly-6E is normally absent but can be highly induced both at the mRNA and surface protein levels by IFN-gamma or IFN-alpha/beta. The combination of the two IFNs was found to result in markedly synergistic Ly-6E induction in this cell line. Moreover, mutants of YAC cells were isolated that did not respond to the Ly-6E-inducing action of IFN-gamma or IFN-alpha/beta alone but did respond to their combination. Such a synergistic interaction is consistent with the notion that the two IFN types utilize different intracellular mechanisms to induce Ly-6E expression. Ly-6E induction mediated by IFN-gamma or IFN-alpha/beta was also enhanced by cotreatment with TNF alpha or IL-1 alpha, which by themselves had no detectable Ly-6E-inducing effect. These two cytokines similarly synergized with IFNs to trigger a response in several Ly-6E-induction-deficient mutants. However, their action could be dissociated in one mutant (B54) where the response to IFN-alpha/beta was enhanced by TNF-alpha, but not by IL-1 alpha. Altogether, these data indicate that Ly-6E antigen expression is regulated by the interaction of several inflammatory cytokines, which may provide a mechanism for the local modulation of T cell activation. The YAC cell mutants described here should facilitate further analysis of the molecular bases of Ly-6E regulation. PMID- 1717167 TI - Collagen-gel-embedded three-dimensional culture of human thyroid epithelial cells: comparison between the floating sandwich method and the dispersed embedding method. AB - Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells. PMID- 1717169 TI - Amphiphile-induced antihaemolysis is not causally related to shape changes and vesiculation. AB - A wide variety of structurally different antihaemolytic amphiphiles were tested for their ability to induce exovesiculation (acetylcholinesterase (AChE) release, transmission electron microscopic (TEM) studies), endovesiculation (fluorescein isothiocyanate conjugated dextran (FITC-dextran) internalization, TEM studies) and shape changes in human erythrocytes at concentrations where they exert maximum protection against hypotonic haemolysis. The results show that vesiculation is a common phenomenon induced by amphiphiles in erythrocytes. Sphero-echinocytogenic amphiphiles induced exovesiculation, whereas stomatocytogenic amphiphiles induced endovesiculation. The antihaemolytic potency of the amphiphiles was not related to their ability to induce exo- or endovesiculation, or to the type or extent of shape changes induced, and it could not be ascribed to any molecular feature of the amphiphiles or to their charge. It is proposed that amphiphiles, when intercalated into the lipid bilayer of the membrane, rapidly induce rearrangements within the bilayer and that these rearrangements are associated with an increase in the permeability of the membrane; it is suggested that a rapid efflux of ions decreases the difference in osmotic pressure between cell interior and hypotonic buffer, thereby protecting cells from being lysed. PMID- 1717168 TI - Repression of integrin beta 1 subunit expression by antisense RNA. AB - A quail cell line (QT6-c) was co-transfected with pTEX vector expressing RNA complementary to chicken integrin beta 1 subunit mRNA (Anti-Int) and pRSVneo vector by a calcium phosphate method. Transfectants showing reduced expression of quail integrin beta 1 subunit were selected with an immunoblot assay, and a few positive clones were examined in detail. Northern blot and immunoblot analyses revealed that the Anti-Int caused a clear reduction of the transcript encoding integrin beta 1 subunit depending on culture conditions. The number of cell surface integrins also decreased in proportion to the decrement of the total amount of integrin beta 1 subunits. When one transfectant (QA23) was cultured in a serum-free medium, cell shape changed from fibroblast-like to neuron-like morphology accompanied by a low growth rate, and the cells did not form focal contact on fibronectin. A similar morphological change occurred in QT6-c cells when the cells were infected with Rous Sarcoma virus, which could produce the Anti-Int. The QA23 cells did not attach to fibronectin as efficiently as did the original QT6-c cells. These data suggest that reduced expression of integrin beta 1 subunit affects cell growth as well as cell morphology by disordering the interaction between integrins and matrix proteins and/or cytoplasmic proteins. PMID- 1717170 TI - [Body language analysis and nursing care of aphasia patients]. PMID- 1717171 TI - Structure, function and biological properties of integrin alpha v beta 3 on human melanoma cells. AB - Human melanoma represents one of the most metastatic cancers in man. The capacity of melanoma cells to invade a variety of tissues and extracellular matrices is, in part, due to their repertoire of adhesion receptors. To this end, human melanoma cells express multiple integrin cell adhesion receptors among these is the vitronectin receptor, alpha v beta 3. This adhesion receptor enables melanoma cells to attach to a wide variety of extracellular matrix components containing the sequence Arg-Gly-Asp. This review will focus on the biosynthetic, biochemical and biological properties of this receptor expressed on the surface of human melanoma cells. PMID- 1717172 TI - Pulmonary toxicity of the combination of bleomycin and peplomycin--an experimental study in rats. AB - Combination of the two drugs bleomycin (BLM) and peplomycin (PEP) may enhance their antineoplastic effects; however, it is not known as to whether this enhancement is accompanied by a concomitant increase in toxicity, especially toxic lung damage. A histologic and stereologic investigation was carried out to compare the pulmonary toxicity of BLM, PEP, and a combination of the two drugs in animals. A total of 180 female Wistar rats were randomly assigned to 4 groups: 45 animals were treated with daily i.p. injections of 4 mg/kg BLM, 45 rats received 2.5 mg/kg i.p. PEP daily, 45 animals were given i.p. injections of a combination of 2.5 mg/kg BLM and 1 mg/kg PEP, and 45 rats received 1 ml 0.9% NaCl solution. Histological examination of the lungs demonstrated varying degrees of exudative and fibrosing alveolitis in animals treated with BLM, PEP, and BLM-PEP. Stereological analysis revealed a significant thickening of the alveolar wall after 20-60 days and a significant decrease in the surface density of alveolar walls after 40-60 days in all treated groups. Both histological examination and stereological parameters indicated more pronounced inflammatory changes in the alveolar walls and a prior loss of alveolar surface after 20 and 30 days in animals receiving PEP and PEP-BLM as compared with those undergoing BLM treatment. After 40-60 days, during which time irreversible fibrotic changes prevailed, significant stereological differences between the three treated groups could not be detected. Thus, our experimental observations did not show any potentiation of the toxic pulmonary effect of BLM and PEP following their combined administration. PMID- 1717173 TI - The Sicilian gambit. A new approach to the classification of antiarrhythmic drugs based on their actions on arrhythmogenic mechanisms. Task Force of the Working Group on Arrhythmias of the European Society of Cardiology. AB - The Queen's Gambit is an opening move in chess that provides a variety of aggressive options to the player electing it. This report represents a similar gambit (the Sicilian Gambit) on the part of a group of basic and clinical investigators who met in Taormina, Sicily to consider the classification of antiarrhythmic drugs. Paramount to their considerations were 1) dissatisfaction with the options offered by existing classification systems for inspiring and directing research, development, and therapy, 2) the disarray in the field of antiarrhythmic drug development and testing in this post-Cardiac Arrhythmia Suppression Trial (CAST) era, and 3) the desire to provide an operational framework for consideration of antiarrhythmic drugs that will both encourage advancement and have the plasticity to grow as a result of the advances that occur. The multifaceted approach suggested is, like the title of the article, a gambit. It is an opening rather than a compendium and is intended to challenge thought and investigation rather than to resolve issues. The article incorporates first, a discussion of the shortcomings of the present system for drug classification; second, a review of the molecular targets on which drugs act (including channels and receptors); third, a consideration of the mechanisms responsible for arrhythmias, including the identification of "vulnerable parameter" that might be most accessible to drug effect; and finally, clinical considerations with respect to antiarrhythmic drugs. Information relating to the various levels of information is correlated across categories (i.e., clinical arrhythmias, cellular mechanisms, and molecular targets), and a "spread sheet" approach to antiarrhythmic action is presented that considers each drug as a unit, with similarities to and dissimilarities from other drugs being highlighted. A complete reference list for this work would require as many pages as the text itself. For this reason, referencing is selective and incomplete. It is designed, in fact, to provide sufficient background information to give the interested reader a starting frame of reference rather than to recognize the complete body of literature that is the basis for this article. PMID- 1717174 TI - [An experimental study on acupoint neiguan-heart short reflex]. AB - It is reported that the dichotomizing afferent fibers supply both the pericardium and the brachium in rat. This finding provides a possible morphological explanation for short reflex which transmits messages only through the connection of peripheral nerve dichotomization. The animals utilized in this study were 15 male or female wistar rats (235-360 g). Rats were anesthetized with 26% urethane (100mg/100g b. wt, i.p.) and then, acute myocardial ischemia (AMI) was produced by pituitrin (1.0-1.5 u/a rat, i.M.). Following anesthetization and AMI, The I lead of electrocardiogram (ECG) was recorded, the superficial electrical resistance and cutaneous temperature of the acupoint Neiguan and Zusali were measured. In order to break off the long reflex which invokes some effects through central nervous system, the dorsal roots were removed of spinal cord segments cervical 6) C6-thoracic 1(T1). In this condition, the AMI can decrease in superficial electrical resistance and cutaneous temperature of the acupoint Neiguan (P less than 0.01). Acupuncture at Neiguan can also influence the superficial electrical resistance of the acupoint Neiguan, improve in the ECG, increase in the heart rate, but acupuncture at Zusali has no these effects. It suggests that acupoint Neiguan and heart can be connected through a short reflex. PMID- 1717175 TI - [Effect of intracerebral injection of substance P on serotonin contents of several brain regions and its relation with pain threshold, electroacupuncture analgesia in rats]. AB - The purpose of this study is to observe the effect of intracerebroventricular (icv) and intra-PAG injection of substance P (SP) on serotonin (5-HT) contents of hypothalamus, hippocampus, striatum and its relation with the change of pain threshold, electroacupuncture (EA) analgesia. The results were as follows: (1) After icv injection of SP, the pain threshold and the 5-HT contents of hypothalamus, hippocampus were significantly increased. After depletion of the 5 HT contents in brain by pCPA, the inhibitor of 5-HT synthesis, the effect of SP on elevating pain threshold and the 5-HT contents of hypothalamus, hippocampus were markedly attenuated, bud did not prevent the analgesic effect of SP (2) The pain threshold and the 5-HT contents of hypothalamus, hippocampus were dose dependently increased by intra-PAG injection of SP. (3) The intra-PAG injection of SP introduced simultaneously with high or low frequency EA did not affect the change of 5-HT contents of three brain regions, but caused a more marked elevation of pain threshold. These results suggest that the serotoninergic system may be activated by PAG for the mediation of SP induced analgesia. There is a synergic action of analgesia between the effects produced by intra-PAG injection of SP and those by high or low frequency EA. PMID- 1717176 TI - Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain. AB - In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured, aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation. PMID- 1717177 TI - Neonatal exposure to substance P alters behavioral and substance P levels in the central nervous system of the adult rat. AB - Substance P (SP) administered subcutaneously to male and female rats during a neonatal period (days 1-7 after birth), produced long-term effects. Thermal/pain perception and elements of both male and female copulatory behavior were altered. A significant increase in the SP level in the dorsal part of the spinal cord was demonstrated by radioimmunoassay and by micro-fluorescence. The present study indicates that exposure to SP during the neonatal period, when the role of SP in transmission is likely to be established, has biochemical and functional consequences for SP systems in the adult. PMID- 1717178 TI - Keratins in the developing olfactory epithelia. AB - At embryonic day 14, the supporting cells of the olfactory epithelium already contained tonofilaments which terminated in the desmosomes, and were stained by antikeratin antibodies of RGE53 and MA902, indicating the presence of 45 and 52.5 kDa keratins. The basal cells were identified at postnatal day 1 by the appearance of a few filaments, and stained by PKK2 antikeratin antibody which reacts with 40, 46, 48, and 54 kDa keratins, and by CKB1 antikeratin antibody which reacts with 50 kDa keratin. At postnatal day 14, the basal cells possessed densely aggregated bundles of filaments and reacted with KL1 and MA902 antikeratin antibodies, indicating the appearance of 56 and 52.5 kDa keratins. The basal cells showed a columnar or pyramidal shape changing into a flat shape during postnatal development. The olfactory cells remained unstained by antikeratin antibodies throughout their development. PMID- 1717179 TI - Interactions between meningeal cells and astrocytes in vivo and in vitro. AB - At the interface between the meninges and the central nervous system there is a characteristic structure known as the glia limitans, consisting of many fine interdigitating astrocyte processes which contain both GFAP and vimentin, and a basal lamina. A similar structure is set up after brain injury where meningeal cells invade the lesion. We have experimentally put astrocytes and meningeal cells in contact with one another, both in vivo and in vitro, to see whether this results in the formation of a glia limitans. Cultured meningeal cells were injected into the hippocampus of adult rats, and from 1 to 12 weeks later brains were stained were stained for GFAP and vimentin. One week after injection there was a widespread astrocytic reaction stretching up to 2 mm from the injection, the cells being stained intensely for both GFAP and vimentin. Over the next 4-6 weeks this widespread reaction subsided, the only remaining vimentin stained astrocytes, apart from those at the normal glia limitans, being in contact with the injected meningeal cells, or with meningeal cells which had migrated into the injection needle track. In vitro a structure reminiscent of the glia limitans formed where patches of astrocytes abutted meningeal cells; the astrocytes formed a layer of fine interdigitating processes all running parallel to the interface between the two cell types, and there was heavy staining for laminin and fibronectin. We conclude that a glia limitans forms wherever astrocytes and meningeal cells come into contact. PMID- 1717180 TI - Substance P-like-immunoreactive sensory neurons in dorsal root ganglia of the chick embryo: ontogenesis and influence of peripheral targets. AB - The expression of substance P (SP) was studied in sensory neurons of developing chick lumbosacral dorsal root ganglia (DRG) by using a mixture of periodic acid, lysine and paraformaldehyde as fixative and a monoclonal antibody for SP-like immunostaining. The first SP-like-immunoreactive DRG cells appeared first at E5, then rapidly increased in number to reach a peak (88% of ganglion cells) at E8, and finally declined (59% at E12, 51% after hatching). The fall of the SP-like positive DRG cells resulted from two concomitant events affecting a subset of small B-neurons: a loss of neuronal SP-like immunoreactivity and cell death. After one hindlimb resection at an early (E6) or late (E12) stage of development (that is before or after establishment of peripheral connections), the DRG were examined 6 days later. In both cases, a drastic neuronal death occurred in the ispilateral DRG. However, the resection at E6 did not change the percentage of SP like-positive neurons, while the resection at E12 severely reduced the proportion of SP-like-immunoreactive DRG cells (25%). In conclusion, connections established between DRG and peripheral target tissues not only promote the survival of sensory neurons, but also control the maintenance of SP-like-expression. Factors issued from innervated targets such as NGF would support the survival of SP expressing DRG cells and enhance their SP content while other factors present in skeletal muscle or skin would hinder SP expression and therefore lower SP levels in a subset of primary sensory neurons. PMID- 1717181 TI - Resistance of the immature hippocampus to seizure-induced synaptic reorganization. AB - Temporal lobe epilepsy is a common form of epilepsy in human adults and is associated with a unique pattern of damage in the hippocampus. The damage includes cell loss of the CA3 and CA4 areas and synaptic growth (sprouting) of mossy fibers in the supragranular layer of the dentate gyrus. Experimental evidence indicates that in adult rats the excitatory amino acid, kainic acid, induces a similar pattern of changes in hippocampal circuitry associated with alterations in perforant path excitation and inhibition. It has been suggested that, in humans, this type of damage may be a result of seizures early in life. In this study we examined the effects of kainic acid-induced status epilepticus on synaptic reorganization and paired-pulse electrophysiology in developing rats and adults. Kainic acid induced more severe seizures in 15-day-old rat pups than in adults. In contrast to adult rats, these seizures did not produce CA3/CA4 neuronal loss, mossy fiber sprouting or changes in paired-pulse excitation or inhibition in the hippocampus of rat pups tested 2-4 weeks after status epilepticus. Our results provide evidence that the immature hippocampus may be more resistant to seizure-induced changes than the mature hippocampus. PMID- 1717182 TI - SMI-32 immunoreactivity in human striate cortex during postnatal development. AB - SMI-32, an antibody which recognizes the non-phosphorylated epitopes on the neurofilament proteins was used to study the morphological changes in the human striate cortex during postnatal development. Striate cortices from 12 autopsied patients with ages ranging from 1 day to 70 years were obtained. Using the avidin biotin-peroxidase method, the first SMI-32 immunoreactive neurons were identified at sublayers Vb/VIa on the first postnatal day. At 5 months, the next group of neurons to develop immunoreactivity were in IVb. By 15 months, SMI-32 immunoreactive neurons were observed at III, IVa, IVb, V and VI. The changes in SMI-32 immunoreactivity (ir) were stabilized from 3 years and after. The SMI-32 ir in the striate cortex could be a useful morphological correlate for studying developmental diseases affecting the neocortex. PMID- 1717183 TI - Effects of dexamethasone on the expression of myelin basic protein, proteolipid protein, and glial fibrillary acidic protein genes in developing rat brain. AB - Effects of dexamethasone (DEX) on the relative abundance of myelin basic protein (MBP), proteolipid protein (PLP) and glial fibrillary acidic protein (GFAP) mRNAs in the developing rat brain were examined. After DEX (1.0 mg/kg body weight) or saline was administered intraperitoneally to 3-day-old rats for 7 consecutive days, wet weight, DNA content and the relative abundance of the glia-specific mRNAs in cerebrum and cerebellum were analyzed at postnatal days (P) 10, 20 and 30. DEX decreased both wet weight and DNA content in cerebellum more profoundly than in cerebrum. The appearance of MBP, PLP and GFAP mRNAs in cerebellum preceded that in cerebrum in the control group. In cerebrum, the relative abundance of MBP and PLP mRNAs was significantly less in the DEX group than that in the control group at P20 and P30. The relative abundance of the GFAP mRNA was significantly less in the DEX group than in the control group at P10 and P20, but there was no significant difference at P30. In cerebellum, a significant decrease in the abundance of MBP, PLP and GFAP mRNAs in the DEX group was observed only at P10 but not at P20 and P30. Our findings indicate that DEX suppresses expression of genes related to glial functions, especially myelination when administered in the early postnatal period. PMID- 1717184 TI - Formation of the cochlea in the chicken embryo: sequence of innervation and localization of basal lamina-associated molecules. AB - The purpose of this study was to determine the temporal and spatial gradients in the innervation of the chicken cochlea, the basilar papilla, as it develops in the early embryo. A series of white Leghorn chick embryos (Hamburger-Hamilton Stage 20-43) were prepared for serial sectioning and stained by Toluidine blue or by antibodies to fibronectin or laminin. Light microscopic observations were made on the first fiber bundles to reach each region of the basilar papilla. There is a distinct temporo-spatial pattern of the ingrowth of fiber bundles to the developing basilar papilla. The primary pattern is not described as any simple linear gradient. Fiber ingrowth begins proximally, shifts to a distal and then to a mid-proximal region. The fiber ingrowth correlates temporally and spatially with disruption of fibronectin and laminin staining of the basal lamina where fiber bundles are penetrating. This pattern may reflect not only the sequence of fiber ingrowth but also the displacement of cells and fibers in the elongating basilar papilla, which grows as a result of a contemporaneous mitotic activity throughout the structure rather than progressing from one end to the other. PMID- 1717185 TI - In vitro and in vivo effects of hypertonic saline/dextran-70 on protein determinations in serum or plasma. PMID- 1717186 TI - Alpha 2 macroglobulin in healthy pregnant women and those with EPH gestosis. AB - The Authors determined alpha 2 macroglobulin in 50 healthy pregnant women and in 50 pregnant women with EPH gestosis, using the technique of nephelometry on the Immunochemistry Analyzer. The assays were done in blood serum of the mother and umbilical cord, urine and in the amniotic fluid of the affected patients. Serum values were expressed in gr/L, and amniotic fluid and urine values in mg/L. Significant reduction was found in the pregnant women with EPH gestosis in serum values of the mothers (healthy pregnant women 2.871 g/L +/- 1.015, with EPH gestosis 2.239 g/L +/- 1.323, p less than 0.05), and in the umbilical cord serum (healthy pregnant women 3.351 g/L +/- 0.859, affected ones 2.032 g/L +/- 0.693 g/L, p less than 0.01). The reduction was also noted in the urine values but was significant one. The importance of these determinations is presented in the discussion. PMID- 1717187 TI - New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response. AB - The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies. PMID- 1717188 TI - Identification of a putative shared epitope between Coxsackie virus B4 and alpha cardiac myosin heavy chain. AB - Molecular mimicry is an important postulated mechanism for autoimmunity in viral myocarditis. The 356-1 monoclonal antibody neutralizes Coxsackie virus B4 by binding to the VP1 protein and cross-reacts with mouse alpha cardiac myosin heavy chain. We used this monoclonal antibody to screen a lambda gt11 expression library made from CD-1 mouse hearts. Of the 48 positive plaques/10(6) recombinant phages examined, 14 of the strongest-reacting clones were purified for additional studies. The inserts were amplified by polymerase chain reaction and the amplified products ranged from about 150 to 1400 bp in size. Northern hybridization using these inserts demonstrated that 11 out of 14 reacted with a message equivalent to that of cardiac myosin in size. Additional Southern hybridization studies suggested that these 11 inserts contained overlapping sequences in the light meromyosin fragment of cardiac myosin. Sequence analysis confirmed that these 11 independent, recombinant clones contained a common sequence representing amino acid residues 1299-1647. Within this fragment only one isoform-specific site matched the observed reactivity pattern of 356-1 among hearts from various species. Thus, we were able to identify a putative shared epitope represented by residues 1632-1647. PMID- 1717189 TI - T cell responses to synthetic thyroid peroxidase peptides in autoimmune thyroid disease. AB - Sixteen peptides, representing four different extracellular regions of thyroid peroxidase (TPO) predicted to contain a high proportion of potential T cell epitopes, were synthesized to investigate which parts of this autoantigen may be targets for the T cell response in thyroid autoimmunity. Compared with 25 controls, peripheral blood T cells from 23-37% of 30 patients with Graves' disease or autoimmune hypothyroidism were stimulated significantly by three peptides, representing amino acids 415-432, 439-457 and 463-481 of the TPO sequence; T cells from individual patients were also stimulated by several other peptides. These results indicate that the T cell response to TPO is directed against several epitopes which may be recognized by different patients. PMID- 1717190 TI - Murine and human B cell epitope mapping of the Mycobacterium tuberculosis 10-kD heat shock protein using overlapping peptides. AB - The human immune response to the 10-kD M. tuberculosis protein was studied by a competition ELISA using monoclonal antibody (MoAb) SA-12. Twenty-five per cent of the sera from 20 patients with tuberculosis and none from 21 control subjects inhibited binding of SA-12 to the 10-kD antigen. To characterize the antigenic parts of the 10-kD antigen, overlapping decapeptides according to the amino acid sequence of the 10-kD protein were synthesized. In total, 91 sequential decapeptides, with an overlap of nine amino acids, were tested in ELISA with MoAb SA-12, human and murine sera (PEP scan). SA-12 recognized the amino acid sequence WDEDGEK (amino acid 50-56). All human sera, from patients with tuberculosis as well as from control subjects, gave almost identical undulating patterns of reactivity with the decapeptides. No relationship was found between the ability of the patients' sera to inhibit binding of MoAb SA-12 and the binding of these sera to the decapeptides comprising the epitope recognized by SA-12 in the PEP scan. Apparently, antibodies in patients' sera against the 10-kD protein are predominantly directed against discontinuous epitopes and, consequently, the continuous epitopes as presented in the PEP scan are not suitable to discriminate between patients with tuberculosis and control subjects. In the PEP scan, sera from BALB/c mice, both non-immunized and immunized with either live M. tuberculosis or the 10-kD protein gave similar patterns of reactivity, albeit different from the patterns obtained with the human sera. However, after immunization of the mice, clearly increased levels of antibodies to primary structures of the 10-kD protein were observed. PMID- 1717191 TI - Analysis of antibodies to RNA in patients with systemic lupus erythematosus and other autoimmune rheumatic diseases. AB - The frequency and clinical associations of anti-RNA antibodies measured by ELISA were assessed in 138 patients with systemic lupus erythematosus (SLE). Of the sera from these patients 9.4% had anti-RNA antibodies but no distinguishing features, clinical, serological or immunogenetic, between those with or without these antibodies could be identified. However, investigations of patients with other autoimmune rheumatic diseases did not reveal any anti-RNA positivity, which indicates a marked disease specificity for anti-RNA antibodies in SLE. The initial anti-RNA antibody screen used a soluble yeast extract as test antigen. The positive sera were further tested against a range of RNAs from 10 different types of rat tissue. In essence few differences were observed, suggesting that the anti-RNA response is directed against common, highly conserved epitopes. PMID- 1717192 TI - Mapping of epitopes on U1 snRNP polypeptide A with synthetic peptides and autoimmune sera. AB - The ability of synthetic peptides encompassing almost the entire sequence of snRNP U1A polypeptide to be recognized in ELISA by sera of autoimmune patients was investigated. Sera from 18 patients with mixed connective tissue disease (MCTD), 145 with systemic lupus erythematosus (SLE) and 120 with other rheumatic autoimmune diseases were tested with 13 overlapping peptides. Among them, peptide 257-282 and, to a lower extent, peptide 1-11 were recognized by MCTD, SLE and Sjogren's syndrome sera. In contrast, peptide 35-58 was recognized by 94% of MCTD and only 19% of SLE sera. It did not react with any of the other patient sera. The ELISA results were compared with the pattern of reactivity observed in immunoblotting. The results indicate that peptide 35-58 probably contains a major epitope recognized by MCTD autoantibodies. It is noteworthy that in snRNP particles, this region of U1A interacts with RNA and presents only limited homology with the corresponding sequence 32-50 of U2B''. PMID- 1717193 TI - Retinoic acid potentiates interleukin-1- and fibroblast growth factor-induced human synovial fibroblast proliferation. AB - All trans-retinoic acid (ATRA) and related compounds, at concentrations ranging from 10(-8) to 10(-6) M, augmented the proliferation of human synovial fibroblasts (HSN) stimulated by human interleukin-1 alpha or -beta (IL-1 alpha, IL-beta) and both the acidic and basic forms of fibroblast growth factor (FGFa, FGFb). In contrast, ATRA had no effect on human tumor necrosis factor alpha (TNF alpha)-induced HSN proliferation. The potentiation of HSN proliferation was completely dependent on the presence of IL-1 or FGF since HSN were unresponsive to ATRA alone. The mechanism by which ATRA enhances IL-1-induced HSN proliferation does not appear mediated by changes in the affinity or number of IL 1 receptors expressed by HSN; however, treatment with dexamethasone (DEX, 10( 6)M) resulted in a twofold increase in IL-1 receptor number. ATRA inhibited both IL-1 beta- and TNF alpha-induced secretion of prostaglandin-E2 (PGE2), a potent feedback inhibitor of cytokine-stimulated HSN proliferation. However, the synergistic effect of ATRA on IL-1- or FGF-induced proliferation did not appear related to the secretion of cyclooxygenase products since ATRA had no effect on TNF alpha-induced HSN proliferation and indomethacin was included in all HSN proliferation experiments. The results of this study suggest that ATRA may contribute to the pathology of chronic arthritic disease by potentially causing increased growth of the joint-destroying pannus. PMID- 1717194 TI - [The distribution of senile plaques and acetylcholinesterase staining in the thalamus in dementia of Alzheimer type]. AB - Recent development of histochemical techniques has demonstrated a significant cholinergic projection from the basal forebrain to the mediodorsal and reticular thalamic nuclei. To determine whether the regional distribution of senile plaques is related to the pattern of cholinergic innervation, we studied the distribution of plaques and changes in acetylcholinesterase (AChE) reactivity in the thalamus of patients with dementia of Alzheimer type (DAT). Brains from 2 age-matched patients without neurologic or psychiatric diseases were used as controls. Eight patients with DAT could be divided to 3 groups according to the distributional pattern of plaques; scarcely distributed, localized and diffusely distributed groups. In general, plaques were preferentially distributed in such subnuclei closely related to the cerebral cortex as anterior, intralaminar, mediodorsal nuclei and posterolateral-pulvinar nuclear complex, rather than in the region that receives projections from the basal forebrain. In addition, the majority of plaques exhibited AChE reactivity, while plaques were less common in the region showing the most prominent AChE reactivity in the thalamus of control cases. The present results provide an evidence against the cholinergic hypothesis of plaque formation and indicate an active involvement of AChE in plaque formation. PMID- 1717195 TI - Pharmacokinetics of rectal drug administration, Part I. General considerations and clinical applications of centrally acting drugs. AB - Generally, oral administration is the route of choice in the daily practice of pharmacotherapy. However, in some circumstances this is impractical or even impossible (during nausea and vomiting or convulsions, in uncooperative patients and before surgery). In these cases, the rectal route may represent a practical alternative and rectal administration is now well accepted for delivering, for example, anticonvulsants, non-narcotic and narcotic analgesics, theophylline, antiemetics and antibacterial agents, and for inducing anaesthesia in children. It may also represent an interesting alternative to intravenous or other injection routes of drug administration. The rate and extent of rectal drug absorption are often lower than with oral absorption, possibly an inherent factor owing to the relatively small surface area available for drug uptake. In addition, the composition of the rectal formulation (solid vs liquid, nature of the suppository base) appears to be an important factor in the absorption process by determining the pattern of drug release. This relation between formulation and drug uptake has been clearly demonstrated for drugs like diazepam, paracetamol (acetaminophen), indomethacin, methadone and diflunisal. Coadministration of absorption-promoting agents (surfactants, sodium salicylate, enamines) represents another approach towards manipulating rectal drug absorption, although this concept requires further research concerning both efficacy and safety. For a number of drugs the extent of rectal absorption has been reported to exceed oral values, which may reflect partial avoidance of hepatic first-pass metabolism after rectal delivery. This phenomenon has been reported for morphine, metoclopramide, ergotamine, lidocaine (lignocaine) and propranolol. Rectal drug delivery in a site- and rate-controlled manner using osmotic pumps or hydrogel formulations may provide opportunities for manipulating systemic drug concentrations and drug effects. The extent of first-pass metabolism may be influenced (lidocaine), depending on the site of drug administration in the rectum. The rate of delivery may determine systemic drug action and side effects (nifedipine), and it may affect the local action of concurrently administered absorption promoters on drug uptake (cefoxitin). Local irritation is increasingly being acknowledged as a possible complication of rectal drug therapy. Long term medication with rectal ergotamine and acetylsalicylic acid, for example, may result in rectal ulceration, and irritation after a single administration of several drugs and formulations has been described. The assessment of tolerability and safety is imperative in the design of rectal formulations. Recent studies corroborate the clinical relevance of rectal drug therapy, and the value of the rectal route as an alternative to parenteral administration has been assessed for several drugs, e.g. diazepam, midazolam, morphine and diclofenac.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717196 TI - Plasma ascorbate levels and inhibition of the antioxidant activity of caeruloplasmin. AB - 1. The copper-containing protein caeruloplasmin has several oxidase activities. 2. Its ability to catalyse the oxidation of ferrous ions to the ferric state (ferroxidase activity) makes it an important antioxidant in vivo. 3. Recent reports have suggested that oral supplementation with vitamin C can inhibit the oxidase activities of caeruloplasmin. 4. As expected, damage to DNA and membrane lipids was stimulated by mixtures of iron salt and ascorbate, and this damage could be inhibited by caeruloplasmin provided the molar ratio of ascorbate to caeruloplasmin was kept sufficiently low. 5. When the molar ratio of ascorbate to caeruloplasmin was greater than 200 substantial loss of ferroxidase antioxidant activity occurred. 6. It is unlikely, however, that oral supplementation with vitamin C can raise plasma levels sufficiently to inhibit caeruloplasmin activity in vivo. PMID- 1717197 TI - Selectivity of ionic channels: as seen through computer simulation. AB - A theoretical approach has been attempted to study the selectivity of ionic channels in membranes. We predict the channel to behave as an allosteric enzyme and have different conformational states that can bind strongly or weakly to a particular ion. The kinetic equation derived for the channel has few empirical parameters like the allostery factor, the probability factor, binding affinity factor and the transporting rate factor, the later two giving an idea of the ion ion interactions and ion-channel interactions. The equation is programmed for an IBM compatible personal computer in MS-FORTRAN and the simulation data has been analysed to explain selectivity of the channels to particular ion. The simulation results show that the ions smaller than the permeable ions tend to act as inhibitors, the amplitude depending on the concentrations of the ions and comparative transport rate of the ion in the channel. The program helps easy study of the different parameters on the conducting rate of the permeable ion through the channel which otherwise would demand intricate experimental set-ups. PMID- 1717198 TI - Modulation of serotonergic and adrenergic systems by long-term antihypertensive treatment with enalapril. AB - Enalapril (Renitec, Merck-Sharp-Dohne, Rahway, USA) administered alone (Group I), or in combination (Group II) in a dosage normalizing blood pressure (BP) was investigated for its ability to inhibit adrenergic and serotonergic systems in a one-year study. BP normalization was achieved. Serotonin (5-hydroxytryptamine, 5HT) levels in platelet rich and poor plasma, urinary excretion of 5HT, adrenaline and noradrenaline decreased. Correlations between the changes in BP and the inhibition of serotonergic and adrenergic systems were found in the acute phase in Group I. In chronic effect the reduction of BP correlated with inhibition of the serotonergic system in both groups. Changes in adrenergic and serotonergic systems correlated in the acute phase in both groups; during chronic treatment only in Group II. It is concluded that a concurrent antihypertensive effect and the inhibition of sympathetic activity and 5HT amplifying mechanism (antiatherogenic effect) are of clinical importance for hypertension treatment and prevention of its complications. PMID- 1717199 TI - FK506 treatment of S-antigen induced uveitis in primates. AB - FK506 is a new immunosuppressive agent which has been found more potent than cyclosporine based on the dosage. FK506 was examined here for its effect on the development of uveitis in primates immunized with S-antigen. FK506 successfully inhibited uveitis in monkeys, even when administered three weeks after the first immunization, at the time when the immunopathogenic mechanism of uveitis is assumed to be developed. All four monkeys injected with 0.5 mg/kg/day of FK506 did not develop uveitis, 2 out of 4 treated with the 0.25 mg and 3 out of 4 of those receiving the 0.125 mg also did not develop disease. FK506 suppressed to some extent the cellular and humoral immune responses to S-antigen. The main side effect of FK506 was weight loss. We consider that this drug may be considered as a new potential therapeutic agent for immune-mediated ocular disease in humans. PMID- 1717200 TI - Localization of acidic fibroblast growth factor in proliferative vitreoretinopathy membranes. AB - The pathogenesis of proliferative vitreoretinopathy (PVR) membranes remains poorly understood. We have studied the presence of acidic fibroblast growth factor (aFGF), a potent mitogen for many cells, within these membranes. We have used affinity purified monospecific anti-aFGF polyclonal antibodies, in conjunction with highly sensitive immunofluorescence techniques. The labelling was exclusively localized to cell bodies and was absent from the extracellular matrix. Double labelling techniques revealed that all cytokeratin positive cells (probably pigmented epithelial cells) and macrophages contained aFGF-like immunoreactivity, whilst glial cells were unlabelled. Appropriate controls indicated the specificity of the antibodies. Hence, the presence of this mitogenic molecule within certain cell types constituting PVR membranes may contribute to the pathogenesis. PMID- 1717201 TI - CD5 B cells in the mouse. PMID- 1717202 TI - Chromosome banding and gene localizations support extensive conservation of chromosome structure between cattle and sheep. AB - By using three gene probes, one derived from the porcine major histocompatibility complex (MHC) and two from bovine cytokeratin genes, type I (KRTA) and type II (KRTB), the hypothesis of conservation of genome structure in two members of the family Bovidae was examined. Gene mapping data revealed the MHC to be in chromosome region 23q15----q23 in cattle (BOLA) and 20q15----q23 in sheep (OLA). KRTA was localized to chromosome region 19q25----q29 in cattle and 11q25----q29 in sheep and KRTB to 5q14----q22 in cattle and 3q14----q22 in sheep. The banding patterns of the chromosome arms to which the loci were assigned were identical in both species. Moreover, the resemblances of GTG- or QFQ-banding patterns between the cattle and sheep karyotypes illustrated further chromosome homologies. These studies, based on gene mapping comparisons and comparative cytogenetics, document that within bovid chromosomes, homology of banding patterns corresponds to a homologous genetic structure. Hence, we propose that gene assignments on identified chromosomal segments in one species of the Bovidae can be extrapolated, in general, to other bovid species based on the banding homologies presented here. PMID- 1717203 TI - Mapping of bovine cytokeratin sequences to four different sites on three chromosomes. AB - The chromosomal location of bovine class I and class II cytokeratin sequences was determined using in situ hybridization and Southern blot hybridization to DNA from hybrid somatic cells. The main signals were found over chromosome region 19q16----qter after in situ hybridization with two probes for the class I cytokeratin gene subfamily (KRT10 and KRT19) and over region 5q14----q23 after hybridization with probes for the class II gene subfamily (KRT1, KRT5, and KRT8). These regions most likely contain the loci of functional cytokeratin genes, with KRT10 and KRT19 mapping to 19q21 and KRT1, KRT5, and KRT8 to 5q21. The in situ hybridization data were corroborated by analysis of a somatic hybrid cell panel. The genes for the class I keratins segregated concordantly with each other and syntenic group U21 but were discordant with the class II keratin genes. The class II keratin genes segregated concordantly with each other and syntenic group U3. Two class II gene probes gave an additional minor signal above chromosome region 5q25----q33 after in situ hybridization, while another class II probe yielded a minor signal above chromosome region 10q31----qter. When the latter probe and an additional linked probe were hybridized to DNAs from a hybrid panel, two independently segregating loci were recognized, one of which cosegregated with the class II subfamily in syntenic group U3 and the other with syntenic group U5. These data confirm the chromosomal assignment of two syntenic groups and allow the assignment of a formerly unassigned syntenic group. PMID- 1717204 TI - Strangers in a strange land: a psychobiological study of infant monkeys before and after separation from real or inanimate mothers. AB - Some rhesus monkey (Macaca mulatta) infants have a "despair" or depression-like response to mother-infant separation, while others do not. The presumed interrelation between early rearing conditions and the neurobiological status of the infant that might lead to increased risk for despair is not understood. In this study, the characteristics of the "mother" were controlled by rearing infant rhesus monkeys with their biological mothers, or with inanimate mothers. Behavioral data were collected before and after separation at 6-7 months of age. The neurobiological status of the infants was evaluated by measuring the concentration of norepinephrine, its major metabolite, and the metabolites of dopamine and serotonin in cerebrospinal fluid. The results suggest that despair is not simply a behavioral response to separation. Instead, despair may reflect the inability to cope with the separation environment. Coping with the separation environment appears to depend on neurobiological and behavioral characteristics of the infant that are related to, if not determined by, characteristics of the mother. PMID- 1717205 TI - [Hepatocellular carcinoma arising in the normal liver. A clinical study and long term prognosis after surgical resection in 12 patients]. AB - Hepatocellular carcinoma mainly affects patients with cirrhosis or with various degrees of fibrosis. From 1979 to 1990, among 87 patients who underwent hepatic resection for non fibrolamellar hepatocellular carcinoma, 12 (14%) had a non fibrolamellar hepatocellular carcinoma developed in a normal liver. There were 8 men and 4 women, aged 29 to 74 years. In 7 patients (58%) hepatocellular carcinoma was associated with clinical manifestations. Serum hepatitis B surface antigen were absent in all patients. Serum alphafetoprotein level was less than 100 ng/ml in 10 (83%), size of the tumor was greater than or equal to 5 cm in 10 (83%) and capsule was present in 10 (83%). Resections included removal of 2 segments or more in 11 (91%). One patient died postoperatively. Actuarial survival rate at 3 and 5 years were respectively 57% and 38%. Intra or extrahepatic recurrence was recognized in 8 (67%), 2 patients were alive respectively 28 and 16 months after treatment of their intrahepatic recurrence (resection in one and intraarterial embolisation in one). In conclusion, our results suggest that aggressive surgical efforts are justified in non fibrolamellar hepatocellular carcinoma arising in normal liver. PMID- 1717206 TI - [The effect of enriched branched chain amino acid solution with hypocaloric TPN on acute phase protein, plasma amino acid profile metabolism in postoperative children]. AB - From 1988 to 1990, 27 postoperative children with aganglionic megacolon were studied prospectively. They were randomly divided into three groups according to the postoperative fluid supply: group A, dextrose, group B, balanced amino acid solution (24% branch chain amino acid); and group C, solution enriched with branched chain amino acid (45% BCAA). The serum acute phase protein and plasma amino acid profile were observed. Nitrogen balance was monitored daily in all groups during the perioperative period. The data showed that prealbumin, transferrin rose more rapidly in group C than in group A and group B. The solution of 45% BCAA was found to be superior to balanced 24% BCAA in normalizing the disorder of plasma amino acid spectrum on the 3rd day and 7th day after operation. Urea nitrogen excretion was reduced in group C more rapidly than in group A and group B on the 4th day after operation. Using solution enriched branched chain amino acid with hypocaloric TPN in postoperative children can achieve an effective nitrogen sparing effect. PMID- 1717207 TI - [Local injection of bleomycin A 5 in children with hemangiomas. Analysis of 210 cases]. AB - 210 children with hemangiomas were treated by local injection of Bleomycin A5. Bleomycin A5 was effective in all patients with strawberry and mixed hemangiomas, 91.2% of patients with cavernous hemangiomas, 44.4% of patients with port-wine stain. There was no response in pampiniform hemangioma. The therapeutic mechanism, indications and complications of the new method for treatment of hemangioma are discussed. PMID- 1717208 TI - [The effect of Inj. Salviae Miltiorrhizae Co. on the retrograde axoplasmic transport in the optic nerve of rabbits with chronic IOP elevation]. AB - With the horseradish peroxidase histochemical technique and electron microscope, the effects of 0.5% timolol, Inj. Salviae Miltiorrhizae Co. and their combination on the retrograde axoplasmic transport in the optic nerve of 230 rabbit models of chronic IOP elevation were studied. The results showed that (1) the nerve damage in chronic glaucoma was due to multiple factors; (2) the use of a drug that improved microcirculation in combination with an IOP depressor better protected the optic nerve functions than did the latter alone; and (3) Inj. Salviae Miltiorrhizae Co. improved the optic nerve axoplasmic transport under chronic IOP elevation. The mechanism of the drug in protecting the optic nerve could be through its actions of improving the local microcirculation and the tolerance of nerve tissues to anoxia. PMID- 1717209 TI - Colorectal cancer in patients over 80 years of age. AB - Between January 1, 1973, and December 31, 1986, 1,734 patients underwent colorectal resections for carcinoma. Patients were divided into two groups: Group I included 163 patients aged greater than or equal to 80 years on first presentation; Group II comprised 1,571 patients aged less than 80 years. The total perioperative mortality rates for the elderly and young group were 15.3 percent and 5 percent, respectively (P less than 0.001). The surgical mortality rates after elective operations in Groups I and II were 7.4 and 4.5 percent, respectively, and were not statistically different. Emergency surgery was associated with a significantly higher incidence of perioperative deaths at any age (P less than 0.001). In the elderly group, most deaths (88 percent) resulted from complications of coexisting medical disorders or thromboembolic complications. The 5-year survival for the young and elderly group were 46.2 percent and 35 percent, respectively (P less than 0.05). However, excluding patients dying from nonmalignant disease, the 5-year survival rate did not differ significantly between the two groups of patients (49.5 percent vs. 41.2 percent). PMID- 1717210 TI - Restaging of colorectal cancer based on the identification of lymph node micrometastases through immunoperoxidase staining of CEA and cytokeratins. AB - The present study was performed to identify tumor cells in lymph nodes from colorectal adenocarcinomas considered free of disease by the classic hematoxylin eosin stain, based on the detection of the carcinoembryonic antigen (CEA) and cytokeratins in neoplastic epithelial cells. For this purpose, 603 lymph nodes from 46 lesions were stained by the peroxidase-antiperoxidase technique. Tumor cells were detected in 22 nodes from 12 patients, mainly in the subcapsular sinuses, permitting a restaging of these patients into two groups: those now considered to have metastatic disease and those free of metastases. However, the 5-year follow-up showed no statistical differences in survival between the two groups. PMID- 1717211 TI - En bloc resection of colon carcinoma adherent to other organs: an efficacious treatment? AB - From 1977 to 1984, 56 patients with colon cancer adherent to other organs were operated upon. Twenty-three (41 percent) underwent palliative treatment without resection. The mean survival in this group was 6 months. The results of en bloc resection were evaluated in 33 patients (59 percent) with colon carcinoma and tumor growth in adjacent organs. Pathologic staging was based on Dukes' (Astler and Coller) classification. Dukes' B carcinoma was shown in 15 patients. Dukes' C in 14 patients, and Dukes' D in four patients. The 4-year survival rate was as follows: Dukes' B, 47 percent; Dukes' C, 29 percent; and Dukes' D, 0 percent. The 4-year survival rate for the whole group was 33 percent. The postoperative morbidity and mortality were 6 percent and 3 percent, respectively. Colon cancer with involvement of adjacent structures should not be regarded as an incurable Dukes' D carcinoma; en bloc resection is indicated and can be performed with acceptable morbidity and mortality. PMID- 1717212 TI - Effects of cyclosporin A and FK506 on ischemia/reperfusion-induced neutrophil infiltration in the cat. AB - Isolated segments of cat small intestine were subjected to 3 hr of ischemia followed by 1 hr reperfusion (I/R). Mucosal biopsies were obtained for measurement of myeloperoxidase (MPO) activity, an index of tissue neutrophil count, and leukotriene B4 (LTB4) production. Animals were pretreated with either cyclosporin A (CsA) or FK506, which are potent immunosuppressants. Both agents significantly attenuated the neutrophil infiltration induced by I/R. FK506, but not CsA, reduced the elevated mucosal LTB4 production normally observed following reperfusion. The results of this study suggest that FK506 and CsA may be important agents in modulating neutrophil infiltration in acute inflammatory conditions. PMID- 1717213 TI - Modification of the pulmonary connective tissue developmental response in the neonatal rat by ciclosporin. AB - Neonatal rat pups were treated either with ciclosporin at 10 mg/kg/day dissolved in olive oil (experimental) or with pure olive oil (control). Lung protein biosynthesis was evaluated in a protocol which involved the measurement of total accumulated protein, collagen and elastin. Four time points were studied in the first 21 days of life, 12 animals contributing to each point (6 control and 6 ciclosporin). Ciclosporin levels in the treated group ranged widely (2,000-4,000 ng/ml). There were significant differences in total body weight and lung weight in treated vs. controls during and after the first week. DNA contents per unit wet weight varied significantly during the second week of life, indicating increased cellularity of the ciclosporin-treated animals. Associated with this was an increase in the lung protein/DNA ratio as well as the elastin/DNA ratio in the control animals, but not in the treated ones. The lung collagen/DNA ratio was not as dramatically affected by the ciclosporin treatment. However, the collagen content per unit wet weight of lung tissue was increased in the ciclosporin treated animals at 15 days of life. We conclude that ciclosporin has a marked effect on lung connective tissue metabolism in early life, the long-term effects of which are unappreciated and undocumented but may well be of vital importance in the lungs of long-surviving organ transplant patients. PMID- 1717214 TI - [Specific suppressor T-cells secrete RNA]. PMID- 1717215 TI - 1,1-Dichloroethylene elicits dose-dependent alterations in covalent binding and glutathione in murine liver. AB - Dose-response studies have been performed using the hepatotoxin 1,1 dichloroethylene (1,1-DCE) to investigate effects of its administration on covalent binding, content of reduced glutathione (GSH), and structural alterations in murine liver. Additionally, these studies have determined if the sites of cellular damage coincided preferentially with the sites of GSH depletion. Treatment with 1,1-DCE evoked dose-dependent alterations in both covalent binding and tissue GSH content. Progressive increases in covalent binding (2.8 +/- 0.8 to 12.5 +/- 1.9 nmol/mg protein) were found within the range of doses tested (75-225 mg/kg). Tissue GSH content (86.4 +/- 6.1 to 35.9 +/- 2.4 nmol/mg protein) declined significantly after 1,1-DCE treatment when compared to that in control mice (128.0 +/- 3.6 nmol/mg protein). Alterations in GSH content due to 1,1-DCE treatment included those associated with significant increases (34 81% of control) in blood volume. Concomitant increases in vascular GSH were observed, and at 225 mg/kg, GSH content comprised 190% of that found in controls. Histochemical staining for GSH exhibited dose-related decreases in staining intensities that were not concentrated in any particular region, but, rather, were uniformly distributed throughout the liver lobule. Liver injury involving centrilobular and midzonal hepatocytes was absent at 75 mg/kg, mild at 125 and 175 mg/kg, and was most severe at 225 mg/kg. The results from these experiments demonstrate dose-dependent relationships between the magnitude of covalent binding of 1,1-DCE metabolite(s), diminished levels of tissue GSH, and hepatocellular necrosis. PMID- 1717216 TI - Clinical pharmacology of 1-beta-D-arabinofuranosyl-5-azacytosine (fazarabine) following 72-hour infusion. AB - The clinical pharmacology of fazarabine (1-beta-D-arabinofuranosyl-5 azacytosine), a structural analogue of 1-beta-D-arabinofuranosylcytosine (ara-C) and 5-azacytidine, was assessed in 14 patients with various malignancies during a phase I trial. Since the starting dose for the protocol was low (0.2 mg/m2/hr over a 72-hr continuous iv infusion), a radioimmunoassay (RIA) using commercially available ara-C antibody and [3H]ara-C was developed to measure the anticipated low plasma drug levels. The assay could be used to measure fazarabine accurately in plasma and urine with a sensitivity of 0.08 ng/ml. The RIA does not require extraction of samples. Using both RIA and HPLC, similar results were obtained in plasma samples from a patient receiving a high dose (180 mg/m2/hr) of fazarabine. The assay is simple, sensitive, reproducible, and specific. Following the infusion, plasma levels declined triphasically with a terminal half-life of 5.7 +/- 2.0 hr. The AUC was linearly related to dose. When the various doses were normalized to 1.75 mg/m2/hr (the maximum tolerated dose as determined from the phase I trial) the mean AUC value was 4232 +/- 987 (ng/ml)hr. Plasma steady-state drug levels (CPss) were achieved in 2-4 hr and were linearly dependent to dose. Also, when normalized, the mean CPss was 58 +/- 13 ng/ml, which is within the reported concentration range necessary for inhibiting malignant cell growth. Total clearance was rapid, 528 +/- 138 ml/(m2.min), and not dose-related. PMID- 1717217 TI - Low dose chlordecone pretreatment altered cholesterol disposition without induction of cytochrome P-450. AB - Pretreatment of mice with low doses of chlordecone (CD) alters the pattern of distribution of a subsequent tracer dose of [14C]CD. We call this preexposure effect a pretreatment disposition response (PDR) and suggest that it reflects important cellular responses to lipophilic compounds. The present study examined three possible mechanisms for CD-induced PDR (CD-PDR). The first was that CD-PDR occurred with induction of the cytochrome P-450 system. A cumulative dose of 45 mg/kg CD caused a PDR, increased the content of cytochrome P-450, and elevated the activities of ethoxyresorufin- and ethoxycoumarin-O-deethylases (EROD and ECOD). A cumulative dose of 10 mg/kg caused a PDR, but did not affect cytochrome P-450, EROD, or ECOD, indicating that an induction of the cytochrome P-450 system in not necessary for PDR. A second possibility examined was that CD-PDR resulted because of an altered affinity of a subcellular fraction. Following a pretreatment regimen designed to produce PDR, amounts of [14C]CD in each fraction paralleled homogenate values in the liver and the kidney. However, when values were calculated as percentages of total label recovered, it was apparent that [14C]CD levels were higher in the microsomal fraction of the liver. Finally, the possibility that CD-PDR occurred because of an interaction of CD with proteins involved in cholesterol synthesis and transport was addressed. CD pretreatment increased disposition of a dose of [14C]cholesterol to the fat at the expense of [14C]cholesterol in the liver and kidney. PMID- 1717218 TI - Mouthwashes in the treatment of oral disease. PMID- 1717220 TI - Antacids and ulcer healing. A review of the evidence. AB - There have been a number of controlled trials of antacids in the treatment of patients with peptic ulcer disease. As a general rule the size of studies has been small and there have been difficulties ensuring adequate binding, because of the formulation and taste of the antacids. Despite these difficulties, antacids appear to be effective ulcer healing agents with efficacies resembling those of other antiulcer drugs. Definite dose relationships are unclear but high doses of buffering capacity over 200 mmol/day appear unnecessary and are associated with increasingly frequent adverse effects. Low dose maintenance treatment is effective at limiting duodenal ulcer relapse. PMID- 1717222 TI - Treatment principles for the use of opioids in pain of nonmalignant origin. AB - Inadequately treated acute and chronic pain remains a major cause of suffering, in spite of enormous advances in pharmacology and technology. Opioids provide a powerful, versatile, widely available means of managing this pain, but their use is too often restrained by ignorance and mistaken fears of addiction. The management of postoperative pain (perhaps the most common form of acute pain) is traditionally attempted with fixed dosages of analgesics by relatively unpredictable routes (e.g. oral, rectal and intramuscular). Intravenous opioid infusions (an improvement) risk respiratory depression and require close monitoring and titration. Patient-controlled analgesia (PCA), by contrast, permits the most efficacious medication (pure opioid agonist) by the optimal route (intravenous) under direct control of the patient, and provides high levels of satisfaction and safety. Ideally, any opioid use should be integrated with a wide spectrum of other analgesic modalities in an anaesthesiology-based 'acute pain service'. The use of opioids for chronic pain of nonmalignant origin remains controversial. There is a perceived conflict between patients' interests and those of society. However, problems (such as tolerance, physical dependence, addiction and chronic toxicity), anticipated from experience with animal experiments and pain-free abusers, seldom cause difficulties when opioids are used appropriately to treat pain (so-called 'dual pharmacology'). With sensible guidelines, and in the context of a multidisciplinary pain clinic, opioids may provide the only hope of relief to many sufferers of chronic pain. PMID- 1717221 TI - Recent developments in insulin delivery techniques. Current status and future potential. AB - Although single or multiple daily subcutaneous injections of insulin are the mainstay of insulin delivery techniques, several other methods of insulin delivery are now available or in development, including: (a) continuous subcutaneous insulin infusion by a wearable infusion pump; (b) total or segmental transplantation of a pancreas; (c) transplantation of isolated islet cells; (d) implantation of a programmable insulin pump; (e) oral, nasal, rectal and transdermal mechanisms of insulin delivery; (f) insulin analogues; (g) implantation of polymeric capsules which give continuous or time-pulsed release of insulin; and (h) implantation of a biohybrid artificial pancreas which uses encapsulated islets. Many of these methods of insulin delivery are aimed at achieving a more physiological means of delivery of the insulin, thus to improve glycaemic control and hopefully minimise the secondary complications of diabetes. Techniques of multiple insulin injections and continuous subcutaneous insulin infusion pumps are already in widespread use and are resulting in improved glycaemic control. With the recent increased use of pancreatic transplantation, the rule of establishing euglycaemia will be elucidated in the treatment and prevention of microvascular and macrovascular complications of diabetes mellitus. Despite these advances, the ideal delivery of insulin to patients has yet to be developed. Subcutaneous methods of insulin delivery do not precisely mimic physiological insulin needs and transplantation requires risky immunosuppression. However, the future does look bright as glucose sensors are developed and insulin analogues synthesised. PMID- 1717223 TI - Roxatidine acetate. A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic potential in peptic ulcer disease and related disorders. AB - Roxatidine acetate is a histamine H2-receptor antagonist which, after almost complete oral absorption (greater than 95%), is rapidly converted to its active metabolite, roxatidine, by esterases in the small intestine, plasma and liver. Roxatidine is a potent inhibitor of basal and stimulated gastric acid secretion in animals and humans and, like most other H2-receptor antagonists, has no anti androgenic effects and does not interfere with the hepatic metabolism of other drugs. Large-scale trials have shown that roxatidine acetate 150mg per day is as effective as standard doses of cimetidine and ranitidine in the treatment of patients with duodenal or gastric ulcer, and that roxatidine acetate 75mg in the evening is likely to become a 'standard' regimen for the prevention of peptic ulcer recurrence. Preliminary data also suggest that roxatidine acetate may be useful in the treatment of reflux oesophagitis and stomal ulcer, and in the prevention of pulmonary acid aspiration. Roxatidine acetate is an H2-receptor antagonist which has been well tolerated in clinical trials. However, broader experience is required before definitive statements about tolerability relative to other H2-receptor antagonists can be made, and before the role of roxatidine acetate in the treatment of reflux oesophagitis and stomal ulcer, and the prophylaxis of acid aspiration pneumonitis, can be clearly defined. PMID- 1717224 TI - Gallium nitrate. A review of its pharmacological properties and therapeutic potential in cancer related hypercalcaemia. AB - Gallium nitrate, a novel drug for the treatment of cancer-related hypercalcaemia, inhibits osteoclast activity but does not affect osteoclast morphology or viability. Limited clinical experience in patients with cancer-related hypercalcaemia indicates that gallium nitrate is effective in restoring normocalcaemia in 75 to 85% of patients and is well tolerated in those with preserved renal function, producing few clinically relevant adverse effects. In comparative clinical trials it proved a more effective antihypercalcaemic agent than calcitonin or etidronate and produced a longer lasting normocalcaemic response. Gallium nitrate would appear to be indicated in symptomatic patients with cancer-related hypercalcaemia who have failed to respond to adequate rehydration. PMID- 1717219 TI - PAF. A review of its effects, antagonists and possible future clinical implications (Part II). PMID- 1717225 TI - Etodolac. A reappraisal of its pharmacology and therapeutic use in rheumatic diseases and pain states. AB - Etodolac is a nonsteroidal anti-inflammatory drug (NSAID) effective in the treatment of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis, and in the alleviation of postoperative pain. Etodolac also provides relief of other types of pain, including that arising from gouty conditions and traumatic injury. In all indications, etodolac appears to be at least as effective as other NSAIDs. The incidence of clinical adverse effects other than abdominal pain and dyspepsia is similar to that observed with placebo, and etodolac has been associated with a low rate of gastrointestinal ulceration and other serious events. Data from preliminary animal studies have suggested that etodolac may provide more selective inhibition of prostaglandin synthesis at sites of inflammation than some other currently available NSAIDs. Thus, available evidence indicates that etodolac, with its low incidence of gastrointestinal events, is an effective and well tolerated alternative to other NSAIDs in the treatment of arthritic diseases and pain of various aetiologies and should be considered a first-line therapy. PMID- 1717227 TI - Effects of lindane on fish carbohydrate metabolism. AB - Exposure of the European eel (Anguilla anguilla) to a high sublethal concentration of 0.335 ppm (0.50 of the 96-hr LC50) of lindane for 6, 12, 24, 48, 72, and 96 hr affected carbohydrate metabolism. Muscle glycogen levels decreased significantly (P less than 0.05) at 6, 12, 24, 48, 72, and 96 hr; liver glycogen content did not decline at any time. Muscle glucose levels in fish were elevated at 6, 12, 24, 48, 72, and 96 hr but in liver, the levels increased only at 96 hr. Mean values of muscle and liver pyruvate were elevated significantly (P less than 0.05) at 6, 12, 24, 48, and 72 hr. Muscle lactate levels increased at 6, 12, 24, and 48 hr in pesticide-treated fish. Liver lactate levels were elevated only at 12, 24, and 48 hr of treatment. The observed effects of lindane on carbohydrate metabolism in fish are discussed in relation to acute stress syndrome. Measurement of carbohydrate metabolites in fish for 6 hr or longer could prove useful as a rapid method for evaluating the toxicity of pesticides and other toxicants. PMID- 1717226 TI - Recombinant granulocyte colony-stimulating factor (rG-CSF). A review of its pharmacological properties and prospective role in neutropenic conditions. AB - Recombinant granulocyte colony-stimulating factor (rG-CSF) is a glycoprotein hormone which has been produced in mammalian cells and, in a nonglycosylated form, in the bacterium Escherichia coli through recombinant DNA technology. It stimulates proliferation, differentiation and activation of cells of the neutrophil-granulocyte lineage and has been investigated as therapy for patients with various neutropenic conditions, both iatrogenic and disease related. rG-CSF is well tolerated, the most frequently reported adverse effect being mild to moderate bone pain. A major use for rG-CSF therapy will be in ameliorating the neutropenia which follows cytoreductive chemotherapy. rG-CSF accelerates neutrophil recovery after chemotherapy, leading to a reduction in duration of the neutropenic phase. Consequently, infection rate is diminished, as is the associated usage of antibiotics and duration of hospitalisation. The implications are that rG-CSF may allow increased dose intensity and stricter adherence to chemotherapy schedules. The increase in neutrophils produced by rG-CSF renders it a useful treatment for conditions such as congenital, acquired and cyclic neutropenias for which current therapy is not very successful. rG-CSF may be an effective therapy in myelodysplasia, although there is concern about acceleration of the possible rate of conversion of this disease to acute myelogenous leukaemia. It is also likely that rG-CSF will be useful in accelerating the recovery of transplanted bone marrow in patients with leukaemia, lymphoma and solid tumour. Furthermore, there is great potential for expansion of the role of rG-CSF as monotherapy or in combination regimens with other cell factors in various haematological disorders such as aplastic anaemia. In summary, while many aspects of its use remain to be clarified, rG-CSF must be seen as an exciting advance in therapeutics. It should rapidly find an important place as an adjunct to cancer chemotherapy, and also appears to have substantial potential in a number of other neutropenic conditions which are currently difficult to treat. PMID- 1717228 TI - EEG in children with spelling disabilities. AB - A total of 23 13-year-old boys with spelling disabilities and 21 matched controls were studied. EEG was recorded for visual and quantitative analysis, including FFT band powers and normalized slope descriptors (NSD). Visual analysis showed general excess of slow activity, as well as an excess of temporal slow wave activity in the index group. Quantitative analysis showed low alpha and beta powers, and low "activity" and high "complexity" (NSD) in parieto-occipital derivations in the index group. Quantitative EEG (qEEG) parameter ratios between temporal and parieto-occipital derivations were increased in the index group, implying a lack of spatial differentiation in these EEGs. In covariance analysis the qEEG parameter differences between the index group and controls were partly explained by the neurotic traits made evident in psychological tests. This implies that psychopathological artifacts should be considered in qEEG examinations of children with cognitive handicaps. Differences in anterior/posterior qEEG ratios were, however, little affected by any confounding factors. Thus these qEEG ratios seem potentially useful in clinical assessments of children with learning disabilities. PMID- 1717229 TI - Significance of positive temporal sharp waves in the neonatal electroencephalogram. AB - We reviewed our computerized neonatal EEG database for records judged to display excessive positive temporal sharp waves (PTS) to determine their electroclinical associations and significance. Typical infants with excessive PTS were: (1) mature, with a mean conceptional age of 41.2 weeks, and (2) neurologically ill, judged by their high rate of associated EEG background abnormality (37 of 46; 80%) and clinical signs of encephalopathy (19 of 29; 66%). In comparison, healthy age-matched control infants never had excessive PTS. We consider an excess of PTS to be pathologic since they arise out of an abnormal EEG background in children exposed to potentially serious central nervous system illnesses and are associated with a high incidence of cerebral structural abnormalities (16 of 25; 64%). PTS may be comparable to the positive rolandic or vertex sharp waves in premature infants with periventricular injury of the deep white matter. PMID- 1717230 TI - Retrospective inventory of EEG abnormalities in partial status epilepticus. AB - In this retrospective study, EEG activity in partial status epilepticus (PSE) was classified into different patterns from analysis of both ictal and interictal discharges. In 64 patients with recorded PSE, continuous seizures and closely spaced seizures interrupted by only brief flat periods were uncommon. PLEDs, defined as classic periodic lateralized epileptiform discharges, and PLEDs Plus, defined as PLEDs associated with stereotyped low amplitude, were the most common abnormalities. PLEDs and PLEDs Plus can each occur alone or sequentially (sequential PLEDs) between consecutive seizures. The quantity of ictal activity was significantly lower with PLEDs, sporadic spikes and with the absence of epileptiform abnormalities than with PLEDs Plus and sequential PLEDs. EEG monitoring is important to gauge the effectiveness of treatment, particularly in patients with patterns associated with a high incidence of seizure activity, namely continuous seizures with or without flat periods, sequential PLEDs and PLEDs Plus. From serial recordings, a sequence was reconstructed which may be relied upon to further assess the need for additional energetic therapeutic measures. The reconstructed sequence differed in patients with chronic lesions since sequential PLEDs and PLEDs Plus were identified exclusively in patients with acute or subacute lesions. PMID- 1717231 TI - Alpha-like EEG activity in non-REM sleep and the fibromyalgia (fibrositis) syndrome. AB - The occurrence and characteristics of alpha-like activity during non-REM (NREM) sleep were examined in 11 subjects suffering from non-inflammatory (non rheumatoid) musculoskeletal pain--fibromyalgia ('fibrositis'), and in 15 symptom free controls. Both groups claimed to be good sleepers. Mean percentage alpha like activity in sleep stages 2, 3, 4 and for NREM as a whole were greatest for the fibromyalgia group, but not significantly different from those of the controls. Overlap in the distribution of NREM alpha-like activity in sleep between the two groups indicated that it is not directly related to musculoskeletal symptoms. Spectral analyses showed a frontal area prevalence of this (kappa?) activity in the fibromyalgia group. PMID- 1717232 TI - Interrelationships between rapid eye and body movements during sleep: polysomnographic examinations of infants including premature neonates. AB - Myoclonic twitching and rapid eye movements (REMs) are believed to occur in close association in animals; but, there have been few studies on their interrelations in humans. Polysomnograms were made from 33 normal infants of 34-84 conceptional weeks of age in order to observe the developmental aspect of the relation between twitching and REMs. We examined the small body movements (BMs), which appeared to be equivalent to twitches in animals and calculated the percentage of BMs that occurred together with the REM bursts in comparison to the total number that occurred during active REM sleep (% BMs in REM bursts). Polysomnograms were also obtained from 5 infant patients whose pathophysiologies were considered to be due to brain-stem immaturity. Whereas the values showed abrupt decreases during early infancy, nearly reaching 0, for the normal infants, they were high in some of the patients' records. These results suggest that few BMs occur during REMs in humans as opposed to animals. The maturation of inhibitory mechanisms, which are located in the brain-stem and act during REMs, may account for the rapid decrease of % BMs in REM bursts during early infancy. Increases of this index may reflect delayed brain-stem maturation. PMID- 1717233 TI - Differential auditory processing continues during sleep. AB - Auditory evoked potentials (AEPs) were used to examine selective stimulus processing in sleep. In waking, repetitive stimuli generate exogenous P1, N1 and P2 components of the auditory evoked potential (AEP). Deviant stimuli generate endogenous cognitive components including the mismatch negativity (MMN), N2 and P3 components. We examined long-latency auditory evoked potentials elicited by repetitive and deviant stimuli during waking and stage II-IV sleep to assess whether stimulus deviance is detected during sleep. The waking P1, N1b and P2 had maximal amplitudes at fronto-central scalp sites, with additional peaks (N1a, N1c) at temporal sites. Deviant tones generated a frontal maximal MMN, and complex novel tones generated an additional P3 component maximal at centro parietal sites. During stages II-IV sleep N1a, b, c amplitudes were reduced. During stage II sleep all stimuli generated increased P2 amplitudes and a late negative component (N340). Deviant stimuli generated greater P2 and N340 amplitudes than frequent stimuli in stage II sleep, as well as an additional P420 component. In stage III-IV sleep the P420 was absent and the AEP was dominated by a negativity of long duration whose amplitude increased in response to deviant stimuli. These data indicate that auditory evoked activity changes from wakefulness to sleep. The differential response to deviant sounds observed during waking and all sleep stages supports the theory that selective processing of auditory stimuli persists during sleep. PMID- 1717234 TI - Reduced attention-related negative potentials in schizophrenic children. AB - ERPs were recorded from normal and schizophrenic children during performance of a reaction time task (RT) followed by a complex visual discrimination, the span of apprehension task (Span), sensitive to vulnerability factors in schizophrenia. Subjects responded rapidly to the onset of the visual arrays in the RT condition and differentially to the presence of 1 of 2 target letters in the Span condition. The EEG was recorded at 19 scalp sites and ERPs included activity 1 sec before through 1 sec after Span array onset. Difference potentials (Span-RT) were computed to remove unvarying exogenous activity, thus isolating endogenous activity associated with the processing demands of the Span task. When RT and Span task ERPs are compared, schizophrenic children produced a significantly smaller than normal increment in endogenous negative activity. This endogenous negativity differed in its topography and time course from the exogenous components (P1, N1 and P2), and most likely reflects attentional effort associated with serial search, pattern recognition and stimulus identification. We believe that the current results support the position that schizophrenics are impaired in their ability to allocate adequate attentional resources for the processing of the Span stimuli. It is important to note that this deficit is apparent quite early in discriminative processing. PMID- 1717235 TI - Localization of the P3 sources using magnetoencephalography and magnetic resonance imaging. AB - In this study, two related issues were addressed: first, whether the P3 component of auditory evoked responses, obtained in the context of an oddball paradigm, and its magnetoencephalographically recorded counterpart (P3m) are generated by the same intracranial sources; and, second, whether these sources, modeled as equivalent current dipoles, can be localized in particular brain structures using magnetic resonance imaging. The study involving 8 normal adult subjects resulted in the following findings. (1) Both the similarities and differences in wave form characteristics of the simultaneously recorded P3 and P3m can be best accounted for by common intracranial sources. (2) Several successively activated single dipolar sources, rather than a single source, account for the entire evolution of the P3m component. (3) Most of these sources were localized in the vicinity of the auditory cortex in all subjects, although some sources appeared to be in deeper structures, possibly the lateral thalamus. (4) The successive activation of sources followed an orderly medial-to-lateral course. These results suggest that activity responsible for the surface-recorded P3 (and P3m) component may be initiated in deep structures, but it quickly spreads over and is sustained in areas near the auditory cortex. PMID- 1717236 TI - Electroencephalography, evoked potentials and MRI brain scans in saturation divers. An epidemiological study. AB - One hundred and fifty-six air and saturation divers, mean age 33.6 (range 21-49) years, were examined. The control group consisted of 100 offshore workers and policemen with the health requirements to have a diving certificate, mean age 34.0 (range 22-48) years. The examination protocol included electroencephalography (EEG), visual evoked potentials (VEPs), brain-stem auditory evoked potentials (BAEPs) and magnetic resonance imaging (MRI) of the brain and brain-stem. Abnormal EEGs, with focal slow waves mostly in the temporal regions and sharp potentials, were found in 18% of the divers and in 5% of the controls (P = 0.003). Abnormal EEGs correlated significantly with the exposure to saturation diving (P = 0.0006) and the prevalence of decompression sickness (P = 0.0102). Alcohol consumption was negatively correlated with abnormal EEGs (P = 0.0006). Mean I-III BAEP latency was increased (P = 0.047) in the diver group. P100 VEP latency decreased with age (21-49 years). High signal intensity changes obtained by MRI were found in 33% of the divers and in 43% of the controls (P = 0.14). It is concluded that the nervous system of saturation divers is influenced by their occupation and that EEG is a useful method in the health examination of divers. PMID- 1717237 TI - Computerized seizure detection of complex partial seizures. AB - In this study, we describe a computerized method that uses 3 quantified EEG features and discriminant analysis to automatically detect seizure EEG. The quantified EEG features were relative amplitude, dominant frequency and rhythmicity. Using EEGs recorded from intracranial electrodes, the seizure detection method was applied to consecutive non-overlapping 2-channel EEG epochs. A seizure detection sensitivity, ranging from 90% to 100%, was associated with a false positive detection rate of 1.5-2.5/h. The performance of the seizure detection method remained stable for EEG recorded over variable time periods. PMID- 1717238 TI - Characterization of immunoreactive insulin-like growth factor-I from leukocytes and its regulation by growth hormone. AB - In the present study, we investigated the production of insulin-like growth factor I (IGF-I) by leukocytes and its production after treatment with GH. Immunoreactive (ir) IGF-I was observed in leukocytes by direct immunofluorescence with fluorescein isothiocynate-conjugated antibodies to IGF-I. Studies using immunoaffinity purification, HPLC and a fibroblast proliferation bioassay suggests that the de novo synthesized leukocyte-derived irIGF-I is similar in mol wt, antigenicity, and bioactivity to serum IGF-I. We also evaluated the effect of GH on the production of leukocyte-derived irIGF-I. Spleen cells cultured for 24 h in the presence of exogenous GH caused a 2-fold elevation of irIGF-I as demonstrated by RIA and immunofluorescence. In order to determine if leukocyte derived irGH can stimulate the production of irIGF-I, we cultured spleen cells for 24 h in the presence of antibodies specific for GH. The data showed a decrease in the number of cells positive for irIGF-I, suggesting that leukocyte derived irGH may stimulate the synthesis of irIGF-I by leukocytes. We also demonstrated that exogenous IGF-I can decrease the levels of leukocyte GH-related RNA and ir protein. Taken together, our data demonstrate the synthesis and secretion of bioactive irIGF-I from leukocytes and suggest a regulatory circuit for leukocyte-derived irGH and irIGF-I within the immune system. PMID- 1717239 TI - Effect of human chorionic gonadotropin on the expression of progesterone receptors and estrogen receptors in rabbit ovarian granulosa cells and the uterus. AB - To investigate whether LH/human CG (hCG) or progesterone acts as a regulator of estrogen receptors (ER) and progesterone receptors (PR) in granulosa cells, we studied the immunohistochemical expression of both ER and PR in the ovary and the uterus of mature rabbits, during the induction of ovulation by FSH followed by administration of hCG, progesterone, or a progesterone antagonist (RU486) and hCG. Granulosa cells pretreated with FSH for 3 days showed ER staining, but negligible PR staining. The staining pattern for ER and PR changed in animals pretreated with FSH followed by hCG injection; by 6 h after hCG injection, we observed the disappearance of ER and the appearance of PR, and by 3 days after hCG injection, we observed the reappearance of ER and the disappearance of PR. However, the expression of ER and PR in the granulosa cells of animals pretreated with FSH followed by progesterone administration instead of hCG was almost the same as that of animals pretreated with FSH alone. In addition, the expression of ER and PR in the granulosa cells of animals pretreated with FSH followed by RU486 and hCG was almost the same as that of animals pretreated with FSH followed by hCG administration. The uterine glandular epithelium, in contrast, began to show decreased appearance of ER and PR by 48 h after hCG injection, and we observed the disappearance of both receptors by 3 days after hCG administration. These results suggest that the expression of ER and PR in granulosa cells is not regulated by the action of progesterone, but by that of LH/hCG. PMID- 1717240 TI - Sexual differences in the distribution of neurons coexpressing galanin and luteinizing hormone-releasing hormone in the rat brain. AB - We have recently reported that a subpopulation of galanin (GAL)-immunoreactive perikarya in the preoptic area near the organum vasculosum of the lamina terminalis (OVLT) has morphological characteristics similar to those of LHRH containing neurons. In fact, both peptides are colocalized in those neurons in the male rat brain. In these studies we describe sexual differences in the incidence of neurons colocalizing GAL and LHRH in this region. In male rats, about 20% of LHRH-immunoreactive cells in the diagonal band of Broca and the medial preoptic area are also immunoreactive for GAL. In contrast, in female rats, about 65% of LHRH-containing perikarya near the OVLT are immunostained for GAL. In addition, GAL and LHRH levels were measured in tissue extracts containing either the OVLT and surrounding areas or the median eminence. The data indicate that the preoptic area, including the OVLT, contains a significantly higher amount of GAL in females killed in proestrus than in those killed in estrus. The amount of GAL measured in males is lower than that in the proestrous females, but somewhat greater than that present in the estrous females. There were no significant differences among the three groups in LHRH content in this region. The GAL content of the median eminence was the highest in proestrous females, followed by estrous females and males. The LHRH content in the median eminence was not significantly different among the three groups. In conclusion, these observations indicate that the coexpression of GAL with LHRH in a discrete subpopulation of LHRH-producing neurons is a sex-related phenomenon. The fact that changes in GAL levels in specific regions can be seen at different times during the estrous cycle suggests that gonadal steroids, possibly estrogens, may physiologically regulate the expression of GAL in this subpopulation of LHRH neurons. PMID- 1717241 TI - Monolayer culture of adult rat pancreatic islets on extracellular matrix: modulation of B-cell function by chronic exposure to high glucose. AB - Studies in vivo indicate that chronic hyperglycemia is deleterious for insulin secretion. We have used an improved islet monolayer culture system to study chronic modulations of B-cell function. Adult rat islets maintained over several weeks on extracellular matrix in the presence of 11.1 mM glucose responded to an acute stimulation with 16.7 mM glucose by a 5- to 8-fold increase in insulin secretion. When cultured in the presence of higher glucose concentrations, the response to an acute glucose stimulus diminished time and dose dependently. In islets desensitized by exposure to 33.3 mM glucose for 1 week, reduction of the glucose level to 11.1 mM reversed the desensitization within 2 weeks. This desensitization was not limited to the glucose stimulus; responses to other nutrient secretagogues, such as glyceraldehyde and alpha-ketoisocaproic acid, were also reduced. In contrast, responses of insulin secretion to nonnutrient stimulators (tolbutamide and quinine) and amplifiers (isobutylmethylxanthine and carbachol) showed no desensitization in islets exposed to 33.3 mM glucose. Desensitization similar to that caused by 33.3 mM glucose could be induced by 11.1 mM glucose together with 0.1 mM isobutylmethylxanthine. High glucose also caused a time-dependent loss in compact monolayer organization with disruption of cell contacts. Our studies suggest that 1) generation of the reduced insulin response may be related to the prolonged high insulin secretion rate; 2) expression of the functional change is specific to the nutrient stimulus secretion coupling; and 3) modifications in intercellular contacts may be involved in B-cell desensitization. PMID- 1717242 TI - Effects of adrenocorticotropin on action potential and calcium currents in cultured rat and bovine glomerulosa cells. AB - Action potentials (APs) and ionic currents were recorded in primary cultured rat and bovine glomerulosa cells by using the whole-cell recording technique. Switching from the current-clamp mode to the voltage-clamp mode allowed recordings of APs and currents in the same cell. APs can be elicited by appropriate stimulation in conditions where the excitability of the cell is increased by blocking a transient outward current. A T-current or a N-current was always present in cells in which APs were recorded; an L-current could also be recorded, but a cell presenting only an L-current was not able to fire an AP. The addition of Bay K 8644 (10(-8) M) induced a dramatic increase in the action potential duration. In the same cells, the analysis of the currents showed that the L-current was increased, whereas the T-current was not significantly affected. The effects of ACTH (10(-8) M) were analysed on APs and currents. On APs, at least two phases could be distinguished, the first corresponded to the reduction of the action potential duration, whereas the second was a huge increase of the plateau duration. The T-current was strongly affected by ACTH as a great inhibition took place in the first seconds after the superfusion with a 10(-8) M ACTH medium. Then a partial recovery of the T-current appeared. The effects of ACTh were reversible on washing. On the contrary, the L-current was increased by ACTH, but this effect was not reversible. The effects of ACTH were mimicked by 8 Bromo cAMP (10(-3) M). Similar results were found in rat and bovine glomerulosa cells. These results suggest that second messengers generated by ACTH would regulate Ca2+ entrance by nondetermined phosphorylation process in the sense of an increase in intracellular Ca2+. PMID- 1717243 TI - Intracerebroventricular atrial natriuretic factor (ANF) antiserum inhibits volume induced ANF in sheep: evidence for the brain's regulation of ANF secretion. AB - Plasma volume expansion stimulates cardiac secretion of atrial natriuretic factor (ANF) and also increases the ANF concentration in cerebrospinal fluid. In order to determine whether brain ANF is involved in the compensatory response to hypervolemia or the regulation of cardiac secretion of ANF, we have studied the integrated hemodynamic, renal, and hormonal response to acute volume expansion (15 ml/kg Dextran over 30 min) in five sheep given nonimmune serum (control) and ANF antiserum by intracerebroventricular (icv) injections on separate days. Dextran loading caused similar decreases in hematocrit and increases in central venous and mean arterial pressures on both study days. Heart rate was higher after antiserum injections (P less than 0.05). Dextran loading increased plasma ANF on the control (20 pmol/liter maximal mean increment above baseline) but not on the antiserum day (P less than 0.01). The diuresis (P less than 0.01) and natriuresis (P less than 0.05) observed on the control day was inhibited by icv antiserum. Plasma aldosterone and cortisol levels showed similar falls in response to the dextran load on both days. These experiments show that icv ANF antiserum inhibits both the increase in cardiac secretion of ANF and the renal response to plasma volume expansion without affecting hemodynamic status. These data support the hypothesis that the brain ANF system is important in the systemic responses to volume loading. PMID- 1717244 TI - Insulin-like growth factor-binding protein-1 modulates blood glucose levels. AB - We have determined the consequences of insulin-like growth factor-binding protein 1 (IGFBP-1) administration alone and in combination with insulin-like growth factor-I (IGF-I). Human recombinant IGF-I, infused as a bolus into male Wistar rats, induced a fall in plasma glucose to 72 +/- 3% of baseline 15 min after injection. Co-infusion of equimolar concentrations of human IGFBP-1 abolished the IGF-I-induced fall (P less than 0.001). Injection of IGFBP-1 alone caused a rise in plasma glucose levels (P less than 0.002). The half life of human IGFBP-1, measured using a primate-specific RIA, was 12.5 +/- 0.7 min and was not influenced by the co-infusion of IGF-I. This study demonstrates that, in the rat, human IGFBP-1 blocks the hypoglycemic response to intravenous IGF-I and increases blood glucose levels when administered alone. Since IGFBP-1 concentrations are dependent on metabolic status, we suggest that fluctuating IGFBP-1 levels might modulate the hypoglycemic activity of unbound IGFs in the circulation. PMID- 1717245 TI - Free alpha molecules from pregnancy stimulate secretion of prolactin from human decidual cells: a novel function for free alpha in pregnancy. AB - Free alpha molecules isolated from pregnancy, as well as highly purified reference preparations of hCG alpha subunit (CR119 or CR123), stimulated the release of prolactin from human decidual cells in culture. The amount of prolactin secreted during a 24 h incubation was concentration-dependent over a range of increasing doses of alpha from 0.2 to 20 ng/ml with an ED50 of about 1.6 ng/ml. These concentrations are well within the physiologic maternal serum free alpha levels which average 350 ng/ml during the third trimester of pregnancy. Incubation of decidual cells with a reference preparation of intact hCG (CR123) at a concentration of 260 ng/ml resulted in stimulated secretion of prolactin, however, the observed stimulation could be attributed to contamination of the preparation with free alpha or dissociated hCG alpha subunit. Purified hCG beta subunit had no stimulatory activity on the decidual cell culture. The effect of alpha subunit on the stimulated release of prolactin was not due to a generalized stimulation of protein synthesis and secretion since no increase was observed in the release of 35S-labeled proteins compared to controls. In addition, the observed increase in prolactin secretion was not due to a toxic effect of the alpha subunit since there was no visible effect on cell viability, and the cellular enzymes, LDH and alkaline phosphatase, were not detected in the culture medium. Addition of exogenous hCG alpha subunit to primary cultures of human trophoblast cultures did not result in stimulated release of human placental lactogen. We conclude that free alpha molecules of pregnancy stimulate release of prolactin from human decidual cells in culture. These results suggest a novel role for free alpha in the paracrine regulation of decidual prolactin secretion. PMID- 1717246 TI - The ovarian expression of the antigonadotropic insulin-like growth factor binding protein-2 is theca-interstitial cell-selective: evidence for hormonal regulation. AB - To begin the process of identification and charactization of rat ovarian insulin like growth factor binding proteins, we have undertaken to explore the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein-2 (IGFBP-2) gene for which an antigonadotropic potential has recently been demonstrated. To this end, a solution hybridization/RNase protection assay was employed wherein total ovarian RNA (20 micrograms) from immature (21-23 days old) female rats was hybridized with a [32P]-labeled IGFBP-2 riboprobe. As in liver, a single protected fragment (550 bases long) corresponding to IGFBP-2 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-2 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm the cellular distribution of the IGFBP-2 protein, media conditioned by cultured granulosa or theca-interstitial cells were subjected to immunoprecipitation using two IGFBP-2-directed polyclonal antisera. Expectedly, both antibodies (but not non-immune rabbit serum) readily immunoprecipitated the 28 kDa rat IGFBP-2 species generated by hepatic BRL-3A cells. Similarly, both antibodies effectively immunoprecipitated an IGFBP the size of rat IGFBP-2 elaborated by theca-interstitial cells. In contrast, neither antibody immunoprecipitated the 28-29 kDa IGFBP species elaborated by granulosa cells otherwise readily apparent in conventional Western ligand blots. Hypophysectomy resulted in a 3-fold decrease (P less than 0.05) in the relative (densitometrically-quantified) abundance of ovarian IGFBP-2 transcripts, a diametrically opposed effect (P less than 0.05) being noted at the level of the liver. In contrast, treatment of immature hypophysectomized rats with a diethylstilbestrol-containing subcutaneous silastic implant for a total of 5 days resulted in a concordant 3-fold increase (P less than 0.05) in the relative abundance of IGFBP-2 transcripts in both ovary and liver when compared with untreated hypophysectomized controls. Taken together, these findings document rat ovarian IGFPB-2 gene expression to be theca-interstitial (rather than granulosa) cell-selective, and subject to upregulatory control by pituitary principle(s) and/or by estrogens. Although equally estrogen-dependent, hepatic IGFBP-2 gene expression proved constitutive in nature and subject to (diametrically opposed) inhibitory control by (potentially distinct) pituitary principle(s). PMID- 1717247 TI - Expressions of sulfated glycoprotein 2 and pSvr-1 genes and involution of steroid hormone-dependent rat tissues. AB - To further survey the molecular mechanisms underlying the involution of steroid hormone-dependent rat tissues, we undertook experiments to test whether or not any significant correlation between the tissue involution and expressions of rat sulfated glycoprotein 2 (SGP-2) and pSvr-1 genes, which had been initially cloned from the Sertoli cells and the seminal vesicles, respectively, and then identified as androgen repressed messages both in the ventral prostate and in the seminal vesicles, could be observed in steroid hormone-dependent rat tissues. Expressions of these genes were stimulated within 48 h after castration of animals both in the ventral prostate and in the seminal vesicles as reported previously, but not significantly altered by ovariectomy in the uterus. Expressions of these genes in the thymus were significantly repressed by the administration of dexamethasone and/or cycloheximide. Although the roles of expressions of SGP-2 and pSvr-1 genes in steroid hormone-dependent tissues remain unclear, their presence might become useful molecular markers of tissue involution not only in androgen-dependent rat tissues but also in glucocorticoid dependent ones, and also provide excellent model systems for the study of negative regulation mechanism of gene expression by steroid hormones. PMID- 1717248 TI - Pharyngo-esophageal prostheses in malignancies of the cervical esophagus. AB - Cervical location of inoperable esophageal carcinoma is usually considered a contraindication of palliative intubation due to technical limitations and complications of the procedure. Between 1978 and 1989, 32 patients with inoperable cancer of the cervical esophagus were treated endoscopically at the Endoscopy Division of the National Cancer Institute, Milan. Eight of them underwent endoscopic intubation. The prostheses were tolerated well and did not cause any respiratory impairment. No complications related to the procedure were observed. In seven of the patients resumption of oral nutrition led to improvement of the general condition. The mean survival time was 5.5 months (range 0.5-23 months). PMID- 1717249 TI - Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine. AB - The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein-A and protein-G affinity chromatography and analyzed by two-dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids. PMID- 1717250 TI - Two-dimensional electrophoresis of human parotid salivary proteins from normal and connective tissue disorder subjects using immobilised pH gradients. AB - Two-dimensional electrophoretic analysis of human salivary proteins using immobilised pH gradients in the first dimension, thin-layer gradient horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second, and modified staining procedures has resulted in a substantial improvement in their resolution. Unlike carrier ampholyte-based techniques, immobilised pH gradients prevent the loss of proteins of pI greater than 8; accordingly, basic components, including basic proline-rich proteins, can now be resolved. A two-dimensional map showing the locations and identities of most of the major proteins has been constructed. Narrow-range pH gradients can be constructed to give increased resolution of proteins of particular interest. By means of a pH 3.5-5.0 gradient, the abnormal salivary proteins associated with connective tissue disorders were found to be a highly heterogeneous group of pI approximately 3.75-4.75 and Mr approximately 32,000; although low levels occurred in some normal individuals, there was less heterogeneity (pI approximately 3.75-4.25). The technique should form a base for future structural, functional, and clinical studies on human salivary proteins. PMID- 1717251 TI - Quantitation of human leukocyte proteins after silver staining: a study with two dimensional electrophoresis. AB - The quantitative attributes of human leukocyte proteins detected by silver staining two-dimensional electrophoresis (2-DE) gels were studied by using computer-assisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15% when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15% when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80% of the patterns) have a linear relationship between the amount of protein loaded onto a 2-DE gel and the spot volume in the final 2-DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein-specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2-DE runs to a level that will allow detection of quantitative changes indicative of biological responses. PMID- 1717252 TI - Electrophoretic and computer analysis of skeletal muscle used for cardiac assistance. AB - Skeletal muscle has an inherent plasticity which allows it to undergo fibre type transformation when induced by a specific stimulus. Electrical stimulation has been used here to induce transformation of a predominantly fast type skeletal muscle towards a slow, more fatigue-resistant phenotype, which is more suitable for use in long-term cardiac assistance. Muscle samples from animals electrically stimulated for periods up to 6 months have been analysed by electrophoresis for myosin heavy chain (MHC) and myosin light chain (MLC) fast and slow isoforms. Densitometry and computer analysis have been used to determine the pattern of transformation of the different myosin subunits over this time period. MHC and MLC 2 fast to slow isoform switching preceded that of the alkali light chains (MLC1 and MLC3). After 3 months of stimulation the MHC slow isoform was found to have doubled in concentration relative to the unstimulated control muscle and by 4 months accounted for almost 50% of the total MHC content. The slow isoform accounted for 75% of the MLC2 after 4 months of stimulation. The protein products of mRNA isolated from stimulated muscle samples, translated in vitro and separated by electrophoresis, showed that transformation at the mRNA level preceded that at the protein level. By 2-4 weeks of stimulation MLC2 slow isoform mRNA represented over 60% of the total MLC2 mRNA population. An understanding of the molecular structure of muscle during transformation provides insight into its haemodynamic performance in cardiac assistance. PMID- 1717253 TI - Production of high purity proteins by elution of individual fractions from two dimensional gels. AB - Results are presented on the production of purified myocardial proteins from two dimensional (2-D) gels. Proteins were fixed in a native condition using potassium acetate and then eluted into an aqueous solution. Homogeneous tropomyosin and myosin light chain fractions and a number of nonidentified myocardial proteins present on 2-D gels were obtained. PMID- 1717254 TI - Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure. AB - The presence of N-linked carbohydrate at Asn58 in the VH of the antigen binding site of an antibody specific for alpha(1----6)dextran (TKC3.2.2) increases its affinity for dextran 10- to 50-fold. Site-directed mutagenesis has now been used to create novel carbohydrate addition sequences in the CDR2 of a non-glycosylated anti-dextran at Asn54 (TST2) and Asn60 (TSU7). These antibodies are glycosylated and the carbohydrates are accessible for lectin binding. The amino acid change in TSU7 (Lys62----Thr62) decreases the affinity for antigen; however, glycosylation of TSU7 increased its affinity for antigen 3-fold, less than the greater than 10 fold increase in affinity seen for glycosylated TKC3.2.2. The difference in impact of glycosylation could result either from the position of the carbohydrate or from its structure; unlike the other antibodies, TSU7 attaches a high mannose, rather than complex, carbohydrate in CDR2. In contrast, glycosylation of TST2 at amino acid 54 inhibits dextran binding. Thus slight changes in the position of the N-linked carbohydrate in the CDR2 of this antibody result in substantially different effects on antigen binding. Unlike what was observed for the anti dextrans, a carbohydrate addition site placed in a similar position in an anti dansyl is not utilized. PMID- 1717255 TI - Functional domains of the granulocyte colony-stimulating factor receptor. AB - The granulocyte colony-stimulating factor (G-CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)-like domain, a cytokine receptor homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G-CSF receptor cDNA into IL-3-dependent murine myeloid cell line FDC-P1 and pro-B cell line BAF-B03, which normally do not respond to G-CSF, enabled them to proliferate in response to G-CSF. On the other hand, expression of the G-CSF receptor cDNA in the IL-2-dependent T cell line CTLL-2 did not enable it to grow in response to G-CSF, although G-CSF could transiently stimulate DNA synthesis. Mutational analyses of the G-CSF receptor in FDC-P1 cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G-CSF-triggered growth signal. PMID- 1717256 TI - The tcl-3 proto-oncogene altered by chromosomal translocation in T-cell leukemia codes for a homeobox protein. AB - The t(10;14)(q24;q11) chromosomal translocation found in malignant cells of 5-10% of patients with T-cell acute lymphoblastic leukemia (T-ALL) involves the T-cell receptor delta chain gene on chromosome 14 and a breakpoint cluster region on chromosome 10. The candidate proto-oncogene tcl-3, thought to be involved in the pathogenesis of t(10;14) T-ALL, was cloned and found to be elevated in expression in leukemic cells harboring the t(10;14) translocation. Sequence analysis revealed that tcl-3 is a new homeobox-containing gene. Comparison of the tcl-3 cDNA and its 5' genomic sequences with DNA sequences from the t(10;14) translocation breakpoints showed that this gene is structurally altered in four patients with t(10;14)(q24;q11) T-ALL. These findings suggest that homeobox containing genes that normally act as transcription factors may contribute to T cell leukemogenesis when abnormally expressed. PMID- 1717257 TI - Analysis of the Rhodobacter capsulatus puc operon: the pucC gene plays a central role in the regulation of LHII (B800-850 complex) expression. AB - Formation of the light harvesting complex B800-850 (LHII) of Rhodobacter capsulatus requires the expression of more than the three known genes specific for that complex (pucA, pucB and pucE) encoding the alpha, beta and gamma subunits of LHII, respectively. In this work evidence is presented that the product of the gene pucC, which is located downstream from pucA, is essential for high-level transcription of the pucBACDE operon and formation of LHII. Plasmids were constructed containing deletions in one or several puc genes and transferred to a pucC::Tn5 mutant in which the puc operon is not expressed. It was found that the LHII- phenotype of the mutant was due to the missing PucC protein and that all known puc genes are located in one operon. To dissect the pucC, pucD and pucE genes from pucB and pucA and independently regulate them, they were placed under control of the nifHDK promoter. Only under nitrogen-fixing growth conditions was the LHII- pucC::Tn5 mutant complemented by this construction. It is concluded that expression of pucC is essential for formation of the LHII complex in R.capsulatus. Analysis of the pucD and pucE genes led to the conclusion that the products of these genes stabilize the B800-850 complex. PMID- 1717258 TI - The Drosophila neurogenic gene neuralized encodes a novel protein and is expressed in precursors of larval and adult neurons. AB - Neuralized belongs to a group of genes involved in neurogenesis in Drosophila. Loss of function mutations lead to an overproduction of neurons at the expense of epidermal tissues. We have cloned the neuralized locus and examined its expression pattern during development. Expression is initially observed during embryogenesis in the neurogenic ectoderm and later in neuroblasts. In addition, transcripts are also found in sensory precursor cells during imaginal disc development. Other tissues that express neuralized include the embryonic mesoderm and specific follicle cells in the ovary. The predicted neuralized gene product is a highly basic protein with a novel motif that bears some resemblance to those found in nucleic acid-binding proteins. The first half of this motif has sequence similarity to the first half of a homeobox whereas the second half is similar to the helix-turn-helix structure of bacterial repressors. PMID- 1717259 TI - In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease. AB - We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed. PMID- 1717260 TI - Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus. AB - Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5-1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non-finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5-1 serves a general biological function involving its RNA binding capacity. PMID- 1717261 TI - Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. AB - Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N terminal globule and rod-like domain and Nd-II corresponding mainly to the C terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes. PMID- 1717262 TI - Disruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice. AB - Previous work in transfected cell lines and in nuclear extracts has led to the identification of two cis-acting elements important for transcription of the human alpha-1-antitrypsin (A1AT) gene, which bind to two liver specific trans acting factors, LF-A1 and LF-B1. Mutations EM3 and PM1, which abolish the binding of LF-A1 and LF-B1 respectively, drastically reduce transcription activity of the A1AT gene in vitro and in cell culture. The same mutants have now been introduced in a larger DNA context and their effect has been tested in transgenic mice. A stretch of DNA was constructed which carries two transcriptional units: 18 kb of the human retinol binding protein (RBP) gene, driving the expression of the bacterial chloramphenicol acetyl transferase, linked to 17.5 kb containing the entire A1AT coding sequence with additional 5' and 3' flanking sequences. Transcription from the RBP promoter was shown to predominate in liver, and could be used as an internal marker of 'active copy number'. Mutations in the A1AT gene promoter were introduced by homologous recombination in bacterial cells. The results show that base pair substitutions in the binding site for LF-A1 and LF-B1 drastically reduce transcription in non-hepatic adult tissues, yolk sac, and fetal liver, whereas only LF-B1 binding site mutations have a marked, albeit variable, effect in adult liver. PMID- 1717263 TI - Cloning, heterologous expression and developmental regulation of a Drosophila receptor for tachykinin-like peptides. AB - We identified clones encoding a Drosophila receptor for tachykinin-like peptides by low stringency screening of an embryonic cDNA library with probes from the bovine substance K receptor. The cDNAs encode a seven transmembrane domain protein (DTKR) of 519 amino acids with 40-48% amino acid identity to mammalian tachykinin receptors within transmembrane regions. Xenopus oocytes injected with DTKR cRNAs showed selective responses to vertebrate substance P, its agonists and not to other vertebrate tachykinin peptides. These responses were eliminated by treatment of oocytes with pertussis toxin. In the adult fly, Northern and PCR analysis demonstrated preferential expression of DTKR in the head; in situ hybridization indicated that DTKR is accumulated in the cell bodies of neurons in the adult CNS. The levels of DTKR transcript are regulated during development. Northern and PCR amplification analysis showed that while DTKR transcripts are present at all stages, high levels of expression occur in later stages of embryogenesis (starting at 10-14 h), coinciding with the beginning of major periods of neural development. Whole mount embryo in situ hybridization demonstrated that DTKR is expressed at these later stages of embryogenesis (11-15 h) in the brain and in a specific subset of neurons in each neuromere of the developing ventral ganglion. The gene encoding DTKR was mapped by in situ hybridization to a single location at 99D on the right arm of chromosome 3. These observations demonstrate that the tachykinin family of peptide transmitters and their receptors represent an evolutionarily ancient form of cellular communication within the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717264 TI - Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803. AB - We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700-chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light-activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild-type cells showed a 4-fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700-apoprotein; greatly reduced whole-chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA-C and PSA-D were not detected in this mutant. ADK9 does assemble near wild-type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6-dichloro-p-benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site-directed mutagenesis of the PS I core. PMID- 1717265 TI - Superhelical stress and nucleosome-mediated repression of 5S RNA gene transcription in vitro. AB - Nucleosomes were assembled on a plasmid carrying a Xenopus somatic 5S RNA gene prepared at different superhelix densities. The gene was preferentially assembled into a positioned nucleosome which was stable to superhelical stress. No evidence for a conformational change in the nucleosome was found, even under extreme negative superhelical stress. Transcription in an extract from Xenopus oocyte nuclei was repressed to a degree which depended on the number of nucleosomes assembled. Topoisomerase activity in the extract was effectively inhibited by camptothecin, which had no effect on transcription. Transcription of reconstitutes remained repressed relative to naked plasmids, and was independent of superhelix density. Transcripts from reconstitutes were derived solely from nucleosome-free genes. Thus, a histone octamer positioned on the gene was sufficient to block its transcription. Tryptic removal of the core histone tail domains had no effect on transcription at any superhelix density. Transcription of reconstitutes containing H3/H4 tetramers was also repressed, but not eliminated (unlike reconstitutes containing octamers), and repression was independent of superhelix density. We suggest that removal of histones H2A/H2B from the nucleosome facilitates activation of transcription in the extract. We conclude that superhelical stress alone does not activate transcription of a 5S RNA gene assembled into a nucleosome in vitro. PMID- 1717266 TI - Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I. AB - Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes. PMID- 1717267 TI - A second pathway of activation of the Torpedo acetylcholine receptor channel. AB - We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that ( ) physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D tubocurarine, alpha-bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FK1 and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 microM range, there exists a further class of low-affinity (Kd approximately mM) sites from which eserine acts as a direct blocker of the acetylcholine-activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel. PMID- 1717268 TI - A rapid, simple enzyme immunoassay for detection of antibody to individual epitopes in the serodiagnosis of tuberculosis. AB - The antibody response to individual epitopes has previously been analysed by competition assay using 125I- or enzyme labelled monoclonal antibodies. A modification of the test is described in which competition of human sera with unlabelled mouse monoclonal antibodies at the limiting dilution is revealed by peroxidase labelled antimouse IgG conjugate. Analysis of 54 sera from patients with pulmonary (36) and extra-pulmonary (18) tuberculosis and 31 controls indicated that the modified test compares favourably with the test based upon directly labelled antibodies. Diagnostic sensitivity for five monoclonal antibodies evaluated was 11.1% (TB78), 35.2% (TB23 and TB68), 37.0% (TB71) and 61.1% (TB72) at 97.5% specificity. For TB72, sensitivity was highest for pulmonary disease (69.7%). The modified assay is also easier to standardise for screening new monoclonal antibodies using a single enzyme-labelled conjugate. PMID- 1717269 TI - Effect of transgenic expression of human alpha 1-acid glycoprotein (AGP) on the glycosylation of human and mouse AGP in various transgenic mouse sera. AB - The occurrence and the glycosylation of human alpha 1-acid glycoprotein (AGP) was studied in two classes of transgenic mice expressing either the A, B and B' genes (ABB'-mice) or only the A gene of human AGP (A-mice). The glycosylation of the human AGP molecules in the transgenic mouse sera was compared with the glycosylation of mouse AGP in the same animal and with human AGP in normal human serum by studying their heterogeneity in binding to concanavalin A (Con A), using crossed affino immunoelectrophoresis (CAIE) with Con A as the affinocomponent in the first dimension gel. Three to four different glycosylated fractions of human as well as mouse AGP were revealed by this method in all the transgenic mouse sera. A close relationship was apparent between the heterogeneities in Con A binding of human and mouse AGP in the same transgenic mouse. The magnitude of this so-called Con A reactivity was, however, strongly dependent on the transgenic mouse studied. Especially within the group of ABB'-mice dramatic changes in Con A reactivity were found when the human AGP genes were expressed. This indicates in the first place that the oligosaccharide chains of the human AGP molecules expressed also mouse-specific features. Secondly, and more importantly, these findings indicate that the expression of the human AGP genes affected the glycosylation process of the transgenic mouse liver. This organ is the source of the AGP forms occurring in serum. We do not know whether this effect has been caused by the introduction or the expression of the human gene(s) or by the presence of human AGP in the Golgi system or in serum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717270 TI - Anti-Coxsackievirus B3 neutralizing antibodies with pathological potential. AB - Adolescent CD-1 mice inoculated with coxsackievirus B3 (CVB3m) will develop acute myocarditis with focal lesions by 7 days post-inoculation (p.i.). Administration of murine sera containing anti-CVB3m-neutralizing antibodies into CVB3m inoculated mice at 3 days p.i. will exacerbate myocarditis, suggesting the presence of pathological antibodies. To study potential pro-inflammatory properties of virus-induced antibodies, a panel of anti-CVB3m-neutralizing monoclonal antibodies (mAbs) was generated. Several studies demonstrated shared epitopes between CVB3m particles and cultured murine cardiac or neonatal skin fibroblasts: (1) one or more mAbs bound to cultured cardiac fibroblasts; (2) several mAbs can participate in complement-mediated lysis of neonatal skin fibroblasts; and (3) at least one mAbs stimulated synthesis of a macrophage chemoattractant from cultured neonatal skin fibroblasts. Injection of one mAb in three doses, each of about 5 micrograms, into adolescent male CD-1 mice induced focal myocarditic lesions which were similar to CVB3m-induced lesions. One mAb induced a diffuse interstitial hypercellularity in most mice and two mAbs did not induce detectable cardiopathology. These data suggest that some anti-CVB3m neutralizing idiotypes (antibodies) which initially can provide protection via virus clearance mechanisms can also bind to cross-reacting epitopes on normal tissues. Binding of antibodies to normal heart tissues could stimulate proinflammatory reactions by several mechanisms and sustain myocarditis. PMID- 1717271 TI - Autoimmune determinants of rheumatic carditis: localization of epitopes in human cardiac myosin. AB - Rheumatic carditis is a sequela of group A streptococcal throat infection. Although the pathogenic mechanisms which lead to heart damage in acute rheumatic fever (ARF) are not well understood, autoimmune processes have been implicated, involving molecular mimicry between streptococci and the human heart. We have studied the immunological cross-reactions between the group A Streptococcus and human heart to understand their molecular and immunological basis. Human and mouse monoclonal antibodies (mAb) and affinity-purified anti-myosin antibodies from acute rheumatic fever sera were characterized and shown to cross-react with group A streptococcal M protein and myosin. Studies of proteolytic fragments of human cardiac myosin identified sites of cross-reactivity in the rod region of the myosin heavy chain. Murine monoclonal antibodies cross-reactive with streptococcal M protein and myosin recognized epitopes located in the S2 and light meromyosin (LMM) subfragments of the heavy chain. None of the cross reactive monoclonal antibodies recognized the S1 subfragment. One broadly cross reactive monoclonal antibody was highly cytotoxic for heart cells in vitro and reactive with the LMM fragment. The data suggest that the cross-reactive epitopes recognized by these antibodies are conformational, dependent upon their alpha helical structures, and potentially damaging to host tissues. PMID- 1717272 TI - Autoantibodies against the beta-adrenergic receptor in human myocarditis and dilated cardiomyopathy: beta-adrenergic agonism without desensitization. AB - The serum of patients with myocarditis and dilated cardiomyopathy contains immunoglobulins capable of causing in vitro acceleration of beating of neonatal rat heart myocytes. This effect is stereoselectively inhibited by (-)-propranolol and is inhibited also by the beta 1-selective adrenergic antagonists, bisoprolol and metoprolol. This effect is not reversed by washing and continues unabated for at least 24 h. PMID- 1717273 TI - Modulation of the inflammatory response in experimental myocardial infarction. AB - The role of inflammation in reperfused, ischaemic myocardium was assessed morphologically in 39 porcine hearts after 24 h (n = 23) and 72 h (n = 16) of reperfusion and after different antiphlogistic treatments. The left anterior descending coronary artery (LAD) was occluded distally for 45 min. Seven pigs received BW755C (10 mg kg-1) i.v. prior to ischaemia (A), eight pigs were given the same dose before 24 h of reperfusion (B), and eight pigs were treated with iloprost (25 ng kg-1 min-1) i.v. before occlusion and continuously for 72 h (C). Two groups of eight pigs each served as controls for 24 h (D) and 72 h (E) of reperfusion. Infarct sizes were determined, myocardium was investigated by light microscopy, and polymorphonuclear leucocytes (PMNs) and macrophages were quantitated after histo- and immunohistochemical staining. Jeopardized myocardium contained 129 neutrophils mm-2 and 120 macrophages mm-2 (D) vs 10 neutrophils mm 2 and 290 macrophages mm-2 (E). Neutrophils and infarct sizes were only significantly decreased in group A (68 neutrophils mm-2, 30% reduction of infarct size). Macrophages infiltration was not significantly affected for all treatment groups (A, B, C). It is concluded that myocardial infarct sizes are neutrophil mediated postischaemic tissue injury can be reduced by BW755C applied prior to ischaemia. Neutrophil-mediated myocardial injury is unlikely to occur beyond 3 days of reperfusion. PMID- 1717274 TI - Molecular cloning of a heart antigen that cross-reacts with a neutralizing antibody to Coxsackievirus B4. AB - A panel of coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies was tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. Immunohistochemical studies revealed an A band pattern of staining of the heart by this antibody. Western immunoblotting of sequential differential extracts of heart showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin. The monoclonal antibody 356-1 was then used to screen a lambda gt11 CD1 mouse heart cDNA expression library. Forty-eight positive plaques were obtained, 14 of which reacted strongly with the antibody and were selected for additional studies. In 13/14 clones, inserts were amplified using the polymerase chain reaction (PCR). The PCR products ranged in size from approximately 150 to 1400 bp. Northern hybridization using these inserts demonstrated that 10/13 recognized a approximately 6.5 kb message in mouse heart total RNA and not in liver total RNA. These amplified inserts were sequenced and were found to contain sequences which encode approximately 1/3 of the amino-terminal end of light meromyosin. By comparison with known sequences of rat alpha and beta cardiac myosin heavy chains, we were able to identify sequences specific for alpha chain as potential cross-reactive autoantigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717275 TI - The possible value of synthetic peptides in the diagnosis and therapy of myocarditis and dilated cardiomyopathy. AB - The adenine nucleotide translocator (ANT) and myosin have been shown to be major autoantigens in myocarditis and dilated cardiomyopathy. We studied the use of synthetic peptides, with sequences derived from ANT and myosin, as antigens in screening tests for autoantibodies in myocarditis (MC) and dilated cardiomyopathy (DCM) and as absorbents for specific elimination of autoantibodies from the sera of patients. Using computer prediction of the secondary structure of the ANT and myosin we identified two sequences of the ANT and three sequences of myosin as possible main antigenic determinants. Using overlapping synthetic peptides and antibodies against them, the antigenicity of the selected determinants was shown. Of 72 sera from patients with MC or DCM 45 (62.5%) bound to the peptides derived from ANT, 32 (44.4%) reacted with the sequences from myosin, in contrast to healthy controls. Using the peptides from the ANT or myosin immobilized on thiopropyl-sepharose, more than 95% of the autoantibodies could be removed specifically from the positive sera. The results demonstrate the usefulness of synthetic peptides as antigens in antibody screening tests in MC and DCM and offers a new approach to the therapy of inflammatory heart disease by specific elimination of autoantibodies. PMID- 1717276 TI - Thromboembolism in gynecologic oncology. AB - The risk of thrombo-embolic complications increases in surgical gynaecological oncology as a consequence of difficult and long-lasting interventions. In gynaecologic operations without any drug prophylaxis thrombosis has been reported by 24-29%, whereas in operations of progressed oncological findings thrombo embolic complications arise in almost every second case. Such complications have to be taken seriously due to difficulty treatable sequelae (post-thrombic syndrome) and due to potentially lethal pulmonary embolism. Furthermore, they are important causes of postoperative early mortality. Diagnosis of a deep thrombosis is insecure even for experienced clinicians. We have various diagnostical means at our disposal, such as phlebography, sounding and 125-iodine-fibrinogen testing. Differentiated drug medication for the prevention and therapy of thrombo embolism is definitely indicated. There are also different kinds of physical and drug-aided means, which can be applied according to the individual situation. PMID- 1717277 TI - Laser palliation of locoregional recurrences of breast cancer. AB - The paper reports on the use of carbon dioxide and Nd:YAG lasers for palliation of locoregional breast cancer recurrences. On the basis of three case reports, pros and cons of laser assisted treatment of loco-regionally recurrent breast cancer are discussed. A carbon dioxide--Nd:YAG combination therapy is proposed as the method best suited. The preliminary results indicate that laser palliation of local relapse and soft tissue metastases might enlarge the therapeutic spectrum. PMID- 1717278 TI - Pretreatment serum levels of C-reactive protein, alpha 1-antitrypsin, haptoglobin, alpha 1-acid glycoprotein and tissue polypeptide antigen in cervical carcinoma. AB - In order to evaluate the potentially additive information of some acute phase reactants to that provided by a general tumour marker, pretreatment concentrations of C-reactive protein, alpha 1-antitrypsin, haptoglobin, alpha 1 acid glycoprotein and tissue polypeptide antigen were determined in serum from healthy women, patients with dysplasia/or carcinoma in situ and patients with primary cervical carcinoma. Specificity varied from 95-100% and sensitivity from 16-29%. A correlation with clinical stage was found for all analytes except for alpha 1-antitrypsin. The latter was the most frequently elevated analyte in early Stages (11/43 in Stage Ib/IIa) and uniquely elevated in 7 cancer patients. Although tissue polypeptide antigen predominantly signaled in advanced stages, 3 women in early stages had elevated tissue polypeptide antigen levels. One of these women died and she was also the only woman with raised alpha 1-antitrypsin who died. It is discussed whether elevated tissue polypeptide levels might represent an unfavourable sign for the individual and if alpha 1-antitrypsin is a favourable sign in early stages of cervical carcinoma. C-reactive protein results were obscured in early stages of disease by the presence of intercurrent illness and the results were regarded as inconclusive. Haptoglobin and alpha 1-acid glycoprotein concentrations provided no additional information to serum alpha 1 antitrypsin levels. However, haptoglobin was elevated in 64% (36/56) of the women with dysplasia/carcinoma in situ of the cervix uteri. PMID- 1717279 TI - Significance of ultrasound appearances in the neurological development and cognitive abilities of preterm infants at 5 years. AB - The developmental outcome at 5 years of age was studied in 93 preterm infants who had been prospectively examined for peri-ventricular leucomalacia (PVL). Standardised neurological examination and developmental assessment, including tests of cognitive function, were carried out. Major sequelae (n = 10) could be ascribed in most cases to the presence of large PVL lesions. Children with normal scans (n = 51), with isolated haemorrhage (n = 15) and with post-haemorrhagic ventricular dilatation (n = 3) had a favourable prognosis. Patients with small focal PVL changes (n = 16) had lower cognitive abilities on the McCarthy scale and presented more abnormal neuromotor signs and more attention deficits when compared to children with normal scans and isolated haemorrhage. Small focal PVL changes seem therefore to interfere with development at 5 years of age and might represent a morphological marker of a more diffuse brain injury. This study demonstrated also the effect of socioeconomic status on outcome at the age of 5 years. PMID- 1717280 TI - Stone incrustation: a relevant complication of the intraprostatic spiral. AB - Six high operative risk patients with urinary retention caused by benign prostatic hyperplasia were managed with an intraprostatic spiral at our hospital. Three of them had severe coronary artery disease, 1 had uremia, 1 had cerebral stroke and 1 had poorly controlled diabetes mellitus. The urinary retention was successfully relieved by the intraprostatic spiral in all patients. No operative mortality or severe complication was encountered. One patient experienced a repeat attack of urinary retention due to proximal migration of the spiral. Four patients complained of urgency, which was relieved by anticholinergic agents. Stone incrustation was found on 2 out of 3 spirals removed (66%), and the stone turned out to be calcium phosphate and struvite by scanning electron microscopy and infrared spectrophotometry. In 1 patient, stone formation was so abundant that it almost obstructed the lumen of the redundant tip of the spiral. From our preliminary results, the intraprostatic spiral seems to be a good alternative to an indwelling catheter for patients awaiting prostatectomy. Nevertheless, the potential complication of stone incrustation should be anticipated and it is suggested to remove the device as soon as possible or to replace it at regular intervals. PMID- 1717281 TI - Structure-function relationship and immunochemical mapping of external and intracellular antigenic sites on the lymphocyte activation inducer molecule, AIM/CD69. AB - Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein structure of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation and is implicated in the induction of T and B cell proliferative responses. Cross-competition monoclonal antibodies (mAb) binding assays have allowed the definition of four antigenic epitopes. Three of them (antigenic sites E1-3) are extracellular while the fourth (site I) is a sequential epitope localized intracellularly and highly conserved interspecies. Site E1 is shown to be an immunodominant antigenic determinant closely related to a functional domain of AIM important for triggering of T cell proliferation. Studies of peptide fragmentation of the two isolated AIM subunits with different proteases have demonstrated that both AIM chains are differentially glycosylated forms of a single 24-kDa core protein. Moreover, the two denatured and isolated AIM chains share common epitope(s) as demonstrated by their reactivity with an mAb by both Western blot analysis and immunoprecipitation of the separated AIM subunits. Biosynthesis studies revealed the rapid appearance of two intermediate precursor forms of 29 and 26 kDa which arise from the 24-kDa unglycosylated AIM polypeptide. This 24-kDa unglycosylated form could be also precipitated from iodinated cells pretreated with tunicamycin, indicating that glycosylation of the protein was neither required for AIM cell surface expression nor for acquisition of external epitopes E1-E3. Cell treatment with pronase resulted in the loss of the external epitopes E1-3 and the generation of a proteolytic peptide of 16 kDa that could be precipitated by the anti-AIM mAb specific for the internal site I. This proteolytic fragment retained the transmembrane and cytoplasmic regions of the molecule where both epitope I and phosphorylation sites reside. These results demonstrate that AIM is an integral membrane homodimeric glycoprotein with a large cytoplasmic domain probably involved in the activation signals transduced through this molecule to lymphocytes. PMID- 1717282 TI - High precursor frequency of human T cells reactive to HLA-DQ molecules expressed on mouse L cell transfectants. AB - In humans, the HLA-DR molecule is a major stimulatory molecule of allogeneic mixed lymphocyte reactions (MLR) and a major restriction molecule for the presentation of soluble antigens to the T cell. Little is known of the biological function of HLA-DQ. To examine the size of the repertoire of precursor T cells recognizing the autologous or allogeneic HLA-DQ molecule, the frequency of T cells reactive to HLA-DQ was estimated in comparison with T cells reactive to HLA DR. We made use of a limiting dilution analysis and mouse L cells transfectants expressing the HLA-DR or -DQ molecule, as stimulators. Human T cells recovered from a primary MLR stimulated with allogeneic peripheral blood lymphocytes (PBL) proliferated in response to L cell transfectants expressing HLA class II genes shared by the stimulator cells in the primary MLR. This observation suggested that the HLA class II molecules on L cell transfectants shared to some extent epitopes for alloreactive T cells with those expressed on human PBL. The precursor frequencies of CD4+ T cells reactive to allogeneic or autologous DQ molecules were as high as those of T cells reactive to allogeneic DR molecules and were estimated to be 1/800-1/1800. The frequency of the T cells reactive to autologous DR molecules was low (1/7200-1/16,000). The biological significance of the high frequency of HLA-DQ-reactive precursor T cells is discussed. PMID- 1717283 TI - Heterogeneous expression of CD23 epitopes by eosinophils from patients. Relationships with IgE-mediated functions. AB - The receptor for IgE (Fc epsilon RII) on human eosinophils presents some common characteristics with CD23, a differentiation marker of B cells. We have used flow cytometry for evaluating the expression of various epitopes of CD23 on purified eosinophils from patients with eosinophilia. A correlation was found between the binding of myeloma IgE protein and the binding of a monoclonal antibody (mAb 135), directed against the IgE-binding site of B cell CD23. Using two additional anti-CD23 mAb, directed (8-30) or not (3-5) against the IgE-binding site, a low expression of these CD23 epitopes was observed on eosinophils from different eosinophilic patients. Northern blot analysis of eosinophil RNA with the cDNA probe of CD23 revealed a low-abundance transcript in three of the six patients expressing membrane CD23. The inhibition by all anti-CD23 mAb of IgE-mediated cytotoxicity and IgE binding to eosinophils clearly indicated the participation of a CD23-related molecule in IgE-dependent eosinophil functions. PMID- 1717284 TI - A possible novel pathway of regulation by murine T helper type-2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase on macrophages. AB - Murine peritoneal macrophages activated with interferon (IFN)-gamma and lipopolysaccharide (LPS) produce high levels of nitric oxide (NO) and are efficient in killing the intracellular protozoan parasites Leishmania major in vitro. Earlier studies have shown that NO, whose synthesis in murine macrophages is catalyzed by an inducible enzyme NO synthase, plays a major effector role in the host resistance against microbial infection. We now shown that both the NO synthesis and the leishmanicidal activity can be inhibited by prior treatment of the cells with recombinant interleukin 4 (IL4). IL4 treatment had no effect on the binding of IFN-gamma to macrophages but prevented the induction of NO synthase in these cells activated with IFN-gamma and LPS. Since IFN-gamma is produced by murine T helper type-1 (Th1) cells, whereas IL4 is secreted by Th2 cells, these results suggest a novel pathway by which Th2 cells regulate an activity of Th1 cells, namely by inhibiting the induction of NO synthase. These results may also account for the mechanism by which the disease-promoting Th2 cells counteract the host-protective effect of Th1 cells in leishmaniasis and other intracellular parasitic diseases. PMID- 1717285 TI - Inhibition of immunological memory and T-independent humoral responses by monoclonal antibodies specific for murine complement receptors. AB - The importance of the complement system for mounting an antibody response in vivo was investigated by down-regulating and blocking the complement receptors (CR) in mice with three different monoclonal rat antibodies (mAb): mAb 8C12 recognized the C3b-binding site of CR1, mAb 7G6 recognized another site of CR1 and the C3d binding site of CR2 and mAb 7E9 again recognized other epitopes on CR1 and CR2. We have earlier shown that 7G6, is the only mAb that completely suppresses the primary antibody response to horse erythrocytes. This antibody was also shown to suppress induction of immunological memory and a secondary antibody response. In contrast to what seems to be the case for thymus-dependent antibody responses, all three mAb could inhibit the antibody response to a thymus-independent antigen, dextran B 1355S. PMID- 1717286 TI - Induction of nitric oxide synthase in rat peritoneal neutrophils and its inhibition by dexamethasone. AB - Rat peritoneal polymorphonuclear leukocytes (PMN) elicited with oyster glycogen contain a Ca(2+)-independent nitric oxide (NO) synthase which is induced in vivo in a time-dependent manner. When washed PMN containing low levels of enzyme activity were cultured ex vivo further expression of NO synthase was observed. This was inhibited by cycloheximide indicating that de novo synthesis of the enzyme occurred during the ex vivo incubation. Enzyme activity was enhanced by interferon (IFN)-gamma, but not by tumor necrosis factor (TNF)-alpha when added ex vivo. However, IFN-gamma and TNF-alpha synergized to increase further the expression of NO synthase. Treatment of rats with dexamethasone inhibited the induction of NO synthase in elicited PMN. This treatment reduced the accumulation of PMN by approximately 30%, without affecting cell viability. Dexamethasone also inhibited the induction of the NO synthase ex vivo in a concentration-dependent manner. Furthermore, the enhanced enzyme activity following treatment of PMN with cytokines was also inhibited by dexamethasone. Once induced, dexamethasone did not affect enzyme activity. These data indicate that PMN elicited in the rat peritoneum with oyster glycogen express an NO synthase in vivo and ex vivo. The induction of the enzyme can be further stimulated ex vivo with IFN-gamma and TNF alpha and inhibited by dexamethasone. The inhibition of the induction of NO synthase in the PMN by dexamethasone may contribute to the anti-inflammatory activity of this and other glucocorticoids. PMID- 1717287 TI - The c-kit-encoded tyrosine kinase regulates the proliferation of early pre-B cells. AB - A monoclonal antibody (mAb; ACK2) recognizing the extracellular domains of the c kit-encoded tyrosine kinase has been employed to demonstrate that c-kit is involved in B lymphocyte development. The c-kit-encoded tyrosine kinase is expressed on the surface of normal DHJH-rearranged murine pre-B cell clones which proliferate continuously at that stage in vitro on stromal cells and in the presence of recombinant interleukin 7. These pre-B cell clones, capable of differentiation to surface immunoglobulin-positive B cells in vitro and in vivo, are inhibited by the mAb in their proliferation while remaining capable of differentiation to surface immunoglobulin-positive B cells. Stimulation of mature B cells by mitogens is unimpaired by the mAb. This indicates that c-kit regulates early antigen-independent, but not late antigen-dependent, B cell development. PMID- 1717288 TI - Thymic epithelial cell-derived supernatants sustain the maturation of human prothymocytes: involvement of interleukin 1 and CD23. AB - During their development, human CD7+ lymphoid stem cells migrate into the thymus where, following intimate contact with thymic tissue, they proliferate and differentiate into functionally mature T lymphocytes. In this study, we investigated the effect of thymic epithelial cell-derived supernatants (TEC-SN) on early CD7+CD2-CD3- thymocytes. Our results indicate that TEC-SN are able to promote CD2 and CD3/TcR alpha/beta expression by CD7+ precursors. This activity correlated with soluble CD23 and interleukin 1 levels in TEC-SN. Furthermore, monoclonal antibodies to these cytokines decreased in vitro maturation of prothymocytes. Thus, in addition to cell-cell interactions, human TEC produce cytokines able to support early steps of thymocyte differentiation. PMID- 1717289 TI - In vivo persistence of a HIV-1-encoded HLA-B27-restricted cytotoxic T lymphocyte epitope despite specific in vitro reactivity. AB - A large number of human immunodeficiency virus type 1 (HIV-1) specific HLA restricted cytotoxic T cell (CTL) epitopes have been mapped, including an HLA-B27 restricted immunodominant epitope within p25gag. Accordingly, this segment of the HIV-1 provirus was amplified by the polymerase chain reaction from DNA derived from fresh uncultured peripheral blood mononuclear cells (PBMC) of four HLA-B27 HIV-1-infected individuals. In all cases the majority of infected PBMC bore sequences encoding the HLA-B27-restricted peptide. CTL escape mutants had not accumulated in vivo 8 and 14 months later despite demonstrable CTL activity in vitro. These data emphasize the importance of silently infected lymphocytes in evading immune surveillance. PMID- 1717290 TI - Tachykinin receptors in the guinea-pig isolated bronchi. AB - The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery. PMID- 1717291 TI - Species differences in affinities of non-peptide antagonists for substance P receptors. PMID- 1717292 TI - The conditions of Ca2+ entry via L-type channels for induction of serotonin release from rabbit hippocampus. AB - The L-type voltage-sensitive calcium channel (VSCC) agonists of the dihydropyridine (DHP) type, Bay K 8644 and (+)-202-791, concentration dependently enhanced the K+ (26.2 mM)-induced 5-HT release from slices of rabbit hippocampus prelabelled with [3H]5-HT when the slices were treated with the monoamine oxidase (MAO) inhibitor, pargyline. The DHP agonists were ineffective on K+ (26.2 mM) induced release in the absence of pargyline. However, when omega-conotoxin GVIA pretreatment of the slices irreversibly blocked N-type VSCCs, (+)-202-791 markedly enhanced the release of 5-HT evoked by 26.2 mM K+. Thus, at this rather strong stimulus intensity either an increase in the (preferentially cytoplasmic) transmitter pool or blockade of N-type VSCCs was necessary in order to unmask agonist-activated L-type VSCCs. Reduction of the depolarization intensity from 26.2 to 17.2 mM K+, given for 8 min, strongly intensified the stimulatory effects of L-type VSCC agonists irrespective of the use of pargyline under these conditions. The concentration-response curve of (+)-202-791 was 'competitively' shifted to the right by the enantiomer, (-)-202-791, with a pA2 value of 8.6. In conclusion, N- and L-type VSCCs seem to differ in their relation to the cellular machinery for 5-HT release, the latter getting markedly operative when a weak and sustained depolarization is applied or when N-type VSCCs are blocked or when the cytoplasmic transmitter pool is expanded by inhibition of MAO. PMID- 1717293 TI - Parathyroid hormone fragment 1-34 inhibits drug-induced inflammation in various experimental models. AB - We investigated the effect of the administration of rat parathyroid hormone-(1 34) on acute or chronic inflammatory processes in different experimental animal models. Fragment 1-34 of parathyroid hormone had an inhibitory effect in all inflammatory acute tests. The dose-response experiments showed that the maximal anti-inflammatory and anti-exudative effects appeared at the dose of 3.30 and 0.33 micrograms/kg, respectively. The anti-inflammatory effect was observed in the first phase of the inflammatory process. In the carrageenin-induced edema test the anti-inflammatory activity began to decline after 180 min. In contrast, this peptide was inactive in the inflammatory chronic test we used. PMID- 1717294 TI - Type-1 and type-2 angiotensin II receptors in fetal rat brain. AB - Brain angiotensin II receptors were located in 18-day-old rat embryos by quantitative autoradiography. In the nucleus of the solitary tract and choroid plexus, binding was displaced by the type-1 antagonist DuP 753. In the inferior olive, paratrigeminal and hypoglossal nuclei, binding was displaced by the type-2 antagonist CGP 42112 A. Meninges and cephalic soft tissues contained predominantly type-2 angiotensin II receptors. Our results indicate a role for type-1 and type-2 angiotensin II receptors during brain development. PMID- 1717295 TI - Effects of the antidepressant drug tianeptine on plasma and platelet serotonin concentrations in the rat. AB - The antidepressant drug tianeptine increased rat plasma 5-hydroxyindoleacetic (5 HIAA) concentrations after single and repeated treatment for 14 days. This effect was not due to an increase in 5-hydroxytryptamine (5-HT) synthesis, as measured by the accumulation of 5-hydroxytryptophan in plasma and brain. A concurrent decrease in platelet-free plasma 5-HT concentrations occurred after subchronic treatment. Tianeptine attenuated the large increase in 5-HT concentrations in platelet-free plasma elicited by the specific 5-HT uptake inhibitors citalopram and paroxetine. These data are compatible with the reported 5-HT uptake-enhancing activity of this antidepressant drug. PMID- 1717296 TI - Effects of the 1-amino-adamantanes at the MK-801-binding site of the NMDA receptor-gated ion channel: a human postmortem brain study. AB - Recent studies from our laboratory have provided evidence that the 1-amino adamantane derivative memantine (1-amino-3,5-dimethyl-adamantane) binds to the MK 801-binding site of the N-methyl-D-aspartate (NMDA)-receptor-gated ion channel. This action has been suggested to account for the antiparkinsonian and antispastic activity of the drug. In the present investigation we have extended our work by testing a series of 1-amino-adamantanes, including amantadine (1 amino-adamantane) and memantine, for their ability to compete with [3H]MK-801 binding in membrane homogenates of postmortem human frontal cortex. The most potent substance (1-amino-3,5-diethyl-adamantane) had a Ki-value of 0.19 +/- 0.06 microM while the weakest substance (1-N-methyl-amino-adamantane) had a Ki-value of 21.72 +/- 1.63 microM. The Ki-value of amantadine was 10.50 +/- 6.10 microM. In agreement with our earlier investigation, the Ki-value of memantine was 0.54 +/- 0.23 microM. The results indicate that 1-amino-adamantanes, in general, may produce their pharmacological effects through an interaction with the NMDA receptor-gated ion channel. The displacement of [3H]MK-801 binding thus may provide the basis to predict the antiparkinsonian and antispastic activity of novel substituted 1-amino-adamantanes and possibly of other drugs. PMID- 1717297 TI - Root resorption after local injection of prostaglandin E2 during experimental tooth movement. AB - The purpose of this study was to investigate the occurrence of orthodontic root resorption in connection with local injection of prostaglandin E2 (PGE2). The material consisted of 25 male Wistar rats. The control group comprised six animals where no force was applied. In five animals 0.1 ml of 0.1 micrograms/microliter PGE2 was injected in the gingival area of the upper right first molar. In one animal no PGE2 was injected. The animals were killed after 3 days. The experimental tooth movement groups consisted of 19 animals. Duration of experiments was 3 days, 7 days, and 10 days. The maxillary first molars on both sides were each moved mesially by means of a coil spring. On the right side 0.1 ml of PGE2 0.1 micrograms/microliters was injected in the gingiva on the buccal side of the upper first molar on days 0, 3, 5, and 7. On the left side no injection of PGE2 was performed. In three animals in the 7-day group the vehicle (Waymouth medium) was injected. There was no significant difference in root resorption between the experimentally moved teeth with and without local injection of PGE2, but a trend towards more root resorption was registered on the teeth where such injections had been performed. PMID- 1717299 TI - The challenge of treating Graves'-Basedow's disease patients with antithyroid drugs (ATD) in a large university hospital. PMID- 1717298 TI - Differences and similarities in the treatment of diffuse goiter in Europe and the United States. AB - In two separate studies, members of the American Thyroid Association (ATA), and the European Thyroid Association (ETA) were surveyed by questionnaire on their management of Graves' disease. The aim was to determine how expert clinical thyroidologists employ three different therapies which are available for this disorder. Herein we summarize, compare and contrast similarities and differences in the results of these surveys. For the index patient, radioiodine (RAI) was the therapy of choice for 69% of ATA respondents but only 22% of ETA respondents. In contrast, only 30.5% of ATA respondents chose antithyroid drugs as first line therapy compared to 77% of ETA respondents. There was consensus on the relative lack of a role for thyroidectomy. The implications of these differing approaches to the treatment of hyperthyroidism due to Graves' disease are discussed. PMID- 1717300 TI - The evolution of the ophthalmopathy during antithyroid drug therapy and its relationship with serum thyroglobulin and antithyroglobulin antibody in patients with Basedow's disease. PMID- 1717301 TI - Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris. AB - Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes. PMID- 1717302 TI - Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of the human factor-dependent cell line, M07e. AB - Murine mast cell growth factor (muMGF), a c-kit ligand, has additive to greater than-additive effects on in vitro colony formation of murine and human myeloid progenitor cells stimulated with erythropoietin, granulocyte-macrophage colony stimulating factor (GM-CSF), and/or interleukin (IL)-3. To confirm direct-acting effects on responding cells, MGF was assessed alone and in combination with other cytokines for effects on the proliferation of the human factor-dependent cell line, M07e. Proliferation was assayed in liquid culture by [3H]thymidine uptake and in semisolid medium by colony formation. Purified recombinant (r) muMGF (25 50 ng/ml) by itself had proliferative activity but less than r human (hu) GM-CSF. In combination with rhuGM-CSF (250 U/ml) or IL-3 (500 U/ml), rmuMGF (25 ng/ml) enhanced [3H]thymidine uptake two- to sevenfold greater than the sum of the effects of each factor alone. Similar enhancement was seen in the number and size of colonies formed. When MGF was used in combination with rhuIL-4 (500-1000 U/ml), rhuIL-6 (5 ng/ml), rhuIL-9 (5-10 U/ml), or rhu interferon gamma (IFN gamma; 250-500 U/ml) (factors that alone stimulate little proliferation), [3H]thymidine uptake and colony formation were respectively increased 2- to 11- and 3- to 55-fold over the sum of each of the effects of the factors alone. Exposure of 5 x 10(5) cells/ml to 50 ng/ml MGF for 24 h, a time during which synergism is noted with MGF plus either GM-CSF or IL-3, did not change GM-CSF or IL-3 receptor binding affinity or the number of binding sites. Exposure of cells to MGF for 48 h did not alter subsequent GM-CSF- or IL-3-stimulated proliferation. The results suggest that M07e cells will be useful as a model for the analysis of intracellular biochemical mechanisms of the direct-acting proliferative and synergistic effects of MGF. PMID- 1717303 TI - Modifications of serotonin-, substance P- and calcitonin gene-related peptide like immunoreactivities in the dorsal horn of the spinal cord of arthritic rats: a quantitative immunocytochemical study. AB - Adjuvant-induced arthritis has been produced in adult rats in order to study the reorganization of serotonergic innervation in the spinal cord dorsal horn in a model of chronic pain. Immunocytochemical detection of CGRP and substance P was quantified with an image analyzer, and we found a transient increase for both peptides at 1 and 2 months, followed by a decrease to control levels after four months. At variance, quantification of serotonergic immunoreactivity showed a significant increase which persisted throughout the study. The significance of this finding is discussed with comparison of other experimental models involving reorganization of primary afferents to the spinal cord. PMID- 1717304 TI - Anatomical plasticity of the tectospinal tract after unilateral lesion of the superior colliculus in the neonatal rat. AB - After unilateral ablation of the superior colliculus (SC) in neonatal or adult rats, the reorganization of the tectospinal tract (TST) was examined using the technique of anterograde transport of horseradish peroxidase to which wheat germ agglutinin had been conjugated (WGA-HRP). In neonatally lesioned rats, aberrant labeled terminals of TST axons were found on the ipsilateral side of the spinal cord. Postnatal development of the TST was then studied by retrograde transport of HRP to determine whether the aberrant tectospinal projections resulted from normally transient ipsilateral projections that persisted in operated rats or were due to collateral sprouting of projections to the contralateral projection field. The results failed to show an ipsilateral projection from the SC to spinal cord in normal neonatal rats. However, in neonatally lesioned rats, aberrant labeled fibers were observed recrossing the midline of the cervical spinal cord. Therefore, the increase in labeled terminals on the ipsilateral side following unilateral SC ablation appeared to originate from collateral sprouting at the spinal cord level of TST fibers from the intact pathway. PMID- 1717305 TI - Ultrastructure and synaptic associations of auditory thalamo-amygdala projections in the rat. AB - Projections from the acoustic thalamus to the lateral nucleus of the amygdala (AL) have been implicated in the formation of emotional memories. In order to begin elucidating the cellular basis of emotional learning in this pathway, the ultrastructure and synaptic associations of acoustic thalamus efferents terminating in AL were studied using wheat-germ agglutinated horseradish peroxidase (WGA-HRP) and Phaseolus vulgaris Leucoagglutinin (Pha-L) as ultrastructural anterograde axonal markers. The tracers were injected into those areas of the thalamus (medial division of the medial geniculate body and posterior intralaminar nucleus, MGM/PIN) known both to project to AL and to receive afferents from the inferior colliculus. Terminals labeled with WGA-HRP or Pha-L in AL contained mitochondria and many small, round clear vesicles and 0-3 large, dense-core vesicles. Most labeled terminals formed asymmetric synapses on unlabeled dendrites; of these the majority were on dendritic spines. These data demonstrate that projections from the acoustic thalamus form synapses in AL and provide the first characterization of the ultrastructure and synaptic associations of sensory afferent projections to the amygdala. PMID- 1717307 TI - Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures. AB - Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA. PMID- 1717306 TI - Avian locomotion activated by brainstem infusion of neurotransmitter agonists and antagonists. I. Acetylcholine excitatory amino acids and substance P. AB - Previous studies have demonstrated that focal electrical stimulation of regions within the brainstem of a decerebrate bird will elicit all the normal patterns of avian locomotion. However, electrical stimulation can activate a variety of neuronal elements within the radius of effective current spread, including axons of passage traversing the stimulation point. To restrict activation to neuronal cell bodies within the immediate vicinity, we have utilized direct intracerebral injection of neurotransmitters, their agonists and antagonists, into identified brainstem locomotor regions. To undertake these studies, birds (geese or ducks) were placed in a stereotaxic frame and decerebrated under halothane anesthesia. After completion of surgery, several discrete locomotor regions were first identified with electrical microstimulation. Acetylcholine (ACh) and excitatory amino acid (EAA) agonists and antagonists, as well as Substance P were then slowly infused into each brainstem region. Any change in locomotor behavior was recorded by electromyographic techniques. When injected into a variety of sites, carbachol (an ACh nicotinic (AChN) and muscarinic (AChM) agonist) and pilocarpine (an AChM agonist) evoked locomotion, whereas atropine (an AChM antagonist) blocked locomotion. N-methyl-D-aspartate NMDA), but not glutamate, also elicited locomotion or reduced the current intensity threshold for electrically-evoked locomotion. The NMDA-induced locomotion evoked locomotion. The NMDA-induced locomotion could be blocked by the injection of glutamic acid diethyl ester (GDEE, an EAA antagonist) or D-2-amino-5-phosphonopentanoic acid (AP5) into the same site. Finally. Substance P also evoked locomotion. The above observations strongly suggest that brainstem electrically-stimulated locomotion in decerebrate birds is not due to activation of fibers traversing a brainstem locomotor region, but instead, is due to the activation of receptors located on neuronal cell bodies, dendrites or presynaptic terminals in the immediate vicinity of the micropipette tip. After correlating our findings with similar lamprey and mammalian studies, the comparable discoveries serve to underscore the suggestion that the neuroanatomical substrates underlying the brainstem control of locomotion appear to be highly conserved in all vertebrates. PMID- 1717308 TI - Loss of hippocampal acetylcholinesterase staining after fornix lesion in the monkey. AB - Cholinergic denervation of the hippocampal formation has been extensively studied in rodents but not in primates. Therefore we studied the changes in acetylcholinesterase histochemical staining of the hippocampus occurring after bilateral transection of the fornices in the cynomolgus monkey. Animals were sacrificed 1.5, 6, 13, and 23 weeks after surgery. We found a 40-50% reduction in the density of acetylcholinesterase-positive fibers in the four analyzed regions (dentate gyrus, CA3, CA1, and subiculum) 1.5 week after surgery and a 60-80% reduction at longer time intervals. The characteristic diffuse AChE staining found in hippocampi from control animals disappeared after fornix lesion, except in the inner third of the molecular layer of the dentate gyrus. We did not find any evidence of spontaneous cholinergic reinnervation over the 6-month period. Thus, as in rats, fornix lesion produces dramatic changes in hippocampal AChE staining, presumably caused by a massive cholinergic denervation. However, in contrast to rodents, spontaneous reinnervation does not seem to occur in the months following the lesion in primates. PMID- 1717309 TI - Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody. AB - The monoclonal antibody (mAB) L1, which binds to the nucleocapsid protein of canine distemper virus (CDV), was shown to bind to avirulent CDV obtained after serial passages in Vero cells, but not to two different virulent demyelinating CDV-strains propagated in dog glial cell cultures. However, when both virulent CDV-strains were passaged through Vero cells they expressed, after a number of passages, an epitope recognized by mAB L1. The occurrence of the L1 epitope appeared to coincide with loss of virulence in animal inoculation experiments. PMID- 1717310 TI - Epidemiological typing of Neisseria gonorrhoeae: a comparative analysis of three monoclonal antibody serotyping panels. AB - Sixteen hundred and thirty seven isolates of Neisseria gonorrhoeae isolated over a two year period were serotyped using three panels of monoclonal antibodies. The isolates comprised 687 serogroup WI strains and 950 serogroup WII/III strains. The antibodies used in the panels were those developed by Pharmacia (Ph) and Genetic Systems (GS). The GS antibodies were used as two separate panels; the American (GS-A) panel which designates serovars by a simple numerical nomenclature and the Swedish (GS-S) panel which designates serovars by a more detailed descriptive nomenclature. Using only a single panel the Ph panel gave the greatest discrimination yielding 42 serovars compared with 28 serovars with the GS-S panel and 23 serovars with the GS-A panel. Using two panels in combination, the GS-A/Ph panel combination gave the greatest discrimination with 81 serovar combinations while a combination of the GS-S and Ph panels yielded 77 serovar combinations. A compilation of antibodies from all three panels yielded 90 serovar combinations. As the combination of GS-A and Ph panels gave a greater degree of discrimination than the combination of the GS-S and Ph panels we advocate that first-stage serotyping should be performed with the more widely used GS-A panel while second-stage serotyping should be performed with the Ph panel. We propose that serovar combinations should be reported using a dual nomenclature e.g. IA-1/Arost, IB-1/Bropt, IB-1/Bropyt etc. The use of such a dual system would allow "core comparison" between all centres while maintaining a degree of flexibility regarding the extent of discrimination required. PMID- 1717311 TI - Use of monoclonal anti-haemagglutinin antibodies for the "in vitro" selection of a sequential influenza virus antigenic variant. AB - A sequential antigenic variant of the A/Texas/77 (H3N2) influenza virus was obtained in vitro using a monoclonal antibody against the haemagglutinin (HA) of the antigenic variant V18 previously selected in vitro from the parental Texas virus. The sequential antigenic variant, designated DV1, the V18 antigenic variant and the parental A/Texas/77 viruses were used to evaluate the frequency of anti-haemagglutinin antibodies in human sera in single radial haemolysis assays. Twenty six of 100 children's sera, which contained antibodies to the parental A/Texas/77 virus, failed to react with the V18 antigenic variant. A higher proportion of sera (42%) failed to react with the DV1 antigenic variant with alterations in two different antigenic determinants with respect to the parental virus. The results are discussed in relation to the mechanism of antigenic drift and to the in vivo reaction of antigenic variants selected in vitro. PMID- 1717312 TI - Folate-deficient human lymphoblasts: changes in de novo purine and pyrimidine synthesis and phosphoribosylpyrophosphate. AB - Peripheral blood lymphocytes from healthy volunteers cultured with phytohaemagglutinin in a folate-deficient medium exhibit megaloblastic maturation and reduced cellular folate content. For these cells, changes in de novo purine and pyrimidine synthesis and cellular phosphoribosylpyrophosphate (PRPP) have been determined. Mitogen-stimulated cells cultured with undialyzed pooled human serum (PHS) exhibit undetectable de novo purine synthesis, a three-fold increase in PRPP content and augmented de novo pyrimidine synthesis; these changes are independent of cellular folate status. Folate-replete cells cultured with PHS which was dialyzed to reduce purine compounds concentrations show markedly increased de novo purine synthesis. The PRPP content and pyrimidine synthesis rates of these cells are similar to those of folate-replete cells cultured with undialyzed PHS. Folate-deficient cells cultured in dialyzed PHS show a 10-fold reduction in purine synthesis and a corresponding increase in PRPP levels. Pyrimidine synthesis was moderately reduced. The purine bases hypoxanthine and adenine markedly reduced the augmented purine synthesis of folate-replete cells or the increased PRPP content of folate-deficient cells cultured with dialyzed PHS. These findings suggest that cellular folate status is critical for de novo purine synthesis only when coupled with purine bases restriction and that the latter are efficient regulators of de novo purine synthesis. PMID- 1717313 TI - A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor. AB - The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutated Torpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the alpha-subunit (alpha T244), beta S250, gamma T253 and delta S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed alpha-helical configuration of the M2-containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the gamma-subunit, are asymmetrically positioned at the channel constriction. PMID- 1717314 TI - Cloning and sequence analysis of a human type A/B hnRNP protein. AB - A cDNA encoding a 284 residue long type A/B hnRNP protein has been cloned. This protein, previously referred to as type C [(1987) J. Biol. Chem. 262, 17126 17137], is an RNA unwinding protein from HeLa 40S hnRNP with a high affinity for G- followed by U-rich sequences. The N-terminal part of the protein contains two consensus RNA binding domains present in a number of other RNA binding proteins. The C-terminal part is glycine-rich and contains a potential ATP/GTP binding loop. The distribution of charged amino acids is highly uneven and there are multiple potential phosphorylation sites. PMID- 1717315 TI - A cyclic-AMP-gated conductance in cochlear hair cells. AB - The patch clamp technique was used to record cAMP-dependent currents of the guinea pig cochlear hair cell plasma membrane. Data obtained indicate that the channels passing this current are moderately selective for monovalent cations and are effectively blocked by L-cis-diltiazem and reversibly blocked by 1 mM Mg2+ or Ca2+. The single-channel unit conductance estimated in the absence of divalent cations is about 16 pS. The results demonstrate that cyclic nucleotide-dependent channels of cochlear hair cells are virtually identical to the photoreceptor and olfactory ones. PMID- 1717316 TI - Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA. AB - The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3' end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A2660, which lies in a sequence that is highly conserved in all species. PMID- 1717317 TI - Nitromethylene actions on in situ and expressed insect nicotinic acetylcholine receptors. AB - Single channel recordings from dissociated housefly (Musca domestica) neurons show that a novel type of nitromethylene insecticide, 2(nitro methylene)tetrahydro-1,3-thiazine (NMTHT) gates a channel, the conductance and open time histogram of which resemble those obtained when acetylcholine is the agonist. Injection into Xenopus oocytes of a locust (Schistocerca gregaria) alpha subunit mRNA results in the expression of functional nicotinic receptors sensitive to NMTHT. Control oocytes injected with distilled water are insensitive to the same concentration of this compound. Thus NMTHT exhibits agonist actions at both in situ and expressed insect nicotinic receptors, and one site of action of this compound is on an insect nicotinic receptor alpha-subunit. PMID- 1717318 TI - Immunopotentiation reverses the embryotoxic effect of serum from women with pregnancy loss. AB - OBJECTIVE: To assess the effect of sera of women with habitual abortions (AB) on attachment and spreading of mouse blastocysts in vitro. DESIGN: Expansion, attachment, and spreading were the mouse blastocyst parameters utilized. Deoxyribonucleic acid (DNA) synthesis and cell markers expression were also assayed by autoradiography analysis and the indirect immunofluorescent technique. SETTING: Sera were drawn from patients attending a habitual AB clinic in a tertiary care university hospital. PARTICIPANTS: Thirty-nine serum samples were drawn from habitually aborting women and the effect compared with 17 control AB sera. INTERVENTION: Habitually aborting women were immunized with paternal leucocytes; 18 post-immunization sera were also assessed. OUTCOME AND RESULTS: After 48 hours, there was delayed attachment and spreading (4% of test blastocysts spread as compared with 50.5% of controls). This was more profound after 72 hours culture (7.5% spread as compared with 72.8% of controls). Experimental sera were capable of reducing DNA synthesis, cytokeratin, fibronectin, or placental alkaline phosphatase expression by blastocyst cells. Leucocyte immunization of women with habitual ABs, clearly reversed the embryotoxic effect of the sera and enhanced cell markers expression. CONCLUSIONS: These data suggest that immunopotentiation may improve blastocyst survival in utero. PMID- 1717319 TI - Rupture of ectopic pregnancy in women with low and declining serum beta-human chorionic gonadotropin concentrations. AB - Rupture of tubal EP in two women whose serum beta-hCG levels were low and declining was reported. It suggests that low and falling serum beta-hCG levels are not always associated with resolution of EP and tubal rupture can still occur. PMID- 1717320 TI - Understanding pathologists (an exercise in communication): 4. Understanding special laboratory procedures. PMID- 1717321 TI - Expression of syndecan gene is induced early, is transient, and correlates with changes in mesenchymal cell proliferation during tooth organogenesis. AB - Syndecan is an integral cell surface proteoglycan which contains an extracellular matrix-binding domain and a cytoskeleton-associated domain and may therefore transfer changes in the extracellular environment to cellular behavior. Changes in syndecan gene expression during embryonic and early postnatal mouse tooth development were analyzed by in situ hybridization and compared with the distribution of syndecan core protein and cell proliferation studied by immunohistochemistry. Syndecan RNA became accumulated in the condensing mesenchymal cells around the invaginating epithelial tooth bud during early development, and this accumulation became more intense when morphogenesis advanced to the cap stage. During the bell stage, when the cuspal pattern of the tooth is established, syndecan transcripts were lost, and RNA was not detected in the terminally differentiated or postmitotic odontoblasts. In the epithelium, syndecan RNA was intensely expressed in the invaginating epithelial bud, but the expression was reduced during the cap and bell stages. However, local stimulation in syndecan gene expression was observed in the epithelial preameloblasts immediately preceding their terminal differentiation into ameloblasts, which was accompanied by a complete loss of transcripts. There was a close correlation between the changes in syndecan transcripts and the distribution of syndecan core protein. Furthermore, analysis of cell proliferation by immunohistochemical detection of BrdU incorporation revealed that in the mesenchyme, but not in the epithelium, syndecan was intensely expressed by proliferating cells. The analysis of mRNA by Northern blot indicated that the transcripts in mesenchymal and epithelial cells were of similar size. In the slot-blot analysis the changes in syndecan transcripts correlated with the overall changes observed in the in situ hybridization analysis. The role of tissue interactions in the regulation of the syndecan gene was studied by using tissue recombination cultures of separated epithelial and mesenchymal components of the early tooth germ. The in situ hybridization and Northern blot analysis of these explants showed that the expression was increased in the mesenchyme cultured in contact with the epithelium. Our results indicate that syndecan gene expression in the embryonic tooth mesenchyme is induced by epithelial-mesenchymal interactions and thereafter expressed stage-dependently and transiently by the differentiating cells during organogenesis. The association of syndecan expression with mesenchymal cell proliferation raises the possibility that, in addition to behaving as a matrix receptor, syndecan may have a role in controlling growth and that syndecan may have different functions in epithelial and mesenchymal cells. PMID- 1717322 TI - Characterization and expression of a gene encoding a 30.6-kDa Strongylocentrotus purpuratus spicule matrix protein. AB - We describe here the isolation and characterization of several cDNA clones that encode a single 30.6-kDa Strongylocentrotus purpuratus spicule matrix protein designated SM30. The clones were isolated by screening a lambda gt11 cDNA library with a rabbit polyclonal antiserum raised against S. purpuratus total spicule matrix proteins. DNA sequencing reveals that the SM30 protein is acidic. RNA blot analysis shows that the cDNAs hybridize to a single 1.8-kb transcript and that there is a sharp increase in the SM30 transcript levels at middle to late mesenchyme blastula stage. SM30 transcript levels remain high through the 3-day pluteus stage. In situ hybridization analysis indicates that, within the embryo, SM30 transcript accumulation is restricted to the primary mesenchyme cells. Quantitations of SM30 transcript levels show that by the prism stage there are about 29,000 SM30 transcripts present per embryo, which averages to approximately 480 transcripts per primary mesenchyme cell. Additionally, RNA blot analysis of total RNA isolated from adult tissues shows that SM30 mRNA accumulates exclusively in mineralized tissues. These findings taken together strongly suggest that the gene corresponding to the SM30 cDNAs does in fact encode a spicule matrix protein. PMID- 1717323 TI - XLPOU 1 and XLPOU 2, two novel POU domain genes expressed in the dorsoanterior region of Xenopus embryos. AB - POU domain genes have been shown to be important in tissue-specific gene regulation during development. We have cloned cDNA's encoding two unique Xenopus laevis POU-domain proteins, XLPOU 1 and XLPOU 2. Sequencing revealed that the POU domain of both of these genes have greater than 90% homology with that of the POU III class of proteins. XLPOU 1 gene expression begins at the neural plate stage, while XLPOU 2 gene expression is first detected at the neurula stage; both XLPOU 1 and XLPOU 2 transcripts continue to be expressed throughout development. In adults, XLPOU 1 expression is restricted to the skin and brain, while XLPOU 2 is observed in the kidney and brain. In dissected embryos, both XLPOU 1 and XLPOU 2 are expressed in the dorsoanterior portion of an early tailbud embryo. Consistent with this localization, uv treatment, a condition that "posteriorizes" embryos, greatly reduces the expression of XLPOU 1 and XLPOU 2. Whole-mount in situ hybridization demonstrates that at the neural fold stage, XLPOU 1 transcripts appear to be localized primarily in the anterior neural plate. In sections of embryos, in situ hybridization shows that XLPOU 1 transcripts are localized mostly in the anterior region of the nerve cord of neurula stage embryos. In tailbud stage embryos, the XLPOU 1 transcripts are found predominantly in the eye and brain, with weak expression along the length of the nerve cord. We believe that XLPOU 1 and XLPOU 2, because of their localized and early expression in embryos, may play an important role in the specification of neuronal phenotypes. PMID- 1717324 TI - Transcription of the embryonic myosin light chain gene is restricted to type II muscle fibers in human adult masseter. AB - We have previously demonstrated that the embryonic myosin light chain (MLC1emb) isoform whose expression is restricted to the early fetal stages in most mammalian skeletal muscles, persists throughout development in human masseter muscle. In order to go further in this study, we have compared the developmental profile of MLC1emb gene transcription in human masseter and quadriceps muscles using both Northern blotting and in situ hybridization techniques. Interestingly, whereas expression of this gene was observed in all fibers during fetal stages in both muscles, transcription in adult masseter was found to be restricted to type II fibers. Existence of a masseter-specific pathway of muscle gene regulation is discussed. PMID- 1717325 TI - Segregation of Na(+)-channel gene expression during neuronal-glial branching of a rat PNS-derived stem cell line, RT4-AC. AB - RT4 is a family of cell lines isolated from an ethylnitrosourea-induced rat peripheral neurotumor. RT4-AC cells express both excitable membrane and glial cell properties. In a process called cell-type conversion, RT4-AC cells segregate these properties to generate three distinct derivative cell types which have been classified as either neuronal (RT4-E and RT4-B) or glial (RT4-D). In this report we demonstrate that: (1) upon cell-type conversion, Na(+)-channel mRNA expression segregates primarily with the RT4 neuronal derivatives, (2) the SkM2 Na(+) channel gene, which was originally isolated from rat muscle cDNA libraries, is the predominant gene expressed by the RT4 neuronal derivatives, (3) the three rat brain Na(+)-channel genes I, II, and III and the muscle-derived SkM1 gene are not the principal Na(+)-channel genes involved in the segregation, although very low levels of message of these genes are detected, and (4) the RT4 glial derivative expresses slightly higher levels of message from rat brain genes I and II than the neuronal derivatives. Since the RT4 cell lines were derived from a peripheral neurotumor these results present the possibility that the SkM2 gene may be important in vivo in the rat peripheral nervous system. PMID- 1717326 TI - Altered cell fate in LiCl-treated sea urchin embryos. AB - Endo16 is a lineage-specific protein expressed during embryogenesis in the sea urchin, Strongylocentrotus purpuratus. We have examined the effects of various concentrations of lithium chloride, a well-known vegetalizing agent, on Endo16 expression in whole sea urchin embryos. Our results show that treatment with LiCl causes increased steady-state levels of Endo16 transcripts. An increase in the number of endodermal cells in treated embryos demonstrates a change in cell lineage. In addition, we observed a delay in development of lithium-treated embryos that is accompanied by a delay in the expression of a late class histone H2B gene. PMID- 1717327 TI - Neurodevelopmental and medical status of low-birthweight survivors of bronchopulmonary dysplasia at 10 to 12 years of age. AB - Thirty low-birthweight (less than 1500g) infants (15 with bronchopulmonary dysplasia (BPD), and 15 controls less than or equal to 5 days O2) and 15 fullterm controls were evaluated at 10 to 12 years of age. BPD children weighted less than fullterm children and had smaller head circumferences than either preterm or fullterm controls. They also had significantly more neurological abnormality than both control groups. BPD children and preterm controls had lower WISC-R arithmetic scores and lower Beery VMI scores, as well as greater need of resources and special education compared with fullterm controls. BPD survivors at 10 to 12 years of age continue to manifest sequelae related to their early pulmonary disease. PMID- 1717328 TI - Hypomelanosis of Ito in three cases with autism and autistic-like conditions. AB - Two girls and a boy showing autistic behaviour and fulfilling the criteria for autistic disorder, Asperger syndrome or atypical autism were diagnosed as having hypomelanosis of Ito syndrome. It is suggested that skin changes indicating underlying neurocutaneous disorders be meticulously looked for in all cases with autism and autistic-like conditions. PMID- 1717329 TI - Inhibitory effect of caerulein on salivary secretion in man. AB - Previous works have documented that pentagastrin has a stimulatory effect on salivary secretion. In this paper, we have studied the action of caerulein, a peptide that has an active gastrin-like terminal tetrapeptide, on salivary secretion in man. Our data documented that caerulein and pentagastrin have similar excitatory effect on gastric secretion in man, but different actions on salivary flow. Caerulein, at increasing doses, inhibits salivary flow, but not the secretion of amylase; this effect is statistically significant at 0.5 micrograms/kg/h which is the same dose that maximally stimulates gastric secretion. The inhibitory effect of caerulein was not reversed by previous injection of papaverin. PMID- 1717330 TI - DC6, a novel type of Dictyostelium discoideum gene regulated by secreted factors but not by cAMP. AB - We have isolated a gene, DC6, which is induced in the early aggregative stages of development in Dictyostelium discoideum. The increase in DC6 expression is dependent on high cell density, indicating that cellular interactions are required for DC6 induction. In low-cell-density cultures, the induction of DC6 occurs if supplied with conditioned medium of developing cells, suggesting that secreted factors are involved in DC6 induction. The expression of DC6 is not affected (1) in the presence of caffeine or adenosine, which block the production or the action of cAMP pulses, (2) in the presence of high concentrations of cAMP, or (3) in mutant strains (Synag7 and FrigidA), which are defective in transduction pathways of cAMP pulse signals. These results indicate that the induction of DC6 does not require extracellular cAMP pulse signals, which are known to regulate the expression of many genes in the early development. Independence of cAMP signals and dependence on other unknown cellular interactions are prominent characteristics of DC6. PMID- 1717331 TI - Induction of NILE/L1 glycoprotein during neuronal differentiation of the embryonal carcinoma cell line EC1003. AB - A new clone of the mouse embryonal carcinoma cell line 1003 (EC 1003.16) can be maintained in an undifferentiated state in serum-containing medium. Shifting these cells to serum-free, hormonally defined medium causes them to differentiate morphologically and acquire a number of molecular properties characteristic of neurons. Whereas undifferentiated cells lack the NILE/L1 glycoprotein, expression of this neuronal cell adhesion molecule is induced in the differentiating cells. Message for NILE/L1 becomes detectable after 5 days in serum-free medium, and cell-surface NILE/L1 can first be seen at this same time. Changes in two other cell adhesion molecules occur in parallel with the induction of NILE/L1. Fibronectin receptor is present on undifferentiated cells, but is down-regulated by the differentiating neurons. The neural cell adhesion molecule (N-CAM) undergoes a shift from the very adhesive adult form to the less adhesive, highly sialylated embryonic form. These changes would appear to emphasize the role of NILE/L1 in adhesive interactions involving differentiating neurons. Some changes in ganglioside expression also occur during EC 1003.16 differentiation. Undifferentiated cells express the D 1.1 ganglioside but lack gangliosides that are reactive with the monoclonal antibody A2B5. Differentiating cells lose D 1.1 and become A2B5-positive. Since D 1.1 is characteristic of undifferentiated neuroepithelial cells and A2B5 reactivity is a marker for several types of differentiated neurons, these changes in vitro appear to mimic events that occur in vivo. PMID- 1717332 TI - Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro. AB - By incubating multilayered primary cultures of human keratinocytes in low-calcium medium the suprabasal cell layers can be stripped off leaving a basal cell monolayer. When this monolayer is re-fed normal calcium medium a reproducible series of cell kinetic, morphological, and biochemical changes takes place resulting in the reestablishment of a multilayered tissue. Analysis of cell-cycle specific proteins indicated that, during regeneration, a large cohort of cells became synchronized undergoing DNA replication after 3 days. Examination of culture morphology at the ultrastructural level confirmed the capacity of the basal cell monolayer to gradually reestablish a multilayered, differentiated epithelium. The ultrastructural appearance at 7 days poststripping was similar to that of unstripped cultures and was indicative of a tissue in steady state. Quantitation of cornified envelope formation at different times during regeneration showed that an increasing proportion of the cells were able to undergo terminal differentiation. In general, the pattern of keratin synthesis in the original epidermal explant labelled in vitro was similar to the pattern observed in human epidermis in vivo; however, in contrast to epidermis in vivo the explant also synthesized the hyperproliferative keratins 6 and 16. The in vitro differentiated keratinocytes showed underexpression of several proteins identified as differentiation markers, whereas several basal cell markers were overexpressed compared to the original explant. In addition, the in vitro differentiated keratinocytes synthesized some new proteins, notably keratins 7, 15 and 19. The basal layer remaining after stripping mainly expressed basal cell markers; however, during recovery, some of the differentiation-specific markers (e.g. keratin 10 and 15) were again expressed together with keratin no. 19, which is also expressed during wound healing in vivo. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model to investigate certain aspects of the regulation of epidermal tissue homeostasis. PMID- 1717333 TI - Antigen specificity of gamma delta T lymphocytes. AB - gamma delta T cells represent a new lymphocyte subset without a definitive functional assignment. Although in many ways similar to alpha beta T lymphocytes, they are clearly distinguished by their expression of a different set of T cell receptor genes, a different distribution in normal tissues, and perhaps also different ligand specificities. Because gamma delta T cells appear to be involved in a variety of human diseases, the determination of their biological role has become an important challenge for immunologists and researchers in related areas. PMID- 1717334 TI - Insulin-induced stimulation of protein phosphorylation in Neurospora crassa cells. AB - 1) Insulin stimulated the phosphorylation of at least 14 discrete proteins in Neurospora crassa cells. Specific proteins were phosphorylated at serine, threonine, and tyrosine residues, as determined by phosphoamino acid analysis of discrete spots on two-dimensional gels. 2) Insulin stimulated the phosphorylation by [gamma-32P]ATP of at least six discrete proteins in solubilized N. crassa membrane preparations at serine and tyrosine residues. 3) A phosphotyrosine containing protein of 38 kDa, pI 7.0-7.2, reacted by both immunoblotting and immunoprecipitation with antiserum to P2, a peptide from the human insulin receptor that contains an autophosphorylated tyrosine residue. In N. crassa cells, therefore, as in mammalian cells, insulin induces a variety of protein phosphorylations, some of which may be part of an evolutionarily conserved signal transduction pathway. PMID- 1717335 TI - Subviral pathogens of plants: viroids and viroidlike satellite RNAs. AB - Contrary to earlier beliefs, viruses are not the smallest causative agents of infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in certain higher plants and cause more than a dozen crop diseases. These RNAs have been termed viroids. Despite their extremely limited information content, viroids replicate autonomously in susceptible cells--that is, they do not require helper functions from simultaneously replicating conventional viruses. Viroids are covalently closed circular molecules with a characteristic rodlike secondary structure in which short helical regions are interrupted by internal and bulge loops. Viroids are not translated; they are replicated by a host enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble viroids in size and molecular structure, but are found within the capsids of specific helper viruses on which they depend for their own replication. These RNAs are of great interest to molecular biology for at least two reasons: 1) they are the smallest and simplest replicating molecules known, and 2) they may represent living fossils of precellular evolution in a hypothetical RNA world. PMID- 1717336 TI - Cytotoxic activity of chimeric proteins composed of acidic fibroblast growth factor and Pseudomonas exotoxin on a variety of cell types. AB - Chimeric proteins composed of acidic fibroblast growth factor (acidic FGF) and several forms of Pseudomonas exotoxin (PE) that cannot bind to the PE receptor have been produced in Escherichia coli by expressing chimeric genes in which DNA encoding acidic FGF is fused to various mutant forms of PE. These acidic FGF-PE fusion proteins were found to be cytotoxic to a variety of tumor cell lines including hepatocellular (PLC/PRF/5 and HEPG2), prostatic (LNCaP), colon (HT29), and breast (MCF-7) carcinomas at concentrations of 1-70 ng/ml. The cytotoxic effects of acidic FGF-PE were FGF-receptor specific as demonstrated by competition with excess acidic FGF and by showing that acidic FGF-PE bound to the FGF receptor with the same affinity as acidic FGF. Furthermore, the cell-killing activity of acidic FGF-PE was toxin-mediated, as an acidic FGF-PE mutant, which does not possess ADP-ribosylation activity, failed to kill cells. These findings demonstrate that acidic FGF-PE is a potent cytotoxic molecule that can be targeted to FGF receptor-bearing cells. Because acidic FGF is a potent angiogenic molecule, cytotoxic acidic FGF-PE chimeras may have utility as anti-angiogenic agents. These molecules could be helpful in determining the functional role of FGF receptors in cellular processes. PMID- 1717337 TI - [Ultrastructural and immunohistochemical studies of hyperplastic endocrine cells of the fundic mucosa in man]. PMID- 1717338 TI - [Role of the enterochromaffin-like cells and histamine in the regulation of gastric acid secretion]. PMID- 1717339 TI - Effect of chronic antigen exposure on growth and intestinal histamine content of sensitized rats. AB - The effects of chronic antigen feeding on systemically sensitized rats were investigated. Findings include a reduction of water and antigen intake in egg albumin (EA), sensitized rats receiving EA in their drinking water for an 8 day period, compared to that of sensitized rats fed bovine serum albumin and of naive rats. Feeding EA to sensitized animals also induced a decrease in daily weight gain. This decline did not seem to be a consequence of a decreased food intake, but might rather reflect a decreased water consumption and an alteration of nutrient absorption in the gut. Indeed, sensitized rats fed EA exhibited a significant increase in jejunal and ileal histamine content compared to control rats, which may indicate the development of an inflammatory reaction in the small intestinal mucosa. Intestinal troubles experienced because of this inflammatory reaction might explain the reduction of antigen and water intake observed in sensitized rats. PMID- 1717341 TI - Laser versus laser plus afterloading with iridium-192 in the palliative treatment of malignant stenosis of the esophagus: a prospective, randomized, and controlled study. AB - Thirty-nine patients with malignant stenoses of the esophagus (22 adenocarcinomas, 17 squamous cell carcinomas) were treated either with Nd:YAG laser recanalization alone (N = 20) or with laser recanalization and subsequent endoluminal afterloading irradiation with iridium-192 at a dose of 3 x 7 Gy (6 x 7 Gy). Squamous cell carcinoma patients in the afterloading group showed a prolonged dysphagia-free first interval (65 vs. 30 days, p less than 0.03), while patients with an adenocarcinoma did not share this benefit, and had a need for more frequent endoscopic procedures (p less than 0.05). The complication of esophagitis was only seen following afterloading treatment (21%, N = 4). Re stenosis occurred in all patients. Neither the duration of relative dysphagia nor survival time was prolonged after endoluminal irradiation in adenocarcinoma or squamous cell carcinoma patients. After prior laser recanalization, palliative afterloading treatment with iridium-192 would seem helpful only in cases of squamous cell carcinoma with a high performance status with the aim of prolonging the first dysphagia-free interval. PMID- 1717340 TI - Effect of protein kinase C on amylase secretion and cyclic AMP production in rat pancreatic acinar cells. AB - The authors examined the effects of protein kinase C on secretin-induced amylase release and cyclic AMP production in rat pancreatic acinar cells. Secretin (10( 6) M) and 12-O-tetradecanoyl-phorbol 13-acetate (TPA) (10(-6) M) induced 53% and 60% increase of amylase release from the basal level, respectively during 10 min. Simultaneous addition of TPA and secretin resulted in 42% amylase release from the basal level for 10 min. Suppression of secretin-induced amylase release was evident within 5 min of pretreatment with TPA. TPA showed the same effect on cyclic AMP production; secretin-induced increase of cyclic AMP was suppressed by pretreatment of TPA for 5 min. To explore the mechanism by which TPA inhibits secretin-induced cyclic AMP production, we also examined the effects of protein kinase C purified from rat brain on adenylate cyclase activity in pancreatic acinar membranes. Basal, forskolin- and secretin plus guanosine 5'-[gamma thio]trisphosphate-stimulated adenylate cyclase activity were inhibited by protein kinase C in the presence of Ca++. These results suggest that protein kinase C might have a role in the inhibitory effect on adenylate cyclase in exocrine pancreas. PMID- 1717342 TI - Analysis of calcium activated chloride current fluctuations in Xenopus laevis oocytes. AB - Fluctuations of calcium activated chloride currents were investigated in oocytes of Xenopus laevis. The method of noise analysis and the model of chloride channels activation by calcium ions were used to estimate the chloride channels lifetime and the average frequency of current fluctuations, which depends on changes of cytoplasmic calcium concentration. This current fluctuations can be evoked by activation of cholinergic receptors or inhibition by Na3VO4 of plasma membrane Ca(2+)-ATPase. The average opening lifetime of chloride channels was approximately 100 ms. The frequency of fluctuations increased with the increasing extracellular calcium concentrations and external ACh concentrations. Caffeine in 2 mmol/l concentration changed the current fluctuations into oscillations with a period of about 18-20s. Ten mmol/l caffeine fully inhibited the oscillation activity. PMID- 1717343 TI - [Oxidative and SOS-response in Escherichia coli and their interference]. AB - Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences. PMID- 1717344 TI - The carboxy-terminal catalytic domain of the GTPase-activating protein inhibits nuclear signal transduction and morphological transformation mediated by the CSF 1 receptor. AB - To determine whether ras p21 products are necessary for signal transduction mediated by the colony stimulating factor-1 receptor (CSF-1R, the c-fms proto oncogene product), we determined whether CSF-1R and ras activate a common nuclear target and whether the interruption of ras action affects CSF-1R signal transduction. Expression of the NVL3 retrotransposon was activated to the same extent in NIH-3T3 cells by both ras and v-fms oncogenes, and the ras-responsive element located in the long terminal repeat of NVL3 was demonstrated to be a common target for oncogene action. Human recombinant CSF-1 stimulated expression of the NVL3 element 30-fold in NIH-3T3 cells that contained human CSF-1R. Expression of the carboxy-terminal 374 amino acid residues of the human ras GTPase-activating protein (GAP) in cells containing CSF-1R was able to inhibit CSF-1 induction of NVL3 expression by 90%. Expression of the catalytic domain of GAP was also able to suppress transformation by either v-fms or ligand-activated CSF-1R. Expression of the c-jun proto-oncogene was activated by CSF-1R but was insensitive to the action of the catalytic domain of GAP. These results provide genetic evidence that in NIH-3T3 cells, ras p21 is involved in signal transduction mediated by CSF-1R. PMID- 1717345 TI - Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. AB - We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2. The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively. Although mHSF1 and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human. The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat. mHSF1 is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures greater than 41 degrees C, the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo. mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at greater than 41 degrees C. Both mHSF1 and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock. However, there is a striking difference in the levels of mHSF1 mRNA in different tissues of the mouse. PMID- 1717346 TI - Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma 54 and modulated by positive and negative feedback mechanisms. AB - The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the sigma-factor sigma 54. Negative regulation is mediated by both PspA and the sigma 32-dependent heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the sigma 32- and sigma E-dependent systems, for genes induced by heat and other stresses. PMID- 1717347 TI - Expression of lacZ gene fusions affects downstream transcription in yeast. AB - Chimeric genes containing Escherichia coli lacZ sequences are often used to characterize gene expression in yeast cells. By Northern analysis, we found that such genes produce multiple transcripts due to inefficient 3'-end formation. The same transcript pattern was found for two related chimeric genes when these genes were cloned separately into the commonly used vector, YIp5, and integrated into the yeast genome at two different locations. Each chimeric gene was composed of promoter and N-terminal coding regions from the yeast SSA1 or SSA2 genes fused in frame to the lac operon. Transcripts were shown to initiate within the yeast promoter fragment, but transcript size indicated that 3' ends were localized to three different regions: within the lac operon near the 3' end of the lacZ gene; near a terminator region previously identified upstream of the URA3 gene in YIp5; and at the URA3 terminator region. Readthrough transcription of the URA3 promoter from upstream lac sequences decreased the basal activity of the URA3 promoter, although induced URA3 transcription levels were unaffected. This readthrough transcription also resulted in a novel, longer URA3 transcript. PMID- 1717348 TI - Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region. AB - A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression. PMID- 1717349 TI - Murine tenascin: cDNA cloning, structure and temporal expression of isoforms. AB - Mouse tenascin (TN)-encoding cDNA clones were isolated from a cDNA library of the 2H6GR mammary tumor cell line. Nucleotide (nt) and deduced amino acid (aa) sequences revealed the characteristic primary structure, which begins with a signal peptide and TN unique sequences, follows with 14 1/2 epidermal growth factor (EGF)-like repeats and 13 fibronectin type-III repeats (FN repeat), and concludes with fibrinogen-homologous sequences. Similar to chicken and human TN, the mouse TN cDNA contains five consecutive insertional FN repeats, as well as eight constitutive FN repeats. Three different cDNA clones that may have been generated by alternative splicing of these insertional FN repeats were identified and characterized. Based upon the deduced as sequence, a polyclonal antibody was produced against a synthetic TN peptide. It specifically recognized two TN isoforms of 230 kDNA and 190 kDa in protein extracts of mouse tissues. The tissue distributions of mouse TN mRNAs, revealed by Northern blot analysis, suggest that there is tissue-specific expression of TN isoforms. Two distinct mRNA transcripts (7 kb and 5.5 kg) were detected in brain, skeletal muscle, digestive tract and bladder, but only one was observed in lung, kidney (7 kg) and thymus (5.5 kg). TN mRNA expression was down-regulated 1 month after birth in most tissues. However, the 5.5-kb transcript persisted in cerebellum, thymus, and colon. The spatial and temporal patterns of TN expression seem to be controlled at the level of transcription, because analysis of various tissues by Western blots showed the same pattern as that seen in Northern blots. PMID- 1717350 TI - Variants of the human high-affinity receptor (Fc gamma RI) for immunoglobulin G. AB - PCR-amplification cloning of the cDNA encoding the human high-affinity receptor for IgG (Fc gamma RI) revealed two splice variants which coincide with domain boundaries predicted by amino acid sequence comparison. Both splice variants maintain the open reading frame. PMID- 1717351 TI - [Results of a study of the pollution of urban air by lead and benzo(a)pyrene emitted by motor transport]. PMID- 1717352 TI - [Morbidity with temporary disability of gas-cylinder bus drivers]. AB - Running of gas-cylinder buses (GCB) fueled with liquefied propane-butane mixture added with ethyl mercaptan odorant leads to undesirable medical and social consequences. Statistically proved was considerably higher morbidity rate among GCS drivers in comparison with petrol-fueled buses' drivers. A questionnaire technique was used to show that most GCB drivers (from 61.8% to 98.2% in different bus depots) had been suffering from the irritating smell of the gaseous fuel and consequent headaches, dizziness, slow reactivity and general weakness, specifically by the end of the working shift. The result of the study proved the necessity to stop further running of GCB until technical improvement of the equipment and normal working conditions for GCB drivers have been attained. PMID- 1717353 TI - Bleomycin, ifosfamide, and cisplatin (BIP) in patients with recurrent and advanced cervical cancer. PMID- 1717354 TI - A primary mucinous cystoadenocarcinoma of the retroperitoneum. AB - A case of primary retroperitoneal mucinous cystoadenocarcinoma of the ovarian type in the presence of normal ovaries is reported. The histogenesis of this rare tumor has been uncertain. Special immunohistochemical stains done on our specimen show that the histogenesis of this tumor is most likely from mucinous metaplasia of coelomic mesothelium. Three cases of primary retroperitoneal mucinous cystoadenocarcinoma of the ovarian type have previously been reported in the English literature and are reviewed. PMID- 1717355 TI - [Effects of NIK-228 on gastric acid secretion in rats using the congo red sprayed method]. AB - We have reported the antiulcer activities of a new compound that we named NIK-228 (3-hydroxy-methyl-2-methylimidazo [2, 1-b] benzothiazole). In the present report, we studied the antisecretory effects of NIK-228 on basal and stimulated gastric acid secretion using the Congo red sprayed method. Male Wistar rats (200 to 250 g) were used after 24 hr of fasting (without water). NIK-228, atropine and cimetidine were administered orally or intravenously 1 hr before operation for Congo red spraying. NIK-228 (100 mg/kg, p.o.), atropine (5 mg/kg p.o.) and cimetidine (100 mg/kg, p.o.) all inhibited basal gastric acid secretion. Oral administration of NIK-228 and atropine inhibited gastrin, 2-deoxy-D-glucose (2 DG) and bethanechol-induced acid secretion, but didn't inhibit histamine-induced acid secretion. Cimetidine inhibited all of histamine, gastrin, 2-DG and bethanechol-induced acid secretion. In vagotomized rats, oral and intravenous administration of atropine both inhibited bethanechol-induced acid secretion, but NIK-228 was not inhibited. These results suggested that antisecretory effects of NIK-228 were caused by the central vagal systems. PMID- 1717356 TI - Effects of calcium channel agonist and antagonists on calcium-dependent events in CA1 hippocampal neurons. AB - The effects of a variety of calcium channel modulators on different calcium dependent events in CA1 pyramidal hippocampal neurons were analysed using intracellular recordings in an in vitro slice preparation. The following substances were tested: the dihydropyridine calcium agonist BAY K 8644, the dihydropyridine calcium antagonist nimodipine, the phenylalkylamine verapamil and the snail toxin omega-conotoxin GVIA (omega-CgTx). BAY K 8644 increased the repolarization time of the after hyperpolarization (AHP) following a spike burst. This effect was antagonized by nimodipine. BAY K 8644 also prolonged the calcium spike and, in some cases, increased the size of the synaptic events resulting from activation of the Schaffer collateral/commissural system. Nimodipine decreased the size of the AHP in some neurons but had no consistent effect on synaptic events. Verapamil at low concentrations (1-10 microM) had no significant effects on the calcium-dependent events in the hippocampus. Increasing the concentration (up to 100 microM) led to a progressive suppression of the AHP and of the slow inhibitory postsynaptic potential (IPSP), probably via an action on potassium conductances. In addition, the baclofen-induced hyperpolarization was blocked by verapamil. Interestingly, at this higher concentration, verapamil could suppress the AHP without depressing the calcium spike. omega-CgTx selectively blocked the synaptic events (especially the IPSPs) but had no effect on non-synaptic events. This last compound exhibits a high degree of selectivity, acting on N-type calcium channels which are involved in neurotransmitter release. Our results provide evidence that different classes of agents which act on calcium channels can be used to discriminate between different calcium-dependent responses in CA1 hippocampal neurons. PMID- 1717357 TI - Efflux of protons from acidic vesicles contributes to cytosolic acidification of hepatocytes during ATP depletion. AB - The objective of this study was to determine the relationship between cytosolic pH and vesicular pH during ATP depletion. Using digitized video microscopy and single, cultured rat hepatocytes, cytosolic pH and vesicular pH were quantitated by ratio imaging of BCECF (2', 7' biscarboxyethyl-5,6-carboxyfluorescein) fluorescence and fluorescein-dextran fluorescence, respectively. Basal value for cytosolic pH was 7.26 and basal value for vesicular pH was 4.86. During ATP depletion by metabolic inhibition with KCN plus iodoacetic acid or antimycin A, cytosolic pH decreased 0.71 units to 6.55. In separate experiments under identical conditions, vesicular pH increased 1.59 units to 6.45, suggesting that protons were leaking from acidic vesicles during ATP depletion. Fluorescein dextran fluorescence remained punctate, indicating that the rise in vesicular pH was due to an efflux of protons from vesicles and not loss of vesicle integrity. To determine whether efflux of protons from acidic vesicles can acidify cytosolic pH, we used two maneuvers that result in leakage of protons from acidic vesicles without significantly decreasing cellular ATP: (a) hypotonic stress in K(+)-free media and (b) exposure of the cells to the H(+)-ATPase inhibitor NBD-Cl. Both hypotonic stress and NBD-Cl decreased cytosolic pH 0.4 units to 6.86 and increased vesicular pH 2.0 units to 6.76, resulting in near-equilibration of cytosolic pH and vesicular pH. Thus an efflux of protons from intracellular compartments will acidify cytosolic pH of hepatocytes (pH 6.86), but not to the same degree as ATP depletion (pH 6.55).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717358 TI - Basaloid-squamous carcinoma of the upper aerodigestive tract and so-called adenoid cystic carcinoma of the oesophagus: the same tumour type? AB - Basaloid-squamous carcinoma of the larynx, pharynx and base of tongue and the so called adenoid cystic carcinoma of the oesophagus are rare but distinctive tumours associated with a grave prognosis. They occur most commonly in elderly males and present at an advanced stage. Our study of four such laryngeal tumours and five such oesophageal tumours shows that they are histologically and immunohistochemically identical, providing support for the idea that they are the same tumour type. They show a biphasic pattern in which basaloid tumour is intimately associated with a neoplastic squamous component which can be invasive or in situ. The basaloid component is in the form of invasive lobules with frequent comedo-necrosis and hyalinization. The constituent cells possess pale pleomorphic nuclei with frequent mitoses. Immunoreactivity for cytokeratin in the basaloid component is remarkable for its absence or weak and focal nature. Review of the literature shows that only a few cases of 'adenoid cystic carcinoma' of the oesophagus are bona fide examples of adenoid cystic carcinoma as it occurs in the salivary glands, while the others are identical to basaloid-squamous carcinoma of the upper aerodigestive tract. Their distinction is important because genuine adenoid cystic carcinoma is much less aggressive than basaloid squamous carcinoma. PMID- 1717359 TI - The L1 antigen and squamous metaplasia in the bladder. AB - The L1 antigen was investigated as a marker of squamous differentiation in urothelium using a monoclonal antibody Mac387, and the results were compared with the expression of high molecular weight cytokeratins. L1 antigen was consistently demonstrated in all instances of partial and complete squamous metaplasia and in squamous carcinomas. In contrast, pure transitional cell carcinomas (except one with minor focal staining), adenocarcinomas and normal and hyperplastic urothelium did not label. In a few squamous carcinomas in situ, the pattern of labelling obtained with Mac387 was different from that seen in invasive squamous carcinomas and metaplasias. Compared with high molecular weight cytokeratins, the expression of L1 was more intense in areas of squamous differentiation. L1 expression, as identified by antibody Mac387, may therefore serve as a useful marker of squamous differentiation in urothelial lesions. PMID- 1717360 TI - Pathology of vulvar intraepithelial lesions and early invasive carcinoma. PMID- 1717361 TI - Lymphoepithelial cyst of the pancreas in a 65-year-old man. AB - A case of an extremely rare cystic lesion of the pancreas is presented. The multilocular cyst was found adjacent to the upper border of the pancreatic body, and the cyst contained bean curd lees-like substances. Histologically, the cyst wall consisted of mature keratinizing squamous epithelium and surrounding lymphoid tissue stroma, and the cyst was filled with keratinized materials. A histopathologic diagnosis of typical lymphoepithelial cyst of the pancreas, proposed by Truong et al (Am J Surg Pathol 11:899-903, 1987), was made. Its histogenesis is still unknown; however, we hypothesize that it might arise from a benign epithelial inclusion of a peripancreatic lymph node, followed by squamous metaplasia of the epithelial inclusion. We recently found a retropyloric lymph node with a squamous epithelial inclusion, which might support this hypothesis regarding the histogenesis of the cyst. PMID- 1717362 TI - Localisation of the gene coding for the haemopoietic stem cell antigen CD34 to chromosome 1q32. PMID- 1717363 TI - High conservation of sequences involved in cystic fibrosis mutations in five mammalian species. AB - Several mutations have been identified in the first nucleotide binding fold (NBF) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We have analyzed the DNA sequences of exons 10 and 11 in five different mammalian species, marmoset, mouse, cow, pig, and sheep; the amino acid conservation studied for nine disease mutations; and two "benign" mutations. For exon 10, 87% homology at the DNA level and 93.5% at the amino acid level were found for these species. For exon 11, the lowest homology (70%) was found in mouse and the highest in marmoset (93%), whereas the amino acid sequence conservation ranged from 82.5 to 100%. All codons involved in CF mutations are highly conserved throughout evolution. PMID- 1717364 TI - Effect of cyclosporine on 3-methylcholanthrene-induced carcinogenesis and immune responses in the rat. AB - Cyclosporine A (CsA) was used to immunosuppress male Sprague-Dawley rats treated with the chemical carcinogen 3-methylcholanthrene (3MC). Rats treated with low doses of CsA (2.5 or 5 mg/kg) given 2 days prior to an injection of 3MC, and then daily for 2 weeks or twice weekly for 10 weeks did not develop tumors. Rats treated with 2.5 mg/kg CsA for 2 weeks beginning 5 days after a single 3MC injection had tumor incidence similar to rats treated with 3MC only. To further examine the effects of CsA on immune function, groups of rats were then treated with 2.5, 5, 10 or 20 mg/kg CsA daily for 14 days and immune function assessed by measuring delayed-type hypersensitivity (DTH), natural killer cell (NK) activity, and production of interleukin 2 (IL-2), interferon (IFN), prostaglandin E2 (PGE2) and specific IgG antibody. Natural killer cell cytotoxicity was enhanced and antibody production was suppressed in rats treated with all doses of CsA tested. Interleukin 2 production was elevated at the two lower doses, but antibody production, DTH reactions and synthesis of IL-2 and IFN were suppressed with the higher dose treatments (10, 20 mg/kg CsA). The enhanced NK activity seen in rats treated with the lower doses of CsA may be due to the increase in IL-2 production, while enhancement of NK activity at higher doses may be due to other mechanisms. The tumor data suggest that CsA does not prevent tumor formation in our chemical-induced model due to an increase in NK activity, since this enhancement was seen even when tumors did develop normally. PMID- 1717365 TI - IL-4 decreases the expression of the monocyte differentiation marker CD14, paralleled by an increasing accessory potency. AB - IL-4 has been found to affect the phenotype and a variety of functions of human monocytes and macrophages and has been discussed as a monocyte activating protein along with other cytokines, such as IL-1 and IL-6. In this study we compared the effects of the cytokines IL-1, IL-6, IL-4, and a combination of IL-1 and IL-6 on the expression of the CD14 antigen, the FcIIIg receptor molecule CD16 and the MHC class II molecules HLA-DR and HLA-DP. These molecules represent characteristic monocyte surface markers. Furthermore, the CD14 molecule has been described as a surface antigen of high in vivo relevance representing an indirect receptor for LPS. We further analyzed the effect of IL-4 on monocytes and macrophages with respect to their accessory function to initiate T-lymphocyte proliferation. Human peripheral blood monocytes strongly express the antigen CD14 and maintain it as a stable surface molecule during their differentiation to macrophages. Flow cytometry analysis of cultured monocytes demonstrated that cells incubated in the presence of IL-4, but not IL-1 and/or IL-6 revealed a reduced expression of the CD14 antigen in a dose- and time-dependent manner. After 3 days IL-4 treated cells were virtually CD14-negative. At the same time the expression of the CD16 antigen (FcRIIIg) was also strongly reduced, whereas the treatment with IL-4 led to an increased expression of MHC class II antigens such as HLA-DR and HLA-DP. The spontaneous low expression of HLA-DQ antigen on monocytes was not affected by any of the cytokines. Functionally, IL-4 treated CD14-negative monocytes exhibited a more than 2-fold higher activity to stimulate an accessory cell dependent T cell proliferation. This was found in a mitogenic assay and in MLC when compared to monocytes cultured in the absence of IL-4. These observations provide further evidence that IL-4 is a major modulator of monocyte surface antigen expression. Moreover, IL-4 has an enhancer-effect on monocytes as accessory cells and therefore may be of considerable importance as a regulatory factor during monocyte development to accessory cells. Inasmuch as the CD14 molecule functions as a receptor for LPS-binding protein, our results suggest that IL-4 might also play an important regulatory role in processes initiated by bacterial lipopolysaccharides during inflammation and sepsis. PMID- 1717366 TI - Murine monoclonal anti-Ba antibody that enhances haemolytic activity of factor B. AB - A murine monoclonal antibody (mAb 20-ET) (IgG1, kappa) was selected from a panel of stable hybridomas produced by fusion of P3-X-63-Ag8-U1 (P3UI) myeloma cells with spleen cells from a BALB/c mouse immunized with human factor B. This antibody was shown by the immune blotting method to be directed against the Ba domain of factor B. The haemolytic activity of factor B was enhanced dose dependently by mAb 20-ET when it was incubated with factor B and EAC4b, 3b cells (sensitized erythrocytes bearing complement fragments C4b and C3b). However, when the antibody was added after factor B had been bound to EAC4b,3b cells and the cells had been washed, it caused little enhancement of the haemolytic activity. The enhancing effect of this antibody was not due to its stabilization of the C3b B complex, because EAC4b,3b dissociated from mAb 20-ET-bound factor B complexes rather more readily than from uncomplexed factor B. The presence of mAb 20-ET in the reaction mixture caused and maintained a much higher steady-state level of binding of factor B with EAC4b,3b cells than that in its absence. Factor P caused delayed dissociation of mAb-bound factor B from EAC4b,3b cells, thus enhancing the haemolytic activity of factor B bound with the mAb. PMID- 1717367 TI - Long-lived reciprocal regulation of antigen-specific IgE and IgG2a responses in mice treated with glutaraldehyde-polymerized ovalbumin. AB - Previously, we discovered that administration of high Mr glutaraldehyde polymerized ovalbumin (OA) to C57BL/6 mice prior to immunization with OA in A1(OH)3 adjuvant resulted in induction of an interferon-gamma (IFN-gamma) dependent, split tolerance in which maximal OA-specific IgE responses were 1-3% of those observed in saline-treated OA-[A1(OH)3] immunized controls. Concomitantly, these mice exhibited up to 10(3)-fold increases in OA-specific IgG2a synthesis. In this report we examine the longevity and resilience of these reciprocal effects on IgE inhibition/IgG2a enhancement over extended periods of time and following multiple re-exposures to the sensitizing allergen. The data indicate that the T-cell mediated changes in responsiveness which are induced upon exposure to glutaraldehyde-modified protein allergen, but not unmodified allergen, are (i) extremely long-lived (greater than 350 days); (ii) resistant to at least five re-immunizations with OA in A1(OH)3 adjuvant; and (iii) antigen specific. The results are consistent with a virtually permanent shift in the OA specific T-cell repertoire in vivo from one dominated by Th2-like patterns of cytokine synthesis (IL-4) to one dominated by Th1-like (IFN-gamma) cytokine production. PMID- 1717369 TI - NK cells inhibit T-cell responses: LFA3+ but not LFA3- T-cell responses are suppressed. AB - There is increasing evidence that natural killer (NK) cells have immunoregulatory effects in addition to their ability to lyse tumour and virus-infected target cells. However, much of the evidence to date is based on the reported effect of adding relatively impure NK cell populations to various in vitro cultures, and the effect on T cells has been contradictory. Here we report the inhibitory effect of highly purified CD16+ NK cells on mixed lymphocyte reaction (MLR), using unseparated peripheral blood mononuclear cells (PBMC) and purified T cells as responders. Marked inhibition was observed (up to 75%) which was proportional to the number of CD16+ cells present, and was abrogated by ultraviolet irradiation. In contrast, the addition of CD16+ cells had no effect on the proliferative responses of five CD4+ anti-DR1 alloreactive T-cell clones. To test the relative sensitivity of previously primed versus virgin T cells to NK cell mediated inhibition, freshly isolated T cells from PBMC were separated into LFA3+ (memory) and LFA3- (virgin) populations. CD16+ cells caused inhibition of proliferation of LFA3+ but not LFA3- cells in an MLR. In addition, the recall response of T cells to influenza was inhibited. These results further illustrate the regulatory potential of CD16+ NK cells, and suggest that previously primed cells are more susceptible to NK-mediated inhibition. However, activated (rather than resting) cells may escape regulation. PMID- 1717368 TI - In vitro regulation of thyroglobulin (Tg) autoantibody production by Tg-specific T-cell lines and hybridomas. AB - To define the interactions between self thyroglobulin (Tg)-reactive T and B we co cultured enriched B cells taken from rat or mouse Tg-primed mice with major histocompatibility complex (MHC) class II-restricted T-cell lines specific for iodinated determinants on self-Tg, or hybridomas derived from those lines. Using two clonally distinct T-cell hybridomas, ADA2 and CH9, in vitro help for Tg autoantibody responses was observed using mouse (M)Tg-primed B cells and a 100 ng/ml MTg challenge. Using rat Tg-primed B cells and the same conditions, only CH9 provided help, indicating that the fine specificity of B cells influences their ability to interact with specific anti-Tg T-cell clones. In contrast to T cell hybridomas, their parent T-cell lines MTg9B3 and MTg12B suppressed Tg autoantibody responses in vitro, although they augmented bystander proliferation of unprimed B cells. The MTg12B cells also (i) diminished the survival of Tg primed B cells, and (ii) inhibited the proliferation of an antigen-presenting B cell hybridoma (LK35.2) in a cytostasis assay. These findings together support the view that their suppressive activity is mediated through cytotoxicity. While the role of class II-restricted cytotoxic cells in thyroid autoimmunity is unknown, the results suggest that such cells may act to suppress autoantibody responses as well as to mediate tissue damage to class II-expressing thyroid cells. PMID- 1717370 TI - Immune recognition of linear epitopes in peptide fragments of epithelial mucins. AB - Anti-human milk fat globule membrane monoclonal antibodies HMFG-1 and HMFG-2 recognize epitopes within the protein core of human polymorphic epithelial mucin (PEM). These have been identified as PDTR and DTR, respectively. Using the solid phase synthesis of immobilized tetrameric peptides, we have systematically investigated the contribution of each amino acid in the immune recognition of the PDTR domain by the antibodies HMFG-1 and HMFG-2. The findings obtained have been interpreted with respect to the presence of, and requirements for elements of secondary structure, which have been identified in this region of the PEM protein core. PMID- 1717371 TI - Cremophor EL as an adjuvant affecting immunoglobulin class switch in the immune response to the thymus-independent antigen alpha(1- greater than 3) dextran B 1355 S. AB - The humoral immune response to the so-called thymus independent antigen dextran B 1355 S in conventionally raised BALB/c mice consists solely of IgM antibodies. Expression of IgG anti-Dex antibodies in these mice is prevented by pre- or perinatally activated idiotype-specific T-suppressor lymphocytes. IgG B-memory cells nevertheless develop during the course of immunization, but are arrested in an anergic state. In the presence of Cremophor EL the induction of this anergic state is inhibited and the immune response shifts fully to an IgG anti-Dex response. PMID- 1717372 TI - Conversion of the human blood group H antigen to A antigen in vitro. AB - A-transferase (N-acetylgalactosaminyl transferase) was purified from human group A plasma using Sepharose 4B affinity chromatography. Human anti-A antibodies were purified from human serum by adsorption to an immunosorbent column and heat elution in order to detect the A antigen. Conditions appropriate for the development of the A antigen on O red cells were examined and several buffer systems were found to be equally effective. Expression of the developed A antigen was found to be similar to that on group A red cells, indicating that the system in vitro has similar activity to the system in vivo. The H antigen from human saliva was coupled to Sepharose 4B or adsorbed to a nitrocellulose membrane. The A antigen was able to be developed on these materials by the action of group A transferase. The procedures enabled the identification in vitro of sugar transferase activities which can be useful in studies within the A,B,H antigen system or other carbohydrate antigen system. PMID- 1717374 TI - Detection of class I MHC antigens on glycosidase-treated cells. AB - Several Class I molecules encoded by the murine H-2K gene have been found to express epitopes with different sensitivities to glycosidases. This result was found to be variably represented by different assays used to measure the binding of monoclonal antibodies (MoAb) specific for Class I H-2Kk molecules. PMID- 1717373 TI - Monoclonal antibodies to secreted antigens of Brugia malayi define a cross reactive non-phosphocholine determinant on helminth parasites. AB - The excretory-secretory (ES) antigens of the filarial parasite Brugia malayi adult (BmA) and microfilariae (MF) were analysed for differences in their protein composition by two-dimensional gel electrophoresis. Both BmA and MF biosynthetically labelled with [3H]-leucine released a 200 kD molecule with pI (isoelectric point) ranging from 5.1 to 6.8. A monoclonal antibody (MoAb) to phosphocholine (PC) immunoprecipitated the 200 kD molecule with a PI of 5.1-5.3 from both BmA and MF. Immunization of CBA/N mice with ES antigens resulted in MoAb that reacted with PC, as well as some that did not (ES-1, ES-2 and ES-3). The epitopes recognized by these non-PC MoAb varied greatly in their molecular weight, heat-resistance, TCA-solubility and metaperiodate-sensitivity, which suggests a proteoglycan nature of the epitope. An antigen capture assay using ES 1 and ES-3 detected a circulating filarial antigen not only in patients infected with Wuchereria bancrofti (77-83% of those with asymptomatic microfilaraemia and 52-60% of those with chronic lymphatic obstruction) but also in serum from patients infected with other helminth parasites (12-100%). Indeed, the epitope recognized by these MoAb was also present on other helminth parasites, which suggests conservation of a carbohydrate-like prosthetic group(s) on diverse helminth species. PMID- 1717375 TI - Identification of class I H-2Db molecules primarily expressed by B lymphocytes in murine spleen. AB - Evidence is presented which supports the phenomenon of heterogeneity amongst H 2Db-encoded Class I molecules. Two monoclonal antibodies (MoAb) called H141-31 and B22-249 were used in these studies. Both bind to the 'private' H-2.2 site of H-2Db-encoded molecules, but the binding of B22-249 is determined by carbohydrate moieties, whereas H141-31 appears to bind to a protein-defined epitope. Some H 2Db molecules, identified by the H141-31 MoAb, are primarily expressed on B lymphocytes and not T lymphocytes in spleen. The number of H-2Db molecules which bind H141-31 on B cells was also found to be three- to four-fold less than the number which bound the B22-249 MoAb. B cells of two mutant strains of mice, B6 C.H-2bm13 and B6-C.H-2bm14 which harbour very few nucleotide changes in the H-2Db gene, also show marked reduction in the binding of both antibodies. This suggests that a single common gene encodes both target molecules and that post translational modifications such as differential glycosylation may account for heterogeneity amongst H-2Db molecules. This would explain the presence of the different H-2Db molecules defined here. It follows that differences in glycosylation evidently occur both within the B cell population, since H141-31 binds to only a subset of H-2Db molecules on B cells, and between T and B lymphocytes, since resting T cells do not bind H141-31 MoAb. PMID- 1717376 TI - The immunosuppressive macrolides FK-506 and rapamycin. AB - Cyclosporin (CsA) and FK-506 are structurally distinct fungal metabolites, which exert powerful inhibitory effects on CD4+ T (helper) cell activation and on the secretion of interleukin-2 (IL-2) and other cytokines, including various cell growth factors and interferon-gamma. Both drugs also inhibit IL-2 receptor expression on T cells. Consequently, when administered from the time of transplant surgery, both CsA and FK-506 inhibit the generation and proliferation of cytotoxic T cells which would otherwise mediate allograft rejection; T-cell dependent antibody responses are also inhibited by both drugs. CsA and FK-506 however, differ markedly in immunosuppressive potency. FK-506 is at least 100 times as potent as CsA in inhibiting human mixed lymphocyte reactions in vitro, whilst the ID50 of FK-506 for inhibition of allograft survival in animals is approximately one tenth that of CsA. FK-506 and CsA, both of which are highly lipophilic molecules, bind to distinct cytosolic proteins, each of which is a peptidyl-prolyl isomerase and the activities of which may play critical roles in signal transduction within activated T cells. The precise molecular mechanism by which these drugs selectively inhibit cytokine gene expression at a pretranscriptional level is not understood but a transcription activator has been implicated as the target. Compared with CsA, the inhibitory action of FK-506 appears more difficult to reverse, e.g., in response to pre-formed IL-2. Both drugs are however, ineffective in inhibiting directly the cytotoxic activity of cytotoxic T cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717377 TI - To be or not to be a responder in T-cell responses: ubiquitous oligopeptides in all proteins. AB - Amino acid sequences of all proteins are essays written in the same language. Accordingly, the same set of words and phrases (oligopeptides) appear in totally unrelated proteins. The reason that only certain individuals of particular major histocompatibility complex (MHC) haplotypes can mount T-cell responses against a given antigen of pathogens is found in the fact that T-cell receptors are designed to recognize 18-20 residue-long peptide fragments sandwiched between two alpha-helices of class I or class II MHC molecules. At this range of peptide lengths, most would appear as self, while nonselfness of the remainders are destined to be quite ambiguous, hence creating responders and nonresponders. PMID- 1717378 TI - Iris neovascularization in Sturge-Weber syndrome. AB - An unusual case of Sturge-Weber syndrome with raised intraocular pressure is presented. The slit lamp examination and anterior segment fluorescein angiography were consistent with iris neovascularization. Cause and effect relationship of iris neovascularization with glaucoma in this syndrome is discussed. PMID- 1717379 TI - Reduction of experimental myocardial infarct size by pre-treatment with magnesium sulfate in rats. AB - Experimental myocardial infarction was induced in albino rats by administration of isoprenaline hydrochloride, 85 mg/kg, sc, daily for two consecutive days. Such rats were pretreated with either saline or magnesium sulfate (60 mg/kg) po, daily for three weeks, to serve as control or treated groups respectively. Heart specimens were taken for gross and histological examination at 24 hr, on 5th day, 12th day and 21st day. Infarct size was significantly reduced in the magnesium treated group (P less than 0.05). We conclude that magnesium sulfate exerted a potent prophylactic effect in limiting infarct size in rats. PMID- 1717380 TI - Neuroimmunomodulation in the intestinal mucosa. AB - The intestine contains major subdivisions of the nervous and immune systems. The lymphoid compartments of the intestine contain functionally distinguishable populations of immunologic cells and are innervated differently. The lamina propria has an extensive network of nerves using the neuropeptides SOM, SP, and VIP. Subpopulations of T cells, B cells, cells of the monocyte/macrophage line, and several other immunologically relevant cells have the ability to recognize and respond to these neuropeptide signals. SOM, SP, and VIP can act as potent regulators of lymphoid cell proliferation and interleukin and immunoglobulin production. The unusual effector lymphocytes in the epithelial layer of the intestine can be exposed to SP and VIP, and their responses may be regulated by these peptides. In the organized lymphoid compartments such as Peyer's patches, the neuropeptides VIP and SP may regulate the accumulation or recirculation of affector lymphocytes from the central compartment of the immune system and their subsequent response to antigens. The large array of immunoregulatory effects that have been found with these neuropeptides suggest that local neurophysiologic signals in the intestinal lymphoid microenvironments can regulate selected aspects of immune responses. The intestine is likely to be a highly specialized venue for neuroimmunomodulation in intact animals, and this has important implications in the physiologic and pathologic responses of the gut. Further investigations of these regulatory pathways will lead to new concepts concerning neural-immune interactions in general and the regulation of mucosal immunology in particular. PMID- 1717381 TI - Mucosal T-cell function. AB - Lymphocytes in the intestinal lamina propria probably differ from lymphocyte populations in the circulation or in other tissue sites in a number of ways. First, lamina propria lymphocytes are phenotypically distinct in that few of these cells normally express the Leu-8 or CD45RA antigens. The presence of these molecules on CD4 T cells correlates with suppressor and suppressor-inducer function, and therefore the majority of CD4 T cells in the intestinal lamina propria have the phenotype associated with high helper activity. A substantial proportion of lamina propria lymphocytes also have evidence of activation, based on expression of the IL-2R alpha chain and HLA-DR molecules. Lymphocytes in the intestinal lamina propria are different in their potential for expression of lymphokine gene products, because activated cells from the lamina propria have high expression of mRNA for IL-2, IL-4, IL-5, and IFN-gamma in comparison to circulating lymphocytes. Mesenteric lymph node T cells also differ from circulating lymphocytes in their high expression of IL-4 and IL-5 mRNA. A further difference between mesenteric lymph node and lamina propria T cells is that the former can proliferate in response to IL-4, whereas the latter cannot. These phenotypic and mRNA differences of lamina propria lymphocytes also correlate well with their high helper activity in vitro for immunoglobulin synthesis in the pokeweed mitogen system. T cells with the potential for cytolytic activity are present in the intestinal lamina propria, although there is no definitive evidence that they are cytolytically active under physiologic or pathologic conditions. Finally, in a model system of intestinal inflammation, lymphogranuloma venereum in nonhuman primates, lamina propria T cells at the site of inflammation were unable to respond to specific antigens with proliferation but did respond with high helper activity. These observations are all consistent with the conclusion that T cells in the lamina propria are pleomorphic but are highly enriched for subpopulations of activated memory cells that are geared for effector functions such as helper and cytolytic functions. These functions are likely to be critical in maintaining normal host defense in the mucosal environment. PMID- 1717382 TI - Distribution of substance P-immunoreactive and calcitonin gene-related peptide immunoreactive nerves in normal human lungs. AB - The distribution of substance P (SP)-immunoreactive (IR) and calcitonin gene related peptide (CGRP)-IR nerves in normal human lungs was immunohistochemically investigated. SP-IR and CGRP-IR nerves were found in lamina propria and adjacent to blood vessels, and also in some of the ganglia and nerve bundles in the submucosal area. Comparison of serial consecutive sections revealed that SP-IR and CGRP-IR nerves represented similar distribution patterns. CGRP-IR nerves were scarce in bronchial smooth muscles, whereas SP-IR nerves were always present therein. Neither SP-IR nor CGRP-IR nerves were found, however, among submucosal glands or in alveolar septa. These localization patterns were closely related to the functions of SP and CGRP in normal lungs. PMID- 1717383 TI - Characterization of methylated bovine serum albumin-induced allergic inflammation in rats. AB - An air pouch type allergic inflammation in rats was induced using an insoluble cationic protein, methylated bovine serum albumin (MeBSA), as an antigen. Changes in vascular permeability, local tissue edema, histamine contents in the pouch fluid, and number of infiltrated leukocytes and chemotactic activity in the pouch fluid were analyzed during an 8-hour period after injecting the antigen solution into the air pouch of the immunized and nonimmunized rats. Vascular permeability during the first 30-min interval in the immunized rats was higher than that in the nonimmunized rats, reflecting a higher histamine level in the pouch fluid. However, both the increase in vascular permeability and histamine level in the immunized rats in this period were much lower than those induced by a soluble, noncationic antigen, azobenzenearsonate-conjugated acetyl bovine serum albumin. In the MeBSA-induced allergic inflammation model, a second peak of vascular permeability was induced at 2 h, and local tissue edema formation became apparent at 2 h, reaching a plateau at 4 h. A prominent increase in leukocyte infiltration, especially neutrophils, into the pouch fluid was induced at 4 h in accordance with an increase in chemotactic activity in the pouch fluid. These observations indicate that the acute phase of MeBSA-induced allergic inflammation is characterized by a weak anaphylactic response and a prominent neutrophil infiltration. PMID- 1717384 TI - TRA-1-60: a new serum marker in patients with germ-cell tumors. AB - TRA-1-60 is a monoclonal antibody (MAb) that recognizes a mucin-like antigenic determinant expressed on the surface of embryonal carcinoma (EC) progenitor cells. In order to determine whether this antigen is released into the serum of patients with a non-seminomatous germ-cell tumor (NSGCT), we developed a sensitive 2-step immunoenzymometric assay. Of 42 EC-positive NSGCT patients tested, 32 (76%) were found to release TRA-1-60-reactive antigen into their serum, in contrast to 1 positive finding in 10 EC-negative NSGCT patients. The marker was found in 67% (10/15) of the EC-positive patients who were negative for both AFP and HCG. Sera from seminoma patients did not contain elevated levels of the TRA-1-60 antigen. Therefore, we propose that the TRA-1-60 antigen is a useful additional serum marker for following the progress of NSGCT(EC+) patients. PMID- 1717385 TI - Activation of an alpha-fetoprotein (AFP)/receptor autocrine loop in HT-29 human colon carcinoma cells. AB - Immunological and morphological approaches have been used to demonstrate, respectively, alpha-fetoprotein (AFP) synthesis and receptor expression in the HT 29 human colon carcinoma parental cell line. HT-29 cells cultivated in the presence of glucose synthesized and secreted AFP in the medium from 48 to 96 hr after seeding, as revealed by monoclonal antibodies (MAbs) to human AFP. Light microscopic observations of cells incubated with fluoresceinated AFP showed that the protein was specifically bound to the cell surface at 4 degrees C, and was internalized in the cytoplasm at 37 degrees C. At the ultrastructural level, horseradish peroxidase (HRP)-conjugated AFP, as well as HRP-transferrin (Tf) (used as a control), appeared to be internalized via coated pits and vesicles before being delivered to endosomes, from which they were apparently recycled back to the cell surface via small vesicles. Our results suggest that a AFP/receptor autocrine pathway might operate in these cells, be preferentially active at the beginning of the exponential phase of cell culture growth, and contribute to cell proliferation. PMID- 1717386 TI - Recombinant human granulocyte colony-stimulating factor inhibits the metastasis of hematogenous and non-hematogenous tumors in mice. AB - We studied the effects of in vivo administrations of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the metastasis of murine hematogenous and non-hematogenous tumors in spontaneous and experimental metastasis models. Spontaneous lung metastasis caused by intra-footpad injections of B16-BL6 melanoma and Lewis-lung-carcinoma (3LL) cells were inhibited by intravenous (i.v.) and subcutaneous (s.c.) injections of rhG-CSF after excision of the primary tumors. Recombinant hG-CSF significantly inhibited liver metastasis when administered i.v. after i.v. injection of L5178Y-ML25 T-lymphoma cells. Multiple i.v. administration of rhG-CSF after the tumor inoculation prolonged the survival times of mice inoculated i.v. with L5178Y-ML25 lymphoma cells. Recombinant hG-CSF did not directly affect the growth of B16-BL6 and L5178Y-ML25 cells in vitro. During the administration periods, both i.v. and s.c. injections of rhG-CSF increased the number of total white blood cells (WBC) in peripheral blood to approximately 3 times the normal level in normal and tumor bearing mice. We also found that the administration of rhG-CSF stimulates neutrophils to become cytostatic against these tumor cells. Our results indicate that the injection of rhG-CSF is effective in inhibiting lung and liver metastases by activating neutrophils and increasing cell number. PMID- 1717387 TI - Tricuspid atresia with common arterial trunk: surgical palliation in a neonate. AB - A two-week-old asymptomatic baby was diagnosed by cross-sectional and Doppler ultrasound to have tricuspid atresia with a common arterial trunk. Successful surgical palliation was undertaken at 17 days of age, by disconnection of the pulmonary arteries from the trunk, and creation of an aortopulmonary shunt. There are no known previous reports of surgical palliation of this lesion. PMID- 1717388 TI - Oxygen free radicals and pelvic adhesion formation: II. The interaction of oxygen free radicals and adhesion-preventing solutions. AB - A three-part animal and in vitro study gives evidence that oxygen free radicals are produced by intraperitoneal macrophages in response to pelvic surgery. These macrophages appear to ingest high-molecular-weight substances used to prevent adhesions (such as 32% dextran 70 and carboxymethylcellulose). However, the production of oxygen free radicals is not enhanced by the presence of 32% dextran 70 or carboxymethyl cellulose. PMID- 1717389 TI - Hepatoid carcinoma of the ovary: a case report. AB - A case of a right ovarian tumor in a 64-year-old patient showing high blood levels of alpha-fetoprotein (AFP) is reported. Histologically, the tumor resembled hepatocellular carcinoma with hyaline globules. Localization of AFP was detected by the immunoperoxidase method. Electron microscopically, the rough surfaced endoplasmic reticulum had developed into a meshwork, and the mitochondria were present within this meshwork. Because a transition from adenocarcinoma to a region resembling hepatocellular carcinoma was observed, this tumor was considered to originate as a common epithelial carcinoma. In the blood, 67% of the AFP was bound with concanavalin A (Con A), and the fraction pattern obtained by lentil agglutinin affinity chromatography (LCA) was of the germ cell type. From these results, the current case may be labeled clinicopathologically a hepatoid carcinoma of the ovary as described by Ishikura and Scully. PMID- 1717390 TI - An antibiotic-free medium for the xenic cultivation of Entamoeba gingivalis. AB - Diamond's TYI-S-33 (Trypticase-Yeast Extract-Iron-Serum) medium was used as the basis for a new antibiotic-free medium for xenic growth of Entamoeba gingivalis. Nutritional requirements of the oral protozoan were determined in an effort to optimize growth. TYI-S-33 medium did not support E. gingivalis growth prior to modification. The changes included: (a) deletion of L-cysteine.HCl and thioctic acid, (b) substitution of glucose for dextran I (mol. wt 185,000) or rice starch, (c) reduction of concentrations of tryptone (2.5 g l-1), yeast extract (1.25 g l 1) and dextran I (1 g l-1), (d) increased concentration of ferric ammonium citrate (0.2 g l-1), and (e) addition of gastric mucin (2.4 g l-1). Dextran I was chosen as the major carbon source; its use in the medium limited growth of accompanying bacteria. This new antibiotic-free medium significantly increased E. gingivalis growth (16-20 E. gingivalis trophozoites observed per field) as compared to growth in Diamond's TYSGM-9 (Trypticase-Yeast Extract-Serum-Gastric Mucin) medium (six to 10 E. gingivalis trophozoites observed per field). PMID- 1717391 TI - Production and characterization of monoclonal antibodies against the excretory secretory antigen of the liver fluke (Opisthorchis viverrini). AB - Monoclonal antibodies (MoAb) were produced against a major soluble metabolic product (excretory-secretory, ES) of Opisthorchis viverrini. The latter was obtained in a form of spent culture medium in which the adult flukes had been maintained in vitro. The MoAb produced were exclusively associated with either IgG or IgM isotypes. When screened against a panel of parasite antigens by indirect ELISA, these MoAb exhibited three patterns of reactivity. Approximately 50% of the MoAb were highly specific for O. viverrini and another 25% cross reacted only with Clonorchis sinensis. The remaining 25% cross-reacted extensively with other parasites. Results from radioimmunoprecipitation and immunoblotting experiments showed all MoAb to react with the 89-kDa glycoprotein. By indirect immunofluorescence, these MoAb reacted almost exclusively with the tegumental surface, tegumental cells, cecum and developing miracidium. Different lines of evidence suggest that these MoAb reacted with different epitopes on the same 89-kDa polypeptide carrier. PMID- 1717392 TI - Conformation and inhibitory properties of peptides based on the tissue kallikrein aprotinin complex. AB - A series of peptides encompassing the primary binding segment (residues 12-19) of aprotinin has been synthesized and tested for their ability to inhibit porcine pancreatic kallikrein. A minimum sequence of five amino acids spanning residues 12-16 of aprotinin is necessary for inhibition of porcine pancreatic kallikrein. An octapeptide homologous with the binding segment of aprotinin has a Ki-value of 1.2 x 10(-4) M. The solution structure of the octapeptide was studied by one- and two-dimensional NMR methods for comparison with the known structure of the segment of aprotinin that contacts tissue kallikrein. NMR experiments suggest that the peptide is either a random coil or that it samples several conformations on the NMR time scale. Analysis of the molecular dynamics trajectory of the octapeptide also suggests that the peptide is highly flexible. Thus, inhibition by the octapeptide occurs because of its homology with residues 12-19 of aprotinin. Moreover, the absence of a stable solution conformation similar to that of the binding segment of aprotinin is consistent with the 150,000-fold increase in Ki of the octapeptide compared to intact aprotinin. PMID- 1717393 TI - Evidence for a glycoconjugate form of glutathione S-transferase pI. AB - The anionic form of glutathione S-transferase from human (GST pi) and rat (GST Yp) sources has been shown to exist in multiple forms which have similar molecular weights but different isoelectric points (pIs). Treatment with endoglycosidase H caused the acidic forms of GST Yp to be converted to proteins with more basic pIs as compared to the untreated control mixtures, suggesting that an N-linked mannose moiety containing acidic residues had been removed. Inability to detect these carbohydrates by techniques requiring unsubstituted vicinal hydroxyls further suggested acidic substitutions on the sugar moiety. GST pi/Yp carbohydrate modifications were also identified by differential staining procedures. These data represent the first indication that glycosylation of GST can occur. Additionally, this may offer an explanation for the often seen microheterogeneity within a class of GST isozymes. PMID- 1717394 TI - Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl phosphate protection. AB - The synthesis of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO3Me2)-OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the -Tyr(PO3Me2)-residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)-containing peptides using t-butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc Tyr(PO3tBu2)-OH was prepared in high yield from Fmoc-Tyr-OH by a one-step procedure which employed di-t-butyl N,N-diethyl-phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)-containing peptides, Ala-Glu-Tyr(P)-Ser-Ala and Ser-Ser-Ser-Tyr(P)-Tyr(P). PMID- 1717395 TI - Vitamin C inhibits DNA, RNA and protein synthesis in epithelial neoplastic cells. AB - Radioactivity measurements and autoradiographic studies of DNA, RNA and protein synthesis using [3H]-thymidine, [3H]-uridine and [3H]-leucine revealed that Vitamin C administration significantly (by 50%) decreases the DNA, RNA and protein synthesis in the neoplastic cells of basal cell carcinomas and squamous cell carcinomas in mice and rats. Basal cell carcinomas and squamous cell carcinomas were induced by a topical application of a chemical carcinogen, 3 methylcholanthrene. The inhibition of DNA, RNA and protein synthesis is accompanied by advanced ultrastructural and cell surface changes. Since the concentrations of Vitamin C were significantly higher in the plasma of Vitamin C treated as compared to that of non Vitamin C-treated animals, these findings demonstrate that Vitamin C inhibits DNA, RNA and protein synthesis in neoplastic epithelial cells, and thus exerts its antineoplastic effect. PMID- 1717396 TI - Effects of cytoskeletal perturbation on the sensitivity of Ehrlich ascites tumor cell surface membranes to mechanical trauma. AB - Evidence presented previously indicated that shape changes in circulating cancer cells within the microvasculature may lead to rapid, lethal rupture of the cell surface membranes, thereby contributing to the inefficiency of this phase of hematogenous metastasis. As the cytoskeleton is known to regulate cell shape and, in at least some cases, to be attached to the surface membrane, we have determined whether or not it plays a role in inhibiting lethal, deformation associated, mechanically induced surface membrane trauma. Thus, Ehrlich ascites tumor (EAT) cells were treated with different cytoskeleton-perturbing agents, and their susceptibility to filtration trauma on passage through Nuclepore membranes was determined. In contrast to the involvement of the cytoskeleton in nonlethal cell deformation, agents which disrupt intracellular networks of microtubules, microfilaments and intermediate filaments had little or no direct effect on EAT cell susceptibility to rapid, mechanically induced, lethal trauma. PMID- 1717397 TI - Expression of tenascin and cellular fibronectin in the rabbit cornea after anterior keratectomy. Immunohistochemical study of wound healing dynamics. AB - Anterior keratectomy (AKE) was done on rabbits, and the appearance of immunohistochemically demonstrable tenascin (TN) or cellular fibronectin (cFN) was studied at different times (5 min to 14 months) after the operation. The substance TN was first observed 12 hr after wounding in the posterior stroma; cFN appeared with the same localization 12 hr later. During postoperative week 1, both TN and cFN immunoreactions shifted to more anterior parts of the cornea, and 9 days after wounding, they were localized in the most anterior part of the stroma only. Thereafter the reactions gradually decreased in intensity but still were visible 3 months after AKE. No reaction for TN or cFN was present 14 months postoperatively. PMID- 1717398 TI - Cloning of alpha 1(IV) and alpha 2(IV) collagen cDNAs from rabbit corneal endothelial cell RNA. AB - To understand the role of type IV collagen in embryogenesis, regeneration, and tissue repair of the cornea at the molecular level, the authors isolated clones coding for rabbit alpha 1(IV) and alpha 2(IV) chains from a cDNA library constructed with rabbit corneal endothelial cell RNA. The isolated alpha 2(IV) cDNA clones encode a part of a 5' untranslated region, the signal peptide, the 7S domain, part of the triple-helical domain, and the entire carboxyl-terminal nontriple-helical (NC1) domain. By cross hybridization, using one of the alpha 2(IV) cDNA inserts, a cDNA clone encoding the alpha 1(IV) chain was isolated from a lambda gt10 library primed with oligo(dT). The clone covered a short portion of the triple-helical domain, the entire NC1 domain, and a short 3' untranslated region. The nucleotide-sequence analysis of these clones provides, for the first time to the authors' knowledge, the primary structure of the carboxyl-terminal portion of both rabbit alpha 1(IV) and alpha 2(IV) collagen chains. Between the alpha 1(IV)NC1 and the alpha 2(IV)NC1, 61% and 66% sequence similarities were observed at the amino acid and nucleotide levels, respectively. The locations of all the 12 cysteinyl residues were conserved in the two NC1 sequences. PMID- 1717399 TI - Comparison of immunofluorescence and an immunohistochemical technique for the detection of perinuclear anti-neutrophil cytoplasmic antibody. AB - Using the standard indirect immunofluorescent (IIF) technique two types of autoantibodies are detected in the sera of patients with vasculitic disorders. These are cytoplasmic or classical antineutrophil cytoplasmic antibody (cANCA) and perinuclear anti-neutrophil cytoplasmic antibody (pANCA). In order to resolve the problems associated with the detection of pANCA an immunocytochemical technique-alkaline phosphatase anti-alkaline phosphatase (APAAP) was developed and used to detect ANCA in various groups of patients. Comparison with the standard immunofluorescence method showed that the results correlated only when immunostaining patterns were of the cANCA type. Detection of pANCA by APAAP was more uncertain than by immunofluorescence because of the greater number of staining patterns seen. Interference from antinuclear antibodies (ANA) appeared to cause more problems with the APAAP technique and sera containing ANA were not distinguished from pANCA using either immunofluorescence or APAAP. In conclusion, it appears that the APPAP technique is not a reliable method of the screening of ANCA and that pANCA has several antigenic specificities. PMID- 1717400 TI - What to do with the patient with a positive HCV antibody test. AB - A test to measure hepatitis C antibody has recently been developed. The development of the antibody test was closely followed by the publication of two trials which demonstrated the efficacy of interferon alfa in the treatment of transfusion-associated hepatitis C. The author discusses management of the patient with a positive hepatitis C virus (HCV) antibody test, including an approach to sorting out those patients who have false positive tests, information about modes of transmission for those patients who are asymptomatic carriers, and guidelines to determine which patients may be candidates for interferon therapy. PMID- 1717401 TI - [Development of the epidermis from fish to human]. AB - The epidermis, the outermost layer of all individuals, is a point of contact between individuals and their surroundings, especially insofar as it mediates sensory stimuli, but it also has the function of separating them from their surroundings and providing protection from harmful environmental influences. This paper traces the adaptation of the epidermis of vertebrates to these multiple functions via various methods of differentiation. In the human, epidermal differentiation is divided into synthesis, transformation, and terminal stages, and the stratum corneum is the final product of this differentiation. The differentiation products (tonofilaments, keratohyalin granules, membrane coating granules, cornified envelopes) are discussed with reference to their molecular bases (especially cytokeratin polypeptides, filaggrin, involucrin, lipids). The importance of these new cell-biological results for the pathogenesis of disorders of keratinization is discussed. PMID- 1717402 TI - [Facial kaposi's sarcoma. Palliative treatment with cryotherapy, intralesional chemotherapy, low-dose roentgen therapy and camouflage]. AB - Facial Kaposi's sarcoma is a severe psychological problem for HIV-infected patients if systemic chemotherapy or interferon therapy is not possible. To help these patients, it is important to offer a palliative treatment that is suitable for outpatients and is not complicated by severe side-effects but still yield satisfactory cosmetic results. A total of 65 patients with 216 facial Kaposi's sarcomas were treated with cryosurgery (92 tumours, 29 patients), intralesional chemotherapy with vincristine (28 tumours, 12 patients), radiotherapy (87 tumours, 15 patients) and camouflage (multiple tumours, 9 patients). Cryosurgery is the treatment of choice for small (less than 1 cm) macular or slightly nodular Kaposi's sarcoma. Larger nodular tumours are better treated by intralesional chemotherapy (single doses of 0.01-0.1 mg vincristine per tumour) or low-dose radiotherapy (3-4 x 4 Gy). These palliative treatment methods are not indicated in cases of rapid tumour progression and dissemination; in such cases, camouflage (covering the tumours with water-resistant make-up) is helpful as a local palliative measure. PMID- 1717403 TI - Sickle cell anemia in the Tunisian population: haplotyping and HB F expression. AB - Thirty-three Tunisian patients, homozygous for Hb S, were examined. Haplotyping using nine restriction sites in the beta-globin gene cluster revealed that the most common type is the Benin type [---- + - + - +] which occurs at a frequency of 0.94% (31 cases); only one patient was homozygous for an atypical haplotype which shows some differences with the Benin haplotype at sites 1, 5, 6, and 8 [+ --- + + + +]; the two remaining patients were assumed to be double heterozygotes for the Benin and atypical haplotypes. The presence of the atypical haplotype suggested a double origin of the beta S gene in Tunisia. Moreover, a heterogeneity in the Hb F production was observed, ranging between 2 to 16%, whereas the G gamma-globin expression was remarkably homogeneous in our patients with a normal amount approaching 40%. These results suggested the presence of a combination of several control factors. PMID- 1717404 TI - Pneumococcal septicemia and meningitis in an infant with Hb S/D-Los Angeles disease: a failure of neonatal hemoglobinopathy screening. PMID- 1717405 TI - A greater than 200 kb deletion removing the entire beta-like globin gene cluster in a family of Irish descent. AB - We describe a new deletional form of gamma delta beta-thalassemia segregating in two generations of a family of Irish descent. Affected family members present with a beta-thalassemia minor phenotype, normal Hb A2 and Hb F levels. Genomic blotting analyses on DNA from affected family members show heterozygosity for a large deletion beginning at least 15 kb upstream of the 5' endpoint of the gamma delta beta-thalassemia-1 deletion, extending through the entire beta-like globin gene cluster, and continuing for at least 10 kb beyond the 3' endpoint of the deletion associated with the Spanish form of delta beta 0-thalassemia. This deletion is among the largest described so far, and removes at least 205 kb encompassing the entire beta-like globin gene cluster on chromosome 11. PMID- 1717406 TI - The G----A mutation at position +22 3' to the Cap site of the beta-globin gene as a possible cause for a beta-thalassemia. AB - We describe the occurrence of a chromosome with a G----A mutation at position +22 relative to the Cap site that was found in five patients with beta-thalassemia. All patients had a common type of beta-thalassemia mutation on the second chromosome, namely the frameshift at codon 8 (-AA), the IVS-I-110 (G----A) and the IVS-II-1 (G----A) mutations. The beta genes of two patients, including the 5' and 3' untranslated regions, were completely sequenced and no other mutations, except a few polymorphic sites, were observed. Dot-blot analyses failed to demonstrate this G----A mutation at +22 in nearly 400 beta-thalassemia chromosomes and 180 normal chromosomes. Heterozygotes have the features of a high Hb A2-beta-thalassemia heterozygosity, although the hematological parameters might be less abnormal than observed in heterozygotes for the more common beta thalassemia mutations. The possibility has been presented suggesting that this mutation might impair the binding of mRNA to ribosomes. Another mutation in this segment of DNA, i.e. a C----G mutation at position +20, is observed exclusively on a chromosome which also carries the C----G mutation at IVS-II-745. It is postulated that the +20 C----G mutation accentuates the beta-thalassemia condition caused by the IVS-II-745 mutation; the mechanism might be similar to that suggested for the G----A at +22 mutation. PMID- 1717407 TI - Re-evaluation of the specificity of adenylyl (beta,gamma-methylene)diphosphonate as a substrate for adenylate cyclase. AB - The conversion of the ATP-analogue adenylyl(beta,gamma-methylene)diphosphonate (AMPPCP) to cyclic AMP by adenylate cyclase of rat liver membranes was demonstrated using a radioimmunoassay for cyclic AMP. The conversion was only insignificantly lower than with adenylylimidodiphosphate (AMPPNP), another ATP analogue which is usually used in the histochemical adenylate cyclase assay. The unspecific phosphate production was lower with AMPPCP as compared to AMPPNP. Therefore AMPPCP is considered to be a more suitable substrate for the histochemical assay. Unspecific phosphate deposition in the histochemical assay was due to ATP:pyrophosphatase activity and could be significantly inhibited by 1 mM NAD. However, a residual phosphate deposition due to cleavage of NAD could not be suppressed. Adenylate cyclase activity could be markedly activated by 5 x 10( 5) M forskolin, an activator of the catalytic subunit of the enzyme, and inhibited by 1 mM 2'5'-dideoxyadenosine, a specific inhibitor of adenylate cyclase. Adenylate cyclase was localized predominantly in the sinusoidal part of the plasma membrane, while ATP-pyrophosphatase seemed to be restricted to the canalicular part. It is concluded that at least three parallel assays are necessary for routine histochemical demonstration of adenylate cyclase, namely (1) basal activity (2) activation by forskolin and (3) inhibition by 2'5' dideoxyadenosine, to demonstrate a specific enzyme reaction. PMID- 1717408 TI - Densitometric analysis of the local bleaching of the Neo-Timm staining pattern following intrahippocampal injection of diethyldithiocarbamate. AB - Treatment with certain metal chelating agents causes a time-dependent bleaching of the Neo-Timm staining pattern of zinc visualized in synaptic vesicles. In the present study, the extent and time course of the reversible chelation of hippocampal vesicular zinc was investigated following intrahippocampal injection of the chelating agent diethyldithiocarbamate. The carbamate (1.0 microliters 45 mg ml-1, 200 mM) was injected unilaterally into the hippocampal region of adult rats, which were allowed to survive 15 min-6 h before sacrifice. Control animals either received injections of distilled water or were untreated. Computerized optical densitometry was performed on cryostat sections of brains stained with the Neo-Timm method. Injection of diethyldithiocarbamate into the hippocampal region resulted in a localized bleaching of the Neo-Timm staining pattern. The extent of the bleaching varied with time being most pronounced at 15 min survival and gradually decreasing with time. After 6 h survival, a faint bleaching of the injected hippocampal region was barely seen. Computerized optical densitometry confirmed and extended the observations providing a semi-quantitative measure of zinc in synaptic vesicles. PMID- 1717409 TI - A histochemical method for the demonstration of unsaturated lipids. AB - A method has been developed for the histochemical demonstration of unsaturated lipids in light microscopy. It is a peracetic acid-thiocarbohydrazide-silver protein sequence followed by a physical development procedure. In the present study on paraffin and cryostat sections of liver, brain and ovary, unsaturated lipids were visualized as distinct reaction products coloured various shades of brown and black. The reaction products are easier to see and the method is more efficient than the peracetic acid-Schiff method. PMID- 1717410 TI - Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures. AB - Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) alpha-naphthylphosphate and Fast Blue BB; (4) beta-glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and beta-glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, an capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections. PMID- 1717411 TI - Neuromodulators of the lingual von Ebner gland: an immunocytochemical study. AB - The serous lingual glands of von Ebner secrete lingual lipase, an enzyme that begins fat digestion in the stomach. The objective of this study was to characterize the neuromodulators in the rat tongue and von Ebner glands using immunocytochemical techniques. Rat lingual tissues were fixed in formalin, embedded in paraffin and sectioned at 4 microns for light microscopic studies. Immunocytochemical localization of neuromodulators was performed with monospecific anti-rat neuromodulator IgG or control (preimmune) IgG as the primary antibody, using the peroxidase-antiperoxidase (PAP) technique. No staining was seen with control anti-rat IgG. Immunospecific staining for vasoactive intestinal peptide (VIP), tyrosine hydroxylase and choline acetyltransferase (CHAT) was observed in nerves in the tongue, and cells containing immunospecific staining for serotonin (5-hydroxytryptamine) were seen in the stroma between the lingual glands. Selected cells in the serous glands stained positively for the presence of substance P and somatostatin. Adrenergic, VIP-containing and cholinergic nerves appear to innervate the tongue and serous glands. Substance P and somatostatin were identified in cells of the lingual serous glands and may be additional local modulators regulating lingual lipase release. PMID- 1717412 TI - Extracellular matrix receptor and platelet antigens on osteoclasts and foreign body giant cells. AB - Osteoclasts (OCs) and other cells of the mononuclear phagocyte system possess receptors for adhesive proteins present in the extracellular matrix. The antigenic phenotype of OCs and foreign body giant cells (FBGCs) was investigated for the presence of several integrin molecules and other largely platelet associated antigens involved in cell adhesion reactions. Both OCs and FBGCs expressed the alpha-chains of the vitronectin receptor (CD51) and of the VLA-2 (CDw49b) and VLA-4 (CDw49d) molecules as well as their respective beta-chains, gpIIIa (CD61) and CD29. OCs and FBGCs also expressed CD9 and CD55 (DAF-Decay Accelerating Factor) and strongly reacted with antibodies directed against fibrinogen, fibronectin and vitronectin; the latter are ligands for several of the above matrix protein receptors. The data suggest that cell-cell and cell matrix interactions involving adhesive proteins may be important in OC and FBGC function. PMID- 1717413 TI - New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining. AB - The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed. PMID- 1717414 TI - Semi-automatic quantitation of dense markers in cytochemistry. AB - The quantitation of electron dense labelling is very tedious when it is done "by hand". Accordingly we developed software allowing, at electron microscopic level, a semi-automatic counting of dense markers in biological specimens. It includes the digitization of images and extraction of dense particles from the grey level of the background. The definition of the areas of interest was carried out by the observer but all quantitative calculations were done automatically. This method was applied to different biological materials (phospholipid and lysozyme labelling in secretory granules of human submucosal bronchial gland cells). The results obtained by this semi-automatic procedure were in good agreement with those obtained by manual counting of colloidal gold labelling (r = 0.97). PMID- 1717416 TI - Epitope mapping of human monoclonal antibodies to HLA-B27 by using natural and mutated antigenic variants. AB - The epitopes defined by three human monoclonal antibodies (mAbs) (Tr3B6, TrCG10, TrBH12) against HLA-B27 have been mapped by flow cytometry. For this purpose we used murine transfected cells expressing at their surface hybrid antigens between HLA-B7 and -B27 and, in addition, Epstein-Barr virus cell lines expressing the six HLA-B27 alleles B*2701 to B*2706. The results indicated that the mAbs are domain specific. TrBH12 recognizes the first external (alpha-1) domain. Residues critical for the TrBH12 epitope are located in the alpha-1 helix and include the polypeptide stretch 63-76 plus a critical amino acid at position 77. Tr3B6 binds the second external (alpha-2) domain, and one mutation (VAL152----GLU152) destroyed its epitope. TrCG10 also binds the alpha-2 domain. PMID- 1717415 TI - Localization of substance P, CGRP, VIP, neuropeptide Y, and somatostatin immunoreactive nerve fibers in the carotid labyrinths of some amphibian species. AB - Immunohistochemical localization of substance P (SP), CGRP, VIP, neuropeptide Y (NPY), and somatostatin (SOM) in the carotid labyrinth were compared in some species of amphibians using the peroxidase-antiperoxidase method. Immunoreactivity of SP, CGRP, VIP, and NPY was found in the nerve fibers distributed in the intervascular stroma of the carotid labyrinth. SP, CGRP, and VIP immunoreactive varicose fibers were densely distributed in the peripheral portion of the carotid labyrinth. Some SP-immunoreactive fibers were distributed similarly to CGRP-immunoreactive fibers. The density of NPY and SOM immunoreactive varicose fibers was low. No immunoreactivity of enkephalins was observed in the labyrinth. The intensities of these peptides were varied from species to species. No glomus cells showed immunoreactivity for any of the 7 peptides studied. These results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, is controlled by the peptidergic mechanisms in addition to regulation through intimate apposition of glomus and smooth muscle cells (g-s connection). PMID- 1717417 TI - Adenosine triphosphatase-positive Langerhans-like cells in the epidermis of the chicken (Gallus gallus). AB - In mammalian epidermis a population of ATPase-positive dendritic cells, identified as Langerhans cells, has been found. Such cells are bone marrow derived and participate in the immunological functions of the skin. We demonstrate the existence of ATPase-positive dendritic cells in separated epidermal sheets of chicken skin, by means of light and electron microscopy. They have a mean distribution of 688 +/- 265 cells/mm2 and showed several features in common with Langerhans cells. Since chickens can develop contact dermatitis, the finding is taken as the first formal demonstration of the presence of Langerhans cells in this group of vertebrates. PMID- 1717418 TI - Autoradiographic analysis of vasoactive neuropeptide uptake by rabbit megakaryocytes and platelets. AB - Autoradiographic analysis has been performed to determine whether or not platelets and megakaryocytes can take up in vitro vasoactive neuropeptides such as [3H]angiotensin II, [3H]neuropeptide Y, [3H]substance P and [3H]vasopressin. Light microscope autoradiography has revealed the active uptake of [3H]angiotensin II by megakaryocytes. [3H]angiotensin II uptake by platelets has also been confirmed by electron microscope autoradiography. Silver grains are preferentially associated with the dense bodies of platelets. Neither megakaryocytes nor platelets show any active uptake of [3H]neuropeptide Y or [3H]substance P. Megakaryocytes are slightly labelled after incubation with [3H]vasopressin. PMID- 1717419 TI - The nucleus of the tractus solitarius of the dog. A morphological and morphometric analysis. AB - The neuronal and fibrous architecture of the nucleus of the tractus solitarius (NTS) of the dog has been studied in transversely cut Nissl, myelin and reduced silver stained serial sections. Eight distinct subdivisions, clearly delimited both by their cytoarchitectonic and fibrous characteristics, have been identified. They are: the commissural, gelatinous, lateral, interstitial, dorsolateral, ventrolateral, intermediate and medial subdivisions. Their rostrocaudal extensions and locations in relation to the obex are summarised in Table 1. A morphometric analysis was additionally done. The frequency distributions of cell areas and cell form factor of each subdivision are represented by histograms in Figures 8 and 9 respectively. PMID- 1717420 TI - Ultrastructural localisation of anionic sites at the dermo-epidermal junction in normal human skin. AB - Glycosaminoglycans have been demonstrated throughout the cutaneous BMZ at the ultrastructural level. Colloidal iron and cationised ferritin proved of limited value, whilst staining with Alcian blue and application of the critical electrolyte concentration principle has provided evidence for the presence of sulphated GAGs at the lamina lucida and lamina reticularis. Digestions with chondroitin ABC lyase and heparin lyase have confirmed the existence of chondroitin and/or dermatan sulphates and heparan sulphates, although the results obtained with hyaluronate lyase have indicated that hyaluronates are also present. PMID- 1717421 TI - A preliminary study of acetylcholinesterase-positive innervation in the human adrenal cortex. AB - We have examined the distribution of probable cholinergic nerves in the human adrenal cortex using acetylcholinesterase (AChE) histochemistry. Adrenal tissue was obtained from three subjects during therapeutic radical nephrectomy for renal neoplastic disease. Light microscopy revealed a heterogeneous pattern of cortical innervation, with nerves most abundant in the head and body and largely absent from the tail. There was frequently a subcapsular plexus of interwoven AChE + ve nerve trunks in close relationship to densely stained (ganglion) cells. Nerves also traversed the zona fasciculata in radial trunks and a further plexus with ganglion cells was observed in the zona reticularis. Nerve trunks were seen to penetrate the medulla. Some nerve trunks appeared to terminate or branch or form individual nerve fibres (often with varicosities) which ramified through the cortical parenchyma. In the medulla, a generalised granular staining was superimposed on nerve trunks which were branched and interwoven to form a plexus around ganglion cells. The presence and distribution of AChE + ve nerve plexuses and varicose nerve fibres is suggestive of a functional intrinsic, probably cholinergic, innervation to the human adrenal cortex, perhaps derived from the splanchnic nerves. PMID- 1717422 TI - Actin, myosin and alpha-actinin containing filament bundles in hyaline cells of the caiman cochlea. AB - Hyaline cells of the auditory organ of the spectacled caiman contain smooth muscle-like filament bundles within their basal cell pole. These bundles were heavily labeled with antibodies to actin, myosin and alpha-actinin (muscular Z line protein). Since hyaline cells are firmly attached to the basilar membrane these cells may actively modify the stiffness of the basilar membrane. A contractile mechanism in hyaline cells might affect frequency tuning of primary auditory afferents. This frequency tuning has been shown to be a temperature dependent process in caimans and other submammalian species. The presence of synaptic contacts between efferent nerve fibres and hyaline cells suggests neural control of hyaline cell activity. PMID- 1717423 TI - Enhanced coupling of adenylate cyclase to lipolysis in permeabilized adipocytes from trained rats. AB - Digitonin-permeabilized adipocytes were used to study the coupling of adenylate cyclase (AC) to lipolysis in exercise-trained rats. Isoproterenol-(IPR) stimulated lipolysis in permeabilized cells was significantly greater in trained than in control rats. Under essentially identical conditions, the dose-response curve for IPR stimulation of AC activity in the absence of 3-isobutyl-1 methylxanthine was similar in trained and control rats. However, the potency of stimulation by IPR as a percentage of the basal level was greater in trained rats. AC activity and lipolysis in the presence of 3-isobutyl-1-methylxanthine were also significantly greater in trained than in control rats. Least-squares analysis by plotting the log AC vs. lipolysis values showed that the regression coefficient was about three-fold greater in trained than in control rats. The concentration of endogenous adenosine 3',5'-cyclic monophosphate (cAMP) needed to produce a half-maximal lipolytic response was 18.58 and 10.81 pmol.min-1.10(6) cells-1 in control and trained rats, respectively. Thus a positive relationship existed between lipolysis and AC activity, with a tighter coupling in trained rats. Lipolysis in response to exogenous cAMP tended to be greater in trained than in control rats, and the difference was statistically significant for 50 microM and 10 mM cAMP. Our finding support the concept that the major mechanism of enhanced lipolysis in trained rats was an increase in the activity of enzymatic step(s) distal to cAMP. PMID- 1717424 TI - Role of neutrophils in endotoxin-mediated microvascular injury in hamsters. AB - The purpose of this study was to examine the role of circulating neutrophils in endotoxin-induced increase in microvascular permeability in vivo. Fifteen hamsters were anesthetized, and a plastic chamber was placed in each cheek pouch to observe the microvasculature. Fluorescein-labeled dextran (FITC-D, 150 kDa) was injected intravenously, and changes in leaky sites and FITC-D clearance were measured in three groups: control (saline, n = 4), endotoxin suffusion (n = 6), and endotoxin suffusion after neutropenia induction (n = 5). We found a significant increase in leaky sites and FITC-D clearance with endotoxin (45 +/- 18/cm2 and 20 +/- 6 x 10(-6) ml/min, respectively; mean +/- SD, P less than 0.05) in comparison to control (7 +/- 6/cm2 and 7 +/- 5 x 10(-6) ml/min) and endotoxin suffusion in neutropenic animals (19 +/- 11/cm2 and 12 +/- 4 x 10(-6) ml/min). There was a significant correlation between the number of leaky sites and FITC-D clearance (r = 0.91, P less than 0.01) and between the number of circulating neutrophils and FITC-D clearance (r = 0.87, P less than 0.01). We conclude that endotoxin-mediated increase in microvascular permeability in the peripheral circulation is dependent in part on circulating neutrophils. PMID- 1717425 TI - Respiratory stimulation by female hormones in awake male rats. AB - The respiratory effect of progestin differs among various animal species and humans. The rat does not hyperventilate in response to exogenous progestin. The present study was conducted to determine whether administration of combined progestin and estrogen prompts ventilatory stimulation in the male rat. Ventilation, blood gases, and metabolic rates (O2 consumption and CO2 production) were measured in the awake and unrestrained male Wistar rat. The combined administration of a synthetic potent progestin (TZP4238) and estradiol for 5 days significantly increased tidal volume and minute expiratory ventilation (VE), reduced arterial PCO2, and enhanced the ventilatory response to CO2 inhalation (delta VE/delta PCO2). On the other hand, respiratory frequency, O2 consumption, CO2 production, and body temperature were not affected. The arterial pH increased slightly, with a concomitant decrease in plasma [HCO3-]. Administration of either TZP4238 or estradiol alone or vehicle (Tween 80) had no effect on respiration, blood gases, and ventilatory response to CO2. The results indicated that respiratory stimulation following combined progestin plus estradiol treatment in the male rat involves activation of process(es) that regulate tidal volume and its augmentation during CO2 stimulus. PMID- 1717426 TI - Growth of distal fetal rat lung epithelial cells in a defined serum-free medium. AB - Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-D-lysine substratum under serum-free culture conditions in Dulbecco's modified minimal essential medium with insulin (50 micrograms/ml), endothelial cell growth supplement (20 micrograms/ml), bovine pituitary extract (100 micrograms/ml), bovine serum albumin (50 micrograms/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 micrograms/ml), transferrin (5 micrograms/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 micrograms/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E1 (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung. PMID- 1717427 TI - Differentiation of pericytes in culture is accompanied by changes in the extracellular matrix. AB - We have previously reported that pericytes derived from retinal and brain microvessels aggregate into nodules soon after reaching confluence. Nodule formation involves a reorganization of the cells resulting in the presence of sparse cells, confluent monolayers, multilayers, sprouts, and nodules within the same culture dish. Extracellular calcification occurs only within the nodules, demonstrating that pericytes are capable of undergoing osteogenic differentiation in culture and that this differentiation is related to nodule formation. Using immunofluorescence we have now studied the distribution of laminin, type IV collagen, type X collagen, and tenascin in pericyte cultures during nodule formation. These matrix macromolecules were also identified by a combination of biochemical techniques, including Northern blot hybridization, immunoblotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecule that seems to be related to type X collagen was demonstrated by the presence of a pepsin resistant, collagenase-sensitive polypeptide of molecular weight approximately 45 kDa. The production of laminin, type X-related collagen, and tenascin by pericytes has not been previously reported. Our results suggest that the synthesis or distribution or both of these molecules is dependent on the state of pericyte differentiation. The expression of laminin, type IV collagen, and type X related collagen was maximal in multilayer areas, sprouts, and nodules. Tenascin appeared homogeneously distributed in monolayer and multilayer areas; when calcified nodules were present, the anti-tenascin serum preferentially decorated a discrete area circumscribing the nodules. Tenascin and type X collagen have been found transiently in vivo preceding calcification; their possible role in this process is not known. Our results also suggest an association between laminin, type IV collagen, and calcification. The in vitro experimental system described here may help to clarify the role of matrix macromolecules in the calcification process. PMID- 1717428 TI - Effects of secretin and caerulein on enzymes of cultured pancreatic acinar cells. AB - We examined the effects of secretin (0 to 200 nM) and caerulein (0 to 100 nM) on rat pancreatic acinar cells cultured 0 to 48 h in serum-free medium. The effects of 100 nM secretin with 1 nM caerulein were also studied because secretin may potentiate the effects of caerulein. Cellular and media (secreted) lipase and amylase were analyzed as were cellular DNA and protein content. Cellular lipase and amylase activities significantly decreased (P less than 0.0001) over time in all treatment groups, whereas media amylase and lipase significantly increased (P less than 0.0001). Neither secretin nor caerulein affected cellular lipase or media amylase. However, secretin significantly increased (P less than 0.04) and caerulein tended to increase (P less than 0.08) media lipase in a dose-dependent manner. At 12 h, 10 nM secretin maximally increased media lipase (58%) suggesting that cultured acinar cells remain responsive to secretin in vitro. Caerulein, at all concentrations, significantly decreased (P less than 0.001) cellular amylase but exhibited a dose-dependent effect only at 24 h when 100 nM caerulein maximally decreased cellular amylase (34%). Secretin (100 nM) did not alter these effects of caerulein. These results support the proposed role of caerulein in the regulation of amylase but not a direct role of secretin in the regulation of lipase. PMID- 1717429 TI - Cellular characteristics of long-term cultured rat parotid acinar cells. AB - We have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes. PMID- 1717430 TI - Isolation and monolayer culture of human fallopian tube epithelial cells. AB - In the present study we have established a pure monolayer culture system of human fallopian tube epithelial cells. The cells were isolated using collagenase digestion, and were cultured in Medium 199 supplemented with 15% fetal bovine serum. The epithelial cells derived from primary and secondary culture were characterized using immunocytochemical staining and electron microscopy. The cells continued to grow for 2 to 3 wk once the monolayer culture of the cells was established. It is currently possible to maintain the cultures until the third generation. Proliferation of these cells was enhanced by epidermal growth factor but not by basic-fibroblast growth factor, insulin, transferrin, estradiol-17 beta, or progesterone. This culture system offers a good model for determining characteristics of the tubal epithelium and would permit effective study of co culture with embryos. PMID- 1717431 TI - Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury. AB - Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated gamma glutamyltranspeptidase in greater than 95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectin Arachis hypogaea (rat proximal tubule) and negatively for Lotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, gamma glutamyltranspeptidase and leucine aminopeptidase, and Na(+)-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium. PMID- 1717432 TI - Cultured proliferating rat mammary epithelial cells. AB - Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occurred as the LA7 cells slowly died from the radiation. By labeling the cultures with 3H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of approximately 1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to approximately 19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco's modified Eagle's medium:Ham's F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no outgrowths resulted from the implantation of cells from Passage 5. The one unusual, feeder independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. PMID- 1717433 TI - Lipid globule staining to aid in differentiating Bacillus species. AB - The use of the lipid globule stain to aid in differentiating the Bacillus cereus group (i.e., B. cereus, B. cereus var. mycoides, and B. thuringiensis) from other Bacillus species was investigated. Smears from colonies grown on suitable agar were made on precleaned slides, stained, and examined microscopically for characteristic deep blue lipid globules. The study included a total of 649 cultures of Bacillus species plus 143 incompletely characterized Bacillus isolates from food. Only B. cereus, B. cereus var. mycoides, B. thuringiensis, B. megaterium, and B. sphaericus were consistently positive for lipid globules, although at times, a few cells of B. aneurinolyticus and B. thiaminolyticus were also positive. The lipid globule stain procedure is of value in differentiating Bacillus species, especially when performed by an experienced analyst and used in conjunction with tests for cell and spore morphology. PMID- 1717434 TI - Evolution of the ferric enterobactin receptor in gram-negative bacteria. AB - Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell envelopes, 59Fe-siderophore uptake studies, and Western immunoblots and cytofluorimetric analyses with monoclonal antibodies (MAbs), we surveyed a panel of gram-negative bacteria to identify outer membrane proteins that are structurally related to the Escherichia coli K-12 ferric enterobactin receptor, FepA. Antibodies within the panel identified FepA epitopes that are conserved among the majority of the bacteria tested, as well as epitopes present in only a few of the strains. In general, epitopes of FepA that are buried in the outer membrane bilayer were more conserved among gram-negative bacteria than epitopes that are exposed on the bacterial cell surface. The surface topology and tertiary structure of FepA are quite similar in E. coli and Shigella flexneri but differ in Salmonella typhimurium. Of the 18 different genera tested, 94% of the bacteria transported ferric enterobactin, including members of the previously unrecognized genera Citrobacter, Edwardsiella, Enterobacter, Haemophilus, Hafnia, Morganella, Neisseria, Proteus, Providencia, Serratia, and Yersinia. The ferric enterobactin receptor contains at least one buried epitope, recognized by MAb 2 (C. K. Murphy, V. I. Kalve, and P. E. Klebba, J. Bacteriol. 172:2736-2746, 1990), that is conserved within the structure of an iron-regulated cell envelope protein in all the bacteria that we have surveyed. With MAb 2, we identified and determined the Mr of cell envelope antigens that are immunologically related to E. coli FepA in all the gram-negative bacteria tested. Collectively, the library of anti-FepA MAbs showed unique patterns of reactivity with the different bacteria, allowing identification and discrimination of species within the following gram-negative genera: Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Haemophilus, Hafnia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia. PMID- 1717435 TI - Dissolution and immunochemical analysis of the sheath of the archaeobacterium Methanospirillum hungatei GP1. AB - The sheath of Methanospirillum hungatei GP1 was degraded by three dissolution techniques, which produced a range of soluble products. By using 0.05 M L arginine buffer (pH 12.6) at 90 degrees C for 10 min, 74% (dry weight) of the sheath was dissolved; however, the solubilized polypeptides were extensively degraded. Treatment with 2% beta-mercaptoethanol and 2% sodium dodecyl sulfate at 90 degrees C in 0.05 M 2(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 9.0) solubilized 42% (dry weight) of the sheath as a group of polypeptides of 30 to 40 kDa. At 100 degrees C for 2 h, 5% beta-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 20 mM EDTA released 74% of the sheath's mass as a group of polypeptides of 10 to 40 kDa. All solubilized products were examined by SDS polyacrylamide gel electrophoresis, and a range of high- and low-molecular-weight polypeptides was identified. None were glycoproteins. Hoops, which comprise the sheath's structure, were seen by electron microscopy after all of the attempted dissolutions. Monoclonal antibodies were produced against the 10- to 40-kDa range of solubilized products and against the approximately 40-kDa polypeptides, and polyclonal antiserum was produced against an 18-kDa polypeptide. These immunological markers were used in Western immunoblotting and protein A-colloidal gold-antibody probing by electron microscopy to identify the structural location of the various polypeptides. Native sheath, which possesses 2.8-nm particles on its outer surface (M. Stewart, T.J. Beveridge, and G.D. Sprott, J. Mol. Biol. 183:509-515, 1985; P.J. Shaw, G.J. Hills, J.A. Henwood, J.E. Harris, and D.B. Archer, J. Bacteriol. 161:750-757, 1985), presented a gentle wave-form surface in platinum-shadowed specimens. In contrast, the inner face of the sheath was highlighted by ridges lying perpendicular to the longitudinal axis of the sheath and likely corresponded to hoop boundaries. Both the polyclonal and monoclonal antibodies were specific for different faces; polyclonal antibodies labeled the inner face, whereas monoclonal antibodies labeled the outer face. Accordingly, the apparent asymmetry of structure between the two faces of the sheath can be correlated by our immunochemical probing with a distinct asymmetry in the distribution of exposed polypeptides between the faces. The possible implications of this asymmetry for growth and maturation of the sheath are explained. PMID- 1717436 TI - Physiological effects of the fructose-1,6-diphosphate aldolase ts8 mutation on stable RNA synthesis in Escherichia coli. AB - The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature. We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation. The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated. This effect is independent of relA and spoT function. Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown. PMID- 1717437 TI - A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus. AB - Staphylococcus aureus exoprotein expression is controlled by a global regulon known as agr. This system activates transcription of some target genes and represses transcription of others. Target genes expressed postexponentially such as alpha-hemolysin (hla) are activated by agr; target genes expressed during exponential phase such as protein A (spa) are repressed by agr. A unique feature of the agr system is that this transcriptional regulation is mediated by a 517 nucleotide transcript, RNAIII. While it is clear that agr differentially regulates the expression of exponential and postexponential exoproteins, the precise role of agr in the temporal control of these events has not yet been explored. In this report, we examine the effects of expressing RNAIII, the agr regulator, under the control of the inducible beta-lactamase (bla) promoter at different times in the growth cycle. We confirm previous results showing that agr is required for postexponential-phase expression of hla and further show that a separate postexponential-phase signal independent of agr function is also needed for activation of hla transcription. We also show that in an agr mutant transcription of spa occurs throughout the growth cycle, is inhibited immediately upon induction of RNAIII, and is thus indifferent to the postexponential signal required for hla activation. PMID- 1717438 TI - Discovery of a rhizobial RNA that is essential for symbiotic root nodule development. AB - All of the Azorhizobium, Bradyrhizobium, and Rhizobium genes known to be involved in the development of nitrogen-fixing legume root nodules are genes that code for proteins. Here we report the first exception to this rule: the sra gene; it was discovered during the genetic analysis of a Bradyrhizobium japonicum Tn5 mutant (strain 259) which had a severe deficiency in colonizing soybean nodules. A DNA region as small as 0.56 kb cloned from the parental wild type restored a wild type phenotype in strain 259 by genetic complementation. The sra gene was located on this fragment, sequenced, and shown to be transcribed into a 213-nucleotide RNA. Results obtained with critical point mutations in the sra gene proved that the transcript was not translated into protein; rather, it appeared to function as an RNA molecule with a certain stem-and-loop secondary structure. We also detected an sra homolog in Rhizobium meliloti which, when cloned and transferred to B. japonicum mutant 259, fully restored symbiotic effectiveness in that strain. We propose several alternative functions for the sra gene product, of which that as a regulatory RNA for gene expression may be the most probable one. PMID- 1717440 TI - Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini. AB - Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+) dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+) dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers. PMID- 1717439 TI - rRNA transcription rate in Escherichia coli. AB - The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. PMID- 1717441 TI - Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains. AB - Bullous pemphigoid antigens are defined as the autoantigens in a blistering skin disease, bullous pemphigoid. One of them, a 230-kDa protein (BPAG1), is associated with hemidesmosomes, attachment complexes at the basal keratinocyte lamina lucida interface within the dermal-epidermal basement membrane zone. The precise functions and cellular compartmentalization of BPAG1 are unknown. In this study, a human keratinocyte lambda gt11 cDNA library was screened for clones corresponding to BPAG1. The composite of overlapping cDNAs delineated 8,930 base pairs of nucleotide sequences that contained an open reading frame encoding 2,649 amino acids. Analysis of the deduced amino acid sequences predicted a putative signal peptide of 43 amino acids and the presence of a membrane-associated sequence of 17 amino acids. Several potential sites for N-glycosylation, as well as for protein kinase C or cAMP- and cGMP-dependent protein kinase-mediated phosphorylation were identified. Three peptide segments were predicted to be highly antigenic, potentially serving as epitopes for the formation of autoantibodies. Eight repeat segments of 38 residues each with a high degree of homology with sequences in desmoplakin I, a component of desmosomal cytoplasmic plaques, were detected in the carboxyl-terminal end of the molecule. In addition, the presence of three subdomains characterized by heptad repeats predicted an alpha-helical coiled coil dimer structure in the central portion of the protein. These data suggest that BPAG1 may be a membrane-associated protein that plays a role in the attachment of basal keratinocytes to the underlying basement membrane. PMID- 1717442 TI - Peptides reproducing the phosphoacceptor sites of pp60c-src as substrates for TPK IIB, a splenic tyrosine kinase devoid of autophosphorylation activity. AB - TPK-IIB, a spleen tyrosine protein kinase devoid of autophosphorylation activity (Brunati, A. M., and Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457), has been purified to near homogeneity and assayed for its ability to phosphorylate the synthetic peptides EDNEYTA and EPQYQPA reproducing the two conserved phosphoacceptor sites of pp60c-src (Tyr-416 and Tyr-527). While EPQYQPA was phosphorylated with low efficiency (Km = 16.7 mM, Kcat = 14.4), EDNEYTA is an excellent substrate displaying a Km value of 58 microM and a Kcat value of 31.2. The single substitution, in the latter peptide, of the glutamic acid adjacent to the tyrosine by alanine to give EDNAYTA caused a 6-fold increase in the Km. The positive influence on the phosphorylation of the acidic residues at -3 and -4 relative to the tyrosine is indicated by comparison of the kinetic constants for peptides EDAAYAA (Kcat = 4.6, Km 0.325 mM) and QNAAYAA (Kcat 2.4, Km 1.7 mM). Furthermore, when residues in the peptide NEYTA were replaced by alanine, the phosphorylation of the peptides NAYTA and AAYAA, was almost negligible (in terms of Kcat/Km ratio). However, AEYTA, NEYAA and AEYAA were still phosphorylated, albeit less efficiently than NEYTA. The probability that these peptides will adopt a beta-turn is EDNAYTA = EDNEYTA, NAYTA greater than NEYTA, and no predicted beta-turn for AEYTA, NEYAA, and AEYAA. Therefore these results support the concept that an amino-terminal acidic residue(s) is strictly required by TPK IIB, irrespective of peptide conformation, although a beta-turn may enhance the phosphorylation of those peptides that satisfy this requirement. Two other spleen tyrosine kinases, TPK-I/lyn and TPK-III, both related to the src family, also have a far greater preference for the peptide EDNEYTA over EPQYQPA. However, they can be distinguished from TPK-IIB by their lower affinity for the peptides EDNEYTA and NEYTA and by their different specificity towards the substituted derivatives of NEYTA. TPK-I/lyn, accepts most of the substitutions that are detrimental to TPK-IIB, the triply substituted peptide AAYAA being actually preferred over the parent peptide NEYTA. The substitution of glutamic acid by alanine is also tolerated by TPK-III, although, in contrast to TPK-IIB, the phosphorylation efficiency is drastically decreased by the substitution of the asparagine at position -2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717443 TI - A novel species of cytochrome P-450 (P-450ib) specific for the small intestine of rabbits. cDNA cloning and its expression in COS cells. AB - We have isolated a cDNA clone for a P-450, designated P-450ib (Ichihara, K., Kusunose, E., Kaku, M., Yamamoto, S., and Kusunose, M. (1985) Biochim. Biophys. Acta 831, 99-105), from a cDNA library of rabbit small intestine mucosa by using synthetic DNA fragment by the polymerase chain reaction, as a hybridization probe. The cDNA with a 1,829-base pair insert encodes a polypeptide of 501 amino acids. The deduced amino acid sequence contains all of the sequences of the NH2 terminal and 14 tryptic fragments from purified P-450ib. As the NH2-terminal methionine was not found in the sequence from the purified protein, the apoprotein of P-450ib is composed of 500 amino acids with a molecular weight of 57,193. P-450ib shows 35-41% sequence similarity with several members of 8 subfamilies in the P-450 II family, whereas it has a less than 30% sequence similarity with other P-450 families, suggesting that this P-450 is the first member of a novel subfamily within the P-450 II family. RNA blot analysis shows that mRNA hybridized to the cDNA is expressed in the small intestine, but not significantly in other tissues including liver, colon, kidney, lung, spleen, brain, stomach, and cecum, indicating that P-450ib is a P-450 specific to the small intestine. The protein expressed in COS-7 cells using the cDNA in an expression vector, pKCRH2, shows benzphetamine N-demethylase activity and gives a band identical with that of P-450ib in its mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis. PMID- 1717444 TI - Structural basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase. AB - Cys-cdc2(8-20), a synthetic peptide derived from p34cdc2, was previously reported to be a specific and efficient substrate of a pp60c-src-related tyrosine kinase isolated from bovine spleen (the spleen tyrosine kinase) (Litwin, C.M.E., Cheng, H.-C., and Wang, J.H. (1991) J. Biol. Chem. 266, 2557-2566). The longer peptide, cdc2(1-24), was found to be phosphorylated by the kinase with similar efficiency, and Tyr15 was the only amino acid residue phosphorylated. This indicated that the amino acid sequence of cdc2(8-20) peptide, EKI-GEGTYGVVYK, contained the structural features important for protein tyrosine kinase substrate activity. A stepwise procedure using synthetic peptides was employed to investigate such structural features. First, a computer search of protein sequences homologous to cdc2(8-20) uncovered five protein kinases containing homologous sequence with tyrosine at a position corresponding to Tyr15 of p34cdc2. Second, a peptide derived from ribosomal S6 protein kinase (rsk(436-456] was synthesized. The rsk(436-456) peptide contained a segment, ETIGVGSYSVCKR, which is highly homologous to that of cdc2(8-20). It was found to be a very poor substrate of the spleen tyrosine kinase. Third, peptide analogs of cdc2(6-20) with single substitutions of amino acid residues Lys9, Glu12, Thr14, Gly16, Val18, and Tyr19 by amino acid residues at corresponding positions of rsk were synthesized and tested as spleen tyrosine kinase substrates. Only Glu12 and Thr14 substituted peptide analogs showed decreased substrate activities. (The substrate activity of a peptide is the ability of the peptide to serve as the substrate of the spleen tyrosine kinase. It was determined of the spleen tyrosine kinase. It was determined either by the kinetic parameters (Km and Vmax) of phosphorylation of the peptide or by the initial phosphorylation rate of the peptide by the spleen tyrosine kinase.) An analog with double substitution at Glu12 An analog with double substitution at Glu12 and Thr14 was found to be almost as poor a substrate as the rsk peptide. In addition, peptide analogs with Tyr15 substituted by Phe or D-Tyr had poor substrate activities as well as weak inhibitory activities. Thus, Glu12, Thr14, and Tyr15 residues of p34cdc2 contained structural components essential for the efficient phosphorylation of the peptides derived from p34cdc2 by the pp60c-src-related spleen tyrosine kinase. PMID- 1717445 TI - The increased potency of cross-linked lymphocyte function-associated antigen-3 (LFA-3) multimers is a direct consequence of changes in valency. AB - We have used chemically cross-linked dimers, trimers, and tetramers of lymphocyte function-associated antigen-3 (LFA-3) to study the role of multivalency in the interaction of the protein with its receptor, CD2. The cross-linked adducts showed enhanced activity in systems where LFA-3 has been shown to (i) block LFA 3/CD2 interactions in a rosetting assay and (ii) provide through the CD2 on peripheral blood lymphocytes a trigger for T-cell proliferation. The level of increase was directly related to the valency state of the multimers. In the rosetting assay, the dimers, trimers, and tetramers, by weight, exhibited 15-, 150-, and 430-fold increases in activity over monomeric LFA-3. In the proliferation assay, the tetramer produced a 6-fold increase in thymidine incorporation at 0.06 micrograms/ml, the trimer was 100 times less active than the tetramer, and the dimer and monomer were inactive. The LFA-3 multimers were generated using a three-step cross-linking chemistry that was targeted at the carbohydrates on LFA-3. With this procedure over 60% of the starting protein was converted into multimers with no effect on function. The cross-linking approach should be applicable to other surface antigens, providing a simple method for analyzing multivalent interactions. PMID- 1717446 TI - Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion. AB - Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. In addition, a UDPGT isoform which is active toward 4-nitrophenol and is induced by 3-methylcholanthrene (3-MC) in normal rats, is produced in a nonfunctional truncated form in Gunn rats due to the deletion of a single guanosine residue in the coding region of its mRNA. The hepatic concentration of bilirubin-UDPGT mRNA was lower in Gunn rats than in congeneic normal rats. However, bilirubin-UDPGT mRNA was of apparently normal length and was induced by clofibrate, a known inducer of bilirubin-UDPGT activity. 3' regions of bilirubin- and 3-MC-inducible UDPGT mRNAs have identical nucleotide sequences; the single base deletion in the 3-MC-inducible UDPGT in Gunn rats occurs within this region. Using oligonucleotide primers corresponding to the identical and unique regions of the two mRNAs, and polymerase chain reaction, we amplified segments of mRNAs for the bilirubin- and 3-MC-inducible UDPGTs from normal and Gunn rat livers. Both amplified DNAs in Gunn rats lacked the restriction site for BstNI. Nucleotide sequence determination revealed that bilirubin- and 3-MC-inducible UDPGT mRNAs in Gunn rats contain an identical frame-shift deletion of a single guanosine residue within the common region of their coding sequences. PMID- 1717447 TI - tRNAs of Trypanosoma brucei. Unusual gene organization and mitochondrial importation. AB - In Trypanosoma brucei the U snRNA B gene (J. C. Mottram, K. L. Perry, P. M. Lizardi, R. Luhrmann, N. Agabian, and R. G. Nelson (1989) Mol. Cell. Biol. 9, 1212-1223) is very tightly linked with other small RNA genes coding for tRNA(ACGArg), tRNA(CUULys), and a approximately 275-nucleotide RNA (RNA X) of unknown function. A similar genomic organization is found at the U6 snRNA locus, where the U6 gene is linked to tRNA(CGUThr) and tRNA(GUATyr) genes. The tRNA(Lys) and tRNA(Arg) genes are members of a multigene family, whereas the tRNA(Thr) and tRNA(Tyr) genes are single copy. Two additional tRNA(CUULys) genes and one tRNA(UUULys) gene were also isolated and sequenced and, together with a sequence previously published (D. A. Campbell (1989) Nucleic Acids Res. 17, 9479), appear to represent the entire gene family. Probes for tRNA(Lys), tRNA(Arg), tRNA(Thr), and tRNA(Tyr) were found to hybridize with mitochondrial and cytoplasmic tRNAs but not with mitochondrial DNA. This supports the hypothesis that mitochondrial tRNAs may be nuclear-encoded and imported from the cytosol into the mitochondrion. PMID- 1717448 TI - Respiratory tract gene transfer. Transplantation of genetically modified T lymphocytes directly to the respiratory epithelial surface. AB - To evaluate the strategy for potentially treating respiratory disorders with genetically modified T-lymphocytes, the interleukin-2 (IL-2)-dependent murine T cell line, CTLL2, was genetically altered with the Escherichia coli beta galactosidase (beta-gal) gene (lacZ) in vitro with a retroviral vector and the modified T-cells were transplanted directly to the respiratory epithelial surface of syngeneic C57Bl/6 mice. Southern and Northern analyses confirmed that the neomycin-selected modified T-cells contained and expressed the lacZ gene. The fate of the modified T-cells (CTLL2/lacZ) was followed by flow cytometry with T cell surface marker Thy1.2 and fluorescent beta-gal analysis. One day after transplantation (7.5 x 10(5) CTLL2/lacZ T-cells/g of body weight), 95 +/- 3% of the Thy1.2+ T-cells recovered from respiratory epithelial lining fluid (ELF) were beta-gal+. Importantly, the modified T-cells remained in the lung for some time; at 3 days, Thy1.2+ beta-gal+ T-cells represented 63 +/- 12% of ELF Thy1.2+ T cells and 59 +/- 6% of Thy1.2+ T-cells recovered from the whole lung. At 7 days, 33 +/- 8% of the Thy 1.2+ cells in ELF and 75 +/- 6% of the Thy1.2+ cells in whole lung were Thy1.2+ beta-gal+. In contrast, the proportion of the Thy1.2+ beta-gal+ T-cells in the spleen, the major extrapulmonary lymphatic organ, never rose above 3 +/- 1% of the total Thy1.2+ cells. The number of Thy1.2+ beta-gal+ T cells in the lung could be modified by the systemic administration of IL-2, with whole lung Thy1.2+ beta-gal+ T-cells increasing 4.6-fold 3 days after transplantation, compared with non-IL-2-treated animals. These studies suggest that direct transplantation of genetically modified T-cells into the lung is feasible and represents a viable strategy for lung-specific gene transfer. PMID- 1717449 TI - Formation of cation channels in planar lipid bilayers by brefeldin A. AB - Brefeldin A (BFA) is a novel agent with the unique property of effecting a rapid increase of Golgi cisternae volume and subsequent loss of a recognizable Golgi apparatus in treated cells. Although a receptor-mediated mechanism has been proposed, the molecular basis of BFA action remains unknown (Lippincott-Schwartz, J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). Since a variety of ionophores distort Golgi architecture by initially causing osmotic swelling of the cisternae (Mollenhauer, H. H., Morre, D. J., and Rowe, L. D. (1990) Biochim. Biophys. Acta 1031, 225-246), Golgi membrane permeabilization by BFA seemed possible. We examined the effects of BFA on the conductance of planar lipid bilayers bathed in several aqueous salt solutions. Addition of BFA (1 microgram/ml) quickly augmented alkali cation conductance (K+ greater than Na+ much greater than Li+) but not anion conductance of the bilayer. Lower concentrations (1 ng/ml) indicated that BFA formed discrete, cation-selective channels in these bilayers. Given that Golgi cisternae volume increases immediately upon treatment with BFA, these findings suggest that alteration of ion gradients or Golgi membrane potential followed by an influx of water may be the mechanism by which BFA initiates disruption of Golgi structural integrity. Subsequent functional perturbations may then ensue either as a consequence of these initial structural changes or by a combination of several distinct mechanisms. PMID- 1717450 TI - Mapping ribosomal protein S20-16 S rRNA interactions by mutagenesis. AB - Specific ribonucleotides within the 5' domain of Escherichia coli 16 S rRNA were altered by deletion and/or substitution by site-directed mutagenesis of cloned DNA to assess the importance of these particular residues in defining the binding site for ribosomal protein S20. Gel filtration and sucrose gradient centrifugation were employed to measure the binding of ribosomal protein S20 to synthetic transcripts spanning the 5' domain of 16 S rRNA containing various alterations. In several cases, the apparent association constants for these interactions were also determined. Three essential results were obtained. First, RNA transcripts containing residues 1-402 are sufficient for high affinity S20 binding. Second, bulges at residues 250-251 and 278-280 in a stem-loop structure extending from residues 240 to 286 are critical for S20 binding, arguing that the "260 stem" directly interacts with S20 at these sites. Third, mutations in a hairpin structure spanning residues 316-337 demonstrate a strong requirement for both the specific A321*G332 bulge and for unpaired residues in the loop itself for proper S20 binding. Our results document the importance of unpaired residues in maintaining the interaction between S20 and 16 S rRNA. PMID- 1717451 TI - Individual embryonic fibroblasts express multiple beta chains in association with the alpha v integrin subunit. Loss of beta 3 expression with cell confluence. AB - The alpha chain of the vitronectin receptor, alpha v, has been found in association with the integrin subunits beta 1, beta 3, or beta 5 on different cell types. We show here that cultured embryonic fibroblasts simultaneously display alpha v beta 3, alpha v beta 1, and alpha v in association with two other beta subunits, one of which is probably beta 5. Polymerase chain reaction analysis of single cells isolated by micromanipulation identified mRNA for alpha v, beta 1, beta 3, and beta 5 in six of eight clones. Immunoprecipitation of iodinated cell surface proteins with a monoclonal antibody to alpha v indicated that the relative proportions of the different beta chains in association with alpha v varied, particularly between two different cell lines. The cytokines platelet-derived growth factor, transforming growth factor beta 1, and tumor necrosis factor alpha did not appear to alter this ratio although tumor necrosis factor alpha increased the surface expression of the alpha v-associated integrins; but overnight culture in basic fibroblast growth factor caused a lower expression of alpha v beta 1 and alpha v beta 5 with no reduction in alpha v beta 3 expression. When the cell cultures were grown to complete confluence, surface expression of beta 3 was abolished, and the expression of an unknown beta chain (beta u) became more prominent. This effect was not overcome by culturing confluent cells with basic fibroblast growth factor. Affinity column chromatography showed that alpha v beta 5 bound to vitronectin but alpha v beta 1 did not, whereas alpha v beta 1 but not alpha v beta 5 bound to fibronectin. These results suggest that, on individual cells, the beta subunits found in association with alpha v may vary according to the proliferative capacity of the cell and that the promiscuous beta 3 subunit is progressively replaced by beta subunits of individual ligand specificity. PMID- 1717452 TI - Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations. AB - Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product. We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions. The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features. Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity. Mutations in the helical domain retain ATP binding activity. Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation. We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations. PMID- 1717453 TI - Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane. AB - We have investigated the characteristics of platelet-activating factor (PAF) stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets. PMID- 1717454 TI - Use of a heat-stable microtubule-associated protein class-specific antibody to investigate the mechanism of microtubule binding. AB - Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding. PMID- 1717455 TI - Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport. AB - The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport. PMID- 1717456 TI - Insulin inhibits transcription of the human gene for insulin-like growth factor binding protein-1. AB - Insulin rapidly lowers serum insulin-like growth factor-binding protein-1 (IGFBP 1) levels in vivo. In studies reported here, HEP G2 cells were used as a model system to investigate how insulin achieves this effect. When HEP G2 cells were incubated with 100 nM insulin for 6, 14, or 24 h, IGFBP-1 protein levels in conditioned medium fell to approximately 50% of control values. This apparently was due to a fall in the rate of IGFBP-1 protein synthesis, since HEP G2 cells incorporated 46% less [35S]methionine into IGFBP-1 during a 4-h incubation with 100 nM insulin. IGFBP-1 mRNA levels were similarly affected by 100 nM insulin, falling to 45% of control values after 2 h, and to 9% of control values after 4 h of incubation with this hormone. The fall in IGFBP-1 mRNA level is consistent with data from nuclear transcription assays. HEP G2 nuclei isolated from cells that were incubated with 100 nM insulin for 2 h synthesized only approximately 1/3 the number of IGFBP-1 transcripts as did control nuclei. Further evidence that insulin decreases IGFBP-1 gene transcription comes from transient transfections using chimeric IGFBP-1 promoter-chloramphenicol acetyltransferase reporter gene constructs. IGFBP-1 promoter activity fell to approximately 50% of control values when HEP G2 cells transfected with a construct containing the first 1205 base pairs of the IGFBP-1 promoter were incubated with 100 nM insulin for 6, 14, or 24 h. Insulin lowered both IGFBP-1 protein levels and promoter activity in a dose-dependent manner. A half-maximal effect was found at approximately 1 nM insulin and a maximal effect was found at approximately 10 nM insulin in each instance. Transfections with constructs containing smaller IGFBP 1 promoter fragments showed that the region spanning from 103 to 529 base pairs 5' to the IGFBP-1 mRNA cap site was necessary to demonstrate the inhibitory effect of insulin. These studies indicate that insulin lowers IGFBP-1 protein levels, at least in part, by rapidly decreasing the rate of IGFBP-1 gene transcription, and suggest that this insulin-mediated fall in transcription is conferred through a specific region of the IGFBP-1 promoter. PMID- 1717457 TI - Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure. AB - We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow. A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211). The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain. This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated). Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment. Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet. Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion. The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar. The presence or absence of the carbohydrate does not influence the results of the various structural analyses. PMID- 1717458 TI - Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes. AB - A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle mediated transport/secretion. PMID- 1717459 TI - Identification of platelet membrane proteins that interact with amino-terminal peptides of pp60c-src. AB - Platelets contain exceptionally high levels of pp60c-src and, thus, provide a convenient system for investigating the physiological function of this protein tyrosine kinase. We have employed chemical cross-linking of myristylated amino terminal peptides of pp60c-src to platelet membranes in order to identify platelet membrane components that interact with pp60c-src to regulate or mediate its activity. We detected specific binding of radioiodinated peptides to platelet membrane proteins of 32, 50, 92, and 105 kDa. The 32-kDa protein may be related to the putative src receptor component recently identified in fibroblast membranes. The most reactive platelet protein, however, is the 50-kDa protein, which is either absent or nonreactive in fibroblast membranes. Binding of src peptides to this protein was saturable, and we estimate the presence of approximately 1 x 10(6) of the 50-kDa binding sites per platelet. The specificity of the peptide binding to the 50- and 32-kDa platelet proteins was analyzed by competition with different peptides. The binding sites displayed an absolute requirement for an N-myristoyl moiety and a strong preference for pp60c-src amino terminal sequences. The identification of these src-interacting proteins may help to decipher the biochemical pathways in which platelet pp60c-src is involved. PMID- 1717460 TI - The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K pumps. AB - Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties. PMID- 1717461 TI - Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. AB - The expression of the cystic fibrosis (CF) gene on its introduction into nonepithelial somatic cells has recently been shown to result in the appearance of distinctive low conductance chloride channels stimulated by cyclic AMP (Kartner, N., Hanrahan, J.W., Jensen, T.J., Naismith, A.L., Sun, S., Ackerley, C.A., Reyes, E.F., Tsui, L.-C., Rommens, J.M., Bear, C.E., and Riordan, J.R. (1991) Cell 64, 681-691; Anderson, M. P., Rich, D.P., Gregory, R.J., Smith, A.E., and Welsh, M.J. (1991) Science 251, 679-682). Since Xenopus oocytes provide a powerful system for ion channel characterization, we have examined whole cell and single channel currents in them after injection of cRNA to program the synthesis of the cystic fibrosis transmembrane conductance regulator (CFTR). This has enabled the direct demonstration that the cyclic AMP activation is mediated by protein kinase A and that CFTR is without effect on the endogenous calcium activated chloride channels of the oocyte, which have been well characterized previously and widely used as reporters of the expression of G-protein-coupled receptors. These findings strengthen the argument that the CF gene codes for a novel regulated chloride channel rather than a regulatory protein which can modulate separate chloride channel molecules. PMID- 1717462 TI - Mutational analysis of the galactose binding ability of recombinant ricin B chain. AB - Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB. PMID- 1717463 TI - Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase. AB - Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5' triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding. PMID- 1717464 TI - Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus. AB - The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2 macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non covalently trapped rather than covalently cross-linked. PMID- 1717465 TI - Structure of the gene for the neuronal intermediate filament protein alpha internexin and functional analysis of its promoter. AB - We have isolated the gene encoding the neuronal intermediate filament protein alpha-internexin using low stringency hybridization conditions and an NF-M (neurofilament middle molecular weight subunit) cDNA probe. This gene consists of three exons and two introns. The sequence data and the exon-intron organization of the gene establish its classification as a type IV intermediate filament gene. Transient transfection experiments showed that up to 5 kilobases of the alpha internexin promoter region transcribed equally well in both the alpha-internexin expressing and -nonexpressing cell lines. The results also demonstrated that the region from -77 to +73 relative to the transcription start site was sufficient for accurate basal transcription, but inclusion of the -254 to -78 region was required for efficient transcription. Sequence analysis shows that the -254 to 78 region contains several potential positive regulatory elements. PMID- 1717466 TI - Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae. AB - It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP 2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP 2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs. PMID- 1717467 TI - Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase. AB - The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH. PMID- 1717468 TI - Arginine-glycine-aspartic acid binding leading to molecular stabilization between integrin alpha v beta 3 and its ligand. AB - Cell adhesion is characterized by an integrin-mediated ligand binding event followed by reorganization of the actin-cytoskeleton leading to cell spreading and/or migration. In this report we examine the role of integrin alpha v beta 3 in mediating cell attachment to vitronectin or a RGD-containing peptide in the presence of cytochalasin B to prevent actin polymerization. Under these conditions cell attachment to a RGD-containing peptide can be dissociated by excess soluble ligand whereas cells attached to vitronectin cannot. These results suggest that alpha v beta 3-mediated cell attachment to vitronectin results in a highly stabilized interaction that is independent of the actin-cytoskeleton. To investigate the molecular nature of this interaction alpha v beta 3 was purified to homogeneity, and its binding properties toward various ligands were measured in a solid-phase receptor assay. The data indicate that alpha v beta 3 binds to vitronectin or fibronectin in a nondissociable manner whereas a RGD-containing peptide derived from vitronectin binds specifically but is completely dissociable with a Kd of 9.4 x 10(-7) M. Moreover, chemical modification of alpha v beta 3 with limited glutaraldehyde treatment allowed vitronectin to bind in a RGD dependent and dissociable manner, suggesting that receptor conformational changes or specific amino acid residues proximal to the ligand binding site(s) are involved in the stabilization event. Thus, in the absence of cytoskeletal proteins or other cellular components, integrin alpha v beta 3-ligand binding involves recognition of the RGD sequence leading to a highly stabilized protein protein association. PMID- 1717469 TI - Identification of two regions within the cytoplasmic domain of the human interferon-gamma receptor required for function. AB - Functionally active human interferon-gamma (IFN gamma) receptors require the presence of at least two polypeptides: the IFN gamma receptor and an accessory molecule encoded by a gene on human chromosome 21. Here we have used a murine L cell line that stably contains human chromosome 21 (SCC16-5) to determine whether the receptor's cytoplasmic domain is important for receptor function. SCC16-5 stably transfected with the full-length human IFN gamma receptor cDNA bound, internalized, and responded to human IFN gamma. In contrast, SCC16-5 expressing human IFN gamma receptors lacking a cytoplasmic domain bound human IFN gamma but did not internalize or respond to it. Using a family of IFN gamma receptor deletion mutants, two functionally important regions within the intracellular domain were identified: (a) a membrane proximal region (residues 256-303) required for ligand processing and biologic responsiveness and (b) the carboxyl terminal 39 amino acids (residues 434-472) needed exclusively for biologic responses. PMID- 1717470 TI - Transgenic mice aberrantly expressing human ornithine decarboxylase gene. AB - We have generated transgenic mice carrying human ornithine decarboxylase gene. Two different transgene constructs were used: (i) a 5'-truncated human ornithine decarboxylase gene and (ii) an intact human ornithine decarboxylase gene. Transgenic mice carrying the 5'-truncated gene did not express human ornithine decarboxylase-specific mRNA. Transgenic mice carrying the intact human ornithine decarboxylase gene expressed human-specific ornithine decarboxylase mRNA in all tissues studied. However, as indicated by actual enzyme assays, the expression pattern was highly unusual. In comparison with their wild-type littermates, the transgenic mice exhibited greatly elevated enzyme activity in almost every tissue studied. Ornithine decarboxylase activity was moderately elevated in parenchymal organs such as liver, kidney, and spleen. Tissues like heart, muscle, lung, thymus, testis, and brain displayed an enzyme activity that was 20 to 80 times higher than that in the respective tissues of nontransgenic animals. The offspring of the first transgenic male founder animal did not show any overt abnormalities, yet their reproductive performance was reduced. The second transgenic founder animal, showing similar aberrant expression of ornithine decarboxylase in all tissues studied, including an extremely high activity in testis, was found to be infertile. Histological examination of the tissues of the latter animal revealed marked changes in testicular morphology. The germinal epithelium was hypoplastic, and the spermatogenesis was virtually totally shut off. Similar examination of male members of the first transgenic mouse line revealed comparable, yet less severe, histological changes in testis. PMID- 1717471 TI - Cytochrome c oxidase subunit VIIa isoforms. Characterization and expression of bovine cDNAs. AB - Subunit VIIa of bovine cytochrome c oxidase occurs in two forms, the so-called heart and liver isoforms, which have been shown by protein analysis to differ in 35% of their amino acids. We have isolated and characterized cDNAs for each isoform. The derived heart-type processed protein is 59 amino acids long, with a 21-residue presequence; the immediate C terminus differs from the established protein sequence. The liver-type processed protein is 60 residues long, with a 23 amino acid presequence. Both presequences are traditional in that they are positively charged and appear amphiphilic when helically arrayed. The presequences are only 22% identical, but they both contain conserved residues indicative of two-step processing of the precursor proteins. Southern blot analysis reveals that the bovine genome contains five to six copies of the liver type gene as opposed to the presence of a single copy heart-type gene. Transcriptional analysis shows that heart-type message is detectable only in heart and skeletal muscle; the liver type is also seen in heart and muscle and, additionally, in the other tissues examined (liver, brain, and lung). The amino acid sequence EKQKLFQED is conserved in rat and in both isoforms in cow and human and may represent a domain of core subunit function. PMID- 1717472 TI - Antimicrobial peptides in the stomach of Xenopus laevis. AB - Antimicrobial peptides are widely distributed in nature and appear to play a role in the host defense of plants and animals. In this study we report the existence of antimicrobial peptides in the stomach of the vertebrate Xenopus laevis, an animal previously shown to store high concentrations of antimicrobial peptides in its skin. Antimicrobial activity was detected in extracts of X. laevis stomach tissue and nine antimicrobial peptides were then purified. A novel 24-amino acid peptide, designated PGQ, was isolated from these extracts, and has the following amino acid sequence: GVLSNVIGYLKKLGTGALNAVLKQ. PGQ is relatively basic and has the potential to form an amphipathic alpha-helix. The other peptides isolated are members of the magainin family of antimicrobial peptides, and include magainins I and II, PGLa, xenopsin precursor fragment, and four caerulein precursor fragments. None of these peptides had been previously identified in tissues other than the skin. The purification of the peptides from stomach extracts and subsequent protein sequence analysis reveals that the peptides have undergone the same processing as their dermal counterparts, and that they are stored in their processed forms. Northern blot analysis indicates that the magainin family of peptides are synthesized in the stomach, and immunohistochemical studies demonstrate that magainin is stored in a novel granular multinucleated cell in the gastric mucosa of Xenopus. This study demonstrates that the magainin family of antimicrobial peptides is found in the gastrointestinal system of X. laevis and offers an opportunity to further define the physiological role of these defense peptides. PMID- 1717473 TI - TEM immunogold staining of C3 from plasma onto titanium oxides. AB - Immunogold staining in conjunction with TEM was used to observe C3 adsorption from plasma in relation to the underlying titanium structure of thermal, anodic, and electropolished oxides. Heat treatments and oxide thickness were found to have no significant effect on the adsorption behavior of C3, while surface oxide type possibly has. Surface concentration of C3 was found to be time- and plasma concentration-dependent. Evidence is given for the possible involvement of C3 in protein exchange, i.e., the Vroman effect. Diluted plasma resulted in a random distribution of gold colloids, whereas clustering occurred with undiluted plasma. Although C3 concentrations present on grain boundaries followed the same trend as that found on the surface, C3 was found to have a higher grain boundary than bulk concentration for 0.1% plasma. PMID- 1717474 TI - Competitive adsorption of vitronectin with albumin, fibrinogen, and fibronectin on polymeric biomaterials. AB - Vitronectin (VN) was competitively adsorbed with human serum albumin (HSA), fibrinogen (FGN), and fibronectin (FN) from binary component mixtures in order to compare the relative affinities of these proteins for various polymer materials. Competitive adsorption was monitored by incubating radiolabeled protein solutions inside 0.125-in. i.d. tubing of the polymers, flushing with buffer, and measuring the adherent radioactivity. Adsorption experiments at equal mass concentrations of the competing proteins revealed that VN comprises at least 75% by weight of the adsorbed protein when competitively adsorbed with HSA and approximately 50% by weight when competitively adsorbed with FGN and FN on all surfaces except a poly(ethylene oxide)-based polyurethane where it comprised closer to 80 wt%. When VN was competitively adsorbed in the presence of increasing amounts of HSA, FGN, and FN, the amount of VN adsorbed on a weight basis was diminished the most by FGN. HSA had the least inhibitory effect at low bulk concentrations and FN had the weakest effect at higher bulk concentration levels. When HSA, FGN, and FN were competitively adsorbed in the presence of increasing amounts of VN, VN diminished their adsorption on a weight basis in the order: HSA greater than FN greater than FGN. PMID- 1717475 TI - The bonding behavior of calcite to bone. AB - Plates of calcite (CaCO3) were implanted in rabbit tibiae, and their biocompatibility and bonding ability to bone were studied. The plates were also implanted subfascially in rabbit muscle for 8 weeks, and changes on their surfaces in the body were examined. Contact microradiography and Giemsa surface stain demonstrated direct bonding between calcite and bone without interpositions. The average failure load of the interface between calcite and bone was 4.11 kg, indicating an adequate strength of bonding. However, a Ca-P rich layer, which formed on the surfaces of other bioactive ceramics in vivo, was not detected by a scanning electron microscope-electron probe x-ray microanalyzer. Scanning electron micrographs of the surface of calcite implanted subfascially for 8 weeks showed marked degradation and a rough surface. However, the surface apatite layer was not detected by thin-film x-ray diffraction analysis and Fourier transform infrared reflection spectroscopy. Calcite is a biodegradable material that bonds to bone without a surface apatite layer. The mechanical bonding provided by the anchoring effect of the newly formed bone into the surface roughness of calcite is considered to be a major factor in calcite bone bonding. PMID- 1717476 TI - Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. AB - We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit. PMID- 1717477 TI - Paxillin is a major phosphotyrosine-containing protein during embryonic development. AB - Phosphotyrosine-containing proteins were immunoprecipitated from embryonic chicken tissue extracts using anti-phosphotyrosine antibody coupled to agarose beads. Major phosphotyrosine-containing proteins of 110, 70, and 50 kD were observed following blotting with anti-phosphotyrosine antibody. The 70-kD band was selectively removed from the samples by precipitation with antibodies to the focal adhesion protein paxillin, therefore identifying paxillin as one of the major tyrosine kinase substrates during chick embryonic organogenesis. The tyrosine phosphorylation of paxillin is regulated developmentally: during embryogenesis, a marked decrease in its phosphotyrosine content was observed, although the total level of paxillin remained essentially constant. Approximately 20% of the paxillin was phosphorylated on tyrosine in the early embryo. In contrast, tyrosine phosphorylation of paxillin was undetectable in the adult. A similar profile of phosphotyrosine-containing proteins was identified in rat embryos. Paxillin was also found to be a major phosphotyrosine-containing protein in the rat embryo. These data suggest that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling cell and tissue cytoarchitecture rearrangement during vertebrate development. PMID- 1717478 TI - Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils. AB - The adhesion of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC) is an early and fundamental event in acute inflammation. This process requires the regulated expression of molecules on both the EC and PMN. EC stimulated with histamine or thrombin coexpress two proadhesive molecules within minutes: granule membrane protein 140 (GMP-140), a member of the selectin family, and platelet-activating factor (PAF), a biologically active phospholipid. Coexpression of GMP-140 and PAF is required for maximal PMN adhesion and the two molecules act in a cooperative fashion. The component of adhesion mediated by EC associated PAF requires activation of CD11/CD18 integrins on the PMN and binding of these heterodimers to counterreceptors on the EC. GMP-140 also binds to a receptor on the PMN; however, it tethers the PMN to the EC without requiring activation of CD11/CD18 integrins. This component of the adhesive interaction is blocked by antibodies to GMP-140 or by GMP-140 in the fluid phase. Experiments with purified GMP-140 indicate that binding to its receptor on the PMN does not directly induce PMN adhesiveness but that it potentiates the CD11/CD18-dependent adhesive response to PAF by a mechanism that involves events distal to the PAF receptor. Tethering of the PMN to the EC by GMP-140 may also be required for efficient interaction of PAF with its receptor on the PMN. These observations define a complex cell recognition system in which tethering of PMNs by a selectin, GMP-140, facilitates juxtacrine activation of the leukocytes by a signaling molecule, PAF. The latter event recruits the third component of the adhesive interaction, the CD11/CD18 integrins. PMID- 1717479 TI - The complement binding-like domains of the murine homing receptor facilitate lectin activity. AB - The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC CAMS) which are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. Previous data have indicated a predominant role for the lectin motif in cell adhesion directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. In this paper, we analyze the functional characteristics of murine HR-IgG chimeras containing the lectin, lectin plus egf, and lectin plus egf plus CB domains. The Mel 14 mAb, an adhesion blocking antibody which recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for a HR construct which lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all showed that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality. PMID- 1717481 TI - Naturally occurring mutations in intestinal sucrase-isomaltase provide evidence for the existence of an intracellular sorting signal in the isomaltase subunit. AB - Mutations in the sucrase-isomaltase gene can lead to the synthesis of transport incompetent or functionally altered enzyme in congenital sucrase-isomaltase deficiency (CSID) (Naim, H. Y., J. Roth, E. Sterchi, M. Lentze, P. Milla, J. Schmitz, and H. P. Hauri. J. Clin. Invest. 82:667-679). In this paper we have characterized two novel mutant phenotypes of CSID at the subcellular and protein levels. The first phenotype revealed a sucrase-isomaltase protein that is synthesized as a single chain, mannose-rich polypeptide precursor (pro-SI) and is electrophoretically indistinguishable from pro-SI in normal controls. By contrast to normal controls, however, pro-SI does not undergo terminal glycosylation in the Golgi apparatus. Subcellular localization of pro-SI by immunoelectron microscopy revealed unusual labeling of the molecule in the basolateral membrane and no labeling in the brush border membrane thus indicating that pro-SI is missorted to the basolateral membrane. Mapping of biosynthetically labeled pro-SI with four epitope- and conformation-specific monoclonal antibodies suggested that conformational and/or structural alterations in the pro-SI protein have prevented posttranslational processing of the carbohydrate chains of the mannose-rich precursor and have lead to its missorting to the basolateral membrane. The second phenotype revealed two variants of pro-SI precursors that differ in their content of mannose-rich oligosaccharides. Conversion of these forms to a complex glycosylated polypeptide occurs at a slow rate and is incomplete. Unlike its counterpart in normal controls, pro-SI in this phenotype is intracellularly cleaved. This cleavage produces an isomaltase-like subunit that is transport competent and is correctly sorted to the brush border membrane since it could be localized in the brush border membrane by anti-isomaltase mAb. The sucrase subunit is not transported to the cell surface and is most likely degraded intracellularly. We conclude that structural features in the isomaltase region of pro-SI are required for transport and sorting of the sucrase-isomaltase complex. PMID- 1717480 TI - Influence of receptor lateral mobility on adhesion strengthening between membranes containing LFA-3 and CD2. AB - We have used an in vitro model system of glass-supported planar membranes to study the effects of lateral mobility of membrane-bound receptors on cell adhesion. Egg phosphatidylcholine (PC) bilayers were reconstituted with two anchorage isoforms of the adhesion molecule lymphocyte function-associated antigen 3 (LFA-3). The diffusion coefficient of glycosyl phosphatidylinositol (GPI)-anchored LFA-3 approached that of phospholipids in the bilayers, whereas the transmembrane (TM)-anchored isoform of LFA-3 was immobile. Both static and laminar flow assays were used to quantify the strength of adherence to the lipid bilayers of the T lymphoma cell line Jurkat that expresses the counter-receptor CD2. Cell adhesion was dependent on LFA-3 density and was more efficient on membranes containing the GPI isoform than the TM isoform. Kinetic measurements demonstrated an influence of contact time on the strength of adhesion to the GPI isoform at lower site densities (25-50 sites/microns2), showing that the mobility of LFA-3 is important in adhesion strengthening. At higher site densities (1,500 sites/microns2) and longer contact times (20 min), Jurkat cell binding to the TM and GPI isoforms of LFA-3 showed equivalent adhesion strengths, although adhesion strength of the GPI isoform developed twofold more rapidly than the TM isoform. Reduction of CD2 mobility on Jurkat cells at 5 degrees C greatly decreased the rate of adhesion strengthening with the TM isoform of LFA-3, resulting in a 30 fold difference between the two LFA-3 isoforms. Our results demonstrate that the ability of a membrane receptor and its membrane-bound counter-receptor to diffuse laterally enhances cell adhesion both by allowing accumulation of ligands in the cell contact area and by increasing the rate of receptor-ligand bond formation. PMID- 1717482 TI - Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile. AB - Nebulin, a family of giant proteins with size-variants from 600 to 900 kD in various skeletal muscles, have been proposed to constitute a set of inextensible filaments anchored at the Z line (Wang, K., and J. Wright. 1988. J. Cell Biol. 107:2199-2212). This newly discovered filament of the skeletal muscle sarcomere is an attractive candidate for a length-regulating template of thin filaments. To evaluate this hypothesis, we address the question of coextensiveness of nebulin and the thin filament by searching for a correlation between the size of nebulin variants and the length distribution of the thin filaments in several skeletal muscles. A positive linear correlation indeed exists for a group of six skeletal muscles that display narrow thin filament length distributions. To examine the molecular and architectural differences of nebulin size-variants, we carried out immunoelectron microscopic studies to map out epitope profiles of nebulin variants in these muscles. For this purpose, a panel of mAbs to distinct nebulin epitopes was produced against rabbit nebulin purified by an improved protocol. Epitope profiles of nebulin variants in three skeletal muscles revealed that (a) nebulin is inextensible since nebulin epitopes maintain a fixed distance to the Z line irrespective of the degree of sarcomere stretch; (b) a single nebulin polypeptide spans a minimal distance of 0.9 microns from the Z line; (c) nebulin contains repeating epitopes that are spaced at 40 nm or its multiples; (d) nebulin repeats coincide with thin filament periodicity; (e) nebulin variants differ mainly at either or both ends; and (f) nebulin remains in the sarcomere in actin-free sarcomeres produced by gelsolin treatment. Together, these data suggest that nebulin is an inextensible full-length molecular filament that is coextensive with thin filaments in skeletal muscles. We propose that nebulin acts as a length-regulating template that determines thin filament length by matching its large number of 40-nm repeating domains with an equal number of helical repeats of the actin filaments. PMID- 1717483 TI - A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment. AB - The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, does not become GPI anchored. We now show that this null sequence for GPI attachment can be converted to a strong GPI signal by mutating a pair of residues (valine-glutamate) in the LDLR sequence at a position corresponding to the normal cleavage/attachment site, to serine-glycine, as found in the DAF sequence. A single mutation (converting valine at the anchor addition site to serine, the normal acceptor for GPI addition in DAF) was insufficient to produce GPI anchoring, as was mutation of the valine-glutamate pair to serine-phenylalanine (a bulky residue). These results suggest that a pair of small residues (presumably flanking the cleavage point) is required for GPI attachment. By introducing the sequence serine-glycine (comprising a cleavage-attachment site for GPI addition) at different positions in the LDLR sequence of the fusion protein, HLD, we show that optimal GPI attachment requires a processing site positioned 10-12 residues NH2-terminal to the hydrophobic domain, the efficiency anchor attachment dropping off sharply as the cleavage site is moved beyond these limits. These data suggest that the GPI signal consists solely of a hydrophobic domain combined with a processing site composed of a pair of small residues, positioned 10-12 residues NH2-terminal to the hydrophobic domain. No other structural motifs appear necessary. PMID- 1717484 TI - Microtubule polymer assembly and transport during axonal elongation. AB - As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown. We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues. Photoactivatable tubulin, microinjected into frog embryos at the two-cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon elongation. This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation. Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone. PMID- 1717485 TI - Fiber type- and position-dependent expression of a myosin light chain-CAT transgene detected with a novel histochemical stain for CAT. AB - We recently generated and characterized transgenic mice in which regulatory sequences from a myosin light chain gene (MLC1f/3f) are linked to the chloramphenicol acetyltransferase (CAT) gene. Transgene expression in these mice is specific to skeletal muscle and graded along the rostrocaudal axis: adult muscles derived from successively more caudal somites express successively higher levels of CAT. To investigate the cellular basis of these patterns of expression, we developed and used a histochemical stain that allows detection of CAT in individual cells. Our main results are as follows: (a) Within muscles, CAT is detected only in muscle fibers and not in associated connective tissue, blood vessels, or nerves. Thus, the tissue specificity of transgene expression observed by biochemical assay reflects a cell-type specificity demonstrable histochemically. (b) Within individual muscles, CAT levels vary with fiber type. Like the endogenous MLC1f/3f gene, the transgene is expressed at higher levels in fast-twitch (type II) than in slow-twitch (type I) muscle fibers. In addition, CAT levels vary among type II fiber subtypes, in the order IIB greater than IIX greater than IIA. (c) Among muscles that are similar in fiber type composition, the average level of CAT per fiber varies with rostrocaudal position. This position-dependent variation in CAT level is apparent even when fibers of a single type are compared. From these results, we conclude that fiber type and position affect CAT expression independently. We therefore infer the existence of separate fiber type-specific and positionally graded transcriptional regulators that act together to determine levels of transgene expression. PMID- 1717486 TI - Astrocyte-derived TGF-beta 2 and NGF differentially regulate neural recognition molecule expression by cultured astrocytes. AB - Because of the importance of neural recognition molecules expressed by glial cells to mediate interactions with neurons, growth factors and cytokines known to be functional during morphogenesis and in diseases of the nervous system were studied for their effects on recognition molecule expression by cultured immature and mature astrocytes from several brain regions. In cultures of immature astrocytes, transforming growth factors-beta 1 (TGF-beta 1) and -beta 2 (TGF-beta 2) and nerve growth factor (NGF) increased expression of the neural adhesion molecule L1, leading to a glia-mediated L1-specific increase in neurite outgrowth of dorsal root ganglion neurons on the astrocyte substrate. L1 expression induced by TGF-beta was inhibited by addition of antibodies to NGF, suggesting that TGF beta influences L1 expression by modulating production of NGF by astrocytes. TGF beta 1 and -beta 2 decreased expression of N-CAM by immature astrocytes. Since N CAM expression was not affected by NGF and antibodies to NGF did not abolish the TGF-beta-induced decrease in N-CAM expression, NGF did not appear to be the mediator for regulating expression of N-CAM. Expression of the adhesion molecule on glia (AMOG) was not affected by any factor. NGF and TGF-beta 2 in latent form, but not TGF-beta 1 were found in the culture supernatants. Addition of interferon gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), platelet derived growth factor (PDGF), or basic fibroblast growth factor (bFGF) to the cultures did not change recognition molecule expression. REcognition molecule expression by mature astrocytes was not found to be modified by any of the factors tested. In view of the observation that levels of L1 and N-CAM expression correlated with the presence of TGF-beta 2 and NGF in the culture supernatants of immature astrocytes, an autocrine regulatory mechanism for recognition molecule expression by these cells is suggested to play a crucial role in regulation of neuron-glia interactions. PMID- 1717487 TI - Hepatocyte adhesion to carbohydrate-derivatized surfaces. II. Regulation of cytoskeletal organization and cell morphology. AB - Rat hepatic lectins mediate adhesion of isolated rat hepatocytes to synthetic surfaces derivatized with galactosides. Initial weak adhesion is followed by rapid adhesion strengthening. After hepatocytes contact galactose-derivatized gels, the hepatic lectins move rapidly into an inaccessible patch at the adhesive surface (Weisz, O. A., and R. L. Schnaar. 1991. J. Cell Biol. 115:485-493). Hepatic lectin patching, which occurs both at 37 degrees C and 4 degrees C, is not responsible for adhesion strengthening, which does not occur at 4 degrees C. Of various cytoskeletal and metabolic perturbants tested, only a combination of hyperosmotic medium, colchicine, and cytochalasin caused a marked (72%) reduction of adhesion strengthening (without reducing weak cell adhesion). Clathrin and actin were readily detected in the adhesive patch by immunofluorescence microscopy. Rat hepatocytes also adhered avidly to surfaces derivatized with asialofetuin, a high-affinity ligand for the rat hepatic lectins. However, hepatic lectin molecules did not migrate into a patch on the asialofetuin derivatized surface, suggesting that hepatic lectin-asialofetuin binding may have resulted in the rapid formation of a ring of essentially irreversibly adherent receptors that prevented diffusion of additional lectin molecules into the contact site. The cells were unable to increase their adhesive contact area by flattening onto the derivatized surface. Treatment of cells with cytochalasin, however, did result in an increase in the size of the contact area. Cells adhering to surfaces derivatized with an adhesion-promoting peptide (containing an arg-gly-asp sequence) had larger contact areas than those adhering to galactoside-derivatized surfaces. A model is proposed in which carbohydrate mediated adhesion causes specific reorganization of cytoskeletal components, leading to strengthened adhesion and a characteristic spherical cell morphology. PMID- 1717489 TI - A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. AB - An antibody was identified previously that recognizes sites of polymerase II transcription on lampbrush chromosomes, puffs on polytene chromosomes, and many small granules in the nucleoplasm of all cells tested. This antibody binds a conserved family of phosphorylated polypeptides in vertebrate and invertebrate cells. We developed a method for purifying these proteins that involves differential solubility in MgCl2. We isolated a Drosophila cDNA encoding one of the proteins using information obtained from microsequencing. In vivo expression studies show that this protein is concentrated on sites of polymerase II transcription and that it is highly phosphorylated. The protein shares a high degree of homology with proteins involved in alternative splicing of pre-mRNA suggesting the possibility that this protein plays a role in pre-mRNA splicing. PMID- 1717488 TI - The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells. AB - Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1 3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium. PMID- 1717490 TI - Developmentally and spatially regulated expression of HNK-1 carbohydrate antigen on a novel phosphatidylinositol-anchored glycoprotein in rat brain. AB - HNK-1 carbohydrate antigen in an epitope expressed commonly in many cell surface adhesion and recognition molecules in the nervous system. We purified and characterized from rat brain a novel phosphatidylinositol (PI)-anchored 150-kD glycoprotein belonging to the HNK-1 family. The molecule (PI-GP150) was detected by combination of PI-specific phospholipase C treatment of brain membranes and Western blot analysis with mAb HNK-1. HNK-1-positive PI-GP150 was purified from the PI-PLC-released materials with three successive chromatographies (Sephacryl S 300, mAb HNK-1-Sepharose 4B, and Mono Q) and proven to be a novel molecule by immunoblot and structural analyses. Polyclonal antibody was raised against PI GP150 and used to show that (a) PI-GP150 is expressed on the surface of neuronal cell bodies and their processes in culture, and (b) PI-GP150 appears during embryonic development and is present throughout all postnatal life in all brain regions. However, the expression of the HNK-1 epitope on PI-GP150 is regulated in both developmental stage-specific and region-specific manners. In newborn rats, the HNK-1 epitope is expressed on PI-GP150 throughout the brain. The level of HNK 1 epitope on PI-GP150 decreases after postnatal day 7 in hindbrain and becomes completely absent in adult myelencephalon and metencephalon. In contrast, HNK-1 epitope on PI-GP150 was constitutively expressed in telencephalon. Thus, while the HNK-1 carbohydrate epitope is strongly coupled to PI-GP150, its expression can be regulated independently of that of PI-GP150. The differential expression of the HNK-1 epitope at different rostro-caudal axial levels was observed also in other HNK-1 family molecules in brain membranes. These results suggest that the HNK-1 epitope plays an important role in adding region-specific and developmental stage-specific modifications on the function of the cell surface molecules. PMID- 1717491 TI - Phorbol esters selectively downregulate contractile protein gene expression in terminally differentiated myotubes through transcriptional repression and message destabilization. AB - Chronic exposure of differentiated avian skeletal muscle cells in culture to the phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (PMA), results in the selective disassembly of sarcomeric structures and loss of muscle-specific contractile proteins, leaving cytoskeletal structures and their associated proteins intact. We demonstrate here that these morphological and biochemical changes are accompanied by dramatic and selective decreases in the level of the mRNAs that encode the contractile proteins. We measured the effects of PMA on the transcriptional activity and mRNA stability of four contractile protein genes (alpha-cardiac and alpha-skeletal actin, cardiac troponin C [cTnC], and myosin light chain lf [MLClf]) and two nonmuscle genes (beta-cytoplasmic actin and the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The transcriptional activity of the alpha-cardiac actin and cTnC genes dramatically decreased by 8 h after the addition of PMA, while other muscle and nonmuscle genes examined showed no change. Pulse-chase experiments of in vivo labeled RNA showed significant reductions in mRNA half-lifes for all the contractile protein mRNAs examined, while the half-lifes of beta-actin and GAPDH mRNA were unchanged. All of the above effects occurred under conditions in which cellular protein kinase C (PKC) levels had been reduced by greater than 90%. The fact that many of the contractile protein genes remained transcriptionally active despite the fact that the cells were unable to accumulate their mRNAs to any significant extent indicated that the treated cells were still committed to skeletal muscle differentiation. The selective changes in the stability of the contractile protein mRNAs suggest that the control of mRNA stability may be part of the normal regulatory program of skeletal muscle differentiation and that this control may be linked to the integrity of the contractile apparatus and mediated by second messenger pathways involving PKC activation. PMID- 1717492 TI - Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions. AB - We have investigated the roles of pp60c-src and p21c-ras proteins in transducing the nerve growth factor (NGF) and fibroblast growth factor (FGF) signals which promote the sympathetic neuronlike phenotype in PC12 cells. Neutralizing antibodies directed against either Src or Ras proteins were microinjected into fused PC12 cells. Each antibody both prevented and reversed NGF- or FGF-induced neurite growth, a prominent morphological marker for the neuronal phenotype. These data demonstrate the involvement of both pp60c-src and p21c-ras proteins in NGF and FGF actions in PC12 cells, and establish a physiological role for the pp60c-src tyrosine kinase in signal transduction pathways initiated by receptor tyrosine kinases in these cells. Additional microinjection experiments, using PC12 transfectants containing inducible v-src or ras oncogene activities, demonstrated a specific sequence of Src and Ras actions. Microinjection of anti Ras antibody blocked v-src-induced neurite growth, but microinjection of anti-Src antibodies had no effect on ras oncogene-induced neurite growth. We propose that a cascade of Src and Ras actions, with Src acting first, is a significant feature of the signal transduction pathways for NGF and FGF. The Src-Ras cascade may define a functional cassette in the signal transduction pathways used by growth factors and other ligands whose receptors have diverse structures and whose range of actions on various cell types include mitogenesis and differentiation. PMID- 1717493 TI - Motility and ultrastructure of large granular lymphocytes on lipid bilayers reconstituted with adhesion receptors LFA-1, ICAM-1, and two isoforms of LFA-3. AB - Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here. PMID- 1717494 TI - Growth regulated expression of B-myb in fibroblasts and hematopoietic cells. AB - The B-myb cDNA has extensive sequence similarities to the c-myb proto-oncogene, but, at variance with c-myb, it is expressed in cells other than hematopoietic cells. In this paper, we show that (1) B-myb is expressed in mouse, human, and hamster fibroblasts; (2) B-myb mRNA levels are growth-regulated in both fibroblasts and peripheral blood mononuclear cells; (3) by its mode of growth regulation (peak of expression, behavior in G1-specific temperature sensitive (ts) mutants and in the presence of cycloheximide), B-myb can be classified, like c-myb, thymidine kinase, PCNA, and others, as a late growth-regulated gene; (4) B myb mRNA levels decrease when HL-60 cells are induced to differentiate; and (5) the increase in mRNA levels in serum-stimulated cells is only partially explained by an increase in rate of transcription. The possibility that the B-myb gene may be the equivalent in fibroblasts and epithelial cells of the c-myb proto-oncogene of hematopoietic cells is discussed. PMID- 1717495 TI - Murine mast cell colony formation supported by IL-3, IL-4, and recombinant rat stem cell factor, ligand for c-kit. AB - Recently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand for c kit. We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin-3 and interleukin-4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue-type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin- and berberine sulfate-positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow-derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin-3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of both CTMC and BMMC. PMID- 1717496 TI - Continuous activation of primitive hematopoietic cells in long-term human marrow cultures containing irradiated tumor cells. AB - Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny. PMID- 1717497 TI - Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3. AB - We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells. PMID- 1717498 TI - Correlation between the proliferative response to granulocyte colony-stimulating factor and the positivity of transferrin receptor in acute myeloblastic leukemia cells. AB - The use of granulocyte colony-stimulating factor (G-CSF) after chemotherapy for acute myeloblastic leukemia (AML) has been reported. However, there is a drawback in that G-CSF may stimulate the proliferation of AML progenitors. To determine the parameter(s) indicative of responsiveness of AML blasts to G-CSF, various surface phenotypes of blasts were examined in relation to the blast colony formation stimulated by G-CSF in 39 AML patients. A correlation was found only with transferrin receptor positivity among the various phenotypes studied. The population mean of percentages of transferrin receptor-positive blasts in the group responding to G-CSF in vitro was significantly higher than that of blasts in the group not responding to G-CSF. A further correlation was found between transferrin receptor positivity and the number of G-CSF receptors on the blasts; that is, blasts expressing more G-CSF receptors have greater transferrin receptor positivity. In our previous study, we observed that blasts with a large number of G-CSF receptors produce more colonies in response to G-CSF. These results indicated that blasts expressing more transferrin receptors have a larger number of G-CSF receptors and may show more active proliferation in response to G-CSF. Therefore, the proliferative response of blasts to G-CSF can be predicted by examining transferrin receptor positivity. The clinical use of G-CSF in AML patients may be recommended when the patient's blasts have a low level of transferrin receptor expression. The measurement of transferrin receptors on blasts, instead of the rather complicated G-CSF receptor determination, would be a useful indicator for the safer application of G-CSF in AML patients. PMID- 1717499 TI - Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro. AB - We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date. PMID- 1717500 TI - Bacterial resistances to mercury and copper. AB - Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanisms of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated. Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals. PMID- 1717501 TI - From growth arrest to growth suppression. AB - Since the introduction of the cell cycle concept two approaches to study growth regulation of cells have been proposed. One claims that cells are naturally quiescent, requiring a stimulatory encouter with growth factors for induction of cell division. The other considers cellular multiplication as the natural steady state; cessation of multiplication is thus a restriction imposed on the system. In the latter case emphasis is mainly on the signals involved in arrest of multiplication. This Prospect focuses on specific events occurring in mammalian cells at growth arrest, senescence, and terminal differentiation, specifically emphasizing the growth inhibitory factors, tumor suppressor genes, and other signals for growth suppression. PMID- 1717502 TI - [Lateral periodontal cyst with keratinization (odontogenic keratocyst). Case report]. AB - The purpose of this study was to present the clinical and histological features of one case of lateral periodontal cyst with keratinization. The incidence, the histological features and the aetiopathogenesis of this type of cyst that is recurred were discussed. PMID- 1717503 TI - Supporting the development of low birthweight infants. PMID- 1717504 TI - Treatment of chronic relapsing experimental allergic encephalomyelitis with the intravenous administration of splenocytes coupled to encephalitogenic peptide 91 103 of myelin basic protein. AB - Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) is an autoimmune demyelinating disease of the central nervous system and serves as an experimental model of human multiple sclerosis. Amino acid residues p91-103 of myelin basic protein are encephalitogenic in SJL mice and transfer of T cell lines that recognize this epitope results in CR-EAE. We show here that coculture of T cells in the presence of p91-103 that has been chemically cross-linked to the antigen presenting cells renders the T cell lines tolerant to the antigen. Injection of p91-103 coupled splenocytes into animals that had received encephalitogenic p91-103 reactive T cells significantly reduced the incidence and severity of EAE. Furthermore, treatment of mice with a single injection of antigen coupled splenocytes after they had recovered from their initial paralytic attack prevented the development of subsequent clinical relapses in all animals. These studies indicate that this effect is long lasting and can be successfully accomplished in an established autoimmune disease. Hence this form of immunotherapy may be considered as a therapeutic modality in the treatment of autoimmune diseases when the autoantigens are known. PMID- 1717505 TI - The refractory phase of experimental allergic encephalomyelitis in the Lewis rat is antigen specific in its induction but not in its effect. AB - Rats that have recovered from experimental allergic encephalomyelitis (EAE) are refractory to attempts to induce further episodes of the disease. The specificity of this refractoriness was examined by determining the susceptibility of such animals to the related disease experimental allergic neuritis (EAN). As EAE and EAN are induced respectively by central nervous system myelin basic protein (MBP) and the P2 peptide of peripheral nerve myelin, it was possible to test whether the refractory phase of EAE represented a generalized hyporeactivity to antigenic challenge or was solely a failure to respond to the antigen with which disease was induced. For some experiments the roles of the two antigens were reversed, i.e. the effects of preimmunizing with P2 peptide on the susceptibility to EAE was examined. Animals preimmunized with MBP or P2 peptide became profoundly refractory to EAE and EAN respectively and rats preimmunized with one antigen but challenged with the other were susceptible to the challenge. It is concluded that refractoriness to EAE and EAN is largely antigen specific. Preimmunization with one antigen followed by challenge with a mixture of both suggested that the immunosuppression responsible for refractoriness, although specific in its induction, was non-specific in its effect. PMID- 1717506 TI - Genetic control of antibody response to bovine rhodopsin in mice: epitope mapping of rhodopsin structure. AB - Inbred strains of mice of independent haplotype were immunized with bovine rhodopsin. All mice tested except SJL developed significant titers of specific antibodies 21 days after a single immunization. Anti-rhodopsin antibody level differed among conventional inbred strains. Comparison of the immune response to rhodopsin of congenic mice on two different genetic backgrounds showed that animals with an A background typically produced higher levels of specific antibody than mice with a B10 background. Titer of specific antibodies in antisera of mice of the same H-2 haplotype but different Igh haplotype differed; e.g. for H-2d haplotype, NZB (Ighn) generated the highest level of antibody with BALB/c (Igha), DBA/2 (Ighc), and B10.D2 (Ighb) strains giving successively lower responses. The location of immunodominant regions of bovine rhodopsin was investigated in primary sera among strains of mice. Sera were tested for their binding of anti-rhodopsin antibodies to synthetic peptides covering the entire primary structure of rhodopsin. From direct binding studies with hydrophilic rhodopsin peptides, the majority of the antigenic binding sites were localized in the sequence of the amino terminus, the II-III loop and the carboxyl terminus. Binding to these antigenic peptides was not strain restricted. Application of the overlapping synthetic peptide strategy of Geysen enabled refinement of these epitopes and determination of an additional major epitope in the hydrophobic sequence 304-310. PMID- 1717507 TI - Pancreatitis following scoliosis surgery in children and young adults. AB - Forty-four patients undergoing single-stage surgery for scoliosis were monitored for biochemical and clinical evidence of pancreatitis. Six patients (14%) developed elevation of both serum amylase and lipase levels. Four of these had symptoms or signs suggestive of pancreatitis. Mean intraoperative blood loss was significantly higher in the group with pancreatitis. No significant differences were noted with regard to age, surgical technique, degree of initial or residual deformity, or length of surgery. The patients with pancreatitis required a longer average period of fasting time. Patients with prolonged ileus or abdominal pain after scoliosis surgery should be investigated for possible pancreatitis. PMID- 1717508 TI - An activated CD8+ lymphocyte appears in lymph nodes of rhesus monkeys early after infection with simian immunodeficiency virus. AB - Although alterations in T lymphocyte subset distribution and function in the peripheral blood of HIV-infected humans are well defined, the extent to which these reflect changes in other lymphoid compartments is unclear. We have characterized the coincident changes in PBL and lymph nodes (LN)1 after simian immunodeficiency virus of macaques (SIVmac) infection of rhesus monkeys. Whereas no consistent change in CD8+ PBL was noted during the first 60 d after infection, CD8+ lymphocytes increased significantly in number in LN. These CD8+ LN lymphocytes exhibited an increased expression of MHC class II and a decreased expression of leukocyte adhesion molecule-1, suggesting that they were activated, but interestingly did not express CD25 (IL-2 receptor). Moreover, there was no evidence that these CD8+ LN cells were proliferating, suggesting that they had migrated to the LN. These changes in the LN CD8+ lymphocyte population preceded any detectable change in the light microscopic appearance of the LN. When SIVmac specific effector T cell responses were assessed, the magnitude of virus-specific effector activity was nearly identical in the PBL and LN of each monkey studied. However, the presence of SIVmac-specific effector cells in the LN did not correlate with the presence of CD8+, MHC class II+ cells. These findings suggest that this numerically important CD8+ lymphocyte subpopulation may serve a regulatory function. PMID- 1717509 TI - Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells. AB - Utilizing cocultures of mouse renal juxtaglomerular cells with bovine microvascular endothelial cells, we have examined whether endothelial cells exert direct influence on renin secretion from renal juxtaglomerular cells. In the presence of endothelial cells both spontaneous and forskolin (10 microM) or isoproterenol (10 microM) stimulated renin release were markedly attenuated. The stimulatory effect of the calmodulin antagonist calmidazolium (10 microM) on renin secretion was not altered by endothelial cells, whereas the stimulatory effect of ethylisopropylamiloride (50 microM) an inhibitor of sodium-proton exchange was enhanced in the presence of endothelial cells. Indomethacin (10 microM) and NG-monomethyl-l-arginine (NMMA) (1 mM) used to inhibit cyclooxygenase activity and production of endothelium-derived relaxing factor (EDRF) decreased spontaneous renin release in the presence of endothelial cells only, but had no effect on forskolin stimulated renin secretion. Endothelin (1 microM) inhibited cAMP stimulated renin release both in the absence and in the presence of endothelial cells. ATP (10 microM) which acts on both endothelial and juxtaglomerular cells via purinergic P2 receptors inhibited cAMP stimulated renin release only in the absence but not in the presence of endothelial cells. This modulatory effect of endothelial cells was no altered by indomethacin nor by NMMA. Taken together, our findings provide first evidence for a local control function of the endothelium on cAMP stimulated renin secretion from renal juxtaglomerular cells, which could in part be mediated by endothelin. PMID- 1717510 TI - A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts. AB - Insulin-like growth factor I and II (IGF-I and IGF-II) associate with specific IGF binding proteins (IGFBPs) present in plasma and extracellular fluids that can modulate the anabolic effects of these peptides. IGF-I has been shown to increase IGFBP concentrations in vivo and in vitro, but the mechanism and significance of this action are unknown. We examined these issues using normal and simian virus 40-transformed adult human fibroblasts (SV40-HF) in culture. Treatment with IGF-I markedly stimulated the appearance of IGFBP-3 (42/38 kD doublet), a 36 kD IGFBP, and 28-32 kD IGFBPs in the medium of these cells, as assessed by Western ligand blotting; IGF-I decreased levels of 24 kD IGFBP in normal HF cultures. The IGF-I induced change in IGFBP levels was not a type I IGF receptor-mediated effect on IGFBP synthesis because (a) high concentrations of insulin did not mimic IGF-I's effect; (b) IGF-II and IGF-I analogues having reduced affinity for the IGF-I receptor were equipotent with IGF-I in increasing medium IGFBPs; (c) [QAYL]IGF-I, and IGF-I analogue having normal receptor affinity and decreased affinity for IGFBPs, had no effect; and (d) alpha IR-3, a monoclonal antibody specific for the type I IGF receptor, did not block IGF-I-stimulated increases in IGFBPs. In physiological studies, preincubation with 1 nM IGF-I had no effect on type I IGF receptor binding in normal HF and SV40-HF. In contrast, preincubation of cells with an equivalent concentration of [QAYL]IGF-I downregulated the receptors 40 50%. Changes in cell surface receptor number were reflected in cell responsiveness to IGF-I-stimulated [3H]thymidine incorporation and [3H]aminoisobutyric acid uptake. In conclusion, IGF-I regulates the availability of specific IGFBPs in cultured human fibroblasts by a novel receptor-independent mechanism. Rapid changes in levels of soluble IGFBPs as a direct response to extracellular IGF-I, in turn, modulate IGF-I peptide and receptor interaction, and may constitute an important level of control in IGF cellular physiology. PMID- 1717511 TI - LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase. AB - LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA in 17 of 20 LKM-1 sera tested. An immobilized, synthetic peptide, DPAQPPRDC, was used to purify LKM-1 antibodies. Affinity purified LKM-1 autoantibodies react on immunoblots with a protein in BHK cells after infection with HSV-1. 11 of 24 LKM-1 sera, including 3 that recognize DPAQPPRD, also exhibit antibodies to the hepatitis C virus (HCV) protein, C100-3. Affinity purified LKM-1 antibodies did not recognize C100-3. However, partial sequence identity was evident between portions of the immunopositive 33-amino acid segment of P450IID6 and other portions of the putative HCV polyprotein. Immune cross-recognition of P450IID6 and HCV or HSV-1 proteins may contribute to the occurrence of LKM-1 autoantibodies. PMID- 1717512 TI - Identification and partial characterization of angiogenesis bioactivity in the lower respiratory tract after acute lung injury. AB - Survival after acute lung injury (ALI) depends on prompt alveolar repair, a process frequently subverted by the development of granulation tissue within the alveolar airspace. Immunohistochemical examination of the intraalveolar granulation tissue confirmed that capillaries as well as myofibroblasts were the principal cellular constituents. We therefore hypothesized that angiogenesis factors would be present on the air-lung interface after ALI. To evaluate this hypothesis, bronchoalveolar lavage fluid from patients with ALI (n = 25) and patient controls (n = 8) was examined for angiogenesis bioactivity by its ability of induce endothelial cell migration. While lavage fluid from controls had no bioactivity, lavage fluid from 72% of patients with ALI promoted endothelial cell migration. Heparin affinity, ion exchange, and gel filtration chromatography resolved the bioactivity into at least two moieties. One appeared identical to the well characterized endothelial cell growth factor, basic fibroblast growth factor. The other was a 150-kD non-heparin binding protein that mediated endothelial cell migration and attachment in vitro, and the growth of new vessels in vivo. These data are consistent with the hypothesis that the growth of capillaries into the alveolar airspace results from angiogenesis factors present on the alveolar surface of the lung after ALI. PMID- 1717513 TI - Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. AB - Two murine monoclonal antibodies (CL-3 and CL-37, both F(ab')2) to human endothelial-leukocyte adhesion molecule-1 (ELAM-1) were found to react immunohistochemically with rat pulmonary artery endothelial cells that had been pretreated with tumor necrosis factor (TNF alpha). CL-3, but not CL-37, blocked in vitro adherence of neutrophils to TNF alpha-treated endothelial cells and the killing of TNF alpha-treated rat endothelial cells by phorbol ester activated neutrophils. In rats treated systemically with CL-3, there was a 70% reduction in accumulation of neutrophils in glycogen-induced peritoneal exudates. Treatment of animals with CL-37 anti-ELAM-1 did not reduce neutrophil accumulation under the same conditions. When IgG immune complex deposition was induced in dermis and in lungs of rats, treatment with CL-3 anti-ELAM-1 markedly reduced vascular injury as measured by changes in vascular permeability (leakage of 125I-albumin) and hemorrhage (extravasation of 51Cr-red blood cells). The protective effects of CL 3 anti-ELAM-1 were related to greatly diminished recruitment of neutrophils (as assessed morphologically, by tissue extraction of myeloperoxidase, and by retrieval, via bronchoalveolar lavage, of neutrophils from lung). CL-37 had no protective effects in vivo after deposition of immune complexes in lung. Using either CL-3 or CL-37 anti-ELAM-1, immunohistochemical analysis of lungs undergoing IgG immune complex-induced injury revealed a striking upregulation of ELAM-1 in the lung vasculature (venules and interstitial capillaries), with a peak intensity developing between 3 and 4 h after deposition of immune complexes in lung. Vascular beds of spleen, liver, and kidney failed to show upregulation of ELAM-1 under these same conditions. The immunohistochemical reactivity of rat lung was abolished if the anti-ELAM-1 preparation was first absorbed with monolayers of human umbilical vein endothelial cells that had been pretreated with TNF alpha. Untreated human endothelial cells failed to cause loss of lung reactivity of the anti-ELAM-1 preparation. These data indicate that ELAM-1 is upregulated in the pulmonary vasculature of rats during deposition of immune complexes and that ELAM-1 appears to play an obligate role in the recruitment of neutrophils. PMID- 1717514 TI - Endothelial leukocyte adhesion molecule-1 mediates antigen-induced acute airway inflammation and late-phase airway obstruction in monkeys. AB - This study examines the role of endothelial leukocyte adhesion molecule-1 (ELAM 1) in the development of the acute airway inflammation (cell influx) and late phase airway obstruction in a primate model of extrinsic asthma. In animals sensitive to antigen, a single inhalation exposure induced the rapid expression of ELAM-1 (6 h) exclusively on vascular endothelium that correlated with the influx of neutrophils into the lungs and the onset of late-phase airway obstruction. In contrast, basal levels of ICAM-1 was constitutively expressed on vascular endothelium and airway epithelium before antigen challenge. After the single antigen exposure, changes in ICAM-1 expression did not correlate with neutrophil influx or the change in airway caliber. This was confirmed by showing that pretreatment with a monoclonal antibody to ICAM-1 did not inhibit the acute influx of neutrophils associated with late-phase airway obstruction, whereas a monoclonal antibody to ELAM-1 blocked both the influx of neutrophils and the late phase airway obstruction. This study demonstrates a functional role for ELAM-1 in the development of acute airway inflammation in vivo. We conclude that, in primates, the late-phase response is the result of an ELAM-1 dependent influx of neutrophils. Therefore, the regulation of ELAM-1 expression may provide a novel approach to controlling the acute inflammatory response, and thereby, affecting airway function associated with inflammatory disorders, including asthma. PMID- 1717515 TI - Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel. AB - Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation. PMID- 1717516 TI - Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat. AB - Previous anatomical and physiological studies have revealed a substantial projection from the periaqueductal gray (PAG) to the nucleus paragigantocellularis (PGi). In addition, physiological studies have indicated that the PAG is composed of functionally distinct subregions. However, projections from PAG subregions to PGi have not been comprehensively examined. In the present study, we sought to examine possible topographic specificity for projections from subregions of the PAG to PGi. Pressure or iontophoretic injections of wheat germ agglutinin-conjugated horseradish peroxidase, or of Fluoro-Gold, placed into the PGi of the rat retrogradely labeled a substantial number of neurons in the PAG from the level of the Edinger-Westphal nucleus to the caudal midbrain. Retrogradely labeled neurons were preferentially aggregated in distinct subregions of the PAG. Rostrally, at the level of the oculomotor nucleus, labeled neurons were i) compactly aggregated in the ventromedial portion of the PAG corresponding closely to the supraoculomotor nucleus of the central gray, ii) in the lateral and ventrolateral PAG, and iii) in medial dorsal PAG. More caudally, retrogradely labeled neurons became less numerous in the dorsomedial PAG but were more widely scattered throughout the lateral and ventrolateral parts of the PAG. Only few retrogradely labeled neurons were found in the ventromedial part of the PAG at caudal levels. Injections of retrograde tracers restricted to subregions of the PGi suggested topography for afferents from the PAG. Injections into the lateral portion of the PGi yielded the greatest number of labeled neurons within the rostral ventromedial PAG. Medially placed injections yielded numerous retrogradely labeled neurons in the lateral and ventrolateral PAG. Injections placed in the rostral pole of the PGi (medial to the facial nucleus) produced the greatest number of retrogradely labeled neurons in the dorsal PAG. To examine the pathways taken by fibers projecting from PAG neurons to the medulla, and to further specify the topography for the terminations of these afferents in the PGi, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoretically deposited into subregions of the PAG that contained retrogradely labeled neurons in the above experiments. These results revealed distinct fiber pathways to the rostral medulla that arise from the dorsal, lateral/ventrolateral, and ventromedial parts of the PAG. These injections also showed that there are differential but overlapping innervation patterns within the PGi. Consistent with the retrograde tracing results, injections into the rostral ventromedial PAG near the supraoculomotor nucleus yielded anterograde labeling immediately ventral to the nucleus ambiguus in the ventrolateral medulla, within the retrofacial portion of the PGi.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717517 TI - Ultrastructural single- and double-label immunohistochemical studies of substance P-containing terminals and dopaminergic neurons in the substantia nigra in pigeons. AB - The vast majority of striatonigral projection neurons in pigeons contain substance P (SP), and the vast majority of SP-containing fibers terminating in the substantia nigra arise from neurons in the striatum. To help clarify the role of striatonigral projection neurons, we conducted electron microscopic single- and double-label immunohistochemical studies of SP+ terminals and/or dopaminergic neurons (labeled with either anti-dopamine, DA, or anti-tyrosine hydroxylase, TH) in pigeons to determine: (1) the synaptic organization of SP+ terminals, (2) the synaptic organization of TH+ perikarya and/or dendrites, and (3) the synaptic relationship between SP+ terminals and TH+ neurons in the substantia nigra. Tissue single-labeled for SP revealed numerous SP+ terminals contacting thin unlabeled dendrites in the substantia nigra, but few SP+ terminals were observed contacting perikarya or large-diameter dendrites. SP+ terminals contained round, densely packed, clear vesicles, and often contained one or more dense-core vesicles. Synaptic junctions between SP+ terminals and their targets were more often symmetric (86%) than asymmetric. In tissue single-labeled for DA, we observed few terminals contacting DA+ perikarya, whereas terminals contacting DA+ dendrites were more abundant. Terminals contacting DA+ structures comprised at least four different morphologically distinct types based on the morphology of the clear synaptic vesicles and the type of synaptic junction. One type of terminal contained round clear vesicles and made symmetric synapses, and thus resembled the predominant type of SP+ terminal. The second type contained round clear vesicles and made asymmetric synapses, the third type contained medium-size pleomorphic clear vesicles and made symmetric synapses, and the fourth type contained small pleomorphic clear vesicles and made symmetric synapses. The presence of contacts between SP+ terminals and dopaminergic dendrites in the substantia nigra was directly demonstrated in tissue double-labeled for SP (by the peroxidase-antiperoxidase procedure, or PAP, with diaminobenzidine) and TH (by either the silver-intensified immunogold procedure or the PAP procedure with benzidine dihydrochloride). SP+ terminals commonly contacted thin TH+ dendrites in the substantia nigra, but few SP+ terminals contacted large-diameter TH+ dendrites or perikarya. Synapses between SP+ terminals and TH+ neurons were always symmetric. TH+ dendrites also were contacted by terminals not labeled for SP, which were more abundant than were SP+ terminals. Non-TH+ neurons were also contacted by both SP+ terminals and non-SP+ terminals.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717518 TI - Gracile projection to the cat medial accessory olive: ultrastructural termination patterns and convergence with spino-olivary projection. AB - The caudal medial accessory subdivision of the inferior olive (cMAO) receives information from the hindlimb from both the gracile nucleus and the lumbosacral spinal cord. This study determined which elements in cMAO serve as the postsynaptic targets of the gracile projection and whether these elements also receive input from the lumbosacral spinal cord. Gracile axons were labeled in cats by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), visualized with tetramethylbenzidine. Convergence of gracile and lumbosacral axons was evaluated by labeling in the same animal, one pathway by WGA-HRP and the other by degeneration. In cMAO, gracile axons synapse with equal probability on dendritic spines and distal dendritic shafts. This termination pattern contrasts markedly with that of other somatosensory inputs to the inferior olive and may account for the greater heterogeneity in responses to somatosensory stimuli displayed by neurons in cMAO. The distal dendritic shafts receiving gracile input were more likely than dendritic spines to receive convergent input from putative inhibitory synapses. The most likely source of these inhibitory synapses is the parasolitary nucleus, a structure that has been shown by others to receive input from the cerebellum. Thus the parasolitary nucleus may serve as an inhibitory relay between the cerebellum and cMAO. The dendritic spines in cMAO that receive input from the gracile nucleus often receive additional input from the lumbosacral spinal cord. This convergence of somatosensory axons on dendritic spines may provide a mechanism through which the unusually complex receptive fields of neurons in cMAO are generated. PMID- 1717519 TI - Single dopaminergic nigrostriatal neurons form two chemically distinct synaptic types: possible transmitter segregation within neurons. AB - These experiments were designed to examine a paradox present in the literature with regard to the fine structure of nigrostriatal dopamine terminals within the rat striatum. Previous studies have shown that anterograde transport of tritiated labeled proteins from the substantia nigra to the striatum over short survival times primarily labels asymmetric synapses (and that these asymmetric synapses are preferentially vulnerable to selective dopaminergic neurotoxins such as 6 hydroxydopamine). In contrast, fine structural immunohistochemical studies with antibodies to tyrosine hydroxylase and dopamine have consistently labeled primarily symmetric synapses en passant within the striatum. We have now confirmed that these two seemingly contradictory types of labeled synapses (radio and immuno-labeled) can both be present, but most often separate from one another, in single ultrathin sections. However, we also found that radiolabeled unmyelinated axons were usually double-labeled by tyrosine hydroxylase immunohistochemistry. Employing longer survival times (10 days after the nigral isotope injections) in order to enhance the ratio of "en passant" to terminal labeling produced a large increase in the occurrence of radiolabeled striatal axonal varicosities with the result that many symmetric synapses en passant were double-labeled with both the autoradiographic and the immunohistochemical markers. Given that more than 95% of the nigrostriatal projection arises from dopamine fluorescent neurons, it would appear that both the asymmetric and symmetric terminals belong to the same type of neuron. Thus, we suggest that single dopaminergic neurons in the substantia nigra make two types of synaptic contact with striatal cells: 1) symmetric synapses en passant, which can be stained with tyrosine hydroxylase and dopamine and which contact dendritic spine necks, and 2) asymmetric terminal boutons of unknown chemical nature which end on dendritic spine heads. We conclude that both the asymmetric terminal and symmetric en passant synapses take origin from a single nigrostriatal dopaminergic neuronal population and that dopaminergic transmitter markers occur only in one of these synaptic types in the rat striatum. PMID- 1717520 TI - Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat. AB - The motor innervation for palatal, pharyngeal, laryngeal, and esophageal muscles originates within the nucleus ambiguus. Although the viscerotopic organization of the upper alimentary tract in the nucleus ambiguus has been extensively studied, little information concerning the dendritic arborization of nucleus ambiguus motoneurons is available. The neural tracer cholera toxin-horseradish peroxidase, which is particularly effective at revealing dendrites of retrogradely labeled neurons, was used to determine the dendritic architecture and organization of nucleus ambiguus motoneurons. In 72 rats, cholera toxin-horseradish peroxidase in volumes of 1.0-18 microliters was directly applied under pressure to the musculature of various sites along the upper alimentary tract. Motoneurons innervating the soft palate, pharynx, cricothyroid muscle, and cervical esophagus were all found to have extensive dendrites that extended into the adjacent reticular formation with a distinct pattern for each muscle group. In contrast, the dendrites of motoneurons innervating the thoracic and subdiaphragmatic esophagus were confined to the compact formation of the nucleus ambiguus. Dendritic bundling within the confines of the nucleus ambiguus was prominent following injection of tracer into the soft palate, pharynx, and esophagus. The bundles were primarily oriented in a rostrocaudal direction. These data suggest that the extensive extranuclear dendritic arborization of motoneurons innervating the soft palate, pharynx, larynx, and cervical esophagus provide a wide ranging target for multiple central afferents that may be involved in the differential control of muscles that participate in multiple complex motor functions. The lack of extensive extranuclear dendrites of motoneurons innervating the distal esophagus suggest that they receive focused central afferents. The prominent bundling of dendrites within the nucleus ambiguus may provide for synchronization of motoneurons innervating a specific muscle and perhaps for synchronization of motoneurons innervating different muscles acting in sequence. PMID- 1717521 TI - Neuropeptide Y (NPY)-immunoreactive neurons in the primate fascia dentata; occasional coexistence with calcium-binding proteins: a light and electron microscopic study. AB - Neuropeptide Y (NPY)-containing neurons are known to be highly vulnerable following sustained electrical stimulation in rats and in humans suffering from temporal lobe epilepsy. This has been related to a strong excitatory input. In contrast, there is evidence that neurons containing calcium-binding proteins exhibit a high resistance under experimental seizure and hypoxia conditions. The aim of this study was to determine the coexistence of NPY and calcium-binding proteins in inhibitory neurons of the primate fascia dentata and their synaptic connections. Vibratome sections of hippocampi of African green monkeys (Cercopithecus aethiops) were immunostained with antibodies against NPY, PARV, and CB. A quantitative coexistence study was performed for NPY and PARV on consecutive semithin sections. In contrast to the rodent hippocampus, NPY immunoreactive neurons were found exclusively in the hilus of fascia dentata with horizontally oriented dendrites which did not extend into the granular and molecular layer. Conversely, PARV-immunoreactive neurons were also present in the granular and inner molecular layer and extended their dendrites far out in the molecular layer and the hilus. Axon terminals immunoreactive for NPY were mostly concentrated in the middle and outer molecular layer and the hilar region and were rare in the granular layer. PARV-immunoreactive boutons were basically restricted to the granular layer where they formed typical baskets. The antibody against calbindin stained almost exclusively granule cells. Coexistence of NPY- and PARV-immunoreactivity was found only in hilar neurons and was rare (9 out of 152 cells analyzed). These results suggest that most NPY-immunoreactive neurons do not contain calcium-binding proteins. NPY-containing neurons exhibited ultrastructural characteristics as described for inhibitory neurons. Their dendrites were only sparsely contacted by mostly asymmetric synaptic terminals, including a very small number of mossy fiber axon terminals. In turn, numerous NPY-immunoreactive axon terminals formed symmetric synapses with spines and dendritic shafts of unlabeled neurons in the middle and outer molecular layer, whereas no contact with granule cell bodies was evident. Thus, we conclude that the vulnerability of NPY-containing inhibitory neurons may be due more to the lack of calcium-binding proteins than to a strong excitatory innervation. As their axons may contribute to the inhibitory control of the major excitatory input from the entorhinal cortex, their loss following overstimulation may play a role in perpetuating hippocampal seizure activity. PMID- 1717522 TI - Peptidergic and serotoninergic innervation of the rat dura mater. AB - The peptidergic and serotoninergic innervation of the rat dura mater was investigated by reacting dural wholemounts immunohistochemically with antibodies to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and serotonin (5-HT). CGRP and SP innervations of the dura were robust and the patterns of distribution of these neuropeptides were essentially the same. The majority of the fibers were perivascular and distributed to branches of the anterior and middle meningeal arteries and to the superior sagittal and transverse sinuses. Other CGRP/SP fibers appeared to end "free" within the dural connective tissue. NPY immunoreactive fibers were extremely numerous and also distributed heavily to the branches of the meningeal arteries, the venous sinuses, and to the dural connective tissue. The pattern of NPY innervation resembled in many ways that of CGRP/SP; however, NPY innervation of the sinuses was greater and NPY perivascular fibers supplying the meningeal arteries formed more intimate contacts with the walls of the vessels. The pattern of VIP innervation was, in general, similar to that observed for the three previous neuropeptides; however, the overall density was considerably less. Small to moderate numbers of serotoninergic nerve fibers were observed in some, but not all, of the duras processed for 5-HT. The latter fibers were almost exclusively perivascular in distribution. Dural mast cells were prominently stained in the 5-HT preparations because of their serotonin content. Mast cells were also labeled in a nonspecific fashion in some of the tissues reacted immunohistochemically for neuropeptides; some of them were located in close apposition to passing nerve fibers. This study represents, to our knowledge, the first comprehensive work on the peptidergic and serotoninergic innervation of the mammalian dura mater. The results should increase our understanding of the roles that these fibers play in normal dural physiology and of their potential interactions in the pathogenesis of vascular headache. PMID- 1717523 TI - Immunohistology of lymph nodes draining local skin reactions (chancres) in sheep infected with Trypanosoma congolense. AB - Marked enlargement of lymph nodes draining local skin reactions (chancres) occurred in sheep following intradermal inoculation of cultured metacyclic forms of Trypanosoma congolense. Histologically, these lymph nodes were characterized by follicular hypertrophy and hyperplasia, compression and relative reduction of the paracortical areas and expansion of the medullary regions. Immunohistochemical staining with monoclonal antibodies to ovine lymphocyte subsets and Fc receptor (FcR) bearing macrophages, revealed increased expression of B cells (CD45R+), major histocompatibility complex (MHC) Class II, FcR+ macrophages, and CD1+ cells in the cortical and paracortical areas. The paracortical areas were found to be sparsely populated by CD5+, CD4+ and CD8+ cells, while the medullary areas contained numerous CD8+ cells and FcR+ macrophages. FcR+ macrophages were also present in cortical trabecular and subcapsular sinuses. As the chancre regressed, lymph node reactivity also subsided and fewer B cell follicles were observed and there was decreased expression of CD45R+ and MHC Class II+ cells. PMID- 1717524 TI - A subpopulation of Langerhans cells (CD1a+Lag-) increased in the dermis of plaque lesions of mycosis fungoides. AB - The population of CD1a+ cells and the quantity of Birbeck granules were evaluated in comparison with the population of T lymphocytes in a variety of clinical lesions of mycosis fungoides. Anti-CD1a and Lag antibodies that specifically react with Birbeck granules and related structures of human Langerhans cells were used immunohistochemically. CD1a+ cells in the dermis of lesions of mycosis fungoides significantly increased in plaques of the plaque stage and in plaques of the tumor stage. They were most frequent in lesions with CD4+ cells ranging in number from 100 to 150/mm2. These lesions were suspected to be progressing from the plaque to the tumor stage. During the course of the disease, most of the dermal CD1a+ cells had few Lag antigens. These results suggest that dermal CD1a+Lag- cells may promote the progression of mycosis fungoides from the plaque to the tumor stage. PMID- 1717525 TI - Bleomycin-lidocaine mixture reduces pain of intralesional injection in the treatment of recalcitrant verrucae. AB - Intralesional bleomycin has been effective treatment of recalcitrant verrucae since 1970, but one major drawback is the moderate to severe pain associated with the injection. To minimize procedural discomfort, bleomycin can be reconstituted in lidocaine. It is effective, associated with minimal morbidity, and well tolerated by most patients. PMID- 1717526 TI - Sweet's syndrome: pathogenesis and associated conditions. PMID- 1717527 TI - Keratinocyte expression of CD36 antigen in benign and malignant epidermal cell derived tumours. AB - Keratinocytes express the macrophage/monocyte antigen CD36 in a variety of inflammatory cutaneous diseases characterised by a T lymphocyte rich infiltrate. Since cell-mediated immune mechanisms also play a role in host responses to skin tumours, we investigated the presence of CD36 antigen on keratinocytes in a range of epidermal cell-derived benign and malignant tumours characterised by a peritumoural, dermal lymphocytic infiltrate. Frozen tissue sections of lesional tissue from a range of epidermally derived tumours were labelled with antibodies to CD1a, CD11b, CD36, and HLA-DR antigens. Benign and malignant squamoproliferative tumour cells exhibited a spectrum of CD36 expression, whereas those of basal cell origin were consistently CD36-. Suprabasal expression of CD36 was present in the normal perilesional epidermis of all tumours studied including basal cell carcinoma. Keratinocyte CD11b expression was not observed. The widespread presence of keratinocyte CD36 positivity in squamoproliferative, but not basal epidermal, tumours suggests its expression may be linked to the degree of keratinocyte differentiation. The stimulus for expression is unknown, but the fact that suprabasal perilesional epidermis expressed CD36 strongly in the absence of infiltrate suggests it may represent a non-specific response by keratinocytes to various stimuli. PMID- 1717528 TI - Effects of granulocyte colony-stimulating factor administration to periparturient cows on neutrophils and bacterial shedding. AB - Administration of recombinant bovine granulocyte colony stimulatory factor to periparturient dairy cows was evaluated as a method to prevent periparturient immunosuppression. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were randomly assigned to four groups in a 2 x 2 factorial design to evaluate the effects of placebo or recombinant bovine granulocyte colony stimulatory factor administration on chronic, subclinically infected and uninfected cows during the periparturient period. Blood neutrophils were isolated and evaluated for phagocytic activities 5 wk before expected parturition through 7 wk postpartum. Administration of recombinant bovine granulocyte colony stimulatory factor (5 micrograms/kg body weight or placebo subcutaneously beginning 14 d prepartum through 10 d post-partum) resulted in a prepartum and postpartum leukocytosis of 35,600/microliters and 53,500/microliters, respectively. This was attributed to a mature neutrophilia of 24,010/microliters during prepartum and 38,080/microliters during postpartum treatment periods (pretreatment baseline = 2330/microliters). Mononuclear cell counts averaged 7610/microliters during prepartum and 9830/microliters during postpartum treatment periods (baseline = 3450/microliters). Neutrophil random and directed migration were reduced during recombinant bovine granulocyte colony stimulatory factor treatment compared with placebo or baseline levels. Ingestion of bacteria and cytotoxicity by neutrophils was increased during recombinant bovine granulocyte colony stimulatory factor therapy compared with placebo or baseline levels. Shedding of S. aureus in lacteal secretions was unaffected by recombinant bovine granulocyte colony stimulatory factor treatment. In summary, administration of recombinant bovine granulocyte colony stimulatory factor increased the number and functional activity of neutrophils and prevented some aspects of periparturient immunosuppression in dairy cows. PMID- 1717529 TI - Expression of OKM5 antigen on keratinocytes in some dermatoses. AB - Employing an avidin-biotin complex immunoperoxidase technique, it was found that there was OKM5 but no OKM1 antigen expression on the keratinocytes of the upper epidermis from all the studied specimens of seborrheic keratosis, verrucous nevus, Bowen's disease, BCC, SCC, MM, lichen planus, psoriasis vulgaris, condyloma acuminatum, and sporotrichosis as well as in two of eight cases of nevus pigmentosus. Our findings indicate that OKM5 antigen expression on epidermal keratinocytes is usually associated with dermal infiltration. It is assumed that human keratinocytes in cutaneous lesions, like the OKM1- OKM5+ monocyte/macrophage lineages, might play certain roles in immune responses and possibly function as antigen-presenting cells. PMID- 1717530 TI - Intravenous immunoglobulin: a new therapeutic approach in steroid-dependent asthma? PMID- 1717531 TI - Common epitopes of birch pollen and apples--studies by western and northern blot. AB - Eighty-three sera from patients with birch-pollen allergy were investigated for IgE antibodies against apple allergens by means of immunoblotting. In immunoblots, 81 patients (97.6%) exhibited IgE directed against the major allergen of birch, Bet v I (17 kd), and these patients also demonstrated IgE binding to apple allergens in the molecular weight range 17 to 18 kd. Inhibition studies by preincubation of sera with birch-pollen extract led to complete blocking of IgE binding to this 17 to 18 kd protein, whereas preincubation with apple extract could not diminish IgE binding to Bet V I. Furthermore, a 17 kd protein in apple extract could be detected by immunoblotting with a Bet v I specific monoclonal antibody. Northern blotting with a Bet v I cDNA clone as a probe revealed cross-hybridization of birch and apple allergen coding nucleic acids under conditions of high stringency, suggesting significant homology of the nucleic acid level. Our results support the concept that antigens in birch pollen and apples share allergenic epitopes leading to IgE cross-reactivities that may cause clinical manifestations when a special threshold level of specific IgE antibodies is reached. PMID- 1717532 TI - Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. AB - To assess whether mast cell and eosinophil (EOS) degranulation occurs in the airway of subjects with moderately symptomatic asthma, we have measured levels of preformed mast cell-derived mediators (histamine and tryptase) and EOS-derived mediators (major basic protein and EOS-derived neurotoxin) in bronchoalveolar lavage fluid (BALF) obtained from patients with symptomatic (N = 14) and asymptomatic asthma (N = 9) and patients without asthma (N = 6). Both the FEV1 (1.52 +/- 0.33 L:55% +/- 15% of predicted FEV1) and the forced expiratory flow at 50% (FEF50) (1.11 +/- 0.62 L/sec:26% +/- 14% of predicted FEF50) in the patients with symptomatic asthma were significantly lower than the corresponding values for FEV1 (3.16 +/- 0.45 L:86% +/- 10% of predicted FEV1) and the FEF50 (4.04 +/- 1.54 L/sec:71% +/- 25% of predicted FEF50) in the patients with asymptomatic asthma. Levels of histamine (4.8 +/- 5.0 ng/ml versus 0.2 +/- 0.2 ng/ml) (p = 0.05), EOS-derived neurotoxin (420.6 +/- 959.4 ng/ml versus 12.6 +/- 7.7 ng/ml) (p = 0.05), major basic protein (31.4 +/- 46.6 ng/ml versus less than 9 ng/ml) (p = 0.05), and percent EOSs (10.6% +/- 7.0% versus 1.1% +/- 0.9% of BAL cells) (p = 0.0006) were all significantly elevated in BALF from symptomatic compared to asymptomatic patients with asthma. The elevated levels of tryptase (13.2 +/- 14.8 ng/ml versus 3.9 +/- 3.9 ng/ml) in BALF from symptomatic compared to asymptomatic patients with asthma approximated, but did not reach, statistical significance. Spontaneous histamine release from BAL mast cells of symptomatic patients with asthma was 46% +/- 5% compared to 5% +/- 2% in asymptomatic patients with asthma. In response to antihuman IgE, histamine release from BAL mast cells recovered from asymptomatic patients with asthma increased to 25% +/- 10%, whereas in BAL mast cells of symptomatic patients with asthma, no anti-IgE potentiation of histamine release occurred. This study suggests that mast cell and EOS degranulation is ongoing in the airway of patients with moderately symptomatic asthma. PMID- 1717533 TI - Novel solid-phase synthesis of thiol-terminated-poly(alpha-amino acid)-drug conjugate. AB - A new method using a controlled pore glass solid support for the preparation of a thiol-terminated-polymerdrug, notably poly-L-glutamate-daunomycin having a terminal thiol group, is described. The method consists of first polymerizing an ester-protected glutamic acid onto an amino-disulfide functionalized controlled pore glass support. The ester protecting group is then removed, freeing the gamma carboxyl groups of the grafted polymer which then allows it to react with daunomycin. Finally, the disulfide bond linking the conjugated polymer-drug to the solid support is broken by thiolysis, thus releasing the desired product. The final product consists of only polymer-drug conjugates with terminal thiol groups (global yield 26%). This novel method is much simpler and more elegant than more conventional preparation methods requiring solution phase techniques. PMID- 1717534 TI - Preganglionic and sensory axons in developing bullfrog sympathetic ganglia express three neuropeptides during early tadpole stages. AB - Retrograde tracing in fixed tissue with the fluorescent carbocyanine dye, DiI, was used to identify neurons that project to paravertebral sympathetic ganglia 9 and 10 in bullfrog tadpoles. Applying DiI to ganglion 9 at stage II labelled spinal preganglionic neurons and sensory neurons in dorsal root ganglia. When examined in a stage V tadpole, the segmental boundaries of the preganglionic cell column which supply the lumbar chain ganglia were identical to that in the adult. Using immunocytochemistry, luteinizing hormone releasing hormone-like immunoreactivity, calcitonin gene-related peptide-like immunoreactivity, and substance P-like immunoreactivity were localized at stage III in axons within sympathetic ganglia 9 and 10. During subsequent stages, the density of fibers containing these peptides increased and it became easier to identify synaptic boutons in contact with postganglionic neurons. These observations demonstrate that projections to the lumbar sympathetic ganglia are already formed by the earliest tadpole stages, they are consistent with the previous physiological observation of nicotinic synapses in stage III ganglia, and they suggest that neuropeptide function may also begin during early stages. PMID- 1717535 TI - Effect of neuropeptide-Y and galanin on autonomic control of heart rate in the toad, Bufo marinus. AB - The effects of neuropeptide-Y (NPY) and galanin (GAL) on the autonomic control of heart rate were investigated in the anaesthetised toad, Bufo marinus. Both vagosympathetic trunks were sectioned to prevent reflex changes in heart rate, and the cardiac responses to electrical stimulation of either the vagal or sympathetic fibres to the heart assessed. Intravenous, bolus doses of 10 or 20 micrograms (2 or 4 nmol) NPY and 5 or 10 micrograms (1.5 or 3 nmol) GAL caused pronounced pressor responses but small direct changes in heart rate. Pulse intervals measured after peptide administration were within 5% of control values. All doses of both peptides caused inhibition of action of the cardiac vagus nerves, the maximum inhibition observed in response to 20 micrograms NPY: mean 49.5 +/- 14% (SEM). No significant changes in cardiac sympathetic nerve action were observed. It is concluded that NPY and GAL have similar, important cardiovascular actions in the toad. Similarities between the responses of toads and mammals to NPY suggest a phylogenetic conservation of function for this peptide. PMID- 1717536 TI - The release of neuronal 5-HT from the intestine of a teleost fish, Platycephalus bassensis. AB - The superfused, isolated intestine of a teleost fish which lacks enterochromaffin cells spontaneously released 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), presumably from enteric neurons. The release of 5-HT, but not 5 HIAA, increased on transmural electrical stimulation. Addition of tetrodotoxin or omission of Ca2+ from the superfusate prevented the increase in 5-HT release on electrical stimulation. Fluoxetine added to the superfusate increased the amount of 5-HT released spontaneously but also prevented the increase in 5-HT release on stimulation. Pretreatment of fish with reserpine markedly reduced tissue levels of 5-HT and 5-HIAA and led to an almost complete loss of the spontaneous release of 5-HT and an elimination of the stimulated release of 5-HT. PMID- 1717537 TI - Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla. AB - The adrenal medulla is innervated by both cholinergic and substance P (SP) containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known. In the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 +/- 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 +/- 19 in T4 to 372 +/- 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 +/- 26, distributed normally about a peak at T8 (33.8 +/- 6.3 cells) and T9 (33.3 +/- 6.8 cells) with the least at T3 (1.5 +/- 0.8) and T13 (5.2 +/- 2.0). No Fast blue-labelled cells were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelled cells were distributed from T7 to T9. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats. PMID- 1717538 TI - Viscerotopic representation of preganglionic efferent vagus nerve in the brainstem of the rat: a Fluoro-Gold study. AB - To investigate the viscerotopic distribution of the cells of origin of preganglionic vagus nerve in rats, Fluoro-Gold was injected into various visceral tissues. After injections into the gastroesophageal junction and the gastric corpus, labelled cells were localized in the medial half of the dorsal motor nucleus of the vagus (dmnX). Cells in the nucleus ambiguous (nA) were also labelled after injections into the gastroesophageal junction. After injections into the pancreatic head and the celiac plexus, labelled cells were located bilaterally in the lateral part of the caudal dmnX. In the rostral dmnX, however, the pancreatic head was represented in the medial segment. After injections into the lung, duodenum, liver and ascending colon, no labelling was observed in the brainstem. PMID- 1717539 TI - Serum amylase determination in the emergency department evaluation of abdominal pain. AB - We hypothesized that selective ordering of serum amylase in the emergency department (ED) is justified because (a) most patients with elevated amylase can be prospectively identified by characteristic clinical findings, and (b) the diagnosis of pancreatitis is usually predominantly based on clinical findings, since amylase is known to be neither sensitive nor specific for pancreatitis. The study population included 133 consecutive patients with a chief complaint of abdominal pain who had amylase drawn over a 2-week period at a university hospital ED. Patients with known major trauma were excluded. Emergency department and hospital charts were reviewed for selected clinical variables. The first part of our hypothesis was evaluated by comparing clinical characteristics of cases (elevated amylase) and controls; the second part was tested by comparing clinical findings and amylase in cases (patients diagnosed as having pancreatitis) and controls. We found that 17 patients with and 116 without elevated amylase were similar with regard to all clinical variables, and that no combination of findings could be used to predict elevated amylase. Amylase level was not predictive of an ultimate diagnosis of pancreatitis, which was, however, strongly related to classical clinical findings. Pancreatitis risk factors, epigastric pain and tenderness, radiation of pain to the back, and nausea and vomiting were each statistically more common in patients diagnosed as having pancreatitis (regardless of amylase) than in patients in whom pancreatitis was excluded despite elevated amylase; all patients diagnosed with pancreatitis had at least two of these. Thus, selective ordering of amylase on the basis of clinical characteristics fails to identify a large proportion of patients with elevated amylase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717540 TI - Serum lipase levels in chronic alcoholics. AB - Using an elevated serum amylase level to diagnose acute pancreatitis in an alcoholic patient with abdominal pain may not be appropriate, because hyperamylesemia is common in asymptomatic alcoholics without acute pancreatitis. To determine whether serum lipase also suffers from the same drawback, we undertook a prospective study involving 202 asymptomatic alcoholics admitted to the detoxification unit of our hospital. Sixty-six of the 202 patients had serum lipase levels above the normal range (0-213 U/L). Of these 66, 55 (83%) had levels that were one to two times normal, while 11 patients had levels ranging between two and three times normal. No patient exceeded three times the normal level. This background information is important in the interpretation of serum lipase levels in alcoholic patients with abdominal pain. PMID- 1717541 TI - Plasma membrane of Leishmania donovani. AB - Plasma membrane of Leishmania donovani promastigotes was isolated by disrupting the cells in Dounce homogenizer and found to be having two fractions M1 and M2. Chemical analysis of the two membrane fractions revealed that M1 had less RNA content and high sterol-phospholipid molar ratio than M2. M1 was also rich in membrane marker enzymes, e.g., 5' nucleotidase and acid phosphatase. Glucose-6 phosphatase, the marker enzyme of endoplasmic reticulum was higher in M2 fraction. The electron micrograph also revealed the presence of plasma membrane vesicles in M1 fraction. PMID- 1717542 TI - The diagnostic value of the assay of des-gamma-carboxy prothrombin in the detection of small hepatocellular carcinoma. AB - Des-gamma-carboxy prothrombin (DCP) assay was performed by a staphylocoagulase method in 35 consecutive patients with small (less than 5 cm), resectable hepatocellular carcinoma (HCC). They also simultaneously received serum alpha fetoprotein (AFP) assay. According to diagnostic strategy, patients were divided into two groups. Group I consisted of eight patients who were candidates for a mass screening project for HCC with elevated AFP levels (greater than 20 ng/ml). Five of these patients had an increased DCP level (greater than 6 U/l). Group II included 27 victims of chronic hepatitis B or cirrhosis whose tumors were detected by ultrasonography during regular follow-up. In this group, increased DCP and AFP levels were observed in 11 and 16 cases, respectively. Of 14 patients with smaller HCC (less than 3 cm), only three had elevated DCP levels, while eight patients had an abnormal AFP level. When these two assays were combined, 18 of 27 patients in group II and nine of 14 patients with smaller HCC (less than 3 cm) revealed elevation of one or both of the two markers. A total of 16 out of 35 patients with small HCC had abnormal DCP levels. In conclusion, DCP assay is less sensitive than AFP assay in the detection of small HCC, and the combination of both markers has little complementary effect. PMID- 1717543 TI - Is autoimmune chronic active hepatitis a HCV-related disease? AB - We evaluated the specificity and clinical relevance of anti-hepatitis C virus antibody positivity in 22 HBsAg-negative patients with autoimmune (anti-nuclear, anti-actin or anti-liver-kidney microsomal antibody positive) chronic active hepatitis. An ELISA anti-HCV test and a recombinant immunoblot assay (RIBA-HCV) were used. Thirteen patients (59%) were anti-HCV positive and five (23%) anti-HCV negative by both ELISA and RIBA-HCV tests. Four patients (18%) were borderline positive by ELISA (OD less than 1.0), and three of them (all with severe disease) were negative by RIBA. Histologic necroinflammation, AST/ALT and gamma-globulins levels were higher and response to prednisolone treatment was better in RIBA anti HCV-negative than in anti-HCV-positive cases. We confirmed with both RIBA and ELISA tests the high prevalence of anti-HCV already reported by ELISA in anti nuclear and anti-liver-kidney microsomal antibody positive chronic active hepatitis. False positive for anti-HCV (i.e., a positive ELISA test not confirmed by RIBA) occurred only among patients with severe disease. Since RIBA-negative subjects showed the best response to corticosteroid, they might represent the only subset of cases of 'true' autoimmune chronic active hepatitis. PMID- 1717544 TI - Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion. AB - Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function. PMID- 1717545 TI - Chondrons from articular cartilage. (IV). Immunolocalization of proteoglycan epitopes in isolated canine tibial chondrons. AB - Chondrons have recently been extracted from adult articular cartilages and techniques developed to study their structure and composition in isolation. This study introduces methods to immobilize isolated canine chondrons in thin layers of agarose gel for immunohistochemistry and future in vitro studies. An antibody to Type VI collagen which stained the chondron in suspension was used to successfully validate the system and its feasibility for immunoelectron microscopy. Monoclonal and polyclonal antibodies to a variety of epitopes on the proteoglycan molecule were tested on fresh and fixed plugs cored from chondron agarose gels. Plugs were immunolabeled with peroxidase-diaminobenzidine before or after digestion with testicular hyaluronidase or chondroitinase ABC. Trypsin/chymotrypsin were used to challenge epitopes of the core protein. The results indicate that epitopes to keratan sulfate, chondroitin sulfate, hyaluronate binding region, and core protein are localized in the chondron. Consistent staining was found in the tail and interconnecting segments between chondrons, whereas staining of the pericellular matrix and capsule adjacent to the chondrocyte varied according to the enzyme pre-treatment employed. We conclude that isolated chondrons are rich in proteoglycan monomer, which is particularly concentrated in the tail and interconnecting segments of the chondron where it could function to protect and stabilize the chondrocyte. PMID- 1717546 TI - Health problems of residents along heavy-traffic roads. PMID- 1717547 TI - Health effects of lead pollution due to automobile exhaust: findings from field surveys in Japan and Indonesia. PMID- 1717548 TI - Affinity enhancement and transmembrane signaling are associated with distinct epitopes on the CD8 alpha beta heterodimer. AB - CD8 is a heterodimeric membrane glycoprotein on MHC class I-restricted T lymphocytes that cooperates with the alpha beta CD3 TCR in the recognition of MHC class I molecules presenting antigenic peptides. Co-operation has two components: enhancement of the affinity of MHC/peptide-TCR interaction, and signal transduction through the T cell membrane. The cytolytic function of CTL is primarily dependent on the affinity-enhancement component of CD8-TCR cooperation whereas activation of resting CD8+ T cells is primarily dependent on transmembrane signaling. Using a panel of mAb, two to the alpha-chain and three to the beta-chain of CD8, we investigated the relationships between epitopes and functional regions of the CD8 molecule. Two of the antibodies, one to the alpha chain and one to the beta-chain of CD8, inhibit the cytolytic function of CTL but not the generation of CTL from resting T cells. Another two antibodies, also one to the alpha- and one to the beta-chain, inhibited the generation of CTL while enhancing the cytolytic function of CTL. These results suggest that both the alpha- and beta-chain of CD8 possess two distinct regions, one involved in affinity enhancement and the other in transmembrane signaling. The former may be the MHC class I-binding region whereas the latter may associate with the alpha beta CD3 TCR. The data can explain the apparent functional equivalence of CD8 alpha alpha homodimers and alpha beta heterodimers. PMID- 1717549 TI - Canine neutrophil margination mediated by lectin adhesion molecule-1 in vitro. AB - The contributions of the canine neutrophil lectin adhesion molecule-1 (LECAM-1) (canine homologue of the murine MEL-14 Ag) in neutrophil-endothelial cell adhesion and transendothelial migration were studied using anti-LECAM-1 mAb, CL2/6, and SL1 under static conditions and at wall shear stresses of up to 1.85 dynes/cm2 (dpc). Both mAb were found to inhibit attachment of neutrophils to cytokine-stimulated canine jugular vein endothelium. The inhibitory effects of the anti-LECAM-1 mAb were more evident at a wall shear stress of 1.85 dpc (greater than 50%) than at 0.23 dpc or under static conditions (approximately 30%). In contrast the anti-CD18 mAb, R15.7, exhibited higher inhibitory ability at the lower shear stress and under static conditions with marginal inhibition of adhesion at 1.85 dpc. Anti-LECAM-1 and anti-CD18 mAb showed additive inhibitory effects at the lower wall shear stress and under static conditions. Chemotactic stimulation of the neutrophils caused rapid down-regulation of LECAM-1 from the neutrophil surface and reduced adhesion by 60% at a wall shear stress of 1.85 dpc. This inhibition was not additive to anti-LECAM-1 mAb. Pretreatment with CL2/6 or SL1 did not affect trans-endothelial migration of adherent neutrophils under any experimental conditions tested. Anti-CD18 mAb, however, blocked transendothelial migration by 98% and 56% under static condition and at a wall shear stress of 0.23 dpc, respectively. The results in this report indicate that canine LECAM-1 is involved in the initial adhesion of unstimulated neutrophils to cytokine-stimulated endothelial cells under flow, but in contrast to CD18 integrins, plays no role in the transendothelial migration. PMID- 1717550 TI - Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy. AB - We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy. PMID- 1717551 TI - Effects of IL-7 and IL-2 on highly enriched CD56+ natural killer cells. A comparative study. AB - IL-7 has been shown to induce low levels of lymphokine-activated killer cell (LAK) activity in bulk PBMC populations. We report here that immunomagnetically purified CD56+ cells from peripheral blood generated high LAK activity in response to IL-7. The LAK activity induced by IL-7 was comparable to, or slightly lower than, the LAK activity induced by IL-2. When analyzing cells from the same donor, no detectable LAK-generating effect of IL-7 was registered in the PBMC population, in contrast to a substantial effect in the CD56+ population. IL-2 induced 8- to 15-fold higher proliferative activity in CD56+ cells, relative to IL-7. At suboptimal concentrations of IL-2, IL-7 had a synergistic effect on the proliferation. IL-2-neutralizing antibodies did not abrogate the IL-7-induced proliferation or LAK generation. Both IL-7 and IL-2 induced comparable levels of 75-kDa TNFR expression, whereas IL-2R alpha expression was higher in IL-7 stimulated CD56+ cells. Low levels of TNF were produced in response to IL-7 at day 5, as opposed to a 50-fold higher TNF production in response to IL-2. No IL-2 or IL-6 production was detected. Our data indicate that IL-7 has profound and direct effects on CD56+ cells. PMID- 1717552 TI - Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay accelerating factor. AB - Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement. PMID- 1717553 TI - Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation. AB - Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+) binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP. PMID- 1717554 TI - Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro. AB - Autologous cell mixtures containing T cells, B cells, and adherent accessory cells from individuals primed to the malaria parasite Plasmodium falciparum by repeated natural infections were investigated for induction of Ig and antibody secretion in vitro. In vitro activation of cell cultures with two synthetic peptides corresponding to immunodominant T cell epitopes of the merozoite Ag ring infected erythrocyte surface Ag (Mr 155,000) (Pf155/RESA), one from its carboxyl terminal repeat and one from its nonrepeated amino-terminal region, gave rise to significant IgG secretion. Supernatants from lymphocyte cultures activated with either one of these peptides contained antibodies reacting with P. falciparum Ag in immunofluorescence assays and with Pf155/RESA peptides in a slot blot assay. No anti-P. falciparum antibodies were induced in the medium controls by lymphocyte stimulation with either tetanus toxoid or PWM. Induction in vitro of anti-Pf155/RESA antibodies was correlated with the presence of such antibodies in the sera of the lymphocyte donors, suggesting that the induction of antibody secretion reflected a secondary response in vitro of in vivo primed cells. Inspection of antibody profiles in individual donors revealed that the peptide corresponding to a sequence in the 3' repeat region induced anti-Pf155/RESA peptide antibodies reacting with identical or related and cross-reacting sequences in the 3' or 5' repeat region of the molecule. In contrast, the peptide corresponding to a nonrepeated T cell epitope in the amino terminus of the molecule only induced antibodies to an immunodominant amino-terminal B cell epitope partly overlapping with the T cell reactive sequence. Similar findings were made in the lymphocyte donors' plasma, frequently displaying significant correlations between antibody reactivities to the repeat peptides but not between these reactivities and those to the amino-terminal peptide. The marked specificity of this antibody formation in vitro suggests an underlying process of cognate recognition involving Ag-specific T and B cells reacting with different segments of the inducer peptide. The present experimental system should be well suited for identification of Th epitopes capable of inducing the production of antibodies of defined specificity in the human system. PMID- 1717555 TI - Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes. AB - CTL constitute an essential part of the immune response against the HIV. CTL recognize peptides derived from viral proteins together with the MHC class I molecules on the surface of infected cells. The CTL response could be important in prevention or control of infection with HIV by destroying virus-producing cells. In this study we have attempted to identify peptide epitopes recognized by HIV-specific CTL. Using synthetic peptides, we have identified six conserved peptidic epitopes on the gp120 envelope glycoprotein recognized by polyclonal human CTL in association with HLA-A2 class I transplantation Ag. These results were confirmed by two approaches: i) blocking of CTL activities with antibodies specific for three of these conserved peptides; and ii) construction of doubly transfected P815-A2 target cells, using deletions of the HIV env gene. Vaccination or immunotherapy in HLA-A2 individuals can thus be considered using highly conserved HIV env peptidic sequences. PMID- 1717556 TI - Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules. AB - A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717557 TI - Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses. AB - Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins. PMID- 1717558 TI - Differential regulation of HLA-A3 and HLA-B7 MHC class I genes by IFN is due to two nucleotide differences in their IFN response sequences. AB - The transcription of HLA-A3 and HLA-B7 class I genes is differentially regulated by IFN-alpha and -gamma, the latter gene being more inducible than the former. To determine the structural basis of this differential response, hybrid genes were constructed in which complete or fragmented HLA-A3 or HLA-B7 promoters were fused to the chloramphenicol acetyl transferase coding sequence. These constructs were tested in transient transfection assays, and the differential response of the HLA A3 and HLA-B7 genes to IFN was correlated with nucleotide differences in their interferon response sequences (IRS). Replacement of two T nucleotides in the HLA A3 IRS by the homologous A and C nucleotides of the HLA-B7 IRS was sufficient to impart IFN inducibility of the HLA-A3 promoter and efficient binding of constitutive and IFN-induced nuclear factors to the IRS of HLA-A3. Since the same two nucleotide differences are shared by all sequenced HLA-A and HLA-B genes, these results suggest that high or low responsiveness to IFN might be a locus related property. PMID- 1717559 TI - Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody. AB - Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL 6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy. PMID- 1717560 TI - Antigen-specific inhibition of ongoing murine IgE responses. II. Inhibition of IgE responses induced by treatment with glutaraldehyde-modified allergens is paralleled by reciprocal increases in IgG2a synthesis. AB - Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent. PMID- 1717561 TI - CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells. AB - CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28. PMID- 1717562 TI - Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells. AB - The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5 triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation. PMID- 1717563 TI - Role of single amino acids in the recognition of a T cell epitope. AB - T cell epitopes can be defined by the use of synthetic peptides, which when added to APC efficiently mimic naturally processed Ag. Free peptide is thought to bind to cell-surface MHC glycoproteins and the TCR then recognizes the resulting complex. The specificity of a tetanus toxin-specific human Th cell clone was investigated using a complete replacement set of peptides in which every amino acid within the minimal T cell epitope was replaced by each of the 19 alternative genetically coded amino acids. Within the minimal epitope, found to be YSYFPSVI (tetanus toxin 593-600), a small number of substitutions could be made without significant loss of activity, defined as substitutions giving peptides whose activity fell within +/- 3 SD of the mean parent response. Y593 could be substituted with F, W, M, L, V, and I; S594 with G and T; Y595, F596, and P597 with no other amino acids; S598 with A; V599 with S, and I600 with L. Rank ordering of the substitutions allowed a precise description to be made of MHC and/or TCR interaction with each amino acid side chain within the epitope. Simplified theoretic calculations based on this study indicate that class II T cell recognition has a specificity greater than 1 in 10(8). Competition experiments indicate that Y595, F596, P597, and I600 are critical for binding of this epitope to its restricting element, HLA DR4Dw14. PMID- 1717564 TI - Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells. AB - The human CD1 locus encodes three nonpolymorphic MHC class I-like cell surface glycoproteins, CD1a-c, which are expressed primarily by immature thymocytes. A mAb and antipeptide antiserum were utilized to determine the tissue distribution of a fourth CD1 molecule, CD1d. Within the lymphoid lineage, CD1d was expressed on B cells but not on thymocytes. Immunoperoxidase staining of fresh frozen intestinal tissues demonstrated that the majority of intestinal epithelial cells, with the exception of cells at the base of some crypts, expressed CD1d. The CD1d staining was observed in the cytoplasm and along the basolateral membranes of the epithelial cells. The intestinal epithelial cell expression of CD1d was confirmed by immunoblotting with a CD1d antipeptide antiserum. Further immunoperoxidase studies indicated that CD1d, unlike murine CD1, was also expressed by nonlymphoid tissues outside of the gastrointestinal tract. The expression of CD1d outside the lymphoid and myeloid lineages clearly distinguishes this molecule from CD1a-c and suggests that it may serve a distinct function. The prominent expression of CD1d by intestinal epithelial cells suggests that this molecule may be an important ligand for T lymphocytes within the gut-associated lymphoid tissue. PMID- 1717565 TI - Catecholamines stimulate the IFN-gamma-induced class II MHC expression on bovine brain capillary endothelial cells. AB - The brain has been considered for a long time as an immunologically privileged site because of the lack of a true lymphatic system and the existence of several barriers that isolate it from the periphery. In the last few years, it became evident that cells in the central nervous system (astrocytes, microglial cells, and brain capillary endothelial cells) can be induced to express class II MHC and present Ag to T lymphocytes. The brain capillary endothelial cells, which are strategically located at the interface between blood and brain, could be involved in the initiation of immune responses within the brain parenchyma. We have previously characterized bovine brain capillary endothelial cells in culture and shown that they maintain in vitro a fully differentiated phenotype associated with the blood-brain barrier endothelium. In order to assess the role of these cells in the development of immune responses in the brain, we initiated the present study on the regulation of their class II MHC surface expression. Our data indicate that this expression on bovine brain capillary endothelial cells is inducible by IFN-gamma and further stimulated by catecholamines through activation of beta-adrenergic receptors. However, this latter effect is not mimicked by forskolin, theophylline, or dibutyryl-cAMP, suggesting the involvement of a cAMP-independent mechanism. PMID- 1717566 TI - Complete sequence of the allergen Amb alpha II. Recombinant expression and reactivity with T cells from ragweed allergic patients. AB - This study defines the complete primary structure of Amb alpha II, an important allergen produced by short ragweed (Ambrosia artemisiifolia). The deduced amino acid sequence derived from the cDNA indicates that Amb alpha II shares approximately 65% sequence identity with the Amb alpha I multigene family of allergens. Full-length cDNA encoding Amb alpha I.1 and Amb alpha II have been expressed in E. coli and purified. An in-frame linker encoding polyhistidine has been added to the 5' end of the cDNA to facilitate purification using Ni2+ ion affinity chromatography, yielding greater than 90% pure recombinant protein in a single step. T cells from patients allergic to ragweed proliferate in response to pollen extract as well as purified recombinant Amb alpha I.1 and Amb alpha II. T cell lines established using either Amb alpha I.1 or II as the stimulating Ag exhibit a high level of cross-reactivity to both proteins. This result is entirely consistent with the extensive primary sequence identity shared by these two proteins. These data suggest that allergic humans recognize shared T cell epitopes on these two related molecules. PMID- 1717567 TI - Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion. AB - The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation. PMID- 1717568 TI - IL-1 receptor antagonist regulation of acute phase protein synthesis in human hepatoma cells. AB - The hepatoma cell line HuH-7 has recently been shown to synthesize serum amyloid A (SAA) in response to IL-1. IL-1 receptor antagonist (IL-1Ra) was able to completely inhibit the response of SAA to IL-1 but not the increase seen in response to IL-6. IL-1Ra was equally effective at inhibiting IL-1 alpha or IL-1 beta. At a 10-fold molar excess of IL-1Ra over IL-1 there was complete inhibition of the SAA response. Removal of IL-1 at 24 h rapidly reduced the SAA secreted over the next 24 h. Addition of IL-1Ra to the cells at this time was as effective as removal of IL-1 at inhibiting the subsequent secretion of SAA. IL-1Ra was less effective at inhibition of IL-1-induced haptoglobin secretion. We would conclude that IL-1Ra may play an important role in the regulation of acute phase protein synthesis. PMID- 1717569 TI - A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma. AB - A 15,000 molecular weight protein (15-kDa), induced and secreted by human PBMC after treatment with IFN-alpha or -beta, was assessed for its ability to modulate cellular function. Although it had no effect on growth or 2'5'-A synthetase activity in Daudi, U-937, or HL-60 cells, when incubated with fresh human PBMC, LPS-induced monocyte cytotoxicity against WEHI-164 target cells was augmented. This stimulation was inhibited by both an antibody against TNF-alpha and a rabbit polyclonal antiserum to the 15-kDa protein. Furthermore, when the 15-kDa protein was added to PBMC an increase in GTP cyclohydrolase I activity, as assessed by neopterin secretion, resulted. Neopterin secretion by PBMC in response to the 15 kDa was increased in a dose-responsive manner up to more than sixfold over baseline, with a 15-kDa concentration of less than 10 ng/ml effective. The 15-kDa protein also stimulated indoleamine 2,3-dioxygenase (IDO) activity in fresh, human PBMC. Induction of neopterin secretion and IDO activity was inhibited by a polyclonal antiserum to 15-kDa. LPS-induced cytotoxic activity was not augmented by 15-kDa pretreatment of purified monocytes, indicating the need for the presence of a second cell population and the indirect action of the 15-kDa on the induction of monocyte activities. When PBMC or purified CD3+ cells, but not purified CD14+ cells, were incubated with the 15-kDa protein, secretion of a factor was induced that resulted in the induction of IDO activity in PMA differentiated THP-1 cells. An antibody to IFN-gamma, but not IFN-alpha, inhibited the induction of IDO activity by this secreted factor. In addition, antiserum to the 15-kDa blocked the secretion of IFN-gamma from the CD3+ cells. Thus, a 15-kDa product of IFN-alpha- and IFN-beta-treated monocytes and lymphocytes can stimulate secretion of IFN-gamma from CD3+ cells. PMID- 1717570 TI - On the interaction of promiscuous antigenic peptides with different DR alleles. Identification of common structural motifs. AB - We have investigated the interaction between DR1 molecules and the two antigenic peptides, tetanus toxoid 830-843 and hemagglutinin 307-319, previously known to bind most DR alleles (degenerate binding) and to be recognized by the same T cell clones in the context of different DR alleles (promiscuous T cell recognition). The DR1 affinity of these two peptides was compared with that of two other different T cell epitopes (pertussis toxin 30-42 and ragweed allergen Ra3 51-65). It was found that degeneracy and promiscuity were associated with high affinity interactions, whereas binding and T cell selectivity were associated with weaker interactions. Thus, the selectivity of DR-peptide interactions, as is commonly observed with the antibody molecule, appears to be inversely correlated to affinity. Several singly substituted analogs of the hemagglutinin 307-319 determinant have also been tested for capacity to bind various DR alleles (DR1, DR2, DR5, and DR7). The results obtained suggest that this determinant may bind the different DR alleles in a similar orientation. Similar conclusions were reached when the interaction between the tetanus toxoid 830-843 determinant and three different DR alleles (DR1, DR2, and DR7) was studied following the same experimental approach. When crucial DR-binding residues of the two peptides were compared, it was found that they were very similar in both chemical nature and spacing in the peptide primary structure, suggesting that the two peptides may bind DR in a very similar orientation. Finally, a putative motif has been derived and shown to be present in a majority of the DR binders tested, but only in a minority of the non-DR binding peptides. PMID- 1717571 TI - Monoclonal antibodies to the leukocyte common antigen (CD45) inhibit IgE-mediated histamine release from human basophils. AB - mAb were selected that inhibited IgE-mediated histamine release from human basophils. The two mAb, HB 9AB6 and HB 10AB2, are of the IgG1 subclass and have a 50% inhibitory concentration of 0.16 to 1.1 micrograms/ml. The mAb required several hours of incubation with the basophils at 37 degrees C to induce maximum inhibition. Neither mAb directly released histamine from human basophils nor did they inhibit release induced by formylmethionine tripeptide, calcium ionophore A23187, or PMA. There was little inhibition of IgE-mediated release when the cells were preincubated with the mAb at 4 degrees C. By FACS analysis the 2 mAb bound to all peripheral blood leukocytes and immunoprecipitated a approximately 200-kDa protein from peripheral blood leukocytes and several cell lines of human origin. In binding studies and by sequential immunoprecipitation the 2 mAb and a known anti-CD45 mAb bound to the same protein. However, the mAb recognized different epitopes. Therefore, mAb to the CD45 surface Ag, a membrane protein tyrosine phosphatase, inhibits IgE-receptor mediated histamine release from human basophils. The data suggest a link between protein tyrosine phosphorylation and high affinity IgE receptor-mediated signal transduction in human basophils. PMID- 1717572 TI - Recognition of the influenza hemagglutinin by class II MHC-restricted T lymphocytes and antibodies. I. Site definition and implications for antigen presentation and T lymphocyte recognition. AB - We have identified the site encompassing residues 126-145 on the A/Japan/57 influenza hemagglutinin molecule that is recognized in association with HLA-DRw11 by a clonal population of human, influenza specific, CD4+ cytolytic T lymphocytes. The critical core sequence of the T cell determinant spans hemagglutinin residues 129-140 and overlaps a putative antibody binding site. Hemagglutinins of influenza field strains that are not recognized by the T cell clones contain sequence alterations within the 129-140 target site of the CD4+ T cells. Functional analyses, with synthetic peptides, of the contribution of each of the residues within the sequence toward the capacity of the antigenic fragment to associate with both the restriction element and the TCR revealed a continuous linear array of residues necessary for MHC binding and/or Ag receptor engagement. At least one residue, the lysine at position 134, was shown to be critical for both DRw11 association and TCR recognition. The significance of these findings for recognition of glycoproteins by human CD4+ T cells is discussed. PMID- 1717573 TI - Identification of an immunodominant B cell epitope on the hepatitis C virus nonstructural region defined by human monoclonal antibodies. AB - Several EBV-transformed B cell lines (BCL) were obtained from two patients with chronic hepatitis C virus (HCV) infection that secreted IgG class antibodies to the HCV nonstructural Ag c100-3. Two cloned BCL, derived from the same parental line, generated stable cloned lines that secreted up to 20 mg/liter of specific IgG1(kappa). Supernatants from oligoclonal and cloned BCL were also analyzed by immunoblot and all strongly reacted with recombinant polypeptides derived from the putative NS4 region of HCV, c100-3 and 5-1-1 (a 42-amino acid fragment of c100-3), whereas no reaction with the viral nucleoprotein, the NS3 nonstructural protein or the superoxide dismutase moiety of the c100-3 fusion protein could be documented. The fine specificity of these antibodies was also evaluated using overlapping synthetic peptides (20-mers) covering the 5-1-1 sequence. All oligoclonal and clonal IgG displayed high affinity binding to peptides covering residues 120-137 of Chiron's c100-3 sequence at the aminoterminus of 5-1-1. In addition, a minimal B cell epitope, N-VLYREF-C, was defined by human oligoclonal and monoclonal antibodies corresponding to residues 132-137. Interestingly, predominant recognition of the N-terminus of 5-1-1 was also observed in more than 80% of sera from patients with HCV infection. In conclusion, we have successfully produced human B cell cloned lines that secrete abundant quantities of IgG1(kappa)-specific for a polypeptide encoded by the NS4 region of HCV. Such antibodies recognize an immunodominant epitope, relative to this region, located at the N-terminus of the 5-1-1 fragment. PMID- 1717574 TI - A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes. AB - BALB/3T3 cells infected with a retroviral vector encoding the influenza virus nucleoprotein (NP) gene are efficiently lysed by CTL generated in BALB/c mice (H 2d background). Cells transduced with a mutant form of NP which contains a frameshift mutation at its NH2 terminus (NPm) do not express biochemically detectable levels of protein but nevertheless present Ag to CTL with high efficiency. Cold target inhibition studies indicate that the same CTL epitope(s) are recognized in cells harboring NP or NPm. L929 cells transduced with the NPm gene also present Ag efficiently to CTL raised in C3H mice (H-2k background). Cells engineered to express 5- to 15-fold lower levels of wild-type NP were not capable of presenting Ag to CTL, arguing against the notion that CTL are able to lyse cells expressing very low levels of Ag which might have resulted from suppression of the frameshift mutation in NPm. Implications to the mechanism of epitope generation in class I MHC-restricted immune responses are considered. PMID- 1717575 TI - Murine T cell-stimulatory peptides from the 19-kDa antigen of Mycobacterium tuberculosis. Epitope-restricted homology with the 28-kDa protein of Mycobacterium leprae. AB - Fifteen overlapping synthetic peptides, spanning the entire amino acid sequence of the Mycobacterium tuberculosis 19-kDa protein, were used to identify epitopes recognized by murine T cells. Five of the 15 peptides tested were able to elicit in vitro lymph node T cell proliferative responses in C57BL/10 mice primed by footpad inoculation with homologous peptide. Analysis in congenic strains of mice revealed H-2 restriction in the response to four peptides. However, one peptide, 19.7 (residues 61 to 80), induced T cell responses in all four haplotypes tested. This peptide was also unique in being able to stimulate lymph node cells from C57BL/10 mice immunized with recombinant 19-kDa protein, killed M. tuberculosis, or live bacillus Calmette Guerin infection. T cell lines specific for peptide 19.7 were of the CD4 phenotype. Significantly, sequence analysis revealed that residues 61 to 80 of the 19-kDa protein exhibited considerable homology with a single 20-amino acid sequence (residues 120 to 140), but not with any other region of the 28-kDa protein expressed in Mycobacterium leprae. This finding is the first evidence of epitope-restricted homology between otherwise structurally unrelated microbial Ag. PMID- 1717576 TI - Induction of T cell CD7 gene transcription by nonmitogenic ionomycin-induced transmembrane calcium flux. AB - The CD7 molecule is a 40-kDa member of the Ig superfamily that has structural homology to the murine Thy-1 molecule and is acquired early in human T cell ontogeny. Previous studies have demonstrated that expression of the CD7 molecule is markedly up-regulated during T cell activation. In this study, we have studied the signals required for CD7 up-regulation on human T cells. We found that nonmitogenic amounts of ionomycin selectively and maximally up-regulated T cell CD7 on mature (peripheral blood) T cells after 24 h. Whereas CD7 expression was increased 78 +/- 25% by 0.5 microM ionomycin, expression of CD25 (IL-2R alpha), class II MHC, 4F2, transferrin receptor, CD2, CD3, CD4, CD5, and CD8 molecules was not increased. Ionomycin-induced CD7 surface expression was associated with peak increases in CD7 mRNA after 4 to 6 h. Transcriptional analysis and CD7 mRNA half-life determination revealed the increase in CD7 mRNA was the result of increased CD7 gene transcription 1 h after ionomycin stimulation and was not due to prolongation of CD7 mRNA half-life. The up-regulation of surface CD7 expression by ionomycin was dependent on extracellular calcium and did not require the activation of T cell tyrosine protein kinase. Mitogenic CD2 and CD3 mAb as well as stimulation of T cells by PHA also up-regulated CD7 expression. CD7 up-regulation by ionomycin was transient (24 to 72 h) and inhibitable by cyclosporin A, whereas CD7 up-regulation by PHA was sustained over 5 to 7 days and was significantly less inhibitable by cyclosporin A. These data demonstrate that induction of a transmembrane calcium flux generates signals that lead to CD7 gene transcription. PMID- 1717577 TI - 1F7 (CD26): a marker of thymic maturation involved in the differential regulation of the CD3 and CD2 pathways of human thymocyte activation. AB - We have recently shown that solid-phase immobilization of anti-1F7 recognizing the 110-kDa CD26 Ag is comitogenic for human peripheral blood T cell activation via both the CD3 and CD2 pathways. We have also demonstrated that binding of anti 1F7 leads to the disappearance of CD26 surface expression, and this anti-1F7 induced modulation results in an increase in anti-CD3 or anti-CD2-mediated peripheral blood T cell activation. In this report, we extended these findings by examining the expression and functional relationship of 1F7 on the CD3 and CD2 pathways of activation of human thymocytes. We now demonstrated that most of the anti-1F7 reactivity is found on medullary thymocytes, the population of thymocytes expressing high level of CD3 (CD3H). We have also shown that binding of anti-1F7 can induce a decrease in CD26 surface expression, with no detectable effect on the surface expression of CD3 or CD2. Most importantly, we showed that solid-phase immobilization of anti-1F7 has a comitogenic effect on thymocyte activation induced by anti-CD3 but not anti-CD2. In addition, anti-1F7-induced modulation of CD26 results in an enhancement in CD3-mediated but not CD2-mediated human thymocyte activation. The observed functional effect of CD26 on the CD3/TCR pathway of activation is mainly restricted to mature thymocytes as distinguished by high surface expression of CD5, although CD26 is also functionally associated with the CD3/TCR pathway on cells expressing low level of CD5. Demonstrating that CD26 involvement in the regulation of human thymocyte activation is restricted mainly to the CD3 pathway, unlike its involvement with both the CD3 and CD2 pathways of mature peripheral blood T lymphocyte activation, our data hence suggested that CD26 may play a role in thymic differentiation and maturation via the differential engagement of the CD3 pathway. PMID- 1717578 TI - Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules. AB - Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules. PMID- 1717579 TI - Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes. AB - The comparative roles of the endothelial cell (EC) adhesion receptors VCAM-1 and ICAM-1 during the adhesion and transendothelial migration of T cells were examined. The adhesion of T cells to IL-1-activated EC was markedly, but not completely, inhibited by mAb to VCAM-1 as well as to its counter-receptor, VLA-4, whereas, T cell binding to IL-1-activated EC was not blocked by mAb to ICAM-1 or to its counter-receptor, LFA-1. In contrast, LFA-1/ICAM-1, but not VLA-4/VCAM-1, mediated much, but not all, of the binding of T cells to unstimulated EC. Activation of T cells with phorbol dibutyrate and ionomycin alter the receptor counter-receptor pairs used for binding to EC. Regardless of the activation status of the EC, the binding of activated T cells was not blocked by mAb to VLA 4 or VCAM-1. Moreover, the binding of activated T cells to EC was blocked to a lesser degree by mAb to LFA-1 than that of resting T cells, and mAb to ICAM-1 blocked binding only modestly. The role of VCAM-1 and ICAM-1 during the transendothelial migration of T cells was also examined. Regardless of the activation status of the T cells or the EC, VCAM-1 was never found to function during transendothelial migration, even when it mediated the binding of resting T cells to IL-1-activated EC. In contrast, ICAM-1 played an important role in transendothelial migration under all of the conditions examined, including situations when T cell-EC binding was not mediated by ICAM-1. Immunoelectron microscopic analysis of transendothelial migration supported the conclusion that ICAM-1 but not VCAM-1 played a central role in this process. Thus, ICAM-1 was prominently and uniformly expressed at all EC membrane sites that were in contact with bound and migrating T cells, whereas VCAM-1 was localized to the luminal surface of IL-1-activated EC, but was often absent from the surface of the EC in contact with T cells undergoing transendothelial migration. These studies confirm that ICAM-1 and VCAM-1 play reciprocal roles in the binding of resting T cells to resting and IL-1-activated EC, respectively, but a less prominent role in the binding of activated T cells. Moreover, ICAM-1 but not VCAM-1 plays a role in transendothelial migration, regardless of the receptor-counter-receptor pairs used for initial binding. PMID- 1717580 TI - Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy. AB - (C57BL/6 x A/J)F1 (B6AF1) mice thymectomized between days 1 and 4 of age develop autoimmune oophoritis (D3TX oophoritis) 4 to 6 wk later. Oophoritis can be adoptively transferred to young recipients, and the disease in D3TX mice is prevented by reconstitution with normal adult spleen cells. The present study was further defined the nature of the effector and suppressor cells. Contrary to an earlier report, oophoritis is transferred to syngeneic and not allogeneic recipients. The spleen cells from D3TX mice when stimulated in vitro with Con A, also transfer oophoritis to adult recipients. The effector cells are CD4+: oophoritis transfer is abrogated by CD4 antibody and not by CD8 antibody and C. Spleen cells from D3TX male mice transfer disease less efficiently than female cells, thus endogenous ovarian Ag may be required for activation of effector T cells. T cells from normal adult spleen that suppress D3TX oophoritis also appear to be of CD4+ phenotype. These cells are likely to be derived from adult thymus because adult thymocytes also suppress D3TX disease. We were unable to substantiate the earlier claim that suppressor cells in normal mice are ovarian Ag specific. Thus male and female spleen cells suppress disease with comparable efficiency, and deprivation of endogenous ovarian Ag by neonatal ovariectomy of cell donors had no observable effect on disease suppression. PMID- 1717581 TI - Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping. AB - Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins. PMID- 1717582 TI - Granulocyte colony-stimulating factor modulation of cytokine receptors on murine bone marrow cells. In vivo and in vitro studies. AB - Human recombinant granulocyte CSF (G-CSF) modulation of cytokine receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo, G CSF reduced 125I-G-CSF binding to BMC by greater than 95% within 30 min, with return to base line after 48 h. Human rCSF-1 binding was reduced greater than 85% after 30 min and failed to recover even after 48 h. Murine rTNF-alpha or recombinant granulocyte/macrophage CSF binding was not significantly altered. However, human rIL-1 alpha binding increased greater than 1.5-fold after 3 h, was elevated greater than 5-fold between 6 and 12 h, and declined to base line after 48 h. In vitro, G-CSF induced a greater than 1.5-fold increase in IL-1 binding to BMC after 8 h, suggesting that up-modulation of IL-1 binding in vivo required G CSF and other influences. Further studies indicated that BMC responded to glucocorticoids and G-CSF with a synergistic increase of IL-1 binding. This synergistic IL-1R modulation was a time- and dose-dependent process and was inhibited by cycloheximide or actinomycin D in a dose-dependent manner. Binding studies further revealed that the synergistic stimulation of IL-1R expression on BMC was probably due to increased receptor number, rather than increased receptor affinity. In addition, this phenomenon was also observed in other hematopoietic cells. Our results demonstrated that G-CSF was capable of stimulating IL-1R expression on BMC both in vivo and in vitro and G-CSF in combination with glucocorticoids synergistically up-modulated IL-1 binding to BMC in vitro. Inasmuch as IL-1 induces the secretion of G-CSF and glucocorticoids in vivo, this synergistic induction may play an important, as yet unknown, role in the inflammatory cascade. PMID- 1717583 TI - Contribution of the repeating domains of membrane cofactor protein (CD46) of the complement system to ligand binding and cofactor activity. AB - Membrane cofactor protein (MCP) (CD46) of the C system binds to C3b and C4b, functions as a cofactor for their cleavage, and protects autologous cells from C mediated injury. The predominant structural motif of MCP is the short consensus repeat (SCR), a repeating domain involved in ligand binding of other related C regulatory proteins. SCR deletion mutants were constructed to determine which of the four SCR of MCP contribute to ligand binding and cofactor activity. ELISA were developed to evaluate binding efficiency of mutants to ligand. Analysis of the deletion mutants indicated that the third and fourth SCR were important for both ligand binding and cofactor activity of C3b (iC3) and C4b. In addition, the same SCR were required for efficient binding of an mAb known to inhibit MCP function. The mutant deleted of SCR-2 bound but lacked cofactor activity for iC3. It did not bind or possess cofactor activity for C4b. Deletion of the first (amino-terminal) SCR had a minimal effect on iC3 binding and cofactor activity but reduced the efficiency of C4b binding. The results identify the SCR of MCP that contribute to ligand binding and cofactor activity. The data also suggest the presence of distinguishable iC3 and C4b binding sites and provide evidence that iC3 binding is not always sufficient for cofactor activity. PMID- 1717584 TI - Characterization of a monoclonal anti-idiotypic antibody that mimics cyclosporine A in a single binding system. AB - BALB/c mice were immunized with a mixture of two anti-cyclosporine (CsA) mAb, H4 1.3 and D3 1.3, which recognize CsA amino acid residues reported to be important for its biologic activity and for its interaction with a CsA-binding protein, cyclophilin. A syngeneic anti-Id mAb, P43E, an IgG1 kappa, was generated. P43E bound to F(ab)'2 fragments of H4 1.3 in an ELISA and showed a dose-dependent inhibition of CsA binding to H4 1.3 and D3 1.3 in ELISA and RIA. The anti-Id antibody could be purified on an affinity column of H4 1.3/D3 1.3, and its binding to H4 1.3 could be inhibited by CsA. Dissociation kinetics and Scatchard analyses of P43E binding to H4 1.3 supported a reciprocal competitive inhibition mechanism for anti-Id binding, excluding a steric hindrance mechanism. P43E did not bind to several other anti-CsA mAb, a rabbit polyclonal anti-CsA antibody preparation, or cyclophilin. It is, therefore, a mimic of CsA but restricted to a single binding system. Our results are in agreement with the epitope specificities of the antibodies, X-ray and electron microscopic analysis of idiotope-anti-idiotope interactions, and recent nuclear magnetic resonance spectrometry studies of a CsA-cyclophilin complex and have implications with respect to the bioeffective epitope of CsA. PMID- 1717585 TI - A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies. AB - We have performed immunofluorescence analysis of COS cells transfected with human CD3 genes and detergent permeabilized to define the specificity of several anti CD3 antibodies. We have found that the mAb OKT3, WT31, UCHT1, and Leu-4 did not stain COS cells singly transfected with the CD3-epsilon chain. However, these antibodies very strongly stained COS cells doubly transfected with a combination of CD3-epsilon plus either CD3-gamma or CD3-delta. By contrast, the antibodies SP34 and APA 1/1, which were raised against isolated SDS-denatured CD3-epsilon protein, gave a strong staining of COS cells singly transfected with CD3-epsilon as well as of the double transfectans. The recognition by this panel of anti-CD3 antibodies of CD3-gamma/epsilon and CD3-delta/epsilon complexes and not of CD3 epsilon alone was assessed by immunoprecipitation. These findings suggest that the most widely used mAb specific for the CD3 complex recognize conformational epitopes on CD3-epsilon, which are expressed when this chain is bound to either CD3-gamma or CD3-delta. It should also be highlighted that antibody WT31 clearly recognizes the CD3 moiety of the TCR/CD3 complex. PMID- 1717586 TI - Lipopolysaccharide-induced stimulation of CD11b/CD18 expression on neutrophils. Evidence of specific receptor-based response and inhibition by lipid A-based antagonists. AB - Gram-negative bacterial septicemia is a common clinical syndrome resulting, in part, from the activation of phagocytic leukocytes by LPS. By using flow cytometry, we have characterized LPS-induced expression of the beta 2 integrin CD11b/CD18. After exposure to Salmonella minnesota R595 LPS, expression of neutrophil CD11b/CD18 is rapidly upregulated, beginning within 5 min and achieving a peak fluorescence (typically two- to threefold over base line) by 30 min. The increase in CD11b/CD18 expression was similar in kinetics and magnitude to that produced by FMLP, PMA, and human rTNF-alpha. Concentrations of LPS necessary to stimulate a response were as low as 1 ng/ml of R595 LPS; a maximal response was observed between 30 and 100 ng/ml. The upregulation of CD11b/CD18 due to LPS was not interrupted by protein synthesis inhibitors. A group of glucosamine disaccharide lipid A-like molecules: Rhodobacter sphaeroides lipid A, lipid IVA, KDO2IVA, and deacylated LPS were able to block the stimulatory effect of LPS. This inhibition was specific for the actions of LPS as stimulation of polymorphonuclear leukocytes (PMN) by FMLP, human rTNF alpha, PMA, and rewarming were not altered by the disaccharide inhibitors. PMN which were exposed to the specific disaccharide LPS antagonists and then washed, were refractory to stimulation by LPS. The monosaccharide lipid A precursor lipid X also blocked stimulation of neutrophils by LPS, although with a 100-fold reduction in potency. Unlike the disaccharide inhibitors, PMN exposed to lipid X were still responsive to LPS stimulation after washing. The PMN response to LPS was less sensitive in the absence of serum, although upregulation of CD11b/CD18 could still be seen using higher concentrations of LPS. Monoclonal antibody directed against CD14 (clone 3C10), also specifically inhibited LPS induced PMN CD11b/CD18 expression both in the presence and absence of serum. These findings support the hypothesis that LPS stimulates neutrophils by interacting with specific cellular receptors. PMID- 1717587 TI - N-glycosylation of HIV-gp120 may constrain recognition by T lymphocytes. AB - The HIV envelope protein gp120 is heavily glycosylated, having 55% of its molecular mass contributed by N-linked carbohydrates. We investigated the role of N-glycosylation in presentation of HIV-gp120 to T cells. T cell clones obtained from humans immunized with a recombinant nonglycosylated form of HIV-gp120 (env 2 3) were studied for their ability to recognize both env 2-3 and glycosylated gp120. We found that 20% of CD4+ T cell clones specific for env 2-3 fail to respond to glycosylated gp120 of the same HIV isolate. Using synthetic peptides, we mapped one of the epitopes recognized by such clones to the sequence 292-300 (NESVAINCT), which contains two asparagines that are glycosylated in the native gp120. These findings suggest that N-linked carbohydrates within an epitope can function as hindering structures that limit Ag recognition by T lymphocytes. PMID- 1717588 TI - Molecular heterogeneity of e antigen polypeptides in sera from carriers of hepatitis B virus. AB - Hepatitis B e Ag (HBeAg) was isolated from pooled sera of carriers, without abnormalities in liver function, by affinity column chromatography with mAb against HBeAg. HBeAg polypeptide with an estimated molecular size of 20,000 Da (p20e) was detected, in addition to regular HBeAg polypeptides (p17e/p18e). p20e, as well as p17e/p18e, did not bind with mAb against the carboxyl-terminal domain of the C-gene product. p20e disclosed an N-terminal sequence of MQLFHLXLII- (X unknown), whereas p17e had that of SKLXLGXLXGMDIDPXKEFG- (X's unknown). By comparing them with the amino acid sequence encoded by the precore region and C gene of hepatitis B virus DNA, p20e was deduced to possess amino acids 1 to 19 of the precore-region product at the N-terminus, which contains signal sequence and usually removed before the secretion of HBeAg. p17e had amino acids 20 to 29 of the precore-region product that continued to the C-gene product. Inasmuch as p20e was invariably detected in HBeAg preparations from carriers without evidence for liver disease, it would not have been released into the circulation from destructed hepatocytes. HBeAg polypeptide bearing an uncleaved signal sequence would help in further understanding the mechanism of HBeAg secretion. PMID- 1717589 TI - Mutational analysis of the cross-reactive idiotype of the A strain mouse. AB - The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each of several monoclonal idiotopes has allowed us to identify more precisely the amino acids responsible for expression of the CRIA. Thus we have shown that expression of the CRIA is equally dependent on amino acids in the second and third CDR of the H chain. Furthermore, the idiotopes studied surround the Ag-binding site and clearly involve multiple interactions in several of the L and H chain CDR. PMID- 1717590 TI - Characterization of the spontaneous mutant H-2Kbm29 indicates that gene conversion in H-2 occurs at a higher frequency than detected by skin grafting. AB - A spontaneous mutation of H-2Kb, Kbm29, was discovered among the progeny of F1 hybrid parents. Unlike other characterized spontaneous class I variants, this mutant was detected with the use of antibody, rather than tissue grafting. Although Kbm29 is serologically indistinguishable from the previously described mutant molecule Kbm3, it is identical to the parental Kb by skin grafting and CTL assays. A full length cDNA of Kbm29 was amplified by polymerase chain reaction with locus-specific primers, cloned, and sequenced. Two nucleotides were found to be mutated, resulting in a single amino acid change (Lys----Ala) at amino acid 89 of the mature glycoprotein. This is consistent with the observed serologic changes, as the same amino acid substitution is responsible for the serologic profile of Kbm3. The occurrence of a mutation which is not detectable by the methods normally used to screen for H-2 mutants provides evidence that the high spontaneous rate of structural mutation described for the Kb molecule is underestimated. PMID- 1717591 TI - The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele specific antibodies. AB - Allelic polymorphism in TCR loci may play an important role in shaping the T cell repertoire and in disease susceptibility. We have used a combination of antibody and sequence analysis to investigate polymorphism in the murine V alpha 11 family. Two different antibodies have been analyzed that recognize particular V alpha 11 family members of the V alpha b and V alpha d haplotypes. One antibody shows J alpha dependency, suggesting a conformational element to the epitope. Investigation of the anti-V alpha 11 staining pattern on different mouse strains indicates that there is a marked influence of MHC haplotype on V alpha 11 selection and that V alpha 11 is preferentially expressed on CD4+ cells. Sequence analysis of V alpha 11 genes from the V alpha a, V alpha b, and V alpha d haplotypes shows two potential regions for the haplotype-specific epitopes. The relatedness of the different V alpha 11 family members from different haplotypes suggests that the V alpha 11.1/11.2 gene duplication is relatively recent, but that V alpha 11.3 separated much earlier. Differences between V alpha 11.3 and V alpha 11.1/11.2 are concentrated in the putative complementarity determining regions (CDR), whereas differences between alleles are not clearly clustered. However, the V alpha 11.1a and V alpha 11.1d alleles differ from V alpha 11.1b and V alpha 11.2b in CDR1. A V alpha 11.2-expressing anti-cytochrome c T cell has the same V-J junction as a V alpha 11.1-bearing cell with a similar fine specificity, indicating that V alpha 11.1b and V alpha 11.2b do not contribute different Ag specificities. PMID- 1717592 TI - Novel methods to rapidly and sensitively analyze antigenic peptide binding to MHC class II molecules. AB - One of the important steps for antigen presentation by MHC class II molecules involves binding of a peptide fragment of the antigen to the class II molecule followed by recognition of the resulting complex by T cells. The most commonly used methods for studying binding of peptide to MHC II are: equilibrium dialysis, gel filtration chromatography, HPLC and polyacrylamide gel electrophoresis. Each of these methods has some limitations and is time consuming. In addition, each requires a considerable amount of native MHC class II, which is always difficult to obtain. In this report, we describe three different sensitive methods using radiolabeled peptide to study peptide binding to murine MHC class II molecules. These are: nitrocellulose filter binding, thin-layer chromatography (TLC) using plate-supported silica gel or PEI cellulose, and paper electrophoresis using Sepraphor cellulose polyacetate paper. All three methods are rapid, highly sensitive and require only ng quantities of affinity pure MHC class II molecules and peptides. These methods can be used to calculate the peptide occupancy of MHC class II molecules. PMID- 1717593 TI - Effects of target antigen density on the efficacy of immunomagnetic cell separation. AB - Immunomagnetic cell separation uses binding of an antibody to its epitope to identify the target cell, which is then removed by attachment to an anti immunoglobulin-coated paramagnetic bead, and passage through a magnetic field. This method has previously been shown to be less sensitive to the effects of low target antigen density than are other cell elimination methods, such as complement-mediated lysis. In this paper we demonstrate that, with certain antibody/target cell combinations, the efficiency of immunomagnetic depletion can be adversely affected by high expression of the target antigen. This can occur by two non-mutually exclusive mechanisms. These are (i) steric hindrance of bead binding due to crowding of monoclonal antibodies on the cell surface; and (ii) binding of the monoclonal antibody molecule in a configuration that is poorly accessible to the anti-immunoglobulin immobilized on the microspheres. The predominant effect operating in any system can be determined by analysis of the cells remaining after the separation procedure. In both cases pre-attachment of the monoclonal to the beads results in improved separation efficiency. These results emphasize the necessity of optimizing experimental conditions in each system that is investigated. PMID- 1717594 TI - Monoclonal antibodies defining distinct epitopes of the human IL-2 receptor beta chain and their differential effects on IL-2 responses. AB - We have established and characterized five new monoclonal antibodies (mAbs) which specifically immunoprecipitate the human interleukin-2 receptor beta chain (IL-2R beta). One of them, TU30, recognizes the intracytoplasmic 'serine-rich region' of IL-2R beta that is critical for IL-2 signal transduction. The others, TU12, TU21, TU23 and TU25, completely inhibit IL-2 binding, as does the previously characterized TU27. However, reciprocal binding competition assays show that the epitopes recognized by the individual mAbs are different from each other. The mAbs inhibit the growth of IL-2-dependent cells. The magnitude of their inhibitory effects is dependent on not only the affinities of the mAbs for IL-2R beta but also upon the number of IL-2R alpha subunits expressed on IL-2-dependent cells. These mAbs should be useful in studying the structure and function of the IL-2R. PMID- 1717595 TI - UV-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides. AB - Detection of peptide-specific antibodies by the conventional ELISA technique is sometimes hampered by the difficulties encountered in immobilizing stretches of amino acids on the solid support. To improve the attachment of synthetic peptides to the solid phase, we have developed a sensitive and rapid immunoassay based on the irradiation of polystyrene plates with UV light prior to coating the target peptide. This pretreatment increases the specific signal in a dose-dependent manner without augmenting the background or altering the specificity of the assay. This simple method was shown to be suitable for the quantitation of murine monoclonal antibodies as well as human and rabbit polyclonal antibodies. It should be applicable to a variety of synthetic peptides and polystyrene ELISA plates. Using this technique, we were able to localize the antigenic motifs recognized by neutralizing monoclonal antibodies generated against the envelope protein gp120 of the human immunodeficiency virus. PMID- 1717596 TI - Practical limitations of estimation of protein adsorption to polymer surfaces. AB - The estimation of protein adsorption to polymeric surfaces is a prerequisite for analysing the biological activity of a coated protein both in the evaluation of polymeric biomaterials and in the standardisation of ELISA assays. Direct quantitative methods utilise either measurements of the physical characteristics of the coated surface (equipment necessary for this is not always available), or measurements of radiolabelled protein. We demonstrate that proteins labelled with 125I using chloramine-T contain a significant proportion of 125I label which is unable to adsorb to polyvinyl microtitre wells. This non-bindable label is not separable from protein by fractionation on Sephadex G-25 but does dissociate upon SDS gel electrophoresis. The proportion of non-adsorbable label increases with storage time at -70 degrees C, the effect being accelerated at higher temperatures. A method is described for estimating protein adsorption which is both independent of the non adsorbable fraction but can also be used to estimate it. The use of biotin as a protein label does not apparently result in deterioration of adsorption with short storage times. The problem of subsequent desorption in ELISA buffers, tissue culture media, or body fluids is discussed. PMID- 1717597 TI - Detection of BLT substrate-specific proteases in individual human peripheral blood leucocytes and bone marrow cells. Application of the method to the classification of leukaemia. AB - A trypsin-like serine esterase (SE) is known to be present in cultured cells with cytolytic potential. The distribution pattern of this enzyme in haematological cells and body tissues has been assessed using a method which permits rapid identification of individual cells. Cells and tissue sections were fixed and immersed in the substrate N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)/Fast Blue BB chromogen solution. To identify the phenotype of SE+ cells the cytochemical stain was followed by the application of monoclonal antibody and alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex immunocytochemical procedures. CD8+ and CD57+ lymphocytes showed SE+ granules. Neutrophil granulocytes and progenitors other than undifferentiated myeloblasts developed a dense stain while eosinophils were negative. 35% of monocytes showed positivity mainly in the area of nuclear indentation. Tumour-infiltrating SE+ lymphocytes could also be demonstrated with this method. PMID- 1717598 TI - Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal antibodies against the 7B2 neuroendocrine protein. AB - Three mouse monoclonal antibodies (Moabs) have been obtained with specificity for the 7B2 protein, a proposed member of the granin family of neuroendocrine proteins. Bacterially produced hybrid proteins of 7B2 were used as immunogens. The Moabs were designated MON-100, MON-101, and MON-102. Furthermore, we report the construction of 35 deletion mutants of the glutathione S-transferase-7B2 (GST 7B2) fusion-gene using recombinant DNA technology. The hybrid proteins encoded by eleven of these mutants were used in epitope mapping experiments and the results of these studies strongly suggested that recognition of 7B2 by all three Moabs involved the same 16 amino acid region of 7B2 (from amino acid residue 128-135). This was further substantiated by the observation that MON-101 and MON-102 specifically recognized a conjugate between bovine serum albumin and the synthetic peptide Phe-Glu-Pro-Glu-His-Asp-Tyr-Pro-Gly-Leu-Gly-Lys based upon the deduced amino acid sequence of the predicted epitope region in 7B2. In an approach to generate a series of 7B2-specific Moabs targeted against another epitope region in the 7B2 protein, the hybrid protein encoded by deletion mutant pPV32 was used as the immunogen. This protein lacked the epitope region recognized by the first series of Moabs. A second series of three Moabs, designated MON-142, MON-143, and MON-144, was obtained and, in all three cases, the region of 7B2 from amino acid residue 64-94 appeared to be involved in specific recognition by the Moabs. The whole panel of six anti-7B2 antibodies appeared to be useful in immunoprecipitation and Western blot analysis of the 7B2 protein and specifically stained neuroendocrine cells in immunohistochemical experiments. Using a double determinant sandwich enzyme immunoassay, 7B2 protein levels in rat pituitary were determined as 20 ng/mg tissue. PMID- 1717599 TI - A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation. AB - Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation. In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical [3H]thymidine assay. Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values. Of these tested fluorochromes H33342 appears to be an appropriate alternative to the [3H]thymidine assay. It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates. The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small. This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments. PMID- 1717600 TI - The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond. AB - The large scale preparation of two second generation immunotoxins containing murine monoclonal antibodies and deglycosylated ricin A chain is described. The procedure for the preparation of immunotoxins consists of the derivatization of antibody with SMPT and reduction of dgA with DTT followed by their reaction to establish a hindered interchain disulfide bond. The purification of the immunotoxin includes affinity chromatography on Blue-Sepharose to remove the free antibody and gel filtration on Sephacryl S-200HR to remove any high molecular weight material and free dgA. The two immunotoxins were prepared by GLP procedures and tested for yield, composition, purity, sterility and biological activity. PMID- 1717601 TI - Limiting dilution analysis of human, tetanus-reactive helper T lymphocytes. A rapid method for the enumeration of helper T lymphocytes with specificity for soluble antigens. AB - The number of helper T lymphocytes (HTL) in human peripheral blood with specificity for the soluble protein, tetanus toxoid, was estimated by limiting dilution analysis (LDA). HTL were detected via antigen-induced interleukin-2 (IL 2) production, as measured by incorporation of tritiated thymidine by an IL-2 dependent indicator cell line, CTLL-20. Culture conditions optimizing assay sensitivity are described, and the ability to detect antigen-specific HTL using this LDA technique are demonstrated. Observed HTL frequencies in healthy human donors tested for tetanus-reactive helper T cells ranged from less than 1 HTL/268,749 peripheral blood mononuclear cells (PBMC) (undetectable) to 1 HTL/1486 PBMC. The LDA technique was also used to detect frequency shifts in human peripheral blood HTL following challenge with antigen. This assay offers distinct advantages over proliferative LDA techniques in that it is rapid (requiring only 2 days), and defines an antigen-reactive T cell subset with defined function (IL-2 secretion). Furthermore, the LDA technique can be adapted for the detection of other soluble protein antigens, such as PPD and Candida albicans. In general, this LDA technique provides a reliable, quantitative index of human HTL reactivity to any of a variety of soluble protein antigens, and has clinical as well as experimental applicability. PMID- 1717602 TI - A re-evaluation of the use of Sephadex for the concentration of dilute protein solutions. PMID- 1717603 TI - In vitro measurement of antigen-specific cell-mediated immune responses using recombinant HIV-1 proteins adsorbed to latex microspheres. AB - Recombinant proteins representing full-length and truncated forms of the human immunodeficiency virus type 1 envelope protein gp160 were produced in E. coli and sf9 insect cells. These proteins were denatured and reduced as a function of purification. We adsorbed these proteins onto latex microspheres and used the protein-coated particles as a vehicle to present the antigen in vitro to splenic mononuclear cells from immune mice. Recombinant proteins presented on the latex particles induced antigen-specific proliferative responses that were dependent on the antigen concentration. The proliferative responses were similar to those produced against an identical protein used in soluble form and equivalent protein concentrations. Latex microspheres coated with recombinant proteins could also induce precursor cytotoxic T lymphocytes to mature to functional effector cells in vitro. The use of the latex microspheres to present recombinant proteins as antigens allowed for the use of denatured proteins in our assay that were not soluble in aqueous solutions, such as cell culture media. This system of delivering recombinant proteins in vitro should greatly facilitate the use of recombinant proteins in assays involving live cells. PMID- 1717604 TI - Immunological and biological characteristics of recombinant human thyrotropin. AB - Analyses of epitopes and biological activity were made on two preparations of recombinant human thyrotropin produced in Chinese hamster ovary cells. Seven monoclonal antibodies recognizing four epitopes on alpha subunit of hTSH (hTSH alpha) and three on beta subunit (hTSH beta) were used for the analysis. Binding activities of the two rhTSH with each antibody were almost identical with that of a pituitary-derived reference hTSH, except at one epitope on hTSH alpha. Their immunoreactivity measured by four commercial immunoassay kits and their bioactivity by thyrotropin dependent FRTL-5 cell system, however, agreed closely with those of the reference hTSH. From these results and its constant availability, rhTSH will be a good candidate for a future standard material in assays for hTSH. PMID- 1717605 TI - A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis. AB - Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (C-ANCA), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in Wegener's granulomatosis (WG). Till now, human neutrophil granulocytes have represented the major antigen source used to detect antibodies in WG by the immunofluorescence technique (IFT). We have tested serum samples of 164 patients with different connective tissue diseases (50 suffering from clinically active WG) performing IFT on a human renal cancer line (SK-RC11) and have found antibodies against the nuclear and cytoplasmic antigens in 39 patients. C-ANCA+ sera displayed a characteristic diffuse cytoplasmic staining pattern. Antibody titers measured with human granulocytes were comparable to titers obtained using culture cells. Antibody binding could be inhibited by preabsorption with an extract of human granulocytes or purified proteinase 3. A protein of 29 kDa MW could be isolated by affinity purification using a SK-RC11 extract and a high-titer C-ANCA+ serum and antigenic identity was further confirmed by IFT using a monoclonal antibody to proteinase 3. Treatment of tumor cells with cytokines (interferon, tumor necrosis factor) led to a time dependent translocation of the antigen into the nucleus and back to the cytoplasm. The antigen was also expressed on the surface of live cells colocalized with MHC II. In addition, 21 WG patients had antibodies to cytoplasmic organelles identified by laser scanning microscopy as secretory vesicles of the Golgi complex, and five had antibodies to nuclear antigens. This is, to the best of our knowledge, the first report of proteinase 3 in human non leukemic cells. Our data demonstrate, that the repertoire of antigens recognized by antibodies in WG sera is not limited to human neutrophils and monocytes and indicates a possible functional role of the antigenic proteins. PMID- 1717606 TI - The application of epitope mapping in the development of a new serological test for systemic candidosis. AB - A new serological test for systemic candidosis was developed by raising a rabbit antiserum probe against a specific epitope on Candida albicans, hsp 90. A major fragment at the carboxy terminal end of this immunodominant candidal antigen was epitope mapped by Geysen's method. An epitope, recognised by all infected patients with antibody to the 47 kDa antigen, was synthesized and conjugated to keyhole limpet haemocyanin. A rabbit was successfully immunized against this synthesized peptide epitope and this antiserum was compared, in a dot immunobinding assay, with unfractionated hyperimmune rabbit antiserum to C. albicans and an affinity-purified rabbit antiserum to the 47 kDa antigen. The epitope-specific antibody probe was more sensitive than the hyperimmune candidal antiserum but less sensitive than the affinity-purified antibody against the 47 kDa antigen, which recognised multiple epitopes. This probe is technically easy to prepare in large amounts and gives no false positives. PMID- 1717607 TI - Expression of simple epithelial keratins 8 and 18 in epidermal neoplasia. AB - A systematic study of keratin expression in epidermal lesions (six actinic keratoses, 10 Bowen's disease, seven squamous cell carcinomas) has been undertaken by using a large panel of monospecific monoclonal antibodies to individual keratins. Expression of differentiation-specific keratins was frequently delayed or lost from dysplastic regions. Novel expression of the embryonic, or simple epithelial, keratins 8 and 18 was widely observed in intradermal areas of poorly differentiated squamous cell carcinomas. In addition, the most proliferative of in situ malignancies (Bowen's disease) also contained small numbers of cells expressing simple epithelial keratins. These observations suggest that the expression of simple epithelial keratins may be of functional importance in malignancy of keratinocytes and could be related to tumor invasion and/or to changes in epithelial-mesenchymal interactions. PMID- 1717608 TI - Responses of human dermal microvascular endothelial cells to histamine and their modulation by interleukin 1 and substance P. AB - The action of histamine on human dermal microvascular endothelial cells and modulation of its effects by the cytokine interleukin-1 and the vasoactive neuropeptide substance P have been investigated. Histamine (10(-6)-10(-3) M) induces release of prostaglandin E2 in a concentration- and time-dependent manner. Prostaglandin E2 release is facilitated principally by histamine H1 receptors as the H1 receptor antagonist pyrilamine attenuates prostaglandin E2 release whereas the H2 receptor antagonist cimetidine only slightly reduces release. In contrast to other cells, the histamine/receptor interaction is not associated with increased intracellular accumulation of the cyclic nucleotides, cyclic AMP, or cyclic GMP. Interleukin-1 induces a concentration-dependent release of prostaglandin E2 following 24 h incubation. However, substance P does not increase release of prostaglandin E2 above baseline. In cells incubated with 1 U/ml human recombinant interleukin 1 alpha for 24 h prior to stimulation with histamine (10(-5)-10(-3) M) for 30 min, there is a significant potentiation of histamine-induced release of prostaglandin E2 (p less than 0.05). Using a solubilized cell sonicate prepared from human dermal microvascular endothelial cells incubated with 1 U/ml human recombinant interleukin 1 alpha for 24 h, conversion of exogenous arachidonic acid into prostaglandin E2 increased by 60.19 +/- 18.28%. Cycloheximide partially reduces the increased conversion but completely blocks interleukin-1-induced release of prostaglandin E2 from intact cells. Substance P does not potentiate histamine-induced release of prostaglandin E2 or increase arachidonic acid conversion. These results demonstrate that human dermal microvascular endothelial cells are responsive to histamine and that interleukin-1, but not substance P, can potentiate histamine-induced release of prostaglandin E2. Interleukin-1 appears to act, at least in part, by regulating the availability of free arachidonic acid. Interactions between histamine and interleukin-1 may be important in the modulation of inflammatory reactions in skin. PMID- 1717609 TI - Activities of human acidic fibroblast growth factor in an in vitro dermal equivalent model. AB - Acidic fibroblast growth factor is a potent mitogen for human dermal fibroblasts in an in vitro three-dimensional collagen matrix, the "dermal equivalent." Both cell numbers and DNA synthesis are optimally stimulated by daily doses of 1 ng/ml of the pure human mitogen in the presence of heparin, which binds to, and stabilizes, the protein. Under daily treatment by 1 ng/ml aFGF, the fibroblast mitogenic response is marked but transient, and decreases steadily when fibroblasts mature in the collagen matrix. aFGF mitogenic stimulation also results in a decrease in cellular volume and inhibition of fibroblast-mediated contraction of the collagen gel. Various dosing regimes indicate that, although the greatest mitotic response was generated by daily dosing, nearly optimal responses can also be achieved with either a short duration of early daily dosing or longer-term intermittent treatment. PMID- 1717610 TI - Hair-specific keratins: characterization and expression of a mouse type I keratin gene. AB - A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae. PMID- 1717611 TI - In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. AB - Extensive full-thickness burns require replacement of both epidermis and dermis. We have described a method in which allogeneic dermis from engrafted cryopreserved cadaver skin was combined with cultured autologous keratinocytes. In the present study we combined human keratinocytes and fibroblasts, and acellular human dermis in vitro and transplanted this "reconstituted skin" into athymic mice. Both human papillary dermis in which the basement membrane zone has been retained and human reticular dermis that has been repopulated with human dermal fibroblasts are good substrates for keratinocyte attachment, stratification, growth, and differentiation. Both of these dermal preparations can be lyophilized and stored at room temperature without losing their ability to support keratinocyte growth. In contrast, human papillary dermis that has been treated with trypsin lacks laminin and collagen type IV in the BMZ and supports keratinocyte attachment and differentiation less well. PMID- 1717612 TI - Junctional epidermolysis bullosa keratinocytes in culture display adhesive, structural, and functional abnormalities. AB - An unusual, elongated, refractile cell morphology was observed in keratinocytes cultured from three patients with non-lethalis forms of junctional epidermolysis bullosa (JEB). To determine whether these changes might be related to altered cell adhesion, keratinocyte strains established from one patient were examined for adhesive, structural, and functional characteristics. JEB keratinocytes expressed keratin tonofilaments, as determined by staining with AE1 monoclonal antibodies and direct observation of tonofilaments by electron microscopy. JEB keratinocytes showed diminished cell-substratum adhesions, judged by interference reflection microscopy. Areas of diminished cell-substratum adhesion corresponded to F-actin-rich cell adhesions (focal adhesions) and not to cellular areas that abundantly express hemidesmosomal antigens. Analysis of cell-substratum adhesion by electron microscopy revealed extensive areas of cell-substratum separation in JEB keratinocytes that were not present in normal keratinocytes maintained in serum-free medium. Normal keratinocytes displayed numerous regions of focal contact between the ventral plasma membrane and the culture substratum, but these structures were not seen in JEB keratinocytes. Bundled actin filaments (stress fibers) were greatly diminished in expected regions of cell-substratum adhesion in JEB keratinocytes and, instead, displayed disorganized individual filaments. The growth rate of JEB keratinocytes was quite slow in culture, with a population doubling time of 2.7 d versus 1.5 d for normal keratinocytes under identical conditions. JEB keratinocytes also displayed a reduced ability to aggregate into colonies upon exposure to medium with increased extracellular calcium. JEB keratinocytes thus display adhesive, structural, and functional abnormalities that suggest this cell type may be central to the pathogenesis of junctional epidermolysis bullosa. Study of affected keratinocytes could be important to characterize associated molecular pathologies. PMID- 1717613 TI - The fever induced by polyriboinosinic:polyribocytidylic acid is not related to interferon synthesis in the rabbit's hypothalamus. AB - We previously demonstrated that direct administration of interferon (IFN) or its inducer polyriboinosinic acid:polyribocytidylic acid [poly(I:C)] into the hypothalamus caused dose-dependent fever in rabbits. It was not clear whether the fever induced by intrahypothalamic injection of poly(I:C) was due to stimulation of IFN or prostaglandin E-2 synthesis in the hypothalamic tissues. Therefore, in the current experiments, we used an established model in which rabbit hypothalamic minces or brain cells were incubated in vitro with poly(I:C) to test for the ability of poly(I:C) to stimulate IFN or PGE-2 synthesis. The results showed that poly(I:C) stimulated PGE-2, but not IFN, synthesis in the hypothalamus. Thus, it appears that the fever induced by poly(I:C) is not related to IFN synthesis in the hypothalamus. PMID- 1717614 TI - The antiviral effect of phorbol ester and calcium ionophore A23187 is not mediated by interferons. AB - Phorbol myristate acetate (PMA) and the calcium ionophore, A23187, produce a dose dependent inhibition of vesicular stomatitis virus (VSV) replication in FL cells, with maximum inhibition when cells are pretreated with 1 ng/ml of PMA or 0.5 microM A23187. No interferon (IFN) activity was detected in culture supernatants from cells treated with these agents, and addition of anti-human IFN antibodies did not prevent the inhibition of VSV replication that they caused. However, their antiviral activities were inhibited by actinomycin D or cyclohexamide treatment. Thus, the antiviral activities of PMA and A23187 involve de novo synthesis of protein but are not mediated through the production of IFN. PMID- 1717615 TI - Interferon-induced and double-stranded RNA-activated enzymes: a specific protein kinase and 2',5'-oligoadenylate synthetases. AB - Treatment of cells with interferon (IFN) results in the induction of two double stranded RNA (dsRNA)-activated enzymes: a specific protein kinase and 2'-5' linked oligoadenylate [pppA(2'p5'A)n referred to as 2-5A] synthetases. The protein kinase, when activated by dsRNA, becomes autophosphorylated and catalyzes and phosphorylation of the protein synthesis initiation factor, eIF2. The 2-5A synthetases, when activated by dsRNA, form 2-5A molecules capable of activating a latent endoribonuclease that degrades RNA. By inhibiting initiation of protein synthesis or by degrading of RNA, these enzymes play key roles in two independent pathways that regulate overall protein synthesis. PMID- 1717616 TI - Effect of microgravity modeling on interferon and interleukin responses in the rat. AB - Rats were placed in whole-body harness suspension in three configurations: antiorthostatic hypokinetic/hypodynamic suspension (AAH) to induce headward body fluid redistribution and unload the limbs, orthostatic hypokinetic/hypodynamic suspension (OHH) to unload the limbs without fluid redistribution, and harness restraint (HR) to produce the restraint stress of the model without fluid redistribution or musculoskeletal disuse. AHH and OHH suspension transiently increased interferon-gamma (IFN-gamma) production in response to the mitogen concanavalin A. Harness restraint alone did not affect IFN-gamma response. However, both suspension modeling and harness restraint caused a transient reduction in interleukin-1 (IL-1) and IL-2 responses to mitogen. This suggests that factors associated with musculoskeletal unloading affected IFN-gamma responses, while IL-1 and IL-2 responses were affected by the physiological stress of restraint. PMID- 1717617 TI - [TIMP family--their structures and functions]. PMID- 1717619 TI - [Physiopathology and therapy of idiopathic thrombocytopenic purpura]. PMID- 1717618 TI - Tumor necrosis factor and immunopathology. PMID- 1717620 TI - VH genes of human autoantibodies. PMID- 1717621 TI - A lavage method for dynamic intraarticular monitoring of animal joints in situ: quantification and release kinetics of histamine after selective synovial mast cell activation by diverse secretagogues. AB - Evidence implicating synovial mast cells in the initiation and perpetuation of arthritis has increased. We have developed a method of joint lavage to monitor dynamic intraarticular events in the intact animal. Lavage was done before and after immunologic (passive sensitization followed by intravenous specific antigen or intraarticular anti-rat immunoglobulin E [IgE] heteroantiserum) and nonimmunologic (intraarticular calcium ionophore A-23187; phorbol 12-myristate, 13-acetate; and compound 48/80) synovial mast cell activation. To quantify and analyze synovial mast cell mediator release kinetics in situ, we measured lavage fluid histamine. With all activation protocols except A-23187, histamine release was evident within 5 minutes after introduction of the stimulus. The quantitative and chronological similarities between immunologically induced and compound 48/80 induced synovial mast cell histamine release kinetics suggested that connective tissue type mast cells are an important source of inflammatory mediators in rat joints. We also measured joint lavage fluid histamine levels in rats immunized with an active sensitization protocol. Histamine levels were determined by the autoanalyzer method and were confirmed by using a commercially available radioimmunoassay that uses a monoclonal antibody against acetylated histamine. We found that in many of these animals, at the peak of the serum IgE response, joint lavage fluid histamine levels were very high even before challenge with specific antigen, and that this increase was not due to diffusion into the joint of abnormally elevated plasma histamine. These data suggested that synovial mast cells are preferentially activated in states of high serum IgE immune responses. We have used a simple, inexpensive, rapid lavage technique to generate the first data on histamine release kinetics after selective synovial mast cell activation in the intact animal. The technique can be adapted for investigation of release kinetics of a variety of other substances from activated synovial cells and can be used in other arthritis models. PMID- 1717622 TI - Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BM5, a murine retrovirus. AB - The actions of retroviral infections, aging, and cocaine and morphine injection on cytokine production were investigated in C57BL/6 female mice. Retroviral infection with LP-BM5 murine leukemia virus was further developed as a model of murine acquired immunodeficiency syndrome (AIDS). The effects of cocaine and morphine on gamma-interferon (IFN) and tumor necrosis factor (TNF) production in vivo and with isolated spleen cells were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) method. Serum IFN was generally not detected in any group except mice injected with saline and young mice infected with LP-BM5 virus. Splenocytes from mice with murine AIDS produced less IFN when stimulated in vitro by ConA. In aged mice, IFN production by spleen cells was severely suppressed by retroviral infection. Cocaine had a tendency to suppress IFN production by stimulated cells in vitro. Morphine tended to reduce IFN production by spleen cells from retrovirally infected animals. The serum TNF level in mice with murine AIDS was elevated creating higher levels in morphine and morphine plus cocaine treated uninfected mice while cocaine injection eliminated serum TNF. When stimulated in vitro by lipopolysaccharides (LPS), splenocytes from mice with murine AIDS also produced more TNF than uninfected controls. TNF production in vitro and in vivo was significantly increased by retroviral infection. Therefore, results indicate that cocaine and retroviral infection modulate TNF and IFN production. PMID- 1717624 TI - A case of intermittent ventricular bigeminy. PMID- 1717623 TI - The liver and the hematolymphoid system: I. The regulation of nylon-passed spleen cell proliferation by active factors released from syngeneic nonparenchymal liver cells. AB - Nylon-passed spleen cells were found to proliferate when cultured with syngeneic nonparenchymal adherent liver cells and their culture supernatants. The supernatants contained IL-1, IL-6, GM-CSF, and IFN (alpha + beta) activities but not IL-2 and IL-3 activities. The IFN level was higher in early culture sup (2-24 hr) than in later culture sup (48-72 hr). Proliferation was greatly increased by anti-IFN (alpha + beta) serum in the spleen cells cultured in the earlier sup. This antiserum increased the spleen cell proliferation only slightly in the later culture sup. This suggests that nonparenchymal liver cells produce two factors, one having a suppressor, and the other an enhancer action, with IFN being one of the suppressor factors. With culture time, DNA synthesis of spleen cells increased and IL-2 and IL-3 activities were generated in the culture sup. Cells proliferated during culture were found to be morphologically lymphocytes, granulocytes, and macrophages. The mechanisms by which nonparenchymal liver cells regulate the hematolymphoid system are discussed based on our observations. PMID- 1717625 TI - In-vivo inhibition of the preovulatory LH surge by substance P and in-vitro modulation of gonadotrophin-releasing hormone-induced LH release by substance P, oestradiol and progesterone in the female rat. AB - The effects of substance P (SP) on the preovulatory surge of LH and on the inhibitory and stimulatory effects of oestradiol-17 beta and progesterone on gonadotrophin-releasing hormone (GnRH)-induced LH release were investigated in vivo and in vitro in the rat. A single s.c. injection of 100 micrograms SP at 12.00 h on the day of pro-oestrus significantly decreased the preovulatory surge of LH. In vitro, the inhibitory effect of oestradiol-17 beta on GnRH-induced LH release was not modified by treatment with SP. The stimulatory effect of progesterone on GnRH-induced LH release was reduced by treatment with SP. It is concluded that SP may play a modulatory role in the neuroendocrine control of the preovulatory LH surge. PMID- 1717626 TI - Effect of oestradiol benzoate and tamoxifen on the growth of and induction of progesterone receptors in the uterus and mammary gland of immature pigs. AB - Intramuscular injection of oestradiol benzoate (0.1, 1 or 10 micrograms/kg per day) and tamoxifen (0.1 or 1 mg/kg per day) to 6-week-old immature pigs for 7 days induced a dose-dependent increase in the wet weight of the uterus and in the total content of uterine DNA, RNA and protein. Both compounds also stimulated a dose-dependent increase in the concentration of progesterone receptors in uterine cytosolic extracts (in terms of either fmol/mg DNA or fmol/g uterus). The concurrent administration of tamoxifen with oestradiol benzoate provoked significant (P less than 0.05) increases in total uterine protein and in the concentration of progesterone receptors (P less than 0.01) compared with treatment with oestradiol benzoate alone. Hence tamoxifen is an oestrogen agonist in the uterus of immature pigs. The effects of oestradiol benzoate and tamoxifen on mammary growth in immature pigs were assessed by image analysis of mammary sections across the gland (in a ventro-dorsal direction through the teat). Oestradiol benzoate at 10 micrograms/kg per day stimulated a fourfold increase in mammary duct area (P less than 0.01), and tamoxifen, at doses of 0.1 or 1 mg/kg per day, stimulated a threefold increase (P less than 0.05). Tamoxifen partially inhibited (P less than 0.05) the effect of oestradiol benzoate. The concentration of progesterone receptors was found to be very heterogeneous in cytosol extracts of mammary tissue of immature pigs and independent of treatment with oestradiol benzoate and/or tamoxifen. PMID- 1717627 TI - Flow cytometric analysis of human dental pulp tissue. AB - Existing knowledge regarding the cellular components of the dental pulp has been derived primarily from classical methods of histology and biochemistry. Since observations made from prepared tissue sections are static, it is not clear whether this accurately reflects the cellular dynamics of living pulp tissue. Therefore, we developed a method to analyze vital human pulpal tissue by flow cytometry. To test this method, two analyses of the prepared pulpal tissue were performed. First, the prepared tissue was stained with monoclonal antibodies to detect lymphocyte subpopulations. Second, the tissue was processed for DNA analysis of individual cells. Results demonstrated that lymphocytes bearing CD4 and CD8 antigens were clearly detected in pulpal tissue by this method. No B cells were found in any sample. DNA analysis revealed two distinct cell populations. Approximately 88% were small and 12% were large. According to DNA content, 90% of all cells were noncycling and 10% were cycling. These results demonstrate the feasibility of using flow cytometric analysis to examine, at a quantitative level, the cellular heterogeneity of the human dental pulp. PMID- 1717628 TI - Effects of exercise, hypoxia and feeding on the gastrointestinal blood flow in the Atlantic cod Gadus morhua. AB - Cardiac output, ventral and dorsal aortic blood pressure, heart rate, and coeliac and mesenteric artery blood flow were recorded simultaneously in the Atlantic cod, Gadus morhua L., at rest, during exercise, during hypoxia and after feeding. In the resting unfed animals, coeliac artery blood flow was 4.1 +/- 0.8 ml min-1 kg-1 and mesenteric artery blood flow was 3.5 +/- 1.1 ml min-1 kg-1 (mean +/- S.E.M., N = 10); together, these flows represent approximately 40% of the cardiac output. Exercise or exposure to hypoxia resulted in increased visceral vascular resistance, leading to reductions in the coeliac and mesenteric artery blood flows. Coeliac and mesenteric blood flows were increased 24 h after feeding and the coeliac and systemic vascular resistances decreased in comparison with the prefeeding values. Phentolamine did not affect the gastrointestinal artery blood flow, but produced a significant decrease in the mesenteric and systemic vascular resistance. Treatment with bretylium and phentolamine revealed differences between the coeliac and the mesenteric vasculature regarding the control mechanisms during hypoxia and during exercise and feeding. During hypoxia, an adrenergic control of the gastrointestinal vasculature with both nervous and humoral components was found, whereas during exercise and after feeding an additional non-adrenergic mechanism controlling gut blood flow was demonstrated. PMID- 1717629 TI - Increased plasma level of substance P in patients with severe congestive heart failure treated with ACE inhibitors. AB - The effects of angiotensin-converting-enzyme (ACE) inhibitors on circulatory regulating mechanisms in congestive heart failure (CHF) were studied by comparison of plasma levels of catecholamines, neuropeptide Y-like immunoreactivity (NPY-LI), substance P (SP-LI), calcitonin gene-related peptide (CGRP-LI), vasopressin (ADH-LI), atrial natriuretic peptide (ANP-LI) and renin activity (PRA) in patients with severe CHF (NYHA III-IV) with (n = 15) or without (n = 17) ACE inhibitors in addition to digoxin and diuretic therapy. Data were also compared with those for healthy subjects (n = 31) and patients with moderate CHF (NYHA I-II). Catecholamines and NPY-LI were increased to the same extent in both groups with severe CHF. CGRP-LI showed no changes relative to controls in any of the patient groups, and was not affected by ACE inhibitors. The SP-LI level was significantly increased in all patient groups. Patients with severe CHF on ACE inhibition had a SP-LI level of 4.05 +/- 0.79 pmol l-1, compared to a concentration of 2.28 +/- 0.30 pmol l-1 (P less than 0.05) in the patient group with a comparable degree of CHF but without ACE inhibition. In the latter group, an inverse relationship appeared between the SP-LI and the serum sodium levels (r = -0.68, P less than 0.05). The patients with severe CHF who received ACE inhibitors had significantly lower ADH-LI levels than the patients with a comparable degree of CHF who were not treated with ACE inhibitors, while the ANP LI levels was increased to a similar extent in both groups. PMID- 1717630 TI - Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. AB - Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR. PMID- 1717631 TI - Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins. AB - The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3 zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed. PMID- 1717632 TI - Antigen-driven bystander suppression after oral administration of antigens. AB - Suppression of experimental autoimmune encephalomyelitis (EAE) in Lewis rats by the oral administration of myelin basic protein (MBP) is mediated by CD8+ T cells that can be isolated from the spleens of MBP-fed animals. These cells adoptively transfer protection to naive animals subsequently immunized with MBP and complete Freund's adjuvant (CFA) and suppress in vitro MBP proliferative responses. Using a transwell system in which the modulator spleen cells from MBP-fed animals are separated by a semipermeable membrane from responder cells, MBP, or OVA-specific T cell lines, we have found that cell contact is not required for in vitro suppression to occur. In vitro suppression is dependent, however, upon antigen specific triggering of modulator T cells. Once antigen-specific triggering occurs, suppression across the transwell is mediated by an antigen-nonspecific soluble factor that equally suppresses an MBP line or an ovalbumin (OVA) line. This phenomenon of antigen-driven bystander suppression was also demonstrated in vivo. Specifically, Lewis rats fed OVA which were then immunized with MBP/CFA plus OVA given separately subcutaneously were protected from EAE. Animals fed OVA and then immunized with MBP/CFA without OVA given subcutaneously were not protected. The protective effect of feeding OVA could be adoptively transferred by CD8+ T cells from OVA-fed animals into MBP/CFA plus OVA-injected animals. Feeding bovine serum albumin (BSA) or keyhole limpet hemocyanin did not suppress EAE in animals immunized with MBP/CFA plus OVA. EAE was suppressed, however, if BSA was fed and animals then immunized with MBP/CFA plus BSA given subcutaneously. Antigen-driven bystander suppression appears to be an important mechanism by which antigen-driven peripheral tolerance after oral administration of antigen is mediated, and presumably occurs in the microenvironment accounting for the antigen specificity of suppression generated by oral tolerization to antigens. PMID- 1717634 TI - Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides. AB - We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product. PMID- 1717635 TI - Determining the viability of early pregnancies: two case reports. PMID- 1717633 TI - Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1. AB - Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM 1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo. PMID- 1717636 TI - Identification of a stretch-activated monovalent cation channel from teleost urinary bladder cells. AB - The urinary bladder of euryhaline teleost is an important osmoregulatory organ which absorbs Na+, Cl-, and water from urine. Using patch clamp technique, single stretch-activated channels, which were permeable to K+ and Na+ (PNa/PK approximately 0.75) and had conductances of 55 and 116 pS, were studied. In excised, inside-out patches which were voltage-clamped in the physiological range of membrane potential, the single-channel open probability (Po) was low (approximately 0.02), and increased to a maximum of 0.9 with applied pipette suction. Single-channel conductance also increased with suction. The channels showed adaptation to applied suction and relaxed to a steady-state activity about 20 seconds after application of suction. The Po increased up to 0.9 with strong membrane depolarization (Vm = 0 to +80 mV); however, there was little dependence of Po on membrane potential in the physiological range. The kinetic data suggest that there is one conducting state and at least two non-conducting states of the channel. The open-time constant increased with suction but remained unchanged with membrane potential (Vm = -70 to +60 mV). The mean closed-time of the channel decreased with suction and membrane depolarization. These results demonstrate the presence of a non-selective monovalent cation channel which may be involved in cell volume regulation in the goby urinary bladder. Additionally, this channel may function as an enhancer of Na+ influx and K+ efflux across the bladder cell as part of transepithelial ion transport if it is located in apical membrane. PMID- 1717638 TI - [Antenatal screening of maternal alpha-fetoprotein with dried-blood spot samples on filter paper]. AB - Abnormal levels of maternal serum alphafetoprotein (AFP) have been used as a good biochemical marker for the screening of neural tube defects (NTD) and Down syndrome. From January 1988 to June 1989, we conducted a community-based maternal AFP screening program in Nan-Tou County, which is located in the middle of Taiwan. A total of 3,776 pregnant women, which accounted for about 42% of the total number of women pregnant during that period of time in Nan-Tou county, were screened using the filter paper blood collecting technique. Of those screened, 5.1% of them were found to be borderline positive for NTD (2.0 less than MoM less than 2.5). The screening positive rates for NTD (MoM greater than 2.5) and Down syndrome (Down's risk greater than 1/100) were 3.7% and 1.7%, respectively. Except for those lost to follow-up, 3 (2.1%) of the borderline cases were confirmed to have serious pregnancy complications (porencephaly, fetal demise, and imminent miscarriage). Eleven (10.8%) of the abnormal pregnancies, which included 1 case of possible hydrocephalus, 2 cases of anencephalus, and 1 case of triploid, were found in the NTD positive group. Six (18.7%) anomalies (1 Down syndrome, 1 hydatidiform mole, and 4 miscarriages) were detected in the increased Down's risk group. Totally, 20 adverse pregnancies, i.e. 0.5% (20/3,776) of the screened women, were confirmed in this screening program. No false-positive cases were reported, or were found among the 1,777 post-delivery questionnaires completed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717637 TI - Granulomatous nasopharyngeal carcinoma: with emphasis on difficulty in diagnosis and favorable outcome. AB - Nasopharyngeal carcinoma (NPC) can provoke a local granulomatous reaction which may cause diagnostic difficulty. To further elucidate this possible diagnostic pitfall, 47 cases who were initially diagnosed as tuberculosis or granulomatous inflammation were reexamined; 7 cases (15%) were found to have NPC. In a routine histological stain, residual malignant tumor cells could be identified in 2 cases. In the remaining 5 cases, malignant tumor cells could only be accurately identified after careful examination. Immunohistochemical staining for keratin easily demonstrated the residual tumor cells engulfed in a granulomatous lesion in all 7 cases. This finding suggests that an immunohistochemical stain for keratin is useful and should be performed for any nasopharyngeal biopsy showing granulomatous lesion with suspicion of malignancy, particularly in countries where NPC is prevalent. The overall 5-year survival rate among these NPC patients was 83%, a most favorable outcome, which suggests that a granulomatous reaction may reflect a favorable local host, and a cell-mediated immune response. PMID- 1717639 TI - Molecular fingerprinting of isolates of the genus Peptostreptococcus using rRNA genes from Escherichia coli and P. anaerobius. AB - Restriction fragment length polymorphisms (RFLPs) of rRNA genes were evaluated as a tool for intra- and interspecies differentiation of Peptostreptococcus isolates. RFLPs from a collection of 20 clinical isolates and five ATCC strains representing five Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. magnus, P. micros and P. prevotii) were obtained by hybridization of Southern blots of HindIII- or EcoRI-digested genomic DNA with three probes: probe A, a 0.98 kb HindIII fragment with a partial 16S rRNA gene sequence from P. anaerobius ATCC 27337; probe B, cloned Escherichia coli rrnB operon in plasmid pKK3535; and probe C, E. coli 16S and 23S rRNA. The hybridization patterns varied, but all yielded RFLPs useful for both intra- and inter-species differentiation. RFLPs of P. asaccharolyticus clinical isolates were closely related to each other and differed significantly from those of the ATCC type strains. The profiles of P. prevotii differed from those of the other four species studied, and based on the HindIII- and EcoRI-generated RFLPs, the strains in this species are more heterogeneous than the other four species studied. PMID- 1717640 TI - Reciprocal phenotype alterations between two satellite RNAs of cucumber mosaic virus. AB - Cucumber mosaic virus Y satellite RNA (Y-satRNA) induces distinctive yellow mosaic symptoms on tobacco, whereas S19 satellite RNA (S19-satRNA) causes an attenuated green mosaic on tobacco, although they show considerable sequence identity. Biological assays of infectious chimeric satellite RNA molecules synthesized from cDNA clones of Y-satRNA and S19-satRNA using common restriction sites showed that the determinant for the induction of yellow mosaic symptoms lies in the BstXI-NheI fragment, in which 14 nucleotide differences are found between the two satellite RNAs. To define more precisely the yellow mosaic determinant(s) in this fragment, several site-directed mutants of Y-satRNA were created. The replacement of AUU, at nucleotides 191 to 193 in Y-satRNA, with GC, which mimics the S19-satRNA sequence at the corresponding site, abolished the ability of Y-satRNA to elicit a yellow mosaic. Conversely, a mutant RNA molecule derived from S19-satRNA in which GC at nucleotides 192 and 193 was changed to AUU induced the yellow mosaic symptoms. Thus, the phenotypes of two satellite RNAs on tobacco can be altered reciprocally by changing the sequences in this limited region. PMID- 1717641 TI - Antigenic structure of chimeras of type 1 and type 3 polioviruses involving antigenic sites 2, 3 and 4. AB - Chimeric polioviruses have been made in which regions of the type 1 Sabin strain corresponding to antigenic sites 2, 3 and 4 have been replaced by the corresponding regions of the type 3 Sabin strain. Manipulation of one site or a component of it generally did not affect the reactions of the others, suggesting that they form independent structural features. The extent to which the inserted site expressed the antigenic properties of type 3 could be assessed by reaction with polyclonal or monoclonal antibodies, or by immunogenicity. Site 2 could be expressed on infectious virus and site 3 on heated non-infectious virus (C antigen), but not on the native virion. The results are consistent with the view that sites consisting of a continuous sequence of amino acids may be presented on chimeras, whereas more complex sites, such as site 4 or site 3 of the native virion, are transferred less readily from type 3 to type 1. PMID- 1717642 TI - Generation of virus genetic lineages during an outbreak of poliomyelitis. AB - Wild poliovirus type 3 isolates collected during the Finnish outbreak (1984 to 1985) in different geographical locations were compared by partial RNA sequencing. The entire 5' non-coding end and a discontinuous part of the capsid coding region were sequenced from 15 isolates. Combining the present sequence data with previously published data and analysing these by the maximum parsimony method showed that the epidemic strains had diverged in cocirculating lineages. Genetic comparison of strains isolated from a single person often revealed a branched structure in the phylogenetic tree indicating high potential for diversification. The extent of variation generated under immunological pressure during an infection lasting for weeks in one person was high as compared with the observed geographical variation. PMID- 1717643 TI - Enzootic nasal tumour of goats: demonstration of a type D-related retrovirus in nasal fluids and tumours. AB - Nasal exudate and tumour tissue from goats with enzootic nasal tumours were shown to contain a reverse transcriptase activity associated with a particle of buoyant density typical of retroviruses. The same particle contained a 25,000 Mr protein that cross-reacted with the p27 of Mason-Pfizer monkey virus (MPMV) and with p25 of sheep pulmonary adenomatosis retrovirus. It also contained a low Mr protein related to p10-12 of MPMV. PMID- 1717644 TI - Spontaneously productive C-type retrovirus infection of fish cell lines. AB - The spontaneous production and release of morphologically typical, 85 to 90 nm diameter C-type retrovirus particles from four cell lines derived from three species of warmwater fish have been identified. Virus pellets from cell culture supernatants showed high levels of Mn(2+)-dependent reverse transcriptase activity at 24 degrees C. Peak enzyme activity was associated with a 1 x 16 g/ml sucrose gradient fraction. All four isolates induced a cytolytic infection of a bluegill fry cell line within 6 to 10 days. PMID- 1717645 TI - The 5'-terminal sequence of potato leafroll virus RNA: evidence of recombination between virus and host RNA. AB - The discrepancy between published sequences of the 5' non-coding regions of RNA of a Scottish (S) and that of Dutch (D), Australian and Canadian isolates of potato leafroll virus (PLRV) was investigated. Reverse transcription followed by amplification by polymerase chain reaction showed that RNA from three distinct Scottish isolates of PLRV contained molecules with 5' ends like that of the original Scottish isolate. However, determination of the 5'-terminal sequences of RNA in two of these preparations showed that most RNA molecules had 5' termini like those of the Dutch and other non-Scottish sequences. Northern blot analysis confirmed that only a small fraction of PLRV RNA contained sequences homologous to the 5'-terminal 119 nucleotides of the PLRV-S sequence. Most PLRV-S RNA molecules therefore have termini like that reported for PLRV-D. The 5'-terminal 119 nucleotides of the minor species of PLRV-S RNA were very similar (109/119 nucleotides were identical) in sequence to an exon of tobacco chloroplast DNA open reading frame 196. The results therefore suggest that recombination has occurred between virus RNA and host RNA. PMID- 1717646 TI - Evaluation of a chimpanzee colony for antibodies to hepatitis C virus. AB - The chimpanzee is the only species other than man that is generally susceptible to infection by hepatitis C virus (HCV). Aspects of future studies on vaccines and therapeutics for HCV may continue to depend on the chimpanzee. In an attempt to determine the HCV status of the animals in a chimpanzee colony, the recently developed enzyme immunoassay (EIA) for antibodies to HCV was used. The results of the assay indicated that only 31.3% of the animals that had previously been inoculated with a non-A, non-B hepatitis agent were currently positive in the assay. A retrospective analysis suggested that an additional 20% of the chimpanzees had been positive at some time following infection. Seroconversion to an anti-HCV antibody response using this assay did not appear to correlate with the severity of the initial disease or the development of chronic liver damage. Examination of the EIA-positive samples using the second generation recombinant immunoblot assay (RIBA) containing four HCV antigens suggested that chimpanzees responded differentially to these antigens. Examination of serum samples from 139 uninoculated animals by EIA revealed seven positive samples and ten samples with borderline values. The nature of the reactivities in most of the positive samples could not be resolved, but analysis by RIBA indicated that at least one animal in the breeder colony had been exposed to HCV. Due to the low seroconversion rate and the uncertainties surrounding many of the positive reactions, this assay cannot be used to determine the initial source or extent of spread of HCV infections in the uninoculated animals. PMID- 1717647 TI - Axotomy accelerates slow component b of axonal transport. AB - Because the integrity of an axon depends on the supply of proteins synthesized in the cell body, we examined the effect of axotomy on the transport of structural proteins in rat motor axons, and the effect of altered transport on the rate of outgrowth after a subsequent testing axotomy. To examine the axonal transport of structural proteins, we labeled newly synthesized proteins with 35S-methionine 7 days after a "conditioning" lesion of the sciatic nerve, and removed the nerve 7 21 days later for SDS-PAGE. Tubulin, actin, calmodulin, and the 68-kD light neurofilament protein (NF-L) were identified by fluorography and removed for liquid scintillation counting. The fastest moving structural proteins were carried by slow component b (SCb) of axonal transport, which advanced 20% faster in conditioned axons: 4.2 versus 3.5 mm/day (p less than 0.01). NF-L was not accelerated, indicating that the motor for subcomponent a (SCa) of slow axonal transport was unaffected by axotomy. To measure outgrowth distances, the testing lesions was made 7 days after the conditioning lesion, and growth cones were located by the fast transport method 3 or 9 days later. The regression analysis of outgrowth distance on time showed that sprouts elongated 25% faster in conditioned axons: 4.0 versus 3.2 mm/day (p less than 0.001). These accelerated sprouts were formed too far from the spinal cord to contain SCb proteins that were synthesized after axotomy. Because the rate of outgrowth correlated closely with the rate of SCb in outgrowing sprouts (McQuarrie and Jacob, J. Comp. Neurol. 305:139-147, 1991), we conclude that SCb is accelerated throughout the length of the axon by 7 days after axotomy. PMID- 1717648 TI - Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex. AB - The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine paracrine system. PMID- 1717649 TI - D1 dopamine receptors of NS20Y neuroblastoma cells are functionally similar to rat striatal D1 receptors. AB - Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-) butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum. PMID- 1717650 TI - Stress- and endotoxin-induced increases in brain tryptophan and serotonin metabolism depend on sympathetic nervous system activity. AB - Stressful treatments and immune challenges have been shown previously to elevate brain concentrations of tryptophan. The role of the autonomic nervous system in this neurochemical change was investigated using pharmacological treatments that inhibit autonomic effects. Pretreatment with the ganglionic blocker chlorisondamine did not alter the normal increases in catecholamine metabolites, but prevented the increase in brain tryptophan normally observed after footshock or restraint, except when the duration of the footshock period was extended to 60 min. The footshock- and restraint-related increases in 5-hydroxyindoleacetic acid (5-HIAA) were also prevented by chlorisondamine. The increases in brain tryptophan caused by intraperitoneal injection of endotoxin or interleukin-1 (IL 1) were also prevented by chlorisondamine pretreatment. The footshock-induced increases in brain tryptophan and 5-HIAA were attenuated by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phenoxybenzamine or the muscarinic cholinergic antagonist atropine. Thus the autonomic nervous system appears to be involved in the stress-related changes in brain tryptophan, and this effect is due to the sympathetic rather than the parasympathetic limb of the system. Moreover, the main effect of the sympathetic nervous system is exerted on beta- as opposed to alpha-adrenergic receptors. We conclude that activation of the sympathetic nervous system is responsible for the stress-related increases in brain tryptophan, probably by enabling increased brain tryptophan uptake. Endotoxin and IL-1 also elevate brain tryptophan, presumably by a similar mechanism. The increase in brain tryptophan appears to be necessary to sustain the increased serotonin catabolism to 5-HIAA that occurs in stressed animals, and which may reflect increased serotonin release. PMID- 1717651 TI - Genomic organization of Drosophila choline acetyltransferase. AB - Choline acetyltransferase (ChAT; acetyl-CoA:choline-O-acetyltransferase; EC 2.3.1.6) is the enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is thus the genetic determinant of neurons with a cholinergic phenotype. We have screened a Drosophila genomic library using a cRNA probe, transcribed from Drosophila ChAT cDNA, and isolated three independent clones representing all the exons of this gene. The gene spans more than 26 kb of DNA and is organized into eight exons containing 594, 80, 192, 759, 408, 147, 201, and 1,612 nucleotides. All inserts that hybridized with a cRNA probe have been subcloned and the sequence of intron/exon boundaries determined. The only part of the ChAT gene not represented in our clones is a part of the first intron. A minimum size for this uncloned DNA has been deduced from Southern analysis of Drosophila genomic DNA. We also have probed the transcripts of the ChAT gene by northern analysis of total Drosophila RNA using two different exon-specific antisense RNA probes. An exon I probe detected two bands of RNA whereas an exon VIII probe hybridized with only the smaller band, previously identified as ChAT mRNA. These results indicate a complex transcription pattern for the ChAT locus in Drosophila. PMID- 1717652 TI - Phosphorylation of nuclear proteins in myelinating oligodendrocytes and its control by cyclic AMP. AB - Oligodendroglial nuclei isolated from rat brains at different stages of myelinogenesis (10, 18, and 30 days of age) were incubated with [gamma-32P]ATP and extracted with 0.75 M perchloric acid to yield a fraction of nonacidic chromatin proteins. The protein extracts were then analyzed by polyacrylamide gel electrophoresis. The phosphorylation pattern of these proteins was found to be different for different age groups. In 10-day-old rat oligodendrocytes the most extensive phosphorylation occurred in low molecular mass species (less than 30 kDa), in contrast to fractions obtained from 18- and 30-day-old rat oligodendrocytes which showed a significantly higher labeling of the proteins with molecular masses greater than 30 kDa. The phosphorylation of the latter species was greatly stimulated by the presence of cyclic AMP in the incubation media. The results suggest that the phosphorylation of specific nuclear proteins, which may play a regulatory role at different stages of oligodendroglial maturation and myelinogenesis, may be at least partially modulated by intracellular cyclic AMP. PMID- 1717653 TI - Monoclonal antibodies against myelin proteolipid protein: identification and characterization of two major determinants. AB - This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes. PMID- 1717654 TI - Internal Ca2+ mobilization by muscarinic stimulation increases secretion from adrenal chromaffin cells only in the presence of Ca2+ influx. AB - The cytosolic free Ca2+ concentration ([Ca2+]in) in single cat and bovine adrenal chromaffin cells was measured to determine whether or not there was any correlation between the [Ca2+]in and the catecholamine (CA) secretion caused by muscarinic receptor stimulation. In cat chromaffin cells, methacholine (MCh), a muscarinic agonist, raised [Ca2+]in by activating both Ca2+ influx and intracellular Ca2+ mobilization with an accompanying CA secretion. In bovine cells, MCh elevated [Ca2+]in by mobilizing intracellular Ca2+ but did not cause CA secretion. The MCh-induced rise in [Ca2+]in in cat cells was much higher than that in bovine cells, but when Ca2+ influx was blocked, the rise was reduced, with a concomitant loss of secretion, to a level comparable to that in bovine cells. Intracellular Ca2+ mobilization due to muscarinic stimulation substantially increased secretion from depolarized bovine and cat cells, where a [Ca2+]in elevated above basal values was maintained by a continuous Ca2+ influx. These results show that Ca2+ released from internal stores is not effective in triggering secretion unless Ca2+ continues to enter across the plasma membrane, a conclusion suggesting that secretion depends on [Ca2+]in in a particular region of the cell. PMID- 1717655 TI - Cyclic AMP accumulation induces a rapid desensitization of the cyclic AMP dependent protein kinase in mouse striatal neurons. AB - Striatal neurons from the mouse brain embryo grown in primary culture express high levels of cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. To study the modulation of PKA in intact neurons, a rapid method based on Zn(2+) protein precipitation was developed. This strategy allowed analysis of the stimulation of PKA under conditions of intracellular cAMP concentration increases. Whereas increases up to 1 microM lead to an activation, large and sustained accumulations of cAMP result in a loss of the enzyme activity. With 8 bromo-cAMP (8-Br-cAMP) at 100 microM, the PKA refractoriness occurs within 2 min. It is rapidly reversible because incubation of treated neurons in fresh medium leads to a complete recovery of PKA activity within 30 min. The decrease in assayable PKA does not involve an activation of phosphatases because the histone dephosphorylation rate is not affected by 8-Br-cAMP treatment. Finally, not only 8-Br-cAMP- but also forskolin- and vasoactive intestinal peptide-induced increases in intracellular cAMP concentration can lead to the PKA desensitization. Altogether, these data unravel a new mechanism of PKA regulation. PMID- 1717656 TI - Regulation of cyclic AMP levels by calcium in bovine adrenal medullary cells. AB - Both nicotine and histamine have been reported to increase cyclic AMP levels in chromaffin cells by Ca(2+)-dependent mechanisms. The present study investigated whether Ca2+ was an adequate and sufficient signal for increasing cyclic AMP in cultured bovine adrenal medullary cells. Depolarization with 50 mM K+ caused a two- to three-fold increase in cellular cyclic AMP levels over 5 min, with no change in extracellular cyclic AMP. This response was abolished by omission of extracellular Ca2+ and by 100 microM methoxyverapamil, and was unaffected by 1 microM tetrodotoxin and by 1 mM isobutylmethylxanthine. Veratridine (40 microM) also increased cellular cyclic AMP levels by two- to fourfold. This response was abolished by either methoxyverapamil or tetrodotoxin. The Ca2+ ionophore A23187 (10-50 microM) had little or no effect on cellular cyclic AMP levels. When the concentration of K+ used to depolarize the cells was reduced to 12-15 mM, the catecholamine release was similar to that induced by 50 microM A23187, and the cyclic AMP response was almost abolished. The results suggest that Ca2+ entry into chromaffin cells is a sufficient stimulus for increasing cellular cyclic AMP production. The possible involvement of a Ca2+/calmodulin-dependent isozyme of adenylate cyclase is discussed. PMID- 1717657 TI - Histamine: a neurotransmitter candidate for Drosophila photoreceptors. AB - Recent experimental evidence suggests that histamine might be the synaptic transmitter used by invertebrate photoreceptors. In the present study, we have examined whether histamine is a transmitter candidate for Drosophila photoreceptors. Our findings are as follows: (a) Large amounts of histamine are synthesized by wild-type heads, whereas heads from the eye-deficient mutants, eyes absent and sine oculis, show reduced histamine synthesis. (b) Histidine decarboxylase activity is approximately 10-fold higher in extracts of normal heads compared with that in the mutants. (c) Histamine taken up by fly heads is metabolized into N-acetylhistamine and imidazole-4-acetic acid. (d) Immunostaining of normal and sevenless heads with histamine-specific antisera demonstrates that histamine is present in photoreceptors R1-6 and R8. (e) Histamine synthesized from exogenously supplied [3H]histidine can be released by depolarization with 50 mM K+, and the release is Ca2+ dependent. These observations strongly suggest that histamine is a major neurotransmitter used by Drosophila photoreceptors. PMID- 1717658 TI - Regulation of five tubulin isotypes by thyroid hormone during brain development. AB - Nucleic acid probes derived from the 3' noncoding region of five tubulin cDNAs were used to study the effects of thyroid hormone deficiency on the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2)- and three beta (beta 2, beta 4, and beta 5)-tubulin isotypes in the developing cerebral hemispheres and cerebellum. The content of alpha 1, which markedly declines during development in both brain regions, is maintained at high levels in the hypothyroid cerebellum, whereas it is decreased in the cerebral hemispheres. The alpha 2 level also declines during development and is decreased in both regions by thyroid hormone deficiency, but only during the two first postnatal weeks. Thyroid hormone deficiency slightly increases at all stages the beta 2 level in the cerebellum, whereas a decrease is observed at early stages in the cerebral hemispheres. The beta 5 level seems to be independent of thyroid hormone in the cerebral hemispheres, whereas it decreases at early stages in the hypothyroid cerebellum. Finally, the expression of the brain-specific beta 4 isotype is markedly depressed by thyroid hormone deficiency, particularly in the cerebellum. These data suggest that the genes encoding the tubulin isotypes are, directly or not, differently regulated by thyroid hormone during brain development. This might contribute to abnormal neurite outgrowth seen in the hypothyroid brain and therefore to impairment in brain functions produced by thyroid hormone deficiency. PMID- 1717659 TI - Phorbol ester pretreatment desensitizes the inhibition of Ca2+ channels induced by kappa-opiate, alpha 2-adrenergic, and muscarinic receptor agonists. AB - Acute treatment of rat spinal cord-dorsal root ganglion cocultured neurons with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known activator of protein kinase C, inhibited the dihydropyridine-sensitive voltage-dependent 45Ca2+ influx measured in these cells (IC50 of approximately 100 nM, 66% inhibition at 1 microM TPA). However, prolonged preincubation (24 h) of the cells with 100 nM TPA followed by extensive washing completely abolished, i.e., desensitized, the capacity of a second application of TPA to inhibit the activity of the voltage dependent Ca2+ channels. Moreover, this treatment also abolished the inhibition of Ca2+ influx produced by kappa-opiate as well as by alpha 2-adrenergic and muscarinic receptor agonists. Substantial desensitization was already observed following a 1-h pretreatment with 100 nM TPA. In contrast to TPA, an inactive phorbol ester (4 beta-phorbol 13-acetate) did not affect the inhibition of the voltage-dependent Ca2+ influx by these receptor agonists. These results suggest that protein kinase C may have a role in the modulation of Ca2+ channels by kappa opiate, alpha 2-adrenergic, and muscarinic receptor agonists. PMID- 1717660 TI - Anatomical basis for a parasympathetic and sensory innervation of the intracranial segment of the internal carotid artery in man. Possible implication for vascular headache. AB - Two ganglionic cell groups, located close together and called the internal carotid ganglion, not described before in man, were demonstrated extradurally on the ventrolateral surface of the human internal carotid artery (ICA), where the greater superficial petrosal nerve is joined by the (greater) deep petrosal nerve to form the vidian nerve. The two ganglionic cell groups have fiber connections to the ICA, and consist of 50-70 cells each. By immunohistochemistry the majority of cells in one of the groups were shown to contain vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT) indicating a parasympathetic function, whereas most cells in the other group contained substance P (SP) and possibly calcitonin gene-related peptide (CGRP), transmitters in pain fibers. Lateral to the intracavernous segment of ICA 10-150 scattered or aggregated VIP- and ChAT-positive cells were found, with fiber connections to the ophthalmic nerve, the ICA, the abducent nerve and the sphenopalatine ganglion. These cells may represent aberrant parasympathetic (sphenopalatine) ganglia, here referred to as cavernous ganglion. By radioimmunoassay substantial amounts of VIP, SP and CGRP were measured in both the extradural and the intracavernous segment of the ICA. Thus, the intracranial segment of the ICA is most likely innervated by parasympathetic and pain fibers from the internal carotid ganglion, sensory fibers from the ophthalmic division of the trigeminal ganglion, and parasympathetic fibers from the sphenopalatine and/or cavernous ganglion. Clinical implications for the activation of these nerves to cause pain, dilatation and edema in this segment of the ICA during attacks of cluster headache and painful ophthalmoplegic syndromes are discussed. PMID- 1717661 TI - Nerve conduction velocity and axonal transport of 6-phosphofructokinase activity in galactose-fed rats. AB - This study examined sciatic motor nerve conduction velocity (MNCV) and the accumulation of 6-phosphofructokinase (PFK) activity proximal and distal to a sciatic nerve ligature in rats fed a diet containing 20% galactose for 4 weeks. MNCV was reduced in the galactose-fed rats to 94% of controls, (P less than 0.05) but PFK activity accumulations were not different from those in controls. Daily administration of the aldose reductase inhibitor ponalrestat throughout the study to another group of galactose-fed rats prevented dulcitol accumulation, myo inositol depletion and increased water content of the sciatic nerve seen in galactose-fed rats. This effective aldose reductase inhibition also prevented the reduced MNCV and had no effect on accumulations of PFK activity. These data support earlier work suggesting that deficits in the axonal transport of PFK activity in diabetic rats are unrelated to either exaggerated flux through the polyol pathway, polyol accumulation or ischemic hypoxia and indicate the possible need for the elucidation of other pathogenic mechanisms which may contribute to the development of diabetic neuropathy. PMID- 1717662 TI - P2 specific lymphocyte transformation in Guillain-Barre syndrome and chronic idiopathic demyelinating polyradiculoneuropathy. AB - Thymidine incorporation proliferation assays to whole bovine P2 protein and its 58-81 and 14-25 synthetic peptides were performed on blood mononuclear cells from ten patients with Guillain-Barre syndrome (GBS), six patients with chronic idiopathic demyelinating polyradiculoneuropathy (CIDP), and age and sex matched normal subjects. The only patients whose cells showed any response were two out of four with very early GBS. One responded to P2 and both synthetic peptides. One responded to P2 but to neither peptide. The results support a role for cell mediated immunity to P2 protein in some patients with Guillain-Barre syndrome. PMID- 1717663 TI - Age-related changes in cerebrospinal fluid protein concentrations. AB - The blood-brain barrier (BBB) has a key role in CSF homeostasis and preservation of normal neuronal function. There has been little investigation of changes in barrier function in elderly subjects without evidence of neurological disease. The ageing brain demonstrates increased vulnerability to a variety of insults, which may reflect a deterioration in barrier performance. Direct measurement of BBB function is difficult but the CSF/serum quotients of individual proteins is the most widely used parameter. The present study sought age related changes in the CSF/serum quotients of prealbumin, albumin, immunoglobulin G and alpha 2 macroglobulin in adults with no neurological deficit. A prospective study was established, and 107 subjects (aged 18-89 yrs) were recruited from patients undergoing spinal anaesthesia or diagnostic myelography. A weak but significant positive correlation was found between the CSF/serum quotients and age for all the proteins studied, but with the exception of alpha 2-macroglobulin, these changes probably reflect age related falls in serum protein concentrations found in this study. alpha 2-Macroglobulin appears to be a sensitive indicator of barrier function for large populations, suggesting a subtle decline of BBB integrity with increasing age. This may be a true age-related change or could reflect an increased incidence of subclinical disease in an ageing population. PMID- 1717664 TI - Apoptosis in the nervous system in experimental allergic encephalomyelitis. AB - We report here for the first time the occurrence of apoptosis of cells in the spinal cord in experimental allergic encephalomyelitis (EAE), an autoimmune, T cell-mediated demyelinating disease. Four different forms of EAE were studied in the Lewis rat: (i) acute EAE induced by inoculation with whole spinal cord and adjuvants; (ii) acute EAE induced by inoculation with myelin basic protein (MBP) and adjuvants; (iii) acute EAE induced by the passive transfer of MBP-sensitized spleen cells; (iv) chronic relapsing EAE induced by inoculation with whole spinal cord and adjuvants followed by treatment with low-dose cyclosporin A. Cells undergoing apoptosis were recognized at light and electron microscopy by the presence of either crescentic masses of condensed chromatin lying against the nuclear envelope or rounded masses of uniformly dense chromatin. They were found in both the white and grey matter of the spinal cord in all 4 forms of this disease. Although it was not possible to identify definitively the types of cells undergoing apoptosis, the size and location of some of the affected cells suggested that they were oligodendrocytes. As there is now a large body of evidence that T-cell-induced target cell death takes the form of apoptosis, it is attractive to hypothesize that oligodendrocyte apoptosis is occurring in EAE as a result of oligodendrocyte-directed T-cell cytotoxicity. However, other apoptotic cells were located within the myelin sheath, meninges and perivascular spaces and were clearly not oligodendrocytes but were most likely blood-derived mononuclear cells. The sparsity of their cytoplasm and the absence of phagocytosed material suggested that they were mainly lymphocytes rather than macrophages. Apoptosis has been shown to be involved in deleting autoreactive T-cells during the normal development of tolerance. Thus apoptotic deletion of myelin/oligodendrocyte specific lymphocytes in the central nervous system in EAE might explain both the subsidence of inflammation and the acquisition of tolerance in this autoimmune disease. PMID- 1717665 TI - In vitro synthesis of antibodies to myelin antigens by Epstein-Barr virus transformed B lymphocytes from patients with neurologic disorders. AB - Anti-myelin antibodies can be found in sera from patients with neurologic disorders of suspected immune-mediated pathogenesis such as multiple sclerosis and inflammatory polyneuropathies. However, the specificity of these findings is controversial. In the present study, in vitro synthesis of antibodies to myelin components was compared to their presence in sera in diverse neurological disorders. Epstein-Barr virus was used to activate B lymphocytes for in vitro antibody production. Anti-myelin basic protein and anti-galactocerebroside antibodies were secreted in vitro by B lymphocytes derived from patients with neurological disorders of various etiologies and pathogenetic mechanisms. Anti myelin basic protein antibodies were detected in many more cell culture supernatants than in sera from the same patients. In vitro secretion of antibodies to myelin antigens, as well as the presence of these antibodies in body fluids, are apparently non-specific for disease type and may be secondary to neural tissue damage. PMID- 1717666 TI - Treatment of osteosarcoma of the extremities with the T-10 protocol, with emphasis on the effects of preoperative chemotherapy with single-agent high-dose methotrexate: a Scandinavian Sarcoma Group study. AB - From 1982 to 1989, 97 patients with extremity-localized, high-grade osteosarcoma were treated according to the T-10 protocol. Two thirds of the patients consisted of the near-complete national patient materials from Norway and Finland. Eighty patients (82%) received four courses of high-dose methotrexate (HD MTX, 8 to 12 g/m2) at weekly intervals as their only preoperative treatment, and 77 patients (79%) were assessable for histologic response grading according to Rosen et al (Cancer 49:1221-1230, 1991). Observed histologic response was no certain chemotherapy effect (grade I) in 21%, grade II effect in 62%, and grade III or IV effect in 17%. Nonresponders had significantly lower serum MTX concentrations after 24 and 48 hours than responders; the significance of the difference at 48 hours was maintained in a multivariate analysis. After a median follow-up of 45 months, projected 5-year overall and relapse-free survival for all patients were 64% and 54%, respectively. Patients with a good response to preoperative chemotherapy (grade III/IV) had a significantly better survival than grade I/II responders, despite a switch to postoperative cisplatin/doxorubicin chemotherapy in the latter group. These results were obtained in a largely nonselected group of patients. We conclude that a good initial chemotherapy effect is important for the final outcome in osteosarcoma, and that HD MTX alone is insufficient preoperative treatment for the majority of patients. The individual MTX excretion rate is of importance for tumor response, suggesting a dose-response relationship for HD MTX treatment. PMID- 1717667 TI - Results of treatment of malignant germ cell tumors in 93 children: a report from the Childrens Cancer Study Group. AB - We report treatment results in 93 children entered on study from 1978 to 1984 with malignant germ cell tumors (MGCTs), excluding dysgerminoma and tumors of the testis or brain. The estimated 4-year survival and event-free survival (EFS) for all 93 patients were 54% and 49%, respectively. For 30 children with ovarian tumors, the estimated 4-year survival was 67% and EFS was 63%. For 63 children with nongonadal tumors, survival and EFS were 48% and 42%, respectively. The comparison of EFS between ovarian and nongonadal tumors was significant at P = .03. The treatment plan included a second-look surgical procedure after 18 weeks of chemotherapy. Over half of 36 patients evaluated as having a residual mass present immediately before second-look surgery had no malignant tumor after review of surgical specimens. Age greater than 11 years at diagnosis, incomplete removal of tumor at first surgery, and more than one structure or organ involved at diagnosis increased the risk for adverse event. The histologic subtype of the primary tumor was not related to outcome. Diagnosis was verified by independent pathologic review, and treatment was uniform. Seventeen percent of all registered patients (21 of 127) were excluded because of ineligible pathologic diagnoses; sixty percent (13 of 21) were immature teratomas. PMID- 1717668 TI - Two types of kainate response in cultured rat hippocampal neurons. AB - 1. Two different types of kainate response were recorded in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. There was an outward rectification in the current-voltage (I-V) plot of the kainate-induced current (type I response) in relatively large neurons bearing a morphological resemblance to young pyramidal cells. In smaller neurons with elliptical somata and fine neurites, the kainate response was characterized by a remarkable inward rectification in the I-V plot of the kainate-induced current and a significant permeability to Ca2+ (type II response). 3. Both type I and type II responses were negligible below 2 microM and almost saturated at 500 microM kainate. The concentrations producing half maximal responses and the Hill coefficients were 68 microM and 1.76 and 56 microM and 1.21 for type I and type II responses, respectively. Both responses were suppressed similarly by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6 cyano-7-nitroquinoxaline-2,3-dione (CNQX). 4. The mean single-channel conductance (gamma) of the type II kainate response was estimated, from the relation between the whole-cell mean currents and current variances, to be 8.7 pS. The power spectrum for the current noise was fitted with the sum of two Lorentzians with cutoff frequencies (fc) of 61.1 +/- 1.4 and 327.8 +/- 10.5 Hz (n = 12).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717669 TI - Rhenium-186(Sn)HEDP for treatment of painful osseous metastases: results of a double-blind crossover comparison with placebo. AB - Rhenium-186 (tin) hydroxyethylidene diphosphonate (HEDP) is a new radiopharmaceutical that simultaneously localizes in multiple skeletal metastases in patients with advanced cancer. A single intravenous administration of 30-35 mCi (1110-1295 MBq) is associated with a prompt, significant relief of osseous pain in about 80% of such patients. The efficacy of this new compound was evaluated further by utilizing a double-blind crossover comparison with 99mTc methylene diphosphonate (MDP) as a radioactive placebo. The new rhenium compound resulted in a significantly (p less than 0.05) greater decrease in pain than did treatment with the radioactive placebo. Rhenium-186(Sn)HEDP appears to be a useful new compound for the palliation of painful skeletal metastases. PMID- 1717670 TI - Dietary tryptophan enhances platelet aggregation in rats. AB - The effect of diets enriched with tryptophan (4 and 10 g/kg) on ADP induced platelet aggregation and on plasma lipids was studied with growing rats, following feeding periods of up to 3 weeks. Dietary tryptophan was found to enhance markedly ADP induced platelet aggregation. It appears that this effect was not related to plasma lipid levels. In vitro studies aimed to clarify the mechanism by which tryptophan exerts its action showed that the latter, at concentrations up to 100 micrograms/ml, had no effect on ADP induced platelet aggregation. On the other hand, serotonin, the metabolic product of tryptophan, increased the ADP induced platelet aggregation in a dose dependent pattern. The possible involvement of serotonin in the observed enhancement of platelet aggregation was further substantiated by the observed high levels of 5 hydroxyindole acetic acid, which is a catabolite of serotonin, in the urine of the experimental animals. It is conceivable that tryptophan enhances platelet aggregation in rats via its metabolite, serotonin, and this may in turn contribute to increased atheroslerotic risk. PMID- 1717671 TI - Schistosoma mansoni: the role of calcium in the stimulation of cercarial proteinase release. AB - We investigated the role of calcium mobilization in the induction of proteinase release from cercarial preacetabular glands. Proteinase release was measured by the ability of cercariae to break down a 3H-labeled proline extracellular fibroblast matrix and calcium influx was measured using 45Ca2+. The role of calcium in the activation of cercarial proteinase was examined by investigating the effects of calcium addition and removal on linoleate-induced matrix degradation, the ability of various calcium modulators (Verapamil, fendiline, nifedipine, SK-525A, BAY K-8644, Ryanodine, and SK-7171A) to stimulate or inhibit linoleate-induced proteinase release, the ability of calcium modulators directly to induce cercarial proteinase release, and the ability of various stimulants of proteinase release to induce calcium influx or efflux from cercariae. The results of these studies indicate that proteinase release is dependent on external calcium concentration, voltage-operated channels are either nonexistent in cercariae or have a minimal role in overall calcium influx, and that activation of Ca2+ influx can be caused by both free fatty acids and calcium modulators by a hypothesized receptor-operated channel. Although calcium uptake is important in cercarial proteinase release, it is not the only factor involved. Calcium uptake alone does not guarantee that proteinase will be secreted. On the other hand, if Ca2+ influx does not occur, proteinase will not be secreted. PMID- 1717672 TI - Carbohydrate epitopes are responsible for antibody cross-reactivity in Trypanosoma cruzi-infected mice. AB - Anti-Trypanosoma cruzi antibodies can be eluted from western blots of T. cruzi antigens and thereby are fractionated on the basis of the electrophoretic mobility of the antigens to which they bind. Antibodies fractionated by these methods can bind antigens with electrophoretic mobility different from those antigens from which they are eluted. Such antibodies thus are considered cross reactive. Studies in which the target antigens are reacted with sodium periodate to destroy carbohydrate epitopes prior to exposure to the eluted antibodies revealed that antibodies are produced that bind to both carbohydrate and noncarbohydrate epitopes on western blots, but that most of the cross-reactive antibodies are directed toward carbohydrate moieties. PMID- 1717673 TI - Odontogenic epithelial hamartoma of the gingiva: a case report. AB - A rare case of odontogenic epithelial hamartoma of the gingiva in a 63-year-old Japanese female is reported. This tumor-like lesion probably originated from the reduced tooth-forming tissues such as rests of dental lamina lying dormant in the gingiva after odontogenesis. The differential diagnosis of this lesion from many other odontogenic and glandular tumors is important; however, through careful clinico-pathological examinations, it would not be a very difficult task. PMID- 1717674 TI - Acute effects of beclamide on brain regional monoamine concentrations, their metabolites and radioligand binding studies. AB - The effects of beclamide on regional brain monoamine levels and radioligand binding have been studied in rats. One hour oral pre-treatment with beclamide (400 mg kg-1) increased rat striatal dopamine turnover by increasing the levels of its major metabolites (DOPAC and HVA) three-fold. Simultaneously the drug reduced the concentration of striatal dopamine by a similar factor, and the concentrations of 5-hydroxytryptamine, (5-HT), 5-hydroxyindoleacetic acid, (5 HIAA) and 3-methoxytyramine in the striatum were reduced below the detection limits of the assay. In the frontal cortex, beclamide depleted the dopamine, 5-HT and 5-HIAA content whilst having no significant effect on the noradrenaline level. The concentrations of bioamines and their metabolites in the hypothalamus were unaffected by such acute beclamide treatment. In radioligand binding studies beclamide lacked affinity and failed to displace radioligands from alpha 2, beta, 5-HT, 5-HT2 and dopamine D2 sites in selective loci of the rat brain. PMID- 1717675 TI - Microemulsions as topical drug delivery vehicles: in-vitro transdermal studies of a model hydrophilic drug. AB - Microemulsions with a 58:42 weight ratio of dioctyl sodium sulphosuccinate: octanol and containing 15, 35, and 68% water have been tested for their ability to transport glucose across human cadaver skin. A flow-through multisample skin diffusion cell showed that both the 35 and 68% water microemulsions caused enhanced (approximately 30-fold) transport of glucose. No transport was discernible for the 15% water microemulsion. Differences in percutaneous glucose transport were shown to parallel differences in the diffusion of water within the microemulsion vehicles before application to the skin. PMID- 1717676 TI - In-vitro interaction of a novel immunosuppressant, FK 506, and antacids. AB - The effect of selected antacids on the amount of FK 506 in solution in simulated gastric juice has been studied. FK 506 (2.5 mg) was incubated in 100 mL simulated gastric fluid (SGF) with the equivalent of 500 mg of various antacids. The addition of Mylanta and Tums resulted in 14 and 30% loss of FK 506, respectively, in 24 h; 98% loss was observed in 12 h in the presence of Mag-Ox; 100% loss was observed in the presence of magnesium oxide powder in 2 h. The loss of FK 506 from these solutions appears to be due to a pH mediated degradation of FK 506. The addition of aluminium hydroxide gel USP (Roxane) to the FK 506 solution resulted in a 35% loss within 2 min but no further loss was noted for 24 h, indicative of adsorption of FK 506. These results suggest that until additional in-vivo studies are carried out, it is prudent not to dose FK 506 and antacids at the same time to avoid potential interactions. PMID- 1717677 TI - Functional interactions between capsaicin-sensitive and cholinergic nerves in the guinea pig bronchus. AB - Electrical stimulation of the right vagus nerve causes a biphasic contraction of the guinea pig isolated right bronchus. The "first-phase" is blocked by hexamethonium or atropine and the "second-phase" is eliminated by capsaicin pretreatment. We investigated a potential interaction between capsaicin-sensitive nerves and cholinergic nerves in the guinea pig bronchus. Hexamethonium (100 microM) abolished the first-phase contraction but had no effect on the capsaicin sensitive second-phase contraction. In the presence of hexamethonium, atropine (0.1 microM) significantly decreased the amplitude of the second-phase contraction by 28%. Similar results were observed with the M3-selective muscarinic receptor antagonist, 4-diphenyl-acetoxy-M-methylpipe-radine, but not with the M2 muscarinic antagonist, AFDX-116. Atropine also reduced contractions induced by exogenously applied neurokinin A. We then analyzed the effect of stimulating capsaicin-sensitive fibers with electrical field stimulation on vagus nerve evoked cholinergic contractions. By reducing the stimulus intensity we were able to evoke vagus nerve-mediated contractions that were exclusively cholinergic in nature. The cholinergic contractions were significantly increased after stimulation of capsaicin-sensitive fibers by about 50%. By contrast, contractions elicited by exogenous methacholine were unaffected after field stimulation of capsaicin-sensitive responses. Our findings indicate that the contractions of the guinea pig bronchus elicited by stimulation of capsaicin-sensitive nerves are due in part to muscarinic cholinergic receptor activation. Secondly, our data demonstrate that the cholinergic contractions elicited by vagal preganglionic nerve stimulation are potentiated by neurotransmitter(s) released from capsaicin sensitive fibers in bronchus. PMID- 1717678 TI - Binding of the novel ligand [4,5-3H-Leu10]substance P to high-affinity NK-1 receptors on guinea pig lung membranes: modulation by GTP analogs and sulfhydryl modifying agents. AB - A novel ligand, [4,5-3H-Leu10]substance P ([3H]SP), with high specific activity (137 Ci/mmole) was utilized to investigate the properties of NK (neurokinin)-1 receptors on guinea pig lung membranes (GPLM) and compared them to NK-1 receptors on rat submaxillary glands (RSGM). In the presence of a neutral endopeptidase inhibitor, thiorphan (100 microM), [3H]SP bound with high specificity (greater than 95%), rapidly (k1 = 0.116 nM-1 x min-1) and in a reversible (k-1 = 0.012 min 1) manner to a single class of high-affinity (Kd = 0.16 nM) and saturable (Bmax = 256 fmol/mg protein) receptors. High specific binding with higher density (5 fold) was also detected in RSGM, albeit with a lower affinity (Kd = 1.36 nM). Guanyl-5'-yl-imidodiphosphate and guanosine-5'-O-3-thiotriphosphate inhibited binding to GPLM (and RSGM) in a concentration-related manner. In GPLM, this effect was mediated by a reduction in affinity, mainly via enhancement of ligand dissociation rates and appearance of a lower affinity state (Kd = 3.4 nM). Preincubation of GPLM with sulfhydryl modifying agents (p-chloromercuriphenyl sulfonic acid and N-ethylmaleimide) reduced receptor density and affinity in a time- and concentration-dependent manner. Competition experiments with tachykinins and analogs illustrated a rank order of potency of: SP greater than or equal to [Sar9,Met(O2)11]SP greater than SP-methyl ester greater than or equal to physalaemin greater than SP(6-11) much greater than kassinin greater than neurokinin A = eledoisin much greater than neurokinin B greater than Nle10-NKA(4 10), clearly demonstrating that these receptors are of NK-1 type. Moreover, analysis of over 30 peptide and non-peptide hormones and antagonists demonstrated exquisite selectivity (greater than 10,000-fold) towards NK-1-selective agonists (vs. other ligands. A highly significant (P less than .005) linear correlation (r = 0.924) exists between agonist affinities in GPLM and RSGM. Combined, the data suggest that [3H]SP labels a nearly homogeneous population of high-affinity, G protein coupled NK-1 receptors on GPLM and RSGM, with very high degree of selectivity. PMID- 1717679 TI - Differential effects of substance P analogs on neurokinin 1 receptor agonists in the mouse spinal cord. AB - Effects of five substance P (SP) analogs on the licking, biting and scratching response induced by neurokinin (NK) 1 receptor agonists such as SP, physalaemin and (p-Glu6,Pro9)-SP (6-11) (septide) were studied after intrathecal injections in mice. Septide brought about a SP-like behavioral response, and was approximately 25 times more potent than the D-Pro9 analog, D-septide. When administered simultaneously with NK-1 receptor agonists, a putative SP antagonist, spantide inhibited SP-, physalaemin- and septide-induced behavioral response in a dose-dependent manner with ED50 values of 1.0, 0.65 and 1.3 nmol/mouse, respectively. Septide-induced response was significantly reduced by lower doses of (D-Arg1, D-Pro2,4, D-Phe7, D-His9, Leu11)-SP than (D-Phe7, D-His9, Leu11)-SP (6-11). In contrast, (D-Arg1, D-Pro2,4, D-Phe7, D-His9)-SP (0.5-1.0 nmol) and (D-Phe7, D-His9)-SP (6-11) (0.5-2.0 nmol) inhibited only SP-induced behavioral response, but not physalaemin- or septide-induced response. The results of this study indicate that NK-1 receptor agonists are not necessarily affected to a same degree by SP analogs containing D-His. These findings may be interpreted as indicative of the existence of different NK-1 receptor subtypes. PMID- 1717680 TI - Nitric oxide synthase inhibitors inhibit interleukin-1 beta-induced depression of vascular smooth muscle. AB - Interleukin-1 beta (IL-1) reduces vascular smooth muscle contractility. The purpose of the present study was to investigate the role of nitric oxide synthesis in mediating this effect of IL-1. We studied the influence of inhibitors of nitric oxide synthesis on the depression of norepinephrine-induced contractions of rat aortic rings by IL-1. Also, we examined the ability of IL-1 to increase the production of nitric oxide by rat aortic smooth muscle cells in culture as determined indirectly by measuring nitrite concentrations. NG-amino-L arginine blocked the effect of IL-1 on norepinephrine-induced contractions of rat aortic rings whereas NG-monomethyl-L-arginine and NG-nitro-L-arginine were considerably less effective. In addition, this effect of IL-1 was prevented by coincubation of the rings with cycloheximide. IL-1 greatly elevated nitrite production by rat aortic smooth muscle cells, and this effect could also be blocked completely by the arginine analogs. NG-amino-L-arginine was the most potent inhibitor of nitrite synthesis (IC50 = 1.7 microM) whereas NG-monomethyl-L arginine and NG-nitro-L-arginine were about 10-fold less potent (IC50 = 16 and 22 microM, respectively). These results suggest that IL-1-induced depression of norepinephrine-induced vascular contraction is mediated by the increased synthesis of nitric oxide synthase by vascular smooth muscle cells. The relative potency of the arginine analogs for the inhibition of nitrite synthesis suggests that the synthase in vascular smooth muscle is similar to the synthase in macrophages. PMID- 1717681 TI - Characterization of endothelium-derived relaxing factor/nitric oxide synthase from bovine cerebellum and mechanism of modulation by high and low oxygen tensions. AB - Experiments were designed to investigate the role of oxygen tension on modulation of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthase activity. EDRF/NO synthase from bovine cerebellum was confirmed to have cofactor and kinetic characteristics similar to that reported in endothelium and other tissues. The effect of oxygen tension on EDRF/NO synthase activity as assessed by L-[3H]citrulline production was investigated. Hypoxia markedly inhibited EDRF/NO synthase activity whereas hyperoxia increased the initial rate of enzyme activity. The inhibition of EDRF/NO synthase activity by hypoxia was reversed by normoxia as well as by hyperoxia. The Km values for L-arginine in hyperoxia, normoxia and hypoxia were 7 +/- 0.7, 4.8 +/- 0.4 and 7 +/- 1.3 microM whereas the Vmax values were 94 +/- 8, 66 +/- 7, and 32 +/- 2 pmol/min/mg of protein, respectively. The effect of oxygen tension on EDRF/NO synthase activity as determined by L-[3H]citrulline production was correlated with EDRF/NO production using a bioassay in which an EDRF/NO synthase preparation was incubated in wells of cultured vascular smooth muscle and cyclic GMP production was measured. Hypoxia almost inhibited the production of cyclic GMP completely, which was comparable to its inhibition of L-[3H]citrulline production. Hyperoxia, however, showed partial inhibition of cyclic GMP accumulation with no significant effect on L-[3H]citrulline production. This cyclic GMP inhibition by hyperoxia was reversed partially by superoxide dismutase. We conclude that hypoxia inhibits EDRF/NO synthase activity primarily through depletion of oxygen, one of the substrates for the enzyme. In hyperoxia, the initial rate of EDRF/NO synthase activity (Vmax) is significantly enhanced with no significant change in enzyme activity at longer time intervals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717682 TI - Effects of charybdotoxin and iberiotoxin on the spontaneous motility and tonus of different guinea pig smooth muscle tissues. AB - Charybdotoxin (ChTX) and iberiotoxin (IbTX), two potent peptidyl blockers of the high conductance Ca(2+)-activated K+ channel (PK,Ca) were used to probe the role of this channel in regulating the contractility of various smooth muscles isolated from the guinea pig. Of the spontaneously contracting tissues that have been investigated, bladder and taenia coli are affected by ChTX, whereas portal vein and uterus are relatively insensitive. In the former two tissues, ChTX (10 100 nM) produces a concentration-dependent increase in contractility, with bladder being most sensitive to action of the toxin. ChTX also causes a contracture of quiescent aortic rings, although not affecting indomethacin treated trachea. In order to demonstrate that the effects of ChTX are due specifically to blockage of PK,Ca, rather than to inhibition of some other K+ channel, two other inhibitors of PK,Ca were monitored. IbTX (10-100 nM), a selective inhibitor of PK,Ca, and tetraethylammonium ion, which blocks PK,Ca at low (0.1-3 mM) concentrations, both increase the myogenic activity of bladder, but not portal vein. In addition, IbTX causes a sustained contracture of aorta. Taken together, these data indicate that the increased contractility of certain guinea pig smooth muscles produced by ChTX, IbTX and tetraethylammonium ion is the result of selective inhibition of PK,Ca. It is suggested that in spontaneously active bladder and taenia coli, PK,Ca provides a repolarization pathway after tissue depolarization, whereas in quiescent aorta, PK,Ca maintains cellular resting potential. In contrast, in indomethacin-treated trachealis muscle, ChTX-sensitive K+ channel pathways are not involved in controlling resting tension.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717683 TI - The vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria. I. Anatomy. AB - Antiserum to arginine-vasopressin has been used to characterise the pair of vasopressin-like immunoreactive (VPLI) neurons in the locust. These neurons have cell bodies in the suboesophageal ganglion, each with a bifurcating dorsal lateral axon which gives rise to predominantly dorsal neuropilar branching in every ganglion of the ventral nerve cord. There are extensive beaded fibre plexuses in most peripheral nerves of thoracic and abdominal ganglia, but in the brain, the peripheral plexuses are reduced while neuropilar branching is more extensive, although it generally remains superficial. An array of fibres runs centripetally through the lamina-medulla chiasma in the optic lobes. Lucifer Yellow or cobalt intracellular staining of single VPLI cells in the adult suboesophageal ganglion shows that all immunoreactive processes emanate from these two neurons, but an additional midline arborisation (that was only partially revealed by immunostaining) was also observed. Intracellularly staining VPLI cells in smaller larval instars, which permits dye to reach the thoracic ganglia, confirms that there is no similar region of poorly-immunoreactive midline arborisation in these ganglia. It has been previously suggested that the immunoreactive superficial fibres and peripheral plexuses in ventral cord ganglia serve a neurohaemal function, releasing the locust vasopressin-like diuretic hormone, F2. We suggest that the other major region of VPLI arborisation, the poorly immunoreactive midline fibres in the suboesophageal ganglion, could be a region where VPLI cells receive synaptic input. The function of the centripetal array of fibres within the optic lobe is still unclear. PMID- 1717684 TI - The current status of myeloid growth factor therapy. AB - Supralethal chemotherapy has been used in an attempt to cure certain malignancies by employing doses of cytotoxic agents beyond conventional limits. The resulting damage to the bone marrow requires rescue with an infusion of autologous or allogenic marrow to repopulate the haemopoietic system. The recent availability of marrow stimulants for clinical use may prove to be a major advance in oncology as early evidence suggests that they can reduce cytotoxic drug induced myelosuppression and possibly allow escalation of chemotherapy with improved tumour responsiveness. Such growth factors may also improve primary marrow disorders. This article outlines the properties of the myeloid growth factors and then reviews the important clinical conditions in which they have been employed so far. Erythropoietin and lymphoid growth factors are not dealt with. PMID- 1717685 TI - Chlorpyrifos toxicosis in two cats. PMID- 1717686 TI - Antiidiotypic antibodies interacting with cGMP-dependent channels of frog retinal rod outer segments. AB - Antiidiotypic approach was used to obtain antibodies interacting with cGMP binding site of the cGMP-activated channel of the photoreceptor cell. Monoclonal anti-BrcGMP antibodies having characteristics of binding of agonist and its analogs close to those for a natural receptor have been obtained. These antibodies were used to raise polyclonal antiidiotypic antibodies capable of interacting with a natural cGMP-receptor. Application of immunoglobulins, isolated from antiidiotypic serum, to inside-out fragments of the rod plasma membrane led to an irreversible increase of the conductance of cGMP-dependent channels. PMID- 1717687 TI - The role of serum and synovial fluid components in the promotion of urate crystal formation. AB - The effect of various components of serum and synovial fluid (SF) on in vitro urate crystal formation was measured. Serum and SF has been shown to promote urate crystal formation. The component(s) responsible for this promotion was found to be a macromolecule, sensitive to heat denaturation. Albumin, alpha and beta globulins and chondroitin sulphate had little effect, whereas gamma globulin markedly promoted crystal formation and insoluble collagen also promoted crystal formation in a dose dependent fashion. PMID- 1717688 TI - Clinical evaluation of a sustained release compared to a standard formulation of tiaprofenic acid (Surgam) in rheumatoid arthritis: a Canadian multicenter study. AB - A double blind, randomized, parallel group clinical therapeutic trial, comparing a sustained release formulation with a standard preparation of tiaprofenic acid (Surgam), was performed in 119 patients with rheumatoid arthritis. The sustained release preparation was prescribed as two 300 mg capsules taken once a day, and the standard formulation as one tablet of 300 mg taken twice a day. From baseline up to the 3rd and 6th week of treatment, a statistically significant difference (p less than 0.001) was observed in both treatment groups for all the variables, indicating objective or subjective changes in the clinical signs and symptoms of the patients. For most of these variables, no significant differences were observed between the sustained release and the standard formulations. Also, no significant differences were observed between the 2 formulations in the incidence of side effects and abnormal laboratory findings. PMID- 1717689 TI - Some monoclonal anti-human lymphocyte and class II MHC antigen antibodies do not stain snap frozen Macaca fascicularis lymphocytes or tissue sections. AB - Experiments were designed to stain snap frozen and fixed Macaca fascicularis peripheral blood mononuclear cell (PBMC) preparations and colon sections assuming that monoclonal mouse anti-human lymphocyte and class II major histocompatibility complex antigen antibodies would cross-react with the monkey counterparts to the human antigens. Most of the monoclonals used in this study did not stain the monkey frozen and fixed tissues in immunoenzymatic protocols despite adequate staining of control human tissues. The fact that snap frozen Macaca fascicularis PBMCs and tissue sections were not always stained is of practical importance for experimental design in Macaca fascicularis using anti-human reagents. PMID- 1717690 TI - Diltiazem at high concentration increases the ionic permeability of biological membranes. AB - The effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 microM. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of 86Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations. These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 microM. PMID- 1717691 TI - Inhibition of inward rectifying tonoplast channels by a vacuolar factor: physiological and kinetic implications. AB - Regulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of young Vigna unguiculata seedlings show no or hardly any channel activity at tonoplast potentials greater than 80 mV, in the vacuole-attached configuration. When the configuration is changed to an excised patch or whole vacuole, a fast (excised patch) or slow (whole vacuole) increase of inward rectifying channel activity is seen. This increase is accompanied by a shift in the voltage-dependent gating to less hyperpolarized potentials. In the whole vacuole configuration the level of inward current increases and also the activation kinetics changes. Induction of channel activity takes up to 20 min depending on the age of the plants used and the diameter of the vacuole. On the basis of the estimated diffusion velocities, it is hypothesized that a compound with a mol wt of 20,000 to 200,000 is present in vacuoles of young seedlings, which shifts the population of channels to a less voltage-sensitive state. PMID- 1717692 TI - Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cryosections. AB - Ultrathin sections of rapidly frozen, briefly pre-treated muscle tissue are cut and thereafter are thawed and contrasted using a negative staining technique. The method has provided micrographs in which the in-vivo order in the muscle fibres has been preserved well enough to enable both a more complete interpretation of X ray diffraction evidence from muscle, and also a gain of new ultrastructural information on aspects of myofibril and myofilament architecture in different types of fibre. Examples here are taken from chicken, rabbit and fish muscles and show both the M-band and the bridge region of the A-band in great detail. To enhance the detail in the original images, one-dimensional (1-D) and 2-D averaging techniques (lateral smearing and step averaging, respectively) are used. Although there is major shrinkage in section thickness to about one-third of its original value, demonstrated here for the first time is the fact that the characteristic A-band lattice planes are preserved in these sections in 3-D. This confirms the usefulness of cryosections not just for 1-D and 2-D image processing, but also for 3-D reconstruction. Thus, in combination with techniques of image processing, cryoultramicrotomy can give the muscle morphologist the detailed data that are needed to match the molecular biologists, biochemists and immunologists in the interpretation of their data about physiological and pathophysiological events in muscle fibres at the macromolecular level. PMID- 1717693 TI - Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli. AB - A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4.5 S RNA (p4.5S). The overall efficiency of cleavage of p4.5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4.5S as substrate has a much lower Km value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4.5S. A model hairpin substrate, based on the sequence of p4.5S, is cleaved with greater efficiency than the parent molecule. The 3' terminal CCC sequence of p4.5 S may be as important for cleavage of this substrate as the 3'-terminal CCA sequence is for cleavage of tRNA precursors. PMID- 1717694 TI - Mutational analysis of the structure and function of the adenine phosphoribosyltransferase enzyme of Chinese hamster. AB - We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed. PMID- 1717695 TI - Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling. AB - The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs. PMID- 1717696 TI - Translation initiation of IS50R read-through transcripts. AB - IS50R (and Tn5) normally transposes at a low frequency, partly because cells containing this insertion sequence synthesize low levels of the transposase protein. Since the 5' end of the transposase gene is located next to the outer end of IS50R (and thus close to flanking host sequences), transposition into actively transcribed genes could result in the production of read-through transcripts that would encode the transposase. We have found that these read through transcripts are made, but are translated poorly. We isolated mutations that increase translation initiation of transposase from read-through transcripts. Most of these mutations destabilize a potential RNA secondary structure in the ribosome binding site that could form in read-through transcripts, but not in normal transcripts. In vitro RNA secondary structure analysis has confirmed the predicted RNA secondary structure and the effects of mutations. We have shown that RNA secondary structure is the major factor limiting transposase expression from read-through transcripts. PMID- 1717697 TI - Novel anticodon composition of transfer RNAs in Micrococcus luteus, a bacterium with a high genomic G + C content. Correlation with codon usage. AB - The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure. PMID- 1717698 TI - Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods. AB - The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors and acceptors of energy which have been linked to the terminal residues of template polynucleotides (poly- and oligo(U) and oligo(A] and 16 S RNA or to SH-groups of ribosomal proteins S1 and S8. The dynamics of mRNA-ribosome interaction have been investigated by the fluorescence stopped-flow technique. It has been shown that the binding to the 30 S subunit of poly(U) with length much shorter (16 nucleotides) than that covered by the ribosome is greatly enhanced by protein S1. However, the final position of oligo(U)16 on the 30 S subunit, which probably includes the ribosomal decoding site, proves to be quite different from that occupied by oligo(U)16 on a free protein S1. Interaction of oligo- and poly(U) with the 30 S subunit occurs in at least two steps: the first one is as fast as the interaction of poly(U) with free S1, whereas the second step represents a first-order reaction. Therefore, the second step may reflect some rearrangement of the template in the ribosome after its primary binding. It is suggested that protein S1 in some cases may fulfill the role of a transient binding site for mRNA in the course of its interaction with the ribosome. The general shape of the template in the mRNA binding region of the ribosome has been studied using various synthetic ribopolynucleotides and has been shown to be similar. It can be represented by a loop(s) or "U-turn(s)". On the basis of estimation of distances from the ends of poly(U) to some well localized points on the 30 S ribosomal surface, a tentative model of mRNA path through the ribosome is proposed. PMID- 1717699 TI - Efficient templates for Q beta replicase are formed by recombination from heterologous sequences. AB - A very efficient replicase template has been isolated from the products of spontaneous RNA synthesis in an in vitro Q beta replicase reaction that was incubated in the absence of added RNA. This template was named RQ135 RNA because it is 135 nucleotides in length. Its sequence consists entirely of segments that are homologous to ribosomal 23 S RNA and the phage lambda origin of replication. The sequence segments are unrelated to the sequence of Q beta bacteriophage genomic RNA. Nonetheless, this natural recombinant is replicated in vitro at a rate equal to the most efficient of the known Q beta RNA variants. Apparently, the structural properties that ensure recognition of an RNA template by Q beta replicase are not confined to viral RNA, but can appear as a result of recombination among other RNAs that usually occur in cells. PMID- 1717700 TI - Combined effects of carbon tetrachloride and chlordecone on calmodulin activity in gerbil brain. AB - The potentiation of carbon tetrachloride (CCl4) toxicity by chlordecone (CD) pretreatment in different animal models is well established. However, these studies have only dealt with hepatotoxicity. The present study was initiated to determine whether CD preexposure potentiates CCl4 neurotoxicity in gerbils. Gerbils were chosen for the reason that the metabolism of CD in gerbil is similar to that of humans. Gerbils (50-80 g), fed on diet without or with CD (10 ppm) for 15 d, were challenged with a single dose of CCl4 (15 microliters, ip). Ca(2+) ATPase and calmodulin (CaM) activities were determined in gerbil brain P2 fraction and cytosol, respectively, at intervals of 0.5, 2, 6, 12, and 24 h after CCl4 administration. Ca(2+)-ATPase and CaM activities were decreased at 0.5 and 2 h in both CD-preexposed and CCl4-treated gerbils. However, CaM activity returned to normal levels after 6 h and Ca(2+)-ATPase activity showed 80% recovery after 2 h. In vitro experiments showed that CCl4 alone at 5 microM concentration inhibited Ca(2+)-ATPase activity up to 50%. Combination of CD (0.5 microM) and CCl4 (1 and 5 microM) on Ca(2+)-ATPase activity showed no additive effect in vitro. Interaction between CCl4 and CaM was studied in the presence and absence of CD by monitoring NPN fluorescence. The decrease in NPN fluorescence observed with CCl4 was not potentiated by CD preincubation. These data suggest that CD does not enhance CCl4-induced alterations of Ca(2+)-ATPase and CaM activities in gerbil brain. PMID- 1717701 TI - Spatial distribution of myelin basic protein mRNA and polypeptide in quaking oligodendrocytes in culture. AB - In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of oligodendrocytes were established from affected and control animals. The cytoarchitecture of the oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP polypeptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture. PMID- 1717702 TI - Substance P gene expression is regulated by interleukin-1 in cultured sympathetic ganglia. AB - We have investigated the effects of interleukin-1 beta (IL-1 beta) on the induction of substance P (SP) in cultured sympathetic ganglia. Northern blot analysis reveals that SP increases are secondary to an increase in mRNA coding for the preprotachykinin (PPT) precursor of SP. Nuclear transcription assays detect an early increase in PPT-specific nascent transcripts, suggesting that the ultimate effect of IL-1 is on transcription itself. Depolarizing agents, interferon-gamma, glucocorticoid hormones, and prostaglandin synthesis inhibitors all diminish the induction of SP and PPT mRNA by IL-1. Since SP has stimulatory effects on the immune system, the IL-1-induced increase in ganglionic SP may be one means by which the nervous and immune systems interact during an acute response to ganglionic injury. PMID- 1717703 TI - Identification of a cDNA clone specific for the oligodendrocyte-derived repulsive extracellular matrix molecule J1-160/180. AB - A cDNA clone specific for the oligodendrocyte-derived extracellular matrix glycoproteins J1-160/180 was obtained from a lambda ZAPII expression library using polyclonal antibodies generated against mouse J1-160. The library was constructed from poly(A)(+)-RNA isolated from O1 antigen-positive rat oligodendrocytes. The cDNA clone expressed a fusion protein that was recognized by the J1-160/180-specific monoclonal antibodies 596, 619, and 620, and, weakly, 597. The fusion protein was not recognized by polyclonal antibodies to mouse J1/tenascin. The cDNA clone with an insert of approximately 5.6 kb in size contained the nucleotide sequence coding for the amino acid sequence of the N terminus of a tryptic peptide derived from mouse J1-160. The developmental and tissue distribution of the mRNA recognized by the cDNA clone is in agreement with the described expression of the J1-160/180 proteins. PMID- 1717704 TI - Axonal transport of cytoskeletal proteins in chemically induced neuropathies. PMID- 1717705 TI - Risk assessment for diesel exhaust and ozone: the data from people and animals. AB - Estimating and setting exposure limits for people exposed to air pollutants are complex processes. This paper discusses the information available from human and animal studies on the potential health effects of inhaled diesel exhaust and ozone. While considerable information is available, additional research is needed to clarify issues relative to establishing exposure standards and estimating risks for these two pollutants. PMID- 1717706 TI - The effect of isoprinosine and levamisole on factors relevant to protection of calves against respiratory disease. AB - Experiments to characterize the effects of two immunomodulators, namely, isoprinosine and levamisole, on factors relevant to the resistance of calves to respiratory infection were undertaken. Daily oral doses of isoprinosine decreased the influx of neutrophils into the respiratory tract, increased membrane immunoglobulin and complement receptor expression on cells from bronchoalveolar lavage samples and decreased the severity of respiratory disease. Additional intravenous doses produced similar effects on neutrophil migration to the respiratory tract and on membrane receptor expression, but the changes were no greater than those seen with oral isoprinosine alone. No significant changes in the anti-bacterial activity of cells in bronchoalveolar lavage samples followed isoprinosine treatment. In vitro incubation of pulmonary alveolar macrophages harvested from normal calves with isoprinosine increased their expression of immunoglobulin and complement receptors. Levamisole did not affect neutrophil migration to the lower respiratory tract or membrane receptor expression by pulmonary alveolar macrophages after in vivo or in vitro treatment. The immunomodulatory effects of isoprinosine beneficially increase the resistance of calves to respiratory disease, and are potentially useful in the control of infectious diseases of farm animals. PMID- 1717707 TI - Isolation of a type D retrovirus from B-cell lymphomas of a patient with AIDS. AB - An atypical syncytial variant of a high-grade Burkitt's-type B-cell lymphoma from a patient with AIDS who was seropositive for human immunodeficiency virus type 1 was studied. A productive type D retrovirus infection was identified in early passage cell lines derived from two lymphomas from this patient. Nucleotide and amino acid sequence analysis as well as immunologic reactivity indicated that the isolated virus was highly related to Mason-Pfizer monkey virus (MPMV). MPMV is an immunosuppressive type D retrovirus that causes an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by polymerase chain reaction generated the appropriate MPMV-specific fragments and indicated that the patient was infected with the MPMV-like retrovirus. In addition, the patient's serum contained antibodies which recognized type D viral env proteins (gp70 and gp20) and gag proteins (p27 and p14). Although there have been reports of human cell lines infected with type D retroviruses and of type D-reactive human sera, this is the first evidence of a type D retrovirus infection in a human confirmed by virus isolation, serum reactivity, and viral DNA identification in tumor tissue. PMID- 1717708 TI - Identification of an immunodominant epitope within the phosphoprotein of rabies virus that is recognized by both class I- and class II-restricted T cells. AB - Immunization of H-2k mice with live rabies virus induces cytolytic T lymphocytes to the phosphoprotein of rabies virus. The antigenic determinant responsible for stimulating this class I-restricted cytolytic response was mapped to 50 amino acids (residues 180 to 229) of the phosphoprotein by using vaccinia virus recombinants expressing either the full-length phosphoprotein or C-terminal truncations of the phosphoprotein. The epitope was more finely mapped to residues 191 to 206 by using synthetic peptides. Several CD4+, class II-restricted T-cell lines were isolated from splenocytes of H-2k mice immunized with the vaccinia virus-rabies virus phosphoprotein recombinant virus. These lines were specifically stimulated by the phosphoprotein, and in addition, each line proliferated and released lymphokines in response to the same synthetic peptide shown to stimulate phosphoprotein-specific, class I-restricted cytolytic T cells. PMID- 1717709 TI - Hepatitis E virus: identification of type-common epitopes. AB - Large epidemic outbreaks of enterically transmitted non-A, non-B viral hepatitis (ET-NANBH) have been documented in developing countries. A molecular clone derived from the causative agent, the hepatitis E virus (HEV), has recently been described (G.R. Reyes, M.A. Purdy, J.P. Kim, K.-C. Luk, L.M. Young, K.E. Fry, and D. Bradley, Science 247:1335-1339, 1990). We now report the isolation, by serologic screening, of two cDNA clones derived from a fecal sample collected during a 1986 outbreak of ET-NANBH in Telixtac, Mexico. The cDNA clones encode epitopes that specifically reacted with acute- and convalescent-phase sera collected during five different ET-NANBH epidemics and represent the initial cloning of the Mexico strain of HEV. Recombinant fusion proteins expressed from these clones were also recognized by antibodies from cynomolgus macaques experimentally infected with HEV. The cDNA clones were shown to be derived from HEV by their specific hybridization to the previously recognized full-length genomic RNA transcript of approximately 7.5 kb. In addition, however, subgenomic polyadenylated transcripts of approximately 2.0 and approximately 3.7 kb were also identified in HEV-infected cynomolgus monkey liver. Sequences homologous to the epitope clones were isolated from the Burma strain of the virus, and these demonstrated reactivity comparable to that seen with the Mexico strain epitopes. When compared with the available full-length sequence of the Burma strain of HEV, it was discovered that the cDNA clones were encoded in different open reading frames (ORFs). The comparison between Mexico and Burma HEV strains indicated amino acid homologies of 90.5 and 73.5% for these epitope-encoding clones derived from ORF2 and ORF3, respectively. The identification of these clones not only has provided insight into the expression strategy of HEV but has also resulted in a source of recombinant protein useful in the diagnosis of HEV-induced hepatitis. PMID- 1717710 TI - An element in the bovine papillomavirus late 3' untranslated region reduces polyadenylated cytoplasmic RNA levels. AB - Expression of the two bovine papillomavirus type 1 (BPV-1) late genes, L1 and L2, coding for the two capsid proteins, is limited to terminally differentiated keratinocytes in bovine fibropapillomas. This pattern of expression is determined both by the activity of the late promoter and by the inhibition of late region expression in less well differentiated cells. Inhibition of L1 and L2 mRNA production in nonpermissive cells must occur since the late region potentially could be transcribed from early region promoters. Nuclear runoff analysis of the late region has demonstrated that up to 95% of transcripts which are initiated in the early region in nonpermissive cells terminate within the late region upstream of the late polyadenylation site (C. C. Baker and J. Noe, J. Virol. 63:3529-3534, 1989). However, very few of the primary transcripts which include the late polyadenylation site are processed into mRNA. In this study, we have used expression vectors to characterize an inhibitory element active in nonpermissive cells which is located in the late 3' untranslated region (3'UTR). While the late polyadenylation site is functional in these cells, a 53-bp element in the late 3'UTR reduces levels of polyadenylated cytoplasmic RNA. This element inhibited chloramphenicol acetyltransferase (CAT) expression 6- to 10-fold when cloned in the sense orientation into the 3'UTR of a CAT expression vector. No block to expression was seen when the fragment was cloned immediately downstream of the poly(A) site, in an intron upstream of the CAT coding sequence, or in an antisense orientation in the 3'UTR. When the same fragment was deleted from a BPV 1 L1 expression vector, a sixfold increase in mRNA levels was seen. Actinomycin D chase experiments using BPV-1 L1 expression vectors indicated that the element does not destabilize cytoplasmic polyadenylated RNA. Therefore, the element must act before the mature mRNA reaches the cytoplasm. The data presented are consistent with effects on nuclear stability and/or inhibition of polyadenylation or nuclear transport. PMID- 1717711 TI - Viral and cellular factors governing hamster cell infection by murine and gibbon ape leukemia viruses. AB - Hamster cells are resistant to infection by most retroviruses, including Moloney murine leukemia virus (MoMLV) and gibbon ape leukemia viruses (GaLVs). We have constructed MoMLV-GaLV hybrid virions to identify viral and cellular determinants responsible for the inability of GaLV and MoMLV to infect hamster cells. The substitution of MoMLV core components for GaLV core components circumvents the resistance of hamster cells to infection by GaLV, demonstrating that hamster cells have receptors for GaLV but are not efficiently infected by this primate retrovirus because of a postpenetration block. In contrast, hamster cells are apparently resistant to MoMLV infection because although they bear a receptor for MoMLV, the receptor is nonfunctional. Treatment of CHO K1 or BHK 21 hamster cells with the glycosylation inhibitor tunicamycin allows the cells to be infected by MoMLV. The construction of MoMLV-GaLV hybrid virions that can efficiently infect resistant cells has allowed the identification of viral and cellular factors responsible for restricting infection of hamster cells by MoMLV and GaLV. PMID- 1717712 TI - A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120. AB - Recombinant native human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp160 and gp120 (residues 1 to 511) expressed in insect cells quantitatively adsorbed the group-specific neutralizing antibodies found in human sera. However, these antibodies were not adsorbed by envelope fragment 1 to 471 or 472 to 857 or by both fragments sequentially, even though together they add up to the full-length gp160 sequence. A hybrid envelope glycoprotein was constructed with residues 342 to 511 of the HIV-1 sequence and residues 1 to 399 of the simian immunodeficiency virus type 1 sequence to vary the HIV-1 sequence while preserving its conformation. This hybrid glycoprotein quantitatively adsorbed human neutralizing antibodies, while native simian immunodeficiency virus type 1 envelope glycoprotein did not. These results identify a new neutralizing epitope that depends on conformation and maps to residues 342 to 511 of gp120. It overlaps the extended CD4-binding site but is distinct from the V3 loop described previously (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989; J. R. Rusche et al., Proc. Natl. Acad. Sci. USA 85:3198-3202). Since it is conserved among diverse HIV-1 isolates, this new epitope may be a suitable target for future vaccine development. PMID- 1717713 TI - Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody. AB - The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host. PMID- 1717714 TI - Transgenic mouse lines that are immunologically tolerant and nontolerant to herpes simplex virus glycoprotein D. AB - A construct containing the gene for glycoprotein D of herpes simplex virus (HSV gD), under the control of the simian virus 40 early promoter, was microinjected into single-cell embryos, and four transgenic mouse lines were established. Three were homozygous (lines 75, 111, and 113) and one was hemizygous (line 108) for the HSV-gD gene. Examination of sera revealed that only one of the lines (line 75) spontaneously produced antibody to HSV-gD. Immunization of the other three lines with vaccinia virus-HSV-gD showed that one of them (line 113) responded by making antibody to HSV-gD, whereas the other two (lines 108 and 111) appeared to be immunologically tolerant. Evidence that tolerance was not absolute was obtained by immunization with infectious HSV, which resulted in an antibody response to HSV-gD in some of the animals from line 111. Examination of organs for HSV-gD mRNA revealed transcripts in the tolerant line (line 108) and in the partially tolerant line (line 111), but not in the nontolerant line (line 113), suggesting that the development of immunological tolerance requires active expression of the HSV-gD gene. PMID- 1717715 TI - Demonstration of two distinct cytopathic effects with syncytium formation defective human immunodeficiency virus type 1 mutants. AB - The mechanism of human immunodeficiency virus type 1 (HIV-1) cytopathicity is poorly understood and might involve formation of multinucleated giant cells (syncytia), single-cell lysis, or both. In order to determine the contributions of the fusion domain to syncytium formation, single-cell lysis, and viral infectivity and to clarify the molecular details of these events, insertion mutations were made in the portion of env encoding this sequence in the functional HIV-1 proviral clone HXB2. Viruses produced from these mutant clones were found to have a partial (F3) or complete (F6) loss of syncytium-forming ability in acutely infected CEM, Sup T1, and MT4 T-cell lines. During the early stage of acute infection by F6 virus, there was a loss of the syncytial cytopathic effect, which resulted in increased cell viability, and a 1.9- to 2.6 fold increase in virus yield in the cell lines tested. In the late stage of acute infection, the single-cell cytopathic effect of F6 virus was similar to that of the parental HXB2 virus. The F3 and F6 viruses were also found to have a 1.7- to 43-fold reduction in infectivity compared with the HXB2 virus. The mutant F3 and F6 and parental HXB2 envelope proteins were expressed in vaccinia virus, and the mutant envelope proteins were observed to be defective in their ability to form syncytia. BSC-40 cells infected with vaccinia virus recombinants revealed no differences in kinetics of cleavage, cell surface expression, or CD4 binding capacity of the mutant and parental envelope proteins. These results demonstrate that a loss of syncytium formation results in an attenuation of infectivity and a loss of the syncytial cytopathic effect without a loss of single-cell lysis. These mutants may reflect in tissue culture the changes observed in the HIV isolates in vivo during disease progression, which exhibit marked differences in syncytium production. PMID- 1717716 TI - Alternate promoter selection within a human cytomegalovirus immediate-early and early transcription unit (UL119-115) defines true late transcripts containing open reading frames for putative viral glycoproteins. AB - The human cytomegalovirus open reading frames (ORFs) UL119 through UL115 (UL119 115) are located downstream of the immediate-early 1 and 2 transcription units. The promoter upstream of UL119 is active at all times after infection and drives the synthesis of a spliced 3.1-kb mRNA. The viral mRNA initiates in UL119, contains UL119-117 and UL116, and terminates just downstream of UL115. True late transcripts that are detected only after viral DNA synthesis originate from this transcription unit. True late mRNAs of 2.1 kb, containing ORFs UL116 and UL115, and 1.2 kb, containing ORF UL115 only, are synthesized. The true late viral mRNAs are 3' coterminal with the 3.1-kb mRNA. This transcription unit is an example of late promoters nested within an immediate-early-early transcription unit. The gene products of UL119-117, UL116, and UL115 are predicted to be glycoproteins. Efficient expression of the downstream ORFs at late times after infection may be related to alternate promoter usage and downstream cap site selection. PMID- 1717717 TI - Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody. AB - While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention. PMID- 1717718 TI - Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly. AB - The retroviruses of the avian sarcoma-leukosis virus group synthesize their viral protease (PR) in two precursor forms--as a carboxy-terminal domain of the Gag precursor and as an embedded domain within the Gag-Pol precursor. We have shown previously that the Gag-derived PR is fully capable of processing the Gag precursor in the absence of the embedded PR (R.P. Bennett, S. Rhee, R.C. Craven, E. Hunter, and J.W. Wills, J. Virol. 65:272-280, 1991). In this study, we examined the question of whether or not the PR domain of Gag-Pol has an essential role in the maturation of the Pol proteins. The Gag-Pol precursor was expressed in the absence of Gag by use of a simian virus 40-based vector in which the gag and pol reading frames were fused. The fusion protein accumulated to high levels in transfected cells without being released into the medium but could be rescued into particles by coexpression of the Gag protein from a second vector. The resulting particles contained mature Gag and Pol proteins and active reverse transcriptase (RT). Using this complementation system, the effects of PR defects in the Gag and/or Gag-Pol proteins on the activation of RT were examined. The results showed that the presence of a functional PR on the Gag precursor, but not on Gag-Pol, was required for full activation of RT. The embedded PR of Gag-Pol was unable to carry out any detectable processing of the Gag precursor and was able to activate RT to only a low level in the absence of a functional Gag PR domain. Finally, some point mutations in the Gag-Pol PR domain inhibited activation of RT in trans by a wild-type PR, suggesting that the correct conformation of the PR domain in Gag-Pol is prerequisite for activation of RT. PMID- 1717719 TI - trans-acting viral protease is necessary and sufficient for activation of avian leukosis virus reverse transcriptase. AB - The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We described previously the construction of PR-defective avian leukosis viruses. These mutant viruses are noninfectious, and their major internal components are the uncleaved gag and gag-pol polyproteins (Pr76gag and Pr180gag pol). The reverse transcriptase (RT) activity associated with the PR-defective virions is approximately 500-fold reduced relative to that of wild-type virions, suggesting that specific cleavages activate RT activity. To gain a better understanding of the role that PR plays in the processing and activation of RT, we performed complementation experiments wherein wild-type or PR mutant gag precursors were separately coexpressed with frame-corrected wild-type or PR mutant gag-pol precursors. The results demonstrate that, as in other retrovirus systems, gag-pol precursors can be assembled into virions only when they are rescued by a gag precursor. If the gag precursor is wild type, then the rescued Pr180gag-pol is completely and properly matured, irrespective of whether its embedded PR domain is wild type or mutant. In both cases, the virions produced are fully and equally infectious. This indicates that an active-site mutation in the PR domain of the gag-pol precursor has no effect on avian leukosis virus infectivity when particles are assembled from wild-type gag precursors. In contrast, if the gag precursor has an active-site mutation in PR or is deleted for PR, then the virions are noninfectious and the gag and gag-pol precursors remain unprocessed, even if the embedded PR domain of Pr180gag-pol is wild type. Thus, in this system, virion-associated Pr180gag-pol displays no detectable cis- or trans-acting PR activity. As assayed with an exogenous template, virions with processed gag-pol polyprotein display high levels of RT activity while those with unprocessed Pr180gag-pol display greatly reduced RT activity. These results demonstrate that during virion assembly, the PR supplied by a gag precursor is both necessary and sufficient for trans-activation of RT through proteolytic maturation of copackaged gag-pol polyprotein. PMID- 1717720 TI - Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus. AB - An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication. PMID- 1717721 TI - Short-term treatment with gamma interferon induces stable reversion of ras transformed mouse fibroblasts. AB - Persistent revertants have been generated from NIH 3T3 cells transformed by an activated human Ha-ras gene after short-term gamma interferon treatment in the presence of the cardiac aminoglycoside ouabain. Normal fibroblastlike morphology and anchorage dependence are restored in revertants. Tumorigenicity in nude mice is abolished. The revertants continue to express high steady-state levels of the ras oncogene. Partial retransformation of reverted cells is induced after 5 azacytidine treatment or after infection with retrovirus vectors carrying the v abl, v-fes, v-myc, or v-src oncogene. The revertants resist the transforming activities of the v-Ha-ras and v-mos oncogenes. PMID- 1717722 TI - Role of conserved gp41 cysteine residues in the processing of human immunodeficiency virus envelope precursor and viral infectivity. AB - All animal retroviruses whose nucleotide sequences have been determined contain two or three closely spaced cysteine residues in the extracellular domain of the env-encoded transmembrane protein. Using human immunodeficiency virus type 1 gp41 as a working model, the functional significance of these highly conserved cysteines was investigated. We report here that substituting the two conserved cysteine residues in this domain of gp41 with glycine residues resulted in the loss of viral infectivity, which could be attributed to severe impairment in the processing of gp160 precursor to gp120. PMID- 1717724 TI - Mitral valvular stromal degeneration. AB - A 35-year-old female who presented with features of mitral valve disease showed mitral stromal deposits which were acidophilic in nature and brilliantly red by Masson trichrome. Stromal GAG (glycosaminoglycan) alterations are considered responsible for the deposits. PMID- 1717723 TI - DNR in the OR. Resuscitation as an operative risk. AB - Should do-not-resuscitate (DNR) orders be routinely rescinded when terminally ill patients undergo palliative surgery? If so, patients will be forced to balance the benefits of palliative surgery against the risks of unwanted resuscitation. If physicians are required to honor intraoperative DNR orders, they may feel unacceptably restrained from correcting adverse effects for which they feel responsible. This dilemma has been overlooked by DNR policies. This article argues for the permissibility of honoring intraoperative DNR orders. The patient's right to refuse treatment outweighs physicians' concerns about professional scrutiny over intraoperative deaths. Physicians' moral concerns about hastening patient death are important but may be assuaged by (1) emphasizing patients' acceptance of operative mortality risk; (2) viewing matters as analogous to surgery on Jehovah's Witnesses who refuse lifesaving transfusion; (3) viewing the patient's intraoperative death as a double effect, that is, an unintended negative effect that is linked to the performance of a good act (palliation); and (4) distinguishing this from assisted suicide. PMID- 1717725 TI - [Drug-induced blood dyscrasia in Okinawa]. AB - Forty one cases of drug-induced blood dyscrasia were seen in the last 10 years in six main hospitals in Okinawa. There were 16 males and 25 females. The average age was 53 year-old. The anticonvulsants were the most common causative drugs (12 cases), followed by the antithyroid drugs (6 cases) and Co-trimoxazole (4 cases). The granulocytopenia was the most common type of blood dyscrasia, comprising 51.0% of all cases. Phenytoin was the most common anticonvulsant (8 cases) and 6 cases received it as a prophylaxis following craniotomy. Three cases of antithyroid drug-induced granulocytopenia developed this complication after readministration of the antithyroid drugs. The intervals between the administration of causative drugs and the onset of blood dyscrasia were less than 3 months, except for alpha-methyldopa, gold, and chlorpromazine. Although 30 cases (73.0%) showed complete recovery, there were 3 fatalities (3.0%) which included bicytopenia due to sodium valproate, aplastic anemia due to Co trimoxazole, and pure red cell aplasia due to aspirin. It is suggested from this study that drug-induced blood dyscrasia is not uncommon in Okinawa. PMID- 1717726 TI - [Hematological effect of 14 days treatment of recombinant human granulocyte colony-stimulating factor for neutropenia in myelodysplastic syndromes]. AB - Nineteen patients with myelodysplastic syndromes (MDS) were treated with a glycosylated recombinant granulocyte colony-stimulating factor (rG-CSF) for improvement of neutropenia. rG-CSF was administrated intravenously at a dose of 5 micrograms/kg/day for 14 consecutive days. Most of patients responded to rG-CSF and an approximately 10 fold increase of the peak neutrophil counts was observed. The neutrophil counts were maintained at high level during the treatment period and returned to pretreatment levels several days after stopping rG-CSF. Consistent with the recovery of neutrophil, infectious complications improved in many cases. Effects of rG-CSF were confined to neutrophils, sparing blast cells and other blood cells. Eruption was observed in one patient as toxicity. We conclude that rG-CSF therapy is effective in improving neutropenia with MDS patients. PMID- 1717727 TI - [A decision analysis model for appropriate laboratory use--should test of urine gram stain be done for selecting antibiotics at the initial visit on women with acute dysuria]. AB - We used a decision analysis model to estimate the clinical and economic implications of testing the gram-stained smears to select the effective antibiotics at the initial visit on women with lower urinary tract infections. A strategy of the initial therapy of antibiotics without routine testing was compared with the initial testing of urine gram stain and selecting antibiotics according to the results. Varying assumptions were used for prevalence of etiologic agents, operating characteristics of gram stain test, treatment efficacy and required days for microbiological tests. We concluded that from the view point of expected direct costs and expected care days, a 90% pretest probability of pathogens to be gram negative rods was the critical point to decide to do or not to do urine gram stain for selection of antibiotics. Sensitivity analysis showed that the gram stain testing strategy was cost effective when the frequency of gram positive coccus infection would be increased or the treatment efficacy after antibiotics therapy would be decreased. PMID- 1717728 TI - A key amino acid determining G3m(b) allotypic markers. AB - A key amino acid substitution specific for allotypic Gm b markers, b0, b3, b5, were determined through sequence analyses of the pFc' fragments of IgG1 (Su) and IgG3 (Ba[Gm(g)], Bu[Gm(b1b3)], and Kam[Gm(b3st)]) myeloma proteins. The results indicate that serine at position 384 is responsible for the specificities. It is considered from crystallographic data of IgG-Fc [Deisenhofer et al. (1981) Biochemistry 20:2361] that two residues, the serine and isoleucine specific for IgG3 subclass at position 422, cause the structural change responsible for b markers. The two residues are close to each other in the CH3 domain. The allocations of the epitopes are estimated to be on two bends (residue no. 382 392, 411-424) between the beta-strands, whose amino acid residues are present in wide contact area [Novotny et al. (1987) Immunol. Today 8:26). PMID- 1717729 TI - [Characterization and localization of alpha 1-adrenoceptors in human prostate- with special reference to the zonal difference in receptor distribution]. AB - Radioligand binding assay and receptor autoradiography were undertaken to characterize alpha 1-adrenoceptors and to clarify their localization in human prostate. Eleven open prostatectomy specimens obtained from patients with benign prostatic hypertrophy were used for radioligand binding assay. The binding of [3H]-prazosin to prostatic crude membrane fraction was a saturable process of high affinity. Scatchard analysis of the specific [3H]-prazosin binding indicated the existence of a single population of receptor sites with an equilibrium dissociation constant of 1.00 +/- 0.19 nM (mean +/- SE) and a maximum binding capacity of 20.8 +/- 3.56 fmol/mg protein (mean +/- SE). alpha-Adrenergic antagonists competed for [3H]-prazosin binding in an order of potency: prazosin greater than or equal to bunazosin greater than phentolamine greater than yohimbine, suggesting that the binding sites for [3H]-prazosin have characteristics of alpha 1-adrenoceptors. To study the distribution of alpha 1 adrenoceptors in prostatic tissues obtained from total cystoprostatectomy specimens of six male bladder tumor patients, we used for autoradiography (+/-) beta-[( 125I]Iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone [( 125I]-HEAT). Peripheral zone (PZ), central zone (CZ) and preprostatic region were dissected from the tissues and incubated with [125I]-HEAT, followed by an autoradiographic procedure. Distribution of alpha 1-adrenoceptors was quantitated by grain counting using a light microscope equipped with a micrometer. The specific binding sites for [125I]-HEAT were demonstrated predominantly in the fibromuscular stroma of CZ, that of PZ, preprostatic sphincter and subordinately in the glandular epithelium of CZ, but not in the glandular epithelium of PZ and periurethral glands.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717730 TI - [Long-term follow up of transurethral resection of the prostate--a review of 137 patients]. AB - For assessing the long-term outcome of patients after transurethral prostatic resection (TUR-P), telephone interview in terms of the urinary symptom and the sexual function was conducted on 191 cases who underwent TUR-P in Hokkaido University Hospital from 1982 to 1988. Adequate replies were obtained from 137 patients (71.7%), whose mean age was 70.2 years old and mean follow up period was 4.8 years. Subjective urinary symptoms, which are mainly classified as dysuria, frequency and incontinence, were improved in 114/120 (95.0%), 99/108 (91.7%), and 20/21 (95.2%) respectively. Overall symptom-free rates of dysuria, frequency, and incontinence were 85.1% (114/134), 86.6% (116/134), and 90.3% (121/134). Morbidity of incontinence following TUR-P was only 6/134 (4.5%). There was one deaths (0.7%) at 2 weeks after TUR-P, but was not attributable to the operative procedure itself. Although 82 cases (59.9%) had risk factors such as the cardiovascular disease, malignancy or other systemic disorders, they did not jeopardize the postoperative course nor were attributed to the mortality. Uninhibited contraction and/or vesical denervation supersensitivity on perioperative cystometrogram were found in 53/84 (63%). These urodynamic abnormalities were not considered to be postoperative urinary symptoms. Postoperatively, the decrease in libido was noted in 12/63 (19%), but its causal relation to the procedure was obscure in most of the patients. We believe TUR-P can offer a satisfactory outcome in the majority of the patients with minimum risk. PMID- 1717731 TI - [The urodynamic effects of an alpha 1-adrenoceptor agonist on the bladder and urethra of female dogs]. AB - It has been known that alpha 1-adrenoceptors play an important role in urethral contraction. The incompetence of the urethral contraction is a cause of stress incontinence. We studied the urodynamic effects of a selective alpha 1 adrenoceptor agonist (midodrine hydrochloride) on the bladder and urethra of female dogs. Under anesthesia with intravenous chloralose, four doses (0.03, 0.1, 0.3 and 1.0 mg/kg) of midodrine were administered intravenously and urodynamic studies including cystometry, urethral pressure profilometry and electromyography (EMG) of the external urethral sphincter were performed. The administration of midodrine induced a significant increase of the maximum closing pressure in the proximal portion of the urethra (p less than 0.05 at 0.03 mg/kg and p less than 0.01 at 0.1, 0.3, 1.0 mg/kg). There were no significant changes in the functional profile length, the closing pressure at the external sphincters, the maximum bladder pressure, bladder capacity and bladder compliance. The administration of 0.3 mg/kg or more of midodrine produced a significant increase in the mean arterial blood pressure. After midodrine administration, transient increases in the external sphincter EMG activities were recognized. The activities showed the synergistic pattern during the bladder contractions. In conclusion, lower dose administration of a selective alpha 1-adrenoceptor agonist (midodrine) specifically produced an increase of the closing pressure in the proximal portion of the urethra without affecting blood pressure. These results suggest that midodrine is useful for the treatment of stress incontinence in humans. PMID- 1717732 TI - Desert Storm: one army nurse's experience in a forward surgical unit during the ground offensive. PMID- 1717733 TI - [Comparative effectiveness of class I anti-arrhythmia drugs and the algorithms of their selection in patients with ventricular arrhythmia]. AB - The efficacy of 7 well-known Class I antiarrhythmic agents were retrospectively and by+crossover compared in 2 groups of patients treated in hospital in different years for ventricular arrhythmias. Class IC agents proved to be the most potent in the two groups of patients. The following algorithm is proposed to choose Class I agents: if quinidine or mexiletine is beneficial in the same patients, the other drugs will be also effective. If disopyramide fails to be beneficial, allapinin , ethacizine , propaphenon will be the most effective. When allapinin produces effects, ethacizine and propaphenon show their highest potency, but when each of them fails, a combination of antiarrhythmic agents should be used. PMID- 1717734 TI - [Study of the dynamics of cardiac rhythm and the ST segment in patients with acute myocardial infarction based on the data of Holter ECG monitoring]. AB - Holter monitoring was performed on days 1-2, 6-9 of the disease and on days 30-60 before their discharge from hospital in 54 patients with acute gross myocardial infarction. The presence of cardiac rhythm and conduction disturbances and ischemic ST-segment depression or elevation was evaluated. The patients having frequent and prolonged (more than 3 hours during a 2-day follow-up) showed a complicated course of the disease: recurrent pain syndrome, signs of heart failure, prolonged cardiac arrhythmias, and fatal outcomes. The patients with uncomplicated acute myocardial infarction had no long-term episodes of ischemic ST-segment depression or elevation, as recorded by Holter monitoring in the first 2 days of the disease. On days 6-9 no cardiac rhythm and conduction disturbances that had been observed in them were recorded. The patients in whom the episodes of silent myocardial infarction remained on their discharge exhibited a high (35%) incidence of myocardial infarction recurrence within a year. PMID- 1717735 TI - [Glucocorticoid function of the adrenal cortex in patients with myocardial infarction: study of elimination of exogenous cortisol]. AB - The paper yields the results of the study into the elimination of exogenous cortisol from the blood bed in 26 patients with myocardial infarction. It is concluded that the nature of cortisol elimination may reflect the natural history and prediction of myocardial infarction. In terms of the body tissue demand, two type of elimination are identified: retarded (a favorable type) and accelerated (an unfavorable type). The data on the nature of cortisol elimination may serve as an indication for glucocorticoid therapy. The baseline cortisol concentrations fail to reflect the nature of glucocorticoid metabolism in patients with myocardial infarction. PMID- 1717736 TI - Dietary protein restriction and glomerular permselectivity in nephrotoxic serum nephritis. AB - We have previously demonstrated that long-term dietary protein restriction ameliorates proteinuria and limits glomerular structural injury in rats with nephrotoxic serum nephritis. In the present study, we examined the influence of short-term dietary protein restriction on glomerular permselectivity. As compared to nephritic rats maintained on a normal protein diet, whole kidney and single nephron hemodynamics were lower in nephritic rats subjected to dietary protein restriction of three days duration (glomerular filtration rate: 0.79 +/- 0.10 vs. 1.46 +/- 0.11 ml/min, P less than 0.003; renal plasma flow rate: 2.50 +/- 0.34 vs. 3.96 +/- 0.38 ml/min, P less than 0.02; glomerular capillary pressure: 44 +/- 1 vs. 53 +/- 1 mm Hg, P less than 0.002; proteinuria: 77 +/- 15 vs. 224 +/- 14 mg/24 hr, P less than 0.01). This was associated with a rise in afferent resistance, from 2.99 +/- 0.77 to 5.45 +/- 0.94 dyn.sec.cm-5, NS. In nephritic rats maintained on 24% protein, fractional clearances were elevated above control values for neutral dextrans with molecular radii exceeding 50 A but were depressed for those with molecular radii below 30 A (P less than 0.05). Dietary protein restriction elevated the fractional clearances of dextrans with radii less than 30 A while depressing the fractional clearances of dextrans with radii greater than 50 A (P less than 0.05). The proportion of glomerular filtrate permeating the shunt pathway was elevated above control values in nephritic rats on the 24% protein diet but declined in those fed the low protein diet (NSN-24%: 0.86%; NSN-6%: 0.31%; control: 0.19%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717737 TI - [Essays on gastric surgery]. PMID- 1717738 TI - [Spongy dressings from alginic acid salts in the treatment of suppurative wounds]. AB - Spongy dressing materials made from alginic acid salts, specifically immobilized with terrilitinum (teralginum) were found to reduce the period of wound purification from suppurative and necrotic tissues and to facilitate more rapid, viz. 1.5-fold compared to the control, patient recovery, particularly after preliminary wound treatment with defocussed CO2 laser beam. In the group on teralginum, the cytological study demonstrated more intensive wound purification from microflora, more active growth and differentiation of reparation cells, specifically of fibroblasts, which influences the results of accelerated wound healing. PMID- 1717739 TI - [Hemodilution in the treatment of central retinal vein thrombosis]. AB - The authors present contemporary opinions on the aetiology, forms and prognosis in central retinal vein occlusion. Methods of treatment by haemodilution, indications and contraindications are discussed. PMID- 1717740 TI - Immunohistochemical study on hepatic tumors--KM01 stains compared with AFP, CEA, CA19-9 and RAS P21. AB - Pathological diagnosis of hepatic tumors is sometimes difficult when performed with only routine examinations such as Hematoxylin and Eosin (H.E.) stain. The diagnostic usefulness of KM01 was compared to that of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9 and ras p21 in this immunohistochemical study. AFP was positive in about half of the cases of hepatocellular carcinoma and hepatoblastoma, and AFP-positive cells were frequently found at the periphery of acini in both diseases. Absorbed CEA stain was mostly negative in hepatocellular carcinoma, but was positive in the cells of mixed hepatocellular and cholangiocellular carcinoma (MHCC) and metastatic liver cancer, especially in their cytoplasm. CA19-9 immunostaining was completely negative, and was only 3% positive in hepatocellular carcinoma. KM01 stain was positive in about half of the cases of hepatocellular carcinoma, hepatoblastoma and MHCC. It was positive in proliferated bile ducts around the capsule in the former two diseases but positive in the tumor cell of both parts of the cytoplasm in the latter. The histological positivity of ras p21 was high in all tumor cells of these three types of tumors. Negative absorbed CEA and KM01 in pseudoglandular hepatocellular carcinoma differentiated from MHCC and metastatic liver cancer. However these tumor markers were occasionally positive and nonspecific in cancer-like lesions, implying no advantage for differential diagnosis between hepatocellular carcinoma and apparent cancer-like lesions. The above results demonstrate that AFP, CEA and KM01 are effective for differentiating hepatocellular carcinoma among various hepatic tumors. PMID- 1717741 TI - The effect of microtubule stabilizer on rat caerulein-induced pancreatitis. AB - Inhibition of pancreatic digestive enzyme secretion in the acinar cell is a significant phenomenon which can trigger acute pancreatitis. It has been shown that microtubules are responsible for the intracellular vesicular transport. The effect of taxol, a microtubule stabilizer, was examined in a model of acute edematous pancreatitis induced in rats by supramaximal stimulation with cholecystokinin analogue, caerulein. Prophylactic administration of taxol ameliorated inhibition of pancreatic digestive enzyme secretion, increased level of serum amylase, pancreatic edema, and histological alterations in this model. Immunofluorescence studies revealed that taxol stabilized the arrangement of microtubules by promoting tubulin polymerization. On the other hand, microtubule disorganization was found in rats without prophylactic taxol treatment. In caerulein-treated rats, there is microtubule disorganization which causes interference with intracellular vesicular transport leading to inhibition of pancreatic digestive enzyme secretion--a forerunner of acute pancreatitis. Taxol was found to prevent these events. PMID- 1717742 TI - [Alkaline phosphatase: methodologic procedures and diagnostic significance (review of the literature)]. PMID- 1717743 TI - [Determination of fecal alpha-amylase activity in the diagnosis of chronic duodenitis]. AB - Amylasorrhea has been detected in 92-97 percent of 190 patients suffering from chronic duodenitis, 40 of these children and 50 ones with concomitant imbalance of colonic microflora, during acutely or moderately manifest catarrhal and erosive mucosal abnormalities. This condition was somewhat more marked in the children and in the patients with colonic microflora imbalance, but less acute than in acute intestinal dysfunctions such as dysentery. A characteristic feature of duodenitis-associated amylasorrhea is the predominance of a thermolabile pancreatic fraction of amylase (a small-intestinal component of amylase); in cases with concomitant colonic microflora imbalance the share of thermostable intestinal fraction (amylase large-intestinal component) grows reaching its maximum in dysentery. In chronic duodenitis amylasorrhea is regularly detectable over the course of many years; whereas in dysentery it is more manifest though not lasting. Transient impaired membranous digestion involving a reduction of the intestinal surface to adsorb pancreatic amylase appears to be responsible for duodenitis associated amylase thin-intestinal component. PMID- 1717744 TI - [The aldehyde dehydrogenase activity in the erythrocytes of alcoholic patients]. AB - Red cell aldehyde dehydrogenase (ALDH, EC 1.2.1.3) activity was measured by spectrophotometry in normal subjects and alcoholics after various periods of alcohol abstinence. ALDH was measured in all red cell hemolysate fractions; red cells were purified from hemoglobin by Sephadex CM-50 chromatography. The enzyme activity was found reduced in alcoholics' red cells as against that in controls. ALDH activities were somewhat increasing and approaching the normal values in the patients not using ethanol for 4 weeks or longer. These results recommend ALDH measurements in human red cells as a test for the detection of subjects abusing alcohol. PMID- 1717745 TI - [Determination of the iron-binding capacity and transferrin in blood]. AB - Though the total iron-binding activity of the blood serum directly depends on blood serum transferrin level, these are different parameters. It is possible to measure transferrin levels with direct estimation of the share of this protein saturation with iron without preliminary measurement of the total iron-binding activity of the blood serum. The degree of transferrin saturation with iron is virtually unrelated to the results of its measurement by various immunochemical methods. PMID- 1717746 TI - [The erythrokaryocyte, the reticulocyte, the normocyte: their place in the scheme of erythropoiesis]. AB - Estimation of reticulocyte and polychromatophil counts has shown that only 28.7 44.9 percent of blood reticulocytes and 61.7 percent of bone marrow reticulocytes are polychromatophilic. Polychromatophil count is approximately equal to the sum of classes I and II reticulocytes. Bone marrow smears staining to detect reticulocytes, followed by staining according to Romanovsky-Giemsa has shown that only solitary erythrokaryocytes did not contain granular or filament reticular substance. The authors analyze the following suppositions: eliminate reticulocytes from the erythropoiesis scheme, denote the former normoblasts erythrokaryocytes but not normocytes, demonstrate the possibility of mature red cell production from oxyphilic erythrokaryocytes and from polychromophilic erythrokaryocytes via polychromatophilic red cells in the scheme. PMID- 1717747 TI - [The surface architectonics of peripheral blood erythrocytes in patients with lung cancer]. AB - Electron microscopic examination of the surface relief of the peripheral blood red cells in 27 untreated male patients with Stages III-IV pulmonary carcinoma, 10 patients with chronic nonspecific pulmonary diseases, and 10 normal subjects has revealed a statistically significant decrease of the discocyte count paralleled by an elevated count of transitory and prehemolytic cellular forms in pulmonary carcinoma patients, as against that in reference groups. Cytometric study of the red cells in patients with tumors has shown an increase in the ratio of the external to internal cellular diameters vs. that in the controls. PMID- 1717748 TI - [Crossed affinity immunoelectrophoresis using lectins for studying acute phase proteins in human blood (review of the literature)]. PMID- 1717749 TI - [A method of determining erythrocyte deformability]. AB - Red cell suspension filtration through synthetic filters is the most prevalent method for estimation of red cell deformability. A modified technique with the use of cellulose acetate filters manufactured by the Polimersintez Research and Production Amalgamation in the town of Vladimir is suggested. Employment of these filters will help introduce this test in clinical practice. PMID- 1717750 TI - [A micromodification of the determination of specific leukocyte lysis in vitro]. AB - A micromodification of the specific leukocyte lysis method was developed; only 0.02 ml of blood is sufficient for the test. The micromodification improved the reproducibility of the technique more than twofold, simplified it, and is recommended for examinations of some populations, including children whose body reactivity is changed and an individual approach to their immunization with TDP vaccine is necessary. PMID- 1717751 TI - [Determination of carcinoembryonic antigen in lung cancer using an immunoenzyme assay]. AB - Elevation of the carcinoembryonal antigen concentration in patients with pulmonary carcinoma is directly dependent on the tumor process dissemination. Measurements of mucinous cancer-associated antigen, neurospecific enolase, CA-125 improve the reliability of enzyme immunoassays of tumor markers of lung cancer. Measurements of carcinoembryonic antigen over the course of treatment may become a valuable test permitting an objective assessment of the treatment efficacy. PMID- 1717752 TI - [Determination of tuberculosis antibodies using an immunoenzyme assay in lyophilized samples of capillary blood]. AB - The aim of this work was to find the technique of enzyme immunoassay (EIA) of capillary blood lyophilized on a filter paper disk. The author has selected a buffer on the basis of egg yolk aqueous extract, that permits blocking of nonspecific binding in EIA but not preventing the specific antigen-antibody interactions. The results of analyses of capillary blood lyophilized drops and of blood serum samples, carried out in 7 tuberculous patients and 6 donors, coincided. The results were tested in 57 blood samples. PMID- 1717753 TI - [The objectives and tasks of clinical microbiology. Outlook for the development of microbiological laboratories]. PMID- 1717754 TI - [The diagnosis of Pneumocystis pneumonia (use of the Bauer reaction for detecting Pneumocystis carinii in pathological material]. PMID- 1717755 TI - [Typing mycobacteria using the specific inhibitor method]. AB - A radionuclide method permits assigning a strain to M. bovis or to M. humanis group, M. bovis R-INH, MOTT on the 4th day. The criterion is the ratio of population growth indices in control cultures and in the presence of an inhibitor, thiophene-2-carboxylic acid. PMID- 1717756 TI - [An evaluation of methods of determining the hemolytic activity of Klebsiella in solid nutrient media]. AB - Hemolytic activities of Klebsiella-237 strains of various origins were measured in solid nutrient media by three methods making use of six different agar bases for the preparation of blood nutrient media. The authors analyze the agar bases, discuss the advantages and shortcomings of all the methods used with consideration for the purpose of the investigation. The findings evidence a higher hemolytic activity in Klebsiella isolated from patients as against the hemolytic activities of reference strains. PMID- 1717757 TI - [Microflora of the cervical canal and the uterine cavity in chronic nonspecific inflammatory diseases of the uterus and the adnexa uteri]. AB - The authors analyze the data pointing to the heterogeneity of the bacteriologic status of the cervical canal and uterine cavity in the women suffering from nonspecific chronic inflammations of the internal genitals. Identification of the bacteriologic contents of the uterine cavity will permit individual antibiotic therapy of the patients. PMID- 1717758 TI - [Cytology of the ascitic fluid in a patient suspected of pseudomyxoma]. PMID- 1717759 TI - [A device for collecting saliva from infants]. PMID- 1717760 TI - [A thermostat with a small-scale chamber for carrying out crystallization of biological fluids]. AB - The authors suggest the use of liquid media of the body after their crystallization under exposure to physical fields. They have developed a small chamber thermostat for crystallization of these media; the chamber may be placed in an external physical field during crystallization. PMID- 1717761 TI - [The work of the Department of Clinical Laboratory Diagnosis of the Ukrainian Institute for Advanced Medical Education over a period of 60 years]. AB - The author reviews the training and research activities of the Chair over the 60 years of its existence and the contributions of the scientists who have worked there. Special attention is given to the activities of one of the heads of the Chair, Professor A. Ya. Althauzen (1890-1960), whose birth centennial was remembered in 1990. PMID- 1717762 TI - [Alternative variants of the provision of reagents for multichannel analyzers]. PMID- 1717763 TI - [Experience in the organization of work and pay by the brigade method in laboratory practice]. AB - A specialized team for specific and nonspecific diagnosis of syphilis was created at the central serologic laboratory of the hospital of the Uzbek Research Institute for Dermatology and Venereology. The laboratory annually analyzes 350,000 serum samples. A principle for estimation of labor contribution coefficient has been developed, based on assessment of labor operations performed by each team member. Work on a team basis helped intensify the labor, improve the quality of serologic diagnosis by introducing present-day methods of investigation. PMID- 1717764 TI - [Experience in the work of a biochemical laboratory in a diagnostic center]. PMID- 1717765 TI - [The control of hydrolysis during studies of blood serum sialic acids]. AB - The author suggests improving the quality of the blood serum sialic acid studies by monitoring and correcting the results via intralaboratory control of the hydrolysis quality. The suggested method has improved the validity of the results: the coincidence of laboratory findings with the clinical data and the results of protein fraction studies has grown from 56.6 to 93.4 percent. PMID- 1717766 TI - Laser bronchoscopy. PMID- 1717767 TI - Effects of serotonin on platelets and blood vessels. AB - We advance the hypothesis that ketanserin, a 5-HT2 receptor antagonist, may provide vascular protection. This is due mainly to inhibition of the effect of serotonin, released by platelets activated at the site of arterial wall injury, on platelets and vascular smooth muscle cells which contribute to vascular smooth muscle cell proliferation and connective tissue synthesis, vasospasms in areas of defective endothelial cell-dependent relaxation and to platelet-dependent thrombus formation in various experimental scenarios. Clinical data obtained so far with ketanserin in patients, subjected to coronary angioplasty, with critical coronary stenosis and at risk for vascular accidents due to atherosclerosis, suggest that, within certain limitations, vascular protection by ketanserin might also occur in man. PMID- 1717768 TI - Serotonin and thrombotic complications. AB - We believe that the abrupt conversion from chronic stable to unstable angina and the continuum to acute myocardial infarction may result from myocardial ischemia caused by progressive platelet aggregation and dynamic vasoconstriction themselves caused by local increases in thromboxane and serotonin at sites of coronary artery stenosis and endothelial injury. Platelet aggregation and dynamic coronary artery vasoconstriction probably result from the local accumulation of thromboxane and serotonin and also relative decreases in the local concentrations of endothelially derived vasodilators and inhibitors of platelet aggregation, such as endothelium-derived relaxing factor (EDRF) and prostacyclin. With severe reductions in coronary blood flow caused by these mechanisms, platelet aggregates may increase, and an occlusive thrombus composed of platelets and white and red blood cells in a fibrin mesh may develop. When coronary arteries are occluded or narrowed for a sufficient period of time by these mechanisms, myocardial necrosis, electrical instability, or sudden death may occur. We believe that unstable angina and acute myocardial infarction are a continuum in relation to the process of coronary artery thrombosis and vasoconstriction. When the period of platelet aggregation or dynamic vasoconstriction at sites of endothelial injury and coronary artery stenosis is brief, unstable angina or non-Q-wave infarction may occur. However, when the coronary artery obstruction by these mechanisms is prolonged for several hours. Q-wave myocardial infarction results. Chronic endothelial injury and coronary artery stenosis are probably associated with the accumulation of platelets, white and red blood cells, and a fibrin mesh at the site of stenosis and endothelial injury.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717769 TI - Implications of thrombosis and vasospasm in peripheral vascular disease. AB - Platelet activation releases not only thromboxane A2 but also serotonin, another vasoconstrictor agent that acts on blood vessels through a specific, 5-HT2 receptor. The development of ketanserin, the selective 5-HT2-receptor blocker, has made it possible to explore the role of serotonin in animal models and patients with endothelial injury and atherosclerotic disease. In animals models, growing collateral arterial vessels are exquisitely sensitive to the vasoconstrictor effects of serotonin, which are reversed by ketanserin. When endogenous platelet activation is induced by endothelial injury, a combination of ketanserin and a thromboxane synthesis inhibitor or antagonist is more effective at reversing collateral arterial vasoconstriction than either agent alone. More recently, studies at the time of angiography in humans with advanced atherosclerosis have shown that more than 50% display a dilator response to ketanserin in low doses: the response primarily involves dilatation of collateral arterial vessels, which is associated with a significant increase in calf blood flow. These findings support existing evidence that platelet products contribute to the ischemia syndromes due to atherosclerosis, and provide a promising approach to improved management. PMID- 1717770 TI - Age-related effects of 5-HT2 antagonists. AB - Ketanserin, an S2 antagonist, has been shown to be an effective antihypertensive drug. Carefully controlled clinical trials have demonstrated that ketanserin is as effective as both metoprolol and thiaside diuretics in reducing blood pressure. However, unlike the beta-blocking drug metoprolol and the diuretics, the response rate to ketanserin is significantly greater in older patients, reflecting perhaps the greater vasoconstrictor effects of 5-hydroxytryptamine in patients with atherosclerotic disease. This enhanced vasoconstrictor response to serotonin in elderly patients may be matched by an increased effect on platelet aggregation, which is also blocked by ketanserin. There is very suggestive evidence from the PACK study that ketanserin may reduce vascular thrombotic episodes in patients with extensive atherosclerosis. This unique characteristic, combined with effectiveness as an antihypertensive agent, makes ketanserin a particularly useful drug in the treatment of elderly patients with hypertension and vascular disease. PMID- 1717771 TI - Platelet serotonin-LDL interaction in essential hypertension. AB - Serotonin (5-HT) released from activated blood platelets plays a pivotal role in the development of thromboembolic complications. Essential hypertension, elevated plasma cholesterol, older age, and smoking are predisposing factors for these vascular events, and they all lead to platelet activation. In hypertensive patients, platelet 5-HT uptake is reduced with increasing age and blood pressure. This uptake inhibition is paralleled by an exaggerated platelet shape change and aggregation response. As a consequence, periplatelet 5-HT plasma concentrations are increased and promote further platelet aggregation and vasoconstriction. Low density lipoproteins (LDL) induce a platelet shape change reaction and amplify the aggregatory response to serotonin. This effect is enhanced in smokers and even greater in hypertensive patients. LDL also inhibit endothelium-dependent relaxations to serotonin thereby unmasking the vasoconstrictor effect. 5-HT2 Receptor blockade with ketanserin interferes with this chain of events at several sites. Ketanserin inhibits platelet 5-HT release and 5-HT-induced platelet aggregation. It exerts a beneficial influence on the lipid profile. Blockade of 5 HT2 receptors leads to direct vascular smooth muscle dilatation as well as unopposed activation of endothelial 5-HT1-like receptors with the subsequent release of endothelium-derived relaxing factor. Taken together, the antihypertensive agent ketanserin may provide a new approach for multiple risk factor intervention therapy, eventually to prevent thromboembolic complications. PMID- 1717772 TI - New approaches to the prevention of thrombotic restenosis after angioplasty or thrombolysis. AB - Percutaneous transluminal coronary angioplasty (PTCA) and early coronary thrombolysis are now widely used to treat selected patients with atherosclerotic coronary artery disease and acute myocardial infarction, respectively. However, the efficacy of these procedures may be limited by the occurrence of thrombotic complications. Restenosis of the dilated vessel, which occurs in about 30% of the patients, is considered the Achilles' heel of PTCA. Experimental and clinical data suggest that restenosis after PTCA may be mediated by platelet attachment at the site of balloon dilation, with a consequent release of platelet-derived mitogen factors able to stimulate intimal hyperplasia. Similarly, a thrombotic reocclusion of the infarct-related coronary artery may significantly reduce the potential benefits of early thrombolysis. This reocclusion occurs in up to 30-35% of the patients successfully reperfused after suspension of the thrombolytic agent, despite the concomitant use of heparin. Increasing evidence indicates that intracoronary platelet activation and aggregation play a major role in the pathophysiology of reocclusion. First, it has been demonstrated that a marked increase in thromboxane A2 production occurs at the moment of reperfusion in patients with acute myocardial infarction undergoing successful thrombolysis. Second, in experimental models of coronary thrombosis, reocclusion after thrombolysis can be prevented or markedly delayed by antiplatelet interventions, such as administration of a monoclonal antibody against the platelet glycoprotein (GP) IIb and IIIa--the putative fibrinogen receptor on platelet membrane whose exposure seems to be an obligatory step for platelet aggregation to occur.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717773 TI - Serotonergic type-2 (5-HT2) antagonists: a novel class of cardiovascular drugs. AB - Serotonin (5-hydroxytryptamine; 5-HT) is widely distributed in the body and subserves many functions. Tissue specificity of action is aided by differential receptor structure and function; the type 2 (5-HT2) receptor mediates arterial constriction and platelet aggregation. Very little serotonin is free in plasma, most being platelet-bound; however, local platelet activation and consequent serotonin release can present free serotonin to peripheral tissues. Serotonin, acting via the 5-HT2 receptor, can contribute to a range of cardiovascular problems, including portal hypertension, Raynaud's phenomenon, carcinoid flushes, preeclampsia, hypertension, arterial atheroma, and restenosis after angioplasty or thrombolysis. 5-HT2 antagonists have a potential therapeutic role in all these conditions. The diversity of such syndromes requires that the term "vascular protection" should not be applied loosely, but must always be precisely defined. Future 5-HT2 antagonists will probably be of two kinds: (a) with weak accompanying alpha 1 antagonism where blood pressure reduction is needed; and (b) as "pure" 5-HT2 antagonists, for use where arterial pressure falls are best avoided. PMID- 1717774 TI - Ketanserin in the treatment of peripheral vascular disease or hypertension in patients with diabetes mellitus: a review. AB - The available data on the use of ketanserin in diabetes mellitus have been reviewed. Ketanserin does not worsen glucose tolerance or destabilize diabetic control. Ketanserin can improve impairment of peripheral blood flow, and especially skin blood flow; the healing of cutaneous ulcers is improved; trophic skin changes are fewer; and there is a trend towards less limb amputations. Walking distance in diabetic claudicants is unaltered by ketanserin. Controlled trials in hypertensive diabetics have provided variable results, with a significant fall in arterial pressure in only one of five studies. As in nondiabetics, ketanserin generally lowers serum cholesterol. The electrocardiographic QTc interval is prolonged. Symptomatic side effects in diabetics are as those in nondiabetic subjects. PMID- 1717775 TI - Platelet-derived serotonin, the endothelium, and cardiovascular disease. AB - The endothelial cells can release both relaxing and contracting substances. The former include prostacyclin and endothelium-derived relaxing factor (EDRF, which most likely is nitric oxide, or a nitrosoderivative releasing nitric oxide, derived from L-arginine). Candidates as endothelium-derived contracting factors (EDCF) include superoxide anions thromboxane A2 and the peptide endothelin. Endothelium-derived relaxing factor causes relaxation of vascular smooth muscle by activation of the soluble form of guanylate cyclase which leads to an accumulation of cyclic GMP; it also reduces platelet adhesion and aggregation. The latter effect is synergistic with the inhibition evoked by prostacyclin. The release of EDRF and prostacyclin plays a key role in the protective role of the endothelium against vasospasm and the unwanted coagulation of blood. Indeed, thrombin and aggregating platelets are potent stimuli for the release of EDRF. The platelet-products responsible are the adenine nucleotides, ADP and ATP, which activate P2y-purinergic receptors on the endothelial cells and 5 hydroxytryptamine (serotonin) that stimulates 5-HT1-like serotonergic receptors. The response to serotonin, but not that to the adenine nucleotides, is mediated by a pertussis toxin-sensitive mechanism. When endothelial cells regenerate, or are cultured, they selectively lose the pertussis toxin-sensitive mechanism of release, which results in a marked decrease in sensitivity to exogenous and platelet-released serotonin. As a consequence, the endothelial cells exhibit a considerably reduced response to aggregating platelets. This phenomenon, which can be exacerbated by hypercholesterolemia, favors ongoing platelet aggregation and vasospasm, and constitutes a first step toward atherosclerosis. PMID- 1717776 TI - Arterial and endocrine effects of a combination of an angiotensin converting enzyme inhibitor and a vasodilator in normotensive healthy volunteers. AB - The aim of the present study was to look for possible additive or synergistic effects of the combined oral administration of a single dose of an angiotensin converting enzyme (ACE) inhibitor, benazepril (10 mg) (B), and a long-acting vasodilator, cadralazine (5 mg) (C), on blood pressure, arterial parameters, and active plasma renin. The study was carried out in eight normotensive subjects according to a double-blind, randomized, placebo-controlled, crossover design. Blood pressure (BP), heart rate, humeral artery diameter (D), carotid-femoral pulse-wave velocity (PWV), finger-pulse ratio (FPR), and plasma active renin (PAR) were measured at baseline and every 2 h over 8 h. A significant treatment effect was observed for supine and tilted BP, FPR, PWV, and PAR. The largest decrease in supine systolic and diastolic BP was observed with the combination (10.0 +/- 6.9/7.2 +/- 3.7 mm Hg). Six hours after drug intake, the mean changes in FPR were 0.05 +/- 0.24 (P), -0.06 +/- 0.30 (C), 0.13 +/- 0.32 (B), and 0.28 +/ 0.34 (B + C), and the mean changes in PWV were 0.14 +/- 0.66 m/s (P), 0.09 +/- 0.54 m/s (C), -0.29 +/- 0.50 m/s (B), and -0.55 +/- 0.48 m/s (B + C). PAR was more markedly augmented with the combination of the two drugs (142 +/- 40 pg/ml) than with benazepril alone (90 +/- 62 pg/ml). It was concluded that a single noneffective dose of a vasodilator administered together with an ACE inhibitor in normotensives can lower blood pressure and increase arterial compliance and plasma active renin. PMID- 1717777 TI - The positive inotropic drugs DPI 201-106, BDF 9148, and veratridine increase ouabain toxicity and [3H]ouabain binding in guinea pig heart. AB - The interaction of three positive inotropic compounds, which are modulators of sodium channels, with the cardiac glycoside ouabain was investigated in isolated guinea pig atria. In the presence of DPI 201-106 (3 x 10(-7) M), of its new acetidine derivative BDF 9148 (10(-7) M), or of veratridine (10(-6) M), the threshold ouabain concentration to induce toxicity was lowered by a factor of 2. This effect can be explained by the observation that specific equilibrium [3H]ouabain binding in intact atria was elevated by these compounds in the appropriate concentrations. The binding results were analyzed by means of a previously established model of "positive cooperative ouabain binding to intact myocardium," which describes the relationship between cellular sodium homeostasis and ouabain binding. The extent to which the compounds increased ouabain binding was in good quantitative agreement with the observed shift in the threshold concentration of ouabain toxicity. The increase of specific [3H]ouabain binding is likely initiated by a gain in cytosolic sodium. It takes place at the cellular level only, since a direct enhancement of [3H]ouabain binding to isolated cardiac membranes was not found. In conclusion, at least under some conditions, new inotropic drugs acting as sodium channel modulators can increase the risk of digitalis toxicity. PMID- 1717778 TI - The antihypertensive property of NIP-121, a novel potassium channel opener in rats. AB - The antihypertensive effects of NIP-121, a novel potassium channel opener, were examined in comparison with cromakalim and its active enantiomer, lemakalim. In experiments by direct blood pressure measurements, orally administered NIP-121 dose-relatedly decreased arterial blood pressure in conscious spontaneously hypertensive rats (SHRs), and the ED20 values (the doses to produce 20% decrease of the mean blood pressure) of NIP-121 and cromakalim were 0.010 and 0.11 mg/kg, respectively, NIP-121 thus being about ten times more potent than cromakalim. The duration of the hypotensive effect by NIP-121 was longer than that by cromakalim. The hypotensive effect of NIP-121 was stronger in SHRs than in normotensive rats. All three drugs showed tachycardia that was antagonized by a beta-blocker, propranolol. Intravenously administered NIP-121 also showed a more potent hypotensive action with longer duration than cromakalim in conscious SHRs. The ED20 values for hypotension by NIP-121, cromakalim, and lemakalim were 0.017, 0.040, and 0.016 mg/kg, respectively. The intravenous hypotensive potency of NIP 121 but not cromakalim was similar to that of p.o. administration. The repeated treatments with NIP-121 (0.025, 0.05, and 0.1 mg/kg p.o. once a day) for 15 days did not modify the degree of the hypotensive action. In the anesthetized SHRs, pretreatment with glibenclamide but not other antagonists (atropine, propranolol, diphenhydramine + cimetidine, or indomethacin) suppressed the decrease in blood pressure induced by NIP-121.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717779 TI - Norepinephrine uptake and efflux from canine peripheral collateral arteries. AB - Collateral arteries from canine hindlimb contain less norepinephrine (NE) than control (normal branch) arteries yet they release more NE per stimulation. This study examines basal neuronal function in developing collateral arteries to determine if the mechanism responsible for this impairment includes limitations on reuptake and/or release of NE. Uptake of NE, determined by accumulation of [14C]l-NE, was significantly less in collateral arteries compared to branch arteries. Cocaine (10(-5)M) decreased the rate of uptake of [14C]l-NE into branch arteries by 60% but had no effect on uptake into collateral arteries. Likewise, normetanephrine (10(-5)M) had no effect on uptake of [14C]l-NE into collateral arteries and only a slight effect on branch arteries. The rate of efflux of [14C]l-NE was greater from collateral arteries. Cocaine decreased the efflux of endogenous NE by 25-30% from collateral arteries while increasing the efflux of endogenous NE from branch arteries by 20-100%. Corticosterone (10(-4) M) and pargyline (10(-4) M) had little effect on endogenous NE efflux. These studies support the hypothesis that, compared with normal branch arteries, the decreased amount of neuro-transmitter in sympathetic neurons of collateral arteries is due to both decreased uptake and increased basal and stimulated efflux. PMID- 1717780 TI - Effect of ramipril, an inhibitor of angiotensin converting enzyme, on the response of rat thoracic aorta to injury with a balloon catheter. AB - We have studied the effect of ramipril (10 mg/kg daily by gastric gavage) on the development of neointima 2 and 14 days after injury to rat aorta with a balloon catheter. In treated animals, there was no significant inhibition of the early mitotic reaction after injury (synthesis of DNA, as reflected by aortic thymidine incorporation on the second day): the mean (95% confidence interval) was 3,553 (892) in the control group vs. 2,853 disintegrations/min/micrograms of DNA (555) in the treated group, 2 p greater than 0.15. However, ramipril decreased the amount of neointima formed 14 days after injury, as characterized by (a) a highly significant decrease of the intima to intima + media areas ratio [21.1 (2.4) vs. 13.7% (2.2), 2 p less than 10(-4]); (b) a significant decrease of intima-media wet weight [35.4 (1.0) vs. 30.9 mg (0.9), 2p less than 0.005]; and (c) without any significant effect on intima-media DNA content [96.3 (7.9) vs. 91.7 micrograms (5.7), 2p greater than 0.3]. These observations suggest that angiotensin converting enzyme inhibitors may not act mainly through an inhibition of smooth muscle cell proliferation. Other effects, such as inhibition of migration, hypertrophy, and matrix synthesis, should also be considered. PMID- 1717781 TI - Angiotensin peptide regulation of bovine brain microvessel endothelial cell monolayer permeability. AB - The passage of fluid-phase endocytosis markers--Lucifer yellow and a fluorescein isothiocyanate-conjugated dextran--across confluent primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers was characterized in the presence of angiotensin II (Ang II) peptides. Exposure to nanomolar concentrations of saralasin, an Ang II agonist, attenuated the passage of the fluorophores across the monolayers by 50-75%. In contrast, exposure to sarathrin, an Ang II antagonist, had no effect on passage of the fluorophores across the BMEC monolayers. Sarathrin, however, did inhibit the saralasin-induced reduction in transendothelial monolayer permeability and suggested specific Ang II binding site mediation. Other experiments performed suggest that saralasin-induced changes in BMEC monolayer permeability can be blocked by pretreatment with inhibitors of prostaglandin synthesis and that substantial saralasin-induced changes in BMEC monolayer permeability occurred only following exposure on the apical side of the endothelial cells. Results were consistent with previous studies demonstrating the existence of specific Ang II binding sites on BMECs and Ang II regulation of fluid-phase endocytosis. In addition, these studies support the role of angiotensin peptides in modulating permeability properties of the blood-brain barrier. PMID- 1717782 TI - Antihypertensive effects of cilazapril, 2.5 and 5 mg, once daily versus placebo on ambulatory blood pressure following single- and repeat-dose administration. AB - Ambulatory blood pressure (ABP) monitoring was used to evaluate the 24-h antihypertensive efficacy of single- and repeat-dose administrations of the nonsulfhydryl-converting enzyme inhibitor cilazapril, 2.5 and 5 mg, versus placebo in patients with mild to moderate essential hypertension. After a 2-week placebo run-in period, patients were randomized to receive, in a double-blind procedure, either 2.5 mg cilazapril (n = 14), 5 mg cilazapril (n = 14), or placebo (n = 14) for 4 weeks. In calculating the systolic/diastolic blood pressure (BP) trough-to-peak (T/P) ratio after subtraction of the placebo effect, 5 mg cilazapril appeared to be more effective than 2.5 mg cilazapril following single- (59/54% vs. 21/48%) and repeat-dose administration (47/76% vs. 31/49%). There were significant differences in 24-h and awake ABP for both 2.5 and 5 mg cilazapril as compared to placebo after single- and repeat-dose administrations. However, there were no significant differences in 24-h and awake ABP reduction between 2.5 and 5 mg cilazapril after single- and repeat-dose administrations. Furthermore, the percentages of "BP load" were identical with both regimens after the first dose (46 vs. 43%) and at steady-state (37 vs. 29%). These data demonstrate that 2.5 and 5 mg doses of cilazapril have equivalent antihypertensive efficacy using ABP and that 24-h ABP monitoring should also be performed in dose-response studies. PMID- 1717783 TI - Interaction of taurine with methionine: inhibition of myocardial phospholipid methyltransferase. AB - Perfusion of isolated, working rat heart with buffer containing 300 microM methionine in the presence of 2.5 U/L insulin led to a 15% decrease in cardiac work and a four-fold decrease in sarcolemmal Na(+)-Ca2+ exchange activity. These effects of methionine were largely prevented by inclusion of 10 mM taurine in the buffer supplemented with methionine and insulin. Taurine also reduced the extent of 3H-methyl group incorporation from radioactive methionine into myocardial phospholipids by approximately 45%. Assays of sarcolemmal phospholipid methyltransferase activity at catalytic sites I, II, and III revealed that taurine inhibited N-methylation activity approximately 30%. The data imply that the ability of taurine to modulate myocardial contraction and calcium transport may be related to taurine-mediated inhibition of phospholipid N-methylation. PMID- 1717784 TI - Combined thrombolytic effects of tissue-plasminogen activator and a fibrinogen degradation product peptide 6A or iloprost. AB - Thrombolytic effects of tissue-type plasminogen activator (t-PA) are limited by in vivo platelet activation and dynamic coronary vasoconstriction. To examine if the concurrent administration of a fibrin(ogen)-degradation product, pentapeptide 6A (Ala-Arg-Pro-Ala-Lys) with t-PA would improve the thrombolytic effects of t PA, dogs with electrically induced coronary thrombus were given t-PA alone or with peptide 6A. In dogs given t-PA alone (0.75 mg/kg over 20 min), coronary blood flow was restored in 69% of animals (9 of 13 dogs), with a mean time to reflow of 21 +/- 10 min and duration of reflow of 35 +/- 18 min. Reocclusion occurred in 77% of dogs (7 of 9 dogs). With concurrent administration of peptide 6A (200 mumol), coronary venous 6-keto-PGF1 alpha concentrations increased from 221 +/- 71 to 422 +/- 161 pg/ml (p less than 0.05), but not with t-PA given alone. Coronary blood flow was restored in 7 of 11 dogs (reperfusion rate 64%), with mean time to reflow of 17 +/- 7 min and duration of reflow of 35 +/- 15 min. The coronary reocclusion rate was 86%. All these indices of thrombolysis were similar to those in dogs given t-PA alone. In ex vivo experiments, we also demonstrated release of endothelium-derived relaxing factor from canine coronary artery rings in response to peptide 6A. To further examine the role of prostacyclin (PGI2) in thrombolytic response to t-PA, eight other dogs were given t-PA with a PGI2 analog iloprost (100 ng/kg/min for 40 min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717785 TI - Interactions between isoprenaline, sodium nitroprusside, and isozyme-selective phosphodiesterase inhibitors on ADP-induced aggregation and cyclic nucleotide levels in human platelets. AB - The interactions between isoprenaline, sodium nitroprusside, and the isozyme selective phosphodiesterase inhibitors OPC 3911 ("cAMP specific") and zaprinast ("cGMP specific") on platelet aggregation induced by adenosine diphosphate (ADP) and on levels of cAMP and cGMP were studied. Isoprenaline at 10(-6)M diminished aggregation by 28%, and this effect was enhanced by 10(-7)-10(-6)M OPC 3911. Neither 10(-6) M isoprenaline nor 10(-7)M OPC 3911 elevated cAMP, but in combination they caused a significant rise in cAMP (27% above the basal level), accompanying the synergistic functional inhibition, without affecting cGMP levels. Sodium nitroprusside at 10(-5) M diminished aggregation by 39%, elevated cGMP levels (81-110%), but also caused a statistically significant increase in cAMP (21-32%), and enhanced the effects of 10(-6)M isoprenaline on cAMP levels. Zaprinast at 10(-5) M caused a modest inhibition of aggregation by 20%, and a small increase in cGMP (20%), and it clearly enhanced the effects of 10(-5)M sodium nitroprusside on both cGMP and cAMP levels, but not on aggregation. The cAMP-increasing effect of sodium nitroprusside might be a consequence of a cGMP mediated inhibition of the "low-Km cGMP-inhibited phosphodiesterase" that is also inhibited by OPC 3911. The effects of all of the drugs on ADP-induced aggregation seem to depend more on their effect on cAMP levels than on the levels on cGMP. PMID- 1717786 TI - Effects of dopamine prodrugs and fenoldopam on glomerular hyperfiltration in streptozotocin-induced diabetes in rats. AB - The effects of dopamine (DA) prodrugs (L-dopa and gludopa) and of a D1-selective agonist (fenoldopam) on glomerular hyperfiltration were studied in the early stage of diabetes in rats. Wistar rats received one injection of streptozotocin (STZ) and were treated 1 week later with L-dopa (2 x 10 mg/kg/day, s.c.), gludopa (2 x 3 or 2 x 10 mg/kg/day, s.c.), or fenoldopam (2 x 0.3 or 2 x 1 mg/kg/day, s.c.). Their renal functions were compared with those of untreated diabetic and nondiabetic control rats. STZ injection led to hyperglycemia that was kept moderate (20-25 mmol/L) by daily insulin therapy (2-4 U of NPH insulin). Within 2 weeks, glomerular hyperfiltration (polyfructosan clearance) developed in diabetic rats (30% increase vs. nondiabetic control). A rise in renal plasma flow (PAH clearance) was sometimes observed. One week of treatment with either L-dopa, gludopa, or fenoldopam normalized the glomerular filtration rate and decreased filtration fraction. These corrections occurred despite similar metabolic disturbance and kidney hypertrophy. Gludopa was less well tolerated by diabetic rats than L-dopa. Results with L-dopa showed that the normalization of glomerular hyperfiltration was linked to DA synthesis and stimulation of D1 receptors, since it was reversed by carbidopa, a dopa decarboxylase inhibitor, and by SCH 23390, a D1-selective antagonist. These data show that DA prodrugs and a D1 agonist can suppress diabetic glomerular hyperfiltration in the very early course of the disease in rats. PMID- 1717787 TI - Acute and long-term efficacy of oral propafenone in patients with ventricular tachyarrhythmias. AB - The class Ic antiarrhythmic agent propafenone was studied by repeated electrophysiologic testing in 54 patients (43 male, aged 54 +/- 10 years, mean ejection fraction of 37.3 +/- 16.9%) with ventricular tachyarrhythmias. Forty patients (74%) had coronary artery disease. Programmed ventricular stimulation (S2, S2S3 during sinus rhythm and/or during S1S1 = 500, 430, 370, and 330 ms) off antiarrhythmic drugs induced sustained ventricular tachycardia, flutter, or fibrillation in all patients. After 450-900 mg of oral propafenone/day for 4-7 days, 51 patients were restudied. In the remaining three patients, spontaneous ventricular tachycardia occurred on drug therapy. Tachycardia induction was suppressed in 9 of 51 patients restudied (18%) and rendered more difficult to induce (basic stimulation drive greater than or equal to 40 beats/min higher than at control study) in another 7 patients (14%) (overall efficacy of 31%). The tachycardia rate decreased from 220 +/- 43 to 177 +/- 44 beats/min (p less than 0.01). The right ventricular effective refractory period increased from 232 +/- 22 to 252 +/- 22 ms (p less than 0.001). Responders to propafenone therapy had higher rates of inducible ventricular tachycardia at control (greater than 230 beats/min: 43%; less than or equal to 230 beats/min: 21%; p less than 0.05), higher ejection fractions, and lower left ventricular end-diastolic pressures than nonresponders. Eleven of the 16 patients showing a positive response to propafenone therapy in electrophysiologic testing were discharged on propafenone alone. During follow-up (17 +/- 12 months), nine patients remained free from ventricular tachycardia, one patient had a relapse, and one patient died of noncardiac death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717788 TI - Effects of components of myocardial ischaemia on cardiac action potentials in vitro. AB - The aim of this study was to examine the effects of hypoxia (PO2 = 34 mm Hg), acidosis (pH 6.8), lactate (10 mM), glucose removal, hyperkalaemia (8 mM), and lysophosphatidylcholine (5-50 microM), alone and in combination, on paced sheep Purkinje fibre action potentials in an attempt to find a combination that simulated the electrophysiological changes associated with the delayed phase of ischaemia-induced arrhythmias. Modification of the physiological salt solution to produce a combination of acidosis, lactate, and lysophosphatidylcholine (5 microM) reduced the resting membrane potential and the maximum rate of depolarisation and prolonged action potential duration, these effects being stable from 60 to 120 min of superfusion. Hypoxia, glucose removal, or elevation of the extracellular concentration of potassium alone or in combination with the above factors caused action potential shortening. Thus, the electrophysiological changes associated with the delayed phase of ischaemia-induced arrhythmias in vivo can be simulated in vitro by a combination of acidosis, lactate, and lysophosphatidylcholine. PMID- 1717790 TI - Altered intra- and extraluminal effects of 5-hydroxytryptamine in hypertensive mesenteric resistance arteries: contribution of the endothelium and smooth muscle. AB - Vasoconstrictor responses to intraluminal and extraluminal 5-hydroxytryptamine (5 HT) were studied in isolated mesenteric resistance arteries of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Third-order branches of mesenteric arteries were dissected free and mounted on glass cannulae in organ chambers. Changes in intraluminal diameter of the perfused and pressurized vessels were measured. Extraluminal 5-HT (10(-8)-10(-4)M) evoked concentration dependent contractions that were augmented after removal of the endothelium. The sensitivity of arteries without but not of those with endothelium to 5-HT was increased in SHRs compared to WKY rats. The pA2 value for ketanserin using 5-HT as an agonist was identical in WKY rats and SHRs. The slope of the Schild plots did not significantly differ from 1. Intraluminal 5-HT caused smaller contractions in arteries with endothelium than extraluminal 5-HT. After endothelial removal, the contractions to intraluminal 5-HT were increased. The contraction induced by intraluminal 5-HT in arteries with endothelium was greater in SHRs than WKY rats. Thus, contractile responses to 5-HT are mediated by homogeneous 5-HT2 receptors in rat mesenteric resistance arteries. In SHRs, the affinity of the receptor to 5-HT is not altered. The sensitivity of vascular smooth muscle to 5-HT is increased and the protective role of the endothelium against intraluminal 5-HT is decreased in SHRs. PMID- 1717789 TI - Personality. Type A behavior, and the effects of beta-blockade. AB - In a previous report, it was shown that a dimension of personality entitled dependence/independence could be used to distinguish the cardiovascular and catecholaminergic response to stress in type A individuals. This study is a continuation of the previous work. It examines the relative effects of beta blockade on stress-induced changes in heart rate, blood pressure, and catecholamines in two groups of type A individuals who differ in reactivity. Sixty-six healthy, middle-aged men with type A behavior were classified as dependent (high reactivity) or independent (low reactivity). The effects of beta blockade on the two groups were tested in a randomized, double-blind, crossover trial utilizing a mental stressor, a physical stressor, and work day monitoring. Results showed that, in comparison with the independent type As, those who had been classified as dependent had higher heart rates in all three stress conditions and had higher epinephrine levels during both the mental and physical stressors. For both groups, beta-blockade lowered the heart rate and systolic blood pressure but increased norepinephrine with the mental stressor and epinephrine and norepinephrine with the physical stressor. It also reduced the mean work day heart rate. For the dependent type As, the drug had a greater effect on increasing epinephrine during the physical stressor and by reducing the percent of the work day spent in high heart rate zones. The results are discussed from the point of view of identifying type As who may be particularly coronary prone and the potential effects of beta-blockade on the reactivity associated with type A behavior. PMID- 1717792 TI - Stimulation of fatty acid oxidation by phosphodiesterase III inhibitors in rat myocytes. AB - In order to study the regulation of fatty acid oxidation in the heart, the effects of some phosphodiesterase III inhibitors, such as enoximone and milrinone, on the oxidation of [1-14C]palmitic acid, [1-14C]octanoic acid, and [2 14C]pyruvate were studied in adult rat myocytes. Enoximone and milrinone, at a concentration of 0.25 mM, increased palmitate oxidation significantly, by 70 and 40%, respectively. Also, enoximone increased octanoate oxidation by 45%. In contrast, pyruvate oxidation was decreased by 60% by enoximone. To investigate the effects of enoximone or milrinone on the pathway of fatty acid oxidation, their effects on the oxidation of either [1-14C]palmitoyl-CoA or [1 14C]palmitoylcarnitine were studied with rat heart homogenates. Neither enoximone nor milrinone had any effects on the oxidation of these compounds. Compounds known to elevate intracellular [Ca2+] or cyclic AMP, such as the calcium ionophore A23187, ionomycin, dibutyryl cyclic AMP, or isoproterenol, had no effect on palmitate oxidation. Enoximone, at a concentration of 0.25 mM, increased palmitate uptake by 40% in rat myocytes. These results suggest that enoximone and milrinone increase fatty acid oxidation in myocytes by increasing their cellular transport, and they also show the usefulness of these compounds as a tool to study the regulation of this vital pathway in heart. PMID- 1717791 TI - Use-dependent actions and effects on transmembrane action potentials of flecainide, encainide, and ethmozine in canine Purkinje fibers. AB - The Cardiac Arrhythmia Suppression Trial implicated flecainide and encainide as proarrhythmic in a specific clinical setting, whereas ethmozine was not. The purpose of the present study was to attempt to understand the cellular basis for these different drug effects by comparing the actions on transmembrane action potential characteristics and onset and offset of use-dependent block in canine Purkinje fibers using standard microelectrode techniques. All drugs caused qualitatively similar changes on action potential characteristics: reduction of action potential amplitude and the maximum rate of depolarization (Vmax) as well as acceleration of repolarization. Ethmozine and the orthodemethyl (O-D) metabolite of encainide had greater effects at all concentrations studied than encainide or flecainide. Although ethmozine induced greater use-dependent reduction of Vmax than flecainide or encainide, it also showed faster onset and recovery from use-dependent block. The time constant of recovery from use dependent block for O-D encainide was about 5 times that of the parent compound. The rates of onset and recovery from use-dependent block for ethmozine were similar to those reported for class IA drugs like disopyramide and much slower than class IB drugs like lidocaine. The faster on and off rates of ethmozine when compared to flecainide and encainide in this study may provide the basis for the differing effects of these drugs on conduction in situ and their proarrhythmic actions. PMID- 1717793 TI - Measurement of tachykinin-induced salivation in conscious rats. AB - A method of quantitatively measuring tachykinin-induced salivation in conscious, male, Sprague-Dawley rats is described. Salivation is quantified by determining the weight of a preweighed, absorbant foam cube after it has been used to swab the oral cavity of a tachykinin challenged rat. Salivation is induced by intravenous (i.v.) injection of sialogogues (microgram/kg) via the lateral tail vein. Measurements are made immediately after injection. Substance P (Sub.P), Sar9, Met (O2) 11Substance P (Sar9 Sub.P), a selective neurokinin (NK) 1 receptor agonist, Physalaemin and Eledoisin are equipotent sialogogues as determined by this method. Neurokinin A (NKA), the endogenous NK2 receptor agonist, is 0.27 (0.14-0.46) times as potent as Sub. P, while (Suc-[Asp6, MePhe8]Substance P(6 11), (senktide), a selective NK3 receptor agonist, only induced salivation at 300 microgram/kg. Acetylcholine (Ach) is only 0.006 (0.002-0.012) times as potent as Sub.P. Treatment with the neurokinin antagonist [D-Arg1, D-Trp7,9 Leu11] Substance P (spantide) dose-dependently inhibits Sub. P stimulated salivation. Atropine dose-dependently inhibits Ach induced salivation but is inactive against Sub.P-induced salivation. These data are consistent with literature values and indicate that this method provides a simple, quantitative model, free of any possible anesthetic side effects, for the measurement of neurokinin stimulated salivation and the assessment of potential neurokinin antagonists in vivo. PMID- 1717794 TI - Endothelial function of human gastroepiploic artery. Implications for its use as a bypass graft. AB - The human gastroepiploic artery has been used as a coronary artery bypass conduit in a limited number of clinical studies. It has been postulated that the capacity of the endothelium to release vasoactive substances may contribute to differing patency rates observed in established bypass grafts. We have now examined endothelial function in the human gastroepiploic artery. Endothelium-dependent relaxations to substance P were observed. A maximum relaxation of 83.25% +/- 8.2% (mean +/- standard error) was attenuated to 48.5% +/- 16.4% in the presence of L NG-monomethyl-arginine, a specific inhibitor of endogenous nitric oxide synthesis. Removal of the endothelium abolished the relaxations. With a specific radioimmunoassay, concomitant changes in levels of cyclic guanosine 3',5' monophosphate, the second messenger that elicits smooth muscle relaxation after release of the endothelium-derived relaxing factor, were measured. It was found that the gastroepiploic artery had significantly higher resting and stimulated levels of cyclic guanosine 3',5'-monophosphate than either the internal mammary artery or the saphenous vein. In the presence of the cyclooxygenase inhibitor indomethacin, and indomethacin plus L-NG-monomethylanginine, the maximum relaxation was decreased to 70% +/- 9.5% and 59% +/- 10.8%, respectively. Our data demonstrate that endothelium-derived relaxing factor and prostacyclin may exhibit synergy in the control of vascular tone in this vessel. It is concluded that the endothelium of the gastroepiploic artery has a strong capacity to secrete vasodilators and inhibitors of platelet activity. This could have important influence on long-term patency. PMID- 1717795 TI - The Blalock-Taussig shunt in the newborn infant. AB - Children with pulmonary oligemia often require palliation in the newborn period. The Blalock-Taussig shunt has been shown to offer adequate palliation in the older child, but its use in the newborn period remains controversial. A retrospective review of 51 neonates younger than age 2 weeks undergoing a Blalock Taussig shunt (or modification) was performed. The operative mortality rate was 5.8%. Six children (15.4%) required reoperation in the first year of life for inadequate shunt function. The modification with interposition grafts necessitated reoperation more often than shunts performed with the subclavian artery. PMID- 1717796 TI - Production of macrophage colony-stimulating factor by human lymphoblastoid cell lines. AB - The in vitro production of macrophage colony-stimulating factor (M-CSF) was studied in 14 human lymphoblastoid cell lines and their lineages were ascertained by surface phenotype analysis. M-CSF gene transcripts were detected in a T lymphocyte-derived cell line (CCRF-CEM) and 3 B-lymphoblastoid cell lines (IM-9, BALL-1, and CCRF-SB) by Northern-blot analysis. The secretion of M-CSF protein into the culture supernatant by each cell line was also studied using an enzyme linked immunosorbent assay for M-CSF. The 4 cell lines which expressed the M-CSF gene secreted considerable amounts of M-CSF into their culture supernatants, while the 10 cell lines without M-CSF gene expression did not do so. The cell lines which constitutively produced M-CSF were then subjected to Southern-blot analysis of the M-CSF gene structure, and all 14 cell lines were examined for infection by the Epstein-Barr virus. Neither structural changes nor amplification of the M-CSF gene were detected, and Epstein-Barr virus infection was found to be not directly related to M-CSF production. PMID- 1717797 TI - Applications of piezoelectric fluid jetting devices to neuroscience research. AB - Piezoelectric pumps or "jets" are used in industry for precise dispensing of small volumes of fluids. In the present work we have tested the feasibility of using these piezoelectric devices for dispensing fluids in neurobiological research. In one experiment, 70 picoliter (pl) droplets of histochemical reagent were jetted onto discrete targets of frozen tissue sections, and qualitative and quantitative histochemical studies were done on the small (170 microns diameter) circle of tissue wet by the droplets. In the second experiment, 70 pl droplets of neuroactive drugs were jetted onto brain tissue slices while recording single neurons extracellularly in vitro, and the effects of the drugs were found to vary systematically as a function of the number of drops and the distance between drop application and the recorded neuron. The results indicate that piezoelectric jets could have wide application for dispensing fluids in neurobiological research. PMID- 1717798 TI - Thymectomy at weaning. An accelerated aging model for the mouse immune system. AB - Mouse thymectomy at weaning induces a long lasting immunodepression which can be measured by in vivo and in vitro experiments. Lymphocyte proliferation and IL2 production in response to a T cell mitogen are greatly diminished during the whole life of the animals, on the contrary B cell proliferation in the presence of lipopolysaccharide is not modified. The lack of effect of surgery on the in vitro T cytotoxic activity compared to the total abolition of in vivo graft versus host reaction shows that these two phenomena are under the control of different immunocompetent cell subsets. Thymectomy induces a stabilization of natural killer cell activity, while during normal aging, this parameter decreases regularly. Surprisingly, the thy 1+ cell level is normal 8-10 months after thymectomy compared to sham operated animals showing that phenotypically normal cells can be dysfunctional. Macrophage activity is not modified either by aging or by thymectomy. So, thymectomized mice can be used after less than 1 year to study immunopharmacology of aging. PMID- 1717799 TI - [Misleading figures concerning PSA and health examination]. PMID- 1717800 TI - Unmasking immunosuppression. PMID- 1717801 TI - Alfuzosin for benign prostatic hypertrophy. PMID- 1717802 TI - Euthanasia. PMID- 1717803 TI - Prevalence of benign prostatic hypertrophy. PMID- 1717804 TI - Pancreatic insufficiency after bone-marrow transplantation. PMID- 1717805 TI - Two-dimensional gel electrophoretic profile of rat basophilic leukemia (RBL) and mast cells. AB - RBL cells are not differentiated, but resemble mucosal mast cells (MMC). Two dimensional (2-D) gel electrophoresis following isoelectric focusing (IEF) was performed using purified rat peritoneal mast cells and RBL cells. Certain similarities were identified with silver staining between mast cells and stationary phase (72 hr) RBL cells. RBL cells were also labelled with [35S] cysteine in order to study the specific expression of proteins during logarithmic or stationary growth phases. Only stationary phase RBL cells appeared to specifically express three proteins of 42, 55 and 93 kD and were still capable of secreting histamine in response to immunoglobulin E (IgE) and specific antigen. These results suggest that specific RBL cell proteins may be used as markers for further analysis of their maturation/differentiation. PMID- 1717806 TI - Study of the relationship between elevated maternal serum alpha-fetoprotein and adverse pregnancy outcome. AB - In a study of 1,703 pregnancies, adverse clinical outcomes associated with unexplained elevated maternal serum alpha-fetoprotein (MSAFP) included intrauterine growth retardation (IUGR), prematurity, IUGR and prematurity, prematurity without IUGR, spontaneous abortion, and stillbirth. These findings have significant implications for careful obstetrical management of patients with elevated MSAFP. PMID- 1717807 TI - Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats. AB - This study examined the effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the levels of two astrocyte mRNAs during responses to unilateral entorhinal cortex lesions (ECL) to identify molecular mechanisms involved in glucocorticoid modulation of astrocyte activation following deafferentation. Both glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) mRNA were increased in the ipsilateral hippocampus 4 days following unilateral ECL. In unlesioned ADX rats CORT replacement decreased both messages in the hippocampus. CORT replacement suppressed the ECL-induced increase of GFAP mRNA in the contralateral, but not ipsilateral hippocampus of ADX rats. In contrast, CORT decreased SGP-2 mRNA both ipsi- and contralaterally. It is clear that several regulatory mechanisms are responsible for maintaining a physiological balance of astrocyte activity in the adult brain, and that changes in circuit integrity and the endocrine milieu can alter this balance. PMID- 1717808 TI - Repeated administration of desmethylimipramine blocks the reserpine-induced increase in tyrosine hydroxylase mRNA in locus coeruleus neurons of the rat. AB - The levels of tyrosine hydroxylase and galanin mRNA were measured by in situ hybridization histochemistry in the rat locus coeruleus after repeated (21 days) administration of desmethylimipramine (10 mg/kg/day), of reserpine (0.25 mg/kg/day), of coadministered desmethylimipramine and reserpine, or of vehicle. Reserpine administration resulted in increased levels of both tyrosine hydroxylase and galanin mRNAs in locus coeruleus neurons as compared to vehicle treated controls. Administration of desmethylimipramine alone failed to alter either the tyrosine hydroxylase or galanin mRNA. However, coadministration of desmethylimipramine with reserpine blocked the elevation in tyrosine hydroxylase mRNA induced by reserpine alone. PMID- 1717809 TI - Evolution of the cetacean mitochondrial D-loop region. AB - We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region. PMID- 1717810 TI - Activation of interferon-inducible genes in vivo by synthetic double-stranded RNA, poly rI:rC. AB - We analyzed the effects of synthetic dsRNA (poly rI:rC) treatment on the expression in vivo of two interferon (IFN)-inducible genes. DBA/2 mice were injected i.p. with poly rI:rC and its effect on the levels of the following mRNAs was determined; 202, 2'-5' oligoadenylate synthetase (2-5A synthetase) and beta actin. After poly rI:rC treatment the levels of the 202 and 2-5A synthetase mRNAs in the spleen and in bone marrow peaked between 12 and 24 h and decreased thereafter, whereas beta-actin levels remained unchained unchanged. Pretreatment of DBA/2 mice with sheep anti-murine IFN-alpha/beta antibodies before rI:rC injection strongly diminished the induction of 202 mRNA indicating that IFN alpha/beta mediated this induction. PMID- 1717811 TI - Sensitivity and specificity of anti-HIV ELISA employing recombinant (p24, p66, gp120) and synthetic (gp41) viral antigenic peptides. AB - We have developed two immunoassay systems, one designated HIV (p24, p66, gp41) ELISA that uses as antigens the immunodominant epitopes mixed from each of three major groups of HIV-1 proteins: the core (p24), the pol (p66) and the env (gp41) gene. The other immunoassay system consists of four separate ELISAs for detection of single antibodies to HIV gag gene (p24), HIV pol gene (p66) and HIV env gene (gp41 and gp120). In the present study 200 specimens from patients with AIDS and 200 specimens from patients with ARC were repeatedly positive by HIV (p24, p66, gp41) ELISA. 1425 specimens from HIV drug addicts positive at W.B. were positive at HIV (p24, gp41, p66) ELISA. In addition, 60 samples that were indeterminate by W.B., were repeatedly positive at HIV (p24, p66, gp41) ELISA. The sensitivity and specificity of HIV (p24, p66, gp41) is estimated to be 100%. In this study 1507 specimens from HIV drug addicts, positive at W.B., were all positive (more than one test positive) at HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA used in combination. 135 samples from HIV positive drug addicts, positive at standard ELISA but indeterminate at W.B., were positive by HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA using the same criteria as in W.B. interpretation. The specificity (defined in terms of percentage of non-reacting persons in a low risk population) of HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA, HIV gp120 ELISA is 100%. In this work we demonstrated that: a) HIV (p24, p66, gp41) ELISA could be used as an adjunct or reliable alternative to standard ELISA for detection or confirmation of HIV antibodies in human sera; b) the specificity and sensitivity of antibodies to p24, p66, gp41, gp120 by ELISA used alone and/or in combination, is equal to or greater than W.B. PMID- 1717812 TI - Mechanism of protective effect of recombinant human granulocyte colony stimulating factor (rG-CSF) on Pseudomonas infection. AB - Decrease in resistance to systemic Pseudomonas infection in cyclophosphamide (CPA)-induced neutropenic mice was prevented by injections of recombinant human granulocyte colony-stimulating factor (rG-CSF). In order to explore mechanism of the prevention of CPA-induced decrease in the anti-infectious resistance by rG CSF, CPA-treated and then rG-CSF-injected mice were inoculated i.p. with P. aeruginosa, and growth of the infecting bacteria and infiltration of leukocytes in the peritoneal cavity were determined. In the mice who had received 4 daily s.c. injections of rG-CSF from the day after CPA-injection, a large number of neutrophils were mobilized into the peritoneal cavity in response to the bacterial inoculation and growth of the infecting Pseudomonas in the cavity was markedly inhibited, whereas in CPA-induced neutropenic mice few neutrophils were mobilized and the infecting bacteria proliferated vigorously in the peritoneal cavity. These results suggest that administration of rG-CSF prevents CPA-induced neutropenia and neutrophils circulating at normal level in the number are normally mobilized into the peritoneal cavity in response to Pseudomonas inoculation, and that the mobilized neutrophils inhibit proliferation of the infecting Pseudomonas. PMID- 1717813 TI - Goals and rationale of cancer treatment. PMID- 1717814 TI - [Malignant diseases. Are vitamins suitable for prevention and therapy?]. PMID- 1717815 TI - Antibodies against core and O antigen epitopes of Escherichia coli O83 in rabbit antisera and in commercial human immunoglobulin. AB - An unexpected serological cross-reaction was detected between two common Escherichia coli O antigens: O83 and O6. It was found that the cross-reacting antibodies were induced by common core epitopes not exposed in naturally occurring smooth strains. Smooth O6 strains would, however, after prolonged immunization of rabbits provoke production of precipitating antibodies against this common core epitope. Routine rabbit O antisera of smooth test strains O1 to O171 did not contain antibodies to the core epitope. Core antisera could readily be produced with an R mutant of the O6 strain. Extracts or Lipopolysaccharides (LPS) of several of the E. coli commonly found in the normal intestine and in extraintestinal diseases would only precipitate in antisera containing anti-core epitope anti-bodies after treatment with deoxycholate or sodium dodecyl sulfate. Other E. coli such as J5 (O111), used extensively for production of antisera intended for protection against endotoxemia, did not react with antibodies against this common core epitope. Another unexpected observation was that normal commercial human immunoglobulin contained precipitating IgG antibodies to the E. coli O83 antigen commonly found in strains from neonatal meningitis and other extraintestinal infections. Significant amounts of precipitating anti-LPS antibodies against other common O antigens (O2, O4, O6, O75 etc.) could not be detected in normal immunoglobulin. PMID- 1717816 TI - [The problem of attempted suicide in accordance with guidelines of the German Society for Humane Dying]. PMID- 1717817 TI - The maize cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family: organ specific expression and genetic analysis. AB - The distribution of the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family (Gpc) in the maize genome was investigated; a genetic variant of glyceraldehyde-3-phosphate dehydrogenase activity is also described. Restriction fragment length polymorphism analysis of an F2 population shows that the variant is not linked to the three known Gpc genes. However, this trait is linked to one of two genomic DNA fragments that hybridize to a fragment of the Gpc3 coding region, implying the existence of a fourth Gpc gene. Antibodies and cDNA clones were used to investigate the organ-specific expression of the Gpc genes. Results were compared with the expression of the Gpc genes. Results were compared with the expression of the alcohol dehydrogenase 1 (Adh1) gene. RNA and protein levels were examined in seedling roots and shoots, as well as the leaves, developing endosperm and embryo, and the aleurone. In general, it was found that Gpc3 expression behaves in parallel with Adh1 in these organs, and protein levels closely parallel that of RNA for each gene examined. Both Gpc3 and Adh1 show a marked increase in expression during endosperm development, reaching a maximum 15 days after pollination, but no expression is detected in the leaf. Gpc1 expression is similar to that of Gpc2, with an overall decrease in the level of RNA during endosperm development. This expression is discussed in terms of the common sequences found upstream of genes expressed in the developing maize seed. PMID- 1717818 TI - Structural and pharmacological characterization of the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts. AB - Previously, we purified the predominant subtype of brain nicotinic acetylcholine receptor (AChR), analyzed its structure, and found that it was composed of two kinds of subunit, with sequences encoded by cDNAs termed alpha 4 and beta 2. Here we express these cDNAs from chicken brain in stably transfected fibroblasts. We demonstrate by synthesis that these cDNAs encode subunit polypeptides of the expected sizes, which coassemble to form receptor macromolecules having the same size as native AChRs. Additionally, we demonstrate that the expressed AChRs exhibit the ligand-binding pharmacology of native brain AChRs and function as acetylcholine-gated ion channels. PMID- 1717819 TI - Transsynaptic activity regulates proenkephalin and tyrosine hydroxylase gene expression and the response to reserpine in the hamster adrenal. AB - Transsynaptic neurogenic activity and reserpine are two signals that cause the proenkephalin (Penk) gene to alter the levels of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides. In the Syrian hamster adrenal, but not in rat adrenal, both of these signals appear to be positive activators of Penk gene expression. The separate and combined effects of reserpine and denervation on EC peptides and catecholamine systems were investigated in the adrenal of the hamster, a species with relatively high medullary PPenk mRNA and EC peptide levels. Unilateral adrenal denervation resulted in a rapid decrease in PPenk mRNA levels of 54% after 2 days, and by 11 days 90% of Penk mRNA had disappeared. After 4 days both EC peptide and PPenk mRNA levels fell in parallel, whereas total RNA and soluble protein levels were unchanged. Denervation had no effect on TH mRNA levels until 8 days after surgery, and after 11 days both TH mRNA and catecholamine levels had decreased by 35-45%. Reserpine produced a dose- and time dependent depletion of EC peptides and catecholamines. One day after 5 mg/kg reserpine (given subcutaneously on each of 2 consecutive days), EC peptides were reduced by 80%, norepinephrine by 79%, and epinphrine by greater than 95%. By 4 days after treatment, EC peptides and catecholamines slightly exceeded or had returned to control (concurrent vehicle treatment) values. PPenk mRNA levels, as measured by solution hybridization, were doubled (206 +/- 17%, mean +/- standard error) by day 4. Tyrosine hydroxylase (TH) mRNA levels were increased nearly 7 fold (686 +/- 71%) 24 hr after the first reserpine dose and declined thereafter. Northern blot analysis demonstrated that reserpine did not alter the size of either PPenk or TH mRNAs. Size exclusion chromatography showed a small (20%) reserpine-induced increase in processing of high molecular weight Penk-like peptides. The effects of reserpine, which increases PPenk mRNA, EC peptides, and TH mRNA, were completely blocked by unilateral denervation, whereas the contralateral innervated gland showed the expected responses. The co-localized EC peptide and catecholamine systems, as reflected in their mRNAs, respond differently in both time sequence and magnitude to reserpine and to denervation. Our results support a critical role, in vivo, for transsynaptic mechanisms in the maintenance of the high levels of Penk gene expression in this species and for the positive activation (mediated by reflex neurogenic stimulation) of reserpine on Penk and TH gene expression. PMID- 1717820 TI - Angiotensin converting enzyme inhibition in Dahl salt-sensitive rats. AB - Angiotensin II has previously been reported to have in vivo and in vitro cardiac hypertrophic effects. We used the salt-sensitive Dahl rat genetic strain to separate mechanical (pressure overload) vs. hormonal (renin-angiotensin system) input in cardiac hypertrophy. Blood pressure was significantly increased and left ventricular hypertrophy, as indexed by LV/BW ratios, was present at 7 and 15 days in rats receiving 4% and 8% NaCl compared to the 1% controls. There was no effect of the angiotensin converting enzyme inhibitor, enalapril maleate, on lowering the blood pressure in 8% NaCl-treated animals, however, there was a significant reduction in LV/BW ratio in 8% NaCl-treated animals that received this drug. Left ventricular angiotensinogen mRNA activity was significantly reduced in rats receiving 4% and 8% NaCl. In this model of hypertension the cardiac hypertrophy which develops is largely dependent on mechanical forces though there remains a significant contribution to this process from either circulating or localized angiotensin II production. Regulation of angiotensinogen gene expression in the hypertrophied left ventricle suggests that volume and electrolyte control of angiotensinogen gene expression in the heart and/or hereditary factors are predominant in the control of regulation of this gene in the left ventricle of Dahl rats. PMID- 1717821 TI - Mechanical transduction by membrane ion channels: a mini review. AB - There are ion channels in the cell membrane that are sensitive to stress in the membrane cytoskeleton. Some channels turn on with stress, others turn off. In specialized receptors such as those involved in hearing, touch, etc. the role of the channels is clear. However, virtually all cells have these channels, and we don't yet know the physiological role of the channels although it is reasonable to suppose that they are involved in the control of cell size, either acutely as in volume regulation, or trophically as in the control of cell division. PMID- 1717822 TI - Characterization of mucin glycoprotein-specific translation products from swine and human trachea, pancreas and colon. AB - RNA was isolated from cultured swine trachea epithelial cells and mucus-secreting tumor cell lines from human pancreas, lung and colon by extraction with guanidine isothiocyanate. Poly(A)+mRNA rich fractions were purified by repeated chromatography on oligo (dT)-cellulose columns and they were translated in a cell free rabbit reticulocyte system. Translation products labelled with 35S methionine were isolated by immunoprecipitation with specific antibodies to the polypeptide chains of mucin glycoproteins and they were analyzed by SDS-PAGE and fluorography. A single principal polypeptide band of 67 kDa was found in all cases when the immunoprecipitates were washed with buffer containing bovine serum albumin and unlabeled deglycosylated mucin glycoprotein. The intensity of the 67 kDa band decreased when unlabeled deglycosylated mucin glycoprotein was added to the translation mixture before immunoprecipitation. Affinity purified monospecific antibodies elicited against chemically deglycosylated polypeptide chains of purified mucin glycoproteins from human and swine trachea and Cowper's gland were all equally effective in immunoprecipitating the 67 kDa translation product. Monospecific antibodies directed against the glycosylated and unglycosylated regions of the polypeptide chain yielded single bands with a molecular size of 67 kDa in each case. Peptide profiles obtained by digestion of the 67 kDa translation product with S. aureus V-8 protease were identical to those obtained with deglycosylated human and swine trachea mucin glycoproteins. These studies clearly demonstrate that the translation product of swine trachea and human lung, colon and pancreatic mucin glycoprotein gene is a single polypeptide chain of 67 kDa. The relative size and properties of the translation products synthesized with poly (A)+RNA isolated from mucus-secreting cells derived from three different tissues are similar to those of mucin glycoproteins purified directly from mucus secretions of human and swine trachea epithelium. PMID- 1717823 TI - AGP/EBP(LAP) expressed in rat hepatoma cells interacts with multiple promoter sites and is necessary for maximal glucocorticoid induction of the rat alpha-1 acid glycoprotein gene. AB - Transcription of the rat alpha 1-acid glycoprotein (AGP) gene is induced by glucocorticoids. In addition to the glucocorticoid response element which maps to bases -120 to -107, sequences located between bases -106 to -42 have been shown to be necessary for hormone induction. We have previously identified multiple sites of C/EBP interaction with the AGP promoter in the region -106 to -64. In this study, we purify and identify a C/EBP family member, AGP/EBP(LAP), present in the rat hepatoma cell line HTC (JZ.1) which also binds to the C/EBP recognition sites in this region. Mutations in the recognition sites that prevent binding are analyzed, and the results suggest a positive as well as a possible inhibitory role for AGP/EBP(LAP) in the glucocorticoid induction of the gene in HTC (JZ.1) cells. PMID- 1717824 TI - Exocrine pancreas transcription factor 1 binds to a bipartite enhancer element and activates transcription of acinar genes. AB - Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer binding activity. It is not identical to previously characterized factors and is termed exocrine pancreas transcription factor 1 (XPF-1). In the highly purified preparation, only a single major protein of 60 kDa was detected by silver staining on sodium dodecyl sulfate-gels and by UV cross-linking. XPF-1 binds to the core enhancer of all tested XP genes and not to a mutant sequence which is inactive in vivo. High-affinity binding sites are bipartite. The results of competition binding and UV-cross-linking assays suggest that XPF-1 interacts with both motifs. XPF-1 selectively stimulates transcription of core enhancer templates in an in vitro transcription system. We hypothesize that XPF-1 plays a role in activation of the transcription of XP-specific genes. PMID- 1717826 TI - The 40-kilodalton to autoantigen associates with nucleotides 21 to 64 of human mitochondrial RNA processing/7-2 RNA in vitro. AB - A 40-kDa To antigen recognized by sera from some patients with autoimmune diseases is an integral component of both human RNase P and mitochondrial RNA processing (MRP) RNase. Human MRP and RNase P RNAs, synthesized in vitro, readily associate with the To antigen present in the HeLa cell extract. Using this in vitro reconstitution system, the binding site of the To antigen is localized to a 44-nucleotide-long sequence corresponding to nucleotides 21 to 64 of the human MRP RNA. UV cross-linking experiments showed that the To antigen binds directly to MRP RNA and to RNase P (H1) RNA through RNA-protein interactions. Although the MRP RNA and RNAse P (H1) RNA show sequence homology in four conserved blocks (H. A. Gold, J. N. Topper, D. A. Clayton, and J. Craft, Science 245:1377-1380, 1989), the To antigen-binding site in MRP RNA does not show any obvious primary sequence homology with H1 RNA. These data suggest that the To antigen binds to a conserved and presumably a common secondary or tertiary structure in human MRP and RNase P RNAs. PMID- 1717825 TI - Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein. AB - GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases. PMID- 1717827 TI - A heat-labile factor promotes premature 3' end formation in exon 1 of the murine adenosine deaminase gene in a cell-free transcription system. AB - An elongation block to RNA polymerase II transcription in exon 1 is a major regulatory step in expression of the murine adenosine deaminase (ADA) gene. Previous work in the laboratory identified abundant short transcripts with 3' termini in exon 1 in steady-state RNA from injected oocytes. Using a cell-free system to investigate the mechanism of premature 3' end formation, we found that polymerase II generates prominent ADA transcripts approximately 96 to 100 nucleotides in length which are similar to the major short transcripts found in steady-state RNA from oocytes injected with ADA templates. We have determined that these transcripts are the processed products of 108- to 112-nucleotide precursors. Precursor formation is (i) favored in reactions using circular templates, (ii) not the result of a posttranscriptional processing event, (iii) sensitive to low concentrations of Sarkosyl, and (iv) dependent on a factor(s) which is inactivated in crude extracts at 47 degrees C for 15 min. The cell-free system will allow further characterization of the template and factor requirements involved in the control of premature 3' end formation by RNA polymerase II. PMID- 1717828 TI - Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides. AB - To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides. PMID- 1717829 TI - Transgenic mouse model for central nervous system demyelination. AB - A common feature of demyelinating diseases such as multiple sclerosis in humans and experimental autoimmune encephalomyelitis in rodents is the marked elevation in the expression of the major histocompatibility complex (MHC) antigens in the involved sites. By specific targeting of a syngeneic MHC class I gene to oligodendrocytes, we have generated transgenic mice which not only exhibit severe involuntary tremors and develop tonic seizures but also show extensive demyelination in both the brain and the spinal cord. The fact that demyelination in these mice occurs in the absence of immune infiltration dismisses an autoimmune involvement but suggests that the MHC class I antigens play a direct role in inducing disease. Our findings lend support to the possibility that demyelinating diseases are induced by infectious agents such as viruses which can either directly activate MHC gene expression in oligodendroglia or indirectly activate expression through the release by reactive T cells of gamma interferon in the brain. PMID- 1717830 TI - Functional expression and RNA binding analysis of the interferon-induced, double stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system. AB - Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors. PMID- 1717831 TI - Low level of Hox1.3 gene expression does not preclude the use of promoterless vectors to generate a targeted gene disruption. off. AB - A variety of experimental approaches have been devised recently to mutate mammalian genes by homologous recombination. In this report, we describe the disruption of the Hox1.3 locus by using two of these approaches, namely, positive negative selection and activation of a promoterless gene. Interestingly, we observe similarly high frequencies of targeted disruption with both procedures. The high frequency of targeted disruption with a promoterless vector was unexpected given the extremely low level of Hox1.3 expression in the embryonic stem cell line used for these studies. These data indicate that minimal expression of the target gene is required to enrich for homologous recombination events with promoterless vectors and thus enhance the promoterless gene approach as a general strategy to mutate mammalian genes by homologous recombination. PMID- 1717832 TI - Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides. AB - Even though the retinoblastoma gene is one of the best-studied tumor suppressor genes, little is known about its functional role. Like all tumor suppressor gene products, the retinoblastoma protein (pRB) is thought to inhibit some aspect of cell proliferation. It also appears to be a cellular target of several DNA tumor virus-transforming proteins, such as adenovirus E1A, human papillomavirus E7, or simian virus 40 large T antigen. To help in the analysis of pRB, we have prepared a new set of anti-human pRB monoclonal antibodies. In addition to being useful reagents for the study of human pRB, these antibodies display several unexpected properties. They can be used to distinguish different subsets of the pRBs on the basis of their phosphorylation states. Some are able to recognize pRB homologs in other species, including mice, chickens, and members of the genus Xenopus. In addition, some of these antibodies can bind directly to other cellular proteins that, like pRB, were originally identified through their association with adenovirus E1A. These immunologically cross-reactive proteins include the p107 and p300 proteins, and their recognition by antibodies raised against pRB suggests that several members of the E1A-targeted cellular proteins form a structurally and functionally related family. PMID- 1717833 TI - Regulation of the alpha inhibin gene by cyclic adenosine 3',5'-monophosphate after transfection into rat granulosa cells. AB - Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. To examine further the transcriptional regulation of the alpha inhibin gene by FSH and cAMP, we have isolated and characterized a genomic clone that contains the entire rat alpha inhibin gene. Sequence analysis of the alpha inhibin promoter region revealed several potential cAMP response elements (CREs) and transcription factor AP2-binding sites that might mediate cAMP regulation. To determine the functional importance of these sequences, fusion genes including the alpha inhibin 5' flanking region linked to a luciferase reporter gene were transiently transfected into primary granulosa cells isolated from immature rats. These fusion genes were both expressed and regulated by the adenylyl cyclase activator forskolin in transfected granulosa cells. Analysis of a series of 5' deletion mutants indicated that a construct containing as little as 170 basepairs up-stream of the alpha inhibin start site, which includes a single imperfect CRE and no AP2 sites, was regulated by forskolin. DNAse footprinting was used to demonstrate that bacterially expressed CRE-binding protein (CREB) binds to this CRE located 122 basepairs up-stream of the alpha inhibin gene transcriptional start site. To investigate further the role of this CRE in alpha inhibin gene expression, site-specific mutagenesis of the CRE was performed. The alpha inhibin promoter containing a mutated CRE was not regulated by forskolin in granulosa cells and did not bind the CREB protein. Interestingly, mutation of the CRE also substantially reduced basal expression of the alpha inhibin promoter. Lastly, a gel mobility shift assay was used to examine CRE-binding proteins from granulosa cell extracts. Granulosa cells contain a protein that specifically interacts with CRE-containing oligonucleotides or with the alpha inhibin promoter and that is recognized by antibodies against the CREB protein. Our results suggest that CREB or related transcription factors play an important role in both basal and cAMP-regulated expression of the alpha inhibin gene in ovarian granulosa cells. PMID- 1717834 TI - Regulation of vasopressin messenger RNA levels in the small cell lung carcinoma cell line GLC-8: interactions between glucocorticoids and second messengers. AB - The role of glucocorticoids and second messenger systems in the regulation of the vasopressin (VP) gene was studied in the human small cell lung carcinoma cell line GLC-8. Small cell lung carcinoma GLC-8 cells express VP mRNA and contain both glucocorticoid and mineralocorticoid receptors. Treatment with the synthetic glucocorticoid dexamethasone when added alone at 10(-8) M had no effect on the VP mRNA level and decreased the level by 30% at 10(-6) M. However, the effect of dexamethasone changed to positive when cells were simultaneously treated with cAMP-enhancing agents. VP mRNA levels, which were elevated by 1.5- to 2-fold by the cAMP-enhancing agents alone, increased a further 1.5- to 3-fold by dexamethasone. Thus, the combined effect of dexamethasone and cAMP stimulation was a 3- to 7.5-fold increase in VP mRNA levels. Long term treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reduced the VP mRNA level by 75%. The TPA-suppressed VP mRNA levels could be up-regulated about 6 fold by simultaneous treatment with 8-bromo-cAMP. Dexamethasone did not alter the TPA-suppressed VP mRNA levels. These results indicate that both cAMP and protein kinase-C pathways as well as glucocorticoid receptors are involved in the regulation of VP mRNA levels and that these factors interact. This leads to a negative or positive response of VP gene expression to glucocorticoids in a state dependent manner. The interactions may be of significance in a physiological context and relate to the different regulation of VP-expressing systems in the brain. PMID- 1717835 TI - In vivo regulation of Zif268 messenger RNA expression by 17 beta-estradiol in the rat uterus. AB - Administration of 17 beta-estradiol (E2) induces a mitogenic response in the rat uterus. Previous studies have shown that this effect involves the transient activation of c-fos and c-myc expression, followed by significant increases in both DNA synthesis and cell proliferation. Zif268 is a zinc finger-containing, DNA-binding transcription factor that has been implicated in the regulation of cell growth and development and has been shown to be coregulated with c-fos in a number of systems. To determine whether Zif268 is also a target for estrogen regulation, we measured the effects of E2 on Zif268 mRNA expression in the uterus of the ovariectomized rat. In this report we demonstrate that although low levels of Zif268 mRNA expression are detectable in the uteri from ovariectomized control rats, treatment with E2 (4, 40, or 400 micrograms/kg BW) induces a rapid and transient 45- to 50-fold increase in the level of Zif268 mRNA 2 h after E2 treatment. The elevated levels of Zif268 mRNA returned to basal 6 h after hormone treatment. Lower doses of E2 (0.004, 0.04, and 0.4 micrograms/kg) had little or no effect on Zif268 mRNA expression, while higher doses of E2 (4-400 micrograms/kg) resulted in maximal increases in Zif268 expression. Dexamethasone, 5 alpha-dihydrotestosterone, and progesterone had no effect on uterine Zif268 mRNA expression, and the induction of Zif268 by E2 was abolished by pretreating the animals with the RNA synthesis inhibitor actinomycin-D. In addition, stimulation of Zif268 mRNA expression was observed with the short-acting estrogen estriol, suggesting that the response may be specific for estrogenic steroids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717836 TI - Immunogenicity of peptides having pre-determined alpha-helical and alpha-alpha fold topologies. AB - A panel of three synthetic peptides based on the 310-327 region of mouse LDH-C4 was used to examine the effect of peptide conformation on immunogenicity. The peptides, without prior conjugation to carrier molecules, were injected into outbred mice and the antisera were assayed for peptide- and LDH-C4-reactive antibodies by ELISA. An 18-residue random coil peptide (alpha N) and an 18 residue amphipathic alpha-helix peptide (alpha 1) were weakly immunogenic. A conformationally stable 40-residue alpha-alpha fold peptide (alpha 3) was highly immunogenic. The antibodies elicited by alpha 3 reacted strongly with the native molecule by ELISA. Solution-phase binding assays were used to further characterize the specificity of the sera from two mice immunized with alpha 3. Antibodies from one of the mice appeared to recognize the helical portion of the peptides, while antibodies from the other mouse reacted only with the immunogen and may be specific for the non-natural beta bend residues or possibly a topographic determinant peculiar to the anti-parallel helices. Serum from neither mouse was able to recognize the native molecule in solution. Peptides intended to mimic topographic determinants for the purpose of synthetic vaccine development may have to be more complex than those used in this study in order to induce high affinity antibodies capable of exerting a significant biological effect. PMID- 1717837 TI - Dextran sulfate specifically interacts with the human LFA-1 molecule (leucocyte function associated antigen-1). AB - We have investigated by flow cytometry the action of dextran sulfate (DxS) on the expression of the LFA-1 molecule in human lymphocytes. This work was undertaken because of the involvement of the LFA-1 molecule in HIV-1 induced syncytia and because of the role of DxS played in the inhibition of syncytia formation. Firstly we detected five distinct topographic regions (epitopes) on the LFA-1 molecule with a panel of 11 monoclonal antibodies (Mabs). Then we demonstrated that DxS interacts with some epitopes mainly present on the alpha chain of the LFA-1 molecule. This inhibition on the LFA-1 expressions by DxS occurs after 1-3 hr of incubation of either 4 or 37 degrees C with complete reappearance of LFA-1 within 1 hr of placing cells in fresh medium. In addition both 5 and 500 kDa have been found to have a similar influence on the inhibition of the LFA-1 expression, while non sulfated dextran have no effect. Other sulfated polyanion (SP) such as heparin and chondroitin sulfate have no effect on the LFA-1 expression. Further at 4 degrees C, DxS does not alter the expression of molecules recognized by Mab such as Leu3a (CD4), Leu2a (CD8), Leu4 (CD3) and Leu5b (CD2). However at 4 degrees C, DxS decreases the expression of CD45R molecule which is recognized by Mab Gap8.3. At 37 degrees C, we observe a decrease also in CD4 expression after DxS exposure. It has also been found that DxS decreases LFA-1 expression to the same extent regardless of the basal expression of LFA-1 in each selected cell subset (LFA-1 low, dim or bright). These results suggest that the inhibitory effect of DxS on the HIV-induced syncytium formation could be due partially to a specific steric hindrance of some LFA-1 determinants. PMID- 1717838 TI - Galactose-containing epitopes on the surface of IgG model immune complexes are accessible for specific binding with the high molecular weight ligand, Ricinus agglutinin, in solution-light-scattering studies. AB - Immunoglobulin G (IgG) molecules contain covalently linked carbohydrate chains with galactose residues in their branched "antennae". The ability of galactose containing epitopes on the surface of IgG model immune complexes (IC) to interact with a high mol. wt ligand in solution has been elucidated. Different types of IgG model IC with pre-determined molecular mass were mixed with Ricinus Agglutinin (RCI), which is known to bind specifically to galactose-containing oligosaccharides. The relative light-scattering increases (delta I) in the reaction mixture were measured as a function of time. The galactose-associated epitopes of the IgG model IC were accessible for binding with RC1. The rate of the interaction between IgG model IC and RC1 was dependent on the molecular mass of the complexes; the larger the model IC molecular mass, the faster the rate of interaction. The binding of RC1 to IgG model IC was highly specific because it was completely abolished in the presence of lactose. The galactose-containing epitopes of monomeric IgG were also able to interact with RC1 but the kinetics of the interaction was much slower. We suggest than an increase in the density of the epitopes on the surface of the model IC, by close attachment of the IgG molecules, mainly determines the ability of galactose-containing epitopes to be recognized by RC1. The data presented support the importance of IgG glycans in recognition events of IgG by biologically active molecules. PMID- 1717839 TI - Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI. AB - Three rat monoclonal antibodies specific for mouse IgE (C12B9, 23G3, and B1E3) were established by using monoclonal anti-DNP mouse IgE (mIgE) as immunogen. These antibodies, as well as a fourth, (R1E4) were characterized. It was found that one antibody (C12B9) recognizes an allotypic determinant (Igh-7a) found on the C epsilon chain of mIgE. Antibody cross-blocking studies and epitope mapping studies using recombinant mIgE indicated that 3 antibodies (C12B9, R1E4 and 23G3) were directed against the C epsilon 3 domain while one (B1E3) was directed against the C epsilon 4 domain. A highly specific sandwich RIA for mIgE was developed using these antibodies. Use of these monoclonal anti-mIgE antibodies in conjunction with recombinant chimeric mIgE-human IgG1 molecules, demonstrated that the C epsilon 3 domain is important in the binding of mIgE to the murine B cell Fc epsilon RII as well as to the murine mast cell F epsilon RI. The presence of the C epsilon 4 domain influenced the binding of the recombinant IgE to the Fc epsilon RII; in contrast to the C epsilon 4 domain had no effect on binding to the Fc epsilon RI. PMID- 1717840 TI - The immune response to synthetic peptides of human protamines HP1 and HP2. AB - Peptides representing the amino-terminal sequence of protamines HP1 (sequence 1 12) and HP2 (sequence 1-11), the two major nuclear proteins of human sperm, have been synthesized. Rabbits were immunized either with peptide conjugated with a carrier or with free peptide. The resulting antisera were examined for their capacities to bind the homologous peptide, other peptides from protamines HP1, HP2, from ram protamine, a protein resembling HP1, and finally with the whole protamine. Only free peptides were immunogenic. Antisera were found to react with the homologous peptide, but also with some other peptides. More especially, antibodies to peptide HP1 1-12 were found to recognize an epitope shared by the homologous peptide, peptide HP1 37-49 and peptide 1-12 of ram protamine. The common antigenic determinant seems to depend on the conformation of the peptides, rather than strictly related to common sequences. Anti-peptide antibodies react poorly and in a non-specific manner with the parent protein. The failure of reactivity with the protamines strongly suggest that these small basic proteins are folded and probably globular molecules in contrast with the totally random model postulated by several previous works. PMID- 1717841 TI - Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef. AB - Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2 pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes. PMID- 1717842 TI - Gene-specific DNA repair in terminally differentiating rat myoblasts. AB - Preferential DNA repair in expressed genes has been well documented in proliferating mammalian cells following ultraviolet irradiation. It was of interest to learn whether excision repair is similarly selective in terminally differentiating cells. We have measured the removal of ultraviolet-induced cyclobutane pyrimidine dimers (detected as T4 endonuclease V-sensitive sites) from various genes in cultured L8 rat skeletal myoblasts. In these cells, the transcription of muscle-specific genes such as the embryonic myosin heavy chain (MHCemb) gene can be regulated by inducing cells to differentiate. L8 myoblasts are somewhat more sensitive than Chinese hamster ovary cells to ultraviolet radiation, and they exhibit relatively poor overall DNA-repair rates throughout differentiation. Irradiation severely reduces the rates of transcription and steady-state RNA levels for the genes studied. Although differences in kinetics are seen between the repair of active and inactive genes, repair rates are low relative to those previously measured in proliferating rodent cell lines. Repair efficiency in the MHCemb gene increases as it is activated during differentiation and, in fact, approaches 100% within 5 days, while that in the silent GAP43 gene is much lower. While repair efficiencies generally correlate with expression in the genes studied, the overall time course of repair appears to be prolonged in these cells compared to that in proliferating cells. These terminally differentiating cells seem to maintain a DNA damage surveillance and repair capacity for selected genes and/or genomic domains. PMID- 1717843 TI - Drosophila methyltransferase activity and the repair of alkylated DNA. AB - The biochemical mechanism and developmental expression for the repair of alkylated DNA has been characterized from Drosophila. As in other organisms, the correction of O6-methylguanine in Drosophila was found to involve the transfer of a methyl group from DNA to a protein cysteine residue. Two methylated proteins with subunit molecular weights of 30 kDa and 19 kDa were identified following incubation with [3H]-methylated substrate DNA and denaturing polyacrylamide gel electrophoresis. Identical molecular weights were found for the unmethylated forms of protein through their reaction to an antibody prepared against the 19 kDa Escherichia coli methyltransferase. Both Drosophila proteins are serologically reactive in adult males and females and most of the other developmental stages tested, with embryos representing the possible exception. The Drosophila proteins do not appear to be induced by sublethal exposures to alkylating agent. PMID- 1717844 TI - Harlequin banding and localisation of sister-chromatid exchanges. AB - Harlequin banding (HB) was standardised on Indian muntjac chromosomes by superimposing harlequin staining or sister-chromatid differentiation and G banding after incorporation of bromodeoxyuridine (BrdU) or cholorodeoxyuridine (CldU), and after treatment with BrdU plus mitomycin C (MMC). SCEs were localized on these chromosomes with the aid of the G-band map. There were more SCEs in G bands than in R-bands in BrdU-incorporated chromosomes. CldU-incorporated chromosomes, however, did not show a preferential localization of SCEs in either G- or R-bands. When BrdU + MMC-induced SCEs were localized in harlequin-banded chromosomes, there was a significantly greater number of SCEs in R-bands; and there was a concomitant reduction in the frequency of SCEs in G-bands, as compared to the SCEs observed in this region after BrdU incorporation alone. Centromeric regions of chromosomes 1 and X had preferred sites for occurrence of SCEs in BrdU-incorporated chromosomes, the preferred sites being more in G-bands after BrdU and CldU incorporation and in R-bands after treatment of BrdU incorporated chromosomes with MMC. Thus the formation of SCEs is not restricted by structure per se as defined by euchromatin or heterochromatin, but depends on the site of lesion production, type of lesion and repair pathway followed. PMID- 1717845 TI - Genotoxicity and cell proliferation activity of omeprazole in rat stomach mucosa. PMID- 1717846 TI - Nucleotide sequence and transcription of a trypomastigote surface antigen gene of Trypanosoma cruzi. AB - In previous studies we identified a 500-bp segment of the gene, TSA-1, which encodes an 85-kDa trypomastigote-specific surface antigen of the Peru strain of Trypanosoma cruzi. TSA-1 was shown to be located at a telomeric site and to contain a 27-bp tandem repeat unit within the coding region. This repeat unit defines a discrete subset of a multigene family and places the TSA-1 gene within this subset. In this study, we present the complete nucleotide sequence of the TSA-1 gene from the Peru strain. By homology matrix analysis, fragments of two other trypomastigote specific surface antigen genes, pTt34 and SA85-1.1, are shown to have extensive sequence homology with TSA-1 indicating that these genes are members of the same gene family as TSA-1. The TSA-1 subfamily was also found to be active in two other strains of T. cruzi, one of which contains multiple telomeric members and one of which contains a single non-telomeric member, suggesting that transcription is not necessarily dependent on the gene being located at a telomeric site. Also, while some of the sequences found in this gene family are present in 2 size classes of poly(A)+ RNA, others appear to be restricted to only 1 of the 2 RNA classes. PMID- 1717847 TI - Inability to attribute susceptibility to primary sclerosing cholangitis to specific amino acid positions of the HLA-DRw52a allele. PMID- 1717848 TI - Biochemical effect of chocolate colouring and flavouring like substances on thyroid function and protein biosynthesis. AB - Synthetic chocolate colourant, flavourant and the mixture of both were administered to healthy adult male albino rats to evaluate their effect on the nucleic acids metabolism, i.e. deoxyribonucleic and ribonucleic acids (DNA and RNA), total serum protein, thyroid hormones (T4 and T3) and nuclease enzymes, i.e. cytoplasmic- and mitochondrial deoxyribonuclease and ribonuclease (DNase and RNase) in brain, liver, and kidneys. Also, the activity of the fundamental enzymes of the oxidative pentose phosphate pathway, i.e. cytoplasmic and mitochondrial glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G-6-PD and 6-PGD), as well as total lipids and cholesterol contents in the same organs were studied. Ingestion of the studied food additives significantly increased serum protein, RNA and T4 hormone, while, DNA and T3 hormone were insignificantly elevated. In connection with this, the hydrolytic enzymes of nucleic acids (DNase and RNase activities) were stimulated by all studied food additives and in all mentioned organs. The activity of G-6-PD and 6 PGD in both cytoplasmic and mitochondrial fractions of all studied organs were increased. The highest increase was noticed in rats fed on diets supplemented with the mixture of both colourant and flavourant followed by colourant then flavourant, respectively. PMID- 1717849 TI - Dysmyelination in transgenic mice resulting from expression of class I histocompatibility molecules in oligodendrocytes. AB - Major histocompatibility complex (MHC) molecules are not normally expressed in the central nervous system (CNS). However, aberrant expression has been observed in multiple sclerosis lesions and could contribute to the destruction of myelin or the myelinating cells known as oligodendrocytes. The mechanism of cell damage associated with aberrant MHC molecule expression is unclear: for example, overexpression of class I and class II MHC molecules in pancreatic beta cells in transgenic mice leads to nonimmune destruction of the cells and insulin-dependent diabetes mellitus. We have generated transgenic mice that express class I H-2Kb MHC molecules, under the control of the myelin basic protein promoter, specifically in oligodendrocytes. Homozygous transgenic mice have a shivering phenotype, develop tonic seizures and die at 15-22 days. This phenotype, which we term 'wonky', is due to hypomyelination in the CNS, and not to involvement of the immune system. The primary defect appears to be a shortage of myelinating oligodendrocytes resulting from overexpression of the class I MHC molecules. PMID- 1717850 TI - Ion channels. Not an open-and-shut case. PMID- 1717851 TI - Domains specifying thrombin-receptor interaction. AB - Platelet activation by the coagulation protease thrombin is central to arterial thrombosis, a major cause of morbidity and mortality. We recently isolated a complementary DNA encoding the platelet thrombin receptor. The extracellular amino-terminal extension of this seven transmembrane domain receptor contains the putative thrombin cleavage site LDPR/S which is critical for receptor activation. By replacing this cleavage site with the cleavage site for enterokinase, we have created a functional enterokinase receptor. This result demonstrates that all information necessary for receptor activation is provided by receptor proteolysis. Nanomolar enterokinase concentrations are required to activate this new receptor, in contrast to the picomolar thrombin concentrations that activate wild-type thrombin receptor. We identified a receptor domain critical for thrombin's remarkable potency at its receptor. This domain resembles the carboxyl tail of the leech anticoagulant hirudin and functions by binding to thrombin's anion-binding exosite. Our studies thus define a model for thrombin-receptor interaction. The utility of this model was demonstrated by the design of novel thrombin inhibitors based on receptor peptides. PMID- 1717852 TI - Growing human B lymphocytes in the CD40 system. AB - One approach to generating human monoclonal antibodies relies on the development of culture techniques allowing the long-term growth of most B lymphocytes, a property of the CD40 system. PMID- 1717853 TI - [Hepatology. New research results in its significance for the understanding of liver diseases]. AB - By two exemplary clinical situations--acute viral hepatitis, acute-phase reaction of the liver--the significance of basic research for the understanding of clinical phenomena and for the development of new diagnostic and therapeutic procedures is demonstrated. The very different phenomena following infection with the hepatitis-B-virus can be explained by the variation in the interactions of virus and liver cell, by the immune reaction of the host, and by mutants of the virus. The reaction of the liver to an extrahepatic infection is mediated by interleukin-6, and characterized by an alteration in protein metabolism. The synthesis of acute-phase proteins is increased. The proteins confine the local injury and establish the homeostasis of the organism. PMID- 1717854 TI - [The morphometric characteristics of the neurons of the reticular nuclei in the brain stem in the kitten]. AB - In the brain stem of 30-day-old kittens three types of Golgi-stained neurons are distinguished: sparsely branched reticular neurons, densely branched arborescent neurons and multipolar giant neurons (according to the classification by Leontovich). Implication of the new method of computer morphometry permits obtaining formalized characteristics of 23 parameters of the above types of neurons. An analysis of the quantitative characteristics of the studied neurons has revealed statistically significant differences in most parameters. This fact permits advancing a conclusion about morphological identity (stability) of each type of the neurons. Structural differences and similarities in reticular nuclei are discussed with respect to their functional features. PMID- 1717855 TI - [The effect of sectioning the lingual nerve on the morphometric characteristics of the reticular nuclei in the brain stem in the kitten]. AB - Three subpopulations of Golgi-stained neurons of the brainstem reticular nuclei have been studied by the method of computer morphometry in 30-day old kittens. 23 parameter of the neuronal geometry were analyzed after uni-and bilateral lingual nerve section 5-7 days after birth. All of cells display statistically valid changes in some parameters, typically differing in every group: for reticular neurons--free distribution of dendrite endings in the dendritic field; for arborescent ones--the length of dendritic segments; for giant multipolar neurons- distribution of foci of maximal dendrite branching in the dendritic field. Unilateral lingual nerve section gives more pronounced quantitative deviations from the normal state, than bilateral one. PMID- 1717856 TI - Do some malignant melanoma cells share antigens with the myeloid monocyte lineage? AB - Melanoma cells freshly isolated from 63 advanced primary lesions and 103 metastases were analyzed by staining with monoclonal antibodies MEM 28 directed against a 200 kDa antigen present on all leukocytes and tissue macrophages (CD 45), MEM 18 directed against a monocyte antigen of 53 kDa, anti CD 14- Immunotech, Marseille and 3.9 directed against a 150 kDa antigen expressed on monocytes and to even greater degree on most tissue macrophages (CD 11 c). All antibodies showed variable reactivity with melanoma cells, percentage of positive tumor cells ranged from 0 to 70. PMID- 1717857 TI - Effects of tissue fixation conditions and protease pretreatment on immunohistochemical performance of a large series of new anti-keratin monoclonal antibodies: value in oncopathology. AB - A comparative study with 21 recently raised monoclonal antibodies (3 of which are reported here for the first time) to human keratin polypeptides was performed on a wide range of paraffin-embedded tissues and tumors, aimed at the examination of effects of four different fixatives and protease pretreatment on the immunohistochemical detection of keratins. Our data demonstrated that: (a) formaldehyde-based fixatives modified by acidification and/or addition of methanol gave results superior to those achieved by routinely used formol saline; (b) relatively rare antibodies (4 out of 21) could be identified which gave reliable immunostaining patterns even on routine formalin-fixed material; (c) a proteolytic digestion step preceding the immunostaining was beneficial for the performance of the majority of antibodies in our panel. Additional options which could potentially lead to further improvement of keratin immunohistochemistry in paraffin embedded specimens are also suggested. This work provides the necessary basis for wider application of the anti-keratin antibodies of the C-series in both routine oncopathology and research-oriented retrospective studies. PMID- 1717858 TI - Transoval administration of opiates into trigeminal cistern for cancer pain. Preliminary report. AB - A new method for administration of opiates into the ventriculo-cisternal system for intractable pain due to cancer is presented. Five patients suffering from such pain underwent the permanent implantation of a subcutaneous reservoir connected to a thin catheter inserted into the trigeminal cistern. The indications are those of the intraventricular way. Percutaneous trigeminal opiates administration (PTO) proved to be a valid and simple alternative method to intrathecal and intraventricular morphine. PMID- 1717859 TI - Relationship between 22Na distribution and cerebral blood flow in ischemic gerbil brain. AB - Sodium transport in the early postischemic period was studied using Mongolian gerbils with right common carotid artery ligation. [22Na]sodium chloride ([22Na]NaCl) was infused immediately after, 10 minutes before, and 4 hours before carotid ligation, and the 22Na distribution was measured in symptomatic animals by autoradiography 1 hour after ischemia. Regional cerebral blood flow was determined by [14C]iodoantipyrine autoradiography. The specific gravity of the brain was measured in symptomatic gerbils 1 and 2 hours after carotid ligation by a gradient column. There was a low uptake of 22Na in the ischemic core and a high uptake in the ischemic periphery when the tracer was given 10 minutes before or immediately after ischemia. In contrast, tracer given 4 hours before ischemia showed an increased radioactivity in both the ischemic core and periphery. It is suggested that increased sodium in the ischemic core is due to a decreased sodium clearance rate and increased sodium in the ischemic periphery is due to some active transport process. PMID- 1717860 TI - Photodynamic therapy using pheophorbide a and Nd:YAG laser. AB - The authors describe a new photodynamic therapy (PDT) method for malignant brain tumors. Pheophorbide a (Ph-a), the photosensitizer, has low toxicity, causes no skin sensitization and is activated with an acoustic Q switched neodymium yttrium argon-garnet (Nd:YAG) laser which achieves deep tissue penetration. The Ph-a distribution in Fisher 344 (F344) rats bearing rat T9 glioma at 24 hours after intravenous injection was very low in the normal brain tissue, but significantly higher in the T9 glioma giving a tumor to normal brain tissue concentration ratio of 7.5:1. The in vitro survival rate of T9 glioma cells pretreated with Ph-a was 68.8 +/- 5.4% after laser irradiation for 20 minutes, significantly lower than in the control groups. This indicates that Ph-a was activated with the acoustic Q switched Nd:YAG laser causing the photodynamic effect. The survival rate after Ph a pretreatment and laser irradiation in a waterbath at 44.0 degrees C was further reduced to 15.8 +/- 3.3%. In vivo PDT studies using T9 glioma cells inoculated into the dorsal region of F344 rats showed tumor eradication in four of six rats. The combination of PDT and laser hyperthermia produced tumor eradication in all six rats. The combination of PDT and hyperthermia is a promising method for tumor treatment. PMID- 1717861 TI - Reversibility of cerebral function assessed by somatosensory evoked potentials and its relation to intracranial pressure--report of six cases with severe head injury. AB - The critical value and duration of intracranial pressure (ICP) causing cerebral function damage was evaluated in six head injury patients by monitoring the first negative cortical component (N20) of the somatosensory evoked potential (SEP). The SEP was elicited by stimulating the median nerve, and N20 (C3' or C4'-Fz on the affected side) and N13 (C2S-Fz) were monitored serially with a signal processor. The auditory brainstem response (ABR) was simultaneously recorded on the affected side (A1 or A2-Cz). A reversible loss of N20 occurred 7 times in six cases. In all cases, the N20 was restored by emergency decompression or hyperosmolar therapy. The minimum ICP at which N20 disappeared was 30 mmHg, and the N20 was restored when decompression was performed within 4.5 hours. However, when the disappearance persisted for more than 1.5 hours, the N20 latency was markedly prolonged after restoration. These changes appeared before the ABR showed definite abnormalities. These results show that the cerebral function may be damaged when ICP exceeds 30 mmHg, and that emergency decompression is required within 4.5 hours, preferably within 1.5 hours, to restore cerebral function. This critical ICP and duration should be of clinical value in patient management. PMID- 1717862 TI - Aneurysm in the horizontal segment of the anterior cerebral artery confirmed by cerebral vasospasm--case report. AB - A rare aneurysm in the horizontal segment (A1) of the right anterior cerebral artery was found in a 58-year-old male presenting with subarachnoid hemorrhage. No obvious bleeding source was observed on the day of onset, but 7 days later, a definite diagnosis was made based on the discovery of cerebral vasospasm by a repeat angiogram. The aneurysm was clipped via the right frontotemporal approach 15 days after onset. He suddenly developed neurological symptoms such as consciousness disturbance, right hemiplegia, and aphasia on the 4th postoperative day, when remission of the cerebral vasospasm was confirmed by transcranial Doppler ultrasound examinations and cerebral angiography. The ischemic symptoms were probably due to cerebral embolus caused by intraluminal thrombi, which had formed during the maximum phase of vasospasm and became detached during the remission phase. PMID- 1717863 TI - Complete remission of metastatic teratoma from malignant testicular tumor using salvage chemotherapy with VP-16 (etoposide), CDDP, and ACNU--case report. AB - A 26-year-old male with an intracranial teratoma, metastasizing from a testicular yolk sac tumor refractory to cis-diamminedichloroplatinum (CDDP), vinblastin, and bleomycin (PVB) therapy, was successfully treated with a combination of etoposide (VP-16), CDDP, and ACNU (salvage chemotherapy). Emergency surgery for subcortical hemorrhage discovered the metastasis, diagnosed as a yolk sac tumor. CDDP chemotherapy failed to prevent recurrence, and total removal was impossible due to subdural invasion. He underwent radiation therapy and salvage chemotherapy. A third operation found only scar tissue. Maintenance salvage chemotherapy was continued. He was doing well 30 months after the first operation. PMID- 1717864 TI - Endodermal epithelial cyst in the prepontine cistern extending into the fourth ventricle--case report. AB - The authors report a case of epithelial cyst, which recurred 32 years after the initial surgical treatment. Computed tomography showed no abnormality, but magnetic resonance (MR) imaging clearly demonstrated a well-demarcated mass in the prepontine cistern, extending into the fourth ventricle. The lesion showed extreme hyperintensity compared with the surrounding brain on both the T1- and T2 weighted images. The ultrastructural features of the cyst suggested an endodermal origin. MR imaging and electron microscopy are essential for correct diagnosis and exact pathogenetic identification of intracranial cystic lesions. PMID- 1717865 TI - Sinus pericranii with severe symptom due to transient disorder of venous return- case report. AB - The authors report a case of sinus pericranii in a 22-year-old female presenting with severe headache, vomiting, bradycardia, and bradypnea following excessive distention of the tumor. After tumor removal, the symptoms were completely relieved. The symptoms were thought to be due to transient impairment of blood flow in the superior sagittal sinus. PMID- 1717866 TI - Hypoplasia of the internal carotid artery--report of an unusual case. AB - We present a rare case of unilateral internal carotid artery (ICA) hypoplasia associated with arterial anomalies in the circle of Willis. The ipsilateral middle cerebral artery was supplied via anomalous arteries from the posterior cerebral artery and the ICA. The ipsilateral common carotid artery also originated from the anomalous brachiocephalic trunk. The etiology of the hypoplastic ICA is uncertain, but the associated multiple vascular anomalies support the congenital origin. PMID- 1717867 TI - Myelopathy due to thickened ligamenta flava and abnormal fibrous tissue of the cervicothoracic junction--case report. AB - A 49-year-old female with subacute myelopathic symptoms due to thickened cervicothoracic yellow ligament and abnormal epidural fibrous tissue is reported. Myelography showed a complete block at the Th3 level. Magnetic resonance imaging demonstrated an extra-axial mass lesion in the spinal canal at the cervicothoracic junction causing the spinal cord compression. Laminectomy with resection of the lesion resulted in good neurological recovery. Histological examination revealed a thickened ligamenta flava and abnormal epidural fibrous tissue without calcification foci. PMID- 1717869 TI - Cholinergic modulation of growth hormone secretion induced by galanin in rats. AB - The effects of cholinergic modulation by hexamethonium (HEX), tetraethylammonium (TEA), pirenzepine (PIR) and neostigmine on growth hormone (GH) secretion induced by galanin (GAL) were investigated in awake, freely moving male rats. Intracerebroventricular (i.c.v.) injection of GAL (0.6 nmol) caused a marked increase in plasma GH levels. Pretreatment with HEX (3 or 15 mg/kg BW), TEA (15 mg/kg BW) or PIR (0.5 mg/kg BW) significantly reduced the GAL-induced GH secretion. Pretreatment of animals with neostigmine (0.05 mg/kg BW) or with a specific antisomatostatin antiserum (10 microliters of 1:10 diluted with saline, i.c.v.) reversed the inhibition of GAL-induced GH secretion by HEX. These findings suggest that both nicotinic and muscarinic cholinergic mechanisms interact with the galanin-induced GH secretion by modulating hypothalamic somatostatin release. PMID- 1717868 TI - [Hemodilution in the treatment of cerebrovascular disorders]. AB - Haemodilution includes intravenous administration of plasma-replacing solutions and/or blood letting for changing the rheological properties of blood, and for improvement of microcirculation. Three methods of haemodilution various plasma substitutes are given: dextran, hydroxyethyl starch (HES), electrolyte solutions (e.g. Ringer's solution, normal saline, 5% glucose, colloidal plasma, protein solutions), plasma, albumins. The controversial results of clinical trials of the method for therapeutic purposes are discussed. In the light of the reports as yet, it seems that the best results are obtained using normovolaemic haemodilution with administration of HES. PMID- 1717870 TI - Inhibitory effects of clonidine and tizanidine on release of substance P from slices of rat spinal cord and antagonism by alpha-adrenergic receptor antagonists. AB - Effects of clonidine and tizanidine, which have antinociceptive and alpha 2 agonistic actions, were studied on the release of substance P from slices of spinal cord from the rat. Veratridine-evoked depolarization induced a 2-3-fold increase in the release of substance P from the slices of spinal cord. Exposure of the cord tissue to 10 microM clonidine and tizanidine significantly reduced the release of substance P. The inhibitory effects of clonidine and tizanidine were attenuated by pre-exposure of the tissue to 10 microM piperoxane, which has alpha 2-antagonistic activity and the inhibitory effect of clonidine was attenuated by 10 microM yohimbine. Moreover, the inhibitory effects of clonidine and tizanidine were also blocked by a small dose of prazosin, an antagonist for alpha 1- and alpha 2B-receptors. None of the antagonists had any effect on release of substance P, when given alone. These results suggest that alpha 2B adrenoceptors are involved in the inhibitory effects of clonidine and tizanidine on the release of substance P. PMID- 1717871 TI - Norepinephrine does not contribute to methamphetamine-induced changes in hippocampal serotonergic system. AB - The purpose of this study was to determine whether norepinephrine (NE) mediated the reduction in the activity of tryptophan hydroxylase (TPH) in the hippocampus and other serotonergic changes, induced by a single or multiple administrations of methamphetamine. The NE in the hippocampus was depleted by injecting rats with DSP4 intraperitoneally, 10 days prior to administration of methamphetamine. A single administration of methamphetamine (15 mg/kg) reduced the activity of TPH to 40% of control after 3 hr. A 90 to 98% reduction in the concentration of NE in the hippocampus, failed to alter this methamphetamine-induced response. The reduction in serotonin (5-HT) in the hippocampus induced by methamphetamine, was not altered by the treatment with DSP4. These observations were confirmed by a lack of effect on methamphetamine-induced changes in 5-HT and TPH after inhibition of the synthesis of NE with U-14,624. The pretreatment with DSP4 also failed to block the decline in activity of TPH in the hippocampus or concentrations of 5-HT measured 18 hr after the last of 4 doses of methamphetamine (15 mg/kg, s.c.). The results presented in this study indicate that NE is not involved in the response of the serotonergic system to methamphetamine. PMID- 1717872 TI - Different role of 5-HT1A and 5-HT2 receptors in spinal cord in the control of nociceptive responsiveness. AB - The effects of the 5-hydroxytryptamine type-2 (5-HT2) receptor agonist (+/-)-1 (2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and the 5-HT1A agonist (+)-8 hydroxy-2-(di-n-propylamino)-tetralin [(+)-8-OH-DPAT] on nociceptive responsiveness were compared in mice. Intrathecal administration of DOI (5-20 micrograms) produced a dose-dependent behavioural syndrome, consisting of biting or licking, directed towards the caudal part of the body and reciprocal hindlimb scratching. However, (+)-8-OH-DPAT (5-20 micrograms) did not produce the biting and scratching behaviour. The response to DOI (20 micrograms) was reversed by treatment with the substance P receptor antagonist, [D-Arg1, D-Trp7,9, Leu11]-SP (Spantide) (5 micrograms). The tail-flick reflex was markedly depressed 5-20 min after administration of (+)-8-OH-DPAT; DOI did not change the tail-flick reflex after 5 min but significantly inhibited the reflex response 10-20 min after injection. The data show that stimulation of 5-HT2 receptors, but not 5-HT1A receptors, induced a behavioural syndrome, which may reflect activation of nociceptive pathways. The tail-flick reflex was more markedly inhibited by stimulation of 5-HT1A than 5-HT2 receptors. Accordingly, 5-HT2 and 5-HT1A receptors seem to have a different function in the modulation of nociceptive responsiveness in the mouse. PMID- 1717873 TI - Combined administration of a non-neurotoxic 3,4-methylenedioxymethamphetamine analogue with amphetamine produces serotonin neurotoxicity in rats. AB - In the present study, a central serotonin neurotoxicity was induced by combining a non-neurotoxic 3,4-methylenedioxymethamphetamine analogue, 5-methoxy-6-methyl-2 aminoindan (MMAI), with the non-vesicular dopamine (DA) releaser, S-(+) amphetamine (Amp). With the multiple dosing regimen utilized neither drug alone resulted in any changes in serotonergic parameters, including 5-HT, 5-HIAA and the number of 5-HT uptake sites. However, MMAI (10 mg/kg) in combination with Amp (2 x 2.5 mg/kg) did result in a long-term 20% decrease in cortical serotonergic parameters. The same dose of Amp plus 20 mg/kg MMAI resulted in a 50 to 60% reduction. Effects in the hippocampus and caudate nucleus were similar. These data support the hypothesis that DA release plays a critical role in the serotonin neurotoxicity of substituted amphetamines. PMID- 1717874 TI - 8-hydroxy-2-(di-N-propylamino)tetralin increases the activity of adenylate cyclase in the hippocampus of freely-moving rats. AB - The present study was designed to examine the effects of intraperitoneal (i.p.) administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on the efflux of cyclic adenosine monophosphate (cAMP) in the extracellular fluid of the dorsal hippocampus, using in vivo microdialysis. One week after implantation of the guide, probes were inserted in conscious rats and perfused with Ringer solution. Steady basal levels of cAMP (2.9 +/- 0.1 pmol/ml, n = 74 rats) were obtained after at least three hours of stabilisation. The 8-OH-DPAT dose dependently increased the basal efflux of cAMP, which was most apparent between 20-40 min after the injection. The largest dose of 8-OH-DPAT (1 mg/kg) tested, induced a maximum response of approximately 50%, whereas injections of saline did not alter the efflux of cAMP. Treatment with (+/-)pindolol (10 mg/kg) did not significantly affect the basal efflux of cAMP, whereas it markedly inhibited the increase in levels of cAMP, induced by 0.5 mg/kg 8-OH-DPAT (injected 40 min later). Simultaneous behavioural observations demonstrated that (+/-)pindolol also attenuated various components of the 8-OH-DPAT-induced behavioural syndrome. Addition of 3-isobutyl-1-methylxanthine (IBMX), forskolin or noradrenaline, to the perfusion fluid, strongly enhanced the levels of cAMP in the extracellular fluid from the hippocampus. Injection of 8-OH-DPAT (1 mg/kg) during perfusion with IBMX induced a similar increase in levels of cAMP, as under normal perfusion conditions. However, 8-OH-DPAT did not significantly alter the efflux of cAMP, when probes were perfused with either forskolin or forskolin and IBMX.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717875 TI - In vivo and in vitro evidence of growth hormone-releasing factor-like produced locally in the adenohypophyseal cells of the rat. AB - In order to characterize immunocytochemically the existence of GRF in the rat adenohypophysis and the origin of this hormone, an immunocytochemical and morphometric study was made of r-GRF-immunoreactive cells from the adenohypophysis of untreated adult rats, adult rats treated intraventricularly with colchicine and in primary cultures of adult rat adenohypophyseal cells that had been incubated with serum devoid of GRF. r-GRF immunoreactive cells were observed in untreated rat pituitaries, both male and female, although the numbers of positive cells were greater in the males (p less than 0.05) and were found to increase in number following treatment with colchicine (p less than 0.01). These cells appeared dispersed throughout the anterior lobe, without forming clusters, and were often close to blood vessels. Additionally, immunoreactive cells appeared in the cultures at 7 days of incubation. The presence of GRF immunoreactive cells in the adenohypophysis of rats previously treated with colchicine suggests the existence of a non-hypothalamic origin for r-GRF; this is confirmed by the findings obtained in the in vitro studies which would corroborate the hypothesis that the origin of the neuropeptide is in the rat adenohypophysis itself. PMID- 1717876 TI - Influence of the replacement of amino acid by its D-enantiomer in the sequence of substance P. 1. Binding and pharmacological data. AB - The D-enantiomer of residues 2, 4, 5, 6, 7, 8, 10 and 11 was introduced in the sequence of Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. The achiral glycine residue was replaced by a D-Ala residue. Regarding NK-1 binding potencies or activities, changing to the D-enantiomer in positions 2, 4 or 5 did not modify the pharmacological patterns of the resulting peptides. Introduction of a D-residue in the 6 to 11 sequence drastically decreased the potency of the D analogues with the exception of [D-Leu10]SP which was found only three times less potent than SP in contracting the guinea-pig ileum. No clear cut evidence between the binding potencies and activities on NK-1, NK-2 and NK-3 assays, was observed which allows a more rational design of tachykinins antagonists. PMID- 1717877 TI - Influence of the replacement of amino acid by its D-enantiomer in the sequence of substance P. 2. Conformational analysis by NMR and energy calculations. AB - The D-enantiomer of residues 2, 4, 5, 6, 7, 8, 10 and 11 was introduced in the sequence of Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH1. The achiral glycine was replaced by a D-Ala residue. The conformations of the D substituted analogues were analysed by NMR and molecular energy calculations. Introduction of a D-amino acid in the address sequence of SP (1 to 5) distorted the helical structure of [D-Pro2]SP and [D-Pro4]SP. A D-glutamine in position 5 hampered the formation of an helix, the core of [D-Gln5]SP was stabilized by the presence of two beta-turns. The exact fitting of both Phe7 and Phe8 in the binding pocket can be achieved by either an alpha- or a 3(10) helix or two beta turns types I and II'. Replacement of an amino acid in the message sequence, 6 to 11, drastically decreased the potencies of the corresponding analogues, different conformational modifications were observed. [D-Gln6]SP presented two beta-turns, however, the types of beta-turns should orientate the side-chains in such a way that [D-Gln6]SP did not fit in the binding site. The conformations of [D-Phe7]SP and [D-Phe8]SP were completely changed, a more or less extended structure being observed. Modifications in the Gly-Leu-Met-NH2 sequence did not affect the helical structure of the core of [D-Ala9]SP, [D-Leu10]SP and [D-Met11]SP, but decreased the percentage of extended structure of the C-terminal tripeptide. PMID- 1717878 TI - The regional distribution of thyrotropin releasing hormone, leu-enkephalin, met enkephalin, substance P, somatostatin and cholecystokinin in the rat brain and pituitary. AB - There was no apparent difference in the regional distribution of neuropeptides in the brain of male and female rats. The highest levels of immunoreactive leu enkephalin, TRH, substance P and somatostatin were found in the hypothalamus, while the striatum and the cerebral cortex had the highest concentrations of met enkephalin and cholecystokinin respectively. The lowest concentrations of these were found in the cerebellum. Enkephalins (cerebral cortex), substance P (cerebral cortex and brain stem), and somatostatin (brain stem and striatum) showed higher level in the female while enkephalin and substance P contents in the anterior pituitary were higher in the male. PMID- 1717880 TI - Plastic metabolism in neurons in the hypochromic type of alterations. PMID- 1717879 TI - Transplantation of human embryonal nervous tissue into the spinal cord of adult rats. PMID- 1717881 TI - Language disturbances from mesencephalo-thalamic infarcts. Identification of thalamic nuclei by CT-reconstructions. AB - The authors report the cases of two patients with CT-documented paramedian mesencephalo-thalamic infarcts, showing language disturbances. The first patient showed a non fluent, transcortical motor-like aphasia, the other had a fluent but severely paraphasic language disorder. The CT study disclosed that it was the dorso-median thalamic nucleus that was mostly involved in both cases. These findings agree with a few previous pathological studies suggesting that the paramedian thalamic nuclei, particularly the dorso-median nucleus may play some role in language disturbances. However the anatomical basis for thalamic aphasia remains speculative, taking into account the importance of cortical connections in the origin of subcortical neuropsychological disturbances. PMID- 1717882 TI - Voltage-dependent calcium currents in dissociated granule cells from rat cerebellum. AB - Voltage-dependent calcium currents were investigated by the patch-clamp technique in whole-cell recording configuration in cultures from 8-day-old rat cerebella, which contained greater than or equal to 90% granule cells. In solutions designed to minimize the sodium and potassium conductances and in 20 mM barium, an inward current activated around -25 mV, reached a peak amplitude at +20 mV and reversed around +80 mV. In 20 mM calcium, this current was approximately 50% of that in barium. From one to three days in vitro only 16% of the cells tested (n = 20) had a current exceeding 50 pA in maximum amplitude, while after four days in vitro the current reached 100 pA in all neurons tested (n greater than 70). Verapamil (50-100 microM) reversibly depressed this current. The dihydropyridine agonist Bay K 8644 (1 microM) enhanced the maximum conductance by 25 +/- 8% (n = 4), caused a negative shift in the activation of 21 +/- 5 mV and a prolongation of the deactivation time course as the voltage was stepped back from +20 to -80 mV. The GABAB agonist baclofen (50 microM) reversibly depressed the current by 27 +/- 8% in 80% of the cells. The effect was similar to that of GABA (10 microM), when the GABAA response (chloride current) was partially blocked by bicucculine. This current can be classified as a dihydropyridine-sensitive high-voltage-activated calcium current. The modulation by GABAB agonists is likely to be significant for presynaptic inhibition. PMID- 1717883 TI - Cryopreservation of postnatal rat retinal ganglion cells: persistence of voltage- and ligand-gated ionic currents. AB - Established methods for cryopreservation of living cells were modified for freeze storage of postnatal retinal ganglion cells from rat. Retinal cell suspensions containing fluorescently labeled ganglion cells were frozen after addition of 8% dimethyl sulfoxide and stored at -80 degrees C for up to 66 days. Viability of identified retinal ganglion cells was assessed by their ability to take up and cleave fluorescein diacetate to fluorescein. No significant difference was found in the number of living retinal ganglion cells when cells obtained from the same dissociation were counted before and after freezing (6.65 +/- 2.37 x 10(4) vs 7.05 +/- 3.67 x 10(4) retinal ganglion cells per ml, respectively; mean +/- S.D., n = 4). In culture following cryopreservation, the cells appeared morphologically normal, and developed neurites and growth cones similar to their freshly dissociated counterparts. Since very little is known about the electrophysiology and membrane properties of neurons after cryopreservation, we used the whole-cell configuration of the patch-clamp technique to study voltage- and ligand-gated conductances in cryopreserved retinal ganglion cells. The cryopreserved retinal ganglion cells studied under current-clamp maintained resting potentials of -60.9 +/- 6.6 mV (n = 10) and upon depolarization fired action potentials. During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na(+) (INa), Ca(2+)-(ICa), and K(+)-currents in all cells tested (n = 122). INa could be reversibly blocked by 1 microM tetrodotoxin added to the external solution. ICa was blocked by external 250 microM Cd2+ or 3 mM Co2+. In some cells, ICa consisted of both a transient and prolonged component. The outward K(+)-current consisted of Ca(2+)-dependent and -independent components. The Ca(2+)-insensitive portion of the K+ outward current was separated into four distinct components based upon pharmacological sensitivity and biophysical properties. In many cells, a rapidly inactivating current similar to the A-type K(+)-current (IA) observed in freshly cultured retinal ganglion cells was isolated by its greater sensitivity to 4-aminopyridine (5 mM) than to tetraethylammonium (20 mM). A tetraethylammonium-sensitive current with a more prolonged time course reminiscent of IK, the delayed rectifier, was also found. When the 4 aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, an additional slowly decaying potassium current became apparent. The inhibitory amino acids, GABA (20 microM) and glycine (100 microM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 microM) and strychnine (5 microM), respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1717884 TI - High resolution radioautographic localization of [125I]FK-33-824-labelled mu opioid receptors in the spinal cord of normal and deafferented rats. AB - Recent data have shown that [125I]D-Ala2, MePhe4, Met(o)ol5-enkephalin (FK-33 824) is a highly selective and specific mu opioid receptor ligand [Moyse et al. (1986) Peptides 7, 351-355]. This probe was used here to investigate the detailed radioautographic distribution of mu sites at various levels of the spinal cord. [125I]FK-33-824 binding sites were localized by both tritium-sensitive film and liquid emulsion radioautography in the spinal cord of naive and deafferented rats. In naive animals, high densities of mu sites were apparent within laminae I II at all levels of the dorsal horn, with higher levels of labelling seen in layer IIi as compared to IIo in the lumbar segment. Laminae III-IV contained about half the quantities of binding observed in superficial layers. Relatively high densities of sites were also seen over lamina VI in the upper cervical cord and throughout Clarke's column. Within the latter, [125I]FK-33-824 binding clearly spared the large perikarya of the spinocerebellar neurons. In the ventral horn, [125I]FK-33-824 binding was mainly concentrated in layer IX, at the level of cervical and lumbar enlargements. Labelled sites were confined to the neuropil, mostly sparing the soma of motoneurons. Significant decreases in [125I]FK-33-824 binding in laminae I-II (55%) and III-IV (28%) were detected four days following cervical (C3-C7) or lumbar (L1-L6) rhizotomies. These decrements were most evident at seven days post-lesion at C3-C7 levels (93 and 76% in laminae I-II and III-IV, respectively) and recovered slightly thereafter up to 28 days post-lesion. In contrast, dorsal rhizotomies did not influence mu labelling in either the ventral horn or Clarke's column. These results confirm the association of mu opioid binding sites with dorsal primary afferent fibres and demonstrate the presence of mu sites in Clarke's column and lamina IX of the ventral horn. These findings suggest that endogenous opioids in the spinal cord play a role in sensory motor integration as well as in the modulation of primary nociceptive inputs. PMID- 1717885 TI - The neurotransmitter role of calcitonin gene-related peptide in the rat and guinea-pig ureter: effect of a calcitonin gene-related peptide antagonist and species-related differences in the action of omega conotoxin on calcitonin gene related peptide release from primary afferents. AB - In the rat and guinea-pig isolated ureter electrical field stimulation of intrinsic nerves (10 Hz for 10 s) produces transient inhibition of evoked (20 mM KCl or 0.1-1 microM neurokinin A) rhythmic contractions by releasing transmitter(s) from peripheral endings of capsaicin-sensitive primary afferents. The C-terminal fragment of human calcitonin gene-related peptide (8-37) blocked the inhibitory effect of electrical field stimulation as well as that produced by exogenous calcitonin gene-related peptide, while leaving unaffected the inhibitory response to isoprenaline. Human calcitonin gene-related peptide (8-37) was devoid of any inhibitory activity of its own but enhanced the amplitude and frequency of KCl-evoked rhythmic contractions in the rat ureter, probably by antagonizing the inhibitory effect of endogenous calcitonin gene-related peptide released by KCl. Omega conotoxin fraction GVIA, a peptide which possesses a potent blocking activity of N-type voltage-sensitive calcium channels, prevented the inhibitory response to electrical stimulation in the guinea-pig ureter, while leaving the response unaffected in the rat ureter. Conotoxin had no effect toward the inhibition produced by exogenous calcitonin gene-related peptide indicating its prejunctional site of action, demonstrated previously in the guinea-pig ureter [Maggi et al. (1990) Neurosci, Lett. 114, 203-206]. Dermorphin, an amphibian peptide with potent agonist activity on mu-type opioid receptors, inhibited the response to electrical stimulation in the guinea-pig ureter but had no effect in the rat ureter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1717886 TI - Delayed response to denervation in muscles of C57BL/Ola mice. AB - Muscle fibre areas and numbers, tetanic force, resident macrophage numbers and acetylcholine sensitivity were measured in normal and denervated soleus muscles of C57BL/Ola mice for comparison with data from outbred and other inbred strains. Serum creatine kinase levels were also measured. The muscles of normal C57BL/Ola mice had more fibres, generated more tension, had fewer resident macrophages and had a lower acetylcholine sensitivity than muscles of other strains. The normal level of serum creatine kinase was lower in C57BL/Ola mice than in Balb/c mice. Following denervation, the mean cross-sectional area of muscle fibres in Balb/c mice started to fall from day 1 but a fall was not seen in muscles from C57BL/Ola mice until day 5. The development of increased acetylcholine sensitivity was slower in the C57BL/Ola mice although the values in all mouse strains had converged by five days. The levels of serum creatine kinase also rose more slowly following denervation in C57BL/Ola mice. The differences between the data from C57BL/Ola mice and others are ascribed to the slow rate of Wallerian degeneration in that strain. The results support the view that the effects of denervation on muscle are due to both inactivity and an inflammatory effect contributed by nerve degeneration. The mutation that causes slow nerve degeneration may, however, also affect muscle directly, making it more resistant to catabolism. PMID- 1717887 TI - Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal. AB - The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals. PMID- 1717888 TI - Responses of functionally identified neurones in the dorsal horn of the cat spinal cord to substance P, neurokinin A and physalaemin. AB - The mammalian tachykinins, substance P and neurokinin A, and the non-mammalian tachykinin, physalaemin, were tested on functionally identified dorsal horn neurones in vivo. The experiments were done on cats which were anaesthetized with sodium pentobarbital or were anaemically decerebrated. Extracellular single-unit recordings were made in the lumbar spinal cord and the tachykinins were applied by iontophoresis. Each neurone was classified functionally as wide dynamic range, non-nociceptive, nociceptive specific or proprioceptive. The response to tachykinin application was determined for each neurone. Application of each of the tachykinins evoked a characteristic excitatory response which was delayed in onset, slow in developing and prolonged: physalaemin excited 99/131 neurones tested, neurokinin A excited 45/63 neurones and substance P excited 32/49 neurones. With two neurones physalaemin evoked a depression of the rate of firing, which may have been caused indirectly by excitation of a neighbouring neurone. Such depression was not elicited by either substance P or by neurokinin A. Physalaemin had a preferential excitatory effect on nociceptive neurones evoking excitation of 76/94 nociceptive neurones compared with 12/23 non nociceptive neurones (chi 2 = 7.9, 1 d.f., P = 0.005). Substance P also caused a preferential excitation, with 30/40 nociceptive neurones being excited while all of the non-nociceptive neurones (n = 7) were unaffected (chi 2 = 11.5, 1 d.f., P = 0.0007). In contrast, neurokinin A failed to have a preferential effect; 32/46 nociceptive and 9/10 non-nociceptive neurones were excited (chi 2 = 1.0, 1 d.f., P = 0.40). Comparing the proportions of nociceptive neurones excited by the different tachykinins indicated that this type of neurone was not differently sensitive to any of the three peptides (chi 2 = 3.2, 2 d.f., P = 0.20). On the other hand, non-nociceptive neurones were preferentially excited by neurokinin A and physalaemin compared with substance P (chi 2 = 13.4, 2 d.f., P = 0.001). With regard to the endogenous tachykinins the results of this study may be interpreted in the following ways. The differential excitatory effect of substance P on nociceptive neurones supports the proposed role for this peptide in the transmission specifically of nociceptive inputs at the first afferent synapse. On the other hand, as neurokinin A excited non-nociceptive as well as nociceptive neurones, there may be a functional role for neurokinin A distinct from that of substance P. PMID- 1717889 TI - Analysis of the galanin-induced decrease in membrane excitability in mudpuppy parasympathetic neurons. AB - Previously, we showed that the neuropeptide galanin hyperpolarizes and decreases membrane excitability of mudpuppy parasympathetic neurons [Konopka L. M., McKeon T. W. and Parsons R. L. (1989) J. Physiol. 410, 107-122]. We also demonstrated that membrane excitability remains depressed when the agonist-induced potential change is negated electrotonically. We hypothesized that galanin inhibits the membrane conductances associated with spike generation. However, we cannot rule out the possibility that the decreased excitability is due to a galanin-induced increase in membrane potassium conductance which reduces the effectiveness of subsequent depolarizing stimuli. Therefore, in the present study we tested, with the galanin-induced hyperpolarization negated, whether the galanin-induced increased membrane potassium conductance was responsible for the decreased excitability. The results showed that the galanin-induced decreased excitability was not dependent on the peak amplitude of the galanin-induced hyperpolarization. Furthermore, the decreased excitability occurred in cells in which there was no measurable galanin-induced hyperpolarization. Moreover, in most cells the galanin induced decrease in input resistance, measured at the peak of the hyperpolarization (3-25 mV), was less than 15% and when the hyperpolarization was negated electronically, the decrease was even less (approximately 2%). These results indicated that when the hyperpolarization was negated, the galanin induced increase in potassium conductance was not responsible for the decreased excitability. In preparations pretreated with 5 mM tetraethylammonium, galanin decreased excitability which indicated that a galanin-induced decrease in the calcium-dependent potassium current was not necessary for the decreased excitability. Galanin also decreased excitability in preparations exposed to either 1-3 microM tetrodotoxin or 100-200 microM cadmium. Following galanin application, the threshold for initiation of tetrodotoxin-insensitive spikes was shifted to more positive membrane potentials. Galanin also decreased the amplitude and hyperpolarizing afterpotential of barium spikes in the absence of any agonist-induced hyperpolarization. These observations confirmed that galanin decreased the voltage-dependent calcium conductance. In the present study, we showed that when the hyperpolarization was negated, galanin decreased excitability by shifting the threshold for spike generation regardless of whether voltage-dependent sodium or calcium currents were primarily responsible for the depolarizing component of the action potential. PMID- 1717890 TI - [Percutaneous cervical cordotomy in cancer pain. Preliminary experience]. AB - Suggestions of percutaneous cervical cordotomy in treatment of advanced cancer pain, limits in treatment of terminal patients (needing pain therapy and total assistance), results and complications of the latest 64 procedures (part of 110 operations performed) are discussed. PMID- 1717891 TI - Antibodies against hepatitis C virus (anti-HCV) in haemodialysis patients: association with hepatitis B serological markers. AB - Hepatitis C virus (HCV) seems to be the main causative agent of the parenterally transmitted non-A, non-B hepatitis and the detection of anti-HCV may be a marker of ongoing infection with this virus. This study was undertaken to determine the frequency of anti-HCV in 51 haemodialysis patients of our renal unit. In addition association of these antibodies to sex, history of blood transfusions, and duration on haemodialysis, as well as to serological markers of hepatitis B virus infection, was applied. Enzyme-linked immunosorbent assay (ELISA), were used for the detection of all serological markers. Nine of the 51 (17.6%) haemodialysis patients had anti-HCV. The presence of anti-HCV was related to male sex. Although seropositive patients were transfused more often than seronegatives, this difference is not statistically significant. The presence of anti-HCV was associated with the duration of haemodialysis. The majority of anti-HCV patients had serological markers of previous HBV infection, in contrast to seronegative patients. PMID- 1717892 TI - Abnormal alanine aminotransferase activity reflects exposure to hepatitis C virus in haemodialysis patients. AB - Prospective studies have shown that the annual incidence of non-A, non-B (NANB) hepatitis may be high in haemodialysis patients. To assess whether hepatitis C virus (HCV), the major causative agent of post-transfusion and community-acquired NANB hepatitis, has a role in the pathogenesis of liver disease in dialysed patients, we have studied the prevalence and significance of antibodies to HCV in a cohort of patients with end-stage renal disease on chronic haemodialysis treatment. Seventy-four (30%) had circulating antibodies to HCV. Statistically significant associations with the anti-HCV carrier status were duration of haemodialysis treatment, blood transfusions, and the finding of abnormally elevated ALT on retrospective analysis. In contrast, only one of 103 dialysis staff members showed transient positivity for anti-HCV, suggesting a low risk of professional exposure to HCV. These findings suggests that HCV infection is relatively frequent in haemodialysis patients and may be responsible for a significant proportion of liver disease in this clinical setting. PMID- 1717893 TI - Ruthenium red decreases capsaicin and citric acid-induced cough in guinea pigs. AB - The influence of aerosols of Ruthenium red (RR) on capsaicin- and citric acid induced cough was investigated in guinea pigs. Aerosols of RR (0.3, 1, 3%) reduced capsaicin-induced cough in dose-dependent manner. Inhalation of RR also reduced cough produced by low, (200 mM) but not high (550 mM), concentrations of citric acid. These data suggest that RR is not a specific capsaicin antagonist and that citric acid and capsaicin share a common mechanism for activation of airway C-fibers that is RR sensitive. Furthermore, high concentrations of citric acid can elicit cough through a RR-insensitive mechanism. PMID- 1717894 TI - Ruthenium red selectively antagonizes capsaicin-induced release of vasoactive intestinal polypeptide (VIP) from the human colon. AB - Ruthenium red (RR) is an inorganic dye that has shown to block the actions of capsaicin on primary sensory neurons in different animal models. The aim of this study was to assess whether RR is able to antagonize the release of vasoactive intestinal polypeptide (VIP) evoked by capsaicin in the human colon. Samples of descending colon were collected from patients undergoing colectomy for carcinoma of the colon. Tissue slices from the muscle of human colon were exposed to either 10 microM capsaicin or an isotonic high K+ medium (KCl 80 mM), in the absence or presence of 10 microM RR. Either capsaicin or high K+ produced a prompt release of VIP. RR (10 microM) completely antagonized the capsaicin-induced release of VIP from muscle of human colon. This effect was elective, since VIP release evoked by high K+ was unaffected by the presence of RR. These findings indicate that RR acts as a selective antagonist of capsaicin in human tissue and that the mechanism underlying peptide release by capsaicin is preserved across species. Multiple mechanisms leading to VIP release exist in the human colon. PMID- 1717895 TI - Localization of the Alz-50 epitope in recombinant human microtubule-associated protein tau. AB - Alz-50 is a monoclonal antibody that stains the neurofibrillary pathology of Alzheimer's disease, as well as apparently normal nerve cells that are at risk of developing neurofibrillary tangles. On immunoblots it recognizes microtubule associated protein tau and proteins of 60-68 kDa that are associated with Alzheimer's disease. We have used recombinant tau proteins expressed in E. coli to map the Alz-50 epitope to amino-terminal residues 2-10, a region common to all known human tau isoforms. A direct correspondence between immunoblots and histological staining was established by the abolition of Alz-50 staining following adsorption with recombinant tau proteins retaining amino-terminal sequences. This suggests that tau pathology represents an early event in the development of the neurofibrillary pathology of Alzheimer's disease. PMID- 1717896 TI - Anatomical and electrophysiological evidence for a glycinergic inhibitory innervation of the rat locus coeruleus. AB - Using a highly specific antiserum to glycine and a very sensitive immunohistochemical technique with streptavidin-horseradish peroxidase, we visualized for the first time a dense plexus of glycine varicose fibers in the locus ceruleus (LC) of the rat. We further demonstrated that iontophoretically applied glycine inhibits the spontaneous LC noradrenergic cell discharge and that this inhibition is blocked by co-iontophoresis of strychnine. These anatomical and electrophysiological results indicate that the rat locus ceruleus receives an inhibitory glycinergic input. PMID- 1717897 TI - Protease nexin-1 activity in cultured Schwann cells. AB - We report that protease nexin-1 (PN-1), a serine protease inhibitor known to have neurite-promoting effects, is made by Schwann cells in tissue culture. Three modalities have been used to demonstrate the presence of PN-1 in Schwann cell cultures. Immunostaining of the cultures with anti-PN-1 antibody gives positive staining over cells and matrix. Western blots of Schwann cell conditioned medium (CM) using anti-PN-1 antibody show a band that co-migrates with the PN-1 standard at 45 kDa. Biochemical assay for protease inhibitory activity shows that CM inhibits thrombin activity in a calorimetric assay. The CM-mediated inhibition of thrombin is reversed if the CM is pre-incubated with anti-PN-1 antibody. PMID- 1717898 TI - Further evidence for the absence of a descending cholinergic projection from the brainstem to the spinal cord in the rat. AB - Serotonergic and catecholaminergic neurons are known to project from the brainstem to the spinal cord. However, evidence for a bulbo-spinal projection that is cholinergic is sparse despite immunocytochemical and physiological evidence for a cholinergic influence on the cord. In this study we examined the possibility of a direct cholinergic bulbo-spinal projection in the rat using a combination of retrograde axonal tracing techniques and choline acetyltransferase immunocytochemistry. Although many cells were found to project to the cord from the brainstem, none were identified as being cholinergic, confirming previous evidence that the cholinergic innervation of the cord is intrinsic. PMID- 1717899 TI - Differential neuropeptide expression after visceral and somatic nerve injury in the cat and rat. AB - The expression of neuropeptides galanin, vasoactive intestinal polypeptide (VIP) and substance P was compared after injury to somatic (sciatic, pudendal) and visceral (pelvic) nerves. Studies in normal rats and the mutant rat 'mutilated foot' suggested that galanin increases in sensory but not sympathetic fibres after sciatic nerve injury, while VIP appears to increase in both sensory and sympathetic fibres, and substance P to decrease in sensory fibres. A direct comparison of neuropeptide changes after somatic and visceral nerve injury was made in the cat dorsal sacral spinal cord, where both pudenal (somatic) and pelvic (visceral) afferents terminate. Four weeks after pudendal nerve transection in the cat there was an increase of VIP and galanin but decrease of substance P in the dorsal sacral cord, similar to the changes in lumbar dorsal cord after sciatic nerve section in the rat. In contrast, 4 weeks after pelvic nerve transection in the cat, galanin was unchanged in the ipsilateral dorsal sacral spinal cord, whereas VIP is known to decrease markedly and substance P to remain unchanged. There is thus differential peptide expression before and after injury in somatic and visceral systems, which may be regulated in part by the target organ. We have proposed that the neuropeptide changes occur in neurons that regulate development, maintenance and repair after injury, processes that may differ in somatic and visceral systems. PMID- 1717900 TI - Demonstration of neurofibrillary tangles in parvalbumin-immunoreactive interneurones in the cerebral cortex of Alzheimer-type dementia brain. AB - The pathological changes occurring in a population of parvalbumin-like immunoreactive cerebral cortical interneurones in postmortem Alzheimer-type dementia brain tissue were determined by double staining cerebral cortical sections for parvalbumin, tau and/or calpain-like immunoreactivities. By counting the number of double immunostained cells it was determined that 4% parvalbumin and 25% of calpain-like immunoreactive cells showed evidence of tau-like immunoreactivity, indicative of neurofibrillary tangles. These results suggest that cerebral cortical parvalbumin-like immunoreactive neurones are relatively resistant to the pathological changes underlying the formation of neurofibrillary tangles associated with Alzheimer-type dementia. PMID- 1717901 TI - Modified Bielschowsky and immunohistochemical studies on senile plaques in aged dogs. AB - Aged dogs developed senile plaques (SP) in the brain which were similar to those occurring in aged humans. These SP were studied with a modified Bielschowsky stain and immunohistochemical methods using polyclonal antibodies raised against beta-protein and glial fibrillary acidic protein (GFAP). Serial sections stained by immunohistochemical and modified Bielschowsky stains showed that all areas of slight beta-protein immunoreactivity were intensely stained by the modified Bielschowsky stain. Most SP were observed in the neocortex of the cerebrum. Plaque densities were highest in the cingulate and temporal cortices. There were occasional SP in the subcortical nuclei including the caudate nucleus and putamen, and hippocampus. Based on the morphological characteristics demonstrated by the modified Bielschowsky stain, SP in the brains of the dogs were grouped into 3 types: diffuse, mature and perivascular plaques, of which diffuse plaques were predominant. Various degrees of astroglial reaction were observed in all subtypes of SP except diffuse plaques. These findings indicate that dog may serve as a model for study of the pathogenesis of SP. PMID- 1717902 TI - Time course of substance P-induced protein extravasation in the rat knee joint measured by micro-turbidimetry. AB - Intra-articular perfusion of the rat knee joint with substance P (SP) resulted in protein extravasation into the synovial cavity. This response was tested over a range of SP concentrations from 100 pM to 100 microM. The response was dose dependent from 10 nM to 10 microM, with each dose producing a steady rise of the protein content in the fluid aspirated from the synovial cavity for 12-16 min and then fell sharply again over an equivalent period despite continuing perfusion with SP. Concentrations below 10 nM gave rise to very brief increases in protein extravasation whose magnitude differed little over the range of 100 pM to 10 nM but differed significantly from control (saline) perfusion. PMID- 1717903 TI - Distribution of axons showing calcitonin gene-related peptide- and/or substance P like immunoreactivity in the sensory trigeminal nuclei of the cat. AB - Distribution of axons with calcitonin gene-related peptide (CGRP)-like and/or substance P (SP)-like immunoreactivity (LI) within the sensory trigeminal nuclei was examined in the cat before and after trigeminal rhizotomy. Axons with CGRP-LI or SP-LI were seen throughout the principal sensory trigeminal nucleus (Vp) and spinal trigeminal nuclei, including the medullary dorsal horn (MDH). They were densely distributed particularly in the dorsolateral part of the dorsal subnucleus of the Vp, ventromedial marginal zone of the ventral subnucleus of the Vp, dorsomedial and ventromedial parts of the oral spinal trigeminal nucleus, ventromedial and lateral marginal zones of the interpolar spinal trigeminal nucleus, and lamina I, outer part of lamina II and lamina V of the MDH. Most of the CGRP-LI axons exhibited SP-LI, while many SP-LI axons did not show CGRP-LI. After trigeminal rhizotomy, almost all CGRP-LI axons disappeared from the ipsilateral sensory trigeminal nuclei, while a considerable number of SP-LI axons remained intact throughout the nuclei; these SP-LI axons did not show CGRP-LI. The results indicate that CGRP-LI axons within the sensory trigeminal nuclei exhibit SP-LI and are of peripheral origin, and that SP-LI axons without CGRP-LI are of central origin. PMID- 1717904 TI - Disposition of amino acid synaptic transmitters, acetylcholine and substance P in the LM-suprageniculate nuclear complex of the cat's thalamus. AB - Immunocytochemical analysis with antibodies raised against aspartate, glutamate, gamma-aminobutyrate (GABA), choline acetyltransferase (ChAT), and substance P (SP) have allowed the transmitter characterisation and distribution of cells of the lateralis medialis-nucleus suprageniculatus (LM-SG) complex to be made at the level of the light microscope. We have found that the intranuclear distributions of aspartate and glutamate differed substantially from that of GABA, as well as there being specific and, in some cases, major differences in the respective populations of cells labelled with all three amino-acid-sensitive antibodies. ChAT-labelled elements were disposed very similarly to acetylcholinesterase (AChE)-positive subregions of the nuclear complex, while SP labelling was comparatively weak, albeit present, throughout the region. These data provide an important first step towards the further understanding of the details of the neurochemical and functional identity of the LM-SG complex. PMID- 1717905 TI - Ultrastructural localization of telencephalin, a telencephalon-specific membrane glycoprotein, in rabbit olfactory bulb. AB - Localization of a telencephalon-specific glycoprotein, telencephalin (TCLN), in the olfactory bulb of the rabbit was studied with an electron microscope. Anti TCLN antisera appeared to stain plasma membrane, Golgi apparatus and multivesicular bodies of granule cells which are local circuit interneurons in the bulb. Principal neurons, mitral and tufted cells, were not immunoreactive. No glial cells showed immunoreactivity. Thus, expression of telencephalin is specific not only to the telencephalic segment of the brain, but also to the neuronal types. PMID- 1717906 TI - Maternal serum alpha 2-macroglobulin and fetal growth retardation. AB - Maternal serum alpha 2-macroglobulin levels were measured twice, at approximately 18 and 30 weeks' gestation, in 289 pregnant women who later delivered at or after 37 weeks. Levels were elevated as early as 18 weeks' gestation in women destined to have a growth-retarded infant, and this elevation persisted through 30 weeks' gestational age. Furthermore, levels were higher in white women than black, in smokers than in non-smokers, and in thin than in heavier women. When the effect of alpha 2-macroglobulin on birth weight was evaluated in a multiple regression analysis adjusting for gestational age, race, body size, smoking, fetal sex, and a history of a low birth weight infant, high alpha 2-macroglobulin levels were associated with a statistically significant decrease in birth weight. The effect was greater in women who smoked. This relationship did not appear to be associated with differences in serum zinc or hematocrit levels. PMID- 1717907 TI - Neoadjuvant chemotherapy for cervical carcinoma. AB - Between October 1986 and August 1988, 33 previously untreated patients with locally advanced cervical carcinoma were studied to evaluate the efficacy and toxicity of a neoadjuvant chemotherapy combination consisting of cisplatin 50 mg/m2 intravenously (IV) on day 1, vincristine 1.4 mg/m2 IV on day 1, and bleomycin 25 mg/m2 IV in a 6-hour infusion on days 1-3. Cycles were repeated every 10 days for a total of three cycles, after which definitive radiation therapy (external and intracavitary) was administered. The median age was 47 years, and distribution by stages (International Federation of Gynecology and Obstetrics) was as follows: IIB, 12 subjects; IIIB, 19; and IVA, two. A multidisciplinary team conducted both staging and assessment of response to induction chemotherapy before the beginning of radiotherapy. Thirty-one women were fully evaluable for response and toxicity. No complete response was observed; seven subjects (23%) experienced a partial response, 18 (58%) had no change, and six (19%) showed progressive disease. Toxicity was mild to moderate and included nausea and vomiting, alopecia, hyperthermia, peripheral neurotoxicity, and anemia. We conclude that this regimen at this dosage and time interval produced a low number of objective regressions with a significant progression rate and is of doubtful value as neoadjuvant chemotherapy. PMID- 1717908 TI - Human papillomavirus DNA in tissue biopsy specimens of vulvar vestibulitis patients treated with interferon. AB - Thirteen women with the diagnosis of vulvar vestibulitis based on clinical symptoms, presence of vestibular tenderness on physical examination, and acetowhite changes of the vulvar vestibule were treated with intradermal injection of alpha-interferon. Biopsies of the acetowhite areas were analyzed for human papillomavirus (HPV) DNA using polymerase chain reaction amplification and dot blot hybridization. Eleven of 13 subjects harbored one of the HPV DNA types; six of these were type 16 and/or 18 and the others were unidentified. Five subjects (all HPV DNA-positive) reported resolution of symptoms with interferon therapy. Our results indicate the presence of HPV DNA in a subset of patients with vulvar vestibulitis, but its presence is not predictive of response to interferon therapy. PMID- 1717909 TI - A comparison of TC7 and 32% dextran 70 for prevention of postoperative adhesions in hamsters. AB - Prevention of postoperative adhesion formation has received considerable attention by infertility specialists. Barrier methods and hydroflotation solutions are currently used for adhesion prevention. The present study compared the efficacy of TC7 and 32% dextran 70 in a hamster model. At laparotomy, 90 golden hamsters had a lesion created on the left uterine horn and repaired with absorbable suture. The first experiment used 3-0 chromic catgut suture in 43 hamsters; the second experiment used 5-0 polyglactin 910 suture in 47 hamsters. The animals were randomly divided into a control group (N = 25) which received no treatment, a TC7-treated group (N = 33), and a 32% dextran 70-treated group (N = 32). The animals were sacrificed between 10-14 days after laparotomy and adhesions were graded (0 = no adhesions; 3 = severe adhesions). TC7 had average adhesion scores of 1.91 for chromic catgut and 2.25 for polyglactin 910; 32% dextran 70 had average adhesion scores of 1.53 and 2.00, respectively. These scores were not different from the control average adhesion score of 1.80 for both chromic catgut and polyglactin 910. We conclude that TC7 and 32% dextran 70 do not appear to be effective agents for preventing postoperative adhesions in this animal model. PMID- 1717910 TI - Diagnosis of ectopic pregnancy. AB - The diagnosis of ectopic pregnancy remains a challenge to the clinician, despite advances in sonographic and biochemical technology. Contemporary practice requires an understanding of the normal sonographic features and hormonal profiles for normal pregnancy as well as the pathogenesis of an ectopic nidation. Frequently, the diagnosis remains uncertain until laparoscopy or dilatation and curettage have been performed. PMID- 1717911 TI - Ectopic pregnancy: common and some uncommon misdiagnoses. AB - Several diseases of the peritoneal cavity may present in manners that are difficult to distinguish from ectopic pregnancy. A careful history and a thorough physical examination are paramount to making the correct diagnosis. Diseases of the gastrointestinal and urinary tracts usually present in specific manner. The difficulty arises when the clinical presentations of these disease processes are atypical. Under such circumstances, other diagnostic modalities, including serial beta-hCG testing and pelvic ultrasound, are extremely useful in distinguishing ectopic pregnancy from other diseases that occur in the abdominal peritoneal cavity. Generous use of the laparoscope facilitates diagnostic exclusion of certain entities, decreases the frequency of misdiagnosis, prevents unnecessary surgical procedures, and reduces morbidity and mortality. PMID- 1717912 TI - [Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)]. AB - Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhailov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin. PMID- 1717913 TI - The effect of transcutaneous electrical nerve stimulation on ocular pain. AB - The effect of transcutaneous electrical nerve stimulation (TENS) on pain originating in the eye was studied in 10 patients. All three patients subjected to TENS during panretinal photocoagulation reported marked reduction in pain. Three of five patients who had persistent pain following scleral buckling and/or vitrectomy reported partial or complete relief of pain during the application of TENS. One patient had equivocal relief of pain when TENS was used during retinal cryopexy; 1 patient with acute pain following paracentesis and 1 patient with persistent pain following cyclocryotherapy had no relief. Among those patients whose pain was reduced by TENS, referred brow pain was relieved more than globe pain. PMID- 1717914 TI - Visual improvement after four laser treatments to foveola for choroidal neovascular membrane. AB - With the advent of new laser technology and refined treatment techniques, it is possible that visual morbidity may be reduced by the treatment of subfoveolar choroidal neovascular membranes. This case report from our prospective study series describes an eye that required four monochromatic green subfoveolar treatments for an active choroidal neovascular membrane, which was ultimately eradicated. Even though the foveola was treated four times, the visual acuity improved from 20/200 preoperatively to 20/40 postoperatively. This case demonstrates that in carefully selected cases, even with multiple subfoveolar treatments, central visual acuity not only can be preserved, but also can actually be improved. PMID- 1717915 TI - Palatal mucosa grafts for oral implant devices. AB - Oral implants placed in the atrophic mandible often lack sufficient surrounding attached keratinized gingiva. Although mobile mucosa surrounding the implant does not necessarily need to be replaced, many oral surgeons and dentists prefer attached, keratinized gingiva, especially in cases where oral hygiene is imperfect. This article discusses a method of palatal mucosa transplantation to improve the implant recipient site. The clinical results of this method used in 30 patients are reported. PMID- 1717916 TI - Clear cell tumors of minor salivary gland origin. An immunohistochemical and ultrastructural analysis. AB - Three cases of monophasic glycogen-rich clear cell tumors of palatal gland origin were examined immunohistochemically and ultrastructurally in attempts to characterize their cellular composition. Despite their histologic resemblances, the clear cells from each case showed different immunohistochemical features. In case 1 the extensive positivity for vimentin and S-100 protein, in addition to the focal expression of actin and glial fibrillary acidic protein, strongly suggested that the clear cells were myoepithelial in nature. In contrast, the clear cells from case 2 exhibited both keratin and epithelial membrane antigen positivity, as well as ultrastructural features that suggested that they were glandular epithelial in nature. In case 3 no special markers except for keratin could be detected, indicating the less differentiated nature of the clear cells. These results show the heterogeneity of the clear cell tumor group of minor salivary glands. PMID- 1717917 TI - Usefulness of antikeratin immunoreactivity in osteosarcomas of the jaw. AB - The immunohistochemical typing of cytoplasmic intermediate filaments has proved helpful to the pathologist in classifying poorly differentiated malignant neoplasms. In general, identification of keratin-type intermediate filaments has been associated with epithelial histodifferentiation, but several exceptions to this generalization have been reported in the literature. A recent report identified false-positive immunostaining for keratin in osteosarcomas of the jaws that was attributed to cross-reactivity induced by enzyme digestion of the tissue specimens before immunostaining. Because the jaws are unique in the skeletal system because of their relatively high incidence of intraosseous epithelial neoplasms, false-positive immunoreactions for keratin could complicate differentiating sarcomatoid epithelial neoplasms from poorly differentiated osteosarcomas. To evaluate this possible pitfall in our laboratory, eight osteosarcomas of the jaws were evaluated for keratin immunostaining with polyclonal and monoclonal antibodies on tissue sections that had been enzymatically treated with protease. No immunostaining was demonstrated in these tumors. Repudiation of the usefulness of antikeratin immunohistochemistry for intraosseous jaw tumors was not confirmed with the procedures used in our laboratory. PMID- 1717919 TI - [Clinical use of interferon]. PMID- 1717918 TI - Recurrence of keratocysts and decompression treatment. A long-term follow-up of forty-four cases. AB - Recurrence was found in eight cases (18%) in a group of 44 patients (22 male) with odontogenic keratocysts treated at the Department of Oral Surgery and Oral Medicine, Odense University Hospital, from 1971 to 1983. All these recurrences were found in cysts with parakeratotic, thin, bandlike epithelium with palisade like basal cells (Forssell group la). In 12 large cysts the use of a polyethylene drainage tube implanted at cystotomy and biopsy some months before primary cystectomy resulted in considerable reduction in the cystic lumen and also in alteration of the thin, fragile cystic epithelium into thick, solid cystic epithelium with no adhesion to the adjoining structures. No recurrence was seen in these 12 patients after an observation period of between 7 and 17 years. The decompression treatment seems to reduce the tendency to recurrence of the odontogenic keratocyst, which is far more important than the advantages to the surgeon of surgical simplicity and safety, and to the patient of less discomfort and pain. PMID- 1717920 TI - [Clinical use of interferon]. PMID- 1717921 TI - [History-making epidemics]. PMID- 1717922 TI - [Amidaron-induced dermatopathy resulting from unnecessary Cordarone therapy of ventricular parasystole]. AB - The case history of a patient is reported who was treated with a variety of antiarrhythmics over a period of years because of refractory ventricular "bigeminy". As the arrhythmia did not respond to any kind of therapy, amiodarone treatment was started, which the patient received in a maintenance dose of 600 400 mg/day for 4 years. More recently, a bluish-grey hyperpigmentation of the face and other areas of the skin exposed to sunlight developed. A cutaneous biopsy of the hand revealed pigment deposits and lamellated lysosomal inclusions characteristic for amiodarone dermatopathy. The interactive, computer-assisted analysis of the ventricular ectopic activity has clearly demonstrated its innocent, parasystolic nature. The differentiation between ventricular extrasystolic and parasystolic activity is essential, because the latter arrhythmia does not require specific antiarrhythmic pharmacotherapy. PMID- 1717923 TI - Expression of p53 in premalignant and malignant squamous epithelium. AB - Using three antibodies (JG8, CM-1 and 1081) directed to the p53 protein, strong positivity was found in 16/47 (34.0%) of mucosal squamous cell carcinomas of the head and neck and in two squamous carcinoma cell lines (LICR-LON- HN5 and HN6Rr). The presence of the mutant p53 was confirmed in the cell lines as substitutions in exon 7 (codon 238, TGT greater than AGT) and exon 5 (codon 152, CCG greater than CTG) respectively. Positive staining was seen only in the undifferentiated cells and progressively lost as the cells keratinized, both in the tumour specimens and in the cell lines. Similar results were seen in areas of dysplasia, well removed from the site of the primary tumour. Staining of epidermal lesions showed positivity in 2/12 (16.6%) cases of Bowen's disease, 0/12 (0.0%) cases of solar keratosis, 0/10 (0.0%) basal cell carcinomas and in 3/20 (15.0%) squamous cell carcinomas. These results are discussed in relation to the multifocal origin of squamous cell carcinomas, the role of p53 mutations in squamous cell carcinomas from different sites and the significance of the 'basal' distribution of p53 as a normal growth regulator. The possible significance of the distribution of p53 in squamous epithelium as it relates to papilloma virus infection is also considered. PMID- 1717924 TI - High frequency of retinoic acid receptor beta abnormalities in human lung cancer. AB - One of the three human retinoic acid receptors, RAR-beta, maps to a region on the short arm of chromosome 3 frequently deleted in lung cancer. Because retinoic acid is required for normal epithelial cell growth and regulation, and loss of a retinoic acid receptor might be expected to contribute to oncogenesis, we examined RAR-beta RNA and DNA in normal lung, 33 lung cancer cell lines and nine primary lung tumors. Normally, RAR-beta is expressed as two transcripts, of sizes 3.1 kb and 2.8 kb, which are strongly induced by retinoic acid. At least 50% of the cell lines and 30% of the tumor samples show altered RAR-beta expression and/or inducibility, including examples of absence or specific loss of one of the RAR-beta transcripts. Abnormalities in the expression patterns of RAR-alpha and RAR-gamma also are found, but at a lower frequency than RAR-beta abnormalities. Southern analysis reveals alteration of the RAR-beta gene in three of the cell lines. Our data suggest that abnormalities in structure and expression of the RAR beta gene may be involved in the pathogenesis of lung cancer. PMID- 1717925 TI - Cloning and sequence analysis of the human acidic fibroblast growth factor gene and its preservation in leukemia patients. AB - Acidic fibroblast growth factor (aFGF), also known as heparin-binding growth factor 1, is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells. Several different aFGF mRNA species resulting from alternative splicing have been reported. These results suggest that the gene structure and regulatory mechanism for gene expression of aFGF are complex. As a first step toward understanding aFGF gene structure, we have isolated nine overlapping genomic DNA clones spanning 54 kbp and determined the complete DNA sequences of all three coding exons. Comparison of the nucleotide sequences between the human and bovine DNA showed that the sequence similarity extended 2400 bp downstream from the coding region. Cloning of the aFGF gene allowed us to characterize this locus in acute nonlymphocytic leukemia (ANLL) patients. A fraction of ANLL patients (10 20%) have a deletion in the long arm of chromosome 5, whose distal breakpoint overlaps the aFGF locus. Therefore, a prospective cohort of eight ANLL patients was screened using three different repetitive sequence-free probes derived from the aFGF locus. Using beta-globin gene as a normalization probe for hybridizing band intensities, we conclude that there is no allelic loss or gross rearrangement within the 40 kbp stretch of the aFGF gene locus in ANLL patients with or without 5q- deletion. Consistent with this observation, the aFGF mRNA was not detected in the mononuclear cells derived from either an ANLL patient or a normal individual as judged by the reverse transcription and polymerase chain reaction. We also identified a DNA fragment, 10.7 kbp upstream from the first coding exon of human aFGF, whose sequence is conserved in both the primate and rodent genomes. Further characterization of this fragment is likely to provide insight into the significance of this high degree of conservation. PMID- 1717926 TI - Detection of retTPC/PTC transcripts in thyroid adenomas and adenomatous goiter by an RT-PCR method. AB - A reverse transcriptase-polymerase chain reaction (RT-PCR) method was adopted for detecting transcripts specific for retTPC/PTC, an activated form of the ret proto oncogene reported to be found specifically in human papillary thyroid carcinomas. By this sensitive method retTPC/PTC transcript could be detected in about 500 fg of total RNA of TPC-1, a retTPC/PTC transcript-positive cell line. In Japanese patients, one of 11 papillary thyroid carcinomas, four of 19 follicular adenomas and one of two adenomatous goiters were positive for the transcript, indicating that the involvement of retTPC/PTC is not specific to papillary thyroid carcinomas. In several independent RT-PCR experiments using different portions of the same positive carcinoma tissue, retTPC/PTC transcript was always detected. On the other hand, the transcript was not always positive in different RNA samples from benign cases, suggesting that positive carcinomas are probably composed of clonal cell populations all expressing retTPC/PTC, whereas adenomas and adenomatous goiter comprise heterogeneous populations: both positive and negative for retTPC/PTC transcript. Activation of the ret proto-oncogene might therefore be involved in malignant conversion to thyroid carcinomas. PMID- 1717927 TI - Direct lysis of Trypanosoma cruzi: a novel effector mechanism of protection mediated by human anti-gal antibodies. AB - Anti-gal antibodies directed against a carbohydrate epitope present in mouse laminin (galactosyl alpha 1-3 galactose) and detected in high levels in sera from patients in the acute phase of Chagas disease are responsible for the direct lysis (DL) of Trypanosoma cruzi blood forms independent of either the classic or alternative complement pathways. Furthermore, the lectins Euonymus europaeus (EE) specific for the carbohydrates gal alpha 1-3 gal present a similar lytic activity against T. cruzi at the same concentrations of purified anti-gal antibodies. The DL activity was tested with several other lectins but Concanavalin A (Con A) specific for alpha-D-mannose and alpha-D-glucose was the only one also presenting lytic activity. The lectins and anti-gal antibodies lytic activity can be inhibited by specific carbohydrates suggesting that this phenomenon is related to the capability of these lectins or anti-gal antibodies to bind to a crucial surface component of T. cruzi. Moreover, the infectivity of T. cruzi blood forms to mice was clearly inactivated by incubation with acute chagasic sera (ACS) but not by ACS absorbed by immunoaffinity chromatography with mouse laminin, a strong evidence that high levels of anti-gal antibodies participate in the decline of the parasitaemia from the acute to the chronic phase in Chagas disease. PMID- 1717928 TI - Identification of phosphorylcholine containing antigens of Fasciola hepatica- successful tolerization against this epitope in experimental animals. AB - Phosphorylcholine containing antigens have been identified in the parasite Fasciola hepatica by immunoblotting and ELISA. Immunoblots probed with polyclonal and monoclonal antibodies indicate that the majority of antigens identified in both the immature and mature parasite contain both phosphorylcholine and non phosphorylcholine epitopes. One antigen of 58 kDa appears to contain predominantly PC epitopes or at least this epitope is the major one responded to by host animals. Successful immunotolerization against the epitope PC was achieved by injecting the PC conjugate, ovalbumin PC, into neonatal rats. Immunotolerization against PC resulted in a 25% reduction in worm burden upon subsequent infection with Fasciola hepatica. PMID- 1717929 TI - Molecular genetics of cystic fibrosis. PMID- 1717930 TI - Developmental rounds: an intervention strategy for hospitalized infants. AB - Infants who experience prolonged or repeated hospitalization are at risk for developmental delays. Federal mandates for early intervention include the initiation of developmental intervention during hospitalization. Developmental Rounds is a creative strategy to address the unique needs of high risk infants and their families and to promote continuity of care and community referrals for developmental intervention. PMID- 1717931 TI - Teaching nurses about neuromotor development: an evaluative study. AB - A neuromotor screening tool, The Chandler Movement Assessment of Infants Screening Test (CMAI-ST), was selected to teach nurses who care for premature infants about neuromotor development. After training with the CMAI-ST, the nurses were more adept at recognizing major components of neuromotor development. PMID- 1717932 TI - G-CSF and GM-CSF. AB - Filgrastim and sargramostim are hematopoietic growth factors that are now produced on a large scale through recombinant DNA technology. Both agents are effective in increasing blood cell counts following chemotherapy and bone marrow transplantations. Investigational work is still being conducted to determine their potential use. PMID- 1717933 TI - Pseudomonas cepacia bacteremia in children with sickle cell hemoglobinopathies. PMID- 1717934 TI - Tissue characterization by transvaginal colour Doppler for the evaluation of gynaecological tumours. 1. Review of the literature. AB - This is the first review published in Hungary about the usefulness of transvaginal colour Doppler (TVCD) in gynaecology. The history of "ultrasound tissue characterization", the conventional and colour Doppler methods are discussed as well as the pathomorphological basis of abnormal Doppler signals. The summary of tumourangiogenesis, of neovascularization and its manifestation in Doppler spectrum are given. The differences between normal and pathological Doppler signals in female small pelvis are shown. It is concluded that the routine application of TVCD will reduce the rate of false positive results and "luxury" operations from screening procedures. PMID- 1717935 TI - Tissue characterization by transvaginal colour Doppler for the evaluation of gynaecological tumours. 2. Clinical experiences. AB - Transvaginal colour Doppler was used to evaluate the blood flow patterns in pelvic vessels in a group of 315 patients including 168 with uterine tumours and 147 with adnexal masses. Neovascularization of malignant tumour tissue was successfully displayed by colour Doppler in the vases of endometrial and ovarian cancers but no abnormal blood supply was observed in the cases of early cervical cancers. A comparison between the characteristics of blood flow within benign and malignant lesions showed lower resistance index in cases of malignancy. The sensitivity, specifity, positive predictive value, negative predictive value and the diagnostic accuracy of this new method in the recognition of endometrial and ovarian cancers are higher than 95%. By the help of transvaginal colour Doppler (together with the classical methods as colposcopy, cytology etc.) it will be possible to establish of complex screening programmes for all types of gynaecological cancers. PMID- 1717936 TI - Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. AB - We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex. PMID- 1717937 TI - Drosophila snRNP associated protein P11 which specifically binds to heat shock puff 93D reveals strong homology with hnRNP core protein A1. AB - We have isolated cDNAs coding for a ribonucleoprotein of Drosophila melanogaster that is distinguished by its nearly exclusive presence at only one of the several heat shock puffs in polytene chromosomes of third instar larvae. We determined the nucleotide sequence and deduced the corresponding amino acid sequence. Its coding capacity for a 39 kDa protein is consistent with the size of the protein detected by the monoclonal antibody P11 used for expression cloning. Our results show that the P11 protein belongs to the category of hnRNP proteins of bipartite structure: the amino-terminal half contains two RNA binding domains and the carboxyterminal half is rich in glycine residues. Analysis of the genomic structure revealed two introns located within the coding portion of the gene and a third one in the 3'untranslated region. We detect two different polyadenylation sites as a result of alternative termination-polyadenylation. Its strong sequence homology with hnRNP A1 protein and its previously shown association with snRNP particles indicates that a typical hnRNP protein may also exist in a complex with snRNP particles. The P11 sequence corresponds to the Hrb87F sequence that was recently described by Haynes et al. (1) as hnRNP A related gene. PMID- 1717938 TI - U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts. AB - The U1snRNP-A (U1-A) protein was used to select specific RNA sequences from a degenerate pool of transcripts using direct RNA binding and polymerase chain reaction amplification (PCR). Sequences were randomized in loops of 10 or 13 nucleotides or as a linear stretch of 25 nucleotides. From all three structural contexts, an unpaired ten nucleotide consensus sequence was obtained. A selected stem-loop structure that resembled the natural U1-A protein binding site on loop II of U1 RNA demonstrated the highest affinity of binding in comparison with the other structural contexts. A data profile of selected sequences identified U1 RNA upon searching the GenBank database. Thus, this method was useful in determining the sequence specificity of an RNA binding protein and may complement the use of phylogenetic comparisons to predict conserved recognition elements. These findings also suggest that the evolutionary conservation of loop II of U1 RNA results from constraints imposed by protein binding. PMID- 1717939 TI - Association of a change in chromatin structure with a tissue-specific switch in transcription start sites in the alpha 2(I) collagen gene. AB - Chick embryonic sternal chondrocytes do not synthesize alpha 2(I) collagen until they are shifted by treatment with 5-bromo-2'-deoxyuridine (BrdUrd) to a fibroblastic phenotype, yet they transcribe this gene as rapidly as BrdUrd treated cells. To examine further this transcription, the DNase I hypersensitive sites were mapped in the 5' region of this gene in chondrocytes, BrdUrd-treated chondrocytes, fibroblasts and three types of non-transcribing cells. A DNase I hypersensitive site at -200 bp, previously shown to be associated with the active transcription of this gene in fibroblasts, is not present in chondrocyte chromatin. The chondrocyte alpha 2(I) gene contains, however, a novel major hypersensitive site in the DNA region corresponding to the fibroblast intron 2, near the chondrocyte-specific transcription initiation site of this gene. This novel hypersensitive site is associated with the use of this alternate start site by chondrocytes, since it is lost when BrdUrd treatment causes these chondrocytes to switch to the initiation of transcription at the fibroblast start site. The BrdUrd-treated chondrocytes contain the same alpha 2(I) hypersensitive sites as fibroblasts, except that fibroblasts have an additional, previously unreported, site at -1000 bp. PMID- 1717940 TI - A rapid microscale procedure for the simultaneous preparation of cytoplasmic RNA, nuclear DNA binding proteins and enzymatically active luciferase extracts. PMID- 1717941 TI - The butyrylcholinesterase gene (BCHE) at 3q26.2 shows two RFLPs. PMID- 1717942 TI - A highly polymorphic probe on 11p15.5: L22.5.2 (D11S774). PMID- 1717943 TI - A MspI-RFLP in the C-terminal part of the gene for desmoglein DGI (DSG). PMID- 1717944 TI - MspI and MboI polymorphisms at the DXS704 locus. PMID- 1717945 TI - A deletion polymorphism in the human alpha-2-macroglobulin (A2M) gene. PMID- 1717946 TI - Alternative tertiary structure attenuates self-cleavage of the ribozyme in the satellite RNA of barley yellow dwarf virus. AB - A self-cleaving satellite RNA associated with barley yellow dwarf virus (sBYDV) contains a sequence predicted to form a secondary structure similar to catalytic RNA molecules (ribozymes) of the 'hammerhead' class (Miller et al., 1991, Virology 183, 711-720). However, this RNA differs from other naturally occurring hammerheads both in its very slow cleavage rate, and in some aspects of its structure. One striking structural difference is that an additional helix is predicted that may be part of an unusual pseudoknot containing three stacked helices. Nucleotide substitutions that prevent formation of the additional helix and favor the hammerhead increased the self-cleavage rate up to 400-fold. Compensatory substitutions, predicted to restore the additional helix, reduced the self-cleavage rate by an extent proportional to the calculated stability of the helix. Partial digestion of the RNA with structure-sensitive nucleases supported the existence of the proposed alternative structure in the wildtype sequence, and formation of the hammerhead in the rapidly-cleaving mutants. This tertiary interaction may serve as a molecular switch that controls the rate of self-cleavage and possibly other functions of the satellite RNA. PMID- 1717948 TI - MspI RFLP in the L1 CAM gene in Xq28. PMID- 1717947 TI - Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. AB - Consistent with the fact that the clinical disorder cystic fibrosis (CF) is manifested on epithelial surfaces, active transcription of the CF transmembrane conductance regulator (CFTR) gene and CFTR mRNA transcripts are detectable in a variety of epithelial cells, suggesting CFTR gene expression might be epithelial cell-specific. However, analysis of the CFTR gene promoter suggests it is a housekeeping gene, implying more widespread expression than only in epithelial cells. To evaluate the latter hypothesis, various human cells of non-epithelial origin, including lung fibroblasts, U-937 histiocytic lymphoma cells, K-562 erythroleukemia cells, HL-60 promyelocytic leukemia cells as well as freshly isolated blood lymphocytes, neutrophils, monocytes, and alveolar macrophages were examined for CFTR gene expression. Although Northern analysis failed to show CFTR mRNA transcripts in these cells, amplification of mRNA (after conversion to cDNA) by polymerase chain reaction combined with Southern analysis demonstrated the presence of CFTR mRNA transcripts at low levels in all cells evaluated except HL 60 cells. Comparative quantitative analysis showed fibroblasts contained 200-400 fold less CFTR mRNA transcripts than the T84 and HT-29 colon carcinoma epithelial cell lines, but had similar levels of CFTR transcripts to those of other epithelial cell lines. Nuclear transcription run-on analyses demonstrated very low level CFTR gene transcription in fibroblasts and U-937 cells, similar to that of other epithelial cells, but lower than the T84 and HT-29 colon carcinoma cell lines. Interestingly, while chromatin DNA of fibroblasts had no DNase I hypersensitivity sites in the 5' flanking region of the CFTR gene, HT-29 chromatin DNA exhibited four DNase I accessible sites in the same region, suggesting that these sites may be related to more active transcription of the CFTR gene in the intestinal epithelial cells than in fibroblasts. PMID- 1717949 TI - An MspI polymorphism at the MX1 locus in 21q22.3. PMID- 1717950 TI - A polymorphism in intron 20 of the CFTR gene. PMID- 1717951 TI - Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord. AB - Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons. PMID- 1717952 TI - Effects of potent bombesin antagonist on exocrine pancreatic secretion in rats. AB - Recent synthesis of specific, potent bombesin receptor antagonists allows examination of the role of bombesin-like peptides in physiological processes in vivo. We characterized effects of [D-Phe6]bombesin(6-13)-methyl-ester (BME) on pancreatic enzyme secretion stimulated by the C-terminal decapeptide of gastrin releasing peptide (GRP-10), food intake, and diversion of bile-pancreatic juice in rats. In isolated pancreatic acini, BME had no agonistic effects on amylase secretion but competitively inhibited responses to GRP-10, yielding a pA2 value of 8.89 +/- 0.19. In conscious rats with gastric, jugular vein, bile-pancreatic, and duodenal cannulas, basal enzyme secretion (bile-pancreatic juice recirculated) was not affected by the antagonist. Maximal amylase response to GRP 10 (0.5 nmol/kg/h) was inhibited dose dependently by BME, reaching 97% inhibition at a dose of 400 nmol/kg/h. The dose response curve of amylase secretion stimulated by GRP-10 was shifted to the right by 40 nmol/kg/h BME, but maximal amylase response was unaltered, suggesting competitive inhibition in vivo. Liquid food intake and bile-pancreatic juice diversion caused substantial increases in amylase secretion; neither response was altered during administration of 400 pmol/kg/h BME. These results demonstrate that BME is a potent, competitive antagonist of pancreatic responses to bombesin-like peptides in vitro and in vivo. Lack of effect of BME on basal pancreatic secretion or responses to liquid food intake or diversion of bile-pancreatic juice in rats suggests that endogenous bombesin-like peptides do not act either directly or indirectly to mediate these responses. PMID- 1717953 TI - Effects of synthetic human pancreastatin on pancreatic secretion and blood flow in rats and dogs. AB - Effects of synthetic human pancreastatin-52 and human pancreastatin-29 on pancreatic secretion and blood flow were examined in rats and dogs. Synthetic human pancreastatin-52 and human pancreastatin-29 were equally potent in suppressing the release of amylase stimulated by cholecystokinin in rats in vivo. However, neither human pancreastatin-52 nor human pancreastatin-29 altered basal and cholecystokinin-stimulated amylase release from isolated dispersed rat pancreatic acini. In studies in dogs, human pancreastatin-29 suppressed releases of amylase and protein stimulated by cholecystokinin, but did not alter pancreatic blood flow. These results suggest that the inhibitory effects of pancreastatin on pancreatic secretion do not involve a direct action on pancreatic acinar cells nor alteration of pancreatic blood flow. Pancreastatin probably is important in regulating exocrine pancreatic secretions as well as endocrine pancreatic secretions. PMID- 1717954 TI - Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion. AB - A novel neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), which has been isolated from ovine hypothalami, shows 68% homology with vasoactive intestinal peptide (VIP). Since VIP stimulates amylase secretion from the pancreas, we investigated the effect of PACAP and VIP on rat pancreatic exocrine secretion after intravenous injections of PACAP-27, PACAP-38, or VIP at doses of 2.5, 5 or 10 nmol/kg. Results showed: 1) Bolus injection of PACAP stimulated pancreatic amylase and protein secretions in a dose-dependent manner; and 2) Stimulation of amylase secretion with 10 nmol/kg of PACAP-27 was greater than that induced with the same dose of VIP or PACAP-38 (p less than 0.05). PMID- 1717955 TI - Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase. AB - Calcitonin gene-related peptide (CGRP) and substance P (SP) are released from sensory nerves upon exposure to irritating stimuli. Neutral endopeptidase (NEP), a membrane-bound peptidase, cleaves many peptides including SP, thereby limiting their biological actions. Recombinant NEP cleaved CGRP1 approximately 88-fold less rapidly than it cleaved SP. The slow cleavage by NEP of CGRP compared to SP suggests that this enzyme is likely to have weaker physiologic effects on CGRP than have been demonstrated for SP. PMID- 1717956 TI - Substance P and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization and effects on uterine contractility. AB - Immunoreactivity to the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, SP immunoreactivity (SP-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns, with nerves immunoreactive for CGRP being the more numerous. Immunocytochemical double labeling studies revealed SP coexisted with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., SP-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. SP (10(-4) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP(1-37) and CGRP(8-37) had no effect on basal uterine tension. While CGRP(1-37) (10(-7) M) reduced SP-stimulated (10(-5) M) uterine contraction by 56%, CGRP(8-37) had no effect on SP-stimulated uterine contraction. However, CGRP(8-37) (10(-6) M) significantly reduced the ability of CGRP(1-37) (10(-7) M) to inhibit SP-stimulated uterine contraction. These results demonstrate that SP- and CGRP-I are present in, and coexist in some uterine nerves, presumably afferent nerves. The first 7 amino acids are necessary for the inhibitory effect of CGRP(1-37) on stimulated uterine contraction. In addition, CGRP(8-37) acted as an antagonist to this inhibitory action. SP and CGRP could be coreleased from afferent fibers in an "efferent fashion" and influence uterine contractility. SP having a contractile effect and CGRP having a relaxing effect. PMID- 1717957 TI - Corticotropin-releasing factor inhibition of substance P-induced vascular leakage in rats: possible sites of action. AB - Substance P (SP), 40 micrograms/kg SC, induced protein leakage in the skin, muscle, trachea and esophagus of the anesthetized rat as measured by Monastral blue B labeling of small blood vessels. CRF, 30 micrograms/kg SC, injected 30 min before SP, decreased the SP-induced dye leakage. To locate where CRF might act, autoradiographic studies of [125I]-CRF binding to esophageal segments were conducted and displaceable binding of [125I]-CRF to submucosal elements in the esophageal epithelium were revealed, suggesting that CRF acts on selective sites to reduce vascular leakage. PMID- 1717958 TI - Interactions between neuropeptides and dexamethasone on the mitogenic response of rabbit spleen lymphocytes. AB - The interactions between vasoactive intestinal peptide (VIP), substance P (SP), a somatostatin analog (SMS 201-995) and dexamethasone have been investigated on the Con A mitogenic response of rabbit spleen cells. The neuropeptide regulatory effects appeared to be time dependent: when added with the Con A mitogen, they inhibited (VIP) or did not modulate (SMS and SP) the rabbit lymphocyte proliferation and did not change the inhibitory effect induced by a dexamethasone preincubation. When added 18 h before the mitogen, they all induced an increase of the proliferative response at high concentration. The mitogenic response observed when adding dexamethasone to lymphocytes previously preincubated in the presence of neuropeptides was not different from control response except with SMS 10(-10) M. The similar lymphocyte responses obtained whatever the neuropeptide suggested that the immunomodulatory effect induced by a neuropeptide preincubation might be mediated by the induction of common effector(s). PMID- 1717959 TI - Expression of glycoprotein hormones and intracytoplasmic distribution of cytokeratin in growth hormone-producing pituitary adenomas. AB - Sixteen growth hormone (GH)-producing pituitary adenomas were studied for the expression of glycoprotein hormone subunits and cytokeratin by light microscopic immunohistochemistry. Cytokeratin immunoreactivity was demonstrated in all adenomas, but its intracytoplasmic distribution showed two distinct patterns; a prominent, dot-like pattern and a diffuse, perinuclear pattern. Seven adenomas (type 1) were exclusively composed of cells with cytokeratin in a dot-like pattern, whereas 9 adenomas (type 2) comprised of cells with cytokeratin of perinuclear distribution. The expression of alpha-subunit of glycoprotein hormone was significantly different between the two types of adenomas; 8 of 9 adenomas of type 2 contained many alpha-subunit immunoreactive cells but none of type 1 adenomas showed any immunoreactivity. Only a small number of adenoma cells were positive for beta-subunit of thyrotropin stimulating hormone in 3 adenomas of type 2. beta-subunits of follicle stimulation hormone and luteinizing hormone were negative in all adenomas. These findings suggest that the expression of glycoprotein hormone subunits in GH-producing adenomas may be closely linked to their types distinguishable by the cytokeratin distribution pattern. PMID- 1717960 TI - Further immunohistochemical study of Crooke's hyalin. AB - For analysis of the cytokeratin (CK) of Crooke's cells, 28 post-mortem pituitary glands with unequivocal Crooke's hyaline change were investigated immunohistochemically using monoclonal antibodies for CK subfamilies. Crooke's hyalin was positive for CK 8 [molecular weight 52.5 kilodalton (KD)] and 18 (45 KD) but negative for skin-, cornea- and esophageal-typed CKs. PMID- 1717961 TI - Immunohistochemical demonstration of insulin-like growth factor I (IGF-1) in normal and pathological human pituitary glands. AB - The authors have studied the presence and distribution of Insulin-Like-Growth Factor-1 (IGF-1) in 5 autopsied normal and 20 surgically removed human pituitary adenomas, employing a peroxidase-anti-peroxidase method. IGF-1 could be demonstrated in all cases, with variation of cells immunostaining from 60% in normal pituitary gland to 100% in corticotroph cell adenoma. PMID- 1717962 TI - Immunohistochemical studies of chromogranins (A and C) in pituitary adenomas. AB - Expression of Chromogranin A and C was examined immunohistochemically on 45 surgically obtained pituitary adenomas. Positive rate of chromogranin A was 40.0% and chromogranin C was positive in 8.0% of 45 pituitary adenomas. Chromogranin A was expressed frequently in adenomas in which at least one of the FSH alpha, FSH beta, LH beta and TSH beta subunits was positive. It also expressed frequently in non-functioning adenomas. The positive rate of chromogranin A was low in GH or PRL positive adenomas. These findings suggest that chromogranin could be a parameter of the functional differentiation of the pituitary adenomas. PMID- 1717963 TI - An ACTH and FSH producing invasive pituitary adenoma with Crooke's hyalinization. AB - We report on a patient with ACTH and FSH producing invasive pituitary adenoma complaining of cutaneous pigmentation. Elevations in plasma ACTH, beta-endorphin and cortisol levels as well as urinary 17-OHCS and cortisol excretion were found. Serum FSH concentration was just within the upper limit of the normal range, whereas serum LH level was reduced and alpha-subunit level was normal. Roentogenographic examination showed an almost complete loss of sellar floor and destruction of the posterior clinoids and dorsum sella. CT scan and MRI demonstrated an enlarged tumor invasion of the clivus and its extension to the sphenoid sinus. After subtotal removal of the large pituitary tumor, serum cortisol and plasma beta-endorphin levels as well as plasma ACTH concentrations returned to normal and serum FSH levels also remarkably decreased. Histologically, the tumor corresponded to a chromophobe, slightly PAS positive adenoma. These tumor cells exhibited positive immunostaining with antibody to ACTH (1-24), beta-LPH, beta-endorphin and FSH, while immunostaining of the adenoma cells was negative for LH, TSH, GH and prolactin. The immunogold technique also demonstrated ACTH and FSH particles in the secretory granules in the cytoplasm of the adenoma cells. Some of the tumor cells disclosed Crooke's hyalinization and type I microfilament occupied most of the cytoplasm. In the present study, a very rare case of ACTH and FSH producing invasive pituitary adenoma is reported. PMID- 1717964 TI - Culture of prostatic epithelial cells from ultrasound-guided needle biopsies. AB - A protocol which was developed for the culture of epithelial cells from radical prostatectomy specimens was slightly modified to permit the culture of cells from ultrasound-guided prostatic needle biopsies. The collagenase digestion step of the standard protocol was omitted, and biopsies were simply minced and allowed to attach to collagen-coated dishes in serum-free medium. Cell outgrowths from biopsies were free of fibroblasts, and expression of keratin, prostate specific antigen, and prostatic acid phosphatase was maintained in vitro. The establishment of a bank of frozen cells from primary cultures permitted repetitive studies with individual cell strains, which could be serially passaged and were capable of clonal growth. The ability to derive cultures from biopsies will facilitate the biological characterization of cells from primary prostate tumors of high malignant grade, which are not commonly available from radical prostatectomy specimens. PMID- 1717965 TI - Relation of endocrine-paracrine cells to cell proliferation in normal, hyperplastic, and neoplastic human prostate. AB - The relative distribution pattern of the pan-endocrine marker Chromogranin A (Chr A) and the proliferation-associated Ki-67 antigen was investigated in 20 prospectively sampled prostatectomy specimens. In cryostat sections, the Chr A immunoreactivity showed evidence of endocrine differentiation in all 15 prostatic adenocarcinomas. Nine tumors displayed a weak, 5 a moderate, and 1 adenocarcinoma a strong endocrine differentiation. These findings highlight the importance of endocrine differentiation in prostate malignancy that histologically resembles ordinary adenocarcinomas. The simultaneous demonstration of Ki-67 and Chr A revealed that in normal, hyperplastic, and neoplastic prostate tissue Chr A positive cells were preferentially situated in proximity to Ki-67-labeled cells. This relative distribution pattern of both markers may indicate that endocrine cells are involved in controlling cell proliferation through a paracrine hormonal mechanism. However, an obvious correlation was not found between the degree of endocrine differentiation and proliferative activity in prostatic adenocarcinomas. Furthermore, a coexpression of Ki-67 and Chr A in the same (tumor) cells was not observed suggesting that the endocrine phenotype is only expressed in the G0 phase of the cell cycle as well as in normal, hyperplastic, and neoplastic conditions. PMID- 1717966 TI - [Immunoregulatory function of macrophages]. AB - This review describes the role of macrophages in antigen processing and cell-cell interactions. The function of macrophages as secretory cells also is discussed. PMID- 1717967 TI - [Peroxisomes and peroxisomal diseases]. AB - This paper is on peroxisomes ("microbodies") dealing with various aspects of their physiological significance including the central question of biogenesis of peroxisomes. Attention is also focused on how peroxisomes are involved in human pathology and the so-called peroxisomal diseases are described. PMID- 1717968 TI - Monokines in growth and development. AB - Stimulation of the immune system results in a series of metabolic changes that are antagonistic toward growth. Monokines, including interleukin-1, tumor necrosis factor, and interleukin-6, are released from cells of the monocyte macrophage lineage after recognition of immunogens. They appear to mediate homeorhetic response, which alters the partitioning of dietary nutrients away from growth and skeletal muscle accretion in favor of metabolic processes which support the immune response and disease resistance. These alterations include 1) decreased skeletal muscle accretion due to increased rates of protein degradation and decreased protein synthesis; 2) increased basal metabolic rate resulting in increased energy utilization; 3) use of dietary amino acids for gluconeogenesis and as an energy source instead of for muscle protein accretion; 4) synthesis by the liver of acute phase proteins; 5) redistribution of iron, zinc, and copper within the body due to the hepatic synthesis of metallothionein, ferritin, and ceruloplasmin; (6) impaired accretion of cartilage and bone; and 7) release of hormones such as insulin, glucagon, and corticosterone. These monokines also influence the differentiation of cells. Tumor necrosis factor suppresses the differentiation of myoblasts and adipocytes whereas the chicken monokine myelomonocytic growth factor induces the differentiation of granulocytes. PMID- 1717969 TI - First-trimester maternal serum unconjugated oestriol and alpha-fetoprotein in fetal Down's syndrome. AB - Maternal sera (MS) taken from 1396 women prior to chorionic villus sampling at 9 12 menstrual weeks were assayed for unconjugated oestriol (uE3) and alpha fetoprotein (AFP). Median levels increased by 41 and 26 per cent per week respectively in normal pregnancies. There were 32 pregnancies with a chromosome abnormality. The median MS uE3 and AFP were 0.73 and 0.75 multiples of the median (MoM) respectively in the ten cases of Down's syndrome (DS) but not decreased in the other abnormalities. These results suggest that both uE3 and AFP may be useful in identifying DS in the first trimester. Additional prospective studies are needed to confirm these findings. PMID- 1717970 TI - Anxiety in women with low maternal serum alpha-fetoprotein screening results. AB - The purpose of this study was to measure anxiety in pregnant women who had low maternal serum alpha-fetoprotein (MSAFP) screening test levels, received genetic counselling and chose to undergo amniocentesis for fetal chromosome analysis. Their anxiety levels were compared with the levels in women undergoing amniocentesis because of advanced maternal age. The results indicate a higher level of anxiety in women with low alpha-fetoprotein (AFP) levels. PMID- 1717971 TI - Screening for Down's syndrome in older women based on maternal serum alpha fetoprotein levels and age: preliminary results. AB - We report the results of screening for Down's syndrome (DS) in older women using published rate schedules based on maternal serum alpha-fetoprotein (MSAFP) and age. Five hundred and seventeen patients aged 35 years and older, who were referred for a mid-trimester genetic amniocentesis, were first tested for MSAFP and then underwent an amniocentesis. Individual risks for DS, combining MSAFP and age, were derived using three different published rate schedules. Theoretical selection for amniocentesis was made using the cut-off level of the average collective risk for a 35-year-old woman (1:380 at live birth or 1:270 at amniocentesis). Six affected pregnancies (five with DS and one with trisomy 18), which were diagnosed prenatally, were all found to be at a higher risk than the specified cut-off. These cases would have been diagnosed in any event, using any of the published rate schedules. According to these rate schedules, between 39 and 45 per cent of the patients would be in the lower risk group and therefore would have been counselled not to undergo amniocentesis. Further studies should be conducted in order to reach conclusive screening policies for DS in older women. PMID- 1717972 TI - [Circulating prostate cancer specific antigens in benign hypertrophy and localized cancer of the prostate]. AB - In benign hypertrophy of the prostate (88 patients) there is a good correlation between the circulating specific prostate antigen (SPA) and the size of the prostate or of the adenoma. This correlation disappears with an adenocarcinoma where tumor volume increases (46 patients). Used as a screening test for cancer, serum levels of SPA, with a threshold value of 2.5 ng/ml, has a 91 per cent sensitivity and a 37 per cent specificity. At 15 ng/ml the sensitivity is 50 per cent and the specificity is 85 per cent. Alone, the SPA level is a poor diagnostic tool: using the low threshold (2.5 ng/ml) leads to needle biopsy in most all benign hypertrophies; with the high threshold (15-23 ng/ml), 50 per cent of the localized cancers go undetected. However, for a level greater than 15 ng/ml, SPA is an argument strongly suggesting prostate adenocarcinoma. The capacity of benign hypertrophy of the prostate to "secrete" SPA is 5 times greater than the normal peripheral prostate; the capacity of cancer is 20 times greater than that of the adenoma. Individual variability in serum levels of SPA, expressed per cm3 of prostate tissue is too great to give a precise interpretation as a function of volume. PMID- 1717973 TI - [A case of Whipple's disease revealed by an isolated inflammatory syndrome]. PMID- 1717974 TI - Comparative investigations of the morphology and chemical composition of the eggshells of Acanthocephala. I. Macracanthorhynchus hirudinaceus (Archiacanthocephala). AB - Eggshells of Macracanthorhynchus hirudinaceus (Archiacanthocephala) were investigated for their fine structure as well as their chemical composition. The acanthor larvae are surrounded by four eggshells (E1-4) separated by interstices of low electron density (G1-4). As these envelopes are secreted in different sequences and are reinforced to different degrees, their appearance varies throughout development. The outermost eggshell (E1) of this species has a tripartite appearance; it contains neither chitin nor keratin. Keratin appears in E2 and E3. It was localized electron microscopically using anti-keratin and, for the first time by fluorescence microscopy with the bromobimane reaction. Keratin occurs in two forms: in the second envelope (E2) it consists of twisted struts of filaments, whereas in the innermost sublayer of the third envelope (E3) it shows a conspicuous cross-striation; in the first and second sublayers of E3, neither keratin nor a discernible structure is present. Chitin occurs in the innermost layer (E4). The interstices G1, G3 and G4 seem to contain glycoproteins, whereas interstice G3 seems to contain some type of carbohydrate. After the extraction of proteins including keratin with sodium dodecyl sulfate (SDS) and dithiothreitol (DTE) only layers E1 and E4 remained. PMID- 1717975 TI - Determination of the primary structure of an alpha-amylase inhibitor from wheat kernel by Edman degradation and fast atom bombardment mass spectrometry. AB - The primary structure of an alpha-amylase inhibitor (coded 0.39) from wheat kernel was determined by fast atom bombardment mass spectrometry and Edman degradation. The sequence is similar to an extent of 97% compared to the other major component of the monomeric isoinhibitor family coded 0.28. The differences consist of the substitution of a single residue and the deletion of two residues in inhibitor 0.39. Neither heterogeneity nor polymorphism were observed in the primary structures of the inhibitors. PMID- 1717976 TI - Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins. AB - The integrin family of cell adhesion receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound influences on cell behavior, it seems likely that integrins transduce biochemical signals across the cell membrane. The nature of these putative signals has, thus far, remained elusive. Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-beta 1 integrin monoclonal antibody for 30 min on ice followed by incubation at 37 degrees C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115- to 130-kDa complex of proteins termed pp130. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, pp130 showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal pp130 phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. pp130 phosphorylation depended on the amount of anti-integrin antibody present. Additionally, the tyrosine phosphorylation of pp130 showed specificity since it was stimulated by antibodies to the integrin alpha 3 and beta 1 subunits but not by antibodies to other integrin alpha subunits or to nonintegrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that pp130 is not itself a beta 1 integrin. It is postulated, therefore, that the integrin-stimulated tyrosine phosphorylation of pp130 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior. PMID- 1717977 TI - Coexpression of alpha 1 with putative beta 3 subunits results in functional Na+/K+ pumps in Xenopus oocytes. AB - The active Na+/K+ pump is composed of an alpha and a beta subunit. Until now, three putative isoforms of the beta subunit have been identified that share sequence similarity. We have expressed the beta 1 and beta 3 isoforms of Xenopus laevis Na+/K(+)-ATPase in Xenopus oocytes to compare functional properties of the Na+/K+ pump, including either of these two isoforms. Na+/K+ pump current, estimated as K(+)-induced outward current in voltage-clamped oocytes, was doubled by coexpression of alpha 1 subunits with either isoform of the beta subunit compared to expression of alpha 1 subunits alone. The kinetics of activation by external K+ and the voltage dependence of the electrogenic activity of the Na+/K+ pump were similar with both beta isoforms, indicating that both beta 1 and beta 3 isoforms can support expression at the oocyte surface of an active Na+/K+ pump with similar functional properties. PMID- 1717978 TI - Retroviral-like element in a marine invertebrate. AB - Retroviral-like elements (RL elements) include retroviruses and long terminal repeat (LTR)-containing retrotransposons. We report the presence of sea urchin RL elements (termed SURL) in eight species of sea urchins and find that these RL elements belong to several subfamilies. The complete DNA sequence of one SURL element in Tripneustes gratilla is 5266 base pairs long, including 254-nucleotide long identical long terminal repeats (LTRs). It contains a single open reading frame nearly 4 kilobases long including the gag and pol genes. Comparison of conserved DNA sequences of RL elements from different sea urchin species indicates that active elements have been inserting copies into echinoid genomes for at least 200 million years. PMID- 1717979 TI - Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. AB - Gap junctions are formed by a family of homologous proteins termed connexins. Their channels are dodecamers, and homomeric forms differ in their properties with respect to control by voltage and other gating stimuli. We report here the properties of coupling from expression of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by injection of DNA oligonucleotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junctions whose conductance exhibited voltage dependence with unusual characteristics suggestive of two gating mechanisms. Junctional conductance (gj) was increased to a small degree by depolarization and decreased by hyperpolarization of either cell in a coupled pair, indicating dependence on the potential between the inside and outside of the cells (Vi-o). These changes were fast compared with the resolution of their measurement (ca. 10 ms). On a slower timescale, large transjunctional potentials (Vj) of either sign caused a more substantial decrease in conductance similar to that previously reported for several other gap junctions. Homotypic junctions formed of another connexin, Cx32, exhibited a similar slow dependence on Vj but no dependence on Vi-o. In contrast, heterotypic junctions between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, which depended, however, on Vj, such that gj increased with relative positivity on the Cx26 side and decreased with relative negativity on the Cx26 side. There was also a large slow decrease in gj in response to Vj for relative positivity on the Cx26 side but not for Vj of the opposite sign. These data indicate that properties of the hemichannels contributed by the two connexins in the heterotypic case were changed from their properties in homotypic junctions. The fast change in gj may involve a mechanism analogous to that at fast rectifying electrical synapses. Experiments in which oocytes expressing Cx32 were paired with oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes expressing both connexins, thereby confirming their potential relevance in vivo, where the same coupled cells are known to express both proteins. PMID- 1717980 TI - Dual activation of a sex pheromone-dependent ion channel from insect olfactory dendrites by protein kinase C activators and cyclic GMP. AB - Olfactory transduction is thought to take place in the outer dendritic membrane of insect olfactory receptor neurons. Here we show that the outer dendritic plasma membrane of silkmoth olfactory receptor neurons seems to be exclusively equipped with a specific ion channel activated by low concentrations of the species-specific sex pheromone component. This so-called AC1 channel has a conductance of 56 pS and is nonselectively permeable to cations. The AC1 channel can be activated from the intracellular side by protein kinase C activators such as diacylglycerol and phorbolester and by cGMP but not by Ca2+, inositol 1,4,5 triphosphate, or cAMP. Our results imply that phosphorylation of this ion channel by protein kinase C could be the crucial step in channel opening by sex pheromones. PMID- 1717981 TI - Characterization of a neu/c-erbB-2 protein-specific activating factor. AB - The neu oncogene encodes a tyrosine kinase with growth factor receptor-like properties. A neu protein-specific activating factor (NAF) was partially purified from medium conditioned by the transformed human T-cell line ATL-2. NAF was able to stimulate the tyrosine-specific kinase activity of the neu protein (p185neu), induce dimerization and internalization, and increase the growth of cells bearing the neu protein. The effects of NAF were mediated by an interaction with the p185neu extracellular domain. NAF had no effect on the epidermal growth factor receptor kinase activity and no effect on cells that express that receptor. Further analysis of NAF and of other recently described neu protein-activating activities should help clarify the role of the neu protein in cell growth and transformation. PMID- 1717982 TI - Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6. AB - Oncostatin M (OSM), a glycoprotein of Mr approximately 28,000 produced by activated monocyte and T-lymphocyte cell lines, was previously identified by its ability to inhibit the growth of cells from melanoma and other solid tumors. We have detected significant similarities in the primary amino acid sequences and predicted secondary structures of OSM, leukemia-inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). Analysis of the genes encoding these proteins revealed a shared exon organization, suggesting evolutionary descent from a common ancestral gene. Using a panel of DNAs from somatic cell hybrids, we have shown that OSM, like LIF, is located on human chromosome 22. We have also demonstrated that OSM has the ability to inhibit the proliferation of murine M1 myeloid leukemic cells and can induce their differentiation into macrophage-like cells, a function shared by LIF, G CSF, and IL-6. We propose that OSM, LIF, G-CSF, and IL-6 are structurally related members of a cytokine family that have in common the ability to modulate differentiation of a variety of cell types. PMID- 1717983 TI - Acidic fibroblast growth factor promotes vascular repair. AB - Intravascular injury to arteries can result in thickening of the intimal smooth muscle layer adjacent to the lumen by migration and proliferation of cells from the underlying medial smooth muscle layer accompanied by deposition of extracellular matrix. This pathological response, which decreases lumen diameter, might, in part, be the result of the access of smooth muscle cells to plasma and platelet-derived growth factors as a consequence of denudation of the overlying confluent monolayer of vascular endothelial cells. Injured rat carotid arteries were treated by i.v. administration of acidic fibroblast growth factor, a heparin binding protein that is chemotactic and mitogenic for vascular endothelial cells. The growth factor treatment resulted in dose-dependent inhibition of intimal thickening with parallel promotion of endothelial regeneration over the injured area. Therefore, acidic fibroblast growth factor might be efficacious in the prevention of restenosis caused by intimal thickening following angioplasty in humans. PMID- 1717984 TI - Mechanistic aspects of the opposing effects of monoclonal antibodies to the ERBB2 receptor on tumor growth. AB - The ERBB2 (also called HER2, neu, and c-erbB-2) gene product, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. An antibody directed to the rat oncogenic receptor has been previously shown to have an antitumor effect in model systems. In an attempt to extend this observation to the protooncogenic human receptor and also to understand the underlying mechanism, we generated a panel of monoclonal antibodies specific to the extracellular portion of the ERBB2 protein. The effects of the antibodies on tumor growth were compared with their cellular and biochemical actions in vitro. Surprisingly, opposing in vivo effects were observed: although some antibodies almost completely inhibited the growth in athymic mice of transfected murine fibroblasts that overexpress Erbb-2, other antibodies either accelerated tumor growth or resulted in intermediate responses. When tested on cultured human breast carcinoma cells or ERBB2 transfectants, the tumor-stimulatory antibody was found to induce significant elevation of tyrosine phosphorylation of the ERBB2 protein. In contrast, only partial correlation was observed between the capacity to restrict tumor growth and the effects of the antibodies on receptor degradation and cellular proliferation in vitro. This suggests that the antitumor antibodies affect both receptor function and host tumor interactions. Our results may help establish experimental criteria for the selection of specific antibodies for use either alone or in conjunction with other molecules as pharmacological antitumor agents. PMID- 1717985 TI - Mutation of the KIT (mast/stem cell growth factor receptor) protooncogene in human piebaldism. AB - Piebaldism is an autosomal dominant genetic disorder characterized by cogenital patches of skin and hair from which melanocytes are completely absent. A similar disorder of mouse, dominant white spotting (W), results from mutations of the c Kit protooncogene, which encodes and receptor for mast/stem cell growth factor. We identified a KIT gene mutation in a proband with classic autosomal dominant piebaldism. This mutation results in a Gly----Arg substitution at codon 664, within the tyrosine kinase domain. This substitution was not seen in any normal individuals and was completely linked to the piebald phenotype in the proband's family. Piebaldism in this family thus appears to be the human homologue to dominant white spotting (W) of the mouse. PMID- 1717986 TI - Galanin inhibits a dihydropyridine-sensitive Ca2+ current in the RINm5f cell line. AB - Mechanisms of action of the neuropeptide galanin, a putative neuromodulator in the central and peripheral nervous systems, have been evaluated extensively in insulin-secreting cells isolated from pancreas and cell lines derived from pancreatic tumors. Galanin inhibits insulin secretion from these cells through several mechanisms, including activation of ATP-dependent K+ channels and inhibition of adenylyl cyclase leading to a decrease in cAMP. Here we report that galanin also inhibits a dihydropyridine-sensitive Ca2+ current. Both electrophysiological actions by galanin would result in less Ca2+ entry, as the action to increase K+ current would hyperpolarize the cells and the decrease in voltage-gated Ca2+ current would decrease Ca2+ influx at depolarized potentials where these channels are activated. These galanin actions would directly counter the two opposing electrophysiological responses to carbohydrate stimulation in RINm5f cells, which are to inhibit K+ current and to stimulate Ca2+ current. Given that stimulation of presynaptic nerve terminals in pancreas releases galanin, these results suggest that Ca(2+)-dependent insulin release from native pancreatic beta cells may also be regulated by similar neuropeptide effects. PMID- 1717987 TI - Retrotransposition of a mouse L1 element. AB - Long interspersed elements (LINEs) of the L1 family represent a major class of mammalian repetitive DNA and are present at copy numbers of between 10(4) and 10(5) elements per genome. Structural similarities between L1 elements and known retrotransposons have led to the suggestion that a subset of L1 elements may function as mobile genetic elements and have thus gained their prominent place in the mammalian genome. We describe a consensus mouse L1 element that was tagged with a heterologous intron and shown to transpose by way of an RNA intermediate when transfected into baby hamster kidney cells, formally establishing L1 elements as retrotransposons. When the putative reverse transcriptase-encoding region of this L1 element was deleted, the element still underwent retrotransposition in hamster cells, suggesting that reverse transcriptase activity can be supplied by an endogenous enzyme. PMID- 1717988 TI - Nonnucleoside reverse transcriptase inhibitors that potently and specifically block human immunodeficiency virus type 1 replication. AB - Certain bis(heteroaryl)piperazines (BHAPs) are potent inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) at concentrations lower by 2-4 orders of magnitude than that which inhibits normal cellular DNA polymerase activity. Combination of a BHAP with nucleoside analog HIV-1 RT inhibitors suggested that together these compounds inhibited RT synergistically. In three human lymphocytic cell systems using several laboratory and clinical HIV-1 isolates, the BHAPs blocked HIV-1 replication with potencies nearly identical to those of 3'-azido-2',3'-dideoxythymidine or 2',3' dideoxyadenosine; in primary cultures of human peripheral blood mononuclear cells, concentrations of these antiviral agents were lower by at least 3-4 orders of magnitude than cytotoxic levels. The BHAPs do not inhibit replication of HIV 2, the simian or feline immunodeficiency virus, or Rauscher murine leukemia virus in culture. Evaluation of a BHAP in HIV-1-infected SCID-hu mice (severe combined immunodeficient mice implanted with human fetal lymph node) showed that the compound could block HIV-1 replication in vivo. The BHAPs are readily obtained synthetically and have been extensively characterized in preclinical evaluations. These compounds hold promise for the treatment of HIV-1 infection. PMID- 1717989 TI - Mouse Erk-1 gene product is a serine/threonine protein kinase that has the potential to phosphorylate tyrosine. AB - Bacterial expression of mouse gene Erk-1 yielded an active kinase with the same substrate specificity shown for ERK1 protein purified from rat cells. Although rat gene ERK1 is believed to encode a serine/threonine kinase based on sequence data and known ERK1 substrate phosphorylation sites, bacterially-produced mouse Erk-1 (bt-Erk-1) autophosphorylated on tyrosine in addition to serine and threonine residues. The bt-Erk-1 protein also had the capacity to reactivate the ribosomal protein S6 kinase (S6KII). Furthermore, treatment of bt-Erk-1 with either serine/threonine-specific phosphatase 2A or tyrosine-specific phosphatase 1B significantly decreased its kinase activity. These findings predict that autophosphorylation may play an important role in Erk-1/ERK1 regulation. PMID- 1717990 TI - Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. AB - The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme (E2) that is required for DNA repair, damage-induced mutagenesis, and sporulation. We have cloned the two human RAD6 homologs, designated HHR6A and HHR6B. The two 152-amino acid human proteins share 95% sequence identity with each other and approximately 70% and approximately 85% overall identity with the homologs from yeasts (S. cerevisiae and Schizosaccharomyces pombe) and Drosophila melanogaster, respectively. Neither of the human RAD6 homologs possess the acidic C-terminal sequence present in the S. cerevisiae RAD6 protein. Genetic complementation experiments reveal that HHR6A as well as HHR6B can carry out the DNA repair and mutagenesis functions of RAD6 in S. cerevisiae rad6 delta mutants. PMID- 1717991 TI - Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides. AB - Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET 1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis. PMID- 1717992 TI - Another discontinuous epitope on glycoprotein gp120 that is important in human immunodeficiency virus type 1 neutralization is identified by a monoclonal antibody. AB - To define the domains in the envelope glycoprotein important for antibody neutralization of the human immunodeficiency virus type 1 (HIV-1), monoclonal antibodies (mAbs) were generated by immunizing mice with purified glycoprotein gp120 of the IIIB isolate. One mAb, G3-4, reacted with the gp120 of homologous (IIIB) and heterologous (RF) isolates. In addition, mAb G3-4 efficiently neutralized both IIIB and RF viruses in vitro, as well as four of nine primary HIV-1 isolates. In competition immunoassays, mAb G3-4 and soluble CD4 were found to inhibit one another in binding to gp120. However, no competition was seen between mAb G3-4 and mAbs directed to the third variable region or the fourth conserved region of gp120. In particular, mAb G3-4 did not compete with our human mAb 15e, which identifies a discontinuous epitope on gp120 involved in group specific neutralization of HIV-1 and in gp120-CD4 binding. Epitope-mapping studies on mAb G3-4 with synthetic or unglycosylated recombinant peptides were negative, suggesting that its epitope may be discontinuous. Indeed, this hypothesis was confirmed by showing the loss of mAb G3-4 serologic reactivity when gp120 was first denatured. We conclude that the site recognized by mAb G3-4 represents another discontinuous epitope on gp120 important for neutralization of HIV-1. PMID- 1717993 TI - A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine rich domain upstream from the human delta-globin gene. AB - To date, DNA-binding factors with a developmental pattern of expression have not been described in human erythroid cells to explain the switch from fetal (gamma-) to adult (delta- and beta-) globin gene expression. Here we describe a factor present in nuclear extracts from adult mouse and human hematopoietic cells that binds to an oligopyrimidine repeat approximately 960 base pairs upstream from the human delta-globin gene. The binding site for the factor is within an unusual 250 base-pair domain that is greater than 95% pyrimidines on one strand. This domain is preferentially sensitive to S1 nuclease in supercoiled plasmids, indicating that it can adopt an alternative non-B-DNA conformation. A number of S1-sensitive sites within the domain, including the factor-binding site, have sequence characteristics associated with the formation of a triple helix (H-DNA). The position of the binding site between the fetal and adult beta-globin-like genes, its potential for adopting an unusual secondary structure, and the restricted activity of the binding factor to adult hematopoietic tissues suggest possible roles in hematopoietic cell development and hemoglobin switching. PMID- 1717994 TI - Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation. AB - We have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34CDC2 kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34CDC2 kinase (65% identical) and more significantly is homologous to Xenopus Eg1 kinase (89% identical), suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34CDC2 kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G1 or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages. PMID- 1717996 TI - Bleomycin-resistance gene derived from the transposon Tn5 confers selective advantage to Escherichia coli K-12. AB - The plasmid pRAB2 contains a silent operon derived from the transposon Tn5 and carrying the gene neo for neomycin-kanamycin resistance and a truncated ble gene (ble333) for bleomycin resistance. Spontaneous mutants that express the two resistances provide Escherichia coli cells an improved fitness during the phase of decline in the absence of the antibiotics. It is shown that the ble333 gene product is responsible for this better fitness. These results can explain a previously described selective advantage attributed to the presence of Tn5. The improved fitness of bleomycin-resistant bacteria is proposed to relate to DNA repair by the ble gene product. The consequences of the presence of an accessory gene improving fitness are discussed in terms of evolutionary stable strategy of a transposon in populations of E. coli. PMID- 1717995 TI - A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. AB - We have cloned a receptor tyrosine kinase cDNA, designated fetal liver kinase 1 (Flk-1), from mouse cell populations enriched for hematopoietic stem and progenitor cells. Sequence analysis of this clone reveals strong homology to the c-Kit subfamily of receptor kinases, and in particular to the Flt gene product. Chromosomal mapping shows that the Flk-1, Kit, and Pdgfra genes are closely linked. Flk-1 mRNA is expressed in primitive and more mature hematopoietic cells as well as in a wide variety of nonhematopoietic tissues. PMID- 1717997 TI - Fyn tyrosine kinase associated with Fc epsilon RII/CD23: possible multiple roles in lymphocyte activation. AB - Expression of low-affinity Fc receptor for IgE (Fc epsilon RII), which is identical to the lymphocyte differentiation antigen CD23, is associated with activation of lymphoid cells. The mechanism of signal transduction through Fc epsilon RII/CD23 was dissected by transfection of cDNA coding for Fc epsilon RII to the YT human natural killer-like cell line, activation of which was easily detected by the induction of interleukin 2 receptor/p55(Tac). Cross-linking of Fc epsilon RII/CD23 with H107 anti-Fc epsilon RII monoclonal antibody markedly enhanced interleukin 2 receptor/p55 expression on the YT cells transfected with Fc epsilon RII cDNA (YTSER cells). Similar induction of interleukin 2 receptor/p55 by the cross-linking of Fc epsilon RII was observed on an Epstein Barr virus-transformed B-cell line, 3B6, and fresh leukemic cells isolated from a patient with B-cell chronic lymphoblastic leukemia. Thus Fc epsilon RII/CD23 provides activation signals not only in YTSER cells but also in activated B cells. A possible involvement of protein-tyrosine kinase in the Fc epsilon RII mediated signal transduction was studied. Fc epsilon RII was physically associated with a src family tyrosine kinase p59fyn and not with p56lck, which was also found in YT cells. Recently it was reported that p59fyn was associated with T-cell antigen receptor. Our results collectively suggest the multiple functions of p59fyn that may be implicated in Fc epsilon RII-mediated activation signal in YT cells. PMID- 1717998 TI - Preferential T-cell receptor beta-chain variable gene use in myelin basic protein reactive T-cell clones from patients with multiple sclerosis. AB - Multiple sclerosis is an autoimmune disease in which T lymphocytes reactive to myelin basic protein (BP) could play a central role. T cells specific for BP were cloned from the blood of multiple sclerosis patients and normal individuals, and expression of T-cell receptor variable region genes was analyzed. A remarkable bias for use of beta-chain variable region (V beta) 5.2 and, to a lesser extent, V beta 6.1 was seen among BP-specific clones from patients but not from controls. The preferential use of V beta 5.2 for BP recognition did not reflect altered expression of this V beta in the peripheral repertoire. Interestingly, shared V beta 5.2 usage was apparent for clones specific for different BP determinants, even when derived from the same individual. The concurrent demonstration by others (J. R. Oksenberg, M. A. Panzara, A. B. Begovich, H. Erlich, R. Murray, M. Sherritt, S. Stuart, C. C. Bernard, and L. Steinman, personal communication) that T cells within demyelinating areas of multiple sclerosis brains preferentially express V beta 5.2 and V beta 6.1 suggests that the BP-specific clones derived from blood may be relevant to disease pathogenesis. These findings may have important implications for the treatment of multiple sclerosis. PMID- 1717999 TI - The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. AB - Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. We describe here a 70-kDa tyrosine phosphoprotein (ZAP-70) that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro. PMID- 1718000 TI - Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. AB - The location of phosphate residues involved in specific centers for binding of metal ions in M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, was determined by analysis of induction of cleavage of RNA by metal ions. At pH 9.5, Mg2+ catalyzes cleavage of M1 RNA at five principal sites. Under certain conditions, Mn2+ and Ca2+ can each replace Mg2+ as the cofactor in the processing of precursor tRNAs by M1 RNA and P RNA, the RNA subunit of RNase P from Bacillus subtilis. These cations, as well as various metal ion inhibitors of the catalytic activity of M1 RNA, also promote cleavage of M1 RNA in a specific manner. Certain conditions that affect the catalytic activity of M1 RNA also alter the rate of metal ion-induced cleavage at the various sites. From these results and a comparison of cleavage of M1 RNA with that of a deletion mutant of M1 RNA and of P RNA, we have identified two different centers for binding of metal ions in M1 RNA that are important for the processing of the precursor to tRNA(Tyr) from E. coli. There is also a center for the binding of metal ions in the substrate, close to the site of cleavage by M1 RNA. PMID- 1718001 TI - Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA. AB - C6-2B rat glioma cells were stably transfected with substance K receptor cDNA and used to study interactions between cAMP and Ca2+ signaling pathways. Activation of the newly expressed receptors by substance K increased the intracellular free Ca2+ concentration, as monitored by single-cell fura-2 imaging, and markedly inhibited agonist-stimulated cAMP accumulation. Blockade of intracellular Ca2+ mobilization abolished the substance K receptor-mediated inhibition of isoproterenol-induced cAMP production. Phosphodiesterase inhibitors, down regulation or inhibition of protein kinase C, and pertussis toxin failed to prevent substance K-induced inhibition of agonist-stimulated cAMP accumulation. An increased intracellular Ca2+ concentration caused by either calcium ionophores or activation of endogenous bradykinin receptors was found to markedly reduce cAMP production in wild-type cells. These results demonstrate that elevated intracellular Ca2+ concentration can negatively modulate agonist-stimulated adenylate cyclase activity in C6-2B glioma cells. PMID- 1718002 TI - Immunocytochemical localization of the cystic fibrosis gene product CFTR. AB - Antisera against two peptides, corresponding to different domains of the cystic fibrosis gene product CFTR, have been raised and extensively characterized. Both antisera recognize CFTR as a 165-kDa polypeptide in Western analysis of cells transfected with CFTR cDNA as well as in epithelial cell lines. The cell and tissue distribution of CFTR has been studied by immunocytochemistry. CFTR is abundant in epithelial cells, including those lining sweat ducts, small pancreatic ducts, and intestinal crypts. Unexpectedly, the level of CFTR in lung epithelia is relatively low, while it is abundant in the epithelia of kidney tubules. The protein appears to be restricted to the apical, rather than basolateral, regions of epithelial cells and at least a proportion is associated with the plasma membrane. The cell and tissue distributions of CFTR are consistent with a function for this protein as a chloride channel or as a regulator of channel activity. PMID- 1718003 TI - The two different receptors for tumor necrosis factor mediate distinct cellular responses. AB - The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses. PMID- 1718004 TI - Human immunodeficiency virus can infect the apical and basolateral surfaces of human colonic epithelial cells. AB - The gastrointestinal tract is considered to be a major route of infection for the human immunodeficiency virus (HIV). To understand the interaction of HIV with epithelial cells of the intestinal mucosa, we have studied the infection of a human colon cancer cell clone HT-29-D4. The enterocyte-like differentiation of this clone can be modulated in vitro according to the concentration of glucose. We show that: (i) undifferentiated HT-29-D4 cells can be infected by HIV types 1 and 2 (HIV-1 and HIV-2) strains with no subsequent effect on cell growth; (ii) undifferentiated HT-29-D4 cells express a CD4-related antigen bearing epitopes of the immunoglobulin-like variable (V) region domains V1 and V2 of CD4 but lacking the epitope known to be involved in HIV envelope recognition; (iii) differentiated HT-29-D4 cells can be infected by HIV after an interaction with either the apical brush border membrane (luminal side) or the basolateral side (serosal side); (iv) the CD4-like molecule is restricted to the basolateral domain of differentiated cells; and (v) the infection is not inhibited by anti CD4 monoclonal antibodies (mAbs) OKT4, OKT4A, Leu-3a, Bl4, 13-B-8-2, S-T4 or S T40. We conclude that epithelial intestinal cells may represent a major site of entry for HIV. Infection of these epithelial cells may occur via the basolateral membrane by HIV-bearing lymphocytes or macrophages of the lamina propria and via the apical membrane by HIV present in the bowel lumen. This infection may remain silent for up to 9 months, and the virus can be rescued by cocultivation with lymphoid cells. These data may give an explanation for the long latent seronegative state that may occur in a HIV-infected individual. PMID- 1718005 TI - Platelet factor 4: production, structure, and physiologic and immunologic action. PMID- 1718006 TI - PADGEM: an adhesion receptor for leukocytes on stimulated platelets and endothelial cells. PMID- 1718007 TI - Patterns of cytokines, plasma endotoxin, and acute phase proteins during the treatment of severe sepsis in humans. PMID- 1718008 TI - Biology of the prostate. PMID- 1718009 TI - Preservation of retroperitoneal lymph nodes in patients with locoregional nonseminomatous germ cell tumors: surveillance and primary chemotherapy. PMID- 1718010 TI - Descending systems activated by morphine (ICV) inhibit kainic acid (IT)-induced behavior. AB - Modulation of spinal systems activated by N-methyl-D-aspartate (NMDA) and substance P administered IT have been an area of interest in several laboratories. In the present investigations, behavior induced by the excitatory amino acid kainic acid, but not quisqualate, is demonstrated to be modulated in a manner similar to that previously observed for NMDA. Biting, scratching and licking behavior was induced by IT injections of excitatory amino acids or substance P in mice. Behavior induced by kainic acid (IT) injection was inhibited in a dose-dependent manner by coadministration of morphine (ICV), norepinephrine (IT), N-ethyl carboxamidoadenosine (NECA) (IT) and agonists interacting at PCP receptors (IT). Kainic acid and NMDA differed, however, in that a dopaminergic agonist, apomorphine, inhibited kainic acid-, but not NMDA-induced behavior and a selective NMDA receptor antagonist inhibits NMDA-, but not kainic acid-induced behavior. Behavior induced by quisqualate (IT) was not inhibited by any treatment and may have nonspecific actions in this type of assay. Our observations support independent spinal sites of action for behavior induced by kainic acid and NMDA, but several similarities were observed in the modulation of spinal systems activated by these agents. PMID- 1718011 TI - From channels to cross bridges. American Physiological Society (APS) Conference. July 13-16, 1991, Bar Harbor, Maine. Abstracts and program. PMID- 1718013 TI - Differential effects of L-type calcium channel blockers and stimulants on naloxone-precipitated withdrawal in mice acutely dependent on morphine. AB - The effects of L-type calcium channel blockers and stimulants on naloxone precipitated withdrawal in mice acutely dependent on morphine were evaluated. Verapamil (10-80 mg/kg), diltiazem (20-120 mg/kg) and nicardipine (20-160 mg/kg), when administered subcutaneously, produced a dose-dependent reduction in forepaw tremor and weight loss during the abstinence reaction; jumping was also reduced by all three drugs, although the effect was not statistically significant in the case of nicardipine. By contrast, the calcium agonist Bay K 8644 (0.5-2 mg/kg, SC) increased forepaw tremor and weight loss, although this latter effect did not reach statistical significance. The effects of the calcium channel active drugs on the rotarod test were also explored, no correlation appearing with the results observed in abstinence (except for the jumping response), which suggests that the withdrawal results are not influenced by motor incoordination or unspecific CNS depression. These findings suggest that L-type calcium channels probably play an important role in withdrawal after acute morphine dependence. Taken together with other observations in chronic models, these results show that calcium channels are similarly involved in morphine abstinence after acute and chronic dependence, in contrast to the differences in the content and uptake of neuronal calcium induced by morphine under both conditions. PMID- 1718012 TI - Failure of gamma-hydroxybutyrate to alter the function of the GABAA receptor complex in the rat cerebral cortex. AB - The present study was designed to evaluate the possible interaction of gamma hydroxybutyrate (GHB) with the GABAA receptor complex in the rat cerebral cortex. To this purpose we studied the effect of in vitro addition and in vivo administration of GHB on the biochemical parameters currently used to evaluate the function of the GABAergic system. In vitro addition of increasing concentrations of GHB failed to modify [3H]flunitrazepam ([3H]FNZ) binding and the modulatory action of GABA on this binding. Moreover, unlike diazepam, GHB did not modify in vitro both muscimol-stimulated 36Cl- uptake and t [35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat cerebral cortex. In vivo administration of sedative and hypnotic doses of GHB (300-750 mg/kg IP) failed to induce in 60 min any significant change in the [35S]TBPS binding to unwashed cortical membranes. Moreover, GHB also failed to antagonize the increase in [35S]TBPS binding (+55%) induced by isoniazid (350 mg/kg SC). In contrast, at the highest doses used, this drug completely antagonized the seizure activity induced by isoniazid. In conclusion, our data show that GHB fails to alter the function of the GABAA/benzodiazepine/ionophore receptor complex in the rat cerebral cortex. PMID- 1718014 TI - Biogenic amines in anorexia nervosa: circadian rhythm in urinary excretion and influence of posture and physical task load on plasma catecholamines. AB - To investigate the sympathetic response in anorexia nervosa (AN) patients to stimuli occurring in normal life, biogenic amines were studied in 10 female outpatients. As control groups 10 lean and 10 normal weight healthy female subjects were included. It was hypothesized that the lean control group would have intermediate values between the AN patients and the normal weight controls. The AN patients and the lean controls had a mean underweight of 33.1% and 13.7%, respectively. For the excretion in 24-hr urine, differences among the groups were observed for several compounds, unexpectedly the values being lowest for the patients and highest for the lean controls. Furthermore, the lean controls had a higher excretion of a number of compounds in diurnal than in nocturnal urine, whereas this effect was absent or reversed for the AN patients and intermediate for the normal weight controls. Plasma norepinephrine was highest in the patients and lowest in the lean controls. The catecholamine response to postural changes and physical exercise did not differ among the groups. The results obtained indicate neither a (linear) relationship between underweight and the metabolism of biogenic amines nor a disturbed response to sympathetic stimulation in AN, but suggest an altered metabolism of biogenic amines in patients suffering from AN. PMID- 1718015 TI - Chronic ICV infusion of neuropeptides alters lymphocyte populations in experimental rodents. AB - The sympathetic nervous system has been shown to influence immune function. Angiotensin II and substance P are two neurally active peptides that have been shown to increase sympathetic nervous system activity when injected centrally. Using osmotic minipumps, we chronically infused angiotensin II (1 microgram/h) and substance P (2 micrograms/h) into the brains of intact Sprague-Dawley rats for a period of 1 month and 2 weeks, respectively. Age-matched control animals were infused with artificial cerebrospinal fluid. We then examined the effect of this infusion on the percentage of different lymphocyte populations in the peripheral blood. The angiotensin II infused animals showed an increase in the percentage of total T-cells and a decrease in the percentage of B-cells relative to controls. The substance P treated animals also showed an increase in the percentage of T-cells present, but failed to show the decrease in the B-cell population seen with the angiotensin II infused group. This study shows that the central nervous system can influence the immune system. As shown in this study, these effects are most likely mediated via the sympathetic nervous system. These results add to the expanding body of data suggesting an important role of the central nervous in regulating immune function and our susceptibility to disease. PMID- 1718016 TI - Motility regulating effects of galanin on smooth muscle of porcine ileum. AB - We studied the effect of synthetic porcine galanin on circular and longitudinal oriented strips of pig ileal muscle. Galanin 10(-11)-10(-6) M had no effect on resting tension in the two layers. In circular muscle precontracted with carbachol 10(-6) M, galanin dose-dependently inhibited the amplitude of contractions to a maximum of 33 +/- 8% at 10(-6) M. In longitudinal muscle the amplitude of contractions induced by carbachol 10(-7) M or transmural field stimulation increased after addition of galanin 10(-9)-10(-7) M to a maximum of 21 +/- 6%, while at higher concentrations inhibition occurred. Maximal inhibition was 36 +/- 14% at galanin 10(-6) M. Tetrodotoxin did not influence the effects of galanin in the preparations. The results indicate that in the homologous species galanin inhibits the circular muscle layer, possibly by a direct action on the smooth muscle. In the longitudinal muscle the effect of galanin is apparently excitatory. The inhibition observed with high concentration of galanin could be due to tachyphylaxis and desensitization. Alternatively, an additional population of low affinity, inhibitory receptors may exist. PMID- 1718017 TI - Galanin inhibits pancreatic amylase secretion in the pentobarbital-anesthetized rat. AB - The potent inhibitory effect of galanin on basal and pentagastrin-stimulated gastric acid secretion in vivo, and the presence of galanin-containing nerves in gastrointestinal tract and pancreas, suggested that this peptide may regulate the exocrine secretion of the GI system. Male rats were anesthetized with pentobarbital and the dose-dependent inhibitory effects of galanin on basal and stimulated pancreatic protein and amylase secretions were investigated in separate experiments. Galanin was administered intravenously in the following doses: 3, 6, 10, 15 and 20 micrograms/kg/h (0.93, 1.86, 3.1, 4.65 and 6.2 nmol/kg/h), and pancreatic secretions measured. The maximal effective dose of galanin (3.1 nmol/kg/h) on basal pancreatic secretions was found, and was used for evaluating the inhibitory effect of galanin on pancreatic protein and amylase secretions stimulated by bombesin, secretin and cholecystokinin. Galanin potently inhibited basal, bombesin-, secretin- and cholecystokinin-stimulated pancreatic protein and amylase secretion. Inhibitory effect of galanin was dose-dependent and biphasic. PMID- 1718018 TI - A quantitative study of neuropeptide immunoreactive cell bodies of primary afferent sensory neurons following rat sciatic nerve peripheral axotomy. AB - Following peripheral axotomy, fluoride resistant acid phosphatase (FRAP) and most neuropeptides are depleted in the central terminals of axotomised nerves and reduced in their corresponding cell bodies (DRG) but vasoactive intestinal polypeptide (VIP) increases. The increase in VIP probably results from a change in gene expression in other ganglion cells which do not normally express VIP. A quantitative study was performed to investigate the proportion of DRG cells immunoreactive for different peptides at increasing times after sciatic nerve section. Retrograde fluorescent neuronal labelling of sciatic nerve cell bodies by injection of fast blue into the proximal stump was combined with unlabelled antibody immunohistochemistry for CGRP and VIP. The proportion of cells immunoreactive for these peptides was quantified between two and fourteen days post-axotomy. The number of VIP immunoreactive profiles increased significantly in the first 4 days post-axotomy, followed by a slight decrease before rising again. In contrast, the number of and CGRP-immunoreactive cell profiles declined to zero by 14 days post-axotomy. 4 days post-axotomy 50% of VIP positive cells were also immunoreactive for CGRP. There was neither colocalisation between VIP and FRAP nor between CGRP and FRAP. It is concluded that many peptidergic DRG cell bodies switch their expression of peptide to VIP after injury, whereas non peptide-containing subpopulations do not. PMID- 1718019 TI - [Biologic response modifiers in the treatment of solid tumors]. AB - This study concerns the clinical applications in Oncology of Biologic Response Modifiers; the authors focus the results reached in neoplasms, such as renal cell carcinoma or melanoma, whose sensitivity to this treatment has been proved. Prospective studies, published in 1990 have been reviewed, considering only those in which route and rate of response (complete and partial) were clearly specified. At present clinical efficacy concerning this topic is controversial and it probably depends on different reasons: immunogenicity of cancer, variable route of administration, lack of predictors. It should be stressed that more in depth studies about cancer immunology are required; a wide spread of immunotherapy will be feasible only by improving therapeutic strategies and limiting adverse effects of this treatment. PMID- 1718020 TI - Surface CD4 density remains constant on lymphocytes of HIV-infected patients in the progression of disease. AB - In an attempt to question the influence of circulating virus, soluble gp120 or CD4 self-reacting antibodies upon results of CD4+ T-cell immunophenotyping in AIDS patients, five anti-CD4 mAb defining several epitopes of the V1 and V2 domains of the CD4 molecule were used to analyse the epitopic density of CD4 on lymphocytes of seropositive patients taken at stages II, III and IV of HIV infection, according to the Centers for Disease Control (CDC, Atlanta) classification. Our results demonstrate that each CD4 epitopic density measured on circulating lymphocytes remains constant at a mean level of 46,000 epitopes per cell whatever the stage of the disease and whatever the serum p25 concentration. These data provide evidence that antibody accessibility to several CD4 epitopes is not altered by putative interactions between CD4 molecules and circulating virus, soluble gp120 or anti-CD4 autoantibodies. If such binding events, as expected, do occur in vivo, they are of too low a magnitude to influence the immunophenotyping. Furthermore, we show that mAb specific for different epitopes in the V1 and V2 domains of the CD4 molecule can be used interchangeably for the biological followup of the CD4+ cell population in blood samples of HIV-infected patients. PMID- 1718021 TI - Visualization of natural autoantibody polyreactivity by rotary metal-shadowing electron microscopy. AB - Immune complexes formed by mouse polyspecific natural autoantibodies and various structurally different antigens, such as DNA, tubulin and myosin, were analysed by rotary-shadowing electron microscopy. Each of the four natural IgM autoantibodies studied (E7, D23, 3C3 and M2-9) recognized multiple epitopes on the myosin molecule. These results, confirmed by immunoblotting experiments using myosin subfragments as antigens, strikingly contrasted with those obtained with an induced myosin-specific IgG antibody which interacted with a single myosin antigenic site. Based on the measurements of the antibody position on the antigen, made on a series of electron micrographs, two negatively charged myosin peptides were prepared by solid phase synthesis. Polymeric forms of one of the two peptides interacted with the positively charged CDR part of E7 and inhibited the binding of E7 and M2-9 to myosin. The importance of charge in the observed cross-reactivities was further supported by enzyme immunoassays showing that most, but not all, antigen/natural autoantibody interactions were sensitive to increasing concentrations of NaCl. PMID- 1718022 TI - Structure and function of the chicken spleen. PMID- 1718023 TI - A pilot clinical and pharmacokinetic study of intracarotid cisplatin and bleomycin. AB - Fifteen patients with progressive primary malignant or metastatic brain tumors were treated on a clinical and pharmacokinetic study with intracarotid cisplatin and bleomycin. Toxicity was tolerable and consisted mainly of nausea and vomiting. Neurologic toxicity included focal seizures (1), leukoencephalopathy (1), and motor weakness (1). Five patients had improvement in CT scans and four patients had stabilization of disease. Recommended dosage for future clinical trials are cisplatin 60 mg/m2 and bleomycin 100 units. Pharmacokinetics of intracarotid cisplatin revealed the jugular vein concentration was twice the peripheral vein level at the end of infusion. Cisplatin is a drug which has demonstrated in vitro activity against malignant gliomas (1). Clinical trials with intravenous administration of cisplatin has shown definite, although limited antitumor activity against primary brain tumors (2,3,4) and metastatic brain tumors (5,6). To enhance its antitumor effect, cisplatin has been administered by the intracarotid route (7,8,9). The results appear encouraging, but neurological and ophthalmological toxicity may occur (8). In our initial study with intracarotid cisplatin, 35 patients with malignant brain tumors (23 with primary brain tumors and 12 with brain metastases) progressing after cranial irradiation +/- chemotherapy were treated. Of 20 evaluable patients with primary tumors, 6 responded to therapy and 5 had stable disease. Five of 10 evaluable patients with brain metastases responded and 2 had stable disease. For responding primary brain tumor patients the median time to progression was 33 weeks. The recommended dose for intracarotid cisplatin was 60-75 mg/m2 administered every 3-4 weeks (7,8). Higher cisplatin doses produced more central neurological toxicity. There is limited data on the central nervous system pharmacology of cisplatin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718024 TI - Treatment of metastatic breast cancer. AB - Relapse of breast cancer connotes a dire prognosis. However, long-term survival with remissions and disease exacerbation are possible using conservative treatment strategies and often sequential hormonal manipulations. Hormonal therapy is the mainstay of metastatic breast cancer treatment for patients with hormonally sensitive tumors; cytotoxic chemotherapy, for patients with hormonally independent tumors. High-dose chemotherapy with autologous bone marrow reinfusion is currently under investigation. PMID- 1718025 TI - T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B, L, and D. AB - We report on a computer algorithm capable of predicting the location of T-helper cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described. PMID- 1718026 TI - There is no correlation between function and lymphokine production of HBs-antigen specific human CD4(+)-cloned T cells. AB - The question whether antigen-specific human CD4+ T cells can be classified on the basis of appropriate and fixed lymphokine production patterns and their corresponding functions still remains to be elucidated. We generated ten CD4+ T cell clones specific for HBsAg from HBsAb-positive but HBsAg-negative individuals. Seven of these clones exhibited helper activity for HBsAb response, while the three other clones did not. Both helper- and non-helper-type T-cell clones produced interleukin 4 (IL-4) after antigenic stimulation. By stimulation with phytohaemagglutinin (PHA) plus phorbol myristate acetate (PMA), three of the seven helper-type clones produced interleukin 2 (IL-2) in addition to IL-4. However, the other four helper-type clones did not produce IL-2 by such stimulation, although they continued the production of IL-4. All non-helper-type T-cell clones produced a large amount of IL-2, and some of them completely became an IL-2 producer after certain stimulation. These results suggested that both helper- and non-helper-type CD4+ T-cell clones specific for HBsAg might have no strict pattern of lymphokine production as in the TH1/TH2 dichotomy of murine CD4+ T cells. The data also revealed that lymphokine-producing capacity of individual cloned T cells is changeable depending upon the sort of activation. PMID- 1718027 TI - Functional epitope analysis of the human CD11a/CD18 molecule (LFA-1, lymphocyte function-associated antigen 1) involved in HIV-1-induced syncytium formation. AB - After binding to the CD4 receptor, the human immunodeficiency virus 1 (HIV-1) may enter the T cell and induce the formation of multinucleated giant cells (syncytia). As well as the CD4 molecule, other molecules, such as the lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18) have been shown to be involved in HIV-1-mediated cell fusion. This study was designed to define regions on the human CD11a/CD18 molecule important for the HIV-1-induced syncytium formation. A CD11a/CD18 MoAb panel discriminating at least five distinct and spatially distant domains on the LFA-1 molecule was used. Comparison of the functional activity of different MoAbs demonstrated that all epitopes of the LFA-1 molecule were not of equal importance in HIV-1-induced syncytium formation between H9.III cells chronically infected with HIV-1 and uninfected CD4+ SupT1 cells. We also demonstrated that CD11a/CD18 MoAbs inhibit syncytia formation only at the level of the uninfected SupT1 cells, suggesting that the LFA-1 molecule expressed on SupT1 cells interacts with ligand(s) expressed on the infected H9.III cells. Two potential LFA-1 receptors on the H9.III cells were tested: the ICAM-1 molecule (intercellular adhesion molecule 1, CD54) and the HIV-1 transmembrane glycoprotein 41 (gp41). A CD54 MoAb (84H10) partially inhibited syncytia formation, thus demonstrating the involvement of the ICAM-1 molecule in the HIV-1 mediated cell fusion. However, the CD11a/CD18 MoAbs do not inhibit binding of the viral envelope glycoprotein gp41 to the cell surface, irrespective of the MoAb concentration used. Although we have not been successful in identifying all candidate fusion receptors for the LFA-1 molecule, these data suggest that some LFA-1 regions are important for syncytium formation and, therefore, in the cell to-cell transmission of virus and in the spread of infection. PMID- 1718028 TI - Human monoclonal antibodies against blood group antigens preferentially express a VH4-21 variable region gene-associated epitope. AB - An anti-idiotypic antibody has been raised which recognizes human immunoglobulins with cold agglutinin activity of anti-I/i specificity. The pattern of reactivity of the antibody indicates that the structural basis for the epitope is located in the VH4-21 gene segment of the VHIV family, which is preferentially utilized by these cold reactive antibodies. Using this antibody, epitope expression was investigated in a panel of 72 human monoclonal allo-antibodies specific for human blood group antigens, as compared with a control panel of 39 randomly selected human monoclonal IgM antibodies of unknown specificities. The anti-blood group panel included 44 IgM and 28 IgG monoclonal antibodies against a variety of blood group antigens including the A antigen, Rh C, c, D, E, e, G antigens, and the Kidd antigens Jka and Jkb. The epitope was expressed by 64% (28/44) of the IgM anti-blood group antibodies and by 21% (6/28) of the IgG antibodies, but by only 7.7% (3/39) of the control IgM antibodies. These data indicate that the human alloimmune response to blood group antigens is biased in the use of VH gene families, with a preference for the VH4-21 gene segment of the VHIV family, or closely related gene segments. The fact that this mirrors the findings for the autoimmune cold agglutinins suggests a link in immunoglobulin gene usage between antibodies against structurally diverse antigens on the red cell surface. PMID- 1718029 TI - [Cryotherapy of an inoperable breast tumor]. AB - The use of cryocauter in inoperable tumor of breast is described. Case-reports are presented of successful approach by cryolysis prior to the final lobe plastic. PMID- 1718030 TI - Demonstration of endogenous lectins in synovial tissue. AB - We have recently shown that synoviocytes and extracellular matrices exhibit distinct patterns of carbohydrate expression. Their biological relevance is however not known. The purpose of the present study was to find out whether human synovial tissue would also show a specific receptor pattern for complex sugar molecules. Endogenous lectins were displayed by means of biotinylated neoglycoproteins and sulfated polysaccharides in paraffin-embedded material or cryosections. In addition to certain carbohydrate components that are known to be constituents of the carbohydrate part of cellular glycoconjugates, our panel included heparin and fucoidan, a sulfated fucose. Binding sites were shown using the avidin-peroxidase technique for light microscopy. The results were compared with immunohistochemical methods and enzyme histochemistry. Our study demonstrates that human synovial tissue contains a complex pattern of endogenous lectins depending on the different types of synovitis. The staining method we used in the investigation allows for precise localization of saccharide binding receptors and is therefore believed to be a reliable technique for further phenotypic characterization of synovial cells. PMID- 1718031 TI - [Pharmacologic effects of biotin on epidermal cells]. AB - Biotin deficiency in animals causes pathological changes of the skin and its appendages including, for example, exfoliative dermatitis, depigmentation, and alopecia. The hooves of biotin-deficient swine are weak, brittle, and often necrotic. These changes disappear after dietary biotin supplementation. Biotin supplementation also noticeably improves the hoof quality of horses, cattle and swine having no apparent biotin deficiency. In order to elucidate the molecular basis of these effects, the influence of biotin on cytokeratin expression in a keratinocyte cell line (Ha-CaT) was investigated using electrophoretic and immunological techniques. Pharmacological biotin concentrations of 1 microM, and 100 microM in the culture medium caused a specific increase in cytokeratins, which are normally induced upon terminal differentiation of epidermal cells in vivo. The expression of cytokeratins occurring in stratified epithelia independent of differentiation were not affected. These findings show that biotin directly stimulates the differentiation of epidermal cells. Such a molecular mechanism revealed in cell culture could provide an explanation for the therapeutic effects of pharmacological doses of biotin on hoof quality in farm animals. PMID- 1718032 TI - Elusive quarry. Researchers are closing in on the stem cell. PMID- 1718033 TI - RNA world. PMID- 1718034 TI - CD14 and immune response to lipopolysaccharide. PMID- 1718035 TI - Suppression of the immune response by a soluble complement receptor of B lymphocytes. AB - The CD19-CR2 complex of B lymphocytes contains proteins that participate in two host-defense systems, the immune and complement systems. The ligand for the subunit of the immune system, CD19, is not known, but the complement receptor subunit, CR2 (CD21), binds activation fragments of the C3 component of the complement system and may mediate immunopotentiating effects of complement. A recombinant, soluble CR2 was prepared by fusing the C3-binding region of the receptor to immunoglobulin G1 (IgG1). The (CR2)2-IgG1 chimera competed with cellular CR2 for C3 binding and suppressed the antibody response to a T cell dependent antigen when administered to mice at the time of immunization. This inhibitory effect of (CR2)2-IgG1 demonstrates the B cell-activating function of the CD19-CR2 complex and suggests a new method for humoral immunosuppression. PMID- 1718036 TI - Neutralization of divergent HIV-1 isolates by conformation-dependent human antibodies to Gp120. AB - The spectrum of human immunodeficiency virus type 1 (HIV-1) isolates neutralized by antibodies from HIV-1-infected humans is broader than the spectrum of isolates neutralized by sera from animals immunized with purified gp120 subunits. This broader neutralization was due, in part, to the presence of antibodies to conserved gp120 conformational epitopes. Purified conformation-dependent gp120 specific human antibodies neutralized a wider range of virus isolates than human antibodies directed to linear determinants in gp120 and were also responsible for the majority of the gp120-specific CD4-blocking activity of HIV-1-infected human sera. A gp120 subunit vaccine that effectively presents these conformation dependent neutralization epitopes should protect against a broader range of HIV-1 variants than a vaccine that presents exclusively linear determinants. PMID- 1718037 TI - A bacterial system for investigating transport effects of cystic fibrosis- associated mutations. AB - LIV-I, a high-affinity system that transports neutral, branched-chain amino acids into Escherichia coli, has two components, LivG and LivF, that are homologous to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). CF associated mutations of human CFTR were introduced into corresponding regions of LivG, and their effects on leucine transport could be grouped into three classes. Mutations were found that (i) abolished LIV-I--directed transport, (ii) retained about a quarter of wild-type activity at the Michaelis-Menten constant (KM), and (iii) had minimal activity at the KM. A mutation equivalent to a benign polymorphism had no effect on transport. The correlation of these mutational phenotypes in LivG and CFTR suggests that the LIV-I prokaryotic transporter is functionally similar to the CF protein and that this similarity can be exploited to clarify the properties of the nucleotide-binding fold in this superfamily of proteins. PMID- 1718038 TI - Regulation of transendothelial neutrophil migration by endogenous interleukin-8. AB - Movement of neutrophils from the bloodstream to inflamed tissue depends on the activation of both the neutrophil and the endothelial cell. Endothelial cells lining the postcapillary venule respond to proinflammatory mediators by expressing adhesion molecules and synthesizing a variety of neutrophil-activating factors. Endothelial cell production of a 77-amino acid variant of interleukin-8 (IL-8) was found to be a requirement for the invasion of neutrophils through a vessel wall model. IL-8 secreted by cytokine- or lipopolysaccharide-stimulated endothelial cells induced the rapid shedding of neutrophil lectin adhesion molecule-1, the up-regulation of leukocyte beta 2 integrins, and the attachment and transmigration of the neutrophils. Thus, endogenous endothelial IL-8 regulates transvenular traffic during acute inflammatory responses. PMID- 1718039 TI - Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. AB - The relation between chromatin structure and transcriptional activity was examined by in vitro transcription analysis of chromatin reconstituted in the absence or presence of histone H1. To maintain well-defined template DNA, purified components were used in the reconstitution of chromatin. Reconstitution of nucleosomal cores to an average density of 1 nucleosome per 200 base pairs of DNA resulted in a mild reduction of basal RNA polymerase II transcription to 25 to 50 percent of that obtained with naked DNA templates. This nucleosome-mediated repression was due to nucleosomal cores located at the RNA start site and could not be counteracted by the sequence-specific transcription activators Sp1 and GAL4-VP16. When H1 was incorporated into the chromatin at 0.5 to 1.0 molecule per nucleosome (200 base pairs of DNA), RNA synthesis was reduced to 1 to 4 percent of that observed with chromatin containing only nucleosomal cores, and this H1 mediated repression could be counteracted by the addition of Sp1 or GAL4-VP16 (antirepression). With naked DNA templates, transcription was increased by a factor of 3 and 8 by Sp1 and GAL4-VP-16, respectively (true activation). With H1 repressed chromatin templates, however, the magnitude of transcriptional activation mediated by Sp1 and GAL4-VP16 was 90 and more than 200 times higher, respectively, because of the combined effects of true activation and antirepression. The data provide direct biochemical evidence that support and clarify previously proposed models in which there is depletion or reconfiguration of nucleosomal cores and histone H1 at the promoter regions of active genes. PMID- 1718040 TI - Brave new (RNA) world. PMID- 1718041 TI - Ion channel research wins physiology Nobel. PMID- 1718042 TI - Primary structure and functional expression of the 5HT3 receptor, a serotonin gated ion channel. AB - The neurotransmitter serotonin (5HT) activates a variety of second messenger signaling systems and through them indirectly regulates the function of ion channels. Serotonin also activates ion channels directly, suggesting that it may also mediate rapid, excitatory responses. A complementary DNA clone containing the coding sequence of one of these rapidly responding channels, a 5HT3 subtype of the serotonin receptor, has been isolated by screening a neuroblastoma expression library for functional expression of serotonin-gated currents in Xenopus oocytes. The predicted protein product has many of the features shared by other members of the ligand-gated ion channel family. The pharmacological and electrophysiological characteristics of the cloned receptor are largely consistent with the properties of native 5HT3 receptors. Messenger RNA encoding this receptor is found in the brain, spinal cord, and heart. This receptor defines a new class of excitatory ligand-gated channels. PMID- 1718043 TI - [PMN-elastase as a marker in diagnosis and follow-up of bone and joint infections]. AB - PMN elastase, a proteolytic enzyme, is a biochemical marker for pathologic granulocyte stimulation. In the presence of sepsis, excessive neutrophil stimulation occurs and significant amounts of PMN elastase are released into the plasma and serve as an indicator for the severity of the disease and the prognosis. PMN elastase is also a useful parameter for preoperative diagnostic management and postoperative follow-up of bone and joint infections. In patients with osteomyelitis and joint empyema (n = 48) PMN elastase had a sensitivity of 77%, which was only exceeded by that of the unspecific erythrocyte sedimentation rate (sensitivity 89%). Sensitivities of other inflammation parameters were lower: C-reactive protein (CRP) 67%, fibrinogen 50%, neopterin 32% and leukocyte count 21%. Determination of PMN elastase levels was also helpful in postoperative follow-up of patients with bone and joint infections. In the early postoperative period PMN elastase levels normalized more quickly than the other parameters unless patients actually developed complications. At the first postoperative determination (day 2-4 after surgery) 38% of the patients (n = 24) already had PMN elastase levels within the normal range (less than or equal to 40 micrograms/l) (CRP 13%). After 10 days PMN elastase was normal in 57% and CRP in 30% of the patients. Later on both parameters reacted similarly: by the time of discharge from hospital levels of PMN elastase were normal in 70% and CRP levels in 74%. PMID- 1718044 TI - Immunology of the maternal-placental interface in normal pregnancy. PMID- 1718045 TI - [The combined radiation and hormonal therapy of prostatic carcinoma: a conceptual analysis]. AB - The concept of a combination of radiotherapy and hormonal therapy for the treatment of locally advanced carcinoma of the prostate was evaluated in view of an improved success rate compared to radiotherapy alone. At present, however, radiotherapy still remains the central mode of therapy in the treatment of the local tumor. The time sequence in a combination with hormonal therapy is of importance. Antecedent hormonal treatment can reduce tumor size, thus improving local conditions for percutaneous--and especially interstitial--radiotherapy; it is indicated in patients with voiding problems. An improvement in survival rates has so far not been achieved, however, local tumor remission rates are better. With concomitant hormonal therapy, compared to radiotherapy alone, tumor progression rates are lower. It remains to be seen, whether these results can be improved by using newer pharmacological ways of androgen blockade. The application of androgen withdrawal as a secondary measure with local tumor persistence or progression after radiotherapy remains a palliative treatment of limited efficacy. Due to the heterogeneity of the available study reports and the concurrent lack of controlled, randomized trials, a conclusive evaluation of the concept of combined radio- and hormonal therapy of prostatic cancer is as yet not possible. PMID- 1718046 TI - Neuroendocrine differentiation and prognosis of extrahepatic biliary tract carcinomas. AB - From 1980 to 1987, 35 patients underwent exploratory surgery for carcinomas of the extrahepatic biliary tract (EBT). Samples from 28 of these tumors (15 gallbladder, 13 bile duct) were assessed by immunohistochemical analysis for exocrine and/or neuroendocrine differentiation. Seven patients were excluded from the study because of insufficient available specimen or loss to follow-up. Paraffin sections were immunostained for neuroendocrine differentiation markers: neuron-specific enolase (NSE), chromogranin-A, synaptophysin, serotonin, somatostatin, substance-P, and glucagon. Additional sections were also stained with monoclonal antibody A-80 that recognizes a glycoprotein related to exocrine differentiation. The tumors were reclassified on the basis of immunophenotyping data: (I) pure exocrine carcinoma (n = 8); (II) predominantly exocrine carcinoma with occasional neuroendocrine cells (n = 9); (III) mixed exocrine-neuroendocrine carcinoma (n = 4); (IV) pure neuroendocrine (n = 2); and (V) predominantly neuroendocrine with occasional exocrine cells (n = 5). Survival time among the two pure neuroendocrine (group IV) and five predominantly neuroendocrine carcinomas (group V) was significantly less than the survival time of patients from the other groups (2.6 +/- 2.2 months vs 13.5 +/- 12.3 months; p = 0.015). No difference was noted between groups in extent of disease, treatment rendered, or location of tumor (bile duct vs gallbladder). This study indicates that (1) the incidence of neuroendocrine differentiation in cancers of the EBT is higher than generally recognized, (2) carcinomas of the EBT may be phenotypically reclassified on the basis of immunohistochemical analysis, and (3) the presence of pure or predominant neuroendocrine differentiation in carcinomas of the EBT is associated with shorter survival time than carcinomas with pure or predominant exocrine differentiation (or mixed exocrine and neuroendocrine factors). PMID- 1718047 TI - Control of cancer pain by epidural infusion of morphine. AB - Pain that cannot be controlled by traditional oral and parenteral methods in those patients with advanced cancer can be alleviated by spinal administration of narcotics. Epidural and intrathecal infusion with morphine causes analgesia by blocking spinal receptors without significant long-term central nervous, gastrointestinal, and genitourinary system effects. Of the total of 33 patients, epidural catheters inserted in 20 patients then connected by a subcutaneous tunnel to a continuous infusion system. Implanted pumps were used in each of these patients. Because of the cost and limitations of the implanted pumps, epidural catheters were connected, either directly or by subcutaneous reservoirs, to external ambulatory infusion pumps in the remaining 13 patients. Patient assessment by a linear analogue scale to measure pain levels determined that 23 of the 33 total patients (70%) had excellent or good relief of pain. The delivery of spinal administration of narcotics to treat intractable cancer pain in patients is safe. Most importantly, this method of delivery can be used in community hospitals, in outpatient settings, and in home health care programs. PMID- 1718048 TI - Bilateral cancer of the breast: a review of clinical, histologic, and immunohistologic characteristics. AB - The clinical, histologic, and immunohistologic characteristics of 19 synchronous and 47 metachronous bilateral breast cancers was compared. Patients with metachronous tumors were 5 years younger and more likely to have a family history of breast cancer than those patients with synchronous cancers. The nondominant synchronous cancer was usually discovered mammographically accounting for small, node-negative tumors, and high prevalence of in situ lesions. The same was true of the second metachronous tumor when discovered mammographically. Patients with metachronous cancers who were not in a follow-up program had second cancers with characteristics similar to incidentally diagnosed unilateral cancer. The mean interval between metachronous cancers was 101 months. Significantly more first metachronous tumors were invasive lobular cancers. Histologic type of the first and second tumor was the same in only 68% of synchronous and 61% of metachronous cancers. Combined histologic evidence and differentiation was concordant in only 13% and 22% of tumors, respectively. Immunoperoxidase studies were performed with two human milk fat globule antibodies. Each antibody reacted similarly in the first and second tumor in less than 50% of tumors and concordance was less than 25% when both antibody reactions were assessed. Differences in histologic evidence, differentiation, and immunohistologic reaction suggest that both synchronous and metachronous cancers are morphologically and functionally dissimilar. PMID- 1718049 TI - Dopaminergic modulation of excitotoxicity in rat striatum: evidence from nigrostriatal lesions. AB - Considerable evidence indicates that dopamine may, under certain circumstances, play a role in the mediation of central nervous system tissue damage. Furthermore, recent studies suggest a synergistic role between the neurotoxic effects of excitatory amino acids and dopamine. To address this issue, rats received a unilateral injection of 6-hydroxydopamine or vehicle into the medial forebrain bundle. After recovery (18 days), both groups of animals received an ibotenic acid injection of the ipsilateral striatum. Seven days later the brains were removed and the size of the striatal lesion was assessed histologically and by means of receptor autoradiography. Regional analysis of profound D1 receptor loss was determined using [3H]SCH 23390, and extent of astrocytic proliferation was examined using autoradiography with the peripheral benzodiazepine receptor ligand [3H]R05-4864. Prior interruption of the nigrostriatal pathway (resulting in dopaminergic denervation of the ipsilateral striatum) partially protected this latter structure from subsequent injection of ibotenic acid (the extent of the lesion was reduced by 28%, P less than .05). The findings indicate that endogenous dopamine release may modulate (and intensify) the excitotoxic effects of ibotenic acid. PMID- 1718050 TI - Structure of vitronectin and its biological role in haemostasis. PMID- 1718051 TI - Leukocyte interactions mediated by selectins. PMID- 1718052 TI - Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction. AB - Platelet glycoprotein Ib (GPIb) acts as a high-affinity thrombin binding site and as a receptor for von Willebrand Factor (vWF). A new anti-GPIb monoclonal antibody (mAB) VM16d was produced that specifically inhibited platelet-thrombin but not platelet-vWF interaction. The epitope for VM16d was located within the 45 kDa N-terminal region of the alpha-chain of GPIb. VM16d inhibited platelet aggregation induced by low dose thrombin (0.05 U/ml) but did not affect platelet aggregation induced by ristocetin, bovine vWF, ADP or collagen. The same inhibitory effects on thrombin-induced platelet aggregation were observed with the whole IgG molecule of VM16d and its F(ab')2 and F(ab') fragments. VM16d also inhibited 14C-serotonin secretion induced by low dose thrombin and binding of 125I-thrombin but not ristocetin-dependent binding of 125I-vWF to platelets. These data indicate that the high-affinity thrombin binding site is located on the N-terminal 45 kDa domain of GPIb and that it is topographically separated from the vWF binding site. PMID- 1718053 TI - Avian permeability barrier function reflects mode of sequestration and organization of stratum corneum lipids: reevaluation utilizing ruthenium tetroxide staining and lipase cytochemistry. AB - The epidermis of avians and terrestrial mammals has evolved distinct, but related mechanisms to survive in a terrestrial environment. In both phyla, stratum corneum lipids form the basis of the cutaneous permeability barrier, but barrier function is less efficient in avians. Whereas in mammals the epidermal lamellar body (LB) secretes its contents into the intercellular spaces, in the feathered epidermis of avians, its distinctive secretory organelle, the multigranular body (MGB), does not secrete its contents into the stratum corneum intercellular spaces. Yet, neutral lipid-enriched droplets, derived from the cytosolic breakdown of MGB, ultimately are squeezed through membrane pores into the stratum corneum interstices. In this study we determined: a) using ruthenium tetroxide (RuO4) fixation, whether these droplets form membrane structures after deposition in the stratum corneum interstices; and b) the similarities and differences between avian MGB and mammalian LB, using enzyme cytochemistry as a marker for secretion, and optical diffraction computer-aided image analysis and reconstruction to compare the internal structure of MGB vs. LB. MGB were shown to possess a similar lamellar substructure to LB in RuO4-fixed specimens, exhibiting comparable dimensions on optical diffraction and computer transform analysis. Moreover, the intercellular lipids of avian stratum corneum lacked membrane substructure, as was present in parallel samples of mammalian stratum corneum. Thus, both the absence of MGB secretion, and the failure of intercellular lipids to form membrane bilayers may explain the inherent differences in barrier function in these two taxa. PMID- 1718054 TI - Immunohistochemical studies on the spinal dorsal horn of the turtle Chrysemys d'orbigny. AB - Immunohistochemical methods were used to characterize some of the systems of nerve fibers occurring in the spinal dorsal horns of the turtle Chrysemys d'orbigny. Substance P (SP), calcitonin gene-related peptide (CGRP) and leuenkephalin (Enk) immunoreactive fibers were found concentrated in the superficial horn region, termed here synaptic field Ia. From this zone the immunoreactive fibers project to deeper dorsal horn regions. Comparison with histological images obtained after HRP labeling of dorsal root axons indicates that SP-, CGRP- and Enk-immunoreactive fibers are small-diameter primary sensory fibers entering the cord via synaptic field Ia. It is posulated here that these three substances may coexist in the same fibers. Enk-positive fibers also occur randomly scattered in the lateral funiculi, showing a conspicuous increase in density at the perimedullary plexus level. Tyrosine hydroxylase (TH) immunoreactive fibers were found in the more compact dorsal horn neuropil (synaptic field II) and also forming bilateral conspicuous bundles in the lateral funiculi. TH-immunoreactive cell bodies were found in the epithelium lining the central canal. Taking into account data derived from Golgi impregnated material it is proposed that they represent epithelial cells undergoing neural differentiation. PMID- 1718055 TI - Regional effects on the cerebral concentration of noradrenaline, serotonin and dopamine in suckling rats after a single dose of lindane. AB - The effect of a low single dose of lindane (gamma-hexachlorocyclohexane) on monoaminergic neurotransmission during brain postnatal development was studied in 8-, 15-, 22- and 29-day-old suckling rats. Concentrations of noradrenaline, serotonin, dopamine and their metabolites were determined in eight cerebral regions 1 h after dosing (20 mg/kg lindane per os). All these aminergic systems were altered in a regional- and age-related pattern. Decreases of noradrenaline in regions rich with noradrenergic terminals together with increases of the ratio 5-HIAA/serotonin and DOPAC/dopamine suggest, as a whole, an enhanced release of the monoamines. This increased neuronal activity does not exclude an initial action of lindane on the inhibitory GABAergic system that would activate the neurotransmitter release in other systems shortly after a single administration of the neurotoxic agent. PMID- 1718056 TI - Peripheral blood neutrophil function in germfree and conventional rats. AB - The superoxide anion production and the myeloperoxidase activity between polymorphonuclear cells (PMNs) from conventional and germfree rats were compared. Furthermore, the response to recombinant human granulocyte colony stimulating factor (G-CSF) between both groups of rats was also examined. Our present investigation showed that: (1) the basal superoxide anion production by PMNs in germfree rats (2.1 +/- 0.5 nmol/min/1 x 10(6) PMNs) is less than one fourth of that of conventional rats (9.5 +/- 2.9 nmol/min/1 x 10(6) PMNs). (2) the myeloperoxidase activity of PMNs from germfree rats (38.1 +/- 5.2 nmol/min/1 x 10(6) PMNs) is 1.7 times that of conventional rats (22.4 +/- 4.9 nmol/min/1 x 10(6) PMNs). (3) G-CSF administration increased the number of PMN, 12.4 times in the germfree rats compared with 3.9 times in the conventional rats. (4) the PMNs induced by G-CSF do not differ in the superoxide anion producing activity. However the myeloperoxidase activity decreased in the G-CSF induced PMNs of both groups of rats. From our data, the conventional rats could have a more effective killing activity than the germfree rats. Furthermore, germfree rats may be used as a powerful tool for assay of other biological substances relating to immune system and its regulation. PMID- 1718057 TI - Immunological effects of retinoids. AB - Vitamin A treatment (100,000 U daily) of systemic lupus erythematosus, non Hodgkin's lymphoma and chronic lymphocytic leukemia patients, children suffering from recurrent respiratory tract infections and healthy controls resulted in an enhancement of antibody-dependent cell-mediated cytotoxicity, natural killer activity and blastogenic response to mitogens. In vitro, retinoids, depending on the concentration, stimulated mitogen- and interleukin-2-induced blastogenesis and lectin-dependent T cell cytotoxicity. Retinoids caused an early plasma membrane hyperpolarization of cells of various origin. This effect was similar to that seen by interferon alpha. Retinoids also slightly inhibited intracellular calcium accumulation. PMID- 1718058 TI - Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on allogeneic bone marrow transplantation in mice. AB - The effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) on the survival rate and hemopoiesis after allogeneic bone marrow transplantation (BMT) was examined. Specific pathogen-free (SPF) C3H mice, kept in a flexible isolator for germ-free animals, received 10Gy of total body irradiation (TBI), and an allogeneic BMT 24 hours later. The 14 day survival rate was 33%. When the mice were received rhG-CSF (30 micrograms/kg/day subcutaneously) daily for 2 weeks following BMT, the 14 day survival rate rose to 79%. The effect of rhG-CSF treatment on hemopoiesis in BMT mice resulted in higher neutrophil counts in the peripheral blood by day 14 post-BMT. Administration of rhG-CSF after allogeneic BMT benefited survival rate and neutrophil recovery without any apparent complications. PMID- 1718059 TI - Suppression of messenger ribonucleic acid for glutathione peroxidase in chemically induced rat hepatocellular carcinoma and its biological significance. AB - Deviations of the enzyme activity, immunoreactivity and messenger ribonucleic acid (mRNA) levels of glutathione peroxidase (GSH-PO) in 3'-methyl-4 dimethylaminoazobenzene- induced hepatocellular carcinoma of the rat were investigated. Enzyme activities of GSH-PO were significantly lower in hepatocellular carcinomas than those in the normal control rat liver. Immunohistochemically, GSH-PO was strongly localized in normal hepatocytes, but was only faintly stained in hepatocellular carcinoma cells. Heterogeneous staining patterns of GSH-PO were observed among individual cancer cells. In Northern blot analysis, GSH-PO mRNA in the cancer tissue was decreased to two thirds of the level in normal hepatocytes. It was suggested that suppressed expression of GSH-PO in carcinogen-induced hepatocellular carcinomas occurred at the level of mRNA transcription. PMID- 1718060 TI - "Mucin types" in normal and neoplastic colonic goblet cells: a mucin histochemical study. AB - Goblet cells in normal colon mucosa from 100 individuals histochemically showed two major "mucin types" in sialic acid composition. One type revealed mixed reactivities for N-acetyl-neuraminic acid (NANA, abbreviated C0), 7-O- acylated NANA (C7) and 9-O-acylated NANA (C9), and represented 25% of the individuals examined. The other type of mucin, seen in 69% of the cases, exhibited reactivity for 8-O-acylated NANA or pluri-O-acylated NANA (C8). C0 and C8 were observed simultaneously in 5% of the individuals. In addition, 8% displayed a mosaic pattern in which isolated "blue" crypts containing sialic acids of the C0 + C7 + C9 type were detected in the "red" colonic mucosa, which was predominantly C8. Little correlation was found between the mucin types and their site in the colon, or with the age, sex, ABO blood groups and degree of sulphation. O-acylated NANA was also observed, though reduced in amount, in all 22 colonic adenocarcinomas in which evaluable numbers of neoplastic goblet cells were found. The mucin types in colon cancer were fundamentally indistinguishable from those seen in the surrounding noncancerous colonic mucosa. PMID- 1718061 TI - [Ribosomal vaccines in oral medicine]. PMID- 1718062 TI - Conjugated superoxide dismutase reduces extent of caudate injury after transient focal ischemia in cats. AB - We tested the efficacy of preischemic and postischemic systemic treatment with 30,000 units polyethylene glycol-conjugated superoxide dismutase in a reperfusion model of focal cerebral ischemia. Forty-one anesthetized cats underwent 2 hours' occlusion of the left middle cerebral artery and both common carotid arteries followed by 4 hours of reperfusion. Cats were blindly assigned to one of three groups: treatment with vehicle (10% polyethylene glycol in saline, n = 17), pretreatment with drug 3 hours before ischemia (n = 12), and posttreatment with drug at the time of reperfusion (n = 12). Size of the ischemic injury was calculated from 2,3,5-triphenyltetrazolium chloride staining. Injury in the caudate nucleus was significantly reduced with pretreatment (28 +/- 6% of ipsilateral caudate volume, mean +/- SEM) compared with the vehicle (56 +/- 8%). Posttreatment did not significantly ameliorate caudate injury (46 +/- 10%). Between the first and second hours of ischemia ipsilateral caudate blood flow determined using microspheres increased significantly from 11 +/- 4 to 16 +/- 5 ml/min/100 g with pretreatment, but blood flow remained constant throughout ischemia with vehicle (8 +/- 2 ml/min/100 g) and posttreatment (10 +/- 3 ml/min/100 g). The size of cortical injury (vehicle, 17 +/- 5%; pretreatment, 11 +/- 3%; posttreatment, 17 +/- 5% of hemispheric volume) did not differ significantly among groups. Somatosensory evoked potential recovery did not differ among groups. We conclude that pretreatment with conjugated superoxide dismutase can ameliorate the extent of injury in an end-artery region, such as the caudate nucleus, in a reperfusion model of focal ischemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718063 TI - Prevention of graft-versus-host disease following allogeneic bone marrow transplantation in rats using FK506. AB - FK506 and cyclosporine were used for the prevention of acute graft-versus-host disease. Acute GVHD was induced in Lewis rats by total-body irradiation and subsequent reconstitution with allogeneic (ACI) bone marrow and spleen cells (BMTx). GVHD was assessed by both clinical and histologic parameters during the experiment duration of 60 days, and longer for selected animals. All untreated BM recipients died within 26 days from severe acute GVHD. GVHD was prevented with CsA during the period of immunosuppressive therapy, but it appeared within a few days afterward. FK506-treated BM recipients were also protected, but they had a markedly prolonged GVHD-free period after therapy was discontinued. Most such animals eventually developed GVHD but with notable exceptions. Maintenance therapy with doses of FK506 as low as 0.1 mg/kg every other day (1/20 of daily induction dose) was infallible insurance against delayed GVHD. The relevance of these findings to GVHD caused by lymphoid-containing solid organs such as the intestine was discussed. PMID- 1718064 TI - The necessity of differential immunosuppression for prevention of immune rejection by FK506 in rat islet allografts transplanted into the liver or beneath the kidney capsule. AB - The purpose of the present study was to achieve prevention of immune rejection in rat islet allografts by FK506. WKA/Qdj (RT1u) islets were transplanted either into the liver via the portal vein (p.v.) or beneath the kidney capsule (k.c.) of streptozotocin (60 mg/kg) induced diabetic Lewis (RT1(1)) rats. Fresh or cultured (24 degrees C, 1 week) islets were used as donors. A miniosmotic pump (0.2 ml, Alzet 2001) containing 5 mg FK was implanted s.c. at the time of transplantation for continuous delivery of FK506 for 7 days after transplantation. The mean survival time (MST) of the fresh p.v. grafts with a pump was offater than 61.4 +/ 37.2 days (mean +/- SD, n = 17) (control 5.5 +/- 0.6, n = 4). Ten out of 17 were normoglycemic for more than 90 days after transplantation. When low-temperature cultured islets were used and FK506 was delivered for 7 days, all the rats were normoglycemic for more than 90 days after transplantation. The MST of the fresh or cultured k.c. grafts with a pump was 22.0 +/- 14.2 or 24.7 +/- 5.0 days, respectively. Long-term administration of FK506 by repeated implantations (5 times; days 0, 7, 14, 21, and 28) of pumps containing 5 mg FK506 produced marked prolongation of the fresh or cultured k.c. graft survival with an MST of greater than 58.7 +/- 22.1 or greater than 56.9 +/- 18.0 days, respectively. These findings clearly demonstrate that the prevention of immune rejection in the islet allografts transplanted into the liver was achieved by short-term post-transplant administration of FK506 and low-temperature culture of donor islets, and also show that long-term continuous administration of FK506 was needed for the prolongation of the graft survival when the renal subcapsular space was the site for implantation of islets. Thus, the present study indicates that in different transplant sites different immunosuppressive regimes are needed for the control of rejection by FK506 in rat islet allografts. PMID- 1718065 TI - The effect of low-potassium-dextran versus Euro-Collins solution for preservation of isolated type II pneumocytes. AB - Limited availability of donor organs is a major factor restricting the clinical application of lung transplantation. Improvements in preservation techniques are essential for prolonging storage time and improving lung function following transplantation. The present investigation used primary cultures of adult rat alveolar type II cells as a model for evaluating lung-preservation solutions. Type II cells were plated onto tissue-culture plastic at a density 5 x 10(5) cells/cm2 and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (D10) for 40 hr. Cells were then exposed to Euro-Collins solution or a low-potassium-dextran solution (LPD). At designated time points, measurements of lactate-dehydrogenase (LDH) release, protein content, and incorporation of 3H-thymidine into cellular DNA were made. During 12 hr of "storage" at 37 degrees C, cells maintained in LPD released less LDH (14.3 +/- 1.2% of cellular total, mean +/- SEM, n = 5) than their counterparts stored in EC (20.6 +/- 1.6%, P less than 0.05). During the 36 hr following a 6-hr exposure to preservative solutions, LPD-treated cells incorporated more thymidine per mg of protein (2566 +/- 419.8 cpm/micrograms protein, mean +/- SEM, n = 6) compared with cells maintained continuously in D10 (1431 +/- 351, P less than 0.05). By contrast, cells exposed to EC incorporated less thymidine (82.2 +/- 62.8 cpm/micrograms protein) than either cells maintained in LPD or D10 (P less than 0.01 for each comparison). These results suggest that LPD solution is less cytotoxic than EC and that LPD enables higher levels of metabolic activity in recovering epithelial cells. In vitro cultures of type II epithelial cells are a useful model system for the study of lung preservation and posttransplantation lung injury. PMID- 1718066 TI - The effect of PGE1 and temperature on lung function following preservation. AB - We studied the effect of a vasodilator (prostaglandin E1) as well as flush (F) and storage (S) temperatures (4 degrees C or 10 degrees C) on lung preservation in an isolated rabbit lung perfusion model. Low-potassium dextran (LPD) or Euro Collins (E-C) solution was used as flush solution. Six groups of six animals were studied: group 1 (LPD, 4 degrees C F-S), group 2 (LPD with PGE1, 4 degrees C F S), group 3 (E-C with PGE1, 4 degrees C F-S), group 4 (LPD, 10 degrees C F-S), group 5 (LPD with PGE1, 10 degrees C F-S), group 6 (E-C with PGE1, 10 degrees C F S). After 18-hr preservation, left lungs alone were ventilated, and reperfused with fresh venous blood. PaO2, PaCO2, pulmonary artery pressure (PAP), tracheal pressure (Pt) during reperfusion, and wet/dry weight (W/D) ratios were measured. PaO2 after LPD with or without PGE1 was significantly higher than after E-C with PGE1 at 4 degrees C (95.8 +/- 11.5 mmHg in group 1 or 102.7 +/- 8.6 in group 2 vs. 41.8 +/- 10.5 in group 3, P less than 0.01) and at 10 degrees C (119.3 +/- 2.3 in group 4 or 131.1 +/- 6.2 in group 5 vs. 54.6 +/- 5.2 in group 6, P less than 0.01). PaCO2, PAP, Pt, and W/D ratios in the LPD groups were lower than in the E-C groups. LPD/PGE1 and LPD alone produced similar pulmonary preservation. PaO2 of lungs flushed with LPD and preserved at 10 degrees C was higher than that of lungs stored at 4 degrees C. We conclude that LPD solution is superior to E-C solution in this ex vivo rabbit lung preservation model, even when PGE1 is used. A moderate dose of PGE1 did not improve the performance of LPD as a flush solution. Pulmonary preservation with LPD at 10 degrees C is superior to preservation at 4 degrees C. PMID- 1718067 TI - OKT3E, an anti-CD3 antibody that does not elicit side effects or antiidiotype responses in chimpanzees. AB - Chimpanzees were injected with OKT3 and two other anti-CD3 antibodies, OKT3D and OKT3E. Both of the new antibodies were of the mouse IgG2b isotype. Administration of the antibodies was identical to the clinical regimen used for OKT3 in humans: 5 mg i.v., daily for 10 consecutive days. All animals were monitored for fever during administration of the antibodies, and blood samples were taken throughout the treatment period for monitoring the effects of the antibodies on peripheral lymphocyte subsets and the appearance of circulating cytokines. OKT3 produced similar clinical effects to those observed in humans; fever (2/3), as well as elevations in cytokines were observed. As in humans, peripheral T cells were cleared with the first dose and remained absent or modulated of their T cell receptor molecules throughout treatment. OKT3D, IgG2b also produced fevers (2/3) and elevations of cytokines. Although it also cleared circulating T cells with the first dose and T cell counts remained low throughout treatment, remaining circulating T cells were coated with administered antibody and were able to reexpress the CD3 antigen. OKT3E, IgG2b produced no temperature elevations and no elevations in cytokines. Although it cleared the circulation of T cells with the first does, cells reappeared during treatment, modulated of their CD3 antigens or coated with the administered antibody. All three antibodies raised antimouse antibodies, and OKT3 and OKT3D also produced blocking antiidiotype antibodies. OKT3E treatment did not result in anti-OKT3E blocking antibodies. PMID- 1718068 TI - Hepatotrophic properties in dogs of human FKBP, the binding protein for FK506 and rapamycin. PMID- 1718069 TI - Postischemic recovery of the lung--biochemical and morphological studies of long term preserved lungs. PMID- 1718070 TI - Examination of the role of the colloids hydroxyethylstarch, dextran, human albumin, and plasma proteins in a modified UW solution. AB - The delayed contralateral nephrectomy procedure (three-step) produced inconsistent results, indicating that the preserved autotransplanted kidney tends to remain unfunctional and to regenerate incompletely unless the demand for work is placed upon it. Omission of HES in UW (UW-plain) did not affect preservation success, but resulted in increased graft edema. Substitution of HES in UW with plasma (SGF-V) or albumin (MAlb) gave significantly worse results than UW-like solutions with or without synthetic colloids. Replacement of HES in UW with UMdex 70 or UMdex-500 gave nonsignificantly worse results than UW-like solutions with or without synthetic colloids. The use of UMdex-40 as the main colloid in UW cheapened the solution, equalled the preservation success of UW and UW-plain but surpassed UW-plain in edema prevention, and exceeded UW concerning recovery of graft microcirculation. PMID- 1718071 TI - Treatment of preservation/reperfusion liver injury by the protease inhibitor aprotinin after cold ischemic storage. PMID- 1718072 TI - Questionable role of leukocyte sticking in the pathogenesis of preservation damage. PMID- 1718073 TI - Early generalized activation of eicosanoid synthesis after liver transplantation. PMID- 1718074 TI - Influence of Iloprost on insulin synthesis in isolated human islets of Langerhans. PMID- 1718075 TI - In situ cooling with use of a new trypsin inhibitor in canine pancreatico duodenal transplantation from non-heart-beating donors. PMID- 1718076 TI - [The use of RNAse A-colloidal gold complexes for research on RNA-containing cell structures: the effect of different factors on the specificity of the labelling]. AB - Effects of various factors on the specificity and intensity of labelling of RNA containing structures on the ultrathin sections by RNase A--colloidal gold complexes were studied. The data obtained show that at the optimal choice and standard conditions of labelling the reproducibility of the results is achieved up to 10-20%. It makes it possible to use RNase A-gold method for the quantitative analysis of RNA distribution in the cells at various stages of cell cycle. PMID- 1718077 TI - [A comparative analysis of the biological activity of an acid-extractable brain protein and fibroblast growth factors]. AB - An acid-soluble protein was isolated from bovine brain tissue using a combination of 5% HClO4 acidic extraction, cation-exchange chromatography and heparin Sepharose affinity chromatography. This preparation had a biological activity similar to that of the fibroblast growth factor (FGF). It has stimulated neurite outgrowth in organotypic culture of chicken embryo dorsal root ganglia and also has stimulated a proliferation of human embryonic fibroblasts in the culture. The biological activity of isolated preparation was totally inhibited by anti-bFGF antibodies, while anti-aFGF antibodies had no effect. The bFGF showed a neurite stimulating activity in the organotypic culture that was completely abolished by the anti-bFGF, IgG, while the aFGF was inactive. This data, as well as cationic properties of isolated protein and its heparin-binding ability, enable us to postulate a high degree of homology of the brain acid-soluble protein and the bFGF. At the same time the relations of the isolated growth factor and other brain-derived heparin-binding growth factors are yet to be established. PMID- 1718078 TI - [The effect of steroid sex hormones on the biosynthesis of alpha 2-macroglobulin and pregnancy-associated alpha 2-glycoprotein in cultures of peripheral blood mononuclear cells]. AB - The peripheral blood mononuclears are capable of intense biosynthesis of alpha 2 macroglobulin and of weak biosynthesis of pregnancy-associated alpha 2 glycoprotein. Sex hormones of men and non-pregnant women exert no influence on the protein biosynthesis. During pregnancy alpha 2-macroglobulin biosynthesis is shortly activated, although it does not depend on the influence of sex hormones. All the steroid sex hormones provide a short-term biosynthesis of this protein during the II trimester of pregnancy, while testosteron inhibits it during the III trimester. Possible mechanisms of control of biosynthesis of these proteins are discussed. PMID- 1718079 TI - Spindle cell squamous carcinoma of the oral region. An immunohistochemical and ultrastructural study on the histogenesis and differential diagnosis with a clinicopathological analysis of six cases. AB - Six cases of spindle cell squamous carcinoma (SCSC) of the oral cavity were studied clinicopathologically, immunohistochemically and ultrastructurally to summarize the clinicopathological features of this rare neoplasm and to discuss the debatable histogenesis of the sarcomatoid component and the differential diagnosis of SCSC. The mean age of the patients was 72 years and the female to male ratio was 1:2. Four of them had a history of irradiation for pre-existing squamous cell carcinoma. One patient died of SCSC. While clinical and histological prognostic factors of SCSC could not be determined, it was shown that radical surgery resulted in good prognosis. The epithelial nature of the sarcomatoid component of SCSC was clearly revealed by a combination of immunohistochemical staining for keratins and electron microscopic demonstration of tonofilament-like filaments and/or desmosome-like structures. Together with electron microscopic evaluation of the tumour cells, immunohistochemical characterization of tumour cells using antibodies to keratin, vimentin, glial fibrillary acidic protein and S-100 protein is very helpful in differentiating SCSC from true spindle cell sarcoma, melanoma and malignant myoepithelioma. In the immunohistochemical differential diagnosis of SCSC, it is important to remember that SCSC should not be ruled out of the differential diagnosis by a positive reaction for vimentin in sarcomatoid tumour cells. Absence of staining for keratin in the sarcomatoid tumour cells does not always exclude SCSC, because some SCSCs show immunoreactivity of keratin in their sarcomatoid components only with some anti-keratin antibodies. Different kinds of anti-keratin antibodies should be applied in the differential diagnosis of SCSC. PMID- 1718080 TI - Peanut lectin: a histochemical marker for phaeochromocytomas. AB - Fifty-five neuroendocrine tumours and 6 adrenocortical tumours were examined immunohistochemically for the expression of neuron-specific enolase (NSE), chromogranin and synaptophysin. The results were compared with the staining patterns obtained with peanut lectin (PNA) using a streptavidin-biotin staining technique. In separate experiments, sections were preincubated with neuraminidase for the demonstration of masked PNA binding sites. Two of the 24 phaeochromocytomas, 1 of the 6 medullary carcinomas of the thyroid gland, 5 out of the 7 islet cell tumours of the pancreas and all 4 extra-adrenal paragangliomas were negative with PNA. When the sections were first incubated with neuraminidase all these tumours were positive with PNA. Six adrenocortical tumours and 7 neuroblastomas were examined and found to be negative with PNA with or without neuraminidase pre-treatment. Seven carcinoid tumours were examined and found to be positive with PNA only in tubular areas and negative in solid areas; pre-treatment with neuraminidase did not alter the staining pattern. Immunoreactivity for NSE was absent in only 1 of the neuroendocrine tumours. A higher proportion of neuroendocrine tumours was positive with anti-chromogranin than with anti-synaptophysin. PMID- 1718081 TI - Changes in the cellular phenotype and extracellular matrix during progression of estrogen-induced mesenchymal kidney tumors in Syrian hamsters. AB - The cellular origin of estrogen-induced kidney tumors in male Syrian hamsters has been repeatedly the subject of controversy. Several authors have proposed that the tumors arise from proximal tubules, from a combination of tubular and interstitial stromal cells, or solely from interstitial cells. Because of the model character of this tumor for hormone-associated cancer, it was further investigated in this study with respect to morphology, enzyme and intermediate filament pattern, the expression of alpha-smooth muscle actin and the extracellular matrix proteins fibronectin and tenascin. These analyses were carried out with early and late tumors as well as metastases to determine possible changes in expression of biochemical parameters during the development and progression of this neoplasm. The enzyme histochemical and intermediate filament patterns were usually the same as those described previously for proliferative foci and early tumors, i.e. highly elevated activities of glucose-6 phosphate dehydrogenase, adenylate cyclase and alkaline phosphatase, a lack of glucose-6-phosphatase and gamma-glutamyltransferase and coexpression of vimentin and desmin, alpha-smooth muscle actin could not be detected in early lesions. In five of 24 advanced tumors inclusions of kidney tubules were found which showed various degrees of alteration in their morphology and enzyme histochemical pattern, but were often directly connected with tubular segments of normal appearance outside the tumor. Like the normal tubules, the enclosed tubular segments were strongly positive for cytokeratin but never expressed vimentin or desmin. Among the 24 tumors studied, two contained cysts which expressed cytokeratin and sometimes also vimentin but not desmin. The enzyme histochemistry of the cells lining the cysts was similar to that of the surrounding tumor mass, except adenylate cyclase was lacking and alkaline phosphatase was not uniformly distributed. In tumors containing cytokeratin-positive cysts, there often were cytokeratin-positive, vimentin-negative and desmin-negative tumor formations in close contact to these cysts. With the exception of cyst formation, the pattern of metastases were identical to that of the primary tumors. All large tumors and the main component of the metastases expressed vimentin, desmin and fibronectin. Mesothelia surrounding metastatic tumor complexes were positive for vimentin, desmin, alpha-smooth muscle actin, fibronectin, cytokeratin and tenascin. It was concluded from these and previous observations on early stages of tumor development that the estrogen-induced hamster kidney tumor originates from mesenchymal interstitial cells (probably pericytes) which may rarely acquire an epithelial phenotype by metaplastic transformation during tumor progression. PMID- 1718082 TI - Molecular cloning and characterization of a novel human gene homologous to the murine ecotropic retroviral receptor. AB - A novel human cDNA homologous to the murine ecotropic retroviral receptor was cloned from a cDNA library derived from a human T-cell line. The human cDNA is highly homologous to the murine counterpart (87.6% amino acid identity), and its sequence predicts a protein with 629 amino acids (approximately 68 kDa), which is 7 amino acids more than the murine counterpart (622 amino acids). The predicted protein is highly hydrophobic and contains 14 potential transmembrane-spanning domains. No other gene and protein with significant homology to the cloned human gene and the predicted protein were identified by a computer-based search of sequence data banks other than the murine T-cell early activation gene (52.5% amino acid identity) and the murine ecotropic retroviral receptor gene. The human gene is ubiquitously expressed in human tissues and conserved among mammalian species. The genomic gene was also isolated from a cosmid library derived from human lymphocytes, and its organization was elucidated. The gene mapped to human chromosome 13. PMID- 1718083 TI - Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene. AB - The conservation of the herpesvirus DNA polymerases has allowed cross hybridization studies to be used for their identification and mapping on the viral genome. With the use of a DNA fragment containing the DNA polymerase gene of human cytomegalovirus (HCMV) as a hybridization probe, we were able to localize the DNA polymerase gene of murine cytomegalovirus (MCMV) to a region within MCMV EcoRI fragment B which spans the HindIII site separating HindIII fragments D and H. This site is colinear with the HCMV strain AD169 DNA polymerase gene. To confirm that this region encoded the MCMV DNA polymerase gene, we sequenced a 5131 nucleotide fragment from the PstI site in HindIII fragment D to a BglII site in HindIII fragment H. Initiating in HindIII fragment D and extending into HindIII fragment H was a long open reading frame (ORF) 1097 amino acids in length with extensive homology to the DNA polymerases of HCMV, herpes simplex virus, and Epstein-Barr virus. Upstream of the polymerase ORF was a reading frame with considerable homology to the carboxy terminal half of the glycoprotein B gene of human herpesviruses. At early times in the infection, we could detect with a probe representing part of the polymerase ORF two 3' coterminal transcripts, 3.9 kb and 1.7 kb in length. S1 nuclease and exonuclease VII analyses indicated that both transcripts were unspliced and initiated at independent sites in HindIII fragment D. By primer extension, we were able to map precisely the 5' end of the 3.9-kb RNA to a site 186 nucleotides upstream of the beginning of the DNA polymerase ORF. PMID- 1718084 TI - Recombinant gp135 envelope glycoproteins of caprine arthritis-encephalitis lentivirus variants inhibit homologous and heterologous variant-specific neutralizing antibodies. AB - The envelope (env) genes of two antigenic variants of caprine arthritis encephalitis virus (CAEV), defined by serum neutralization, were expressed in vaccinia virus. Recombinant gp135 envelope glycoprotein competitively inhibited neutralizing activity of serum from CAEV-infected goats, indicating gp135 is a dominant target antigen of CAEV neutralizing antibody. In addition, type-specific neutralizing activity of goat serum directed against one variant was inhibited by both homologous and heterologous variant recombinant gp135. Hence, a CAEV variant env gene encodes type-specific neutralization epitopes of both variants. The results indicate that antigenic variation of CAEV involves env gene mutations encoding amino acid differences outside conserved neutralization epitopes affecting epitope exposure to the immune system. PMID- 1718085 TI - Synthetic peptides of the E2 glycoprotein of Venezuelan equine encephalomyelitis virus. II. Antibody to the amino terminus protects animals by limiting viral replication. AB - A peptide composed of the amino-terminal 25 amino acids of the E2 glycoprotein of the virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalomyelitis virus was found to protect peptide-immunized mice from lethal TRD virus challenge (Hunt et al., 1990). Viral growth in peptide-immunized animals was found to be limited in comparison to that in nonimmunized controls. Although both treated and control groups of mice responded to virus challenge by producing neutralizing antibody, only immunized mice with preexisting antipeptide antibody survived. Polyclonal antipeptide sera as well as a monoclonal antipeptide antibody were able to passively protect naive mice from TRD virus challenge, despite the fact that these antibodies were nonneutralizing. Passive transfer of antipeptide antibody to immunosuppressed recipients was not protective, thus indicating that survival of TRD virus challenge required an in situ immune response as well as preexisting antipeptide antibody. Binding studies of both polyclonal and monoclonal antipeptide antibodies indicated that they recognize only epitopes present on virus-infected cells or denatured virus. PMID- 1718086 TI - Purification and partial characterization of equine infectious anemia virus reverse transcriptase. AB - Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme. We have now fractionated EIAV RT by immunoaffinity chromatography with immobilized C2003 antibody. The procedure yielded an equimolar mixture of two proteins of 66 and 51 kDa associated with both DNA polymerase and RNase H activities. When the EIAV RT proteins were examined by in situ activity gel assays, polymerase activity was found to be principally associated with the 66-kDa component. The fidelity of DNA synthesis by EIAV RT was found to be equivalent to that of HIV-1 RT and lower than that of AMV RT. These observations indicate that the RTs of EIAV and HIV-1 share similar structural and functional properties. PMID- 1718087 TI - In vivo neutralization of duck hepatitis B virus by antibodies specific to the N terminal portion of pre-S protein. AB - The neutralization of duck hepatitis B virus (DHBV) infection using antibodies directed against the N-terminal portion of the large surface protein was examined in vitro and in vivo. We demonstrate here that a monoclonal antibody, directed against an epitope mapped between aa 77 and aa 100 on the DHBV pre-S, exerts a similar neutralizing activity (77%) both in vivo and in vitro. Furthermore, we have found that a polyclonal antiserum raised against the bacterially expressed 131 first amino acids of the DHBV pre-S region abolished the infectivity of DHBV in ducklings. Therefore, antibodies against a peptide representing most of the DHBV pre-S region (1-131) or a monoclonal antibody specific to an epitope within this region neutralizes in vivo DHBV infectivity. PMID- 1718088 TI - Use of synthetic peptides to map sequential epitopes recognized by monoclonal antibodies on the bovine leukemia virus external glycoprotein. AB - Six sequential epitopes (A, B, B', D, D', E) were previously defined on the bovine leukemia virus (BLV) envelope glycoprotein gp51 by their reactivity with monoclonal antibodies. A panel of synthetic peptides covering almost the entire sequence of gp51 was tested in enzyme-linked immunosorbent assays in order to localize these epitopes. E was shown to be included in peptide 142-161 (MCF4), B and B' in peptide 195-205, D and D' in peptide 218-237 (MCF6), and A in peptide 249-268 (MCF7). These results extend and confirm previous observations suggesting that the sequential epitopes recognized by our battery of monoclonal antibodies are located in the carboxylic half of BLV gp51. PMID- 1718089 TI - Nucleotide sequence and transcriptional mapping of the major capsid protein gene of pseudorabies virus. AB - The gene encoding the 142-kDa major capsid protein (MCP142) of pseudorabies virus (PrV) was isolated and sequenced. Nucleotide sequence analysis revealed that the MCP142 gene has a single open reading frame of 3993 nucleotides (nt) encoding 1330 amino acids. The 4400-nt major RNA from the MCP142 gene was detected in PrV infected cells. The 5' end of the transcript was located 60 nt upstream of the initiation codon. The 3' end of the transcript was located 18 nt downstream of a putative poly(A) signal sequence TATAAA and 133 nt downstream of the termination codon. In comparing amino acid sequence homology between MCP142 of PrV and other available herpesviruses MCP was shown to have 58% homology with herpes simplex virus type 1 and varicella-zoster virus, 27% with Epstein-Barr virus, and 24% with human herpesvirus 6 and human cytomegalovirus. It has greater homology with those of the alpha-herpesviruses than with those of the beta-herpesviruses and the gamma-herpesviruses. PMID- 1718090 TI - Rat monoclonal antibodies to nonoverlapping epitopes of human immunodeficiency virus type 1 gp120 block CD4 binding in vitro. AB - Monoclonal antibodies (MAbs) to a recombinant form of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1 IIIB) were raised in rats and screened for their ability to block recombinant gp120 binding to recombinant, soluble CD4 (sCD4) in vitro. Four such MAbs were identified and characterised. Each MAb bound strongly to gp120 from eight widely divergent HIV-1 strains from the United States and Africa. Two MAbs were mapped to the fourth conserved (C4) region of gp120, whereas the other two recognised an as yet undefined, conformationally sensitive epitope. MAbs to the latter epitope were the more potent in blocking the gp120-sCD4 interaction. None of the MAbs, however, had potent neutralising activity. PMID- 1718091 TI - A new qualitative variant of the RhE antigen revealed by heterogeneity among anti E sera. AB - We have recently detected a new qualitative variant of the RhE antigen characterised by negative reactions with a minority of anti-E sera, and an inability of the relevant cells to completely absorb most, if not all, anti-E samples that we tested. In tube tests, using a two-stage enzyme technique, cells from the proposita (V.R.) were completely agglutinated by 8 out of 12 undiluted polyclonal sera, weakly agglutinated (2+ and 3+ reactions) by 2 sera, but not agglutinated by the 2 remaining sera. Cross-absorption tests using 1 non reacting, 1 weakly reacting and 4 strongly reacting polyclonal anti-E sera showed that V.R.'s cells only slightly reduced their anti-E titres versus r"r cells, whilst completely removing their reactivity against her own cells. Absorptions with R2R2 cells abolished activity against both cell types. The cells of V.R. failed to absorb or elute anti-Ew, and had normal expression of c. Three monoclonal human anti-E reagents were tested by fluorescence flow cytometry; one reagent failed to react with the cells of V.R., a second bound as strongly with V.R.'s cells as with R1R2 controls while the third bound much more weakly with V.R. than with any other E+ sample. The variant of RhE in the V.R. family is therefore distinct from Ew, and lacks an epitope recognised by a major constituent of most anti-E sera. This epitope is unlikely to be ET, since all of 91 samples from E-positive Australian aborigines (many of which are expected to lack ET) were agglutinated by a serum that failed to react with V.R.'s cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718092 TI - 13-Hydroxyoctadecadienoic acid (13-HODE) metabolism and endothelial cell adhesion molecule expression: effect on platelet vessel wall adhesion. AB - Endothelial cells synthesize two important fatty acid metabolites, PGI2, which is synthesized from arachidonic acid via the cyclooxygenase pathway, and 13-HODE, which is synthesized from linoleic acid via the lipoxygenase pathway. PGI2 is synthesized following cell activation or injury while 13-HODE is synthesized in the unstimulated cell. While the role of PGI2 in platelet vessel wall interactions has been studied extensively, the role of 13-HODE in platelet vessel wall interactions is just now being understood. The present evidence suggests that 13-HODE is continuously synthesized in "resting" vessel wall cells and is in close juxtaposition with the ubiquous integrin adhesion molecule, the vitronectin receptor. The observation that the endothelial cell is not adhesive when 13-HODE and the vitronectin receptor are in close association and becomes adhesive when these two moieties dissociate and the vitronectin receptor relocates on the surface of the cell, provides further evidence that 13-HODE may induce conformational changes in the vitronectin receptor to reduce its ability to recognize its adhesive ligands. The additional observations that 13-HODE levels in both human and animal vessel walls are inversely related with vessel wall adhesivity, and that this adhesivity can be altered by altering 13-HODE synthesis, provides evidence that 13-HODE down-regulates the thrombogenecity of the injured vessel wall surface. PMID- 1718093 TI - [Myocarditis and leukocyte-endothelium interaction]. PMID- 1718094 TI - [Self-expanding and expandable bile duct prostheses]. AB - The main problem of conventional endoscopic or percutaneous biliary drainage is the clogging of plastic endoprostheses. Therapeutic advances may be achieved by self-expanding or balloon-expandable braided or slit metal stents due to their large lumen and small surface area. Preliminary clinical studies show excellent early results but a divergent long-term clinical outcome depending on the selection of patients, the implantation technique or the type of the stent. If a long-distance overlap of biliary stenoses is achieved the metal stents may be superior to plastic prostheses due to a reduction of the risk of bile encrustation. PMID- 1718095 TI - [The delta sleep-inducing peptide as a factor enhancing the content of substance P in the hypothalamus and the resistance of rats to emotional stress]. AB - The influence of the delta-sleep inducing peptide (DSIP, 60 and 120 nmol/kg, intraperitoneally) on the content of substance P (SP) in rats hypothalamus was studied on males of August line. DSIP administration significantly increased the mean SP content in the hypothalamus and also its content in animals, stable and predisposed to emotional stress. Daily DSIP administration before putting the rats in conditions of stress increased the SP content in the hypothalamus decreased at the emotional stress. Preliminary single DSIP administration to the animals subjected to stress also increased the SP content. Single DSIP administration in a dose of 60 nmol/kg sharply reduced classical stress manifestations, such as hypertrophy of adrenals and thymus involution. PMID- 1718096 TI - [The extracellular content of dopamine, its metabolites and 5-hydroxyindoleacetic acid in the amygdala of free-moving rats during an act of interspecific aggression]. PMID- 1718097 TI - [Early educational support and pediatric psychiatry]. PMID- 1718098 TI - [Neurochemistry of the NMDA receptor complex]. PMID- 1718099 TI - [Effect of different media on long-term cultivation of human synovial macrophages]. AB - Cells of rheumatoid synovial tissue were bred in dispersion culture. In order to investigate the influence of three different culture media on the maturation and vitality of synovio-macrophages, Ham F10 (+fetal calf serum) and RPMI 1640 (+pooled AB Rh+ human serum) were compared with the serum- and cytokin-free solution AIM V. Combining light microscopic, transmission electron, and scanning electron microscopic methods (backscatter-mode), the enzyme and antibody features of synoviocytes were detected. The ratio of macrophages were determined in the light microscope by marking with CD 14. The functional maturation was shown by the amboceptor test. This study demonstrates that RPMI 1640 (+AB Rh+ human serum) is favorable for cultivating human synoviomacrophages. Under serum-free conditions AIM V is especially appropriate. PMID- 1718100 TI - Microbial transformation of hexachlorocyclohexane. AB - Some microorganisms are able to accumulate and to metabolize the pesticide lindane (gamma-hexachlorocyclohexane, gamma-HCH) and other HCH-isomers. Microbial activities, detected metabolites, transformation rates, concentrating factors are reviewed. The results are discussed in respect to use microbiological processes for purification of contaminated water and soil and for recycling of waste HCH isomers. PMID- 1718101 TI - Protective activity to murine experimental brucellosis conferred by monoclonal antibody ISS/32 anti-B. abortus. AB - The protective properties of the monoclonal antibody ISS/32 anti-B. abortus were estimated by splenic infection with B. abortus 544. Five groups of Balb/c mice were used: two groups, previously vaccinated with a 45/20 antigen and a-LPS antigen, were challenged after 30 days intravenously by inoculation of 2.10(5) cells of B. abortus 544, one group was challenged with the same dose of B. abortus 544 preincubated with MAb-ISS/32 and another one with B. abortus 544 incubated with negative serum; the fifth group infected with B. abortus 544 only served as control. The results, expressed as an index of splenic infection, show significant protective properties of monoclonal antibody ISS/32. The infection index in the MAb-ISS/32 group of mice was a bit lower than in B. abortus 45/20 vaccine group. PMID- 1718102 TI - Polyadenylated RNAs as error sources in ribosomal RNA turnover analyses. AB - An approach to ribosomal RNA turnover studies in which cytoplasmic RNA was extracted and subsequently fractionated to isolate ribosomal RNA is reported. The presumption that the pool of 28S and 18S RNAs represented ribosomal RNA, exclusively, proved false and led to erroneous results of ribosomal RNA turnover. Polyadenylated RNAs exhibited a heterogeneous size distribution and, although constituting only 3% (w/w) of the cytoplasmic RNA extract, accounted for fully 10% of radioactivity of the presumptive ribosomal RNA pool. Profiles from the radioactivity data suggested that the discrepant results were due to these polyadenylated RNAs. An additional analytical procedure, an oligo (dT) cellulose column chromatography of the RNA extract prior to the sucrose density gradient fractionation step, performed as described in this paper, proved an effective remedy for this error. PMID- 1718103 TI - Surgical relocation of retracted eponychion. AB - Burn wound contracture of the dorsal side of fingers often leads to retraction of the nailfold with exposure of the growth zone of the nail. Disfigured nails, extremely annoying to the patients, are the results. A surgical procedure and clinical results of 50 cases (fingers) are reported and commented on. PMID- 1718104 TI - Revascularization of avascular spongy bone and head of the femur in transplantation of vascular bundle (An experimental and clinical study). AB - Transplantation of a vascular bundle into an avascular spongiosa replant and the head of the femur after interruption of its blood supply was carried out in experiment (66 experiments on 34 dogs). The periods of observation reached 72 weeks. The mechanisms of revascularization of bones devoid of vessels and the results of morphometric analysis were investigated. It has been established that revascularization of a spongiosa replant sets in after 3 weeks and that of the femoral head after 12-16 weeks. Clinical observations (69 cases) were concerned with transplantation of a vascular bundle in the treatment of patients with aseptic osteonecrosis. PMID- 1718105 TI - Concentrations of selenium and lipid peroxides and glutathione peroxidase activities in plasma of thermally injured pigs. AB - The mechanism of behaviour of parameters involved in protection of the cell from oxidative damage in burn disease remains unclear. Therefore, selenium and lipid peroxide (MDA) levels, and glutathione peroxidase (GSH-Px) activities have been measured for one month in plasma of thermally injured young pigs. Immediately post-burn mean MDA level and GSH-Px activity decreased, while Se concentration increased. After 3 week lipid peroxide levels reached top concentration. Only the enzyme activity returned to initial value at the end of the study, whereas the other two parameters were below the content noted at the beginning. The Se concentration was significantly and positively correlated with GSH-Px activity, and negatively with MDA levels. The similar relationship was also showed for the enzyme and lipid peroxides. The results indicate an interesting role of the studied agents in burn disease, but still suggestions of treatment of severely burned patients with antioxidants need to be supported by detailed studies. PMID- 1718106 TI - The surgical repair of a defect of tendinous distortion of the patella and of the joint capsule during an arthrolysis of the knee joint. AB - The suture of tissues resected during surgical tendomyolysis and arthrolysis of the knee joint required by its extension contracture is carried out during tibial flexion at an angle of 90 degrees. This can lead to the development of a diastasis of the tendinous distortion of the patella and of the joint capsule which prevents the suture of the wound. It is suggested to repair the resulting tissue defect with the use of a split flap of local tissues with a wide base situated in front of the defect. The flap is dissected from the lateral part of the tendinous distortion and of the capsule. The flap is turned by 180 degrees around its base and extended to cover the defect with its subsequent suture to the opposite edge of the defect. The described supplementary surgical procedure is technically uncomplicated and results in stable anterior and lateral joint structures, in a reduction of the risk of complications due to infection, and allows an early initiation of active kinesitherapy in the early postoperative period. PMID- 1718107 TI - Penetration power of antiseptic creams ("in vitro" model with pig skin). AB - By means of an "in vitro" method using pig skin, the authors determine the penetration power of some antiseptic creams in order out the most effective one from this point of view in the treatment of subscar-located infections. The following antiseptic creams were studied: 1% Silver Sulfadiazine, 1% Silver Sulfadiazine with 2.2% Cerium Nitrate, 2.2% Cerium Nitrate, 10% Iodine Povidone, 0.2% Nitrofurazone 0.1%, 0.5% and 1% Chlorhexidine. These products were faced with 17 microorganisms isolated from burn wounds and a control one. The minimal inhibitory concentrations (MIC) obtained after passing through the penetration power of some antiseptic creams in order to find out the most effective one from this point of view in the treatment of subscar-located infections. PMID- 1718108 TI - Aetiological, modifying and lethal factors in cleft lip and palate. AB - Prenatal factors influencing cleft lip (CL), cleft lip and palate (CLP) and isolated cleft palate (CP) may account for the development of the defect (aetiological factor) for a change in the degree or type of the defect (modifying factor), or for the death of the embryo (lethal factor). Each active factor appears to act in all three directions, all be it at different ratios. This is used for analysis of groups of cleft-effected individuals registered at the Department of Plastic Surgery, Prague, from the catchment area of Bohemia. In terms of cleft defect teratogenesis (aetiology, modification, lethality) a "protective influence" appears probable in female embryos, in blood group A (ABO system), in HLA antigens A9, A11, B35. An "embryotoxic influence" i.e., an increase in all the three influences is seen in male embryos, regional influences, in the B and AB blood groups, in HLA antigens B17, in primiparae, in multiple pregnancies, in older mothers and in cytomegalovirus infections. The Epstein-Barr virus seem to increase, in particular, CLP embryo lethality. The activity of the factors was rated by means of correlations between the cleft frequency in first-degree relatives and the frequency of the factor in six subgroups classified by the cleft defect subtypes, by means of interactions between two and more factors and by a study of the differences between male and female probands. A model of the threshold of CL and CP teratogenesis was proposed, a model in agreement with prenatal reciprocal equilibrium. PMID- 1718109 TI - External ear reconstruction. AB - Plastic reconstruction of the external ear designed to achieve a near-natural shape is now a practical proposition even though only few clinical centres can afford to perform it. The present communication is based on experience in the practical application of this new otoplastic technique in 28 patients aged 7 to 25 years. The method involves the making of an auricular structure on a flat cartilaginous groundwork on which to form the helix, anthelix as well as the concha situated in the horizontal, vertical and saggital planes in the shape of two successive steps similarly as in the cartilage of a normal external ear. The proposed technique eliminates the need to excise the block of cartilage from two ribs, and for that reason is less traumatizing and better suited for going ahead with the otoplasty in children of younger age (7-8 years). Given the present rate of incidence of auricular deformity and incomplete development of facial bones and jaws, osteoplasty precedes otoplasty. PMID- 1718110 TI - Unexpected gastrointestinal complications in severely burned children. AB - The authors refer to the frequently difficult diagnosis of gastrointestinal complications of major burns, particularly in childhood, to weight the factors co responsible for the development of those complications and to stress the importance of the patient's history. They report on the case of three and a half year-old child to demonstrate the fatal course of a late diagnosed complications - perforating cholecystitis. Autopsy showed the chronic nature of the disease with acute exacerbation in the course of treatment. No case similar to this one has yet been seen at the Prague Burns Centre in any age category. PMID- 1718111 TI - Metaplastic breast carcinoma. A diagnostic problem in fine needle aspiration biopsy. AB - Fine needle aspiration (FNA) was used to evaluate a breast lump and enlarged lymph nodes in a woman with a prior history of lumpectomy on the contralateral breast and a recent negative mammogram. The FNA cytologic findings included markedly atypical fibroblast-like cells lying singly and in groups in a myxoid background, highly atypical multinucleated cells and numerous mitoses, features that were interpreted as a high-grade malignant mesenchymal tumor. The carcinomatous cells in the aspirates were only fully appreciated after histologic examination of the mastectomy specimen and the axillary lymph node metastases showed a dual differentiation consisting of both epithelial and mesenchymal components, leading to a final diagnosis of metaplastic carcinoma. Electron microscopic study of histologic samples confirmed the dual differentiation, and both keratin and vimentin intermediate filaments were recognized by immunohistochemical staining. The regional lymph node metastases were predominantly sarcomatous, which apparently is a rare event. The entity of metaplastic carcinoma is discussed in relation to other mixed epithelial mesenchymal lesions of the breast, and the previous literature on this entity is briefly reviewed. PMID- 1718112 TI - Fine needle aspiration of Actinomyces infection of the breast. A novel presentation of thoracopleural actinomycosis. AB - A case of Actinomyces infection of the breast secondary to thoracopleural disease was initially diagnosed by fine needle aspiration biopsy. The tender, hard, swollen left breast was clinically suspected of harboring an inflammatory carcinoma. Cell block sections of the aspirate showed polymorphonuclear leukocytes surrounding a typical "sulphur granule" composed of branching filaments. The diagnosis was confirmed by culture of material obtained at subsequent surgery. PMID- 1718113 TI - Cytology of lumpectomy specimens. AB - Residual microscopic disease after lumpectomy may cause significant local recurrence. This study evaluated the use of touch preparation cytology to assess lumpectomy margins. For 90 specimens, the findings on Diff-Quik-stained touch preparations (rendered intraoperatively within 15 minutes after the lumpectomy) were correlated with the gross findings, frozen-section results and, later, the findings in the permanent histologic sections. Three specimens were cytologically unsatisfactory, 68 yielded benign findings, and 19 were suspicious or diagnostic for malignancy. The margins showed tumor involvement in 5 lumpectomy samples by gross examination, in 13 by frozen-section evaluation and in 17 by the study of permanent sections. Touch preparation cytology was putatively falsely positive in two cases while frozen-section evaluation was falsely negative in four cases. Cytology had a sensitivity of 100%, a specificity of 97.1% and a diagnostic accuracy of 97.7%. These results demonstrate that touch preparation cytology rapidly and reliably evaluates lumpectomy margins and can overcome some sampling errors and artifacts related to frozen-section analysis. Touch preparation cytology is now being used to complement frozen-section evaluation of lumpectomy margins as part of a protocol aimed at reducing the local recurrence rate. PMID- 1718114 TI - Fine needle aspiration cytology of papillary endometrioid carcinoma of the prostate. The grooved nucleus as a cytologic marker. AB - The fine needle aspiration (FNA) cytologic findings of an endometrioid carcinoma of the prostate are presented, along with the histologic, immunohistochemical and endoscopic features. Cystoscopy of an elderly male patient with hematuria and symptoms of bladder outlet obstruction showed the delicate papillary growths at the verumontanum that are characteristic of this lesion. Transrectal FNA of the prostate produced samples that included clusters of malignant cells with crowding and overlapping of hyperchromatic nuclei containing prominent nucleoli and a loss of polarization and cohesion. Many of the groups of tumor cells suggested papillary structures. A novel finding in the aspirate was a grooved nucleus in 10% of the tumor cells. Immunohistochemical staining of biopsy sections of the papillary growths for prostate-specific antigen was strongly positive. It is important to recognize this variant of prostatic carcinoma since its behavior and response to therapy are not yet established. PMID- 1718115 TI - Diagnosis of cystic pancreatic lesions by cytologic examination and carcinoembryonic antigen and amylase assays of cyst contents. AB - The contents obtained by fine needle aspiration (FNA) from 41 pancreatic cysts in 32 patients were studied cytologically and assayed for amylase and carcinoembryonic antigen (CEA) levels, which have been shown to discriminate pancreatic pseudocysts from mucinous cystic neoplasms and necrotic cystic carcinomas. The results were correlated with the histopathologic findings following surgery or with a clinical and radiologic follow-up of up to two years. The clinical, radiologic and cytologic characteristics did not discriminate pseudocysts from cystic neoplasms. The amylase content of cysts was high in pseudocysts, cystic carcinomas and mucinous cystic neoplasms. The mean CEA content was highest in cystic carcinomas and mucinous cysts and low in pseudocysts. The cytologic diagnosis of mucinous cystic neoplasms and carcinomas had a sensitivity of 54% and a specificity of 91%. The diagnosis of these lesions based on a CEA level greater than 10 ng/ml had a sensitivity of 100% and a specificity of 81%. The adjunctive use of CEA content analysis enhanced the sensitivity of the cytologic diagnosis of mucinous cystic neoplasms and carcinomas to 100%. PMID- 1718116 TI - False-positive fine needle aspiration cytologic diagnosis of a Warthin's tumor with squamous metaplasia as a squamous-cell carcinoma. PMID- 1718117 TI - Histologic reprocessing of unreadably thick cytologic preparations. PMID- 1718118 TI - Cytology of polychrome-stained equine synovial fluid smears. Comparison with clinical findings, histologic specimens, Wright-Giemsa-stained smears and outcome. AB - Polychrome-stained equine synovial fluid specimens from 34 normal joints and 129 joints with clinical abnormalities were examined cytologically. The smears from joints with abnormalities were categorized as within normal limits (4.7%), slight abnormality (27.9%), proliferative synovitis (21.7%), neutrophilic pattern (20.2%), elongated cell pattern (10.1%), other moderate to marked abnormality (11.6%) and unsatisfactory (3.9%). Cytologic abnormalities that were not restricted to a single category included spindle cells, crystals, stellate cells and cartilage fragments. Multinucleate cells and mononucleate cells with dense cytoplasm and a delicate periphery were seen in smears from cases with clinical diagnoses of osteochondrosis or fracture; interpretation of these cells as osteoclasts and their mononucleate precursors was supported by positive staining with tartrate-resistant acid phosphatase. Smears within the same cytologic category were not found to correspond with a single clinical diagnosis. The identification of several cytologic patterns in cases with the same clinical diagnosis suggests that multiple stages of disease were sampled. Except in cases with the cytologic neutrophilic pattern, there was not a consistent relationship between the histologic features in synovial biopsy specimens and the cytologic findings; the morphologic variation within synovial membrane sections and between sections from different locations was sometimes marked. When compared with air dried, Wright-Giemsa-stained smears, the polychrome-stained smears were more sensitive in the detection of cytologic abnormalities and were less often falsely negative or unsatisfactory. Following surgery, cases with clinical diagnoses of osteochondrosis (29 cases) and fracture (25 cases) were analyzed according to clinical outcome and cytologic category. While 80% of the horses with proliferative synovitis in cytologic specimens were sound, only 67% of those with the elongated cell pattern, 50% of those with slight abnormality and 33% of those with other moderate to marked abnormality were sound. A statistically significant relationship (P less than .02) was found in cases with a diagnosis of osteochondrosis: animals with a proliferative synovitis pattern were almost three times as likely to be sound as compared to those with slight abnormality. These findings indicate that polychrome-stained equine synovial fluid smears (1) provide information that is different from that found in corresponding histologic sections and (2) are superior to air-dried, Wright-Giemsa-stained smears for cytologic examination. The polychrome-stained equine synovial fluid smears were found to provide information supportive of clinical, radiographic and prognostic data. PMID- 1718119 TI - Reactions to intradermally injected substance P and topically applied mustard oil in atopic dermatitis patients. AB - Skin reactions and itch or burning pain sensations following intradermal injection of the neuropeptide substance P and topical application of the substance P releasing agent mustard oil were studied in 20 atopic dermatitis patients and 20 healthy controls. Changes in skin blood flow were measured with a Laser Doppler flowmeter. Areas of wheal and flare reactions were evaluated planimetrically. Simultaneous with Laser Doppler flowmeter measurements, subjective itch and burning pain ratings were verbally reported on a category partitioning scale at 10-second intervals. Substance P evoked dose-dependent wheal, flare, and itch reactions in both patients and controls. However, substance P doses of 10(-9) -10(-11) mol elicited smaller flares in patients than in the controls whereas the wheal sizes were similar in both groups. Substance P induced itch ratings were lower in patients at a dose of 10(-10) mol, and the onset of itching was delayed at all substance P levels applied. Mustard oil elicited similar neurogenic inflammatory reactions in both groups, although pain sensations were significantly delayed in atopic dermatitis patients at two mustard oil concentrations, which is further indication of a desensitization of afferent nerve endings contributing to the neurogenic inflammatory reactions in the skin of these patients. PMID- 1718120 TI - Inflammatory linear verrucous epidermal naevus (ILVEN) versus linear psoriasis. A clinical, histological and immunohistochemical study. AB - Inflammatory Linear Verrucous Epidermal Nevus (ILVEN) has been suggested to be a separate disease entity. However, the distinction from linear psoriasis has been discussed in the literature over recent decades. The aim of the present study was to investigate, in addition to the clinical and histological criteria, the immunohistochemical aspects of inflammation, epidermal proliferation and keratinization. From a clinical and histological point of view, ILVEN and psoriasis, according to the established criteria, have been proved to overlap. The immunohistochemical study suggests that the following procedures have an additional diagnostic impact: assessment of elastase-positive cells, assessment of keratin 16 and of keratin 10. PMID- 1718121 TI - Binding studies of gold labelled lectins on carbohydrate compounds of the flask cells in claw-frog kidney. AB - The carbohydrate compounds of the mucus of flask cells in the kidney of claw frogs (Xenopus laevis) were studied by gold marked lectins (WGA, RCA, L, LCA, HPA, PNA). We used a post-embedding technique. Seminthin or ultrathin sections of Lowieryl K H M-embedded kidney tissue were incubated. For light microscopy, a gold-silver technique was used. The mucus of the flask cells reacted strongly with WGA, RCA L and HPA, whereas LCA and PNA showed no binding. The Golgi apparatus and small cytoplasmatic vesicles reacted also positively with WGA, RCA L and HPA. The autoradiographically detected secretion routes of the glycosaminoglycan-rich secretion of flask cells are also demonstrable by lectins. PMID- 1718122 TI - Localization of calmodulin in epidermis and skin glands: a comparative immunohistological investigation in different vertebrate species. AB - The study deals with the immunolocalization of calmodulin-reactive epithelial cells in different vertebrates (Tinca tinca, Ambystoma mexicanum, Xenopus laevis, Rana ridibunda, Columba domestica, Sus scrofa domestica, Homo sapiens sapiens). The immunoperoxidase technique was performed on acetone fixed frozen sections using monoclonal (BF8) and polyclonal (ACAM) anti-calmodulin antibodies. We were able to differentiate 2 major types of staining patterns: 1. A more superficial epidermal staining in species adapted to an aqueous environment and 2. a staining along the epidermal-dermal junction in species adapted to a terrestrial environment. It seems most likely that epithelial cells immunoreactive for calmodulin are involved in skin permeability control. PMID- 1718123 TI - [Histochemical model investigations of the action mechanisms of lathyrogenic substances]. AB - In histochemical model studies, aminoacetonitrile and 3-aminopropionitrile can form hydrolysis-stable azomethines (Schiff bases) with periodate-induced aldehyde groups of tissue slices. Both substances and triethylentetramine do not inhibit the histochemical monoaminoxidase activity, they even can act as substrates of this enzyme. These substances cause an inhibition of the histochemical aminopeptidase M activity, but this inhibition was recognized as a methodological error due to the formation of complexes between diazonium salts and aliphatic amines. The results indicate that the inhibition of the lysyl oxidase will not be the only mechanism of action of lathyrogenic substances. PMID- 1718124 TI - The histochemical localization of reduced glutathione in skeletal muscle under different pathophysiological conditions. AB - The localization of reduced glutathione in skeletal muscle fibres of patients with inherited or acquired neuromuscular diseases and of subjects with no apparent disease of the neuromuscular system was studied histochemically. In healthy human skeletal muscle fibres, the level of reduced glutathione is higher in aerobic type I fibres than in anaerobic type II fibres. This finding suggests that glutathione in these healthy fibres is held in the reduced state chiefly by the activity of the decarboxylating and NADPH regenerating enzyme NADP(+) dependent isocitrate dehydrogenase. In diseased muscle fibres, there is generally a positive relationship between the activity of the NADPH producing enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the level of reduced glutathione. This positive relationship suggests that glutathione in these diseased fibres is held in the reduced state chiefly by the activity of both enzymes of the pentose phosphate pathway. PMID- 1718125 TI - A modified method for the detection of nucleolar organizer regions (AgNORs). AB - A modified method for the detection of argyrophilic nucleolar organizer regions (AgNORs) is described. By combination of the original silver staining with a Feulgen staining at the same specimen both the AgNORs and the nuclei of the cells under study are sufficiently contrasted, so that a partial automated counting and measurement of these structures can be done using a computerized microscope image analysis system. PMID- 1718126 TI - Computer-assisted image analysis of nucleolar organizer regions (NORs): a pilot study of astrocytomas and glioblastomas. AB - Computer-assisted image analysis was used to measure nucleolar organizer regions (NORs) of 22 astrocytomas and glioblastomas. Image analysis provides reproducible information about number and size of NORs together with further karyometric data, which can be compared and processed with other patient-related data. Our study exhibits a statistical relationship between number and size of NORs and malignancy of the measured gliomas. PMID- 1718127 TI - Granular cell brain tumors of the laboratory rat: an immunohistochemical approach. AB - We have studied paraffin-embedded specimens of 17 rat granular cell brain tumors (GCBT) from four long-term drug safety carcinogenicity studies by peroxidase antiperoxidase (PAP) immunohistochemistry with either polyvalent or monoclonal antibodies against glial fibrillary acidic protein (GFAP), S-100 protein (S-100), Leu-7 epitopes, vimentin (VIM), keratin, desmin, and myelin basic protein. We have found that 9 of the 17 GCBT contained GFAP-positive, S-100-positive, and VIM positive astrocytes, while GFAP-positive and VIM-positive granular cells were observed in 5 of these 9 tumors. Our findings indicate that astroglial cells are involved in rat GCBT and suggest that an astrocytic origin should be considered for these neoplasms. PMID- 1718128 TI - Late-infantile Gaucher disease in a child with myoclonus and bulbar signs: neuropathological and neurochemical findings. AB - Clinical, neurochemical and neuropathological findings on a case of late infantile Gaucher disease with oculomotor apraxia, progressive myoclonus and prominent bulbar signs are reported. There was a marked increase in glucosylceramide in cerebral cortex and cerebellum; the increase was more in the range of that seen in the Norrbottnian type III than in type II Gaucher disease. Cerebral cortical changes were characterized by a band-like intraparenchymal accumulation of Gaucher cells in lamina IV with an accompanying astrogliosis. In the cerebellum, a focal severe loss of granule cells and a global loss of dentate nucleus neurons was recorded. Milder changes were seen in thalamus and brain stem where perivascular accumulation of Gaucher cells was present in all regions. The cerebral cortical changes resembled those seen in type II Gaucher disease and was much more marked than in the Norrbottnian type III, whereas the changes in the dentate nucleus were more severe than in both type II and type III. The phenotypic variability with different patterns of clinical symptoms and neuropathological changes in neuronopathic Gaucher disease is discussed. PMID- 1718129 TI - Infratentorial ependymomas of childhood. Correlation between histological features, immunohistological phenotype, silver nucleolar organizer region staining values and post-operative survival in 16 cases. AB - We have examined pathological criteria in 16 cases of infratentorial ependymomas of childhood using a conventional histological approach, with immunohistochemistry and silver nucleolar organizer region staining (AgNORs). We have found that some of these criteria are of prognostic value. The following histological features were evaluated in each case: cellular density, cellular or nuclear pleiomorphism, mitosis, focal necrosis, endothelial proliferation and complete loss of differentiation. The expression of the following antigens was also studied: epithelial membrane antigen (EMA), human natural killer (HNK1), glial fibrillary acidic protein (GFAP) and vimentin. Only three histological criteria have been retained as indicative of bad prognosis, i.e., high mitotic index, a large amount of necrosis and complete loss of differentiation. These criteria distinguish ependymomas from anaplastic ependymomas. GFAP was expressed in all tumors while other antigens were more variable. In addition tumors expressing large amounts of GFAP were statistically associated with a better prognosis. Increased vimentin expression associated with a decrease of GFAP immunoreactivity correlated with anaplasia and short survival. EMA was not directly correlated with postoperative survival but may be considered as a further prognostic factor. Finally AgNORs values were not statistically correlated with postoperative survival. PMID- 1718130 TI - A case of hepatocellular carcinoma in pregnancy detected by routine screening of maternal alpha-feto-protein. AB - A rare case of hepatocellular carcinoma in a 31-year-old woman is reported. The diagnosis was made at 16 weeks of pregnancy when very high levels of alpha-feto protein were found upon routine examination. The pregnancy was terminated and cytostatic therapy was given. The patient died one year later. PMID- 1718132 TI - Morphology of human vestibular ganglion. AB - Vestibular ganglion cells of mammals have been considered to be myelinated like spiral ganglion cells. Recently, electron microscopic studies reported that the ganglion cells of human vestibular ganglion were mostly unmyelinated unlike in other mammals. The purpose of this study was to find the myelin sheaths of human vestibular ganglion cells by light and laser microscopy. PMID- 1718131 TI - Mechano-electrical transduction in the chicken hair cell. PMID- 1718134 TI - Cell culture study of the vestibular ganglion cells. Morphology and immunohistochemical activity. AB - Incubation of vestibular ganglion cells from the rat fetus and chick embryos was successfully done demonstrating bipolar cells and two types of multipolar cells, small round cells and large cells, in the cell cultures produced. Vestibular ganglion cells were found to be highly irregular in size. Furthermore, the presence of neurotransmitters (choline acetyltransferase and substance P) was confirmed immunohistochemically. Substance P positive cells had many bipolar cells and some multipolar cells. However, choline acetyl transferase positive cells had some small multipolar cells but few bipolar cells. These findings suggest that all vestibular ganglion cells do not have the same function. PMID- 1718133 TI - GABA distribution in the central vestibular system after retroauricular galvanic stimulation. An immunohistochemical study. AB - The changes of the neurotransmitter (GABA) distribution in the brain stem of rats by retroauricular galvanic stimulation were investigated using the immunohistochemical method. In the lateral vestibular nucleus GABA-like immunoreactivity was more intensive on the side ipsilateral to the anodal stimulation than on the other side. It is concluded that retroauricular galvanic stimulation causes some changes in the inhibitory activity of the lateral vestibulo-spinal tract and of the spinal motor neuron. PMID- 1718135 TI - Immunohistochemical examination of S-100 protein and substance P in the inner ear of the rat. AB - S-100 protein and substance P in the inner ear of the rats on decalcified specimens was investigated immunohistochemically. Immunoreactivity of S-100 protein and substance P was found in various parts of inner ear tissues. These results suggest the possibility of immunohistochemical study of decalcified and paraffinized inner ear tissues. PMID- 1718136 TI - Coexistence of substance P and calcitonin gene-related peptide-like immunoreactivities in the rat vestibular endorgans. AB - The immunocytochemical distribution and coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the rat vestibular endorgans were investigated. SP-like immunoreactivity was found in nervous elements beneath and around hair cells. CGRP-like immunoreactivity was also abundantly distributed beneath and within the sensory epithelia. In the present study, double-staining immunocytochemistry revealed that three different types of immunoreactivities: SP positive/CGRP-negative, SP-negative/CGRP-positive, and SP/CGRP-positive immunostaining can be distinguished. SP/CGRP-immunoreactive fibers were localized within as well as beneath the sensory epithelia. These fibers often penetrated the epithelia and nearly reached the surface. The present immunocytochemical evidence suggests that different types of peripheral nervous systems may exist in the vestibular periphery. PMID- 1718137 TI - Retinal ganglion cells projecting to the nucleus of the optic tract in the rat. AB - The nucleus of the optic tract (NOT) has been identified as the link between the retina and the premotor nuclei in the pathway mediating the optokinetic nystagmus (OKN). However, it is still unclear from what parts of the retina and what kinds of retinal ganglion cells the NOT receives visual inputs related to OKN. In the present study, horseradish peroxidase conjugated with wheatgerm agglutinin (WGA HRP) was injected into the NOT of hooded rats to investigate retrogradely labeled cells in the retina. A solution of WGA-HRP was injected into the NOT through a glass micropipette. Injection sites of the brain and the retina were processed by tetramethyl benzidine protocol and Hanker-Yates method. The labeled cells were observed in the whole area of the retina contralateral to the injection site, while located only in the ventral region of the retina ipsilateral to the injection site. In the retina contralateral to the injection site, a large number of small-sized cells and a few medium-sized cells were labeled diffusely except for surroundings of the optic disc, whereas in the ipsilateral retina there were small and medium-sized cells in the ventral part, being about 8 times greater than those in the contralateral retina. The present study indicates that visual signals responsible for OKN are conducted through the retinal ganglion cells mainly contralaterally, and only from the ventral area of the retina ipsilateral to the injection site. PMID- 1718138 TI - WGA-HRP studies on the fiber connections from the spinal cord to the Deiters' nucleus. AB - The labyrinthine-spinal reflexes are influenced by the inputs from the cervical and lumbal propriocepters. We studied the afferent route by the retrograde WGA HRP method in cats. After the injection of WGA-HRP into either the dorsal or the ventral Deiters' nucleus, labeled neurons were investigated in the spinal cord, the cerebellum, the contralateral vestibular nucleus complex and the brain stem nucleus. In this paper, we report the results in the spinal cord of cats. i) When WGA-HRP was injected into the dorsal Deiters' nucleus, labeled neurons in the spinal cord were found mainly extending from the cervical to the lumbosacral area of the spinal cord. A number of labeled cells were located predominantly in the contralateral cervical segments, while a small number of labeled cells was found ipsilaterally in the lumbosacral segments. ii) In the case of the ventral Deiters' nucleus, labeled neurons were found extending from the cervical to the upper thoracic area of the spinal cord. Localization of labeled neurons in the spinal cord was limited mainly to Rexed's laminae VII and VIII. These results suggest that the afferent fibers from the spinal cord to the Deiters' nucleus are closely related to the labyrinthine-spinal reflex. PMID- 1718139 TI - [Role of chemotherapy in squamous cell tumors of the larynx]. PMID- 1718140 TI - [Carcinoma of the larynx: palliative radiotherapy]. PMID- 1718141 TI - Insulin-like growth factor (IGF) binding proteins: the role of serum IGFBPs in regulating IGF availability. PMID- 1718142 TI - [Fuzzy cluster for analysis of the relationship between the structure of cephalosporins and immune cross-reaction]. AB - Six parameters (molecular negentropy, acidic group number, basic group number, proton donor group number, proton acceptor group number, and a ratio of C atomic group number to total atomic group number) for characterizing the structure of an antibody combining site in a R1 chain of cephalosporins were selected. Although 12 parameters characterized the site A and site B in a R1 chain were used in fuzzy cluster, Fischer weighting ratio (Fi) indicated that only 5 parameters, 4 of them characterized the structure of site A, play an important part in the cluster. Therefore it was speculated that the site A was the major combining site in the antigen-antibody interaction. According to the similarity of the R1 chains, cephalosporins could be clustered into 4 groups among which less cross reaction took place. Using the "relative Hamming distance" of the R1 chains for description of their similarity, we found that the intensity of the cross reaction assayed by immune tests had a close correlation with the "relative Hamming distance", so the distance was used for prediction of the intensity of the cross-reaction of cephalosporins. PMID- 1718143 TI - [Effect of bleomycin A5 with calmodulin inhibitor on the proliferation of S-180 cells in vitro]. AB - The effect of bleomycin A5 (BLM) alone and along with calmodulin inhibitor N-(4 aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13) on the proliferation of S 180 cells in vitro were studied. IC50 of BLM alone to the cells was about 2.63 micrograms/ml, which was decreased to 1/3.8 and 1/9.5 of 2.63 micrograms/ml when plus W-13 1, 5 micrograms/ml respectively. The results indicated that nontoxic doses of W-13 enhanced the inhibition of cell proliferation under the condition of BLM 0.5-2.5 micrograms/ml. In colony forming test, the survival fraction of S 180 cells treated with BLM plus W-13 was decreased to 1/87-240 of the cells treated with BLM alone. The results suggest that W-13 can enhance antitumor activity of BLM in vitro and may be used as an enhancer of BLM in vivo. PMID- 1718144 TI - Drug responses in antigen-induced contractions of tracheal strips of ovalbumin sensitized guinea pigs. AB - Guinea pig trachea strips were isolated after 21 d of ovalbumin-sensitization. Challenging the preparations with antigen induced a double-phase contractile response (DPCR) which consisted of a rapid contraction (RC) and a tonic contraction (TC). The effects of tetrodotoxin (TTX), atropine, hexamethonium, diphenhydramine and a substance P (SP) antagonist on the DPCR were observed. DPCR was insensitive to atropine or hexamethonium at 1 mumol/L. Diphenhydramine 2 mumol/L abolished RC, and inhibited TC by 48.0 +/- 5.6%. TTX 1 mumol/L and lidocaine 2 mumol/L almost completely abolished TC and reduced RC by 49.6 +/- 6.7% and 44.6 +/- 8.4%, respectively. The substance P antagonist, (D-Arg1, D-2,4 diCl2-Phe5, Asp6, D-Trp7,9, Nle11)-SP 10 mumol/L, inhibited RC by 49.9 +/- 6.1% and TC by 90.7 +/- 9.3%. The results suggest that SP sensory nerve fibers in respiratory tract play an important role in the pathogenesis of DPCR, especially in that of TC. PMID- 1718145 TI - [Histamine and early ischemic arrhythmia in anesthetized cats]. AB - Ligation of left anterior descending coronary artery caused various arrhythmias and reduced histamine content in ischemic myocardium in anesthetized cats. Intracoronary injection of compound 48-80 100 micrograms shortened the onset of VT and VF from 13 +/- 5, 18 +/- 5 min (n = 7) in control to 7.2 +/- 1.1 (P less than 0.01), 11 +/- 5 min (P less than 0.05) (n = 6) respectively, elevated the histamine concentration of plasma after acute coronary artery occlusion (15 +/- 3, 26 +/- 10 ng/ml, P less than 0.05, before and after ligation respectively). Iv chlorpheniramine (5, 10 mg/kg) or cimetidine (20, 40 mg/kg) dose-dependently reduced arrhythmia score, incidence of VF and mortality after myocardial ischemia, but with little influence on histamine content in ischemic myocardium and plasma. These results suggest that release of histamine from the ischemic myocardium is involved in the generation of early arrhythmias through H1 and H2 receptors in anesthetized cats subjected to acute coronary artery occlusion. PMID- 1718146 TI - Immune enhancement of a polysaccharides peptides isolated from Coriolus versicolor. AB - A protein-bound polysaccharides (PSP) isolated from Coriolus versicolor in Shanghai, at the concentrations of 100-800 micrograms/ml promoted lymphocyte proliferation. PSP 25 mg/kg ip into mice for 5 d antagonized the inhibition of IL 2 production by cyclophosphamide from activated T lymphocytes and restored the suppressed T-cell-mediated delayed, type hypersensitivity response to normal. PSP 10-1000 micrograms/ml induced interferon alpha and gamma production from human peripheral leukocytes 4 and 8 times respectively higher than that of the control groups. Moreover, PSP also increased phagocytic functions of host reticulo endothelial system. The results suggest that the anti-tumor effects of PSP may be related to its potentiation of host immunological responses. PMID- 1718147 TI - 4-Aminopyridine activates an inhibitory alpha 1-adrenergic mechanism unmasked by nifedipin on the electrically stimulated rat vas deferens. AB - 4-Aminopyridine (4-AP) acts by blocking voltage dependent potassium channels in excitable membrane thus increasing the influx of extracellular (Ca2+) in response to an action potential. 4-AP exerts either in vivo or in vitro effects in many organs. The present report deals with a study on the electrically stimulated rat vas deferens of the action of 4-AP on Ca2+ mediated mechanism related to the adrenergic transmission. To avoid the interference of the non-adrenergic components we used the epididymal portion of the vas. 4-AP was shown to enhance the electrically-stimulated contractions in a way which was inversely related to external Ca2+. This effect was abolished by nifedipine (1 x 10(-6) M). Unexpectedly, however, 4-AP acted synergistically with nifedipine by enhancing the inhibition of the electrically-induced tension sustained by nifedipine. This property of 4-AP was shared by noradrenaline, phenylephrine, clonidine, serotonin, morphine and ATP. Their inhibitory effects were antagonized by prazosin (1 x 10(-8) M), yohimbine (2.5 x 10(-5) M), methysergide (1 x 10(-4) M), naloxone (1.2 x 10(-7) M), and theophylline (6 x 10(-6) M) respectively. The inhibitory effect of 4-AP was antagonized only by prazosin (1 x 10(-8) M) and was not observed in vas deferens of rats pretreated with reserpine. It is concluded that 4-AP is able not only to activate excitatory mechanisms but also to stimulate inhibitory alpha 1-adrenergic mechanisms. PMID- 1718148 TI - Lack of retrograde axonal transport of the heparin-binding growth factors by chick ciliary neurones. AB - In view of the likelihood that the heparin-binding growth factors of fibroblast growth factors are neurotrophic for the neurones of the chick ciliary ganglion a study has been made of the retrograde axonal transport of the acidic and basic fibroblast growth factors by these neurones. No high capacity retrograde axonal transport of these molecules was seen. The amount of transport was equivalent to the low level seen with many proteins such as horse radish peroxidase or bovine serum albumin rather than the convincing transport seen for nerve growth factor in the sympathetic system. The iodinated proteins retained their ability to bind to neuronal receptors. Thus, if the fibroblast growth factors are neurotrophic in the ciliary ganglion, they may exert their action by mechanisms other than the retrograde axonal transport of the factor itself. PMID- 1718149 TI - Development of vessels in the foetal cortical transplant depending on the place of grafting in the rat brain. AB - Formation of new blood vessels within the graft is crucial for the survival of brain grafts. Moreover, it must occur rapidly to prevent ischaemic changes in the grafted neurons. A study was made of the development of the vascular system in the foetal cortical grafts depending on the place of grafting in the rat brain. Pieces of neocortical tissue from an 18-day old rat foetus were transplanted into the lateral ventricle, the striatum or the corpus callosum of 2 months old Wistar rats. The vascular system of the graft was visualized from coronal sections of the brain by means of Pickworth's technique 3, 7, 14 and 28 days after transplantation. After 3 days the vessels in the graft were absent. After 7 days the vessel pattern was poor and very simple and after 14 days the vessels formed large number of branches in the graft. After 28 days the pattern of the vascular network in the graft was similar to that of the vessels of the host brain. The size and branching of the vessels showed considerable variations depending on the localization of the graft. PMID- 1718150 TI - Kawasaki syndrome. PMID- 1718151 TI - [Social pediatric developmental rehabilitation and responsibilities of the social pediatric centers in Germany]. AB - This survey sheds light on the historical background and contemporary situation of social paediatrics as an area of responsibility and a principle of integration in modern paediatric practice. The structures, workings and goals of the concept of centres for social paediatrics in the context of developmental rehabilitation are outlined here. PMID- 1718152 TI - [The change in immunohistochemical localization of basic fibroblast growth factor (b-FGF) around the lens capsule after extracapsular extraction]. AB - It has been reported that basic fibroblast growth factor (b-FGF) accelerates proliferation of mesenchymal cells and epithelial cells of cornea and crystalline lens in vitro. The immunohistochemical localization of b-FGF on guinea pig lens capsule after extracapsular extraction (ECE) of the lens was studied in order to investigate the role of b-FGF on posterior capsular opacification after ECE and neovascularization. On the 12th postoperative day, immunohistochemical staining of b-FGF was recognized along the posterior capsule on the cortical side, but not along the anterior capsule. On non-operated eyes, scanty immunoreactive materials were seen in cells at the germinative center of the equatorial region. More than two months after ECE, almost no immunoreactive staining could be recognized. In this study, immunoreactive staining of b-FGF was recognized along the posterior capsule on cortical side after operation. It has also been reported that b-FGF stimulates proliferation of the lens epithelial cells in vitro. Therefore, it is suggested that b-FGF plays an important role on posterior capsular opacification after cataract formation. PMID- 1718153 TI - Changes in the values of immunoglobulins and antiprotease activity accompanying the death of children with cystic fibrosis. PMID- 1718154 TI - [The effects of mepartricin on the symptomatology of prostatic hypertrophy- double-blind controlled trial]. AB - Seventy one patients were treated with mepartricin or placebo in three urological centres for a mean duration of 102 days (extremes: 60 and 142 days). An analysis of the results was carried out for 34 patients in the placebo group and 36 patients in the mepartricin group. The results indicate a significant improvement in both the placebo group and the mepartricin group. The irritative and obstructive symptoms are improved in the active treatment group with a response rate of the order of 70%, compared to approx. 45% in the placebo group. An improvement of the values on the flow meter, though not statistically significant, is observed following treatment with mepartricin, compared to the placebo group. There were no significant differences in the evolution of the prostate gland volume, determined by ultrasound in the placebo group and the active treatment group. Side-effects were minor and only one patient reported epigastric pain. PMID- 1718155 TI - Crusted (Norwegian) scabies. AB - Crusted (Norwegian) scabies, a rare variant of ordinary scabies, is a highly contagious infection in which the skin is infested with thousands to millions of mites. The infection is frequently overlooked because of its atypical presentations. Patients with cognitive deficiency or an immunodeficiency disorder (including immunosuppressive therapy) are predisposed to developing crusted scabies. The infection often presents as generalized dermatitis with crusted hyperkeratosis on the palms and soles. Diagnosis is made by examining skin scrapings from the crusted lesions. Lindane is the scabicide most widely used in the treatment of crusted scabies. Eradication frequently requires repeated applications, and care must be taken to avoid lindane toxicity. Permethrin cream is as efficacious as lindane in the treatment of ordinary scabies. Because of its wider margin of safety, permethrin may become the preferred treatment for crusted scabies. PMID- 1718156 TI - Spontaneous variability of ventricular arrhythmias in patients with chronic heart failure. AB - Spontaneous variability of ventricular arrhythmia in patients with chronic heart failure is not well described. We measured this variability in 23 consecutive patients with chronic heart failure who were prospectively enrolled in the placebo limb of a trial concerned with treatment of heart failure. Patients underwent from one to three periods of ambulatory monitoring separated by 1 to 3 months while they were not receiving antiarrhythmic drug treatment. The variability in frequency of premature ventricular complexes (PVCs) was determined at interrecording intervals of 1, 2, and 3 months. The percentage reductions in total PVCs required to exceed the 95% confidence limits of spontaneous variability at these intervals were 91%, 90%, and 97%, respectively. Corresponding values for repetitive beats (beats in couplets and beats in ventricular tachycardia events) were 98%, 80%, and 97% and for ventricular tachycardia events 98%, 83%, and 98%, respectively. The percentage increases in total PVCs, repetitive beats, and ventricular tachycardia events required to identify aggravation of arrhythmia in this study population were 1301%, 4050%, and 6147%, respectively, at 1-month intervals and 2950%, 2868%, and 5938%, respectively, at 3-month intervals. The percentage reductions required to show a true drug effect at 2- and 3-month intervals were 63% and 84% for patients with an ejection fraction less than 0.22 and 89% and 98% for those with an ejection fraction greater than or equal to 0.22 (p less than 0.05 for both). Ventricular arrhythmia would have been missed in 6 (26%) of the 23 patients if only one screening ambulatory recording was available. Thus marked variability in PVCs occurs in patients with chronic heart failure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718157 TI - Distribution patterns of ventricular premature complexes in long-term electrocardiographic recordings and their usefulness in disclosing modulated parasystole. AB - Long-term electrocardiographic recordings from 25 patients with abundant ventricular premature complexes (VPCs) were screened with a computer program for sequences of interectopic intervals with the number of sinus beats consistent with manifest or concealed bigeminy/trigeminy. On average, 19.0% of VPCs in this patient series were followed by an uninterrupted sequence of greater than or equal to 10 interectopic intervals with concealed bigeminy. The corresponding figure for concealed trigeminy was 2.2%, for manifest bigeminy 9.6%, and for manifest trigeminy 0.8%. The longest sequence with concealed bigeminy comprised 149 interectopic intervals. The corresponding figure for concealed trigeminy was 135 interectopic intervals. The sequences of concealed bigeminy occurred at significantly lower heart rates than those of concealed trigeminy. These findings can be explained by the entrainment phenomena described in experimental models of modulation of a ventricular parasystolic focus across a zone of impaired conduction. PMID- 1718158 TI - Prevalence, characteristics and significance of ventricular premature complexes and ventricular tachycardia detected by 24-hour continuous electrocardiographic recording in the Cardiac Arrhythmia Suppression Trial. CAST Investigators. AB - The prevalence, characteristics and significance of ventricular arrhythmias detected by ambulatory electrocardiography were evaluated in 1,498 patients who were randomized to encainide, flecainide or placebo in the Cardiac Arrhythmia Suppression Trial. The mean ventricular premature complex (VPC) frequency at baseline was 133 +/- 257 VPCs/hour. Nonsustained ventricular tachycardia (VT) (rate greater than or equal to 120 beats/min) was present in 22% of patients. Accelerated idioventricular rhythm (rate less than 120 beats/min) occurred in 22% of subjects. There were 63 deaths/resuscitated cardiac arrests in the active treatment (encainide/flecainide) group and 26 in the placebo group. In the treatment group mortality increased with increasing VPC frequency, (p = 0.006), whereas in the placebo group such a relation was not present. Mortality/resuscitated cardiac arrest increased in patients with greater than or equal to 2 VT episodes than in those with less than or equal to 1 episode in the active treatment group (p = 0.04). There was no significant association between VT and mortality/resuscitated cardiac arrest in the placebo group. The presence of accelerated idioventricular rhythm was not associated with increased mortality/resuscitated cardiac arrest in either the active treatment or placebo groups. However, mortality was lower in patients with accelerated idioventricular rhythm rates less than 100 beats/min than in those with rates greater than or equal to 100 beats/min (p = 0.05). Thus, in the Cardiac Arrhythmia Suppression Trial the previously described association between mortality/resuscitated cardiac arrest and ventricular arrhythmias (VPC and VT) were only observed in the active treatment group. In addition, based on the results obtained in this highly selected population, it is suggested that the definition of accelerated idioventricular rhythm should be a rate less than 100 beats/min, and at a rate greater than or equal to 100 beats/min it should be categorized as VT. PMID- 1718159 TI - Giardia and immune deficiency. PMID- 1718161 TI - Isoenzyme analysis of Pseudomonas cepacia as an epidemiologic tool. AB - Multilocus enzyme electrophoresis has successfully been used to establish basic marker systems for the epidemiologic analysis of a variety of bacterial pathogens. This study was done to determine the efficacy of this technique for characterizing Pseudomonas cepacia, using 31 known-related strains isolated during an outbreak of infections involving intrinsically contaminated povidone iodine solution, and five outbreak-unrelated strains used in serotyping of P. cepacia. Crude cell extracts were analyzed by starch gel electrophoresis for electrophoretic variants using 13 enzyme substrates; esterase bands were detected using an additional four substrates. The 31 outbreak strains had identical isoenzyme patterns for all enzymes examined. Five electrophoretic types were obtained for the serotyping strains; electrophoretic mobilities of one of the five strains corresponded to the patterns obtained for the outbreak strains. These results suggest that enzyme electrophoretic typing may be a useful adjunct to other typing methods used in epidemiologic analyses of P. cepacia infections. PMID- 1718160 TI - Mapping of epidermolysis bullosa simplex mutation to chromosome 12. AB - Epidermolysis bullosa simplex (EBS) is a dominantly inherited genodermatosis characterized by intraepidermal blister formation. Recent reports have suggested that EBS mutations may relate to keratin abnormalities. In this study, we conducted RFLP analyses to test the hypothesis that EBS is linked to one of the keratin gene clusters on chromosome 12 or chromosome 17. Although these keratin gene loci are not defined by RFLPs, several mapped RFLPs in the same chromosomal regions could be tested for linkage. A large EBS family with 14 affected and 12 unaffected individuals in three generations was analyzed for RFLP inheritance. Within this family there was no evidence for linkage of the EBS mutation to markers on chromosome 17q. However, there was evidence for close linkage to D12S17 located on chromosome 12q, with a maximum LOD score of 5.55 at theta = 0. Mapping of this mutation to chromosome 12 defines an EBS locus distinct from both EBS1 (Ogna) and EBS2 (Koebner), which are on chromosomes 8 and 1, respectively. Further mapping will determine whether this EBS locus on chromosome 12 resides within the keratin gene cluster at 12q11-q13. PMID- 1718162 TI - The effects of tactile defensiveness and tactile discrimination on in-hand manipulation. AB - The purpose of this study was to compare in-hand manipulation efficiency in children with and without tactile defensiveness and low tactile discrimination. Fifty children, aged 4 to 6 years, were tested with the use of three subtests of the Southern California Sensory Integration Tests (SCSIT) (Ayres, 1980), which measured tactile function, and three in-hand manipulation tasks. Tactile defensiveness was rated during performance of the selected SCSIT subtests. Nine of the children had mild developmental delays and 41 were without delays. Low correlations between scores on tactile defensiveness and tactile discrimination suggested that these two aspects of tactile function are separate but related phenomena. Children with both defensiveness and discrimination problems demonstrated the least efficiency on all of the in-hand manipulation tasks and had significantly higher time scores on the turn and translation in-hand manipulation tasks. Poor discrimination or tactile defensiveness alone did not relate to poor in-hand manipulation. The results suggest that a child's tactile function should be considered in therapy to improve manipulation skill. Strategies to decrease tactile defensiveness and improve tactile discrimination may facilitate achievement of higher levels of in-hand manipulation. PMID- 1718163 TI - Serum response heterogeneity among nonsmall cell lung cancer cell lines. AB - This study examined the morphology, in vitro growth, and two genetic responses to serum stimulation in the nonsmall cell lung cancer (NSCLC) cell lines SK-Lu-1, SK MES-1, A427, and A549. Morphologically, all four were NSCLC: SK-Lu-1 was undifferentiated, the remainder were adenocarcinoma variants. SK-Lu-1 and SK-MES 1 were slow growing with low-anchorage independent growth capacity; the A427 and A549 lines were fast growing with high-anchorage independent growth capacity. All of the lines expressed basic fibroblast growth factor (bFGF) as a dominant 7.1 kb transcript at amounts significantly lower than in control human lung fibroblasts. bFGF expression could be upregulated by serum exposure in several nontransformed human cell lines, but only the SK-Lu-1 NSCLC cells increased bFGF after serum exposure (482%) compared with a peak increase of 1222% in the fibroblast controls. All of the NSCLC cell lines increased c-fos in response to the same serum stimulations. These results show that growth-factor gene expression can be modulated in NSCLC, and that significant differences exist among NSCLC cell lines commonly used as laboratory correlates of human disease. PMID- 1718164 TI - Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells. AB - The early genetic response to hyperosmotic stress remains to be elucidated in eukaryotes. We observed that hyperosmotic NaCl in Madin-Darby canine kidney cells increased levels of mRNA encoding the immediate early gene (IEG) transcription factors Egr-1 and c-fos at 2 h of treatment by two- and threefold, respectively. Sham treatment and hyperosmotic glycerol, and ineffective osmole, had no effect. Hyperosmotic NaCl, but not glycerol, also increased the mRNA level of the stress protein HSP70 by four- to fivefold at 2, 6, and 24 h. These changes occurred despite inhibition of total RNA transcription rate and DNA synthesis rate by NaCl. Neither NaCl nor glycerol treatment manifested significant cytotoxicity. NaCl, and to a lesser extent glycerol, suppressed protein synthesis, a phenomenon previously correlated with IEG superinduction. Therefore, hyperosmotic stimuli with different physiological effects result in differential expression of IEGs and the stress protein HSP70; induction of the former may govern the ensuing program of gene expression that culminates in the osmolyte response, while the latter may serve as a temporizing protective measure. PMID- 1718165 TI - Effect of mucosal halides on Ca(2+)-blockable currents through the skin of Rana ridibunda. AB - The present study deals with the interaction of mucosal anions with apical Ca(2+) blockable cation channels of the skin of Rana ridibunda. The intracellular potential was depolarized by exposing the basolateral membranes to K2SO4 Ringer solution. The apical bathing medium consisted of nominal Ca(2+)-free K+ or Na+ solutions with SO4(2-), Cl-, Br-, or I- as the major anion. The effects of mucosal anion substitutions were studied by analyzing 1) the fluctuation in K+ current across the apical membrane driven by imposed transepithelial clamping potentials and 2) alterations of the transepithelial current (It) and conductance (Gt) as well as the Lorentzian parameters in response to anion substitution in the mucosal bathing solution. It and current noise spectra were recorded at different transepithelial potentials (Vt). A Lorentzian component was present in the power density spectrum when Vt was clamped at mucosa-positive voltages. Such noise components were never observed with mucosa-negative potentials. These findings suggest a rectifying behavior of the transepithelial cation currents. The Lorentzian noise component and the inward-oriented cation currents were depressed by the addition of micromolar concentrations of Ca2+ to the apical solutions as well as by replacing mucosal K+ or Na+ by N-methyl-D-glucamine. The Ca(2+)-blockable current and Lorentzian noise plateau (So) were gradually increased by raising Vt. Both parameters, as well as the corner frequency (fc), depended strongly on the major anion species in the apical solution; replacing mucosal SO4(2-) by one of the halides tested reduced fc and elevated So, It, and Gt considerably. PMID- 1718166 TI - Synthesis and action of nitric oxide in rat glomerular mesangial cells. AB - Macrophages and certain tumor cell lines can be induced to synthesize nitric oxide (NO) from L-arginine after stimulation with lipopolysaccharide (LPS) or cytokines. In the present study, we have found that culture medium collected after 24 h from unstimulated rat mesangial cells (MC) contains 6.3 +/- 1.2 microM of NO3-/NO2- (the degradation products of NO). These levels were significantly increased when MC were incubated with LPS (10 micrograms/ml) for 24 h (23.9 +/- 4.1, P less than 0.05). The specific inhibitor of NO synthesis, NG-monomethyl-L arginine (L-NMMA) completely inhibited LPS-stimulated production of NO3-/NO2-, confirming that the NO3-/NO2- was derived from NO within the MC. Recent studies suggest that endothelium-derived relaxing factor (EDRF) produced by vascular endothelium is also NO, and we have previously shown that both EDRF and NO stimulate increases in MC guanosine 3',5'-cyclic monophosphate (cGMP). Thus we sought to determine whether NO synthesized by the MC could affect cGMP levels within the same cells. After 24-h incubation with LPS (10 micrograms/ml), intracellular cGMP level within the MC was 706.3 +/- 197 (SE) compared with 40.5 +/- 7 fmol/micrograms protein in control MC incubated in media alone (P less than 0.01). The changes in cGMP in response to LPS were inhibited by greater than 90% by L-NMMA. Similar to LPS, incubation of MC with the cytokine gamma-interferon also increased NO3-/NO2- in the culture media and increased cGMP levels within MC. The induction of NO synthesis within MC and the concomitant stimulation of MC cGMP may be important in the modulation of the effects of endotoxemia, as well as inflammation, within the glomerulus. PMID- 1718167 TI - Ca2+ channels and aldosterone secretion: modulation by K+ and atrial natriuretic peptide. AB - Two populations of voltage-dependent Ca2+ channels, T-type and L-type, are present in bovine adrenal glomerulosa cells. Activation of these channels by cell depolarization with the resultant increase in Ca2+ influx may be one way in which agonists regulate aldosterone secretion. In addition, these channels may be the site of antagonist action. In the present study, we have demonstrated that atrial natriuretic peptide (ANP), an antagonist of aldosterone secretion, alters only the voltage dependence of inactivation of the T-type channel while enhancing the voltage dependence of activation of a subpopulation of L-type channels. These patch-clamp data, which demonstrated contrasting effects of ANP on the activity of T- and L-type Ca2+ channels correlated with changes induced in cytosolic calcium [( Ca2+]i). In the weakly depolarized cell, ANP (greater than 30 pM) lowered [Ca2+]i, in contrast to the strongly depolarized cell, in which ANP (greater than 10 pM) raised [Ca2+]i. Similar alterations in the level of [Ca2+]i in the stimulated cell were induced by the Ca(2+)-channel blocker nitrendipine and the L-type channel agonist, (-)BAY K 8644. With increasing concentrations of extracellular K+ (3.5-60 mM) the rate of aldosterone secretion rose nonmonotonically. ANP inhibited secretion over this broad range of K+ concentrations; however, its potency as an inhibitor of secretion was diminished in the strongly depolarized cell. These data are discussed in the context of a model that proposes a role for sustained Ca2+ influx in cell activation. PMID- 1718168 TI - Endothelin 1 increases cell calcium in mouse collecting tubule cells. AB - Effects of endothelin 1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) were examined in superfused single-nephron segments dissected from mouse kidney. ET-1, 10(-9) to 10(-6) M, caused a biphasic increase in [Ca2+]i consisting of an initial rapid rise followed by a second more sustained elevation in [Ca2+]i in cortical collecting tubules (CCT), outer medullary CT (OMCT), and inner medullary CT (IMCT). The magnitude of the response was dose dependent and was greater in CCT than in OMCT or IMCT. Additional studies using CCT revealed that Ca2+ removal from the superfusate resulted in attenuation of the second phase of [Ca2+]i with approximately 50% reduction in the height of the initial [Ca2+]i peak in response to 10(-6) M ET-1. Ca2+ channel blocker nicardipine had little effect on ET-1-evoked changes in [Ca2+]i. BAY K 8644 and high superfusate K+ also did not affect [Ca2+]i. Addition of ET-1 and arginine vasopressin (AVP), 10(-6) M each, showed the presence of homologous desensitization but the absence of heterologous desensitization in [Ca2+]i changes. There was no additive effect of ET-1 and AVP on [Ca2+]i when they were added together. These data show that ET 1 evokes a biphasic increase in [Ca2+]i of collecting tubules and suggest that the initial peak of the ET-1-evoked rise in [Ca2+]i is largely due to cell Ca2+ release and that the second sustained rise in [Ca2+]i is largely due to increased Ca2+ influx. Data also suggest that ET-1 and AVP may act in the collecting tubules through different receptors. PMID- 1718169 TI - Contractile activity modulates myosin heavy chain-beta expression in neonatal rat heart cells. AB - To determine whether spontaneous contractile activity affected the expression of myosin heavy chain isoenzymes in cultured neonatal rat heart cells, ventricular myocytes were isolated from 2-day-old rat pups by collagenase digestion and cultured for 24-96 h in the presence and absence of verapamil (10 microM), KCl (50 mM), or dihydropyridine receptor antagonists that produced contractile arrest. Inhibition of spontaneous contractile activity was associated with significant reductions in total myosin heavy chain (MHC) content and synthetic rates. Electrophoretic analysis of MHC isoenzymes indicated that MHC-beta protein rapidly disappeared from arrested cells, whereas MHC-alpha isoenzyme levels were less affected. In association with these protein changes, mRNA transcript levels for MHC-beta were markedly reduced in quiescent cells, whereas mRNA transcript levels for several other contractile protein genes were relatively less affected. Inhibition of contractile activity and MHC-beta expression were reversible upon removal of the arresting agents. Furthermore, the decrease in MHC-beta mRNA levels in arrested myocytes could be prevented by direct activation of protein kinase C with phorbol 12-myristate 13-acetate (without restoration of contractile activity). Conversely, MHC-beta mRNA levels in beating cells were reduced by treatment with staurosporine (a selective protein kinase C inhibitor). Thus contractile arrest (produced by either L-channel blockade or membrane depolarization) inhibited the accumulation of MHC-beta in cultured neonatal rat heart cells via a pretranslational mechanism. These effects may occur in response to the modulation of signaling system(s) involving mechanical "stretch" transduced via protein kinase C. PMID- 1718170 TI - BAY K 8644 and nifedipine alter halothane but not caffeine contractures of malignant hyperthermic muscle fibers. AB - The purpose of these experiments was to determine if the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine alter the mechanical responses of malignant hyperthermia-susceptible (MHS) skeletal muscle to halothane and caffeine. Muscle fiber bundles were dissected from MHS porcine skeletal muscle and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), low-Ca2+ media [Ca2+ replaced by 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], or diltiazem (30 microM) administered alone and with halothane (3%) or caffeine (0.5 0.8 mM). When administered alone, both halothane and BAY K 8644 evoked a significant change in resting tension (i.e., contracture) of 193.7 +/- 61.0 and 51.9 +/- 21.5 mN/cm2, respectively. When administered in combination, BAY K 8644 had no effect on the magnitude of the halothane contracture (195.2 +/- 58.6 mN/cm2) but reduced its onset time from 306.7 +/- 36.3 to 105.9 +/- 8.9 s. Nifedipine, low Ca2+, and diltiazem significantly reduced the halothane contracture (103.1 +/- 30.3, 123.1 +/- 20.6, and 112.6 +/- 16.2 mN/cm2, respectively) but had no effect on its onset time. In addition, low Ca2+ reduced the magnitude of the BAY K 8644 contracture (8.2 +/- 2.1 mN/cm2). BAY K 8644 also increased contractures induced by low caffeine concentrations (0.5-2.0 mM) but did not alter contractures induced by 4.0 and 8.0 mM caffeine, whereas nifedipine, low Ca2+, and diltiazem had no effect on these contractures. These results suggest that extracellular Ca2+ influx may have some influence on halothane but not on caffeine contractures of MHS skeletal muscle. PMID- 1718171 TI - A 90-kDa surface antigen of immature smooth muscle cells is ICAM-1. AB - A monoclonal antibody, designated 10F3, that reacts with an antigen of molecular mass 90,000 Da has been developed by immunization of BALB/c mice with smooth muscle cells in long-term culture. The cells were originally isolated from fetal human aorta. The 10F3 was identified as an antibody that reacts with the ICAM-1 molecule. ICAM-1 is a mesenchymal antigen that is lost during differentiation of cells other than endothelium but is reexpressed by the intimal cells of vessels involved in atherogenesis. PMID- 1718172 TI - Control of growth in the neonatal pig heart. AB - The newborn heart is an excellent model in which to study cardiac growth because the neonatal period is a normal situation in which the left ventricle (LV) grows rapidly and the right ventricle grows slowly. Accelerated LV growth is in response to mechanical, neural, and endocrine changes at birth. Faster growth of the LV is accounted for by greater capacity for protein synthesis, as evidenced by greater RNA content. At 18 h of life, ribosomes are formed in preference to total heart protein, but at 48 h of life, faster rates of both ribosome formation and total protein synthesis are observed. In the LV of hearts from 2-day-old pigs, these rates are insensitive to the addition of glucagon, 1-methyl-3 isobutylxanthine, or a combination of norepinephrine and propranolol. These observations could result because of maximal growth stimulation already present in the LV of the newborn heart. To restrain LV growth in the neonatal period, we treated pigs with enalapril maleate, an angiotensin II-converting enzyme inhibitor. Enalapril blocked growth of the LV as well as the increase in RNA content. When hearts from enalapril-treated pigs were perfused in vitro, rates of protein synthesis and ribosome formation in the LV were lower. These studies suggest that angiotensin II is an important factor accounting for rapid growth of the neonatal heart in response to pressure overload at birth. PMID- 1718173 TI - Expression of cytokeratin 8 in human aortic smooth muscle cells. AB - An immunofluorescence method was used to study the expression of cytokeratin 8 in human aortic smooth muscle cells (SMCs) during prenatal development and in atherosclerotic plaques. Aortic SMCs from a 10-wk-old fetus contained cytokeratin 8 in additional to vimentin and a small amount of desmin, whereas, in the cells from a 25-wk-old fetus, cytokeratin 8 was not detected. Cytokeratin 8 was found in the SMCs from intimal thickenings, fatty streaks, and atherosclerotic fibrous plaques. Clusters of cytokeratin 8-positive cells were more abundant in rather advanced lesions (fibrous plaques) that contained at least some amount of lipid. Expression of cytokeratin 8 in the cells of human atherosclerotic lesions probably reflects general rearrangement of gene expression in the intimal cells. PMID- 1718174 TI - Compulsive ritualistic behavior: purposive and personological. PMID- 1718175 TI - Extramammary Paget's disease arising in mature cystic teratoma of the ovary. AB - We report a case of extramammary Paget's disease in ovarian mature cystic teratoma. The patient was a 70-year-old Japanese woman who complained of lower abdominal pain. Examination showed elevation of carcinoembryonic antigen and CA 19-9. Ultrasonography and computer tomography revealed a cystic tumor of the left ovary. The gross appearance of the resected ovary was typical for mature cystic teratoma. Microscopic observation revealed a lesion of Paget's disease within the squamous epithelium. The tumor cells had intracytoplasmic mucin and positive immunoreactivity for carcinoembryonic antigen, epithelial membrane antigen, and cytokeratin; but they were negative for S-100 protein and vimentin. On multiple and serial sections, underlying adenocarcinomas were not found either in the ovary or other primary sites. From these pathological findings, we concluded that the disease was an intraepithelial adenocarcinoma, possibly derived from multipotential cells in squamous epithelium of ovarian mature cystic teratoma. This is the first reported case, to our knowledge, of extramammary Paget's disease arising in mature cystic teratoma of the ovary. PMID- 1718176 TI - Epithelioid angiosarcoma of deep soft tissue: a distinctive tumor readily mistaken for an epithelial neoplasm. AB - We report eight cases of epithelioid angiosarcoma arising in deep, usually intramuscular soft tissue. All the patients were men (mean age, 58). All the lesions arose in a limb or limb girdle. Cardinal morphologic features were the diffuse, sheetlike growth pattern, with only focally apparent vascular differentiation, and epithelioid tumor cells with a degree of intracytoplasmic vacuolation/lumen formation. Immunohistochemically, all eight cases coexpressed keratin as well as endothelial markers. In three cases, endothelial differentiation was confirmed ultrastructurally. Clinically, deep-seated epithelioid angiosarcomas are high-grade neoplasms that rapidly develop metastases. These findings expand the range of recognized epithelioid endothelial tumors and provide further evidence of keratin expression by such lesions. The presence of intracytoplasmic lumina/vacuoles (sometimes containing red blood cells) combined with the characteristic reticulin pattern and striking positivity for Factor VIII-RAg provide the clearest means of distinction from an epithelial metastasis. PMID- 1718177 TI - The lipid-rich epithelioid glioblastoma. AB - The authors add to the literature an account of four aggressive glial neoplasms characterized by diffuse cytoplasmic lipidization and a cohesive architectural disposition in epithelioid nests and sheets. These neoplasms arose in the cerebral hemispheres of adults and tended to a circumscribed neuroradiologic presentation that in two instances prompted an unrewarding preoperative search for an extracranial primary. One represented recurrent disease in a patient being followed for a biopsy-proven low-grade astrocytoma. Three cases were collected by way of consultation from pathologists uncertain as to their primary versus metastatic derivation. The apparent expression of cytokeratins and epithelial membrane antigen further conspired to obscure the glial lineage of these peculiar neoplasms, which are best regarded as tumors of the astrocytic series. PMID- 1718178 TI - Undifferentiated carcinoma of the vulva mimicking epithelioid sarcoma. AB - We report an undifferentiated sweat gland carcinoma of the vulva in an 80-year old woman. The tumor, which was located in the right labium majus, resembled an epithelioid sarcoma histologically; it had a granulomatous appearance with multiple tumor nodules containing epithelioid tumor cells. The tumor also contained rhabdoid cells; a large cluster of them showed histological features indistinguishable from those of a malignant rhabdoid tumor. Immunohistochemically, the tumor cells reacted not only for epithelial markers such as cytokeratins, EMA, and CEA, which are known to be expressed by epithelioid sarcoma, but also for CA125 and with monoclonal antibodies recognizing sweat gland structures--namely, EKH5 and EKH6. For comparison, two epithelioid sarcomas and two extrarenal malignant rhabdoid tumors were also studied. Of these tumors, only one extrarenal rhabdoid tumor reacted with EKH5, and none reacted for CA125. Electron-microscopic examination of the present tumor showed the presence of discontinuous basal laminae and tonofibril-like structures as well as primitive cell junctions and interdigitating filopodia. From these findings, we conclude that the tumor was an undifferentiated sweat gland carcinoma mimicking an epithelioid sarcoma. Findings in this case support the idea of the diverse histogenesis of extrarenal malignant rhabdoid tumors and indicate that electron microscopy is important for differentiating epithelioid sarcoma from skin adnexal carcinoma. PMID- 1718179 TI - Plasmodium falciparum reinfection in children from a holoendemic area in relation to seroreactivities against oligopeptides from different malaria antigens. AB - The rate and densities of Plasmodium falciparum reinfections were investigated in children five to 14 years old from one village in Tanzania with a high transmission rate. Initial parasitemias were eradicated by a curative treatment with quinine, a drug with a short elimination half-life, to minimize the effects of residual drug on reinfection. The seroreactivities to seven oligopeptides, representing T and B cell epitopes from the ring erythrocyte surface antigen (Pf155/RESA), the clustered arginine-rich protein antigen (CARP), and the circumsporozoite (CS) proteins were determined in the children at the start of the study and after 28 days. All children were reinfected within 42 days (mean 27 days). The geometric mean maximum parasite density at reinfection was 308 parasites per microliter (range 4-13, 920). The antipeptide antibody levels showed high interindividual variation, with a significant mean decrease (16%) between days 0 and 28 for the blood stage antigens, but not for the (NANP)6 peptide from the CS protein. This suggests that the absence of blood stage antigenic stimulation had already influenced the antibody levels within this short period of time. The mean reinfection day was not influenced by the levels of antibodies to any of the peptides. However, the children with higher antibody levels to (EENVEHDA)2(EENV)2 developed significantly lower parasitemias than those with lower antibody levels (P less than 0.05). This suggests that this subunit of the Pf155/RESA molecule is an important B cell epitope for protective antiparasitic immunity. PMID- 1718180 TI - Melanoma metastatic to stomach, small bowel, or colon. AB - Approximately 60% of patients who die from melanoma have gastrointestinal (GI) metastases at autopsy, yet antemortem diagnosis is uncommon. A retrospective review was completed on 32 patients who underwent an operation at Memorial Sloan Kettering Cancer Center between 1977 and 1987 for complications of melanoma metastatic to the stomach, small bowel, or colon. Operations were most often performed on an emergent basis, and indications included bleeding or anemia in 12, obstruction in 10, abdominal pain in 8, intestinal perforation in 1, and acute GI bleeding with obstruction in 1. GI involvement was the first sign of metastatic disease in 10 patients. Median survival after operation was 6.2 months (range: 1 to 42 months). Five patients were alive 2 years after operation, although only one remains free of disease 39 months after complete resection of a single site. Operative mortality was 3%, and 94% of patients were discharged from the hospital. Due to the low operative mortality, surgical palliation should be considered for those in whom the quality of life may be improved. PMID- 1718181 TI - Laser palliation for colorectal carcinoma. AB - A review was conducted of 27 patients with colorectal carcinoma treated palliatively with endoscopic neodymium:yttrium-aluminium-garnet (Nd:YAG) laser. There were 25 rectal carcinomas and 2 primary invasive sigmoid colon carcinomas. Of the 25 rectal carcinomas, there was 1 carcinoma in situ, 16 primary cancers, and 8 recurrent rectal carcinomas. The level of the lesions from the anal verge ranged from 0 to 25 cm, with a mean of 7.2 cm. The length of the lesions ranged from 1.5 to 8.5 cm, with a mean of 5 cm. The mean number of Nd:YAG laser treatments was three, with a range from one to nine. The duration of the treatments ranged from 30 to 90 minutes, with a mean of 40 minutes. Four of 27 patients (15%) developed complications. The success rate in terms of the relief of symptoms was established in 23 of the 27 patients. PMID- 1718182 TI - Pain management in advanced carcinoma of the head and neck. AB - Although pain is one of the most feared consequences of cancer, pain management is rarely discussed in the literature on head and neck cancer. The pain experienced by patients with head and neck malignancies, of a biologic origin, is compounded by the emotional distress caused by alterations in function and cosmesis. Control of pain is possible, but an effective program must include more than pain medication. A current treatment program is presented, based on scientific study and clinical experience. The most helpful pain medication is immediate-release, liquid morphine sulfate (20 mg/mL) administered every 4 hours. A nonsteroidal anti-inflammatory drug may also be used and it may decrease the amount of morphine necessary. Stool softeners must be provided, and anti-nausea medication is often given. Steroid drugs are regularly used to increase appetite, decrease edema, and enhance the patient's sense of well-being. Factors related to the selection and dosage of medications are discussed. PMID- 1718183 TI - Cortical cytoarchitectural and immunohistochemical studies on Zellweger syndrome. AB - In two cases of Zellweger syndrome, Golgi studies revealed an irregular neuronal arrangement, the presence of immature neurons, poor dendritic arborization and poor spine development, all of which suggest abnormal morphogenesis and delayed maturation. In immunohistochemical studies with antisera against human catalase, negative staining of neurons suggested a decrease of catalase due to defects of microperoxisomes, and positive staining of myelination glia only in the internal capsule may have been related to delayed myelination. Abnormal peroxisomal membrane or its related metabolites may cause a migration disorder in intrauterine development and myelination disturbance in perinatal maturation. PMID- 1718184 TI - Connective tissue nevi collagens. Study with picrosirius red and polarizing microscopy. AB - Biopsy specimens of five connective tissue nevi were examined under crossed polars after staining with Picrosirius red. One biopsy specimen was from a solitary nevus, another from a Shagreen patch. The other three specimens were of erupted nevi. In all cases, thick (as well as thin) collagen fibers appeared green to yellow. In contrast, thick fibers of normal human dermis appeared orange to red. The findings indicate that the collagen of collagenous connective tissue nevi is less well packed than normal collagen. Examination of the polarization colors of Picrosirius red-stained sections is a useful procedure for diagnosing collagenous connective tissue nevi. PMID- 1718185 TI - Naturally occurring aliphatic polyamines-induced histamine release from rat peritoneal mast cells. AB - Rat peritoneal mast cells were incubated with different concentrations of naturally occurring aliphatic polyamines, spermine and spermidine, at 0.1-10 mM and the amount of histamine release into the supernatant solutions was measured. The addition of each polyamine to the suspensions of the mast cells caused a histamine release in a dose-dependent manner. The effect of 10 mM spermine and spermidine was as much as that of 0.5 microgram/ml compound 48/80. The histamine release from the cells incubated with each polyamine was rapid and the amount of histamine release into the supernatant solutions reached a maximum at 1 min with the incubations. 0.1 mM spermine, which in itself could not cause a significant histamine release, showed a tendency to enhance anti-IgE-induced histamine release from the mast cells. PMID- 1718186 TI - Imbalance of CD45R and CDw29 helper cell subsets after seven days of culture from penicillin hypersensitive patients. AB - Specific PBMC proliferation to penicillin represents one of the in vitro diagnostic procedures to show antigenic sensitization. Nine patients with suggestive clinical history of penicillin hypersensitivity, significant stimulation index in the lymphocyte transformation test (LTT), and increased CD25 expression were selected. Their phenotypic expression of T-cell subset markers was compared with that of six normal subjects after 7 days of culture. Higher percentages of CD45R and CDw29 positive cells were found in penicillin hypersensitive patients. This group of patients showed a significantly (P less than 0.01) higher percentage of CD45R positive cells compared with normal subjects, after 7 days of culture. The percentage of CD45R cells did not differ between patients and normal subjects at day 0, and the progressive decrease of CD45R positive cells in culture of normal subjects was not observable in our patients. These results suggest that the patients with penicillin hypersensitivity have an imbalance of CD45R/CDw29 ratio after 7 days of culture, with a higher expression of CD45R positive cells. PMID- 1718187 TI - Tandem mass spectrometry of peptides using hybrid and four-sector instruments: a comparative study. AB - Product-ion spectra produced by high- and low-energy collisionally activated dissociation (CAD) of [M + H]+ ions of a series of peptides (Mr 550-2500) have been compared on four-sector and hybrid tandem mass spectrometers, respectively. The fast atom bombardment product-ion spectra obtained for the smallest peptide analyzed (methionine-enkephalin) were remarkably similar, but substantial differences in fragmentation were observed for the heavier analytes. For peptides with Mr greater than 1000, more complete sequence information was obtained from high-energy CAD on the four-sector instrument. Nevertheless, low-energy CAD on the hybrid mass spectrometer was able to produce partial sequence information even for the largest of the peptides compared. Limits of analysis, defined as the least quantities of analyte for which product-ion spectra of essentially uncompromised quality could be obtained, were similar (ca. 15 pmol) for small peptides, but lower limits were achieved for larger peptides (Mr greater than 1000) with the four-sector instrument. High-energy CAD spectra were found to be highly reproducible, with qualitatively similar spectra obtained over a wide range of operating conditions. In contrast, it was necessary to carefully control collision gas pressures and collision energies in order to obtain good reproducible data for low-energy CAD. Experimental procedures for obtaining reproducible spectra with good sensitivity for peptides on the hybrid instrument are presented. PMID- 1718188 TI - Tenascin localization in skin wounds of the adult newt Notophthalmus viridescens. AB - Earlier studies have shown that the extracellular matrix (ECM) protein tenascin (TN) is present between uninjured epidermal cells of urodele appendages, but is absent from most of the mesenchymally derived ECM. Following appendage amputation, this distribution is reversed. TN is lost from the epidermis and appears in the ECM of the stump and the regeneration blastema. In the present study, monoclonal and polyclonal antibodies to TN were used to localize this protein immunohistochemically in limbs of the adult urodele Notophthalmus viridescens at various stages following skin removal with or without damage to underlying muscle to determine 1) if the loss of TN by the epidermis and its gain by mesenchymal tissues occurs in wounds that do not require regulation by epigenetic mechanisms, and 2) if TN is present in the provisional wound matrix beneath migrating epidermal cells. In addition, skin explants were cultured on TN coated dishes to learn if TN possesses active sites that can support epidermal cell migration. The results indicate that simple wounding leads to the same TN patterns as occurs following limb amputation. Tenascin loss from the epidermis could be seen as early as 6 hr after wounding, a time during which migrating epidermal cells are moving over the wound bed. During this period, there was no evidence of TN in the provisional wound matrix. In contrast to collagen, which supports considerable epidermal cell migration from skin explants, TN allowed no more migration than did the inactive protein, myoglobin. PMID- 1718189 TI - Motor neurons of the laryngeal nerves. AB - The present study was undertaken to determine the relationship between the motor neurons of the superior and recurrent laryngeal nerves within the nucleus ambiguus. The retrograde transport of horseradish peroxidase was utilized to identify the motor neurons subsequent to its application to the proximal transected end of the superior and recurrent laryngeal nerves. Labeled superior laryngeal motor neurons were distributed ventrolaterally in the rostral portion the nucleus. The recurrent laryngeal motor neurons were distributed throughout the nucleus with two distinct populations: a rostral group and a caudal group. The rostral group overlaps the motor neurons of the superior laryngeal nerve. The caudal group occupies that portion of the nucleus that is classically described for the recurrent laryngeal nerve. Additional superior laryngeal nerve labeled perikarya were found in the dorsal motor nucleus of the vagus. This study defines the rostral distribution of the recurrent laryngeal nerve motor neurons and suggests that this rostral group is a component of the neuroanatomical substrate that is involved in the co-activation of the laryngeal abductors controlling the laryngeal aperture. PMID- 1718190 TI - Modulation of platelet surface adhesion receptors during cardiopulmonary bypass. AB - Alterations in platelet receptors critical to adhesion may play a role in the pathogenesis of the qualitative platelet defect associated with cardiopulmonary bypass. Using flow cytometry, we measured changes in the following platelet surface adhesive proteins: the von Willebrand factor receptor, glycoprotein Ib; the fibrinogen receptor, glycoprotein IIb/IIIa; the thrombospondin receptor, glycoprotein IV; the adhesive glycoprotein granule membrane protein 140, whose expression also reflects platelet activation and alpha-granule release; and, as a control, the nonreceptor protein HLA, A,B,C. Glycoprotein Ib decreased during cardiopulmonary bypass (P less than 0.05) and reached a nadir at 72% (P less than 0.05) of its baseline value at 2-4 h after bypass. This decrease correlated (r = 0.76) with the magnitude of platelet activation (alpha-granule release) in any given patient, but even platelets that were not activated demonstrated a decrease in glycoprotein Ib expression. Glycoprotein IIb/IIIa also decreased in both the activated (47% of baseline, P less than 0.01) and unactivated (63% of baseline, P less than 0.01) subsets of platelets at the end of cardiopulmonary bypass. Glycoprotein IV and HLA A,B,C did not decrease, but instead increased 2-4 h after cardiopulmonary bypass (P less than 0.05). We conclude that cardiopulmonary bypass produces selective decreases in surface glycoproteins Ib and IIb/IIIa as well as in platelet activation; that these two alterations are temporally but not necessarily mechanistically linked; and that these changes have the potential to adversely affect platelet function. PMID- 1718191 TI - Molecular epidemiology of Pasteurella multocida in turkeys. AB - Pasteurella multocida isolated from turkeys during an outbreak of fowl cholera was characterized by serotype and heterogeneity of genes encoding rRNA (ribotype) to investigate the epidemiology of the organism. Isolates were collected between October 1985 and July 1986. The M9 or Clemson University fowl cholera vaccine like strain was detected in 17% of the flocks with fowl cholera. One particular strain, isolated only from breeder flocks, was recovered from 7 of the 10 breeder flocks examined in this study. Intracompany transmission appeared to be common, implying a failure in biosecurity. Circumstantial evidence indicated that in the field; the incubation period of P multocida in a turkey flock may be between 2 to 7 weeks. Wildlife did not appear to be an important reservoir of P multocida for turkeys during this study period. Ribotyping results tended to discount several of the possible interflock transmissions, as suggested by examination of serotyping results alone; however, serotyping in combination with ribotyping proved helpful in understanding the epidemiology of P multocida in turkeys. PMID- 1718192 TI - Quantitative assessment of lung pathology in idiopathic pulmonary fibrosis. The BAL Cooperative Group Steering Committee. AB - The diagnosis and classification of most interstitial lung diseases requires histologic evaluation of lung tissue, obtained by an open lung biopsy to confirm the diagnosis. In addition, it is generally accepted that response to therapy in idiopathic pulmonary fibrosis (IPF) is related to the relative degree of cellularity and fibrosis present. Because only a qualitative assessment of the relative extent and severity of these changes is generally provided, correlation with clinical and physiologic alterations is difficult. This report describes results of a semiquantitative assessment by four pathologists of inflammatory/exudative changes, fibrotic/reparative changes, and airway alterations, in addition to an overall assessment of cellularity and fibrosis in 50 patients with IPF. In 10 randomly selected biopsies examined twice in a blinded fashion, absolute agreement between assessments for a given pathologist varied between 54 and 64% (mean = 57.5%) and in the majority of instances the agreement was greater than would have occurred by chance. There was good agreement for most variables across the four raters on the 101 samples. The mean score for some of the parameters reported by a given rater deviated occasionally from those of the other raters, but no single rater was consistently different from the other raters. A principal component factor analysis revealed that the pathologic features fell into four general groupings: alveolar wall metaplasia, fibrosis, honeycombing, smooth muscle, and vascular changes fell into one group; severity and extent of cellularity in the alveolar wall into a second group; severity and extent of cellularity in the alveolar space into a third group; and interstitial young connective tissue along with granulation tissue in the airways formed the fourth group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718194 TI - Squamous carcinoma of the mid-esophagus. A survival study. AB - A survival study for squamous carcinomas of the mid-esophagus treated by Southern California Permanente Medical Group in the interval of 1954 to 1988 was undertaken. Radiation therapy and surgery were equally efficacious in terms of 5 year survival for patients without distant disease and performance status sufficient to tolerate treatment (11% and 16%, respectively). There was no survival benefit for patients treated with palliative surgery. Less invasive endoscopic means along with chemotherapy and radiation for palliation are recommended except for special circumstances. Optimal treatment combinations remain to be discovered. PMID- 1718193 TI - Comparison of acridine orange stain with culture and gram stain of needle aspirate in experimental Pseudomonas pneumonia. AB - The sensitivity and specificity of culture, acridine orange stain, and Gram stain were determined using needle aspiration (NA) material obtained from 82 rats with acute Pseudomonas aeruginosa pneumonia and 18 control rats. Lungs were then processed for either bacterial quantitation or histopathologic examination. NA culture proved to be the most sensitive and specific (55 and 100%, respectively). Sensitivity of acridine orange stain was 40%, whereas Gram stain was only 29%. The specificity of each stain was at least 94%. Lung bacterial concentrations influenced the sensitivities of all three techniques, with better sensitivity found in NA samples obtained from lung with bacterial concentration of at least 10(4) colony-forming units (cfu) of P. aeruginosa. Acridine orange and Gram stain results were similar except in NA samples from lung with bacterial concentration of less than 10(4) cfu in which acridine orange stain was more sensitive. The presence of stains identifying bacteria collected from animals with sterile NA culture was found in a small but significant number of samples, suggesting the presence of nonviable though stainable organisms. Use of all three techniques (culture, acridine orange stain, and Gram stain) increased sensitivity to approximately 70% with minimal decrease of specificity. PMID- 1718195 TI - Impact of splenectomy on survival following gastrectomy for adenocarcinoma. AB - From 1972 to 1985, 161 palliative and 99 curative gastrectomies were performed, including splenectomy (SPL), in 49 and 42 patients, respectively. The relative contribution of tumor histology and location of primary tumor within the stomach to median survival times in months (MST) was examined by chi-square analysis for patients who had SPL and those who did not. MST for the 40 patients treated by curative gastrectomy and SPL was 33 months compared with 43.5 months in the 56 patients treated by curative gastrectomy without SPL (P = .24). Three postoperative deaths were excluded from this analysis. Neither tumor location nor the histological type had a statistically significant impact on MST within these two groups. SPL was not found to have statistically significant impact on survival following curative gastrectomy. In 46 patients treated by palliative gastrectomy with SPL the MST was 10.5 months compared with 15.5 months in 110 patients who were treated by palliative gastrectomy without SPL (P = .007). Five postoperative deaths and three patients in which primary tumor location could not be accurately determined were excluded from this analysis. The MST of 23 patients treated by SPL who had poor or undifferentiated histology was 8 months, compared with 15 months in 73 patients with similar histology not treated by SPL (P = .02). There was no statistically significant difference in MST between palliative gastrectomy patients who had well or moderately differentiated histology treated with (n = 23) and without (n = 37) SPL (P = .11). The impact of SPL on MST following palliative gastrectomy was not significantly influenced by tumor location in the stomach.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718197 TI - ["Quadrangular" Le Fort II osteotomy for hypoplasia of the midface. Apropos of 28 cases]. AB - In 1971, Kufner described the "quadrangular" osteotomy of the midface, which he used to correct asymmetry in patients with recession of the midface and a normal nasal skeleton. We have used this method in 28 patients, 5 women and 23 men. The surgical technique is described and the indications, the approach and the stability of the postoperative results are discussed. The conclusion is that the "quadrangular" osteotomy is a very satisfactory method for the aesthetic and functional rehabilitation of a certain group of midfacial anomalies. PMID- 1718196 TI - Major chest wall resection for recurrent breast carcinoma. AB - Local recurrence of breast cancer is a relatively common entity. Occasionally, the management of such recurrences necessitates full-thickness chest wall resection. Although the chance for cure in such circumstances is small, achieving local control remains a desirable goal to maintain comfort and hygiene. This study evaluates the utility and morbidity of full-thickness chest wall resection in the control of symptomatic local recurrence of breast carcinoma. Twelve patients, undergoing such resections, were identified from hospital records. The resections included an average of three ribs (range, two to five) and, in seven cases, part or all of the sternum. A variety of rigid and soft tissue chest wall reconstructive techniques were utilized. Of the 11 patients available for follow up, ten reported good overall function postoperatively. There were no postoperative deaths and only one patient developed a major complication requiring prolonged hospital stay. The patients spent an average of 6.6 hours in surgery, 14.6 hours intubated, and 20 days in the hospital. There were ten patients available for long-term follow-up. At the time of this study, 70 per cent were still alive, with or without disease, with a mean survival of 27 months (range, 3-71 months). Forty per cent were alive, disease-free, with a mean survival of 36 months (range, 3-71 months). This series demonstrates low morbidity, improved quality of life, and the possibility of long-term, disease free survival after full-thickness chest wall resection for recurrent breast carcinoma. PMID- 1718198 TI - [Vascularization and innervation of the sternocleidomastoid muscle and its cutaneous covering: anatomical bases for its use in plastic surgery]. AB - The commonest use of the sternocleido-mastoid muscle (SCM) and its cutaneous recovering in plastic surgery remains the rotation flap. With the improvement in microsurgery, it is tempting to anticipate the free transfer of the muscle with vascular and nervous anastomosis. 40 SCM muscles from fresh human adult cadavers were studied. The authors stress the richness of the arterial blood supply of the upper portion of the muscle. They give precise details about its venous drainage, the arterial anastomosis, the blood supply of the superficial cutaneous planes and the motor and sensory innervation. PMID- 1718199 TI - [Our own experience in the treatment of hypotrophy and ptosis of the breast]. AB - The outcome of surgical of small, ptotic breasts is reported in a retrospective series of fifteen patients operated between 1983 and 1989. Seven patients underwent breast augmentation by insertion of breast implant supplemented in one case by a dermopexy while eight patients underwent cutaneous and glandular remodeling alone. Simple breast augmentation with breast implant gave good results with mild to moderate ptosis (i.e. when the distance between the inferior margin of the clavicle and the upper margin of the areola was equal to or less than 17 cm) provided that: the subareolar segment III did not exceed 5 cm, the skin had good static qualities for breast suspension, the amount of glandular, adipose and cutaneous tissue present allowed the prothesis to be covered by a sufficiently thick layer. With breast ptosis greater than 17 cm or when segment III exceeded 5 cm, cutaneous remodeling by dermopexy was associated with insertion of breast implant. In this group of patients treated, either by breast implant alone, or associated with dermopexy, results were good in 33%, and satisfactory in 50% of cases. Better results were obtained in patients with marked ptosis and breasts which, although hypotrophic, conserved a amount of adipose and glandular tissue sufficient to allow breast reconstruction by soft tissue remodeling alone (without the insertion of an implant). Patients in this group were treated by glandular and cutaneous remodeling with good to excellent results in 80% of cases. PMID- 1718200 TI - [Evaluation of results of a series of 58 breast reconstruction after cancer]. AB - The evaluation of 58 breast reconstructions operated in Boucicaut Hospital demonstrated positive result. The authors review the techniques used for contralateral breast symmetrization. The policy generally adapted has been to perform two sequential operations: 1st step: implantation of a prefilled prosthesis and contralateral breast symmetrization; 2d step: reconstruction of areolo-nipple complex and refinement of contralateral breast. Radiotherapy seems to be a problem in reconstruction. A 12% recurrence rate has been quoted in this group. Tram flap has been exceptionaly used by our team, as we tend to prefer the latissimus dorsi myocutaneous flap. Very few expanders have been implanted. PMID- 1718201 TI - [Musculocutaneous island flap using the upper gluteus maximus muscle]. AB - The authors report 24 cases treated with an gluteus maximus musculocutaneous flap. The gluteus maximus is generally used as a VY flap. Our technique uses the superior part of the muscle with the overlying skin as a rotation flap. The upper part of the muscle is supplied by the superior gluteal artery. A good knowledge of the anatomy makes this flap easy to perform. The skin part of the flap is drawn over the trochanter. It is generally a 8 cm diameter circle. We then create a subcutaneous tunnel to prepare the rotation. The muscle is then freed from its lateral origin. The separation from the gluteus medius is made by blunt dissection and the superior gluteal artery can then be seen. The myocutaneous flap can now be raised and transferred to the defect. This flap has, in our experience, many advantages especially in paraplegic patients: large skin defects can be covered with a single flap, the perisacral skin is free of any scar, the lower part of the muscle can still be used to cover ischial ulcers. Since 1987 we have treated 24 patients with good results. The reliability and the great technical simplicity makes us think that the superior gluteus maximus musculocutaneous island flap is optimal for the coverage of sacral pressure sores. PMID- 1718202 TI - [Sacral bedsore: an evaluation of 10 years' treatment with the gluteus maximus muscle]. AB - The authors review the notes of 40 patients who underwent surgery for large sacral pressure sore over the last 10 years. In most cases a gluteus maximus flap with skin graft has been employed to cover sacral sore, as originally described by Ger in 1971. The results of this series are then compared with those published by other authors using different techniques, with particular attention to gluteus maximus musculo-cutaneous island flaps and purely cutaneous flaps. All procedures carry a small mortality rate because of the advanced age and frail conditions of most patients. In our series 2 patients died in the postoperative period; in all the others we eventually achieved a sound healing of the sacral sore. We conclude that the gluteus maximus rotation flap is a safe and effective for the treatment of this condition. Its only major drawback is represented by the prolonged period required for the epithelisation by secondary intention of some fairly frequent areas of failure on the skin graft. The importance of a multidisciplinary team approach in the management of these patients cannot be overemphasised. PMID- 1718203 TI - [Sagittal rhinoplasty for broad noses]. AB - Normal profile wide noses, without osteo-cartilaginous bump, are rare and very few cases have been described in the literature, to our knowledge. Based on a short experience of 4 cases, we describe a surgical variant of the classic rhinoplasty technique. The resection of two osteo-cartilaginous symmetrical quadrilaterals resolves this specific problem with no risk of profile modification with sagging and necessity of reinclusion. PMID- 1718204 TI - [Double triangular resection technique at the feet of the cartilaginous arch in the correction of the nasal tip. "Do not touch the dome"]. AB - Retropulsion and modelling of the nose tip can be obtained very simply without touching the dome by means of double triangular resection at the feet of the cartilaginous arch formed by the two alar cartilages. Static and dynamic effects are obtained retropulsion, elimination of the prominence of the superior border of the alar cartilage (without shortening resection) allowing creation of a finer nose tip; rounding of the nasal orifices, improving respiratory function; suppression of labial dependence of the tip a factor responsible for secondary ptosis of the tip. PMID- 1718205 TI - [The mongoloid nose]. AB - The more precise term "mongoloid" should be used in the place of "oriental" and "asian". The nose of the Han race constitutes the most typical example. The description of "intermediary", usually used to describe this nose is insufficient, as it has a very particular type which contributes to the "Asian triad". A study conducted by Chengdu tries to overcome this deficiency. This information is particularly useful in that aesthetic rhinoplasty, performed increasingly frequently for this type of nose, raises specific problems and requires particular caution to avoid excessive correction which may be disastrous in a small face. PMID- 1718206 TI - [Coronal approach of zygoma and direct osteosynthesis in post-traumatic dysmorphic sequelae]. AB - A direct approach to the zygomatic arch can be a useful alternative for primary or secondary deformities in which simple well known technics are ineffective. This approach is very limited because of the anatomical elements located directly over the zygomatic region. The exposure of the fracture site is performed by an unusual surgical approach which allows visualization of both zygomatic arches simultaneously, to correct all deformities and to perform osteosynthesis. Four cases using this technique are reported and the value of this approach is assessed. PMID- 1718207 TI - [Cutaneous dehiscences during tissue expansion: use of surgical adhesive plastic film]. AB - Cutaneous expansion, especially in the lower limb, is sometimes complicated by the development of partial dehiscence of the surgical scar or a small area of necrosis over an angle of the prosthesis. The expander implant can be salvaged by closing the dehiscence with surgical adhesive film which allows continuation of expansion while preventing herniation of the prosthesis. PMID- 1718208 TI - [Verrucous epidermal nevus of the face]. AB - The authors report a case of extensive verrucous epidermal naevus of the face in a 15 year old Senegalese boy. This is the second reported case in Western Africa following the case presented to the French Language African Medical Society in 1984 (B. Ndiaye). The skin lesion in the form of a naevus of variable dimensions is an essential manifestation of the epidermal naevus syndrome described by Solomon, Fretzin and Dewald in 1968. This syndrome consists of a variable but inconstant association of dysembryoplastic abnormalities affecting the central nervous system (epilepsy, mental retardation, hydrocephalus, localized central deficits), the eye (fibrous conjunctival tumours, corneal opacities, colobomas) and the bones (spine, clavicle, pelvis, limb bones). The bones may be affected by malformations or hypoplasia. The epidermal naevus generally has a linear verrucous appearance, but it is not exceptional to find Jadassohn's sebaceous naevus or even localized erythroderma ichthyosiformis. Mucosal lesions, especially oral, well described in 1960 by Brown and Gorlin, correspond to a particular localization of epidermal naevus and must be differentiated histologically from white sponge naevus, which has a fairly similar clinical appearance. This non-hereditary disease must be systematically investigated looking for visceral abnormalities which are very common. Lastly, in terms of therapy, surgery may be justified when the facial lesions are unsightly, extensive or disabling. Various techniques may be applied depending on the extent and the site of these naevi. PMID- 1718209 TI - [Is it reasonable to perform any esthetic surgery in HIV positive patients?]. AB - HIV seropositivity is becoming an increasingly frequent problem amongst candidates for aesthetic surgery. Although a routine screening test for HIV is not legally compulsory, it should nevertheless be requested prior to any aesthetic surgery. Is it reasonable to take the risk of aggravating the disease in order to perform purely elective aesthetic surgery? The answer to this question is not unequivocal, but must tend towards non-intervention, the reasons for which must be clearly explained to the patient in order to obtain his "informed consent". PMID- 1718210 TI - [Superficial temporal venous drainage and its risks. Surgical implications]. AB - Certain failures in scalp surgery due to defective venous drainage have led the authors to reconsider the classical anatomical data concerning the temporofrontal venous network. The study conducted in the anatomy laboratory concerned 110 superficial temporal territories injected with various coloured polymerizable substances in order to visualize this network in the skin. This study enabled the authors to describe nine types of venous drainage, the surgical implications of which are discussed. PMID- 1718211 TI - Palliative gastrojejunostomy for advanced carcinoma of the stomach. AB - The outcome of 51 patients who underwent gastrojejunostomy for unresectable gastric cancer with outlet obstruction was studied to evaluate the palliative benefit. The operative mortality and postoperative complication rates were 22% and 55% respectively. The mean duration of postoperative hospital stay was 13 days and median survival period 14 weeks. Delayed gastric emptying occurred in 21%, mostly in patients with extensive tumour involvement of the stomach and was associated with a poor outcome. Among the survivors, 87.5% were able to take a soft diet by the eighth postoperative day and 60% could cope with the progression of the disease at home. We conclude that gastrojejunostomy offers satisfactory palliation in patients with advanced gastric cancer. PMID- 1718212 TI - In vitro demyelination by serum antibody from patients with Guillain-Barre syndrome requires terminal complement complexes. AB - Serum from 7 patients who had acute-phase Guillain-Barre syndrome with high anti peripheral nerve myelin antibody activity (54 to 210 units/ml) was compared with serum from 3 patients in the recovery phase (0 to 17 units/ml) and serum from 7 disease control subjects (0 to 24 units/ml) and 7 normal control subjects (0 to 7 units/ml) for its ability to demyelinate rodent dorsal root ganglion cultures. The demyelinating capacity of each serum was quantitated by counting the percent of damaged internodal segments in each of four cultures. All sera from patients in the acute phase GBS caused 50 to 78% demyelination, in contrast with 6 to 19% by the sera from all 3 patients in the recovery phase and all other control subjects. The degree of demyelination correlated with anti-peripheral nerve myelin antibody activity of the sera and demyelination was complement-dependent. Further, cultures were treated with an immunoglobulin M (IgM) fraction of an acute-phase Guillain-Barre syndrome plasma plus normal human serum depleted of complement component C7. Only those cultures treated with IgM and C7-depleted human serum reconstituted with purified C7 resulted in 50.8% demyelination, which was significantly greater than the 14.2 to 16.2% demyelination observed in the presence of heat-inactivated, C7-depleted human serum plus purified C7 or in the absence of C7 or antibody. In summary, our work suggests that anti-peripheral nerve myelin antibody in Guillain-Barre syndrome mediated complement dependent demyelination of rodent dorsal root ganglion cultures. Further, this in vitro demyelination required generation of activation complexes of the terminal complement cascade. PMID- 1718213 TI - Use of intravenous gamma globulin to passively immunize high-risk children against respiratory syncytial virus: safety and pharmacokinetics. The RSVIG Study Group. AB - Infants with cardiopulmonary disease develop severe illness from respiratory syncytial virus (RSV) infection. Safety, feasibility, and pharmacokinetics of intravenous gamma globulin (IVIG) to prevent RSV illness were studied in 23 high risk infants in a phase I trial. IVIG with an RSV neutralizing antibody titer of 1:1,100 in 5% solution was given monthly over a 2- to 4-h period in a clinical setting during the RSV season. The first group (n = 7) received 500 mg/kg of body weight, the second group (n = 9) received 600 mg/kg, and the third group (n =7) received 750 mg/kg. Serum was drawn prior to infusion and 2, 14, and 30 days after infusion. Total immunoglobulin G and RSV A2 and RSV B neutralizing antibody levels were obtained after the first IVIG infusion. Two children developed mild reversible pulmonary edema (group receiving 600 mg/kg per dose), and one developed hives and wheezing during one infusion (group receiving 500 mg/kg per dose). Twelve children developed subsequent RSV infection during two RSV seasons (November to April) over a 2-year follow-up period; 9 of 12 developed infection during the infusion year. Eleven illnesses were mild; one child died of progressive RSV illness (group receiving 500 mg/kg per dose). A cumulative infusion effect was not observed. IVIG appears safe and feasible in an outpatient setting, and at 750 mg/kg per dose, a target RSV antibody level of greater than or equal to 1:100 was achieved. PMID- 1718215 TI - Immunologic relatedness of extracellular ligninases from the actinomycetes Streptomyces viridosporus T7A and Streptomyces badius 252. AB - Four isoforms of the extracellular lignin peroxidase of the ligninolytic actinomycete Streptomyces viridosporus T7A (ALip-P1, P2, P3, and P4) were individually purified by ultrafiltration and ammonium sulfate precipitation, followed by electro-elution using polyacrylamide gel electrophoresis. Three of the purified peroxidases were compared for their immunologic relatedness by Western blot analysis using a polyclonal antibody preparation produced in rabbits against pure isoform P3. The anti-P3 antibody was also tested for its reactivity towards a lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and another ligninolytic actinomycete Streptomyces badius 252. Results showed that peroxidases ALip-P1 through ALip-P3 are immunologically related to one another. The peroxidases of S. badius, but not the peroxidase of P. chrysosporium, also reacted with the antibody, thus indicating that the lignin peroxidases of S. viridosporus and S. badius are immunologically related. Based upon its specific affinity, lignin peroxidase isoform ALip-P3 of S. viridosporus was readily purified using an anti-P3 antibody affinity column. PMID- 1718214 TI - Pseudomonas pseudomallei resistance to beta-lactam antibiotics due to alterations in the chromosomally encoded beta-lactamase. AB - Pseudomonas pseudomallei, the causative agent of melioidosis, is generally susceptible to some of the newer extended-spectrum cephalosporins or to combinations of a beta-lactam and clavulanic acid, a beta-lactamase inhibitor. Resistance to these agents may, however, emerge during treatment. We report on alterations in the chromosomal beta-lactamase associated with the development of resistance. Three resistance patterns resulted from three different mechanisms in the strains investigated. Derepression of the chromosomal enzyme resulted in a general increase in the MICs of all of the beta-lactams tested. The second mechanism observed was an insensitivity to inhibition of the beta-lactamase by clavulanic acid. In this case, the level of susceptibility to beta-lactams as independent entities remained unchanged. The final "resistance" pattern occurred in a patient treated with ceftazidime and resulted in a beta-lactamase that was capable of hydrolyzing this antibiotic at detectable levels, but with reduced efficacy against other beta-lactams. The net result was a strain that was generally susceptible to all of the beta-lactams tested except ceftazidime. In all cases, the level of susceptibility to antibiotics other than beta-lactams remained unchanged. Such variability found within one genus over a relatively short time course suggests that treatment of infections caused by this organism should be carefully monitored to detect susceptibility alterations to the chosen therapy. PMID- 1718216 TI - Protein-protein interactions and structural entities within the sea urchin extraembryonic matrix, the hyaline layer. AB - We have investigated the effects of a variety of experimental conditions on the structural integrity of the sea urchin extraembryonic matrix, the hyaline layer. Removal of Ca2+ resulted in the quantitative release of hyalin from isolated layers. Protein gel blot analyses indicated that, in the absence of Ca2+, hyalin was also quantitatively released from the layers surrounding 1-h-old embryos. However, no other polypeptides of the hyaline layer were released in significant amounts. The layers remaining after removal of hyalin were refractory to digestion with proteinase K and dissociated only in the presence of chaotropes. These results provide insights into the structural organization within the hyaline layer as well as the role of the embryonic cell surface in maintaining the structural integrity of this extraembryonic matrix. PMID- 1718217 TI - Rat sulfite oxidase antibodies cross-react with two gene family-related proteins: albumin and vitamin D-binding protein. AB - Screening lambda cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be 1/5 for rat serum albumin and 1/10 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related. PMID- 1718218 TI - [Combined preoperative treatment with bilateral intra-arterial chemotherapy (CBDCA + PEP) and radiotherapy of oral cancer on the midline]. AB - Intra-arterial chemotherapy from bilateral superficial temporal arteries and radiotherapy were carried out preoperatively on two patients with oral cancer on the midline. Total doses of preoperative chemo- and radio-therapies were 160 mg/m2 of carboplatin, 50 mg of peplomycin and 20 Gy of 60Co-irradiation, respectively. Therapeutic effect of preoperative chemo- and radio-therapies was evaluated on the resected materials from histological point of view. In case 1, the effect was judged as Grade II A in Oboshi's classification, which indicated a mild destruction of architecture of tumor tissue and a few viable tumor cells, but an extreme reduction of the primary lesion was observed on clinical appearance. In case 2, the therapeutic effect was regarded as Grade II B, which indicated a severe destruction of architecture of tumor tissue and few viable tumor cells. Concerning toxicity, mucositis and slight thrombocytopenia (96,000/mm3) in case 1, and mucositis and leukopenia (2,300/mm3) in case 2 appeared. However, they soon recovered after termination of the preoperative therapies. From the above results, it was considered that a combination of bilateral intra-arterial chemotherapy and radiotherapy was quite effective as a preoperative treatment for oral cancers on the midline at the same doses of anti neoplastic agents and irradiation as for the other unilateral oral cancers. PMID- 1718219 TI - Extensive calcinosis as a late complication of pentazocine injections: response to therapy with steroids and aluminum hydroxide. PMID- 1718220 TI - A special conditions register. AB - A special conditions register (SCR) linked to the child health system's register of all children has been in use in West Sussex since 1977. This paper describes the aims, organisation, and use of the SCR and gives examples of the aggregated data that may be obtained. Of the 155,000 children aged 0-17 resident in West Sussex in 1990, 4.3% were included on the SCR. Altogether 45.7% of children on the SCR had physical conditions with mild or no disability and 17.2% had moderate educational problems. The prevalence of severe hearing loss as defined was 1.7 per 1000 aged 5-17. The prevalence of diabetes mellitus was 1.2 per 1000 children aged 0-17. Validation of the SCR for diabetes mellitus found 35/36 of the eligible children were correctly registered and no child was incorrectly included. The conflicting priorities for maintaining a register for the care of individual children, for service planning, and for epidemiological research are discussed. PMID- 1718221 TI - Prognosis of motor development and joint hypermobility. AB - In a study of 59 infants aged 18 months there were 20 with joint hypermobility and delayed motor development, 19 with joint hypermobility and normal motor development, and 20 normal controls. They were reassessed for motor function 3.5 years later at the age of 5 years. Both gross and fine motor performance were significantly delayed in the group of children who exhibited joint hypermobility and motor delay in infancy. No significant delay was evident in those with joint hypermobility only. Joint hypermobility resolved more frequently in children who presented normal motor development at age 18 months. Infants with joint hypermobility and motor delay are a subgroup associated with a less favourable motor outcome and careful follow up is indicated. PMID- 1718222 TI - Progressive renal toxicity due to ifosfamide. AB - A prospective and follow up study of renal tubular and glomerular function in 11 children receiving ifosfamide treatment was conducted. Each child received between four and 14 courses of ifosfamide, given as a continuous infusion of 3 g/m2 over 24 hours for two or three days. Evidence of renal toxicity was seen in all patients. There was a treatment related rise in urinary tubular markers (N acetyl-glucosaminidase and alpha 1 microglobulin). Recovery was limited, so that by the fourth course of treatment all values remained abnormal. There was an associated treatment related reduction in plasma phosphate concentration. Urinary albumin also showed a treatment related rise, but with fewer abnormal values. Electrophoresis was used to confirm tubular or glomerular patterns. Glomerular toxicity was less severe and occurred in fewer patients. The follow up study showed persistence of tubular damage in all seven patients examined, and there was evidence of glomerular damage in five of the seven children. Children receiving ifosfamide need to be carefully monitored for renal toxicity both during treatment and at follow up. PMID- 1718223 TI - Heart-lung transplantation: all the facts. AB - Of 27 children referred for assessment of suitability for heart-lung transplant, 10 (37%) were actually transplanted. Six are still alive from three months to three years since operation. Two thirds of the cohort have died at various stages during referral, assessment, and transplant. While the transplant has offered miraculous new life to a few children, many more have experienced increased and unnecessary suffering. Planning of transplant programmes must take all facts into account. The possibility of heart-lung transplant must not deter further efforts to control chronic lung diseases medically and must not influence appropriate terminal care. PMID- 1718224 TI - Evaluation of paediatric intensive care in a regional centre. AB - All 162 consecutive admissions to a multidisciplinary paediatric intensive care unit in the UK have been prospectively evaluated in terms of therapeutic intention, sickness levels, age, utilisation of resources, and outcome. For 101 (62.3%) of the children admitted the aim of treatment was to cure the condition but for 30 (18.5%) ultimately only a palliative option was available. Five children were admitted to avail of specialised monitoring facilities. One half of the children admitted were physiologically unstable. The majority (102, 62.9%) were age 12 months or less. Resource utilisation, which was not affected by therapeutic intention, was greatest for the sickest patients, those age 1 month or less and non-survivors. Mortality rate overall was 17.9%. Mortality was unaffected by age and therapeutic intention and was inversely related to level of sickness. The information provided by this study forms a basis for medical audit within the unit and is essential for meaningful comparisons of standards of care and outcome with other units. PMID- 1718226 TI - Cellular and subcellular distribution of 2,4-dinitrophenyl groups in mouse epidermis and regional lymph nodes after epicutaneous application of 2,4 dinitrofluorobenzene. AB - The cellular and subcellular distribution of 2,4-dinitrophenyl (DNP) groups in the epidermis and regional lymph nodes of the mouse was investigated after epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to sensitized and non sensitized mice. The peroxidase-antiperoxidase method and the immunogold technique were used to visualize the DNP groups at both light and electron microscopic levels. The highest intensity of immunolabelling was found on tonofilaments of keratinocytes present in the upper layers of the epidermis. On the other hand, in vitro experiments showed that DNFB has the capacity to bind keratin which, together with immunocytochemistry, suggests that this molecule may be one of the skin protein carriers for DNFB. In addition, intense immunostaining for DNP was observed in the Golgi area of some epidermal Langerhans cells. Cells immunoreactive to DNP were also observed in the marginal sinus of cervical lymph nodes 6, 12 and 24 h after challenge. Immunoelectron microscopy revealed immunoreactive DNP groups in phagosomes of Langerhans cells at this site. The present findings support the hypothesis that the hapten DNFB penetrates passively into the cytoplasm of Langerhans cells, concentrates in the Golgi area and, during the migration of Langerhans cells to the lymph nodes, it is probably processed in the lysosomes before its presentation to T lymphocytes. PMID- 1718225 TI - Radiotherapy in paediatric practice. PMID- 1718229 TI - Advantages of early relief of subaortic stenosis in single ventricle equivalents. AB - Thirteen patients with single ventricle equivalents and subaortic stenosis underwent relief of the stenosis and subsequent Fontan operation. Nine patients, group 1, had the obstruction relieved at 3.6 +/- 1.6 years of age whenever the pressure gradient became apparent. Four patients, group 2, had the subaortic stenosis operated on at the neonatal period, 10.5 +/- 10 days old, before hemodynamic evidence of obstruction. Preoperative pressure gradient across the outflow tract was 44.2 +/- 4.7 mm Hg in group 1 versus 4.7 +/- 5 mm Hg in group 2 (p = 0.002). Ventricular muscle mass was 186% +/- 18% in group 1 versus 114% +/- 5% of normal in group 2 (p = 0.0001), and mass/volume ratio was 1.12 +/- 0.62 in group 1 versus 0.62 +/- 0.16 in group 2 (p = 0.003). Relief of subaortic stenosis was achieved by proximal pulmonary artery to ascending aorta or aortic arch anastomosis and by systemic to distal pulmonary artery shunt. There was no hospital mortality or complication related to the procedure. At evaluation before Fontan operation, 4.3 +/- 1.6 years after relief of subaortic stenosis in group 1 and 3.2 +/- 0.9 years in group 2, the pressure gradient across the ventricular outflow tract was 4 +/- 3 mm Hg in group 1 versus 3 +/- 2 mm Hg in group 2 (p = not significant), ventricular muscle mass was 184% +/- 31% in group 1 versus 114% +/- 5% of normal in group 2 (p = 0.003), and the mass/volume ratio was 1.17 +/- 0.2 in group 1 versus 0.62 +/- 0.2 in group 2 (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718228 TI - Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and profilaggrin. AB - The antiperinuclear factor, an autoantibody specific for rheumatoid arthritis, was found in 51/63 (81%) patients with rheumatoid arthritis by indirect immunofluorescence on human buccal mucosa cells. The sensitivity of the antiperinuclear factor test was increased by pretreating the buccal mucosa cells with 0.5% Triton-X100. The specificity of the test for rheumatoid arthritis as compared with control serum samples was maintained. The localisation of the perinuclear factor in the keratohyalin granules of the buccal mucosa cells was verified by immunoelectron microscopy. The perinuclear factor was found to be an insoluble protein whose antigenicity was sensitive to various fixation procedures. In serum samples from patients with rheumatoid arthritis there was a positive correlation between the presence of antiperinuclear factor and the presence of the so called antikeratin antibodies as detected by immunofluorescence on unfixed rat oesophagus cryostat sections. No relation was found between the presence of the perinuclear factor and either the rheumatoid factor, Epstein-Barr virus components, or any cytokeratin. By double immunofluorescence an exact colocalisation of the perinuclear factor and profilaggrin was found. Although the precise biochemical identity of the perinuclear factor remains unclear, our results suggest that it is a protein only present in the fully differentiated squamous epithelial cell layer. PMID- 1718227 TI - Comparative cytokeratin analysis of sweat gland ducts and eccrine poromas. AB - Human eccrine sweat gland ducts and benign and malignant eccrine poromas were studied for the expression of various cytokeratins (CK) and vimentin by applying immunoperoxidase and immunofluorescence microscopy to frozen or paraffin-embedded sections, and using two-dimensional gel electrophoresis and immunoblotting. In acrosyringia and dermal eccrine ducts, the luminal cells exhibited intense staining for CKs 1/10/11 and 19. The periluminal cell layers of acrosyringia contained CKs 1/10/11, while CK 5 was absent. In contrast, the basal cell layer of dermal ducts was only positive with the antibody against CK 5, i.e. a pattern resembling that seen in epidermal basal cells. CK 9 was detected only in keratinocytes peripherally surrounding acrosyringia. In benign poromas, gel electrophoresis revealed that CKs 5 and 14 were predominant, with CKs 6, 16 and 17 being minor components. At the immunohistochemical level CKs 1/10/11 and 19 could be further detected with varying frequency in scattered or clustered cells and/or duct-like structures. Occasionally, CK 9-positive cells were observed. Malignant poromas displayed a similar overall gel-electrophoretic pattern. Their immunohistochemical staining patterns were also similar to (albeit rather more variable than) those seen in benign poromas. Our results show that, with respect to their CK expression pattern, the majority of poroma cells resemble the basal cells of both the dermal ducts and the epidermis, while only minor and variable subpopulations acquire features present in ductal/acrosyringial luminal cells that would be indicative of poral differentiation. Thus, the matrix cells of poromas seem to be most closely related to basal cells located at the transition between the glandular epidermal ridge and dermal eccrine duct, being in no way analogous to the cells of the adult acrosyringium above the basal cell level. PMID- 1718230 TI - Blood saving in Jehovah's Witnesses. PMID- 1718231 TI - Effect of aprotinin to improve myocardial viability in myocardial preservation followed by reperfusion. AB - Isolated canine hearts were preserved at 4 degrees C with multi-dose cardioplegic solution every hour for 6 hours. Reperfusion was observed for 2 hours under cross circulation without cardiotonic drugs. The aprotinin group (n = 8), which received cardioplegic solution with added aprotinin (150 KIU/mL), was compared with the control group (n = 6). The increase in tissue adenosine triphosphate and total adenine nucleotide content during reperfusion was significant in the aprotinin group; there was no change in the control group, and the levels at the end of reperfusion tended to be higher in the aprotinin group than in the control group. Tissue adenosine diphosphate levels remained unchanged in both groups. Tissue adenosine monophosphate levels declined during reperfusion in both groups and were slightly lower in the control group. Tissue levels of cyclic adenosine monophosphate remained unchanged in the aprotinin group whereas they increased during ischemia and declined significantly during reperfusion in the control group. Tissue levels of cyclic guanosine monophosphate declined during reperfusion in both groups without difference. Creatine phosphate levels recovered in both groups without difference. Serum cyclic guanosine monophosphate concentration tended to be lower in the aprotinin group than in the control group. Serum creatine kinase-MB level increased slightly during reperfusion in both groups without difference. N-acetyl-beta-D-glucosaminidase levels were significantly suppressed during reperfusion in the aprotinin group as compared with the control group. These results suggest that aprotinin is effective in preserving adenine nucleotide and adenosine triphosphate levels and in stabilizing tissue cyclic adenosine monophosphate levels in prolonged hypothermic cardioplegic preservation followed by reperfusion. PMID- 1718232 TI - [Post-infarction recurrent myocardial ischemia. Characterization with Holter ECG and exercise test]. AB - In order to characterize postinfarction ischemia, 68 patients were studied by 24 hours Holter-monitoring and exercise testing. Twenty-four (35%) patients in Holter-monitoring and 26 (38%) in exercise testing had transient ischemic episodes. A significant coefficient of agreement was found between both tests. Nineteen (79%) of the patients had only silent ischemic episodes in Holter monitoring, and 87% of all episodes were asymptomatic. Twenty-two (87%) of the patients during positive exercise testing had silent ischemia. Eleven (46%) patients had transient ischemia at low and also at high heart-rates. Ten (37%) patients had ischemic episodes at lower charges than 100 watts, and all of them had more than 60 min of total ischemic burden in Holter-monitoring. A significant correlation was found between total ischemic burden and maximum ST segment shifts. The number of ischemic episodes were significantly higher during morning hours. A significantly higher rate of ventricular extrasystoles was found in recurrent ischemic patients, however, no difference was found in complex arrhythmias. After 1 year follow-up, 3 residual ischemic patients have died. The morbidity-calculated relative-risk is 13.9 times higher in patients with recurrent ischemia. PMID- 1718233 TI - [Changes of parotid gland in rat caerulein-induced acute pancreatitis: study on in vitro system]. AB - To explore the relationship between exocrine pancreas and parotid gland, we measured the changes of parotid gland in in-vitro system at an acute pancreatitis induced by supramaximal dose of caerulein (5 micrograms/kg/h for 3.5 hours) in rats. Both the serum amylase levels and parotid gland amylase content in rats with acute pancreatitis increased significantly compared with normal rats. The dry/wet weight ratio also decreased significantly and LDH discharge from parotid acini as well as lysosomal enzyme leakage from lysosomes in acini increased significantly compared with normal rats. In addition, redistribution of lysosomal enzyme in parotid acini was seen in acute pancreatitis. These results indicate the edema and congestion of amylase in parotid gland, and furthermore the increased cellular and lysosomal fragility of parotid gland at an acute pancreatitis. Thus, there seems to be the intimate organ relationship between exocrine pancreas and parotid gland as well as the important roles of gut hormones such as caerulein in the pathophysiology of parotid gland. PMID- 1718234 TI - Structural studies on Hafnia alvei 114-60 O-antigen. AB - The structure of Hafnia alvei 114-60 O-antigen has been established using sugar and methylation analyses as well as 1H-NMR spectroscopy. The results obtained proved that the repeating unit of Hafnia alvei 114-60 O-antigen is identical to that of Hafnia alvei ATCC 13337 standard strain and has the following structure: [formula: see text] PMID- 1718235 TI - Capsular and somatic antigens of Klebsiella bacilli. AB - Capsular and somatic antigens were determined in 100 Kl. oxytoca strains isolated from patients with the respiratory tract inflammations. K antigens were assigned by the capsula swelling test using 77 specific anti-capsular sera. Most frequent were: K14, K2, K55, K8 and K16 antigens. Positive reaction was noted with 64 strains in 2 or more sera. Somatic antigens of Klebsiella oxytoca bacilli were tested by the test tube agglutination reaction. Of 63 strains tested with anti-01 Kl. pneumoniae and Kl. oxytoca sera, all reacted positively in anti-0 Kl. oxytoca serum and 77% strains in anti-01 Kl. pneumoniae serum. Of 29 strains agglutinating in anti-03 sera, 65.5% agglutinated with anti-0 Kl. oxytoca serum and 76% with anti-03 Kl. pneumoniae. The results have revealed that Kl. oxytoca strains investigated have more complicated capsular antigens and different frequency of their occurrence. The most commonly encountered somatic antigen is antigen 01, next in turn is antigen 03, these antigens proved nonidentical with their 01 and 03 counterparts in Kl. pneumoniae strains. PMID- 1718236 TI - Surface markers on human activated T lymphocytes. IV. Comparison of high-affinity E-rosette receptor expression with the expression of other activation markers (receptor for interleukin 2, MHC class II (antigens). AB - In order to re-examine the value of high-affinity E rosette receptor (Eh-R) as an activation marker of human T lymphocytes, its existence on resting and activated T cells was compared with the expression of such known activation markers as receptor for interleukin 2 (IL-2R; Tac antigen) and MHC class II antigens (DR/DP and DQ). To this aim expression of the above surface markers on lymphocytes of TEe subset, derived from early E rosettes (Eh-R+) and on lymphocytes of TEl subset, derived from late E rosettes (Eh-R-), was examined immediately after purification of the cells from peripheral blood as well as during cell activation with PHA. The phenotypic studies were done by using monoclonal antibodies and indirect immunofluorescence technique. We confirmed previous observation that in the course of PHA stimulation Eh-R like IL-2R marked currently activated T cells. However, it was also found that in the long-term cultures of lymphocytes activated with PHA, the expression of Eh-R was sustained on the cells which lost their IL-2R and DR/DP antigens. The above findings and the fact that TEe cell subset consisting of Eh-R+ lymphocytes was almost completely depleted from cells bearing IL-2R and MHC class II antigens allowed us to conclude that this subset of peripheral blood T lymphocytes represented not currently activated cells but the cells which had been previously activated in vivo. PMID- 1718237 TI - HMB-45 detection in adenocarcinomas. AB - Several studies have suggested that HMB-45 is a specific marker for melanoma, presumably due to its ability to detect a glycoprotein that is present in premelanosomes. The present study was conducted to evaluate whether HMB-45 is an absolutely specific antigenic determinant for melanoma and the role that testing with this antibody has in the differential diagnostic workup of amelanotic melanoma vs adenocarcinoma. Formaldehyde solution-fixed, paraffin-embedded tissue samples from 52 adenocarcinomas (primary or metastatic) and five melanomas (two primary and three metastatic) were immunostained with the use of a commercially available monoclonal antibody (MoAb), ie, HMB-45 (Enzo), a polyclonal antibody to S100 protein, a wide-spectrum keratin polyclonal antibody, and a keratin MoAb, ie, AE1/AE3. Approximately 10% (ie, 9.6%) of the adenocarcinomas (five cases) expressed HMB-45 with varied intensity and distribution. Positive primary tumors (n = 3) included one each from the breast, colon, and kidney; positive metastatic tumors (n = 2) included one each from the breast and endometrium. Fifty-two percent of the adenocarcinomas were positive for S100 protein. One renal carcinoma was negative for both keratins when tested with the AE1/AE3 MoAb and polyclonal antibody (Dako). This was the only adenocarcinoma that was negative when the keratin polyclonal antibody (Dako) was used. All but one additional adenocarcinoma demonstrated keratin expression when the AE1/AE3 MoAb was used for testing. This study showed that HMB-45 is not absolutely specific for melanoma. HMB-45 may react with some adenocarcinomas, at least when tested with the commercially available MoAb (Enzo). This fact, in conjunction with aberrant keratin expression by some melanomas and S100 protein expression by adenocarcinomas and other neoplasms other than melanomas, should be considered when antibody panels are evaluated in the workup of poorly differentiated tumors. However, HMB-45 appears to be the most specific marker that is available at the present time for supporting a diagnosis of melanoma. PMID- 1718238 TI - Extrauterine mixed mesodermal tumors. An immunohistochemical study. AB - Mixed mesodermal tumors are uncommon outside the uterus. Nine extrauterine mixed mesodermal tumors (eight ovarian and one extragenital) were selected for histochemical and immunoperoxidase study. In eight cases, both epithelial and mesenchymal elements were malignant (chondroid in six, rhabdomyoid in four, and osteoid in two). One ovarian tumor was an adenosarcoma. All cases were stained with periodic acid-Schiff with and without diastase and for alpha 1-antitrypsin, myoglobin, keratin, vimentin, muscle-specific actin, and alpha 1 antichymotrypsin, by using the avidin-biotin-immunoperoxidase method. The periodic acid-Schiff-positive, diastase-resistant droplets in several of the tumors showed peripheral alpha 1-antitrypsin positivity. Keratin delineated epithelial areas well in seven cases, and rhabdomyoid differentiation was confirmed with myoglobin in four cases. However, squamous elements in one tumor were falsely positive for myoglobin. We concluded that despite occasional cross reactivity, carefully interpreted immunoperoxidase stains can be useful in distinguishing epithelial and mesenchymal elements in these tumors. PMID- 1718239 TI - Galanin immunoreactivity in neuroendocrine tumors. AB - We investigated immunoreactivity for galanin, a 29-amino acid peptide, in formalin-fixed, paraffin-embedded sections of 123 neuroendocrine tumors. Galanin immunoreactive cells were found in one of 12 hypothalamic gangliocytomas, nine of 18 adrenal pheochromocytomas, nine of 14 pituitary corticotroph adenomas, and one of two thymic endocrine tumors. In pheochromocytomas, galanin-immunoreactive cells were seen either singly or in clusters. In corticotroph adenomas, many tumor cells were positive for galanin, indicating colocalization of corticotropin and galanin in the same tumor cells. No galanin-immunoreactive cells were noted in four extra-adrenal paragangliomas; 10 medullary carcinomas of the thyroid; 35 endocrine tumors arising in the lung, pancreas, and gastrointestinal tract; and 28 pituitary adenomas composed of cells other than corticotrophs. In nontumorous counterparts of these neuroendocrine tumors, galanin immunoreactivity was observed in nerve cells of the hypothalamus, nerve fibers of the duodenum, and adenohypophyseal cells corresponding to corticotrophs. These findings indicate that galanin expression in neuroendocrine tumors is uncommon and restricted to some tumor types. PMID- 1718240 TI - Black bones following long-term minocycline treatment. AB - During a surgical procedure, black vertebrae were observed in a 42-year-old white woman. An undecalcified iliac crest bone biopsy specimen revealed intense fluorescence compatible with tetracycline labeling and osteoporosis. A urinary screening test was negative for amino acids. The patient had been treated with minocycline hydrochloride (100 to 300 mg/d) for at least 6 years. Since minocycline is known to discolor many body tissues, it is likely that the black discoloration of bone in our patient was caused by the long-term intake of the antibiotic. PMID- 1718241 TI - Amylase-producing multiple myeloma. AB - We managed a case of amylase-producing multiple myeloma with extensive extramedullary spread. We reviewed five cases of amylase-producing multiple myeloma, including this case. This type of multiple myeloma has shown unique clinicopathologic features. (1) A distinct elevation of the serum amylase activity was demonstrated in all five patients. The amylase isozyme was of the S type without exception. (2) Extensive extramedullary spread with extramedullary tumors and/or myelomatous pleural effusions or ascites was seen in all five patients during the course of illness. (3) In three of four cases in which it was mentioned, extensive destruction of multiple bones was demonstrated roentgenographically. (4) In four patients, excepting one with a solitary bone lesion, the survival from the initial therapy was shorter than 1 year. (5) Myeloma cell lines were established in three cases. A common feature of these three cell lines was a translocation of chromosome 1, which supplied the amylase gene. This finding may be pathogenetically related to this entity. PMID- 1718242 TI - Multicentric papillary-cystic neoplasm of the pancreas. AB - Two distinct papillary-cystic neoplasms were found in the pancreas of a young black woman. She presented to the hospital in her first trimester of pregnancy with the chief complaint of sharp right upper quadrant abdominal pain that radiated to the right shoulder. This was associated with jaundice, vomiting, and pruritus. On examination, a large, nontender, midepigastric abdominal mass was palpated. Serum liver enzyme levels were moderately to markedly elevated. An abdominal computed tomographic scan revealed a 9-cm solid and cystic mass located within the head of the pancreas with associated marked bile duct dilatation. A total pancreatectomy was performed. Gross examination of the specimen revealed two separate well-circumscribed tumors of unequal size. The larger one was found within the head of the pancreas and contained multiple hemorrhagic, cystic cavities. The smaller one, located within the tail, consisted primarily of solid tissue. Microscopic examination of both lesions revealed papillary-cystic neoplasms. To our knowledge, this is the first report of two synchronous papillary-cystic tumors of the pancreas and the first reported demonstration of the potential of this tumor for multicentricity. PMID- 1718243 TI - 7.5% sodium chloride/dextran for resuscitation of trauma patients undergoing helicopter transport. AB - To evaluate the use of hypertonic saline/dextran solutions in the prehospital resuscitation of severely injured patients, we administered 250 mL of either 7.5% sodium chloride/dextran 70 (HSD) (n = 83) or lactated Ringer's solution (n = 83), followed by conventional isotonic fluids, to 166 trauma patients with systolic blood pressures less than or equal to 100 mm Hg, in a prospective, randomized, double-blinded clinical trial. Patients in the sodium chloride/dextran 70 group required less fluid before hospitalization and arrived in the emergency department with higher systolic blood pressures than patients in the lactated Ringer's solution group. The rate of survival to hospital discharge for the entire cohort was 64% for patients in the sodium chloride/dextran 70 group vs 59% for patients in the lactated Ringer's solution group. The rate of survival to hospital discharge for the patients with severe head injuries was 32% for the sodium chloride/dextran 70 group vs 16% for the lactated Ringer's solution group. Actuarial survival for patients with severe head injuries in the sodium chloride/dextran 70 group compared with patients with severe head injuries in the lactated Ringer's solution group did not quite reach statistical significance. There were no adverse side effects associated with sodium chloride/dextran 70 administration. Administration of small volumes of sodium chloride/dextran 70 before hospitalization increased the blood pressure of severely injured patients more effectively than did lactated Ringer's solution and showed tendencies toward improving survival in the patients with severe head injuries. PMID- 1718246 TI - The stimuli releasing histamine from murine bone marrow-derived mast cells. 2. Mechanisms involved in histamine release induced by extracellular ATP and its metabolites. AB - Extracellular ATP stimulated histamine release and generation of leukotrience C4 (LTC4) accompanied with the formation of inositol phosphates and a rapid increase in intracellular Ca2+ ([Ca2+]i) in mouse bone marrow-derived cultured mast cells (BMMC). The rank order of histamine-releasing potency of ATP and its metabolites is ATP greater than ADP greater than AMP greater than adenosine. Nonhydrolyzable ATP analog, adenosine-5'-O-[2-thiotriphosphate] (ATP-S) released more histamine from the cells than ATP. On the other hand, simultaneous addition of adenosine analogues at micromolar concentrations potentiated histamine release from the cells induced by ATP (50 microM) or DNP-HSA antigen (0.1 ng/ml) in the following rank order: adenosine greater than AMP much greater than ADP = ATP. Histamine release potentiated by adenosine was blocked by the treatment with pertussis toxin, whereas histamine release induced by ATP was not affected by the toxin, suggesting that extracellular ATP stimulate histamine release from BMMC probably via mechanisms independent of the potentiation of histamine release induced by adenosine. PMID- 1718244 TI - Modified-live infectious bovine rhinotracheitis virus vaccine expressing monomer and dimer forms of foot-and-mouth disease capsid protein epitopes on surface of hybrid virus particles. AB - Modified-live, attenuated infectious bovine rhinotracheitis (IBR) hybrid virus vaccines have been constructed by inserting in the major IBRV glycoprotein gIII gene chemically synthesized deoxyribonucleotide sequences encoding the bovine growth hormone signal sequence and monomeric or dimeric forms of the foot and mouth disease virus (FMDV) VP 1 epitope sequences. The foreign DNA sequences were inserted at the N-terminal end of the IBRV gIII coding sequence and were driven by the IBRV gIII promoter. The sequences encoding the first 38 and the first 21 amino acids of the IBRV gIII were deleted from the hybrid viruses containing inserts of the monomeric and dimeric FMDV epitope sequences, respectively, to avoid redundant signal sequences. Plaque immunoassay experiments with guinea pig and bovine anti-FMDV peptide antisera, and with anti-IBRV gIII monoclonal antibodies demonstrated that IBRV-FMDV fusion proteins were expressed in virus infected MDBK cells. Immunoelectron microscopy analyses demonstrated that the IBRV-FMDV fusion proteins were expressed as repeated structures on the surface of virus particles. Experiments showed that the recombinant IBRV-FMDV viruses protected cattle from IBRV (Cooper) challenge and induced anti-FMDV peptide antibodies, thereby demonstrating that the FMDV epitopes were expressed in vivo. PMID- 1718245 TI - Effect of SN-408 (salmeterol hydroxynaphthoate) on passive cutaneous anaphylaxis and anaphylactic chemical mediator release in rats and guinea pigs. AB - The influence of SN-408 (salmeterol hydroxynaphthoate), a new selective beta 2 stimulant, on homologous passive cutaneous anaphylaxis (PCA), histamine- or serotonin-induced skin reaction and anaphylactic chemical mediator release was investigated in rats and guinea pigs, and its efficacy was compared with that of salbutamol, isoproterenol or disodium cromoglycate (DSCG). Intravenous administration of SN-408 (0.1-10 micrograms/kg) 1 min before, the antigen challenge dose-dependently inhibited rat homologous PCA to a similar extent to salbutamol. The inhibitory effect of these beta-agonists was quickly attenuated along with time between the administration of the drug and the antigen challenge. SN-408, however, still showed significant inhibition at the time of administration 5 min before the antigen challenge at which other agonists did not significantly affect any more. Ten micrograms/kg of SN-408 exhibited approximately 50% inhibition of skin reaction induced by either histamine or serotonin in rats. In addition, the compound represented concentration-dependent inhibition of the anaphylactic histamine release from the isolated rat peritoneal exudate cell, indicating that the inhibitory effect of SN-408 on rat homologous PCA is due to the suppression of mediator release in addition to the antagonism to mediator(s) released. The anaphylactic release of either histamine, immunoreactive (i-) leukotriene (LT)B4 or i-LTC4 from guinea pig lung fragments was concentration-dependently inhibited by 10(-10)-10(-7) g/ml SN-408, and the inhibitory potency appeared to be enhanced by prolongation of the pretreatment interval with the drug.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718247 TI - Tritiated imipramine binding. A peripheral marker for serotonin in Parkinson's disease. AB - Tritiated imipramine binding in platelets has been used to evaluate serotonin activity in depression in previous studies. This article examined this marker as a possible measure of central nervous system serotonergic activity for depression in patients with Parkinson's disease (PD). The number of binding sites was significantly lower in depressed patients with PD than in a healthy control group. Patients with PD who were not depressed had lower values than the comparison group, but this difference was not significant. We also found a significant correlation between the receptor site values in platelets and cerebrospinal fluid levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (r = .59), but this was independent of a diagnosis of depression. Receptor site values were examined to identify appropriate cutoff scores to predict depression in the group of patients with PD. A maximum sensitivity of 50% was achieved with a specificity of 64%. Our results strongly support a generalized alteration in serotonin metabolism in depressed patients with PD, but tritiated imipramine binding in platelets is not a useful diagnostic tool for depression. PMID- 1718248 TI - Pretreatment fundus characteristics as predictors of recurrent choroidal neovascularization. PMID- 1718249 TI - Management of subfoveal choroidal neovascularization. PMID- 1718250 TI - Laser photocoagulation of subfoveal neovascular lesions in age-related macular degeneration. Results of a randomized clinical trial. Macular Photocoagulation Study Group. AB - A clinical trial has been conducted to evaluate the effect on vision of laser treatment (argon green or krypton red lasers, assigned randomly) compared with no treatment of eyes with subfoveal choroidal neovascularization associated with age related macular degeneration. Three months after enrollment, the visual acuity of 37 (20%) of 184 laser-treated eyes had decreased 6 or more lines from the baseline level, while only 19 (11%) of 178 untreated eyes had suffered such large decreases (P = .01, chi 2 test). However, 24 months after enrollment, the visual acuity of only 23 (20%) of 114 laser-treated eyes had decreased by 6 or more lines from baseline compared with 41 (37%) of 112 untreated eyes (P less than .01, chi 2 test). On average, after 24 months, visual acuity of laser-treated eyes had decreased 3 lines from baseline, and visual acuity of untreated eyes had decreased 4 lines (P = .003, t test). Laser-treated eyes retained contrast threshold for large letters at or near baseline levels through 36 months of follow-up examinations, while the contrast threshold of untreated eyes worsened. Persistent or recurrent neovascularization was observed in 51% of the laser treated eyes by 24 months after initial treatment but was not associated with decreased visual acuity. None of the findings suggested a reason to favor either the argon green or krypton red wavelength. Laser treatment of subfoveal neovascular lesions that meet the eligibility criteria for this study is recommended if both the patient and the ophthalmologist are prepared for a large decrease in visual acuity immediately after treatment. PMID- 1718251 TI - Laser photocoagulation of subfoveal recurrent neovascular lesions in age-related macular degeneration. Results of a randomized clinical trial. Macular Photocoagulation Study Group. AB - In a randomized clinical trial, the effects of laser treatment of subfoveal recurrent neovascularization have been compared with no additional laser treatment. Among eyes with age-related macular degeneration assigned to laser treatment of recurrent neovascularization (n = 97) and those assigned to no additional laser treatment (n = 109), average visual acuity was 20/125 (Snellen equivalent) at entry. Both at 3 and 24 months after enrollment, the average visual acuity of laser-treated eyes was 20/250, indicating a decrease in visual acuity of 3 lines immediately after treatment, but little further decrease in acuity thereafter. In contrast, the average visual acuity of eyes assigned to no additional treatment decreased by 2 lines to 20/200 by 3 months after entry. After 24 months, acuity had decreased an additional 2 lines to 20/320. By 24 months after enrollment, only three (9%) of 35 laser-treated eyes, but 13 (28%) of 46 untreated eyes, had decreased visual acuity of 6 or more lines from baseline (P = .03, chi 2 test). On average, treated eyes maintained the initial contrast threshold for large letters, while the contrast threshold of untreated eyes worsened steadily throughout 24 months of follow-up. This study was halted before the target number of patients had been enrolled because of the similarity of these findings to findings from a related Macular Photocoagulation Study clinical trial of laser treatment of subfoveal neovascularization secondary to age-related macular degeneration in eyes without previous laser treatment. Laser treatment is recommended for subfoveal recurrent neovascularization in eyes with age-related macular degeneration that meet the eligibility criteria for this study. PMID- 1718252 TI - Subfoveal neovascular lesions in age-related macular degeneration. Guidelines for evaluation and treatment in the macular photocoagulation study. Macular Photocoagulation Study Group. AB - The Macular Photocoagulation Study (MPS) guidelines for interpreting angiograms of eyes with subfoveal choroidal neovascularization (CNV) secondary to age related macular degeneration and for treating these lesions are described to assist ophthalmologists in applying the results of the MPS clinical trials of laser treatment. The MPS criteria for treatment of subfoveal neovascular lesions require the following conditions: (1) the presence of classic CNV, (2) well demarcated lesion boundaries, and (3) size less than or equal to 3.5 disc areas (if no previous treatment of CNV in the macula was performed). In subfoveal recurrent CNV, size had to be such that after treatment of the recurrence, the final treatment scar (prior treatment scar and newly treated area) would be no larger than 6 disc areas and would spare some retina within 1500 microns of the center of the foveal avascular zone. Treatment of all classic and occult CNV and areas in which the boundaries of CNV may be obscured is recommended, as it treatment extending at least 100 microns beyond the peripheral boundaries of the lesion. In subfoveal recurrent CNV, treatment should also extend at least 300 microns into the previous treatment scar and cover any feeder vessels. The desired end point for the intensity of the laser burns is a uniformly white lesion. PMID- 1718253 TI - Perifoveal laser treatment for subfoveal choroidal new vessels in age-related macular degeneration. Results of a randomized clinical trial. AB - In a controlled clinical trial, 160 eyes underwent "perifoveal" laser photocoagulation. All patients had age-related macular degeneration and subfoveal neovascularization (0.5 to 2.5 disc diameters), without detectable fibrous tissue, and visual acuity from 20/100 to 20/1000. At least 1 year of follow-up has been completed in 127 eyes. Visual acuity was maintained or improved in 12 (20.3%) of 59 untreated eyes vs 28 (41.1%) of 68 treated eyes (P = .04). Reading visual acuity (J4) with the use of low-vision aids was retained in 28 untreated eyes (47.4%) vs 50 treated eyes (73.5%) (P less than .01). Automated static perimetry showed an increased scotomatous area and/or depth with eccentric fixation in all but four patients. A flat atrophic scar was achieved in 53 of 68 treated eyes. Statistical analysis indicated that perifoveal photocoagulation has been effective in the short-term preservation of visual acuity. PMID- 1718254 TI - Hypertonic saline dextran for post-injury resuscitation: experimental background and clinical experience. PMID- 1718255 TI - Second twin: quality of survival if born by breech extraction following internal podalic version. AB - The intrapartum management of the vertex-breech and vertex-transverse twin gestation is controversial. The fall in perinatal mortality rate to a low level has resulted in this parameter failing to be an adequate gauge of the safety of breech extraction and the answer lies in the quality of survival of the infants. Fifty-one twin pairs, collected over 12 years at the Mercy Hospital for Women, Melbourne, occurred where twin 2 was born by breech extraction following internal inversion and the control (twin 1) did not have this procedure performed. In 8 pairs either a stillbirth or neonatal death occurred; in one pair childhood death due to an accident (fire) occurred; in 4 pairs the parents refused entrance to the study as they perceived both twins to be similar; in 2 sets the assessment was incomplete; 11 sets were untraceable leaving 25 sets fully assessed as children ranging in age from 2 to 12 years. Growth, and psychological scores were not significantly different between twins 1 and 2 but 2 children had cerebral palsy and both were born by breech extraction following internal version at 29.2 and 30.1 weeks' gestation, respectively. Because of small numbers the results failed to achieve statistical significance and this study was unable to answer the question regarding the safety of breech extraction following internal version but did show that the majority of infants so born do well. PMID- 1718256 TI - Coupling of reverse transcriptase and RNase H during HIV-1 replication. AB - The retroviral replicating enzyme reverse transcriptase (RT) is associated with an RNase H which specifically hydrolyzes RNA in RNA-DNA hybrids. The RNase H exhibits endo- as well as exonuclease activity when analyzed with in vitro synthesized viral RNA hybridized to a shorter synthetic DNA oligonucleotide. In the presence of deoxyribonucleotides the RT synthesizes DNA in a concerted action with the RNase H, which simultaneously hydrolyzes the RNA template. Mutants of the RNase H derived by site-directed mutagenesis of a conserved histidine 539 are preferentially impaired in their exo- and less in their endonuclease activity. A specific polypurine-rich oligonucleotide created at the polypurine tract (PPT), serves as a primer for plus-strand DNA synthesis. Initiation of DNA synthesis at the PPT-primer can be demonstrated for the wt enzyme in vitro in contrast to the mt enzymes which do not recognize this sequence. A replication model based on these results is presented. PMID- 1718257 TI - Functional aspects of a tyrosine kinase encoding protooncogene, the c-src gene. AB - To date the src gene family consists of at least 9 closely related protein tyrosine kinases belonging to the non-receptor type of kinases: c-src, c-yes, c fgr, fyn, lyn, lck, hck, tkl and bkl. We have intensively studied the expression of the c-src gene during evolution and with respect to its possible functions in the processes of cellular differentiation and proliferation. From our results we conclude, that the c-src encoded tyrosine kinase could play a role in the development and/or maintenance of the multicellular organisation of primitive organisms like sponges or coelenterates. With respect to its tissue-specific expression pattern with neuronal cells always displaying elevated levels of pp60c src and the observation that synaptophysin, the major constituent of the synaptic vesicle membrane protein, is phosphorylated by the c-src encoded tyrosinekinase in vitro and in intact synaptic vesicles, we suggest an essential role for pp60c src in signal transduction pathways and/or axonal transport mechanisms in neurons. PMID- 1718258 TI - Molecular cloning of a human cannabinoid receptor which is also expressed in testis. AB - A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s). PMID- 1718259 TI - Regulation of amylase and chymotrypsinogen expression by dexamethasone and caerulein in serum-free-cultured pancreatic acinar AR4-2J cells. Influence of glucose. AB - The direct effects of dexamethasone and caerulein on two pancreatic enzymes, amylase and chymotrypsin, were determined in AR4-2J cells cultured under serum free conditions at two glucose concentrations (1.0 and 4.5 g/l). In the absence of any hormone, the higher glucose concentration resulted in a 1.6-1.8-fold increase in the basal levels of amylase and chymotrypsinogen. Dexamethasone (50 nM) increased the biosynthesis and mRNA levels of both enzymes at both glucose concentrations. However, dexamethasone had a more pronounced effect on amylase biosynthesis (5-fold induction) than on chymotrypsinogen biosynthesis (1.8-fold induction). The parallel increases in mRNA and protein indicated the existence of pre-translational regulation. This is in contrast with what was observed in serum containing media, where a translational regulation of amylase biosynthesis took place, probably under the control of both glucose and some serum factors. By contrast, caerulein (10 nM) exerted a more specific action on chymotrypsinogen. The increases in chymotrypsinogen mRNA were 2.2- and 2.1-fold, and increases in chymotrypsin activity were 1.6- and 2.9-fold at 1.0 and 4.5 g of glucose/litre respectively. Thus the regulation by caerulein occurred mainly through the enhancement of chymotrypsinogen transcription and/or mRNA stabilization. PMID- 1718260 TI - Definition of surface-exposed epitopes on the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum. AB - Epitopes for monoclonal antibodies binding to the native (Ca(2+)-Mg2+)-ATPase have been defined by studying binding to sets of hexameric peptides synthesized on plastic pegs. Epitopes have been confirmed by demonstrating the binding of anti-peptide antibodies to the ATPase. A method is presented for definition of surface-exposed epitopes using polyclonal antibodies. Three surface-exposed epitopes have been defined in the nucleotide-binding domain of the ATPase, suggesting considerable surface exposure of this region. Other surface-exposed epitopes have been located in the region of the fourth stalk domain. PMID- 1718261 TI - Regulation by forskolin of cyclic AMP phosphodiesterase and protein kinase C activity in LLC-PK1 cells. AB - Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC PK1 protein kinase C activity can be attenuated by staurosporine. Both 5 pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13 acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells. PMID- 1718263 TI - Nomenclature errors. PMID- 1718262 TI - Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells. AB - Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID- 1718264 TI - Effect of dextran on synthesis, secretion and deposition of type III procollagen in cultured human fibroblasts. AB - When subconfluent cultures of primary human skin fibroblasts are incubated for 20 h in the presence of 5% neutral dextran, the newly synthesized procollagens are shifted from the medium into the pericellular matrix fraction. This is accompanied by an overall decrease by 30-50% in the secretion rates of these proteins, as indicated by the incorporation of tritiated proline into collagenase sensitive macromolecules and by radioimmunoassays for the propeptide regions of type I and III procollagens. Inhibition of tyrosine sulphation in newly formed type III procollagen by NaClO3 does not change the distribution of this protein between the medium and matrix fractions either in the presence or in the absence of the polymer. Processing of type III procollagen at its N-terminus is incomplete in this situation, irrespective of whether the protein is present mainly in the medium or in the pericellular matrix. The concentrations of the mRNAs for the pro alpha 1(I)- and pro alpha 1(III)-chains of procollagens and for actin were monitored for up to 20 h; in the dextran-treated cultures these are not different from the corresponding concentrations in control cultures, indicating that the rapid down-regulation of the synthesis of type I and III procollagens in response to the enhanced pericellular matrix deposition induced by dextran is not due to transcriptional mechanisms. PMID- 1718265 TI - Structural characterization of neutral oligosaccharides with blood-group A and H activity isolated from bovine submaxillary mucin. AB - In this study we investigated the structures of 11 neutral oligosaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by h.p.l.c. One hexa-, one penta-, three tetra-, four tri- and two di saccharides containing core types 1, 2, 3 or 4 were obtained. We report their structures, determined by a combination of one- and two-dimensional 1H n.m.r. spectroscopy at 270 MHz and methylation analysis involving g.l.c.-m.s., along with their approximate molar ratios. Only three of these oligosaccharides have previously been reported in this source. Of the new oligosaccharides, one contains the blood-group-A antigenic determinant, two contain the blood-group-H type 2 determinant, while another contains the blood-group-H type 3 determinant. The oligosaccharide GlcNAc beta (1----6)[GlcNAc beta (1----3)]GalNAcol, although previously found as a core structure, has been isolated here as a novel trisaccharide. PMID- 1718266 TI - Molecular basis of acid sphingomyelinase deficiency in a patient with Niemann Pick disease type A. AB - Niemann-Pick disease, an autosomal recessive lysosomal storage disorder, is caused by deficiency of acid sphingomyelinase. Sequence analysis of mRNA and genomic DNA of fibroblasts of a type A patient showed a single G1729 to A nucleotide transition. This mutation resulted in a substitution of serine for normal glycine at position 577 of the peptide sequence. Amplification of the genomic DNA region around the mutation and subsequent sequencing yielded exclusively the same base change found at the cDNA level. Expression studies with this abnormal cDNA in COS-1 cells revealed a complete loss of enzymatic activity of the mutated protein. These findings indicate that this mutation is responsible for the clinical disease of the patient. PMID- 1718267 TI - Molecular cloning, structural characterization and functional expression of the human substance P receptor. AB - A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-1-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP much greater than neurokinin A greater than neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver. PMID- 1718268 TI - cDNA cloning of a novel testis-specific calcineurin B-like protein. AB - A cDNA clone encoding a novel calcineurin B-like protein (CBLP) was isolated from a rat testis cDNA library by cross-hybridization with a calcineurin B cDNA probe. CBLP is composed of 176 amino acids and contains four putative Ca(2+)-binding domains. The overall predicted amino acid sequence homology between CBLP and calcineurin B is 82%. Northern blots showed that transcripts for CBLP are specifically and highly expressed in the testis. PMID- 1718269 TI - Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis. AB - Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity. PMID- 1718270 TI - Isolation and expression of a guanylate cyclase-coupled heat stable enterotoxin receptor cDNA from a human colonic cell line. AB - Heat stable enterotoxins (STs) are low molecular-weight peptides secreted by enterotoxigenic bacteria. One type of these enterotoxins (STa) induces intestinal secretion leading to acute diarrhea by binding to a membrane form of guanylate cyclase. We have isolated a cDNA from a human colonic cell line, T84, encoding for a guanylate cyclase-coupled enterotoxin receptor (STaR). The predicted amino acid sequence of the human STa receptor is 81% identical with the previously cloned enterotoxin receptor (GC-C) from rat intestine. COS-7 cells transiently transfected with the cloned cDNA expressed specific concentration-dependent response to STa as measured by cyclic GMP accumulation and is about 20 times more sensitive to the stimulation by STa than has been shown for GC-C. PMID- 1718271 TI - Identification of the cDNA for xlcaax-1, a membrane associated Xenopus maternal protein. AB - xlcaax-1 is a cDNA coding for a CAAX box containing protein in Xenopus laevis that undergoes isoprenylation and palmitoylation. Here we report on the confirmation that this clone (formerly xlgv7) codes for a 110 kDa membrane associated protein and not an 80 kDa nuclear protein as originally believed (1). The reason for the misidentification was the presence of a common epitope on these two proteins recognized by the monoclonal antibody 37-1A9. We clarified the discrepancy by raising polyclonal antibodies against the xlcaax-1 protein produced in a bacterial expression system and demonstrating that these antibodies only recognize the 110 kDa protein on western blots of oocyte extracts. During early development xlcaax-1 protein starts reaccumulating from the neurula stage. In the adult frog both the xlcaax-1 protein and its cognate mRNA are highly enriched in the kidney. Consistent with the presence of CAAX box at the C terminus this protein is associated with the membranes in Xenopus tissue culture cells (XTC). PMID- 1718272 TI - Class II antigen-associated invariant chain mRNA in mouse small intestine. AB - MHC class II antigen-associated invariant (Ii) chain mRNA appears in mouse small intestine during postnatal development. Ii chain cDNA hybridizes to RNA from epithelial sheets dissociated from the lamina propria with EDTA. Of several mouse organs tested, only bone marrow and spleen contain higher levels of Ii chain mRNA than small bowel. Ii chain mRNA is not detected in stomach, colon, duodenum, testis, liver, submandibular gland, or L-cell RNA; brain contains a cross reactive but uncharacterized sequence. cDNA amplification using primers specific for both Ii31 and Ii41 chain mRNAs showed that both forms occur in small intestine. These results support the conclusion that regulation of the class II Ii chain gene is associated with the ontogeny of intestinal immunity. PMID- 1718273 TI - Effect of substance P/bombesin antagonists on the release of growth hormone by GHRP and GHRH. AB - The substance P(SP)/bombesin (Bn) antagonists [DArg1DTrp7,9Leu11] SP(P-7482), [DArg1-DPro2DTrp7,9Leu11]SP (P-7483), [DArg1DPhe5DTrp7,9Leu11]SP(P-7492), and the growth hormone releasing hormone (GHRH) antagonist [DArg2Ala8,9,15]GHRH(1 29)(DC21-366) were tested for their in vitro effects on the release of growth hormone (GH) in the presence of GHRH and growth hormone releasing peptide, HisDTrpAlaTrpDPheLysNH2(GHRP). P-7492, P-7483, and P-7482 decreased, dose dependently, the release of GH by GHRP (IC50 = 0.2 microM, 0.85 microM, and 6 microM, respectively). These antagonists had only a 10-15% inhibitory effect on the stimulated GH release of GHRH even at high dosage. DC21-366 decreased the stimulated release of GH by GHRH (IC50 = 0.16 microM) but not by GHRP. Neither SP nor Bn had GH releasing or inhibitory effects in this system. PMID- 1718274 TI - Cortisone and thyroxine modulate intestinal lactase and sucrase mRNA levels and activities in the suckling rat. AB - Glucocorticoids and thyroxine modulate postnatal intestinal sucrase and lactase activities. Whether changes in enzyme activity are accompanied by changes in enzyme mRNA levels were determined in day 6 rats given thyroxine, cortisone, or thyroxine plus cortisone and killed 3 days later. Cortisone induced precocious expression of jejunal sucrase activity which was enhanced when cortisone plus thyroxine was administered; sucrase mRNA changed in parallel. Jejunal lactase activity was unaffected by thyroxine and was increased after cortisone, but not after thyroxine plus cortisone. Jejunal lactase mRNA levels increased equally after cortisone or after cortisone plus thyroxine. Thus, cortisone induces coordinated increases in sucrase and lactase activities and in corresponding mRNA levels. Thyroxine only enhances cortisone induced sucrase expression and antagonizes cortisone by depressing lactase activity post-translationally. PMID- 1718275 TI - Rat follistatin: ontogeny of steady-state mRNA levels in different tissues predicts organ-specific functions. AB - Follistatin (FS), a monomeric glycoprotein which specifically binds activin, is expressed in many tissues. This study investigated 1) the ontogeny of the steady state FS mRNA levels in different extragonadal tissues and 2) whether the ratio of the differential splicing products, FS 344 or its carboxy-truncated form FS 317, is changed during postnatal development. Whereas the levels of FS mRNA 344 in the kidney showed a profound increase from the day of birth to adulthood, the levels in the muscle peaked during the infantile period and then declined. Brain cortex, heart and thymus also showed tissue specific expression in the steady state mRNA level of FS during postnatal development. None of the tissues showed a measurable change in the ratio of the mRNA for FS 344 and FS 317. The FS mRNA 344 levels in male and female kidney were not different. It is concluded that the ontogeny of steady state FS mRNA varies in a tissue specific manner during postnatal development of the rat and may be involved in modulating the outcome of activin. PMID- 1718276 TI - Antigenic properties associated with Vinca alkaloid resistance in ovarian cancer cells: identification of a 92,000 Da protein. AB - In order to characterize the membrane changes related to Vinca alkaloid resistance, we raised monoclonal antibodies (mAbs) against a Vincristine resistant subline (OV1/VCR) derived from a human ovarian adenocarcinoma cell line (OV1/p). Among three monoclonal antibodies selected for a higher binding to OV1/VCR than to OV1/p cells, one designated OVR09, recognized a Mr 92,000 protein. This protein appears to be gradually overexpressed along the drug resistance establishment in vitro, and to decrease slowly in absence of drug. Further, mAb OVR09 showed a much higher binding to the vinblastine resistant epidermoid tumor cell line KbV1 than to its parental counterpart. The Mr 92,000 protein was also detected in various tumor cell lines and in an ovarian carcinoma surgical sample. PMID- 1718277 TI - High intracellular pH in CFPAC: a pancreas cell line from a patient with cystic fibrosis is lowered by retrovirus-mediated CFTR gene transfer. AB - Expression of CFTR from a retroviral vector in CFPAC, a pancreatic adenocarcinoma cell line derived from a patient with Cystic Fibrosis, causes a decrease in the average intracellular pH (pHi) in these transduced clones (PLJ-CFTR), as compared to CFPAC or CFPAC transduced with control virus (PLJ clones). Whereas the average pHi calculated based on results obtained in two PLJ-CFTR clones, PLJ-CFTR-20 (n = 2) and PLJ-CFTR-6 (n = 5), was 7.46 +/- 0.07, the average pHi calculated from results obtained in CFPAC (n = 13), PLJ-6 (n = 11) and PLJ-10 (n = 3) was pH 7.83 +/- 0.11. This finding suggests that CFTR may be involved, directly or indirectly, in the regulation of pHi in the pancreas. PMID- 1718278 TI - Increased serum levels of basic fibroblast growth factor in patients with renal cell carcinoma. AB - The serum level and urinary output of basic and acidic fibroblast growth factors (FGFs) were measured by sandwich enzyme immunoassay (EIA) in patients with renal cell carcinoma. In over fifty percent (16/31) of renal cell carcinoma patients, basic FGF was elevated (greater than 30 pg/ml) in their sera. There is relatively good correlation between serum levels of basic FGF and tumor stage or grade, while urinary daily output of basic FGF did not correlate with increased malignancy. The present results indicate that serum basic FGF level of patients with renal cell carcinoma is a useful diagnostic and prognostic marker for renal cell carcinoma. On the other hand, acidic FGF was not detectable in all sera and urine. PMID- 1718279 TI - Retinoylation of cytokeratins in normal human epidermal keratinocytes. AB - Retinoylation (retinoic acid acylation) is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in undifferentiated and squamous-differentiated normal human epidermal keratinocytes were retinoylated after treatment with [3H]retinoic acid. The major retinoylated proteins were identified as cytokeratins based on their profile in two-dimensional gel electrophoresis and their immunoreactivity with anti-keratin monoclonal antibodies. The covalently bound [3H]retinoic acid was not removed by mild hydrolysis with methanolic-KOH indicating that it is not linked to the cytokeratins by a thioester bond. The results raise the possibility that retinoylation of cytokeratins is involved in some of the effects of retinoic acid on keratinocytes. PMID- 1718280 TI - Studies on the relationship between cell proliferation and cell death: opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in PHA-stimulated human lymphocytes. AB - To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle. PMID- 1718281 TI - Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland. AB - The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins. PMID- 1718282 TI - Statherin: a major boundary lubricant of human saliva. AB - The lubricating properties of human submandibular-sublingual salivary fractions were examined using a servohydraulic model of mandibular movement. Fractions containing statherin exhibited a strong tendency to boundary lubrication. The lubricity of purified statherin was confirmed and compared to the amphipathic molecules gramacidin S and sodium dodecyl sulfate. Contact angle measurements of statherin paralleled the other amphipathic molecules. The helical content of statherin increased in trifluoroethanol indicating the presence of amphipathic helical regions. CD studies and hydrophobic moment calculations indicated that statherin adopts an amphipathic helical conformation at the N-terminus. An energy minimized model of the polar N-terminal residues 1-15 suggested that this domain could be positioned in space to interact with a hydroxyapatite substrate. These data imply that under appropriate conditions statherin may display an amphipathic nature which enables it to function as a boundary lubricant on enamel. PMID- 1718283 TI - Stimulation of nucleic acid and protein synthesis in mungbean (Vigna radiata L.) seeds by uv irradiation. AB - The effect of a range of ultraviolet (uv) irradiation doses on nucleic acid and protein synthesis has been studied during seed germination and seedling growth in mungbean (Vigna radiata L). The treatment of seeds with low dose irradiation were stimulative for the synthesis of these molecules. PMID- 1718284 TI - Increasing the plasma half-life of trichosanthin by coupling to dextran. AB - Trichosanthin (TCS) is a plant protein which has a wide spectrum of pharmacological activities. It was demonstrated recently that this compound suppressed the replication of human immunodeficiency virus (HIV-1) in vitro. The mechanism of action is believed to be inhibition of protein synthesis. Trichosanthin is a low molecular weight protein which is expected to be easily filtered and eliminated through the kidney. To minimize renal loss, the molecular size of trichosanthin can be increased by coupling to dextran. The larger complex will not undergo glomerular filtration and therefore renal loss can be prevented. This study investigates the kidney's role in trichosanthin elimination and the beneficial effect afforded by coupling to dextran in prolonging plasma half-life. For this purpose, a radioimmunoassay has been developed to determine the concentration of TCS in plasma and urine. The sensitivity of this assay is in the nanogram range. Trichosanthin was coupled to dextran T40 by a dialdehyde method and successful coupling was confirmed by gel filtration chromatography. The complex retained specific binding to trichosanthin antibodies with decreased affinity which can be partially reversed after incubation with dextranase; an enzyme that digested dextran. The pharmacokinetics of intravenously administered trichosanthin (0.75 mg/kg) was compared between two groups of rats with normal and impaired renal function (bilateral renal arterial ligation). Rats with ligation showed a decrease in plasma clearance from 4780 +/- 570 to 220 +/- 20 microL/min and an increase in the mean residence time from 9 +/- 1 to 145 +/- 16 min. Despite the several-fold difference in these parameters, recovery of trichosanthin from normal rat urine was only 0.38 +/- 0.05%. This value can be increased by using higher injection doses. The data indicate that the kidney is an important organ for the elimination of trichosanthin. When the dextran trichosanthin complex was injected into normal rats trichosanthin activity was not detected in the urine. All the pharmacokinetic parameters suggest that the dextran-trichosanthin complex stayed longer in the body and maintained a much higher plasma concentration than trichosanthin. PMID- 1718285 TI - Human basophil releasability. VIII. Increased basophil releasability in patients with scleroderma. AB - We evaluated basophil releasability in 16 female patients with scleroderma (systemic sclerosis) and in 16 normal age- and sex-matched donors. Basophils from patients with scleroderma released significantly more histamine "spontaneously" than did those from normal donors (12.9 +/- 2.1% versus 4.5 +/- 0.7%; P less than 0.0005). Basophil reactivity (maximal percentage histamine release) to anti-IgE was higher in patients with scleroderma than in controls (57.0 +/- 7.5% versus 35.4 +/- 7.8%; P less than 0.05). Basophil sensitivity (the concentration of anti IgE that causes 40% of maximal percentage histamine release) to anti-IgE in scleroderma patients was similar to that found in controls (4.6 +/- 2.8 x 10(-2) micrograms/ml versus 2.3 +/- 1.0 x 10(-1) micrograms/ml; P not significant). Scleroderma patients also showed enhanced releasability compared with that of the controls when challenged in vitro with interleukin-3 (8.3 +/- 1.7% versus 3.2 +/- 0.6%; P less than 0.01). Releasability induced by the formyl-containing tripeptide, f-met peptide, was significantly higher in the scleroderma patients than in the controls at the 2 lower concentrations used. No differences in basophil reactivity and sensitivity to f-met peptide and calcium ionophore A23187 were found between patients and normal donors. These results show that spontaneous basophil releasability and releasability in response to IgE cross linking and activation of interleukin-3 receptors are increased in patients with scleroderma. PMID- 1718286 TI - Antibodies to retroviral proteins and reverse transcriptase activity in patients with essential cryoglobulinemia. AB - Human T cell lymphotropic virus type I (HTLV-I) gag and env protein-specific antibodies were identified in 6 of 13 patients with essential cryoglobulinemia (EC), by Western blot and radioimmunoprecipitation analysis. Supernatants of cells from 2 of the 5 EC patients tested showed reverse transcriptase activity. DNA sequences homologous to HTLV-I could not be detected by polymerase chain reaction, thus excluding the presence of prototype HTLV-I in each patient with EC. The data suggest that retroviral proteins distinct from but related to HTLV-I may be involved in the pathogenesis of EC in some patients. PMID- 1718287 TI - Predominant role of neutrophils in the inactivation of alpha 2-macroglobulin in arthritic joints. AB - We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in SF was due to the concerted action of both reactive oxygen species and lysosomal proteinases. PMID- 1718288 TI - Isolation of Yersinia-specific T cell clones from the synovial membrane and synovial fluid of a patient with reactive arthritis. AB - Synovial fluid (SF) mononuclear cells from patients with reactive arthritis (ReA) proliferate in vitro when challenged with ReA-associated bacteria, the maximal response being for the organism causing the triggering infection. We report the results of a study of the antigenic specificity of synovial T lymphocytes from an HLA-B27 positive ReA patient whose SF mononuclear cells responded preferentially to Yersinia antigens. This is the first report of the isolation of Yersinia specific T cell clones from synovial membrane (obtained by closed-needle synovial biopsy). We present a detailed analysis of these clones, together with others obtained from the SF. PMID- 1718289 TI - Expression and function of surface antigens on scleroderma fibroblasts. AB - Dermal fibroblasts from patients with systemic sclerosis (SSc) bound a much greater number of T lymphocytes than did normal dermal fibroblasts. Monoclonal antibodies (MAb) against classes I and II antigens of the major histocompatibility complex (MHC) and their receptors, CD8 and CD4, had no effect on T cell interaction with SSc and normal cells, while MAb against lymphocyte function-associated antigen type 3 (LFA-3) and CD2 both strongly inhibited lymphocyte attachment. MAb against intercellular adhesion molecule type 1 (ICAM 1) and LFA-1 also prevented binding of T lymphocytes, but had a more marked effect on adhesion to SSc fibroblasts than to normal fibroblasts; they also completely abolished the increased binding to fibroblasts treated with interleukin-1 alpha, tumor necrosis factor alpha, and interferon-gamma. No difference was found in the proportion of normal and SSc fibroblasts that expressed MHC classes I and II and LFA-3, but more SSc cells expressed ICAM-1, and at a higher level, than did normal fibroblasts. These results show that cultured SSc cells have elevated binding to T lymphocytes, which possibly results from expansion of a subset of fibroblasts that produces high levels of ICAM-1. PMID- 1718290 TI - Potential antidepressant activity and enhancement of serotonin uptake of a new dibenzothiadiazepine derivative. AB - A molecule, 6-methyl-6,11-dihydro-11-[(N,N-dimethylamino) acetyl]dibenzo[c,fl [1,2,5]thiadiazepine 5,5-dioxide, (IM/P/3/4, CAS 128377-70-8), was identified in a screening program, which had the scope of finding compounds with antidepressive potential without the common sideeffects of existing antidepressive medication. IM/P/3/4 was found active a) in antagonizing apomorphine (16 mg/kg) and reserpine induced hypothermia in mice; b) in potentiating yohimbine-induced lethality in mice; c) in reducing immobility of rats forced to swim and of mice suspended by the tail. IM/P/3/4 does not affect a) apomorphine-induced stereotypy; b) amphetamine-induced hypermotility; c) haloperidol-induced catalepsy and water induced grooming and d) does not induce stereotypy or alter motor activity. The compound also a) reduced the beating of rat right heart atria only at a concentration of 3 x 10-4 mol/l; b) had weak anticholinergic activity; c) antagonized electroshock-induced convulsions and d) prevented indometacin-induced duodenal ulcers. IM/P/3/4 does not have good affinity for noradrenergic, serotonergic, dopaminergic, histaminergic or muscarinic receptors and does not displace imipramine, desipramine and mianserine from their binding sites. IM/P/3/4 increases 5-hydroxyindolacetic acid content and 3H-serotonin uptake in the hypothalamus. The present results suggest that IM/P/3/4 is a potential antidepressant with reduced side effects and with a mechanism of action which is different from that of other antidepressants. PMID- 1718291 TI - Aberrant neuronal development in hemimegalencephaly: immunohistochemical and Golgi studies. AB - Immunohistochemical and Golgi studies were performed on 6 patients with hemimegalencephaly. Immunohistochemical staining demonstrated glial, neuronal, and myelin heterotopia in the leptomeninges, cortex, and white matter with glial fibrillary acidic protein, myelin basic protein, and synaptophysin antisera. Golgi studies revealed small and deformed neurons in the superficial layers around abnormal sulci, and hypertrophic neurons with an increased number of dendrites and spines in the deeper cortex. The coexistence of a cell migration disorder, proliferation, and hypertrophy in each patient may imply a growth factor disturbance that controls cell proliferation. These unilateral cerebral malformations suggest that early surgical excision may be beneficial. PMID- 1718292 TI - The Apgar score: evolution, limitations, and scoring guidelines. AB - The Apgar score has been useful for nearly four decades in focusing on five physiological signs (heart rate, respiratory effort, reflex irritability, muscle tone, color) that denote the condition of an infant during the first critical minutes of life. Before the development of the system, narcotic analgesia and sedation during labor, and general anesthesia for vaginal deliveries were commonly used. Research of the scoring method has focused on the effects of these interventions on the fetus and newborn and has been a major impetus for change in obstetric practices. The Apgar score has been used as a predictive index for neonatal mortality and morbidity and for later neurologic or developmental disability. Both the one- and five-minute scores are predictors of mortality in normal-birthweight infants, whereas in high-risk low-birthweight infants their score is limited. The score is an insensitive predictive index of long-term neurologic or mental handicap, and lacks both sensitivity and specificity to reflect accurately the degree of acidosis. It can be used effectively, however, if these limitations are understood and considered. PMID- 1718293 TI - [Analysis of the psychodynamic aspects of the dentist-patient relationship. 1. Symbolism of the oral cavity]. AB - Authors carry out an analysis about odonto-stomatologist-patient relationship, from a psychoanalytic approach. This first part deals about the symbolic contents of the oral cavity which enhance the emergency of regression in the patient. PMID- 1718294 TI - Multiparametric evaluation of flow cytometric synthesis phase fraction determination in dual-labelled breast carcinomas. AB - Multiparametric, two-color DNA and cell cycle analyses were performed on 112 consecutive mechanically dissociated, ethanol-fixed breast carcinomas using a dual-label method with monoclonal antibodies (CAM 5.2) to cytokeratin (CK) and leukocyte common antigen (LCA) with propidium iodide (PI) staining. There was marked intertumoral variation of CK-positive (range, 3-87%; mean, 40%) and LCA positive (range, 1-28%; mean, 6.5%) events in DNA histograms. Approximately 70% of DNA aneuploid cells were CK positive. CAM 5.2-stained (avidin-biotin technique) Cytospin preparations correlated with flow cytometric (FCM) detection of CK-positive cells in 15/21 (71%) cases. In each discrepant case, FCM detected greater numbers of CK-positive cells. Cytospin controls of tumor suspensions revealed that cytoplasmic loss was the major cause of decreased CK staining. Synthesis phase fraction (SPF) calculation from CK-gated histograms resulted in kinetic indices (mean ungated, 12.3%, vs. mean CK-gated, 16.8%; P less than .01) with improved statistical correlations with tumor grade and estrogen receptor (ER) status. Differences between ungated vs. CK-gated SPF were greatest in cases having less than 20% CK-positive events (P less than .05). Cases with lower CK staining events generally had higher SPF and were more often high grade (below median CK staining, 61% high grade, vs. above median CK staining, 31% high grade) and ER-negative (below median CK staining, 55% ER negative, vs. above median CK staining, 12% ER negative).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718295 TI - A comparative study of quantitative stains for DNA in image cytometry. AB - In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide. PMID- 1718296 TI - Planimetry of bronchoalveolar macrophages. Importance of preparation and staining techniques. AB - Bronchoalveolar lavage seems a well-established, valuable research tool in the study of alveolar macrophages. The influence of fixation, cytocentrifugation and staining procedures on the cellular and nuclear size has been investigated by planimetry. As a reference, mean profile areas of 109 and 39 microns 2 for cell and nucleus, respectively, were measured for alveolar macrophages suspended in the hemocytometer. For comparison, stained Cytospin preparations were measured. Unfixed cells were compressed during cytocentrifugation. The cellular profile areas for Cytospin preparations increased about 15% and 70% after May-Grunwald Giemsa and Feulgen staining, respectively. The nuclear area was approximately 25% larger for both staining procedures as compared to the hemocytometer values. When the cells had been fixed prior to cytocentrifugation, these differences were less conspicuous. No significant differences were observed after May-Grunwald-Giemsa staining, showing a cellular area of 114 microns 2 and a nuclear area of 45 microns 2. Depending on the staining procedure, low nucleus:cell ratios (31%) were observed after Feulgen staining, while higher values (about 43%) were measured after May-Grunwald-Giemsa staining, regardless of which fixation or centrifugation procedure had been followed. In conclusion, these findings indicate that fixation should be carried out in order to prevent cell changes resulting from cytocentrifugation. Moreover, different staining procedures considerably influence the measurement of cellular and nuclear profile areas and the determination of nucleus:cell ratios. PMID- 1718297 TI - Three-dimensional reconstruction from serial sections. V. Calibration of dimensional changes incurred during tissue preparation and data processing. AB - Three-dimensional (3-D) reconstructions from serial tissue sections produce a table of x, y, z coordinates (i.e., a numerical description of the object) that will support 3-D computer graphics displays and morphometric analyses. While measures such as volume or surface area can be generated interactively, almost instantaneously, they are usually of unknown accuracy due to artifacts that may be introduced at two different stages: (1) tissue shrinkage during dehydration and polymerization of a plastic and compression or expansion during sectioning and mounting and (2) geometric and intensity distortions during image capture and data processing. This paper describes simple methods for (1) introducing fiducials (reference marks) that allow measurement of the net distortion incurred during tissue preparation and (2) estimating the amount of geometric distortion arising during image capture and data processing. Application of these methods showed that the net areal change introduced in dog and sheep hearts during tissue processing amounted to +/- 5%. Apparently, the substantial shrinkage that occurs during tissue processing is largely compensated for by the expansion during tissue sectioning and mounting. The methods described may have application to other semisolid tissues. PMID- 1718298 TI - Quantitative study of KI-67 antibody staining in non-Hodgkin's lymphomas using image analysis. AB - In sections from 32 B malignant lymphomas (ML), the total KI-67 stained area was compared to the number of KI-67 positive cells in order to demonstrate the reliability of using image analysis to quantify the proliferative activity. The total KI-67 area percentage correlated highly with the number of KI-67 positive cellular profiles (r = .93). Significant differences were found between low- and high-grade ML according to the Kiel classification (mean values +/- SD, respectively, of 7.7 +/- 3.81% and 16.6 +/- 6.23%), and between low-, or intermediate- and high-grade ML only, according to the International Working Formulation. Within the Working Formulation, the statistical analysis grouped the diffuse large cell subtype of intermediate grade with the immunoblastic high grade subtype. A wide range of KI-67 area percentage values was noted, particularly in follicular ML; for these follicular ML, considering follicular areas only, values were comparable to high-grade ML (14.8 +/- 6.60%). In conclusion, the KI-67 area percentage is a reliable alternative method to manual cell counting, and image analysis allows quicker measurements appropriate to large and strictly lymphomatous areas, using a greater number of cells than in manual cell counting. PMID- 1718299 TI - Morphometric analysis of borderline atypia in prostatic aspiration biopsy specimens. AB - In a prior study of 411 fine needle aspirates of the prostate (FNAPs), we identified a subset of 50 (12%) aspirates in which the findings were designated cytologically atypical. These equivocal cases formed a heterogeneous group composed of both histologically benign and malignant proliferations in which cutting needle biopsy was necessary to further delineate the nature of the abnormality. In an attempt to define quantitative and morphologic criteria for the separation of these proliferations into benign and malignant categories and to reduce the need for diagnostic cutting needle biopsies, we performed a cytomorphometric analysis of smears from 12 FNAPs (6 histologically proven benign and 6 histologically proven malignant) with equivocal cytologic atypia. We were unable to define any cytomorphometric criteria that accurately subdivided these cases into benign and malignant categories. PMID- 1718300 TI - Cytomorphometry of uveal melanoma. Comparison of fine needle aspiration biopsy samples with histologic sections. AB - A number of approaches are being investigated to increase the prognostic accuracy for uveal melanoma patients; the standard deviation of nucleolar area measurements and the DNA content appear to correlate better with survival than do classic histologic parameters. The utility of performing cytomorphometric measurements on fine needle aspiration (FNA) biopsy samples was prospectively analyzed for 24 eyes containing uveal melanomas that were examined with both 25 gauge FNA biopsy and standard histologic techniques. "Masked" analysis of the cellular composition of the 24 cases showed the presence or absence of epithelioid cells to be accurately predicted on the FNA samples in all cases. Image analysis cytomorphometric measurements of nucleolar area showed marked variability (with r less than 0.4) when FNA and histologic samples from the same case were compared. The relationship between these measurements was affected by cell type, sampling, specimen processing and investigator experience. PMID- 1718301 TI - The putative role of members of the CEA-gene family (CEA, NCA an BGP) as ligands for the bacterial colonization of different human epithelial tissues. AB - Immobilized purified CEA (carcinoembryonic antigen), NCA (non-specific crossreacting antigen) and BGP I (biliary glycoprotein I) bind strains of E. coli (including EPEC) and some Salmonella species (including S. typhi, S. paratyphi A + B and S. java) while Shigella-, Yersinia- and Bacteroides- strains showed no adhesion. The binding was of high avidity, heat sensitive, dose dependent, saturable and nearly completely abolished in the presence of 10 mM alpha methylmannoside. From inhibition studies with aromatic mannose compounds, it was suggested that in contrast to Salmonella strains E. coli strains exhibit a higher hydrophobicity in the binding region adjacent to the CEA-, NCA- and BGP-binding site. By further inhibition experiments it could be demonstrated that E. coli and Salmonella strains bind to high-mannose type oligosaccharides of these molecules via lectins on bacterial type I fimbriae. We conclude that the expression of products of this gene family on different human epithelial cells (colon-, bile canaliculi, uroepithel etc.) may function as ligands for bacterial colonization of epithelial tissues. PMID- 1718302 TI - Visualization of endo-beta-N-acetylglucosaminidase, lysozyme, and lysostaphin after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. AB - Bacteriolytic enzymes of different bond specificities, denatured by sodium dodecyl sulphate (SDS), were electrophoresed in polyacrylamide gels containing bacterial cells, then renatured after removal of SDS by diffusion. Enzyme activity was seen in sharp transparent bands resulting from bacteriolysis in the gels, while these sections containing bacterial cells appeared cloudy. Bacteriolytic enzymes including staphylococcal endo-beta-N-acetylglucosaminidase, lysozyme (N-acetylmuramidase), and lysostaphin (endopeptidase) were detected. The major bacteriolytic enzymes of Staphylococcus spp. were identified in gels after electrophoresis of crude enzyme preparations. This demonstrates the wide applicability of this method to the study of staphylococcal bacteriolytic enzymes. However, it should be noted that the method will fail to detect activities of bacteriolytic enzymes which are irreversibly inhibited by SDS. PMID- 1718303 TI - Comparative biotyping, bacteriocin typing, and serogrouping (O-antigens) of Serratia liquefaciens. AB - A total of 83 clinical isolates of Serratia liquefaciens from 81 patients were biotyped, bacteriocin typed (with group A phage tail bacteriocins from S. marcescens), and serogrouped. Biotyping afforded least discrimination, because 71 of the 83 isolates (85.5%) comprised 2 biotypes; the remainder were of 5 different biotypes. Bacteriocin typing differentiated 70 of the 83 isolates (84.3%) into 20 types. Polyclonal rabbit anti-O immune sera identified 16 O antigens, and all 83 isolates could be serogrouped. PMID- 1718304 TI - Serotyping of Serratia liquefaciens: H-antigens. AB - Polyclonal anti-H rabbit immune sera differentiated 12 flagellar (H) antigens among 79 motile of 83 clinical isolates of Serratia liquefaciens from 81 patients. Seven of the anti-H S. liquefaciens sera cross-reacted with H-antigens of S. marcescens, specifically anti-S. liquefaciens H6, H7, H8, H9, H10, H11, and H12 with S. marcescens H-antigens H7, H8, H12, H14, H5, H3, and H17, respectively. These cross-reactions were reciprocal. PMID- 1718305 TI - Serum resistance in different serotypes of Escherichia coli. AB - Resistance to complement-mediated serum activity is an important virulence factor in E. coli isolated from extraintestinal infections. Because there are no reports about the percentage of serum-resistant E. coli strains in common O serogroups, the study was carried out using Taylor's method (75% serum) for the determination of serum resistance of 253 E. coli strains, which had been isolated from urinary tract infections. The strains belonged to 8 common serogroups (O1, O2, O4, O6, O9, O16, O18, and O75) with a frequency of 6 to 24%, 218 (86%) were encapsulated. Among 26 different K antigens, K1 and K5 could be found in 32 and 33%. 25% of all strains investigated were found to be serum-resistant. The percentage of serum resistant strains was between 11% and 63% in the different O serotypes, the highest frequency was found in O6 (63%) and O2 strains (43%). Among all serum resistant strains carrying 13 different K antigens, K1 and K5 were the most common ones, with a percentage of 62% altogether. Serum resistance can be expected in strains from urinary tract infections, with a quite varying frequency depending on the O serotype, certain K antigens and other factors. PMID- 1718306 TI - Conspecificity of Hanseniaspora nodinigri and Hanseniaspora vineae: comparison by immunodiffusion and immunoelectrophoresis. PMID- 1718307 TI - Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus. AB - FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (Kd = 0.4 nM) and rapamycin (Kd = 0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFV- LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino acid sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other posttranslational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaert et al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent beta strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes. PMID- 1718308 TI - Kinetic comparison of caiman epsilon-crystallin and authentic lactate dehydrogenases of vertebrates. AB - Kinetic comparison of epsilon-crystallins isolated from the avian and reptilian species and the authentic lactate dehydrogenases (LDHs) was undertaken in order to clarify the identities of these structural lens proteins in relation to their enzymatic activity. Caiman epsilon-crystallin similar to the previously characterized duck epsilon-crystallin appeared to possess a genuine and stable LDH activity as detected by nitro blue tetrazolium staining on polyacrylamide gels and conventional kinetic assays. Kinetic parameters for pyruvate, L-lactate, NAD+, and three structural analogues of the coenzyme in this epsilon-crystallin catalyzed reaction were also determined and compared. Despite the structural similarities between epsilon-crystallins and chicken heart LDH, differences in charge and kinetic properties have been revealed by native isozyme electrophoresis and kinetic analysis as examined by initial velocity and substrate inhibition studies. It is found that the kinetic data analyzed for caiman epsilon-crystallin were more fitted with a compulsory ordered Bi-Bi sequential mechanism similar to those for the authentic LDHs and duck epsilon crystallin. Caiman epsilon-crystallin has for the first time been established as a heart-type LDH based on the kinetic analysis and comparison with the authentic heart- and muscle-type LDHs from pig and chicken. PMID- 1718310 TI - Enzymatic semisynthesis of aprotinin homologues mutated in P' positions. AB - The replacement of amino acids in the P'1 and P'2 position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the "modified" inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys15-Ala16, the residues P'1 (Ala16) and P'2 (Arg17) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a "one pot" reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala16 Arg17 was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg17 Ile18 peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys15 and Xaa16 are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P'1 residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced. PMID- 1718309 TI - Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies. AB - Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A2 (PLA2S) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA2. Antibodies neutralizing lethal or enzymatic effects of PLA2 did not recognize linear epitopes. PMID- 1718311 TI - Comparison of the antigenic peptides between human prostatic and lysosomal acid phosphatases. AB - The relative antigenicity (capacity to bind antibodies raised against the intact prostatic acid phosphatase) of the selected peptides from human prostatic and lysosomal acid phosphatases was evaluated in a competitive assay. Both prostatic and lysosomal acid phosphatases were shown to possess similar antigenic determinants on both terminal regions, along with more similarity on NH2-terminal peptide than COOH-terminal site. At least one additional antigenic site is present at the internal region of prostatic acid phosphatase, since the mixture of both amino- and carboxyl-terminal peptides exhibited only 70% inhibition. PMID- 1718312 TI - Cytoskeletal proteins in chronic villitis of unestablished etiology. AB - Cytoskeletal proteins (i.e., cytokeratins, vimentin, actin, and desmin) are normally present in the placental chorionic villi and are related to the maintenance of the villous shape, and to the ability of the villi to contract and permit a normal blood flow. In areas of villitis of unestablished etiology, the normal reactivity of these proteins in the villous core around fetal stem vessels disappears, and an increased number of cytokeratin positive cells is identified. The vascular damage in stem villi with villitis could develop ischemia in the villous tree promoting cytotrophoblast proliferation. Increased numbers of these areas could be related with the appearance of abnormal pregnancies. PMID- 1718313 TI - Cytokines: an overview. AB - Cytokines are a broad, heterogeneous group of proteins and polypeptides that regulate intercellular communication. Examples of cytokines include interleukins, interferons, colony-stimulating factors, and a variety of growth factors. The preparation of large quantities of highly purified recombinant cytokines has provided a basis for their biological and physicochemical characterization. The pleiotropic biological effects of these factors are expressed through binding to specific, high-affinity cell-surface receptors. Although they are different in amino acid sequence, cytokines have a number of biological and physicochemical properties in common. PMID- 1718314 TI - Analysis of major histocompatibility complex gene products in tissues isolated from the developing human nervous system. AB - We have examined the expression of the major histocompatibility complex (MHC) class I and II gene products in the developing human fetal peripheral nervous system. As determined by RNA blot hybridization analysis, MHC class I RNA was readily detectable in extracts prepared from dorsal root ganglia (DRG) obtained from aborted human fetal material. However, utilizing similar methodology, it was not possible to detect MHC class II RNA. In conjunction with these studies, expression of MHC class I and II proteins in primary human fetal DRG tissue was examined by fluorescence-activated flow cytometry and protein immunoblotting. Consistent with the detection of MHC-specific RNA, the accumulation of MHC class I-specific protein was readily detectable in human fetal DRG neural cell populations with little, if any, accumulation of MHC class II-specific protein evident. These studies suggest that MHC gene products may be expressed early in the development of the human nervous system resulting in the generation of specific immunocompetent neural cell populations. PMID- 1718315 TI - Identification of linear epitopes of the BPV-1 L1 protein recognized by sera of infected or immunized animals. AB - Sera from cattle that had been inoculated with BPV-1 virions or with recombinant L1 proteins and serum from a rabbit that had been immunized with SDS-denatured virions were evaluated for their reactivity with 466 overlapping synthetic peptides corresponding to 95% of the BPV-1 L1 protein. The late serological response of cattle to both intact virions and recombinant L1 proteins exhibited a similar profile of reactivity with approximately 70% (7 of 10) of L1 antigenic sites. However, the L1 serological response of the rabbit to SDS-denatured virions exhibited a significant difference from bovine serum antibodies in the profile of epitopes recognized, including a relative lack of response to major bovine epitopes located between L1 amino acids (AAs) 300-400. Importantly, only the sera from animals inoculated/immunized with intact virions was capable of neutralizing BPV-1 infectivity of murine C127 cells, suggesting that nonlinear epitopes are important for papillomavirus neutralization. PMID- 1718316 TI - cDNAs encoding members of a family of proteins related to human sterol carrier protein 2 and assignment of the gene to human chromosome 1 p21----pter. AB - Sterol carrier protein 2 (SCP2) is believed to play a key role in intracellular lipid movement. Here we report the cloning and nucleotide sequences of cDNAs encoding SCP2-related proteins of 58.85 kD and 30.8 kD and the assignment of the SCP2 gene to human chromosome 1 p21-pter. The SCP2-related proteins share common deduced carboxyl amino acid sequences with SCP2 and the cDNAs have a common 3' untranslated nucleotide sequence. The mRNAs encoding these proteins increased in a coordinate fashion as human placental cytotrophoblasts differentiated into syncytiotrophoblasts in culture. Our observations document the existence of a family of related proteins encoded by the human SCP2 gene. PMID- 1718317 TI - Molecular cloning and expression of ferritin mRNA in heavy metal-poisoned Xenopus laevis cells. AB - In a search for genes transcriptionally regulated by metal ions, we have isolated a Xenopus laevis ferritin cDNA clone, XL2-17, from cadmium-poisoned XL2 cells. The large size of the corresponding ferritin mRNA (1.4 kb) is due to the presence of a 629-nucleotide 5'-untranslated region. The Xenopus ferritin sequence is highly isologous with other vertebrate ferritins. In particular, there is a complete sequence identity for the iron-responsive element (IRE) located in the 5'-untranslated region in both XL2-17 and Rana catesbeiana ferritin mRNAs. The position of this IRE is unusual since it is located 489 nucleotides from the 5' end of the ferritin mRNA. Our analysis of phylogenetic relationships among ferritins indicates that all amphibian ferritins thus far sequenced would be more closely related to the mammalian H-type ferritin than to the L-type. The level of ferritin mRNA in XL2 cells rises 10- to 15-fold following exposure of cells to cadmium or copper. This increase is due to both transcriptional and translational regulation. A 10-fold increase was also found at the protein level. These results suggest that ferritin may be a primary detoxification response to heavy metals in Xenopus cells. PMID- 1718318 TI - Differential distribution of immunohistochemical markers in the bed nucleus of the stria terminalis in the human brain. AB - A variety of histochemical findings have contributed to a more differentiated architectonical description of the bed nucleus of the stria terminalis (BNST) in the mammalian brain. However, in the human brain investigations of the chemoarchitecture of this nucleus have been rare. Therefore we chose this region in six human autopsy brains in order to map the distribution patterns of 13 immunohistochemical markers for neurotensin (NT), neuropeptide Y (NPY), somatostatin (SOM), enkephalins (ENK), vasoactive intestinal polypeptide (VIP), substance P (SP), neurophysins (NPH), glial fibrillary acid protein, 3-fucosyl-N acetyl-lactosamine epitope, myelin basic protein (MBP), calbindin (CAB), synaptophysin (SYN) and chromogranin-A (CHR-A). Three chemoarchitectonically distinct areas could be defined. The lateral subdivision of the BNST contained high amounts of NPY and SP-fibre immunoreactivity and was further characterized by the occurrence of neurons labelled for NPY. The central subdivision of the BNST appeared as a histochemically clearly circumscribed compartment with massive fibre immunoreactivity for SOM, ENK, VIP, SYN, CHR-A, CAB as well as SOM, ENK, NT and CAB positive cells but lacked cytosolic or fibre-like immunolabel for NPY and SP. This structure was also ensheathed by myelinated fibres identified by means of MBP immunohistochemistry. The medial subdivision of the BNST showed moderate to high SP and NPY fibre immunoreactivity but lacked immunolabelled neurons and was only scarcely supplied with varicose or punctiform ENK immunoproduct. In the most posterior levels of our sections a cell group labelled for NPH was located lateral to the fornix columns. The lateral subdivision of the BNST (with NPY, SYN) and mainly the central BNST (with SOM, ENK, VIP, SYN and CHR-A) contributed to ventrolateral extensions of dense patchy fibre immunoreactivity throughout the basal forebrain region. PMID- 1718319 TI - Cytotoxic T-cell recognition of HIV proteins and peptides. PMID- 1718320 TI - Evaluation of monoclonal antibodies to HIV-1 by neutralization and serological assays: an international collaboration. Collaborating Investigators. AB - In a National Institutes of Health (NIH)/World Health Organization (WHO) sponsored collaboration, 26 laboratories characterized a coded panel of monoclonal antibodies (MAb) to HIV-1 envelope protein. The MAb were evaluated by serological [radioimmunoprecipitation, immunoblot, enzyme-linked immunosorbent assay (ELISA) and peptide mapping] and neutralization assays. Although laboratories used diverse neutralization assays that vary considerably in sensitivity, qualitatively similar data were obtained. The MAb were classified into three neutralization specificities: type-specific for MN and SF2, type specific for IIIB, and group-specific for MN, SF2, and IIIB. The group-specific MAb displayed much lower neutralizing titers than the type-specific MAb. The specificity of MAb for neutralization was greater than for serological recognition of gp120 protein or peptide epitopes. Some MAb that bound to the same or closely overlapping linear epitopes had very different neutralization properties. The distinction between serological recognition and neutralization may result from differences in affinity of the MAb or may indicate that MAb can neutralize by interactions at a site distinct from the antibody binding site. PMID- 1718321 TI - How to minimize pulpal death. PMID- 1718322 TI - Estrogens and benign prostatic hyperplasia: rationale for therapy with aromatase inhibitors. PMID- 1718323 TI - Recent development in elucidation of the diagnostic and aetiologic problems of very early pregnancy. PMID- 1718324 TI - Endemic goitre--iodine deficiency disorders. AB - Endemic goitre occurs when the prevalence of thyroid enlargement in the population of an area exceeds 10%. With few exceptions its cause is iodine deficiency superimposed on other goitrogenic factors normally present and responsible for sporadic goitre. Iodine deficiency causes significant health problems and so, the term iodine deficiency disorders (IDD) has been introduced. The earliest sign of IDD is goitre, but these disorders also include cretinism, neonatal hypothyroidism and congenital defects, as well as retardation of mental and physical development etc. IDD are a worldwide problem: WHO estimates that substantially more than 800 million people are at risk and more than 190 millions suffer from IDD; over 3 million people have cretinism and in the largest and worst affected areas many millions suffer from mental and physical developmental defects. IDD can be totally eliminated by prophylaxis using iodine administered in salt, oil or some other vehicle. Problems over preventing iodine deficiency relate to difficulties in the handling and distribution of the iodized vehicle in some parts of the world and on the political will to introduce preventive schemes. In only a very few areas does the presence of goitrogenic agents in the environment cause endemic goitre despite adequate iodine supply. In a limited number of places excessive iodine from seaweed used as staple food results in endemic goitre. PMID- 1718325 TI - Milk proteins in the etiology of insulin-dependent diabetes mellitus (IDDM). AB - The etiology of insulin-dependent diabetes mellitus (IDDM) is multifactorial. The final cause of the disease, the specific destruction of the islet beta-cells, is the result of a cellular/humoral autoimmune process that operates in individuals with a particular genetic background in response to an external triggering factor(s). The most likely environmental triggers are virus infections and dietary factors. Among the latter group dietary proteins, mainly cow milk proteins, have been found to be important. Elimination of intact cow milk proteins from the diet significantly reduced the incidence of IDDM in the spontaneously diabetic BB rat, the elimination being most effective when it occurs during the pre-weaning period. Conversely, in newly discovered diabetics (both rats and children) increased levels of antibodies to cow milk proteins as compared with non-diabetic controls were found. These higher titres of antibodies were against beta-lactoglobulin and anti-bovine serum albumin. In further studies we found that antibodies to bovine serum albumin cross-react with a beta-cell membrane protein of Mr 69,000 and that this protein is likely induced by interferon. At the molecular level, a region of the bovine serum albumin has distinct homology to the beta-subunits of the MHC class II proteins Ia, DQ and DR, and antibodies raised against this bovine serum albumin region identified the same 69K beta cell membrane protein, in the same manner as antibodies to the third hypervariable region of DR-beta did. Our hypothesis is that bovine milk proteins (mainly bovine serum albumin) might be an important environmental factor providing specific peptides that share antigenic epitopes with host cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718326 TI - Association between keratin staining patterns and the structural and functional aspects of palatine tonsil epithelium. AB - The keratin composition of stratified squamous epithelia has a complex pattern, which varies in different regions and as a result of pathological developments. The exact factors responsible for the characteristic keratin composition in a given epithelium are unknown. However, the environment, including factors from the connective tissue, is known to influence epithelial morphology and keratin composition. We here report that the reticulated squamous epithelium of the crypts of palatine tonsils shows an extensive staining for keratins 5 and 19 in basal as well as suprabasal cells, in contrast to neighbouring non-reticulated crypt epithelium and the epithelium at the tonsillar surface, in which staining is restricted to basal cells. The reticulation of the crypt epithelium is thought to be initiated by infiltration of immune-related cells in a preexistent non reticulated epithelium. The extensive staining for keratins 5 and 19 in reticulated crypt epithelium correlates with the presence of numerous immune system-related cells and marked expression of intercellular adhesion molecule-1 (ICAM-1), thought to be involved in inflammatory and immunological responses. The results suggest that the massive lymphocytic traffic in the reticulated crypt epithelium and the overall distinct immune environment are responsible for the unique keratin staining pattern observed. PMID- 1718327 TI - RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. AB - Fixed and paraffin-embedded tissues from pathology department archives can be available for RNA expression analysis. In this report, we show that RNA isolated from surgical or autopsy tissues, routinely processed by fixation and paraffin embedding, is not completely degraded. RNA fragments around 100-200 bases in length are still present even in organs late fixed and very rich in RNase, such as the pancreas. Here we describe a general protocol to obtain RNA from single 6 8-microns tissue sections. The RNA extracted can be analyzed for the presence of specific sequences by reverse transcription and amplification with the PCR. We studied the retinoblastoma gene expression in 38 human pancreas specimens from surgical or autopsy origin. PMID- 1718328 TI - Rapid, small-scale RNA isolation from tissue culture cells. AB - A rapid and simple protocol for the isolation of RNA from transfected tissue culture cells is described. The protocol employs a guanidinium thiocyanate/phenol mixture to lyse cells directly from tissue culture plates and extract the total RNA. A total of six simple steps, which can be accomplished within 2.5 hours, are required. The protocol reproducibly yields 20-40 micrograms RNA from 0.5 x 10(6) 1 x 10(6) cells per sample. The quality of the RNA obtained is sufficient for reverse transcriptase assays such as oligonucleotide-directed primer extension and random-primed cDNA synthesis. PMID- 1718329 TI - Cytometrically coherent transfer of receptor proteins on microporous membranes. AB - A new method (Freeze-Transfer) is described for performing high-resolution immunocytochemistry for soluble cell proteins on frozen sections of biological tissues that involves thaw-mounting frozen tissue sections directly onto the surface of nitrocellulose thin films instead of directly onto glass slides. This technically straight-forward change in methodology resulted in chromogenic immunocytochemical assays for Her-2 and EGF receptors that were 1-2 orders of magnitude more sensitive while still fully utilizing the diagnostic resolving power of light microscopy. The effects of membrane pore size and surface chemistry on the resolution and intensity of Her-2 signal suggest that the enhanced sensitivity of Freeze-Transfer was caused by the cytologically coherent transfer of target molecules normally lost from cut surfaces of cells mounted on nonporous glass during assay. PMID- 1718330 TI - Cloning and expression of antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen in Escherichia coli. AB - Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease. The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D'. All the epitopes were subcloned and expressed as fusion proteins with the glutathione S-transferase in Escherichia coli using the novel expression system pGEX that allows very high yields of recombinant proteins after a single-step purification. The sera of patients with the autoimmune disease were analyzed for the expressed recombinant proteins by an immunoblotting technique. All positive sera showed a patient-specific behavior and could be divided into four groups regarding recognition of the four antigenic epitopes of the 68-kDa (U1) ribonucleoprotein antigen. The epitope B' was reactive to all patient sera positively tested and classified as the marker antigenic epitope for the mixed connective tissue disease. PMID- 1718331 TI - Unique 3'-untranslated sequence of insulin-like growth factor-I isolated from human placenta. AB - A cDNA clone of 525 bp corresponding to the 3'-untranslated region of insulin like growth factor-I was isolated from a human placenta library. The sequence of this clone extended 200 nucleotides downstream from the previously reported 3' end of IGF-IA cDNA, indicating the existence of IGF-IA transcripts having an even larger 3'-untranslated region. By using this clone for RNA transfer blot hybridization, it was shown that this longer 3'-untranslated region is included in the 7.5- and 5.0-kb transcripts, but not in the 1.1- and 0.9-kb transcripts. It is also apparent that transcripts bearing the extended 3'-untranslated sequence are highly expressed in human placenta. PMID- 1718332 TI - Cyclic appearance of cytoplasmic NOR-silver-stained particles in sea urchin embryos. AB - When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos. PMID- 1718333 TI - Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin. AB - To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence. Electron microscopy revealed that the affected cells failed to segregate large axons and initiate a myelin spiral despite having formed a basal lamina, which normally triggers Schwann cell differentiation. Cells infected with the MAG sense virus formed normal compact myelin. These observations strongly suggest that MAG is the critical Schwann cell component induced by neuronal interaction that initiates peripheral myelination. PMID- 1718334 TI - Both NMDA and non-NMDA subtypes of glutamate receptors are concentrated at synapses on cerebral cortical neurons in culture. AB - N-methyl-D-aspartate (NMDA) and non-NMDA receptors play a key role in synaptic transmission and plasticity in the vertebrate central nervous system. Previous studies have suggested that although both receptor types are present at synapses, the NMDA receptors may be relatively uniformly distributed. We have combined iontophoretic mapping of NMDA and non-NMDA receptors with immunohistochemical localization of synaptic vesicles along dendrites of single neocortical neurons to determine the relationship between NMDA and non-NMDA receptor distribution and the location of synapses. We find that when corrections for glutamate diffusion are made, NMDA responses are concentrated at focal "hot spots" that coincide with non-NMDA hot spots and that there is an excellent correlation between these hot spots and synapses. PMID- 1718335 TI - Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. AB - Nitric oxide is a free radical that has been recently recognized as a neural messenger molecule. Nitric oxide synthase has now been purified and molecularly cloned from brain. Using specific antibodies and oligonucleotide probes, we have localized brain nitric oxide synthase to discrete neuronal populations in the rat and primate brain. Nitric oxide synthase is exclusively neuronal, and its localization is absolutely coincident with NADPH diaphorase staining in both rat and primate. PMID- 1718336 TI - Vascular cell adhesion molecule-1 and eosinophil adhesion to cultured human umbilical vein endothelial cells in vitro. AB - Cultured human umbilical vein endothelial cell (EC) monolayers stimulated with 10 ng/ml tumor necrosis factor demonstrate a time-dependent increase in the expression of the vascular cell adhesion molecule-1 (VCAM-1) with maintained maximal expression at 24 h following EC activation. A monoclonal antibody (mAb) directed against VCAM-1 (1G11) significantly inhibited the adhesion of eosinophils, but not neutrophils, to EC, which had been activated by tumor necrosis factor-alpha for 24 h, but only when eosinophils had been pretreated with an mAb directed against the common beta chain of the CD11/CD18 complex. In the absence of pretreatment with anti-CD18, mAb 1G11 had no significant effect on eosinophil adhesion. These results suggest that eosinophils bind to VCAM-1. However, the functional capacity in this model of the eosinophil receptor for VCAM-1 is likely to be minor compared with the activity of the CD11/CD18 leukocyte adhesion molecules. PMID- 1718337 TI - Hypoxia-induced inhibition of tropoelastin synthesis by neonatal calf pulmonary artery smooth muscle cells. AB - Animals chronically exposed to hypoxia develop characteristic structural changes in the pulmonary arterial vasculature including cell hypertrophy, hyperplasia, and increased deposition of extracellular matrix proteins. The medial smooth muscle cells' (SMC) increase in tropoelastin mRNA expression and elastin deposition as determined by in situ hybridization and histologic examination appears to contribute significantly to this increase in matrix protein accumulation. The primary stimulus for the increased tropoelastin production, which persists in vitro, is unknown but mechanical forces and hypoxia seem to play a role. In order to determine the direct effects of hypoxia on tropoelastin production by pulmonary artery SMC, cultured neonatal bovine pulmonary artery SMC were exposed to 3%, 10%, and 21% O2 concentrations for 48, 72, and 120 h and soluble tropoelastin was measured by direct immunoassay. Tropoelastin mRNA levels were also determined by Northern and slot blot analysis after 48 h of incubation under hypoxic conditions. SMC cultured in 3% and 10% O2 for 120 h showed dose dependent decreases (11-fold and 2-fold, respectively) in measured tropoelastin levels compared with SMC cultured in 21% O2 conditions. This decrease was not due to cell damage or accumulation of toxic metabolites while under hypoxic conditions nor to a change in tropoelastin partitioning between the cell and media. Tropoelastin mRNA levels were also decreased under hypoxic conditions. Secreted, cell layer, and total protein synthesis determined by L-[3H]leucine incorporation again showed a dose-dependent decrease under hypoxic conditions but not to the same extent as tropoelastin production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718338 TI - Thrombospondins. AB - The thrombospondins are a family of proteins generated by alternative splicing and gene duplication, which contain binding sites for many soluble proteins and up to five cellular receptors. This family of modular proteins functions in regulation of cellular migration and proliferation as manifested in development, wound healing, angiogenesis and tumorigenesis. PMID- 1718339 TI - Anti-adhesive molecules of the extracellular matrix. AB - The prototype extracellular matrix glycoproteins had been identified on the basis of their activity in promoting cell adhesion and spreading. Recently, more and more evidence is accumulating that the reverse effect of extracellular matrix proteins, namely the inhibition of cell adhesion and spreading, may be equally important for proper cell function during morphogenesis and development. Several anti-adhesive proteins have been described and their mechanisms of action are being investigated. PMID- 1718340 TI - Haplotype specific homology scanning algorithm to predict T-cell epitopes from protein sequences. AB - We present a homology scanning microcomputer program to predict functional T-cell epitopes within proteins. By taking into account particular human or mouse restriction elements the predictions are made haplotype-specific. The generality of this approach is confirmed by (i) identification of well-characterized immunogenic T-cell determinants in lysozyme (ii) search for potential T epitopes on unanalysed proteins like the human beta 2-adrenoreceptor (iii) modification of non-immunogenic peptide sequences in order to generate T-cell determinants. PMID- 1718341 TI - Relationship of the chemical structure and immunobiological activities of lipoteichoic acid from Streptococcus faecalis (Enterococcus hirae) ATCC 9790. AB - Two molecular species of lipoteichoic acid (LTA 1 and LTA 2) were isolated from whole cells of Streptococcus faecalis (Enterococcus hirae) ATCC 9790 by hydrophobic chromatography on Octyl Sepharose CL-4B. Chemical analysis revealed that LTA 1 and LTA 2 contained two and four acyl lipid anchors respectively. LTA 1 was less active than LTA 2 in inducing cytokines, except interleukin-1 (IL-1), but their in vivo antitumour effects were similar. LTA 2 was a potent inducer of tumour necrosis factor (TNF) and interferon (IFN) production and had excellent antitumour activity against Meth A fibrosarcoma established in mice. Deacylation of LTA 2 by alkaline hydrolysis abolished these biological activities. The phosphatidylglycolipid fraction derived from LTA 2 after acid hydrolysis could also induce TNF, IFN, and IL-1 production, as well as having antitumour activity against Meth A fibrosarcoma. Therefore, the lipid anchor portion of S. faecalis LTA may play an important role in the manifestation of these various biological activities. PMID- 1718342 TI - Stimulation of ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate generation by pyrroline-5-carboxylate in mouse liver in vivo: evidence for a regulatory role of ribose-5-phosphate availability in nucleotide synthesis. AB - Pyrroline-5-carboxylase (P5C), a physiological stimulator of hexose-monophosphate pentose pathway activity, was found before to increase 5-phosphoribosyl-1 pyrophosphate (PRPP) generation and nucleotide synthesis in human erythrocytes and cultured fibroblasts. We now report the stimulation of PRPP generation by P5C also in mouse liver in vivo. In addition we demonstrated a simultaneous elevation in ribose-5-phosphate (R5P) concentration, which was relatively smaller and transient. The demonstrated effect of P5C on liver R5P and PRPP content in vivo provides strong evidence for the physiological role of R5P availability in the regulation of PRPP and purine production. PMID- 1718344 TI - Peptides mimicking selected disulfide loops in HIV-1 gp120, other than V3, do not elicit virus-neutralizing antibodies. AB - The positions of all 9 intrachain disulfide bonds within the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1) have been established recently. Peptides expected to mimic some of the disulfide-bonded domains [(120-133)-(203-221); (133-138)-(164-203); (224-254); (391-425) and (385 392)-(425-452)] were synthesized. All peptides, except (120-133)-(203-221), elicited in immunized rabbits relatively high levels of antibodies reacting with gp120 in enzyme-linked immunosorbent assay (ELISA) and/or Western immunoblot assays. However, these antibodies failed to neutralize the infectivity of HIV-1. Combined with earlier reports concerning other gp120 loop peptides, these results confirm the uniqueness of the V3 (303-338) loop in encompassing a principal determinant(s) involved in virus neutralization. PMID- 1718343 TI - Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro. AB - Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied. The inhibitory effect was significantly weaker in HIV-2 systems. However, the reverse transcriptase activities of both types of viruses were inhibited by Evans blue. Another polyanionic compound, phosphorothioate 28-mer cytidine homopolymer (SdC28) was shown to inhibit syncytium formation induced by HIV-1-and HIV-2-infected cells in an identical manner. Evans blue showed partial blocking of gp120 binding to CD4 in a solid phase enzyme-linked immunosorbent assay (ELISA). These results suggest that the polyanionic dyes may exert their antiviral effects, at least in part, by interfering with the binding and fusion of HIV with susceptible T cells. PMID- 1718345 TI - Predictive B- and T-cell linear epitopes in structural proteins of HTLV-I, HTLV II, and STLV-I. AB - The primary amino acid sequences derived from the gag, pol, and env gene products of human T-cell lymphotropic virus type I (HTLV-I) and type II (HTLV-II) and the env protein of simian T-cell lymphotropic virus type I (STLV-I) were aligned and computer algorithms were used to predict B- and T-cell epitopes. Structural B- and T-cell motifs that showed amino acid sequence conservation of antigenic determinants in HTLV-I, HTLV-II, and STLV-I, as well as different antigenic determinants of HTLV-I and HTLV-II, were identified. Several of these B and T epitopes have been shown experimentally to be immunodominant and two of the B epitopes have been used for type-specific serology. These predictive epitopes provide a guide to develop improved diagnostic assays and for the development of potential subunit vaccines for HILV-I and HTLV-II. PMID- 1718347 TI - In vitro assays for detecting neutralizing and fusion-inhibiting antibodies to SIVMAC251. AB - Sensitive and reproducible assays for SIV infection and syncytium formation have been developed in which high titers of neutralizing and fusion-inhibiting antibodies can be recorded. These assays will contribute toward our understanding of the role of humoral responses in SIV vaccine strategies. PMID- 1718346 TI - Brefeldin A inhibits the processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1. AB - The processing and secretion of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected T cells and in primary macrophages treated with an antiviral antibiotic brefeldin A (BFA). BFA blocks the egress of proteins from the endoplasmic reticulum and has a profound effect on the structure of cis/medial Golgi. In MOLT-3 cells infected with the IIIB strain of HIV-1 (MOLT-3/IIIB), BFA inhibited the intracellular processing of gp160. The secretion of envelope proteins from these cells was significantly inhibited in the presence of BFA. The gag proteins, on the other hand, were processed and secreted normally. BFA also inhibited the proteolytic processing of gp160 in primary macrophages infected with HIV-1. The infectivity of virus pelleted from the medium of MOLT-3/IIIB cells treated with BFA was markedly lower than that obtained from untreated cells. These results demonstrate that the proteolytic processing of gp160 in HIV-1-infected cells takes place after the glycoprotein exists the endoplasmic reticulum and that the transport of glycoprotein to the cell surface is required for assembly of complete HIV-1 particles. PMID- 1718348 TI - Randomised EORTC head and neck cooperative group trial of preoperative intra arterial chemotherapy in oral cavity and oropharynx carcinoma. AB - Between February 1978 and January 1984, 222 eligible patients were randomised in a multicentre trial of preoperative intra-arterial chemotherapy in the treatment of oral cavity and oropharynx carcinoma. Patients were randomised between either surgery or preoperative chemotherapy. This latter group received vincristine and bleomycin for 12 days. Patients were stratified according to the primary site: floor of the mouth (FM) versus posterior oral cavity or oropharynx (POC) and institution. The FM group received postoperative radiotherapy depending upon quality of the margins and lymph-node pathological involvement, when it was systematically applied in the POC group. Tumour regression after chemotherapy either complete (CR) or partial (PR greater than 50%) was observed in 48% in the FM group and 41% in the POC group, and lymph-node regression (CR + PR) was respectively 15% and 23%. Some discrepancies appeared between clinical regression and pathological response, and the number of cases without histological response was clearly higher than the number of cases without clinical response. The overall survival showed a statistically significant difference (P = 0.048) between FM and POC groups. In the FM group, median survival in the chemotherapy arm was estimated at 7 years compared with 3 years in the surgery arm. In the POC group, median survival was estimated at 3 years in both treatment arms. Chemotherapy lowered the uncontrolled disease and local recurrence in the FM group. These differences do not exist in the POC group, which may be due to the systematically postoperative radiotherapy. PMID- 1718349 TI - Long-term survivors after salvage high dose chemotherapy with bone marrow rescue in refractory germ cell cancer. AB - Between April 1984 and May 1985, 17 heavily pretreated patients with relapsing or refractory germ cell tumours were treated with cisplatin 40 mg/m2/day, days 1-5; etoposide 350 mg/m2/day, days 1-5; cyclophosphamide 1600 mg/m2/day, days 2-5 and autologous bone marrow transplantation on day 8 as consolidation of conventional salvage chemotherapy. None of the 11 refractory patients and 4 of the 6 responders to prior salvage treatment are long-term survivors at 68, 72, 74 and 74 months. Mean aplasia duration was 17 days and there were 7 documented episodes of septicaemia in 17 febrile patients. 1 patient died of treatment. Among the 4 survivors, 2 patients have a sustained grade II invalidating neuropathy. We conclude that this regimen is not recommended as salvage therapy in refractory patients but may be a useful consolidation treatment in patients responding to conventional salvage chemotherapy. PMID- 1718350 TI - Understanding and managing bone metastases. PMID- 1718351 TI - Obstruction of the GI tract. PMID- 1718352 TI - [Quality in nursing. Professional regulation in nursing--a project in the international nursing organization]. PMID- 1718354 TI - Radiolunate arthrodesis in rheumatoid wrist (21 cases). AB - Surgical radiolunate arthrodesis appears to be an appropriate procedure to stabilize ulnar translation of the carpus, to correct radial deviation of the wrist and consequently ulnar drift of the fingers and restore neutral orientation of the lunate when collapse occurs. From 1979 to 1988, radiolunate arthrodesis was performed on 21 unstable rheumatoid wrists with subluxation of the lunate (20 cases combined with an ulnar head resection and one case without). Average follow up was 4 years and 1 month. Wrist collapse increased in 3 cases and remained stable in 6 cases (9 documented cases). When ulnar drift of the fingers is present (6 cases), the ulnar angulation of the third finger shows a 14-degree average improvement. Thirteen wrists were painfree, one presented a painful click, 4 patients were dissatisfied because of pain and recurrence of the disease. Three patients had died. Average ROM was 41 degrees in extension, 28 degrees in flexion, 8 degrees radial deviation and 23 degrees ulnar deviation. Grip, measureable in 9 wrists, showed improvement with an average range of 12,6 kg on right side and 9 kg on left side. Surgical procedure and complications are described. PMID- 1718353 TI - Cyclosporin treatment does not impair the release of nitric oxide in human coronary arteries. AB - OBJECTIVE: It has been hypothesised that compromised endothelial function can contribute to the toxic manifestations associated with cyclosporin therapy. In vitro animal studies have implicated inhibition of release of the endothelium derived relaxing factor, nitric oxide; however, this has not been investigated in human tissue. The present study investigated the effect of cyclosporin A on nitric oxide release in human coronary arteries. DESIGN: Study of in vitro organ bath preparations and in vivo angiographic measurements in the coronary circulation. PATIENTS: For the in vitro experiments coronary arteries were harvested from the excised hearts of 10 patients requiring transplantation for reasons other than ischaemic heart disease. Three of these patients were being re transplanted for obliterative bronchiolitis and had been receiving cyclosporin for a mean of 22 months. The in vivo study was performed on a group of 12 cardiac transplant recipients who were clinically well 1-5 years postoperatively and were not undergoing allograft rejection at the time of assessment. RESULTS: Isolated vessel segments in vitro relaxed in a dose dependent manner in response to substance P (10(-11)-10(-7) mol/l). The maximum response was 76.6 (7.4)% of the response to 1 microgram/ml glyceryl trinitrate. Incubation with 1000 and 2000 ng/ml cyclosporin reduced the response to 63.0 (11.5)% and 62.2 (11.1)% respectively; this was not statistically significant. In segments taken from the explanted hearts of three patients requiring re-transplantation, the mean maximum response was 78.0 (11.0)% and there was no correlation between maximum response in segments from each patient and the duration of cyclosporin therapy. The effect of intracoronary substance P in 12 cardiac transplant recipients was also examined (mean cyclosporin blood concentration 228.9 (42.8) ng/ml). The mean maximum dilatations measured as the percentage diameter change induced by substance P and isosorbide dinitrate were 22.1 (3.2)% and 26.0 (2.5)% respectively. There was no correlation between the degree of endothelium mediated vasodilatation in response to substance P and cyclosporin concentration. CONCLUSIONS: The nitric oxide response was preserved in the coronary arteries of patients exposed to cyclosporin. The mechanisms that initiate cyclosporin associated toxicity remain to be elucidated. PMID- 1718355 TI - Radio-scapho-lunate partial wrist arthrodesis following comminuted fractures of the distal radius. AB - Painful radiocarpal arthritis following comminuted fractures of the distal radius may be treated either by total wrist fusion or by procedures which preserve movement. The authors have reviewed 15 patients with such fractures who have undergone radio-scapho-lunate partial arthrodesis. They report the results with an average follow-up of 23.8 months. Pain was abolished in 7 patients and resolved virtually completely in 4 cases. Restored grip strength averaged 49% of the contralateral side. There was considerable limitation of postoperative range of motion which was restricted to an oblique plane extending dorso-radial to palmar-ulnar. Most patients did not report this as a problem. Two cases of non union were reported as well as a 35.7% incidence of secondary degenerative change in the midcarpal joint. This feature casts doubt on the predictability of outcome of this procedure. PMID- 1718356 TI - Carpal tunnel syndrome in the elderly. "Beware of severe cases". AB - This study compares the clinical and electrophysiological features of Carpal Tunnel Syndrome (CTS) in elderly (over 70 years of age) and middle age populations (50-60 years of age). Our study shows a severe (95%) electrophysiological motor and sensory denervation of CTS, in 60% of the elderly group and only 18% of the middle aged group (P much much less than 10(-3). Clinically, the presence of thenar wasting was the only reliable sign of significant denervation, Phalen's test, Tinel sign and painful paresthesia being equivocal. Some 18% of the elderly were asymptomatic despite 25% of these patients having severe neurological impairment. It was noted that exclusively diurnal paresthesiae were confined to the elderly group (23%) (P much less than 10(-3)). To avoid permanent disability the elderly should be viewed as a high risk group of severe cases even in the absence of firm clinical evidence and should undergo objective electrophysiological assessment with a view to surgical intervention as a priority. PMID- 1718357 TI - Preiser's disease. AB - Nine patients with the diagnosis of unilateral Preiser's disease were seen between 1970 and 1987. The mean age of four male and five female patients was 37 years (range, 20 to 70 years). The diagnosis was based on radiographic evidence of sclerosis, fragmentation, erosion, and collapse of the proximal pole of the scaphoid. Onset was usually insidious but two had a preexisting radial hypoplasia, a third had a modest scaphoid malunion, and a fourth had a recent fall. Treatment consisted of scaphoid excision and silicone rubber prosthetic replacement in three, debridement of necrotic bone fragments in one, and different periods of cast support and observation in the remaining five. No relationship to ulnar variance was seen. Mean follow-up was 6.9 years. Of the scaphoid implants, two had subluxated, but only one was painful. In the more conservatively treated group, pain and function were only modestly limiting in four, who returned to their original occupations as did the one treated by curettage of necrotic bone in the proximal pole. One patient (case 4) was severely incapacitated because of associated severe congenital anomalies of the lower extremities. Polyaxial tomography permitted a better assessment of the degree of involvement and carpal alignment. Preiser's disease is a rare affliction of the carpal scaphoid which may involve the entire bone in avascular changes but primarily leads to fragmentation and collapse of the proximal pole. A conservative approach to treatment is favored based on this experience. PMID- 1718358 TI - Factors influencing elbow arthrolysis. AB - Over the period 1982 to 1988, 31 consecutive patients at the Hand Surgery Unit of the Sheba Medical Centre were subjected to elbow joint arthrolysis to treat restriction of range of motion solely due to trauma. This retrospective study aims to evaluate the relative influence of the followings factors on functional outcome: sex, age, type of original injury and initial management, presence of para-articular ossification, delay between injury and arthrolysis, and the use of manipulation and a continuous passive motion device (CPM) following surgery. The range of motion was recorded prior to arthrolysis and after operation (excluding one patient who subsequently underwent arthrodesis for intractable pain). Follow up averaged 15.3 months (+/- 5.4). In the 24 patients with extension deficit (greater than 20 degrees), the mean improvement was of 26.9 degrees (greater than 23.1 degrees); in the 21 patients with flexion deficit the mean improvement was of 21.2 degrees (greater than 18 degrees). The mean improvement for total range of motion in the series overall was of 35.2 degrees (+/- 23.8 degrees). 90% showed an improvement of at least 10 degrees and 30% attained normal ROM. All of these improvements in range were statistically highly significant (p less than 0.0001). None of the variables had predictive value with regard to improvement of flexion. With regard to improvement in extension, the only variable of value was the use of a continuous passive motion device following surgery; those patients subjected to CPM showed a mean improvement of 32.6 degrees (+/- 19.0 degrees), while those without averaged 12.8 degrees (+/- 27.5 degrees) (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718359 TI - The rheumatoid elbow: patterns of joint involvement and the outcome of synoviorthesis. AB - Rheumatoid arthritis of the elbow is a common condition. A group of 86 patients has been reviewed. Patterns of joint involvement are described. These patients have been subjected to 137 radioisotopic synoviortheses. 72% achieved a result classed as "good" and this review underlines the efficacy of the procedure of synoviorthesis. Radiological staging has been shown to have prognostic significance; 89% of good results were obtained in management of Steinbrocker grade I. No complications were recorded. PMID- 1718360 TI - The TEC treatment (continuous extension technique) for severe Dupuytren's contracture of the fingers. AB - TEC used for 2 weeks preoperatively has eliminated finger amputation in severe cases of Dupuytren's disease. It must be continuous to be effective. The apparatus and technique are described. PMID- 1718361 TI - The vascular rein technique: a new way for thumb reconstruction. AB - Many surgical procedures have already been published for reconstruction of an amputated thumb. A new technique is proposed consisting of a composite island transfer of part or of the whole distal phalanx of the middle finger. A double vascular bundle is provided and microsurgery is not necessary. 13 cases have been carried out with a maximum follow up of 6 years. PMID- 1718362 TI - Carpal tunnel syndrome due to an haemangioma of the median nerve in a 12-year-old child. AB - A case of carpal tunnel syndrome due to an haemangioma of the median nerve in a 12-year-old male is reported. Radiological and histological data support a venous origin of the tumour. Interfascicular dissection and resection were performed, and 6 months later the patient remains symptoms free. PMID- 1718363 TI - Metaphors of bleeding in women. AB - All women experience bleeding throughout their life cycle, usually in a normal context. However, throughout history bleeding has been described metaphorically in negative ways. This paper presents an anthropologist's viewpoint concerning the presentation of bleeding in the literature. PMID- 1718364 TI - Effect of cisapride on morphine absorption after oral administration of sustained release morphine. AB - We investigated the effect of cisapride 20 mg given orally with MST 20 mg on the absorption of morphine in a double-blind, placebo-controlled study. Cisapride increased significantly both plasma concentrations of morphine after 1 h and peak concentrations. There was no significant change in time to peak concentrations, sedation scores or percentage decrease in pupil diameters. Plasma concentrations of amylase were increased in three patients in the MST-placebo group and three in the MST-cisapride group. One patient in the MST-cisapride group developed acute pancreatitis. PMID- 1718365 TI - Reduction of chromium(VI) in Chinese hamster V-79 cells. AB - The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50-500 microM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI). PMID- 1718366 TI - Copper supplementation effects on indicators of copper status and serum cholesterol in adult males. AB - Two 6-wk double-blind studies evaluated the effects of supplements of 2 or 3 mg Cu/d on serum copper, ceruloplasmin, red-blood-cell super oxide dismutase (RBC SOD), total serum cholesterol, and serum lipoprotein-cholesterol fractions in adult males. Study I had 6 supplemented and 8 placebo subjects, whereas study II had 7 and 6, respectively. Copper supplementation did not appear to affect serum copper levels, RBC-SOD, hematocrit, and ceruloplasmin levels when assayed by radial immunoassay diffusion. Supplementation with 2 mg Cu/d produced an increase in LDL cholesterol and the percentage of cholesterol as LDL at wk 4 compared to the placebo group, and a concomitant decline in VLDL-cholesterol levels and the percentage of cholesterol as VLDL. At wk 6, the percentage of cholesterol as LDL increased and that of cholesterol as VLDL decreased compared to baseline values in the supplemented group. Supplements of 3 mg Cu/d increased hemoglobin levels, ceruloplasmin activity, and serum total-cholesterol levels at wk 6 compared to placebos. Differences in cholesterol may be partly explained by variability in the placebo groups in both studies. Copper supplementation effects on cholesterol deserves further investigation. PMID- 1718367 TI - Parenteral supplementation with zinc in surgical patients corrects postoperative serum-zinc drop. AB - Zinc has been known for a long time to facilitate wound healing. But, so far, supplementation trials in patients treated by major severity surgery gave either partial or controversial results. In a double-blind, randomized study including 30 patients, we show that zinc supplements (30 mg/d for 3 d) administered by a drip correct postoperative drop of serum zinc, that this correction concerns the available part of serum zinc (i.e., zinc that is bound to compounds other than alpha-2 macroglobulin in serum), and that this supplementation can improve clinical wound healing. Possible influence of increased urinary output after the intervention is discussed, and we found that serum cortisol remains stable when zinc/albumin ratio is stable, and increases sharply when the same ratio drops. Cortisol, therefore, seems to play a major role in zinc redistribution after surgery. PMID- 1718368 TI - In ovo administration of boron alters bone mineralization of the chicken embryo. AB - It has been hypothesized that boron (B) is an essential element for animals, especially in bone metabolism. In this study, the influence of in ovo boron administration was assessed in the chicken. At 8 d of embryogenesis, carrier or B (0.1, 0.5, or 1.0 mg) was injected on to the chorioallantoic membrane of fertile eggs. At hatching, body weights were recorded and tissue samples collected. Although boron failed to alter bone mineralization, it decreased (p less than 0.05) dried bone weight, suggesting a reduction in the bone organic matrix. Furthermore, 1 mg boron decreased (p less than 0.05) hatchability and increased (p less than 0.05) the height of the proliferative zone in the growth plate, indicating an unfavorable effect on bone elongation of the developing chick. PMID- 1718369 TI - Does dietary magnesium modulate blood lipids? AB - In a randomized, single-blind, controlled study (400 patients aged 25-63 yr; 374 males, 26 females), 206 subjects were administered a magnesium-rich diet, and 194 subjects their usual diet, for 6 wk. Age, sex, body weight, hypertension, hyperlipidemia, smoking, obesity, diuretic therapy, and diabetes were comparable between the two groups, as were laboratory data at entry to the study. Intervention-group A received a significantly higher amount of dietary magnesium and potassium compared to group B, which received its usual diet. After 6 wk, there was a significant fall in total serum cholesterol (228.5 +/- 46.2 mg/dL), LDL cholesterol 146.5 +/- 75.5 mg/dL), and triglyceride (143.8 +/- 40.5 mg/dL) in group A compared to serum cholesterol (242.5 +/- 58.2 mg/dL), LDL cholesterol (157.0 +/- 78.4 mg/dL), and triglyceride (156.5 +/- 60.0 mg/dL) at entry to study, but no such changes in group-B subjects. HDL cholesterol showed a marginal mean decrease of 0.8 mg/dL in group B and a 2.5 mg/dL increase in group A. The changes in blood lipids were consistent with an increased intake of magnesium and with a rise in serum levels. Although a general blood-lipid-reducing effect of such a diet cannot be excluded, it is possible that dietary magnesium may have contributed to the reduction of total serum cholesterol, LDL cholesterol, and triglyceride, and the marginal rise in HDL cholesterol. More studies with longer follow-up periods are needed to confirm this observation. PMID- 1718371 TI - Maternal hair zinc concentration in neural tube defects in Turkey. AB - Hair zinc concentration was measured in samples taken from 57 mothers who delivered infants with neural tube defects (NTD) (mainly anencephaly). Control groups consisted of 30 healthy mothers with normal offspring and 37 nonpregnant women from middle-income backgrounds. Zinc concentration was also measured in the hair of eight infants with NTD (four being anencephalic). The mean maternal hair zinc concentration in the NTD group (128.2 +/- 38.9 micrograms/g) was lower than that of the control women (p less than 0.001), whereas the mean hair zinc level of malformed babies (250.4 +/- 85.2 micrograms/g) was significantly higher than that of normal infants (193.4 +/- 39.2 micrograms/g) (p less than 0.05). Maternal nutritional zinc deficiency was thought to be one of the factors responsible for NTD in Turkey. PMID- 1718370 TI - Effects of low copper and high zinc intakes and related changes in Cu,Zn superoxide dismutase activity on DMBA-induced mammary tumorigenesis. AB - The effect of low copper and high zinc intakes on Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity and mammary tumorigenesis induced by 9,10-dimethyl-1,2 benzanthracene (DMBA) was investigated. Groups of 40 weanling female Sprague Dawley rats were fed a modified AIN-76 diet containing the following (/kg diet): 1 mg Cu (0.016 mmol) and 30 mg Zn (0.459 mmol); 6 mg Cu (0.094 mmol) and 30 mg Zn (0.459 mmol) (control); or 6 mg Cu (0.094 mmol) and 150 mg Zn (2.295 mmol) for 21 wk. At 5 wk, 30 rats/group were given 4 mg (15.6 mumol) DMBA in corn oil intragastrically, and controls (10/group) received corn oil alone. Erythrocyte Cu,Zn-SOD activity was measured at 3, 5 (just before DMBA), 9, 13, 17, and 21 wk. The group fed the high-Zn diet had a slightly lower weight gain and food consumption. DMBA treatment had no effect on these parameters. Plasma and liver Cu concentration decreased in the low-Cu group. Femur zinc was significantly elevated in the high-Zn group. Erythrocyte Cu,Zn-SOD activity was decreased in the low-Cu group from 3 to 21 wk and was significantly elevated in the high-Zn group at 3 and 5 wk. In the low-Cu group, there were 5 nonmalignant adenomas and 3 malignant adenocarcinomas; in the control group, there were 4 adenomas and 3 adenocarcinomas; in the high-Zn group, there were 5 adenomas and 3 adenocarcinomas. No relationship between Cu,Zn-SOD activity and the presence of tumors could be found. PMID- 1718372 TI - Dietary phytate x calcium/zinc millimolar ratios and zinc nutriture in some Ontario preschool children. AB - Millimolar ratios of phytate/Zn, Ca x phytate/Zn, and Ca x phytate/Zn per 4.2 MJ were calculated from 3-d weighed-food records collected from 62 male (M) (mean age: 58 +/- 7 mo [mean +/- SD]) and 44 female (F) (mean age: 58 +/- 6 mo) preschool children from Southern Ontario. Food-composition values for phytate were based on laboratory analysis and the literature. No gender differences existed for median millimolar ratios of phytate/Zn (median: M, 5.3; F, 5.3), and Ca x phytate/Zn per 4.2 MJ (M, 68.1; F, 59.5), but median intakes of phytate (M, 399; F, 333 mg/d) and median millimolar ratios of Ca x phytate/Zn (median: M, 102.1; F, 72.3; p less than 0.01) were higher for boys than for girls. Of the children, only two (1M, 1F) and 22 (17 M, 5F) had millimolar ratios of phytate/Zn and Ca x phytate/Zn per 4.2 MJ, respectively, that were above critical values. Millimolar ratios of Ca x phytate/Zn (p = 0.06) and Ca x phytate/Zn per 4.2 MJ (p = 0.05) were higher in boys with hair zinc less than 1.07 mumol/g v greater than or equal to 1.07 mumol/g. Analysis of variance showed that height was influenced by an interaction between millimolar ratios of Ca x phytate/Zn per 4.2 MJ and sex (p = 0.0007), when age and midparent height were treated as covariates. Results suggest that dietary Ca x phytate/Zn millimolar ratios, when expressed per 4.2 MJ, influenced the zinc nutriture of these Southern Ontario boys. PMID- 1718373 TI - Products of the reaction of selenite with intracellular sulfhydryl compounds. AB - The usual first step in the intracellular metabolism of exogenous selenite is its chemical reaction with glutathione to form selenodiglutathione (1). We have investigated whether selenite also reacts intracellularly with other SH compounds. HeLa cells were exposed to [75Se]selenite and lysed with SDS. Cellular proteins and nucleic acids were precipitated with trichloroacetic acid, and the acid-soluble fraction was analyzed by ion-exchange thin-layer chromatography (ion exchange TLC) and autoradiography. In control cells, the major [75Se]-containing species detected can be identified by its mobility as selenodiglutathione. Two other species were detected, which can be identified as selenodimercaptoethylamine and the mixed selenotrisulfide of mercaptoethylamine and glutathione. In contrast, in cells that were depleted of glutathione (by treatment with buthionine sulfoximine), very little, if any, selenodiglutathione was detected. However, new [75Se]-containing species were detected, which can be identified as selenodicysteine and the mixed selenotrisulfide of cysteine and glutathione. The same species were detected when [75Se]selenite was added to the acid-soluble fraction of a cell extract (as opposed to living cells), confirming that these compounds can be formed by nonenzymatic reactions. PMID- 1718375 TI - Lymphokines and cytokines. PMID- 1718376 TI - The role of colony stimulating factors in cancer therapy. PMID- 1718374 TI - New anticancer agents. PMID- 1718378 TI - Bleomycins. PMID- 1718377 TI - Genitourinary cancer. PMID- 1718379 TI - Malignant melanoma. PMID- 1718380 TI - Supportive care. PMID- 1718381 TI - Surgical treatment of gastric cancer. PMID- 1718382 TI - Nonvascular intervention in the genitourinary tract. AB - The past few years have seen significant expansion in interventional genitourinary radiology. Balloon dilatation of the prostate is a new, safe procedure that may be offered as a surgical alternative to high-risk patients as well as to younger patients wishing to avoid surgical complications. The procedure is contraindicated in patients with middle lobe hypertrophy, carcinoma, very large glands, or a history of retention. Because of the lack of large randomized controlled studies, the procedure should still be considered investigational. Transcervical fallopian tube recanalization for proximal tubal occlusion is another new outpatient procedure with low morbidity and cost compared with the more expensive alternatives of in vitro fertilization and tubal microsurgery. Pregnancy rates approach 60% in selected subgroups of patients. Furthermore, selective salpingography may provide diagnostic information not available with conventional hysterosalpingography. The most recent advance in renal parenchymal biopsy has been the development of an automated biopsy gun, which, in conjunction with real-time ultrasonographic monitoring, has increased the ease, speed, and accuracy of the procedure and significantly decreased complications. PMID- 1718383 TI - AIDS surveillance in the Americas. PMID- 1718384 TI - Which immunomodulator? PMID- 1718385 TI - Cytokines in dermatology. AB - Cytokines are soluble factors that are secreted by one cell and affect the function of other cells. The ability to clone genes for cytokines and produce large quantities of biologically active proteins has allowed for better characterization of cytokines and paved the way for their therapeutic use. The vast number of compounds initially described on the basis of their functional activities are now understood to be members of a more limited number of cytokine families, many of which are either interleukins, interferons, or colony stimulating factors. This article summarizes the effects of these cytokines in vitro, their relationship to various dermatologic disorders, and some potential therapeutic uses relevant to dermatology. PMID- 1718387 TI - International Symposium on Interferon-Alpha Therapy in Malignant Diseases. Athens, November 1-4, 1990. Proceedings. PMID- 1718386 TI - Loss of monomorphic and polymorphic HLA antigens in metastatic breast and colon carcinoma. AB - MHC class I antigens are intimately involved in intercellular communication, and recognition by cytotoxic T cells. Thus tumour cells that fail to express them may be at a growth or metastatic advantage. A series of ten colorectal and ten breast carcinomas, and their respective lymph node metastases, were examined immunohistologically using monoclonal antibodies (mAb) against both monomorphic and A2 polymorphic determinants, and beta-2-microglobulin (beta 2m). Four colon polypoid adenomas stained positively throughout, but 6/10 primary tumours had partial or complete loss of expression of monomorphic determinants using mAb W6/32: two node and the liver metastasis showed less, four more expression. Similar results were seen for beta 2m. HLA-A2 expression was absent or reduced in 4/4 colon tumours and all their metastases. Among the breast tumours, W6/32 staining was absent or reduced in 2/10, and node deposits showed two with less reactivity than their primary. Beta 2m staining was reduced or absent in 8/10 primaries and all the node metastases; in every case in which beta 2m was detected in the primary tumour their corresponding lymph node metastasis showed a decreased expression. HLA-A2 expression was absent or reduced in 3/4 primary breast carcinomas, and all their metastases. These results show that individual human colon and breast carcinomas often have a reduced HLA class I antigen expression, which apparently confers a metastatic advantage. PMID- 1718388 TI - Antitumour effects of interferons: past, present and future. PMID- 1718389 TI - Hairy cell leukaemia: review of treatment. AB - The cardinal features of hairy cell leukaemia are: (i) cytopenias, (ii) splenomegaly, and (iii) mononuclear cells of B-cell origin with cytoplasmic projections and tartrate-resistant acid phosphatase-positivity. The most common complication is infection. In the past, the mainstay of therapy has been splenectomy, and this procedure is still often suggested as a first-line approach. However, research during the last decade has resulted in three new, highly effective therapies for hairy cell leukaemia: interferon-alpha (IFN alpha), 2'-deoxycoformycin (DCF) and 2-chlorodeoxyadenosine (2CDA). IFN-alpha is currently approved for this indication. About 90% of patients have a durable haematologic recovery, and complete remission rates range from less than 5% to greater than 40% in different series. It should be noted that patients with partial remissions generally have normal or near-normal blood counts, and can live indefinitely without disease-related problems, despite a few remaining hairy cells in the bone marrow. In this paper we will discuss the various therapeutic modalities available for patients with hairy cell leukaemia. PMID- 1718390 TI - Retinoids and interferon: a new promising combination? AB - The retinoids: all-transretinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin) and the aromatic retinoids etretinate and acitretin have a preventive and therapeutic effect on chemically-induced tumours. Clinically, retinoids have shown variable effectiveness in therapy and/or prevention of oncological diseases of skin, head and neck, lung, bladder, vulva and bone marrow. With a few exceptions, monotherapy with retinoids has not been satisfactory. Similarly, monotherapy with interferon alpha has been used successfully only for some specific indications. Retinoids have a marked differentiation-inducing effect which may contribute to their therapeutic effect. Experiments were carried out in transformed cell lines to test the combination of retinoids with interferon alpha and other cytokines on differentiation. In HL-60 cells, an acute promyelocytic leukaemia cell line, induction of differentiation was determined by induction of an oxidative burst potential. Retinoids showed the following order of activity: tretinoin greater than isotretinoin greater than acitretin. Cytokines had no differentiation-inducing effect by themselves. However, the addition of the following cytokines to retinoids potentiated the retinoid-induced differentiation: IFN alpha, IFN beta, IFN gamma, TNF alpha, G CSF, IL-1 alpha and IL-4. In experiments with HL-60 or other cell lines, the pattern of differentiation-induction was always dependent on the particular retinoid/cytokine combination. IFN alpha provoked a marked potentiation of retinoid-induced differentiation. The combination of the antiproliferative and differentiation-inducing effect of the retinoids together with the antiproliferative, immunostimulatory and differentiation-potentiating effects of IFN alpha suggests that this combination might be a particularly promising treatment for neoplastic diseases. PMID- 1718391 TI - ABO blood grouping of saliva from mixed body fluids by sandwich methods using monoclonal antibodies to tissue specific epitopes on blood group substance in saliva. AB - In this paper methods for ABO blood grouping of saliva from mixed body fluids have been established. Monoclonal antibodies to tissue specific epitopes on blood group substances in saliva were used as solid phase antibodies to catch the blood group substances. ABO blood grouping of saliva could be performed by these methods without interference from other body fluids (eg. semen, vaginal secretion, urine, sweat and serum). At least 16,000 and 3,000 fold dilutions of secretor saliva were sufficient for ABO blood grouping by sandwich ELISA and sandwich absorption-elution test, respectively. PMID- 1718392 TI - Production and characterization of monoclonal antibodies against tissue specific epitopes on ABO blood group substances in saliva. AB - Mouse monoclonal antibodies (P4-2F, P4-5C) against ABO blood group substances in saliva were produced by immunization with ABO blood group active-glycoprotein after ethanol precipitation from heated saliva. These antibodies bound to saliva, irrespective of the ABO blood group and secretor status. Saliva diluted at least 3.2 x 10(4)-fold could be detected by ELISA using these antibodies. Tissue and species specificity of the antibodies was tested by ELISA and counterimmunoelectrophoresis and showed that the antibodies were specific for human saliva. By immunoblotting of the deglycosylated ABO blood group substances it was evident that the epitopes for the antibodies were localized on the core protein of blood group substances in saliva. These antibodies could be extremely suitable reagents for the identification of saliva in medico-legal examinations. Furthermore, they may be used as capture antibodies in sandwich methods for ABO blood grouping of saliva from mixtures of body fluids. PMID- 1718393 TI - Inter-alpha-trypsin inhibitor (ITI): a useful genetic system in paternity testing. Evidence for polymorphic occurrence of ITI*3 and existence of a new allele, ITI*4. AB - The genetic polymorphism of human inter-alpha-trypsin inhibitor (ITI) has been investigated in sialidase-treated samples by isoelectric focusing on polyacrylamide gels with a pH range 3.5-9.5 followed by passive blotting with enzyme immunoassay. In 400 blood donors from western Japan, 8 simplified band patterns were observed, 6 of which could be explained by the previously described 3 polymorphic alleles, ITI*1, ITI*2, and ITI*3. The others were products of a new and rare fourth allele designated ITI*4, whose expression is also consistent with autosomal codominant inheritance. The frequency of these alleles was 0.440, 0.526, 0.030 and 0.004, respectively. The theoretical exclusion rate for putative fathers in paternity cases was calculated to be 0.228. The ITI system is a useful genetic marker for forensic hemogenetics in Japanese and in Europeans. PMID- 1718394 TI - The examination of the ITI system in disputed paternities. AB - 106 paternity cases with a total of 114 putative fathers were examined in the inter-alpha-trypsin inhibitor (ITI) system. Analysis was performed by isoelectric focusing (IEF) of untreated sera on polyacrylamide gels. From 39 paternity exclusions, determined in other genetic systems, 7 were confirmed in the ITI system. In 75 expertises the alleged man was not excluded from fatherhood; in 68 cases the probability of paternity was W greater than 99.73%. The practical exclusion rate in the ITI system was therefore calculated to be 10.45%. The theoretical exclusion rate was determined to be 19.3%. In one paternity case the alleged father and the child showed inverse homozygosity in the ITI system, while the man was not excluded from fatherhood in 28 additional marker systems. The calculated probability of his paternity was 99.99%. The assumption of an incomplete expression of the ITI phenotypes in infants is supported by a significant deviation between the observed and expected ITI distributions at population equilibrium. PMID- 1718395 TI - Automated cytometry of muscle fibre sections: size and spatial distribution of the four fibre types in young and adult rats. AB - An automated cytometry program was applied to the extensor digitorum longus muscle of young and adult rats. Lesser diameter and spatial distribution of about 4000 fibres were measured in digital images from ATPase-stained muscle sections. All fibre types grow thicker with age, but the coefficient of variation of the diameter is age-independent. At both ages, 2B fibres have the largest mean diameter and are most frequent. 2A fibres in young rats present a diameter smaller or equal to type 1 but then show a faster increase in size; their relative number increases from 20 to 28%. Consequently type 2A displays the most important change with age. The spatial distribution of fibres is mathematically expressed; most images show a random distribution of type 1 and type 2 fibres. Taking into account the variation of fibre size of each type, the number of fibres which should be measured in order to reach a specified precision was calculated. PMID- 1718396 TI - Influence of fixation and immunohistological technique on accuracy, precision and inter-observer reproducibility of plasma cell counting. AB - Recently two highly sensitive and specific diagnostic criteria for Sjogren's syndrome based on percentages of IgA-, IgG-, and IgM-containing plasma cells measured in immunohistologically stained labial salivary gland tissue have been described. The reliability of such a criterion is dependent on the accuracy, precision and inter-observer reproducibility in plasma cell counting. The present study evaluates the effect of tissue fixation and immunohistological procedures on the aforementioned factors. Immunoglobulin (Ig)-containing plasma cells in sections of lamellated submandibular salivary gland tissue, alternately fixed in a 4% buffered formol solution or formol-sublimate solution and stained with an indirect immunoperoxidase and unlabelled peroxidase anti-peroxidase (PAP) method respectively, were enumerated by three independent observers. Relative numbers of Ig-containing plasma cells appeared to be less sensitive for systematic errors due to tissue fixation and immunohistological procedure than absolute numbers of Ig-containing plasma cells. The best inter-observer reproducibility of plasma cell counts was obtained in sections from formol sublimate-fixed specimens stained according to the PAP procedure. PMID- 1718397 TI - Efficacy of disopyramide and mexiletine used alone or in combination in the treatment of ventricular premature beats. AB - The efficacy of oral disopyramide and mexiletine used alone or in combination was studied in 75 patients with frequent ventricular premature beats (VPBs). The efficacy was evaluated with 24-hour ambulatory ECG and greater than or equal to 75% reduction in the number of VPBs was defined as effective. When disopyramide or mexiletine were ineffective or not tolerated, the alternative drug was administered and the efficacy was again evaluated. If the single administration of neither drug was effective, the combination of disopyramide and mexiletine was then given. Either disopyramide or mexiletine was effective in 48 patients, and neither drug was effective in 19 patients. In 19 patients unresponsive to both drugs, combination therapy was effective in six patients (32%). Both drugs caused side effects or one drug caused side effects and another drug was ineffective in eight patients. In five out of those patients, we attempted combined therapy with a reduced dosage of those drugs that caused side effects. This therapy was effective in two patients without intolerable side effects. Thus, when the single use of neither disopyramide nor mexiletine single-drug therapy is effective, it is worthwhile to try combination therapy. Also, combination therapy with a reduced dosage of the drugs that caused side effects might be the therapy of choice in patients who have developed dose-dependent side effects. PMID- 1718399 TI - Hyperfractionated accelerated radiotherapy for carcinoma of the oesophagus. AB - We report on 48 patients with carcinoma of the oesophagus treated by hyperfractionated accelerated radiotherapy. The patients, aged 46 to 93 years, were considered suitable for radiotherapy on their performance status irrespective of the presence of metastases. The radiotherapy was given three times a day over 2 weeks with a minimum of 3 h between treatments. The treatment was well tolerated acutely and to date there have been no unacceptable long-term side-effects. Dysphagia was improved in 39 (81.2%) patients. Product-limit survival was 35.7%, 18.5% and 12.3% at 1, 2 and 3 years. We conclude that this regime is feasible within the normal working day, well tolerated, effective and the shorter overall treatment duration desirable. PMID- 1718398 TI - Primary non-Hodgkin's lymphoma of the central nervous system: phase II study of chemotherapy (BVAM) prior to radiotherapy. AB - Ten patients were diagnosed as having primary non-Hodgkin's lymphoma of the central nervous system at University Hospital, Nottingham, between September 1986 and April 1989. None had clinical evidence of HIV-1 infection. All the patients started treatment with chemotherapy (BVAM), designed to cross the blood-brain barrier, followed by radiotherapy. Seven patients completed both chemotherapy and radiotherapy. Dose reduction during chemotherapy was necessary in three patients because of neutropenia. In two of the six patients with solitary tumours, complete resection was achieved surgically prior to treatment. Five of the remaining eight patients (63%) had radiological evidence of a complete response with chemotherapy. The other three patients had no response to chemotherapy but one had a complete response after radiotherapy. The two-year cause-specific survival of the 10 patients was 37%. Two of the three patients who had a postoperative performance status of 0 or 1 (ECOG/WHO) are alive and disease-free at 26 and 46 months from diagnosis. The median survival of the seven patients with a performance status of 2-4 was 10 months with two patients alive and disease-free at 19 and 26 months. The two-year cause-specific survival of these seven patients was 22%. PMID- 1718400 TI - Palliative radiotherapy for bronchial carcinoma: science or art? PMID- 1718401 TI - Risk-related adjuvant chemotherapy for stage I non-seminoma of the testis. AB - Seventy-Two patients with stage I testicular non-seminoma presenting between January 1984 and December 1988 were managed either by adjuvant chemotherapy in the presence of histological adverse prognostic factors or by surveillance if none of these factors were present. The determining factors were vascular invasion, lymphatic invasion, involvement of the epididymis, involvement of the rete testis. Thirty patients were treated with three courses of platinum, vinblastine and bleomycin (PVB) and 42 were managed by surveillance. All 72 patients are alive and have been free of disease from 12-60+ months. One of 42 patients managed by surveillance relapsed 16 months after orchidectomy and he has been disease-free for 32+ months after chemotherapy. No relapses occurred in patients treated with adjuvant chemotherapy. The results confirm the suggestion by Peckham (Hoskin et al., 1986) that histopathological analysis of the primary tumour can provide the basis for subsequent management either by surveillance or chemotherapy. PMID- 1718402 TI - Retinal neuromodulation: the role of dopamine. AB - Dopamine exerts multiple effects on retinal horizontal cells. Dopamine, via cyclic AMP and protein kinase A, reduces the light responsiveness of horizontal cells and the electrical coupling between the cells. The gating kinetics of both gap-junctional and glutamate channels are altered as a result of phosphorylation by protein kinase A. Dopamine also causes a reversible retraction of neurites of horizontal cells maintained in culture. Diacylglycerol analogues as well as phorbol esters mimic this effect of dopamine, but not cyclic AMP analogues or Forskolin. The results suggest that dopamine causes neurite retraction by the activation of protein kinase C via diacylglycerol. PMID- 1718403 TI - The rod bipolar cell of the mammalian retina. AB - Three approaches to study the function of mammalian rod bipolar cells are described. Extracellular recordings from the intact cat eye under light- and dark adapted conditions showed that in dark-adapted retina all light responses can be blocked by 2-amino-4-phosphonobutyrate (APB). Immunocytochemical staining with an antibody against protein kinase C (PKC) labeled rod bipolar cells in all mammalian retinae tested. When rat retinae were dissociated, PKC immunoreactivity was also found in isolated bipolar cells and could be used for their identification as rod bipolars. Patch-clamp recordings were performed from such dissociated rod bipolar cells and their responses to APB were measured. APB closed a nonselective cation channel in the cell membrane. The actions of GABA and glycine were also tested and both opened chloride channels in dissociated rod bipolar cells. These results suggest that rod bipolar cells are depolarized by a light stimulus and that GABA as well as glycine modulate their light responses. PMID- 1718406 TI - Angiogenic factors: regulators of blood supply-side biology. FGF, endothelial cell growth factors and angiogenesis: a keystone symposium, Keystone, CO, USA, April 1-7, 1991. PMID- 1718405 TI - HLA polymorphism and T cell recognition of a conserved region of p190, a malaria vaccine candidate. AB - We have examined T cell recognition of a recombinant polypeptide (190L), corresponding to a 175-amino-acid-long conserved region of the major surface antigen (p190) of Plasmodium falciparum merozoites. We show that 190L contains a variety of T cell epitopes, and can be recognized in association with many different MHC class II molecules, including HLA-DR, DP, and DQ antigens. Most of the epitope-containing peptides are able to bind to more than one DR, and a single DR molecule can bind to different peptides. These findings, together with the fact that humans are generally heterozygous at the DR, DQ, and DP beta chain loci, suggest that MHC restriction should not be a major constraint in the development of malaria subunit vaccines. PMID- 1718404 TI - Complete sequence of the genes encoding the VH and VL regions of low- and high affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient. AB - We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity. PMID- 1718407 TI - Effects of inhibitors of intracellular messenger systems on amylase release from rat parotid gland. AB - Effects of a series of novel inhibitors of calmodulin or protein kinases on amylase release were studied in rat parotid slices. Amylase release induced by a cholinergic agonist, carbamylcholine, was inhibited by N-(6-aminohexyl)-5-chloro 1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, 1-(5-chloronaphthalen-1 sulfonyl)-1H-hexahydro-1, 4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor, and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of Ca(2+)-activated, phospholipid-dependent protein kinase (protein kinase C), while N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), an inhibitor of cyclic AMP-dependent protein kinase (protein kinase A), did not inhibit the release. On the other hand, amylase release induced by a beta-adrenergic agonist, isoproterenol, was inhibited only by H-8, but not by W-7, ML-9 or H-7. These results suggest that cholinergic stimulation evokes amylase release via the Ca(2+)-dependent system which involves calmodulin, MLCK and protein kinase C, while beta-adrenergic stimulation via the cyclic AMP-dependent system involves protein kinase A. PMID- 1718408 TI - Human monocytes in a long-term culture with interleukin-2 show high tumoricidal activity against various tumor cells. AB - We compared the tumoricidal activity of human monocytes cultured with interleukin 2 (IL-2) or human recombinant interferon-gamma (IFN-gamma) alone, or IFN-gamma in combination with a small amount of lipopolysaccharides (LPS). Human monocytes cultured with IL-2 for 7 days or longer, termed lymphokine-activated macrophages (LAMs), showed higher tumoricidal activity than those cultured for 1 day. In contrast, monocytes cultured with IFN-gamma alone or in combination with LPS for 7 days or longer showed lower tumoricidal activity. LAMs were identified as macrophages by nonspecific esterase staining and immunofluorescence staining with anti-CD14 antibody. LAMs were not induced in fetal calf serum-containing medium, but they were induced when colony-stimulating factor-1 was added to the medium. LAMs showed high tumoricidal activity against all human and murine tumor cell lines tested, although they showed no cytotoxic activity against human normal cells. During incubation with IL-2, tumoricidal activity of LAMs was maximal at days 8-16 and was sustained until day 28. The difference in tumoricidal mechanism between LAMs and lymphokine-activated killer (LAK) cells was also shown by using two kinds of cytotoxic assay systems. LAMs require a long incubation time to kill tumor cells, but LAK cells can kill them immediately. Furthermore, LAMs kill tumor cells with complete DNA degradation, whereas LAK cells can induce significant but not complete DNA degradation. These results indicate that LAMs and LAK cells have different tumoricidal mechanisms for killing target cells, although they were induced by incubation with the same lymphokine, IL-2. PMID- 1718409 TI - [Morphological possibilities for objectification of the prognosis for invasive ductal carcinoma of the breast]. AB - Tissue sections were taken from 187 cases of invasive ductal mammary carcinoma (among them 94 cases with lymph node metastases, average follow-up period being 44 months) and, after Feulgen staining, were investigated by automated microscopic image analysis. A comparison was made, with reference to survival periods, between the relevance to prognosis of nuclear image analysis and results obtained from histopathological grading, according to Bloom and Richardson (1957), as well as findings recorded from image analysis of DNA. Histopathological grading was found to be of poor reproducibility (71.1%). While statistically significant differences were found to exist between survival curves of Grades I and III patients, grading proved to be of minor importance to prognostic assessment of the individual case, with the number of G-II cases being relatively high (42.7%). Results of better reproducibility were achievable from the quantitative morphological method of nuclear image analysis. Its forecasting value with regard to good or poor prognosis was clearly higher than that of histopathological grading, and the number of cases with obscure or dubious prognosis was clearly lower (18.9%). DNA ploidy measurement, using DNA histogram types according to Auer (1980), has proved to be another method by which prognostically favourable and unfavourable cases can be distinguished from each other with statistical significance, though its classification quality (70.5%) still was unambiguously below that achievable by nuclear image analysis (79%). PMID- 1718410 TI - [Frequency distribution, microscopic image analysis and grading of mammary carcinoma]. AB - The frequency of diagnoses of breast diseases in 1685 patients was examined on the basis of biopsy material at the Institute of Pathology of Charite Hospital in Berlin, during 1984-1987, with particular consideration of histological types of mammary carcinomas. Breast cancer was detected in 562 patients of whom 429 had undergone operations according to standardized recommendations by the Charite Department of Surgery. Histological tumor classification, postoperative staging and, in cases of invasive ductal carcinoma, also histological grading were part of the pathologico-anatomic investigation. Reproducibility of grading was 72.7% for intra-observer examination and 63.3% for evaluation by different pathologists. A group of 20 premenopausal patients with tumors staged as pT1, pN0 was selected for morphometric studies and objectivation of tumor grading. HE stained paraffin sections were made for conventional grading according to Bloom and Richardson (1957), whereas Feulgen reaction in 4 microns thick sections was used for quantitative examination by means of a microscopic image analysis system (Robotron A6471 with AMBA/R software). The measuring program used was based mainly on evaluation of karyometric features. Measurements were concentrated on analysis of the chromatin structure of tumor cell nuclei. Correlations with histological grading were found to exist for 16 features out of 28 parameters selected for statistical analysis. Some new parameters of the texture of tumor cells nuclei were demonstrated to be strongly correlated with the degree of tumor differentiation. The prognostic significance of nuclear texture features has to be proved by further studies in which clinical data of the course of the tumor disease must be included. PMID- 1718411 TI - Effect of protein deprivation on insulin-like growth factor-binding proteins in rats. AB - The effect of protein deprivation on plasma concentration of insulin-like growth factor-binding proteins (IGFBP) was studied in rats. A significant decrease in the concentration of IGFBP of molecular weight (mass) approximately 40 kDa was observed in protein-deprived rats. There was no prominent effect of protein deprivation on the concentration of IGFBP with molecular weights of about 30 kDa or 22-24 kDa. The binding capacity to plasma IGFBP of exogenously-added 125I labelled insulin-like growth factor-1 (125I-IGF-1) was also studied. IGFBP of molecular weight about 30 and 22-24 kDa (the native form of this protein is presumed to be 29 kDa) in protein-deprived rat plasma bound more 125I-IGF-1 than those in protein-fed rat plasma. This suggested that these IGFBP in protein deprived rat plasma are relatively unsaturated by endogenous IGF-1. The response of IGFBP to protein deprivation which was elucidated in the present investigations add further evidence to our previous assumption that IGFBP play an important role in protein nutrition. PMID- 1718412 TI - The influence of mechanical injury on the metabolic activity of transplanted cerebral cortex. AB - The authors studied the metabolic activity of rat embryonic cerebral cortex grafts (ED 15-16) implanted into rat brains immediately (TR0) and 14 days (TR14) after cavity formation. Over a period of two months, the ATP, lactate and glucose concentration in TR0 transplants remained at the same level as observed in the intact cortex, whereas in TR14 transplants the ATP and glucose concentration fell significantly and the lactate concentration rose. The DNA concentration rose in both types of transplants, but the increase was more pronounced in TR0 grafts. Choline acetyltransferase activity (a neuron marker) fell significantly in both cases, but the decrease was greater in TR14 transplants. The results indicate that grafts implanted into the brain immediately after cavities had been formed have better metabolic activity and are capable of longer survival than grafts implanted 14 days after cavitation. PMID- 1718413 TI - Purification of rabbit and human serum paraoxonase. AB - Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed. PMID- 1718414 TI - Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin. AB - We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin. PMID- 1718415 TI - Differences in deoxyribonuclease I hypersensitive sites in phenobarbital inducible and constitutive rabbit P450IIC genes. AB - DNase I hypersensitivity of nuclear chromatin near the rabbit cytochrome P450IIC genes was investigated by indirect end-labeling genomic Southern analysis. Major DNase I hypersensitive sites were observed in proximal (-200 base pairs) and distal (-2000 to -2200 base pairs) regions of the genes. The presence of the proximal site correlated well with the expression states of the individual genes. In contrast, the distal site was present in DNA from both liver and kidney nuclei and in untreated and phenobarbital-treated animals irrespective of the expression state of the genes. However, the distal site was present only in genes that respond to phenobarbital (cytochromes P450IIC1 and P450IIC2) and was not detected in the constitutive cytochrome P450IIC3 gene. The nucleotide sequences of 500 base pairs in the distal site regions of cytochromes P450IIC1 and P450IIC2 were 67% similar, and hepatocyte nuclear factor 1 like motifs were conserved in these two sequences. These results are consistent with the hypothesis that distal regions cooperate with the proximal promoter regions in the regulation of cytochrome P450IIC gene expression. PMID- 1718416 TI - Low-temperature crystallographic analyses of the binding of Hoechst 33258 to the double-helical DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G. AB - The crystal structure of the complex of Hoechst 33258 and the DNA dodecamer C-G-C G-A-A-T-T-C-G-C-G has been solved from X-ray data collected at three different low temperatures (0, -25, and -100 degrees C). Such temperatures have permitted collection of higher resolution data (2.0, 1.9, and 2.0 A, respectively) than with previous X-ray studies of the same complex. In all three cases, the drug is located in the narrow central A-A-T-T region of the minor groove. Data analyses at -25 and -100 degrees C (each with a 1:1 drug/DNA ratio in the crystallizing solution) suggest a unique orientation for the drug. In contrast, two orientations of the drug were found equally possible at 0 degrees C with a 2:1 drug/DNA ratio in solution. Dihedral angles between the rings of Hoechst 33258 appear to change in a temperature-dependent manner. The drug/DNA complex is stabilized by single or bifurcated hydrogen bonds between the two N-H hydrogen bond donors in the benzimidazole rings of Hoechst and adenine N3 and thymine O2 acceptors in the minor groove. A general preference for AT regions is conferred by electrostatic potential and by narrowing of the walls of the groove. Local point-by-point AT specificity follows from close van der Waals contacts between ring hydrogen atoms in Hoechst 33258 and the C2 hydrogens of adenines. Replacement of one benzimidazole ring by purine in a longer chain analogue of Hoechst 33258 could make that particular site GC tolerant in the manner observed at imidazole substitution for pyrrole in lexitropsins. PMID- 1718417 TI - Abortive products as initiating nucleotides during transcription by T7 RNA polymerase. AB - The kinetics of formation of abortive initiation products during transcription of a synthetic template (encoding the transcript GAUGGC) by T7 RNA polymerase have been determined. This study revealed that while total RNA was formed in the reaction as expected, the levels of the dinucleoside tetraphosphate guanylyl 3',5'-adenosine-5'-triphosphate (pppGpA) and trinucleoside pentaphosphate guanylyl-3',5'-adenosine-3',5'-uridine-5'-triphosphate (pppGpApU) formed by premature termination of transcription reached a maximum after 10 min, and then decreased. Transcription of the same template, in the presence of either [gamma 32P]GTP and ATP, or GTP and [alpha-32P]ATP, gave the 32P-labeled dinucleotides *pppGpA and pppG*pA. Incorporation of each of these substrates into longer RNA transcripts in the same enzyme-template system was demonstrated. The incorporation was shown to require the presence of template in the reaction mixture. The requirement for base complementarity restricts the position of incorporation to that of initiating (5') nucleotide. Transcription of a second template, which encodes an RNA transcript having the partial sequence GpA at two internal positions, in the presence of each of the labeled dinucleoside tetraphosphates, failed to bring about the synthesis of significant yields of any longer radiolabeled transcripts. It is concluded that dinucleoside tetraphosphate (and perhaps trinucleoside pentaphosphate) can function as initiating nucleotides when complementary to the nucleotide sequence at promoter regions. However, a dinucleotide is not used as substrate for subsequent chain elongation in T7 RNA polymerase catalyzed transcription reactions. PMID- 1718418 TI - A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase. AB - With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990). PMID- 1718419 TI - Functional expression and characterization of the interferon-induced double stranded RNA activated P68 protein kinase from Escherichia coli. AB - The P68 protein (referred to as P68 on the basis of its molecular weight of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by double-stranded (ds) RNAs. Although extensively studied, little is currently known about the regulation of kinase function at the molecular level. What is known is that activation of this enzyme triggers a series of events which lead to an inhibition of protein synthesis initiation and may, in turn, play an integral role in the antiviral response to interferon. To begin to understand P68 and its biological functions in the eukaryotic cell, we have expressed the protein kinase in Escherichia coli under control of the bacteriophage T7 promoter. In rifampicin-treated cells, metabolically labeled with [35S]methionine and induced by IPTG, the P68 kinase was the predominant labeled product. Further, P68 was recovered from extracts as a fully functional enzyme, shown by its ability to become activated and phosphorylate its natural substrate, the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). Moreover, P68 was phosphorylated in vivo in E. coli, providing conclusive evidence that the kinase has the capacity to phosphorylate and activate itself in the absence of other eukaryotic proteins. In contrast, a mutant P68 protein, containing a single amino acid substitution in the invariant lysine in catalytic domain II, was completely inactive. Interestingly, both the mutant and wild-type protein kinases efficiently bound activator dsRNAs despite the fact that only the latter was activated by these RNAs. Finally, the expressed kinase could be isolated from contaminating E. coli proteins in an active form by immunoaffinity chromatography with a monoclonal antibody specific for P68. PMID- 1718420 TI - Assignment of the natural abundance 13C spectrum of proteins using 13C 1H detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon hydrogen coupling constants. AB - Proton-detected heteronuclear multiple-bond 1H-13C correlations (HMBC) previously have been used for assignment purposes in a variety of isotopically enriched proteins. In the present study it is demonstrated that the technique yields an almost complete assignment of the natural abundance 13C spectrum of the protein basic pancreatic trypsin inhibitor (BPTI). In addition, the technique permits additional 1H assignments to be made for this well-studied protein. The intensities of observed correlations permit rough estimates to be made of 2J(C,H) and 3J(C,H) coupling constants. These couplings can be used for conformational studies of both the side chains and the backbone. Intra- and interresidue coupling between C alpha H and the carbonyl carbon provides information about the backbone angles psi and phi. Side-chain conformations can be determined from both two- and three-bond carbon-hydrogen coupling constants. The present study of BPTI together with its known high-precision solution structure yields an experimental correlation between resonance intensities and secondary structure. The spectra show the potential of the method in analyzing 13C NMR spectra of nonenriched proteins. The method yields 13C NMR chemical shifts, which are versatile parameters to be used to monitor structural changes, titrations, etc. PMID- 1718421 TI - Sequential NMR resonance assignment and structure determination of the Kunitz type inhibitor domain of the Alzheimer's beta-amyloid precursor protein. AB - Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove. PMID- 1718422 TI - Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity. AB - The RNA- and DNA-dependent DNA polymerase activities of two point mutants of HIV 1 reverse transcriptase lacking ribonuclease H activity have been compared to the wild-type enzyme activities using substrates consisting of an oligodeoxynucleotide primer hybridized to either a RNA or a DNA template. The RNase H phenotype had a negligible effect on the steady-state kinetics and processivity of reverse transcription of a homopolymer template-primer [poly(A).oligo(dT)]. However, analysis of the distribution of DNA products indicated that the ability of the mutants to reverse-transcribe a specifically primed 345-nucleotide heteropolymeric RNA template derived from the gag region of HIV-1 was impaired relative to the wild-type enzyme. Although the wild-type and mutant enzymes shared the same pause sites of synthesis along the RNA template, certain prematurely terminated nascent primer chains were poorly extended by the mutant enzymes and hence accumulated, suggesting that a catalytically functional RNase domain facilitated reinitiation of DNA synthesis at specific pause sites along a heteropolymer template. In contrast, the processivity and product distribution of DNA synthesis directed by a heteropolymer gag DNA template of the same nucleotide sequence were not significantly influenced by the RNase H phenotype of the mutants. PMID- 1718423 TI - Reverse transcriptase from human immunodeficiency virus: a single template-primer binding site serves two physically separable catalytic functions. AB - The binding of substrates to recombinant reverse transcriptase from human immunodeficiency virus (HIV) and the natural enzyme from avian myeloblastosis virus (AMV) has been examined by analyzing both the ribonuclease H and the RNA dependent DNA polymerase activities. With 3'-end-labeled globin mRNA hybridized to (dT)15 as the substrate in the ribonuclease H reaction, the enzymes partially deadenylated the mRNA in a distributive manner. Under these conditions, there was a rapid initial burst followed by a prolonged, but much slower, steady-state rate. The biphasic reaction made possible determinations of kinetic constants as follows: values for Km, KD, and kcat were, respectively, 27 nM, 11 nM, and 5 x 10(-3) s-1 for the HIV enzyme and 30 nM, 9 nM, and 5 x 10(-3) s-1, respectively, for the avian enzyme. These constants were used to derive other parameters: The rate of association of the template-primer with reverse transcriptase was approximately 2 x 10(5) M-1 s-1, and the rate of dissociation was approximately 2 x 10(-3) s-1, regardless of the source of the enzyme. The rate of release of the product was essentially equivalent to the value of kcat indicated above for each of the enzymes. The polymerase reaction was evaluated under processive conditions of synthesis; values of Km and kcat of approximately 6 nM and approximately 2.5 s 1, respectively, for the human enzyme, and approximately 10 nM and approximately 2 s-1, respectively, for the avian enzyme were observed. The interaction of substrates with HIV reverse transcriptase was characterized further with the aid of ribonucleoside-vanadyl complexes. These complexes inhibited the polymerase and ribonuclease H activities of the enzyme competitively with respect to globin mRNA.(dT)15. Values of Ki ranging from 1 to 3 mM were obtained. With respect to deoxyribonucleoside triphosphate substrates in the polymerase reaction, mixed inhibition was observed. Deoxyribonucleoside triphosphates had no effect on kinetic parameters governing the ribonuclease H activity of the HIV enzyme but apparently facilitated the formation of active enzyme. These data fit a model in which one template-primer binding site serves both the polymerase and the ribonuclease H catalytic sites. PMID- 1718424 TI - Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding. AB - Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component. Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template. The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300. This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase. PMID- 1718425 TI - A protease activity associated with acetylcholinesterase releases the membrane bound form of the amyloid protein precursor of Alzheimer's disease. AB - Amyloid deposits in the brains of patients with Alzheimer's disease (AD) contain a protein (beta A4) which is abnormally cleaved from a larger transmembrane precursor protein (APP). APP is believed to be normally released from membranes by the action of a protease referred to as APP secretase. Amyloid deposits have also been shown to contain the enzyme acetylcholinesterase (AChE). In this study, a protease activity associated with AChE was found to possess APP secretase activity, stimulating the release of a soluble 100K form of APP from HeLa cells transfected with an APP cDNA. The AChE-associated protease was strongly and specifically inhibited by soluble APP (10 nM) isolated from human brain. The AChE associated protease cleaved a synthetic beta A4 peptide at the predicted cleavage site. As AChE is decreased in AD, a deficiency of its associated protease might explain why APP is abnormally processed in AD. PMID- 1718426 TI - Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text]. AB - Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence. PMID- 1718427 TI - Membrane fluidity and lipid hapten structure of liposomes affect calcium signals in antigen-specific B cells. AB - Antigen-specific B-cell clones directed against a 2,4,6-trinitrophenyl (TNP) hapten have been established [Hamano et al. (1990) J. Immunol. 144, 811-815]. We measured here the cytosolic free calcium ion concentration ([CA2+]i) in these B cell clones after antigen stimulation. Trinitrophenylated liposomes with different length spacers between TNP and phosphatidylethanolamine (TNP-Cn-PE) increased cytosolic free calcium concentration in TNP-specific B cells (clone TP67.21). The magnitude of calcium signals depended on the length of the spacer. TNP-C6-PE in dipalmitoylphosphatidylcholine (DPPC) liposomes triggered larger calcium signals in B cells than TNP-Cn-PE with n = 0, 4, 8, or 12. The magnitude of the calcium signals was strongly dependent on the fluidity of the liposome membranes. TNP-C6-PE in the solid DPPC liposomes triggered the calcium signals in B cells 50-100 times as efficiently as TNP-C6-PE in the fluid dimyristoylphosphatidylcholine liposomes. The difference between the solid liposomes and the fluid liposomes was more pronounced in triggering calcium signals in B cells than in antibody binding to these liposomes. PMID- 1718428 TI - Characterization of the solubilized charybdotoxin receptor from bovine aortic smooth muscle. AB - Monoiodotyrosine ([125I]ChTX) binds with high affinity to a single class of receptors present in bovine aortic smooth muscle sarcolemmal membranes that are functionally associated with the high-conductance Ca(2+)-activated K+ channel [maxi-K channel; Vazquez, J., et al. (1989) J. Biol. Chem. 265, 20902-20909]. Cross-linking experiments carried out with this preparation in the presence of [125I]ChTX and disuccinimidyl suberate indicate specific incorporation of radioactivity into a protein of Mr 35,000. The smooth muscle ChTX receptor can be solubilized in active form in the presence of selected detergents. Treatment of membranes with digitonin releases about 50% of the ChTX binding sites. The solubilized receptor retains the same biochemical and pharmacological properties that are characteristic of toxin interaction with membrane-bound receptors. The solubilized receptor binds specifically to wheat germ agglutinin-Sepharose resin, suggesting that it is a glycoprotein. Functional ChTX binding sites can also be solubilized in 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS). Sucrose density gradient centrifugation of either digitonin or CHAPS extracts indicates that the ChTX receptor has a high apparent sedimentation coefficient (s20,w = 23 and 18 S, respectively). Cross-linking experiments indicate that the appearance of the 35-kDa membrane protein correlates with ChTX binding activity after both wheat germ agglutinin-Sepharose and sucrose density gradient centrifugation steps. Given the high apparent sedimentation coefficient of the ChTX receptor, the 35-kDa membrane protein may be a subunit of a higher molecular weight complex which forms the maxi-K channel in smooth muscle sarcolemma. PMID- 1718429 TI - Two-dimensional diffusion of F1F0-ATP synthase and ADP/ATP translocator. Testing a hypothesis for ATP synthesis in the mitochondrial inner membrane. AB - We report here the first experimentally determined lateral diffusion coefficients of the F1F0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F1F0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the alpha-beta doublet, gamma and delta subunits of F1 and subunits two, three and four of F0. This IgG, conjugated with lissamine rhodamine, was used as a fluorescent probe to monitor the diffusion of the synthase in the membrane. IgG to cytochrome bc1 complex, prepared and labeled similarly, was used as a fluorescent probe for diffusion of this redox component. Eosin maleimide was determined to specifically label the ADP/ATP translocator in the isolated inner membrane and was used as a specific probe for the diffusion of the translocator. Using fluorescence recovery after photobleaching, the experimental average lateral diffusion coefficient of the F1F0-ATP synthase was determined to be 8.4 x 10(-10) cm2/s or twice that of cytochrome bc1 complex while the diffusion coefficient of the ADP/ATP translocator was 1.7 x 10(-9) cm2/s or four times that of cytochrome bc1 complex suggesting that all three components are independent two-dimensional diffusants. Using these diffusion coefficients and applying a number of basic assumptions, we calculated the theoretical two-dimensional diffusion-controlled collision frequencies and derived collision efficiencies (protons transferred per collision) between each of the three proton-transferring redox complexes and both the F1F0-ATP synthase and ADP/ATP translocator by treating the redox components as proton donors and the synthase and translocator as proton acceptors. These collision efficiencies support the physical possibility of a diffusion-based, random collision process of proton transfer and ATP synthesis in the mitochondrial inner membrane. PMID- 1718430 TI - Synthesis of acylated gramicidins and the influence of acylation on the interfacial properties and conformational behavior of gramicidin A. AB - Five gramicidin A analogs were synthesized in which various acyl chains, differing in length and unsaturation, were covalently coupled to the C-terminal ethanolamine group. The analogs were characterized by various spectroscopic techniques and their molecular properties were investigated using monolayer techniques and circular dichroism. It is demonstrated that neither the interfacial properties nor the conformational behavior of gramicidin A at the air/water interface are seriously affected upon acylation. It is proposed that at the limiting area the gramicidin molecule is oriented with its C-terminus towards the subphase with the covalently coupled acylchain located parallel to the helical axis in between the protruding tryptophans. Circular dichroism experiments, in which gramicidin-containing vesicles were prepared from different organic solvents, indicate that the presence of a covalently coupled fatty acylchain tends to stabilize the beta 6.3 helical conformation. It is demonstrated that, like for gramicidin A, also for the acylgramicidins the single stranded beta 6.3 helical conformation, or channel conformation, is the preferred conformation upon incorporation in bilayers. PMID- 1718431 TI - Permeation of Pb2+ through calcium channels: fura-2 measurements of voltage- and dihydropyridine-sensitive Pb2+ entry in isolated bovine chromaffin cells. AB - Fura-2 was used to monitor Pb2+ entry into isolated bovine chromaffin cells exposed to micromolar concentrations of Pb2+ in media containing basal or high concentrations of K+. The entry of Pb2+ consists of voltage-independent and voltage-dependent (K(+)-stimulated) components. The voltage-dependent Pb2+ entry is enhanced by Ca2+ channel agonist BAY K 8644 and blocked by the channel antagonist nifedipine, suggesting the involvement of the L-type Ca2+ channels. In contrast to the transient, K(+)-depolarization-dependent increase in [Ca2+]i, the increase in [Pb2+]i is sustained over a period of several minutes, suggesting the absence of channel inactivation and/or the saturation of Pb(2+)-buffering capacity of the cell cytosol. PMID- 1718432 TI - Hypotonic shock activated Cl- and K+ pathways in human fibroblasts. AB - The exposure of human fibroblasts to hypotonic medium (200 mosmolal) evoked the activation of both 36Cl- influx and efflux, which were insensitive to inhibitors of the anion exchanger and of the anion/cation cotransport, and conversely were inhibited by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 36Cl- efflux was linked to a parallel efflux of 86Rb+; thus conductive K+ and Cl- pathways are activated during volume regulation in human fibroblasts. This conclusion is supported by evidence that, in hypotonic medium, 36Cl- influx and 86Rb+ efflux were both enhanced by depolarization of the plasma membrane. Depletion of the intracellular K+ content, obtained by preincubation with the ionophore gramicidin in Na(+)-free medium, had no effect on Cl- efflux in hypotonic medium. This result has been interpreted as evidence for independent activation of K+ and Cl- pathways. It is also concluded that the anion permeability is the rate-limiting factor in the response of human fibroblasts to hypotonic stress. PMID- 1718433 TI - Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase. AB - Bovine myelin basic protein (MBP), charge isoform 1 (C1) was carboxylmethylated by the enzyme D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate and/or asparagine residues which are recognized by this methyltransferase. Native MBP, C1 was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, C1 which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, C1 at Gly-127-Gly-128 and of the carboxylmethylated MBP, C1 at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His Lys-Gly-Leu-Lys- Gly-His-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ileu-Phe-Lys-. This sequence matches MBP residues 125-154. As a result of these findings, Asp-132 and Asp-144 were identified as two of the modified (isomerized or racemized) methyl accepting L-aspartates in MBP. The results of the proteolysis experiments wherein the sequencing grade staphylococcal V8 proteinase was used at the rarely tested pH of 4.0, rather than at its commonly tested pH of 7.8, also disclose that the proteinase totally failed to recognize and hence cleave the two Glu-X bonds (Glu 82-Asn-83 and Glu-118-Gly-119) of MBP, preferring to cleave the protein at a number of hitherto unreported sites. PMID- 1718434 TI - Functional cholecystokinin receptors are distinguished kinetically by biotinyl Tyr-Gly-(Thr28,Nle31)CCK(25-33) in rat pancreatic acini. AB - Biotinyl-tyrosine-glycine(Thr28,Nle31)CCK(25-33) (BTG-TN-CCK-9) promoted amylase secretion and phosphatidylinositol (PI) metabolism with the same potency and efficacy as TN-CCK-9 in dispersed rat pancreatic acini. A 1 min preincubation of the ligand with a 20-fold excess of streptavidin completely suppressed this biological activity. On the other hand, amylase secretion and PI metabolism prestimulated with BTG-TN-CCK-9 were blocked within 1-5 min after streptavidin addition. [125I]BTG-TN-CCK-9 bound to high (Kd 0.17 nM) and low (Kd 13 nM) affinity receptors. Its dissociation, in the presence of either streptavidin or TN-CCK-9, showed a rapid component and a slow component. The proportion of tracer dissociating slowly increased with increasing preincubation time as did the proportion of tracer that could not be washed away quickly by acidic treatment, in parallel experiments. This phenomenon occurred less readily at 4 degrees C or in the presence of 1 mM CCCP. In acini preincubated for 30 min with 0.3 nM [125I]BTG-TN-CCK-9 and various concentrations of unlabelled BTG-TN-CCK-9, then washed at neutral pH (in order to eliminate rapidly dissociating ligand preferentially), the tracer displacement curve was shifted leftward, suggesting that rapidly dissociating receptors corresponded to low affinity receptors. When acini were preincubated for 1 min with BTG-TN-CCK-9, then washed at neutral pH with buffer only, we observed residual stimulated secretion over the next 30 min period, that correlated with the BTG-TN-CCK-9 concentration offered during the short preincubation period. This phenomenon was inhibited by streptavidin suggesting that intracellularly accumulated intact BTG-TN-CCK-9 (as shown, by radio-HPLC) promoted residual secretion when free to bind again to cell surface receptors in the absence of streptavidin. Taken collectively, these data suggest the coexistence of at least 2 types (or states) of CCK receptors. PMID- 1718435 TI - Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative. AB - The peptide SAEEEDQYN, corresponding to the carboxyl-terminal tryptic fragment of rat progastrin, whose penultimate tyrosyl residue is sulphated in the native peptide, is phosphorylated with Km values of 120 and 180 microM by two spleen tyrosine protein kinases, termed TPK-IIB and TPK-III, respectively. Another spleen tyrosine protein kinase related to the src family (TPK-I/lyn) is poorly active toward this peptide, displaying a Km 6.5 mM. The Tyr-phosphorylated peptide is recognized by an antibody (L304), which reacts with both sulphated and unmodified peptides, while it is not recognized by a second antibody (L303), which reacts with unmodified peptide yet not with the sulphated derivative. These data, in conjunction with previous observations (Hofsteenge, J., Stone, S.R., Donella-Deana, A. and Pinna, L.A. (1990) Eur. J. Biochem. 188, 55-59) support the view that phosphotyrosine is an effective surrogate for sulphotyrosine in a wide spectrum of biological activities. PMID- 1718436 TI - Tyrosyl phosphorylation and activation of the myelin basic protein kinase p44mpk during sea star oocyte maturation. AB - The most prominent tyrosyl-phosphorylated protein in maturing sea star oocytes was identified as the 44 kDa myelin basic protein (MBP) kinase p44mpk. Immunoblotting studies with anti-phosphotyrosine PY-20 antibody and phosphoamino acid analysis of in vivo [32P]phosphate-labelled p44mpk showed that the tyrosyl phosphorylation of the kinase correlated with a greater than 10-fold stimulation of its MBP phosphotransferase activity. The activation of p44mpk was reversed almost completely by purified preparations of the protein-tyrosyl phosphatases CD45 and 1B. Purified p44mpk has previously been shown to undergo autophosphorylation in vitro on seryl residues and this was associated with further enhancement of its MBP phosphorylating activity (Sanghera et al. (1991) J. Biol. Chem. 266, 6700-6707). p44mpk also underwent seryl phosphorylation during oocyte maturation, and the protein-seryl/threonyl phosphatase 2A reversed partially the maturation-associated stimulation of its MBP kinase activity. The properties of p44mpk resemble the murine 42 kDa mitogen-activated protein kinase (p42mapk). While p44mpk may feature the phosphorylatable tyrosyl residue that is critical for activation in p42mapk, it lacks the upstream threonyl phosphorylation site that is also required for p42mapk activity (Payne et al. (1991) EMBO J: 10, 885-892). These findings indicate partial differences in the regulatory mechanisms that govern the activities of these isozymes. PMID- 1718437 TI - Nucleotide and nucleic acid metabolism in rat thymocytes during cell cycle progression. AB - A complete cell cycle of mature, concanavalin A (Con A) stimulated rat thymocytes was documented by analyzing the cell number as well as the content and synthesis of DNA and RNA. Cell cycle progression is accompanied by an elevation of class I, II and III RNA polymerase activities (about 10-fold) in the S phase maximum, 48 h after stimulation. Moreover, maximal cellular contents of DNA, ATP, ADP and AMP were observed at this culture period, whereas the RNA level peaked at 60 h. The synthesis of purine and pyrimidine nucleotides de novo was detected by use of [14C]HCO3-. Maximal incorporation rates of [14C]HCO3- into nucleotides (de novo synthesis) and of [3H]adenine into adenylates ('salvage pathway') occur during the S phase. However, the de novo synthesis rates were markedly lower than those of the 'salvage pathway'. The highest cellular level of the nucleotide precursor 5-phosphoribosyl-1-pyrophosphate (8.4-fold increase) also coincided with the S phase. PMID- 1718439 TI - Phenotypic heterogeneity in cultured skin fibroblasts from patients with disorders of peroxisome biogenesis belonging to the same complementation group. AB - We have studied fibroblast cell lines derived from a control subject (cell line 85AD5035F) and three patients clinically described as having the Zellweger syndrome (cell line W78/515), the infantile form of Refsum disease (cell line BOV84AD) and hyperpipecolic acidaemia (cell line GM3605), respectively. The mutant cell lines belonged to the same complementation group. The fibroblasts were cultured under identical conditions and were harvested at different time intervals after reaching confluence. Several peroxisomal parameters were determined. In agreement with previous reports, a lowered enzymic activity of acyl-CoA: dihydroxyacetonephosphate acyltransferase and a decrease in latent catalase clearly distinguished the patient cell lines from the control cell line. However, the cell lines exhibited a phenotypic heterogeneity. This was most strikingly encountered when cells were processed for indirect immunofluorescence microscopy and stained with anti-(catalase). The control cells exhibited a punctate fluorescence, which is indicative of the presence of catalase in peroxisomes. In the mutant cell line W78/515 a diffuse fluorescence was observed, indicative of the presence of catalase in the cytosol. In the other two mutant cell lines a punctate fluorescence was observed in some of the cells. Moreover, clear differences in the extent of proteolytic processing of acyl-CoA oxidase were detected. The mutant cell line BOV84AD displayed a control-like pattern with all molecular forms of acyl-CoA oxidase (72, 52 and 20 kDa) present, whereas in the W78/515 cell line only the 72 kDa component could be visualised. The GM3605 cell line was intermediate in this respect. PMID- 1718438 TI - Glucocorticoids inhibit the induction of nitric oxide synthase and the related cell damage in adenocarcinoma cells. AB - Lipopolysaccharide (LPS) induced a time-dependent synthesis of nitric oxide (NO) in EMT6 adenocarcinoma cells, assayed by accumulation of NO-derived nitrite in the medium. The induction of NO synthesis was inhibited in a concentration dependent manner by the glucocorticoids dexamethasone (IC50 = 5 nM) and hydrocortisone (IC50 = 20 nM) and this effect was partially antagonized by progesterone and cortexolone. If addition of dexamethasone was delayed 6 h or more, inhibition of nitrite accumulation over 24 h was substantially reduced, indicating a lack of direct effect of glucocorticoids on the NO synthase. Nitrite accumulation was accompanied by cell damage, which was increased by L-arginine and inhibited by NG-monomethyl-L-arginine (L-NMMA) and dexamethasone. These data show that NO is a primary cytotoxic mediator and that suppression of its formation by glucocorticoids explains some of their anti-inflammatory and cytoprotective effects. PMID- 1718440 TI - Long-term plasma exchange in a case of Refsum's disease. AB - Refsum's disease (Heredopathia atactica polyneuritiformis) is caused by accumulation of phytanic acid in all body tissues due to an inherited failure of alpha-oxidation of branched chain fatty acids. Plasmapheresis has been reported to be beneficial by removal of phytanic acid from the blood. We describe a patient with Refsum's disease in whom long-term plasmapheresis did not improve clinical, biochemical or electrophysiological parameters. PMID- 1718441 TI - Electrophoresis and detection of nanogram quantities of exogenous and endogenous glycosaminoglycans in biological fluids. AB - Glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate types I (corneal) and II (cartilage) added to buffer, plasma and urine were enzymatically depolymerized. Enzymes, including chondroitin ABC lyase (chondroitinase ABC), heparin lyase (heparinase), heparan sulfate lyase (heparitinase), endo-beta-galactosidase and keratanase were used to depolymerize each GAG. Depolymerized GAGs and GAG mixtures were fractionated using gradient polyacrylamide gel electrophoresis. Staining with alcian blue dye resulted in a distinctive and well resolved banding pattern for each GAG. When these same gels were silver stained, an increase in detection sensitivity of 1000-fold was obtained. Picogram quantities of an oligosaccharide standard in buffer could be detected with silver staining while nanogram quantities could be detected in urine or plasma. The banding pattern observed for each depolymerized GAG was well resolved from contaminants found in these biological fluids and from intact GAGs. Endogenous GAGs present in samples of human urine and plasma were first concentrated and then enzymatically depolymerized. Chondroitin or dermatan sulfates, heparan sulfate and keratan sulfate were each detected in both concentrated plasma and urine samples. PMID- 1718442 TI - [Schistosoma mansoni and human immunodeficiency virus: new research avenues]. PMID- 1718443 TI - Flow cytometric nuclear DNA content of fresh and paraffin-embedded tissues of breast carcinomas and fibroadenomas. AB - Flow cytometric analysis of DNA ploidy level in fresh tissue samples of human breast tumors has been carried out extensively. Recently, investigations regarding the prognostic value of nuclear DNA content have been facilitated by using nuclei isolated from paraffin-embedded tissues. The aim of our study was to monitor the possible differences between the analysis in fresh and fixed paraffin embedded tissues in the same tumor and to investigate the possible prognostic implications obtained with this new approach. Nuclei suspensions were obtained, according to the method proposed by Hedley et al. with minor modifications, from 45 carcinomas and 5 fibroadenomas. Flow cytometric analysis revealed diploidy in 57% of carcinomas, while the remaining 43% showed cytometric aneuploidy. Corresponding results were observed between fresh and paraffin-embedded tissue in 26/35 cases. Moreover, a fairly good correlation between the DNA indices of fresh and paraffin-embedded carcinoma samples was observed. Furthermore, the frequency of recurrence was higher in the aneuploid group. Finally, 4 fibroadenomas were diploid and one was aneuploid. Our results confirm that this approach permits retrospective studies to evaluate the potential prognostic significance of nuclear DNA content monitored by flow cytometry. PMID- 1718444 TI - Preliminary findings on the nucleus of large neurons in Lophius piscatorius L. (Osteichthyes, Lophiiformes). AB - Some Teleosts belonging to the orders Batrachoidiformes, Lophiiformes, Perciformes and Tetraodontiformes show a cluster of large neurons dorsally located at the boundary between the spinal cord and medulla oblongata. These elements have traditionally been grouped with the supramedullary neurons aligned in the dorso-medial region of the spinal cord of other teleosts (see Marini and Benedetti, in press). However, recent morphological and immunohistochemical studies have suggested that the neurons grouped in a cluster on the spinal cord of Lophius piscatorius may not be of the same nature as the supramedullary neurons aligned within the dorsal gray matter of the spinal cord (Benedetti and Mola, 1988; Mola, 1990). Over the course of these previous investigations, it seemed that the nucleus of the neurons in the cluster showed more abundant chromatin than other neurons in the dorsal spinal cord. This prompted a series of investigations on the nucleus of these neurons. PMID- 1718445 TI - Specificity of commercially available Entamoeba histolytica antigen in enzyme linked immunosorbent assay (ELISA) to sera from dogs with various parasitic infections. PMID- 1718446 TI - Identification of Escherichia coli O2 antigen in chickens by avidin-biotin peroxidase complex and immunogold-silver methods. PMID- 1718447 TI - [A new method of isolation and a new form of transketolase from baker's yeast]. AB - A new procedure for the isolation of homogeneous transketolase from baker's yeast based on the use of enzyme-specific antibodies immobilized on a insoluble matrix has been developed. The enzyme yield is 90% of its total content in the original yeast extract. The eluate from the immunocolumn was found to contain a previously unknown form of transketolase which represents an enzyme-RNA complex. PMID- 1718448 TI - [Immunochemical study of tryptophanyl-tRNA-synthetase]. AB - The immunochemical approach used extensively in medicine and in some fields of biology has not yet been systematically applied to enzymology, in particular, to the study of a large, functionally significant group of enzymes, such as aminoacyl-tRNA synthetases (EC 6.1.1). The present investigation was aimed at the analysis of applicability of polyclonal and monoclonal antibodies against bovine tryptophanyl-tRNA synthetase in the study of functional properties of this enzyme as well as of its distribution inside the cell and among organs and tissues of various animals. The general conclusions one may draw from these data are as follows. i) Tryptophanyl-tRNA synthetase of eukaryotes, eubacteria and archaebacteria share one common structural element (antigenic determinant) that is not essential for the catalytic activity. The evolutionary conservative nature of this element suggests that the enzyme may implement functions other than catalysis of tryptophanyl-tRNA formation. ii) Tryptophanyl-tRNA synthetase shows an anomalous distribution among mammalian organs: its content is far greater in the exocrine part of the ruminant animal pancreas in comparison with their other organs (liver) or with other mammalian orders. This finding suggests that the enzyme or its fragments may play a role in the digestive function of ruminant animals. iii) Tryptophanyl-tRNA synthetase was found in considerable quantities in diffuse chromatin of mammalian cell nuclei. This fact indicates that the enzyme may participate in such processes in the nucleus as transcription, processing, transport, etc. It may thus be concluded that tryptophanyl-tRNA synthetase of higher organisms, besides catalyzing the formation of aminoacyl tRNAs can exert some other, yet unknown, noncanonical functions. PMID- 1718449 TI - [Dependence of Bacillus subtilis cell respiration on monovalent cations]. AB - Nigericin, monensin, valinomycin + carbonyl-cyanide-m-chlorophenylhydrazone and gramicidin inhibit the respiration of Bacillus subtilis cells incubated with NAD dependent substrates or succinate, but not with ascorbate + N,N,N',N'-tetramethyl p- phenylene-diamine. The level of inhibition was decreased by potassium ions and, in a lower degree, by sodium or ammonium ions. The results obtained suggest that the respiration of Bacillus subtilis depends on the presence of monovalent cations whose effects seem to be directed at complexes I, III and probably complex II of the respiratory chain. PMID- 1718450 TI - [Supermolecular organization of apolipoprotein E in solution]. AB - The aggregation behaviour and stability in solution of apolipoprotein E (apoE) isolated from human blood plasma very low density lipoproteins were investigated. The equilibrium denaturation of fluorescein-labeled apoE by guanidine hydrochloride determined by anisotropy and overall intensity of fluorescence, shift of the emission spectrum maximum and gel-chromatographic behaviour was characterized by reversibility, biphasity, apoE concentration dependence and the existence of native structure of the apoE monomer. The contribution of the long living component to the kinetic dependence of fluorescence anisotropy in the presence of the 6 M denaturant increased with an increase in apoE concentration. The data obtained fit into the following scheme: oligomer (upon aging of the preparation) in equilibrium tetramer in equilibrium native monomer in equilibrium denaturated monomer. The presence in the tetrameric structure of apoE of two domains is postulated; one of those is formed by lipid-binding fragments during aggregation of individual molecules of apoE. Monoclonal antibody 3D12F11 (subclass IgG1) showed a high affinity for the apoE (Kd = 3.5 +/- 0.5 nM) without any effect on the apoprotein binding to heparin-Sepharose and apoE-induced destruction of dipalmitoylphosphatidylcholine liposomes. It is concluded that the 3D12F11 epitope is localized outside heparin- and lipid-binding sites of the apoprotein molecule. PMID- 1718451 TI - [Expression of glial fibrillary acidic protein in the developing human brain]. AB - It was shown that the glial fibrillary acidic protein (GFAP) content in developing (fetal) human brain is sharply increased. The expression of GFAP was observed already on the 7th-8th week after gestation, the GFAP concentration being less than 0.05% in comparison with adult brain. GFAP can be immunohistochemically detected in radial glial cells. At early stages of development the presence of antigenic determinants of 68 kDa and 100 kDa polypeptides interacting with monoclonal antibodies alongside with native GFAP (51 kDa) and its low molecular weight forms was demonstrated. These antigenic determinants cannot be detected at later stages of development and are absent in adult brain. The data obtained testify to changes in the gene expression of intermediate filament proteins at early stages of human brain ontogenesis. PMID- 1718452 TI - [Assessment of the composition and structure of covalent complexes of superoxide dismutase with aldehyde dextran by analytical ultracentrifugation]. AB - Superoxide dismutase was covalently coupled wih aldehyde dextran, a polymeric carrier of molecular mass of 70 kDa. Modification produced an increase in the enzyme thermostability. Modified preparations retained a high specific activity. The composition of the thus obtained conjugates was analyzed by the ultracentrifugation and diffusion methods. The protein induced the destruction of aldehyde dextran, the enzyme being modified by its fragments. The presence of aldehyde dextran excess in the incubation medium promoted superoxide dismutase dissociation into individual subunits. At the enzyme/carrier ratio of 1:02 modification occurred as covalent coupling of the biocatalyst subunits and its one-point modification. PMID- 1718453 TI - Regulation of c-myc and ornithine decarboxylase expression by distinct protein kinase systems in IL-3-dependent myeloid cells. AB - We have investigated whether PK-C-regulated events are independent of those biochemical events related to IL-3-induced tyrosine kinase activation by 32Dcl cells. The depletion of functional PK-C isoform activity by prolonged PMA treatment reduced the proliferative response to IL-3 by half that of untreated control cells. PK-C-deficient 32Dcl cells were unable to respond to PMA for the induction of c-myc and ODC mRNA accumulation. PK-C down-regulation did not affect IL-3-induced tyrosine phosphorylation and inhibited IL-3-regulated c-myc and ODC mRNA expression by only 30%. However, PK-C down-regulation had a pronounced inhibitory effect on IL-3 regulation of ODC enzymatic activity. While a PK-C dependent and -independent pathway for the regulation of c-myc and ODC mRNA expression could be demonstrated, the regulation of ODC enzymatic activity appeared to require an intact PK-C system. The data suggest that the optimum biological and biochemical responses to IL-3 requires both pathways intact, however, tyrosine kinase activation and significant increases in gene products associated with proliferation can be achieved in the absence of a functional PK-C system. PMID- 1718454 TI - [The symbolic payment: desirable money]. AB - Because of the "free" therapy provided in the health and social services network, it is often difficult to make patients realize they ought to invest in their treatment. The authors first introduce the concepts supporting the idea of a "symbolic payment", designed to help solve some of these difficulties. Also, they recount anecdotes resulting from the application of this approach in clinics. In conclusion, the authors point to avenues that could lead to a broadening of the concepts underlying this clinical tool. PMID- 1718455 TI - [An etiological study of eosinophilic granuloma]. AB - By means of the proof for the enzyme phenol oxidase (PO; EC 1.14.18.1) was investigated, whether the eosinophilic granuloma correspond to a cell mediated immunological defence reaction. The method allowed to determine the eosinophils rapidly and surely and give an overview on the frequency and distribution of these cells. By electron microscopy was Langerhans-cells identified in close contact to many eosinophils. Their granula shows manifold changes in their structure. The eosinophilic granuloma is a cell mediated defence reaction approximate to the type of a delayed hypersensitivity. PMID- 1718456 TI - [Value of fibergastroscopy and biopsy in the diagnosis and developmental control of Whipple's disease]. PMID- 1718457 TI - The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis. AB - Auto-antibodies to the nicotine acetylcholine receptor (AChR) cause the disease myasthenia gravis (MG). Animals immunized with AChR or receiving anti-AChR antibodies acquire MG symptoms. The majority of the monoclonal antibodies (mAbs) raised in rats against intact AChR bind to a region on the extracellular side of the AChR's alpha-subunit, the main immunogenic region (MIR). The major loop of the overlapping epitopes for several anti-MIR mAbs has been localised between residues 67-76 of the alpha-subunit. Anti-MIR mAbs are very potent in accelerating AChR degradation (antigenic modulation) in muscle cell cultures and transferring experimental MG in animals. Fab fragments of single anti-MIR mAbs when bound to the AChR inhibit two-thirds of the MG patients' antibodies from binding and from inducing antigenic modulation of the AChR. This suggest that the majority of the human MG antibodies are also directed against the MIR. It has however to be verified by direct experiments. PMID- 1718458 TI - Analysis of the mechanism of graft-versus-host-like disease in B6 mice with transferred B6-lpr spleen cells. AB - Irradiated C57BL/6(B6) mice, when they were injected with spleen cells of C57BL/6J-lpr/lpr(B6-lpr) mice, developed splenomegaly at 2 weeks post-transfer, but afterward displaced by GVH-like disease. At 2 weeks the enlarged spleen in the chimeric mice, designated as [B6-lpr----B6] chimera, contained about 70% of the total cell population as CD8-positive T cells. Spleen cells from [B6-lpr--- B6] chimeras were unresponsive to Con A and LPS stimulation and suppressed the mitogenic response of B6, B6-lpr, and C3H spleen cells to Con A. However, they had no cytotoxic activity towards Con A blasts of B6 and B6-lpr spleen cells. The suppressor activity found in the [B6-lpr----B6] spleen cells was removed by pretreatment of them with anti-Thy-1.2 or anti-CD8(Lyt2.2) plus complement. The present experiment showed that enormous proliferation of CD8-positive suppressor T cells was induced in the [B6-lpr----B6] chimeras. These cells were probably responsible for the GVH-like lymphoid atrophy observed in these [B6-lpr----B6] chimeras. PMID- 1718459 TI - Self reactive repertoire of tight skin mouse: immunochemical and molecular characterization of anti-topoisomerase I autoantibodies. AB - Tight skin (TSK) mice develop cutaneous hyperplasia accompanied by histopathological alterations of skin and collagen metabolism similar to those described in human scleroderma. Diffuse scleroderma, the most severe form of progressive systemic sclerosis, is associated with the production of autoantibodies specific for Scleroderma 70 antigen (topoisomerase I). Our studies show that there is an increase in the level of serum anti-topoisomerase I (topo I) autoantibodies in aged TSK mice. The monoclonal antibodies isolated from TSK mice bind to epitopes which interact with autoantibodies from scleroderma patients. A significant number of TSK monoclonal anti-topo I antibodies and serum immunoglobulin (Ig) from aged TSK mice bear a cross reactive idiotype (Id) recognized by a syngeneic monoclonal anti-Id antibody obtained from a 2 month-old TSK mouse. Analysis of V gene usage by monoclonal anti-topo I antibodies showed that the majority of these antibodies are encoded by VH genes derived from VHJ558 family pairing with VK genes from various families in a stochastic manner. PMID- 1718460 TI - Idiotypic characterisation of monoclonal antibodies with restricted epitope specificity for retinal S-antigen. AB - This paper describes the idiotypic specificities of eight murine monoclonal antibodies directed to three independent epitopes on retinal S-antigen. The antigenic sites recognised by these monoclonal antibodies have previously been localised to a small region near the C-terminal of bovine S-antigen. Xenogeneic, site-related anti-idiotypes prepared against each of the monoclonal antibodies recognised common idiotypes only amongst those monoclonal antibodies which reacted with the same epitope on S-antigen. Two of the three idiotypes were detected in the sera of BALB/c mice but not in two strains of rat immunised with xenogeneic S-antigen and none could be detected in the sera of patients with anti photoreceptor autoantibodies. Our results demonstrate that the idiotypes of murine monoclonal antibodies to retinal S-antigen exhibit restricted epitope specificity but are species-restricted and imply that the S-antigen lacks a dominant antigenic epitope. PMID- 1718461 TI - Translocation of the nuclear autoantigen La to cell surface: assembly and disassembly with the extracellular matrix. AB - La (SS-B) protein is known as one major antigenic target for autoantibodies from patients with certain autoimmune diseases such as Sjogren's syndrome or Lupus Erythematosus. La protein belongs to the so called "extractable nuclear antigens". Here we report that La antigen is not restricted to the nucleus as one might deduce from the exclusive nuclear staining pattern of patient anti-La antibodies but after stimulation of serum-starved cells with 10% fetal calf serum (FCS) appears and stays for at least 45 min at the outer surface of CV-1 cells being available for binding of anti-La antibodies. In addition we found that a minor part of La antigen associates with the extracellular fibronectin network. After addition of 10% FCS to serum starved cells this extracellular autoantigen disassembled from the extracellular matrix and was taken up again by the cells. Incubation of serum starved cells with mercuric chloride, a known potent inducer of autoantibodies, also resulted in a detachment of the extracellular matrix associated La protein. From our studies it becomes likely that La protein itself is the antigen during autoimmunization. Moreover, once developed, anti-La antibodies might be able to bind to cell surface expressed La protein resulting in a damage of these cells leading to the inflammational events known to occur during disease. PMID- 1718462 TI - Protection against viral infections with the aid of synthetic peptides. Foot-and mouth disease as an example. AB - The problems involved in the use of synthetic peptides as antiviral vaccines are reviewed. The possibilities of protection against virus-induced disease are demonstrated in relation to foot-and-mouth disease. PMID- 1718463 TI - Latex-agglutination analysis of human recombinant interleukin-2 with monoclonal antibodies. AB - Hybridomas producing monoclonal antibodies (Mabs) to human interleukin-2 have been obtained. The antibodies have been characterised in terms of binding constant, subclass, and cross-reaction with proteins from Escherichia coli and human blood serum. The epitope for the monoclonal antibody LNKB-2, namely the 66 72 fragment of the interleukin-2 molecule, has been located. From the antibodies obtained, two capable of reacting simultaneously with the monomeric form of interleukin-2 have been selected and a rapid simple Mab-based latex-agglutination assay which can detect as little as 1.5 ng ml-1 interleukin-2 has been developed. PMID- 1718464 TI - Oligopeptides in the regulation of feeding and avoidance behaviour in the land snail (Helix lucorum). AB - The effects of the oligopeptides pentagastrin, cholecystokinin octapeptide, substance P, and FMRF-amide on the feeding and avoidance behaviour of the land snail Helix lucorum were studied. Administration of 1 microM pentagastrin to fasted snails immediately before feeding led to a significant increase in the amount of food consumed in the first 20 min of the feeding period, although there was no change in the total amount of food consumed. Prefeeding injections of 0.05 microM pentagastrin or 0.05 microM or 1 microM solutions of cholecystokinin octapeptide, substance P, or FMRF-amide had no effect on feeding behaviour. Pentagastrin and cholecystokinin octapeptide (0.05 microM solutions) invoked a significant reversible decrease in the generalised avoidance reaction thresholds in fasted but not in prefed snails, whereas the same concentration of FMRF-amide reduced these thresholds only in prefed snails. PMID- 1718465 TI - The use of aprotinin in thrombocytopenic patients: a preliminary evaluation. AB - Previous studies have shown that high dose aprotinin successfully reduces blood loss in patients undergoing cardiac or vascular surgery, but the use of this approach to reduce bleeding associated with thrombocytopenia has not been studied. We report the results of high dose aprotinin treatment in five patients with thrombocytopenia of differing aetiology. Aprotinin was effective in controlling bleeding in all five patients, some of whom would otherwise have had a poor prognosis. These results suggest that this agent may have a role in the supportive treatment of thrombocytopenia and point to the need for controlled trials of high dose aprotinin treatment in such individuals. PMID- 1718466 TI - Heparin cofactor II: an acute phase reactant in patients with deep vein thrombosis. AB - In human plasma, heparin cofactor II (HCII) is a thrombin inhibitor which displays similarities with antithrombin III (ATIII). As previously reported for hereditary ATIII deficiency, cases of recurrent thrombosis were reported in patients with hereditary HCII deficiency. Here, plasma HCII activity was studied in 372 patients with a history of thrombosis, classified according to their anticoagulant therapy. The mean plasma HCII level was significantly higher in patients with acute deep vein thrombosis (DVT) under heparin therapy than in patients with a history of thrombosis, who were studied more than 3 months after the acute event, and were either on, or had been on, oral anticoagulant therapy. HCII and fibrinogen were significantly correlated in all three groups of patients. These results were strengthened by those of a follow-up study in 23 patients with acute DVT. Changes in plasma HCII activity paralleled those of fibrinogen. This suggests that HCII might behave like an acute phase reactant in patients with thrombosis and that the measurement of its plasma level as a risk factor for thrombosis should be performed some time after the acute episode. In conclusion, the prevalence of HCII deficiency in patients with a history of thrombosis might have been underestimated in series which included patients with acute thrombosis. PMID- 1718467 TI - Open channel noise. VI. Analysis of amplitude histograms to determine rapid kinetic parameters. AB - Recently we reported that rapid fluctuations of ion currents flowing through open gramicidin A channels exceed the expected level of pure transport noise at low ion concentrations (Heinemann, S. H. and F. J. Sigworth. 1990. Biophys. J. 57:499 514). Based on comparisons with kinetic ion transport models we concluded that this excess noise is likely caused by current interruptions lasting approximately 1 microsecond. Here we introduce a method using the higher-order cumulants of the amplitude distribution to estimate the kinetics of channel closing events far below the actual time resolution of the recording system. Using this method on data recorded with 10 kHz bandwidth, estimates for gap time constants on the order of 1 microsecond were obtained, similar to the earlier predictions. PMID- 1718468 TI - Single-channel dose-response studies in single, cell-attached patches. AB - A method for carrying out dose-response studies of ion channel currents in cell attached patches has been devised. Patch pipettes are filled at the tip with a solution containing one concentration of ligand and then backfilled with another. The concentration of ligand at the membrane is described as a function of time by the equation for diffusion in a cone, allowing response vs. time data to be transformed into a dose-response curve. For Xenopus myocyte cholinergic receptors, examples of the use of this method are given for several concentration dependent reactions including blockade by the local anesthetic QX-222, activation by acetylcholine, and modulation of current amplitude by sodium ions. Several methods of analyzing the nonstationary channel kinetics are presented, including a pseudo-stationary approach that uses interval likelihood maximization. PMID- 1718469 TI - Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor. AB - The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine. PMID- 1718471 TI - Fluorescence, CD, and NMR studies on spantide, a bombesin and substance P antagonist. AB - The conformational properties of spantide [(D-Arg, D-Trp, Leu) substance P] have been studied by fluorescence, CD, and nmr techniques. The fluorescence, CD, and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for bombesin. A preliminary investigation on [D Pro] spantide is also reported. PMID- 1718470 TI - Synthesis and mutagenicity of trans-dihydrodiol metabolites of benzo[b]naphtho[2,1-d]thiophene. AB - The syntheses of potentially important metabolites of benzo[b]naphtho[2,1 d]thiophene ([2,1]BNT)--trans-1,2-dihydroxy-1,2-dihydrobenzo[b]naphtho[2,1- d]thiophene ([2,1]BNT-1,2-diol) and trans-3,4-dihydroxy-3,4 dihydrobenzo[b]naphtho[2,1-d]thiophene ([2,1]BNT-3,4-diol)--are described. The syntheses involved preparation of the appropriate 1-(3-benzo[b]-thiopheneyl)-2 (methoxyphenyl)ethylenes followed by photocyclization to methoxy-[2,1]BNTs, hydrolysis to hydroxy-[2,1]BNTs, oxidation to [2,1]BNT-diones, and NaBH4 reduction. The dihydrodiols were tested for mutagenicity in Salmonella typhimurium TA 100 with activation; [2,1]BNT-3,4-diol, which can form a bay region diol epoxide, was as mutagenic as [2,1]BNT whereas [2,1]BNT-1,2-diol was inactive. These results suggest that the metabolic activation of [2,1]BNT proceeds partially via formation of a bay region diol epoxide. PMID- 1718472 TI - Toward nonpeptidal substance P mimetic analogues: design, synthesis, and biological activity. AB - 1,4-Piperazine and 4-hydroxyproline, two small cyclic polyfunctional systems with defined stereochemistry, were introduced as "molecular scaffolds." We define a "bioactive topology," which is a derived putative low-energy conformation obtained through theoretical conformational analysis of substance P. Substitution of these molecular scaffolds by pharmacophors characteristic of the bioactive topology of the C-terminal hexapeptide of substance P resulted in active, partially nonpeptidal substance P mimetic agonists. The study discusses the concepts and tools used to achieve this structural transformation, and points out the need to address flexibility-rigidity issues in an attempt to maintain sufficient molecular plasticity. PMID- 1718473 TI - Backbone cyclization: A new method for conferring conformational constraint on peptides. AB - This article describes a new concept of medium- and long-range cyclization of peptides through "backbone cyclization." In this approach, conformational constraints are conferred on a peptide by linking omega-substituted alkylidene chains replacing N(alpha) or C(alpha) hydrogens in a peptidic backbone. Backbone cyclization, which is divided into N-backbone and C-backbone cyclizations, allow for new modes of cyclization in addition to the classical ones that are limited to cyclization through the side chains and/or the amino or carboxyl terminal groups. The article also describes the application of the N-backbone cyclization to the active region of substance P. Conformational constraints of this peptide by the classical cyclization modes led to inactive analogues whereas N-backbone cyclization provided an active, selective, and metabolically stable analogue. PMID- 1718474 TI - Pharmacology of neurokinin receptors. AB - The neurokinins are a group of naturally occurring peptides with the common C terminal sequence Phe-X-Gly-Leu-Met.NH2. They include substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). SP and NKA are coded on the same gene, the PPT-A, while NKB is coded on a separate gene, the PPT-B. Neurokinins are present in the central nervous system and in peripheral organs where they exert various actions. They act on three receptors--NK-1, NK-2, and NK-3- characterized through pharmacological, biochemical, and histochemical studies. Selective agonists for each neurokinin receptor were developed and evaluated on isolated smooth muscle preparations containing only one neurokinin receptor type. All three neurokinin receptors were cloned and expressed in Xenopus oocytes. Relative affinities of those receptors to neurokinins are the same as in their respective smooth muscle preparation. Finally, the mechanism of action of SP on histamine release from rat peritoneal mast cell has been studied and a direct activation of G proteins by peptides with basic amino acids is proposed as a working hypothesis. PMID- 1718475 TI - The cytological diagnosis of Pneumocystis carinii by fluorescence microscopy of Papanicolaou stained bronchoalveolar lavage specimens. AB - In a retrospective and prospective analysis fluorescence microscopy of Papanicolaou stained bronchoalveolar lavage specimens has been applied to the diagnosis of Pneumocystis carinii (PC) in routine cytology. The pneumocysts presented as circular structures of 5 microns in diameter and of brilliant green yellow fluorescence surrounding two mirror image reniform structures. Fluorescent inclusions of 1-3 microns diameter within the alveolar macrophages could be identified as remnants of pneumocysts by a follow-up of all steps of degradation ending in very small irregular granules. By applying both criteria, i.e. pneumocysts with reniform bodies and degradation inclusions within macrophages, Pneumocystis carinii pneumonitis (PCP) could be detected in 100% of cases. Transbronchial biopsy permitted the correct diagnosis in only 65.2% of cases. Retrospective analysis of slides is possible after a long period as no significant loss of fluorescence occurs after 4 years. Thus fluorescence microscopy permits the diagnosis of Pneumocystis carinii without any additional staining or loss of time. PMID- 1718476 TI - The effects of aprotinin on hemostatic function during cardiac surgery. AB - The mechanism of action by which large doses of aprotinin decrease blood loss during cardiac surgery is not completely understood. In a prospective, controlled study, 30 patients undergoing cardiac surgery were given high-dose aprotinin in accordance with a commonly used regimen. Twenty untreated but otherwise comparable patients served as the control group. The effects of aprotinin therapy during cardiopulmonary bypass on coagulation parameters, the kallikrein-kinin system, fibrinolysis, platelet stimulation, and the release of elastase from neutrophils were studied. The fibrinolysis parameters were the only measurements that showed clear and significant differences between the two groups. Aprotinin almost completely inhibited the formation of fibrin and fibrinogen degradation products. It is assumed that inhibition of systemic fibrinolysis and suppression of local fibrinolysis contribute to the hemostatic action of aprotinin. The study did not demonstrate a significant protective effect of aprotinin on platelets. In addition, the dose of aprotinin administered did not affect the kallikrein-kinin system of elastase. Therefore, these data suggest that the previously demonstrated hemostatic effects of aprotinin derive primarily from its antifibrinolytic action. PMID- 1718477 TI - Narcotic-induced histamine release: a comparison of morphine, oxymorphone, and fentanyl infusions. AB - This study, using an improved histamine assay, repeated previous studies that demonstrated large doses of morphine for induction of anesthesia in patients undergoing coronary artery bypass grafting were associated with histamine release. Thirty randomized patients received infusions of either morphine, 1 mg/kg, oxymorphone, 0.2 mg/kg, or fentanyl, 50 micrograms/kg, over a 10-minute period for induction of anesthesia prior to surgery. There were no significant changes in plasma histamine levels in individual patients or among drug groups. The discrepancy between the present histamine results and those previously reported using similar protocols is due, in part, to variations in plasma histamine measurements that can occur using the less reproducible, older assays for histamine. During routine inductions, large doses of morphine, oxymorphone, or fentanyl administered by infusion do not appear to stimulate release of clinically significant plasma levels of histamine. PMID- 1718479 TI - Circadian oscillations of nuclear-encoded chloroplast proteins in pea (Pisum sativum). AB - The diurnal and circadian expression of light-inducible chloroplast proteins, i.e. light-harvesting chlorophyll a/b protein (LHCP), early light-inducible protein (ELIP) and Fe-S Rieske, has been studied in young pea plantlets at the level of mRNA integrated into polysomal complexes and at the level of proteins. Under light-dark as well as constant light conditions the levels of the three nuclear-encoded chloroplast proteins oscillate while the investigated plastid encoded proteins, large subunit of ribulose-1,5-bisphosphate carboxylase (LSU), reaction center protein D1 and cytochrome f, do not show oscillations at the protein level. The levels of the nuclear-encoded polysome-bound mRNAs fluctuate in parallel with the changes in the levels of poly(A) RNA which were described previously. Under constant light conditions the oscillation at the level of polysomal bound mRNA is readily dampened while the steady-state levels of the investigated nuclear-encoded proteins still fluctuate. We conclude that the extent of expression of the genes for the nuclear-encoded chloroplast proteins studied is controlled by a circadian ocillator primarily, but not exclusively, at the level of transcription. PMID- 1718478 TI - A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP specific glutamate dehydrogenase(s) in Chlorella sorokiniana. AB - Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit. PMID- 1718480 TI - The protein-encoding gene T-urf13 is not edited in maize mitochondria. AB - RNA editing of T-urf13, a gene specific to the mitochondria of cytoplasmic male sterile, type-T (cms-T) maize, and an adjacent, cotranscribed gene orf221, have been studied by cDNA sequencing. No editing was detected in 22 cDNA clones. This is the only report of a polypeptide-encoding gene in higher-plant mitochondria that is not edited. T-urf13 may not be edited because it is derived largely from the coding and flanking regions, which are rarely edited, of a ribosomal RNA gene. orf221 is edited; however, the similarity between the predicted amino acid sequences of orf221 in cms-T and normal cytoplasms is not increased. PMID- 1718481 TI - Survey of plastid RNA abundance during tomato fruit ripening: the amounts of RNA from the ORF 2280 region increase in chromoplasts. AB - A comprehensive survey of the levels of plastid RNAs at progressive stages of tomato fruit ripening was conducted by hybridizing total RNA with labeled Pst I fragments that cover almost the entire tomato plastid genome and with gene specific probes. Two different cultivars of tomato (Lycopersicon esculentum Mill.) were examined, Traveler 76 and Count II. One of the tomato probes, P7, revealed a pronounced increase in the amount of an 8.3 kb RNA in ripe fruit. The homologous region of the tobacco plastid genome contains several genes for ribosomal proteins and a large unidentified open reading frame (2280 codons). Little change was observed in the levels of many transcripts during ripening. However, in some cases (e.g. psbA and psbC/D) the amount of RNA decreased during ripening of Count II but showed little or no change in Traveler 76. The contrast between Traveler 76 and Count II tomatoes shows that the level of plastid transcripts can vary substantially during fruit ripening with no obvious effect on the chloroplast to chromoplast transition. The large RNA from the P7 region may encode a protein that functions predominantly in chromoplasts. PMID- 1718482 TI - The potential use of T cell epitopes to alter the immune response. AB - Recent advances in the understanding of T cell specificity and activation have lead to the design of T cell specific immunomodulators. T cell epitope containing peptides have been proposed as agents which may either enhance or dampen the immune response. In this review, we examine two systems which can benefit from the application of this novel technology. Vaccine development is moving toward the use of defined cloned or synthetic molecules. T cell epitope identification and design can be used to augment the ability of a weak antigen to generate an immune response. In contrast, traditional allergy immunotherapy has been shown to alter the immune response to the allergenic antigen. T cell epitope approaches to allergy desensitization offer a new therapeutic modality. PMID- 1718483 TI - T cell inducing determinants contain a hierarchy of residues contacting the T cell receptor. AB - T cells through their antigen specific T cell receptor recognize a bi-molecular ligand composed of an antigenic peptide bound to a MHC molecule. Several T cell inducing determinants have been extensively characterized by single amino acid substitutions. In this review, we have summarized our characterization of four immunodominant determinants. Each of these determinants possessed a single amino acid residue which was absolutely critical for the recognition by T cells. From these data we propose a hypothesis that there is a hierarchy in the T cell contact residues of a determinant, composed of a single primary residue, and a few secondary residues. PMID- 1718484 TI - Trimolecular interactions in experimental autoimmune demyelinating disease and prospects for immunotherapy. AB - Experimental allergic encephalomyelitis (EAE) is a T cell mediated model of demyelinating disease that develops as a result of an autoimmune response to the myelin structural antigen, myelin basic protein (MBP). Much information has accumulated on the nature of trimolecular interactions which lead to the activation of self-reactive T lymphocytes in this disorder. Many parallels exist in the etiopathogenesis of EAE and multiple sclerosis (MS). Based upon these similarities selective immunotherapy that targets either class II molecules of the major histocompatibility complex or T cell receptor variable region genes will be described for EAE with consideration given to application of these principles in MS. PMID- 1718485 TI - Competitive inhibition of antigen presentation in animal models of autoimmune disease. AB - Competition between peptides for binding to class II major histocompatibility complex (MHC) molecules has been demonstrated both in vitro and in vivo. Peptide competition may provide a way to interfere with T cell activation in the treatment of autoimmune diseases. It may be possible to provide a substitute 'blocking' peptide to compete for presentation of an autoantigenic peptide to T cells. The approach described is a general one, which may be applicable to a number of T cell mediated MHC-linked autoimmune diseases, and to other undesirable immune responses. So far, peptide competitors have only been successfully used in the prevention of experimental autoimmune encephalomyelitis (EAE). Whether or not this approach will work in treating spontaneous disease models remains to be tested, although work in other test systems is encouraging. PMID- 1718486 TI - [Thymus hyperplasia after chemotherapy in a case of Hodgkin's disease]. PMID- 1718487 TI - Diamond-blackfan anemia: in vitro response of erythroid progenitors to the ligand for c-kit. AB - To provide insights into the pathogenesis of Diamond-Blackfan anemia, we examined the in vitro response of erythroid progenitors to the recently isolated ligand for c-kit (stem cell factor, SCF). For these studies, marrow or blood mononuclear cells from 10 Diamond-Blackfan patients were cultured with erythropoietin (Ep), Ep and interleukin-3, Ep and granulocyte-macrophage colony-stimulating factor, or Ep and lymphocyte conditioned media (LCM). These combinations were tested in the presence or absence of SCF. The mean number of cells per erythroid burst increased 5 to 50-fold in cultures containing SCF. Furthermore, many additional erythroid bursts were seen (mean increment 3.2 x baseline values). Although burst forming unit-erythroid (BFU-E) from all patients responded, there were differences among individuals in the sensitivity of their BFU-E to SCF. In six patients and all control studies, plateau frequencies of erythroid bursts were achieved with less than or equal to 10 ng/mL SCF, whereas in studies from the other four patients, over 50 ng/mL SCF was required. These data invite speculation that the c-kit receptor/ligand axis is involved in the pathogenesis of Diamond-Blackfan anemia. More importantly and regardless of whether the observed patterns of response reflect the primary defect or an epiphenomenon, our data strongly support a therapeutic trial of SCF in patients with Diamond Blackfan anemia. PMID- 1718488 TI - In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia. AB - Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL 6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte-conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation. PMID- 1718489 TI - Diamond-Blackfan anemia: heterogenous response of hematopoietic progenitor cells in vitro to the protein product of the steel locus. AB - Diamond-Blackfan anemia is a congenital disorder of erythropoiesis in humans, characterized by a macrocytic anemia often associated with physical anomalies. Mutations at either the W or Steel loci in the mouse also leads to a severe macrocytic anemia, as well as other developmental abnormalities. The W locus encodes the proto-oncogene c-kit, a member of the receptor tyrosine kinase family, while the Steel locus encodes a potent hematopoietic growth factor that is the ligand for c-kit. Growth of clonogenic marrow erythroid progenitor cells in vitro in the presence of the recombinant hematopoietic growth factors interleukin-3 (IL-3) and Steel was used to characterize this disease at the cellular level. Three patterns of in vitro marrow response to both recombinant IL 3 or Steel were observed among 10 Diamond-Blackfan patients: those that responded quantitatively and qualitatively almost as well as cells from normal marrow, those that responded at an intermediate level, and those that did not respond at all. These results provide evidence for cellular heterogeneity underlying the pathogenesis of this disorder and therefore raise the possibility that there may be more than one underlying molecular basis for the disease. No gross abnormalities in the structure of either the c-kit or Steel loci were observed in these patients. The normal response in culture of the progenitor cells from at least some patients to Steel with or without IL-3 raises the possibility of using this novel growth factor as a therapeutic agent in Diamond-Blackfan anemia. PMID- 1718490 TI - Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential. AB - The replating capability of human multipotential (colony-forming unit-granulocyte erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was assessed in vitro as a potential measure of self-renewal using purified, recombinant (r) human (hu) or murine (mu) mast cell growth factor (MGF), a ligand for the c-kit proto-oncogene receptor. Primary cultures of human umbilical cord blood or adult human bone marrow cells were initiated in methylcellulose with erythropoietin (Epo) alone or in combination with rhu interleukin-3 (IL-3) or MGF. Individual day 14 to 18 CFU-GEMM or BFU-E colonies were removed from primary cultures and reseeded into secondary methylcellulose cultures containing a combination of Epo, MGF, and rhu granulocyte-macrophage colony-stimulating factor (GM-CSF). The data showed a high replating efficiency of cord blood and bone marrow CFU-GEMM in response to Epo + MGF in terms of the percentage of colonies that could be replated and the number of secondary colonies formed per replated primary colony. The average number of hematopoietic colonies and clusters apparent from replated cultures of cord blood or bone marrow CFU-GEMM stimulated by Epo + MGF was greater than with Epo + rhuIL 3 or Epo alone. Replated cord blood CFU-GEMM gave rise to CFU-GEMM, BFU-E, and GM colony-forming units (CFU-GM) in secondary cultures. Replated bone marrow CFU GEMM gave rise mainly to CFU-GM in secondary cultures. A more limited capacity for replating of cord blood and bone marrow BFU-E was observed. These studies show that CFU-GEMM responding to MGF have an enhanced replating potential, which may be promoted by MGF. These studies also support the concept that MGF acts on more primitive progenitors than IL-3. PMID- 1718491 TI - Interleukin-7 induces the proliferation of normal human B-cell precursors. AB - In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL 7 acts as a growth factor during the ontogeny of human B lymphocytes. PMID- 1718492 TI - Bidirectional effects of transforming growth factor beta (TGF-beta) on colony stimulating factor-induced human myelopoiesis in vitro: differential effects of distinct TGF-beta isoforms. AB - Transforming growth factor-beta (TGF-beta) has potent antiproliferative effects on human hematopoietic progenitor cells. We report here that TGF-beta 1 and -beta 2 also exert bimodal dose-dependent stimulation of granulocyte-macrophage colony stimulating factor (CSF) and granulocyte-CSF-induced day 7 granulocyte-macrophage colony-forming units. This increase in colony formation was restricted to low doses (0.01 to 1.0 ng/mL) of TGF-beta 1 and was due to increased granulopoiesis, showing that TGF-beta can affect the differentiation as well as the proliferation of hematopoietic progenitors. Furthermore, TGF-beta 3 was found to be a more potent inhibitor of hematopoietic progenitor cells than TGF-beta 1 and -beta 2. In contrast to the bidirectional proliferative effects of TGF-beta 1 and -beta 2, the effects of TGF-beta 3 on human hematopoiesis were only inhibitory, showing for the first time that TGF-beta isoforms differ not only in potencies but also with regard to the nature of the response they elicit. PMID- 1718493 TI - Alpha 2-macroglobulin binds and inhibits activated protein C. AB - In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine-Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo. PMID- 1718494 TI - Alpha-2-macroglobulin may provide protection from thromboembolic events in antithrombin III-deficient children. AB - Antithrombin III (ATIII) deficiency has been implicated in adults as a predisposing factor to thrombosis; however, thromboembolic complications are rare in children with the same deficiency. We hypothesized that because of the elevated levels of plasma alpha-2-macroglobulin (alpha 2M) throughout childhood, plasmas of ATIII-deficient children inhibit thrombin more efficiently than those of ATIII-deficient adults. In total, 14 ATIII-deficient adults (ages 25 to 46 years), 13 ATIII-deficient children (ages 2 to 13 years), 9 normal children (ages 3 to 15 years), and 16 normal adults were studied. We measured thrombin inhibition in these plasmas, as well as the contributions of ATIII, alpha 2M, and heparin cofactor II (HCII) as thrombin inhibitors in each plasma. 125I-alpha thrombin, 25 nmol/L, was added to each plasma (defibrinated with Arvin at 37 degrees C), and 90 seconds later the free thrombin and thrombin-inhibitor complexes were quantitated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric scanning. Plasma from ATIII deficient adults inhibited significantly less thrombin (12.8 +/- 0.6 nmol/L) than both normal adults (16.1 +/- 0.3 nmol/L, P less than .01), normal children (15.7 +/- 0.4 nmol/L, P less than .01), or ATIII-deficient children (15.5 +/- 0.3 nmol/L, P less than .01). There was no significant difference between the total concentration of thrombin inhibited by ATIII-deficient children and either normal adult or normal children groups. In addition, plasmas of ATIII-deficient children inhibited thrombin significantly more efficiently than plasma of ATIII-deficient adults (P less than .01). In the ATIII-deficient patients there was a significant correlation between the alpha 2M level and ability to inhibit thrombin (P less than .01), but no correlation between either ATIII or HCII levels and thrombin inhibition. On the addition of heparin (0.4 U/mL) to plasma, all four types of plasma inhibited thrombin to the same extent. Although ATIII was the predominant inhibitor in all heparinized plasmas, HCII inhibited more thrombin in the ATIII deficient patients than in normal patients (2.8 +/- 0.3 v 1.2 +/- 0.2 nmol/L, P less than .01). We hypothesize that the lower risk of thromboembolic complications in ATIII-deficient children may be due in part to the protective effect of elevated alpha 2M levels during childhood. PMID- 1718495 TI - Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s. AB - MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems. PMID- 1718496 TI - Natural inhibitors of T-cell activation in Hodgkin's disease. AB - Secondary immunodeficiency is frequently observed in Hodgkin's disease (HD) and is due in part to impaired T-cell function. Using monoclonal antibodies that bind to triggering molecules of human T lymphocytes (CD3/Ti antigen receptor; CD2 E rosette receptor) and exert functional effects on T-cell activation, we have investigated in vitro immune responses of circulating lymphocytes from patients with HD in progression (n = 9) and in remission (n = 14). In patients with progressive HD, a severe dysfunction of the alternative CD2-mediated T-cell activation pathway was detected (49.3 +/- 14.2 v 9.4 +/- 5.1 cpm x 10(-3), in controls, P less than .01; n = 9) that parallels the reduced capacity of T lymphocytes to form rosettes with sheep red blood cells. Diminished alternative pathway activation in HD is not only due to a defect at the cellular level but also due to soluble mediators in the patients' plasma. Plasma from patients in progression markedly reduces CD2 mediated activation (P less than .01). These activities interfere, at least in part, with CD2/CD58 interactions and, therefore, reduce T-lymphocyte triggering through this amplifier mechanism. PMID- 1718497 TI - Transcriptional regulation of two myeloid-specific genes, myeloperoxidase and lactoferrin, during differentiation of the murine cell line 32D C13. AB - The transcriptional regulation of myeloperoxidase (MPO) and lactoferrin (LF) was examined during terminal myeloid differentiation of the murine cell line 32D C13. The rates of transcription initiation for MPO and LF, determined by an in vitro nuclear run-on assay, increased approximately ninefold. The accumulation of MPO mRNA in 32D C13 cells, determined by Northern blot analysis, correlated temporally with the observed increase in MPO transcription initiation. On the other hand, accumulation of LF mRNA lagged behind the observed increase in LF transcription initiation. In mouse L cells, the LF gene was transcribed more frequently than the MPO gene, though neither mRNA accumulated. Finally, murine MPO transcription is shown, by Northern blot and primer extension analysis, to initiate at multiple sites. These results indicate that whereas transcription induction may largely account for the accumulation of MPO mRNA during terminal myeloid differentiation, both transcriptional and posttranscriptional mechanisms operate to allow accumulation of LF mRNA. The 32D C13 cell system will be a useful model for elucidating these mechanisms. PMID- 1718498 TI - Synthetic peptides homologous to human glycophorins of the Miltenberger complex of variants of MNSs blood group system specify the epitopes for Hil, SJL, Hop, and Mur antisera. AB - The antigenic epitopes of the MNSs blood groups are localized on alpha and delta glycophorins (glycophorins A and B) of the erythrocyte surface. Hil, SJL, Mur, and Hop antisera define the Miltenberger (Mi) complex of MiV, MiJ.L., MiIII, and MiVI variant serologic phenotypes of this blood group system. We report here the location of the epitopes for antibodies in these antisera. The antigens of these Mi classes are variant glycophorins that are hybrids of alpha and delta glycophorins in alpha-delta and delta-alpha-delta arrangements. The hybrid junctions give rise to novel polypeptide sequences not present in the parent glycophorins; in MiIII and MiVI this also includes an expressed sequence of the delta pseudoexon. These sequences are identical in the above Mi-glycophorins occurring in erythrocytes that share a common Mi determinant. Four peptides of 10 to 14 amino acids each were constructed to be homologous to the identical sequences; they were designated, "Hil", "SJL", "Mur", and "Hop" to reflect the common determinant. The peptides were tested for inhibition of reaction of appropriate cells with the relevant antisera. The Hil peptide, outlining the alpha-delta s junction region in MiIII, MiV, and MiVI glycophorins, inhibited the reaction of respective erythrocytes (red blood cells [RBCs]) with anti-Hil. The SJL peptide, which differs from the Hil peptide by a single Thr----Met substitution, was specific for inhibition of the reaction of MiJ.L. RBCs with anti-SJL (an example of anti-S specific for such RBCs). The Hop peptide, which corresponds to the delta-alpha junction in MiVI glycophorin, inhibited the hemagglutination of MiVIII RBCs by anti-Hop. MiVI and MiVIII glycophorins share an identical sequence at that site. The Mur peptide, corresponding to a portion of the expressed pseudoexon sequence in MiIII and MiVI glycophorins, was specific for inhibition of the reaction of MiIII and MiVI RBCs with anti-Mur. The peptides had no effect on the hemagglutination of control MNSs RBCs by their respective antisera nor of unrelated Mi classes RBCs by antisera that distinguish these classes. We conclude that the alpha-delta junction in MiIII, MiV, and MiVI glycophorins outlines the epitopes for anti-Hil, the alpha-delta junction in MiJ.L. outlines the epitope for anti-SJL, the delta-alpha junction in MiVI constitutes the epitope for anti-Hop, and the expressed delta pseudoexon sequence in MiIII and MiVI constitutes the epitope for anti-Mur. PMID- 1718499 TI - Reduction of postischemic reperfusion injury by the vasoactive drug buflomedil. AB - The effect of buflomedil on postischemic reperfusion injury was studied in the dorsal skin fold chamber preparation of awake hamsters. Microvascular events were investigated in the striated skin muscle by means of intravital fluorescence microscopy prior to 4 h of pressure-induced ischemia and 30 min, 2 and 24 h after reperfusion. In untreated control animals, ischemia and reperfusion provoked marked leukocyte sticking and macromolecular leakage while functional capillary density was reduced. Treatment with buflomedil (3 mg/kg b.w. in 0.3 ml saline, administered as bolus of 0.1 ml 10 min prior to release of ischemia followed by i.v. infusion of 0.2 ml during the first 20 min of reperfusion) significantly reduced leukocyte sticking and macromolecular leakage, while functional capillary density was effectively preserved. No differences in macro- and microhemodynamic parameters were observed between buflomedil-treated and untreated animals. These findings support the concept that activated leukocytes are involved in the microvascular manifestation of reperfusion injury and indicate that leukocyte sticking and its sequelae can be efficiently prevented by treatment with buflomedil. PMID- 1718500 TI - Bone mass in disorders of calcium metabolism and abnormal parathyroid function. PMID- 1718501 TI - Methodology for studies of antral motility. PMID- 1718502 TI - Oesophageal function tests. PMID- 1718503 TI - The determination of gastric emptying rate. PMID- 1718504 TI - Methods for studying motility in the biliary tract. PMID- 1718505 TI - Methodology for motility studies on the small intestine: a Scandinavian consensus. PMID- 1718506 TI - Methods for the investigation of colonic motility. PMID- 1718507 TI - Ano-rectal physiology measurements. PMID- 1718508 TI - High-tech comfort: ethical issues in cancer pain management for the 1990s. PMID- 1718509 TI - Replication of a plant virus satellite RNA: evidence favors transcription of circular templates of both polarities. PMID- 1718510 TI - Symptom-modulating properties of peanut stunt virus satellite RNA sequence variants. AB - The symptom-modulating properties of three peanut stunt virus (PSV) satellite RNA (satRNA) sequence variants were studied. The (V)-satRNA did not affect symptom development in tobacco plants infected with PSV. The (G)- or (WC)-satRNA, on the other hand, attenuated the symptoms. In these plants, the symptoms of PSV were restricted primarily to the inoculated leaves, and in some cases, a few leaves above the inoculated leaf showed small chlorotic areas. Northern blot analysis of total nucleic acids from PSV-infected plants containing the (V)-satRNA revealed the presence of both satellite and viral RNAs in inoculated leaves as well as in systemically infected leaves. On the other hand, satellite and viral RNAs were detected in the inoculated but not in the noninoculated leaves from infected plants containing either (G)- or (WC)-satRNA. Although a decrease in the quantities of genomic RNAs 1, 2, and 3 was characteristic of all satRNA containing plants, this effect was more evident in the case of (G)- and (WC) satRNAs. The complete nucleotide sequences of the three satRNAs were determined and compared to the published sequence of PSV satRNA. The (V)-satRNA differed from the published sequence at two positions, whereas the (G)- and (WC)-satRNAs differed at six and eight positions, respectively. Comparison of the nucleotide sequence of the satRNA having no effect on PSV-induced symptoms with those reducing virus symptoms suggests that a single nucleotide change or as many as five nucleotide changes may distinguish between attenuating and nonattenuating satRNAs. PMID- 1718511 TI - Laser endoscopic treatment for upper gastrointestinal cancers. AB - We report the effective clinical use of endoscopic laser in Japan using the results of a nationwide survey and our own experience with more than 100 cases. The Nd:YAG laser and argon dye laser with hematoporphyrin derivative (photodynamic therapy) were most commonly used in digestive endoscopy and were investigated as new modalities of cancer therapy. Photodynamic therapy was fairly effective, especially in superficial esophageal cancer and the ill-defined lesions of early gastric cancer. Endoscopic laser treatment was carried out on 80 patients with 86 lesions of early gastric cancer at our hospital, and the following tumor types were proven highly curable by this means: focal cancer, IIa and so-called "gastritis-like" tumors less than 2 cm in size. The Nd:YAG laser provides a new approach to palliative treatment, such as recanalization of neoplastic obstruction in the advanced stage of gastrointestinal cancers. PMID- 1718512 TI - Effects of monochlorophenols and p-chloroaniline on nucleic acid synthesis in microbial growth process. PMID- 1718513 TI - Strategy for a healthy environment. PMID- 1718514 TI - Effect of culture duration on hepatocyte subcellular membranes involved in endocytosis. AB - The influence of culture duration on some characteristics of hepatocyte subcellular membranes involved in endocytosis was investigated. Activity of enzymes located in plasma membrane, Golgi apparatus and lysosomes increases with time. These modifications are accompanied with several changes in the sedimentation properties of these organelles. Endocytosis of [14C]sucrose and [14C]sucrose-LDL is not affected by culture age. On the contrary, [14C]sucrose ASF endocytosis strongly decreases in these conditions. These modifications are delayed to some extent by lowering the temperature. Addition to the culture medium of 3-methyladenine (an inhibitor of autophagy), sodium butyrate, dimethylsulfoxide, phenobarbital or nicotinamide does not prevent the decrease of ASF endocytosis caused by culture duration. These results indicate that one must be cautious when extrapolating to liver in vivo, observations on endocytosis obtained with primary culture of hepatocytes. PMID- 1718515 TI - Variations in polymer fitness at elevated mutation rates. AB - Two series expressions were obtained that give the first and second order rates of change in population fitness during competitive replication at elevated mutation rates. At their zero-error limit, the respective power series reduces to the second (Fisher's theorem) and third moments of the fitness distribution. The first series maximized the variation in mean polymer fitness, for a given amount of population covariance. From experimental results reported by Spiegelman's group on evolution in vitro among Q beta RNA variants, it was demonstrated: (i) terms in the (second) series fall-off at a rate broadly equal to the replicase error (epsilon congruent to 10(-4); (ii) the rate of change in mean RNA fitness (polymer formation rate constant) corresponds to the variance in fitness; and (iii) agreement exists between second order rate changes in fitness and the third moment (skewness) regression line, extending over 20 successive replication reactions. The impact of these findings on the standard model of evolution has been discussed. PMID- 1718516 TI - Immunotherapy after bone marrow transplantation. AB - Residual disease after bone marrow transplantation (BMT) can either cause relapse or persist in a balanced state in which immune mechanisms keep control over malignant cell proliferation. After allogeneic BMT the latter mechanism [summarized as graft-versus-leukemia (GVL)] is probably supported by cellular (e.g. T lymphocytes, natural killer cells) and humoral (e.g. cytokines) effectors and often linked to clinical manifestations of GVHD. Preclinical and clinical studies are now emerging in which cytokines, such as interleukin-2 or interferons, are given after BMT to support cellular immune function particularly in situations in which GVL does not occur. We summarize here results from both pre-clinical and clinical studies that are relevant to the design of protocols seeking to improve elimination of residual malignant disease in BMT patients by modulation of the immune system. PMID- 1718517 TI - Autologous bone marrow transplantation as adjuvant treatment for high-risk Hodgkin's disease in first complete remission after MOPP/ABVD protocol. AB - Fifteen patients with very poor prognosis Hodgkin's disease in remission after MOPP/ABVD regimen, were treated with high-dose chemotherapy (HDC) and autologous marrow transplantation (ABMT) immediately after achieving complete remission (CR). Thirteen patients (86.6%) remain alive in unmaintained CR at a median time of 36 months (range 10-64 months) post-transplant. In the other two patients reasons for failure included relapse of Hodgkin's disease (one patient) and death due to interstitial pneumonitis secondary to carmustine therapy. These patients were compared with a historical control group consisting of 24 patients with the same poor prognostic factors, who achieved CR with MOPP/ABVD and did not receive other treatment. Eight out of 24 patients (33%) remain alive and well in unmaintained CR at a median time of 42 months (range 19-83 months). The administration of MOPP/ABVD combined with HDC and ABMT was not associated with an increased incidence of major toxicity. The results achieved support the early sequential treatment of a highly effective drug combination followed by HDC/ABMT that can substantially improve the likelihood of cure in these advanced stage very poor prognosis Hodgkin's disease patients. PMID- 1718518 TI - Historical perspectives of medical intelligence. PMID- 1718519 TI - Narrative and procedural discourse after closed head injury. AB - Aspects of productivity, content, and cohesion in the narrative and procedural discourse of 11 closed head-injured (CHI) adults and 21 normal adults were examined. Two narrative tasks, one involving retelling a story heard and the other formulating a story based on a comic strip, and one procedural task of telling how to buy groceries were administered to each subject. CHI subjects consistently produced fewer words, spoke slower, used more mazes (dysfluencies), produced fewer target content units, and used fewer cohesive ties per utterance, as compared to the normal subjects. Other significant differences in discourse occurred between the two groups, but these varied from task to task. Normal subjects varied characteristics of their discourse performance according to the discourse task. Significant differences across tasks occurred on seven of the 13 discourse measures. The CHI subjects, however, showed more limited variation in that their performance varied on only three of the 13 measures. Correlations among discourse, language, and memory measures were examined and discussed. The results of this study indicate that analysis of CHI narrative and procedural discourse has important clinical and theoretical implications. PMID- 1718520 TI - Ketamine potentiates 5-HT3 receptor-mediated currents in rabbit nodose ganglion neurones. AB - The interaction of ketamine with the 5-HT3 receptor of rabbit nodose ganglion neurones is described. Ketamine (3-30 microM) enhanced 5-HT3 receptor-mediated currents recorded under voltage-clamp conditions. This action did not appear to be related to the known effect of ketamine of inhibiting 5-HT uptake. PMID- 1718521 TI - Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. AB - 1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Isc) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in Isc. 3 The rank order of potency of purine- or pyrimidine-induced changes in Isc of normal human nasal epithelium when applied to the apical membrane (UTP greater than or equal to ATP greater than ATP gamma S greater than 2MeSATP greater than ADP beta S much greater than beta gamma MeATP greater than or equal to alpha beta MeATP) or basolateral membrane (2MeSATP greater than UTP greater than ATP greater than ATP gamma S greater than alpha beta MeATP greater than beta gamma MeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP greater than or equal to ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium. PMID- 1718522 TI - Heterogeneous involvement of endothelium in calcitonin gene-related peptide induced relaxation in coronary arteries from rat. AB - 1. The effects of rat- and human-CGRP and capsaicin were studied in isolated rings of rat proximal epicardial (PC) and distal intramyocardial (DC) coronary arteries. 2. The relaxing effect of rat-CGRP was dependent on the level of vessel tone induced by prostaglandin F2 alpha (PGF2 alpha) in PC but not in DC arteries. Submaximally contracted DC and PC arteries were more sensitive to rat- than human CGRP. There was no difference in sensitivity to rat- and human-CGRP between PC and DC arteries. 3. Substance P elicited a small relaxation only in 4 of the 6 PC arteries tested. PC and DC arteries were concentration-dependently relaxed by capsaicin. The relaxation was partly inhibited by ruthenium red, thus suggesting that capsaicin causes specific release of CGRP from sensory nerve endings in rat coronary arteries. 4. The relaxant effect of rat-CGRP was antagonized by endothelium removal and indomethacin but not methylene blue in endothelium-intact PC arteries. The relaxation in DC arteries was not affected by any of these treatments, indicating a heterogeneous involvement of the endothelium in CGRP mediated coronary vasodilatation and the release of a cyclo-oxygenase product in PC arteries in rats. 5. Glibenclamide had no inhibitory effect on the CGRP induced relaxation of PC and DC arteries, thus excluding the involvement of glibenclamide-sensitive K(+)-channels in the mechanism of action of CGRP in rat coronary arteries. PMID- 1718523 TI - Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor. AB - 1. The identity of the neuropeptide Y (NPY) receptor associated with the observed inhibition of neuronal Ca2+ currents (ICa) in rat dorsal root ganglion (DRG) cells has been established on the basis of agonist responses to analogues and carboxy terminal (C-terminal) fragments of the NPY molecule. 2. Whole cell barium currents (IBa) in DRG cells were reversibly inhibited by 100 nM NPY, 100 nM PYY and C-terminal fragments of NPY in a manner that correlated with the length of the NPY fragments (for inhibition of the IBa NPY = PYY greater than NPY2-36 greater than NPY13-36 greater than NPY16-36 greater than NPY18-36 much greater than NPY25-36). 3. C-terminal fragments of NPY were also effective in reversibly reducing the ICa, the associated increase in the intracellular Ca2+ concentration [( Ca2+]i) and the increased [Ca2+]i produced by evoked action potentials in the DRG cells. In addition, a Ca(2+)-activated Cl- conductance was also reversibly reduced by NPY fragments only when accompanied by a reduction in Ca2+ entry. 4. We conclude that the Y2 receptor for neuropeptide Y is coupled to inhibition of Ca2+ influx via voltage-sensitive calcium channels in DRG cells. PMID- 1718524 TI - Alkali cation permeability and caesium blockade of cromakalim-activated current in guinea-pig ventricular myocytes. AB - 1. The sensitivity of cromakalim-activated current (Icrom) to manipulations of extracellular cationic composition was examined in whole-cell voltage clamp recordings from freshly-dispersed, adult guinea-pig ventricular myocytes. In bathing media with different concentrations of K+ (1, 2.5, 5.4 and 12 mM) the Icrom reversal potential (Erev) varied in strict correspondance with the K+ equilibrium potential and inward Icrom amplitude was proportional to the external K+ concentration. 2. Replacement of 12mM K+ with 12mM Rb+ induced a slight positive shift of Erev indicating that PRb+/PK+ = 1.06. K+ replacement with 12mM Cs+ reduced or abolished inward Icrom and produced a negative shift of Erev by at least 50 mV; an upper limit of PCs+/PK+ was fixed at 0.18. 3. Addition of Rb+ (1 30 mM) to 2.5 mM K(+)-containing medium produced a concentration-dependent increase in inward Icrom and positive shift of Erev suggesting that K+ and Rb+ have similar permeabilities and conductivities and do not interfere with each other in the channel. 4. CS+ (0.01-30 mM), added to medium containing 12 mM Rb+, induced a potent, voltage-dependent inhibition of inwardly rectifying current (IK1; IC50 = 0.2-3 mM). Voltage-dependent inhibition of inward Icrom was observed only at considerably higher CS+ concentrations (IC50 = 4-30 mM). Extracellular Rb+ and CS+ did not substantially alter the amplitude of outward Icrom. 5. The results support the contention that the ATP-sensitive K+ channel is the primary target of cromakalim action in ventricular myocytes. PMID- 1718525 TI - Guinea-pig isolated trachealis: the effects of charybdotoxin on mechanical activity, membrane potential changes and the activity of plasmalemmal K(+) channels. AB - 1. A study has been made, in guinea-pig isolated trachealis, of the effects of charybdotoxin in modulating (a) the activity of large conductance K(+)-channels, (b) the spontaneous electrical activity of intact cells and (c) the mechanical effects of some bronchodilator drugs. 2. Single smooth muscle cells were isolated from guinea-pig trachealis by enzymic digestion and were studied by the patch clamp recording technique. Recordings were made from outside-out plasmalemmal patches when the medium bathing the external surface of the patches contained 1.2 mM Ca2+ and 6 mM K+ while that bathing the cytosolic surface contained 0.1 microM Ca2+ and 140 mM K+. Charybdotoxin (100 nM), applied to the external surface of patches held at 0 mV, abolished the unitary currents associated with the opening of large conductance K(+)-channels. 3. Opened segments of guinea-pig trachea were used for the simultaneous recording of membrane potential and tension changes. In these experiments charybdotoxin (100 nM) caused the conversion of spontaneous electrical slow waves into spike-like action potentials. This effect was accompanied by a very small reduction in resting membrane potential. 4. Tissue bath recording showed that charybdotoxin (100 nM) increased the spontaneous mechanical tone of the tissue, antagonized (2.8 fold in each case) the relaxant actions of isoprenaline and theophylline but did not antagonize the relaxant actions of cromakalim or RP 49356. 5. It is concluded that charybdotoxin is an effective inhibitor of large conductance K(+)-channels in guinea-pig trachealis cells. The ability of charybdotoxin to convert spontaneous slow waves into spike like action potentials suggests that the large, charybdotoxin-sensitive, K+ channels play an important role in determining the strong outward rectifying behaviour of the cells. The ability of charybdotoxin to antagonize isoprenaline and theophylline, but not to antagonize cromakalim and RP 49356, suggests that opening of the large conductance, charybdotoxin-sensitive K+-channel is implicated in the action of the former but not the latter pair of bronchodilator drugs. PMID- 1718526 TI - Selective stimulation of glucagon secretion by beta 2-adrenoceptors in isolated islets of Langerhans of the rat. AB - 1. In rat isolated islets of Langerhans the selective beta 2-adrenoceptor agonist, clenbuterol (1 to 20 microM), significantly increased the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) within 2 min of incubation. 2. The cyclic AMP response to clenbuterol was inhibited in the presence of the selective beta 2 adrenoceptor antagonist, ICI 118551 (0.1 or 10 microM) but remained unchanged when the beta 1-antagonist, atenolol (0.1 microM) was administered. 3. Despite causing an elevation in cyclic AMP, clenbuterol (up to 20 microM) failed to influence insulin secretion at any glucose concentration tested, even in the presence of a phosphodiesterase inhibitor. 4. By contrast, clenbuterol elicited a dose-dependent rise in the rate of glucagon secretion; the maximal agonist-induced increase in secretion was two fold, a response equivalent to that observed with 20 mM L-arginine. 5. ICI 118551 significantly inhibited the rise in glucagon secretion induced by clenbuterol (up to 20 microM). 6. The results indicate that the rat islet A cell population is equipped with functional beta 2-adrenoceptors which influence glucagon secretion via the second messenger cyclic AMP, but that the B cells are deficient in functional beta-receptors. PMID- 1718527 TI - Histopathology of benign prostatic hyperplasia after failure of hyperthermia treatment. AB - We have used a fine morphometric method to evaluate hyperplastic prostate tissue after treatment by local hyperthermia and compare it with untreated specimens. Local hyperthermia was delivered to the prostate gland by a transrectal applicator using microwaves at 915 MHz. Prostatic tissue was obtained from 13 patients who had completed a course of local hyperthermia treatment and who underwent prostatectomy 3 to 12 months later because the treatment had failed. Prostatic tissue from 9 patients who had undergone prostatectomy with no previous treatment served as a control. A significant reduction in the volume fraction of fibrous tissue was found in the study group (37%) compared with the general population (48%). No inter-group difference was observed in the volume fractions of vascular or glandular tissue. We suggest that the difference observed in the fibrous elements may be the reason for the failure of treatment and that the histological composition of the diseased gland may, therefore, be a key factor in determining the outcome. PMID- 1718528 TI - Late complications of Prostakath treatment for benign prostatic hypertrophy. AB - The self-retaining intra-urethral device "Prostakath" was inserted in a consecutive series of 29 men with obstructive benign prostatic hypertrophy. Fifteen patients were relieved of prostatic symptoms for an observation period of 22 weeks (range 2-60). The spiral was removed in 14 cases (48%) at an average of 14 weeks (range 1-82) after insertion; in 9 patients this was because of urinary retention and in 5 it followed dislocation of the stent into the bladder. Five stents removed after 44 weeks of treatment (range 21-82) were severely calcified. Light microscopic investigation of the Spirals that were removed revealed no damage to the gold-plated surface. All patients with calcification had chronic urinary infection resistant to antibiotic treatment. We believe that infected urine is the major factor responsible for the calcification. We suggest that patients with recurrent urinary infection after insertion of the Prostakath should be closely followed up and checked for stone formation by a plain X-ray of the bladder region. It may be advisable to change the Prostakath in patients with resistant urinary tract infection at 6-monthly intervals. PMID- 1718529 TI - Evaluation of palliative surgical procedures in unresectable pancreatic cancer. AB - To update our experience with palliative surgical procedures in unresectable adenocarcinoma of the pancreas, the records of 297 patients surgically treated at Memorial Sloan-Kettering Cancer Center were reviewed. Between October 1983 and November 1989, 117 patients underwent exploratory laparotomy as a single procedure: 24 patients had gastric bypass, 38 biliary bypass, and 118 both gastric and biliary bypass. The postoperative in-hospital mortality rate was 4.4 per cent. Overall morbidity was 29.7 per cent; the morbidity rate in patients with a double bypass was 29.7 per cent. Median (s.e.m.) survival was 231(20.3) days. Statistical analysis showed a significantly increased risk of morbidity in patients who underwent one therapeutic and one prophylactic bypass. Survival was decreased in patients who had a therapeutic gastric bypass (median(s.e.m.) survival 136(70.2) days) or a combination of two therapeutic bypasses (median(s.e.m.) survival 93(85.9) days). These data emphasize the poor prognosis of patients with pancreatic adenocarcinoma who cannot be resected. The need for therapeutic double bypass is a bad prognostic indicator, and a prophylactic bypass added to a therapeutic bypass increases morbidity without prolonging survival. PMID- 1718530 TI - Substance P-containing neurons in the mesopontine tegmentum are severely affected in Parkinson's disease. AB - Substance P immunoreactive (SP+) neurons were analysed quantitatively in serial sections of the mesopontine tegmentum in 6 patients with idiopathic Parkinson's disease and 5 age-matched normal controls. In the tegmentum of the Parkinson's disease brains many SP+ neurons contained swollen, twisted neuronal processes as well as Lewy bodies. There were significant reductions in the total number of SP+ neurons in the pedunculopontine tegmental nucleus (loss 43%), in the laterodorsal tegmental nucleus (loss 28%), in the oral pontine reticular nucleus (loss 41%) and in the median raphe nucleus (loss 76%). It was the large SP+ (greater than 20 microns) neurons that were particularly affected. In our control group we did not document a significant relationship between age at death and number of SP+ neurons in these tegmental nuclei or between age at death and number of pigmented neurons in the locus coeruleus. In contrast, in patients with Parkinson's disease, there was a strong inverse relationship between age at death and numbers of SP+ and pigmented neurons. Our findings suggest an interaction between the pathophysiological mechanisms initiated by Parkinson's disease and other processes related to ageing. Since tegmental SP+ neurons are affected by the primary pathological processes underlying Parkinson's disease as severely as catecholamine-synthesizing neurons are affected, theories of pathogenesis and therapeutic strategies in Parkinson's disease will need to take into account the involvement of these SP+ neurons. PMID- 1718531 TI - The noun-verb problem in Chinese aphasia. AB - Previous studies have shown that Broca's aphasics experience a selective difficulty with action naming inside or outside of a sentence context. Conversely, it has been suggested that Wernicke's aphasics are particularly impaired in object naming. A number of explanations have been offered to account for this double dissociation, including grammatical accounts according to which the main verb problem in agrammatic Broca's aphasics is viewed as a by-product of their syntactic and/or morphological impairment, due perhaps to the greater morphological load carried by verbs (compared with nouns). In the Chinese language, there are no verb conjugations and no declensions. Hence there is no reason to expect a relationship between morphological impairment and deficits in action naming. We examined comprehension and production of object and action names, outside of a sentence context, in a sample of Chinese-speaking Broca's and Wernicke's aphasics. There was an interaction between patient group and object/action naming, but no corresponding interaction on the comprehension task. We conclude that action-naming deficits in Broca's aphasia (and/or the corresponding sparing of action names in Wernicke's aphasia) cannot be attributed to morphological differences between nouns and verbs. We also found a sublexical variant of the noun/verb dissociation applied to the internal structure of compound words made up of a verbal and a nominal element: Broca's aphasics tended to lexicalize the verbal portion of these words more often than the nominal compound, while Wernicke's showed the opposite pattern. These sublexical effects are difficult to explain in syntactic terms nor do they fit the standard lexical view. A modified lexical account is proposed, emphasizing semantic/conceptual effects in a distributed lexicon. PMID- 1718532 TI - Sentence interpretation in normal and aphasic Hindi speakers. AB - In interpreting a sentence, listeners rely on a variety of linguistic cues to assign grammatical roles such as agent and patient. The present study considered the relative ranking of three cues to agenthood (word order, noun animacy, and subject-verb agreement) in normal and aphasic speakers of Hindi. Because animacy plays a grammatical role in Hindi (determining the nature and acceptability of sentences without accusative marking), this language is relevant to the claim that Broca's aphasia involves a dissociation between grammar and semantics. Results of Study 1 with normal Hindi-dominant speakers showed that animacy is the strongest cue in this language, while agreement is the weakest cue. In Study 2, Hindi-English bilinguals were tested in both their languages. Most showed the normal animacy-dominant monolingual pattern in Hindi, with a mixture of strategies from both languages in their interpretation of English. A substantial minority showed mixed strategies in both languages. Only 5 of 48 subjects displayed a complete separation between languages, with animacy dominance in Hindi and word order dominance in English. In Study 3, two Hindi-English bilinguals with Broca's aphasia were tested in both languages. Results indicate (a) greater use of animacy in Hindi than in English and (b) greater use of word order in English than in Hindi. The strategies displayed by these patients fall well within the range observed among bilingual normals. We conclude that the use of animacy in sentence interpretation by these aphasic patients reflects preservation of normal, language-specific processing strategies; it cannot be interpreted as a nonlinguistic strategy developed to compensate for receptive agrammatism. Results are discussed in light of other cross-linguistic evidence on sentence comprehension in monolingual and bilingual aphasics. PMID- 1718533 TI - Speech-related body movement in aphasia: period analysis of upper arms and head movement. AB - The effects of aphasia on coverbal body movement have important implications for the understanding of both normal and pathological speech processes. The related findings were often inconsistent, partly due to inherent methodological difficulties which could be reduced by the use of advanced techniques of movement monitoring (Hadar, 1991). The present study employed a new computerized system, CODA-3, which locates small prismatic markers and computes by triangulation their three-dimensional position at 100 Hz. Movement of the head and the upper arms was monitored in 15 aphasic and normal subjects engaged in speech during a naturalistic interview. Movement analysis was based on automatized identification of successive movement extrema ("period analysis") and the computation of amplitude, duration, and velocity of each period. The results showed higher incidence and amplitude of all body movement in the aphasic population. Fluent aphasics showed this particularly with "symbolic," content-bearing movements, while nonfluent aphasics were higher than controls in both symbolic and "motor" (simple and small) movements. No deficit in the internal organization of movement was seen in the aphasic population. These results indicate that aphasics increase their coverbal movement in compensation for their speech impairment: fluent aphasics compensate primarily for a symbolic impairment, while nonfluent aphasics compensate more for a motor impairment. PMID- 1718534 TI - Auditory lateralization for speech in language-impaired children. AB - The ability of five language-impaired (LI) children and five matched controls, aged 7-10 years, to discriminate natural pairs of consonant-vowel syllables contrasted on place of articulation and voicing, presented to the right or left ear with white noise in the contralateral ear, was investigated. The general pattern of errors indicated that LI children had more difficulty than controls in discriminating place of articulation contrasts only when they were presented to the left ear, as well as a difficulty in discriminating voice contrasts selective to the right ear. The results are discussed in terms of acoustic integration and suggest that bihemispheric dysfunction is a basis for specific language impairment. PMID- 1718535 TI - Errors resembling semantic paralexias in Spanish-speaking aphasics. AB - Forty-one Spanish-speaking left-hemisphere-damaged patients were selected and divided into seven groups (transcortical, Broca's aphasia, conduction aphasia, Wernicke's aphasia, anomic aphasia, alexia without agraphia, and global aphasia). A reading battery composed of eight different subtests was given to each patient (reading of letters, reading of syllables, reading of pseudowords, reading of words, reading of sentences, understanding commands, reading and comprehension of texts, and logographic reading). Different types of reading errors were analyzed. Only in the logographic reading subtest were some word-recognition errors found, resembling semantic paralexias. It is proposed that semantic paralexias in English (and other languages) depend upon the partial logographic nature of the reading system. The importance of cross-linguistic analysis of reading errors, taking into account reading system idiosyncracies, is emphasized. PMID- 1718536 TI - The internal validity of efficacy studies: design and statistical power in studies of language therapy for aphasics. AB - In this study the internal validity of efficacy studies of language therapy for aphasic patients is discussed. The lack of sufficient internal validity is demonstrated with respect to research designs used in these studies and the statistical power of their statistical significance tests. The internal validity problems are viewed as the major cause of the conflicting conclusions in the efficacy studies in the past three decades. PMID- 1718537 TI - Lithium augments pilocarpine-induced fos gene expression in rat brain. AB - Lithium salts are considered the most effective agents used in treating manic depression. Previous studies in PC12 pheochromocytoma cells indicate that lithium has a dramatic augmenting effect on expression of the fos proto-oncogene, a component of the AP-1 transcription factor. Although fos expression is activated by agonists that function through different signal transduction pathways, the lithium augmenting effect appears to be specific for receptor and post-receptor stimulators of protein kinase C (PKC). In particular, fos induction mediated by the m1 muscarinic receptor linked to PKC activation was found to be exquisitely sensitive to lithium enhancement. We now show that a similar augmenting effect can be demonstrated in rat brain. Following treatment with the muscarinic agonist pilocarpine, fos mRNA accumulates in the cortex, an effect that is blocked by the m1 antagonist pirenzepine. Rats treated with a single intraperitoneal injection of lithium chloride exhibited a substantial increase in pilocarpine-mediated fos expression. In contrast, fos expression induced in several brain regions by a single electroconvulsive shock is not augmented by lithium. The finding that short-term treatment with lithium enhances fos expression in the brain suggests a mechanism for its therapeutic action. PMID- 1718538 TI - Cardiovascular effects of the local injection of 5,7-dihydroxytryptamine into the nodose ganglia and nucleus tractus solitarius in awake freely moving rats. AB - The role of the nucleus tractus solitarius (NTS) serotonergic afferents in cardiovascular (CV) regulation is yet to be established. However, several findings suggest that in this nucleus the serotonergic endings coming from the nodose ganglia (NG) are involved in the control of blood pressure (BP). The purpose of the present study was to identify the CV effects of the destruction of this NG-NTS serotonergic pathway. For that, the BP, BP variability (BPV) and heart rate (HR) effects of the local microinjection of 5,7-dihydroxytryptamine (5,7-DHT), into the NG and NTS were investigated in awake freely moving rats. The local degeneration of serotonergic elements was associated with a significant decrease in the 5-HT and 5-hydroxyindole acetic acid levels within the NG and NTS in 5,7-DHT treated rats. In addition, the microinjection of the neurotoxin in both structures produced a transient and significant increase in BP. This effect was of greater amplitude and associated with an increase in BPV in NG lesioned rats. These results may indicate that the NG-NTS serotonergic pathway participates in the transfer of the messages arising from the aortic baroreceptors. However, the vagal component of the baroreflex assessed with the phenylephrine test was not significantly modified in NG lesioned animals as compared to controls. Consequently, if the present data suggest that the NG-NTS serotonergic pathway plays a depressor role in BP regulation, its involvement in the reflex CV responses triggered by the stimulation of the aortic baroreceptors has yet to be established. PMID- 1718539 TI - Characterization of axonally transported [125I]PYY binding sites in rat vagus nerve. AB - Quantitative receptor autoradiography was used to characterize [125I]PYY binding to slide-mounted sections of rat cervical vagus nerve which had been ligated 24 h prior to extraction. Saturation and competitive binding characteristics were determined for the accumulation of binding sites detected in the segment of the nerve proximal to the ligature. High and low affinity binding components were resolved (Kd = 93 pM and 2.1 nM). The rank order of potency for competing ligands was PYY greater than NPY much greater than PYY13-36 much greater than [Pro34]NPY suggesting that vagal PYY/NPY binding sites are PYY-preferring and of the Y2 type. PMID- 1718540 TI - Membrane currents elicited by the epileptogenic drug pentylenetetrazole in the native oocyte of Xenopus laevis. AB - The effects of the epileptogenic agent pentylenetetrazole (PTZ) on membrane currents of native oocytes of Xenopus laevis were studied. PTZ elicits a response that consists of two inward currents. The first one is interpreted to be due to a decrease of potassium permeability since: (1) the input resistance is increased; (2) the equilibrium potential is near that of potassium; (3) the current is decreased during administration of potassium channel blocking agents; and (4) the PTZ response can be mimicked by blocking potassium channels without PTZ application. The second one is interpreted to be due to an increase of chloride permeability since: (1) the input resistance is decreased; (2) the equilibrium potential is near that of chloride; and (3) the response is decreased during administration of chloride blocking agents. These findings correspond to some extent with those made in neurons. PMID- 1718541 TI - Inhibition of Ca-channels by diazepam compared with that by nicardipine in pheochromocytoma PC12 cells. AB - The effects of diazepam on voltage-gated Ca channels were studied in PC12 pheochromocytoma cells using whole-cell voltage-clamp techniques. An inward current activated by a depolarizing voltage step to +10 mV from a holding potential of -60 mV in 10.8 mM Ba was larger than that activated in 10.8 mM Ca. The Ba current was completely blocked by a low concentration of Cd (30 microM) and was also sensitive to nicardipine (100 nM to 10 microM). Diazepam (1-100 microM) inhibited the Ba current in a concentration-dependent manner. Neither diazepam nor nicardipine affected the current-voltage relationship or the dependence on holding potentials of the Ba current. Both slightly accelerated the inactivation time course of the Ba current. When diazepam was applied to the cells in combination with nicardipine, the observed inhibition agreed with a value predicted assuming independent blockade by diazepam and by nicardipine. These results suggest that diazepam inhibits Ca channels in a manner similar to nicardipine, but that the binding sites for diazepam are different from those for nicardipine. PMID- 1718542 TI - Inhibition of cAMP production by alpha 2-adrenoceptor stimulation in rabbit retina. AB - Adenylate cyclase activity in rabbit retinal homogenates can be stimulated directly by forskolin or through a receptor-mediated mechanism by vasoactive intestinal peptide (VIP). In contrast the alpha 2-adrenoceptor agonists clonidine and UK-14,304 reduce the basal cAMP level slightly. This was more evident following application of forskolin and VIP where the decrease of cAMP caused by clonidine and UK-14,304 is dose-dependent. The alpha 2-adrenoceptor agonist response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor, isobutylmethylxanthine, suggesting the involvement of a Gi-protein. Clonidine and UK-14,304 attenuation of elevated cAMP levels can be inhibited by the alpha 2-receptor antagonist yohimbine and phentolamine but not by the specific alpha 1-receptor antagonist, prazosin. Serotonergic, cholinergic and beta-adrenergic receptor antagonists were without effect. The results demonstrate that alpha 2-adrenergic receptors in the retina exert inhibitory effects on adenylate cyclase activity mediated by an inhibitory guanine nucleotide regulating protein. PMID- 1718543 TI - Role of voltage-sensitive calcium channels in mitogenic stimulation of neuroblasts. AB - The present study examines the role of Ca2+ in the regulation of sympathetic neuroblast mitosis. Employing a fully defined neuroblast culture system, we previously found that insulin growth factors (IGFs), depolarization and vasoactive intestinal peptide (VIP) regulated precursor mitosis. We now report that Ca2+ entry via voltage-sensitive channels was required for depolarization stimulated mitogenesis. Ca2+ channel blockade with nitrendipine completely inhibited the increase in [3H]thymidine incorporation elicited by depolarizing stimuli including 30 mM KCl and the Na+ channel agonist veratridine. However, Ca2+ channel activity was not involved in the stimulation of DNA synthesis by IGFs or VIP. Thus, neuroblast mitosis may be regulated by multiple intracellular as well as extracellular signals. PMID- 1718544 TI - Intrinsic membrane potential oscillations in hippocampal neurons in vitro. AB - Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus. PMID- 1718545 TI - Organization of claustro-cortical projections to the primary somatosensory area of primates. AB - Different fluorescent dyes were injected in the face (S1fa) and hand (S1hn) representations of the primary somatosensory cortex, involving both areas 3b and 1. Claustral neurons labeled by either S1fa or S1hn were divided in two populations. One population was located in the dorsal part of the nucleus: neurons labeled from S1fa were placed laterally to those labeled from S1hn. The second population was located more ventrally, with a rostro-caudal distribution of S1fa vs S1hn neurons. These findings demonstrate the existence of ordered and possibly multiple somatosensory representations in the monkey claustrum. PMID- 1718546 TI - Morphology and distribution of efferent vagal innervation of rat pancreas as revealed with anterograde transport of Dil. AB - Vagal efferent innervation of the pancreas was labeled by anterograde transport of Dil injected into the dorsal motor nucleus (dmnX). While over the entire organ only 19 +/- 3 (or 8 +/- 1%) of the 231 +/- 17 interlobular ganglia received Dil labeled vagal fibers and terminals, the proximal duodenal lobe (or head) was significantly more densely innervated. Laser scanning confocal microscopy revealed further morphological details of the vagal terminals and their target ganglion cells. No vagal fibers or terminals were found in islets and acinar tissue. PMID- 1718547 TI - Tracing SCN graft efferents with Dil. AB - The suprachiasmatic nuclei (SCN) regulate circadian rhythmicity in many biological and behavioral responses. Hamsters are made permanently arrhythmic by bilateral destruction of the SCN. Circadian locomotor rhythmicity is restored by fetal tissue transplants placed in the 3rd ventricle (3V). If intact animals are implanted with fetal SCN grafts, they maintain locomotor activity rhythms when the host SCN are subsequently destroyed. The mechanism(s) whereby the SCN (either grafted or in situ) regulate locomotor rhythmicity is not known. Evidence from other graft models point to the possibility of efferents to appropriate targets in the host. In the present study, efferent connections of transplanted fetal SCN were examined using the carbocyanine dye, Dil. Intact or SCN-lesioned animals were sacrificed 7 or 40 days after receiving fetal SCN grafts into 3V. Dil crystals were placed on the grafts in fixed brains which were then incubated for 3-6 weeks before sectioning. Sections bearing Dil-labelled efferents from the graft were photographed and then stained for immunoreactive VIP and NP cells to locate donor SCN. Although labelled efferents were observed in a majority of the grafts, most were confined to the limits of the graft. The few labelled efferents that entered the host tissue when the graft seemed to merge with the host did not extend very far regardless of whether the graft contained immunohistochemical evidence for donor SCN or not. The observation of limited graft-host connectivity suggests either that a limited number of efferents is sufficient to support circadian locomotor rhythmicity, or that the mechanism mediating restoration of function entails a diffusible substance. PMID- 1718548 TI - Lindane may enhance nocturnal pineal N-acetyltransferase activity via beta adrenergic receptors. AB - Lindane, a chlorinated hydrocarbon pesticide, was previously shown to enhance the nighttime rise in pineal N-acetyltransferase (NAT) activity and melatonin as well as serum melatonin levels. The purpose of the present study was to test whether lindane acts on the pineal gland by means of a beta-adrenergic receptor mechanism. Whereas lindane (total dose 17.8 mg/kg b.wt. over 6 days) by itself significantly augmented the nocturnal levels of pineal NAT activity in otherwise untreated rats, the pesticide was ineffective in reference to this enzyme when it was given in conjunction with the beta-adrenergic receptor antagonist propranolol (20 mg/kg b.wt., one hour before lights off). The augmentation of NAT activity by lindane also caused significant reductions in pineal serotonin (5-HT) and 5 hydroxyindole acetic acid (5-HIAA); again, both these responses were blocked by propranolol treatment. Neither pineal 5-hydroxytryptophan nor pineal or serum melatonin levels were significantly changed as a result of either lindane or propranolol treatment. The results are consistent with the idea that lindane influences pineal 5-HT metabolism either at the level of the beta-adrenergic receptor or via the sympathetic innervation to the pineal gland. PMID- 1718549 TI - Substance P- and calcitonin gene-related peptide-like immunoreactivities in the human carotid body studied at light and electron microscopical level. AB - The distribution of substance P- (SP-) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) was studied in the human carotid body at the light and electron microscopic level. Of the different compartments of the carotid body, the glomic lobules received the densest innervation by SP/CGRP double labelled axons, followed by interlobular arteries and interlobular connective tissue. Ultrastructurally, preterminal SP/CGRP-LI axons are unmyelinated and measure 0.09-1.02 micron in diameter with a median diameter of 0.28 micron (calculated conduction velocity: 0.5 m/s). Within the glomic lobules, vesicle containing varicosities are numerous around glomus cells, but only few of them directly contact (non-synaptically) glomus cells. The findings are consistent with the existence of two populations of SP/CGRP-LI C-fibers, one of them probably representing chemoreceptor C-fibers, the other belonging to the ubiquitous vascular sensory innervation. In addition to its localization in nerve fibers, CGRP-LI also occurs in dense core vesicles of a subpopulation of glomus cells. In this regard, the human carotid body is unique among mammalian species studied so far, which severely hampers the interpretation of pharmacological data obtained in laboratory animals in terms of human physiology. PMID- 1718550 TI - Excitatory amino acid immunoreactivity in vestibulo-ocular neurons in gerbils. AB - Retrogradely labeled vestibulo-ocular neurons (VOR) that also stain with antibodies for the excitatory amino acid neurotransmitters glutamate (GLU-LI) or aspartate (ASP-LI) were studied. VOR neurons that contained GLU-LI or ASP-LI label were identified in the medial (MVN) and superior (SVN) vestibular nuclei, and cell group Y. More than half of the VOR cells in MVN were also GLU-LI positive, but less than half of the VOR cells in SVN were double labeled. PMID- 1718551 TI - GABA-induced potentiation of neuronal excitability occurs during contiguous pairings with intracellular calcium elevation. AB - The temporal convergence of neuronal signals is commonly considered as a likely prerequisite for enhanced neuronal excitability underlying the induction of associative memories. Here we report that transmitter application on presynaptic terminals of the Hermissenda Type B photoreceptors, when paired with depolarization, results in a potentiation of the excitability of the B-cell which derives from an increase in input resistance across the B-cell soma membrane. Pressure microapplication of gamma-aminobutyric acid (GABA) (12.5 microM) on the terminal branches of the Hermissenda Type B photoreceptors results in the fast (less than 1 s) activation of an inward Cl- conductance, characterized by a decrease in neuronal membrane resistance and an accompanying hyperpolarization (3 6 mV) of the B-cell. A slower effect of GABA, characterized by a slight depolarization (2-4 mV) and increase in resistance was observed approximately 2 min after GABA application. Following bath application of the Cl- channel blocker picrotoxin (100 microM), this increase in resistance was observed within 20 s of GABA application, suggesting that it was normally masked by the faster Cl- conductance. The magnitude of the resistance increase in response to GABA was enhanced when the B-cell was held at depolarized membrane potentials (-40 to -20 mV), but was eliminated if Ca2+ was removed from the extracellular bath, or if the non-specific protein kinase inhibitor H7 (100 microM) was added to the extracellular bath. In a final experiment, GABA application was paired with a transient (10 s) depolarization of the B-cell (to -20 mV).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718552 TI - Thalamically projecting cells of the lateral cervical nucleus in monkey. AB - The number, location, and morphology of thalamically projecting lateral cervical nucleus (LCN) cells were determined in monkey using retrograde transport of wheatgerm agglutinin-conjugated horseradish peroxidase. These data were compared to the total population of LCN neurons as determined by Nissl stain. In 4 Macaca fascicularis and one Saimiri sciureus the average size of the thalamic projection from LCN was found to be 506 +/- 94 cells contralateral to the injections. Thalamically projecting LCN neurons were located between the lower medulla and the third cervical segment; approximately 90% of these cells were in the first two cervical segments. Morphologic analysis of thalamically projecting LCN cells showed that they were smaller in size, and more oblong in shape in caudal regions of the nucleus. In 3 macaques, the average total number of LCN cells was determined to be 1617 +/- 908 on one side, in Nissl material. In these Nissl stained preparations LCN neurons were found as far caudal as the fourth cervical segment; 68% were located in the first two cervical segments. Hence, thalamically projecting LCN neurons in the monkey are located in the rostral portion of the nucleus and comprise about one-third of the total population. Comparison of these data with reports in the literature imply that, unlike the cat, the major projection from LCN in monkeys is to the mesencephalon rather than to the thalamus. PMID- 1718553 TI - Localization of substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion. AB - The immunocytochemical distribution of substance P (SP) in guinea pig vestibular endorgans and the vestibular ganglion was investigated. Two kinds of SP immunoreactive fibers were distinguished. Most were thick, and found around or beneath sensory hair cells. These SP-immunoreactive fibers were distributed predominantly on the slope of the crista and the peripheral region of the macula. By electron microscopy, we confirmed this type of SP-like immunoreactivity to be restricted within primary afferent neurons. Some vestibular ganglion cells also showed SP-like immunoreactivity, suggesting that SP is present in some primary afferent neurons, and is involved in afferent neurotransmission. The characteristic distribution of SP may indicate functional differences within each endorgan. The other group of immunoreactive nerve fibers, varicous thin fibers, could be found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels of the vestibular ganglion. These fibers may have a different origin, and have an influence on blood flow and certain other functions. PMID- 1718554 TI - Histochemically demonstrable phosphotyrosine protein phosphatase in the rat hippocampal formation. AB - Using o-phospho-L-tyrosine as substrate, a possible localization of phosphotyrosine protein phosphatase (PTPPase) activity was histochemically demonstrated in the rat hippocampal formation. The PTPPase activity was found in almost all layers of the hippocampal formation, with a high activity in the stratum moleculare. The activity was inhibited by vanadate and molybdate, but not by NaF and Zn2+. The activity was localized in the dendritic cytoplasm, particularly on the postsynaptic density, of hippocampal neurons. PMID- 1718555 TI - Serotonergic sprouting is induced by dopamine-lesion in substantia nigra of adult rat brain. AB - We have previously extracted a serotonin (5-HT) neurotrophic supernatant from the 5,7-DHT lesioned hippocampus. The current study shows that a new 5-HT neurotrophic signal was monitored in the striatum and nigra after DA-denervation. Such a signal may be involved in the heterotypic sprouting. Dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected directly into the substantia nigra of adult rats. Two months after surgery, immunocytochemical staining showed that tyrosine hydroxylase (TH)-positive cell bodies had mostly disappeared in the substantia nigra, and TH-positive terminals in the striatum were almost completely depleted. Meanwhile, the 5-HT fibers, which exist in the same areas with low density, sprouted in the nigra as well as in the striatum and became dense. Normally 5-HT fibers innervate the striatum sparsely and the globus pallidus densely with sharp delineation (in the control side), and become dense across both areas with no appreciable delineation (in the lesion side). The increase of 5-HT fibers was more prominent in the posterior than in the anterior striatum. A significant increase in 5-HT and 5-HIAA levels was also evident in the posterior striatum when the decrease in DA level exceeded 90% in the nigra and striatum. In addition, we found that induction of 5-HT sprouting requires a greater than 90% decrease of DA level. Current data support that 6-OHDA injection in the substantia nigra of adult rats triggered a trophic signal or removed an inhibition for the growth of 5-HT neurons which responded with sprouting in the nigra as well as in the striatum. PMID- 1718556 TI - Antagonistic effects of somatostatin and substance P on respiratory regulation in the rat ventrolateral medulla oblongata. AB - Substance P (SP) in the dose range 0.75-1.5 nmol exerts a potent stimulatory effect on ventilation after microinjection into the rat ventrolateral medulla oblongata (VLM; n. reticularis lateralis, n. paragigantocellularis lateralis). A significant but less pronounced effect is also seen in the dorsal medulla (DM; n. tractus solitarius). Somatostatin (0.6-1.8 nmol) inhibited ventilation and induced apnoea after microinjection into the VLM but not the DM. Serial microinjections of the two peptides showed a reciprocal antagonistic action in the VLM but not in the DM. The apnoea-inducing effect of SOM was blunted by SP while SOM reduced the ventilatory stimulation induced by SP. Extracellular single unit recordings were performed following the microiontophoretic application of SP and/or SOM to respiratory-related and non-respiratory-related neurons in the VLM and DM. Although a heterogeneous population of neurons were recorded from, the majority of respiratory-related units in the VLM responded with excitation to SP and inhibitory to SOM. A direct interaction between the peptides was seen in some respiratory-related units. The neurons not responding to either of the peptides were usually non-respiratory. Dorsal to the VLM, the type of response to the two peptides was less likely to be antagonistic and a wider distribution of response types were recorded. The results indicate a direct physiological antagonism between SP and SOM regarding their effects on respiratory regulation elicited in the VLM. PMID- 1718557 TI - Substance P (neurokinin-1) receptor mRNA is selectively expressed in cholinergic neurons in the striatum and basal forebrain. AB - In the striatum substance P (neurokinin-1) receptor, mRNA is selectively localized in large neurons that also express mRNA encoding choline acetyltransferase (ChAT) by in situ hybridization histochemistry. Substance P receptor mRNA is also localized in ChAT mRNA-containing neurons in the medial septum and basal forebrain cell groups. Thus, in the rat forebrain the substance P receptor appears to be expressed selectively by cholinergic neurons. Striatal neurons that contain substance P also utilize gamma-aminobutyric acid (GABA) as a transmitter. These neurons make synaptic contact with striatal cholinergic neurons, which are shown here to express the substance P receptor, and with other GABAergic neurons in the striatum and substantia nigra, which express GABA receptors but not substance P receptors. This suggests that individual striatal neurons may differentially affect target neurons dependent on the receptors expressed by those target neurons. PMID- 1718558 TI - Morphology of rostral medullary neurons with intrinsic pacemaker activity in the rat. AB - Neurons with regular ongoing activity attributable to intrinsic pacemaker properties were recorded in coronal tissue slices within the nucleus reticularis rostroventrolateralis of the rat medulla oblongata (RVL). The cells were injected with horseradish peroxidase or Lucifer yellow and their dendritic and proximal axonal characteristics were investigated (n = 15). These small-to-medium-sized neurons had a simple dendritic arborization (3-6 primary dendrites branching up to 3 times) apparently confined within the limits of nucleus RVL and with limited extension in the rostrocaudal direction. Their axons originated either from the cell body or from a primary dendrite and coursed in a dorsomedial direction without giving rise to local arborizations. It is concluded that RVL pacemaker neurons, presumed to represent a non-adrenergic class of sympathoexcitatory premotor neurons, exhibit characteristics reminiscent of the archetypal 'reticular core' neurons. PMID- 1718559 TI - Immortalized retinal neurons used as immunogen for the generation of cell specific antisera. AB - We have recently described a class of immortalized neurons which were derived from retinal tumors induced in PNMT-SV40 transgenic mice (TgBri59)9. These neurons possess a differentiated neuronal phenotype which includes the elaboration of extensive neurite processes and the expression of markers specific for amacrine and horizontal neurons, as well as the expression of the neurofilament triplet proteins. As these 'RT-1' neurons are derived from a restricted set of retinal neurons, they represent an enriched source of immunogen for the production of cell-specific antisera. Therefore, we have used RT-1 cell cultures to generate polyclonal antisera in rabbits. Two of these antisera have been characterized in immunocytochemical and Western blotting experiments using normal mouse and rat tissues. The antisera recognize neurons in the inner nuclear layer and particularly in the ganglion cell layer in the normal retina and cells of the adrenal medulla. These data indicate that specific cell lines derived from transgenic animals provide a rich source of antigen for the production of cell specific antibodies. These antibodies should prove valuable for studies of retinal development and function. PMID- 1718560 TI - Effects of conditioned taste aversion on extracellular serotonin in the lateral hypothalamus and hippocampus of freely moving rats. AB - This study used microdialysis to monitor extracellular levels of 5-HT and its metabolite, 5-hydroxyindole acetic acid (5-HIAA) in the lateral hypothalamus (LH) and hippocampus of freely moving rats that had developed a CTA to a 2.5 mM saccharin solution (CS) following its pairing with illness induced by lithium chloride (US). Results showed that oral infusion of the saccharin CS significantly enhanced extracellular LH 5-HT in animals that had developed a taste aversion compared with control groups, including unconditioned (CS-no US) and pseudoconditioned (no CS-US) subjects. As an anatomical control, the hippocampus was identified based on previous research suggesting that it is not integrally involved in CTA learning or retrieval and that 5-HT in this brain site does not directly mediate feeding behavior but is closely correlated with arousal. In contrast with the results obtained in the LH, hippocampal 5-HT was not preferentially elevated in subjects in the CTA group but rather was increased to the same extend in both CTA and control groups after saccharin infusion. Moreover, the increase in LH 5-HT for the CTA group was nearly twice that observed in the hippocampus for any group. Acute administration of LiCl elevated extracellular 5-HT to similar levels in both sites, well above the changes observed following conditioning. 5-HIAA was unaffected in either brain site by oral infusion of saccharin solution or injection of LiCl.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718561 TI - Neurofilament reassembly in vitro: biochemical, morphological and immuno-electron microscopic studies employing monoclonal antibodies to defined epitopes. AB - The reassembly process of purified native (phosphorylated) and enzymatically dephosphorylated bovine neurofilament (NF) subunits was studied to delineate how NF triplet proteins assemble together into intermediate-size filaments in vitro. We determined the time course for reassembly, the ultrastructural characteristics of reassembled NFs, and the topographical disposition of NF protein subdomains within reassembled NFs using quantitative biochemical techniques, negative staining and immunoelectron microscopy. Our data indicate that: (1) approximately 50% of the purified NF subunit proteins assembled within 30 min from the start of reassembly into 10- to 12-nm filaments, and by 90 min approximately 85-90% of the NF proteins reassembled, (2) low concentrations (0.15-0.5 mg/ml) of purified NF proteins were able to reassemble into long filaments, (3) the rate and ability of native phosphorylated and dephosphorylated NF proteins to assemble into NFs were comparable, (4) negative staining revealed a periodicity of approximately 18-22 nm and a protofilamentous substructure in reassembled NFs, (5) immunoelectron microscopy using domain specific anti-NF monoclonal antibodies (mAbs) to all 3 NF proteins demonstrated specific labeling patterns corresponding to the spatial relationships of subdomains within reassembled NFs, and (6) negative staining and immunolabeling revealed that reassembled NFs are very similar to isolated native NFs. We conclude that purified mammalian axonal NF triplet proteins, independent of their phosphorylation state, rapidly and efficiently reassemble in vitro to generate characteristic 10-nm filaments. Furthermore, immunological analysis reveals that the rod domains of NF-H, NF-M and NF-L are buried within the reassembled NF, whereas the head domain of NF-M and the tail domains of all 3 NF proteins remain exposed following reassembly. PMID- 1718562 TI - Individual neurofilament subunits reassembled in vitro exhibit unique biochemical, morphological and immunological properties. AB - Purified bovine neurofilament (NF) subunit proteins were reassembled in vitro to form either homopolymeric or heteropolymeric intermediate-sized filaments using single or paired combinations of NF triplet proteins. Using conditions established for the reassembly of bovine NF triplet proteins, we demonstrated that the low Mr NF subunit (NF-L) alone and in combination with the middle Mr NF subunit (NF-M) reassembled very efficiently, i.e. greater than 95% of these proteins formed filaments within 90 min from the start of reassembly. In contra distinction, the high Mr NF subunit (NF-H) alone and in combination with NF-M or NF-L underwent reassembly to a lesser extent, i.e. 62-88% of these proteins reassembled within 90 min. Immunolabeling of the reassembled NF polymers revealed striking differences in the organization of rod domain determinants. Specifically, antibodies specific for epitopes in the rod domains of NF-H, NF-M and NF-L failed to bind heteropolymeric filaments but recognized rod domains in the homopolymers. In contrast, antibodies specific to head and tail domains of all NF proteins labeled the reassembled hetero- and homopolymeric NFs. Double labeling of heteropolymers demonstrated that pairs of different NF subunits coassembled into intermediate-sized filaments. Our results also showed that only copolymeric filaments of NF-L and NF-M, but not NF-L/NF-H and NF-M/NF-H were able to form long and stable 10-nm wide filaments. These observations provide new insights into the requirements for stable filament formation from NF subunits. In particular, they support the notion that only NF-L/NF-M, but not NF-L/NF-H or NF M/NF-H might assemble into a stable filamentous network in vivo. PMID- 1718563 TI - Origin of cuneate projections to the anterior and posterior lobes of the rat cerebellum. AB - The present study was carried out to analyze the topography of projections from external cuneate nucleus to the anterior and posterior lobes of the cerebellum and to investigate whether projections to the two lobes come from different cuneocerebellar neurons or from branching axons of the same cells. We used retrograde double-labeling techniques to estimate the incidence of cuneocerebellar neurons projecting to both anterior and posterior lobes via axon collaterals. Cells sending their axons to the lobus anterior were about twice the number of those projecting to the posterior lobe. The double-labeled cells were about 1/7 of all labeled neurons and were located mainly in the more lateral half of the nucleus. Therefore, the two lobes of the cerebellum are likely to receive common information from these cells, but different information from the separate populations of cuneocerebellar neurons that project only to one lobe or the other. PMID- 1718564 TI - Pargyline and gamma-butyrolactone enhance tyrosine hydroxylase immunostaining of nigrostriatal axons. AB - Administration of pargyline or gamma-butyrolactone enhanced immunostaining of tyrosine hydroxylase immunoreactive axons in the striatum. The qualitative characteristics of the enhancement were compound dependent and the enhancement was not associated with an increase in the amount of tyrosine hydroxylase within the striatum. These results demonstrate the pharmacological enhancement of immunostaining for neurotransmitter synthetic enzymes in the absence of increased synthesis. PMID- 1718565 TI - Presence of calpain II immunoreactivity in senile plaques in Alzheimer's disease. AB - Calpains are calcium-dependent neutral cysteine proteinases. We utilized a specific anti-calpain II antibody to examine immunohistochemically whether calpain II is associated with pathological changes in Alzheimer's disease (AD). Calpain II was mainly expressed in the neurons in control human brains. In AD brains, intense immunoreactivity was present in the dystrophic neurites of senile plaques. These results suggest that calpain II is involved in the pathogenesis of AD. PMID- 1718566 TI - Localization of the substance P-induced cardiovascular responses in the rat hypothalamus. AB - Intracerebroventricular injection of substance P (SP) has been reported to induce a typical cardiovascular defense response characterized by an increase in blood pressure, heart rate, sympathetic efferent activity, hindlimb vasodilatation and mesenteric vasoconstriction. In this study we employed microinjections of SP to localize the hypothalamic areas in which SP elicits the activation of the cardiovascular system. SP (550 pmol) injected into the anterior hypothalamus (AH) produced, after a short latency, a marked increase in mean arterial pressure and heart rate. In the ventromedial hypothalamus, the magnitude of the cardiovascular response to SP was identical to that in the AH, but the response was delayed. SP injected into the posterior hypothalamus failed to induce any cardiovascular response. These results suggest that the anterior and ventromedial parts of the hypothalamus are responsible for eliciting the central cardiovascular effects of SP in conscious rats. PMID- 1718567 TI - Spinocerebellar projection in the meander tail mutant mouse: organization in the granular posterior lobe and the agranular anterior lobe. AB - The cerebellum of the mutant mouse, meander tail, is characterized by normal cytoarchitecture posteriorly and abnormal, agranular cortex anteriorly. Anterograde WGA-HRP tracing analysis of the spinocerebellar projection reveals typical mossy fiber labeling posteriorly in lobule VIII. However, in the anterior cortex, a finer, more diffuse pattern of labeling is seen, unlike the distinct banded pattern of mossy fiber rosettes which characterizes the spinocerebellar projection in the normal animal. PMID- 1718568 TI - Acetylcholine-activated ionic currents in isolated paratracheal ganglion cells of the rat. AB - The electrophysiological property of acetylcholine (ACh)-induced current (IACh) was studied in paratracheal ganglion cells freshly isolated from rat trachea under whole-cell voltage-clamp condition. IACh consisted of an initial transient peak component and a successive steady-state plateau one. The peak component increased in a sigmoidal fashion with increasing ACh concentration. The IACh was mimicked by nicotine. The current-voltage relationship for the IACh showed inward rectification at the positive membrane potentials beyond the reversal potential (EACh). In a K(+)-free solution, the EACh was close to the Na+ equilibrium potential. The IACh was blocked by either D-tubocurarine or atropine. The ion selectivity of ACh-activated channels to various monovalent cations was weak, and similar to those of other preparations. It was concluded that the IACh in rat paratracheal ganglion cells was mediated by nicotinic receptor activation. PMID- 1718569 TI - Demonstration of a novel neurofilament associated antigen with the neurofibrillary pathology of Alzheimer and related diseases. AB - A monoclonal antibody, termed NFT200, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer brain. The antigen to which NFT200 is directed was expressed in the paired helical filaments of NFT in sporadic and familial Alzheimer disease (AD), in the straight filaments of NFT in AD, progressive supranuclear palsy and of Pick bodies, and the NFT in several other conditions such as Parkinson-dementia complex of Guam and subacute sclerosing panencephalitis. Granulovacuolar degeneration of AD was also labeled with NFT200. Hirano bodies and amyloid deposits in AD, as well as Lewy bodies of idiopathic Parkinson disease lacked in the antigen. The NFT200-antigen was also expressed as a phosphatase-insensitive antigen in normal neurofilaments found in spinal cord and peripheral nerve axons but was absent from the perikaryal accumulation of neurofilaments induced by aluminum intoxication. Nevertheless, immunoblot studies failed to detect the NFT200 in isolated preparations of the neurofilament proteins, MAP-2, tau, ubiquitin or A4-amyloid peptide. The results indicate that the NFT200 monoclonal antibody is directed against a phosphatase-insensitive epitope of an axonal protein associated with neurofilaments but is labile to isolation and expressed as a stable epitope of a 200 kDa component of NFT. PMID- 1718570 TI - Microinjections of calcitonin gene-related peptide within the trigeminal subnucleus caudalis of the cat affects adrenal and autonomic function. AB - To assess the role of calcitonin gene-related peptide (CGRP) within the trigeminal subnucleus caudalis (Vc) on adrenal and autonomic function, microinjections were directed at different laminae of Vc in chloralose anesthetized cats. Microinjections of CGRP (5 pmol, 100 nl) into laminae I-II increased significantly the adrenal secretion of epinephrine, adrenal blood flow, adrenal vascular conductance, mean arterial pressure and heart rate. Injections of CGRP into laminae V-VI decreased significantly the adrenal secretion of epinephrine, however, other measured variables were not affected. To examine if CGRP interacts with substance P within Vc to modify adrenal and autonomic function, subthreshold doses of each peptide were injected alone and simultaneously. Combined subthreshold doses of CGRP and substance P injected into laminae V-VI, but not into laminae I-II or III-IV, evoked increases in arterial pressure and in heart rate that exceeded the responses seen after injection of either peptide alone. The adrenal secretion of catecholamines was not affected by individual or combined subthreshold doses of either peptide, regardless of the laminar site of injection. These data suggest that release of CGRP within laminae I-II of Vc alters adrenal and autonomic function via mechanisms separate from those that mediate substance P-evoked responses. In contrast, CGRP and substance P may act, at least in part, through a common neural substrate within the deeper laminae of Vc to modify arterial pressure and heart rate. Thus, multiple subpopulations of peptide-responsive neurons in the medullary dorsal horn likely contribute to the reflex adrenal and autonomic responses that often accompany nociception. PMID- 1718571 TI - SP-like immunoreactivity in the primary trigeminal neurons projecting to the nucleus tractus solitarii. AB - Using the double-labeling method of retrograde transport of HRP combined with PAP immunocytochemistry, the neurons containing SP-like immunoreactivity (SPLI) of the trigeminal nerve projecting to the nucleus tractus solitarii (NTS) were examined. HRP-SP double-labeled neurons in the trigeminal ganglion, mainly small or middle-sized, were found after the NTS or the lingual nerve was injected with HRP followed by SP-immunoreaction. These findings demonstrate that the projection of the trigeminal nerve to the NTS contains SPLI. PMID- 1718572 TI - Persistent elevation in GABAA receptor subunit mRNAs following social stress. AB - Stress is associated with alterations in GABA/benzodiazepine binding and function. We evaluated effects of social stress on GABAA receptor subunit (alpha 1 and gamma 2) mRNAs by Northern hybridization. In cortex, no change was observed in either subunit mRNA immediately after stress, but a 4 hours mRNAs for both subunits were increased. These changes persisted for 72 hours after stress, and returned to baseline levels at 7 days. No changes in mRNAs were observed in sham treated mice. No changes in either subunit mRNA were observed in stressed or sham treated mice in cerebellum or hippocampus. In undefeated resident mice, mRNAs for both subunits in cortex were unaffected at 24 hours after the stress episode. Social stress is associated with increases in GABAA receptor alpha 1 and gamma 2 subunit mRNAs in cortex. PMID- 1718573 TI - Anterograde transport of lucifer yellow-dextran conjugate. PMID- 1718574 TI - Thalamic afferents of the rat infralimbic and lateral agranular cortices. AB - The infralimbic cortex is a visceromotor area of the cortex. To define the thalamic afferents of this area, contrast them with those of the lateral agranular cortex, a somatic motor region, and assess the degree to which the thalamus might coordinate the activity of these cortical areas through axon collaterals, we conducted a retrograde fluorescent double labeling study using bisbenzimide and Fast Blue. Injections into infralimbic cortex resulted in labeling in the mediodorsal, intralaminar, and midline nuclei. Injections into lateral agranular cortex resulted in labeling in the ventrolateral, ventrobasal, ventromedial, and intralaminar nuclei. There was almost no overlap in the thalamic labeling following injections into these two cortical areas. The pattern of labeling following infralimbic injections is discussed in terms of the possible function of the midline thalamic nuclei as a relay for visceral sensory information. The labeling in mediodorsal nucleus following infralimbic cortex argues for including this area in the definition of rodent prefrontal cortex. In addition, the results suggest that the role of the thalamus in coordinating the activity of these cortical areas is minimal. PMID- 1718575 TI - Pyramidal cells in rat temporoauditory cortex project to both striatum and inferior colliculus. AB - A retrograde fluorescent double-labeling technique was employed to examine whether the cortico-striate fibers are collaterals of the corticofugal fibers directed to the inferior colliculus in the rat. Following injections of two different fluorescent tracers into the striatum and the inferior colliculus, double-labeled cells were found in layer V pyramidal cells of the temporal cortex. These double-labeled cells were located mostly in the area corresponding to the rat primary auditory cortex, and constituted 6.4% of the pyramidal cells projecting to the inferior colliculus. This study has revealed the existence of a common innervation of the basal ganglia and the auditory system by the pyramidal cell of the auditory cortex. PMID- 1718576 TI - Direct amygdaloid projections to the superior salivatory nucleus: a light and electron microscopic study in the cat. AB - Amygdaloid projections to the superior salivatory nucleus (SSN) were investigated in the cat by using the anterograde and retrograde tracing techniques of horseradish peroxidase (HRP). After HRP injections were made into the lingual nerve, retrogradely labeled SSN neurons were located in the lateral tegmental field medial to the spinal trigeminal nucleus from the middle level of the superior olivary nucleus to the caudal level of the facial nucleus. These labeled neurons, triangular, oval or polygonal in shape, were small to medium-sized (12 29 microns) and formed loosely packed clusters. In further HRP studies, HRP injections were made into the amygdala and in the reticular formation containing the SSN neurons. The results suggested that the SSN receives direct afferents from the central nucleus of the amygdala with ipsilateral predominance. Final proof of such direct connections from amygdala to the SSN can be obtained only by electron microscopic study. Therefore, HRP injections were made into the lingual nerve and in the amygdala in the same animal and electron microscopic observations were carried out on the SSN. It appeared that anterogradely labeled amygdalo-tegmental fibers formed axosomatic and axodendritic synaptic contacts with retrogradely labeled SSN neurons. PMID- 1718577 TI - BOMM regimen for the treatment of advanced head and neck carcinoma. AB - Eighty patients with Stage III and IV head and neck cancer were treated with a four-drug regimen consisting of bleomycin, vincristine, methotrexate, and mitomycin-C (BOMM regimen). Of 69 patients evaluable for response, 7 complete and 40 partial responses were achieved, with an overall response rate of 68%. The median duration of response was 13 weeks (range 3-41 weeks). Anorexia, lassitude, febrile reactions, and myelosuppression were major side effects. The BOMM regimen produced a response rate significantly better than VCR, BLM, and MTx combination (VBM) which we previously reported with improved response duration. This study demonstrates that the BOMM regimen, which can be administered on an outpatient basis, achieved a response rate comparable to cisplatin-containing regimens. The BOMM regimen may be beneficial to patients with poor general condition and/or marginal renal dysfunction who are in high risk of toxicity-associated cisplatin containing regimens. PMID- 1718578 TI - BOMM: new solution or old problem? PMID- 1718579 TI - Agranulocytosis associated with anti-thyroid drug in patients with Graves' thyrotoxicosis--report of 11 cases. AB - Retrospective analysis of 11 Chinese patients with Graves' thyrotoxicosis developing agranulocytosis during anti-thyroid treatment was done. Seven of them received methimazole and 4 received carbimazole. None of the 11 patients had taken propylthiouracil. The major chief complaints were high fever (100%), chillness (91%), and sore throat (73%). The duration of drug treatment prior to the detection of agranulocytosis ranged from 13 to 63 days (mean +/- 1SE: 33.1 +/ 16.1). At the time of agranulocytosis detected, the peripheral leukocyte counts were 0.5 to 2.1 X 1000/mm3 (mean +/- 1SE: 1.05 +/- 0.47 X 1000/mm3), absolute neutrophil counts 0 to 450/mm3 (mean +/- 1SE: 54.27 +/- 132.12/mm3), and hemoglobin 8.2 to 15.9 g/dl (mean +/- 1SE: 11.85 +/- 2.24 gm/dl). Three of the 11 patients had positive bacterial blood cultures. The recovery time of absolute neutrophil counts above 500/mm3 ranged from 3 to 25 days (mean +/- 1SE: 10.5 +/- 6.6) after discontinuation of antithyroid drugs. Mortality was found in 2 of them (18%). PMID- 1718580 TI - Acute basophilic leukemia. A case report. AB - A case of acute nonlymphocytic leukemia (ANLL) with primitive basophilic differentiation is presented. The patient had no antecedent history or concomitant presence of chronic myelogenous leukemia. The leukemic blasts constituted 83% of the peripheral white blood cells and more than 90% of the marrow nucleated cells. Cytoplasmic vacuoles were found in some leukemic cells. About half the leukemic cells showed a few azurophilic granules stained with Wright's stain, whereas exhibited a faint pinkish hue around the cells without cytoplasmic granules (water-soluble granules) by Riu's stain. The cytoplasmic granules failed to be stained with peroxidase but stained positively with toluidine blue. The former result could lead one to misclassify the case as lymphoid leukemia, but the characteristic finding of basophilic cells in Riu's stain should direct one to make the diagnosis of ANLL with basophilic differentiation. The cytochemical findings of this case suggested that basophilic differentiation should be considered when leukemic cells show peroxidase-negative granules. Riu's stain and toluidine blue stain are useful to make the correct diagnosis. PMID- 1718581 TI - The role of axonal transport in neurodegenerative disease spread: a meta-analysis of experimental and clinical poliomyelitis compares with amyotrophic lateral sclerosis. AB - ALS symptom spread results from local spread of the neuronal degeneration because contiguous areas are more quickly involved than non-contiguous areas. Local spread to contiguous areas of motor neuron dysfunction is faster at the brainstem, cervical and lumbar regions than spread to non-contiguous areas. The time for caudal-rostral symptomatic spread of ALS to involve a distant region is a function of the distance of that region from the site of onset. The time for spread to the bulbar region is shorter following arm onset than leg onset. Spread to non-contiguous areas is faster within the spinal cord than from the spinal cord to the bulbar region. These kinetics are consistent with axonal transport of the etiological agent in a manner similar to spread of poliovirus in poliomyelitis patients. Spread from the bulbar region to the spinal cord, on the other hand, occurs faster than symptom spread from the limb region to the bulbar region in limb onset patients. This rapid limb involvement following bulbar onset is more dramatic in males compared with females. Females with leg onset, on the other hand, show more rapid involvement of the opposite leg, either arm or bulbar structures than males. Gender effects may determine the course of ALS depending on the original site of onset. PMID- 1718582 TI - Second-hand tobacco smoke: early insights. PMID- 1718583 TI - Helix pomatia agglutinin binding activity is a predictor of survival time for patients with gastric carcinoma. AB - The authors used helix pomatia agglutinin (HPA) staining to examine 163 primary gastric carcinoma isolates, using the avidin-biotin-peroxidase complex method. The positive HPA staining rate was 59% (96/163) for the primary tumors, and the positive staining correlated well (with statistical significance) with tumor enlargement, penetration, lymphatic invasion, and metastasis (P less than 0.01), and with infiltrative spread (P less than 0.05). Patients with gastric cancer showing positive HPA staining had a lower survival rate (P less than 0.001), particularly when the cancer cells were present on the serosal surface. Careful follow-up and intensive postoperative therapy are required for patients with an HPA-positive advanced gastric carcinoma. PMID- 1718584 TI - Inhibition of cell proliferation, protein kinase C, and phorbol ester-induced fos expression by the dihydropyridine derivative B859-35. AB - The dihydropyridine derivative B859-35 inhibits phospholipid- and calcium dependent protein kinase C (PKC) in cell-free extracts from NIH3T3 cells. Inhibition is competitive with regard to phosphatidylserine. At 1 microM phosphatidylserine, half-maximal inhibition (IC50) is obtained at approximately 2.5 microM B859-35. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-dependent activation of the Na+/H+ antiporter was used to determine whether the enzyme is also affected in intact cells. The activity of the antiporter was monitored by following the dimethylamiloride-sensitive cytosolic alkalinization. It is demonstrated that B859-35 depresses the TPA-induced alkalinization with an IC50 of 5 microM, indicating that PKC in intact cells and the enzyme in cell-free extracts are equally sensitive to the drug. TPA-induced expression of the c-fos gene was used as an additional marker for intracellular PKC activity. Activation of c-fos expression was determined by measuring chloramphenicol acetyltransferase (CAT) activity in cells transfected with a c-fosCAT construct in which the CAT gene is expressed under the control of the endogenous human c-fos promoter. The studies revealed that 2.5 microM B859-35, a concentration equivalent to the IC50 in cell-free extracts, significantly depresses TPA-induced c-fosCAT expression. B859-35 inhibited cellular proliferation of NIH3T3 cells with an IC50 of approximately 5 microM. This is close to the IC50 for the anti-PKC activity of B859-35. It is suggested that the inhibition of PKC contributes to the growth inhibition following exposure to B859-35. PMID- 1718585 TI - The breast tumor-associated epitope defined by monoclonal antibody 3E1.2 is an O linked mucin carbohydrate containing N-glycolylneuraminic acid. AB - The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5 acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins. PMID- 1718586 TI - Insulin-like growth factor I rapidly induces tyrosine phosphorylation of a Mr 150,000 and a Mr 160,000 protein in highly metastatic mouse colon carcinoma 26 NL 17 cells. AB - Insulin-like growth factor I (IGF-I) stimulates the proliferation of highly metastatic NL-17 cells to a greater extent than poorly metastatic NL-44 cells, both of which are derived from mouse colon carcinoma 26. The NL-17 cells have been compared with NL-44 cells for the signal transduction pathway of IGF-I. IGF I receptors of both cell types were identified by affinity labeling, and there was no significant difference between the two cell types in the amount or the autophosphorylation activity of the IGF-I receptors. However, when IGF-I dependent tyrosine phosphorylation of cellular components was examined, remarkable tyrosine phosphorylation of proteins with molecular weights of 150,000 (pp150) and 160,000 (pp160) was found in NL-17 cells. In contrast, this phosphorylation stayed at significantly lower levels in NL-44 cells than in NL-17 cells. The phosphorylation of pp150 and pp160 was induced within 10 s after the addition of IGF-I and reached its maximal level by 30 s. After the removal of IGF I, the phosphorylation of pp150 and pp160 was reduced to the basal level within 30 min. This phosphorylation was not induced by platelet-derived or epidermal growth factor. The pp150 and pp160 were not absorbed by wheat germ agglutinin agarose. They were found in the soluble fraction of cytoplasm but not in the membrane or the cytoskeleton. The pp150 and pp160 might be endogenous substrates of IGF-I receptor kinase. These results suggest that tyrosine phosphorylation of pp150 and pp160 mediates the higher proliferative response of NL-17 cells to IGF I. PMID- 1718587 TI - alpha-Interferon down-regulates epidermal growth factor receptors on renal carcinoma cells: relation to cellular responsiveness to the antiproliferative action of alpha-interferon. AB - The CaKi-I line of renal carcinoma (RC) cells is highly sensitive to the antiproliferative effect of human leukocyte interferon (IFN-alpha). These RC cells express high numbers of cell surface receptors for epidermal growth factor (EGF), and EGF stimulates their proliferation. IFN-alpha blocks EGF-stimulated proliferation of these cells and down-regulates EGF receptors (EGFR) by inhibiting EGFR synthesis. Although EGF stimulates the proliferation of RC cells resistant to the antiproliferative action of IFN-alpha, IFN-alpha treatment does not block the EGF-stimulated proliferation of these cells and has no effect on EGFR expression. Thus, the down-regulation of EGFR is specific for RC cells sensitive to IFN-alpha. While IFN-alpha does not affect the level of total cellular message or total polyadenylated message for the EGFR, IFN-alpha treatment decreases the level of cytoplasmic EGFR message. Analysis of polysome distribution of cellular mRNAs indicates that IFN-alpha treatment results in an accumulation of EGFR mRNA in lighter polysome fractions, consistent with a partial block in translational elongation. Thus, IFN-alpha regulates the expression of EGFR and possibly other growth-related proteins by post transcriptional mechanisms, which may play an important part in the complex inhibitory action of IFN-alpha on RC proliferation. PMID- 1718588 TI - Combination therapy: lonidamine, hyperthermia, and chemotherapy against the RIF-1 tumor in vivo. AB - Lonidamine enhances the cytotoxicity in vitro of several conventional antitumor drugs as well as that of hyperthermia (HT). We have investigated the possibility that such enhancement can also be demonstrated in vivo against the RIF-1 tumor system. Two assays were used to examine antitumor activity: tumor growth delay and clonogenicity of cells obtained from tumors from treated animals. We used drug (and HT) doses that by themselves did not achieve significant cell killing. The drugs whose interaction with lonidamine was tested were: cis diamminedichloroplatinum (CDDP), mitomycin C (MMC), bleomycin, 5-fluorouracil, and nitrosourea. Of these only CDDP and MMC yielded positive data. Both assays gave essentially the same results, showing that antitumor activity reflected direct cell killing. CDDP and MCC activity was also enhanced by HT. When we combined all three modalities, however, the results of the trimodality therapies were no better than that of individual bimodality treatments. These last results suggest that lonidamine and HT have similar mechanisms, most likely inhibition of repair of DNA damage. Our data do suggest that lonidamine may have a role in multidrug therapies that include either CDDP or MMC as a component of the treatments. PMID- 1718589 TI - Development of resistance to the growth inhibitory effects of transforming growth factor beta 1 during the spontaneous transformation of rat liver epithelial cells. AB - The temporary maintenance of a rat liver epithelial cell population at confluence before passaging followed by periods of rapid proliferation resulted in the generation of spontaneous transformants after about 108 population doublings. The appearance of morphologically aberrant transformants correlated directly with an increased resistance of the population to the growth inhibitory effects of transforming growth factor beta 1 (TGF-beta 1). Clonal cell lines derived from the transformants were resistant to TGF-beta 1 dependent inhibition of DNA synthesis. These cell lines were also highly tumorigenic and aneuploid, with characteristic gross chromosomal abnormalities, and they expressed a number of phenotypic markers common to rat liver epithelial cells transformed by oncogenes or chemicals. In contrast, apparently normal looking cell lines cloned from the same population were nontumorigenic and near diploid, with few chromosomal abnormalities, and they were as sensitive to TGF-beta 1 as early passage normal rat liver epithelial cells. Morphologically normal late passage rat liver epithelial cells were sensitive to transformation by the DNA hypomethylating agent 5-aza-2-deoxycytidine, in contrast to earlier passage cells, and this transformation was accompanied by the development of resistance to the growth inhibitory effects of TGF-beta 1. These findings suggest that acquisition of resistance to the effects of growth inhibitors such as TGF-beta 1 is an important and possibly essential stage in the spontaneous transformation of rat liver epithelial cells. PMID- 1718590 TI - Coexpression of cytokeratins characteristic for myoepithelial and luminal cell lineages in rat 13762NF mammary adenocarcinoma tumors and their spontaneous metastases. AB - We used immunohistochemical procedures to study the cellular expression and distribution of cytokeratins (CKs) in rat 13762NF mammary adenocarcinoma cells growing at mammary fat pad sites and at spontaneous lymph node and lung sites. In order to establish CK distribution in normal rat mammary epithelia, immature, resting, and lactating rat mammary glands were probed with a panel of monospecific antibodies that recognize individual CKs. Basal/myepithelial cells were distinguished by expression of CKs 5 and 14 and coexpression of vimentin from luminal cells, which expressed CKs 8, 18, and 19. Antibody to CK 7 recognized luminal epithelium of immature and resting, but not lactating, mammary glands. Myoepithelial cells of lactating mammary gland were weakly recognized by antibodies to CKs 7 and 19. Tumors formed by cell lines and clones derived from parental 13762NF tumor (MTPa, MTC, MTA, and MTF7) were not recognized by any of the anti-CK antibodies. Only vimentin was expressed in these tumors and their metastases. In tumors and metastases generated from cell lines and clones derived from lymph node (MTLY) and lung metastases (MTLn2 and MTLn3) of the 13762NF tumor we observed heterogeneous CK phenotypes. Expression of CKs 5 and 18 was greatly reduced or lacking, while CK 14 was coexpressed with CKs 7, 8, and 19 with or without vimentin. Tumors from the highly metastatic clone MTLn3 had a dominant cellular phenotype, expressing CKs 7, 8, 14, and 19 and vimentin, a pattern that did not match normal mammary epithelia, whether luminal, basal/myoepithelial, or the dual-phenotype stem cell, in which CKs 5, 8, 14, and 18 were coexpressed. MTLn3 lymph node and lung metastases expressed the same cellular phenotype as the s.c. growing MTLn3 tumor. The results appear to contradict the belief that malignant mammary tumors may be distinguished from benign tumors or hyperplastic growths by the lack of basal/myoepithelial markers. PMID- 1718591 TI - Involvement of transforming growth factor alpha/epidermal growth factor receptor autocrine growth mechanism in an ovarian cancer cell line in vitro. AB - Although transforming growth factor (TGF) alpha and epidermal growth factor (EGF) receptor (EGFR) autocrine mechanism is widely demonstrated in many kinds of cancers, its biological significances still remain circumstantial. We critically assessed the significance of this mechanism on the growth of an ovarian cancer cell line. Northern blot analysis in polyadenylated RNA isolated from cells by using 32P-labeled pre-TGF alpha, EGRF, and prepro-EGF complementary DNAs as probes revealed that pre-TGF alpha and EGFR but not prepro-EGF gene transcripts were expressed in the cell. TGF alpha and EGFR but not EGF proteins were observed by immunocytochemical stainings, using monoclonal antibodies against human TGF alpha, EGFR, and EGF, respectively. This cell line possessed a class of high affinity EGF receptor by 125I-EGF binding studies; Kd being 2.9 x 10(-10) M and Bmax to be 7.7 x 10(4) sites/cell. As much as 1.12 +/- 0.14 ng (SD; n = 3)/10(7) cells/24 h of TGF alpha was secreted in the conditioned media. These results suggested the expression of a TGF alpha/EGFR autocrine mechanism in this cell line. We, therefore, assessed the biological significance of this mechanism on the growth of this cell line in serum-free monolayer cell cultures. Although 0.1, 1.0, and 10 nM concentrations of TGF alpha did not show significant growth promotion, monoclonal antibodies against TGF alpha and EGFR but not EGF significantly inhibited cell growth. All these data suggested the biological importance of a TGF alpha/EGFR autocrine mechanism on the growth of this cell line in vitro. PMID- 1718592 TI - Norethindrone scatters silver-stained nucleolar organizer regions of Ishikawa cells. AB - We investigated the effects of sex steroids on silver-stained nucleolar organizer regions (Ag-NORs) and DNA/RNA kinetics in Ishikawa cells. Norethindrone and its isomer norethynodrel exclusively caused Ag-NORs to scatter in the nuclear matrix, from the nucleolus. No such effect occurred with the other sex steroids tested, including progestational, androgenic, and estrogenic compounds. Nuclear argyrophilic substances induced by norethindrone, as well as nucleolar ones, were neither DNA nor RNA but protein. Electron microscopy showed that norethindrone caused nucleolar segregation, in which the fibrillar components disappeared, and it produced islets, consisting of dense fibrillar materials, in the nucleoplasm. Ag-NORs observed on the fibrillar components in control nucleoli were translocated onto the dense fibrillar materials in the nucleoplasm. Although scattering was preferentially found in the cells synthesizing DNA, the scintillation assay of DNA/RNA kinetics suggested that scattering was related to the inhibition of RNA synthesis. These results imply that norethindrone preferentially interacts with intranucleolar DNA when its duplication is occurring and then interferes with rRNA synthesis. Scattering of Ag-NORs might not be caused by the hormonal activity of these agents but by a pharmacological effect derived from their molecular structures. PMID- 1718593 TI - Human colorectal adenocarcinoma cells: differential nitric oxide synthesis determines their ability to aggregate platelets. AB - The existence and role of an L-arginine:nitric oxide (NO) pathway in two human colorectal adenocarcinoma cell lines, SW-480 and SW-620, were investigated. Both cell lines, which derive from the same patient, SW-480 from the primary tumor and SW-620 from its metastatic lesion, were shown to have a cytosolic, Ca(2+) independent, NADPH-dependent NO synthase, the activity of which was lower in the cytosol of SW-620. These cells were more potent inducers of platelet aggregation. In contrast, SW-480, which had more NO synthase activity, were less potent inducers of platelet aggregation. Pretreatment of both cell lines with NG monomethyl-L-arginine, an inhibitor of NO synthase, potentiated their proaggregating effect and made them equally active. Exogenous L-arginine, NO, and related nitrovasodilators all inhibited platelet aggregation induced by SW-620. The antiaggregating activity of NO was further potentiated by prostacyclin and by M&B22948, a selective inhibitor of cyclic GMP phosphodiesterase. We propose that the generation of NO by tumor cells inversely correlates with their metastatic potential. Furthermore, we show that the lower activity of NO synthase in metastatic cells is due to the presence in these cells of a low molecular weight inhibitor of the NO synthase. In addition, agents which modulate platelet function by a cyclic GMP-dependent mechanism may be useful in the prevention of tumor metastasis. PMID- 1718594 TI - Action of 2',2'-difluorodeoxycytidine on DNA synthesis. AB - The action of the new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) on DNA synthesis was investigated in whole cells and in vitro assay systems with purified DNA polymerases. DNA synthesis in human lymphoblastoid CEM cells was inhibited by dFdC in a concentration-dependent manner that could not be reversed by exogenous deoxynucleosides. The analogue was incorporated into cellular DNA; most of the incorporated dFdC 5'-monophosphate (dFdCMP) residues were in internucleotide linkage. In vitro DNA primer extension assays demonstrated that dFdC 5'-triphosphate (dFdCTP) competed with deoxycytidine triphosphate for incorporation into the C sites of the growing DNA strand. The ratios of the apparent Km values for the incorporation of dFdCTP and dCTP into a C site of M13mp19 DNA were 21.8 and 22.9 for DNA polymerases alpha and epsilon, respectively. The apparent Ki values of dFdCTP were 11.2 microM for DNA polymerase alpha and 14.4 microM for polymerase epsilon. After dFdCMP incorporation, the primer was extended by one deoxynucleotide before a major pause in the polymerization process was observed. This was in contrast to the action of arabinosylcytosine 5'-triphosphate, which caused both DNA polymerases alpha and epsilon to pause at the site of incorporation. The 3'----5' exonuclease activity of DNA polymerase epsilon was essentially unable to excise nucleotides from DNA containing dFdCMP at either the 3'-end or at an internal position, whereas arabinosylcytosine monophosphate was removed from the 3'-terminus at 37% the rate for deoxynucleotides. The cytotoxic activity of dFdC was strongly correlated with the amount of dFdCMP incorporated into cellular DNA. Our results demonstrate qualitative and quantitative differences in the molecular actions of dFdC and arabinosylcytosine on DNA metabolism, but are consistent with an important role for such incorporation in the toxicity of dFdC. PMID- 1718595 TI - Neural cell adhesion molecule expression and messenger RNA splicing patterns in lung cancer cell lines are correlated with neuroendocrine phenotype and growth morphology. AB - Diverse histological types of lung cancer express neuroendocrine (NE) markers. We studied the expression and alternative splicing forms of the neural cell adhesion molecule (NCAM) NKH-1, a member of the immunoglobulin superfamily, in 56 lung cancer cell lines representing all histological types. We found a strong correlation between expression of NCAM with both NE phenotype and lack of substrate adhesion in culture. Several cell lines expressed high levels of the leukocyte antigen Leu-7 (HNK-1) but were negative for NCAM antigen and mRNA, indicating that the Leu-7 antigen is distinct from NCAM. All of the NCAM-positive cell lines demonstrate a single 6.2-kilobase mRNA, and analysis of the known 3' alternative splices shows predominant expression of only the membrane form with the small intracytoplasmic domain. We conclude that (a) expression of NCAM is associated with NE phenotype regardless of the histological type of lung cancer; (b) these cell lines share a single form of NCAM; (c) with few exceptions, NCAM expression is associated with cell to cell adhesion and lack of substrate adhesion (growing as floating clusters); and (d) Leu-7 antigen is distinct from NCAM. This form of NCAM may play a functional role in NE differentiation or may be a part of the NE program expressed by these cells. PMID- 1718596 TI - Characterization of human autotumor-reactive T-cell clones obtained from tumor infiltrating lymphocytes in liver metastasis of gastric carcinoma. AB - Three autotumor-reactive T-cell clones have been established from tumor infiltrating lymphocytes isolated from a metastatic lesion of human gastric carcinoma in the liver. The clones all were shown to be CD3+, CD8+, CD4-, CD16-, T-cell receptor alpha/beta +, and T-cell receptor gamma/delta-, and they have retained both their autotumor reactivity and the same phenotype for over a year in culture. Each clone had a different rearrangement of the T-cell receptor gamma chain genes as indicated by Southern blot analysis. Tested against a panel of 18 tumor cell targets, the clones preferentially lysed autologous tumor (AuTu) cells, but each clone also showed weak cytotoxicity against one allogeneic cholangiocarcinoma cell line. At the same time, each clone showed appreciable cytotoxicity against K562 targets. In blocking experiments, anti-CD3, anti-WT31, anti-CD8, or anti-HLA class I monoclonal antibodies blocked AuTu cytotoxicity but not cytotoxicity against K562. In contrast, allocytotoxicity against the cholangiocarcinoma was blocked only by anti-CD3 monoclonal antibody. All 10 subclones of one T-cell clone had high levels of AuTu cytotoxicity but variable levels of anti-K562 cytotoxic activity. Proliferation of the T-cell clones was significantly stimulated by the addition of irradiated autologous but not allogeneic tumor cells. Preincubation of cultured AuTu cells with tumor necrosis factor alpha or gamma-interferon (IFN-gamma), but not with IFN-alpha, increased their susceptibility to lysis by the T-cell clones; however, it increased resistance of AuTu to lysis by interleukin 2-activated natural killer cells. The expression of an adhesion molecule, intercellular adhesion molecule 1, on the surface of AuTu was also up-regulated by tumor necrosis factor alpha or IFN gamma, but not by IFN-alpha. All three cytokines up-regulated HLA-class-I antigens on AuTu. Pretreatment of K562 targets or allogeneic cholangiocarcinoma cells with the same cytokines increased their resistance to lysis by the T-cell clones. Overall, the results indicate that these T-cell clones show specificity for AuTu but also independently recognize a limited number of allogeneic tumor targets and lyse K562 targets. The mechanisms involved in the recognition by the T-cell clones of autologous, allogeneic, and K562 tumor targets appeared to be distinct. PMID- 1718597 TI - Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor. AB - Basic fibroblast growth factor (bFGF) is a potent angiogenic mitogen. To elucidate the effect of bFGF inhibitors in vivo, anti-bFGF immunoneutralizing monoclonal antibody was prepared. One monoclonal antibody against human bFGF, obtained by cell fusion and designated 3H3, completely inhibited bFGF-induced proliferation of human umbilical vein endothelial cells at a concentration of 100 ng/ml. 3H3 did not bind to acidic fibroblast growth factor or HST1 protein, indicating high specificity for bFGF. Furthermore, the immunoneutralizing activity of 3H3 was examined in vivo. K1000 cells (a BALB/c 3T3 transformant in which the leader sequence-fused bFGF gene was transfected) were transplanted s.c. into BALB/c nude mice. Growth of the tumor cells was inhibited by i.v. treatment with 3H3 at a concentration of 200 micrograms/mouse. Histological observation showed that the antitumor effect of 3H3 was due to the inhibition of bFGF-induced angiogenesis. This experiment provides direct causal evidence for the hypothesis that tumor growth is angiogenesis dependent. This finding could also have implications for the development of novel therapeutic approaches to angiogenic solid tumors. PMID- 1718598 TI - Correspondence Re: E. Gorelik et al., Effect of ultraviolet irradiation on MCA102 tumor cell immunogenicity and sensitivity to tumor necrosis factor. Cancer Res., 51: 1521-28, 1991. PMID- 1718599 TI - An immunotoxin prepared with blocked ricin: a natural plant toxin adapted for therapeutic use. AB - Ricin, the cytotoxic protein isolated from castor beans, is composed of two subunits, A-chain and B-chain. Ricin intoxicates cells by binding through its B chain to galactose-terminated oligosaccharides found on the surface of all eukaryotic cells and then transferring its A-chain to the cytosol where it disrupts protein synthesis by inactivating ribosomes. In addition to binding, the B-chain plays an important, but not yet understood, role in the translocation of the A-chain through a cellular membrane to the cytosol. Blocking the two galactose-binding sites of native ricin by chemical modification with affinity ligands created an altered toxin, called blocked ricin, that has at least a 3500 fold lower binding affinity and is more than 1000-fold less cytotoxic than native ricin for Namalwa cells (a Burkitt's lymphoma line) but that has maintained the translocation function of the B-chain and the catalytic activity of the A-chain. Conjugation of blocked ricin to monoclonal antibodies that bind to cell surface antigens creates new cytotoxins that approach the potency of native ricin. These cytotoxins incorporate the three essential functions of natural toxins, i.e., binding to cells, transport through a membrane, and catalytic inactivation of an essential cellular process; but in addition they possess a defined cellular target specificity. Such potent immunotoxins may play an important therapeutic role in cancer treatment. Clinical trials with an anti-CD19-blocked ricin and an anti-CD33-blocked ricin conjugate against B-cell cancers and acute myeloblastic leukemia have begun. PMID- 1718600 TI - Clinico-biological behavior of germ-cell tumors. AB - Fifty-five patients with intracranial germ-cell tumors were treated in Kobe University Hospital between 1972 and 1989. Thirty-four patients were male and 21 were female, and all patients were between 0 and 39 years of age. There was a peak incidence between 16 and 20 years in male patients and 11 and 15 years in female patients. The tumors occurred most frequently in the pineal region (25 cases), followed by the suprasellar (17), both pineal and suprasellar (6), basal ganglia (5), and frontal and temporal lobe (2) regions. There was a male predominance in the incidence of germ-cell tumors except for the patients with suprasellar tumors. Initial symptoms and neurological signs differed according to the location of the tumor. The symptoms of increased intracranial pressure and Parinaud's sign were most frequently seen in patients with a pineal tumor, while diabetes insipidus and visual disturbance were most common in patients with suprasellar tumors. Human chorionic gonadotropin were positive in the serum, cerebrospinal fluid and/or tumor cyst in 14/26 patients, and alpha-fetoprotein in 6/22 patients examined. There were 45 patients with pure germinoma both histologically verified and clinically diagnosed. The 5-year survival rate of the patients with germinoma was 69% in cases in which tumor marker was negative and unknown cases, and 86% in cases where the tumor marker was positive. The authors discuss management of the patients with intracranial germ-cell tumors. PMID- 1718601 TI - The effects of N-glycosylation on the lectin activity of recombinant ricin B chain. AB - Soluble, biologically-active recombinant ricin B chain has been produced by expressing B chain-encoding DNA in heterologous eukaryotic or prokaryotic hosts. N-Glycosylated recombinant ricin B chain expressed in Xenopus oocytes bound to both immobilized asialofetuin and immobilized lactose. Non-glycosylated ricin B chain expressed in either E. coli or in tunicamycin-treated oocytes did not bind to immobilized lactose. However, it did bind to asialofetuin, and increasing concentrations of free lactose did not reduce this asialofetuin binding dramatically, in contrast to the effect of free lactose on the binding of either glycosylated recombinant B chain or native ricin B chain. PMID- 1718602 TI - Review of neuromuscular blockers. AB - Neuromuscular blockers are primarily used as adjuncts in procedures requiring general anesthetics. Some agents have had a long, romantic history while others are relatively new or still in clinical trials. The following is a brief review of the history, mechanism of action, and potential adverse effects of neuromuscular blockers. PMID- 1718603 TI - A new visualization principle of cerium phosphate reaction product of phosphatases in cryotome sections for light microscopy--the cerium-oxalate-osmium silver (Ce-Ox-Os-Ag) method. AB - A new visualization principle for the detection of cerium phosphate reaction product of phosphatases in light microscopy is described. The new mode is based on the conversion of cerium phosphate into cerium oxalate. The latter is able to react with OsO4. In this way osmium black is formed, staining enzymatic activity sites grey or greyish-black. Utilizing the argyrophilia of osmium black, the staining contrast could be remarkably intensified by posttreatment with Ag proteinate (Ce-Ox-Os-Ag procedure). This procedure is of similar sensitiveness in comparison to the DAB-Ni method, proposed earlier. The advantages are a very pale background staining and a complete suppression of the cerophilia. Moreover, it substitutes to DAB, a probable potent carcinogen. The method is time-saving when the processing of the reactions was stimulated in a microwave oven. PMID- 1718604 TI - Slow axonal transport models come full circle: evidence that microtubule sliding mediates axon elongation and tubulin transport. PMID- 1718605 TI - Cycles of progressive realignment of gRNA with mRNA in RNA editing. AB - We characterized numerous partially edited NADH dehydrogenase 7 and ATPase 6 cDNAs. Most of these have a stretch of incompletely edited sequence at the junction of mature and unedited sequences. The characteristics of the junctions suggest editing of sites multiple times and that editing within each junction does not proceed precisely 3' to 5'. Analyses of gRNAs and corresponding junction sequences predict a series of progressively more stable, but incompletely base paired, interactions in the junction region. The predicted interactions suggest that the gRNA is progressively realigned with the mRNA being edited. We suggest that gRNA interactions with the mRNA result in regions of lower thermodynamic stability that are selected for editing, thus driving toward the most stable structure, the complete gRNA/mRNA duplex. PMID- 1718606 TI - Nucleoside triphosphates are required to open the CFTR chloride channel. AB - The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF associated mutations in the NBDs block Cl- channel function. PMID- 1718607 TI - Effect of antigen challenge on lymph node lymphocyte adhesion to vascular endothelial cells and the role of VLA-4 in the rat. AB - Lymphocytes from antigen-stimulated lymph nodes avidly migrate from the blood to cutaneous sites of inflammation such as DTH reactions or contact sensitivity. One of the initial steps in this migration is the adhesion of the lymphocyte to endothelial cells (EC); therefore, the adhesion of lymphocytes from antigen stimulated lymph nodes to microvascular EC in the rat was examined. Two to five days after subcutaneous immunization with antigen, lymphocytes that adhered to unstimulated and IFN-gamma-, TNF-alpha-, IL-1 alpha-, and LPS-treated EC were increased in the regional lymph nodes. The enhanced adhesion was attributable to low-density lymphoblast-enriched lymph node cells while small high-density lymphocytes displayed little or no increase in their adhesion. Lymphoblast adhesion required the stimulation of the EC with 10 times the concentrations of IFN-gamma and TNF-alpha required for peritoneal exudate lymphocyte adhesion. There was a synergistic increase in the adhesion of the low-density lymphocytes to EC stimulated with combinations of IFN-gamma and TNF-alpha. Antibody to VLA-4 inhibited about 40% of the stimulated adhesion to EC treated with IFN-gamma, TNF alpha, or LPS. In vivo anti-VLA-4 inhibited lymphoblast migration to IFN-gamma, TNF-alpha, LPS, and DTH reactions by 60%. Thus antigen stimulates the generation of low-density lymphoblasts that have an enhanced adherence to cytokine- and LPS treated EC through a partially VLA-4-dependent mechanism and the migration of these cells to cutaneous inflammatory reactions is dependent upon VLA-4. PMID- 1718608 TI - Human thymic epithelial cells are frequently transformed by retroviral vectors encoding simian virus 40. AB - The thymic microenvironment contains a mixture of phenotypically distinct epithelial cells of varied functions, some of which are unknown. In an attempt to understand their relevance to T cell differentiation in the thymus, human thymic epithelial cell clones from both fetal (SM3-SM5) and postnatal (SM6) thymus were produced by using a defective recombinant retroviral vector encoding the simian virus 40 large T antigen and the neomycin resistance gene. The presence of keratins 8 and 18, desmosomes, and tonofilaments confirmed the epithelial origin of the cell strains. The cells expressed Thy-1 and HLA-Class I at high levels, showed weak-expression antigens defined by TE3B and A2B5, and low to negligible levels of the MR19-defined molecule. When compared with the phenotype of thymic epithelial cells in situ, the cell strains appear to be derived from neuroendocrine components in the outer cortical region of the human thymus. The use of retroviral vectors to transform human thymic epithelium was considerably more efficient than transfection with a plasmid carrying the origin of replication-defective SV40 large T gene. In the latter case, only two cell strains with subcapsular epithelial phenotypes were derived from fetal thymus. With the retroviral vectors, epithelial cell strains could, for the first time, be generated from human postnatal thymus as well as from fetal thymus. PMID- 1718609 TI - Characterization of a yeast nuclear gene, AEP2, required for accumulation of mitochondrial mRNA encoding subunit 9 of the ATP synthase. AB - The temperature-conditional pet mutant, ts379, of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature due to mutation of a single nuclear locus, AEP2. The inability to synthesize subunit 9 correlates with a lowered accumulation of the cognate oli1 mRNA indicating that the AEP2 product is involved in oli1 transcript maturation or stabilization. The AEP2 gene has been isolated in this study from a wild-type yeast genomic library by genetic complementation of ts379 at the restrictive temperature. A 1,740 nucleotide open-reading frame was observed that encodes a basic, hydrophilic protein of 67,534 Da which possesses a putative mitochondrial address signal. Disruption of chromosomal DNA within this reading frame produced a non-conditional respiratory mutant unable to synthesize subunit 9, identifying the AEP2 gene. Hybridization analyses indicate that AEP2 is located on chromosome XIII and produces a 2.1 kb poly(A)+ transcript. Two additional open-reading frames were found in close proximity to that of AEP2. The three open-reading frames shared no significant homology with entries in several data bases. PMID- 1718610 TI - Isolation of transcripts preferentially expressed during fruit body primordia differentiation in the basidiomycete Agrocybe aegerita. AB - An Agrocybe aegerita cDNA library, constructed from fruit body primordia poly(A)+ RNAs, was screened by differential colony hybridization. Clones which preferentially hybridized to poly(A)+ RNA sequences from fruit body primordia, versus poly(A)+ RNAs from mycelium, were isolated. Eight of these clones (EMAa-1 to EMAa-8) encoded eight different poly(A)+ RNAs which were demonstrated to be undetectable in the four stages preceding primordia formation and to be concomitantly accumulated when primordia differentiate, suggesting that EMAa gene products are closely involved in the morphogenesis of primordia. The eight EMAa cDNAs hybridize to at least seven unique regions distributed randomly in the A. aegerita genome. The expression of two EMAa cDNA sequences in E. coli led to the isolation of their gene products as fusion proteins. PMID- 1718612 TI - Two promoters within the psbK-psbI-trnG gene cluster in tobacco chloroplast DNA. AB - Transcription of the 2.6 kbp psbK-psbI-trnG cluster in tobacco chloroplasts has been studied. This cluster contains, in linear sequence, the genes encoding two low-molecular-mass polypeptides, K and I, of photosystem II (psbK and psbI, respectively), and tRNA(Gly) (UCC) (trnG). Northern blot hybridization revealed that the largest transcript (2.6 kb) hybridizes to psbK, psbI and trnG, but not to the following trnR-UCU. Ten other transcripts ranging from 0.1 to 1.3 kb were also detected. Three of these transcripts overlap the divergent transcript arising from trnS-GCU located on the opposite DNA strand. S1 mapping and primer extension experiments showed that these multiple transcripts comprise eight distinct 5' ends. By in vitro capping assays two of them were determined to be transcriptional initiation sites; one is located 163 bp upstream of psbK and the other is 6 bp upstream of trnG. The 3' ends of transcripts were determined by S1 mapping; one lies between psbI and trnG and the other is at the end of trnG. The presence of dual promoters of trnG is discussed. PMID- 1718611 TI - Sequence analysis of wheat mitochondrial transcripts capped in vitro: definitive identification of transcription initiation sites. AB - To identify transcription initiation sites in wheat mitochondria, the nascent 5' ends of transcripts were specifically labeled by incubation of wheat mitochondrial RNA with [alpha-32P]GTP in the presence of the enzyme guanylyltransferase. After separation of the resulting capped transcripts by electrophoresis in polyacrylamide gels, individual RNAs were recovered and directly sequenced. Four RNA sequences obtained in this way were localized upstream of the protein-coding genes atpA, coxII, coxIII and orf25. Comparison of mRNA and gene sequences allowed precise positioning of transcription initiation sites for these four genes. Sequence similarities immediately upstream of these sites define a conserved motif that we suggest as a candidate regulatory element in wheat mtDNA. The relationship between this motif and putative mitochondrial promoters in other plant species is discussed. PMID- 1718613 TI - The first analysed archegoniate mitochondrial gene (COX3) exhibits extraordinary features. AB - The first mitochondrial-encoded gene of an archegoniate has been identified, cloned and sequenced. The cytochrome oxidase III gene (cox3) of the moss Physcomitrella patens consists of a 618 bp open reading frame with high homology (around 72%) to known cox3 sequences of higher plants. Nevertheless, it is a quarter shorter than these. The cox3 gene of P. patens contains no introns and reveals a G + C-content of 41.3%. The region containing the cox3 gene exists as a single copy in the mitochondrial genome as shown by restriction mapping. In the 5' flanking sequence a putative ribosome binding site and a putative secondary structure were found. Two main transcripts of 2.4 kb and 2.6 kb were detected indicating a complex mitochondrial transcription pattern possibly due to co transcription. Additional open reading frames were found downstream from, as well as upstream of, the cox3 gene. In Western blots a polyclonal cox3 antibody from yeast detected one single band with an apparent molecular weight of 22 kDa. PMID- 1718614 TI - The rice mitochondrial nad3 gene has an extended reading frame at its 5' end: nucleotide sequence analysis of rice trnS, nad3, and rps12 genes. AB - The nucleotide sequences of the tRNASer (trnS), pseudo-tRNA, NADH dehydrogenase subunit 3 (nad3), and ribosomal protein S12 (rps12) genes from rice mitochondrial DNA (mtDNA) were determined. Both trnS and nad3 were confirmed to be single copy genes by Southern blot analysis. The nad3 and rps12 genes were arranged in tandem, and the two were co-transcribed. The order of the above four genes in rice mtDNA differed from the linear order observed for the wheat and maize genes. In rice mitochondria, the trnS and pseudo-tRNA genes were found upstream of the cytochrome c oxidase subunit I gene, instead of the nad3 and rps12 genes as observed in maize and wheat. Additionally, while the rice nad3 and rps12 genes remain paired, they too are in a different sequence environment from the wheat and maize genes. The apparent split of the two pairs of genes indicates the occurrence of a mitochondrial intramolecular recombinational event. Another peculiarity is that the sequence upstream of the translational initiation codon of the rice nad3 gene is different from that of the wheat and maize versions. The ATG initiation codon of wheat and maize nad3 is replaced by TTG in the rice nad3. A subsequent deduction of the amino acid sequence, accompanied by a primer extension analysis, indicates that the predicted rice NAD3 protein has an additional 37 amino acid residues at its N-terminus compared to the wheat and maize NAD3 proteins. cDNA sequence analysis showed no introns or the occurrence of RNA editing at the newly replaced TTG codon. PMID- 1718615 TI - Metabolism and alkylating activity of thio-TEPA in rat liver slice incubation. AB - Precision-cut rat-liver slices were used to study the metabolism of the alkylating agent N,N',N''-triethylenethiophosphoramide (thio-TEPA). Exposure to high concentrations (1-10 mM) of thio-TEPA for 6 h did not prove to be toxic to the liver slices as indicated by insignificant leakage of potassium from the cells. The time course of the disappearance of thio-TEPA (initial concentration, 5.2 microM) from the buffer during incubation followed first-order kinetics. Formation of N,N'N''-triethylenephosphoramide (TEPA) apparently accounted for the elimination of thio-TEPA. Pretreatment of the rats with phenobarbital significantly increased the reaction rate. Conversely, pretreatment with the cytochrome P-450 inhibitor allylisopropylacetamide significantly reduced the metabolic rate. The elimination of thio-TEPA and formation of TEPA occurred independently of thio-TEPA concentration, which ranged from 5.2 to 104 microM. Thio-TEPA's oxo-analogue TEPA, which was not further metabolized, was the only metabolite identified. However, a significantly time-related increase in 4 (nitrobenzyl)-pyridine (NBP) alkylating activity was observed following incubation of liver slices with thio-TEPA but not after their incubation with TEPA. This may possibly indicate the formation of unknown active metabolites. PMID- 1718616 TI - Temporal alterations in cytokeratin expression during experimental oral mucosal carcinogenesis. AB - Precancerous lesions and squamous cell carcinomas (SCCs) were induced in the oral mucosa of outbred male Sprague-Dawley rats by repeated application of the carcinogen 4-nitroquinoline-1-oxide. Temporal alterations in the pattern of cytokeratins expressed by epithelial cells in these developing neoplasms were investigated by means of one- and two-dimensional polyacrylamide gel electrophoresis and by probing electrophoretic blots of these gels with anticytokeratin antibodies. Progressive diminution in the expression of a 58.3 kDa cytokeratin was detected in moderately dysplastic epithelium and subsequent lesions, as was reduced expression of a 53.5 kDa cytokeratin after a stage of severe dysplasia had been induced. Analysis of two-dimensional electrophoretograms indicated an alkaline shift of acidic variants of a 49.0 kDa cytokeratin in moderately dysplastic epithelium; this shift was more pronounced in the induced, severely dysplastic epithelium and SCCs. The latter finding of an alkaline shift in cytokeratins of dysplastic epithelial cells has not been previously reported in preneoplastic lesions. PMID- 1718617 TI - Synthetic murine epidermal growth factor sequence 20-31 is mitogenic and angiogenic. AB - Epidermal growth factor (EGF) and its homologue, transforming growth factor alpha (TGF alpha), are mitogenic, angiogenic and tumour-promoting polypeptides. Much effort has therefore been directed towards the development of EGF/TGF alpha antagonists as a potential cancer therapy. Initial reports that some EGF/TGF alpha synthetic fragments possess EGF-receptor binding activity have not been confirmed in subsequent studies. We have found, however, that the murine EGF B loop sequence: Ac-[(S-acetamidomethyl)-Cys20,31]-EGF-(20-31)-NH2 [(mEGF-(20-31)] produces biological effects consistent with the parent molecule in bovine, murine, chick and human, but not rat, model systems. In parallel experiments, both mEGF and mEGF-(20-31) elicit migratory, cytoprotective, growth-stimulatory, growth-inhibitory and angiogenic responses. The reverse B-loop sequence, mEGF-(31 20), is also mitogenic and angiogenic. The C-loop sequence, mEGF-(33-42), has no mitogenic or angiogenic activity when applied alone, does not block the mitogenic effect of mEGF, but does block the angiogenic effect of mEGF. It has not been established that the EGF receptor is the target for these fragments, but the results suggest that the residual biological activities of EGF fragments merit further investigation. PMID- 1718618 TI - DNA adduct formation in liver following the administration of [3H]2-nitrofluorene to rats in vivo. AB - The formation of RNA and DNA adducts by the environmental pollutant 2 nitrofluorene (2-NF) has been investigated in rat liver in vivo. The adduct pattern was studied after trifluoroacetic acid hydrolysis of DNA or RNA, followed by analysis of the adducts by HPLC. This was also done by enzymatic hydrolysis of DNA, followed by 32P-postlabeling. Both after oral and i.v. administration of [3H]2-NF, one major adduct was found. This adduct did not co-migrate with one of the known adducts of 2-(acetyl)-aminofluorene, N-deoxyguanosin-8-yl-2 aminofluorene (dG-C8-AF), which could have been formed after nitroreduction of 2 NF. 32P-Postlabeling revealed that two minor adducts were also formed, one of which was dG-C8-AF. The observation that the major adduct was also formed after i.v. administration of 2-NF to bile duct-catheterized rats makes a role for the intestinal microflora in the formation of this adduct very unlikely. In vitro experiments with inhibitors of the enzyme epoxide hydrolase indicated that epoxidation of 2-NF may play a role in the microsomal bioactivation of this compound. PMID- 1718619 TI - Development of two types of hepatocellular carcinoma in transgenic mice carrying the SV40 large T-antigen gene. AB - We produced transgenic mice by introducing a fusion gene (ST) comprising of the promoter for human serum amyloid P component (SAP) and the coding region of the simian virus 40 (SV40) large tumor antigen (Tag). The ST transgenic mice developed hepatocellular carcinomas (HCC) between 7 and 12 weeks of age. Clinically and pathologically these HCC were classified into two types: diffuse and focal. In the diffuse type Tag is expressed in almost all hepatocytes and HCC has presumably developed from these T-antigen-positive cells. The focal type, which resembles human HCC, is only Tag positive in the neoplastic nodules. These are surrounded by Tag-negative normal hepatocytes that form normal lobular structures. As the human SAP promoter can direct adult liver-specific expression of heterologous genes, it is assumed that Tag gene expression in the transgenic animals is increased sharply after birth. Interestingly, a similar level of Tag expression was observed in both the cytoplasm and the nucleus. Our results suggest that spatial differences in Tag expression result in the generation of two types of HCC in transgenic mice and that the ST mouse can serve as a model for human hepatocarcinogenesis. PMID- 1718620 TI - Altered regulation of growth and expression of differentiation-associated keratins in benign mouse skin tumors. AB - Alterations in the pattern of epidermal cell differentiation and proliferation in mouse skin and benign skin tumors were studied by two-color immunofluorescence using monospecific antibodies. Replicating cells were identified by 5-bromo deoxyuridine (BrdU) pulse-labeling and differentiating cells by keratins K1 and K10. In normal mouse skin, pulse-chase experiments for 120 h revealed that replication was restricted to a single layer of basal cells. Replicating cells did not express K1 or K10, but these keratins were sequentially expressed in post mitotic basal cells 18 and 24 h following DNA synthesis respectively, and cells expressing these keratins migrated into the suprabasal layers. In phorbol-ester- or cantharidin-stimulated hyperplastic skin, replicating cells were also confined to the basal cell compartment and suprabasal cells expressed keratins 1 and 10. In papillomas induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate, replication occurred predominantly in cells in an expanded basal cell compartment (two to four layers above the basement membrane). Cells in these basal layers did not express K1 or K10, but more superficial cells did. After a 1 h pulse of BrdU, replication was also identified in suprabasal cells expressing the differentiation-associated keratins. These and other results suggest that benign tumor cells escape the obligatory growth arrest associated with differentiation. Replication of K1- and K10-expressing suprabasal cells may represent an early alteration during mouse skin carcinogenesis. PMID- 1718621 TI - Bypass of a hydrocarbon adduct in an oligonucleotide template mediated by mispairing adjacent to the adduct. AB - The action of DNA polymerase (Sequenase Version 2.0) on an oligonucleotide template containing a 7-bromomethyl-benz[a]anthracene-deoxyadenosine adduct flanked by thymidine residues was investigated. The polymerase incorporated deoxyadenosine or deoxyguanosine residues opposite the thymidine 3' to the adduct with similar efficiencies. Whereas the normal A.T base pair led to arrest of polymerase progression along the template, formation of the G.T mismatch allowed incorporation of thymidine opposite the adduct and further primer extension. This mispair-mediated bypass was also seen with AMV reverse transcriptase and may represent a novel mechanism for overcoming the replication block of a bulky carcinogen--DNA adduct. PMID- 1718622 TI - Mode of action of 2-(diethylamino)-7-ethoxychromone on human platelets. AB - The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident. PMID- 1718623 TI - Force-interval relations of twitches and cold contractures in rat cardiac trabeculae. Effect of ryanodine. AB - The twitch force (Ft)-interval relation of cardiac muscle reflects recovery of calcium release from the sarcoplasmic reticulum (SR). The calcium content of the SR is thought to be reflected by force developed during a contracture (Fc), induced by rapid cooling to near 0 degrees C. In right ventricular trabeculae of rat, under control conditions, the Ft-interval relation consisted of recovery of Ft to steady state (early recovery), followed by a secondary increase of Ft up to a maximum at an interval of approximately 100 seconds (rest potentiation) and a decline of Ft at intervals greater than 100 seconds (rest depression). The mechanisms that may underlie recovery of force after the last twitch at short intervals are 1) time-dependent transport of Ca2+ from the uptake compartment of the SR to the release compartment, 2) recovery of slow inward Ca2+ current during the action potential, and 3) recovery of the Ca2+ release channels in the SR. The Fc-interval relation was similar to the Ft-interval relation in that both a rest potentiation and a rest depression phase were present. However, at short interstimulus intervals (less than 1 second), Fc was independent of time, suggesting that the mechanism underlying early recovery was bypassed. Ryanodine (0.1-10 nM) reduced rest potentiation in a dose-dependent manner and accelerated rest depression of both Ft and Fc. At high ryanodine concentration, a significant Fc could only be induced after short intervals. Significant acceleration of rest depression was observed at low ryanodine concentrations, when Ft at intervals of 5 seconds was kept constant by increasing the stimulus frequency of [Ca2+]o, suggesting that the ryanodine effect was enhanced by increased [Ca2+]i. Ryanodine also increased the rate of decay of postextrasystolic potentiation in a dose dependent manner. A significant effect was observed in 10 nM ryanodine. The twitch was not prolonged by ryanodine at these concentrations. These results suggest that the small magnitude of the twitch at short intervals is due to the finite time required by SR Ca2+ release channels to fully recover after a twitch. Furthermore, the results offer support for the hypothesis that ryanodine (in the nanomolar range) promotes Ca2+ leak from the SR in a dose-dependent manner and thereby limits Ca2+ accumulation during the interstimulus interval. Therefore, it may be expected that the negative inotropic effect of ryanodine is due to the SR Ca2+ depletion, and it is not necessary to postulate that ryanodine "blocks" the Ca2+ release channels in the SR. PMID- 1718624 TI - Hysteresis in the excitability of isolated guinea pig ventricular myocytes. AB - Hysteresis phenomena were demonstrated in the excitability of single, enzymatically dissociated guinea pig ventricular myocytes. Membrane potentials were recorded with patch pipettes in the whole-cell current-clamp configuration. Repetitive stimulation with depolarizing current pulses of constant cycle length and duration but varying strength led to predictable excitation (1:1) and nonexcitation (1:0) patterns depending on current strength. However, transition between patterns depended on the direction of current strength change, and stable hysteresis loops were obtained in stimulus-response pattern versus current strength plots in 31 cells. Increase of pulse duration and decrease of stimulation rate contributed to a reduction in hysteresis loop areas. In addition, at the abrupt transitions from 1:0 to 1:1 patterns, a latency adaptation phenomenon was consistently observed. Bath application of tetrodotoxin (30 microM) produced no change of hysteresis, whereas hysteresis was substantially decreased in cobalt (2 mM) superfusion experiments. Analysis of the changes in amplitude and shape of the subthreshold responses during the transitions from one stable pattern to the other suggested that activity led to an increase in membrane resistance, particularly in the voltage domain between resting and threshold potentials. We therefore modeled the dynamic behavior of the single cells, using an analytical solution aimed at calculating the recovery of activation latency as a function of diastolic membrane resistance. Numerical iteration of the analytical model equations closely reproduced the experimental hysteresis loops in both qualitative and quantitative ways. The effect of stimulation frequency on the model was similar to the experimental findings. The overall study suggests that the excitability pattern of guinea pig ventricular myocytes is responsible for hysteresis and bistabilities when current intensity is allowed to fluctuate around threshold levels. PMID- 1718625 TI - Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells. AB - During early development, rat cardiac muscle cells actively proliferate. Shortly after birth, division of cardiac muscle cells ceases, whereas DNA synthesis continues for approximately 2 weeks at a progressively diminishing rate. Little DNA synthesis or cell division occurs in adult cardiocytes. Thus, developing cardiac muscle cells are an ideal system in which to examine the expression of cell cycle-regulated genes during development. We chose to examine proliferating cell nuclear antigen (PCNA), a gene expressed at the G1/S phase boundary of the cell cycle. Northern blots of RNA from cardiac muscle cells from 18-day-old rat fetuses and from day 0, 5, and 14 neonatal as well as adult rat hearts revealed that the PCNA mRNA was found in cardiac muscle cells from all ages. However, because it was possible that this was a result of fibroblast PCNA gene expression, we used reverse transcription followed by polymerase chain reaction to see if it was possible to detect the message for PCNA in cardiac muscle cells from all ages. Because of the great sensitivity of this technique, RNA was recovered from 25 isolated adult cardiac muscle cells. Polymerase chain reaction amplification products for PCNA produced from the RNA isolated from these cells conclusively demonstrated that mRNA for this gene, which normally is associated with proliferating cells, is expressed in adult cardiac muscle cells that no longer divide. Furthermore, Western blot analysis demonstrated that the PCNA protein was found only in embryonic and neonatal cells and not in adult rat cardiac muscle cells. Therefore, it might be inferred from these data that PCNA might be regulated at the posttranscriptional level in adult cardiac muscle cells. PMID- 1718626 TI - Isovolumic loading prevents atrophy of the heterotopically transplanted rat heart. AB - Heterotopically (infrarenal) transplanted cardiac isografts are histologically normal, spontaneously beating, hemodynamically unloaded hearts that undergo rapid and predictable atrophy. To study the factors that regulate cardiac growth we examined the effect of left ventricular (LV) isovolumic balloon placement on the weight, in vitro protein synthesis rate, and LV RNA content of the transplanted heart. At day 0 the donor LV weight was 367 +/- 8 mg, which decreased to 226 +/- 14 mg (p less than 0.001) after transplantation. Isovolumic loading with a latex balloon (volume, 111 +/- 10 microliters) significantly increased the LV weight of the transplanted heart to 397 +/- 13 mg when compared with that of the unloaded heart 14 days after surgery and prevented the atrophy when compared with the LV weight at day 0. Sham-loaded transplants had LV weights of 256 +/- 17 mg, which did not differ from the LV weight of unloaded transplanted hearts. Protein synthesis rates (nmol lysine/LV/hr) and total LV RNA and LV 18S RNA content were significantly greater in the balloon-loaded hearts when compared with the unloaded isografts and returned to levels similar to those measured in the in situ hearts. The ability of isovolumic loading of the LV to prevent atrophy via increased protein synthesis and the maintenance of LV RNA in the heterotopically transplanted heart further supports the important role of cardiac work in the regulation of cardiac growth. PMID- 1718627 TI - Responses of atherosclerotic human coronary arteries in vivo to the endothelium dependent vasodilator substance P. AB - BACKGROUND: The effects of the endothelium-dependent vasodilator substance P (SP) on atherosclerotic human coronary arteries was studied. METHODS AND RESULTS: [125I]-SP binding to luminal cells was shown to be preserved in the atherosclerotic epicardial coronary arteries of four patients. No binding to medial smooth muscle cells was demonstrated. Intracoronary infusions of SP were undertaken in patients with coronary artery disease. SP was infused for 2-minute periods starting at a dose of 2.8 pmol/min rising by doubling increments to 22.4 pmol/min. Analysis of the epicardial coronary artery diameter, using a computerized analysis system (CAAS) of the angiograms, was performed at the end of each infusion. Analysis of seven smooth vessel segments from seven coronary vessels, which were stenosed at more proximal sites, was performed. Significant dose-dependent dilatation was seen (p = 0.04), which was maximal at 5.6 pmol/min SP. No additional dilatation was produced with 2 mg intracoronary isosorbide dinitrate (ISDN). Two of these seven patients showed no response to SP, and only one of these appeared to sustain dilatation with ISDN (2 mg intracoronary). In a second group of six patients with discrete coronary stenoses, analysis at the site of the stenosed segments appeared to reveal dilatation in response to SP in only one instance. One other stenotic segment dilated with isosorbide dinitrate but failed to dilate with SP; the remaining four were fixed. The segment immediately proximal to the stenosis preserved a dose-dependent vasodilator response. CONCLUSIONS: These findings demonstrate that the endothelium-dependent vasodilator substance P can still produce epicardial vasodilatation in vivo in the presence of coronary atherosclerosis. PMID- 1718628 TI - Function of the anatomic pulmonary valve in the systemic circulation. AB - The anatomic pulmonary valve, which has thin leaflets with little elastic tissue in the normal heart, must function as the neoaortic valve after arterial switch operation (ASO) for transposition of the great arteries, palliative surgery for hypoplastic left heart syndrome (HLHS), and pulmonary artery-to-aortic (P-A) anastomosis for complex heart disease with subaortic obstruction. The long-term function of this valve under these circumstances is not known. To investigate the function of this valve in the systemic circulation, the follow-up echocardiograms, catheterization data, and angiograms were reviewed for 189 patients at our institution after an ASO (n = 112), palliative surgery for HLHS (n = 45), or P-A anastomosis (n = 32). In addition, the effect on valve function of preoperative anatomy, prior placement of a pulmonary artery band (PAB), and length of follow-up was examined. Neoaortic regurgitation was present in 41% of patients after an ASO (mean +/- SD) follow-up (20 +/- 20; range, 2.2-80.5 months), 60% of patients after an HLHS repair (21 +/- 15; range, 3.7-62.4 months) and 50% after a P-A anastomosis (27 +/- 21; range, 2.6-89.4 months). Only eight patients had more than trivial/mild regurgitation. No neoaortic stenosis was observed. Minor preoperative valve abnormalities did not influence postoperative valve function. Prior PAB placement significantly increased the likelihood of postoperative neoaortic regurgitation after a two-stage ASO but not after a P-A anastomosis. In the ASO group, patients with an intact ventricular septum had a significantly higher prevalence of neoaortic regurgitation than those with a ventricular septal defect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718629 TI - Echocardiographic comparison of postoperative left ventricular contractile state between one- and two-stage arterial switch operation for simple transposition of the great arteries. AB - Left ventricular contractile function was compared between primary and two-stage arterial switch operation (ASO) in neonates and infants with simple transposition of the great arteries (TGA). Eight patients (group 1) underwent primary ASO at 13 42 (23 +/- 10 [mean +/- SD]) days of age, and five patients (group 2) underwent two-stage ASO after pulmonary artery banding and Blalock-Taussig shunting at 11 26 (15 +/- 3) months of age. Postoperative echocardiographic studies were undertaken at 6-48 (22 +/- 16) months of age in group 1 and 25-45 (32 +/- 7) months of age in group 2. The results were compared with those obtained from nine age-matched normal children (group N). To compare left ventricular contractile state among the patients, the deviations from the mean normal values in relations of end-systolic wall stress to fractional shortening and to rate-adjusted mean velocity of circumferential shortening (the stress-shortening index and stress velocity index, respectively) were obtained for each patient. The stress shortening index was significantly lower in group 2 (-3.1 +/- 3.1) than in group 1 (1.3 +/- 1.7, p less than 0.05); the stress-velocity index was significantly lower in group 2 (-3.3 +/- 1.8) than in group 1 (1.5 +/- 1.6, p less than 0.01) or in group N (-0.4 +/- 0.5, p less than 0.05). These results suggest that postoperative left ventricular contractility can be well preserved in patients after primary ASO for simple TGA in neonates, but there is a possibility of depressed contractility in patients after two-stage ASO. PMID- 1718630 TI - Natural history of apical left ventricular to aortic conduits in pediatric patients. AB - To determine the long-term efficacy of apical left ventricular to aortic conduits in palliating complex left ventricular outflow obstruction, we reviewed our entire experience of 20 pediatric patients who underwent placement of a composite porcine valve-bearing conduit from the left ventricular apex to the aorta from November 1977 to April 1987. There were two early postoperative deaths, both in infants less than 2 months of age at the time of surgery. The remaining 18 patients had successful conduit placement with significant relief of the left ventricular outflow obstruction. Conduits have remained functional from 3 to greater than 9 years after initial placement. Long-term maintenance of a well functioning conduit was limited by conduit heterograft valve dysfunction secondary to degeneration and calcification, with a 3-year conduit survival of 80 +/- 9% and a 7-year survival of 52 +/- 11%. Clinical identification of conduit valve insufficiency was found to be a useful early indicator of impending conduit failure, frequently preceding other signs and symptoms of conduit valve dysfunction. Eight patients required reoperation for conduit failure, and there were two late deaths associated with conduit valve dysfunction. Other long-term complications were rare. No thromboembolic events occurred despite avoidance of anticoagulant therapy, and there were no documented episodes of bacterial endocarditis. Thus, apical aortic conduit placement is an effective surgical technique for intermediate palliation of complex left ventricular outflow obstruction and may be useful in selected pediatric patients as a staging procedure before left ventricular outflow reconstruction. PMID- 1718631 TI - Initial studies with FK506 in renal transplantation. AB - FK506 is a novel immunosuppressive agent which is approximately 100 times as potent as cyclosporine in vitro. In this initial trial, 65 renal transplant patients of high complexity received primary FK506 immunosuppression. Overall, graft and patient survival rates are 80% and 98.5%, respectively. A major advantage of FK506 is its potency with relatively few side effects, which has permitted elimination of steroids in 31 (60%) of these patients. Because of these encouraging results, a randomized trial comparing the therapeutic efficacy and toxicity of FK506 and cyclosporine is currently underway at our institution. PMID- 1718632 TI - Temperature-dependent immunoreactive assay to screen for digoxin-like immunoreactive factor(s). AB - Endogenous circulating digoxin-like immunoreactive factors (DLIF) are known to cross-react with antibodies to digoxin and to inhibit Na+/K(+)-transporting ATPase (Na+K+ATPase; EC 3.6.1.37). Moreover, increasing the immunoassay temperature from 4 to 37 degrees C markedly decreases DLIF from human cord serum. We tested several compounds, including hormonal steroids, bile salts, lipids, and methionine-enkephalin, for their ability to cross-react with two commercially available 125I digoxin RIAs, to inhibit porcine Na+K+ATPase, and to see whether they present the same incubation temperature dependence as human cord serum. Except for methionine-enkephalin, all compounds were inhibitors of Na+K+ATPase in the range of 1-10 mmol/L. Progesterone exhibited the highest cross-reactivity in the two RIAs. The apparent digoxin immunoreactivity for the majority of the cross reacting steroids, bile salts, and linoleic acid was markedly decreased by increasing the incubation temperature from 4 to 37 degrees C, whereas estriol, pregnanediol, and nonspecific compounds (e.g., ethanol, human serum albumin) did not appear to be temperature-sensitive. Both lysophosphatidyl lipids gave an increased apparent digoxin concentration with increasing incubation temperature. Our data suggest that numerous weakly cross-reactive compounds can parallel the response of human cord serum. However, the temperature-dependent effect could be an additional criterion for identifying DLIF. PMID- 1718633 TI - Rapid detection of 1717-1G----A mutation in CFTR gene by PCR-mediated site directed mutagenesis. PMID- 1718634 TI - Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle. AB - Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined. PMID- 1718635 TI - Quantitative analysis of human erythrocyte spectrin by sodium dodecyl sulfate polyacrylamide gel electrophoresis using pancreatic trypsin inhibitor (Kunitz) as an internal standard. PMID- 1718636 TI - Treatment of alopecia totalis with a combination of inosine pranobex and diphencyprone compared to each treatment alone. AB - Recent developments in alopecia areata have included the use of oral inosine pranobex and the introduction of diphencyprone as a contact sensitizer. Good results have been claimed for these treatments even in severe forms of the disease. We performed a study to investigate the efficacy of a combination of these treatments in the most severe form of alopecia areata. Thirty-three patients suffering from alopecia totalis were enrolled. Subjects were divided into three groups matched for age and sex. One group received treatment with inosine pranobex (50 mg/kg/day) for 6 months. The second was sensitized to diphencyprone and treated for 6 months by maintenance of contact allergic dermatitis on the scalp. The third received both treatments. There was no evidence of response to inosine pranobex in any of the 22 subjects who received this treatment. Only two of 22 patients responded to diphencyprone. Patients with long-standing alopecia totalis contemplating diphencyprone therapy should be advised that the chances of success are only around 10%. Inosine pranobex does not appear to improve the response rate. PMID- 1718637 TI - Bleomycin-induced flagellate erythema. AB - A 56-year-old woman who developed widespread pruritus and flagellate erythema after attempted pleuredesis with bleomycin is described. The raised linear lesions of flagellate erythema could not be reproduced by scratching, and histopathological examination revealed a lymphocytic vasculitis. The rash faded spontaneously over several weeks to leave streaks of post-inflammatory melanoderma which remained for 6 months. The role of scratching and dermographism in the pathogenesis of the bleomycin-specific eruption is discussed. PMID- 1718638 TI - Aberrant T-regulation in rheumatoid arthritis and IgA nephropathy affects CD5+ and CD5- B lymphocytes equally. AB - T-suppressor function and T-helper function in healthy adults, elderly patients with non-immune diseases, and patients with rheumatoid arthritis (RA) and IgA nephropathy (IgAN) were titrated by adding graded concentrations of CD8+ cells to autologous CD8-depleted peripheral blood mononuclear cells (PBMC), or CD4+ cells to CD8- 4- PBMC, respectively. Following culture with pokeweed mitogen (PWM), numbers of CD5+ and CD5- immunoglobulin-secreting cells were determined using a combination of rosetting with anti-CD5-coated Dynabeads and reverse haemolytic plaque formation (Jones, 1990). Of 11 RA patients studied, eight had slightly reduced suppressor activity for CD5+ and CD5- IgM-secreting cells, and three with active disease and high serum levels of C-reactive protein, could not suppress IgG, IgA or IgM secretion by either B subset. Helper activity for both CD5+ and CD5- B cells was slightly but significantly increased in RA patients. One of eight patients with IgAN could not suppress IgG, IgA or IgM production by CD5+ or CD5- B cells, and all IgAN patients required strikingly fewer CD4+ cells for PWM induced activation of CD5+ and CD5- B cells than controls. It was concluded that in two immunologically mediated diseases in which some patients have raised numbers of circulating CD5+ B cells, aberrant T-regulation affects CD5+ and conventional CD5- B cells equally. PMID- 1718639 TI - The effect of FK506 and cyclosporin A on antigen-induced arthritis. AB - FK506 and cyclosporin A inhibited the development of antigen-induced arthritis in the rat and rabbit. FK506 was five times more potent than cyclosporin A in the rat and approximately 20 times more potent in the rabbit. FK506 was effective in both species if administered either from the day of intra-articular administration of antigen or when the arthritis was established. In the rabbit, arthritis returned when administration of FK506 was stopped. FK506 (10 mg/kg/day) caused renal damage which was not observed at a dose of 2.5 mg/kg/day. Both of these doses were equally effective at inhibiting the arthritis. The conclusion from these studies is that FK506 is a more effective anti-arthritic agent than cyclosporin A and that a pronounced therapeutic effect can be achieved at non toxic doses of the drug. PMID- 1718640 TI - Separation of self from non-self in the complement system: a role for membrane cofactor protein and decay accelerating factor. PMID- 1718641 TI - Reaction of complement with endothelial cells in a model of xenotransplantation. AB - We review our studies on the role of complement (C) as mediator of xenograft hyperacute rejection using an in vitro model consisting of porcine endothelial cells as target and human serum as source of natural antibodies and C. Cytotoxicity of endothelial cells required IgM antibodies to porcine endothelial cells, and the classical pathway and membrane attack complex of C. These findings correlated with in vivo results of porcine organs transplanted into rhesus monkeys, which showed a) co-deposition of IgM, C3, C4 and C9, along blood vessels of rejecting organs, with trace deposits of factors B or P, and b) minimal deposition of IgM and C components in transplants with prolonged survival that were performed in rhesus monkeys depleted of natural antibodies but with normal C levels. Human serum causes activation of porcine endothelial cells manifested by release of heparan sulfate proteoglycan. Heparan sulfate release was induced by C5a alone. A new approach to avert xenograft hyperacute rejection was tested. To inhibit cytotoxicity of porcine endothelial cells by human C, the membrane associated C inhibitor decay-accelerating factor (DAF) of human origin was incorporated into endothelial cells. Human DAF was able to efficiently inhibit C mediated killing of porcine endothelial cells, suggesting that the use of DAF and other C inhibitors could be used to interfere with C-mediated xenograft hyperacute rejection. PMID- 1718643 TI - Anti-inflammatory and immunosuppressive effects of recombinant soluble complement receptors. AB - CR1 and CR2 have served as unusual probes for the analysis of the two major functions of the immune system involving inflammation and the immune response, respectively. CR1, or some construct containing its active site SCRs, may find a role in the therapy of complement-dependent tissue injury, and may be used to define which diseases are caused by the inappropriate or excessive activation of this system. Although soluble forms of CR2 may be shown to have potential clinical utility when foreign antigen is given prospectively, as in monoclonal antibody therapy, perhaps the most important finding emanating from the analysis of this receptor is the recognition of a previously unrecognized membrane protein complex whose role in B cell development is yet to be determined. It is reasonable to predict that the function of the CD19 complex will be significant as it serves as the link between two evolutionarily distinct systems that share a common purpose of anti-microbial host defense. PMID- 1718642 TI - Paroxysmal nocturnal hemoglobinuria and glycosyl phosphatidylinositol anchored proteins that regulate complement. PMID- 1718644 TI - Speculations on the ataxia-telangiectasia defect. AB - Ataxia-telangiectasia (A-T) is inherited as an monogenetic autosomal recessive disease. Ataxia appears around 1 year of age and progresses until the patient becomes wheelchair-bound, usually by age 10. This progress correlates with deterioration of Purkinje cells in the cerebellum. Sinopulmonary infections are common in patients from some countries but not others. One-third of the patients develop a neoplasm, usually lymphoid, sometime during their shortened lives. Conventional doses of radiation therapy for such cancers are contraindicated since A-T patients are hypersensitive to ionizing radiation. Five complementation groups have been described, based on correction of radioresistant DNA synthesis of fused fibroblasts from pairs of patients. Chromosomal translocations are found in 5-10% of peripheral T cells from most patients and the translocation breakpoints involve sites of normal somatic DNA rearrangement. Thus, the A-T gene(s) effects several cell lineages, suggesting that it is a "housekeeping" gene. Other speculations on "candidate genes" are considered. Recent progress localizing A-T to chromosome 11q23 is reviewed. PMID- 1718645 TI - Effect of prenatal ethanol exposure on postnatal neural gene expression in the rat. AB - To examine the effects of ethanol exposure on neural development, pregnant rats were fed a liquid diet in which 37.5% of the total caloric content was ethanol derived. The developmental appearance of the messenger RNAs coding for preprosomatostatin, glial fibrillary acidic protein, and proteolipid protein was examined by Northern blotting of total cellular RNA obtained from forebrain and hindbrain at various times after birth. In general, there was a delay in the developmental pattern of appearance of these mRNAs which was most noticeable at the early postnatal times. These results suggest that the previously reported delay in neural maturation is reflected at the level of the gene expression. PMID- 1718646 TI - Developmental regulation of the sheep beta-lactoglobulin gene in the mammary gland of transgenic mice. AB - beta-Lactoglobulin (BLG) is the most abundant whey protein in sheep milk but it is not present in mouse milk. We have previously shown that transgenic mice carrying the BLG gene express it specifically in the mammary gland and secrete BLG into milk at high concentrations. Here we demonstrate that BLG transcription is correctly initiated in mice and that BLG synthesis is restricted to the secretory epithelial cells of the mammary gland. We have also determined the temporal pattern of milk protein gene expression and find that the BLG transgene is regulated coordinately with mouse beta-casein and that the patterns of regulation of BLG in mouse and sheep share some similarities. PMID- 1718647 TI - Several testis-expressed genes in the mouse t-complex have expression differences between wild-type and t-mutant mice. AB - The t-complex of the mouse occupies the proximal half of chromosome 17 and contains genes which have profound effects on spermatogenesis. Mutations of several loci in the t-complex appear to interact to cause male sterility or transmission ratio distortion (TRD). By cDNA screening or chromosomal walking we have identified seven genes, which are expressed in the germ cells of testis and map to various regions of the t-complex. These genes were named t-complex testis expressed (Tctex) genes. An analysis of their expression patterns in testes from +/+, +/t, and t/t mice was done by in situ hybridization and by northern blotting. Six genes begin to be expressed at the pachytene stage: Three of them are more abundant at pachytene stage, while three others are more abundant at postmeiotic stages. One gene is expressed at all the stages of spermatogenesis. Interestingly, four Tctex genes show differences in the amount of transcript between wild-type and t-mutant testes. The chromosomal location and expression pattern imply that Tctex genes might be candidate genes for sterility or TRD. PMID- 1718648 TI - Immunotherapy of malignant melanoma. AB - The growth of melanoma in humans depends not only on the malignant potential of the tumor but also on the patient's immune response to the melanoma. As a result, a variety of strategies have evolved to treat melanoma by stimulating antitumor immune responses to melanoma. This article reviews the different approaches being taken and the results obtained to date. PMID- 1718649 TI - Advances in the treatment of nonmelanoma skin cancer. AB - Nonsurgical and nonradiation therapy for nonmelanoma skin cancer is focusing on immunotherapy, recombinant human gene products, localized delivery of radiant energy, photodynamic therapy, and retinoids. Although they are not currently practical for widespread application to nonmelanoma skin cancer, there is good reason to hope for significant therapeutic advances in these areas, which will make successful nonsurgical therapy widely available. PMID- 1718650 TI - High levels of substance P in rheumatoid arthritis synovial fluid. Lack of substance P production by synoviocytes in vitro. PMID- 1718651 TI - Comparison of phosphatase isoenzymes PAP and PSA with bone scan in patients with prostate carcinoma. AB - The aim of this study was to assess the diagnostic value of five biological markers--prostate acid phosphatase (PAP), prostate specific antigen (PSA), tartrate resistant (Tr-ACP), and tartrate labile (TI-ACP) acid phosphatases, and alkaline phosphatase bone isoenzyme (B-ALP)--for the detection of bone metastases in patients with prostate carcinoma. Using the Tc-99m HMDP bone scans of 80 patients scored from 0 (normal) to 2 (diffuse bone involvement) as the "gold standard," a receiver operating characteristic (ROC) analysis was performed. This method allows the determination of different threshold values (corresponding to different couples of sensitivity and specificity) for the assays. An ROC curve comparison was also performed. Results show that B-ALP is the best test for such detection (area under the ROC curve = 0.93; Spearman Rank correlation with bone scan r' = 0.81). Among the other markers, PSA was found to be the best (area under the ROC curve = 0.81; Spearman Rank correlation with bone scan r' = 0.58). In addition to the prostatic tumor markers (PSA and PAP), we suggest the use of the low-cost B-ALP assay in the follow-up of prostate carcinoma patients to determine the optimum moment to perform a bone scan. A normal result of this assay indicates a very low probability of bone metastasis; conversely, raising of B-ALP concentration must lead to a bone scan. PMID- 1718652 TI - High performance liquid chromatography (HPLC): a simple method to quantify Hb C, O-Arab, Agenogi and F. AB - Differentiation of some abnormal haemoglobins, such as Hb C, O-Arab, Agenogi, E, O-Indonesia, C-Harlem, and Siriraj, is difficult and quantitation of the various fractions is impossible with cellulose acetate electrophoresis. The authors report 13 cases of Hb C, 10 of Hb O-Arab and 5 of Hb Agenogi whose haemoglobin fractions were quantitated by HPLC during a thalassaemia screening programme. Hb F was determined by both Betke's method and HPLC. Analysis of data by linear regression demonstrates that the methods furnish overlapping results. Our findings show that HPLC is a rapid and easily reproduced method which allows quantitative and qualitative discrimination of the various haemoglobin fractions, making it a valid tool in screening programmes for haemoglobinopathies. PMID- 1718653 TI - Flow cytometric reticulocyte analysis using thiazole orange; clinical experience and technical limitations. AB - Flow cytometric (FCM) reticulocyte analysis using thiazole orange (TO) is becoming an increasingly popular method for routine quantification of reticulocytes. The methodology is accurate, cost-effective and shows a high correlation with manual techniques. We describe our experience with the clinical application of FCM reticulocyte analysis in a general hospital setting over a 20 month period with special emphasis on technical limitations. PMID- 1718655 TI - Monitoring HLA alloimmunization. Analysis of HLA alloantibodies in the serum of prospective transplant recipients. AB - The identification of serum HLA alloantibody in clinical transplantation can only be considered meaningful if the information derived from clinical tests can be used to predict crossmatch outcome. Drawing on the foundations and principles of blood transfusion medicine, this article presents a strategy for efficient, cost effective patient HLA antibody monitoring that relies on a screening technique identical in sensitivity to the final donor crossmatch method. Application of this approach to the successful transplantation of highly immunized patients using HLA disparate cadaver renal allografts supports the contentions advanced within this article. PMID- 1718654 TI - [Telethermography in the early diagnosis and clinico-therapeutic monitoring of Sudeck's disease]. AB - In order to evaluate the potential value of telethermography in the early diagnosis of Sudeck's disease, the authors examined 10 patients presenting with this condition. Mean disease duration was 3.2 months and algodystrophic lesions in all patients were localized in one of the lower extremities. Ten healthy subjects, with mean age and sex distribution similar to those of the patients with Sudeck, were chosen as controls. Clinical examination, laboratory tests and telethermography were performed every two weeks for three months; X-rays of the affected limbs were also performed at the beginning and at the end of the study. All patients with algodystrophy were treated with salmon calcitonin (100 U.I./die/i.m. during the first 2 months and 100 U.I. on alternate days during the last month). Clinical-therapeutic thermographic monitoring showed that the localized hyperthermic pattern, initially shown in all patients (temperature levels at least three centigrades above normal values), later underwent a progressive time-related reduction leading to normalization. These results enable the authors to confirm the potential value of telethermography in the early diagnosis of Sudeck's disease and in its clinical monitoring, particularly in relation to therapy. PMID- 1718657 TI - Ion conductances in identified leech neurons. PMID- 1718656 TI - The effects of streptozotocin induced diabetes mellitus and fish oil compound on gene expression of atrial natriuretic peptide in rat. AB - 1. Adult male Wistar rats were injected with streptozotocin (STZ: 55 mg/kg) for inducing diabetes. Then blood and atria for RNA extraction were withdrawn from rats treated 3 and 11 weeks previously with STZ respectively. Atrial total RNA were extracted with cold phenol method. The ANP mRNA contents were determined using Dot blot hybridization technique with alpha-32-P-labelled r-prepro ANP cDNA probe. 2. Plasma glucose was increased and plasma immunoreactive insulin was lowered in rats at 3 and 11 weeks after injection of STZ. ANP gene expression in diabetic rats was depressed. ANP mRNA contents in rats treated 3 and 11 weeks with STZ were 86.4% and 31.7% of that of control rats. 3. Three weeks after treatment of STZ, the rats were gastrically perfused with FOC (Fish Oil Compound) (0.355 ml/kg) once a day successively until 11 weeks. This treatment induces lower blood pressure in rats. ANP gene expression in FOC group was apparently recovered which had been decreased because of the effect of diabetes mellitus. PMID- 1718658 TI - An extended antigenic scheme for Yersinia pseudotuberculosis. PMID- 1718659 TI - Somatic and flagellar antigens of Yersinia enterocolitica and related species. PMID- 1718660 TI - Fetal alcohol exposure and adverse pregnancy outcomes. PMID- 1718661 TI - Chemical xenogenization: antigenic changes of cancer cells induced by triazene compounds. PMID- 1718662 TI - DNA probes: applications of the principles of nucleic acid hybridization. AB - Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates. PMID- 1718663 TI - Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. AB - The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies--examining the critical parameters and variations and their widespread applications--giving the strengths and limitations of these methodologies. PMID- 1718664 TI - Atopic dermatitis and food hypersensitivity in children. PMID- 1718665 TI - Antigenic variance. PMID- 1718666 TI - Symbolic functioning in very young children: understanding of pictures and models. AB - Before one can understand or use any symbol, one must first realize that it is a symbol, that is, that it stands for or represents something other than itself. This article reports 4 studies investigating very young children's understanding of 2 different kinds of symbolic stimuli--scale models and pictures. The data replicate previous findings that 2.5-year-old children have great difficulty appreciating the relation between a scale model and the larger space it represents, but that they very readily appreciate the relation between a picture and its referent. This result is interpreted in terms of the dual orientation hypothesis. Models are difficult for young children because they require a dual representation--a child must think about a model both as an object itself and as a representation of something else. Because pictures are not salient as real objects, they do not require a dual representation. Several kinds of evidence supporting the dual orientation hypothesis are presented. An additional result was the occurrence of a transfer effect: Prior experience with a picture task led to better performance on a subsequent model task. This finding suggests that experience with a symbolic medium they understand can help young children figure out a different, unfamilar medium that they would otherwise not understand. PMID- 1718667 TI - Protein phosphorylation and cell transformation. PMID- 1718668 TI - Synthesis and biochemical studies of dithioate DNA. AB - Dithioate DNA was synthesized and used for various biochemical studies. Results from these studies indicate that dithioate DNA is a potent inhibitor of HIV Reverse Transcriptase, activates endogenous RNase H in HeLa cell nuclear extracts, and is a useful probe for studying protein-DNA interactions. PMID- 1718669 TI - Self-assembly in supramolecular systems. AB - The complex structural and functional properties of many natural molecules have spawned innumerable attempts to understand and mimic biological activity. This has often involved preparing extremely complex structures of carefully designed geometries. In natural systems the primary structure of a protein (the amino acid sequence) establishes all of the structural relationships within the molecule, although many of these are not apparent until the molecule folds, coils, or otherwise adopts the appropriate conformation. Nature has selected suitable amino acid sequences for various applications during eons of evolution. In this paper, we report our efforts to achieve similar results by providing all of the required structural elements on a flexible framework. This concept is illustrated in three ways: the design and preparation of a redox-switched vesicle and a small-molecule molecular receptor (both based on the ferrocene system) and of a functional cation channel. PMID- 1718670 TI - [Titanium-nickel alloy stand for urethrostenosis caused by prostatauxe]. AB - Self-made spiral tubular stand of titanium-nickel alloy, characterized by shape memory effect and good biocompatibility, was used in 15 patients with urethro stenosis caused by prostatauxe. Of these patients, 14 restored normal urination and 1 failed because of technical problems. With the shape memory feature of titanium-nickel alloy, the stand may be easily inserted and long-term set in the prostatic urethra. This safe, simple method is particularly suitable to the aged patients. PMID- 1718671 TI - Calibration of fluorescence intensities to quantify antibody binding surface determinants of cell subpopulations by flow cytometry. AB - Quantitative indirect immunofluorescence analysis by flow cytometry was used to determine the mean number of antibody binding sites per cell in a small subpopulation of rat brain cells expressing low levels of a cell surface differentiation antigen recognized by monoclonal antibody (Mab) RB13-6 (Kindler Rohrborn et al.: Differentiation 30:53-60, 1985). For these non-disjunct distributions of fluorescence intensities, the cut-off border between antigen positive and antigen-negative cells was defined by a statistical test. To eliminate the influence of accidental disturbances leading to incorrect statistical decisions, the curves for antigen-negative cells were fitted according to cell number and shape. The flow cytometer was calibrated with the use of a clonal cell line for which the average number of Mab RB13-6 binding sites per cell had previously been determined by radioimmunoassay and Scatchard plot analysis. Using this analytical procedure, both the proportion of Mab binding brain cells and the mean number of Mab binding sites per Mab binding cell could be determined as a function of developmental stage. PMID- 1718672 TI - Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki-67 or bromodeoxyuridine). AB - Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)-stimulated normal human blood lymphocytes for the double staining of 1) Ki-67 antigen and DNA and 2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd-labeled cells. With respect to the discrimination between antigen-positive and -negative subpopulations, there was no statistically significant differences between the results from direct (Ki-67) or indirect (Ki-67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells. PMID- 1718673 TI - Cell kinetic analysis of mixed populations using three-color fluorescence flow cytometry. AB - The development of antibodies to DNA-incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours. Selecting out cells from the mixture required the application of three-color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd-DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC-conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE-conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid-S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately. A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid-S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors. PMID- 1718675 TI - [Tumor follow-up in colorectal carcinoma]. PMID- 1718674 TI - Temporal studies of the inhibition of voluntary ethanol intake in the rat induced by intermittent ethanol treatment and some long term neurochemical consequences. AB - Male rats were injected with ethanol (groups 3 and 5; 2.0 g/kg i.p.) or saline (groups 2 and 4) once a week for 52 weeks. The rats had access to ethanol as a voluntary choice for 24 h either once 6 days after the injection (groups 2 and 3) or twice 3 and 6 days after the injection (groups 4 and 5). At the beginning of the treatment ethanol injections inhibited voluntary ethanol intake if tested 6 days later (groups 3 and 5), but a tolerance developed to this inhibition. During tolerance development the rats in group 5 also drank less ethanol on day 3 than on day 6. No corresponding behaviour was seen in group 4. Thus part of the tolerance was a gradual reduction of the duration of inhibition. During the evaluation period (25 weeks) after the treatment, ethanol exposure (20 weeks) consisted of a continuous choice between ethanol and water. Of different ethanol concentrations both ethanol-injected groups (3 and 5) took the same voluntary dose of ethanol independent of the offered concentration. After 5 weeks without ethanol all rats were killed and a number of neurochemical variables were determined. Compared with almost unexposed rats (group 1) changes were seen in inositol phospholipid breakdown, muscarinic binding sites in hippocampus, noradrenaline concentrations in frontal cortex, hippocampus and hypothalamus, dopamine concentration in frontal cortex and 5-hydroxytryptamine concentration in hypothalamus. In most cases the largest changes were seen in group 5. None of the variables had a constant relation to ethanol intake in the total population. However, significant correlations were found in some of the groups. PMID- 1718676 TI - Localized and inducible expression of Xenopus-posterior (Xpo), a novel gene active in early frog embryos, encoding a protein with a 'CCHC' finger domain. AB - Xenopus-posterior (Xpo) is a gene that is activated at or shortly after the midblastula transition (MBT). The RNA accumulates to a relatively low level, which remains constant until gastrulation, then rapidly and transiently increases in posterior ectoderm and mesoderm. A single copy of a putative finger motif, of the 'CCHC' type, is located near the carboxyl terminus. One or two copies of similar sequence motifs are found in the nucleocapsid protein of retroviruses where they are involved in protein-RNA interactions, and in cellular nucleic acid binding protein (CNBP), a protein that binds to the sterol regulatory element. Xpo expression is induced in ectodermal explants by treatment with basic fibroblast growth factor (bFGF) and with polypeptide growth factors found in medium conditioned by the Xenopus XTC cell line (XTC-CM). Taken together, these properties suggest a possible role for Xpo in the organization of the anteroposterior axis during development. PMID- 1718677 TI - The expression of tenascin by neural crest cells and glia. AB - The extracellular matrix glycoprotein tenascin is concentrated in both the embryo and adult in regions where cell motility is taking place. For example, during avian neural crest morphogenesis tenascin is concentrated in the rostral half of the sclerotome, precisely where the neural crest cells themselves are found. Previous in vitro studies indicated that somite cells were the source of this tenascin, implying a role for tenascin in directing the ventral migration of neural crest cells and thus the establishment of the periodic arrangement of the PNS. In this study, we have used a cDNA probe to identify the source of tenascin found along the pathways of the neural crest using in situ hybridization. In tissue sections, individual cells found along the neural crest migratory pathways, both before entering the somites and within the somites, are strongly labelled by the tenascin cDNA. In vitro neural crest cells are more strongly labelled with the tenascin probe than somite cells. Finally, western blotting has been used to identify tenascin in culture medium conditioned by neural crest cells. This indicates that neural crest cells themselves are the source of much of the tenascin found lining their migratory pathways, and that interactions with somite cells may not be needed to induce the expression of tenascin. We have also studied the distribution of tenascin mRNA in the developing spinal cord and spinal ganglia. At embryonic days 7 and 10, tenascin cDNA hybridizes within cells that appear to be migrating from the ependymal layer to the white matter, as well as within cells in the dorsal roots.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718678 TI - The L5 epitope: an early marker for neural induction in the chick embryo and its involvement in inductive interactions. AB - The pattern of expression of the carbohydrate epitope L5 was studied during early development of the chick neuroepithelium. Immunoreactivity first appears during gastrulation, at mid-primitive streak stage, and persists until at least 3.5 days of development. The epitope is expressed on all the components of the developing nervous system, both central and peripheral. In immunoblots, the antibody recognises a major component of about Mr 500,000 and several more minor components of lower molecular mass. If a Hensen's node from a donor embryo is transplanted into the area opaca of a host embryo, L5 immunoreactivity appears in the epiblast surrounding the graft. If hybridoma cells secreting the antibody are grafted together with Hensen's node into a host chick embryo, the induction of a supernumerary nervous system is inhibited. We suggest that the L5 epitope is an early and general marker for neural induction and that it may be involved directly in inductive interactions. PMID- 1718679 TI - Pleiotropic effect of okadaic acid on maturing mouse oocytes. AB - Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion. PMID- 1718680 TI - Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila. AB - Metamorphosis in Drosophila is triggered by a pulse of the steroid hormone ecdysone at the end of larval development. Ecdysone initiates a genetic hierarchy that can be visualized as a series of puffs in the larval salivary gland polytene chromosomes. The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. Here we describe the spatial and temporal patterns of E74A RNA and protein expression at the onset of metamorphosis. We use in situ hybridization, antibody stains, and western and northern blot analyses to follow E74A expression from its initial appearance as nascent transcripts on the polytene chromosomes, to spliced mRNA, to post-translationally modified nuclear E74A protein. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. We discuss possible mechanisms for post-transcriptional regulation of E74A expression and suggest that the unusually long and complex 5' leader in the E74A mRNA may regulate its translation. PMID- 1718681 TI - Optimum use of growth hormone in children. PMID- 1718682 TI - Formoterol. A review of its pharmacological properties and therapeutic potential in reversible obstructive airways disease. AB - Formoterol, a long-acting beta 2-selective adrenoceptor agonist, produces dose proportional bronchodilation in patients with obstructive airways disease with a reversible component. A significant effect occurs within minutes of inhalation of a therapeutic formoterol dose and persists for approximately 12 hours. Oral formoterol has a slower onset of action than the inhaled formulations, but also produces prolonged bronchodilatory effects. Inhaled formoterol has shown a therapeutic efficacy equivalent to or better than comparable dosages of the conventional beta 2-agonists salbutamol, fenoterol and terbutaline in short and long term trials, in both adults and children with asthma. Its prolonged duration of action permits a twice-daily dosage regimen and results in improved control of nocturnal symptoms by reducing the 'morning dip'. Formoterol also compares well with oral slow release theophylline. In addition, significantly more patients with chronic obstructive airways disease (COAD) had an improvement in symptoms when treated with formoterol compared with salbutamol or fenoterol. Noncomparative studies indicate formoterol also provides effective prophylaxis of exercise-induced asthma. Development of tachyphylaxis has not been observed. Formoterol is generally well tolerated. Adverse effects observed represent predictable extensions of its pharmacology. Tremor and palpitations are most frequently reported. The incidence of adverse events is dose-proportional and therefore related to the route of administration, being more frequent following oral than inhalation therapy. The long-acting beta 2-agonists, including formoterol, represent a significant advance over current maintenance or prophylactic bronchodilator therapy with intermediate-acting beta 2-agonists such as salbutamol, fenoterol and terbutaline, predominantly because of the twice daily administration regimen. However, comparisons with other long-acting beta 2 agonists, such as salmeterol, evaluation of its role in improving symptom control in patients failing to respond to prophylactic therapy, and clarification of the optimal role of beta 2-agonists in asthma maintenance therapy are required to fully determine the value of formoterol in the management of obstructive airways disease. PMID- 1718683 TI - Omeprazole. An updated review of its pharmacology and therapeutic use in acid related disorders. AB - Omeprazole is the first of a new class of drugs, the acid pump inhibitors, which control gastric acid secretion at the final stage of the acid secretory pathway and thus reduce basal and stimulated acid secretion irrespective of the stimulus. In patients with duodenal or gastric ulcers, omeprazole as a single 20 mg daily dose provides more rapid and complete healing compared with ranitidine 150 mg twice daily or 300 mg at nighttime, or cimetidine 800 or 1000 mg/day. Patients poorly responsive to treatment with histamine H2-receptor antagonists respond well to omeprazole--most ulcers healed within 4 to 8 weeks of omeprazole 40 mg/day therapy. Omeprazole 20 or 40 mg/day has been administered as maintenance therapy for peptic ulcer disease for up to 5.5 years with very few ulcer recurrences. In patients with erosive or ulcerative oesophagitis, omeprazole 20 or 40 mg/day produces healing in about 80% of patients after 4 weeks, and is superior to ranitidine with respect to both healing and symptom relief. Healing rates of greater than 80% are achieved after 8 weeks in patients with severe reflux oesophagitis unresponsive to H2-receptor antagonists. Maintenance therapy with a daily 20 mg dose prevents relapse in about 80% of patients over a 12-month period. Omeprazole is considered to be the best pharmacological option for controlling gastric acid secretion in patients with Zollinger-Ellison syndrome. Daily dosages of 20 to 360 (median 60 to 70 mg successfully reduce basal acid output to target levels (less than 10 mmol/h or less than 5 mmol/h in patients with severe oesophagitis or partial gastrectomy) during treatment for up to 4 years. Omeprazole is well tolerated in short term studies (up to 12 weeks); the reported incidence of serious side effects (about 1%) being similar to that seen in patients treated with an histamine H2-receptor antagonist. The longer term tolerability of omeprazole has been investigated in patients treated for up to 5.5 years. Slight hyperplasia, but no evidence of enterochromaffin-like (ECL) cell dysplasia or neoplasia or ECL cell carcinoids has been reported. ECL cell carcinoids have been observed in rats after life-long treatment with high doses of omeprazole or ranitidine, or in rats with partial corpectomy; the weight of experimental evidence indicates that this is a result of prolonged hypergastrinaemia.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718685 TI - Optimal drug therapy in the treatment of testicular cancer. AB - Although testes cancer is the most common malignancy affecting young men, dramatic survival rates are now possible with the development of optimal individualised drug therapy. Human chorionic gonadotropin and alpha-fetoprotein are important tumour markers associated with testes cancer, and can provide essential information about prognosis and treatment efficacy. For treatment purposes, testicular germ-cell malignancies are broadly classified as seminomatous or non-seminomatous. Early stage seminomas are treated with radiotherapy, while more advanced disease requires systemic chemotherapy. Stage I nonseminoma patients can now be offered the option of retroperitoneal lymph node dissection (RPLND) or close clinical observation, while patients with stage II or III nonseminoma should generally be treated with chemotherapy. The dramatic survival rates now apparent with chemotherapy are due in large part to the introduction of cisplatin (cisplatinum II)-based chemotherapy and to the optimisation of therapy based on pretreatment risk analysis. The most common chemotherapeutic regimen for standard risk patients includes cisplatin and etoposide (VP 16213) and long term disease-free survival rates exceed 80%. A subset of poor risk patients with significantly reduced survival can be defined. These patients, and patients with relapsed or refractory disease, should receive more aggressive regimens, and ifosfamide (isophosphamide) is proving to be a particularly promising new agent in this regard. High-dose carboplatin with autologous bone marrow rescue is another encouraging alternative currently being investigated for these patients. Chemotherapy, despite substantial effectiveness, is not without toxicity, which consists primarily of myelosuppression, nausea and emesis, and renal toxicity. With careful monitoring and prophylaxis, however, these toxicities can generally be ameliorated or avoided. PMID- 1718684 TI - Management of nephrotic syndrome in childhood. AB - Nephrotic syndrome is defined as proteinuria sufficiently severe to result in hypoalbuminaemia, oedema and hyperlipidaemia. The early modern history of this illness was characterised by the serendipitous development of renal biopsy technique at approximately the same time as the use of corticosteroids for nephrotic syndrome. The coincidence of these events set the stage for evaluating therapeutic response to corticosteroids and cytotoxic agents in relation to renal histology and ultimate clinical outcome. The International Study of Kidney Disease in Children (ISKDC) was initiated in the 1960s as a multicentre study examining these relationships in children. Over the next decade this study, as well as contributions from other investigators, helped define optimum therapy for these children. It was determined that a child with nephrotic syndrome under the age of 6 years, who did not present with hypertension, azotaemia, hypocomplementaemia or signs of systemic illness, had an approximately 85% chance of responding to corticosteroid therapy. If only those children who had minimal change histology on biopsy were considered, 94% responded. The original regimen which is still used today, was 60 mg/m2 bsa/day prednisone administered on a 3 times per day dosage schedule for 4 weeks, followed by an additional 4 weeks of therapy at a dose of 40 mg/m2 bsa given as a single oral dose every other day. Of those who respond roughly one-third will have no relapses, while almost half will have frequent relapses (greater than or equal to 2 in 6 months) and the rest will have infrequent relapses. Patients in relapse are treated as at presentation but are usually converted to the 40 mg/m2 bsa dose when the urine has been free of protein for 3 days, and are then tapered off or maintained on this dose for several weeks, depending on the individual's history of relapses and incidence of side effects from corticosteroids. For those children who are suffering frequent relapses and severe corticosteroid side effects (e.g. growth failure, morbid obesity, aseptic necrosis of bone), cytotoxic agents were identified as providing long term remission. After inducing remission with conventional corticosteroid dosages, cyclophosphamide is administered at a dose of 2 mg/kg/day given as a single dose for 8 weeks. This regimen was shown to lead to approximately 70% of patients being in remission 2 years after completion of this course of therapy. Chlorambucil given at a dose of 0.2 mg/kg/day as a single oral dose has been equally efficacious.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718686 TI - Pravastatin. A review of its pharmacological properties and therapeutic potential in hypercholesterolaemia. AB - Pravastatin is an HMG-CoA reductase inhibitor which reduces plasma cholesterol levels by inhibiting de novo cholesterol synthesis and increasing the receptor mediated catabolism of low density lipoprotein (LDL). Several large multicentre placebo-controlled trials have shown that pravastatin reduces total and LDL cholesterol levels in a dose-proportional manner in patients with familial or nonfamilial hypercholesterolaemia. Reductions in LDL-cholesterol levels reported in the largest study were 18% (10 mg/day), 23% (20 mg/day) and 31% (40 mg/day) after 12 weeks. Once-daily administration appears to be as effective as two daily doses. Pravastatin consistently increases HDL-cholesterol levels and decreases levels of total triglycerides but these changes are not dose dependent. At the study dosages used, the antihypercholesterolaemic effects of pravastatin were superior to those of bezafibrate and clinofibrate, and were similar to those of simvastatin, lovastatin, gemfibrozil and cholestyramine although in some studies a trend towards a superior effect with pravastatin was seen. Pravastatin did not reduce HDL-cholesterol like probucol, or increase triglyceride levels like cholestyramine. Combined treatment with pravastatin and cholestyramine or colestipol enhances the cholesterol-lowering effects of either drug administered alone and offsets the increase in total triglyceride levels seen with cholestyramine or colestipol therapy. Pravastatin is well tolerated during treatment of up to 24 months but longer term tolerability has not yet been established. The effect of provastatin on cardiovascular events related to elevated plasma cholesterol levels is under investation in several large scale regression and primary and secondary prevention trials.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718687 TI - Platelet activating factor (PAF). A review of its effects, antagonists and possible future clinical implications (Part I). AB - This review is an attempt to summarise recent data on platelet activating factor (PAF) and PAF antagonists from 1988 to the present. This period saw a burst in research activity focused predominantly on the effect of PAF in various organs. The effect of PAF and its antagonists was further intensively studied in vitro on isolated platelets, leucocytes, macrophages and endothelial cells. From these and earlier data, based on the catastrophe theory of Thom and Zeeman, a new concept on the interaction between PAF and various cytokines could be recognised as an important mechanism of action of the phospholipid mediator, suggesting the existence of an autocatalytic feedback network through which PAF can influence cellular function under certain pathophysiological conditions. This mechanism can be regarded as the culmination of our recent knowledge on the role of PAF, and may influence the possible clinical implications of PAF antagonists in the near future. It is recognised that PAF is released in shock and ischaemic states, and that PAF antagonists can protect the heart and brain against ischaemic injury. Therefore, in contrast to the previous period, which was predominantly devoted to the elucidation of the role of PAF in immediate hypersensitivity reactions, studies performed on cerebral, myocardial and intestinal ischaemia as well as in various shock conditions have concentrated on entirely new aspects of the effect of PAF antagonists, emphasising the significance of the inflammatory process and cell-to-cell interactions in these pathophysiological states. This has led to a re-evaluation of the experimental data previously accumulated. At the same time, these new trends in PAF and PAF antagonist research have explored further possibilities for the application of PAF antagonists in clinical practice. Attention has been focused on the physiological role of PAF as a signal molecule, especially between the neuroendocrine system and related sensory organs. The recognition of the significance of PAF in mammalian reproduction is fascinating and may lead to new clinical applications of PAF antagonists. It appears probable that, like eicosanoids, PAF is involved in a great variety of membrane-dependent processes that play a fundamental role in the maintenance of homeostasis. PAF research has provided several potent natural and synthetic antagonists which may facilitate the clinical application of these drugs in the near future. PMID- 1718688 TI - Perindopril. A review of its pharmacological properties and therapeutic use in cardiovascular disorders. AB - Perindopril is a long acting angiotensin converting enzyme (ACE) inhibitor, which displays similar pharmacodynamic properties to other agents in this class. In common with enalapril, it is also a prodrug. After absorption, perindopril is hydrolysed to the active metabolite, perindoprilat, and with once daily administration adequate 24-hour inhibition of ACE is obtained. Perindopril 4 to 8mg once daily is usually effective for blood pressure control in patients with mild to moderate essential hypertension. Those patients who do not respond adequately to monotherapy with perindopril usually respond with the addition of a second agent, such as a thiazide diuretic. General practice trials indicate that perindopril is at least as effective and as well tolerated as usual therapeutic dosages of captopril, atenolol or hydrochlorothiazide plus amiloride in mild to moderate essential hypertension. Preliminary results indicate that perindopril may also be effective in patients with severe hypertension or congestive heart failure. Perindopril is generally well tolerated and has an adverse effect profile similar to that of other ACE inhibitors. It further clinical experience confirms initial findings, perindopril is likely to represent a useful alternative to other members of the ACE inhibitor class in all grades of hypertension and congestive heart failure. PMID- 1718689 TI - Calcium antagonists and myocardial microperfusion. AB - Recent studies have improved our understanding of the beneficial actions of calcium antagonists on myocardial microcirculation and metabolism. The effect of calcium antagonists on the microcirculation of the left ventricular rat myocardium was studied using in vivo microscopic techniques. Intravenous verapamil 0.3 mg/kg and nifedipine 75 micrograms/kg produced a 15 to 18% increase in the diameter of larger A1 and A2 coronary arterioles (range 31 to 300 microns); diameters of terminal (A4) arterioles and capillaries did not change significantly. Furthermore, verapamil significantly (p less than 0.001) increased the ratio of capillaries filled with red cells to those containing plasma alone. Verapamil pretreatment produced a similarly selective dilatation of larger coronary arterioles and protected against the ischaemia-induced fall in capillary red cell content. Spectroscopic data show that verapamil also produces an increase in myocardial phosphocreatine and preservation of adenosine triphosphate (ATP) during ischaemia in the Langendorff-perfused heart. In patients with exercise-induced angina, gallopamil decreased global myocardial 201Tl and 123I phenylpentadecanoic acid (IPPA) uptake due to a reduction in myocardial oxygen consumption. Regional 201Tl and IPPA uptake as well as IPPA clearance in post stenotic areas tended to rise after gallopamil treatment. Thus, the beneficial effects of calcium antagonists such as verapamil or gallopamil in patients with ischaemic heart disease may result from dilatation of predominantly larger arterioles. Consequently, there is an improvement in regional perfusion and free fatty acid utilisation in reversibly ischaemic regions. PMID- 1718691 TI - Aspects of the medical therapy of angina pectoris. AB - Patients with stable and unstable angina may experience, in addition to a fixed decrease in their coronary flow reserve, transient impairment of coronary flow, which may precipitate spontaneous angina attacks. In stable angina the cause of impairment of flow is vasoconstriction at the site of stenosis or in distal vessels. In unstable angina the cause of impairment of flow is dynamic thrombosis and vasoconstriction. In variant angina, angiographic studies with vasoactive substances indicate that the coronary spasm is caused by a localised alteration in which a segment of a coronary artery becomes temporarily hyperreactive to constrictor stimuli. Patients with chronic stable angina may also experience a variable ischaemic threshold. A recent study in patients with occlusion of a single coronary artery indicates that, in stable angina, the constriction of distal and/or collateral coronary vessels may be an alternative pathogenic mechanism, in addition to constriction at the stenotic site. Calcium antagonists have a well established role in the treatment of angina pectoris. In order to optimise their effectiveness in the various syndromes of angina, further elucidation of the mechanisms by which vasoconstriction can interfere with coronary flow is required. PMID- 1718692 TI - Effect of gallopamil on myocardial ischaemia during percutaneous transluminal coronary angioplasty. AB - This report summarises selected preliminary results from an ongoing study designed to investigate the effect of the calcium antagonist gallopamil on myocardial ischaemia during percutaneous transluminal coronary angioplasty (PTCA). To date, 12 adult males with coronary artery disease and significant proximal stenosis of the left anterior descending coronary artery (LAD) have been randomly assigned to gallopamil or placebo under double-blind conditions. Patients with recent myocardial infarction, apparent collateralisation of the LAD, myocardial failure, sinoatrial or atrioventricular block, severe hepatic disease or renal failure were excluded from the study. PTCA was performed using at least 2 balloon inflations, each of 2 minutes' duration. Gallopamil 0.4 mg or placebo (normal saline) were administered during the 10-minute interval between the 2 inflations. Blood samples were taken simultaneously from the coronary sinus and the femoral artery before and immediately after each inflation. Lactate concentration and the relative amount of activated neutrophils were selected for trend analysis. Furthermore, ECG changes were analysed by calculating the sum of the absolute ST-segment deviations (80 msec after J point, maximal T deviation) of leads I, II, III, V2, V4 and V6. In the presence of gallopamil, the degree of ST-segment/T-wave changes induced by balloon inflation was reduced. Additionally, gallopamil attenuated myocardial lactate release and appeared to prevent the increase in activated neutrophils observed during control inflations. These preliminary results suggest a beneficial effect from intracoronary administration of gallopamil during PTCA, achieved by attenuation of the ischaemic reaction during coronary occlusion. PMID- 1718690 TI - Protective effects of calcium antagonists against ischaemia and reperfusion damage. AB - The most positive results in this area have been those of the second Danish Study Group on Verapamil in Myocardial Infarction (1990) which assessed the benefit of treatment with verapamil from the second week after myocardial infarction. Verapamil produced a significant reduction in both mortality and reinfarction rates. Consequently, it may be concluded that treatment with calcium antagonists, such as verapamil and diltiazem, should not be used in the acute phase of myocardial infarction, but rather as prophylaxis to prevent reinfarction by protecting against myocardial ischaemia. The lack of reported cardioprotective efficacy with calcium antagonists, which contrasts with experimental predictions, can be explained by the inappropriate timing of administration and the use of dihydropyridine, which can be detrimental in myocardial infarction. These is little or no evidence to show that calcium antagonists are cardioprotective in patients with myocardial infarction or unstable angina. Thus, the randomised trials studying acute myocardial infarction reveal no overall effect of treatment on mortality in the short or long term. The prototype calcium antagonists differ in their effects on the reinfarction rate in these patients. With verapamil there is a small tendency for a reduction in reinfarction, with nifedipine a clear worsening, and with diltiazem a reduction almost reaching statistical significance. The general lack of protective efficacy is presumably a result of the drugs being administered too late after the onset of ischaemia. PMID- 1718693 TI - Comparison of dihydropyridine and phenylalkylamine calcium antagonists in patients with coronary heart disease. AB - To evaluate possible differences between dihydropyridine and phenylalkylamine calcium antagonists in the setting of chronic stable angina, 2 placebo controlled, double-blind, crossover trials were conducted comparing the effects of gallopamil and nifedipine on exercise tolerance and ischaemic ST depression, using standard as well as slow release formulations. In the first study, 30 patients received standard formulations of gallopamil (50mg 3 times daily) and nifedipine (20mg 3 times daily). This trial was stopped after 9 patients had been enrolled, because of severe exacerbation of angina in 3 nifedipine recipients. 21 patients then entered a second protocol in which the nifedipine dose was reduced to 10mg 3 times daily. Compared with the preceding placebo periods, time to angina onset and total exercise time were statistically significantly (p less than 0.01) prolonged by gallopamil (by 30 and 18%, respectively), and nonsignificantly prolonged by nifedipine (by 20 and 13%, respectively), after 4 weeks' treatment. Increases in heart rate and rate-pressure product at maximal comparable workloads were less with gallopamil than with nifedipine (p less than 0.01). In contrast to nifedipine, gallopamil was associated with very few side effects. The second trial comprised 24 patients who received slow release formulations of gallopamil (100mg twice daily) and nifedipine (20mg twice daily) over 2 weeks. Again, both drugs exhibited significant anti-ischaemic efficacy, as evidenced by reductions in ST depression at maximal comparable workloads and increases in exercise time compared with placebo, but the differences between the treatments were not statistically significant. Side effects were more frequent with nifedipine, but less severe than with the standard formulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718695 TI - Silent ischaemia in the 1990s. AB - Silent myocardial ischaemia (significant ST depression without chest pain) is a common occurrence in most forms of coronary heart disease and can be associated with permanent changes in myocardial structure. The haemodynamic and ECG manifestations of silent episodes are similar to those observed in painful ischaemia. Exercise testing is the most appropriate method for assessing the severity of coronary artery disease; increased sensitivity can be obtained by combining it with radionuclide scintigraphy or ventriculography. Ambulatory ECG monitoring may fail to detect ischaemic changes revealed by exercise provocation. The treatment approach should depend on the degree of ischaemia. Numerous clinical investigations in stable and unstable angina and in patients with a previous myocardial infarction indicate that the prognosis of patients with myocardial ischaemia does not depend on whether the ischaemia is silent or symptomatic. Silent and symptomatic episodes alone represent the same degree of coronary disease. Moreover, it appears that ischaemic episodes are a more powerful adverse prognostic influence than left ventricular function or the extent of coronary artery disease. All anti-ischaemic agents, such as beta blockers, calcium antagonists and nitrates, and interventions such as coronary balloon angioplasty or coronary bypass surgery, are very effective treatments for myocardial ischaemia. All efforts should be made to prevent ischaemic episodes, whether silent or symptomatic, since the severity of disease rather than the presence or absence of symptoms more accurately reflects the need for intervention. PMID- 1718694 TI - Intrarenal infusion of gallopamil in acute renal failure. A preliminary report. AB - In order to ascertain the protective role of a potent calcium entry blocking agent in human acute renal failure, 10 patients were randomised to treatment with either intrarenal gallopamil plus intravenous furosemide (frusemide) 0.5 mg/kg/h for 24 hours, or furosemide alone. Gallopamil was infused into each kidney at the rate of 40 to 80 micrograms/min for 4 hours. During 7 days of post-treatment follow-up, the gallopamil treatment group exhibited a significantly higher urine output [257 ml/h vs 81 ml/h (p less than 0.001) after 2 days, and 199 ml/h vs 120 ml/h (p less than 0.005) after 7 days] and creatinine clearance [20 vs 4 ml/min (p less than 0.005) after 2 days, and 38 vs 14 ml/min (p less than 0.001) after 7 days] than the furosemide-only control group. Furthermore, gallopamil treatment accelerated the decline of serum creatinine after renal failure and reduced the requirement for dialysis. Although patient numbers were small, these results indicate that the addition of intrarenal gallopamil to intravenous furosemide treatment enhances the recovery of renal function after acute renal failure. PMID- 1718696 TI - Local myocardial biochemical and ionic alterations during myocardial ischaemia and reperfusion. AB - Acute myocardial ischaemia and reperfusion result in a series of inhomogeneous metabolic, ionic and neurohumoral events that explain the associated mechanical and electrical events, including cardiac death. The time course of the hydrolysis of high energy phosphates, the rise in extracellular potassium and the fall in intracellular and extracellular pH induced by acute no-flow ischaemia have been well characterised. However, the time course of the changes in intracellular sodium, calcium and magnesium levels is less clear. It appears that the changes in intracellular calcium may be pivotal to many of the biochemical and electrophysiological changes produced by the abrupt cessation of coronary arterial inflow and the associated interruption of venous washout. Consequently, agents that modify the handling of calcium by the sarcolemma and the sarcoplasmic reticulum have a significant impact on many of the metabolic, ionic and electrical abnormalities characterising acute ischaemia and reperfusion. PMID- 1718697 TI - The pathophysiology and epidemiology of myocardial infarction. A review. AB - Myocardial infarction continues to represent a major cause of death in the Western world, and although there have been significant reductions in its incidence in recent years, some countries such as Scotland and Finland still have high mortality rates. Thrombotic occlusion, in association with varying degrees of plaque disruption and coronary artery spasm, represents the major cause of acute myocardial infarction (AMI). At the cellular level, this results in a shift towards anaerobic metabolism, depletion of energy stores, disrupted membrane integrity, alterations in ionic gradients, myocyte oedema, inhibition of contraction and a proarrhythmic potential. Reperfusion can exacerbate the damage, producing calcium ion accumulation and free radical generation. Infarct expansion and ventricular remodelling can often follow AMI as can additional necrosis, in the form of infarct extension/reinfarction. Rational and optimal treatment of AMI should be based on an understanding of the epidemiological influences and the pathophysiological processes involved. This review considers some of the important features in the pre-, peri- and postinfarction periods. PMID- 1718698 TI - Basic mechanisms involved in the protection of the ischaemic myocardium. The role of calcium antagonists. AB - A sudden, severe and sustained reduction in coronary blood flow triggers progressive damage to the myocardium, starting with a loss of adenosine triphosphate, creatine phosphate, potassium ions and active tension-generating capacity, and proceeding until the myocytes are swollen, acidotic, incapable of maintaining ionic homeostasis, depleted of the purine precursors needed for energy (as adenosine triphosphate) repletion, and showing signs of structural disorganisation. In addition, the cells become electrically unstable and unable to relax. The early development of raised end-diastolic resting tension probably reflects the early rise in cytosolic Ca++ which occurs in part because of the generation of free radicals. The calcium antagonists can protect against the ischaemia-reperfusion-induced injury, provided they are used prophylactically, and adequate plasma levels are attained. The basis of this protective effect is complex and includes an energy-sparing effect, a slowed loss of adenosine precursors, a slowed rise in cytosolic Ca++, and sometimes, a slowed release of endogenous noradrenaline (norepinephrine). In the case of verapamil, evidence of its protective effect includes reduced creatine kinase release, reduction in infarct size, improved recovery of contractility, maintenance of energy-rich phosphates, preservation of ultrastructure, and maintenance of mitochondrial function. Prophylactic therapy with calcium antagonists has additional advantages in that these agents slow the development of atherogenic plaques, protect the vascular endothelial cells and, in some instances, protect against lipid peroxidation caused by free radicals. PMID- 1718700 TI - Myocardial infarction. Secondary prevention with nifedipine. AB - The rationale for the use of nifedipine in patients with acute myocardial infarction (MI) is based on the various cardiovascular actions of the compound: reduction of myocardial oxygen consumption by attenuation of cardiac and vascular smooth muscle tension; augmentation of oxygen and substrate supply after increased coronary blood flow with dilatation of epicardial coronary arteries (particularly in coronary obstructions) and dilatation of coronary resistance and collateral vessels; myocardial 'protection', i.e. reduction of myocardial damage via a complex intracellular mechanism, the primary outcome of which is the maintenance of an energy level sufficient to preserve the ionic homeostasis of the myocyte. The effect of nifedipine on reinfarction and mortality rates was evaluated in 6 well designed studies involving 8670 patients with evolving or established acute MI. Compared with placebo, short term therapy (for up to 6 months) with nifedipine 30 to 120 mg/day initiated, in some patients, as early as 3 hours after the onset of symptoms did not reduce either reinfarction rate or mortality. In one study (SPRINT I) [Israeli Sprint Study Group 1988], where a regimen of nifedipine 30 mg/day was only started 7 to 21 days after infarction, the exceptionally low mortality rate (5.7%) over 10 months in the placebo group precluded the demonstration of a beneficial effect of nifedipine. These results collectively suggest that nifedipine does not prevent the 'secondary' coronary events of plaque rupture and thrombus formation associated with MI and sudden cardiac death. However, the suppression of early lesions by nifedipine (as demonstrated in the INTACT study [Lichtlen et al. 1990]) might reduce 'primary' progression and improve the long term survival after MI. PMID- 1718699 TI - Management of acute non-Q-wave myocardial infarction. The role of prophylactic diltiazem therapy and indications for predischarge coronary arteriography. AB - Non-Q-wave myocardial infarction (MI) differs from Q-wave MI in 3 important respects: a smaller infarct size, possibly due to early reperfusion resulting from spontaneous thrombolysis, relief of spasm, or both; more frequency patency of the infarct-related artery; and a larger residual mass of viable but jeopardized myocardium within the perfusion zone of the infarct-related vessel. Left ventricular function is generally better unless impaired by previous MI. After the acute phase, the prognosis is worse when residual ischaemia is present, and reinfarction rates during hospitalisation and in the subsequent year of follow-up are higher. As myocardial ischaemia is potentially reversible, its presence should be actively sought in all patients with recognised non-Q-wave MI. On the basis of current knowledge and available data, the following guidelines for the management of non-Q-wave MI patients can be recommended: (1) diltiazem and aspirin should be administered to all patients as soon as the diagnosis is established, unless contraindications exist; (2) patients who develop early recurrent ischaemia on therapy (i.e. angina with associated ST-T-wave changes) should undergo prompt cardiac catheterisation and myocardial revascularisation; and (3) patients with entirely uncomplicated hospital histories who are asymptomatic should undergo exercise stress testing, preferably in conjunction with 201Tl perfusion scintigraphy, before hospital discharge. Only those patients with evidence of significant residual ischaemia need cardiac catheterisation and myocardial revascularisation. PMID- 1718701 TI - Treatment with verapamil after an acute myocardial infarction. Review of the Danish studies on verapamil in myocardial infarction (DAVIT I and II). AB - The effect of verapamil on death, reinfarctions, and major events, i.e. death or reinfarction after a myocardial infarction has been investigated in 2 Danish double-blind placebo-controlled verapamil infarction trials (DAVIT I and II). DAVIT I was an early intervention trial that demonstrated a statistically nonsignificant reduction in mortality and reinfarction after 6 months of treatment. DAVIT II was a later intervention trial that demonstrated a nonsignificant reduction in the 18-month mortality rate [p = 0.11, hazard ratio 0.80; 95% confidence limits (CL) 0.61 to 1.05], a significant reduction in the reinfarction rate (p = 0.04, hazard ratio 0.77; CL 0.58 to 1.03), and in the major event rate (p = 0.03, hazard ratio 0.80; CL 0.64 to 0.99) in the verapamil group compared with the placebo group. In patients without heart failure in the coronary care unit, a statistically significant reduction in the 18-month mortality rate (p = 0.02, hazard ratio 0.64; CL 0.44 to 0.94), the reinfarction rate (p = 0.02, hazard ratio 0.67; CL 0.46 to 0.97), and the major event rate (p = 0.01, hazard ratio 0.70; CL 0.52 to 0.93) was observed in the verapamil group compared with the placebo group. No significant differences were found in patients with heart failure. Meta-analyses of DAVIT I (for patients alive at day 8) and DAVIT II showed a statistically significant reduction in the odds ratio of mortality (22%), reinfarctions (27%), and the major event rate (21%) in verapamil treated patients. It is concluded that long term treatment with verapamil after an acute myocardial infarction is associated with a significant reduction in overall mortality, major events, and reinfarctions, with the greatest effect in patients without heart failure. PMID- 1718702 TI - Calcium antagonists in secondary prevention after myocardial infarction. AB - A reliable theoretical background exists to support a secondary preventive effect of calcium antagonists after myocardial infarction. Recent studies also indicate that positive results can be achieved with diltiazem and, in particular, verapamil, provided that they are not given to patients suffering clinically manifest myocardial failure during the acute phase of the disease. When the results of treatment with verapamil and diltiazem are compared with comparable studies with beta-blockers, there is no convincing difference as to their effect on a reduction in mortality. However, studies on nifedipine have shown a negative trend, indicating that the calcium channel blockers should not be regarded as one entity in this respect. PMID- 1718704 TI - XIIth International Congress of Electroencephalography and Clinical Neurophysiology. Rio de Janeiro, Brazil, January 14-19, 1990. Abstracts. PMID- 1718703 TI - An overview of therapeutic interventions in myocardial infarction. Emphasis on secondary prevention. AB - The high risk of subsequent mortality and morbidity following the occurrence of a myocardial infarction (MI) underlies the importance of instituting effective preventive regimens as part of the overall management of these patients. The aim of such treatment is to prolong survival by preventing sudden and non-sudden death and further major cardiovascular events. Currently available data from randomised, controlled clinical trials in MI patients indicate that early treatment with thrombolytic agents [streptokinase, anisolyated plasminogen streptokinase activator complex (APSAC) or recombinant tissue-type plasminogen activator (rt-PA)] in the first few hours after onset of symptoms significantly reduces the short term mortality following MI, with follow-up studies at 1 year indicating that the early benefits persist long term. The optimum time for initiation of thrombolytic therapy is within 6 hours of onset, but treatment started between 7 and 24 hours after onset can also be beneficial. Similarly, there is good evidence that beta-blockers (e.g. propranolol, timolol, metoprolol, atenolol) and aspirin are effective in reducing both the mortality and reinfarction rate following MI. With beta-blockers, a policy of starting treatment early intravenously and continuing orally for 2 to 3 years seems likely to save more lives than either strategy alone; however, contraindications to beta blockade reduce the number of patients eligible to receive this treatment. In the case of aspirin, the optimum dosage has yet to be determined, but on the basis of present evidence, it seems reasonable to start treatment early with doses of 160 to 325 mg daily and continue for a year or 2 after recovery. Other studies have shown that long term oral anticoagulant therapy is also effective in reducing the mortality and reinfarction rate after MI. Subcutaneous heparin therapy given for 10 days in patients with acute anterior MI reduces the frequency of left ventricular mural thrombosis, while intravenous heparin given for greater than or equal to 4 days improves coronary artery patency rates following administration of a thrombolytic agent. In a comparison with low dose aspirin, intravenous heparin proved more effective in maintaining coronary artery patency rates after rt-PA thrombolysis. There is also evidence that oral nitrates may reduce mortality in MI patients, especially in those with heart failure. However, current data on the use of antiarrhythmic drugs do not support the routine of these drugs following MI. Similarly, with lipid-lowering agents, the evidence that lowering cholesterol is beneficial in reducing mortality after MI is at present inconclusive. Further studies with these groups of drugs are awaited with interest.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718705 TI - Quantitative EEG during progressive hypocarbia and hypoxia. Hyperventilation induced EEG changes reconsidered. AB - To investigate the role of cerebral hypoxia as a causative factor in the alteration of the qEEG during hyperventilation, qEEG changes caused by progressive hypocapnia were compared with qEEG changes due to progressive normobaric hypoxia in two parallel groups of 12 and 10 healthy male subjects (age 20-27 years), respectively. In the first group, qEEG records were obtained before and during hyperventilation to pCO2 levels of 4.0, 3.0 and 2.0 kPa. In the second group, the qEEG samples were taken before and during hypoxia with hemoglobin oxygen saturations of 80, 70 and 60%. In both groups, blood flow velocity in the middle cerebral artery was also recorded. Hyperventilation caused an exponential increase in slow activity and a decrease in alpha power. No shift in the alpha mean frequency and alpha peak frequency was observed, except with the pCO2 level of 4.0 kPa, which caused an increase in both variables. Hypoxia with a hemoglobin oxygen saturation of 60% caused a much less pronounced increase in slow activity. No change in total power in the alpha band was found, but both the alpha peak frequency and alpha mean frequency decreased. Lesser degrees of hypoxia caused only minimal EEG changes. Blood flow velocity was decreased by hyperventilation but increased by hypoxia. It is concluded that the EEG changes observed during hyperventilation must mainly or totally be attributed to factors other than cerebral hypoxia. PMID- 1718706 TI - Serial EEG in Alzheimer's disease: 3 year follow-up and clinical outcome. AB - We describe EEG findings and clinical outcomes of 24 Alzheimer (AD) patients over a 3 year follow-up period. Three records, baseline, 1 year and 3 year, were available for 13 patients. Although the majority of the patients showed slowing of the EEG over 3 years, evolutions of the EEG changes were not equal in all patients. In 12 (50%) of the patients the EEG from T6-O2 derivation was normal or slightly abnormal in the initial record and remained stable during 1 year. At year 3, only 2 patients still had normal EEGs and mild dementia, probably representing a subgroup of AD with a benign course, 7 patients needed institutional care and 3 had died. EEG slowing at the time of diagnosis was predictive of poor outcome at year 3; absolute theta amplitude was the best discriminating variable of outcome. The results support the idea of heterogeneity of AD patients and suggest that EEG may provide valuable information for prediction of outcome. PMID- 1718707 TI - Development of a novel EEG rating scale for head injury using dichotomous variables. AB - We developed a new EEG rating scale for electrographic assessment of head injured patients. Phenomena present in posttraumatic EEG were scored as dichotomous variables (present or absent). These phenomena included background activity (alpha, beta, theta, delta), sleep spindles, focal abnormalities, reactivity and variability, epileptiform activity, and specific comatose patterns. Each variable was weighted according to its perceived prognostic value: i.e., normal alpha 10, flat EEG -10, spindles 4, etc. Combinations of possible scores ranged from +23 to -10. Fifty-seven EEGs from different head injured patients were independently and retrospectively analyzed by two investigators. There was a high correlation for intra- (r = 0.95) and inter- (r = 0.85) observer rating using the dichotomous test. When patients with scores over 15 (i.e., with reactive alpha) and patients with scores of -10 (i.e., ECI records) were excluded, the intra-rater and inter rater correlations were still high (0.81 and 0.76, respectively). There was a high correlation between Glasgow outcome score at discharge and the dichotomous EEG score. This EEG scale scores most major categories of EEG activity, utilizes a multipoint scale for correlation purposes, and allows data to be analyzed in sub-categories (i.e., spindles in coma). The separate weighting score allows for refinement of the scale after data collection (i.e., to fit prospective outcome). We feel that this scale is reproducible and valid, and may be applicable to other patient groups with severely altered EEGs. PMID- 1718708 TI - Facial muscle activity and EEG recordings: redundancy analysis. AB - The present study explored the use of redundancy analysis, a multivariate technique for assessment of interset association, to examine facial muscle contamination of EEG recordings in studies involving covert levels of emotional expression. Redundancy analyses were performed on simultaneously recorded EEG and facial EMG data obtained in an emotion induction paradigm. Redundancy indices obtained suggest that (1) the amount of variance in EEG activity that can be explained by facial muscle activity under such conditions is minimal, and (2) the EEG alpha band may be at least as or even more susceptible to muscle contamination as the beta band. PMID- 1718709 TI - The weighted average reference montage. AB - In this article we describe the implementation of a linearly weighted average reference montage. This montage differs from the commonly used source derivation montage in that the reference includes all the scalp electrodes of the 10-20 system; and it differs from all other montages implemented thus far in that the weighting of the reference electrodes is based on directly measured interelectrode distances. Using EEG and computer generated signals we have been able to demonstrate that the weighted average reference montage combines topographic selectivity and accuracy in the display of both focal and regional background changes. In comparison to the source derivation montage, ectopic peaks and troughs of localized potential fields are less prominent and interhemispheric symmetry is better preserved. In comparison to the common average reference montage the frequent prominent ectopic displacement of high amplitude (e.g., vertex waves) or widespread potentials (e.g., alpha background activity) is suppressed. We believe that the spatial filtering characteristics of the weighted average reference montage, which are intermediate between those of the common average reference montage and the source derivation and Laplacian montages, will make it a useful alternative for topographic analysis. Our results also indicate that actual scalp measurements should be used to calculate reference electrode weighting factors because such measurements yield values that are substantially different from those derived from other methods of estimation presented thus far. A weighted average montage derived from pooled scalp measurements can be easily implemented using the weighting factors provided herein. PMID- 1718710 TI - Chaos or noise in EEG signals; dependence on state and brain site. AB - EEG signals have been considered to result either from random processes or to be generated by non-linear dynamic systems exhibiting chaotic behaviour. In the latter case, the system may behave as a deterministic chaotic attractor. The complexity of the attractor can be characterized by the correlation dimension that can be computed from one signal generated by the system. A new procedure was developed and applied in order to test whether the correlation dimension, calculated from an EEG epoch, may correspond to a chaotic attractor or to a random process. This procedure was applied to EEG signals recorded from different sites of the limbic cortex of the rat during different states: wakeful rest, locomotion and in the course of an epileptic seizure induced by kindling. The signals recorded during the first two states had high dimensions and could not be distinguished from random noise. However, during an epileptic seizure the correlation dimension became low (between 2 and 4) indicating that in this state the networks behave as chaotic systems. A low correlation dimension appeared at different times and brain sites during an epileptic seizure. These results show that the computation of the correlation dimension may be useful in order to obtain insight into the dynamics of the propagation of an epileptic seizure in the brain. PMID- 1718711 TI - Test-retest reliability in EEG frequency analysis. AB - This study was performed to gain a better understanding of EEG frequency analysis test-retest reliability in normal healthy adults, and to evaluate factors which could influence the measured inter-record differences. Nineteen subjects underwent serial EEG recordings at 5 min and 12-16 week intervals. Records were visually edited using a standardized protocol, and FFT frequency analysis performed on segments of 60, 40, or 20 sec total length. Correlation coefficients for broad band features averaged 0.92 over the 5 min retest interval and 0.84 over the 12-16 week interval. There was essentially no difference between correlation coefficients of absolute and relative power features. Coefficients based on 60 sec records were marginally higher than those of 40 or 20 sec records. On the other hand, test-retest percent differences were typically lower for relative as opposed to absolute power features, and 60 sec records showed consistently lower percent differences than did 40 or particularly 20 sec records. Peak alpha frequency and mean frequency were the most stable EEG features at either interval. Montage had significant effects on test-retest differences at the 12-16 week interval. A significant association between intra record and inter-record variability could not be demonstrated. PMID- 1718712 TI - A statistical evaluation of the main interpolation methods applied to 3 dimensional EEG mapping. AB - This paper is a review of the main interpolation methods applicable to 3 dimensional EEG mapping. The use of simple statistical comparison methods on recorded EEG maps allowed us to evaluate the qualities of interpolation methods belonging to 3 mathematical families (barycentric, polynomial, spline). A combination of a 3-dimensional representation of EEG maps and a reliable interpolation method makes it possible to obtain better spatial resolution than with standard planar mapping. PMID- 1718713 TI - Radial coherence, wave velocity and damping of electrocortical waves. AB - Mean squared coherence was calculated as a function of frequency (1-32 Hz) and electrode separation (1-8 mm) from 64-channel extradural arrays on occipito parietal association cortex of cats. A 2-parameter theoretical function was then fitted to sets of pooled estimates. The theoretical function described coherences between recording sites of small separation for linear, non-dispersive, dissipative waves moving on an infinite homogeneous plane medium, and driven by spatio-temporally noisy inputs. Residuals of fit were then plotted as a function of frequency and distance, and were found to show no systematic trends with frequency, but an irregular and generally increasing relation to distance. This was the result predicted for linear non-dispersive waves on a surface actually folded, and with significant additional wave action generated between electrodes. Further recordings of coherence from more widely separated electrodes indicated that boundary conditions were absorbing or remote, rather than closed or reentrant. The phase velocity for electrocortical waves obtained from autoregression estimates of temporal damping and parameters of fit to coherence, was found in the range 0.1-0.29 m/sec, and appeared independent of the direction of electrode alignment. This compares with the velocity of 0.33 m/sec for alpha waves earlier found by Lopes da Silva and Storm van Leeuwen. PMID- 1718714 TI - Artifactually high coherences result from using spherical spline computation of scalp current density. AB - Coherence computed from common reference montages inextricably confounds true coherence with power and phase at the recording and reference electrodes. Direct measurement of coherence requires reference-free EEG data, such as data from EEG scalp current densities (SCDs), which estimate the potential gradient perpendicular to the scalp. Perrin et al. (1989) presented a method for computing SCDs by taking the Laplacian of the scalp potential surface generated by spherical spline interpolation. When this method of computing SCDs was applied to EEG data gathered from young adults, very high values were observed for inter electrode coherences computed from the spherical spline derived SCD data but not from coherences computed from the common reference data. These high coherences prompted further examination of the properties of the spherical spline function and of spherical spline derived SCDs. Simulated data were constructed, and coherence was computed on the simulated data and on the SCDs derived from the spherical spline procedure and from the Hjorth (1980) procedure. The results of those simulations are presented, which demonstrate that a major artifact is introduced by using the spherical spline procedure. This artifact results from the spline weighting matrix used to derive the SCDs and strongly inflates the inter-electrode coherences of the SCD transformed data. PMID- 1718715 TI - The effects of changing state on elicited ponto-geniculo-occipital (PGO) waves. AB - Waves similar to ponto-geniculo-occipital (PGO) waves occurring spontaneously in the lateral geniculate body (LGB), pons, and occipital cortex during rapid eye movement (REM) sleep can be elicited in the LGB and the cortex by tones in waking (W), non-rapid eye movement sleep (NREM), and REM. In W, the elicited waves (PGOE) sometimes accompany orienting responses (OR). We have hypothesized that REM is a state resembling exaggerated "orienting" in part because spontaneous PGO waves similar to PGOE accompanying OR are constantly observed in REM. The present experiment tested whether: (1) PGOE and OR were strongly correlated in W across a large number of tone presentations as might be predicted if PGOE were central wave form markers for a state of orienting; and (2) recovery of responsiveness of PGOE to tones would then be greater in REM than NREM, as might be expected if REM but not NREM were a state in which central mechanisms of orienting were highly active. Tones were presented in W and then in REM and NREM to six cats in order to measure the degree of habituation of OR and PGOE simultaneously. PGOE and OR exhibited a degree of independence: the former were readily produced in W despite the rapid decline in OR across trials. Recovery in the amplitude of PGOE occurred in both NREM and REM. The recovery tended to be greater in REM than NREM, although this was not statistically significant. Refinements of the theory that REM represents a state of exaggerated internal orienting are discussed. PMID- 1718716 TI - Arousal, performance and absence seizures in rats. AB - Rats of the WAG/Rij strain show bilateral symmetrical spontaneous spike-wave discharges in the EEG, with clinical concomitants. The present experiment investigated whether, during a learning task, the number of discharges would be diminished compared to a period of rest. Additionally, it was investigated whether behavioural differences would be noticed within the task in trials with and without spike-wave discharges. The length of the post reinforcement pause in a fixed interval task was used as a performance index. Eleven rats were extensively trained to press a lever for food in a fixed (60 sec) interval task until a stable response pattern emerged: a post-reinforcement pause of about half the interval. Next, EEG electrodes were implanted and baseline EEGs were made, before and after the first and fifth test sessions. In addition, the behavior of the animals in the task was monitored when an EEG was recorded. During the task, a significantly smaller number of spike-wave discharges was found, compared to the preceding and succeeding baseline hours. This reduction is probably related to a higher level of vigilance during the task compared to the rest hour. Furthermore, the post-reinforcement pause was significantly enhanced in trials with spike-wave discharges compared to trials without discharges, indicating a clear change in performance. Both results are in agreement with what could be expected in patients with absence epilepsy and provide further evidence for the validation of the spike-wave discharges as genuine epileptic phenomena. PMID- 1718717 TI - A new method to measure the distribution of motor conduction velocity in man. AB - A new method of measuring the distribution of conduction velocities in human motor fibers is described. In this method, a modification of Kimura's collision technique is combined with Hopf's technique. This enabled us to determine the collision end-point in Hopf's technique, from which the minimum velocity is derived. The size of the distorted compound muscle action potential (CMAP) measured with Hopf's technique is corrected using the CMAP size with the modified Kimura technique. This resolves the problem of CMAP distortion due to transient change in muscle conduction in Hopf's technique. In addition, a new equation to correct for the refractory period was developed. This can be applied even if there is stimulus spread. Using our method, one can clearly determine the maximum and minimum velocities. The former corresponds to the motor conduction velocity as measured by the conventional method. PMID- 1718718 TI - Multichannel measurements of magnetic compound action fields of the median nerve in man. AB - Magnetic compound action fields (CAFs) evoked by electrical stimulation of the median nerve at the wrist were recorded with 7-channel 2nd-order SQUID gradiometers. CAFs measured over the elbow and upper arm were biphasic with field patterns and polarities corresponding to the depolarization and repolarization fronts of the action potential volley. PMID- 1718719 TI - Myoclonus and sensorimotor integration in a patient with Ramsay Hunt syndrome. AB - Clinical and neurophysiologic studies were done on a patient with action myoclonus secondary to Ramsay Hunt syndrome (dyssynergia cerebellaris myoclonica). Myoclonic jerks in the arms were much more common during movements directed to a target than in other movements. They appeared to be triggered primarily by external sensory inputs relevant to the movement rather than by the motor activity itself. Both somatosensory and visual inputs appeared able to trigger the myoclonic jerks. Myoclonic jerks in the deltoid muscle followed finger contact with a target by approximately 100 msec. Electrical stimuli delivered to the fingers during a reaching movement also triggered myoclonic jerks with a similar latency and also evoked giant cortical potentials which preceded the myoclonic jerks in deltoid by 15-20 msec. Our results suggest that during sensory guided movements, sensory inputs relevant to successful completion of the movement may have access to motor systems controlling the muscles involved. In our patient, who likely has lesions involving the cerebellar nuclei and/or cerebellar cortex, these sensory inputs appeared to result in an excessive motor response, possibly through mechanisms involving cerebellar-motor cortex connections. PMID- 1718720 TI - Silent period induced by cutaneous stimulation. AB - An electrical stimulus applied to a cutaneous nerve during isometric muscle contraction causes a suppression of EMG activity (silent period) followed by a rebound. The extent of inhibition is related to the stimulus intensity as the silent period is more evident when stimulation is perceived as painful. The silent period is present in different limb and cranial muscles after stimulation of the same cutaneous nerve and in the same muscle after stimulation of distant cutaneous nerves. It also occurs synchronously in antagonist muscles. Within the silent period induced after cutaneous stimulation the maximal inhibition on the opponens pollicis motor neuron pool, as tested by the motor response evoked after transcranial cortical stimulation, occurs between 50 and 70 msec. Using the double stimulus technique to study the recovery cycle, the silent period is present at interstimulus intervals as low as 100 msec, and does not habituate with trains of stimuli at frequencies up to 5 Hz. Our results suggest that motor neuron inhibition from nociceptive stimulation may be mediated by Renshaw cells directly activated by high threshold cutaneous afferents. PMID- 1718721 TI - Long latency postural responses are functionally modified by cognitive set. AB - We examined how cognitive set influences the long latency components of normal postural responses in the legs. We disturbed the postural stability of standing human subjects with sudden toe-up ankle rotations. To influence the subjects' cognitive set, we varied the rotation amplitude either predictably (serial 4 degrees versus serial 10 degrees) or unpredictably (random mixture of 4 degrees and 10 degrees). The subjects' responses to these ankle rotations were assessed from the EMG activity of the tibialis anterior, the medial gastrocnemius, and the vastus lateralis muscles of the left leg. The results indicate that, when the rotation amplitude is predictable, only the amplitude of the long latency (LL) response in tibialis anterior and vastus lateralis varied directly with perturbation size. Furthermore, when the rotation amplitude is unpredictable, the central nervous system selects a default amplitude for the LL response in the tibialis anterior. When normal subjects are exposed to 2 perturbation amplitudes which include the potential risk of falling, the default LL response in tibialis anterior appropriately anticipates the larger amplitude perturbation rather than the smaller or an intermediate one. PMID- 1718722 TI - Percutaneous magnetic coil stimulation of human cervical vertebral column: site of stimulation and clinical application. AB - In order to understand which neural elements are excited after percutaneous magnetic coil (MC) stimulation over the cervical vertebral column we have performed such study in 8 normal subjects and 4 patients. On moving the coil rostrocaudally up to 3 cm and horizontally up to 2 cm from the midline we found no change in the latencies of the compound muscle action potentials to biceps, deltoid, abductor pollicis brevis (APB) and abductor digiti minimi muscles indicating a fixed site of excitation of the spinal roots within the intervertebral foramina. F latencies to APB after stimulation of the median nerve at the wrist were always longer than the direct latencies obtained after cervical vertebral stimulation. The mean difference between indirect latency based on F technique and direct latency to APB was 0.45 msec which represented a distance of 2.7 cm distal to the anterior horn cells assuming a conduction velocity of 60 m/sec. MC stimulation in 2 patients suggested a diagnosis of cervical radiculopathy which was confirmed by imaging studies or operative findings. Both MC and needle root stimulation in one patient with diabetic brachial plexopathy and in another with diabetic polyneuropathy suggested that the needle stimulation occurred about 1.2-1.8 cm proximal to MC stimulation. PMID- 1718723 TI - Attenuation in detection of somatosensory stimuli by transcranial magnetic stimulation. AB - Effects of magnetic stimulation (MS) of the scalp and direct cortical electrical stimulation on detection of an electrical stimulus to the index finger (S1) were studied in 7 normal volunteers and a patient with epilepsy. Detection of somatosensory stimuli was attenuated when MS was delivered 200 msec before S1, was blocked when MS was delivered simultaneously to and 20 msec after S1, and was fully recovered when MS was delivered 200 msec after S1. This effect showed topographic specificity, being produced by scalp stimulation of restricted scalp positions contralateral to the finger stimulated, was maximal with low intensities of finger stimulation and high intensities of MS (usually over that required for motor threshold), and could also be produced in the absence of motor evoked responses in a peripheral hand muscle. These results show that a focal cortical stimulus can briefly attenuate detection of somatosensory stimuli before, during, and after cortical arrival of a somatosensory afferent volley. Several different mechanisms probably contribute to this phenomenon. PMID- 1718724 TI - Transcranial stimulation of motor cortex in upper motor neurone syndrome: its relation to the motor deficit. AB - The purpose of this investigation was to clarify the functional significance of the fastest cortico-motoneuronal connections in chronic upper motor neurone syndromes. Using magneto-electrical stimulation of motor cortex the intactness of cortico-motoneuronal connections was assessed in 51 patients presenting with variable degrees of impairment. There was a gross correlation between clinical impairment of the patient and the degree of pathology of cortico-motoneuronal efferents. Covariation of clinical data with transcranial stimulation was better than covariation with the size of lesion on CT scans. In some patients, however, definite clinical impairment, especially affecting distal fractionated movements, was associated with completely normal responses. There was no evidence of response abnormality in distal muscles ipsilateral to the hemispheric lesion. The data indicate that motor deficit can exist in the presence of normal cortico motoneuronal conduction times, showing that intactness of these connections is not a sufficient condition for preservation of voluntary motor activities. This underlines the importance of other pathways for the pathogenesis of upper motor neurone syndromes. PMID- 1718725 TI - Electrical and magnetic transcranial stimulation in patients with corticospinal damage due to stroke or motor neurone disease. AB - Twenty patients with hemiplegia and 13 patients with motor neurone disease were studied with electrical and magnetic transcranial stimulation. Motor evoked potentials were recorded from the biceps, thenar and tibialis anterior muscles. In both groups of patients magnetic stimulation with a Novametrix stimulator revealed fewer abnormalities than electrical stimulation with a Digitimer D180 stimulator. In patients with hemiplegia, motor evoked potentials after electrical stimulation were absent in 70% of muscles, delayed in 22% and normal in 8%; after magnetic stimulation, they were absent in 53% of muscles, delayed in 28% and normal in 19%. In patients with motor neurone disease, motor evoked potentials after electrical stimulation were absent in 62% of muscles, delayed in 10%, and normal in 29%; after magnetic stimulation, they were absent in 45% of muscles, delayed in 15%, and normal in 40%. The reason why magnetic stimulation reveals fewer abnormalities than electrical stimulation could be that magnetic stimulation repetitively discharges the pyramidal cells and, because of temporal summation mechanisms, produces more powerful excitatory potentials at the lower motoneurone synapse. PMID- 1718726 TI - Further observations on the facilitation of muscle responses to cortical stimulation by voluntary contraction. AB - The effect of voluntary contraction on the discharge of single motor units following electrical and magnetic stimulation of the motor cortex was examined using the post-stimulus time histogram (PSTH) technique. The latencies of responses in single motor units of the first dorsal interosseous muscle to cortical stimulation were 2-4 msec shorter when the muscle was contracting than when at rest in 9 of 10 units studied. These latency differences are comparable with those recorded by surface electromyography for compound muscle action potentials following cortical stimulation in relaxed and active muscles. The new findings are that the intensity of cortical stimulation required to discharge a resting motor unit to produce a single PSTH peak produced multiple PSTH peaks when the same unit was contracting. The timing of the PSTH peak of relaxed motor unit discharge corresponded to one of the later PSTH peaks (usually the second) when the motor unit was voluntarily activated. These findings are in keeping with our previous suggestions that the longer latency of responses in relaxed muscles is due to the time taken for temporal summation of multiple descending corticospinal volleys at the cortico-motoneurone synapse. Facilitation produced by voluntary contraction occurs at least in part at the level of the spinal cord by lowering motoneurone threshold to enable discharge on the initial descending volley. The higher threshold of relaxed muscles is related to the higher intensities of stimulation needed to recruit multiple descending volleys and discharge resting motoneurones. PMID- 1718727 TI - Manganese superoxide dismutase: a hepatic acute phase protein regulated by interleukin-6 and glucocorticoids. AB - The superoxide dismutases (SODs) are important metallo-enzymes which scavenge and dismutate the superoxide free radical. They are thought to be the main enzymes in the antioxidant defense system. Identification of stimuli that control transcription of the SOD genes is essential for understanding SOD gene regulation. In this study we show that manganese SOD (MnSOD) mRNA levels are elevated by lipopolysaccharide, a bacterial endotoxin, in rat liver. However, neither lipopolysaccharide nor tumor necrosis factor-alpha had an effect on MnSOD mRNA expression in cultured primary hepatocytes. On the other hand, the inflammatory cytokines, interleukin-1 (IL-1) and IL-6 did increase MnSOD mRNA levels, either 2- or 15-fold, respectively, over a 20-h period in hepatocytes. The IL-6-induced increase in MnSOD mRNA levels was attenuated by dexamethasone, a glucocorticoid, in hepatocytes cultured for less than 16 h. In contrast, in hepatocytes originally cultured for more than 16 h, IL-6 and dexamethasone produced a synergistic increase in MnSOD mRNA levels. The induction of MnSOD expression by IL-6, which is a known inflammatory cytokine, suggests that MnSOD may play a role in the inflammation process. Since inflammation is known to result in oxidative damage to cells, the role of MnSOD may be to protect cells from inflammation-mediated oxidative damage. PMID- 1718728 TI - Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells. AB - The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1 34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation. PMID- 1718729 TI - The effects of thyroid hormone on insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression in the neonatal rat: prolonged high expression of IGFBP-2 in methimazole-induced congenital hypothyroidism. AB - In the rat a developmental switch in the serum insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) profile takes place during the first 3 postnatal weeks. The fetal expression pattern of high IGF-II and IGFBP-2 is replaced by the adult pattern of low levels of IGF-II and IGFBP-2 and high levels of IGF-I and IGFBP-3. The regulatory mechanisms mediating these changes are unknown, but may include perinatal changes in endocrine function. To study the effects of thyroid function and the perinatal thyroid secretory burst on IGF and IGFBP expression, we established a rat model of congenital hypothyroidism, leading to marked postnatal growth retardation during the perinatal period. The hypothyroid animals lacked the steep rise in serum IGF-I levels normally occurring during the third week of life, showing only a modest rise to approximately 50% of control levels. The pattern of serum IGF-II decline in hypothyroid animals was slightly different from that in controls, with lower IGF-II levels during the second week of life and a slower decline down to the very low final levels. The hypothyroid pups continued to express high levels of IGFBP-2 up to the age of 19 days, while the control animals, after a slow initial decline, showed an abrupt fall of IGFBP-2 serum levels during the third week of life. Liver IGFBP-2 mRNA levels reflected the serum changes, with elevated IGFBP-2 mRNA in hypothyroid animals. The expression of other IGFBPs did not differ from that in the control group. At the age of 18 days, serum GH levels in the hypothyroid animals were approximately one third of control GH levels, which suggests a role for GH as a possible mediator of thyroid hormone actions on the IGF system. The changes in growth parameters and in the IGF and IGFBP profile of hypothyroid pups could be abolished by thyroid hormone replacement from birth. We conclude that thyroid hormone is, directly or indirectly, essential for some of the neonatal changes in IGF and IGFBP profiles. PMID- 1718730 TI - Evaluation of the developmental and nutritional changes in porcine insulin-like growth factor-binding protein-1 and -2 serum levels by immunoassay. AB - Porcine serum contains five insulin-like growth factor-binding proteins (IGFBPs), whose regulation has been studied by ligand blotting. To more accurately quantify changes in two specific forms of IGFBP a heterologous RIA for porcine (p) IGFBP-2 was developed, and IGFBP-1 levels were analyzed by immunoblotting. By RIA, postnatal hypophysectomy caused a 7-fold increase in serum pIGFBP-2 levels compared to controls (2,622 +/- 378 vs. 382 +/- 10 ng/ml, respectively). Fetal pIGFBP-2 levels were higher at 110 vs. 45 days gestation (1,074 +/- 214 vs. 418 +/- 30 ng/ml, respectively), rose to 1,905 +/- 167 ng/ml within 12 h after birth, then decreased to 1,010 +/- 10 ng/ml at 48 h. By immunoblot analysis, bovine IGFBP-2 antiserum reacted with a 34,000 mol wt (Mr) IGFBP and did not react with other forms of IGFBP detected by ligand blotting. Serum levels of the 34,000 Mr IGFBP, as detected by ligand blot analysis, are decreased when neonatal pigs are fasted for 48 h. In contrast, by RIA, pIGFBP-2 concentrations increased 4-fold. Immunoblots of these sera showed two lower Mr (22,000 and 14,000 Mr) bands that did not bind either [125I]IGF-I or [125I]IGF-II and were distinct from a smaller (20,000 Mr) IGFBP which bound only [125I]IGF-II. These two bands were increased in serum of 48-h fasted compared to fed piglets, suggesting that they are proteolytic fragments of pIGFBP-2. In vitro incubation of 48-h fasted pig serum with intact IGFBP-2 failed to reveal proteolytic fragments, indicating that the IGFBP-2 fragments were not generated by a protease that was released into the serum. Analysis performed with human IGFBP-1 antiserum revealed a 29,000 Mr immunoreactive band whose abundance was increased by either postnatal hypophysectomy or fasting. No fragments of IGFBP-1 were found in any serum tested. We conclude that heterologous antibodies can be used to identify and quantify IGFBP-1 and IGFBP-2 in porcine serum. Changes in pIGFBP-2 levels measured during fasting are due to the combination of changes in intact 34,000 Mr IGFBP-2 and smaller non-IGF-binding fragments. Changes in levels of specific forms of IGFBP as well as the presence of fragments have the potential to modulate the transport of IGF-I and -II out of the vasculature. PMID- 1718731 TI - Galanin and galanin extended at the N-terminus with seven and nine amino acids are produced in and secreted from the porcine adrenal medulla in almost equal amounts. AB - Galanin is present in high concentrations in porcine adrenals, but nothing is known about the processing and secretion of other products of the 123-amino acid precursor preprogalanin. Using, in combination, RIA against galanin, a variety of chromatographic procedures, mass spectrometry, and amino acid sequencing, we studied the processed and the secreted products of preprogalanin. From the tissue extracts we isolated in equimolar amounts and sequenced two major pools of galanin immunoreactive peptides: galanin and two N-terminally extended forms, preprogalanin-(24-61) and preprogalanin-(26-61). The same peptides were identified upon gel chromatography and analytical HPLC in effluents collected during electrical stimulation of the intact splanchnic nerve supply of an isolated perfused preparation of porcine adrenals. The processing of preprogalanin in porcine adrenals thus includes the formation and release of galanin, preprogalanin-(24-61), and preprogalanin-(26-61). The signal peptidase cleaves the preprogalanin at either Gly23 or Gly25. PMID- 1718732 TI - Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. AB - In the ovary, greater than 99% of the follicles present at birth are destined to degenerate during life. In humans, less than 400 of the more than 400,000 follicles found at puberty will eventually ovulate whereas the overwhelming majority of follicles undergo atresia. Although follicular atresia plays a critical role during the recruitment of follicles for ovulation, the exact mechanism of this process is unknown. In chicken and porcine ovaries, atretic follicles can be morphologically distinguished from their healthy counterparts of the same size. Adapting a sensitive 3'-end labeling method for DNA analysis, we identified internucleosomal cleavage of cellular DNA in atretic (but not normal) follicles of both animal species, resembling that found during programmed cell death in embryogenesis, autoimmune T-cell removal and prostate regression. The present findings provide a basis for elucidating the hormonal signals involved in the initiation of follicular atresia during follicle recruitment, reproductive aging and premature ovarian failure. PMID- 1718733 TI - Use of conditioned distributions in the analysis of ion channel recordings. AB - A method to test the Markov nature of ion channel gating is proposed. It makes use of singly and doubly conditional distributions. The application of this method to recordings from single BK channels provides evidence that at least two states of the underlying kinetic scheme are left at a constant rate. Moreover, the probabilities, when leaving a state, of reaching another given state are shown to be constant for all the states of the system. PMID- 1718734 TI - Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting. AB - Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination. PMID- 1718735 TI - Barley cultivar discrimination: II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients. AB - Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol-soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2-11 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitol-soluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected. PMID- 1718736 TI - A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity. AB - A novel multiphasic buffer system for high resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively. PMID- 1718737 TI - A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining. AB - Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma alpha 2-macroglobulin, hemocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels. PMID- 1718738 TI - Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells. AB - Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied. PMID- 1718739 TI - Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube. AB - Murine and human sequences homologous to the paired box of the Drosophila segmentation gene paired have been reported previously. Here we describe a zebrafish (Brachydanio rerio) paired box-containing clone, pax[zf-a], which is clearly distinct from reported vertebrate Pax genes. The putative protein encoded by pax[zf-a] contains a paired box and a paired-type homeobox separated by a glycine-rich, acidic linker and a carboxy-terminal end which is remarkably rich in serine, threonine and proline residues. By in situ hybridization to embryonic tissue sections and whole mount embryos, pax[zf-a] transcripts were found within restricted regions of the central nervous system and the eye. In contrast to the murine Pax genes recently characterized, pax[zf-a] is not expressed in the segmented mesoderm. At the 17 h stage, pax[zf-a] expression is detected in a defined area of the diencephalon which circumscribes the presumptive thalamus. This suggests an involvement of pax[zf-a] in pattern formation in the rostral brain. The pax[zf-a] gene is also expressed throughout the hindbrain and spinal cord. This hybridization signal is restricted to a longitudinal column which includes the basal plate. Later in development, at 36 h post-fertilization, pax[zf-a] transcripts are no longer restricted to a specific region of the diencephalon, but are distributed over the entire developing brain. PMID- 1718740 TI - Ligand stimulation of transfected and endogenous growth factor receptors enhances cytokine production by mast cells. AB - IL-3 dependent mast cell lines produce cytokines in response to Fc receptor cross linkage or to ionomycin. In this study we have observed that cells pre-cultured in IL-3 produce 10-100 times more cytokine after receptor cross-linkage in comparison with IL-4 pre-cultured cells. Although several hematopoietin receptors, including those for IL-3, IL-4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL-3 dependent mast cell line, CFTL-15, to produce cytokines was analyzed. The CFTL-15 cells were transfected with growth factor receptors containing ligand-inducible tyrosine kinase domains (EGFR and PDGFR, and CSF-IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL-3 or to their respective growth factor and to produce IL-3 in response to IgE receptor cross-linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL-3, IL-6, and GM-CSF. Stimulation of the CSF-1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co-stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross-linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross-linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718741 TI - Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells. AB - Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3 dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype. PMID- 1718742 TI - Transient and locally restricted expression of the ros1 protooncogene during mouse development. AB - The ros1 gene was detected originally by virtue of its transforming potential; the cDNA of the human protooncogene was isolated from a tumor cell line expressing the gene ectopically. It encodes a receptor-type tyrosine specific protein kinase which is closely related to sevenless in Drosophila. Here we report the novel and remarkable in vivo expression pattern of c-ros1, which was determined in the mouse. By a combination of RNase protection and in situ hybridization, we find transient c-ros1 expression during development in the kidney, intestine and lung, coinciding with major morphogenetic and differentiation events in these organs. This temporally restricted nature of expression is unusual for tyrosine kinase receptors and suggests a role for ros1 during development. Furthermore, in kidney development c-ros1 transcripts are confined to subgroups of ureter cells known to be involved directly in inductive interactions between ureter epithelium and metanephric mesenchyme. Thus, this study implicates for the first time a tyrosine kinase receptor in mesenchymal epithelial interactions and suggests a molecular basis for these important inductive events in development. PMID- 1718743 TI - Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein. AB - The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb-responsive genes during myeloid differentiation. To identify such myb-regulated genes, and to explore the mechanisms by which v-myb affects their expression, we have established a conditional expression system for v-myb. We have converted the v myb protein to an estrogen-inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell-line causes estrogen-dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen-dependent v-myb expression system to identify a novel v-myb regulated gene. PMID- 1718744 TI - Molecular characterization of the mouse beta 3-adrenergic receptor: relationship with the atypical receptor of adipocytes. AB - The gene encoding the murine beta 3-adrenergic receptor (beta 3AR) has been isolated. It translates into a polypeptide of 388 amino acid residues which shows 82% overall homology with the human beta 3AR. In Southern blot experiments, a probe derived from the murine beta 3AR gene hybridizes to a unique restriction fragment in the murine and human genomes. In both species, the beta 3AR gene is located on chromosome 8, in regions (8A2----8A4 in mouse, and 8p11----8p12 in man) which are conserved between mouse and man. The pharmacological profile of the mouse beta 3AR strongly resembles that of the human beta 3AR. It is characterized by a low affinity toward the radiolabelled beta-adrenergic antagonist [125I]Iodocyanopindolol and a low efficiency of other antagonists such as propranolol, ICI 118551 or CGP 20712A to inhibit cAMP production induced by isoproterenol. Another salient feature shared by the murine and the human beta 3ARs is the very potent effect of the lipolytic compound BRL 37344 on cAMP accumulation and the partial agonistic effect of the beta 1- and beta 2 adrenergic antagonists CGP 12177A, oxprenolol and pindolol. These properties are very close to those ascribed to the atypical beta AR of rodent adipocytes. In addition, Northern blot analyses indicate that the beta 3AR gene is mainly expressed in mouse brown and white adipose tissues, suggesting that the murine beta 3AR described here is the atypical beta AR involved in the control of energy expenditure in fat tissue. PMID- 1718745 TI - Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. AB - We have purified and determined functional parameters of reconstituted, recombinant HIV-1 reverse transcriptase (RT) heterodimers within which either the p66 or p51 polypeptide was selectively mutated in one or both aspartic acid residues constituting the proposed polymerase active site (-Y-M-D-D-). Heterodimers containing a mutated p51 polypeptide retain almost wild type levels of both RNA-dependent DNA polymerase and ribonuclease H (RNaseH) activity. In contrast, heterodimers whose p66 polypeptide was likewise mutated exhibit wild type RNaseH activity but are deficient in RNA-dependent DNA polymerase activity. These results indicate that in heterodimer RT, the p51 component cannot compensate for active site mutations eliminating the activity of p66, indirectly implying that solely the p66 aspartic acid residues of heterodimer are crucial for catalysis. PMID- 1718746 TI - Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. AB - Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level. PMID- 1718747 TI - Tumor secretion of growth factors. AB - An increasing number of polypeptide growth factors have been identified that regulate not only cell proliferation but also an extraordinary range of cell activities, including matrix protein deposition and resolution, the maintenance of cell viability, cell differentiation, inflammation, and tissue repair. Normal cells appear to require growth factors for proliferation and for maintenance of viability. Cells that secrete a polypeptide growth factor have an advantage in growth. These factors can act either externally through cell surface receptors or internally during the transport of receptors and growth factors through the endoplasmic reticulum and Golgi apparatus, causing autocrine stimulation of cell growth. Depending on the cell type, growth factors can also be potent inhibitors of cell growth rather than stimulators of growth, and the effect can depend on the presence or absence of growth factors. Among the growth factors considered, IGFs are unusual in that they function both as endocrine and as autocrine/paracrine agents. IGF-II, which is associated with fetal growth, is the IGF most frequently expressed by tumors. There is now convincing evidence that some tumors secrete sufficient IGF-II to have systemic endocrine effects as recognized as nonislet cell tumor hypoglycemia. PDGF is normally highly concentrated in platelets and has major significance in stimulation of cellular proliferation in inflammation and wound repair. Normally, this proliferation is self-limited, but the secretion of PDGF by tumors and its effects on cell proliferation of tumors persist. The fact that PDGF B monomer has an identical structure with that of the proto-oncogene C-cis further strengthens the connection between PDGF and tumor growth. EGF has a restricted role in normal physiology, but its close relative, TGF-alpha, is widely distributed in normal and neoplastic tissues. The common receptor for EGF and TGF-alpha is present in many normal and neoplastic cell types. The EGF receptor is the product of the C erb gene. The oncogene V-cis is a truncated form of the EGF receptor whose tyrosine kinase activity is not dependent on ligand binding. TGF-beta exists in multiple forms. Although it can transform the morphology of certain cell lines in culture, it probably does not act generally as a mitogenic agent. Its major physiologic role in the body appears to be the stimulation of mesenchymal matrix formation. It is of special importance in the regulation of bone matrix formation. Its expression is increased in many tumors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718748 TI - Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. AB - Highly degenerate oligonucleotide primers designed from regions conserved between protein-serine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encoding putative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein-serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP-dependent protein kinase (PKA) families and its gene has thus been termed pkb. Expression of the pkb gene is general but highest in brain, heart and lung. Translation of pkb RNA in vitro generated a 57-kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60-kDa protein which was partially purified by Mono Q anion-exchange chromatography. Column fractions containing PKB-immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase. PMID- 1718749 TI - Analysis of the complex transcription termination region of the Escherichia coli rrnB gene. AB - The complex terminator region of the Escherichia coli rrnB gene was analyzed by subcloning the terminators T1 and T2 and the inverted repeats IR1 and IR2 individually, or in various combinations, in a normal or inverted orientation into a terminator probe vector. The in vivo terminating efficiency was assayed by measuring the galactokinase activity encoded by the downstream galK gene. Termination efficiencies of all fragments were compared in two constructs, differing in the presence or absence of readthrough translation over the investigated terminator signal. The following main conclusions were drawn. (a) T1 and T2 are both efficient terminators in isolated forms. (b) IR1 and IR2 have some terminating effect (much lower than the proper terminators), especially in the inverted orientation. Their presence modifies the effect of the proper terminators in a quite unpredictable way, especially if these regions are translated. (c) The terminators are not symmetrical; in the inverted orientation T1 is practically inactive and T2 termination is reduced. (d) Translation radically decreases the efficiency of the terminators. (e) Several sequences in the rrnB gene, upstream of the terminator region (one in the 16S RNA and one in the 5S RNA coding region), are very efficient in vivo terminators in the inverted orientation. PMID- 1718750 TI - 1H-NMR conformational analysis of a high-affinity antigenic 11-residue peptide from the tryptophan synthase beta 2 subunit. AB - Two synthetic peptides from the beta 2 subunit of tryptophan synthase have been studied by 1H-NMR spectroscopy at 300 MHz. One peptide, His-Gly-Arg-Val-Gly-Ile Tyr-Phe-Gly-Met-Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native beta 2 subunit. The second peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe (peptide 8) reacts very weakly with the antibody. The 1H-NMR spectra of the two peptides have been assigned from two-dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide-proton exchange rates and their temperature coefficients, and chemical shifts. In aqueous solvent, the C terminal part of peptide 11 presents some structure centered around residues Phe Gly-Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed. PMID- 1718751 TI - Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator. AB - 1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl) 5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported. PMID- 1718752 TI - Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin. AB - Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments. PMID- 1718753 TI - Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G. AB - The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor. Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity. Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10(-8) M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier. PMID- 1718754 TI - Triple immunofluorescence confocal laser scanning microscopy: spatial correlation of novel cellular differentiation markers in human muscle biopsies. AB - Two different methods for triple immunofluorescence imaging with a confocal laser scanning microscope (CLSM) are described. The methods enable spatial "mapping" of 3 different epitope distribution patterns simultaneously in one tissue section. The key to triple imaging includes: (a) specific immunolabeling with 3 different mouse monoclonal antibodies (mAbs), (b) localization of the antibody binding sites by 3 different dyes, (c) spectral isolation of each dye by using selective band pass or long pass emission filters, (d) computerized imaging of the fluorescences as colored overlays or as selective signals in each optical section through the tissue in the z-direction. Method 1 consists of the combination of fluorescein isothiocyanate (FITC), phycoerythrin R (PE), and Texas Red (TR) as fluorescent markers. These dyes can be imaged by using 488 nm and 543 nm excitation laser lines. In method 2 aminomethyl coumarin acetic acid (AMCA) was combined with FITC and PE. For this application the CLSM was adapted to ultraviolet microscopy that enabled the use of 3 laser lines (364 nm, 488 nm, 543 nm) for excitations. Cryostat sections of diagnostic human muscle biopsies (n = 9) were studied which were normal by ordinary light microscopic examination. Sections were incubated with mAbs specific for: (1) the fast myosin heavy chain (My32); (2) the major histocompatibility class II antigen HLA-DR; (3) the lymph node homing receptor Leu8, and (4) the cell adhesion receptor OKM5 (CD36). By combining these mAbs in triple staining procedures, 3 capillary types and 4 different phenotypes expressed by muscle fibers were identified simultaneously. The mAbs Leu8 and OKM5, widely used as leukocyte typing antibodies in the blood, exhibit hitherto unrecognized specificities for antigens displayed by muscle fibers. At the level of these markers, specific spatial correlations between OKM5 reactive capillaries and both OKM5 reactive and nonreactive muscle fiber types become visible. The presented results provide direct evidence for cellular complexity and novel insight into the immunoanatomical architecture of skeletal muscle. The methods may be of general significance for the construction and quantification of three-dimensional multiparameter "maps" of cells and tissues. PMID- 1718755 TI - Cultivation of HL-60 cells in a serum-free medium containing granulocyte/macrophage colony-stimulating factor. AB - In order to develop a defined cultivation medium for HL-60 cells, we cultivated these cells in a serum-free suspension medium and tested the effect of various growth factors. Of the factors tested, granulocyte/macrophage colony-stimulating factor was most active in growth stimulation. A much lower effect was obtained with granulocyte colony-stimulating factor and transferrin. No effect was found with interleukin-3 and insulin. Granulocyte colony-stimulating factor was the only growth factor tested that also induced differentiation as judged by the nitroblue tetrazolium test. Growth of HL-60 cells in medium containing granulocyte/macrophage colony-stimulating factor (125 U/ml) and transferrin (5 micrograms/ml) as the only protein factors was similar to growth in medium containing 10% serum. No increase in spontaneous differentiation of HL-60 cells in this defined medium was observed. Physiological concentrations of retinol bound to retinol-binding protein and retinyl ester in chylomicron remnants reduced proliferation as well as the level of c-myc oncoprotein and induced differentiation of HL-60 cells cultivated in defined medium. Hence, this defined medium may be useful when studying the function of retinoids in HL-60 cells. PMID- 1718757 TI - Endogenous nitric oxide: physiology, pathology and clinical relevance. PMID- 1718756 TI - Treatment of hemorrhagic shock with intraosseous or intravenous infusion of hypertonic saline dextran solution. AB - The efficacy of intravenous or intraosseous infusion of 250 ml of 7.5% NaCl and 6% dextran 60 (H/H) was compared with intravenous Ringer's lactate (RL) for the initial treatment of patients with hemorrhagic shock due to upper gastrointestinal bleeding. 49 patients were randomly assigned to receive either H/H (n = 26) or RL (n = 23). In the first 16 patients with H/H and in all RL patients, solutions were infused by the intravenous route, while the intraosseous route through sternal puncture was chosen for the last 10 H/H subjects. H/H patients were analyzed together since no differences were noticed between the routes of infusion. The H/H group also received 2.3 +/- 0.7 liters of intravenous crystalloid solutions in the first hour and 4.4 +/- 0.1 liters in the 24-hour period, while RL received 3.3 +/- 0.7 and 7.3 +/- 2.4 liters, respectively. Blood pressure (BP) increased during the first 15 min in the H/H group (from 61 +/- 17/30 +/- 12 to 85 +/- 30/48 +/- 14 mm Hg) and thereafter, while remaining unchanged in the RL group (from 75 +/- 18/40 +/- 12 to 75 +/- 17/40 +/- 14 mm Hg; p less than 0.05). The differences between groups were significant throughout 24 h. Urine output and improvement of the Glasgow Coma Score were also higher in H/H patients than in the control group (p less than 0.05). There were 5 deaths in RL group and 1 in the H/H group. Sternal of peripheral vein infusion of 250 ml of 7.5% NaCl/6% dextran 60 is an effective initial treatment of hemorrhagic shock. PMID- 1718758 TI - Major histocompatibility complex controls clonal proliferation of CD5+ B cells in H-2-congenic New Zealand mice: a model for B cell chronic lymphocytic leukemia and autoimmune disease. AB - By employing H-2-congenic NZB, NZW and (NZB x NZW)F1 mice with either the homozygous H-2d/H-2d, H-2z/H-2z or heterozygous H-2d/H-2z haplotype, we found that in the spleen of all the congenic strains homozygous for H-2z, there were extremely high frequencies of CD5+ B cells. These cells eventually proliferated in an oligoclonal or even monoclonal fashion, and B cell-chronic lymphocytic leukemia (B-CLL) developed in some cases. Because this feature was not observed in H-2d/H-2d homozygotes or H-2d/H-2z heterozygotes, the high CD5+ B cell frequencies are apparently controlled by the homozygosity of a locus or cluster of loci closely linked to H-2z complex of NZW strain. As the CD5+ B cell frequencies in the peritoneal cavity did not differ among the H-2-congenic strains, the frequencies of these cells in the peritoneal cavity and in the spleen appear to be at least in part under separate control. Flow cytometry and Southern blot analyses using an immunoglobulin gene JH probe revealed that the H 2z/H-2z homozygotes, there was a propagation of distinct clonal populations between the spleen and the peritoneal cavity, a finding which suggested that in the major histocompatibility complex (MHC)-related microenvironments for CD5+ B cell propagation differ between the two compartments. All our findings taken together imply that certain different but related MHC haplotypes may predispose either to B-CLL or to autoimmune disease, in close relatives. PMID- 1718759 TI - Comparison of class I- and II-restricted T cell recognition of the identical peptide. AB - There is structural and functional evidence that both class I- and II-restricted T cells recognize short processed peptides bound to MHC molecules. Although the structural conformation of bound peptides remains unknown, no evidence of distinct structural motifs of class I- or class II-restricted peptides has been described. Conversely, two algorithms proposed to predict T cell epitopes, and based on primary amino acid sequence or tertiary structure, are both compatible with many observed class I- and class II-restricted peptides. We previously identified eight class I-restricted peptides which were also recognized by class II-restricted T cells. Based on functional and direct binding studies, additional examples of peptides with both class I and II restrictions have been identified. In this study, we have directly compared the fine specificity of T cell recognition of a single epitope in a single mouse strain in the context of both class I- and class II-restricted responses. Based on a panel of analogue peptides with amino acid substitutions and peptides of various lengths, we observed several striking similarities in the recognition patterns of both class I- and class II-restricted T cells. In addition, some characteristics of recognition were different in the two systems indicating that the recognition processes were similar but not identical. PMID- 1718761 TI - Rejection of bone marrow cell allografts by natural killer cell subsets: 5E6+ cell specificity for Hh-1 determinant 2 shared by H-2d and H-2f. AB - The 5E6 antigen, defined by anti-5E6 mAb, is expressed on one-half of murine natural killer (NK) cells, and we have previously demonstrated (C. L. Sentman et al., J. Exp. Med. 1989. 170: 1991) that 5E6+ NK cells are necessary for the rejection of BALB/c (Hh-1d) but not C567BL/6 (Hh-1b) bone marrow cells (BMC). In experiments described here, we have characterized the specificity of 5E6+ and 5E6 NK cell subsets for hemopoietic histocompatibility-1 (Hh-1) antigens. Prospective recipient mice were treated with anti-5E6 mAb and challenged with BMC from a variety of donors. In addition, H-2d/Hh-1d C.B-17 scid 5E6+ or 5E6- NK cells were adoptively transferred into irradiated, NK cell-depleted hosts and challenged with H-2b/Hh-1b BMC. The data indicate that the 5E6+ NK cells are necessary for the rejection of only those BMC that express the Hh-1 determinant 2 shared by H-2d and H-2f haplotypes of strains BALB/c (d), A.Ca (f), and B10.M (f). No reactivity to other Hh-1 antigens resides in the 5E6+ population. In contrast, the ability of NK cells to lyse H-2d or H-2b tumor cells was independent of 5E6 expression. These results suggest that the 5E6 molecule is likely to be important in the specific recognition and rejection of BMC that express Hh-1 determinant 2, and is probably not involved in recognition of "tumor target cell structures". PMID- 1718760 TI - In vivo induction of gamma/delta T cells with highly potent and selective anti tumor cytotoxicity. AB - High frequencies of CD5+TcR alpha/beta- T cells were induced in the peritoneal cavity of rats immunized with syngeneic W439 lymphoma cells. These TcR alpha/beta cells expressed TcR delta mRNA as analyzed by the polymerase chain reaction technique. The delta + (TcR gamma/delta +) T cells were of the CD2+, CD3+, CD4-, CD8+, CD45RB+ phenotype and showed stronger anti-tumor cytotoxicity compared to the TcR alpha/beta + T cells. The cytotoxic effects of both alpha/beta and gamma/delta T cells were selective for the W439 lymphoma cells and were not directed to other syngeneic tumors, natural killer targets and syngeneic or allogeneic normal cells. T cells, including both alpha/beta and gamma/delta cells, were induced when WF rats were immunized with allogeneic BN spleen cells. In this case the gamma/delta T cells showed allo-selective cytotoxicity, although weaker compared to the TcR alpha/beta + T cells. The gamma/delta T cells, induced by immunization with either W439 cells or BN spleen cells, were selective for the immunogen used and had no effect on irrelevant target cells, indicating that these effector cells were not activated by a shared gamma/delta T cell-related superantigen. Since highly potent tumor-selective gamma/delta cytotoxic T lymphocytes could be induced by syngeneic lymphoma cells, we suggest a role for gamma/delta T cells in the defense against certain types of tumors. PMID- 1718762 TI - The use of the polymerase chain reaction to map CD4+ T cell epitopes. AB - CD4+ T cells recognize processed exogenous antigen in the form of peptides bound to syngeneic major histocompatibility complex class II molecules on antigen presenting cells. We have developed a novel and convenient method to synthesize and map CD4+ T cell epitopes of cloned antigens using polymerase chain reaction (PCR)-directed construction of genes expressing recombinant protein fragments. Unique restriction sites incorporated into the PCR primers were employed for the unidirectional cloning of gene fragments into a bacterial expression vector that can be induced to high-level expression. The bacterial lysate could be used directly in T cell proliferation assays. Overlapping recombinant fragments spanning the entire protein were generated and tested. The length of the sequence containing the epitope was further reduced by utilizing PCR to generate 3' truncations. Finally, a small number of overlapping peptides spanning a sequence of 39 amino acids were synthesized to identify a thirteen-amino acid peptide epitope within chicken transferrin that stimulates the T helper cell clone D10.G4.1. PCR-directed construction of fragments of antigen allows for optimal design of strategies for the mapping and analysis of CD4+ T cell epitopes. PMID- 1718763 TI - Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA-4 promotes CD3-dependent T cell proliferation. AB - Vascular cell adhesion molecule, VCAM-1, is an adhesion molecule expressed on activated endothelium thought to play a role in leukocyte migration to sites of inflammation. VCAM-1 adheres to leukocytes through the VLA-4 integrin. Recombinant soluble VCAM-1 (rsVCAM) and anti-CD3 mAb OKT3 were utilized to address the role of the VCAM-1/VLA-4 pathway in antigen-dependent T cell activation. Monocyte-depleted T cells proliferated upon exposure to co immobilized OKT3 and rsVCAM but to neither alone. In contrast, an anti-VLA-4 mAb HP1/2 failed to co-activate with OKT3, despite the fact that both rsVCAM and HP1/2 support T cell adhesion comparably. These data indicate that adhesive function is not sufficient for co-stimulatory activity. They also reveal that VCAM-1 may play a role in regulating T cell immune responses as well as migration in vivo. PMID- 1718764 TI - Exact prediction of a natural T cell epitope. AB - T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351:290). These allele specific motifs should allow exact prediction of natural T cell epitopes. H-2Kb restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb-restricted epitope of ovalbumin, thought to be contained in the 19-mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible. PMID- 1718765 TI - The effects of pregnancy and fluoride on orthodontic tooth movements in rats. AB - The present investigation was undertaken in order to study the velocity of orthodontic tooth movement in rats and the effect of the hormonal changes that occur during pregnancy or with fluoride, which is one of several trace elements that affect hard tissue metabolism. Adult, female Sprague-Dawley rats were separated into three groups: non-pregnant, pregnant, and non-pregnant NaF supplied. All rats were treated with a fixed orthodontic appliance which moved the upper first molars in a buccal direction during 21 days. The appliances delivered an initial force of 150 mN. Repeated intra-oral, standardized radiographs were taken during the experimental period, and at the end of the experiment the maxillae were examined histologically. The velocity of tooth movement was calculated after measurement of the radiographs. The first molars were moved in a buccal direction in all groups. The mean value of the expansion from day 0 to 21 was significantly higher among the pregnant rats (0.64 mm) compared to the non-pregnant, control animals (0.46 mm) while the NaF-supplied rats had a significantly lower expansion (0.22 mm) compared to the control animals. In the histological examination of the pressure sides of the PDL of the upper first molars, the mean value of osteoclasts per microns x 10(-3) increased non-significantly in the group of pregnant rats and decreased significantly among the NaF-supplied animals compared to the non-pregnant control rats. The present experiment in rats indicated that the velocity of orthodontic tooth movement is influenced by hormones as well as trace elements. PMID- 1718766 TI - Different secretory response of pancreatic isolated lobules and dissociated acini from hypothyroid rats to exogen TRH. AB - This paper analyses the effect of hypothyroidism on pancreatic TRH and somatostatin concentrations, as well as the action of exogen TRH on pancreatic amylase secretion from isolated lobules and dissociated acini of both healthy and hypothyroid rats. In the hypothyroid group, pancreatic TRH and somatostatin increased. In the pancreatic lobules of untreated animals, bethanechol produced stimulatory action that was inhibited by TRH. On the other hand, lobules from hypothyroid rats did not respond to bethanechol stimulation. Acini amylase secretion after bethanechol stimulation was similar in both groups, although hypothyroid animals were more sensitive to the inhibitory effect of TRH. These findings suggest the existence of a factor blocking the amylase secretion in pancreatic lobules. This agent, probably TRH, could be eliminated in the experimental model of dissociated acini. PMID- 1718767 TI - Characterization of Neisseria meningitidis isolated by ribosomal RNA gene restriction patterns and restriction endonuclease digestion of chromosomal DNA. AB - The use of ribosomal RNA (rRNA) gene restriction patterns to study the molecular epidemiology of Neisseria meningitidis was investigated. Ninety-four isolates of Neisseria meningitidis were characterized by their rRNA gene restriction patterns with 16 + 23 S rRNA from Escherichia coli as a probe. Thirteen rRNA gene restriction patterns were recognized; each of these patterns represented between 1 and 30 isolates. Isolated with the outbreak-associated phenotype B15P1.16 (sulphonamide resistant) all gave a single rRNA gene restriction pattern but this pattern also contained isolates with other phenotypes. Further discrimination between isolates was achieved by comparison of banding patterns resulting from restriction endonuclease digestion of chromosomal DNA with Bgl II. This gave a banding pattern consisting of about ten bands which was simple to interpret. Using this technique 94 isolates were classified in 54 patterns containing between 1 and 14 isolates. Restriction endonuclease analysis with Bgl II characterized outbreak-associated isolates with the phenotype B15P1.16 and enabled strains not typable by conventional methods to be identified as probable outbreak-associated isolates. The techniques should prove useful for epidemiological studies. PMID- 1718768 TI - A numerical analysis of ribosomal RNA gene patterns for typing clinical isolates of Corynebacterium group D2. AB - Restriction digest fragments of DNA from 46 clinical isolates identified as Corynebacterium group D2, were separated by electrophoresis, Southern blotted onto nylon membranes and hybridized to a ribosomal RNA gene probe. The resulting band patterns were subjected to unweighed pair-group cluster analysis. Representative strains from the main clusters were compared with similarly prepared band patterns from type strains of human Corynebacterium species. The results indicate that strains identified as Corynebacterium group D2 represent a unique taxon and that computer-assisted analysis of rRNA gene restriction fragment polymorphism (ribotyping) could be a useful technique in epidemiological studies of these bacteria. PMID- 1718769 TI - HSP 90, yeasts and Corynebacterium jeikeium. AB - Recovery from disseminated candidosis is associated with seroconversion to a 47 kDa breakdown product of the Heat Shock Protein (HSP) 90 of Candida albicans. Cloning, sequencing and epitope mapping has allowed the delineation of the immunodominant epitopes LKVIRKNIVKKMIE and STDEPAGESA. Monoclonal and polyclonal antibodies specific to these epitopes are used to show that all strains of C. albicans tested produce HSP 90 in both the yeast and mycelial phases. Homologous proteins are demonstrated in Saccharomyces cerevisiae, Candida parapsilosis and Corynebacterium jeikeium but not in Torulopsis glabrata. Evidence is presented for the existence of two distinct HSP 90s in C. albicans. The first of these is expressed constitutively whilst the second is produced on heat shocking the yeast from 23 to 37 degrees C. PMID- 1718770 TI - A dot-blot hybridization procedure for the detection of astrovirus in stool samples. AB - We have developed a nucleic acid dot-blot hybridization test for the detection of astroviruses in stool samples. The test was not as sensitive as electron microscopy for the detection of low numbers of well preserved astrovirus particles, but was able to identify astroviruses in stools containing particles of indistinct morphology. In total, this procedure identified astroviruses in more samples than did electron microscopy, and the data indicate that the incidence of astroviruses may be substantially underestimated. PMID- 1718771 TI - Effect of systemic acetazolamide on the fluid movement across the aqueous vitreous interface. AB - In an attempt to study the effect of systemic acetazolamide on the fluid flow between the aqueous and vitreous in normal eyes, 50 mg kg(-1) acetazolamide was given intravenously every hour for 3 hr and the time change of the aqueous flow rate was calculated in two groups of rabbits, applying one of the following two different methods to each: the fluorescein method II of Jones and Maurice, a classical fluorometric method, or the more recently developed Johnson-Maurice method which entails intravitreal injection of FITC-dextran and measurements of its concentration in the anterior chamber many days after the injection. The flow rate after acetazolamide calculated by the fluorescein method II of Jones and Maurice in one group of rabbits was reduced to 55 +/- 5% (mean +/- S.E. n = 9) of the control on the average. When calculated by the Johnson-Maurice method in another group of rabbits, the reduction was to 80 +/- 4% (n = 11) of the control rate. The difference between the above figures was significant (P less than 0.005). Furthermore, the effect of acetazolamide calculated by the first procedure was significantly greater than that calculated by the second at 1 and 2.33 hr and at later times after the acetazolamide injection (P less than 0.05 0.01). On the other hand, the outflow pressure was reduced by 53-60% in both groups. The difference between the flow rates after acetazolamide determined by the above two methods was best explained by assuming that the FITC-dextran movement from the vitreous into the aqueous was reduced by about 25% after acetazolamide administration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718773 TI - Calcitonin gene-related peptide is a potent vasodilator of bovine retinal arteries in vitro. AB - Calcitonin gene-related peptide (CGRP) invariably induced a slow acting but potent relaxation of bovine retinal small arteries contracted with PGF2 alpha. Maximal relaxation obtained was 93% and 96% with a pD2-value of 8.97 and 8.86 for rat and human CGRP, respectively; thus the bovine retinal arteries cannot discriminate between CGRP from these two species. The CGRP-induced relaxation was reversible. Substance P was without effect on retinal arteries contracted with PGF2 alpha. Bradykinin relaxed 4 of 18 vessels tested in the concentration range of 11(-11)-10(-8) M whereas the vessels were contracted again at 3 x 10(-8) M. Bradykinin was without effect in the remaining 14 vessels. None of the peptides had a contractile effect on retinal arteries kept relaxed in normal buffer solution. Capsaicin 3 x 10(-5) M induced a relaxation comparable to that obtained by 10(-9) M of CGRP. The capsaicin-induced relaxation was reproducible and it was concentration dependently inhibited by ruthenium red which suggests that capsaicin releases CGRP in the arterial wall. The results indicate that CGRP has a powerful relaxing effect on the retinal vasculature indicating a role for CGRP in ocular blood flow regulation. PMID- 1718772 TI - Cytokine regulation of C3 and C5 production by human corneal fibroblasts. AB - Recent investigations have suggested that cytokines play important roles during inflammation and host defense, primarily by regulating the diverse functions of immunologic cells (e.g. lymphocytes and monocytes). However, much less is known about the capacity of cytokines to also regulate the functions of resident tissue cells. We hypothesize that during inflammation, cytokines (e.g. monokines and lymphokines) directly regulate the expression of inflammatory precursors and mediators, such as the third and fifth complement components, by resident ocular cells and are therefore important in the local regulation of ocular inflammation. To test this hypothesis we developed an in vitro culture system utilizing isolated human corneal fibroblasts and examined the effects of specific cytokines, i.e. interleukins and interferons, on the production of the third and fifth components of the complement system. Human corneal fibroblasts were cultured in the presence of varying concentrations (1-500 U ml-1) of interleukin 1 alpha, interleukin 1 beta, interleukin 2, interferon alpha and interferon gamma for 48 hr at 37 degrees C, 5% CO2. The supernatants were then evaluated for antigen levels for the third and fifth components of complement using specific enzyme-linked immunospecific assays. These studies revealed that both interleukin 1 alpha and interleukin 1 beta induced seven to tenfold increases in the levels of the third component. Similarly interferon alpha and interferon gamma stimulated an approximate four and ninefold dose-dependent increase, respectively, in the production of the third component. Analysis of the effect of interleukin 2 on third component production demonstrated that higher concentrations (100 U ml-1) were required to induce a fivefold increase in the production of the third component.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718774 TI - Loss of fluorescein across the conjunctiva. AB - The rate of disappearance from the tear film of fluorescein and rhodamine dextran instilled together into the human eye was compared. In many cases fluorescein disappeared more rapidly, which was attributed to its penetration across the conjunctival surface. A corresponding mean fluorescein permeability across this surface of 2.5 x 10(-5) cm min-1 was calculated. This route of loss of fluorescein from the tears leads to an average overestimate of tear turnover of 25%. PMID- 1718775 TI - Regulation of calcitonin release from the 6.23 rat C-cell line by cyclic nucleotide analogues and pharmacological mediators. AB - Calcitonin release from 6.23 rat medullary thyroid carcinoma C-cells was stimulated by dibutyryl cyclic AMP and inhibited by dibutyryl cyclic GMP in concentration dependent fashion. Histamine, isoproterenol, prostaglandin E2 and Bay K 8644 stimulated calcitonin release, while acetylcholine and serotonin had no significant effect on CT release. PMID- 1718776 TI - Proliferation and transformation of cultured liver fat-storing cells (perisinusoidal lipocytes) under conditions of beta-D-xyloside-induced abrogation of proteoglycan synthesis. AB - Fat-storing cells (perisinusoidal lipocytes, Ito cells) are the major connective tissue-producing cell type in liver. In areas of necroinflammation the cells proliferate and transform into desmin and smooth muscle alpha-actin-positive myofibroblast-like cells which synthesize a broad spectrum of significant amounts of collagens, proteoglycans, and matrix glycoproteins. Available data suggest a central role for these cells in the pathogenesis of fibrosis. Beta-D-Xyloside, an artificial initiation site for galactose-linked glycosaminoglycans, thereby uncoupling the synthesis of core protein and GAG, was used as a probe to study main cellular functions under conditions of abrogated proteoglycan synthesis. The exposure for 48 hr of fat-storing cells to p-nitrophenyl beta-D-xyloside (PNP Xyl) increased dose-dependently the synthesis of [35S]sulfate-labeled medium GAG. Maximum stimulation of fivefold above normal was reached at 1.0 mM PNP-Xyl. Higher concentrations of PNP-Xyl progressively decreased the stimulatory effect on GAG synthesis. The relative composition of GAG in medium (60% chondroitin sulfate, 34% dermatan sulfate), at the cell surface, and intracellularly (mainly heparan sulfate) was not changed significantly by PNP-Xyl. The amounts of intracellular and cell surface-bound GAG were reduced by 40 and 30%, respectively, by PNP-Xyl leading to a depletion of heparan sulfate at the cell surface. Pulse-chase experiments revealed that xyloside-initiated GAG were secreted immediately after synthesis into the medium. GAG synthesized in the presence of 1 and 5 mM PNP-Xyl were free of core protein, and the molecular size of the GAG chains was smaller than that of GAG obtained from beta-eliminated proteoglycans synthesized in control cultures. At concentrations above 3 mM PNP Xyl generated a dose-dependent inhibition of cell proliferation, which was at any stage of culture fully reversible upon removal of the drug. Viability and general protein synthesis were not reduced, but fat-storing cell transformation and deposition of matrix glycoproteins were retarded. Only a very small fraction of drug-treated cells (5 mM PNP-Xyl) did express on the 11th culture day smooth muscle iso-alpha-actin- and desmin-containing cytoskeletal filaments, which are important indicators of transformation into myofibroblast-like cells. Furthermore, the synthesis of hyaluronan and the expression of immunostained fibronectin, laminin, and tenascin were reduced in cultures exposed to 5 mM PNP Xyl. The described cellular functions were not affected by exposure of fat storing cells to p-nitrophenyl beta-D-galactoside.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718777 TI - Factors contributing to the inhibition of HIV reverse transcriptase by chain terminating nucleotides in vitro and in vivo. AB - Arguments are presented leading to the conclusion that two major factors contribute to the potency of inhibition of DNA-polymerase activity by chain terminating nucleotides. The relative significance of these factors varies with the reaction conditions, particularly with the length of the template and the concentration ratio of enzyme (reverse transcriptase or other DNA polymerase) to primer. It is concluded that potent inhibition of HIV-reverse transcriptase activity under typical in vitro and in vivo conditions arises from different features of the interaction of chain terminators with the enzyme. A new method of testing for the parameter important under in vivo conditions is suggested. PMID- 1718778 TI - Widespread tissue distribution, species distribution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthases. AB - The distribution of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase (NOS) was studied in rabbits and in control and endotoxin-treated rats and guinea pigs. There was a widespread localization of NOS which differed for the two forms of the enzyme and which showed marked differences between species. Endotoxin induced the activity of the Ca(2+)-independent NOS in many tissues and also increased the activity of Ca(2+)-dependent NOS in the rat ileum and caecum. These results demonstrate the differential distribution of NOSs in control and endotoxin-treated animals and emphasize the widespread biological role of nitric oxide (NO). PMID- 1718779 TI - Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1. AB - Cyclic Arg-Gly-Asp-Phe-Val peptides with either D-Phe or D-Val residues were 20- to more than 100-fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly-Arg-Gly-Asp-Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding of platelet receptor alpha IIb beta 3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all-trans conformation with a beta II' and gamma turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity of inhibition. PMID- 1718780 TI - Augmentation of haptoglobin production in Hep3B cell line by a nuclear factor NF IL6. AB - The nuclear factor NF-IL6 had been suggested to be responsible for the IL-6 mediated induction of several acute-phase proteins. To obtain evidence for the involvement of NF-IL6 in the induction of acute-phase proteins, we introduced the NF-IL6 gene and its truncated mutant (delNFIL6) gene into a hepatoma cell line Hep3B. Then, we examined the effect of the overproduced NF-IL6 and delNFIL6 on the expression of haptoglobin, fibrinogen and albumin. As a result, basal production as well as induction of haptoglobin by IL-6 were augmented by the expression of NF-IL6, whereas delNFIL6 blocked the production of haptoglobin, fibrinogen and albumin. PMID- 1718781 TI - Mycoplasma gallisepticum strain S6 genome contains three regions hybridizing with 16 S rRNA and two regions hybridizing with 23 S and 5 S rRNA. AB - Southern hybridization and cloning experiments revealed existence of 3 regions hybridizing with 16 S rRNA and 2 regions hybridizing with 23 S and 5 S rRNA in Mycoplasma Gallisepticum strain S6 genome thus forming 4 separate contiguous regions. One set of a putative rRNA genes is organized classically for eubacteria order 16 S-23 S-5 S. The other two 16 S rRNA and the other one 23 S-5 S rRNA hybridizing regions are separated from each other and from the complete rRNA operon for a distance of more than 6 kb. PMID- 1718782 TI - Functional expression of a human C5a receptor clone in Xenopus oocytes requires additional RNA. AB - cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes. PMID- 1718783 TI - Site-directed mutagenesis of the N-terminal region of IGF binding protein 1; analysis of IGF binding capability. AB - To define domains involved in IGF binding 60 N-terminal amino acid residues of IGFBP-1 were deleted. This deletion resulted in loss of IGF binding suggesting that the N-terminus may enclose an IGF binding domain. However, most point mutations introduced in this region did not affect IGF binding. In contrast to Cys-34, only substitution of Cys-38 for a tyrosine residue abolished IGF binding. With the determination that all 18 cysteine residues are involved in disulphide bond formation our data suggest that, although not all cysteines contribute to the same extent, the ligand binding site may be spatially organized. PMID- 1718784 TI - F(ab')2 segment is the active component of immunoglobulin G autoantibody generation in patients with endometriosis. AB - OBJECTIVE: To determine if the low titer antiendometrial antibody detection by indirect immunofluorescence (IIF) in the sera of patients with endometriosis is because of specific antigen-antibody interactions. DESIGN: Sera of selected patients subjected to separation of immunoglobulin fragments by ammonium sulfate precipitation and pepsin digestion and tested by IIF. In a comparative method, a fragment, crystallizable (Fc) flooding technique was used to determine the active component. SETTING: Sera obtained preoperatively in the outpatient and results matched with surgical findings. PATIENTS/PARTICIPANTS: Patients 45 years of age or less presenting for gynecological surgery who had signed an informed consent for the drawing of preoperative blood. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Indirect immunofluorescence was graded according to the intensity of staining and the lowest dilution at which staining occurred. RESULTS: All of the sera of the 11 patients with endometriosis exhibited F(ab')2 specific staining but none exhibited Fc fragment staining. None of the 7 control patients displayed F(ab')2 or Fc fragment staining. CONCLUSIONS: F(ab')2 fragment is the active component of an immunologically specific interaction when antiendometrial antibodies are detected in the sera of patients with endometriosis. PMID- 1718785 TI - Determination of neutrophil concentration in semen by measurement of superoxide radical formation. AB - OBJECTIVE: To develop an assay that measures the concentration of functional neutrophils in human semen. DESIGN: Human blood neutrophils were first isolated to establish linearity and proportionality for the determination of functional neutrophil concentration. Thereafter blood neutrophils in seminal plasma and neutrophils in semen of patients were measured. SETTING: Blood and human samples were obtained from clinics of the Royal Victoria Hospital. PATIENTS, PARTICIPANTS: Blood from normal men or from patients presenting with chest pain or trauma were used. Semen samples were also obtained from healthy fertile donors or from unselected patients attending the infertility clinic. MAIN OUTCOME MEASURES: Active neutrophil concentration in semen by a colorimetric assay. RESULTS: The assay developed is based on the reduction of nitroblue tetrazolium of pale yellow color to blue formazan by the superoxide anions produced by stimulated neutrophils. The intensity of the derived blue color is proportional to the concentration of active neutrophils. This assay is simple, requires only 10 minutes of preparation time, and detects neutrophil concentrations greater than 0.5 x 10(6) neutrophils/mL of semen, independent of sperm concentration. PMID- 1718786 TI - Recognition of carbohydrate antigen epitopes by sperm-immobilizing antibodies in sera of infertile women. AB - STUDY OBJECTIVE: To study carbohydrate natures in the antigen epitopes corresponding to sperm-immobilizing antibodies in infertile women. DESIGN: Antibody absorption with human sperm and seminal plasma before and after treatments with trifluoromethanesulfonic acid or sodium metaperiodate. PATIENTS: Thirty-three patients who showed a positive sperm immobilization test provided their sera for the experiment. RESULTS: In 25 patients' sera whose sperm immobilizing antibodies were absorbed with human seminal plasma, the antibody absorbing capabilities were completely abolished by deglycosylation treatment with trifluoromethanesulfonic acid. The sperm-immobilizing antibodies in 4 patients' sera were absorbed out with sperm membrane fraction before the treatment but not after the treatment with trifluoromethanesulfonic acid. In some patients' sera, the antibody-absorbing capabilities of ejaculated sperm were markedly reduced by sodium metaperiodate treatment. CONCLUSION: The majority of sperm-immobilizing antibodies in infertile patients might be generated to carbohydrate structures of the sperm-coating antigens or sperm membrane antigens. PMID- 1718787 TI - Staining of the inner acrosomal membrane of human spermatozoa with concanavalin A lectin as an indicator of potential egg penetration ability. AB - OBJECTIVE: To determine the sensitivity and functional significance of the fluorescein isothiocyanate concanavalin A (FITC-ConA) staining method of assessment of acrosomal status. DESIGN: Treatments were assessed for their ability to induce human sperm acrosomal loss. Penetration of zona-free human eggs by treated spermatozoa was subsequently determined. SETTING: Zona-free human eggs were obtained from the Infertility Medical Centre, Richmond, Victoria, Australia. PATIENTS, PARTICIPANTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosomal loss of human spermatozoa was determined at 4 hours and 10 hours of treatment incubation. Penetration of zona-free human eggs was assessed 16 hours after reinsemination. RESULTS: Human spermatozoa incubated in a strontium- or lanthanum-based medium, or T6 + 10% maternal human serum (HS) supplemented with 12 mM 8-bromo cyclic guanosine 3,5'-monophosphate and 10 mM imidazole for a 4 hour period before transfer to fresh T6 + 10% HS for a further 6 hours, demonstrated a significant increase (P less than 0.05) in acrosomal loss compared with T6 + 10% HS for a total 10-hour incubation. This increase in acrosomal loss with test treatments correlated with an increase in the development of pronuclei of zona-free human eggs (r = +0.98). CONCLUSIONS: The FITC-ConA staining procedure therefore reflects biological function as assessed by the penetration of zona-free human eggs and consequently provides a further research tool for the investigation of the human sperm acrosome reaction. PMID- 1718788 TI - Hypothermia and rewarming after cardiac operation. AB - Those caring for patients who undergo cardiac surgery share a dilemma. Complications associated with hypothermia encourage the care giver to rewarm the patient quickly, but complications associated with rapid temperature changes and hyperthermia can threaten the patient's hemodynamic stability. What are adequate rewarming measures, when to terminate active rewarming measures to avoid hyperthermia, and what is the optimal rate of rewarming are all unanswered questions. Continued nursing research will expand the science of nursing and provide care givers with knowledge for expert care. As the turn of the century approaches and surgical techniques advance, high-risk patients who undergo reoperation and multi-valve cardiac surgery require expert nursing care. PMID- 1718789 TI - Evidence that metformin ameliorates cellular insulin-resistance by potentiating insulin-induced translocation of glucose transporters to the plasma membrane. AB - To examine the cellular mechanism of the antihyperglycemic action of metformin we studied the effect of metformin on various functional and molecular parameters involved in the pathogenesis of insulin resistance. Isolated rat adipocytes were incubated with or without metformin (1-100 micrograms/ml) for 2 hours at 37 degrees C followed by an incubation with or without insulin (10 ng/ml). Metformin treatment had no significant effect on basal 3-0 methylglucose uptake. In contrast, Metformin increased insulin-stimulated glucose transport in a dose dependent manner up to 43 +/- 7%. This effect was neither associated with a significant effect of Metformin on trace insulin binding, 1.74 +/- .2% (-M) vs. 1.89 +/- .3% (+M), P greater than 0.05, nor with an effect of metformin on in vivo activation of insulin receptor kinase activity as measured by 32P incorporation into the 95 kDa beta-subunit of the insulin receptor and an exogenous substrate, histone 2B. Determination of glucose transporter numbers in subcellular membrane fractions, plasma membranes and low-density microsomes, using cytochalasin B binding and immunodetection revealed that metformin's effect to increase insulin-stimulated glucose transport is associated with a potentiation (38 +/- 5%) of insulin-induced translocation of glucose transporters from the intracellular pool to the plasma membrane, while the basal state was not significantly affected. Determination of specific glucose transporter mRNA-levels revealed that this metformin effect is not associated with an increase in glucose transporter gene expression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718790 TI - Immunocytochemical markerprofile of endometriotic epithelial, endometrial epithelial, and mesothelial cells: a comparative study. AB - A comparative immunocytochemical study was performed on endometriotic epithelial versus endometrial epithelial and normal mesothelial cells in order to obtain further evidence for either the endometrial implantation or mesothelial metaplasia theory. The three cell types could not be distinguished by keratin subtyping, using monoclonal antibodies (MAbs) to keratins 5, 7, 8, 14, 18, and 19. The epithelial markers HMFG-2 and BW 495/36, and a newly developed MAb NEND-3 (against endometrial cells) discriminated between generally negatively reacting mesothelial cells and positively reacting endometrial and endometriotic epithelial cells. The MAb NEND-3 appeared to be specific for endometrial (and endometriotic) epithelial cells since no reactivity with other epithelial cell types was found. Typing with MAbs against ovarian carcinoma related antigens (OV TL 3, OV-TL 10 and OC 125) did not permit sufficient distinction. The marker profile of cultured endometrial, endometriotic and mesothelial cells confirmed the immunocytochemical findings on frozen sections. Although the data are consistent with the endometrial implantation theory, mesothelial metaplasia can not be excluded with regard to the histogenesis of endometriosis since metaplastic mesothelium may express different antigen markers. PMID- 1718791 TI - An elevated maternal serum alpha-fetoprotein leading to the diagnosis of an immature teratoma. AB - A case report is presented of a woman with a maternal serum alpha-fetoprotein 17.46 multiples of the median (MOM), who was found to have an ovarian immature teratoma. It is suggested that patients who present with a maternal serum alpha fetoprotein value greater than 9 multiples of the median receive a more comprehensive evaluation remembering that the alpha-fetoprotein in the adult can be a tumor marker. PMID- 1718792 TI - Dual control by androgens and peptide growth factors of prostatic growth in human benign prostatic hyperplasia. PMID- 1718793 TI - Differential effect of glycosylation on the expression of antigenic and bioactive domains in human thyrotropin. AB - Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone. PMID- 1718794 TI - Identification and regulation of insulin-like growth factor binding proteins produced by hormone-dependent and -independent human breast cancer cell lines. AB - Using molecular hybridization, ligand blotting and immunoprecipitation, our studies were designed to identify the insulin-like growth factor binding proteins (IGFBPs) produced by human breast cancer cells in culture and evaluate their regulation by estradiol (E2) and polyamines (PA). We demonstrate that the hormone dependent MCF-7 and -independent BT-20 cell lines express the mRNA for IGFBP-2 (1.7 kb) and secrete this BP (31 kDa) in the conditioned medium. In contrast, the hormone-independent MDA-MB-231 cell line does not express the IGFBP-2 gene, while synthesizing and secreting IGFBP-1. E2 administration (10(-9) M) did not significantly influence IGFBPs secretion in MCF-7 cells. Addition of alpha difluoromethylornithine (DFMO, 4 mM), an inhibitor of PA biosynthesis, consistently lowered IGFBP-2 mRNA in the MCF-7 and BT-20 cell lines and IGFBP-1 mRNA in MDA-MB-231 cells. Surprisingly, however, this compound either did not influence IGFBPs secretion in MCF-7 cells or actually increased their secretion in the BT-20 and MDA-MB-231 cell lines. PA involvement in IGFBPs production by breast cancer cells is complex and may involve differential regulation of transcriptional and post-translational events. PMID- 1718795 TI - Insulin-like growth factor-binding protein-1: a role in glucose counterregulation? PMID- 1718796 TI - Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme. AB - In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P 450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P 450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes. PMID- 1718797 TI - Prolactin activates protein kinase C and stimulates growth-related gene expression in rat liver. AB - We have examined the effect of prolactin (PRL) on growth-related gene expression, protein kinase C (PKC) activity and diacylglycerol (DAG) mass in rat liver. Hepatic levels of messenger (m)RNA for c-myc, ornithine decarboxylase (ODC) and beta-actin increased in a dose-dependent manner within 1 h after PRL administration. Prolactin also caused a transient elevation of liver DAG levels and particulate-associated PKC activity. The PRL-provoked increases in DAG mass and particulate PKC activity were coincident and maximal at 20 min and began declining toward control levels by 30 min. These results suggest a temporal relationship between PRL-stimulated DAG accumulation and PKC activation. Furthermore, the subsequent rapid induction of growth-related gene expression provides new information on the role of PRL as a hepatic mitogen. PMID- 1718798 TI - Synthesis of tubulin during early postgastrula development of Artemia: isotubulin generation and translational regulation. AB - Isotubulin diversity and the synthesis of tubulin were examined during development of the brine shrimp, Artemia. It was found, by Northern and dot-blot analyses, that Artemia possess constant amounts of one size class of mRNA each for alpha- and beta-tubulin during the first 24 hr of postgastrula development. Two-dimensional gel electrophoresis and fluorography, following the in vitro translation of developmentally staged poly(A)+ mRNA, yielded one alpha- and one beta-tubulin. Clearly, the isotubulin diversity seen on Coomassie blue-stained two-dimensional gels of Artemia tubulin is not generated by differential gene transcription during postgastrula growth, nor is development accompanied by synthesis of novel isotubulins resolvable by the methods employed. Characterization of polysomal poly(A)+ mRNA, and of proteins synthesized in vivo, indicated very little tubulin was synthesized in Artemia as they developed from gastrula to first instar larvae. The results suggest control of tubulin synthesis in Artemia by a mechanism that restricts binding of the message to ribosomes. Of general significance, it appears that a complex metazoan animal is able to undergo extensive growth with limited tubulin synthesis and in the absence of differential expression of tubulin genes. Moreover, the capacity of microtubules to assume changing and/or increased functions associated with cellular development is seemingly not dependent on the synthesis of new tubulin isoforms. PMID- 1718799 TI - Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization. AB - We previously showed that tenascin, a large, extracellular matrix glycoprotein, exhibits a temporally and spatially restricted distribution during urodele limb regeneration. To further investigate the role of tenascin in regeneration, we cloned a newt tenascin cDNA, NvTN.1, that has 70% homology to the chicken tenascin sequence. A deduced amino acid sequence of NvTN.1 showed a modular structure unique to tenascin characterized by epidermal growth factor-like and fibronectin type III repeats. To determine the cellular origin of tenascin protein during limb regeneration, we localized tenascin transcripts by in situ hybridization using a riboprobe synthesized from NvTN.1. Transcripts could not be detected in normal limb tissues but first became detectable in the wound epithelium at 2 days and in the distal mesoderm at 5 days after amputation. These wound epithelial cells are probably the source of tenascin protein found within and immediately underneath the wound epithelium. At preblastema stages, hybridization was seen in cells associated with most of the distal mesodermal tissues but not in dermis. At blastema stages, essentially every mesenchymal cell contained tenascin transcripts. Thus, regardless of origin, blastemal mesenchymal cells may share a common regulatory mechanism that results in tenascin gene transcription. Finally, during redifferentiation stages of regeneration, tenascin gene transcription was associated with both differentiation and growth. The results show that initiation of tenascin gene expression is an early event in regeneration and continued tenascin gene transcription is associated with some of the important processes of regeneration, namely wound epithelial-mesenchymal interactions, dedifferentiation, initiation of cell cycling, blastema outgrowth, and cellular differentiation. PMID- 1718800 TI - CD5+ B lymphocytes in high-risk islet cell antibody-positive and newly diagnosed IDDM subjects. AB - Human CD5+ B lymphocytes produce autoantibodies that bind to self- and exogenous antigens. Extremely high percentages of CD5+ B lymphocytes are present in the fetal and newborn periods, whereas they constitute only a minority of B lymphocytes in healthy adults. Increased percentages of circulating CD5+ lymphocytes have previously been demonstrated in several autoimmune diseases, including rheumatoid arthritis, progressive systemic sclerosis, Graves' disease, and Sjogren's syndrome. We measured the percentages of B lymphocytes that expressed the CD5 determinant in 93 control subjects (age range 1 day to 59 yr, mean +/- 22.6 +/- 17.7 yr), 17 subjects with newly diagnosed insulin-dependent diabetes mellitus (IDDM; range 5-29 yr, mean +/- SD 13 +/- 5.9 yr), 31 high-risk islet cell antibody (ICA)-positive nondiabetic subjects (range 4-45 yr, mean +/- SD 19.8 +/- 14.1 yr), and 13 subjects with IDDM of greater than 5 yr duration (range 10-43 yr, mean +/- SD 24.2 +/- 9.9 yr). We report that CD5+ B-lymphocyte percentages are strikingly age dependent in healthy control subjects, declining progressively from the newborn period to the middle-age years (r = -0.75, P = 0.0001). In ICA+ nondiabetic and recent-onset IDDM subjects less than 29 yr of age, the percentage of circulating CD5+ B lymphocytes fell within the 95% confidence intervals established for control subjects. However, the age-dependent rate of decline in the percentage of CD5+ B lymphocytes within the control range was slower in ICA+ and newly diagnosed IDDM subjects than in control subjects. PMID- 1718801 TI - Adhesion molecules in human islet beta-cells. De novo induction of ICAM-1 but not LFA-3. AB - Understanding how T lymphocytes recognize beta-cell autoantigens is essential for the elucidation of the pathogenesis of insulin-dependent diabetes mellitus. The increased and ectopic expression of HLA class I and II molecules detected in human beta-cells may facilitate this interaction. T-lymphocyte recognition of surface antigens also involves adhesion accessory molecules: intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 3 (LFA 3). These molecules not only allow cell contact but can also provide costimulatory signals for T-lymphocyte activation. Levels of ICAM-1 and LFA-3 expression in normal human islet cells and regulation of their expression by cytokines interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and IL-6 have been studied by two-color immunofluorescence staining of pancreatic cryostat sections and fluorescence activated cell sorter analysis. Neither ICAM-1 nor LFA-3 could be demonstrated in sections or in fresh cell preparations, but after 18 h of culture, beta-, alpha-, and delta-cells expressed spontaneously moderate levels of ICAM-1 (but not LFA 3). IFN-gamma and TNF-alpha alone or in combination strongly enhanced this spontaneous expression of ICAM-1 in a time- and/or dose-dependent and additive manner but had no effect on LFA-3. An SV40-transformed islet cell line showed high basal levels of both ICAM-1 and LFA-3, but the response to cytokines followed the same pattern as primary cultures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718802 TI - Identification of G protein alpha-subunits in RINm5F cells and their selective interaction with galanin receptor. AB - Galanin, an inhibitor of insulin secretion in pancreatic beta-cells, exerts its multiple effects through mechanisms that are sensitive to pertussis toxin (PTX). G proteins have been characterized in RINm5F cells. By ADP ribosylation and immunoblotting, the alpha-subunits of Gi1, Gi2, Gi3, and two forms of Go were identified, Gi alpha 2 being predominant. As expected from a G protein-linked receptor, GTP and its nonhydrolyzable analogue GTP-gamma-S decreased tracer galanin binding to cell membranes. This resulted from a change in receptor affinity without any modification in the number of sites. Selective antibodies against the COOH-terminal decapeptide of the alpha-subunits of the Gi and Go proteins were used to block G protein interaction before we studied galanin binding. Antibody AS, which selectively recognizes Gi alpha 1 and Gi alpha 2, decreased tracer galanin binding to membranes at concentrations where there were no effects of other antibodies specifically directed against Gi alpha 3 or G alpha o. These data suggest that Gi1 and/or Gi2 interact with the galanin receptor and probably mediate the effects of galanin in pancreatic beta-cells. PMID- 1718803 TI - Treatment of acquired aphasia and epilepsy. PMID- 1718804 TI - Porcelain-composite interface microleakage with various porcelain surface treatments. AB - This study evaluated the effects of various porcelain surface treatments on the microleakage of the porcelain-composite interface. The experimental model isolated and evaluated only the porcelain-composite interface without the presence of a bond to tooth structure. Four types of porcelain were fired into circular porcelain tabs 1.0 mm thick by 8.5 mm in diameter. The groups of porcelain were divided into subgroups for treatment with hydrofluoric acid etching and silane. A jig standardizing composite thickness to 0.2 mm was used to photopolymerize composite to porcelain. The margins were finished and polished with burs and disks. Samples were stored in 37 degrees C water thermocycled 1000X, placed in AgNO3 solution, embedded in epoxy, and cross-sectioned every 90 degrees for measurement of stain penetration at the composite-porcelain interface. Occasional crazing of porcelain from composite polymerization shrinkage was observed. Porcelain surface treatment significantly increased the specimens' ability to withstand water storage and thermal-cycling procedures. Porcelain surface treatment with silane alone did not reduce microleakage, but, in combination with etching, reduced microleakage significantly. PMID- 1718805 TI - Sequences of islet amyloid polypeptide precursors of an Old World monkey, the pig tailed macaque (Macaca nemestrina), and the dog (Canis familiaris). AB - The 37-amino acid islet amyloid polypeptide represents the major protein component present in islet amyloid deposits. Although the presence of islet amyloid is a characteristic pathological feature of the islets of humans, monkeys and cats with Type 2 (non-insulin-dependent) diabetes mellitus, it is not found in the islets of diabetic rats, mice or dogs. To further explore the molecular basis for these species differences in amyloid deposition we have used a polymerase chain reaction based method to clone cDNAs encoding the monkey (Macaca nemestrina) and dog (Canis familiaris) islet amyloid polypeptide precursors. The predicted amino acid sequence of the monkey precursor is 96% identical to that of the human protein; differences include one replacement in the signal peptide and three in the islet amyloid polypeptide domain. The sequence of the dog precursor is most closely related to that of the cat protein (85% identity); the sequences of dog and cat islet amyloid polypeptide differ only at two positions and are identical in the region of amino acids 20-29, the region thought to be primarily responsible for amyloidogenesis. Thus, amino acid residues in addition to those at positions 20-29 may facilitate the aggregation of islet amyloid polypeptide. The presence of amyloid deposits in some dog pancreatic endocrine tumours suggests that the dog protein can be amyloidogenic, perhaps due to elevated expression of islet amyloid polypeptide by the tumours relative to normal islets. PMID- 1718806 TI - Calcitonin gene-related peptide and substance P decrease in the rabbit colon during colitis. A time study. AB - The sensory neuropeptides, substance P and calcitonin gene-related peptide, have been implicated in inflammatory reactions in several tissues. An immune-complex model of colitis was used in rabbits to determine the colonic content (nmol/g protein) of immunoreactive substance P and calcitonin gene-related peptide at various times after induction of inflammation to assess changes in these neuropeptides during the inflammatory response. Calcitonin gene-related peptide content was decreased by 66% 4 hours after induction of inflammation and reached a maximum of 80% at 48 hours. The substance P content was decreased at 8 hours, with a maximum decrease of 64% at 48 hours. Substance P decrease was detected in the muscle layer. The amounts of substance P in the mucosal/submucosal layer extracts were too low to allow accurate measurements. Calcitonin gene-related peptide decreased both in the muscle and the mucosal-submucosal layers. Immunohistochemical analysis showed that calcitonin gene-related peptide and substance P innervation patterns were comparable in normal and inflamed colon, even though there appeared to be a decrease in density and intensity of the staining, particularly for calcitonin gene-related peptide at 48 hours. The early decrease of calcitonin gene-related peptide and substance P during the time course of colitis might be due to release from nerve terminals of the gut during the inflammatory response. The profound changes in colonic calcitonin gene related peptide and substance P content during colitis may have important implications during inflammation and subsequent tissue repair and may also lead to disturbances in gut motility. PMID- 1718807 TI - Effect of phospholipase C on cholesterol solubilization in model bile. A concanavalin A-binding nucleation-promoting factor from human gallbladder bile. AB - Human bile contains a phospholipase C activity. To examine its pathophysiological importance, the effect of phospholipase C on the dynamics of lipid solubilization and nucleation (cholesterol crystal formation) were investigated in model bile. Phospholipase C from gallbladder bile from patients with gallstones was partially purified by competitively eluting from a concanavalin A (con A)-Sepharose (Sigma, St. Louis, MO) column and incubating with Pronase (Calbiochem, Behring Diagnostics, La Jolla, CA). Phospholipase C activity was resistant to Pronase digestion. When this fraction (concentrated to half the original volume) was mixed with model bile (1:1, vol/vol), a transfer of cholesterol and phospholipid from the micellar to the vesicular phase and an accelerate nucleation time were found concomitant with phospholipid hydrolysis. These effects were prevented by inhibiting the phospholipase C activity with ethylenediaminetetraacetic acid. To confirm that the results were caused by phospholipase C activity and not some other nucleation-promoting factor within the biliary con A preparation, model bile was incubated with bacterial phospholipase C. An identical cascade of events to that found with the partially purified biliary enzyme was observed. Further purification of the con A-bound proteins on DEAE-Sephadex (Pharmacia, Uppsala, Sweden) did not resolve any separate nucleation-promoting activity to that associated with phospholipase C activity. In conclusion, this study has identified phospholipase C as a/the con A nucleation-promoting activity in human gallbladder bile and has characterized a possible molecular mechanism by which cholesterol nucleation is stimulated by this fraction. PMID- 1718808 TI - Lipase/amylase ratio. A new index that distinguishes acute episodes of alcoholic from nonalcoholic acute pancreatitis. AB - Because of observations that patients with acute episodes of alcoholic pancreatitis had high serum lipase levels whereas patients with gall stone pancreatitis had high serum amylase levels, a prospective study was undertaken to determine whether the ratio of serum lipase to serum amylase, a newly computed ratio, would discriminate between acute episodes of alcoholic and nonalcoholic pancreatitis. In phase one, 30 consecutive patients with acute pancreatitis were entered into the study and divided into groups A and B. Patients with renal failure were excluded from the study. Group A consisted of 20 patients in whom the etiology of pancreatitis was alcohol. Group B consisted of 10 patients whose pancreatitis was nonalcoholic in etiology (predominantly gallstones). Serum lipase values in group A ranged 492 to 25,706 U/L (median, 3433 U/L) and in group B from 711 to 31,153 U/L (median, 1260 U/L). These differences were not significant statistically. Serum amylase values in group A ranged from 104 to 2985 U/L (median, 331 U/L) and in group B from 423 to 13,000 (median, 1187 U/L). Although these figures were statistically different (P less than 0.005), there was a considerable degree of overlap in the values between the two groups. The lipase/amylase ratio calculated from the blood sample obtained at presentation appeared to be a promising discriminatory index. The lipase/amylase ratio was calculated by using the amylase and lipase levels expressed as multiples of the upper limit of normal in each case. The lipase/amylase ratios in the alcoholic group ranged from 2.2 to 14.8, whereas the lipase/amylase ratio in nonalcoholic pancreatitis ranged from 0.31 to 1.93. These differences were statistically significant (P less than 0.005). A lipase/amylase ratio of greater than 2 was indicative of an alcoholic etiology, and a ratio of less than 2 suggested that the pancreatitis was nonalcoholic in nature. In phase two, this lipase/amylase ratio of 2 was applied prospectively to an unselected population of 21 consecutive patients with acute pancreatitis. Thirteen patients had a lipase/amylase ratio of greater than 2; in 11 of them, the etiology of the pancreatitis was alcohol. Eight patients had a lipase/amylase ratio of less than 2; of them, only 1 patient had an alcoholic etiology for the pancreatitis. These differences were statistically significant (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718809 TI - A shift in balance between profibrinolytic and antifibrinolytic factors causes enhanced fibrinolysis in cirrhosis. AB - The aim of this study was to assess the cause of enhanced fibrinolysis in cirrhosis by studying the balance between profibrinolytic and antifibrinolytic proteins in 24 patients with mild or severe cirrhosis. Antigen levels of both tissue-type plasminogen activator and plasminogen-activator inhibitor 1 were increased in mild and severe cirrhosis. Activity levels showed a very wide variability, but median activity levels of both proteins were normal. In most patients, the increase in tissue-type plasminogen activator was counterbalanced by the increased levels of plasminogen-activator inhibitor 1, but in a subgroup of patients the change in balance resulted in extremely high tissue-type plasminogen-activator levels. The specific activity of both proteins (activity/antigen quotient) was reduced in either mild or severe cirrhosis. This finding indicates either that more enzyme-inhibitor complexes circulate in the blood of patients with cirrhosis than in normal individuals or that dysfunctional molecules circulate. Plasminogen and alpha 2-antiplasmin antigen and activity levels were decreased in both mild and severe cirrhosis. The binding of alpha 2 antiplasmin to fibrin was decreased in severe cirrhosis, making fibrin clots more susceptible to lysis. Clot lysis experiments were performed to see if equal decreases in plasminogen and alpha 2-antiplasmin levels, as found in cirrhosis, result in a change in the rate of fibrinolysis. It was found that the proportionate decreases led to enhancement of fibrinolysis, indicating that the inhibitor depletion is more important than the proenzyme depletion. The authors conclude that enhanced fibrinolysis frequently found in cirrhosis may be attributed to an increased tissue-type plasminogen-activator activity relative to plasminogen-activator-inhibitor activity and decreased levels of alpha 2 antiplasmin, resulting in a reduced binding of alpha 2-antiplasmin to fibrin. PMID- 1718810 TI - [Treatment of fallopian tube pregnancies with prostaglandins]. AB - In a study, the clinical use of prostaglandin F2 alpha in local and systemic application in women with ectopic pregnancies were studied (1, 9). Two different treatment schedules were defined and applied. In group A, patients with a diagnosed ectopic and beta-HCG level lower than 850 mIU/ml were treated with prostaglandin F2 alpha i.m. injected only. In group B, prostaglandin F2 alpha were injected in the chorionic cavity of the ectopic by laparoscopy after localisation with a thin needle. In spite of prostaglandin F2 alpha treatment, 6 of 30 patients (20.0%) had to be operated by microsurgery because of increasing serum beta-HCG levels. A control of tubal patency 6 month later showed one closed tube only (4.5%). Up to now, 8 spontaneous intrauterine pregnancies occurred in our study groups after successful prostaglandin F2 alpha treatment; one pregnancy was seen in a women with a single fallopian tube. The conserving treatment of one ectopic pregnancy using prostaglandin F2 alpha yields positive results, if serum beta-HCG levels are below 2000 mIU/ml. PMID- 1718811 TI - [The HLA system]. PMID- 1718812 TI - Comparison of the effects of milrinone and OPC 3911 with those of isoprenaline, forskolin and dibutyryl-cAMP in rat aorta. AB - 1. OPC 3911 and milrinone, inhibitors of a cyclic nucleotide phosphodiesterase, and isoprenaline were more potent against contractions induced by phenylephrine (1 microM) than by K+ (60 mM). A similar tendency was found for dibutyryl-cAMP (db-cAMP) and forskolin, while the opposite was evident for nifedipine and diltiazem. 2. Contractions induced by Bay K 8644 (0.1 microM), in the presence of K+ (12 mM), were abolished by OPC 3911 (10 microM), milrinone (10 microM), db cAMP (100 microM) and forskolin (1 microM). These agents had little effect on contractions induced by Bay K 8644 in the presence of K+ (20 mM), whereas diltiazem (10 microM) caused complete inhibition. 3. In nominally Ca(2+)-free medium, OPC 3911 (10 microM), milrinone (10 microM), db-cAMP (100 microM) and forskolin (1 microM) reduced phenylephrine-induced contractions. Db-cAMP and forskolin also attenuated contractions elicited by caffeine (20 mM). 4. Pretreatment by ryanodine (10 microM) reduced the effects of OPC 3911 (10 microM), milrinone (10 microM) and forskolin (1 microM) on phenylephrine-induced contractions. PMID- 1718813 TI - Effects of the calcium channel agonist, Bay K 8644, on the mechanical output of skeletal muscle fibers. AB - 1. The effects of the calcium agonist, Bay K 8644, on the mechanical output of skeletal muscle were studied in frog semitendinosus fiber bundles and in whole sartorius muscles. 2. Low concentrations of Bay K 8644 (less than or equal to 1 microM) had no significant influence on isometric twitches. Concentrations between 5-20 microM increased peak tension by 28.6 +/- 2.9% while higher concentrations (greater than or equal to 50 microM) initially increased then depressed twitches by 70 +/- 3.5%. 3. 10 microM Bay K 8644 also increased peak tension developed during low frequency stimulation (i.e. less than or equal to 40 Hz), slightly depressed high frequency contractions (i.e. greater than or equal to 80 Hz) but did not reduce maximal tetanic tension which occurred at about 60 Hz. 4. Potentiation of twitches and low frequency tetani and depression of high frequency tetani by Bay K 8644 were partially antagonized by nifedipine (10 microM), low extracellular calcium and D-600 (5 microM). These conditions did not, however, block the depressant actions of greater than or equal to 50 microM Bay K 8644. 5. In skinned fibers, 10 microM Bay K 8644 had no effect on resting or maximal Ca2+ activated tension. Also, 10 microM Bay K 8644 had no effect on caffeine contractures when added to the previous Ca2+ loading solution. 6. These results suggest that Bay K 8644 has both positive and negative inotropic actions on isolated skeletal muscle, which are dependent on drug concentration and muscle activation pattern. PMID- 1718814 TI - Some substances with proposed digitalis-like effects evaluated on platelet functions sensitive for cardiac glycosides. AB - 1. The effects of progesterone, corticosterone 21-acetate, chlormadinone acetate, dehydroepiandrosterone 3-sulfate and lysophosphatidylcholine were tested on 86Rb uptake, 3H-5-HT-uptake, ADP-induced aggregation and 5-HT-induced shape change in human platelets. Ouabain and digoxin were used for reference. 2. Ouabain and digoxin 10(-5) M inhibited 86Rb-uptake by more than 85%, and chlormadinone acetate 10(-5) M by 20%. The other substances had no effects. 3. Ouabain and digoxin were potent inhibitors on 3H-5-HT-uptake, whereas chlormadinone acetate had no effect. 4. Ouabain and digoxin increased ADP-induced aggregation but chlormadinone acetate decreased it. 5-HT-induced shape change was decreased by ouabain and digoxin, and to a lesser extent by chlormadinone acetate and its vehicle (ethanol 1.0%). PMID- 1718815 TI - Tachykinins and intestinal motility in different fish groups. AB - The presence and function of tachykinins were studied in the intestine of hagfish (Myxine glutinosa), lamprey (Lampetra fluviatilis), starry ray (Raja radiata), lungfish (Lepidosiren paradoxa), bichir (Polypterus senegalensis), and rainbow trout (Oncorhynchus mykiss), which represent different systematic groups of fish. Immunohistochemistry revealed cells containing substance P (SP)-like material in the intestine of lamprey and lungfish, and in the stomach of the ray. The intestinal motility was studied using isolated muscle strip preparations. SP had no effect on hagfish or lamprey intestine. In the other four species SP produced intestinal contractions. In ray, bichir, and lungfish the tachykinins may be released from endocrine cells and act, at least in the bichir and lungfish, directly onto the smooth muscle cells. In the rainbow trout intestine, where SP like material may be released from both nerve fibres and endocrine cells, it is indicated that the contractile effect is in part direct upon the smooth muscle and in part via stimulation of cholinergic and serotonergic neurons. PMID- 1718816 TI - A phylogenetic analysis of an atypical leuconostoc: description of Leuconostoc fallax sp. nov. AB - The 16S rRNA sequence of an unknown leuconostoc originally isolated from sauerkraut was investigated by reverse transcription. A comparison of the sequence with those from other lactic acid bacteria revealed the unknown organism represents a new albeit peripheral line within the genus Leuconostoc sensu stricto. A new species, Leuconostoc fallax, is proposed for this organism. The type strain is DSM 20189. PMID- 1718817 TI - The epitope structure in lipopolysaccharide recognized by the serotype 2-specific monoclonal antibody of Actinobacillus pleuropneumoniae. AB - The polysaccharide structure recognized by a monoclonal antibody specific to serotype 2 lipopolysaccharide of Actinobacillus pleuropneumoniae was investigated using an enzyme-linked immunosorbent assay inhibition test. Lipopolysaccharide obtained from serotype 2, strain SH-15, was hydrolysed with acetic acid to liberate the polysaccharide portion, and the polysaccharide mixture was fractionated by gel filtration. The longer polysaccharide, composed of O antigenic polysaccharide and core, fully inhibited the binding of monoclonal antibodies to a whole cell antigen of strain SH-15, whereas the core oligosaccharide without O-polysaccharide did not. No inhibition was observed with the monosaccharides which were the components of serotype 2 LPS. Enzyme-linked immunosorbent assay inhibition ability of O-polysaccharide was completely lost only by O-deacetylation. These results demonstrate that the epitope of the serotype-specific monoclonal antibody resided in O-polysaccharide of LPS and that the O-acetyl group was essential for the epitope structure. PMID- 1718818 TI - Spontaneous mutation at the mtr locus of Neurospora: the spectrum of mutant types. AB - We have isolated 135 strains of Neurospora which have mutations at the mtr locus resulting from independent spontaneous events. mtr is the structural gene for the neutral amino acid permease. The mutants have been characterized by their reversion behavior (both spontaneous and induced) and by hybridization studies of restriction digests of their DNA. About half of the mutants (54%) appear to result from single base-pair substitutions. Thirty-four percent have deletions, including some which extend into neighboring genes. Most of the remaining mutants have insertions. Several of the insertions are tandem duplications of 400-1000 bp and these mutants are unstable, reverting to mtr+ with a high frequency. When tandem-duplication mutants go through a cross, they are modified: the mutant progeny are fully stable. This modification is probably due to RIP (repeat induced point mutation). This process has an important bearing on the comparison of germinal to somatic mutation. PMID- 1718819 TI - IS406 and IS407, two gene-activating insertion sequences for Pseudomonas cepacia. AB - We have determined the nucleotide sequences of IS406 (1368 bp) and IS407 (1236 bp), two insertion sequence (IS) elements isolated from Pseudomonas cepacia 249 on the basis of their abilities to activate the expression of the lac genes of Tn951. IS406 and IS407 when inserted into the lac promoter/operator region of Tn951 generated, respectively, duplications of 8 and 4 bp of target DNA. IS406 had 41-bp terminal inverted repeat (IR) sequences with eleven mismatches. IR-L (left) contained a 12-bp motif present at the ends of Tn2501. In other respects, IS406 was distinct from previously described bacterial IS elements listed in the GenBank and EMBL databases. IS407 had 49-bp terminal IRs with 18 mismatches. IR-R (right) contained an outwardly directed sigma 70-like promoter. IS407 was closely related to IS476 and ISR1 from Xanthomonas and Rhizobium sp., respectively. PMID- 1718820 TI - Two French genotypes of hepatitis C virus: homology of the predominant genotype with the prototype American strain. AB - A hepatitis C virus (HCV) cDNA covering part of the nonstructural region, NS3, was amplified from the serum of 50 out of 76 French non-A, non-B hepatitis patients by the nested polymerase chain reaction (PCR). Determination of a 407-bp sequence from four such cases revealed the presence of two different virus genotypes, F1 and F2, which exhibited 19-20% sequence divergence. F1 was represented by three of the four isolates and showed a sequence homology of about 97.5% to the prototype American HCV isolate, but of only 79% to a reported Japanese isolate. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype American HCV. PCR products from the 50 samples were hybridized with labeled F1 and F2 fragments under stringent conditions; results indicated the F1-related strain(s) as the major HCV genotype. Furthermore, a total of 1477 bp of sequence has been determined for one of the isolates belonging to the F1 category. These results will have implications for the PCR detection of HCV infection and production of HCV vaccines, especially for European countries. PMID- 1718821 TI - DNA-dependent RNA polymerase from bacteriophage T3 transcribes and amplifies an RNA template in vitro. AB - RNA-based amplification systems that have been recently described are dependent upon the presence of more than one enzyme. In an attempt to minimize the number of polymerases required for efficient amplification, we have studied the template specificity of bacteriophage T3 RNA polymerase. A synthetic bacteriophage T3 promoter was covalently attached to an RNA template. The T3 promoter-RNA complex was found to be selective for its native polymerase, and dependent upon the presence of all four ribonucleoside precursors. The product of the RNA-directed transcription is complementary to the initial template. PMID- 1718822 TI - Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell line. AB - We have isolated two novel serpin-encoding sequences from EB22, a chondrocytic cell line derived from a mouse teratocarcinoma. Both sequences fall within the Spi-2 sub-family, and are related to the gene encoding human alpha 1 antichymotrypsin (ACT), a major acute-phase reactant. Considerable amplification of the Spi-2 gene family in the mouse has occurred, hindering the identification of a functional equivalent of the human gene. However, one of the sequences described here, EB22/4, exhibits several features which indicate that it may represent the physiological rodent equivalent of ACT. The sequence is expressed in the liver, as expected, and is induced several-fold during the acute-phase response. The P1 amino acid residue, which is primarily responsible for inhibitor specificity, is Met rather than the human Leu, most probably a functionally conservative substitution. Analysis of the orthologous sequence in related rodents demonstrates conservation of the predicted reactive centre-encoded specificity. The second isolated cDNA, EB22/3, encodes an unexpected Cys residue at the P1 position in the reactive centre, and represents a novel sub-class of the Spi-2 serine proteinase inhibitor (serpin)-encoding gene family. At least one of the sequences appears to be expressed at sites of skeletal deposition during the later stages of mouse foetal development, indicating a role for serpins during development. PMID- 1718823 TI - Constipation prevention: empiric use of stool softeners questioned. PMID- 1718824 TI - Gel injection adjustable keratoplasty. AB - Gel injection adjustable keratoplasty (GIAK) is a new refractive surgical procedure designed for the correction of myopia by injection of a gel substance into the peripheral corneal stroma. This paper describes the GIAK technique and reports the results obtained in 21 fresh cadaveric eyes using the procedure. After a deep interlamellar canal has been dissected with a helicoid spatula surrounding the visual axis, the gel is injected under keratometric control. In the 1st group of 14 eyes, the degree of correction varied from 2.2 to 12.8 D; there was a direct relationship between the amount of gel injected and the keratometric change. In the 2nd group of 7 eyes, the adjustability of the procedure was demonstrated. Through partial extraction of the gel and subsequent modification of the corneal curvature, the previously induced keratometric changes could be reversed or altered to a specific extent. Following the initial injection of gel to a targeted flattest meridian power of 35 D, an average value of 35.8 +/- 0.5 D was achieved in these eyes. We subsequently attempted to increase the flattest meridian to 40 D by partial removal of the gel and achieved a mean value of 40.2 +/- 0.4 D. Average presurgical astigmatism of 1.497 +/- 0.737 D was reduced to a postsurgical reading of 0.941 +/- 0.590 D (P = 0.005, Student's paired t-test), indicating an autocorrection by autodistribution of the gel inside the canaliculus (Laplace's law). GIAK is a simple, inexpensive procedure designed for the correction of myopia that has the added advantage of reducing preexisting astigmatism without encroaching on the visual axis. PMID- 1718825 TI - Immunohistochemical localization of epidermal growth factor receptor in a human epiretinal membrane. AB - Cell growth in proliferative vitreoretinopathy (PVR) is activated by naturally occurring mitogenic substances; thus, growth factors seem to play an important role in PVR. Cell activation by growth factors requires the presence of specific cell-bound receptors by which the mitogenic signal is transmitted. Using the indirect immunoperoxidase method, we investigated frozen sections of 15 epiretinal membranes in PVR for the presence of epidermal growth factor receptor (EGFR), which is commonly utilized by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This receptor could be localized only in one epiretinal membrane. The receptor-bearing cells stained positively for vimentin, cytokeratin, and macrophage marker Ki-M7, indicating that they most probably correspond to transformed macrophages. They stained negatively for desmin and glial fibrillary acidic protein. The cells were embedded in a fibronectin-positive intercellular matrix and were in an actively proliferating state as demonstrated by positive staining for the nuclear proliferation marker Ki-67. We propose that EGFR might be expressed only in certain as yet undefined stages of PVR. PMID- 1718826 TI - A note on the inhibition of DT-diaphorase by dicoumarol. AB - The participation of DT-diaphorase or NAD(P)H:(quinone acceptor) oxidoreductase (E.C. 1.6.99.2) in metabolism or in events leading to toxicity is often implied on the basis of the inhibitory effects of dicoumarol. DT-diaphorase functions via a ping pong bi-bi kinetic mechanism involving oxidized and reduced flavin forms of the free enzyme. Dicoumarol, a potent (Ki = 10 nM) inhibitor, binds to the oxidized form of the enzyme, competitively versus reduced pyridine nucleotide. Inhibition is effectively complete at 1 microM dicoumarol in typical studies using DCPIP, one of the best known substrates for the enzyme, as electron acceptor. The antitumor quinone Diaziquone (AZQ) is a poor substrate for DT diaphorase relative to DCPIP, but effective inhibition of its reduction requires ten-fold higher concentrations of dicoumarol than for inhibition of DCPIP reduction under otherwise similar conditions. The variable inhibition of DT diaphorase by dicoumarol dependent on the efficiency of the electron acceptor can be explained on the basis of the complete rate equation describing its ping pong type kinetic mechanism. Thus, the concentration of dicoumarol used to inhibit DT diaphorase must be chosen carefully and consideration should be given to the efficiency of the electron acceptor. The absence of an inhibitory effect using low doses of dicoumarol cannot rule out a reaction mediated by DT-diaphorase. Although higher doses of dicoumarol may be required to inhibit DT-diaphorase mediated metabolism of less efficient electron acceptors, the use of such doses in cells may also affect biochemical processes other than DT-diaphorase and should be approached with caution. PMID- 1718827 TI - Mycosis fungoides: a therapeutical review. AB - In this review of current therapy for mycosis fungoides, the analytical data have been extrapolated from the literature. We have considered the various therapeutic modalities such as topical therapy, photochemotherapy, electron beam radiation therapy, systemic chemotherapy, the combined modalities, as well as the use of interferon and other alternative biological approaches. We conclude with a final section on comments and recommendations that may prove useful in the design of appropriate therapeutic strategies. PMID- 1718828 TI - [How long is a musculocutaneous microvascular transplant flap dependent on its vascular pedicle?]. AB - Two cases of open fractures of the lower leg with microvascular flap reconstruction for soft-tissue coverage illustrate the time span necessary for neovascularisation of the flap. In the first case, the supplying artery of the flap had to be ligated due to a septic aneurysm 17 days after operation. Because of sufficient blood circulation between the flap margin and the surrounding tissue, there was no ischemic damage to the flap. A similar observation was made in a second case, where more than one year postoperatively, venous congestion was managed with leeches. Careful consideration must be given to incisions of these microvascular flaps prior to any further surgical procedure, particularly if the traumatised region is badly vascularised and the flap consists only of a split thickness skin grafted muscle. PMID- 1718829 TI - Language rehabilitation of aphasics based on Pavlov's conditioned reflexes and signal systems. PMID- 1718830 TI - An enzyme-linked immunosorbent assay (ELISA) for prostate-specific antigen. AB - A sandwich ELISA for human prostate-specific antigen (PSA) is described. Optimal assay conditions, resulting in a sensitive assay with a low background, are presented. The method uses a hyperimmune antiserum produced in the New Zealand white rabbit, against human semen PSA. The IgG fraction of the antiserum was conjugated with horseradish peroxidase and used in the sandwich ELISA method. The anti-PSA IgG showed no cross reactions with saliva, normal blood, female urine, vaginal fluid, or menstrual blood. On occasions, a blood sample showed a non specific cross-reaction, which was detected by non-immune rabbit IgG. This reaction could be caused by rheumatoid factors, as indicated by experiments with a series of known IgG and IgM rheumatoid antibodies, although other heterophilic antibodies could not be eliminated. The recovery of PSA added to blood plasma, saliva and vaginal fluid was affected by three factors; (a) protein concentration (dilution) of body fluid; (b) nature of the protein; and (c) amount of PSA added. PMID- 1718831 TI - Identification of saliva stains by determination of the specific activity of amylase. AB - The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added. PMID- 1718832 TI - A case of fibrous hamartoma of infancy: an immunohistochemical study. AB - To clarify the histogenesis and immunohistochemical nature of fibrous hamartoma of infancy, we have studied a typical case of this disease using an immunohistochemical technique with a large panel of antibodies. It was found that immature-appearing stellate and spindle-shaped cells were mesenchymal in nature, but it was not ascertained whether those cells were differentiated into various specialized connective tissues corresponding with embryonic stages of connective tissue differentiation. These results reveal the immunohistochemical nature of tissue components of fibrous hamartoma but poorly support the previously described concept that fibrous hamartoma of infancy is a hamartomatous disease. PMID- 1718833 TI - Detection of proliferating liver cells in various diseases by a monoclonal antibody against DNA polymerase-alpha: with special reference to the relationship between hepatocytes and sinusoidal cells. AB - Proliferating cells in liver specimens from patients with various diseases were detected by use of a monoclonal antibody against human DNA polymerase-alpha, which is present in the nuclei of cells in the G1, S, M and G2 phases of the mitotic cell cycle and absent in the G0 phase, to clarify the kinetics and morphological characteristics of these cells. This monoclonal antibody was supernatant derived from clone CL22-2-42B, and the peroxidase antiperoxidase method was used. Not only epithelial cells (hepatocytes, biliary epithelial cells and hepatocellular carcinoma cells) but also nonepithelial cells (Kupffer cells and other macrophages, endothelial cells, fat-storing cells, lymphocytes and fibroblasts) were stained for DNA polymerase-alpha. In acute viral hepatitis with confluent necrosis, small hepatocytes with basophilic cytoplasm next to the necrosis accounted for most of the proliferating cells. In these areas, Kupffer cells and other macrophages and lymphocytes had often proliferated. Hepatocellular carcinoma cells were frequently stained for DNA polymerase-alpha, in addition to endothelial cells, macrophages and lymphocytes. These nonepithelial cells were stained more frequently in specimens with many stained carcinoma cells than in those with only a few cells stained. In fibrotic areas, fibroblasts were often stained for this enzyme. In proliferating bile ducts, both small epithelial cells and large mature cells were stained. The differences between stained and nonstained cells that were not hepatocytes could not be defined by their ultrastructural characteristics. From these findings, it seemed possible that sinusoidal cells, especially Kupffer cells and other macrophages, might be much involved in hepatocytic proliferation during regeneration of the liver and also in the occurrence of malignant tumors. PMID- 1718834 TI - Histochemical demonstration of sinusoidal gamma-glutamyltransferase activity by substrate protection fixation: comparative studies in rat and guinea pig liver. AB - Most histochemical methods for the detection of an enzymatic activity are preceded by tissue fixation with chemical agents that partially inactivate the enzymes. It is well known that substrates exert a marked protection against fixative-induced inactivation. The conventional histochemical methods for the demonstration of hepatic gamma-glutamyltransferase activity have not been successful in detecting the activity of the enzyme on the sinusoidal side of the hepatocytes despite mounting biochemical evidence for its presence on that pole of the hepatocyte. Under conventional fixation the enzymatic activity in hepatocytes is only seen on the bile canalicular side. This may be the result of a preferential protective effect of gamma-glutamyltransferase by its normal substrate, glutathione, present in the bile canaliculus at concentrations 500 times higher than in the sinusoidal lumen (8 mmol/L vs. 10 to 20 mumol/L). To test this hypothesis and to reduce the degree of fixative-induced inhibition of the enzyme activity, glutathione was either incorporated in the fixative solution or the livers were perfused with high concentrations of glutathione (10 mmol/L) before fixation. Our results histochemically demonstrate, in the normal adult rat liver, the existence of gamma-glutamyltransferase activity not only on the bile canalicular pole but also on the sinusoidal pole of the hepatocytes. Visualization of the enzyme activity on the sinusoidal pole is dependent on glutathione protection. Guinea pig livers, which present a 10-fold higher gamma glutamyltransferase activity than rat livers (similar to that in human beings), showed marked sinusoidal gamma-glutamyltransferase activity even in the absence of glutathione protection. Glutathione protection further increased this sinusoidal activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718835 TI - The role of capillarization in hepatic failure: studies in carbon tetrachloride induced cirrhosis. AB - During the cirrhotic process, the hepatic microvascular phenotype is transformed from sinusoids (discontinuous capillaries) into continuous capillaries. This transformation has been termed capillarization. Many hepatic functions depend on the rapid, bidirectional exchange of macromolecules between plasma and hepatocytes. To determine whether capillarization contributes to hepatic failure in cirrhosis, we decided to study the plasma clearance (125I) and hepatocyte uptake (electron microscopy) of three tracers in normal and cirrhotic rats. The tracers chosen were a hemeundecapeptide with peroxidatic activity (fluid-phase pinocytosis), asialofetuin (receptor-mediated endocytosis of a medium size protein) and ferritin (receptor-mediated endocytosis of a large size protein). The results demonstrate a decreased hepatocyte uptake of hemeundecapeptide; a significant delay in plasma clearance of asialofetuin; and a minor delay in plasma clearance of ferritin, but a striking trapping of ferritin in the cirrhotic capillary basement membrane. The delayed plasma clearance in cirrhosis cannot be ascribed to a decreased number of surface receptors because, in isolated hepatocytes, the number of molecules bound per cell was equivalent in normal and cirrhotic livers. These findings support the concept of capillarization, with the formation of continuous diffusion and filtration barriers between plasma and hepatocytes, representing a significant hindrance to the bidirectional macromolecular exchange normally taking place between these two compartments. Furthermore, at least in the case of ferritin, the capillary basement membrane of cirrhotic livers seems to be the major filtration barrier. This hindrance to hepatocyte uptake, and presumably also to secretion, may be the cause (or at least a major determinant) of the hepatic failure characteristic of cirrhosis. PMID- 1718836 TI - Dexamethasone modulates alpha 2-macroglobulin and apolipoprotein E gene expression in cultured rat liver fat-storing (Ito) cells. AB - Fat-storing (Ito) cells are perisinusoidal liver cells thought to play a central role in vitamin A metabolism and fibrongenesis. Glucocorticoids have been shown to be beneficial in the treatment of certain types of liver diseases by delaying the development of cirrhosis. To study the regulatory effects of dexamethasone on Ito cell gene expression, Ito cells were isolated from normal rat liver and primary cultures were established. The effect of dexamethasone on the synthesis of alpha 2-macroglobulin, apolipoprotein E, fibronectin and actin was examined. Protein synthesis was studied both at the protein level and at the RNA level by means of biosynthetic labeling, immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Northern blot analysis of total RNA. After exposure to dexamethasone for 20 hr, alpha 2-macroglobulin protein synthesis was increased threefold, whereas apolipoprotein E expression was decreased 80%. Biosynthesis of fibronectin remained unaffected by hormone treatment. The dexamethasone effect became detectable 5 hr after beginning the exposure. Deinduction kinetic experiments showed that the glucocorticoid effect was detectable more than 12 hr after the replacement of the dexamethasone containing culture medium by medium without the hormone. Corresponding to the data obtained at the protein level, dexamethasone increased the steady-state levels of alpha 2-macroglobulin-specific messenger RNA and reduced apolipoprotein E-specific transcripts, whereas fibronectin and actin messenger mRNA remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718839 TI - Useful HIV disease multimedia resources for hospices. PMID- 1718837 TI - A phase II trial of ProMACE-CytaBOM in previously untreated non-Hodgkin's lymphoma of intermediate- or high-grade histology. AB - Between November 1985 and June 1989 the aggressive combination chemotherapy programme ProMACE-CytaBOM was used at a community-based hospital as primary treatment for non-Hodgkin's lymphoma (NHL) of intermediate or high-grade histology in Ann-Arbor stages IB-IV. The 53 patients entering the study represented 90 per cent of all consecutive eligible patients with NHL diagnosed during the time period considered. Their median age was 54 years and median observation time was 36 months. Of 50 patients evaluable for response, 35 (70 per cent) achieved complete remission (CR), seven (14 per cent) partial remission, and five (10 per cent) were refractory. Treatment was given on an outpatient basis. Actually delivered drug doses ranged from 88 per cent to 97 per cent of the theoretical doses. Life-threatening toxicity was experienced by four patients. Treatment was stopped in three cases (6 per cent) because of toxicity and there was one treatment-related death. Actuarial 2-year disease-free survival of patients in CR was 73 per cent. Overall actuarial 3-year survival and disease free survival were 67 per cent and 51 per cent respectively. High LDH level was a significant adverse prognostic factor both for achievement of CR (P less than 0.005) and for survival (P less than 0.0002). Age was of no prognostic importance. We conclude that ProMACE-CytaBOM is an effective, easy to administer and well-tolerated regimen for patients with aggressive NHL. PMID- 1718838 TI - Silver staining of nucleolar organizer regions in prostatic lesions. AB - Variations in the number of silver-stained nucleolar organizer region-associated proteins (AgNORs) were studied in paraffin sections of 42 benign prostatic lesions, comprising four cases of granulomatous prostatitis, five of squamous or transitional metaplasia, eight of atypical and 25 of regular hyperplasia, and 37 of prostatic adenocarcinoma, with their metastases. There was a significant difference between the mean AgNOR counts of the benign and malignant prostatic lesions (1.58 +/- 0.26 v. 4.34 +/- 1.53; P less than 0.01). The mean AgNOR counts significantly increased with increasing Gleason's grade (P less than 0.01) and clinical stage (P less than 0.05) of the tumours. AgNOR counting may contribute to the conventional diagnostic and prognostic indices of cancer of the prostate. PMID- 1718840 TI - Issues in the current treatment of hospice patients with HIV disease. AB - Hospice administrators and clinicians face many complex issues regarding the treatment of persons terminally ill with AIDS, at one end of the spectrum of HIV disease. Among these issues are: whether the hospice model applies to persons with AIDS; at what point does the person with AIDS receive palliative rather than curative therapy; and what alternatives exist for hospice care if the AIDS patient has no primary care provider or no home in which to receive care. This article delineates and discusses these issues. PMID- 1718841 TI - The ITI system in South Koreans and Iranians analysed by an improved classification procedure. Distribution of alleles and description of "new" phenotypes. AB - Phenotype and gene frequency distributions of the inter-alpha-trypsin inhibitor (ITI) system were analysed in populations from southern Korea and from Iran. The gene frequencies of the common alleles ITI*I and ITI*2 were 0.532 and 0.422, respectively, in southern Korea, and 0.612 and 0.354, respectively, in Iran. The postulated third allele, ITI*3, was found in the homozygous form. Gene frequencies of this rare allele were calculated to be 0.042 and 0.029 in Korea and Iran, respectively. Two additional rare alleles, ITI*4 and ITI*5, determine further phenotypes found in the population from Taejon (Korea) and Iran, respectively, in combination with the common ITI*2 allele. Gene frequencies of ITI*4 and ITI*5 were calculated to be 0.006 and 0.005, respectively. For phenotype classification, untreated sera were separated by isoelectric focusing (IEF) on polyacrylamide gels followed by immunoblotting. PMID- 1718842 TI - A neutralizing monoclonal antibody binds to an epitope near the amino terminus of murine granulocyte-macrophage colony-stimulating factor. AB - A rat anti-murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) monoclonal antibody, A2, that neutralizes bioactivity in vitro was isolated. The binding epitope recognized by this antibody was identified using human-murine hybrid GM-CSF proteins. A2 was unable to immunoprecipitate a hybrid (hm7) protein containing the human GM-CSF sequence for the first 11 amino terminal amino acids, and the mGM-CSF sequence for amino acids 12-124. In contrast, A2 did recognize a hybrid which substitutes human GM-CSF amino acids 23-36 in the murine sequence. These data suggest that this neutralizing antibody recognizes an epitope at the amino terminus of mGM-CSF. Because hm7 did maintain in vitro bioactivity, it is probable that the epitope recognized by the neutralizing antibody is not itself part of the receptor-binding domain of mGM-CSF; rather, it is likely that neutralization occurs as a result of antibody binding near the receptor-binding site, with steric inhibition of mGM-CSF binding to its receptor. Interestingly, monoclonal antibody A2 does not recognize mGM-CSF glycosylation species corresponding to predicted maximal O-glycosylation variants. The presence of O glycosylation sites within the antibody-binding epitope was confirmed using site directed mutagenesis. Potential O-glycosylation sites in native mGM-CSF were removed by introducing conservative amino acid substitutions, and expected molecular weight reductions were obtained. These findings are consistent with previous reports that suggest the importance of the integrity of residues near the amino terminus to GM-CSF bioactivity. PMID- 1718843 TI - Monoclonal antibodies to sweet taste proteins. I. Analysis of antigenic epitopes on thaumatin by competitive inhibition assays. AB - Using a competitive inhibition binding immunoassay, we have examined some of the antigenic epitopes present on thaumatin, an intense sweet tasting protein from the African fruit katemfe. We have developed a library of monoclonal antibodies which react with different surface antigenic epitopes on thaumatin. Some of these monoclonal antibodies also cross-react with monellin, another unrelated sweet tasting protein. The competitive binding immunoassay examines the immunoreactivity of both solid-phase and liquid-phase monoclonal antibodies. At least six major antigenic epitopes on thaumatin were identified by our library of monoclonal antibodies. This type of competitive binding analysis may prove useful in the discovery of "sweet taste determinants" on plant proteins and in the development of tandem immunoassays for quantitation of sweet tasting proteins in plant extracts. PMID- 1718844 TI - Recognition of a single hsp-60 epitope by an entire subset of gamma delta T lymphocytes. AB - We can conclude that a large subset of gamma delta cells, present in both murine newborn thymus and in adult spleen, respond to the stress protein, hsp60. hsp60 seems to be stimulatory whether it is derived from a foreign pathogen such as mycobacteria, or whether it originates from the mouse's own cells. The gamma delta cells that respond to this antigen bear very similar receptors, all expressing V gamma 1 and most expressing V delta 6, although their junctional variations indicate that not all members of the subset stem from clonal expansion of only one or a few cells. The hsp60-reactive subset has not at this time been shown to "home" to an epithelial location, in contrast to other known gamma delta cell subsets, and may rather carry out its functions while in circulation. Whether the hsp60 antigen requires a "presenting" molecule remains at this point unclear, but because the gamma delta cells all respond to a synthetic peptide representing an epitope of hsp60, presentation is implied. Human gamma delta cells that respond to PPD from mycobacteria, as do the mouse hsp60-reactive gamma delta cells, have also been described, many as members of a major subset in peripheral blood, although only rarely have these been reported to respond to mycobacterial hsp60. The antigenic source in PPD for these cells has not yet been determined, but as for the mouse, a low molecular weight peptide appears to be sufficient for stimulation (P. Brennan and R. Modlin, personal communication). The PPD-reactive gamma delta cells, when their receptors have been characterized, have been found to express a V gamma 9+ chain. Some evidence indicates that these cells can also recognize self hsp60; hence, in several ways, this human subset has characteristics similar to the mouse hsp60-reactive subset. Perhaps gamma delta cells that respond to hsp60 play an important role, in both mice and humans, in the detection of transformed self cells or cells containing intracellular pathogens, that escape detection by alpha beta T cells. PMID- 1718845 TI - [Changes in the ions and protein concentrations and flow rate of parotid saliva by tongue sour stimulation]. AB - Changes in the concentrations of ions (K+, Cl-, Na+), total protein, amylase activity and flow rate of parotid saliva were observed by tongue stimulation with 3% tartaric acid various intervals in a subject after about one hour rest. The results may be summarized as follows: 1) After about one hour rest state, the values of each item were K+: 42-56 mEq/l, Cl-: 8-28 mEq/l, Na+: 1-10 mEq/l, total protein: 5.7-11 mg/ml, amylase activity: 2-6.5 x 10(3)U/ml and flow rate: 0.03 0.05 ml/min. 2) The changes were classified into two groups based on the recovery from peak to rest values. One group showed short recovery times (Cl-: 20 min., Na+: 8 min. and flow rate: 2 min.) and the other group had rather longer recovery times (K+: 40 min., total protein: 55 min. and amylase activity: 40 min.). The differences between these two groups were clear when the stimulus intervals were 30 sec. to 5 min. 3) When the tongue was stimulated every 15 sec. all items showed first responses, after that, the all values except the flow rate remained unchanged. 4) Different values in ions and protein concentrations and amylase activity were observed when the flow rate was similar before and after stimulation. 5) The recovery time of the responses of protein concentration and amylase activity to stimulation was about one hour, and a rest period of more than one hour is necessary to analyze the concentration of total protein and amylase activity in parotid saliva. PMID- 1718846 TI - Enhancement of lymphocyte-mediated K562 cytotoxicity by antibodies against complement membrane cofactor protein (CD46) and decay-accelerating factor (CD55). AB - A natural killer (NK) target, K562 (a human erythroblastoid cell line), was found by flow cytometry to express three complement regulatory membrane proteins on its surface: decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and p18 (CD59). We examined the effects of F(ab')2 of polyclonal antibodies raised against DAF and MCP and monoclonal antibody to p18 on the susceptibility of K562 to cytolysis by homologous lymphocytes, granulocytes and complement. C3bi deposition was induced on K562 when the cells were treated with both anti-DAF and anti-MCP. Lymphocyte-mediated K562 cytolysis was markedly enhanced by these two antibodies whereas anti-p18 barely affected the degree of cytolysis. Complement immunocytolysis, on the other hand, became highest by combination treatment with the three antibodies, although anti-p18 and either anti-DAF or anti-MCP induced little potentiation of cytolysis. Granulocytes showed the least cytolysis and minimal potentiation of lysis by treatment with both anti-DAF and anti-MCP. PMID- 1718847 TI - Failure of Met-enkephalin to enhance natural killer cell activity. AB - Several papers have reported the enhancing effects of opiate peptides, like Met enkephalin, on natural killer (NK) cell activity. We examined the actions of Met enkephalin on NK activity in blood obtained from 18 different donors, of different ages, many of them on several occasions, at several ratios of effector to target cells, several concentrations of peptide, in several types of flasks, with the purity and identity of the pentapeptide verified by chromatography, in a system responsive to interferon, and with results calculated in different ways. No significant increase was found for the peptide for any ratio of cells, any concentration of peptide, or any single subject, even when the subjects with the lowest baseline NK cell activity were used or when the subjects were more than 60 years old. Instead of an increase, the combined results for all subjects at all ratios at all concentrations of Met-enkephalin showed an overall decrease of 31.3% specific cytotoxicity. These results fail to support the reports of an enhancing effect of Met-enkephalin on NK cell activity. PMID- 1718848 TI - Characterization of B cells in human thymus. AB - The surface characteristics of B cells present in the human thymus were investigated. Cytofluorometrical and immunohistological studies, using anti-human IgM or anti-B cell monoclonal antibodies (mAbs; anti-Leu 12Ab, anti-Leu 16Ab, or L26), revealed that a small number of B cells are present in the human thymus. The thymic B cells were detected only in a low-density cell population, whereas in a high-density cell fraction, only T cells were found. In 15 cases, all of which the thymi were histologically normal, the percentages of B cells in the low density fraction were 0.28% to 50% (6.8% in average), and Leu 1+ (CD5+) B cells in the low-density fraction were 0.1% to 26% (3.5% in average); approximately 50% of the thymic B cells were Leu 1+ B cells. These results indicate that B cells, especially Leu 1 (CD5)+ B cells, are also present in the human thymus, as suggested from our previous reports on mice. PMID- 1718849 TI - Analysis of human T-cell epitopes in the 19,000 MW antigen of Mycobacterium tuberculosis: influence of HLA-DR. AB - The potential number of T-cell epitopes in the 19,000 molecular weight (MW) antigen has been investigated using overlapping peptides which comprise the complete sequence. Sixteen potential epitopes could be deduced from the responses to these peptides by polyclonal T cells derived from 22 antigen-responsive donors. The majority of epitopes were not predicted by either of the major paradigms, the Rothbard motif and the amphipathic helix. A hierarchy of epitopes was indicated by the responses, which ranged from strong and frequent in the N terminal region, to moderate or weak elsewhere. Some epitopes were restricted by single HLA-DR determinants, or families of determinants sharing structural features in common, whilst the two N-terminal peptides were recognized by donors with a diversity of DR types. The high degree of T-cell recognition of the N terminal region may be of relevance to the design of a sub-unit vaccine capable of priming T cells against Mycobacterium tuberculosis. PMID- 1718850 TI - Blood dendritic cells carry terminal complement complexes on their cell surface as detected by newly developed neoepitope-specific monoclonal antibodies. AB - Blood dendritic cells carry the terminal complement complex (TCC) on their surface, as detected by three monoclonal antibodies (mAb). Two of these mAb were generated by immunizing mice with the terminal complement complex, whereas the third was generated by immunizing mice with blood dendritic cells. All three mAb are directed against neoepitopes on the C9 molecule, as assessed by binding and blocking experiments and studies with several forms of denatured C9 and C9 depleted serum. Only one of these mAb binds to soluble polymerized C9. All three mAb allow the quantification of human TCC in sensitive ELISA procedures and could be used as markers for the evaluation of the functions of non-lytic TCC on dendritic cells. PMID- 1718851 TI - Junin virus-induced non-specific suppressor cells interact with unrelated antigen specific suppressor cells. AB - Junin virus (JV) infection of adult (resistant) BALB/c mice induces antigen non specific delayed-type hypersensitivity (DTH) suppressor T cells, termed Tsv, bearing the Thy-1+, Ly-1+2- phenotype. These cells may be related to survival to infection since DTH reaction is associated with lethal meningoencephalitis. Employing several xenogeneic red blood cell (RBC) sensitization schedules to induce different cell subpopulations, we have attempted to establish the target of JV-induced suppressor cells (Tsv). The target of Tsv cells was actually included in the antigen-specific suppressor cell compartment, as demonstrated for the RBC system. Tsv cells were able to trigger suppressor cells to act without loss of their specificity. The presence of two sets of sheep RBC-induced DTH suppressor cells bearing the Ly-1+2- and Ly-1-2+ phenotypes was disclosed in low (10(6))-dose sensitized mice. Both sets were simultaneously required by Tsv to achieve DTH suppression. In contrast, in high (10(8] SRBC-dose sensitized animals treated with cyclophosphamide (doses of 50 mg/kg), a single Ly-1-2+ suppressor cell was required. PMID- 1718852 TI - Autoantibody to heat-shock protein 90 can mediate protection against systemic candidosis. AB - Epitope mapping shows that patients recovering from systemic infection with Candida albicans produce antibodies against both fungal-specific and conserved epitopes of the heat-shock protein (hsp) 90. In a mouse model of systemic candidosis, mortality was halved by prior administration of sera from two infected patients containing antibodies to hsp 90. One of these patients had no other candidal antibodies detectable on immunoblotting. The protective effect was mediated by the immunoglobulin fraction of the immune serum. It was not observed with a normal human serum. A mouse monoclonal antibody raised against one of the conserved peptide epitopes suggested that an autoantibody to hsp 90 could mediate protection against systemic candidosis in the animal model. PMID- 1718853 TI - Protective effect of isoprinosine in genetically susceptible BALB/c mice infected with Leishmania major. AB - The effects of an immunopotentiating drug Inosine Pranobex (isoprinosine) were investigated in an experimental cutaneous leishmaniasis model. The highly susceptible BALB/c mice treated orally with isoprinosine developed significantly delayed onset of disease when infected with Leishmania major compared to untreated mice. The drug itself is not toxic to the parasite up to millimolar levels in vitro. The increase in resistance to L. major infection is accompanied by a marked decrease in the CD4+/CD8+ ratio and the leishmanial antigen-specific proliferative response of the spleen cells of isoprinosine-treated mice compared to untreated mice. There was a significant increase in the production of IFN gamma but a decrease in the secretion of IL-3 and IL-4 by the spleen cells of isoprinosine-treated mice in response to concanavalin A with or without L. major infection compared to untreated controls. There was, however, no significant difference in the level of IL-2 production by the spleen cells between mice with or without isoprinosine treatment. These data are consistent with the interpretation that isoprinosine potentiates the resistance to leishmanial infection by up-regulating the host-protective Th1 cells and down-regulating the disease-promoting Th2 cells or, alternatively, by increasing CD8+ T-cell function. PMID- 1718854 TI - In vivo effects of anti-idiotype on Plasmodium chabaudi infection in mice. AB - A polyclonal anti-idiotype was raised in rabbits following immunization with a murine monoclonal antibody which recognized a 250,000 MW antigen of Plasmodium chabaudi-infected erythrocytes. The monoclonal antibody, NIMP M23 (clone 3,) has been shown to protect mice against homologous parasite challenge. Following purification, the anti-idiotype was shown to bind only the immunizing idiotype and to recognize antigen-binding site-associated anti-idiotype. Mice primed with anti-idiotype and challenged with live parasites had an altered course of infection, with significant reduction in their peak parasitaemia levels. Anti idiotype priming did not induce an antigen-reactive antibody response in vivo but a population of T cells capable of proliferating in vitro to P. chaubaudi infected red cells was stimulated. These data are discussed in the context of possible idiotypic interaction in murine malaria. PMID- 1718855 TI - Murine anti-human IL-6 monoclonal antibody prolongs the half-life in circulating blood and thus prolongs the bioactivity of human IL-6 in mice. AB - Human interleukin-6 (hIL-6) injected into mice increases antigen-specific antibody production. In the present study, we examined the possibility that anti hIL-6 murine monoclonal antibody (MH-166) could neutralize this hIL-6 activity in vivo. Although MH166 completely neutralized hIL-6 activity in vitro, treatment in vivo with hIL-6 and MH166 combined unexpectedly increased both anti-dinitrophenyl (DNP) and anti-sheep red blood cell (SRBC) antibody production more than treatment with IL-6 alone did. To explain this phenomenon, the serum hIL-6 level was monitored following the MH166 administration. The hIL-6 level was significantly higher in the mice treated with both hIL-6 and MH166 than in the mice treated with hIL-6 alone during the 24 hr following hIL-6 administration. These results indicate that MH166 prolongs half-life of a xenogeneic hIL-6. PMID- 1718856 TI - Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus. AB - In a previous study, two murine T-cell hybridomas generated after immunization with infectious bronchitis virus (IBV) were shown to be responsive to the internally localized viral nucleocapsid protein. In the present study, the antigenic determinants were mapped using recombinant expression products and synthetic peptides. Both hybridomas recognized the region spanning amino acid residues 71 to 78 of the nucleocapsid protein. The experimentally determined epitope corresponded with predicted motifs. Both an I-Ed binding motif and a predicted cleavage site for the aspartyl protease cathepsin D were contained within the sequence. The epitope was shown to prime cellular immune responses to IBV in the chicken. PMID- 1718857 TI - An HLA-DR alpha promoter DNA-binding protein is expressed ubiquitously and maps to human chromosomes 22 and 5. AB - The class II major histocompatibility complex antigens are a family of integral membrane proteins whose expression is tissue-specific and developmentally regulated. Three consensus sequences, X1, X2, and Y, separated by an interspace element, is found upstream from all class II genes. Deletion of each of these sequences eliminates expression of class II genes in vitro or in transgenic mice. Here we further characterize the expression of a cDNA encoding a DNA binding protein (human X-box binding protein, hXBP-1) which, like the proteins in whole nuclear extract, recognizes both the X2 promoter element of the human DR alpha and DP beta and mouse A alpha genes. The hXBP-1 cDNA hybridizes to human RNA species of approximately 2.2 kilobases (kb) and 1.6 kb, which are expressed in class II negative as well as class II positive cells. hXBP-1 transcripts are present in several class II deficient mutant B cell lines, although in one such line, 6.1.6, levels were somewhat reduced. Chromosome mapping studies demonstrate that hXBP-1 arises from a small gene family, two of whose members map to human chromosomes 5 and 22. Taken together, these data suggest a high degree of complexity in the transcriptional control of the class II gene family. PMID- 1718858 TI - A role for the interferon response DNA sequence in directing transcription of the T18d Tla gene. AB - T18d of BALB/c mice is a member of the Tla category of class I genes of the major histocompatibility complex of the mouse and is highly restricted in expression. Deletion analysis implies that an element essential to T18d expression resides within the region -4 to +54. The homologous region of T3d, a Tla gene which normally is not expressed in BALB/c mice, also has promoter activity. Thus the expressibility of T18d vs T3d is unlikely to be due to sequence differences in this region. A DNA-binding protein, factor VI, was found to bind to the region 33 to +54. DNase I footprinting analysis indicated that the DNA fragment 5' ACTATAGTTTCACTTTTT-3' (+3 to +20) was protected by factor VI. This region includes the interferon response sequence (IRS). Homologous DNA segments of other class I genes, Ld and Dd, competed for factor VI in DNA-protein binding assay with lower affinity as compared with T18d. In mutation analysis, the 3' portion of the IRS is more important than the 5' portion with respect to binding affinity of factor VI and to transcriptional activity in transfected cells. This result signifies a role of IRS in T18d transcription and suggests that the mechanism of T18d transcription might be unusual. PMID- 1718859 TI - Gomori-positive astrocytes: biological properties and implications for neurologic and neuroendocrine disorders. AB - Granule laden astrocytes exhibiting an affinity for chrome alum hematoxylin and aldehyde fuchsin (Gomori stains) have been described in the periventricular brain of all terrestrial vertebrate species examined to date including humans. The astrocytic inclusions are rich in sulfhydryl groups, emit an orange-red autofluorescence, and stain intensely with diaminobenzidine, a marker of endogenous peroxidase activity. The distinct autofluorescence pattern and the absence of neutral lipid, acid phosphatase, and beta-glucuronidase activity exclude lipofuscin or lysosomes as components of these astrocytic granules. The emission of orange-red autofluorescence and the nonenzymatic nature of the peroxidase activity implicate the presence of porphyrins and metalloporphyrins such as heme as major constituents of these cytoplasmic gliosomes. The role of Gomori-positive astrocytes under normal and pathologic conditions is incompletely understood. In vivo, numbers of astrocytic granules increase as a function of advancing age, in response to chronic estrogen stimulation, and following X irradiation. In vitro, these cells accumulate with increasing time in culture and following exposure to the sulfhydryl agent, cysteamine. Gomori-positive astrocytes may supply heme to neurons for the synthesis of cytochromes, catalases, and other heme enzymes. They may play a role in photostimulation of sexual cyclicity, the promotion of neuritic development, the degradation of toxic lipoperoxides, and the metabolism of various neurotransmitters. Conversely, these cells may contribute to the pathogenesis of several neurologic and neuroendocrine disorders. Examples of the latter include a) augmentation of goldthioglucose neurotoxicity, b) induction of hypothalamic anovulation and reproductive failure, c) exacerbation of porphyric encephalopathy, and d) potentiation of parkinsonism and other free radical-related neurodegenerations. PMID- 1718860 TI - FGFs stimulate IGF binding protein synthesis without affecting IGF synthesis in rat astroblasts in primary culture. AB - The production of insulin-like growth factor (IGF)-I and -II and their binding proteins (BPs) has been studied in new-born rat astroblasts at confluency in primary culture. Under the influence of fibroblast growth factors (FGFs) (acidic and basic), the morphology of the astroblasts was altered, 125I-deoxyuridine incorporation was increased, and glutamine synthetase activity was stimulated. IGF production and IGF mRNA expression remained unchanged. Production of the 32 kDa BP (IGFBP-2), the sole or predominant form under base-line conditions, was enhanced and the 43-39 kDa forms (IGFBP-3) appeared or were increased. Epidermal growth factor (EGF) also stimulated production of these BPs, whereas thrombin and db-cAMP had no effect. Our data suggest that a relationship exists between FGF induced maturation of astroblasts and the forms of BP they produce. The data also indicate that some factors may act specifically on BP synthesis, without affecting IGF synthesis, and in this way play a role in regulating the bioavailability of the IGFs. PMID- 1718861 TI - Glial fibrillary acidic protein in the fish optic nerve. AB - The intermediate filament glial fibrillary acidic protein (GFAP) is the predominant cytoskeletal protein of mature glial cells in the mammalian nervous system. The nervous systems of lower vertebrates, such as fish, have been examined for the presence of GFAP and several investigators have shown that goldfish (Carassius auratus) brain contains GFAP-positive astrocytes. The same studies have demonstrated that, in contrast to the brain, the optic nerve of goldfish did not show any GFAP immunoreactivity, suggesting that this intermediate filament protein is not expressed in fish optic nerve astrocytes. The present study shows, however, that the monoclonal antibodies to porcine GFAP react with the optic nerve of carp (Cyprinus carpio), another member of the goldfish family. These antibodies to porcine GFAP cross react with rat brain and carp optic nerve, yielding a band of approximately 52 kDa in both species. Northern blot analysis using mouse GFAP DNA probe revealed that carp optic nerve RNA contains two transcripts of 2.3 and 2.1 kb, which hybridize with the mouse GFAP probe. Injury to the carp optic nerve was followed by a decrease of GFAP immunoreactivity from neural tissue and a strong expression around blood vessels and connective tissues. On the basis of these observations and within the limitation of the techniques it is reasonable to conclude that the carp optic nerve expresses GFAP immunoreactivity and that the pattern of expression of this intermediate filament protein is altered after injury. Such an alteration might be relevant to the process of regeneration. PMID- 1718862 TI - Modulation of intracellular Ca++ in cultured astrocytes by influx through voltage activated Ca++ channels. AB - Fura-2 and indo-1 fluorescence measurements were used to examine intracellular Ca++ concentration ([Ca++]i) and its modulation by voltage-activated influx in murine cortical astrocytes in primary cell culture. Extracellular K+ was increased from 5 to 50 mM to depolarize cells to determine if Ca++ influx through voltage activated Ca++ channels could alter [Ca++]i. In confluent 4 to 6 weeks in vitro astrocyte cultures 50 mM K+ increased [Ca++]i 3-4-fold (from 150 nM up to 550 nM); this increase was blocked by nifedipine and enhanced by BayK 8644 indicating that influx was through L-type channels. However, in 1 to 2 weeks in vitro astrocyte cultures, high K+ reduced [Ca++]i. L-type channels were apparently present in these cells because high K+ in combination with BayK 8644 increased [Ca++]i. Following pretreatment of 1 to 2 weeks in vitro astrocytes with dibutyryl cAMP (dbcAMP) high K+ increased [Ca++]i in the absence of BayK 8644 indicating enhanced activity of Ca++ channels in agreement with previous voltage-clamp studies. Ca++ influx through voltage-activated channels in cultured cortical astrocytes can substantially increase [Ca++]i and these channels can be dynamically modulated by dihydropyridines. Immature astrocytes may express 'silent' or inactive Ca++ channels or have a much lower number of channels. PMID- 1718863 TI - Stretch-activated channels in human retinal Muller cells. AB - The cell-attached and excised patch configurations of the patch clamp technique were used to study stretch-activated ion channels in Muller glial cells that were obtained from postmortem adult human retinas and were maintained in culture. A stretch-activated channel permeable to monovalent and divalent cations was found. Ion channels of this type have not been demonstrated previously in glial cells, though indirect evidence has suggested that such stretch-activated channels may help mediate a compensatory response of glia to swelling. Consistent with a possible role for volume regulation is the finding that the activation of calcium permeable stretch-sensitive channels is associated with an increase in the activity of calcium-activated potassium channels. Activation of potassium channels to produce an efflux of potassium with a subsequent loss of anions and cell water could be an effective mechanism to decrease glial cell volume. PMID- 1718864 TI - Ion channels in cultured adult human Schwann cells. AB - Single channel currents have been recorded from cultured adult human Schwann cells. In both cell-attached and -excised (inside-out) patches, openings from a high-conductance (360 pS) channel were observed; measurements of the zero-current potential indicated that the channel was predominantly selective for chloride. Depolarizing and hyperpolarizing voltage steps activated the anion channel, which subsequently reverted to a closed state even in the presence of the maintained step. A second channel, with a conductance near 20 pS and with a current amplitude that increased with patch hyperpolarization, passed inward K+ currents in both cell-attached and inside-out patches. The mean open times for this channel were near 20 ms at the cell resting potential and decreased with patch hyperpolarization. The presence of these anion and cation selective channels in the human Schwann cell membrane would be consistent with a role for the cells in the regulation of extracellular K+. PMID- 1718865 TI - Technologies to improve weaning foods in developing countries. PMID- 1718866 TI - Growth faltering and developmental delay in children with PEM. AB - Anthropometric measurements, Somatic Quotient (SQ), Development Quotient (DQ), Motor Quotient (MoQ) and Mental Quotient (MeQ) in 136 children in the age group 1 24 months with varying degrees of protein energy malnutrition (PEM) were compared with an equal number of comparable well nourished children. There was a progressive reduction in SQ, DQ, MoQ and MeQ as the degree of PEM advanced. There was a direct linear correlation between SQ and DQ and between height and DQ in 4 degrees PEM. However, there was no direct correlation between head circumference and either DQ or MeQ. PMID- 1718867 TI - Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae. AB - The production of diphtheria toxin and siderophore by the Corynebacterium diphtheriae regulatory mutant C7(beta)hm723 is resistant to the inhibitory effects of iron, and the mutant strain is defective for function of the regulatory gene dtxR. A 2.8-kb HindIII fragment carrying the C7(beta)hm723 dtxR allele was cloned and characterized in Escherichia coli. The restriction endonuclease maps of the 2.8-kb HindIII fragment from C7(beta)hm723 and the corresponding fragment from wild-type C. diphtheriae C7 were identical. RNA dot blot analysis with total RNA isolated from wild-type C. diphtheriae C7 and C7(beta)hm723 indicated that the dtxR gene was transcribed at very low but equivalent levels in both strains and was not regulated by iron. beta Galactosidase synthesis from a tox-lacZ translational fusion construct in E. coli in high-iron medium was not repressed by the C7(beta)hm723dtxR allele, but was strongly repressed by the wild-type dtxR gene. The 28- to 29-kDa polypeptide expressed from the mutant dtxR allele in E. coli had the same electrophoretic mobility as the wild-type dtxR gene product in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nucleotide sequence of the coding region and the 5' upstream region of the C7(beta)hm723 dtxR allele was determined and compared with the wild-type nucleotide sequence. The dtxR allele from C7(beta)hm723 contained a single-base change located 140 nucleotides from the 5' start of the gene, which resulted in replacement of arginine in the wild-type sequence by histidine in the mutant protein. These data demonstrate that C7(beta)hm723 expresses a mutant DtxR repressor protein that is severely defective in repressor activity. PMID- 1718868 TI - Relatedness of O-specific lipopolysaccharide side chain genes from strains of Shigella boydii type 12 belonging to two clonal groups and from Escherichia coli O7:K1. AB - The O-specific lipopolysaccharide side chains of Escherichia coli O7 and Shigella boydii type 12 possess similar but not identical chemical structures. We investigated the genetic relatedness between the O-specific side chain genes in members of these two species. Examination of outer membrane protein and lipopolysaccharide (LPS) banding patterns demonstrated that five strains which had been identified as S. boydii type 12 fell into two clonal groups, SB1 and SB2. Hybridizations with O7-specific radiolabeled probes derived from the chromosomal DNA of an E. coli O7 strain detected identical fragments among the three SB1 strains of S. boydii type 12 and the two E. coli O7 reference isolates. The two other S. boydii type 12 strains, which belonged to the SB2 clone, did not show homologies with the O7 probe under high-stringency conditions of hybridization. The homology between the O7 and type 12 LPS gene regions from the SB1 strains was further confirmed by the construction of O-specific side chain deficient mutations in these strains by homologous recombination of a suicide plasmid containing O7-specific DNA sequences. Immunoblot experiments with O7 antiserum gave a weak cross-reaction with LPS purified from the SB2 strains but a very strong cross-reaction with the LPS from SB1 isolates. Antiserum raised to one of the SB2 strains cross-reacted only with S. boydii type 12 LPS from the SB1 clone but failed to react with O7 LPS. PMID- 1718869 TI - Comparative susceptibility to mouse interferons of Rickettsia tsutsugamushi strains with different virulence in mice and of Rickettsia rickettsii. AB - Three strains of Rickettsia tsutsugamushi (Karp, Gilliam, and TA716, representing three virulence types in mice) were examined for their sensitivity to the inhibitory effects of recombinant gamma interferon (IFN-gamma) and purified IFN alpha/beta in two cultured mouse fibroblast cell lines. The susceptibilities of another species, Rickettsia rickettsii, and of encephalomyocarditis virus (EMCV) were also tested for comparative purposes. IFN-gamma inhibited rickettsial replication in only one of the six combinations of R. tsutsugamushi strains and mouse cells (strain Gilliam and the BALB/c mouse-derived cell line). In contrast, R. rickettsii and EMCV replication were markedly inhibited in both cell types, but to a greater extent in the BALB/c line than in the C3H cells. IFN-alpha/beta (300 to 450 U/ml) was uniformly ineffective in three of the combinations of R. tsutsugamushi strains and mouse cells (Gilliam in C3H cells and Karp in both C3H and BALB/c cells); in the remaining sets, IFN-alpha/beta-mediated inhibition of rickettsial replication was variable and in no case was it very pronounced. The tests with R. rickettsii in both cell types also indicated slight, variable sensitivity to IFN-alpha/beta. EMCV, on the other hand, was very susceptible to IFN-alpha/beta, confirming the potency of the preparation used; as with IFN gamma, virus replication was inhibited to a greater degree in the BALB/c cell line than in the C3H cultures. These results are discussed in terms of their relationship to the virulence properties of the R. tsutsugamushi strains in BALB/c and C3H mice and to the known IFN-sensitivities of the more widely studied Rickettsia prowazekii. PMID- 1718870 TI - Functional and structural mapping of Chlamydia trachomatis species-specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies. AB - Three monoclonal antibodies (MAbs), E4, L1-4, and L1-24, to the major outer membrane protein (MOMP) of Chlamydia trachomatis were identified that neutralized in vitro the infectivity of members of the B- and C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, L1-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by the technique of pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VD IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and L1-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine. PMID- 1718871 TI - Expression of adhesion molecules in leprosy lesions. AB - Leprosy presents as a clinical spectrum that is precisely paralleled by a spectrum of immunological reactivity. The disease provides a useful and accessible model, in this case in the skin, in which to study the dynamics of cellular immune responses to an infectious pathogen, including the role of adhesion molecules in those responses. In lesions characterized by strong delayed type hypersensitivity against Mycobacterium leprae (tuberculoid, reversal reaction, and Mitsuda reaction), the overlying epidermis exhibited pronounced keratinocyte intracellular adhesion molecule 1 (ICAM-1) expression and contained lymphocytes expressing the ICAM-1 ligand, LFA-1. Conversely, in lesions in which delayed-type hypersensitivity was lacking (lepromatous), keratinocyte ICAM-1 expression was low and LFA-1+ lymphocytes were rare. Expression of these adhesion molecules on the cells within the dermal granulomas was equivalent throughout the spectrum of leprosy. The percentage of lymphocytes in these granulomas containing mRNA coding for gamma interferon and tumor necrosis factor alpha, synergistic regulators of ICAM-1 expression, paralleled epidermal ICAM-1 expression. In lesions of erythema nodosum leprosum, a reactional state of lepromatous leprosy thought to be due to immune complex deposition, keratinocyte ICAM-1 expression and gamma interferon mRNA+ cells were both prominent. Antibodies to LFA-1 and ICAM-1 blocked the response of both alpha beta and gamma delta T-cell clones in vitro to mycobacteria. Overall, the expression of adhesion molecules by immunocompetent epidermal cells, as well as the cytokines which regulate such expression, correlates with the outcome of the host response to infection. PMID- 1718872 TI - Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells. AB - Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding. PMID- 1718873 TI - Mapping T-cell epitopes in group A streptococcal type 5 M protein. AB - Group A streptococcal cell surface M proteins elicit highly protective, serotype specific opsonic antibodies and many serotypes also elicit host cross-reactive antibodies, which may contribute to the pathogenesis of poststreptococcal autoimmune disease. To date, studies aimed at designing safe (non-host-cross reactive, defined-epitope) M vaccines have focused almost exclusively on antibody epitopes. Here we identify T-cell epitopes recognized by T cells from BALB/c, C57BL/6, and CBA/Ca mice immunized with purified, recombinant serotype 5 M protein (rM5). The responses of rM5-specific, major histocompatibility complex class II-restricted, T-cell clones to synthetic peptides representing most of the M5 sequence identified at least 13 distinct T-cell recognition sites, including sites recognized by more than one major histocompatibility complex haplotype of mice. Although none of these sites appeared to be strongly immunodominant, an N terminal peptide, sM5[1-35], was recognized by lymph node T cells of rM5 immunized mice and by a larger proportion of rM5-specific T-cell clones than any other individual peptide. The fine specificity of these clones was mapped with subpeptides to a single site at or overlapping the sequence ELENHDL at residues 21 to 27, which is in close proximity to previously mapped protective antibody epitopes. Other T-cell recognition sites are distributed throughout the M protein and include several in the highly conserved C-terminal region of the molecule. One of these C-terminal sites, located within residues 300 to 319, was recognized by a significant proportion of T-cell clones from two strains of mice. Helper T cell epitopes located in the C-terminal region of M5 are likely to be widely conserved between different M serotypes and could be particularly useful in designing multivalent, defined-epitope M vaccines. PMID- 1718874 TI - Epitope mapping of the alpha-toxin of Clostridium perfringens. AB - A panel of monoclonal antibodies specific for the Clostridium perfringens alpha toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin. PMID- 1718875 TI - Distinct pattern of antibody reactivity with oligomeric or polymeric forms of the capsular polysaccharide of Haemophilus influenzae type b. AB - The chain length of oligosaccharides required for antibody binding has been studied by using the capsular polysaccharide from Haemophilus influenzae type b or oligosaccharides derived from it. The concentration of competing antigens required to achieve a 50% inhibition of antibody binding by human polyclonal antisera in an in vitro competition enzyme-linked immunosorbent assay decreased progressively from greater than 10(-3) to 5 x 10(-7) M as the inhibiting saccharide chain length increased from 1 to 262 repeat units. Even small oligosaccharides (one or two repeat units) are potentially capable of competing to a significant level if a high enough concentration of saccharides is used. A similar pattern of reactivity was seen with a monoclonal anti-polyribosyl ribitol phosphate antibody, suggesting that the differences in the avidity of the antibody subpopulations in the polyclonal antisera do not contribute to the binding patterns observed. The binding reaction was specific as evaluated with pneumococcal saccharides. Furthermore, an oligosaccharide-protein conjugate binds antibody better than the free oligosaccharides do. Such a difference in binding was not observed between the polysaccharide and a polysaccharide-protein conjugate. Overall, the data suggest that identical epitopes are expressed by oligomeric and polymeric forms of the antigen and that a particularly more stable conformation in polysaccharides is preferred by antibodies. Covalent coupling of oligomers to protein increases the expression of stable conformation of epitopes. The data further suggest that this kind of antigenic analysis may be important for the design and synthesis of glycoconjugate vaccines. PMID- 1718876 TI - Identification of a virulence-associated antigen of Toxoplasma gondii by use of a mouse monoclonal antibody. AB - A monoclonal antibody generated against the mouse-lethal RH strain of Toxoplasma gondii was developed. Tachyzoites of virulent and avirulent T. gondii isolates grown in permanent macrophage cell cultures were examined for differences in reactivity with this antibody. Virulence of these Toxoplasma isolates was quantified by injecting different numbers of tachyzoites into NMRI mice and observing the animals for signs of infection or death. The monoclonal antibody identified a 23-kDa antigen expressed by the mouse-lethal strains BK and RH, whereas this antigen was not detected in low-mouse-virulent strains, which were all clinical isolates from Europe. Using Western blot (immunoblot), immunofluorescence, and immunoelectron microscopy, we localized the 23-kDa antigen to the membrane compartment. From these results, we suggest that this 23 kDa antigen is a marker of strain virulence upon which a virulence classification of T. gondii may be based. PMID- 1718877 TI - Interactions of Neisseria gonorrhoeae with human neutrophils: studies with purified PII (Opa) outer membrane proteins and synthetic Opa peptides. AB - We investigated the role of gonococcal outer membrane protein PII (also called Opa protein) in nonopsonic adherence to human neutrophils. Gonococcal outer membranes, purified Opa in detergent (Opa), purified Opa in liposomes (Opa+ lips), and peptides composing the second hypervariable (HV2) region of OpaB (strain FA1090) in liposomes (pepHV2 lips) were tested for their abilities to inhibit subsequent gonococcal adherence to human neutrophils. Outer membranes from gonococci possessing adherent Opa, liposomes containing adherent Opa, purified adherent Opa, and two of three liposome preparations (pepHV2 lips) containing peptides from the HV2 region of an adherent Opa inhibited subsequent adherence to neutrophils of homologous Opa+ gonococci. On the other hand, outer membranes from Opa- gonococci, outer membranes containing a nonadherent Opa (OpaA from strain FA1090), purified OpaA, and OpaA lips had little or no inhibitory effect. Outer membranes containing adherent Opas, purified adherent Opas, and liposomes containing such Opas all bound to neutrophils, whereas preparations containing OpaA or no Opa protein did not. The results indicate that (i) Opa proteins can bind to neutrophils in a partially purified or purified form and (ii) the HV2 region of Opa appears to at least partially mediate Opa's biological role. PMID- 1718878 TI - Immunopathology of experimental African sleeping sickness: detection of cytokine mRNA in the brains of Trypanosoma brucei brucei-infected mice. AB - The immunopathology of Trypanosoma brucei brucei in the central nervous system was studied by using an experimental model of chronic meningoencephalitis in outbred CD-1 mice. Mice infected with T. b. brucei were treated with a subcurative dose of the trypanocidal compound diaminazine aceturate. These mice relapsed and were again drug treated. The brains were examined histologically and by immunocytochemistry to identify activated astrocytes. The polymerase chain reaction was used to detect cytokine RNA transcripts. The infected and treated animals developed severe chronic meningoencephalitis characterized by large numbers of inflammatory cells and widespread astrocyte proliferation. In uninfected controls, only interleukin 1 and beta-actin RNA transcripts were detected while transcripts for beta-actin, tumor necrosis factor alpha, macrophage inflammatory protein 1 and interleukins 1 and 4 were demonstrated in the brains of infected animals. Gamma interferon and interleukin 6 were also detected in a few of the infected animals, but interleukin 2 was not found in any of these animals. PMID- 1718879 TI - Comparison of immune repertoires of Chinese and Philippine patients infected with Schistosoma japonicum. AB - Chinese and Philippine patients exhibited detectable titers of serum antibodies to Schistosoma japonicum worm and egg antigens. Western blot (immunoblot) data revealed differing antigenic profiles. Antigen inhibition studies showed low and high levels of cross-reactivity with anti-worm and anti-egg antibodies, respectively, derived from both Chinese and Philippine patients. Thus, anti-egg antibodies and egg antigens are more conserved than anti-worm antibodies and worm antigens. PMID- 1718880 TI - Enhanced resistance against Listeria monocytogenes achieved by pretreatment with granulocyte colony-stimulating factor. AB - Phagocytosis, H2O2 production, Mac-1 expression, and in vivo elimination of Listeria monocytogenes were enhanced in granulocyte colony-stimulating factor (G CSF)-treated mice. Transfer of polymorphonuclear leukocytes prolonged survival of mice infected with a lethal dose of L. monocytogenes. G-CSF augments the functions of polymorphonuclear leukocytes and thus plays a role in protection. PMID- 1718881 TI - Early development of mast cells. AB - Mast cells originate from pluripotential cells in the bone marrow. Specifically, human mast cells originate from CD 34-positive progenitor cells. Mast cell proliferation requires IL-3. In the mouse, additional mast cell growth is achieved by the addition of IL-4, and GM-CSF prevents mast cell proliferation. Early bone-marrow-derived mast cells can be identified by their IgE receptors, although they may not yet have the characteristic morphology of mature mast cells. Whether these early cells may by themselves have a physiologic role, remains to be determined. Mast cells persist in culture on fibroblast monolayers, in part due to the production of soluble factor(s) from the fibroblasts themselves. Final mast cell phenotype appears dependent upon the local tissue environment. PMID- 1718882 TI - Histamine-releasing factors and inhibitory factors. AB - Peripheral-blood mononuclear cells (MNC) synthesize several histamine-releasing factors (HRF) spontaneously and when stimulated. Some of the characterized cytokines have histamine-releasing activity, especially connective tissue activating peptide III, neutrophil-activating peptide 2 and interleukin-3 (IL-3). At least two species of HRF remain to be characterized. MNC also secrete a histamine release-inhibitory factor (HRIF), which is a highly specific inhibitor, because it antagonizes only HRF. IL-8 resembles the low-molecular-weight species of HRIF in terms of size and specificity. PMID- 1718883 TI - IgE-dependent histamine-releasing factors. A brief review. AB - A cytokine, termed histamine-releasing factor (HRF) and produced by many cell types, has become the focus of research by many investigators due to its potential importance as a stimulus in chronic inflammation. We are producing and characterizing an HRF which causes IgE-mediated histamine release from human basophils. Following extensive purification procedures, the molecule will be sequenced and synthesized. A functional heterogeneity of IgE molecules was revealed by these studies. We are currently producing IgE antibody in vitro and testing the hypothesis that differential glycosylation is the basis for the heterogeneity. Knowledge of the structures and interactions of these molecules should advance our understanding of allergic and more chronic diseases. PMID- 1718884 TI - Histamine-releasing factors. AB - Histamine-releasing factors (HRF) are cell-derived products which cause histamine release from basophils and/or mast cells. We have isolated HRF from human mononuclear cells and platelets and have purified 3 molecular species having molecular weights of 8-10, 15-17 and 35-41 kilodaltons (kDa). We prepared monoclonal antibodies to the 8- to 10-kDa form and have isolated it by affinity chromatography. A broad band was seen upon sodium dodecyl sulfate gel electrophoresis in 15% gels as well as immunoblotting, and the band was divided into an upper and a lower half. Amino acid sequence analysis of the upper half indicated that it is closely homologous to connective-tissue activating peptide III (CTAP III). The lower half also aligned with CTAP III beginning with amino acid 16; thus, proteolysis and occurred removing the N-terminal 15 amino acids. This corresponds to neutrophil-activating peptide 2. Both appear to be active on basophils with a dose-response between 250 ng up to 10 micrograms. Although interleukin-3 and granulocyte/macrophage-colony-stimulating factor have similar histamine-releasing capability at lower effective concentrations, they do not account for HRF activity in mononuclear cell/platelet supernatants, and the 15- to 17 and 40- to 41-kDa moieties appear to be unique gene products unrelated to previously described cytokines. PMID- 1718885 TI - The characteristics of antigen define the cellular source and kinetics of histamine-releasing factor induced by human peripheral-blood mononuclear cells. AB - Peripheral-blood leucocytes from Dermatophagoides pteronyssinus- and/or Lolium perenne-allergic patients produce in vitro histamine-releasing factor (HRF) in an allergen-specific and concentration-dependent relationship. Maximum production of HRF occurred in cultures containing as little as 10-100 pg/ml crude allergen extract, although a second peak occurred at higher concentrations (10-100 micrograms/ml). While HRF was detectable in 1-hour cultures, maximal production required 24 h in culture. In contrast, HRF induced by streptokinase/streptodornase (SK/SD) was generally maximal after 1 h. Allergen induced HRF production was almost exclusively associated with monocytes and B cells. In contrast, peripheral-blood T cells were the major source of induced HRF production in cultures containing SK/SD. Histamine-releasing cytokines are apparently produced by different cell populations, the activation of which may be dependent upon the characteristics of the stimulating antigen. PMID- 1718886 TI - Regulation of mediator release by human basophils: importance of the sequence and time of addition in the combined action of different agonists. AB - Biologically active molecules affecting basophil function can be divided into 4 groups according to their capacity to induce basophil degranulation and/or leukotriene generation: (1) full agonists such as anti-IgE or fMLP, which induce both histamine and leukotriene release; (2) partial agonists such as C5a, which induces degranulation only; (3) incomplete agonists such as neutrophil-activating peptide-1, platelet-activating factor or C3a, which induce mediator release only after cytokine preincubation, and (4) basophil response modifiers, such as interleukin-3, interleukin-5 and granulocyte/macrophage- colony-stimulating factor, which (a) enhance the releasability to all basophil agonists, (b) change the mediator profile, (c) enhance the rate of mediator release, (d) render basophils responsive to lower agonist concentrations and (e) render basophils responsive to incomplete agonists. We demonstrated that histamine release and de novo synthesis of lipid mediators are clearly separately regulated, and that combined actions of different molecules are of importance. In particular, the type(s), the time interval and the sequence of action of basophil-activating molecules are crucial for the final outcome of the basophil release reaction. PMID- 1718887 TI - Use of antibodies as carriers for T-cell epitopes. PMID- 1718888 TI - Effect of nitric oxide generators on ischemia-reperfusion injury and histamine release in isolated perfused guinea pig heart. AB - In an ischemia-reperfusion model obtained in isolated perfused guinea pig heart by means of a double ligature of the left anterior descending coronary artery, the reperfusion of the ischemic myocardium leads to a release of lactate dehydrogenase and histamine, related to a decrease in the microdensitometry of cardiac mast cells and to a tissue calcium overload. The perfusion of the heart with L-arginine and with nitric oxide donors significantly reduces the release of histamine, the loss of mast cell metachromasia and calcium overload. These effects were potentiated by superoxide dismutase. PMID- 1718889 TI - Inhibition of antigen-induced late asthmatic response and bronchial hyperresponsiveness by cyclosporin and FK 506. AB - Using a guinea pig model of asthma, we have shown that the administration of cyclosporin, a T-lymphocyte-selective immunosuppressive agent, from the beginning of the immunization period inhibits the development of the late asthmatic response and bronchial hyperresponsiveness after antigen challenge. Similar results were obtained with FK 506, a new potent immunosuppressive agent. Since these compounds have been shown to suppress the activation of guinea pig T lymphocytes, the present data suggest that T lymphocytes may be important for the elicitation of the late asthmatic response and bronchial hyperresponsiveness. PMID- 1718890 TI - Time course of immediate release and relationships between thrombin-like proteinase, other proteinases, histamine, protein and dye extravasation in rat passive peritoneal anaphylaxis. AB - A thrombin-like proteinase (THROLP) was detected colorimetrically as the main proteolytic activity (PROA) in the lavage fluid some minutes after antigen challenge of rats for passive peritoneal anaphylaxis (PPA) reaction. THROLP is equivalent to histamine (H) as parameter for the early phase of PPA: both increased significantly after 6 min, but decreased to prechallenge levels 20 min after challenge. H was released from serosal mast cells, in contrast THROLP originates predominantly from plasma leakage and activation of the clotting reaction in PPA. These findings underline the importance of PROA in the sequence of allergic reactions. PMID- 1718891 TI - Definition of platelet-derived histamine-releasing factor and histaminergic receptors modulating platelet aggregation. AB - Endogenous and exogenous free radical scavengers significantly decrease mast cell histamine release induced by coincubation with resting, activated platelets or with platelet-derived supernatant. Histamine and pyridylethylamine dose dependently enhance platelet aggregation; the effect is potentiated by ranitidine and blocked by mepyramine. Histamine increases also cytosolic calcium concentration in platelets stimulated with thrombin, and binding sites for [3H] mepyramine are present on platelet membranes. PMID- 1718892 TI - Neurogenic inflammation in airways. AB - Neurogenic inflammation, due to release of neuropeptides from sensory nerves, has been demonstrated in airways of several species, particularly rodents, and may contribute to the inflammatory response in asthmatic airways. Tachykinins (substance P and neurokinin A) and calcitonin-gene-related peptide released from airway sensory nerves may cause bronchoconstriction, vasodilatation, plasma exudation and mucus secretion. Sensory nerves may become sensitised by inflammatory products and triggered by mediators such as bradykinin, resulting in exaggerated inflammation. The effects of tachykinins may be further amplified by loss of the major degrading enzyme, neutral endopeptidase, from epithelial cells. Several strategies for reducing neurogenic inflammation are possible. PMID- 1718893 TI - Neuropeptide-induced secretion from human skin mast cells. AB - Unlike human mast cells associated with mucosal surfaces such as lung, adenoids, tonsils and intestine, skin mast cells may be stimulated to release histamine by the neuropeptides substance P, vaso-active intestinal polypeptide and somatostatin or by other basic secretagogues such as morphine and compound 48/80. Release of histamine by neuropeptides is rapid and accompanied by minimal generation of the eicosanoids prostaglandin D2 and leukotriene C4. Transient elevations of intracellular calcium are associated with mediator secretion induced by both immunological and non-immunological stimulation, that induced by anti-IgE being derived from extracellular sources through channels in the plasma membrane while that stimulated by neuropeptides is mobilized intracellularly. Similarly, elevations of intracellular cyclic AMP induced by anti-IgE occur only in the presence of extracellular calcium, whereas with substance P elevations are apparent even in the absence of extracellular calcium. With the latter stimulus, histamine release is complete before the peak cyclic AMP is achieved. Despite these biochemical and temporal differences, degranulation induced by both secretagogues proceeds by compound exocytosis which is indistinguishable under the electron microscope. From these results we suggest that IgE-dependent and neuropeptide stimulation of human skin mast cells proceed by distinct biochemical pathways which eventually merge to produce exocytosis of their preformed granule associated mediators. PMID- 1718894 TI - Peptidase modulation of the pulmonary effects of tachykinins. AB - The physiological effects of the tachykinin peptides substance P (SP) and neurokinin A (NKA) are limited by their microenvironmental degradation. We used the isolated tracheally superfused guinea pig lung to examine the importance of various degradative enzymes in limiting the physiological effects of exogenously administered and endogenously released tachykinins. When SP and NKA are administered via the airway epithelium, neutral endopeptidase (NEP; EC 3.4.24.11) is the major degradative enzyme as indicated by the effects of NEP inhibitors alone compared to the effects of a NEP inhibitor along with a cocktail of other peptidase inhibitors. The effects of enzyme inhibitors on physiological responses is mirrored in the amounts of peptide recovered from lung perfusates as determined using an enzyme-linked immunosorbent assay. We found similar effects when SP and NKA were released endogenously by the acute infusion of capsaicin. These data indicate that NEP is the predominant degradative enzyme modulating the effects of SP and NKA administered via the airways. PMID- 1718895 TI - Mucosal nerves in endobronchial biopsies in asthma and non-asthma. AB - To investigate neural events within the airways in asthma, endobronchial biopsies were obtained by fibre-optic bronchoscopy from 8 atopic asthmatic subjects and 8 non-atopic healthy controls. The biopsies were immediately fixed on sampling and subsequently analysed for nerves using specific indirect immunofluorescence with antisera to the neural marker PGP 9.5 and to the neuropeptides vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). Nerves were present in all the biopsies from both subject groups, with no significant difference between the asthmatic and non-asthmatics. VIP immunoreactive nerves were equally present in both subject groups, being localized to smooth muscle and glandular sites. No immunoreactive nerves to SP or CGRP could be identified in any biopsy at any location. These in vivo findings do not identify an anatomical neuronal imbalance in asthma. PMID- 1718896 TI - The possible role of substance P in the allergic reaction, based on two different provocation models. AB - It was shown in two different provocation models (nasal and bronchial provocation) that substance P (SP) may play an important role in the neurogenic inflammatory response in upper and lower airway disease. (1) Pretreatment with SP augments the antigen challenge response of the nasal mucosa. (2) The baseline bronchoalveolar lavage (BAL) concentrations of SP are elevated 8-fold in allergies (pollen asthma) as compared with normals, even outside of season. (3) The SP concentration in BAL increases significantly (p less than 0.05) after bronchial allergen provocation. These findings support a previous hypothesis of an abnormally elevated activity of nonadrenergic-noncholinergic excitatory nerves and are in accordance with the results of a decreased activity of neutral endopeptidase exaggerating neurogenic inflammatory responses in the airways, including bronchomotor tone hyperresponsiveness. PMID- 1718897 TI - Allergenicity and physicochemical characterization of house dust mite derived amylase. AB - The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus. In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I. Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000. The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGXRSVITXLME. It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic. The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds. PMID- 1718898 TI - Ion influx as a transduction signal in mast cells. AB - Stimulation of mast cells leads to a transient increase in the concentration of free intracellular calcium, which, in a fraction of cells, is followed by a plateau of elevated calcium. The early part of this signal is due to release of calcium from intracellular stores. It is not important for secretion. The late plateau phase, if present, greatly accelerates secretion. It is mediated by two influx pathways, one of which can be identified as a nonspecific cationic channel. PMID- 1718899 TI - Regulation of airway mucosal ion transport. AB - The secretion of fluid into the airway lumen is due in part to active chloride secretion across the epithelium. Changes in Cl secretion require coordinated changes in the turnover of several transport proteins in order to avoid lethal changes in cell volume. Several different Ca- and cAMP-dependent pathways effect this coordination. Many mediators raise cyclic AMP which activates protein kinase A which in turn opens the apical membrane Cl channel. Cyclic AMP also raises Cai (without change in levels of inositol triphosphate, IP3), and this Ca is probably responsible for activation of basolateral K channels and NaK2Cl cotransporters. Other mediators raise Cai by elevating IP3, and seem to stimulate chloride secretion entirely by Ca-dependent pathways with no change in cyclic AMP levels. Protein kinase C and arachidonic acid inhibit Cl secretion. PMID- 1718900 TI - Inhibitors of protein and RNA synthesis block the sensitization of murine peritoneal mast cells. AB - Addition of the protein synthesis inhibitor cycloheximide (CX, 1 microgram/ml) and the RNA synthesis inhibitor actinomycin D (AD, 0.1 microgram/ml) to unfractionated mouse peritoneal mast cells simultaneously with IgE anti-DNP, for 24 h prior to challenge, completely blocked antigen-induced 5-HT release. Responses to anti-IgE were strongly abrogated whereas responses to the calcium ionophore A23187 were not affected. When CX and AD were added to presensitized cells their effects on antigen and anti-IgE-induced release were much reduced. These results suggest a requirement for protein synthesis during mast cell sensitization. PMID- 1718902 TI - Changes in filament actin accompanying IgE-dependent and -independent histamine release from IL-3-dependent cultured human basophils. AB - When cord blood mononuclear cells were cultured in the presence of rhIL-3 for 5 weeks or more, 40-90% of cultured cells became morphologically mature basophils. We analyzed the kinetics of histamine release, changes in filament actin (F actin), and movement of intracellular Ca2+ ([Ca2+]i) induced by IgE-dependent (anti-IgE) and -independent (fMLP) stimuli in these cultured basophils. Anti-IgE and fMLP released 24.5 +/- 5.4% and 14.5 +/- 4.5% histamine from the cells, respectively. Anti-IgE caused actin polymerization with a peak response at 15 min, which began much later than the elevation of [Ca2+]i. In contrast to anti IgE stimulation, fMLP induced rapid actin polymerization with a peak response at 30 s in correlation with kinetics of histamine release. Our results indicate that cord blood-derived cultured basophils show similar cell functions to mature basophils, and are useful models with which to investigate the mechanisms of degranulation, specifically when a large amount of highly purified cells are required. PMID- 1718901 TI - Extracellular ATP stimulates interleukin-dependent cultured mast cells and eosinophils through calcium mobilization. AB - We examined the effect of ATP and related nucleotides on the changes in intracellular calcium ([Ca2+]i) in murine bone marrow-derived mast cells (BMMC) and human cord blood-derived eosinophils (EO) cultured in the presence of interleukins. ATP, ADP and AMP released a substantial amount of histamine and leukotriene C4 from BMMC, and EO showed locomotive activity in response to ATP, ADP and GTP. These reactions were accompanied with an increase in [Ca2+]i in BMMC and in EO. The rise in [Ca2+]i in BMMC induced by ATP or antigen at optimal concentrations was inclined to be persisting. On the other hand, these nucleotides induced a rapid and transient rise in [Ca2+]i in EO. Purified human peripheral EO also exhibited locomotive activity and an increase in [Ca2+]i in response to ATP. These results indicate that extracellular ATP activates interleukin-dependent cultured mast cells and EO through Ca2+ mobilization, and suggest that ATP, which is known to be released from activated platelets or autonomic nerves, may stimulate in vivo counterparts of these cultured inflammatory cells. PMID- 1718903 TI - Ciclosporin A inhibits mediator release from human Fc epsilon RI+ cells by interacting with cyclophilin. PMID- 1718904 TI - Elucidation of the epitope locations of human autoanti-IgE: recognition of two epitopes located within the C epsilon 2 and the C epsilon 4 domains. AB - IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma, where it is suggested to be potentially involved in modulating IgE mediated hypersensitivity. Using a series of overlapping recombinant human epsilon-chain peptides, we have shown that circulating IgG anti-IgE antibodies recognise at least 2 epitopes located within the C epsilon 2 and the C epsilon 4 domains, respectively. The C epsilon 2 recognition site is located within the C terminal portion of the C epsilon 2 domain (i.e. aa301-339) which is thought to contribute residues to the Fc epsilon RI-binding site on IgE. The recognition by autoanti-IgE of an effector function site of such pivotal importance in IgE mediated hypersensitivity suggests that it plays a possible modulatory role during mast cell and basophil activation. PMID- 1718905 TI - The immune response to anti-idiotype antibodies bearing an internal image epitope of tetanus toxin/toxoid. II. Comparison of the primary humoral immune response to xenogeneic Ab2 beta 1 and Ab2 beta 2 internal image anti-idiotype antibodies. AB - Mouse monoclonal (Ab1) anti-tetanus toxin/toxoid antibodies were used to raise Ab2 beta (tetanus toxin/toxoid internal image bearing) anti-idiotype antibodies in rabbits. Those rabbit serum antibodies (Ab2 beta) that did not bind to mouse serum proteins on an affinity column gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response in mice. Rabbit serum antibodies that did bind to the affinity column, when eluted and used to inoculate mice also gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response. It is suggested that one population of rabbit Ab2 beta anti-idiotype antibodies (unbound fraction) bears a partial or complete internal image of a tetanus epitope (Ab2 beta 1) while others (bound fraction) bear a complete or partial mirror image of a mouse immunoglobulin epitope as well (Ab2 beta 2). PMID- 1718906 TI - Antiasthmatic effects of Picrorhiza kurroa: androsin prevents allergen- and PAF induced bronchial obstruction in guinea pigs. AB - In the Ayurvedic medicine, Picrorhiza kurroa Royle ex Benth. is used for the treatment of liver and lung diseases. Using different chemical and pharmacological methods, we could identify the phenol glycoside androsin as active compound preventing allergen and platelet-activating factor induced bronchial obstruction in guinea pigs in vivo (10 mg/kg p.o.; 1 h prior to the inhalation challenge). Histamine release from human polymorphonuclear leukocytes in vitro was inhibited by other compounds yet to be identified. PMID- 1718907 TI - Stimulation of human peripheral blood lymphocytes with chironomid hemoglobin allergen (Chi t I). AB - Hemoglobins (Chi t I) of the dipteron species Chironomus thummi thummi are known to cause severe allergic diseases in humans. We tested the allergen-specific stimulation of human peripheral blood lymphocytes (PBL) by Chi t I and its nine main components. Further, we applied fragments of the well-analyzed component III, obtained by cleavage with trypsin as well as arginine protease. In this way, we screened the molecule in order to identify T-cell epitopes. The whole component was found to be immunogenic and to have regions demonstrating varying PBL stimulation. In addition, interindividual patterns of reactivity, probably due to genetic restriction, were found. A T-cell epitope could be shown to be within the site 98-111, as predicted by application of Rothbard's algorithms. PMID- 1718908 TI - Effect of neutral endopeptidase inhibition on substance-P-induced increase in short-circuit current of canine cultured tracheal epithelium. AB - We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussing's short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP. PMID- 1718909 TI - Bleomycin-induced pulmonary fibrosis in genetically mast cell-deficient WBB6F1 W/Wv mice and mechanism of the suppressive effect of tranilast, an antiallergic drug inhibiting mediator release from mast cells, on fibrosis. AB - It has been well known that the number of mast cells increases during the development of fibrosis in various tissues including the lung. However, the role of mast cells in fibrosis still remains obscure. In the present paper, we evidenced that pulmonary fibrosis could be induced in genetically mast cell deficient WBB6F1-W/Wv mice as well as WBB6F1-(+/+) mice having mast cells normally by the treatment with bleomycin (BLM, 5 mg/kg, i.v., 10 days), and there was not much difference in the histological changes of lungs between the two strains. An increase in the hydroxyproline content of the lung of WBB6F1-W/Wv mice was rather higher than that of WBB6F1-(+/+) mice. Previously, we reported that tranilast, an antiallergic drug inhibiting chemical mediator release from mast cells, suppressed the development of BLM-induced pulmonary fibrosis in ICR mice, suggesting the possibility that mast cells play certain roles in fibrosis. However, it was evidenced in the present report that tranilast suppressed BLM induced fibrosis in WBB6F1-W/Wv mice. Tranilast neither suppressed the cytotoxic activity of BLM against KB cells and L-929 cells in vitro, nor inhibited the antitumor activity of BLM against Sarcoma-180 transplanted subcutaneously into ICR mice. Tranilast may act through suppressing BLM-induced activation of lymphoid cells including macrophage and neutrophil. These results indicate an inconsequential role of mast cells in the development of fibrosis. Increases in the number of mast cells and in histamine content of the lung, which were widely reported in the lungs of BLM-treated mice, may be the result of fibrosis. PMID- 1718910 TI - Lysosomal enzyme activities in experimental granulomatous inflammation. AB - Foreign-body (dextran beads) and hypersensitivity (antigen-coupled agarose beads) lung granulomas were induced in BALB/c mice by the intratracheal injection of beads. Large granulomas developed, which reached peak intensity within 3 days and declined in size thereafter. Aqueous extracts of both granulomas contained high levels of lysosomal enzymes N-acetyl-beta-D-glucosaminidase (NAG) and lysozyme. Lysosomal enzyme activities in the extracts correlated with granuloma sizes. Dispersed granuloma cells were able to produce these enzymes. These results suggest that lysosomal enzymes may reflect the activity/size of granulomatous inflammation. PMID- 1718911 TI - Faecally derived hydrolytic enzymes from Dermatophagoides pteronyssinus: physicochemical characterisation of potential allergens. AB - The previous findings that the group I and III mite allergens, and amylase present in mite faeces are hydrolytic enzymes has prompted a study to determine whether this material contains other enzymes which could be allergenic. Thus, spent growth medium devoid of whole Dermatophagoides pteronyssinus mites was shown to contain glucoamylase, lipase and lysozyme in addition to the cysteine protease, serine protease and amylase activities associated with the above allergens, respectively. All of these enzymes are probably associated with mite digestive processes. They were rapidly solubilised, heterogeneous with regard to charge (pI in the range 4-8) and demonstrated maximum biochemical activity in the neutral pH range. Three serine proteases were detected and comprised a chymotrypsin-like, a trypsin-like and an unclassified enzyme with pI of 4.1 and 5.3, 8.5 and 7.1, respectively. Only one cysteine protease was observed, which paralleled immunochemically identified Der p I in a variety of assays. It was shown to cleave at lysyl residues and could be inhibited by the specific cysteine protease inhibitor, E-64. The remaining serine proteases, glucoamylase, lipase and lysozyme represent potential allergens. PMID- 1718912 TI - Tissue concentrations of prostate-specific antigen in prostatic carcinoma and benign prostatic hyperplasia. AB - Prostate-specific antigen (PSA), as measured in peripheral blood, is currently the most widely used marker for the assessment of tumor burden in the longitudinal study of patients with carcinoma of the prostate (PCA). Studies from other laboratories have led to the conclusion that a given volume of PCA causes a much higher level of PSA in the peripheral circulation of patients than a similar volume of prostate without carcinoma. We have evaluated PSA in the resected tissues immunohistochemically and in extracts of PCA and of prostates resected because of benign prostatic hyperplasia (BPH) with an enzyme-linked immunosorbent assay. Immunohistochemical results were less quantitative than but consistent with the results of the ELISA of tissue extracts. Immunohistochemically, there was considerable heterogeneity in the expression of PSA by both PCA and BPH both within and among prostatic tissues from different patients. While the levels of expression of PSA in these tissues overlap broadly, PSA is expressed at a lower level in PCA than in BPH when PSA is expressed as a function of wet weight of tissue (p = 0.0095), wet weight of tissue/% epithelium (p less than 0.0001), protein extracted from the tissue (p = 0.0039), or protein extracted/% epithelium (p less than 0.0001). PMID- 1718913 TI - Mucin production by colon cancer cells cultured in serum-free medium. AB - Although many colon cancer cell lines are available for study, few of them exhibit differentiated properties. When cultured in medium containing fetal bovine serum, WiDr cells (WiDr-FBS) show an undifferentiated phenotype: growth as a multilayer of cells adherent to plastic and lack of polarization, brush border, and mucin vacuoles. In contrast, WiDr cells cultured in a chemically-defined serum-free medium containing insulin, transferrin and selenium (WiDr-ITS) grow as clusters of nonadherent cells with abundant desmosomes and tight junctions, microvilli and electron-lucid vacuoles. As WiDr-FBS cells, WiDr-ITS are not polarized. WiDr-ITS cells show a marked enhancement in mucin synthesis as demonstrated by: periodic acid-Schiff and Alcian blue stains, electron microscopy, immunohistochemistry using monoclonal antibodies (MAbs) reactive with mucin-associated epitopes, immune electron microscopy and immunochemical analysis using Western blots. In comparison with WiDr-FBS cells, WiDr-ITS cells showed strong expression of Tn, sialyl-Tn, blood group A and CEA. When mouse MAbs were used, higher levels of the MUCI gene product were detected in WiDr-ITS than in WiDr-FBS cells. The full spectrum of phenotypic changes was observed after I month of culture in ITS medium, and transfer of WiDr-ITS cells to FBS medium was accompanied by a partial phenotypic reversal, suggesting that these phenotypic changes result from an adaptative--rather than selective--process. PMID- 1718914 TI - QUADRAVOC: an apparatus for audio recording of speech and voice. PMID- 1718915 TI - 'To be the best or not to be, that is the question ...' On enactment, play and acting out. PMID- 1718916 TI - Current nursing courses and nursing textbooks. AB - This study has two parts. In the first part, current nursing courses in 18 baccalaureate nursing programs of Canada are examined to find out how traditional nursing courses have been changed. Major trends of nursing courses are identified, and two major types of current nursing courses are identified. In the second part, 16 nursing textbooks [child nursing (8); medical-surgical nursing (8)] are examined to see how they have changed to reflect the newer nursing curricula. Textbook and chapter content organizers commonly used in nursing textbooks are identified. Contents organizers currently used in nursing textbooks are argued to be ineffective for students' knowledge integration and knowledge development within the nursing profession. This position and its implications are discussed, and two approaches to developing textbooks are suggested. PMID- 1718917 TI - The longitudinal effectiveness of osseointegrated dental implants. The Toronto Study: peri-implant mucosal response. PMID- 1718918 TI - Zn, Ca and Na levels in the prostatic secretion of patients with prostatic adenoma. AB - Zinc, calcium and sodium levels of the prostatic secretions in patients with prostatic adenoma were studied. In a control group of 20 patients, two samples of the secretion, taken on the first and the seventh day, were compared and found to show no difference. In the treated group of 20 patients, where a 7-day therapy of ERU (extractum radix urticae) followed upon the first sampling, the 7th-day specimen presented a significant drop in the Zn level. Correlation was found to exist between the Zn and Ca levels. From literary data the inference was drawn that ERU is producing a derangement in the zinc-testosterone metabolism and diminishes the zinc secretion in adenomatous tissue. PMID- 1718919 TI - Immunohistochemical detection of beta-human chorionic gonadotropin in urothelial carcinoma. AB - Histologic sections from the specimens of 60 patients with urothelial carcinoma were stained immunohistochemically to search for beta-human chorionic gonadotropin (B-HCG) production. There were 6 doubtful, 5 weak and 1 strong positive B-HCG stainings among 65 sections from 60 patients. De novo acquisition of B-HCG production capability of tumour cells seemed to be predictive for early haematogenous dissemination of the disease, but the data obtained in this particular study were insufficient to suggest B-HCG as a routine prognostic marker. PMID- 1718920 TI - Experience with the Peponen capsule in the management of benign prostatic hyperplasia. AB - Sixty patients in Stages I and II of benign prostatic hyperplasia were treated with Peponen capsule. Out of them 26 took the drug for 10 months, 22 for at least 7, and 12 for at least 4 months. The daily dosage was 3 x 2 capsules in the first month and 3 x 1 capsule for the rest of the time. On the ground of urodynamic test results and changes in subjective complaints, more than 80% of the patients experienced improvement. The therapy intensified the uroflow, appeased dysuria, the difficult and painful discharge, and reduced the frequency of nocturnal urination. PMID- 1718921 TI - Evaluation of uroflowmetry in different groups of patients after transurethral resection of prostatic adenoma. AB - One hundred patients after transurethral prostatectomy (TURP) were examined. The patients' age, presence or absence of urinary tract infection, time after TURP, and the size of adenoma were taken into consideration in the assessment of uroflowmetry. It was found that with the passage of time all parameters have improved. Comparison of the results of uroflowmetry performed in patients with large or small adenomas showed that TURP was successful in both groups. Infection was found to affect the volume of the bladder, but it had no significant influence on other uroflowmetry parameters or on residual urine. The age of the operated patients seemed to have no influence on the results of flowmetry after TURP. PMID- 1718922 TI - Multiple marker evaluation in prostatic cancer with prostatic acid phosphatase, gamma-seminoprotein and prostate-specific antigen. AB - Serum prostatic acid phosphatase (PAP), gamma-seminoprotein (gamma-Sm), and prostate-specific antigen (PA) levels were measured in 63 untreated patients with prostatic cancer. The sensitivities of PAP, gamma-Sm, and PA as markers of malignancy were 68%, 83%, and 77%, respectively. The latter two markers were more sensitive than PAP, especially in stage B disease. The specificities of PAP, gamma-Sm, and PA were 95%, 93%, and 93%, respectively. Patients with multiple positive markers were very likely to have prostatic cancer. In reactivation of the disease, positive rates for gamma-Sm and PA were higher than for PAP, indicating that the former two markers are more reliable for monitoring prostatic cancer. PMID- 1718923 TI - Experience with prostate-specific antigen in prostatic carcinoma. AB - A total of 71 prostatic tumour patients and 45 prostatic adenoma patients were tested for prostate-specific antigen (PSA), immunological prostatic acid phosphatase (PAP) concentration as well as serum prostatic phosphatase (SPP) and enzymic serum phosphatase. It was found among untreated patients that PSA showed the highest percentage of pathologic affection in each stage. PSA, on the evidence of clearance test in the initial days of therapy and after a follow-up period of several months, gave a good picture of the course that the disease had taken. PMID- 1718924 TI - Differentiation in cultured limbal epithelium as defined by keratin expression. AB - The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells. PMID- 1718925 TI - Towards partnership in palliative and cancer care. PMID- 1718926 TI - [Ichthyosis and alopecia after maprotiline: corneolysis caused by temporary disorder of keratinization]. AB - A 37-year-old woman developed ichthyosiform desquamation of the skin and a severe diffuse alopoecia 3 weeks after taking the antidepressant maprotilin. No signs of inflammation were present. Histology revealed acanthosis with preserved stratum granulosum, follicular hyperkeratosis and dystrophic changes of the hair follicle. Electron microscopy revealed rarefication of tonofilaments and necrobiotic changes of epidermal keratinocytes with vacuolar degeneration of the cytoplasm and disorganization of the organelles. Pathogenetically this disease represents a drug-induced transitory disorder to keratinization, which had resulted in desquamation of the stratum corneum and alopecia. The authors propose the designation corneolysis for this pathogenetic principle. PMID- 1718927 TI - A dual staining method for identifying mucins of different gastric epithelial mucous cells. AB - A dual staining method has been developed to identify two types of mucous secreting cells in the gastric mucosa of human and rat in one and the same tissue section. Sections were stained first using the galactose oxidase-cold thionin Schiff (GOCTS) procedure and then with paradoxical Concanavalin A staining (PCS). Surface mucous cell mucin stained blue with GOCTS, whereas gland mucous cell mucin stained brown with PCS. This method enabled us to differentiate these two types of mucins not only in gastric epithelial cell cytoplasm but also in the extracellular space. Sugar residues detected by GOCTS were explored by employing four species of lectins, which were peanut and Allomyrina dichotoma agglutinins for beta-galactose and Vicia villosa and Wistaria floribunda agglutinins for beta N-acetylgalactosamine. The effect of oxidation with galactose oxidase was also examined on the affinities of reactive sites for these lectins. The results indicated that, in the human stomach, the sugar residues responsible for this reactivity were most likely beta-N-acetylgalactosamine and beta-galactose in specimens lacking secretion of blood group determinants and beta-N acetylgalactosamine in those showing the secretion. In the rat stomach, on the other hand, sugar residues responsible for GOCTS were not elucidated by these lectins. PMID- 1718928 TI - Double lectin and immunolabelling for transmission electron microscopy: pre- and post-embedding application using the biotin-streptavidin system and colloidal gold-silver staining. AB - Pre- and post-embedding methods are described that can be used for consecutive localization of two intracellular cytoplasmic binding sites in cells and tissues embedded in acrylic plastic for transmission electron microscopy. Both applications make use of the biotin-streptavidin system with colloidal gold detector particles and involve silver staining of the first gold signal to a predetermined size. Silver augmentation effectively masked any free binding sites on the biotinylated molecule and on the streptavidin complex of the first labelling reaction, thereby allowing a second cycle with the same detection system. Excellent ultrastructural localization was obtained with silver lactate as the silver ion donor in the developing solution, and the enhancement treatment did not destroy or even visibly reduce target site reactivity for the subsequently applied probe. Using these methods it was possible to achieve specific double lectin and immunological labelling; they could, however, be adapted to dual or multiple-labelling procedures with any biotinylated molecules. PMID- 1718929 TI - A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation. AB - A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed. PMID- 1718930 TI - Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states. AB - Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2-3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200-800 ng dry weight. At saturating cyclic AMP concentrations (46 microM) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 microM) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 microM) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus. PMID- 1718931 TI - A method for quantitative autoradiography over stained sections of tumors exposed in vivo to radiolabeled antibodies. AB - Quantitative autoradiography can be an important element in calculating dose patterns delivered to tumors and normal tissues of animals injected with radiolabeled antibodies. In this article we develop a computer algorithm that permits quantitative evaluation of grain density over large areas of stained tissue with minimal grain counting. This algorithm, using spectral classification of brightfield and darkfield images, image ratios, and manual counting of small sections of an autoradiograph, yields images defined in terms of grain density, independent of staining artifacts. These quantitative images give a precise indication of intratumor radioimmunoglobulin targeting, and can serve as source functions for dosimetric calculations. PMID- 1718932 TI - Inhibition of angiogenesis by 15-deoxyspergualin. PMID- 1718933 TI - Chronic heat stress and prenatal development in sheep: II. Placental cellularity and metabolism. AB - Aspects of placental protein and energy metabolism were examined in pregnant ewes subjected to either thermoneutral (TN, 18 to 20 degrees C, 30% humidity, n = 7) or hot (H, 30 to 40 degrees C, 40% humidity, n = 5) temperatures through mid and late gestation. Fetal and placental weights and total content of protein, RNA, and DNA were reduced (P less than .001) in H ewes. Placental protein and RNA concentrations (mg/g) were not different, and DNA concentrations were slightly greater (P less than .1), in H vs TN ewes. Thus, heat seemed to greatly reduce total cell number and placentome size and only slightly decrease cell size. Ratios of RNA to DNA indicated a reduced capacity for protein synthesis in H placenta. However, in vitro fractional rates of protein synthesis in tissue slices from the fetal and maternal placenta and from the myoendometrium were not different between TN and H ewes. The H ewes had greater placental protein concentrations of hydroxyproline and glycine, perhaps suggesting a greater collagen content. In vitro oxygen consumption of fetal placenta, but not of maternal placenta or myoendometrium, was lower in H than in TN ewes. This lower oxygen consumption was partially due to a lower Na+,K+ ATPase-dependent oxygen consumption. PMID- 1718934 TI - Impact of dietary tryptophan and behavioral type on behavior, plasma cortisol, and brain metabolites of young pigs. AB - The behavioral reactivity in an "open-field" test and plasma cortisol levels were studied in 72 pigs from 12 litters fed for 3 wk one of three diets with different levels of tryptophan: deficient (.14%), adequate (.23%), or excess (.32%). "Open field" tests were performed three times: 5 d (day W + 5), 23 d (day W + 23) and 45 d (day W + 45) after weaning. The exploration time and the number of grunts provided an adequate measure of the individual emotional reactivity at day W + 5. Significant correlations were obtained between exploration time and the number of grunts at each time (r = -.83 at day W + 5; r = -.46 at day W + 23; r = -.71 at day W + 45). The distinction between animals remained (P less than .05) in terms of exploration time at both 23 and 45 d after weaning. At day W + 23, exploration time was lower in the group fed the adequate diet than in the two other groups. This effect was maintained subsequently after feeding all pigs the same adequate diet (day W + 45). In 36 pigs slaughtered at day W + 23, brain TRP concentration was higher with the excess dietary TRP than with deficient or adequate levels. Conversely, other plasma amino acids (particularly threonine) accumulated only in the brains of pigs fed the deficient diet. Plasma cortisol level assayed at weaning (W) and 2 wk later increased with age and was higher in 16-h fasted (day W + 15) than in 3-h fasted (day W + 17) pigs. Correlations were observed within litters in the fasting state, between the cortisol level and behavioral traits measured at day W + 23 (r = .70 for number of grunts, r = -.60 for exploration time). Dietary TRP did not affect the plasma cortisol level irrespective of the nutritional state after weaning. However, an interaction was noted between plasma cortisol and TRP status (P less than .05). Although dietary TRP induced large variations in brain amino acids and 5-hydroxyindole concentrations, changes in behavioral and cortisol responses were relatively minor. PMID- 1718935 TI - Regulation of lipoprotein lipase in muscle and adipose tissue during exercise. AB - Lipoprotein lipase (LPL) is regulated in a tissue-specific manner; exercise increases LPL activity in muscle at the same time it is reduced in adipose tissue. The purpose of this study was to determine the relationship between LPL activity and LPL mRNA in muscle and adipose tissue in rats exposed to one bout of exercise. Immediately after a 2-h swim, LPL activity [pmol free fatty acids (FFA).min-1.mg tissue-1] in the exercised animals was reduced 43% in adipose tissue (110 +/- 26 to 63 +/- 17) and increased almost twofold in the soleus muscle (203 +/- 26 to 383 +/- 59) compared with sedentary control animals. At the same time, LPL mRNA was reduced 42% in adipose tissue and increased 50 and 100% in the red vastus and white vastus muscles, respectively. Twenty-four hours after the swim, LPL activity had returned to control levels in adipose tissue and the soleus muscle. At hour 24 of recovery, LPL mRNA was still reduced 23% in the adipose tissue of exercised animals but was not significantly different between exercised and control animals in any of the muscle tissues analyzed. Changes in total RNA concentration could not account for the changes in relative LPL mRNA expression. The relationship between LPL enzyme activity and LPL mRNA in muscle and adipose tissue was +0.86 and +0.93 at 0 and 24 h postexercise, respectively. Thus the tissue-specific changes in enzyme activity induced by exercise could be mediated, in part, through pretranslational control. PMID- 1718936 TI - Ultrastructural appearances of pulmonary capillaries at high transmural pressures. AB - Electronmicroscopic appearances of pulmonary capillaries were studied in rabbit lungs perfused in situ when the capillary transmural pressure (Ptm) was systematically raised from 12.5 to 72.5 +/- 2.5 cmH2O. The animals were anesthetized and exsanguinated, and after the chest was opened, the pulmonary artery and left atrium were cannulated and attached to reservoirs. The lungs were perfused with autologous blood for 1 min, and this was followed by saline-dextran and then buffered glutaraldehyde to fix the lungs for electron microscopy. Normal appearances were seen at 12.5 cmH2O Ptm. At 52.5 and 72.5 cmH2O Ptm, striking discontinuities of the capillary endothelium and alveolar epithelium were seen. A few disruptions were seen at 32.5 cmH2O Ptm (mostly in one animal), but the number of breaks per millimeter cell lining increased markedly up to 72.5 cmH20 Ptm, where the mean frequency was 27.8 +/- 8.6 and 13.6 +/- 1.4 (SE) breaks/mm for endothelium and epithelium, respectively. In some instances, all layers of the blood-gas barrier were disrupted and erythrocytes could be seen moving into the alveolar spaces. In about half the endothelial and epithelial breaks, the basement membranes remained intact. The average break lengths for both endothelium and epithelium did not change significantly with pressure. The width of the blood-gas barrier increased at 52.5 and 72.5 cmH2O Ptm as a result of widening of the interstitium caused by edema. The cause of the disruptions is believed to be stress failure of the capillary wall. The results show that high capillary hydrostatic pressures cause major changes in the ultrastructure of the walls of the capillaries, leading to a high-permeability form of edema. PMID- 1718937 TI - Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation. AB - The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+. PMID- 1718938 TI - New chemotherapeutic agents for non-Hodgkin's lymphomas. AB - Combination chemotherapy regimens achieve complete remissions in 60% to 80% of patients with non-Hodgkin's lymphomas; however, the majority of patients will relapse, and resistant disease remains a problem. Attempts to identify new, effective chemotherapy agents have primarily focused on the development of analogues that, unfortunately, have uniformly failed to provide a substantial therapeutic advantage. Drugs with a unique mechanism of action are more likely to be successful; among these are the purine analogues (e.g., fludarabine, 2' deoxycoformycin, 2-chlorodeoxyadenosine) and agents that can reverse clinical drug resistance. The number of patients who can be cured can be increased only by incorporating new agents into front-line regimens through carefully designed clinical trials. PMID- 1718939 TI - Biologic agents and approaches in the management of patients with lymphoma. A critical appraisal. AB - There are many new approaches to cancer treatment emerging as a result of improvements in our understanding of tumor biology and the host response to cancer. At this time, none of these new approaches are sufficiently active or well developed to replace or improve upon standard combination chemotherapy in the management of patients with lymphoma. PMID- 1718940 TI - Use of hematopoietic growth factors in patients with HIV disease. PMID- 1718941 TI - A development-specific protein in Myxococcus xanthus is associated with the extracellular fibrils. AB - We have been using monoclonal antibodies (MAbs) as probes to study developmentally relevant cell surface antigens (CSA) that may be required for cellular interactions in Myxococcus xanthus. Three independently isolated MAbs, G69, G357, and G645, isolated by Gill and Dworkin recognize a CSA detectable only on developing cells (J. S. Gill and M. Dworkin, J. Bacteriol. 168:505-511, 1986). The CSA is made within the first 30 min of submerged development and increases until myxosporulation. The CSA is also produced at low levels after 24 h in shaken-starved cultures and during glycerol sporulation. No antigen can be detected in lysed, vegetative cells, and expression of the antigen is blocked in the presence of rifampin or chloramphenicol. The antigen is expressed in submerged, developmental cultures of asg, bsg, csg, dsg, and mgl mutants and is not expressed in a dsp mutant. All of the three MAbs immunoprecipitate the same protein of approximately 97,000 Da from lysed developmental cells. Competitive immunoprecipitations suggest that they recognize at least two different epitopes on the CSA. The epitopes recognized by MAbs G69, G357, and G645 are sensitive to protease digestion, whereas the epitopes recognized by MAbs G357 and G645 are resistant to periodate oxidation. The epitope recognized by MAb G69 is sensitive to periodate oxidation. Fractionation of lysed developing cells shows that most of the antigen is localized in the pellet after centrifugation at 100,000 x g. To determine whether the antigen is expressed on the cell surface, we labeled developing whole cells with either MAb G69, G357, or G645 and gold-labeled anti mouse immunoglobulin G. Low-voltage scanning electron microscopy of labeled cells shows that the antigen is associated with the fibrillar matrix that surrounds the cells and that the antigen is retained on isolated, developmental fibrils from M. xanthus. The CSA has been designated dFA-1, for developmental fibrillar antigen 1. PMID- 1718942 TI - Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane. AB - Pseudomonas paucimobilis UT26 is capable of growing on gamma hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID- 1718943 TI - Procaine, a local anesthetic, signals through the EnvZ receptor to change the DNA binding affinity of the transcriptional activator protein OmpR. AB - Local anesthetics are known to reduce the level of OmpF and increase the synthesis of OmpC in the outer membrane of Escherichia coli K-12. It has been shown that the anesthetics procaine and phenethyl alcohol (PEA) act at the transcriptional level for ompF and ompC and that in the case of procaine, its action is dependent on EnvZ, the membrane-bound signal transducer required for ompF and ompC expression. In an effort to further understand how anesthetics regulate ompF and ompC expression, we have analyzed the DNA binding properties of OmpR (the transcriptional activator protein for ompF and ompC genes) from cells treated with procaine or PEA. Treatment of a wild-type cell with either anesthetic converted OmpR from a low-affinity DNA binding form to a high-affinity DNA binding form. The change in DNA binding affinity was correlated with alterations in outer membrane porin profiles and could occur in the absence of protein synthesis. A strain lacking EnvZ was unable to respond to procaine to produce either the shift in the OmpR DNA binding property or cause any change in the outer membrane porin profile. PEA treatment was also dependent on EnvZ for the alteration in the OmpR DNA binding property, but it could induce ompC expression in the absence of EnvZ. Further studies suggest that the amino terminal region of EnvZ is responsible for the procaine signalling. Our results indicate that procaine and PEA regulate ompF and ompC expression by modifying the DNA binding properties of OmpR through EnvZ signal transduction. PMID- 1718944 TI - Cloning and sequencing of a multigene family encoding the flagellins of Methanococcus voltae. AB - The flagellins of Methanococcus voltae are encoded by a multigene family of four related genes (flaA, flaB1, flaB2, and flaB3). All four genes map within the same region of the genome, with the last three arranged in a direct tandem. Northern (RNA) blot and primer extension analyses of total cellular RNA indicate that all four genes are transcribed. The flaB1, flaB2, and flaB3 flagellins are transcribed as part of a large polycistronic message which encodes at least one more protein which is not a flagellin. An intercistronic stem-loop followed by a poly(T) tract located between the flaB2 and flaB3 genes appears to increase the resistance of the flaB1/flaB2 portion of this polycistronic message to digestion by endogenous RNases. The flaA gene, located approximately 600 bp upstream from the tandem, is transcribed as a separate message at very low levels. The four open reading frames encode proteins of molecular weights 23,900, 22,400, 22,800, and 25,500, much less than the Mr values of 33,000 and 31,000 for the flagellins calculated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated flagellar filaments. Each flagellin contains multiple eukaryotic glycosylation signals (Arg-X-Ser/Thr), although they do not appear to be glycoproteins, and each has an 11- or 12-amino-acid leader peptide. The N termini of all four flagellins (amino acids 1 through 47 of the mature protein) are very hydrophobic, and this region shows a high degree of homology with the flagellins from Halobacterium halobium. PMID- 1718945 TI - Evidence for two sets of structural genes coding for ribulose bisphosphate carboxylase in Thiobacillus ferrooxidans. AB - Previously, we reported the cloning of the ribulose-1,5-bisphosphate carboxylase genes (rbcL1-rbcS1) of Thiobacillus ferrooxidans Fe1 (T. Kusano, K. Sugawara, C. Inoue, and N. Suzuki, Curr. Microbiol. 22:35-41, 1991). With these genes as probes, a second set of ribulose-1,5-bisphosphate carboxylase genes (rbcL2-rbcS2) was identified in the same strain and cloned. rbcL1 and rbcL2 encode the large subunits, and rbcS1 and rbcS2 encode the small subunits. Similar restriction patterns between these gene sets suggested a high level of sequence homology. In fact, sequence analysis showed that a 2.2-kb region, including the entire large and small subunit structural genes, was totally conserved in rbcL1-rbcS1 and rbcL2-rbcS2. The rbcL1 (rbcL2) and rbcS1 (rbcS2) genes were 1,422 and 333 bp in length and encoded 473- and 110-amino-acid proteins, respectively. The genes were separated by a 90-bp spacer sequence and were preceded by possible ribosome binding sites. The N-terminal amino acid sequences of the subunit proteins, synthesized in Escherichia coli, were determined by Edman degradation and found to agree with the deduced amino acid sequences, except for the N-terminal methionine residue. The transcriptional start site of the rbc genes was determined by primer extension, and the size of the rbc transcript was estimated to be about 2.1 kb, suggestive of the cotranscription of rbcL1-rbcS1 and/or rbcL2 rbcS2 mRNAs. Comparisons of amino acid sequences of both subunits with those of other organisms revealed that the ribulose-1,5-bisphosphate carboxylase of T. ferrooxidans, a chemoautotrophic bacterium, is phylogenetically closer to the photosynthetic bacterium Chromatium vinosum than to another chemoautotrophic bacterium, Alcaligenes eutrophus. PMID- 1718946 TI - Identification and cloning of the glnR locus, which is required for transcription of the glnA gene in Streptomyces coelicolor A3(2). AB - Six Streptomyces coelicolor mutants that required glutamine for growth at the wild-type rate on all nitrogen sources (Gln-) were isolated. The phenotypes of all six mutants were similar. The glutamine synthetase (GS) levels were 20- to 100-fold lower in extracts of the Gln- mutants than in extracts of their parents. The reduced levels of GS activity in the Gln- mutants were not due to adenylylation of the GS protein, because GS activity in Gln- extracts did not increase after snake venom phosphodiesterase treatment. No transcripts of the GS structural gene (glnA) could be detected in RNA isolated from the Gln- mutants in primer extension experiments. All six gln mutations mapped adjacent to adeA. S. coelicolor chromosomal DNA complementing the Gln- mutants was isolated from a library of S. coelicolor chromosomal DNA constructed in the low-copy-number S. coelicolor plasmid pIJ922. Subcloning experiments showed that a 1.45-kb DNA fragment could complement all six Gln- mutants. This DNA fragment did not hybridize with either the cloned S. coelicolor glnA gene or the cloned S. viridochromogenes GSII gene in Southern blots. Since glnA transcription was restored in the Gln- mutants containing the complementing DNA, the gln mutations appear to lie in one or more closely linked genes that are required for glnA transcription in S. coelicolor. PMID- 1718947 TI - The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn glycerophosphoryl diester phosphodiesterase. AB - The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', and insertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphism among different staphylococcal isolates. We sequenced and characterized the hypervariable region of the methicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bp hypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNA sequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. The dru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open reading frame that codes for 145 amino acids (ORF145), whose deduced amino acid sequence showed 57% amino acid sequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) of Escherichia coli. PMID- 1718948 TI - cAMP negatively regulates mRNA levels of actin and tropomyosin in rat cultured vascular smooth muscle cells. AB - The isoform expression patterns of actin and actin-binding proteins have been reported to be good markers for phenotypic modulation of rat vascular smooth muscle cells. In order to elucidate the regulatory mechanism of actin and tropomyosin isoform expression on a molecular basis, we examined the effects of various agents on the isoform expression patterns of actin and tropomyosin at the mRNA level in smooth muscle cells. We found that cAMP-elevating agents induced drastic decreases in the amounts of alpha-smooth muscle type actin and alpha tropomyosin transcripts, the expression of other actin and tropomyosin isoforms being also repressed, but to a lesser extent. The results of the experiment involving RNA synthesis inhibitors strongly suggest that activation of some mRNA specific degradation machinery by cAMP might be responsible at least for the rapid disappearance of alpha-smooth muscle type actin and alpha-tropomyosin transcripts in smooth muscle cells. PMID- 1718949 TI - Characterization of rat factors X and Xa: demonstration of factor Xa in rat plasma. AB - We have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl2 eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a Ki of 2.2 x 10(-11) M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6 x 10(4) M-1.min-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat alpha-1-antitrypsin and alpha-2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1718950 TI - Synthesis of protein C in human umbilical vein endothelial cells. AB - By monitoring the activation of protein C and the regulation of factor Xa catalyzed thrombin formation by the activated protein C (APC) on the surface of human umbilical vein endothelial cells (HUVEC), we found that functional protein C was synthesized in cultured HUVEC and expressed thereon in the presence of vitamin K. Furthermore, without exogenously added protein S, time-dependent and saturable accumulation of APC (20 fmol APC/10(5) cells) on the surface of HUVEC was observed. During prothrombin activation by the complex of membrane-bound factor Xa and endogenous factor Va formed on the surface of HUVEC, APC was generated, and the rate of thrombin formation decreased. Treatment of HUVEC with an antibody that inhibits the APC-catalyzed inactivation of endogenous factor Va clearly quenched the activity of surface-associated APC. Immunostaining of HUVEC with a horseradish peroxidase (HRP)-conjugated antibody that solely recognizes human protein C confirmed the presence of protein C on the surface of HUVEC. Northern blot analysis revealed that an about 1.8 kb mRNA species derived from HUVEC was hybridized with 32P-labeled protein C cDNA, as in the case of those from HepG2, which are known to synthesize normal protein C. The increase in the amount of protein C mRNA in HUVEC in parallel with cell growth provided supporting evidence for the synthesis of protein C during the culture of HUVEC. These results indicate that blood coagulation is regulated by endogenously generated and activated protein C, together with or without protein S, through inactivation of factor Va on the surface of endothelial cells. PMID- 1718951 TI - Human heterophile antibodies recognizing epitopes present on insect glycolipids. AB - As a consequence of detecting an IgM M-protein (naturally occurring diseased state monoclonal antibody) immunoreactive to insect acidic glycolipids in a patient with demyelinating peripheral neuropathy, normal human sera were examined for the occurrence of heterophile antibodies directed against carbohydrate epitopes present on glycosphingolipids of Calliphora vicina (Insecta: Diptera). The insect glycolipids can be separated into neutral, zwitterionic, and acidic types, according to whether the oligosaccharide chains consist of neutral monosaccharides only, or carry an additional phospho-ethanolamine side chain and/or a beta-glucuronic acid residue, respectively. Natural antibody activity to these three classes of insect glycosphingolipids was detected in all normal human sera examined. The antibody activities were separated by sequential chromatography on affinity columns of octyl-Sepharose 4B-bound neutral and zwitterionic glycolipids into three populations with differing epitope-type specificities. As expected for heterophile antibodies, they are mainly of the IgM class. Population I recognized epitopes present on the three types of insect glycolipids, i.e., the neutral oligosaccharide chain backbone, the main determinant of which contains a terminal N-acetylhexosamine. Immunoreactivity is separable into at least four subpopulations of differing carbohydrate epitope specificity. Population II recognized epitopes containing phosphoethanolamine in zwitterionic and some acidic insect glycolipids. There are two subpopulations, the majority of which require the free amino group of phosphoethanolamine for immunoreactivity. Population III antibodies showed immunoreactivity to terminal beta-glucuronic acid-containing epitopes present only on acidic insect glycolipids. PMID- 1718952 TI - Microbial endo-beta-N-acetylglucosaminidases acting on complex-type sugar chains of glycoproteins. AB - The activity and substrate specificity of endo-beta-N-acetylglucosaminidase [glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl beta-glucosamino-hydrolase, EC 3.2.1.96] obtained from Mucor hiemalis (Endo-M) was compared with that of the enzyme obtained from Flavobacterium meningosepticum (Endo-F), which is the only enzyme available that acts on the complex oligosaccharides of asparagine-linked sugar chains in glycoproteins. They showed almost the same activities toward DNS-ovalbumin glycopeptide containing high mannose and hybrid asparagine-linked oligosaccharides. However, Endo-M showed high activity towards DNS-asialotransferrin and DNS-transferrin glycopeptides, which contain complex biantennary oligosaccharides. Endo-M could weakly act even on DNS-asialofetuin glycopeptide containing complex triantennary oligosaccharides, while Endo-F could not. SDS-denatured asialotransferrin was deglycosylated by both enzymes in the presence of non-ionic detergent (NP-40) and EDTA, and the deglycosylated protein migrated to a lower molecular weight position than asialotransferrin on SDS-PAGE. However, even in the absence of detergent, Endo-M deglycosylated native asialotransferrin and transferrin. Deglycosylation of asialotransferrin was confirmed by means of Con A-Sepharose 4B column chromatography and SDS-PAGE. PMID- 1718953 TI - Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium. AB - A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434 amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer. PMID- 1718954 TI - Stimulation of 45Ca uptake and amylase release by cationic amino acids in parotid cells. AB - L-arginine, L-ornithine, L-lysine and L-histidine (each 10 mM) stimulated amylase release from rat parotid cells. The secretory response to the cationic amino acids was suppressed in the absence of extracellular Ca2+ and, at physiological Ca2+ concentration, coincided with stimulation of 45Ca net uptake by the parotid cells. All cationic amino acids also accumulated inside the parotid cells. Nevertheless, the concept that the stimulation of amylase release is merely attributable to depolarization of the plasma membrane, secondary to the accumulation of these positively charged amino acids in the parotid cells, is questioned in view of both the inverse correlation found between their secretory effects and degree of ionization and the knowledge that parotid cells are electrically inexcitable. PMID- 1718955 TI - Cytokeratin profiles in oral epithelial: a review and a new classification. AB - This article proposes a classification of oral epithelial and summarizes recent literature of cytokeratin expression in the oral cavity. The oral epithelial are subdivided into two major groups: superficial and deep epithelia. Epithelial lining the oral cavity, i.e., superficial, are essentially stratified squamous epithelia, with the exception of taste buds. The epithelium covering the dorsal tongue is a combination of keratinized and non-keratinized epithelia. Deep epithelia are of two kinds, odontogenic and glandular epithelia. This study provides a classification of the cytokeratins expressed in the oral cavity in healthy and pathological situations based on published data and our own studies. The profiles of these polypeptides in different oral epithelia should provide information that may be used in various disciplines of oral medicine. PMID- 1718956 TI - Acinic cell carcinomas of salivary glands histoprognosis. Value of NORs stained with AgNOR technique and examined with semi-automatic image analysis. AB - Twenty one patients surgically treated for acinar cell salivary gland carcinomas were studied retrospectively. Four cases were excluded from the results because of an inadequate follow-up time (less than 5 years). The rest was classified into two groups: eleven patients with a favorable outcome were still alive without any recurrence after a mean follow-up period greater than 10 years; in six other patients, recurrences and metastases occurred followed by death in four cases. Previously for such tumors a high degree of histological undifferentiation usually gave rise to a poor prognosis, although some exceptions were noted. Thus, in an attempt to find a more reliable prognosis criterion, we evaluated by semi quantitative image analysis nucleolar organizer regions by means of AgNOR count and mean area in all these cases. Our results significantly correlated with the clinical prognosis: a high count (more than 3) and a small mean area were always found in tumors with an unfavorable outcome whereas lower counts and larger mean areas were found in tumors with a favourable clinical course. Thus, this method showed promise as establishing the prognosis of acinar cell tumours. PMID- 1718957 TI - Substrate phosphorylation specificity of the human c-kit receptor tyrosine kinase. AB - The chimeric EK-receptor (EK-R), consisting of the epidermal growth factor receptor (EGF-R) extracellular binding domain and p145c-kit cytoplasmic signal generating sequences, was fully functional in forming high and low affinity EGF binding sites and in ligand-regulated receptor and substrate phosphorylation activities. Relative to EGF-R, EK-R activation stimulated kit-characteristic phosphorylation of human 293 fibroblast substrate polypeptides. Transient coexpression of EK-R with candidate substrates resulted in ligand-induced phosphorylation of phospholipase C gamma and guanosine triphosphatase-activating polypeptide. The RAF-1 serine/threonine kinase was shown to be associated with activated EK-R, but no tyrosine phosphorylation could be detected. The faithfulness of EK-R substrate phosphorylation specificity was confirmed with stem cell factor-stimulated p145c-kit. PMID- 1718958 TI - A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor. AB - The Nb2 cell line is a pre-T rat lymphoma that is dependent on prolactin (PRL) for mitogenesis. Two forms of PRL receptor (PRL-R), which differ in the length of their cytoplasmic domains have been identified in different tissues and species. In the present study we have cloned the cDNA and characterized the mitogenic form of PRL-R in Nb2 cells. Polymerase chain reaction amplification of first strand cDNA prepared from Nb2-11C (PRL-dependent) and Nb2-Sp (PRL-independent) cell lines was performed using oligonucleotide primers specific for the binding domain, the short form of the PRL-R, and the cytoplasmic domain of the long form of the PRL-R. These studies indicate that both cell lines express a novel form of PRL-R. A cDNA was isolated from an Nb2-Sp cDNA library, which contains 1446 base pairs identical to the nucleotide sequence of the long form of the rat PRL-R. However, the cDNA sequence is missing 594 base pairs in the cytoplasmic domain compared with the long form of the PRL-R. The cDNA encodes a protein of 393 amino acids, lacking 198 amino acids in the cytoplasmic domain. Scatchard analysis of 125I-labeled ovine prolactin (oPRL) binding to microsomes prepared from transiently transfected COS-7 cells with either PRL-R long form cDNA or Nb2 PRL-R cDNA indicates that the long form of PRL-R binds oPRL with high affinity (K alpha = 8.8 x 10(9) M-1), while the Nb2 PRL-R showed a 3.3-fold increased affinity for PRL (K alpha = 29.1 x 10(9) M-1). In addition, immunoblot analysis of these microsomes using 125I-labeled monoclonal antibody (U6) to the PRL-R demonstrates a Mr of approximately 82,000 for the long form and approximately 62,000 for the Nb2 form of PRL-R. Polymerase chain reaction amplification of genomic DNA prepared from PRL-dependent and -independent cell lines suggests that this form of PRL-R results from a deletion in the PRL-R gene. The identification of a modified long form of PRL-R in the Nb2 cell line should help localize domains of the PRL-R involved in signal transduction and further the investigation of prolactin's role in immune cell proliferation. PMID- 1718959 TI - Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-helical model peptides of various chain lengths. AB - Basic amphipathic alpha-helical peptides Ac-(Leu-Ala-Arg-Leu)3 or 4-NHCH3 (4(3) or 4(4)) and H-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)2 or 3-OH (4(5) or 4(6)) were synthesized and studied in terms of their interactions with phospholipid membranes, biological activity, and ion channel-forming ability. CD study of the peptides showed that they form alpha-helical structures in the presence of phospholipid liposomes and thus they have amphipathic distribution of the side chains along the axis of the helix. A leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides possess a highly potent ability to perturb the membrane structure. Membrane current measurements using the planar lipid bilayer technique revealed that the peptide 4(6), which was long enough to span the lipid bilayer in the alpha-helical structure, formed cation-selective ion channels at a concentration of 0.5 microM in a planar diphytanoylphosphatidylcholine bilayer. In contrast, other shorter peptides failed to form discrete and stable channels though they occasionally induced an increase in the membrane current with erratic conductance levels. The probability of detecting a conductance increase was in the order of 4(6) greater than 4(5) greater than 4(4) greater than 4(3), which corresponds to the order of the peptide chain lengths. Furthermore, 4(6) but not 4(5) showed an antimicrobial activity against both Gram-positive and -negative bacteria. The structure of ion channels formed by 4(6) and the relationship between the peptide chain length and biological activity of the synthetic peptides are discussed. PMID- 1718960 TI - Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase. AB - Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5 trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5 phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase. PMID- 1718961 TI - Transcriptional regulation through cytokine and glucocorticoid response elements of rat acute phase plasma protein genes by C/EBP and JunB. AB - Independent of de novo protein synthesis, interleukin-1, interleukin-6, and dexamethasone caused immediate stimulation of transcriptional activity of most major acute phase plasma protein genes in the rat hepatoma H-35 cells. However, activation of alpha 2-macroglobulin and alpha 1-acid glycoprotein genes were delayed by 2-4 h and required ongoing protein synthesis. The hormones also increased transiently the transcription of the junB gene and the amounts of JunB, C/EBP, and C/EBP-like mRNA. To identify whether JunB and C/EBP have the ability to control both the early and late acute phase reactants, expression vectors for mouse C/EBP and JunB together with reporter gene constructs containing recognized hormone-specific regulatory elements were introduced into hepatoma cells. C/EBP displayed prominent transactivation activity with the interleukin-1 and glucocorticoid regulatory elements of alpha 1-acid glycoprotein, the interleukin 1 regulatory element of haptoglobin gene, and the interleukin-6 regulatory element of beta-fibrinogen. The interleukin-6 regulatory elements of the first two genes and the glucocorticoid response element of the third gene were not affected by C/EBP. These data suggest that normal hormone activation of these three acute phase reactant genes might involve, in part, C/EBP-related factors which have a broad range of specificity. H-35 cells stably transformed with a mouse C/EBP expression vector showed an elevated basal level as well as cytokine inducible expression of some but not all acute phase reactants. Cotransfected JunB resulted in reduced activity of cytokine-responsive constructs and in lower transactivation by C/EBP. JunB appears to function as a modulator of plasma protein expression during the course of acute phase response. PMID- 1718962 TI - Interleukin-11 regulates the hepatic expression of the same plasma protein genes as interleukin-6. AB - Treatment of rat hepatoma H-35 cells with purified human recombinant interleukin 11 (IL-11) resulted in the stimulated production of several major acute phase plasma proteins. The qualitative and quantitative changes were comparable to those mediated by IL-6 or leukemia inhibitor factor (LIF). Like IL-6, IL-11 acted synergistically with IL-1 on type 1 acute phase proteins. The combination of IL 11 and dexamethasone yielded a magnitude of stimulation which was more similar to the combination of LIF and dexamethasone than IL-6 and dexamethasone. IL-11 elicited in treatment of primary cultures of rat hepatocytes a qualitative change of plasma protein production which was similar to that in H-35 cells. Comparison of rat and human hepatoma cells indicated that the IL-11 response did not correlate with that of IL-6 or LIF, suggesting that the action of IL-11 was mediated by an IL-11-specific receptor system. However, the intracellular transduction of the IL-11, IL-6, and LIF signals to the acute phase protein genes seems to rely, in part, on common elements as judged from their stimulatory effects on the transfected expression vector containing the IL-6 response element of the rat beta-fibrinogen gene. The finding that IL-11 shares liver-regulating properties with IL-6 and LIF suggests that IL-11 has the potential of contributing to the control of systemic homeostasis and hepatic acute phase response. PMID- 1718963 TI - Purification and characterization of two DNA helicases from calf thymus nuclei. AB - Two DNA helicases from calf thymus nuclei have been purified and characterized. The two proteins, designated as nuclear DNA helicase I and II, were copurified on Bio-Rex 70, DEAE-Sepharose, phosphocellulose and subsequently separated from each other on a heparin-Sepharose column. Final purification of DNA helicase I was achieved on single-stranded DNA-cellulose and that of DNA helicase II on ATP agarose. On denaturing polyacrylamide gels, nuclear DNA helicase I displayed two polypeptide bands of 170 and 200 kDa; nuclear DNA helicase II also consisted of two bands, in this case of 100 and 130 kDa. Both enzymes catalyzed the unwinding of a DNA primer from a single-stranded DNA template but had different nucleotide requirements for their energy supply. While nuclear DNA helicase I preferred to hydrolyze ATP or dATP to support its unwinding activity, nuclear DNA helicase II could utilize all four rNTPs or dNTPs, though ATP or dATP were still preferred to other nucleotides. ADP, AMP, or adenosine 5'-O-(thiotriphosphate) could not be used by either enzyme in the unwinding reactions. A divalent cation was essential for activity of both enzymes. Nuclear DNA helicase I required 3-5 mM Mg2+ or 1 mM Mn2+ for optimal unwinding. In contrast, nuclear DNA helicase II displayed high activity even at very low concentrations of Mg2+. Nuclear DNA helicase I was stimulated by NaCl, KCl, or potassium acetate up to concentrations of 150 mM; in contrast, nuclear DNA helicase II was strongly inhibited at salt concentrations over 75 mM. Both DNA helicases had an associated ATPase activity. However, while the ATPase activity of nuclear DNA helicase I was stimulated only in presence of single-stranded DNA, the ATPase activity of the nuclear DNA helicase II was stimulated by single-stranded DNA and, even more efficiently, by RNA. Finally, the translocation of both DNA helicases had a polarity from 3' to 5' with respect to the single-stranded DNA template to which the enzymes were bound. A comparison of these DNA helicases with the other reported mammalian DNA helicases will be given. The significance of the association of the two DNA helicases during the process of the purification will be discussed. PMID- 1718964 TI - DNA hypomethylation and germ cell-specific expression of testis-specific H2B histone gene. AB - Testis-specific H2B (TH2B) histone gene of rat is expressed during meiotic event of spermatogenic differentiation. The gene is unusual in that it has conserved the regulatory elements involved in the S phase-specific transcription of somatic H2B genes as well as the S phase-specific stabilization of histone mRNA. Genomic sequencing revealed that all analyzed CpG sites in the promoter region of TH2B gene are methylated in somatic tissues but not in testis. During spermatogenesis, these CpG sites are unmethylated as early as spermatogonia type A and up to sperm. Thus, there is a good correlation between DNA hypomethylation and germ cell-specific expression of TH2B gene. Results obtained from in vivo DNase footprinting and DNA mobility shift experiments are consistent with the hypothesis that DNA methylation inhibits gene activity by preventing the binding of transcription factors to their recognition sequences. The results show that (i) the binding of ubiquitous transcription factors to the promoter region of TH2B gene may be blocked in nuclei of liver, and (ii) DNA methylation can directly interfere with the binding of transcription factors recognizing a hexamer (ACGTCA) motif. In vitro DNA methylation and transfection experiments demonstrated that expression of TH2B gene is inhibited by DNA methylation in vivo. These findings indicate that DNA methylation may play a key role in the transcriptional repression of germ cell-specific TH2B gene. PMID- 1718965 TI - Cholesterol-mediated suppression of alpha 1-inhibitor III, a plasma alpha macroglobulin family protein. AB - Using differential hybridization techniques we have isolated a hamster cDNA encoding a cholesterol-regulated protein. By sequence homology we concluded that the isolated cDNA encodes alpha 1-inhibitor III (alpha 1 I3), a protein of the alpha-macroglobulin (alpha M) family. When hamsters were fed diets rich in cholesterol, cholic acid, or chenodeoxycholic acid, the amount of alpha 1I3 RNA was reduced between 5- and 10-fold. Drugs that lower plasma cholesterol levels, such as colestipol and mevinolin, increased alpha 1I3 RNA between 2- and 3-fold. Additionally, plasma alpha 1I3 protein levels, as measured by immunoblotting techniques using an anti-human alpha 2M antibody, correlate well with alpha 1I3 RNA levels in those hamsters. Plasma alpha 1I3 protein was inversely proportional to plasma cholesterol levels in those hamsters. The observed suppression of alpha 1I3 expression by cholesterol mimics the cholesterol-mediated regulation of other genes that maintain cholesterol homeostasis, such as 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and low density lipoprotein receptor. We hypothesize that alpha 1I3 may play a role in the onset of atherosclerosis and may provide a link between cholesterol and the clotting system. Furthermore, the availability of another sterol-regulated gene, like alpha 1I3, should help elucidate the molecular mechanisms of cholesterol mediated regulation of gene transcription. PMID- 1718966 TI - Translational control of globin chain ontogeny in hamster yolk sac erythroid cells. AB - Prior research has demonstrated that globin ontogeny of hamster proceeds nearly to completion during the several days that yolk sac erythroid cells (YSEC) circulate in the embryo; synthesis of embryonic globin chains gives way to synthesis of adult globin chains in these primitive cells. In the present study, we translated total cell RNA extracted from YSEC on days 9-13 of gestation in wheat germ cell-free extract, expecting to observe the same progressive rise that occurs in vivo in rates of translation of alpha- and beta-globin mRNA during ontogeny. The opposite occurred; translation rates of both globins decreased sharply. This disparity between synthesis of alpha- and beta-globins in vivo and in vitro suggested an element of control of translation attributable to the YSEC cytoplasm. We therefore assayed the effect of RNA-free clarified YSEC cytoplasm on cell-free translation of YSEC RNA. A repression of translation was detected of alpha- and beta-globin mRNA (not of embryonic globin mRNA), exercised strongly by cytoplasm from YSEC early in ontogeny (gestational day 9), and weakening as ontogeny progressed. The same effect was noted on alpha- and beta-globin mRNA of adult hamster and of rabbit. Heat treatment of cytoplasm abolished the greater part of the translation regulation, suggesting that the active agent is protein. Further characterization of this translational regulator included: (a) it binds to globin poly(A) mRNA but not to poly(A), (b) it was not detected in cell lysate of adult hamster brain, lung, or erythrocytes, and (c) it did not inhibit translation of adult hamster brain and liver RNA. We conclude that hamster globin ontogeny is substantially modulated by this translational regulation of alpha- and beta-globin expression. PMID- 1718967 TI - Mutational and structural analysis of the nitrate reductase heme domain of Nicotiana plumbaginifolia. AB - We have analyzed four Nicotiana plumbaginifolia null mutants presumably affected in the heme domain of nitrate reductase. The DNA sequence of this domain has been determined for each mutant and for the wild type. Two mutations were identified as single base changes leading to, respectively, the substitution of a histidine residue by an asparagine (mutant E56) and to the appearance of an ochre stop codon (mutant E64). Based on the amino acid sequence homology between the nitrate reductase heme domain and mammalian cytochrome b5, we have predicted the three dimensional structure of this domain. This showed that the nitrate reductase heme domain is structurally very similar to cytochrome b5 and it also confirmed that the residue involved in E56 mutation is one of the two heme-binding histidines. The two other mutations (mutants A1 and K21) were found to be, respectively, -1 and +1 frameshift mutations resulting in the appearance of an opal stop codon. These sequence data confirmed previous genetic and biochemical hypotheses on nitrate reductase-deficient mutants. Northern blot analysis of these mutants indicated that mutant E56 overexpressed the nitrate reductase mRNA, whereas the nonsense mutations present in the other mutants led to reduced levels of nitrate reductase mRNA. PMID- 1718968 TI - A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active. AB - We report here a human immunodeficiency virus type 1 (HIV-1) recombinant ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its isolation by affinity chromatography. The purified protein is active in hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of recent reports of similar HIV-1 RNase H domains which were enzymatically inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R., Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80; Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that a stretch of 20-30 residues immediately upstream of the polymerase-RNase H junction (residues 440 441 of HIV-1 reverse transcriptase) may be required for productive binding and alignment of the hybrid RNA-DNA substrate. The active HIV-1 RNase H domain is suitable for structural analysis, thereby providing a unique active molecule to better understand the structural basis for the functional organization of RNase associated with the HIV-1 reverse transcriptase. PMID- 1718969 TI - Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver. AB - To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells. PMID- 1718970 TI - Protein kinase C down-regulation enhances cAMP-mediated induction of urokinase type plasminogen activator mRNA in LLC-PK1 cells. AB - Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase. PMID- 1718971 TI - Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function. AB - In the present work, a method based on an epitope-tagged ubiquitin derivative is described that allows for the unambiguous detection of ubiquitin-protein conjugates formed in vivo or in vitro. Expression in the yeast Saccharomyces cerevisiae of ubiquitin that has been tagged at its amino terminus with a peptide epitope results in the formation of tagged ubiquitin-protein conjugates that are detectable by immunoblotting with a monoclonal antibody that recognizes the tag. The expression of tagged ubiquitin has no adverse effect on vegetative growth and, moreover, can suppress the stress-hypersensitive phenotype of yeast lacking the polyubiquitin gene UBI4. We also show that tagged ubiquitin is correctly conjugated in vivo and in vitro to a short-lived test protein and can be covalently extended into the multimeric ubiquitin chain that is normally required for the degradation of this protein. Surprisingly, however, conjugation of tagged ubiquitin inhibits proteolysis. These and related results suggest that the amino terminal region of ubiquitin is important in protease-substrate recognition and that the multiubiquitin chain is a dynamic transient structure. The potential of tagged ubiquitin for the identification and isolation of ubiquitin-protein conjugates and ubiquitin-related enzymes, and as a tool in mechanistic studies is discussed. PMID- 1718972 TI - Functional analysis of the trans-acting factor binding sites of the mouse alpha fetoprotein proximal promoter by site-directed mutagenesis. AB - The trans-acting factors of the mouse alpha-fetoprotein proximal promoter (-202 base pairs) are aligned as follows: regions Ia (HNF-1), Ib (C/EBP), II (NF-1 or C/EBP), II' (NF-1 or HNF-1), III (NP-III), IV (NP-IV), Va (NP-Va), and Vb (C/EBP). Site-specific mutation abolished protein binding to the corresponding mutated site with the exception of the NF-1 site, in which mutation causes partial protection. Transient expression analyses indicate that chloramphenicol acetyl-transferase (CAT) activity is reduced by mutations in regions Ia, II', Ib, II, and IV. Mutation of region III causes an increased activity and mutation of regions Va and Vb shows a slight inhibitory effect. Linking alpha-fetoprotein enhancer I to the wild type promoter resulted in a 12-fold stimulation of CAT activity. The activity of promoters with mutated C/EBP-binding sites (Ib, II, and Vb), was slightly above controls, indicating that enhancer I can reverse the effect of these mutations. Inhibition or stimulation of promoter activity resulting from mutations of the HNF-1 or NP-III binding sites, respectively, persisted when enhancer I was linked to the promoters, indicating that enhancer I cannot rescue these mutations. Mutation of both HNF-1-binding sites resulted in greater than 90% inhibition of CAT expression with and without enhancer I, indicating these sites are essential for promoter activity. The stimulation of promoter activity by mutation of the NP-III site suggests that this site may be essential for repression or attenuation of the alpha-fetoprotein gene. Our studies indicate that regulation of the alpha-fetoprotein gene requires the combinatorial effect of multiple cis- and trans-acting elements in the proximal promoter and that enhancer I may provide a factor(s) that specifically rescue the promoter from the inhibitory effect of mutation in the C/EBP-binding sites. PMID- 1718973 TI - 39-kDa protein modulates binding of ligands to low density lipoprotein receptor related protein/alpha 2-macroglobulin receptor. AB - A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2 macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2 macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues. PMID- 1718974 TI - Functional insertion of the SV40 large T oncogene in cystic fibrosis intestinal epithelium. Characterization of CFI-3 cells. AB - Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro. PMID- 1718975 TI - The "lamin B-fold". Anti-idiotypic antibodies reveal a structural complementarity between nuclear lamin B and cytoplasmic intermediate filament epitopes. AB - Previous studies have shown that nuclear lamin B binds specifically to the C terminal domains of type III intermediate filament (IF) proteins under in vitro conditions. To further explore such site-specific interactions, we have used a two-step anti-idiotypic antibody approach. First, a monoclonal antibody disrupting the cytoplasmic IF network organization of living cells (mAb7A3) (Matteoni, R., and Kreis, T. E. (1987) J. Cell Biol. 105, 1253-1265) was characterized. Epitope mapping demonstrated that this antibody recognized a site located in the C-terminal domains of vimentin and peripherin (type III IF proteins). mAb7A3 was able to inhibit more than 80% of the in vitro binding of nuclear lamin B to PI, a synthetic peptide modeled after the C-terminal domain of peripherin that comprises a lamin B-binding site (Djabali, K., Portier, M. M., Gros, F., Blobel, G., and Georgatos, S. D. (1991) Cell 64, 109-121). In a second step, animals were immunized with mAb7A3 and the resulting anti-idiotypic sera were screened. Two of these antisera reacted specifically with nuclear lamin B but not with type A lamins or cytoplasmic IF proteins. The anti-lamin B activity of one of the antisera was isolated by affinity chromatography using a lamin B agarose matrix. The reaction of these affinity-purified antibodies with lamin B was inhibited by mAb7A3. Furthermore, the anti-lamin B antibodies reacted with Fab fragments of mAb7A3 and abolished binding of lamin B to PI. From these data we conclude that anti-idiotypic antibodies against the paratope of mAb7A3 recognize specific epitopes of the lamin B molecule that have shapes complementary to the one of the C-terminal domain of type III IF proteins. We speculate that these (regional) conformations, which we term the "lamin B-fold," may also occur in non-lamin proteins that mediate the anchorage of IFs to various membranous organelles. PMID- 1718976 TI - Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis. AB - Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes. PMID- 1718977 TI - Mitogenic repression of myogenin autoregulation. AB - The muscle-specific helix-loop-helix proteins MyoD and myogenin have been shown to positively autoregulate their own expression and to cross-activate one another's expression following transfection into a variety of nonmyogenic cell lines. We show that the ability of an exogenous myogenin expression vector to activate the endogenous myogenin allele in 10T1/2 fibroblasts is dependent on serum withdrawal. Repression of myogenin autoregulation by serum can be observed with a reporter gene linked to myogenin upstream sequences, indicating that repression occurs at the level of transcription. Like MyoD, myogenin exhibits a short half-life (approximately 20 min), which may serve to maintain the autoregulatory loop without allowing excess accumulation of the protein. PMID- 1718978 TI - Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B. AB - The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease. PMID- 1718979 TI - Molecular cloning and analysis of a cDNA coding for chorismate synthase from the higher plant Corydalis sempervirens Pers. AB - Chorismate synthase catalyzes the last common step in the biosynthesis of the three aromatic amino acids in microorganisms and plants. We have cloned a cDNA for this enzyme from the higher plant Corydalis sempervirens. This is the first chorismate synthase cDNA from a eukaryotic organism. The nucleotide sequence was determined and the identity of the cDNA was confirmed by the amino acid sequence of tryptic peptides obtained from purified chorismate synthase. The homology to the two known bacterial sequences is about 48%. The cDNA contains an open reading frame of 1341 base pairs, encoding a protein of 447 amino acids. This protein with a molecular mass of 48,100 daltons resembles a chorismate synthase precursor targeted for chloroplast import. Multiple sites of polyadenylation were observed in chorismate synthase mRNAs. PMID- 1718980 TI - Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes. AB - The effect of hydroxy substitution on 1,4-naphthoquinone toxicity to cultured rat hepatocytes was studied. Toxicity of the quinones decreased in the series 5,8 dihydroxy-1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone greater than 1,4-naphthoquinone greater than 2-hydroxy-1,4-naphthoquinone, and intracellular GSSG formation decreased in the order 5,8-dihydroxy-1,4 naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone much greater than 1,4 naphthoquinone much greater than 2-hydroxy-1,4-naphthoquinone. The electrophilicity of the quinones decreased in the order 1,4-naphthoquinone much greater than 5-hydroxy-1,4-naphthoquinone greater than 5,8-dihydroxy-1,4 naphthoquinone much greater than 2-hydroxy-1,4-naphthoquinone. Treatment of the hepatocytes with BSO (buthionine sulfoximine) or BCNU (1,3-bis-2-chloroethyl-1 nitrosourea) increased 5-hydroxy-1, 4-naphthoquinone and 5,8-dihydroxy-1,4 naphthoquinone toxicity, whereas neither BSO nor BCNU largely affected 1,4 naphthoquinone and 2-hydroxy-1, 4-naphthoquinone toxicity. Dicumarol increased the toxicity of 1,4-naphthoquinone dramatically and somewhat the toxicity of 2 hydroxy-1,4- naphthoquinone, whereas 5-hydroxy-1,4-naphthoquinone and 5,8 dihydroxy-1,4-naphthoquinone toxicity increased only slightly. The toxicity of 5,8-dihydroxy-1,4-naphthoquinone decreased dramatically in reduced O2 concentration, whereas 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 2 hydroxy-1,4-naphthoquinone toxicity was not largely affected. It was concluded that 5,8-dihydroxy-1,4-naphthoquinone toxicity is due to free radical formation, whereas the toxicity of 1,4-naphthoquinone and of 5-hydroxy-1,4-naphthoquinone also has an electrophilic addition component. The toxicity of 2-hydroxy-1,4 naphthoquinone could not be fully explained by either of these phenomena. PMID- 1718981 TI - Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins. AB - Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1718982 TI - Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes. AB - Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing. PMID- 1718983 TI - Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants. AB - In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D. R., and Stanley, P. (1990) J. Biol. Chem. 265, 1615-1622). One of these transfectants was used to make a genomic DNA library in lambda ZAP-II from EcoRI-digested, size selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified. After in vivo excision, two plasmids with DNA inserts greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants. A partial restriction map of this clone revealed an approximately 3.6-kb PstI fragment that contained an Alu sequence. This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(1,3)-Fuc-T gene (Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484). Both approximately 3.6-kb fragments gave identical restriction patterns. In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4)[Fuc alpha(1,3)]GlcNAc beta 1 but not the alpha(2,3)-sialyl-Lex determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex is a carbohydrate ligand recognized by ELAM-1. Surprisingly, however, an open reading frame encoded within the approximately 3.6 kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356). Possible explanations for these apparently disparate results are discussed. PMID- 1718984 TI - The substitution of arginine for glycine 85 of the alpha 1(I) procollagen chain results in mild osteogenesis imperfecta. The mutation provides direct evidence for three discrete domains of cooperative melting of intact type I collagen. AB - We report a case of mild osteogenesis imperfecta in a 56-year-old male undergoing aortic valve replacement surgery. The primary defect in this patient was the substitution of arginine for glycine 85 in one of the two chains of alpha 1(I) procollagen. The thermal stability of the type I collagen synthesized by the patient's cultured skin fibroblasts was examined by enzymatic digestion. Digestion of the mutant type I collagen with trypsin and chymotrypsin at increasing temperatures sequentially generated three discrete collagenous fragments, approximately 90, 170, and 230 amino acids shorter than normal type I collagen. This incremental thermal denaturation is indicative of cooperative melting blocks within the type I collagen. This is the first demonstration of such cooperative blocks of melting in intact, essentially normal post translationally modified type I collagen. This direct evidence for cooperative melting domains of uncut type I collagen suggests that discrete blocks of amino acids function as core sites stabilizing the collagen helix. The location of mutations of the alpha chains of type I collagen relative to these discrete blocks of amino acids may influence the severity of the disease phenotype. PMID- 1718985 TI - A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences. AB - cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849 13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus. PMID- 1718986 TI - Amyloid-beta peptide, substance P, and bombesin bind to the serpin-enzyme complex receptor. AB - During the formation of an inhibitory complex with neutrophil elastase, alpha 1 antitrypsin (alpha 1 AT) undergoes a structural rearrangement and the resulting alpha 1 AT-elastase complex becomes endowed with chemoattractant activities, mediates an increase in synthesis of alpha 1 AT, and is rapidly cleared from the circulation. In previous studies we have provided evidence that these biological activities involve the recognition of a conformation-specific domain in the alpha 1 AT molecule by a cell surface receptor on human hepatoma HepG2 cells and human monocytes. The receptor has been termed the serpin-enzyme complex (SEC) receptor because it also recognizes complex of serpins antithrombin III, alpha 1 anti chymotrypsin, and C1 inhibitor with their cognate enzymes. Because a pentapeptide domain of alpha 1 AT (amino acids 370-374, Phe-Val-Phe-Leu-Met) is sufficient for binding to the SEC receptor and the sequence of this domain is remarkably similar to those of substance P, several other tachykinins, bombesin, and the amyloid beta peptide, we have examined the possibility that these other ligands bind to the SEC receptor. The results indicate that substance P, several other tachykinins, and bombesin compete for binding to, and cross-linking of, the SEC receptor. The SEC receptor is distinct from the substance P receptor by several criteria. There is no substance P receptor mRNA in HepG2 cells; the SEC receptor is present in much higher density on receptor-bearing cells and binds its ligands at lower affinity than the substance P receptor; the SEC receptor is much less restricted in the specificity with which it recognizes ligand; ligands for the SEC receptor including peptide 105Y (based on alpha 1 AT sequence 359-374), alpha 1 AT-protease complexes, and bombesin do not compete for binding of substance P to a stable transfected cell line expressing the substance P receptor. Finally, we show here that the amyloid-beta peptide competes for binding to the SEC receptor but does not bind to the substance P receptor, therein raising the possibility that the SEC receptor is involved in certain biological activities, including the recently described neurotrophic and neurotoxic effects ascribed to the amyloid-beta peptide. PMID- 1718987 TI - The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus. AB - The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane. PMID- 1718988 TI - Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels. AB - High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1. PMID- 1718989 TI - The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase. AB - The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3 kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids. PMID- 1718990 TI - Peptide:N-glycosidase activity found in the early embryos of Oryzias latipes (Medaka fish). The first demonstration of the occurrence of peptide:N-glycosidase in animal cells and its implication for the presence of a de-N-glycosylation system in living organisms. AB - The recent discovery of free oligosaccharides typical for the complex type of glycan chains terminating with a free di-N-acetylchitobiosyl structure in certain fish eggs and early embryos (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) led us to find an enzyme responsible for detachment of N-linked glycan chains from glycoproteins by hydrolyzing the beta aspartyl-glucosylamine linkage in Oryzias latipes embryos. The enzyme, peptide-N4 (N-acetyl-beta-glucosaminyl) asparagine amidase or peptide:N-glycosidase (PNGase), was partially (2090-fold) purified, and the reaction site at which this enzyme acts was specified by analysis and identification of the reaction products. This is the first demonstration showing PNGase in animal sources, although the presence of PNGases was reported in a variety of plant extracts and bacteria. Thus, the commonality of this type of enzyme is now demonstrated, and the possible physiological role of PNGase in de-N-glycosylation as a basic biologic process is proposed. PMID- 1718991 TI - Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance. AB - It has been widely assumed that the interaction of transforming growth factor beta 1 (TGF-beta 1) with its serum-binding protein, alpha 2-macroglobulin (alpha 2M), mediates the rapid clearance of TGF-beta 1 from the circulation. To test this, we have analyzed the effect of TGF-beta 1 binding on the conformational state of alpha 2M. Our results demonstrate that the binding of TGF-beta 1 to alpha 2M does not lead to the conformational change in the alpha 2M molecule that is required for the clearance of the alpha 2M.TGF-beta 1 complex via the alpha 2M receptor. Furthermore, endogenous TGF-beta 1 is associated with the conformationally unaltered slow clearance form of alpha 2M. Clearance studies in mice show that the half-life of 125I-TGF-beta 1 in the circulation (1.6 +/- 0.71 min) is not affected by blocking the alpha 2M receptor with excess conformationally altered alpha 2M. These results suggest that TGF-beta 1 is rapidly cleared from the circulation after injection by a pathway not involving alpha 2M. PMID- 1718992 TI - Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140. AB - GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition. PMID- 1718993 TI - Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase. AB - The major soluble protein in the lenses of most birds and reptiles is delta crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2 crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity. PMID- 1718994 TI - Modulation of insulin-like growth factor-II/mannose 6-phosphate receptors and transforming growth factor-beta 1 during liver regeneration. AB - Transforming growth factor-beta 1 (TGF-beta 1) is a potent mito-inhibiting substance that is thought to play an important function in regulating hepatocyte proliferation during liver regeneration. In this investigation, we have shown by immunohistochemistry that hepatocytes containing significant intracellular concentrations of TGF-beta 1 12 h after a two-thirds partial hepatectomy. This increase in hepatocyte TGF-beta 1 concentration was initially confined to those cells that resided in the periportal region of the liver. The elevation of intracellular TGF-beta 1 was, however, transient, and within 36 h, the hepatocytes positive for TGF-beta 1 had changed in a wavelike fashion from the periportal to the pericentral region of the liver lobules. By 48 h, most hepatocytes no longer contained TGF-beta 1. Interestingly, this temporary increase in TGF-beta 1 always preceded the onset of hepatocyte replication by approximately 3-6 h. Since TGF-beta 1 mRNA has been shown to be absent from hepatocytes normally and throughout liver regeneration, these results imply that the increase in intracellular TGF-beta 1 resulted from an augmented uptake. We have further shown that the insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man-6-P) receptors were up-regulated during liver regeneration and that the increased expression of this receptor co-localized in those hepatocytes containing elevated concentrations of TGF-beta 1. The latent TGF-beta 1 phosphomannosyl glycoprotein complex has been shown to bind to the IGF-II/Man-6-P receptor. Therefore, our data are consistent with the hypothesis that this latent complex is internalized through the IGF-II/Man-6-P receptor to the intracellular acidic prelysosomal/endosomal compartments where the mature TGF-beta 1 molecule could be activated by dissociation from the latent complex. PMID- 1718995 TI - Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases. AB - Molecular cloning of a full-length cDNA for human lysosomal acid lipase/cholesteryl ester hydrolase (EC 3.1.1.13) reveals that it is structurally related to previously described enteric acid lipases, but lacks significant homology with any characterized neutral lipases. The lysosomal enzyme catalyzes the deacylation of triacylglyceryl and cholesteryl ester core lipids of endocytosed low density lipoproteins; this activity is deficient in patients with Wolman disease and cholesteryl ester storage disease. Its amino acid sequence, as deduced from the 2.6-kilobase cDNA nucleotide sequence, is 58 and 57% identical to those of human gastric lipase and rat lingual lipase, respectively, both of which are involved in the preduodenal breakdown of ingested triglycerides. Notable differences in the primary structure of the lysosomal lipase that may account for discrete catalytic and transport properties include the presence of 3 new cysteine residues, in addition to the 3 that are conserved in this lipase gene family, and of two additional potential N-linked glycosylation sites. Transfection of the cDNA into Cos-1 cells resulted in the expression of acid lipase activity with the substrate range of the native enzyme at a level that was greater than 40 times the endogenous activity. PMID- 1718996 TI - Molecular basis for a functionally unique cytochrome P450IIB1 variant. AB - Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16 hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793 2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM. PMID- 1718997 TI - Protein kinase C-independent expression of stromelysin by platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C. AB - Changes in the expression of several genes play critical roles in cell growth and tumor transformation. A number of proteases are increased in some tumors, and the level of these enzymes correlates with the metastatic potential of several cancer cell lines. Stromelysin, with the widest substrate specificity, can degrade the extracellular matrix conferring metastatic potential to tumor cells. The mechanisms whereby growth factors and oncogenes control the expression of stromelysin are beginning to be characterized. In the study shown here we also identify a region in the stromelysin promoter which is involved in the induction of stromelysin in response to platelet-derived growth factor, phosphatidylcholine hydrolyzing phospholipase C, and ras oncogene. Our results are consistent with the notion that platelet-derived growth factor/phosphatidylcholine-hydrolyzing phospholipase C induces stromelysin gene expression through a phorbol myristate acetate/protein kinase C-independent mechanism by acting through elements in the stromelysin promoter distinct from the 12-O-tetradecanoylphorbol-13-acetate responsive element. PMID- 1718998 TI - Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u globulin DNA. AB - Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses. PMID- 1718999 TI - Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator. AB - Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland. PMID- 1719000 TI - Evidence for localized calcium mobilization and influx in single rat gonadotropes. AB - Dynamic video-imaging microscopy was used to investigate the spatial and temporal nature of Ca2+ mobilization and Ca2+ influx in acutely dissociated, fura-2 loaded, rat gonadotropes. Addition of luteinizing hormone-releasing hormone (LHRH) to an isolated gonadotrope stimulated a wave of Ca2+ originating from a specific locus of the cell. This probably reflects Ca2+ mobilization from an intracellular store, since this response was unaffected by the removal of extracellular Ca2+. Application of the dihydropyridine-sensitive Ca2+ channel agonist Bay K 8644 (Bay K) stimulated a rise in cytosolic free Ca2+ concentration in the rat gonadotrope. This response was blocked by the removal of extracellular Ca2+ and probably reflects the influx of Ca2+ across the cell membrane. High speed (30 frames.s-1) imaging of the Bay K-induced Ca2+ influx revealed a wave of Ca2+ originating from a localized part of the cell membrane, which, in general, was spatially distinct from the LHRH-induced Ca2+ wave produced in the same cell. This suggests that Ca2+ channels in the cell membrane may be clustered in a specific area of the cell membrane. The velocity of the LHRH-induced Ca2+ mobilization wave was faster (mean = 79 +/- 5 microns.s-1, n = 9) than the Bay K induced Ca2+ influx wave (39 +/- 7 microns.s-1, n = 9) (p less than or equal to 0.01, Wilcoxon signed rank test) measured in the same cells. Thus, both Ca2+ mobilization from intracellular stores and Ca2+ influx through the cell membrane appear to be spatially localized in the rat gonadotrope. These findings may have important implications in the intracellular regulation of Ca(2+)-dependent cell functions such as hormone biosynthesis and secretion. PMID- 1719001 TI - A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites. AB - To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR. PMID- 1719002 TI - Transitional change of colony stimulating factor requirements for erythroid progenitors. AB - The course of the differentiation and proliferation of the human erythroid burst forming units (BFU-E) to colony-forming units (CFU-E) was directly investigated using a combination of highly purified BFU-E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU-E with a purity of 45-79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU-E required not only rEP but also rIL-3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 +/- 20-fold above the starting cells, while erythroid progenitors increased 156 +/- 74-fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rEP combined with or without rIL-3 showed the following: 1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. 2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rIL-3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU-E. These studies provide a quantitative assessment of the loss of IL-3 dependency by BFU-E and indicate that the size of the generated erythroid colonies and their IL 3 requirement correlate with the erythroid differentiated state. PMID- 1719003 TI - Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: binding, internalization, degradation, and biological effects. AB - Vasculotropin/vascular endothelial cell growth factor (VAS/VEGF) is a newly purified growth factor with a unique specificity for vascular endothelial cells. We have investigated the interactions of VAS/VEGF with human umbilical vein endothelial cells (HUVE cells). 125I-VAS/VEGF was found to HUVE cells in a saturable manner with a half-maximum binding at 2.8 ng/ml. Scatchard analysis did show two classes of high-affinity binding sites. The first class displayed a dissociation constant of 9 pM with 500 sites/cell. The dissociation constant and the number of binding sites of the second binding class were variable for different HUVE cell cultures (KD = 179 +/- 101 pM, 5,850 +/- 2,950 sites/cell). Half-maximal inhibition of 125I-VAS/VEGF occurred with a threefold excess of unlabeled ligand. Basic fibroblast growth factor (bFGF) and heparin did not compete with 125I-VAS/VEGF binding. In contrast, suramin and protamin sulfate completely displaced 125I-VAS/VEGF binding from HUVE cells. VAS/VEGF was shown to be internalized in HUVE cells. Maximum internalization (55% of total cell associated radioactivity) was observed after 30 min. 125I-VAS/VEGF was completely degraded 2-3 hr after binding. At 3 hr, the trichloroacetic acid (TCA)-soluble radioactivity accumulated in the medium was 60% of the total radioactivity released by HUVE cells. No degradation fragment of 125I-VAS/VEGF was observed. Chloroquine completely inhibited degradation. VAS/VEGF was able to induce angiogenesis in vitro in HUVE cells. However, it did not significantly modulate urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t PA), plasminogen activator inhibitor (PAI-1), and tissue factor (TF). Prostacyclin production was only stimulated at very high VAS/VEGF concentrations. Taken together, these results indicate that VAS/VEGF might be a potent inducer of neovascularization resulting from a direct interaction with endothelial cells. The angiogenic activity seems to be independent of the plasminogen activator or inhibitor system. PMID- 1719004 TI - Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: effects of flow rate, thrombin, and cytochalasin D. AB - We have applied a multiple isotope dilution technique to examine junctional permeability of human umbilical vein endothelial cells (HUVEC) in vitro. Primary cultures were grown to confluence on porous Cytodex-3 microcarrier beads, packed into 0.3 ml columns (3 x 10(6) cells) and perfused at varying flow rates (0.3-1.2 ml/min) with HEPES-buffered Tyrodes solution containing unlabeled cyanocobalamin, insulin, and albumin. Columns were challenged periodically with mixtures of radioactive tracers of different molecular size. Permeability to 22Na+, [57Co]cyanocobalamin (1.3 kD), [125I]insulin (6 kD) or [125I]albumin (66 kD) was assessed relative to [131I]IgG (160 kD, impermeant reference tracer) by comparing column elution profiles. Although the single passage extraction of [125I]albumin by beads alone approximated 40%, the presence of confluent HUVEC rendered these beads effectively impermeable to albumin. High junctional extractions were measured for cyanocobalamin (0.79 +/- 0.02, n = 28) and insulin (0.51 +/- 0.05, n = 14) in cultures perfused at 0.3-0.4 ml/min, and tracer extraction decreased as perfusion rates increased. Permeability coefficients for cyanocobalamin (9.66 x 10(-5) cm/s) and insulin (4.18 x 10(-5) cm/s) increased significantly during perfusion with thrombin (10 U/ml) or cytochalasin D (1 microgram/ml), whereas permeability to albumin (0.39 x 10(-5) cm/s) remained unchanged. Morphological studies, using the glycocalyx stain ruthenium red, revealed that thrombin or cytochalasin D increased the penetration of the stain into junctions between endothelial cells. PMID- 1719005 TI - Rapid epithelialisation of fetal wounds is associated with the early deposition of tenascin. AB - Wound healing is a complex process involving the interaction of many cell types with the extracellular matrix (ECM). Fetal skin wound healing differs from that in the adult in that it occurs rapidly and without scar formation. The mechanisms underlying these differing processes may be related to the fetal environment, the stage of differentiation of the fetal cells or the ECM deposited in the wound. The spatial and temporal distribution of two components of the ECM, fibronectin and tenascin, were studied by immunostaining of cryosections from trunk wounds of fetal and adult sheep. Epithelialisation was complete earlier in the fetal wound than in the adult. The distribution of fibronectin was similar in fetal and adult wounds but tenascin was present earlier in the fetal wound. Fibronectin has several roles in wound healing including acting as a substratum for cell migration and as a mediator of cell adhesion through cell surface integrins. The attachment of fibroblasts to fibronectin is inhibited by tenascin and during development the appearance of tenascin in the ECM of migratory pathways correlates with the initiation of cell migration. Similarly, the appearance of tenascin in healing wounds may initiate cell migration. Tenascin was present in these wounds prior to cell migration and the rapid epithelialisation of fetal wounds may be due to the early appearance of tenascin in the wound. PMID- 1719006 TI - Cytokeratins are exposed on the outer surface of established human mammary carcinoma cells. AB - Normal human mammary epithelial cells and established tumour cells of the same origin express three to eight cytokeratins, which are distributed throughout the cytoplasm in the form of intermediate filaments. The combined use of the iodogen and the two-dimensional gel electrophoresis methods has allowed us to demonstrate the presence of cytokeratins 8, 18 and 19 on the outer surface of established human mammary carcinoma cells, in particular MCF-7 cells, while they were absent from the surface of normal mammary cells in primary culture. By ultrastructural immunocytochemistry, these cytokeratins were localized on blebs formed by the cell surface. Cytokeratins 8, 18 and 19 were also detected in the culture medium of mammary carcinoma cells. PMID- 1719007 TI - Distinct effects of cell-cell communication and corticosteroids on the synthesis and distribution of cytokeratins in cultured rat hepatocytes. AB - Cytokeratins CK 8 and CK 18 are the two keratins expressed in the liver. They are known to undergo extensive changes in expression with alteration of the hepatocyte phenotype in vitro. In this study, we have investigated the variation in levels of these two cytokeratins in hepatocytes selected from different situations in vivo. The amounts of corresponding transcripts were compared; cytokeratin 8 and 18 mRNAs were present at similar levels in hepatocytes freshly isolated from adult liver and, unexpectedly, from 17-day-old foetuses and newborn rats, whereas they were markedly higher in regenerating hepatocytes isolated early after partial hepatectomy. In order to investigate whether the different factors that can promote hepatocyte differentiation also produce a similar set of cytoskeletal changes, we have analysed both the expression and the distribution of cytokeratins in hepatocytes under different culture conditions allowing modulation of differentiation. Establishment of cell-cell contacts and addition of glucocorticoids were used as two modulating factors. Coculturing hepatocytes with rat liver epithelial cells (RLEC), which favours active expression of liver specific genes, resulted in a gradual decline of cytokeratin mRNAs, whereas pure hepatocyte cultures, which exhibit rapid phenotypic changes, expressed increasing levels of CK 8 and CK 18 transcripts. Furthermore, intracellular CK distribution was dramatically modified in parallel: the CK-positive material formed a fine network of fibrils uniformly distributed in the cytoplasm of hepatocytes in pure culture, whereas in cocultured cells CK immunofluorescence appeared principally located at the cellular periphery and it was regularly arranged in long fibrils just beneath the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719008 TI - Fusion proteins containing a minimal GPI-attachment signal are apically expressed in transfected MDCK cells. AB - We have shown that addition of the C-terminal 37 amino acids of decay accelerating factor (DAF) to secretory proteins leads to glycosyl-phosphatidyl inositol (GPI) anchoring and apical surface expression in MDCK cells. Theoretically, transferred apical sorting information may reside in the glycolipid-anchor moiety or the DAF sequence (9 amino acids) that remains after signal cleavage and GPI attachment. We show here that removal of eight of these nine remaining amino acids, thereby creating a minimal GPI-attachment signal, results in apical expression of GPI-anchored human growth hormone. These data argue that the apical sorting information conveyed by the C terminus of DAF is related to its ability to direct GPI attachment, rather than to a specific sequence that remains in the fusion protein. PMID- 1719009 TI - The microglial reaction in the rat dorsal hippocampus following transient forebrain ischemia. AB - We have examined the distribution and time course of the microglial reaction in the rat dorsal hippocampus after 25-min transient forebrain ischemia (four-vessel occlusion model). Microglial cells were visualized in brain sections using lectin staining with the Griffonia simplicifolia B4-isolectin following intervals of reperfusion ranging from 20 min to 4 weeks. Increased staining of microglial cells was detected in the dentate hilus and area CA1 as early as 20 min after reperfusion. These same regions demonstrated more intense microglial staining after 24 h. The strongest microglial reaction was observed 4-6 days after reperfusion when reactive microglia were abundant throughout all laminae of CA1 and the dentate hilus. Following longer reperfusion intervals, the microglial reaction became less intense; however, it remained above normal levels until the end of the fourth week. At all time points examined, microglial reactivity in the CA3 pyramidal and dentate granule cell layers was considerably lower than that observed in CA1 and dentate hilus. Our results are consistent with the known serial pathological changes associated with cerebral ischemia, but, in addition, show that the examination of the microglial reaction provides an extremely sensitive indicator of subtle and morphologically nonapparent neuronal damage during the early stages of injury. PMID- 1719010 TI - Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports. AB - The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared. PMID- 1719011 TI - Staining and quantification of proteins separated by polyacrylamide gel electrophoresis. AB - The present review concentrates on techniques for the staining and quantification of proteins separated by polyacrylamide gel electrophoresis. Staining with organic dyes has been used for approximately thirty years; the silver staining technique was introduced in 1979. The problems of silver staining are presented separately because the mechanism of this staining is in principle different from staining with organic dyes. Less attention has been devoted to quantification of two-dimensional gels, because this autoradiography is preferred because of its high sensitivity and fewer problems with accurate quantification in contrast to silver staining. PMID- 1719012 TI - Clinical applications of lectin affinity electrophoresis. AB - Principles and several modifications of lectin affinity electrophoresis are described. The results obtained using these newly developed techniques are reviewed for individual glycoproteins, the altered lectin reactivities of which have some clinical implications, showing different lectin reactivities, which occur not only on malignant transformation but also in association with inflammatory process and hormonal action. PMID- 1719013 TI - Effect of glutaraldehyde-fixation on the immunogenicity, particle stability and antigenic reactivity of alfalfa mosaic virus, and the specificity of elicited antibodies. AB - Glutaraldehyde-fixation was shown to stabilize the structural integrity of alfalfa mosaic virus (AMV) particles as well as to increase their immunogenicity and antigenic reactivity. The antigenic reactivity of the particles was substantially increased irrespective of whether the antibodies were from animals immunized with native or fixed AMV, or preparations of coat protein subunits isolated from the virus. No significant changes in the antigenic specificity of AMV particles were detected following glutaraldehyde-fixation. Thus it is possible to raise antisera to AMV with higher titres by using fixed virus as immunogen. PMID- 1719014 TI - Characterization of two monoclonal antibodies to Epstein-Barr virus diffuse early antigen which react to two different epitopes and have different biological function. AB - Five monoclonal antibodies (mAbs) were identified using immunofluorescence that were specific for the Epstein-Barr virus (EBV) encoded 52/50 kDa early antigen (EA-D) protein complex. Evidence to suggest that these mAbs react with the same 52/50 kDa EA-D protein was obtained by Western blotting, immunoprecipitation and ELISA. Two of the mAbs, 90E2 and 214A9, neutralized EBV DNA polymerase activity. The 214A9 mAb also inhibited the activity of bacteriophage T4 DNA polymerase while the 90E2 mAb did not. These data suggest that the 90E2 and 214A9 mAbs recognize two different epitopes on the 52/50 kDa EA-D protein. The high frequency of recovery of hybridomas producing anti 52/50 kDa EA-D mAbs suggest that this protein may have an important role in EBV pathogenesis/replication. PMID- 1719015 TI - A semiautomated multiparameter approach for anti-HIV drug screening. AB - We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3' dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited. PMID- 1719016 TI - Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study. AB - To evaluate the hypothesis that endocrine profiles change with aging independently of specific disease states, we examined the age trends of 17 major sex hormones, metabolites, and related serum proteins in 2 large groups of adult males drawn from the Massachusetts Male Aging Study, a population-based cross sectional survey of men aged 39-70 yr conducted in 1986-89. Group 1 consisted of 415 men who were free of obesity, alcoholism, all prescription medication, prostate problems, and chronic illness (cancer, coronary heart disease, hypertension, diabetes, and ulcer). Group 2 consisted of 1294 men who reported 1 or more of the above conditions. Each age trend was satisfactorily described by a constant percent change per yr between ages 39-70 yr. Free testosterone declined by 1.2%/yr, and albumin-bound testosterone by 1.0%/yr. Sex hormone-binding globulin (SHBG), the major serum carrier of testosterone, increased by 1.2%/yr, with the net effect that total serum testosterone declined more slowly (0.4%/yr) than the free or albumin-bound pools alone. Among the major androgens and metabolites, androstane-3 alpha,17 beta-diol (androstanediol; 0.8%/yr) and androstanediol glucuronide (0.6%/yr) declined less rapidly than free testosterone, while 5 alpha-dihydrotestosterone remained essentially constant between ages 39-70 yr. Androstenedione declined at 1.3%/yr, a rate comparable to that of free testosterone, while the adrenal androgen dehydroepiandrosterone (3.1%/yr) and its sulfate (2.2%/yr) declined 2-3 times more rapidly. The levels of testosterone, SHBG, and several androgen metabolites followed a parallel course in groups 1 and 2, remaining consistently 10-15% lower in group 2 across the age range of the study. Subgroup analyses suggested that obese subjects might be responsible for much of the group difference in androgen level. Serum concentrations of estrogens and cortisol did not change significantly with age or differ between groups. Of the pituitary gonadotropins, FSH increased at 1.9%/yr, LH increased at 1.3%/yr, and PRL declined at 0.4%/yr, with no significant difference between groups 1 and 2.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1719017 TI - Abnormal regulation of insulin-like growth factor binding proteins in adolescents with insulin-dependent diabetes. AB - We have measured fasting 0800 h insulin-like growth factor binding proteins (IGFBP)-1 and IGFBP-3, in 52 diabetic adolescents and 74 puberty-matched control subjects with short stature and normal hormonal status. We have also measured overnight hourly profiles of IGFBP-1, glucose, free insulin, and GH in 12 of the diabetic adolescents. With advancing age and pubertal status, IGFBP-1 declined and IGFBP-3 increased significantly in the control, but not the diabetic group. Fasting IGFBP-1 levels were elevated 4-fold compared to controls. Median IGFBP-3 was significantly lower in the diabetic compared to the control group in pubertal stages III and V. Elevated IGFBP-1 was significantly correlated with metabolic control in poorly controlled subjects (mean 12-month glycosylated haemoglobin greater than 8.5%). In the overnight profiles, mean hourly IGFBP-1 was inversely related to insulin, but not glucose. As free insulin levels declined, IGFBP-1 rose, associated with rising predawn blood sugars. The integrated 3-h IGFBP-1 value (0500-0800 h) was significantly correlated with the corresponding glucose integrated value. IGFBP-1 area under the curve for the whole overnight profile was significantly correlated with glycosylated hemoglobin in 11 of the 12 subjects. IGFBP-1 from diabetic adolescents has been shown to inhibit IGF-I bioactivity. We postulate that IGFBP-1 may have a role in growth impairment of poorly controlled diabetes and may contribute to the dawn phenomenon. PMID- 1719018 TI - Quality of oocytes from superovulated rhesus monkeys. AB - The parameters, serum oestradiol (E2) response, follicle size and cumulus morphology, which are commonly used to determine in-vivo oocyte maturation in human in-vitro fertilization and embryo transfer (IVF-ET) programmes, were shown to be unreliable predictors of maturation of rhesus oocytes. In two groups of rhesus, one stimulated with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) and the other with human menopausal gonadotrophin (HMG) and HCG, the three parameters varied widely within and between protocols. Triple fluorochrome staining (TFS) for chromatin, microtubules and filamentous actin (f-actin) in oocytes at the time of collection and following 24 h in culture showed major differences in their maturation both in vivo and in vitro following priming with PMSG and HMG. In evaluating IVF protocols, TFS provides a valuable assay for the meiotic status of fixed oocytes of non-human primates. PMID- 1719019 TI - Characterization of the steroid-dependence of insulin-like growth factor-binding protein-2 synthesis and mRNA expression in cultured human endometrial stromal cells. AB - The insulin-like growth factors (IGF-I and -II) are believed to be important in endometrial differentiation and blastocyst nidation, and proteins that regulate IGF action (IGF-binding proteins, IGFBPs) are hormonally regulated in endometrium during the menstrual cycle. To characterize further steroid-dependence of the IGFBPs, we established endometrial stromal cells in culture in the absence and presence of oestradiol (E2) and progesterone (P) and examined the conditioned medium for IGFBPs by Western ligand blot analysis. Stromal cells constitutively synthesized IGFBP-3, IGFBP-2, a 27 kd, and a 24 kd IGFBP. In the presence of E2 and P, a 10- to 15-fold increase in IGFBP-2 was detected in the conditioned medium beginning after about 7 days in culture, when cells decidualized and steroid-mediated prolactin secretion began. Withdrawal of steroids resulted in a marked decrease in IGFBP-2, comparable to control levels, and cells increased their IGFBP-2 production when rechallenged with E2 and P. Total RNA was isolated from stromal cells, and Northern blot analysis using a cDNA probe specific for IGFBP-2 revealed differential expression of a 1.4 kb mRNA transcript in steroid treated compared to control cells. The effects of progestational agents alone on IGFBP synthesis was also examined. Progesterone, medroxyprogesterone acetate and norethindrone all stimulated IGFBP-2 synthesis 12- to 15-fold compared to controls, and a progesterone receptor antagonist, RU 486, blocked the stimulatory effect of progesterone. IGFBP-2 synthesis was increased two-fold above controls by 17-alpha-hydroxyprogesterone, and RU 486 alone and hydrocortisone were without effect. Identification of IGFBP-2 in conditioned medium was made using IGFBP specific antiserum. These data show that (a) endometrial stromal cells synthesize and secrete IGFBP-2, (b) IGFBP-2 protein synthesis is controlled by steroid hormones, (c) P, by interacting with its receptor, modulates IGFBP-2 synthesis and (d) expression of IGFBP-2 mRNA is controlled by sex steroids. PMID- 1719020 TI - Evaluation of a simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli which is based on the O antigen. AB - A simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens was developed and tested for its applicability as a routine typing method. The assay involves the sensitization of erythrocytes with an antigenic extract and performance of a slide agglutination assay with specific antisera. In order to simplify the typing system to a greater extent, the standard typing antisera were pooled into nine groups for C. jejuni and four groups for C. coli. The five antiserum samples allocated to each pool were selected so that pairs or groups of cross-reacting antisera were included in the same pool. When this system was tested with the serotype reference strains, it was found that, in most cases, a strain reacted in only one pool. The specific serotype of that strain could then be further defined by typing in each of the antisera belonging to that pool. To evaluate the specificity of the simplified method, 246 clinical isolates of C. jejuni and 57 clinical isolates of C. coli were typed at the same time by the standard passive hemagglutination assay and by the rapid slide agglutination system. Although both schemes effectively differentiated isolates and results from both schemes were generally very similar, differences were noted for a few isolates. On the basis of these findings, the simplified procedure may be recommended as an alternative means for serotyping these species for epidemiological purposes. PMID- 1719021 TI - 16S rRNA sequences of Bartonella bacilliformis and cat scratch disease bacillus reveal phylogenetic relationships with the alpha-2 subgroup of the class Proteobacteria. AB - The primary structures of 16S rRNAs of Bartonella bacilliformis, an isolate of the cat scratch disease (CSD) bacillus, and a strain phenotypically similar to the CSD bacillus were determined by reverse transcriptase sequencing. These microorganisms were found to be members of the alpha-2 subgroup of the class Proteobacteria. The sequence from B. bacilliformis was most closely related to the rRNA of Rochalimaea quintana (91.7% homology), the etiologic agent of trench fever. The sequence from the isolate of the CSD bacillus showed the greatest homology with Brucella abortus (89.7%) and, when compared with oligonucleotide catalog data, formed a cluster with Rhodopseudomonas palustris, Pseudomonas carboxidovorans, Nitrobacter species, and Bradyrhizobium species. The 16S rRNA sequence was also determined for the Cleveland Clinic isolate, which was previously shown to be phenotypically similar to and approximately 30% related, by DNA hybridization, to the CSD bacillus. The Cleveland Clinic isolate was isolated from a patient not diagnosed with CSD. The rRNAs from these bacteria exhibited 98.2% homology, confirming that this isolate is a second species in the same genus as the CSD bacillus. Our data suggest that neither B. bacilliformis nor the CSD bacillus is the etiologic agent of bacillary epithelioid angiomatosis. PMID- 1719022 TI - Use of synthetic oligonucleotide DNA probes for identification and direct detection of Bacteroides forsythus in plaque samples. AB - Oligonucleotide DNA probes complementary to the hypervariable region of the 16S rRNA of Bacteroides forsythus were tested for their specificity and sensitivity against reference and clinical isolates of 73 different species of bacteria. The probes were used to detect the organism directly from nucleic acid extracts of subgingival plaque samples, and the results were compared with results of detection by culture methods. The data demonstrate that the probes are very specific (100%) and sensitive (100% when they are compared with those obtained by the culture method) and could reliably detect the organism in plaque samples. When a probe to a conserved region of the 16S rRNA (UP9A) was used to estimate the quantity of bacteria present in plaque samples, it gave results comparable to those of culture methods. The data suggest that total microbial load may be a parameter in periodontal disease. PMID- 1719023 TI - Epstein-Barr virus nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors. AB - The entire amino acid sequence of the unique region of the EBNA 1 protein was synthesized as a set of 41 20-residue peptides with an overlap of 10 amino acids. The peptides were tested in the enzyme-linked immunosorbent assay for reactivity with immunoglobulin A (IgA) and IgG in sera from 50 patients with nasopharyngeal carcinoma (NPC) as compared with 36 serum samples from healthy Epstein-Barr virus (EBV)-seropositive donors and 5 serum samples from EBV-negative donors. The most immunoreactive peptide for both IgA and IgG binding was localized to the glycine alanine repeat domain of the antigen. In the unique regions, 16 immunoreactive peptides were found. Of these, four were reactive with IgG but not IgA and three peptides were reactive with IgA but not IgG in NPC sera. In addition, several IgA and IgG epitopes on the carboxy-terminal region were specifically reactive with NPC sera, but unreactive with sera from healthy EBV-positive donors. The results suggest that EBV serology specific for individual epitopes may provide additional useful information not available by conventional serology with whole antigens or the EBNA complex. PMID- 1719024 TI - Glycoinositol phospholipids from American Leishmania and Trypanosoma spp: partial characterization of the glycan cores and the human humoral immune response to them. AB - The glycoinositol phospholipid (GIPL) profiles of American Leishmania spp. (L. mexicana and L. braziliensis), Leishmania donovani, and American Trypanosoma spp. (T. cruzi and T. rangeli) were compared. The major GIPLs in these parasites include tetraglycosyl-, pentaglycosyl-, and hexaglycosylphosphatidylinositol. These were partially identified by their comigration by high-performance thin layer chromatography with purified L. major GIPLs, gas-liquid chromatography of the monosaccharides released after aqueous HF treatment, N-acetylation and methanolysis, sensitivity to exoglycosidases, and antibody absorption on several specific natural haptens. Members of the genus Leishmania have two other highly polar glycolipids, while the T. rangeli glycolipid profile was quite different from those of other kinetoplastids that were studied. On a weight basis, the glycan core of L. major GIPL-1 is the most reactive, followed by GIPL-3 and GIPL 2. Antibodies to the core glycans of GIPL-1, GIPL-2, and GIPL-3 were present at a low titer in the serum of every normal individual studied, while elevated GIPL-2 antibody levels were present in 80 to 100% of T. cruzi-, T. rangeli-, or L. donovani-infected patients, with lower values being found for GIPL-3 (30 to 60%) and GIPL-1 (30 to 50%). Except for GIPL-2 antibodies, which were mainly located on immunoglobulin G (IgG) and IgM, GIPL-1 and GIPL-3 antibodies were mainly distributed in IgM, with lower reactivity present in IgG. Antigen-antibody binding was very selectively blocked with Gal(alpha 1-3)Man, or Gal(beta 1-4)Man, Gal(alpha 1-3)Gal, and Gal(alpha 1-6)Gal for GIPL-1, GIPL-2, and GIPL-3 antibodies, respectively. PMID- 1719025 TI - Evaluation of first- and second-generation RIBA kits for detection of antibody to hepatitis C virus. AB - Samples reactive by first-generation recombinant immunoblot assay (RIBA) to detect antibody to hepatitis C virus (anti-HCV) (RIBA-1 [Chiron, Calif.]) remained reactive by a second-generation test (RIBA-2) for HCV antibodies. A total of 75% of specimens indeterminate by RIBA-1 became reactive, 12.5% were nonreactive, and 12.5% remained indeterminate by RIBA-2. Among RIBA-1-nonreactive specimens, 12.0% became positive and 5.1% became indeterminate by RIBA-2. The antigens c33c and c22-3 have increased the sensitivity of RIBA-2. PMID- 1719026 TI - Humoral immune response of tuberculous patients against the three components of the Mycobacterium bovis BCG 85 complex separated by isoelectric focusing. AB - An isoelectric-focusing technique followed by Western blot (immunoblot) analysis was used to investigate the immunoglobulin G response of tuberculous patients against each of the three components of the Mycobacterium bovis BCG antigen 85 complex. The 85A component was stained by the tuberculous as well as the non tuberculous sera. In contrast, the 85B and the 85C proteins of the complex were not stained by the control sera but were stained by 20 of 28 tuberculous serum samples. PMID- 1719027 TI - Gen-Probe Rapid Diagnostic System for distinguishing between Mycobacterium avium and Mycobacterium paratuberculosis. PMID- 1719028 TI - Structurally different Drosophila striated muscles utilize distinct variants of Z band-associated proteins. AB - Monoclonal antibodies raised against four proteins from insect asynchronous flight muscle have been used to characterize the cross-reacting proteins in synchronous muscle of Drosophila melanogaster. Two proteins, alpha-actinin and Z(400/600), are found at the Z-band of every muscle examined. A larger variant of alpha-actinin is specific for the perforated Z-bands of supercontractile muscle. A third Z-band protein, Z(210), has a very limited distribution. It is found only in the asynchronous muscle and in the large cells of the jump muscle (tergal depressor of the trochanter). The absence of Z(210) from the anterior four small cells of the jump muscle demonstrates that cells within the same muscle do not have identical Z-band composition. The fourth protein, projectin, greater than 600 kDa polypeptide component of the connecting filaments in asynchronous muscle, is also detected in all synchronous muscles studied. Surprisingly, projectin is detected in the region of the thick filaments in synchronous muscles, rather than between the thick filaments and the Z-band, as in asynchronous muscles. Despite their different locations, the projectins of synchronous and asynchronous muscles are very similar, but not identical, as judged by SDS-PAGE and by peptide mapping. Projectin shows immunological cross-reactivity with twitchin, a nematode giant protein that is a component of the body wall A-band and shares similarities with vertebrate titin. PMID- 1719029 TI - Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. AB - Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide. PMID- 1719031 TI - Establishment of an enzyme-linked immuno-sorbent assay for urinary trypsin inhibitor by using a monoclonal antibody. AB - Monoclonal antibodies against inter-alpha-trypsin-inhibitor (ITI) were produced. One clone showing specificity for urinary trypsin inhibitor (UTI), a proteolytic fragment of ITI, which is excreted into urine, was selected for the establishment of an enzyme-linked immuno-sorbent assay (ELISA). The ELISA for the quantification of UTI was shown to work reproducibly in the range between 0.5 and 10 ng UTI/ml urine. Urines of several patients suffering from different lung diseases were screened for UTI using the established ELISA. Highest UTI levels were found in the urine of patients with lung empyema. A more moderate increase was observed in patients suffering from lung tuberculosis and from secondary and primary lung tumors. PMID- 1719032 TI - Combined chemotherapy and radiotherapy for advanced maxillary ameloblastoma. A case report. AB - A case of advanced maxillary ameloblastoma was successfully treated with combined intra-arterial chemotherapy (Peplomycin, 85 mg) and radiotherapy (Co60, 7080 r). The tumour showed a remarkable shrinkage, and the patient survived. He is still alive and well at the time of this report. Carefully applied chemotherapy combined with radiotherapy has a useful role in the management of ameloblastoma especially in an advanced maxillary tumour. This report presents a typical example, which indicates that the ameloblastoma may not be an inherently radioresistant and chemoresistant tumour. PMID- 1719030 TI - A lymphocyte homing receptor (L-selectin) mediates the in vitro attachment of lymphocytes to myelinated tracts of the central nervous system. AB - Lymphocytes enter lymph nodes by first adhering to high endothelial venules, an adhesive event mediated by a lectinlike lymphocyte receptor (L-selectin). Previously, it was shown with an in vitro assay that lymphocytes preferentially adhere to myelin-rich regions in brain sections. Here, using a recombinant form of L-selectin as an immunohistochemical reagent, we demonstrate potential ligands for L-selectin in myelinated regions of the central but not the peripheral nervous system. Using several antibodies and phorbol ester downmodulation of the receptor, we establish that L-selectin on human lymphocytes has a primary involvement in lymphocyte adherence to the myelinated regions. On mouse lymphocytes, the contribution of L-selectin appears to be partial. These findings raise the possibility that leukocyte targeting to myelin-rich regions, via a L selectin dependent mechanism, may be a factor in the pathogenesis of certain central nervous system demyelinating diseases. PMID- 1719033 TI - The Minnesota Child Development Inventory: validity and reliability for assessing development in infancy. AB - The concurrent validity and reliability of the Minnesota Child Development Inventory (MCDI) was assessed by comparing the MCDI general development index score, and each of the seven subscale scores, with the mental and psychomotor age equivalents achieved on the Bayley Scales of Infant Development. In addition, the co-positivity, co-negativity, positive and negative predictive values of the MCDI in identifying infants with a mental development index (MDI), or psychomotor development index (PDI) of greater than 2 SD below the mean were assessed. Subjects were 101 infants (8 to 19 months old) who were seen at a neonatal developmental follow-up clinic after discharge from the neonatal intensive care unit. Correlations were obtained for the entire sample as well as for the two chronological age groups (i.e., 8 to 10 months and 17 to 19 months) within the sample. A strong correlation between the MCDI scales and the Bayley Mental and Psychomotor Scales was documented for the entire population as well as for the individual age groups. The overall validity of the MCDI in identifying infants with a MDI or PDI of greater than 2 SD below the mean was limited due to relatively poor co-positivity and positive predictive value. Although the MCDI may yield consistent information about the development of an infant's skills, this research suggests the MCDI has limited capacity to discern infants having delayed development. PMID- 1719034 TI - Developmental disabilities associated with congenital nystagmus. PMID- 1719035 TI - Effects of volume loading on left atrial systolic time intervals. AB - The effects of volume loading on the left atrial preejection period (LAPEP) and left atrial ejection time (LAET) were examined in 24 patients with various heart diseases using pulsed Doppler echocardiography. In response to volume loading, the left atrial dimension before atrial contraction significantly increased from 30.6 mm +/- 5.8 mm to 32.4 mm +/- 5.4 mm and the change in the left atrial dimension during atrial contraction tended to increase. The peak velocity in the atrial contraction phase significantly increased from 58 cm/s +/- 14 cm/s to 63 cm/s +/- 13 cm/s, and the integral of the atrial contraction phase tended to increase. LAPEP significantly decreased from 114 ms +/- 16 ms to 104 ms +/- 14 ms and LAET significantly decreased from 128 ms +/- 15 ms to 124 +/- 12 ms. The relation between LAET and left ventricular end-diastolic pressure, and that between LAPEP and mean pulmonary capillary wedge pressure, shifted downward to the right after volume loading. Thus, left atrial ejection is augmented by volume loading according to the Frank-Starling mechanism, while LAPEP decreases due to an increase in preload and LAET decreases due to an increase in afterload. PMID- 1719036 TI - Recurrent hepatocellular carcinoma: usefulness of ultrasonography compared with computed tomography and AFP assay. AB - Follow-up studies using monitoring with alpha1-feto protein (AFP), ultrasonography (US), and computed tomography (CT) were carried out in 75 patients who had prior partial hepatectomy for hepatocellular carcinoma (HCC). Recurrence in the remaining liver was confirmed in 31 patients (41.3%) during the 4 month to 3 years 7 month period. Ultrasonography detected recurrence in 29 cases (sensitivity: 93.5%), CT in 26 (83.9%), and AFP assay in 12 (38.7%). In 4 patients, ultrasonography detected four recurrent nodules that CT missed. In 1 patient, two subphrenic nodules were detected with CT but not with ultrasonography. The specificity of US, CT, and AFP assay was 90.9%, 95.5%, and 93.2% respectively. Frequent follow-up study with ultrasonography in combination with CT and AFP assay should be recommended for the early detection of recurrent HCC. Ultrasonography is mandatory for the follow-up of patients with a prior hepatectomy for HCC. PMID- 1719037 TI - Distribution of galanin-like immunoreactivity in the brain of Rana esculenta and Xenopus laevis. AB - The immunocytochemical distribution of galanin-containing perikarya and nerve terminals in the brain of Rana esculenta and Xenopus laevis was determined with antisera directed toward either porcine or rat galanin. The pattern of galanin like immunoreactivity appeared to be identical with antisera directed toward either target antigen. The distribution of galanin-like immunoreactivity was similar in Rana esculenta and Xenopus laevis except for the absence of a distinct laminar distribution of immunoreactivity in the optic tectum of Xenopus laevis. Galanin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the pars medialis of the amygdala and the preoptic area. In the diencephalon, immunoreactive perikarya were detected in the caudal half of the suprachiasmatic nucleus, the nucleus of the periventricular organ, the ventral hypothalamus, and the median eminence. In the mesencephalon, immunoreactive perikarya were detected near the midline of the rostroventral tegmentum, in the torus semicircularis and, occasionally, in lamina A and layer 6 of the optic tectum. In the myelencephalon, labelled perikarya were detected only in the caudal half of the nucleus of the solitary tract. Immunoreactive nerve fibers of varying density were observed in all subdivisions of the brain with the densest accumulations of fibers occurring in the pars lateralis of the amygdala and the preoptic area. Dense accumulations of nerve fibers were also found in the lateral septum, the medial forebrain bundle, the periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the mesencephalic central gray, the laminar nucleus of the torus semicircularis, several laminae of the optic tectum, the interpeduncular nucleus, the isthmic nucleus, the central gray of the rhombencephalon, and the dorsolateral caudal medulla. The extensive system of galanin-containing perikarya and nerve fibers in the brain of representatives of two families of anurans showed many similarities to the distribution of galanin-containing perikarya and nerve fibers previously described for the mammalian brain. PMID- 1719038 TI - The earliest ultrastructural development of the tangential vestibular nucleus in the chick embryo. AB - The tangential nucleus is a primary vestibular nucleus located where the vestibular fibers enter the medulla. It is composed of neurons that migrate between 5 and 8 days in the chick embryo. Although primary vestibular fibers enter the medulla at 3 days, the first synapses are formed at 5 days on the processes of neuron precursors by longitudinally coursing fibers. Since the major components, or their precursors, are present at 3 days within the presumptive nucleus, we are interested in determining what cellular interactions occur among these structures following their entry and during the time leading up to synapse formation. At 2 days, prior to the appearance of VIIth and VIIIth nerve fibers in the medulla, the tangential nucleus anlage contained processes and endfeet of primitive epithelial cells, separated from each other by enlarged extracellular spaces. Longitudinal fibers first appeared within these spaces coincident with the appearance of root fibers, including some identified VIIth motor axons, associated with the primordial VII/VIIIth ganglia. By 3 days, some vestibular and VIIth nerve fibers could be identified by their ultrastructure and relative positions within the marginal zone and nerve roots. However, it was not until 4 days that the presumptive tangential nucleus acquired its orderly, characteristic organization. Although synapses were rare from 2 to 4 days, attachment plaques and coated pits were observed commonly between structures, especially between future synaptic structures. Thus, we confirm that synapse formation begins at 5 days. This represents the first detailed ultrastructural study of cranial sensory nerve ingrowth into the medulla. PMID- 1719039 TI - Ipsilateral cortical connections of granular frontal cortex in the strepsirhine primate Galago, with comparative comments on anthropoid primates. AB - Modern studies of granular frontal cortex (GFC) in large-brained, anthropoid primates, such as Macaca, indicate that this region is comprised of many areal subdivisions. These areas vary in their architectonic appearance and each has a distinctive, diverse set of corticocortical connections. The great extent of the GFC region in anthropoids, and its high degree of areal parcellation, suggest that some GFC areas may be specializations of anthropoids, not found in other mammals. To investigate this possibility, we studied the corticocortical connections of GFC in the relatively small-brained, strepsirhine primate Galago, with a series of eight tracer injections in the frontal cortex, and an additional eight injections of parietal and temporal cortex. Tracers used were wheat-germ agglutinin conjugated to horseradish peroxidase and tritiated amino acids. Our results indicate that Galago GFC has strong, reciprocal connections with the parietal area-7 complex and with higher-order temporal areas; there are additional connections with extrastriate visual cortex, parahippocampal, and cingulate areas, and frontal cortex. Thus GFC has an extremely diverse array of cortical connections in Galago, as in Macaca. However, we also found that the pattern of parietofrontal connections is simpler in Galago than in Macaca. Specifically, parietal areas project to fewer discrete zones within the GFC of Galago, consistent with the view that these animals have fewer GFC areas than Macaca. In addition, Galago GFC possesses connections that specifically resemble those of Macaca arcuate cortex, but lacks connectional patterns that are characteristic of principalis cortex. These results are in accord with our previous architectonic studies, which indicated that Galago does not possess homologues of principalis areas. We conclude that the arcuate areas are common elements of primate GFC organization, while the areas located within and adjacent to the principal sulcus are anthropoid specializations. PMID- 1719040 TI - Morphological differentiation of distinct neuronal classes in embryonic turtle cerebral cortex. AB - As a starting point for understanding the development of the cerebral cortex in reptiles and for determining how reptilian cortical development compares to that in other vertebrate classes, we studied the appearance and morphological differentiation of cerebral cortical neurons in embryonic turtles. 3H-thymidine birthdate labeling and focal injections of horseradish peroxidase (HRP) in in vitro cortical slices revealed that replicating cells occupy the outer ventricular zone, and subsequently migrate to the ventricular surface where they divide. Postmitotic neurons begin differentiating and elaborating neurites while migrating back through the ventricular zone. On their arrival at the top of the ventricular zone, pyramidal and nonpyramidal neurons can be distinguished morphologically. Cells with multipolar apical dendritic tufts ascending in the marginal zone resemble immature pyramidal neurons. Neurons morphologically similar to these early pyramidal cells were retrogradely labeled by injections of the lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (diI) in a known pyramidal cell target, the thalamus. Nonpyramidal neurons, resembling Cajal-Retzius cells, had horizontally oriented long axons and dendrites coursing in the plexiform primordium, the future marginal zone. With further development morphological differences between cell types became accentuated, and pyramidal cell somata were segregated into a single cellular layer flanked by zones containing predominantly nonpyramidal cells. Axon elaboration occurred early in embryonic development, as pyramidal cells sent axonal branches to the septum, thalamus, and cortical targets soon after their generation, and the intracortical axonal plexus became increasingly dense during embryonic life. Over a similar time course the distribution of projecting neurons labeled by thalamic diI injections changed from an initial homogeneous distribution to a preferential location in the superficial half of the cellular layer. Results from this study demonstrate several features of cortical differentiation that are conserved in reptiles and mammals, including similar early morphological differentiation events, the early distinction of principal cell types, and the parallel development of pyramidal and nonpyramidal neurons. The context in which these similar developmental events occur, however, differs profoundly in reptiles and mammals, with differences in the timing and location of neurite elaboration and differences in the appearance and architectonic organization of the cortex. Comparison of cortical developmental patterns between reptiles and mammals shows that similar functional cortical circuits with balanced excitation and inhibition can emerge in diverse cortical structures. PMID- 1719041 TI - Organization of visceral and limbic connections in the insular cortex of the rat. AB - The anterograde and retrograde transport of horseradish peroxidase was used to study the anatomical organization of visceral and limbic terminal fields in the insular cortex. Following injections into the ventroposterolateral parvicellular (VPLpc) and ventroposteromedial parvicellular (VPMpc) visceral relay nuclei of the thalamus, dense anterograde and retrograde labeling was present in the posterior granular and dysgranular insular cortices, respectively. The parabrachial nucleus had extensive connections with the posterior dysgranular cortex and to a lesser degree with the anterior dysgranular and granular cortices. In contrast, injections into the medial prefrontal cortex and mediodorsal nucleus of the thalamus resulted in dense anterograde and retrograde labeling primarily in the anterior agranular cortex, whereas injections in the amygdala resulted in axonal labeling in the agranular and dysgranular insular cortices. Injections into the lateral hypothalamic area resulted in dense anterograde and retrograde labeling mainly in the agranular and dysgranular cortices and moderate to light labeling in the granular cortex. Our results indicate that ascending visceral afferents, VPLpc, VPMpc, and parabrachial nuclei, are topographically organized in the granular and dysgranular fields of the insular cortex, whereas the agranular cortex appears to receive highly integrated limbic afferents from the infralimbic cortex and the mediodorsal nucleus of the thalamus. Although these visceral and limbic inputs to the insular cortex are segregated for the most part into different longitudinally oriented strips of cortex, limbic input from the lateral hypothalamic area and the amygdala, which have extensive autonomic as well as limbic connections, are more diffusely distributed over the different regions of the insular cortex. This organization may subserve a role for the insular cortex in integration of autonomic response with ongoing behaviour and emotion. PMID- 1719042 TI - Cytoarchitecture and intrafrontal connections of the frontal cortex of the brain of the hamadryas baboon (Papio hamadryas). AB - A study was made of the cytoarchitecture of the lateral and medial frontal cortex in the hamadryas baboon (Papio hamadryas). The frontal cortico-cortical connections of areas 46, 8, 6, and 4 were investigated by injection of wheat-germ agglutinine conjugated to horseradish peroxiase (WGA-HRP) into different regions of areas 46, 8, and 6. The lateral region of the frontal lobe of the baboon consists of broad areas of motor (area 4), premotor (area 6), and the dorsolateral prefrontal cortex, each of which is further divided into subdivisions with distinct cytoarchitectural features: areas 4a, 4b, 4c; 6 a alpha, 6a beta, 6a gamma, and 6b beta; 8A and 8B; 45; 46 and 46ps; 9; 10; and 12. Although the frontal cortex of the baboon brain exhibits the same basic cytoarchitectural features as the frontal corticies of the cercopithecus (campbelli?) (Vogt and Vogt, '19) or the macaque (Walker, '40; Barbas and Pandya, '87, '89), the baboon frontal cortex is very different from that of the macaque and cercopithecus in terms of cytoarchitecture: (1) the baboon frontal cortex has an additional area, termed here "6a gamma", within area 6, which has cytoarchitectural characteristics that are intermediate between those of areas 6 and 8; (2) the aggregation of giant pyramidal cells (greater than 50 microns in diameter) is found only in area 4a in the baboon, whereas such aggregates are found in areas 4a and 4b and, occasionally, in area 4c in the macaque; and (3) area 46 of the prefrontal cortex of the baboon can be subdivided into the cortex that surrounds the principal sulcus (area 46) and the upper and lower banks of the principal sulcus (area 46ps). Retrogradely WGA-HRP labeled cells and anterogradely WGA-HRP labeled terminals coexisted in the frontal cortex in a columnar fashion, indicative of a reciprocity among the connections. The frontal cortico-cortical connections of areas 46, 8, 6, and 4 in the hamadryas baboon were organized as follows: (1) areas 46, 8, and 6 were connected to one another, (2) area 4 was connected only to area 6, and (3) these connections showed a gross ventrodorsal topography: the ventral regions of each of areas 46, 8, and 6 were connected more strongly to the ventral than the dorsal regions of the other areas; the dorsal regions of each of areas 46, 8, and 6 were connected more strongly to the dorsal than the ventral regions of the other areas.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1719043 TI - Compartmental organization of the thalamostriatal connection in the cat. AB - The compartmental organization of the thalamostriatal connection in the cat was studied by labelling thalamic fibers in anterograde axonal transport experiments and comparing their striatal distributions with the arrangement of striosomes and matrix tissue identified by histochemical staining methods. When analyzed according to their principal compartmental targets in dorsal striatum, the thalamic deposits indicated the existence of medial and lateral divisions within the thalamostriatal projection. Nuclei of the medial division, which includes parts of the thalamic midline, projected primarily to striosomes. The lateral division, which embraces the anterior and posterior intralaminar groups, the rostral ventral tier nuclei, and parts of the posterior lateral nuclear complex, predominantly innervated matrix tissue. In the dorsal division of the nucleus accumbens, the medial system preferentially terminated in zones that stain heavily in butyrylcholinesterase and substance P preparations, but fibers from both the medial and the lateral systems largely avoided the histochemically marked compartments such as the border islands of the nucleus accumbens that are seen elsewhere in the ventral striatum. Medial division: Thalamic deposits involving the paraventricular and rhomboid nuclei of the thalamic midline elicited labelling of striosomes and, invariably, ventral extrastriosomal matrix, the nucleus accumbens, and the amygdala. This projection was topographically organized: rostral thalamic deposits elicited labelling in the medial caudate nucleus and the medial nucleus accumbens. More caudal injections produced more lateral labelling. Lateral division: The lateral division is composed of at least three projection systems distinguished by their patterns of matrix innervation. Deposits involving the anterior intralaminar nuclei and the striatally projecting cells located lateral to the stria medullaris (anterior intralaminar complex) produced an even, diffuse labelling of the matrix tissue and weak labelling of the striosomes. Injections placed in the ventroanterior, ventrolateral, and ventromedial nuclei (rostral ventral complex) elicited fibrous labelling of matrix tissue that often showed nonstriosomal inhomogeneities. Deposits involving the centromedian and parafascicular nuclei (posterior intralaminar complex) produced a highly variable pattern of matrix labelling that included both homogeneous and decidedly patchy innervations of the extrastriosomal matrix. Each of these lateral thalamostriatal systems showed a similar spatial organization, whereby dorsoventral and mediolateral thalamic axes were roughly preserved in the projection to striatum. PMID- 1719044 TI - Origin of neuronal inputs to the region of the tuberomammillary nucleus of the rat brain. AB - The origin of afferent connections of the hypothalamic tuberomammillary nucleus has been examined by using retrograde and anterograde tracing techniques. Retrogradely labeled neurons were found in about 70 cell groups of the forebrain and brainstem after injection of tracer into the ventral subgroup of the tuberomammillary nucleus. The majority of the labeled neurons were seen in the forebrain, with particularly large numbers in the infralimbic cortex, lateral septal nucleus, and preoptic region. The anterograde tracing experiments supported the general results of the retrograde tracing experiments. However, we did not observe any single cell group that selectively projected to the cell-rich core of the nucleus. In general, only a few fibers entered the core, whereas many labeled fibers seemed to terminate immediately adjacent to the cell group. Thus the target for the afferents is not primarily the perikarya of the neurons of the tuberomammillary nucleus, but either dendrites radiating out from the nucleus or neurons not belonging to the tuberomammillary nucleus. The results of the present study demonstrate that the histaminergic tuberomammillary nucleus derives its main input from the limbic forebrain. Through their widespread projections, the histaminergic neurons may transmit information originating from the limbic system to most if not all parts of the brain. PMID- 1719045 TI - Light microscopic Golgi study of mitral/tufted cells in the accessory olfactory bulb of the adult rat. AB - Mitral/tufted cells (MTCs) of the accessory olfactory bulb (AOB) of adult rats were investigated light microscopically with the rapid Golgi method. The somata of the MTCs, appearing ovoid or triangular in shape, are distributed throughout the external plexiform layer. The soma size varies from small to large (12-26 microns). Apical dendrites originating from the soma enter the glomerular layer to provide branches that form the glomerular arbors. After making a glomerular arbor, some dendrites develop a second arbor (en passant and terminal arbors, respectively). The MTCs have a very diverse dendritic branching pattern and most have a variable number of glomerular arbors per cell (up to 6); we have tentatively classified the MTCs into simple, intermediate, and complex. Of the glomerular arbors, 80% have a diameter of less than 50 microns. The glomerular arbors have been classified as baskets (small spherical or ovoid) with short loopy processes; balls of yarn (large and nearly spherical) with loosely intermingled thick loops; and bushes (small to large and rather polymorphic) with irregular processes. The MTCs send dendritic arbors to terminate in one or more glomeruli where they are arranged in several different types of endings. Since it is generally believed that the dendrites of mitral and tufted cells of the main olfactory bulb terminate in only one glomerulus, the difference in the termination of the dendrites of the MTCs may represent a morphological characteristic that is relevant to the coding and/or integration of sensory information. PMID- 1719046 TI - Retinal ganglion cell terminals in the hamster superior colliculus: an ultrastructural study. AB - The distribution, cross-sectional area, and presynaptic and postsynaptic characteristics of retinal ganglion cell axon terminals in the superior colliculus of normal adult female Syrian hamsters were investigated by quantitative ultrastructural techniques. After an intravitreal injection of horseradish peroxidase, most labelled axon terminals were found in the stratum griseum superficiale and stratum opticum of the contralateral superior colliculus. However, a small proportion (approximately 2%) of retinal ganglion cell axon terminals were located in deeper layers of the superior colliculus between the stratum opticum and the periaqueductal grey matter. Terminals were smaller in the upper two-thirds of the stratum griseum superficiale than in the lower one-third of this layer, the stratum opticum, and the stratum griseum intermedium. Presynaptic characteristics such as the length and number of contacts and the postsynaptic neuronal domains (somata, dendritic spines, or shafts) contacted by retinal ganglion cell axons in the superior colliculus were similar in all layers. PMID- 1719047 TI - Two types of chloride channels in hen colon epithelial cells identified by patch clamp experiments. AB - Freshly isolated epithelial cells from hen colon were investigated using the patch-clamp technique. The aim of this investigation was to characterise the cellular conducting site for Cl- secretion. In cell-attached mode two types of Cl(-)-channels were found. Both showed distinct outward rectification. The channel types differed in single channel conductances and the marked voltage dependence of the open probabilities. A low conductance Cl(-)-channel was observed with a mean conductance at negative holding potentials of g- = 9 pS, and of g+ = 34 pS at positive potentials. This channel was predominantly open at negative potentials, corresponding to cell hyperpolarization. The second channel type observed had conductances of g- = 35 pS and g+ = 77 pS, and showed increasing open probabilities with increasing holding potentials (cell depolarisation). Both channel types were blockable by the Cl(-)-channel blocker NPPB. These data in combination with previously published transepithelial transport data on hen colon indicate that these channels are the Cl- secretory sites in colon epithelium. PMID- 1719048 TI - Adenosquamous carcinoma of the skin: a report of 10 cases. AB - Cutaneous squamous carcinoma with true glandular differentiation has only rarely been documented. Ten patients with such tumors are presented. There were six men and four women, aged 48 to 87 years. The tumors were located on the central face (eight), scalp (one), and hand (one) and consisted of minimally elevated, indurated, keratotic plaques, up to 6 cm in size. Microscopically, the neoplasms exhibited multifocal origin from the epidermis; deep, dispersed, infiltrative growth; perineural invasion; and stromal desmoplasia. Squamous differentiation was most marked superficially. Glandular differentiation was more obvious in deeper areas. Lumens typically developed within squamous nests and were often lined by cells with cytoplasmic vacuoles, some of which contained mucin. The neoplastic cells had obvious cytologic atypia and easily identified mitotic figures. Immunohistochemically, nine neoplasms studied contained carcinoembryonic antigen in glandular foci. Each patient had one or more surgical resections, and six also received radiation and/or chemotherapy. Five patients died with uncontrolled local recurrence, and two are alive with extensive disease and clinical evidence of regional lymph node involvement. Two individuals with small, superficial neoplasms that could be completely removed are disease free. One patient died of unrelated causes shortly after diagnosis. Cutaneous adenosquamous carcinoma is more aggressive than the usual carcinoma of the skin. It must be distinguished from the cytologically bland, microcystic adnexal (sclerosing sweat duct) carcinoma which is capable of recurring but rarely, if ever, proves fatal. The question of whether adenosquamous carcinoma is an epidermally derived squamous tumor with divergent differentiation or should be viewed as a newly recognized adnexal carcinoma remains to be resolved. PMID- 1719049 TI - Ki 67 immunostaining in melanocytic skin tumors. Correlation with histologic parameters. AB - A total of 145 melanocytic tumors (nevus, 38; primary malignant melanoma, 72; metastatic malignant melanoma, 35) were stained with Ki 67 monoclonal antibody using a three-step immunoperoxidase technique. For each case, mean numerical density and maximum numerical density of Ki 67 positive nuclei (number per mm3) were quantitatively evaluated using interactive image analysis. Maximum numerical densities revealed highly significant differences. Within the group of primary malignant melanomas, there was a significant correlation between proliferative activity and maximum tumor thickness. Further, a 'Ki 67-prognostic index' was assessed in each case of primary malignant melanoma, calculating the product of the Breslow index and maximum numerical density/1000 (103 +/- 12; range 1-694). In a prospective, short-term evaluation of primary malignant melanomas, there was a significant difference concerning 'Ki 67-prognostic index' between disease-free survival and occurrence of metastases. After a follow-up time of 24 months, only 63% of the patients with a 'Ki 67-prognostic index' greater than 25 were disease free, whereas no patient with a 'Ki 67-prognostic index' less than 25 was found to have metastases. We conclude: assessment of the maximum numerical density of Ki 67 reflects the degree of malignancy in melanocytic skin tumors; within primary malignant melanomas, maximum numerical density of Ki 67 positive cells correlates with well-established prognostic parameters (tumor thickness, level of invasion, mitotic rate); assessment of the 'Ki 67-prognostic index' may be of additional prognostic value for patients with primary malignant melanoma. PMID- 1719050 TI - Fibrous papule of the nose with granular cells: two cases. AB - Two cases of fibrous papule of the nose (FPN) with granular cell features are presented and discussed. Both lesions have classic architectural features of FPN; however, the main stromal cells show large cytoplasmic granules of the type seen in granular cells. We are uncertain of the significance of these findings. Our hypotheses include a perifollicular granular cell reaction and a granular degenerative change of local dermal dendrocytes. PMID- 1719051 TI - Age-related changes in mucins from human whole saliva. AB - The predominant mucins in human whole saliva, MG1 and MG2, serve to protect and to lubricate the oral cavity. In this study, both unstimulated and stimulated whole salivas were collected from two groups of subjects: young (18-35 years of age) and aged (65-83 years of age). The subjects were in apparent good health. Saliva samples from each subject were analyzed by SDS-PAGE. The gels were stained with Stains-all, and both MG1 and MG2 were quantitated by video-image densitometry. The protocol gave reproducible values for each mucin. The stimulated and unstimulated salivas from aged subjects showed significant reductions in concentrations of both MG1 and MG2, as quantitated in mucin dye binding units. Possible associations of these reductions with the aging process are discussed. PMID- 1719052 TI - Trichilemmal tumor arising in a seborrheic keratosis: analysis of cell kinetics by BrdU staining. AB - We report a case of a 74-year-old male with a trichilemmal tumor arising in a seborrheic keratosis on the buttock and the results of a cell kinetic study of this tumor using a BrdU staining method. The incidence of trichilemmal tumor arising in a seborrheic keratosis seems to be extremely rare. The labeling index of this tumor was 12.0%; this was a level intermediate between normal epidermis and a variety of hyperproliferative skin diseases such as squamous cell carcinoma, Bowen's disease, and psoriasis vulgaris. DNA replicating cells were present in the germinative layers in normal epidermis and the benign hyperproliferative skin diseases, psoriasis vulgaris. In contrast, DNA replicating cells were found throughout the entire epidermis in premalignant and malignant tumors such as in Bowen's disease and squamous cell carcinoma. In this case, DNA replicating cells were localized mainly in the basal and parabasal cell layers, but also seen in the upper squamous layers. These findings suggest that this trichilemmal tumor had a malignant tendency, though it was slow-growing and relatively benign in nature. PMID- 1719053 TI - Recurrent malignant fibrous histiocytoma with expression of cytokeratin. AB - A case of locally recurrent malignant fibrous histiocytoma was documented in a 70 year-old man. He first noticed a subcutaneous nodule forty years previously. The tumor was surgically removed four times during the last four years with local recurrence on every occasion. In the recurrent tumors, the tumor cells almost completely replaced the whole dermis and invaded skeletal muscles. They were composed of pleomorphic spindle cells arranged in a storiform pattern and bizarre histiocytic cells, which were present principally in the deeper portions of the tumor. Both types of tumor cells showed marked nuclear atypicality. In the primary tumor, surrounding a large necrotic area, spindle-shaped cells were arranged in a storiform pattern. These tumor cells exhibited only mild nuclear atypia. The recurrent tumor was strongly positive for vimentin and alpha-1 antichymotrypsin. Most tumor cells were also weakly positive for KL1, a monoclonal antibody for keratin. A Western-blot analysis revealed the presence of two bands (62 and 69 Kd) reacting with KL1 in the fractions which were obtained from the tumor according to the method for keratin extraction. PMID- 1719054 TI - Shear-wave detection of structural effects in aqueous solutions of bovine serum albumin and hemoglobin. AB - Using cylindrical quartz crystal torsional resonators operating at 39 and 75 kHz to generate shear waves in aqueous solutions of the proteins bovine serum albumin and hemoglobin and the polypeptide poly l-glutamic acid, it has been possible to determine the complex dynamic shear viscosities of the solutions. The effects of concentration, pH, and denaturation using various agents have been studied. It is possible to relate the viscosity and configurational elasticity of the solutions, to the intramolecular and intermolecular forces associated with the of the proteins at frequencies between 60 and 400 kHz and attributed to conformational changes of bovine serum albumin and the quaternary doublet interactions of hemoglobin have been confirmed and emphasized by the use of shear waves. PMID- 1719055 TI - The evocative power of enactments. AB - The inevitability of analytic enactments, defined as symbolic interactions between patient and analyst, is discussed. Clinical material from the psychoanalysis of a latency-age child is presented to illustrate the role of enactments and to demonstrate their usefulness in furthering the analytic work. PMID- 1719056 TI - Biochemical assessment of the genotoxicity of the in vitro interaction between chlordecone and carbon tetrachloride in rat hepatocytes. AB - The genotoxic potential of the administration of carbon tetrachloride alone or carbon tetrachloride to chlordecone-pretreated rats was investigated using an in vivo-in vitro animal model and a battery of biochemical assays to measure DNA repair in rat hepatocytes. Whereas carbon tetrachloride alone was not genotoxic, chlordecone or chlordecone in combination with carbon tetrachloride was genotoxic. The need for further investigation into the mechanism underlying the interaction between chlordecone and carbon tetrachloride is indicated strongly by the results of the present study. PMID- 1719057 TI - A simple and rapid experimental protocol for studies of nucleic acids metabolism and their base composition. AB - A suitable, simple and rapid protocol for metabolic studies of nucleic acids and determining their base composition, using reversed-phase high-performance liquid chromatography is described. Modified classic methods of isolation of the nucleic acids fraction from a biological material, in our particular case Artemia sp., were used. Then analysis of their constituents and the incorporated radioactivity, after hydrolytic processes, was performed by high-performance liquid chromatography under isocratic conditions, with 9 min total retention time. This method may be applied in several aspects of nucleic acids research, such as molecular cloning or metabolic and phylogenetic studies. PMID- 1719058 TI - Sympathetic influence on carotid chemoreceptor response to substance P in the cat. AB - Previous studies have suggested that substance P (SP) may play a role in the carotid chemoreceptor response to hypoxia. Given the data from these studies we speculated that within the carotid body hypoxia might release SP which then acts on the chemosensitive unit. Concomitantly SP might be released in the superior cervical ganglion (SCG) and increase sympathetic outflow to the carotid body by interacting with acetylcholine in the SCG. The resulting vasoconstriction in the carotid body would further increase neural output from the carotid body. Hence we hypothesized that the exogenous SP on the carotid chemoreceptor neural activity would decrease after eliminating preganglionic inflow into the SCG. The hypothesis was tested using anesthetized, paralyzed and artificially ventilated cats. Neural activity from the carotid body (carotid chemoreceptor activity) or from the SCG (ganglioglomerular efferent nerve activity (GGN)) was measured. Close intra-arterial administration of SP (10 micrograms) caused a sustained stimulation of the carotid chemoreceptor activity which was accompanied by a fall in arterial blood pressure. The magnitude and time-course of the carotid body responses were extremely variable among the cats. The duration of increased chemoreceptor activity was significantly shortened after a transection of the cervical sympathetic nerve (CVSN). As a control, the duration of carotid body stimulation produced by the second injection of SP in a group of sham-operated cats was measured. This was essentially the same as the first injection, suggesting that the tachyphylactic effect of SP was negligible. The effects of the commonly used pharmacological agents (nicotine, cyanide, dopamine) on carotid chemoreceptor activity were not affected by the transection of the CVSN, GGN activity was also increased by exogenous SP. These results suggest that the effect of exogenous SP on carotid chemoreceptor activity consists of two components: (1) an initial direct excitatory effect; (2) a slowly developing excitatory effect mediated by the sympathetic outflow to the carotid body. The effects could be augmented by the accompanying hypotension. PMID- 1719059 TI - Comparison of cyclocytidine and sympathetic nerve induced changes in norepinephrine and adrenoceptors in salivary glands. AB - Norepinephrine (NE) concentration of parotid and submandibular glands of young rats was reduced 51% and 39%, respectively at 1 h, and 60% and 47% at 2 h after i.p. administration of a single dose (500 mg/kg body weight) of the anti-tumor agent, cyclocytidine (CC). For adult rats, the reductions were 44% and 46%, respectively, at 1 h and 54% and 49% at 2 h. This decrease from controls was generally similar to the decrease induced following 1 and 2 h of electrical stimulation (square wave pulses of 4 V, 5 ms duration, and frequency of 16 Hz) of the sympathetic innervation to these glands (young rats, 59% and 58% at 1 h; 66% and 63% at 2 h; for adult rats, 51% and 55% at 1 h and 69% and 53% at 2 h for parotid and submandibular, respectively). The changes in density of beta adrenoceptors induced by direct nerve stimulation also corresponded to the changes induced by CC (CC induced a decrease in parotid of 12%, compared with a decrease of 11% with electrical stimulation; a 15% and 18% reduction in number of beta-adrenoceptors of submandibular gland was found at 1 h after CC and electrical stimulation, respectively). Compelling evidence for the mechanism of CC action was thus established, showing that CC mimics effects of sympathetic nerve stimulation (inducing reduction in NE concentration and transient change in beta-adrenoceptor density) by causing release of NE from sympathetic nerve endings. PMID- 1719060 TI - Effects of biological response modifiers on lung natural killer activity. AB - Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rIL-2), purified rat interferon alpha/beta (IFN-alpha/beta), or murine recombinant tumor necrosis factor alpha (rTNF-alpha) resulted in a dose dependent increase in LLNK activity. The maximum stimulation was similar for rIL 2 and IFN-alpha/beta, although a much higher concentration of IFN-alpha/beta was required to reach this level of stimulation. The maximum response to rTNF-alpha treatment was about half that seen with rIL-2 or IFN-alpha/beta and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rIFN-gamma) or murine recombinant interleukin 1 (rIL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-alpha/beta and rTNF-alpha that had little enhancing effect were able to synergize with a suboptimal dose of rIL-2, whereas rIL-1 and rIFN-gamma failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments. PMID- 1719061 TI - Inhibitory effect of MY-1250 on histamine release from rat peritoneal mast cells and guinea pig lung fragments: the elucidation of the mechanism. AB - When the effect of MY-1250 (5,6-dihydro-7,8-dimethyl-4,5-dioxo-4 H-pyrano [3,2-c] quinoline-2-carboxylic acid) on histamine release from rat peritoneal mast cells induced by compound 48/80 was studied, MY-1250 caused a significant inhibition of histamine release at concentrations higher than 3 microM. Furthermore, the compound inhibited not only 45C a uptake into the mast cells but also Ca2+ release from the intracellular Ca store at a concentration of 10 microM in both cases. By contrast, MY-1250 did not affect either histamine release from permeabilized mast cells induced by TPA, IP3 and GTP gamma S or Ca2+ release from the endoplasmic reticulum induced by IP3. In the chopped lung preparations, MY 1250 at doses of 10 and 100 microM caused a significant inhibition in histamine release from the pieces of actively sensitized guinea pigs exposed to antigen and simultaneously prevented a decrease in intracellular cAMP contents taking place in association with antigen-antibody reaction. No significant changes were effected by MY-1250 in alpha-chymotrypsin activity and phospholipase A2 activity. Also, no antagonistic effects on LTD4 and PAF were observed. PMID- 1719062 TI - The immunohistochemical evidence of amyloid diffuse deposits as a pathological hallmark in Alzheimer's disease. AB - We performed an immunocytochemical study of cerebral cortex from cases of Alzheimer's disease and from aged nondemented controls, using periodic acid pretreatment and polyclonal beta-protein antibodies. In addition to senile plaques (SP) and amyloid angiopathy (AA), the beta-protein antibodies detected band-like deposits present throughout the cortical layers. Moreover, large plaque like infiltrations with diffuse and amorphous characteristics were observed in the cortical gray and white matter, and these deposits were often associated with capillaries. Our results suggest that an abundance of these lesions, which were detected only with this immunostaining procedure in Alzheimer cortex, may be characteristic of Alzheimer's disease. PMID- 1719063 TI - Effect of long-term vigabatrin therapy on selected neurotransmitter concentrations in cerebrospinal fluid. AB - Ten patients, suffering from drug-resistant complex partial seizures were treated for a period of up to 3 years with vigabatrin (Sabril). Vigabatrin is a novel antiepileptic agent, whose action is based on the inhibition of gamma aminobutyric acid (GABA) aminotransferase, the enzyme responsible for the catabolism of the neurotransmitter GABA. Samples of lumbar cerebrospinal fluid were obtained from the patients prior to commencing vigabatrin therapy, and thereafter at 6 months, 1 year, 2 years, and up to 3 years following the initiation of vigabatrin treatment. The influence of vigabatrin on the cerebrospinal fluid concentrations of free and total GABA, homocarnosine, homovanillic acid, 5-hydroxyindoleacetic acid, and 3-methoxy-4 hydroxyphenylethylene glycol, as well as of the drug itself, was assessed. All patients demonstrated a clinical response to vigabatrin, and the drug was well tolerated over the entire observation period. Mean (+/- SD) reduction of seizure frequency was 65% +/- 23% (range, 26% to 100%) when comparing the end of the treatment period to the previgabatrin baseline. The cerebrospinal fluid concentrations of both free and total GABA and of the dipeptide homocarnosine showed approximately 2- to 5-fold increases over baseline values, with free GABA and homocarnosine being the more sensitive variables. Cerebrospinal fluid concentrations of homovanillic acid, 5-hydroxyindoleacetic acid, and 3-methoxy-4 hydroxyphenylethylene glycol were not altered in a significant manner over the observation period. These findings support the concept that the effects of vigabatrin are restricted to an effect on GABA catabolism and do not extend to the neurotransmitters dopamine and norepinephrine. Clinical efficacy and elevation of GABA and homocarnosine concentration were sustained over the period of observation. PMID- 1719064 TI - Sudden infant death syndrome: altered aminergic-cholinergic synaptic markers in hypothalamus. AB - Alterations of sleep are reported to occur in sudden infant death syndrome (SIDS). It is well established that the hypothalamus mediates the onset, maintenance, and timing of sleep, and does so via serotonergic and cholinergic mechanisms. We have investigated serotonergic and cholinergic synaptic markers in the hypothalamus from eight SIDS infants and six age-matched non-SIDS infants between 3 and 7 months of age. By use of established methods, we observed a number of chemical alterations in SIDS hypothalamus: (1) tryptophan content was increased and serotonin content was decreased, (2) serotonin binding was increased and imipramine binding was unchanged, (3) monoamine oxidase-A activity was increased without an effect on monoamine oxidase-B, and (4) choline acetyltransferase activity was decreased and acetylcholinesterase activity was unchanged. PMID- 1719065 TI - Sensory and motor fiber differentiation with Karnovsky staining. AB - We examined four acetylthiocholine methods based on Karnovsky's procedure--two fast-acting requiring 1 hour and two slow-acting requiring 24 hours. We compared these with our modification, which requires less than an hour and is simple to use. Rabbit sciatic nerves and spinal cords were used to compare methods. Our modification showed clearer differentiation than other fast-acting methods and staining identical to slow-acting methods. In blind examination of radial nerve specimens stained with our method, motor and sensory fascicles were correctly identified, showing sensitivity and specificity of 100%. In 12 clinical cases, our method produced staining in the proximal stump as long as 16 months after injury and in the distal stump as long as 5 days after injury. In 10 of 12 patients, this staining helped in aligning motor fascicles to motor fascicles and sensory fascicles to sensory fascicles. PMID- 1719066 TI - Quality of gastric ulcer healing: a new, emerging concept. AB - Assessment of gastric ulcer healing is usually based on a visual examination (by endoscopy in patients, or the evaluation of ulcer size in experimental studies), and not on histologic and ultrastructural assessment of subepithelial mucosal healing. This approach has led to the assumption that the mucosa of grossly "healed" gastric and/or duodenal ulcers returns to normal, either spontaneously or following treatment. However, the re-epithelialized mucosa of grossly "healed" experimental gastric ulcer has recently been found to have prominent histologic and ultrastructural abnormalities, including reduced height, marked dilation of gastric glands, poor differentiation and/or degenerative changes in glandular cells, increased connective tissue, and disorganized microvascular network. It has been postulated that these residual abnormalities might interfere with mucosal defense and may be the basis of ulcer recurrence. In the present article, the ulcer healing process and the role of luminal factors, transitional zone at the ulcer margin, and granulation tissue are discussed. The healing of an ulcer is accomplished by filling of the mucosal defect with epithelial cells and connective tissue to reconstruct mucosal architecture. Under influence of growth factors [predominantly epidermal growth factor (EGF) and transforming growth factor (TGF alpha)], the epithelial cells at the ulcer margin dedifferentiate and proliferate, supplying cells for re-epithelialization of the mucosal scar surface and reconstruction of glandular structures. Granulation tissue at the ulcer base supplies connective tissue cells to restore the lamina propria and endothelial cells and microvessels for mucosal microvasculature reconstruction. The final outcome of healing reflects a dynamic interaction between an "epithelial" component from the ulcer margin and a connective tissue component including microvessels originating from granulation tissue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719067 TI - Use of acetic orcein as a fast method to relax, fix, clear and stain cercariae for light microscopy. AB - Acetic orcein stain may be dropped onto the specimen before applying a cover slip. This single reagent reveals cercarial and metacercarial structures that would otherwise be made visible only by complicated and time consuming procedures. PMID- 1719068 TI - Functional Golgi units in microtubule-disrupted cultured atrial myocytes. AB - We examined the effects of disassembly of microtubules (MT) on the structure and the functions of the Golgi apparatus (GA) in cultured atrial myocytes. MT disassembly with nocodazole led to fragmentation of the GA into small units. The fragmented Golgi units retained their cis-trans polarity and post-cisternal elements, including the trans-Golgi network (TGN). Neither endocytosis of lectin labeled membrane nor its delivery to the fragmented Golgi units was interrupted by fragmentation of the GA after MT disassembly with nocodazole treatment. A fraction of the secretory granules associated with the fragmented Golgi units was also labeled with the internalized tracer. These results suggest that in nocodazole-treated cultured atrial myocytes, the fragmented Golgi units appear to be structurally and functionally intact despite the altered geometric arrangement of the GA in the cells. PMID- 1719069 TI - A specific ultrastructural method to reveal DNA: the NAMA-Ur. AB - We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10 12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level. PMID- 1719070 TI - Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase. AB - We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites. PMID- 1719071 TI - Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study. AB - The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions. PMID- 1719072 TI - Use of the dinitrophenyl hapten sandwich staining procedure (DHSS) to localize estrogen receptors in paraffin-embedded tissues. AB - To date, reliable and sensitive methods to localize the estrogen receptor (ER) in rat tissues and human breast cancers have required the use of frozen sections. This not only incurs poor tissue structure but also precludes the study of small breast lesions that are usually paraffin embedded for histological evaluation. We have developed and optimized a dinitrophenyl hapten sandwich staining (DHSS) immunocytochemical procedure to demonstrate ER in paraffin-embedded, hormone sensitive tissues of the rat and in human breast cancers. The method was applicable to formalin- and Bouins-fixed material, with trypsinization of sections being essential. The immunocytochemical system utilized a dinitrophenyl (DNP) hapten-labeled monoclonal antibody to the receptor. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase complex, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This highly sensitive method localized the ER within paraffin-embedded rat uterus, fallopian tube, vagina, and normal and cancerous mammary gland. Furthermore, excellent staining was generated in human breast cancers in accordance with their ER-ICA status. Control sections involving simultaneous incubation with DNP-labeled and unlabeled H222 were background free, while uteri from castrated rats demonstrated reduced receptor immunostaining. Staining was also absent in ER-negative human breast tumors. PMID- 1719073 TI - Localization and synthesis of alpha-fetoprotein in chorioallantoic membrane from chick embryo. AB - Alpha-fetoprotein (AFP) is a major globulin of the embryonic serum of mammals, birds, and other vertebrates. It is synthesized chiefly by the liver and/or the yolk sac. The aim of this work was to confirm the occurrence of AFP in the chorioallantoic membrane (CAM) from 14-day chick embryo. AFP had previously been detected by immunoelectrophoresis in CAM extracts under the suspicion that it could be a mere artifact resulting from blood contamination. The immunohistochemical study of the CAM carried out for this purpose revealed the protein to be solely located in the mesodermal layer. The joint use of organ culture and immunoperoxidase techniques has enabled us to find evidence for the synthesis of AFP in the cells of this layer. These results confirm the occurrence of such a significant carrier globulin to embryonic development in one more tissue that can be added to the short list of AFP-producing tissues. PMID- 1719074 TI - Pertussis toxin prevents the induction of peripheral T cell anergy and enhances the T cell response to an encephalitogenic peptide of myelin basic protein. AB - In a murine model of T cell-mediated autoimmune disease, experimental autoimmune encephalitis (EAE), 80% of all encephalitogenic T cell clones in H-2u mice use the V beta 8.2 TCR element. To induce EAE in susceptible strains of mice either heat-killed Bordetella pertussis organisms or Bordetella pertussis toxin (PT) must be injected in addition to Ag in CFA. We investigated the mechanisms by which PT facilitates the induction of EAE. Our data show, that PT interferes with the induction of Ag-induced peripheral T cell anergy. Furthermore it has a specific adjuvanticity for the autoantigen pAc1-11 in vivo and acts as a selective mitogen in vitro. We also tested the hypothesis that PT is a bacterial superantigen that specifically expands the V beta 8.2+ subset of T cells, thereby expanding the encephalitogenic T cell clones that are contained in this subset, so that the number of autoreactive T cells is brought over a critical threshold, necessary to induce autoimmune disease. Our data show that PT is not a superantigen. Staphylococcal enterotoxin B, a V beta 8.2-specific superantigen, does not enhance the immune response to the encephalitogenic peptide. PMID- 1719075 TI - Development of a bone marrow culture for maintenance and growth of normal human B cell precursors. AB - The absence of long term bone marrow cultures for studying the growth and differentiation of human B cell precursors (BCP) has placed restrictions on the ability to analyze the early stages of human B cell ontogeny. We now describe a bone marrow-derived adherent cell microenvironment that maintains human BCP for several weeks in vitro. The adherent cells are maintained in a serum-free tissue culture medium, and consist of a predominant population of CD10+ fibroblast-like cells and a minor population of CD10+/nonspecific esterase+ macrophages. Adherent cell cultures seeded with fresh or cryopreserved fetal bone marrow, or purified CD10+/surface IgM- cells, provide a supportive microenvironment for lymphoid cells with a predominant phenotype of CD10+/CD19+/HLA-DR+/surface IgM-. Supplementation of the adherent cell cultures with human IL-7 induces active growth of BCP during the first 14 to 21 days of culture. However, the expansion of these cells does not continue past 21 days, and the cultures undergo a steady decline in BCP. Analysis of adherent cell conditioned medium revealed the presence of an unidentified soluble factor (or factors) that acts in concert with IL-7 to promote the growth of CD10+/surface IgM- cells. This culture system will be useful in elucidating the patterns of gene expression and growth factor requirements that characterize normal human B cell ontogeny, and perturbations of normal B cell ontogeny that lead to immunodeficiency and leukemia. PMID- 1719076 TI - A monoclonal antibody (OT145) specific for the T cell antigen receptor V beta 6.7a allele detects an epitope related to a proposed superantigen-binding site. AB - A mAb, OT145, recognizes a TCR allotype encoded by one of two alleles of the V beta 6.7 gene. The peptide products of the two V beta 6.7 alleles differ because of nonconservative amino acid substitutions at positions 38 and 72. V beta 6.7a encodes ser38 and gly72, whereas V beta 6.7b encodes arg38 and glu72. We show here that the binding of mAb OT145 ot the beta-chain of TCR is lost when residue 72 of V beta 6.7a is mutated from gly to glu. The binding of OT145 is not affected by mutation of residue 38 from ser to arg. Thus, OT145 recognizes an epitope related to position 72. Residue 72 of the beta-chain of the TCR is located at a putative superantigen-binding site. PMID- 1719077 TI - Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation. AB - The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL 2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis. PMID- 1719078 TI - Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophil degranulation. AB - Stimulation of human normodense eosinophils with immobilized secretory IgA (sIgA) or IgG, or with the soluble stimulus, FMLP, triggers the exocytotic release of the granule protein, eosinophil-derived neurotoxin (EDN). In this report, we demonstrate that these stimuli also provoke an increase in phospholipase C mediated phosphoinositide breakdown in eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) for 2 h irreversibly abolished the increases in phospholipase C activity and EDN release induced by immobilized sIgA or FMLP. In contrast, PTX treatment only transiently inhibited eosinophil activation induced by immobilized IgG. Maximal inhibition of IgG-stimulated phosphoinositide hydrolysis and EDN release occurred after 2 h of PTX pretreatment with PTX, followed by a gradual recovery of cellular responsiveness to immobilized IgG as the duration of PTX pretreatment was extended to 16 h. Activated PTX catalyzed the in vitro ADP-ribosylation of 41- and 44-kDa proteins in eosinophil membranes. A 2-h pretreatment of intact cells with PTX markedly reduced the pools of unmodified 41- and 44-kDa substrates available for subsequent ADP-ribosylation in vitro, suggesting that both proteins were substrates for PTX in intact eosinophils. Continuous exposure of eosinophils to PTX for times ranging from 2 to 15 h resulted in the gradual reappearance of unmodified 44-kDa protein, whereas the levels of unmodified 41-kDa protein were persistently reduced in PTX treated cells. The time course of the decline and reappearance of unmodified 44 kDa substrate in PTX-treated eosinophils closely paralleled the changes in the responsiveness of these cells to immobilized IgG. These results suggest that the receptors for sIgA, FMLP, or IgG transduce activating signals for eosinophil degranulation through differential coupling to at least two PTX-sensitive G proteins. PMID- 1719079 TI - Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes. AB - Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8 37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function. PMID- 1719080 TI - The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics. AB - We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity. PMID- 1719081 TI - Vaccine-induced CD4+ T cells against the simian immunodeficiency virus gag protein. Epitope specificity and relevance to protective immunity. AB - We have examined the induction and epitope specificity of T cells for the simian immunodeficiency virus (SIV) gag p27 protein in macaques immunized with either a recombinant SIV gag protein or an inactivated SIV vaccine. CD4+ MHC class II restricted T cell lines and clones derived from five immunized macaques recognized a total of seven peptides in three immunodominant regions of p27. Two T cell clones generated from one of the lines, recognized a single 20 amino acid peptide that overlapped with a region previously shown to include a CTL epitope from SIV-infected macaques. Although this epitope is in a conserved region of the gag protein of SIV, its recognition by a CD4+ T cell clone was abrogated by sequence variation in the equivalent HIV protein. The specificity of the T cell lines for synthetic peptides demonstrated considerable overlap between T cells generated by immunization with the recombinant gag protein and inactivated SIV. However, in contrast to the protective efficacy of the whole virus vaccine in the syntex adjuvant formulation, immunization with the p27 protein with alum failed to generate a protective immune response. Furthermore, despite the consistent gag specific T cell responses induced by the recombinant protein, there was no evidence of an enhanced antibody response to envelope (env) after live SIV challenge. PMID- 1719082 TI - Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. AB - The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein. PMID- 1719083 TI - The CD19 complex of B lymphocytes. Activation of phospholipase C by a protein tyrosine kinase-dependent pathway that can be enhanced by the membrane IgM complex. AB - We have investigated the mechanism by which the membrane protein complex of the B lymphocyte that contains CD19 and CR2 activates phospholipase C (PLC) to induce a rise in [CA2+]i. The CD19 complex resembled the membrane IgM complex in that three protein tyrosine kinase inhibitors suppressed increases in [Ca2+]i and inositol bisphosphate and inositol triphosphate generation. However, the activation of PLC by the CD19 complex could be distinguished from that by the membrane IgM complex by slower kinetics of generation of inositol phosphates, resistance to inhibition by activators of protein kinase C, and different pattern of tyrosine-phosphorylated cellular substrates. Western blot analysis of lysates from cells stimulated by the CD19 complex demonstrated a single new phosphotyrosine-containing protein of 85 kDa, whereas multiple other phosphotyrosine-containing proteins were present in cells activated by the mIgM complex. In particular, PLC-gamma 1, which is a substrate for the protein tyrosine kinase activated by the mIgM complex, was not tyrosine-phosphorylated in cells stimulated by the CD19 complex. Cross-linking the two complexes together caused a synergistic increase in [CA2+]i which was neither suppressed by activation of protein kinase C nor associated with increased tyrosine phosphorylation of PLC, characteristic of the CD19 pathway. Therefore, the B cell has two signal transduction complexes, associated with membrane IgM and CD19, that activate PLC by different mechanisms and that can synergistically interact to enhance this function by the CD19 pathway. PMID- 1719084 TI - Identification and characterization of a T cell-inducing epitope of bovine ribonuclease that can be restricted by multiple class II molecules. AB - An immunodominant epitope of bovine RNase restricted by I-Ek molecules was identified using a T cell hybridoma recognizing RNase. This epitope was localized to the peptide RNase(90-105). Single conservative amino acid substitutions were made at each of the positions 94 through 105. It was found that only at one position, Asn-103, were conservative substitutions not allowed. This residue was shown to be the critical residue in determining T cell specificity. The ability of RNase(90-105) and the well-defined T cell epitope, HEL(46-61) to stimulate mouse strains expressing different independent H-2 haplotypes was examined using a T cell proliferation assay. The response to HEL(46-61) was completely restricted to mice expressing an I-Ak molecule. In striking contrast, 6 of 10 different mouse strains, H-2b,f,k,q,s,u, mounted vigorous T cell responses to RNase(90-105). The response was restricted to both I-A and I-E molecules, including I-Ab, I-Af, I-Ek, I-Aq, and I-As. H-2d mice were nonresponders to RNase(90-105), which was shown to be due to the failure of RNase(90-105) to bind to I-Ad molecules. A variant RNase(90-105) peptide was generated, containing an I Ad binding motif, that could bind to I-Ad molecules. Despite its ability to bind, this variant peptide was not able to stimulate a response in H-2d mice. This result demonstrates that the ability of a peptide to bind to an Ia molecule is necessary but not always sufficient for a response to occur. Thus, in contrast to the highly restricted HEL(46-61) determinant, the RNase(90-105) determinant is permissive in its binding to Ia molecules. These results show that in the universe of T cell inducing epitopes contains both highly restricted and broadly restricted epitopes are found. PMID- 1719085 TI - Induction of phorbol ester responsiveness in conventional B cells after activation via surface Ig. AB - The signals required to induce S phase entry in murine splenic B cells were found to be altered by prolonged treatment with low doses of anti-Ig antibody. Whereas fresh splenic B cells are stimulated by the combination of a phorbol ester protein kinase C agonist plus a calcium ionophore, anti-Ig-treated splenic B cells were stimulated by phorbol ester alone, in the absence of a comitogen. The majority of these phorbol ester responsive B cells expressed CD5. The phorbol ester responses of anti-Ig-treated splenic B cells paralleled those previously reported for untreated peritoneal CD5+ B cells in a number of respects: responses were not idiosyncratic to phorbol esters but occurred with nonphorbol protein kinase C agonists; phorbol ester responses were enhanced by IL-4; and, phorbol ester responses occurred rapidly and were greater at 24 than at 48 h. However, the effect of agents that act to raise intracellular levels of cAMP distinguished between anti-Ig-treated splenic B cells and untreated peritoneal B cells in that the phorbol ester responses of the former were enhanced whereas the responses of the latter were inhibited. The present results add a functional dimension to the phenotypic similarity between splenic B cells treated with anti-Ig and resident peritoneal B cells that constitutively express CD5; however, some differences in behavior were noted. PMID- 1719086 TI - CD14 contributes to the adherence of human monocytes to cytokine-stimulated endothelial cells. AB - Monocyte adherence to endothelial cells (EC) is selectively increased during inflammation. The mechanisms underlying monocyte-EC interaction indicated the involvement of surface-adhesion molecules on monocytes and EC. In earlier studies we noticed that the monocyte-specific mAb, designated mAb 63D3, in contrast to mAb against the beta 2-integrin molecules, inhibited the monocyte binding to monolayers of rIL-1 alpha-stimulated venous EC. The aim of the present study was to further characterize the Ag recognized by mAb 63D3 and to investigate the specific contribution of this Ag to the adherence of monocytes to cultured human macrovascular venous or arterial EC. Flow cytometric analysis demonstrated that the 63D3 Ag is expressed exclusively on the surface of peripheral blood monocytes. SDS-PAGE analysis of mAb 63D3 immunoprecipitates of 125I-labeled human monocyte surface proteins revealed that the target Ag for mAb 63D3 is a 52- to 55 kDa molecule identical to the myeloid differentiation protein CD14. Stimulation of EC with rIL-1 alpha or rTNF-alpha for 4 or 24 h or rIFN-gamma for 24 h increased (p less than 0.005) the number of monocytes bound to both types of EC. This cytokine-induced increase in monocyte adherence was significantly (p less than 0.0005) inhibited when the monocytes were coated with various mAb against CD14. The binding of monocytes to nonstimulated venous or arterial EC was not inhibited by anti-CD14 mAb. Our results lead to the conclusion that CD14 molecules, which on basis of their structure and m.w. are not related to the beta 2-integrin family of heterodimeric leukocyte adhesion molecules, participate in the binding of monocytes to cytokine-stimulated EC. PMID- 1719087 TI - Biochemical analysis of the immune B cell defect in xid mice. AB - Previous studies have shown that B cells from xid immune defective CBA/N mice that are unresponsive do not proliferate after stimulation with unconjugated anti Ig. The experiments in this manuscript demonstrate that dextran-anti-Ig conjugates, which induce extensive and prolonged sIg cross-linking, are able to stimulate proliferation of xid B cells. The ability of these conjugates to stimulate proliferation of xid B cells is not related to their ability to stimulate higher levels of PIP2 breakdown. Thus, high concentrations of unconjugated anti-Ig antibody, which are nonmitogenic for xid B cells, stimulate higher levels of PIP2 breakdown and of calcium transients than lower concentrations of dextran-conjugated anti-Ig, which are mitogenic. Although unconjugated anti-Ig does not provide a fully competent signal to stimulate proliferation of xid B cells, it induces a sufficiently stimulatory signal to enable them to enter DNA synthesis in the presence of the protein kinase C activator, indolactam. This suggests that the extent or duration of activation of protein kinase C by anti-Ig may be limiting in xid B cells. To examine whether another recently described pathway of B cell activation is defective in these mice, we studied the induction of early anti-Ig-mediated tyrosine kinase activity in xid B cells. Both unconjugated and dextran-conjugated anti-Ig antibody stimulated comparable but not identical patterns of tyrosine phosphorylation. These data taken together with other findings that the combination of phorbol ester and calcium ionophore stimulates high levels of proliferation in xid B cells suggests that the immune defect of xid B cells may be distal to surface Ig mediated activation of tyrosine kinase and of PIP2 breakdown but proximal to PKC activation. Alternatively, the xid immune defect may not result from abnormalities in the early signal transduction pathways, but rather from more distal and/or as yet undefined pathways leading to B cell activation. PMID- 1719088 TI - Hyperstimulatory CD1a+CD1b+CD36+ Langerhans cells are responsible for increased autologous T lymphocyte reactivity to lesional epidermal cells of patients with atopic dermatitis. AB - We studied whether abnormalities in epidermal APC could be responsible for intracutaneous T cell activation in atopic dermatitis (AD). In the absence of added Ag, patients' peripheral blood T cells demonstrated significantly increased proliferation to their autologous lesional epidermal cells (mean +/- SEM = 19,726 +/- 9,754 cpm [3H]TdR uptake) relative to epidermal cells from uninvolved AD skin (2179 +/- 697 cpm) (n = 10) (p = 0.0001, log transformed data). AD T cell proliferative responses to autologous epidermal cells were dependent upon cells expressing HLA-DR, CD1a, and CD36, and not upon keratinocytes or their cytokines. Ultrastructurally, these cells ranged from typical Langerhans cells to indeterminate cells with irregular nuclear contours. Enriched populations of lesional AD Langerhans cells were highly stimulatory for autologous T cells, whereas equal numbers of Langerhans cells from non atopic epidermis were poor stimulators, even at high concentrations. The dermal perivascular dendritic cell markers CD36 and CD1b, not usually present on normal epidermal APC, were expressed by 40 and 60% of lesional AD CD1a+ epidermal Langerhans cells, respectively. Addition of anti-CD1b to cocultures of AD epidermal cells and autologous T lymphocytes augmented T cell activation, suggesting that the expression of CD1b by AD Langerhans cells may represent over expression of a molecule functionally linked to the enhanced T cell stimulatory capacity of these cells. Thus, stimulatory signals for T cells contained within AD epidermis are carried by cells in an abnormal differentiation state as indicated by expression of phenotypic characteristics of both epidermal and dermal antigen presenting cells (HLA-DR+, CD1a+, CD1b+, CD36+). We propose that activation of autologous T cells by an altered cutaneous APC population may represent a mechanism for the hyperactive and disordered cell-mediated immune response that characterizes the dermatitic lesions of AD. PMID- 1719089 TI - Location of a new encephalitogenic epitope (residues 43 to 64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in PL/J and (SJL x PL)F1 mice. AB - Synthetic peptides of proteolipid protein (PLP) were screened for their ability to induce experimental autoimmune encephalomyelitis (EAE) in SJL/J, PL/J, and (SJL x PL)F1 mice, and T cell lines were selected by stimulation of lymph node cells with PLP peptides. PLP 141-151 was found to be less encephalitogenic in SJL/J mice than PLP 139-151, due to deletion of two amino acids from the amino terminal end. PLP 139-151 immunization induced relapsing EAE in SJL/J and F1 mice but not PL/J mice. In contrast, PLP 43-64 induced relapsing EAE in PL/J and F1 mice but not SJL/J mice. F1 T cell lines specific for either PLP 43-64 or PLP 139 151 adoptively transferred demyelinating EAE to naive F1 recipients. Haplotypes H 2s and H-2u appear to be immunologically co-dominant in F1 mice in the PLP EAE system, which differs from the H-2u dominance in F1 mice in the myelin basic protein EAE system. The identification of a PLP peptide that is encephalitogenic in PL/J mice, in addition to the previous demonstration of PLP peptides that are encephalitogenic for SWR mice (PLP 103-116) and SJL/J mice (PLP 139-151), lends support to a role for PLP as a target Ag in autoimmune demyelinating diseases. PMID- 1719090 TI - IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells. AB - We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma. PMID- 1719092 TI - Involvement of the CD11b/CD18 integrin, but not of the endothelial cell adhesion molecules ELAM-1 and ICAM-1 in tumor necrosis factor-alpha-induced neutrophil toxicity. AB - TNF-alpha can incite neutrophil-mediated endothelial cell damage and neutrophil H2O2 release. Both effects require adherent neutrophils. Using specific mAb, we showed in this in vitro study that the CD18 beta 2-chain and the CD11b alpha M chain of the CD11/CD18 integrin heterodimer have a major role in both TNF-alpha induced neutrophil-mediated detachment of human umbilical vein endothelial cells and H2O2 release by TNF-alpha-activated human neutrophils. In contrast to anti CD18 mAb, which consistently prevented neutrophil activation, anti-CD11a mAb and two of three anti-CD11b mAb did not reduce endothelial cell detachment and neutrophil H2O2 release, although they decreased neutrophil adhesion to human umbilical vein endothelial cells. mAb 904, directed against the bacterial LPS binding region of CD11b, reduced endothelial cell detachment for about 40% and neutrophil H2O2 release for more than 50%, demonstrating that CD11b/CD18 is engaged in TNF-induced neutrophil activation. Dependence on CD11b/CD18 could not be overcome by CD18-independent anchoring of neutrophils via PHA. Additionally, neither induction of increased expression of the endothelial cell adhesion molecules ICAM-1 and ELAM-1, nor subsequent addition of specific mAb, influenced endothelial cell injury or H2O2 release by TNF-activated neutrophils. Interaction with ICAM-1 and ELAM-1 therefore appears not to induce additional activation of TNF-stimulated neutrophils. These studies suggest that a specific, CD11b/CD18 mediated signal, instead of adherence only, triggers toxicity of TNF-activated neutrophils. PMID- 1719091 TI - Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells. AB - Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag. PMID- 1719093 TI - Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope. Production, characterization, and interaction with the kinin receptor. AB - mAb against bradykinin, the prototypic member of the kinin family of vasodilator peptides, were generated by somatic cell fusion. The antibodies were isotyped as IgG1, kappa-type, and their target epitopes mapped with bradykinin, lysyl bradykinin (kallidin), kinin receptor antagonists, and fragments thereof, revealing three distinct sets of mAb, i.e., mAb against bradykinin (MBK)1, MBK2, and MBK3. Comparison of the immunologic binding affinities and the known pharmacologic binding specificities of bradykinin derivatives disclosed a striking similarity in the binding profiles of mAb MBK3 and the B2 type of the kinin receptor. Anti-idiotypic antibodies against MBK1, MBK2, and MBK3 were raised in rabbit and sheep. Inhibition and competition experiments on the level of the Ag (ligand), the idiotype, and the anti-idiotype demonstrated the mutual specificity of the network system components. Anti-idiotypic antibodies against MBK3 recognized a particular idiotope that was conformation-dependent and associated with the Ag binding site of the antibody. Binding of anti-idiotypic antibodies to the B2 receptor expressed by human foreskin fibroblasts and guinea pig ileum demonstrated that the anti-idiotypes cross-react with the corresponding receptor across species. Specific stimulation of the inositol phosphate pathway in human fibroblasts and of the PG pathway in mouse fibroblasts, respectively, and inhibition of the latter effect by the B2 kinin receptor antagonist NPC 567 indicated that the anti-idiotypes bear the internal image of a bradykinin epitope. Furthermore, antibodies of the third generation (anti-anti-idiotypic antibodies) recognized the authentic Ag, i.e., bradykinin. Hence, the anti idiotypic approach provides powerful tools to probe for the hitherto poorly characterized B2 kinin receptor. PMID- 1719094 TI - Complement activation induces the expression of decay-accelerating factor on human mesangial cells. AB - In the present study we evaluated the effect of complement activation by immune complexes (IC) on the expression of decay-accelerating factor (DAF) on human mesangial cells (MC). MC in culture were incubated with an Ag (DNP-Gelatin) that binds to fibronectin present in the MC matrix. Subsequently, MC were incubated with anti-DNP antibodies in the presence of human serum. By immunoperoxidase staining we showed that these incubations resulted in IC formation and deposition of human C3 and terminal complement components (C5b-9) on the mesangial matrix and on the surface of MC. By immunoperoxidase staining and by RIA we showed that IC formation and complement activation significantly increased DAF expression on the MC plasma membrane. The induction of DAF expression was a consequence of deposition of terminal complement components on the MC because, zymosan-activated serum and IC formation in the presence of C5- or C8-deficient serum failed to increase MC DAF expression. Furthermore, the observed increased DAF expression was the consequence of increased DAF synthesis by MC. Thus, both cycloheximide and actinomycin D blocked the increase on MC DAF observed after incubation with IC and serum. MC DAF had biophysical and functional characteristics similar to DAF in other cells. Thus, 1) MC DAF was resistant to trypsin but was removed from the MC membrane by pronase; 2) phosphatidylinositol-specific phospholipase C removed 48 +/- 4% of MC DAF indicating that MC DAF is anchored in the cell membrane by phosphatidylinositol groups; 3) DAF isolated from MC-inhibited complement-mediated hemolysis and demonstrated a molecular mass of 83 kDa. In conclusion, deposition of terminal complement components on human MC trigger new synthesis and membrane expression of DAF. Because DAF protects cells against complement-mediated lysis, we postulate that DAF may protect glomerular cells during IC and complement-mediated glomerulonephritis. PMID- 1719095 TI - Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. I. Role in the anti-S. mansoni antibody response. AB - Previous data have shown that from an antiparasitic IgE mAb (mAb1), antianti-Id IgG and IgE antibodies (Ab3) could be prepared. These Ab3 demonstrated the same functional properties as the Ab1, such as in vitro cytotoxic activity toward schistosomula and in vivo a protective effect against Schistosoma mansoni infection. To study the possible interactions between the idiotypic network and the regulation of isotypic expression, we focused on Id-specific T cells obtained by immunization with Ab2. Both Ab2 idiotopes and native schistosomula Ag were able to stimulate the proliferation of anti-Ab2 T cells in vitro. The activation of anti-Ab2 T cells by Ab2 shared the classic characteristics of Th cells, namely, it was MHC-restricted and required APC. A T cell line could be maintained in long term culture by stimulation with schistosomula Ag. The adoptive transfer of cells from this line to 26-kDa Ag-immunized or S. mansoni-infected rats led to a dramatic increase in the specific humoral response. This effect was restricted to antibodies specific for 26- and 56-kDa Ag (the targets of the mAb1) and was observed for the two isotypes tested, i.e., IgG and IgE. Finally, the helper effect on the antibody response could be further amplified by cooperation of anti Ab2 T cells with Id-specific cells of the first generation (anti-Ab1 cells). Together with Ag-specific Th cells, the Id-specific T cells may, due to their specificity and their functional properties, play a major role in the induction and more importantly, in the maintenance of the immune response. PMID- 1719096 TI - Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. II. Induction of protective immunity in experimental rat schistosomiasis. AB - In our previous work on the idiotypic network in the rat model of schistosomiasis we showed that immunization with an IgE mAb specific for 26/56-kDa parasitic Ag resulted in the production of anti-anti-Id antibodies of both the IgG and IgE classes. Further studies demonstrated that anti-Ab2 T cell lines, obtained by immunization with Ab2 antibodies, functioned as conventional Th cells; they were MHC-restricted and required APC to proliferate in the presence of the native schistosomula Ag and the Ab2 antibodies. We report the involvement of these anti Ab2 cells in the regulation of protective immunity. The transfer of long term culture anti-Ab2 T cell lines into LOU/M rats, followed by a challenge infection by Schistosoma mansoni 1 day after the cell transfer led to a slight increase in the worm burden. On the contrary, the transfer of anti-Ab2 T cells 90 days before S. mansoni infection induced a significant reduction of the worm burden (up to 57%). T cells recovered from the protected rats were stimulated by the native schistosomula Ag as well as by tryptic fragments of IgG isolated from the Ab2 sera, in the presence of irradiated thymic cells as APC. We also analyzed the humoral response developed by the rats after transfer with the anti-Ab2 T cell lines. The sera induced various inflammatory cells into cytotoxic effectors against the larvae of S. mansoni, arguing for the presence of functional IgE in the sera. Moreover, when these sera were passively transferred into rats infected 1 day later, a significant reduction of the worm burden was observed. However, antibody-dependent cytotoxic mechanisms efficient 10 days after the anti-Ab2 T cell transfer did not correlate with the protective immunity which required a 90 day delay to be established. These data suggest that the protective immunity induced by the anti-Ab2 cells is supported both by the cellular and humoral components and that in a future vaccinating strategy the idiotypic network may play a crucial role. PMID- 1719097 TI - Analysis of cis-acting elements present in the CD20/B1 antigen promoter. AB - A genomic clone spanning a large portion of the 5' untranscribed region of the CD20 gene was isolated. Deletion analysis of subcloned fragments identified several regulatory elements. A major positive cis-acting element was localized between base pairs -290/-186. A second positive regulatory element was localized between -454/-280 and negative regulatory elements were present in the region between bp -828/-454. The sequence -280/-186 conferred B cell-specific expression on a heterologous, TATA box containing c-fos promoter. Electrophoretic mobility shift assays with overlapping oligonucleotide probes spanning -280/-186 revealed that a 25-bp probe (-225/-201) bound a nuclear protein present in B cell lines expressing the CD20/B1 antigen but not in Jurkat (T cell), U937 (promonocytic), U251 (glioma), or HeLa cells. To confirm the functional significance of this sequence, a trimer of this region was subcloned into the c-fos promoter containing CAT plasmid. Expression was observed only in BJA-B and HS-Sultan cells but not in CD20/B1- cell lines. This sequence element is also important in phorbol ester-induced CD20 expression in the pre-B cell line BP-697. These results partially characterize several regulatory elements present in the CD20 promoter that are likely important in the B cell-specific expression of the CD20 gene. PMID- 1719098 TI - The use of peptide-mediated electrofusion to select monoclonal antibodies directed against specific and homologous regions of the potyvirus coat protein. AB - Whilst monoclonal antibodies (Mab) to potyviruses have been generated, it has not been possible to produce molecules with high specificity or broad reactivity to defined conserved amino acid sequences. In the current study, peptide-mediated electrofusion was used to select for high efficiency antibody-secreting hybridomas after mice were immunized with highly immunogenic viral coat protein. Mice were immunized with coat protein from either one potyvirus (potato virus Y, PVY-D) or a mixture of five distinct potyviruses. Two well-defined peptides were used for selective electrofusions. Peptide-1 was selected from the highly specific N terminal region of PVY-D and peptide-2 from the highly conserved N terminal/core junction region of Johnson grass mosaic virus (JGMV). Conventional PEG-mediated fusions using mice immunized with these peptides did not result in hybridoma formation. On the other hand, electrofusions using biotin-streptavidin to bridge peptide-specific B cells to myeloma cells produced hybridomas secreting antibodies either highly specific to PVY-D or cross-reactive with all potyviruses, depending on the peptide used. PMID- 1719099 TI - Characterization of two monoclonal antibodies against the COOH-terminal part of the human epidermal growth factor receptor and potential clinical use. AB - Two monoclonal antibodies of the IgG2 subclass, designated A 01 and B 11, were prepared against two synthetic peptides corresponding to the COOH-terminal sequence of the human epidermal growth factor receptor (EGF-R), in order to detect EGF-R in a radioimmunometric assay and by immunohistochemistry. Characterization of these Mabs showed that they recognized two different eptiopes on the original peptides with Kd of 1.7 x 10(-8) M and 1.3 x 10(-7) M, respectively, without crossreaction. The A 431 antigen recognized by A 01 and B 11 had an apparent molecular weight of approximately 170,000 and was able to specifically link to EGF. Thus, A 01 and B 11 are directed against an antigenic site on the human EGF-R. With Western blot analysis and immunostaining, A 01 was shown to be EGF-R specific. In addition to the EGF-R, B 11 recognized two unidentified soluble proteins present in the cytoplasm of the SKBR-3 cell line but different from the c-erb B-2 oncoprotein expressed by these cells. Mabs A 01 and B 11 were used in an IRMA for the determination of EGF-R using the A 431 cell line as a source of EGF-R. Mab A 01 was also shown to be a useful tool for immunohistochemical detection of EGF-R. PMID- 1719101 TI - Transurethral or transvesical prostatectomy. Are they the same patients? PMID- 1719100 TI - Determining the extent of labeling for tetramethylrhodamine protein conjugates. AB - A new, relatively simple, spectrophotometric technique has been developed which is useful for accurately determining the extent of chromophore labeling of proteins. Often the absorbance spectra and extinction coefficients of dye/protein conjugates are strongly affected by changes in the chromophore microenvironment that may occur at high dye/protein ratios. In the method being presented, the microenvironment effects have been significantly reduced by denaturing the dye/protein complex in 6 M guanidine hydrochloride prior to making the necessary spectrophotometric measurements. With this approach, extinction coefficients were obtained under native and denatured conditions for tetramethylrhodamine isothiocyanate (TRITC) when bound to a model protein receptor, the sugar binding protein concanavalin A (ConA). The extinction coefficients used for TRITC/ConA conjugates under native and denaturing conditions were 6.52 x 10(4) M-1 cm-1 and 6.96 x 10(4) M-1 cm-1, respectively. These values were obtained from a model dye complex formed between TRITC and epsilon-amino-n-caproic acid which closely resembles the sidechain of lysine residues. Additional dye/ConA conjugates were prepared with tetramethylrhodamine succinimidyl ester (RHS) and eosin isothiocyanate (EITC), and the effects of microenvironment changes on these conjugates were examined. Extinction coefficients for these dyes in native and denaturing conditions, as a function of the degree of labeling, were not appreciably different indicating that changes in the microenvironment did not have a significant affect on the spectral properties of these two dyes. In summary, with this new approach it is quite easy to accurately determine the dye/protein ratio for TRITC conjugates. Also, it is expected that RHS would be a better dye than TRITC for protein conjugation because more accurate values for dye/protein ratios can be obtained under native conditions. PMID- 1719102 TI - [Cervico-prostatic incision in the treatment of benign prostatic hypertrophy. Apropos of 40 cases]. AB - The authors report on the results of 40 cases of cervico-prostatic incision for benign prostatic hypertrophy. Evaluation of the prostatic gland was based only on rectal examination. Operative technique is simple. We have treated patients with prostatic hypertropy less than 30 g. Young men with prostatic hypertrophy greater than 30 g have been treated by this technique, to preserve antegrade ejaculation. Success rate was 90% at 6 months and 85% at 18 months. Retrograde ejaculation was observed in 21% of the patients following treatment. PMID- 1719104 TI - Annual meeting of the International Society for Interferon Research, ISIR 91. Nice, France, 3-8 November 1991. Program and abstract book. PMID- 1719103 TI - Granulocyte colony-stimulating factor enhances pulmonary host defenses in normal and ethanol-treated rats. AB - Ethanol suppresses functions of the polymorphonuclear leukocyte (PMNL), seriously compromising normal host defenses against pneumonia. Because granulocyte colony stimulating factor (G-CSF) augments the number and function of PMNL, the effect of G-CSF on the antibacterial defenses of the lung in normal and acutely intoxicated rats was studied. Animals received G-CSF or vehicle twice a day for 2 days, then ethanol or saline, followed by challenge with Klebsiella pneumoniae. K. pneumoniae elicited an intrapulmonary influx of PMNL in control rats that was markedly suppressed by prior ethanol administration. G-CSF augmented the recruitment of PMNL into the lungs of control rats and significantly attenuated the adverse effects of ethanol on PMNL entry into the lung. G-CSF enhanced intrapulmonary bactericidal activity against this pathogen in normal and ethanol treated rats. All intoxicated rats pretreated with the vehicle died, while greater than 90% of rats pretreated with G-CSF survived. These findings suggest a potential role for G-CSF in mitigating the adverse effects of ethanol on PMNL delivery and pulmonary host defenses. PMID- 1719105 TI - Concentrations of neuropeptides substance P, neurokinin A, calcitonin gene related peptide, neuropeptide Y and vasoactive intestinal polypeptide in synovial fluid of the human temporomandibular joint. A correlation with symptoms, signs and arthroscopic findings. AB - Arthroscopy was performed on 18 patients (19 joints) with temporomandibular joint arthropathy. Arthroscopic investigation revealed that 12 patients had disk derangement, including 3 patients with rheumatoid arthritis. Six patients had osteoarthrosis, including one patient with rheumatoid arthritis. Synovial fluid content of substance P-like immunoreactivity (SP-LI), neurokinin A (NKA-LI), calcitonin gene-related peptide (CGRP-LI), neuropeptide Y (NPY-LI) and vasoactive intestinal polypeptide (VIP-LI) were analysed using radioimmunoassay technique. All peptides analysed were found, although in various concentrations, in the different joints. There were no significant differences in concentrations of the peptides in the synovial fluid between patients in the various groups. No significant correlation was found between clinical symptoms and signs, arthroscopic findings, or use of analgesic/anti-inflammatory medication versus concentrations of peptides in the synovial fluid. In comparison with earlier findings in the knee joint significantly higher concentrations of SP-LI, CGRP-LI and NPY-LI were found in the TMJ. PMID- 1719106 TI - Normal brain response after interstitial microwave hyperthermia. AB - The effects of interstitial hyperthermia were assessed in the normal brain after a single 30 min treatment using 2450 MHz microwaves. A single helical coil microwave antenna was inserted into the frontal white matter and reference temperatures of 40, 41, 42, 43 and 44 degrees C maintained for 30 min along a temperature sensing probe 5 mm away and parallel to the antenna. The extent of hyperthermia damage was quantified weekly for 6 weeks using computed tomography (CT), and changes in regional cerebral blood flow (rCBF), tissue vascularity and mean transit time were determined using ultrafast CT. Qualitative histopathological analysis was carried out on tissues at various times after heating. Heat lesions were radiographically characterized by rapid development and resolution and consisted of an area of focal low density surrounded by a ring of contrast enhancement. Histologically, the focus of low density corresponded to regions of tissue necrosis, whereas the ring enhancement showed local reactive changes, including endothelial cell proliferation and infiltration with macrophages. Tissue necrosis occurred at temperatures greater than 43.9 +/- 1.5 degrees C (mean +/- standard deviation), and volumes of necrosis and ring enhancement were at a maximum 1 week following treatment. Relative to the contralateral, unheated hemisphere, rCBF in the heated brain appeared to be reduced for the first 3 weeks after treatment but approached normal by week 4. The mean transit time of blood was increased for weeks 1-3 compared to the untreated hemisphere, and tissue vascularity reached a maximum, 3 weeks after treatment. The rapid CT changes together with ultrafast CT and histopathological findings suggest that focal heat lesions in the brain stimulate a significant and rapid vascular response. PMID- 1719107 TI - [Intraarterial combination chemotherapy consisting of bleomycin, vincristine, mitomycin-C, and cisplatin (BOMP) for cervical cancer recurrence in the pelvis]. PMID- 1719109 TI - A different approach to determining platelet thrombospondin receptors. PMID- 1719108 TI - Cystic fibrosis transmembrane regulator mRNA expression relative to ion-nutrient transport in spontaneously differentiating human intestinal CaCo-2 epithelial cells. AB - The relative abundance of cystic fibrosis transmembrane regulator (CFTR) message at various stages of postconfluence development was compared with evolving cellular ion-nutrient transport properties of CaCo-2 human intestinal cells. Initially these cells demonstrate electrogenic Cl secretion manifested by secretagogue-induced changes in short-circuit current. Over time, however, the secretory characteristics of CaCo-2 monolayers diminish, and brush border hydrolase activities and glucose-dependent and amiloride-sensitive Na transport increase. With a polymerase chain reaction-derived cDNA probe to CFTR exon 13, two distinct mRNA transcripts of 6.5 and 4.3 kb were found. No significant differences in their abundance were noted in cells at 6 and 28 days after confluence. These data suggest two possible interpretations for the role of CFTR protein. First, if CFTR is membrane Cl channel, its protein expression or activity could be differentially regulated during CaCo-2 cell development. Alternatively, CFTR may not be a Cl channel but may serve as an important regulatory membrane protein. PMID- 1719110 TI - North Shore cascade. Freevision. PMID- 1719111 TI - Detection of the apoB-3500 mutation (glutamine for arginine) by gene amplification and cleavage with MspI. AB - A single primer-template mismatch 2 bp from the apoB-3500 (G to A) mutation permits introduction of a cleavage site for MspI (C/CGG) in normal alleles but not in mutant alleles (CCAG). After amplification, cleavage, and polyacrylamide gel electrophoresis, normal and mutant alleles could be unambiguously distinguished. We constructed a positive (homozygous mutant) standard by site directed mutagenesis. A negative standard was DNA from a homozygous normal subject. The method enables us to screen for the mutation with 12 microliters of spotted whole blood as the source of DNA. PMID- 1719112 TI - Enhanced phagocytic cell respiratory burst induced by spinal manipulation: potential role of substance P. AB - The effect of spinal manipulation on the respiratory burst of polymorphonuclear neutrophils (PMN) and monocytes from treated adults was measured by zymosan stimulated chemiluminescence (CL). Peripheral blood was collected 15 min before and 15 min after treatment (sham manipulation, thoracic spine manipulation, or soft tissue manipulation), the cells were isolated, challenged with a standardized, opsonized luminol-containing suspension of zymosan, and monitored for CL. Plasma from two subsets of subjects was radioimmunoassayed for Substance P (SP). PMN were also preincubated with SP in vitro over the dose range 5 x 10( 12) M to 5 x 10(-8) M and the CL response monitored. The CL responses of both PMN and monocytes from subjects who received spinal manipulation were significantly higher after than before treatment, and significantly higher than the response in sham or soft-tissue treated subjects. Measurement of the force applied by sham and spinal manipulation suggested a force threshold for the enhancement of the CL response. Plasma levels of SP before and after treatment in sham treated subjects did not differ significantly; however, elevated plasma SP was observed in subjects after spinal manipulation. Preincubation of PMN with 1 x 10(-11) M, 5 x 10(-11) M or 1 x 10(-10) M SP in vitro primed PMN for an enhanced respiratory burst when the cells were subsequently challenged. PMID- 1719113 TI - Detection of estrogen and progesterone receptor bindings in mammary carcinoma cells: a comparative study using immunohistochemical and biochemical methods. AB - A comparative study of estrogen and progesterone receptor bindings of breast carcinoma tissue was done by immunoperoxidase and dextran-coated charcoal (DCC) methods. Fifteen cases of paraffin embedded formalin fixed tissue of mammary carcinomas which had previously been evaluated by the DCC method were selected. Twelve cases were ductal carcinoma and 3 were of lobular origin. Lymph node tissue showing metastasis was available in 3 cases. By immunoperoxidase technique, 12 and 10 (80 and 66.7%) cases were positive for estrogen and progesterone receptor respectively compared with 8 and 3 (53.3 and 20%) cases by the DCC technique. Corresponding results of both methods to detect estrogen and progesterone bindings were 9 and 8 (60 and 53.3%) of all cases, respectively. Five cases for estrogen and 6 cases for progesterone positive by immunoperoxidase could not be detected by the DCC technique. Only one case of estrogen negative by the immunoperoxidase gave a positive result with the DCC technique. Variability of staining occurred between primary and metastatic lesions, 2 out of 3 cases displayed positive staining in both sites; one remaining case was positive only in the lymph node metastasis. Immunoperoxidase is a relatively simple, swift and inexpensive technique in comparison to the DCC technique. Using fixed embedded tissue makes it possible for retrospective studies and providing a permanent record for reevaluation. Moreover, morphology of the tumor can be determined at the same time as detection of hormonal receptor bindings. PMID- 1719114 TI - Serotoninergic stimulation of aldosterone secretion in the rat in vivo: role of the renin-angiotensin system. AB - Serotoninergic control of aldosterone secretion in vivo was investigated in conscious rats with indwelling arterial cannulae. Serial blood samples were taken from the animals before and after i.p. administration of 1 ml (4 g/l) 5 hydroxytryptophan (5-HTP), the precursor of serotonin, or saline and they were analysed for 5-HTP, serotonin, 5-hydroxyindoleacetic acid, plasma renin activity (PRA), corticosterone, aldosterone, sodium and potassium concentrations. The role of the renin-angiotensin system was investigated in animals pretreated for 1 week with the angiotensin-converting enzyme inhibitor captopril (25 mg/day). 5-HTP caused a significant increase in all parameters within 45 min except for sodium and potassium. Saline administration showed no significant effect. Captopril pretreatment did not impair the increase in any parameter by 5-HTP, with the exception of the aldosterone response which was significantly attenuated, though not completely. The results show that administration of 5-HTP, which increases serum serotonin levels, stimulates PRA, aldosterone and corticosterone secretion. Captopril pretreatment inhibits the aldosterone response, suggesting that the aldosterone stimulatory properties of 5-HTP require the presence of angiotensin II, although it is unclear whether it acts in a mediatory or permissive capacity. The failure of captopril to inhibit the aldosterone response completely suggests the involvement of other mechanisms such as the hypothalamo-pituitary adrenal axis or a direct action of serotonin on the adrenal. PMID- 1719116 TI - Facile separation of the subunits of ovine and bovine gonadotrophins by high performance liquid chromatography: microheterogeneity and biological activity. AB - The alpha and beta subunits of ovine and bovine gonadotrophins which undergo instant dissociation in 0.1% (v/v) trifluoroacetic acid in water could be separated into their multiple components by reverse-phase high-performance liquid chromatography on analytical and preparative scales. Several batches of highly purified ovine LH (oLH) separated into four alpha components (alpha 1 to alpha 4) and five beta components (beta 1 to beta 5). While the major alpha component (alpha 3) remained the same in all, the beta subunit was variable in all these preparations of oLH of equal biological and immunoreactivity. Various manipulations, such as prolonged incubation in dilute acid or alkali or conditions prone to air oxidation, did not change elution patterns or produce interconversions among the alpha or beta components, suggesting that microheterogeneity arose in the pituitary itself or during its collection. The subunits obtained on a preparative scale were of high yield and quality and recombined (100%) to produce fully active LH or FSH. Chemical deglycosylations in which 75-80% of the peripheral sugars were removed shifted the elution of all components (alpha as well as beta) slightly but modifications to the protein backbone by other reagents changed the profile of the alpha subunit. Obtaining the various alpha and beta components in high yield would permit a complete analysis of gonadotrophin microheterogeneity. PMID- 1719115 TI - Administration of insulin-like growth factor-I, but not growth hormone, increases maternal weight gain in late pregnancy without affecting fetal or placental growth. AB - During late pregnancy in the rat, circulating levels of insulin-like growth factor-I (IGF-I) and some IGF-binding proteins (IGFBP) decline. The aim of the present study was to determine the relationship of GH to circulating IGF and IGFBP in the late-pregnant rat and to examine the effects on maternal, fetal and placental growth of preventing the decline in serum IGF and IGFBP concentrations. During the first 9 days of pregnancy, IGF-I concentrations increased from 340 to 500 micrograms/l. Recombinant human (rh) GH at 2.4 mg/kg per day and rhIGF-I at 1.4 mg/kg per day were infused into pregnant rats via osmotic mini pumps during the second half of pregnancy. After pump implantation on day 11 of pregnancy, only IGF-I infusion significantly increased circulating IGF-I. A maximum IGF-I concentration of 907 micrograms/l was measured on day 14 during treatment with IGF-I, after which the serum concentration decreased to 510 micrograms/l by day 20 of pregnancy. The serum IGFBPs were examined using a Western ligand blot technique. Infusion of neither GH nor IGF-I returned the IGFBPs to non-pregnant levels. Administration of IGF-I slightly increased IGFBP-3 and a smaller 32 kDa IGFBP at days 17 and 20 of pregnancy. Neither fetal nor placental weight was significantly different between treatment groups. However, administration of IGF I significantly increased maternal weight gain during the 10-day treatment period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719117 TI - Galanin in the normal human pituitary and brain and in pituitary adenomas. AB - Galanin-like immunoreactivity (IR) was measured by radioimmunoassay in extracts of non-tumorous and tumorous human pituitaries and in multiple sites in the human brain. Galanin-IR was present in considerable quantities in the non-tumorous pituitaries (21.4 +/- 1.2 pmol/g wet weight; mean +/- S.E.M., n = 30). In 25 pituitary tumours, galanin-IR was detectable in extracts of only nine, with a mean concentration of 11.5 +/- 4.4 pmol/g. Galanin-IR was undetectable in the remaining 16. Of ten brain sites, galanin-IR was detected only in the hypothalamus, where the concentration was 9.1 +/- 1.8 pmol/g (n = 5). On fast protein liquid chromatography of the non-tumorous pituitary extracts, galanin-IR mostly eluted in a peak with a retention time similar to that of synthetic porcine galanin. On gel permeation chromatography, galanin-IR eluted as a peak with an elution coefficient (Kav) of 0.72, also similar to that of porcine galanin, with additional preceding (Kav 0.62) and following (Kav 0.77) peaks of galanin-IR. These results show that healthy human pituitary and hypothalamus contain substantial amounts of galanin, whereas it is present in variable amounts or not at all in pituitary tumours. Chromatographic analysis suggests that pituitary galanin is present in three molecular forms, with the majority corresponding to synthetic porcine galanin. PMID- 1719118 TI - The induction of a specific protease for insulin-like growth factor binding protein-3 in the circulation during severe illness. AB - The insulin-like growth factors (IGF-I and IGF-II) are almost completely bound in the circulation to specific binding proteins (IGFBPs). These IGFBPs appear to play a pivotal role in maintaining circulating levels and modulating the delivery of the IGFs to the tissues. A large proportion of the circulating IGFs are bound with high affinity to one of the binding proteins. IGFBP-3. The mechanism by which these IGFs are transferred from the circulatory pool to the tissue receptors is at present unclear. Recent studies in late pregnancy have demonstrated the presence of specific proteases which may modify the IGFBPs such that their affinities for the IGFs are reduced. In this paper, we have demonstrated the presence of a heat-sensitive cation-dependent proteolytic enzyme specific for IGFBP-3 in the serum of five severely ill patients. The activity of this protease was found to vary in these patients, becoming more apparent during fasting than when studied after commencement of parenteral nutrition, indicating that one of the influencing factors in the activity of this protease is the nutritional intake of the patient. Age- and sex-matched healthy adults were also studied in a similar protocol, but no proteolytic modification of any of the IGFBPs was found in any of the samples examined. As the levels of both IGF-I and IGF-II were found to be low in the patients, the presence of a circulatory protease suggests that this may be an adaptive response to increase the bioavailability of the IGFs and possibly to improve the nitrogen retention and counter the catabolic state in severe illness. PMID- 1719119 TI - The purification and development of a radioimmunoassay for beta-core fragment of human chorionic gonadotrophin in urine: application as a marker of gynaecological cancer in premenopausal and postmenopausal women. AB - The beta-core fragment of human chorionic gonadotrophin (hCG) is a major part of the immunoreactive hCG-like material found in the urine of normal pregnant women. Patients with non-trophoblastic gynaecological malignancies have been found to have raised levels of urinary beta-core. We describe the purification of beta core, the preparation of a polyclonal sheep antiserum and the development of radioimmunoassay. The minimum detection limit of this assay was 0.025 micrograms beta-core/l. There was no significant cross-reaction with the free alpha-subunit, hLH, hFSH and hTSH (less than 0.7%), and only partial cross-reaction with intact hCG and free beta-subunit of hCG (6.9 and 18%). Within-assay variability ranged from 2.03 to 12.5% and between-assay variability from 2.25 to 13.4%. The assay was applied to urine samples from 92 normal non-pregnant premenopausal women, 54 normal postmenopausal women and 65 women with active gynaecological disease (47 postmenopausal and 18 premenopausal). In normal premenopausal women the values ranged from less than 0.025 to 0.62 micrograms beta-core/l (median 0.043 micrograms beta-core/l). The values for normal postmenopausal women ranged from less than 0.025 to 0.64 micrograms beta-core/l (median 0.26 micrograms beta core/l). Postmenopausal women with gynaecological malignancy had values which ranged from less than 0.025 to 4.0 micrograms/beta-core/l (median 0.31 micrograms beta-core/l); premenopausal women in this group had values which ranged from less than 0.025 to 1.15 micrograms beta-core/l (median 0.12 micrograms beta-core/l). On molecular sieve chromatography, the material found in the urine of normal postmenopausal women showed the physicochemical characteristics of authentic beta core.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719120 TI - Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro. AB - Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony stimulating factor-driven granulocyte colony formation in vitro. PMID- 1719121 TI - Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ precursor cells. AB - The search for human hematopoietic stem cells has been hampered by the lack of appropriate assay systems. Demonstration of the ability of precursor cell candidates to give rise to T cells is of significant difficulty since dissociated in vitro cultured thymus stroma cells lose their ability to sustain thymocyte maturation. To define further the differentiative capacities of the rare human fetal liver and bone marrow cells that express the CD34 surface antigen and exhibit in vitro myeloid and pre-B cell activities, we have microinjected them into HLA-mismatched fetal thymus fragments, partially depleted of hematopoietic cells by low temperature culture. In vitro colonized thymuses have then been allowed to develop upon engraftment into immunodeficient SCID mice. Using this modification of the SCID-hu system, we show that low numbers of fetal CD34+ progenitor cells can repopulate the lymphoid compartment in the human thymus. PMID- 1719122 TI - Calcium regulation of epidermal cell differentiation in the frog Xenopus laevis. AB - Adult frogs have a stratified epidermis with a keratinized stratum corneum. Since the extracellular calcium concentration is known to regulate differentiation of mammalian epidermal cells in vitro, we studied the effects of calcium on the terminal differentiation of frog epidermal cells. Exposure of the epidermal cells to a high concentration of calcium (greater than 0.2 mM) induced cornification and the synthesis of a 51 Kd acidic keratin. These data are very similar to the results from mammalian epidermal cell cultures, suggesting that the mechanism of terminal differentiation is conserved throughout the evolution of terrestrial vertebrates. PMID- 1719123 TI - Region-specific expression of scutate scale type beta keratins in the developing chick beak. AB - This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed. PMID- 1719125 TI - Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation. AB - The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin-permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control." PMID- 1719124 TI - Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin. AB - Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1719126 TI - Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes. AB - The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side. PMID- 1719127 TI - Calcium feedback and sensitivity regulation in primate rods. AB - Membrane current was recorded from a single primate rod with a suction pipette while the cell was bath perfused with solutions maintained at a temperature of approximately 38 degrees C. A transient inward current was observed at the onset of bright illumination after briefly exposing the outer segment in darkness to Ringer's (Locke) solution containing 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cGMP phosphodiesterase. After briefly removing external Na+ from around the outer segment in darkness, a similar current was observed upon Na+ restoration in bright light. By analogy to amphibian rods, this inward current was interpreted to represent the activity of an electrogenic Na(+)-dependent Ca2+ efflux, which under physiological conditions in the light is expected to reduce the free Ca2+ in the outer segment and provide negative feedback (the "Ca2+ feedback") to the phototransduction process. The exchange current had a saturated amplitude of up to approximately 5 pA and a decline time course that appeared to have more than one exponential component. In the absence of the Ca2+ feedback, made possible by removing the Ca2+ influx and efflux at the outer segment using a 0 Na(+)-0 Ca2+ external solution, the response of a rod to a dim flash was two to three times larger and had a longer time to peak than in physiological solution. These changes can be approximately accounted for by a simple model describing the Ca2+ feedback in primate rods. The dark hydrolytic rate for cGMP was estimated to be 1.2 s-1. The incremental hydrolytic rate, beta*(t), activated by one photoisomerization was approximately 0.09 s-1 at its peak, with a time-integrated activity, integral of beta*(t)dt, of approximately 0.033, both numbers being derived assuming spatial homogeneity in the outer segment. Finally, we have found that primate rods adapt to light in much the same way as amphibian and other mammalian rods, such as showing a Weber-Fechner relation between flash sensitivity and background light. The Ca2+ feedback model we have constructed can also explain this feature reasonably well. PMID- 1719128 TI - Mapping of the epitopes of Epstein-Barr virus gp350 using monoclonal antibodies and recombinant proteins expressed in Escherichia coli defines three antigenic determinants. AB - The Epstein-Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro. The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome. To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus. The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay. One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins. The data identify three antigenic determinants on gp350. DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment. In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones. Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies. PMID- 1719129 TI - Protective effects of monoclonal antibodies against lethal canine distemper virus infection in mice. AB - Monoclonal antibodies (MAbs) against the haemagglutinin (H), fusion protein (F) and nucleoprotein of canine distemper virus (CDV) were examined for their ability to protect mice against lethal CDV infection. One MAb against H and two of six MAbs against F protected mice, the protective effect of the anti-H MAb being stronger than that of the anti-F MAbs. The anti-H MAb showed virus neutralizing activity, but the two anti-F MAbs, which recognized the same epitope, did not. Protection by the anti-F MAbs correlated with cell fusion inhibition, but not with complement-dependent neutralization, complement-dependent cytolysis or antibody-dependent cell-mediated cytotoxicity. These results suggest that neutralization by antibody against H and cell fusion inhibition by antibody against F play important roles in the protective mechanism against CDV infection. PMID- 1719130 TI - Activation of cellular oncogenes by clinical isolates and laboratory strains of human cytomegalovirus. AB - The effect on cellular (c) oncogene RNA levels was investigated after infection of permissive cells with cell culture adapted strains (AD-169, C-87, Davis) and unadapted clinical isolates (82-1, 84-2, 85-1) of human cytomegalovirus (HCMV). The results indicate that both adapted and unadapted strains of HCMV induce substantial increases in c-oncogene RNA levels for fos, jun, and myc measured by Northern blot hybridization. Elimination of immediate early (IE) protein synthesis between 0 and 3 hrs or reduction of virus infectivity (99.99%) by UV irradiation did not reduce the increase in c-oncogene RNA levels. Inhibition of viral and cellular protein synthesis by cycloheximide resulted in a high abundance (superinduction) of specific RNAs which hybridized to c-oncogene probes after infection with either adapted or unadapted strains of HCMV. These data suggest that IE viral gene expression is not essential for activation of c oncogenes. Inhibition of DNA-dependent RNA synthesis by blocking RNA elongation with actinomycin-D or by inhibiting the activity of RNA polymerase II with alpha amanitin significantly reduced the increase in c-oncogene RNA levels, suggesting that activation of cellular genes by HCMV is controlled at the level of transcription. Activation of c-oncogenes by HCMV may be particularly important because their protein products appear to be involved in initiation and regulation of viral and cellular gene expression. PMID- 1719131 TI - Synaptic regulation of immediate early gene expression in primary cultures of cortical neurons. AB - Neuronal stimulation can rapidly activate several immediate early genes that code for transcription factors. We have used primary cortical cultures to study the regulation of four of these genes, c-fos, c-jun, jun-B, and zif268. Immunocytochemical studies with antibodies to Jun-B, c-Jun, and c-Fos demonstrate intense staining in the nuclei of a subset of cortical neurons in mature cultures (21-25 days in vitro) but not young cultures (3-7 days in vitro). To assess whether this immunoreactivity may be induced by spontaneous synaptic activity that develops with a similar profile, we examined the effects of agents that reduce this synaptic activity. Tetrodotoxin or N-methyl-D-aspartate receptor antagonists suppress basal immunoreactivity to Jun-B and c-Fos, but not c-Jun, indicating that the basal level of c-Jun expression is not dependent on electrical activity. Picrotoxin, an agent that increases synaptic excitation indirectly by blocking inhibitory synaptic currents mediated by gamma aminobutyric acidA receptors, markedly increases the percentage of neurons displaying immunoreactivity to c-Fos, c-Jun, Jun-B, and Zif268. Northern analysis suggests that the increases in immunostaining induced by picrotoxin are secondary to a rapid increase in mRNA for these proteins. These findings provide evidence for rapid transcriptional regulation of immediate early genes in cortical neurons by synaptic activity. PMID- 1719132 TI - The susceptibility of cerebral endothelial cells to astroglial induction of blood brain barrier enzymes depends on their proliferative state. AB - Primary cultures of brain capillary endothelial cells (BCECs) were used to investigate the induction of blood-brain barrier (BBB) characteristics in vitro. Enzymatic activities of gamma-glutamyltranspeptidase (gamma-GT) and alkaline phosphatase (ALP) were taken as indicators for the expression of the BBB phenotype. We were able to show that a coculture system with a direct cell-cell contact between astroglial cells and BCECs is the necessary precondition for an increase of these enzyme activities that are lost in pure BCEC cultures. Coculture with both astrocytes and C6-glioma cells reestablishes the BBB phenotype whereas conditioned media as well as an astrocyte-derived extracellular matrix were ineffective. The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs. Cells in an early highly proliferative culture phase were stimulated to express an enzymatic activity level similar to the in vivo situation. Confluent BCEC monolayers were not induced at all. With the ALP we observed a spatial induction within a BCEC colony. Astrocyte-induced ALP activity was first observed at an outer belt of BCEC colonies in direct contact with the astrocyte layer. However, this signal is transferred to the center of the colony with time in culture. We conclude that direct contact of BCECs with astroglial cells is necessary for the induction of the BBB phenotype in cultured BCECs and that this signal may be transferred from induced to noninduced BCECs. PMID- 1719133 TI - Cyclic AMP modulates differentially the release of dopamine induced by hypoxia and other stimuli and increases dopamine synthesis in the rabbit carotid body. AB - We have investigated the effects of different treatments that increase cyclic AMP levels on the in vitro synthesis and release of catecholamines in the rabbit carotid body. We also measured the rate of 45Ca2+ efflux from previously loaded carotid bodies under different conditions. Forskolin produced a dose-dependent increase in the release of [3H]dopamine elicited by a hypoxic stimulus of medium intensity (PO2 = 33 mm Hg) without altering basal [3H]dopamine release (100% O2 equilibrated medium). At a concentration of 5 x 10(-6) M, forskolin increased the release of [3H]dopamine induced by hypoxic stimuli of different intensities; the increase was maximal (498%) at the lowest intensity of hypoxic stimuli (PO2 = 66 mm Hg), averaged 260% for hypoxic stimuli of intermediate intensity and 2 x 10( 4) M cyanide, and was 150% under anoxia. Dibutyryl cyclic AMP (2 mM) and 3 isobutyl-1-methylxanthine (0.5 mM) mimicked forskolin effects under hypoxic stimulation. Forskolin (5 x 10(-6) M) also increased (180%) the release of [3H]dopamine induced by 20% CO2/pH 6.6, 2.5 x 10(-4) M dinitrophenol, and 3 x 10( 5) M ionomycin. Forskolin and 3-isobutyl-1-methylxanthine were without effect on the release of [3H]dopamine elicited by 30 mM extracellular K+. Forskolin (5 x 10(-6) M) augmented significantly the rate of 45Ca2+ efflux induced by hypoxic stimuli (PO2 of 33 and 66 mm Hg) and 2 x 10(-4) M cyanide and showed a tendency to increase (20%) the 45Ca2+ efflux induced by dinitrophenol and low pH and to decrease (21%) the efflux induced by 30 mM K+ without altering the rate of efflux under basal conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719134 TI - Synergistic regulation of cytosolic Ca2+ concentration in mouse astrocytes by NK1 tachykinin and adenosine agonists. AB - The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9] substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9] substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2 chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine. PMID- 1719135 TI - Brain metabolism in Alzheimer's dementia: studies of 11C-deoxyglucose accumulation, CSF monoamine metabolites and neuropsychological test performance in patients and healthy subjects. AB - Thirteen patients with dementia of Alzheimer type and nine age-matched control subjects were examined by a battery of neuropsychological tests and by positron emission tomography (PET) with 11C-deoxyglucose as a tracer for regional glucose metabolism in the brain. Concentrations of the monoamine metabolites HVA, MHPG and 5-HIAA were determined in the CSF from patients and controls. In the patients there was a diminished glucose metabolism in posterior parietal and superior temporal cortex areas to 60% of control levels. Other cortical areas showed similar changes, whereas the pre- and postcentral area, the cerebellum, the hippocampus and the basal ganglia showed less or no change. The decline in cortical metabolism in the patients was symmetrical but the variation in the left/right ratio was greater than in the controls. The CSF levels of monoamine metabolites did not differ between patients and controls. High levels of the metabolites were associated with low rates of glucose metabolism, possibly due to inhibitory influences of monoaminergic pathways upon cortical and subcortical neurons. The rate of glucose metabolism correlated positively with the neuropsychological test performance in both patients and controls. Verbal and memory performances were associated with greater left hemisphere metabolism in the patients, but not in the controls, whereas non-verbal abilities tended to be associated with right hemisphere metabolic dominance. PMID- 1719136 TI - Limb apraxia without aphasia from a left sided lesion in a right handed patient. AB - A right handed man had a massive left middle cerebral artery stroke. CT and MRI revealed extensive destruction of both anterior and posterior areas typically associated with language. There was, however, no aphasia, but instead a marked limb apraxia, dyscalculia, dense right visual neglect, and anosognosia. These uncommon dissociations and associations support the hypothesis that cerebral control of motor function of the limbs is not fundamentally related to the motor control involved in speech, and the notion that handedness is related to laterality of motor control, and only accidentally to laterality of language control. PMID- 1719137 TI - Intractable seizures from infancy can be associated with dentato-olivary dysplasia. AB - Five children with severe developmental delay had intractable fits of various types but tonic, often extensor, seizures were prominent from an early stage. Onset was in the neonatal period in 4 cases. EEGs were severely abnormal and showed a "burst-suppression" pattern in the first months of life. There were no metabolic or consistent neuroradiological abnormalities. A distinctive form of dentato-olivary dysplasia was found in all cases. Inferior olives were hook shaped, coarse and lacking undulations, while dentate nuclei showed a compact arrangement of interconnected islands. The clinico-pathological findings form a novel nosological entity. PMID- 1719138 TI - Penetration and internalization of plasma proteins in the human spinal cord. AB - In animal studies, motoneurons take up plasma proteins including immunoglobulins at their terminals. These proteins are then transported back to cell bodies in the spinal cord. To determine if these processes also occur in humans, we localized several different plasma proteins in autopsied spinal cords from 13 patients without neurological disease. As in animals, plasma proteins are associated with vascular and pial structures. Motoneurons, particularly large cervical and lumbar motoneurons, frequently showed immunoreactivity within their cytoplasm to several plasma proteins. Motoneuron labeling was more consistent with antisera against plasma proteins of lower molecular weights such as IgG, IgA and transferrin, than with antisera against higher molecular weight proteins such as IgM and alpha-2-macroglobulin. Other large neurons without connections outside the blood-brain barrier such as those of Clarke's column also occasionally labeled with antisera against all plasma proteins tested. Our results are compatible with the concept that motoneurons take up and transport plasma proteins. These neurons can be distinguished from cells which internalized extravasated serum proteins before and after death. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease. PMID- 1719139 TI - Three-dimensional fine structure of cytoskeletal-membrane interactions at nodes of Ranvier. AB - Cytoskeleton-membrane-extracellular matrix interactions at the node of Ranvier were examined in both central and peripheral axons by combining three different methods for tissue preparation with three different electron microscopic techniques for imaging supramolecular structure. Conventional and three dimensional high voltage electron microscopy of thin and semithick sections of tissues stained en bloc with ferric chloride revealed the presence of transcellular structures across the nodal gap traversing the paranodal glial axonal junction. These structures penetrate both axonal and glial membranes and are further traced to the cortical axoplasm. This observation was verified by an examination of similar regions in rapidly-frozen freeze-substituted fresh axons. The filamentous nature of these structures, their focal attachment to the external true surface of the nodal and paranodal axolemma and their association with membrane particles were visualized in deep etch rotary-shadow replicas. At the node, both extracellular gap-crossing filaments and membrane-cytoskeletal linkers in the nodal axoplasm are joined to one of the prominent membrane particles of the nodal axolemma. At the paranodal axo-glial junction, the anchoring site of these membrane-cytoskeleton linkers are found on the linear arrays of 16 nm particles. Thus, cytoplasmic filaments and extracellular filaments or bridge structures are involved in the membrane-cytoskeletal interaction at the node and paranode. Some of these membrane particles are known to play a role in ionic conductances known to occur at this site. An additional role in cell adhesion or maintenance of the membrane specialization of this functionally important site of axolemma is now indicated. PMID- 1719140 TI - Ultrastructure of afferent axon endings in a neuroma. AB - Injured sensory axons with endings trapped in a nerve-end neuroma become a source of abnormal impulse discharge and neuropathic pain. We have examined the ultrastructure of such endings anterogradely transported WGA-HRP and freeze fracture replication, with emphasis on the postinjury period during which the abnormal neural discharge is maximal. Most axons ended in a terminal swelling, depleted of myelin but surrounded by Schwann cell processes. These 'neuroma endbulbs' were richly packed with membrane-bound organelles, and had a smoothly undulating surface with (in neuromas of several weeks standing) a moderate number of short filopodia. Massive sprouting did not occur until several months postinjury. Both p- and e-faces of endbulb axolemma had larger intramembranous particles, on average, than corresponding internodal membrane of control axons. This change, interpreted as indicating remodelling of axolemmal channel (and perhaps receptor) content, may be related to the abnormal electrical behavior of neuroma afferents. PMID- 1719143 TI - Going home. PMID- 1719141 TI - Treatment of HTLV-I-associated myelopathy with high-dose intravenous gammaglobulin. AB - Fourteen patients with HTLV-1-associated myelopathy were treated with high-dose intravenous gammaglobulin (IVGG). Ten received 10 g/day of IVGG and 4 received 400 mg/kg of body-weight/day of IVGG for 5 consecutive days. Improvement of spastic paraparesis was observed in 10 within 7 days of the commencement of IVGG. The therapeutic effects were sustained for more than 3 weeks in some patients. There were no side effects. Analysis of factors of relevance to the clinical improvement with IVGG showed that the beneficial response was preferentially found in patients having a high CSF titre of anti-HTLV-I antibodies, a high CSF IgG level and a marked brain MRI abnormality. PMID- 1719142 TI - Chemotherapy of advanced dysgerminoma: trials of the Gynecologic Oncology Group. AB - Between 1984 and 1989, 20 assessable patients with incompletely resected ovarian dysgerminoma were treated on two protocols of the Gynecologic Oncology Group (GOG). All patients received cisplatin, bleomycin, and either vinblastine or etoposide. More recent patients also received consolidation chemotherapy with vincristine, dactinomycin, and cyclophosphamide (VAC). Eleven patients had clinically measurable disease, and 10 responded completely. Fourteen second-look procedures were done, and all were negative. Currently, 19 of 20 patients are disease-free with median follow-up of 26 months. Cisplatin-based chemotherapy is highly effective in patients with advanced dysgerminoma. PMID- 1719144 TI - Defensive charting--offensive coding. PMID- 1719145 TI - Endodontic mishaps: perforations. PMID- 1719146 TI - What causes peri-implantitis? PMID- 1719147 TI - Porcelain laminate veneers. PMID- 1719148 TI - AIDS: the ultimate occupational accident. PMID- 1719149 TI - Ending a shortage. PMID- 1719150 TI - Craniofacial imaging. PMID- 1719151 TI - The TMDevil's TMDictionary. PMID- 1719152 TI - Abuse and neglect as a component of pediatric treatment planning. PMID- 1719153 TI - The formocresol pulpotomy revisited: looking at alternatives. PMID- 1719154 TI - Use of glass ionomer cements in pediatric dentistry. PMID- 1719155 TI - Molecular cloning and sequencing of general odorant-binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: comparisons with other insect OBPs and their signal peptides. AB - Odorant-binding proteins (OBPs) are small, water-soluble proteins uniquely expressed in olfactory tissue of insects and vertebrates. OBPs are present in the aqueous fluid surrounding olfactory sensory dendrites and are thought to aid in the capture and transport of hydrophobic odorants into and through this fluid. OBPs may represent the initial biochemical recognition step in olfaction, because they transport odorants to the receptor neurons. Insect OBPs are represented by multiple classes: pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBP1 and GOBP2). PBPs associate with pheromone-sensitive neurons, while GOBPs associate with general odorant-sensitive neurons. Analysis of N terminal amino acid sequences of 14 insect OBPs isolated from six species indicated that the PBPs were variable and the GOBPs were highly conserved. However, inferred properties of these proteins were based only on partial sequence data. We now report the full-length sequences of a GOBP1 and GOBP2 from the moth Manduca sexta and compare these sequences with those of PBPs from three species, including M. sexta, Antheraea polyphemus, and A. pernyi. We also compare these with a GOBP2 of A. pernyi, previously identified only as a novel OBP. These comparisons fully support our N-terminal analysis. The signal peptide sequences of seven insect OBPs reveal conserved sequences within OBP classes, but not between OBP classes even within the same animal species. This suggests that multiple OBPs may be coexpressed in the same cell type, but differentially processed in a class-specific manner. Properties of the GOBPs suggest that general olfaction is broadly receptive at the periphery. Properties of the PBPs suggest that pheromone olfaction is discriminatory at the periphery, and that the initial biochemical steps in pheromone detection may play an active role in odor perception. PMID- 1719156 TI - Induction of the myelin proteolipid protein (PLP) gene in C6 glioblastoma cells: functional analysis of the PLP promotor. AB - The terminal differentiation of postmitotic oligodendrocytes is marked by the induction of myelin-specific genes. In this report, we demonstrate that culture conditions that induce oligodendrocyte differentiation of glial progenitor cells also induce differentiation of C6 glioblastoma cells, as monitored by activated transcription of the gene encoding proteolipid protein (PLP), the major myelin protein of the CNS. When assayed by transfections of hybrid reporter plasmids, the transcriptional control region of the PLP gene is preferentially active in differentiated C6 cells and contains both positive and negative cis-regulatory elements. In general, functional identification of these elements is well correlated with the binding sites of glial nuclear proteins, as visualized by the presence of DNase I-protected footprints. A sequence within one positive cis regulatory element of the PLP gene is conserved in the control regions of three other myelin-specific genes, suggesting that their coordinate transcription may involve a common regulatory mechanism. PMID- 1719157 TI - Behavioral sensitization to kainic acid and quisqualic acid in mice: comparison to NMDA and substance P responses. AB - Substance P (SP) and the excitatory amino acid (EAA) agonists NMDA, kainic acid (KA), or quisqualic acid (Quis) each produce a transient, caudally directed biting and scratching response (CBS) in mice after their intrathecal injection. We have previously shown that repeated injections of SP result in a decrease in the intensity of CBS, or desensitization. The goals of the present study were (1) to determine whether desensitization also develops to the CBS behavior produced by EAAs in the spinal cord, (2) to characterize the role of interneurons in desensitization, and (3) to examine possible interactions between EAAs and SP. While injection of NMDA at 2 min intervals resulted in desensitization to its CBS behavioral effect, behavioral responses to repeated injections of KA or Quis increased in intensity, exhibiting sensitization. The NMDA antagonist DL-2-amino 5-phosphonovaleric acid failed to alter sensitization to either KA or Quis but inhibited behaviors produced by SP and NMDA, suggesting an NMDA-mediated component in SP-induced behavior. Concanavalin A, which is reported to block desensitization to the electrophysiologic effect of Quis, blocked sensitization to the behavioral effects of both Quis and KA. Strychnine, bicuculline, and 5 aminovaleric acid each inhibited desensitization to SP and NMDA, supporting the notion of recruitment of inhibitory transmitters in the attenuation of NMDA and SP activity. Pretreatment with capsaicin selectively inhibited the development of behavioral sensitization to KA, suggesting an involvement of small-diameter C fibers in the enhancement of responsivity to KA. Consistent with this, pretreatment with SP selectively potentiated the CBS response to KA. The potentiation of KA effects by SP and dependence of KA behavioral sensitization on C-fiber activity suggest a possible mechanism by which EAAs and SP may be involved in the mediation of pain. PMID- 1719158 TI - Inositol 1,4,5-trisphosphate-gated channels in cerebellum: presence of multiple conductance states. AB - The mechanism by which inositol 1,4,5-triphosphate (InsP3) induces calcium (Ca) release from the reticulum of canine cerebellum was examined. Reticular membrane vesicles used in these experiments accumulated Ca in the presence of ATP and then released approximately 30% of the accumulated Ca upon addition of micromolar concentrations of InsP3. When these membrane vesicles were incorporated into planar lipid bilayers, InsP3-gated Ca channels were observed. Up to four current amplitudes were observed at a given voltage, yielding conductances of 20, 40, 60, and 80 pS with 50 mM Ca as the current carrier. Thus, the cerebellar InsP3-gated Ca channel exhibits four conductance levels that are multiples of a unit conductance step. Moreover, examination of the single-channel records showed both openings directly to each of the current levels and rapid transitions between current levels. These four conductance steps may reflect the interaction among the four InsP3 receptors thought to comprise the InsP3-gated Ca channel in these tissues. Examination of the InsP3 dependence of channel openings and Ca release from vesicles, however, yielded Hill coefficients of 1-1.3. Thus, we hypothesize that it takes only one molecule of InsP3 to open the channel. The observation that the conductance of the InsP3-gated Ca channel assumes four levels that are multiples of a unit conductance suggests that the number of interacting InsP3 receptors in one complex can vary from one to four and supports the hypothesis that the channel is a tetramer. PMID- 1719159 TI - Two novel kinases phosphorylate tau and the KSP site of heavy neurofilament subunits in high stoichiometric ratios. AB - We have identified, purified, and characterized two neurofilament/tau kinases from bovine brain, PK36 and PK40, with apparent Mr of 36,000 and 40,000 and with novel biochemical properties. A specially designed immunoassay for phosphorylated epitopes in neurofilament (NF) proteins was used in the early stages of the purification. Neither kinase is closely associated with the cytoskeleton. Both kinases phosphorylate bovine intermediate (NF-M) and heavy (NF-H) NF subunits and also bovine tau at the expected KSP sequences, though other sites cannot be ruled out. In human paired helical filaments, tau, phosphorylated at these same KSP sites, is a major characterized constituent. Neither kinase is activated by the usual second messengers. Tau and the above NF subunits are phosphorylated in high stoichiometric ratios. In the intermediate NF subunit, all the expected sites appear to be phosphorylated, but in the heavy NF subunit only 7 out of the greater than 40 expected sites can be phosphorylated by our kinases. We demonstrate that both kinases can induce considerable shifts of apparent Mr with SDS-PAGE for tau and, for the first time in vitro, also for the intermediate NF subunit. Interestingly, PK36 and particularly PK40 are strongly inhibited by an excess of free ATP. We propose that during normal aging, and in Alzheimer's disease, age-related mitochondrial dysfunction would reduce ATP levels, which in turn might release the neurofilament/tau kinase from inhibition with consequent paired helical filament formation. PMID- 1719160 TI - Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. AB - The extracellular matrix (ECM) molecules chondroitin-6-sulfate proteoglycan (CS PG) and cytotactin/tenascin (CT), present on subpopulations of astroglia or their precursors during development, can inhibit neurite outgrowth in vitro. However, it is not known whether these molecules are expressed within the mature CNS following injury, where they could contribute to regenerative failure. Thus, the expression of various ECM molecules that affect axon growth was examined in areas of reactive gliosis caused by implanting a piece of nitrocellulose into the cortex of neonatal and adult animals. The expression of these molecules was compared to the amount of neurite outgrowth that occurred in vitro when the damaged CNS tissue from animals of various ages was removed intact and used as a substrate in explant culture. The results demonstrate that the growth-promoting molecules laminin, collagen type IV, and fibronectin were present around the implant in all experimental groups. In comparison, CS-PG and CT were present within and around the area of the lesion only in adult animals. In vivo, these molecules were colocalized with intensely glial fibrillary acidic protein (GFAP) positive astrocytes in and immediately adjacent to the scar, but not with other equally intensely GFAP-positive astrocytes in the cortex away from the site of injury. CT and CS-PG were present in gray matter areas of the cortex that had been directly damaged during the implant procedure and in the corpus callosum when lesioned during implantation. In vitro, the glial tissue removed from the lesion site of neonatal animals supported neurite outgrowth, while scars removed from adult animals did not. The inability of the adult glial scar tissue to support neurite outgrowth was best correlated with the expression of CS-PG and CT, suggesting that these molecules may be involved in limiting the growth of regenerating axons in the CNS after injury. PMID- 1719161 TI - Characterization of quisqualate receptor desensitization in cultured postnatal rat hippocampal neurons. AB - The quisqualate class of glutamate receptors is thought to play an important role in excitatory synaptic transmission, synaptic plasticity, and neuronal death. Since desensitization is a prominent feature of the responses mediated by this class of receptors, we have characterized the rapidly desensitizing quisqualate response in cultured postnatal rat hippocampal neurons using the whole-cell patch clamp technique. Quisqualate and its structural analogs elicit a peak current that rapidly decays to a steady-state level. In contrast, currents induced by kainate, NMDA, and their structural analogs exhibit either no decay or a much slower decay. The biophysical and pharmacological properties of the peak and steady-state quisqualate currents indicate that both are mediated by an ionotropic quisqualate receptor. Quisqualate currents desensitized monoexponentially by approximately 70% with a time constant near 80 msec. Both the rate and percentage of desensitization showed slight voltage dependence and were concentration dependent, reaching maximal values at saturation. Additionally, the overlap of the dose-response curves for activation of the steady-state current and desensitization of the peak current by a conditioning dose suggests that the two processes are related. Furthermore, desensitizing quisqualate currents were observed when Ca2+, Mg2+, Na+, K+, and Cl- were removed from the extracellular solution or their concentrations greatly reduced. These results suggest that the decline in the response is not caused by a simple open channel block mechanism. Despite the lack of desensitization by kainate, our observations are consistent with the hypothesis that quisqualate and kainate act at a single receptor-channel complex. Kainate and quisqualate appeared to interact competitively when applied simultaneously and noncompetitively when quisqualate was applied first. In addition, saturating doses of quisqualate and kainate gave steady-state currents of equal amplitude in neurons treated with the lectin WGA, an inhibitor of quisqualate receptor desensitization. PMID- 1719162 TI - The generation of monoclonal antibodies that bind preferentially to adrenal chromaffin cells and the cells of embryonic sympathetic ganglia. AB - Adrenal chromaffin cells, sympathetic neurons, and small intensely fluorescent (SIF) cells are each derived from the neural crest, produce catecholamines, and share certain morphological features. These cell types are also partially interconvertible in cell culture (Doupe et al., 1985a,b; Anderson and Axel, 1986). Thus, these cells are said to be members of the sympathoadrenal (SA) lineage and could share a common progenitor. To investigate the origins of this lineage further, we used the cyclophosphamide immuno-suppression method (Matthew and Patterson, 1983) to generate five monoclonal antibodies (SA1-5) that bind strongly to chromaffin cells, with little or no labeling of sympathetic neurons or SIF cells in frozen sections from adult rats. Competition experiments indicate that these antibodies bind to at least three distinct epitopes in tissue sections. The SA antibodies also label most of the cells of embryonic sympathetic ganglia and adrenal primordia. Labeling of sympathetic ganglia appears as the cells initially coalesce and express high levels of tyrosine hydroxylase (TH). Not all TH+ cells in the embryo are SA 1-5+, however; carotid body SIF cells, nodose ganglion TH+ cells, and the transiently TH+ cells in the dorsal root ganglia do not display detectable SA 1-5 labeling. Thus, the expression of these markers for the SA 1-5 lineage is selective. SA antigen expression is hormonally controlled; removal of glucocorticoid and addition of NGF to cultured adrenal chromaffin cells result in the loss of SA 1-5 labeling. These results suggest that the presumed precursors for sympathetic neurons and SIF cells initially express chromaffin cell markers. PMID- 1719163 TI - Isolation of the progenitor cells of the sympathoadrenal lineage from embryonic sympathetic ganglia with the SA monoclonal antibodies. AB - Our previous articles in this series described the production of five monoclonal antibodies (SA1-5) that bind to adrenal chromaffin cells and to cells in embryonic sympathetic ganglia and adrenal primordia (Carnahan and Patterson, 1991), and the downregulation of the sympathoadrenal (SA) antigens in vivo as neuronal markers begin to be expressed (Anderson et al., 1991). These results support the hypothesis that sympathetic neurons and adrenal chromaffin cells are derived from a common embryonic progenitor that displays both neuron- and chromaffin cell-specific markers. We have taken advantage of the fact that at least some of the SA antigens are expressed on the cell surface to isolate SA+ cells from embryonic day 14.5 rat superior cervical, sympathetic ganglia by fluorescence-activated cell sorting. This population of cells is significantly enriched in the expression of markers (tyrosine hydroxylase and neurofilament) found in the putative progenitors in situ. Growth in glucocorticoid maintains the expression of the SA antigens in the sorted cells and induces the chromaffin cell marker enzyme phenylethanolamine N-methyl transferase. In contrast, growth of the sorted cells in basic fibroblast growth factor, NGF, and insulin results in the rapid loss of SA 1 expression and the outgrowth of neurites. The ability to manipulate the fate of the SA+ cells in vitro confirms the suggestion from the in vivo observations that the SA+ cells in the ganglia are at least bipotential progenitors, capable of differentiating along the chromaffin or neuronal pathways. PMID- 1719164 TI - Characterization of potential precursor populations in the mouse olfactory epithelium using immunocytochemistry and autoradiography. AB - There are at least two basal cell populations in the olfactory epithelium that could give rise to olfactory neurons during development, in the normal adult, and after experimentally induced receptor cell death. These populations have been subdivided as horizontal (HBC) and globose (GBC) basal cells on the basis of morphological criteria and by staining with antibodies against cytokeratin. HBCs are positive for cytokeratin while GBCs are negative. We have studied which cell type is induced to divide during receptor cell regeneration stimulated by olfactory bulbectomy using a combination of immunocytochemistry and autoradiography. By examining which population increases its labeling index with 3H-thymidine (3H-TdR) at various times after bulbectomy, it is shown that there is an increase in 3H-TdR uptake in the cytokeratin-negative GBCs with no change in the cytokeratin-positive HBCs. This suggests that the GBCs are specifically induced to divide in response to cues that accompany receptor cell death, and it is thus concluded that these cells are among the precursors of new olfactory receptor neurons. PMID- 1719165 TI - Single odor-sensitive channels in olfactory receptor neurons are also gated by cyclic nucleotides. AB - Olfactory transduction is thought to occur by processes that are mainly restricted to the specialized cilia emanating from the distal end of the receptor neuron's single dendrite. The involvement of a cAMP-based second messenger system seems likely, and a cyclic nucleotide-sensitive current has been recorded in patches of membrane from the cilia. However, the small diameter of the cilia and the high density of channels within the membrane limit the application of the patch recording technique in the cilia. We have found that the cAMP-sensitive channels also exist at a much lower density within the far more accessible dendritic membrane. Recording from on-cell patches, we have observed single channel activity in response to extracellularly applied odor substances. The channels have a single-channel conductance of 40 pS and a reversal potential near 0 mV. These same channels are activated by treatments that elevate intracellular cyclic nucleotide concentrations. The results provide a direct demonstration that the cyclic nucleotide-gated channel is the conductance pathway for the odor elicited current. PMID- 1719166 TI - Analysis of single cyclic nucleotide-gated channels in olfactory receptor cells. AB - In the accompanying article (Firestein et al., 1991b), we have demonstrated that odor- and cyclic nucleotide-sensitive channels exist at a low density in the dendritic membranes of isolated salamander olfactory receptor neurons. Here, we analyze the cyclic nucleotide sensitivity of these channels using the inside-out patch recording technique. Both cAMP and cGMP, at micromolar concentrations, are capable of inducing channel openings. The biophysical parameters of channel activity are nearly the same in response to either ligand. The unitary conductance is about 45 pS, the reversal potential of single-channel currents is +5 mV, and the I/V relation is linear over the range -80 to +80 mV. The channel activity shows no obvious voltage dependence in divalent cation-free symmetrical solutions. The channel shows no desensitization, even to agonist exposures lasting 15 sec. Mean open time is about 1.5 msec; the closed time distribution is best fit by two exponentials with a fast time constant in the submillisecond range (ca. 0.15 msec) and a slower time constant in the millisecond range (ca. 1.5 msec). The only clear difference in the activity of the two ligands is in their affinity constants. The K1/2 for cAMP is 20 microM; that for cGMP is 4 microM. In both cases, the Hill coefficient is greater than 2, suggesting that channel opening requires the cooperative action of three ligand molecules. PMID- 1719167 TI - Decussation of hind-limb and fore-limb fibers in the monkey corticospinal tract: relevance to cruciate paralysis. AB - Cruciate paralysis is a clinical entity in which patients with trauma to the anterior cervicomedullary junction present with weakness of the upper extremity greater than that of the lower extremity. The underlying mechanism of this paralysis is commonly thought to be selective damage affecting the upper extremity nerve fibers in the pyramidal decussation. The authors examined the anatomical basis of cruciate paralysis in six New World squirrel monkeys and two Old World cynomolgus monkeys. No evidence for a differential decussation of fore limb and hind-limb fibers was found. Thus, there is no obvious anatomical explanation for cruciate palsy. The results do suggest two alternative explanations for cruciate paralysis: 1) selective damage to neural areas involving the internuncial cells, the central gray area, and the cuneate nucleus, or 2) injury to the ventral corticospinal tract. PMID- 1719168 TI - Age and protein restriction followed by balanced refeeding affect pancreatic digestive enzyme outputs and turnover times in rats. AB - Outputs and turnover times of trypsinogen 2, chymotrypsinogen 1, lipase and amylase were determined in pancreatic juice of growing male Wistar rats at various times during protein restriction (5% protein) followed by balanced refeeding (20% protein). In control rats fed a 20% protein diet, trypsinogen 2, chymotrypsinogen 1 and amylase outputs increased progressively with age, those of lipase remained constant and the turnover times of the four hydrolases were shortened. With time, protein restriction induced the most rapid decrease in trypsinogen 2 output, followed by that of amylase, then by those of trypsinogen 1 and lipase. Compared with controls, protein restriction enhanced specific radioactivity in total proteins and each hydrolase and diminished the enzyme turnover times at the beginning of the experiment. Refeeding returned each hydrolase output to control values, but the turnover times remained shortened to the end of experiment. Pancreatic lobules were isolated from control and protein depleted rat pancreata after 23 d of dietary treatment. After a pulse of [3H]leucine, lobules were incubated in Krebs-Ringer-bicarbonate-HEPES. Basal and stimulated (50 pmol cholecystokinin/L) secretions of total proteins, amylase and radioactive proteins were measured. Protein restriction resulted in a lower response of acinar cells to cholecystokinin. The reduced ability of cholecystokinin to stimulate secretion may be attributable to a lower number and/or to the reduced affinity of cholecystokinin receptors in acinar cells. Therefore, the observed output and turnover changes during protein restriction could be partly due to an impairment of cholecystokinin efficiency. PMID- 1719170 TI - Is palliation "medicine"? Ethical and epistemological problems. PMID- 1719169 TI - The ethics of palliative care. PMID- 1719171 TI - Family doctor and palliative care team: 1988 versus 1990. PMID- 1719172 TI - Parenteral hydration of terminally ill cancer patients. PMID- 1719173 TI - Palliative management of dyspnea. PMID- 1719174 TI - X-linked dilated cardiomyopathy with neutropenia, growth retardation, and 3 methylglutaconic aciduria. AB - Seven boys with an apparently X-linked syndrome of dilated cardiomyopathy, growth retardation, neutropenia, and persistently elevated urinary levels of 3 methylglutaconate, 3-methylglutarate, and 2-ethylhydracrylate were studied. The natural history of the disorder was characterized by severe or lethal cardiac disease and recurrent infections during infancy and early childhood but relative improvement in later childhood. The initial presentation of the syndrome varied from congenital dilated cardiomyopathy to infantile congestive heart failure to isolated neutropenia without clinical evidence of heart disease. The excretion of 3-methylglutaconate and 3-methylglutarate appeared to be independent of the metabolism of leucine, the presumed precursor of these organic acids in humans. Although the cause of the organic aciduria remains obscure, the constellation of biochemical and clinical abnormalities forms a distinct syndrome that may be a relatively common cause of dilated cardiomyopathy or neutropenia in boys during infancy and childhood. PMID- 1719175 TI - Granulocyte and granulocyte-macrophage colony-stimulating factors for treatment of neutropenia in glycogen storage disease type Ib. AB - Two children with glycogen storage disease type Ib associated with numerous recurrent bacterial infections as a result of neutropenia and neutrophil dysfunction were treated with recombinant human granulocyte colony-stimulating factor (G-CSF). One of the two patients was previously treated with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); therapy had to be discontinued because of severe local side effects. Both colony-stimulating factors at dosages of 3 and 8 micrograms/kg/per day, respectively, increased the average neutrophil counts from less than 300 cells/microliters to more than 1200 cells/microliters. Two subpopulations of neutrophils could be identified by their capacity to produce H2O2: one subpopulation generated H2O2 normally and a second was defective in H2O2 production. The doses of G-CSF effectively enhanced and maintained that subpopulation of neutrophils which produced normal amounts of H2O2. Moreover, these colony-stimulating factor-induced neutrophils demonstrated effective phagocytosis of zymosan particles and killing of staphylococci. Chemotaxis was decreased and could not be normalized by treatment with G-CSF. We conclude that maintenance treatment with G-CSF improved the quality of life in both patients: The number and severity of bacterial infections decreased markedly during treatment. Long-term treatment with G-CSF (12 and 10 months, respectively) was well tolerated, and no adverse clinical events were observed. PMID- 1719176 TI - Erosions of the angelchik prosthesis in pediatric-sized developmentally disabled patients. AB - We reviewed case histories of 40 pediatric-sized developmentally disabled patients who had previously participated in a study comparing the Nissen fundoplication with the Angelchik prosthesis for the surgical treatment of severe gastroesophageal reflux. Five of these patients had experienced erosions of the prosthesis into the gastrointestinal tract. These erosions were diagnosed between 2 years and 2 years 8 months following surgical insertion of the device. Erosions were associated with a variety of symptoms including vomiting, increasing discomfort, melena, anemia, coffee ground gastric residuals, and repeated small bowel obstructions. In no case was erosion associated with the development of peritonitis. Despite the documented advantages of the Angelchik prosthesis, the 12.5% erosion rate in this patient population is excessive. We recommend that use of the Angelchik prosthesis is not advisable in pediatric-sized developmentally disabled patients. PMID- 1719177 TI - Correlates of concern in parents of high-risk infants at age five. AB - Parents whose children have experienced a high-risk birth may be in double jeopardy due to the biomedical effects of the birth trauma as well as parent perceptions and feelings regarding medical vulnerability and the subsequent impact on caregiving. The present study determined whether the developmental, social, and medical concerns reported by 223 parents of high-risk infants at age 5 were congruent with the child's medical history and developmental performance at age 5. Results indicated that parents with the highest levels of developmental, social, and medical concerns had children with lower birth weights who were born earlier and spent longer periods of time in the hospital at birth. These children also had lower outcomes on the cognitive ability measures at age 5, and were more likely to demonstrate potential behavior problems. Present findings suggest the degree of concern reported by parents of high-risk infants is congruent with the birth history and the developmental status of their child. PMID- 1719178 TI - Antigens of diagnostic value in three isolates of Paracoccidioides brasiliensis. AB - Yeast cellular extracts of three isolates of Paracoccidioides brasiliensis were tested by the Western-blot technique against 53 sera from patients with paracoccidioidomycosis (PCM). Numerous antigens were recognized by the sera but only five were specific for (58, 57, 48, 44 and 23 kDa). These specific antigens had the same relative molecular mass in the different isolates but their reactivity with sera from PCM patients was variable. An antigen in isolate 688, which was specific for PCM, had an apparent molecular mass of 48 kDa. Although a 48 kDa antigen was also present in isolates B339 and 1789.88 it was not specific for PCM, demonstrating antigenic variability among isolates. The 44 kDa antigen in isolate B339 and the 44 and 48 kDa antigens in isolate 688 reacted with a rabbit antiserum raised against a 43 kDa glycoprotein, a specific antigen used in the diagnosis of PCM. There was no correlation between the specific antigen(s) detected and the clinical form of the disease. PMID- 1719179 TI - Identification of germ tube cell wall antigens of Candida albicans. AB - The reactivity of affinity-purified antibody to two components of germ tube cell wall extracts of Candida albicans showed that the components shared a common determinant(s). Surface expression of at least one of these determinants was demonstrated by indirect immunofluorescence where antibody binding was observed only on the hyphal extension of the organism. PMID- 1719180 TI - Exoantigen test for the rapid identification of Exophiala spinifera. AB - Exophiala spinifera, one of the etiologic agents of subcutaneous phaeohyphomycosis, may be wrongly identified as Exophiala jeanselmei because of the morphologic similarity of the two species. A reagent containing a precipitin specific for E. spinifera was produced by absorbing antiserum to E. spinifera with E. jeanselmei serotype 1 antigen. Using this absorbed antiserum, we were able to correctly identify isolates of E. spinifera and differentiate them from those of Exophiala alcalophila, E. jeanselmei (serotypes 1, 2 and 3), Exophiala moniliae, Exophiala pisciphila, Exophiala salmonis, Phaeoannellomyces werneckii and Wangiella dermatitidis. PMID- 1719181 TI - Aggressive excision of pulmonary metastases is warranted in the management of childhood hepatic tumors. AB - Although most children who die of liver malignancies do so as the result of complications of pulmonary metastases, little has been published regarding the efficacy of surgically excising such lesions. To the 12 previously reported cases of children who have undergone excision of pulmonary metastases of hepatic tumors, are added 5, 4 with hepatoblastoma and 1 with hepatocellular carcinoma. Total excision of a primary hepatic tumor leads to survival much more frequently than does incomplete excision. No patient had metastases at diagnosis. The length of time between resection of the primary tumor and the development of pulmonary disease resistant to chemotherapy is available for 9 of the 17 children; it was under 6 months for the 2 who died but over 6 months for the 7 who survived. Postoperative alpha-fetoprotein (AFP) levels accurately predicted the development of metastases in our 5 patients. Resection of metastases benefitted the 4 whose AFP levels had declined to less than 25 ng/mL following initial chemotherapy and who underwent operation before their levels increased above 1,000 ng/mL. They are alive and free of disease 4 to 83 months following excision of their lesions. Resection did not benefit the 1 nonsurvivor whose AFP level fell only to 5,000 ng/mL before beginning to increase, eventually reaching 58,000 ng/mL at the time of operation. Incomplete resection of metastases unresponsive to chemotherapy predictably leads to death. Multiple thoracotomies were successful in achieving the long-term survival of 4 children in this series.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719182 TI - Multidrug resistance in human neuroblastoma cells. AB - Neuroblastoma remains a significant problem in pediatric oncology. Recently a "multidrug-resistance" gene that may cause cells to become resistant to various chemotherapeutic agents has been cloned. The gene encodes the high-molecular weight plasma membrane protein known as P-glycoprotein. To study the expression of this gene in cells exhibiting the multidrug-resistant phenotype, a panel of sublines selected with several different natural product drugs was established. The drug-sensitive parental BE(2)-C cells were clonally isolated from the human neuroblastoma SK-N-BE(2) line and exhibit a 150-fold increase in the copy number of the N-myc protooncogene. Sublines were selected by stepwise increases in the concentration of actinomycin-D, doxorubicin, vincristine, or colchicine. Gene amplification was assessed using Southern analysis, and RNA levels were determined by Northern and dot-blot analysis. Western blotting was used to determine protein levels. N-myc amplification and expression were simultaneously determined to assess possible alterations associated with development of multidrug resistance. Amplified P-glycoprotein-encoding genes were not seen in control lines but were clearly present in those that had undergone exposure to each of the chemical agents. Similarly, steady-state messenger RNA and protein levels were greatly increased in the drug-resistant sublines. We conclude that human neuroblastoma cells can acquire the multidrug-resistant phenotype after exposure to various chemotherapeutic agents. PMID- 1719183 TI - Life-threatening hematochezia from a rectosigmoid vascular malformation in Klippel-Trenaunay syndrome: long-term palliation using an argon laser. AB - Recurrent severe hemorrhage from intestinal vascular malformations, although extremely rare, may be associated with significant morbidity and mortality. The treatment may require extensive surgical resection, vascular embolization, and repeated blood transfusions. An adolescent boy with the Klippel-Trenaunay syndrome involving the pelvis and left leg presented with recurrent life threatening hematochezia associated with defecation. Endoscopy documented that the bleeding originated from the submucosal venous angiomata in the region of the hemorrhoidal plexus. An argon laser was used to systematically coagulate the venous angiomata involving the distal 7 cm of the anorectal canal. Postoperative minor rectal bleeding and rectal tenesmus resolved in a few days. The patient has had only one brief episode of hematochezia in more than 4 years of follow-up. The use of the argon laser has provided effective palliation of colorectal vascular malformations with minimal morbidity but more importantly has allowed the preservation of normal anorectal function. PMID- 1719184 TI - IgG- and IgE-mediated histamine release from superfused guinea-pig airway tissues. AB - Anaphylactic histamine release and the inhibition by the beta-adrenoceptor agonist fenoterol has been investigated using lung and tracheal tissues from two groups of guinea-pigs, differently sensitized to respond with IgG or IgE antibodies, respectively. A superfusion method was introduced and compared with classical batch incubation. The difference between IgG- and IgE-mediated histamine release during superfusion of both tissues was much greater than the difference obtained during batch-incubations. Fenoterol inhibited IgG-mediated histamine release during superfusion at lower concentrations and to a larger extent than the release from IgE-sensitized tissues. The inhibition by fenoterol was less pronounced after batch-wise incubations, preincubation at 0 degrees C abolished the quantitative difference of IgG- and IgE-mediated histamine release from lung slices as well as the difference in beta-adrenergic inhibition. It is concluded that the new superfusion procedure for airway tissues enhances the sensitivity of antigen-induced histamine release for pharmacological modulation, compared with batch-wise incubation. In addition, the effects of 0 degrees C pretreatment show that cooled transport and storage of airway tissue should be considered with care. PMID- 1719185 TI - The effect of aminoguanidine treatment, with or without food restriction on the liver, stomach and small intestine of the rat. AB - A comparative investigation has been made into the effects of aminoguanidine treatment (60 mg kg-1 day-1) with or without dietary restriction (i.e. 50% reduction in food intake) on the protein, RNA and DNA composition of the liver, small intestine and stomach of young rats (0.1 kg body weight). After 3 weeks of dietary restriction the wet weights of the liver and small intestine decreased by 56 and 52%, respectively. There were significant reductions (approx 50%) in total hepatic and intestinal protein, RNA and DNA. Changes in ratios of RNA/protein, RNA/DNA and protein/DNA were only significant for intestinal RNA/DNA, where a 15% reduction was observed. In contrast, stomach wet weight and total protein content were unaltered by dietary deprivation. Stomach RNA and DNA contents were reduced by only 18-21%, and the protein/DNA ratio increased by 22%. Similar responses of liver, small intestine and stomach to dietary deprivation were observed in aminoguanidine-treated rats. Aminoguanidine-treatment of rats on an unrestricted diet for three weeks had no effect on the wet weights, total protein, RNA or DNA contents of the liver, stomach or small intestine. In dietary-restricted rats, and stomach were unaffected by the treatment. However, aminoguanidine treatment of dietary-restricted rats caused significant increases in the amounts of intestinal protein, RNA and DNA by approximately 15%. The treatment abolished the dietary restriction-induced decrease in total intestinal DNA/body weight. The wet weights of the lung, diaphragm, kidney, spleen and testes of both fed and dietary restricted rats were also unaffected by aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719186 TI - [Determinants of left ventricular inflow: the relation of the transmitral velocity profile to loading conditions]. AB - To clarify the factors controlling left ventricular inflow, hemodynamics and Doppler-derived indices were analyzed in six anesthetized open-chest dogs with intact pericardia. Low molecular dextran was intravenously infused at 1 L/hr. During rapid infusion, an electrocardiographic lead, left ventricular pressure curve, and a transmitral Doppler signal were recorded at various heart rates produced by right atrial pacing. In five of the six dogs, the relationship of the ratio of peak velocity during atrial contraction to that during rapid filling (A/R) and left ventricular end-diastolic pressure (LVEDP) showed a non-linear quadratic curve concave to the LVEDP axis. Data from the ascending and descending limbs of the A/R-LVEDP relationship were subjected to stepwise multiple linear regression analysis (criterion variables: A/R; explanatory variables: peak dP/dt, maximum left ventricular pressure, time constant T, minimum left ventricular pressure, LVEDP, and heart rate). In both limbs, the A/R correlated positively with maximum left ventricular pressure (left ventricular afterload) and negatively with LVEDP (left ventricular preload). The time constant T was selected as a positive correlate only in the ascending limb. The transmitral flow velocity profile is determined in a complex manner by multiple factors, including left ventricular loading conditions and heart rate, as well as the left ventricular diastolic property. It was suggested that the A/R is not altered unidirectionally by the changes in cardiac function and loading conditions, but that it returns to the initial value accompanied by systolic and diastolic cardiac dysfunction. PMID- 1719187 TI - [Pre-ejection flow in the left ventricular outflow tract: a pulsed Doppler echocardiographic study]. AB - Pre-ejection flow (PEF) has been recognized as the flow of short duration toward the cardiac base in the left ventricular outflow tract. In the present study, PEF was analyzed by pulsed Doppler echocardiography to determine the contribution of left ventricular contraction to the genesis of PEF. The study subjects consisted of 20 patients with lone atrial fibrillation (Af) and 30 patients with premature ventricular contractions (PVC) without other cardiovascular diseases. Phonocardiogram and electrocardiogram were simultaneously recorded with Doppler signal of PEF. PEF was clearly detected in only 18 patients with PVC; in the remaining 12, it was not recorded or could not be differentiated from the signals of the preceding left ventricular diastolic filling. Results were as follows: 1. PEF in the cases with sinus rhythm appeared before the Q wave; whereas, it appeared after the Q wave in the cases with Af or PVC, and the peak of PEF coincided with the first heart sound on the phonocardiogram. 2. The duration of PEF was 93.6 +/- 15.4 msec in sinus rhythm, 60.6 +/- 14.2 msec in Af, and 87.5 +/ 19.4 msec in PVC. 3. Peak velocities of PEF in Af and PVC were significantly less than peak velocity of PEF in sinus rhythm. 4. Peak velocity of PEF in PVC with left bundle branch block tended to be less than that in PVC with right bundle branch block.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719188 TI - Three-color cytofluorometric analysis of CD8 cell subsets in HIV-1 infection. AB - Previous studies have shown that CD8 cell subsets, some expressing activation markers, are elevated in human immunodeficiency virus (HIV) infection. To assess the overlap of these subsets, we used three-color flow cytometry to phenotype CD8 cells in cryopreserved mononuclear cells from uninfected controls and from people infected with HIV, in CDC classes II, III, and IV (n = 12 per group). There were several CD8 subset changes observed in association with HIV infection. A shift from a naive (CD45RA+CD45RO-) to a memory (CD45RA-CD45RO+) phenotype occurred in the CD8 subset, but the intermediate phenotype (CD45RA+CD45RO+) was unchanged. Increases in DR+CD8 and CD38+CD8 cells were noted in both naive and memory CD8 subsets, defined by CD45RA or CD45RO expression. Both the CD57+ and CD57- subsets of DR+CD8 cells were increased, whereas only the CD57+ subset of CD38+CD8 cells was elevated. The increase in CD57+CD8 cells reflected a selective rise in CD57+CD8 cells coexpressing CD38, DR, and CD45RO. The CD38+ DR+ CD8 subset was markedly increased and was apparently derived from both the CD38-DR-CD8 and CD38+DR-CD8 subsets. Compared with classes II and III, the CDC class IV group showed an increased proportion of CD8 cells expressing CD38; higher percentages of CD38+DR+CD8, CD38+CD45RA-CD8, and DR+CD45RO+CD8 subsets; and decreased percentages of CD38-CD45RA+CD8 and CD38-CD57-CD8 cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719189 TI - Evidence for an HIV-1 mediated suppression of in vitro growth of enriched (CD34+) hematopoietic progenitors. AB - The effect of HIV-1 on the in vitro growth of CD34+ cells, purified from bone marrow of normal donors, was studied. HIV-1 treated CD34+ cells exhibited a progressive and significant decrease of cell viability in liquid cultures and a reduced percentage of committed progenitors in the absence of viral infection. The same results were obtained treating CD34+ cell cultures with recombinant gp 120 alone. These results point to a direct cytotoxic activity of gp120 for CD34+ cells. PMID- 1719190 TI - [Receptor mechanisms underlying the actions of substance P and related neuropeptides]. AB - The excitability of peripheral and central neurons is regulated by two types of ion channels, voltage- and ligand-operated ones. Recent studies have revealed that the activities of these ion channels are under the control of a variety of classical neurotransmitters and neuropeptides. In particular, certain ion channels such as voltage-dependent Ca and K channels are reciprocally regulated by excitatory and inhibitory neurotransmitters, leading to the excitation and inhibition of nerve cells: for example, 1) the activation of voltage-dependent K channel is facilitated by somatostatin and inhibited by substance P, and 2) the opening of voltage-gated Ca channel is augmented by substance P and suppressed by somatostatin and certain opioid peptides. The ligand-gated ion channel, nicotinic acetylcholine receptor is also controlled by the actions of neuropeptides, substance P and calcitonin gene related peptide (CGRP). The regulation of ion channels with neuropeptides may contribute not only to the control of neuronal excitability but also to the plasticity of the nervous system. PMID- 1719191 TI - Effects of guanidino compounds on the endothelium-derived relaxing factor inhibitor NG-monomethyl L-arginine. AB - The vasoconstrictor effect of NG-monomethyl L-arginine (L-NMMA) is believed to be due to the inhibition of the synthesis of endothelium-derived relaxing factor (EDRF) from L-arginine. Here, we tested a series of guanidino compounds other than L-arginine on the rat aorta preparation with and without endothelium present. None of the compounds promoted vascular relaxation like N alpha-benzoyl L-arginine ethyl ester or elicited vasoconstriction like L-NMMA. We discovered that the two guanidino compounds, L-homoarginine (L-HA) and L-amino-tau-guanidino butyric acid (L-AGBA), behave like L-arginine and reversed the vasoconstrictor effect of L-NMMA. This effect was stereospecific and concentration-dependent. The order of potency to overcome the effects of L-NMMA was L-HA, followed by L-AGBA. This was the same order of potency for overcoming the inhibitory effects of L NMMA on cyclic GMP formation. Two related compounds, L-amino guanidino propionic acid and guanidine, were ineffective. Furthermore, high performance liquid chromatography analysis showed that rat aortic vessels contain the same amount of L-arginine in the presence or absence of endothelium, and no detectable amount of L-citruline is formed during endothelium-dependent relaxation. We conclude that the reversal of the effects of L-NMMA by L-HA and L-AGBA is due to their structural similarities to L-NMMA and not to synthesis of an EDRF-like material from these guanidines. We suggest that some of the inhibition of L-NMMA by L arginine may have a similar basis of structural antagonism. PMID- 1719192 TI - Rate-dependent effects of lidocaine on His-Purkinje conduction in the intact neonatal heart--characterization and amplification by N-acetylprocainamide. AB - According to mathematical models of antiarrhythmic drug-receptor interactions, lidocaine binds preferentially to the sodium channel when the membrane is depolarized (i.e., the "inactivated" channel state). Therefore, the effect of lidocaine on conduction should be greater when the action potential duration is prolonged. To test this prediction in vivo we evaluated the rate-dependent effects of lidocaine on His-Purkinje conduction in the intact newborn canine heart. Lidocaine's effect was assessed alone and then when given in combination with N-acetylprocainamide, a Class III antiarrhythmic agent. Utilizing intracardiac electrical stimulation and electrogram recording techniques, changes in the steady-state His-Purkinje conduction time during atrial pacing at increasingly rapid cycle lengths, changes in the conduction time of pacing trains delivered directly to the His bundle and changes in conduction time during His bundle extra stimulation were measured. After bilateral sectioning of the vagi and the administration of propranolol (1.0 mg/kg i.v.), His-Purkinje conduction was assessed in newborn canines (ages 5-15 days) under the following conditions: 1) control (n = 18); 2) after an i.v. infusion of lidocaine HCl (serum concentration 3.7 +/- 0.8 micrograms/ml) (n = 12); and 3) after the combined administration of lidocaine and N-acetylprocainamide (serum concentration, 26.4 +/- 6.3 micrograms/ml) (n = 12). Rate-dependent changes in His-Purkinje conduction time were observed in the newborn in response to lidocaine at rapid paced cycle lengths. These changes were significantly amplified by the coadministration of N-acetylprocainamide. Furthermore, the time constant of recovery from rate-dependent conduction delay, determined during His bundle extra stimulation, was 45 msec, which is notably shorter than values reported for lidocaine in the adult.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719193 TI - Methylxanthine treatment of smooth muscle cells differentially modulates adenylate cyclase responsiveness. AB - The DDT1 MF-2 smooth muscle cell line was used to study regulation of the A1 and A2 adenosine receptor (AR)-adenylate cyclase system by two different methylxanthines. 3-isobutyl 1-methylxanthine (IBMX) is both an AR antagonist and a phosphodiesterase inhibitor, while xanthine amine congener is an AR antagonist without phosphodiesterase activity. Incubation of cells for 18 hr with 100 microM IBMX produced a significant (P less than .05) decrease in the basal, isoproterenol- and sodium fluoride-stimulated adenylate cyclase activity. This generalized decrease in adenylate cyclase activity was associated with a significant decrease in the quantity of alpha s (Gs) as determined by Western blotting. In contrast, no alteration in alpha i (Gi) was observed in these same membranes. A significant increase in both the quantity of A1AR and the receptors' affinity for agonist occurred; however, no alteration in the ability of an A1AR selective agonist to inhibit adenylate cyclase activity was observed. Treatment for 18 hr with 50 nM xanthine amine congener, conversely, resulted in an increase in basal and isoproterenol stimulated adenylate cyclase activity with no change in membrane alpha s (Gs). With IBMX, there was an increase in agonist affinity for the A1AR without an associated change in the effect of adenosine agonists on adenylate cyclase activity. These data indicate that methylxanthine analogs which lack the ability to inhibit phosphodiesterases regulate receptor-mediated transmembrane signaling systems quite differently from those possessing such characteristics. The more prototypic methylxanthines regulate both receptors and G proteins in these smooth muscle cells. PMID- 1719194 TI - Reproducibility of two malignancy grading systems with reportedly prognostic value for oral cancer patients. AB - Supplementary prognostic factors should be added to the TNM classification for oral squamous cell carcinomas in order to optimize its clinical value. We have recently published two prognostically valuable malignancy grading systems based on histopathology and immunohistology of the most invasive cells in oral squamous cell carcinomas (OSCCs). However, a major problem with classifications based on histologic features is frequent lack of interobserver agreement which limits the clinical value of subjective histologic classifications. Thirty-eight file cases of OSCCs were therefore graded by three pathologists according to criteria of the histologic malignancy grading system which includes 5 morphologic features, each graded from 1 to 4. Agreement was calculated by kappa statistics, which showed that interobserver agreement was not optimal, but significantly better than by chance alone. We also studied the reproducibility of grading of immunohistologic membrane expression of a tumor-associated marker (blood group antigen H), and found a similar level of agreement. We conclude that the clinical value of our grading systems will increase by improving reproducibility. PMID- 1719195 TI - Cellular differentiation and morphologic heterogeneity in polymorphous low-grade adenocarcinoma of minor salivary gland. AB - Histologic diversity is intrinsic to salivary gland tumors, but it is a particular feature of polymorphous low-grade adenocarcinoma as denoted by the terminology. Application of immunohistochemistry and electron microscopy to three examples allowed the study of some aspects of tumor cell differentiation in this minor salivary gland lesion. In one case where the tumor cells were cytologically of one type, immunohistochemistry clearly identified both luminal and nonluminal tumor cells, but the latter showed no evidence of myoepithelial cell differentiation. A second case revealed differentiation only of luminal-type tumor cells, while a third example was largely differentiated as myoepithelial cells but again immunohistochemistry confirmed focal formation of duct-like structures by luminal epithelium. These cases show a considerable range of tumor cell heterogeneity as well as variations in their organization. This variation in differentiation characteristics underlies the histology of polymorphous low-grade adenocarcinomas, and likely occurs in other salivary gland tumors. To establish specific and reliable diagnostic criteria for such tumors requires awareness of this neoplastic process. The limited malignant potential and excellent survival of patients with polymorphous low-grade adenocarcinoma is apparently little affected by patterns of differentiation in this particular neoplasm. PMID- 1719196 TI - Relative color stability of ceramic stains subjected to glazing temperatures. AB - Ceramic stains are routinely used to modify Hue or characterize ceramic dental restorations. Subjective opinion has led to the hypothesis that certain stains are not color stable when subjected to glazing temperatures. This study tested the individual stains in nine different ceramic staining kits for color stability when subjected to glazing temperatures. Ceramic disks were made with gingival porcelain and coated with individual stains. Colorimetric recordings were made before and after glazing and the color difference (delta E) was calculated. Significant color changes were noted for specific individual stains from each of the ceramic staining kits tested. PMID- 1719197 TI - Abdominal instillation of high-molecular-weight dextran or lactated Ringer's solution after laparoscopic surgery. A randomized comparison of the effect on weight change. AB - Abdominal fluid retention after the instillation of lactated Ringer's solution into the abdomen after operative laparoscopy was evaluated by comparing the serial weights of patients receiving lactated Ringer's after surgery to those of patients treated with the abdominal instillation of high-molecular-weight dextran and to those of negative controls. Twenty-four patients were randomized to receive either lactated Ringer's or high-molecular-weight dextran after operative laparoscopy. Patients undergoing only diagnostic laparoscopy served as negative controls. Patients receiving either lactated Ringer's or high-molecular-weight dextran had increased weights as compared to the negative controls for at least 36 hours (P less than .5), although the weight gain in the treatment groups did not differ statistically significantly. The weight gain remained significantly greater than in the negative controls on postoperative days 3 and 4 in patients treated with dextran. Since the "flotation" effect of dextran in preventing pelvic adhesions is likely to be most pronounced in the immediate postoperative period, the findings suggest the need for a reinterpretation of adhesion prevention studies in which the use of dextran was compared to that of lactated Ringer's solution or to saline as a negative control. PMID- 1719198 TI - Who should have a prostatectomy? A survey of the management of patients presenting with bladder outflow obstruction. AB - A postal questionnaire was sent to 33 urologists and to 15 general surgeons who perform prostatic surgery in Scotland. Forty-six out of 48 surgeons replied. The waiting time for outpatient consultations and waiting list statistics of the respondents were compared. Differences in access to and use of imaging, laboratory and urodynamic facilities are reported. Waiting times were affected by the individual surgeon's policy. In busy units, the desire to achieve acceptable waiting times may lead to rationing of treatment to only the most severe cases. Better provision and use of modern investigational facilities might better select those patients who will benefit most from surgical management, leading to more effective use of resources. Medical audit of surgical patients must start from the time of their referral and not confine itself to patients undergoing treatment. PMID- 1719199 TI - Children's psychological health status--the impact of liver transplantation: a review. AB - Medical audit needs to encompass physical, psychological and social aspects to patient functioning. The aim of this paper is to review the psychosocial impact of liver transplantation on children and their families. Evidence suggests that end stage liver diseases are associated with developmental delays. Emotional and behavioural problems are common and have been found to relate to the child's developmental status, physical appearance as well as family functioning. Post transplant, children and families often experience an exacerbation of preexisting emotional problems. One year post transplant, children typically show continuing developmental delays. Longer term assessments of quality of life suggest that children may experience fewer hospital contacts and that over time psychological difficulties reduce. Many families continue to experience problems in normalizing their interactions within and outside the family. The findings demonstrate the importance of including psychosocial outcomes in auditing the efficacy of medical interventions. PMID- 1719200 TI - Congenital insensitivity to pain. PMID- 1719201 TI - Cutaneous aspects of Refsum's disease. PMID- 1719202 TI - Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies. AB - The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis. PMID- 1719203 TI - The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction. AB - Nucleotide sequence data for bacterial 16S ribosomal RNA was used to identify oligodeoxyribonucleotide primers suitable for probing the rRNA gene of mycobacteria and related organisms, with the polymerase chain reaction. The method enabled us to distinguish mycobacteria from other closely related genera, and to differentiate between slow- and fast-growing mycobacteria. Mycobacterium leprae fell within the slow-growing group of mycobacteria but there are significant differences between the sequence of the M. leprae 16S rRNA gene and that of other slow-growing mycobacteria. These differences were used to devise a rapid, non-radioactive method for detecting M. leprae in infected tissue. PMID- 1719204 TI - SIV of stump-tailed macaque (SIVstm) is a divergent Asian isolate. AB - Analysis of molecularly cloned DNAs of SIVs isolated from Asian rhesus macaque (Macaca mulatta; SIVmac) and pig-tailed macaque (Macaca nemestrina; SIVmne) has indicated a high degree of sequence homology between these viruses. Thus SIVmac and SIVmne might have originated from the same or very closely related viruses. We have cloned and sequenced a PCR-amplified segment containing the LTR sequences of SIV originating from a stump-tailed macaque (Macaca arctoides; SIVstm). Comparative sequence analysis indicates that SIVstm belongs to the SIV/HIV-2 group; however, it is genetically distinct from the other members. PMID- 1719205 TI - Characterization of T- and B-cell epitopes of a simian retrovirus (SRV-2) envelope protein. AB - Synthetic envelope peptides of a simian retrovirus (SRV-2) were used to define both T- and B-cell epitopes of the envelope protein. The SRV-2 peptide 100-106 specifically blocks rhesus anti-SRV-2 neutralizing antibody activity, and a peptide 100-106 keyhole limpet hemocyanin conjugate induces a strong antipeptide antibody response. SRV-2 peptide 100-106 and 233-249 induces good T-cell proliferation of murine spleen cells immunized with the SRV-2 virus. Thus, SRV-2 envelope peptide 100-106 represents both a T- and B-cell epitope, and peptide 233 249 a T-cell epitope. PMID- 1719206 TI - Neutralization epitope in the envelope glycoprotein of simian retrovirus-1 (SRV 1) and identification of the virus receptor. AB - We previously demonstrated that the area encompassing residues 147-162 of the envelope protein of simian retrovirus serotype-1 (SRV-1) is the target for rhesus anti-SRV-1 neutralizing antibodies. A peptide representing amino acids 142-167 of envelope protein (gp70) is capable of inducing virus-neutralizing antibodies in mice. The virus receptor was immunoprecipitated from Raji cells using gp70 as the ligand. Antibodies to peptide 142-167 inhibit the immunoprecipitation, indicating that the receptor recognizes residues 142-167 of the viral envelope protein. PMID- 1719207 TI - Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence. AB - The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes. PMID- 1719208 TI - Accumulation of fluorescent non-cationic probes in mitochondria of cultured cells: observations, a proposed mechanism, and some implications. AB - Cultured rat fibroblasts were exposed to millimolar concentrations of forty-four non-cationic fluorescent probes, of very varied physico-chemical properties. Mitochondrial staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non-ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge less than or equal to 0; log P (less ionized form) less than 0; and pKa approximately 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion-trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here. PMID- 1719209 TI - Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA. AB - Ribosome-inactivating proteins (RIPs) are known to inactivate eukaryotic ribosomes, which results in the inhibition of protein synthesis, but there has been no evidence that they inactivate the ribosomes of Escherichia coli. Recently, Mirabilis antiviral protein (MAP), a RIP, has been shown to inhibit the protein synthesis of E. coli as well as eukaryotes. To elucidate its mechanism, E. coli ribosomes treated with MAP were analyzed by polyacrylamide/agarose composite gel electrophoresis and RNA sequencing using reverse transcriptase with DNA primer. The 23 S rRNAs, with an A260 value for ribosomes of 15, were completely cleaved in vitro by a 30 minute treatment with MAP at a concentration of 100 nM at 37 degrees C and a subsequent treatment with aniline. However, they were not affected by ricin A-chain under the same conditions. The primer extension of DNA polymerization stopped before A2660 of 23 S rRNA in RNA sequencing. Furthermore, both 16 S and 23 S rRNAs were cleaved by the MAP and aniline treatments when naked E. coli rRNAs were used as substrates, and the primer extension stopped before bases A2660 and A1014, respectively, in RNA sequencing. As the A2660 region has been shown to interact with the elongation factors EF-Tu and EF-G these results indicate that MAP cleaves the N-glycosidic bond at A2660 in E. coli 23 S RNA resulting in the inactivation of the ribosome. PMID- 1719210 TI - Different intrastrand and interstrand contributions to stacking account for roll variations at the alternating purine-pyrimidine sequences in A-DNA and A-RNA. AB - An explanation is suggested for the roll alternation between low and high values in A-type nucleic acid duplexes containing alternating sequences of purine and pyrimidine residues. The explanation combines two points. (1) Roll inevitably occurs in A-type duplexes due to geometrical reasons. (2) Intrastrand base stacking is much more impaired by roll than interstrand base stacking in A-type duplexes. Therefore purine-pyrimidine steps, whose bases mainly exhibit an intrastrand stacking, resist roll and decrease it. By contrast, bases at pyrimidine-purine steps exhibit a significant interstrand stacking that is tolerant to roll in A-type nucleic acid duplexes. In consequence, it is favourable if the purine-pyrimidine and pyrimidine-purine steps adopt low and high rolls, respectively in A-conformations of DNA and RNA molecules containing alternating purine-pyrimidine sequences. This is actually observed in the relevant molecular crystal structures. PMID- 1719211 TI - In vitro assembly of a positioned nucleosome near the hypersensitive region in simian virus 40 chromatin. AB - Previous studies have identified a nucleosome near a potential late boundary for the nuclease-hypersensitive region in simian virus 40 chromatin. We have performed in vitro reconstitution analysis to determine whether the underlying DNA sequence encodes for the assembly of this nucleosome and applied hydroxyl radical and DNase I footprinting techniques to examine the structure of the reconstituted nucleosome. Both methods revealed the formation of a precisely positioned nucleosome in vitro, on a fragment spanning the strong in vivo nucleosome location site determined previously in the viral chromatin. The center of the positioned nucleosome maps between nucleotide 384 and 387 on simian virus 40 DNA. The corresponding nucleosome core includes the major-late transcription site (12 base-pairs within the core), the MspI site, and a segment shown previously to adopt a bent structure in the absence of proteins. The hydroxyl radical produces a strikingly well-defined cleavage pattern over the bent DNA incorporated in nucleosomes. The dominant periodicity of DNA in this nucleosome is 10.26 base-pairs per turn. The distribution of the .OH cut sites in the positioned nucleosome provides strong support for models in which the minor grooves of the A/T-rich tracts are oriented toward the histone core while the minor grooves of the G/C-rich sequences are facing outward. PMID- 1719212 TI - Cloning, sequence and overexpression of NADH peroxidase from Streptococcus faecalis 10C1. Structural relationship with the flavoprotein disulfide reductases. AB - DNA fragments encoding streptococcal NADH peroxidase (NPXase) have been amplified, cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The NPXase gene (npr) comprises 1341 base-pairs and is preceded by a typical ribosome binding site. Upstream from the structural gene, putative -10 and -35 promoter regions have been identified, as has a possible factor-independent terminator that occurs in 3'-flanking sequences. The deduced relative molecular mass (Mr = 49,551), amino acid composition and isoelectric point of NPXase are in good agreement with previous values obtained with the purified enzyme. In addition, three sequenced peptides totaling approximately 20% of the protein were located in the npr gene product. From the sequencing data the deduced NPXase sequence shares low but significant homology with the flavoprotein disulfide reductase class of enzymes ranging from 21% for glutathione reductase (GRase) to 28% for thioredoxin reductase. Alignment of NPXase to Escherichia coli GRase allowed the identification of three previously reported fingerprints for the FAD, NADP+ and central domains of GRase, in the peroxidase sequence. In addition, Cys42 of NPXase, which is present as an unusual stabilized cysteine-sulfenic acid in the oxidized enzyme, aligns favorably with the charge-transfer cysteine in E. coli GRase, and both residues closely follow FAD-binding folds found near their respective amino termini. Such sequence characteristics can also be seen in mercuric reductase, lipoamide dehydrogenase and trypanothione reductase, suggesting that all these enzymes may have originally diverged from a common ancestor. Sequences that are on average 50% identical with three previously reported peptides of the related streptococcal NADH oxidase were also identified in the NPXase primary structure, suggesting a strong similarity between these flavoenzymes. Using the E. coli phage T7 expression system the npr gene has now been overexpressed in an E. coli genetic background. The resultant overexpressing clone produced a recombinant NPXase that was catalytically active and immunoreactive to NPXase antisera. PMID- 1719213 TI - Secondary structure as a constraint on the evolution of a plant viral satellite RNA. AB - The genetic variability and evolution of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) was analyzed. Twenty-five CMV-satRNAs compared clustered into three main groups, and no correlation was found between genetic proximity and other characteristics (pathogenicity, geographical origin) of the satRNAs. Values for the number of nucleotide substitutions per site between any two satRNAs suggest that divergence is checked by functional constraints. The analysis of mutations relative to an ancestral sequence, and the number of substitutions per site at first, second and third positions of codons in putative open reading frames, show that the variation of CMV-satRNAs does not follow a pattern typical of coding sequences, and indicates that preservation of the sequence of encoded products is not a constraint to evolution. On the other hand, when the observed variation was analyzed relative to a secondary structure model proposed for CMV satRNAs, several lines of evidence indicated that the maintenance of the secondary structure is a constraint to evolution: the number of substitutions per site, the number of point insertions and deletions and the number of base substitutions that would disrupt base-pairing were significantly higher for unpaired than for base-paired positions. Also, compensatory mutations at base paired positions occurred more frequently than expected from random. The results suggest that CMV-satRNAs are non-coding, functional RNAs whose biology would be determined by their direct interaction with components of the host and/or the helper virus. PMID- 1719214 TI - Secondary structure of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase in solution using three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. AB - The solution structure of the ribonuclease H domain of HIV-1 reverse transcriptase has been investigated by three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. The domain studied has 138 residues and comprises residues 427 to 560 of the 66 kDa reverse transcriptase with an additional four residues at the N terminus. Initial studies on the wild-type protein were hindered by severe differential line broadening, presumably due to conformational averaging. Mutation of the single tryptophan residue located in a loop at position 113 (position 535 in the reverse transcriptase sequence) to an alanine resulted in much improved spectral properties with no apparent change in structure. 1H, 15N and 13C backbone resonances were assigned sequentially using a range of three-dimensional double and triple resonance heteronuclear experiments on samples of uniformly (greater than 95%) 15N and 15N/13C-labeled protein, and the secondary structure was elucidated from a qualitative analysis of data derived from three-dimensional 15N and 13C-edited nuclear Overhauser enhancement spectra. The secondary structure comprises three alpha-helices and five strands arranged in a mixed parallel/antiparallel beta-sheet with a +1, +1, -3x, -1x topology. The C-terminal region from residue 114 onwards appears to be conformationally disordered in solution as evidenced by an almost complete absence of sequential and medium range nuclear Overhauser effects. PMID- 1719215 TI - Structure and assembly of the Escherichia coli transcription termination factor rho and its interaction with RNA. I. Cryoelectron microscopic studies. AB - Cryoelectron microscopy has been used to visualize the Escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants. In the absence of RNA cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microM). One of the most common structural motifs we see is a six-membered ring of rho subunits (present as either a closed or "notched" circle), which corresponds to the predominant hexameric association state of the protein. Also visible are smaller oligomeric structures, present as curved lines of rho subunits, which probably represent the lower association states of the protein that coexist with the hexamer at these protein concentrations. Addition of oligomers of ribocytosine (rC) of defined lengths (23-mers and 100-mers) results in the generation of more homogeneous populations of rho oligomers. In the presence of (rC)23, all identifiable particles appear either as closed or as notched hexameric circles. A small fraction of these particles are of visibly higher density, and are identified with the dodecamers expected as a subpopulation of rho under these conditions. Binding of (rC)100, an oligomer of length greater than that needed to span the entire hexamer binding site, results in a uniform population of closed circular hexamers. In some images additional features are visible at either the centers or the peripheries of the particles. These features may correspond to the excess length of the rC strands bound to the hexamers. The distributions of particles observed under the various experimental conditions used correlate well to those deduced from physical biochemical studies Seifried et al., accompanying paper). PMID- 1719216 TI - Structure and assembly of the Escherichia coli transcription termination factor rho and its interactions with RNA. II. Physical chemical studies. AB - Transcription termination factor rho from Escherichia coli is comprised of a hexamer of identical protein monomers. Hydrodynamic and light-scattering studies have shown the fully assembled rho to be a doughnut-shaped structure. Semi denaturing gels, protein crosslinking, and spectroscopic studies, as well as other functional and binding determinations have established that the rho hexamer displays D3 symmetry (i.e. it exists as a trimer of dimers). In the accompanying paper we visualize rho directly in the absence of cofactor and show that binding of RNA it into the hexameric form. In this paper we examine the pathway and association constants involved in rho oligomer assembly. Sedimentation and fluorescence-detected size exclusion chromatography are used to demonstrate three steps in the assembly process. These steps can be differentiated by subunit association affinity and kinetic properties. The kinetics of the monomer-dimer equilibrium are fast and an apparent association constant of 1.3 x 10(6) M-1 is measured for this process. In contrast, the dimer-tetramer and tetramer-hexamer association processes appear to be slower (of the order of seconds) and to involve association constants that are smaller than that of the monomer-dimer reaction. This behaviour is consistent with a hexamer of D3 symmetry. Such a particle displays two kinds of subunit interactions; one associated with an intra dimer A:A interface and the other with an inter-dimer B:B interface. The closure of the circular hexamer does not appear to contribute additional free energy to the assembly process. Fluorescence and sedimentation studies show the association steps to be sensitive to salt concentration. Consistent with earlier work, we find that assembly to the hexameric state is driven by RNA binding. PMID- 1719217 TI - Mutations in the MotA protein of Escherichia coli reveal domains critical for proton conduction. AB - The MotA protein of Escherichia coli is an essential component of the torque generating units that drive the flagellar rotary motor. A variety of evidence indicates that MotA is involved in transmembrane proton conduction. We have now mapped a number of MotA mutants, focusing primarily on those previously shown to be dominant. Fifty-six mutations (all dominant), each causing severe or complete impairment of function, were sequenced and found to correspond to 31 different alleles. All except two of these encoded amino acid substitutions clustered in four hydrophobic, presumably membrane-spanning segments, that together make up only one-third of the length of the polypeptide chain. In contrast, eight mutations (5 dominant), each causing only slight impairment of function (slow alleles), were sequenced and found to specify amino acid substitutions in three hydrophilic domains. The clustering of the mutations provides independent support for the suggestion that MotA is a transmembrane proton channel and places significant constraints on models for the molecular mechanism of ion conduction. PMID- 1719218 TI - Preliminary X-ray investigation of a bifunctional inhibitor from Indian finger millet (ragi). AB - A bifunctional alpha-amylase/trypsin inhibitor that has two binding sites has been purified from ragi. The inhibitor has been crystallized from its ammonium sulphate solution by the vapour diffusion method. The crystals belong to the orthogonal space group P2(1)2(1)2(1) with unit cell dimensions a = 30.49 A, b = 56.30 A, c = 73.65 A and Z = 4. PMID- 1719219 TI - On the nature of spontaneous RNA synthesis by Q beta replicase. AB - Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs. PMID- 1719220 TI - Specificity of antitermination mechanisms. Suppression of the terminator cluster T1-T2 of Escherichia coli ribosomal RNA operon, rrnB, by phage lambda antiterminators. AB - Transcription of the ribosomal RNA operons (rrn) in Escherichia coli is subject to an antitermination mechanism whereby RNA polymerase is modified to a termination-resistant form during transit through the rrn leader region. This antitermination mechanism is unable to overcome the T1-T2 terminator cluster located at the end of an rrn operon, such as rrnB. We have tested the specificity with which the T1-T2 terminators override an antitermination mechanism, by placing the terminator cluster downstream from the nut and qut sites recognized by phage lambda N and Q gene antiterminators, respectively. Measurement of downstream gene expression shows that RNA polymerase modified by either N or Q reads through the T1-T2 terminators quite efficiently. This supports the view that T1-T2 are not superterminators, and that the rrn antitermination mechanism may have a restricted terminator specificity. PMID- 1719221 TI - Effect of captopril on isoproterenol-induced myocardial ornithine decarboxylase activity. AB - Polyamines are thought to play an essential role in cellular hypertrophy and proliferation. Ornithine decarboxylase (ODC) catalyzes the first and probably the rate-limiting step in biosynthesis of polyamines. In this study, we evaluated the pathophysiological role of the renin-angiotensin system in isoproterenol-induced cardiac hypertrophy, using myocardial ODC activity as an indicator of cardiac hypertrophy. Isoproterenol caused an eight-fold increase of myocardial ODC activity in normotensive Wistar rats within 4 h after injection. Captopril suppressed the induction of ODC by isoproterenol to two-thirds of the control level. These results indicate that the renin-angiotensin system may participate in the induction of myocardial hypertrophy by isoproterenol. PMID- 1719222 TI - Atrial natriuretic peptide inhibits oxidant-induced increases in endothelial permeability. AB - Chemically and enzymatically generated oxidants alter endothelial cell shape, increase macromolecular permeability across endothelial cell monolayers, and increase lung microvascular permeability. We examined the effect of ANP (atrial natriuretic peptide) on oxidant-induced injuries to bovine aortic endothelial cell monolayers and to isolated, perfused rabbit lungs. Treatment of cultured endothelial monolayers with glucose oxidase (1.4 U/ml) caused changes in cell shape characterized by a retraction of cells and the formation of numerous intercellular gaps. Glucose oxidase treatment also caused a reduction in F-actin stress fibers visualized by rhodamine-phalloidin fluorescence. Pretreatment (5 min) of the endothelial monolayers with ANP (10(-7) M) attenuated the oxidant induced changes in cell shape and reduction in F-actin staining. In addition, ANP significantly (P less than 0.05) reduced increases in endothelial monolayer permeability to albumin resulting from glucose oxidase treatment. Oxidant-induced injury of isolated, perfused rabbit lungs produced pulmonary edema measured as an increase in lung weight. This increase in weight was significantly (P less than 0.05) inhibited by pretreatment of lungs with ANP (10(-7) M). Collectively, these results suggest that ANP may act to preserve endothelial barrier function and reduce edema formation caused by oxidant injury. PMID- 1719223 TI - Failure of iloprost to protect the regionally ischemic, reperfused porcine heart. AB - The effect of iloprost (Schering AG, Berlin), a stable prostacyclin analogue, was investigated in ischemic, reperfused porcine hearts. The left anterior descending coronary artery (LAD) was distally occluded in 18 pigs for 45 min followed by 3-d of reperfusion. Nine pigs were continuously treated with iloprost at a dose of 25 ng/kg per min. Treatment was started as intracoronary infusion into the proximal LAD 10 min before occlusion. The intercoronary infusion was replaced by an intravenous infusion after 45 min of reperfusion, which was continued until the end of the experiment. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Myocardial concentrations of adenosine triphosphate were evaluated at the end of the experiment. Generation of free radicals by stimulated polymorphonuclear neutrophils was determined by luminol enhanced chemiluminescence. Histologic and immunohistologic techniques were applied to characterize the myocardial inflammatory response. Global hemodynamics did not differ between the two groups. Neither infarct size (control group 68 +/- 18%, treated group 74 +/- 14%), recovery of systolic shortening (control group 3 +/- 6%, treated group 6 +/- 6%), nor myocardial adenosine triphosphate concentrations were improved by iloprost treatment. Myocardial inflammatory response remained unaffected by this treatment. The capacity of coronary venous, stimulated polymorphonuclear neutrophils to generate free radicals was slightly suppressed in the treated group before ischemia, at the end of ischemia and during early reperfusion. In this preparation, iloprost did not exhibit any beneficial effect on infarct size, recovery of systolic shortening and myocardial adenosine triphosphate concentrations. PMID- 1719224 TI - Angiotensin II and left ventricular growth in newborn pig heart. AB - The left ventricle of the neonatal pig heart is a model of rapid physiological cardiac growth that is dependent upon accelerated ribosome formation and increased RNA content. The goals of the present study were to investigate the role of angiotensin II in this rapid growth. Hearts from 3 d old control piglets or piglets that were treated with enalapril maleate, an angiotensin converting enzyme inhibitor, or DuP 753, an angiotensin II receptor antagonist, were used for measurements of left ventricular mass, RNA, DNA and protein. Hearts from enalapril-treated pigs also were used for measurements of rates of ribosome formation and total protein synthesis during perfusion as modified Langendorff preparations. Treatment of piglets with enalapril maleate resulted in decreased left ventricle/body wt ratio, RNA content, total RNA and total protein in the left ventricle. These parameters were unaffected in the right ventricle. In vitro perfusion of hearts from enalapril-treated piglets revealed decreased ribosome formation and total protein synthesis in the left ventricle. Piglets treated with DuP 753 had decreased left ventricle/body wt ratio as well as decreased RNA content, total RNA and RNA/DNA ratio in the left ventricle. These results suggest that angiotensin II may be required for rapid growth of neonatal pig hearts. PMID- 1719225 TI - Studies on eye irritation caused by chemicals in rabbits--II. Structure-activity relationships and in vitro approach to primary eye irritation of salicylates in rabbits. AB - Structure-activity relationships and in vitro evaluation of eye irritation potential of salicylates in rabbits were studied. The primary eye irritation potential of ten salicylates was evaluated according to Draize method. The effects of chemicals on model protein and lipid were investigated in vitro. The effects of chemicals on the protein could be detected by the production of aggregates of human serum gamma-globulin (HSG) and a good correlation was obtained between the ability of salicylates to produce aggregation of HSG and the potential of corneal irritation. The effects on the lipid could be detected by the adhesion potential of chemicals on lipid membrane and a linear correlation was not obtained between the adhesionary effects of salicylates on lipid membrane and the potential eye irritation. The corneal irritation and protein aggregation potential of salicylates were correlated with the acid dissociation constant more closely than octanol/water partition coefficient. The destruction of alpha-helix of proteins in corneal surface by salicylates were observed from the nondestructive structural analysis of corneal surface by Fourier Transform (FT) IR spectroscopy. These results suggest that eye irritation caused by salicylates are mainly the results of denaturation of proteins in ocular tissue and that the effects on protein depend on the dissociation potential of molecules. PMID- 1719226 TI - Hypertonic saline dextran resuscitation fails to improve cardiac function in neonatal and senescent burned guinea pigs. AB - Our previous studies suggested that the greater diminution in burn-induced cardiac contractile function which occurs in young and elderly subjects compared with adult subjects is related to differences in intracellular calcium availability to the myofilaments. We recently showed that improved cardiac function after hypertonic saline dextran (HSD) resuscitation from burn injury in adults was related to enhanced intracellular calcium content. In the study presented here, 126 hearts isolated from neonatal, adult, and senescent guinea pigs were used to evaluate age-related differences in cardiac contractile response to HSD resuscitation from burn injury. Scald burn was induced in 30 adult, 18 neonatal, and 30 senescent guinea pigs; within each age group, half of the burned animals were resuscitated with lactated Ringer's (Parkland formula, 4 mL/kg/% burn for 24 hours); half received an initial bolus of HSD (4 mL/kg, IV) plus lactated Ringer's (1 mL/kg/% burn for 24 hours). An additional 16 animals from each age group served as sham burn controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719227 TI - Molecular anatomy of viral persistence. PMID- 1719228 TI - The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress. AB - A recombinant virus from which the start codon and 53% of the UL20 open reading frame had been deleted was constructed and characterized. We report the following: (i) The UL20- mutant formed small plaques in 143 tk- cells but failed to form plaques in Vero cells. Virus yields were approximately 10- to 100-fold lower than those of wild-type virus in all cell lines tested. (ii) Electron microscopic examination of Vero cells infected with the UL20- mutant revealed that enveloped and unenveloped capsids accumulated in the cytoplasm, possibly in the space between the inner and outer lamellae of the nuclear membrane, and that virtually no virus was present in the extracellular space. (iii) Glycoproteins B, C, D, E, H, and I recovered from lysates of cells infected with the UL20- mutant could not be differentiated from those present in lysates of cells infected with the wild-type parent virus with respect to the electrophoretic mobility of mature and precursor forms. (iv) Repair of the deleted sequences restored the wild-type phenotype. (v) The gene product of the UL20 gene was shown to be associated with cellular membranes and to possess characteristics of integral membrane proteins. We conclude that the UL20 gene encodes an integral membrane protein with a hitherto unrecognized function in that it enables the transit of virions to the extracellular space. The function of the UL20 gene product is complemented by some cell lines but not by Vero cells. The vesicles which serve to transport virions may have an origin different from those associated with transport of normal cellular proteins. PMID- 1719229 TI - Regulation of polyadenylation of hepatitis delta virus antigenomic RNA. AB - Hepatitis delta virus (HDV) is a subviral agent with a small RNA genome that is replicated in the nucleus of an infected cell. During genome replication, there is the synthesis of a complementary RNA, known as the antigenome, and also of a smaller complementary species that is polyadenylated and acts in the cytoplasm as the mRNA for the only known HDV protein, the delta antigen. We have carried out an examination of the cis- and trans-acting elements that regulate the polyadenylation process involved in the synthesis of this mRNA for the delta antigen. Our experimental approach has been to study the processing of nascent antigenomic RNA as it occurs in transfected cells via DNA-directed RNA synthesis, in the absence of genome replication. Three conclusions have been made. (i) The polyadenylation process occurs independent of the functionality of a unique self cleavage domain located just 3' of the polyadenylation site. (ii) RNA transcripts that proceed beyond the polyadenylation site can be stabilized by the self cleavage reaction. Thus, a single transcription initiation event can lead not only to the mRNA species but also to at least one more stable RNA species. (iii) If the nascent RNA species can fold on itself, into the so-called rodlike structure, then the presence of the delta antigen leads to a major suppression of polyadenylation. These results are incorporated into a more detailed model of the replication of the HDV genome. PMID- 1719230 TI - Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus. AB - The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells. This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study. PMID- 1719231 TI - Human-murine chimeras of ICAM-1 identify amino acid residues critical for rhinovirus and antibody binding. AB - Human ICAM-1 is the cellular receptor for the major group of human rhinoviruses (HRVs). Previous studies have suggested that the N-terminal domain of ICAM-1 is critical for binding of the major group rhinoviruses. To further define the residues within domain 1 that are involved in virus binding, we constructed an extensive series of ICAM-1 cDNAs containing single and multiple amino acid residue substitutions. In each case, substitutions involved replacement of the human amino acids with those found in murine ICAM-1 to minimize conformational effects. To facilitate the mutagenesis process, a synthetic gene encompassing the first two domains of ICAM-1 was constructed which incorporated 27 additional restriction sites to allow mutagenesis by oligonucleotide replacement. Each of the new constructs was placed into a Rous sarcoma virus vector and expressed in primary chicken embryo fibroblast cells. Binding assays were performed with six major group HRVs, including one high-affinity binding mutant of HRV-14, and two monoclonal antibodies. Results indicated that different serotypes displayed a range of sensitivities to various amino acid substitutions. Amino acid residues of ICAM-1 showing the greatest effect on virus and antibody binding included Pro 28, Lys-29, Leu-30, Leu-37, Lys-40, Ser-67, and Pro-70. PMID- 1719232 TI - Splice site skipping in polyomavirus late pre-mRNA processing. AB - Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site. PMID- 1719233 TI - Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action. AB - Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1 associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope. PMID- 1719234 TI - Antigenic and immunogenic epitopes shared by human papillomavirus type 16 and bovine, canine, and avian papillomaviruses. AB - All types of papillomaviruses (PV) share common, so-called group-specific epitopes. To identify the major group-specific epitopes, we immunized 26 guinea pigs or rabbits with purified bovine PV type 1 (BPV), canine PV, or avian PV from the common chaffinch. The resulting hyperimmune sera, as well as a commercially available rabbit antiserum to BPV and seven monoclonal antibodies to BPV, were tested in an enzyme-linked immunosorbent assay with a set of 66 overlapping 20 amino-acid peptides representing the complete sequence of the major capsid proteins (L1 and L2) of human PV type 16 (HPV 16). Sera from the same animals before immunization were used as controls. The minimal reactive epitopes within each peptide were further characterized by testing of truncated peptides. The cross-reactive epitopes were clustered in two regions of L1, an internal region (at positions 171 to 235), which contained three epitopes, and the more reactive region at the carboxy terminus (at positions 411 to 475), which contained six epitopes. The most reactive of the HPV 16 broadly cross-reactive epitopes was a carboxy-terminal epitope which had the sequence DTYRF and which reacted with nine of the antisera to BPV, canine PV, or avian PV, with the commercially available rabbit antiserum to BPV, and also with a mouse monoclonal antibody to BPV. Antipeptide antisera to all of the HPV 16 L1 peptides and to the most antigenically reactive of their truncated analogs were made in guinea pigs. Antipeptide antisera reactive with BPV were obtained for three of the cross reactive epitopes, and one of these antisera allowed highly sensitive detection of group-specific PV antigen by immunoperoxidase staining. PMID- 1719235 TI - Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV. AB - The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1 transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59. PMID- 1719236 TI - Human T-cell leukemia virus type I Tax induces expression of the Rel-related family of kappa B enhancer-binding proteins: evidence for a pretranslational component of regulation. AB - The Tax protein of the human T-cell leukemia virus type I (HTLV-I) serves as a potent transcriptional activator of its own long terminal repeat as well as select cellular genes, including interleukin-2 and the alpha subunit of the interleukin-2 receptor. Tax activation of these two growth-related genes appears to involve the induced nuclear expression of DNA-binding proteins that specifically engage related kappa B enhancer elements present in the 5' regulatory regions of these genes. In human T cells, kappa B enhancer-binding activity has been discerned as an unexpectedly large family of UV cross-linked nucleoprotein adducts, termed p50, p55, p75, and p85. The protein components of each of these DNA-protein adducts have been shown to share structural similarity with the v-rel oncogene product. The p55 adduct is composed of the 50-kDa subunit of NF-kappa B derived from a 105-kDa precursor polypeptide, while the p50 adduct contains a smaller protein that is closely related to NF-kappa B p50. The p75 adduct contains the 65-kDa subunit of NF-kappa B, while the p85 adduct is composed of the human c-rel proto-oncogene product. We now demonstrate that HTLV I Tax, in the absence of other viral pX gene products, is capable of inducing the nuclear expression of all four of these kappa B-binding proteins in human T cells, with most marked effects involving c-Rel and NF-kappa B p65. Tax induction of the nuclear expression of c-Rel and NF-kappa B p50 is regulated, at least in part, at a pretranslational level involving increases in c-rel and NF-kappa B p105 mRNA expression. To study the pattern of expression of these kappa B specific proteins in cells infected with the whole HTLV-I, seven cloned HTLV-I infected T-cell lines were established from the peripheral blood of patients with adult T-cell leukemia. Of note, only three of these seven cell lines produced Tax, and c-rel mRNA and nuclear protein expression was confined to these three cell lines. In contrast, NF-kappa B p50 and NF-kappa B p65 were constitutively expressed in the nuclei of all seven of the HTLV-I-infected cell lines, even in the absence of detectable Tax or other viral gene expression. These findings raise the possibility of an alternate, Tax-independent pathway for the induced nuclear expression of NF-kappa B p50 and NF-kappa B p65 following HTLV-I infection. PMID- 1719237 TI - A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies. AB - Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein. PMID- 1719238 TI - Purification and characterization of recombinant equine infectious anemia virus reverse transcriptase. AB - A 1.67-kb segment of the equine infectious anemia virus pol gene, encoding a 66 kDa reverse transcriptase (RT), was cloned and expressed in Escherichia coli. Recombinant RT, purified by a combination of metal chelate affinity chromatography and ion-exchange chromatography, displays both RNA-dependent DNA polymerase and RNase H activity. The affinity of purified RT for its replication primer, tRNA(3Lys) was equivalent to that observed for human immunodeficiency virus RT. Our data suggest that an additional domain between RT-RNase H and integrase on the equine infectious anemia virus pol open reading frame is not an integral component of the RT polypeptide. PMID- 1719239 TI - Use of a lambda gt11 expression library to localize a neutralizing antibody binding site in glycoprotein E2 of Sindbis virus. AB - The Sindbis virus envelope contains two species of integral membrane glycoproteins, E1 and E2. These proteins form heterodimers, and three dimeric units assemble to form spikes incorporated into the viral surface which play an important role in the specific attachment of Sindbis virus to host cells. To map the neutralization epitopes on the surface of the virus, we constructed a lambda gt11 expression library with cDNA inserts 100 to 300 nucleotides long obtained from randomly primed synthesis on Sindbis virus genomic RNA. This library was screened with five different neutralizing monoclonal antibodies (MAbs) specific for E2 (MAbs 50, 51, 49, 18, and 23) and with one neutralizing MAb specific for E1 (MAb 33). When 10(6) lambda gt11 plaques were screened with each antibody, four positive clones that reacted with E2-specific MAb 23 were found. These four clones contained overlapping inserts from glycoprotein E2; the domain from residues 173 to 220 of glycoprotein E2 was present in all inserts, and we concluded that this region contains the neutralization epitope recognized by the antibody. No clones that reacted with the other antibodies examined were found, and we concluded that these antibodies probably recognize conformational epitopes not present in the lambda gt11 library. We suggest that the E2 domain from residues 173 to 220 is a major antigenic determinant of Sindbis virus and that this domain is important for virus attachment to cells. PMID- 1719240 TI - A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response. AB - We used 18 monoclonal antibodies against B19 parvovirus to identify neutralizing epitopes on the viral capsid. Of the 18 antibodies, 9 had in vitro neutralizing activity in a bone marrow colony culture assay. The overlapping polypeptide fragments spanning the B19 structural proteins were produced in a pMAL-c Escherichia coli expression system and used to investigate the binding sites of the neutralizing antibodies. One of the nine neutralizing antibodies reacted with both VP1 and VP2 capsid proteins and a single polypeptide fragment on an immunoblot, identifying a linear neutralizing epitope between amino acids 57 and 77 of the VP2 capsid protein. Eight of nine neutralizing antibodies failed to react with either of the capsid proteins or any polypeptide fragments, despite reactivities with intact virions in a radioimmunoassay, suggesting that additional conformationally dependent neutralizing epitopes exist. PMID- 1719241 TI - Combination chemotherapy with methotrexate, bleomycin and cisplatin for advanced squamous cell carcinoma of the male genital tract. AB - A total of 14 men with inoperable or metastatic squamous cell carcinoma of the genital tract received methotrexate, bleomycin and cisplatin. In 12 patients the penis was the primary site. Metastases were usually advanced, and 4 patients had ulceration and infection in the inguinal region or perineum secondary to tumor. Two patients had tumor-related hypercalcemia. Of the patients 11 received the 3 drugs intravenously, whereas 3 received methotrexate intravenously, and bleomycin and cisplatin intra-arterially for large unilateral nodal metastasis. Of the 14 patients 10 responded, for a response rate of 72% (95% confidence interval 57 to 92%) and the median response duration was 6 months (range 4 to 24 months). Two patients (14%) who were treated intravenously achieved complete responses lasting 6 and 24+ months. Responses occurred in 3 patients with infection and in both patients with hypercalcemia. The combination of methotrexate, bleomycin and cisplatin has significant activity in patients with advanced squamous cell carcinoma of the male genital tract. This chemotherapy regimen should be evaluated in earlier disease in the adjuvant or neoadjuvant setting. PMID- 1719242 TI - Adjuvant chemotherapy of metastatic stage II nonseminomatous testis tumor. AB - In a prospective randomized trial, 225 patients with stage IIB nonseminomatous testis tumor after radical retroperitoneal lymph node dissection received 2 versus 4 courses (arms 1 and 2, respectively) of adjuvant chemotherapy with cis platinum, vinblastine and bleomycin. With a median followup of 43 months, a total of 7 relapses occurred; 6 in arm 1 and 1 in arm 2. Three patients died: 2 during adjuvant chemotherapy and 1 of progressive disease. The difference in relapse rates between arms 1 and 2 is not statistically significant. Patient compliance differed: chemotherapy was administered according to protocol in 83% and 50% of the cases in arms 1 and 2, respectively. Most frequent side effects observed were nausea, vomiting and alopecia. No significant differences regarding these or other side effects were obtained. Patients with stage IIB nonseminomatous testis tumor after retroperitoneal lymph node dissection are treated sufficiently with 2 courses of adjuvant cis-platinum-containing chemotherapy. PMID- 1719243 TI - Systematic prostatic biopsies in 100 men with no suspicion of cancer on digital rectal examination. AB - A total of 100 men with a mean age of 63 years underwent, in the following order, prostate specific antigen (PSA) assay (radioimmunometric assay, normal less than 2.5 ng./ml.), rectal examination, transrectal ultrasonography with a 7 MHz. probe, measurement of the prostatic volume, and 6 ultrasound-guided randomized biopsies and biopsies of any hypoechogenic zones. All men with a suspicious prostate on rectal examination (nodule, induration or firm zone) were excluded from the study. There were 14 prostatic cancers detected: 3 (8.5%) in men less than 60 years old, 4 (11%) in men between 60 and 70 years old and 7 (24%) in men more than 70 years old. No cancer was detected in men with a PSA level of less than 10 ng./ml., 5 (26%) were detected in 19 men with a PSA level of 10 to 19 ng./ml., 4 (40%) were detected in 10 men with a PSA of 20 to 29.9 ng./ml. and 5 (100%) were detected in 5 men with a PSA of 30 or more ng./ml. A total of 66 men (66%) had a PSA level of less than 10 ng./ml. There were 18 (18%) hypoechogenic zones detected: 2 (11%) were positive for cancer but, over-all, the hypoechogenic zones revealed cancer in only 2 of 100 cases (2%). In 12 of the 14 cancers detected (86%) with no clinical suspicion the PSA level was higher than the maximal PSA level related to the prostate weight. We conclude that systematic randomized prostatic biopsies are the best method of early diagnosis, detecting 41% of all prostatic cancers in men with a normal rectal examination when the PSA level is 10 ng./ml. or more. The real question is to determine whether this early diagnosis is useful for the patient, since presently, there is no certainty of the therapeutic benefit in terms of quantity and quality of life. PMID- 1719244 TI - Biopsy after external beam radiation therapy for adenocarcinoma of the prostate: correlation with original histological grade and current prostate specific antigen levels. AB - We obtained post-irradiation biopsies in 37 men with initially stage T3 prostatic adenocarcinoma treated by external beam radiotherapy. Eligibility for post irradiation biopsy included no clinical local failure, interval since treatment of 24 months or more and no endocrine therapy. Of the 37 patients 23 (62%) had negative biopsies while 14 (38%) had positive biopsies. Of 23 patients whose original cancer was well or moderately differentiated 18 (78%) had negative biopsies, compared to only 5 of 14 (36%) of those with poorly differentiated cancer (p less than 0.03). Among 19 patients whose current serum prostate specific antigen (PSA) value is less than 2.5 ng./ml. 15 (79%) had negative biopsies, compared to only 4 of 14 (29%) with a PSA level of greater than 2.5 ng./ml. (p less than 0.02). These results strongly suggest that there is a low probability of positive post-irradiation biopsy regardless of its significance in men with a normal prostate by palpation and a normal serum PSA value. However, short followup since biopsy precludes analysis of the predictive value of post irradiation biopsy on long-term local and distant disease status. PMID- 1719245 TI - Dynamics of micturition in benign prostatic hypertrophy patients with compensated obstruction of the vesical outlet: a denervation supersensitivity-related energy saving mechanism. AB - Infravesical obstruction was assessed by pressure flow study in 40 benign prostatic hypertrophy patients with a fully compensated bladder (20 stable and 20 unstable). The degree of obstruction was defined by the minimum contraction strength needed by the detrusor to start the urinary flow (opening contraction power). There were significant positive correlations among opening contraction power, maximum flow rate and maximum external voiding power, and a negative correlation between opening contraction power and voiding duration (r = -0.54, p less than 0.001). Such features suggest a decreased electrical resistance between the detrusor smooth muscle cells (due to an obstruction-related state of denervation supersensitivity) and would mirror an energy-saving mechanism. This probably works only when there is no heavy detrusor collagenosis upsetting the depolarization wave. Within physiological muscle mechanical limits, severely obstructed bladders could thus empty completely despite even highly increased amounts of work needed per unit of volume voided. PMID- 1719246 TI - Elevation of prostate specific antigen from bacillus Calmette-Guerin-induced granulomatous prostatitis. PMID- 1719247 TI - Prostatic acid phosphatase, beta-glucuronidase and prostate specific antigen assays in fine needle aspirates from benign and malignant prostates. AB - Enzymatic assays for tartrate-sensitive acid phosphatase and beta-glucuronidase, and radio-immunoassay for prostate-specific antigen, were modified for application to fine-needle aspirate samples from benign and malignant human prostates. When compared to samples from benign prostates, the ratio of acid phosphatase to beta-glucuronidase activities was significantly decreased in needle aspirates from malignant prostates. Prostate-specific antigen values in the aspirates did not correlate with malignancy. PMID- 1719248 TI - An improved vasoactive drug combination for a pharmacological erection program. AB - Papaverine hydrochloride (smooth muscle relaxant), phentolamine mesylate (alpha adrenergic blocking agent) and prostaglandin E1 (vasodilator and smooth muscle relaxant) were combined to produce a potent vasoactive drug therapy for use in a pharmacological erection program. Doses of 2.5 cc papaverine (30 mg./cc), 0.5 cc phentolamine (5 mg./cc), 0.05 cc prostaglandin E1 (500 micrograms./cc) and 1.2 cc 0.9% normal saline were combined to produce a vial of 4.25 cc for patient convenience. Twenty unit vials were made from the 1 cc vial of prostaglandin E1, the most expensive ingredient. The solution is physiologically active for at least 6 months and can be stored at room temperature although refrigeration is recommended. The pH of the solution is 4. This vasoactive drug combination has been used in 116 patients for diagnostic testing and subsequent treatment. A dose of 0.25 cc has been effective for diagnosis and treatment in the majority of patients with mild to moderate arteriogenic and/or venogenic and diabetic impotence. For patients with neurogenic dysfunction 0.1 to 0.125 cc was the usual dose. Two patients had a prolonged erection requiring irrigation, 1 on the day of initial testing and 1 on home therapy. Pain at the site of injection or during intercourse was noted in only 2 patients and to date no fibrosis or plaques have been found. PMID- 1719249 TI - Re: Transurethral incision of the bladder neck and prostate. PMID- 1719250 TI - Re: Prostate specific antigen for assessing response to ketoconazole and prednisone in patients with hormone refractory metastatic prostate cancer. PMID- 1719251 TI - Mechanical irritation induces neurogenic inflammation in the rat urethra. AB - A catheter was inserted into the urethral meatus of urethane-anaesthetized rats and rotated (30 rotations/minute) during a three minute period. One hour later, microvascular permeability in the distal urethra was evaluated by means of the Evans Blue leakage technique. Dye extravasation increased significantly (74 +/- 12 ng./mg. of wet tissue weight, p less than 0.05), as compared to control values (18 +/- 2 ng./mg.). The effect of catheterism was prevented by about 50% by systemic pretreatment with capsaicin performed in either adult or newborn rats, as well as by bilateral removal of pelvic ganglia. Furthermore, pretreatment with capsaicin of adult rats, combined to pelvic ganglionectomy, virtually abolished the inflammatory response produced by mechanical irritation of the urethra. These results indicate that: 1) the increase of vascular permeability produced by mechanical irritation is nerve-mediated, 2) capsaicin-sensitive afferents participate in the inflammatory process and 3) capsaicin-insensitive nerves, which pass through the pelvic ganglia, contribute to the overall response. PMID- 1719252 TI - Comparison of 15 monoclonal antibodies against tumor-associated antigens of transitional cell carcinoma of the human bladder. AB - Quantitative urinary immunocytology with our monoclonal antibody (mab) 486p 3/12 proved to be valuable for diagnostic use in bladder-cancer patients' urine, especially in the followup of patients with superficial bladder carcinoma. To evaluate the use of other monoclonal antibodies in bladder cancer, we compared 15 mabs directed against bladder-tumor-associated antigens from seven research groups in a broad panel of cellular and tissue specimens (bladder tumor, prostatic adenoma, and kidney stone). Quantitative evaluation was done in cytocentrifuged preparations and tissue specimens. None of the 15 mabs was bladder-tumor-specific. All 15 stained normal urothelium to some extent and six stained granulocytes. Each of the 15 seemed to identify a different cellular antigen, as can be clearly demonstrated by the staining pattern of different regions in the normal kidney. The sensitivity of quantitative urinary immunocytology in bladder-tumor patients can be improved by using a panel, rather than one mab in bladder-tumor patients, but specificity decreases simultaneously. A main reason for the poor specificity of quantitative urinary immunocytology with all 15 mabs is that false-positive results are obtained with all mabs in kidney-stone patients. Our quantitative urinary immunocytology method is a general tool for the diagnostic use of all mabs in bladder-tumor patients. Mabs that have a high sensitivity might be useful in the followup of patients with superficial bladder carcinoma. None of the 15 mabs (because of their poor specificity) seems to be helpful in quantitative urinary immunocytology for screening a population for bladder carcinoma. PMID- 1719253 TI - The innervation of the human prostate gland--the changes associated with benign enlargement. AB - Different regions of the prostate gland, namely prostatic capsule, peripheral prostate and central prostate (subdivided into proximal (near the bladder neck), distal (near the verumontanum) and midway between these areas) were obtained from 32 obstructed (stable obstructed, n = 8; unstable obstructed, n = 13; acute retention, n = 11) and five control patients. The innervation of these tissues was studied both histochemically to localise acetylcholinesterase activity and immunohistochemically for dopamine-beta-hydroxylase, 5-hydroxytryptamine, vasoactive intestinal polypeptide, neuropeptide Y, leu- and met-enkephalin, calcitonin gene-related peptide, substance P and somatostatin. In control patients the greatest density of nerves was found in the proximal central prostate, followed by the anterior capsule and distal central prostate, with the least density in the peripheral prostate. The greatest density of nerves were acetylcholinesterase positive and immunoreactive to neuropeptide Y followed (in decreasing order) by nerves immunoreactive to: vasoactive intestinal polypeptide and dopamine beta-hydroxylase; leu-enkephalin and 5-hydroxytryptamine; calcitonin gene-related peptide; met-enkephalin; substance P; somatostatin. In addition a group of periacinar 5-hydroxytryptamine-immunoreactive cells and ganglia containing acetylcholinesterase, dopamine beta-hydroxylase and all of the peptides studied except somatostatin were identified. In the prostate gland from obstructed patients there was a significant reduction in the density of acetylcholinesterase-positive nerves (p less than 0.001) when compared with the controls. A similar trend was found for dopamine beta-hydroxylase, 5 hydroxytryptamine and all of the putative neuropeptides in most areas of the prostate, the most notable exceptions being in the peripheral prostate, with an increase in dopamine beta-hydroxylase- and leu-enkephalin-immunoreactive nerves in all three groups of obstructed patients an an increase in vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerves in those presenting in urinary retention. The functional significance of these findings is discussed. PMID- 1719254 TI - Harry M. Vars Research Award. Arginine supplementation improves histone and acute phase protein synthesis during gram-negative sepsis in the rat. AB - Mechanisms of nutrient alteration of hepatic protein synthesis during sepsis are unclear. In vitro, arginine downregulates endotoxin-stimulated hepatocyte protein synthesis but in vivo effects are unknown. This study evaluated the effects of supplemental arginine or glycine on fibrinogen (acute-phase protein), histone, albumin, and liver protein synthesis after Gram-negative sepsis in the rat. Adult rats (225 g, n = 36) were randomized to receive isonitrogenous isocaloric total parenteral nutrition supplemented with 264 mg of N per kilogram per day as either arginine or glycine. On day 5, each group was further randomized to control or sepsis. Sepsis was induced by injection of 8 x 10(7) Escherichia coli per 100 g body weight, and then a continuous infusion of [1-14C]leucine was started. The rats were sacrificed 4 hours later. The fractional protein synthesis rates (percent per day) of histone, fibrinogen, albumin, and liver were determined. Supplemental arginine led to significantly increased histone (p less than 0.05, analysis of variance) and fibrinogen (p less than 0.01, analysis of variance) synthesis in the septic rats compared with all other groups. Histone and albumin synthesis were also significantly increased (p less than 0.05) in the arginine supplemented control group compared with the glycine-supplemented control group. Arginine supplementation during sepsis significantly increased (p less than 0.05) albumin and liver protein synthesis compared with controls. Histones which are involved in DNA synthesis and are rich in arginine may play a role in the host response to stress and sepsis. These in vivo results appear to contradict hepatocyte-Kupffer cell coculture studies perhaps because of the hormonal and cytokine responses to nutrient substrate and acute septicemia. PMID- 1719255 TI - [Bone marrow transplantation in a pediatric case of acute nonlymphocytic leukemia with hepatitis C]. AB - An allogenic bone marrow transplantation (BMT) in an acute nonlymphocytic leukemia (ANLL) patient with post-transfusion hepatitis C is presented. A 13-year old girl was admitted to our hospital on May 1988, and diagnosed as having ANLL M2 according to the FAB classification. During the induction and post-induction chemotherapy, 116 units of blood products were transfused to her as the supportive therapy until October 1988, when non-A non-B hepatitis developed. As the persistent liver dysfunction interfered with anti-leukemic chemotherapy on the protocol, allogeneic BMT from her HLA identical MLR nonreactive brother was done on July 1989. Preconditioning regimen consisted of busulfan and cyclophosphamide. GVHD prophylaxis consisted of cyclosporine A and short term methotrexate. After the BMT, her liver dysfunction once improved; her serum amino transferase levels were normal for about 3 months. Soon after discontinuation of cyclosporine A, however, her liver function deteriorated again. The examination of hepatitis C virus antibody in her sera, which had been harvested sequentially and stored at -40 degrees C, on November 1989 revealed that she had been already seropositive at the time of BMT. The BMT-induced immunologic changes may have influenced the natural course of hepatitis C virus infection in the patient. PMID- 1719256 TI - [Effects of maintenance treatment after high-dose intravenous gamma-globulin for idiopathic thrombocytopenic purpura]. AB - High-dose intravenous immunoglobulin (IVIG) for idiopathic thrombocytopenic purpura (ITP) produces a dramatic and substantial increase in platelet count, but the increased count tends to return rapidly to its pretreatment level. We studied the effects of immunosuppressive treatment aimed at the maintenance of platelet counts following the IVIG administration in ITP. Thirty-five patients with ITP were treated with IVIG, and then thirty-two of them with an immunosuppressant (azathioprine) and a glucocorticoid (prednisolone). After IVIG, the platelet count increased significantly. With immunosuppressive therapy after IVIG, most patients had a tendency to maintain the counts. In particular, this maintaining effect was remarkable in those patients who had been responsive to the standard prednisolone therapy while non-responders to the prior prednisolone failed to maintain the counts. When prednisolone was given after IVIG, the effect of maintaining platelet counts was dose-dependent. The treatment with azathioprine and prednisolone after IVIG appears to be effective in maintenance of platelet counts. PMID- 1719257 TI - [Factor VIII epitopes recognized by inhibitors in hemophiliacs]. AB - A 44-year-old male hemophiliac with high titer anti-factor VIII antibody (66 bethesda units/ml) was admitted on November 11, 1989 because of epigastralgia and melena. A gastric ulcer with a spurting artery was revealed by an upper gastrointestinal endoscopy. Infusion of activated prothrombin complex concentrates and endoscopical ethanol injection to the bleeding vessel were ineffective. After clipping of the vessel, the bleeding was completely ceased. The inhibitor antibody was purified by Sephacryl S 200 and Protein A cellulofine column chromatography. Purified IgG showed factor VIII inhibitor activity. Factor VIII epitopes recognized by the inhibitors was examined by western blotting. Factor VIII concentrate purified by the antigen. This factor VIII preparation was composed of a doublet of light chains (M.W. 80 kD) and 3 heavy chains (M.W. 160 200 kD) when examined by SDS-PAGE followed by immunoblotting using monoclonal antibodies against factor VIII light and heavy chains. The inhibitor in this case reacted to the heavy chains of factor VIII, whereas antifactor VIII antibody in the other case reacted to the light chain of factor VIII. PMID- 1719258 TI - [IBL-type lymphadenopathy after infection of rubella virus]. AB - A 52-year-old woman presented slight fever, diffuse papular skin rash and painful cervical lymph node swelling. Her lymph node swelling generally up to 3 cm in diameter, with petechiae on the lower legs and hepato-splenomegaly within a few weeks. ESR was 45 mm/h, Hb 10.0 g/dl, RBC 345 x 10(4)/microliter, WBC 22,600/microliter (atypical lymphocyte 47%), PLT 1.0 x 10(4)/microliter, GPT 91 U/L, gamma-globulin 34.3%, EBV-VCA x 2,560, EBNA x 20, and anti-rubella antibody x 512. The biopsied cervical lymph node showed histologic features of effacement of nodal architecture by an exuberant vascular proliferation accompanied with infiltration of the immunoblasts, and was diagnosed as immunoblastic lymphadenopathy (IBL)-type lymphadenopathy. The pulse therapy of methylprednisolone and high dose of gamma-globulin improved lymphadenopathy, thrombocytopenia and anemia. IBL-type lymphadenopathy after infection of rubella virus may be different from true IBL, but is important to discuss the pathogenesis of IBL. PMID- 1719259 TI - Clinical effect of granulocyte colony-stimulating factor on neutrophils and leukemic cells in myelogenous leukemia: analysis. AB - Clinical experiences with recombinant granulocyte colony-stimulating factor (rhG CSF) in 13 acute (AML) and four chronic (CML) myelogenous leukemia patients are reported. Sixteen patients received rhG-CSF in support of treatment for life threatening infections and one CML patient in support of induction chemotherapy. After their first induction chemotherapy, six out of eight AML patients showed a rapid increase of neutrophils, recovered from infections and achieved complete remission (CR). One patient, in whom both neutrophils and blasts had increased during rhG-CSF administration, achieved CR through the next administration of chemotherapy (CR rate 87.5%). The last of the eight AML patients showed no increase of neutrophils, and died of interstitial pneumonitis. Two of five AML patients who received rhG-CSF after reinduction chemotherapy for relapsed or refractory leukemia achieved CR, a rate of 40%. In one of the two, the administration of rhG-CSF prior to induction chemotherapy seemed advantageous in achieving CR. During rhG-CSF administration, an increase of blastic cells in peripheral blood was observed in four out of all 13 AML patients. One of three CML patients, with a lymphoid crisis, showed an increase only of neutrophils, and recovered from infection. The other two showed increases of both neutrophils and blasts. One patient with CML in blastic crisis, undergoing induction chemotherapy with rhG-CSF administration, returned to the chronic phase. These clinical experiences suggest rhG-CSF to be effective in supporting infection therapy and in possibly enhancing the sensitivity of myelogenous leukemic blasts to antileukemic agents. PMID- 1719260 TI - Identity of brain-associated small cell lung cancer antigen and the CD56 (NKH 1/Leu-19) leukocyte differentiation antigen and the neural cell adhesion molecule. AB - Previous studies have demonstrated the monoclonal antibody, TFS-4, to recognize a cell surface antigen, 124,000 Daltons, of molecular weight expressed selectively on small cell lung cancer but not on non-small cell lung cancer, and to cross react with the human brain, cardiac muscle and some smooth muscle cells. The cross-reactivity of TFS-4 with peripheral blood lymphocytes was examined by two color immunofluorescence analysis, using monoclonal antibodies to leukocyte differentiation antigens. Flow cytometric analysis revealed an identical subset of cells to express both BASCA and CD56(NKH-1 antigen). The immunofluorescence profiles for both TFS-4 and NKH-1 were, furthermore, identical to those of the background controls, identicating identical quantities of the antigens to be present on each cell within the population. Since CD56 has been show to be identical to the neural cell adhesion molecule (N-CAM), we determined the amino terminal amino acid sequence of BASCA purified from the human brain. The amino terminal amino acid sequence of BASCA was identical to that of N-CAM. PMID- 1719261 TI - [Mirror-writing in the aged]. AB - Mirror-writing is script that runs in the direction opposite to normal, with the individual letters also reversed. Although mirror-writing is well recognized as occurring in the presence of central nervous system damage, and is especially seen in association with hemiplegia, its mechanism has not yet been elucidated. The purpose of the present study is to document a high incidence of mirror writing among patients aged 65 years or more, and to investigate the relationship of mirror-writing with brain damage and the degree of cognitive dysfunction. The subjects analyzed in this study were 112 patients (44 males and 68 females) and their average age was 73.8 years. Hasegawa's Dementia Scale (HDS) was used to evaluate their cognitive function. We could find no cases of mirror-writing with the right hand. A high incidence of mirror-writing was found in patients who could use their left hand. Mirror-writing was seen in 67% of patients with cerebral lesions on CT scan. However, we could not observe any relationship between the incidence of mirror-writing and damage to a given circumscribed area of the brain. More than 90% of demented patients, whose HDS scores were less than 20, showed mirror-writing with their left hand. The mean score on the HDS for those with a high incidence of mirror-writing was significantly lower than the score for those without mirror-writing. These results indicate that a high incidence of mirror-writing among aged patients is related to cerebral damage and cognitive dysfunction. PMID- 1719262 TI - Hemorrhagic cystitis caused by bleomycin treatment. PMID- 1719263 TI - A slow voltage-dependent Na(+)-current induced by 5-hydroxytryptamine and the G protein-coupled activation mechanism in the ganglion cells of Aplysia. AB - Application of 5-hydroxytryptamine (5HT) induces a slowly depolarizing response in the neurons of Aplysia abdominal ganglion. In voltage-clamped cells, 5HT induced a slow inward current that increased steeply with membrane depolarization from -85 mV showing a negative slope conductance, but never reversed into outward when hyperpolarized beyond the equilibrium potential for K+. The 5HT-induced response was markedly augmented in Ca(2+)-free media, but depressed in Na(+)-free media, and unaffected by a change in external potassium. Intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) significantly depressed the 5HT response in a dose-dependent way. Injection of cholera toxin (CTX) selectively blocked the 5HT-induced response, the effect being irreversible. Neither 3'-deoxyadenosine, an inhibitor of adenylate cyclase, nor H-8, an inhibitor of protein kinase A, depressed the 5HT response. 3-Isobutyl-1 methylxanthine (IBMX) did not augment the 5HT response appreciably. The 5HT responses were not depressed at all during a saturated response to Br-cyclic AMP injected intracellularly. It was concluded that the 5HT response is produced by opening of the voltage-dependent Na(+)-channels with activation of CTX-sensitive G-protein but not necessarily with an increase in intracellular cyclic AMP. PMID- 1719264 TI - [Histological effects of hormono-chemotherapy on prostatic cancer]. AB - Using new criteria for histological effects of anti-cancer treatment, the effects of hormono-chemotherapy on 10 patients with prostatic cancer not previously treated were compared with those on 10 patients who received conventional hormone therapy. Marked effects were observed in 4 (40%) patients received hormono chemotherapy but not observed in patients who received conventional hormone therapy (chi 2 test, p less than 0.05). All four cases who showed marked effects were in stage B at the beginning of treatment. Hormonal effects were more obvious in well differentiated cancer, and the effects of chemotherapy were observed in some cases with moderately and poorly differentiated cancer. Therefore, the addition of chemotherapy is recommended as the initial therapy on prostatic cancer to reduce the relapsing rate, especially for patients with poorly and moderately differentiated cancer. PMID- 1719265 TI - Dual mechanism of angiotensin II inhibits ANP-induced mesangial cGMP accumulation. AB - To evaluate an interaction between vasoconstrictive (Ang II) and vasodilating (ANP) peptides, we examined the effect of Ang II on ANP-induced accumulation of cGMP in cultured glomerular mesangial cells. ANP rapidly increased intracellular cGMP levels, with a peak stimulation at one minute in the absence of IBMX and at ten minutes in the presence of IBMX. The ANP-induced cGMP accumulation was significantly inhibited when the cells were treated with Ang II simultaneously with ANP for one minute in the absence of IBMX. This inhibitory effect of Ang II was completely abolished by IBMX and significantly reduced in calcium-free media or by W7, but not affected by H7. Similar inhibitory effect was observed when cells were treated with A23187 but not with TPA for one minute. In the presence of IBMX, Ang II inhibited ANP-induced cGMP accumulation when cells were treated with Ang II for 15 minutes prior to the stimulation by ANP. This inhibition by Ang II was blocked by H7. ANP-induced increase in particulate guanylate cyclase activity was significantly reduced in the cells treated with Ang II or TPA. This reduction of enzyme activity was also prevented by H7. These results indicate that Ang II inhibits ANP-induced cGMP accumulation in cultured glomerular mesangial cells through at least two mechanisms; one is the activation of calcium dependent, calmodulin-stimulated cyclic nucleotide phosphodiesterase in the initial phase, and the other is the inhibition of guanylate cyclase resulting from protein kinase C activation in the maintenance phase. PMID- 1719266 TI - Effects of hemodialysis on platelet-derived thrombospondin. AB - The effects on platelet-derived thrombospondin (TSP) of hemodialysis with a cellulose membrane were studied in patients during routine hemodialysis and in normal subjects using an ex vivo model. Plasma and platelet-bound TSP were determined pre- and post-dialysis, in blood entering and leaving the dialyzer after 1, 3, 5, 15, and 30 minutes of dialysis, and in blood leaving the ex vivo module after 5, 10, 15, 20, and 25 minutes of perfusion. Plasma concentrations of beta-thromboglobulin (beta TG) and thromboxane B2 (TxB2), and platelet membrane expression of the alpha-granule protein GMP-140, were also measured. Significant increases in plasma concentrations of TSP and beta TG occurred between the inlet and outlet of the dialyzer after 5, 15, and 30 minutes of dialysis, accompanied by a slow, but significant, increase in their arterial plasma concentrations. In contrast, initiation of dialysis was associated with an immediate increase in plasma TxB2 concentration between the inlet and outlet of the dialyzer and an abrupt increase in arterial plasma TxB2 concentration which plateaued at 250% of the pre-dialysis value after five minutes. Transit of platelets through the dialyzer had no effect on platelet-membrane-associated TSP or GMP-140. Plasma TSP and beta TG concentrations at the outlet of the ex vivo module also increased significantly during perfusion, but plasma TSP concentrations were twofold greater than those during hemodialysis. In vitro stimulation of platelets with thrombin and immunoblotting studies of platelet release proteins showed reduced TSP release by platelets of hemodialysis patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719267 TI - [Treatment of non-testicular germ cell tumors in children and adolescents with BEP and VIP: initial results of the MAKEI 89 therapy study]. AB - The pilot protocol of the German Society of Pediatric Oncology for treatment of non testicular germ cell tumors was initiated in November 1987. The final protocol was started at 1. 1. 89. Different therapy was administered depending on histology, primary localisation or stage of tumors. Patients with malignant germ cell tumors such as dysgerminomas, embryonal carcinomas, yolk sac tumors or chorio carcinomas received BEP (Bleomycin 15 mg/m2/days 1-3, Etoposide 100 mg/m2/days 4-8, Cisplatinum 20 mg/m2/days 4-8), followed by VIP (Vinblastine 3 mg/m2/days 1 + 2, Ifosfamide 1500 mg/m2/days 1-5 including Mesna uroprotection and Cisplatinum 20 mg/m2/days 1-5). Patients with ovary tumors of stage 1 were treated with 3 courses of BEP, patients with ovary tumors stage II and extragonadal localisation received 3 courses of VIP in addition to 3 courses of BEP. In cases of extended tumors 4 courses of BEP were followed by delayed resection of tumors and 4 courses of VIP. Patients with intracranial germinomas were treated with 30 Gy of craniospinal radiation therapy and additional 15 Gy as a tumor boost. Since some cases of spinal extension were reported a spinal radiation therapy seems to be indispensable. Patients with intracranial embryonal carcinomas, yolk sac tumors or chorio carcinomas tumors were given 2 courses of BEP and VIP followed by 30 Gy of craniospinal radiation therapy and additional 20 Gy as a tumor boost. Patients with immature teratomas of the ovary grade 1-3 and grade 3 of tumors with extragonadal localisation were treated with 3 courses of BEP after resection of tumors. Until 1. 1. 1991 92 patients were reported to the study--27 with intracranial and 65 with extracranial primary localisation of tumors. 43 patients suffered from teratomas (including 20 immature teratomas grade 1-3), 18 from germinomas (seminomas/dysgerminomas) and 31 from malignant non-seminomatous germ cell tumors. After an observation period of 29 months disease-free survival rate was 80% (79/92 patients, Kaplan-Meier Statistics). Outcome of intracranial tumors was death or relapse in 2/9 patients with malignant non-seminomatous germ cell tumors, in 2/14 patients with intracranial germinomas, in 2/4 patients with teratomas. Patients with extracranial localisation of tumors suffered from death or relapse in 1/21 cases with non seminomatous tumors, in 0/4 cases with dysgerminomas and 5/39 cases with teratomas. During pilot study one infant with a malignant non-seminomatous germ cell tumor died of a pneumopathia. Therefore infants treated according to the final protocol did not receive Bleomycin.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1719268 TI - [The prognostic significance of serum alpha 1-fetoprotein in children and adolescents with malignant extracranial non-testicular germ cell tumors]. AB - The cooperative therapy study MAKEI 83/86 included an examination of the prognostic value of the AFP in children and adolescents with extracranial non testicular yolk sac tumors. The serum AFP values of 72 protocol- and follow-up patients were documented at diagnosis and up to the ninth month of treatment. 32 of these patients had saccrococcygeal tumors, 27 had tumors of the ovary and 13 suffered from extragonadal germ cell-tumors. 4 children showed progressive disease under initial chemotherapy and 1 patient died of therapy, 10 of 72 patients relapsed. The AFP measurements were plotted on semilogarithmic charts. They were compared to the measurements of healthy children up to the age of 1 year. According to the development of the patients' AFP values compared to the reference curves the following classifications could be made: 1. Patients with a normal AFP-decrease id est 50% in less than or equal to 6 days during the 1st month of therapy: 48/72 patients 2. Patients with slow AFP-decrease: 17/72 patients 3. Patients with transient AFP-decrease: 5/72 patients 4. Patients with no AFP-decrease: 2/72 patients According to Kaplan-Meier life table analysis, patients with a normal AFP-decrease had an event-free survival of 89% +/- 4%, whereas all other patients showed an event-free survival of 63% +/- 10% (p less than 0.05). Regarding primary therapy id est tumor resection or preoperative chemotherapy an equal distribution of the patients among those with a normal and slow AFP-decrease was observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719269 TI - Anti-angiogenesis as a new concept for the therapy of neovascular diseases. PMID- 1719270 TI - Short-term effects of oral enoximone on hemodynamics, exercise capacity, anaerobic threshold, and arrhythmias in congestive heart failure. AB - Enoximone, a phosphodiesterase-inhibitor, is a potent inotropic vasodilator agent that causes a marked improvement in hemodynamics in patients with congestive heart failure. The acute effects of oral enoximone on rest and exercise hemodynamics, ejection fraction, aerobic metabolism, exercise capacity, and arrhythmias were studied in 11 patients with moderate to moderately severe dilative cardiomyopathy after 8 days of enoximone (100 mg tid) in addition to baseline therapy (diuretics and digitalis). The cardiac index increased from 2.44 +/- 0.45 to 2.72 +/- 0.50 l/min/m2 (p less than 0.01) at rest and from 4.00 +/- 0.96 to 4.75 +/- 0.95 l/min/m2 (p less than 0.005) during exercise. Pulmonary wedge pressure decreased from 16.8 +/- 7.3 to 12.5 +/- 6.5 mmHg (p less than 0.005) at rest and from 28.2 +/- 8.0 to 24.5 +/- 10.3 mmHg (p less than 0.05) during exercise. Systemic vascular resistance decreased from 1608 +/- 243 to 1495 +/- 300 dynes*sec*cm-5 (p less than 0.05) at rest and from 1152 +/- 155 to 1027 +/- 236 dynes*sec*cm-5 (ns) during exercise. The anaerobic threshold, which was recorded simultaneously, increased from 13.2 +/- 2.7 to 15.5 +/- 2.5 ml/kg/min VO2 (p less than 0.02). The radionuclide ventriculography ejection fraction improved from 21.7 +/- 5.0 to 28.1 +/- 9.1% (p less than 0.01) during exercise; the changes at rest were not significant (20.8 +/- 6.2 vs 25.8 +/- 8.4%). Exercise tolerance showed an increase of 16% (492 +/- 133 to 573 +/- 135 sec, p less than 0.005). The resting heart rate remained unchanged (81.8 +/- 13.4 vs 81.8 +/- 11.9). Interestingly, 24-h Holter monitoring revealed more or new repetitive arrhythmias in 9/11 patients. PMID- 1719271 TI - [Intravenous administration of immunoglobulin preparations in clinical practice ( review of foreign literature)]. PMID- 1719272 TI - The efficacy and safety of chlorpyrifos (Dursban) for control of Myobia musculi infestation in mice. AB - Mite infestation in laboratory mice is a common, but troublesome problem in animal facilities. Recommended treatment regimens are frequently ineffective because of the short period of exposure to the control agent. In an effort to develop a time-release approach, we have investigated the use of Dursban granules applied in animal bedding. Initial toxicity studies indicated that this pesticide can be added to shoebox cage litter at levels three times that used for outdoor application (6 g per 27 by 48 cm shoebox cage) without producing clinical signs of toxicity. Metabolism studies demonstrated that although individual mice showed decreased brain acetylcholinesterase activity following treatment, liver cytosolic glutathione-S-transferase, liver microsomal aminopyrine N-demethylase, or aryl hydrocarbon hydroxylase were not induced after 1 week of exposure. Parasitological studies indicated elimination of mites and itching in an experimental infestation, as well as reduction of itching in severely symptomatic, naturally infested mice, following treatment with the granules. These studies demonstrate the nontoxic efficacy of Dursban in the control of Myobia musculi. PMID- 1719273 TI - An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice. AB - Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice. PMID- 1719274 TI - Column extraction of chlorpyrifos from contaminated fish. AB - A method for measuring chlorpyrifos in fish, which combines extraction, filtration, and cleanup in one step, is described. Minced fish samples were mixed with potassium dihydrogen phosphate and disodium hydrogen phosphate, ground with anhydrous sodium sulfate, and eluted from a prepacked chromatographic column containing silica gel. The endogenous coextractives were retained by the column while chlorpyrifos was quantitatively eluted with 40 mL of 5% ether in hexane. Recoveries averaged 86.8% for unexposed fish fortified with 2-12 ppm of chlorpyrifos. The method was applied to the analysis of fish from a lagoon contaminated with chlorpyrifos by a spray treatment of a wooden bridge for termites. PMID- 1719275 TI - Iron chelation with a deferoxamine conjugate in hemorrhagic shock. AB - Oxygen-derived radicals are cytotoxic, highly reactive molecules that contribute to cellular death and injury in hemorrhagic shock. Iron released into the plasma in hemorrhagic shock may contribute to cellular damage by catalyzing lipid peroxidation of cell membranes. Deferoxamine (DFO) chelation of transitional metal ions prevents formation of these radicals and may diminish reperfusion injury. The conjugation of DFO to pentastarch (PS) decreases DFO toxicity and extends its half-life making it a potentially useful resuscitative fluid. A porcine hemorrhagic shock model was used to evaluate the effects of five resuscitative fluids on survival and hepatic function. Swine (11-16 kg) underwent splenectomy, liver biopsy, and placement of arterial and venous catheters. Awake animals were bled at 1 ml/kg/min to a MAP of 45 mm Hg, maintained for 1 hr, and resuscitated over 30 min with one of five fluids: Lactated Ringer's (LR); LR + free DFO 2.5 mg/ml (LR + DFO) (n = 6); 5% PS in LR (PS) (n = 6); 5% PS + free DFO (PS + DFO) 7.5 mg/ml (n = 6); 5% PS/DFO conjugate (7.5 mg/ml) in LR (n = 6). LR and LR + DFO received 3 ml/ml shed blood; PS, PS + DFO, and PS/DFO received 1 ml/ml shed blood. No shed blood was returned to the animals. There was no significant differences between groups in MAP, HR, CVP, and T pre- and post resuscitation. No LR lived to sacrifice at 24 hr. Thirty-three percent of LR + DFO and PS + DFO animals died within minutes of receiving the free DFO containing resuscitative fluid, presumably from acute DFO toxicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719276 TI - Nebivolol increases survival in cardiomyopathic hamsters with congestive heart failure. AB - The genetically inbred cardiomyopathic Syrian hamster provides a valuable model of congestive cardiomyopathy: myocytolytic necrosis at 30-50 days of age is followed by cardiac hypertrophy at 150-250 days, and finally by congestive failure and death at 250-350 days. Successful drug treatment has been reported in the prenecrotic stage, but not when started during congestive failure. The present study evaluated survival of the cardiomyopathic hamster treated with the new beta-adrenoceptor blocker nebivolol, which was initiated during congestive failure. Fifty animals (BIO82.62, either sex, age 200 days) were acclimated to an environmentally controlled room for 20 days, receiving food and water ad libitum; seven hamsters died, indicating development of congestive failure. The remaining animals were then randomly assigned to one of three groups: 15 animals received control food; food for the others was supplemented with nebivolol, yielding a daily intake of either 0.1 mg/kg (n = 14) or 1 mg/kg (n = 14). At the lower nebivolol dose, death rate was unaltered in comparison with controls. However, at 1 mg/kg, survival was markedly improved (p = 0.042). Nebivolol treatment, started during congestive failure, thus significantly delays mortality in the cardiomyopathic hamster. PMID- 1719277 TI - Postsynaptic alpha 1-blockade with terazosin does not modify insulin sensitivity in nonobese normotensive subjects. AB - Plasma insulin levels and the sensitivity of peripheral tissue to insulin (SI) have pathophysiological, therapeutic, and possibly also prognostic relevance. To investigate the effects of short-term selective alpha 1-adrenergic receptor blockade in nonobese normotensive humans on glucose and lipoprotein homeostasis, we assessed SI (determined by the minimal model method of Bergman), before and after glucose load plasma, glucose, and insulin levels, serum total triglycerides and lipoprotein cholesterol fractions, and some other variables in 20 healthy young men (26 +/- 1 years old, mean +/- SEM) during placebo and after 5 weeks of terazosin administration at a dose up to 10 mg once daily. Measurements were made after 3 days of standard diet (2,500 kcal/day, 45% carbohydrates, 40% fat, and 15% proteins) and after an overnight fast. Compared to placebo, terazosin decreased the upright systolic blood pressure (placebo vs. terazosin: 125 +/- 2 vs. 117 +/- 2 mm Hg, p less than 0.05) and increased supine (63 +/- 2 vs. 70 +/- 1 beats/min, p less than 0.05) and upright (77 +/- 2 vs. 88 +/- 2 beats/min, p less than 0.01) heart rates, while the body weight was unaltered. Terazosin did not significantly modify fasting plasma glucose (5.08 +/- 0.09 vs. 5.23 +/- 0.08 mmol/L, respectively), or insulin (8.9 +/- 0.5 vs. 8.6 +/- 0.6 microU/ml), SI (14.3 +/- 1.8 vs 11.8 +/- 1.5 x 10(-4)/min/microU/ml), the areas under the insulin or glucose curves, serum total triglycerides, and cholesterol or lipoprotein cholesterol fractions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719278 TI - Effects of amiodarone with and without polysorbate 80 on myocardial oxygen consumption and coronary blood flow during treadmill exercise in the dog. AB - Since amiodarone has been reported to possess antianginal activity, this study examined the effects of amiodarone on coronary blood flow and myocardial oxygen consumption during exercise. Studies were performed in 14 chronically instrumented dogs trained to run on a motor-driven treadmill. Left circumflex coronary artery blood flow was measured with an electromagnetic flowmeter while aortic and coronary sinus catheters allowed measurement of myocardial oxygen extraction. During control conditions, graded exercise resulted in progressive increases in heart rate, aortic pressure, and coronary blood flow. Two preparations of amiodarone, 5 mg/kg, one dissolved in sterile water and the other in 10% polysorbate 80, were given intravenously to separate groups of dogs. Amiodarone in sterile water caused no hemodynamic changes at rest. However, the increase in heart rate during exercise was blunted after amiodarone, so that heart rate during the heaviest level of exercise was significantly less than during control exercise. Coronary blood flow and myocardial oxygen consumption were unchanged. Amiodarone with polysorbate 80 also blunted the increase in heart rate during exercise, but in addition caused a significant decrease in aortic pressure both at rest and during exercise. Myocardial oxygen consumption and coronary blood flow were significantly decreased after administration of amiodarone with polysorbate 80 at rest and during all exercise levels. Amiodarone with or without polysorbate 80 did not change myocardial oxygen extraction. These data demonstrate that amiodarone exerts a negative chronotropic effect during exercise. However, the decreased arterial pressure and myocardial oxygen consumption were not due to amiodarone, but was seen only with the combination of amiodarone dissolved in polysorbate 80. PMID- 1719279 TI - Coronary thrombolytic properties of a novel recombinant plasminogen activator (BM 06.022) in a canine model. AB - We studied the thrombolytic dose-response relationship of a recombinant plasminogen activator (rPA) (BM 06.022) compared with alteplase in a canine model of coronary artery thrombosis. BM 06.022 consists of the kringle 2 and protease domains of human tissue PA (tPA) and lacks oligosaccharide side chains because of its expression in Escherichia coli. Thrombus formation in anesthetized, open chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery in the presence of a critical stenosis. Intravenous bolus injection of BM 06.022 (50, 100, 140, and 200 kU/kg) or of alteplase (200, 800, 1,130, and 1,600 kU/kg) 30 min after coronary occlusion to six heparinized dogs per group achieved a dose-dependent increase in reperfusion rate and decrease in residual thrombus wet weight. Vehicle-treated dogs did not reperfuse. Semilogarithmic regression analysis showed that the effective dose that produced 50% reperfusion of BM 06.022 (83 kU/kg) was 11.6-fold lower than that of alteplase (951 kU/kg). Comparison with infusion experiments showed that intravenous bolus injection of 140 kU/kg of BM 06.022 was equieffective to a 90 min infusion of 800 kU/kg (= 1 mg/kg) of alteplase as a standard treatment regarding reperfusion rate (66%) and time to reperfusion (15 +/- 6 vs. 18 +/- 8 min). Pharmacokinetic analysis for functionally active BM 06.022 or alteplase in plasma revealed a total plasma clearance of 4.1-6.6 ml/min/kg for BM 06.022 and of 12.6-42.3 ml/min/kg for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719280 TI - Different potency of endothelium-derived relaxing factors against thromboxane, endothelin, and potassium chloride in intramyocardial porcine coronary arteries. AB - Endothelium-derived relaxing factors (EDRFs) and prostacyclin (PGI2) released from endothelial cells are potent vasodilators; endothelin-1 and thromboxane A2 may be their physiological antagonists. Interactions between these vasodilators and vasoconstrictors were studied in isolated intramyocardial porcine coronary arteries suspended in myographs for isometric tension recording. Endothelium dependent relaxations to bradykinin and serotonin were reduced to a similar extent in arteries contracted with endothelin-1 and KCl as compared to those contracted with the thromboxane analog U 46619 (p less than 0.05; n = 5-6). In contrast, relaxations to the nitric oxide donor SIN-1 were comparable. PGI2 was most potent in arteries exposed to U 46619, while its effects were inhibited in the presence of endothelin-1 or acetylcholine and prevented by KCl (p less than 0.05-0.0001; n = 5). At high concentrations PGI2 evoked contractions in arteries contracted with endothelin-1, acetylcholine, or KCl, but not in those with U 46619, which were prevented by the thromboxane receptor antagonist SQ 30741 (p less than 0.05; n = 5), indicating that PGI2 is a partial agonist for the thromboxane receptor. Thus, in intramyocardial porcine coronary arteries, contractile agonists differently interact with the release and action of EDRFs and PGI2. Both are most effective against contractions induced by thromboxane. In contrast, endothelin-1 and particularly KCl reduce the potency of these endogenous vasodilator systems. PMID- 1719281 TI - Milrinone inhibits sympathetic-mediated tachycardia by a postjunctional action independent of cyclic AMP. AB - In pithed rats with stimulated sympathetic outflow, the phosphodiesterase inhibitor milrinone (0.3 mg/kg, i.v.) decreased the peak tachycardiac response produced by both sympathetic nerve stimulation (15 s at 0.5-3 Hz) and norepinephrine administration (0.3-5 micrograms/kg, i.v.). However, another phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 0.5 mg/kg, i.v.) had no effect on the peak tachycardic response to sympathetic stimulation. Similarly, in isolated rat atria, milrinone (9 mumol/L) inhibited the tachycardia produced by norepinephrine, whereas IBMX (1 mumol/L) had no effect. The inhibitory effect of milrinone on sympathetic responses was not due to changes in norepinephrine release since milrinone (9 mumol/L) increased norepinephrine release in isolated rat atria incubated with [3H]norepinephrine. When the duration of the tachycardia (rather than the peak tachycardic response) produced by sympathetic nerve stimulation was measured, it was found to be prolonged by both milrinone and IBMX, suggesting that in this case cyclic AMP was involved. Furthermore, in contrast to its inhibitory effects on norepinephrine-induced tachycardia in rat atria, milrinone enhanced the tachycardia produced by the adenylate cyclase activator forskolin. These results suggest that milrinone has complex actions on sympathetic control of heart rate and that beta-adrenoceptor tachycardia occurs by mechanisms dependent on and independent of cyclic AMP. PMID- 1719283 TI - Responses of coronary arteries to neurotransmitters: changes with sexual maturity in the female rabbit. AB - Vasomotor responses of isolated coronary arteries to peptide and nonpeptide neurotransmitter agents changed with the development of sexual maturity in female New Zealand white rabbits aged 4-12 months. There was a significant reduction from 4- to 12-month-old animals in both the direct smooth muscle vasodilator responses to calcitonin gene-related peptide (p less than 0.01) and vasoactive intestinal polypeptide (p less than 0.001), and in the endothelium-mediated response to substance P (p less than 0.005). Vasodilator responses to concentrations of acetylcholine (ACh) greater than 0.1 microM were virtually absent in the 6- and 12-month-old animals. No change in maximal relaxation to noradrenaline was seen with maturation, although there was a small significant increase in potency. The contractile response of the smooth muscle to 30 mM KCl declined steadily as the animals matured, but the maximal contraction to ACh (p less than 0.05), neuropeptide Y (p less than 0.02), and serotonin (p less than 0.05) increased significantly between 4 and 12 months of age. These results indicate that following sexual maturation in the female rabbit, the epicardial coronary artery shows a significant increase in maximum responses to vasoconstrictor neurotransmitter agents and, at the same time, a marked decline in responses to some vasodilator agents, both those acting directly on the smooth muscle and those acting via the endothelium. PMID- 1719282 TI - Effects of pentisomide and E-4031 on canine atrial flutter due to reentry: a comparative study with disopyramide and propafenone. AB - Effects of new antiarrhythmic drugs, pentisomide [3.5 +/- 0.5 mg/kg intravenously (i.v.) n = 8], and E-4031 (5.6 +/- 1.0 micrograms/kg, n = 8), a class III drug, on atrial flutter (AF) caused by reentry were compared with those of disopyramide (1.6 +/- 0.2 mg/kg, n = 8) and propafenone (2.2 +/- 0.2 mg/kg, n = 8). AF was induced with burst atrial pacing after we made an intercaval crush in anesthetized, open-chest dogs. Termination of AF did not differ among test drugs (8 of 8 with disopyramide, 7 of 8 with propafenone, 6 of 8 with pentisomide, and 8 of 8 with E-4031). Cycle length (CL) of AF was prolonged more with propafenone (57 +/- 10%) and pentisomide (41 +/- 5%) than with E-4031 (12 +/- 3%, p less than 0.05). This was also true for increase in interatrial conduction time determined at a pacing CL of 150 ms. Increase in atrial effective refractory period (ERP) determined at a basic pacing CL of 300 ms did not differ among test drugs. Changes in CL of AF correlated significantly with those in interatrial conduction time (r = 0.84, p less than 0.001), but not with those of ERP (r = 0.10, NS). Reinitiation of AF was significantly greater in propafenone (7 of 7) and pentisomide (5 of 6) groups than in disopyramide (1 of 8) and E-4031 (0 of 8) groups (p less than 0.001). Pentisomide and E-4031 were effective in terminating canine AF due to reentry, as were disopyramide and propafenone. Reinitiation of AF was greater in dogs treated with antiarrhythmic drugs that had more prominent effects on conduction time than on ERP. PMID- 1719284 TI - Desensitization of alpha-adrenergic receptor-mediated smooth muscle contraction: role of the endothelium. AB - Desensitization of alpha-adrenergic receptor-mediated smooth muscle contraction occur in aortas from New England Deaconess Hospital (NEDH) rats harboring pheochromocytoma (PHEO) and following chronic exposure to the alpha-adrenergic agonist phenylephrine in vitro. Endothelium is known to release an endothelial cell-derived relaxing factor that promotes smooth muscle relaxation. We wondered if the endothelium might contribute to the desensitization of contraction. The role of the endothelium in desensitization was studied using aortic rings with endothelium [E(+)] and with endothelium removed [E(-)]. Maximal phenylephrine (PE)-induced contraction (Emax) for E(+) was 1.7 +/- 0.3 g in controls and 0.4 +/ 0.1 g in PHEO (p less than 0.001), demonstrating desensitization; however, for E(-), Emax was 2.4 +/- 0.2 g in PHEO vs. 2.5 +/- 0.2 g in controls, demonstrating restoration of maximal contraction when the endothelium was removed. However, sensitivity [-log EC50(M)] to PE in E(-) remained significantly lower in PHEO compared to controls (6.94 +/- 0.12 vs. 7.51 +/- 0.14, respectively, p less than 0.001). Similarly, in aortic ring segments desensitized in vitro with phenylephrine, the maximal contraction in phenylephrine-exposed aortas was 60% of that seen in controls. Removal of the endothelium from the vessels pretreated with phenylephrine fully restored the maximal response and sensitivity of these vessels. Treatment of desensitized vessels with hemoglobin (5 x 10(-5) M) restored the maximal contraction and sensitivity to phenylephrine. When the endothelium was removed prior to chronic exposure to phenylephrine, the sensitivity to phenylephrine decreased while the Emax remained similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719285 TI - Evidence for medial-mass regression in the vascular wall of Dahl hypertensive rats by cicletanine treatment. AB - We designed experiments to investigate the effects of cicletanine, a novel antihypertensive drug, on medial hypertrophy in Dahl rats susceptible to salt induced hypertension (Dahl S rats). Cicletanine treatment (500 mg of cicletanine/kg of chow) for 6 weeks lowered blood pressure by 19% in Dahl S rats challenged with a high-salt (4%) diet. The blood pressure reduction was associated with a significant decrease in weight of the aortic vessels. Morphological examination revealed that this treatment decreased medial hypertrophy and expansion of intimal tissue, in concert with resolution of the periarteritis in the intrarenal arteries. In fact, the content of actin in the aortic wall, analyzed by SDS-PAGE, was decreased significantly with this treatment and myosin content was reduced to the same extent as well. Moreover, cicletanine per se lowered protein synthesis in randomly cycling cultured vascular smooth muscle cells (VSMCs) from Sprague-Dawley rats. Actin formation by VSMCs was decreased with cicletanine. Thus, these data indicate that chronic cicletanine treatment produces regression of the medial hypertrophy in Dahl S rats. Direct inhibitory effects on cytoskeleton protein synthesis, as well as its antihypertensive action, are partly responsible for this regression in vivo. PMID- 1719286 TI - Cellular electrophysiological effects of the class III antiarrhythmic agents sematilide and clofilium on rabbit atrial tissues. AB - Sematilide (N-[2-(diethylamino)ethyl]-4- [(methylsulfonyl)amino]benzamide HCl) is a new class III antiarrhythmic agent that has been shown to be effective in preventing reentrant ventricular arrhythmias in experimental animals and humans. In this study, we examined the in vitro effects of sematilide (1-100 microM) on isolated sinoatrial (SA) node, atrioventricular (AV) node, and atrial muscle. These results were then compared to another class III agent, clofilium (1-30 microM). In SA nodal tissue, sematilide increased the action potential duration (APD) and spontaneous cycle length (SCL) in a concentration-dependent manner (EC20% = 15 +/- 3 and 54 +/- 13 microM, respectively). In addition, there was a slight reduction in maximum diastolic potential at 100 microM. Clofilium had similar class III effects, but was approximately 3 to 18 times more potent (EC20% = 6 +/- 2 and 3 +/- 1 microM for the APD and SCL, respectively). Neither agent had a significant effect on the slope of phase 4 nor on other action potential parameters. Results in AV nodal preparations were similar. Both sematilide and clofilium increased the APD and SCL in a concentration-dependent manner, with clofilium being approximately four to six times more potent than sematilide (EC20% for the APD and SCL for sematilide = 12 +/- 4 and 12 +/- 8 microM, respectively, and for clofilium = 2 +/- 1 and 3 +/- 2 microM, respectively). No significant effects were observed on other action potential parameters. Sematilide and clofilium increased the APD and effective refractory period (ERP) in atrial trabeculae in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719287 TI - The novel cardiotonic agent EMD 53 998 is a potent "calcium sensitizer". AB - EMD 53 998, a novel thiadiazinone derivative, increases the contractile force of cardiac tissue in vitro through both an inhibition of phosphodiesterase III (PDE III) and a sensitization of cardiac contractile proteins to Ca2+. Guinea pig ventricular PDE III is selectively inhibited by EMD 53 998 (IC50 = 60 nM) without major effects on other PDE isoenzymes. Consonant with this is an increase in cAMP content of rat ventricular cells and a potentiation by EMD 53 998 of the cAMP elevating action of isoprenaline (increase by 50% at 1.3 microM). Sensitization to Ca2+ by EMD 53 998 (3-30 microM) finds its expression in a leftward shift of the Ca2+ response curve for force generation in skinned fibers from porcine ventricular muscle and failing human heart as well as for activation of bovine cardiac myofibrillar actomyosin ATPase. Interestingly, EMD 53 998 elevates the maximum of the Ca(2+)-response curve for both parameters. Pimobendan studied under identical conditions was 100 times less potent than EMD 53 998. EMD 53 998 increases force development of guinea pig papillary muscle in a concentration dependent manner with an EC50 of 3.6 microM, thus being 10 times more potent than pimobendan. In contrast to pimobendan, the positive inotropic effect of EMD 53 998 is barely affected by carbachol. Further evidence for a Ca(2+)-sensitizing effect of EMD 53 998 is provided by an additional increase in force generation in the presence of supramaximal isoprenaline concentrations. It is concluded that the positive inotropic action of EMD 53 998 is mediated through both cAMP independent and cAMP-dependent mechanisms, with the former probably prevailing. We are not aware of other compounds with a similarly high Ca(2+)-sensitizing potency. On these grounds. EMD 53 998 appears to be a promising inotropic agent. PMID- 1719288 TI - Oral pharmacokinetics of bisoprolol in resting and exercising healthy volunteers. AB - The effects of exercise on bisoprolol oral pharmacokinetics were studied in eight healthy male volunteers in an open, randomized, three-period, crossover trial. Oral bisoprolol (20 mg) was given either at rest during 24 h or with iterative stress tests before and 2.5, 5, 10, and 24 h after dosing. Exercise tests were repeated on a third placebo period. Bisoprolol was assayed in plasma and urines, and plasma catecholamines were measured before and after stress tests, Cmax, Tmax, elimination t1/2, and renal clearance of bisoprolol were not significantly modified by exercise. AUC0-infinity significantly decreased by 7.5% (p less than 0.05) with stress test resulting in an increase in apparent oral clearance. The beta-blocking effect peaked at 2.5 h, lasted greater than 24 h, and was related to plasma levels. The exercise-induced increase in plasma norepinephrine levels was significantly augmented with bisoprolol. These results suggest that repeated exercise tests exerted only limited effects on the oral pharmacokinetics of bisoprolol. PMID- 1719289 TI - The effect of pentoxifylline (Trental) and two analogues, BL 194 and HWA 448, on the release of plasminogen activators and von Willebrand factor in rats. AB - The effects on fibrinolytic components of pentoxifylline (Trental), of its first metabolite BL 194 (penthydroxyfylline), and of its analogue HWA 448 (torbafylline) were studied in rats. BL 194, though not pentoxifylline and HWA 448, significantly enhanced the induced release by platelet-activating factor (PAF) of tissue-type plasminogen activator (tPA) from isolated perfused rat hindlegs. In contrast, the simultaneously induced release of von Willebrane factor (vWF) was reduced by BL 194. The effect of BL 194 on PAF-induced release of tPA and vWF could be mimicked by isobutyl-methylxanthine (IBMX), an inhibitor of phosphodiesterases. In vivo, BL 194 and pentoxifylline did not affect baseline levels of plasma tPA and PA inhibitor activity, nor did these compounds affect the in vivo induction of tPA release by PAF. Similarly, the induction by endotoxin of PA inhibitor activity was not influenced by pentoxifylline or BL 194. By its opposite effects on tPA and vWF release. BL 194 might favrorably influence the thrombotic balance. PMID- 1719290 TI - Heterogeneity of postjunctional alpha-adrenoceptors in isolated mesenteric resistance arteries from rats, rabbits, pigs, and humans. AB - Mesenteric resistance arteries (internal diameter 174-337 microns) from rats, rabbits, pigs, and humans were isolated and mounted in a myograph. In all preparations, phenylephrine (alpha 1-adrenoceptor agonist) evoked concentration dependent contractions that were antagonized by prazosin (alpha 1-adrenoceptor antagonist). B-HT 933 (alpha 2-adrenoceptor agonist), however, did not evoke contractions in any rat or rabbit vessels. In contrast, this agonist elicited contractions in some (five of 15) porcine and all (18) human vessels; in the vessels responsive to B-HT 933, the maximum responses amounted to 53 +/- 7 and 79 +/- 6%, respectively, of the responses to high potassium. The affinities of yohimbine (alpha 2-adrenoceptor antagonist) for the receptors mediating responses to B-HT 933 were 7.46 +/- 0.13 (pKB) and 7.57 +/- 0.10 (pA2), respectively. Prazosin (10(-8) M) antagonized the responses to norepinephrine in all preparations, whereas yohimbine (10(-7) M) caused significant inhibition of these responses in human vessels only; moreover, the inhibition in these vessels of the responses to norepinephrine caused by the combination of these two antagonists was greater than the antagonism caused by either antagonist alone. These results suggest the presence of postjunctional alpha 1-adrenoceptors that are involved in responses to norepinephrine in mesenteric resistance arteries from all species examined. Functional, postjunctional alpha 2-adrenoceptors appear to be present in porcine and human vessels, but this receptor seems to be involved in responses to norepinephrine in human vessels only. PMID- 1719291 TI - Endothelin-1 has a dilator effect on neonatal pig pulmonary vasculature. AB - Endothelin-1 (ET-1), a 21-residue potent vasoconstrictor peptide produced by endothelial cells, was reported to cause vasodilation in the systemic and pulmonary vascular beds. Therefore, in isolated perfused lungs from 7-day-old piglets, we studied the effects and the mechanisms responsible for the dilator effect of ET-1. ET-1 produced a mild transient decrease in perfusion pressure at low doses (less than 10(-7) M/g dry lung); at higher doses, a potent long-lasting vasoconstriction was noted. Indeed, the constrictor effect of ET-1 was at least equal to or greater than that of U-44069 and prostaglandin D2 (PGD2). When the vascular tone of the preparation was increased with U-46619, another stable endoperoxide analogue, the dilator response to low doses of ET-1 was increased, while the constrictor response remained unchanged. Indomethacin (2.8 x 10(-6) M) and glybenclamide (an ATP-sensitive potassium channel inhibitor) (10(-5) M) did not alter the responses to ET-1. The endothelium-derived relaxing factor (EDRF) inhibitor Nw-nitro-L-arginine (2 x 10(-4) M) not only inhibited the dilator response to ET-1 almost completely, but also potentiated the constrictor response. Finally, Nw-nitro-L-arginine alone had a mild vasoconstrictor effect in newborn pig lung. The results of these studies indicate that ET-1 has both vasodilator and vasoconstrictor activity in neonatal pig pulmonary vascular bed. This vasodilator activity may be mediated by EDRF.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719292 TI - Effects of hyperkalemia, acidosis, and hypoxia on the depression of maximum rate of depolarization by class I antiarrhythmic drugs in guinea pig myocardium: differential actions of class Ib and Ic agents. AB - Standard microelectrode methods were used to record intracellular action potentials from strips of guinea pig right ventricular myocardium superfused with either standard physiological saline (pH 7.3; PO2 greater than 650 mm Hg; [K+] = 5.6 mM) or the same solution modified to produce either hyperkalemia ([ K+] = 11.2 mM), acidosis (pH = 6.3), or hypoxia (PO2 = 60 mm Hg). The effects on action potential parameters of three therapeutic concentration of lidocaine, flecainide, and encainide were studied under all four conditions at four different drive rates (interstimulus interval = 2,400, 1,200, 600, and 300 ms). Hyperkalemia in the absence of drugs produced reductions in resting potential (-87.9 +/- 3.8 to 74.6 +/- 3.3 mV), maximum rate of depolarization (316 +/- 68 to 240 +/- 12 V/s), and action potential duration (178 +/- 21 to 165 +/- 27 ms). All three drugs produced increased depression of Vmax in hyperkalemia compared to control conditions but, at all three concentrations and all four rates, this enhancement of effect was greater for lidocaine than for either of the other two agents (which did not differ significantly from each other; p less than 0.001). Similar though less marked effects were produced by acidosis (3.5 mV depolarization and 19% reduction in Vmax), and once again the depression of Vmax by lidocaine was enhanced more by this intervention than were the actions of encainide or flecainide (p less than 0.01). Hypoxia had no effect on action potential parameters other than duration and no significant modulation of drug actions was seen for this intervention.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719293 TI - Lidocaine and cardiovascular reflex responses to simulated orthostatic stress in normal volunteers. AB - The interactions of lidocaine with baroreceptor reflexes during simulated orthostatic stress or moderate volume depletion were investigated in healthy volunteers using the lower body negative pressure (LBNP) test. Cardiopulmonary baroreceptors were selectively unloaded at low levels of LBNP (-15 mm Hg) since the central venous pressure (CVP) decreased (-1.4 +/- 0.3 mm Hg) without changes in arterial pressure (AP) and heart rate (HR) but significant increases in forearm vascular resistance (FVR) and plasma noradrenaline (PNA). Cardiopulmonary as well as arterial baroreflexes were both unloaded at higher levels of LBNP (-40 mm Hg) since the CVP (-3.4 +/- 0.4 mm Hg) and systolic AP (-10 +/- 3 mm Hg) decreased whereas FVR, PNA, and plasma renin activity (PRA) increased further (all p less than 0.05). Following lidocaine infusion (serum level of 3.8 +/- 0.2 micrograms/ml), the AP, HR, FVR, and PNA increased and CVP decreased (p less than 0.05). Compared to LBNP performed under saline, lidocaine did not alter the LBNP (-15 mm Hg)-induced changes in cardiovascular and biological parameters but significantly decreased the induced rises in HR, FVR, PNA, and PRA at a LBNP of 40 mm Hg (p less than 0.05). In an additional study, it was also demonstrated that lidocaine significantly decreased the sensitivity of the reflex-mediated bradycardia following phenylephrine injection and attenuated the vasoconstrictor response to the cold pressor test taken as a nonspecific somatic pressor reflex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719294 TI - Inhibition of thrombin-induced endothelium-dependent relaxation after coronary ischemia in the dog: possible role of the coagulation cascade. AB - Myocardial ischemia inhibits endothelium-dependent relaxation stimulated by the coagulant peptide, thrombin. To investigate whether activation of endogenous thrombin contributed to this reduction in relaxant sensitivity, the effects of pretreatment of dogs with the coumarin anticoagulant, brodifacoum, were studied. Experiments were performed in both normal coronary vasculature and coronary vasculature exposed to 90 min of myocardial ischemia, with or without 60 min of subsequent reperfusion. Ischemia was induced in the left anterior descending artery (LAD); nonischemic vessels from the left circumflex (LCX) artery of the same animals were used as control. Thrombin caused dose-dependent relaxation in isolated LCX preconstricted with prostaglandin F2 alpha (Emax of 89.1 +/- 2.33%). Relaxation was reduced by 90 min of ischemia (Emax of 27.5 +/- 8.0%; p less than 0.05), and further reduced after subsequent reperfusion (Emax of 8.7 +/- 8.7%). However, maximum relaxations to acetylcholine, calcimycin, and isoproterenol were unchanged after ischemia (Emax greater than 90% in all groups). Brodifacoum had no effect on thrombin-induced relaxation in control vessels (Emax of 83.0 +/- 3.5%), or on relaxation in response to acetylcholine, calcimycin, or isoproterenol (Emax greater than 90%). In contrast, brodifacoum markedly reduced thrombin-induced relaxation after ischemia (Emax of 3.3 +/- 3.3%; p less than 0.05) yet significantly preserved the relaxant response to thrombin after ischemia and reperfusion (Emax of 36.6 +/- 4.3%). Infusion of the thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK), during ischemia and reperfusion also preserved in part the relaxant response induced by thrombin (Emax of 30.0 +/- 5.1%; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719295 TI - Effects of tetrodotoxin and imipramine on the cardiac responses to nicotine in isolated, blood-perfused canine heart preparations. AB - The effects of nicotine on the sinus rate and atrial or left ventricular contractile force were investigated in the isolated, blood-perfused dog right atrium and left ventricle. Nicotine (3-300 nmol) in the right atrium induced dose dependent negative followed by positive chronotropic and inotropic responses, whereas nicotine caused only a positive inotropic response in the left ventricle. The negative responses to nicotine were blocked by atropine, hexamethonium (C6), and tetrodotoxin (TTX). The positive effects of nicotine were abolished by propranolol and C6. TTX significantly inhibited the positive responses to nicotine by about 50% in the atrial preparation and totally suppressed them in the left ventricle. Imipramine inhibited the positive cardiac responses to nicotine and tyramine, but potentiated the responses to noradrenaline (NA) in atrial and ventricular preparations. These results suggest that (a) nicotine induces negative and positive cardiac effects mediated by parasympathetic ganglionic nicotinic receptors and presynaptic nicotinic receptors of the postganglionic sympathetic nerves, respectively, in the dog heart; (b) there are few parasympathetic ganglionic cells in the dog left ventricle; (c) the positive cardiac responses to nicotine are caused by both TTX-sensitive and TTX insensitive NA release mechanisms; and (d) imipramine inhibits the positive cardiac responses to nicotine at the presynaptic nicotinic receptor sites of the postganglionic sympathetic nerves. PMID- 1719296 TI - Amrinone reduces pulmonary vascular resistance elevated by U46619 in isolated perfused lungs. AB - The effects of amrinone on pulmonary vascular resistance (PVR) were studied in an isolated, perfused rabbit lung model where all the major determinants of PVR were controlled. In this preparation, the alveolar oxygen and carbon dioxide tensions, vascular pH and vascular oxygen and carbon dioxide tensions, and zonal conditions of the lung and phasic variations of pulmonary artery pressures could be precisely measured and controlled. Measurements of PVR were made by a complete determination of the pulmonary pressure-flow curve and determination of the PVR under identical flow conditions for all studies. This approach allowed a more precise determination of the primary effects of amrinone on normal and elevated PVR than has been previously possible. We found that amrinone in final concentrations of either 4 or 8 micrograms/ml had no effect on basal PVR and no effect on lung water weight to dry ratios. When PVR was elevated by the addition of the thromboxane A2 mimetic U46619, amrinone reduced the PVR by 27% at a final concentration of 4 micrograms/ml and by 74% at a final concentration of 8 micrograms/ml. We conclude that in the doses tested, amrinone has no effects on basal PVR but is able to reduce elevated PVR in a dose-dependent manner. These results are the first to demonstrate clearly that amrinone has the ability to reduce elevated pulmonary vascular tone through a direct mechanism and not through secondary effects on other determinants of PVR such as left atrial pressure (Pla), increased cardiac output with resultant vascular recruitment, or increases in mixed venous oxygen tension. The possible implications for the clinical use of amrinone in situations of elevated PVR are discussed. PMID- 1719297 TI - Beneficial effects of transforming growth factor-beta and tissue plasminogen activator in splanchnic artery occlusion and reperfusion in cats. AB - We studied the effects of transforming growth factor-beta (TGF-beta), tissue plasminogen activator (tPA), and their combination in cats subjected to splanchnic artery occlusion (SAO) with reperfusion. Untreated anesthetized cats subjected to total occlusion of the celiac, superior, and inferior mesenteric arteries for 120 min, followed by reperfusion, uniformly died within 120 min after reperfusion. The mean survival time was 75 +/- 8 min. Plasma amino-nitrogen concentrations and cathepsin D and myocardial depressant factor (MDF) activities were markedly elevated following reperfusion. Superior mesenteric artery (SMA) rings isolated from cats subjected to SAO with reperfusion exhibited a significant loss of vasorelaxation to the endothelium-dependent dilators acetylcholine and A-23187. Administration of tPA (1 mg/kg) intravenously just before reperfusion did not prolong survival time (81 +/- 10 min) nor did it influence any biochemical or cardiovascular responses following reperfusion or ameliorate the depressed endothelium-dependent relaxation of SMA rings. In contrast, TGF-beta (50 micrograms/cat) ameliorated the SAO postreperfusion state in terms of survival rate and plasma MDF activity, and protected against depressed endothelium-dependent relaxation of SMA rings. TGF-beta alone slightly increased the survival time to 102 +/- 11 min. However, combined treatment with tPA (1 mg/kg) and TGF-beta (50 micrograms/cat) preserved endothelium-dependent relaxation and prevented increases in plasma amino-nitrogen more prominently than TGF-beta given alone and significantly increased the survival time to 118 +/- 3 min (p less than 0.01). These results indicate that TGF-beta exerts beneficial effects in SAO followed by reperfusion in cats, and tPA has an augmenting action on some of the beneficial effects of TGF-beta. These findings suggest that TGF beta alone or in combination with tPA may be potentially useful therapeutic regimens in splanchnic ischemia shock by preserving splanchnic parenchymal and endothelial cells. PMID- 1719298 TI - Domain specialization in voltage-dependent Na+ and Ca2+ channels. PMID- 1719299 TI - A new method for the analysis of the dynamics of the molecular genetic control systems. I. Description of the method of generalized threshold models. AB - In the molecular system of coding polymers and metabolites a control subsystem has been singled out that forms controlling variables showing the action of regulatory molecules and a controlled subsystem where, depending on the values of controlling variables controlled variables are formed, i.e. concentrations of DNA, m-RNA, proteins and metabolites. Relationships have been obtained which enable controlling variables to be found. Equations showing the dynamics of molecular genetic control systems' components have been obtained. A method of generalized threshold models that enables kinetic curves to be obtained by pure mathematical means for macromolecular components (DNA, RNA, proteins) of molecular genetic control systems of varying complexity is suggested. PMID- 1719300 TI - A model of the phase-sensitivity of the pacemaking cell in the bullfrog heart. AB - In this study, mathematical models of the bullfrog sinus venosus (SV) pacemaker cell (Rasmusson et al., 1990, Am. J. Physiol. 259, H352-H369) and the ACh sensitive K+ channel (Shumaker et al., 1990, Biophys. J. 57, 567-576) are combined to simulate the response of the SV myocyte to brief hyperpolarizing currents or acetylcholine (ACh) pulses. These simulations provide an ionic basis for the interpretation of the response of this pacemaker cell to either single perturbation or periodic stimuli. The model predicts that the effects of ACh stimulation on the pacemaker cycle length are dependent both on the phase and temporal characteristics of the [ACh] waveform. For example, the simulations show that (1) although ACh normally has an inhibitory effect on the pacemaker model, for cases where the rise time and duration of the [ACh] waveform are sufficiently brief, ACh can paradoxically accelerate the beat in which a single stimulus is given; (2) the SV pacemaker normally exhibits type 1 (odd) phase-resetting in response to ACh delivery, however type 0 (even) phase-resetting behavior may be exhibited when the [ACh] waveform is large enough and has a very fast rise time; and (3) the SV pacemaker may become phase-locked to a repetitive ACh stimulus applied with either a constant period or coupling interval. In the latter case, this entrainment phenomenon has implications for the control of the cardiac pacemaker by a neural oscillator (e.g. located in the medullary cardiovascular control center) which provides input to the pacemaker cell via the vagus nerve. In these regions of capture, repetitive ACh stimulation produces a well-known paradoxical accelerative effect on the SV pacemaker cell, similar to that seen in a variety of other species. PMID- 1719301 TI - An analytical model of ionic movements in airway epithelial cells. AB - A new mathematical model of ion movements in airway epithelia is presented, which allows predictions of ion fluxes, membrane potentials and ion concentrations. The model includes sodium and chloride channels in the apical membrane, a Na/K pump and a cotransport system for Cl- with stoichiometry Na+:K+:2Cl- in the basolateral membrane. Potassium channels in the basolateral membrane are used to regulate cell volume. Membrane potentials, ion fluxes and intracellular ion concentration are calculated as functions of apical ion permeabilities, the maximum pump current and the cotransport parameters. The major predictions of the model are: (1) Cl- concentration in the cell is determined entirely by the intracellular concentration of negatively charged impermeable ions and the osmotic conditions; (2) changes in intracellular Na+ and K+ concentrations are inversely related; (3) cotransport provides the major driving force for Cl- flux, increases intracellular Na+ concentration, decreases intracellular K+ concentration and hyperpolarizes the cell interior; (4) the maximum rate of the Na/K pump, by contrast, has little effect on Na+ or Cl- transepithelial fluxes and a much less pronounced effect on cell membrane polarization; (5) an increase in apical Na+ permeability causes an increase in intracellular Na+ concentration and a significant increase in Na+ flux; (6) an increase in apical Cl- permeability decreases intracellular Na+ concentration and Na+ flux; (7) assuming Na+ and Cl- permeabilities equal to those measured in human nasal epithelia, the model predicts that under short circuit conditions, Na+ absorption is much higher than Cl- secretion, in agreement with experimental measurements. PMID- 1719302 TI - Prebiotic co-evolution of self-replication and translation or RNA world? AB - A prebiotic scenario is proposed, based on the recent "domain hypothesis" model (Lahav, 1989, J. molec. Evol. 29, 475-479), suggested for domain propagation of RNA-like molecules in a fluctuating environment. The same system is suggested now not only for the evolution of ribozymes, but also for the evolution of directed peptide synthesis, as follows: Short, self-structured strands (termed prebioectons), each possessing a templatable domain which is chargeable by an amino acid, are the predecessors of tRNA (proto-tRNA). Complementary domains are formed on these prebioectons during an environmental cycle such as wetting drying, followed by their dissociation from their template domain and ligation, to form the predecessor of mRNA (proto-mRNA). The evolution of directed peptide synthesis is suggested to be based on the ability of the charged prebioectons to attach preferentially to their complementary domains on the proto-mRNA. Two stages of this process are envisioned, namely: (a) Template-directed, random peptide synthesis taking place when non-specifically-charged prebioectons are sequentially attached each to its complementary domain on the proto-mRNA, followed by peptide bond formation. (b) Template-and-sequence-directed peptide synthesis, which can be realized after the "invention" of a catalytic molecule capable of specifically charging a proto-tRNA by an amino acid; this is the crucial evolutionary stage, where a crude genetic code becomes functional. Gradually, catalytic peptides and ribozymes are selected for their functions and evolve, while being encoded in the primitive "memory" of the emerging system. Thus, rather than the RNA monopoly postulated by the RNA World hypothesis, an early co-evolution of primitive enzymes and ribozymes is suggested.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719303 TI - Low-dose aprotinin also allows reduction of blood loss after cardiopulmonary bypass. PMID- 1719304 TI - Increased anticoagulation during cardiopulmonary bypass by aprotinin. PMID- 1719305 TI - Light and electron microscopical visualization of anterogradely labelled corticospinal growth cones using a new combination of HRP staining techniques. AB - Up until now, the ultrastructural visualization of growth cones of developing long fibre tracts could only be achieved by horseradish peroxidase (HRP) application 'en route', resulting in axonal damage, which in turn may affect growth cone morphology. Besides, this technique results in labelling of passing fibres, thus hampering the identification of axon origin as well as the interpretation of growth cone configuration. In the present investigation a new combination of HRP staining and intensification techniques is presented which makes it possible to visualize anterogradely labelled corticospinal growth cones over long distances in developing rat spinal cord at the light as well as the electron microscopical level. HRP was applied to the originating cells of the corticospinal tract, located in the sensorimotor cortex, and after 24 h was visualized using a procedure which essentially consists of 3 subsequent steps: first a tetramethylbenzidine (TMB)/ammoniumheptamolybdate (AHM) reaction; second diaminobenzidine (DAB)/nickel (Ni) stabilization and finally glucose oxidase intensification. As was verified at the EM level, the staining procedure here described reveals a complete intense black staining of HRP-labelled growth cones of outgrowing corticospinal axons. Therefore, the method described here guarantees a correct analysis of growth cone morphology at the light microscopical and the ultrastructural level. The present procedure is especially valuable in studying the development of long central nervous fibre systems. PMID- 1719306 TI - A simple and inexpensive slicer for preparation of brain slices. AB - A simple and inexpensive slicer has been developed for the preparation of slices of mouse or rat brain. The instrument consists of razor blades, separated by an 0.5 mm thick polyethylene sheet (1 x 1 cm), mounted on metal screws through a hole in the center of the polyethylene sheet. Using this slicer, 6-8 uniform slices of 500 microns thickness were obtained from mouse or rat brain. These brain slices were incubated in a medium consisting of artificial cerebrospinal fluid for 1 h at 37 degrees C under an oxygen atmosphere and the activities of various subcellular marker enzymes were assayed. The slice weights and the activities of the enzymes did not vary significantly in different batches of slices. Morphological evaluation of the slices revealed well-preserved neurons. Histochemical staining for mitochondrial enzymes revealed intense staining of neuronal cells and lighter staining of the white matter in all the regions examined. These slices could serve as a useful in vitro model for studying brain function and the effect of various toxicants on the brain. PMID- 1719307 TI - Enhancement of horseradish peroxidase histochemistry and/or uptake with radiocontrast media. AB - The effects of dissolving horseradish peroxidase (HRP) or HRP conjugated to wheatgerm agglutinin (WGA-HRP) in radiocontrast media (MD76) on the intraaxonal transport and enzymatic activity of these tracers were evaluated both in vivo and in vitro. The in vivo studies showed that more reaction product was visible in the L4 dorsal horn following soaking of the sciatic or peroneal nerve, or following cutaneous injection of WGA-HRP, when WGA-HRP was dissolved in MD76, as compared to dissolving WGA-HRP in distilled water. Additionally, there appeared to be an enhancement of anterograde transport and a reduction of retrograde transport of WGA-HRP when this tracer was dissolved in MD76, as compared to dissolving it in distilled water. The in vitro studies indicated that radiocontrast media increased the enzymatic activity of both WGA-HRP and HRP as compared to their enzymatic activity in distilled water when assayed spectrophotometrically. Data are presented that indicate some binding (about 4%) of HRP with MD76. This binding of HRP with organically bound iodine may account for the enhancement of anterograde transport and/or increased enzymatic activity. PMID- 1719308 TI - Granulocyte-macrophage colony-stimulating factor and interleukin-3 protect leukemic blast cells from ara-C toxicity. AB - The blast cells of acute myeloblastic leukemia (AML) usually require growth factors for optimum proliferation in cell culture. Growth factors also affect the sensitivity of AML blast cells to cytosine arabinoside (ara-C). Others have reported that factor-treated cells are more ara-C sensitive than blasts in culture without factors. These authors have reported previously that AML blasts grown with rG-CSF, with or without GM-CSF, are more sensitive than cells in GM CSF alone. This paper reports experiments which show that changes in the ara-C sensitivities of blast cells in different growth factors are not explained by changes in the percentage of cells in the DNA synthesis (S) phase of the cycle. Blasts freshly obtained from five AML patients were cultured in either rG-CSF, rGM-CSF, or rIL-3; they were then exposed to 20 min pulses of either high specific activity tritiated thymidine (3HTdR) or a high concentration of ara-C. Regardless of the factor present, the pulse of 3HTdR decreased the number of clonogenic cells by about 50%, the result expected for actively proliferating cells with an S phase occupying about half the cycle time. The same result was found for four of the five blast cell populations grown in G-CSF and pulsed with ara-C; in contrast, clonogenic cells grown in GM-CSF or IL-3 from these four populations were not killed by ara-C. The blasts from the fifth patient were ara C resistant under all conditions. It was concluded that exposure to GM-CSF or IL 3 decreased ara-C sensitivity in blasts that were actively making DNA. The observation was explored in more detail using a cell line (OCI/AML-1a) that is both ara-C sensitive and growth factor dependent. These studies showed that about 15 h of growth in factor are required for a change in ara-C sensitivity. PMID- 1719309 TI - Effect of protein kinase inhibitors on the proliferation of leukemic cells stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor or interleukin-3. AB - The effects of the inhibitor for protein kinase A or C, or tyrosine kinase (H-8, staurosporine, or genistein, respectively) on the proliferation of leukemic and normal bone marrow cells stimulated by granulocyte colony-stimulating factor (G CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) were studied using the MTT assay. These inhibitors suppressed the proliferation of leukemic and normal bone marrow cells in a dose-dependent manner. Although the suppressive effect of each inhibitor on cell proliferation was varied in each instance, the effects were almost similar whichever CSF was added. A significant difference was not recognized between leukemic and normal bone marrow cells in terms of sensitivity to these inhibitors. The data indicate that protein kinase inhibitors have an inhibitory effect on leukemic and normal hematopoietic cell proliferation and that further studies are required to determine if this effect is due to the inhibition of protein kinases acting as the second messenger of CSFs. PMID- 1719310 TI - Co-ordinate expression of T-cell antigens on acute myelogenous leukemia and of myeloid antigens on T-acute lymphoblastic leukemia. Speculation on a highly balanced bilinearity. PMID- 1719311 TI - Medical mythology: bekokko. PMID- 1719312 TI - [The 1991 Nobel Prize in medicine: discovery of the function of cellular ion channels]. PMID- 1719313 TI - [Instructive pictures can help the patient to understand the cause of his dyspepsia]. PMID- 1719314 TI - [Angiogenesis--a new field for cancer therapy]. PMID- 1719315 TI - Immediate hypersensitivity in colon of children with chronic Trichuris trichiura dysentery. AB - There are few data on mucosal immune responses to intestinal helminths in human beings, especially those involving the IgE system, which is thought to be important in parasite expulsion. We sought evidence of an immediate hypersensitivity reaction in the colon of children with chronic dysentery due to Trichuris trichiura. 28 children with Trichuris dysentery syndrome (TDS) were compared with 16 control children (with no TDS or worms visible on colonoscopy). All children were aged 1-11 years. Rectal biopsy samples were taken before and after expulsion of the worms by means of mebendazole treatment. Children with TDS had significantly greater numbers than controls of mast cells (mean [SD] 10.9 [1.3] vs 3.9 [0.6]% of all cells; p less than 0.0003) and of cells with surface IgE (median [range] 11.1 [7.5-11.6] vs 1.0 [0-1.5]%; p less than 0.001) in the subepithelial region of the mucosa. On electronmicroscopy, degranulating mast cells were prominent in parasitised children. In culture, rectal biopsy samples from parasitised children showed high rates of spontaneous histamine release, but only low rates of antigen-specific release. After treatment, spontaneous histamine release was significantly reduced and antigen-specific histamine release could be provoked. Thus, an IgE-mediated immune mucosal response to a helminth infection does occur in human beings but is not sufficient to cause appreciable parasite expulsion. PMID- 1719316 TI - Persistent anogenital warts. PMID- 1719317 TI - Euthanasia. PMID- 1719319 TI - Spirochaetes in periodontal disease. PMID- 1719318 TI - Screening for prostatic hyperplasia. PMID- 1719320 TI - Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. AB - Only small numbers of cells from solid tumours are needed for haematogenous metastasis. Detection is difficult because existing techniques are not sensitive enough. We have used reverse transcriptase to make complementary DNA from peripheral blood messenger RNA, and the polymerase chain reaction (PCR) to amplify cDNA specific for a gene actively transcribed only in the tumour tissue type. We prepared cDNA from peripheral blood of seven patients with malignant melanoma, four patients with other metastatic cancers, and four healthy subjects, as well as from several melanoma-derived cell lines. PCR was used to amplify the gene for tyrosinase, a tissue-specific gene in melanocytes. Since normal melanocytes are not thought to circulate in peripheral blood, detection of tyrosinase transcription in peripheral blood should indicate the presence of circulating cancer cells. The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood. Blood samples from four of the seven patients with malignant melanoma gave positive results, whereas all eight control subjects gave negative results. This method does not depend on the characterisation of cancer-specific genetic abnormalities and can be applied to any cancer for which tissue-specific genes can be identified, including epithelial cancers. It could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment. PMID- 1719321 TI - Effects of cyclosporin, FK506, and rapamycin on graft-vessel disease. AB - Graft-vessel disease (GVD) limits the long-term survival of heart-transplant patients, and this effect has not been altered by use of cyclosporin for immunosuppression. We compared the effects of the immunosuppressants cyclosporin, FK506, and rapamycin on GVD in a rat-heart transplantation model. Allografted hearts from rats treated with 1 mg/kg FK506 for 50 days showed the same degree of myocardial rejection but a significantly worse (p less than 0.05) grade of GVD compared with grafted hearts from rats treated with 1.5 mg/kg cyclosporin for the same time. 2 mg/kg FK506 for 50 days prevented cellular rejection but GVD was as severe as that found with 1 mg/kg FK506. Moderate GVD was present in two of five allografted hearts after treatment with 4 mg/kg FK506. 1.5 mg/kg rapamycin for 50 days was an effective inhibitor of rejection and GVD. Based on our results in rats, the possibility that GVD may occur in human heart-transplant recipients treated with FK506 cannot be excluded. PMID- 1719323 TI - Directory of Otolaryngological Societies. PMID- 1719322 TI - Microvascular phenomena during pancreatic islet graft rejection. AB - Transplantation of insulin secreting tissue as a free graft has the potential to become a safe and simple procedure to cure diabetes. However, clinical results, i.e. achievement of insulin independency, are poor, in spite of the use of immunosuppressive regimens, which are regularly successful in whole organ transplantation. In contrast to whole organ grafts, which are revascularized immediately after transplantation, free pancreatic islet grafts require the process of revascularization in order to establish a microvascular network, sufficient for the nutritional blood supply. We have demonstrated for the first time in vivo images of the process of revascularization of free islet xenografts including microvascular phenomena during graft rejection. Rat islet xenografts were isolated by collagenase digestion and transplanted into hamster dorsal skinfold chambers. After 6, 10 and 14 days the microvasculature of the islet grafts was analyzed by means of intravital fluorescence microscopy. Xenogeneic grafts were revascularized during the first 6 days similarly compared to syngeneic grafts; however, on day 10 after transplantation a reduction in size of the microvascular network as well as a decrease in functional capillary density and a reduction in capillary red blood cell velocity were observed, accompanied by microvascular rejection phenomena, such as an increase of microvascular permeability, edema formation, capillary widening and intravascular accumulation of white blood cells (WBCs) with concomitant WBC-endothelium interaction in post capillary venules. Treatment with 2.5 mg/kg/d (+/-)-15-deoxyspergualin could not completely alleviate these microvascular rejection phenomena.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719324 TI - Neurokinin receptors antagonists: old and new. AB - Four neurokinin antagonists of different size have been used to counteract the myotropic effects of substance P, neurokinin A and neurokinin B in isolated organs containing a single receptor type (monoreceptor systems). These are: the dog carotid artery, the rabbit jugular and cava veins and the guinea pig ileum (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3). Undeca and octapeptides containing 2 D-Trp residues in their sequences were slightly more active on the NK-1, than on the NK-2 and NK-3 receptors and showed little selectivity. In contrast, compound AcThr-D.Trp(For)-Phe.NMe Bz was found to be as good an antagonist as the larger compounds and showed some selectivity for the NK 1 receptors. When tested against kinins or angiotensin, all compounds were found to be inactive, suggesting that they are specific for neurokinins. The present results show that NK-1 receptor antagonism can be obtained with compounds of different size, including tripeptides and nonpeptides. PMID- 1719325 TI - Enzymatic dispersion of mast cells from human sinus mucosa: characterization of histamine release and its comparison with chopped fragments of the tissue. AB - Functional characteristics of mast cells in chopped fragments from sinus mucosa, which was dissected from patients with chronic sinusitis, were compared with those from dispersed cells prepared by enzymatic treatment. The results obtained in this study were the following. (1) Both chopped fragments and dispersed cells released histamine in a dose-dependent manner when incubated with anti-IgE. However, higher histamine release was always observed in dispersed cells. (2) Although no differences in the ability to reduce histamine release with salbutamol or forskolin could be observed between chopped fragments and dispersed cells, staurosporin and p-bromophenacyl bromide were more active on dispersed mast cells than chopped fragments. (3) Passive sensitization of dispersed cells with an allergic serum containing IgE to mite could be achieved only after elution of IgE on the cells with lactic acid. PMID- 1719326 TI - Protective effects of gabexate mesilate (FOY) against impaired pancreatic energy metabolism in rat acute pancreatitis induced by caerulein. AB - A supramaximal dose of caerulein (5 micrograms/kg.hr for 3.5 hours) caused edematous acute pancreatitis in rats, characterized by portal hyperamylasemia (32 +/- 3 U/ml) and pancreatic edema (pancreatic water content, 86 +/- 2%) [control group: amylase, 8 +/- 1 U/ml; water content, 74 +/- 2%]. In this model, increased portal levels of malate dehydrogenase (148 +/- 25 U/ml), increased mitochondrial fragility and impaired pancreatic energy charge level (0.77 +/- 0.05) were also observed [control group: malate dehydrogenase, 54 +/- 11 U/ml; energy charge level, 0.94 +/- 0.03]. Administration of gabexate mesilate, FOY, in a dose of 50 mg/kg.hr for 2 hours before and during the caerulein infusion had a significant protective effect against these pancreatic injuries (portal amylase level, 11 +/- 2 U/ml; MDH level, 72 +/- 19 U/ml; E.C., 0.89 +/- 0.02; water content, 76 +/- 2%). FOY in a dose of 20 mg/kg.hr was partially protective. These results indicate that subcellular organelle fragility and malfunction are closely related to the pathogenesis of acute pancreatitis and suggest the usefulness of FOY in the treatment of this disease. PMID- 1719327 TI - Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro. AB - Exposure of rat peritoneal mast cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca2+. These observations suggest that magnesium may decrease the secretory response by displacing the cellular calcium which is utilized in stimulus-secretion coupling. PMID- 1719328 TI - Disposition of fluorescein-labelled dextran (150 kD) in isolated perfused livers from control and diabetic rats. AB - Isolated perfused livers were used to study the hepatic disposition of fluorescein-labelled dextran with a MW of 150 kD (FD-150), in control and streptozotocin-induced diabetic rats. A 100-microliter volume of FD-150 solution (10%, w/v) was injected as a rapid bolus dose into the inlet catheter of the isolated livers, and samples were collected from the outlet catheter in 2-sec intervals for 80 sec. Statistical moment theory was used to calculate the distribution and elimination parameters of the tracer based on the concentrations of FD-150 in the outflow. The values (mean +/- SD) of mean transit time, volume of distribution, and extraction ratio of FD-150 in the isolated livers from control rats were 16.3 +/- 2.00 sec, 0.298 +/- 0.054 ml/g liver, and 0.24, respectively. Similar values were obtained in diabetic livers, suggesting that streptozotocin-induced diabetes does not affect the hepatic disposition of FD 150. PMID- 1719329 TI - Alpha-fetoprotein: a sensitive avidine-biotin assay. AB - A conventional enzyme-linked immunosorbent assay (ELISA) for alpha-fetoprotein was modified to incorporate the avidine-biotin complex, in order to enhance sensitivity. Five modifications of this assay were compared, of which the 2-step noncompetitive avidine-biotin assay (NABA) was found to be the most rapid and sensitive (detection limit 0.05 ng/ml). Levels of AFP in human cord serum measured by the 2-step NABA method showed good correlation (r = 0.965) with measurements by radio-immuno assay, and the assay can be performed within one hour. The simplicity and sensitivity of this assay should make it a reliable method of choice for determination of alpha-fetoprotein. PMID- 1719330 TI - Serum prostatic acid phosphatase activity and prostate specific antigen when the prostate gland is enlarged. AB - Serum prostatic acid phosphatase activity and prostate specific antigen levels were measured in the serum of 113 patients undergoing transurethral resection of an enlarged prostate gland, and the results compared with the histological diagnosis. Prostate-specific antigen was found to be the more sensitive indicator of malignancy, but prostatic acid phosphatase was more specific. PMID- 1719332 TI - [Local electromagnetic hyperthermia of prostatic adenoma]. AB - The paper is concerned with the results of therapy of 43 patients with prostatic adenoma using local electromagnetic hyperthermia (LEH) on a Yakhta-3 unit of 915 MHz. LEH can be one of the promising methods of conservative therapy of prostatic adenoma. Therapeutic efficacy grows with increasing warming and the number of LEH sessions. LEH repeated courses of not lead to side-effects and improve the patients' local status. PMID- 1719331 TI - [Radioimmunological determination of tumor markers in the diagnosis of recurrences of rectal cancer]. AB - The levels of tumor markers were determined in 173 patients with rectal cancer recurrences by radioimmunoassay. An increase in a CEA level was observed most frequently (92.5%). An increase in the levels of alpha-fetoprotein, ferritin and beta 2-microglobulin was observed in 61.7, 56.6 and 46.3%, respectively. CA-19-9, a carbohydrate antigen, was of no importance for the detection of cancer of this site, and an increase in its titer was observed in 15.5% only. Thus the most specific and effective diagnostic test for the diagnosis of rectal cancer recurrences is the determination of a CEA level. PMID- 1719333 TI - Magnetic resonance imaging of the efficacy of specific inhibition of 5 alpha reductase in canine spontaneous benign prostatic hyperplasia. AB - A leading role for prostatic levels of dihydrotestosterone (DHT) in the pathogenesis of benign prostatic hyperplasia is well established, if incompletely understood. The present study provides initial confirmation that 5 alpha reductase inhibition alone is sufficient to prevent prostatic accumulation of DHT and to produce epithelial regression in the canine prostate. In dogs treated with the specific 5 alpha-reductase inhibitor finasteride, prostatic volume decreased to one-third of the baseline volume, while the prostatic concentration of DHT fell fivefold: both were constant in placebo control dogs. Demonstration that MR imaging can serve as accurate modality to assess prostatic volume was provided by serial measurements of the canine prostate and by correlation of the last imaging measurement with the weight of the excised prostate. Significant intensity changes were observed in T2-weighted images measured post-treatment; these changes correlated with the histopathology of the prostate. These results suggest that beyond quantifying regression, multiecho T2 measurements can be useful in probing accompanying changes occurring on the cellular level. PMID- 1719334 TI - Autocrine activity of interleukin 6 secreted by hepatocarcinoma cell lines. AB - Among several rat hepatoma cell lines known to secrete interleukin 6 (IL6), the HTC.JZ1 line stands out as a high-level producer. HTC.JZ1 cells were stimulated to secrete up to fourfold increased amounts of IL6 over 24 hours by treatment with lipopolysaccharides (LPS). Both functional IL6 levels, measured as hepatocyte stimulating factor (HSF) activity, and IL6 mRNA concentrations were increased proportionally by exposure to LPS. Similarly, IL6 mRNA was induced by LPS treatment in cultured primary rat hepatocytes. The induction of Il6 mRNA by LPS was inhibited both in primary hepatocyte and hepatoma cell cultures by treatment with the synthetic glucocorticoid dexamethasone, consistent with the known analogous repression of the IL6 gene by dexamethasone in macrophages, monocytes and fibroblasts. IL6 secreted by HTC.JZ1 cells was utilized as an autocrine inducer of endogenous acute phase gene expression: HTC cells expressed constitutive levels of alpha 2-macroglobulin (alpha 2M) mRNA specified by the major rat acute phase gene, the alpha 2M gene, which is known to be regulated by IL6. By contrast, normal rat liver biopsy material and a number of other rat hepatoma cell lines lacked endogenous IL6 production and showed very low to zero expression of endogenous alpha 2M mRNA. Expression of alpha 2M mRNA in HTC.JZ1 cells was inducible by treatment with LPS. The constitutive and the LPS-induced production of alpha 2M mRNA were significantly reduced (up to 50% inhibition) by addition of an anti IL6 serum to the culture medium and removal of the immune complexes. However, complete neutralization of the alpha 2M-inducing HSF activity could not be obtained with anti-IL6 serum alone, probably because HTC.JZ1 cells secrete comparable quantities of a second HSF activity. This activity, the cytokine leukemia inhibitory factor (LIF), is also known to stimulate transcription of the rat alpha 2M gene but was not reactive with anti-IL6 sera. The induction of IL6 mRNA in HTC cells by LPS was regulated at the transcriptional level, as demonstrated by a series of mutagenesis and transfection experiments. Progressive deletion of 5' flanking sequences from the IL6 gene promoter region reduced the basal level, and the LPS-induced promoter activity after transfection into HTC.JZ1 hepatoma cells. IL6 has been shown to act as an autocrine regulator of growth for certain B lymphoid cell lines derived from human multiple myelomas. The results presented here establish that IL6 secreted by certain hepatoma cell lines also acts in an autocrine fashion to induce expression of the endogenous acute phase alpha 2M gene. PMID- 1719335 TI - The three-dimensional structure of the regular surface protein of Comamonas acidovorans derived from native outer membranes and reconstituted two-dimensional crystals. AB - The three-dimensional structure of the regular surface protein (p4 symmetry, lattice constant a = b = 10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three-dimensional reconstructions were performed using native outer membranes and artificial two-dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two-fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain. PMID- 1719336 TI - Classification of micronuclei in murine erythrocytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA. AB - Micronuclei (MN) in erythrocytes of mouse bone marrow cells were induced in vivo by the spindle poisons colchicine (COL) and vinblastine (VBL), by hydroquinone (HQ) and by the alkylating agent mitomycin C (MMC). Two different methods were applied to detect whole chromosomes with centromeric proteins or chromatin in MN to discriminate between spindle damaging or clastogenic activity of these chemicals. One method determined the fraction of MN with centromeric chromatin by immunofluorescent staining using antikinetochore antibodies (CREST staining). The other method applied non-radioactive in situ hybridization with a novel DNA probe. The fractions of MN that showed positive signals by either technique thus indicating with a high probability the presence of whole chromosomes instead of acentric fragments, were in good agreement for COL, VBL and HQ. After application of MMC, however, 4.5% of the MN were CREST-positive, while 29% gave a positive hybridization signal. The results suggest, that kinetochores may have lost certain centromeric antigens due to treatment with MMC so that MN containing whole chromosomes appear CREST-negative. The presented in situ hybridization scheme using satellite DNA is a more direct detection and is advantageous to the CREST staining technique in that it is unaffected by damage of kinetochore or centromeric function. PMID- 1719337 TI - In vitro micronucleus test with kinetochore staining: evaluation of test performance. AB - In the framework of the coordinated programme 'Genomic Mutations' sponsored by the Commission of European Communities, eight known or suspected spindle poisons (cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vinblastine) were tested in a modified in vitro micronucleus test in Chinese hamster cells. Micronuclei (MN) with or without kinetochore were analyzed by staining of the kinetochore with an antikinetochore antibody (CREST staining). Mitotic index and ana-telophase:mitosis ratio were also recorded to evaluate cytotoxicity and c mitotic effects of tested chemicals. CREST-positive MN were induced by diazepam, thiabendazole and vinblastine. Hydroquinone, pyrimethamine, econazole and cadmium chloride induced MN that contained both entire chromosomes and acentric fragments. Negative results were obtained with thimerosal. The results obtained indicate that the detection of CREST-positive MN is a powerful assay to identify spindle poisons. Furthermore, this method provides a useful tool to ascertain the origin(s) of induced MN. PMID- 1719338 TI - Function of the growth hormone-insulin-like growth factor I axis in the profoundly growth-retarded diabetic child: evidence for defective target organ responsiveness in the Mauriac syndrome. AB - Mauriac syndrome (MS) consists of a triad of poorly controlled diabetes, profound growth retardation, and hepatomegaly. The mechanisms involved in the growth retardation of those patients are not well understood. In an attempt to determine whether the growth retardation was secondary to somatroph secretory failure, abnormal pulsatile secretion, deletion of the growth hormone (GH) receptor, inadequate insulin-like growth factor I (IGF-I) generation, or abnormal IGF-I binding proteins (IGFBPs) two patients with MS were studied and their results compared with those of age-matched diabetic boys of similar glycemic control who were growing well. Overnight GH profiles in the MS and normally growing diabetics were analyzed by the CLUSTER program. The mean 12-hour GH concentrations, pulse amplitude, and pulse frequency were not different in either group of patients and did not change during acute normalization of the serum glucose overnight in the MS patients. The GH-binding proteins (GHBPs) relative binding were found to be the same in both groups of patients and did not differ from normal nondiabetic sera (62% +/- 8.0% relative specific binding in MS patients, v 53% +/- 4.3% in diabetic controls). The IGF-I concentrations were normal and comparable in both groups of patients (1.1 +/- 0.1 U/mL MS, v 1.1 +/- 0.3 diabetic controls). The IGFBPs were comparable in both groups of patients as well. One of the patients with MS had no meaningful increase in his growth velocity after 1 year on GH therapy despite good compliance. In conclusion, our data show normal hypothalamic pituitary function, normal GHBP, IGF-I generation, and IGFBPs in two patients with MS when compared with normally growing diabetic children. These data, and the lack of linear growth in response to exogenous GH therapy in one patient, suggest a GH-resistant state, either secondary to impaired bioactivity of IGF-I, or a defect at or distal to the IGF-I receptor. PMID- 1719339 TI - Generation and use of anti-phosphotyrosine antibodies for immunoblotting. AB - The techniques of detecting phosphotyrosine-containing proteins by AIA and purifying these proteins by affinity chromatography with anti-phosphotyrosine monoclonal antibodies are both reliable and consistent. Although not every tyrosine-phosphorylated protein can be detected and purified by using these techniques, a majority can. No doubt future studies will employ these approaches both for analyzing the specific role that tyrosine protein phosphorylation plays in regulating cell division and for investigating the properties of heretofore uncharacterized proteins that contain phosphotyrosine. PMID- 1719340 TI - Sequence analysis of phosphotyrosine-containing peptides. PMID- 1719341 TI - Determination of phosphoamino acid composition by acid hydrolysis of protein blotted to Immobilon. PMID- 1719342 TI - Synthesis of O-phosphotyrosine-containing peptides. AB - As a consequence of developments in peptide synthesis, it is now well established that Tyr(P)-containing peptides can be prepared in high yield by the use of Boc Tyr(PO3Me2)-OH in Boc solution or solid-phase peptide synthesis or Fmoc Tyr(PO3Me2)-OH in Fmoc/solid-phase peptide synthesis. It is considered that with further developments in solid-phase synthesis techniques and the use of alternative phosphate-protecting groups, the synthesis of large and complex Tyr(P)-containing peptides will be routine. PMID- 1719343 TI - Comparison of three methods for detecting tyrosine-phosphorylated proteins. PMID- 1719344 TI - Separation of phosphotyrosine, phosphoserine, and phosphothreonine by high performance liquid chromatography. PMID- 1719345 TI - Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors. PMID- 1719346 TI - Purification of protein-tyrosine phosphatases from human placenta. PMID- 1719347 TI - Purification and assay of CD45: an integral membrane protein-tyrosine phosphatase. PMID- 1719348 TI - Generation and use of anti-phosphotyrosine antibodies raised against bacterially expressed abl protein. AB - Polyclonal antibodies for phosphotyrosine can be obtained by immunization with a t/abl fusion protein. These antibodies work well in immunoblotting and immunostaining experiments to identify P-Tyr proteins. For example, a 95-kDa thrombin-induced P-Tyr protein in platelets is recognized by these polyclonal antibodies but reacts poorly with other types of anti-P-Tyr antibodies (A. Golden and J.S. Brugge, 1988, personal communications). To obtain large quantities of anti-P-Tyr antibodies for preparative purposes, it is much easier to use hybridomas. An attempt to isolate hybridoma anti-P-Tyr antibodies by immunization with t/abl protein was unsuccessful, possibly because anti-P-Tyr antibodies were produced by a small percentage (approximately 2%) of the immune cells responsive to the t/abl fusion protein. Monoclonal anti-P-Tyr antibodies, however, have been produced using other types of immunogens (see Chapters [8] and [9] in this volume). The different preparations of anti-P-Tyr antibodies appear to have distinct characteristics. Test several preparations to determine the optimal reagents for each experimental need. PMID- 1719350 TI - Generation of monoclonal antibodies against phosphotyrosine and their use for affinity purification of phosphotyrosine-containing proteins. PMID- 1719349 TI - Preparation and use of anti-phosphotyrosine antibodies to study structure and function of insulin receptor. PMID- 1719351 TI - Isolation of tyrosine-phosphorylated proteins and generation of monoclonal antibodies. AB - The methods used above allow one to generate the tools to study individual phosphotyrosine-containing proteins. While all of the substrates we have identified in this way contain phosphotyrosine, this cannot be assumed. Some polypeptides may bind to the anti-phosphotyrosine column because of association with other phosphotyrosine-containing proteins or by nonspecific binding to the anti-phosphotyrosine affinity column. The antibodies generated to these substrates allow one to assess potential questions directly. At a minimum, phosphoamino acid analysis must be performed on the candidate substrate labeled in intact cells with 32PO4 and precipitated with the antibody to the substrate. Methods for this can be found in Cooper and Hunter. PMID- 1719352 TI - Expression of foreign polypeptides at the Escherichia coli cell surface. PMID- 1719353 TI - Endothelial surface charge of intestinal mucosal capillaries and its modulation by dextran. AB - Capillary endothelial surface charge was investigated by perfusing the intestinal circulation of anesthetized rats in situ with 0.1 or 10 mg/ml native ferritin (NF, pI = 3.8-4.2) or with 0.1 mg/ml cationized ferritin (CF, pI greater than 10.0) for 5 min, with or without 5% Dextran 40. Ferritin binding was quantified by electron microscopy. All electron micrographs of capillaries perfused with NF showed some NF binding. Mean NF particle densities (particles/microns) were significantly greater at vesicle necks (PDv) than elsewhere on the endothelial surface (PD). Capillaries perfused with CF showed binding in only 60% of the transverse sections examined. The binding was very marked in a large proportion of these vessels. Mean CF particle densities were significantly greater at fenestrae (PDf) than elsewhere. These results demonstrate that mucosal capillaries have a variable negative electrostatic charge on the endothelial surface and support the hypothesis that some vesicle diaphragms act as preferential attractors for anionic macromolecules. Such structures could promote transendothelial vesicular transport of albumin. Dextran significantly decreased PD for NF from 25.4 +/- 2.4 (SEM) to 13.1 +/- 0.9 and PD, PDv, and PDf for CF from 45.9 +/- 4.6 to 31.0 +/- 2.7, from 62.1 +/- 6.2 to 43.6 +/- 5.4, and from 152.9 +/- 15.5 to 114.5 +/- 8.6, respectively. The above responses result from dextran's weak interaction with the endothelial surface. Dextran reduces access of ferritin molecules to the cell surface by steric hindrance and/or electrostatic shielding of the glycocalyx. PMID- 1719354 TI - Macrophages secrete a heparin-binding inhibitor of endothelial cell growth. AB - Macrophages may play an important role in the regulation of angiogenesis by secreting modulators of endothelial cells (EC) proliferation. To investigate this, human mononuclear cells were plated in culture, and the conditioned media of these cells were analyzed by heparin-Sepharose affinity chromatography. The fractions were tested for modulation of EC growth, as determined by endothelial cell number in proliferation assays. A single peak of EC growth-inhibitory activity was found to elute from heparin-Sepharose with 1.0 M NaCl. Secretion of this EC inhibitor persisted for many weeks in cell culture, at which point the cultures consisted of adherent macrophages only. This activity was therefore designated as macrophage-derived endothelial cell inhibitor (MD-ECI). Analysis using specific neutralizing antisera as well as comparative heparin affinity analysis showed that MD-ECI was distinct from the known EC inhibitors TGF-beta and TNF-alpha. MD-ECI inhibits basal EC growth as well as FGF-stimulated EC growth. Its effect on EC is dose-dependent, nontoxic, and reversible. PMID- 1719355 TI - Experimental determination of the linear correlation between in vivo TV fluorescence intensity and vascular and tissue FITC-DX concentrations. AB - A novel in vivo calibration procedure was developed to determine the microvascular and tissue concentration of fluorescein isothiocyanate-labeled dextrans (FITC-Dx) in the hamster cheek pouch, using intravital fluorescence microscopy with manually controlled TV camera gain and threshold value. Two FITC dextrans (70,000 and 150,000 MW) were used as tracers. Five minutes after the tracer was administered, selected venules (diameter 20-50 microns) were videotaped, and intravascular gray levels were obtained by digital image processing. Simultaneously, arterial blood samples were taken to measure vascular FITC-Dx concentrations with a spectrofluorometer. The gray levels and the concentrations were used to produce a calibration curve for the vascular FITC-Dx concentration. A similar calibration curve for the interstitial FITC-Dx concentration was obtained by first video recording interstitial space areas saturated with the tracer. After flushing out the tracer in the vessels, the hamster cheek pouch was then cut, weighted, and homogenized. The interstitial FITC-Dx concentration was finally measured with a spectrofluoromet. The gray levels and the concentrations were used to produce a calibration curve for the interstitial FITC-Dx concentration. The gray level was found to vary linearly with both the FITC-Dx vascular concentration (range 0.4-3.0 mg/ml) and the interstitial FITC-Dx concentration (0.12-1.50 mg/ml) in the hamster cheek pouch. PMID- 1719356 TI - A new model for the study of angiogenesis in connective tissue repair. PMID- 1719357 TI - Rapid method for isolation and staining of bacterial lipopolysaccharide. AB - Classically bacterial lipopolysaccharide (LPS) purification and silver staining take several days. We designed a simple and fast method for LPS isolation which when combined with silver staining using Pharmacia PhastSystem both can be completed in few hours. The purity of LPS isolated by this simple method may not be comparable to that by the phenol-water method hence we recommend this rapid isolation and staining procedures for simple and fast study of LPS patterns in gels. PMID- 1719358 TI - Possible presence of a capsule in Branhamella catarrhalis. AB - Clinical isolates of Branhamella catarrhalis from patients with respiratory infections were used in this study. Electron microscopic observation after treating Branhamella catarrhalis with immune serum and ruthenium red revealed the capsule. In the phagocytosis test, most organisms were not ingested by human polymorphonuclear neutrophils in the presence of normal rabbit serum (NRS), while organisms were primarily cell associated and apparently ingested in the presence of immunized rabbit serum (IRS). The capsule may be one of the virulence factors in this bacteria. This study demonstrates the possible presence of a capsule in Branhamella catarrhalis. PMID- 1719359 TI - Intractable chronic lymphocytic leukemia and interferon. AB - The hypothesis that interferon may be useful for intractable chronic lymphocytic leukemia (CLL) is based on two incidental findings: first, the similarity in the in vitro radioresistance of the blood lymphocytes of intractable CLL and of hairy cells; secondly, the similarity of the incidence of intractable CLL patients and the incidence of CLL patients who respond to interferon treatment. PMID- 1719360 TI - Palliative care in a general teaching hospital. 2. Establishment of a service. AB - OBJECTIVE: To assess the first year of operation of a palliative care service in a general teaching hospital. DESIGN, SETTING, PATIENTS: A retrospective analysis of 241 terminally ill patients referred to the Austin Hospital Palliative Care Service during its first year of operation. MAIN OUTCOME MEASURES: The occurrence and relief of pain, the occurrence and management of social and psychological problems, involvement of allied health services and the place of death. MAIN RESULTS: Unrelieved pain was the most frequent medical problem but, with appropriate medical management as well as input from other members of the multidisciplinary team, all patients achieved satisfactory pain control. The occurrence and severity of social and psychological problems was greater than expected. Most of the patients were discharged home and referred to domiciliary palliative care services; one-quarter of patients were able to die at home, one third died in an inpatient hospice, and the remainder died in hospital. CONCLUSIONS: The Service was able to achieve most of its stated aims and has generated a much improved appreciation of the palliative care needs of patients with terminal illness and their families within the Hospital. A multidisciplinary approach to the management of terminally ill patients is crucial, and this Service has been successful despite limited staffing because of the willing involvement of other Units and Services in the Hospital. The difficulties inherent in the evaluation of a palliative care program (quality of life, cost effectiveness) are discussed. PMID- 1719361 TI - Palliative care in a general teaching hospital. 1. Assessment of needs. AB - OBJECTIVE: To assess the palliative care needs and the results of treatment of patients with terminal cancer admitted to a general teaching hospital. DESIGN, SETTING, PATIENTS: A retrospective analysis of 110 consecutive patients with terminal cancer admitted to the Austin Hospital. MAIN OUTCOME MEASURES: The occurrence and relief of pain, the use of allied health services and the place of death. MAIN RESULTS: Pain was the most common symptom and was satisfactorily improved in only two-thirds of the patients. Allied health services were used sporadically and appeared to be underused. Psychological problems were documented in very few patients. Only seven patients died at home, the remainder dying in hospital (82) or in a hospice (21). CONCLUSIONS: One-third of patients with terminal cancer in a general teaching hospital received inadequate pain relief; the reasons for this included lack of medical expertise in the use of analgesics for chronic cancer pain and the frequent use of analgesia given only "as required". The underuse of allied health services, the infrequent documentation of psychological issues and the observation that only a small proportion of patients were able to die outside hospital all underline the need for a coordinated multidisciplinary approach to the management of patients with terminal cancer. PMID- 1719363 TI - A patient's right to a good death. PMID- 1719362 TI - Legal aspects of the management of chronic pain. AB - OBJECTIVE: To review the legal provisions which control the prescription of opioid analgesics in Australia, and to summarise the areas in which practitioners who treat patients with chronic pain may expect to become involved with the legal system. DATA SOURCES: The relevant legislation was reviewed, and a selective review was undertaken of literature dealing with the legal aspects of pain and suffering which may form a basis for personal injury claims. Case law which deals with issues of consent to treatment was also examined. DATA SYNTHESIS: Statutory requirements which control the prescription of opioids were summarised. Leading cases on patient consent were discussed to clarify for the practitioner the principles which the Courts use in the assessment of the validity of the consent given by patients for treatment. The assessment of the pain patient involved in litigation was briefly discussed. CONCLUSIONS: The prescription and administration of opioid analgesics must be in accordance with the legislative provisions. Treatment options must be discussed and explained to patients so that valid consent can be obtained. Patients' questions must be answered in full, and documentation in the clinical record is required. PMID- 1719364 TI - [Serum pancreatic enzymes and fecal chymotrypsin before and after glyco-metabolic control in diabetic patients]. AB - The aim of the present study was to ascertain whether secretory capacity of the pancreas, evaluated by assaying serum total amylase (TA) and pancreatic amylase activity (PA) and fecal chymotrypsin excretion (FCT), is impaired in diabetic and to what extent it is influenced by the degree of glyco-metabolic control. TA, PA and FCT were assayed in 40 patients affected with type II diabetes mellitus in secondary insufficiency, both at hospitalization and after metabolic control assessment; 43 hospitalized patients constituted the control group. A statistically significant difference was found between the metabolic failure phase values of diabetics patients and those of the control group for TA (p less than 0.0005), PA (p less than 0.025) and FCT (p less than 0.0005); between metabolic control phase values and control group fo TA (p less than 0.0005), and FCT (p less than 0.005) but not for PA values. PA values were statistically significant, within diabetic group, before and after metabolic control assessment. A statistically significant result was obtained by correlating C peptide and FCT values (p less than 0.01), C-peptide and PA values (p less than 0.001); glycaemia and PA values (p less than 0.05). Our data suggest that in diabetic patients there is an impairment in secretory capacity of the pancreas and that the the PA is the more sensitive enzyme to the local levels of insulin. PMID- 1719365 TI - [Monoclonal antibody MIA15-5, which inhibits cell motility and metastatic potential, has a great effect on the prognosis of the post-surgical patients with lung cancer]. AB - The functional monoclonal antibody, selected based on the inhibition of cell motility, was found to be directed to specific carbohydrate structure Fuc alpha 1 ---2 Gal beta 1----R. This monoclonal antibody, MIA15-5 was established after immunization of mice with adenocarcinoma line of the lung PC7, and selected based on inhibition of U937, HEL, and MAC10 cell migration due to the transwell assay. MIA15-5 reacted with 30-40% of high metastatic variant BL6 of mouse melanoma B16 line and metastatic deposition to lung after injection of BL6 cells was strongly inhibited if MIA15-5 was injected within 3 hours, but was not inhibited by injection of other anti-H antibodies under the same conditions. Injection of MIA15-5 had much less inhibitory effect if administered 1 day after BL-6 injection, and no effect at 3 days. In immunohistochemical staining of 149 formalin fixed specimens of patients with lung cancer, especially adenocarcinoma has the highly frequency of MIA15-5 (65.4%, 49 of 75 cases) and the five year survival rate of the patients with negative staining of MIA15-5 was 58.6% and much better than one with positive staining, 20.9%. PMID- 1719366 TI - Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12. AB - The nucleotide sequence of the Escherichia coli K12 beta-methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome. PMID- 1719367 TI - Characterisation of saporin genes: in vitro expression and ribosome inactivation. AB - A. Saponaria (soapwort) genomic library was screened with a PCR-derived saporin specific gene probe. The nucleotide sequences of three saporin genomic clones were determined. One of the clones contained a full-length saporin coding sequence whilst the other two were truncated. A hybrid full-length saporin coding sequence was constructed using the two truncated clones. An SP6 promoter sequence and in-frame initiation codon was added to each of the coding sequences using PCR. In vitro translation of saporin coding sequence transcripts in rabbit reticulocyte lysates resulted in the specific depurination of 28S RNA. This indicated that the saporin sequences encoded functional polypeptides with RNA N glycosidase activity. PMID- 1719368 TI - Different modes of regulation for receptors activating phospholipase C in the rat pancreatoma cell line AR4-2J. AB - The inositol phosphate responses to substance P, bombesin, cholecystokinin, and the muscarinic cholinergic agonist methacholine were examined in the rat pancreatoma cell line AR4-2J. It was found that each agonist produced a distinct temporal pattern of inositol phosphate formation. Furthermore, these different response patterns resulted, at least in part, from different patterns of homologous receptor desensitization. The response to substance P desensitized rapidly and completely within 90 sec. After a 10-15-min refractory period, the response recovered with a t1/2 of approximately 1 hr. The response to methacholine also completely desensitized. However, in this case desensitization developed slowly over the course of 40 min, and no recovery of responsiveness was detected for up to 45 min after the cessation of stimulation. The inositol phosphate responses to bombesin and cholecystokinin were similar to one another and appeared to be composed of two phases. Initially, there was a robust activation of phospholipase C. This initial phase was followed within 20 sec by a second phase of lesser magnitude. For bombesin, attenuation of the initial phase was due to rapid, but only partial, desensitization of the response. Furthermore, the concentration of bombesin required to maintain the second phase of the response was about 100-fold lower than that required to maximally activate the initial phase of the response. These results may indicate multiple mechanisms for the regulation of different phospholipase C-linked receptors in this cell line. PMID- 1719369 TI - Pharmacological, radioligand binding, and electrophysiological characteristics of FPL 64176, a novel nondihydropyridine Ca2+ channel activator, in cardiac and vascular preparations. AB - The pharmacological, radioligand binding, and electrophysiological properties of FPL 64176, a new nondihydropyridine Ca2+ channel activator, were studied in rat tail artery, cardiac membranes, and A7r5 smooth muscle cells. FPL 64176 induced a contractile response, with an EC50 value of 2.11 x 10(-7) M. The maximum tension response to FPL 64176 was approximately 2-fold higher than that to (S)-Bay K 8644. FPL 64176 showed no significant inhibitory activity at concentrations up to 10(-5) M. The Ca2+ channel antagonists nifedipine, verapamil and diltiazem noncompetitively antagonized and completely relaxed the responses induced by FPL 64176. IC50 values of these three drugs were 5.22 x 10(-9), 1.31 x 10(-7), and 1.95 x 10(-7) M, respectively, for relaxing submaximum contractile responses to FPL 64176 (5 x 10(-7) M). The washout time for FPL 64176 was about 40 min, which was much longer than that for (S)-Bay K 8644 (within 1 min). FPL 64176 weakly inhibited (+)-[3H]PN 200-110, [3H]D888, and [3H]TA-3090 binding in rat cardiac membranes, with IC50 values of 1.04 x 10(-5) M and 7.03 x 10(-6) M for inhibition of (+)-[3H]PN 200-110 and [3H]TA-3090 binding, respectively, and with 23% inhibition of [3H]D888 binding at a FPL 64176 concentration of 1 x 10(-5) M. Dissociation kinetics of the three radioligands were allosterically accelerated by FPL 64176. Electrophysiological studies on the A7r5 smooth muscle cell line directly confirmed a large (approximately 14-fold) stimulatory effect on L-type Ca2+ current amplitude. The results suggest that FPL 64176 is a new type of Ca2+ channel activator with higher efficacy and a mechanism and site of action that are distinct from those for (S)-Bay K 8644. PMID- 1719370 TI - Mechanisms of sensitivity and natural resistance to antifolates in a methylcholanthrene-induced rat sarcoma. AB - A methylcholanthrene-induced rat sarcoma that can be propagated in vitro or in vivo was evaluated for resistance to antifolates and was found to be relatively resistant to methotrexate and 10-ethyl-10-deazaaminopterin but sensitive to trimetrexate. Rat sarcoma cell extracts contained low levels of dihydrofolate reductase activity, the target enzyme of methotrexate, and inhibition of this enzyme by these three antifolates was similar. Transport studies showed poor uptake of both methotrexate and 10-ethyl-10-deazaaminopterin. In contrast, trimetrexate achieved high intracellular levels. The poor uptake of methotrexate was not due to lack of polyglutamylation. Thus, the basis for natural resistance to methotrexate and 10-ethyl-10-deazaaminopterin, compared with trimetrexate, in this rat sarcoma cell line was due to decreased transport of these drugs. PMID- 1719371 TI - [Cloning preprolactin cDNA from Pacific salmon in Escherichia coli]. AB - Prolactin coding mRNA was shown to be a prevalent part of chum salmon (Oncorhynchus keta) pituitary poly(A)-RNA during the spawning period. Clone lambda gtPrk12 was selected from the pituitary cDNA library by means of hybridization with the prolactin probe, and a nucleotide sequence of the insertion was determined and compared to the prolactin coding sequences from rainbow trout and Pacific chinook salmon, which had been published earlier. The sequences compared exhibited a significant homology. The deduced amino acid sequence of the chum salmon prolactin differed from a sequence determined directly in a single position. The prolactin-coding sequence can be used for constructing the bacterial strain producing prolactin. PMID- 1719372 TI - Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527). AB - Repression of the tyrosine kinase activity of the cellular src protein (pp60c src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr 527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed. PMID- 1719373 TI - Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation. AB - Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined. PMID- 1719374 TI - A position-dependent silencer plays a major role in repressing alpha-fetoprotein expression in human hepatoma. AB - A large percentage of human hepatomas produce alpha-fetoprotein (AFP), but the levels of AFP expression vary greatly among hepatomas. To understand the molecular basis for this variation, we analyzed transcriptional regulatory activities associated with the 5'-flanking region of the AFP gene in two human hepatoma cell lines, HuH-7 and huH-1/cl-2, which produce a high and a low level of AFP, respectively. We found that the low level of AFP production in huH-1/cl-2 is due to the action of at least two silencer regions located between the enhancer and the promoter of the AFP gene. In contrast, no silencer activity is expressed in HuH-7. We identified 5'-CTTCATAACTAATACTT-3' to be a core sequence responsible for the negative regulatory activity. This sequence is repeated four times in a strong, distal silencer region, Sd, whereas one copy is present in a weak, proximal silencer region, Sp. The silencer reduces transcriptional initiation by blocking enhancer activation of the AFP promoter in a position dependent manner. The silencer functions in the presence of positive transcription factors and may play a key role in developmental repression as well as variable expression of the AFP gene in hepatomas. PMID- 1719375 TI - tpr-met oncogene product induces maturation-producing factor activation in Xenopus oocytes. AB - tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway. PMID- 1719376 TI - RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B. AB - Ribosome binding to mRNA requires the concerted action of three initiation factors, eIF-4A, eIF-4B, and eIF-4F, and the hydrolysis of ATP in a mechanism that is not well understood. Several lines of evidence support a model by which these factors bind to the 5' end of mRNA and unwind proximal secondary structure, thus allowing 40S ribosomal subunits to bind. We have previously used an unwinding assay to demonstrate that eIF-4A or eIF-4F in combination with eIF-4B functions as an RNA helicase. To elucidate the molecular mechanism of RNA unwinding, we used a mobility shift electrophoresis assay which allows the simultaneous analysis of unwinding and complex formation between these factors and RNA. eIF-4F forms a stable complex (complex A) with duplex RNA in the absence of ATP. Addition of eIF-4B results in the formation of a second complex (complex B) of slower mobility in the gel. In the presence of ATP, both complexes dissociate, concomitant with the unwinding of the duplex RNA. We present evidence to suggest that unwinding occurs in a processive as opposed to distributive manner. Thus, we conclude that helicase complexes that are formed in the absence of ATP on duplex RNA translocate processively along the RNA in an ATP-dependent reaction and melt secondary structure. These helicase complexes therefore represent intermediates in the unwinding process of mRNA that could precede ribosome binding. PMID- 1719377 TI - Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro. AB - The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation. PMID- 1719378 TI - Complex transcriptional regulation of myc family gene expression in the developing mouse brain and liver. AB - myc family genes (c-, N-, and L-myc) have been shown to be differentially expressed with respect to tissue type and developmental stage. To define and compare the regulatory mechanisms governing their differential developmental expression, we examined the transcriptional regulation of each myc family member during murine postnatal brain and liver development. Nuclear run-on transcription assays demonstrated that both the rate of transcriptional initiation and the degree of transcriptional blocking contribute in a complex manner to the regulation of all three genes. During postnatal brain development, the relative contribution of each transcriptional control mechanism to the regulation of myc family gene expression was found to be different for each gene. For instance, while modulation of transcriptional attenuation did not appear to contribute to the down-regulation of L-myc expression, attenuation was found to be the dominant mechanism by which steady-state N-myc mRNA levels were down-regulated. Different transcriptional strategies were found to be employed in newborn versus adult developing liver for repression of N- and L-myc expression. Undetectable steady state N- and L-myc mRNA levels in newborn liver were associated with a very low rate of transcriptional initiation, whereas the lack of N- and L-myc expression at the adult stage was accompanied by a high rate of initiation and a striking degree of transcriptional attenuation. Transcriptional attenuation in the N-myc gene was found to map to a region encoding a potential stem-loop structure followed by a thymine tract within the first exon and was not dependent on the use of a specific transcriptional start site. PMID- 1719379 TI - A human alpha-fetoprotein enhancer-binding protein, ATBF1, contains four homeodomains and seventeen zinc fingers. AB - We have isolated a full-length cDNA encoding a protein (ATBF1) that binds to an AT-rich motif in the human alpha-fetoprotein gene enhancer. The amino acid sequence deduced from the cDNA revealed that this is the largest DNA-binding protein (306 kDa) known to date, containing four homeodomains, 17 zinc finger motifs, and a number of segments potentially involved in transcriptional regulation. Although the exact function of this protein has not been determined, these structural features suggest that ATBF1 plays a transcriptional regulatory role. PMID- 1719380 TI - A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi. AB - A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons. PMID- 1719381 TI - Small differences in Drosophila tropomyosin expression have significant effects on muscle function. AB - The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function. PMID- 1719382 TI - Differential effect of 5 alpha-reductase inhibition and castration on androgen regulated gene expression in rat prostate. AB - Castration reduces prostate size and causes intraprostatic testosterone (T) and dihydrotestosterone (DHT) to fall to very low levels. 5 alpha-Reductase inhibition also reduces prostate size, but results in a marked increase in intraprostatic T levels. To compare the effects of 5 alpha-reductase inhibition and castration on prostate physiology, male Sprague-Dawley rats were left intact, castrated, or given the selective 5 alpha-reductase inhibitor finasteride for up to 9 days. To be sure that finasteride itself did not directly affect gene expression, an additional group of rats was castrated and given finasteride for 4 days. The prostates were weighed, intraprostatic RNA, DNA, and androgen levels were measured, and mRNAs for two androgen-regulated genes, prostate steroid binding protein (PSBP; an androgen-induced gene) and testosterone-repressed prostate message (TRPM-2), were quantitated by Northern and slot blot analyses. Finasteride caused a 95% reduction in intraprostatic DHT levels and a 10-fold increase in intraprostatic T levels. Finasteride, as expected, caused a pronounced decrease in prostate weight (45% on day 4). DNA content fell correspondingly (48% on day 4). Intraprostatic DNA (micrograms of DNA per gland) on day 4 was 328 +/- 53 in control rats, 171 +/- 10 in finasteride-treated rats (P less than 0.001 compared to controls), 115 +/- 2 in castrated rats (P less than 0.05 compared to finasteride), and 107 +/- 43 in finasteride-treated plus castrated rats (P = NS compared to castration alone). There were no significant differences in DNA levels among the groups when expressed per mg prostate tissue, indicating that mean prostate cell size was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719383 TI - Isolation and molecular cloning of insulin-like growth factor-binding protein-6. AB - Insulin-like growth factors (IGFs) together with their binding proteins (BPs) are potential regulators of folliculogenesis in mammalian ovary. To identify the various species of IGFBPs present in the ovary, we have undertaken a comprehensive purification scheme using gel filtration, ligand-affinity chromatography, and several steps of reverse phase HPLC to isolate all of the BPs in pig ovarian follicular fluid. Our effort yielded five distinct IGFBPs, and upon analysis, they were found to correspond to the previously identified human and rat IGFBP-2, -3, -4, -5, and -6. IGFBP-1 was not found in the pig ovarian follicular fluid under our experimental procedure. Of the six known classes of IGFBPs, the complete primary structures of the first five have been determined, but not IGFBP-6. Using amino acid sequence information from a tryptic fragment of pig IGFBP-6 to prepare a probe, cDNA clones encoding rat and human IGFBP-6 have been isolated and characterized. The deduced amino acid sequence revealed that rat IGFBP-6 contains 201 amino acids with a calculated mol wt of 21,461, while the human homolog contains 216 amino acids with a calculated mol wt of 22,847. In addition, a distinctive feature of human and rat IGFBP-6 is that they lack, respectively, two and four of the 18 homologous cysteines that are present in all other five IGFBPs. The missing cysteines in IGFBP-6 resulted in the absence of the invariant Gly-Cys-Gly-Cys-Cys sequence in the amino-terminal region of the molecule. Human IGFBP-6 possesses a single Asn-linked glycosylation site near the carboxyl-terminal, whereas no potential Asn-linked glycosylation sites are present in the rat sequence. A single 1.3-kilobase IGFBP-6 mRNA was detected by Northern analysis in all rat tissues examined, including testis, intestine, adrenal, kidney, stomach, spleen, heart, lung, brain, and liver, indicating that this BP is a ubiquitous protein. The chromosome location of the IGFBP-6 gene in human has been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that it is located on chromosome 12. PMID- 1719384 TI - Mutations in the C-terminal part of insulin-like growth factor (IGF)-binding protein-1 result in dimer formation and loss of IGF binding capacity. AB - In an attempt to define domains in insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) that are involved in IGF binding, we subjected the carboxyl end of the coding region of IGFBP-1 cDNA to mutagenesis. Mutant cDNAs were isolated, characterized by sequencing, and cloned in an expression vector under control of the simian virus-40 (SV40) early promoter. The constructs were transfected into COS-1 cells, and the mutant proteins, secreted into the culture medium, were analyzed for IGF binding by ligand blotting. The results obtained show that deletion of the C-terminal 20 amino acids or introduction of frame shifts in this region resulted in loss of IGF binding and for some mutants in the formation of dimeric IGFBP-1 molecules. These dimers are probably formed when cysteine-226 (Cys-226) is missing, and its putative partner is able to form intermolecular disulfide bonds. Site-directed mutagenesis demonstrated that most of the introduced point mutations in the C-terminal region did not affect IGF binding. Only mutation of Cys-226 to tyrosine completely abolished IGF binding, as did the introduction of a negatively charged amino acid in the vicinity of this residue. Again, dimers were observed, supporting that Cys-226 is essential for the conformation of IGFBP-1. In addition, our data suggest that an IGF binding domain may be located in the vicinity of the intramolecular disulfide bond formed by Cys-226 and its putative partner. PMID- 1719385 TI - Insulin-like effects of inositol phosphate-glycan on messenger RNA expression in rat hepatocytes. AB - The ability of an inositol phosphate-glycan (IPG) to mimic the effects of insulin on regulation of the expression of specific mRNAs was studied in isolated hepatocytes from normal and diabetic rats. Incubation of normal liver cells with IPG (10 microM) during 90 min produced a 5-fold decrease in phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels, which had been previously increased about 10 fold by incubation with 8-bromo-cAMP (0.1 mM). The effect of IPG was dose dependent and could not be reproduced by galactose, glucosamine, or myo-inositol. IPG reduction of PEPCK mRNA is primarily due to a decrease in the rate of transcription of the gene, as judged by nuclear run-on transcription experiments performed in rat hepatoma H4IIE cells. In hepatocytes isolated from diabetic rats, treatment with 5 microM IPG for 15 min caused a 4-fold induction in the expression of alpha 2-microglobulin mRNA concomitantly with a 2.5-fold decrease in the level of PEPCK mRNA. Cleavage of IPG with nitrous acid abolished both the increase and the decrease in specific mRNAs levels. Glycosyl phosphatidylinositol, the lipid precursor of IPG, did not modify either PEPCK or alpha 2-microglobulin mRNA levels. These data indicate that both positive and negative effects of insulin on the regulation of gene expression are mimicked by IPG. PMID- 1719386 TI - Insulin rapidly inhibits insulin-like growth factor-binding protein-1 gene expression in H4-II-E rat hepatoma cells. AB - The insulin-like growth factor-binding proteins (IGFBPs) are thought to determine the distribution of IGF-I and IGF-II between the blood and tissue compartments and to modulate their biological activities. A dynamic metabolic role for one of the IGFBPs, IGFBP-1, is suggested by the fact that plasma IGFBP-1 was increased after fasting and diabetes and rapidly decreased by refeeding or insulin treatment, respectively. IGFBP-1 mRNA also is increased in the livers of diabetic rats and decreased by insulin treatment. To understand the molecular basis for this regulation, we have examined the effects of insulin on IGFBP-1 and IGFBP-1 mRNA in the H4-II-E cell line derived from the well differentiated H35 rat hepatoma. IGFBP-1, identified by ligand blotting and immunoblotting, is the major IGFBP in H4-II-E cells. Incubation of H4-II-E cells with insulin for 24 h decreased IGFBP-1 in the culture medium by approximately 50%. Inhibition was observed at physiological concentrations of insulin (ED50, less than 0.5 nM), but not at higher concentrations of IGF-II. These results, together with the fact that H4-II-E cells do not possess IGF-I receptors with which insulin might cross react, suggest that insulin acts via the insulin receptor. Insulin inhibited IGFBP-1 in the medium by 80% in the absence of glucose, suggesting that the inhibition is a direct effect of insulin; glucose exerted a smaller independent effect in the absence of insulin. Insulin decreased IGFBP-1 mRNA in H4-II-E cells by 50% within 1 h and by 90% after 2-12 h of incubation. Nuclear run-on transcription assays indicated a corresponding decrease in the rate of IGFBP-1 gene transcription. Pretreatment of H4-II-E cells with dexamethasone stimulated IGFBP-1 transcription and increased steady state IGFBP-1 mRNA; stimulation was abolished by insulin treatment, indicating that inhibition by insulin was dominant over induction by dexamethasone. Thus, insulin, acting through the insulin receptor, rapidly decreases the abundance of IGFBP-1 mRNA in H4-II-E cells. Regulation occurs at least in part at the level of gene transcription. We propose that regulation of IGFBP-1 synthesis is an important component of the regulation of IGFBP-1 by insulin in vivo. PMID- 1719387 TI - [Study of the antigenic structure of the E1 glycoprotein of the Venezuelan equine encephalomyelitis virus using monoclonal antibodies]. AB - The collection of eight rat and mouse hybridomas secreting the high affinity monoclonal antibodies to glycoprotein E1 of the Venezuelan equine encephalomyelitis has been obtained. The antigenic structure of E1 protein has been studied with the use of these antibodies for the strains Trinidad, TC-83 and 230 of the virus. Antigenic map of glycoprotein E1 based on competition radioimmunoanalysis is proposed. Five sites are mapped including eight epitopes binding monoclonal antibodies. Antibodies to sites E1-1, E1-3 and E1-5 are crossreactive in interaction with the virus of Venezuelan equine encephalomyelitis, while antibodies to site E1-5 interact also with the virus of tick-borne encephalitis. Antibodies to site E1-1 possess the protective effect and lack the neutralizing effect in tissue cultures. Antibodies to all sites of E1 protein are devoid of ability to neutralize the Venezuelan equine encephalitis virus. PMID- 1719388 TI - [Cycle dependent expression of the gene coding for B2mRNA]. AB - The relative contents of individual tissue specific RNA B2mRNAx was studied in the population of nuclear and poly(A)+ cytoplasmic RNA from the livers of intact, falsely operated and having suffered the partial liver resection rats. Dot hybridization technique was used to study this transcript containing the transcribed copy of the B2 repetitive genetic element of rats. The expression of the gene coding for B2mRNAx takes place at the lower level in the liver induced to proliferation as compared with the one in the liver of intact animals. It is changed reproducibly in antiphase with the c-fos RNA and with major inclinations at the moments of cellular phases switch. PMID- 1719389 TI - The non-genotoxicity to rodents of the potent rodent bladder carcinogens o anisidine and p-cresidine. AB - The two potent rodent bladder carcinogens o-anisidine and p-cresidine, and the structurally related non-carcinogen 2,4-dimethoxyaniline, have been extensively evaluated for genotoxicity to rodents and found to be inactive. Most data were generated on o-anisidine, an agent that is also only marginally genotoxic in vitro. The two carcinogens induced methaemoglobinaemia in rodents indicating that the chemicals are absorbed and metabolically oxidized. Despite their total lack of genotoxicity in vivo, the two carcinogens have the hall-marks of being genotoxic carcinogens given that most test animals of both sexes of B6C3F1 mice and F344 rats are reported to have succumbed rapidly to malignant bladder cancer. No reasons for this dramatic conflict of test data are so far apparent. The experiments described involve, in one or other combination, 2 strains of mice (including B6C3F1) and 4 strains of rat (including F344), the use of oral and i.p. routes of exposure and observations made after 1, 3 or 6 doses of test chemical. 6 tissues (including the rat bladder) were assayed using 3 genetic endpoints (unscheduled DNA synthesis, DNA single-strand breaks and micronuclei induction). Aroclor-induced rats were employed in one set of experiments with o anisidine. In the case of one set of mouse bone-marrow micronucleus experiments the same batch of the 3 chemicals as used in the cancer bioassays, and the same strain of mouse, were used. Possible further experiments and the implications of these findings are discussed. PMID- 1719390 TI - Biological and chemical monitoring of occupational exposure to ethylene oxide. AB - Studies were carried out on two populations occupationally exposed to ethylene oxide (EtO) using different physical and biological parameters. Blood samples were collected from 9 hospital workers (EI) and 15 factory workers (EII) engaged in sterilization of medical equipment with EtO and from matched controls (CI and CII). Average exposure levels during 4 months (the lifespan of erythrocytes) prior to blood sampling were estimated from levels of N-(2-hydroxyethyl)valine adducts in hemoglobin. They were significantly enhanced in EI and EII and corresponded to a 40-h time-weighted average of 0.025 ppm in EI and 5 ppm in EII. Exposures were usually received in bursts with EtO concentrations in air ranging from 22 to 72 ppm in EI and 14 to 400 ppm in EII. All samples were analyzed for HPRT mutants (MFs), chromosomal aberrations (CAs), micronuclei (MN) and SCEs. MFs were significantly enhanced by 60% in EII but not in EI. These results are the first demonstration of mutation induction in man by ethylene oxide. CAs were significantly enhanced in EI and EII by 130% and 260% respectively. MN were not enhanced in EI but significantly in EII(217%). The mean frequency of SCEs was significantly elevated by 20% in EI and by almost 100% in EII. SCE was the only parameter that allowed distinction between daily and occasionally exposed workers in EII. An interesting finding in exposed workers was the large increase of the percentage of cells with high frequencies of SCE (3-4 times in EI and 17-fold in EII). The relative sensitivity of endpoints for detection of EtO exposure in the present investigation was in the following order: HOEtVal adducts greater than SCEs greater than chromosomal aberrations greater than micronuclei greater than HPRT mutants. PMID- 1719391 TI - Protective effects of sodium selenite on killing and mutation by N-methyl-N' nitro-N-nitrosoguanidine in E. coli. AB - Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU). The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA. A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU. Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection. PMID- 1719392 TI - The induction and repair of (6-4) photoproducts in Neurospora crassa. AB - The (6-4) photoproduct lesion found in DNA after UV irradiation is repaired by germinating Neurospora crassa conidia. Wild-type Neurospora removes 80% of the (6 4) photoproduct in approximately 20 min and maximal repair is accomplished by 30 min with approximately 89% of the original lesions removed. Mutagen-sensitive Neurospora mutants belonging to the established excision repair epistasis group, UVS-2, are not defective in the removal of cyclobutane pyrimidine dimers. Furthermore, we find these mutants capable of removing (6-4) photoproducts from their DNA at a rate similar to wild type. Comparable kinetics are also observed in key members of the other two epistasis groups. PMID- 1719393 TI - Elevated levels of DNA double-strand breaks (dsb) in restriction endonuclease treated xrs5 cells correlate with the reduced capacity to repair dsb. AB - Recently we have reported the kinetics of DNA double-strand breaks (dsb) induced in electroporated mammalian (CHO) cells that had been treated with the restriction endonuclease PvuII, as measured by the filter elution assay at the non-denaturing pH of 9.6. A gradual accumulation of dsb was observed over a 24-h incubation period following the restriction endonuclease (RE) treatment and this was attributed to a competition between incision of the DNA by PvuII and dsb repair. In order to test this 'competition' hypothesis we have carried out similar experiments in the radiosensitive xrs5 mutant cell line, which has been shown to be deficient in dsb repair. The levels of dsb monitored by the non denaturing filter elution assay in the xrs5 cell line treated with PvuII was found to be 3-4 times higher than that found for the wild-type CHO K1 cell line. Levels of dsb were also significantly raised in xrs5 cells treated with BamHI, as compared with the background levels observed in the CHO line. These data lend strong support to the competition hypothesis of simultaneous incision and repair of RE-induced dsb. PMID- 1719394 TI - The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks. AB - The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents. This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent. A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR). The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents. There were no significant differences in the binding of these agents to the DNA of WS or WR cells. All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks. WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells. Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink. Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic. Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants. The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells. In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells. The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair. PMID- 1719395 TI - DNA repair and mutant frequency in schizophrenia. AB - The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes was determined with a cloning assay. T-lymphocytes were obtained from 14 individuals diagnosed with schizophrenia and 5 controls. No significant difference in mutant frequency was observed between the 2 groups. In addition, DNA-repair capacity was measured with the unscheduled DNA synthesis technique in lymphocytes from 7 individuals diagnosed with schizophrenia and 7 controls. Repair capacity was determined following treatment with MMS, MNNG, and 20 J/m2 ultraviolet light. No significant differences in DNA repair were observed between the patient and control groups in response to any of the 3 DNA-damaging agents. These results argue against differences between normal and schizophrenic individuals with respect to in vivo mutant frequency or their capacity to repair DNA lesions induced by MMS, MNNG, or ultraviolet radiation. PMID- 1719396 TI - Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene. AB - Alkylating agent damage was quantified in human T-lymphocytes by calculating gene specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0, 6, and 24 h), T-lymphocyte DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N methylpurines. The methoxyamine-treated aliquot provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt greater than dhfr greater than dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene than in the inactive X-linked dmd gene. The lesion frequency found in the male's lone hprt allele was the highest observed. These lesion frequency differences are discussed in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes. PMID- 1719397 TI - Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells. AB - The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences. PMID- 1719398 TI - Cross-adaptive response in Escherichia coli caused by pretreatment with H2O2 against formaldehyde and other aldehyde compounds. AB - A cross-adaptive response (CAR), defined as a reduction of the effects of an agent by pretreatment with another agent, was demonstrated when E. coli WP2 cells were pretreated with hydrogen peroxide (H2O2) followed by challenging treatment with aldehyde compounds. Pretreatment with a sublethal dose (60 microM) of H2O2 for 30 min made WP2 cells resistant to the killing effects of formaldehyde (FA), and 4 other mutagenic aldehydes: glutaraldehyde, glyoxal, methyl glyoxal and chloroacetaldehyde. CAR was also observed in WP2uvrA (uvrA-) and ZA12 (umuC-) cells, but not in ZA60 (recA-) and CM561 (lexA- (Ind-] cells. A role of recA and lexA in CAR was further suggested by the lack of beta-galactosidase induction in recA- and lexA- cells by H2O2. CAR and beta-galactosidase induction, however, were found to be separate events since CAR was recovered by introducing the recA+ gene into lexA- cells, but no induction of beta-galactosidase by H2O2 was observed in cells with the same gene transfer. These results suggest that H2O2 has the capacity to induce a function which reduces the killing effects of aldehydes, and the function is controlled by the recA gene without involvement of SOS response. PMID- 1719399 TI - Enhancement of repair of UV-irradiated plasmids in cultured fish cells by fluorescent light preillumination and growth arrest. AB - The UV-irradiated plasmid pBSCATSV, which could express chloramphenicol acetyltransferase (CAT) in the presence of SV40 early promoter, was transfected into RBCF-1 cells derived from the goldfish (Carassius auratus). The cells were incubated in the dark for 24 h and then the CAT activity was measured. CAT expression relative to non-irradiated control was calculated. The CAT expression of the exponentially growing cells transfected with UV-irradiated plasmid was enhanced by fluorescent light (FL) preillumination of the cells 8 h before transfection. The efficiency of photorepair (PR) measured by CAT expression was also enhanced by the same FL preillumination. This suggests that FL preillumination enhances both photorepair and dark repair of RBCF-1 cells for UV damaged plasmid transfected into the cells. The enhancement of repair of UV damage by FL preillumination was also observed in survival assays. When the UV irradiated pBSCATSV was transfected into growth-arrested cells in confluent culture, CAT expression was less sensitive to UV irradiation, and FL preillumination was much less effective in enhancing photorepair and dark repair. PMID- 1719400 TI - Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells. AB - Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive diseases with clinical and cellular hypersensitivity to UV radiation, differ in ability to repair UV DNA photoproducts in their overall genome: normal repair in CS, defective repair in XP. In order to characterize a DNA repair defect in an active gene in CS, we measured the capacity of cells from patients with CS and XP to reactivate 2 major types of UV-induced DNA damage, photoreactivatable (i.e., cyclobutane pyrimidine dimers) and non-photoreactivatable (primarily pyrimidine (6-4)pyrimidone photoproducts), in the actively transcribing chloramphenicol acetyltransferase (cat) gene of the plasmid expression vector pRSV-cat. Epstein Barr virus-transformed lymphoblast lines from 4 normal persons and from 3 patients with CS and from two with XP were transiently transfected with the plasmid, and the cat activity in cell extracts was determined. When the cells were transfected with UV-irradiated plasmid, expression was abnormally decreased in both the CS and XP cells. When the cyclobutane pyrimidine dimers in the UV irradiated plasmid were removed by photoreactivation prior to transfection, cat expression in the CS, but not in the XP, lines reached normal levels. These data imply that both the XP and CS cells are unable to repair normally the cyclobutane pyrimidine dimer photoproducts which block transcription of cat. However, the CS, but not XP, cells can repair normally the other UV-induced photoproducts which block transcription. The ability of CS, but not XP, cells to repair these non dimer photoproducts indicates that the active gene repair mechanism treats the cyclobutane pyrimidine dimer differently from the non-dimer photoproducts. PMID- 1719401 TI - Biochemical mechanisms by which reutilization of DNA 5-methylcytosine is prevented in human cells. AB - The salvage metabolism of 5-methyldeoxycytidine 5'-monophosphate (5MedCMP) was studied in human promyelocytic leukemia (HL-60) cells and in PHA-stimulated human lymphocytes. To this end [5'-32P]5MedCMP was synthesized by a novel postlabeling procedure. At low substrate concentrations (less than 100 microM), the enzyme(s) present in crude HL-60 whole-cell extract deaminated 5MedCMP faster than they did dCMP. Although the phosphorylation of dCMP to dCDP was easily demonstrable with both kinds of cell extracts, no phosphorylation of 5MedCMP to 5MedCDP (5 methyldeoxycytidine 5'-diphosphate) was observed. This phenomenon was confirmed using HL-60 cells made permeable to nucleotides with Tween 80. In view of the substantial 5MeCyt (5-methylcytosine) content of DNA and the degradation of DNA that occurs in cells, it is conceivable that 5MedCyd (5-methyl-2'-deoxycytidine) and 5MedCMP are available for reutilization in DNA synthesis. This would have devastating effects on cellular control and gene expression. The results of the present investigation indicate that rapid deamination at the monophosphate level and, in particular, stringent discrimination of 5MedCMP by cellular monophosphokinase(s) are the key mechanisms by which reutilization of DNA 5MeCyt is prevented in human hematopoietic cells. PMID- 1719402 TI - The genetic toxicology of ortho-toluidine. AB - ortho-Toluidine, a monosubstituted aniline and an intermediate in the dyeing industry, with a number of uses in other fields such as rubber processing and pharmaceutical production, has been in production for over 100 years. It is metabolised in vivo into a number of compounds, some of which are active genotoxins. It has been demonstrated to be a carcinogen in mice and rats and is a suspected human carcinogen. o-Toluidine has a wide range of genetic effects. It is a weak bacterial, fungal and mammalian mutagen, although the conditions required are stringent. The metabolising system used is of particular importance. o-Toluidine is also a clastogen, generally on prolonged exposure. It induces aneuploidy in both fungi and mammalian cultured cells. It also produces DNA damage (single-strand breaks and unscheduled DNA synthesis, UDS) and causes cell transformation. o-Toluidine can be considered a general genotoxin demonstrable under special conditions, particularly with regard to metabolism. PMID- 1719403 TI - The detection of chemically induced chromosomal malsegregation in Saccharomyces cerevisiae D61.M: a literature survey (1984-1990). AB - Our objective is to summarize the published data obtained with a recently developed tester strain suitable for the detection of chromosomal malsegregation in yeast. Results from 25 papers were reviewed in which numerical data for 111 chemicals tested in Saccharomyces cerevisiae D61.M are reported (a total of 316 independent tests; 279 acceptable, 37 not meeting our criteria). Of the 111 compounds analyzed 43 compounds are positive for chromosomal malsegregation, 56 compounds are negative and 12 compounds do not meet our criteria for acceptance (inconclusive). Of the 43 compounds judged positive 5 (acetone, acetonitrile, benzonitrile, ethylacetate and propionitrile) were only positive using a cold interruption protocol. Recommendations are made for standardization of methods and protocols for screening purposes. Finally, a comparison with in vitro tubulin assembly data using mammalian tubulin is presented. PMID- 1719404 TI - Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity. AB - OPP: This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline). A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative and/or quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the OPP approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1719405 TI - Genotoxic, carcinogenic, and teratogenic hazards in the marine environment, with special reference to the Mediterranean Sea. AB - Genotoxic, carcinogenic, and teratogenic hazards arising out of pollution in the marine environment are discussed in this article, with special reference to the situation in the Mediterranean area. A number of chemical compounds or complex mixtures relevant to marine pollution, either natural or of anthropogenic origin, are tentatively listed, along with protective factors which may play a counteracting role in the same environment. Harmful substances tend to undergo interactions and transformations in seawater, sediments, and marine biota, due to physical, chemical, microbial, or light-mediated mechanisms. Bioaccumulation phenomena in marine organisms may result from food-chain biomagnification processes or from concentration of pollutants by filter feeders. A variety of sources can account for marine pollution by genotoxic, carcinogenic, and teratogenic compounds, but there is a relative paucity of analytical data concerning the Mediterranean. Metabolic transformations of xenobiotics occur in all marine organisms, the biochemical mechanisms in fish being comparable to those which have been extensively investigated in mammals. Induction of metabolic pathways, and especially of the mixed-function oxygenase system, represents the earliest warning signal of exposure to pollutants. Occurrence of neoplastic diseases is documented by experimental and field studies in marine vertebrates as well as in invertebrates. The association with local pollution phenomena has been recognized in several studies, but other etiopathogenetic factors may be also involved, and in some cases tumors have been reported to be unrelated to chemical pollution. Genotoxic agents have been detected by means of suitable techniques in seawater, sediments, and marine organisms. Several studies have investigated the presence of carcinogen-DNA adducts, DNA damage and repair processes, and cytogenetic alterations, such as chromosomal aberrations, sister-chromatid exchanges, and micronuclei, in tissues of marine organisms. However, monitoring of these end-points under field conditions encounters some limitations and problems. Even more fragmentary is the information on teratogenic effects in marine organisms, although interesting test systems have been set up. On the whole, a quite extensive database on all these toxicological issues is already available in the literature, but further studies are warranted for an adequate assessment of genotoxic, carcinogenic, and teratogenic hazards, and possibly counteracting factors in the marine environment, and specifically in the Mediterranean Sea. PMID- 1719407 TI - Evaluation of frequency of micronucleated oral mucosa cells as a marker for genotoxic damage in chewers of betel quid with or without tobacco. AB - The frequency of micronucleated cells (MNC) derived from exfoliated human oral mucosal cells has been measured to assess genotoxic damage in chewers of betel quid with tobacco (BQT) and tobacco with lime (T). Significantly elevated frequencies of MNC were observed in the exposed groups (BQT = 4.83 +/- 0.70; T = 5.20 +/- 0.66 per 1000 cells) compared to the control group (C = 2.59 +/- 0.37) although the levels observed were lower than those reported in the literature. No correlation was seen between age, duration and frequency of habits and the frequency of MNC in the 2 habit groups. Clastogenic agents in betel quid possibly involved in micronucleus formation are discussed. PMID- 1719406 TI - Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid. AB - Nordihydroguaiaretic acid (NDGA), which occurs in the resinous exudates of many plants is used as an antioxidant in fats and oils. In this study we show that NDGA inhibited the mutagenicity of methyl methanesulfonate, benzo[a]pyrene (BP), 2-aminofluorene, and aflatoxin B1 in Salmonella typhimurium strain TA100 or TA98 in the absence and presence of rat hepatic microsomal activation system. The addition of NDGA during and after nitrosation of methylurea (MU) resulted in a dose-dependent inhibition of mutagenicity induced by nitrosation products of MU. In a two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O tetradecanoylphorbol-13-acetate (TPA) as tumor promoter, pretreatment of animals with NDGA prior to DMBA application, afforded significant protection against skin tumorigenicity in female SENCAR mice. In additional studies, skin application of NDGA also inhibited the binding of topically applied [3H]BP and [3H]DMBA to epidermal DNA. When assessed in the anti-tumor promotion protocol, pretreatment of animals with NDGA before each application of TPA in DMBA-initiated mouse skin, resulted in 72% decrease in the total number of tumors when compared to non-NDGA pretreated animals. The possible mechanism(s) of the antimutagenic and anti tumorigenic activities may be due to the multiple effects of NDGA as inhibitor of the carcinogen metabolism and DNA-adduct formation, scavenger of carcinogen free radicals, and as inhibitor of TPA-induced ornithine decarboxylase activity. PMID- 1719408 TI - Induction of numerical chromosomal aberrations during DNA synthesis using the fungicides nimrod and rubigan-4 in root tips of Vicia faba L. AB - The 2 fungicides nimrod and rubigan-4 were tested for genotoxicity using Vicia faba root tips as the biological test system. Treating lateral roots with different concentrations of each fungicide for different periods showed that both fungicides were able to produce numerical but not structural chromosomal aberrations. The percentage of total aberrations in root tips exposed to nimrod reached 54.39% at 250 ppm for 4 h, and 64.69% in root tips exposed to rubigan-4 at 250 ppm for 6 h. The types of numerical chromosomal aberrations produced by both fungicides included: binucleate cells, c-metaphases, sticky chromosomes, polyploid cells, and laggards. Recovery experiments for 24, 48, and 96 h showed no significant differences between the percentage of total aberrations in treated and control groups. PMID- 1719409 TI - Clastogenic effect of pesticides in peripheral lymphocytes of cotton-field workers. AB - We studied clastogenic effects in peripheral lymphocytes of cotton-field workers who were exposed to different pesticides. All the cells were grown in RPMI 1640 medium for 48 and 72 h. The type of aberrations observed in the exposed group are gaps, breaks, dicentrics, exchanges, rings and polyploidy. The frequency of total chromosomal aberrations increased significantly in male pesticide applicators when compared to controls. A significant decrease in mitotic index was observed in the exposed group as compared to the control group. The 48-h cultures showed high incidence of chromosomal aberrations and low mitotic index when compared to 72-h cultures. The difference in chromosomal aberrations between 48- and 72-h cultures was not significant. 24 out of 26 individuals showed ill health effects such as severe giddiness and nervous disorders. PMID- 1719410 TI - Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse. AB - The mutagenicity and toxicity of diethylstilbestrol (DES), 17 beta-estradiol and zeranol on the male mouse germ cells were investigated with meiotic micronucleus assays in vivo and in vitro, sperm-head abnormality test and morphometry. Further, the developmental effects of DES on testicular morphology were explored. Micronucleus induction was observed at 10(-7) M concentration of DES and 17 beta estradiol in vitro, but other treatments yielded negative results. The micronucleus assay in vivo revealed a small number of micronuclei in early haploid spermatids 17 days after a single subcutaneous injection of DES 50 mg/kg, whereas estradiol and zeranol gave negative results. The sperm-head abnormality rates were significantly elevated 5 weeks after treatments with high doses of DES, 17 beta-estradiol and zeranol, and testicular morphometry revealed transient changes in the volume densities of testicular tissue components. Prenatal and neonatal estrogen administration resulted in permanent alterations in seminiferous epithelium and dilatation of the rete testis, but did not affect micronucleus or sperm-head abnormality rates. The mutagenicity and toxicity of hormones in the mouse testis paralleled the hormonal activity of these compounds. Early estrogenization was the most sensitive toxicity test, followed by in vitro meiotic micronucleus induction, whereas the sperm-head abnormality assay and morphological analysis did not reveal subtle changes. PMID- 1719411 TI - Sister-chromatid exchanges in cannabis smokers. AB - The genotoxicity of cannabis smoking was evaluated by means of the sister chromatid exchange (SCE) test. The SCE test is considered to be a sensitive tool for the discovery of genotoxic agents in the environment. Twenty-two tobacco smokers and 22 persons smoking both tobacco and cannabis were compared. Our findings showed that smoking in itself enhanced the SCE level significantly (18.5%) compared to a group of non-smokers, but adding smoking of cannabis to tobacco smoking did not affect the SCE level further. Based on our observations cannabis smoking could not be considered genotoxic. PMID- 1719412 TI - Evaluation of secondary nitroalkanes, their nitronates, primary nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay. AB - The secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2 methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10-80 mumoles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species. PMID- 1719413 TI - Lack of mutagenicity of ochratoxin A and B, citrinin, patulin and cnestine in Salmonella typhimurium TA102. AB - The Aspergillus mycotoxins ochratoxin A and B, citrinin and patulin as well as combinations of ochratoxin A and citrinin did not induce reverse mutations in Salmonella typhimurium strain TA102. Therefore there is no indication for the induction of oxidative damage or crosslinks. The same is true for cnestine, a compound extracted from the plant Cnestis glabra. PMID- 1719414 TI - Cytogenetic effects in rotogravure printers exposed to toluene (and benzene). AB - Comparing 21 rotogravure printers exposed to toluene (medians: time-weighted air level 150 mg/m3, blood toluene 1.6 mumole/l) and 21 unexposed controls (median blood toluene less than or equal to 0.01 mumole/l) there was a significant increase in the frequency of micronuclei (MN) in pokeweed mitogen (PWM) stimulated peripheral blood lymphocytes in the printers, as compared to the controls (2.8% vs. 1.5%; p = 0.03; all p adjusted for age and smoking). The frequency of small MN (size ratio MN/main nucleus less than or equal to 0.03) in PWM-stimulated lymphocytes was associated with the exposure (1% vs. 0.3%; p = 0.05). Furthermore, among the exposed subjects there was an association between blood toluene and small MN (0.17% per mumole/l; p = 0.0005). Small MN in phytohemagglutinin (PHA) cultures displayed no association with any exposure parameter. However, in the printers, an estimated cumulative exposure index was weakly correlated with the frequency of total MN in PHA-stimulated cells (0.00003% per mg/m3 x year; p = 0.07). Among the printers, chromosomal breaks in PHA-stimulated cells were associated with the duration of earlier benzene exposure (0.03% per year; p = 0.01). The results of this study strongly indicate that toluene causes a clastogenic effect on the B-cells even at low exposure levels. Further, earlier benzene exposure seems to have caused chromosomal breaks in T-cells. PMID- 1719415 TI - Biomarkers in styrene-exposed boatbuilders. AB - 14 fiberglass-reinforced plastics (FRP) boatbuilders were compared with 9 unexposed controls with respect to several chemical specific and nonspecific biomarkers measured in peripheral blood. Biomarkers included styrene-hemoglobin adducts (styrene-Hb), sister-chromatid exchanges (SCEs), micronuclei (MN), single strand breaks (SSBs) and N-acetoxy-2-acetylaminofluorene-induced DNA binding (NA AAF binding) as a measure of susceptibility to DNA damage. Workers' exposures averaged 11 ppm (8-h TWA; geometric mean) and ranged from 0.6 to 44 p.p.m. Mandelic acid levels were measured in end-of-shift urine samples and reflected an average styrene exposure equivalent to 15 p.p.m. There was a large though not significant difference in levels of styrene-Hb adducts among exposed workers and controls, largely the consequence of a single heavily-exposed individual with an extremely high level of adducts. Significant differences between biomarker levels in exposed workers and controls were observed with MN, SSBs and NA-AAF binding. No significant differences were seen in mean levels of SCEs nor in the incidence of cells with a high frequency of SCEs. The data suggest that exposure to levels of styrene in occupational settings near or below the current OSHA standard (50 p.p.m.) can induce damage at the cellular/molecular level. Appropriately-selected panels of biomarkers can be useful in identifying potentially harmful exposures. PMID- 1719416 TI - A Plasmodium falciparum malaria vaccine candidate which contains epitopes from the circumsporozoite protein and a blood stage antigen, 5.1. AB - The previously described Plasmodium falciparum blood stage antigen, 5.1 (also referred to as exp-1) was expressed at a high level in Escherichia coli. Saimiri monkeys immunised with purified recombinant antigen 5.1 were partially protected from P. falciparum blood stage parasite challenge. The gene coding for 5.1 was combined with DNA coding for an (Asn-Ala-Asn-Pro)19 sequence (abbreviated (NANP)19 in the one-letter amino acid code). To facilitate purification of the recombinant protein, DNA coding for a hexahistidine (His6) sequence was introduced at the 5' end of the gene (proteins containing His6 have high affinity for Ni(2+)-chelate columns even in the presence of 6 M guanidine HCl). The recombinant protein, His6-5.1-(NANP)19 with an apparent molecular size of 40 kDa could be highly purified by a combination of 4 steps: (1) release and solubilization of the recombinant fusion protein from E. coli in the presence of 6 M guanidine-HCl; (2) precipitation of over 60% of the bacterial proteins by the addition of ammonium sulphate to 50% saturation; (3) affinity chromatography on a Ni(2+)-chelate column in the presence of 6 M guanidine-HCl; (4) adsorption onto a cation exchange resin in the presence of 6 M urea, and elution with an increasing NaCl gradient. Compared with the previously tested tetanus toxoid-(NANP)3 malaria vaccine, this protein elicits an anti-(NANP)n response which more closely resembles that evoked by native sporozoites. The recombinant vaccine also induces the production of antibodies against the blood stages of the malaria parasite. PMID- 1719417 TI - Biochemical characterization of the protective membrane glycoprotein GP46/M-2 of Leishmania amazonensis. AB - Biochemical features of the immunologically protective, membrane glycoprotein GP46/M-2 of Leishmania amazonensis have been investigated. The protein appears to have a single carbohydrate side chain of approximately 3 kDa, representing 7% of the mass of the mature GP46/M-2 protein. Experiments removing this carbohydrate side chain from GP46/M-2 indicate that the carbohydrate is not involved in the epitope recognized by the monoclonal antibody, M-2. As this monoclonal antibody recognizes a species-specific epitope, these data suggest that this determinant is defined by the polypeptide portion of the molecule. Studies employing the VSG lipase as well as anti-CRD antibody clearly indicate that the molecule is anchored to the surface membrane of the promastigote via a phosphatidylinositol linked lipid anchor. Neither the carbohydrate side chain nor the lipid anchor appear to be responsible for the apparent refractoriness of this protein to protease digestion, suggesting that properties of the polypeptide itself may be responsible. These data are discussed in terms of recent DNA-derived protein sequence of the GP46/M-2. PMID- 1719418 TI - Strategies for the global eradication of poliomyelitis by the year 2000. PMID- 1719419 TI - Chronic granulomatous disease presenting in a 69-year-old man. PMID- 1719420 TI - Cell adhesion. A birth certificate for CD2. PMID- 1719421 TI - Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. AB - Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro. PMID- 1719422 TI - A cyclic GMP-activated channel in dissociated cells of the chick pineal gland. AB - Phototransduction in the vertebrate retina is dependent in part on a cyclic GMP activated ionic channel in the plasma membrane of rods and cones. But other vertebrate cells are also photosensitive. Cells of the chick pineal gland have a photosensitive circadian rhythm in melatonin secretion that persists in dissociated cell culture. Exposure to light causes inhibition of melatonin secretion, and entrainment of the intrinsic circadian oscillator. Chick pinealocytes express several 'retinal' proteins, including arrestin, transducin and a protein similar to the visual pigment rhodopsin. Pinealocytes of lower vertebrates display hyperpolarizing responses to brief pulses of light. Thus it is possible that some of the mechanisms of phototransduction are similar in retinal and pineal photoreceptors. We report here the first recordings of cyclic GMP-activated channels in an extraretinal photoreceptor. Application of GMP, but not cyclic AMP, to excised inside-out patches caused activation of a 15-25 pS cationic channel. These channels may be essential for phototransduction in the chick pineal gland. PMID- 1719423 TI - Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. AB - A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor. PMID- 1719424 TI - Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells. AB - Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3) sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell. PMID- 1719425 TI - Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes. AB - Listeria monocytogenes is a Gram-positive bacterium which grows in the cytoplasm of eukaryotic cells and can cause severe disease in immunocompromised individuals. In murine systems CD8+ T lymphocytes have been shown to be important effectors of acquired protective immunity against L. monocytogenes. Class I MHC restricted CD8+ cytotoxic T lymphocytes (CTL), which lyse J774 macrophage-like targets infected with L. monocytogenes, are induced following in vivo injection of live organisms. Natural peptide epitopes derived from L. monocytogenes can be acid-extracted from heavily infected BALB/c spleens and detected by CTL. A CTL clone, B9, derived from a (BALB/c x C57BL/6)F1 (H-2dxb) mouse, recognizes one of these natural epitopes in an H-2Kd-restricted fashion. B9 also recognizes P815 (H 2d) mastocytoma cells transfected with the listeriolysin gene. To identify the region of the listeriolysin recognized by CTL we used the H-2Kd peptide-binding motif described by Rammensee and colleagues to synthesize 11 nonamer peptides. One of these peptides, listeriolysin 91-99, was recognized very efficiently by B9. This represents the first identified class I MHC-restricted epitope of bacteria and demonstrates the utility of the allele-specific motif for predicting CTL epitopes. PMID- 1719426 TI - A de novo Alu insertion results in neurofibromatosis type 1. AB - Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder with a high mutation rate and variable expression, characterized by neurofibromas, cafe au-lait spots, Lisch nodules of the iris, and less frequent features including bone deformities and learning disabilities. The recently cloned NF1 gene encodes a transcript of 13 kilobases from a ubiquitously expressed locus on chromosome 17. Most NF1 patients are expected to have unique mutations, but only a few have so far been characterized, restricting genetic and functional information and the design of DNA diagnostics. We report an unusual NF1 mutation, that of a de novo Alu repetitive element insertion into an intron, which results in deletion of the downstream exon during splicing and consequently shifts the reading frame. This previously undescribed mechanism of mutation indicates that Alu retrotransposition is an ongoing process in the human germ line. PMID- 1719427 TI - Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex. AB - The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein. PMID- 1719428 TI - Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. AB - Existing methods for the synthesis and screening of large numbers of peptides are limited by their inability to generate and screen the requisite number (millions) of individual peptides and/or their inability to generate unmodified free peptides in quantities able to interact in solution. We have circumvented these limitations by developing synthetic peptide combinatorial libraries composed of mixtures of free peptides in quantities which can be used directly in virtually all existing assay systems. The screening of these heterogeneous libraries, along with an iterative selection and synthesis process, permits the systematic identification of optimal peptide ligands. Starting with a library composed of more than 34 million hexa-peptides, we present here the precise identification of an antigenic determinant recognized by a monoclonal antibody as well as the straightforward development of new potent antimicrobial peptides. PMID- 1719429 TI - Carbachol-induced nonspecific desensitization in guinea-pig ileum. AB - The effects of repeated stimulation by carbachol on force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. Carbachol was applied at 20 degrees C for 5 min. Each application was followed by a 25-min washout period and the desensitization was expressed by the decline of the maximal force development. Three hours after the first carbachol-induced contraction the peak amplitude was about 40% of the initial value. Increasing the frequency of application, thereby decreasing the washout time, enhanced the desensitization, while the presence of the competitive blocker atropine reduced the phenomenon. At 35 degrees C no desensitization could be observed. Blocking the Na+/K+ pump by ouabain or by K(+)-free solution reduced the force development to less than 20%. Increasing [K+]0 in the washout solution at 20 degrees C reduced the desensitization phenomenon, while decreasing [K+]0 resulted in an enhanced desensitization as expressed by a decline of the force development. The total cellular Na+ content after various stimulation sequences was determined at 20 degrees and 35 degrees C from the 22Na+ effluxes. At 35 degrees C the cellular Na+ content did not change significantly during stimulation for 10 min with 10( 4) mol/l carbachol. At 20 degrees C the resting Na+ content was significantly increased, and it doubled during carbachol stimulation for 10 min. Furthermore, the recovery of the cellular Na+ content after washout proceeded extremely slowly at that temperature. The appearance of desensitization was increased by 10 mumol/l ryanodine, while it was reduced by adding the Ca2+ agonist Bay K 8644.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719430 TI - Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria. AB - The effect of Bay K 8644 (a dihydropyridine Ca(2+)-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [3H]-noradrenaline. Bay K 8644 (3 mumol/l) significantly increased atrial rate from 206 +/- 7 to 259 +/- 9 beats.min-1 (P less than 0.05) and also tritium outflow (expressed as fractional rate of loss in min-1 x 10(3)) from 6.49 +/- 0.35 to 8.61 +/- 0.74 (P less than 0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([3H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria. PMID- 1719431 TI - The effect of sodium deoxycholate and other surfactants on the mucosal surface pH in proximal jejunum or rat. AB - The mucosal surface pH (acid microclimate) and nucleotide levels of rat proximal jejunum were measured in vivo under various conditions which included exposure to pharmacological agents and to surfactants. Mucosal surface pH was unaffected by sodium nitroprusside, A23187 and amiloride, as was mucosal cGMP content, although amiloride and A23187 reduced cAMP content. In contrast, surfactants elevated the pH of the mucosal surface significantly (P less than 0.001): control value 6.23 +/- 0.02 (n = 12); Lubrol PX 0.8% (v/v) 6.98 +/- 0.02 (n = 5); sodium deoxycholate 2 mmol/l 6.67 +/- 0.04 (n = 5); Triton X-100 0.5% (v/v) 7.41 +/- 0.03 (n = 5). No significant changes in cGMP levels were noted after surfactant treatment, although DOC and Triton X-100 reduced cAMP levels. The ability of higher concentrations of surfactant to elevate the mucosal surface pH beyond neutrality to values similar to plasma pH contrasts with the action of Escherichia coli heat-stable (STa) enterotoxin which at high concentrations could not elevate the mucosal surface pH beyond neutrality. Consistent with the known effects on tight junction permeability, surfactants may act by allowing plasma like subepithelial fluid to neutralise the microclimate. PMID- 1719432 TI - Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: involvement of 5-HT3 receptors. AB - Isolated segments of the guinea-pig small intestine were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Release of acetylcholine from isolated superfused intestinal segments was determined as outflow of [3H]radioactivity from preparations preincubated with [3H]choline. Cisplatin (3 microM) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 microM cisplatin decreased the outflow of 5-HT and its metabolite by 40%-50%. The stimulatory effect of cisplatin was consistently observed only when the bicarbonate-phosphate buffer of the Tyrode's solution was replaced by HEPES-buffer. The stimulatory effect of cisplatin was abolished in the absence of extracellular calcium or presence of tetrodotoxin (1 microM). The stimulatory effect of cisplatin was also prevented by hexamethonium (100 microM) or scopolamine (100 nM). The 5-HT3 receptor antagonists ondansetron and ICS 205-930 in concentrations as low as 1 pM also abolished the stimulatory effect of cisplatin. The 5-HT3 receptor antagonist MDL 72222 prevented the stimulatory effect of cisplatin only at a concentration of 1 microM. None of the 5-HT3 receptor antagonists alone significantly altered the outflow of 5-HT and 5-HIAA. Cisplatin (3 microM) enhanced the outflow of [3H]radioactivity from intestinal segments and caused longitudinal muscle contractions that were abolished by 100 nM scopolamine. In conclusion, cisplatin, at concentrations which occur during anti-cancer therapy in humans and induce emesis, increases the release of 5-HT from the enterochromaffin cells of the small intestine of the guinea-pig.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719433 TI - In vivo modulation of the histamine release in the hypothalamus by adrenoreceptor agonists and antagonists. AB - The modulation of the histamine release from histaminergic neurons by noradrenergic neurons was investigated by the push-pull technique. The posterior hypothalamus of the conscious, freely moving rat was superfused with artificial CSF through a push-pull cannula and the release of endogenous histamine was determined in the superfusate. Hypothalamic superfusion with a potassium-rich CSF enhanced the release rate of histamine. Superfusion with the alpha 2-agonists noradrenaline or clonidine diminished the release rate of histamine. Moreover, clonidine abolished the potassium-induced increase in the histamine release. Superfusion with the alpha 2-antagonists yohimbine or idazoxan enhanced the release rate of histamine. It is concluded that noradrenaline released from noradrenergic neurons of the hypothalamus modulates the release of histamine from histaminergic neurons by stimulating alpha 2-adrenoreceptors located on histaminergic nerve terminals. PMID- 1719434 TI - Subtypes and excitation-contraction coupling mechanisms for neurokinin receptors in smooth muscle of the guinea-pig Taenia caeci. AB - This study investigated the subtype and coupling mechanisms mediating the direct contractile response to tachykinins in the guinea-pig Taenia caeci preparation in vitro. Coupling of neurokinin receptors was compared throughout with coupling of muscarinic receptors. The smooth muscle neurokinin receptors seem to be predominantly of the NK-1 subtype. Thus, the relative activities of the common naturally-occurring tachykinins fell within one order of magnitude, and the selective NK-1 receptor agonist substance P methyl ester was high in activity (0.38 relative to substance P). Some contribution from NK-3 receptors is, however, possible in view of the appreciable activity of the selective NK-3 agonist succ-[Asp6, N-MePhe8]-SP(6-11) (senktide; activity 0.004 relative to substance P), and NK-2 or NK-3 receptors in view of the higher activity of the D isomer of [Glp6, *Pro9]-SP(6-11) as compared to its NK-1 selective L-isomer (D/L activity ratio 1.53). Contractile actions of tachykinins were compared with carbachol for reliance on membrane-potential dependent (electromechanical) and membrane-potential independent (pharmacomechanical) coupling mechanisms. Log concentration-response curves to carbachol and substance P in normal Krebs' medium were compared with curves obtained in a high-K+ solution where processes dependent on changes in membrane potential could play no part in excitation. In the high-K+ depolarizing solution, a concentration-related relationship was maintained, though with some diminution in the maximal additional tension generated: the maximum tension with carbachol was under both conditions greater than that with substance P. The relative effects of several tachykinins and carbachol in producing receptor-mediated changes in membrane permeability through presumed receptor-operated ion channel opening, was estimated in terms of the ability to increase 86Rb-efflux, as a marker for K+, in a high-K+ depolarizing solution. Carbachol (10 microM) consistently increased 86Rb-efflux. In contrast, no permeability increase could be detected with any tachykinin tested (substance P, eledoisin, substance P methyl ester, neurokinin A, neurokinin B, 1 or 10 microM). Tachykinins and carbachol were compared in terms of ability to increase phosphatidylinositol hydrolysis. Both substance P and carbachol showed a concentration-related increase in accumulation of total inositol phosphates; though the maximal response to carbachol was considerably greater than that to any tachykinin (substance P, eledoisin, substance P methyl ester, senktide, neurokinin A, neurokinin B), or combination of two tachykinins (substance P and eledoisin, senktide and substance P methyl ester).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1719435 TI - On-line measurement of nitric oxide release from organic nitrates in the intact coronary circulation. AB - This study determines the release of nitric oxide (NO) from the coronary circulation of Langendorff hearts of rabbits, subsequent to administration of glyceryl trinitrate (GTN) and SIN-1. NO was measured on-line in the coronary effluent by the oxyhaemoglobin technique. Infusion of either GTN (10-40 mumoles/l) or SIN-1 (0.1-2.3 mumoles/l) into the coronary inflow resulted in a concentration-dependent NO release into the coronary effluent and a decrease in the coronary vascular resistance. NO generation from SIN-1 was identical with and without passage of the coronary circulation whereas NO generation from GTN was only detected after passage of the coronary vascular bed. NO generation by both substances was in the same range as endogenous NO release by two endothelium dependent vasodilators, bradykinin (0.05 mumoles/l) and substance P (0.05 mumoles/l). Oxyhaemoglobin used for the assay of NO, inhibited the relaxation by SIN-1, but did not reduce vessel relaxations induced by GTN or iloprost, a stable prostacyclin analogue. Removal of the coronary endothelium by trypsin or pretreatment with L-NG-Monomethylarginine (30 mumoles/l) did neither affect NO release from GTN and SIN-1 nor the vasodilatory effect of both substances. These data are the first to directly demonstrate endothelium-independent NO release from organic nitrates during passage of an intact organ circulation. They additionally suggest a subendothelial site of metabolic NO formation from GTN. PMID- 1719436 TI - Properties of palytoxin-induced whole cell current in single rat ventricular myocytes. AB - We examined the properties of the current induced by palytoxin in single ventricular cells of rats. The current was measured by a whole cell voltage clamp method. When the cell was held at -75 mV, palytoxin induced a sustained inward current in a concentration-dependent manner (2-100 pmol/l). The time-course of the inward current paralleled that of the depolarization. At a holding potential of +50 mV, it caused an outward current. Palytoxin-induced current reversed at 0 mV and its current-voltage relation was almost linear at either negative or positive voltage. Substitution of external NaCl with choline-Cl suppressed the palytoxin-induced inward current but not the outward current, by shifting the reversal potential to levels more negative than -50 mV. A cardiac glycoside, cymarin (10 and 100 mumol/l) partially inhibited the palytoxin-induced current without changing the reversal potential, only when applied before palytoxin. Palytoxin decreased the nicardipine-sensitive Ca2+ current. These data suggest that palytoxin-induced inward current is carried by extracellular Na+ and the outward current is carried mainly by intracellular K+, and that the inward current is responsible for the toxin's depolarizing action. The antagonism by cysmarin indicates that the site of action of palytoxin is in the vicinity of the binding site of cardiac glycosides. PMID- 1719437 TI - [Cell biology from a medical viewpoint. IV. The cell nucleus and the action of the genome]. PMID- 1719438 TI - [The aphasic patient]. PMID- 1719439 TI - [Pathogenesis of amyloidosis: a role for anti-proteases?]. AB - A new type of amyloidosis has been observed which appears in long-term survivors on haemodialysis, with beta 2-microglobulin identified as its major protein constituent. We recently identified alpha 2-macroglobulin, the major serum anti protease, in amyloid deposits of haemodialysis patients. It has also been reported that amyloid fibrils can be redissolved in vitro by proteases. These findings, coupled with the facts that alpha 2-macroglobulin blocks the proteases and decreases macrophage production of proteases, suggest that alpha 2 macroglobulin may prevent the degradation of the amyloidogenic protein allowing amyloid deposit formation and/or persistence. In our opinion, alpha 2 macroglobulin and possibly other antiproteases as well, may play a key role in amyloidogenesis in dialysis patients. PMID- 1719440 TI - Loss of axonal contact causes down-regulation of the PLP gene in oligodendrocytes: evidence from partial lesions of the optic nerve. AB - We have examined the influence that axons may have on the expression of proteolipid protein (PLP), the major myelin protein of the CNS. Partial transections were made of the optic nerve of adult rats to produce approximately a 50% loss of axons. Twenty-eight days after lesioning, sections of the distal nerves were immunostained for GFAP and neurofilament protein and hybridized for PLP mRNA. The area of astrocytosis, as defined by GFAP immunostaining, usually exceeded the extent of axonal loss. PLP mRNA expression in oligodendrocytes in the denervated area of the lesioned nerve was reduced compared to the innervated zones of the lesioned nerve and the contralateral intact nerve. This down regulation correlated with the axonal loss rather than the area of astrocytosis. The data support the contention that axons are necessary for oligodendrocytes to maintain full expression of their major myelin protein genes. PMID- 1719441 TI - Multiplication of rubella and measles viruses in primary rat neural cell cultures: relevance to a postulated triggering mechanism for multiple sclerosis. AB - Rubella virus multiplied to low titre and produced a partial cytopathic effect in rat glial cell cultures. Anti-galactocerebroside staining showed that this cytopathic effect involved the disintegration of oligodendrocytes. A similar effect was produced following infection of myelinating neural cell cultures with rubella virus, but virus multiplication could not be detected in pure neuron cultures. Measles virus was found to multiply and produce a cytopathic effect in primary cultures of both neurons and glial cells. These results are discussed in relation to the ability of measles and rubella viruses to trigger human multiple sclerosis. PMID- 1719442 TI - Demonstration of insulin-like growth factors I, II and heterogeneous insulin-like growth factor binding proteins in the cyst fluid of patients with craniopharyngioma. AB - Insulin-like growth factor (IGF)-II and its binding proteins were demonstrated to be present in human craniopharyngioma cyst fluid using gel filtration and ligand blot analyses. Immunoreactive IGF-II in 3 patients was found to be 274, 232 and 310 ng/ml after gel chromatography whereas IGF-I concentrations were 13, 8 and 15 ng/ml. The IGF-II levels were severalfold higher in cyst fluid than in spinal fluid while the IGF-I levels in both fluids did not differ significantly. The binding proteins showed high affinities for [125I]IGF-II which could be displaced by unlabelled IGF-II. With the ligand blot analysis, [125I]IGF-II shows bands at 300, 175 and 46/43 kilodaltons probably representing IGF-II receptor and IGFBP-3. IGFBP-1 levels 17, 22 and 45 ng/ml, respectively, were undetectable by ligand blot. PMID- 1719443 TI - Somatostatin-28 (1-12)-like immunoreactivity in the cat diencephalon. AB - Using an indirect immunoperoxidase technique, the location of somatostatin-28 (1 12)-like immunoreactive fibres and cell bodies in the cat diencephalon was studied. The hypothalamus was richer in somatostatin-28 (1-12)-like immunoreactive structures than the thalamus. A high density of immunoreactive fibres was observed in the nuclei habenularis lateralis, paraventricularis anterior (its caudal part), filiformis, hypothalami ventromedialis, and regio praeoptica, whereas a moderate density was found in the nuclei paracentralis, supraopticus, supra chiasmaticus, hypothalamus posterior and area hypothalamica dorsalis. The nuclei lateralis dorsalis, lateralis posterior, medialis dorsalis, rhomboidens, centralis medialis, ventralis medialis, reuniens, anterior dorsalis, parataenialis, interanteromedialis, hypothalamus lateralis, hypothalamus dorsomedialis and arcuatus had the lowest density of immunoreactive fibres. In addition, a high or moderate density of somatostatin-28 (1-12)-like immunoreactive cell bodies was observed in the nuclei paraventricularis hypothalami, supraopticus, supra chiasmaticus, area hypothalamics dorsalis, subparafascicularis, hypothalamus posterior and hypothalamus anterior, whereas scarce immunoreactive perikarya were visualized in the nuclei lateralis dorsalis and parafascicularis. The distribution of somatostatin-28 (1-12)-like immunoreactive structures is compared with the location of other neuropeptides in the cat diencephalon. PMID- 1719444 TI - Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists. AB - Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L 659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations. PMID- 1719445 TI - Receptor-selective, peptidase-resistant agonists at neurokinin NK-1 and NK-2 receptors: new tools for investigating neurokinin function. AB - The pharmacological profiles of two novel neurokinin agonists have been investigated. delta Ava[L-Pro9,N-MeLeu10]SP(7-11) (GR73632) and [Lys3,Gly8-R gamma-lactam-Leu9] NKA(3-10) (GR64349) are potent and selective agonists at NK-1 and NK-2 receptors respectively. In the guinea-pig isolated trachea preparation, contractions induced by these agonists were largely unaffected by inclusion of peptidase inhibitors in the bathing medium, indicating that these agonists are resistant to metabolism by peptidases. In the anaesthetised guinea-pig, both agonists were more potent bronchoconstrictor agents than either NKA or the SP analogue, SP methylester. In the anaesthetised rat, the NK-1 agonist, GR73632 was more potent than SP, NKA or NKB at causing the histamine-independent extravasation of plasma proteins into the skin after intradermal administration. The NK-2 agonist, GR64349 and the NK-3 agonist, senktide were without significant effect in this model. These agonists are useful tools for characterizing neurokinin receptor-mediated actions both in vitro and in vivo. PMID- 1719446 TI - Somatostatin 28 interacts with CCK receptor in brain and pancreas. AB - The ability of somatostatin analogs to interact with the binding of cholecystokinin has been studied in pancreatic and brain cortical membranes. Only the 28 amino-acid forms of somatostatin (S28), [Nle8]S28 and [Des Lys14,DTrp22]S28 were found to inhibit the binding of cholecystokinin to rat pancreatic plasma membranes and to increase the amylase release from pancreatic acini. This effect was independent of somatostatin receptor and resulted from an interaction between S28 and CCK receptor. This interaction was not observed with [Leu8, DTrp22, Tyr25]S28, indicating that this analog does not possess the biological activity of the native peptide and that the iodinated peptide could not label specific S28 receptors. S28 interacted also with CCK receptors in cortical brain membranes. Our results support the concept that S28, but not S14, may function as a regulatory molecule at CCK receptors and emphasize that S28 and S14 may be distinct neuromodulators. PMID- 1719447 TI - The emergence of architectonic field structure and areal borders in developing monkey sensorimotor cortex. AB - Adult monkey sensorimotor cortex consists of several structurally and functionally distinct areas. The developmental sequence through which the characteristic architectonic features and the borders of these areas become resolved was examined in a series of fetal, postnatal and adult monkeys by using Nissl staining, cytochrome oxidase and acetylcholinesterase histochemistry, and immunocytochemistry for GABA and the neuropeptides somatostatin, neuropeptide Y, substance P and cholecystokinin. At the youngest fetal age examined (E110), the pre- and postcentral gyri possess six clearly delineated cellular layers; populations of GABA- and neuropeptide-immunoreactive cells can be identified, but their somatic sensory cortex at E110 lacks areal cytoarchitectonic parcellation. Despite the apparent homogeneity in the cytoarchitecture of the somatic sensory cortex, incipient areal borders are revealed by staining for cytochrome oxidase and acetylcholinesterase activity, and by staining immunocytochemically for several neuropeptides. The motor cortex at E110 differs from that in adults by the presence of a prominent layer IV; a clear cytoarchitectonic border between areas 3a and 4 is detectable at E110, which is also revealed by chemoarchitectonic markers. With increasing age, the characteristic architectonic features gradually emerge and areal cytoarchitectonic borders appear, becoming adult-like by early postnatal ages. The gradual changes in cytoarchitecture are paralleled by redistributions of GABA- and neuropeptide-immunoreactive cells and fiber plexuses. The data demonstrate that the progressive refinement in cytoarchitectonic features and in the distributions of neurotransmitter- and peptide-containing cells occurs primarily during the latter third of gestation. The major changes are temporally coincident with the ingrowth of afferent axonal systems, suggesting that the establishment of connectivity may be capable of modulating finer details of structural or molecular phenotype, particularly intra areal cytoarchitectonic features and neurotransmitter or peptide expression. PMID- 1719448 TI - Spatial distributions of cytoskeletal proteins and the nerve growth factor receptor in septal transplants in oculo: protection from abnormal immunoreactivity by hippocampal co-grafts. AB - Intraocular grafts of embryonic rat septum and co-grafts of septum plus hippocampus were studied with immunohistochemical markers after one and six months (short term) and 12 months (long term) of survival. Neurons in all the septal tissues expressed the epitope for the rat beta-nerve growth factor receptor in sections reacted with the monoclonal antibody 192-IgG. Stained fibers traversed the interface of the short and long term co-grafts and 192-IgG-positive processes were most prominent in the septum when combined with the hippocampal formation. In contrast, labeled processes were sparse and the perikarya of positive neurons appeared shrunken in the long term single septal transplants. Axon and dendrite profiles in the grafts were examined with antibodies that recognize the phosphorylated heavy neurofilament unit (RT97) and the high molecular weight microtubule-associated protein termed MAP 2, respectively. In the short term single and double grafts, characteristic arrays of RT97-positive processes defined the tissues and axonal tracts connecting the septum with the hippocampus. Typical immunostaining of the neuronal somas and the dendrite arbors were were outlined with the MAP 2 antibody. After one year in oculo, extensive changes in the patterns of axonal and dendritic immunoreactivity were noted in the isolated septal grafts. Abnormalities identified with the RT97 antibody included hypertrophied axons, short fragments of kinked axons and neurofilaments in the neuronal perikarya. The formation of circular "abnormal fiber aggregates" composed of densely packed abnormal and normal axonal processes were also distinctive in only the long term single septal transplants. In addition, a reduction in the density of dendrites and the presence of truncated arbors stained with the MAP 2 antibody suggested that regression of the dendrites had occurred. These spatial modifications in axonal and dendritic staining were not present in the septal portion of the combined preparations. In astrocytes, an increase in the antigenicity to glial fibrillary acidic protein paralleled the age of the transplant and was most extensive in the septal grafts. The results illustrate that intraocular co-grafts of hippocampus protect septal neurons and glial cells from abnormal changes in immunoreactivity to antibodies directed against cytoskeletal proteins and exemplify the long term supportive effects of the hippocampus on the morphology of septal neurons, including neurons that express the receptor for nerve growth factor. PMID- 1719449 TI - Histamine neurons in human hypothalamus: anatomy in normal and Alzheimer diseased brains. AB - The anatomy of histamine-immunoreactive cell bodies in normal adult human brain was examined in detail. In addition, the distribution of these cells in three cases of Alzheimer's disease was compared to the distribution of neurofibrillary tangles. Histamine-immunoreactive cell bodies were confined to the tuberal and posterior hypothalamus, forming the tuberomammillary nuclear complex. Most of the about 64,000 histamine neurons were large and multipolar. They comprised four distinct parts: (i) a major ventral part corresponding to the classical tuberomammillary nucleus, (ii) a medial part including the supramammillary nucleus and part of the posterior hypothalamic area, (iii) a caudal paramammillary part, and (iv) a minor lateral part. The parts showed some similarity with the subgroups in rat. In human, as compared to rat, the histamine neurons occupy a larger proportion of the hypothalamus. Numerous neurofibrillary tangles were found in the Alzheimer hypothalami, concentrated in the tuberomammillary area. Most of them were of globular type and extracellular, and only a minority were histamine immunoreactive. They may represent remnants of degenerated tuberomammillary neurons. PMID- 1719450 TI - On the role of NK-2 tachykinin receptors in the mediation of spinal reflex excitability in the rat. AB - The effects of intrathecal administration of neurokinin A, substance P and [Tyr5, D-Trp6,8,9 Arg10]neurokinin A-(4-10) (Men 10207), a specific NK-2 receptor antagonist, on the spinal nociceptive flexor reflex were studied in decerebrate, spinalized, unanesthetized rats. Intrathecal neurokinin A and substance P facilitate the flexor reflex in a similar manner. The reflex facilitation to intrathecal neurokinin A, but not substance P, is dose-dependently blocked by pretreatment with Men 10207. The NK-2 receptor antagonist by itself facilitates the flexor reflex with a potency about 10 times less than that of neurokinin A, indicating a partial agonistic property. Reversible depression of the flexor reflex, which is not due to nonspecific spinal blockade, is observed after 700 pmol Men 10207. Further increasing the dose of Men 10207 to 7 nmol for 20 s at an intensity that activates unmyelinated (C) fibers stimulation of peripheral nerves at 1 Hz for 20 s at an intensity that activates unmyelinated (C) fibers facilitates the ipsilateral flexor reflex. The duration of the facilitation after conditioning stimulation of the cutaneous sural nerve is several minutes and about 1 h after conditioning stimulation of the gastrocnemius muscle nerves. Pretreatment with Men 10207 (70-700 pmol) has no effect on facilitation by the sural nerve conditioning stimulation, but effectively blocks the long-term reflex facilitation to the gastrocnemius nerve stimulation. The present results indicate a distinct role for NK-2 tachykinin receptors in mediation of spinal reflex excitability in the rat. Neurokinin A may be involved in the long-term increase of spinal reflex excitability after activation of unmyelinated fibers innervating muscle. PMID- 1719451 TI - Citrulline in the rat brain: immunohistochemistry and coexistence with NADPH diaphorase. AB - The presence in the brain of the urea cycle intermediate citrulline in the absence of a complete urea cycle has never been adequately explained. In an attempt to clarify this problem, we developed antibodies to citrulline and determined the distribution of citrulline-immunoreactivity in fixed sections of rat brain using immunoperoxidase and indirect immunofluorescence techniques. Citrulline-positive neurons were found to have a restricted distribution within the brain. A few cells were present in the cortex and corpus callosum. A large population of strongly stained cells was diffusely scattered throughout the striatum, nucleus accumbens and olfactory tubercle. Less strongly stained cells were detected in the supraoptic and paraventricular nuclei of the hypothalamus, the dorsal raphe, and the laterodorsal and pedunculopontine tegmental nuclei of the pons. The citrulline-immunoreactive cells were similar to those previously shown to contain NADPH-diaphorase activity, and double staining experiments indicated that citrulline-immunoreactivity was present in a subpopulation of NADPH-diaphorase-positive neurons. We have recently identified NADPH-diaphorase as a nitric oxide synthase. Thus the presence of citrulline in these cells suggests that it is formed within the brain as a coproduct during nitric oxide formation from arginine. PMID- 1719452 TI - Centrally administered galanin inhibits osmotically stimulated arginine vasopressin release in conscious rats. AB - The effect of centrally administered galanin on arginine vasopressin (AVP) release was investigated in conscious rats. Intracerebroventricular injection of porcine galanin suppressed hypertonic saline-induced increase in plasma AVP in a dose-dependent manner (12.5-100 pmol/rat) at 10 min after the injection. Pretreatment with subcutaneous injection of naloxone (1 mg/100 g b.wt.) partially blocked the galanin-induced effect on plasma AVP. These results suggest that central galanin inhibits osmotically stimulated AVP release and endogenous opioids are, at least in part, involved in the mechanism. PMID- 1719453 TI - Galanin stimulates acetylcholine release in the rat striatum. AB - The effect of the neuropeptide galanin (GAL) on the basal and the evoked release of acetylcholine (ACh) was investigated in the rat striatum using microdialysis and HPLC techniques. GAL (0.3, 1 and 3 nmol), applied in the lateral ventricle (10 microliters), was found to cause a dose-dependent stimulation of the basal ACh release. The stimulating effect of GAL on ACh release was longlasting (greater than 90 min) and reached its peak 30 min after i.c.v. administration. GAL failed to affect the scopolamine (0.25 and 0.5 mg/kg, i.p.) stimulated release of ACh. Possible mechanisms behind the GAL-stimulated ACh release in the rat striatum are discussed. It may involve effects on GAL receptors in the striatum or indirect effects via stimulation of GAL receptors in the substantia nigra resulting in inhibition of striatal dopamine (DA) transmission. PMID- 1719454 TI - NMDA receptor antagonist blocks the facilitation of the tail flick reflex in the rat induced by intrathecal administration of substance P and by noxious cutaneous stimulation. AB - This study examined effects of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), on facilitation of the tail flick reflex (1) by intrathecal administration of 6.5 nmol of substance P at the lumbar spinal level in awake rats and (2) by noxious cutaneous stimulation in anesthetized rats (by immersing the tip of the tail in hot water at 55 +/- 1 degrees C for 1.5 min). Reaction time was decreased by about 70% by intrathecal administration of substance P and by about 40% by tail immersion. Intrathecal administration of APV (2 nmol) or cerebrospinal fluid (CSF) failed to alter the baseline responses. However, APV but not CSF blocked the facilitation induced by intrathecal administration of substance P and by tail immersion. These results indicate that while NMDA receptors do not appear to be involved in mediating the tail flick reflex, they may be involved in expression of the facilitation of this reflex by substance P and/or by a related peptide. PMID- 1719455 TI - Serotonin hyperinnervation after foetal nigra or raphe transplantation in the neostriatum of adult rats. AB - Embryonic nigral or raphe grafts implanted in the dopamine-depleted neostriatum contain serotonergic neurones and give rise to a serotonergic hyperinnervation of the host striatum. No similar pattern of hyperinnervation is observed in rats with striatal tissue grafts, rats with nigral grafts implanted in the intact striatum, or rats with lesions alone. These observations suggest that striatal dopamine denervation induces some target-derived trophic factors that stimulate fibre growth from serotonergic as well as dopaminergic neurones. PMID- 1719456 TI - [A forgotten language]. PMID- 1719457 TI - [Diversion and relaxation within the hospital ... what an idea]. PMID- 1719458 TI - Antibiotics for otitis media. PMID- 1719459 TI - Ultrasound detection of neural tube defects in patients with elevated maternal serum alpha-fetoprotein. AB - We conducted this study to determine the accuracy of ultrasound in the prenatal diagnosis of neural tube defects in women with elevated maternal serum alpha fetoprotein (MSAFP). Among 905 pregnancies, 49 neural tube defects were correctly diagnosed by ultrasound alone; one was not. Ultrasound scanning had 98% sensitivity and 100% specificity for the detection of neural tube defects. The predictive value of a positive ultrasound diagnosis was 100% and of a negative ultrasound 99.9% for neural tube defects. Forty-three other structural abnormalities were also detected in patients with elevated MSAFP, including 19 abdominal wall defects, seven chromosomal abnormalities, five urinary tract abnormalities, one cardiac abnormality, and 11 others. Two chromosomal abnormalities were not detected. We suggest that ultrasound can be used reliably to detect neural tube defects, thereby avoiding the risks of amniocentesis. PMID- 1719460 TI - Acidic FGF and other growth factors in preretinal membranes from patients with diabetic retinopathy and proliferative vitreoretinopathy. AB - The development and extension of fibrovascular or fibroglial membranes onto the retinal surface are a major cause of visual loss in diabetic patients with proliferative retinopathy and in patients suffering from retinal detachment with proliferative vitreoretinopathy. The pathogenesis of these proliferative diseases, however, remain poorly understood and the nature of growth-promoting mediators implicated in these phenomena has not been determined yet. Using indirect immunofluorescence procedures, three different growth factors known to be mitogenic for various cell components of preretinal membranes, acidic fibroblast growth factor, epidermal growth factor and insulin-like growth factor type I, were sought in 14 specimens of preretinal proliferative tissues. Similar results were obtained in diabetic preretinal membranes and tissues from patients with proliferative vitreoretinopathy. The three different growth factors were found diffusely in the connective stroma and around new blood vessels within the vascular walls. Some fibroblast-like and pigment epithelial-derived cells more markedly reacted with anti-growth factor antibodies. These results provide indications on the eventual involvement of three potent growth factors in intraocular proliferative diseases, but whether or not these mediators play an active role in the development of preretinal membranes remains to be determined. PMID- 1719461 TI - Double hapten challenge in guinea pigs undergoing an ocular late-phase reaction. AB - Clinical and histological profiles of guinea pig conjunctiva undergoing late phase reaction (LPR) were evaluated before and after additional antigenic challenge. Four groups of animals were passively sensitized with IgG1 antibodies, challenged with the specific hapten di-DNP-lysine, and rechallenged during LPR either with di-DNP-lysine, or with PBS, or with an aspecific antigen to characterize the reactivity of the inflamed conjunctival tissues. The course of the clinical anaphylactic responses was modified slightly only by challenge with a specific hapten during LPR. However, no significant changes in the inflammatory cell profile were observed in conjunctiva undergoing LPR after an additional challenge. This suggests the participation of mechanisms which may mediate the attenuation of the anaphylactic response in physiologic conditions of persistent antigen provocation. PMID- 1719462 TI - Differential expression of the jun family members in rat brain. AB - The transcription factor AP-1 is phorbol ester-regulated and, as such, is considered to be a nuclear target of the signal transduction pathway involving protein kinase C. AP-1 is constituted by the various products of the jun and fos gene family members. These genes belong to the early response class and are inducible in different ways by growth factors, phorbol esters and depolarization. We studied the transcript distribution of c-jun, junB and junD in the rat brain. Our results show that the transcripts for these three genes are differentially distributed in various neuronal tissues. We also provide evidence for developmentally regulated expression of jun genes in post-natal brain. The spatiotemporal pattern of expression of c-jun, junB and junD offers clues to the understanding of the links between gene regulation and neuronal processes. PMID- 1719463 TI - Changes in phosphorylation of myc oncogene and RB antioncogene protein products during growth arrest of the murine lymphoma WEHI 231 cell line. AB - The expression of the protein products of the c-myc oncogene and retinoblastoma susceptibility gene (RB) was investigated during either goat anti-mouse immunoglobulin (GaMIg)- or phorbol ester (TPA)-induced growth arrest of the murine B-lymphoma cell line WEHI 231. Previously we have demonstrated that c-myc mRNA levels increase within 1-2 h of treatment, return to control levels by 4 h, and decline below these values by 24 h of treatment. Here we demonstrate that the level of c-myc protein synthesis and mRNA change in parallel. The predominant c myc protein expressed during the time course is the one initiated at the AUG codon (P2). The myc protein synthesized following 1-2 h of anti-immunoglobulin or TPA treatment migrates more slowly in a polyacrylamide gel as a result of increased phosphorylation. This hyperphosphorylation was no longer detectable by 4-6 h of treatment. Furthermore, the hyperphosphorylated myc protein appears to be more readily extractable with salt than the hypophosphorylated form. The product of the RB gene is present in multiple phosphorylation states in exponentially growing WEHI 231 cells. By 8 h of GaMIg or TPA treatment, a hypophosphorylated form begins to be detectable and significant levels were seen by 15 h. Thus post-translational control of both c-myc and RB expression occurs during the growth arrest of WEHI 231 cells. These changes in phosphorylation may play a role in mediating the cessation of proliferation of these cells. PMID- 1719464 TI - Effect of herbimycin A on growth and pp60c-src activity in human colon tumor cell lines. AB - The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125ng ml-1 dose of the antibiotic caused greater than 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dose-dependent inactivation of pp60c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60c-src kinase activity preceded changes in pp60c-src steady-state levels. Growth and pp60c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60c-src tyrosine kinase activity may be important in growth control of colon tumor cells. PMID- 1719465 TI - Expression of the Met/HGF receptor in normal and neoplastic human tissues. AB - The MET oncogene encodes a transmembrane tyrosine kinase receptor. Recently, hepatocyte growth factor (HGF), a potent growth factor for hepatocytes involved in liver regeneration, has been proposed as a ligand. In this paper, the physiological role of the human Met/HGF receptor is investigated by studying its specific distribution in normal and neoplastic tissues. Northern blot analysis has shown that the MET gene is selectively expressed in several epithelial tissues. High levels of MET mRNA have been found in liver, gastrointestinal tract, thyroid and kidney. Western blot analysis has shown that the levels of the Met protein generally correspond to those of the mRNA. However, in the thyroid, where there is a high level of MET mRNA, the protein was barely detectable, suggesting translational or post-translational regulation. The protein was also detected in the brain. Normal or increased levels of MET mRNA and Met protein were consistently found in fresh samples of carcinomas as well as in epithelial tumor cell lines. In thyroid carcinomas of a specific histiotype the amount of Met protein, almost undetectable in the normal counterpart, was found to be increased more than 100-fold. The tissue distribution of the Met/HGF receptor indicates that this molecule is involved in growth control of epithelial cells other than hepatocytes and suggests that its increased expression may confer a growth advantage to neoplastic cells. PMID- 1719466 TI - Different usage of two polyadenylylation signals in transcription of the N-myc gene in rat tumor cells. AB - The complete nucleotide sequence of a rat genomic DNA fragment of 6.9 kbp containing the entire N-myc gene was determined. A unique structural feature of the rat N-myc gene is the presence of two polyadenylation signals in the exon 3 region resulting in formation of two poly(A)+ N-myc mRNAs of 2.9 and 2.2 kb length. Elevated expression of the 2.9 kb mRNA, which was not due to gene amplification, was observed in normal rat tissues such as brain, adrenal and testis, and in rat tumor cells such as ascites hepatoma AH130, AH70Btc and AH7974 cells, and pituitary tumor GH3 cells. In contrast, expression of 2.2 kb mRNA, for which transcription was terminated at the upstream polyadenylation site, was observed only in GH3 cells. PMID- 1719467 TI - Nuclear localization is essential for the activity of p53 protein. AB - p53 appears to be a growth regulator, the perturbation of which induces changes in normal cell proliferation. Wild-type p53 protein is thought to function as a growth arrest gene, whereas mutant p53, which accumulates in transformed cells, has been shown to enhance malignant transformation. Both wild-type and mutant p53 migrate into the cell nucleus by means of identical nuclear localization signals (NLS) inherent in their primary sequences. Results presented here show that the suppressive activity of wild-type p53 measured as the reduction of transformation of primary rat fibroblasts induced by co-transfection with ras and either E1A or mutant p53, as well as the transformation enhancement of mutant p53 estimated by cooperation with ras in transformation of primary rat fibroblasts, is dependent upon nuclear localization signals in p53 protein. While transfection of unmodified wild-type p53 significantly reduces the number of rat embryonic fibroblast-transformed foci induced by E1A and ras or mutant p53 and ras, the wild-type p53 protein without NLS has completely lost this suppressive activity. Partially defective NLS wild-type p53, with a reduced nuclear accumulation ability, still exhibits some suppressive activity. In addition, we found that plasmids coding for intact mutant p53 protein efficiently cooperate with the ras oncogene, whereas the corresponding plasmids without NLS are totally inert. On this basis we conclude that nuclear localization of both wild-type and mutant p53 is a fundamental feature for manifesting the activities of these proteins. Both the suppressor activity mediated by the wild-type p53 and enhancement of transformation mediated by the mutant p53 require nuclear localization of the proteins to function. PMID- 1719468 TI - Population genetics of Plasmodium falciparum within a malaria hyperendemic area. AB - Serotyping with monoclonal antibodies was used to estimate the number and frequencies of allelic variants of two merozoite surface proteins, MSP1 and MSP2, and an exported protein Exp-1, in a sample of 344 clinical isolates of Plasmodium falciparum from an urban region of The Gambia. Represented among the isolates were 36, 8 and 2 alleles of the MSP1, MSP2 and Exp-1 loci respectively. Relative frequencies of these alleles remained stable in the parasite population over the 2 years of the study. A computer program was used to calculate from the frequencies of individual alleles at the three loci, the probable number of different genotypes in samples from the population, assuming random assortment among the loci. No significant difference was found between the expected and the observed genotype diversity. It is concluded that recombination among unlinked loci is a common consequence of sexual reproduction of P. falciparum in The Gambia. Slightly lower genotype diversity was observed in each of two villages, which may be a consequence of smaller population size compared with the urban region. PMID- 1719469 TI - Developmental and behavioral consequences of prenatal drug and alcohol exposure. AB - This article summarizes what is known about the effects of cocaine, opiates, marijuana, and alcohol on neonatal and postnatal growth and development. The development of a child affected by prenatal exposure to drugs and alcohol is best understood through a multifactorial model consisting of interrelated prenatal and postnatal factors. The article also describes the prenatal effects of drugs and alcohol on the newborn, especially on central nervous system functioning, which is seen as creating a biologic vulnerability that renders a child more vulnerable to the effects of poor caretaking. PMID- 1719470 TI - Pitfalls in developmental diagnosis. AB - The more common and more glaring pitfalls in developmental diagnosis encountered during infancy and early childhood have been outlined. Much of the ability to avoid these traps depends on a comfortable understanding of the four spheres of early child development and a sound familiarity with the principles of developmental assessment, especially the separation of intellectual and motor entities. Motor milestones are excellent indicators of motor competence but correlate poorly with intellectual capacity. Language and problem-solving milestones in infancy provide the best insights into a child's intellectual potential, and their evolution is independent of motor competence. They may be obscured by motor disability and as a result may be more difficult to demonstrate, but that is a separate issue. In that instance there is nothing subtle about the fact that one is already dealing with a disabled infant. Psychosocial abilities (affective milestones) are critical in understanding the whole child and in making a meaningful statement about behavior, but they lend little additional information to the assessment of intellectual and motor competence. For physicians the "curb-side consult" is a highly efficient tool that has great practical application to developmental concerns and especially to the avoidance of the pitfalls described. Every practitioner should have a resource in developmental and behavioral pediatrics with whom he or she can communicate in an informal fashion. This is especially valuable in situations in which the urgency or even the need for referral (a time-consuming, expensive, and often anxiety-provoking process) is not clear. PMID- 1719471 TI - Effectiveness of developmental intervention in the first five years of life. AB - Developmental intervention in the first 5 years of life is an expanding, complex enterprise. Documenting efficacy by traditional scientific methods has proven to be elusive for a number of practical reasons, e.g., target population heterogeneity, methodology variability, inadequate outcome measures, and cost of longitudinal cohort designs. Nevertheless, despite these shortcomings, there is accumulating research information as to which types of intervention approaches are likely to be most beneficial to specific groups of infants and children and their families. It is quite clear that preventive strategies for at-risk children and families are different than ameliorative strategies for children with established disabilities. It is also clear that comprehensive evaluation of effectiveness must include consideration of both functional child gains (e.g., social, communication, mobility, and adaptive skills) and enhancement of family function. It is the pediatrician's responsibility to be adequately informed about contemporary developmental interventions in order to balance parental hopes and needs with potential benefits. PMID- 1719472 TI - [The use of proteolytic inhibitors in the comprehensive treatment of children with chemical burns of the esophagus]. PMID- 1719473 TI - Emission controls on cars and accidental deaths from car exhaust. PMID- 1719474 TI - Postnatal induction and neural regulation of inward rectifiers in mouse skeletal muscle. AB - The whole-cell voltage-clamp technique was used to examine developmental changes of inward rectifier currents in fibres of the flexor digitorum brevis muscle acutely isolated from mice on postnatal day 0 (P0) to P36. Neither a steady-state component (Is-s) nor a slowly activated component (Irise) of inward rectifier currents were observed in fibres of P0 and P4 mice. Both Is-s and Irise became apparent between days P8 and P16. The specific amplitudes of Is-s and Irise measured at a test-pulse potential of -100 mV at 20 mM extracellular K+ [( K+]o) increased to their respective platcau values of -68 +/- 10 and -15 +/- 7 microA/cm2 at P20. In fibres denervated on day P4 the developmental increase of Is-s was suppressed, its specific amplitude at P20 being one-tenth of that in the corresponding normal fibres. Irise did not appear in P4-denervated fibres throughout the development. In muscle fibres denervated at P16 or P20, the specific amplitudes of Is-s and Irise decreased, reaching the levels of P4 denervated fibres in 2-4 days after denervation. We conclude that Is-s and Irise develop within 3 weeks after birth, and suggest that innervation plays a key role in their induction. PMID- 1719475 TI - [The surgical treatment of spinal metastases in Scandinavia]. AB - The article consists in a review of the findings in a questionnaire study, which indicate that the adoption of new surgical techniques varies from one centre to another. Several prognostic factors have been identified, that enable the differentiation of treatment from palliation to reconstruction. The new fixation methods yield improved functional outcome without a significant increase in surgical trauma. An active surgical approach is outlined which, in conjunction with overall oncological care, may improve the patient's quality of life. PMID- 1719476 TI - Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco. AB - Three cDNA clones coding for eukaryotic translation initiation factor 4A, eIF-4A, were isolated from a Nicotiana plumbaginifolia root cDNA library by heterologous screening. The clones comprise two distinct gene classes as two clones are highly similar while the third is divergent. The genes belong to a highly conserved gene family, the DEAD box supergene family, although the divergent clone contains a DESD box rather than the characteristic DEAD box. The two clones are representatives of separate small multigene families in both N. plumbaginifolia and N. tabacum. Representatives of each family are coordinately expressed in all plant organs examined. The 47 kD polypeptide product of one clone, overexpressed in E. coli, crossreacts immunologically with a rabbit reticulocyte eIF-4A polyclonal antibody. Taken together the data suggest that the two Nicotiana eIF 4A genes encode translation initiation factors. The sequence divergence and the coordinate expression of the two Nicotiana eIF-4A families provide an excellent system to determine if functionally distinct eIF-4A polypeptides are required for translation initiation in plants. PMID- 1719477 TI - Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants. AB - Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents. PMID- 1719478 TI - Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo. AB - In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro. PMID- 1719479 TI - MspI polymorphism of the human CYP2E gene. PMID- 1719480 TI - MspI RFLP of the human TCR gamma chain variable gene V9 (TCRGV9). PMID- 1719481 TI - Ara-ATP impairs 3'-end processing of pre-mRNAs by inhibiting both cleavage and polyadenylation. AB - Previous studies have demonstrated that Ara-ATP can inhibit poly(A) polymerase activity by competing with ATP. To elucidate the mechanism of action of this compound, its effect on the cleavage and polyadenylation of two specific substrates, SV40L and adenovirus L3 pre-mRNAs, was studied in HeLa nuclear extracts. Unlike cordycepin 5' triphosphate, Ara-ATP inhibited both cleavage and poly(A) addition. Addition of poly(A) polymerase fraction devoid of any other factors required for the processing reactions overcame the inhibitory effect on cleavage as well as polyadenylation of pre-mRNAs. These data suggest that Ara-ATP inhibits both cleavage and polyadenylation reactions by interacting with the ATP binding site on poly(A) polymerase, the activity of which is essential for the cleavage reaction. Ara-ATP also blocked formation of the post-cleavage and polyadenylation-specific complexes, which further confirmed the inhibitory effect of the ATP analog on the two tightly coupled 3'-end processing reactions. PMID- 1719482 TI - Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. AB - Antibody-binding epitopes in the central helical region of the muscular dystrophy protein, dystrophin, have been mapped using a new strategy of transposon mutagenesis. Tn1000 transposons carrying translation termination codons were introduced randomly by bacterial mating into a large fragment of dystrophin cDNA in a pEX2 plasmid to produce a library of transformants expressing truncated dystrophin fusion proteins. Epitopes were progressively lost as the expressed sequences were shortened, enabling the epitopes recognised by 22 monoclonal antibodies to be placed in order along the dystrophin molecule without in vitro manipulation of DNA. The C-terminus of each truncated fusion protein was precisely located within the dystrophin sequence by direct sequencing of pEX2 transformants using transposon-specific primers. Sequences as short as 7 and 17 amino-acids have been identified as essential for antibody binding in this way. Nineteen of the 22 monoclonal antibodies had been selected for their ability to bind both native and SDS-denatured dystrophin and 15 of these bind to one sequence of 74 amino-acids (residues 1431-1505 of the 3684 residue sequence). This may be an area of high immunogenicity or of close structural similarity between native dystrophin and the SDS-treated recombinant fragment used for immunization. PMID- 1719483 TI - A thermodynamic study of unusually stable RNA and DNA hairpins. AB - About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs. PMID- 1719484 TI - Two distinct human DNA diesterases that hydrolyze 3'-blocking deoxyribose fragments from oxidized DNA. AB - Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis. Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells. The two activities were purified to differing degrees from HeLa cells. One enzyme had the properties of the known HeLa AP endonuclease (Mr approximately 38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein. The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity. This second enzyme may have been detected in other studies but never characterized. In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3' deoxyribose diesterase activities. It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected. The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused. PMID- 1719485 TI - Analysis of the gene encoding the RNA subunit of ribonuclease P from T. thermophilus HB8. AB - The gene for the RNA subunit of ribonuclease P from the extreme thermophilic eubacterium T. thermophilus HB8 was cloned using oligonucleotide probes complementary to conserved regions of RNase P RNA subunits from proteobacteria. The monocistronic gene and its flanking regions were sequenced. The gene is enclosed by a promoter and a rho-independent terminator. Nuclease S1 protection analyses showed that the primary transcript is identical with the mature RNA, i.e. no processing events are involved. The stem and loop structure of the terminator remains part of the mature molecule. In vitro transcription of the cloned gene with purified RNA polymerase from T. thermophilus yields the same RNA product as in vivo, indicating that no other components except RNA polymerase are involved in the synthesis of the RNA. RNase P RNA from T. thermophilus cleaved a pre-tRNA(Tyr) from E. coli with highest efficiency between 55 degrees C and 65 degrees C. The T. thermophilus RNA, which has a G-C content of 86% in helical regions, displays several structural idiosyncrasies, although its secondary structure is similar to that of proteobacteria. Numerous invariable nucleotides in the structural core of eubacterial RNase P RNAs are also conserved in the RNA from the extreme thermophilic eubacterium. PMID- 1719486 TI - Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry. AB - We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9 dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports. PMID- 1719487 TI - Specific incorporation of glycine into bacterial lipopolysaccharide. Novel function of specific transfer ribonucleic acids. AB - It has been found that the bacterial endotoxins (lipopolysaccharides, LPSs) contain some amino acids and glycine is the most abundant amino acid in the polysaccharide core preparations of LPSs of gram-negative bacteria. Until now nothing was known about the mechanism of amino acid incorporation into the lipopolysaccharide core. We found that one out of three glycyl-tRNAs(Gly) from Escherichia coli is the donor of amino acid and is the substrate for a putative aminoacyl-tRNA:LPS transferase. We have isolated, purified this tRNA and determined its nucleotide sequence to be major E.coli tRNA(3Gly). This tRNA(Gly) (anticodon GCC) conserved the tRNA structural features. The assay for determination of the specific incorporation of glycine into the lipopolysaccharide was also invented and described. PMID- 1719488 TI - Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood. PMID- 1719489 TI - An efficient method for isolation of RNA from tissue cultured plant cells. PMID- 1719490 TI - AhaII polymorphism in human X-linked proteolipid protein gene (PLP). PMID- 1719491 TI - A polymorphic DNA probe (D22S263) mapping to 22q12.1-q13.1. PMID- 1719492 TI - The patient with ventricular arrhythmias can be offered optimal treatment on the basis of simple clinical variables. PMID- 1719493 TI - Recovery of sinus node function after pacemaker implant for sinus node disease following cardiac transplantation. AB - In a 4-year interval, 3/55 (7.8%) heart transplant recipients developed (greater than 4 weeks) severe sinus node dysfunction. An epicardial ventricular pacemaker was implanted at 90, 40, and 38 days after operation. Follow-up ranged from 7 to 20 months. In two of the three cases, failure of stimulation or sensing was detected at the 3rd and 4th month after pacemaker implantation. Sinus rhythm reappeared in two of the three patients at the 2nd and 4th months after transplantation. Severe sinus node disease requiring pacemaker implantation can improve following the first 3 months after transplantation. Failure of stimulation or sensing may not be uncommon. PMID- 1719494 TI - Pacemaker syndrome in a patient with DDD pacemaker for long QT syndrome. AB - A patient with long QT syndrome was treated with beta blockers and had a permanent DDD pacemaker implanted. The lower rate was set to 85 beats/min because this provided the best shortening of QT interval at the lowest paced heart rate. The atrioventricular (AV) delay was programmed to 250 msec to allow native AV conduction. Patient returned complaining of symptoms suggestive of pacemaker syndrome. ECG during one of these episodes showed AV sequential pacing. Doppler echocardiography of hepatic vein flow suggested atrial contraction against a closed tricuspid valve. Endocardial electrogram telemetry demonstrated ventriculoatrial (VA) conduction with the retrograde atrial electrogram falling within the atrial refractory period and thus was not sensed. The following atrial stimulus did not capture because of the atrial refractoriness. Ventricular pacing proceeded after the programmed AV delay. Reprogramming the AV delay to 200 msec restored AV synchrony by allowing the atrial stimulus to capture by placing it outside of the refractory period of the atrium. No further symptoms reported during six months of follow-up. PMID- 1719495 TI - Active fixation of endocardial pacing leads: the preferred method of pediatric pacing. AB - Pacing system failure due to lead related problems may necessitate repositioning or explantation of the problem lead. Pediatric patients with permanent pacemakers have additional considerations that necessitate revision or explantation of pacing leads. Active fixation type leads appear to offer the physician advantages over passive fixation leads that may make them the lead of choice for use in children. We reviewed our experience with active fixation type leads to determine whether the ease with which these leads could be revised or explanted justified recommending their use in our patients. Eleven patients underwent 13 lead revisions. The time from implant to revision was a mean of 12.3 months. Six patients had previously undergone repair of a congenital heart defect. Modes of pacing were: DDD (seven); AAI (three); and VVI (one). Exposed, isodiametric leads accounted for 11/13 leads. Leads were successfully explanted in nine cases and repositioned in four cases. The only lead that could not be revised and resulted in retention was a nonisodiametric, retractable helix lead at the junction of the subclavian vein and clavicle. We conclude isodiametric active fixation leads can be safely repositioned or explanted in children and should be considered the preferred method for endocardial pacing in children. PMID- 1719496 TI - Low energy catheter ablation of a posteroseptal accessory pathway associated with a diverticulum of the coronary sinus. AB - A 23-year-old man was resuscitated from ventricular fibrillation and subsequently shown to have the Wolff-Parkinson-White syndrome. Electrophysiological study demonstrated a posteroseptal accessory pathway, and coronary sinus angiography demonstrated that this was associated with a diverticulum of the coronary sinus. Catheter ablation was performed using a new low energy system. Five shocks were delivered within the coronary sinus diverticulum, with a cumulative energy of 39 joules (J). Accessory pathway conduction was blocked successfully, and there were no complications. PMID- 1719497 TI - Experimental study about removal of the implanted tined polyurethane ventricular lead by radiofrequency waves through the lead. AB - Polyurethane pacemaker leads are widely used nowadays. However, only a few studies have been done to investigate the fixation mechanism of polyurethane leads. To elucidate how pacemaker leads are fixed at the early phase after implantation, polyurethane-insulated tined ventricular leads were implanted in seven mongrel dogs. One to 4 months later, tips of the leads were anchored among the trabeculae and the distal part of the leads were encapsulated by whitish fibrous tissue. It was found that not organized thrombi, but cell reaction with various stages of inflammatory cells was responsible for forming the fibrous tissue. We attempted to remove the lead by delivering radiofrequency wave through the lead. However, no lead could be removed. PMID- 1719498 TI - Diagnostic value of synchronized transesophageal atrial pacing. AB - Synchronized transesophageal atrial pacing (single and double extrastimuli) was used in 137 patients with various tachycardias inducible by atrial pacing during transesophageal electrophysiological study (EPS). This pacing mode in five patients initiated atrioventricular tachycardias with ipsilateral bundle branch block not seen when using other pacing modes. During the tachycardia, single or double extrastimuli caused ipsilateral bundle branch block disappearance in two patients with atrioventricular tachycardia, and changed AV activation ratio in one patient with atrioventricular junctional reentrant tachycardia. This pacing mode causes very little discomfort, what is important in children, and enhances diagnostic abilities of transesophageal EPS. So, this pacing mode should be used routinely as one of the steps of transesophageal EPS. PMID- 1719499 TI - Orthodromic and antidromic pacemaker circus movement tachycardia. AB - Pacemaker circus movement tachycardia (PCMT) during DDD pacing is usually sustained by retrograde natural and antegrade electronic atrioventricular (AV) conduction. As PCMT is often initiated by a ventricular premature beat (VPB) one method of its prevention is the programming of an atrial stimulus synchronously following a ventricular extrasystole. A patient is described with preserved antegrade, but without retrograde, i.e., VA, conduction. The optional pacemaker mode of synchronous atrial stimulation following a VPB caused an unusual PCMT sustained by retrograde electronic and antegrade natural AV conduction. This PCMT is similar to a natural reentry tachycardia, the most common variety of which (based on retrograde conduction) is termed antidromic and that which we describe is orthodromic. PMID- 1719500 TI - Effects of lidocaine on defibrillation threshold in the pig: evidence of anesthesia related increase. AB - Some antiarrhythmic drugs may influence the ability of a shock to defibrillate a patient but the effect of lidocaine on defibrillation efficacy has been controversial, suggesting multiple influencing factors. We determined the effects of three doses of lidocaine on defibrillation thresholds, using single and sequential shocks, in 36 open chest halothane-anesthetized pigs. An additional eight pigs were anesthetized with barbiturate and received the highest infusion regime of lidocaine. Shocks were delivered through three mesh electrodes sutured over the anterior right ventricle, posterior right ventricle, and lateral left ventricle for sequential pulse shocks and between a lateral right to a lateral left ventricular mesh electrode for single pulse shocks. Triplicate defibrillation thresholds (DFTs) were obtained before and after lidocaine (n = 32) or saline (n = 12) administration, either with halothane or barbiturate anesthesia. Lidocaine did not alter DFT at any dose with either the single pulse (control 17.4 +/- 3.7 joules [J], highest dose of lidocaine 13.5 +/- 1.7 J, P = NS) or sequential pulse shocks (control 7.6 +/- 0.8 J, highest dose lidocaine 6.6 +/- 0.7 J, P = NS), when halothane anesthesia was used. Similar results were obtained with lower doses. In contrast, lidocaine in pentobarbital anesthetized pigs produced a significant increase of the DFT with single (control 13.7 +/- 1.9, during lidocaine 16.6 +/- 3.1, P less than 0.01) and sequential shocks (control 11.1 +/- 2.2, during lidocaine 14.5 +/- 3.4, P less than 0.01). Interaction between barbiturates and lidocaine, and/or pH may account for the inconsistency in previous studies and must be considered for animal and clinical experiments. PMID- 1719501 TI - Transesophageal echocardiography to improve positioning of radiofrequency ablation catheters in left-sided Wolff-Parkinson-White syndrome. AB - Two patients with the Wolff-Parkinson-White syndrome who underwent successful radiofrequency catheter ablation of their left-sided bypass tracts are described. Transesophageal echocardiography, a relatively new echocardiographic technique, was utilized in both patients and provided excellent visualization of intracardiac anatomy as well as the catheter tip. Transesophageal echocardiography was also synergistic with fluoroscopy and the intracardiac electrogram in providing more precise catheter placement. In addition, the use of transesophageal echocardiography may reduce fluoroscopic exposure and shorten the procedure time. PMID- 1719502 TI - Complications associated with retained pacemaker leads. AB - Retention of functionless pacemaker leads may occur following mechanical or infective problems (potentially or definitely infected) or after electrical failure of the lead. One hundred nineteen patients with a pacemaker lead (or leads) retained between 1970 and 1990 were reviewed retrospectively. Lead retention after an intervention dictated by potential or definite infection of the pacing system resulted in complications in 27 of 53 patients (51%), which in 22 patients (42%) were major (septicemia, superior vena cava syndrome, and further surgery under general anesthesia for recurrent "infective" problems) including three deaths. Complications were less likely if lead retention occurred after electrical failure with three minor and two major (surgery under general anesthesia, superior vena cava syndrome) complications in 66 patients (P less than 0.001). Bacteriology of swabs taken at the time of retention in the patients with potential or definite infection was unhelpful in predicting future complications: 8/18 patients (44%) whose swabs were negative had complications of which 5/18 (28%) were major. In our experience retention of functionless pacemaker leads after an intervention dictated by potential or definite infection of the pacing system, is associated with significant morbidity and mortality and should be avoided. PMID- 1719503 TI - Practical aspects of rate adaptive atrial (AAI,R) pacing: clinical experiences in 44 patients. AB - Forty-four patients with sinus node disease and chronotropic incompetence but no evidence of AV conduction disturbances were treated with rate adaptive atrial (AAI,R) pacemakers. Medtronic Activitrax and Siemens Sensolog activity sensing single chamber pulse generators were used. Twenty-four patients (55%) had the bradycardia-tachycardia syndrome. The mean follow-up time is 20 +/- 14 months (range 1-48, median 17 months). All patients remain alive. Two patients were reoperated upon for lead problems without change of pacing mode. One patient developed symptomatic second-degree Wenckebach block during follow-up, and received a DDD,R system. Although 22 of the patients were treated with antiarrhythmic drugs postoperatively, no further cases of significant AV conduction disturbances were seen. During rapid atrial pacing, exercise-induced enhancement of AV conduction was a consistent finding, although less pronounced in patients treated with beta-blocking drugs. One patient developed permanent atrial fibrillation with an adequate ventricular rate. By systematic reprogramming procedures, QRS complex sensing through the atrial electrode could be demonstrated in 25 patients (23/28 with unipolar and 2/16 with bipolar leads). It could be counteracted effectively by pulse generator program selection in all cases. Forty-two of 44 patients (95%) remain in AAI,R pacing with normal function. Rate adaptive atrial pacing can be successfully applied in this patient group. PMID- 1719504 TI - Automatic recognition of ventricular arrhythmias using temporal electrogram analysis. AB - Future antitachycardia devices must be able to deliver a variety of therapies according to the requirements of the underlying arrhythmia. To ensure that appropriate treatment is prescribed the device must use a detection algorithm that is able to discriminate between multiple arrhythmias. Current criteria such as rate, change of rate, duration at high rate, and high rate stability are inadequate for this purpose. Many algorithms that evaluate the morphology of the endocardial electrogram are of too great a complexity to be incorporated in implantable devices that require real-time analysis without undue power consumption. In this study the sensitivity of a simple morphological technique (temporal electrogram analysis) is examined. The method sets threshold 'rails' above and below the isoelectric line and classifies ECG complexes according to the sequence and duration of rail excursions. A total of 27 ventricular tachyarrhythmias were induced in 25 patients (17 with a history of recurrent arrhythmias and eight undergoing risk stratification postmyocardial infarction). Temporal electrogram analysis (TEA), initially detected the onset of the ventricular arrhythmia in all patients whose surface ECG showed polymorphic or right bundle branch block pattern tachycardia, in 5/8 of cases with left bundle branch block pattern and in 4/5 of patients with concordant complexes across the precordial leads. After minor modifications the overall sensitivity of the method was improved to 95% (26/27 arrhythmias detected). TEA is a technique of low computational demands, which in this study, reliably discriminated between resting sinus rhythm and ventricular arrhythmias. PMID- 1719505 TI - Persistent atrial standstill--clinical, electrophysiological, and morphological study. AB - Persistent atrial standstill (PAS) is a rare disorder characterized by absence of atrial activity on the surface and intracavity electrograms, absence of atrial mechanical activity, and inability to electrically stimulate the atria. Four patients (ages 18-60 years) with PAS were evaluated. One of these (no. 3) only had right atrial (RA) standstill, whereas left atrium (LA) showed spontaneous activity and could be stimulated electrically. As RA biopsy is not possible, right ventricular (RV) endomyocardial biopsy (EMB) was obtained to identify possible atrial pathology that revealed inflammatory myocarditis, 2; amyloidosis, 1; and myocardial hypertrophy with fibrosis, 1. Three patients were given permanent pacemakers. One of these with amyloidosis died suddenly. One is lost to follow-up. The others cases are persisting with PAS. PMID- 1719506 TI - Paced beats following single nonsensed complexes in a "codependent" cardioverter defibrillator and bradycardia pacing system: potential for ventricular tachycardia induction. AB - Patients with implantable defibrillators often require bradycardia pacemakers. Adverse interactions between separate defibrillator and bradycardia pacing units have occurred, including failure to detect ventricular fibrillation due to persistent bradycardia pacing during the arrhythmia. A device with combined bradycardia pacing and antitachycardia therapy capability may obviate adverse device interactions. We describe a previously unrecognized phenomenon that may occur in a combined device when the algorithms for sensing bradycardia and tachycardia are "codependent"; that is, the circuitry for brady- and tachyarrhythmia detection relies on the same automatic gain sense amplifier. Three of 37 patients in whom the device was implanted had ventricular tachycardia initiated when bradycardia pacing stimuli were delivered by the device after probable nonsensed sinus beats. In each case, nonsensed beats appeared to have a markedly diminished amplitude, occurred after ventricular premature depolarizations that produced large amplitude electrograms, and had an electrogram morphology that matched that of sinus rhythm. In each case, the bradycardia pacing interval was at least 1,200 msec (range 1,200 to 1,714 msec). In two of the three patients, large amplitude ventricular premature depolarizations or nonsustained ventricular tachycardia caused an adjustment of the gain control that potentiated the failure to sense the subsequent lower amplitude signal. In all three patients, the induced arrhythmia was rapidly terminated by pacing or cardioversion. Decreasing the bradycardia pacing interval by 110-514 msec has prevented recurrence during short-term follow-up. Our findings suggest that codependent bradycardia and antitachycardia devices may have their own unique potential difficulties in adapting to rapid changes in rate and signal amplitude. PMID- 1719507 TI - Experience with a new implantable pacer-, cardioverter-defibrillator for the therapy of recurrent sustained ventricular tachyarrhythmias: a step toward a universal ventricular tachyarrhythmia control device. AB - Ten consecutive patients (mean age 57.9 +/- 7.6 years) were treated with an investigational tachyarrhythmia control device, the implantable Medtronic Pacer-, Cardioverter-, Defibrillator model 7216A or 7217B. All patients had coronary artery disease with old myocardial infarctions and presented hemodynamically significant sustained ventricular tachyarrhythmias not suppressed by antiarrhythmic drug therapy and unrelated to acute myocardial infarction. In two patients a nonthoracotomy lead system was implanted. Lowest effective defibrillation energy ranged from 5 to 18 joules (mean 12.2 +/- 4 joules) for the epicardial bielectrode systems and were 15 and 18 joules for the nonthoracotomy lead system implants. The postoperative periods were unremarkable. Follow-up ranged from 7 to 19 months (mean 13.8 +/- 4.5 months). Spontaneous tachyarrhythmia episodes were detected and treated by the device in six patients, five of them received staged therapies. No deaths occurred and no hospital admissions were necessary for device related or ventricular tachyarrhythmia related complications. In conclusion, this integrated device represents a major step toward the development of a universal ventricular arrhythmia control device. PMID- 1719508 TI - Clinical applications of external pacing: a renaissance? AB - It is nearly 40 years since the first reports of noninvasive external pacing for Stokes-Adams syncope. Despite the ease and safety, this method of pacing has yet to flourish despite a recent interest by several authors. At present, external pacing seems best suited for temporary pacing situations that arise as an emergency or for purely prophylactic indications. External pacing is the preferred method of pacing recommended in the advanced cardiac life support guidelines. However, most emergency room and prehospital cardiac arrest trials have not shown any significant benefit from early application of external pacing. The indications have been broadened to include symptomatic bradycardia and termination of some ventricular tachycardias. It may be useful for the termination of AV reciprocating tachycardia and AV nodal reentrant tachycardia. There is a vision that external pacing may be used for serial electrophysiological testing of antiarrhythmic agents. However, there is little data in this regard. More importantly, the external pacing thresholds must be reduced further to allow for sophisticated pacing protocols to be implemented. For practical purposes, external pacing does not capture the atrium. Since the left atrium is easily captured by esophageal pacing, it is likely that noninvasive external pacing will be combined with transesophageal pacing to perform noninvasive electrophysiological testing. The future for external pacing remains in limbo because of the discomfort associated with skeletal muscle contraction. If technical advances can reduce or eliminate this problem, then external pacing may find broader application for bradycardia and tachycardia. PMID- 1719509 TI - A teaching booklet for patients receiving high dose rate brachytherapy. AB - A teaching booklet developed for patients with lung cancer who were about to receive high dose rate (HDR) brachytherapy is described and presented. The booklet was developed by nurses assigned to a radiation oncology center and details the procedure using text and photographs. PMID- 1719510 TI - Reconstituted basement membrane promotes morphological and functional differentiation of primary human prostatic epithelial cells. AB - Prostatic epithelial cells undergo rapid proliferation and lose their ability to synthesize and secrete prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) under standard tissue culture conditions. Herein, we compared the morphology, growth, secretory activity, and intermediate filament expression of human prostatic epithelial cells cultured on either standard tissue culture plastic or reconstituted basement membrane. Epithelial cells grown on plastic exhibited a 10-fold increase in proliferation and a higher percentage of cells in the S-phase of the cell cycle compared to cells cultured on basement membrane. However, cells grown on basement membrane secreted markedly higher levels of PSA and PAP. The basement membrane-induced enhancement of secretory activity was potentiated by dihydrotestosterone (DHT) and prostate stromal cell conditioned medium. Morphological studies showed that cells plated on basement membrane formed organoid-like clusters and maintained several aspects of differentiated epithelium including abundant secretory vesicles, microvilli, and desmosomes with associated cytoskeletal elements. Cultivation of epithelial cells on basement membrane components also suppressed the expression of vimentin, a mesenchymal intermediate filament polypeptide. However, cytokeratin expression was abnormal in cells grown on either surface. These results indicate that the differentiated properties of prostatic epithelial cells are promoted by cultivation on reconstituted basement membrane in the presence of DHT and stromal cell conditioned medium. PMID- 1719511 TI - Endogenous protein substrates for prostatic acid phosphatase in human prostate. AB - In order to identify the endogenous phosphoprotein substrates for human prostatic acid phosphatase (PAP), cellular proteins of human normal, benign, and malignant prostatic tissues as well as carcinoma cell lines were phosphorylated by the cellular kinases in the presence of (gamma-32P)-ATP and then were subjected to dephosphorylation reaction by PAP. Of several endogenous phosphoproteins, PAP preferentially dephosphorylated a cytosolic protein of Mr 83 kDa. The dephosphorylation of the 83 kDa phosphoprotein (designated pp83) by PAP was uniformly observed in all cells/tissues of prostate origin, and was completely inhibited by L(+)-tartrate, the classic inhibitor of PAP. Phosphoamino acid analysis revealed that pp83 was a tyrosine-poor phosphoprotein and was mostly dephosphorylated by PAP at serine/threonine residues rather than tyrosine residues. Further comparison of dephosphorylation rate with that of an endogenous phosphotyrosine-containing phosphoprotein (pp53) revealed that PAP possessed both phosphoserine/threonine protein phosphatase and phosphotyrosine protein phosphatase activity. These results demonstrate that pp83 apparently is an endogenous substrate of PAP in human prostate, and that, instead of a phosphotyrosine protein specific phosphatase, PAP is a universal protein phosphatase hydrolyzing equally well the phosphotyrosine, serine, and threonine residues. PMID- 1719512 TI - Antidepressant drugs and ventricular arrhythmias. PMID- 1719513 TI - Counteraction by nicardipine of the ionophoretic effect of gliclazide: a mitochondrial study. AB - Using mitochondria, we demonstrate that gliclazide exhibits a calcium ionophoretic activity. Indeed, gliclazide induces a decrease in the respiratory control of mitochondria; this effect is increased by addition of Ca2+ and corrected by ruthenium red, all characteristics of a calcium ionophoretic compound. Our results, on a biological membrane, confirm previous findings in vitro. Moreover, we show that nicardipine counteracts the action of gliclazide. Besides the effect on ATP-stimulated K+ channels, the ionophoretic effect of gliclazide may play a role in its hypoglycaemic effect, thus this counteracting action of nicardipine might induce drug interaction during the concomitant clinical administration of gliclazide and nicardipine. PMID- 1719514 TI - Effects of cilazaprilat and enalaprilat on experimental dermatitis in guinea pigs. AB - Two non-sulfur containing ACE-inhibitors were tested concerning their local effect on experimental dermatitis in ovalbumin-sensitized guinea pigs. Enalaprilat but not cilazaprilat potentiated the ovalbumin-evoked inflammatory response. Furthermore, enalaprilat clearly enhanced the erythema evoked by substance P, whereas cilazaprilat did not. Concerning, the bradykinin-evoked erythema, enalaprilat significantly potentiated the response, whereas cilazaprilat only caused a slight increase. Our results suggest that different affinities for peptidases involved in degradation of inflammatory peptides can explain differences between the pro-inflammatory properties of enalaprilat and cilazaprilat. PMID- 1719515 TI - Inhibition of cell growth and DNA, RNA, and protein synthesis in vitro by fentanyl, sufentanil, and opiate analgesics. AB - We have studied the cytotoxic nature of two groups of narcotic analgesics. Group 1 consists of the opioids, morphine, codeine, hydromorphone, thebaine, and etorphine. Group II contains but two phenylpiperidine-type narcotics, fentanyl and sufentanil. To measure cytotoxicity, three different bioassays were employed using an established line of human cells. Specifically, the effects of narcotic analgesics on DNA, RNA, and protein synthesis were measured by following the uptake and incorporation of radiolabeled thymidine, uridine, and amino acids, respectively. Inhibition of cell growth also was studied by measuring population doubling times of logarithmically growing cells in the presence (or absence) of the test compounds. Lastly, cloning efficiencies of cells were determined in the presence of both groups of compounds. Group I compounds were significantly less inhibitory than Group II compounds by all three bioassays. Moreover, flow cytometric DNA analysis of cells treated with 100 and 320 microM etorphine HCl showed essentially no effects on cell cycle distribution. These in vitro results thus suggest that (1) fentanyl and sufentanil are inherently more cytotoxic than the opioid narcotics in Group I, and (2) the highly potent morphinoid drug etorphine HC1 appears to have special promise as a transdermal narcotic to control pain. PMID- 1719516 TI - Dual effect of magnesium on compound 48/80-induced histamine secretion from rat peritoneal mast cells. AB - The effect of magnesium on the secretory response to compound 48/80 from rat peritoneal mast cells was studied. The decrease in secretion caused by calcium deprivation was enlarged by magnesium. Glucose partially counteracted the decrease caused by calcium deprivation but not the one caused by magnesium. The addition of calcium to the cells simultaneously with compound 48/80 completely restored the secretory response if magnesium was present. The response was only partially restored in a magnesium- and glucose-free medium, whereas it was almost completely restored if glucose was present. Magnesium had a considerable effect on the restoration of the secretory response of EGTA-treated cells, whereas the effect of glucose was minimal indicating that an effect on the energy metabolism was of minor importance. The secretory response could also be restored by an exposure of the cells to calcium prior to stimulation with compound 48/80. This was, however, only observed if magnesium was present and glucose had no effect. The influence of magnesium on the restoration of the secretory response may partly occur by an effect on the energy metabolism, partly by an effect on the stimulus-secretion coupling. We propose that insufficient supply of Mg2+ to the G protein during activation by compound 48/80 might cause a suboptimal signal transduction. PMID- 1719517 TI - [Postoperative spindle cell nodules of the bladder and prostate]. PMID- 1719518 TI - [Electron microscopy studies of the surface epithelium of the stomach mucosa in man with reference to secretory function and cell desquamation]. PMID- 1719519 TI - [Diseases observed after return from travels outside Europe. 109 cases]. AB - We report a prospective study of travel-associated illnesses observed after their return in 109 French travellers, including 86 tourists. Sixty-three were returning from Africa and 84 percent had been abroad for less than 4 weeks. The percentages of travellers immunized against tetanus, poliomyelitis and typhoid fever were 70, 63 and 36 percent respectively. Malaria prophylaxis was well adjusted to current recommendations in only 19 patients; for 9 patients it was a routine visit. One hundred patients reported 105 diseases. The diagnosis was undetermined in 31 patients, including 19 with diarrhoea and 8 with fever, and it was determined in 74 patients who were found to have malaria (14), cutaneous myiasis (12) or bacterial skin infections (12). PMID- 1719520 TI - [Diagnosis of premature membrane rupture by searching for alpha-fetoprotein in vaginal secretions]. PMID- 1719521 TI - [Cytokeratin expression in fetal lung--immunohistochemical studies]. AB - This paper is to give information about the pattern of expression of cytokeratin in bronchial mucosa, alveolar epithelium, lung stroma and pleura during the prenatal development of the lung. Our immunohistochemical investigations occurred in the lung of a 6 week old embryo and in 21 lungs of intrauterine died fetuses from the 12th to the 36th week of gestation according to the modified avidin biotin-method. Findings of investigations will be compared with expression patterns in tumors and normal lungs of adults. In summary the investigations showed the following results: 1. Immunohistochemically in fetal lungs proteins are demonstrable that are used in the pathology of adults as markers for the histogenetic differentiation of various cell forms. 2. An expression of cytokeratins we could constantly prove in epithelial cells of fetal lungs after the 6th week of gestation. 3. Cytokeratins are mostly found at the cell membranes. 4. With increasing differentiation in direction to alveolar epithelium changes the composition of cytokeratin in the cells. 5. Cells of the pleura mesothelium are expressing cytokeratins in a pattern similar to that of bronchiolar epithelium. All in all the pattern of antigenic expression in fetal lungs considerably assimilates to that in adult lungs during the fetal lung development. PMID- 1719522 TI - The ileum and carbohydrate-mediated feedback regulation of postprandial pancreaticobiliary secretion in normal humans. AB - We investigated the effect of perfusing carbohydrate into the ileum on postprandial pancreaticobiliary secretion and the relationships among carbohydrate in the ileum, pancreaticobiliary secretion, and gastric emptying. Eighteen healthy volunteers were intubated with a multilumen oroileal tube. A metal labeled with 111In-diethylenetriamine-pentaacetic acid (400 calories; 60% carbohydrate, 20% protein, 20% fat) was then infused into the stomach, and a carbohydrate solution (rice starch + glucose) was perfused into the terminal ileum at rates (mg/min) of 0 (saline; n = 6), 12.5 (n = 4), 25 (n = 4), 50 (n = 2), and 100 (n = 2). To prevent digestion of the carbohydrate in the ileum, an amylase inhibitor (3.3 mg/ml) was added to the perfusate used in half of the subjects. Postprandially, we measured outputs of amylase, trypsin, and bile acids in the duodenum, the amount of carbohydrate in the ileum, and gastric emptying. During the second postprandial hour the rate of gastric emptying was inversely related to the amount of carbohydrate in the ileum (p less than 0.01) and was directly correlated with pancreaticobiliary secretion (p less than 0.05). However, as the amount of unabsorbed carbohydrate in the ileum increased, the ratio of amylase to trypsin secretion increased (p less than 0.005). Postprandially carbohydrate in the ileum induces changes of upper-gut function that should increase digestion and absorption of carbohydrate since gastric emptying (and delivery of carbohydrate to the duodenum) slows and pancreatic amylase secretion increases relative to trypsin secretion and gastric emptying. PMID- 1719523 TI - Growth and differentiation of pancreatic acinar cells: independent effects of glucocorticoids on AR42J cells. AB - Dexamethasone (DEX) inhibits growth and induces differentiation in rat pancreatic acinar AR42J cells. We wished to determine whether growth and differentiation are mutually exclusive in AR42J cells and whether DEX effects on growth and differentiation are mutually dependent or independent. Inhibition of DNA synthesis, assessed by [3H]thymidine incorporation, was detectable after 6 h, half-maximal after 12 h, and complete after 18-h DEX treatment, at which time incorporation was reduced to 9.0% of control. The half-maximal effective dose for inhibition of DNA synthesis was 0.5 nM, and maximal inhibition was achieved with 10 nM DEX. This dose-response was similar to that previously reported for DEX induced parameters of differentiation. The rank order of potency for inhibition of DNA synthesis by various steroid hormones was DEX greater than corticosterone greater than aldosterone greater than progesterone. Hydroxyurea or serum starvation inhibited growth to the same extent as DEX but did not induce differentiation. Moreover, hydroxyurea or serum starvation did not block the ability of DEX to induce differentiation. Addition of either EGF or insulin significantly reversed the growth inhibitory effects of submaximal (1 nM) DEX. In cultures released from growth inhibition, 1 nM DEX increased cellular amylase content 5.9- to 6.5-fold, similar to the amylase increase in growth-inhibited cultures. Therefore, growth inhibition and differentiation are independent delayed events regulated by DEX in AR42J cells. PMID- 1719524 TI - Effect of ethanol on pancreatic exocrine secretion in rats. AB - The effect of ethanol on pancreatic exocrine secretion was studied in isolated rat pancreatic acini. Ethanol caused a dose-dependent stimulation of amylase release, and a twofold increase of amylase release was observed with 600 mM ethanol. Ethanol inhibited cholecystokinin octapeptide (CCK-8)- and carbamylcholine-stimulated amylase release and similarly inhibited binding of [125I]CCK-8 and [N-methyl-3H]scopolamine to isolated rat pancreatic acini in a dose-dependent manner. The inhibitory effect of ethanol was fully reversible with respect to CCK-8-induced amylase release. On the other hand, ethanol potentiated secretin- and vasoactive intestinal peptide (VIP)-stimulated amylase release. Ethanol induced a small but significant increase in Ca2+ efflux, whereas CCK-8 induced an immediate and large increase, but ethanol significantly inhibited CCK 8-stimulated Ca2+ efflux. The present study clearly demonstrates the dual effects of ethanol on pancreatic exocrine function: stimulation and inhibition. We suggest that mobilization of intracellular Ca2+ may be involved in the mechanism of ethanol's action on isolated rat pancreatic acini. PMID- 1719525 TI - Effect of insulin administration on contents, secretion, and synthesis of pancreatic lipase and colipase in rats. AB - After insulin administration in vivo, changes in pancreatic lipase, colipase and amylase contents and outputs were assayed and quantitatively compared. The incorporation of [35S]cysteine into individual enzymes was measured. The mRNA coding for lipase and amylase were determined by dot-blot hybridization. It was found that insulin dose-dependently decreased lipase and colipase contents, but only slightly decreased amylase content. Four hours after insulin administration (0.5 U/100 g), the contents of lipase and colipase decreased 80 and 72%, respectively, while amylase content decreased only about 25%. The decrease in amylase content was accompanied by a 21% increase in its output. The outputs of lipase and colipase only increased transiently and then sharply decreased to a level much lower than control. Total outputs of lipase and colipase could not quantitatively explain the great loss of lipase and colipase contents caused by insulin administration. After insulin injection, the incorporation of [35S]cysteine into amylase increased by 21%, whereas incorporation into lipase and colipase decreased by 18 and 25%, respectively. Dot-blot hybridization with cDNA probes revealed that lipase mRNA decreased by 50% 4 h after insulin administration, whereas mRNA for amylase did not significantly change. The results indicate an inhibitory effect of insulin administration on synthesis of pancreatic lipase and colipase, with the inhibition of lipase synthesis being at pretranslational level. PMID- 1719526 TI - Potentiating effect of CCK and secretin on rat exocrine pancreas and its cholinergic dependence. AB - We investigated whether physiological doses of cholecystokinin (CCK) potentiate the stimulating effect of a physiological dose of secretin on exocrine pancreatic secretion, and the effect of atropine on this potentiating action in rats. Pure pancreatic juice was collected from anesthetized rats prepared by pancreatic duct and bile duct cannulation. Intravenous infusion of CCK-8 in three different doses, 0.03, 0.06, and 0.12 micrograms/kg/h, significantly increased pancreatic juice volume and amylase output, dose-dependently. Simultaneous infusion of CCK-8 in graded doses with secretin in a dose of 0.03 CU/kg/h, produced a dose-related increase in pancreatic secretory response significantly greater than the response to CCK-8 alone (p less than 0.05) and greater than the sum of the response to secretin alone and CCK-8 alone. The incremental pancreatic secretion, including juice volume and amylase output, in response to intravenous infusion of CCK-8 with secretin, was significantly suppressed by intravenous administration of atropine in a dose of 100 micrograms/kg/h (p less than 0.01). Thus, it is concluded that CCK-8 and secretin in physiological doses potentiate each other's stimulatory action on exocrine pancreatic secretion and this potentiating action appears to be cholinergic-dependent. PMID- 1719527 TI - A somatostatin analogue is protective against retrograde bile salt-induced pancreatitis in the rat. AB - Despite the proposal that somatostatin or its stable analogue, octreotide (SMS 201,995), may exert an ameliorating effect on acute pancreatitis, data concerning its beneficial effect in this situation are conflicting. This study examines the effects of octreotide on acute pancreatitis, resulting from the retrograde injection of a bile salt (taurocholate) plus saturating trypsin into the common bile-pancreatic duct of the rat. Octreotide given before the induction of pancreatitis significantly reduced the levels of serum amylase and lipase, ascites amylase concentration, degree of leukocyte infiltration, and focal areas of pancreatic tissue necrosis. In contrast, administration of octreotide as soon as 5 min following induction had no demonstrable ameliorating effects on the pancreatitis. These results indicate that octreotide may have application to prophylaxis of acute pancreatitis in cases where bile salts may play a role in pathogenesis, but may not be beneficial in established acute pancreatitis. PMID- 1719528 TI - Effect of Urey-Bradley-Shimanouchi force field on the harmonic dynamics of proteins. AB - A normal mode analysis of bovine pancreatic trypsin inhibitor is carried out by using a Urey-Bradley-Shimanouchi potential energy function. The density of vibrational states, the magnitudes, and time scales of the atomic fluctuations are compared with experimental and theoretical results obtained by the more commonly used potential energy functions. The atomic fluctuations of Lys-15 are subject to extensive considerations as this residue is buried in the trypsin specificity pocket. It is found that Arg-17 is likely to be of importance in order to understand the way BPTI binds on trypsin. PMID- 1719529 TI - Detection of vitronectin mRNA in tissues and cells of the mouse. AB - Mouse vitronectin (Vn) was isolated from serum by heparin affinity chromatography. The purified protein (Mr 71,000) supported adhesion of mouse and human cells in an Arg-Gly-Asp-dependent manner and bound to type 1 plasminogen activator inhibitor with kinetics similar to those observed using human and bovine Vn. To further characterize murine Vn and its biosynthesis in vivo, a mouse Vn cDNA was isolated from a liver cDNA library. The amino acid sequence of mouse Vn was deduced from the cDNA and was aligned with that of human Vn. Based on this alignment, mouse Vn was inferred to be 457 amino acids long and to have extensive (82%) homology with human Vn. Northern blot hybridization analysis of RNA from mouse tissues, using the mouse Vn cDNA as a hybridization probe, revealed the presence of a single transcript of 1.7 kilobases in mouse liver. Vn mRNA was not detectable in heart, lung, kidney, spleen, muscle, brain, thymus, testes, uterus, skin, adipose tissue, and aorta. The cellular localization of liver Vn mRNA was studied by in situ hybridization. Strong staining was observed only in hepatocytes, suggesting that these cells are the primary source of Vn in vivo. PMID- 1719530 TI - Structure of the human hexabrachion (tenascin) gene. AB - The structure of the gene encoding human hexabrachion (tenascin) has been determined from overlapping clones isolated from a human genomic bacteriophage library. The genomic inserts were characterized by restriction mapping, Southern blot analysis, PCR, and DNA sequencing. The coding region of the hexabrachion gene spans approximately 80 kilobases of DNA and consists of 27 exons separated by 26 introns. The exon-intron structure supports a hypothesis based on the cDNA sequence that the hexabrachion gene is an assembly of DNA modules that are also found elsewhere in the genome. Single exons may encode a module, a portion of a module, or a group of modules. The 15 type III units similar to those found in fibronectin are each encoded either by a single exon or by two exons interrupted by an intron. All type III units known to be spliced out of the smaller forms of the protein are encoded by one exon. The fibrinogen-like domain of 210 amino acids is encoded by five exons. The 14.5 epidermal growth factor-like repeats are all encoded by a single exon. PMID- 1719531 TI - Nonresponsiveness to an immunodominant Epstein-Barr virus-encoded cytotoxic T lymphocyte epitope in nuclear antigen 3A: implications for vaccine strategies. AB - An immunodominant Epstein-Barr virus (EBV)-encoded cytotoxic T lymphocyte (CTL) epitope has been mapped to the EBV nuclear antigen 3A. The epitope, represented by the peptide sequence AWNAGFLRGRAYGLD (hereafter termed AWNA), is restricted through the HLA-B8 allele and is expressed by type A but not type B-infected transformants. Herein, we show that EBV-specific memory CTLs from an HLA-B8+ healthy virus carrier, JS, did not respond in vitro to AWNA, even though that individual's endogenously infected transformants processed and presented the natural equivalent of this peptide to AWNA-specific CTLs from another B8+ individual. Instead, an epitope, represented by the peptide sequence QLSDTPLIPLTIFVGENTGV, was the dominant EBV-specific CTL epitope in donor JS. This epitope mapped to EBV nuclear antigen 2A, was restricted by an HLA-A2 subtype, and specifically associated with type A strains of EBV. No AWNA-specific CTL precursors were detected by limiting dilution analysis of peripheral blood mononuclear cells from donor JS whereas the precursor frequency of AWNA-specific CTLs from a responder donor, LC, was estimated at 1:4500. The presentation in vivo of an immunogenic epitope-HLA antigen complex is clearly insufficient to guarantee an effective memory CTL response to that foreign epitope. Thus, vaccination strategies based on peptides inducing CTL responses may need to take into account not only the polymorphism of HLA antigens but also possible allelic variation in the repertoires of T-cell receptors. PMID- 1719532 TI - Limitations in plasticity of the T-cell receptor repertoire. AB - How constrained is T-cell recognition? Is a truncated T-cell receptor (TCR) repertoire, missing half of its V beta components (where V indicates variable), still broad enough to produce an antigen-specific T-cell response to all determinants? These questions can be answered for certain T-cell antigenic determinants whose response in the wild type is limited to specific gene segments. Our results show that mice with such a deletion in their TCR V beta genes (V beta truncated haplotype, Va beta) are unable to respond to two antigen determinants (sperm whale myoglobin 111-121/I-Ed and myelin basic protein 1-11/I Au) whose response in the wild type is restricted to the missing V beta (V beta 8.2 in the case of 111-121/I-Ed and V beta 8.2 and V beta 13 in the case of 1 11/I-Au) gene segments. Fundamentally, this restriction could have been attributed to another aspect of immunodominance--that a favored TCR with high affinity would dominate the response, but in its absence, a hierarchy of T cells with lesser efficiency and expressing alternate TCR V genes could take over. However, from our experiments it has become evident that there is an absolute limit to the flexibility inherent in the TCR repertoire. Since it is clear that mouse populations have many ambient deletion ligands (such as self-superantigens) that can result in the loss of multiple V beta gene segments during normal T-cell development, these deletions can have serious consequences, such as unresponsiveness to the antigen as a whole--a hole in the repertoire--if a dominant determinant of that antigen normally shows restricted TCR V beta gene usage. PMID- 1719533 TI - Cross-reactions and specificities of monoclonal antibodies against myelin basic protein and against the synthetic copolymer 1. AB - Antibody cross-reactivity is here demonstrated between basic protein (BP), the encephalitogenic molecule of myelin, and copolymer 1 (Cop 1), the synthetic amino acid copolymer, which has a suppressive effect on experimental allergic encephalomyelitis and is effective in reducing the number of relapses in exacerbating-remitting multiple sclerosis. This cross-reactivity is conclusively established using mouse monoclonal antibodies (mAbs). About a third of anti-rat BP mAbs and most of anti-mouse BP mAbs cross-reacted with Cop 1. This cross reactivity could be demonstrated with anti-BP mAbs of different specificities. In addition, several anti-Cop 1 hybridomas cross-reacted with BP. This cross reactivity was verified in several assay systems, including competitive inhibition experiments. Moreover, some anti-BP mAbs and anti-Cop 1 mAbs reacted in a heteroclitic manner and favored the cross-reactive antigen over the immunogen. In contrast to the mAbs, no cross-reactivity could be demonstrated with the antisera of immunized mice. This observation may reflect the different B cell populations expressed in the mAb response as compared to the polyclonal response. Thus, the use of mAbs has uncovered specificities that are not evident in antisera and has revealed pronounced cross-reactivity between BP and Cop 1 at the B-cell level. These results further establish the immunological interrelationships between Cop 1 and BP, demonstrated earlier at the T-cell level. PMID- 1719534 TI - Identification of a continuous and cross-reacting epitope for Plasmodium falciparum transmission-blocking immunity. AB - Identification of continuous epitopes in the target antigens of Plasmodium falciparum transmission-blocking antibodies is likely to facilitate the production of a subunit peptide vaccine. Two such epitopes shared among several sexual-stage antigens were identified with murine monoclonal antibodies. An epitope recognized by four monoclonal antibodies capable of blocking infectivity of gametocytes in the mosquitoes is shared among three antigens (230, 48/45 doublet, and 27 kDa). These antigens are synthesized at different times during the development and maturation of gametocytes, and the blocking epitope appears conserved among parasites from diverse geographical locations. Immune response against such a unique epitope (continuous, cross-reacting, and conserved) is likely to be boosted by natural infection. The 27-kDa protein is reported here as a target of malaria transmission-blocking monoclonal antibodies, and the cross reacting epitope represents an attractive candidate for a transmission-blocking vaccine. PMID- 1719535 TI - Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus. AB - The herpes simplex virus type 1 (HSV-1)-mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human alpha 2 interferon (HuIFN-alpha 2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIV alpha 1 cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIV alpha 1 cells, HuIFN-alpha 2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIV alpha 1 cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIV alpha 1 cells. No major differences could be detected in the expression of HSV-1 immediate-early (IE) genes IE175 and IE110 (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIV alpha 1 cells to account for the inability of HSV-1 to induce HIV-1 in HNHIV alpha 1 cells. However, major differences were observed in the binding pattern of NF-kappa B-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-kappa B-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIV alpha 1 cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes. PMID- 1719536 TI - A single amino acid change in a myelin basic protein peptide confers the capacity to prevent rather than induce experimental autoimmune encephalomyelitis. AB - Experimental autoimmune encephalomyelitis (EAE) is an experimental demyelinating disease of rodents. In (PL/J x SJL) F1 mice, it is induced by immunization with the myelin basic protein peptide Ac1-11. Ac1-11 [4A], a myelin basic protein peptide analog with a single amino acid substitution, (i) binds to class II major histocompatibility complex molecules and stimulates encephalitogenic T cells in vitro better than Ac1-11, (ii) is nonimmunogenic and nonencephalitogenic in vivo in (PL/J x SJL)F1 mice, (iii) prevents EAE when administered before or at the time of immunization with Ac1-11, and (iv) prevents EAE when administered later, near the time of disease onset. Initial studies suggest that Ac1-11 [4A] does not prevent EAE by competitive inhibition or by activation of regulatory cells. Thus, substitution of a single amino acid in a myelin basic protein peptide confers the capacity to prevent rather than induce EAE, even after peptide-specific encephalitogenic T cells have been activated. PMID- 1719537 TI - Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts. AB - Insulin-like growth factor binding protein 3 (IGFBP-3) mRNA levels were consistently higher in both senescent normal human diploid fibroblasts (HDFs) at late passage (old cells) and prematurely senescent HDFs from a subject with Werner syndrome (WS) during serum depletion and repletion of growth medium and during proliferation from sparse to high-density inhibited cultures, compared to normal early-passage (young) HDFs. However, IGFBP-3 protein accumulated to higher levels in conditioned medium of old cells than in medium of WS and young cells, in that order, under the same conditions. Insulin-like growth factor I (IGF-I) was not detected in naive medium or in any of the media conditioned by these three cell types, whereas IGF-II was detectable in serum-repleted medium and remained relatively constant. Thus, molar ratios of IGFBP-3/IGF-II were consistently higher in old and WS cells and increased substantially as all three cell types became quiescent, due to either serum depletion or high cell density. These data are consistent with either an adaptive or a causal role for IGFBP-3 protein in the senescent and quiescent growth arrest of HDFs. PMID- 1719538 TI - DNA.RNA helicase activity of RAD3 protein of Saccharomyces cerevisiae. AB - The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV damaged DNA and is essential for cell viability. The RAD3 protein exhibits a remarkable degree of sequence homology to the human excision repair protein ERCC2. The RAD3 protein is a single-stranded DNA-dependent ATPase and a DNA helicase capable of denaturing long regions of duplex DNA. Here, we demonstrate that RAD3 also possesses a potent DNA.RNA helicase activity similar in efficiency to its DNA helicase activity. The rad3 Arg-48 mutant protein, which binds but does not hydrolyze ATP, lacks the DNA.RNA unwinding activity, indicating a dependence on ATP hydrolysis. RAD3 does not show any RNA-dependent NTPase activity and, as expected, does not unwind duplex RNA. This observation suggests that RAD3 translocates on DNA in unwinding DNA.RNA duplexes. That the rad3 Arg-48 mutation inactivates the DNA and DNA.RNA helicase activities and confers a substantial reduction in the incision of UV-damaged DNA suggests a role for these activities in incision. We discuss how RAD3 helicase activities could function in tracking of DNA in search of damage sites and effect enhanced excision repair of actively transcribed genes. PMID- 1719539 TI - Reverse transcriptase encoded by a retrotransposon from the trypanosomatid Crithidia fasciculata. AB - The long interspersed nuclear element (LINE)-like elements are a distinct family of eukaryotic transposons that contain a long open reading frame with limited sequence homology to retroviral reverse transcriptases. Unlike many retrotransposons, they lack long terminal repeats. The mechanism by which LINE like elements move within the genomes of their hosts remains speculative. We have used an unusual approach to express and detect enzymatic activities associated with Crithidia retrotransposable element 1 (CRE1), a site-specific LINE-like element found in the insect trypanosomatid Crithidia fasciculata. A chimeric gene fusing the yeast retrotransposon Ty1 and the CRE1 open reading frame is constructed and then overexpressed in yeast. Fusion proteins are packaged into virus-like particles, which can be partially purified and directly analyzed for enzymatic activity. Here we demonstrate that CRE1 encodes an RNA-directed DNA polymerase. These data provide direct biochemical evidence that this widely distributed class of retrotransposons encodes reverse transcriptase and sets the stage for a detailed understanding of the mechanisms involved in LINE-like element transposition. PMID- 1719540 TI - A region of the insulin receptor important for ligand binding (residues 450-601) is recognized by patients' autoimmune antibodies and inhibitory monoclonal antibodies. AB - Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 monoclonal antibodies that inhibit insulin binding were found to recognize an epitope contained within residues 450-601 of the alpha subunit of the receptor. In contrast, 2 monoclonal antibodies that do not inhibit insulin binding were found to recognize the cysteine-rich region of the alpha subunit. Chimeric insulin receptors that had residues 450-601 replaced with the homologous residues of the insulin-like growth factor I receptor exhibited a decreased ability to bind insulin. In contrast, insulin-like growth factor I receptors that have had the comparable region replaced with that of the insulin receptor showed no decrease in their ability to bind ligand. These results indicate that residues 450-601 of the insulin receptor are important for insulin binding and include the major site for recognition by inhibitory monoclonal antibodies and patients' autoimmune anti-receptor antibodies. PMID- 1719541 TI - Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium. AB - Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels. PMID- 1719542 TI - Chimeric human immunodeficiency virus type 1/type 2 reverse transcriptases display reversed sensitivity to nonnucleoside analog inhibitors. AB - Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), an important therapeutic target in the treatment of AIDS, is effectively inhibited by a class of nonnucleoside analog compounds that includes nevirapine (BI-RG-587) and tetrahydroimidazo[4,5,1-jk]-[1,4]benzodiazepin-2(1H)-one and -thione. We show that both tyrosine residues at positions 181 and 188 flanking the putative catalytic site of HIV-1 RT are required for sensitivity of the enzyme to these compounds. HIV-2 RT, which does not have tyrosines at these positions, is resistant to these nonnucleoside analog inhibitors. Substitution of the HIV-2 RT amino acid residues at position 181 or 188 into HIV-1 RT results in an enzyme that is resistant to these compounds while retaining sensitivity to 3'-azido 2',3'-dideoxythymidine triphosphate. HIV-2 RT substituted with amino acids 176 190 from HIV-1 RT acquires sensitivity to these nonnucleoside analog inhibitors. PMID- 1719543 TI - Effect of CP-96,345, a nonpeptide substance P receptor antagonist, on salivation in rats. AB - CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl) methyl]-1 azabicyclo[2.2.2]octan-3-amine) antagonism of substance P-stimulated salivation was investigated in pentobarbital-anesthetized rats. Administered either intraperitoneally or orally, CP-96,345 produced dose-dependent inhibition of the sialogogic response elicited by substance P, with a median effective dose of 12 24 mumol/kg (5-10 mg/kg) of body weight, but had no effect on acetylcholine stimulated salivation. CP-96,345 produced concentration-dependent inhibition of [3H]substance P binding to rat submaxillary gland membranes, with a median effective concentration of 34 +/- 3.6 nM. These biological activities were confined to CP-96,345 in that the 2R,3R enantiomer (CP-96,344) was without effect. PMID- 1719544 TI - Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA. AB - Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process. PMID- 1719545 TI - A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. AB - A panel of human monoclonal antibody Fab fragments has been generated against the surface glycoprotein gp120 of type 1 human immunodeficiency virus (HIV) by antigen selection from a random combinatorial library expressed on the surface of filamentous phage. The library was prepared from 5 ml of bone marrow from an asymptomatic individual who has been HIV-positive for 6 years. The antibodies have high affinity for antigen (mostly with affinity constants of greater than 10(8) M-1) and notable sequence diversity. Given appropriate donor selection, the methods described should allow the generation of antibodies for the evaluation of passive immunization as a therapy for AIDS. PMID- 1719546 TI - Macrophages and smooth muscle cells express lipoprotein lipase in human and rabbit atherosclerotic lesions. AB - Lipoprotein lipase (LPL; EC 3.1.1.34) may promote atherogenesis by producing remnant lipoproteins on the endothelial surface and by acting on lipoproteins in the artery wall. In vitro, smooth muscle cells and macrophages synthesize LPL, but in human carotid lesions only a few smooth muscle cells were reported to contain LPL protein. Endothelial cells do not synthesize LPL in vitro, but in normal arteries intense immunostaining for LPL is present on the endothelium. We used Northern blot analysis, in situ hybridization, and immunocytochemistry of human and rabbit arteries to determine cellular distribution and the site of the synthesis of LPL in atherosclerotic lesions. Northern blot analysis showed that LPL mRNA was detectable in macrophage-derived foam cells isolated from arterial lesions of "ballooned" cholesterol-fed rabbits. In situ hybridization studies of atherosclerotic lesions with an antisense riboprobe showed a strong hybridization signal for LPL mRNA in some, but not all, lesion macrophages, which were mostly located in the subendothelial and edge areas of the lesions. Also, some smooth muscle cells in lesion areas also expressed LPL mRNA. Immunocytochemistry of frozen sections of rabbit lesions with a monoclonal antibody to human milk LPL showed intense staining for LPL protein in macrophage-rich intimal lesions. The results suggest that lesion macrophages and macrophage-derived foam cells express LPL mRNA and protein. Some smooth muscle cells in the lesion areas also synthesize LPL. These data are consistent with an important role for LPL in atherogenesis. PMID- 1719547 TI - Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain. AB - Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain. PMID- 1719548 TI - Mutator activity in maize correlates with the presence and expression of the Mu transposable element Mu9. AB - Mutator is a powerful system for generating new mutants in maize. Mutator activity is attributable to a family of transposable, multicopy Mu elements, but none of the known elements is an autonomous (regulatory) element. This paper reports the discovery of Mu9, a 4942-base-pair Mu element that was cloned after it transposed into the Bronze-2 locus. Like other Mu elements, Mu9 has approximately 215-base-pair terminal inverted repeats and creates a 9-base-pair host sequence duplication upon insertion. A small gene family of elements that cross-hybridize to Mu9 has been found in all maize lines, and one of the other known Mu elements, Mu5, probably arose as a deletion of Mu9. Mu9 has several of the properties expected for the proposed regulator of Mutator activity. (i) The presence of Mu9 parallels the presence of Mutator activity in individuals from a line that genetically segregates for the Mu regulator. (ii) Lines that transmit Mutator to greater than 90% of their progeny have multiple copies of Mu9. (iii) Most maize lines that lack Mutator activity and that are not descended from Mutator lines lack the Mu9 element. (iv) Transcripts that hybridize to Mu9 are abundant in active Mutator lines, but they are absent from lines that have epigenetically lost Mutator activity. These correlations suggest that Mu9 is a candidate for the autonomous Mutator element. PMID- 1719549 TI - Pharmacological properties of a potent and selective nonpeptide substance P antagonist. AB - We describe here the pharmacological properties of RP 67580 [(3aR,7aR)-7,7 diphenyl-2-[1-imino-2-(2-methoxyphenyl)ethyl] perhydroisoindol-4-one], a nonpeptide antagonist of substance P (SP). In vitro, the compound was found to inhibit in a competitive manner (Ki = 4.16 +/- 0.59 nM) [3H]SP binding to neurokinin receptors type 1 (NK1 receptors) in rat brain membranes. Contractions induced by SP and septide (a selective NK1 agonist) in guinea pig ileum were competitively inhibited by RP 67580 (pA2 = 7.16 and 7.59, respectively). Moreover, RP 67580 displayed the profile of a specific antagonist of NK1 receptors: it was not active on NK2 and NK3 receptors as seen in binding assays and in isolated preparations of rabbit pulmonary artery and rat portal vein. In the rat, low intravenous doses of RP 67580 totally inhibited the plasma extravasation induced by SP in the urinary bladder (ED50 = 0.04 mg/kg i.v.) and by antidromic electrical stimulation of the saphenous nerve in the hind paw skin (ED50 = 0.15 mg/kg i.v.). This compound was also active in two classical analgesic tests in mice: phenylbenzoquinone-induced writhing (ED50 = 0.07 mg/kg s.c.) and the formalin test (ED50 = 3.7 mg/kg s.c.). Its potency was of the same order as that of morphine. Thus we conclude that RP 67580, a SP antagonist, belongs to a class of drugs that may be useful in the management of various clinical pathologies where pain and neurogenic inflammation are involved. PMID- 1719550 TI - Agonist-independent activation of acetylcholine receptor channels by protein kinase A phosphorylation. AB - Protein phosphorylation is a ubiquitous and one of the most effective means of regulating protein activity. Receptor phosphorylation is a key event in signal transduction. The question, therefore, that arises is whether this modulatory mechanism might produce functional changes in a membrane receptor in the absence of its naturally occurring ligand. To examine this issue, single-channel properties of purified acetylcholine receptors (AChRs) from Torpedo californica reconstituted in lipid bilayers were studied in the absence of ACh in both unphosphorylated preparations and after in vitro phosphorylation by a purified catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A). Notably, the spontaneous open-channel probability of phosphorylated AChRs is significantly higher than that of unphosphorylated AChRs. Channel activation by protein kinase A is correlated with AChR phosphorylation and is abolished by alpha-bungarotoxin. Analysis of probability distributions of the open dwell times indicates that, similar to unphosphorylated AChR has two distinct open states, short- and long-lived. The frequency of occurrence of the long openings over the short and the magnitude of both time constants increase after phosphorylation, as they do with agonist concentration. Thus, phosphorylation of AChR gamma and delta subunits activates AChR channel opening in the absence of ligand binding. This result is compatible with the notion that protein phosphorylation may effectively act as an intracellular ligand with the phosphorylation sites envisioned as cytoplasmic ligand binding sites. PMID- 1719551 TI - cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins. AB - We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways. PMID- 1719552 TI - Expression of acidic and basic fibroblast growth factors in the substantia nigra of rat, monkey, and human. AB - The distribution of acidic (aFGF) and basic (bFGF) fibroblast growth factor mRNA and protein were examined in mesencephalon by immunohistochemistry, immunoblot analysis, in situ hybridization histochemistry, and RNA analysis. Coexistence of aFGF or bFGF with tyrosine hydroxylase protein in nigral cells was observed with immunohistochemistry. Both aFGF and bFGF mRNAs were found in the substantia nigra. Unilateral 6-hydroxydopamine lesions of nigrostriatal neurons resulted in a loss of aFGF and tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] mRNA-positive neurons on the lesioned side. The distribution of aFGF mRNA in monkey brain was similar to that seen in the rat. RNA and immunoblot analysis confirmed the presence of both aFGF and bFGF mRNAs and proteins in the substantia nigra of rat, monkey, and human. PMID- 1719553 TI - Thyrotropin-releasing hormone induces opposite effects on Ca2+ channel currents in pituitary cells by two pathways. AB - Thyrotropin-releasing hormone (TRH) stimulates pituitary secretion by steps involving a cytosolic Ca2+ rise. We examined various pathways of Ca2+ elevation in pituitary GH3 cells. By using the patch clamp technique in the whole-cell configuration and Ba2+ as divalent charge carrier through Ca2+ channels, TRH (1 microM) reversibly reduced the current by about 55%. This hormonal effect was prevented by infusing guanine 5'-[beta-thio]diphosphate (GDP[beta S]) intracellularly but not by pretreating the cell with pertussis toxin (PT). Since PT-insensitive guanine nucleotide-binding regulatory (G) proteins are known to mediate a hormone-stimulated inositol trisphosphate-mediated Ca2+ release from intracellular stores, we assume that the inhibitory effect of TRH on Ba2+ currents through Ca2+ channels is caused by the increased intracellular Ca2+. To prevent a Ca(2+)-release-dependent inhibition of Ca2+ channels, we preincubated GH3 cells in a medium free of divalent charge carriers and measured the Na+ current through Ca2+ channels. When fura-2 was used as indicator for the cytosolic Ca2+, TRH induced a release from intracellular stores only once and had no effect on the intracellular Ca2+ concentration during further applications. In line with this observation, TRH initially reduced the Na+ current through Ca2+ channels but stimulated it during subsequent applications. The stimulation was sensitive to GDP[beta S] and was abolished by pretreatment with PT, suggesting that the stimulatory action of TRH is mediated by a G protein different from the one that functionally couples the receptor to phosphatidylinositol 4,5 bisphosphate hydrolysis. In conclusion, the present data suggest that TRH increases the intracellular Ca2+ concentration by two interacting pathways, that release from intracellular stores causes a secondary blockage of Ca2+ channels, and that, especially with empty intracellular Ca2+ stores, Ca2+ channels are stimulated by a PT-sensitive G protein. PMID- 1719554 TI - Defective membrane expression of human growth hormone (GH) receptor causes Laron type GH insensitivity syndrome. AB - Mutations in the growth hormone receptor (GHR) gene can cause growth hormone (GH) resistance. Given the sequence homology between the extracellular domain of the GHR and a soluble GH-binding protein (GH-BP), it is remarkable that GH-BP binding activity is absent from the serum of patients with Laron-type GH insensitivity, a hereditary form of severe dwarfism. We have previously identified a mutation within the extracellular domain of this receptor, replacing phenylalanine by serine at position 96 of the mature protein, in a patient with Laron syndrome. We have now investigated the effect of this Phe----Ser substitution on hormone binding activity by expressing the total human GHR cDNA and mutant form in eukaryotic cells. The wild-type protein expressed was able to bind GH but no plasma membrane binding was detectable on cells transfected with the mutant cDNA; this was also the case of cells transfected with a Phe96----Ala mutant cDNA, suggesting that the lack of binding activity is not due to a posttranslational modification of serine. Examination of the variant proteins in subcellular fractions revealed the presence of specific GH binding activity in the lysosomal fraction, whereas immunofluorescence studies located mutant proteins in the cytosol. Our findings suggest that these mutant GHRs fail to follow the correct intracellular transport pathway and underline the potential importance of this phenylalanine residue, which is conserved among the GH, prolactin, and erythropoietin receptors that belong to the same cytokine receptor superfamily. PMID- 1719556 TI - Structural requirements for the carbohydrate ligand of E-selectin. AB - The acute inflammatory response requires that circulating leukocytes adhere to, and then migrate through, the vascular wall at the site of injury or infection. Several receptors have been implicated in this adhesion and migration process, including the selectins, a family of carbohydrate-binding proteins. The ligand for one of these proteins, E-selectin (LECAM-2, ELAM-1) has been described by several groups to contain a polylactosamine structure bearing a terminal sialic acid residue and at least one fucose residue. We report here a more detailed investigation into the minimum structural requirements for carbohydrate recognition by E-selectin. Using both direct binding and inhibition studies we demonstrate that the sialyl Lewisx tetrasaccharides Sia(alpha 2-3)Gal(beta 1 4)[Fuc(alpha 1-3)]GlcNAc, and Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc are the smallest oligosaccharides recognized by the lectin. In addition, an oligosaccharide containing the sialyl Lewisa epitope is also recognized, but less avidly. We propose a structural model of functional groups necessary for recognition by E-selectin, based on these data and additional experiments on modifications of sialic acid and the reducing terminal saccharide. PMID- 1719555 TI - Specific lysis of human immunodeficiency virus type 1-infected cells by a HLA A3.1-restricted CD8+ cytotoxic T-lymphocyte clone that recognizes a conserved peptide sequence within the gp41 subunit of the envelope protein. AB - A HLA-A3.1-restricted CD8+ cytotoxic T-cell clone, E7.20, that lyses cells infected with human immunodeficiency virus type 1 was isolated from an infected individual. The epitope was localized to amino acids 768-778 (RLRDLLLIVTR, NL43 env sequence) of the cytoplasmic domain of gp41 by successive use of a panel of recombinant vaccinia viruses that express truncated env genes and synthetic peptides. The epitope is conserved on 7 (NL43, BRU, HXB2, BRVA, SC, JH3, and JFL) of 13 human immunodeficiency virus type 1 isolates from North America. Synthetic peptides of this region of strains RF and CDC4 are also recognized by E7.20 despite a nonconservative Thr----Val or Thr----Ala change at amino acid 777; however, an MN peptide, which has four amino acid substitutions, was not reactive. The epitope recognized by E7.20 has a predicted hydrophobic alpha helical structure, with three contiguous Leu residues followed by Ile and Val at amino acids 772-776. Cytotoxicity was restricted by HLA-A3.1 using allogeneic target cells that shared HLA class I antigens with the donor and an HLA-A and -B negative human plasma cell line transfected with the HLA-A3.1 gene. The transfected cells were infectable by human immunodeficiency virus type 1 strains IIIB and MN but only the former virus sensitized them to killing by E7.20. The ability of E7.20 to specifically lyse a human lymphocyte line infected with a human immunodeficiency virus type 1 strain carrying the conserved epitope is consistent with an important role for cytotoxic T cells in controlling infection. PMID- 1719557 TI - Immunochemical dissection of the Ultrabithorax homeoprotein family in Drosophila melanogaster. AB - The homeotic gene Ultrabithorax (Ubx) specifies metameric identities in multiple tissues of the thorax and abdomen in Drosophila melanogaster. Alternatively spliced Ultrabithorax mRNAs encode five protein isoforms that differ in internal sequences immediately adjacent to a homeodomain DNA-binding motif. Each of these proteins is phosphorylated in vivo at multiple serine and threonine residues. An extensive panel of monoclonal antibodies was raised against the Ultrabithorax proteins, including antibodies specific for individual isoforms and antibodies that discriminated between different phosphorylation states. Characterization of these antibodies provided insights into shared and isoform-specific features of Ultrabithorax protein structure that may be functionally important. Immunohistochemical staining experiments demonstrated that each isoform is expressed in a different stage- and tissue-specific pattern and suggested that Ultrabithorax protein phosphorylation is also developmentally regulated. These results support the hypothesis that alternative splicing and phosphorylation modulate developmentally specific functions of the Ubx gene. PMID- 1719558 TI - Ligand-stimulated signaling events in immature CD4+CD8+ thymocytes expressing competent T-cell receptor complexes. AB - During thymic selection of the developing T-cell repertoire, the fate of individual CD4+CD8+ thymocytes is determined by the specificity of the T-cell antigen receptors (TCRs) they express. Paradoxically, most CD4+CD8+ thymocytes express few TCR molecules, and those they express are essentially incapable of transducing intracellular signals as measured by intracellular calcium mobilization. However, both TCR number and calcium-signaling capability are significantly induced in CD4+CD8+ thymocytes when the cells are released from intrathymic inhibitory signals that are mediated by their CD4 molecules. Here, the response to ligand engagement of TCR on "induced" CD4+CD8+ thymocytes that have been released from CD4-mediated inhibition was examined and was found to result in internalization of surface TCR complexes and rephosphorylation of zeta chains of the TCR complex. In addition, a proportion of induced CD4+CD8+ thymocytes were found to fragment their DNA upon ligand engagement. Thus, this study describes early events in immature CD4+CD8+ thymocytes resulting from TCR mediated signals. PMID- 1719561 TI - Four letters in the genetic alphabet: a frozen evolutionary optimum? AB - Piccirilli et al. (Nature, Lond. 343, 33-37 (1990)) have shown experimentally that the replicatable introduction of new base pairs into the genetic alphabet is chemically feasible. The fact that our current genetic alphabet uses only two base pairs can be explained provided that this basic feature of organisms became fixed in an RNA world utilizing ribozymes rather than protein enzymes. The fitness of such ribo-organisms is determined by two factors: replication fidelity and overall catalytic efficiency (basic metabolic or growth rate). Replication fidelity is shown to decrease roughly exponentially, and catalytic efficiency is shown to increase with diminishing returns, with the number of letters for a fixed genome length; hence their product, i.e. fitness, gives rise to a set of values with an optimum. Under a wide range of parameter values the optimum rests at two base pairs. The chemical identity of the particular choice in our genetic alphabet can also be rationalized. This optimum is considered frozen, as currently the dominant catalysts are proteins rather than RNAs. PMID- 1719560 TI - Steady-state currents through voltage-dependent, dihydropyridine-sensitive Ca2+ channels in GH3 pituitary cells. AB - Ca2+ influx via voltage-dependent Ca2+ channels is known to be elicited during action potentials but possibly also occurs at the resting potential. The steady state current through voltage-dependent Ca2+ channels and its role for the electrical activity was, therefore, investigated in pituitary GH3 cells. Applying the recently developed 'nystatin-modification' of the patch-clamp technique, most GH3 cells (18 out of 23 cells) fired spontaneous action potentials from a baseline membrane potential of 43.7 +/- 4.6 mV (mean +/- s.d., n = 23). The frequency of action potentials was stimulated about twofold by Bay K 8644 (100 nM), a Ca(2+)-channel stimulator, and action potentials were completely suppressed by the Ca(2+)-channel blocker PN 200-110 (100 nM). Voltage clamping GH3 cells at fixed potentials for several minutes and with 1 mM Ba2+ as divalent charge carrier, we observed steady-state Ca(2+)-channel currents that were dihydropyridine-sensitive and displayed a U-shaped current-voltage relation. The results strongly suggest that the observed long lasting, dihydropyridine sensitive Ca(2+)-channel currents provide a steady-state conductivity for Ca2+ at the resting potential and are essential for the generation of action potentials in GH3 pituitary cells. PMID- 1719559 TI - Augmentation of synthesis of plasminogen activator inhibitor type 1 by insulin and insulin-like growth factor type I: implications for vascular disease in hyperinsulinemic states. AB - Accelerated atherosclerosis accompanying diabetes mellitus, obesity, and some types of hypertension has been associated with hyperinsulinemia, augmented plasma plasminogen activator inhibitor type 1 (PAI-1), or both. We hypothesized that insulin and insulin-like growth factor type I (IGF-I) can influence synthesis of PAI-1, thereby potentially attenuating fibrinolysis. In HepG2 cells used as a model system, concentrations of insulin and IGF-I consistent with those seen in plasma independently stimulated PAI-1 synthesis. Accumulation of PAI-1 protein in conditioned medium over 24 hr was stimulated more with insulin alone than with the combination. Synergistic increases were evident, however, in the accumulation of PAI-1 protein over 48 hr with a concomitant increase in PAI-1 mRNA. A 10- to 20-fold increase in IGF binding protein I mRNA was seen 16-48 hr after exposure of the HepG2 cells to insulin and IGF-I, an increase abolished by cycloheximide. The results obtained are consistent with the hypothesis that hyperinsulinemia coupled with physiologic concentrations of IGF-I may attenuate fibrinolytic activity in vivo, thereby contributing to accelerated atherosclerosis. PMID- 1719562 TI - Modification of cyclocytidine-induced changes in [3H]thymidine incorporation and weight of parotid gland by partial sialoadenectomy. AB - Cyclocytidine (CC), a potent antitumor agent, caused a 2.3- to 5.0-fold increase in [3H]thymidine uptake of rat parotid gland after 3 days of daily administration of 500 mg/kg body wt. Gland weight also showed a 47-67% increase from that of controls. Ablation of the submandibular-sublingual glands prior to initiation of the CC regimen prevented the usual CC-induced increase in [3H]thymidine uptake, but did not inhibit the increase in gland size. It is postulated that CC-induced parotid hyperplasia requires an initial release of growth factors from the submandibular gland; however, enlargement of the parotid gland by CC is independent of such factors. PMID- 1719563 TI - Zinc depletion modifies CD5 expression by 70Z/3 murine pre-B leukemia cell line. AB - CD5 expression on B cells is regulated by certain humoral factors. In a pre-B leukemia cell line 70Z/3, we found that interleukin 4 down-regulates it. Herein, we report that zinc influences spontaneous CD5 expression by this cell line as well as actions of these factors on CD5 expression considerably. In zinc-depleted culture media, spontaneous CD5 expression by 70Z/3 cells was enhanced. In contrast, the down-regulatory action of interleukin 4 was significantly reduced under culture conditions of zinc depletion. The supplementation of zinc to physiologic concentrations (1 to 2 microM) abolished such effects of zinc depleted medium. The reduction of the suppressive action of interleukin 4 was observed at the level of gene expression. However, CD5 mRNA expression enhanced by lipopolysaccharide or NZB-SF was not further enhanced under conditions of zinc deficiency. These observations may suggest that CD5 expression by malignant or even normal B cells may be influenced by cellular/serum zinc levels. PMID- 1719564 TI - Reduced anaphylactic responsiveness of strain 2 guinea pigs. AB - Strain 2 guinea pigs have been shown to have diminished anaphylactic responsiveness. In the present study, experiments were conducted comparing various characteristics of the anaphylaxis-resistant Strain 2 guinea pigs to those of an outbred anaphylaxis-prone Dunkin-Hartley strain. To bypass the possibility that differences in antibody titers accounted for the difference in anaphylactic reactivity, both strains of guinea pig were passively sensitized with the same amount of IgG antibody to ovalbumin. Measures of anaphylactic responsiveness to subsequent antigen challenge with ovalbumin included (i) systemically induced respiratory responses; (ii) isolated cardiac responses; and (iii) cutaneous responses. In all cases, using an amount of antibody sufficient to sensitize Dunkin-Hartley guinea pigs, the anaphylactic responses of the Strain 2 guinea pigs were either nonexistent or significantly less than those of the Dunkin-Hartley strain. To further determine which factors might be responsible for this difference, tissue histamine content, histamine releasability, and histamine responsiveness of the two strains were measured. The results of these studies indicated that the respiratory hyporesponsiveness of the Strain 2 guinea pigs may be due to a low pulmonary histamine content combined with reduced pulmonary responsiveness to histamine. However, since the cardiac histamine content and the responsiveness of the Strain 2 guinea pigs were not different from those of the Dunkin-Hartley strain, these factors cannot contribute to the reduced Strain 2 cardiac anaphylactic responsiveness. Compound 48/80 released equal quantities of histamine from the isolated hearts of the Strain 2 and the Dunkin-Hartley animals, but antigen challenge evoked histamine release only from the isolated Dunkin-Hartley hearts. We conclude that the cardiac anaphylactic hyporesponsiveness of the Strain 2 guinea pigs may be due to an inability of antigen to evoke release of anaphylactic mediators such as histamine. PMID- 1719565 TI - Role of hepatocyte proliferation in alpha-hexachlorocyclohexane and phenobarbital tumor promotion in B6C3F1 mice. PMID- 1719566 TI - Leukotriene C4 formation by enriched human basophil preparations from normal and asthmatic subjects. AB - Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively. PMID- 1719567 TI - Effect of the prostacyclin analogue iloprost in sodium-depleted rats pretreated with captopril. AB - Previous studies have shown that administration of captopril to sodium-depleted rats decreases the glomerular filtration rate (GFR) and blunts the increase in glomerular prostacyclin synthesis normally occurring in response to sodium depletion. To clarify the relationship between these two responses, iloprost, a stable analogue of prostacyclin, was administered to Na-depleted, captopril treated (LNC) rats. At a dosage not affecting systemic blood pressure (12.5 ng/kg/min), iloprost increased GFR in LNC rats by 25% (from 0.26 +/- 0.03 to 0.35 +/- 0.03 ml/min/100 g body wt, P less than 0.01), without significant effects on renal plasma flow. No effect was observed in control rats. The results suggest that altered prostacyclin synthesis could contribute to the decrease of GFR in this model. PMID- 1719568 TI - Bay K 8644 potentiates the anxiolytic effect of ethanol. AB - The possible role of voltage-sensitive calcium channels (VSCCs) in the anxiolytic effect of ethanol was investigated using three different doses of ethanol (0.5, 1.0 and 2.0 g/kg) with calcium agonist Bay K 8644 (0.5 mg/kg) and calcium antagonist nifedipine (5 mg/kg) in rats. Ethanol produced an anxiolytic effect in a dose-dependent manner. The Bay K 8644-potentiated anxiolytic effect of ethanol, however, Bay K 8644 did not alter anxiety when used alone. Nifedipine itself showed an anxiolytic effect but did not change the ethanol-induced anxiolytic effect. This finding may lead to the consideration of the neurochemical mechanisms of the anxiolytic effects of ethanol and nifedipine as they vary from each other. PMID- 1719569 TI - Comparative behavioral and neurochemical studies with striatal kainic acid- or quinolinic acid-lesioned rats. AB - In the present studies the effects of kainic acid (KA)- or quinolinic acid (QA) induced striatal lesions were compared in different behavioral tests in rats. Both KA- and QA-lesioned animals had ipsilateral barrel-rotation (BR). The KA lesioned rats, however, had contralateral, while the QA-lesioned rats had both ipsi- and contralateral turning activity. The KA-lesioned animals showed increased open-field activity as well as increased percentage of entries, and time spent in the open arms of Montgomery's conflict test. Learning of an active avoidance response was strongly inhibited by both striatal QA- or KA-induced striatal lesions. The QA-lesioned animals showed less pronounced behavioral changes than KA-lesioned animals in most of the tests, and had a smaller loss of body weight. There was no significant difference in the extent of the KA- and QA induced substance P (SP) and GABA depletions in striatum, however, the depletions with QA lesions were slightly greater. These findings show that KA-induced striatal lesions produce more pronounced behavioral effects than QA lesions of similar size. It is possible that the differential effects of KA versus QA on striatal interneurons may result in its more marked behavioral effects. PMID- 1719570 TI - Streptozotocin-induced diabetes in mice reduces the nociceptive threshold, as recognized after application of noxious mechanical stimuli but not of thermal stimuli. AB - We report herein that streptozotocin (STZ)-induced diabetes selectively alters the nociceptive threshold with respect to noxious mechanical stimuli. Mice were rendered diabetic by an injection of STZ (200 mg/kg, IV). In the tail-pinch test, the latency of the biting response to forceps was significantly decreased in animals with diabetes of 2 weeks and 8 weeks duration as compared to that in age matched controls. However, the nociceptive threshold, as determined by the tail flick test, was not significantly altered. The level of substance P in the spinal cord was significantly increased in mice that has been diabetic for 2 weeks, while, there was a significant decrease, as compared to control levels, in level of substance P in mice diabetic for 8 weeks. However, the level of somatostatin was not significantly altered in mice diabetic for either 2 weeks or 8 weeks. These data suggest that STZ-induced diabetes selectively alters a neuronal system that involves substance P but not somatostatin in the spinal cord. PMID- 1719571 TI - Essential role of ATP and possibility of activation of protein kinase C in Ca(2+) dependent histamine release from permeabilized rat peritoneal mast cells. AB - To elucidate the role of ATP in histamine release, the present study was performed using beta-escin-permeabilized rat peritoneal mast cells. Ca(2+) induced histamine release from permeabilized cells is totally dependent upon exogenous ATP in the medium. In the presence of Ca2+, ATP caused histamine release concentration-dependently at concentrations ranging from 0.01 to 5 mmol/l. The maximum release was achieved at 3 mmol/l of ATP in the medium. When the other adenosine nucleotides (AMP, ADP), or nonhydrolyzable ATP analogues (adenylylimidodiphosphate, beta, gamma-methylene ATP) were added in place to ATP, no histamine release took place. Other ribonucleoside triphosphates (GTP, ITP, UTP and CTP) had little effect at the same concentration range. When the ribonucleoside triphosphate content of mast cells was determined by means of HPLC, ITP and CTP were not detectable. A millimolar range of the ATP content was determined in mast cells, but the amounts of other ribonucleoside triphosphates (GTP and UTP) were remarkably lower than that of ATP. These results seem to indicate that the ATP molecule plays a crucial role in histamine release from rat mast cells in association with its concurrent hydrolysis. Furthermore, 12-O tetradecanoylphorbol-13-acetate and 1-oleoyl-2-acetylglycerol enhanced histamine release elicited in the presence of Ca2+ (0.1 mumol/l) and ATP (3 mmol/l). Calphostin C, a potent inhibitor of protein kinase C, inhibited Ca2+/ATP dependent histamine release by approximately 60%. At the same concentration, calphostin C inhibited by 95% protein kinase C activity in the crude extract obtained from rat mast cells. It was suggested that protein kinase C activation took place in the Ca2+/ATP-dependent histamine release from permeabilized rat mast cells. PMID- 1719572 TI - The Florey Lecture, 1991. The colony-stimulating factors: discovery to clinical use. AB - The four colony-stimulating factors, GM-GSF, G-CSF, M-CSF and Multi-CSF, are specific glycoproteins with a likely common ancestral origin which interact to regulate the production, maturation and function of granulocytes and monocyte macrophages. Each has been purified and produced in active recombinant form. Animal studies have shown the ability of injected CSF to increase the production and functional activity of granulocytes and macrophages in vivo and to enhance resistance to infections. These studies have led to the current extensive clinical use of CSFs to promote the formation and function of granulocytes and macrophages in a wide variety of disease situations in which there is an associated risk of serious infections. Although our knowledge of the control of haemopoiesis remains incomplete, the approaches used to develop the CSFs can be used to extend this knowledge, with the promise of the introduction into clinical medicine of additional effective therapeutic agents. PMID- 1719573 TI - [The anatomic sapheno-femoral intersection, a source of varicose recurrence?]. PMID- 1719574 TI - The stuck slide: how to unstick it. AB - A longitudinally folded 3 x 5 card or similar-sized stiff piece of paper can be used to release a slide stuck in a carousel without removing the carousel or its locking ring. PMID- 1719575 TI - Origins of the afferent fibers to the cat superior cervical ganglion. AB - The origins of the afferent fibers to the cat's superior cervical ganglion (SCG) were demonstrated by using the retrograde horseradish peroxidase tracing method. We found that the preganglionic neurons were located in the spinal segments C8 T5, particularly in T1-T3. These neurons were situated mainly in the intermediolateral column. The extra-SCG neurons along with the cervical sympathetic trunk originated ipsilaterally from the middle cervical and stellate ganglia, and contralaterally from the caudal part of the SCG. Labeled neurons also originated from the mandibular division of the trigeminal ganglion. Our results demonstrated that many fiber sources projected to the SCG, which plays a complicated synaptic role in controlling the visceral organs of the head and neck region. PMID- 1719576 TI - [Characteristics of the development and mental status of siblings of neurotic children]. AB - 150 children were examined. They were divided into two groups. The first group was composed of the children hospitalized with the diagnosis of neurotic disorder, the second one of their brothers and sisters of similar age (7-13) nontreated psychiatrically. There were statistically significant differences between these two groups in the following ways: 1) pathology of the pre- and postnatal periods, 2) psychomotor development and somatic state during the infantile age, 3) emotional development during the preschool period. PMID- 1719577 TI - CSF GABA in depressed patients and normal controls. AB - The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) has been implicated in the pathophysiology of depression. Therefore, we examined cerebrospinal fluid (CSF) levels of GABA in depressed patients (N = 25) and normal controls (N = 20). There was no significant difference between the groups. However, among the depressed patients the subgroup of unipolar melancholic patients (N = 13) had significantly lower CSF levels of GABA than the rest of the depressed patients (N = 12). There was no significant difference for CSF levels of GABA between depressed patients who were (N = 14) or were not (N = 11) cortisol non-suppressors. It was of interest that among the controls there was a significant negative correlation between CSF levels of GABA and CSF levels of norepinephrine. PMID- 1719578 TI - Radioresistant derivatives of radiosensitive CHO cells obtained following treatment with 5-azacytidine retain their sensitivity to cisplatin. AB - Treatment of the radiation-sensitive CHO mutant, xrs-5, with the demethylating agent 5-azacytidine results in the complete conversion to wild-type levels of X ray resistance in 50% of the colonies examined (10/20). In addition to being sensitive to X rays, xrs-5 is also sensitive to the killing effects of the crosslinking agent cisplatin. The 5-azacytidine-treated xrs-5 cells which exhibit wild-type survival levels following exposure to X rays failed to demonstrate conversion to wild-type levels of resistance to cisplatin. These results support the hypothesis that increases in gene expression can alter the radioresistance of xrs-5 cells without influencing the cells' survival after exposure to cisplatin. PMID- 1719579 TI - [Results of experimental radiobiological studies made within the 10-kilometer zone of the Chernobyl AES accident]. AB - A study was made of the effect of high radioactive contamination on the animal organism (C57BL/6 mice) and HeLa cell culture within the ten-kilometer zone of the Chernobyl A.P.S. accident. The total radiation dose, as calculated by a gamma component, was 0.09 to 2 Gy. A long-term exposure of mice within the zone (cumulative dose of 1.8 to 2 Gy) caused a significant decrease in bone marrow stem potencies and changes in the brain vascular system; subsequent acute exposure of animals increased interferon titres in the serum to a much greater extent than a single acute exposure did. As to HeLa cells, irradiation there of with doses of 0.09 to 0.4 Gy during 15-20 postirradiation generations caused a decrease in the proliferative activity, an emergence of cells with micronuclei and of giant cells, and remote cell death. PMID- 1719580 TI - [Antimutagenic activity of dextran gammaphos derivatives]. AB - In experiments with V-79 Chinese hamster cell culture the influence of dextran gammaphos derivatives on the mutagenic effects of gamma-radiation was studied by the number of cells with micronuclei and fragmented nuclei. Products of interaction between gammaphos and dialdehyde dextran were shown to have a higher antimutagenic activity than gammaphos. PMID- 1719581 TI - Towards a molecular approach in teratology. PMID- 1719582 TI - Alveolocapillary remodeling in bleomycin-induced rat lung injury: interpretation from lectin-binding studies. PMID- 1719583 TI - [Recent advance of research for hepatitis C virus]. PMID- 1719584 TI - [Hematopoiesis: I. Cytokines involved in its regulation]. AB - The study of the direct involvement of colony stimulating factors, interleukins, and other purified factors in distinct steps of hematopoiesis has been complicated by the in vitro presence of non-hematopoietic cells which can intermediate the effects observed on hematopoietic precursors. The review covers the recent finding that the CD34 antigen is expressed on the membranes of essentially all pluripotent stem cells, but is lacking in the majority of the differentiated blood and stromal bone marrow cells. This finding allowed in vitro experiments with selected CD34+ hematopoietic precursors, and a consequent reevaluation of the participation of different factors in their differentiation. The role of interferons, tumor necrosis factors, and transforming growth factors beta in the negative regulation of hematopoiesis is also analysed. PMID- 1719585 TI - [Scintigraphy using 99m-TC HM-PAO labeled autologous granulocytes in the diagnosis of Whipple's disease]. AB - Whipple's disease is a systemic disease, mainly localized in the small intestine, that even today shows some difficulties about its etiopathogenesis and diagnosis. The surveys usually used in the diagnosis, among which the biopsy is an indispensable test, have some limits particularly in follow-up. The 99mTc-HM-PAO labelled granulocytes scintigraphy may be a useful alternative method in the evaluation of location and extension of the Whipple's disease. Furthermore, as it is not an invasive method, 99mTc granulocyte scintigraphy may be an important means in establishing the term of the therapy mainly when other methods are not able to exactly confirm the remission of the disease. PMID- 1719586 TI - Immunogenetics of rheumatoid arthritis and juvenile arthritis. AB - Starting with the historical background and ending with the most recent data obtained by DNA typing, using PCR and oligonucleotide probes, the role of HLA antigens in rheumatoid arthritis (RA) and in several forms of juvenile arthritis (JA) is reviewed. RA is thought to be associated with an epitope of the third hypervariable region of DRB1 which is shared by several alleles including DR4 Dw4, Dw14, Dw15, DR1, DRw14.2, and DRw10. Rheumatoid factor-positive JA is also associated with DR4, but in rheumatoid factor-negative JA DR4 is absent, or markedly decreased, suggesting that it has a protective effect. Typing for the HLA-DP alleles has confirmed the association of pauciarticular JA with DPBI*0201. Recent studies in the author's laboratory have shown that DPB1*0301 is the main susceptibility factor for rheumatoid factor-negative polyarticular onset JA. It is of interest that also adult rheumatoid factor-negative RA patients have an increase of DPB1*0301, suggesting that these two clinical subsets may represent related diseases. PMID- 1719587 TI - [Educational-vocational development of young aphasic patients: results of a catamnestic study]. AB - 60 adolescents whose course of recovery from their aphasic disorders had already been studied during the postacute treatment phase in a rehabilitation clinic were catamnestically assessed by a questionnaire. The aim of the study was to gain information about their further linguistic, educational, vocational and social development, and to evaluate the validity of the recommendations made at discharge from the rehabilitation clinic. The results show that inspite of the generally good improvement of aphasic disorders in adolescents, there hardly ever is complete recovery; and they mainly make clear the severe consequences on the further educational and vocational development, which seem to be underestimated as far as institutional help is concerned. Moreover the consequences of the language disturbances on social relations seem to be felt as even harder to cope with. The educational and vocational recommendations at the end of the postacute rehabilitation phase turned out to be valid to a surprisingly high degree. Concluding, some possible consequences the results could have on improving the rehabilitation process are discussed. PMID- 1719588 TI - Specific interaction between Listeria monocytogenes and glycosylated albumins. AB - We have previously shown that Listeria monocytogenes serovar 1/2b can bind strongly to bovine albumin (BA) glycosylated by glucosamine or fucosylamine with about 20 to 30 carbohydrate residues per albumin molecule. We now show that the binding is time-dependent, reversible, saturable and specific. The two glycosylated compounds inhibit each other competitively. Scatchard analysis showed that about 100 molecules of BA-glucosamide (heptameric configuration) and 14,300 molecules of BA-fucosylamide (monomeric configuration) bound per bacterial cell. The apparent dissociation constants for BA-glucosamide and BA-fucosylamide were found to be 3.9 x 10(-14) M and 3.5 x 10(-13) M, respectively. PMID- 1719589 TI - Ca2+/calmodulin-binding proteins in Dictyostelium discoideum. AB - We have initiated a systematic study of Ca2+/calmodulin-regulated enzymes in the cellular slime mold Dictyostelium discoideum. Using 125I-labelled D. discoideum calmodulin (CaM) as a functional probe, several Ca2+/CaM-binding proteins were detected in crude cell lysates. Proteins with apparent molecular weights of 22 kDa and 78-80 kDa, respectively, were found in the soluble fraction. In addition, membrane-bound high molecular weight CaM-binding proteins were identified. Binding of CaM to all of the proteins required the presence of Ca2+ ions and competed efficiently with nonradioactive CaM from both Dictyostelium and bovine brain. The CaM antagonists melittin, W-7 and R24571 inhibited CaM binding. With a functional cloning approach, we previously obtained cDNA clones by screening a lambda gt11 lysogen expression library; in this paper, we report the analysis of CaM-binding activity by one of the recombinant cDNA clones in Escherichia coli. When rabbit antiserum was raised against it, the antiserum recognized a 78-80-kDa protein in Dictyostelium extracts which comigrated on SDS-polyacrylamide gels with 78-80-kDa CaM-binding activity. PMID- 1719590 TI - Lack of the alpha-1,2-linked N-acetyl-D-glucosamine epitope in the outer core structures of lipopolysaccharides from certain O serogroups and subspecies of Salmonella enterica. AB - A total of 176 strains of Salmonella enterica representing 116 serotypes were tested for the presence of the T6 epitope of the alpha-1,2-linked N-acetyl-D glucosamine residue by reaction with a murine monoclonal antibody T6 specific for this structure in the Salmonella Ra core lipopolysaccharide (LPS). All 20 serotypes (70 strains) belonging to serogroups A to E were positive for the T6 epitope while 29% of the 96 serotypes (106 strains) belonging to O serogroups F to 67 were negative; 12 serotypes (12 strains) of subspecies IIIb Salmonella were positive for the T6 epitope, but 10 serotypes (11 strains) of subspecies IIIa Salmonella were found to lack this epitope. In T6-positive strains, the epitope was accessible to antibody binding in both the unsubstituted free rough core LPS and in the rough core LPS substituted with a few repeating units of O side chains. The presence or absence of the T6 epitope in Salmonella strains was not affected by culture conditions, the source of the isolate, the age of the culture or the presence of fimbriae antigens. PMID- 1719591 TI - Effect of marine coral prostanoids, clavulones, on spontaneous beating rate of cultured myocardial cells from fetal mouse hearts. AB - We examined effects of newly discovered marine coral prostanoids, clavulones, isolated from the Japanese stolonifer Clavularia viridis, on the spontaneous beating rate of cultured myocardial cells from fetal mouse hearts. Clavulone caused positive chronotropic action at 2-5 min after addition of clavulone (0.45 microM) to the reaction media of cardiac cells. This effect induced by clavulone was clearly different from the positive inotropic effects of ouabain (10 microM) and Bay K 8644 (0.1 microM) as judged by photoelectric recordings of beating. These results suggest a new biological action of clavulone that has positive chronotropic action on the cultured mouse myocyte preparation. PMID- 1719592 TI - Doxapram inhibits the in vitro oxidation of hexobarbital. AB - The effect of doxapram on the mouse liver microsomal hexobarbital metabolism in vitro was studied. Doxapram inhibited hexobarbital oxidase in a competitive manner, with inhibition constants between 2 and 10 mM. Doxapram induced a reverse type I spectral change with a spectral dissociation constant of about 0.1 microM. These results indicate that doxaprame is an inhibitor of microsomal drug metabolism in the mouse. PMID- 1719593 TI - Physiological effects of a perfluorochemical blood substitute in beagle dogs. AB - Perfluorochemical (PFC) emulsions have been examined for use as erythrocyte substitutes in the treatment of various disease states. The physiological changes induced by PFC infusion would be an important determinant of successful clinical therapy. Previous studies have reported PFC induced changes in the disposition of drugs. This report describes some physiological (hematology, cardiovascular, liver enzyme) changes resulting from a 30% blood exchange with a PFC emulsion in Beagle dogs. A 30% blood exchange with hydroxyethylstarch (HES) also was evaluated and compared to the PFC emulsion exchange. The blood pressure was markedly reduced shortly after PFC infusion while HES infusion produced only minor changes. Changes in the heart rate following blood replacement were similar for PFC and HES treated dogs. Hematology profiles also were similar for the PFC and HES treatment groups. The liver enzyme levels in PFC treated dogs showed marked elevations beginning shortly after PFC infusion and remained elevated for months after the initial PFC blood replacement. In contrast, HES treated dogs exhibited no observable changes in liver enzyme levels over the time course of the study. PMID- 1719594 TI - Chrysotile asbestos-induced membrane damage in human erythrocytes. AB - Chrysotile asbestos causes the release of K+ and hemoglobin from erythrocytes. At short incubation times K+ release is slightly higher than hemoglobin release but for incubation times of 10 min or longer the percentage K+ release, and the percentage hemolysis are the same. The results suggest that asbestos causes a sudden and complete disruption of the membrane permeability for K+, which is probably followed by colloid osmotic hemolysis. Polyanions inhibit asbestos induced hemolysis completely. Leaching of asbestos diminishes its hemolytic potency, as does a decrease in pH. The results support the view that positive charges, probably of magnesium, play a predominant role in asbestos-induced hemolysis. PMID- 1719595 TI - Strategy for diagnosis of Toxoplasma gondii in stereotactic brain biopsies. AB - The colloidal gold method using polyclonal antitoxoplasma antibodies was used to detect Toxoplasma organisms in stereotactic brain biopsies of patients with AIDS. For comparison, conventional histologic staining and additional other immunohistochemical methods were also studied in the same cases. Compared to the other stains, a reliable diagnosis of Toxoplasma gondii could be established with the colloidal gold method in all patients investigated. This method was highly specific and sensitive and stained both the encysted and the tachyzoite forms of the organism. Stronger contrast of the single parasite and better identification is possible if it is applied together with silver enhancement. Conventional staining and immunohistochemical peroxidase techniques are not advantageous, because they allow a diagnosis only by observation of the rare pseudocysts and not by free parasites in tissue, so the colloidal gold method appears to be the preferred method for a rapid diagnosis of cerebral toxoplasmosis, especially in cases where only small amounts of tissue can be obtained. PMID- 1719596 TI - [The value and limits of subtotal gastrectomy in gastric cancer]. AB - In the interval 1970-1988, in 321 of 670 patients with gastric neoplasm admitted to the 1st Surgical Clinic of Iasi a subtotal gastrectomy was performed. Most patients were males (68.9%), more commonly aged between 50 and 70 years and in advanced evolutive stages. The gastric neoplasm were sited in order of frequency in the antrum (46.9%), body of the stomach (34.6%), eso-cardio-tuberosity (11.5%), linitis (4%), gastric stump and malignant ulcer (3%). Surgery was possible in only 59% of the cases (25% curative and 34.1% palliative). The surgical technique and its difficulties are detailed. In the 321 subtotal gastric resections 20 complications, 4 deaths (1.2%) and 20% survivals over 5 years were recorded. It is concluded that radioendoscopic and microscopic investigations, with their possibility of an early diagnosis, may cause a limitation of the indications of total gastric resection in the treatment of gastric neoplasm in favour a subtotal resection, more satisfactory from the functional viewpoint. PMID- 1719597 TI - Laser treatment of the urethra and prostate. AB - For most applications in the urethra, lasers simply represent an alternative to existing technology. Most studies conducted to date can be considered "feasibility studies," ie, they demonstrate that the technique can be performed without clearly showing whether it holds any clear advantages or indications. We have found laser treatment of condyloma acuminata of the urethral meatus as well as more proximal portions of the urethra to be easy and to provide cosmetic results that appear to be superior to those obtained with alternative techniques. In more proximal portions of the urethra, scarring is less than that with an electrocautery and the incidence of treatment induced urethral strictures may be lower with laser. The recurrence rate remains high and is probably unrelated to the device or technique used in treating gross lesions. PMID- 1719598 TI - [Allergic and pseudo-allergic reactions in the perioperative period]. AB - Most drugs or infusions are potent inducers of allergic or pseudoallergic reactions. During the perioperative period the patient is usually on multiple medication. Thus, life-threatening reactions can be expected. In this publication these adverse reactions are considered from an allergological point of view. A review of the literature is given. Four case reports point out the variety of possible allergens, the difficulties in definite causal identification and the necessity of referring the patient to an allergological work-up. PMID- 1719599 TI - [The introduction of interleukins to the world]. PMID- 1719600 TI - [Palliative care: fashion or necessity?]. PMID- 1719601 TI - [A day of reflection on palliative care (Vevey, June 1990). From diagnosis to prognosis]. PMID- 1719602 TI - [Analysis of needs: the physician's viewpoint]. PMID- 1719603 TI - [Role of a social worker of the Vaud League against Cancer in palliative care]. PMID- 1719604 TI - [Palliative care: where and how?]. PMID- 1719605 TI - [Palliative care and use of measures]. PMID- 1719606 TI - [Pain, suffering, limitations in palliative care]. PMID- 1719607 TI - [Palliative care unit in a district hospital: analysis of 2 years of experience. Multidisciplinary palliative care teams]. PMID- 1719608 TI - [Palliative care: control of nausea and vomiting]. PMID- 1719609 TI - Epitope analysis of recombinant human thyrotropin. AB - Recombinant proteins and peptides are anticipated to become promising tools as reagents for immunoassays because of their constant availability and structural uniformity. However, careful assessment of their immunological properties is necessary prior to practical use. Epitope analysis of recombinant human thyrotropin (rec-hTSH) was recently performed in our laboratory. Details are to be published elsewhere (T. Kashiwai, K. Miyai et al.) but the results are briefly shown here. PMID- 1719610 TI - Tumour markers in prostatic cancer. AB - Prostate cancer is now the third commonest cancer in men. Extensive clinical trials comparing acid phosphatase, alkaline phosphatase (ALKP) and prostate specific antigen (PSA) have shown that PSA is the most sensitive and specific of the tumour markers available for prostate cancer. Caution is needed when comparing the results from different assay methods, there is no international standard for PSA. In the management of localised disease, radical treatment can reduce the PSA levels to less than 0.4 ng/ml, similar results can be obtained for a varying duration in patients sensitive to androgen withdrawal. Raised levels greater than 0.4 ng/ml after radical prostatectomy are indicative of residual disease. PSA is valuable in monitoring deferred treatment or the effects of hormone manipulation and give an indication of the prognosis and early warning of recurrence. In extensive metastatic disease the combination of PSA and ALP reflects the tumour activity. Less than 15 hot spots on the scintigram at presentation and a PSA less than 10 ng/ml 3 to 6 months after commencing treatment is associated with prolonged survival. The role of PSA in population screening for early prostatic cancer is uncertain; early results suggest it can be used in combination with digital rectal examination and ultrasonic examination of the prostate. The effect of a PSA decision level at 4 or 10 ng/ml has a considerable influence on the pick up rate. PMID- 1719611 TI - Tumour markers in gynaecologic oncology. AB - Human chorionic gonadotropin is a highly sensitive and specific tumour marker for gestational trophoblastic disease and reflects accurately tumour volume and clinical course of the disease. Alpha-fetoprotein is usually a reliable marker for endodermal sinus tumours and embryonal carcinomas, and also predicts the presence of yolk sac elements in mixed germ cell tumours. Squamous cell carcinoma antigen can be useful in the detection of recurrent cervical carcinoma. Carcinoembryonic antigen determination can be of help to distinguish adenocarcinomas of the cervix from those of the endometrium. Thus far, CA125 is the most widely used tumour marker in gynaecologic oncology. Its main area of application is epithelial ovarian carcinomas where it is useful for disease monitoring during and after therapy. The specificity of the CA125 test is too low for use in preoperative diagnosis. The utility of CA125 for screening depends on combinations with other diagnostic methods, such as pelvic examination and ultrasound. No combination of tumour markers has so far proven superior to CA125 alone. PMID- 1719612 TI - Interferons and the colony-stimulating factors IL-3 and GM-CSF enhance the IFN alpha response in human blood leucocytes induced by herpes simplex virus. AB - Human peripheral blood mononuclear cells (PBMC) were stimulated to produce interferon-alpha (IFN-alpha) by glutaraldehyde-fixed Herpes simplex virus type 1 (HSV)-infected WISH amnion cells in vitro. Different cytokines were included during the stimulation and tested for their ability to enhance the IFN-alpha response which occurs in the natural IFN-alpha producing (NIP) leucocytes. The total production of IFN-alpha and the numbers of IFN-alpha producing cells (IPCs) were increased by interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Their most marked effect was to reduce the time required for induction of the IPC by the HSV-infected cells, thereby causing both an earlier peak of IPC numbers and secretion of IFN-alpha. Addition of IFN-alpha 2b did not alter the kinetics of the IFN-alpha response in the same way as the two CSFs, but instead generally increased the IPC numbers and the production of IFN-alpha. The IL-3 and GM-CSF, especially in combination with IFN-alpha, had the most pronounced enhancing effects on IPC numbers when PBMC were induced at low cell concentrations. The cytokines IL-1, IL-2, IL-4, IL-6 or tumour necrosis factor-alpha (TNF-alpha) had no detectable effects on the IFN-alpha response. The results suggest that cytokines such as the CSFs and IFNs may be involved in the regulation of NIP cell functions. PMID- 1719613 TI - Flow cytometric analysis of natural interferon-alpha producing cells. AB - Herpes simplex virus-infected cells induce high interferon-alpha (IFN-alpha) production in infrequent cells among peripheral blood mononuclear cells (PBMC), designated natural IFN-alpha producing (NIP) cells. The properties of such NIP cells were compared with defined populations of leucocytes by means of flow cytometric analysis and sorting. The NIP cells are characterized as a discrete population of cells with high forward and low to intermediate orthogonal light scattering, similar to that of early progenitors of myeloid and lymphoid cells. However, they appear to lack the stem cell-associated molecule CD34. Furthermore, NIP cells cannot be localized to the myeloid line of cell differentiation, because they do not express the CD33, CD13, CD11b, CD15 or CD14 antigens. Neither do they express CD10 and CD19 antigens which are present in all stages of B-cell differentiation plasma cells excepted, nor CD7 antigens expressed on early T cells. In combination with previous results, our data support the view that the NIP cell is a unique and distinct cell type in peripheral blood, possibly with a physiological role in the defence against certain viral infections. PMID- 1719614 TI - Characterization of monoclonal anti-alpha 1-microglobulin antibodies: binding strength, binding sites, and inhibition of lymphocyte stimulation. AB - Eleven monoclonal antibodies (MoAb) directed against the immunoregulatory plasma glycoprotein alpha 1-microglobulin were characterized. The MoAb were produced in mice immunized with a mixture of alpha 1-microglobulin homologues from man, guinea pig, rat and rabbit. Using radioimmunoassay, western blotting, affinity chromatography, and Scatchard analysis, the affinities and binding sites of the MoAb were analysed. All antibodies were more or less cross-reactive, but most showed a major specificity for one or two of the alpha 1-microglobulin homologues. None of the antibodies was directed against the carbohydrate moiety of alpha 1-microglobulin. Six of the MoAb had high affinity for the antigen and four of these were directed towards the same part of the molecule though differing in their species specificity. Five showed lower affinity for the antigen and were mainly directed towards epitopes on other parts of the molecule. Only some of the antibodies could block the proliferation of lymphocytes induced by human alpha 1-microglobulin. The blocking efficiency of the different antibodies was similar when tested on the stimulation of human or mouse lymphocytes, suggesting that the same part of the alpha 1-microglobulin molecule is responsible in both species. The magnitude of blocking by the different MoAb was not related to their affinities, emphasizing the importance of where on the alpha 1-microglobulin molecule, rather than how strongly, they bind. The binding of the strongest blocking antibody was shown to be directed to a C-terminal peptide of rat alpha 1-microglobulin, indicating that this part of alpha 1 microglobulin is important for the mitogenic effects. Thus the panel of anti alpha 1-microglobulin MoAb should be a valuable tool for structural and functional studies of alpha 1-microglobulin. PMID- 1719615 TI - Kinetics and regulation of NK activity by interleukin-2 and interferon in acute toxoplasmosis. AB - Natural killer (NK) activity against Toxoplasma gondii tachyzoites and tumour cells during acute toxoplasmosis was investigated using a single-cell NK assay. During the course of infection the percentage of lymphocytes binding tachyzoites and tumour cells did not vary significantly and NK activity was enhanced due to an increase in the specific cytolytic capacity per cell. To determine whether regulatory mechanisms mediated by cytokines might explain the increased NK activity, the kinetics of interleukin-2 (IL-2) and interferon (IFN) production and the correlation between their concentrations and the level of NK activity were analysed at the same time. As the infection progressed NK activity increased in spite of the fact that IL-2 production decreased (except for a small increase during the first day of infection). However, IFN production increased gradually in close temporal and quantitative association with the overall increase in NK activity. These results suggest that T. gondii, via its ability to produce interferon, enhances NK activity against itself and other cells. PMID- 1719616 TI - Transrectal ultrasound, digital rectal examination, and prostate-specific antigen: preliminary results of an early detection program for prostate cancer. AB - Three hundred and ninety eight self-referred men with no histories of prostate problems were followed once each year for up to four years to determine the feasibility of early prostate cancer detection by digital rectal examination, transrectal ultrasound, and prostate-specific antigen. Evaluation of prostate specific antigen was based on a polyclonal level of normal of 2.6 nanograms per milliliter by the Yang assay. Biopsies were performed when indicated by either transrectal ultrasound or digital rectal examination. The overall cancer detection rate for the four year period was 6.3 percent. A 3:1 cancer detection advantage of transrectal ultrasound over digital rectal examination was shown. Transrectal ultrasound and prostate-specific antigen each detected 92 percent of the proven cancers, and were complementary when either test was normal, together detecting 100 percent of the cancers. Thirty two percent (8/25) of all cancers were detected by digital examination, with digital exam having no predictive power after two study years. Prostate-specific antigen as an initial screening test for early prostate cancer may identify a suspicious group, whom may further be evaluated by transrectal ultrasound and digital exam. Results of this study lend credibility to the large scale randomized screening study proposed by the U.S. National Institutes of Health in which prostate-specific antigen and digital rectal examination are to be used as initial tests for prostate cancer detection. PMID- 1719617 TI - Transrectal sonography. A personal review and recent advances. PMID- 1719618 TI - Color Doppler as an adjunct to prostate ultrasound. AB - Endorectal ultrasound of the prostate with color Doppler is a new technique to identify internal vasculature and flow. In a period of one year more than 500 men have been examined with this technique. The normal prostate will demonstrate flow within the surrounding vasculature; there is no distinguishing flow among the various zones. In prostate cancer there is an increased flow within or surrounding the lesion. Diffuse prostatitis exhibits a diffusely abnormal flow; in focal prostatitis the flow is not significantly different from cancer. In benign prostatic hyperplasia a significant abnormal flow was seen, usually in the inner gland, and flow was frequently identified in the area of the surgical capsule. Color Doppler can provide additional physiologic information for prostate ultrasound and may increase the specificity and sensitivity of the examination in the evaluation of potential prostatic malignancy. PMID- 1719619 TI - Reproducibility of transrectal ultrasound of prostatic disease. AB - One hundred patients with suspicion of prostatic disease were examined by transrectal ultrasound. Two urologists with experience in this technique performed the examination separately for each patient. In the present study the differential diagnosis between normal prostate, benign hypertrophy and prostate cancer was almost perfectly reproducible. However, prostatitis was a difficult diagnosis by transrectal ultrasound. Prostatic cysts and stones were readily recognised. Although not perfectly reproducible, the transverse dimensions of the prostatic adenomas were reasonably comparable. Measurement of the longitudinal diameter of prostatic adenomas was difficult and poorly reproducible in our hands. PMID- 1719620 TI - Antimicrobial prophylaxis in transurethral resection of the prostate. With special reference to preoperatively sterile urine. AB - The literature on antimicrobial prophylaxis in connection with transurethral resection of the prostate (TURP) is reviewed, and it is concluded that there is no proof of clinically significant beneficial effect of prophylaxis when the urine is sterile preoperatively. Prophylaxis is indicated when bacteriuria or an indwelling urethral catheter is present at the time of operation. Other possible risk factors, such as diabetes mellitus, neurogenic bladder dysfunction, immunosuppression, earlier coronary bypass operation and the presence of prosthetic devices, need further investigation. PMID- 1719621 TI - Evaluation of outpatient assessment after transurethral prostatectomy. AB - The need for a three-month visit at the outpatient clinic for assessment including uroflowmetry was retrospectively evaluated in 78 patients who underwent transurethral resection for benign prostatic hypertrophy during 1986. One patient died before discharge and six were lost to follow-up. Eleven patients requested appointments earlier than three months, and 10 of these needed further treatment; four were operated on and six were treated with drugs. The remaining 60 patients attended as arranged. Thirty-nine were discharged, but 21 required further treatment. Eleven were operated on, four were given drugs, one had an external catheter because of incontinence, and five needed further visits to the outpatient clinic for other reasons. It was not possible to predict the necessity for follow-up from preoperative symptoms, cystitis, maximum urinary flow (ml/s), blood loss during operation, or length of stay in hospital after operation. We conclude that 35% (95% c.l. 23-48%) of the arranged outpatient visits three months after transurethral resection for benign prostatic hypertrophy are necessary and that the need is not predictable. PMID- 1719622 TI - Healing of ligaments in synovial fluid. An experimental study in rabbits. AB - To investigate the importance of nutritive environment in the healing of reconstructed ligaments, the morphology of sutured free patellar ligaments in synovial fluid of the contralateral knee joint, without re-establishment of microcirculation, was studied in rabbits. After two weeks (n = 6) and one (n = 6) and three (n = 5) months, the sutured ligament was removed from the joint and fibroblasts were seen growing into the suture gap. The ligaments were enveloped in a capsule of fibroblasts, which was more developed in those ligaments that were studied three months after operation. The collagen of the piece of ligament was disorganised at all time points, and tensile strength (n = 6) was low compared with non-sutured patellar ligament. Some ligaments disappeared or were partly attached to the synovial membrane and revascularised. The findings indicate that diffusion of nutrients may be important for the survival and healing of reconstructed ligaments, as the ligaments can heal in synovial fluid without re-establishment of the microcirculation. PMID- 1719623 TI - Correlation between sensory loss, functional disability and short-latency somatosensory evoked potentials in strokes. AB - Short-latency somatosensory evoked potentials (SEPs) were reviewed for their correlation with the clinical features and functional deficit of 64 patients with supratentorial deep or superficial ischemic strokes. Abnormal SEPs correlated with lesions of proprioceptive pathways and with clinical sensory impairment. 87% of patients with abnormal SEPs had sensory loss and 88% of those with normal SEPs had normal sensation. The reliability of the SEPs made them useful in an objective demonstration of an abnormality in sensory system function. However, they are not sensitive enough to be used as a method of prognostication because 1) the SEPs abnormalities depend on the involvement of their generators and therefore may be normal in some thalamic or cortical lesions associated with severe disability, 2) 48.4% of the patients studied had normal sensation, and 3) in the group with absent SEPs, the functional disability was poorer than in the group with normal SEPs, not in relation to the type of the sensory loss but to the volume and location of the infarcted area. PMID- 1719624 TI - Guillain-Barre syndrome in a 93-year-old woman: rapid improvement of neurologic function following intravenous immunoglobulin. AB - We treated a 93-year-old woman with Guillain-Barre syndrome (GBS) with high-dose intravenous immunoglobulin (IgIV). The patient improved within 48 hours of the onset of treatment. There were no adverse side effects. PMID- 1719625 TI - Occlusion of the carotid artery as presenting symptom of acute promyelocytic leukemia. AB - In a 44-year-old female acute promyelocytic leukemia (APL) presented with abrupt onset of right hemiplegia and aphasia due to occlusion of the left carotid artery at bifurcatio. There was laboratory evidence of disseminated intravascular coagulation (DIC). Thrombotic complications are unusual in APL, even in cases with evidence of DIC. This report aims at underlying the important implication of a correct timely diagnosis in young patients presenting with stroke. PMID- 1719626 TI - [Narcolepsy and activity monitor]. AB - By means of a wrist accelerometer, motor activity of 14 narcoleptic patients was continuously monitored. The accelerometer registered accelerations exceeding a threshold value of 0.1 gr and stored activity integrated over 3 minutes. After a registration period of 20 days, the data were read out, graphically displayed and compared to the patient's diary. Interruptions of night sleep and some sleep attacks during day correlated closely with the patient's diary. However, sleep attacks of less than about 5 minutes duration were not identified, because they could not be differentiated from normal rest. Except for that, activity monitoring with this accelerometer is a useful and simple method for ambulatory activity recording in narcoleptics. PMID- 1719627 TI - [Cerebral consequences of stenosing and occlusive diseases of the interior carotid artery. Importance of transcranial Doppler examination. 1]. AB - By means of transcranial Doppler (TCD), the author reports intracranial haemodynamic consequences of 57 stenosis or occlusions of the internal carotid artery (ICA), 13 dissections and 44 atheromatous lesions, in 50 patients 32 of whom having had angiography. If a stenosis is significant, the curve of the middle cerebral artery (MCA) becomes dampened and PTI (Pulsatility Transmission Index) diminishes. In this series, the association MCA curve dampened and PTI lower than 0.90 is the evidence of a more than 90% stenosis, except in 2 atheromatous and symptomatic occlusions. No precise explanation for these exceptions is given by literature. An ICA stenosis has also an influence on communicating arteries. In this study, collateralisation by the circle of Willis exists in 14% of 50-75% stenosis, 72.7% of 75-90% stenosis and 100% of more than 90% stenosis. It can depend on anterior communicating artery (ACoA) alone, posterior communicating artery (PCoA) alone or both. Detailed study of the 3 groups leads to the hypothesis of a hierarchy in the function of the communicating arteries, ACoA having the priority. PMID- 1719628 TI - Modern psychiatric classification in research and clinical practice. AB - After general "antipsychiatric" criticism and with empirical results, which demonstrated partly very low interrater reliabilities of conventional psychiatric diagnosis a new appreciation of nosological difficulties in psychiatry arose. Based on philosophical concepts of logical empiricism which had been established in philosophy at the beginning of our century the operational psychiatric diagnosis was developed. The first system, the Research Diagnostic Criteria (RDC), was used for scientifical purpose in the early seventies. Based upon this system the American Psychiatric Association (APA) introduced DSM-III in 1980, which had a very strong influence on psychiatric diagnosis and classification during the last ten years. Operational psychiatric diagnosis is demanding precise criteria for inclusion and exclusion and definite diagnostic rules. The tenth revision of the International Classification of Diseases (ICD-10) of the WHO is in preparation and will also contain an operational approach. This new instrument will be introduced from 1992 in WHO member states. As a result of a number of empirical studies an improvement of interrater reliability in psychiatric diagnosis using DSM-III, DSM-III-R and the drafts of ICD-10 as well was demonstrated. An other important innovation of modern classification systems is the multiaxial diagnosis. These approaches have a number of advantages for clinicians, for communication in health service systems as well as for epidemiological and biological psychiatric research. PMID- 1719629 TI - [Post-amnesia syndrome of transient global amnesia. A psychopathometric study]. PMID- 1719630 TI - [Affective and schizophrenic syndromes in multiple sclerosis. Review of the literature and case reports]. AB - Based on reports of three cases, the manic, depressive, schizophrenic and organic mental symptoms coincident to multiple sclerosis (MS) are described. The literature indicates an increased prevalence of psychiatric disturbances in MS. However, the relation between the psychiatric and the neurological disorders remains speculative. The temporal covariance and responsiveness to pharmacotherapy of the psychiatric and neurological symptoms of MS as well as the localisation of MS foci in diagnostic procedures (CT, MRI) are discussed in the light of the literature. The present authors hypothesize that every disturbance of the central nervous system (CNS), especially if chronic-inflammatory and multilocal, increases the probability that psychiatric symptoms will arise (as measured on a continuum ranging from "psychically conspicuous" to "psychically inconspicuous"). To this extent, then, MS would be comparable to other chronic inflammatory CNS disorders such as AIDS, neuroborrelioses, syphilis and certain forms of viral encephalitis. PMID- 1719631 TI - [Heparin-binding growth factors]. PMID- 1719632 TI - On the trail of the errant T cells of multiple sclerosis. PMID- 1719633 TI - Phosphorylation of c-Src on tyrosine 527 by another protein tyrosine kinase. AB - The protein tyrosine kinase activity of the cellular Src protein is negatively regulated by phosphorylation at tyrosine residue 527 (Tyr527). It has not been established whether this regulatory modification of Src is mediated by autophosphorylation or by another cellular protein kinase. The phosphorylation of a modified form of c-Src that lacks kinase activity was examined in mouse cells that do not express endogenous Src (because of the targeted disruption of both src alleles). Phosphorylation of the inactive form of Src on Tyr527 occurred to a similar extent in cells lacking endogenous Src as it did in cells expressing Src. Therefore, Tyr527 phosphorylation, and thus negative control of Src kinase activity, is mediated by another cellular protein tyrosine kinase. PMID- 1719634 TI - Long-range structure in ribonuclease P RNA. AB - Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA. PMID- 1719635 TI - GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. AB - Binding of ligand or antibody to certain cell-surface proteins that are anchored to the membrane by glycophosphatidylinositol (GPI) can cause activation of leukocytes. However, it is not known how these molecules, which lack intracellular domains, can transduce signals. The GPI-linked human molecules CD59, CD55, CD48, CD24, and CD14 as well as the mouse molecules Thy-1 and Ly-6 were found to associate with protein tyrosine kinases, key regulators of cell activation and signal transduction. A protein tyrosine kinase associated with the GPI-linked proteins CD59, CD55, and CD48 in human T cells, and with Thy-1 in mouse T cells was identified as p56lck, a protein tyrosine kinase related to Src. This interaction of GPI-linked molecules with protein tyrosine kinases suggests a potential mechanism of signal transduction in cells. PMID- 1719636 TI - Rusting of the lock and key model for protein-ligand binding. PMID- 1719637 TI - Clinical investigations with ateroid in old-age dementias. PMID- 1719638 TI - Behavioral, endocrine, and neurochemical effects of sulfomucopolysaccharide treatment in the aged Fischer 344 male rat. AB - 1. Daily treatment of male 19- to 22-month-old Fischer 344 male rats with Ateroid (20 mg/kg/day, p.o.), beginning one month before and continuing throughout testing, resulted in a significant partial reversal of age-related deficits in: a) Conditioned one-way (spatial, unsignaled) avoidance acquisition and retention b) Conditioned two-way (nonspatial, signaled) avoidance acquisition. 2. Ateroid reversed the age-related reductions in nucleus accumbens DOPAC and HVA levels, but not the age-related decrease in neostriatal HVA content or concomitant increase in 5-HIAA levels. Reduced dopamine turnover in the nucleus accumbens may underlie, at least in part, age-related deficits in conditioned avoidance learning and retention. Thus, the behavioral effects of Ateroid observed in the present study may be due to its normalizing influence on dopamine neurotransmission in the nucleus accumbens. 3. Stress-induced corticosterone secretion was greater in the old than in the young vehicle-treated rats. Ateroid treatment normalized this exacerbated corticosterone response to stress. 4. Daily Ateroid treatment did not affect any of the parameters measured in the young (5-8 months) F344 male rats. 5. Ateroid treatment did not affect the age-related reductions in exploratory behavior. The aged and young animals did not differ in their swimming ability. Thus, the effect of Ateroid on learning and memory processes does not appear to be due to an effect on locomotor or performance skills. 6. The age-related deficits in conditioned avoidance learning were not associated with abnormal basal (morning trough) plasma corticosterone levels, and Ateroid did not affect basal plasma corticosterone concentrations. PMID- 1719639 TI - Experimental and clinical pharmacology of pentosan polysulfate. PMID- 1719640 TI - Interferon: advances in biotherapy. PMID- 1719641 TI - The role of interferon and interleukin-2 in the immunotherapeutic approach to renal cell carcinoma. AB - Approximately 24,000 cases of renal cell carcinoma are expected in the United States in 1990. Although approximately 50% of patients with local disease are cured by surgery, in patients with metastatic disease the median survival is only approximately 10 months. Neither chemotherapy nor radiation therapy has been shown to be effective against metastatic renal cell carcinoma. Immunotherapy has come to the forefront of clinical research for the treatment of metastatic renal cell carcinoma. In the past decade, the development of recombinant DNA techniques has enabled the production of large quantities of biologic response modifiers such as the interferons and interleukins. Following initial reports in 1983 by the University of California-Los Angeles (UCLA) group and the investigators at M. D. Anderson Hospital, in Houston, TX, numerous trials have demonstrated a reproducible objective response rate to interferon of 15% to 20%. These responses are independent of the interferon preparation used, and optimal dosage/schedule has not been determined. In general, responses have been correlated with the following patient factors: previous nephrectomy, good performance status, a long disease-free interval, and lung-predominant disease. Median response durations of from 8 to 10 months can be expected. The addition of vinblastine, gamma interferon, or aspirin has not improved the therapeutic index. Interleukin-2 therapy has produced encouraging results in 10% to 15% of patients. Although high dose therapy is associated with substantive side effects, a small cohort of patients have been in continuous remission for extended periods of time, raising the possibility of "true" complete remissions of clinical significance. Recent trials, including our trials at UCLA, have combined the interleukins and interferons in this patient population. This combination has a sound scientific basis and the results are encouraging, especially when the toxicity profile is considered. Most patients receive these combinations as outpatients and have not required hospitalization nor suffered the toxicities of the high-dosage regimens. Complete pathologic remissions have been observed using this lower dosage, outpatient schedule. Clinical trials suggest that interferon and interleukin-2 may have an expanding role in metastatic kidney cancer both as single agents and in combination outpatient biologic therapy. The future clinical trials of kidney cancer will continue to incorporate these biologic response modifiers into the therapeutic strategies of the 1990s. PMID- 1719642 TI - Phase I/II trials of alpha-interferon alone or in combination with zidovudine as maintenance therapy following induction chemotherapy in the treatment of acquired immunodeficiency syndrome-related Kaposi's sarcoma. AB - Kaposi's sarcoma (KS) is a malignant neoplasm that develops in 20% to 30% of all acquired immunodeficiency syndrome (AIDS) cases. Kaposi's sarcoma primarily involves the skin, but can progress to involve the lungs, gastrointestinal tract, and liver. alpha-Interferon alone or in combination with zivoduvine has activity in acquired immunodeficiency syndrome-related KS, especially in patients with limited disease and CD4 lymphocyte counts over 400/mm3. Patients with progressive or symptomatic visceral disease, however, can be treated more effectively with cytotoxic chemotherapy. We have used a combination of doxorubicin, bleomycin, and vincristine (ABV) and have achieved response rates of over 80%. Discontinuation of therapy, however, is associated with relapse shortly after response (2 to 3 months). Thus, we have begun studies to define a safe and effective maintenance therapy. Such therapies should include antiretroviral agents since most patients succumb to other human immunodeficiency virus complications, and since human immunodeficiency virus directly, through viral proteins, and indirectly, through the induction of cellular genes, induces KS growth. Additionally, agents with antitumor activity and possible antiviral activity, such as alpha-interferon, may be potentially effective in maintenance therapies. We recently studied 21 patients in a phase I study of recombinant interferon alpha-2b (INTRON-A, Schering-Plough Corp, Kenilworth, NJ) alone following ABV chemotherapy. A dose of 10 million units, given in daily subcutaneous injections, was the maximal tolerated dose; higher doses were associated with intolerable fatigue, diarrhea, and fevers. We are currently conducting a phase I/II trial studying the combination of zivoduvine (500 mg/d) and recombinant interferon alpha-2b (5, 10, and 15 million units) as maintenance in patients with advanced or progressive KS. PMID- 1719643 TI - Treatment options for hairy-cell leukemia. AB - In 1991, there are numerous proven therapies for the treatment of hairy-cell leukemia. Since none have been proven to be curative, it is important that all be considered, as individual patients might need to undergo a series of sequential treatments with each. In fact, some elderly patients with minimal splenomegaly and relatively normal blood counts might require no therapy. Splenectomy has a role in rapid reversal of severely depressed blood counts in association with a systemic infection; however, growth factors might soon replace this role of emergency splenectomy. Treatment with recombinant interferon results in few complete remissions, but with normalization of peripheral blood counts in over 80% of patients. In the original interferon alfa-2b study of 195 patients, only three of the 159 patients achieving a normalization of their blood counts have subsequently died. Treatment with deoxycoformycin either after interferon or initially has resulted in an apparent complete remission rate of approximately 60%, but recent follow-up analyses suggest that hairy cells persist within the bone marrow; however, patients seem to remain in a clinical remission. Treatment with 2-chlorodeoxyadenosine shows little toxicity and a high apparent complete remission rate. However, review of the posttreatment bone marrow specimens remains to be done, and longer follow-up is necessary to assess true differences in the degree of response from interferon or deoxycoformycin treatment. Advances in therapy over the past decade have led to significant benefits for patients with hairy-cell leukemia. PMID- 1719644 TI - Mechanisms of interaction of interferon and 5-fluorouracil in solid tumors. AB - Recent clinical trials demonstrate the combined activity of 5-fluorouracil (5-FU) and interferon (IFN) in advanced colon cancer. Several possibilities exist for explaining the interaction. Interferon may alter the pharmacokinetics of 5-FU infusion by increasing the steady state concentration. Interferon enhances the inhibitory effects of 5-FU for tumor cells in culture. This enhancement is blocked by thymidine. Interferon reduces the concentration of thymidylate synthetase, and this may account for the thymidine-reversible interaction. An alternative mechanism invokes the immunomodulatory effects of IFN. Interferon augments the activity of killer cells with possible anti-tumor activity, both in vitro and in vivo. Also, by increasing the expression of human leukocyte class I antigens, IFN reduces the sensitivity of tumor cell lines to cell-mediated killing, an effect termed resistance. 5-Fluorouracil reverses the resistance in a time- and dose-dependent manner. The effect is mediated through inhibition of protein synthesis, since thymidine cannot reverse it. Fluorouridine is more active in reversing resistance than fluorodeoxyuridine. 5-Fluorouracil also reverses the induction of human leukocyte antigens by IFN. Studies in the resistance model suggest that high doses of 5-FU by infusion for several days might be the optimal method for modulation of IFN-induced effects. PMID- 1719645 TI - Respiratory tract side effects of angiotensin converting enzyme inhibitors: current knowledge. PMID- 1719646 TI - A comparative evaluation of a new immunoenzymatic test (RREID) with currently used diagnostic tests (DME and FAT) for dog rabies. AB - Diagnosis of rabies in dogs was performed in microplates which had been coated with immunoglobulin G previously sensitized to purified rabies virus antinucleocapsids. Homogenized brain suspensions were incubated in the plates and the specific binding rabies antigen was revealed by the use of the same IgG conjugated with horseradish peroxidase. Samples from the same specimens were subjected to standard rabies diagnostic tests--the direct microscopic examination (DME) or Sellers staining for Negri bodies and the fluorescent antibody test (FAT). FAT was used as the reference test or gold standard because of its proven sensitivity and accuracy. The concordance of FAT with RREID was 98.89% while that with DME was 96.67%. Sensitivity of both DME and RREID compared with FAT in this study was 100% while specificity of RREID versus FAT was 98.46% as compared with 95.38% DME versus FAT. The positive predictive value of RREID versus FAT was 96.15% while that of DME versus FAT was 89.29% although the negative predictive value of both RREID and DME compared with FAT was 100%. In the overall assessment, RREID results were demonstrated to approximate closely those of FAT. It is therefore concluded that RREID can be used in diagnostic laboratories to corroborate DME and where MIT and FAT cannot be done. RREID would also be useful in epidemiological studies where large samples are tested. PMID- 1719647 TI - Case report: septicemic melioidosis following near drowning. PMID- 1719648 TI - Characterization of a Plasmodium falciparum epitope recognized by a monoclonal antibody with broad isolate and species specificity. PMID- 1719649 TI - [The radiation and combined chemoradiation therapies of malignant thymus tumors after palliative surgical interventions]. PMID- 1719650 TI - Randomized comparison of endoscopic palliation of malignant esophageal stenoses. AB - In a randomized study, palliative therapy of malignant esophageal and gastric stenosis was investigated by a comparison of endoscopic laser therapy (ELT) with palliative endoscopic perturbation (PEP). A total of 124 patients exhibiting a malignant stenosis in the esophagus and proximal stomach were referred to our unit between January 1, 1987, and March 31, 1990. Criteria for randomization were: (1) inoperable malignant stenosis, (2) dysphagia enabling the ingestion of semi-solid food, (3) the possibility of performing ELT and PEP, and (4) the absence of fistula formation. Only 40 patients met these criteria; the remaining 84 subjects were assigned to an escape group whose treatment consisted of ELT, PEP, percutaneous endoscopic gastrostomy, transnasal feeding tube, radiotherapy, and endoscopic bougienage. We found no significant difference between ELT and PEP with regard to survival, food passage, or quality of life. We recommend the application of PEP in patients exhibiting advanced tumor disease and a poor general condition and favour the use of ELT combined with afterloading in patients with a life expectancy of greater than or equal to 3 months. PMID- 1719651 TI - [Intravenous mitoxantrone use and simultaneous radiotherapy as a palliative treatment in recurrent head and neck tumors]. AB - After preceding radiotherapy, chemotherapy and/or surgery the therapeutic options are limited in case of recurrence. In order to enhance the effect of a reduced radiation dose we have combined intravenous mitoxantrone and simultaneous irradiation. From 1988 to 1991 22 patients with recurrent head and neck carcinomas were treated with this combined regimen. 20 patients had already been irradiated with 50 to 70 Gy. The second treatment course was given with 19.8 to 56.0 Gy and simultaneous application of one to three courses of mitoxantrone. Eleven (50%) patients achieved a clinical complete remission. A partial remission was seen in nine patients. PMID- 1719652 TI - [Nursing. Ambulatory surgery for cataract]. PMID- 1719653 TI - [Surgical department should also perform optimally]. PMID- 1719654 TI - Use of the prostacyclin analogue iloprost in the treatment of patients with critical limb ischaemia. AB - Five large, placebo-controlled, randomised prospective multicentre trials have been completed in several European countries, including a total of 728 patients with critical limb ischaemia (CLI). 593 had ulceration or gangrene and it is these which will be analysed in detail. Only patients with CLI who were unsuitable for further reopening procedures were entered and approximately a third of the patients had already had attempts at surgical revascularisation or interventional radiology. The maximum tolerated dose up to 2 ng/kg/min was determined during the first three days and then continued as a six-hourly intravenous infusion every day for two to four weeks, depending on the study. Pooled results showed a significant overall 21% increase in ulcer healing rate due to iloprost (p less than 0.001) compared with placebo. The improvement was greater in the three studies when treatment was continued for four weeks. The very hard end point of a major amputation or death during a 3 to 6 month follow up was available in three of the studies. Analysis of these together demonstrated a significantly lower incidence of major amputations after iloprost treatment (p less than 0.05). Thus the existing weight of evidence from a large number of patients suggests that a course of intravenous iloprost is useful in the management of patients with CLI and trophic skin changes, who are unsuitable for reconstructive surgery or catheter procedures. PMID- 1719655 TI - Components of coagulation and fibrinolysis during thrombosis prophylaxis with a low molecular weight heparin (Enoxaparin) versus Dextran 70 in hip arthroplasty. AB - In a prospective randomized study heptest, thrombin-antithrombin complexes (TAT), D-dimer, and t-PA:ag were analysed pre- and postoperatively in 206 consecutive patients undergoing hip arthroplasty during thromboprophylaxis with either a LMW heparin (Enoxaparin) or Dextran 70. Deep vein thrombosis (DVT) developed in 6 of 102 (6%) Enoxaparin and in 21 of 104 (20%) Dextran patients diagnosed by bilateral phelobography. In the Enoxaparin group heptest showed a significant increase from the pre- to the postoperative level opposed to a significant decrease in the Dextran group. Postoperative levels of TAT, D-dimer, and t-PA:ag were significantly increased in both groups, however, TAT was significantly higher in patients in the Dextran group than in the Enoxaparin patients. D-dimer was significantly higher in Dextran patients with DVT postoperatively compared with patients without DVT. No differences concerning TAT or t-PA:ag were observed between patients with and without DVT in any of the groups. PMID- 1719656 TI - [Esophageal cancer. Treatment and results]. AB - Between 1979 and 1989, 38 patients were treated for cancer of the oesophagus. 25% (21/38) of the patients underwent resection of the oesophagus with either curative or palliative measures. 12 patients were treated radically, of whom two (17%) have lived free of recurrence for more than five years. Radical thoracoabdominal oesophageal resection is recommended as long as the operative mortality is low. Careful preoperative evaluation is necessary to select patients who should be treated by palliative procedures, such as oesophageal resection, by pass procedures, laser coagulation and/or local irradiation. PMID- 1719657 TI - Major coagulation disorders when using aprotinin--observations on a case. AB - Intraoperative application of the proteinase inhibitor aprotinin allows to drastically reduce blood loss during and after cardiopulmonary bypass operation. The side effects of this therapy (if any) have not been systematically registered and a possible interaction with heparin is not excluded. Such an interaction seems to have occurred in one of our patients. He developed a resistance to heparin shortly after prophylactic administration of aprotinin and maintained it for about one hour after discontinuation of the aprotinin. Prophylactic and therapeutic implications of this observation are briefly exposed. PMID- 1719658 TI - HLA class II specificities and haplotypes in South Africa detected using polymerase chain reaction and sequence-specific oligonucleotide typing. AB - DNA sequencing of HLA class II alleles has revealed a degree of polymorphism much greater than was expected on the basis of the standard serological typing methods. Amplification of the polymorphic second exon of the class II genes using the polymerase chain reaction, followed by hybridization with sequence-specific oligonucleotide probes, allows the unambiguous identification of alleles which could not be detected previously. Using the protocols of the Eleventh International Histocompatibility Workshop, we have applied this procedure for the typing of several individuals and their families with suspected alleles that had been observed using serology, cellular typing and restriction fragment length polymorphism (RFLPs). These included an allele related to DRw8 and DRw14, which has only been observed in the mixed ancestral South African population. In addition, unusual combinations of class II genes forming unique haplotypic associations were seen. PMID- 1719659 TI - [Defense mechanisms of the bovine mammary gland]. AB - In the mammary gland of cattle there is a complex defense system of non-specific and specific reactions available preventing the invasion of pathogenic bacteria. Most infections occur via the teat canal, so teat canal keratin (SKK) is of particular importance in non-specific defense of the gland. The SKK serves as a physical barrier, and bacteriostatic and/or bactericidal effects of SKK lipids and proteins against certain mastitis bacteria could be demonstrated. By increasing the concentrations of lactoferrin and lysozyme in milk a reduction of mastitis frequencies could be observed. However, those high concentrations in the proteins occur only during the dry period of the cow. An improvement of the mastitis situation would also appear possible by increasing phagocytosis. The numerous trials intended to reduce mastitis by improving specific protection showed no significant success. Therefore, the most successful and cheapest means to achieve udder health remains the strict and consistent hygiene of housing, animals and mammary glands. PMID- 1719660 TI - Prevention of posttransfusional non-A, non-B hepatitis by a screening test for hepatitis C virus antibody of donor bloods. AB - The preventive effect on posttransfusional non-A, non-B hepatitis (PTH) of screening out HCV-Ab positive blood and the prevalence of HCV-Ab-positive donor blood were examined. The incidence of HCV-Ab-positivity in donor blood A was 0.9% and that in donor blood B was 1.35%. The mean ALT and guanase levels were 11.5 +/ 5.8 and 0.58 +/- 0.24 IU/l in HCV-Ab negative blood and 17.3 +/- 7.9 and 0.84 +/ 0.23 IU/l in HCV-Ab-positive blood. Both levels were significantly higher in HCV Ab-positive blood. These differences were considered to be nonspecific, but there may be some relationship between the levels of ALT and guanase in donor blood and the HCV carrier status. After adoption the screening test for HCV-Ab positive blood, there was no case of a definite diagnosis of PTH, although 4 patients (6.6%) suspected of developing PTH. So, the incidence of PTH was clearly lower than the lowest incidence before adoption of this test. Therefore, we conclude that screening for HCV-Ab in donor blood should be routinely used for prevention of PTH. PMID- 1719661 TI - Lead, a major environmental pollutant, is immunomodulatory by its differential effects on CD4+ T cells subsets. AB - Studies were undertaken to address the necessity of B-T cell contact for the enhancement of B cell differentiation caused by the heavy metal lead (Pb). Membrane segregated cultures were used so that the influences of direct B-T cell contact and T cell factors on B cell differentiation could be independently evaluated. B-T cell contact was not absolutely required for Pb's enhancement of B cell maturation to antibody forming cells (AFCs); however, enhancement of the AFC response by Pb was optimal when B-T cell interactions were allowed. These results were corroborated by use of anti-L3T4 (mouse CD4) to block CD4+ T cell-B cell interaction. Blockade of B-T cell contact with anti-L3T4 did not inhibit the enhancement of the AFC response by Pb. Additional experimentation showed that Pb enhanced the AFC response and Ig production in the presence of antigen-specific T cell help, suggesting that Pb enhances B cell differentiation by augmenting cognate help rather than by inducing a response to Pb-altered-self. In studies employing antigen-specific T cell clones, Pb was found to differentially modulate antigen presentation to TH1 versus TH2 T cell clones, in that TH1 activation was inhibited and TH2 activation was enhanced by Pb. PMID- 1719662 TI - Induction of hepatic metallothionein by nonmetallic compounds associated with acute-phase response in inflammation. AB - Induction of hepatic metallothionein (MT) synthesis by several nonmetallic compounds and its relationship to an acute-phase response in inflammation were studied in mice. Subcutaneous injections of menadione, paraquat, carbon tetrachloride (CCl4), and several organic solvents caused an increase of hepatic MT concentration. This MT contained only zinc. Menadione and n-hexane caused the greatest accumulation of hepatic MT among these nonmetallic compounds (about 13 fold). The concentration of Zn was significantly decreased in plasma in contrast to liver after an injection of these nonmetallic compounds. When 65ZnCl2 was injected iv after these injections, uptake of 65Zn to the liver was increased. This effect was not observed after treatment with cycloheximide. The association with inflammation of this induction of MT accumulation was examined by determination of acute-phase proteins. The concentration of fibrinogen in the plasma was significantly increased following injection of those nonmetallic compounds which caused marked hepatic MT accumulation. An injection of 1 N NaOH, 1 N HCl, turpentine oil, or endotoxin caused a significant increase in the plasma concentration of fibrinogen and in the hepatic MT concentration. Injections of n hexane as well as turpentine oil significantly increased hepatic MT concentration and plasma concentration of fibrinogen and ceruloplasmin with time. The concentration of fibrinogen was significantly correlated (r = 0.789) with the concentration of hepatic MT. Neither adrenalectomy nor pretreatment with dexamethasone prevented hepatic MT accumulation caused by these compounds. These results indicate that induction of hepatic MT synthesis by these nonmetallic compounds is associated with an acute-phase response in inflammation and is independent of glucocorticoids. PMID- 1719663 TI - Effects of acute nickel toxicity upon plasma and liver metal homeostasis as a function of sex. AB - The effects of an acute dose of nickel chloride (4 mg Ni/kg body wt) upon liver and plasma essential metal homoeostasis were studied in male and female rats. Total levels of copper and zinc in the tissues and in the fractions of chromatographic profiles (Sephadex G-100 and G-150) were determined. Plasma and liver levels of both metals rose significantly. The higher levels of copper in plasma are associated with increased ceruloplasmin activity and the initial increase of zinc in plasma is due to higher zinc content in the plasma albumin fraction. In the liver, the higher levels of both metals similarly affected all the metal-containing chromatographic fractions, although a significant increase is only observed in metallothionein-containing fractions, which agrees with previous reports on increased levels of metallothionein after nickel treatment. Regarding the sex-dependent changes, both sexes showed the same alterations, yet males recovered faster than females from all the nickel-induced changes in metal homoeostasis. PMID- 1719664 TI - An enzyme-linked immunosorbent assay for diagnostic detection of Taenia saginata copro-antigens in humans. AB - An immunodiagnostic sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of soluble Taenia saginata antigens in stool samples (copro-antigens) of infected humans, using affinity-purified polyclonal antibodies obtained from rabbits hyperimmunized with excretory/secretory antigens derived from T. saginata maintained in vitro. Investigation of operating characteristics showed very low cross-reactivity with crude antigens from helminths other than Taenia, including Dipylidium caninum and Diphyllobothrium latum. The specificity of the assay was 95% when testing stool samples from 100 persons who were either infected with Ascaris lumbricoides, Trichuris trichiura, hookworms, Enterobius vermicularis or Hymenolepis nana, or who had no intestinal helminthosis detected. Analysis of diagnostic sensitivity demonstrated that in 85% of 34 samples from 23 untreated persons with intestinal T. saginata infection (selected by previous proglottid and/or egg detection) copro-antigens were detected by the T. saginata ELISA. In the same samples, Taenia eggs were detected in 62%. Only 41% of the samples reacted positively in a heterologous T. hydatigena ELISA. Post-treatment control revealed a high concentration of T. saginata copro-antigens for 1-4 d after administration of niclosamide or praziquantel, and negative values 9-17 d after treatment. The Taenia copro antigens remained detectable by ELISA even after storage of untreated faeces at 25 degrees C for at least 5 d. PMID- 1719665 TI - Do second messengers play a role in interferon signal transduction? AB - The signalling pathway by which the binding of interferons (IFNs, alpha and beta) to their receptor elicits its biological activity, the activation of the transcription of a distinct set of genes called the IFN-stimulated genes (ISG), is far from clear. Debate continues about whether interferon-receptor interaction results directly in gene activation or if second messengers are involved. In this article, we will discuss the evidence that rapid and transient changes in lipid metabolism and the activation of specific isoforms of protein kinase C (PKC) are involved in the initial signalling of interferon activation. PMID- 1719666 TI - Morphology of acute rejection and corresponding cytological findings in exocrine secretion after pancreas transplantation in the rat. AB - Reliable and timely rejection diagnosis still represents a major problem of pancreas allotransplantation. The aim of this study was to confirm the clinical findings of exocrine function impairment and pancreatic juice cytology during rejection, to refine the latter by means of flow cytometry, and to correlate these changes with graft histology. Heterotopic pancreatic transplants were performed in a modified technique in Lewis rats rendered diabetic by means of streptozotocin from LEW donors (group I, n = 10), Brown Norway rats without immunosuppression (group II, n = 16), and from BN rats where recipients were given cyclosporine 12 mg/kg/BW (group III, n = 10). Pancreatic juice was obtained by daily aspiration from a self-made fully implantable catheter reservoir system. In group II animals acute rejection diagnosed on histomorphological grounds was clearly associated with a decrease in the amount of exocrine secretion and its enzyme content from day 8 on. In contrast to groups I and III, a significant increase in lymphocytes in the pancreatic juice up to 13.5% occurred in group II between days 5 and 7. Activated lymphocytes increased from 7% to 13%, pan-T cells from 193 to 340 events. Histology revealed three distinct phases of acute rejection--phase I: diffuse infiltration of acinar structures; phase II: destruction of interlobular ducts; phase III: vasculitis associated with islet cell damage. The anatomy of the pancreas with the slackness of its highly vascularized interstitial connective tissue facilitates early infiltration of inflammatory cells and migration of these cells into the lumen of the pancreatic duct. Thus pancreatic juice cytology together with an impaired exocrine graft function is highly indicative of acute rejection. PMID- 1719667 TI - HLA epitope matching. Contribution of matched residues to epitopes recognized by alloantibodies. AB - Applying absorption-elution techniques with homozygous typing cells and flow cytometry, a number of alloantibodies that recognized HLA-B62 but not B46 were identified. B62 and B46 are identical except in amino acids 66-76, which are probably recognized by the B62-specific antibodies. The patients who made these antibodies, however, had HLA antigens sharing amino acids 66-76 with B62, indicating that residues that are identical to the patient's own contribute to the antigenic determinants of foreign HLA molecules. Fine specificity analysis of most of these antibodies revealed that they recognized residues in the 66-76 segment in addition to other residues which were located in close proximity to this segment. We conclude that mismatched amino acid residues located in one part of the HLA molecule can interact with residues that are not different from those in the patient's own HLA molecules to form epitopes recognizable by alloantibodies. These findings should be helpful in improving our understanding of how to use current knowledge at the molecular level for the purpose of matching transplant donors and recipients. PMID- 1719668 TI - Immunosuppressive properties of thalidomide. Inhibition of in vitro lymphocyte proliferation alone and in combination with cyclosporine or FK506. PMID- 1719669 TI - Capsaicin: gaps in our knowledge start to be filled. PMID- 1719670 TI - Color and the integration of motion signals. PMID- 1719671 TI - A noseful of odor receptors. PMID- 1719672 TI - Concanavalin A: a tool to investigate neuronal plasticity. AB - Neuronal plasticity is the ability of neurons to alter their cellular properties in response to changes in their environment. These changes are typically triggered by the binding of specific ligands, such as neurotransmitters, growth factors or other neuromodulators, to receptors on the neuronal membrane surface. Since the extracellular domains of many of these receptors are glycosylated, they can also be bound by lectins--proteins with high affinity binding sites for polysaccharides. Different lectins have different affinities for various sugar residues. This feature has made lectins useful in the investigation of the regional localization and relative mobility of different classes of glycosylated membrane receptors, and in the subsequent purification of the receptors. This article reviews some of the different kinds of neuronal plasticity produced by the plant lectin concanavalin A (Con A), such as enhancement of neurite outgrowth, modulation of neurotransmitter responses, and alteration in the specificity and strength of synaptic connections. PMID- 1719673 TI - Neuroscience in the former GDR. AB - Until 1945, when Germany was subdivided into four occupied territories, scientific traditions and science policy were the same throughout Germany. Then came 45 years of increasing separation and diverging development. Now, the East and West are again formally united and their inhabitants are experiencing, with an intensity that was not anticipated, just how different their countries have become. PMID- 1719674 TI - Recognizing the visual stimulus from neuronal discharges. AB - In this article, experiments are reviewed that have a bearing on the question of how 'meaning' is related to neuronal activity in the mammalian cortex; there is evidence that the excitation time pattern plays a major role. We suggest that 'objective meaning' is related to spatiotemporal activity distributions, whereas spike counts indicate the importance of the contribution of a given neurone to that meaning. Such a 'dual-coding' principle allows the visual system to emphasize selected activity components by central messages. The feature to be noted is that the objective meaning remains largely unaltered. PMID- 1719675 TI - Opening the grey box. AB - The single neurone has been the guiding light for generations of neuroscientists. Now there are signs from experimental and theoretical work on the neocortex that we are on the threshold of a revolution in which the hegemony of the single neurone will be replaced by much more circuit-oriented concepts. We consider here why traditional views of the significance of single neurones are fading in power, and consider the problem of deciding on the form of a new order. PMID- 1719676 TI - cGMP: the wayward child of the cyclic nucleotide family. AB - An informal poll of neurobiologists indicates the following widely-held misconceptions about cGMP: (1) we know very little about it; (2) it must not be very different from cAMP; and (3) no new biological principles are likely to emerge from studying it. In fact, despite these prejudices, our understanding of the cGMP second messenger cascade has increased dramatically in the last few years. We now know that it is very different from the cAMP system in almost every particular, and the differences reveal interesting and novel solutions to the biological problem of receptor-effector coupling. PMID- 1719677 TI - The dopamine hypothesis of the reinforcing properties of cocaine. AB - A variety of evidence suggests a 'dopamine hypothesis' for the reinforcing properties of cocaine. This hypothesis proposes that cocaine binds at the dopamine transporter and mainly inhibits neurotransmitter re-uptake; the resulting potentiation of dopaminergic neurotransmission in mesolimbocortical pathways ultimately causes reinforcement. This model suggests potential medications for treatment of cocaine abuse and dependence. Some, but not all, pharmacological data in humans support the hypothesis and additional experimentation is needed. PMID- 1719678 TI - Target attraction: are developing axons guided by chemotropism? AB - A century has elapsed since Ramon y Cajal proposed his chemotropic theory of axon guidance, i.e. the attraction of developing axons by diffusible molecules emanating from their targets. Although the precise contribution of axonal chemoattractants to guidance in vivo remains to be established, two lines of investigation have provided evidence for their existence and importance. First, concentration gradients of nerve growth factor (NGF) have been shown to orient the growth of regenerating sensory axons in vitro. Although NGF does not appear to guide axons during development, these studies show that growth cones can orient in gradients of diffusible molecules. Second, the cellular targets of several different classes of developing neurons have been shown to secrete as yet unidentified diffusible factors that can orient axons. We review these studies and discuss the potential contribution of chemotropism to the establishment of axonal projection patterns in vertebrates. PMID- 1719679 TI - The identity of the calcium-storing, inositol 1,4,5-trisphosphate-sensitive organelle in non-muscle cells: calciosome, endoplasmic reticulum ... or both? AB - Although the initial phase of receptor-mediated Ca2+ signaling, involving Ca2+ release from intracellular stores by inositol 1,4,5-trisphosphate, is relatively well characterized, the nature of the organelle releasing Ca2+ is a controversial subject. At issue is the question of whether Ca2+ is released from the endoplasmic reticulum, or from a more specialized organelle called the 'calciosome'. In this review, we attempt to analyse the arguments for and against these two views, and attempt to reconcile some of the apparently conflicting findings by proposing a hypothetical model of the inositol 1,4,5-trisphosphate sensitive Ca2+ pool. PMID- 1719680 TI - Ion channels that are directly activated by cyclic nucleotides. PMID- 1719681 TI - Abnormal prothrombin (PIVKA-II) and hepatocellular carcinoma. PMID- 1719682 TI - [Filgrastim--a hematopoietic growth factor (r-metHuC-CSF) for treatment of neutropenia caused by cytostatics]. PMID- 1719683 TI - Thresholds for premature ventricular contractions in frog hearts exposed to lithotripter fields. AB - Piezoelectrically generated lithotripter shocks were shown to produce premature ventricular contractions of the frog heart. Anesthetized grass frogs, Rana pipiens, were studied following implantation of an aortic catheter and EKG leads. The most sensitive phase of the heart cycle for the generation of premature ventricular contractions with lithotripter shocks at 30 MPa peak pressure was found to be the T-P segment. During this phase of the heart cycle, the minimum peak-positive pressure shock wave necessary to produce a premature ventricular contraction in a frog heart was between 5 MPa and 10 MPa. PMID- 1719684 TI - Change of external urethral sphincter function in prostatic patients. AB - External urethral function was urodynamically examined in 13 patients with benign prostatic hypertrophy (BPH) associated with chronic urinary retention and in 5 volunteers. Prevoiding drop in external urethral sphincter pressure was noted in all the volunteers, whereas it was not found in 6 of the 13 cases of BPH. Bladder neck opening pressure was higher in these 6 cases (p less than 0.05). After administration of phentolamine, prevoiding drop was noted in 5 of these 6 cases, and bladder neck opening pressure decreased so much that there was no significant intergroup difference. The above results mean that the increase in alpha adrenergic receptors makes the prostate, which has been already hypertrophied, less elastic, inhibiting external urinary sphincter function. PMID- 1719685 TI - Urodynamic assessment and quantification of prostatic obstruction before and after transurethral resection of the prostate: standardization with the aid of the computer program CLIM. AB - The clinical manifestations of benign prostatic hypertrophy (BPH) are ascribed entirely to the infravesical obstruction. Therefore relief of symptoms is pursued by surgical removal of obstruction, i.e. prostatectomy. On the other hand, it is generally accepted that the conventional clinical signs and symptoms do not correlate with obstruction as urodynamically defined. In the present study, objective obstruction and detrusor contractility parameters were calculated from detrusor pressure and flow rate measurements in 22 patients before and after transurethral resection of the prostate (TURP) using the computer program CLIM. The results indicate: (1) that CLIM supplies reliable obstruction and contractility parameters for preoperative assessment and follow-up in patients with BPH and (2) that greater than 25% of the patients, who underwent TURP, were objectively not obstructed. These findings form strong arguments for including CLIM parameters in the preoperative assessment to perform a TURP. PMID- 1719686 TI - [The clinical use of the prostate-specific antigen in patients with prostatic cancer]. AB - Radioimmunoassay determination of prostatic-specific antigen (PSA) was conducted in 71 patients with prostatic cancer varying in stages. 85 serum samples were investigated. Of 21 newly diagnosed cases PSA was elevated in 16 (76.2%). No significant elevation of the marker was noticed in stage II patients, while at stage III and IV PSA levels were universally increased. The mean PSA level for stage III was 149.5 ng/ml, for stage IV 291.4 ng/ml. Two stage II patients in remission out of ten presented with high PSA concentrations, while in partial remission this was observed in 3 cases out of 9.50% occurrence of PSA elevated level was registered for stage III patients in remission, 100% occurrence in progression. Prostatic cancer stage IV manifested high PSA levels in 33% of cases under stabilization and in 100% in progression. PMID- 1719687 TI - [Ureteral stents]. AB - Due to the increasing clinical importance of ureteral stents, the advantages and disadvantages of the different materials used are described and discussed. The spectrum of indications includes palliative, curative and auxiliary uses of ureteral stents. In spite of the low complication rate, the advisability of clinical application of stents should always be critically considered before the final decision is made. The possibility of treating the basic disease should be considered in each patient, to lessen the likelihood of "permanent stent patients". PMID- 1719688 TI - Palliative percutaneous and endoscopic urinary diversion for malignant ureteral obstruction. AB - We performed a retrospective analysis of 22 patients with malignant ureteral obstruction who underwent palliative urinary diversion by retrograde ureteral stenting or nephrostomy tube placement. The average duration of survival after diversion was 526 days and was unrelated to tumor type, patient's age or sex, renal function, or indications for diversion. As a group, patients without previous hormonal or chemotherapy survived longer. Morbidity related to the urinary diversion was low. The majority of patients (77%) were discharged from the hospital, and this group spent 86 percent of their survival time at home. We conclude that modern palliative urinary diversion can be performed with low morbidity and can result in long-term survival and improved quality of life. Predictions or assumptions concerning survival of individual patients should be made with caution. PMID- 1719689 TI - Myoepitheliomas in inbred laboratory mice. AB - Myoepitheliomas are subcutaneous tumors that arise from myoepithelial cells of various exocrine glands. In a retrospective study of 142 tumors observed over a period of 3 years, myoepitheliomas occurred spontaneously in A/HeJ, A/J, BALB/cJ, BALB/cByJ, LLC.A/Ckc, and NOD/Lt inbred strains of mice. Tumors presented primarily in the subcutaneous tissues of the ventral neck (74% of the myoepitheliomas evaluated) but were observed in several other subcutaneous locations, including the head, perineum, and ventral abdomen. These areas were adjacent to salivary, mammary, clitoral, preputial, and Harderian glands. Forty myoepitheliomas were tested by the avidin-biotin complex technique with a panel of antisera specific for mouse keratins, intermediate filaments, and other cytoskeletal proteins to determine the cell type from which this neoplasm originated. Antibodies directed against the specific mouse keratins K5, K6, and K14, and a broadly cross-reactive cytokeratin antibody stained acinar and ductal myoepithelial cells in normal mammary, salivary, and Harderian glands, and neoplastic cells in all cases. Antisera directed against a smooth muscle actin (anti-alpha-sm-1) stained acinar myoepithelial cells of the glands and vascular smooth muscle but neither ductular myoepithelial cells nor tumor cells. This supports the notion that these tumors originate from extraglandular ductular myoepithelial cells. Southern blots, prepared from DNA extracted from nine myoepitheliomas, did not show restriction fragment length polymorphisms when mouse mammary tumor virus (MMTV) cDNA or Int-1 genomic DNA probes were used; this implies that a retrovirus is not the etiologic agent. PMID- 1719690 TI - Differentiation of parapoxviruses by application of orf virus-specific monoclonal antibodies against cell surface proteins. AB - Monoclonal antibodies were produced against orf virus-specified cell surface proteins in an attempt to develop reagents capable of differentiating between members of the Parapoxviridae. Two immunization protocols were used to induce an anti-orf response in BALB/c mice, one of which resulted in virus replication in the recipient. The monoclonal antibodies produced were tested for crossreactivity with bovine papular stomatitis virus (BPS) and milker's node virus (MNV) by indirect immunofluorescence assay (IFA) and immunoblotting. The results indicate that significant antigenic overlap exists between isolates of orf, MNV and BPS, even at the level of specificity provided by monoclonal antibodies. One monoclonal antibody reacted strongly in IFA with orf virus isolates, very weakly with MNV, and not at all with BPS. On immunoblots this same antibody recognized a 40-43 kDa protein in orf virus-infected cells, and also a 45-48 kDa protein in cells infected with MNV or BPS virus. The data suggest that it may be possible to define parapoxvirus strains on the basis of small variations in specific virus directed cell surface proteins. PMID- 1719691 TI - Avian leucocyte common antigens: molecular weight determination and flow cytometric analysis using new monoclonal antibodies. AB - New leucocyte common antigens expressed on all the normal chicken leucocytes have been characterized using two monoclonal antibodies designated as K-11 and K-55. These monoclonal antibodies stained virtually 100% of the leucocytes derived from various lymphoid organs including the spleen, thymus, bursa of Fabricius, caecal tonsil and peripheral blood, as well as a monocytic cell line (MC29), a B cell line (LSCC-RP9), and a T cell line (CU12). However, they did not stain mature erythrocytes, intestinal epithelial cells, or chicken embryonic fibroblasts. The two monoclonal antibodies showed different staining patterns and detected non overlapping epitopes on MC29 cells in two color immunofluorescence analysis. Western blot analysis under non-reducing conditions showed that the monoclonal antibody K-11 recognized three splenic leucocyte proteins with molecular weights of 92, 42 and 41 kDa, whereas the monoclonal antibody K-55 recognized two proteins with molecular weights of 97 and 42 kDa. The data indicate that the monoclonal antibodies K-11 and K-55 recognize novel leucocyte-common antigens which have lower molecular weights than the previously reported leucocyte-common antigen family. PMID- 1719692 TI - Antigen specificity of antibody responses to Corynebacterium pseudotuberculosis in naturally infected sheep with caseous lymphadenitis. AB - Constituents of Corynebacterium pseudotuberculosis were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of sonicated whole bacterial cells and ether-extracted cells revealed more than 35 bands in silver-stained gels. SDS-PAGE analysis of concentrated culture filtrates with exotoxin activity demonstrated more than 15 bands. Sera from sheep with C. pseudotuberculosis-induced disease of variable severity were used to probe immunoblots of electrophoresed ether-extracted cells and culture filtrates. Twenty or more corynebacterial molecules, ranging in molecular weight from 20 to 112 kDa, in ether-extracted cells were recognized by antibodies in the sera of naturally exposed sheep with positive ELISA titers. These sera also recognized up to six molecules, ranging from, 20 to 68.1 kDa, on immunoblots of ammonium sulfate-concentrated culture filtrate. There was no apparent relationship between the stage of disease and the response to specific corynebacterial antigens in these animals. PMID- 1719693 TI - Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies. AB - Monoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these anti-Id antibodies as Ab2 gamma or Ab2 beta. By inhibiting experiments it was shown that these anti-Id antibodies did not recognize interspecies cross-reactive idiotopes, but recognized private idiotopes, uniquely associated with the Id of the anti-CPV MoAb used for immunization. This classifies these anti-Id antibodies as non-internal image Ab2 gamma. The potential use of these non-internal image anti-Id antibodies for the induction of antiviral antibodies in the CPV system is discussed. PMID- 1719694 TI - Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens. AB - The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the right udder with HKS at 1 month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral (draining) and contralateral (non-draining) suprammammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory cell migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, immunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals. PMID- 1719695 TI - Application of lectin and B-lymphocyte-specific monoclonal antibodies for the demonstration of human microglia in formalin-fixed, paraffin-embedded brain tissue. AB - To evaluate the usefulness of microglial markers for routine neuropathological material, we studied formalin-fixed, paraffin-embedded human brain tissue with the immunoperoxidase method using the lectin Ricinus communis agglutinin (RCA-1) and four monoclonal antibodies (LN-1, LN-2, LN-3, anti-HLA-DR/alpha). RCA-1 stained resting microglia, but the staining intensity was mostly weak. LN-1 also stained resting microglia in paraffin sections first treated with protease. In contrast to LN-1, RCA-1 stained blood vessels heavily. LN-1 stained resting microglia more markedly than RCA-1 in brains fixed for a prolonged period of time. However, LN-1 recognized a small number of astrocytes in routine paraffin sections. LN-3 reactivity was detected on a few resting microglia, but was intensely expressed on large numbers of reactive microglia in many neurological diseases. Both LN-2 and anti-HLA-DR/alpha labelled microglia, but the reactions were inconsistent. This study suggests that the monoclonal antibodies LN-1 and LN 3 are useful for the demonstration of microglia in paraffin sections, and a combination of these antibodies and the antibody to glial fibrillary acidic protein is recommended in attempting to identify microglia. PMID- 1719696 TI - Prevention of bleomycin-induced fibrosing alveolitis with indomethacin: stereological studies on rat lungs. AB - The prevention of the pulmonary toxicity of bleomycin (BLM) has been investigated in experimental models where pulmonary damage was induced with one intra-tracheal dose of BLM. The present investigation was carried out as a pre-clinical study in which BLM was administered systemically. The non-steroid anti-inflammatory drug indomethacin (INDO) was chosen as a possible candidate for pulmonary protection. Twenty female Wistar rats were treated daily with 4 mg/kg (7.3 units) BLM intra peritoneally for 50 days and 20 rats with BLM and with 1 mg/kg INDO subcutaneously for 62 days. There were 20 animals as controls. Histological examination revealed fibrosing alveolitis in the BLM-treated group which was markedly suppressed in the combination group. Quantitative morphological (stereological) parameters demonstrate that BLM induced alveolar wall thickening (+45%), pulmonary fibrosis (+110%), and an increase of alveolar wall nuclei and of intra-alveolar macrophages (volume densities +43% and +133%, P less than 0.001). In contrast, after combination with INDO significant differences to the control group could not be detected except for a slight increase of intra alveolar macrophages (+62%). Thus, INDO is a highly efficient agent in the prevention of BLM-induced pulmonary damage. PMID- 1719697 TI - Morphologic correlation between liver epithelium and mesenchyme allows insight into histogenesis of focal nodular hyperplasia (FNH) of the liver. AB - The microanatomic organization of focal nodular hyperplasia (FNH) of the liver was analyzed to obtain information about the histogenesis of this tumor-like lesion. All of the 11 examples of FNH studied showed subdivision into multiple pseudolobules, which were characterized by fibrovascular and ductular areas radiating from perilobular septa, and an expanding periphery of normal appearing hepatocytes. Immunohistochemical analysis showed continuous transitions from normal hepatocytes in the periphery of the pseudolobules, which expressed only the keratins 8 and 18, to small hepatocytes and ductular aggregates in the center of the pseudolobules, both of which also expressed the keratins 7 and 19. Ductular metaplasia of hepatocytes was always accompanied by sinusoidal endothelial cells stained by the endothelial markers BMA 120, M 616, and by an increase in collagenous fibers especially of type III. Further development of this fibrovascular and ductular transformation lead to subdivision of the involved pseudolobules. The pseudolobules had similar mean sizes, irrespective of their site in the periphery or the center of the FNHs, showing that proliferation, fibrovascular and ductular transformation and subdivision of these micronodules are a basic histogenetic phenomenon in FNH. The findings indicate that local changes in the interrelations between liver epithelial and mesenchymal cells influence substantially the abnormal but nevertheless regulated growth of liver parenchyma which gives rise to FNH. PMID- 1719698 TI - [A comparative study of alpha2-macroglobulin and pregnancy-associated alpha2 glycoprotein and protein A as possible analogs]. AB - Pregnancy-dependent protein A, alpha 2-glycoprotein as well as alpha 2 macroglobulin were isolated from retroplacental blood serum by means of affinity chromatography. These proteins proved to be similar in their structure, amino acid and carbohydrate compositions as well as in their properties to bind proteinases and to inhibit the enzymatic activity. The hypothesis on possible integration of these proteins into a group of high molecular inhibitors of proteinases is discussed. PMID- 1719699 TI - [Use of two-dimensional O'Farrell electrophoresis for studying polymorphism of blood proteins]. AB - Blood plasma of 50 healthy volunteers was analyzed using two-dimensional electrophoresis by O'Farrell. After staining with Coomassie R-250 and subsequent silver treatment of gels 200 protein fractions were detected on typical electrophoregrams. About 20 groups, belonging to known polymorphic systems, were identified. Dissimilar types of proteins electrophoretic variants were found in the preparations studied. PMID- 1719700 TI - HLA-DR: serology versus restriction fragment length polymorphism. AB - The results of the serological typing and the patterns of restriction fragment length polymorphisms for the detection of HLA-DR gene products are compared. The data shown demonstrate that the use of DNA typing gives a clearer definition of the HLA-DR antigens and that the HLA-DR polymorphism is greater than detected by serology. PMID- 1719701 TI - Immunochemical characterisation of the low-incidence antigen, Dha. AB - Immunoblotting with two examples of anti-Dha to the electrophoretically separated components of antigen-positive membranes gave a positive reaction with a component of the same apparent Mr (40,000) as sialoglycoprotein beta (SGP beta, syn: glycoconnectin, glycophorin C). The Dha antigenic determinant was sensitive to trypsin, but resistant to chymotrypsin and Endo F. By immunoblotting, one anti Dha failed to react with sialidase-treated Dh(a+) cells, whilst the other gave a positive result. In contrast, neither antibody agglutinated sialidase-treated red cells. SGP beta was precipitated from Dh(a+) and Dh(a-) phenotype red cells by monoclonal anti-beta (NBTS/BRIC 10). SGP beta from Dh(a+) but not from Dh(a-) red cells was stained by immunoblotting with anti-Dha. These results assign the Dha antigenic epitope to SGP beta. PMID- 1719702 TI - [Natural killer cells in autoimmune diseases]. PMID- 1719704 TI - [Pain therapy and terminal care]. PMID- 1719703 TI - The structure and function of gift giving in the patient-nurse relationship. PMID- 1719705 TI - [Comments on the debate about euthanasia]. AB - On the one hand euthanasia raises the question of humane dying (1). On the other side it is connected with the question of "wrongful existence" (2). The ambiguity of the term "human/humane life" might be explained in the following way: in respect of group 1 one might say that nobody is privileged to define materially or pragmatically the one who holds the dignity of man. In consequence no one may be killed because he lacks this type of criteria. Concerning group 2: a) No one may be expected to kill an innocent, defenceless human being; and there does not exist any right of demanding such a thing. b) Everyone has the right to get medicine for lessening pain. c) In case of b) death may not be intended on purpose, but may be accepted as a side-effect of medical treatment. d) Though you can formulate a rule of thumb there will occur tragical dilemmas with regard to cases of coma. PMID- 1719706 TI - [Current knowledge on the formation of nitric oxide in endothelial cells of blood vessels, in nerve cells and macrophages as well as its significance in vascular dilatation, information transmission and damage of tumor cells]. AB - The vasculature endothelium cells and the nerve cells of several regions of the brain and the autonomous nerve system contain a nitric oxide (NO)-synthase, that forms NO from arginine. The NO-synthase is stimulated by bradykinin, histamine and acetylcholine and is especially active in the coronary and brain vessels. In the vasculature smooth muscle NO activates the guanylate cyclase: The increase in the concentration of cGMP induces a relaxation and in this way a vasodilatation. In the nerve cells NO is active as a neuromodulator. The activation of macrophages by gamma-interferon or by lipopolysaccharides induces the formation of a NO-synthase, that has other properties than the enzyme of the endothelium cells. The macrophages secrete NO and inhibit the metabolism of tumour cells, especially enzymes of the respiratory chain and of the citric acid cycle as well as the DNA-synthesis. Trinitroglycerin and amyl nitrite form with thiol-compounds S-nitroso-compounds, the decomposition of these forms NO. PMID- 1719707 TI - [Assessment of fetal kidney function by determining the microprotein levels (alpha-1 and beta-2 microglobulin) in fetal blood]. AB - Fetal renal function cannot be assessed with clearance methods. Fetal urea and creatinine serum levels correspond to maternal values, the fetus being hemodialyzed by his mother till birth. To predict glomerular filtration independently of maternal renal function, levels of alpha 1-microglobulin (MG) and beta 2MG and creatinine were routinely determined in cord blood of 375 newborns (GA 25.-42 weeks) in cord blood and in 154 cases in corresponding maternal blood samples. alpha 1MG was determined in a radial immunodiffusion method, beta 2MG by radioimmunoassay, creatinine enzymatically. The mean alpha 1MG concentration fell from 3.1 mg/dl in 25-31 weeks prematures to 2.3 mg/dl in 38-42 week group (p less than 0.005), beta 2MG concentration fell from 3.9 mg/l to a mean of 2.6 mg/l (p less than 0.0005), while in maternal blood, levels rose from 4.0 to 4.3 mg/dl (alpha 1MG) and from 1.7 to 1.8 mg/l (beta 2MG) (n.s.). Creatinine levels rose from 0.63 to 0.75 mg/dl in both fetal and maternal blood. Parallel to the rise in GFR, microprotein levels fall during intrauterine development, while maternal serum levels increase. There is no correlation between maternal and cord blood alpha 1MG or beta 2MG concentrations. This is distinct in cases with severe maternal or fetal renal insufficiency (n = 10). Thus microprotein determination in fetal serum can be used to detect severe renal function disturbances and to estimate glomerular filtration rate independently of maternal renal function. PMID- 1719708 TI - [Changes in plasma coagulation and fibrinolysis following cesarean section and relationship to deep venous thrombosis. Results of a randomized prospective comparative study with 6% hydroxyethyl starch 0.62 and low-dose heparin as thrombosis prophylaxis]. AB - Routine postoperative monitoring of plasma coagulation and fibrinolysis system values after cesarean delivery in 191 women who did not develop thrombosis and 16 who did revealed a preoperative defect in the fibrinolysis (PAI, TAT) and inhibitor (AT III) systems. Significant postoperative correlations in the drop in antithrombin levels could not be explained solely by hemodilution. Moreover, a disturbance of the equilibrium of the alpha-2 increase and plasminogen was observed, favouring alpha-2 antiplasmin. Hydroxyethyl starch is capable of simultaneously influencing hypercoagulability and postoperative status. The only difference noted between the two groups of drugs was in the course of the PAI concentration (P less than 0.02). It would therefore appear logical in clinical practice to extend preoperative tests to include determination of the plasminogen activator inhibitor and the thrombin-antithrombin complex. PMID- 1719709 TI - [Prevalence of late potentials in high frequency signal-averaged electrocardiography and arrhythmias in long-term ECG in healthy probands]. AB - In a prospective study 79 symptom-free persons (41 females; 38 males) with an age range of 22-69 (mean 44) years were investigated by 48-h continuous ambulatory electrocardiography and by the signal-averaging ECG according to Simson's technique after having normal findings with echocardiography, standard ECG at rest and exercise stress test. Late potentials were defined according to Denes criteria (40 Hz highpass-filter); at least two out of the following three criteria had to be fulfilled for a correct positive finding: 1) QRS duration (QRSdur) greater than 120 ms; 2) root mean square of the last 40 ms (RMS 40) less than 20 microV; 3) mean duration of terminal low-amplitude signals (LAdur) greater than 39 ms. With long-term ECG 25% of the test subjects had no ventricular extrasystoles (VES), 28% had uniform VES, 33% multiform VES, 10% couplets, and 4% short runs of ventricular tachycardia during 48-hour recordings. Only 19% of them showed more than 48 VES/48 h. Individuals of advanced age demonstrated arrhythmias of higher Lown classes, as well as more frequent VES. By applying the signal-averaging technique 12.6% of the apparently healthy individuals showed late potentials, but none had LAdur greater than 45 ms. Individuals of higher age had not more late potentials than the younger ones. However, individuals with incomplete right bundle branch block pattern (n = 6) demonstrated with 50% significantly more often later potentials in comparison to 9.6% of those without this pattern (n = 73) (P less than 0.05). There was no correlation between late potentials and spontaneous arrhythmias, neither with regard to Lown classes, nor with regard to the frequency of VES. In conclusion, late potentials may occur in some individuals without apparent cardiovascular disease; they are unrelated to age as well as to spontaneous ventricular arrhythmias in normals. PMID- 1719711 TI - [Care of the dying. Experiences from a hospice--correct care of the dying]. PMID- 1719710 TI - [The reorganization of the structure of the vascular bed in different functional states of the body]. PMID- 1719712 TI - [Synthesis and anti-retroviral activity of various new 2',3'-dideoxynucleoside analogs]. AB - About 40 new 2',3'-dideoxynucleoside analogues with different basic structures have been synthetized with the aim of finding more potent and selective inhibitors of HIV and the associated pathogenicity. Some new synthesis techniques were developed and existing ones were used for the preparation of these analogues. Among the 3'-fluorinated 2',3'-dideoxynucleosides several potent inhibitors of HIV were found and especially 3'-fluoro-2',3'-dideoxy-5-chloro uridine (81) is endowed with a high selectivity index, comparable to the selectivity displayed by 3'-azido-3'-deoxythymidine (AZT). Because of its substantially reduced toxicity profile compared to AZT, this compound deserves further evaluation as a possible alternative for the treatment of AIDS. Likewise, the 5-chlorinated cytidine analogues 102 and 103 should be further examined because of their lack of toxicity in vitro. PMID- 1719713 TI - Primary bovine viral diarrhoea virus infection in calves following direct contact with a persistently viraemic calf. AB - Six calves, aged 24 to 58 days and not previously exposed to bovine viral diarrhoea virus (BVDV), were infected with this agent by nose-to-nose contact with a persistently BVDV viraemic calf. The study was conducted in two trials, using 3 calves in each. All 6 calves showed a peak interferon level in serum at 4 days post infection (dpi), and they seroconverted to BVDV at 16-21 dpi. The calves in trial 1 had diarrhoea for 2 or 3 days between 2 and 6 dpi and one calf again from 9 to 11 dpi. During the periods of fever, the calves were slightly depressed. Those in trial 2 were more depressed and their oral and nasal mucous membranes were reddened but they never had diarrhoea. In both trials, fever (up to 41.3 degrees C) was a prominent symptom at 8 to 9 dpi and 2 calves showed a diphasic fever course. Respiratory affection was mild and no medical treatment was required. Haematological assessment demonstrated a transient but significant leukopenia and lymphopenia at 4 dpi (P less than 0.01 and P less than 0.05 respectively) and 11 dpi (P less than 0.05 and P less than 0.01 respectively). A significant decrease in thrombocyte count was seen at 4 dpi (P less than 0.05, n = 3). This study has demonstrated that nose-to-nose contact is an effective way of transmitting BVDV from persistently infected to susceptible cattle. PMID- 1719714 TI - Studies on linear epitopes of Semliki Forest virus in protection studies against lethal challenge virus infection. AB - Purified reduced and non-reduced glycoproteins E1 and E2 of Semliki Forest Virus (SFV) were used to investigate the protection potency to prevent clinical disease after lethal virus challenge. In parallel synthetic oligopeptides deduced from conserved regions of the nucleotide sequences coding for the glycoproteins E1 and E2 were included. It could be demonstrated that both reduced and non-reduced glycoprotein preparations induced protection against lethal virus challenge, whereas the oligopeptides did not. The role of linear epitopes in immunity and their potential use as synthetic vaccines against Alphaviruses are critically discussed. PMID- 1719715 TI - [The immunogenic and serological properties of the O-specific polysaccharide (L hapten) in Shigella]. AB - O-specific polysaccharide (L-hapten) was isolated earlier (Zh. mikrobiol. epidemiol. immunobiol., 1989, No. 11, pp. 8-11). In this paper L-hapten was shown to be unable, even at high concentrations (up to 2,000 micrograms/ml), to sensitize sheep red blood cells for passive hemagglutination by O-antibodies. At the same time classical LPS and heat-activated LPS were active at concentrations ot 32 and 8 micrograms/ml respectively. The O-antibody-neutralizing activity of L hapten was lower than that of LPS 10(3)-10(4) times in the passive hemagglutination test and 25-50 times in competitive ELISA. The immunogenicity of isolated L-hapten was very weak: primary response in mice to the i.v. injection of 1-10 micrograms of L-hapten was similar to the effect produced by 10(-3)-10( 4) micrograms of LPS. No protective activity of L-hapten was noted in mice when the challenge dose of virulent shigellae was 16 LD50 or more, and only a weak protective effect was observed with a low challenge dose (8 LD50). The molecular basis of low serological and biological activity of L-hapten is discussed. The most probable explanation of the results obtained in this study is that L-hapten contains some nonspecific carbohydrates, inserted in or complexed with the O-side chain. Despite its low immunogenicity, L-hapten can be an important component of effective bacterial vaccines provided it is included into a suitable delivery system as is the case with Shigella ribosomal vaccine. PMID- 1719716 TI - [The characteristics of the reactogenicity and immunological activity of a new cholera bivalent chemical vaccine based on the results of controlled trials]. AB - The reactogenic properties and immunological potency of modified cholera chemical vaccine (choleragen-toxoid + O-antigens Inaba and Ogawa) were tested in 278 volunteers aged 18 years and over in comparison with those of a commercial batch of monovalent cholera vaccine (choleragen-toxoid + O-antigen Inaba). The cholera vaccine, enriched with O-antigen Ogawa, was found to be safe; vaccination with this vaccine was not accompanied by the development of systemic and local reactions whose frequency and intensity met the requirements for the reactogenic properties of commercial cholera vaccine. The immunological potency of the bivalent vaccine with respect to strain Inaba was not inferior to that of the commercial vaccine; at the same time in persons immunized with the new preparation the titers of vibriocidal antibodies to strain of serovar Inaba were five-fold higher. The conclusion on the expediency of using cholera chemical vaccine enriched with O-antigen Ogawa was made. PMID- 1719717 TI - [The immunogenic properties of the endotoxin protein: serum antibodies in animals and man]. AB - Endotoxin protein or lipid A-associated protein (LAP) from Shigella sonnei was isolated and characterized earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 47-50). In this investigation serum antibodies against LAP were studied in ELISA Anti-LAP antibodies were detected in high titers in the sera of nonimmunized mice, guinea pigs, rabbits, monkeys and healthy adults. We suppose that normal anti-LAP antibodies resulted from interaction between the immune system and environmental endotoxin. Parenteral injections of LAP to different animals induced intensive antibody response with a 100- to 1000-fold increase in the serum anti-LAP antibody level and a significant rise in the serum O-antibody level. The latter is seemingly due to the contamination of LAP with minute amounts of O-antigen (0.12% or less) and to the amplification of its immunogenicity by LAP. Both antigenic and amplifying activity of LAP was destroyed by proteinase K. The biological function of LAP and its possible use as a component of bacterial vaccines are briefly discussed. PMID- 1719718 TI - [The serological characteristics of the O antigens of Pseudomonas aeruginosa isolated from patients with a Pseudomonas infection during immunotherapy]. AB - Serologic characteristics of P. aeruginosa O-antigens isolated from patients with P. aeruginosa infection were studied over the course of treatment with anti-P. aeruginosa sheep immunoglobulin. The preparation was used in 54 patients with nongeneralized forms of P. aeruginosa infection (infected wounds, pleural empyema) externally or intraperitoneally. From the clinical material collected from the patients a total of 54 P. aeruginosa strains were isolated. Serologic typing of the isolated strains with factor or group diagnostic agglutinating sera has revealed the O-group composition of the isolated strains; 66% of them were classified with O-groups 2,3, and 6. Serologic variants of the strains isolated from patients proved to be stable over the course of the disease immunotherapy. Analysis of the results of bacteriologic control of immunotherapy. efficacy and the clinical data has demonstrated the efficacy of immunotherapy in 61.1% of cases and its partial effect in 20.4% of cases of P. aeruginosa infection. PMID- 1719719 TI - [The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis]. AB - After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours. PMID- 1719720 TI - Changes in the chemical composition of fat body during the last larval instar of Manduca sexta. AB - The fat body increases in weight throughout the last larval instar in spite of the loss in total body weight during the wandering and prepupal stages. The protein content of fat body increases dramatically and is greatly responsible for the increase in fat body weight in the wandering and prepupal stages. Lipids do not contribute significantly to the fat body weight gain except during the feeding stage. The amount of total fat body RNA increases until wandering then drops abruptly in the prepupal stage. The total amount of fat body DNA peaks before the onset of wandering and prepupal stages. PMID- 1719721 TI - Hepatitis C and its causative agent. PMID- 1719722 TI - Comparison of smears and cell blocks in the fine needle aspiration diagnosis of recurrent gynecologic malignancies. AB - A retrospective, seven-year study was conducted to evaluate the value of cell blocks as an adjunct to smears in the fine needle aspiration (FNA) diagnosis of recurrent gynecologic malignancies. Eighty-four FNAs were performed on patients with previously diagnosed malignancies of the cervix (39 cases), ovary (27), uterus (14), vulva (2) and vagina (2). Material for the preparation of cell blocks was available in all cases. Smears and cell blocks were reviewed separately, and the findings were categorized as positive, negative, suspicious or unsatisfactory. Identical smear and cell block results were reported in 71 (84.5%) of the 84 cases (45 positive, 20 negative, 1 suspicious and 5 unsatisfactory). In 12 cases (14.3%) the smear was superior to the cell block in detecting malignant cells; while all 12 smears were positive, 8 cell blocks were negative, and 4 were suspicious. In no case was the cell block positive with a negative smear; in one (1.2%) the cell block was positive and the smear suspicious. The results of this study indicate that the additional study of cell blocks is of little benefit in the FNA cytodiagnosis of recurrent disease in patients with documented gynecologic malignancies. PMID- 1719723 TI - Ethanol-induced growth inhibition of erythroleukemia stem cells. Cell cycle effects. AB - Friend erythroleukemia stem cells are strongly growth inhibited by 60 mM ethanol. The expression of this inhibition requires several days to develop fully, and is not relieved by washing into new ethanol-containing medium even in the presence of excess folic acid. Removal of the fully inhibited cells from ethanol results in rapid growth recovery, with the onset of recovery occurring within a few hours. Cell cycle analysis reveals a G1 delay which is evident within a few hours after initial ethanol exposure. Bivariate RNA-DNA analysis indicates that this G1 delay results from restriction in late G1. It is unclear at present whether this delay can account for all the observed growth inhibition. PMID- 1719724 TI - Fetal red cell in Thai thalassemia trait patients. PMID- 1719725 TI - In vivo effect of human granulocyte colony-stimulating factor derivatives on hematopoiesis in primates. AB - Using cynomolgus monkeys, we studied the hematopoietic effect and antigenicity of the human granulocyte colony-stimulating factor (hG-CSF) derivatives mutated at N terminal amino acids (NC-59 and KW-2228) and that lacking N-terminal amino acids (M-7) in comparison with intact hG-CSF. These compounds were subcutaneously administered daily into the back of cynomolgus monkeys at the doses of 1 and 100 micrograms/kg for 2 weeks, and withdrawn for 2 weeks (1st course). After that, the same schedule was repeated (2nd course), and intact hG-CSF and KW-2228 were further administered for 2 weeks (3rd course). In the first course, all of the G CSF derivatives showed excellent dose-dependent granulopoietic activities. In the 2nd course, however, the activities of NC-59 and M-7 were decreased and the elevations of binding and neutralizing antibody titers were observed. On the other hand, KW-2228 showed granulopoietic activities equal to or better than that of intact hG-CSF and had low antibody titer throughout all the courses. PMID- 1719726 TI - Human recombinant granulocyte colony-stimulating factor for the treatment of autoimmune neutropenia. AB - We have recently treated a case of autoimmune neutropenia in a 57-year-old male. Because neutropenia persisted despite the administration of prednisolone for 30 days, daily subcutaneous injection of human recombinant granulocyte colony stimulating factor (rhG-CSF) at a dosage of 100 micrograms was started. Neutrophil count increased gradually and reached a plateau of 5,000/microliters by day 25 after administration of rhG-CSF. This observation suggests that rhG-CSF is effective for the treatment of autoimmune neutropenia. PMID- 1719727 TI - New approaches in the treatment of MDS: an overview of current trials. PMID- 1719728 TI - [Kinetic effects of 6% hydroxyethyl starch 200.000/0.6-0.66 in hypervolemic hemodilution]. AB - An open study was carried out in order to examine therapeutic effectivity of repeated administration of 6% hydroxyethyl starch 200.000/0.60-0.66 (HES). 10 patients aged 63 to 77 years with peripheral arterial occlusive disease Stage II b were given 500 ml Elohaest as a hypervolemic hemodilution for 12 days. Fasting levels of hydroxyethyl starch (HES) in serum, hematocrit, and serum-alpha-amylase were measured. During the first 4 treatment days, HES levels and serum-alpha amylase increased in a significant manner; then it fluctuated towards a plateau without significant differences. The hematocrit decreased significantly from the beginning to the end of therapy. So it was possible to show, that during repeated application with the chosen dosage of 6% HAES 200.000/0.6-0.66 there is no dangerous cumulation of HAES. PMID- 1719729 TI - [Hyper- or isovolemic hemodilution in patients with stage II peripheral arterial occlusive disease]. AB - The influence of hyper- and isovolaemic hemodilution using HES 200/0.5 6% (HES steril, Fresenius AG) on pain-free walking distance in patients with peripheral arterial occlusive disease stage II according to Fontaine was tested in a randomised investigation involving three groups. The increase in pain-free walking distance was significant both in the hypervolaemic and isovolaemic hemodilution group. The increase in both dilution groups did not differ but compared to a control group (infusion of Ringer lactate) it was significantly higher. Considerable changes concerning rheological parameters (decrease in haematocrit and plasma viscosity) were observed after isovolaemic hemodilution. PMID- 1719730 TI - [Hemodilution in peripheral arterial occlusive disease. Placebo controlled randomized double-blind study with hydroxyethyl starch or dextran]. AB - Hemodilution is often recommended for peripheral arterial occlusive disease, yet the ultimate proof of efficacy is lacking in two randomized, placebo-controlled, double-blind, crossover trials (one using Dextran 40 and the other 10% hydroxyethyl starch 200) it has been shown to clinically benefit claudicants. During the hemodilution phases of these trials, where a 500 ml volume was exchanged against 500 ml blood, there was a significant prolongation of the walking distances. With sham-hemodilution there were no beneficial effects. These studies suggest furthermore that responders and non-responders to hemodilution can be identified prior to therapy by angiogram. A theory is developed which implies that low shear forces in vivo are a precondition for the clinical effectiveness of hemodilution. The experimental evidence supporting this theory is reported. Dextran and hydroxyethyl starch do not seem to differ with respect to their clinical response. It is concluded that hemodilution is effective for peripheral arterial occlusive disease stage II and probably even more effective in more advanced stages. PMID- 1719731 TI - [Hypervolemic hemodilution with medium molecular-weight hydroxyethyl starch: effect of duration of administration on viscoelasticity of blood in IIa and IIb peripheral arterial occlusive disease]. AB - 45 consecutive patients suffering from peripheral arterial occlusive disease (PAOD) stage IIa and b were integrated in a study about the effect of hemorheological parameters due to different application times. The patients were divided into 3 subgroups with different application times: group A received the infusion during 2, group B during 4, and group C during 6 hours. The used substance was 500 ml 6% HES 200,000/0.6-0.66. Except elasticity of group A all hemorheological parameters from group A to group C show a decrease after 6 hours, the trend to the same phenomenon could be observed after 24 hours in group B and C, while group A except plasma viscosity showed even a trend to increasing values compared to onset. As a conclusion of these results, long-time application should be preferred. PMID- 1719732 TI - [Hydroxyethyl starch-induced transient renal failure in preexisting glomerular damage]. AB - Hemorheological therapy through hemodilution has been gaining importance for several years and been applied to an ever increasing degree in stationary as well as in ambulant treatment. While renal insufficiency without previously established nephropathy is known to be a side effect of dextrans, cases of HES induced nephropathy have so far not been reported. Two cases are presented in which in the course of stationary hemodilution therapy with HES an acute deterioration of an already exiting nephropathy was noted. Possible pathophysiological causes for such a deterioration are most likely to be found in an increased permeability of the glomerular basal lamina. Hydroxyethyl starch molecules are filtered above the physiological renal threshold which increases the viscosity of the primary urine. This can be counteracted by increasing diuresis. This conclusion can be drawn from our own observations which proved that renal insufficiency can be avoided through sufficient fluid intake (approx. 3 liters/day). In patients with creatinine values above 1.5/dl and arterial hypertension the indication for hemodilution therapy must be analysed carefully. If hemodilution therapy proves to be necessary, sufficient fluid intake must be guaranteed. Retention parameters must be controlled every other day in the course of the therapy. As an alternative, the administration of gelatin preparations should be considered as it does not cause cumulation. PMID- 1719733 TI - [Value of hemodilution therapy in pregnancy]. AB - The ability to produce a large increase in plasma volume is one of the hallmarks of a successful pregnancy. Data from Garn et al. (5), Knottnerus et al. (14) and Murphy et al. (15) have shown, that hemoglobin levels above 13 g/dl or hematocrit 38% before admittance to hospital are associated with a high incidence of IUGR (intrauterine growth retardation), gestational hypertension and with a greater perinatal mortality. Patients with an elevated viscosity and hematocrit have increased perinatal risks. In these cases the hemodilution with hydroxy- ethylstarch (HES) improves the blood flow and decreases the incidence of dysmature babies and pregnancy complications. The effect of HES on certain coagulation assays seems qualitatively similar to those of Dextran. In a prospective trial we evaluated the effect of HES in the incidence of thrombosis after cesarean section and we found a 5.9% incidence of thrombosis in patients treated with 6% HES 0.62 compared with a 7.8% incidence in heparin treated patients. Treatment with 1500 ml 6% HES 0.62 before and after cesarean section is similarly effective in preventing deep vein thrombosis as heparin prophylaxis. PMID- 1719734 TI - [Hemodilution therapy of acute inner ear damage]. AB - Two independently conducted randomized clinical studies (sudden deafness; acute acoustic trauma) showed that in terms of hearing gain and the elimination of tinnitus low-molecular dextran 40 and 6% hydroxyethyl starch 40/0.5 do not produce any significant difference in therapy. Clear significant results were obtained with patients who suffered from acute acoustic trauma, when only NaCl infusions were applied. Both rheologically effective substances show side effects, but they lie in the per-mil-range. On the grounds of measured clinical results we refuse zero or next to zero therapy of patients with acute inner ear damage. In our opinion, the two rheologically effective infusion solutions are equivalent. PMID- 1719735 TI - [Hemodilution in acute arterial circulatory disorders of the retina]. AB - 20 patients (age: 68 +/- 9 years) with an acute central artery obstruction of the retina were examined in a prospective study. All patients received a hyper- or isovolemic hemodilution depending on the haematocrit value for a period of 10 days. Clinical, hemodynamical and rheological data of these patients were recorded before therapy, after 10 days and after 6 weeks. Under this therapy, 10 patients presented an improved central vision by 2 or more stages. The degree of the distributed circulation can be determined by means of the arteriovenous passage time (AVP) and the dye bolus velocity (MDV). Compared to a control group, a significantly increased arterious-venous passage time and decreased dye bolus velocity can be observed initially. The values after 10 days of therapy and after 6 weeks were significantly improved (p less than 0.01) compared to the initial values. PMID- 1719736 TI - [Hemorheologic and circulatory effects of hemodilution with medium molecular weight hydroxyethyl starch in two concentrations (HAES 200/0.62 10% and 6%)]. AB - This study was designed to investigate whether the positive effect occurring after infusion of a 10% hydroxyethyl starch solution 200.000/0.62 on cutaneous capillary blood flow could still be increased by using a 6% solution having a lower initial viscosity (HES 6% 200.000/0.62). A single blind study of 10 female volunteers was carried out to study the influence of a hypervolemic hemodilution using 500 ml of a 10% solution hydroxyethyl starch solution at first and that of 6% hydroxyethyl starch solution on macro- and microcirculation, transcutaneous oxygen partial pressure and blood fluidity. After hydroxyethyl starch infusion blood flow in the macro- and microvessels increased significantly. This increase occurred considerably earlier after administration of 6% solution than after infusion of 10% solution. Thus blood flow had already increased after 1 hour and continued to show a tendency of increase over about three hours. 6 hours after end of infusion blood flow had almost dropped to its initial value. The volume effect of the 6% solution immediately after infusion was less pronounced than that after infusion of the 10% solution, but it reached the value of the 10% solution at a later point in time. The course of the haematocrit reflected this process. Average decrease in haematocrit value compared to that following the 10% solution was not as pronounced at hour 3. After 6 hours the decreases were comparable. Similar observations were made of plasma viscosity and platelet aggregation. One hour after infusion plasma viscosity and erythrocyte aggregation had hardly changed but haematocrit had clearly dropped. Thus, blood viscosity at this point in time was essentially lowered.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719737 TI - Compartmentalized mast cell degranulations in the ovarian hilum, fat pad, bursa and blood vessel regions of the cyclic hamster: relationships to ovarian histamine and blood flow. AB - Ovaries from hamsters on each day of the oestrous cycle at 09.00 h were observed for the number of mast cells, the pattern of mast cell degranulation, histamine concentration and blood flow. On day 4 (pro-oestrus), ovaries were also observed at 9.00, 15.00 and 21.00 h. Mast cell degranulation was evaluated by 3 criteria: (1) no degranulation = less than 5 granules dispersed from the cell; (2) moderate degranulation = 5 or more granules dispersed but less than 15, and (3) extensive degranulation = 15 or more granules released. Blood flow was determined using radio-active microspheres in anaesthetized animals. Mast cells were observed in fat pad (beyond 2 mm of the bursal mesothelium), bursa (within 2 mm of the bursal mesothelium), hilum and near ovarian blood vessels (these 4 regions are collectively called the ovarian complex). The distribution of ovarian mast cells was not uniform. Most mast cells were near ovarian blood vessels (42.2%) and in the fat pad (37.2%). A moderate number of cells were in the bursal wall (20%) and only a few cells were observed in the hilum (0.64%). Mast cell number remained unchanged on days 1-4 of the cycle in each ovarian compartment. However, summation of the number of mast cells in the entire ovarian complex revealed a significant decline in number at 15.00 h on pro-oestrus. Alterations in mast cell degranulation were primarily restricted to 2 periods of the cycle (pro-oestrus and di-oestrus). An increase in moderate but not extensive degranulation was observed in only the fat pad and bursa on day 2 when compared with day 1 values. In most ovarian compartments on pro-oestrus, degranulation was higher than on any other day of the cycle. At 15.00 h on pro-oestrus, extensive degranulation in bursa, fat pad and blood vessel regions (but not hilum) coincided with an increase in ovarian histamine and decline in number of mast cells; ovarian blood flow also increased at the time but remained unchanged the remainder of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719738 TI - Disturbances of speech prosody following right hemisphere infarcts. AB - The ability to perceive and express emotional, as well as number of linguistic prosodic qualities of speech was tested in 20 Swedish-speaking patients with right-sided cortical, as well as purely subcortical brain infarcts, and in 18 normal controls. The infarcts were assessed by clinical neurological examination, and by CT, EEG, and measurements of regional cerebral blood flow (rCBF). In the patients the identification of emotional messages was disturbed, as well as the identification and production of several linguistic prosodic qualities. The study supports the claim that prosodic impairment could be linguistic in nature, and not secondary to affective disorder. The total degree of anatomical and functional disturbance of the right hemisphere played a role for both the ability to identify emotional messages and for identification of two of the linguistic prosodic qualities tested. However, it was not possible to find support for the hypothesis that the organization of prosody in the right hemisphere mirrors that of propositional speech on the left side. PMID- 1719739 TI - P2-peptide induced experimental allergic neuritis: a model to study axonal degeneration. AB - In experimental allergic neuritis (EAN) severity of clinical disease and pathology correlate with the dose of antigen (Hahn et al., Lab Invest 59:115-125, 1988). To avoid axonal membrane contamination of the antigen, EAN was induced with a synthetic peptide, corresponding to residues 53-78 of bovine P2 myelin protein. Severity of EAN correlated with the dose of peptide in the inoculate. The relationship between demyelination, inflammation and axonal degeneration was studied. Low doses resulted in pure demyelination. Axonal degeneration occurred only with high doses of antigen and in association with very active mononuclear inflammation. The role of macrophages in producing axonal damage is discussed. PMID- 1719740 TI - Immunochemistry of ethylnitrosourea-induced rat neurinomas, the RN6 neurinoma cell line and their transplantation tumors. AB - The expression of glial fibrillary acidic protein (GFAP), vimentin, S-100 protein (S-100), HNK-1, myelin basic protein (MBP) and fibronectin was investigated immunohistochemically in 51 ethylnitrosourea (ENU)-induced neurinomas of the rat. Additionally, 90 transplantation tumors derived from ENU-induced neurinomas and the RN6 rat neurinoma cell clone were studied. Vimentin immunoreactivity was shown in 50/51 primary neurinomas and 60/90 transplantation tumors. In contrast, GFAP was expressed in only 23/51 primary tumors and in 5/90 transplantation tumors. In the RN6 neurinoma clone, vimentin and GFAP could be demonstrated both in vivo and in vitro. GFAP expression varied depending on the tumor localization, i.e., tumors of distal portions of peripheral nerves were more frequently GFAP positive than tumors of the spinal roots or of cranial nerves. The same tendency was observed for S-100. In the series of transplantation tumors S-100 and GFAP immunoreactivity decreased with increasing numbers of transplantation passages. Only individual cells in 5 primary tumors were HNK-1 positive and no MBP immunoreactive cells were observed. Our results demonstrate that the expression of differentiation antigens in ENU-induced experimental neurinomas parallels the results reported for human neurinomas. PMID- 1719741 TI - Heterogeneity of xenografted osteosarcoma. A human sarcoma transplanted into nude mice. AB - Human osteoblastic osteosarcoma transplanted into nude mice developed two different subtypes of non-osteogenic sarcoma: one solid and the other cystic. This could reflect the heterogeneity of osteoblastic osteosarcoma; various tumor regions have different characteristics. PMID- 1719742 TI - Histological and immunohistochemical observations of dedifferentiated leiomyosarcoma of the uterus. AB - A case of dedifferentiated leiomyosarcoma of the uterus was examined using immunohistochemistry. The tumor arose in the myometrium, and was a whitish large nodule with hemorrhage and necrosis. Histologically it was a well differentiated leiomyosarcoma with foci showing epithelioid pattern, and in part resembling malignant fibrous histiocytoma (MFH) and giant cell tumor (GCT). Additionally, small round neoplastic cells arranged in an alveolar manner, simulating alveolar rhabdomyosarcoma, were seen in some areas. Neoplastic cells in well differentiated areas expressed desmin, muscle-specific actin and LeuM1, whereas those in epithelioid and poorly differentiated areas lacked these antigens. Instead, tumor cells in epithelioid and small round cell areas were positive for keratin. Interestingly, most tumor cells in well differentiated, epithelioid and small round cell areas were also positive for MB1. However, tumor cells in GCT- and MFH-like areas reacted with none of the antibodies used. Ultrastructurally, some tumor cells possessed various amounts of microfilaments with or without dense patches, whereas others lacked them. These findings suggest that the divergent antigen expression was attributable to different levels of differentiation, and that poorly differentiated components had lost their native features. PMID- 1719743 TI - B-cell lymphoma with vimentin-positive cytoplasmic inclusions. AB - A 60-year-old woman complaining of cervical lymphadenopathy was admitted to Keiyu General Hospital, Yokohama. Malignant lymphoma involving systemic lymph nodes and the bilateral tonsils was suspected by computed tomography. The biopsy diagnosis of the cervical lymph nodes was B-cell lymphoma, diffuse medium-sized cell type. The cleaved centrocytic lymphoma cells were immunoreactive for CD20 and CD22 but negative for immunoglobulins. Characteristically, a considerable number of neoplastic lymphocytes possessed eosinophilic round inclusions in the cytoplasm. The inclusions were green in color by Papanicolaou staining, whereas they appeared vacuole-like in Giemsa-stained preparations. Ultrastructural study confirmed the presence of aggregates of intermediate-sized filamentous structures mainly in the perinuclear area. The rough endoplasmic reticulum and Golgi apparatus were poorly developed. Immunocytochemical staining using cytologic specimens and fresh-frozen sections disclosed that the inclusions were composed of vimentin filaments. Morphologic similarities to signet ring cell lymphoma are discussed. PMID- 1719744 TI - [Combined pharmacokinetic and pharmacodynamic model analysis for procainamide and its metabolite]. AB - The pharmacokinetic and pharmacodynamic profiles of procainamide (PA) and its major metabolite, acetylprocainamide (NAPA), were analyzed by extended combined pharmacokinetic and pharmacodynamic model in rabbits. The pharmacodynamic parameters Keo, S, Ce(50), Emax for PA were 0.023 +/- 0.005 min-1, 3.9 +/- 1.1, 3.6 +/- 0.9 micrograms.ml-1, 37 +/- 10 ms respectively and for NAPA were 0.061 +/ 0.017 min-1, 2.2 +/- 0.4, 6.2 +/- 1.7 micrograms.ml-1, 53.6 +/- 2.5 ms. Following PA iv to rabbit both PA and NAPA were involved in the QTc prolongation of the initial period, but the later action was mainly associated with NAPA. Differences of pharmacokinetic and pharmacodynamic parameters between PA and NAPA were found. PMID- 1719745 TI - Long-term effect of immunization with neural and nonneural antigens on the noradrenaline and serotonin levels of discrete brain areas in mice and the relationship between neurotransmitter levels and antibody production. AB - The effect of immunization with neural and nonneural antigens on hypothalamic and mesencephalic neurotransmission and antibody response have been investigated. Treatment of SJL/N mice with bovine myelin basic protein (BMBP), mouse spinal cord homogenate (MSCH) or bovine serum albumin (BSA) produced no significant changes in the average hypothalamic and mesencephalic noradrenaline (NA) or serotonin (5-HT) content. In Balb/c mice, however, treatment with BMBP caused a significant increase in mesencephalic NA, and treatment with MSCH caused a significant increase in hypothalamic 5-HT 3 weeks after the first antigenic challenge. The SJL/N strain showed a high antibody response to BMBP and BSA, and a moderate one to MSCH, while in Balb/c mice, there was a low response to BMBP, a moderate one to MSCH and a high response to BSA. Significant, positive correlations were found between the level of antigen-specific serum antibodies and the following neurotransmitters: hypothalamic NA in the BMBP and MSCH-treated groups, hypothalamic 5-HT in the MSCH-treated group, mesencephalic NA and 5-HT in the BMBP-treated group, and mesencephalic 5-HT in the MSCH-treated SJL/N animals. No significant correlations between the levels of antibodies and neurotransmitters have been found either in the BSA-immunized SJL/N animals, or in any of the groups of Balb/c mice treated with BMBP, MSCH or BSA. PMID- 1719746 TI - Galanin and neuropeptide Y in chromaffin granules from the guinea-pig. AB - We have examined the subcellular distribution of galanin-like immunoreactivity, neuropeptide Y-like immunoreactivity and the catecholamines noradrenaline and adrenaline in the adrenal medulla from guinea-pigs. By differential centrifugation of the adrenal medulla homogenate the neuropeptides as well as the catecholamines sedimented in a 10,000 g pellet. This pellet was resuspended and further examined in discontinuous and continuous density gradients. In the discontinuous gradient the catecholamines peaked in the heavy bottom fraction, assumed to contain chromaffin granules. Galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity were also enriched in this fraction. However, both neuropeptides showed high levels of sedimentable material also in a fraction of intermediate density. In the continuous density gradient, the sum of sedimentable and soluble catecholamines showed peak values in two fractions corresponding to 1.07 and 1.47 M sucrose, respectively. The NA peak in the denser fraction was more pronounced than the corresponding A peak. Galanin-like immunoreactivity showed only one peak, in the fraction corresponding to 1.07 M sucrose. The data suggest that galanin-like immunoreactivity and neuropeptide Y like immunoreactivity are partly stored with catecholamines in chromaffin granules. However, galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity was also found in fractions lighter than those containing the bulk of the catecholamines. PMID- 1719747 TI - Gut blood flow in the estuarine crocodile, Crocodylus porosus. AB - Simultaneous recordings of blood flow in the right and left aorta and carotid and coeliac artery were made in the crocodile, Crocodylus porosus at rest and during various stimuli. In resting animals the right aorta and carotid artery flow profiles resembled the recordings obtained in the caiman (Axelsson et al. 1989a), with an anterograde blood flow throughout the cardiac cycle. As in the caiman, the left aorta flow profile was complex with both anterograde and retrograde blood flow during the cardiac cycle, and a net left aorta blood flow near zero at rest. The coeliac artery blood flow profile did not show the complex pattern seen in the upper aorta, immediately suggesting that most of the coeliac artery blood originates elsewhere. We believe that coeliac artery blood flow in the resting animal derives from the right aorta via the abdominal anastomosis between the two aortas. Feeding induced an increase in the coeliac artery and left aorta blood flow, probably due to a decrease in visceral vascular resistance, and hence coeliac arterial and the left aorta blood pressure, which facilitates blood flow (from right to left aorta) through the foramen of Panizza. During short 'fright dives', heart rate fell and there was a decrease in the recorded blood flows: carotid artery blood flow did not decrease to the same extent as the RAo and coeliac artery flow, indicating some capacity for redistribution of blood to the cephalic circuits during diving. Similarly, a near-unimpaired carotid artery blood flow was maintained after adrenaline injection. Substance P increased the coeliac artery blood flow and produced a right-to-left cardiac shunt, probably by construction of the pulmonary vasculature. PMID- 1719748 TI - Effects of hydrocephalus on the sympathetic nerves of cerebral arteries, investigated with WGA-HRP anterograde tracing in the rat. AB - The effects of hydrocephalus on the plexus of sympathetic nerves of the intracranial vessels were investigated using wheat germ agglutinin combined with horseradish peroxidase (WGA-HRP). Hydrocephalus was induced by injection of kaolin into the cisterna magna of rats. Three weeks later the superior cervical ganglion (SCG) of one side received WGA-HRP. Three days later the circle of Willis and the contralateral superior cervical ganglion were dissected out. The intensity of labelling and density on the cerebral vessels and the number of labelled neurons on the contralateral superior cervical ganglion were calculated. The intensity of labelled nerves and thick bundles were significantly decreased, although tracing the nerve fibers throughout the length of the vessels was possible. The number of labelled neurons in the contralateral superior cervical ganglion indicated that the injection technique of WGA-HRP into the ganglion was correct in all rats. These results are in favour of the assumption, that the hydrocephalus related incomplete adrenergic denervation of the sympathetic perivascular nerve plexus was mainly due to neuropraxia of the nerve fibers rather than anatomical interruption of the axons. PMID- 1719749 TI - Immunopharmacology and immunotoxicology. PMID- 1719750 TI - Neuroimmunopharmacologic effects of drugs of abuse. AB - Direct studies linking drugs of abuse with changes in neurotransmitters and subsequent effects on the immune system are not abundant. One can, however, hypothesize that an indirect effect can occur since a variety of neurotransmitters known to be acted on by various drugs of abuse can, in turn, be correlated with changes in immunity. These changes most likely are mediated via alterations in the autonomic nervous system or the ratio of hormones regulated by the pituitary gland. In addition, there is sound evidence to suspect that the immune system might be capable of altering either the induction of tolerance or the severity of withdrawal symptoms. An increasing body of evidence indicates that IL-1, IFN-alpha, as well as C3a and C5a of the complement cascade, are capable of acting on central catecholamines within the brain. The possibility that immune system peptides are capable of regulating neurotransmitters is further suggested by the evidence of neuropsychiatric side effects during the course of clinical trials. Since a variety of drugs of abuse can directly alter immunocompetence as evidenced by the results of in vitro protocols described elsewhere in this volume, one could speculate that certain behavioral manifestations of drug addiction may be modulated, in part, by immunologic status. PMID- 1719751 TI - The effects of ethanol, tumor necrosis factor, and granulocyte colony-stimulating factor on lung antibacterial defenses. AB - In summary, evidence is emerging indicating that alcohol-abusing hosts are seriously undermined by profound disturbances in cytokine production and activity. These alterations likely play a critical role in the development and clinical sequelae of their immunosuppressed status. Recombinant technology currently provides the clinician with the potential to immunologically reconstitute and restore host defenses in these hosts. While the ultimate role of these agents in patients will require extensive clinical investigations into their multiple biologic effects and interactions, cytokines, when properly employed, will likely have a major impact on the prevention and treatment of many life-threatening diseases. For the first time, we possess the potential to regulate essential functions of the host defense system which may prevent the development and mitigate the severity of infections in these and other immunocompromised hosts. PMID- 1719752 TI - Cystic fibrosis--a strategy for the future. AB - The identification of the cystic fibrosis locus must be regarded as a major triumph for human molecular genetics. In less than five years, it has been possible to move from the designation of the chromosomal location of CF to the identification of the previously unknown protein which is mutated. This work is a credit to all of the groups which participated in it, and particularly to the Toronto and Ann Arbor groups, which identified the sequence coding for the cystic fibrosis transmembrane regulator as the mutated gene. However, as powerful as molecular genetics is, it can only provide the first step towards understanding the correlation between structure and function for CFTR, analysing the clinical correlates between the gene and pathology, gaining knowledge of the population genetics of CF (including the numbers of mutations, as well as the question of whether or not there is a carrier advantage), and progressing to prevention through carrier testing in the community and to treatment through the development of pharmaceuticals and (ultimately) somatic gene therapy. The solutions to these problems will require the recruitment of new groups, with different techniques, to the CF research community. PMID- 1719753 TI - Ion transport in normal and CF airway epithelia. PMID- 1719754 TI - Some properties of sodium and chloride channels in respiratory epithelia of CF- and non-CF-patients. PMID- 1719755 TI - Roles of Ca and cAMP on C1 channel activity in cystic fibrosis sweat clear cells as studied by microsuperfusion and cell volume analysis. AB - In an attempt to study regulation of C1 channels in intact sweat secretory coils in cystic fibrosis and controls in the least invasive manner, isolated secretory coils were superfused with various drugs and K-efflux was determined as an indirect measure of C1 movement. C1 channel activity was also determined from the drug-induced cell volume increase in gramicidin (GC)-treated dissociated eccrine clear cells. We observed that while MCh-induced K-efflux from the CF secretory coils was entirely normal, K-efflux in the presence of isoproterenol (ISO), forskolin (FK), or IBMX was absent in CF, suggesting that these agents failed to stimulate C1 movement. C1 channel activity of dissociated CF clear cells, as studied by cell volume analysis, was entirely normal when stimulated by Ca elevating agents but was defective when stimulated by cAMP-elevating agents. TPA (phorbol ester) does not appear to stimulate C1 channel activity nor does it modify the effect of other agents. The following observations from the present and previous studies are not necessarily consistent with the traditional thesis that the observed C1 movement is due to cAMP: CT-cAMP had no effect on cell swelling or on K-efflux; ISO is more potent in accumulating tissue cAMP than IBMX yet the latter is more potent in stimulating K-efflux; IBMX increases cytoplasmic [Ca] yet is unable to stimulate K-efflux in CF; K-efflux stimulated by cAMP elevating agents was inhibited by removal of Ca from the bath; and, cell swelling of GC-treated cells in response to cAMP elevating agents was inhibited by removal of Ca. The inability of IBMX to stimulate C1 channels in the face of elevated cytoplasmic [Ca] and cAMP in CF cells deserves further scrutiny. PMID- 1719756 TI - Regulation of absorption in the human sweat duct. PMID- 1719757 TI - Altered biochemical regulation of secretion in cystic fibrosis epithelial cells. PMID- 1719758 TI - The CF gene product as a member of a membrane transporter (TM6-NBF) super family. PMID- 1719759 TI - Regulation of epithelial chloride channels: roles of protein kinases and arachidonic acid. PMID- 1719760 TI - Cytosolic inhibition and excision activation of epithelial chloride channels. PMID- 1719761 TI - Purification of the epithelial Cl channel. PMID- 1719762 TI - Regulation of expression of CFTR in human intestinal epithelial cells. AB - As a first step in our efforts to delineate the role of CFTR in cellular phenotypes we have studied its expression in cultured human intestinal epithelial cells. In particular we have examined the effect of cellular differentiation on CFTR gene expression. CFTR mRNA was measured by quantitative densitometry of Northern blots and normalized to the amounts of pyruvate dehydrogenase message. We have found that in T84 cells the levels of CFTR mRNA do not change as the cells grow to confluence. In contrast, levels of CFTR mRNA increase by a factor of 10-20 as Caco2 cells grow after subculture. This change in the levels of CFTR mRNA is correlated with the morphological differentiation that occurs in Caco2 cells during culture. The potential significance of this observation is discussed. PMID- 1719764 TI - Regulation of ion conductance in human skin fibroblasts. PMID- 1719763 TI - Cystic fibrosis, the CFTR, and rectifying Cl- channels. AB - The human genetic disease cystic fibrosis is caused by a single defective gene on chromosome 7 that codes for a 1480 amino acid protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The defect causes a profound reduction of Cl- permeability in several tissues, which in turn impairs salt absorption and fluid secretion. A 25-80 pS, rectifying Cl- channel has been targeted as the exclusive or primary channel affected in CF. However, we have found no evidence for significant activation or spontaneous activity of this channel in cell-attached patches of normal lymphoblasts or dog tracheal cells. However, in dog tracheal cells, we find lower conductance, linear Cl- channels that are spontaneously active in unstimulated cells and may show increased activity in stimulated cells. Attempts to correlate the expression of mRNA for the CFTR protein in various types of cells with the presence of the rectifying Cl channel show a lack of correlation: i.e., depolarization-activated rectifying Cl channesl have been found in excised, inside-out patches from all cell types that we have examined to date, but the CFTR mRNA has so far only been detected in a subset of epithelial cells. PMID- 1719765 TI - Chloride transport in the cystic fibrosis enterocyte. AB - Molecular mechanisms of intestinal chloride channel regulation and potential abnormalities in electrogenic chloride secretion in intestinal epithelium from cystic fibrosis (CF) patients were investigated by a combination of Ussing chamber, vesicle transport and off-cell patch-clamp analysis. Short circuit current (Isc) measurements in normal and CF rectal biopsies provided evidence for i) a defect in the cAMP-provoked activation of chloride secretion and a (hyper)expression of cAMP-dependent potassium secretion in all CF patients examined (n = 11); ii) a defect in the carbachol-provoked chloride secretion and a (hyper)expression of carbachol-induced potassium secretion in 6/11 patients; iii) a residual (but still impaired) carbachol-induced chloride secretion in 5/11 CF patients (including 2 sibs). The latter class of CF patients appeared to consist genetically of compound heterozygotes for the major delta-F508 deletion, suggesting a correlation between the nature of the mutation in the CF gene and the severity of the chloride secretory defect in CF intestine. In our search for a regulatory function of GTP-binding (G-) proteins detected previously in the luminal membrane of rat and human intestinal epithelial cells, evidence was found for the presence of a GTP[S]-activatable- and GDP[S]-inhibitable chloride conductance in the apical membrane of rat enterocytes and human colonocytes. In excised patches of human colonocyt membranes, this G-proteine-sensitive chloride conductance was identified further as a novel type of chloride channel (20pS; inwardly rectifying) that was different from the 33pS outwardly rectifying chloride channel activatable by cAMP-dependent proteinkinase (PK-A) and voltage depolarization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719766 TI - Chloride transport pathways in human keratinocytes. PMID- 1719767 TI - Genetic analysis of cystic fibrosis. PMID- 1719768 TI - Regulation of lymphocyte chloride channels. PMID- 1719769 TI - Molecular and genetic analyses at the CF locus. PMID- 1719770 TI - A deletion mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) locus: Delta I507. AB - During the development of an amplification refractory mutation system (ARMS) assay for the detection of the Delta F508 mutation and corresponding normal locus in cystic fibrosis we discovered a family in which a further variant of the sequence exists. PCR amplification and direct sequencing of a region of exon 10 of the CFTR locus indicated the deletion of the three base pairs encoding isoleucine or isoleucine, or possibly the presence of a single base substitution in conjunction with the Delta F508 mutation. The resulting protein has a deletion of an isoleucine residue at position 507 as opposed to the previously described deletion of phenylalanine at position 508. We conclude that the loss of an isoleucine residue at position 507 (Delta I507) is another defective variant of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene product. PMID- 1719771 TI - Identification of cystic fibrosis mutations. PMID- 1719772 TI - Molecular genetics of cystic fibrosis. PMID- 1719773 TI - Genotype-phenotype correlations in cystic fibrosis patients. AB - Genetic and biomedical data from 346 cystic fibrosis patients of German origin have been evaluated. We demonstrated an age dependent distribution of CFTR genotypes, and confirmed the previously reported association between the dF508 mutation in the CFTR gene and pancreatic insufficiency. However 3 out of 22 pancreatic sufficient patients were dF508 homozygous. When patients were grouped with respect to height development, significant differences were seen in the distribution of J3.11-MspI alleles. We conclude that genetic determinants in and around the CFTR gene contribute to the variability in the clinical course of the disease. PMID- 1719774 TI - Decrease of duodenal calcitonin gene-related peptide- and substance P-like immunoreactivity in rat duodenal ulcers. AB - We have investigated the endogenous levels of duodenal calcitonin gene-related peptide- (CGRP) and Substance P- (SP) like immunoreactivity (li) following the induction of duodenal ulcers in rats. Using three duodenal ulcerogens, namely cysteamine, dulcerozine or mepirizole given in a single oral dose, a decrease of duodenal CGRP-li and SP-li was observed. Time-relationship studies of this phenomenon show that CGRP-li and SP-li were decreased concomitantly to the formation of gastroduodenal ulcers after the administration of cysteamine (900 mg/kg p.o.). Pretreatment with the selective sensory neurotoxin capsaicin induced CGRP-li decrease in the duodenum, which was not further decreased by an ulcerogenic dose of cysteamine, indicating that cysteamine induced a release of CGRP-li of capsaicin-sensitive origin. Otherwise duodenal SP-li was not sensitive to capsaicin pretreatment and its duodenal content decreased by cysteamine originates from an intrinsic source. Our observations indicate that CGRP and SP may play an important local role in duodenal ulcerogenesis. PMID- 1719775 TI - Relationship between sensory neuropeptides and other local vasoactive mediators in modulating gastric mucosal integrity. PMID- 1719776 TI - Effect of substance P and related neurokinins on gastric acid secretion. AB - Substance P and a series of related neurokinins having various degrees of selectivity for tachykinin receptors have been studied for their effects on gastric acid secretion both "in vitro" and "in vivo". In the isolated gastric fundus from immature rats, substance P, the C-terminal heptapeptide of neurokinin A, NKA (4-10), [Arg]NKB and two synthetic analogues of NKA (4-10), namely, [beta Ala8]NKA (4-10) and [Ala5]NKA (4-10) (compounds marked Men 10210 and Men 10209, respectively) had no effect on spontaneous secretion but enhanced the secretory response to histamine. All the different neurokinins were effective in the range of concentrations 10(-7) - 10(-6) M. In the conscious cat with gastric fistula, substance P dose-dependently increased basal acid secretion, whereas Men 10210 was absolutely ineffective. Men 10209 caused a slight increase in acid output which, however, was only 10% of that induced by dimaprit or pentagastrin. The secretory effect of dimaprit and pentagastrin was not affected by the different neurokinins, conversely the response to 2-Deoxy-D-glucose was slightly reduced by Men 10210 (10 nmol/kg/h). The above data suggest that the natural and synthetic neurokinins studied have negligible effects on gastric acid secretion, thus the gastro-protective effect observed in some experimental conditions is unlikely to be related to an antisecretory effect of these compounds. PMID- 1719777 TI - Studies on secretomotor effects of galanin on various "in vivo" or "in vitro" preparations. PMID- 1719778 TI - Efferent function of capsaicin-sensitive nerves and neurogenic vasodilation in rat mesenteric circulation. PMID- 1719779 TI - Sensory denervation with capsaicin reduces the liver collagen deposition induced by common bile duct obstruction in rats. PMID- 1719780 TI - Occurrence and distribution of substance P- and CGRP-containing nerve fibers in gastric mucosa: species differences. PMID- 1719781 TI - Histamine releasing factors and cytokine-dependent activation of basophils and mast cells. PMID- 1719782 TI - Dissociation between antiinflammatory action of tilorone and its interferon inducing activity. AB - A relation between antiinflammatory action of tilorone and its interferon inducing activity was studied in rats. Tilorone (20-100 mg/kg, p.o.) was dose dependently effective on both carrageenin oedema and passive Arthus, suggesting that the pharmacological property of the compound is different from nonsteroidal antiinflammatory medicaments which were weak inhibitors of passive Arthus. The antiinflammatory action of tilorone had a time-lag and priming with tilorone 24 hrs prior to inflammatory insult was required to demonstrate maximal efficacy. This time course change was similar to that of serum interferon level. In addition, a high correlation was observed between the antiinflammatory activity and the serum interferon level. However, the antiinflammatory action was scarcely affected even if the serum interferon level was remarkably lowered by treatment of an inhibitor of protein biosynthesis, cycloheximide. These findings suggested that the antiinflammatory action of tilorone could be dissociated from its interferon inducing activity. PMID- 1719783 TI - Inhibition of rat paw oedema and pleurisy by the extract from Mandevilla velutina. AB - This study reports the oral anti-inflammatory profile of the crude extract (CE) of Mandevilla velutina, a plant which has been previously demonstrated to selectively antagonize bradykinin response of the isolated tissues on rat paw oedema and pleurisy caused by different phlogistic agents. The CE (50 to 200 mg/kg), given 60 min before, inhibited in a dose-dependent manner bradykinin (BK) and cellulose sulphate-induced paw oedema, maximal inhibition of 59% and 65%, respectively. In the same range dose the CE also significantly antagonized pleural exudate and cell infiltration caused by these substances, maximal inhibition of 34% and 46%, respectively. In addition, the CE (100 and 200 mg/kg) also inhibited paw oedema induced by serotonin, PAF-acether and zymosan, maximal inhibition of 55%, 38% and 46%, respectively, but enhanced histamine oedema. However, the CE revealed only partial or no inhibition in pleural exudate caused by these agents. The CE (100 and 200 mg/kg) also inhibited in a dose and time dependent manner carrageenan-induced paw oedema with a maximal inhibition of 44%, but only partially affected carrageenan-induced pleural exudate. The CE also partially inhibited dextran oedema, but even at a higher dose (400 mg/kg) it failed to interfere with Bothrops Jaracaca-induced paw oedema. The CE inhibited BK and to a lesser extent cellulose sulphate-induced cell migration, but failed to interfere with the differential leukocyte migration in the pleural cavity. These findings provide evidence that the CE from M. velutina, besides antagonizing kinin action, exhibit an oral anti-oedematogenic activity against a variety of phologistic agents, but it was more effective in inhibiting those models where kinins are more involved. PMID- 1719784 TI - Interactions between human and porcine interferons. AB - To investigate the possible interactions between human and porcine interferons (IFNs) in vitro, human transformed (FL) and nontransformed (HEF) cells were treated with either HuIFN alpha and/or gamma and porcine alpha and/or gamma. In both cases the antiproliferative activity was measured to determine the effects of different combinations between human and porcine IFNs on cell proliferation. Combinations of human and porcine IFNs acted mostly antagonistically with exception of IFN combination Hu-alpha/Po-gamma which showed a synergic cooperativity in therms of antiproliferative activity on human transformed cells. PMID- 1719785 TI - A related epitope is consistently present on glycoprotein C of herpes simplex virus type 1 and 2. AB - A monoclonal antibody (VE8) directed to glycoprotein C of herpes simplex virus type 1 (HSV-1) cross-reacted with all HSV type 2 (HSV-2) strains tested. Positive reaction was also observed with all investigated HSV-1 strains, indicating that the related epitope is consistently present in HSV-1 and HSV-2. PMID- 1719786 TI - Precise localization of the epitope of major BLV envelope protein. PMID- 1719787 TI - Percutaneous drainage of hepatic abscesses: comparison of results in abscesses with and without intrahepatic biliary communication. AB - Results of percutaneous drainage performed in eight patients with eight liver abscesses with intrahepatic biliary communication and 22 patients with 26 liver abscesses without biliary communication were analyzed to determine whether the presence of an intrahepatic biliary communication affected the outcome of treatment. The clinical features and response to treatment of both groups were compared. The presence or absence of biliary communication was determined by injection of contrast material into the abscess under fluoroscopic guidance either during or several days after initial drainage. Duration of drainage was longer (p less than .05) in patients with communication (range, 7-44 days; mean, 22 days) than in patients without communication (range, 1-33 days; mean, 13 days). Percutaneous drainage was curative in five (63%) and palliative or temporizing in one (13%) of eight patients with communication. It was curative in 15 (68%) and palliative or temporizing in five (23%) of 22 patients without communication (p = .317). Liver abscesses with intrahepatic biliary communication did not require percutaneous transhepatic biliary diversion for cure. Despite longer duration of drainage for abscesses with intrahepatic biliary communication, the cure rates of percutaneous drainage for both groups were similar. Patients in whom an intrahepatic biliary communication was shown did not require alternative interventional or surgical measures for cure. PMID- 1719788 TI - Correct placement of epidural steroid injections: fluoroscopic guidance and contrast administration. AB - We prospectively evaluated 316 caudal-approach epidural steroid injections given by staff radiologists and residents in our department over a 1-year period. Needle placement was checked with fluoroscopy and corrected if necessary. When the needle tip was within the sacral canal, nonionic contrast material was injected. If epidural contrast was not observed, the needle tip was repositioned. Of 111 procedures performed by physicians who had given fewer than 10 epidural steroid injections, 53 (47.7%) resulted in correct nonfluoroscopically directed placement of the needle. For physicians who had performed between 10 and 50 such procedures, 62 (53.4%) of 116 had correct nonfluoroscopically directed placement. For staff physicians, 55 (61.7%) of 89 placements were correct. Even when the sacral hiatus was easily palpated and a staff physician was confident that he or she was within the epidural space, fluoroscopy revealed incorrect placement 14.2% of the time (seven of 49 procedures). In addition, when the needle was positioned within the sacral canal and no blood was evident on Valsalva maneuver or aspiration, the injection was venous in 29 of 316 procedures (9.2%). The presence of blood on the needle stylus was not a reliable indicator of venous placement of the needle. Our findings indicate that fluoroscopy is essential for correct placement of epidural steroid injection. Contrast administration is necessary to avoid venous injection of steroids. PMID- 1719789 TI - Mother-to-child transmission of HIV-1 infection. Epidemiological and experimental studies. PMID- 1719790 TI - Current approaches to vaccination against human immunodeficiency viruses. PMID- 1719791 TI - An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals. AB - The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6 hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers. PMID- 1719792 TI - Epidermal ridge formation in the human fetus: a correlation to the appearance of basal cell heterogeneity and the expression of epidermal growth factor receptor and cytokeratin polypeptides in the epidermis. AB - This paper aims to clarify an expression of epidermal growth factor receptor (EGFR) and cytokeratin 10 and/or 11 in relation to primary and secondary epidermal ridge formation of the human fetus. Firstly, scanning electron microscopy revealed heterogeneity in basal cell morphology during epidermal ridge formation. Basal cells had a uniform, smooth, and polygonal dermal surface until formation of the primary epidermal ridges. Thereafter, the dermal surface became ruffled and elliptic except at the primary epidermal ridges. Secondly, EGFR was detected by monoclonal antibody and autoradiography using 125I-EGF. The antibody reacted with primary epidermal ridge, stratum basale, stratum intermedium, and outer layer of sweat duct. The reactivity became stronger at the primary epidermal ridge than at the secondary one. The binding of 125I-EGF was concentrated in the primary epidermal ridge and sweat duct. Thirdly, cytokeratin 10 and/or 11, a maturation marker of keratinocytes, was detected by monoclonal antibody. The antibody reacted only with the stratum intermedium before secondary epidermal ridge formation. Afterward, it also reacted with the stratum basale of the secondary epidermal ridge but never reacted with that of primary epidermal ridge. The results indicate that basal cells of the secondary epidermal ridge enter the maturation process and suggest a localization of epidermal stem cells on the primary epidermal ridges. Concerning epidermal ridge formation, we suppose that the formation of the primary epidermal ridge causes the segregation of the epidermal stem cells, and that the increased density of the basal cells between the two primary epidermal ridges brings about the change in their dermal surface shape and the formation of the secondary epidermal ridge. PMID- 1719793 TI - Survival and analysis of failure following hydroxyurea, 5-fluorouracil and concomitant radiation therapy in poor prognosis head and neck cancer. AB - Thirty-nine patients with head and neck cancer were entered into Phase I-II study of simultaneous radiation therapy with continuous infusion fluorouracil at 800 mg/m2/day and escalating doses of hydroxyurea. Twenty of these patients had recurrent disease after previous surgery and/or radiation therapy (group 1). Nineteen patients had not received prior local therapy but had advanced-stage disease (group 2). Cycles were repeated every other week until the completion of radiation therapy. The median follow-up was 32 months. Patients with recurrent disease were generally treated with palliative doses of radiation (median dose 5,000 cGy) while previously untreated patients received radiation with curative intent (median dose 7,040 cGy). The response rate for 15 evaluable patients with recurrent disease was 93% with 40% of patients achieving a complete response. For 17 evaluable patients without recurrent disease the response rate was 100%, with a complete response rate of 71%. This regimen exhibited a high activity and significant palliative benefit in group 1 patients. However the local control rate was 25% (5/20) because the majority of patients in this group eventually developed a local recurrence. The local control rate for group 2 patients was 84% (16/19). The higher local failure rate in group 1 patients appeared to be attributable to the palliative doses of radiation delivered and the fewer cycles of treatment received. We conclude that this regimen has palliative benefit in patients who have failed prior local therapy and has the potential for cure in patients with poor prognosis advanced stage disease as well. PMID- 1719794 TI - Atypical intraepithelial lesions of the prostate gland. PMID- 1719795 TI - Fungi in megakaryocytes. An unusual manifestation of fungal infection of the bone marrow. AB - When fungi infect the bone marrow, typically they are associated with granuloma formation and/or necrosis, and the fungi are found within histiocytes or admixed with necrotic debris. Recently two bone marrow biopsy specimens were encountered in which fungi were confined to the cytoplasm of megakaryocytes, a finding not previously reported in the literature. The first case was that of a 46-year-old man with pulmonary histoplasmosis and no known immunodeficiency. The second was that of a 38-year-old man with the acquired immune deficiency syndrome and cryptococcal meningitis. In the first case, many megakaryocytes contained fungal forms consistent with Histoplasma. In the second, one small cluster of megakaryocytes contained several budding yeast consistent with Cryptococcus. Neither marrow biopsy specimen had necrosis, granulomas, or histiocytic infiltration. In both cases, because of the unusual localization of the fungi, they were initially overlooked. The bone marrow may contain fungi even in the absence of abnormalities suggesting fungal infection on routinely stained sections. A silver stain or a periodic acid--Schiff stain should be performed on all marrow biopsy specimens in cases of known or suspected fungal infection outside the marrow. The phenomenon of megakaryocyte emperipolesis is well known, and this process may be responsible for the apparent ability of megakaryocytes to internalize fungi. PMID- 1719796 TI - Metastatic carcinoma in lymph nodes simulating "syncytial variant" of nodular sclerosing Hodgkin's disease. AB - The authors report the histories of two patients with undifferentiated carcinoma metastatic to lymph nodes simulating the "syncytial variant" of nodular sclerosing Hodgkin's disease. One of the patients initially was treated for Hodgkin's disease, but the clinical evolution was more typical of carcinoma. Both lesions were characterized histologically by noncohesive aggregates of large neoplastic cells with abundant eosinophilic cytoplasm and conspicuous nucleoli. Although cells compatible with diagnostic Reed-Sternberg cells were identified in an "appropriate" cellular background in both patients, the diagnosis of carcinoma was supported by intense cytokeratin immunoreactivity. Subtle histologic clues that should suggest the possibility of metastatic carcinoma in a patient whose morphologic data suggests the syncytial variant of nodular sclerosing Hodgkin's disease include sinus infiltration, phagocytosis of neutrophils by tumor cells, marked nuclear anaplasia, and the presence of spindle-shaped tumor cells. PMID- 1719797 TI - How vitronectin binds to activated glycoprotein IIb-IIIa complex and its function in platelet aggregation. AB - Vitronectin, which is present in both plasma and extracellular matrix, inhibits the complement cascade and promotes the growth and attachment of cells in vitro. Like other adhesive proteins such as fibrinogen, von Willebrand factor, and fibronectin, vitronectin contains the sequence Arg-Gly-Asp and binds to some members of the family of receptors called integrins. Platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) is well known as a member of integrins that bind to vitronectin as well as to fibrinogen, von Willebrand factor, and fibronectin. The interaction of vitronectin with GPIIb-IIIa was studied. Vitronectin bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with a molecular weight of 290 nmol/L and 9,100 sites per platelet. The binding was inhibited by the other adhesive proteins with IC50s of 0.078-0.15 mumol/L. The binding also was inhibited by the tetrapeptide Arg-Gly-Asp-Ser and the monoclonal antibody to GPIIb-IIIa (LJ-CP8). Vitronectin inhibited thrombin-induced platelet aggregation in a dose-dependent manner and fibrinogen enhanced platelet aggregation. These results suggest that vitronectin might modulate platelet aggregates by interfering with the interaction of fibrinogen with thrombin-activated GPIIb-IIIa. PMID- 1719798 TI - Lipase and pancreatic amylase activities in tissues and in patients with hyperamylasemia. AB - Lipase, pancreatic amylase, and total amylase activities were measured in nondiseased and diseased human pancreatic tissues and in six different locations of the human digestive system. In addition, it was determined whether serum lipase and pancreatic amylase tests could replace the total amylase test to improved diagnostic efficiency in the evaluation of acute pancreatitis in hyperamylasemia patients. Nondiseased pancreatic tissue contained 4.5 times more lipase activity than total amylase activity. Diseased pancreatic tissue contained less activity for both lipase and total amylase compared to normal tissue. The total amylase activity of the pancreas was comprised solely of pancreatic amylase. Tissue obtained from six different anatomic locations in the digestive system contained 35 to 45 times less lipase and total amylase activity compared to the pancreas. Total amylase activity of the digestive system tissues were comprised of 25% pancreatic and 75% salivary isoamylases. Lipase, pancreatic amylase, and total amylase levels also were determined in serial serum samples from 17 consecutive hyperamylasemia patients admitted with possible acute pancreatitis. The serum lipase level remained higher than normal longer than either the total amylase and pancreatic amylase levels. In patients with hyperamylasemia of pancreatic origin, a poor correlation was observed at admission between serum pancreatic amylase and serum lipase. Not all patients with elevated lipase had an elevated pancreatic amylase level and vice versa. However, in every patient pancreatic disease would have been detected by the elevation of either lipase or pancreatic amylase levels. Diagnostic efficiency for pancreatic disease using serum pancreatic amylase, lipase, and total amylase tests was 94.1%, 76.5%, and 64.7%, respectively. These data suggest that lipase and pancreatic amylase tests are specific for the pancreas and might be considered replacements for total amylase as the stat or routine laboratory test for the diagnosis of pancreatic tissue injury. PMID- 1719799 TI - Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents. AB - Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens. PMID- 1719800 TI - Morphonuclear relationship between prostatic intraepithelial neoplasia and cancers as assessed by digital cell image analysis. AB - Using digital cell image analysis performed on Feulgen-stained nuclei, the nuclear characteristics of prostatic neoplasia, ranging from benign (benign prostatic hyperplasia [BPH]), through dysplastic (prostatic intraepithelial neoplasia [PIN] 1-3), to carcinoma were studied. Four histopathologic groups were studied: group IA (18 samples) contained BPH, PIN 1, and PIN 2 lesions that were from 9 prostate samples free of cancer. Group IB (23 samples) was identical to group IA, contained also BPH, PIN 1, and PIN 2 lesions, but lesions that were from 7 prostate samples where malignant foci were detected elsewhere. Group II (11 samples) were PIN 3 specimens. Group III (24 samples) were carcinomas. Features of neoplastic nuclei were quantified objectively through morphometric (nuclear size), densitometric (nuclear DNA content), and textural (chromatin organization and heterogeneity) parameters. Cell kinetic parameter, i.e., cell proliferation index, was assessed from the densitometric measurement. The proliferation index was significantly higher in PIN 3 and cancers as compared to BPH, PIN 1, and PIN 2 tissues. Morphonuclear characteristics were also dramatically distinct among the four groups. Indeed, the nuclear size and the hyperchromatism of severe prostatic dysplasia were similar to those of carcinomas, these two lesion types showing mean parameter values that were higher as compared to BPH, PIN 1, and PIN 2 lesions. Finally, benign tissues related to mild or moderate dysplasia taken in histologic material in which cancer was present already share the morphonuclear characteristics of severe dysplasia, although they are nonproliferating. PMID- 1719801 TI - Assessment of antigen damage in immunohistochemistry. The vimentin internal control. AB - Monitoring of the quality of antigen preservation and the uniformity of tissue fixation in paraffin-embedded, formaldehyde-fixed tissues is facilitated by the routine use of an antibody to an epitope of vimentin that is partially susceptible to formaldehyde fixation. Other diagnostically useful molecules often show fixation- or processing-induced alterations that parallel those of the vimentin epitope. Thus, the use of vimentin as an internal control, or reporter molecule, and its extrapolation to other antigens allow for better interpretation of results, improved selection of fields optimal for diagnostic immunohistochemistry, and more rational application of compensatory procedures, such as protease digestion. PMID- 1719802 TI - Keratins in soft-tissue sarcomas--common phenomenon or technical artifact? PMID- 1719803 TI - Pediatric germ cell and human chorionic gonadotropin-producing tumors. Clinical and laboratory features. AB - Germ cell tumors may cause various aberrations in pubertal development. In prepubertal boys, these tumors may secrete human chorionic gonadotropin, resulting in precocious puberty. Human chorionic gonadotropin and alpha fetoprotein are both useful as germ cell tumor markers in the diagnosis and detection of recurrence. Pregnancy-specific beta 1-glycoprotein, another oncoplacental antigen, has been used as a tumor marker for trophoblastic neoplasms, but not previously for human chorionic gonadotropin-producing tumors associated with precocious puberty. Patients with germ cell tumors may also have abnormal karyotypes. Herein, we describe six male pediatric patients with germ cell tumors and pubertal derangements seen during an 8-year period. We confirm the high incidence of associated sexual precocity, the usefulness of alpha fetoprotein, human chorionic gonadotropin, and pregnancy-specific beta 1 glycoprotein as tumor markers in the diagnosis and follow-up of these patients, and the occurrence of sex chromosomal abnormalities. PMID- 1719804 TI - Distribution of substance P and vasoactive intestinal peptide in the human liver: light and electron immunoperoxidase methods of observation. AB - The localization of substance P (SP) and vasoactive intestinal peptide (VIP) in 12 normal human liver tissues was examined by light and electron immunohistochemistry using immunoperoxidase methods. SP and VIP immunoreactive nerve fibers were observed around portal veins, bile ducts, and hepatic arteries in portal areas, along sinusoids and hepatocytes in hepatic lobules, and around central veins. More SP and VIP immunoreactive nerve fibers were present in the portal areas than in other regions. Moreover, SP and VIP containing nerve endings were localized close to myofibroblasts, Ito cells, fibroblasts and endothelial cells of blood vessels, and sinusoids. The results suggested that part of the innervation of the human liver may be related to the contraction and relaxation of the cells close to nerve endings, and to the regulation of hemodynamic processes by the neurotransmitters such as SP and VIP at the hepatic lobular level. PMID- 1719805 TI - Hyperamylasemia and pancreatitis in leptospirosis. AB - Hyperamylasemia has been documented in up to 65% of our patients with leptospirosis and jaundice. However, pancreatitis is an uncommon complication of leptospirosis. Three patients with leptospirosis and pancreatitis are described and compared with two leptospirosis patients who had hyperamylasemia but in whom the diagnosis of pancreatitis could not be substantiated. The cause of the hyperamylasemia in the latter patients was nonpancreatic. The elevation of the amylase in these latter two patients could not be explained by renal insufficiency, because the level of the amylase was greater than three to four times the normal value, the upper limit to which amylase rises in renal failure. Thus, hyperamylasemia in patients with leptospirosis can be from pancreatic and nonpancreatic sources. Leptospirosis should be considered in the differential diagnosis of hyperamylasemia and pancreatitis. PMID- 1719806 TI - Incidence of surgically treated benign prostatic hypertrophy and of prostate cancer among blacks and whites in a prepaid health care plan. AB - The incidence of surgically treated benign prostatic hypertrophy and of prostate cancer was examined to December 1987 in 14,897 men (2,175 blacks and 12,722 whites) who received multiphasic health checkups during 1971-1972 while members of the Kaiser Permanente Medical Care Program (San Francisco-Oakland, California). Prostate cancer incidence was higher in blacks than in whites for all age groups (age-adjusted relative risk (RR) = 1.8, 95% confidence interval (CI) 1.4-2.3). The incidence of benign prostatic hypertrophy was somewhat higher in blacks than in whites until age 65 years, after which it was higher in whites. In contrast to the risk of prostate cancer, the age-adjusted risk of benign prostatic hypertrophy was the same for blacks as for whites (RR = 1.0, 95% Cl 0.8 1.2). PMID- 1719807 TI - Hb S/beta zero-thalassemia due to the approximately 1.4-kb deletion is associated with a relatively mild phenotype. AB - We report a relatively mild phenotype associated with two siblings who are compound heterozygotes for Hb S and a beta zero-thalassemia mutation due to a approximately 1.4-kb deletion of the 5' region of the beta-globin gene. Each is found to have unusually high levels of Hb A2 and Hb F, accounting for more than 20% of the total hemoglobin. These may interfere with intracellular Hb S polymerization, thus leading to a mild clinical course. PMID- 1719808 TI - Significance of hemostatic molecular markers during disseminated intravascular coagulation in patients with liver cirrhosis treated by endoscopic embolization for esophageal varices. AB - We studied the quantitative changes of hemostatic molecular markers with time during the course of disseminated intravascular coagulation (DIC) induced by endoscopic embolization using thrombin for esophageal varices in nine patients with liver cirrhosis. The plasma levels of D-dimer, a product of plasmin degradation of cross-linked fibrin, and thrombin-antithrombin-III complex (TAT) were significantly higher in patients before treatment when compared with 60 healthy individuals. The plasma levels of TAT, D-dimer, and plasmin alpha 2 plasmin inhibitor complex (PIC) increased significantly 5-15 min after thrombin injection into the varices, earlier than the changes of conventional coagulofibrinolytic factors, reached a maximum level after 180 min, and started to decline after 1 day. Although the plasma PIC level returned to normal after 7 days, both TAT and D-dimer were still above the pretreatment level. Although there was no change in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) increased significantly after 5 min. The plasma level of plasminogen activator inhibitor type 1 (PAI-1) showed only a slight elevation after treatment. We propose that the hemostatic molecular markers TAT, D-dimer, and PIC are suitable for the early diagnosis of DIC after endoscopic embolization using thrombin in patients with liver cirrhosis and that the increase of PAI-1 is too small for the regulation of fibrinolysis due to t-PA in DIC occurring in liver cirrhosis. PMID- 1719809 TI - In vivo efficacy of intravenous gammaglobulins in patients with lupus anticoagulant is not mediated by an anti-idiotypic mechanism. AB - The authors have investigated the presence in commercially available intravenous gammaglobulins (IVIg) of anti-idiotypic antibodies directed to Lupus Anticoagulant (LA). In vitro incubation of 4 LA plasmas with increasing concentrations of IVIg (from 0 to 39 mg/ml) resulted in a dose-dependent inhibition of LA activity (the highest inhibitions ranged from 14.0 to 53.4%). Similar results were obtained when patients' plasma was substituted with total IgG (the highest inhibitions ranged from 43.0 to 55.0% and were obtained at IgG:IVIg molar ratios ranging from 1:15 to 1:50). Also the incubation of patients' F(ab')2 with F(ab')2 from IVIg produced a similar dose-dependent inhibition of LA activity. These data are suggestive of an in vitro idiotypic anti-idiotypic interaction between LA and IVIg. However, when injected in patients with LA, IVIg do not seem to operate by this mechanism of action. In fact, reduction or disappearance of LA was only observed in 2 out of 4 patients; also the quick reappearance of LA activity was not consistent with the time course of anti-idiotypic response. Finally, this effect was reached by half the IVIg concentrations necessary to produce an appreciable inhibitory effect on LA activity in vitro. Thus, it is concluded that, even if IVIg contain anti idiotypic antibodies reacting with LA, the clinical efficacy of IVIg treatment in patients with these autoantibodies should be attributed to other mechanisms. PMID- 1719810 TI - Induction of carbonic anhydrase I isozyme precedes the globin synthesis during erythropoiesis in K562 cells. AB - Induction of carbonic anhydrase isozyme I (CA-I) by erythropoietin or hemin was investigated using erythroleukemia (K562) cells. Immunological estimation and purification of carbonic anhydrases showed that untreated K562 cells contained only carbonic anhydrase isozyme II(CA-II), while incubation of the cells with 2 units of erythropoietin (EP) per ml of the incubation medium or with 50 microM hemin resulted in the induction of CA-I. The purified CA-I induced in K562 cells was enzymatically and immunologically identical to that from mature erythrocytes. Flow cytometric analysis showed that incubation of K562 cells with EP as well as hemin induced CA-I at the 3rd h, while alpha-globin was detected at the 8th h. Northern blot analysis of CA-I mRNA using a cloned genomic DNA as a probe showed that mRNA of CA-I was induced by EP. These results suggest that induction of CA-I is regulated at the transcriptional level during developmental changes of erythroid cells, and that CA-I may play a physiologically important role during erythroid differentiation. PMID- 1719811 TI - Autosomal dominant macrocephaly: benign familial macrocephaly or a new syndrome? AB - During the course of a clinical study of Sotos syndrome, six out of 79 probands failed to fit the phenotype of Sotos syndrome but showed remarkable similarities to each other and to a further 11 first- and second-degree relatives. Clinical features in these index cases included macrocephaly, greater than +3.5 SD; normal or near normal birth weight and length with subsequent relative obesity; variable developmental delay; and typical face, characterised by a square outline with frontal bossing, a "dished-out" mid-face, biparietal narrowing, and long philtrum. All recorded bone ages were less than the 75th centile, and two were below the 10th centile. The authors believe the original diagnosis was incorrect and that these cases likely represent a previously undescribed autosomal dominant macrocephaly syndrome. PMID- 1719812 TI - Interstitial deletion of chromosome 18[del(18)(q11.2q12.2 or q12.2q21.1]. AB - A 27-month old boy with mild developmental delay, growth delay, strabismus, midface hypoplasia, relative telecanthus, downslanting palpebral fissures, epicanthal folds, dental hypoplasia, and cardiac defects was found to have an interstitial deletion of chromosome 18 involving band q12.1 or q12.3 PMID- 1719813 TI - Limb body-wall complex in association with sirenomelia sequence. AB - Limb body-wall complex and sirenomelia sequence are uncommon birth defects and their association is extremely rare. Their overlapping manifestations and their concurrence in our patient suggest that they share a common cause and belong to a group of pathologically closely related conditions. Embryonic vascular disruption may be a common pathogenesis in both anomalies. PMID- 1719814 TI - Human chorionic gonadotrophin and trisomy 18. AB - Estimation of maternal serum beta-hCG is used in conjunction with alpha fetoprotein (AFP) and estriol (E3) for estimating the risk of Down syndrome (DS) affected fetuses. However, low hCG levels have not been regarded as having clinical significance. We report on 2 patients with trisomy 18 fetuses in whom antenatal screening showed extremely low hCG levels (0.05 and 0.15 MOM). Low hCG levels might indicate increased risk for trisomy 18 despite low estimated risk for DS. PMID- 1719815 TI - High resolution replication banding combined with in situ hybridization for the delineation of a subtle chromosome rearrangement. AB - Molecular cytogenetic techniques were used to delineate a subtle chromosome rearrangement in an infant with growth and psychomotor retardation, abnormal scalp hair pattern, narrow palpebral fissures, broad nasal bridge, bulbous nose, small nostrils, thin lips in a cupid's bow configuration, bilateral simian creases, and unilateral cryptorchidism. Analysis using GTG-banded chromosomes at about 400 band level showed no obvious abnormality. Prometaphase analysis at about 600 band level showed an extra band at 14q32 on GTG-banding. The father had the same extra band suggesting a reciprocal translocation but the second chromosome involved in the translocation could not be identified. High resolution replication banding on the father's lymphocytes showed a balanced reciprocal translocation 46,XY,rcp(8;14)(q24.1;q32.1). The translocation was confirmed by in situ hybridization with an immunoglobulin heavy chain probe which maps to 14q32.3. The infant therefore had duplication of 8q24.1----qter and deficiency of 14q32.1----qter. His phenotype resembled that of patients with partial duplications of the distal long arm of chromosome 8. PMID- 1719816 TI - Risk of open spina bifida. PMID- 1719817 TI - Development of a treatment rating in school systems: service determination through objective measurement. AB - This paper introduces the Capital Area Treatment Rating (CATR) being developed by the occupational and physical therapists of the Capital Area Intermediate Unit in Harrisburg, Pennsylvania. This rating assists therapists in identifying children in special education who require occupational or physical therapy treatment. The CATR consists of two measurement categories - functional levels and clinical judgment factors - each of which is rated on a 4-point ordinal scale. The scores from each of these categories are then added together for a final rating score. A pilot test involving 180 children in special education indicated that the need for treatment is most consistently associated with a score of 28 points or above on the CATR. A survey of 17 pediatric occupational therapists revealed strong support for the instrument's content and resulted in a revised version. Suggestions for future research and ongoing development are discussed, as are general guidelines for use of the instrument in special education occupational therapy programs. PMID- 1719818 TI - Nonradioactive RNA in situ hybridization detection of human papillomavirus 16-E7 transcripts in squamous cell carcinomas of the uterine cervix using confocal laser scan microscopy. AB - Paraffin-embedded squamous cell carcinomas of the uterine cervix selected for the presence of human papillomavirus (HPV) genotype 16 (n = 19) by polymerase chain reaction, were studied for transcription of the early open reading frame E7 (ORF E7). Nonradioactive RNA in situ hybridization (RISH) was performed using in vitro generated biotinylated probes. Hybrids were visualized by streptavidin gold and silver enhancement staining in combination with confocal laser scan microscopy. Quality of mRNA was verified by detection of beta-actin gene transcripts before E7 expression was studied. In all carcinomas containing HPV 16 DNA and showing beta-actin mRNA signals (n = 13), clear E7 ORF transcription could be found. Additional RNA-PCR on purified cytoplasmic RNA of snapfrozen tissue of identical carcinomas (n = 7) showed E6-E7 specific transcripts in all E7 RISH positive samples. These results indicate continuous expression of E7 ORF in all cervical carcinomas containing HPV 16 DNA and support an active role of the E7 ORF in the pathogenesis of cervical cancer. PMID- 1719819 TI - Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. AB - The ultrastructural localization of the CD68 antigen, a 110-kd intracellular glycoprotein associated with myeloid cells and with monocytes/macrophages, was investigated in human neutrophil granulocytes by postembedding immunogold staining, using monoclonal antibody KP1. The antigen was found in the primary granules of neutrophils, although not all primary granules were labeled. It was absent from the plasma membrane. In monocytes, it was also detected within cytoplasmic granules, colocalized with lysozyme and myeloperoxidase. This observation confirms and completes results obtained by immunofluorescence and other light-microscopic methods. Moreover this study shows that the CD68 epitope recognized by antibody KP1 is able to resist fixation and embedment and therefore emphasizes the value of using KP1 as a marker for this macrophage-associated molecule. PMID- 1719820 TI - Tenascin expression in human dermis is related to epidermal proliferation. AB - The extracellular matrix glycoprotein tenascin is sparsely distributed in normal human dermis. The authors have shown that in a number of skin diseases (psoriasis, skin tumors), tenascin expression is strongly increased. In this immunohistochemical study, using polyclonal and monoclonal antisera, we have tested the hypothesis that tenascin expression in vivo is linked to epidermal proliferation. Using the sellotape stripping model in normal human skin, which causes a rapid recruitment of keratinocytes into the cell cycle, induction of tenascin expression was found in the upper dermis within 24 hours after stripping. In contrast, in normoproliferative monogenic disorders of keratinization (X-linked recessive ichthyosis, autosomal dominant ichthyosis vulgaris, non-erythrodermic lamellar ichthyosis), no increase in tenascin expression was found compared with normal skin. These findings demonstrate a relationship between epidermal proliferation and metabolic alterations in the dermal compartment. PMID- 1719821 TI - Chloride-mediated junction potentials in circular muscle of the guinea pig ileum. AB - Junction potentials were recorded from circular muscle cells of the guinea pig ileum at various sites oral and anal to a transmural stimulus in the presence of atropine, apamin, and substance P antagonism (desensitization) at 30 degrees C. A short train of pulses produced an inhibitory junction potential (slow IJP), which preceded an excitatory junction potential (slow EJP). The slow IJP was observed up to 56.4 +/- 2.9 mm oral and 65.4 +/- 2.2 mm anal to the stimulus. The slow EJP was observed up to 50.4 +/- 1.9 mm oral and 58.3 +/- 2.1 mm anal to the stimulus. Hexamethonium (400 microM) decreased the amplitudes of the slow IJP and slow EJP at all sites. After hexamethonium, the slow IJP was observed up to 37.3 +/- 2.3 mm oral and 39.8 +/- 2.1 mm anal to the stimulus and the slow EJP was 44.2 +/- 2.5 mm oral and 43.3 +/- 2.6 mm anal to the stimulus. The slow IJP and slow EJP were associated with an increase and decrease in membrane resistance, respectively. Conditioning depolarizations of the circular muscle cells reduced the amplitudes of the slow IJP and slow EJP. Both were abolished at a membrane potential of approximately -25 mV. Conditioning hyperpolarizations increased the amplitude of both the slow IJP and slow EJP. Ionic substitution experiments with low external chloride solution (12.4 mM) resulted in an immediate increase in slow IJP and slow EJP amplitudes, whereas more prolonged perfusion resulted in a significant decrease in slow IJP and slow EJP amplitudes. 4,4' Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) resulted in decreases in slow IJP and slow EJP amplitudes. These results suggest that the slow IJP and slow EJP are due to a decrease and increase in membrane chloride conductance, respectively. The noncholinergic neural pathways responsible for the slow IJP and slow EJP extend approximately 40 mm orally and anally along the longitudinal axis of the guinea pig ileum. PMID- 1719822 TI - H(+)-K(+)-ATPase contributes to regulation of pHi in frog oxynticopeptic cells. AB - The effect of intracellular acidosis on luminal H+ secretion and the role of H(+) K(+)-ATPase in regulation of intracellular pH (pHi) in oxynticopeptic cells (OPC) (measured with a pH-sensitive fluorescent dye) were examined in intact sheets of in vitro frog (Rana catesbeiana) gastric mucosa. Intracellular acidosis of OPC induced by decreasing pH in the serosal solution (pHs) from 7.2 to 6.0 reversibly increased forskolin-stimulated H+ secretion without increasing endogenous histamine release. The observed increase in H+ secretion was unaffected by either 1 mM cimetidine or 1 mM histamine, but was accentuated by 1 mM amiloride, an effect abolished by 0.3 mM omeprazole. Steady-state pHi values in stimulated or resting OPC at pHs 7.2 were not significantly different. However, pHi in OPC was significantly higher in stimulated than in resting tissues at pHs 6.9, a difference accentuated by decreasing pHs to 6.4 or by 1 mM amiloride. Amiloride completely prevented recovery from intracellular acidosis induced by pHs 6.4 or 6.9 in omeprazole-treated tissues, but only partially mitigated recovery in cimetidine- or forskolin-treated tissues. At pHs 6.4, high luminal [K+] (100 mM) increased H+ secretion and hastened recovery of pHi in cimetidine-treated tissues in the presence of amiloride. These results suggest that, in intact sheets of in vitro frog gastric mucosa, 1) intracellular acidosis stimulates luminal H+ secretion via histamine-independent mechanisms and 2) H(+)-K(+)-ATPase contributes to the recovery of OPC from intracellular acidosis. PMID- 1719823 TI - Profound increase in viscosity and aggregation of pig gastric mucin at low pH. AB - Epithelial mucins are glycoproteins of very large molecular weight that provide viscoelastic and gel-forming properties to mucus, the jellylike protective layer covering epithelial organs. In the mammalian stomach the mucus gel layer protects the underlying epithelial cells from HCl in the lumen. We report here that pig gastric mucin undergoes a 100-fold increase in viscosity in vitro when pH is lowered from 7 to 2. Sedimentation velocity and dynamic light-scattering measurements revealed the formation of extremely large aggregates at low pH consistent with the observed increase in viscosity. Aggregation of mucin at low pH was prevented by increasing the ionic strength, suggesting the involvement of electrostatic interactions. Trypsin digestion and thiol reduction, but not enzymatic removal of neuraminic acid, prevented aggregation at low pH. This implies that the peptide core rather than the oligosaccharide side chains of the molecule is involved in the aggregation of mucin at low pH. Increased aggregation and viscosity at low pH were also observed in a solvent made to mimic the ionic composition of gastric juice, indicating the physiological relevance of our findings. Our observations suggest that one mechanism of gastric protection may be the ability of gastric mucin to undergo aggregation with a marked increase in viscosity at low pH. PMID- 1719824 TI - Characterization of prazosin-sensitive alpha 2 B-adrenoceptors expressed by cultured rat IMCD cells. AB - alpha 2-Adrenoceptor subtype expression was investigated in cultured rat inner medullary collecting duct (IMCD) cells using radioligand binding studies, Northern blot analysis, and adenosine 3',5'-cyclic monophosphate (cAMP) assays. [3H]rauwolscine bound to a single class of alpha 2-adrenoceptors with high affinity [Kd = 1.7 +/- 0.3 nM, maximum binding (Bmax) = 45.2 +/- 10.8 fmol/mg protein]. alpha 2-Adrenoceptor ligands inhibited [3H]rauwolscine binding with a rank order of potency characteristic of interaction with the alpha 2B adrenoceptor [inhibitory constant (Ki) values (in nM) rauwolscine (1.95) greater than ARC-239 (8.52) greater than prazosin (237) greater than oxymetazoline (30,000)]. Northern blot analysis was performed using poly(A)+ RNA isolated from 90% confluent rat IMCD cells and probes derived from alpha 2-adrenoceptor DNA sequences from the rat nonglycosylated alpha 2B-adrenoceptor and the human alpha 2A-adrenoceptor. The alpha 2B probe hybridized to a 4.2-kb band under high stringency conditions, but the alpha 2A-adrenoceptor probe did not hybridize to this band. In functional studies, the full alpha 2-adrenoceptor agonists epinephrine and UK-14,304 potently inhibited vasopressin-stimulated cAMP accumulation by 50 to 70% [half-maximal response (EC50) (in nM) epinephrine = 11.2, UK-14,304 = 6.4]. Guanabenz and clonidine were partial agonists, inhibiting cAMP accumulation by 30 to 40% and were less potent than the full agonists [EC50 (in nM) 56.0 guanabenz and 94.5 clonidine]. Epinephrine-induced inhibition of cAMP accumulation was blocked by rauwolscine, prazosin, and ARC-239 but not by the alpha 1-adrenoceptor antagonist corynanthine. We conclude that rat IMCD cells in primary culture express functional alpha 2-adrenoceptors of the alpha 2B subtype. PMID- 1719825 TI - Ion channel activities of cultured rat mesangial cells. AB - We used the patch-clamp technique to clarify the nature of ion channels in renal mesangial cells in culture. In the cell-attached mode most patches were silent in the absence of agonists. In some patches a 25-pS nonselective channel was observed. This 25-pS cation channel was consistently observed in inside-out patches, and it was activated by intracellular Ca2+. Excised patch experiments also revealed the existence of a 40-pS K+ channel, which was activated by intracellular Ca2+. This 40-pS K+ channel was observed infrequently in the cell attached mode. The activities of both channels were increased by arginine vasopressin or angiotensin II, resulting from an increase in intracellular Ca2+ concentration. PMID- 1719826 TI - Possible mechanisms underlying differences in force production between normal and cardiomyopathic hamster atria. AB - The rate of recovery of force after a long rest interval is lower than normal in left atria from 80- to 85-day-old cardiomyopathic (CM) golden Syrian hamsters. To determine whether this difference was due to a reduced amount of Ca2+ available for release with each beat, we manipulated the amount of Ca2+ entering excised atria using the Ca2+ agonist BAY K 8644 and the antagonist nifedipine. We also simulated altered Ca2+ influx in a recent model of cardiac excitation-contraction coupling (V. J. A. Schouten, J. K. Van Deen, P. de Tombe, and A. A. Verveen Biophys. J. 51: 13-26, 1987) by varying the parameter representing Ca2+ influx and observing the effect on the force it predicted. Steady-state force of normal and CM atria was recorded in response to 1-Hz stimulation and recovery of steady state force was monitored after a 600-s rest interval. Ca2+ fluxes were manipulated by raising external Ca2+ or by the presence and absence of drug. The recovery of force after a 600-s rest interval was digitized and fitted with an exponential function, and the time constant and steady-state force to which the muscle recovered after the pause were compared. Inclusion of the Ca2+ agonist BAY K 8644 (0.25 or 2.0 microM) made the response of CM atria similar to that of normal, while inclusion of the Ca2+ antagonist nifedipine (0.8 microM) made the response of normal atria similar to that of the CM. Similarly, decreasing the simulated Ca2+ influx in the model produced all of the differences observed between normal and CM muscle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719827 TI - Differential effects of cytokines on thermosensitive neurons in guinea pig preoptic area slices. AB - This study was undertaken to determine whether the reported different courses of the febrile responses to the cytokines interleukin-1 beta (IL-1), interferon alpha 2 (IFN), and tumor necrosis factor-alpha (TNF) might have neuroelectrophysiological correlates. The reactions of individual thermosensitive neurons in the preoptic area (POA) were evaluated by recording their extracellular single-unit firing rates (FR) in slices of guinea pig POA perfused with artificial cerebrospinal fluid (aCSF), human recombinant IL-1 (50-500 ng), IFN (1,000-8,000 U), and TNF (400-5,000 ng) (all doses per min/ml aCSF); thermosensitivity was assessed by FR responses to changes of perfusate temperature (32-42 degrees C). Overall, these cytokines depressed the FR of warm sensitive units and excited those of cold-sensitive units, in agreement with expectations. However, the responses of individual neurons treated with two or all three cytokines were dissimilar: 61% of the units tested reacted differentially to two or three cytokines, 32% exhibited identical responses, and 7% had no response to any cytokine. These results support the possibility that IL 1, IFN, and TNF may affect not the same but rather distinct neurons functionally connected to common pyrogenic effectors. Thus they suggest that differential neuronal substrates may be utilized by each cytokine to exert its pyrogenic effect. PMID- 1719828 TI - Channel arrest: implications from membrane resistance in turtle neurons. AB - A widespread defense strategy used by hypoxia-tolerant animals is metabolic depression. One possible mechanism for metabolic depression is "channel arrest." This hypothesis predicts that ion leakage through plasma membrane leakage channels is reduced during an anoxic episode. The decreased ion flux would result in the conservation of energy through the reduction of ATP-demanding ion pumping. We tested this hypothesis with the anoxia-tolerant turtle (Chrysemys picta) as a model system. With intracellular recording used in cortical slices, whole cell input resistance and specific membrane resistivity were monitored under control and anoxic conditions. There were no significant changes in resistance, indicating that the channel arrest defense mechanism was not utilized for energy conservation during short-term anoxia (less than or equal to 120 min). PMID- 1719829 TI - Metaphor and psychotherapy. AB - The authors describe the role of metaphor in the theory and practice of psychotherapy. Metaphors are considered fundamental elements of our worlds of language and concepts and not just figures of speech, poetic devices, parables, or creative ways to make interpretations. Metaphors shape the process of therapy by structuring the therapist's perceptions, stance, and attitude. They also organize the way problems are discussed as well as the solution that are seen as effective. The authors review the literature on metaphor in psychotherapy and explore common psychotherapeutic metaphors such as "psychotherapy is war," "the mind is a brittle object," "the conduit metaphor," and a variety of somatic metaphors. They conclude with a discussion of how metaphors function in the process of psychotherapy. PMID- 1719830 TI - Sclerosing Sertoli cell tumor of the testis. A report of 10 cases. AB - Sex cord-stromal tumors of the testis are uncommon and have been less well characterized than similar tumors of the ovary, with a much greater proportion of them falling into the "unclassified" category. We report the clinical and pathological features of 10 Sertoli cell tumors of the testis with prominent sclerosis, representing a distinctive, heretofore undescribed subtype of Sertoli cell tumor in the human. The patients were mostly in their 3rd and 4th decades (median age, 30 years; range, 18-80 years). One tumor occurred in a cryptorchid testis, and one patient had had an orchidopexy several years prior to his presentation. There was no evidence of estrogen production by the tumor in any case. The tumors occurred equally often in each testis and were small (0.4-1.5 cm) in diameter, except for two tumors, which were 4.0 cm in diameter. All of them were centered in the testicular parenchyma and were well-demarcated, hard, yellow-white to tan nodules. They were characterized histologically by solid and hollow, simple and anastamosing tubules, large irregular aggregates, and thin cords of Sertoli cells in a prominent collagenous background. The tumor cells were of medium size and had pale cytoplasm, which sometimes contained large lipid vacuoles; the round nuclei varied from small and dark to large and vesicular. Follow-up information on five patients--including the only one whose tumor had malignant features histologically--showed no evidence of recurrence or metastasis 3-10 years (mean, 5.8 years) after orchidectomy alone. PMID- 1719831 TI - Solitary fibrous tumor of the upper respiratory tract. A report of six cases. AB - We report six cases of a neoplasm that arose in the upper respiratory tract and had a histological appearance indistinguishable from that of solitary fibrous tumor of the pleura (SFT, so-called fibrous mesothelioma). The patients were adults who presented with nasal obstruction. The lesions lacked the characteristic features of other recognized neoplasms that occur in this region. The tumor cells were immunoreactive for vimentin but not for keratin. The occurrence of SFT in this location further supports the argument that SFT is a tumor of mesenchymal and not mesothelial origin. None of the tumors in this series had the histologic features of malignancy described for SFT in other locations, and there was no aggressive behavior in limited follow-up. Until more cases of SFT in unusual locations have been studied, we recommend that the same criteria used for assessing aggressiveness in SFT of the pleura be applied to them. PMID- 1719832 TI - An immunohistochemical study of normal endometrial stroma and endometrial stromal neoplasms. Evidence for smooth muscle differentiation. AB - To determine the immunohistochemical staining profile of endometrial stromal cells, we analyzed a series of formalin-fixed, paraffin-embedded normal endometrial tissues, stromal nodules, and stromal sarcomas for immunoreactivity with a panel of eight antibodies. Normal proliferative-phase (five cases) and secretory-phase (five cases) endometrial stromal cells showed the following immunopositivity: vimentin 10 of 10, muscle-specific actin (MSA) 10 of 10, alpha smooth muscle actin (alpha sm) 10 of 10, desmin nine of 10, cytokeratin (AE1/AE3 and CAM 5.2) zero of 10, epithelial membrane antigen (EMA) zero of 10, and S-100 protein zero of 10. Antibodies to vimentin, MSA, and alpha sm stained a greater number of proliferative-phase stromal cells as compared with secretory-phase cells. Only rare stromal cells were immunoreactive for desmin, except for one case in which predecidual cells were diffusely positive. Both endometrial stromal nodules reacted with antibodies to MSA, alpha sm, and desmin, and one was vimentin positive. Each was unreactive for epithelial markers and S-100 protein. The 12 endometrial stromal sarcomas had the following immunopositivity: vimentin 11 of 12, MSA 10 of 12, alpha sm 10 of 12, desmin seven of 12, AE1/AE3 one of 12, CAM 5.2 two of 12, EMA zero of 12, and S-100 protein zero of 12. The antibodies to MSA and alpha sm usually stained a greater number of cells than did the desmin antibody. Three stromal sarcomas had sex cord-like areas, one of which exhibited focal CAM 5.2 positivity. These immunohistochemical findings for normal and neoplastic endometrial stromal cells indicate smooth muscle differentiation and are similar to those of smooth muscle neoplasms and myofibroblastic cells. PMID- 1719833 TI - Primary prostatic Wilms' tumor. AB - The first case of primary Wilms' tumor of the prostatic gland is described. It occurred in a 32-year-old man. Histologically, it consisted of a triphasic tumor; tubular and glomeruloid structures were identified, among prominent blastematous sheets and in an edematous stroma. No teratomatous components were encountered. We propose that this prostatic primary, nonteratomatous Wilms' tumor can arise from persistent, nephrogenic, blastematous rests in the prostate, in relation to the Wolffian duct system. PMID- 1719834 TI - Immunization of owl monkeys with a recombinant protein containing repeated epitopes of a Plasmodium falciparum glycophorin-binding protein. AB - A Plasmodium falciparum glycophorin binding protein (GBP-130) has been implicated in protective immunity to malaria. The gene for GBP-130 encodes a protein containing 11 tandemly repetitive 50 amino acid units. We report an immunization trial in Aotus monkeys using a recombinant DNA protein containing three of these 50 amino acid repeats. When administered with aluminum hydroxide, this antigen induced low levels of antibodies that reacted with the recombinant protein by ELISA and with parasite antigens in immunoblot and immunofluorescence assays, but not by immunoprecipitation. When administered with Freund's complete adjuvant, this antigen induced high levels of antibodies that reacted in ELISA, immunoblot, immunofluorescence, and immunoprecipitation assays. Serum from immunized monkeys did not inhibit parasite growth, and protection from intravenous challenge with P. falciparum-infected erythrocytes was not observed in any experimental group. These results suggest that the repetitive region of GBP-130 is not a useful vaccine candidate. PMID- 1719835 TI - Effects of N-acetyl-L-cysteine and ascorbic acid on mutagen-induced chromosomal sensitivity in patients with head and neck cancers. AB - The protective effect of N-acetyl-L-cysteine (NAC) and ascorbic acid on mutagen induced chromosomal breakage was determined using human lymphoblastoid cell lines as well as freshly cultured lymphocytes from patients with head and neck malignancies and healthy control subjects. Mutagen sensitivity was determined using the previously described bleomycin exposure assay. The toxicities of different concentrations of NAC and ascorbic acid, as well as both the preincubation and dose-dependent protective effects of these two agents, were analyzed. Both test drugs proved to be effective in diminishing mutagen-induced chromatid breakage in established lymphocyte cell lines. In freshly cultured lymphocytes, NAC given in doses ranging from 0.1 to 10 mmol/L decreased the number of mutagen-induced breaks per cell in a range from 23% to 73%, and ascorbic acid decreased chromosomal breakage by 21% to 58% in a dose range from 0.01 to 1 mmol/L. The results of this study demonstrate the protective effect mediated in vitro by both NAC and ascorbic acid against mutagen-induced chromosomal damage. A similar in vivo phenomenon may explain the differences in occurrence of head and neck cancer between populations with different dietary backgrounds. PMID- 1719836 TI - Role of surgical intervention in the management of intestinal metastases from malignant melanoma. AB - Malignant melanoma is the most common metastatic lesion of the intestine. Surgical consultation is often sought when bowel metastases become symptomatic. To determine the role of surgical intervention in such cases, a database of 6,000 melanoma patients was examined, and a subset of 102 patients with small intestinal or colonic metastases were identified premortem. Common presenting features included abdominal pain with or without acute symptoms (29% of patients), obstruction or intussusception (27%), and bleeding (26%). The presence of metastatic lesions was confirmed by surgical exploration in 80% of patients, endoscopic procedures in 11%, and percutaneous biopsy in 5%. Cure was achieved in 36 patients by resection, which resulted in the removal of all demonstrable disease. The subsequent mean length of survival in this group was 31 +/- 5.2 months. Forty-two patients underwent palliative enteric bypass or debulking procedures, and 24 patients received either chemotherapy alone or symptomatic treatment. The average length of survival in these latter groups was 9.6 +/- 15.9 and 9.6 +/- 3.6 months, respectively, both of which were significantly less than the duration of survival in the complete resection group (p less than 0.05). Small or large bowel resection for bleeding or obstruction and enteric bypass for obstruction provided symptomatic relief in 92% of patients thus treated. There was no operative mortality in the series. An aggressive search for resectable disease in patients with symptoms secondary to intestinal metastases from malignant melanoma should be performed. Surgical intervention may then allow the palliation of pain, obstruction, and bleeding. Survival can be significantly prolonged if it is possible to remove all demonstrable disease. PMID- 1719837 TI - [Serum trophoblastic beta 1-glycoprotein and alpha fetoprotein levels in physiological pregnancy]. AB - Measurements of maternal blood serum trophoblastic beta 1-glycoprotein and alpha fetoprotein, carried out over the course of normal pregnancy, have demonstrated a progressive increase of trophoblastic beta 1-glycoprotein up to the 36th week, though its level somewhat reduced during the 28th and 32nd weeks. After week 39 the level of this protein in maternal blood serum progressively lowered. alpha Fetoprotein level was increasing over the course of pregnancy as long as up to the 32nd-34th weeks, then lowering the rest of the term up to delivery. These data permit a conclusion that the time course of the afore-said specific protein concentrations in pregnancy sufficiently well reflects the processes of fetoplacental system establishment and functioning, this permitting the use of these protein measurements in monitoring the course of gestation. PMID- 1719838 TI - [The role of biologically active substances in the pathogenesis of dysfunctional uterine hemorrhage in the climacteric period]. AB - The activities of the blood kallikrein-kinin system components (plasma kallikrein, alpha 1-antitrypsin, alpha 2-macroglobulin), and gonadotropin levels in various phases of the menstrual cycle were measured in 35 women ob various age groups. The same parameters were investigated in 28 women with dysfunctional uterine bleedings of the climacteric period. Phasic changes in the blood kallikrein-kinin system components, closely related to the hormonal status, were revealed over the course of the onto-genesis. Climacteric dysfunctional uterine bleedings were found associated with an elevated activity of the plasma kallikrein, parallelled by a relative insufficiency of the blood plasma inhibitor potential. A relationship between the activities of the blood kallikrein-kinin system components and endometrial function has been revealed. These findings necessitate search for approaches to pharmacologic regulation of the blood kallikrein-kinin system activity and to pathogenetic therapy of the climacteric dysfunctional uterine bleedings. PMID- 1719839 TI - Carcinogenesis in porokeratosis. Evidence for a role relating to chronic growth activation of keratinocytes. AB - Porokeratoses are known to give rise to squamous and basal cell carcinomas. In this study, we examined 15 lesions of porokeratosis immunohistochemically for evidence of aberrant keratinization using several markers of keratinocyte (KC) maturation and differentiation, including involucrin, filaggrin, cytokeratins, and the growth activation marker psi-3. The staining patterns obtained were compared with several non-premalignant parakeratotic skin lesions including psoriasis, pityriasis rosea, pityriasis rubra pilaris, irritated seborrheic keratosis, atopic dermatitis, seborrheic dermatitis, and verruca vulgaris. The centers of porokeratoses stained in a pattern identical to that observed in other premalignant keratinocytic lesions including actinic keratoses, recessive dystrophic epidermolysis bullosa, and nonhealing wounds. KCs beneath the cornoid lamella (CL) stained in a pattern similar to that observed in squamous cell carcinomas. KCs peripheral to the CL in the epidermis showed a normal staining pattern. The control non-premalignant parakeratotic lesions displayed a variety of staining patterns, but none showed a pattern identical to that observed in porokeratosis. The failure of KCs in porokeratoses to mature and differentiate normally may be related to the increased incidence of carcinomas associated with these lesions. PMID- 1719840 TI - Immunohistochemical study of human eccrine sweat ducts with anti-keratin antibodies. Presence of a layer between luminal and peripheral cell layers. AB - It is known that human eccrine sweat ducts are composed of luminal cells and peripheral cells. In this study, the immunohistochemical staining properties of human eccrine sweat ducts were investigated using seven different anti-keratin antibodies by light microscopy. Anti-keratin antibody MA904, which reacts with 68 kDa keratin peptide specifically stained an intermediate cell layer between the luminal cell layer and the peripheral cell layer in the ducts. Anti-keratin antibody CK8,60 stained both the luminal cell layer and the intermediate cell layer. Anti-keratin antibody MA903 stained all of the layers. Anti-keratin antibodies CK4.26, PKK1, and MAK-6 weakly to faintly stained the luminal cells. Anti-keratin antibody PKK3 stained no cells in the ducts. These results suggest that each cell layer has its own characteristic staining pattern with anti keratin antibodies. Moreover, the presence of an intermediate cell layer was confirmed by immunoelectron microscopy using anti-keratin antibody MA904. PMID- 1719842 TI - Isoelectric focusing electrophoresis of lignin. AB - Isoelectric focusing is introduced as a technique for the analysis of macromolecular lignin. The analysis is performed in a pH gradient from 3.5 to 10. Separated lignin fragments are visualized under uv light or by silver staining. The method can be used to distinguish between differently processed lignin preparations and to identify their components. Even the slight modification resulting from attack by ligninolytic enzymes could be detected. PMID- 1719841 TI - [Organ blood supply and oxygenation during limited isovolemic hemodilution with 6% HES 200/0.62 and 6% Dextran 70]. AB - Oxygen delivery (systemic oxygen transport) is directly dependent upon cardiac output and oxygen content of the blood. The rheology of blood, however, represents a co-determinant of oxygen delivery. It has recently been argued that the increase in plasma viscosity occurring under hemodilution with dextran could be detrimental to blood flow and, hence, tissue oxygenation. METHODS. Twelve splenectomized beagles (12.5 +/- 1.7 kg) were anesthetized and randomly assigned to hemodilution to 20 vol% hematocrit (hct) with 6% hydroxyethyl starch (HES 200/0.62) or 6% dextran-70 (DX-70). The effects of hemodilution (HD) upon macrohemodynamics, plasma and blood volumes (131I dog albumin distribution), organ blood flow (radioactive-labelled microspheres, phi 15 microns), and local tissue oxygenation (pO2 multiwire surface electrode) were evaluated with special reference to any actual plasma viscosity. RESULTS. Moderate HD with either solution resulted in equivalent changes in macrohemodynamics and plasma and blood volumes. Tissue oxygen extraction increased (p less than 0.05) due to a small rise (maximally 28%) in cardiac output. HD with either solution resulted in an increase in plasma viscosity that was more pronounced in the DX-70 group (1.45 +/ 0.07 mPa.s) as compared to HES-diluted animals (1.16 +/- 0.04 mPa.s). Blood flow increased (p less than 0.01) in all organs after HD independently of the diluent. Both higher pO2 values on the surface of liver and skeletal muscle (p less than 0.01) as well as a shift of the pO2 histograms to the right indicated a more homogeneous tissue perfusion during HD. CONCLUSIONS. In normotensive animals without peripheral arterial occlusive disease undergoing moderate hemodilution, organ blood flow was independent of plasma viscosity. Systemic oxygen transport was not affected by plasma viscosity changes, but is primarily determined by systemic hct. Local surface tissue oxygenation on skeletal muscle and liver was not impaired, but rather improved during hemodilution despite raised plasma viscosity. Of the rheological factors influencing oxygen delivery, hct thus plays the predominant role while plasma viscosity is of minor importance. PMID- 1719843 TI - Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans. AB - A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels. PMID- 1719844 TI - Gel electrophoresis in the presence of soluble, aqueous polymers: horizontal sodium dodecyl sulfate-polyacrylamide gels. AB - Because little is known about the use of aqueous polymers in polyacrylamide gel electrophoresis, we undertook a feasibility study that enables the discontinuous Laemmli-formulated system of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to be performed in a horizontal format by the addition of large sized aqueous polymers (i.e., dextrans and methylcelluloses). We studied four parameters: the cross-linking agent (bisacrylamide vs AcrylAide) and the polymer concentration, nature, and size. Three concentrations of each polymer were used. The best differentiation between the standard markers and the sharpest bands were obtained using concentrations of 2.5 and 0.06% (w/v) for Dextran T-500 and methylcellulose 4000, respectively. There was no predictable pattern to the variation in the plots of log Mr vs Rf caused by varying the concentration and length of the dextrans; however, the methylcellulose patterns suggest that gel viscosity is important. The results suggest that the combination of 0.06% methylcellulose 4000 polymers with bisacrylamide is a convenient and inexpensive means of performing flatbed Laemmli SDS-polyacrylamide gel electrophoresis. PMID- 1719845 TI - Isolation of functional RNA from plant tissues rich in phenolic compounds. AB - A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation. PMID- 1719846 TI - Re-examination of the topographical distribution of motoneurons innervating the digastric muscle in the rabbit and guinea pig. AB - The topographical distribution of motoneurons innervating the digastric muscle in the rabbit and guinea pig was re-examined by the retrograde tracing method of HRP (horseradish peroxidase). Motoneurons innervating the anterior belly of the digastric muscle of the rabbit and guinea pig constituted a longitudinal cell column in the ventromedial part of the motor nucleus of the trigeminal nerve. Motoneurons innervating the posterior belly of the digastric muscle were localized in the accessory facial nucleus. No motoneurons supplying the digastric muscle were found within the main facial nucleus. PMID- 1719847 TI - Hypertonic/hyperoncotic fluid resuscitation after hemorrhagic shock in dogs. AB - We compared canine systemic and cerebral hemodynamics after resuscitation from hemorrhagic shock with 4 mL/kg (a volume approximating 12% of shed blood volume) of 7.2% saline (HS; 1233 mEq/L sodium), 20% hydroxyethyl starch (HES) in 0.8% saline, or a combination fluid consisting of 20% hydroxyethyl starch in 7.2% saline (HS/HES). Eighteen endotracheally intubated mongrel dogs (18-24 kg) were ventilated to maintain normocarbia with 0.5% halothane in nitrous oxide and oxygen (60:40). After a 30-min period of hemorrhagic shock (mean arterial blood pressure = 40 mm Hg), extending from time T0 to T30, animals received one of three randomly assigned intravenous resuscitation fluids: HS, HES, or HS/HES. Data were collected at baseline, at the beginning and end of the shock period (T0 and T30), immediately after fluid infusion (T35), and at 60-min intervals for 2 h (T95, T155). After resuscitation, mean arterial blood pressure and cardiac output increased similarly in all groups, but failed to return to baseline. Intracranial pressure decreased during shock and increased slightly, immediately after resuscitation in all groups. During shock, cerebral blood flow (cerebral venous outflow method) declined in all groups. After resuscitation, cerebral blood flow increased, exceeding baseline in the HS and HS/HES groups but remaining low in the HES group (P less than 0.05 HS vs HES at T35). We conclude that small-volume resuscitation (4 mL/kg) with HS, HS/HES, or HES does not effectively restore or sustain systemic hemodynamics in hemorrhaged dogs. In dogs without intracranial pathology, the effects on cerebral hemodynamics are also comparable, except for transiently greater cerebral blood flow in the HS group in comparison with the HES group. PMID- 1719848 TI - The effects of halothane on ventricular tachycardia in intact dogs. AB - Halothane has either proarrhythmic or antiarrhythmic effects in a variety of clinical circumstances. This investigation tested the hypothesis that halothane would display different effects on ventricular tachycardia (VT) produced by different electrophysiologic mechanisms in intact dogs. Four models of VT produced by abnormal automaticity, reentry, delayed-afterdepolarization-induced triggered activity, and early-afterdepolarization-induced triggered automaticity (groups 1-4, respectively) were studied. In groups 1 and 2, the left anterior descending coronary artery (LAD) was ligated. In group 1 (n = 5), 24 h after LAD ligation and infarction, all dogs demonstrated incessant VT with 94.7 +/- 2.3% of beats of ventricular origin. This ectopy presumably was due to abnormal automaticity. Halothane reduced the frequency of ventricular ectopy until at 2% halothane only 34.8 +/- 15% of beats were of ventricular origin. One week after LAD ligation, programmed stimulation produced nonstimulated extrasystoles of presumably reentrant origin in six dogs. In three, halothane 1% abolished extrasystoles while increasing the ventricular refractory period by 23 +/- 3.8% (P less than 0.05). In the three other dogs, halothane had no effect (two dogs) or worsened the severity of VT (one dog), while the refractory period increased by 7.7% (P greater than 0.05). In group 3 dogs, ouabain was infused until VT secondary to triggered activity occurred. Halothane restored sinus rhythm in 4 of 5 dogs. Overall the percentage of sinus beats increased from 11.1 +/- 2.8 to 97.4 +/- 2.6% when halothane 2% was added during ouabain toxicity. Cesium chloride infusion increased the QT interval and produced complex VT in 5 dogs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719849 TI - [Joubert syndrome: report of a case of electrocardiographic anomalies]. PMID- 1719850 TI - [Guillain-Barre syndrome: response to high dose intravenous gammaglobulin therapy in a 12-year old girl]. PMID- 1719851 TI - Pulmonary physiology and pharmacology of neuropeptides. PMID- 1719853 TI - hrIL-8-induced hyperresponsiveness in guinea pig perfused lungs. PMID- 1719852 TI - Pharmacological studies of tachykinin receptors mediating contraction of isolated airway smooth muscle. PMID- 1719854 TI - The role of eosinophils in the pathophysiology of asthma. AB - From current information, a number of conclusions can be drawn. Antigen activation of the allergic reaction in the airways is associated with an immediate rise in mast cell derived mediators, including histamine and tryptase. Associated with antigen activation of the allergic reaction is recruitment of eosinophils to the airways. This can best be seen in the airway lavage 48 hours after challenge with antigen. An increased presence of eosinophils suggests that they are an important contributor to the late allergic reaction and may be one of the major constituents in the development of bronchial inflammation. Although many factors participate in the late allergic inflammatory response, eosinophil derived proteins are known to cause airway injury. Regulation of eosinophils in this process is not clearly established; however, our findings of increased IL-5 in relationship to the presence of eosinophils and their granular proteins suggests that this cytokine may be an important modulator of eosinophil function and activation following allergen challenge. However, much remains unknown in understanding bronchial inflammation and the eosinophil's role in the process. In conclusion, the eosinophil is a major cellular participant in late phase allergic airway disease. Its presence and known functions suggest that the eosinophil is a significant cellular factor in the development of allergic airways disease in asthma. Further advances in this area will follow continued studies, particularly those which involve biopsy and correlation with airway physiology. PMID- 1719855 TI - Substance P and Related Peptides: Cellular and Molecular Physiology. July 18-21, 1990, Worcester, Massachusetts. Proceedings. PMID- 1719856 TI - Developmental and hormonal regulation of the sex difference in preprotachykinin gene expression in rat anterior pituitaries. PMID- 1719857 TI - Regulation of substance P expression in sympathetic neurons. PMID- 1719858 TI - Substance P induced inhibition of potassium channels via a pertussis toxin insensitive G protein. PMID- 1719859 TI - Pharmacological and biochemical evidence for multiple types of tachykinin NK2 receptors. PMID- 1719860 TI - Strategies to detect heterologously expressed tachykinin receptors in Xenopus Oocytes. PMID- 1719861 TI - Concepts in characterization of tachykinin receptors. PMID- 1719862 TI - Myoinositol uptake in rat parotid gland. A selective bioassay for NK1 receptors. PMID- 1719864 TI - Effects of lymphokines on substance P in injured ganglia of the peripheral nervous system. PMID- 1719863 TI - Neurokinin agonists and antagonists. PMID- 1719865 TI - Tachykinins and related peptides in the substantia nigra and neostriatum. PMID- 1719866 TI - On the role of substance P, galanin, vasoactive intestinal peptide, and calcitonin gene-related peptide in mediation of spinal reflex excitability in rats with intact and sectioned peripheral nerves. PMID- 1719867 TI - Tachykinin-evoked release of neurotransmitters from isolated spinal cord of the newborn rat. PMID- 1719868 TI - Substance P actions on sensory neurons. AB - SP is involved in sensory transmission as a mediator of excitation at target tissues following its release from the terminals of certain primary afferent fibers. In view of the pronounced SP effects on the perikarya of sensory neurons, it seems reasonable to suggest that its actions may extend to autoreceptors and to the enhancement of the excitabilities of sensory neurons that do not synthesize the peptide. This hypothesis would be strengthened by the demonstration of nonsynaptic release of SP within sensory ganglia, which would provide another site for interaction of SP with the various component cells of the sensory system. PMID- 1719869 TI - Effects of substance P on secretion of catecholamines from populations of bovine chromaffin cells and on calcium transients in individual cells. PMID- 1719870 TI - Substance P interactions with the nicotinic response. PMID- 1719871 TI - Substance P and the inflammatory and immune response. AB - These findings suggest that SP may have proinflammatory actions in both the peripheral tissue and the central nervous system after tissue injury. Although the possibility that the same neuropeptide could have actions in both the brain and the peripheral tissues is certainly not without precedent, there is a key difference in the source of the ligand in these tissues. Unlike peripheral tissues such as the gastrointestinal tract or skin, where there is a dense innervation by SP-containing dorsal root ganglion neurons, the brain lacks such a sensory innervation. This important difference raises the question as to the possible origin of the SP that could occupy the SP receptors expressed by the CNS glia after neuronal injury. Whereas the answer to this question is currently unknown, an important clue may be the findings that circulating leukocytes have been reported to synthesize neuropeptides such as ACTH, opiates, and SP. To begin to fully understand the role that SP may play in coordinating the inflammatory and immune response to tissue injury, we must first understand where SP fits into the cascade of events that occur after tissue injury, what events lead to nociceptor sensitization (which may lead to an increase in SP release), and what regulates SP receptor expression (which may be involved in the direction of leukocytes to the site of injury, plasma extravasation, or the proliferation/hypertrophy of reactive astrocytes). Although this may seem like a daunting task, several recent advances including the cloning of the three mammalian tachykinin receptors and the introduction of highly potent and specific SP receptor antagonists should make this a highly fruitful field of investigation. PMID- 1719872 TI - The tachykinin neuroimmune connection in inflammatory pain. PMID- 1719873 TI - Substance P afferents regulate ACTH-corticosterone release. PMID- 1719874 TI - Substance P and related peptides associated with the afferent and autonomic innervation of the uterus. PMID- 1719875 TI - Neuroanatomical relationships of substance P and sex steroid hormone-sensitive neurons involved in sexual behavior. PMID- 1719876 TI - Effects of various tachykinins on pituitary LH secretion, feeding, and sexual behavior in the rat. AB - Our investigations of the four tachykinines tested have shown that NPK characteristically evoked a spectrum of biological effects in male and female rats. NPK suppressed pituitary LH release by inhibiting the release of hypothalamic LHRH, presumably by activation of NK-2 tachykinin receptor subtypes. However, NPK may also act at the level of gonadotrophs to stimulate LH release in male rats. Central injection of NPK rapidly disrupted copulatory behavior in sexually active male rats. NPK also suppressed feeding, but, in this case, peripheral injections were more effective than central injections. Taken together, these observations strongly imply that NPK may be an inhibitory messenger molecule in the hypothalamic control of reproduction, sexual, and feeding behaviors. PMID- 1719877 TI - Significance of substance P- and enkephalin-peptide systems in the male genital tract. AB - The plexi of the male reproductive tract have components of both the autonomic and sensory nervous systems. Rat epididymis was found to be a rich source of substance P. Substance P levels in the epididymis were higher by about 2.8 and 19.3 times than those in the prostate and seminal vesicles, respectively. Seminal vesicles were found to be a rich source of enkephalins. They had about 2.9 and 2.6 times higher leucine enkephalin levels than epididymis and prostate, respectively. Human seminal plasma contained about 47 times higher levels of leucine enkephalin than substance P. Using the split ejaculate technique, it has been demonstrated that early fractions of the human ejaculate contain fluids from prostate (and possibly epididymis), whereas later fractions represent seminal vesicle secretions. A low exogenous concentration of substance P (400 nM) increased sperm motility, whereas leucine enkephalin (100 microM) depressed it. Substance P (1-10 micrograms/mL) and muscarinic agonists enhanced the adrenergic transmission of the rat vas deferens to electrical stimulation. Leucine enkephalin (1-10 micrograms/mL) depressed adrenergic transmission and antagonized the effects of substance P and muscarinic agonists. These studies suggest that substance P-like tachykinins may play a role in sperm maturation, in expulsion of fluid from the epididymis, and in initiation of motility, whereas leucine enkephalin-like peptides may contribute to the orgasmic experience and detumescence. PMID- 1719878 TI - 125I-substance P binding sites in rat spinal cord in a chronic constriction injury and following dorsal rhizotomy. PMID- 1719879 TI - NK-1 receptors and vascular permeability in rat airways. PMID- 1719880 TI - Activation of primary afferents in the rabbit ear by noxious heat. PMID- 1719881 TI - Substance P is distributed between somatotrophs and thyrotrophs in a sexually dimorphic manner in rat. PMID- 1719883 TI - Substance P-containing and calcitonin gene-related peptide-containing neurons in the human trigeminal ganglion. Immunohistochemical detection, morphometric characterization, and coexistence of peptides. PMID- 1719882 TI - Multiple neurokinin receptor subtypes are present in the colon of the cat and rat. PMID- 1719884 TI - An examination of the proposed functional interaction of substance P and 5-HT in the guinea pig ileum. PMID- 1719885 TI - Modulation of endogenous levels of substance P in rat CNS tissues by N,N diethyldithiocarbamate. PMID- 1719886 TI - Evidence supporting a mitogenic role for substance P in amphibian limb regeneration. Involvement of the inositol phospholipid signaling pathway. PMID- 1719887 TI - Evidence for two types of tachykinin receptors on cholinergic neurons of the guinea pig ileum myenteric plexus. PMID- 1719888 TI - Novel ligands confirm NK-1 receptor-mediated modulation of neurotransmission in the guinea pig vas deferens preparation. PMID- 1719889 TI - Effects of substance P and SP(1-7) on dopamine release from rat substantia nigra. PMID- 1719890 TI - Characterization of substance P receptors in human astrocytoma cells. PMID- 1719891 TI - The role of substance P in regulation of blood pressure and hypertension. PMID- 1719892 TI - Modulation of sialyl-glycoconjugate secretion in cultures of immortalized lymphocytes by treatment with substance P and interleukin-2. PMID- 1719893 TI - Effects of serotonin and opioid agonists on tachyphylaxis to SP-induced vasodilatation. PMID- 1719894 TI - Allosteric interaction of heparin with NK-1 receptors in rat striatal membranes. PMID- 1719895 TI - Substance P is present in cholinergic paravertebral sympathetic neurons innervating exocrine sweat glands. PMID- 1719896 TI - Neurokinin innervation of the rat median raphe nucleus does not originate in the brain stem. PMID- 1719898 TI - The glycine-extended SP precursor, SP-G, is normally expressed in SP-containing neurons. PMID- 1719897 TI - Substance P and somatostatin levels in rheumatoid arthritis, osteoarthritis, and psoriatic arthritis synovial fluid. PMID- 1719899 TI - Neurokinin receptor-mediated regulation of [Ca]i and Ca-sensitive ion channels in mammalian colonic muscle. AB - In conclusion, these findings suggest the following: (1) NK receptor activation results in [Ca2+]i oscillations; (2) the receptor-mediated [Ca2+]i increase is partially due to influx of Ca2+ through L-type Ca2+ channels and partially to release from intracellular stores; (3) the receptor-mediated depolarization results from activation of a Cl- channel at the cell resting potential; (4) NK receptor-mediated release of [Ca2+]i may play a role in Cl- channel activation; (5) there is no evidence for multiple NK receptor types involved in cell activation. PMID- 1719900 TI - The effect of substance P on guanine nucleotide--activated striatal adenylate cyclase. PMID- 1719901 TI - SP immunoreactivity in the dental pulp and periodontium during tooth movement. PMID- 1719902 TI - Pharmacological properties of the tachykinin receptor subtype in the endothelial cell and vasodilation. PMID- 1719903 TI - Endogenous opioids and ruthenium red inhibit the flare reaction in the pig skin by different mechanisms. PMID- 1719904 TI - Endogenous 5-hydroxytryptamine modulates the release of tachykinins and calcitonin gene-related peptide from the rat spinal cord via 5-HT3 receptors. PMID- 1719905 TI - Neurokinin-1 receptors in the human eye. Characterization and autoradiographic distribution. PMID- 1719906 TI - Substance P and antagonists. Surface activity, conformations, and lipid binding. PMID- 1719907 TI - Immunochemical characterization of SP processing intermediates in sensory ganglia. PMID- 1719908 TI - Tachykinin receptor subtype. Central cardiovascular regulation of tachykinin peptides. PMID- 1719909 TI - Functional role of substance P for respiratory control during development. PMID- 1719911 TI - Effect of acupuncture on release of substance P. PMID- 1719910 TI - Serotonin innervation affects SP biosynthesis in rat neostriatum. PMID- 1719912 TI - Molecular characterization of the three tachykinin receptors. PMID- 1719913 TI - Molecular and genetic characterization, functional expression, and mRNA expression patterns of a rat substance P receptor. PMID- 1719914 TI - Role of inositol phosphates in the actions of substance P on NK1 receptors in exocrine gland cells. PMID- 1719915 TI - Mitochondrial DNA polymorphisms among Hindus: a comparison with the Tharus of Nepal. AB - The polymorphisms of mitochondrial DNA for the restriction enzymes HpaI, BamHI, HaeII, MspI, AvaII and HinecII were studied in a sample of 79 Hindus, 45 from New Delhi (India) and 34 from Terai (Nepal), both to characterize another Caucasian population and to investigate some possible Hindu component in the genetic structure of the Tharus, a Nepalese population, the anthropological position of which is still disputed. 1. A new BamHI polymorphism was detected: about 5% of the Hindu mtDNAs have lost the site at 14258 bp and lack any BamHI site. Once again a BamHI polymorphism was found in a Caucasian population. 2. New site mutations were found to yield morphs previously described (MspI-7, AvaII-18). 3. Variant morphs for two different enzymes were found due to a shared mutation (morphs BamHI-0/AvaII-30 and morphs MspI-7Hindu/AvaII-18Hindu). 4. Comparison between Hindu and Tharu data does not show any evidence of a specific Indian component in the Tharu genetic structure and allows us to conclude that Tharus are clearly differentiated from modern Hindus. PMID- 1719916 TI - Segmental spinal muscular atrophy and dermatological findings in a patient with chromosome 18q deletion. AB - We have evaluated a young woman with segmental spinal muscular atrophy, who has a deletion of a portion of the long arm of chromosome 18. She also has vitiligo and lichen sclerosis et atrophicus. She has neither the facial dysmorphism nor the mental deficit usually associated with the 18q- syndrome. Magnetic resonance imaging scan of her brain demonstrates high signal intensity consistent with abnormal myelination. Southern blot analysis of her DNA demonstrates that the deletion includes the gene for human myelin basic protein. Neither spinal muscular atrophy nor this patient's skin manifestations have been previously reported in association with 18q-. PMID- 1719917 TI - Hepatic dysfunction in paediatric typhoidal salmonellosis. AB - Although hepatic dysfunction has been described among adults with typhoid, there are few reports of significant hepatic functional impairment in children with typhoid. Of 435 children with culture-proven typhoid seen at the Aga Khan University Hospital, hepatomegaly was noted in 125 (29%) and isolated right hypochondrial tenderness in three (7%). Liver function tests performed on 156 of these children were normal in 121 (78%) and showed significant hepatic dysfunction in 35 (22%). Patients with significant hepatic dysfunction were more toxic (p less than 0.001) at presentation and had higher mortality (p less than 0.002) than those with normal liver function. Two children presented with a picture of fulminant hepatic failure with fatal outcome. PMID- 1719918 TI - Fulminant hepatitis in children: report of 12 cases. AB - Twelve cases of childhood fulminant hepatitis seen over a 4-year period are described. Six had hepatitis A, five hepatitis B and one non-A non-B hepatitis. Encephalopathy, the cardinal feature of fulminant hepatitis, was usually evident within 2 weeks of onset of illness, and the median duration of illness in fatal cases was 19 days. Deep jaundice, prolongation of the prothrombin time and raised serum ammonia were invariable. Eight children died and the four survivors were critically ill before recovering. Acute viral hepatitis is generally a benign illness in childhood. Although infrequently recorded, fulminant hepatitis may, however, ensue and is associated with a high mortality. PMID- 1719919 TI - Acute renal failure in non-fulminant hepatitis A infection. AB - A 7-year-old boy developed acute renal failure during the icteric phase of non fulminant hepatitis A infection. He needed peritoneal dialysis for 3 days, which was followed by a rapid recovery in renal function which was normal when he was discharged 4 weeks later. PMID- 1719920 TI - Scorpion sting in children in the Jerusalem area: a review of 54 cases. AB - Fifty-four children from the Jerusalem area were studied prospectively following scorpion envenoming. Their ages ranged from 11 months to 10 years. Severe symptoms (convulsions, brain oedema, shock, respiratory distress and myocarditis) were encountered in 19. Respiratory distress was the main feature in 17 of the children, in two cases owing to pulmonary oedema and in a third because of adult respiratory distress syndrome and myocarditis; mechanical ventilation was required in three cases. The severity of the symptoms and signs was not related to sex, age, weight, interval between scorpion sting and admission or to the type of offending scorpion; it was most likely dependent upon the susceptibility of the individual and/or the dose of venom injected by the scorpion. Intravenous antivenom quickly reversed the symptoms, and no side-effects were seen in the patients studied. The two patients who died had not received the antivenom intravenously. We recommend that specific antivenom should be given intravenously in all children who show significant symptoms. Furthermore, a longer period of observation is necessary following scorpion sting in this age group. PMID- 1719921 TI - Acute rheumatic fever during childhood in Saudi Arabia. AB - During a 5-year period ending in December 1989, 73 episodes of acute rheumatic fever in 67 children aged 4-14 years were prospectively studied to ascertain the clinical profile of the disease in initial attacks and recurrences, and to compare the findings with those from other countries. Among 51 children with a first episode of acute rheumatic fever, 76% had arthritis and 43% had carditis. In 22 children with recurrences, arthritis was present in 45% and carditis in 91%. Carditis was more severe among the cases with recurrences. Mitral insufficiency was the most common valvular lesion, but no case of mitral stenosis was detected. Chorea, subcutaneous nodules, and Erythema marginatum were relatively rare. The demographic, clinical and laboratory findings of this study resemble those from Western countries, in contrast with data from tropical countries. Efforts aimed at prompt recognition and adequate treatment of streptococcal pharyngitis and maintenance of anti-streptococcal chemoprophylaxis would be rewarding in reducing the incidence of this disease and its sequelae. PMID- 1719922 TI - Infective endocarditis in children in the Guinea savannah of Nigeria. AB - Thirty-two children with 33 episodes of infective endocarditis were admitted into the paediatric unit of Ahmadu Bello University Teaching Hospital, Zaria during an 8-year period (January 1982-December 1989). Thirty (94%) had underlying heart disease. Rheumatic heart disease was the pre-existing anomaly in 21 (66%) while congenital cardiac anomalies were detected in nine (28%). Cardiac failure, changing murmur or persisting fever drew attention to the disease. Bacterial isolation was achieved in 19 patients (58%), staphylococci in 11, and salmonella was found in three children. Others included Acinetobacter spp. in two patients, one of whom had a mixed infection involving alpha haemolytic streptococcus whereas three children had Klebsiella, pseudomonas or alpha haemolytic Streptococcus, respectively. Only six patients (18%) recovered. Abscondment rates were high (28%) and overall hospital mortality was 47%. Intractable cardiac failure and neurological complications were the most important events heralding death. There is a need for increased awareness and improved facilities for prompt and effective treatment. PMID- 1719923 TI - Cerebrospinal fluid investigations in tuberculous meningitis. AB - The results of conventional cerebrospinal fluid (CSF) investigations (CSF cell count, protein and glucose concentrations and Pandy's test for CSF globulin) obtained on admission and sequentially from weekly follow-up lumbar punctures for 4 weeks were evaluated in 99 children (median age 28 months) with stage II (50 children) and stage III (49 children) tuberculous meningitis. On admission, six children (6%) had a CSF cell count greater than 500 x 10(6)/l and nine (9%) a polymorphonuclear predominance. A CSF protein less than 0.8 g/l was found in 17 children (18%) of 97 in whom CSF protein was evaluated. Globulin was either absent or present as a trace only in 26 children (27%). CSF glucose was less than 2.2 mmol/l in 58 cases (60%) and less than 2.5 mmol/l in 67 (69%). In 63 children weekly CSF specimens obtained for the 1st 4 weeks of therapy showed an uninterrupted decline in cell count in 23 (37%), a fluctuating downward trend in 27 (43%) and a fluctuating upward trend in 13 (21%). Sequential CSF protein values in 57 children showed an uninterrupted rise in three (5%), a fluctuating upward course in 19 (33%), an uninterrupted downward trend in seven (12%), and a fluctuating downward course in 28 (49%). Of the 61 children in whom sequential CSF glucose concentrations were available, 11 (18%) experienced fluctuating concentrations, values falling to less than 2.2 mmol/l after being greater than 2.2 mmol/l on admission or after having risen to greater than 2.2 mmol/l.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1719924 TI - Effect of pneumococcal vaccine on morbidity from acute lower respiratory tract infections in Papua New Guinean children. AB - The effect of a 14-valent pneumococcal polysaccharide vaccine on morbidity from acute lower respiratory tract infection (ALRI) was determined in a randomized double-blind controlled trial in children under the age of 5 years living in the Paupa New Guinea highlands. The vaccine did not protect against mild ALRI. Vaccine efficacy in the study as a whole was 28% for moderate/severe ALRI, which was not statistically significant though consistent with the significant effect on mortality. Children entered the trial in five separate cohorts 4 months apart. The incidence of disease and vaccine efficacy varied between cohorts and with age. There was no vaccine effect in the first cohort, which had a much higher proportion of older children. The effect was greatest and statistically significant among those groups encountering an epidemic of moderate and severe ALRI at a young age. It was therefore in children at the most vulnerable age in times of greatest incidence of disease that the vaccine had its most potent effect. It is postulated that the efficacy of pneumococcal vaccine is dependent on the predominant invading serotypes in the period after vaccination, the age at which children develop immunocompetence to specific vaccine serotypes, and the levels of naturally acquired specific immunity already present in children at the time of vaccination, and that for all of these conditions there will be a cohort effect. PMID- 1719925 TI - Increased incidence of hyperbilirubinaemia in 'unchallenged' glucose-6-phosphate dehydrogenase deficiency in term Saudi newborns. AB - The incidence of severe hyperbilirubinaemia was significantly higher among the G6PD-deficient Saudi infants born at term than in non-deficient babies (34% vs 9%) (p less than 0.005). No apparent offending factors were detected in either the babies or their mothers. All babies who developed hyperbilirubinaemia did so during the 1st week of life. The highest mean bilirubin level for all jaundiced G6PD-deficient babies was recorded on the 4th postnatal day. Although the incidence of severe hyperbilirubinaemia among our neonates was relatively high, only two of them (7%), a boy and a girl, required exchange transfusions. Five of 29 jaundiced babies with G6PD deficiency were readmitted after discharge because of significant jaundice. One required exchange transfusion. Since G6PD deficiency seems to be a relatively common cause of neonatal jaundice in Saudi newborns, early detection of this enzymopathy by cord blood screening is justified to avoid morbidity and deaths. PMID- 1719926 TI - Congenital malaria with chloroquine resistance. AB - A preterm infant with possible congenital clinical malaria is described. The infant developed persistent pyrexia, hyperbilirubinaemia, anaemia, increasing gastric residuals and hepatosplenomegaly from the 7th day of life. Thick and thin smears of the infant's blood were heavily loaded with various asexual stages of Plasmodium falciparum. The parasite exhibited R1 resistance. There was no satisfactory response to chloroquine, but response to intravenous quinine therapy was achieved on day 15. The initial 6-month follow-up period was uneventful. The mother had apparently had chloroquine-resistant malaria which responded to sulfadoxine-pyrimethamine (Fansidar). PMID- 1719927 TI - Invasive candidiasis in infants: experience from Saudi Arabia. AB - There are few reports about invasive candidiasis in infants in the tropics in general and in the Kingdom of Saudi Arabia in particular. Two Saudi infants with invasive candidiasis are reported and their clinical features and response to treatment are compared with that found in the paediatric literature, mainly from the developed world. Prematurity, low birthweight, invasive procedures, long hospital stay and prolonged use of broad-spectrum antibiotics were found to be predisposing factors in the two patients, and we believe that a lack of awareness of these by the referring physicians led to a delay in diagnosis. The need for greater awareness and vigilance, and the dangers inherent in overlooking isolates of candida from clinical materials are emphasized. PMID- 1719929 TI - Dietary manipulation for treating infants with prolonged dehydrating diarrhoea: a comparison of four different formulae. AB - Dietary manipulation is often the first step in the treatment of infants with persistent acute dehydrating diarrhoea. This usually entails elimination of lactose, but other disaccharides or whole protein may be causing the disease. Serial elimination of these takes time and it may be preferable to use a whole protein and disaccharide-free formula as the first feed change. This study assessed the effect on stool weight of a change from cow's milk formula feeds to one of four different formulae in infants with severe diarrhoea persisting after 3 days in hospital. Two feeds were lactose-free soy formulae containing sucrose, one was disaccharide-free soy formula and one a disaccharide-free protein hydrolysate. Regardless of which feed the infants received, diarrhoea resolved in approximately 50% following the change in diet. Comparing those who got better with those who did not, the former were generally better nourished and had an initial lower stool output, but it was impossible to predict on clinical grounds which individual would respond to the removal of cow's milk. The results suggest that elimination of lactose in infants with persistent severe diarrhoea will benefit a significant number in the early stage of the disease. As there is no additional benefit from eliminating sucrose or whole protein at this stage, the cheapest available lactose-free formula should be used initially. PMID- 1719928 TI - Cryptosporidiosis in children in Gaza. AB - A prospective survey of children admitted with gastro-enteritis to the Nasser Children's Hospital, Gaza revealed that 19% were excreting cryptosporidium, a significantly (p less than 0.001) greater percentage than that (7%) observed in children admitted for other reasons. Detection of cryptosporidium decreased when the change from hot dry to colder wetter weather occurred. Although Salmonella spp were isolated more frequently than cryptosporidium in children with diarrhoea (20% of cases), there was no statistically significant association between excretion of Salmonella spp and gastro-enteritis. A follow-up study of a cohort of the children with cryptosporidiosis indicated that over three-quarters were dehydrated and all were below their expected weight-for-age. There was a statistically significant association between cryptosporidium gastro-enteritis and evidence of respiratory tract infection. PMID- 1719930 TI - Late-onset hypolactasia in Hong Kong school children. AB - Three hundred and twenty Chinese school children aged between 6 and 19 years from six schools in Hong Kong were tested for their lactose digestion status. After an overnight fast, the children were challenged with cow's milk, 5 ml/kg bodyweight (i.e. lactose approximately 0.25 g/kg). Malabsorption was assessed by measuring hydrogen concentration from end-expiratory breath samples taken in duplicate before and at 90 and 180 minutes after the challenge. On average, 10% of the children showed an increase in breath hydrogen excretion within 3 h after the challenge, indicating malabsorption of lactose. None of the children complained of gastro-intestinal symptoms or showed any clinical sign of intolerance to the milk. The number of malabsorbers increased significantly (p less than 0.001) with age, starting at about 3% at the age of 8 and reaching about 27% at the age of 18 years. The sharpest rise occurred between 14 and 15 years. It is concluded that, despite the high prevalence of hypolactasia, Hong Kong Chinese children can consume normal amounts of milk without developing any untoward clinical symptom or sign. PMID- 1719931 TI - Fatal respiratory obstruction due to Ascaris lumbricoides--a case report. PMID- 1719932 TI - The impact of medical services on trachoma in a Gambian village: antibiotics alone are not the answer. AB - We have measured the prevalence of active trachoma in children aged less than 15 years in the Gambian village of Keneba, which has had excellent free medical care and a continuous supply of antibiotics since 1974. The prevalence was 13%, with the peak prevalence (20%) occurring in the 2 to 3-year age group. Of 71 cases diagnosed, only 23 (33%) had complained of ocular symptoms in the previous 3 months, in spite of the fact that 66 (94%) had attended the clinic. Only five had been diagnosed as having trachoma by the duty paediatrician (7%). Compliance with treatment was poor, with only 29 subjects returning for continued treatment (41%), and at follow-up 16 months later 22 of 64 subjects still had active disease (34%). We conclude that the widespread use of antimicrobial agents does not preclude the persistence of endemic disease. Socio-economic improvement or behavioural changes appear necessary for the control of trachoma in endemic areas. In the meantime there is a need for greater awareness of the disease both among clinicians in endemic areas and among the communities afflicted. PMID- 1719933 TI - Successful pregnancies following remission in childhood acute lymphoblastic leukaemia--case report. AB - We report two successful pregnancies in a 17-year-old Arab girl who had received modern combination chemotherapy and central nervous system minimal disease therapy for childhood acute lymphoblastic leukaemia at the age of 9.5 years. PMID- 1719934 TI - Wolman's disease in a Jordanian infant. AB - We report a case of Wolman's disease that is apparently the first to be reported in a Jordanian infant. The clue to diagnosis was the radiological evidence of bilateral adrenal calcifications and foam cells in bone marrow. The disease was confirmed by skin fibroblast culture which showed decreased 'acid esterase' activity. PMID- 1719935 TI - Development of a mechanism of action-based screen for anthelmintic microbial metabolites with avermectinlike activity and isolation of milbemycin-producing Streptomyces strains. AB - A high-volume screen for anthelmintic microbial metabolites with an avermectinlike mode of action was developed. The primary screen used the free living nematode Caenorhabditis elegans in a whole-organism assay. The specificity for avermectinlike compounds resides in the secondary screen, which takes advantage of the chloride channel-opening properties of the avermectins. By using standard microelectrode techniques, membrane conductance changes following exposure to extracts of microbial cultures were measured in the walking leg stretcher muscle fibers of the lined shore crab Pachygrapsus crassipes. The avermectins and related milbemycins give a characteristic response of rapid loss of membrane resistance coupled with a slight hyperpolarization of the membrane. This is partially (near 50%) reversible with the chloride channel blocker picrotoxinin. Four morphologically similar cultures that produced avermectinlike activities were identified by this screen. Isolation of the active components from one of these cultures (strain UC 8984) followed by nuclear magnetic resonance spectroscopy resulted in the identification of milbemycins alpha 1 and alpha 3. These metabolites are members of a large family of milbemycins produced by Streptomyces hygroscopicus subsp. aureolacrimosus NRRL 5739. Systematic studies revealed that strain UC 8984 is also a S. hygroscopicus strain, but which is taxonomically distinct from NRRL 5739. PMID- 1719936 TI - Effect of human, recombinant interleukin 2 on Punta Toro virus infections in C57BL/6 mice. AB - The effect of human recombinant interleukin-2 (rIL-2) on Punta Toro virus (PTV) infection was investigated in C57BL/6 mice. Immunologic and viral parameters were assessed after mice were treated i.p. with rIL-2 for 5 days. Treatment of mice with 25000 and 12500 units/mouse of rIL-2 resulted in significant inhibition of the disease as indicated by increases in survival of mice as well as decreases in liver and serum virus titers. Serum glutamic oxalic acid and pyruvic acid transaminase levels were also lowered indicating reduced liver damage. Murine IL 2 production returned to normal or above-normal levels in rIL-2 treated mice. Natural killer cell activity was also moderately stimulated by rIL-2 treatment. Significant amounts of interferon were not detected in the sera of treated mice. Weight gain and survival rates were similar for both toxicity and normal controls indicating that rIL-2 treatments had no toxic effect. PMID- 1719937 TI - Audiovisual specialists can make life in the OR easier. PMID- 1719938 TI - Detection of multiple antigenically related proteins from various rat tissues by monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase. AB - Mouse hybridomas were prepared by fusing myelomas and spleen cells from mice immunized with purified rat 3 alpha-hydroxysteroid dehydrogenase. Hybridomas secreting monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase were selected by indirect enzyme-linked immunoassay and then subcloned by limiting dilution. From two mice we have obtained four positive hybridomas, three secreting high affinity immunoglobulin (Ig) G1 and one secreting IgM. Only two of these monoclonal antibodies (MAbs 3G6 and 7D3, both IgG1) recognized denatured enzyme and, therefore, were used for further immunoblotting experiments. MAb 7D3 recognized a structurally related mouse enzyme, but not the human enzyme, whereas monoclonal antibody 3G6 recognized a human enzyme, but not the mouse enzyme. When these two monoclonal antibodies were used in immunoblotting to survey the expression of 3 alpha-hydroxysteroid dehydrogenase in rat liver and a number of other tissues, striking differences were found in the protein band patterns in kidney, lung, and testis. Both MAbs 7D3 and 3G6 recognized 3 alpha-hydroxysteroid dehydrogenase, a 34-kDa 7D3 recognized a protein of the same size as the liver protein, whereas MAb 3G6 recognized a 34-kDa protein plus another protein of 36 kDa. In kidney only MAb 3G6, but not MAb 7D3, recognized a 34-kDa protein. Conversely, the 34-kDa protein in testis was recognized by MAb 7D3, but not by MAb 3G6. These findings suggest the existence of multiple antigenically related proteins in different tissues. PMID- 1719939 TI - [Superficial bladder cancer: prophylaxis of recurrence and progression]. AB - Superficial bladder cancer has a good prognosis compared with invasive bladder cancer. However, recurrence of the tumor is frequent and tumor stage and/or grade progress at the time of recurrence in many cases. Intravesical chemotherapy has been employed as a prophylactic method after trans-urethral resection (TUR). Although intravesical chemotherapy has been proved to be effective in delaying the first recurrence of tumor after TUR, it cannot improve the ultimate prognosis of superficial bladder cancer. Many primary, solitary, non-invasive (Ta) and grade 1 tumors do not recur or progress in stage and grade. In these cases, prophylactic intravesical chemotherapy is not essential. Bacille Calmette-Guerin (BCG) should be considered superior overall, to any chemotherapeutic agents. Comparative studies will give information about the best clinical schedule for the treatment of superficial bladder cancer. PMID- 1719940 TI - [Prostate cancer--treatment of hormone independent cancer]. AB - At the present time in Japan, the androgen ablation therapy, such as the surgical castration, estrogen therapy, antiandrogen therapy and LHRH agonist therapy, is mainly used for the treatment of advanced prostate cancer as well as for early prostate cancer. Ten to twenty percent of advanced prostate cancer do not respond to the initial endocrine therapy. The most of advanced prostate cancer relapse to androgen independent state within several years after the initial endocrine therapy. This characteristic of prostate cancers to develop resistance to androgen ablation therapy is the main problem in the treatment of prostate cancer. We surveyed the literatures regarding the treatments of the hormone independent prostate cancer. The results of bilateral adrenalectomy or antiandrogen therapy for patients who had relapsed to standard hormone therapy was disappointing. These data showed that the absence of testes and adrenals is not sufficient to stop the progression of the hormone independent cancer cells. Theoretically, the chemotherapeutic agents will be expected to be active agents for the hormone independent prostatic cancer. However, none of the products available are particularly active and the objective response rate is less than 10%. Therefore, the least toxic agents should be used. The treatment of painful metastasis in the terminal stage patients with hormone independent prostate cancer should be positively achieved. The external beam irradiation is useful for palliation of local bone pain of prostate cancer. Analgesics including morphine should be also positively used for the relief of pain in the terminal stage patients with prostate cancer. PMID- 1719941 TI - [A case report of relapsed stage D2 prostate cancer successfully treated with intraarterial chemotherapy]. AB - A 70-year-old Japanese male was first diagnosed as poorly differentiated adenocarcinoma of the prostate with bone metastasis in 1983. He received chemoendocrine therapy with both DESD and HCFU following subcapsular orchiectomy since 1983. As a result of the treatment, the prostate cancer was stabilized. At the end of 1988, the serum levels of PA were elevated. Diagnostic imaging revealed a local recurrence in the prostate. The pathological analysis of the prostate revealed undifferentiated adenocarcinoma. Intraarterial chemotherapy with a reservoir system was carried out. Doses of CDDP, ADM and MTX were 75 mg, 30 mg and 50 mg, respectively. Eight weeks after intraarterial chemotherapy, the regression rate was 60%, and serum PA titer improved to within normal limits. In this case, as the initial clue for suspecting recurrence, periodic detection of PA was useful, and the intraarterial chemotherapy was considered useful for the control of the locally recurrent prostate carcinoma. PMID- 1719942 TI - Autosomal recessive hypoparathyroidism with renal insufficiency and developmental delay. AB - Four children (two boys and two girls) with hypoparathyroidism, renal insufficiency, and developmental delay are described. They were the products of consanguineous marriages in three related Asian families presenting over a six year period. All the children died within the first 15 months of life despite treatment. Postmortem examination on one child showed absent parathyroid glands. We believe these children represent a previously undescribed syndrome that appears to be inherited in an autosomal recessive manner. PMID- 1719943 TI - Toxicity of four common pollutants to the freshwater macroinvertebrates Chironomus riparius Meigen (Insecta:Diptera) and Gammarus pulex (L.) (Crustacea: Amphipoda). AB - The lethal toxicities of the four pollutants 3,4-dichloroaniline (DCA), atrazine, copper, and lindane were determined for the 2nd larval instar of the insect Chironomus riparius Meigen and the juvenile stage (2nd or 3rd moult) of the crustacean Gammarus pulex (L.). Median lethal concentrations (LC50s) were determined over a 240 h test period. The order of toxicity of the test chemicals is different for each species. For C. riparius, lindane was the most toxic, followed by copper, DCA, and atrazine. During the first 96 h of exposure, the order for G. pulex was copper, lindane, then DCA and atrazine with similar LC50 values. However, at 240 h lindane replaced copper as the most toxic chemical to G. pulex. The relative sensitivity of the two species was dependent on both the toxicant and the exposure period. The lethal concentrations determined for the four chemicals are compared to the results of other toxicity studies and discussed with respect to current standard test methods. PMID- 1719944 TI - Conization for CIN associated with human papillomavirus infection. AB - We report results of follow-up by PAP smears and colposcopy in 116 women treated since 1981 by conization for cervical intraepithelial neoplasia with human papillomavirus infection (HPV-CIN). The mean follow-up time was 31.9 (SD 19) months. Preoperative diagnosis was HPV-CIN II (with extension into the endocervix) in 11 cases and HPV-CIN III in 103 cases; two diagnostic conizations were performed. The histological examination of the cone biopsies showed complete excision of CIN in 109 cases (94%). Three patients underwent hysterectomy after conization; one had microinvasion in the cone biopsy, one had suspicion of microinvasion and one had non-radical conization. Three patients (2.6%) were lost to follow-up. After excluding these six patients the primary cure rate of HPV lesions (normal cytological and colposcopical finding after conization) was 82.7%. Four patients (4.6%) had residual CIN after conization. During the follow up 15 patients had recurrence of HPV infection, only one had HPV-CIN I. HPV 16 was the most common HPV type (56/116, 48.2%) in the conization group and also in the recurrent cases (9/15, 60%). The results support the role of HPV 16 in cervical carcinogenesis. PMID- 1719946 TI - [Use of 5-fluorouracil, bleomycetin and platidiam in combined treatment of localized cancer of the larynx]. AB - Preliminary results of chemoradiotherapy in 30 patients with the larynx cancer T3T4N0M0 are presented. Two treatment schemes were applied. According to scheme I, the treatment was started with chemotherapy with 5-fluorouracil in doses of 1000 mg on days 1, 2 and 3, bleomycetin in doses of 15 mg on days 1, 2 and 3 and platidiam in doses of 120 mg/m2 on day 4 followed by radiotherapy to the total dose of 66 to 70 Gy. Scheme II included two analogous courses of the chemotherapy, one prior to the radiotherapy and the other after irradiation in a dose of 38 to 40 Gy, after a two-week interval the radiotherapy was continued to doses of 66 to 70 Gy. 15 patients were treated in accordance with each scheme. Complete resorption of the tumor was observed in 66.6 and 83.3 per cent of the patients treated according to schemes I and II, respectively. The results showed that the use of 5-fluorouracil, bleomycetin and platidiam in combination with radiotherapy was promising in treatment of patients with local cancer of the larynx. PMID- 1719945 TI - Duodenal ulcer. Discovery of a new mechanism and development of angiogenic therapy that accelerates healing. AB - The complete purification of the first angiogenic molecule, basic fibroblast growth factor (bFGF), was carried out in the authors' laboratory in 1983. Application of this peptide to chronic wounds enhances angiogenesis and accelerates wound healing. The authors showed that an acid-stable form of bFGF (i.e., bFGF-CS23) could be administered orally to rats with duodenal ulcers. The peptide promoted a ninefold increase of angiogenesis in the ulcer bed and accelerated ulcer healing more potently than cimetidine. Basic fibroblast growth factor did not reduce gastric acid. The authors now show that bFGF exists as a naturally occurring peptide in rat and human gastric and duodenal mucosa. This endogenous bFGF is present also in the bed of chronic ulcers in rats. Sucralfate binds bFGF and protects it from acid degradation. The sucralfate is angiogenic, based on its affinity for bFGF. When sucralfate is administered orally to rats, it significantly elevates the level of bFGF in the ulcer bed. Cimetidine, by its capacity to reduce gastric acid, also elevates bFGF in the ulcer bed. A hypothetical model is proposed in which prevention of ulcer formation or accelerated healing of ulcers by conventional therapies may be FGF dependent. Acid-stable bFGF-CS23 may be considered as a form of replacement therapy in the treatment of duodenal ulcers. PMID- 1719947 TI - [Relation between interferon-inducing activity of natural interferon inducers and their chemical structure]. AB - The interferon-inducing activity of new derivatives of gossypol, a low molecular weight natural polyphenol, and its model compounds was studied in regard to their chemical structure. The active groups required for development of optimal interferon inductors on the basis of the natural polyphenols were defined. As a result of the studies the new interferon inductor AC-1 which is a condensation product of gossypol, a natural polyphenol, and a cellulose derivative was isolated. The interferon-inducing activity of AC-1 was investigated with respect to its dose and the administration route. High interferon-inducing activity of the interferon inductor after its administration by any route in doses of 10 to 100 mg/kg was demonstrated. The serum interferon titers reached 2560 units/ml. PMID- 1719948 TI - Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. AB - The xylA and xylB genes of Bacillus subtilis BR151 encoding xylose isomerase and xylulokinase, respectively, were disrupted by gene replacement rendering the constructed mutant strain unable to grow on xylose as the sole carbon source. The Bacillus megaterium encoded xyl genes were cloned by complementation of this strain to xylose utilization. The nucleotide sequence of about 4 kbp of the insertion indicates the presence of the xylA and xylB genes on the complementing plasmid. Furthermore, a regulatory gene, xylR, is located upstream of xylA and has opposite polarity to it. The intergenic region between the divergently oriented reading frames of xylR and xylA contains palindromic sequences of 24 bp spaced by five central bp and 29 bp spaced by 11 bp, respectively, and two promoters with opposite orientation as determined by primer extension analysis. They overlap with one nucleotide of their--35 consensus boxes. Transcriptional fusions of lacZ to xylA, xylB and xylR were constructed and revealed that xylA and xylB are repressed in the absence and can be 200-fold induced in the presence of xylose. The increased level of xylAB mRNA in induced and its absence in repressed cells confirms that this regulation occurs on the level of transcription. Deletion of the xylR gene encoding the Xyl repressor results in constitutive expression of xylAB. The transcription of xylR is autoregulated and can be induced 9-fold by xylose. The mechanism of this regulation is not clear. While the apparent xyl operator palindrome is upstream of the xylR promoter, the potential recognition of another palindrome downstream of this promoter by Xyl repressor is discussed. PMID- 1719949 TI - [The morphogenesis of polyps of the large intestine]. AB - The proportion of so-called hyperplastic polyps among other colon epithelial tumours remains unclear. Colon polyps are studied in 400 patients: hyperplastic polyps are found in 107, a combination of hyperplastic polyp with tubular adenoma in 143, tubular adenoma in 72, villous adenoma in 41 and tubulovillous adenoma in 37 cases. Hyperplastic structures and formation of adenomatous structures were, as a rule, observed in a polyp tip, this corresponding to the increase of nuclei volume and DNA amount in the tip colonocytes. The determination of karyometric coefficient (the ratio of the colonocyte nuclei volume in the polyp tip to colonocytes in the intact mucosa) is proposed as a quantitative criterion of proliferative and dysplastic processes. PMID- 1719950 TI - [The use of monoclonal antibodies to cytokeratin peptides for the diagnosis of basal cell carcinoma of the skin]. AB - 25 skin basaliomas were studied immunohistochemically using 6 different monoclonal antibodies (MAB) to the low-molecular cytokeratins N 8, 18 (according to Moll's catalog) as well as to the cytokeratins N 1, 2, 9, 10, 11 and N 17. Prekeratin N 17 was found in all tumours while cytokeratin N 18 was found in no tumour. Prekeratin N 8 was expressed in 24% of tumours. MAB EE 21-06 to the cytokeratin N 1, 2, 9, 10, 11 are reactive with tumour cells in 20% of cases. Basalioma cells (apart from trichobasalioma) did not react with MAB G 36-19. These results suggest a dissimilar origin of basaliomas and may help in the differential diagnosis of the skin malignant tumours. PMID- 1719951 TI - Serum immunoglobulin concentrations in genetically different types of suckling beef calves in a tropical environment. AB - Factors influencing the serum concentrations of gamma-globulin (gamma-G) during the neonatal period were studied in Shorthorn x Hereford (SH), Africander x SH and Brahman x SH calves born to cows grazing in a tropical environment. There were no significant effects of age of dam, sex or breed of calf on the gamma-G concentrations of calves from birth to 48 hours of age. Concentrations of gamma-G fell within two ranges: group A, 10 to 20 g/l and group B, 35 to 70 g/l. The number of calves in each group was not significantly different between breeds and overall 30% of calves were in group A. Body weight gain from birth to 10-day-old was greater (P less than 0.01) in calves in group B than in group A. Plasma cholesterol concentrations in 10-day-old calves were higher in group B than in group A calves supporting the interpretation that group B calves had higher milk intakes. Identification of calves receiving adequate amounts of colostrum has fundamental significance for the efficient production of cattle in the tropics. PMID- 1719952 TI - Structural polypeptides of type II avian adenoviruses analyzed by monoclonal and polyclonal antibodies. AB - Polypeptides of hemorrhagic enteritis virus (HEV) of turkeys and marble spleen disease virus (MSDV) of pheasants were analyzed by immune precipitation and immunoblot assays. A total of 11 polypeptides ranging in molecular weight from 14,000 to 97,000 were detected in lysates of HEV-infected turkey cells analyzed by immunoblot assay using a polyclonal antibody against HEV. Identical patterns were observed with preparations of MSDV. Five monoclonal antibodies (MAbs) against HEV were chosen based on their virus neutralization activity and used for identification of neutralizing epitopes of these two viruses. Three MAbs precipitated a single 97,000-molecular-weight hexon polypeptide in an immune precipitation assay. PMID- 1719953 TI - Angiogenesis in the adult heart. AB - We have studied the development of the collateral circulation in the heart in response to gradual and progressive coronary artery occlusion. When the coronary stenosis becomes critical, tissue ischemia occurs, which we believe leads to the production (and probably to release from storage sites) of tissue hormones (mitogens) that lead to mitosis of endothelial and smooth muscle cells. We have identified from hearts several known mitogens (aFGF, bFGF), non-mitogenic angiogenic factors (TGF-beta), a new anti-mitogen, and a new myocyte-derived growth factor (structures of the last two not yet elucidated). An important principle in the development of collaterals is the remodeling of pre-existing small vessels into the much larger vascular structure. To accommodate new cells old structures have to be removed by controlled proteolysis (tPA, uPA, elastase). PMID- 1719954 TI - Cellular mechanisms controlling EDRF/NO formation in endothelial cells. AB - We investigated the molecular mechanisms whereby Ca2+ enters the endothelial cytosol and regulates endothelial nitric oxide synthesis L-arginine-dependent nitric oxide synthesis by isolated endothelial cytosol as quantified by activation of a purified soluble guanylate cyclase was concentration-dependently enhanced by free Ca2+ (EC50 0.3 microM). The Ca(2+)-dependent activation was inhibited by the calmodulin antagonists mastoparan, melittin, and calcineurin (IC50 450, 350, and 60 nM, respectively) in a calmodulin-reversible manner. After removal of endogenous calmodulin the Ca(2+)-dependency of endothelial NO synthase was lost, but could be reconstituted with exogenous calmodulin. The results indicate that Ca(2+)-calmodulin directly activates the endothelial nitric oxide synthase, thereby transducing agonist-induced increases in intracellular free Ca2+ concentration to nitric oxide formation from L-arginine, K(+)-induced depolarization of the endothelial cells markedly inhibited the sustained, but not initial phase of the intracellular Ca2+ response to bradykinin, indicating that K(+)-induced depolarization depresses the transmembrane Ca2+ influx. On the contrary, the K+ channel activator Hoe 234 which elicits hyperpolarization of the endothelial cell membrane, augmented the sustained phase of the agonist-induced intracellular Ca2+ signal, but not the resting intracellular Ca2+ level. The effects of K+ and Hoe 234 on the agonist-induced Ca(2+)-response were reflected by corresponding changes in agonist-induced EDRF/NO release. From these data, we suggest that the endothelial membrane potential may play an important role for the extent of agonist-induced Ca2+ influx and, thereby, the endothelial EDRF/NO synthesis. PMID- 1719955 TI - Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous beta 1 subunits of the sodium pump. AB - Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Hauptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte. PMID- 1719956 TI - Certain changes in ornithine decarboxylase gene methylation accompany gene amplification. AB - The ornithine decarboxylase (ODC; EC 4.1.1.17) gene in parental, dexamethasone resistant and 2-difluoromethylornithine (DFMO)-resistant human IgG-myeloma-cell lines was studied with the aid of methylation-sensitive restriction endonucleases and probes recognizing different parts of the gene. In all cell lines the promoter region of the ODC gene appeared to be heavily methylated, whereas the first long intron was unmethylated. Methylation analyses of several clones from the parental cell line revealed that these cells are heterogeneous with respect to the methylation status of the ODC gene, whereas all clones from DFMO-resistant cell lines displayed the same methylation pattern. Two of the parental clones represented a hypomethylated type very close to that exclusively found among the DFMO-resistant clones with ODC gene amplification. This typical methylation pattern was due to decreased methylation of a few CCGG sequences in the 3' flanking region of the gene. It is possible that this kind of hypomethylation favours the initiation of the gene-amplification process in certain individual cells. This hypothesis was supported by the finding that no hypomethylation was present in the ODC gene of another human myeloma cell line that had acquired resistance to DFMO without gene amplification. In a dexamethasone-resistant cell line that overproduced ODC mRNA at normal gene dosage there were some minor differences between the methylation pattern of the ODC gene of different clones, but no such hypomethylation could be found in clones from the parental cell line. In dexamethasone-resistant cells the ODC gene was hypomethylated around the two HpaII sites and three CfoI sites in the coding region and also, as well as in cells with amplified ODC sequences, in the 3'-flanking region of the gene. Some hypomethylation in the distant 5'-flanking region was also observed. PMID- 1719957 TI - Enzymic synthesis of 1-O-(indol-3-ylacetyl)-beta-D-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme. AB - The enzyme indol-3-ylacetylglucose synthase (UDP-glucose:indol-3-ylacetate beta-D glucosyltransferase) catalyses the reaction: [formula: see text] This is the first step in the series of reactions leading to the indol-3-ylacetic acid conjugates found in maize. Previous attempts to purify this enzyme from the liquid endosperm of kernels of Zea mays (sweet corn) were not entirely successful owing to the lability of partially purified preparations during column chromatography. Thus this enzyme has not previously been purified to homogeneity. During the present study it was found that retention of enzyme activity required the combined presence of glycerol and dithiothreitol. Adding these requirements permitted purification of the enzyme to homogeneity with retention of catalytic activity. These purified preparations were used for preparation of rabbit polyclonal antibodies to the enzyme. Antibodies to the Zea mays endosperm enzyme cross-react with the enzyme from Zea mays vegetative tissues and with an enzyme from the liquid endosperm of oak acorns (Quercus sp). In this paper we report a simplified purification procedure adaptable to the preparation of milligram amounts of the enzyme. PMID- 1719958 TI - Effects of human recombinant interleukin-1 beta on protein synthesis in rat tissues compared with a classical acute-phase reaction induced by turpentine. Rapid response of muscle to interleukin-1 beta. AB - The early time course (1, 3, 9, 24 h) of changes in rates of protein synthesis (ks) in liver and three different muscles (gastrocnemius, soleus and heart) was investigated after injection of saline, interleukin-1 beta (IL-1) or turpentine in rats. IL-1 injection induced a consistent increase in body temperature of about 3 degrees C between 3 and 5 h, but thereafter a hypothermic response occurred. With turpentine, a delayed fever response with a peak value by 9 h was observed. Both IL-1 and turpentine had no effect on protein synthesis in the small intestine, but produced a significant increase in ks in the liver at 9 h. By 24 h in IL-1-treated animals, liver ks had returned back to control values, whereas the turpentine-treated group showed a progressive rise in ks. Gastrocnemius and soleus muscles exhibited a significant fall in ks at 9 h after IL-1 and turpentine injection compared with the control. In contrast, the ks of heart muscle increased at 3-9 h after IL-1 injection, but there was no effect of turpentine. Thus for the first time a marked decrease of protein synthesis in skeletal muscle in response to IL-1 could be demonstrated. PMID- 1719959 TI - Glycosylation and transmembrane topography of bovine chromaffin granule p65. AB - The bovine homologue of p65, a calmodulin-binding protein located in the membranes of synaptic vesicles and endocrine secretory granules, has been studied by the use of monoclonal antibodies directed against this antigen and against dopamine beta-mono-oxygenase. The protein (apparent molecular mass 67 kDa; pI = 5.5-6.2) is partially degraded by treatment with neuraminidase or endoglycosidase F. Trypsin treatment of intact adrenal chromaffin granules or of granule membranes releases a soluble 39 kDa fragment of p65 which corresponds to the whole of its cytoplasmic domain. This domain contains both the epitope for the monoclonal antibody cgm67 and the calmodulin-binding site. The 20 amino acids at the N-terminus of this fragment are identical to part of the rat p65 sequence. PMID- 1719960 TI - Recognition of chylomicron remnants and beta-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver alpha 2-macroglobulin-recognition site. AB - The uptake in vivo of chylomicrons and beta-migrating very-low-density lipoprotein (beta-VLDL) by rat liver, which is primarily carried out by parenchymal cells, is inhibited, 5 min after injection, to respectively 35 and 8% of the control values after preinjection of lactoferrin. The decrease in the uptake of lipoproteins by the liver caused by lactoferrin is a specific inhibition of uptake by parenchymal cells. Competition studies in vitro demonstrate that chylomicron remnants and beta-VLDL compete for the same recognition site on parenchymal cells. Data obtained in vivo together with the competition studies performed in vitro indicate that chylomicron remnants and beta-VLDL interact specifically with the same remnant receptor. Hepatic uptake of 125I-labelled-alpha 2-macroglobulin in vivo, mediated equally by parenchymal and endothelial cells, is not decreased by preinjection of lactoferrin and no effect on the parenchymal-cell-mediated uptake is found. In vitro, alpha 2-macroglobulin and chylomicron remnants or beta-VLDL show no cross-competition. Culturing of parenchymal cells for 24-48 h leads to a decrease in the cell association of alpha 2-macroglobulin to 26% of the initial value, while the cell association of beta-VLDL with the remnant receptor is not influenced. It is concluded that beta VLDL and chylomicron remnants are recognized by a specific remnant receptor on parenchymal liver cells, while uptake of alpha 2-macroglobulin by liver is carried out by a specific receptor system (presumably involving the LDL-receptor related protein) which shows properties that are distinct from those of the remnant receptor. PMID- 1719961 TI - Calcium phosphate-induced fusion of human erythrocyte ghosts monitored by dilution of a membrane bound fluorescence probe. AB - Calcium-phosphate induced fusion of human erythrocytes was investigated using an assay based on the relief of the fluorescence selfquenching of a fluorescent amphiphile incorporated into ghost membranes as it occurs when labeled membranes are fused with unlabeled membranes. Measuring ghost fusion up to Ca(2+) concentrations of 4 mM in the presence of 10 mM phosphate, both an acceleration of the fusion process and an enhancement of the fusion extent with increasing amounts of calcium were observed. Fusion takes place even in the case when calcium-phosphate complexes were formed in the suspension medium prior to the addition of ghosts. These results are in contrast to previous observations, obtained by an internal aqueous content mixing assay, where fusion of ghosts was inhibited at Ca(2+)-concentrations greater than 1.75 mM, and preformed calcium phosphate complexes were not able to induce fusion. The results point to the necessity of utilizing content mixing assays in conjunction with the lipid dilution assay to get a more detailed insight in membrane fusion mechanism(s). PMID- 1719962 TI - Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro. AB - When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibitory effect of anti-FGF antibody was dose-dependent and maximal inhibition was observed at 2.0 micrograms/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor. PMID- 1719963 TI - Further characterization of a high affinity thyrotropin binding site on the rat thyrotropin receptor which is an epitope for blocking antibodies from idiopathic myxedema patients but not thyroid stimulating antibodies from Graves' patients. AB - Cysteine 390 of the rat thyrotropin (TSH) receptor, when mutated to serine, results in a receptor with a reduced ability of TSH to bind and increase cAMP levels but a preserved ability of thyroid stimulating autoantibodies (TSAbs) from hyperthyroid Graves' patients to increase cAMP levels. The ability of receptor autoantibodies from hypothyroid patients with idiopathic myxedema to inhibit the TSAb activity which is preserved is, however, like TSH binding, significantly reduced. Cysteine 390, together with tyrosine 385, thus appears to be an important determinant in a high affinity TSH binding site which is an epitope for receptor autoantibodies which block TSH or TSAb action and cause hypothyroidism rather than TSAbs which increase cAMP levels and are associated with hyperthyroidism. Threonine 388 and aspartic acid 403 may contribute to this ligand interaction site. PMID- 1719964 TI - On the spontaneous adherence of myelin basic protein to T lymphocytes. AB - We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation. PMID- 1719965 TI - Differential effects of all-trans and 13-cis-retinoic acid on mRNA levels of nuclear retinoic acid receptors in rat lung and liver. AB - The effects of three retinoids, all-trans-retinoic acid (all-trans-RA), 13-cis RA, and etretin were examined on mRNA abundance of nuclear retinoic acid receptors (RAR-alpha, beta, and gamma) in lung and liver of retinol deficient and chow fed rats. All-trans-RA increased lung RAR-beta mRNA levels 5 or 11-fold in chow fed and retinol deficient rats, respectively. Similarly to lung, liver RAR beta mRNA levels were 3-fold higher in retinol deficient rats fed all-trans-RA than the rats fed cottonseed oil. Lung RAR-gamma mRNA levels were also induced 2 fold by all-trans-RA. In contrast to this, 13-cis-RA and etretin at equimolar doses failed to enhance lung or liver RAR-beta or lung RAR-gamma mRNA levels in retinol deficient rats. These data for the first time show that all-trans-RA is more effective than its 13-cis-isomer in regulating the expression of RAR-beta and gamma transcripts in adult animal. PMID- 1719966 TI - Inhibitory binding of adenosine diphosphoribosyl transferase to the DNA primer site of reverse transcriptase templates. AB - Purified adenosine diphosphoribose transferase protein binds to RNA-DNA hybrid templates of reverse transcriptase at the DNA primer site and inhibits RT activity of HIV and MMu RTs. This action is prevented by auto-poly-ADP ribosylation of the transferase but is reinduced by inhibitory ligands of the enzyme. PMID- 1719967 TI - Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin. AB - A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species. PMID- 1719968 TI - Effects of a variety of cytokines and inducing agents on vascular permeability factor mRNA levels in U937 cells. AB - Vascular permeability factor (VPF) is an approximately 40-kDa disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth and angiogenesis. Little is known about VPF gene regulation. In this study, we investigated the effects of a variety of cytokines and inducing agents on VPF mRNA levels in the monocyte-like U937 cell line. Transforming growth factor-beta 1 caused a 1.8-fold increase in VPF mRNA levels after 4 hours, followed by a decline to basal levels by 18 hours. Phorbol 12 myristate 13-acetate, a potent inducer of the differentiation of U937 cells, caused a 12.5-fold increase in VPF mRNA levels at 24 hours, coinciding with the differentiation of these monocyte-like cells into macrophage-like cells. PMID- 1719969 TI - A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts. AB - Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue. PMID- 1719970 TI - Single cell analysis of changes in cytosolic calcium induced by vitamin D3 metabolites in cultured rat mesangial cells. AB - The acute effects of 1,25-Dihydroxy-vitamin D3 [1,25(OH)2D3] on the concentration of cytoplasmic ionized calcium [Ca2+] of cultured rat mesangial cells were studied at the single cell level by microspectrofluorometry of fura-2-loaded cells. Addition of 1,25(OH)2D3 produced an immediate increase of [Ca2]+. This rise in [Ca2+] was sustained and similar to that caused by the Ca2+ channel agonist BAY K 8644. Comparable changes were also observed in cultured human mesangial cells. The effects of the hormone (10 (-10)-10(-7) M) were dose dependent (62% and 285%). Only 30-40% of the cells responded to stimulation with 1,25(OH)2D3. 25OHD3 also increased Ca2+ whereas 24,25(OH)2D3 and 1aOHD3 were inactive. Addition of 1 mM CoCl2 or 2-5 microM nifedipine largely blocked the effects of 1,25(OH)2D3 suggesting the involvement of Ca2+ channel activation in the rapid 1,25(OH)2D3-induced increase in mesangial cell [Ca2+]. 45Ca uptake studies are consistent with This interpretation. PMID- 1719971 TI - The determination of absolute electron affinities of the purines and pyrimidines in DNA and RNA from reversible reduction potentials. AB - We report the reversible reduction potentials of the purines and pyrimidines of DNA and RNA. These were determined in dimethylsulfoxide using cyclic voltammetry. The absolute electron affinities have been determined from these reduction potentials by calibration with the absolute electron affinities for acridine and anthracene measured in the gas phase. These are the first experimentally determined values of the electron affinities of these purines and pyrimidines and are: Guanine = 1.51 eV, Adenine = 0.95 eV, Uracil = 0.80 eV, Thymine = 0.79 eV and Cytosine = 0.56 eV. PMID- 1719972 TI - FKBP, the binding protein for the immunosuppressive drug, FK-506, is not an inhibitor of protein kinase C activity. AB - Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C. PMID- 1719973 TI - Amplification and sequencing of mRNA encoding acidic fibroblast growth factor (aFGF) from porcine heart. AB - Progredient stenosis of coronary arteries can induce angiogenic processes, which are probably regulated by polypeptide growth factors like aFGF. Using applications of reverse transcription-polymerase chain reaction, we amplified and sequenced an mRNA encoding aFGF in the porcine myocardium. A DNA fragment of expected size encoding aFGF was amplified with human and bovine aFGF specific oligonucleotide primers in porcine heart. Identity of amplified PCR product to aFGF sequence was confirmed by internal reamplification, Southern hybridization and sequencing of asymmetrically amplified PCR products. The nucleotide sequence analysis of porcine aFGF revealed a homology of 94% to the human and 92% to the bovine cDNA sequences respectively. The amino acid sequence was homologous to the known sequences except for three alterations in the human and thirteen in the bovine aFGF sequences. PMID- 1719974 TI - The crucial role of cyclic GMP in the eclosion hormone mediated signal transduction in the silkworm metamorphoses. AB - The signal transduction of the peptide, eclosion hormone, in the silkworm Bombyx mori appears to be mediated via the second messenger cyclic GMP throughout their life cycle. Injection of 8-bromo-cGMP induced the ecdysis behavior in pharate adults with similar latency to eclosion hormone-induced ecdysis; the moulting occurred 50-70 min after the injection. The potency of 8Br-cGMP was 10(2) fold higher than that of cGMP and the efficacy was increased by the co-injection of the phosphodiesterase inhibitor IBMX. On the other hand, in the silkworm pupal ecdysis the eclosion hormone and also 8Br-cGMP induced the moulting behavior in a dose-dependent manner. The adult development of the ability to respond to 8Br cGMP took place concomitantly with the response to the eclosion hormone. Both the developmental time courses were shifted by a shift of light and dark cycles. Accordingly, the sensitivities to the peptide and cyclic nucleotide developed correspondently under the light and dark circadian rhythm. Thus throughout the silkworm life cycle, eclosion hormone is effective to trigger the ecdysis behavior and cGMP plays a crucial role as the second messenger in the eclosion hormone-mediated signal transduction. PMID- 1719975 TI - Induction kinetics of RNA and proteins in exponentially growing organisms. AB - A mathematical model of the induction kinetics of RNAs and proteins in exponentially growing organisms is derived, and the cellular concentrations of the induced macromolecules at a given time after induction are related to three parameters: the fraction of the synthesis of these macromolecules in total synthesis, the half life of the inducible macromolecules, and the generation time of the organisms. The model predicts that the concentrations of the inducible macromolecules reach one half of the maximum induction level within one generation time after the onset of the induction. The model also predicts that induction curves of proteins are parabolic when their mRNAs are short-lived, but sigmoid when they are stable. Observed induction curves of beta-galactosidase in Escherichia coli cells fit in the theoretical induction curves. PMID- 1719976 TI - Synthesis of nitric oxide in the bovine retina. AB - In the absence of light, high concentrations of cGMP open ion channels in the plasma membranes of rod outer segments. The source of stimulation of retinal guanylate cyclase is not known. Nitric oxide is a potent stimulator of guanylate cyclase in other cell systems. The present data demonstrate that nitric oxide synthase, an enzyme responsible for the production of nitric oxide, is present in retina, and specifically in the rod outer segments. This enzyme uses L-arginine as a substrate and is NADPH- and calcium- dependent. L-arginine-derived nitric oxide may be a source of activation of guanylate cyclase in the retina. PMID- 1719977 TI - Expression of Id, a negative regulator of helix-loop-helix DNA binding proteins, is down-regulated at confluence and enhanced by dexamethasone in a mouse osteoblastic cell line, MC3T3E1. AB - The message of Id (for "inhibitor of DNA binding") was expressed in an osteoblastic cell line, MC3T3E1, when the cells were in early cultures, while undetectable after the cells became confluent. The abundance of Id message in MC3T3E1 cells in early cultures was increased when the cells were treated with dexamethasone. This effect was time and dose dependent in a range between 10( 11)M and 10(-7)M. Id message expression was not enhanced by TGF-beta, 1,25 dihydroxyvitamin D3, retinoic acid, interleukin 1-alpha, interleukin 6, tumor necrosis factor-alpha or parathyroid hormone. These observations indicate for the first time the presence of HLH protein family member, Id, in osteoblast-like cells and its regulation by dexamethasone. PMID- 1719978 TI - Influence of internucleotide phosphate linkage on relative base stacking in 3'-5' and 2'-5' RNA: a circular dichroic spectroscopic study of RNA hexamer AACCUU. AB - The variations in base stacking interactions of two isomeric RNA hexamers, 3'-5'r (AACCUU) and 2'-5'r' (AACCUU), have been studied using temperature dependent CD spectroscopy. Both RNA hexamers, in single strand form, exhibited a right handed helical sense. Van't Hoff analysis of the CD spectral results, derived from a two state model, gave a higher enthalpy of stacking for 3'-5' RNA than for 2'-5'RNA. The results suggest that 3'-5' linkage in RNA facilitates formation of better helical stacks in relation to an isomeric 2'-5' linkage. PMID- 1719979 TI - Cloning and characterization of cDNA encoding human A-type endothelin receptor. AB - A cDNA coding for the human A-type endothelin receptor (ETA) was cloned from a human placenta cDNA library. The cDNA contained the entire coding sequence for the 427 amino acid protein with a relative Mr of 48,722. The deduced amino acid sequence of the human ETA was, respectively, 94% and 93% homologous with the sequence of bovine ETA and rat ETA, but was only 64% homologous with that of the human ETB receptor. Upon expression in COS-1 cells, the human ETA receptor showed binding activity to ETA, with the highest selectivity to ET-1. Northern blot analysis showed that the mRNA of human placenta ETA consists of one species 5 kilo-nucleotides in length, and the same analysis for the uterus, testis, heart and adrenal gland of Cynomolgus monkey showed that the cognate mRNAs are widely distributed. PMID- 1719980 TI - Purification and kinetic characterization of equine infectious anemia virus reverse transcriptase. AB - The reverse transcriptase of Equine Infectious Anemia Virus (EIAV) was partially purified from virus particles and appeared to be a heterodimer with subunit molecular masses of 70 kdal and 59 kdal. The polymerase activity of this enzyme had an absolute requirement for a divalent cation, preferring Mg++ over Mn++. Addition of a monovalent cation to the reaction mixture enhanced, but was not required for enzyme activity. Kinetically, the reverse transcriptase of EIAV is similar to the reverse transcriptase of Human Imunodeficiency Virus Type 1 (HIV 1). Both enzymes have similar Km values for 2'-deoxynucleoside-5'-triphosphates on the synthetic template/primers tested, both exhibit substrate inhibition, and both are inhibited to similar extents by most nucleoside-triphosphate analogs. The results of this study suggest that the reverse transcriptase of EIAV may be a good model for studying structure/function relationships of retroviral reverse transcriptases. PMID- 1719981 TI - Regulation of endogenous chloride conductance in Xenopus oocytes. AB - Radiotracer (86Rb, 125I) efflux measurements and intracellular microelectrode recording were performed to study the cellular mechanisms that regulate the endogenous ionic conductances in Xenopus oocytes. Addition of isoproterenol (Iso, 10(-5) M) caused a marked increase in 86Rb efflux, with a time course that is in good agreement to Iso-elicited membrane hyperpolarization. Thus, radiotracer efflux measurement appears to be a sensitive assay method to study stimulus secretion coupling in oocytes. 125I efflux was suppressed by the C1- channel blocker diphenylamine-2-carboxylate, but was insensitive to bumetanide. Elevation of ambient [Ca2+] from 0.4 to 10 mM resulted in an eminent increase in 125I efflux for up to approximately 20 min. Acetylcholine (10(-5) M), which mobilizes cell Ca2+, also enhanced 125I efflux. Iso although increased intracellular cAMP level approximately 2-fold, but showed no stimulatory effect on 125I efflux. Addition of 8-(-4-chlorophenylthio)-cAMP (1 mM), or of forskolin (10(-5) M) plus the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (2 x 10(-4) M), also failed to enhance 125I efflux. These results suggest that, in sharp contrast to the mechanisms for Cl-conductance regulation in mammalian Cl-secreting epithelia, the endogenous Cl- conductance in Xenopus oocytes is, under normal physiological conditions, primarily regulated by intracellular Ca(2+)- rather than a cAMP mediated signaling mechanism. PMID- 1719982 TI - Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase. AB - Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation. PMID- 1719983 TI - Potent anti-inflammatory action of calcitonin gene-related peptide. AB - Calcitonin gene-related peptide (CGRP), but not substance P (SP), was found to inhibit edema-promoting actions of inflammatory mediators (histamine, leukotrine B4, 5-hydroxytryptamine) in vivo in the hamster cheek pouch, human skin, and rat paw. The effect of CGRP was present in the low nanomolar dose range, and it was mimicked by activation of sensory nerves with capsaicin which caused release of endogenous CGRP-like immunoreactivity (IR). The findings provide new information on the potential impact of sensory nerve activation during inflammatory processes by indicating that sensory nerves may play an anti-inflammatory role. PMID- 1719984 TI - Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site. AB - Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) gamma 1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC gamma 1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC gamma 1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC gamma 1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site. PMID- 1719985 TI - The carboxyl terminal amino acid residues of Pseudomonas aeruginosa exotoxin A involved in cell toxicity and pathogenesis, characterized by a neutralizing human monoclonal antibody. AB - Human monoclonal antibody HI-1A4 (IgG3, lambda) neutralized a toxicity caused by pseudomonal exotoxin A (Ex-A) in cell culture and in vivo, and was effective in experimental Pseudomonas aeruginosa infections in mice. HI-1A4 inhibited an Ex-A catalyzed ADP-ribosylation of elongation factor 2 but did not inhibit an incorporation of toxin into a target cell at all. One molecule of HI-1A4 neutralized at least 2 molecules of Ex-A. HI-1A4 retained its binding activity at pH 4.0. The epitope region for HI-1A4 was demonstrated to be a carboxyl terminal end of amino acid residues 591-613 of Ex-A. HI-1A4 might bind to Ex-A carboxyl terminal region outside a target cell, be incorporated into cells as a complex with Ex-A, and inhibit the intracellular function in which the carboxyl terminal part of Ex-A was involved, resulting in the interruption of intoxication of Ex-A. PMID- 1719986 TI - Human placenta DNA primase: purification of enzyme and analysis of RNA primer synthesis. AB - The immunoaffinity purification of human placenta DNA primase devoid of DNA polymerase alpha activity is described. Primase consists of 52 and 59 kDa polypeptides. They form a single protein of 330 kDa under native conditions. The polypeptide structure of primase is believed to be (52 + 59)3. Primase synthesizes the oligoribonucleotides 2-9 monomers long and multimeric oligoribonucleotides of a modal length of about 10 monomers. The following model of RNA primer synthesis is proposed: 1) primase, being in free state or in complex with Pol alpha, forms a protein trimer or another structure that includes several primases; 2) primase synthesizes de novo only the oligonucleotides 2-10 monomers in length; 3) the newly synthesized oligonucleotides dissociate in solution or translocate to either Pol alpha or a neighbouring primase unit to be further elongated with the next 7-10 mononucleotides. PMID- 1719987 TI - Quantitation of IgM- and IgG-secreting B cells in the peripheral blood of patients with systemic lupus erythematosus. AB - An enzyme-linked immunospot assay was used to quantitate the number of autoantibody-secreting B cells in the peripheral blood of 67 patients with systemic lupus erythematosus. These patients had 1.5-4-fold more lymphocytes secreting IgG and IgM per million peripheral blood lymphocytes than did normal controls. There was a concomitant increase in the number of B cells secreting antibodies reactive with a diverse panel of foreign and self antigens (including actin, myosin, tri-nitrophenylated keyhole limpet hemocyanin, ovalbumin, and retroviral gp160). By comparison, the number of B cells producing anti-DNA antibodies was increased disproportionately. The magnitude of this anti-DNA response correlated significantly with disease activity. Thus, B cell activation in human systemic lupus erythematosus had characteristics of both generalized (polyclonal) B cell activation and (auto)antigen-specific immune stimulation. PMID- 1719988 TI - Measurement of an adhesion molecule as an indicator of inflammatory disease activity. Up-regulation of the receptor for hyaluronate (CD44) in rheumatoid arthritis. AB - The hyaluronate receptor (CD44) molecule is a multifunctional cell surface protein involved in T cell activation, monocyte cytokine release, fibroblast locomotion, and lymphocyte binding to high endothelial venules. To study the roles CD44 molecules play in inflammatory synovitis, we measured expression of CD44 in inflamed and noninflamed synovial fluid and tissue, using indirect immunofluorescence assays on tissue sections and quantitative Western blot analysis. The ability of purified CD44 protein to modulate T cell responses was tested in T cell activation assays in which CD44-containing liposomes were added in vitro. CD44 was widely expressed on many synovial cell types, and synovial tissue from rheumatoid arthritis (RA) patients contained 3.5 times more CD44 than tissue from osteoarthritis patients and 10.7 times more than tissue from patients with joint trauma. The level of soluble CD44 in RA synovial fluid was elevated only in fluids with low cell counts (less than or equal to 7,000/mm3), and not in RA synovial fluid with higher cell counts. Soluble purified CD44 protein in liposomes partially suppressed T cell activation in vitro. These data demonstrate that CD44 is up-regulated on many synovial cell types in patients with RA, and that the level of CD44 present in synovial tissue is related to the degree of synovial inflammation. Determination of ways to inhibit the proinflammatory functions of immune cell membrane CD44 molecules may provide new therapeutic modalities for RA. PMID- 1719989 TI - Interactions of macrophages and monocytes with granulocytes in asthma. AB - The pathology of bronchial asthma demonstrates a multicellular process. The airway mucosa is infiltrated with both mononuclear cells and granulocytes, of which the eosinophil is particularly prominent. In an attempt to further the understanding of the interactions between different cells, we have elected to study the effects of monocyte/macrophage-derived cell products on granulocytes, because of the evidence for monocyte/macrophage activation in bronchial asthma. PMID- 1719990 TI - The direct determination of phase invariants provided by diffraction data measured at two different temperatures. AB - A procedure is described for the determination of the crystal structure phase invariants of a compound based on diffraction data measured at two different temperatures. This temperature difference replacement (TDR) technique is shown to provide phase-invariant information from experimentally measured X-ray diffraction data for two different test structures. Although the new method does not appear to be as powerful as single-derivative isomorphous replacement (SIR) phasing, it does appear to be capable of reliably determining a limited number of negative as well as positive phase-restricted invariants for structures containing as many as 300 non-H atoms in the asymmetric unit. PMID- 1719992 TI - Overview of Italian practice on setting priorities of chemicals hazards. PMID- 1719991 TI - Insights on the amino acid side-chain interactions of a synthetic T-cell determinant. AB - The effect of single amino acid substitutions at positions 18 and 20 on the T cell determinant (TD) character of peptide p12-26 from lambda repressor protein and on its recognition by a monoclonal antibody was studied by means of 40 synthetic peptides of a length of 15 amino acids. ELISA competition experiments showed that the identity of amino acid at position 20 is very important for antibody recognition, whereas that of amino acid at position 18 is much less important. In contrast, both Leu 18 and Ala 20 are important residues in defining the TD character of peptide p12-26. The most tolerated replacements, ordered in increasing disrupting power are: Ala 20 by Cys, Ser or Gly and Leu 18 by Ile or Val. Any other amino acid replacement completely abolishes the TD capacity of peptide p12-26. The peptides used in this study were synthesized using a multiple solid-phase peptide synthesizer newly designed. Their purity was very high as shown by amino acid sequence experiments. PMID- 1719993 TI - [Evaluation of methods for determination of enteric phages in water]. PMID- 1719994 TI - [Review of the distribution of Listeria monocytogenes and listeriosis]. PMID- 1719995 TI - [Blood pressure of elementary school children in Perugia]. PMID- 1719996 TI - [A service for the family: the health assistant visitor]. PMID- 1719997 TI - [The problem of drug dependence in Italy: trends of the phenomenon and proposal for a health-hygiene protocol for community health centers]. PMID- 1719998 TI - [Neonatal infections with Streptococcus group B: epidemiological, diagnostic and preventive aspects]. PMID- 1719999 TI - [The physician and the environment]. PMID- 1720000 TI - [Hantaan virus infection]. PMID- 1720001 TI - [Survival among the AIDS patients registered in Campania from January 1, 1985 to March 31, 1990]. PMID- 1720002 TI - Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification. AB - Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications. PMID- 1720003 TI - Effect of nucleotide concentration on specificity of sequence amplification. PMID- 1720004 TI - Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA. AB - A very simple procedure for the simultaneous preparation of genomic DNA and total RNA is described. The procedure is essentially the same for eukaryotes and prokaryotes except for the lysis buffer and can be used for small or large numbers of cells. Mammalian cells are lysed in sodium dodecyl sulfate and bacterial cells are lysed in Triton X-100, both in the presence of EDTA. RNA is obtained in the aqueous phase after phenol (acidic pH):chloroform:isoamyl alcohol extraction. DNA is eluted out of the organic phase (and the interface) into the aqueous phase by increasing the pH with highly basic 1 M Tris solution. The method is extremely rapid for small or large numbers of cells, and several large samples can be processed in one day. The qualities of both nucleic acids are excellent and the yield is high. PMID- 1720005 TI - Human alpha-fetoprotein purified from amniotic fluid enhances growth factor mediated cell proliferation in vitro. AB - Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly. PMID- 1720006 TI - Ion channels in boar sperm plasma membranes: characterization of a cation selective channel. AB - Plasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high-pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head. PMID- 1720007 TI - Expression of SV40-lacZ gene in mouse preimplantation embryos after pronuclear microinjection. AB - In order to study the expression of an exogenous gene in developing mouse embryos during the preimplantation period, DNA carrying the SV40 early promoter fused with the Escherichia coli beta-galactosidase gene (lacZ) was microinjected into the pronucleus of fertilized mouse eggs. Expression of lacZ gene was detected by staining embryos with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a substrate at pH 7.2. The embryos expressing the lacZ gene showed various intensities of blue staining, all showing a mosaic pattern. The exogenous gene was expressed from the 4-cell stage until the blastocyst stage. The proportion of embryos expressing the lacZ gene was maximal (38%) at the morula stage, and the expression was dependent on the presence of the SV40 promoter. PMID- 1720008 TI - Effects of actinomycin D and cycloheximide on transcript levels of IGF-I, actin, and albumin in hepatocyte primary cultures treated with growth hormone and insulin. AB - The stability of several RNA transcripts in cultured hepatocytes is known to increase when serum is omitted from the culture medium. In order to investigate possible mechanisms for this phenomenon, we examined the effects of actinomycin D and cycloheximide on the levels of actin, albumin, and insulin-like growth factor I transcripts in primary cultures incubated in serum-free medium. The levels of IGF-I and albumin transcripts per culture increased for the first 4 hours following addition of actinomycin D and then declined. The levels of actin transcripts and total RNA per culture declined immediately following actinomycin D addition in a manner consistent with exponential decay. IGF-I and albumin transcript levels were relatively unaffected by cycloheximide, while actin transcript levels increased 7-fold over 7 hours. The half-lives of actin transcripts and total RNA were calculated to be 4.6 to 7.7 hours and 11 to 19 hours, respectively, with no statistically significant correlation with hormone treatment. The data suggest that the stability of albumin and IGF-I transcripts, but not actin transcripts, is controlled in part by an actinomycin D-sensitive process. PMID- 1720009 TI - Peptidergic nerve terminals associated with the central lacteal lymphatics in the ileal villi of dogs. AB - Nerve fibers in the villi of the canine ileum were studied with special reference to their relation to the central lacteal. Immunohistochemically demonstrable nerve fibers containing substance P (SP) and calcitonin gene-related peptide (CGRP) were rather more numerous in the villi of the ileum than in those of the duodenum, as observed in our previous study (Ichikawa et al., 1991). They were distributed beneath the epithelium and associated with smooth muscle fibers. Besides these localizations, immunoreactive fibers were gathered, especially at the middle of the height of the villus, close to the endothelial cells of the central lacteal. This particular distribution of nerves was more evident in the ileum than in the duodenum. Electron microscope observation indicated beaded fibers containing large cored (peptidergic) and small clear vesicles coursing closely under the lacteal endothelium, partly intercalated by a basement membrane and partly in direct contact. The nerve fibers often penetrated the endothelial cell, being directly surrounded by its cytoplasm. Although the above-described findings essentially coincide with our previous observations in canine duodenum (Ichikawa et al., 1991), the present study in the ileum demonstrated occasional nerve fibers protruding into the lacteal lumen with a knob-like swelling. It is suggested that the SP and CGRP-containing nerves in problem might be sensory in nature, possibly monitoring mechanical information from the lumen and wall of the central lacteal. At the same time, these nerves are suggested to be secretory in nature, releasing the peptides to exert unknown effects upon the lacteal wall and its vicinity, presumably in response to luminal and mural stimuli. PMID- 1720010 TI - Substance P-immunoreactive neurons in the retina of two lizards. AB - The dendritic morphology and retinal distribution of substance P(SP) immunoreactive neurons was determined in two Australian lizard species Pogona vitticeps and Varanus gouldii, by using immunohistochemistry on retinal wholemounts and sectioned materials. In both species, two classes of SP immunoreactive neurons were described in the inner nuclear layer (INL) and classified as amacrine cells (types A and B). Type A amacrine cells had large somata and wide-field, bistratified dendrites branching in sublaminas 1 and 5 of the inner plexiform layer (IPL). Their morphology and retinal distribution differed between the two species. Type B amacrine cells in both species had small somata and small-field dendritic branching. A population of SP-immunoreactive neurons with classical ganglion cell morphology were identified in the ganglion cell layer (GCL). Immunostained ganglion cells occurred in larger numbers of Varanus gouldii than in Pogona vitticeps. In both species type B SP cells were the most numerous and were estimated to be about 60,000-70,000. They were distributed non-uniformly with a high density band across the horizontal meridian of the retina, from where the density decreased towards the dorsal and ventral retinal margins. In both species type A amacrine cells occurred in small numbers distributed sparsely in the peripheral retina. The faint immunostaining of SP immunoreactive neurons in the GCL, did not allow us to reliably determine their numbers and retinal distribution. The functional significance of SP immunoreactive amacrine and ganglion cells in the lizard retina remains to be determined. PMID- 1720011 TI - Management of breast cancer with bone metastases. PMID- 1720012 TI - Treatment of skeletal disease in breast cancer with clodronate. AB - Complications of breast cancer involving the skeleton include hypercalcaemia, bone pain and fracture. These complications arise because of progressive osteolysis which is in turn dependent on the activation of osteoclasts by tumour and host tissues. Clodronate is a powerful inhibitor of osteoclastic bone resorption which led us to evaluate its potential in metastatic breast cancer. When given intravenously it lowers serum calcium in the majority of hypercalcaemic patients. A convenient regimen is 600 mg iv as a single dose infused over several hours. We have additionally shown in a double-blind cross over study that this regimen also has a significant effect on bone pain. This had led us to assess the longer term effects of clodronate by mouth in a prospective double-blind study of patients with established skeletal metastases. These studies are not yet complete but the agent appears to prevent hypercalcaemia and trends are emerging which indicate that the incidence of bone pain and fractures may also decrease. PMID- 1720013 TI - An evaluation of the potential cost reductions resulting from the use of clodronate in the treatment of metastatic carcinoma of the breast to bone. AB - The reported studies of clodronate in the management of osteolytic bone metastases suggest a significant palliative role for this drug. In this paper we report on analysis of the hospital costs associated with the management of osteolytic metastatic disease, and an estimate of the potential cost/benefit impact of clodronate therapy. Two separate patient populations were assessed retrospectively. The first, a sample of 120 patients with symptomatic bone metastases who had died from metastatic breast cancer over the period 1980-1990, was used to define the natural history of the disease. A second non-concurrent patient group of 337 patients was evaluated to determine the mean cost of all hospital admissions for patients with bone metastases from breast carcinoma. The length of stay and costs for hospital admissions related to the bone metastases were also assessed, in addition to the cost of out-patient radiation therapy. Our cost/benefit value analysis suggests that there are significant savings to be gained from the use of clodronate if a 20% or greater reduction occurs in the incidence of fractures, hypercalcaemia, and hospital-based treatment for pain control (via radiotherapy). We also speculate that the quality of life of patients with osteolytic bone metastases may be improved with this agent. PMID- 1720014 TI - Clodronate: the potential for the future. AB - Osteolytic bone metastases secondary to breast cancer are extremely common, occurring in more than 50% of breast cancer patients. The resulting increased bone resorption leads to significant symptomatic morbidity caused by bone pain, hypercalcaemia and pathological fracture. Clodronate, an anti-osteolytic agent, inhibits osteoclastic bone resorption and has considerable therapeutic value. Recent studies have shown that clodronate is effective in the treatment of malignancy hypercalcaemia, relief of bone pain and decreases the risk of pathological fracture. The use of clodronate in the future, other than as a palliative therapy, may focus upon the prevention of osteolytic bone metastases at the time of primary diagnosis or later in the disease progression in those patients at risk, for example, those with non-osseous relapse. Since patients with bone metastases secondary to breast cancer often have an increased duration of survival, any agent that would decrease the symptomatic morbidity would have a significant impact upon quality of life, even more so if the actual development of osteolytic bone metastases was delayed or prevented. PMID- 1720015 TI - Computer-based tools for assessment and remediation of speech. AB - The electrolaryngograph (ELG), first conceived in the 1970s, has been used extensively as a tool in both research and clinical practice. Recent software developments have extended its clinical utility for both quantitative assessment of voice production and as an interactive visual display for remediation and teaching. In this paper, we discuss applications of the electrolaryngograph, highlighting general principles of computer-based assessment and remediation. PMID- 1720016 TI - Pigment content of cultured human melanocytes does not correlate with tyrosinase message level. AB - Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that posttranscriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes. PMID- 1720017 TI - Secretion and movement of wingless protein in the epidermis of the Drosophila embryo. AB - The segment polarity gene wingless encodes a cysteine rich protein which is essential for pattern formation in Drosophila. Using polyclonal antibodies against the product of the wingless gene, we demonstrate that this protein is secreted in the embryo and that it is taken up by neighbouring cells. The protein can be found two or three cell diameters away from the cells in which it is synthesized. We discuss the possible mechanisms which are responsible for this spatial distribution and its regulation during embryogenesis. PMID- 1720018 TI - 5-fluorouracil + folinic acid with cisplatinum and bleomycin in the treatment of advanced head and neck squamous cell carcinoma. AB - Thirty-six patients with advanced squamous cell carcinoma of the head and neck (SCCHN) were treated with a regimen including cisplatinum (CP) 30 mg/m2 i.v., 5 fluorouracil (5-FU) 500 mg/m2 i.v. bolus, folinic acid (FA) 200 mg/m2 i.v. in a continuous one-hour infusion, and bleomycin (B) 15 mg i.m. on the first and second days and repeated every 28 days. Thirty-three patients (25 with recurrent disease and 8 untreated) are evaluable for objective response. Of these, 4 (12%) achieved CR and 15 (45%) PR. All of the untreated patients responded. The mean duration of response in the patients with recurrent or metastatic disease was 5.5 months (range 2-10+). Remission of symptoms, such as pain and dysphagia, was obtained in 58% and in 44%, respectively. Subjective remission occurred almost exclusively in objectively responsive patients. The major side effects were leukopenia (55%) and nausea/vomiting (58%). This regimen is active in the treatment of advanced SCCHN. The quality of life may be improved in responsive patients. PMID- 1720019 TI - A modified Arg-Asp-Val (RDV) peptide derived during the synthesis of Arg-Glu-Asp Val (REDV), a tetrapeptide derived from an alternatively spliced site in fibronectin, inhibits the binding of fibrinogen, fibronectin, von Willebrand factor and vitronectin to activated platelets. AB - Characterization of a side-product obtained during the synthesis of Arg-Glu-Asp Val (REDV) with inhibitory activity in thrombin-activated platelet aggregation was carried out. The semipreparative column fractionation of REDV peptide was rechromatographed on an analytical HPLC column and revealed two peaks which were re-tested for inhibitory activity. Using amino acid analysis with sequencing and fast atom bombardment mass spectrometry (FABMS), the first peak was determined to be REDV with molecular mass of 517 Da, and the second peak was determined to be a modified RDV with a mass of 608 Da. The modified RDV peptide inhibited thrombin induced platelet aggregation with an IC50 of 200 microM, and complete inhibition occurred at 600 microM. However, the REDV peptide did not inhibit platelet aggregation up to 1 mM concentration. The modified RDV peptide eluted platelet glycoprotein IIb-IIIa complex that had been bound to GRGDSP-agarose. These studies show that the modified RDV peptide interacts with the platelet glycoprotein IIb-IIIa complex. Based on the collision-induced dissociation (CID) mass spectral data analysis, the modified RDV peptide has been characterized to contain an N-terminus blocking group on the Arg residue. The origin of this blocking group is presumed to have originated from decomposition products of the phenylacetamidomethyl (PAM) resin used in the solid-phase synthesis of the target peptide Arg-Glu-Asp-Val. PMID- 1720020 TI - Interaction of a brain extracellular matrix protein with hyaluronic acid. AB - A glial hyaluronate-binding protein (GHAP) was isolated from bovine spinal cord and partially characterized. Bovine GHAP consisted of three immunologically related polypeptides with molecular masses of 76, 64, and 54 kDa and isoelectric points of 4.1, 4.2, and 4.4, respectively. Peptide mapping and partial amino acid sequencing showed that all three polypeptides derive from the same protein. The protein was localized immunohistochemically with rabbit antisera in the white matter surrounding the myelinated axons. Sugar analyses indicated that the three polypeptides are glycosylated and the sugar residues account for at least 30% of their weight. After enzymatic deglycosylation, the apparent molecular mass of the bovine GHAP was reduced to 43 kDa. The biochemical properties of bovine GHAP were compared to those of human GHAP. Initial peptide mapping indicated similarities between bovine and human GHAP. Partial amino acid sequencing of bovine GHAP showed a striking identity (up to 90%) with human GHAP and with the hyaluronate binding domain of the large human fibroblast proteoglycan, versican. Bovine and human GHAP were demonstrated to bind specifically to hyaluronic acid (HA) with one protein molecule binding to an average 17 disaccharide repeating units. The binding of bovine and human GHAP was inhibited by oligosaccharides of HA and specifically by the octamer. Salt concentrations of up to 1 M NaCl had very little effect on the binding of the GHAP to HA. The GHAP-HA interaction was pH dependent. Dissociation only took place at low pH (less than 3.5). Analysis of several polypeptides derived from GHAP by limited proteolysis allowed us to conclude that one of the tandem repeated sequences is sufficient for HA binding and that the aminoterminal domain (which contains an immunoglobulin-like fold) is not involved in the GHAP-HA-binding event. PMID- 1720021 TI - Effect of limited proteolysis in the 8th loop of the barrel and of antibodies on porcine pancreas amylase activity. AB - The porcine pancreatic alpha-amylase is a (beta/alpha)8-barrel protein, containing domains A and B (peptide sequence 1-403) and a distinct C-domain (peptide sequence 404-496). Separation of the terminal C-domain from the A and B domains has been attempted by limited proteolysis in the hinge region. Subtilisin was found to hydrolyse amylase between residues 369 and 370 situated in the loop between the eighth beta-strand and alpha-helix. The cleaved amylase was isolated by chromatofocusing and found to retain about 60% of the activity of the native enzyme, while the isolated fragments were inactive. Antigen binding fragments prepared from polyclonal antibodies to native amylase and the CNBr-fragment P1 (peptide sequence 395-496) respectively, were tested for influence on the enzyme activity. Antibodies directed against P1 had no effect whereas antibodies against the peptide sequence 1-394 and amylase respectively inhibited hydrolysis of substrates having four or more glucose residues but not of shorter oligomaltosides. Crystallographic analysis revealed that changes in the region of residue 369 might affect the conformation of the active site as well as of a second binding site. This site, located on the enzyme surface, is proposed to be required for the hydrolysis of larger substrates. PMID- 1720022 TI - Effect of alpha-fetoprotein and albumin on the uptake of polyunsaturated fatty acids by rat hepatoma cells and fetal rat hepatocytes. AB - The uptake of polyunsaturated fatty acids by rat hepatoma cells and fetal hepatocytes has been studied using albumin and alpha-fetoprotein (AFP) as carrier proteins. Both types of cells took up linoleic (18:2(n-6)) and linolenic (18:3(n 3)) fatty acids at the same rate when they were added complexed either to albumin or AFP at a 1:1 molar ratio. At 37 degrees C a greater incorporation of arachidonic acid (20:4(n-6)) and mainly docosahexaenoic acid (22:6(n-3)) was observed when these acids were bound to albumin (6.5 nmol 20:4(n-6); 6.4 nmol 22:6(n-3) /million cells) as compared to AFP (5.5 nmol 20:4(n-6); 4.3 nmol 22:6(n 3)/million cells). The 20:4(n-6) and 22:6(n-3) uptake seems to be inversely related to the apparent association constants (k'a) between these fatty acids and both proteins (1.3 and 1.5 x 10(-7) M-1 20:4(n-6) and 22:6(n-3) for albumin; 6.4 and 54.0 x 10(-7) M-1 20:4(n-6) and 22:6(n-3) for AFP). Experiments carried out at 4 degrees C indicated that binding of 20:4(n-6) (0.83 nmol/million cells in presence of albumin; 2.16 nmol/million cells in presence of AFP) and 22:6(n-3) (0.83 nmol/million cells in presence of albumin; 1.32 nmol/million cells in presence of AFP) by cell membranes was also inversely related to the k'a of these proteins. At 4 degrees C, the k'a of AFP and albumin for 20:4(n-6) and 22:6(n-3) changed considerably (12.7 and 9.6 x 10(-7) M-1 20:4(n-6) and 22:6(n-3) for albumin; 3.9 and 14.6 x 10(-7) M-1 20:4(n-6) and 22:6(n-3) for AFP) with respect to the k'a calculated at 37 degrees C. Hence, k'a values were higher for albumin and lower for AFP than the corresponding values at 37 degrees C. It was concluded that uptake by cells and interaction of fatty acids with cell membranes depend mainly on the k'a of fatty acids and carrier proteins at equilibrium. PMID- 1720023 TI - Differential expression in Escherichia coli of the alpha and beta forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure. AB - Synthetic DNA fragments encoding the entire open-reading frame of human heparin binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed. When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield. However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector. Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter. PMID- 1720024 TI - Immunoglobulin variable gene expression in human autoantibodies. PMID- 1720025 TI - Molecular mimicry of self-antigens. PMID- 1720026 TI - Immunodominant determinants of thyroglobulin associated with autoimmune thyroiditis. PMID- 1720027 TI - The natural anti-Gal antibody: evolution and autoimmunity in man. PMID- 1720028 TI - Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies. AB - BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells. PMID- 1720029 TI - Molecular recognition between ligands and nucleic acids: novel pyridine- and benzoxazole-containing agents related to Hoechst 33258 that exhibit altered DNA sequence specificity deduced from footprinting analysis and spectroscopic studies. AB - The syntheses of certain analogues of the DNA minor groove binding agent Hoechst 33258 designed to exhibit altered sequence recognition are described. The structural modifications include the following: substitution of pyridine for the benzene ring of the benzimidazole moiety, replacement of one benzimidazole unit by a benzoxazole in the two possible orientations with respect to the DNA receptor, and a synthesis of 2,2'-m-phenylene-bis[6-(4-methyl-1 piperazinyl)benzimidazole]. Sequence recognition of these agents on a HindIII/EcoRI fragment of pBR322 DNA was determined by MPE footprinting procedures. Some of the analogues exhibited altered DNA sequence preference compared with Hoechst 33258. In particular, a structure bearing a benzoxazole moiety with the oxygen oriented inward to the minor groove together with an inward-directed pyridine nitrogen appears to confer the property of recognition of a GC base pair within the binding sequence. The possible factors, structural, stereochemical, and electrostatic, contributing to the altered DNA sequence recognition properties are discussed. PMID- 1720030 TI - Systemic tumor embolism following thoracotomy partially masked by postoperative epidural analgesia. PMID- 1720031 TI - Aprotinin and thrombus formation on pulmonary artery catheters: a piece of the coagulation puzzle. PMID- 1720032 TI - Early formation of thrombi on pulmonary artery catheters in cardiac surgical patients receiving high-dose aprotinin. PMID- 1720033 TI - Cardiovascular effects of large doses of pentamorphone in the dog. AB - The cardiovascular effects of large doses of pentamorphone were evaluated in nine mongrel dogs basally anesthetized with sodium thiopental, 25 to 30 mg/kg, intravenously. All dogs were mechanically ventilated with 100% oxygen, and the PaCO2 was maintained between 35 and 40 mm Hg. Mean arterial pressure (MAP), central venous pressure, heart rate (HR), cardiac output (CO), pulmonary artery pressure, and pulmonary artery occluded pressure were measured, and stroke volume and systemic and pulmonary vascular resistances were calculated. Baseline measurements were obtained, then pentamorphone, 10 micrograms/mL, was given as an intravenous infusion at 2.5 micrograms/kg/min. Additional data were obtained after infusion of 25, 50, 75, 100, 125, 150, 200, 250, 300, and 350 micrograms/kg of pentamorphone. The inspired gases were then changed to 50% nitrous oxide in oxygen, and after a 20-minute equilibration period, an additional set of data was collected. Pentamorphone, 25 micrograms/kg, decreased HR 50%, MAP 65%, and CO 54%. No further changes in any measured or calculated variables were observed with additional doses of pentamorphone. The addition of 50% nitrous oxide to the inspired gas mixture had no effect on any measured or calculated hemodynamic variable. The minimal hemodynamic effects of pentamorphone in the dog suggest that further investigation into its use as an anesthetic is warranted. PMID- 1720034 TI - Granulocyte colony-stimulating factor and its receptor. PMID- 1720035 TI - Adhesive functions of platelets lacking glycoprotein IV (CD36). AB - Glycoprotein IV (GPIV; CD36 or GPIIIb) is a cell surface glycoprotein that has been proposed as mediating a number of physiologically important processes such as the adhesion of platelets to thrombospondin (TSP) and collagen, the cytoadherence of Plasmodium falciparum-infected erythrocytes, and the TSP dependent interaction of monocytes with platelets and macrophages. Because platelets of the Naka-negative phenotype have recently been shown to lack detectable GPIV, their availability offered the opportunity to test directly these hypotheses regarding its adhesive functions. It has been found that Naka negative platelets and monocytes do not support cytoadherence of P falciparum infected erythrocytes. Naka-negative platelets are deficient in the initial stages of their adhesion to fibrillar collagen and this defect is most marked under Mg(2+)-free conditions. Finally, the ability of Naka-negative platelets to bind TSP before or after activation is unimpaired as compared with normal controls. These results do not support a role for GPIV as the TSP receptor. PMID- 1720036 TI - Proliferation of human myeloid leukemia cell line associated with the tyrosine phosphorylation and activation of the proto-oncogene c-kit product. AB - We investigated the expression, degree of phosphorylation, and activation of the proto-oncogene c-kit product before and after stimulation with the c-kit ligand in a human factor-dependent myeloid leukemia cell line, MO7E. The culture supernatant of the BALB/3T3 fibroblast cell line, which contains the ligand for the murine c-kit product, was found to stimulate proliferation of the MO7E cell line in a dose-dependent manner. The proliferation was significantly inhibited by a tyrosine kinase inhibitor, genistein. An immunoblot technique with a monoclonal antibody specific for phosphotyrosine, showed that there was rapid, dose dependent tyrosine-phosphorylation of the c-kit product in response to murine c kit ligand. Furthermore, the murine c-kit ligand increased autokinase activity of the c-kit product in vitro. Similar results were obtained with human stem cell factor (SCF), a recombinant human ligand for the c-kit product. These results suggest that the phosphorylation and activation of the c-kit product are involved in proliferative signals of some human leukemia cells, as well as of normal hematopoietic cells. PMID- 1720037 TI - In vivo interleukin-1 (IL-1) administration indirectly promotes type II IL-1 receptor expression on hematopoietic bone marrow cells: novel mechanism for the hematopoietic effects of IL-1. AB - Interleukin-1 (IL-1) has profound stimulatory effects on hematopoiesis but the mechanism(s) of action remain unknown. The direct action of IL-1 on hematopoietic progenitor cells requires the presence of a specific IL-1 receptor (IL-1R). In this report, we tested the effect of in vivo IL-1 treatment on the expression of IL-1R on bone marrow (BM) cells. Injection of mice with IL-1 results in a marked upregulation of IL-1R on light-density BM cells as on a subpopulation enriched for myeloid precursors. Pretreatment of mice with anti-type I IL-1R antibody (35F5), which has been shown to prevent the radioprotective effect of IL-1, also blocked IL-1-induced IL-1R expression on BM cells. This antibody did not directly bind and block IL-1 binding to the type II IL-1R expressed on hematopoietic cells, suggesting that IL-1R upregulation by IL-1 is indirect. It is therefore possible that IL-1 acts on type I IL-1R-expressing accessory cells such as stromal cells or T cells to induce production of hematopoietic growth factors (HGFs). In support of this, granulocyte colony-stimulating factor administration can induce the increase of IL-1R on BM cells. Thus, the increased expression of IL-1R on hematopoietic BM cells by IL-1 is indirect, probably mediated in part through endogenous HGF production. These results also suggest that the restorative hematopoietic effect of IL-1 occurs through both indirect and direct mechanisms. PMID- 1720038 TI - CD34 expression by stromal precursors in normal human adult bone marrow. AB - Normal bone marrow cells were isolated by fluorescence-activated cell sorting (FACS) on the basis of CD34 antigen expression and then assayed in vitro for colonies of fibroblastic cells (fibroblast colony-forming units [CFU-F]). Greater than 95% of detectable CFU-F were recovered in the CD34+ population, while their numbers were markedly depleted in the CD34- population. Additional experiments showed that the majority of CFU-F exhibited high forward and perpendicular light scatter and low-density CD34 antigen. Growth of sorted cells in medium optimized for long-term marrow culture (LTMC) produced a complex mixture of adherent stromal elements including fibroblasts, adipocytes, smooth muscle cells, and macrophages. Monoclonal antibody STRO-1, which identifies bone marrow stromal cells, reacted with approximately 5% of CD34+ cells, which included all CFU-F and stromal precursors in LTMC. Experiments using soybean agglutinin (SBA) further showed that these stromal elements were restricted to a population of bone marrow cells with the phenotype CD34+/SBA+. These properties of stromal precursors are quite distinct from those of primitive hematopoietic progenitors, showing that although the precursors of the hematopoietic and stromal systems share expression of CD34, they are otherwise phenotypically distinct cell types. PMID- 1720039 TI - Effect of c-kit ligand on in vitro human megakaryocytopoiesis. AB - An evaluation of the effects of a recombinant, soluble form of the c-kit ligand alone and in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) on the regulation of human megakaryocytopoiesis was performed using a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of the c-kit ligand on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit megakaryocyte (BFU-MK), and the more differentiated colony-forming unit megakaryocyte (CFU-MK) were determined. The c-kit ligand alone had no megakaryocyte colony-stimulating activity (MK-CSA) but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. The range of synergistic interactions of c kit ligand varied with the class of MK progenitor cell assayed. In the case of the BFU-MK, the c-kit ligand synergistically augmented the numbers of colonies formed in the presence of IL-3, but not GM-CSF, but increased the size of BFU-MK derived colonies cloned in the presence of both of these cytokines. However, at the level of the CFU-MK, c-kit ligand synergized with both GM-CSF and IL-3 by increasing both colony numbers and size. Although the c-kit ligand alone exhibited limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3, but not GM-CSF, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that the c-kit ligand plays a significant role in this process by amplifying the MK-CSA of both GM-CSF and IL-3. PMID- 1720040 TI - Expression and functional role of the proto-oncogene c-kit in acute myeloblastic leukemia cells. AB - The c-kit proto-oncogene encodes a receptor tyrosine kinase that is thought to play an important role in hematopoiesis. In a series of human acute myeloblastic leukemia (AML), the expression of the c-kit proto-oncogene and its product was studied by means of Northern blot and immunoblot analyses. The c-kit mRNA was expressed in 20 of 25 cases of AML, and in those cases the product of the c-kit proto-oncogene was detected by immunoblotting with anti-c-kit antibody. The expression of c-kit transcripts and protein was barely detectable in normal bone marrow cells as a control. The expression of c-kit transcript did not correlate with the French-American-British classification nor clinical manifestations. In 6 of 11 cases that expressed c-kit product, AML cells were found to proliferate in response to recombinant human stem cell factor (rhSCF), the ligand for c-kit, and the synergistic stimulation of AML cells was observed by rhSCF and granulocyte macrophage colony-stimulating factor. Immunoblotting with anti-phosphotyrosine antibody showed that the c-kit receptor protein was detectably phosphorylated in 7 of 12 cases tested before the stimulation with rhSCF, while the rhSCF treatment resulted in an increased tyrosine phosphorylation of c-kit in AML cells. These results indicate that c-kit proto-oncogene is expressed in most cases of AML and is functional in terms of supporting proliferation. PMID- 1720042 TI - The 1990 Nordic Oncology Seminar. Regional treatment of cancer research frontiers and direction of progress. Storlien, Sweden, March 21-24, 1990. Proceedings. PMID- 1720041 TI - Characterization of the complement sensitivity of calcium loaded human erythrocytes. AB - A deficiency of membrane proteins having a glycosylphosphatidylinositol (GPI) anchor is characteristic of the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) and is currently believed to be the basis for the enhanced susceptibility to lysis by activated complement observed in these cells. Our recent observation that GPI-anchored proteins are preferentially lost into membrane vesicles shed from normal erythrocytes after calcium loading led us to examine the hypothesis that the remnant erythrocytes might also have increased sensitivity to complement-mediated hemolysis. Indeed, red blood cells treated in such a manner became more sensitive to lysis by antibody and complement or to lysis initiated by activated cobra venom factor complexes (CoFBb). As a consequence of membrane vesiculation, the erythrocytes lost up to approximately 50% of their immunoreactive decay-accelerating factor and 25% to 30% of their immunoreactive membrane inhibitor of reactive lysis (MIRL). Closer examination of the defect responsible for the marked increase in sensitivity to CoFBb-initiated hemolysis seen in calcium-loaded erythrocytes showed that a complex combination of factors produced the defect. These included a decrease in both functional and immunoreactive MIRL and depletion of intracellular potassium and adenosine triphosphate (ATP). These results suggest the possibility that loss of DAF and MIRL via membrane vesiculation, as well as decreases in intracellular potassium and/or ATP, might contribute to the phenotype of PNH erythrocytes. Further, normal or pathologic red blood cells might develop a PNH-like defect after membrane vesiculation if sufficient decreases in potassium and ATP also occurred. PMID- 1720043 TI - Oncogenes and growth factors--an updating. PMID- 1720044 TI - Anti-vascular cancer therapy: is it a dream or reality? PMID- 1720045 TI - Pharmacokinetic rational for regional chemotherapy. PMID- 1720046 TI - How to improve regional chemotherapy by influencing tumor blood flow--an overview. PMID- 1720047 TI - Mechanisms of tumor cell resistance to cancer chemotherapeutic drugs. PMID- 1720048 TI - Cellular pharmacologic strategies for overcoming drug resistance: potential applications to regional therapy. PMID- 1720049 TI - Liposomes for drug targeting in cancer chemotherapy. PMID- 1720050 TI - Carriers in cancer treatment: monoclonal antibodies. PMID- 1720051 TI - Monoclonal antibodies for therapy of human malignant tumours. PMID- 1720052 TI - Liver surgery for liver metastases. PMID- 1720053 TI - Cytoreductive surgery and intraperitoneal chemotherapy with peritoneal spread of cystadenocarcinoma. PMID- 1720054 TI - Intraperitoneal antineoplastic agents in the management of ovarian cancer. PMID- 1720055 TI - Biological response modifiers in regional cancer therapy. PMID- 1720056 TI - Tumor biology--colorectal cancer as an illuminating example. PMID- 1720057 TI - Trophic response and morphological changes in pancreas of caerulein treated rats: dose and time dependent effects. AB - The present study was performed to determine the effect of the duration of chronic caerulein administration given at different doses, on the rat pancreas. Four groups of rats, one treated with 0.9% NaCl (control) and the others with caerulein 2, 5 and 10 micrograms/Kg twice a day i.p. were used. After a treatment period of 15, 30 and 60 days, 6 rats from each group were anesthetized, the pancreas was removed, and growth and composition of pancreatic tissue were determined. Small samples were taken for histological examination. Caerulein induced pancreatic hyperplasia and hypertrophy. The dose of caerulein used and the length of the treatment did not significantly modify the trophic effect. Focal perivascular and periductular lymphomonocytic infiltrates associated with cellular abnormalities were seen at 30 and 60 days. The results suggest that 1) the trophic effect of caerulein is not dose-and-time dependent and 2) morphological abnormalities can appear during long term treatment with CCK analogous. PMID- 1720058 TI - Relationship between alpha-fetoprotein serum levels, tumour volume and growth rate of hepatocellular carcinoma in a western population. AB - In order to detect a possible relationship between alpha-fetoprotein (AFP) levels, total tumour volume at the moment of the discovery and the tumour volume doubling time, we studied a population of 138 patients, affected by Hepatocellular Carcinoma (HCC) discovered at abdominal ultrasound (US) examination and confirmed by liver biopsy in all cases. In each patient the serum AFP level was determined within a week before or after the US examination. A small therapy-free subgroup of 23 patients, was also serially observed for a mean period of 4 months, so making possible the evaluation of the tumour volume doubling time and its relationship with the initial value of AFP. In 81 patients (58.7%) the serum AFP resulted less than 20 ngr/ml in 21 (15.2%), between 20 and 200 and in 36 (26.1%) greater than 200ngr/ml. No statistical correlation was found between the tumour volume calculated on the basis of the US image at the moment of the discovery and the AFP level, even though very high levels (greater than 3000) were found only in large tumours. Furthermore the tumour doubling time was not correlated with the initial value of AFP. PMID- 1720059 TI - Localization of hepatitis C virus (HCV) antigen by immunohistochemistry on fixed embedded liver tissue. AB - An immunohistochemical method of staining HCV-related antigens in fixed-embedded liver biopsies is described. Two primary antisera were used: 1) a high titre anti HCV human IgG separated from an anti-HCV positive serum; and 2) rabbit anti-HCV antibodies. In our experience this method proves to be reproducible and shows a good correlation with serologic results. PMID- 1720060 TI - [Epidemiological studies of air pollution and health effects in areas near roadways with heavy traffic in Tokyo]. AB - Recent concern regarding health effects of air pollution in Japan has concentrated mainly on traffic-induced air pollution and its health effects in large cities. In Japan, where many people in large cities have been living near major roadways, the increase of automobile exhaust due to heavy traffic congestion will predictably cause a greater impact on people living near major roadways. We surveyed the characterization of residential suspended particulate matter (SPM) and nitrogen dioxide (NO2) concentrations along the major roadways in Tokyo, along with a health survey on the respiratory conditions of residents living in the same area, to examine the relationships between indoor pollutant levels, prevalence of respiratory symptoms and distance from roadways. The environmental monitoring was conducted in five phases. Using a newly developed SPM sampler and NO2 filter badge, continuous 4 day (96 hours) measurements were conducted in two hundred residential homes for four weeks. NO2 was measured in the living room, kitchen and outside of each home, while SPM was monitored in the living room. Health information was collected in October 1987 using ATS-DLD self administered questionnaires. Of the 1,093 homes investigated, responses from 805 homes were received. The following results were obtained. SPM and NO2 concentrations showed large variations. Indoor pollution levels mostly depended on indoor sources, i.e. cigarette smoking and unventilated space heaters, and the effects of those indoor sources were influenced by the building structure with respect to air tightness. An association between increase in pollutant levels and the distance from the roadway was observed. However its effect is small compared to indoor source effects. The prevalence rate of respiratory symptoms was higher in those areas nearest roadways with heavy traffic both in children and adults. These results suggest the presence of a relationship between automobile exhaust and health effects. PMID- 1720061 TI - [Current status of air pollution in Sao Paulo, Brazil: effects and problems associated with the introduction of ethanol-fueled motor vehicles]. AB - Recently suggestions have been advanced that alternative fuels including ethanol, methanol or methane instead of so called "fossil fuels" may help improve the current conditions of air pollution. According to results of general survey in Sao Paulo, since their introduction in 1978, ethanol-fueled cars have increased their share to almost 50% of all light vehicles in 1983. The current status of air pollution in Sao Paulo metropolitan area (SPMA) is described in relation to the use of such alternative fuel. The average concentrations in air of SO2 and lead have been decreasing drastically during the period of 1982-88, whereas non methane hydrocarbon, NO2 and O3 levels have been increasing to attain the worst levels in the world as indicated in Fig. 2. The use of ethanol-fuel, which contains less sulphate and lead, is thought to have contributed more or less to the above reductions of SO2 and lead in the air. However, the pollutants that have increased may derive mainly from diesel and gasoline exhausts of heavy vehicles. The general state of air pollutions appears not to have been improved, suggesting the difficulty in resolving air pollution issues. On the other hand, a current problem specific to ethanol-fuel is the aldehydes or other carcinogenic components in exhaust. Peak formaldehyde concentration, for example, have been reported to have reached 159 ppb in SPMA, which may be one of the highest levels shown in ambient air.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720063 TI - Aprotinin and cardiac surgery. PMID- 1720062 TI - Ion channels: this year's Nobel prize in physiology or medicine. PMID- 1720064 TI - Local infiltration of an angiogenic growth factor does not stimulate the delay phenomenon. AB - The role of angiogenesis in the delay phenomenon is unclear. In this study a potent angiogenic growth factor, basic fibroblast growth factor (bFGF) was used to ascertain the importance of angiogenesis in this phenomenon. bFGF (100 micrograms) was infiltrated beneath the panniculus carnosus on the dorsum of 50 rats. Another 50 rats received saline vehicle infiltration only. Ten days later a modified McFarlane flap (10 x 3 cm) was elevated and biopsies collected. Flap blood flow was determined by laser Doppler before and after elevation. The mean surviving length (Group I--71.3 +/- 4.6 mm and Group II--73.4 +/- 5.5 mm) and Doppler flow measurements were comparable between the two groups. Animals treated with bFGF showed marked perivascular changes and proliferation of fibroblasts, but no increase in the number or size of capillaries was seen. This lack of angiogenesis suggests pharmacologically mediated delay may require more than just an angiogenic stimulus. PMID- 1720065 TI - [Advances in basic and clinical researches on liver cancer]. PMID- 1720066 TI - Effects of cyclic adenosine 3',5'-monophosphate on amino acid transport and incorporation into protein in the rat aorta. AB - To assess the effects of cyclic AMP on amino acid transport and incorporation into aortic tissue protein, rat aortic rings were incubated with the cyclic AMP analog, N6-monobutyryl cyclic AMP (MBcAMP), the phosphodiesterase inhibitor, 3 isobutyl-1-methylxanthine (MIX), and radiolabeled amino acids. Subsequently, the aortic rings were homogenized in 5% trichloroacetic acid (TCA) and processed for liquid scintillation counting. Radioactivity present in the TCA supernatant following centrifugation was used to estimate amino acid transport. TCA precipitable radioactivity was used as a measure of amino acid incorporation into protein. MBcAMP induced an increase in the uptake of [3H]alpha-aminoisobutyric acid into aortic rings and an increase in the incorporation of radiolabeled proline and leucine into TCA-precipitable protein. Similar effects were observed with low concentrations of MIX (0.025-0.25 mM); however, at higher concentrations of MIX, there was an attenuation of the effect or frank inhibition. Maximum stimulation of transport was observed within 90-120 min of the addition of MIX or MBcAMP to the incubation medium, whereas the effect on amino acid incorporation was not detectable until after 12 h of exposure to MIX or MBcAMP. The effects of cyclic AMP on transport were observed in both the tunica media and the tunica adventitia, whereas the effects on amino acid incorporation into protein were observed only in the tunica media. These data are consistent with a possible role for cyclic AMP in promoting changes in the tunica media that could lead to the development of vascular hypertrophy. PMID- 1720067 TI - Use-dependent decline in rat aorta sensitivity to contraction by potassium. AB - Aortic rings excised from rats at 12 weeks of age showed a decrease in responsiveness during repeated contraction by increasing potassium concentration. By comparison, aortic rings obtained from rats at 22 and 26 weeks exhibited less loss or an increase in responsiveness to high potassium concentration during repeated contraction. The decrease in responsiveness to potassium in aortae of young rats was not due to the extended interval of incubation of these tissue in vitro. Aortic rings incubated without stimulation for 4 h following a reference contraction showed no change in contractile response to potassium. However, the magnitude of loss in responsiveness to potassium did appear to be related to the potassium concentration and the length of time the tissues were exposed to the high potassium solutions. Contraction of the tissue at 60 versus 30 mM KCl or extending the interval in depolarizing solution from 15 to 60 min significantly enhanced the decline in tissue responsiveness to potassium. The interruption of a series of potassium-induced contractions by exposure of the tissue to contractile (serotonin, norepinephrine) or relaxant (acetylcholine, isoproterenol) stimuli had no effect on the loss in responsiveness to potassium. However, injection of the calcium channel agonist, Bay K 8644, into the incubation media restored responsiveness to potassium. Concentration-response curves indicated that both sensitivity and the maximal response to potassium were reduced in aortic rings repeatedly contracted with potassium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720068 TI - Characterization of bradykinin receptors in peripheral organs. AB - Bradykinin (BK) and related kinins are potent stimulants of the rabbit jugular vein, the hamster urinary bladder, and the guinea pig trachea. The characterization of kinin receptors in these tissues was made with agonists and antagonists. Results obtained with agonists indicate that bradykinin and kallidin are much more active than des-Arg9-BK and suggest the presence of B2 receptors in the three organs. Some new agonists were also tested and the BK analogue, [Hyp3,Tyr(Me)8]BK, was found to be a potent and selective stimulant of the three preparations, with pD2 values of 8.56, 8.00, and 8.39, respectively, but inactive on the rabbit aorta (a B1-receptor system). Contractile effects of kinins in the rabbit jugular vein and hamster urinary bladder were reduced or eliminated by B2 receptor antagonists but at different concentration levels; e.g., acetyl-D Arg[Hyp3,D-Phe7]BK showed pA2 values of 7.78 on the rabbit jugular vein but only 5.72 on hamster urinary bladder. This compound contracted the guinea-pig trachea and was found to be inactive as an antagonist on this preparation. Contractions of the hamster urinary bladder and the guinea-pig trachea in response to bradykinin were markedly reduced or eliminated by indomethacin and by BW 755C, while those of the rabbit jugular vein were not modified. The present findings indicate that the myotropic effect of kinins on the rabbit jugular vein depends on the activation of B2 receptors and suggest that B2 receptors are largely responsible also for the response of the hamster urinary bladder. B2 receptors and (or) a nonreceptor mechanism appear to be involved in the stimulant effects of the kinin agonists and some antagonists in the guinea-pig trachea. PMID- 1720069 TI - A role for xanthine oxidase in the loss of cytochrome P-450 evoked by interferon. AB - It has been suggested that the loss of cytochrome P-450, which is mediated by interferon and its inducers, can result from the generation of free radical species by the enzyme xanthine oxidase. Cytochrome P-450, aminopyrine N demethylase, and ethoxyresorufin deethylase were depressed by 35, 36, 38%, respectively, in the livers of hamsters 24 h following the administration of a synthetic interferon (IFN-alpha-Con1) which contains the most frequent amino acid sequences of the human subtypes. Interferon increased the activities of the D and O forms of xanthine oxidase by 65 and 74%, respectively, in the same animals. The induction of the D form of xanthine oxidase, which is the precursor of the O form, preceded the loss in cytochrome P-450. The protein synthesis inhibitor, actinomycin D, prevented the interferon-induced loss of drug biotransformation and the increase in xanthine oxidase. The free radical scavenger, alpha tocopherol, and the xanthine oxidase inhibitor, allopurinol, also prevented the loss of cytochrome P-450 mediated by the interferon inducer poly rI.rC. In chickens in which xanthine oxidase cannot be formed, poly rI.rC had no effect on cytochrome P-450 levels. These results suggest that xanthine oxidase induction may play some role in the interferon-mediated loss of cytochrome P-450. PMID- 1720070 TI - Some patients with primary antiphospholipid syndrome have increased circulating CD5+ B cells that correlate with levels of IgM antiphospholipid antibodies. AB - Antibodies against bromelain-treated erythrocytes occurring in normal mice are germline gene-encoded IgM natural autoantibodies that are secreted by CD5+ B cells, and react primarily with phosphatidylcholine (PTC), but may crossreact with cardiolipin (aCL). Some of the IgM antiphospholipid antibodies (aPL) present in patients with the recently described primary antiphospholipid syndrome (PAPS) also react with PTC and could thus be natural autoantibodies akin to those found in mice. Patients with PAPS (n = 20) were found to have increased total B cells, as well as CD5 + B cells, in their peripheral blood, but normal total lymphocytes, as well as CD4 and CD8 T lymphocytes, compared to normal controls. The 6 PAPS patients with increased CD5+ B cells were found to have increased levels of IgM aPL, including aPTC. In them we also found a correlation between the number of CD5+ B cells and the levels of IgM aCL. Our findings suggest that within the family of aPLs present in patients with PAPS there may be some that could be IgM natural autoantibodies secreted by CD5+ B cells. PMID- 1720071 TI - Screening for fetal and genetic abnormalities. AB - Screening for genetic abnormalities is an integral part of obstetrics. Prior to initiating screening, however, several prerequisites must be met: (i) capacity to alter clinical management, (ii) cost effectiveness, (iii) reliable means (usually assays) of assessment, and (iv) capacity to handle problems. In all pregnancies one should determine in systematic fashion whether family history places a pregnant woman at increased risk over the background risk of 2-3% congenital anomalies. All women over age 35 years at delivery should be offered prenatal cytogenetic testing, and women of all ages should be offered maternal serum alpha fetoprotein screening for neural tube defects. Screening ostensibly normal populations is appropriate in certain ethnic groups to determine heterozygosity for selected disorders: Blacks for sickle-cell anaemia, Mediterranean people for beta-thalassaemia, Southeast Asians and Filipinos for alpha-thalassaemia, Ashkenazi Jews and perhaps French-Canadians for Tay-Sachs disease. Cystic fibrosis screening (delta F508 mutations) is not currently recommended for the general populations, but should be offered to relatives of an individual having delta F508 cystic fibrosis. Irrespective of the extent of screening programmes for Mendelian traits, the mutant allele will remain in the general population because by far the greatest genetic load lies in clinically normal heterozygotes, affected contributing far less to the load despite the obvious clinical effect. PMID- 1720072 TI - From medical certainty to medical amulets: three aspects of ancient therapeutics. PMID- 1720073 TI - Dietetic and pharmacological therapy: a dilemma among fourteenth-century Jewish practitioners in the Montpellier area. PMID- 1720074 TI - The history of therapeutics. PMID- 1720075 TI - Characteristic features of eighteenth-century therapeutics in Germany. PMID- 1720076 TI - The origins of homoeopathy in Germany. PMID- 1720077 TI - Science, healing, and the physician's identity: a problem of professional character in nineteenth-century America. PMID- 1720078 TI - "To operate or not to operate?" Scientific and extraneous factors in therapeutical controversies within the Swiss Society of Surgery 1913-1988. PMID- 1720079 TI - Phentolamine mesylate: the antidote for vasopressor extravasation. PMID- 1720080 TI - Role of iron and proteins of iron metabolism in cell growth. Potential for manipulations of cellular iron metabolism in modulating cell proliferation. PMID- 1720081 TI - Characterization of the murine monoclonal antibody NL07 specific for the human thrombospondin receptor (CD36 molecule). PMID- 1720082 TI - Preliminary characterization of a panel of monoclonal antibodies to human factor VII. PMID- 1720083 TI - PADGEM, a leukocyte receptor on activated platelets. Biology and application to in vivo medical diagnostics. PMID- 1720084 TI - The role of Rhipicephalus appendiculatus and R. evertsi evertsi males in inducing resistance in laboratory animals: preliminary studies. AB - Guinea-pigs infested with male ticks of the species Rhipicephalus appendiculatus, and rabbits infested with R. evertsi evertsi, acquired immunity to conspecific female ticks. The hosts were first infested with male ticks and thereafter were challenged with males and females of the same species. The mean weight of the engorged females of R. appendiculatus fed on guinea pigs previously infested with male ticks was 509.0 (+/- 41.4) mg compared with that of females fed on control guinea pigs (651.2 +/- 31.8 mg). Similar weight differences were observed for R.e. evertsi females which fed on rabbits previously infested three times with male ticks. The mean weight of the female ticks which fed on these rabbits was 520.1 (+/- 29.8) mg compared with 640.7 (+/- 30.2) mg of R.e. evertsi females which fed on control hosts. The concentration of gammaglobulins in the sera of rabbits was monitored at various intervals after the first infestation. It was found, for the first time, that infestation of laboratory animals with male ticks conferred immunity, but to a lesser degree than infestation with both sexes. It was also shown that the level of gammaglobulins increased from 3.4 +/- 0.28 g l-1 to 7.3 +/- 0.24 g l-1 in sera of rabbits hosts as a result of the feeding activity of males, but to a lesser extent than in sera of rabbits on which both sexes had fed (10.8 +/- 2.4 g l-1). PMID- 1720085 TI - [Preparation and study of cytotoxic properties of the immunotoxin anti-CD7/A chain of ricin]. PMID- 1720086 TI - Thermoluminescence dating of Neanderthal and early modern humans in the Near East. AB - Archaeological excavations in Europe provide no evidence for the first modern humans pre-dating Neanderthal man. In the Near East, however, a quite different sequence seems to have pertained. Thermoluminescence dating indicates that at some sites the modern humans were settled some 30,000 years before the Neanderthals. This raises the possibility of two lines of descent from a common ancestor. PMID- 1720087 TI - Prolonged fasting reduces rat hepatic beta 1 thyroid hormone receptor protein without changing the level of its messenger ribonucleic acid. AB - The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the beta 1-receptor (beta 1-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-beta 1 TR immunoglobulin G preparation. Although no changes in beta 1-TR mRNA concentration were noted, we observed a 60% fall in total binding capacity. beta 1-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in beta 1-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein. PMID- 1720088 TI - Immunocytochemical localization of the glucocorticoid receptor in rat brain, pituitary, liver, and thymus with two new polyclonal antipeptide antibodies. AB - The intracellular localization of the glucocorticoid receptor (GR) was studied in male rat brain, pituitary, liver, and thymus. Two new polyclonal anti-GR antibodies, GR 57 and GR 59, raised against two synthetic peptides (346-357 and 245-259) that correspond to unique regions of the amino-terminus of human GR were used. Vibratome sections (30-50 microns) of perfused brain and frozen sections (6 8 microns) of pituitary, liver, and thymus fixed in paraformaldehyde were incubated in preimmune serum, immunoserum, epitope-purified immunoserum, or peptide-absorbed immunoserum of either GR 57 or GR 59 and immunostained by the avidin-biotin peroxidase method. GR immunoreactivity (GR-ir) was primarily nuclear in brain, pituitary, liver, and thymus sections from intact rats. Adrenalectomy caused nuclear GR-ir to decrease and cytoplasmic GR-ir to increase. When adrenalectomized rats were treated with corticosterone (100 micrograms and 1 mg) or dexamethasone (1 microgram, 100 micrograms, and 1 mg), GR-ir was again predominantly nuclear. One microgram of corticosterone failed to cause nuclear GR ir when administered to adrenalectomized rats. Immunoreactive neurons and glial cells were extensively distributed, with varied intensity, throughout the rat forebrain. The areas include cortex, septum, hippocampus, amygdala, thalamus, and hypothalamus. Cells with the strongest GR-ir were located in the caudate putamen, paraventricular, arcuate, and central amygdala nuclei, areas CA1-CA2 of the hippocampus, and laminae 4 and 5 of the cortex. In the pituitary, cells of the anterior and posterior lobes were GR immunoreactive, while those in the intermediate lobe were not. Hepatocytes of the liver and thymocytes and reticuloepithelial cells of the thymus were GR immunoreactive. The results show that GR can be localized immunocytochemically in numerous rat tissues using antipeptide polyclonal antibodies and correlated with the results of biochemical and ligand receptor studies. PMID- 1720089 TI - 1,25-Dihydroxyvitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts. AB - 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation and inhibits proliferation in many cell types including bone cells. These effects may be mediated by the modulation of the insulin-like growth factor (IGF) regulatory system. Therefore we investigated the effects of 1,25-(OH)2D3 on transcript and protein levels of both IGF-I and IGF binding proteins (IGFBPs) in clonal mouse osteoblasts. Subconfluent cultures were treated in serum-free medium with 1,25 (OH)2D3. Secreted IGF-I was measured using a RIA under conditions eliminating the interference of IGFBPs. 1,25-(OH)2D3 (10(-11)-10(-8) M) inhibited IGF-I release in a dose dependent manner at 24 h (maximally to 30 +/- 5% of control, mean +/- SEM of seven independent experiments). In a time course study IGF-I increased in the media of control cultures over a 48-h period, while IGF-I secretion was completely prevented from 6 h onward in 1,25-(OH)2D3 treated cultures. Northern blot analysis revealed four IGF-I transcripts of 0.9, 1.8, 4.4, and 7.5 kilobases (kb). 1,25-(OH)2D3 decreased levels of the 7.5 kb IGF-I transcript from 4-48 h, with maximal inhibition occurring at 24 h (25% of control). Western ligand blots of the culture medium demonstrated secretion of a 25-kilodalton IGFBP, which comprised greater than or equal to 90% of the secreted IGFBPs. The 25-kilodalton IGFBP had previously been shown to have sequence similarity with IGFBP-4, a binding protein which inhibits the action of IGFs on bone cells. 1,25-(OH)2D3 treatment increased secretion of IGFBP-4 up to 14-fold over 24 h. 1,25-(OH)2D3 also increased IGFBP-4 (2.2 kb) transcript levels within 30 min, with the maximal stimulation of 8-fold occurring after 8 h. [3H]Thymidine incorporation into cells was inhibited by 1,25-(OH)2D3 both under basal and serum-stimulated conditions. Our results are consistent with the hypothesis that the effects of 1,25-(OH)2D3 on osteoblast proliferation may be mediated in part by decreased levels of IGF-I and increased concentrations of inhibitory IGFBP-4. It is proposed that this alteration in the IGF system may be an important functional autocrine or paracrine switch in the transition of osteoblasts from states of proliferation to differentiation. PMID- 1720090 TI - Interleukin-1 beta-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase. AB - The aim of this study was to characterize the dynamics and functional relevance of interleukin-1 beta (IL-1 beta)-induced nitric oxide production in isolated pancreatic islets. Thus, islets were isolated from adult rats, precultured for 3 5 days in medium RPMI-1640 plus 10% fetal calf serum, and then exposed to IL-1 beta for different time periods, after which islet nitrite production and aconitase activity were determined. IL-1 beta (5 ng/ml) did not increase islet nitrite production during the first hour of incubation. Moreover, the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (Meth-arg; 5 mM) failed to prevent the initial (90 min) IL-1 beta-induced increase in islet insulin release. After 4, 7, and 24 h, however, nitrite production was increased by 50%, 93%, and 139%, respectively. Islet aconitase activity and glucose oxidation rates were decreased by 70% after incubation for 24 h with IL-1 beta. Both Meth-arg and N alpha-p-tosyl-L-lysine chloromethyl ketone (0.1 mM), a protease inhibitor, could completely counteract the IL-1 beta-induced increases in nitrite production and inhibition of aconitase activity and glucose oxidation rates. In a separate series of experiments, islets were incubated for 60 min with or without IL-1 beta and the RNA synthesis inhibitor actinomycin-D (5 micrograms/ml) and subsequently incubated for another 9 h without any additions. The presence of actinomycin-D during the 1-h IL-1 beta incubation period prevented the IL-1 beta-induced rise in nitrite production and the IL-1 beta-induced inhibition of aconitase activity and insulin release. It is concluded that IL-1 beta-induced nitric oxide production is a late event which requires gene transcription and does not mediate the initial stimulatory effects of IL-1 beta on beta-cell function. However, the gradually augmented rate of nitric oxide production may inhibit the enzyme aconitase, leading to a suppressed mitochondrial activity and a defective insulin release in response to nutrient secretagogues. PMID- 1720091 TI - The timing of progesterone-induced ribonucleic acid and protein synthesis for augmentation of luteinizing hormone secretion. AB - Progesterone addition to pituitary cells pretreated with estradiol leads within 45 min to an unambiguous augmentation of pulsatile GnRH-stimulated LH secretion. To investigate this rapid action, we established the kinetics of early events through manipulation of RNA synthesis, protein synthesis, and progesterone receptor binding. Female rat pituitary cells cultured in medium containing charcoal-treated serum plus 0.2 nM estradiol were changed to 0.1% BSA-medium +/- 200 nM progesterone at time 0; at 90 and 150 min the cells were challenged with 1 nM GnRH 15-min pulses. The 3-fold augmentation of GnRH-stimulated LH secretion induced by progesterone was inhibited completely by simultaneous addition of 1 microM actinomycin D or emetine as was GnRH self-priming. In another series, the ability of cycloheximide to completely block progesterone augmentation was gradually diminished with delay of addition, but even 90 min after progesterone (30 min before GnRH pulse) cycloheximide resulted in 50% blockade of augmentation. In contrast, inhibition of RNA synthesis 60-90 min after progesterone introduction had little or no effect on progesterone augmentation. The temporal profile of inhibition by the progesterone antagonist RU486 was indistinguishable from that resulting from blockade of RNA synthesis and suggests that continual activation of the receptor is required for continued RNA synthesis. In summary: 1) both RNA and protein synthesis are required for GnRH self-priming; and 2) progesterone augmentation of GnRH-stimulated LH secretion requires RNA synthesis and synthesis of protein(s) which appear to be turning over rapidly, accumulating slowly, or both. PMID- 1720092 TI - Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3. AB - Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3E. coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3E. coli or hIGFBP-3CHO produced a dose-dependent inhibition of IGF-I but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGAFBP 3E. coli or hIGFBP-3CHO potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3E. coli for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3H]AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3E. coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 Mr hIGFBP-3E. coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3E. coli forms of 12,000-27,000 Mr. Cell-associated IGFBP-3E. coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3E. coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF I action. PMID- 1720093 TI - Regulation of peptide-YY synthesis and secretion in fetal rat intestinal cultures. AB - The regulation of intestinal peptide-YY (PYY) synthesis and secretion has not been well studied. We have used fetal rat intestinal cells in culture to examine the intra- and extracellular factors controlling the production of PYY. Immunohistochemical analysis demonstrated a distinct population of cells containing immunoreactive PYY (IR-PYY). When examined by HPLC, the IR-PYY stored and secreted by fetal rat intestinal cell cultures eluted as a single moiety with the same elution time as synthetic rat PYY. Pro-PYY mRNA transcript levels and secretion of IR-PYY into the cell medium were increased by activation of protein kinase-A with either a long-acting cAMP analog or forskolin. In contrast, IR-PYY secretion only was stimulated in a synergistic fashion through calcium- and protein kinase-C-dependent pathways (stimulated with A23187 and phorbol myristate acetate, respectively). The intestinal endocrine peptide, gastric inhibitory peptide, and the neurocrine peptide, gastrin-releasing peptide, were found to stimulate IR-PYY secretion in a dose-dependent fashion, with significant effects observed at concentrations as low as 10(-8) and 10(-12) M, respectively (P less than 0.05-0.001). Cholecystokinin and vasoactive intestinal peptide were without effect on IR-PYY secretion at doses of 10(-12)-10(-6) M. The fatty acid sodium oleate and the cholinergic agonist bethanechol were also found to stimulate IR PYY secretion, each at a concentration of 10(-4) M (P less than 0.001). The results of the present study indicate that the synthesis and secretion of PYY by the rat intestine is under the regulatory control of a wide variety of extracellular agents, mediated by several intracellular signalling pathways. PMID- 1720094 TI - Maternal insulin-like growth factor-binding protein messenger ribonucleic acid during rat pregnancy. AB - Pregnancy is associated with rapid growth of maternal and fetal tissues. Insulin like growth factors (IGF-I and -II) have roles in mediating both fetal and placental growth. In this study serum IGFs and IGF-binding proteins (IGFBPs) were characterized, IGFBP protease activity was quantified, and hepatic IGFBP-1, -2, 3, and -4 mRNA were investigated throughout rat pregnancy. IGF-I in maternal serum was elevated (P less than or equal to 0.001) on days 5 and 10 (d5 and d10) of gestation compared to levels in nonpregnant controls (NP), but was significantly decreased below NP levels (P less than or equal to 0.001) after d10 of pregnancy. Serum IGF-II levels were unaffected by pregnancy. Using Western ligand blotting (WLB), six IGFBP bands were visualized in NP, d5, and d10 pregnancy rat sera. At 15 and 20 days gestation, the IGFBP-3 bands were no longer detectable by WLB. Using an in vitro IGFBP protease assay, sera from rats at 15 and 20 days gestation proteolyzed 63 +/- 4% and 81 +/- 5% of recombinant human IGFBP-3, respectively. Regression analyses demonstrated that serum IGF-I was positively correlated with serum IGFBP-3 (r2 = 0.73; P = 0.001), whereas serum IGFBP-3 (r2 = -0.85; P = 0.001) and serum IGF-I (r2 = -0.78; P = 0.001) were negatively correlated with serum protease activity. In addition, no change was observed in liver IGFBP-3 mRNA during pregnancy, further suggesting that protease activity is primarily responsible for the decrease in serum IGFBP-3. However, IGFBP-1 and -4 mRNA levels were increased 3- to 11-fold after d5 of gestation. The hormonal and/or metabolic regulators of hepatic IGFBP-1 and -4 expression during rat pregnancy remain to be determined. PMID- 1720095 TI - Primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) and effects of ovine PACAP on frog pituitary. AB - Pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily, was isolated in pure form from the brain of the European green frog, Rana ridibunda. The primary structure of the peptide indicates that evolutionary pressure to conserve the complete amino acid sequence has been very strong. Frog PACAP comprises 38 amino acid residues and contains only 1 substitution (isoleucine for valine at position 35) compared with human/ovine/rat PACAP. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, synthetic ovine PACAP-(1-38) produced a dose dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 2.1 +/- 0.6 x 10(-7) M; mean +/- SE; n = 6). Maximum stimulation (an approximately 8-fold increase in concentration over basal values) was produced by 10(-6) M peptide. The truncated form of PACAP [PACAP-(1-27)] also produced a dose-dependent increase in cAMP in frog anterior pituitary fragments, and the potency of the peptide (ED50 = 5.9 +/- 0.6 x 10(-8) M) was comparable to that of PACAP-(1-38). The data suggest, therefore, that the function as well as the structure of PACAP have been conserved during the evolution of amphibia to mammals. PMID- 1720096 TI - Blood lead levels in South African inner-city children. AB - Little is known about childhood lead absorption in South Africa. In this study a cross-sectional analytic survey was carried out to determine the blood lead levels and associated risk factors for inner-city, first-grade schoolchildren. Blood lead analyses, hematological and anthropometric measurements were conducted, and a pretested questionnaire was administered to parents to identify risk factors for lead exposure. In a detailed environmental study, daily air and dust samples were collected over a period of 1 year from several sites in the study area, contemporaneously with the blood and questionnaire surveys. Spatial and temporal variations in atmospheric lead were determined. It was found that 13% of mixed race children, but no white children, had blood lead levels greater than or equal to 25 micrograms/dL, the U.S. action level. Air lead levels averaged around 1 microgram/m3, and dust lead levels ranged from 410 to 3620 ppm. Environmental lead levels were significantly elevated near heavy traffic, where Environmental Protection Agency standards were exceeded mainly during winter months. Baseline exposure was of significance in influencing blood lead levels of children attending schools in direct proximity to heavy traffic, where blood lead levels were elevated irrespective of other influencing factors. Primary and secondary preventive measures are urgently needed in South Africa to reduce environmental lead exposure. At the time of the study, South Africa had one of the highest levels of lead in gasoline in the Western World, namely, 0.836 g/L. Although levels have subsequently been reduced, this is typical of the situation in many African countries today. PMID- 1720097 TI - Effect of preschool integration for children with disabilities. AB - This study examined the effects of integration and segregation in a special education preschool program for children with mild to moderate disabilities to determine whether initial level of development differentially influenced gains achieved. No main-effect differences between the two groups appeared on several pretest and posttest measures. Aptitude-by-Treatment analyses revealed than higher performing students gained more from integrated classes, whereas lower performing students gained more from segregated classes. The data suggest careful monitoring of lower functioning students to ensure appropriate academic and social stimulation. PMID- 1720098 TI - Role of timing of administration in the cardioprotective effect of iloprost, a stable prostacyclin mimetic. AB - We administered iloprost, a stable prostacyclin mimetic, 27 nM, to isolated and perfused rabbit hearts submitted, after 60 min of equilibration, to an ischaemic period (60 min at a coronary flow of 1 ml/min) followed by a period of reperfusion (30 min at a coronary flow of 25 ml/min). Iloprost was delivered at different times during the experimental protocol: 60 min before ischaemia, at the onset and after 30 min of ischaemia and only during reperfusion. The iloprost cardioprotective effect was evaluated in terms of recovery of left ventricular pressure developed during reperfusion, creatine phosphokinase (CPK) and noradrenaline release, mitochondrial function (expressed as yield, RCI (respiratory control index), QO2, ADP/O), ATP and creatine phosphate (CP) tissue contents, calcium homeostasis and by measuring several parameters of oxidative stress: reduced and oxidized glutathione release and tissue contents, Mn and Cu Zn superoxide dismutase activities; glutathione reductase and peroxidase activities. Our data show that the cytoprotective action of iloprost is closely related to the time of administration. Optimal myocardial preservation was achieved when it was given before or at the onset of ischaemia. Iloprost administration 30 min after the onset of ischaemia was still beneficial, although to a lesser extent. Iloprost lost its protective effect when given only on reperfusion. The data suggest that the iloprost cardioprotective effect is related to maintainance of membrane integrity. PMID- 1720099 TI - Thiokynurenates: a new group of antagonists of the glycine modulatory site of the NMDA receptor. AB - Several substituted derivatives of kynurenic acid were tested on the N-methyl-D aspartate (NMDA) receptor/ion channel complex present in the guinea pig myenteric plexus, on the binding of [3H]glycine and of [3H]N-[1-(2 thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat cortical membranes and on the depolarization of mice cortical wedges induced by NMDA or quisqualic acid (QA). Kynurenic acid derivatives, having a chlorine (CI) or a fluorine atom in position 5 or 7 but not in position 6 or 8 had significantly lower IC50s than the parent compound when tested on the antagonism of glutamate-induced ileal contraction and in the glycine binding assay. A further significant increase in potency was obtained by substituting a thio group for the hydroxy group in position 4 of kynurenic acid: the IC50 was 160 +/- 20 microM of kynurenic acid and 70 +/- 15 microM of thiokynurenic acid in the myenteric plexus whereas these IC50s for glycine binding were 25 +/- 3 and 9 +/- 2 microM respectively. Several thiokynurenic acid derivatives were synthetized and showed an increased affinity for the glycine recognition site over the corresponding kynurenic acid derivatives. Glycine competitively antagonized the actions of the thiokynurenates in the ileum, in cortical wedges and on [3H]TCP binding. In this preparation, 7 Cl-thiokynurenic acid had an IC50 of 8 microM for antagonizing 10 microM NMDA induced depolarization while 50% of the 10 microM QA depolarization was antagonized at 300 microM. Thus thiokynurenic acid derivatives seem to be a new group of potent and selective antagonists of strychnine-insensitive glycine receptors. PMID- 1720101 TI - A question of ethics. Does a patient have to know their diagnosis to provide informed consent for hospice services? PMID- 1720100 TI - Oral N-acetylcysteine reduces bleomycin-induced collagen deposition in the lungs of mice. AB - N-acetylcysteine (NAC) has been employed in the treatment of acute lung injury but its therapeutic value is as yet unproven. In the present study we examined the effect of both L- and D-forms of NAC as inhibitors of bleomycin-induced fibrosis in mice. We hypothesized that, because of the D-form is not metabolized, it may be more effective than the L-form in ameliorating lung injury and fibrosis. Both drugs were given daily in the drinking water at a dose of approximately 400 mg.kg-1 body weight commencing one week prior to a single intratracheal instillation of bleomycin at a dose of 6 mg.kg-1 body weight. Lung injury was assessed 35 days later by measuring total lung collagen content, lung wet weight and examination of tissues by light and electron microscopy. Total collagen content and lung wet weight of animals receiving bleomycin together with L-NAC were 2.90 +/- 0.03 mg and 0.23 +/- 0.01 g, respectively. The values for collagen content, but not wet weight, were significantly less (p less than 0.05) than those given for bleomycin alone (3.90 +/- 0.02 mg and 0.260 +/- 0.005 g, respectively), but greater than (p less than 0.05) controls (2.10 +/- 0.01 mg and 0.160 +/- 0.002 g, respectively). Values for collagen content and wet weight of animals given bleomycin together with D-NAC (3.10 +/- 0.02 mg and 0.21 +/- 0.01 mg, respectively) were both significantly greater than values for control animals but lower than animals given bleomycin alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720102 TI - Number and topography of epitopes of human chorionic gonadotropin (hCG) are shared by desialylated and deglycosylated hCG. AB - A previously established map of the surface epitopes of human chorionic gonadotropin (hCG) served as template for the present study in which we investigated the antigenic surfaces of two glycosylation variants of hCG, i.e. desialylated hCG (asialo-hCG) and deglycosylated hCG (degly-hCG). This map allocates five epitopes to the alpha subunit, five to the beta subunit and four alpha beta epitopes to structures formed only by the alpha/beta heterodimer holo hCG (Schwarz et al. (1986) Endocrinology 118, 189-197; Berger et al. (1990) J. Endocrinol. 125, 301-309). Here it is described that both variants complied with this template: each of the 14 distinct monoclonal antibodies with which the epitopes of hCG were defined reacted with radiolabeled asialo-hCG and degly-hCG as well and generally bound degly-hCG with greater affinity than hCG. Moreover, every combination of capture and radiolabeled detection antibody that was either compatible or incompatible on unlabeled hCG was so also on unlabeled asialo-hCG and degly-hCG. It thus appears that alterations of the carbohydrate structure of hCG can be associated with a change in affinity between some antibodies and their respective epitopes but not with a loss of an epitope or with a change in the topographical relationships of the 14 epitopes. PMID- 1720103 TI - Decreased mitochondrial gene expression in isolated islets of rats injected neonatally with streptozotocin. AB - The aim of the present study was to evaluate the possible role of the expression of the mitochondrial genome for the regulation of insulin production in the pancreatic Beta cell. For this purpose, islets of Langerhans were isolated from adult control rats and rats injected neonatally with streptozotocin and the islet contents of specific mitochondrial DNAs and RNAs together with nuclear-encoded RNAs were determined. The contents of mitochondrial cytochrome b mRNA, the mitochondrial 12 S rRNA and insulin mRNA were all 30-40% lower in islets isolated from the streptozotocin-treated rats as compared to islets from control rats. In contrast, the nuclear mRNA coding for the mitochondrial adenine nucleotide translocator was not decreased in the streptozotocin-treated rats. Contents of mitochondrial DNA, as assessed by the Southern blotting technique, were markedly decreased in the streptozotocin islets. Sequence analysis of mitochondrial DNA from streptozotocin islets and control islets however, did not reveal any differences in nucleotide sequences. In control islets the contents of mitochondrial cytochrome b mRNA increased in response to a high glucose concentration during a 4-h incubation period. Serum deprivation or the addition of theophylline or 4-phorbol 12-myristate 13-acetate failed to affect the cytochrome b mRNA contents in vitro. It is concluded that islets of streptozotocin-treated rats contain low contents of mitochondrial DNA and RNA. Since a lower mitochondrial RNA content may result in a diminished oxidative capacity, it is conceivable that a deficiency of this messenger may contribute to the development of insulin deficiency. PMID- 1720104 TI - Galanin inhibition of vasoactive intestinal polypeptide release and circular muscle motility in the isolated perfused canine ileum. AB - The role of porcine galanin, infused arterially into isolated perfused canine ileal segments, in modulating the tonically elevated neural release of vasoactive intestinal polypeptide and possible concomitant motor actions dependent on vasoactive intestinal polypeptide modulation was studied. Galanin infusions (9 minute) inhibited vasoactive intestinal polypeptide release in a concentration dependent manner (maximum during minutes 8-10) irrespective of the absence (quiescence) or presence of phasic circular muscle contractions induced by local electrical field stimulation of nerves. During quiescence, galanin induced phasic contractions in four of five segments beginning in the 8th minute of the infusion. During stimulated contractions, galanin inhibited phasic motor activity within 2 minutes of initiation of the infusion; this inhibition may result from direct smooth previously reported muscle inhibition. Thus galanin may inhibit both neural release of vasoactive intestinal polypeptide and circular muscle motility directly. The delayed period of phasic activity initiated by galanin during quiescence may be related to inhibition of vasoactive intestinal polypeptide release, freeing the muscle from tonic inhibition by vasoactive intestinal polypeptide. Because galanin and vasoactive intestinal polypeptide are colocalized in some enteric nerves, galanin may regulate vasoactive intestinal polypeptide release by negative feedback. PMID- 1720105 TI - Differential effect on neuropeptide release of different concentrations of hydrogen ions on afferent and intrinsic neurons of the rat stomach. AB - In the muscle layer of the glandular portion of the rat stomach, in vivo capsaicin pretreatment markedly reduced calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) but did not affect substance P-like immunoreactivity (SP-LI). Accordingly, in vitro superfusion of slices of this tissue with capsaicin (10 mumol/L) released CGRP-LI but not SP-LI, whereas both neuropeptides were released by 80 mmol/L K+. Exposure to relatively low-pH (pH 6) physiological salt solution induced an increase in the CGRP-LI outflow that was reduced by 70% in a Ca(2+)-free medium and was completely abolished by a previous exposure to capsaicin. However, superfusion with pH-6 medium did not produce any detectable SP-LI release. After exposure to pH-6 medium, both capsaicin and high-K+ medium were still able to release a consistent quantity of CGRP-LI and SP-LI, respectively. Increased mucosal blood flow induced by acid back-diffusion is considered a protective mechanism against mucosal gastric lesion. The present findings suggest that hydrogen ions diffusing into the gastric wall may promote protective vasodilatation by activating the "efferent" function of capsaicin sensitive nerves without affecting the secretory process of other intrinsic peptidergic neurons. PMID- 1720106 TI - Antibodies to hepatitis C virus in low-risk blood donors: implications for counseling positive donors. AB - The significance of antibodies to hepatitis C virus (HCV) found by screening enzyme-linked immunoassay testing in a low-risk blood donor population is unclear. The rate of false positivity in this group as well as the usefulness of supplemental testing were examined by correlating the results of two screening enzyme-linked immunoassays (Ortho Diagnostics, Raritan, NJ, and Abbott Laboratories, North Chicago, IL) with supplemental antibody testing by the recombinant immunoblot assays (1 and 2) (Ortho) and neutralization assay (Abbott). Polymerase chain reaction was used to detect HCV genomic RNA to confirm viremia. Among 11.243 volunteer donors who were screened for the presence of antibodies to HCV by enzyme-linked immunoassay, 60 (0.53%) sera were repeatedly reactive. Twenty-five of these 60 sera were available for further testing. Seven sera were reactive by both screening enzyme-linked immunoassays, as well as by both recombinant immunoblot and neutralization assays. Six of these seven sera had detectable HCV genomic RNA by polymerase chain reaction. Among the remaining 18 sera, none were reactive by either recombinant immunoblot assays, whereas two sera were reactive by the neutralization assay only. None of the 18 samples had detectable HCV genomic RNA. Five of the six sera with elevated aminotransferase levels were among the seven sera reactive by all immunoassays. It is concluded that there is a significant false positivity rate associated with screening enzyme-linked immunoassay testing in a low-risk blood donor population. Supplemental testing correlates well with detection of hepatitis C genomic material by polymerase chain reaction and identifies donors who are truly infected. PMID- 1720107 TI - Palliation of advanced esophageal cancer: a (laser) light at the end of the tunnel. PMID- 1720108 TI - Investigating the HLA component in rheumatoid arthritis: an additive (dominant) mode of inheritance is rejected, a recessive mode is preferred. AB - We examined the mode of inheritance of rheumatoid arthritis (RA) and estimated the genetic contribution of the HLA-linked locus to the development of RA using data from 111 multiplex families (54 London, 57 Cleveland), and 43 randomly ascertained patients (Seattle). HLA-DR4 was present in 78 multiplex probands (70%); a further 16 probands who were negative for DR4 were positive for DR1. Both DR4 and DR1 were significantly in excess when compared to control population frequencies (P less than 0.001); an additional finding was an excess of DR7, although the numbers of probands with DR7 were small. Despite the well established HLA association with RA, neither recessive nor additive (dominant) modes of inheritance, nor any intermediate models have been ruled out using affected sib-pair and antigen genotype frequency among patients (AGFAP) methods. However, in our study the AGFAP data for HLA-DR4 and DR1 were close to recessive expectations (P = ns) while an additive (dominant) mode of inheritance was rejected (P less than 0.001). The same results were obtained by an independent method which considered HLA-DR transmission from affected parents to their affected children. The affected sib-pair haplotype sharing method showed deviation from random expectations but did not allow discrimination between recessive and additive (dominant) modes. The effect of the HLA-linked locus on familiarity accounted for only a 1.61-fold increased risk to sibs over the population prevalence, compared to an observed value of 3.90. This indicated that there could be at least one other non-HLA locus predisposing to RA with a weight that is slightly greater than that of HLA. PMID- 1720109 TI - Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy. AB - Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed. PMID- 1720110 TI - Effect of differentiation agents on expression of CA 125, alkaline phosphatase, and cytokeratins in human ovarian adenocarcinoma cells (OVCA 433). AB - A number of chemical agents have been found to influence the proliferation, morphology, enzymatic activity, and antigen expression of neoplastic cells toward a more differentiated phenotype. We studied the effects of differentiating agents retinoic acid, sodium butyrate, and dibutyryl cyclic AMP on the expression of the tumor-associated antigen CA 125 and several biochemical markers of differentiation in cultured OVCA 433 ovarian cancer cells. Treatment of OVCA 433 cells with these agents for 96 hr reduced cellular proliferation and altered cellular morphology. Quantitation of cell surface CA 125 using flow cytometry revealed that CA 125 expression was reduced by 35-50%. The amount of CA 125 antigen shed into the culture media was reduced to a similar degree. In addition, differentiation inducers markedly enhanced cellular alkaline phosphatase activity and induced the expression of a 65-67-kDa cytokeratin. These findings provide support for the induction of a more differentiated phenotype by these agents. PMID- 1720111 TI - [Serrated dwarfpalm in the therapy of benign prostate hypertrophy. Portrait of a plant medicine]. PMID- 1720112 TI - Effect of L-dopa loading on 5-HTP decarboxylation in rat brain areas. AB - The time course of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5 hydroxyindoleacetic acid (5-HIAA) concentrations in four rat brain areas (hypothalamus, hippocampus, striatum and olfactory bulbs) were investigated after treatment with L-dopa (125 mg/kg, ip) + benserazide (50 mg/kg, ip). 5-HTP levels increased as early as 0.5 h, showed maximum accumulation at 1.5 h and returned to control levels within 4 h, while 5-HT was markedly decreased in all four structures, with a maximum effect at 1.5 h (approximately -70%) in the four areas. The decrease in 5-HT was not accompanied by changes in 5-HIAA levels. In agreement with previous studies, these data demonstrate that L-dopa loading interferes with serotonin metabolism in the rat brain. However, in addition to the releasing action of newly-synthesized dopamine, the accumulation of 5-HTP and the parallel decrease in 5-HT indicate a reduction in 5-HT synthesis. This inhibition could be explained by a competitive effect of L-dopa for aromatic aminoacid decarboxylase activity. PMID- 1720113 TI - A new approach to intravital videomicroscopy of rat spinotrapezius muscle. AB - A modified preparation of the rat spinotrapezius muscle is described in which optimal conditions for intravital microscopy can be achieved while the supplying blood vessels are left fully intact, and mechanical stress to the muscle during preparation is reduced to an unavoidable minimum. The viability of the preparation is demonstrated using the response of arterial microvessels to endothelium-dependent and -independent dilators and to changes of ambient PO2, the presence of spontaneous vasomotion, and histochemical analysis of pertinent enzyme systems. The preparation is viable for much longer experimental time periods (up to 10 hours) than reported previously, provided the intensity of illumination is kept at a very low level. If the latter prerequisite is met, tissue edema, maximal vasodilation, and the associated loss of responsiveness to vasoactive stimuli of arterioles is reliably avoided. PMID- 1720114 TI - Consideration on two cases of dystonia-parkinsonism. AB - We describe two sporadic cases of dystonia-parkinsonism at different stages of disease progression. The two girls, first seen at the ages of 10 and 12 years, have been followed for 9 and 2 years respectively. In both patients L-dopa 60 mg + carbidopa 6 mg brought about a swift remission of symptoms, which persists to date. All examinations, including CT and MR brainscans, were normal. The CSF and urine levels of HVA and 5HIAA were low in one case and normalized with treatment. This finding might provide a fairly valid predictive index of responsiveness to L dopa. PMID- 1720115 TI - Detection of serum alpha-fetoprotein in dogs with hepatic tumors. AB - Serum alpha-fetoprotein (AFP) concentration was detected by use of 2 commercially available kits containing antibodies to human AFP--a radioimmunoassay and an enzymetric test. Using neonatal canine serum (a source high in AFP), it was determined that reagents from both kits were able to bind to canine AFP, but a significant difference was detected in AFP concentration. The enzymetric test was superior in detecting canine AFP. Sera from dogs were classified into 6 groups: from dogs with primary hepatic tumors only (group 1); from dogs with primary hepatic tumors and other tumors (group 2); from dogs with normal liver but with other types of neoplasia (group 3); from dogs with nonneoplastic hepatic disease and tumors originating in other organs (group 4); from dogs with nonneoplastic hepatic disease only (group 5); and from clinically normal dogs (group 6). Serum biochemical determinations (alkaline phosphatase, alanine transaminase, albumin, total protein, total bilirubin, and serum bile acids) and values from the 2 AFP assays were obtained for all dogs. Serum AFP concentration detected by the enzymetric test was significantly higher in dogs with hepatocellular carcinoma and cholangiocarcinoma. Values greater than 250 ng/ml were detected in 5 of 9 dogs with cholangiocarcinoma and in 3 of 4 dogs with hepatocellular carcinoma. High serum AFP concentration also was indicative of liver involvement in 2 of 3 dogs with primary hepatic lymphosarcoma; 2 dogs had values greater than 225 ng/ml. Serum AFP concentration in dogs with other types of hepatic tumors was less than 250 ng/ml, and serum AFP concentration could not be correlated with such tumors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720116 TI - Comparative pharmacokinetic properties of murine monoclonal antibody A7 modified with neocarzinostatin, dextran and polyethylene glycol. AB - The murine monoclonal antibody A7 (Mab A7) was chemically modified with several macromolecules: dextran, polyethylene glycol and the anti-cancer polypeptide neocarzinostatin. The pharmacokinetic properties of the combinations were subsequently examined. Radioimmunoassay revealed that all preparations retained their antigen-binding activities. The Mab A7-neocarzinostatin conjugate was cleared from the blood circulation with a kinetic pattern almost identical to that of the parent Mab A7. Of the three preparations, Mab A7-dextran (A7-Dx) was removed the most rapidly from the circulation. Mab A7-polyethylene glycol (A7 PEG) exhibited the slowest blood clearance curve, with twice the half life of the parent Mab A7 in the circulation. In normal organ distributions, A7-Dx exhibited the highest liver, spleen and kidney uptake, and A7-PEG showed the lowest uptake, when expressed as tissue:blood ratio. Although A7-Dx exhibited lower tumor uptake, there was no significant difference among the three conjugates in tumor bearing nude mice. A7-PEG seems to be a good candidate for targeted cancer therapy using antibody due to its high blood retention but low normal organ uptake. PMID- 1720117 TI - The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces. AB - Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1 methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces. PMID- 1720118 TI - Arthroscopic lavage of osteoarthritic knees. AB - A strong clinical impression exists that joint lavage often provides symptomatic relief for painful osteoarthritis of the knee. A controlled trial was conducted to test this hypothesis. A group of 37 painful osteoarthritic knees were treated by arthroscopic lavage and physiotherapy, and a control group of 24 knees were treated by physiotherapy alone. There was better relief of pain in the lavage group, and the effect was still present at one year. An improvement in the signs of inflammation lasted for about three months. Pain was relieved more effectively in patients with slight radiographic changes than in those with advanced changes. Our results confirm the effectiveness of joint lavage in the management of painful osteoarthritis of the knee. PMID- 1720119 TI - Dystrophin is a component of the subsynaptic membrane. AB - A subsynaptic protein of Mr approximately 300 kD is a major component of Torpedo electric organ postsynaptic membranes and copurifies with the AChR and the 43-kD subsynaptic protein. mAbs against this protein react with neuromuscular synapses in higher vertebrates, but not at synapses in dystrophic muscle. The Torpedo 300 kD protein comigrates in SDS-PAGE with murine dystrophin and reacts with antibodies against murine dystrophin. The sequence of a partial cDNA isolated by screening an expression library with mAbs against the Torpedo 300-kD protein shows striking homology to mammalian dystrophin, and in particular to the b isoform of dystrophin. These results indicate that dystrophin is a component of the postsynaptic membrane at neuromuscular synapses and raise the possibility that loss of dystrophin from synapses in dystrophic muscle may have consequences that contribute to muscular dystrophy. PMID- 1720120 TI - Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4). AB - Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1 expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection. PMID- 1720121 TI - Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin. AB - Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells. PMID- 1720122 TI - Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface. AB - During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface. PMID- 1720123 TI - Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. AB - The functional organization of the nucleus was studied using a fluorescence microscopy approach which allowed integration of positional information for RNA, DNA, and proteins. In cells from sea urchin to human, nuclear poly(A) RNA was found concentrated primarily within several discrete "transcript domains" which often surrounded nucleoli. Concentrations of poly(A) RNA were coincident with snRNP antigen clusters, providing evidence for the localization of pre-mRNA splicing at these sites. The spatial relationship of transcript domains with respect to various classes of DNA was established, in that the poly(A) RNA-rich regions coincided with discrete regions of low DNA density and were non-randomly distributed with respect to specific DNA sequences. Centromeric DNA and late replicating DNA did not overlap transcript domains, whereas a subset of early replicating DNA may. Results indicate that transcript domains do not result directly from a simple clustering of chromatin corresponding to metaphase chromosomes bands. Finally, observations on the reassembly of these domains after mitosis suggest that the clustering of snRNP antigens may be dependent on the reappearance of pol II transcription. Implications of these findings for overall nuclear structure and function are considered, including a discussion of whether transcript domains may be sites of polymerase II transcription reflecting a clustering of active genes. PMID- 1720124 TI - Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus. AB - The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed. PMID- 1720125 TI - Somatostatin regulation of glycoprotein hormone and free subunit secretion in clinically nonfunctioning and somatotroph adenomas in vitro. AB - There is increasing evidence that clinically nonfunctioning pituitary tumors produce and secrete glycoprotein hormone and/or free alpha- and beta-subunits. In addition, hypersecretion of free alpha-subunit occurs in up to 37% of patients with somatotroph adenomas. An understanding of glycoprotein hormone regulation is important in developing effective therapeutic strategies for patients with tumors associated with intact glycoprotein hormone and free subunit hypersecretion. We investigated glycoprotein hormone and free subunit secretion by somatostatin in primary dispersed cultures of pituitary tumor cells from 23 patients with pituitary adenomas. Fifteen tumors from patients with clinically nonfunctioning adenomas (group 1) and 8 tumors from patients with somatotroph adenomas and cosecretion of alpha-subunit (group 2) were studied. Cultures were incubated with control or somatostatin-supplemented media for 24 h. Media samples from group 1 tumors were assayed for intact glycoprotein hormones and free alpha- and beta subunits secretion levels, while media samples from group 2 cultures were assayed for alpha-subunit and GH secretion levels. Significant (P less than 0.05-0.001) inhibition of secretion of 1 or more intact hormones and/or free subunits was found in 10 of the 15 group 1 tumors. SRIF[10(-7) M] suppressed intact gonadotropin secretion in 60% of FSH-producing tumors and 30% of LH-producing tumors. Media concentrations of FSH beta and LH beta were decreased in 31% and 50% of group 1 tumors, respectively, following somatostatin treatment in those tumors which secreted free beta-subunits. alpha-Subunit was secreted by 12 of the 15 tumors, but significant (P less than 0.02-0.01) inhibition by somatostatin was observed in only 2 tumors. In contrast, significant (P less than 0.05-0.001) inhibition of alpha-subunit in the somatotroph adenomas was found in 6 of the 8 tumors. Significant decreases in alpha-subunit were observed only in those tumors where GH was also significantly inhibited by somatostatin. We conclude that 1) somatostatin inhibits intact glycoprotein or free subunit secretion in the majority of clinically nonfunctioning pituitary tumors in vitro and 2) alpha subunit secretion is suppressed in 17% and 69% of clinically nonfunctioning and somatotroph adenomas, respectively, consistent with a differential regulation of alpha-subunit by somatostatin in these two tumor types. PMID- 1720126 TI - Clonal composition of pituitary adenomas in patients with Cushing's disease: determination by X-chromosome inactivation analysis. AB - The synthesis and secretion of anterior pituitary hormones are subjected to a variety of positive and negative feedback mechanisms. Aberrancies of these highly regulated phenomena may lead to hyperplasia involving multiple cells of the anterior lobe. Alternatively, a rare genetic mutation in a single cell may precede its clonal expansion. Which of these mechanisms is operative in the development of corticotroph adenomas is not known. To examine this question, we studied the clonal composition of ACTH-producing pituitary adenomas from female patients with Cushing's disease by using X-chromosome inactivation analysis. Nine of 27 patients examined were heterozygous at 1 of the 2 X-chromosome-linked polymorphic loci, hypoxanthine-phosphoribosyl-transferase and phosphoglycerate kinase. The methylation patterns of the hypoxanthine-phosphoribosyl-transferase and phosphoglycerate-kinase genes, distinguishing between the active and inactive alleles, were analyzed in DNA extracted from the central part of the tumor and compared with those of autologous lymphocyte DNA. Six tumors (4 microadenomas and 2 macroadenomas) showed a single active allele of the X-chromosome-linked genes and were monoclonal in nature. The other 3 pituitary adenomas (1 microadenoma and 2 macroadenomas, 1 from a patient with Nelson's syndrome) revealed a polyclonal pattern of X-chromosome inactivation. Our data demonstrate that corticotroph adenomas of the pituitary may arise from a single cell or from more than one cell. Whether fundamentally different endocrine mechanisms underlie the two processes remains to be seen. PMID- 1720127 TI - Induction of HLA-DR expression in endometrial epithelial cells by endometrial T cells: potential regulatory role of endometrial T-cells in vivo. AB - Endometrial epithelial cells express HLA-DR molecules of the major histocompatibility complex in vivo adjacent to aggregates of T-cells in the endometrial stroma. To test whether HLA-DR expression on endometrial epithelium is mediated by T-cells, endometrial T-cells were isolated from human endometrium by the sheep red blood cell rosetting technique. In contrast to the resting T cells from peripheral blood and similar to the peripheral blood T-cells activated with Concanavalin-A, endometrial T-cells formed colonies in vitro. Direct addition of the endometrial T-cells to epithelial cell cultures derived from autologous glands induced both morphological changes as well as HLA-DR molecules in the epithelial cells. The intensity of the immunostained HLA-DR molecules in the epithelial cells as well as the percentages of the HLA-DR-positive epithelial cells correlated with the number of T-cells added to the epithelial cell cultures. T-Cells were bound to the HLA-DR-positive epithelial cells as single cells or aggregates, and they were not found bound to the HLA-DR-negative epithelial cells. The supernatant of the endometrial T-cells induced expression of HLA-DR molecules and morphological changes in cultures of HLA-DR-negative epithelial cells. This expression could be inhibited by a neutralizing antiserum to interferon-gamma. These findings are consistent with the hypothesis that endometrial T-cells are activated and suggest that the expression of HLA-DR molecules in glandular epithelium in vivo is mediated by the interferon-gamma secreted by the endometrial T-cells. PMID- 1720128 TI - Insulin-like growth factor (IGF) binding protein-3 in pregnancy serum binds native IGF-I but not iodo-IGF-I. AB - Although serum immunoreactive insulin-like growth factor binding protein-3 (IGFBP 3) increases during pregnancy, radioligand binding methods such as ligand blotting with iodinated IGFs fail to detect the protein in pregnancy serum. Since IGFBP-3 must bind IGF-I or IGF-II to form a complex with the acid-labile subunit (alpha-subunit), we have used ternary complex formation from [125I]alpha-subunit as a measure of IGF binding to serum IGFBP-3. High-pressure liquid chromatography fractions containing IGFBP-3 from pregnancy serum did not bind [125I]IGF-I, although the equivalent fractions from nonpregnancy serum showed dose-dependent binding. In contrast, IGFBP-3 fractions from nonpregnancy and pregnancy sera both bound [125I]alpha-subunit in the presence of either exogenous IGF-I or endogenous serum IGFs, implying that non-iodinated IGFs could bind to the IGFBP-3. Substitution of nonradioactive iodo-IGF-I for native IGF-I in the complex formation assay confirmed that the pregnancy-induced alteration in IGFBP-3, probably resulting from proteolysis, prevents it from binding iodo-IGF-I while having little effect on its binding of the native peptide. This provides an explanation for the failure to detect IGFBP-3 in pregnancy by radioligand binding methods, and raises the question of the significance of proteolysis of IGFBP-3. PMID- 1720129 TI - Insulin-like growth factors and their binding proteins in human follicular fluid. AB - Increasing evidence suggests that insulin-like growth factors I and II (IGF-I, IGF-II) have a regulatory role in animal granulosa cells. This study was undertaken to investigate the presence of IGF-I and IGF-II, as well as that of their binding proteins (BP), IGFBP-1 and IGFBP-3 in human serum and follicular fluid (FF). Preovulatory FF was obtained from 51 patients undergoing in-vitro fertilization. The IGFBP-1 level was found to be significantly higher (P less than 0.01) in FF than in serum, whereas IGF-I and IGFBP-3 values remained markedly lower (P less than 0.01) in FF. Serum IGF-II levels were slightly but not significantly elevated compared to values obtained in the FF of patients. A positive correlation (P less than 0.001) between individual serum and FF levels was observed only for IGF-I. When a group of poor responders was compared to patients with normal stimulation characteristics, no significant difference was found in either IGF or IGFBP levels in the FF. It is concluded that IGFBP-1 is produced locally, whereas the serum may possibly be the major source of IGF-I. No clear conclusions can be drawn regarding the source of FF IGF-II and IGFBP-3. Neither the absolute level nor the relationship of IGFs to their transport proteins could explain the poor response to ovarian stimulation. PMID- 1720130 TI - T cell vaccination does not induce resistance to experimental autoimmune neuritis. AB - The effectiveness of T cell vaccination was analyzed in experimental autoimmune neuritis (EAN) that can be induced by immunization with bovine P2 protein or a peptide representing the amino acids 53-78 of P2 (P2 53-78). Lewis rats were vaccinated with glutaraldehyde-fixed lymph node cells which had been primed in vivo with P2 protein or P2 53-78 and had been activated in vitro with concanavalin A. Vaccinated animals were not protected from EAN induced by immunization with P2 protein in complete Freund's adjuvant (CFA). In a second set of experiments Lewis rats were vaccinated with irradiated or fixed P2-specific T cell lines of different specificity and neuritogenicity and were subsequently challenged with P2 53-78 in CFA. Likewise, severity of P2 53-78-induced EAN was not different between naive and T line-vaccinated groups. In spleens of vaccinated animals a substantial suppressive activity was demonstrated which was positively correlated with a weak anti-ergotypic response of these spleen cells. The fact that development of actively induced EAN was not prevented or even mitigated by T cell vaccination, in spite of an apparent vaccination-induced response to and on T lymphocytes, suggests that protection from disease is not readily induced in every autoimmune disease model. PMID- 1720131 TI - Antibodies to thymic epithelial cells in myasthenia gravis. AB - The presence of anti-thymus antibodies was investigated in the serum of 36 patients with myasthenia gravis (MG). Using an immunofluorescence technique on frozen thymic sections, we found 45% of patients sera reacting with normal or MG thymuses. Staining was confined to subcapsular and medullary keratin-positive epithelial cells. Thirty-five out of 36 sera from healthy controls and all 15 sera from patients presenting another autoimmune disorder were negative. Antibodies to thymic epithelial cells were almost exclusively detected in patients presenting thymic hyperplasia and did not disappear after thymectomy. They were not clearly associated with antiacetylcholine receptor antibody titer, nor with disease severity. Their strong association to thymic abnormalities highlights the role of the thymus in pathogenesis of MG. The reasons for the appearance of these antibodies, the structure they recognize on thymic epithelial cells and their possible etiological role are discussed. PMID- 1720132 TI - Glial fibrillary acidic protein immunoreactivity in adrenocortical and Leydig cells of the Syrian golden hamster (Mesocricetus auratus). AB - In an immunocytochemical investigation of the expression of glial fibrillary acidic protein (GFAP) in non-nervous system tissues ten anti-GFAP antibodies were used on a range of normal adult organs from different species. All four polyclonal and six monoclonal antibodies revealed the expression of GFAP in cells of the zona fasciculata and reticularis of the adrenal cortex and Leydig cells of the Syrian hamster. The Chinese hamster, mole, rat, mouse, guinea pig, rabbit, pig, duck and man were negative. Co-expression of immunoreactivity for GFAP and vimentin was observed in adrenocortical and Leydig cells of the Syrian hamster but there were differences in the staining patterns of these intermediate filament proteins. Expression of GFAP in adrenal cortex of Syrian hamster is confirmed by immunoblot and limited proteolysis analysis which reveal a light form which is immunochemically indistinguishable from its counterpart in the central nervous system. The results presented here suggest a new model for the study of the possible role of GFAP expression in cells known to be sites of steroid synthesis. PMID- 1720133 TI - Suppression of experimental allergic encephalomyelitis by the encephalitogenic peptide, in solution or bound to liposomes. AB - We have investigated the ability of liposome-bound encephalitogenic peptide to suppress experimental allergic encephalomyelitis (EAE) in the guinea pig. EAE was induced by challenge with the encephalitogenic peptide, residues 113-122 of human myelin basic protein (MBP) in complete Freund's adjuvant. The peptide was acylated with stearic acid in order to anchor it to the lipid bilayer. The liposomal-bound peptide effectively suppressed clinical signs of EAE at relatively low doses, when given subcutaneously or intraperitoneally without incomplete Freund's adjuvant, several days after challenge. In vitro proliferation of lymphocytes from treated, protected animals in response to the peptide was greatly decreased but that to the purified protein derivative of tuberculin antigen was not, indicating an antigen-specific effect. However, histological signs of EAE were not reduced. The free peptide in solution was somewhat less effective when given intraperitoneally but was as or nearly as effective as liposome-bound peptide when given subcutaneously. Binding to liposomes may decrease the rate of clearance or degradation of the peptide when given intraperitoneally. PMID- 1720134 TI - Expression of immunomolecules on microglial cells following neonatal sciatic nerve axotomy. AB - We have examined the microglial reaction accompanying motor neuron death following sciatic nerve crush in the newborn rat using lectin staining with the Griffonia simplicifolia B4-isolectin, as well as immunocytochemistry with a panel of monoclonal antibodies directed against brain macrophage antigen (ED2), and various immunologically important surface molecules (immunomolecules), such as major histocompatibility complex (MHC) class II (Ia) antigen (OX-6), CR3 complement receptor (OX-42), CD4 antigen (W3/25), and leukocyte common antigen (OX-1). The lectin histochemical method provided the earliest indication of a microglial response by demonstrating increased microglial density and clustering around dying motoneurons as early as 2 days after lesioning. Most immunomolecules were largely undetectable in the normal and early post-lesion spinal cord; however, at post-lesion day 5 localized expression of Ia antigen was visualized in the area of degenerating motor neurons. Ia expression preceded the appearance of other immunomolecules at day 8. No increase in staining with the ED2 antibody for macrophage antigen could be detected at any post-lesion interval. When compared to the microglial activation that occurs after axotomy in adult animals, our results show a similar onset in microglial activation in neonatal animals; however, the duration of immunomolecule expression is much briefer. PMID- 1720135 TI - Autoantigen-induced self lysis of human myelin basic protein-specific T lymphocytes. AB - Cytotoxic T cells reactive with myelin basic protein (MBP) may be isolated from most human subjects. Since activated T cells express major histocompatibility complex (MHC) class II antigens, we assessed whether MBP-specific, CD4+ T cells could present MBP or synthetic MBP peptides to themselves and whether this provoked self lysis. We examined two MBP-specific cell lines and eight T cell clones recognizing four different MBP epitopes. All T cell populations presented MBP as well as synthetic peptides to themselves eliciting self lysis of the T cell clones. CD4+ T cell populations recognizing another central nervous system (CNS) protein, proteolipid protein (PLP), or the recall antigen, Candida, did not exhibit this antigen-induced, autocytolytic activity. However, activated, PLP reactive T cells were susceptible to lysis by cytotoxic MBP-specific T cells in the presence of MBP. These results suggest that antigen-induced self lysis of activated human T cells might limit an autoimmune response within a target organ independent of other immunoregulatory mechanisms. PMID- 1720136 TI - Non-specific oligodendrocyte cytotoxicity mediated by soluble products of activated T cell lines. AB - Supernates from myelin basic protein (MBP)-reactive T cell lines have been tested by a battery of assays for cytotoxicity against oligodendrocytes in vitro. All supernates tested demonstrated cytotoxic activity in both a dose- and time dependent manner that ranged from 29.1% to 55.8% after 48 h incubation. Oligodendrocyte cytotoxicity mediated by MBP-reactive T cell lines was not antigen specific since lines reactive with purified protein derivative (PPD) of tuberculin showed similar cytotoxicity. Supernates cytotoxic to oligodendrocytes were equally effective against syngeneic and non-syngeneic target cells, but had no effect upon astrocytes. Neutralization studies revealed that oligodendrocyte cytotoxicity mediated by MBP-reactive T cell supernates could only be partially attributed to lymphotoxin/tumor necrosis factor activity, and was not associated with perforin, serine proteinase or N-methyl-D-aspartate (NMDA) receptor agonists. PMID- 1720137 TI - Relative susceptibility of SJL/J and B10.S mice to experimental allergic encephalomyelitis is correlated with high and low responsiveness to myelin basic protein. AB - SJL/J mice are highly susceptible to actively induced experimental allergic encephalomyelitis (EAE), whereas B10.S mice are not. Yet both strains share the H 2s major histocompatibility complex (MHC) haplotype. In order to help determine the cellular basis for the disparate susceptibility to EAE, the antigen-specific in vitro proliferative responses of lymph node (LN) T cells from SJL/J and B10.S mice primed with porcine myelin basic protein (MBP) were assessed. The results indicated that SJL/J mice were high responders and B10.S mice were low responders to both porcine and murine MBP, as demonstrated by limiting dilution analyses and cloning efficiency analysis of MBP-reactive T cells. The low response of B10.S mice to MBP was not due to elevated suppressor cell activity or to a discernible defect in antigen-presenting cell activity. Rather, it appeared to be due to a paucity (or defect in function) of high affinity MBP-reactive T cells in B10.S as compared to SJL/J mice. This difference in MBP responsiveness must, by necessity, be linked to non-MHC background genes. Therefore, assuming that the relative number of MBP-reactive T cells parallels that of EAE-effector T cells in SJL/J and B10.S mice (as separate in vivo studies indicate), the present results suggest that differences in the T cell repertoire for the encephalitogenic determinants of MBP may contribute significantly to the observed differences in antigen reactivity, and may relate to differences in susceptibility to EAE. PMID- 1720138 TI - Myelin basic protein infused into cerebrospinal fluid suppresses experimental autoimmune encephalomyelitis. AB - We have evaluated the antibody and the effector T-cell responses to a single cerebrospinal fluid (CSF) infusion of myelin basic protein (MBP) in Lewis rats by measuring serum anti-MBP antibodies and clinical signs of experimental autoimmune encephalomyelitis (EAE), respectively. Some rats developed anti-MBP antibodies, but none manifested EAE in response to the primary infusion. Antibody responses to an EAE challenge 3 weeks after CSF infusion were normal, but clinical symptoms of EAE were markedly suppressed. Brain trauma at the time of MBP pretreatment enhanced this suppression. The CSF route of MBP administration is more effective in inducing suppression of EAE than peripheral routes. PMID- 1720139 TI - Subarachnoidal macrophages share a common epitope with resident non-cerebral macrophages and show receptor-mediated endocytosis of albumin-gold and IgG-gold complexes. AB - Cerebral macrophages are supposed to exploit a pivotal role in scavenger functions of the central nervous system. We have examined the in vivo uptake of serum albumin and IgG conjugated with colloidal gold by subarachnoidal macrophages. These serum-borne proteins are endocytosed by receptor-mediated endocytosis by varying kinetics. Albumin-gold conjugates were found to be associated to a significant amount with coated pits and coated vesicles 1 min after superfusion of the cerebral surface. Within 25 min the major fraction of albumin-gold was transferred to the lysosomal compartment. IgG-conjugates revealed a less pronounced uptake. The uptake of albumin-gold could be competed by saturating of the 'receptor sites' with free albumin. It is suggested that the described receptor-mediated uptake of serum-borne proteins by subarachnoidal macrophages serves a cleansing function following blood-brain barrier disruption during acute or subacute inflammatory reactions. PMID- 1720140 TI - Rod-signal interneurons in the rabbit retina: 1. Rod bipolar cells. AB - The cellular morphology and topographic distribution of the rod bipolar cells in the rabbit retina have been investigated by selective labelling with protein kinase C-immunohistochemistry (Negishi et al., Neurosci, Lett. 94:247-252, 1988) and by Lucifer Yellow injection of microscopically identified cells in a superfused retinal preparation. The distribution of the rod bipolar cells parallels that of their input neurons, the rod photoreceptors, in that the rod bipolars reach maximum densities of 5,000-7,000 cells/mm2 on the inferior and superior flanks of the visual streak, dropping to slightly lower densities at the peak visual streak. The centre-to-periphery density gradient of the rod bipolars is about 2.5:1, and the density ratio of rods to rod bipolars shows little variation across the retina, ranging from 43:1 in superior retina to 58:1 in inferior retina. The dendritic field area of the rod bipolar cells increases from 600 microns2 on the visual streak to 1,200 microns2 in the far-superior retina, with each point on the retina overlapped by 2.5-3.5 dendritic fields. The axonal field area of the rod bipolar cells increases from about 100 microns2 at the peak visual streak to about 250 microns2 at the retina edge, and the axonal field coverage ranges from 0.55 in the visual streak to about 0.8 in peripheral retina. Although there appear to be gaps in the local array of rod bipolar somata, these areas are covered by the axonal arbours of neighbouring rod bipolar cells. PMID- 1720141 TI - Immunolocalization studies of putative guidance molecules used by axons and growth cones of intersegemental interneurons in the chick embryo spinal cord. AB - The earliest developing interneurons in the chick spinal cord can be divided into two groups: neurons in the ventral region whose axons pioneer the primitive longitudinal pathway (PL-cells) and neurons whose axons project circumferentially (C-cells) along the lateral marginal zone and join the ipsilateral or contralateral ventrolateral longitudinal pathways. To begin to examine the molecular cues for axonal pathway formation of these interneurons, we screened a variety of molecules from embryonic day (E) 2 to E6.5 [stage 14-30 of Hamburger and Hamilton (1951) J. Morphol. 88:49-92]. These include cell adhesion and related molecules (G4, F11, neurofascin, N-cadherin, TAG-1-like molecule), extracellular matrix (ECM) molecules (laminin, fibronectin, heparan sulfate proteoglycan, laminin-heparan sulfate proteoglycan complex, and collagen type IV), and receptors for ECM molecules (beta 1-class integrin). PL-cells first expressed neurofascin at stage 14+ before the onset of axonogenesis. When the PL cells began to extend their axons at stage 15, they expressed G4 and avian TAG-1 like molecules, as well as neurofascin, on both cell bodies and longitudinal axons. In the following stages, PL-cells continued to strongly express neurofascin and G4 on their fasciculating axons, suggesting the involvement of these glycoproteins in growth and fasciculation. C-cells began to express G4 and TAG-1-like molecules on cell bodies and axons at stage 15-16 shortly after axonal growth. In the following stages, C-cells expressed several cell adhesion molecules differentially on their axonal segments. The proximal segment of C axons in the circumferential pathway strongly expressed a TAG-1-like molecule, whereas the distal segment in the longitudinal pathway strongly expressed G4 and neurofascin. The commissural axonal segment in the floor plate expressed TAG-1 like molecule, neurofascin, N-cadherin, and beta 1-class integrin. The basement membrane around the spinal cord was enriched with ECM glycoproteins (laminin, fibronectin, heparan sulfate proteoglycan, and collagen type IV) during the stages examined (stage 15-27), and commissural C-cell axons became strongly integrin positive in the floor plate where they contacted the basement membrane. These data indicate that interneurons may use multiple molecules during axonal pathway formation, depending on cell type, pathway position, and developmental stage. PMID- 1720142 TI - Postembedding immunocytochemistry for GABA and glycine reveals the synaptic relationships of the dopaminergic amacrine cell of the cat retina. AB - Postembedding electron microscope immunocytochemistry of glycine and GABA conjugated to colloidal gold has been applied to pre-embedded cat retina stained with the antibody against tyrosine hydroxylase (Toh+). Toh+ stained cells are the equivalent of A18 amacrine cells of Golgi descriptions (Kolb et al., '81). The dendrites of Toh+ cells synapse upon several different types of glycine-positive amacrine cell bodies. We suggest that these are the A8, A3/A4, and AII amacrine cell varieties by analogous immunocytochemical staining intensity, to glycine autoradiographic labeling intensity (Pourcho and Goebel, '85). The greatest number of synapses from Toh+ dendrites are directed at the least glycine-positive amacrine, which is the AII cell by all morphological criteria. A few glycine positive profiles are also presynapatic to the Toh+ stained cell body itself. Toh+ profiles are also presynaptic to GABA-positive amacrine cell bodies. The commonest amacrine synapsed upon is very heavily labeled with GABA immunocytochemistry. We consider it to be the A17 amacrine cell, which is known to label strongly by [3H] muscimol autoradiography (Pourcho and Goebel, '83). The cell body of the Toh+ amacrine cell also receives many synapses, which appear to be GABA-positive, and Toh+ profiles running in stratum 1 of the inner plexiform layer (IPL) are both pre- and postsynaptic to GABA-positive amacrine cell profiles. In addition, the cell body and primary dendrites of the Toh+ cell receive input from a bipolar type and GABA- or glycine-negative profiles. GABA positive profiles, belonging to the interplexiform cell (IPC), are synapsed upon by Toh+ profiles that run in the outer plexiform layer (OPL).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720143 TI - Corticomotoneuronal connections in the rat: evidence from double-labeling of motoneurons and corticospinal axon arborizations. AB - In order to investigate the possibility of direct corticomotoneuronal (CM) connections in the rat, an anterograde-retrograde double-labeling method was developed. Phaseolus vulgaris-leucoagglutinin (PHA-L) anterograde tracing of corticospinal axons was combined with retrograde labeling of spinal motoneurons either by a conjugate of choleragen subunit B with horseradish peroxidase (CB HRP) or by wheat germ agglutinin (WGA). The location of PHA-L injection unilaterally in the forelimb area of sensorimotor cortex and the CB-HRP or WGA injections in corresponding contralateral wrist or digit extensors or flexors were determined and matched on the basis of movement responses elicited by intracortical microstimulation. Light microscopic observation showed, in addition to the main contralateral dorsal corticospinal tract (CST), the presence of four other CST minor components in the contralateral lateral, ipsilateral ventral, and ipsilateral dorsal funiculi of the cervical spinal white matter and at the base of contralateral dorsal horn of the gray matter, respectively. PHA-L-labeled CST axonal arbors were observed from Rexed's lamina I through lamina X of contralateral spinal gray matter, most extensively in laminae VI and VII; some CST axons reached the zone of motoneuronal somata in lamina IX and a few of them also entered the lateral and occasionally the ventral funiculi, ramifying in the white matter. Between the zones of PHA-L-labeled CST axonal arbors on the one hand and CB-HRP/WGA labeled spinal motoneuronal somata with their extensive dendritic trees on the other, there was a large overlap, covering partly both the gray and the white matter. PHA-L-labeled axonal boutons (en passant or terminaux) were seen to contact the dendrites or even the somata of motoneurons in the gray matter, according to light-microscopic criteria for identification of synaptic contacts. Axodendritic CM contacts were occasionally observed in the lateral funiculus of the white matter as well. In general, only a single contact was observed between an individual PHA-L-labeled CST axon and a given retrogradely labeled motoneuron. In contrast to the common notion that direct CM connections are a specialty of primates, the present morphological data support the presence of direct CM connections also in some other mammals, such as the rat. PMID- 1720144 TI - Differential complementary localization of metabolic enzymes for quinolinic acid in olfactory bulb astrocytes. AB - The cellular localizations of the synthetic [3-hydroxyanthranilic acid oxygenase (3HAO)] and degradative [quinolinic acid phosphoribosyltransferase (QPRT)] enzymes of the endogenous excitotoxin quinolinic acid were studied in the adult rat main olfactory bulb by immunohistochemical techniques. 3HAO and QPRT were expressed only in astrocytes. The two enzymes were differentially expressed by astrocytes in a complementary pattern: 3HAO staining was strongest at the glomerular-external plexiform layer junction; QPRT staining was strongest at the glomerular-olfactory nerve layer junction. The complementary distributions of these metabolic enzymes suggests that there could be a gradient of quinolinic acid across the glomerular layer of the main olfactory bulb. Such a gradient could function to restrict the ingrowth of new olfactory axons to the glomeruli and/or to stabilize the formation of new synapses. PMID- 1720145 TI - Nigropedunculopontine projection in the rat: an anterograde tracing study with phaseolus vulgaris-leucoagglutinin (PHA-L). AB - The termination of the substantia nigra pars reticulata efferents in the nucleus tegmenti pedunculopontinus was studied in the rat by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Both large and small injections of PHA-L in various portions of the substantia nigra pars reticulata labeled varicose fibers in the ipsilateral and contralateral nucleus tegmenti pedunculopontinus, subnucleus dissipatus as well as in the ipsilateral nucleus tegmenti pedunculopontinus, subnucleus compactus. However, the bulk of the nigral fibers appeared to terminate in the medial two-thirds of the ipsilateral subnucleus dissipatus of the pedunculopontine nucleus and exhibited a discrete dorsoventral topographical pattern. The terminal plexus displayed patches of uneven density, which was partly due to the numerous fiber bundles passing through the pedunculopontine nucleus, but also to an obvious preference of nigral fibers for some cells. Electron microscopic examination confirmed that nearly all of the varicosities observed in the light microscope contained synaptic vesicles and represented either terminal boutons or boutons en passant. The labeled boutons were elongated (average length: 1.5 microns) and consistently contained a prominent group of mitochondria. The results suggest that the nigral input to the nucleus tegmenti pedunculopontinus may be directed toward specific subpopulation(s) of pedunculopontine neurons and may influence not only cells in the subnucleus dissipatus, but also in the subnucleus compactus. PMID- 1720146 TI - Sensory innervation of the rat kidney and ureter as revealed by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from dorsal root ganglia. AB - The sensory innervation of the rat kidney and ureter was investigated in wholemount preparations and sectioned materials by labeling the afferent nerve fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from dorsal root ganglia. Labeled fibers were seen in large numbers in the ureter and in the lining of the renal pelvis, where they were located in the adventitia, smooth muscle, subepithelial connective tissue, and epithelium. Most of the fibers in the ureter and ureteropelvic junctional zone traveled parallel to the long axis of the organ. In contrast, fibers in the widest part of the funnel-shape renal pelvis were oriented predominantly in a circumferential fashion. Many of the pelvic afferents were extremely fine and appeared to terminate as free nerve endings. Modest networks of labeled axons were also observed around branches of the renal artery; the greatest innervation was supplied to the distal portions of the interlobar arteries and to the arcuate arteries. Only single axons were observed around the interlobular arteries, and very few fibers were seen around afferent arterioles or near glomeruli. In contrast to the arteries, branches of the renal vein were relatively sparsely innervated. Occasional labeled fibers entered the renal cortex and formed intimate associations with renal tubules; however, the vast majority of renal tubular elements were not contacted by labeled sensory fibers. Labeled fibers were never observed in the renal medulla or in the papilla. The present study represents the first time that the sensory innervation of the kidney and ureter has been investigated by using a highly specific anterograde nerve tracing technique. The pattern of innervation demonstrated here reveals an anatomical configuration of ureteral and renal pelvic sensory nerves consistent with a role in detection of ureteral and pelvic pressure and chemical changes and a renal vascular sensory innervation that may monitor changes in renal arterial and venous pressure and chemical content. Still other renal afferent nerve endings may signal renal pain. PMID- 1720147 TI - Ipsilateral cortical connections of primary somatic sensory cortex in rats. AB - The organization of ipsilateral cortical connections of the rat primary somatic sensory area (SI) was analyzed following small injections of multiple fluorescent tracers in the same case, into two or three SI body representations identified electrophysiologically. Labeling patterns were studied in tangential cortical sections and in flattened reconstructions from coronal sections. The cytochrome oxidase staining in tangential sections served as a control for injection location and to position labeling patterns found within granular portion of SI. The results show that most connections made with SI are reciprocal. Their topographical organization show different degrees of precision in the different areas. Homotypical and heterotypical connections were defined, the latter being more evident within the granular portion of SI. The findings: (1) were consistent with subdividing rat SI into four distinct areas with each having its own pattern of connections, (2) revealed two topographically organized regions in parietal cortex lateral to SI called second somatosensory (SII) and parietal ventral (PV) areas, (3) confirmed a topographical pattern in motor cortex and suggested an organization for connections between SI and an agranular medial field, and (4) demonstrated three more regions in parietal cortex connected to SI: posterior to SI called parietal medial; lateral to PV called parietal rhinal; posterior to SII called parietal lateral. Differences were noted in the distinctions between and within the maps when label distributions were plotted separately from supra- and infragranular layers. These findings agree with previous parcellations of the rat SI (Chapin et al., '87: J. Comp Neurol 263:326-346), squirrel PV and SII (Krubitzer et al., '86: J. Comp Neurol 250:403-430), and the organization of rat corticospinal neurons in many of the same areas (Li et al., '90: Somat Motor Res 7:315-335). PMID- 1720148 TI - Histamine-releasing factors and inhibitors: historical perspectives and possible implications in human illness. AB - The initiation of allergic reactions with the bridging of surface-bound IgE antibodies on mast cells and basophils by allergens is well recognized. However, it is clear that other factors most likely play a role in regulating these cells. A number of cytokines have been identified that modulate the secretory response of mast cells and basophils. Among the well-characterized cytokines, interleukin 3 and connective tissue-activating peptide III (or its degradation product, neutrophil-activating peptide 2) can increase the secretory response, whereas interleukin-8 specifically inhibits the response to cytokines. Additional factors are currently under investigation. Preliminary studies suggest an important role for these histamine-releasing factors in atopic disorders, as well as in other conditions in which an IgE-dependent mechanism is not demonstrable. Furthermore, these cytokines may modulate the response of basophils and mast cells in physiologic conditions, such as tissue repair and host defense. PMID- 1720149 TI - 13-cis retinoic acid enhances in vivo B-lymphocyte differentiation in patients with common variable immunodeficiency. AB - Retinoic acid (RA) has been demonstrated to drive both phenotypic and functional in vitro differentiation of B cell hybridomas from patients with common variable immunodeficiency (CVI) who manifest an "intrinsic" defect in terminal B cell differentiation (J Exp Med 1988;168: 55-71). Therefore, we conducted an open trial to determine the effects of oral 13-cis RA (0.5 mg/kg/day; 12 weeks receiving and 12 weeks without drug) on in vivo B cell differentiation in subjects with CVI. At various times before, during, and after drug administration, patients' B cells were tested for changes in cell-surface phenotype and in vitro immunoglobulin production in response to recombinant cytokines. Before 13-cis RA, all patients had decreased Leu-8 coexpression on CD20+ cells. Seven of eight subjects demonstrated "normalization" of this phenotype after 8 to 16 weeks of 13-cis RA administration. Patients whose B cells demonstrated more than normal CD20 display also had a fall toward normal in this parameter. These effects persisted for 6 to 12 weeks after drug was stopped. It appears that 13-cis RA drives B cells of patients with CVI to express a more differentiated cell-surface phenotype and may promote functional differentiation in some patients. PMID- 1720150 TI - Mononuclear cells from patients with the hyper-IgE syndrome produce little IgE when they are stimulated with recombinant human interleukin-4. AB - To investigate whether B cells from patients with the hyper-IgE syndrome are more sensitive to the effects of interleukin-4 in vitro than B cells of normal or atopic individuals, we stimulated blood mononuclear cells (MNC) with varying doses of recombinant human interleukin 4 (rhIL-4) and measured supernatant IgE concentrations after 18 days of culture. Geometric mean spontaneous IgE synthesis after 18 days of culture without rhIL-4 was low (less than 3 ng/ml) and similar for MNCs from nine patients with the hyper-IgE syndrome, nine atopic and nine normal subjects. As found in our previous studies, MNCs from the nine atopic and the nine normal donors produced significant and similar quantities of IgE (geometric mean maximum IgE, 25.2 and 18.7 ng/ml, respectively) when MNCs were stimulated with rhIL-4. MNCs from both donor groups had similar sensitivity to the concentration of IL-4 eliciting the IgE response. In striking contrast, MNCs from the nine patients with the hyper-IgE syndrome failed to produce significant IgE over that produced spontaneously when MNCs were stimulated by a wide range of rhIL-4 concentrations. Coculture of B cell-enriched subpopulations from patients with the hyper-IgE syndrome with T cell-enriched subpopulations from nonatopic and atopic donors failed to restore responsiveness to rhIL-4. The addition of anti-CD40 monoclonal antibody to MNC cultures did result in enhancement of rhIL-4 IgE synthesis by MNCs from patients with the hyper-IgE syndrome, but the concentration of anti-CD40 required to elicit this enhancement was tenfold higher than for control MNCs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720151 TI - Human immunodeficiency virus reverse transcriptase T helper epitopes identified in mice and humans: correlation with a cytotoxic T cell epitope. AB - T cell immunity may be critical to development of a vaccine for human immunodeficiency virus (HIV-1). T helper epitopes were identified in three predominantly conserved regions in the HIV-1 reverse transcriptase by using reverse transcriptase-immunized mice of five major histocompatibility complex haplotypes. One peptide (residues 38-52) that stimulated H-2k T cells also contained an epitope recognized by cytotoxic T cells from the same mice and from HIV-infected patients. Such concordance between helper and cytotoxic T lymphocyte epitopes, observed in four cases, may be important in vaccine development. Peptide 36-52 was recognized by interleukin-2-producing peripheral blood T cells from 9 of 17 HIV-seropositive humans studied, of multiple human leukocyte antigen DR and -DQ types. The broad recognition of this peptide by both helper and cytotoxic T cells substantiates its potential importance in a vaccine. PMID- 1720153 TI - Anti-human immunodeficiency virus type 1 activity of sulfated monosaccharides: comparison with sulfated polysaccharides and other polyions. AB - Previously, the anti-human immunodeficiency virus type I (HIV-1) activities were reported of four sulfated polysaccharides: dextran sulfate, pentosane polysulfate, chondroitin sulfate, and heparin sulfate. In the present study, the anti-HIV-1 activities of several other sulfated polysaccharides, monosaccharides, neutral polysaccharides, and polypeptides were evaluated. Anti-HIV-1 activities of these various agents were measured by four different assays: (1) HIV-1-induced syncytia formation; (2) infectivity of cell-free HIV-1 after preincubation with the putative anti-HIV-1 agent; (3) protective ability of the agents for target CD4+ cells, and (4) anti-reverse transcriptase activity. In addition, potential toxicity of the putative anti-HIV-1 agents was measured by their effects on cellular proliferation, cytotoxic effects, and effects on coagulation processes. These data indicate that only sulfated polysaccharides and one sulfated monosaccharide, glucosamine 6-sulfate, have significant anti-HIV-1 activity. The therapeutic potentials of these agents are also discussed, with special reference to absorption of glucosamine 6-sulfate through the gastrointestinal tract. PMID- 1720152 TI - Detection of mutations associated with zidovudine resistance in human immunodeficiency virus by use of the polymerase chain reaction. AB - A sensitive and specific polymerase chain reaction (PCR)-based assay was developed for four mutations in the reverse transcriptase gene of human immunodeficiency virus type 1 that have been associated with zidovudine resistance. These mutations were correlated in 366 specimens with zidovudine chemotherapy and resistance. Mutations at these four codons were detected only after zidovudine therapy. The usual sequence of appearance of mutations was codons 215, 70, 67, and 219, although individual variations occurred. The degree of resistance was proportional to the number of mutations present, although variable susceptibilities with identical patterns of mutations suggested the likelihood that additional mutations contribute to resistance. The existence of both phenotypic and genotypic mixtures was documented as was the occasional selection of subpopulations with passage of virus in vitro. The many complexities of zidovudine resistance render the assay of limited use for application to individual patients; however, it could prove useful for correlating disease or therapy with the emergence of resistance. PMID- 1720154 TI - Mouse hematopoietic stem cells and the interaction of c-kit receptor and steel factor. AB - Hematopoietic stem cells (HSCs) are distinguished from other hematopoietic progenitors in bone marrow by their unique ability to undergo multilineage differentiation and self-renewal. Two mouse mutations, dominant spotting (W) and steel (Sl), have pleiotropic effects on hematopoiesis, gametogenesis, and melanoblast development. These two mutations have been shown to be intrinsic (W) and microenvironmental (Sl) defects. Recently, molecular studies revealed that the W and Sl loci encode the c-kit receptor and steel factor (SLF), respectively. The c-kit receptor is expressed on HSCs and hematopoietic progenitors, while SLF is produced by stromal cells. SLF acts on hematopoietic progenitors synergistically with other growth factors. Here we review the effect of these mutations on mouse hematopoiesis, and show that SLF acts on HSCs and other myeloerythroid progenitors, but that it, in our hands, does not play a critical role in HSC generation or self-renewal. Rather, SLF is the most potent co-mitogen (with IL-1, IL-3, IL-6, G-CSF, GM-CSF, or M-CSF) found that acts on these cells, but the effect of such treatments is the rather specific and massive expansion of myeloerythropoiesis, not lymphopoiesis, and perhaps at the expense of HSC self renewal. PMID- 1720155 TI - Isolation and characterization of the CD34+ hematopoietic progenitor cells from the peripheral blood of patients with chronic myeloid leukemia. AB - A modified version of the original reported panning technique was used to separate CD34+ cells from the peripheral blood of patients with chronic myeloid leukemia (CML). In 13 out of 23 separations, populations of cells were obtained in which CD34+ cells constituted greater than 50% of the cells present. The best recovery and enrichment of the CD34+ cells was achieved when cells were obtained from patients in the accelerated phase of CML, when the cells were processed on the same day they were obtained from patients, and when adherence to soybean agglutinin flasks was used as a pre-enrichment step. In suspension culture, the CD34+ cells were capable of extensive proliferation and differentiation. In semi solid culture, the number of colony-forming units (CFUs) directly correlated with CD34 positivity. The number of clonogenic cells/CD34+ cells was highest at the time of initial diagnosis of CML, fell during the chronic phase (CP) of the disease, and rose at the time of disease acceleration. This observation suggests that therapy during the CP of the disease produces a greater reduction in clonogenic cells than in the number of CD34+ cells. This effect disappears at the time of disease acceleration, presumably because of the development of drug resistance in the clonogenic cells. PMID- 1720156 TI - Continuous subcutaneous infusion of hyoscine butylbromide reduces secretions in patients with gastrointestinal obstruction. AB - We describe the use of hyoscine butylbromide as a subcutaneous infusion in 3 patients with inoperable malignant bowel obstruction. An objective reduction of drainage from the gastrointestinal tract was observed with the hyoscine butylbromide infusion (60-120 mg/day). We suggest that this effect can be useful in the palliative treatment of vomiting in inoperable bowel obstruction. PMID- 1720157 TI - Phenotype of HML-1-positive T cells in the human intestinal lamina propria. PMID- 1720158 TI - Production of inflammatory cytokines in the intestinal lamina propria. PMID- 1720159 TI - Multiple roles of Leu-8/MEL-14 in leukocyte adhesion and function. AB - Leu-8 and its murine homologue MEL-14 are members of a new family of adhesion molecules encoded on chromosome-1 that share common structural features, including lectin-like domains and tandem repeats homologous to complement binding proteins. The expression of Leu-8 is rapidly down-regulated during cell activation, both at the transcriptional level, and by a rapid post-translational event at the cell membrane, probably involving direct cleavage of the molecule from the cell surface. Lymphocytes that express Leu-8/MEL-14 bind selectively to HEVs in peripheral lymph nodes, and MEL-14 on neutrophils is thought to be important in the initial localization of neutrophils to sites of inflammation. In addition to its role in leukocyte adhesion, there is evidence that the Leu-8 molecule plays a role in cell function. Anti-Leu-8 monoclonal antibody increases suppressor activity of CD4+, Leu-8+ T cells for immunoglobulin synthesis, and anti-Leu-8 directly inhibits differentiation of Leu-8+ B cells. Together these findings indicate that the Leu-8 molecule in common with other cellular adhesion molecules is important not only in cellular adhesion, but also in modification of cell function. PMID- 1720160 TI - Cross-linking the Leu-8 lymph node homing receptor on B cells inhibits immunoglobulin synthesis. AB - About one half of circulating human B cells express the Leu-8 peripheral lymph node homing receptor that has been implicated in adhesion of lymphocytes and neutrophils to vascular endothelium. A novel and unique function of the Leu-8 antigen has been found in the present study: anti-Leu-8 monoclonal antibody directly inhibits B cell antibody production induced by SAC + IL-2, without affecting B cell proliferation or other early steps of B cell activation. This effect of anti-Leu-8 is unique in that other antibodies that inhibit B cell differentiation are not selective and also inhibit B cell proliferation. The inhibitory effect is not reversed by addition of T cells, monocytes, or recombinant IL-1 or IL-6 and is not blocked by anti-TGF-beta. Thus, the natural ligand(s) for Leu-8 may be capable of transducing regulatory signals that have a selective effect on B cell differentiation. PMID- 1720161 TI - Molecular characterization of the mitochondrial autoantigens in primary biliary cirrhosis. PMID- 1720162 TI - [Diagnosis of skeletal metastases by serum tumor markers]. AB - Levels of serum tumor markers including CEA, AFP, CA 15-3, CA 19-9, CA 125 and TPA were measured in 26 patients with bone metastases and in 9 patients with primary bone tumors. TPA was the most sensitive marker to detect skeletal metastasis being elevated in 15 of the 22 patients (68.2%). High sensitivity was observed in CEA (46.1%), CA 15-3 (40%), and CA 125 (35%), and AFP showed relatively low sensitivity (4.3%). When elevation of TPA only or elevation of more than two tumor markers including TPA was used as a screening test for skeletal metastasis, over-all sensitivity, specificity, and accuracy were 73.1%, 88.9%, and 81% respectively. No definite correlation between the markers could be seen in this study. A combination of serum tumor markers was useful in the differential diagnosis of bone metastases from primary bone lesions. However, organ specificity of the markers were relatively low. PMID- 1720163 TI - Neurochemical changes correlated with behavior maintained under fixed-interval and fixed-ratio schedules of reinforcement. AB - Key pecking of 4 pigeons was maintained under a multiple 3-min fixed-interval, 30 response fixed-ratio schedule of food presentation. Only one schedule was in effect during an experimental session, and each was correlated with a different keylight stimulus and location (left vs. right). The different schedule components alternated across days or weeks. Cerebrospinal fluid was collected from chronically implanted intracerebroventricular cannulae following sessions with the different schedules, as well as following sessions in which reinforcement was withheld (extinction), when response-independent food was delivered, and when the experimental chamber was dark and there were no scheduled events. Metabolites of the neurotransmitters serotonin, norepinephrine, and dopamine were assayed in cerebrospinal fluid using high-performance liquid chromatography with electrochemical detection. Compared to the fixed-ratio condition, responding maintained under the fixed-interval schedule resulted in consistently higher levels of the serotonin metabolite 5-hydroxyindoleacetic acid and of the dopamine metabolite homovanillic acid in all pigeons. Levels of 3 methoxy-4-hydroxyphenylethylene glycol, a metabolite of norepinephrine, and dihydroxyphenylacetic acid, another dopamine metabolite, were also higher in 3 of the 4 pigeons following exposure to the fixed-interval schedules when compared to levels of these metabolites after exposure to the fixed-ratio schedule. Extinction of fixed-ratio responding resulted in large increases in 5 hydroxyindoleacetic acid compared to levels of this metabolite under the fixed ratio schedule, whereas this serotonin metabolite decreased during extinction of responding under the fixed-interval schedule. Control procedures suggested that the neurochemical changes were not related to the rate of responding but were a function of the specific experimental conditions. Distinctive neurochemical changes that accompany schedule-controlled responding show the sensitivity of the neurochemical environment to behavioral contingencies and demonstrate further the profound impact that such contingencies have on biobehavioral processes. PMID- 1720164 TI - [Effect of combinations of anticancer drugs with interferons on human lung cancer cell lines evaluated by human tumor clonogenic assay]. AB - Using the double agar layer method of human tumor clonogenic assay, the anticancer effect of different combinations of anticancer drugs and interferons was tested on 3 lung cancer cell lines, PC-13, PC-14, and Calu-1. The anticancer drugs and the concentrations used in this study were cisplatin (1.0 microgram/mL), adriamycin (1.0 microgram/mL), mitomycin C (0.2 microgram/mL), VP 16 (5.0 micrograms/mL) and 5-FU (5.0 micrograms/mL). Three kinds of interferon, alpha, beta and gamma in 5,000 units/mL, were tested in combination or in sequence with other anticancer drugs on lung cancer cell lines. The results demonstrate an enhanced anticancer effect on PC-14 only with sequential or simultaneous combination of VP-16 with alpha, beta and gamma interferons; and on Calu-1, only with sequential use of adriamycin and beta-interferon. Our results indicate that there is no unique way of combining anticancer drugs and interferons which can obtain an enhanced anticancer effect on all lung cancer cell lines. The best combination of interferon and anticancer drugs seems to be influenced by the biological characteristics of the cancer cells. PMID- 1720165 TI - Cloning, characterization and taxonomic significance of genes for the 5S ribosomal RNA of Leptonema illini strain 3055. AB - The genes encoding the 5S ribosomal RNA (rRNA) for Leptonema illini strain 3055 were isolated and sequenced. The 5S RNA molecule encoded was 117 nucleotides long. The genome of strain 3055 contained two genes for 5S rRNA that were located close together. The nucleotide sequences of the Leptonema illini genes exhibited less similarity to the rRNA gene of Leptospira interrogans strain Moulton and also to those of typical eubacterial genes than did the rRNA genes of other leptospires. However, the overall secondary structure of the 5S rRNA encoded exhibited a strong similarity to that of typical eubacterial 5S rRNA. Southern hybridization of the 5S rRNA gene probe with the genomic DNA of strain 965, which is currently classified as Leptospira biflexa, showed the latter to have close similarity to that of strain 3055. The physical map of strain 965 was quite similar to that of strain 3055 and was greatly different from that of any other strains of L. biflexa. In the organization of 5S rRNA genes, strain 965 is sufficiently different from other members of the genus Leptospira to be regarded as a member of the genus Leptonema. PMID- 1720166 TI - Chlamydia trachomatis major outer membrane protein epitopes expressed as fusions with LamB in an attenuated aro A strain of Salmonella typhimurium; their application as potential immunogens. AB - The major outer-membrane protein (MOMP) of Chlamydia trachomatis is the focus of attention for chlamydial vaccine design, particularly those serovar- and subspecies-specific epitopes which provoke neutralizing immune responses. Selected surface-exposed B-cell epitopes of MOMP, incorporating B-subspecies specificities, were expressed as fusions with LamB, an inducible outer-membrane transport protein of Escherichia coli. These recombinant chlamydial-LamB proteins were correctly transported to the outer membrane of both E. coli and an aro A mutant of Salmonella typhimurium. The immunogenicity of the constructs was investigated in a mouse model of chlamydial salpingitis. After oral immunization, recombinant S. typhimurium were recovered from the livers of mice for up to two weeks, and a serum IgG response was induced both to the Salmonella and to the inserted chlamydial epitopes. By contrast, intravenous inoculation was ineffective. Although these LamB fusions proved only weakly immunogenic, this approach should be useful for investigating the ability of attenuated S. typhimurium vaccines incorporating chlamydial epitopes to stimulate protective mucosal immunity in the mouse model of chlamydial salpingitis. PMID- 1720167 TI - Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei. AB - The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%. PMID- 1720168 TI - Bacterial metabolism of 3-chloroacrylic acid. AB - Two bacterial strains were isolated with 3-chloroacrylic acid (CAA) as sole source of carbon and energy. Strain CAA1, a Pseudomonas cepacia sp., was capable of growth with only the cis-isomer of CAA. Strain CAA2, a coryneform bacterium, utilized both isomers of CAA as sole source of carbon and energy. Strain CAA1 contained cis-CAA hydratase and strain CAA2 contained two hydratases, one with cis-CAA hydratase activity and one with trans-CAA hydratase activity. The product of the hydratase activities with CAA was malonate semialdehyde. In both strains malonate semialdehyde was subsequently decarboxylated by a cofactor-independent decarboxylase yielding acetaldehyde and CO2. PMID- 1720169 TI - Evaluation of a bone substitute prepared from alpha-tricalcium phosphate and an acid polysaccharide solution. AB - Tissue response to a readily consolidating material prepared by mixing alpha tricalcium phosphate (alpha-TCP) powder with a glycolic acid dextran solution and to this consolidating material combined with particulate hydroxylapatite (HA) was studied after implantation in the subperiosteal space of the mandible in rabbits. Active new bone formation comparable to that seen on HA implants was observed around the two compounds. The newly formed bone was in direct contact with the HA as well as the readily consolidated material and little adverse effect resulting from the glycolic acid and dextran was observed. Because the readily consolidating material was firm and could be contoured into any shape during the process of consolidation, it may be quite useful as a bone substitute and as an adherent for HA particles for reconstructive bone surgery, overcoming the disadvantages inherent to the particulate form of HA. PMID- 1720170 TI - Characterization of integrin chains in normal and neoplastic human pancreas. AB - Integrins are a complex family of non-covalently linked heterodimeric glycoproteins which function as cell adhesion molecules, interacting with extracellular matrix molecules such as laminin, fibronectin, vitronectin, and collagen, and also having a role in intercellular adhesion. Each integrin subfamily is characterized by a common beta chain associated with variable alpha chains. We have examined, using immunohistological methods, the expression of the VLA (very late activation) family comprising beta 1 in association with alpha 1 6, and also alpha 6 in association with beta 4, the LFA beta chain beta 2, and the vitronectin receptor, in association with beta 1 or beta 5 and as the complex alpha v beta 3. Cryostat sections of normal pancreas, pancreatic adenocarcinomas, and ampullary tumours were studied together with six pancreatic carcinoma cell lines. Normal pancreas showed expression of beta 1 in all parenchyma. alpha 2 and alpha 6 had a similar distribution whereas alpha 3 expression was confined to ducts, including the very smallest radicles. Staining along the basement membranes of ducts was seen with beta 4 and the anti-vitronectin alpha v chain receptor antibody 13C2. Islet cells failed to stain with any antibody. No staining of epithelial components was seen with antibodies to alpha 1, alpha 4, alpha 5, or to the alpha v beta 3 form of the vitronectin receptor (beta 3 and alpha v beta 3 using the antibody 23C6). Pancreatic adenocarcinomas and ampullary tumours showed expression of alpha 2, alpha 3, alpha 6, beta 1, beta 4, and the vitronectin receptor (alpha v associated with beta 1 or beta 5).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720171 TI - Helium-neon laser irradiation of rat liver mitochondria gives rise to a new subpopulation of mitochondria: isolation and first biochemical characterization. AB - An experiment was performed to isolate the small atypical mitochondria produced during the irradiation of normal mitochondria with an He-Ne laser. Rat liver mitochondria were irradiated with a low-power continuous-wave He-Ne laser (energy dose, 5 J cm-2), followed by isolation using a sucrose gradient. In the irradiated sample, two bands were observed, one corresponding to normal mitochondria and the other to atypical mitochondria. Certain biochemical features of the mitochondria were investigated: mitochondrial enzyme activity and the presence of DNA and RNA were demonstrated. Hybridization experiments carried out with labelled mitochondrial probes, containing the genes for cytochrome oxidase subunit I and 12S rRNA, confirmed the mitochondrial nature of the isolated RNA. PMID- 1720172 TI - The effect of phorbol esters on the expression of mRNA for the alpha subunit of calcium/calmodulin dependent protein kinase II in rabbit renal proximal tubules. AB - Calcium-calmodulin dependent protein kinase II (CaM-KII) has been implicated in the inhibition of Na(+)-H+ exchange activity in the brush border of the renal proximal convoluted tubule. Conversely, the activity of the antiporter is stimulated in response to phosphorylation by calcium-phospholipid dependent protein kinase (PKC). In these experiments, we explored the potential for direct interaction between these two protein kinases by determining the effect of PKC activation by tumor promoting phorbol esters on the expression of mRNA for CaM KII in the rabbit renal proximal tubule. The results indicate that activation of PKC reduced the steady-state levels of the mRNA for the alpha subunit of CaM-KII in a dose and time dependent manner. This suggests a novel mechanism by which PKC can antagonize the action of CaM-KII in selected tissues. PMID- 1720173 TI - [Ultrasonic diagnosis of hepatocellular carcinoma]. PMID- 1720174 TI - Preoperative versus postoperative dextran 70 for preventing adhesion formation. AB - Since serosal drying and tissue abrasion play an important role in adhesion formation, we tested the hypothesis that the peritoneal instillation of 32% high molecular-weight dextran 70 (H) before, rather than after, a surgical procedure results in less postoperative adhesion formation and reformation. Twenty rabbits were subjected to a standardized surgical injury on one ovary, the ipsilateral uterine horn and adjacent parietal peritoneum. Three weeks later the animals underwent a second laparotomy to blindly score the adhesions and subsequently lyse them using microsurgical techniques. The animals were randomly assigned to one of two treatment groups, with H administered either before or at the end of each surgical procedure. Three weeks after the second surgical procedure, the animals were killed to blindly score adhesions. There was no difference in the mean adhesion scores between the two groups after either the first (2.0 versus 2.9, NS) or second surgical procedure (5.5 versus 5.1, NS). Thus, we conclude that preoperative instillation of H does not offer any advantage over postoperative instillation in the prevention of either adhesion formation or reformation. PMID- 1720175 TI - Activity of acyclic 6-(phenylselenenyl)pyrimidine nucleosides against human immunodeficiency viruses in primary lymphocytes. AB - Several 6-phenylselenenyl-substituted acyclouridine derivatives were prepared for evaluation as antiviral agents. Lithiation of the tert-butyldimethylsilyl protected acyclonucleosides 4a-f with lithium diisopropylamide at -78 degrees C, followed by reaction with diphenyl diselenide as an electrophile, and subsequent removal of the protecting group with tetra n-butylammonium fluoride gave 1-[(2 hydroxyethoxy)methyl]-6-(phenylselenenyl)uracils 6a-f in 50-70% overall yield. The potency and spectrum of activity of compounds 6a-f against HIV-1 in vitro was similar to HEPT (1), a related 6-phenylthio acyclonucleoside. However, whereas HEPT inhibited HIV-1 reverse transcriptase, the selenium-containing derivatives were ineffective, suggesting a different mechanism of action. Of significance was the finding that the 6-phenylselenenyl acyclonucleosides inhibited also HIV-2 in primary human lymphocytes. PMID- 1720176 TI - Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers. AB - Unitary currents through cGMP-dependent channels of retinal rods are observed following incorporation into planar lipid bilayers of native vesicles from purified rod outer segment membranes washed free of soluble and peripheral proteins. The influence of the concentration of cGMP, inhibitors (cis-diltiazem, tetracaine and Ag+) and divalent cations (Ca2+, Mg2+, and Co2+) on the conductance and open probability of the channel is described, as well as the voltage dependence of these effects. The cGMP dependence suggests the existence of four binding sites for cGMP and reveals that sequential binding of four cGMP molecules corresponds to the opening of four discrete conductance levels. Finally, we provide conclusive evidence that activated G-protein does not directly inactivate the cGMP-dependent channels of bovine retinal rods. PMID- 1720177 TI - Ion flow through membranes and the resting potential of cells. AB - A knowledge of the relationship between ion flow, both passive and active, ionic concentration, and membrane potential is essential to the understanding of cellular function. The problem has been analyzed on the basis of elementary physical and biophysical principles, providing a theoretical model of current flow and resting potential of cells, including those in epithelia. The model assumes that the permeability of the ion channels is not voltage dependent, but applies to gated channels when the gates are open. Two sources of nonlinearity of the current-voltage relationship are included in the analysis: ionic depletion and accumulation at the channels' mouths, and channel saturation at higher concentrations. The predictions of the model have been quantitative, validated by comparison with experiment, which has been limited to the only two cases in which adequate data was found. Application of the theory to the scala media of the mammalian cochlea has explained the source of its high positive potential and provided estimates of the Na+ and K+ permeabilities of the membranes of its marginal cells. This analysis provides a theoretically sound alternative to the widely used Goldman equation, the limited validity of which was emphasized by Goldman (D.E. Goldman, 1943, J. Gen. Physiol, 27:37-60), as well as its derivatives, including the Goldman-Hodgkin-Katz equation for resting potentials. PMID- 1720178 TI - Maximizing communication skills in graduate and postgraduate health-care education through medical writing. AB - Graduate and postgraduate health-care professional training and postdoctoral fellowship programs that deny trainees opportunities to practice both oral and written communication skills produce an incompletely trained health-care provider unable to compete for faculty positions at university hospitals and affiliated staffs. Therefore, it is imperative that program directors make medical writing a prerequisite to successful completion of postgraduate training programs. To make trainees as well as administrators and faculty aware of the importance of oral and written communication skills, a variety of oral abilities needed for presenting medical findings prior to publication are detailed. The use of 2 x 2 slides to support a presentation as well as transparencies, movies, and videotapes are considered. The poster session/scientific exhibit, now becoming more visible because of increasing attendance at professional meetings, is also explained. Written communication abilities are discussed. Consideration is given to the writing of professional manuscripts for publication in a refereed journal. Other types of written communication include case reports, clinicopathological conferences, letters to the editor, book reviews, books, and book contributions. The opportunity to learn needed skills must be offered in the postgraduate health care curriculum. Mandatory medical writing will maximize the marketability of black health-care professionals for faculty staff placement. Moreover, the establishment of a "track record" early in a professional career will increase the likelihood that black health providers are awarded grants for research. PMID- 1720179 TI - [Effects of ethanol and low-carbohydrate diet on contents of noradrenaline, dopamine, serotonin and their metabolites in rat brain]. AB - Effects of ethanol consumption and intake of low-carbohydrate (low-CHO) diet on noradrenaline (NA), dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), serotonin (5HT) and its metabolite, 5 hydroxyindoleacetic acid (5HIAA) contents in six brain regions of rats were investigated. 1) Change of DA neuron Ethanol-containing control diet (hypercaloric ethanol diet) did not affect DA content in any area of brain, but decreased HVA in cortex and hypothalamus and increased DOPAC and HVA in midbrain. Low-CHO diet increased DA content in striatum, DOPAC and HVA in midbrain, but decreased DOPAC in hippocampus and hypothalamus, and HVA in cortex, pons and medulla, hippocampus and hypothalamus. Ethanol-containing low-CHO diet (isocaloric ethanol diet) increased DA level in striatum, DOPAC and HVA in midbrain, but decreased HVA in cortex, hippocampus, striatum and hypothalamus. These results suggest that i) hypercaloric ethanol diet has an opposite effect to carbohydrate on DA metabolism: hypercaloric ethanol diet and lowered carbohydrate intake per se enhance DA metabolism in midbrain, whereas inhibit it in cortex and hypothalamus, ii) lowered carbohydrate intake also declines DA metabolism in pons and medulla and hippocampus, whereas enhances DA synthesis in striatum, iii) the combined effect of ethanol and carbohydrate intake on DA metabolism is inhibited each other in the rats of isocaloric ethanol diet feeding, and this diet decreased DA metabolism in striatum. 2) Change of 5HT neuron Hypercaloric ethanol diet did not affect the contents of 5HT and 5HIAA in any region of brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720180 TI - Analysis of common epitopes on gag proteins of HIV-1, HIV-2 and SIV[AGM] using monoclonal antibodies against HIV-1. AB - Five monoclonal antibodies (MoAbs) to gag proteins of HIV-1 were prepared in mice. Western blot analyses showed that three clones recognized p24 and the other two p17. Among the three MoAbs recognizing p24, all recognized two of three strains of HIV-2. The spectra of reactions to SIV[AGM] of these MoAbs against p24 were different from one to another; K3-24 recognized all four strains of SIV[AGM], L6-24 three of them, and K5-24 none of them. Of the two MoAbs recognizing p17, K7-17 recognized two of the three strains of HIV-2 but not any SIV[AGM] strain, and the other clone, L14-17 recognized none of analogous proteins of HIV-2 nor of SIV[AGM]. These results demonstrate that the gag proteins of HIV-2 and SIV[AGM] share some common epitopes with those of HIV-1 which are heterogenic in some degree among the different isolates. PMID- 1720181 TI - Heat-stable and heat-labile components of nonspecific acid phosphatase detected in Pseudomonas pseudomallei. AB - In a whole cell assay system with p-nitrophenyl phosphate as substrate, strains of Pseudomonas pseudomallei showed a two-peak pattern in pH activity curve of acid phosphatase, suggesting the presence of two enzyme components different in pH optimum (4.2 and 5.2). The component of 5.2 pH optimum was detected in the outer membrane fraction and the activity was resistant to heating at 70 C for 30 min. The other component of 4.2 pH optimum was heat-labile. No substantial difference was observed in the enzymatic activity between R and S type colonies. PMID- 1720182 TI - Growth and survival of Pseudomonas pseudomallei in acidic environments. AB - A study was made on the growth and survival of Pseudomonas pseudomallei in culture environments differing in nutrients, initial pH, and aeration, in comparison with Pseudomonas cepacia and Pseudomonas aeruginosa. The observations led us to a view that P. pseudomallei has the highest adaptability to acidic environments among the three species. Unlike the other species, it grew in heart infusion broth of initial pH 4.5 under aeration and survived keeping a high level (10(9) per ml) of viable counts for as long as 30 days. This sort of adaptation was found to be more evident in the media of poor nutrition and under limited aeration. PMID- 1720183 TI - The Health Information Video Project. PMID- 1720184 TI - [Rhinitis medicamentosa--a revived disease?]. PMID- 1720185 TI - Down with double projection. PMID- 1720186 TI - A class of gyrB mutants, substantially unaffected in DNA topology, suppresses the Escherichia coli K12 ftsZ84 mutation. AB - Previous work in our laboratory suggested that DNA topology could be implicated in the regulation of the division gene ftsZ. To settle this question, we have selected and characterized mutants in the gyrB gene able to phenotypically suppress the defects of the ftsZ84 mutation. No strict correlation was found between the degree of plasmid DNA relaxation and the level of suppression of the thermosensitivity of the ftsZ84 strain. Interestingly, the class of mutants that shows maximal suppression is substantially unaffected in DNA topology. In addition, the amount of ftsZ-specific mRNA in this class of mutants is comparable to that present in the ftsZ84 strain. These results hint that the ability of these gyrB mutants to correct the effects of the ftsZ84 mutation is largely unrelated to the function of the GyrB (as a part of DNA gyrase) in the control of DNA superhelicity and suggest hitherto unsuspected interaction between the ftsZ and gyrB gene products. PMID- 1720188 TI - Preparation and use of protein kinase C subspecies-specific anti-peptide antibodies for immunostaining. PMID- 1720187 TI - Purification of mitogen-activated protein kinase from epidermal growth factor treated 3T3-L1 fibroblasts. PMID- 1720189 TI - Generation and use of anti-peptide antibodies directed against catalytic domain of protein kinases. PMID- 1720190 TI - Isolation of cDNA clones that encode active protein-tyrosine kinases using antibodies against phosphotyrosine. PMID- 1720191 TI - Production of p60c-src by baculovirus expression and immunoaffinity purification. PMID- 1720192 TI - Use of recombinant baculoviruses and 1H nuclear magnetic resonance to study tyrosine phosphorylation by a soluble insulin receptor protein-tyrosine kinase. PMID- 1720193 TI - Nonradioactive assays of protein-tyrosine kinase activity using anti phosphotyrosine antibodies. PMID- 1720194 TI - Poster presentations: another way to share nursing research. PMID- 1720195 TI - Medical-surgical nursing: a lifetime of challenges and accomplishments. PMID- 1720196 TI - A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples. AB - The umu-microtest is a miniaturized automated short-term test version proposed for screening of umuC-dependent mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the SOS/umu-test and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the beta galactosidase, which reflects umuC induction, is determined colorimetrically. The incubation of the bacteria in the presence of the test compounds as well as the assessment of beta-galactosidase activity takes place in 96-well microplates, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within 8 h. Computer-controlled automation is possible by using a laboratory workstation. PMID- 1720197 TI - Radiation dosimetry by automatic image analysis of dicentric chromosomes. AB - A system for scoring dicentric chromosomes by image analysis comprised fully automatic location of mitotic cells, automatic retrieval, focus and digitization at high resolution, automatic rejection of nuclei and debris and detection and segmentation of chromosome clusters, automatic centromere location, and subsequent rapid interactive visual review of potential dicentric chromosomes to confirm positives and reject false positives. A calibration set of about 15,000 cells was used to establish the quadratic dose response for 60Co gamma irradiation. The dose-response function parameters were established by a maximum likelihood technique, and confidence limits in the dose response and in the corresponding inverse curve, of estimated dose for observed dicentric frequency, were established by Monte Carlo techniques. The system was validated in a blind trial by analysing a test set comprising a total of about 8000 cells irradiated to 1 of 10 dose levels, and estimating the doses from the observed dicentric frequency. There was a close correspondence between the estimated and true doses. The overall sensitivity of the system in terms of the proportion of the total population of dicentrics present in the cells analysed that were detected by the system was measured to be about 40%. This implies that about 2.5 times more cells must be analysed by machine than by visual analysis. Taking this factor into account, the measured review time and false positive rates imply that analysis by the system of sufficient cells to provide the equivalent of a visual analysis of 500 cells would require about 1 h for operator review. PMID- 1720198 TI - Quantification of the predictivity of some short-term assays for carcinogenicity in rodents. AB - A statistical procedure is described for assessing the predictive performance of short-term tests for carcinogenicity in which the actual number of chemicals tested is taken into consideration. The method is then applied to several widely used short-term assays. PMID- 1720199 TI - Environmental Mutagen Society of Japan. 19th annual meeting. 29-31 October 1990, Fukuoka, Japan. Selected abstracts. PMID- 1720200 TI - Intermediate filaments. Getting under the skin. PMID- 1720201 TI - High prevalence of circulating antibodies to MuLV p30 antigen in human sera. An autoimmune response? AB - To study the possible involvement of a murine leukemia virus (MuLV) related agent in human cancer, an extensive immunoblotting analysis of human sera (cancer, autoimmune as well as control normal ones) for the presence of antibodies to MuLV structural proteins was performed. Out of 350 sera, 89 reacted with gag precursor Pr65, 72 reacted with major viral core protein p30 and five with the matrix protein p15. Antibody reactivity to the env-encoded glycoprotein gp70 was detected in 7 cases out of 16 sera tested. There were no significant differences between pathological and normal sera concerning the patterns and the frequency of the reactivity. Sera from patients with various malignancies (mainly with breast cancer) generally displayed more intensive signals to MuLV p30 than normal sera. Epitope mapping revealed that MuLV p30-reactive antibodies recognize an antigenic determinant(s) located at the carboxyterminus of the protein. PMID- 1720202 TI - Functional characteristics of the peritoneal membrane in long-term continuous ambulatory peritoneal dialysis. AB - Peritoneal transport characteristics of 20 long-term (LT) patients with a mean duration on continuous ambulatory peritoneal dialysis (CAPD) of 60 months were compared with those of 20 matched patients who recently started (RS) CAPD (mean 39 days, range 11-63). Mass transfer area coefficients (MTC) of creatinine, glucose and inulin were higher in the LT group than in the RS group (12.1 versus 9.2 ml/min, p less than 0.01; 9.9 versus 8.3 ml/min, p less than 0.05; 4.1 versus 3.5 ml/min, p less than 0.05). The MTC of alpha 2-macroglobulin were lower in the LT group (13 versus 25 microliters/min; p less than 0.01). The size selectivity of the membrane for the transport of macromolecules, determined as protein MTC ratios, showed a more restricted passage for macromolecules in the LT group. Net fluid removal using glucose 3.86% was lower in the LT patients (487 versus 826 ml/4 h; p less than 0.001). The results indicate the development of a larger effective peritoneal surface area combined with a less permeable peritoneal membrane after many years of CAPD. PMID- 1720203 TI - Detection of polymorphonuclear cells, superoxide dismutase and poly C9 in glomeruli of patients with IgA nephropathy. PMID- 1720204 TI - Angiographic morphology of the posterior communicating artery and basilar in patients with ICA-PComA aneurysm. AB - The relationships between the angiographic morphology of the posterior communicating artery (PComA) and the basilar artery (BA) and saccular aneurysms at the internal carotid artery (ICA)-PComA junction were evaluated in 23 patients with ICA-PComA aneurysm and 46 controls. No significant differences were found in the height of the basilar top, the dislocation and inner diameter of the BA, and the distance between the basilar top and the ICA-PComA junction. However, the angle between the PComA and C2 portion of the ICA was larger and the PComA straighter in ICA-PComA aneurysm patients. Tension in the PComA and mechanical damage to the divergent angle of the PComA are probably important factors in the development of ICA-PComA aneurysms. PMID- 1720206 TI - Persistent primitive hypoglossal artery aneurysms--report of two cases. AB - The authors present two patients with subarachnoid hemorrhage caused by ruptured intracranial saccular aneurysms of the persistent primitive hypoglossal artery. A standard unilateral suboccipital approach in one patient resulted in incomplete neck clipping since the operative field was restricted by a protruding jugular tubercle. Successful aneurysmal neck clipping was achieved in the second patient via a unilateral-transcondylar-suboccipital approach with resection of the jugular tubercle and rim of the foramen magnum. PMID- 1720205 TI - Outcome of primary central nervous system lymphoma--a study of 32 patients. AB - The outcomes in 32 cases of histologically diagnosed primary central nervous system lymphoma were investigated. The 1-, 2-, and 5-year survival rates were 54, 36, and 8%, respectively. Good outcome was indicated by extensive surgical removal with 50-Gy irradiation and lower ages. 61% of patients receiving radiation therapy suffered recurrence within 1 year. The incidence of multiple lesions increased at recurrence. These lesions were almost all remote from the initial site in the brain, occurring more frequently in the central part of the supratentorial regions near the ventricle. Multiple lesions recurred more rapidly than single lesions. Longer survival times were indicated by a long tumor-free period after initial treatment. Extensive surgical removal results in long survival times for patients with a localized single tumor in the early stage. Radiochemotherapy should be given as part of the initial treatment. PMID- 1720207 TI - True aneurysms of the middle meningeal artery associated with cavernous hemangioma of the skull--case report. AB - A rare case of true aneurysms of the middle meningeal artery (MMA) associated with cavernous hemangioma in a 47-year-old male is presented. Angiography showed aneurysmal dilatation of the MMA, which was a tumor feeder. Histological examination confirmed the true aneurysmal character of the dilatation. The aneurysms were considered to result from increased hemodynamic stress and medial defects in the MMA. PMID- 1720208 TI - Unusual spontaneous entrapment of a dissecting aneurysm of the vertebral artery- case report. AB - The authors report a case of subarachnoid hemorrhage due to spontaneous dissection of the intracranial segment of the left vertebral artery. Serial angiography demonstrated spontaneous entrapment of the lesion 4 months after ictus. PMID- 1720209 TI - Intracranial cystic hemangiopericytoma--case report. AB - A rare case of intracranial hemangiopericytoma associated with a large cyst was treated by gross total removal and local irradiation. The tumor has not recurred for 16 months, although the effectiveness of radiation therapy for hemangiopericytoma is unclear. Histological examination of the tumor specimen showed aggregation of the microcystic components, possibly contributing to the cyst formation. Hemangiopericytoma should not be classified as meningioma because of the different neoplastic and cytological properties. Complete surgical removal is essential for this tumor because of its malignancy. PMID- 1720210 TI - Ring-enhanced primary intracranial malignant lymphoma--report of two cases. AB - Two nonimmunosuppressed patients with primary intracranial malignant lymphoma demonstrated ring-enhanced lesion on postcontrast CT scans. One patient underwent total removal of the tumor and the other patient biopsy, both followed by whole brain irradiation. Histological examination revealed the tumors contained diffuse large cells and necrosis in the central region. The possible mechanisms of ring enhancement may represent central necrosis of the rapidly growing tumor, but is probably not related to the prognosis. PMID- 1720211 TI - Malignant fibrous histiocytoma of the occipital bone with intracranial extension- case report. AB - The authors present a rare case of malignant fibrous histiocytoma originating in the cranial bone. A 72-year-old male was admitted with a diffuse painless swelling in the left occipital region but no neurological abnormality. Plain skull x-ray films and computed tomographic scans showed a large tumor in the left temporo-occipital bone. The tumor invading subcutaneous tissue was totally excised and histologically diagnosed as malignant fibrous histiocytoma. Postoperatively, 40-Gy irradiation was given to the left temporo-occipital region. Several months later, however, the tumor recurred in the posterior fossa. Neuroradiological examination showed tumor extension into the occipital bone and muscle and the subdural space of the posterior fossa. A second operation extirpated all tumors except in the cerebellum. He died of pneumonia on the 14th postoperative day. Autopsy revealed malignant fibrous histiocytoma invading into the bilateral cerebellar hemispheres. Radiation and chemotherapy should be given as soon as possible following extensive surgery for malignant fibrous histiocytoma of the cranial bone. PMID- 1720212 TI - Bilateral microphthalmos with orbital cyst--case report. AB - A 3-month-old girl presented with a rare occurrence of bilateral microphthalmos associated with orbital cysts. She underwent subtotal removal of the right orbital cyst. Histological examination was compatible with microphthalmos with orbital cyst. Although microphthalmos associated with orbital cyst is rarely encountered, it must be considered in the differential diagnosis of orbital cystic lesions. PMID- 1720213 TI - Partial corpus callosotomy beneficial for Lennox-Gastaut syndrome--report of two cases. AB - Seizures in patients with Lennox-Gastaut syndrome are very resistant to drug therapy. Division of the anterior half of the corpus callosum was performed in two patients with Lennox-Gastaut syndrome incapacitated by frequent atonic seizures leading to sudden falls. Postoperatively, the atonic seizures were completely eliminated and their mental reactivities remarkably improved. It is clear that they have benefited from a lower frequency of seizures and reduced dosages of anticonvulsant medication, which outweight the acquired disabilities. PMID- 1720214 TI - Changes in phosphorus-31 magnetic resonance spectra caused by epidural balloon in cats. AB - Changes in brain high energy phosphate metabolite and pH levels were studied using serial phosphorus (P)-31 magnetic resonance spectroscopy (MRS) in an epidural balloon cat model. The balloon was inflated with 1, 2, 3, or 4 ml of water at 1 ml/sec and deflated after obtaining a spectrum 4 minutes later. Serial spectra, intracranial pressure (ICP), physiological parameters, and the pupil sizes were observed for 1 hour. The mean ICP after balloon inflation increased to between 36 +/- 3 and 130 +/- 6 mmHg. Some animals showed oculomotor paralyses. Phosphocreatine, adenosine triphosphate, phosphodiester, and phosphomonoester levels suddenly decreased after inflation and gradually recovered after balloon deflation. Acidosis progressed with cerebral compression for 4 minutes and gradually recovered. All changes and recovery were volume related. This study demonstrates the potential of P-31 MRS for noninvasive, serial, in vivo measurements of critical high energy phosphate metabolites and intracellular pH after head injury. PMID- 1720215 TI - Clinical application of proton magnetic resonance spectroscopy to cerebral ischemia. AB - Proton magnetic resonance spectroscopy (MRS) with the depth resolved surface coil spectroscopy technique and the 1331-2662 water suppression method was used to examine two cerebral ischemia patients and 10 normal volunteers. In all cases, N acetyl-aspartate, creatine, phosphocreatine, and residual lipid were clearly observed. No lactic acid peak was observed in normal volunteers, but a large lactic acid peak appeared in the early stage of cerebral ischemia. This MRS abnormality was observed before abnormalities appeared in conventional imaging such as computed tomographic scanning and magnetic resonance imaging. PMID- 1720216 TI - Diffuse axonal injury and early intracranial sequelae in severe head injury. AB - The importance of diffuse axonal injury (DAI) and early intracranial sequelae was studied in 107 patients with diffuse and focal brain injuries. Comprehensive neuropathological study was also undertaken in 24 fatal patients. The mortality rate was clearly the highest in traumatic subarachnoid hemorrhage, followed by acute subdural hematoma, cerebral contusion with delayed hematoma formation, traumatic intracerebral hematoma, diffuse cerebral swelling, DAI with classical features, and finally nearly normal on computed tomographic scans. The mean flow velocities in the middle cerebral artery recorded by transcranial Doppler ultrasound were variable in diffuse brain injury, but commonly decreased on the hematoma side depending on increased intracranial pressure and decreased cerebral perfusion pressure in focal brain injury. Deep-seated hemorrhagic lesions did not expand in diffuse brain injury, but sizable hematoma developed within 24 hours in focal brain injury. The platelet count was significantly lower in patients with poor outcomes in focal brain injury. Histological evidence of classical DAI was found in eight (50%) of 16 cases with focal brain injury. DAI of varying severity is the common subjacent lesion in patients with severe head injury, but the final outcome varies greatly with different lesion types. PMID- 1720217 TI - Continuous alprenolol infusion for control of hypertension in the acute stage of ruptured intracranial aneurysms. AB - The clinical benefits and hemodynamic effects of continuous alprenolol infusion for control of hypertension in the acute stage of ruptured cerebral aneurysms were investigated. Twenty-five patients manifesting systemic hypertension (greater than 160/100 mmHg) were treated with alprenolol, a beta-adrenergic antagonist, phentolamine, an alpha-adrenergic antagonist, and trimethaphan camsilate, a ganglionic blocker, given intravenously. All drugs decreased the mean arterial blood pressure significantly. However, alprenolol decreased the heart rate and cardiac index while phentolamine increased them. Alprenolol also decreased arterial catecholamine and renin activity, but caused no change in central venous pressure, pulmonary capillary wedge pressure, pulmonary vascular resistance, and systemic vascular resistance. The results indicate the usefulness of continuous alprenolol infusion for the control of acute hypertension in hemorrhagic cerebrovascular disease. The mode of action of alprenolol is also discussed. PMID- 1720218 TI - Embolization of intramedullary spinal arteriovenous malformation fed by the anterior spinal artery with monitoring of the corticospinal motor evoked potential--case report. AB - Intramedullary spina AVMs fed by the anterior spinal artery cannot be embolized without risking unacceptable motor deficits, since the feeding arteries may supply the corticospinal tract (CST). An 8-year-old boy underwent successful embolization of such an AVM under general anesthesia using intermittent infusion of embolic material with monitoring of the CST integrity with the corticospinal motor evoked potential (MEP). This case illustrates the value of corticospinal MEP monitoring during therapeutic procedures under general anesthesia which risk interrupting the blood supply to the CST. PMID- 1720219 TI - Hemifacial spasm caused by CP angle AVM associated with ruptured aneurysm in the feeding artery--case report. AB - A 66-year-old male presented with clinical features of hemifacial spasm. Cerebral angiograms disclosed an arteriovenous malformation (AVM) in the cerebellopontine angle. The hemifacial spasm was caused by a dilated feeding artery of the AVM compressing the facial nerve at the root exit zone. Surgery was not initially performed because of his age and absence of AVM rupture. However, the AVM was associated with a small aneurysm in the feeding artery, which rapidly grew during 20 days after discharge and ruptured causing subarachnoid hemorrhage. The aneurysm was clipped and the feeding artery of the AVM partially obliterated. Careful angiographic examination for associated aneurysms and consequent surgical obliteration to prevent hemorrhage are suggested in cases of AVM. PMID- 1720220 TI - Hemifacial spasm due to cerebellopontine angle epidermoid tumor--case report. AB - The authors report a case of cerebellopontine angle epidermoid presenting as typical hemifacial spasm. A 33-year-old male had experienced intermittent right hemifacial spasm for 2 years. Cranial nerve examination was otherwise normal, including auditory and trigeminal nerve functions. Metrizamide computed tomographic cisternography and magnetic resonance imaging demonstrated a characteristic epidermoid tumor. The tumor was totally removed. Postoperatively, no facial spasm or other facial nerve dysfunction was noted. PMID- 1720221 TI - Cystic cavernous angioma--case report. AB - A rare case of a cystic cavernous angioma in a 20-year-old female was diagnosed preoperatively by magnetic resonance imaging and computed tomography. Total surgical removal resulted in a successful recovery. Cystic cavernous angioma is benign and can be completely-removed. The importance of magnetic resonance imaging in the differential diagnosis is emphasized. PMID- 1720222 TI - Neurosurgical application of a flow-directed oximetry thermodilution catheter for evaluation of cerebral blood flow--technical note. AB - The neurosurgical application was evaluated of a flow-directed oximetry thermodilution catheter for measurement of oxygen saturation in the jugular vein, which reflects cerebral blood flow (CBF). The catheter allows estimation of changes in CBF during carotid endarterectomy and therapeutically induced hypertension in the management of delayed vasospasm after subarachnoid hemorrhage. PMID- 1720223 TI - [A case of Steel-Richardson-Olszewski syndrome]. AB - A case of Steele-Richardson-Olszewski disease is reported calling attention to diagnostic difficulties in cases of chronic dementia with neurological signs. The authors propose including therapy with antiviral and antiparkinsonian drugs in S R-O disease. The presented case confirms the effectiveness of this treatment. PMID- 1720224 TI - Topographic segregation of corticostriatal projections from posterior parietal subdivisions in the macaque monkey. AB - The distribution of corticostriatal projections from areas 7m, 7a, 7b and 7ip of the posterior parietal cortex was studied in rhesus monkeys using horseradish peroxidase conjugated with wheat-germ agglutinin as an anterograde tracer. All parietal subdivisions project bilaterally over a broad anteroposterior expanse of the caudate nucleus and putamen; however, the zones of densest terminal labeling varied for each parietal subdivision. Thus, area 7m projects preferentially to dorsal and dorsolateral portions of the head and anterior part of the body of the caudate nucleus. The main striatal target of area 7a is also in the head and anterior portion of the body of the caudate nucleus, but at dorsal and dorsomedial zones. The preferential target region of area 7ip in the striatum is in the posterior two-thirds of the body of the caudate nucleus, where the labeled terminals spare only the medial border. In contrast to the other parietal subdivisions, 7b projects preferentially to the putamen. In this nucleus, the location of labeling after 7b injections appears to correspond to the zones containing the representations of the distal forelimb and head. Each parietal subdivision projects to a rather extended anteroposterior domain in the contralateral neostriatum, the projection zones being always less extensive than in the ipsilateral side, but with a similar topographic distribution. Because we have shown previously that each parietal subdivision is part of a distinct distributed corticocortical network, the neostriatal territories innervated by each subdivision can be correlated with the corresponding network, thus providing insight into the functional specializations of the striatum. PMID- 1720225 TI - Spantide II, a novel tachykinin antagonist, and galanin inhibit plasma extravasation induced by antidromic C-fiber stimulation in rat hindpaw. AB - The effect of intradermal injection of Spantide II, a novel tachykinin antagonist, and the neuropeptide galanin on neurogenic plasma extravasation induced by antidromic stimulation of C-fibers in the sciatic nerve was examined in the hindpaws of rats. Activation of C-fibers by antidromic sciatic nerve stimulation (2 Hz, 5 min) consistently evoked a localized plasma extravasation of Evans Blue in the skin area of the hindpaw innervated by the sciatic nerve. Intradermal injection of 3 nmol Spantide II significantly inhibited this response. The plasma extravasation was nearly totally abolished when the concentration of Spantide II was increased to 9 nmol. Intradermal injection of 1.5 and 15 nmol galanin also inhibited plasma extravasation. Intradermal injection of 9 nmol Spantide II effectively blocked the plasma extravasion in the hindpaw induced by 8 nmol intravenous substance P. Plasma extravasation induced by intravenous substance P was also inhibited by the higher, but not by the lower, dose of galanin injected intradermally. The present results indicate that Spantide II, a potent non-toxic tachykinin antagonist, effectively blocks the neurogenic plasma extravasation induced by antidromic C-fiber stimulation, thus supporting the view that tachykinins play an important role in this neurogenic inflammatory process. It is further shown that galanin, a naturally occurring neuropeptide present in primary afferents, also inhibits C-fiber activation evoked plasma extravasation, indicating an interaction between galanin and tachykinins in the peripheral terminals of primary afferents, possibly through both pre- and postsynaptic mechanisms. PMID- 1720226 TI - Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures. AB - The subnuclear and synaptic distribution of substance P immunoreactivity was examined in the rat interpeduncular nucleus at the light and electron microscope level. The nucleus possessed a prominent substance P-immunoreactive axonal plexus in the lateral and dorsomedial subnuclei, and in the dorsal cap of the rostral subnucleus. The density of substance P-immunoreactive axons in the remaining subnuclear divisions was sparse to moderate. Terminals of immunoreactive axons contained spherical vesicles and formed asymmetric contacts on dendritic processes exclusively. Immunoreactive neurons, restricted to the rostral subnucleus, possessed long, sparsely branched dendrites. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P immunoreactive dendritic profiles. Axodendritic and axosomatic synapses containing substance P immunoreactivity pre- and postsynaptically were not observed. Ultrastructural evidence for synaptic relationships between substance P containing profiles and those containing either choline acetyltransferase or glutamate decarboxylase was obtained by means of double antigen immunohistochemistry. Terminals of fasciculus retroflexus axons stained for choline acetyltransferase immunoreactivity formed asymmetric synaptic contacts with substance P-immunoreactive dendritic profiles. Few substance P-positive dendrites in the rostral subnucleus received terminals possessing glutamate decarboxylase activity. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P- and glutamate decarboxylase immunoreactive dendritic profiles simultaneously. Terminals possessing either substance P or glutamate decarboxylase immunoreactivity formed synaptic contacts with dendritic processes of neurons in the lateral subnucleus. Many of the neurons within this subnuclear division contained glutamate decarboxylase. This study provides direct evidence of synaptic relationships between choline acetyltransferase-immunoreactive axons and substance P-immunoreactive dendritic profiles, and between substance P-positive axons and glutamate decarboxylase immunoreactive dendrites. These findings reveal that two types of transmitter specific axons of the fasciculus retroflexus innervate neuronal populations of the interpeduncular nucleus stained immunohistochemically for either substance P or glutamate decarboxylase. PMID- 1720227 TI - Distribution, morphology and number of monoamine-synthesizing and substance P containing neurons in the human dorsal raphe nucleus. AB - The distribution, morphology and number of serotonin-, catecholamine- and substance P-containing neurons in the human dorsal raphe nucleus were studied. Parallel series of sections were prepared from 10 human brainstems obtained at autopsy from patients without neurological disease aged between 42 and 88 years. The neurons were identified using immunohistochemistry with antibodies raised against phenylalanine hydroxylase (tryptophan hydroxylase-containing, serotonin neurons), tyrosine hydroxylase (catecholamine neurons) and substance P. A reference series of Nissl-stained sections was also prepared and data published separately were used to delineate the subnuclear divisions of the dorsal raphe nucleus and to establish the total number of neurons in each subnucleus. The following principal findings emerged. (1) Serotonin-synthesizing neurons are present in all regions of the dorsal raphe nucleus and their total number is 165,000 +/- 34,000. The same types of neurons as those seen in Nissl material characterize each of the five subnuclei (caudal, dorsal, ventral, ventrolateral and interfascicular). (2) Substance P-containing neurons mostly occupy the rostral part of the nucleus and their number is 74,600 +/- 17,600. (3) Catecholamine cells are only found in the rostral part of the dorsal raphe nucleus and their number is 5600 +/- 3400. (4) In the ventral and interfascicular subnuclei the combined number of serotonin-synthesizing and substance P containing neurons exceeds the total number of Nissl-stained neurons suggesting that serotonin and substance P co-exist in a substantial part of the cell population of the dorsal raphe nucleus. This is further supported by the highly similar morphology and size of these neurons. It is concluded that there are demonstrable chemical differences between the various subregions of the human dorsal raphe nucleus. These differences are in harmony with the results of hodological studies in animals, which have demonstrated differential projection pathways emerging from this nucleus. PMID- 1720228 TI - Immunohistochemical localization of insulin receptors and phosphotyrosine in the brainstem of the adult rat. AB - Previous studies have demonstrated that insulin receptors are widely distributed throughout areas of the forebrain in the adult rat that are involved in modulating neuroendocrine functions and feeding behaviour. In addition, a recent investigation showed that there is a good correlation between the presence of the insulin receptor and phosphotyrosine-containing proteins in these regions, indicating a possible functional activity of insulin receptors in vivo. It is unknown whether neural connections between specific brainstem nuclei to forebrain regions may also be under direct regulation of insulin or related factors. In order to test this possibility, the distribution of insulin receptors and phosphotyrosine was mapped throughout the hindbrain of the adult rat by immunocytochemistry, using specific antibodies against the beta-subunit of the insulin receptor as well as against phosphotyrosine. Both markers showed a high degree of overlap throughout numerous distinct anatomical regions of the hindbrain. In the mesencephalon, insulin receptor and phosphotyrosine-positive neurons were found in the precommissural nucleus, the lateral and dorsal part of the central gray, the mammillary bodies and the interpeduncular nucleus. In addition, immunoreactivity was found in the subependymal layer around the aqueduct along fibres and nerve cells possibly contacting the cerebrospinal fluid. In the pons and medulla, dense immunoreactivity was seen in the lateral superior olive, nucleus of the solitary tract, spinal trigeminal nucleus and nucleus ambiguous. Scattered cells were found in the pontine and vestibular nuclei, as well as in the reticular formation. The cerebellum contained moderately dense immunoreactivity in the granule cell and molecular cell layer of the cortex, as well as in the deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720229 TI - Identification and immunohistochemistry of cholinergic and non-cholinergic circular muscle motor neurons in the guinea-pig small intestine. AB - Motor neurons which innervate the circular muscle layer of the guinea-pig small intestine were retrogradely labelled, in vitro, with the carbocyanine dye, DiI, applied to the deep muscular plexus. By combining retrograde tracing and immunohistochemistry, the chemical coding of motor neurons was investigated. Five classes of neuron could be distinguished on the basis of the co-localization of immunoreactivity for the different antigens; the five classes were also characterized by different lengths and polarities of their axonal projections and by their cell body shapes. Two classes with local or orally directed axons were immunoreactive for choline acetyltransferase and substance P and are likely to be cholinergic excitatory motor neurons. Two other classes had anally directed axons; they were immunoreactive for vasoactive intestinal polypeptide and are likely to be inhibitory motor neurons. A small proportion of neurons with short projections to the circular muscle were immunoreactive for neither substance P nor for vasoactive intestinal polypeptide, but are likely to be cholinergic. The morphological and histochemical identification of excitatory and inhibitory motor neurons provides a neuroanatomical basis for the final motor pathways involved in the polarized reflex motor activity of the gut. PMID- 1720230 TI - Quantitative study of neuronal degeneration induced by Ricinus toxin and crush of postganglionic nerves in the ciliary ganglion of quail. AB - The effects of Ricinus toxin on the neurons of the ciliary ganglia were investigated in the quail. The neuronal death and the morphological alterations of the ganglionic cells were assessed following injection of the toxin in the anterior chamber of the eye or after application of the toxin on the postganglionic nerves at a crush site. A 45% loss of choroid neurons without loss of ciliary neurons was observed after postganglionic nerve crush alone. Injection of the toxin in the anterior chamber of the eye led to a selective loss of ciliary neurons (38%). Application of the toxin to the crushed postganglionic nerves led to a loss from both neuronal populations (40% of total neurons). This work indicates that different procedures result in selective lesion of the different neuronal populations in the ciliary ganglion. PMID- 1720231 TI - Myelin basic protein-messenger RNA (MBP-mRNA) expression during triethyltin induced myelin edema. AB - Triethyltin (TET) is a neurotoxicant that produces severe but transient cerebral edema, characterized ultrastructurally by vacuolation of the intraperiod line of central nervous system (CNS) myelin. TET has been reported to depress levels of myelin basic protein (MBP), a protein thought to play a critical role in myelin compaction. In the present study, the genomic expression (i.e., mRNA) of MBP was monitored throughout the pathogenesis of TET-induced myelin edema and recovery in Sprague-Dawley rats given a single injection of a neuropathic (8.0 mg/kg) or non neuropathic (0.8 mg/kg) dose of TET-bromide. Levels of MBP-mRNA from the anterior and posterior brain were collected 1 hr, 3 hr, 2d, and 7d, postexposure. The optic nerve and caudal brainstem, representing anterior and posterior brain sites, respectively, were examined at the same time-points for ultrastructural evidence of edema and recovery. Our data indicate that neuropathic doses (8.0 mg/kg) of TET significantly stimulated MBP transcript throughout the brain at all exposure time-points. The magnitude and time-course of this stimulation differed in the anterior and posterior brain, with the latter region showing higher levels of MBP-mRNA. In the posterior brain, the highest levels of mRNA correlated with the appearance of edema in the caudal brainstem. In the anterior brain, MBP-mRNA levels were only marginally increased over controls. Ultrastructural evidence of myelin edema was confined to the brainstem in rats treated with neuropathic dose of TET. Intralamellar vacuolation appeared at 3 hr and 2d postexposure and could be correlated with peak levels of MBP transcript, whereas, recompacted myelin, which appeared by 7d postexposure, was associated with declining levels of the mRNA. Ultrastructural changes in the oligodendroglia were suggestive of metabolic stimulation and correlated with high MBP-mRNA levels. In summary, these data indicate that an initial genomic event in TET-induced myelin edema is stimulation of MBP transcript. PMID- 1720232 TI - [Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]. AB - Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated. PMID- 1720233 TI - Decreased stimulated GM-CSF production and GM-CSF gene expression but normal numbers of GM-CSF receptors in human term newborns compared with adults. AB - We investigated cord and adult production of granulocyte-macrophage colony stimulating factor (GM-CSF), expression of GM-CSF mRNA from unstimulated and activated mononuclear cells, and the affinity and presence of GM-CSF receptors on mature effector cells in an attempt to better understand the underlying pathophysiology of altered neonatal host defense. Utilizing 125I-GM-CSF as a ligand, Scatchard analysis revealed the presence of a single class affinity GM CSF receptor with similar binding characteristics on both cord and adult peripheral PMN (kd = 44 and 39 pM) for adult and cord, respectively. Additionally, there was no significant difference in the number of GM-CSF receptors on cord versus adult neutrophils. Using a sandwich ELISA for measuring GM-CSF levels, we found nondetectable levels from supernatants of unstimulated cord and adult mononuclear cells and serum from cord and adult peripheral blood. However, there was a significant difference between cord and adult GM-CSF production from stimulated phytohemagglutinin and phorbol-12-myristate-6-acetate mononuclear cells (p less than 0.02). Additionally, GM-CSF mRNA expression from activated cord mononuclear cells was significantly reduced after 6 h of stimulation compared with adults. Nuclear run-on experiments revealed no difference in transcriptional activation from activated cord and adult mononuclear cells. Actinomycin D transcriptional decay studies, however, demonstrated reduced GM-CSF half-life from activated cord versus adult mononuclear cells (t1/2 30 versus 100 min). These results suggest normal affinity and numbers of GM-CSF receptors on peripheral mature effector cells but decreased GM-CSF production and GM-CSF mRNA expression from activated cord versus adult mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720234 TI - Prediction of developmental outcome using a perinatal risk inventory. AB - An effective perinatal developmental screening that predicts developmental outcome of high-risk neonates is currently not available. One hundred twenty-five high-risk infants were evaluated prior to discharge from the neonatal intensive care unit using a newly developed perinatal risk inventory, family status index, and abbreviated neurobehavioral assessment scale. All infants had been evaluated using the Bayley Mental and Motor Scales of the Stanford-Binet. They were also evaluated by a pediatrician, audiologist, and ophthalmologist. Fifteen infants had been evaluated using 9-month Bayley Scales of Infant Development, 74 had been evaluated using the Bayley at 9 and 18 months, and 36 children had been assessed using the Stanford-Binet at 36 months. The total score of the perinatal risk inventory demonstrated a significant correlation with the infants' last score on the Bayley and Stanford-Binet (r = .55, P less than .001). The abbreviated neurobehavioral assessment scale correlated with the infants' IQ and developmental quotient score (r = .3, P less than .001); the family status index did not correlate well with the developmental outcome. Using a score of 10 on the perinatal risk inventory provided a sensitivity of 0.76, specificity of 0.79, positive predictive value of 0.475, and negative predictive value of 0.929. Twelve of the 13 infants with cerebral palsy were identified as being potentially developmentally abnormal prior to discharge. It appears that it is possible to predict the developmental outcome of high-risk neonates using a perinatal risk inventory. PMID- 1720235 TI - Cardiac sequelae of Kawasaki disease in Japan: statistical analysis. AB - The proportions of Kawasaki disease patients with cardiac sequelae in Japan were analyzed using nationwide survey data from the 6 1/2-year period July 1982 through December 1988. Of 46,864 cases of Kawasaki disease reported in the surveys, 7637 or 16.3% had cardiac sequelae such as dilation or stenosis of coronary arteries, myocardial infarction, and valvar lesions 1 month or more after onset. The prevalence of cardiac sequelae was particularly high in males, infants younger than 1 year, and children older than 5 years of age. In sequential observation, there was no correlation between the prevalence of cardiac sequelae and periods of high or low incidence of the disease. The prevalence of cardiac sequelae overall declined steadily over the observation period, perhaps as a consequence of increasing use of intravenous gamma globulin. However, children older than the age of 5 years manifested increasing prevalence of cardiac sequelae over the observation period, probably as a result of lower rates of intravenous gamma globulin administration. PMID- 1720236 TI - American Academy of Pediatrics Committee on Genetics: Maternal serum alpha fetoprotein screening. PMID- 1720237 TI - Concomitant inheritance of homozygous alpha-thalassemia-2 with homozygous IVS-I-5 G-C beta gene mutation does not influence the severity of the thalassemic phenotype. AB - An Asian Indian child presented the severe transfusion dependent form of beta thalassemia major. Sequencing data and gene mapping analysis revealed the concomitant presence of homozygous alpha-thal-2 type 3.7 kb deletion with homozygous beta+ thal IVS-1-5 G-C mutation. Further studies at the DNA level demonstrated that he was XmnI negative at the -158 G gamma site and had the haplotype + ----- +. These results indicate that in the absence of high HbF determinants, concomitant inheritance of homozygous alpha-thal-2 with homozygous severe beta+ thal does not influence the severity of the thalassemic phenotype. PMID- 1720238 TI - Expression of adult and tadpole specific globin genes from Xenopus laevis in transgenic mice. AB - Transgenic mice were generated which carried the adult alpha and beta-globin genes and the major tadpole specific beta-globin gene of Xenopus laevis. The adult specific alpha and beta genes were found to express in erythroid tissues in adult mice, while the major tadpole specific beta gene (beta T1) was expressed in blood from 12.5 day embryos. The pattern of expression of the beta T1 gene during mouse development was consistent with its being regulated as an embryonic globin gene in the mouse. This observation suggests that some of the factors mediating globin switching have been conserved during the evolution of modern amphibia and mammals and raises interesting questions concerning the evolution of vertebrate globin gene switching. PMID- 1720239 TI - Genomic organization and complete sequence of the human gene encoding the beta subunit of the cGMP phosphodiesterase and its localisation to 4p 16.3. AB - As part of the search for the Huntington disease (HD) gene we have cloned and sequenced 34 kb of genomic DNA containing the full-length gene for the beta subunit of the human cGMP phosphodiesterase (beta-cGMP PDE). This gene is localized to 4p16.3 about 700 kb proximal to the 4p telomere and represents the most telomeric gene characterized on 4p to date. We show that this gene is comprised of 22 exons spanning approximately 43 kb of genomic DNA. We also provide 400 bp immediately 5' to the putative initiator methionine and 700 bp of 3' flanking sequences. Northern blot analysis of several human tissues revealed a highly abundant 3.5 kb transcript and a minor signal of 4.5 kb in retinal tissue. Alignment of the deduced amino acid sequence to the previously identified beta subunits of the cGMP PDEs of mouse and cow demonstrates highly significant similarities and, therefore, confirms the identity of the cloned gene. A defect in the beta-subunit of the cGMP PDE gene has been shown recently to be the cause for the retinal degeneration in the rd mouse. The cloning of the human homolog and the knowledge of its genomic organization with exon/intron boundaries will allow rapid assessment of the role of this gene in the causation of human retinopathies. PMID- 1720240 TI - Leishmania tarentolae minicircles of different sequence classes encode single guide RNAs located in the variable region approximately 150 bp from the conserved region. AB - The complete sequences of three kinetoplast DNA minicircles (B4, D3 and D12) from Leishmania tarentolae are reported. All L. tarentolae minicircles encode single gRNAs localized within the variable region approximately 150 bp from the conserved region. The 5' termini and tentative 3' termini of the new gRNAs were determined and the gene sequences and flanking sequences of all minicircle gRNA genes compared for conserved motifs of possible transcriptional regulatory significance. All minicircle gRNAs possess 3' oligo-[U] tails of variable length similar to maxicircle gRNAs. A role for the D3 minicircle gRNA in the editing of the 5' pan-edited MURF4 mRNA was suggested by sequence analysis, and a role for the D12 minicircle gRNA in the editing of the COIII mRNA and another minicircle gRNA (Lt154) in the editing of the pan-edited G6 mRNA have been previously reported. The cryptogene mRNAs edited by the B4 and Lt19 minicircle gRNAs are yet undetermined. PMID- 1720241 TI - Mechanism of DNA cleavage by cationic manganese porphyrins: hydroxylations at the 1'-carbon and 5'-carbon atoms of deoxyriboses as initial damages. AB - Cationic manganese-porphyrin complexes, free or targetted with an intercalating agent, are able to cleave DNA using oxygen atom donors like potassium monopersulfate or magnesium monoperphthalate as coreactants. Detailed studies of the cleavage of calf thymus DNA, before and after a heating step, show that free bases and 5-methylene-2-furanone are the main reaction products, indicating that hydroxylation at the 1'-carbon atom is the main target of these chemical agents. These data confirm that metalloporphyrin derivatives interact with the minor groove of double-stranded DNA. Hydroxylation of one of the two C-H bonds at position-5' is another initial DNA damage, characterized by the formation of furfural as sugar degradation product. Besides these two main initial damage sites, a low contribution of a hydroxylation reaction at C4' can not be definitively discounted, while an hydroperoxidation route at C4' can be excluded. PMID- 1720242 TI - Analysis of the expression immunoglobulin gene repertoire by screening libraries derived from PCR-amplified cDNA. PMID- 1720243 TI - SSCP-polymorphism in intron 12 of the CFTR gene recognized by BcII. PMID- 1720244 TI - Two additional MspI RFLPs revealed by MC.34 (D2S63). PMID- 1720245 TI - A new sequence variant of the human DNA ligase type I gene (LIG I). PMID- 1720246 TI - Organ growth and digestive enzyme levels to fifteen days of age in lines of chickens differing in body weight. AB - Weights of internal organs and levels of digestive enzymes were obtained through the first 15 days posthatch for cockerels from three lines of chickens known to differ greatly in body weight. On Day 15 body weights from the fastest growing line were eight times greater than those from the slowest growing line. Differences among lines were found for weights at hatching and for growth patterns (both absolute and relative to body weight) of the vitelline residue, heart, lungs, liver, pancreas, crop, proventriculus, gizzard, and segments of the small intestine. Line differences were also evident for levels of trypsin, chymotrypsin, and amylase in the pancreas and contents of the small intestine. Ranking of lines for these traits varied with age. In all lines weights of the small intestine, liver, and pancreas increased relatively more than did total body weight during the 1st wk posthatch, after which the relationship reversed. PMID- 1720247 TI - Amylase activity increases in the yolk of fertilized eggs during incubation in chickens. AB - Amylase activity was detected in the egg of chickens. In unfertilized eggs, no change of amylase activity was observed during 22 days incubation, however, in fertilized eggs, amylase activity increased markedly during embryonic development. The increase depended upon an increase of amylase in the yolk, not in the albumen. Isoamylases in the yolk were electrophoretically identical to those in the pancreas of the embryo or the hen. PMID- 1720248 TI - Axonal transport of cadmium in the olfactory nerve of the pike. AB - 109Cd2+ was applied in the olfactory chambers of pikes (Esox lucius) and the dynamics of the axoplasmic flow of the metal was determined in the olfactory nerves by gamma spectrometry and autoradiography. The results showed that the 109Cd2+ is transported at a constant rate along the olfactory nerves. The profile of the 109Cd2+ in the nerves showed a wave front of transported metal followed by a saddle region. When the nasal chambers were washed 2 hr after application of the 109Cd2+ well-defined transport peaks for the metal were seen in the olfactory axons. The maximal velocity for the transport of 109Cd2+, which corresponds to the movement of the wave front, was 2.38 +/- 0.10 mm/hr (mean +/- S.E.) at the experimental temperature (10 degrees C). The average velocity for the transport of the 109Cd2+, which corresponds to the peak apex movement of the wave, was 2.18 +/- 0.05 mm/hr (mean +/- S.E.) at 10 degrees C. The transported 109Cd2+ was strongly accumulated in the anterior parts of the olfactory bulbs, whereas in other brain areas the levels of the metal remained low. Autoradiography of a pike exposed to 109Cd2+ via the water showed a strong labelling in the receptor-cell containing olfactory rosettes, whereas other structures in the olfactory chambers were only weakly labelled. The accumulation and axonal transport in the olfactory neurons may be noxious and constitute an important component in the toxicology of cadmium in fish, and this may apply also to some other heavy metals. PMID- 1720250 TI - [Membranes in taste and olfaction]. PMID- 1720249 TI - "America Responds to AIDS": its content, development process, and outcome. AB - During the 1987-90 period, five phases of new AIDS information materials were released to the general public in the ARTA campaign, including a national mailer. The five were "General Awareness: Humanizing AIDS" in October 1987, "Understanding AIDS," the national mailout, April 1988, "Women at Risk/Multiple Partner, Sexually Active Adults," October 1988, "Parents and Youth," May 1989, and "Preventing HIV Infection and AIDS: Taking The Next Steps," July 1990. From planning to implementation to evaluation, ARTA is based on well-established theory and practice. Initially, the campaign was a response to an immediate crisis. It has evolved into the deliberate and systematic development of objectives to combat a chronic problem. ARTA represents one of the most comprehensive formative research processes in the history of public service campaigns. The dynamic process of carefully developing each new phase to include such important entities as State and local health agencies and community-based organizations is at least as important as the quality of the end materials. The objectives of each new phase are based on the needs of the public and of specific audiences. Maximum input from all relevant constituencies is obtained to ensure that they support the campaign's objectives and implementation strategy. PMID- 1720251 TI - [Membrane damage induced by biologically active peptides]. PMID- 1720252 TI - Antimineralocorticoids. AB - A number of chemical modifications in the spironolactone molecule were attempted over the last decade to synthesize ligands with high affinity for the mineralocorticoid receptor (MCR), and for possible use in the clinical control of the hypertensive disease. ZK 91587 has been commercialized as the 'ideal' ligand for the MCR, replacing the natural hormone aldosterone. None of the derivatives was retained for possible clinical use as an improvement over Canrenone or Spironolactone. No apparent correlation could be drawn between affinity for the MCR in vitro and biological potency in vivo. Such considerations challenge classical notions regarding the receptor mediated hormone action. PMID- 1720253 TI - Biological properties of a renotropic protein present in plasma of uninephrectomized rats. AB - The biological characteristics of a kidney growth factor (KGF) from uninephrectomized rat plasma have been studied. A crude preparation of this factor [Nephrol. Dial. Transplant. 4: 334-338, 1989] was further purified by hydrophobic interaction HPLC and gel filtration. KGF was found to be a heat- and trypsin-resistant protein. This factor stimulated dose-dependently DNA synthesis by the mouse kidney in vivo, and by either rat renal tubules or serum-deprived LLC-PK1 cells, in vitro. KGF also increased protein synthesis in these cells, in a dose-dependent manner. Moreover, KGF stimulated sodium uptake by these cells, associated with the maximal increase of protein synthesis. Our findings indicate that KGF is a potent renotropic protein which can play a key role in the renal compensatory growth after uninephrectomy. PMID- 1720254 TI - Tamm-Horsfall protein excretion during chronic alterations in urinary concentration and protein intake in the rat. AB - Tamm-Horsfall protein (THP), a normal constituent of mammalian urine, has been determined in rat urine under various conditions in an attempt to elucidate the physiological role of this glycoprotein. Experiments were designed to assess whether THP production is related to the process of urine concentration or to the transport activity of the thick ascending limb of the loop of Henle (TAL), the nephron segment where it is produced. For this purpose, THP excretion was measured, by radioimmunoassay, in adult male rats under 4 different conditions induced by the following chronic treatments: (1) furosemide (12 mg/day in osmotic minipumps); (2) increased water intake; (3) antidiuretic hormone (ADH) infusion (50 ng DDAVP/day in osmotic minipumps) in rats of the Brattleboro strain with hereditary hypothalamic diabetes insipidus; (4) high-protein (32% casein) versus low-protein diet (10% casein). Each experiment included 6 experimental and 6 control rats. After treatment for 1-3 weeks, 24-h urines were collected for determination of urine flow rate, osmolality, and creatinine and THP concentrations. No significant changes in THP excretion were observed in experiments (1) and (2) despite 5- to 7-fold-differences in urine flow rate. Antidiuretic hormone treatment in (3) slightly lowered THP excretion (287 +/- 53 vs. 367 +/- 41 micrograms/day per 100 g body weight; p less than 0.005), whereas high-protein diet, in experiment (4), led to a 50% increase in THP excretion (446 +/- 57 vs. 304 +/- 79 micrograms/day per 100 g body weight; p less than 0.001). Expressing THP excretion relative to that of creatine did not change these findings. These results show (1) that chronically established changes in the level of diuresis, chronic furosemide-induced blockade of the Na,K,Cl cotransporter or the absence of ADH in Brattleboro rats have little or no impact on the level of THP production, and (2) that THP production is independent of the intensity of transport in the TAL, since two conditions which both are known to increase the transport rate of solutes in the TAL (ADH infusion and high-protein diet), resulted in opposite changes in THP excretion. It is concluded that the rate of THP synthesis is neither linked to the process of urine concentration nor to the ion transport activity of the TAL. PMID- 1720255 TI - The Munich Wistar Fromter rat: proteinuria and blood pressure in correlation to the number of superficial glomeruli. AB - Rats with a high number of superficial nephrons (MWF/Ztm) also show an elevated urinary protein excretion and a high systolic blood pressure. To investigate a possible correlation between the number of superficial glomeruli and these physiological changes, MWF/Ztm rats were crossed and backcrossed to Wistar cryptorchic (WC/Ztm) animals with no superficial nephrons in order to produce genotypes with differing numbers of superficial glomeruli. In the parental strains, the F1 hybrids and the 8 possible backcrosses, the number of superficial glomeruli, the distance of the 10 most superficial glomeruli to the renal surface, and the diameter of Bowman's capsules were determined by morphometric analysis. The excretion of total protein, in detail low molecular weight proteins, albumin, and high molecular weight proteins were measured quantitatively in 5 males of each genotype. Systolic blood pressure was determined by a tail-cuff method in conscious rats. Means of each variate of the 12 available genotypes were linearly correlated and demonstrate a close correlation between the amount of superficial nephrons and the observed physiological changes, i.e. the more superficial the glomeruli the higher the urinary protein excretion, especially albumin, and the higher the systolic blood pressure. PMID- 1720256 TI - Adenosine triphosphate stimulates thymidine incorporation but does not promote cell growth in primary cultures of renal proximal tubule cells. AB - Acute addition of adenosine triphosphate (ATP) stimulated thymidine incorporation in confluent, quiescent primary cultures of rabbit renal proximal tubule cells in a dose-responsive manner. Similar increases in thymidine incorporation was observed with adenosine diphosphate and adenosine monophosphate but not with adenosine. The effect of chronic administration of ATP, however, suppressed cell growth. This suppression appears to be due to an effect of ATP to cause detachment of cells from culture plates, resulting in an increase in thymidine incorporation acutely but in suppression of cell growth chronically. ATP is, therefore, not a direct growth promoter of renal proximal tubule cells in primary culture. PMID- 1720257 TI - Elevation of renal glutathione enhances ischemic injury. AB - In a previous study, we tested the hypothesis that an elevated level of renal glutathione (GSH) would protect the kidney from ischemic injury. However, prior elevation of GSH with GSH monoethylester enhanced then injury induced by 35 min of ischemia and blood reflow [Scaduto RC Jr, Gattone VH, Grotyohann LW, et al; Effect of an altered glutathione content on renal ischemic injury. Am J Physiol 1988;255:F911-F921]. Additionally, GSH monoethylester produced morphologic alterations in the absence of ischemia. Thus the greater ischemic injury observed after GSH ester pretreatment could have been due to a synergistic effect between the events caused by ischemia and the pretreatment. The present study was conducted to evaluate the utility of elevating renal GSH levels by administration of GSH. Administration of GSH (1 mmol/kg body weight) caused a 3-fold elevation of renal GSH levels and a 6-fold elevation of renal cysteine levels after 60 min without causing changes in renal morphology or GFR. After 35 min of renal artery occlusion and 90 min of blood reflow, animals pretreated with GSH had a much greater decline in GFR than untreated control animals. This enhancement of renal ischemic injury in GSH-treated animals was similar to that observed following administration of GSH monoethylester. We conclude that administration of GSH is the method of choice for elevation of renal GSH and that elevation of renal GSH leads to an enhanced ischemia-induced injury which is independent of the method employed to elevate renal GSH. PMID- 1720258 TI - [Artificial nutrition in surgery: status, current problems and perspectives]. AB - Parenteral and enteral nutrition are safe and efficacious substitutes of normal oral intake even for prolonged periods of time and offer adequate treatment of protein-calorie malnutrition and effective palliation of cancer cachexia. Perioperative nutritional support reduces operative morbidity and mortality in malnourished patients. Furthermore, parenteral and enteral nutrition are an effective primary treatment for acute inflammatory bowel disease, short bowel syndrome and small bowel fistulae. Enteral nutrients specifically support structure and function of both the intestinal mucosa and the mucosa-associated lymphatic tissues: Recent clinical data emphasize the need to feed enterally whenever possible, especially in critically ill patients. Continuing efforts to include new substrates and to create more balanced and disease-adapted solutions of parenteral and enteral nutrition will improve the available systems. In addition, parenteral and enteral nutrition as a component of new complex treatment modalities including growth factors and cytokines will improve treatment and patient care in general surgery, oncology and traumatology. PMID- 1720259 TI - [What is the latest in anti-arrhythmia therapy?]. AB - Ventricular premature beats are an independent risk factor for sudden cardiac death. Class Ic antiarrhythmic drugs suppress these premature contractions. The Cardiac Arrhythmia Suppression Trial (CAST-Trial) tested the hypothesis that the suppression of ventricular premature beats after myocardial infarction reduces the incidence of cardiac death. The results of treatment in this low risk group were, however, disappointing. Cardiac mortality (mortality related to arrhythmia and myocardial infarction) was higher in the group treated with flecainide or encainide than in the placebo group. After a treatment period of 10 months, 89 of 1498 patients had died, 63 (8.3%) of them in the flecainide/encainide and only 26 (3.5%) in the placebo-treated group. Drugs that increase the refractory period of a myocardial substrate are obviously more effective in suppressing cardiac death than drugs that delay conduction. In the Basel Antiarrhythmic Study of Infarct Survival (BASIS study) patients with complex ventricular arrhythmias after infarction were treated with low dose amiodarone (200 mg/day). After a follow-up period of 12 months, mortality was lower in the amiodarone treated group (5/98 patients, 5%) than in the group that received no antiarrhythmic treatment (15/114 patients, 13%). Amiodarone was also more beneficial than individual antiarrhythmic therapy (mortality 10/100 patients, 10%). In the past few years no new antiarrhythmic drugs have been registered in Switzerland. Our pathophysiological and clinical knowledge has, however, increased and we know that the asymptomatic patient with low grade ventricular ectopies after myocardial infarction need not be treated. However, if we decide to use antiarrhythmic drugs, strict quality control of the effects of the treatment is mandatory.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720260 TI - A layer by layer look at the skin blister disease. PMID- 1720261 TI - Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities. AB - Epidermolysis bullosa simplex (EBS) is characterized by skin blistering due to basal keratinocyte fragility. In one family studied, inheritance of EBS is linked to the gene encoding keratin 14, and a thymine to cytosine mutation in exon 6 of keratin 14 has introduced a proline in the middle of an alpha-helical region. In a second family, inheritance of EBS is linked to loci that map near the keratin 5 gene. These data indicate that abnormalities of either of the components of the keratin intermediate filament heterodipolymer can impair the mechanical stability of these epithelial cells. PMID- 1720262 TI - The use of high dose aprotinin in liver transplantation: the influence on fibrinolysis and blood loss. AB - Orthotopic liver transplantation (OLT) is frequently associated with systemic fibrinogenolysis and diffuse bleeding. At present antifibrinolytic treatment has not been initiated routinely in OLT. Therefore the influence of high dose aprotinin in OLT (2 million kallikrein inactivator units (KIU) given after induction of anesthesia followed by a 500,000 KIU/h infusion throughout the operation) on intraoperative blood loss and fibrinolysis was studied in 25 patients compared to 25 patients without aprotinin. The incidence of fibrinolysis shown in thrombelastography was 72% in the control group versus 16% in the aprotinin group. Oozing after reperfusion of the graft caused by severe fibrinolysis defined as a clot lysis index below 15% was only observed in the control group (42.8%). In contrast no significant difference was found between the groups in the course of fibrin and fibrinogen degradation product levels (FbDP, FgDP) although the mean concentrations of both parameters were evidently lower in the aprotinin treated patients. Levels of tissue-type plasminogen activator (t-PA) activity were initially high in both groups and peaked during and after the anhepatic period. After aprotinin there was a trend of lower t-PA levels which reached significance at the time of reperfusion (p less than 0.02). In both groups the course of thrombin antithrombin complex was in line with the variations of FbDP and FgDP. No correlation between thrombin formation and t-PA activity was found. Mean homologous blood requirement was reduced by 50% (5.6 +/- 4.0 vs. 11.2 +/- 8.6 units, p less than 0.005). The blood saving effect was more pronounced in the postanhepatic period (p less than 0.000001). In conclusion high dose aprotinin inhibits hyperfibrinolysis and reduces intraoperative homologous blood requirement. Therefore its routine use in OLT is recommended. PMID- 1720263 TI - [Systemic juvenile chronic arthritis]. AB - Juvenile chronic arthritis, systemic onset, is a form of juvenile chronic arthritis with severe systemic and constitutional involvement. We discuss the clinical findings, therapy and outcome of a series of forty patients we have seen in our hospital from 1979-1991. PMID- 1720264 TI - [Heat-shock proteins and arthritis]. AB - Heat-shock proteins are a category of proteins which are synthesized under stressful conditions (such as increased temperatures) both by prokaryotic and eukaryotic cells. Heat-shock proteins are a major target of the immune response and thus can be considered dominant antigens. Under physiological circumstances the response to heat-shock proteins is considered to play a role in overall defence against bacterial infections. An aberrant immune response against heat shock proteins may lead to autoimmunity; adjuvant arthritis in Lewis rats. Current evidence also points towards a role of T cell immunity against heat-shock proteins in the etiology and pathogenesis of human autoimmune diseases such as juvenile chronic arthritis. PMID- 1720265 TI - [The relationship between the neuroendocrine system and juvenile chronic arthritis: a hypothesis]. AB - During the last decennium it has become clear that the course of an inflammatory reaction is not only governed by the immune system. In this paper we describe which other organ systems than the immune system may influence chronic inflammatory responses such as observed in Juvenile Chronic Arthritis (JCA). Attention will be focussed on the role of glucocorticoids, catecholamines and of the neurotransmitter substance P. PMID- 1720266 TI - Alterations in the metabolism of serotonin and dopamine in the mouse brain following a single administration of allylnitrile, which induces long-term dyskinesia. AB - The effects of allylnitrile (ALN), which induces a long-term dyskinesia in mice, on the metabolism of serotonin (5-HT) and dopamine (DA) were studied after a single administration. One day after injection, ALN produced a significant increase in the levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA): 5-HT in the brain cortex, medulla oblongata plus pons, hypothalamus and midbrain; 5-HIAA in the cortex, medulla oblongata plus pons, striatum, hypothalamus and midbrain; the ratio of 5-HIAA/5-HT in the medulla oblongata plus pons, striatum and midbrain; HVA in the cortex and midbrain. These changes were not seen 10 and 35 days after injection when the animals were showing behavioral abnormalities. The present findings suggest that changes in 5 HT and DA metabolism are involved in the appearance of the dyskinetic syndrome. PMID- 1720267 TI - Diagnostic tests for Trichinella spiralis infection in pigs. A comparative study of ELISA for specific antibody and histamine release from blood cells in experimental infections. AB - A study on the histamine release test (HR) for the demonstration of infections with Trichinella spiralis in pigs was carried out on 18 pigs, six infected with 200 larvae, six infected with 5000 larvae and six non-infected (control group). The results obtained by HR during a 7 week infection were compared with those of the enzyme-linked immunosorbent assay (ELISA). All inoculated pigs were found to be positive on Day 40 post-inoculation (p.i.) by necropsy examination of selected muscle groups, with mean recoveries of 7.9 and 225 larvae g-1 of tissue in the low- and high-dose group, respectively. At this time, all animals of the high dose group and five out of six animals of the low-dose group were antibody positive in ELISA with any of three coating antigens employed (a crude muscle larva extract, an excretory/secretory (ES) antigen and a purified 45 kDa antigen). HR performed on whole blood was positive in four out of six pigs of the high-dose group and one out of six pigs of the low-dose group. The earliest ELISA seroconversions took place at Day 15 p.i. with crude and ES antigens. The earliest measurable reaction in HR performed on whole blood was found on Day 19 p.i. There was considerable individual variation regarding which test was the most sensitive for the early detection of infection. Washing of the blood cells prior to antigen provocation led to a markedly improved sensitivity of HR, all animals of the high-dose and three out of six animals of the low-dose group being positive by Day 40 p.i. The time course of the development of ELISA titres and HR reactivity indicated that this effect is due to the removal of blocking antibodies. PMID- 1720268 TI - Anticoagulation and Iloprost for heparin-induced thrombocytopenia: a case report. AB - In patients who receive heparin for anticoagulation, there can be a mild, transient thrombocytopenia which is usually devoid of complications. However, 6% of these patients experience serious heparin-induced thrombocytopenia (HIT), which can result in coagulation problems or death. Iloprost, a recently developed drug, given in conjunction with heparin, can be used to manage the patient with HIT. Two cases reported are of patients diagnosed with HIT and their management with Iloprost. PMID- 1720269 TI - Neonatal follow-up of very low birthweight/extremely low birthweight infants to school age: a critical overview. AB - Neonatal follow-up studies of school age children, published in the last decade, were critically reviewed. Nine studies examined extremely low birthweight infants (less than or equal to 1000 g) and 16 involved very low birthweight infants (less than or equal to 1500 g). The majority of children had age appropriate I.Q. scores, however, there was a greater variability of test scores. There was an increased need for special education or remedial therapy. Visual-motor integration deficits were frequently reported. Behavioural difficulties were described. Fine and gross motor incoordination was identified. There was no conclusive correlation between perinatal course and school outcome. Gender did appear to influence outcome, in the small percent of studies which examined this variable, with females generally faring better. Low socioeconomic status was the most frequently reported predictor of poor outcome. Identified methodological limitations included heterogeneous samples, lack of control groups, high attrition, variable diagnostic criteria and lack of consensus regarding correction for prematurity. PMID- 1720271 TI - [Detection of early prostatic cancer in mass screening program]. AB - Since 1975 up to 1988, we have performed a mass screening program (MS) for prostatic diseases using transrectal ultrasonography (TRS) in the primary study. In our study, 42 cases of prostatic cancer (0.6%) was detected among 6,674 examinees. Out of 42 cases of cancer, 24 (57.1%) were diagnosed as early prostatic cancer (Stage A 1, Stage B 23). The detection rate of cancer in MS and the ratio of early stage of cancer among them were higher than those in the outpatient clinic of our department. Diagnostic accuracy of TRS, palpation and tumor markers in MS were studied respectively through our series of MS. TRS was useful for MS especially in low false negative rate. On the other hand, palpation was characteristic in low false positive rate. Prostatic specific antigen (PSA) among tumor markers was effective to detect early prostatic cancer. However, there were some problems about distinguishing early prostatic cancer from BPH, because of the high false positive rate. PMID- 1720270 TI - [Experimental studies on the antitumor activity of monoclonal antibody--bleomycin A6 conjugate against human liver cancer]. AB - Bleomycin A6 (A6), a single component of bleomycin complex, is highly active against human liver cancer cells in vitro and xenografts in nude mice. A6 was conjugated to monoclonal antibody H111 directed against human hepatoma BEL - 7402 cells, using Dextran T40 as an intermediate. The conjugate consisted of a coupling molar ratio of 1:264 for H111 and A6, and retained 6.3% of A6 activity. As determined by clonogenic assay with hepatoma BEL - 7402 cells exposed to the agents for 1 h, the IC90 values for H111 - A6 conjugate, free A6 and M3 - A6 conjugate (an irrelevant conjugate) were 0.17 mu mol/L, 17 mu mol/L and 7 mu mol/L respectively. The cytotoxicity of Hill - A6 conjugate to target cells was markedly blocked by unconjugated H111 but not by irrelevant monoclonal antibody M3. The H111 - A6 conjugate exhibited 78% inhibition on the growth of hepatoma BEL - 7402 xenografts in nude mice, whereas the equivalent doses of free A6, M3 - A6 conjugate and H111 plus A6 mixture showed approximately 30% inhibition. Histopathological examination showed no toxic changes in the liver, lung, kidney and bone marrow in the H111 - A6 conjugate--treated animals. These results suggest that the conjugate of monoclonal antibody and bleomycin A6 exhibits specific cytotoxicity to target liver cancer cells and the conjugate is highly effective against liver cancer xenografts in nude mice with more marked tumor inhibition than free A6 at comparable dose levels. PMID- 1720272 TI - [Hormonal treatment of carcinoma of the prostate]. AB - Efficacy of orchiectomy and intravenous administration of diethylstilbestrol diphosphate (DESP) for the treatment of prostatic carcinoma was evaluated on 184 patients treated between 1975 to 1989. The patients were between 49 to 88 years old with a mean age of 73.4 +/- 8.3 years. Clinical stage was A in 9.8%, B in 12%, C in 26.6% and D in 51.6%. The histologically well, moderately and poorly differentiated adenocarcinoma were observed in 20.9, 29.4 and 49.7%, respectively. The 5-year survival rate of stage A, B, C and D calculated with the Kaplan-Meier method were 90, 49, 60 and 34%, respectively. The 5- and 10-year survival rate of the patients who had received orchiectomy was 53 and 24%, respectively, while that of the patients without orchiectomy was 38 and 14%, respectively. The 5 and 7-year survival rate of the patients treated with intravenous administration of DESP was 54 and 34%, respectively while that of the patients without DESP was 46 and 31%, respectively. These findings suggest that orchiectomy and intravenous administration of DESP in any form prolonged patient survival compared with only oral administration of estrogens or antiandrogens. Reactivation was seen in 24 (40%) of the 60 patients under sufficient observation. Clinical relapse occurred within an average of 32.3 +/- 26.4 months after primary hormonal manipulation. The average time to relapse in stage D was shorter than that in stage B and C. Reactivation was observed in the patients on interrupted treatment earlier than in the patients on continuous administration of drugs. Cardiovascular death followed by endocrine therapy was 7.4% in this study. PMID- 1720273 TI - [Chemotherapy of prostatic cancer]. AB - CDDP combined chemotherapy was performed in 55 cases out of 229 prostatic cancer patients who were treated in Nara Medical University and Nara Prefectural Nara Hospital between January 1979 and August 1989. The previously untreated 33 patients received chemotherapy with anti-androgen treatment as an initial treatment, as well as 7 cases of unresponsive to antiandrogen treatment, 14 relapsing cases and one case with recurrence after total prostatectomy. The major regimens of chemotherapy were cis-diammine dichloroplatinum (CDDP) alone in 16 cases, PVB regimen (bleomycin or peplomycin + vincristine + CDDP) in 19 cases, and CAP regimen (cyclophosphamide + adriamycin + CDDP) in 16 cases. Complete response was not achieved or partial response was observed in 20 cases (34%), no change was seen in 20 cases (34%), and progression was seen in 19 cases (32%). Among each evaluable lesion, effects (CR + PR) were observed in 40% in the prostate, in 18% in the bone lesions, in 44% in the soft tissue lesions, and in 42% in the prostatic tumor marker. The 7-year survival rate of the chemotherapy group (35.6%) was better than that of the antiandrogen treatment group (26.6%) in stage D patients, but was not significant statistically When evaluated by the regimen, a partial response was observed in 56% of CDDP alone, in 21% of PVB regimen, and in 38% of the CAP regimen. However, there was no significant difference in survival rate among the regimens. As an adverse effect, myelosuppression and renal toxicity seemed to be dose limiting factors of CDDP combined chemotherapy for advanced prostatic cancer patients. PMID- 1720274 TI - [Thermochemotherapy of prostatic cancer]. AB - We performed hyperthermia concomitantly with the use of anticancer agents (etoposide and peplomycin) for the treatment of 13 patients with prostatic cancer. Seven of them were new cases and the others were recurrent ones. After intravenous administration of etoposide and peplomycin, hyperthermia was applied twice a week for 10 times in total. Clinical efficiency was evaluated by CT, ultrasound, prostatic biopsy. Tumor regression were observed in 12 cases. According to the General Rule for Clinical and Pathological Studies on Prostatic Cancer by Japanese Urological Association and the Japanese Pathological Society, one case of Ef2 and 5 cases of Ef1 were obtained with this treatment. Side effects caused by hyperthermia were urethral pain (1 case) and skin burning (1 case) noted. PMID- 1720275 TI - [Clinical evaluation on serum osteocalcin in advanced prostate cancer patients]. AB - The clinical significance of osteocalcin as a marker for advanced prostate cancer was examined. Osteocalcin is produced by osteoblasts and is also detected in the blood. Its change is a good index of osteometabolic diseases and especially of the osteoblastic activity. In the present study, we examined the serum osteocalcin concentration of those patients with urogenital tumor, especially prostate cancer, who had been confirmed for multiple bone-metastasis by clinical examination. These patients comprised an untreated group (15 cases) of patients with prostate cancer presenting confirmed bone-metastasis, and a group of patients without bone-metastasis. The respective serum osteocalcin concentrations of these two groups were compared with 51 cases of prostate hypertrophy used as the control group. The findings revealed that the serum osteocalcin concentration demonstrated high values in the first group with a tendency toward lowering during treatment. Neither the latter group nor the control group showed high values. On the other hand, false-positive cases (8%), and false-negative cases (20%) were found. In the case of bone-metastasis, these results suggest that measurement of serum osteocalcin concentration is useful for clinical periodical observation about the activity of the bone metastatic focus. PMID- 1720276 TI - [A mass screening of the prostatic diseases and serum prostate specific antigen]. AB - We examined the values of prostate specific antigen (PSA) with a RIA kit (Pros Check PSA) and an EIA kit (Market F PA), measuring prostate weights of 125 participants in a mass screening of the prostatic diseases. A mild positive correlation (r = 0.467) between values of PSA (RIA) and prostate weights was found in the participants in whom the prostate cancer was not detected. Since serum PSA levels measured by RIA of 45 normal subjects were 1.8 +/- 1.5 ng/ml (Mean +/- S.D), the upper limit of the normal range was set at 6.3 ng/ml. The participants whose PSA levels exceeded this upper normal range and also whose prostate weights were under 30 g were found in 11 of the 122 subjects (9%). On the contrary, the abnormal values (EIA) were found in only two subjects (one, a prostate cancer and the other, a benign prostate hypertrophy). We, further, examined the PSA values (EIA) in 415 subjects in whom the prostate cancer was detected in 5 (1.2%). The abnormal values were found in 8 (4 prostate cancer, 3 benign prostate hypertrophy and one without prostatic disease). As the false positive rate was very low, the use of PSA is recommended in the first screening of the check up program of the prostatic disease. PMID- 1720277 TI - Postextrasystolic sinoatrial exit block in human sick sinus syndrome: demonstration by direct recording of sinus node electrograms. AB - Ten patients with sick sinus syndrome having repetitive sinus node electrograms during long postpacing pauses were studied during programmed atrial stimulation. Sinus node activity was recorded using a percutaneous catheter electrode. A sinus node electrogram was recorded before the return atrial beat in seven patients; it was similar to the sinus node electrogram observed during postpacing pauses and is clearly identified because sinoatrial conduction time was markedly prolonged following the atrial extra beat. Complete sinoatrial exit block occurred in four patients. (1) Sinus node electrograms were thus validated both during postpacing pauses and during programmed atrial stimulation in most patients with sick sinus syndrome. (2) Sinoatrial conduction time was markedly prolonged after one extrasystole, accounting for supracompensatory atrial return cycles. (3) If it were cumulative following multiple extrasystoles, this effect could constitute the electrophysiologic link between an abnormal response during programmed atrial stimulation and the complete sinoatrial block recorded during the pauses that follow rapid atrial pacing. PMID- 1720278 TI - Central nervous system involvement in patients with diffuse aggressive non Hodgkin's lymphoma. AB - Central nervous system (CNS) involvement was evaluated in 277 consecutive patients with aggressive non-Hodgkin's lymphoma treated by the Nebraska Lymphoma Study Group. Three patients (1.1%) developed CNS involvement at presentation and 11 (4.0%) at relapse. The involvement was meningeal in 8 patients and documented by CSF cytology; it was parenchymal in 2 patients and proven by biopsy; and it was in the cauda equina in 1 patient at autopsy. Factors significantly associated with a greater likelihood of CNS relapse were age less than 60 years and epidural disease. Other factors, including tumor histology, extranodal disease at presentation, response to therapy, sex, and symptom type, were not significantly associated with a higher risk of CNS relapse. Survival of the patients presenting with CNS disease (6, 26, and 27+ months) was longer than patients whose CNS disease relapsed (median 2 months). PMID- 1720279 TI - Treatment of advanced refractory lymphomas with a combination of carmustine, bleomycin, teniposide, dexamethasone, and cisplatin (the BBVDD regimen). An ECOG pilot study. AB - Forty-four patients with relapsed, refractory malignant lymphomas (12 Hodgkin's disease, 32 non-Hodgkin's lymphoma) were treated with a combination of carmustine, bleomycin, teniposide, dexamethasone, and cisplatin (BBVDD regimen). Patients had failed at least one, and frequently two, chemotherapy regimens before admission to the study. Of the patients with Hodgkin's disease, 2 (17%) achieved complete response (CR), and 3 (25%) attained a partial response (PR) for an overall response rate (CR + PR) of 42%. Among the patients with non-Hodgkin's lymphoma there were 6 CR (19%) and 12 PR (37%), for an overall response rate of 56%. Median durations of response ranged from 2.5 months for nodular non Hodgkin's lymphoma in PR to 28.5 + months for Hodgkin's disease in CR. In these heavily pretreated patients, the incidence of toxic effects was grade 3 (48%), grade 4 (23%), grade 5 (2%). The one death (grade 5 toxicity) was attributed to pulmonary impairment due to bleomycin. BBVDD is a moderately effective regimen for the palliation of patients with refractory lymphomas and merits further study. PMID- 1720280 TI - Congenital heart block: successful prophylactic treatment with intravenous gamma globulin and corticosteroid therapy. AB - In mothers with anti-Ro-positive antibodies whose previous pregnancies have ended in deliveries of infants with congenital heart block, prophylactic therapeutic strategies are used to try to diminish the production and passage into fetal circulation of autoantibodies. Intravenous gamma globulin was given at 14 and 18 weeks' gestation and prednisone was given from 14 weeks' gestation to a woman with Sjogren's syndrome. The pregnancy ended with delivery of an infant without congenital heart block. PMID- 1720281 TI - The effect of gestational age on the detection rate of Down's syndrome by maternal serum alpha-fetoprotein screening. AB - Low levels of maternal serum alpha-fetoprotein are currently being used to screen for Down's syndrome in midpregnancy. Because of the possibility that gestational age may affect the detection rate of Down's syndrome, we analyzed maternal serum AFP levels and gestational age in 51 Down's syndrome pregnancies that had been confirmed by amniocentesis or at birth, and we compared these pregnancies with 3239 screened singleton pregnancies with known normal outcomes. The highest yield of a low risk for Down's syndrome associated with maternal serum alpha fetoprotein occurred at 16.5 to 17.5 weeks' gestation. Our data suggest that maternal serum alpha-fetoprotein screening for Down's syndrome should be done between 16 and 18 weeks' gestation, which is the gestational age currently recommended for neural tube defect screening. PMID- 1720282 TI - Neovascularization of the iris in rhegmatogenous retinal detachment. AB - To identify conditions associated with neovascularization of the iris in rhegmatogenous retinal detachment, we examined 36 eyes with this disorder seen at our hospital between 1979 and 1990. Clinical courses of disease were divided into the following three groups: (1) neovascularization of the iris without a history of a vitreoretinal operation (four eyes), (2) neovascularization of the iris after an unsuccessful vitreoretinal operation (26 eyes), and (3) neovascularization of the iris after surgical complications (six eyes). In all eyes of Groups 1 and 2, retinal detachment persisted at the onset of iris neovascularization; however, in six eyes, iris neovascularization subsided after retinal reattachment. Characteristic features of Groups 2 and 3 were patient age of 50 years or more, severe myopia, a history of increased intraocular pressure, a history of choroidal detachment, and a large scleral buckle. PMID- 1720283 TI - New instruments for submacular surgery. PMID- 1720284 TI - Dust from carpeted and smooth floors. IV. Solid material, proteins and allergens collected in the different filter stages of vacuum cleaners after ten days of use in schools. AB - The accumulation of dust, proteins and allergens from alder, birch, timothy, cat, dog, mite, hen egg white, codfish and mould in schools was investigated by analysing the content of vacuum cleaners after 10 days of use. The main goals were to compare the dust accumulation on carpeted and smooth floors and to estimate to what degree the three vacuum cleaner filter stages (i.e. the disposable bag, the main filter and the microfilter) collected dust, proteins and allergens. Carpeted floors accumulated more dust, proteins and allergens per unit area than smooth floors. Histamine release studies of some of the dust extracts showed that the dust from carpeted floors released histamine from passively sensitized basophils at concentrations for which dust from smooth floors gave low or no histamine release. The analyses showed that most of the dust, proteins and allergens were retained in the dust bags. Less than 1% of the vacuumed material had accumulated in the main filters, which, according to the manufacturer, detain 99.5% of particles greater than 2 microns. By the use of a scanning electron microscope (SEM), particle deposits were observed in the microfilters. These deposits, which represented less than 0.1% of the total mass, showed no significant allergenic activity. Thus, for the field conditions of this study, the microfilters were not needed for cleaning the exit air of allergens, although they were useful for removing fine (less than 2 microns) particles. PMID- 1720285 TI - Total serum IgD is increased in atopic subjects. AB - We have developed a sandwich-type ELISA system for measuring total IgD levels in the serum of atopics and non-atopic controls. In this ELISA system, affinity purified goat anti-human IgD was used for capture. Results were superior to those obtained with monoclonal anti-human IgD antibody. No cross-reactivity could be demonstrated to IgG, IgM, IgA or IgE. The assay showed minimal non-specific binding even with initial serum dilutions of 1:2. The results obtained were reproducible among replicates (Mean CV +/- SEM = 0.03 +/- 0.002; n = 251), between dilutions (CV = 0.08 +/- 0.006; n = 108), and between assays (CV = 0.05 +/- 0.12; n = 5). We used routine radioimmunoassay for measuring total serum IgE. Using these assays total serum IgD and IgE levels were measured in 75 atopic patients and 33 normal subjects. None of the atopics had recent immunotherapy. As expected, the geometric mean serum IgE in atopics (373 ng/ml) was significantly higher than that in normal subjects (49 ng/ml) (P less than 0.01). However, geometric mean serum IgD was also significantly higher in atopics (20.3 micrograms/ml) than that in normal subjects (8.4 micrograms/ml) (P less than 0.02). In both atopic and normal groups, mean serum IgD level did not differ significantly on the bases of age, sex or asthmatic status. Furthermore, total serum IgD was not significantly correlated with total serum IgE (r = 0.14; P = 0.14; n = 108), indicating that immunoregulatory control of the basal levels of the two isotypes is not linked.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720286 TI - Study of the effects of paf-acether on human nasal airways. AB - In 6 normal subjects and 6 patients with allergic rhinitis, nasal response to insufflation of paf-acether (paf, platelet-activating factor), lyso-paf and histamine was evaluated. Nasal challenge with paf, at doses of 300 and 600 nM, induced nasal obstruction, associated with an increase in nasal airway resistances, measured by anterior passive rhinomanometry. Maximum increase in nasal airway resistance was observed at 30 min after challenge (mean percent change + 481 with 600 nM paf; P less than 0.05). Other symptoms induced by paf insufflation were rhinorrhea (6 out of 12 subjects), itching (8 out of 12), sneezing (4 out of 12) and a burning sensation (6 out of 12). No differences were observed between normal and rhinitic subjects, concerning nasal sensitivity to paf. Neither nasal symptoms nor changes in nasal airway resistance were observed after nasal challenge with lyso-paf (300 and 600 nM); by contrast, histamine (100 nM) induced sneezing, nasal obstruction, itching and rhinorrhea in all the studied subjects, associated with an increase in nasal airway resistance (maximum 5 min after challenge; percent change + 358; P less than 0.02). Nasal effects of paf were not mediated by histamine, since no increase in histamine levels was observed in nasal washings following paf insufflation. We conclude that paf may have pathogenetic relevance in allergic rhinitis. PMID- 1720287 TI - Three predominant prostatic proteins. AB - Prostatic acid phosphatase (PAP), prostate-specific antigen (PSA; or gamma seminoprotein), and beta-microseminoprotein (beta-MSP; PSP94 or beta-inhibin) are the three predominant proteins secreted by the normal human prostate gland. In the epithelium of normal and hyperplastic prostatic acini and ducts PAP, PSA and beta-MSP have an identical immunohistochemical localization. Highly differentiated (grade I) carcinomas contain an almost equal number of PAP-, PSA- and beta-MSP-immunoreactive cells; the incidence of these cells is lower and they display a greater staining variability in the moderately and poorly (grade II III) differentiated tumours. Especially in poorly differentiated tumours PSA seems to be a more sensitive immunohistochemical marker than PAP or prostatic carcinomas. Moreover, the use of PAP as a marker for prostatic carcinomas is complicated by the reported structural similarities between the prostatic secreted acid phosphatase and lysosomal acid phosphatase occurring in all tissues. The use of beta-MSP as a marker for prostatic carcinomas may be limited by indications of non-prostatic production of this protein. PMID- 1720288 TI - Dynamics of a human seminal vesicle specific protein. AB - The present paper is concerned with the temporal alterations and tissue localization of a seminal antigen secreted by the human seminal vesicle. This antigen is recognized by antibody MHS-5, which is one of a set produced in mice by immunization with human sperm. The respective clone produced an antibody of the IgG1 subtype, which reacted with seminal fluid from over 400 normal donors and 21 semen samples from vasectomized men. Incubation of seminal vesicle secretion with either prostatic fluid or prostate specific antigen (PSA) resulted in degradation on the antigen. The experiments showed that MHS-5 antigen is a substrate for the serine protease PSA: Immunohistochemical studies suggested that MHS-5 is a "sperm-coating" antigen and is exclusively synthesized and secreted by the seminal vesicle. PMID- 1720289 TI - Monoclonal antibody to the prostate specific antigen. AB - Somatic cell hybrids were made from mouse myeloma cells and spleen cells derived from BALB/c mice immunized with homogenized epithelial fractions of BPH. The screening by immunoperoxidase staining on human prostate and non-prostate tissue resulted in one monoclonal antibody identifying a prostate specific antigen. Upon SDS-PAGE and Western blot this antigen exhibited a single band at the position of 34 kDa molecular weight. The immunoreactivity of the prostate antigen was found to be localized exclusively in the epithelial lining of ducts and secretions of normal prostate, BPH and prostate cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule and inhibited the binding of Anti PSA antibody to PSA by about 80 to 90% in the RIA. PMID- 1720290 TI - Swiss chard hypersensitivity: clinical and immunologic study. AB - Allergy to vegetables and fruits seems to be more prevalent in atopics, especially in birch pollen-sensitized individuals. We report a case of a grass pollen-sensitized woman, in whom the inhalation of vapor from boiling Swiss chard precipitated rhinoconjunctivitis and asthma. Type I hypersensitivity to Swiss chard was demonstrated by means of immediate skin test reactivity, specific IgE determination by RAST, basophil degranulation, histamine release test, and an immediate bronchial provocation test response to Swiss chard extract. The controls did not react to any of these tests. RAST inhibition assays suggest the presence of some cross-reactivity among Swiss chard and grass pollen antigens, as well as cross-reactivity between vegetables and weed pollens of the chenopod family. PMID- 1720291 TI - Monoclonal antibodies against protease inhibitors: the case of the bovine pancreatic trypsin inhibitor. AB - A monoclonal antibody (MoAb) directed against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a small protein made up by 58 aminoacids, has been produced. Mice were immunized with the native form of the protein and gave low antibody response. After somatic hybridization of spleen cells from immunized mice, few clones secreting antibodies against BPTI have been found and just two of them were stable. Both secreted IgM. One (ICI) produces a monoclonal antibody which binds to BPTI with an equilibrium dissociation constant, Kd, of 6.1 x 10( 7) M at pH 7.4. Competition experiments demonstrated that ICI recognizes an epitope close to the reactive site of BPTI. Furthermore, Kd of ICI with an isoinhibitor similar to BPTI (S.I. II) but with few differences at the active site is higher, confirming specificity of binding. PMID- 1720292 TI - Phosphotyrosine antibodies as probes for activated oncogene products endowed with tyrosine kinase activity. AB - The usefulness of phosphotyrosine antibodies for the detection of physiologically regulated or deregulated kinases is shown in this paper. This rather rare enzymatic activity is shared by receptors for some polypeptide growth factors and by the products of class 1 oncogenes. The antibodies are able to detect proteins phosphorylated on tyrosine in fibroblasts stimulated with growth factors, such as EGF and PDGF. The major phosphorylated protein species are the receptors themselves, which undergo phosphorylation only following the addition of the exogenous factor and only transiently. Phosphotyrosine antibodies were able to detect the products of the retroviral class 1 oncogenes, which are endowed of deregulated tyrosine kinase activity. In fact, in these cases a constitutive phosphorylation of the relevant proteins was observed, which occurred continuously and independently of the presence or lack of the growth factor. A tyrosine kinase constitutively activated in human gastric carcinoma cells was detected by P-Tyr antibodies. This molecule has been characterized at molecular level and the mechanisms responsible for its enzymatic activation has been investigated. The question of whether the tyrosine kinase identified is responsible for the induction and the maintenance of the transformed phenotype in gastric carcinomas remains to be answered. It is reasonable to suggest that this might be the case by analogy with other known pathologies, such as class 1 oncogenes activated by transduction by retroviruses, abnormal expression of EGF receptors or deregulated activity of c-abl encoded proteins in CML and ALL. Thus, the search for deregulated kinases by means of phosphotyrosine antibodies seems to be useful for identifying new activated oncogenes in clinical oncology. PMID- 1720294 TI - Identification of a specific Mycobacterium tuberculosis protein able to activate T lymphocytes. AB - The recent advances in the field of immunology and molecular biology allow to consider not distant the identification of protective antigens to be used in new prophilactic and diagnostic tools. In our laboratory we have analysed a M. tuberculosis expression library by murine monoclonal antibodies, human sera and human T lymphocytes. We have identified a 35 Kd protein present only on the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum). This 35 Kd protein is also detected, by immunohistological analysis, inside the alveolar macrophage of pulmonary tuberculosis patients. Both recombinant beta galactosidase and MS2 polymerase-fused proteins have been produced and used to perform western blotting and T cell proliferation assay. As the cloned fragment contains an internal EcoRI restriction site, it was possible to identify two fragments of 1.7 and 2.2 kb respectively. Southern blot analysis showed that both the fragments hibridized with the genomic DNA from M. tuberculosis complex but not with the DNA from other mycobacterial isolates from 12 pulmonary tuberculous patients hibridized with the 1.7 kb fragment. The possible use of this insert for the molecular diagnosis of tuberculosis is under investigation. PMID- 1720293 TI - Use of monoclonal antibodies for the characterization of allergenic extracts. AB - The diagnosis of allergic diseases is commonly carried out by using poorly standardized preparations containing allergenic and non-allergenic components. Recently, monoclonal antibody technology has been applied to the biochemical characterization and to the purification of allergens, in order to evaluate their potential role in the standardization of diagnostic and therapeutic extracts. Three main results have been achieved: 1) the characterization of allergenic epitopes recognized by IgE and IgG from patients' sera; 2) the purification by affinity chromatography of relevant allergenic components; 3) the development of immunoassays for the quantitation of allergens in the commercially available extracts used for diagnosis and therapy. PMID- 1720295 TI - Identification of T cell epitopes on hepatitis B surface antigen. AB - The antigenic sites for human T lymphocytes on hepatitis B surface antigen (HBsAg) have been studied using synthetic peptides. The results indicate that aminoacid residues 24-28 of HBsAg (located near the amino terminus of the HBsAg molecule) constitute an immunodominant "helper" determinant, whereas residues 17 18 and/or 34-36 represent a major "suppressor" epitope. However, non responsiveness to currently used hepatitis B virus (HBV) vaccines (employing the whole HBsAg molecule) does not depend, in the vast majority of cases, on suppressor T cells. In vivo experiments with synthetic peptide vaccines are needed to verify the possibility of enhancing the production of protective antibodies against the hepatitis B virus. PMID- 1720296 TI - Reversibility of age associated NK defect by endocrinological and/or nutritional intervention. AB - Several abnormalities arise at the level of NK cell function in both rodents and humans with increasing age. The age-related changes are not irreversible alterations since they can be reversed either by endocrinological or nutritional approaches, suggesting that age-related microenvironmental changes may play a relevant role in the age-related immune deterioration. Whether endocrine and nutritional factors have an additive effect or act through the same intracellular mechanisms remains to be established, though the first possibility seems more likely since the action of TSH and thyroid hormones is specifically directed towards lymphokine-boosted NK activity, while AL are able to prevent age associated defect of basal NK cytotoxicity. PMID- 1720297 TI - Increased urinary transferrin excretion in exercising normoalbuminuric insulin dependent diabetic patients. AB - The median rate of urinary transferrin excretion is greatly increased by exercise in subjects with uncomplicated type 1 (insulin-dependent) diabetes. This increase is proportionally far greater than that seen in urinary albumin excretion rate after the same exercise. Non-diabetic control subjects showed no rise in urinary transferrin excretion rate following exercise. N-acetyl-beta-D-glucosaminidase excretion rate was higher in diabetic than control groups but did not rise with exercise. Our results suggest that patients with apparently uncomplicated diabetes have abnormal renal function. In this group an elevated urinary transferrin excretion rate appears to be a more sensitive indicator of altered renal function than is elevated albumin excretion rate. The mechanism underlying exercise-induced urinary loss of transferrin remains to be elucidated. PMID- 1720298 TI - Primary hepatocellular carcinoma in Zimbabwe: frequency of abnormal biochemical investigations. PMID- 1720299 TI - Relative sensitivity of serum and urinary retinol binding protein and alpha-1 microglobulin in the assessment of renal function. PMID- 1720300 TI - T cell receptor expression in Sjogren's syndrome. AB - T lymphocytes expressing the gamma/delta T cell receptor and B lymphocytes expressing CD5 are known to occur in expanded numbers in the peripheral blood of patients with primary Sjogren's syndrome. The cellular infiltrates for the surface phenotypic markers for alpha/beta and gamma/delta T cell receptors, CD4, CD8, CD45, and CD5 were examined in lip biopsy specimens from two patients with primary Sjogren's syndrome, six with secondary Sjogren's syndrome, and seven healthy controls. Most of the Sjogren's lip biopsy cellular infiltrates were T lymphocytes of the CD4 subset expressing the alpha/beta T cell receptor (mean 70%). The low prevalence of gamma/delta T cell receptor bearing cells in lip biopsy specimens is maintained in Sjogren's syndrome (mean 1.5%), and thus it seems unlikely that these lymphocytes bearing the gamma/delta T cell receptor have a major role in the immunopathology of Sjogren's syndrome. Over 70% of cells within the lesional infiltrate of primary and secondary Sjogren's syndrome expressed the CD5 and CD45 cell surface molecules. PMID- 1720301 TI - Candida arthritis: cellular immune responses of synovial fluid and peripheral blood lymphocytes to Candida albicans. AB - A case of septic Candida albicans arthritis of the knee in a patient with systemic candidiasis is presented. Systemic and intra-articular cellular immune responses to C albicans and various bacterial antigens were monitored for 15 weeks. It is shown that the candida induced blastogenesis of synovial fluid lymphocytes was much more stimulated than that of peripheral blood lymphocytes, and that the proportion of activated cells expressing HLA class II antigens was markedly increased in the synovial fluid. Strong cellular immune responses to Candida albicans could still be shown many weeks after the synovial fluid aspirates had become sterile. For the first time synovial fluid derived, CD4 positive T lymphocyte clones with specificity for candida antigens were characterised and further propagated in vitro. PMID- 1720302 TI - Characterization of purified gp 51 from bovine leukemia virus integrated into iscom. Physicochemical properties and serum antibody response to the integrated gp51. AB - It is proposed that the envelope glycoprotein, gp 51, is the protective antigen of bovine leukemia virus (BLV). An experimental iscom vaccine has been prepared from immunoaffinity purified gp 51. To overcome the problem of integrating a nonamphipathic protein, gp 51 was partially denatured at pH 2.4 before integration into the iscom. The recovery of gp 51 into the iscom was calculated to be 85%. The gp 51 incorporated into iscom retained its physicochemical properties and the neutralizing epitopes F, G and H were found to be intact. The iscom preparation was shown to induce a specific immune response to gp 51 after inoculation into mice and calves, as tested by ELISA and Western blotting. Sera from the immunized calves specifically inhibited the VSV-(BLV) pseudotypes. Thus the gp 51-iscom preparations appear to be highly immunogenic and to induce a gp 51 specific response. PMID- 1720303 TI - [Antigenic determinants on ovalbumin and ovomucoid: comparison of the specificity of IgG and IgE antibodies]. AB - To delineate the antigenic determinants on ovalbumin (OA) and ovomucoid (OVM) recognized by specific IgG and IgE antibodies in sera from patients with allergy to hen's egg, we studied the binding activities of IgG and IgE antibodies to native OA or OVM and seven different OA or OVM preparations, i.e. heated OA or OVM (100 degrees C for 3 min and 80 degrees C for 30 min) or 0.3 M NaOH, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), 6 M urea or HCl treated OA or OVM. Eight patients with IgE anti-OA antibodies and 12 patients with IgE anti-OVM antibodies were used in these studies. The binding activities of IgG and IgE antibodies to physically or chemically denatured OA were partially decreased compared with those to native OA, whereas IgG and IgE antibodies in sera from patients bound well to denatured OVM with similar binding activities to native OVM. These results strongly suggest that antibodies to OA recognize partially conformational antigenic determinants on OA, whereas antibodies to OVM mainly recognize sequential antigenic determinants on OVM, and that antigenic determinants of OVM recognized by antibodies in sera from patients are more stable than those of OA under these denaturation conditions. In addition, the binding activities of IgE antibodies to denatured OA or OVM were significantly different from those of IgG antibodies to these OA or OVM, suggesting that the specificities of IgE antibodies to OA or OVM may be different from those of IgG antibodies to OA or OVM. PMID- 1720304 TI - [Mechanism of ethanol-induced bronchoconstriction in Japanese asthmatic patients]. AB - Acute ingestion of alcoholic beverages causes no changes in the expiratory airflow rates of most Occidental asthmatic patients. However, asthmatic symptoms are worsened after drinking small amounts of alcohol in Japanese asthmatic patients. Our laboratory results showed that the ingestion of pure ethanol caused a fall in FEV1.0 in about half of the Japanese asthmatic patients tested. The mechanism of ethanol-induced bronchoconstriction remains unclear. In order to investigate this mechanism, we performed oral-provocation tests with 10% ethanol in vivo, and leukocyte histamine release assay induced by ethanol and acetaldehyde in vitro. 55% of asthmatics showed a significant fall in FEV1.0 after ingestion of 10% ethanol (responder). There was no difference in the rise of blood ethanol concentration between the responder group and non-responder group. Acetaldehyde and histamine concentration in the responder group were significantly higher than in the non-responder group. Leukocyte histamine release assay showed that acetaldehyde (2 microM-100 microM) caused dose-dependent histamine release, whereas, ethanol (2 mM-100 mM) had no effect on histamine release. Our data indicate that histamine release from mast cells (or basophils) caused by acetaldehyde may play an important role in ethanol-induced bronchoconstriction. This is the first report on the mechanism of ethanol-induced bronchoconstriction in Japanese asthmatic patients. PMID- 1720305 TI - Molecular basis of an adult form of beta-hexosaminidase B deficiency with motor neuron disease. AB - A patient (KL) with progressive motor neuron disease associated with partial Hex A (alpha beta) and no Hex B (beta beta) activity, synthesized beta-chains which only associated with alpha-chains. To identify the molecular basis of this inability of beta-chains to self associate, RNA from cultured fibroblasts was reverse transcribed, the cDNA encoding the beta-chain amplified by polymerase chain reaction, subcloned, and sequenced to reveal two types of single missense mutation. The first mutation, (Type I) 619A----G, was paternally inherited and converted a 207IIe----Val in a highly conserved region believed to be associated with catalytic activity and activator protein binding. Biochemical evidence for impaired activator protein binding was obtained by purifying Hex A from KL urine and demonstrating a greater than 50% reduction of in vitro GM2 hydrolysis compared to normal urinary Hex A. In other cDNA species (Type II), a maternally inherited 1367A----C mutation converted 456Tyr----Ser in another highly conserved region of the beta-chain and we propose that this mutation leads to the inability of the beta-chains to self associate and thus reach maturity. These same cDNA species contained a second 362A----G mutation which converted 121Lys----Arg, but is apparently a polymorphism since it also occurs in some normal subjects. We propose that the patient is a compound heterozygote in which a combination of no self-association of the mutant beta-chains and impaired activator protein binding to alpha-beta (mutant) (Hex A) required for GM2 hydrolysis result in total beta Hex B deficiency and slow accumulation of GM2 ganglioside, primarily in motor neurons. PMID- 1720306 TI - Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A. AB - Human members of a family of structurally related cytokines, which play a role as effectors of inflammation, were analyzed for their expression and regulation in T lymphocytes. Members of this gene family include Platelet Basic Protein (PBP); Platelet Factor 4 (PF-4); IL-8/NAP-1; IP-10, a gamma interferon induced protein; GRO; pAT 464 and pAT 744. In resting T lymphocytes the RNAs of the individual genes could not be detected, but all genes were induced upon stimulation with PHA or with PHA/PMA. The induction of five genes was blocked by the immunosuppresive drug cyclosporin A (CSA), which appears to affect initial events in T cell activation. This expression in T lymphocytes, especially the sensitivity to CSA, indicates a common immunmodulatory role of these structural related proteins. PMID- 1720307 TI - Isolation and sequence determination of cDNA encoding P2 protein of human peripheral myelin. AB - A full length cDNA of P2 protein of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 2150 base pairs (bp) in length and contains a 393 bp open reading frame encoding a polypeptide of 131 residues. The deduced amino acid sequence is highly homologous to P2 protein from other species. PMID- 1720308 TI - Expression of liver type pyruvate kinase in insulinoma cells: involvement of LF B1 (HNF1). AB - The messages for LF-B1, which interacts with the cis-acting element of PKL-I to play an essential role in expression of L-type pyruvate kinase (PK) in the liver, and L-type PK were found to be present in RIN-m5F insulinoma cells as well as the liver, kidney and small intestine, although the levels of the two mRNAs in these tissues were not correlated. Gel retardation assay suggested that similar nuclear proteins bound to two other cis-acting elements, PKL-II and PKL-III, were expressed in both liver and insulinoma cells, and that additional PKL-III-binding proteins were present only in RIN-m5F cells. Thus, we suggest that the mechanism of L-type PK expression in pancreatic B cells is similar to that in the liver. PMID- 1720309 TI - Distribution of plural HCV types in Japan. AB - A detection system was developed to distinguish the four different HCV genomes [HCV-J, HCV-US, HCV-K2 and group II HCV (HCV-GII)], involving reverse transcription followed by a nested polymerase chain reaction using specific primers for each HCV type. The putative non-structural (NS) 5 regions of HCV-J, HCV-US and HCV-K2 and the putative NS3 region of HCV-GII were amplified. Of 95 specimens from patients with acute hepatitis, chronic hepatitis, liver cirrhosis or hepatocellular carcinoma, 67 specimens were positive for HCV-J, 2 for HCV-US, 23 for HCV-K2 and 11 for HCV-GII. About half the specimens that were positive for HCV-K2 or HCV-GII were coinfected with HCV-J and all those that were positive for HCV-GII were also positive for HCV-K2. Nucleotide sequence analysis of several amplified cDNA products revealed that HCV-K2 and HCV-GII could each be classified into two groups, and the pattern of classification of HCV-K2 was identical with that of HCV-GII. Therefore, our results strongly suggest that HCV-K2 is the same as HCV-GII. PMID- 1720310 TI - Promyelocytic leukemia cell line, HL-60, produces human hepatocyte growth factor. AB - The human promyelocytic leukemia cell line, HL-60, stimulated with PMA, produced human HGF-like immunoreactivity (HL-60 HGF), which was detected with human HGF specific ELISA. The purified HL-60 HGF was indistinguishable from human HGF in the plasma of patients with fulminant hepatic failure by studies of subunit constitution and amino acid composition. The HL-60 HGF mRNA corresponded to 6 kb, which was consistent with previous reported data in rat and human HGF mRNA, was detected in stimulated HL-60, by northern hybridization analysis using human HGF cDNA probe. These findings indicated that HL-60 HGF was identical to, or closely resembled, human plasma HGF. The HL-60 cell is an attractive model for studies of HGF-producing mechanisms, the manner of secretion and the nature of induction signals. PMID- 1720311 TI - CFTR: development of high- affinity antibodies and localization in sweat gland. AB - Rabbit antisera were raised to six synthetic peptides corresponding to amino acid sequences contained in the protein product of the cystic fibrosis gene, CFTR. For two peptides, [Lys102]CFTR(102-116) and CFTR(1468-1480), antibody-peptide binding was of high affinity in that half-maximal binding occurred at peptide concentrations below 10 nM. Monospecific antibodies were prepared using these peptides, and these antibodies were used to stain human skin. Specific staining was detected in the cells lining the reabsorptive duct of the sweat gland. Within these lumenal cells, staining was most prominent at the apical domain but was also detected near the basolateral surface. This finding agrees well with predictions based on the effects of cystic fibrosis on sweat gland function, and suggests that these antibodies will be useful for studying CFTR in other human tissues. PMID- 1720312 TI - Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia. AB - L-glutamate, N-methyl-D-aspartate (NMDA), kainate, quisqualate and sodium nitroprusside increased cyclic GMP (cGMP) level on rat whole brain cell culture. The accumulation of cGMP evoked by L-glutamate was inhibited by a NMDA antagonist MK-801, an inhibitor of guanylate cyclase methylene blue and two nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine (NO2Arg). The inhibition of L-NMMA on cGMP level was reversed partially by addition of L-arginine. Although MK-801 was able to protect cells from neuronal injury induced by L-glutamate or by 5 h hypoxia, L-NMMA and NO2Arg were ineffective. The present study suggests that cGMP elevation mediated by NO following activation by L-glutamate is not involved in neuronal cell injury. PMID- 1720314 TI - Retinoic acid decreases thyroid hormone receptor expression in pituitary GH1 cells. AB - Retinoic acid produced a time and dose-dependent depletion of thyroid hormone receptors in GH1 cells without modifying their affinity for triiodothyronine (T3). A maximal decrease (50-70%) was obtained after 24-48 h incubation with 5-10 microM retinoic acid. Treatment with 0.8 nM T3 for 24 h caused a similar reduction and did not potentiate the decrease produced by these concentrations of retinoic acid. However, the combination of sub-maximally effective doses of both ligands had an additive effect on receptor levels. The reduction of receptor caused by retinoic acid is accompanied by a decreased expression of c-erbA alpha 1 and alpha 2 mRNAs, but the retinoid did not reduce the abundance of c-erbA beta mRNA. In contrast, T3 decreased the levels of both alpha and beta transcripts. PMID- 1720313 TI - Sequence conservation of the catalytic regions of amylolytic enzymes in maize branching enzyme-I. AB - We have identified cDNA clones encoding branching enzyme-I (BE-I) from a maize kernel cDNA library. The combined nucleotide sequence of the cDNAs indicates that maize BE-I is initially synthesized as a precursor protein with a putative 64 residue transit peptide at the amino terminus, and that the mature enzyme contains 759 amino acid residues with a calculated molecular mass of 86,236 Da. The four regions, which constitute the catalytic site of amylolytic enzymes, are conserved in the sequences of BE-I and bacterial branching enzymes. This result demonstrates that branching enzyme belongs to a family of the amylolytic enzymes. The BE-I gene is highly expressed in the early stages of kernel development, and the level of the message concentration decreases slowly as kernel maturation proceeds. PMID- 1720315 TI - The tilorone-induced mucopolysaccharidosis in rats. Biochemical investigations. AB - The tissues of rats chronically treated with tilorone exhibited a significant accumulation of acid glycosaminoglycans (GAGs): In the liver, the GAG concentration was found to be elevated by a factor of 38, in the spleen by a factor 15 and in the kidneys by a factor of 5. Furthermore, the renal excretion of GAGs was increased 32-fold as compared to control animals. Dermatan sulphate was predominant among the GAGs stored in the three organs; chondroitin sulphate and heparan sulphate were found in smaller amounts. GAG storage was accompanied by accumulation of the drug within the tissues: the molar ratio of tilorone per disaccharide unit of GAG was calculated to be one to two in each tissue. According to previous reports, tilorone-induced mucopolysacchariodosis is due to impaired lysosomal degradation of GAGs. The present results give support to the hypothesis that an interaction between the polyanionic GAGs and the dicationic drug may lead to GAG-drug complexes which cannot be digested by lysosomal enzymes. PMID- 1720316 TI - Detection of cerebrospinal fluid antibodies against myelin basic protein in patients with AIDS dementia complex. AB - We evaluated cerebrospinal fluid (CSF) antibody levels against a lipid-free, denatured form of myelin basic protein (LF-MBP) in 11 patients with AIDS dementia complex (ADC) by using an enzyme-linked immunosorbent assay (ELISA). In 9 out of 11 patients, anti-LF-MBP antibody levels were significantly higher than those observed both in 15 human immunodeficiency virus (HIV)-infected patients without neurological disorders and in 9 anti-HIV-negative subjects affected by other neurological diseases. Furthermore, we followed up anti-MBP levels in 5 out of the 11 ADC patients and detected a strict relationship with the encephalopathy progression. At the same time, with the aim to detect early demyelinating events we investigated CSF antibody levels against a lipid-bound, native-like form of MBP (LB-MBP). Results did not show any significant difference between LF-MBP and LB-MBP in terms of antibody reactivity. The detection of anti-MBP antibodies in CSF may provide the opportunity to assess a diagnostic tool for discovering demyelinating lesions in ADC patients. PMID- 1720317 TI - Serum levels of alpha-1 microglobulin and beta-2 microglobulin in bone marrow transplant recipients treated with cyclosporin A. AB - The levels of alpha-1 microglobulin (alpha 1m) and beta-2 microglobulin (beta 2m) in serum were estimated in 77 bone marrow transplant recipients. In comparison to pre-transplant levels, the highest levels of alpha 1m and beta 2m were found during impairment of renal function, i.e., during cyclosporin-induced nephrotoxicity and during treatment with other nephrotoxic drugs (P less than 0.001). The alpha 1m levels were less elevated during infections and acute graft versus-host disease (P less than 0.01), while beta 2m levels were markedly elevated during the same conditions (P less than 0.001). The linear correlations between serum creatinine and alpha 1m and creatinine and beta 2m were r = 0.7 and 0.8, respectively (P less than 0.001). The overall correlation between alpha 1m and beta 2m was 0.4 (P less than 0.001). It is concluded that alpha 1m might be a complement to serum creatinine levels in monitoring renal function after bone marrow transplantation. PMID- 1720318 TI - Does antibody-dependent epitope masking permit progressive tumour growth in the face of cell-mediated cytotoxicity? AB - It is not clear how immunogenic tumours can grow from small inocula in syngeneic hosts that mount both T-cell and B-cell responses. In this article Lionel Manson argues that antibody coats the tumour cells, protecting them from attack by cytotoxic lymphocytes, and thus permits the tumour to continue to grow and overwhelm the host. These observations may explain the paradox that an immunogenic tumour overwhelms the tumour-bearing host in the face of an ongoing anti-tumour immune response. PMID- 1720319 TI - Immunohistochemical localization of coagulation, fibrinolytic and antifibrinolytic markers in adenocarcinoma of the lung. AB - Extravascular coagulation and fibrinolysis are intimately involved in and modulate cancer cell growth, invasion and metastasis. Samples from resection specimens of patients with primary lung cancer (adenocarcinomas) were tested with monoclonal (MAb) and polyclonal (PAb) antibodies against various factors of the coagulation or fibrinolysis systems, or against antigens of inflammatory or proliferating cells. MAb Ki-67 specific to nuclear antigens of proliferating cells showed a distinct but variable staining of cell nuclei throughout the tumor tissue. Nests of tumor tissue stained with cytokeratin-specific antibodies (PKK1), whereas other parts were negative. Fibrin(ogen) and fibronectin were found throughout the tumor tissue stroma and in the alveolar lining, and the most densely stained areas were at the transition zone between normal and tumor tissue. Fibrinolytic system components like tissue plasminogen activators (t-PA), and urokinase (u-PA), and their inhibitors PAI-1 and PAI-2 were all studied. All specimens were negative for t-PA (except endothelial linings), whereas urokinase specific antibodies stained loosely packed tumor cells and macrophages within the tumor stromal tissue and alveolar septa. Both PAI-1 and PAI-2 were most prominently expressed within interstitial and alveolar macrophages. A weaker staining of tumor tissue cells was demonstrated. Inflammatory cells like macrophages and T lymphocytes were located in aggregates or diffusely spread within tumor stromal tissue. The inflammatory reaction was most intense at the border between normal lung and tumor tissue. PMID- 1720320 TI - Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization. AB - Gene expression for tropoelastin, the proprotein for elastin, was examined in the rat lung from 17 days of gestation (pseudoglandular stage) to adulthood by in situ hybridization using a rat-specific 35S-radiolabeled riboprobe. The tropoelastin message was present in vascular and airway smooth muscle, endothelial, septal interstitial, alveolar wall, and mesothelial cells but not in epithelial cells. With alveolar septal formation, the message in the interstitium increased progressively from 17 days of gestation, reaching a peak at 7 to 11 days postnatal. The signal in the arterial walls, in contrast, peaked between 19 days of gestation to 1 day postnatal and thereafter declined first from the outer media. The signal in general declined significantly by 21 days postnatal, and elastogenesis was virtually absent in the adult. These results support the idea that tropoelastin gene expression in the interstitium is closely associated with the centripetal progression of alveolarization, and the early postnatal decrease of tropoelastin expression in blood vessels corresponds with the sudden postnatal changes in the pulmonary hemodynamics. Furthermore, in the rat fetus and neonate, endothelial cells expressed the gene for tropoelastin and hence probably play a significant role in the formation of internal elastic lamina in vivo. PMID- 1720321 TI - A phase II study of treatment of painful multifocal skeletal metastases with single and repeated dose samarium-153 ethylenediaminetetramethylene phosphonate. AB - A phase II study of 153Sm ethylenediaminetetramethylene phosphonate (153Sm-EDTMP) palliative treatment was conducted on 23 patients with painful disseminated skeletal metastases. The administered activity of 153Sm-EDTMP was determined by prospective dosimetry and the radiation absorbed dose to bone marrow was fixed at 2 Gy. Symptomatic relief of bone pain was experienced by 14 of 23 evaluable patients (61%) with a median duration of 8 weeks (range 0-40). Toxicity was limited to myelosuppression with median nadir counts of leucocytes 3.3 x 10(9)/1 (range 1.0-7.5) and platelets 133 x 10(9)/1 (range 24-176) occurring at 2 weeks and 4 weeks, respectively. Retreatment with 153Sm-EDTMP was studied in 15 patients, including in 4 of the 23 patients treated with a single dose. The retreatment median radiation absorbed dose to red marrow was 1.9 Gy given at a median of 9 weeks (range 6-38) after initial treatment. Good control of pain was obtained in 13 of these patients (87%). Both the median duration of pain control (24 weeks) and survival (9 months) in the retreated group were substantially greater than for patients treated with a single dose, where duration was 8 weeks and survival 4 months. Additional toxicity in the retreated patients was confined to anaemia which required blood transfusion in 9 of the 15 patients (60%). PMID- 1720322 TI - Prediction of long-term gonadal toxicity after standard treatment for testicular cancer. AB - Long-term post-treatment gonadal toxicity was examined (median 3 years after treatment discontinuation) in 125 testicular cancer patients treated with standard regimens: no radiotherapy or chemotherapy (36 patients), infradiaphragmatic radiotherapy (38 patients), and 3-4 cycles of cisplatin-based chemotherapy (51 patients). Radiotherapy and chemotherapy had no impact on serum testosterone, but led to a slight increase in serum follicle-stimulating hormone (FSH). The lowest median value of post-treatment sperm cell count was observed after infradiaphragmatic radiotherapy, the highest value after standard chemotherapy. After more intensive cytotoxic treatment recovery of the gonadal function seemed to be delayed. In testicular cancer long-term post-treatment gonadal toxicity is correlated to the patient's pretreatment gonadal function and age rather than to the standard treatment of the malignancy. In patients with pretreatment normal gonadal function the risk of permanent treatment-induced toxicity is minimal after present standard treatment. PMID- 1720323 TI - Comparison of prostate specific antigen assays. PMID- 1720324 TI - Autoantibodies against platelet glycoprotein Ib/IX: a frequent finding in autoimmune thrombocytopenic purpura. AB - Many autoantibodies involved in the pathogenesis of autoimmune thrombocytopenic purpura (AITP) are directed against epitopes on platelet glycoproteins (GP). These autoantibodies are a specific diagnostic characteristic of patients with AITP. In this study, the relative frequency of antibodies against GPs IIb/IIIa and Ib/IX was assessed in sera from 81 AITP patients with a glycoprotein-specific enzyme immunoassay (MAIPA assay) using monoclonal antibodies against these platelet GPs. All sera contained platelet-specific antibodies which had been detected by platelet immunofluorescence. Of the 81 antibodies tested, 58 (72%) reacted with at least one of the platelet GPs studied. Autoantibodies against GPIb/IX were as common as antibodies against the GPIIb/IIIa complex. The same ratio of specificities was observed on autologous platelets of an independent cohort of 29 patients. The epitope of three autoantibodies against GPIb/IX and of mab Gi10, a monoclonal antibody, which inhibits binding of these autoantibodies, was further characterized. Severity of thrombocytopenia was not related to the GP specificity of the autoantibody. The observation that in 23 (28%) of these sera the antigenic determinants could not be assigned to the glycoproteins under investigation suggests that platelet autoantibodies may react with other GPs or other membrane constituents, e.g. glycolipids. PMID- 1720325 TI - The Corfu delta beta thalassaemia mutation in Greece: haematological phenotype and prevalence. AB - The Corfu delta beta thalassaemia mutation, a 7.2 kb deletion partially removing the delta-globin gene and a single nucleotide mutation (G----A) at intervening sequence I (IVSI-n5) in the beta-globin gene in cis, was first described in a family from Corfu; the carriers for this mutation had the unusual haematological phenotype of heterozygous beta-thalassaemia with normal levels of HbA2. To investigate the frequency and haematological characteristics of Corfu delta beta thalassaemia in Greece we analysed 25 unrelated normal HbA2 type 2 beta thalassaemia heterozygotes and their 23 clinically affected offspring. Gene mapping demonstrated that nine (36%) of the 25 normal HbA2 beta-thalassaemia heterozygotes were in fact Corfu delta beta thalassaemia heterozygotes and of the 23 patients, two were Corfu delta beta thalassaemia homozygotes and five compound heterozygotes for Corfu delta beta thalassaemia and another beta-thalassaemia defect. Detailed haematological analysis demonstrated that: the Corfu delta beta thalassaemia mutation does not completely abolish the expression of the beta globin gene; the HbA2 levels are slightly lower (P less than 0.01) and the HbF levels slightly higher (P less than 0.01) in Corfu delta beta thalassaemia heterozygotes compared to beta-thalassaemia heterozygotes with the normal HbA2 type 2 phenotype who do not have the Corfu delta beta chromosome. PMID- 1720326 TI - Epitope mapping of the anti-human progesterone receptor monoclonal antibody, AB 52. AB - The monoclonal antibody AB-52 has high affinity and specificity for the two natural human progesterone receptor forms, receptor A (hPRA) and receptor B (hPRB), but it does not bind the PR of chick, mice, rats or rabbits. We have used a novel method to map its epitope. Based on a series of site-directed mutants of hPRA, together with immunoblotting, DNA gel mobility shift and antibody super shift assays, we have mapped the epitope of AB-52 to a 17 amino acid sequence lying between Val221 and Leu237 of the 933 amino acid hPRB protein. This N terminal sequence is common to both hPRB and hPRA but is missing in chick PR and differs extensively from mouse PR. No anti-rabbit PR antibodies map to the homologous rabbit PR sequence which differs from hPR by four amino acids, suggesting that one or more of these four amino acids form a critical subset of residues in hPR that define the human specificity of AB-52. Knowledge of the AB 52 epitope is useful in structural analysis of PR, and in competition studies. Additionally, this 17 amino acid peptide whose antigenicity would not be predicted from computer analysis, should be useful for generating additional hPR specific antibodies. PMID- 1720327 TI - Analysis of oestrogen receptor dimerisation using chimeric proteins. AB - Sequences essential for dimerisation have been identified in the hormone binding domain of the mouse oestrogen receptor by insertional and point mutagenesis and sequence comparisons reveal that equivalent residues may be conserved in other members of the nuclear hormone receptor superfamily. To assess functional compatibility of this region between members of the receptor superfamily, peptide sequences corresponding to the equivalent regions of the human androgen receptor and retinoic acid receptor have been substituted for the dimerisation domain of the mouse oestrogen receptor. The resulting chimeric proteins were analysed for high affinity DNA binding using a gel retardation assay and shown to bind with reduced affinity compared to the wild type oestrogen receptor. The reduction in DNA binding observed may result from the intramolecular incompatibility of functional elements within the hormone binding domain of nuclear hormone receptors. PMID- 1720328 TI - [Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex]. AB - The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720329 TI - [Establishment and characterization of a human undifferentiated carcinoma cell line (HMG)]. AB - The undifferentiated carcinoma cell line (HMG) was established from a nude mouse tumor which had been produced by transplantation of a intraperitoneal tumor of 27 year-old woman. The HMG cell line has the following biological properties. 1. The HMG cells are round to oval in shape and grow as floating cell aggregates like a rouleau or a cluster of grapes. 2. 100 passages have been carried out over a year, and the population doubling time is about 17 hrs. 3. In the original tumor, keratin and vimentin were expressed simultaneously, in HMG cells, however, only localization of vimentin was confirmed. 4. By chromosomal analysis, over 90% of the cells revealed 46, XX, with no karyological abnormalities, at passage 82. 5. When heterotransplanted into the subcutis of a nude mouse, HMG cells produced a undifferentiated carcinoma resembling the original tumor. PMID- 1720330 TI - Low-pressure perfusion results in effective microvascular perfusion of isolated rabbit hearts during hypothermic preservation for twenty-four hours. AB - Hypothermic, low-pressure coronary artery perfusion with oxygenated electrolyte solutions containing oncotic agents has resulted in successful orthotopic transplantation of hearts after extended preservation periods. Coronary flow may not, however, be consistently maintained during preservation. Previous research has demonstrated that coronary flow during preservation is related to contractile function after preservation. Specific mechanisms leading to reduced coronary flow during preservation remain undefined. This study was designed to determine whether low-pressure perfusion is a mechanism for decreased coronary flow and decreased microvascular perfusion during preservation. With India ink used as a marker of flow, microvascular perfusion was measured in rabbit hearts immediately after isolation (controls) or after 24 hours of hypothermic perfusion at 13 mm Hg (preserved hearts). There were no differences in the percentage of perfused microvessels in the control hearts perfused at either 13 or 80 mm Hg (94% +/- 2% and 99% +/- 1%, respectively). Nor was there a difference in the percentage of perfused microvessels in hearts perfused at either 13 or 80 mm Hg after 24 hours of hypothermic, low-pressure perfused preservation (67% +/- 9% and 74% +/- 6%, respectively). There was a significant difference, however, between the percentage of perfused microvessels in control hearts and in hearts preserved for 24 hours. These differences were independent of the pressure at which the India ink solution was administered. A perfusion pressure of 13 mm Hg is as effective as is 80 mm Hg in providing perfusion of the coronary microvascular beds during long-term hypothermic perfused preservation. PMID- 1720331 TI - Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line. AB - NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system. PMID- 1720332 TI - Comparative analysis of the influences of IL-1, IL-3 and GM-CSF on the commitment of granulocyte-macrophage progenitors in vitro. AB - Our experiments were directed towards the detection of the influence of interleukin-1 (IL-1); interleukin-3 (IL-3), and granulocyte-macrophage colony stimulating factor (GM-CSF) on the generation of granulocyte-macrophage progenitor cells. We also set out to examine whether this process is connected with changes within the early precursor cell compartment. Bone marrow suspension cultures (12 days) supplemented with these cytokines were tested for the presence of GM colony-forming cells (GM-CFC) in a colony-forming unit assay. The percentage of CD34+ and HLA-DR+ as well as the number of blasts and promyelocytes were estimated cytofluorometrically and morphologically. The proliferative effect of GM-CSF was associated with a net increase of GM-CFC and HLA-DR+ myeloid cells and a decrease in the percentage of CD34+ early precursor cells. IL-3 acted similarly and also caused an absolute decrease of CD34+ cells in the cultures. IL 1 did not stimulate the generation of blasts or GM-CFC but elevated the number of CD34- as well as HLA-DR-expressing cells in the cultures. These results imply that GM-CSF supported the maintenance of hematopoiesis in vitro. The transition from early precursor cells to committed myeloid progenitor cells (GM-CFC) and more mature precursor cells (G-CFC, M-CFC) may be supported by GM-CSF without affecting the self-renewing capacity of CD34+ early precursors. In contrast, the blast-generating and proliferation-inducing action of IL-3 is associated with a drop in the total number of CD34+ stem cells. An efficient renewal of this population obviously depends on the presence of IL-1. PMID- 1720333 TI - Stimulation of insulin release from pancreatic islets by quinones. AB - Coenzyme Q (CoQ0) and other quinones were shown to be potent insulin secretagogues in the isolated pancreatic islet. The order of potency was CoQ0 congruent to benzoquinone congruent to hydroquinone-menadione. CoQ6 and CoQ10 (ubiquinone), duroquinone and durohydroquinone did not stimulate insulin release. CoQ0's insulinotropism was enhanced in calcium-free medium and CoQ0 appeared to stimulate only the second phase of insulin release. CoQ0 inhibited inositol mono , bis- and trisphosphate formation. Inhibitors of mitochondrial respiration (rotenone, antimycin A, FCCP and cyanide) and the calcium channel blocker verapamil, did not inhibit CoQ0-induced insulin release. Dicumarol, an inhibitor of quinone reductase, did not inhibit CoQ0-induced insulin release, but it did inhibit glucose-induced insulin release suggesting that the enzyme and quinones play a role in glucose-induced insulin release. Quinones may stimulate insulin release by mimicking physiologically-occurring quinones, such as CoQ10, by acting on the plasma membrane or in the cytosol. Exogenous quinones may bypass the quinone reductase reaction, as well as many reactions important for exocytosis. PMID- 1720334 TI - Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena. AB - Cellular events are accomplished by the coordinated interactions of cellular components within the three-dimensional context of a cell. Simultaneous observation of multiple components in three dimensions can be essential for understanding such interactions. Toward this end, we have developed a computerized microscope workstation capable of recording three-dimensional images of multiple cellular components in fixed and living cells. All aspects of microscope control, data collection, image processing and analysis can be performed on the one workstation. In this report, we describe the components and capabilities of this integrated system. In addition, we discuss some general problems of multiple-wavelength, three-dimensional imaging and our application of this technology to the analysis of chromosome organization in Drosophila melanogaster. Three-dimensional imaging of fixed embryos stained by indirect immunofluorescence has revealed the structural organization of chromosomes, microtubules, and the nuclear lamins. Imaging of living embryos injected with fluorescently labelled proteins has confirmed and extended these results by allowing the study of these structures throughout the cell cycle. The combination of the molecular specificity of fluorescence microscopy and the three-dimensional structural information obtained by our workstation has provided novel insights into the dynamic aspects of chromosome behavior during the cell cycle. We believe this system has many important applications in the study of the molecular basis of cellular events. PMID- 1720335 TI - Probing the structure and function of the nuclear pore complex. AB - Nucleocytoplasmic transport is a bi-directional process mediated by the nuclear pore complex (NPC), which results in a segregation of cytoplasmic and nuclear macromolecules within cells. Some progress has been made in understanding the mechanistic basis of this selective transport phenomenon. In particular, cryo electron microscopy of frozen-hydrated nuclear envelopes coupled with image processing and labeling studies, has provided a glimpse of the transporter at the center of the NPC. PMID- 1720336 TI - Acute plasmapheresis during cardiac surgery: volume replacement by crystalloids versus colloids. AB - Acute plasmapheresis (APP) is an additional tool for blood conservation during cardiac surgery. In a randomized study of 60 aortocoronary bypass patients undergoing APP, the influence of replacement of the withdrawn autologous plasma (10 mL/kg) by either colloids (low molecular weight hydroxyethyl starch solution [6% HES 200/0.5]) or crystalloids (Ringer's solution) was investigated. APP was performed by means of a centrifugation technique producing platelet-poor plasma. During and after cardiopulmonary bypass (CPB), either a cell saver (CS) or a hemofiltration (HF) device was also used for blood concentration. Almost three times as much crystalloid as HES solution was necessary for replacement of autologous plasma. Fluid balance during CPB was significantly more positive in the crystalloid patients, particularly when a CS was used. Blood loss was highest in the crystalloid patients in whom a CS was used in addition to APP, and these were the only patients who needed packed red cells. The platelet count, AT-III and fibrinogen plasma concentrations, colloid osmotic pressure, albumin, and total protein were significantly less compromised in the patients with colloid volume replacement. These parameters were closest to control values in patients receiving colloid replacement and HF. It is concluded that colloid is preferred for replacement of autologous plasma withdrawn by APP, and HF is superior to the CS when the combined technique for blood conservation is used. PMID- 1720337 TI - Double minute chromosomes and homogeneously staining regions in tumors taken directly from patients versus in human tumor cell lines. AB - There is increasing evidence that copies of amplified oncogenes or drug-resistant genes located on extrachromosomal DNA (e.g. double minutes and/or episomes) can be eliminated from mammalian tumor cell lines by treatment of the cells with low concentrations of hydroxyurea. However, amplified oncogenes or drug-resistant genes located in an intrachromosomal site (such as in a homogeneously staining region (HSR)) cannot be eliminated from the cells. A question which arises is do primary human tumors have extrachromosomal DNA present often enough to make elimination of that extrachromosomal DNA a potentially important therapeutic strategy? To address that question we have reviewed published cytogenetic analyses of 200 tumors taken directly from patients to determine the percentage of primary human tumors which have amplified genes present on extrachromosomal DNA (present in the form of double minutes [DMs]) vs the percentage of tumors which have amplified genes located on an intrachromosomal site (in the form of HSRs). Of the 200 primary human tumors reviewed, 91% contained DMs only, 6.5% contained HSRs, and 2.5% contained both. Of interest, in a parallel review of 109 cell lines with cytogenetic and/or molecular evidence of gene amplification, 60.6% contained DMs, 26.6% contained HSRs, and 12.8% contained both. These data indicate that DMs are the predominant cytogenetic marker for gene amplification in patients, but are present less frequently in established cell lines. These findings indicate that ongoing efforts to eliminate amplified drug-resistant genes or oncogenes contained on DMs (or precursors of DMs) from tumor cells may be relevant for in vivo situations. PMID- 1720338 TI - HLA typing using IL-2 activated T lymphocytes: usefulness in pediatric candidates for allogeneic bone marrow transplantation. AB - Following a preliminary study in healthy blood donors, we have performed serological HLA-A, B, C, DR and DQ typing using recombinant IL-2 activated T lymphocytes (IL-2.aTLs) in pediatric candidates for allogeneic bone marrow transplantation. In such patients, it is often difficult to obtain the quantity of lymphocytes required for HLA typing, particularly for class II typing using B lymphocytes, considering the timing of sampling and the volume of blood to be collected. Peripheral blood mononuclear cells (PBMCs) were activated and expanded with IL-2 until a sufficient number of IL-2.aTLs of good viability were available for the typing. In the first 10 cases, analyses of surface markers (CD2, CD20, CD25, CD36, HLA-DR and HLA-DQ, CD2/HLA-DR: two color) of IL-2.aTLs were done using flow cytometry at the time of HLA typing and indicated that IL-2.aTLs expressed HLA-DR and DQ antigens sufficient for evaluation. A small number (less than 10(6] of fresh or cryopreserved PBMCs, even those containing leukemic blast cells, were sufficient to induce and expand IL-2.aTLs for HLA typing. To date we have been able to successfully HLA-A, B, C, DR and DQ type 20/20 pediatric candidates. The HLA antigens identified on the patients' IL-2.aTLs were confirmed by a family study. PMID- 1720339 TI - Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in serum in bone marrow transplanted patients. AB - Colony-stimulating factors (CSF) are being increasingly used to accelerate hematopoietic recovery after bone marrow transplantation. To study the endogenous serum levels of CSF in bone marrow transplanted patients we have used immunoassays measuring granulocyte-macrophage colony-stimulating factor (GM-CSF) with a sensitivity of 0.10 ng/ml and granulocyte colony-stimulating factor (G CSF) with a sensitivity of 0.05 ng/ml. Serum samples, taken from the conditioning treatment until engraftment, were analysed in 13 patients receiving allogeneic transplants and in eight patients receiving autologous transplants. Ten patients had acute myeloid leukemia, seven acute lymphoblastic leukemia, one acute undifferentiated leukemia, two non-Hodgkin's lymphoma and one multiple myeloma. Samples were taken 1-2 times before transplantation and 1-2 times per week after transplantation (median of 46 days in allotransplant recipients and 32 days in autotransplant recipients); 17% of the allogeneic transplanted patients and 35% of the autologous transplanted patients had detectable levels of G-CSF. In both types of transplantation the G-CSF concentrations were low: median 0.06 (range 0.05-0.14) and 0.08 (range 0.05-0.40) ng/ml respectively. GM-CSF was detected only in one analysed sample in all patients. There was no evidence of increased CSF levels related to engraftment or documented infections. PMID- 1720340 TI - Quality of care dependent on nurses' specialized skills. PMID- 1720341 TI - Effects of fibroblast growth factors and platelet-derived growth factor on food intake in rats. AB - In the present study, the relations between acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), platelet-derived growth factor (PDGF), and food intake were studied. When aFGF-, bFGF-, and PDGF-like activity in cerebrospinal fluid (CSF) was examined by bioassay, the activity of those factors significantly increased in postfeeding CSF, compared to prefeeding CSF. Injections of aFGF, bFGF, aFGF (synthetic amino-terminal peptide of aFGF), and PDGF into the third cerebral ventricle decreased food intake, and injections of anti-aFGF, anti-bFGF, and anti-aFGF antibodies into the lateral hypothalamus (LHA) increased food intake. The activity of LHA glucose-sensitive neurons was inhibited by electrophoretic application of aFGF. These results suggest that aFGF, bFGF and PDGF have in vivo physiological roles in the central nervous system, distinct from those as mitogens. PMID- 1720342 TI - [Detection of calcium in tissues using alizarin red S and optical properties of the reaction product]. AB - A histological method of staining calcium deposits in organs and tissues is presented. Staining at different pH provides a certain differentiation of the characteristics of calcium deposits. Further findings concern optical properties of the reaction product of staining observed at examination in polarized light, differential interference contrast (OPTON), and by fluorescence microscopy. The chemical nature of the Alizarin red S reaction product in sites of calcium deposits is discussed. PMID- 1720343 TI - Serum biochemical and histological changes in caerulein-induced acute pancreatitis in rats: a correlative study. AB - We investigated correlations between serum biochemical changes and pathological alterations in caerulein-induced acute pancreatitis in rats. Seven consecutive doses of caerulein consisting of 50 mu g/kg, were given intraperitoneally, in 40 male Sprague-Dawley rats at hourly intervals. Serum biochemical and histological changes of the rats were studied at hours 7, 9, 12, 18, 24 and 36 after the first injection, as well as day 3 and 5 respectively. Serum amylase in the controlled rats (n = 6) was 2549 +/- 221 U/L (mean +/- SE). It was elevated to twenty times that of the baseline value (52630 +/- 11397 U/L) at the seventh hour, declined to about two times the control (5520 +/- 800 U/L) at the 18th hour, and returned to the baseline level at the 24th hour after the first caerulein injection. Serum lipase, elevated since the 7th hour, reached its peak value (631 +/- 34 U/L) at the 12th hour, then declined abruptly to the baseline value (0 U/L) at the 24th hour, after the first injection. However, severe histological changes of the pancreas were apparent at the 7th hour, reaching maximal destruction at the 18th hour, with severe inflammatory changes at the 24th hour; all after the first injection. The frank inflammation did not subside until 5 days after the first injection. These results suggest that, in the case of acute pancreatitis, normalization of serum biochemistries does not indicate recovery of the pancreas from acute inflammation. PMID- 1720344 TI - Structure of the 021 antigen from Serratia marcescens. AB - Lipopolysaccharide was isolated from both phases of an aqueous-phenol extraction of defatted cell walls from the reference strain for Serratia marcescens serogroup 021. The product from the aqueous phase was of the R type, lacking a polymeric side-chain. The polymeric fraction of the lipopolysaccharide from the phenolic phase (the 021 antigen) had a disaccharide repeating-unit with the following structure: ----4)-alpha-D-Glcp-(1----4)-beta-D-ManpNAc-(1----. PMID- 1720345 TI - Maltotetraose-forming, amylase-mediated, p-nitrophenyl alpha- and beta maltopentaoside formation in an aqueous-organic solvent system: a substrate for human amylase in serum. PMID- 1720346 TI - Somatic antigens of pseudomonads: structure of the O-specific polysaccharide chain of Pseudomonas syringae pv. syringae (cerasi) 435 lipopolysaccharide. PMID- 1720347 TI - Somatic antigens of pseudomonads: structure of the O-specific polysaccharide chain of Pseudomonas syringae pv. lachrymans 7591 (serogroup IX) lipopolysaccharide. PMID- 1720348 TI - Somatic antigens of the pseudomonads: structure of the O-specific polysaccharide chain of Pseudomonas syringae pv. tabaci 225 (serogroup VIII) lipopolysaccharide. PMID- 1720349 TI - Somatic antigens of pseudomonads: structure of the O-specific polysaccharide chain of Pseudomonas gladioli pv. alliicola 8494 (serogroup X) lipopolysaccharide. PMID- 1720350 TI - Therapeutic recommendations for aphthous ulcerations in children. AB - The purpose of this article is to formulate a systematic means for treatment of recurrent aphthous ulcerations in children. Remedies showing the most merit and ease of use while at the same time exhibiting minimal side effects were evaluated for applicability in a systematic approach to the management of aphthous ulcerations. These remedies include nutritional and vitamin supplementation, topical anesthesia, and debriding, anti-inflammatory, and protective agents. A protocol for the management is presented. Both systemic and topical management techniques are outlined. Many products are available and their rationale and mechanism of action are described. PMID- 1720351 TI - [Ca2+]i recordings and the inactivation of the high-voltage activated Ca2+ currents in the adult rat sensory neuron. AB - Fast, single cell, measurement of the average cytosolic [Ca2+]i with the Fura-2 technique suggests that the depolarization induced [Ca2+]i rise is entirely due to entry through the voltage-activated Ca2+ channels. Involvement of a Ca(2+) induced Ca(2+)-release process is not evident. Under physiological cytosolic buffering the current-induced [Ca2+]i rise persists for seconds and decays exponentially (tau = 7 s). Analysis of the [Ca2+]i changes during two-pulse protocols indicates that the purely voltage-dependent inactivation of the high voltage-activated (HVA) channels, in the range -80/+70 mV, is a slow process (0.2 1 s) which removes at most 40% of the current. On the contrary, Ca(2+)-dependent inactivation acts in a fast way and it is therefore responsible for the fast inactivating phase of the current; this phase disappears under sustained [Ca2+]i loads, and reappears when redistribution of free Ca2+ takes place. A suitable correction may be devised to compensate for the Ca(2+)-dependent inactivation. PMID- 1720352 TI - Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. AB - Pemphigus vulgaris (PV) is a life-threatening skin disease in which autoantibodies against a keratinocyte cell surface 130 kd glycoprotein, PV antigen (PVA), cause loss of cell-cell adhesion, with resultant epidermal blisters. We used affinity-purified PV IgG to isolate cDNA, containing the entire coding sequence for PVA, from human keratinocyte expression libraries. Northern blot analysis indicated PV mRNA expression only in stratified squamous epithelia. The deduced amino acid sequence of PVA was unique but showed significant homology with members of the cadherin family of Ca(2+)-dependent cell adhesion molecules, most markedly to desmoglein I. These findings demonstrate that a novel epithelial cadherin is the target of autoantibodies in PV. PMID- 1720353 TI - prospero is expressed in neuronal precursors and encodes a nuclear protein that is involved in the control of axonal outgrowth in Drosophila. AB - Neurogenesis in Drosophila begins with the formation of neuronal precursors, which give rise to neurons of individual identity. To find out whether there are genes that are expressed in most or all neuronal precursors and are involved in controlling particular aspects of neuronal differentiation, we used the enhancer trap method to screen for such "neuronal precursor genes." One gene of this group is prospero. Our mutant analysis indicates that prospero regulates other neuronal precursor genes and is essential for the axonal outgrowth and pathfinding of numerous central and peripheral neurons. prospero encodes a large nuclear protein with multiple homopolymeric amino acid stretches and is expressed in neuronal precursors early during their formation. It is probably generally required for proper neuronal differentiation. PMID- 1720354 TI - RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos. AB - In Drosophila embryos, graded activity of the posterior determinant nanos (nos) generates abdominal segmentation by blocking protein expression from maternal transcripts of the hunchback (hb) gene. When active inappropriately at the anterior pole, nos can also block expression of the anterior determinant bicoid (bcd). We show that both regulatory interactions are mediated by similar sequences in the 3' untranslated region of each transcript. These nos response elements (NREs) are both necessary and sufficient to confer nos-dependent regulation, the degree of regulation determined by the number and quality of the elements and the level of nos in vivo. Based on these and other results, we argue that nos acts as a morphogen, controlling hb expression (and hence abdominal pattern) as a function of its concentration-dependent interaction with the NREs. PMID- 1720355 TI - Nuclear targeting of the transcription factor PTF1 is mediated by a protein subunit that does not bind to the PTF1 cognate sequence. AB - The pancreas-specific transcription factor PTF1 is a heterooligomer that exists as two variants, alpha and beta, both of which bind DNA. The nucleus contains exclusively alpha while the cytoplasm contains both forms. Alpha and beta differ in protein composition. Reconstitution of alpha in vitro requires, in addition to the DNA-binding subunits common to both forms, a 75 kd glycosylated protein that apparently does not bind DNA. Here we show that this protein is essential for targeting PTF1 to the nucleus. Upon injection into frog oocytes, alpha is translocated quantitatively to the nucleus while beta remains in the cytoplasm. However, if beta is coinjected with purified 75 kd protein or a particular size fraction of pancreatic mRNA, it can be converted to alpha and imported into the nucleus. PMID- 1720356 TI - Influenza surveillance. AB - The main objectives of influenza surveillance are: collection of influenza virus isolates and analysis of their antigenic characteristics so that the most appropriate virus variants can be recommended as constituents of influenza vaccines for use during the next epidemiological season; collection and analysis of information on influenza morbidity and mortality; and earliest possible detection of influenza epidemics. Exact estimates of the specific morbidity and mortality due to influenza are now being carried out in only certain countries. Simple notification of clinical cases and deaths without laboratory confirmation is unsatisfactory and leads to errors in interpretation. The methods used to predict baseline mortality may be inaccurate, resulting in underestimation of mortality associated with influenza virus infection. Comparison of the impact of influenza in different countries is also difficult owing to a variety of methods used for the estimation of mortality and morbidity. Although the laboratory aspects of influenza epidemiology are more uniformly covered worldwide than the statistical aspects, it is still necessary to increase laboratory coverage of some parts of the world and to improve the techniques for the isolation and characterization of not only influenza viruses but also other acute respiratory viruses. The systems and methods for influenza surveillance should be improved and standardized. PMID- 1720357 TI - [Experimental study of schistosome immune RNA on transfer of cellular and humoral immunity to normal mice]. AB - The present paper reported the extraction of immune RNA (iRNA) from spleen cells of Schistosoma japonicum infected rabbits and the activity of this schistosome iRNA (SiRNA) on transfer of cellular immunity and humoral immunity using macrophage migratory inhibition test (MMIT) and Dot-ELISA. The results indicated that specific cellular immunity could be transferred to normal mice by SiRNA either via in vivo or in vitro sensitization. Intraperitoneal inoculation of SiRNA to normal mice may result in production of specific antibodies. PMID- 1720358 TI - Coronary vascular response to endothelin in isolated perfused hearts of spontaneously hypertensive rats. AB - 1. The coronary vasoconstrictive response to endothelin (ET-1) was evaluated using the isolated perfused hearts of 15 week old spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY). Endothelin produced marked increases in perfusion pressure (PP) in both SHR and WKY. The effects of ET-1 were more potent than those of acetylcholine, vasopressin and angiotensin II. The vascular response to ET-1, expressed as the increase in PP, was greater in SHR than in WKY. 2. Nicardipine (10(-8) mol/L) shifted the concentration-PP response curve for ET-1 to the right. The extent of the rightward shift was greater in SHR than in WKY. Additionally in SHR, Bay K-8644 elicited a dose-dependent increase in PP, the effect being more potent than that in WKY. 3. The increased response of the coronary vasculature to ET-1 was observed after 15 weeks of age but not at 6 weeks, indicating that enhancement of the response develops with ageing in SHR. 4. Enhancement of the vascular response to ET-1 in SHR was prevented by chronic (10 weeks) treatment with enalapril (10 mg/kg per day), but not by hydralazine (30 mg/kg per day). 5. These results indicate that the coronary vascular response to ET-1 increases with age in SHR. The mechanism of the enhanced response may involve the activation of dihydropyridine-sensitive Ca2+ channels, however, this type of mechanism may also be modulated at least in part by the renin-angiotensin system. PMID- 1720359 TI - A B cell line from a patient with pure red cell aplasia produces an immunoglobulin that suppresses erythropoiesis. AB - A 4-year-old boy with pure red cell aplasia was investigated. Immunophenotypic analysis of peripheral blood lymphocytes revealed a marked increase of CD20+ cells, which fell from 25.9% in the active stage to 9.7% in remission. The plasma contained a suppressive activity against CFU-e and BFU-e formation by the patient's bone marrow cells, which disappeared when the disease went into remission. Prednisone (2 mg/kg/day) therapy was tried for 5 weeks, but produced no improvement. Subsequently, high-dose gamma-globulin therapy induced complete remission of anemia. A lymphoblastoid B cell line obtained from the patient before therapy produced a factor that suppressed erythropoiesis but not granulopoiesis. The suppressive activity resided in the immunoglobulin fraction and was adsorbed by an anti-immunoglobulin column. These results indicate that expansion of B cells producing an immunoglobulin which suppressed erythropoiesis was involved in the pathogenesis of the disease in this patient. PMID- 1720361 TI - Abnormalities in CD4+ T lymphocyte subsets in patients with common variable immunodeficiency. AB - In order to investigate whether deficient immunoglobulin production in common variable immunodeficiency (CVI) patients was related to defective T cells functions, phenotype and proliferative responses to mitogen of peripheral blood mononuclear cells (PBMC) were investigated in 9 patients with CVI. The results were compared to those of 12 age- and sex-matched normal controls. The numbers of CD3+ and CD8+ T cells in the patients were not different from those in the control group, but the numbers of CD4 T cells were decreased (511 +/- 237 vs 844 +/- 247/mm3; P less than 0.01). The decrease in CD4 T cells was due to a dramatic deficiency in the CD4+ CD45RA+ subset, observed as an absolute value of blood lymphocytes (126 +/- 91 vs 384 +/- 142; P less than 0.001) and as a percentage (9.0 +/- 7.1 vs 18.8 +/- 5.0; P less than 0.01). In contrast, the CD4+ CD29+ T cell subset was not different in CVI from those in the control group. Moreover, there was a strong positive correlation between the number of percentages of CD4+ CD45RA+ blood T cells and the proliferative response of PBMC to PHA (respectively, P less than or equal to 0.02 and P = 0.05) and to Con A (P less than or equal to 0.02). The decrease of CD4+ CD45RA+ T cells could reflect an abnormality in the physiological status of T cells and could be of critical importance in the antibody deficiency. PMID- 1720360 TI - Antigenic domains on the U1 small nuclear ribonucleoprotein-associated 70K polypeptide: a comparison of regions selectively recognized by human and mouse autoantibodies and by monoclonal antibodies. AB - Antigenic regions on the U1 small nuclear ribonucleoprotein (snRNP)-associated 70K polypeptide recognized by human and mouse autoantibodies or by monoclonal antibodies were identified and compared. Using a set of 70K fusion proteins as antigen in enzyme-linked immunosorbent assay and immunoblotting revealed that serum autoantibodies of human and of MRL/Mp mouse origin recognized a common region of the 70K polypeptide. Monoclonal anti-70K antibodies derived from a patient with mixed connective tissue disease, from an autoimmune MRL/Mp mouse, and from a BALB/c mouse immunized with purified U1 snRNP were all shown to bind to a part of the 70K polypeptide rich in charged residues and different from the region recognized by most human and MRL/Mp mouse serum autoantibodies. PMID- 1720362 TI - Changes in serum protein profile of healthy adult Nigerians after three decades. AB - Serum protein profile was recently determined in 55 healthy adult Nigerians and compared with values obtained 3 decades ago. The total serum protein levels were similar, but an increase in titre was recorded in albumin, alpha 1-globulin and beta-globulin. The gamma-globulin level fell markedly in the present study, suggesting improvement in health care and quality of life in the last 3 decades. Changes seen in the serum protein pattern of healthy adult Nigerians were within the normal range compared with values recorded in other Africans and Black Americans. These changes should be considered in the interpretation of laboratory results. PMID- 1720363 TI - Microbumintest and non-albumin proteinuria in diabetes. AB - Bedside methods for the detection of microalbuminuria such as Microbumintest (TM) have the advantage of simplicity but not the specificity of radio-immunoassay. In the present study we assessed whether apparently inappropriate positive Microbumintest results in the presence of low urinary albumin concentrations could be accounted for by non-albumin proteinuria of glomerular or renal tubular origin. Urinary albumin and transferrin were considered to indicate glomerular proteinuria, and alpha-1-microglobulin and N-acetyl-beta-D-glucosaminidase to reflect tubular proteinuria. Microbumintest had a sensitivity of 100% and specificity of 67% to detect a urinary albumin concentration of 40 mg/l. Samples with albumin concentration less than 40 mg/l contained more total protein: 110 (78-155) v 60 (35-104) mg/l p less than 0.0001 (geometric means with 1 SD range), more albumin: 11.7 (5.1-26.8) v 5.4 (2.8-10.4) mg/l p less than 0.005 and more transferrin: 496 (191-1284) v 174 (78-389) micrograms/l p less than 0.001, in those testing positive with Microbumintest than in those testing negative. Microbumintest had a sensitivity of 82% and specificity of 75% to detect an albumin concentration of 20 mg/l. In samples containing less than or equal to 20 mg/l albumin, the mean albumin concentration was no greater in those testing positive compared with those testing negative. However, total protein: 108 (72 161) v 60 (34-105) mg/l p less than 0.001, and transferrin: 326 (148-715) v 157 (78-316) micrograms/l p = 0.01 both remained increased in samples testing positive compared with those testing negative.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720364 TI - Hypothalamic regulatory peptide disturbances in the spontaneously obese JCR: LA corpulent rat. AB - Central and lateral hypothalamic concentrations of 9 regulatory peptides implicated in the control of feeding behaviour were measured in corpulent (cp/cp) JCR:LA-cp rats which develop spontaneous obesity, hyperinsulinaemia and hyperlipidaemia, and in lean (+/?) controls. In female cp/cp rats, central hypothalamic levels of neuropeptide Y (NPY), neurotensin, somatostatin and substance P were significantly lower (p less than 0.02) than in lean female controls. Following food restriction with a 16% reduction in body weight, these differences were apparently reversed and there were also significant rises in the lateral hypothalamic concentrations of neurotensin and of galanin. The other 4 peptides examined (bombesin, calcitonin gene-related peptide, neuromedin B and vasoactive intestinal peptide) did not differ significantly between cp/cp and lean females, either fed freely or food-restricted. Male cp/cp rats showed no significant differences from lean males in central or lateral hypothalamic concentrations of any of the 9 peptides. NPY and galanin are powerful and specific central appetite stimulants, whereas neurotensin, substance P and somatostatin inhibit feeding when injected centrally. Disturbances in these putative appetite-regulating peptides may be involved in the hyperphagia and other hypothalamic abnormalities in this spontaneous obesity syndrome. The apparent absence of differences between the male corpulent and lean groups may relate to sexual dimorphism of the syndrome, which is more marked in the females. PMID- 1720365 TI - Preparation and characterization of a monoclonal antibody against bovine CD5 lymphocyte surface antigen. AB - The M1 monoclonal antibody (mAb) was proved to recognize 51-70% of Bovine peripheral blood lymphocytes (PBL). The M1+ cells were SIg-. In spleen and lymph nodes, the M1 positive lymphocytes were located within the T cell areas. All the lymphoid follicles remained negative. In the thymus, 10% of thymocytes were M1+, most of them were located in the medulla. The M1 mAb did not inhibit spontaneous rosette formation by sheep erythrocytes and bovine lymphocytes. On the other hand, biochemical analysis of membrane antigen with bovine thymic tumor cell line LB203 gave a molecular weight of 75 kDa. Despite a slight difference in biochemical results (75 vs 67-69 kDa). Our data permit us to consider M1 mAb as a possible homologous of human anti-CD5 mAb. Finally, M1 cross-reacted with sheep peripheral blood T lymphocytes. PMID- 1720366 TI - Changes in ovalbumin and protein synthesis in vivo in the magnum of laying hens during the egg formation cycle. AB - 1. The present study was carried out to investigate whether or not the rate of synthesis of total protein in various oviducal segments and ovalbumin, a major egg white protein, in the magnum fluctuated during the egg formation cycle in laying hens. 2. Synthesis of total protein and ovalbumin was measured in vivo by the incorporation of [15N]methionine after a primed continuous infusion of tracer for 3 hr. 3. Protein and ovalbumin contents in the magnum and the entire oviduct decreased sharply when an ovum moved down from the magnum to the isthmus, probably due to the secretion of egg white proteins. 4. In contrast, total protein and ovalbumin synthesis in the magnum was significantly higher when an ovum was in there than when it was in any other segments. Fluctuations of ovalbumin synthesis and total protein synthesis in the magnum were roughly parallel to those of total protein synthesis in the entire oviduct. 5. It was concluded, therefore, that the changes seen in total protein synthesis in the whole oviduct during the egg formation cycle were mainly attributable to those in magnum protein synthesis, of which a significant portion was accounted for by the synthesis of ovalbumin. PMID- 1720367 TI - Resuscitation fluids for the treatment of hemorrhagic shock in dogs: effects on myocardial blood flow and oxygen transport. AB - BACKGROUND AND METHODS: The efficacy of using colloids vs. crystalloids in the treatment of hemorrhagic shock remains controversial. An important aspect in the treatment of hemorrhagic shock is the reestablishment of normal myocardial blood flow after fluid resuscitation. This study, therefore, was designed to investigate the effect of resuscitation with different plasma substitutes on myocardial blood flow and oxygen transport after acute hemorrhage in dogs. Forty three dogs were anesthetized and bled into a heparinized Wiggers' reservoir to a mean arterial pressure of 35 mm Hg. The animals were maintained at this level of hypotension for 90 mins, whereupon the animals were infused with one of five randomly selected fluids: a) succinylated gelatin (Gelofusine); b) urea-linked gelatin (Haemaccel); c) 6% hetastarch (Hespan); d) lactated Ringer's solution; or e) shed blood. Myocardial blood flow was measured using the radiolabeled microsphere technique. RESULTS: Resuscitation with succinylated gelatin, urea linked gelatin, and hetastarch resulted in significant hemodilution. However, infusion of these fluids resulted in a compensatory hyperemia that increased myocardial blood flow and maintained oxygen transport at preshock values. No hyperemia was observed with reinfusion of shed blood. Resuscitation with lactated Ringer's solution produced significant hemodilution without hyperemia and, consequently, a significant decrease in oxygen transport. CONCLUSIONS: These results suggest that in lieu of blood, the artificial colloids are more effective than crystalloids in restoring myocardial blood flow and oxygen transport after acute experimental hemorrhage in dogs. PMID- 1720368 TI - Regulation of antioxidant enzyme expression in LPS-treated bovine retinal pigment epithelial and corneal endothelial cells. AB - Retinal pigment epithelial (RPE) and corneal endothelial (CE) cells, because of their locations and functions, are continuously exposed to toxic oxidants. Protection from these toxic materials may be due, in part, to the action of endogenous antioxidant enzymes. We have established the presence of mRNAs that encode antioxidant enzymes in bovine RPE and CE cells and have determined the effect of bacterial lipopolysaccharide (LPS) on their expression. The most striking change in antioxidant enzyme expression is an increase in the level of mitochondrial manganous superoxide dismutase (MnSOD) mRNA in the LPS-treated RPE and CE cells. This increase in mRNA expression is accompanied by a slight increase in MnSOD activity as determined by SOD activity gels. PMID- 1720369 TI - The optimal treatment of malignant pleural effusions. A continuing dilemma. PMID- 1720370 TI - Intrapleural therapy for malignant pleural effusions. A randomized comparison of bleomycin and tetracycline. AB - Between December 1985 and August 1988, there were 115 patients at 13 centers who were entered on a randomized comparison of tetracycline and bleomycin for treatment of malignant pleural effusions. Fifteen patients were not treated, primarily due to rapid progression of systemic cancer. Fifteen patients entered on a high-dose regimen of bleomycin (120 units) were excluded from this analysis (following early closure of that arm), leaving 85 patients randomized to low-dose bleomycin (60 units; 44 patients) or tetracycline (1 g; 41 patients). Patients were required to have a cytologically positive pleural effusion, good performance status (0, 1, or 2), lung reexpansion following tube thoracostomy with drainage rates of 100 ml/24 or less, no prior intrapleural therapy, no prior systemic bleomycin therapy, no chest irradiation, and no recent (four weeks) change in systemic therapy. A total of 11 patients (five with bleomycin and six with tetracycline) were not evaluable due to technical problems with tube drainage (one), loss to follow-up (two), sudden death due to pulmonary embolus (one), and rapid progression of systemic disease (seven). There were no clinically significant differences in demographic factors, primary site, performance status, or presence of metastases other than pleural effusion. Overall survival did not differ between the two groups. Median time to recurrence or progression of the effusion was 32 days for tetracycline-treated patients and at least 46 days for bleomycin-treated patients (p = 0.037). The recurrence rate within 30 days of instillation was 36 percent (10/28) with bleomycin and 67 percent (18/27) with tetracycline (p = 0.023) (not all patients were restudied in the first 30 days). By 90 days the corresponding recurrence rates were 30 percent (11/37) for bleomycin and 53 percent (19/36) for tetracycline (p = 0.047). Toxicity was similar between groups. PMID- 1720371 TI - Pulmonary hyalinizing granuloma presenting as multiple cavitary calcified nodules. AB - We describe a patient with PHG who presented with multiple cavitary calcified nodules. Laboratory evaluations revealed that she had serum immune abnormalities, and a histoplasmin skin test yielded positive results. Her Histoplasma infection may have produced a hyperimmune reaction that resulted in PHG and the calcified nodules. PMID- 1720372 TI - Palliation of left main bronchus compression due to malignant tumor by intubation via a tracheostomy tube. AB - Intubation of the left main bronchus via a tracheostomy tube was performed in a patient with local recurrence of lung cancer associated with invasion and obstruction of the left main bronchus after right sleeve pneumonectomy. The result was satisfactory not only for preventing asphyxia, but also for maintaining the patency of the airway after extubation of the endotracheal tube. PMID- 1720373 TI - Binding and multiple hydrolytic sites in epitopes recognized by catalytic anti peptide antibodies. AB - Autoantibodies purified from humans catalyse the hydrolysis of the neurotransmitter, vasoactive intestinal peptide (VIP). Evidence that the hydrolysis of VIP is due to antibodies includes: the antibody preparations are free of detectable non-immunoglobulin (non-Ig) contamination; the hydrolytic activity is removed by precipitation with anti-human IgG antibody; human B lymphoblastoid cells transformed with Epstein-Barr virus secrete hydrolytic antibodies in culture; the Fab fragments of the antibodies exhibit VIP hydrolysis; and affinity chromatography on immobilized VIP permits purification of specific antibodies with greatly enriched hydrolytic and binding activities. One of the catalytic antibody preparations hydrolyses the Gln-16-Met-17 bond. Studies with synthetic VIP fragments showed that the epitope recognized by this antibody is formed by VIP(15-28). Important binding interactions are contributed by VIP(22-28), a sequence four residues distant from the scissile bond. Antibodies from a second subject hydrolyse six peptide bonds in VIP, clustered between residues 14 and 22. These bonds link amino acids of different charge, size and hydrophobicity, suggesting that the hydrolytic repertoire of the antibodies is considerable. The antibodies do not hydrolyse peptides unrelated in sequence to VIP. Cleavage of several peptide bonds in VIP by polyclonal antibody preparations may be due to several antibodies, each with a unique cleavage specificity. Alternatively, a single antibody may make catalytically productive contact at multiple peptide bonds in the substrate, because of conformational flexibility of VIP or of the antibody active site. Purified light chains from the catalytic antibodies hydrolysed VIP more rapidly than did intact antibodies. The residues constituting the catalytic site of an antibody may be encoded in germline V-region genes or may arise during maturation of the antibody response. PMID- 1720374 TI - Endoscopic Nd:YAG laser in the palliative treatment of advanced low rectal carcinoma in Singapore. AB - We used the Nd:YAG laser to palliate symptoms of bleeding and obstruction in 27 cases of rectal carcinoma. Twenty of these patients had advanced inoperable rectal carcinoma, three were at high surgical risk, and four refused surgery. Obstructive symptoms were the main complaint in 10 cases, while 17 patients presented with bleeding. Good palliation of obstructive symptoms was achieved in all obstructive cases with one laser treatment session. However, bleeding tumors required an average of two sessions for complete hemostasis. There were no major complications; minor complications of bleeding after treatment occurred in two patients. Good symptomatic relief was achieved in all cases. The mean survival for all patients was five months. Nd:YAG laser therapy is a safe and efficacious means for palliation of obstructive symptoms and bleeding in advanced rectal carcinoma. PMID- 1720375 TI - [Primary duodenal carcinoma]. PMID- 1720376 TI - [Nobel Prize for Medicine and Physiology 1991. Analysis of the function of single ion channel]. PMID- 1720377 TI - Is there a rational use for n-3 fatty acids (fish oils) in clinical medicine? PMID- 1720378 TI - Calcium antagonists in patients with heart failure. A review. AB - The presence of calcium ions is essential to the normal function of the cardiovascular system. Drugs such as calcium antagonists can modulate the interaction between these ions and specific cells at different levels, interfering with myocardial contraction and relaxation, vascular tone, specific conduction tissues and neuromuscular function. Vascular beds play a crucial role in adjustment of myocardial function to different body oxygen requirements; compensatory mechanisms in congestive heart failure (CHF) involve the vascular system to a large extent and paradoxically may worsen myocardial performance. Vasodilating drugs represent an important step forward in achieving better symptomatic results in CHF patients, and may also increase their survival. Of the different classes of vasodilator drugs calcium antagonists may represent an attractive alternative due to their anti-ischaemic and antiarrhythmic effects. Despite the overall good response to the acute use of these drugs in CHF, long term studies in which first generation calcium antagonists (nifedipine, diltiazem, verapamil) were used have produced disappointing results. Their main drawbacks were negative inotropism, lack of preload reduction and activation of neurohormonal mechanisms with a subsequent adverse effect on cardiovascular function, the latter effect being the most significant. A few long term studies, of between 1 and 52 months, have not demonstrated a consistent improvement in functional class in spite of apparently good initial results. The second generation of calcium antagonists have more potent and selective vasodilating properties with less negative inotropic effects; these properties might justify their use in the therapy of CHF, but no clear recommendations can be given due to the lack of large, long-term, controlled studies. Overall, the existing clinical trials with calcium antagonists in CHF have not proved the superiority of this group of drugs when compared to other vasodilators. If the aetiology of CHF is related to the presence of coronary artery disease or arterial hypertension, calcium antagonists might be considered as additional therapeutic options. Diastolic dysfunction may be corrected or improved and coronary tone may be diminished, both of which may lead to a better myocardial oxygen supply. Systolic myocardial function must be evaluated in CHF patients before starting therapy with calcium antagonists in order to avoid possible deleterious effects. Further studies may shed more light on this matter and may indicate decisively whether or not calcium antagonists should play a role in the therapeutic pharmacological arsenal of selected CHF patients. PMID- 1720379 TI - The clinical use of barbiturates in neurological disorders. AB - Barbiturates retain an important place in clinical neurological practice. They are used as both diagnostic and therapeutic drugs, their most common uses being as anticonvulsant and anaesthetic agents. This article explores the current theories explaining the mechanism of action of the barbiturates, with special emphasis on their anaesthetic and anticonvulsant effects. The primary mechanism of action of barbiturates is to increase inhibition through the gamma aminobutyric acid (GABA) system. Anaesthetic barbiturates also decrease excitation via a decrease in calcium conductance. Phenobarbital (phenobarbitone), the primary anticonvulsant barbiturate, is effective for partial, complex partial and secondarily generalised seizures. While no longer the drug of choice for all these seizure types, it remains an important and useful agent. Mysoline has been shown to be useful in the treatment of essential tremor and several other movement disorders, and as an anticonvulsant. Barbiturates are also used for their sedative-hypnotic properties. A relatively new use is in the evaluation of patients with medically intractable seizure disorders for possible surgical therapy. The roles of methohexital and amobarbital (amylobarbitone) are discussed in the section on barbiturates used as diagnostic agents. The experimental use of barbiturates is also commented on; the most important of these is perhaps the use of barbiturates in cerebral resuscitation. PMID- 1720380 TI - Schistosomiasis drug therapy and treatment considerations. AB - Schistosomiasis, a grave and debilitating disease of socioeconomic importance, is increasing in incidence despite concerted efforts to control and contain the disease in all the endemic areas. While a multipronged method of control using health education, sanitation and snail control has been used, chemotherapy and chemoprophylaxis play the most important and crucial role in containing/preventing the transmission of the disease. Schistosomicides such as antimonials were introduced, as early as the 1990s as the drugs of choice and continued to be used until the early 1960s. The antimonials were administered intravenously, and produced severe side effects; the various variables that determined their effects at the site of action made their application difficult and adversely affected their use in large scale chemotherapy. The antimonials were then replaced by hycanthone and lucanthone which were administered intramuscularly. These drugs produced immediate side effects such as hepatotoxicity and gastrointestinal disturbances, and were consequently withdrawn. It was then decided that the alternative was to produce synthetic drugs that could be administered orally. Niridazole, oxamniquine, and metrifonate were introduced as schistosomicidal agents, with drugs like oltipraz and amoscanate still at clinical trial phase. Therapeutic doses of drugs like hycanthone, niridazole and amoscanate have been found to cause many major side effects. A significant advance in the control of schistosomiasis chemotherapy is the introduction of a relatively safe, effective, broad spectrum oral helminthic agent, praziquantel. Studies have also shown that oxamniquine is as effective as praziquantel in eliminating intestinal S. mansoni infection, and metrifonate is as effective as praziquantel in eliminating urinary S. haematobium and S. mansoni infections. Praziquantel has been found to be effective in treating S. haematobium infections compared with metrifonate and more effective in treating S. mansoni infection when compared with oxamniquine. Because the drug is effective even when treating advanced hepatosplenic schistosomiasis, with few side effects, praziquantel is currently the drug of choice for the treatment of any kind of schistosomiasis. The only limitation is the cost which restricts its use in many developing countries. While effective, safe drugs for mass chemotherapy are being developed, the problem of therapeutic failure and drug resistance is being reported from certain developing countries. Under these circumstances, alternative drugs must be resorted to. Mass treatment, a crucial goal in the eventual control of schistosomiasis, awaits a well-tolerated and nontoxic drug that will ultimately prove to be effective where cure is definite. Until such a time, while eradication of the disease is a near impossibility, reducing the intensity of infection can ultimately reduce morbidity and even mortality.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1720381 TI - Herpes simplex virus infections of the central nervous system. Encephalitis and neonatal herpes. AB - Herpes simplex virus (HSV) infections are among the most commonly encountered in humans. Fortunately, the resulting diseases are more usually nuisances, such as recurrent fever blisters, rather than life threatening or morbidity inducing. Nevertheless, HSV can result in disease of the central nervous system (CNS) with attendant neurological complications. Examples of the latter include herpes simplex encephalitis (HSE) or neonatal HSV infection. The past decade has witnessed significant advances in our understanding of the pathogenesis of these 2 forms of disease and, even more importantly, their amenability to treatment. This review summarises our current understanding of the natural history, pathogenesis, presentation, and treatment of HSV infections of the CNS. Because of the life-threatening nature of herpes simplex infections of the CNS, particular attention is paid to clinical presentation and differential diagnosis of confounding entities which mimic herpes simplex encephalitis. The controversy of brain biopsy versus alternative noninvasive diagnostic procedures is discussed. Clinical presentation and, importantly, the lack of uniform clinical presentation, as well as the value of intervention with appropriate antiviral drugs such as aciclovir and vidarabine (adenine arabinoside, ara-A), are stressed. The clinical outcome of herpes simplex virus infections of the CNS with therapy is particularly relevant. In spite of early intervention with selective and specific inhibitors of viral replication, return to normal function is not always achieved. At the conclusion of this review, the reader should be aware of the potential value of therapy as well as the problems encountered with diagnosis and management of patients with herpes simplex virus infections of the CNS. PMID- 1720382 TI - Ifosfamide/mesna. A review of its antineoplastic activity, pharmacokinetic properties and therapeutic efficacy in cancer. AB - Ifosfamide is an oxazaphosphorine alkylating agent with a broad spectrum of antineoplastic activity. It is a prodrug metabolised in the liver by cytochrome P450 mixed-function oxidase enzymes to isofosforamide mustard, the active alkylating compound. Mesna, a uroprotective thiol agent, is routinely administered concomitantly with ifosfamide, and has almost eliminated ifosfamide induced haemorrhagic cystitis and has reduced nephron toxicity. Therapeutic studies, mostly noncomparative in nature, have demonstrated the efficacy of ifosfamide/mesna alone, or more commonly as a component of combination regimens, in a variety of cancers. In patients with relapsed or refractory disseminated nonseminomatous testicular cancer, a salvage regimen of ifosfamide/mesna, cisplatin and either etoposide or vinblastine produced complete response in approximately one-quarter of patients. As a component of both induction and salvage chemotherapeutic regimens, ifosfamide/mesna has produced favourable response rates in small cell lung cancer, paediatric solid tumours, non-Hodgkin's and Hodgkin's lymphoma, and ovarian cancer. Induction therapy with ifosfamide/mesna-containing chemotherapeutic regimens has been encouraging in non small cell lung cancer, adult soft-tissue sarcomas, and as neoadjuvant therapy in advanced cervical cancer. As salvage therapy, ifosfamide/mesna-containing combinations have a palliative role in advanced breast cancer and advanced cervical cancer. Ifosfamide/mesna can elicit responses in patients refractory to numerous other antineoplastic drugs, including cyclophosphamide. With administration of concomitant mesna to protect against ifosfamide-induced urotoxicity, the principal dose-limiting toxicity of ifosfamide is myelosuppression; leucopenia is generally more severe than thrombocytopenia. Reversible CNS adverse effects ranging from mild somnolence and confusion to severe encephalopathy and coma can occur in approximately 10 to 20% of patients after intravenous infusion, and the incidence of neurotoxicity may be increased to 50% after oral administration because of differences in the preferential route of metabolism between the 2 routes of administration. Other adverse effects of ifosfamide include nephrotoxicity, alopecia, and nausea/vomiting. In general, intravenously administered mesna is associated with a low incidence of adverse effects; however, gastrointestinal disturbances are common following oral administration. Thus, ifosfamide/mesna is an important and worthwhile addition to the currently available range of chemotherapeutic agents. It has a broad spectrum of antineoplastic activity and causes less marked myelosuppression than many other cytotoxic agents. At present, the role of ifosfamide/mesna in refractory germ cell testicular cancer is clearly defined; however, its overall place in the treatment of other forms of cancer awaits delineation in future well-controlled comparative studies. PMID- 1720385 TI - Experimental hypersensitivity pneumonitis: suppressor cell influences. AB - Experimental hypersensitivity pneumonitis (EHP) can be transferred to strain 2 guinea pigs by lymph node cells (LNC) cultured in vitro with antigen. Using mixtures of cell populations, we sought to determine if functional suppressor cells were present in our system. We also characterized the composition of cell populations that were capable (blast 10 micrograms/mL Micropolyspora faeni from 2 week donor animals) and incapable (blast 0 micrograms/mL M. faeni from 2-week donor animals; blast 10 micrograms/mL from 8-week donor animals) using flow cytometry, anti-Ig and monoclonal antibody 8BE6 (T cell marker) of transferring EHP. Two groups of donors were used: animals sensitized with Freund's adjuvant and M. faeni and challenged with either two or eight weekly intratracheal (IT) injections of M. faeni (2- and 8-week groups). LNC from donor animals were cultured with a soluble extract of M. faeni (10 or 0 micrograms/mL) blasts isolated and transferred IV to syngeneic recipients. Control animals received media IV. Recipients were challenged IT with M. faeni 48 h after the cell transfer and sacrificed 4 days thereafter. All animals were maintained in HEPA filtered air. Randomly selected microscopic fields of the lung (250/animal) were judged to be normal or abnormal without knowledge of treatment. There was a low level of pulmonary response to an IT challenge of M. faeni in media recipients. There was a substantial increase (P less than .01) in pulmonary abnormalities in the animals receiving blasts from the 10-micrograms/mL M. faeni 2-week group. Addition of cells from incompetent cell populations (0 micrograms/mL M. faeni 2 week donors or 10 micrograms/mL M. faeni 8-week donors) did not alter the ability of competent populations to transfer EHP. Cells cultured with antigen had a decreased proportion of T cells and an increased proportion of SIg+ and large cells. Competent and incompetent cell populations did not differ in regard to proportion of large cells, surface Ig+, or T cells. We conclude that the inability of certain cell populations to transfer EHP is not associated with the appearance of functional suppressor cells. Differences of ability to transfer EHP do not correlate with differences of size distribution or T and B cell composition. PMID- 1720384 TI - Benazepril. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in hypertension and congestive heart failure. AB - Benazepril is a nonsulfhydryl ACE inhibitor prodrug, which is converted in vivo to its active form, benazeprilat. Data from clinical studies have indicated that benazepril 5 to 80 mg (usually 10 to 20 mg), administered as a single daily dose, effectively decreases blood pressure in patients with mild to moderately severe hypertension. In a small number of comparative studies, the anti-hypertensive efficacy of benazepril appeared to be at least equivalent to that of captopril, enalapril, hydrochlorothiazide, nifedipine, nitrendipine or propranolol at usual therapeutic doses. Combinations of benazepril and hydrochlorothiazide or nifedipine achieved a greater lowering of blood pressure than benazepril alone, and this approach may be suitable for patients with more severe hypertension. Benazepril is reported to have beneficial effects on various indices of cardiac function and to improve clinical symptoms and exercise capacity in patients with congestive heart failure. The tolerability of benazepril in clinical trials has been very good, with an incidence of adverse effects similar to that observed in placebo recipients. Thus, benazepril appears to be an effective alternative to other members of its class for the management of hypertension, and further studies will accurately define its usefulness in congestive heart failure. PMID- 1720386 TI - Serum and peritoneal fluid amylase and lipase reference values in horses. PMID- 1720387 TI - Development of very low birth weight infants: a regional study of 371 survivors. AB - We re-examined 371 infants with birth weights less than 1501 g at a corrected age of 18-20 months. This sample amounted to 91% of such infants admitted to one of the six neonatal intensive care units in Hamburg between July 1983 and 1986. The neurological examination and a developmental evaluation using the Griffith Developmental Scale revealed higher rates of abnormalities than in most other studies. Fifty-five children (14.8%) suffered from cerebral palsy, classified in 45 as spastic diplegia, in 5 as spastic tetraplegia, in 1 as spastic hemiplegia and in 4 as dystonia. Of the children, 41 (11%) showed minor neurological deviations (hyperactivity, clumsiness, intention tremor). The development of 30 children (8%) without neurological abnormalities was moderately retarded (DQ 80 89, corrected for gestational age [GA]). Nineteen children (5%) were severely retarded (DQ less than 80, corrected for GA) and four children (1.5%) were blind due to retrolental fibroplasia. An isolated delay of speech development was found in 5 children. Seventy children (18.9%) had a major and 87 children (23.5%) a minor handicap. PMID- 1720383 TI - Atenolol. A reappraisal of its pharmacological properties and therapeutic use in cardiovascular disorders. AB - Atenolol is a selective beta 1-adrenoceptor antagonist with a duration of activity of at least 24 hours. The scope of therapeutic use of the drug has been expanded and become better defined since it was first reviewed in the Journal in 1979. Atenolol is effective and generally well tolerated in patients with all grades of hypertension. Data from comparative studies show that when administered orally, atenolol reduces blood pressure to a similar extent, and in a similar proportion of patients, as usual therapeutic doses of other beta-adrenoceptor antagonists (such as acebutolol, celiprolol, betaxolol, indenolol, metoprolol, nadolol, pindolol, propranolol, tertatolol), angiotensin converting enzyme (ACE) inhibitors (e.g. captopril, enalapril and lisinopril), calcium antagonists (e.g. amlodipine, diltiazem, felodipine, isradipine, nitrendipine, nifedipine, verapamil), doxazosin, ketanserin and alpha-methyldopa. Atenolol effectively lowers blood pressure in elderly patients with hypertension and in women with hypertension associated with pregnancy, and improves objective and subjective indices in patients with stable angina pectoris. Oral atenolol is used for preventing recurrence of supraventricular arrhythmias once control is achieved by intravenous administration of atenolol. Early intervention with intravenous atenolol followed by oral maintenance therapy reduces infarct recurrence and cardiovascular mortality in patients with known or suspected myocardial infarction. There is also encouraging evidence of reduced mortality from cardiovascular disease during long term therapy with atenolol in patients with hypertension. Atenolol is well tolerated in most patients. Increases in plasma levels of both total triglycerides and very low density lipoprotein (VLDL) triglycerides have accompanied atenolol therapy although the clinical relevance, if any, of longer term metabolic effects has yet to be determined. Its low lipid solubility and limited brain penetration results in a lower incidence of central nervous system effects than that associated with propranolol. After many years of clinical usage atenolol is a well established treatment option in several areas of cardiovascular medicine such as mild to moderate hypertension and stable angina pectoris. Furthermore, it has also shown potential in the treatment of some cardiac arrhythmias and has been associated with reduced cardiovascular mortality in patients with hypertension and in patients with myocardial infarction. PMID- 1720388 TI - Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments. AB - alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments. PMID- 1720389 TI - Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents. AB - In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxyellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants, ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc. PMID- 1720390 TI - Subclones of C6 rat glioma cells differing in intermediate filament protein expression. AB - The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation. PMID- 1720391 TI - Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells. AB - Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells. PMID- 1720392 TI - Dissection of murine lymphocyte-endothelial cell interaction mechanisms by SV-40 transformed mouse endothelial cell lines: novel mechanisms mediating basal binding, and alpha 4-integrin-dependent cytokine-induced adhesion. AB - Lymph node-derived endothelial cells were immortalized by infection with SV40 virus and subclones expressing the marker MECA 325 specific for high-endothelial venules (HEV) were selected. These transformed mouse endothelial (TME-) cell lines grow permanently without requirement for special growth factors. Staining of the selected clones with endothelium-specific antibodies and with anti-von Willebrand factor antiserum and uptake of acetylated low-density lipoprotein provide evidence for their endothelial origin. The vascular addressins identified by mAbs MECA 79 and MECA 367 on HEV are not detectable, indicating that the phenotype of the cells differs from that of HEV-type endothelium. The TME cells display a constitutive capacity to bind lymphocytes. An additional binding component is induced by treatment of the TME cells with TNF alpha. Antibodies against the homing receptor LECAM-1 (lectin-related leucocyte-endothelial cell adhesion molecule 1), alpha 4-integrins, vascular addressins, LFA-1, or ICAM-1 known to block lymphocyte interaction with particular types of HEV were unable to inhibit the basal adhesion to TME cells, indicating that a further binding mechanism in mice is displayed by this cell type. The adhesion component induced by TNF alpha is mediated by alpha 4-integrins since enhanced binding could be blocked by an antibody against mouse alpha 4 (lymphocyte-Peyer's patch adhesion molecule 1/2). TME cell lines therefore seem to be a useful model for the dissection and analysis of hitherto poorly characterized murine lymphocyte/endothelial cell interaction mechanisms. PMID- 1720393 TI - Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences. AB - The technique of nonradioactive in situ hybridization has been used to visualize the DNA and mRNA expression of human cytomegalovirus (HCMV) immediate early antigen (IEA) in a transfected rat fibroblast cell line. Expression of the transfected HCMV immediate early DNA can be induced by a cycloheximide treatment and is S-phase-dependent. In addition to cytoplasmic mRNA localization, a nuclear RNA hybridization signal was found. In a substantial part of the cells the nuclear signal was in the form of a "track," possibly showing transport of IEA mRNA from the site of transcription to the cytoplasm. The use of PCR-generated intron- and exon-specific probes in a double hybridization revealed that intron and exon mRNA sequences coexist in the nuclear RNA signal. This shows the applicability of multiple-color fluorescence hybridization to obtain information about the site of pre-mRNA splicing in the nucleus. In addition, by combining the technique of in situ hybridization with an immunocytochemical procedure we illustrate the possibility of visualizing transcribed mRNAs simultaneously with their translation products. PMID- 1720394 TI - Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism during the life cycle. AB - All four life cycle stages (bloodstream, procyclic, epimastigote, and metacyclic) of Trypanosoma congolense IL 3000 were assayed with an oxygen electrode (polarograph) for the presence of terminal oxidases and carbon-source preference. In addition, these stages were used for histochemical analysis of mitochondrial activity using rhodamine 123, nitroblue tetrazolium, and diaminobenzidine. Morphometry was used to compare mitochondrial volumes and surface area among the different life cycle stages. It was found that in contrast to epimastigote forms, which were metabolically almost identical to procyclic forms, metacyclic forms showed characteristics of, and seemed preadapted to, differentiation into the bloodstream stage. While mitochondrial NAD+ diaphorase activity and an electrochemical potential were detected in all life cycle stages, metacyclic metabolism was glucose-based and terminal oxidase activity was primarily dependent upon the trypanosome alternative oxidase with the contribution of cyanide-sensitive respiration accounting for only 20-30% of the total respiratory capacity. PMID- 1720395 TI - Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation. AB - Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded. PMID- 1720396 TI - Plasmodium falciparum: intragenic recombination and nonrandom associations between polymorphic domains of the precursor to the major merozoite surface antigens. AB - Extensive allelic polymorphism in the Plasmodium falciparum major merozoite antigen precursor (MSP1/PMMSA) is partly due to intragenic recombination events within a short region near the 5' end of the gene. Newly described allelic sequences from this region of the gene are compared to those previously published, revealing additional sites of intragenic recombination. Epitopes recognised by monoclonal antibodies on the protein have been assigned on the basis of correlations between serology and amino acid sequence polymorphisms among different allelic types of MSP1. Serological analyses of MSP1 from 567 wild isolates from The Gambia, Nigeria, and Brazil reveal that certain pairs of epitopes, although sited on MSP1 domains separated by known sites of intragenic recombination, are highly significantly associated on parasites in endemic populations. Most associations are similar in the three countries. These associations are discussed with respect to the intragenic recombination hypothesis. PMID- 1720397 TI - Induced hypertension as an approach to treating acute cerebrovascular ischaemia: possibilities and limitations. AB - As, after an stroke, the autoregulation of the cerebral vessels in the ischaemic region is disturbed to a high degree, it is, on principle, possible to improve the blood flow particularly in the zone surrounding the infarct (penumbra) by raising the systemic blood pressure. During a basic treatment with low-molecular dextrans (infukoll M40), 37 patients with an acute ischaemic cerebral stroke multiply underwent elevations in blood pressure up to systolic values of about 210 to 220 mmHg. A comparison with a control group (n = 44) who were treated with low-molecular dextrans revealed no differences in lethality on the 21st day after the stroke. However, a very good acute effect in terms of a short-term improvement was remarkable a result that is noteworthy also in future. PMID- 1720398 TI - Multiple cDNAs of phosphoenolpyruvate carboxylase in the C4 dicot Flaveria trinervia. AB - We have isolated and characterized cDNA clones for the leaf-specific C4 phosphoenolpyruvate carboxylase (PEPCase) from the dicotyledonous C4 plant Flaveria trinervia. The isolation of multiple cDNAs indicates that in this plant the C4 isoform is encoded by a small subgroup of the PEPCase gene family. The deduced amino acid sequence reveals a higher degree of similarity to the CAM and C3 isozymes of the dicotyledonous, facultative CAM plant Mesembryanthemum crystallinum than to the C4 PEPCases of monocotyledonous origin. PMID- 1720399 TI - Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco. AB - An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization. PMID- 1720400 TI - Crystallization and preliminary X-ray analysis of methylamine-treated alpha 2 macroglobulin and 3 alpha 2-macroglobulin-proteinase complexes. AB - Crystals of methylamine-treated alpha 2-macroglobulin (alpha 2M-MA), alpha 2 macroglobulin in complex with two molecules of trypsin, alpha 2M-T2, one molecule of plasmin, alpha 2M-PL, and one molecule of plasmin followed by methylamine treatment, alpha 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of alpha 2M-MA diffracted to at least 9 A resolution, crystals of alpha 2M-T2 diffracted to 10 A resolution and crystals of alpha 2M-PL and alpha 2M-PL(MA) diffracted to 11 A resolution. For alpha 2M-MA the cell parameters were determined as: a=b=257 A, c=555 A; and for alpha 2M-T2 as: a=b=247 A, c=559 A. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of alpha 2M-MA and alpha 2M-T2 contain one 360 kDa alpha 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6 A3/Da. The crystal parameters of alpha 2M-PL and alpha 2M-PL(MA) were not determined. PMID- 1720401 TI - Switching of bovine cytochrome c oxidase subunit VIa isoforms in skeletal muscle during development. AB - A cDNA encoding the liver isoform of bovine cytochrome c oxidase subunit VIa (VIaL) was cloned from bovine liver RNA by reverse transcription and the polymerase chain reaction. The nucleotide and deduced amino acid sequences show high conservation with the corresponding rat and human liver subunits. The sequence similarity between beef heart and beef liver VIa is 60%. Northern analyses of the steady-state levels of the VIa-heart (VIaH) and VIa-liver (VIaL) transcripts showed that adult liver and brain contained only VIaL transcripts, the VIaH transcript predominated in heart with a small amount of VIaL also present, while in adult skeletal muscle VIaH was present exclusively. The VIaL transcript was found in heart with a small amount of VIaL also present, while in adult skeletal muscle VIaH was present exclusively. The VIaL transcript was found in fetal heart and skeletal muscle from 104-215-day-old fetuses, in as much as 25% of the amount of VIaH transcript. The down-regulation of VIaL transcript in skeletal muscle at or close to birth may be correlated with a change in amount of cytochrome c oxidase relative to the bc1 complex (complex III) observed spectrally when fetal and adult muscle samples were compared. PMID- 1720402 TI - Phorbol 12-myristate-13-acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c-fos mRNA in a human neuroblastoma cell line. AB - Regulation of cholecystokinin (CCK) and the proto-oncogene c-fos mRNA expression was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated either with the tumor promoting phorbol-ester phorbol-12-myristate-13-acetate (PMA), the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c-fos mRNA was determined by Northern-blot analysis with CCK and c-fos specific antisense RNA probes after 4 24 h of drug treatment. Treatment with PMA and IBMX for 4-24 hours transiently raised the CCK mRNA level approximately 1.5-3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK mRNA abundance 1.5-6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c-fos mRNA level approximately 3-5.5 times. The drug combination showed a pronounced synergistic effect and raised the c-fos mRNA level approximately 3-20 times as compared to controls. Apparently, CCK and c-fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP-dependent mechanisms in SK-N-MC cells. PMID- 1720403 TI - Treatment of limb threatening ischaemia with intravenous iloprost: a randomised double-blind placebo controlled study. U.K. Severe Limb Ischaemia Study Group. AB - A number of patients (151) with ischaemia of the lower limb presenting as ulcers or gangrene and/or rest pain were entered into a multicentre randomised double blind controlled study of intravenous iloprost or placebo given for 14-28 days. Patients were assessed for evidence of ulcer healing as judged by reduction in size with granulation at the base and relief of rest pain sufficient for discharge from hospital. Based on these criteria, 45% in the iloprost and 29% in the placebo group showed evidence of improvement of clinical status at the end of treatment (p less than 0.05). At 6 months follow-up improvement was maintained in 42% of iloprost patients and 26% of placebo patients (p less than 0.01). At this follow up 64% of the iloprost patients and 42% of the placebo patients were alive with a viable limb. Thirty-one per cent of the iloprost patients and 47% of the placebo patients underwent major amputation (p less than 0.05). It has been shown that iloprost significantly improves patients with ischaemic ulcers or gangrene compared with placebo. This improvement is maintained for up to 6 months after treatment resulting in a reduced major amputation rate. PMID- 1720404 TI - Treatment of digital ischaemia associated with chemotherapy using the prostacyclin analogue iloprost. AB - Digital ischaemia is a recognised complication of both malignant disease and chemotherapy. Its onset may prevent further treatment and lead to tissue loss. We report a case of severe digital ischaemia in a patient with advanced breast carcinoma undergoing cytotoxic chemotherapy. This was successfully treated with Iloprost, a prostacyclin analogue. PMID- 1720405 TI - Differentiation of mouse embryonic palatal epithelium in culture: selective cytokeratin expression distinguishes between oral, medial edge and nasal epithelial cells. AB - During normal murine palatogenesis, regional specific differentiation of the epithelium results in three cell phenotypes: nasal (ciliated pseudostratified columnar cells), oral (stratified squamous cells) and medial edge (migratory, epithelio-mesenchymally transformed cells). We have developed a defined, serum free, culture system which supports the growth and differentiation of isolated murine embryonic palatal epithelia in vitro. Using immunofluorescence microscopy, an established panel of antibodies was used to characterise the cytokeratin intermediate filament profile of palatal epithelial sheets at a precise developmental stage, following culture in serum-free medium with and without either transforming growth factor alpha (TGF alpha) or 10% donor calf serum (DCS). The morphologically discernable oral, medial edge and nasal phenotypes exhibited distinctive cytokeratin profiles, which remained consistent for all culture conditions, and which correlated with the known differentiation states of the epithelial types. The oral epithelia stained positively for cytokeratin 19 and cytokeratins characteristic of multilayered epithelia (1, 5, 14). Nasal epithelia stained similarly but in addition expressed the simple-epithelial cytokeratin pair, 8 and 18. Medial edge epithelia also expressed cytokeratins 1, 5 and 14 but with the exception of a few isolated cells there was no staining for cytokeratins 8 and 18. Cytokeratin 19 was absent specifically from the medial edge epithelial cells: this result may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformations. By exhibiting a complexity of expression linked to differentiation state and independent of culture conditions, cytokeratins constitute useful markers of palatal epithelial differentiation in vitro as well as in vivo. PMID- 1720406 TI - 1,25-Dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes. AB - Human keratinocytes grown on deepidermized dermis (DED) are able to reconstruct a morphologically normal stratified and keratinized epidermis. This culture system is suitable for studying in vitro the effects of various hormones and factors on epidermal differentiation, and the goal of the present work was to study the effect of vitamin D. We found that the hormonal form of vitamin D3, 1,25 dihydroxyvitamin D3, produced very specific alterations in epidermal architecture in a dose-dependent manner, consisting of significant reduction of the nucleated layers of the epithelium, but not of the stratum corneum, which was instead slightly thickened. The study of stage-specific differentiation markers showed that the two extreme layers of epidermis, i.e. the basal layer and the stratum corneum, were unaffected by the hormone, but that the reduction involved specifically the intermediate differentiation compartment, i.e. the spinous and granular layers. It was shown that the reduction of the intermediate compartment provoked by 1,25-dihydroxyvitamin D3 is not due to a block in the proliferation of basal cells or to inhibition of their differentiation into suprabasal cells, but to stimulation of the terminal differentiation of suprabasal cells into corneocytes. PMID- 1720408 TI - Cross-reactions between respiratory and food allergens. AB - Cross-reactions between inhaled and food allergens are usually attributed to pollen hypersensitivity associated with fruit and vegetable allergy. However, other allergens are involved in these types of cross-reactions. In a few cases, there is a complete similarity between the inhaled and food allergens (garlic, crustacea proteins). More frequently, partial similarity has been demonstrated: whole inhaled allergens are included in ingested substances. Moreover, immunological techniques can demonstrate common antigenic epitopes in organic substances without any apparent relationship. This has been demonstrated by RAST inhibition and/or immunoblot techniques, using sera from patients cross sensitized to (1) pollens and fruits or vegetables or (2) avian sera and eggs. Respiratory sensitization always seems to precede food allergy symptoms. PMID- 1720407 TI - [Preinduction in the treatment of patients with malignant lymphoma]. AB - Prospective clinical study of ninety-eight patients with untreated malignant lymphoma were randomly assigned to be treated wether with combined chemotherapy: cyclophosphamide, hidroxidaunil adriamycin, vincristine, prednisone and bleomycin (CHOP-Bleo) or with two low-dose of methotrexate as preinduction regimen and the same chemotherapy: CHOP-Bleo. The number of early deaths afterwards the group was less with preinduction, also more of them had complete remission, it lasted longer and survival was better than in the group just treated with conventional chemotherapy. Toxicity, secondary to preinduction chemotherapy. Toxicity, secondary to preinduction regimen was acceptable, while it was similar in both groups receiving conventional chemotherapy. Supported on these results it is considered that the use of preinduction regimen, as herein described, is useful in patients with malignant lymphoma and should be applied for the therapeutic approach of these neoplasms. PMID- 1720409 TI - [Transurethral balloon dilatation in benign prostatic hypertrophy]. PMID- 1720410 TI - [Terminal cancer care and QOL]. PMID- 1720411 TI - [Establishment of cell lines of human germinal tumors]. AB - Human germinal tumor cells have been studied in cancer research because of their characteristics of the multi-potentiality and the ability to produce marker proteins such as AFP and SP1. Although human germinal tumor cell colonies had usually been maintained by serial transplantation into nude mice, we established in vitro culture method of cells derived from human malignant germ cell tumors. Thirty-two cell lines were established in vitro, and AFP and SP1 produced in culture medium of those cell line were demonstrated immunochemically. PMID- 1720412 TI - [Electrophoretic variants of rat alpha-fetoprotein]. AB - It has been well documented that rat AFP is separated into two discrete fractions electrophoretically and by ion exchange chromatography and it is generally accepted that the two forms of AFP, "Slow" and "Fast" variants, have different charges and molecular sizes. In this paper, the molecular basis of electrophoretic variants of rat alpha-fetoprotein (AFP) was studied. Carbohydrate free rat AFP was electrophoretically homogeneous. Stepwise conversion of molecular sizes by deglycosylation with glycopeptidase F and the specific activities of the variants of which sugar chains were radiolabelled suggest that "Slow" and "Fast" variants have two and one sugar chains per molecule, respectively. PMID- 1720413 TI - Significance and specificity of antibodies to neutrophils detected by western blotting for the serological diagnosis of primary sclerosing cholangitis. AB - Antibodies against neutrophils have been detected in sera from patients with primary sclerosing cholangitis and inflammatory bowel diseases either by immunofluorescence or by enzyme-linked immunosorbent assay. To assess primary sclerosing cholangitis-specific antibodies, we examined sera from 30 patients with clinically and morphologically well-established primary sclerosing cholangitis by Western blotting against neutrophils and compared these results with those obtained by testing sera from patients with inflammatory bowel diseases. By Western blot using sonified neutrophils, 24 (80%) of 30 primary sclerosing cholangitis sera were positive. Five antigenic determinants at 95, 60, 55, 40 and 30 kD were visualized. Twenty-eight of the primary sclerosing cholangitis sera also showed the characteristic perinuclear fluorescence pattern by immunofluorescence on neutrophils. Thus a serological diagnosis of primary sclerosing cholangitis could be made in 80% of patients based on these two methods. In contrast, only 9% of 23 patients with ulcerative colitis and 10% of 60 patients with Crohn's disease were positive by Western blot, and these patients also showed positive perinuclear fluorescence pattern by immunofluorescence, suggesting an overlap between inflammatory bowel diseases and primary sclerosing cholangitis. Although some patients with classical primary biliary cirrhosis and autoimmune chronic active hepatitis had antibodies against primary sclerosing cholangitis epitopes, none of the patients with obstructive bile duct disorders, collagen diseases, Wegener's granulomatosis or other hepatic and nonhepatic disorders were positive by Western blot, indicating the specificity of these five primary sclerosing cholangitis-related neutrophilic epitopes. PMID- 1720414 TI - Monoclonal antibodies which identify carbohydrate-defined MHC class I epitopes. AB - Eleven different monoclonal antibodies specific for H-2K- and H-2D-encoded Class I molecules have been screened to determine Class I epitopes dependent on both carbohydrate and protein structures. Monoclonal antibodies have been identified which bind to carbohydrate-defined antigens encoded by both the H-2K and H-2D gene regions. Sensitivity to glycosidases versus pronase has been used to classify antigens both expressed as cell surface molecules and when prepared as detergent solubilized antigen. Several simple sugars have also been found to act as inhibitors of antibodies which bind to carbohydrate-defined sites. The genetic control of carbohydrate antigen expression by H-2K- and H-2D-linked genes has been verified since a specific antibody does not bind to H-2Kb or H-2Db molecules encoded by several mutant strains of mice containing single amino acid substitutions in their protein product. All of these data are consistent with Class I antigenic structures being encoded in carbohydrate as well as protein moieties. PMID- 1720415 TI - Human IgE-binding synthetic peptides of bovine beta-lactoglobulin and alpha lactalbumin. In vitro cross-reactivity of the allergens. AB - The allergenicity of cow's milk whey proteins, purified by high performance liquid chromatography (HPLC), was examined by the radio-allergosorbent test (RAST) against the sera of children immediately hypersensitive to milk. beta lactoglobulin and alpha-lactalbumin bound specific IgE in the sera of 63% and 75% of these patients respectively. These allergens were tested for cross reactivity with each other by RAST inhibition. Both inhibited the binding of IgE, in the sera of allergic patients, to the other protein. Two possible determinant peptides, one from beta-lactoglobulin and one from alpha-lactalbumin, were selected by computer prediction of antigenic sites and synthesized by the fluorenylmethoxycarbonyl (FMOC)-polyamide method. The peptides were adsorbed to nitrocellulose discs and used in further RAST studies with sera from the allergic children. Both peptides bound specific IgE in the RAST assay. PMID- 1720416 TI - Induction of anti-idiotypic T cells through a network mechanism. AB - BALB/c mouse T cells that recognized the idiotype expressed on M104E(mu, lambda 1) were induced by immunization with Dextran B-1355. T cells derived from mice immunized with 1 mg of Dextran B-1355 showed a marked proliferative response against M104E, whereas T cells from mice immunized with Ficoll or smaller amounts of Dextran B-1355 did not. BCL1Id, which had an identical isotype, did not induce proliferation of T cells. The T cell proliferative response against the idiotype on M104E required macrophages as antigen-presenting cells. The proliferative response was inhibited when antigen-presenting cells were treated with NH4Cl or chloroquine, which are antigen-processing inhibitors. These results indicate that anti-idiotypic T cells which recognized processed idiotopes could be induced physiologically through a network mechanism. PMID- 1720417 TI - The immunosuppressive agent FK506 inhibits in vitro expression of membrane-bound and soluble interleukin-2 receptors on resting but not on activated human lymphocytes. AB - FK506 is a recently introduced immunosuppressive agent synthesised by the microorganism Streptomyces tskubaensis. It has been found to be more potent than Cyclosporin A in inhibiting T cell activation. We investigated its effects on the expression of membrane bound as well as soluble interleukin-2 receptors on human lymphocytes. The membrane-bound IL-2 receptor expression was inhibited by FK506 in resting lymphocytes at a concentration of 1 pmol/l. At 10 nmol/l no further inhibition was seen. In activated lymphocytes FK506 exerted no inhibitory effect on the IL-2 receptor expression. The release of soluble IL-2 receptor showed a pronounced decline in the concentration interval between 10 pmol/l and 0.1 nmol/l. Above a concentration of 10 nmol/l, no further decrease was seen. In activated lymphocytes the expression of soluble IL-2 receptors was unaffected by FK506 incubated up to 72 h. Pretreatment of the lymphocytes with the compound did not further depress the expression of the membrane-bound or the soluble receptor. Our results also indicate that the expression of the membrane-bound receptor is more sensitive to the drug than the soluble form of the receptor. PMID- 1720418 TI - Monoclonal antibody 117,C-11 recognizes three exposed regions on the surface of the Lathyrus ochrus isolectin I. AB - A mouse IgA monoclonal antibody, MoAb 117,C-11, was produced against the glucose/mannose specific isolectin I (LoLI) from Lathyrus ochrus seeds, a legume two-chain lectin comprising two heavy subunits non covalently associated to two light subunits. The MoAb reacted in ELISA with three different peptides representing three distinct amino acid stretches present on the surface of the heavy subunits of LoLI. Since these three stretches are too far apart to build a discontinuous/conformational epitope, it is presumed that they act as continuous/sequential epitopes. The presence of a complete or partial TGNV sequence within these epitopes could account for their reactivity towards the MoAb, as shown by inhibition studies using a chemically synthesized TGNV peptide. MoAb 117,C-11 also reacted with closely related two-chain lectins. PMID- 1720419 TI - Rapid "tea-bag" peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins. AB - The role of individual amino acids in binding human and macaque antibodies were determined in the human immunodeficiency virus type 1 (HIV-1) gp41, residues 594 613, and for human antibodies in the hepatitis B (HB) virus core/e antigens (HBc/eAg), residues 121-140. Decapeptides with 9 amino acids (aa) overlap were synthesised using a rapid method for simultaneous multiple peptide synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) protection for the alpha-amino group of the aas. One coupling cycle including washing steps was performed within 60-90 min. The crude products were analysed by reversed-phase HPLC and PD-mass spectrometry. With the 11 decapeptides covering residues 594-613 of HIV-1 gp41, the sequences SGKLI at aa 599-603 was found to be the main recognition site for 19 human anti HIV positive sera. Two macaques repeatedly immunized with a peptide covering aa 594-613 of gp41, preferentially recognised the sequence CTTAVPW at residues 604 610 after 1-2 months of immunisation. One macaque also recognised the sequence CSGKLI, with sera sampled greater than 10 months after start of immunisation. Out of 9 human sera from patients with chronic HB, and reactive to a peptide covering residues 121-140 of HBc/eAg, 8 were found to recognise the sequence TPPA at residues 128-131, with an individual variation within residues 125-133 in regard to N- and C-terminal ends of the recognised antigenic site. Thus, human recognition of this antigenic site overlaps the reported T- and the B-cell recognition site found in mice. We believe that this simple and rapid approach to obtain large numbers of immunologically active peptides can be useful for most laboratories interested in the immunological characterisation of proteins. PMID- 1720420 TI - Multiple antigen peptides for specific detection of antibodies to a malaria antigen in human sera. AB - Multiple antigen peptides (MAP), consisting of a number of peptide copies synthesized on a branching lysyl core, offer a novel approach for rendering small peptides immunoreactive in solid-phase immunoassays. An octameric MAP, carrying 6 repeats of the sequence -N-A-A-G-, tandem repeated in the immunodominant region of the circumsporozoite (CS) protein of Plasmodium malariae, was used as a model to evaluate the suitability of the MAP system in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against a parasite antigen in individuals exposed to natural infection. The reaction of endemic sera in ELISA on MAP8-(NAAG)6 was related to that obtained in immunofluorescence on sporozoites, indicating the specificity of the antibody-MAP interaction. The reactivity of immune sera was found to be directed only against the (NAAG)6 moiety of the MAP and not against the lysyl core, since antibody binding to MAP8 (NAAG)6 was completely inhibited by (NAAG)6-NA monomer, but remained uninfluenced when lysyl core was used as competing ligand. The levels of antibodies to MAP8 (NAAG)6, in two groups of individuals naturally exposed to malaria infection, appeared to be related to their respective exposure to the parasite. PMID- 1720421 TI - Hyperamylasaemia & related enzyme factors in renal failure associated with benign & malignant conditions. AB - Total serum amylase activity, its isoenzymes and pancreatic to salivary amylase (P/S) ratio were studied in 40 normal subjects and 47 patients with renal failure, 32 with benign and 15 with malignant conditions. Amylase to creatinine clearance (Cam/Ccr) was studied in 17 normals and 14 patients with renal failure, 10 benign and 4 with malignant diseases. Total amylase activity, and pancreatic and salivary fractions were found to be increased by about 3.4 times the normal in both benign and malignant conditions producing renal failure though the P/S ratio was within the normal range. However, the increase in the urea and creatinine levels in patients could not be related to the increase in serum total amylase. Besides the Cam/Ccr ratio was elevated in patients with both benign and malignant conditions producing renal insufficiency whereas the Cam and Ccr were individually found to be decreased. Why patients with chronic renal failure in both conditions without clinical evidence of pancreatitis should have elevated Cam/Ccr ratio is not clear. PMID- 1720422 TI - Rapid technique for the detection of carcinogens using mice liver microsomes. AB - Mice liver microsomes were prepared at low g (10,000 g) force by Ca(2+) aggregation method and were used for the detection of carcinogens by degranulation technique. RNA/protein ratio of these microsomes was 0.177 which was comparable to rat liver microsomes. Per cent degranulation with two carcinogens (O-dianisidine and benzidine) at 20 micrograms concentration was 21 per cent on the basis of RNA/protein ratio basis with both the carcinogens. At 40 micrograms concentration the per cent degranulation was observed to be almost double. Both the carcinogens required NADPH for their activation. PMID- 1720423 TI - Endogenous regulation of rat brain mast cell serotonin release. AB - Mast cells are involved in allergic reactions where they release numerous vasoactive and other mediators in response to IgE and antigen. They are also activated by neuropeptides and are found in close contact with neurons. Mast cell heterogeneity has now been documented for mucosal mast cells and connective tissue mast cells. Rat brain mast cells were studied in a perfusion system and were shown to release serotonin in response to the mast cell secretagogue compound 48/80 (C48/80). High-potassium neuronal depolarization also released serotonin, but this was calcium dependent, not associated with beta hexosaminidase, and was unaffected by prior treatment with C48/80. Neuronal depolarization, however, was associated with somatostatin secretion and substantially reduced subsequent C48/80 stimulation, an effect abolished by neonatal treatment of the animals with capsaicin. Perfusion with somatostatin and substance P also induced brain mast cell serotonin release. C48/80 stimulation of combined thalamic and hypothalamic slices after neuronal depolarization substantially reduced the C48/80 effect, suggesting the possible presence of endogenous inhibitors released from the hypothalamus. Finally, the alpha 2 receptor agonist clonidine had a slight stimulatory effect. These results indicate that brain mast cell serotonin release may be regulated by endogenous neurotransmitters and/or neuromodulators. PMID- 1720424 TI - Identification of reactive synthetic gliadin peptides specific for coeliac disease. AB - Gluten intolerance (coeliac disease) is characterised by the development of a small intestinal lesion following exposure to the gliadin fraction after consumption of wheat and related cereals. Cellular immune mechanisms are thought to be responsible for gliadin toxicity, but the toxic sequence/s within gliadin have not been clearly established. A panel of synthetic gliadin peptides was tested using peripheral blood mononuclear cells from coeliac patients and two assays for cell-mediated immunity. Using the indirect leucocyte migration inhibition factor and the macrophage procoagulant activity assays, gliadin peptides which were located in the aminoterminal or the proline-rich domain of the alpha/beta gliadin molecule were coeliac-active. Peptides predicted by T cell algorithms or on the basis of homology to adenovirus Ad12 Elb protein and which were located in the proline-poor gliadin domains were inactive. Protein sequence studies which indicate significant homology in the proline-poor gliadin domains with a number of non-coeliac-toxic seed proteins also supported the hypothesis that the proline-rich domains may be more important in the pathogenesis of coeliac disease. PMID- 1720425 TI - Expression of beta 1-integrins, H-CAM (CD44) and LECAM-1 in primary gastro intestinal B-cell lymphomas as compared to the adhesion receptor profile of the gut-associated lymphoid system, tonsil and peripheral lymph node. AB - beta 1-Integrins (VLA-1 to -6) are cell-surface molecules binding to matrix molecules such as collagen, fibronectin and laminin. VLA-4 is the human homologue to the murine Peyer's patch homing receptor mediating cell/cell adhesion required for lymphocyte extravasation or "homing". Other structures which have a homing receptor function through recognition of venular endothelium are H-CAM (CD44) and LECAM-1 (LAM-1, human MEL-14 equivalent). In order to elucidate whether these adhesion receptors are expressed in primary gastro-intestinal malignant B-cell lymphomas (GI BmL) which, in this case, might contribute to the initial confinement to this extranodal site, 31 extensively characterized tumors were examined together with reactive lymphoid tissues from small and large intestine, tonsil and lymph node using monoclonal antibodies (MAbs) against these receptors. All types of adhesion receptors were differentially expressed in the cytologically and microtopographically defined B-cell subsets [follicular center cells (FC), mantle-zone cells (MZ), extrafollicular cells (EF) and plasma cells (PC)] of the normal B-cell system. With the exception of differences in LECAM-1 levels among EF and PC of intestinal vs. nodal vs. tonsillar sites, receptor profiles were almost identical in different lymphoid organs. The expression pattern of these molecules in GI BmL was markedly heterogeneous, mimicking to some extent the receptor equipment of their reactive cellular counterpart. Thus, we failed to find a unifying adhesion receptor profile indicative of a tissue specific homing of reactive and neoplastic B-cells. PMID- 1720426 TI - Integrin expression in human melanoma cell lines: heterogeneity of vitronectin receptor composition and function. AB - Ten human melanoma cell lines were examined for integrin-receptor expression using a panel of antibodies directed against different integrin subunits. Considerable heterogeneity was detected for levels of expression of 7 integrins, including the alpha v beta 3 vitronectin receptor where a correlation between tumorigenic capacity in athymic nude mice and alpha v beta 3 levels was found. Detailed analysis of the vitronectin receptor on these lines revealed heterogeneity of composition. In one cell line, VUP, an alpha v beta 1 association was detected and, by antibody-inhibition studies, this receptor was shown to bind vitronectin as its ligand. In another line, DX3, evidence was obtained which indicated that apart from the alpha v beta 3 receptor the alpha v was able to associate with another beta subunit which was not beta 3. The existence of these alternative forms of the vitronectin receptor in this small sample of tumours of common origin might explain why the capacity to bind to fibrinogen and vitronectin substrates by these cells did not necessarily correlate with alpha v beta 3 levels. PMID- 1720427 TI - Estradiol and EGF requirements for cell-cycle progression of normal human mammary epithelial cells in culture. AB - The purpose of our study was to show the feasibility of accurately investigating the factors likely to control cell proliferation of normal human mammary epithelial (HME) cells, using a scanning cytometric method. The methodology was previously developed with the SAMBA 200 cell image processor to characterize in situ the cell-cycle phases of HME cells. Since various compartments constitute the mammary epithelium, cells obtained after reduction mammoplasty were cultured in a medium with a low calcium content (0.06 mM) to provide proliferating normal HME cells while maintaining their differentiation characteristics. Estradiol and EGF requirements for cell-cycle phase progression of these cells were examined. Then, we showed that 2 normal HME cell cultures displaying different phenotypic characteristics may differently progress through the cell cycle under the same hormonally defined conditions. Cell-cycle progression of the epithelial cells presenting luminal phenotype was induced only by sequential stimulation/pretreatment with estradiol followed by EGF treatment; this progression was enhanced when estradiol was maintained during EGF treatment. This definite order demonstrated that estradiol could have a permissive effect on EGF mitogenic activity. In contrast, epithelial cells likely to be localized in the basal position in the mammary gland showed the same proliferating activity whatever the estradiol and EGF treatment. These cells progressed profusely in cell cycle, independent of exogenous estradiol and EGF contribution, but they remained sensitive to estradiol regarding EGF-receptor detection. Our results suggest autonomous proliferation of these cells through an autocrine pathway, in the absence of negative regulators. PMID- 1720428 TI - Ultrastructural study of the initial phases of pancreatic carcinoma induced by BOP in the Syrian golden hamster. AB - A serial study was carried out on the lesions induced by N-nitroso-bis(2 oxopropyl)amine (BOP) in the Syrian golden hamster until the appearance of pancreatic ductal carcinomas. During the initial phase, first findings were cytolysis of acinar cells close to blood vessels and other cells, together with a loss of zymogen granules from the cytoplasm, and an increase in the diameters of the acinar lumens. After week 11 a proliferation of ductule-like cells was observed. We consider that a minimum proliferation of cells at the acinus-ductule junction would give rise to pseudoductules composed of remains of acinar cells together with ductule-like cells. PMID- 1720429 TI - Orbital phlebography and signs of inflammation in episodic and chronic cluster headache. AB - One of our 7 patients (14%) with chronic cluster headache had an abnormal orbital phlebogram; this was significantly less than the 61% encountered in our 13 patients with active episodic cluster headache who had this test done. There were no pathologically increased values for serum haptoglobin or orosomucoid in our 9 patients with chronic cluster headache, again significantly less than in our 43 patients with active episodic cluster headache, 51 percent of whom had pathologically increased values of haptoglobin or orosomucoid. These inflammatory signs decreased after the episodic cluster headache was over. Episodic cluster headache we suggest to be due to temporary sympathicoplegia caused by venous vasculitis in the cavernous sinus region; chronic cluster headache we attribute to permanent post-inflammatory sympathicoplegia in the middle fossa. PMID- 1720430 TI - The use of RNA/DNA ratio measurements to assess rifampicin-induced growth inhibition of Escherichia coli. AB - Growth rates and cellular levels of RNA, DNA and protein were studied in Escherichia coli during and following exposure to rifampicin, and in the transition from stationary to exponential phase growth in drug-free medium. At rifampicin concentrations of up to twice the MIC, significant changes in growth rates were only apparent after several generations. At higher rifampicin concentrations growth was terminated much more rapidly. In every case changes in growth rates were closely paralleled by corresponding changes in RNA/DNA ratios. These findings suggest the possibility of using RNA/DNA ratio measurements to monitor the drug-induced growth inhibition of other bacteria. PMID- 1720431 TI - Anti-ampicillin monoclonal antibodies and their cross-reactivities to various beta-lactams. AB - Two cell lines producing monoclonal antibodies, Abp4 (IgM) and Abp7 (IgG1) against ampicillin were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. Abp4 showed broad cross-reaction to human serum albumin (HSA) conjugates of several beta-lactams, 6-aminopenicillanic acid and 7-aminocephalosporanic acid. Abp7 reacted only with ampicillin and cephalexin, which have the same acyl side chain. In ELISA inhibition tests, Abp4 inhibited binding to ampicillin-HSA by benzylpenicilloyl-epsilon-amino-n-caproic acid, and strongly inhibited the binding by penicillamine. Abp7 strongly inhibited the reaction by aminobenzylpenicilloyl-epsilon-amino-n-caproic acid, but benzylpenicilloyl-epsilon-amino-n-caproic acid was less effected. These data suggest that Abp4 recognizes the thiazolidine ring and Abp7 recognizes the acyl side chain. Therefore, the thiazolidine ring-epitope acts in broad cross-reaction among beta-lactams, and the acyl side chain acts only in the cross-reactivity between penam and cephem, which have a similar acyl side chain. PMID- 1720432 TI - Effect of combination therapy with recombinant human granulocyte colony stimulating factor (rG-CSF) and antibiotics in neutropenic mice unresponsive to antibiotics alone. AB - The effect of recombinant human granulocyte colony-stimulating factor (rG-CSF) in enhancing antimicrobial chemotherapy was investigated. Combined treatments of rG CSF with cefotaxime, cefazolin, fosfomycin, gentamicin or amphotericin B were evaluated in systemic infections with Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Staphylococcus aureus and Candida albicans in leucopenic mice induced by pretreatment with cyclophosphamide. Administration of appropriate antibiotics afforded a dose-related inhibition of death from infection in normal mice and mice treated with rG-CSF after cyclophosphamide injection. In cyclophosphamide-treated mice, even larger doses of the antibiotics failed to provide protection against infection with the same inoculum size. These results suggest the possibility that rG-CSF could be of help in treatments with antimicrobial agents against infections in leucopenic patients. PMID- 1720433 TI - Human dermal fibroblasts express multiple bFGF and aFGF proteins. AB - We investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions. PMID- 1720434 TI - Chronic myelomonocytic leukaemia (an analysis of fourteen consecutive cases). AB - Fourteen consecutive cases of chronic myelomonocytic leukaemia aged 6 to 73 (mean 40.5) years were reviewed to define the natural history of the disease and the risk of acute transformation. The common presenting features included anaemia, fever, purpura, and bleeding tendencies. Abnormal karyotypes were seen in 4 of 6 patients subjected to cytogenetic analysis. Low dose cytosine arabinoside achieved complete remission in two and partial remission in one, of the four patients treated with this modality. The mean survival was 5.6 (range 2-12) months and two patients) evolved to acute myeloid leukaemia. The long term survival with the present form of therapy in chronic myelomonocytic leukaemia is poor. PMID- 1720435 TI - Beta 2-glycoprotein-1 (apolipoprotein H) excretion in chronic renal tubular disorders: comparison with other protein markers of tubular malfunction. AB - Urinary beta 2-glycoprotein-1 was measured in 60 patients with conditions recognised as causing renal tubular impairment and compared with established markers of early tubular malfunction. Increased beta 2-glycoprotein-1 excretion was found in 49 (82%) of the subjects; raised excretion of alpha 1-microglobulin, retinol-binding protein, and beta 2-microglobulin was found in 46 (77%), 45 (75%), and 31 (52%), respectively, and increased urinary N-acetyl-beta-D glucosaminidase activity in 32 of 54 of the subjects (59%). The increase was particularly pronounced in those with proximal tubule malfunction, although considerable variation occurred. beta 2-glycoprotein-1 was shown to be stable in urine over the physiological pH range, and it is concluded that its measurement provides a means of detecting chronic malfunction of the renal tubules that is marginally more sensitive than assays of alpha 1-microglobulin or retinol-binding protein, and more reliable than assays of beta 2-microglobulin or N-acetyl-beta-D glucosaminidase. PMID- 1720436 TI - Early and late results of the modified Fontan procedure for double-inlet left ventricle: the Mayo Clinic experience. AB - Between May 1974 and March 1989, 155 patients with double-inlet left ventricle had the Fontan procedure performed at the Mayo Clinic. Age at operation ranged from nearly 2 to 41 years (median 10). The operative mortality rate from 1974 through 1980 (39 patients) was 21%, but from 1981 through 1989 (116 patients) it was reduced to 9%. The 17 late deaths were secondary to reoperation (n = 8), progressive myocardial failure (n = 5), sudden arrhythmia (n = 3) and bleeding varices (n = 1). Neither operative nor late mortality rate was significantly related to age at operation. At follow-up of 6 months to 11 years (mean 4.9 years) in 111 patients, 88% were in good or excellent condition and 12% were in fair or poor condition. The Fontan operation can be performed with a mortality risk of less than 10% in properly selected patients with double-inlet left ventricle. Late results are encouraging when contrasted with the clinical course of patients before this operative approach was utilized. PMID- 1720437 TI - Hormonal mechanisms of postprandial hypotension. PMID- 1720438 TI - Benign prostatic hyperplasia: pathogenesis and medical therapy. AB - Epidemiologic studies in castrates strongly support the key role of the testis in the pathogenesis of benign prostatic hyperplasia (BPH). Since the testis secretes androgen and estrogen, both of these hormones have been implicated in BPH. Much information supports the important role of dihydrotestosterone (DHT) in BPH, including intraprostatic activities of enzymes that regulate DHT. Although controversy still exists, DHT levels in BPH may be higher than in normal prostate tissue. Based upon these findings and the ability to quantitate prostate size and function with reliable new techniques, suppression of androgen-mediated action has been tested to assess the validity of the DHT theory. A variety of drugs has been demonstrated to decrease prostate size by approximately 30% by either blocking secretion of circulating testosterone and adrenal androgen, inhibiting 5 alpha-reductase to prevent DHT formation, or blocking DHT binding to androgen receptors. Accompanying these changes in size was significant improvement in clinical symptoms of prostatism in about 50% of patients, when double-blind, large multicenter studies were conducted with one of these drugs. Although these results suggest a very important role for androgen, particularly DHT, in the pathogenesis of BPH, other abnormalities may coexist since significant numbers of patients do not show a total reversal of disease. There is strong indirect evidence for a possible role for estrogen in the pathogenesis of BPH, and studies are under way to test the effects of estrogen withdrawal on prostate size and symptoms. Similarly, dynamic aspects of prostatic obstruction, which are under alpha-adrenergic regulation, may also be a component of this disease and amenable to therapy with alpha-adrenergic blockers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720440 TI - Biological studies and markers in suicide and attempted suicide. PMID- 1720439 TI - Combination therapy with ARA-AMP and interferon of chronic active hepatitis B. Interim analysis of an ongoing study. PMID- 1720441 TI - Investigation and prevention of artifactual staining in flow cytometric analyses of whole blood samples from patients treated with H65-RTA, an anti-CD5 monoclonal antibody conjugated to ricin A chain. AB - To assess the biological effect of therapeutic monoclonal antibodies (mAbs) immunoconjugates, flow cytometric assays are often performed on patients' blood samples. We report here that, using standard protocols, staining whole blood samples with mAb-fluorochromes can result in erroneous immune cell phenotyping. Blood samples were obtained from patients treated with H65-RTA, a murine IgG1 anti-CD5 mAb conjugated to ricin A chain. While a transient decrease in CD4+ and CD8+ cells was observed during the 5 days of therapy, alterations in lymphocyte phenotype were also noted, beginning around days 15-30. The most prominent effect was an apparent loss of CD4+ cells. However, if the cells were separated from plasma prior to staining, the patients' samples demonstrated their pre-treatment lymphocytic phenotypes. Co-incubation of post-treatment (day greater than or equal to 15) patients serum with lymphocytes from normal donors also resulted in artifactual staining. The effects of the post-treatment serum could be correlated with the onset of a human immune response directed against H65-RTA. Moreover, co incubation of normal PBMC with goat anti-mouse immunoglobulin could replicate the artifactual staining patterns. Even though the human immune response was generated against a murine IgG1 mAb, there were sufficient human antibodies crossreactive with other murine IgG isotypes to induce artifactual staining with all mAb-fluorochromes tested. To minimize artifacts, cells should be separated from plasma prior to flow cytometric analysis as well as for other immunoassays involving murine mAbs. PMID- 1720442 TI - A monoclonal antibody sandwich immunoassay for serum amyloid A (SAA) protein. AB - An antibody sandwich immunoassay using two purified rat monoclonal antibodies to human serum amyloid A was developed and used to measure serum amyloid A in human serum. The assay was specific, sensitive, reproducible, and reliable and does not require denaturation of the specimen prior to assay. Serum amyloid A purified by hydrophobic interaction chromatography of acute phase human serum afforded a reliable standard for the assay. A significant (r = 0.69) correlation for SAA and C reactive protein values was found for 180 patient samples analyzed. PMID- 1720443 TI - New antigenic determinants revealed on human IgG by binding to immunoblotting membranes [corrected]. AB - During immunoblotting for detection of human IgG, rabbit antibodies to goat IgG reacted with human IgG, Fc fragments and gamma chains when these proteins were bound to nitrocellulose or Immobilon-P membranes. This reactivity could not be adsorbed with human IgG-agarose beads or blocked with fluid phase human IgG. It was readily abrogated with human IgG adsorbed to one of these membranes, indicating that new antigenic determinants were exposed and accounted for the cross-reactivity. PMID- 1720444 TI - Copper stained protein gels can be efficiently used for immunoblotting. AB - We have found that the previously described fast and sensitive copper staining of proteins resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis does not interfere with the subsequent electrotransfer of these proteins to a solid support and their detection by specific antibodies. After the gel is copper stained and photographed it is simply destained and then equilibrated in transfer buffer prior to immunoblotting. We find that this treatment has no significant effect on transfer efficiency or band sharpness and is compatible with all common detection methods for the blotted proteins. It thus permits the separation of proteins to be checked in a simple way before immunoblotting is performed. PMID- 1720445 TI - [Arachidonic acid metabolism and effectiveness of aprotinin treatment during cardiopulmonary bypass]. AB - Arachidonic acid metabolism was investigated in 30 open heart cases, utilizing nonpulsatile cardiopulmonary bypass (CPB), consisted of 15 untreated cases (Group I) and 15 cases treated with aprotinin mostly given into CPB circuit during CPB (Group II). In group I, arterial blood concentration of thromboxane B2 (TXB2, stable metabolite of thromboxane A2, pg/ml) significantly increased from 45.9 +/- 40.5 preoperatively to 560.2 +/- 381.5 (p less than 0.01) at 30 minutes of CPB (total bypass) and to 830.5 +/- 591.1 (p less than 0.005) at the end of CPB (partial bypass). TXB2 levels in pulmonary artery (PA) and left atrium (LA) did not significantly increase just before, 5 minutes of CPB as compared with preoperative value. At the end of CPB TXB2 levels in PA (625.0 +/- 186.3) and LA (817.0 +/- 320.0) were significantly higher than preoperative value. However there was no significant difference between PA and LA values. Contrarily in group II TXB2 levels were significantly suppressed as compared with the value at each corresponding time in group I. beta-thromboglobulin levels also changed almost parallel to TXB2 levels in both groups. In conclusion, arachidonic acid metabolic disorders could occur in CPB circuit rather than in pulmonary circulation during CPB. Aprotinin administration into CPB circuit suppressed to some extent the platelet activation. PMID- 1720446 TI - [Staining of bone canaliculi using decalcified bone tissues]. AB - Recently we were able to stain bone canaliculi using the decalcified bone tissues, according to a modified method of the Bodian staining method, which is commonly used for staining the nerve fibers with silver impregnation of protargol. The bone tissues from normal rabbits, rats, and humans were decalcified with buffered EDTA solution after 10% formalin fixation. The tissues were embedded in paraffin and were sliced at 4 microns then the sections were attached to the gelatin-coated glass slides. These sections were treated first with potassium dichromate followed by 2% protargol solution containing copper. After gold chrolide treatment, they were immersed in oxalic acid. Both the lacunae and canaliculi of osteocytes were stained black in sharp contrast to bone matrix of red purple. Using the present staining method, the bone canaliculi were clearly detectable in fine details without producing apparent artificial damage of soft tissues. The canaliculi showed also markedly complex fine structures. By combining the present staining method with eosin or toluidine blue staining, we were able to recognize even immature osteocytes in enchondral ossification at growth cartilage with fine bone canaliculi. From these results, we conclude that the present method is very useful in histopathological studies of the lacuna canalicular system of osteocytes in the decalcified bone tissues. PMID- 1720447 TI - Cytochrome P450 isoforms. Regulation during infection, inflammation and by cytokines. AB - Multiple isoforms of cytochrome P450 (P450) have been discovered that are important biomedically to drug pharmacokinetics, chemical carcinogenesis, and metabolism of endogenous agents such as steroids, arachidonic acids, and prostaglandins. P450 level and activity are altered during infection and inflammation, apparently by cytokines released during activation of the immune system. Cytokines shown to affect P450 are interferon (IFN), interleukin 1 (IL 1), tumor necrosis factor (TNF), and interleukin 6 (IL-6). Understanding the enzymology and regulation of the P450 monooxygenation system in animals and humans may allow more accurate extrapolation of toxicity and cancer data from laboratory animals to clinical medicine as well as more rational selection of therapeutic regimens. PMID- 1720448 TI - Reconstitution of an epithelial chloride channel. Conservation of the channel from mudpuppy to man. AB - We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species. PMID- 1720449 TI - Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model. AB - cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents. PMID- 1720450 TI - Immunocytochemical analysis of glial cells in the hypomyelinated optic nerve of the BW mutant rat. AB - The Browman-Wyse (BW) rat is a mutant with structural defects of the visual system, including a failure of the proximal (retinal) end of the optic nerve to myelinate. This latter abnormality is correlated with an absence of CAII+ oligodendrocytes, but we have previously shown that astrocytes are normally distributed, as judged by morphological characteristics of GFAP+ cells in vivo. We have further examined in vitro the immunohistochemical characteristics of macroglia isolated from the BW optic nerve, either as cell suspensions or after 4 days in culture. Cell cultures derived from the hypomyelinated proximal segment of BW optic nerves contained very few 0-2A progenitor cells (from which oligodendrocytes and cells with the GFAP+/A2B5+ phenotype develop), whereas over 90% of the glia were Schwann cells. A proportion of these few 0-2A progenitor cells differentiated normally after 4 days in vitro into both progeny phenotypes in appropriate media. Accordingly, we conclude that the myelination deficiency in the BW optic nerve could be explained as a failure of 0-2A progenitor cells to populate fully the proximal extremity of the nerve during development. Since most glia isolated from adult optic nerves did not adhere to the culture substrate, we analysed the phenotypes of freshly isolated cells in suspension. Comparing optic nerves of normal adult rats with those of BW mutants, a significantly higher fraction of the GFAP+ cells reacted with A2B5 in cell suspensions of the latter. The double-labelled cells which are present in abnormally high numbers may be the differentiated progeny of 0-2A progenitors in the hypomyelinated segment of nerve. One explanation for these findings is that Schwann cells within the BW nerve induce the differentiation of 0-2A progenitor cells to the GFAP+/A2B5+ phenotype. We investigated this possibility using conditioned medium from cultured Schwann cells which increased tenfold the frequency of GFAP+/A2B5+ cells in normal neonatal rat optic nerve cultures. Oligodendrocyte numbers showed a concomitant decline with increasing concentration of Schwann cell conditioned medium. Hypomyelination in the BW rat optic nerve may therefore arise because Schwann cells, present in the proximal segment of the nerve, not only impede the migration of 0-2A progenitor cells but also release a factor which induces those 0-2A progenitor cells which arrive in the proximal segment of the nerve to differentiate into GFAP+ cells at a critical stage in oligodendrocyte development. PMID- 1720451 TI - P0 gene expression in cultured Schwann cells. AB - This study examines the expression of the major myelin protein gene P0 in cultured Schwann cells, grown on their own or in association with neurons. Many freshly dissociated Schwann cells from actively myelinating nerves express Po mRNA in high abundance. If neurons are not present, signal intensity falls markedly with time so that by 7 days in culture only a basal expression is evident which is negligible compared to the level in vivo. Dorsal root ganglia from embryo day 16 (E16) rats contain no significant levels of Po mRNA but when grown in full myelinating medium (containing serum and embryo extract) increasing expression is seen from 4 to 5 days onward even though myelination does not occur until after the second week. In this intervening period the intensity of P0 mRNA expression is lower than that found in the actively myelinating cell. Neurons from sympathetic ganglia are also capable of inducing P0 mRNA expression. Schwann cells in dorsal root ganglia explants grown in serum-free defined medium do not assemble a basal lamina and will not wrap or myelinate axons. Nevertheless P0 mRNA, but not protein, is expressed in levels similar to those found in full myelinating medium prior to myelination. Such Schwann cells also exhibit galactocerebroside and the sulphatide recognised by the 04 antibody. It appears that in defined medium or in myelinating medium prior to myelination axonal signals can induce P0 mRNA expression to a certain degree. However, full up regulation is usually associated with the rapid membrane expansion accompanying myelination. Whether this augmented up-regulation is due to further axonal signalling or events in the Schwann cell is unknown, but the results suggest that P0 expression can be regulated at several stages of synthesis. PMID- 1720452 TI - Effective treatment of unresectable or metastatic hepatoblastoma with cisplatin and continuous infusion doxorubicin chemotherapy: a report from the Childrens Cancer Study Group. AB - The Childrens Cancer Study Group (CCSG) undertook a study (CCG-823F) to test the feasibility of administering continuous infusion doxorubicin (CI DOX) and cisplatin (CDDP) in patients with unresectable or incompletely resected hepatoblastoma (HB) or hepatocellular carcinoma (HCC). Chemotherapy consisted of CI DOX 20 mg/m2/d for days 1 to 4 and CDDP 100 mg/m2 on day 1 followed by a 21 day rest period. Second-look surgery was performed after the administration of four chemotherapy courses. Forty-seven (47) assessable patients were entered on study, 33 with HB and 14 with HCC; of these, 34 (26 HB and eight HCC) completed the initial four courses of chemotherapy. Of the 26 HB patients, 25 were evaluated as responding to chemotherapy before the scheduled second-look procedure and were considered surgically resectable at that time. Surgery was performed on 22 patients; three patients refused the second-look surgery. Nine patients had no evidence of residual malignant disease, seven underwent surgical resection of remaining tumor, four were left with microscopic residual disease, one had a partial resection with gross tumor left behind, and one remained unresectable. Nine HCC patients completed four chemotherapy courses. Eight patients achieved a partial remission and second-look surgery was attempted on seven. Only two had all malignant disease removed at the second procedure. Data from 225 courses of chemotherapy were evaluated for toxicity. Neutropenia (absolute granulocyte count less than 500/mL) was observed in 68 courses, and five of these episodes were associated with sepsis. Severe mucositis was documented in 21 courses, and hypomagnesemia (magnesium less than 1.2 mg) was noted in 30 patients. Two patients developed decreased left ventricular shortening fraction, which resolved when chemotherapy was discontinued. In summary, CI DOX plus CDDP is a well-tolerated and effective regimen in inducing surgical resectability in HB patients who are unresectable at diagnosis and significantly improves survival for this group of patients to 66.6%. PMID- 1720453 TI - Long-term follow-up of ifosfamide renal toxicity in children treated for malignant mesenchymal tumors: an International Society of Pediatric Oncology report. AB - The renal function of 74 children with malignant mesenchymal tumors in complete remission and who have received the same ifosfamide chemotherapy protocol (International Society of Pediatric Oncology Malignant Mesenchymal Tumor Study 84 [SIOP MMT 84]) were studied 1 year after the completion of treatment. Total cumulative doses were 36 or 60 g/m2 of ifosfamide (six or 10 cycles of ifosfamide, vincristine, and dactinomycin [IVA]). None of them had received cisplatin chemotherapy. Ages ranged from 4 months to 17 years; 58 patients were males and 42 females. The most common primary tumor site was the head and neck. Renal function was investigated by measuring plasma and urinary electrolytes, glucosuria, proteinuria, aminoaciduria, urinary pH, osmolarity, creatinine clearance, phosphate tubular reabsorption, beta 2 microglobulinuria, and lysozymuria. Fifty-eight patients (78%) had normal renal tests, whereas 16 patients (22%) had renal abnormalities. Two subsets of patients were identified from this latter group: the first included four patients (5% of the total population) who developed major toxicity resulting in Fanconi's syndrome (TDFS); and the second group included five patients with elevated beta 2 microglobulinuria and low phosphate reabsorption. The remaining seven patients had isolated beta 2 microglobulinuria. Severe toxicity was correlated with the higher cumulative dose of 60 g/m2 of ifosfamide, a younger age (less than 2 1/2 years old), and a predominance of vesicoprostatic tumor involvement. This low percentage (5%) of TDFS must be evaluated with respect to the efficacy of ifosfamide in the treatment of mesenchymal tumors in children. PMID- 1720454 TI - Intensive weekly combination chemotherapy for patients with intermediate-grade and high-grade non-Hodgkin's lymphoma. AB - High response and overall survival rates have been reported for second- and third generation combination chemotherapy regimens used in the treatment of advanced intermediate- and high-grade non-Hodgkin's lymphoma (NHL). Results with methotrexate with leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) chemotherapy have been particularly impressive, although this regimen produces considerable toxicity. We have devised a similar regimen, which differs from previously reported weekly regimens in that it includes etoposide given at 14-day intervals. The doses of methotrexate and prednisolone were lower in our regimen than those used in MACOP-B. Alternating cycles of cyclophosphamide, doxorubicin, and etoposide (week 1) and methotrexate, bleomycin, and vincristine (week 2) were given for a total of 12 weeks, with continuous oral prednisolone and prophylactic antibiotics. We report here the first 61 patients entered onto this study. The overall response rate is 84% (57% complete remission [CR], 27% partial remission [PR]). With a median follow-up of 32 months for surviving patients, the actuarial overall survival at 3 years is 47%, and the failure-free survival is 45%. The dose-limiting toxicity of this regimen was mucositis. Five deaths occurred during chemotherapy, two of which were due to sepsis. The dose intensities of cyclophosphamide and doxorubicin in this regimen are considerably lower than those in MACOP-B. However, because of the inclusion of etoposide, the projected average relative dose intensity for our regimen is higher than that for MACOP-B. Our regimen has produced inferior results to those reported for MACOP-B. This may be because the addition of etoposide has failed to compensate for the lower doses of doxorubicin and cyclophosphamide. Alternatively, it may reflect differences in the presenting features of the patient populations. PMID- 1720455 TI - Effects of drugs on pituitary fine structure in laboratory animals. AB - Although an increasing number of chemicals are reported to affect endocrine glands, only a few studies are dealing with their toxic effect on pituitary. The drugs can induce lesions acting directly on endocrine cells or indirectly by interfering with the regulation of their endocrine activities. Some drugs stimulate pituitary cell proliferation leading to hyperplasia and tumor formation; other chemicals have an inhibitory effect on adenohypophysial cells; and only one drug, hexadimethrine bromide, has been found to induce pituitary necrosis. Although complex toxicologic studies have been carried out on many chemicals, the mechanism of action of most drugs is not completely elucidated and further studies are necessary to establish structure function correlations. PMID- 1720456 TI - Outcome of apparently stillborn infants. PMID- 1720457 TI - Medical and psychosocial outcome of children with congenital central hypoventilation syndrome. AB - We report the long-term medical and psychosocial outcome of 13 children with congenital central hypoventilation syndrome. One child (8%) died before initial hospital discharge. Of the remaining 12 children, 11 (92%) have been successfully cared for in their natural or foster parents' homes. Home ventilatory support was provided with positive-pressure ventilation, negative-pressure ventilation, or diaphragm pacers. After an initial lengthy hospitalization, children spent little time in the hospital. Severe medical complications were uncommon but included cor pulmonale (one child), poor growth (two children), and seizure disorder (three children). Most children functioned in the slow-learner range of mental processing, with a composite score (Kaufman Assessment Battery for Children) of 78 +/- 20 (SD); two were mentally retarded, and one functioned above the normal range. The children's care givers were assessed as having low levels of psychologic distress (Symptom Checklist 90--Revised) and good coping resources (Coping Resources Inventory) but a high level of marital discord. The children were able to attend school and partake in normal childhood activities. We conclude that with modern techniques for home ventilation, children with CCHS can have a good long-term medical and psychosocial outcome. We speculate that early diagnosis and the prevention of intermittent hypoxia will improve their physical and mental outcome. PMID- 1720458 TI - Prospective treatment of urea cycle disorders. AB - We present a diagnostic and therapeutic protocol designed to prevent clinical expression of inborn errors of urea synthesis in the neonatal period, and discuss the long-term developmental outcome of survivors. The families of 32 infants, among 43 identified prenatally as being at risk for a urea cycle disorder, chose to have their infants treated according to a diagnostic and therapeutic protocol, beginning at birth. The therapy was effective in avoiding neonatal hyperammonemic coma and death in seven patients with carbamoyl phosphate synthetase deficiency, argininosuccinate synthetase deficiency, and argininosuccinate lyase deficiency. When treated prospectively, five of eight patients with ornithine transcarbamylase deficiency avoided severe hyperammonemia and survived the neonatal period. Two patients with carbamoyl phosphate synthetase deficiency and two with ornithine transcarbamylase deficiency have subsequently died; three additional patients with the latter disorder have received orthotopic liver transplants. Our experience suggests that these surviving patients have had a more favorable neurologic outcome than patients rescued from neonatal hyperammonemic coma. However, all of them require a burdensome medical regimen and may have handicaps that include impairment of development and recurrent episodes of hyperammonemia. Further, those with deficiency of carbamoyl phosphate synthetase or ornithine transcarbamylase have a high mortality rate. PMID- 1720460 TI - Palliative care. Guidelines for good practice and audit measures. Report of a Working Group of the Research Unit, Royal College of Physicians. PMID- 1720459 TI - WISC-test scores at the age of 10 for children born to women with risk pregnancies. AB - The subjects (N = 50) were born to mothers who had earlier participated in an extensive clinical investigation during their pregnancies. Maternal serum hormone levels were investigated from pregnancy week 20 and on to partus. Fourteen children were born small-for-gestational age (SGA) and eight pre-term appropriate for-gestational age (AGA). Each SGA child had two control children, born to mothers with normal or high serum levels of alpha-fetoprotein (AFP) in pregnancy week 16-17. Elevated serum levels of AFP was considered to be a sign of fetal stress. All 50 children were administered the WISC-test at 10 years of age. The SGA children had lower scores than control children in Performance and Full scale scores. The pre-term SGA children had lower scores than the controls in Verbal, Performance and Full scales. That was not the case for on-time SGA and pre-term AGA children. Girls born to smoking mothers performed less well than girls of non smoking mothers on the Verbal scale. Positive correlations of maternal serum hormone levels (oestriol, hCG, and hPL) and WISC-test scores were present for girls. For boys a single maternal hormone in pregnancy (prolactin) was correlated with WISC-test scores at 10 years of age. PMID- 1720461 TI - Endometrial T-lymphocyte subset infiltration during the ovine estrous cycle and early pregnancy. AB - T-lymphocytes were quantitated within luminal, stromal and glandular areas of ovine endometrium. In experiment 1, ovariectomized (OVX), estrus (E) and day 13 (D13) ewes (six/group) received 500 micrograms of phytohemagglutinin (PHA) or vehicle in ligated right and left uterine horns, respectively. At 48 h, uteri were removed for the immunohistochemical evaluation of T-lymphocyte subsets. In experiment 2, T-lymphocytes were quantitated within non-pregnant and pregnant uterine horns on day 19. For experiment 1, mean numbers of T4 and T8 lymphocytes within luminal and stromal areas of PHA-treated horns were greatest (P less than 0.05) for D13 ewes and least (P less than 0.05) for E ewes. Numbers of T6 lymphocytes for these same areas were greatest (P less than 0.05) for PHA-treated horns of OVX ewes. Overall, the T4/T8 ratio (P less than 0.004) and mean number of T19 cells (P less than 0.009) were increased by PHA. Numbers of CD45R lymphocytes were not affected by PHA but were greater (P less than 0.05) in glandular and luminal than stromal areas. For experiment 2, mean numbers of endometrial T4, T6, T8 and T19 lymphocytes were similar (P greater than 0.05) between non-pregnant and pregnant horns; however, the number of CD45R lymphocytes was greater (P less than 0.05) in endometrial tissue of pregnant than non pregnant horns. The data indicate that the in vivo response of specific ovine T lymphocytes to PHA was generally dependent upon reproductive stage and the presence of conceptus tissue influenced the infiltration of CD45R lymphocytes. PMID- 1720462 TI - Fate of the junction phosphate in alternating forward and reverse self-splicing reactions of group II intron RNA. AB - The RNA-catalysed self-splicing reaction of group II intron RNA is assumed to proceed by two consecutive transesterification steps, accompanied by lariat formation. This is effectively analogous to the small nuclear ribonucleoprotein (snRNP)-mediated nuclear pre-mRNA splicing process. Upon excision from pre-RNA, a group II lariat intervening sequence (IVS) has the capacity to re-integrate into its cognate exons, reconstituting the original pre-RNA. The process of reverse self-splicing is presumed to be a true reversion of both transesterification steps used in forward splicing. To investigate the fate of the esterified phosphate groups in splicing we assayed various exon substrates (5'E-*p3'E) containing a unique 32P-labelled phosphodiester at the ligation junction. In combined studies of alternating reverse and forward splicing we have demonstrated that the labelled phosphorus atom is displaced in conjunction with the 3' exon from the ligation junction to the 3' splice site and vice versa. Neither the nature of the 3' exon sequence nor its sequence composition acts as a prominent determinant for both substrate specificity and site-specific transesterification reactions catalysed by bI1 IVS. A cytosine ribonucleotide (pCp; pCOH) or even deoxyoligonucleotides could function as an efficient substitute for the authentic 3' exon in reverse and in forward splicing. Furthermore, the 3' exon can be single monophosphate group. Upon incubation of 3' phosphorylated 5' exon substrate (5'E-*p) with lariat IVS the 3'-terminal phosphate group is transferred in reverse and forward splicing like an authentic 3' exon, but with lower efficiency. In the absence of 3' exon nucleotides, it appears that substrate specificity is provided predominantly by the base-pairing interactions of the intronic exon binding site (EBS) sequences with the intron binding site (IBS) sequences in the 5' exon. These studies substantiate the predicted transesterification pathway in forward and reverse splicing and extend the catalytic repertoire of group II IVS in that they can act as a potential and sequence-specific transferase in vitro. PMID- 1720463 TI - Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. AB - Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule. PMID- 1720464 TI - Distribution and expression of two interactive extracellular matrix proteins, cytotactin and cytotactin-binding proteoglycan, during development of Xenopus laevis. I. Embryonic development. AB - An immunohistochemical study of the localization of cytotactin and cytotactin binding (CTB) proteoglycan throughout embryonic development of the anuran Xenopus laevis reveals that both appear in a restricted pattern related to specific morphogenetic events. CTB proteoglycan expression is first detected during gastrulation at the blastopore lip. Later, it is seen in the archenteron roof around groups of cells forming the notochord, somites and neural plate. Cytotactin first appears after neurulation, and is restricted to the intersomitic regions. Both molecules appear along the migratory pathways of neural crest cells in the trunk and tail. Later, cytotactin is present at sites where neural crest cells differentiate, around the aorta and in the smooth muscle coat of the gut; CTB proteoglycan is absent from these sites. In the head, cytotactin is initially restricted to the regions between cranial somites, while CTB proteoglycan is distributed throughout the cranial mesenchyme. The expression of both molecules is later associated with key events in chondrogenesis during the development of the skull. After chondrogenesis, CTB proteoglycan is distributed throughout the cartilage matrix, while cytotactin is restricted to a thin perichondrial deposit. Both molecules are expressed in developing brain. These findings are compared to studies of the chick embryo and although distinct anatomical differences exist between frog and chick, the expression of these molecules is associated with similar developmental processes in both species. These include mesoderm segmentation, neural crest cell migration and differentiation, cartilage development, and central nervous system histogenesis. PMID- 1720465 TI - Distribution and expression of two interactive extracellular matrix proteins, cytotactin and cytotactin-binding proteoglycan, during development of Xenopus laevis. II. Metamorphosis. AB - During metamorphosis of Xenopus laevis the extracellular matrix (ECM) proteins cytotactin and cytotactin-binding (CTB) proteoglycan and the cell adhesion molecules N-CAM and Ng-CAM, appear in highly restricted patterns determined by immunofluorescence histology. During limb development, cytotactin appears from the earliest stages in a meshwork of ECM fibrils associated with migrating mesenchymal cells forming the limb bud. Cytotactin also appears in the ECM between the apical limb ectoderm and mesenchyme. Later, both cytotactin and CTB proteoglycan appear co-localized within the central (prechondrogenic) limb mesenchyme. During chondrogenesis in these areas, cytotactin becomes restricted to perichondrium, while CTB proteoglycan is expressed throughout the cartilage matrix. The premyogenic mesenchyme surrounding the chondrogenic areas expressed N CAM. Later, N-CAM is concentrated at the myogenic foci where cytotactin appears at sites of nerve/muscle contact and in tendons. Expression of these molecules in the blastemas of regenerating limbs was also studied, and during development of the central nervous system, stomach, and small intestine. Analysis of the expression patterns of cytotactin and CTB proteoglycan throughout development and metamorphosis reveals several consistent themes. The expression of these molecules is highly dynamic, often transient, and associated with key morphogenetic events. Cytotactin appears at multiple sites where cells undergo a transition from an undifferentiated, migratory phenotype to a differentiated phenotype. One or both molecules appear at several sites of border formation between disparate cell collectives, and CTB proteoglycan expression is associated with chondrogenesis. PMID- 1720466 TI - Rearrangements of mitochondrial transfer RNA genes in marsupials. AB - The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNA(Tyr) gene at the same position as in placental mammals and Xenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and; in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNA(Tyr) gene has been evolving under more constraints than the other tRNA genes. PMID- 1720467 TI - Long-term dietary study and development of no-observed-effect level (NOEL) of technical HCH to rats. AB - Daily feeding of technical hexachlorocyclohexane (HCH) (0.5, 5, 25, 250, and 500 mg/kg diet/d) for a period of 360 d to male rats elicited a dose-dependent toxicity, while the dose of technical HCH (0.5 mg/kg diet/d) did not produce signs of intoxication, mortality, organ body weight ratio, enzymatic, and pathomorphological changes. Other doses (5, 25, 250, and 500 mg/kg diet/d) produced significant changes in one or the other parameters studied. Based on this study, it may be suggested that the lowest dose of technical HCH (0.5 mg/kg diet/d) could be considered as the no-observed-effect level in experimental rats. PMID- 1720469 TI - [Enzyme inhibitors related to the nucleic acids biosynthesis and gene expression]. PMID- 1720468 TI - Flow cytometric assessment of platelet function in patients with peripheral arterial occlusive disease. AB - This study compared new and traditional measures of platelet function in 16 patients with severe peripheral arterial occlusive disease and 15 age-matched controls. Circulating platelets were characterized by the use of fluorescence flow cytometry to assess platelet aggregate formation and expression of the secretion-dependent alpha granule membrane protein GMP-140, by measurement of plasma beta-thromboglobulin (beta-TG), and by performance of platelet-rich plasma aggregation studies. In addition, blood samples were treated with graded concentrations of adenosine diphosphate (ADP; 0 to 10 mumols/L) to characterize by fluorescence flow cytometry the secretory and aggregatory responses to mild stimulation. No differences were detected between the two groups with regard to platelet function in unstimulated circulating blood by use of these techniques. Values (mean +/- SEM) observed were: GMP-140-positive platelets, 11% +/- 3% versus 13% +/- 2%; platelet aggregates in circulating whole blood, 4% +/- 1% versus 9% +/- 3%; plasma beta-TG, 92 +/- 12 versus 94 +/- 22 ng/ml; and ED50 (concentration of ADP required to produce half maximal aggregation), 3.8 +/- 1.1 versus 3.1 +/- 0.5 mumol/L in the patients with peripheral arterial occlusive disease and controls, respectively. Treatment with ADP caused a dose-related increase in GMP-140 expression in both groups, without significant differences in this parameter between the groups at any given concentration. However, stimulation with ADP concentrations greater than 1 mumol/L resulted in more frequent aggregate formation in the control than in the peripheral arterial occlusive disease group (25% +/- 4% versus 11% +/- 2%, respectively at 5.0 mumols/L, p = 0.002).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720470 TI - [Inhibitors of HIV-1 reverse transcriptase and protease as chemotherapeutics for AIDS]. PMID- 1720471 TI - [Protease inhibitors in shock]. PMID- 1720472 TI - [Protease inhibitors in the treatment of pancreatitis]. PMID- 1720473 TI - Dynamic changes in the rate of amylase release induced by various secretagogues examined in isolated rat parotid cells by using column perifusion. AB - Isolated parotid acinar cells were perifused in small columns by embedding them in Bio-Gel P-2 beads as an inert supporting matrix, and the effect of carbamylcholine, substance P, and isoproterenol on the rate of amylase release was examined by measuring amylase activity in the effluent. Amylase release by continuous stimulation with carbamylcholine and substance P was biphasic. They caused a rapid and large increase in the rate of amylase release that reached maximum 30 to 60s after the onset of stimulation, followed by a rapid decline to a lower sustained level that was maintained as long as the agonists were present. The rapid decline in the rate of amylase release was due to rapid development of refractoriness. Repeated 1 min pulse stimulation with these secretagogues showed that recovery from refractoriness was also rapid in onset, and 1 min of washout was sufficient to cause significant recovery from refractoriness for both carbamylcholine and substance P. Recovery, however, was not complete after 10 min of washout. Amylase release by continuous stimulation with isoproterenol, on the other hand, developed more slowly with the peak rate being attained at about 6 min after the onset of stimulation. Refractoriness was not observed in the effect of isoproterenol. The maximum effect in the rate of amylase release attained by carbamylcholine or substance P was higher than that by isoproterenol. These results suggest that the apparent small effect of carbamylcholine and substance P on amylase release reported earlier by using batch systems is probably due to the rapid development of refractoriness to these secretagogues, but not to isoproterenol. PMID- 1720474 TI - Factors influencing the acute phase protein levels in patients with esophageal cancer. AB - Based on the findings that the enhancement of serum alpha 2-macroglobulin (A2M) is associated with the occurrence of infectious complications following surgery in patients with esophageal cancer, we examined possible factors which could contribute to the alterations of serum acute phase protein levels in patients with this disease. A multiple linear regression analysis was made for 71 patients with esophageal cancer and 58 with gastric cancer. In patients with esophageal cancer, protein calorie malnutrition (PCM) and age factors more strongly contributed to the alteration of 6 acute phase protein levels than did the malignant tumor when compared to those with gastric acner. PCM was negatively associated with A2M levels while it was positively associated with alpha 1 acidglycoprotein (A1AG) and haptoglobin (Hp) levels. Age did not contribute to the A2M levels but did have a negative effect on the Hp, ceruloplasmin (Cp) and fibronectin (Fn) levels. On the other hand, the malignant tumor was positively related only to the A1AG levels. Since none of these factors contributed to the elevation of A2M levels, it is suggested that the presence of chronic infection might be a factor contributing to the A2M increase which was associated with the occurrence of postoperative infectious complications in patients with this disease. PMID- 1720475 TI - [Determination of plasma atrial natriuretic peptide in follow-up of hypervolemic hemodilution in patients with retinal circulatory disorders]. AB - We measured plasma levels of atrial natriuretic peptide (ANP) in 20 patients with impaired retinal circulation in order to monitor hypervolemic hemodilution. In 18 control subjects, plasma ANP levels averaged 37.4 +/- 7.3 pg/ml and 30.0 +/- 5.4 pg/ml before and after an i.v. infusion of 250 ml of HAES 6% over 3 hours. In 8 of 20 patients, plasma ANP exceeded the highest value of 87 pg/ml in the controls at the end of the first infusion. In the group B, ANP increased from 103 +/- 21.6 pg/ml to 142 +/- 12 pg/ml before and after the infusion. In the remaining 12 patients (group A), plasma levels of ANP were 41.1 +/- 8.6 and 44.8 +/- 6.3 pg/ml at the start and the end of the first infusion of HAES. All patients received 500 ml of HAES daily for 8 days. In group B, hemoglobin was significantly lower on day 8 (11.7 +/- 0.48 versus 13.2 +/- 0.38 mg/100 ml) compared to group A (p less than 0.005). Similarly, hematocrit and serum protein was reduced to a greater extent in group B compared to group A indicating intravascular volume expansion. Measuring plasma ANP-levels might be of value to identify patients with reduced tolerance to hemodilution therapy. PMID- 1720476 TI - Calcium antagonists as first-line therapy in hypertension: results of the Swiss Isradipine Study. Swiss Hypertension Society. AB - In this study of the efficacy and safety of isradipine as first-line therapy in hypertension, 1,647 patients enrolled; 1,472 completed the 4-week placebo run-in period and began treatment with isradipine at 2.5 mg twice daily for 4 weeks. During placebo, 11% (n = 175) of the 1,647 patients withdrew because of normalization of blood pressure, side effects, noncompliance, violation of the study protocol, side effects from concomitant therapy, or other reasons. During isradipine therapy (n = 1,376), blood pressure decreased from 168 +/- 18/102 +/- 8 mm Hg at the end of the placebo period to 155 +/- 17/94 +/- 9 mm Hg after 2 weeks (p less than 0.001) and 151 +/- 16/92 +/- 9 mm Hg after 4 weeks (p less than 0.001). During active treatment, 6.4% (n = 94) were withdrawn because of flushing, headache, edema, palpitations, gastrointestinal side effects, skin rashes, or other side effects, and two patients because of lack of efficacy. The side effect score in the remaining patients worsened for flushing, remained unchanged for edema, but significantly improved for palpitations, fatigue, dizziness, headache, and nervousness. After 4 weeks, 60% of patients had diastolic blood pressures of less than or equal to 90 mm Hg. Thus, isradipine is effective and safe as first-line therapy in patients with primary hypertension as seen in general practice. PMID- 1720477 TI - Comprehensive cardiovascular care with a first-line antihypertensive agent. Proceeding of a symposium. Basel, Switzerland, February 13-15, 1991. PMID- 1720478 TI - Evaluation of isradipine and captopril alone or in combination for the treatment of hypertension. AB - The antihypertensive effects of isradipine and captopril were studied in 231 patients with mild-to-moderate hypertension in a double-blind, randomized, between-patient trial. Treatment was started with 1.25 mg of isradipine or 12.5 mg of captopril twice daily which, if normotension was not obtained, was increased after 4 weeks to 2.5 mg or 25 mg twice daily, respectively. If the maximum dose of each drug alone did not result in normotension, captopril (12.5 mg or, if necessary, 25 mg twice daily) was added to the isradipine regimen, and isradipine (1.25 mg or, if necessary, 2.5 mg twice daily) to the captopril regimen. Following 24 weeks of active treatment, systolic blood pressure (SBP) was significantly (p less than 0.001) reduced by isradipine (170 +/- 17 vs. 153 +/- 17 mm Hg) and by captopril (170 +/- 19 vs. 155 +/- 19 mm Hg). Diastolic blood pressure (DBP) also fell significantly (p less than 0.001) in both groups (isradipine: 106 +/- 5 vs. 93 +/- 8 mm Hg; captopril: 106 +/- 5 vs. 95 +/- 10 mm Hg). After isradipine as monotherapy, DBP was normalized in 49% of patients compared with 52% after captopril monotherapy. Combination of both drugs increased the normalization rate to 84%. The results indicate that combined treatment with a calcium antagonist and an angiotensin-converting enzyme (ACE) inhibitor is effective in lowering blood pressure, and is well tolerated as long term therapy. PMID- 1720479 TI - The Multicenter Isradipine/Diuretic Atherosclerosis Study: a study of the antiatherogenic properties of isradipine in hypertensive patients. MIDAS Research Group. AB - Hypertension is a risk factor for the development of atherosclerosis and its complications, which are among the major causes of morbidity and mortality. Although recent clinical trials indicate that antihypertensive treatment reduces morbidity and mortality associated with stroke, congestive heart failure, and renal insufficiency, questions remain as to whether such treatment also prevents coronary heart disease (CHD) mortality. The observed reduction in CHD mortality from pooled clinical trial data was 10-14% and was much less than the expected 20 25% reduction for a 5-6 mm Hg reduction in diastolic pressure. One explanation may be that subtle adverse metabolic effects of treatment may have blunted the beneficial effects. Isradipine, a dihydropyridine calcium antagonist, is a potent antihypertensive drug with antiatherogenic properties in animal models. Therefore, we hypothesized that isradipine may be appropriate for testing the efficacy of antihypertensive treatment in retarding the progression of atherosclerosis in humans. The Multicenter Isradipine/Diuretic Atherosclerosis Study (MIDAS) is a clinical trial designed to compare the efficacy of isradipine (2.5 or 5 mg b.i.d.) with hydrochlorothiazide (12.5 or 25 mg b.i.d.) in retarding the progression of early carotid atherosclerosis as monitored by high-resolution B-mode ultrasonography. PMID- 1720480 TI - Coronary vascular changes in the progression and regression of hypertensive heart disease. AB - Coronary hemodynamics (blood flow, coronary reserve, myocardial oxygen consumption) were analyzed in both experimental and clinical hypertension. Significantly reduced coronary reserve was found in hypertensive patients with left ventricular hypertrophy. Medial hypertrophy of small coronary vessels associated with a marked increase in the wall thickness/radius ratio was considered sufficient to explain the impaired coronary flow in hypertensive left ventricular hypertrophy. After long-term pharmacotherapy, there was normalization of both medial hypertrophy and coronary reserve. This small-vessel abnormality correlates well with clinical findings in hypertensive heart disease (angina and electrocardiographic changes despite normal coronary arteriogram). Moreover, this structural adaptation of the small vessels may carry the inherent risk of an impaired oxygen supply to the hypertrophied myocardium. Thus, late cardiac failure of the hypertrophied heart in hypertension may be attributed, in part, to this microcirculation disorder. Conversely, reversal of left ventricular hypertrophy and of hypertrophy of vascular smooth muscle by specific pharmacotherapy can be considered a possible rational approach to the prevention of cardiac failure in hypertensive patients. Controlled clinical trials are needed to confirm these findings with regard to prevention of heart failure, and pharmacotherapeutic studies are necessary to define the optimal drug regimen for reversal of vascular smooth muscle hypertrophy. PMID- 1720481 TI - Reversal of left ventricular hypertrophy following treatment of hypertension with isradipine. AB - In order to complement earlier short-term observations, we studied the effects of isradipine (1.25 or 2.5 mg twice daily) on blood pressure as well as its action in reversing cardiac hypertrophy in 25 moderately hypertensive patients. We observed that the treatment produced short-term (3 month) and longer-term (9 month) control of blood pressure [decreases in mean arterial pressure (MAP) from 128 +/- 2.3 to 112 +/- 2.7 mm Hg and to 105.5 +/- 2.9 mm Hg; p less than 0.001] while heart rate remained constant throughout the study (from 76.6 +/- 2.3 to 74.7 +/- 2.4 beats/min; NS). Reversal of left ventricular hypertrophy (LVH) obtained after 3 months of treatment (LV mass index from 173.7 +/- 8.8 to 135.7 +/- 4.5 g/m2; p less than 0.001) was accentuated with continued therapy (to 131.0 +/- 4.0 and 124.4 +/- 3.1 g/m2 at 6 and 9 months, respectively; p less than 0.01). These results indicate that significant regression of LVH can be obtained with short-term treatment of hypertension with isradipine and that this effect will be fully obtained with longer-term (9 month) therapy. PMID- 1720482 TI - Serotonin and platelet activation during treatment with isradipine. AB - The effect of the calcium antagonist isradipine on serotonin metabolism and platelet aggregation was studied in 17 patients with essential hypertension. Platelet serotonin content, plasma serotonin, 5-hydroxyindoleacetic acid levels, and platelet aggregation [induced ex vivo by serotonin and low-density lipoprotein (LDL)] were measured after a 4-week placebo period and after 12 weeks of oral treatment with isradipine. Isradipine treatment significantly inhibited platelet aggregation induced by LDL and serotonin; the amplifying effect of LDL on serotonin-induced aggregation seen with placebo was not observed after 12 weeks of treatment with isradipine. Platelet serotonin content increased significantly during isradipine treatment; this increase was inversely related to the pretreatment content of serotonin in platelets. The results indicate that treatment with isradipine restores the impaired handling of platelet serotonin as well as the platelet response to serotonin and LDL in hypertensive patients. This effect of isradipine may be regarded as one of the cellular mechanisms of thrombovascular protection and may be of clinical significance in terms of platelet and vessel wall interaction. PMID- 1720483 TI - Does antihypertensive therapy affect the natural protection against thrombosis? AB - Fibrinolytic activity was measured in a 14-day placebo-controlled study of propranolol and the calcium antagonist isradipine in 20 mildly hypertensive patients (diastolic blood pressure 95-115 mm Hg) compared with 24 healthy volunteers. The parameters under study included euglobulin clot-lysis time, and tissue-plasminogen activator activity and its inhibitor (PAI). The two drugs exerted equal antihypertensive effects in the patients who had raised blood pressure, but had markedly different actions on the fibrinolytic system. Propranolol substantially reduced the fibrinolytic activity in both the hypertensive and healthy control groups. Isradipine, on the other hand, had no effect on fibrinolytic activity in the controls, but augmented the activity in the hypertensive subjects. The possible mechanisms for the different actions of the two agents may be related to the vascular endothelium. PMID- 1720484 TI - Isradipine, a new calcium antagonist: effects on maternal and fetal hemodynamics. AB - To assess the effect of isradipine on blood pressure, and uteroplacental and fetal blood flows in pregnancy-induced hypertension, 41 women with a diastolic blood pressure greater than or equal to 95 mm Hg were included in our study. Of these, 27 received isradipine 5 mg twice daily. Uteroplacental blood flow index was calculated from the activity-time curve of the very low radiation from the placenta after intravenous injection of 18.5 MBq indium-113m. Blood flow velocity was measured in the uterine and umbilical arteries, and fetal aorta, using the pulsed Doppler technique. Investigations were performed before and after 1 week of isradipine. A control group of 14 women was examined in the same way. The isradipine group showed a significant reduction in mean arterial pressure (from 117 to 112 mm Hg) whereas there was no change in the controls. Uteroplacental blood flow indices were similar before treatment in both groups and did not change during treatment. Furthermore, the pulsatility indices were the same in both groups at the first examination and also did not change. Isradipine had no adverse effects on the fetus. In conclusion, isradipine lowered blood pressure without altering uteroplacental or fetal blood flows. PMID- 1720485 TI - First clinical experience with isradipine in the treatment of hypertension in Portugal. AB - The efficacy and safety of isradipine and nifedipine retard were compared in 51 patients with mild-to-moderate essential hypertension. A 4-week placebo run-in period was followed by an 8-week course of treatment. Patients were randomly allocated to either isradipine 1.25 mg twice daily (n = 24) or nifedipine 20 mg twice daily (n = 826); dosages were doubled if blood pressure was not normalized [diastolic blood pressure greater than or equal to 90 mm Hg) after 4 weeks of active treatment. Systolic/diastolic blood pressures were significantly reduced (p less than 0.01/p less than 0.01) by isradipine from 162/103 to 145/89 mm Hg, and by nifedipine from 162/104 to 143/88 mm Hg. Normalization rates were 79% with isradipine and 67% with nifedipine. It was necessary to double the dosage in seven of the patients taking isradipine and in three of those taking nifedipine; the mean final dosages were 1.63 mg and 22.4 mg twice daily, respectively. Heart rate did not change significantly with either treatment. There were drug-related adverse events in five patients (21%) taking isradipine (2 edema, 2 headache, 2 palpitations, 1 flushing) and in eight (30%) of those taking nifedipine (5 edema, 2 headache, 1 palpitations). Therapy was withdrawn in one patient in the isradipine group (1 headache) and two patients in the nifedipine group (1 edema, 1 headache). We conclude that isradipine is a highly effective and well tolerated antihypertensive agent. PMID- 1720486 TI - Sleep disturbances in hypertension: a double-blind study between isradipine and metoprolol. AB - This was a comparison of the effects of isradipine and metoprolol on sleep apnea syndrome in 12 hypertensive men who were habitual snorers. Each patient received double-blind isradipine 1.25-2.5 mg twice daily or metoprolol 50-100 mg twice daily after a 4-week placebo period. Static charge-sensitive bed examination was performed during the placebo period and 5-7 weeks after starting treatment. The number of obstructive breathing patterns was increased by metoprolol in five patients, remained the same with isradipine in four, and was decreased by metoprolol in one patient and by isradipine in two. Obstructive patterns increased in the metoprolol group from 24 +/- 26% to 32 +/- 31% and decreased in the isradipine group from 21 +/- 23% to 19 +/- 25% (p less than 0.05, Mann Whitney U test). Neither drug had a significant effect on blood pressure values. There was no significant difference in oxygen desaturation or in the amount of quiet sleep in either treatment group. On the basis of these results, it would appear that isradipine is more suitable than metoprolol for the treatment of hypertension in patients who are habitual snorers. PMID- 1720487 TI - Swedish Isradipine Study in Hypertension: evaluation of quality of life, safety, and efficacy. SWISH Group. AB - This was a double-blind multicenter study to compare the efficacy, tolerability and effects on the quality of life with isradipine and atenolol in the treatment of essential hypertension. Of 588 patients entering the 6-week placebo run-in period, 549 were eligible for randomization to receive either isradipine or atenolol for 8 weeks. If, at the end of this period, diastolic blood pressure (DBP) remained greater than 90 mm Hg, then both agents were given in combination for a further 10 weeks. Tolerability and quality of life were assessed repeatedly during the placebo and active-treatment phases. A subgroup of 30 patients were followed by 24-h ambulatory blood pressure monitoring, and their results are now being analyzed. In another subgroup of 26 patients, maximum exercise capacity, as determined by ergometer bicycle-testing, was measured once during placebo and twice during active treatment. At the end of the 24-week study period, both isradipine and atenolol as monotherapy had produced significant decreases in blood pressure. There were no significant differences overall between the compounds in quality-of-life and side-effect profiles, although there was a relative absence of ankle edema and headache with isradipine. Furthermore, patients receiving isradipine had no change in performance on exercise testing whereas patients on atenolol had a significant decrease (p less than 0.01). PMID- 1720488 TI - The antihypertensive action of isradipine in mild essential hypertension. AB - The antihypertensive effect of isradipine compared with placebo was assessed in 28 male patients (aged 40-64 years) with mild-to-moderate essential hypertension. After withdrawal of all previous antihypertensive treatment, these patients were entered into a 4-week placebo period followed by randomization to receive double blind, for 8 weeks, either placebo (n = 14) or isradipine at 1.25-2.5 mg twice daily (n = 14). Twenty-four-hour ambulatory blood pressure was measured by Accutracker at the end of the placebo period and at the end of active treatment. In the isradipine group, both systolic and diastolic blood pressures decreased significantly (p less than 0.0001) whereas blood pressure increased in those taking placebo. In addition, isradipine as monotherapy controlled blood pressure throughout the day and night, and especially during the early morning, in these patients. PMID- 1720489 TI - Estimation of interleukin 6 production by reverse transcriptase-polymerase chain reaction in four human myeloma cell lines. AB - Although an autocrine growth mechanism through interleukin 6 has been advocated in human myeloma cells, reports of IL-6 production by cells from established myeloma cell lines are rare. In the present study, we examined whether or not a minute amount of interleukin 6 is produced in 4 human myeloma cell lines. IL-6 production was not detected in any of the 4 lines by enzyme immunoassay, bioassay with two interleukin 6-dependent murine hybridoma cell lines and Northern hybridization. However, we detected interleukin 6 mRNA in one (U266) of the 4 lines by the reverse transcriptase-polymerase chain reaction. Nevertheless, the proliferation of all 4 lines was not inhibited by an anti-interleukin 6 antibody. These results suggest that autocrine stimulation by interleukin 6 is not involved in the majority of human myeloma cell lines. PMID- 1720490 TI - The product of the proto-oncogene c-kit (P145c-kit) is a human bone marrow surface antigen of hemopoietic precursor cells which is expressed on a subset of acute non-lymphoblastic leukemic cells. AB - A monoclonal antibody (17F11) was raised by immunization of a Balb/c mouse with leukemic blasts from a patient with acute non-lymphocytic leukemia (ANLL). This antibody recognizes most leukemic blasts of myeloid but not of lymphoid lineage and no peripheral blood cells. By screening NIH-3T3 fibroblasts transfected with the human proto-oncogene c-kit (NIH-3T3/hckit) it could be shown that 17F11 specifically recognizes the gene product P145c-kit. Immunofluorescence analysis on normal hemopoietic cells revealed that 17F11 weakly stains 1-3% of bone marrow mononuclear cells (BMMNC). By FACS sorting and colony assays it could be shown that granulocyte--macrophage progenitor cells could be enriched 10-20-fold, granulocyte progenitors 50-80-fold, and erythroid and multipotential progenitor cells 15-20-fold, in the 17F11 positive fraction. Double fluorescence analysis revealed that P145c-kit is co-expressed on 40-60% of the CD34 positive BMMNC. Finally, these data show that P145c-kit is expressed on blast cells from most patients with ANLL (26/30) and chronic myeloid leukemia in blast crisis (7/9), but is absent on blasts from patients with acute lymphoblastic leukemia expressing the T-, B-lineage, or common ALL phenotypes. PMID- 1720491 TI - Myeloid differentiation associated tyrosine protein kinase activity in WEHI-3B murine monomyelocytic leukemia cells. AB - Tyrosine protein kinase activity was examined during the induction of granulocytic differentiation of WEHI-3B murine monomyelocytic leukemia cells by retinoic acid and aclacinomycin A. Tyrosine kinase activity was found to increase throughout the period of induced maturation. The specificity of this increase in enzymatic activity for the differentiated state was demonstrated by the findings that (a) it was independent of the inducer used, and (b) the treatment of a differentiation-resistant subline of this murine leukemia with an inducer did not produce a significant elevation of tyrosine kinase activity. To determine whether tyrosine protein kinase activity was involved in the differentiation process itself or whether it was a product of the mature state, a series of experimental approaches was employed. Kinetic analyses showed that tyrosine protein kinase activity continued to increase beyond the peak in the level of differentiation. In addition, the total cellular protein phosphotyrosine content, measured by immunoblotting and flow cytometric analysis with anti-phosphotyrosine antibodies, did not increase in accord with the elevation of tyrosine kinase activity. Increases in protein phosphotyrosine content, which were dependent upon the length of exposure to the inducing agent, were observed when cultured cells were treated with the phosphotyrosine phosphatase inhibitor, sodium orthovanadate. Thus, the effect of the increasing tyrosine kinase activity in maturing cells appeared to be negated by competing protein phosphotyrosine phosphatase activity. Finally, the inhibitors of tyrosine kinase activity, genistein and PKI-23, did not interfere with the induction of differentiation by retinoic acid. These findings support the concept that myeloid differentiation associated tyrosine protein kinase activity may not be involved in the initiation of the differentiation process itself. This conclusion deviates from previous assumptions, based on earlier work in this laboratory as well as in that of others, that the differentiation associated kinase activity has an essential role in the initiation of the maturation process. An attractive alternative speculation is that this activity may have a functional role in the mature myeloid cell. PMID- 1720492 TI - [Gynecomastia following cytotoxic treatment of testicular cancer]. AB - A case of post-chemotherapy gynecomastia is reported in a 40 years old male patient with a tumor of seminomatous germinal cells with no evidence of tumoral reappearance or augmentation of the chorionic gonadotropin hormone. The existence of primary hypogonadism was tested. A differential diagnosis was performed and possible physiopathological mechanisms of the gynecomastia were analyzed in a patient previously treated for testicular cancer. PMID- 1720493 TI - [Brain metastasis of solid neoplasms: prognostic factors and therapy]. PMID- 1720494 TI - [Antineoplastic chemotherapy in digestive tumors]. PMID- 1720495 TI - An automated microassay for enzyme inhibitory effects of M2 antibodies in primary biliary cirrhosis. AB - In primary biliary cirrhosis (PBC), autoantibodies are produced to the M2 group of mitochondrial antigens, of which a major constituent is the E2 subunit of the pyruvate dehydrogenase complex (PDC). These antibodies, in addition to conventional reactivities with PDC, characteristically inhibit the catalytic function of PDC in vitro, as judged by a macroinhibition assay based on spectrophotometry. We describe a microinhibition assay adapted for microtitre plates and for an automated readout of results by an ELISA plate reader. We show that this microassay has a sensitivity similar to that of the macroassay. In a study of 83 sera, from PBC and other diseases, the inhibitory microassay proved specific for PBC. This automated inhibitory microassay for PBC-sera could become a primary laboratory procedure for the diagnosis of PBC, particularly because it may identify antibodies to the actual autoepitope on the PDC-E2. PMID- 1720496 TI - Changes of cytokeratin filament organization in human and murine Mallory body containing livers as revealed by a panel of monoclonal antibodies. AB - Mallory bodies (MBs) are characteristics morphologic features of alcoholic hepatitis and can be produced in mouse hepatocytes by chronic griseofulvin (GF) intoxication. The formation of MBs, which share some immunological, biochemical, and ultrastructural features with cytokeratin (CK) filaments of normal liver, is accompanied by derangement and even loss of the CK cytoskeleton of hepatocytes ("empty cells") as revealed by immunofluorescence microscopy. To clarify whether this diminution or lack of CK-related staining of MB-containing hepatocytes was due to loss of CK filaments or changes in antigenicity or accessibility of antigenic determinants immunohistochemical studies using a battery of monoclonal and polyclonal CK antibodies were performed. It could be shown that all these antibodies directed against different CK polypeptide components and antigenic determinants of CKs revealed a highly reduced or even undetectable cytoplasmic CK meshwork in most cells with fully developed large MBs. In the light of our present knowledge of the organization of CK intermediate filaments, these results indicate that the phenomenon of the "empty cells" reflects a diminution of CK meshwork rather than altered antigenic determinants. PMID- 1720497 TI - Intraoperative radiotherapy in the multidisciplinary treatment of bone sarcomas in children and adolescents. AB - From September 1984 to December 1989, 38 patients of pediatric age with localized bone sarcomas received intraoperative radiotherapy (IORT) as part of a multidisciplinary treatment program. The age ranged from 6 to 21 years. The tumor histologies were 22 osteosarcomas and 16 Ewing's sarcomas. Thirty-four had initial primary disease (90%) and 4 were treated for local recurrence (10%). IORT was used on 32 untreated patients and in 6 previously treated with external beam radiotherapy (EBR). The IORT field included the surgically exposed tumor bed area. Single radiation doses ranging from 10 to 20 Gy were delivered, using 6-20 MeV electron beams. The median follow-up time for the entire group is 25 months (2-65+ months). The projected 5-year disease-free and overall survival rates are 65% and 69%, respectively. One patient developed a local recurrence in each histological group: one chondroblastic osteosarcoma and one cervical Ewing's sarcoma. Six patients died from metastatic progression: 3 initially recurrent tumors and three primary disease cases. Severe neuropathy and soft tissue necrosis were seen in some patients as IORT related complications. IORT is a feasible technique to be integrated in multidisciplinary programs that may promote local control in pediatric and adolescent patients with bone sarcomas. Peripheral nerves are dose-limiting tissue structures for IORT. PMID- 1720498 TI - Early childhood screening. PMID- 1720499 TI - Update: cholera--Western Hemisphere, 1991. AB - The epidemic of cholera that began in Peru in January 1991 continues to spread. Most recently, Bolivia, El Salvador, Honduras, Nicaragua, and Panama were added to the list of countries reporting cholera cases (1-3). PMID- 1720500 TI - Analysis of interleukin 6 (IL-6)/IL-6 receptor system using monoclonal anti-IL-6 antibodies. AB - To investigate the IL-6/IL-6 receptor system, we obtained five murine monoclonal antibodies against human IL-6, which neutralize its biological activity. We classified them into two groups (Type I mAb and Type II mAb) according to the epitopes they recognized. These two types of antibodies showed no difference in the manner in which they neutralized IL-6 activity, but they differed in the way they inhibited the binding of IL-6 to its receptor. While Type I mAb inhibited IL 6 binding to its receptor completely, Type II mAb inhibited only partially, even at a concn of Type II mAb sufficient to neutralize biological activity. Scatchard plot analysis revealed that in the case of the human myeloma cell line, the U266 cell, which showed two-phase binding of IL-6 to its receptor, the high affinity binding disappeared and the affinity of the low affinity binding decreased in the presence of Type II mAb. These results suggest that Type I mAb neutralizes IL-6 activity by the blocking of IL-6 binding to its receptor directly. In contrast, Type II mAb neutralizes IL-6 activity not by direct blocking of IL-6 binding to its receptor, but by modulating the binding affinity of IL-6 to its receptor, that is, inhibiting the formation of high affinity binding in IL-6 receptor system. This also indicates that the formation of high affinity may be necessary to transduce the IL-6 signal. PMID- 1720501 TI - Human recombinant dimeric IL-6 binds to its receptor as detected by anti-IL-6 monoclonal antibodies. AB - Three different epitopes of the human IL-6 (IL-6) molecule were recognized by the mAb B-E4 (IgG2b), B-E8 (IgG1) and B-F6 (IgG1). The affinities of these three mAb for IL-6 differ little in several assays but if ranked by affinity they fall into the following order B-E8 greater than B-E4 greater than B-F6. B-E4 and B-E8 mAb, recognizing two different epitopes, are inhibiting mAb in the bioassay with the IL-6 depending cell line B9, however B-E8 has an inhibiting activity higher than B-E4. Both human natural IL-6 (HnIL-6) and human recombinant IL-6 (HrIL-6) were inhibited but not the murine natural IL-6 (MnIL-6). Surprisingly, not only the non-inhibiting mAb (B-F6) recognizes the HrIL-6 fixed to the receptor but also the inhibiting mAb B-E4 and B-E8. This together with the results obtained in a sandwich ELISA where the same mAb was used as both catcher and tracer to detect HrIL-6, it was concluded that dimeric HrIL-6 is able to fix the IL-6 receptor. Competition studies between monomeric HnIL-6 and dimeric HrIL-6 showed that the affinity of the dimeric HrIL-6 for the receptor was higher than that of HnIL-6. PMID- 1720502 TI - The influence of the adjuvant Quil A on the epitope specificity of meningococcal lipopolysaccharide anti-carbohydrate antibodies. AB - Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates. PMID- 1720503 TI - Immunoglobulin VH and VK genes of the BALB/c anti-foot-and-mouth disease virus (O1) VP1 response: cloning, characterization and transgenic mice. AB - Hybridomas producing monoclonal antibodies of different isotypes were isolated from BALB/c antibody responses to the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) strain O1. According to antigen binding measured by ELISA a weak-binding (81D10, IgM) and a strong-binding antibody (113C12, IgG2a) were selected. As RNA sequencing of productive immunoglobulin VH and VK genes turned out, both chains of the weak-binding antibody (81D10) are encoded by germline (i.e. not mutated) genes whereas the gene encoding the strong-binding antibody (113C12) k chain is mutated at several sites. Therefore, rearranged VH and VK genes of 81D10 were cloned, expressed in immunoglobulin non-producing plasmacytoma cells, and mice transgenic for the 81D10 k gene were produced. These mice provide a first step in the development of a transgenic mouse model for genetical investigations in the affinity maturation of anti-viral immunoglobulin variable genes. PMID- 1720504 TI - Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides. AB - Fourteen synthetic peptides of 15 amino acid residues length, overlapping by five residues and spanning the entire sequence of the major allergen Der p II from the house dust mite Dermatophagoides pteronyssinus were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope mapping studies using human IgE antisera. These antibodies bound predominantly to the peptide comprising residues 65-78, the binding of which was inhibited by native Der p II. In addition these antisera bound, to a lesser extent, to the peptide that comprised residues 1-15, which binding was not inhibited by native Der p II. Thus, we found one sequential epitope for a number of IgE sera. PMID- 1720505 TI - Epitope expression and partial structural characterization of F62 lipooligosaccharide (LOS) of Neisseria gonorrhoeae: IgM monoclonal antibodies (3F11 and 1-1-M) recognize non-reducing termini of the LOS components. AB - F62 LOS of Neisseria gonorrhoeae consists of two components. The higher molecular weight (MW) component is recognized by monoclonal antibody (MAb) 1-1-M and the smaller MW component by MAb 3F11. Epitope expression of the two LOS components and their partial structures were investigated by treating the F62 LOS with several glycosidases and then monitoring their antigenicity with the two mouse IgM MAbs. The 1-1-M-defined LOS component was cleaved with both beta-N acetylhexosaminidase and endo-beta-galactosidase, and each cleavage resulted in the loss of expression of the 1-1-M-defined epitope. The N-acetylhexosamine (HexNAc) released by the hexosaminidase was found to be GalNAc, and the smaller oligosaccharide released by the endo enzyme was identified to be a dimer GalNAc beta----Gal. In contrast, the MAb 3F11-defined LOS component was not digested by the endo galactosidase, but it was cleaved with alpha and beta-galactosidase, and expression of the MAb 3F11-defined LOS epitope expression of the MAb 3F11-defined LOS was abolished by the treatment with each of two exo enzymes. MAb 3F11 bound to the 1-1-M-defined LOS component resulting from the removal of the beta-GalNAc residue, and the resulting LOS was further cleaved with beta-galactosidase, but not with alpha-galactosidase. From these results, we conclude the following: (1) MAbs 1-1-M and 3F11 both recognize the non-reducing termini of the LOS components; (2) the 1-1-M-defined LOS component has the GalNAc beta----Gal beta 1 ---4-Glc (or GlcNAc) structure, and the GalNAc beta----Gal residue is involved in the MAb 1-1-M-defined epitope; (3) the MAb 3F11-defined LOS component may not have a Gal beta 1----4GlcNAc beta 1----4Gal beta 1----4Glc structure within the molecule. However, it has beta-Gal residue at its non-reducing terminus, and this residue is involved in the MAb 3F11-defined epitope; (4) the two LOS components share a similar antigenic structure, and the 3F11-defined epitope structure is present in the MAb 1-1-M-defined LOS component. Expression of this epitope within the 1-1-M-defined LOS molecule is blocked by the beta-GalNAc residue; however, the beta-GalNAc residue at the non-reducing end may be not the only structural difference between the two components. PMID- 1720507 TI - Fighting measles through child-to-child education. PMID- 1720506 TI - Mapping of two immunodominant structures on human interferon alpha 2c and their role in binding to cells. AB - Structure-function studies of human recombinant interferon (IFN) alpha 2c were performed using a panel of specific monoclonal antibodies in the binding and neutralizing assays. Two immunodominant structures, designated sites I and II, were detected and localized within two conserved hydrophilic regions of IFN-alpha molecule. Using the NK2 antibody as a marker, site I was mapped into a carboxy terminal domain around residues 112-148. This site was shown to be, most probably, responsible for inducing the antiviral and antiproliferative activities of the receptor-bound IFN-alpha 2c in the cell. Site II that mapped into the amino-terminal domain of IFN-alpha 2c was, at least partially, formed by the amino acid residues 36-41. This region was shown to be most probably involved in the binding of IFN to its cellular receptor. These findings fit with Sternberg and Cohen's model (Int. J. Biol. Macromol. 4, 137-144, 1982) for the tertiary structure of human IFN-alpha. PMID- 1720508 TI - MHC class I surface expression in embryo-derived cell lines inducible with peptide or interferon. AB - It has long been recognized that the absence of expression of products of the major histocompatibility complex (MHC) during early development might allow the fetus to escape recognition by maternal lymphocytes. In addition to the MHC class I heavy chain and beta 2-microglobulin, antigenic peptide is an essential structural component of the class I molecule. Indeed, there is evidence that MHC linked genes encoding peptide transporter molecules and possibly components of a proteolytic complex are necessary for MHC class I assembly and stability at the cell surface. Here we demonstrate that embryonic cells in general show a defect in MHC class I assembly. Surface expression was rescued in the presence of an appropriate antigenic peptide, or by treatment with interferon. Consistent with this, HAM1 messenger RNA was not constitutively expressed, but was inducible by interferon, and during differentiation in vitro. Thus, tolerance of the fetal allograft may in part be controlled at the level of peptide-dependent MHC class I assembly. PMID- 1720509 TI - YY1 is an initiator sequence-binding protein that directs and activates transcription in vitro. AB - Regulation of eukaryotic messenger RNA transcription is governed by DNA sequence elements that serve as binding sites for sequence-specific transcription factors. These include upstream and downstream promoter-proximal elements, enhancers, repressors, and silencers, which modulate the rate of specific initiation by RNA polymerase II. In addition, the promoter-proximal region between -45 and +30 (relative to the start of initiation) contains two highly conserved motifs, the TATA sequence at around -30 and CA at +1. Although the TATA element-binding factor TFIID has been purified and cloned from several organisms and has provided invaluable insight into the process of transcription initiation and its regulation, little is known about factors that interact at the +1 region. We have recently shown that the adeno-associated virus type 2 P5 promoter +1 region (P5 + 1 element) binds transcription factor YY1. We report here that this sequence is necessary and sufficient for accurate basal transcription. Further, partially purified YY1 can restore basal level transcription from a P5 + 1 element in a HeLa extract depleted for YY1 or a Drosophila embryo extract devoid of YY1 activity, whereas a YY1-specific antibody can block the reactivation. Finally, using electrophoretic mobility shift assay, we have identified YY1-related factors that bind to two other transcription initiators in cellular genes. PMID- 1720510 TI - Voltage-clamp experiments reveal receptor type-dependent modulation of chloride secretion in the guinea pig colonic mucosa by intestinal opioids. AB - The influence of four opioid antagonists on short circuit current (Isc), transepithelial potential difference (Pdo) and tissue conductance (Gt) in the guinea pig colonic mucosa was investigated in vitro under both basal and PGE1 plus theophylline-stimulated conditions. The experiments aimed at identifying the opioid receptor type(s) endogenously activated to control chloride secretion. Under blockade of sodium-dependent Isc by amiloride (100 mumol/l), net anion secretion was regarded to equal the lumen-negative shift in Isc upon addition of 1 mumol/l PGE1 plus 100 mumol/l theophylline. It was significantly elevated by 100 nmol/l of the kappa-selective antagonist nor-binaltorphimine (nor-BNI). This augmenting effect was totally abolished in amiloride-free buffer or by omission of chloride. 1 mumol/l TTX completely prevented the effect of both PGE1 plus theophylline and nor-BNI. Both the kappa agonist U 69593 (10 nmol/l) and the calcium channel agonist Bay K 8644 (1 mumol/l) significantly depressed net anion secretion stimulated by PGE1 plus theophylline. Nor-BNI at 10 nmol/l prevented the suppressive effect of both Bay K 8644 and U 69593. This suggests release of endogenous opioids by the calcium channel agonist Bay K 8644 and competition between the kappa agonist U 69593 and the kappa antagonist nor-BNI. In contrast to the kappa antagonist nor-BNI, the mu antagonist CTOP-NH2 at 100 nmol/l significantly impaired, while the mu-selective agonist DAGO at 0.2 nmol/l augmented, net anion secretion stimulated by PGE1 plus theophylline. The effect of CTOP-NH2 was abolished in chloride-free buffer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720511 TI - FK-506 shows similar graft survival rate to cyclosporine but fewer side effects: Pitt transplant team. PMID- 1720512 TI - The discovery of FK-506 and U.S. development. PMID- 1720513 TI - The influence of age on neurotransmitter turnover in the rat's superior colliculus. AB - Measurements of the turnover of dopamine, noradrenaline and serotonin and their metabolites have been performed in the superior colliculus of adult and aged rats. The turnover of dopamine, noradrenaline and their metabolites after pargyline treatment was significantly lower in aged rats than in adults. On the contrary, the synthesis rate of serotonin (measured by accumulation of 5 hydroxytryptophan after decarboxylase blockade) and the turnover rate of serotonin (after pargyline treatment) did not change during aging. These findings suggest that aging has a different effect on catecholamines and serotonin turnover in the superior colliculus of the aged rats. PMID- 1720514 TI - A silver impregnation method for labeling both Alzheimer paired helical filaments and their polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AB - The Gallyas silver impregnation which is specific to neurofibrillary changes of paired helical filaments (PHF) and 15 nm straight filaments, was adapted to stain polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both PHF and tau polypeptides were readily and consistently stained by the Gallyas stain. This technique stained PHF greater than tau greater than high-molecular-weight microtubule-associated polypeptides (MAPS). Tubulin was stained only weakly. Neurofilament triplet, ubiquitin, bovine serum albumin and histones were unstained. The staining of PHF and tau polypeptides by Gallyas silver stain is consistent with the presence of tau in PHF. PMID- 1720515 TI - [Antalgic therapy in terminal patients: our experience]. PMID- 1720516 TI - [Giant diverticulum of the bladder. A case report]. AB - Vesical diverticula are a common pathology of the urinary bladder, generally secondary to cervico-urethral obstruction. Frequency of giant diverticula is not reported in the literature. The Authors present a case of giant vesical diverticulum (20 x 30 cm in diameter) due to prostatic adenoma in which any referable symptomatology was absent. The Authors debate the reasons for which progressive development of the diverticulum has been related with the improvement of the frequency and urgency previously presented by the patient. PMID- 1720517 TI - Presenting problems in black children referred for outpatient psychological consultation in South Africa. AB - An examination of outpatient referrals for psychological consultation at a psychiatric hospital in South Africa was undertaken. Over an 18-month period, 87.1% of a total of 31 children were referred with problems of poor school performance. Behaviour problems were underrepresented. The results are discussed in the context of inadequate community mental health services, which are implicated in the under-detection of childhood psychopathology. PMID- 1720518 TI - Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye, fura-2. AB - Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca2+]i, was monitored with a fluorescent Ca2+ dye, fura-2. The fluorescence intensity ratio (I340/I380) was used as an index of [Ca2+]i. [Ca2+]i of the cells suspended in the nominally Ca(2+)-free solution was estimated at 52 +/- 3 nM. Ca2+ uptake was followed for as long as 5 min in the presence of 100-1000 microM added CaCl2. Most of the experiments were performed at 200 microM CaCl2. The Ca2+ uptake was abolished by 0.8 mM Ni2+ and 50 microM Mn2+ and partitally antagonized by 50 microM verapamil and 50 microM diltiazem but not affected by 20 microM nifedipine. The Ca2+ entry was reduced by increasing concentrations of extracellular K+ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca2+ transport doubled in the presence of Bay K8644 (8 microM), a Ca2+ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 microM nifedipine, 10 microM verapamil or 10 microM diltiazem and was relatively independent of extracellular K+ concentration. These results suggest that there are at least two distinct Ca2+ transport systems in the rat ileal epithelial cells, one resistant to organic Ca2+ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential. PMID- 1720519 TI - Effects of arylaminobenzoate-type chloride channel blockers on equivalent short circuit current in rabbit colon. AB - Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (Vte) and resistance (Rte) were measured and the equivalent short-circuit current (Isc = Vte/Rte) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 mumol/l) Isc was -71 +/- 11 (n = 118) microA/cm2. Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to +32 +/- 4 (n = 118) microA/cm2. In the presence of amiloride and indomethacin, prostaglandin E2 (1 mumol/l, s), known to induce Cl- secretion, generated an Isc of -143 +/- 8 (n = 92) microA/cm2. The arylaminobenzoate and Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) reduced Isc reversibly with a half-maximal inhibition (IC50) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC50 to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-L-cysteine (m) preincubation. Pretreatment with N-acetyl-L-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and L-homocysteine (m) were more effective and reduced IC50 to approximately 50 mumol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/-1 phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl- conductance in the thick ascending limb of the loop of Henle (TAL).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720520 TI - Inhibiting synthesis of extracellular matrix improves patch clamp seal formation. AB - The ability to form gigaohm seals is essential for patch-clamp studies. Cells with otherwise useful properties must be abandoned for electrophysiological studies if seal formation is not possible. We have found that by inhibiting the growth of extracellular matrix with beta-D-xyloside, the success of forming gigaohm seals increased from near 0% to near 100%. Treated cells remained viable and appeared morphologically similar to untreated cells. Prototype treatment protocols are given for the renin secreting cell line As4.1. PMID- 1720521 TI - Regulations by the Health Care Financing Administration will affect the practice of cardiac pacing. PMID- 1720522 TI - Crosstalk with external bipolar DVI pacing: a case report. AB - An increase of the basic atrial pacing rate from a preset value of 70 beats/min up to 91 beats/min was recorded in a patient with an external bipolar dual chamber pacing system. This observation could be explained by the occurrence of crosstalk; this specific manifestation of crosstalk was the result of the use of an uncommitted DVI pacing mode. PMID- 1720523 TI - Renal extracorporeal shock wave lithotripsy performed in patient with implantable cardioverter defibrillator. AB - The effect of extracorporeal shock wave lithotripsy on the automatic implantable cardioverter defibrillator is unknown. To evaluate what effect might occur, a non implanted automatic implantable cardioverter defibrillator was subjected to a full course of extracorporeal shock wave lithotripsy while inactive. Bench testing by the manufacturer after lithotripsy demonstrated normal function of the device. A patient with an automatic implanted cardioverter defibrillator who required contralateral extracorporeal shock wave lithotripsy then underwent this procedure. The right renal calculus was destroyed successfully with no apparent damage to the automatic implantable cardioverter defibrillator. A test of the automatic implantable cardioverter defibrillator after lithotripsy demonstrated normal sensing and conversion of induced ventricular tachycardia. PMID- 1720524 TI - Implantable cardioverter defibrillator proarrhythmia: case report and review of the literature. AB - A 31-year-old man who received an automatic cardioverter defibrillator subsequently underwent exercise testing. During exercise, a sinus tachycardia resulted above his device detect rate prompting two shocks, the second of which produced an unstable polymorphous ventricular tachycardia. In this article, we review the literature on automatic cardioverter defibrillator-induced ventricular tachyarrhythmias as well as the management of exercise testing in patients with these devices. PMID- 1720525 TI - Hemodynamic effect of physiological dual chamber pacing in a patient with end stage dilated cardiomyopathy: a case report. AB - I report a case of end-stage dilated cardiomyopathy with first-degree atrioventricular (AV) block, which had been resistant to intensive medical therapy and was eventually treated by DDD pacemaker. The optimal AV interval setting was decided using invasive right-heart catheterization and Doppler echocardiography. At a pacing rate of 92/minute, an AV interval setting of between 200 and 100 msec increased left ventricular filling and enhanced myocardial contractility. An AV interval setting of 50 msec increased the left ventricular filling further. However, this resulted in deteriorated left ventricular function. Based on these findings, the pacemaker was programmed at an optimal AV delay of 100 msec, a rate of 82-150 beats/min and a DDD mode, resulting in a good clinical course for 4 months after the therapy. Thus, it is suggested that in patients with end-stage dilated cardiomyopathy and first-degree AV block, an optimal AV delay setting using a DDD pacemaker can improve deteriorated myocardial function probably by increasing the left ventricular filling, and thus promote utility of the Frank-Starling mechanism. PMID- 1720526 TI - Impedance measurements in the human right ventricle using a new pacing system. AB - A promising new pacemaker that provides on-line measurements of right ventricular (RV) impedance was evaluated in ten patients with symptomatic second- or third degree atrioventricular (AV) block. We tested the assumption that if changes in RV impedance represented changes in RV stroke volume (SV), conditions known significantly to affect RV SV should be accompanied by significant changes in RV impedance. One week after pacemaker implantation, RV impedance was measured noninvasively during normal respiration in the supine (baseline), left lateral, right lateral, sitting, and standing positions. In addition, all patients performed a Valsalva maneuver test. The amplitude of the impedance signal was different during in- and expiration in every body position studied. At baseline, the amplitude of the signal was 18.80 +/- 2.24 mm; in the right lateral position 16.75 +/- 3.24 mm (P = 0.04) and 17.80 +/- 2.35 mm in the left lateral position (P = 0.04). The amplitude of the signal in the sitting position was 16.65 +/- 2.89 mm (P = 0.07) and in the standing position 16.95 +/- 3.44 mm (P = 0.11). The most impressive change in amplitude was noted during performance of the Valsalva maneuver. During this test the amplitude decreased to 13 +/- 2.81 mm and rose to 20 +/- 2.66 mm (P = 0.002) afterwards. These results strongly support the assumption that changes of RV impedance as measured by this catheter represent changes in RV SV. This new pacing system is the first pacemaker that reports on the hemodynamic response of every heartbeat by measuring RV impedance. PMID- 1720527 TI - Intravenous 3-methoxy-O-desmethyl-encainide in reentrant supraventricular tachycardia: a randomized double-blind placebo-controlled trial in patients undergoing EP study. AB - Encainide is an agent effective in atrioventricular and atrioventricular nodal reentrant tachycardia. The metabolites O-desmethyl encainide and 3-methoxy-O desmethyl encainide (MODE) are responsible for the clinical effects of encainide in most patients. In this study, intravenous MODE was evaluated in eight patients with reentrant supraventricular tachycardia undergoing electrophysiological testing. After tachycardia was induced at least twice to ensure reproducibility, MODE (30 micrograms/kg/min x 15 min, then 7.5 micrograms/kg/min) or placebo was administered in a double-blind fashion. If tachycardia remained inducible, the infusion was unblinded; in nonresponding subjects who received placebo, MODE was then administered. Placebo was ineffective in 3/3 patients. MODE prevented tachycardia induction in 5/8 patients and increased the tachycardia cycle length from 302 +/- 38 to 413 +/- 67 msec in the other three. At a mean concentration of 774 +/- 229 ng/ml, MODE prolonged PR, AH, HV, QRS, and QT intervals, right ventricular and accessory pathway effective refractory periods, and slowed or blocked antegrade accessory pathway conduction. Changes in intracardiac conduction were rate independent between cycle lengths 400 to 600 msec, while changes in ventricular effective refractory periods were most pronounced at rapid pacing rates. No adverse effects, hemodynamic changes, or conduction disturbances occurred. Thus, MODE can modify or suppress induction of reentrant atrioventricular or atrioventricular nodal tachycardia. The study design used here is well suited for the evaluation of newer antiarrhythmic agents by electrophysiological testing. PMID- 1720528 TI - Fourier analysis in patients with different pacing modes. AB - The purpose of this study was to evaluate the usefulness of phase analysis in detecting the altered activation sequence induced by different pacing modes. Radionuclide ventriculography and planar gated blood pool scintigraphy were performed at rest in 56 patients with different pacemakers. This method permitted us to localize the pacemaker impulse site in the right ventricle and its diffusion in the heart. In patients with VVI pacemaker, this technique showed an evident asynchronism of contraction and relaxation of each ventricle and the standard deviation of phase angle (sigma), calculated by computer, is greater during pacing than sinus rhythm for left (LV) and right (RV) ventricles (LV sigma: 17 degrees +/- 4 vs 11 degrees +/- 3, less than 0.001; RV sigma: 31 degrees +/- 7 vs 14 degrees +/- 4, P less than 0.001). In the patients with VVI rate responsive pacemakers, the LV sigma changed from 18.5 +/- 3 under pacing to 11 degrees +/- 3 in sinus rhythm, P less than 0.001, while the RV sigma changed from 30 degrees +/- 8 to 14 degrees +/- 4, P less than 0.001. Instead in the patients with DDD pacemakers, the LV sigma changed from 15.5 degrees +/- 2 under pacing to 11 degrees +/- 3 in sinus rhythm, P less than 0.05, while the RV sigma changed from 29.1 degrees +/- 6 to 14 degrees +/- 4, P less than 0.001.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720529 TI - Transesophageal atrial pacing--stimulation and discomfort thresholds: the role of electrode configuration and pulse width. AB - A balloon catheter with six electrodes has been developed for transesophageal atrial stimulation of the human heart. Introduction is easy and its positioning is simple with the help of six unipolar atrial electrograms. In a group of 20 healthy volunteers, stimulation and discomfort thresholds (intolerable discomfort) were measured for three levels of pulse widths (12, 16, and 20 msec) and for five electrode configurations. Stimulation thresholds were below discomfort thresholds in each case. The stimulation threshold depended on pulse width and not on electrode configuration. The discomfort threshold, however, depended on the electrode configuration and not on the pulse width. A moderate but potentially important increase of the ratio between stimulation threshold and discomfort threshold could be achieved by combining a long pulse width (20 msec) and avoiding the largest distance between the active (cathode) and the passive (anode) electrode. Transesophageal atrial stimulation promises to be a practical noninvasive tool for the termination of regular supraventricular tachycardias, basal electrophysiological studies, and controlled acceleration of the heart rate in the study of myocardial ischemia. PMID- 1720530 TI - Can we treat carotid sinus syndrome? PMID- 1720531 TI - MCL1 and MCL6 compared to V1 and V6 in distinguishing aberrant supraventricular from ventricular ectopic beats. AB - Use of V1 and V6 has been suggested for distinguishing aberrant supraventricular from ventricular ectopy. For two decades, "modified" leads MCL1 and MCL6 have been widely used as V1 and V6 substitutes for bedside monitoring, but their use has never been validated. To determine the value of MCL1 and MCL6, 81 morphologically distinct wide QRS ectopic beats were recorded from 46 patients during cardiac electrophysiological study. As determined by the His-bundle electrogram, 31 of the ectopics were aberrant supraventricular, 50 were ventricular. A new criterion, measurement of QRS onset to the predominant peak or nadir of the complex, was valuable in diagnosing wide complexes in MCL6 and V6. An interval of 50 msec or less predicted aberrant supraventricular ectopy; an interval of 70 msec or more predicted ventricular ectopy. There was agreement between the modified and conventional precordial leads regarding which QRS patterns were useful in distinguishing aberrant supraventricular from ventricular ectopy. A greater proportion of wide complexes in MCL1 and V1 exhibited patterns useful in making the diagnosis compared to MCL6 and V6. Using well-established criteria, the proportion of correct diagnoses that was made from individual leads was: MCL1 = 86%, V1 = 85%, MCL6 = 72%, V6 = 67%. The bedside leads (MCL1 and MCL6) were not statistically different in diagnostic accuracy from their conventional lead counterparts (V1 and V6); however, MCL1 and V1 were superior to MCL6 and V6. When the new criterion was added to make the diagnosis from MCL6 and V6, no difference in diagnostic accuracy was present between the four leads. PMID- 1720532 TI - Tachycardia recognition and diagnosis from changes in right atrial pressure waveform--a feasibility study. AB - Implantable defibrillators either monitor heart rate or use a probability density function to detect ventricular fibrillation/tachycardia. As a result, they are unable to discriminate sinus tachycardia and atrial arrhythmias from malignant ventricular rhythms. We have assessed high fidelity fiber-optic pressure recordings in the right atrium during cardiac arrhythmias in 23 patients (mean age 44 years, 11 females) undergoing electrophysiological study. The unfiltered pressure signal was amplified and recorded on paper. During sinus rhythm, a constant amplitude deflection occurred during atrial systole (a wave). A characteristic waveform pattern was observed during each of the studied tachyarrhythmias, which included atrial flutter and fibrillation, atrioventricular nodal reentrant tachycardia, atrioventricular reentrant tachycardia, and ventricular tachycardia with and without ventriculoatrial conduction. The waveform pattern allowed clear visual discrimination of the underlying arrhythmia. Mean atrial pressure was increased during all arrhythmias and did not allow discrimination of the nature of the tachycardia. High fidelity pressure recordings produced characteristic appearances for pattern recognition of each arrhythmia studied. They allowed determination of the temporal relation between electrical and mechanical cardiac events and may have potential in the detection and recognition of cardiac arrhythmias. PMID- 1720533 TI - Variability in the measurement of human ventricular refractoriness. AB - The degree of variability in ventricular refractoriness and factors potentially affecting this variability were evaluated in 80 patients undergoing an electrophysiological study. Each of seven variables (stimulation current, coupling interval of the basic drive train to spontaneous rhythm, pause between determinations, bipolar pacing configuration, bipolar vs unipolar pacing, atrioventricular synchrony, and autonomic tone) was evaluated in a group of ten patients to determine its effects on the reproducibility of refractoriness. Measurements were repeated ten times in every patient under each of two conditions. Five variables had significant effects on the reproducibility of measurements. Pacing at 10 mA was associated with less variability in the determination of ventricular refractoriness than pacing at twice threshold (within-subject variance component 4.5 vs 10.1 msec; P less than 0.001). The mean difference between the longest and shortest determinations of refractory periods (range) was 6.2 msec at 10 mA and 8.6 msec at twice threshold. The use of a conditioning period of pacing and continuous trains (eight beats with a 3-sec pause) rather than a variable pause between serial trials reduced the mean within subject variance component from 16.5 to 3.3 (P less than 0.001) and the mean range of refractory period determinations from 10.8 to 4.8. The use of the distal rather than the proximal pole as the cathode decreased the mean within-subject variance component from 9.4 to 3.3 (P less than 0.001) and the range of determinations from 6.4 to 5.8 msec. Unipolar pacing was associated with less variability than bipolar pacing (mean within-subject variance component 4.6 vs 6.4; P less than 0.05, mean range 5.0 vs 7.6 msec).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720534 TI - Comparative efficacy of subcutaneous mesh and plate electrodes for nonthoracotomy canine defibrillation. AB - To determine the optimal configuration for the subcutaneous placement of electrodes for the performance of ventricular defibrillation without thoracotomy, internal defibrillation using four different subcutaneous electrodes was performed in 13 anesthetized dogs (7-12 Kg, mean +/- SD: 9.2 +/- 1.5 Kg). An electrode (7 cm2) was positioned transvenously in the superior vena cava with the following electrodes randomly implanted subcutaneously on the left chest: small mesh electrode (14 cm2), large mesh electrode (28 cm2), small titanium plate electrode (14 cm2), and large plate electrode (28 cm2). Ventricular fibrillation was induced by applying alternating current; a monophasic defibrillation wave was administered between the superior vena cava and the subcutaneous electrodes 10 seconds later. The energy level associated with a 50% successful defibrillation, as predicted by logistic regression analysis, was defined as the ED50. After the completion of the defibrillation protocol using the four subcutaneous electrodes, the small mesh electrode was sutured to the epicardium and the ED50 measurements were repeated. Energy ED50s were lower when the superior vena cava electrode was used as the cathode rather than as the anode. Of the subcutaneous electrodes, the large plate electrode showed the lowest energy ED50 (3.3 +/- 0.9 joules). The plate electrodes had lower energy ED50s than the mesh electrodes, and the large electrode had a lower energy ED50 than the small electrodes. Using the epicardium electrode, transient arrhythmias and ST elevation were observed following successful defibrillation; however, no arrhythmias or ST-T changes were observed following defibrillation using the subcutaneous electrodes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720535 TI - Unipolar versus bipolar pacing--poles apart. PMID- 1720536 TI - Epidural lead fracture caused by material processing fault. AB - Fracture of the epidural lead (Pisces Quad 3487) is documented in four out of eight patients with an implanted Itrel pacing system for treatment of peripheral vascular disease. In two patients, lead fracture was established during x-ray fluoroscopy. In the remaining two patients, x-ray examination did not reveal any fracture, due to proximity of the fragments. Microscopic examination of the extracted lead, however, confirmed lead fracture, as well as the presence of tissue fluid and thrombus between the two ends of the spiral shaped lead, but no insulation defect was observed. A cross-sectional area on the fracture line of the broken lead was examined using scanning electron microscopy. It was found, by tracing the radial marks to their point of convergence, that the initial microcrack started from a large inclusion of the calcium-silicon type at the lead surface. The initial microcrack was propagated by the fatigue mechanism. The presence of a large inclusion at the surface suggests that the main cause of the failure of the investigated epidural leads could be improper fabrication of the material. The high incidence of epidural lead fracture in our group suggests that this complication should be considered as a possible cause of epidural spinal electrical stimulation pacing system dysfunction. PMID- 1720537 TI - Control of refractory ventricular tachycardia with biventricular assist devices. AB - A 65-year-old man developed incessant ventricular tachycardia following coronary artery bypass grafting while being weaned from cardiopulmonary bypass. The arrhythmia was refractory to procainamide, lidocaine, bretylium, and magnesium. Ventricular tachycardia subsided following reinitiation of cardiopulmonary bypass. Ultimately, the patient required ventricular assist devices to control his arrhythmia. This case is unique as the ventricular assist devices were used not for hemodynamic support, but for arrhythmia control. The mechanism of arrhythmia suppression may be related to contraction-excitation coupling. PMID- 1720538 TI - Possible involvement of (2'5')oligoadenylate synthetase activity in pre-mRNA splicing. AB - The first step in splicing of pre-mRNA involves an intermediate lariat structure, in which a 2'5' phosphodiester bond between the 5' terminal guanosine residue of the intron and a specific adenosine residue near the 3' end of the intron is formed. A mammalian enzyme that generates 2'-5' phosphodiester bonds is (2' 5')oligoadenylate synthetase [(2'-5')OASE]. Although the expression of this enzyme is induced by interferon, low constitutive levels can be detected in untreated cells and tissues. The structural similarity between the lariat branch point and the 2'-5' phosphodiester bond generated by (2'-5')OASE prompted the experiments described here which suggest that this enzyme is involved in pre-mRNA splicing. (i) We show that a (2'-5')OASE activity is associated with 60S spliceosomes in an ATP- and RNA-dependent manner and that it can be indirectly immunoprecipitated by anti-Sm antibodies. (ii) Antibodies against (2'-5')OASE inhibit the lariat formation in the first step of splicing when added directly to a splicing reaction in vitro. (iii) HeLa cell nuclear extracts immunodepleted of (2'-5')OASE activity were also deficient in splicing activity. PMID- 1720539 TI - Two additional protein-tyrosine kinases expressed in human lung: fourth member of the fibroblast growth factor receptor family and an intracellular protein tyrosine kinase. AB - The expression of protein-tyrosine kinases (PTKs; ATP:protein-tyrosine O phosphotransferase, EC 2.7.1.112) was studied in normal human lung and various tumors by PCR followed by molecular cloning and sequence analysis. Six known PTKs (YES, FGR, LYN, HCK, PDGFB-R, and CSF1-R), as well as two additional members of this enzyme family, were detected in lung. One of the newly discovered sequences appears to represent a group of cytosolic PTKs. The cDNA sequence of the second unknown PTK revealed that it is a fourth member of the fibroblast growth factor receptor family. It was therefore called TKF (tyrosine kinase related to fibroblast growth factor receptor). Among a wide variety of cells and tissues tested, including human lymphocytes and macrophages, TKF was only found expressed in lung. Apart from normal lung, TKF expression could be demonstrated in some tumors of lung origin, but also in malignancies not derived from lung tissues. As fibroblast growth factors are generally involved in a variety of functions such as mitogenesis, angiogenesis, and wound healing, the specific expression of a receptor-related gene in lung only may point to yet another special function of this group of proteins. PMID- 1720540 TI - A dominant positive and negative selectable gene for use in mammalian cells. AB - We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells. PMID- 1720541 TI - t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. AB - The t(8;21)(q22;q22) translocation is a non-random chromosomal abnormality frequently found in patients with acute myeloid leukemia (AML) with maturation (M2 subtype). We report here the cloning of a gene, named AML1, on chromosome 21 that was found to be rearranged in the leukemic cell DNAs from t(8;21) AML patients. The breakpoints in 16 out of 21 patients were clustered within a limited region of AML1, and detailed analysis in 3 patients revealed that the breakpoints occurred in the same intron of the gene. Sequencing of cDNA clones identified a long open reading frame encoding a 250-amino acid protein. Northern blot analysis detected four constant mRNA species in t(8;21) leukemic and normal cells; the largest species was more abundant in the leukemic cells than in normal cells. In addition, two mRNA species limited to the leukemic cells were found. These findings indicate that the AML1 gene may be involved in neoplastic transformation of AML with the t(8;21) translocation. PMID- 1720542 TI - Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. AB - The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to approximately 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation of L-citrulline from L arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium denuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity. PMID- 1720543 TI - Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding. AB - A cloned yeast TOP2 gene was modified to produce yeast DNA topoisomerase II (EC 5.99.1.3) epitopically labeled at its amino or carboxyl terminus. Limited digestion with SV8 endoprotease shows three distinct protease-sensitive sites in each polypeptide of the dimeric enzyme. These sites were mapped by immunostaining of the end-labeled proteolytic fragments resolved by SDS/polyacrylamide gel electrophoresis; two of the mapped locations were confirmed by sequencing the amino ends of two unlabeled peptic fragments. Proteolytic cleavage by SV8 endoprotease at a pair of sites corresponding to the carboxyl sides of Glu-411 and Glu-680 is modulated by the binding of the nonhydrolyzable ATP analogs adenosine 5'-[beta, gamma-imido]triphosphate (5'-adenylyl imidodiphosphate) and adenosine 5'-[gamma-thio]triphosphate: in their absence cleavage occurs predominantly at Glu-411; in the presence of either analog, cleavage occurs predominantly at Glu-680. These results are interpreted in terms of allosteric interdomainal movements in the type II DNA topoisomerase following the binding of ATP. PMID- 1720544 TI - Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction. AB - Mutant mitochondrial DNA with large-scale deletions (delta-mtDNA) has been frequently observed in patients with chronic progressive external ophthalmoplegia (CPEO), a subgroup of the mitochondrial encephalomyopathies. To exclude involvement of the nuclear genome in expression of the mitochondrial dysfunction characteristic of CPEO, we introduced the mtDNA of a CPEO patient into clonal mtDNA-less HeLa cells and isolated cybrid clones. Quantitation of delta-mtDNA in the cybrids revealed that delta-mtDNA was selectively propagated with higher levels of delta-mtDNA correlating with slower cellular growth rate. In these cybrid clones, translational complementation of the missing tRNAs occurred only when delta-mtDNA was less than 60% of the total mtDNA, whereas accumulation of delta-mtDNA to greater than 60% resulted in progressive inhibition of overall mitochondrial translation as well as reduction of cytochrome c oxidase activity throughout the organelle population. Because these cybrids shared the same nuclear background as HeLa cells, these results suggest that large-scale deletion mutations of mtDNA alone are sufficient for the mitochondrial dysfunction characteristic of CPEO. PMID- 1720545 TI - Chronic lithium regulates the expression of adenylate cyclase and Gi-protein alpha subunit in rat cerebral cortex. AB - A possible role for adenylate cyclase and guanine nucleotide-binding proteins (G proteins) in contributing to the chronic actions of lithium on brain function was investigated in rat cerebral cortex. It was found that chronic treatment of rats with lithium (with therapeutically relevant serum levels of approximately 1 mM) increased levels of mRNA and protein for the calmodulin-sensitive (type 1) and calmodulin-insensitive (type 2) forms of adenylate cyclase and decreased levels of mRNA and protein for the inhibitory G-protein subunits Gi alpha 1 and Gi alpha 2. Chronic lithium did not alter levels of other G-protein subunits, including Go alpha, Gs alpha, and G beta. Lithium regulation of adenylate cyclase and Gi alpha was not seen in response to short-term lithium treatment (with final serum levels of approximately 1 mM) or in response to chronic treatment at a lower dose of lithium (with serum levels of approximately 0.5 mM). The results suggest that up regulation of adenylate cyclase and down-regulation of Gi alpha could represent part of the molecular mechanism by which lithium alters brain function and exerts its clinical actions in the treatment of affective disorders. PMID- 1720546 TI - Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src. AB - Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr 527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein. PMID- 1720547 TI - Detection of homeobox genes in development and evolution. AB - The homeobox genes encode a family of DNA-binding regulatory proteins whose function and genomic organization make them an important model system for the study of development and differentiation. Oligonucleotide primers corresponding to highly conserved regions of Antennapediaclass homeodomains were designed to detect and identify homeobox sequences in populations of DNA or RNA by means of the polymerase chain reaction (PCR). Here we present a survey of sequences detected by PCR using an initial set of primers (HoxA and HoxB) based on an early nucleotide consensus for vertebrate Antennapedia-class homeodomains. Several novel sequences are reported from both mouse genomic DNA and RNA from the developing mouse telencephalon. Forebrain-derived clones are similar to the chicken CHox7, Drosophila H2.0, and mouse Hlx genes. PCR also proved to be a rapid method for identifying homeobox sequences from diverse metazoan species. Cloning of three Antennapedia-related sequences from cnidarians provides evidence of ancient roles for homeobox genes early in metazoan evolution. PMID- 1720548 TI - Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells. AB - A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possibly passage number, influences the recovery of CFTR-targeted cells. PMID- 1720549 TI - Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. AB - Recurring chromosomal translocations involving chromosome 11, band q23, have been observed in acute lymphoid leukemias and especially in acute myeloid leukemias. We recently showed that breakpoints in four 11q23 translocations, t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13.3), were contained within a yeast artificial chromosome clone bearing the CD3D and CD3G gene loci. We have identified within the CD3 yeast artificial chromosome a transcription unit that spans the breakpoint junctions of the 4;11, 9;11, and 11;19 translocations, and we describe two other, related transcripts that are upregulated in the RS4;11 cell line. We have named this gene MLL (myeloid/lymphoid, or mixed-lineage, leukemia. PMID- 1720550 TI - Beta 3-adrenergic receptor stimulation restores message and expression of brown fat mitochondrial uncoupling protein in adult dogs. AB - Brown adipose tissue (BAT) is present throughout life in rodents and plays an important role in energy balance. However, whereas BAT is clearly recognizable in the neonates of larger mammals (including dogs, cats, sheep, cattle, and humans), it is undetectable or present in only small quantities in adults of these species and is replaced by a tissue with the gross characteristics of white adipose tissue. Here we provide evidence that treatment of adult dogs with a beta 3 adrenergic receptor agonist (ICI D7114) that has thermogenic and antiobesity properties leads to the appearance of BAT at several anatomical sites. The presence of BAT was primarily demonstrated by monitoring the inner mitochondrial membrane uncoupling protein and its mRNA, which are unique to the tissue. Neither message nor protein was detected in adipose tissue samples from control dogs but both were detected in samples from dogs treated with ICI D7114. The data suggest that stimulation of beta 3-adrenergic receptors can reactivate nascent BAT (which has the appearance of white adipose tissue) by increasing expression of the gene coding for uncoupling protein or lead to the recruitment of fully differentiated BAT from preadipocyte precursor cells. PMID- 1720551 TI - Characterization of the two size forms of the alpha 1 subunit of skeletal muscle L-type calcium channels. AB - The molecular properties of two size forms of the alpha 1 subunit of purified skeletal muscle calcium channels were analyzed. The minor, full-length, form, alpha 1(212), was found to have an apparent molecular mass of 214 kDa by Ferguson plot analysis, while the major, truncated, form, now designated alpha 1(190), had an apparent molecular mass of 193 kDa. Antibody mapping of the C-terminal region of alpha 1(190) with 10 anti-peptide antibodies placed the C terminus between residues 1685 and 1699. Three consensus sites for cAMP-dependent protein phosphorylation are present in the C-terminal region of alpha 1(212) but not in alpha 1(190), and they may be important for the regulation of the ion conductance activity of the calcium channel. PMID- 1720552 TI - Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils. AB - The rate of metabolic acid generation by neutrophils increases greatly when they are activated. Intracellular acidification is prevented in part by Na+/H+ exchange, but a sizable component of H+ extrusion persists in the nominal absence of Na+ and HCO3-. In this report we determined the contribution to H+ extrusion of a putative H+ conductive pathway and its mode of activation. In unstimulated cells, H+ conductance was found to be low and unaffected by depolarization. An experimental system was designed to minimize the metabolic acid generation and membrane potential changes associated with neutrophil activation. By using this system, beta-phorbol esters were shown to increase the H+ (equivalent) permeability of the plasma membrane. The direction of the phorbol ester-induced fluxes was dictated by the electrochemical H+ gradient. Moreover, the parallel migration of a counterion through a rheogenic pathway was necessary for the displacement of measurable amounts of H+ equivalents across the membrane. These findings suggest that the H+ flux is conductive. The effect of beta-phorbol esters was mimicked by diacylglycerol and mezerein and was blocked by staurosporine, whereas alpha-phorbol esters were ineffective. Together, these findings indicate that stimulation of protein kinase C induces the activation of an H+ conductance in the plasma membrane of human neutrophils. Preliminary evidence for activation of a separate, bafilomycin A1-sensitive H+ extrusion mechanism, likely a vacuolar type H(+)-ATPase, is also presented. PMID- 1720553 TI - Deletion of the c-kit protooncogene in the human developmental defect piebald trait. AB - The protooncogene c-kit is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. Homozygous mutations of this gene in the mouse cause anemia, infertility, and albinism, whereas heterozygous mutant mice usually exhibit only a white forehead blaze and depigmentation of the ventral body, tail, and feet. The heterozygous mouse phenotype is very similar to human piebald trait, which is characterized by a congenital white hair forelock and ventral and extremity depigmentation. To investigate the possibility that alterations in the human c-kit gene may be a cause of piebald trait, DNA from seven unrelated affected individuals was examined by Southern blot analysis. One subject, although cytogenetically normal, has a heterozygous deletion of the c kit protooncogene. This deletion encompasses the entire coding region for c-kit and also involves the closely linked gene for platelet-derived growth factor receptor alpha. Fluorescence in situ hybridization of genomic c-kit probes to metaphase chromosomes independently confirmed the deletion in this case. These findings provide molecular evidence mapping piebald trait to the c-kit locus on chromosome 4. Although we cannot exclude the involvement of other closely linked genes, the demonstration of a genomic c-kit deletion in one subject with piebald trait and the marked concordance of the human and mouse phenotypes provide strong evidence for the role of c-kit in the development of human melanocytes and in the pathogenesis of piebald trait. PMID- 1720554 TI - Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution. AB - Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment. One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate resolution structure determination. The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics. These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process. PMID- 1720555 TI - Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C. AB - cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA sequence encoding human PDI. The polypeptide encoded by a clone designated B2 consisted of 512 amino acids and was characterized by a 24-amino acid hydrophobic leader sequence, two regions with absolute identity to the vertebrate PDI active site (Ala-Pro-Trp Cys-Gly-His-Cys-Lys), and a C-terminal endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu). The overall identity of the B2 sequence to that of human PDI was 35% at the amino acid level (79% when conservative substitutions were included) and 39% at the nucleotide level; this included homology between B2 and the region of human PDI believed to be involved in binding estrogens. The deduced amino acid sequence of B2 was also 35% identical to that of a rat form I phosphatidylinositol-specific phospholipase C. Lysates from Escherichia coli cells harboring an expression plasmid bearing the B2 sequence contained significantly elevated levels of PDI activity. Southern analysis indicated the presence of a small PDI-related gene family in alfalfa, of which B2 appeared to correspond to a single gene. An approximately 2-kilobase B2 transcript was expressed in all alfalfa organs tested. In alfalfa cell suspension cultures, B2 transcripts were strongly induced by tunicamycin but not by exposure to fungal elicitor. PMID- 1720556 TI - Two distinct alpha beta T-cell lineages can be distinguished by the differential usage of T-cell receptor V beta gene segments. AB - Avian T cells can be divided into three subpopulations based on their expression of distinct T-cell receptors (TCR1, TCR2, and TCR3), ontogeny, and tissue distribution. The TCR1 cells appear to be the equivalent of mammalian gamma delta cells, but the derivation of cells expressing TCR2 and TCR3 has been unclear. Here we report that chickens contain two families of TCR beta variable (V) gene segments, V beta 1 and V beta 2. Furthermore, TCR2 and TCR3 represent subsets of alpha beta cells that are defined by mutually exclusive usage of these two families of V beta gene segments. Sequence comparisons of V beta 1 and V beta 2 with mammalian TCR beta V segments reveal that V beta 1 gene segments encode the conserved amino acids used to define the mammalian V beta consensus subgroup I, while V beta 2 encodes the amino acids used to define the mammalian V beta subgroup II. Although the beta chains of TCR2 and TCR3 cells are encoded by the same diversity (D), joining (J), and constant (C) region segments, V beta 1 gene segments undergo rearrangement before V beta 2 gene segments during T-cell development. This may result from the fact that TCR2 cells undergo V-DJ joining by deletional rearrangement, whereas TCR3 cells undergo V-DJ joining by inversional rearrangement. These data suggest that the TCR alpha beta cells can be divided into two distinct and evolutionarily conserved lineages based on V beta gene segment usage. The clear-cut separation of these lineages in the chicken may help to define their immunologic role. PMID- 1720558 TI - Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1. AB - The effects of the viral gene nef on human immunodeficiency virus type 1 (HIV-1) replication in culture were investigated using nef alleles of the HIV-1 IIIB and ELI strains. The results demonstrate significant allelic variation in the effect of nef on virus replication in both an established human CD4+ T-cell line and primary human lymphocytes. In the context of the HXB2 virus, the ELI nef allele but not the IIIB nef allele permits initiation of efficient low-multiplicity infection in primary peripheral blood mononuclear cells, including unfractionated peripheral blood lymphocytes, T cells, and monocyte/macrophages. Within the same genetic context, the IIIB nef allele slightly retards replication of the virus in a T-cell line, whereas the ELI nef allele accelerates replication of the virus. Sequences in the IIIB and ELI genomes outside of nef also moderate the effects of nef on HIV-1 replication. nef did not appear to determine the host-cell preference of the virus. These studies may help to reconcile apparently conflicting reports on the role of nef in HIV-1 replication and suggest that HIV 1 nef may play an important role in viral pathogenesis. PMID- 1720557 TI - M-15: high-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus, locus coeruleus, and spinal cord. AB - The 20-amino acid peptide M-15 binds with high affinity (IC50 approximately 0.1 nM) to 125I-labeled galanin (125I-GAL) binding sites in membranes from the ventral hippocampus, midbrain, and rat spinal cord. Receptor autoradiographic studies show that M-15 can displace 125I-GAL from all labeled sites. M-15 acts as a reversible high-affinity antagonist in blocking the inhibitory effects of GAL on the evoked release of acetylcholine in vivo in the hippocampus and on the GAL induced hyperpolarization of locus coeruleus neurons in slices. M-15 also blocks the facilitatory effects of GAL on the spinal flexor reflex. Thus, the chimeric peptide M-15 [GAL-(1-13)-substance P-(5-11)amide] represents the first antagonist to the neuronal actions of GAL. PMID- 1720560 TI - Mast cell degranulation in hemorrhagic shock in rats and the effects of vasoactive intestinal peptide, aprotinin and H1 and H2-receptor blockers on degranulation. AB - Various stressful stimuli cause mast cell degranulation. Hemorrhagic shock is one such stressful stimulus which may cause mast cell degranulation and histamine release. Histamine may be involved in the pathophysiology of hemorrhage. It was reported that there are large amounts of histamine in the anterior and posterior lobes of the pituitary and the adjacent median eminence of the hypothalamus. Most of the histamine in the posterior pituitary is in mast cells. In addition, both vasoactive intestinal peptide (VIP) and histamine-containing neurons are available in the hypothalamus. It therefore seems reasonable to suppose that these three systems (i.e., mast cells, VIP-containing neurons, and histamine containing neurons) may play an important role in the progression of hemorrhagic shock. 66 albino rats (200-250 g) of either sex were used. The presence of mast cells was examined by light microscopy. Hemorrhage caused mast cell degranulation in a correlation with the amount of blood loss. In all cases, the most intense degranulation was observed in the hypothalamus, especially the nucleus arcuatus, and in the subcutaneous tissue. The intensity of degranulation gradually decreased in the peripheral blood vessel, peritoneum and omentum, in this order. VIP prevented degranulation, but aprotinin and H1 and H2 receptor blockers did not. PMID- 1720559 TI - Effect of the steroidal alkaloid buxaminol-E on blood pressure, acetylcholinesterase activity and (3H)quinuclidinyl benzilate binding in cerebral cortex. AB - Pharmacological properties of the steroidal alkaloid buxaminol-E isolated from Buxus sempervirens varietas bullata were studied in relation to the cholinergic nervous system. In anaesthetised cats buxaminol-E (1.25 mumol/kg) initially induced a small short-lasting increase in blood pressure followed by marked hypotension. Atropine (1.47 mumol/kg) inhibited the hypotensive effect almost completely and methylatropine (1.47 mumol/kg) partially. Buxaminol-E and the muscarinic agonists McN-A-343 and bethanechol inhibited the binding of the muscarinic antagonist (3H)quinuclidinyl benzilate in cat and rabbit cerebral cortex membranes in decreasing order of potency. In about 10-fold higher concentrations they also inhibited acetylcholinesterase activity in cat cerebral cortex in the same order of potency. The results suggest that the hypotensive effect of buxaminol-E could be due to both central and peripheral activation of muscarinic receptors, and to a lesser degree to inhibition of acetylcholinesterase activity. PMID- 1720562 TI - Antigenic variation in Trypanosoma equiperdum. AB - Trypanosoma equiperdum is an African trypanosome that causes dourine in horses. Like the other African trypanosomes, T. equiperdum escapes elimination by the immune system of its host by using an elaborate system of antigenic variant. The trypanosomes are covered by a coat consisting of a single protein called the variable surface glycoprotein (VSG) that acts as the major trypanosome immunogen. As the host responds to one VSG, trypanosomes covered with another VSG become dominant. There is a loose order of appearance of these VSG during the infection. The factors that affect the timing of VSG expression and the effective size of the VSG repertoire in T. equiperdum are reviewed. The VSG genes are generally activated by a process of duplicative transposition involving the duplication of a silent VSG gene and inserting a copy of the gene into an expression site. The order of VSG expression is related to the amount of homology between the silent gene and the expression site. The genes expressed late in infection lack extensive homology with the expression site and depend on homology with the gene in the expression site. The genes coding for VSG expressed late in infection are hybrid genes because of this mode of transfer. This transfer mechanism allows the trypanosome to create complex VSG genes from parts of several different silent genes that are each pseudogenes. Additionally, data are presented showing that only a limited portion of the VSG is actually seen by the host immune system. These factors indicate that the effective VSG repertoire is greater than the number of VSG genes in the trypanosome genome. PMID- 1720561 TI - [Bilateral interpleural analgesia in a patient with multiple myeloma]. AB - The case of one patient with multiple myeloma and thoracic pain during myeloptysis is described. Bilateral interpleural analgesia was accomplished with 10 ml of 0.375% bupivacaine with adrenaline (5 micrograms/ml) in both hemithorax and bilateral infusion at a rate of 3 ml/h started immediately and maintained during 24 hours. Analgesia was then obtained by means of a bolus pattern 10 ml/6 h until myeloptysis finished. Analgesic effectiveness was evaluated through visual analogue scale (VAS). Plasmatic levels of bupivacaine were found below toxic levels; maximum concentration was (1.12 microgram/ml) at the steady state (5 x t 1/2). Difficulty in treatment of pain and utility of interpleural analgesia is suggested. PMID- 1720563 TI - Visual results after laser treatment for peripapillary choroidal neovascular membranes. AB - To investigate the potentially harmful effects of laser photocoagulation in the papillomacular bundle (PMB), the records of patients treated for idiopathic neovascular membranes or membranes secondary to histoplasmosis extending into the PMB were reviewed. Twenty-eight eyes of 27 patients were identified. Most eyes (75%) were treated with the krypton red laser, while the remainder (25%) were treated with either argon green (18%) or a combination of the two (7%). After treatment 21 eyes (75%) had stable or improved visual acuity and three eyes (11%) lost more than three lines. Four eyes (14%) had changes in the optic disc and one eye developed a permanent arcuate scotoma. These data suggest that severe visual loss and extensive visual field defects occur rarely after photocoagulation of peripapillary choroidal neovascular membranes. PMID- 1720564 TI - Disc neovascularization in patients with AIDS and cytomegalovirus retinitis. AB - The authors report two patients with AIDS and cytomegalovirus retinitis who developed disc neovascularization. The disc neovascularization regressed spontaneously over several months and did not cause vitreous hemorrhage or visual loss. PMID- 1720565 TI - Clinicopathologic studies of treated choroidal neovascular membranes. A review and report of two cases. AB - The previously reported and two additional clinicopathologic studies of treated choroidal neovascular membranes were reviewed. Laser photocoagulation can obliterate choroidal neovascular membranes, but persistent and recurrent neovascularization contiguous with the treated area or the development of a new area of neovascularization contiguous or not contiguous with the treated area was seen histopathologically in nine of 12 (75%) lesions of 10 eyes. The scar that ensues after photocoagulation resembles the naturally occurring scar associated with choroidal neovascularization. Portions of the scar are comprised of hyperplastic retinal pigment epithelium. The inner retinal layers are more likely to be preserved after krypton red photocoagulation. Full-thickness destruction of the retina occurs with argon blue-green and argon green photocoagulation at levels of energy in which the end point is a uniform, white lesion. PMID- 1720566 TI - Axonal projections from the pontine pneumotaxic region to the nucleus raphe magnus in cats. AB - In 15 pentobarbital anesthetized and vagotomized cats, 60 non-respiratory units recorded from the medial parabrachial and Kolliker-Fuse nuclear complex (NPBM KF), were found to be antidromically activated by electrical stimulation of the nucleus raphe magnus (NRM). Seven respiratory units (6 inspiratory, 1 expiratory), comprising 8.0% of the 87 respiratory units examined, were also antidromically activated by stimulation of the NRM. The antidromic latencies ranged from 0.4 to 2.5 ms (mean 1.2 ms). In 6 cats, following injection of WGA HRP (wheat germ agglutinin-conjugated horseradish peroxidase) into the NRM, a number of retrogradely labelled neurons were observed mainly in the NPBM-KF complex, and some in subcoeruleus nucleus and adjacent tegmental field. These results demonstrate that predominantly non-respiratory and a portion of respiratory neurons in the rostral pons, especially in the NPBM-KF complex, send a monosynaptic axonal projection to the NRM. It is suggested that the NPBM-KF to NRM pathway could be, in part, involved in the control of respiration as well as nociception control. PMID- 1720567 TI - Use of recombinant interferons and hematopoietic growth factors in patients infected with human immunodeficiency virus. AB - The recombinant cytokines are increasingly important therapeutic agents for patients with AIDS. Recombinant interferon-alpha has demonstrated antitumor and antiretroviral activities in patients with Kaposi's sarcoma. Limited studies with interferon-beta suggest that it also has antitumor effects in patients with Kaposi's sarcoma, but interferon-gamma appears to be ineffective in controlling this tumor. The hematopoietic growth factors, including erythropoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF), have been evaluated in several populations of human immunodeficiency virus (HIV)-infected individuals. The combination of G-CSF and recombinant human erythropoietin completely reversed the zidovudine-induced neutropenia of AIDS patients but was only partially effective in reversing anemia. In several clinical trials, GM-CSF induced marked increases in leukocyte counts and improved neutrophil function in some AIDS patients. In severely immunocompromised patients with disease caused by HIV who were receiving therapy with either G-CSF or GM-CSF, opportunistic infections continued to occur despite increases in circulating white blood cell counts. Recombinant cytokines may be used in the future in AIDS patients as adjunctive treatment with myelosuppressive antibiotics and chemotherapeutic drugs, as a possible means of enhancing host defense, or as agents of immune reconstitution. PMID- 1720568 TI - Use of cytokines during prolonged neutropenia associated with autologous bone marrow transplantation. AB - Molecular cloning, expression, and formulation of recombinant myeloid colony stimulating factors (CSFs) have afforded the potential to reduce therapy associated toxicity in patients who receive intensive chemotherapy. The use of granulocyte CSF and granulocyte-macrophage CSF has resulted in acceleration of hematopoietic recovery in patients undergoing autologous bone marrow transplantation. This acceleration is associated with a reduction of treatment related toxicity, although a period of absolute leukopenia prevails despite infusion of bone marrow and recombinant CSFs. Addition of CSF-primed peripheral blood progenitor cells to bone marrow in the supportive care of these patients has provided a further reduction in the duration of absolute leukopenia and an associated reduction in the incidence of infections and other complications associated with bone marrow transplantation. The ability of CSFs to mobilize hematopoietic progenitor cells offers the possibility of utilizing these cells for a variety of medical purposes, including modification of the therapeutic approach to malignant disease. PMID- 1720569 TI - Folding of circularly permuted transfer RNAs. AB - All of the ribose-phosphate linkages in yeast tRNA(Phe) that could be cleaved without affecting the folding of the molecule have been determined in a single experiment. Circular permutation analysis subjects circular tRNA molecules to limited alkaline hydrolysis in order to generate one random break per molecule. Correctly folded tRNAs were identified by lead cleavage at neutral pH, a well characterized reaction that requires proper folding of tRNA(Phe). Surprisingly, most of the circularly permuted tRNA molecules folded correctly. This result suggests that the tRNA folding motif could occur internally within other RNA sequences, and a computer search of Genbank entries has identified many examples of such motifs. PMID- 1720570 TI - Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia. AB - The translocation t(15;17) associated with acute promyelocytic leukemia results in the fusion of the retinoic acid receptor alpha (RARA) gene to the PML gene. Characterization of PML revealed that it is a putative zinc finger protein and potential transcription factor that is commonly expressed, with at least three major transcription products. PML breakpoints cluster in two regions on either side of an alternatively spliced exon. Although leukemic cells with translocations characteristically express only one fusion product, both PML/RARA (on the 15q+ derivative chromosome) and RARA/PML (on the 17q- derivative) are transcribed. PMID- 1720572 TI - A requirement for the intercellular messenger nitric oxide in long-term potentiation. AB - Long-term potentiation (LTP) of synaptic transmission is a widely studied model of neuronal plasticity. The induction of LTP is known to require processes in the postsynaptic neuron, while experimental evidence suggests that the expression of LTP may occur in the presynaptic terminal. This has led to speculation that a retrograde messenger travels from the post- to the presynaptic cell during induction of LTP. Extracellular application or postsynaptic injection of two inhibitors of nitric oxide synthase, N-nitro-L-arginine or NG-methyl-L-arginine, blocks LTP. Extracellular application of hemoglobin, which binds nitric oxide, also attenuates LTP. These findings suggest that nitric oxide liberated from postsynaptic neurons may travel back to presynaptic terminals to cause LTP expression. PMID- 1720571 TI - Identification of a competitive HGF antagonist encoded by an alternative transcript. AB - We identified a naturally occurring hepatocyte growth factor (HGF) variant, whose predicted sequence extends only through the second kringle domain of this plasminogen-related molecule. This smaller molecule, derived from an alternative HGF transcript, lacked mitogenic activity but specifically inhibited HGF-induced mitogenesis. Cross-linking studies demonstrated that the truncated molecule competes with HGF for binding to the HGF receptor, which has been identified as the c-met protooncogene product. Thus, the same gene encodes both a growth factor and its direct antagonist. PMID- 1720574 TI - Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. AB - Conflicting three-dimensional structures of charybdotoxin (Chtx), a blocker of K+ channels, have been previously reported. A high-resolution model depicting the tertiary structure of Chtx has been obtained by DIANA and X-PLOR calculations from new proton nuclear magnetic resonance (NMR) data. The protein possesses a small triple-stranded antiparallel beta sheet linked to a short helix by two disulfides and to an extended fragment by one disulfide, respectively. This motif also exists in all known structures of scorpion toxins, irrespective of their size, sequence, and function. Strikingly, antibacterial insect defensins also adopt this folding pattern. PMID- 1720573 TI - Functional contribution of neuronal AChR subunits revealed by antisense oligonucleotides. AB - Although multiple related genes encoding nicotinic acetylcholine receptor (AChR) subunits have been identified, how each of these subunits contributes to AChRs in neurons is not known. Sympathetic neurons express four classes of AChR channels and six AChR subunit genes (alpha 3, alpha 4, alpha 5, alpha 7, beta 2, and beta 4). The contribution of individual subunits to AChR channel subtypes in these neurons was examined by selective deletion with antisense oligonucleotides. An alpha 3 antisense oligonucleotide decreased the number and altered the properties of the normally expressed ACh-activated channels. The remaining AChR channels have distinct biophysical and pharmacological properties that indicate an important functional contribution of the alpha 7 subunit. PMID- 1720575 TI - Modifications of health behaviour in response to air pollution notifications in Copenhagen. AB - Ambient air quality is a major issue today in large cities all over the world. On the theoretical background of the health belief model and the health locus of control model, we studied the knowledge and beliefs about air pollution and the modifications of health behaviour brought about by information to the public about projected levels of air pollution, with special emphasis on reduction of outdoor activity and avoidance of car driving. Data were collected with a questionnaire among a sample of residents in the Copenhagen area. The respondents were almost universally knowledgeable about the prime emission source and concerned about the possible health effects of the air pollution in the area. Avoidance of outdoor activity was associated with personal experiences of symptoms ascribed to the air pollution, employment status, and with female sex, but not with knowledge or beliefs about the degree or health implications of the air pollution. The willingness to avoid car driving was positively associated with the belief that one can oneself influence one's health and with female sex. Lung diseased respondents were generally more prone to protect themselves than the healthy, both by avoiding outdoor activity and by being less willing to avoid car driving. The present study was conducted in an only moderately polluted city, and it is not clear whether the findings and conclusions can be generalized to more polluted cities. The study partly supported the underlying theories of the determinants of health behaviour, but also indicated a need for a broader theoretical framework, incorporating aspects of the respondents' life situation and personal experience which would be relevant to the specific type of health behaviour under study. PMID- 1720576 TI - Neurochemical analysis of focal ischemia in rats. AB - BACKGROUND AND PURPOSE: Increases in uric acid follow experimental stroke, which may be related to free radical formation by xanthine oxidase. The present study examined the time course of changes in xanthine and uric acid and their relationship to changes in the free radical scavengers glutathione, cysteine, and ascorbic acid. METHODS: Focal ischemia was induced by occluding the middle cerebral artery, followed by transient occlusion of the common carotid arteries for 60 minutes. At varying time points, animals were sacrificed, and ischemic cortex was dissected. Neurochemical measurements were made by high-performance liquid chromatography with 16-sensor electrochemical detection. RESULTS: Marked increases in uric acid were seen at all time points, with a maximal increase at 1 day and a persistent increase lasting up to 21 days. There were smaller reciprocal decreases in xanthine. Glutathione, cysteine, and ascorbic acid showed significant decreases, consistent with the generation of free radicals. Reductions in levels of cysteine and glutathione were significantly correlated with increases in uric acid levels. CONCLUSIONS: These findings confirm marked alterations in purine metabolism following focal ischemia and suggest that xanthine oxidase contributes to the generation of free radicals. PMID- 1720577 TI - Scrapie and GSS--the importance of protein. PMID- 1720578 TI - How chicks make memories: the cellular cascade from c-fos to dendritic remodelling. AB - Training chicks on a one-trial passive avoidance task results in a cellular cascade over the subsequent hours. Phosphorylation of the presynaptic phosphokinase C substrate B-50 is followed by immediate-early gene expression and increased synthesis of pre- and postsynaptic glycoproteins, increases in dendritic spine densities, synapse and synaptic vesicle numbers, and a prolonged increase in neuronal bursting. Many of these effects have been localized to two forebrain regions: the left intermediate medial hyperstriatum ventrale and the lobus parolfactorius. Pretraining lesions in the left intermediate medial hyperstriatum ventrale, or post-training lesions in the lobus parolfactorius result in amnesia. These and related results lead to models of memory storage based on multiple representation by way of synaptic stabilization through glycoprotein synaptic recognition molecules. PMID- 1720579 TI - Correlated activity in the CNS: a role on every timescale? AB - Until recently, correlated neuronal activity was seen by many as an arcane subject, of interest only to those with mathematical minds and access to elaborate electronics. However, the list of situations in which correlated activity is known or strongly suspected to be highly influential now embraces almost every branch of neuroscience, including perception, memory and the development and plasticity of structural and functional linkages throughout the CNS. Previous reviews in TINS have covered several specific roles of correlated activity in detail. Here, my aim is to explore their diversity, emphasizing the organizing potential of correlation across timescales ranging from the momentary to the evolutionary. PMID- 1720580 TI - Magnetic brain stimulation: a tool to explore the action of the motor cortex on single human spinal motoneurones. AB - The human brain can be stimulated by a single intense magnetic pulse over the scalp. Currents induced within the cranium excite the motor cortex and cause limb muscles to contract. The discharge of single motor units, the firing of which is maintained by voluntary effort, can be modulated by magnetic stimuli. Peri stimulus time histograms suggest that after a cortical stimulus spinal motoneurones are induced to fire by a sequence of EPSPs arising from a train of impulses transmitted monosynaptically over fast-conducting corticospinal fibres. In multiple sclerosis both dispersion of this descending volley and partial transmission failure can impair motoneurone excitation and may explain motor symptoms in these patients. PMID- 1720581 TI - Fatty acid elevation and brain seizure activity. PMID- 1720582 TI - Reflex control of magnocellular vasopressin and oxytocin secretion. AB - Reflex control of magnocellular vasopressin and oxytocin secretion has captured the curiosity and investigative imagination of neuroendocrinologists for nearly 50 years. While it may seem obvious that brisk elevations in circulating levels of vasopressin in response to hemorrhage, or of oxytocin in response to suckling, must of necessity arise from magnocellular neurosecretory neurons in the hypothalamus, the central pathways mediating these reflexes have, until quite recently, remained elusive. In this brief review, ongoing attempts to delineate these pathways are summarized. Evidence for plasticity and local modulation of magnocellular reflexes in response to prolonged stimulation, such as chronic dehydration and lactation, is also presented. PMID- 1720583 TI - Pancreatic B cells are bursting, but how? AB - Insulin secretogogues have long been known to stimulate and modulate bursting electrical activity in pancreatic islet B cells and thereby supply extracellular Ca2+ for the exocytosis of insulin. Recent results have ruled out a long-held hypothesis for the mechanism of burst formation that postulated key roles for intracellular Ca2+ accumulation and activation of Ca(2+)-activated K+ channels. Here, we present an alternative hypotheses based on a persistent Ca2+ conductance and, possibly, phasic activation of ATP-sensitive K+ channels. These hypotheses are compared with mechanisms of bursting proposed for invertebrate and mammalian neurons. PMID- 1720584 TI - Is the histaminergic neuron system a regulatory center for whole-brain activity? AB - Recent immunocytochemical studies have demonstrated the existence of histaminergic neurons in the brain, which are concentrated in the tuberomammillary nucleus of the posterior hypothalamus, and which project efferent fibers to almost all parts of the brain. Three subtypes of histamine receptors are widely distributed in the brain, not only on neurons but also on astrocytes and blood vessels. Consistent with its wide-ranging output, the histaminergic neuron system regulates various activities of the brain, such as the arousal state, brain energy metabolism, locomotor activity, neuroendocrine, autonomic and vestibular functions, feeding, drinking, sexual behavior, and analgesia--this regulation is possibly achieved by the histaminergic system as a whole. PMID- 1720585 TI - Phocine distemper virus is phylogenetically related to canine distemper virus. PMID- 1720586 TI - Leiomyoma of the small colon in a horse. AB - A leiomyoma of the small colon was discovered incidentally in a 4-year-old Thoroughbred gelding during colic surgery to correct large colon displacement. The mass and 20 cm of small colon were resected, and an end-to-end anastomosis was performed. A postoperative fecal impaction proximal to the anastomosis responded after 5 days to administration of intravenous fluids, analgesics, and stool softeners. PMID- 1720587 TI - Distribution of extracellular matrix proteins in odontogenic tumours and developing teeth. AB - The distribution of two cellular fibronectins (cFn), tenascin, laminin, as well as type VII collagen was studied in 14 benign odontogenic tumours of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origins, as well as in developing human teeth by immunocytochemical means using monoclonal antibodies (Mabs). An extradomain sequence-A-containing form of cFn (EDA-cFn) was seen in the extracellular matrix (ECM) of all tumours studied and in the mesenchyme of the developing tooth germs, indicating that cFn in these tissues are predominantly produced locally. A form of cFn containing an oncofetal domain (Onc-cFn), hitherto found only in carcinomas, was detected focally in the stroma of most ameloblastomas but was absent from ameloblastic fibromas and tooth germs. Tenascin was strongly expressed in the basement membrane (BM) zone of all odontogenic tumours and in that of the early tooth germs. Focal absence of laminin and type VII collagen from the BM of some ameloblastomas and the presence of Onc-cFn in the ECM of most ameloblastomas may correlate with their aggressive behaviour. The results also suggest that EDA-cFn and tenascin are involved in epithelial-mesenchymal interactions during tooth development and in odontogenic tumours. PMID- 1720588 TI - Keratin and carcinoembryonic antigen (CEA) in human melanoma cells. AB - Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin. PMID- 1720589 TI - Protection of BALB/c mice from respiratory syncytial virus infection by immunization with a synthetic peptide derived from the G glycoprotein. AB - A synthetic peptide homologous to amino acids 174-187 of the G glycoprotein of the A2 strain of human respiratory syncytial (RS) virus (G/174-187) was shown to induce protection from live virus challenge of BALB/c mice after immunization with three doses of 50 micrograms of peptide coupled to keyhole limpet hemocyanin. Immunized mice showed high levels of circulating RS-specific antibodies as detected by ELISA assay; however, no neutralizing antibodies were found. Moreover, an important short-term cytotoxic T-cell response was observed with lymphocytes isolated from the lungs but not from the spleen of immunized mice. This response was lost 24 weeks after immunization; however, mice remained protected against challenge with live RS virus. In addition, a monoclonal antibody that specifically binds to peptide G/174-187 was found efficient in conferring passive protection from challenge: this data further supports our results on the importance of the 174-187 region in protection. Another peptide, spanning amino acids 144 to 159, was shown to induce neutralizing antibodies but did not confer protection. PMID- 1720590 TI - Binding to CD4 of synthetic peptides patterned on the principal neutralizing domain of the HIV-1 envelope protein. AB - The interaction between the viral envelope protein gp120 and the cellular surface antigen CD4 is a key event in HIV-1 infection. Reciprocal high affinity binding sites have been located in the first domain of CD4 and in the carboxy-terminal region of gp120, respectively. Upon infection, the membranes of the target cells fuse; sites of CD4 and gp120, distinct from their high affinity binding sites, play a role in the post-binding events leading to syncytia formation. We have studied the interactions of CD4 with gp120 and gp120-derived peptides using an in vitro assay based on immobilized recombinant soluble CD4 (sCD4). In this system CD4 binds to recombinant soluble gp120 and to anti-receptor peptides derived from the high affinity CD4-binding site of gp120, as well as to peptides corresponding to the principal neutralizing domain (PND) of the envelope protein, i.e., to the domain required for HIV-1-mediated syncytium formation. Competition experiments performed using epitope-specific mAbs and a variety of peptides indicated that PND-derived peptides are specifically recognized by a CD4 site adjacent to, but distinct from, the high affinity gp120-binding site of CD4. Synthetic peptides patterned on the PND of different viral isolates were retained onto sCD4-based affinity columns at different extent; some of the structural requirements for binding were analyzed. Studies performed on CD4+ T-cells showed that PND-derived peptides also interact with CD4 in its native membrane-bound conformation. These results indicate that a direct contact takes place between CD4 and the gp120 domain participating in HIV-induced syncytia formation. PMID- 1720591 TI - Sequence of the genes encoding the structural proteins of the low-virulence tick borne flaviviruses Langat TP21 and Yelantsev. AB - The structural protein coding regions of the genomes of Langat virus (strain TP21) and Yelantsev virus, which was originally described to be a low virulence natural isolate of tick-borne encephalitis virus, were cloned and sequenced. These viruses had both been used as experimental live vaccines against tick-borne encephalitis in Czechoslovakia and Russia, respectively. Peptide mapping and monoclonal antibody binding experiments yielded identical reaction patterns for Langat virus and Yelantsev virus which were distinct, however, from the pattern obtained with tick-borne encephalitis virus. Sequence analysis confirmed this distinctiveness and proved that the vaccine strain Yelantsev was also Langat virus. The envelope protein E of both viruses exhibits an 88% amino acid sequence homology with that of tick-borne encephalitis virus. Assessment of the antigenic reactivity and sequence comparison with the E protein of tick-borne encephalitis virus revealed several differences affecting epitopes involved in virus neutralization. These observations suggest that Langat-like virus-based vaccines may not represent the most effective means to achieve protection against tick borne encephalitis virus. PMID- 1720592 TI - Selected human immunodeficiency virus replicates preferentially through the basolateral surface of differentiated human colon epithelial cells. AB - We have used HIV1-NDK-infected HT29 cells grown on permeable substratum to study the polarity of virus maturation in human intestinal cells. When cultured in glucose-containing medium, these cells are mostly undifferentiated. The removal of glucose from the medium allowed the emergence of a selected differentiated subpopulation which continued to produce viral particles in the culture supernatant. The polarity of viral production was evaluated by harvesting virus from the two sides of the monolayer. Seventy-five percent of released HIV was found on the basolateral side of the monolayer. Mature viral particles were observed by electron microscopy near the apical (luminal) and the basolateral (serosal) membrane. These data suggest that epithelial cells of the colon productively infected by a selected strain of HIV are able to produce the virus through both sides of the epithelium but mainly through the serosal side. PMID- 1720593 TI - Interferon treatment inhibits early events in vaccinia virus gene expression in infected mice. AB - We have analyzed the role of exogenous administration of mouse interferon (IFN alpha + beta) on the replication of vaccinia virus in peritoneal cells and in the spleen of Balb/c mice. Mice were pretreated for 16 hr with IFN and then infected with a vaccinia virus recombinant expressing luciferase under an early or late virus promoter, and the enzyme activity was measured in the course of virus infection. A dose of IFN as low as 10(3) units/mouse abolished the appearance of luciferase activity in cells of the peritoneal cavity and in spleen cells. The IFN-mediated inhibition of luciferase activity was observed even when mice were infected 4 days after the administration of IFN. The IFN-treated animals were considered free of virus since neither luciferase nor viral proteins were detected in target cells several days after virus infection. Despite a severe IFN mediated inhibition of luciferase activity, the appearance of luciferase on mRNA levels was not inhibited 6 hr after virus infection. Our finding revealed that replication of vaccinia virus in Balb/c mice is exquisitively sensitive to inhibition by IFN and that this effect occurs at early times postinfection, most likely as a result of a translational block. PMID- 1720594 TI - [Clinical evaluation of serum elastase-1 levels in patients with acute pancreatitis]. AB - The prospective examination and quantitative determination of concentration of serum elastase 1 using RIA method were performed in 75 patients with pancreatic diseases at the Clinic of General and Vascular Surgery and the Institute of Nuclear Medicine of the M.M.A. in the period 1988-1989. The values of serum elastase 1 concentration were compared with values of amylase in serum and urine. The greater diagnostical importance of serum elastase 1 was found compared to values of amylase in serum and urine, not only in patients with acute pancreatitis but also in exacerbation of chronic pancreatitis. The level of serum elastase 1 concentration enables more reliable follow up of the evolution of the disease and therapeutical effects in patients with acute pancreatitis. PMID- 1720595 TI - The carbohydrate-deficient glycoprotein syndrome. A new inherited multisystemic disease with severe nervous system involvement. PMID- 1720596 TI - Contribution of galanin to stress-induced impairment of insulin secretion in swimming mice. AB - This study examines the potential role of the neuropeptide, galanin, in stress induced inhibition of insulin secretion in swimming mice. Firstly, the pancreatic and adrenal content of galanin-like immunoreactivity was determined in mice after swimming stress. It was found that pancreatic content was significantly lower in stressed mice than in resting controls, both after 2 (P less than 0.05) and 6 (P less than 0.025) minutes of swimming, suggesting partial release of pancreatic galanin during stress. In contrast, the adrenal content of galanin-like immunoreactivity did not change during the swimming stress. Gel filtration of tissue extracts indicated that (1) mouse pancreas contains two forms of galanin like immunoreactivity; one co-eluting with synthetic porcine galanin (centered on Kav of 0.70) and another with a larger molecular weight (centered on Kav of 0.30), and (2) mouse adrenal contains a small void volume-peak and a larger peak of immunoreactivity, the latter co-eluting with synthetic galanin. Secondly, the effects of swimming stress on plasma glucose and insulin levels were compared in mice that received high titre rabbit anti-galanin serum with those in mice receiving normal rabbit serum. In normal rabbit serum-pretreated swimming mice, glucose-induced insulin levels were only 50% of resting controls (P less than 0.01). Immunoneutralization of galanin with specific antiserum abolished this swimming stress-induced inhibition of glucose-stimulated insulin levels. This was accompanied by a modestly enhanced rate of glucose disappearance. These findings suggest that pancreatic galanin is released during swimming stress in mice and that endogenous galanin makes a major contribution to stress-induced impairment of insulin secretion. PMID- 1720597 TI - Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy. AB - Vitamin C deficiency is associated with defective connective tissue, particularly in wound healing. Ascorbate is required for hydroxylation of proline residues in procollagen and hydroxyproline stabilizes the collagen triple helical structure. Consequently, ascorbate stimulates procollagen secretion. However, collagen synthesis in ascorbate-deficient guinea pigs is decreased with only moderate effects on proline hydroxylation. Proteoglycan synthesis, which does not require ascorbate, also is decreased and both effects are correlated with the extent of weight loss during scurvy. Fasting, with ascorbate supplementation, produces similar effects. Both functions are inhibited in cells cultured in sera from either scorbutic or starved guinea pigs and inhibition is reversed with insulin like growth factor (IGF)-I. The inhibitor appears to consist of two IGF-binding proteins induced during vitamin C deficiency and starving and may be responsible for in vivo inhibition of collagen and proteoglycan synthesis. PMID- 1720598 TI - Comparative study of the anti-HIV activities of ascorbate and thiol-containing reducing agents in chronically HIV-infected cells. AB - To elucidate the action of vitamin C on pathogenic human retroviruses, we investigated and compared the effects of noncytoxic concentrations of ascorbic acid (AA), its calcium salt (Ca-ascorbate), and two thiol-based reducing agents [glutathione (GSH) and N-acetyl-L-cysteine (NAC)] against human immunodeficiency virus (HIV)-1 replication in chronically infected T lymphocytes. Ca-ascorbate reduced extracellular HIV reverse transcriptase (RT) activity by about the same magnitude as the equivalent dose of AA. Long-term experiments showed that continuous presence of ascorbate was necessary for HIV suppression. NAC (10 mmol/L) caused less than twofold inhibition of HIV RT and conferred a synergistic effect (approximately eightfold inhibition) when tested simultaneously with AA (0.426 mmol/L). In contrast, nonesterified GSH (less than or equal to 1.838 mmol/L) had no effect on RT concentrations and did not potentiate the anti-HIV effect of AA. These results further support the potent antiviral activity of ascorbate and suggest its therapeutic value in controlling HIV infection in combination with thiols. PMID- 1720599 TI - An immunocytochemical study of the development of the olfactory system in the three-spined stickleback (Gasterosteus aculeatus L., Teleostei). AB - Antisera against a variety of substances have been found to produce an identical immunoreaction in the developing olfactory system of a teleost, the three-spined stickleback (Gasterosteus aculeatus). The label is localized in the olfactory placode, the olfactory nerve and those parts of the secondary olfactory tracts which constitute the dorsal descending fascicles and the ventral descending fibers of the medial olfactory tract. The label was first detected 3 days after fertilization (3D) in the olfactory placode where labeled supporting cells were observed. At 4D, the label was observed at the site of the developing olfactory bulbs. At 7D, the olfactory placode lost the direct contact with the brain and the labeled olfactory nerve became visible. At the same time, the medial olfactory tract emerged from the bulbs, and contacts with cells in the nucleus of the terminal nerve were observed. The development of the medial olfactory tract proceeded caudally, and by the end of 10D, the olfactory tract reached the periventricular hypothalamus. Pre-absorption of the antisera with the respective antigens did not abolish the capacity of the antisera to produce the label. The immunoreaction is thus not specific for the antigens against which the antisera have been raised. Yet the label produced by the immunoreaction is an extremely reliable marker for the primary olfactory tract, and the only existing marker by which secondary olfactory tracts can be visualized. PMID- 1720600 TI - A new method for evaluation of the acrosome reaction in viable human spermatozoa. AB - The acrosome reaction of human spermatozoa was induced by changes of temperature. Spermatozoa were collected from fertile donors and a patient group, and selected by the "swim-up" method. The spermatozoa were treated in two different ways: Protocol I: 24 hours at room temperature followed by additional incubation at 37 degrees C for 3 hours (control), and protocol II: 24 hours at 4 degrees C followed by additional incubation at 37 degrees C for 3 hours. The acrosome reaction of the viable spermatozoa was evaluated by a new method utilizing indirect immunofluorescence with anti-outer acrosomal membrane antibodies and exposure to a hypo-osmotic medium. In fertile donors as well as in the patient group, significant induction of the acrosome reaction (20%) was evident after exposure to low temperature (4 degrees C). The spontaneous rate of acrosome reaction in the control group was below 7%. PMID- 1720601 TI - Experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects. AB - Viral respiratory infections exacerbate asthma in many patients. We hypothesized that one mechanism by which this effect occurs may include potentiated or altered mediator release by mast cells and/or basophils to favor the development of late phase asthmatic reaction (LAR). Therefore, we studied eight subjects with allergic rhinitis before and during an experimentally induced rhinovirus 16 (RV16) infection. We determined levels of plasma histamine and tryptase, and we observed the associated patterns of airway obstruction that developed following inhaled antigen challenge. Bronchial responsiveness to histamine, methacholine, and antigen were all significantly increased during the RV16 illness. Further, the incidence of LAR was significantly higher (five of eight) during the infection than before (one of eight; p = 0.014). In addition, in those patients whose pattern of response following antigen challenge converted from an immediate response only before infection to a dual response (immediate + late phase) during infection, plasma histamine concentrations after challenge were significantly greater than in those whose pattern of response did not change. We conclude that one mechanism by which RV16 infection increases the likelihood of LAR could include enhanced mediator release from pulmonary mast cells or from circulating or recruited basophils. PMID- 1720602 TI - Different hemodynamic responses between acute and chronic infusion of iloprost (prostacyclin-stable analogue) in severe pulmonary hypertension. AB - We report the hemodynamic and clinical effects of acute and chronic administration of iloprost in two patients with severe pulmonary hypertension caused by toxic oil syndrome. We tested the acute effect of progressive increments of iloprost, followed by long-term infusion of the drug during 14 days. The acute response produced an increase in cardiac output and moderate reduction in pulmonary vascular resistance, with no change in pulmonary artery pressure. Nevertheless, a maintained reduction in pulmonary artery pressure and resistance, as well as clinical improvement, was observed after chronic infusion. We conclude that (1) the acute effect of iloprost does not necessarily predict long-term hemodynamic response, and (2) iloprost given in long-term infusion seems to have been an efficacious and safe drug in our two patients, and it opens a new line of treatment. PMID- 1720603 TI - Evidence for a model of exocytosis that involves calcium-activated channels. PMID- 1720604 TI - G-protein modulation of [3H]serotonin release through the L-type calcium channel. PMID- 1720605 TI - Modulation by voltage of calcium channels and adrenal catecholamine release. PMID- 1720606 TI - Possible role for neurosecretory granule channel that resembles gap junctions. PMID- 1720607 TI - Effect of calcium on presynaptic inhibition in the isolated spinal cord of newborn rats. PMID- 1720608 TI - msDNA and bacterial reverse transcriptase. PMID- 1720609 TI - RNA editing in trypanosomatid mitochondria. PMID- 1720610 TI - Using research-based interventions to decrease patient falls. AB - The purpose of this study was to decrease patient falls by applying relevant interventions found in the nursing research literature. Clinical nurse specialists assisted staff with the application of selected research-based patient fall program interventions on two adult medical-surgical specialty units in a tertiary care facility. The fall rate on these two units decreased during the study year, while the all-hospital patient fall rate increased. To encourage comparison of fall data among institutions, a standard method for calculating fall rate is presented. PMID- 1720611 TI - The role of acetaldehyde in the pathogenesis of acute alcoholic pancreatitis. AB - Acetaldehyde (AA), the first product of ethanol metabolism, has been suggested as an important mediator in alcoholic pancreatitis, but experimental evidence has not been convincing. Prior work using the isolated perfused canine pancreas preparation has suggested that toxic oxygen metabolites generated by xanthine oxidase (XO) may mediate the early injury in pancreatitis. Xanthine oxidase is capable of oxidizing AA, and during this oxidation free radicals are released. The hypothesis that acute alcoholic pancreatitis may be initiated by AA in the presence of active XO (converted from xanthine dehydrogenase [XD]) was tested in the authors' experimental preparation by converting XD to XO by a period of ischemia, and infusing AA. Control preparations remained normal throughout the 4 hour perfusion (weight gain, 7 +/- 4 g; amylase activity, 1162 +/- 202 U/dL). One hour of ischemia or infusion of AA at 25 mg/hr or at 50 mg/hr without ischemia did not induce changes in the preparation. Acetaldehyde at 250 mg/hr induced minimal edema and weight gain (16 +/- 4 g; p less than 0.05), but not significant hyperamylasemia. Changes also were not observed when 1-hour ischemia was followed by a bolus of ethanol (1.5 g) or sodium acetate (3.0 g), or by infusion of 25 mg/hr of AA. One hour of ischemia followed by infusion of AA at 50 mg/hr or at 250 mg/hr induced edema, hemorrhage, weight gain (22 +/- 7 g [p less than 0.05] and 26 +/- 17 g [p less than 0.05]) and hyperamylasemia (2249 +/- 1034 U/dL [p less than 0.05] and 2602 +/- 1412 U/dL [p less than 0.05]). Moreover infusion of AA at 250 mg/hr after 2 hours of ischemia potentiated the weight gain (62 +/- 20 g versus 30 +/- 14 g [p less than 0.05]), but not the hyperamylasemia (3404 +/- 589 U/dL versus 2862 +/- 1525 U/dL) as compared with 2 hours of ischemia alone. Pancreatitis induced by 1 hour of ischemia followed by AA at 50 mg/hr could be inhibited by pretreatment with the free radical scavengers superoxide dismutase and catalase and ameliorated with the XO inhibitor allopurinol. The authors conclude that AA, in the presence of active XO, can initiate acute pancreatitis in the isolated canine pancreas preparation and may be important in the initiation of acute alcoholic pancreatitis in man. Toxic oxygen metabolites appear to play an important intermediary role. PMID- 1720612 TI - Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide-coupled receptor, but are differentially regulated by guanine nucleotides. AB - Bombesin (BB), neuromedin C (NMC) and neuromedin B (NMB) stimulated amylase secretion to similar maximum levels, with EC50 values (concentrations causing 50% of maximum effect) of 0.2, 0.3 and 2 nM respectively. Treatment of pancreatic acini with BB or NMB (10 nM) for 30 min resulted in cross-desensitization of secretory responses to subsequent BB and NMB, but not to acetylcholine, which suggests that NMB and BB activate the same receptor. BB, NMC and NMB stimulated production of similar maximum amounts of inositol mono-, bis- and tris phosphates, with EC50 values of 3, 5 and 141 nM respectively. The bombesin receptor antagonist [Leu13-psi(CH2NH)Leu14]BB inhibited stimulation of amylase secretion and inositol phosphate formation by BB, NMC and NMB. Binding of 125I labelled gastrin-releasing peptide (GRP; 200 pM) to rat pancreatic membranes at 22 degrees C was inhibited with relative potencies and IC50 (concn. causing 50% of maximal inhibition; nM) as follows: NMC (0.4) = BB (0.5) greater than NMB (1.8 = GRP (2.6). IC50 values for BB, NMC and NMB inhibition of 125I-GRP binding to intact acini were 5-, 19- and 68-fold higher than their respective values in membranes. The guanine nucleotide analogue guanosine 5'-[beta gamma imido]triphosphate (Gpp[NH]p) produced rightward shifts of NMC and NMB competition curves by 3.5- and 16-fold respectively, but had little effect on the BB and GRP curves. Elevation of the temperature to 37 degrees C or inclusion of NaCl (40 mM) produced quantitatively similar effects to those of Gpp[NH]p. In the presence of both NaCl and Gpp[NH]p the affinities of peptides for membrane receptors were similar to those for intact cells. Modulation of NMB competition curves by Gpp[NH]p was not attenuated by prior treatment of acini with activated pertussis toxin. These results suggest that BB, NMB and NMC stimulate pancreatic secretion by interaction with a common phosphoinositide-linked receptor. Differences in guanine nucleotide regulation suggest that secretagogue-induced receptor-protein interactions may not be identical for NMB and BB. PMID- 1720613 TI - Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119. AB - The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR). The gene is transcribed into a 1250-base transcript. The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron. The PCR amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli. High concentrations of flavodoxin were found (20% of total protein). The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin. PMID- 1720614 TI - Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A. AB - A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy. PMID- 1720615 TI - Determination of the epitope for the inhibitory monoclonal antibody 5-B6 on the catalytic subunit of gastric Mg(2+)-dependent H(+)-transporting and K(+) stimulated ATPase. AB - The monoclonal antibody 5-B6, directed against the alpha-subunit of pig gastric H+,K(+)-ATPase (Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase), was shown to be a potent inhibitor of the K(+)-ATPase activity, thereby binding to the cytoplasmic side of the alpha-subunit of the enzyme [Van Uem, Peters & De Pont (1990) Biochim. Biophys. Acta 1023, 56-62]. In order to define the epitope for 5-B6 on pig gastric H+,K(+)-ATPase more precisely, the alpha-subunit of the enzyme was subjected to limited proteolysis followed by chemical cleavage. Restricted proteolysis with papain followed by sequence analysis yielded an immunoreactive fragment of 27 kDa beginning at Ser379. This fragment was water soluble and possessed the fluorescein isothiocyanate-reaction site. Limited tryptic digestion in the presence of K+ gave rise to an immunoreactive 56 kDa fragment beginning at Ile456, thus restricting the location of the epitope from Ile456 to the C-terminal end of the 27 kDa fragment (around residue 620). Further degradation of the 27 kDa fragment by means of formic acid cleavage at Asp-Pro bonds resulted initially in the formation of two non-immunoreactive fragments of 17 kDa and 11 kDa, indicating that the epitope for 5-B6 has to be localized around the chemical cleavage sites Asp507 and/or Asp510. Comparison of the primary structure of the alpha-subunits of gastric H+,K(+)-ATPase and non immunoreactive rat kidney Na+,K(+)-ATPase shows almost no similarity for the sequence containing these formic acid cleavage sites (Thr504-Leu-Glu-Asp-Pro-Arg Asp-Pro-Arg512), whereas the adjacent sequences are nearly 100% identical. These findings strongly suggest that the epitope for 5-B6 includes (part of) this sequence. PMID- 1720616 TI - Induction of acyl-CoA oxidase and cytochrome P450IVA1 RNA in rat primary hepatocyte culture by peroxisome proliferators. AB - We have characterized the induction of acyl-CoA oxidase and cytochrome P450IVA1 RNAs in a primary hepatocyte culture system in vitro, using a sensitive and specific RNAse protection assay. Hepatocytes were cultured with a maximal inducing dose of the peroxisome proliferator clofibric acid (1 mM), or vehicle control, for 4 days, and the level of RNAs compared with the level in rats which had been treated with corn oil or clofibric acid (300 mg/kg) for 4 days. The level of acyl-CoA oxidase and P450IVA1 RNAs in 4-day-old control hepatocytes was less than 2% of that in control liver. However, the level of these RNAs in RNA from treated hepatocytes was 61% of that in liver RNA from treated rats. Hepatocytes were treated with the potent peroxisome proliferator methylclofenapate (100 microM), and the induction of RNAs determined at various times after exposure. P450IVA1 RNA was significantly induced 1 h after dosing, rising to 34-fold above control after 8 h, whereas acyl-CoA oxidase RNA was not significantly induced until 4 h, increasing to 5.2-fold above control after 8 h. A similar time course of induction was seen after treatment of hepatocytes with 100 microM-nafenopin, 100 microM-methylclofenapate, 1 mM-clofibric acid or 1 mM mono(ethylhexyl) phthalate, suggesting that the differential time course of induction of P450IVA1 and acyl-CoA oxidase RNAs is not related to the esterification, structure or potency of the peroxisome proliferator, but is intrinsic to the process of peroxisome proliferation. Hepatocytes were treated with methylclofenapate in the presence and absence of cycloheximide. P450IVA1 RNA was significantly induced by methylclofenapate in the presence of cycloheximide, rising to 17-fold above control after 8 h. However, no induction of acyl-CoA oxidase RNA was detected in the presence of cycloheximide. Therefore we characterize the induction of acyl-CoA oxidase and P450IVA1 RNAs in primary hepatocyte culture in vitro as a faithful model of the induction response in rat liver, and suggest that induction of P450IVA1 RNA is a primary event in the process of peroxisome proliferation. PMID- 1720617 TI - A vitronectin-receptor-related molecule in human placental brush border membranes. AB - The heterodimeric vitronectin receptor (VNR) and platelet glycoprotein IIb/IIIa (GPIIb/IIIa) are two members of the integrin family of cell adhesion receptors that share the same beta subunit (GPIIIa). These proteins are involved in binding to vitronectin, fibrinogen and fibronectin and in cytoskeleton-membrane interactions. The present study shows that the human placental syncytiotrophoblast brush border membrane contains a heterodimer of subunit Mr values of 140,000 and 90,000 (non-reduced) or 125,000 and 100,000 (reduced). This protein was recognized by a monoclonal antibody to GPIIIa, rabbit antisera to the VNR and a human alloantiserum to GPIIIa. Brush border VNR-related protein bound to an immobilized peptide containing the Arg-Gly-Asp sequence and, less avidly, to immobilized fibrinogen. Only a small fraction of brush border VNR was associated with a cytoskeleton fraction. Membrane-bound brush border GPIIIa was distinct from that of platelets in its resistance to digestion by trypsin and Staphylococcus aureus V8 protease, and had a slightly lower mobility on SDS/PAGE. In addition, lectin-binding studies indicate glycosylation differences between microvillar and platelet GPIIIa heterodimers. Thus, although placental syncytiotrophoblast expresses a beta 3 integrin in its apical brush border, differences in protease sensitivity and carbohydrate content suggest that it may lack or mask certain antigenic determinants. This may be beneficial in avoiding harmful maternal alloantibody responses during pregnancy. Immunohistology showed that the VNR was present in syncytiotrophoblast apical but not basal plasma membranes, and was absent from other forms of trophoblast. The brush border VNR could function in localizing Arg-Gly-Asp-sequence-containing plasma proteins to the materno-trophoblastic interface. PMID- 1720618 TI - Isoforms of nitric oxide synthase. Characterization and purification from different cell types. PMID- 1720619 TI - Relationship between second trimester maternal serum alpha-fetoprotein and umbilical artery Doppler velocimetry and their association with preterm delivery. AB - One explanation for an abnormal maternal serum alpha-fetoprotein (MSAFP) may be an abnormal placenta. A specimen for MSAFP and a series of umbilical artery waveforms were obtained prior to amniocentesis from 144 consecutive women referred for either maternal age (n = 85), a persistently elevated MSAFP unassociated with a structural abnormality (n = 42), or a low MSAFP (n = 17). Almost 50% of deliveries before 37 weeks and 60% of neonatal birthweights below the tenth percentile occurred in the high MSAFP group. We observed that the systolic-diastolic ratio (S/D), pulsatility index, and resistance index tended to be higher in women referred for an elevated MSAFP. Confining analyses to the elevated MSAFP group, the MSAFP in multiples of the median correlated with birthweight independent of the gestational age at delivery (p less than 0.001). In addition, the S/D related directly to MSAFP (p less than 0.03) and indirectly to the gestational age at delivery (p less than 0.04). None of these relationships was observed in the groups of women referred for either maternal age (who had a normal MSAFP) or a low MSAFP. Employing a stepwise multiple linear regression, we found that the gestational age at delivery could be predicted during the midsecond trimester using a combination of the umbilical artery S/D and MSAFP. When the analyses and predictions were limited to subjects whose MSAFP still exceeded 2 multiples of the median at the time of amniocentesis, the positive predictive value for preterm delivery was 100% and the negative predictive value was 93%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720620 TI - Structure of respiratory allergens. AB - Respiratory allergens from one of the most important group of allergens. Except for professional allergens they are characterized by an enormous heterogeneity including a restricted number of major allergens. By means of modern immunological, chemical and genetic techniques some of them are, at least partially, characterized. Immunogenicity depends on a restricted area of the molecules. These epitopes seem to be in principle different for B cells (antibodies) and T cells. The strategies to improve immunodiagnosis are, therefore, not necessarily identical with those for the development of immunotherapy. The present data on selected respiratory allergens are summarized. PMID- 1720621 TI - Distinct subsets of stromal cells confined to unique microenvironments in human endometrium throughout the menstrual cycle. AB - Human endometrial stroma exhibits rather uniform morphology throughout the endometrium. However, predecidualization develops characteristically around vessels and subsequently around glands and under surface epithelium, demonstrating existence of regional differences among stromal cells. Immunoreactivity of stromal cells in endometrial tissues from various phases of the menstrual cycle, as elucidated by employing monoclonal antibodies to cytokeratin, vimentin, very late antigen-1 (VLA-1), Ber-EP4, and HLA-DR, revealed presence of phenotypically distinct subsets of stromal cells confined to unique microenvironments throughout the menstrual cycle. All stromal cells strongly expressed vimentin and weakly expressed cytokeratin. However, Ber-EP4 positive stromal cells were distinctly confined around glands and to the subluminal regions of the surface epithelium. The intervening stromal cells were Ber-EP4 negative. The HLA-DR positive stromal cells were characteristically present in three different locations: around glands and under surface epithelium, around blood vessels and around HLA-DR positive lymphoid cells. From all antigens studied, only expression of VLA-1 in the stromal cells showed a characteristic change throughout the menstrual cycle. Stromal cells in the proliferative and early secretory phases were VLA-1 negative. However, VLA-1 characteristically developed initially in the HLA-DR positive cells around vessels and then in HLA DR/Ber-EP4 positive cells around glands and under surface epithelium. Eventually, all stromal cells in the upper functionalis expressed VLA-1 in the late secretory phase of the menstrual cycle. These data underscore a heterogeneity in stromal cells not exemplified by their morphology. Also, they provide a basis for understanding the differences that the stroma exhibits in morphologic and functional differentiation throughout the menstrual cycle. PMID- 1720622 TI - Single primer-mediated polymerase chain reaction: application in cloning of two different 5'-untranslated sequences of acidic fibroblast growth factor mRNA. AB - Polymerase chain reactions (PCR) are used to generate specific DNA sequences from minute amounts of DNA templates using a pair of oligonucleotide primers. To amplify regions of unknown sequence, methods such as inverted PCR, Alu PCR, and rapid amplification of cDNA ends (RACE) have been developed. These methods require several enzymatic manipulations of DNA which are either tedious or only suitable for certain special conditions. We have explored the possibility of PCR using a single primer. This method takes advantage of the fact that partial complementarity provides sufficient affinity for the oligonucleotide primer to anneal to a secondary, imperfect binding site. Thus, no modification of DNA template was required for the single primer-mediated PCR. We have used this method to generate two different aFGF cDNA clones containing different 5' untranslated sequences. PMID- 1720623 TI - [Studies of the manifold of amylase]. AB - alpha-Amylase (1.4-alpha-D-glucan-glucanohydrolase, E.C 3.2.1.1) is distributed widely in animal and plant kingdoms. A number of properties of this enzyme have been recognized molecular biologically using animal organs. On the other hand, physiological roles and specificities of serum amylase are not known. The source organs of serum amylase have not been confirmed in every animal. The purposes of the experiments are to find out the specificities and varieties of amylase in some kinds of animals. The following results were obtained. 1. Amylase activities in the sera (body fluids) of some animals (Mammals, Birds, Amphibians, Fishes, Insects and Shellfish, 25 kinds altogether) were quite different from each other. The highest amylase activity except insects was observed in the serum of hamster (400 units) and the lowest was in the serum of horse (0.4 units). The activity of locust and oriental longheaded locust, eating grain mainly, was high (locust, 1965 units). 2. Five isoamylases were detected in the serum of rat. Four of them migrated to anode. 2-5 isoamylases were observed in other subjects and the mobility of isoamylases was different from each other. This seemed to be caused by the differences of isoamylase proteins. 3. Amylase activities in the brain, parotid gland, submandibular gland, sublingual gland, tongue, lung, heart, liver, stomach, spleen, pancreas, adrenal, serum and urine of hamster, rat, mouse and rabbit were measured. The activity was especially high in the parotid gland and pancreas. In rabbit, however, amylase activity in these organs was lower than that of other animals. 4. Isoamylases in some organs of four kinds of animals (hamster, rat, mouse and rabbit) were separated electrophoretically. Isoamylases in the serum of rat, hamster and mouse were similar to those in the parotid, submandibular and sublingual glands, respectively. In rabbit, it was difficult to separate isoamylases in the parotid gland and pancreas. 5. Amylases in the parotid gland and locus body were purified by column chromatography (potato starch). PMID- 1720624 TI - Parotid protein secretion from the rabbit during feeding. AB - Rabbits were trained to accept a standardized feeding regime. Under anaesthesia both parotid ducts were cannulated in a retrograde direction and saliva was subsequently collected in feeding sessions involving pellets and carrots before and after the administration of propranolol. Salivary total protein and amylase concentrations were assayed and protein analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Propranolol produced a reduction in protein secretion but not fluid secretion, indicating that protein secretion is partly under beta-adrenergic control. A transient increase in protein secretion was seen 8 h after the administration of propranolol and suggested the existence of different neural mechanisms involved in protein secretion compared to synthesis. Protein output (with high fluid secretion) during feeding on pellets was higher than on carrots (with high protein concentration) and suggested a significant role of the parasympathetic nervous system. The pattern of protein secretion as seen by SDS-PAGE was similar in individual rabbits and remained largely unchanged with the different foods and in the presence of propranolol. PMID- 1720625 TI - P0 is an early marker of the Schwann cell lineage in chickens. AB - We have generated a monoclonal antibody, termed 1E8, that is specific for myelinating and nonmyelinating Schwann cells in mature chickens. 1E8 first stains cells at the edge of the neural crest; later, cells located between the neural tube and somites and in the sclerotome are immunopositive. Double labeling with HNK-1 indicates that these 1E8-positive cells represent a subset of neural crest cells in the ventral migratory pathways. 1E8-positive cells are later associated with the dorsal and ventral roots and with extending nerve trunks. In Western blots, 1E8 reacts with proteins comigrating with P0. Immunodepletion experiments establish that all P0 molecules carry the 1E8 determinant. The developmental distribution of P0, as determined by 1E8 immunoreactivity, differs from that reported for P0 in mammals and suggests that, in chicken, P0 is an early marker for the Schwann cell lineage. PMID- 1720626 TI - The role of complex carbohydrates in adhesion of the myelin protein, P0. AB - The most abundant protein of peripheral nerve myelin, a glycoprotein termed P0, is believed to be involved in the compaction of the myelin sheath and is postulated to be the closest relative to the ancestral gene for the immunoglobulin superfamily. Recently, P0 has indeed been shown to behave like a homophilic adhesion molecule via interactions of its extracellular domains. Here we demonstrate the importance of the oligosaccharide moieties of P0 in its functioning as a homophilic adhesion molecule. Expression of the complex form of P0 glycoprotein in transfected Chinese hamster ovary cells greatly increased the adhesiveness of those cells, whereas expression of the high-mannose form of P0 glycoprotein did not. This is the first step in the dissection of P0-P0 interaction at the molecular level. PMID- 1720627 TI - Identification of conserved residues in the human immunodeficiency virus type 1 principal neutralizing determinant that are involved in fusion. AB - The principal neutralizing determinant of the human immunodeficiency virus type 1 (HIV-1) is located within the V3 loop of the surface glycoprotein gp120. Recently a mutational approach was used to demonstrate that the tip of the V3 loop is involved in cell fusion mediated by the HIV-1 envelope glycoproteins. Here these results are extended by introducing seven additional single amino acid mutations in the V3 loop. Mutations at highly conserved amino acids in the left stem, tip, and right stem of the V3 loop blocked or greatly reduced cell fusion without affecting envelope glycoprotein processing, transport, or binding to the CD4 receptor molecule. This study further characterizes the involvement of the V3 loop in cell fusion mediated by the HIV-1 envelope glycoproteins and identifies residues involved in the fusion reaction. PMID- 1720628 TI - Antibody responses of chimpanzees immunized with synthetic peptides corresponding to full-length V3 hypervariable loops of HIV-1 envelope glycoproteins. AB - Immunization of primates or humans with human immunodeficiency virus type 1 (HIV 1) glycoproteins usually elicited moderate immune responses to the principal neutralizing determinant (PND) located within the V3 hypervariable loop of gp120. Since an antibody response to the PND appears to be protective, experiments were carried out to determine the responsiveness of chimpanzees to immunization with synthetic peptides corresponding to the full-length V3 loop. Seven chimpanzees (4 preimmunized with gp160, 2 preimmunized with HIV-1 antigens unrelated to gp160, and 1 unimmunized) were vaccinated with a mixture of full-length V3 loop peptides from 21 distinct HIV-1 isolates (clones) either in unconjugated form or linked to carrier proteins from HIV-1 nef and gag P18, respectively. Six chimpanzees developed high levels of antibodies to the peptides (dilution endpoints 1: greater than 25,000), and 5 had high levels of antibodies to gp120 from HIV-1IIIB (endpoint titers 1: greater than 500,000). Chimpanzees immunized with peptide carrier conjugates (4) had antibodies to the carrier proteins nef and gag P18, respectively (endpoint titers 1: greater than or equal to 35,000). Virus neutralizing (VN) antibodies were detected in sera of 5 of 7 chimpanzees, but were present at titers of 1: greater than or equal to 400 only in sera of 2 chimpanzees. One of these was challenged with HIV-1 and was protected against infection, as reported elsewhere. The antibodies were primarily specific for the HIV-1 isolate used for primary immunization before boosting with peptides. The relatively low dilution endpoints of VN antibodies as compared with endpoints determined by site-specific immunoassays probably can be ascribed to imperfect mimicry of conformational epitopes by synthetic peptides. Nevertheless, sequential or simultaneous immunization with recombinant envelope glycoproteins of HIV-1 and selected synthetic peptides offers an approach for eliciting protective immunity against HIV-1. PMID- 1720629 TI - Characteristics of the principal neutralizing determinant of HIV-1 prevalent in Japan. AB - The principal neutralizing determinants (PNDs) of 29 human immunodeficiency virus type 1 (HIV-1) isolates in Japan were analyzed using polymerase chain reactions. The viruses were isolated from 16 hemophiliacs, 11 individuals infected by their sexual transmission and 1 patient infected by blood transfusion (total 28 patients). Two virus isolates which were obtained from the same individual at different periods were also analyzed. All individuals were Japanese except one. The results produced 32 different PND sequences. A highly conserved central core sequence (GPG) was present in 27 of 32 patients, similar to the number reported in the United States, despite the marked heterogeneity in flanking regions of PNDs. The PNDs of all the 16 HIV-1 isolates obtained from patients with coagulation disorders had GPG sequences. Secondary structure prediction of PNDs by a joint method suggested that they were composed of coil-beta strand-coil-beta strand-alpha helix. It is suggested that the conserved core sequence has a type I turn. These findings may be useful in planning further clinical trials for passive vaccination. PMID- 1720630 TI - High prevalence of antibodies to the gp120 V3 region principal neutralizing determinant of HIV-1MN in sera from Africa and the Americas. AB - Neutralizing antibodies (NA) against HIV-1MN and HIV-1IIIB, and antibodies binding to synthetic peptides (BA) derived from the gp120 envelope V3 region principal neutralizing determinants (PND) of the HIV-1MN, HIV-1IIIB, and HIV-1Z3 virus strains were assayed in HIV-1 antibody-positive sera from the United States, Haiti, Brazil, Zaire, and Zimbabwe. The ability of soluble PND peptide to block neutralization of the corresponding virus by representative sera was also tested. In each country, NA and BA titers were highest against the HIV-1MN strain, and compared with other countries, NA and BA titers against HIV-1MN were higher in sera from the United States and Haiti. When NA titers were compared with BA titers against either HIV-1MN or HIV-1IIIB, no correlation was found for the HIV-1IIIB strain, but there was a significant correlation for HIV-1MN. Addition of the HIV-1MN strain peptide to a neutralization assay for HIV-1MN resulted in a four- to tenfold reduction in NA titers in sera from the United States, Zaire, and Brazil. The results suggest that HIV-1MN and closely related variants are prevalent in many parts of the world, and that antibodies directed against the PND account for most of the neutralizing activity in sera of infected individuals. PMID- 1720631 TI - N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding. AB - Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120 CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain. PMID- 1720632 TI - Site-directed serology using synthetic oligopeptides representing the C-terminus of the external glycoproteins of HIV-1, HIV-2, or SIVmac may distinguish subtypes among primate lentiviruses. AB - In this study the presence of a highly immunogenic domain located at the C terminus of the external glycoproteins (EGP) of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIVMAC) is shown using synthetic oligopeptides as antigens in enzyme immunoassays. This epitope is probably located within the last 13 and 15 residues of the EGP of HIV-1 and HIV-2, respectively. The C terminal epitope of the EGP of SIVMAC may involve residues located more upstream. Among the HIV-2/SIV serotype, we observed that the reactivity to the C-terminal epitope of gp120 was dependent of both species and geographical origin of the samples tested. It seems that this gp 120 C-terminal epitope could distinguish subtypes among the HIV-2/SIV serotype. Further studies, using site-directed enzyme immunoassays with synthetic peptides representing the C-terminus of the EGP derived from a wide variety of HIV2/SIV strains must be performed to confirm this observation. These kinds of assays may constitute important tools for use in seroepidemiological studies and broaden our understanding of the distribution and phylogenetic relationship of primate lentiviruses. PMID- 1720633 TI - Palliation of malignant obstruction--use of lasers and radiotherapy in combination. PMID- 1720634 TI - Alternating cycles of PVB and BEP in the treatment of patients with advanced seminoma. AB - 33 patients (median age 39 years) with advanced seminoma were treated with 4 courses of alternating cisplatin-containing chemotherapy PVB/BEP (cisplatin, vinblastine and bleomycin; bleomycin, etoposide and cisplatin). Patients were classified as stage IIC (n = 7), IID (n = 9), III (n = 13) and IV (n = 4). 8 had had prior radiotherapy; 9 had an elevated beta human chorionic gonadotropin (beta HCG). 30 patients were evaluable for response and 33 for toxicity. During chemotherapy 3 patients died, 1 due to malignant disease, another due to a cardiac arrest, and 1 patient of a bleomycin pneumonitis. 13 (43%) had a complete remission and 17 (57%) had a clinical partial remission (residual radiographic mass). At a median follow-up of 28 months (range 16-88), 3 patients relapsed, 6-8 months after entry. After completion of therapy there were 2 deaths, 1 due to bleomycin pneumonitis and 1 neither tumour nor treatment related. 26 of 33 (79%) patients achieved a continuously disease-free status. Leucocytopenia and thrombocytopenia of WHO grade 3/4 occurred in, respectively, 32/33 (97%) and 20/33 (61%) of the patients. This study shows that alternating PVB/BEP in this group yields comparable response rates with non-alternating schedules but at the expense of considerable toxicity. PMID- 1720635 TI - Undergraduate education about cancer. AB - The quality, quantity and balance of undergraduate cancer teaching in Australian Medical Schools were investigated by a survey, using a self-administered questionnaire, of recent graduates from all Australian medical schools. Stratified random cluster sampling was used and a response rate of 84% (389 respondents) was achieved. The results revealed substantial differences in knowledge, experience in, and rating of teaching between the medical, surgical, radiotherapeutic and palliative components of cancer management. The proportions of graduates who had never attended radiotherapy and palliative care clinics or units (42.3% and 49.9%, respectively) were more than double the proportion who had never attended medical and surgical cancer clinics or units (17.5% and 10.9%, respectively). More than twice as many graduates rated their instruction in the palliative management of cancer as poor or very poor (29.4%) compared with those rating their instruction as poor or very poor in both cancer prevention (8.4%) and treatment for cure (14.6%). The respondents displayed a considerable lack of knowledge about radiotherapy treatment options, and reported a lack of perceived competence in doing cervical smears. Their answers to questions about 5-year survival of selected cancers, about the existence of screening tests validly shown to reduce mortality, and the ages at which breast and cervical cancers are likely to develop all revealed worrying levels of incorrect knowledge. There was some important disturbing variation in levels of knowledge, experience and rating of cancer instruction between states and between universities. PMID- 1720636 TI - Micrometastatic tumour cells in bone marrow of patients with gastric cancer: methodological aspects of detection and prognostic significance. AB - Monoclonal antibodies (Mab) are potent probes to identify individual tumour cells or small tumour cell clusters in bone marrow. In the present study, various antibodies directed against either cell surface or intracytoplasmic antigens of epithelial cells were assessed for their ability to detect such cells in bone marrow of patients with breast, colorectal and gastric cancer. According to the presented data, monoclonal antibodies against intracellular cytokeratin (CK) components are superior in terms of specificity and sensitivity to antibodies reacting with epitopes of the cell membrane. Using a monoclonal antibody against the cytokeratin polypeptide 18 in connection with the alkaline phosphatase anti alkaline phosphatase detection system (APAAP), we could detect tumour cells in bone marrow of 34 out of 97 patients with gastric cancer examined at the time of primary surgery. The incidence of positive findings was correlated to established risk factors, such as histological classification and locoregional lymph node involvement. Clinical follow-up studies on 38 patients demonstrated a significantly increased relapse rate in patients presenting with CK-positive cells in their bone marrow at the time of primary surgery. Thus the described technique may help to identify patients with gastric cancer carrying a high risk of early relapse. PMID- 1720637 TI - [Immunohistochemical study of glial cytoplasmic inclusion in multiple system atrophy]. AB - Recently, glial cytoplasmic inclusion (GCI) has been demonstrated to be argyrophilic cytoplasmic body by silver staining in the oligodendroglia of patients with multiple system atrophy. We observed such GCIs in all 20 cases of multiple system atrophy. No GCI was noticed in all 6 cases of hereditary spinocerebellar degenerations. Immunohistochemically, GCI was stained positively with antibodies to ubiquitin, alpha-tubulin, and beta-tubulin, of which characteristics is consistent with previous reports. In addition, GCI was first demonstrated to react with an antibody to microtubule-associated protein-1B(5), which is one of the proteins of cytoskeleton organization and a component of cross-bridges between microtubular assembly. The result suggests strong relationship between the formation of the OCI and immunohistochemical expression of MAP-1B(5). PMID- 1720638 TI - [A case of temporal lobe epilepsy with recurrent dysphasic seizures]. AB - We reported a 40-year-old right-handed female with temporal lobe epilepsy manifesting recurrent dysphasic seizures. At age 25, the patient developed a complex partial seizure, who subsequently showed frequent auditory seizures that often evolved to complex partial or secondarily generalized seizures at age 25 27, and dysphasic seizures (DSs) at age 27 -40. DSs were characterized by total aphasia without impairment of consciousness, which were often accompanied by functional hallucination. Brain CT, cerebral angiography, and brain MRI demonstrated no abnormal findings. At age 39 (May 20, 1989), recurrent aphasic state was unexpectedly observed during medical examinations. An EEG was performed immediately, and the EEG seizure pattern (duration: ca. 8-17 sec) in which 15 -16 Hz spikes began in the left posterior temporal region and spread rapidly to the left midtemporal, inferior frontal, and central regions was detected 14 times within 30 minutes. During the seizure patterns, the patient was aphasic. PMID- 1720639 TI - Drug-induced terminal sedation for symptom control. PMID- 1720640 TI - Changes of zinc values in children during malignant disease. AB - In 47 children with malignancy, zinc status, growth, and performance during standard treatment were compared with those in controls. At diagnosis, growth was retarded and hair zinc values were high, 2.4 +/- 0.7 mumol/g, as in chronic deficiency. During induction therapy, serum declined to 10.4 +/- 2.3 mumol/L and urinary excretion increased to 5.3 +/- 2.8 mumol/mol creatinine, as in acute exacerbation of deficiency. Control CSF values in children in remission, 0.04 +/- 0.01 mumol/L, were lower than reference values in adults. No difference in mean CSF zinc was observed during therapy, or in those with acute lymphoblastic leukemia (1) at high risk, (2) with central nervous system involvement, (3) with low performance, or (4) resistant to therapy. In six children unexplained values, up to 0.28 mumol/L during induction, were measured. No correlations between the various zinc parameters were found. PMID- 1720641 TI - Suboptimal levels of dietary copper vary immunoresponsiveness in rats. AB - The effects of severe, moderate, and mild copper deficiencies on cellular and humoral immunity were studied. Fifty male Sprague-Dawley rats, 5 wk of age, were fed diets containing 0.5, 2.0, 3.5, or 5.0 micrograms Cu/g for either 4 or 8 wk. Ten of the rats were fed the control diet, but were pair-fed with the 0.5 micrograms/g treatment group. All rats were immunized once with sheep red blood cells. Mean plasma-copper concentration reflected the dietary levels of copper, and ceruloplasmin activity correlated highly to plasma copper. Rats consuming suboptimal levels of copper responded differently to the deficiencies, so copper status varied among those animals. After 8 wk, cell proliferation, when stimulated by phytohemagglutinin, was dependent on the copper status of the animal. Severely deficient rats had consistently lower lymphocyte stimulation indexes for phytohemagglutinin and concanavalin A, but specific antibody response was not reduced. Immunoglobulin G (IgG) concentrations were variable for all rats, and immunoglobulin M (IgM) concentrations were lower for the severely deficient rats. Suboptimal dietary copper may alter immune function in rats, depending on the ensuing effect on copper status. PMID- 1720642 TI - Developmental toxicity evaluation of orthovanadate in the mouse. AB - Sodium orthovanadate in deionized water was administered once daily by gavage on gestational days 6-15 to mice at doses of 0, 7.5, 15, 30, and 60 mg/kg. Dams were killed on day 18 of pregnancy, and fetuses were examined for external, visceral, and skeletal defects. Maternal toxicity was observed at the highest doses of sodium orthovanadate, as evidenced by a significant number of deaths (60 and 30 mg/kg/d) and reduced weight gain and food consumption (30 and 15 mg/kg/d). Embryolethality and teratogenicity were not observed at maternally toxic doses and below, but fetal toxicity was evidenced by a significant delay in the ossification process of some skeletal districts at 30 mg/kg/d. The no-observed adverse-effect level (NOAEL) for maternal toxicity was 7.5 mg/kg/d, and 15 mg/kg/d represented a NOAEL for developmental toxicity in mice under the conditions of this study. PMID- 1720643 TI - The effects of selenium on gestation, fertility, and offspring in mice. AB - Supplementation of mice from 22 d old with the K-Selenocarrageenan (0.25 ppm Se) in drinking water reduced gestation period by 3.2 d. Selenium supplementation increased litter size by 53.8% and average litter weight by 5%. Continuous supplementation with selenium (0.25 ppm) of mice until the age of 50-56 d significantly increased the concentration of selenium and the glutathione peroxidase activity in whole blood and liver. In serum, fluorescent peroxidized lipid products were decreased by 22% and reducing sugar was decreased by 16% compared to unsupplemented controls. In whole blood of young mice, collagen was increased by 14%. IR differential spectra of whole blood show strong absorption at the acrylamide band, suggesting a role of selenium in preventing lipid peroxidation, as well as a stabilizing effect on blood proteins. PMID- 1720644 TI - In vitro OKT3-induced mitogenesis in selenium-deficient patients on a diet for phenylketonuria. AB - Patients with phenylketonuria (PKU) are frequently deficient in the essential trace element selenium (Se), because of their very low protein diet. Using two approaches to investigate T-cell response to proliferative signaling, viz, mitogenesis caused by the monoclonal antibody OKT3 and the plant lectin phytohaemagglutinin (PHA), we demonstrated significantly reduced responses to optimal concentrations of OKT3 in a group of PKU patients with reduced serum Se compared with a normal group (p = 0.0005) and with a group of PKU patients whose serum Se was normal (p = 0.0023). The response of the Se-deficient group to optimal levels of PHA did not differ from that of the normal controls or from that of Se-normal PKU patients. A dose-dependent relationship between serum Se levels and mitogenic response was evident for OKT3 (r = 0.34, p = 0.0154), but not for PHA (r = -0.02, p = 0.9086). We suggest that the reduced response to OKT3 mitogenesis in Se-deficient PKU patients is possibly the consequence of impaired Se-dependent metabolic activity, which affects mitogenic signaling via the T cell antigen receptor (TCR/CD3) complex. PMID- 1720645 TI - Radioimmunoassay of metallothionein in rabbit, rat, mouse, Chinese hamster, and human cells. AB - We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample. PMID- 1720646 TI - Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes. AB - The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis, as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range of 10 500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide. PMID- 1720647 TI - Biliary excretion of barium in the rat. AB - Biliary excretion of barium was studied in Sprague-Dawley bile-duct-cannulated rats injected intravenously with 1.8 micrograms Ba/rat as 133Ba-labeled barium chloride. Approximately 0.5% of the barium dose was excreted into bile within 2 h. The time-course profile of biliary excretion of the radiotracer closely reflected that of plasma concentrations. Biliary barium levels reached their peak in the first 15-min period after administration and rapidly declined thereafter. The plasma-to-bile barium-concentration ratio was approx 1 at 2 h after injection. There was no tendency of barium to concentrate in liver, and the 133Ba levels in stomach and small intestine largely exceeded hepatic levels. There is evidence indicating that barium is predominantly excreted with feces following parenteral administration in rats and humans. The results of this study suggest that biliary excretion is of little quantitative importance and that physiological routes other than bile contribute to elimination of barium by the digestive tract. PMID- 1720648 TI - Calcium and phosphorous in teeth from children with and without cystic fibrosis. AB - The objective of this study is to analyze the percentage of Ca and P in teeth from children with and without cystic fibrosis with respect to different variables, namely, gender, age, type of teeth, area, fluoridation of water supply, term of pregnancy, and maternal smoking and drinking habits. The method of analysis is proton-induced X-ray emission and proton-induced gamma emission on tooth-crown samples. T-test results show less Ca in the teeth of the population of CF + NT (cystic fibrosis+ nontetracycline antibiotics) than in that of NCF (noncystic fibrosis) for incisors and the total tooth population. It also shows greater Ca for incisors than for nonincisors for NCF. Teeth from urban CF + NT have less Ca than those from urban NCF. This holds for the total area population. Both Ca and P in teeth of NCF population living in fluoridated areas are greater than in those living in nonfluoridated area. Ca is depleted in the teeth of CF + NT children whose mothers smoke and P is depleted in the teeth of NCF children whose mothers drink. High and low Ca and P values for individual teeth are reported. PMID- 1720649 TI - Toxic effects of ingested lead shots in domestic fowls. AB - Lead poisoning from ingested shots is thought to be a major cause of high mortality in waterfowls throughout the world, and some millions of fowls die each year. However, there have been no other Japanese studies regarding lead toxicity in birds from ingested lead shots. We used domestic fowls instead of waterfowls as the experimental birds, in order to make clear the distribution and the toxic effects of lead shot in the birds. In a 1-wk follow-up study, two, four, and eight #4 lead shots were administered orally. A dose-dependent increase of the lead concentrations in blood, brain, liver, kidney, lung, spleen, bone, and epidermis of the gizzard was observed. In the 12-wk follow-up study, twenty domestic fowls were used and eight #4 lead shots were administered to the experimental birds. Lead concentrations in brain, liver, kidney, bone, ovary, fat tissue, and breast muscle increased more than in the 1-wk follow-up study. The observed lead concentrations of organs in the domestic fowls were lower than those of the other species used in past studies. The blood lead concentrations increased up to the third week and a remarkable suppression of delta aminolevulinic acid dehydratase activity in red blood cells and elevation of free erythropoietic protoporphyrin were observed in the exposed group. Body weight loss, loss of hair, and neurological symptoms were also observed. However, there were no mortalities during the 1- and 12-wk studies. PMID- 1720650 TI - World malaria situation in 1989. Part II. PMID- 1720651 TI - Imported dengue, 1990. PMID- 1720652 TI - Identification of a plant-derived mollicute as a strain of an avian pathogen, Mycoplasma iowae, and its implications for mollicute taxonomy. AB - Strain PPAV, a filamentous but nonhelical mollicute, was isolated from aborted apple seeds in France in late 1979. This organism grew well in SP-4 broth, fermented glucose, and required sterol for growth, and most of its properties suggested that it belonged to the genus Mycoplasma. However, it was serologically distinct; in addition, unlike other Mycoplasma species, genome measurements consistently yielded values of about 1,000 MDa (ca. 1,500 kbp), and the organism had a growth temperature optimum of 43 degrees C. A comparison of strain PPAV 16S rRNA sequences with those of other mollicutes revealed a high degree of sequence similarity to a strain of Mycoplasma iowae, which is commonly encountered in poultry. This relationship was confirmed by performing a restriction endonuclease pattern analysis and DNA-DNA hybridization tests. The genome size of type strain 695 of M. iowae was determined to be about 1,000 MDa (1,500 kbp) by renaturation kinetics, a value which is much higher than any other value known in the genus. Additional measurements by pulsed-field gel electrophoresis yielded values of 1,300 kbp for both strain PPAV and M. iowae. Subsequent phenotypic comparisons supported this relationship. Serologic tests with strain PPAV and other strains of M. iowae confirmed the findings of other investigators that this species is serologically heterogeneous. The high optimum temperature for growth of strain PPAV was also shared by a number of M. iowae isolates. Genome size is an inappropriate character for taxonomic assignment to the family Mycoplasmataceae because strain PPAV and other established species in this family are now known to have genomes ranging in size from 1,000 to 1,400 kbp. PMID- 1720653 TI - Intraspecies variations in nutritionally variant streptococci: rRNA gene restriction patterns of Streptococcus defectivus and Streptococcus adjacens. AB - The rRNA gene restriction patterns of two species of nutritionally variant streptococci, Streptococcus defectivus and Streptococcus adjacens, were determined, and the results were compared with the electrophoretic migration profiles of penicillin-binding proteins. Reference strains belonging to various streptococcal species were used as controls. Our results correlated with the results of DNA-DNA hybridization experiments and confirmed the delineation of these two species. Moreover, they demonstrated that intraspecies variations occur and suggested that there are two subspecies of S. defectivus. PMID- 1720654 TI - Intrageneric structure of Streptococcus based on comparative analysis of small subunit rRNA sequences. AB - The partial 16S rRNA sequences of 24 Streptococcus species were determined by reverse transcription. A comparative analysis of these sequences and the sequences of seven previously studied streptococcal species revealed the presence of several clusters within the genus. The clusters obtained from the sequence analysis agreed in general with the groups outlined on the basis of the results of nucleic acid hybridization studies, but there were some exceptions. The pyogenic group was extended to include Streptococcus agalactiae, S. parauberis, S. porcinus, and S. uberis. Four oral groups were discerned; these four groups centered on S. mutans, S. salivarius, S. anginosus, and S. oralis. Some species (e.g., S. suis and S. acidominimus) did not cluster with any particular group. Our findings are discussed in the context of data from other genetic and chemotaxonomic studies. PMID- 1720655 TI - Prolonged disease-free survival after high-dose sequential chemo-radiotherapy and haemopoietic autologous transplantation in poor prognosis Hodgkin's disease. AB - Although effective in achieving durable remission in selected tumors incurable by conventional-dose chemotherapy, high-dose regimens requiring bone marrow transplantation remain too toxic for widespread use. With the aim to improve the therapeutic index of high-dose therapy, we have developed a novel program whereby several non-cross-resistant agents (including total body irradiation at myeloablative dose) were delivered sequentially rather than concurrently. The regimen has been tested in 25 patients with Hodgkin's disease refractory to primary chemotherapy (MOPP and ABVD) or relapsed within 12 months after first complete remission. The efficacy of the sequential regimen (72% complete response rate, 49% event-free survival and freedom from progression at four years, with a median observation of 3.5 years for patients remaining alive) compares favorably with the activity so far reported for the most effective high-dose regimens. Toxicity was low; no toxic deaths occurred and only three life-threatening adverse events were documented after autografting. The excellent tolerability of the final myeloablative course most likely reflects the speed and completeness of haematologic recovery that followed the use of peripheral blood progenitors as the sole or additional source of myeloid stem cells. The reduction of haematologic toxicity was further emphasized when rhG-CSF was employed to both shorten post-chemotherapy neutropenic intervals and to collect large amounts of peripheral blood stem cells. We expect that the use of growth factors will have a major impact on the therapeutic index of high-dose regimens. This, in turn, will confirm their safety as primary treatment in selected high-risk subsets. PMID- 1720656 TI - VAB-6 and cisplatin-cyclophosphamide combinations in the treatment of metastatic seminoma patients: the U.S.S.R. experience. AB - In a non-randomized study the treatment results of 59 patients with disseminated seminoma were evaluated: 21 patients were treated with a VAB-6 combination and 38 with a CP (cyclophosphamide and cisplatin) combination. After VAB-6 CR was observed in 8 patients and 6 achieved CR with additional treatment: 1 with chemotherapy (PVB) and 5 with radiotherapy (RT). The final CR rate was 67%. At a median follow-up of 38 (11-70) months 15 (71%) are alive, and 11 of them (52%) are NED; 6 have died. Of the 38 patients treated with CP alone only 18 achieved CR and 9 had a CR after additional RT and 1 chemotherapy (VAB-6), the overall CR rate was 72%. The median follow-up is 24 (4-55) months, 28 (74%) are alive, 24 (66%) are currently NED, and 9 have died. Both regimens were well tolerated, the main toxicity being leukopenia: 48% (WHO grade 111-1V-5%) for VAB-6, and 59% (13%) for CP. Hearing loss was registered in 8 patients receiving CP and in 2 receiving VAB-6. There were no fatal toxicities. Thus, VAB-6 and CP regimens seem to have compatible and high activity in disseminated seminoma. PMID- 1720657 TI - Quality of life and palliative care. PMID- 1720658 TI - The role of palliative radiotherapy in malignant mesothelioma. AB - Chest pain is often a major problem in patients with malignant pleural mesothelioma but there are few data regarding the usefulness of radiotherapy (RT) in its palliation. Following a recent retrospective report which suggested that wide-field RT frequently affords pain relief in this disease, we prospectively assessed the effect of hemithorax irradiation on pain control. Twenty-two patients with chest pain due to mesothelioma received 30 Gy in 10 daily fractions to the involved hemithorax. The patients' symptoms were assessed before RT, 1 month after RT, and then 2 monthly. Symptoms were graded by the clinician and the patient, and analgesic requirements were noted at each assessment. Performance status and respiratory status were recorded by WHO and MRC criteria respectively. Nineteen assessable patients have been followed for at least 3 months after RT. The treatment was well tolerated, with nausea and vomiting in only 1 patient. Pain control improved in 13/19 patients at 1 month, but 9/12 patients had worsening chest pain at 3 months, and at 5 months pain control had deteriorated in 6/7 patients. Although partial regression of chest wall masses was seen in 5/9 patients, RT did not appear to delay the progression of respiratory symptoms or radiological changes. The median duration of survival after RT was 4 months. Radiotherapy can relieve pain due to malignant pleural mesothelioma but its effect at this dose is short-lived. PMID- 1720659 TI - Immunohistochemical demonstration of nerve-Merkel cell complex in fetal human skin. AB - Using Merkel cell specific antikeratin antibodies and neurofilament antibody the nerve-Merkel cell relationship was studied with a double staining method on frozen sections. Merkel cells were stained with monoclonal anti-cytokeratin CK-5 and CAM 5.2 which react against human cytokeratin polypeptide 45 kDa and 52.5 kDa, respectively. Peripheral nerves were stained with monoclonal murine antibody Neurofilament 70 and 200 kDa. Epidermal Merkel cells were recognized in 12-week fetuses in the plantar skin. In 15-week fetuses dermal Merkel cells were found. Most of the dermal Merkel cells initially lacked a close association with immunoreactive nerve fibers. In 16-week fetuses immunoreactive small nerves reached the epidermis and a few dermal Merkel cells became heavily entangled with the meshwork of nerve endings. Nerve-Merkel cell complex in the dermis was confirmed by electron microscopy. The appearance of epidermal Merkel cells preceded the attachment of immunoreactive nerve endings to the epidermis. In an analysis of 448 dermal Merkel cells the nerve-Merkel cell complex became more frequent as the age of fetus advanced. It was concluded that Merkel cells do not arrive at the epidermis with peripheral nerves. Rather, the peripheral nerves are attracted to the dermal Merkel cells which originated in the epidermis. PMID- 1720660 TI - Dynamics and binding mode of Hoechst 33258 to d(GTGGAATTCCAC)2 in the 1:1 solution complex as determined by two-dimensional 1H NMR. AB - We have investigated the interaction of the bisbenzimidazole derivative Hoechst 33258 with the self-complementary dodecadeoxynucleotide duplex d(GTGGAATTCCAC)2 using one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance (1H NMR) spectroscopy. To monitor the extent of complex formation, we used the imino proton region of the 1D 1H NMR spectra acquired in H2O solution. These spectra show that the DNA duplex loses its inherent C2v symmetry upon addition of the drug, indicating that the two molecules form a kinetically stable complex on the NMR time scale (the lifetime of the complex has been measured to be around 450 ms). We obtained sequence-specific assignments for all protons of the ligand and most protons of each separate strand of the oligonucleotide duplex using a variety of homonuclear 2D 1H NMR experiments. The aromatic protons of the DNA strands, which are symmetrically related in the free duplex, exhibit exchange cross peaks in the complex. This indicates that the drug binds in two equivalent sites on the 12-mer, with an exchange rate constant of 2.2 +/- 0.2 s-1. Twenty five intermolecular NOEs were identified, all involving adenine 2 and sugar 1' protons of the DNA and protons in all four residues of the ligand, indicating that Hoechst 33258 is located in the minor groove at the AATT site. Only protons along the same edge of the two benzimidazole moieties of the drug show NOEs to DNA protons at the bottom of the minor groove. Using molecular mechanics, we have generated a unique model of the complex using distance constraints derived from the intermolecular NOEs. We present, however, evidence that the piperazine group may adopt at least two locally different conformations when the drug is bound to this dodecanucleotide. PMID- 1720661 TI - Session VII: Physiologic and psychological growth and development in pediatric heart transplant recipients. PMID- 1720662 TI - Aminoacylation of tRNAs as critical step of protein biosynthesis. AB - Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted. PMID- 1720663 TI - Is there a unique ribosome phenotype for naturally occurring Escherichia coli? AB - We have compared the growth characteristics of natural isolates of E coli with the kinetic properties of their ribosomes in vitro. The variability of the different performance characteristics of ribosomes isolated from natural isolates of E coli show that there is not a unique wild-type ribosome phenotype just as there is not a unique growth phenotype for the bacteria. In addition, there is a strong correlation between the growth rates and the efficiency of the kinetic interaction between the ribosomes and the EF-Tu-GTP-aminoacyl-tRNA ternary complex in vitro. No such correlation is seen between the growth rate and the maximum turnover rate of the ribosomes in vitro. These data suggest that the codon-programmed ribosomes are not kinetically saturated with ternary complexes in vivo. PMID- 1720664 TI - Translation in vitro of codon UGA as tryptophan in Mycoplasma capricolum. AB - The in-frame UGA codons in the synthetic messenger RNA were translated in the cell-free system of Mycoplasma capricolum. The result, together with the occurrence of codon UGA at tryptophan sites in the genes and the presence of tRNA(UCATrp) pairing with UGA, clearly indicated that UGA is a tryptophan codon in this bacterium. PMID- 1720665 TI - The binding of thiostrepton to 23S ribosomal RNA. AB - The antibiotic, thiostrepton, binds to 23S ribosomal RNA from E coli with a dissociation constant (KD) of 2.4 x 10(-7) M. The specificity of the interaction was established using 16S rRNA and modified or mutationally-altered 23S rRNA. Thus, no binding was detected with rRNA from the 30S subunit nor with rRNA modified in vitro by the thiostrepton resistance methylase. Mutant 23S rRNA, altered at residue 1067 in each of the 3 possible ways, showed reduced binding affinity for thiostrepton. The KD for the G mutation was 3.5 x 10(-6) M; for the C mutation, 2.4 x 10(-5) M; and for the U mutation, 4.8 x 10(-5) M. This reduction in drug binding is compatible with functional analyses; the C or U mutation results in ribosomal particles which are poorly inhibited by the drug compared with wild-type, whereas the G mutation results in an intermediate response to the drug in protein synthesis. The smallest 23S rRNA fragment used here that was capable of binding thiostrepton, in a nitrocellulose filter binding assay, comprised residues 1052-1112 and the dissociation constant was 3.0 x 10( 7) M, ie virtually indistinguishable from that with intact 23S RNA. However, the drug was incapable of binding to the 5'-moiety of this fragment (ie residues 1052 1084) or to an RNA transcript complementary to 1052-1112. PMID- 1720666 TI - Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome. AB - Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site. PMID- 1720667 TI - Binding sites of the antibiotics pactamycin and celesticetin on ribosomal RNAs. AB - The binding sites of the antibiotics pactamycin and celesticetin on the rRNAs of Escherichia coli ribosomes were investigated by a chemical footprinting procedure. Pactamycin protected residues G-693 and C-795 in 16S RNA which are located in an important functional region of the 30S subunit participating in initiation complex formation and ribosomal subunit interaction. Celesticetin altered the reactivities of 5 residues A-2058, A-2059, A-2062, A-2451 and G-2505 within the central loop of domain V of 23S RNA which has been implicated in peptidyltransferase activity. Inferences are drawn concerning the mode of action of the antibiotics. PMID- 1720668 TI - Thermus thermophilus ribosomes for crystallographic studies. AB - Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies. PMID- 1720669 TI - Frozen spin targets in ribosomal structure research. AB - Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation. A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction. Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins. Pure deuteron spin labels and proton spin labels are created by NMR saturation. We report on results obtained from the large subunit of E. coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN. The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation. Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K. At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target). Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively. The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature. This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs. PMID- 1720671 TI - RNA-protein interactions in the Escherichia coli ribosome. AB - Over the last two decades essentially three different approaches have been used to study the topography of RNA-protein interactions in the ribosome. These are: (a) the analysis of binding sites for individual ribosomal proteins or groups of proteins on the RNA; (b) the determination of protein footprint sites on the RNA by the application of higher order structure analytical techniques; and (c) the localisation of RNA-protein cross-link sites on the RNA. This article compares and contrasts the types of data that the three different approaches provide, and gives a brief and highly simplified summary of the results that have been obtained for both the 16S and 23S ribosomal RNA from E coli. PMID- 1720670 TI - Localization of a segment of 16S RNA on the surface of the small ribosomal subunit by immune electron microscopy of complementary oligodeoxynucleotides. AB - Oligonucleotides that complement Escherichia coli 16S ribosomal RNA residues 685 696 and 694-705 have been synthesized so as to incorporate antibody-recognizable markers: a 3'-terminal residue of N6-delta 2-isopentenyladenosine, a 5' dinitrophenyl group, or both. Each oligonucleotide is able to bind RNA within the small ribosomal subunit, whether free or in 70S ribosomes. Immune electron microscopy places probes at nucleotides 685, 694 and 705 within a single area, at the tip of the subunit platform, very near the position of the 3'-end of the 16S RNA. PMID- 1720672 TI - A consonant model of the tRNA-ribosome complex during the elongation cycle of translation. AB - Chemical and photochemical affinity techniques have been used extensively to determine the positions of the tRNA binding sites on the Escherichia coli ribosome. Recent advances in our understanding of ribosome structure and function prompted us to critically review the data that have accumulated on tRNA-ribosome cross-links. As a result, we propose a new model of the tRNA-ribosome complex that accounts for nearly all of the pertinent evidence. PMID- 1720673 TI - Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers. AB - Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of cDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNAPhe. Only the binding of cDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAPhe or tRNAfMet alone. The poly(AGU)/tRNAfMet ligand pair caused an additional decline in the binding of cDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [1]. PMID- 1720674 TI - Topography of the Escherichia coli ribosomal 30S subunit-initiation factor 2 complex. AB - The specific effect of the binding of initiation factor IF2 on E coli 16S rRNA within the [IF2/30S/GTP] complex has been probed by crosslinking experiment with trans-diamminedichloro platinum (II) and by phosphate alkylation with ethylnitrosourea. Several 16S rRNA fragments crosslinked to IF2 have been identified and are mostly located in the head and the lateral protrusion of the 30S subunit. The study of the effect of IF2 binding to the 30S subunit reveals that the factor does not tightly bind to the 16S rRNA and induces both isolated reductions and enhancements of phosphate reactivity in the 16S rRNA. Several of them are located near the binding site of IF2 and weak effects are observed in distant parts of the subunit. These results are discussed in the light of current knowledge of the topographical localization of IF2 with the 30S subunit and of its relation with function. PMID- 1720675 TI - [Nutritional support in non-Hodgkin's lymphoma treated with polychemotherapy MACOP-B cycle]. AB - The collateral effects of antineoplastic therapy often lead to a deterioration of the cachectic condition induced by the presence of the tumour itself. This study analysed the effects of a programme of dietary surveillance/support in patients with non-Hodgkin's lymphoma undergoing a cycle of MACOP-B polychemotherapy. During the entire course of therapy patients were followed weekly by a nutritional specialist and a dietician in order to assess and if necessary modify food intake, also in relation to the onset of collateral effects. Using this programme it was observed that a satisfactory nutritional state was maintained during the entire cycle, with an increased food intake compared to the start of the cycle and to conditions of good health. PMID- 1720676 TI - Effect of plasma glow, glutaraldehyde and carbodiimide treatments on the enzymic degradation of poly (L-lactic acid) and poly (gamma-benzyl-L-glutamate) films. AB - The hydrolytic and enzymic degradation of poly(L-lactic acid) (PLA) and poly(gamma-benzyl L-glutamate) (PBGA) films, together with a series of surface treatments, were studied, as a function of exposure time. The degradation of these polymers was monitored by weight loss, contact angle, pH changes and tensile strength studies. Glutaraldehyde treatment retained the maximum strength of PLA in buffer, followed by carbodiimide, compared with control films. On the other hand, plasma glow reversed the effect. The ability of alpha-chymotrypsin, carboxypeptidase, ficin, esterase, bromelain and leucine aminopeptidase to modulate the degradation of PLA and PBGA was also investigated. Addition of these enzymes to the polymer-buffer system reduced the tensile strength of these polymers variably. Among the six enzymes studied, leucine aminopeptidase showed the highest enzymic effect on the degradation of the glutaraldehyde-treated and bare PLA or bare PBGA films. However, glutaraldehyde-cross-linked PLA demonstrated maximum stability in buffers or in all other enzyme systems studied compared with bare PLA. It is conceivable that surface treatments on these polymers might have altered their physical and chemical configuration and the subsequent degradation properties. Surface modifications may provide new ways of controlling the biodegradation of polymers for a variety of biomedical applications. PMID- 1720677 TI - Multiple epitopes on cartilage type II collagen are accessible for antibody binding in vivo. AB - Monoclonal mouse antibodies specific for the major epitopes on mouse type II collagen (CII) were biotinylated and injected into neonatal and adult mice. Anti CII antibodies, specific for four different epitopes on the CII molecule, could be shown to bind specifically to joint surfaces in the paws of 2-day-old syngeneic DBA/1 mice after an intraperitoneal injection of 100 micrograms of biotinylated antibody. The anti-CII antibodies did not bind to cartilage from DBA/1 mice in vitro, unless the sections were pretreated with hyaluronidase or the specimens decalcified prior to freezing, showing that the epitopes are accessible in vivo but not in vitro. By analyzing the in vivo binding capacity for a number of monoclonal anti-CII antibodies which represented different IgG subclasses, it could be demonstrated that binding to the same epitopes occurred independent of IgG subclass. However, one epitope (denoted "B1") was only weakly detected, possibly due to the fact that the antibody used (CIIB1) crossreacts with type I collagen and C1q. Monoclonal anti-CII antibodies, injected into neonates or adult mice, bound specifically to most, but not all, tissues containing CII; including hyaline joint cartilage, fibrous sternal and costal cartilage, tracheal cartilage and fibrous cartilage in the spine but not to CII containing structures in the eye. The finding that CII, while present in cartilage, is accessible for antibody binding in vivo may have important implications for the availability of CII for the immune system and for the understanding of the development of pathological autoimmunity leading to collagen induced arthritis in mice. PMID- 1720678 TI - A statistical method for the comparison of a discrete diagnostic test with several continuous diagnostic tests. AB - In this paper we study a statistic that is suitable for comparing a discrete diagnostic marker to one or more continuous diagnostic markers. Test procedures and confidence intervals are based on asymptotic normality. The statistic is applicable for correlated data in which all the markers are obtained for each subject. The statistic was studied for use in comparing two markers for rectal bleeding. Examples for this application and two more general applications are presented. PMID- 1720679 TI - Formation of ion-conducting channels by the membrane attack complex proteins of complement. AB - The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b 9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different. PMID- 1720680 TI - Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel. AB - This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction. PMID- 1720681 TI - Effect of Bay K8644 on Ca2+ channel gating charge. PMID- 1720682 TI - Visualization of RNA transcription and processing. AB - Electron microscopic visualization of transcriptionally-active chromatin dispersed by the Miller spreading technique allows a unique view of in vivo genetic events on an individual gene basis. We have used the method to ultrastructurally analyze transcription, ribonucleoprotein assembly and early RNA processing events on the pre-messenger RNA transcripts of Drosophila melanogaster Pol II genes. Our findings are surprising in two regards--splicing as a rule initials co-transcriptionally and is frequently complete before polyadenylation, and cleavage at poly(A) sites, at least for a few specific genes, occurs post transcriptionally. PMID- 1720683 TI - Spatial organization of nucleic acid sequences within cells. AB - High-resolution in situ hybridization has allowed the distribution of specific nucleic acids to be visualized within cells. This has revealed the extent to which nucleic acids exhibit intracellular spatial organization. Hybridization of nonisotopically labeled probes can be detected with high resolution using fluorescence or alkaline phosphatase for light microscopy or colloidal gold for electron microscopy. Results of this approach have shown that messenger RNAs for specific proteins, nuclear transcripts, or the gene for those transcripts all can exhibit non-random intracellular locations. Actin mRNA can be seen localized in the periphery of motile cells, just proximal to the lamellipodia. Highly localized distributions of EBV primary transcripts can be visualized in some cases creating an elongated track of specific RNA. These nuclear transcripts could be seen associated with the nuclear matrix. Further evidence indicates that the genes from which specific RNAs derive may themselves show defined spatial organization. This work sheds light on a level of cellular organization indicating that nucleic acid sequences contain spatial positioning information and therefore can influence the genesis of cellular structure and function. PMID- 1720684 TI - [Relationships between Czechoslovakia and the French-speaking countries in the domain of physiology]. PMID- 1720685 TI - [The hundredth anniversary of the birth of Vilem Laufberger, Czech physiologist]. PMID- 1720686 TI - Brain stem mechanisms of conditioned taste aversion learning in rats. AB - Acquisition of conditioned taste aversion (CTA) in rats is not prevented by functional decortication, anesthesia or hypothermia applied after intake of the flavored fluid and maintained throughout the action of the poison but is disrupted by bilateral application of 10 ng tetrodotoxin (TTX) into the parabrachial nuclei. The blockade is directly proportional to TTX dosage, indirectly proportional to distance of the injection site from parabrachial nuclei and equally affects CTAs using different CS (saccharin, NaCl) and different US (LiCl, carbachol, amphetamine, cycloheximide). CTA is disrupted by TTX applied up to 4 but not 8 days after a single CS-US pairing. TTX fails to disrupt overtrained CTA and elicits only a weak anterograde amnesia when applied 1 but 2 or more days before CTA acquisition. It is concluded that the parabrachial nuclei and the adjacent reticular formation probably represent the neural substrate of the permanent CTA engram the protracted consolidation of which is disrupted by prolonged cessation of impulse which is disrupted by prolonged cessation of impulse activity in the information storing network. PMID- 1720687 TI - Epileptic phenomena in the immature brain. PMID- 1720688 TI - [The thalamic reticular nucleus: its involvement in the control of thalamic interactions]. PMID- 1720689 TI - [Peptide YY and neuropeptide Y in the intestine: availability, biologic effects and epithelial receptors]. PMID- 1720690 TI - [Neuropeptide Y in the central nervous system: an approach to the mechanisms of neuronal plasticity]. PMID- 1720691 TI - [The nucleus tractus solitarius: neuroanatomic, neurochemical and functional aspects]. AB - The nucleus tractus solitarii (NTS) has long been considered as the first central relay for gustatory and visceral afferent informations only. However, data obtained during the past ten years, with neuroanatomical, biochemical and electrophysiological techniques, clearly demonstrate that the NTS is a structure with a high degree of complexity, which plays, at the medullary level, a key role in several integrative processes. The NTS, located in the dorsomedial medulla, is a structure of small size containing a limited number of neurons scattered in a more or less dense fibrillar plexus. The distribution and the organization of both the cells and the fibrillar network are not homogeneous within the nucleus and the NTS has been divided cytoarchitectonically into various subnuclei, which are partly correlated with the areas of projection of peripheral afferent endings. At the ultrastructural level, the NTS shows several complex synaptic arrangements in form of glomeruli. These arrangements provide morphological substrates for complex mechanisms of intercellular communication within the NTS. The NTS is not only the site of vagal and glossopharyngeal afferent projections, it receives also endings from facial and trigeminal nerves as well as from some renal afferents. Gustatory and somatic afferents from the oropharyngeal region project with a crude somatotopy within the rostral part of the NTS and visceral afferents from cardiovascular, digestive, respiratory and renal systems terminate viscero-topically within its caudal part. Moreover the NTS is extensively connected with several central structures. It projects directly to multiple brain regions by means of short connections to bulbo-ponto-mesencephalic structures (parabrachial nucleus, motor nuclei of several cranial nerves, ventro-lateral reticular formation, raphe nuclei...) and long connections to the spinal cord and diencephalic and telencephalic structures, in particular the hypothalamus and some limbic structures. The NTS is also the recipient of several central afferent inputs. It is worth to note that most of the structures that receive a direct projection from the NTS project back to the nucleus. Direct projections from the cerebral cortex to the NTS have also been identified. These extensive connections indicate that the NTS is a key structure for autonomic and neuroendocrine functions as well as for integration of somatic and autonomic responses in certain behaviors. The NTS contains a great diversity of neuroactive substances. Indeed, most of the substances identified within the central nervous system have also been detected in the NTS and may act, at this level, as classical transmitters and/or neuromodulators.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1720692 TI - [Physiology of fecal continence and defecation]. AB - Defaecation is a complex function that requires interactions between the somatic and autonomic nervous systems. Moreover, social living standards lead to self control of continence involving supraspinal nervous structures. After a brief description of the anatomy of the hind gut, the various reflex mechanisms underlying faecal continence and defaecation are exposed; the specificities of the nervous control of the smooth and striated muscles brought into play in these events are detailed. Then the main useful investigations allowing to localize the nervous structures at the origin of the anorectal disorders are reviewed. PMID- 1720693 TI - [Role of substance P in the nervous system control of digestive motility]. AB - Substance P is a 11 amino-acids peptide which belongs to the tachykinins, a family of peptide which induces a rapid contraction of the smooth muscle of the digestive tract. The occurrence of substance P has been demonstrated by immunohistochemical and radioimmunological techniques in most parts of the central and peripheral nervous system. Substance P exerts on the smooth muscle of all the areas of the digestive tract a strong excitatory effect which is either direct or relayed by the cholinergic intramural neurones. Numerous electrophysiological, pharmacological and immunohistochemical data lead to the conclusion that substance P is released by intrinsic neurones of the digestive tract or by extrinsic nerves (vagus and splanchnic nerves, etc...). This release is enhanced by acetylcholine, cholecystokinin, serotonin and neurotensin, it is reduced by opioid peptides and noradrenaline. Substance P participates in the intestinal peristaltic reflex by the activation of the smooth muscle cells of the intestine, either directly or through the activation of the cholinergic intrinsic neurones. Substance P is also involved in the genesis of a non-cholinergic ascending excitatory activity likely occurring during vomiting. Lastly, substance P participates in the reflex contraction of the lower oesophageal sphincter following acidification of the distal part of the oesophagus. PMID- 1720694 TI - [Relationship between postural support and intentional movement: biomechanical approach]. AB - The presentation is in two parts. The first, more theoretical, section reviews the basic biomechanical features underlying the movement of all bodies, both animate and inanimate, being in contact with the ground. Application of biomechanical laws shows that intentional movement can only be executed if the external environment can react to the movement of members relative to the body. Thus, it is theoretically necessary that the part of the body, including the body segment(s) that are located between the ones which are voluntarily moved and the supporting surfaces, ie, the "postural support", is involved in the motor activity. The general equilibrium conditions of a complex mechanical system such as the human body are then examined to show why movement itself disturbs balance stability and that postural reactions are, a priori, required to preserve this stability. The second sections includes an analysis of postural activities for several types of sensory-motor tasks. They are shown to be synergistically organized, and the major features of this organisation are described. The anticipatory postural adjustments (APA) which occur BEFORE the start of voluntary movement, and are thus "preprogrammed", are then examined. It is shown that they depend on the parameters of the planned movement, posture and the uncertainty about the tasks. This is followed by a discussion of the reasons why APA may be considered as a counter-perturbation, opposing, in advance the perturbation of equilibrium brought about by the voluntary movement. The presentation concludes with several hypotheses for explaining the organisation of postural activity associated with voluntary movement. The most likely appears to be a process of "parallel" command of the voluntary and postural components. PMID- 1720695 TI - Acquired aplastic anemia and paroxysmal nocturnal hemoglobinuria: studies on clonality. AB - We used X-chromosome methylation patterns to study clonality in aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). AA is usually not considered to be a clonal stem cell disorder, although this has not been directly investigated. PNH is generally assumed to be a clonal disorder, although there is contradictory evidence. Methylation analysis was performed on DNA from separated granulocytes and mononuclear cells, using the M27 beta and hypoxanthine phosphoribosyl transferase (HPRT) probes. Six of seven AA patients showed a polyclonal pattern of X inactivation. In contrast, five of five PNH patients showed a monoclonal pattern. These results imply that at least 80% of the cell population derives from a single stem cell. Because this high proportion of PNH cells might be considered surprising, three patients were studied for membrane expression of decay accelerating factor (DAF). In support of the DNA data, more than 95% of the granulocytes were DAF--ve in all three cases. We conclude that AA is predominantly a polyclonal disorder, whereas PNH is a clonal stem cell disorder. Our data support a model in which a single PNH stem cell has a growth advantage over other remaining stem cells and eventually dominates hematopoiesis. PMID- 1720696 TI - c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides. AB - The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors. PMID- 1720697 TI - Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, monocyte-CSF, and interleukin-3. AB - CD34+ cells isolated from bone marrow or umbilical cord blood from healthy donors were studied for proliferation and differentiation in liquid cultures in the presence of recombinant human granulocyte-monocyte colony-stimulating factor (GM CSF), granulocyte CSF (G-CSF), monocyte CSF (M-CSF), and interleukin-3 (IL-3), followed by immunophenotyping for myeloid and myeloid-associated cell surface markers. IL-3, either alone or together with GM-CSF, G-CSF, or M-CSF, induced, on average, 50-fold cell multiplication, GM-CSF five fold to 10-fold, and G-CSF and M-CSF less than fivefold. Cells from cultures stimulated with GM-CSF, G-CSF, or M CSF alone contained cells with a "broad" myeloid profile, "broader" than observed in cultures with IL-3. However, since IL-3 induced rapid cell multiplication, high numbers of cells expressing early (CD13, CD33) and late myeloid markers (CD14, CD15) were recovered. The presence of other CSFs together with IL-3 did not alter the IL-3-induced effect on the cells. When 5,000 CD34+ cells were cultured with IL-3 alone, the cultures still contained 2,000 to 5,000 CD34+ cells after 14 days of culture, while cells cultured with GM-CSF, G-CSF, or M-CSF contained less than 1,000 CD34+ cells. Furthermore, 1,000 to 3,000 cells were positive for the megakaryocytic lineage marker CD41b after cultures with GM-CSF or IL-3, while cultures with G-CSF or M-CSF did not contain detectable numbers of CD41b+ cells. Finally, erythroid cells could also be generated from purified CD34+ cells. The results show that IL-3 and GM-CSF can induce rapid proliferation of purified CD34+ cells in vitro with differentiation to multiple myeloid lineages, while certain subsets maintain expression of CD34. PMID- 1720698 TI - Potential use of human stem cell factor as adjunctive therapy for human immunodeficiency virus-related cytopenias. AB - Hematopoietic dysfunction with peripheral cytopenias is a common complication of human immunodeficiency virus (HIV) infection. Symptomatic anemia is the most common cytopenia and occurs in the presence and absence of myelosuppressive drug therapy such as zidovudine. Drug-induced neutropenia and immune thrombocytopenia are also frequent and occur in up to 50% of acquired immunodeficiency syndrome (AIDS) patients. Attempts to reduce the impact of bone marrow failure have focused on dose reduction of zidovudine, ganciclovir, and chemotherapy, and the use of recombinant hematopoietic hormones such as erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF). Despite these maneuvers, approximately 30% of patients with AIDS receiving zidovudine will become transfusion-dependent. This has led to investigations of other cytokines that may increase blood cell formation. The recent identification of decreased number and proliferation of hematopoietic progenitors in patients with HIV infection suggests that agents which have activity on progenitor cell pools may have clinical utility. We demonstrate that human stem cell factor (HuSCF) increases burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte-monocyte (CFU-GM), and CFU-Mix formation in vitro in normal and HIV-infected individuals. HuSCF also decreases the sensitivity of BFU-E to inhibition by zidovudine without altering HIV replication in lymphocytes or monocytes, altering peripheral blood mononuclear cell proliferation to phytohemagglutinin (PHA) and interleukin-2 (IL 2) or altering the effectiveness of zidovudine or dideoxyinosine in inhibiting HIV replication in lymphocytes or monocytes. These studies suggest that HuSCF may have clinical utility in HIV infection as an adjunctive treatment for HIV-related cytopenias. PMID- 1720699 TI - Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes. AB - Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding. PMID- 1720700 TI - Inhibitory effect of granulocyte-macrophage colony-stimulating factor therapy on the generation of natural killer cells. AB - We investigated the effects of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human granulocyte colony stimulating factor (rhG-CSF) therapy on the natural killer (NK) cell lineage in patients with aplastic anemia and myelodysplastic syndrome. Selected bone marrow (BM) cells were prepared by the elimination of nylon wool-adherent cells and mature T and NK cells from BM cells. The frequency of BM NK progenitors relative to BM cells selected was significantly decreased 4 weeks after the start of rhGM CSF therapy (P less than .01), while the peripheral blood NK cell count and NK activity were also significantly decreased (P less than .05). A return to the pretreatment levels was seen 4 weeks after the cessation of treatment in all cases. No suppressive effect was noted in the patients who received rhG-CSF therapy. These results suggest that rhGM-CSF therapy suppresses the generation of NK cells from human BM NK progenitors. PMID- 1720701 TI - Modulating activity of interferon-gamma on endotoxin-induced cytokine production in cancer patients. AB - Intravenous (IV) administration of purified lipopolysaccharide (LPS) from Salmonella abortus equi to cancer patients induces the formation of high amounts of endogenous cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). On repeated administration of LPS at 2-week intervals, a marked downregulation of the cytokine response was observed, especially between the first and the second challenge. This study sought to determine whether it would be possible to prevent this downregulation by pretreating patients with interferon-gamma (IFN-gamma), which is known to enhance cytokine production by monocytes and macrophages in vitro. Ten patients with disseminated cancer received a first injection of 4.0 ng LPS/kg. Thereafter, patients were divided into two groups. One group received two further LPS injections (4.0 ng/kg) at 2 week intervals. The second group was pretreated (-12 hours) with 50 micrograms IFN-gamma subcutaneously (SC) before the second and third LPS challenge. To prevent constitutional side effects such as fever and chills, patients received 1,600 mg ibuprofen orally before LPS injection. The results of the current study demonstrate that apart from TNF-alpha and IL-6, two other cytokines, interleukin 8 (IL-8) and granulocyte colony-stimulating factor (G-CSF) are produced in cancer patients in response to LPS. LPS application at 2-week intervals resulted in a transient attenuation of all cytokines (TNF-alpha, IL-6, IL-8, G-CSF) on the second challenge. In the case of TNF-alpha, IL-6, and G-CSF, pretreatment with IFN-gamma not only prevented the downregulation, but enhanced the production of these cytokines to levels higher than those obtained after the first LPS challenge. In contrast, the downregulation of IL-8 remained unaffected by IFN gamma pretreatment. Further studies are warranted to determine whether the prevention of cytokine downregulation by IFN-gamma following repeated LPS injections is of clinical relevance in respect to the antitumor activity of LPS. PMID- 1720702 TI - Biochemical studies on red blood cells from a patient with the Inab phenotype (decay-accelerating factor deficiency). AB - A 38-year-old Russian woman (KZ) has been identified as the fourth proposita with the Inab blood group phenotype. Like the first two propositi, she has a chronic intestinal disorder and, as shown for the third proposita, her Inab phenotype is demonstrably inherited. KZ's serum contained anti-IFC, which reacted with a red blood cell (RBC) membrane component with an Mr of 70,000, which is decay accelerating factor (DAF). Her RBCs lacked all Cromer-related blood group antigens and DAF. Her RBCs were no more susceptible than normal control RBCs to lysis in acid lysis or in rabbit or human antibody-initiated complement lysis tests. Northern blots of total RNA isolated from KZ's Epstein-Barr virus transformed lymphoblasts showed a marked reduction of DAF mRNA when compared with normal. Polymerase chain reaction (PCR) amplification of cDNA confirmed this reduced level of DAF mRNA. Sequencing of the PCR product showed a 44-nucleotide deletion in the mRNA close to the short consensus repeats IIIa/IIIb intron/exon boundary. This deletion results in a change in the reading frame that places a termination codon six amino acids after the deletion. The putative translation product would lack a glycosyl phosphatidyl-inositol linkage site and, therefore, would not be membrane-bound in the RBC. PMID- 1720703 TI - Polar axis fixation in Fucus zygotes: components of the cytoskeleton and extracellular matrix. AB - Polar axis formation and polar axis stabilization (or fixation) can be separated and analyzed in synchronously developing zygotes of the brown alga Fucus. Extensive experimental evidence points to a role for both the cytoskeleton and the extracellular matrix (ECM) in the process of axis fixation in Fucus. A structural complex composed of the cytoskeleton and the ECM has been postulated to stabilize membrane asymmetries generated as a result of axis-forming vectors. This axis stabilizing complex (ASC) may take the form of transmembrane connections between the cytoskeleton on the cytoplasmic face and the ECM on the external side of the plasma membrane, similar to focal contacts in animal cells. At present we know of two components in the proposed ASC of Fucus: an adhesive sulfated glycoprotein which is localized in the ECM, and an actin network which is localized on the adjoining cytoplasmic face. This preliminary report describes evidence for the presence of molecules in two-celled Fucus embryos that are similar to those found in focal contacts in animal cells, i.e. vinculin, integrin and vitronectin. However, their localization and interaction with each other relative to the polar axis has yet to be determined. These initial observations will provide the basis to pursue further an analysis of these components in the process of polar axis fixation. PMID- 1720704 TI - Projection of nodose ganglion cells to the upper cervical spinal cord in the rat. AB - Afferent fibers mediating pain from myocardial ischemia classically are believed to travel in sympathetic nerves to enter the thoracic spinal cord. After sympathectomies, angina pectoris still may radiate to the neck and inferior jaw. Sensory fibers from those regions are thought to enter the central nervous system through upper spinal cord segments. We postulated that axons from nodose ganglion cells might project to cervical cord segments. The purpose of this study was to determine the density and pathway of vagal afferent innervation to the upper cervical spinal cord. Following an injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord, approximately 5.8% of cells in the nodose ganglion contained reaction product. Cervical vagotomy did not diminish the density of WGA-HRP labeled cells in the nodose ganglion. However, a spinal cord hemisection cranial to the injection site eliminated labeling of nodose cells. These data indicate that a portion of vagal afferent neurons project from the nodose ganglion to the upper cervical spinal cord. In addition, vagal afferent fibers reach the spinal cord via a central route rather than through dorsal root ganglia. PMID- 1720705 TI - Astrocytes colonize dorsal root ganglia transplanted into rat brain. AB - Fragments of dorsal root ganglia (DRG) were grafted into rat brain and examined one month later. The autografts were similar to their normal counterparts when stained with toluidine blue or by indirect immunofluorescence with laminin and neurofilament antibodies. However, a major difference was observed with antibodies to the glial fibrillary acidic protein (GFAP). Normal DRG were GFAP negative while the autografts were intensely and diffusely stained. The GFAP antibodies used in this study did not decorate Schwann cells or satellite cells in peripheral nerve and DRG, and thus appeared to recognize the "central" form of GFAP (17). Thus reactive astrocytes appear to be capable of migration into grafted nervous tissues without producing apparent neuronal damage. PMID- 1720706 TI - Production and characterization of monoclonal antibodies that localize human thymidylate synthase in the cytoplasm of human cells and tissue. AB - Thymidylate synthase (TS; EC 2.1.1.45) is an important cellular enzyme that converts dUMP to dTMP, which is essential for DNA biosynthesis. In addition, TS is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid tumors. We have generated five monoclonal antibodies against human TS using a recombinant human TS enzyme. These antibodies react specifically with human TS and display negligible cross-reactivity with other cellular proteins found in human cells. Binding affinity studies demonstrate that all antibodies form a tight interaction with recombinant human TS enzyme (Kd range = 0.3-11.0 nM). All antibodies display reactivity on enzyme linked immunosorbent assay and immunoprecipitation. On Western blot analysis each detects a protein of approximately 36 kDa molecular mass under denaturing conditions. In addition to their reactivity on immunoprecipitation and Western analysis, two of the antibodies, TS 106 and TS 109, are reactive on immunohistochemical staining of human colon carcinoma cell lines and tissue, producing a granular cytoplasmic staining pattern. Specificity for TS is demonstrated by the lack of staining with preimmune IgG and the disappearance of the signal when the antibodies are preabsorbed with recombinant human TS enzyme. Quantitation of TS by Western blot analysis and biochemical FdUMP binding assay in 5-fluorouracil-resistant colon carcinoma cell lines (NCI H630R10, NCI H630R1) and a sensitive colon carcinoma cell line (NCI H630) revealed a 36- and 6-fold increase in TS in the resistant cell line as measured by the biochemical assay compared to a 39- and 10.6-fold increase as measured by densitometric analysis of the Western blot. These comparative studies of immunohistochemical, Western, and biochemical analyses reveal that the immunological detection of TS in human colon cell lines is a sensitive and quantitative assay. Thus the ability of these antibodies to detect TS in human cancer cells and tissue may allow measurement of TS in human tissues by quantitative immunohistochemistry in studies of drug resistance and for determination of proliferative rates. PMID- 1720707 TI - Circadian rhythm of tryptophan hydroxylase activity in chicken retina. AB - 1. Retinal tryptophan hydroxylase activity in chickens (1-4 weeks old and embryos) was estimated by determination of levels of 5-hydroxytryptophan (5HTP) in retinas at defined intervals after inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine (NSD1015). 2. The relationship of tryptophan hydroxylase activity to photoperiod was explored. In chickens maintained on a 12-hr light: 12-hr dark cycle, a diurnal cycle in tryptophan hydroxylase activity was observed. Activity during middark phase was 4.4 times that seen in midlight phase. Cyclic changes in tryptophan hydroxylase activity persisted in constant darkness with a period of approximately 1 day, indicating regulation of the enzyme by a circadian oscillator. The phase of the tryptophan hydroxylase rhythm was found to be determined by the phase of the light/dark cycle. The relationship of the tryptophan hydroxylase rhythm to the light/dark cycle mirrors previously described rhythms of melatonin synthesis and serotonin N acetyltransferase (NAT) activity in the retina. 3. Light exposure for 1 hr during dark phase suppressed NAT activity by 82%, while tryptophan hydroxylase activity was suppressed by only 30%. 4. Based on the differential responses of retinal NAT activity and tryptophan hydroxylase activity to acute light exposure during dark phase, it was predicted that exposure to light during dark phase would divert serotonin in the retina from melatonin biosynthesis to oxidation by MAO. In support of this, levels of 5-hydroxyindole acetic acid (5HIAA) in retina were found to be elevated approximately two-fold in chickens exposed to 30 min of light during dark phase. In pargyline-treated chickens, 2 hr of light exposure during dark phase was found to increase retinal serotonin levels by 64% over pargyline-treated controls. 5. Cyclic changes in tryptophan hydroxylase activity and NAT activity persisted for 2-3 days in constant light. Tryptophan hydroxylase activity at mid-night gradually decreased on successive days in constant light; on the first day of constant light, tryptophan hydroxylase activity at mid-night was 70% of activity seen during middark phase of the normal light/dark cycle and decreased further on subsequent days. In contrast, on each of 3 days of constant light, NAT activity at mid-night was approximately 15% of normal middark phase activity. 6. Cycloheximide completely inhibited the nocturnal increase in tryptophan hydroxylase activity when given immediately before light offset. The nocturnal increase in NAT activity was inhibited in a similar fashion. 7. Like the development of the NAT rhythm, cyclic changes of tryptophan hydroxylase activity in the retinas of chickens began on or immediately before the day of hatching. hatching.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1720708 TI - Pain, tenderness, wheal and flare induced by substance-P, bradykinin and 5 hydroxytryptamine in humans. AB - The algesic effect of substance-P with and without the addition of bradykinin or 5-hydroxytryptamine was studied in 13 healthy volunteers. Test substances dissolved in saline were injected into the temporal muscle and the forearm skin and the effects compared with those of saline. In the temporal muscle, none of the test substances induced more pain than saline, but substance-P with bradykinin lowered the pressure pain threshold by 18% (p less than 0.02). All test substances induced pain wheal and flare in the forearm skin. Substance-P induced a more pronounced flare reaction than bradykinin, whereas the latter induced more pain than substance-P. This dissociation between pain and flare may indicate that C-fibres in the human skin represent more than one type of nociceptor. PMID- 1720709 TI - Ultrastructural effects of colchicine on perikarya and initial segment of rat retinal ganglion cells. AB - Our ultrastructural study was focused on the perikaryal region and initial segment of the axon of rat retinal ganglion cells in controls and after intraocular injections of colchicine. In control rats that region contained, among other organelles, elements of the Golgi complex and, close to them, short isolated microtubules oriented preferentially toward the axon where they funnel and aggregate in bundles. One day after sufficient doses of colchicine to inhibit axoplasmic transport (2-20 micrograms) these cytoplasmic microtubules were absent, whereas some axonal microtubules were still present but reduced in number. In addition, colchicine induced an altered distribution of organelles, leaving empty spaces in the periphery and most organelles concentrated in the perinuclear region, especially around Golgi elements where numerous vesicles and tubules accumulate at the trans face of Golgi elements. These results suggest that the vesicles that leave the Golgi and have been directed towards axoplasmic transport may need the cytoplasmic microtubules located between Golgi elements and the axonal initial segments to reach the axon. PMID- 1720710 TI - Intensive chemotherapy for adult lymphoblastic lymphomas. AB - A total of 20 adults patients presenting with previously untreated lymphoblastic lymphoma underwent an intensive chemotherapy protocol. Either the BACOP or the m BACOD regimen was used for induction. If the patients achieved a complete clinical remission (CR) after three courses, they were given intensive consolidation and maintenance chemotherapy based on a protocol that was modified from the L10/L17M regimen of the Memorial Sloan-Kettering group for acute lymphoblastic leukaemia and lymphoblastic lymphoma. Patients exhibiting localised areas of bulky disease were given additional involved-field radiotherapy. In all, 15 (75%) men and 5 (25%) women were entered in this study. Their median age was 28 years (mean, 30 years; range, 12-64 years). Overall, 3 (15%) had stage II disease, 3 (15%) had stage III disease and 14 (70%) had stage IV disease; 7 (35%) patients exhibited B symptoms and 4 (20%) had bulky disease. The overall (CR) rate was 10/20 (20%), and that following BACOP and m-BACOD therapy was 4/8 (50%) and 6/12 (50%), respectively. In all, 7 of the 10 complete responders (70%) relapsed. The disease-free survival of the ten who achieved a CR was 23% at 3 years. The overall survival of all 20 patients at 3 years was only 37%, and there were very few long-term survivors. More effective treatment for adult lymphoblastic lymphoma is required. PMID- 1720711 TI - Neuroendocrine responses to hypertonic saline/dextran resuscitation following hemorrhage. AB - The neuroendocrine responses to resuscitation with 7.5% hypertonic saline/6% Dextran-70 (HSD) following hemorrhagic hypotension were evaluated in conscious swine. Following hemorrhage (37.5 ml/kg/60 min) animals received 4 ml/kg of HSD (n = 6) or 0.9% saline (n = 8). Administration of normal saline did not alter cardiovascular function nor attenuate an increase in hormones. HSD rapidly improved cardiovascular function and acutely decreased ACTH, plasma renin activity (PRA), cortisol, norepinephrine (NE), epinephrine (E), aldosterone, and lysine vasopressin levels (LVP). The initial decreased in ACTH, cortisol, and aldosterone levels was due primarily to hemodilution associated with the expansion of plasma volume. The reductions in NE, E, LVP, and PRA were greater than those attributed to hemodilution alone. Values for LVP, NE, and E remained at values below those at the end of hemorrhage, but greater than basal levels, while PRA returned to values similar to these at the end of hemorrhage. The decrease in LVP, NE, and E following HSD resuscitation for the treatment of hemorrhagic hypotension may result from and contribute to the rectification of cardiovascular and metabolic function. PMID- 1720712 TI - Effects of dibutyryl cyclic AMP, ouabain, and xanthine derivatives on crossbridge kinetics in rat cardiac muscle. AB - In a previous communication, we showed that beta-adrenergic stimulation of cardiac muscles was associated with an increase in the rate of cycling of crossbridges as measured by perturbation analysis in the frequency domain. In this analysis, the frequency at which dynamic stiffness is a minimum (fmin) is taken as a measure of the rate of crossbridge cycling. In this paper, we test the hypothesis that the beta-adrenergic receptor-induced increase in crossbridge cycling rate is mediated by elevation of the intracellular level of cyclic AMP. The approach taken is to compare the effects on fmin in rat papillary muscles during Ba(2+)-activated contractures of 1) an agonist of cyclic AMP that can easily penetrate the cell, namely, dibutyryl cyclic AMP, 2) agents that block cyclic AMP phosphodiesterase, namely, the xanthine derivatives isobutylmethylxanthine and caffeine, and 3) an inotropic agent that does not affect the intracellular level of cyclic AMP, namely, ouabain. Our results showed that dibutyryl cyclic AMP at a dose of 5 mM has the same actions as beta adrenergic stimulation: it potentiated the isometric twitch force, reduced the time to peak tension and time to half relaxation, and shifted fmin by a factor of 1.8 +/- 0.1 (n = 5). Isobutylmethylxanthine at up to 1.1 mM also acted in the same manner, increasing fmin by a factor of 1.8 +/- 0.2 (n = 6), but ouabain, at a dose (0.03 mM) sufficient to potentiate twitch force by 40 +/- 2% (n = 4), was without effect on the time course of the twitch nor was fmin changed (n = 4). Our findings support the hypothesis that a beta-adrenergic receptor-mediated increase in crossbridge cycling rate is due to an increase in intracellular cyclic AMP level and illustrate the usefulness of the frequency domain analysis approach in the analysis of the mechanism of action of inotropic agents. PMID- 1720713 TI - Repetitive reentrant and non-reentrant ventriculoatrial synchrony in dual chamber pacing. AB - Repetitive retrograde ventriculoatrial (VA) conduction in patients with dual chamber pacemakers may cause two forms of VA synchrony. (1) Endless loop tachycardia (pacemaker-mediated tachycardia) or repetitive reentrant VA synchrony occurs when the pacemaker senses retrograde P waves. Appropriate programming can prevent pacemaker reentrant tachycardia in almost all cases. However, the measures used to control tachycardia may themselves create new problems. (2) AV desynchronization arrhythmia or repetitive non-reentrant AV synchrony occurs when the pacemaker does not sense retrograde P waves. In this form of VA synchrony, the atrial stimulus is ineffectual because it falls in the atrial myocardial refractory period generated by the preceding unsensed retrograde P wave. A long atrioventricular interval and a relatively fast lower rate (or sensor-driven rate with DDDR pacing) favor the development of AV desynchronization arrhythmia and its unfavorable hemodynamic consequences. PMID- 1720714 TI - A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation. PMID- 1720715 TI - Human immunodeficiency virus infection of human bone marrow stromal fibroblasts. PMID- 1720716 TI - Symptoms, diagnosis and treatment of thyroid disease. PMID- 1720717 TI - Crossed anomic aphasia: mild naming deficits following right brain damage in a dextral patient. AB - A detailed case study is reported of crossed aphasia (CA) in a dextral patient, bearing upon such controversial issues as intrahemispheric localisation of language function and hemispheric reversal of nonverbal function. DA, a man aged 37, developed a mild naming problem due to right temporal lobe haematoma. Apart from a mild acquired stutter, his continuous speech was fluent and had a normal proportion of open to closed class lexical items. His naming deficit appears to originate in the 'blocking' or 'disconnection' of the phonological lexicon: he could usually give a functional definition of un-named items and retrieve them with the help of a phonemic cue. Lexical retrieval appears his only language deficit, as he had no comprehension or phonological discrimination deficits. DA showed no visuo-spatial or auditory-nonverbal deficits, suggesting the complete reversal of hemispheric specialisation. PMID- 1720718 TI - [Normalization of synthesis and structure of dystrophic neurons after the effects of hypoxia, 10% NaCl and organospecific RNA]. PMID- 1720719 TI - The early pregnancy factor (EPF) in pregnancies of women with habitual abortions. AB - The EPF activity of 67 sera, obtained during the 6th to 9th week of pregnancy, was determined using the rosette inhibition test. The sera of uncomplicated pregnancies (n = 9) and of women with habitual abortions who came to delivery during the most recent pregnancy (n = 10) contained the highest EPF activity during the 6th week of gestation. When pregnancies ended in another abortion (n = 10) the EPF was never detected, or disappeared at least 1-2 weeks before abortion. In the absence of EPF activity the relative risk of abortion was 7.6 (first determination) or 20.0 (second determination 1 week later). In comparison, the risk was lower when the more commonly used pregnancy parameters (HCG 4.4 (5.7), estradiol 2.6 (4.9), or progesterone (2.7 (2.7)) were depressed. Our results suggest that the EPF determination has high prognostic value in patients with high risk pregnancies and in pregnancies which follow treatment for sterility. PMID- 1720720 TI - Evoked potentials. PMID- 1720721 TI - Cerebral magnetic fields to lingual stimulation. AB - We recorded cerebral magnetic fields to electric stimulation of the tongue in 7 healthy adults. The two main deflections of the response peaked around 55 msec (P55m) and 140 msec (N140m). During both of them the magnetic field pattern, determined with a 7- or 24-channel SQUID magnetometer, suggested a dipolar current source. The topography of P55m can be explained by a tangential dipole at the first somatosensory cortex (SI) in the posterior wall of the central sulcus. The equivalent source of N140m is, on average, about 1 cm lateral to the source of P55m. The reported method allows non-invasive determination of the cortical tongue representation area. PMID- 1720722 TI - Sensorimotor central conduction time in comatose patients. AB - Motor evoked potentials (MEPs) following magnetic stimulation were recorded in 22 patients comatose as a result of head injury (13 cases), stroke (7 cases) or anoxia (2 cases). Somatosensory evoked potentials (SEPs) from median nerve were recorded as well in 19 cases in the same session. Thirteen patients died or remained vegetative (59.1%), 3 were severely disabled (13.6%) and 6 showed a good recovery (27.3%). MEPs were significantly related to the outcome; they appeared to be a more accurate prognostic indicator than the Glasgow Coma Scale (GCS). However, 1 out of 6 patients with bilaterally absent MEPs (16.7%) showed a good recovery. SEPs were significantly related to the outcome as well, but the combined use of SEP and MEP improved the outcome prediction, decreasing the rate of false negatives. Two patients had normal sensorimotor function, 13 a combined sensorimotor dysfunction, while 4 had a pure motor dysfunction. Our results suggest that SEPs and MEPs may improve the assessment of sensorimotor dysfunction in comatose patients. A significant relationship between MEPs and outcome appears to exist, but the assessment of MEP reliability requires further study. PMID- 1720723 TI - The human cervical and lumbo-sacral evoked electrospinogram. Data from intra operative spinal cord surface recordings. AB - We have undertaken the analysis of the human 'evoked electrospinogram' during intra-dural surgical explorations in 20 patients. Averaged spinal cord surface evoked potentials to peripheral nerve electrical stimulation were obtained from various restricted loci on the pial surface of the cervical and lumbo-sacral spinal cord. The brachial plexus P9 potential and its lumbo-sacral counterpart P17 were recorded as ubiquitous initial far-field positivities. The pre-synaptic compound action potentials N11 and N21 dwelt on the ascending slope of N13 and N24 respectively. They were composed of 1-5 sharp peaks and collected from the dorsal and dorso-lateral positions mainly, on the cervical and lumbo-sacral cord respectively. They are thought to be generated in the proximal portion of the dorsal root, the dorsal funiculus and the afferent collaterals to the dorsal horn. Compound action potentials could also be gathered from the surface of the dorsal roots, the cervical N10 and lumbo-sacral N19 potentials. The large cervical N13 and lumbo-sacral N24 waves originate from a dorso-ventral post synaptic dipole, generated in deep laminae of the dorsal horn during the activation of large diameter afferent fibers. These waves were maximal on the main entry cord segments of the stimulated nerves and fell off on the 1-4 more rostral and caudal segments. The N2 wave is the dorsal component of another post synaptic dorso-ventral dipole generated in deep laminae of the dorsal horn but activated by medium diameter afferent fibers. The latest event was the N3 wave, also possibly part of a dorso-ventral post-synaptic dipole, and generated by cells in the dorsalmost and deep dorsal horn laminae during the activation of small diameter afferent fibers. The P wave was a prolonged positive deflection which carried the N2 and N3 waves. It is the manifestation of pre-synaptic inhibition on primary afferent fibers. A supra-segmental ascending spinal cord volley was also described, composed of a long succession of sharp and low voltage peaks. PMID- 1720724 TI - Cerebral evoked potentials after rectal stimulation. AB - We obtained reproducible cortical evoked potentials (EPs) in response to electrical stimulation of the rectum with 1 Hz frequency. We found 2 distinctly different EPs in response to rectal stimulation. In 5 females, the EP had an early onset latency (mean 26 msec) with multiple positive and negative peaks. In 10 females, the EP had a later onset latency (mean 52 msec) and a trifid configuration, having a very prominent negative peak. The early onset EPs after rectal stimulation appeared very similar to the wave form of the cortical EPs recorded after pudendal nerve stimulation. Finding similar interpeak latencies in the early onset EP after rectal stimulation and the EP after pudendal nerve stimulation suggests that either the same pathway was used or that rectal stimulation also stimulated the pudendal nerve. It appears that we stimulated visceral afferents when we recorded late onset EPs, because the large EP amplitude declined rapidly with faster stimulation rates and also with greater number of averaging, and the sensation threshold was very unstable, all different to somatosensory EPs. PMID- 1720725 TI - Nasopharyngeal recordings of somatosensory evoked potentials document the medullary origin of the N18 far-field. AB - Because the nasopharyngeal electrode provides non-invasive access to the ventral brain-stem at the medullo-pontine level we used it for recording somatosensory evoked potentials (SEPs) to median nerve stimulation (non-cephalic reference). After the P9 and P11 far-fields, the nasopharyngeal SEPs disclosed a negative going component which was interpreted as the near-field equivalent of the P14 scalp far-field generated in the caudal part of the medial lemniscus. Nasopharyngeal SEPs also revealed a large N18 with voltage and features strikingly similar to those of the scalp-recorded N18 far-field. These results suggest that N18 is generated in the medulla and not more rostrally in the brain stem. The use of a nasopharyngeal electrode as reference for topographic brain mapping is discussed. The paper documents the feasibility and relevance of nasopharyngeal recordings for non-invasive analysis of short-latency SEPs. PMID- 1720726 TI - Right or left ear reference changes the voltage of frontal and parietal somatosensory evoked potentials. AB - Short-latency cortical somatosensory evoked potentials (SEPs) to left median nerve stimulation were recorded with either the left or right earlobe as reference. With a right earlobe reference the voltage of the parietal N20 and P27 was reduced while the voltage of the frontal P20 and N30 was enhanced. The effects were consistent, but their size varied with the SEP component considered and also among the subjects. Analysis of SEPs at different scalp sites and at either earlobe suggested that the ear contralateral to the side stimulated picked up transient potential differences, depending a.o. on side asymmetry and geometry of the neural generators as disclosed in topographic mapping. For example, the right ear potential can be shifted negatively by the right N20 field evoked by left median nerve stimulation. The changes involve the absolute potential values, but not the time features or the gradients of potential fields. Scalp current density (SCD) maps are not affected. The results are pertinent for current discussions about which reference to use and document the practical recommendation of recording short-latency cortical SEPs with a reference at the ear ipsilateral (not contralateral) to the side of stimulation. PMID- 1720727 TI - SEPs to finger joint input lack the N20-P20 response that is evoked by tactile inputs: contrast between cortical generators in areas 3b and 2 in humans. AB - A method using a DC servo motor is described to produce brisk angular movements at finger interphalangeal joints in humans. Small passive flexions of 2 degrees elicited sizable somatosensory evoked potentials (SEPs) starting with a contralateral positive P34 parietal response thought to reflect activation of a radial equivalent dipole generator in area 2 which receives joint inputs. By contrast, electric stimulation of tactile (non-joint) inputs from the distal phalanx evoked the usual contralateral negative N20 reflecting a tangential equivalent dipole generator in area 3b. Finger joint inputs also evoked a precentral positivity equivalent to the P22 of motor area 4, and a large frontal negativity equivalent to N30. It is suggested that natural stimulation allows human SEP components to be differentiated in conjunction with distinct cortical somatotopic projections. PMID- 1720728 TI - Gating of the early components of the frontal and parietal somatosensory evoked potentials in different sensory-motor interference modalities. AB - Three different interfering conditions were studied during the recording of pre- and postcentral somatosensory evoked potentials (SEPs) following median nerve stimulation at the wrist in 16 normal subjects: active finger movement (MVT), light superficial massage (LSM) and deep muscular massage (DMM) of the hand. Special attention was focused on selective effects on individual SEP components. The frontal N30 component showed the most significant amplitude reduction during the three interfering conditions (76.4% of reduction in MVT, 36.4% in DMM and 32.9% in LSM). In contrast the frontal N23 was not significantly changed and the preceding P22 component was only reduced in the MVT condition. Postcentral N20 was unchanged by the three conditions while P27 was clearly gated by movement but not significantly by LSM and DMM. The three interfering conditions enhanced the parietal N32 and had no significant effect on the parietal P45. An important point was the interindividual variability of these effects and it appeared that group average wave forms would therefore be confusing. The peak latency of some SEP components was changed during the interfering conditions. The most important effect was an increase of postcentral P45 latency which was found to be related to the amplitude enhancement of N32. PMID- 1720729 TI - Recovery functions of somatosensory vertex potentials in man: interaction of evoked responses from right and left fingers. AB - Somatosensory vertex potentials (SVPs) were examined in 12 healthy subjects in response to painful electrical stimulation of the finger. SVPs consisted of N1, P1, and N2. The average latencies of the 3 peaks were 150, 225, and 350 msec, respectively. The latency and amplitude of each potential were reproducible for each subject. Recovery functions of the SVPs were analyzed in 10 subjects. A pair of stimuli were delivered to the right or left finger with interstimulus intervals (ISIs) of 50, 100, 150, 200, 350, 500 and 650 msec. SVPs partially recovered with the shortest ISI (50 msec). Full recovery could not be obtained even with the longest ISI (650 msec). Differences in recoveries within 650 msec of ISI were not observed between right and left stimulations. To examine the interaction between SVPs evoked by right and left finger stimulation, recovery functions from prior contralateral finger stimulation were analyzed with the same ISIs. SVP recoveries for right after left or left after right patterns of stimulus delivery were nearly the same as those for ipsilateral ones. It is suggested that SVPs are generated at nearly the same site in the sensory pathway regardless of the side stimulated. PMID- 1720730 TI - SEPs in two patients with localized lesions of the postcentral gyrus. AB - Scalp distributions of median nerve SEPs were studied in normal controls and 2 patients with localized lesions of the postcentral gyrus. In controls, parieto occipital electrodes registered N20-P27 while frontal electrodes registered P20 N27. Other small components, parieto-occipital P22 and frontal N22, were recognized in about half of the control records. The wave forms at a frontal and a parieto-occipital electrode, both distant from the central region, formed exact mirror images of each other concerning N20-(P22)-P27 and P20-(N22)-N27. Electrodes near the central region contralateral to the stimulation registered cP22-cN30 (central P22 and central N30). When the postcentral gyrus was damaged, N20/P20-P27/N27 and cP22-cN30 were eliminated and the only remaining components were a frontal negative wave (frN) and a contralateral parieto-occipital positive wave (poP). Digital nerve stimulation also evoked poP and frN in both cases. In case 2, poP coincided with P22 of the non-affected side. The following generators were proposed; N20/P20-P27/N27: area 3b, cP22-cN30: areas 1 and 2, poP/early frN (= P22/N22): area 4 at the anterior wall of the central sulcus (due to direct thalamic inputs to motor cortex), late frN: uncertain (SMA?, SII?). PMID- 1720731 TI - Changes in BAEP under hypoglycemia: temperature-related? AB - Latencies of the brain-stem auditory evoked potentials were observed to increase in subjects whose plasma glucose levels were reduced. These changes appeared to be attributable to reduced body temperature, rather than direct effects of hypoglycemia on the auditory nerve or the brain-stem. The results suggest the need for caution in interpreting evoked potential measurements under hypoglycemia. PMID- 1720732 TI - Tetrahedral recording of 3-D BAEPs: evidence for the centered dipole model. AB - Three-dimensional brain-stem auditory evoked potentials (3-D BAEPs) were recorded from 12 normal subjects using a new tetrahedral montage, as well as two other bipolar montages previously described for 3-channel Lissajous' trajectories (3 CLTs). Mean responses, as well as between-subject and within-subject variability were described. A mathematical transformation was applied to the recorded trajectories to render them in a common canonical form to test the assumption that the BAEP conforms to a centrally generated dipolar field. Apex, segment, and plane orientations were measured for each trajectory, and discrepancies between montages were evaluated to judge the adequacy of the centered dipole model. For the vector means of apices, segments, and planes, median angles of discrepancy between montages ranged from 10 to 23 degrees. These results support the validity of a centered dipole model for the BAEP and affirm the rationale for employing the 3-channel recording technique. Among the montages studied, the tetrahedron provided maximum economy by using fewer electrodes, avoided certain problematic recording sites, and produced less variable data. PMID- 1720733 TI - P3 responses to prosodic stimuli in adult autistic subjects. AB - Autistic persons are known to have serious abnormalities in speech prosody. The present study attempted to ascertain whether autistic persons could discriminate and/or recognize prosodic contrasts in auditory stimuli. A group of 11 adult autistic subjects with normal IQ and an age-matched group of normal subjects were studied electrophysiologically and behaviorally during presentations of prosodic and phonemic stimuli. The cognitive P3 potential was recorded in response to rare (20%)/frequent (80%) presentations of phonemic stimuli, 'ba/pa,' linguistic prosodic stimuli, 'Bob.' (statement)/'Bob?' (question), and emotional-prosodic stimuli, 'Bob' (happy)/'Bob' (angry). Behaviorally, auditory discrimination was tested by requiring a button-press response to each presentation of the rare target stimulus and cognitive association was tested by requiring a match between the verbalized stimulus and an appropriate picture/word. Contrary to our hypothesis, the autistic subjects generally showed normal P3 responses to all stimuli and performed at a normal level in all behavioral tests. However, a significant autistic P3 response to the phoneme 'pa' was not demonstrated. This surprising result was reexamined and shown to reflect an unusually large autistic response to 'pa' as the frequent stimulus in the first recording block, this initial hyper-reactivity prevented a 'frequent/rare' differential when 'pa' was presented as the rare stimulus in a later recording block. In the P3 latency window, both the autistic and control groups showed the largest amplitude responses to emotional-prosodic stimuli; neither the N1 nor P2 showed these stimulus effects. Thus, 'emotional sounds' appear to be particularly effective in activating the neural substrate of the P3 generator system. Overall, these data indicate remarkably normal P3 and behavioral processing of prosodic stimuli by the high-functioning autistic subjects of this study. PMID- 1720734 TI - The effects of digital filtering on feline auditory brain-stem evoked potentials. AB - The power spectrum of the feline auditory brain-stem evoked potentials (ABEPs) consists of 3 frequency bands, similar to the human wave form, but differing in range. The frequency bands in the feline spectra were separated by notches at 326 Hz and 732 Hz. Click-evoked ABEP from 15 cats were digitally filtered in 3 passbands: (1) below 326 Hz ('slow filter'), (2) between 326 and 732 Hz ('medium filter'); and (3) between 732 and 1790 Hz ('fast filter'). Filtering in each of these bands differentially affected the ABEP components. The vertex positive components are labeled by their order of appearance, i.e., 1, 2, ... 5. Peak 1 is subdivided into 2 subcomponents labeled 1a and 1b. The slow filter was associated with the loss of all components leaving a slow potential shift, i.e., the 'pedestal' peaking at the latency of peak 4. The medium filter was associated with the loss of components 1a, 1b and 2, sparing 3 and 4. The fast filter was associated with the loss of 1b and a diminution of 2. Comparing cat and human ABEP, feline components 2, 3 and 4 behaved precisely the same as the human II, III and V. In contrast to the human I, the feline first component (1a) was not detected with the medium filter. No feline component, following peak 1 in the unfiltered wave form, disappeared with the slow and medium filters, and reemerged with the fast filter (as human IV does).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720735 TI - Voltage dependent calcium channels in cerebellar granule cell primary cultures. AB - Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K 8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than 15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture. PMID- 1720737 TI - Local hyperthermia in the treatment of benign prostatic hyperplasia. Assessment of 100 patients. AB - This study includes our first 100 patients who received local prostatic hyperthermia treatment for benign prostatic hyperplasia. Subjective symptoms such as nycturia, stream, urgency, and objective facts like urine flow and postmicturition residue were monitored before treatment and 3 months after. The clinical (subjective) symptoms improved in 76 patients. Urinary flow increased in 63 patients, and the postmicturition urinary residue decreased in 32 patients. We were able to show that local prostatic hyperthermia is a valid option for the treatment of benign prostatic hyperplasia although it is still too early to assess the long-term results. PMID- 1720736 TI - Seizure-induced protein tyrosine phosphorylation in rat brain regions. AB - Phosphorylation of protein tyrosines is an important modulatory process for cell signaling and other cellular functions. Rat brain regions were examined for altered protein phosphotyrosines, using Western blot analysis and microwave irradiation to limit postmortem alterations, after administration of two convulsants: lithium plus pilocarpine or kainic acid (KA). Most phosphotyrosine proteins were unaltered by these treatments, but there was a large, specific increase in the tyrosine phosphorylation of a 40-Kd protein. This increase was evident in all three regions examined: cerebral cortex, hippocampus, and striatum; it occurred abruptly with onset of generalized status epilepticus (SE) and remained elevated for at least 90 min. Most of the tyrosine phosphorylated 40 Kd protein was in the cytosolic fraction. These results demonstrate a large, specific effect of chemically induced seizures on a single phosphotyrosine protein in rat brain. PMID- 1720738 TI - Intravenous infusion of iloprost in arterial occlusive disease: dose-dependent effects on skin microcirculation. AB - Transcutaneous oxygen pressure (tcPo2), laser Doppler flux and capillary microscopy have been used to examine the forefoot skin in 5 healthy men and 8 patients with severe peripheral arterial occlusive disease in order to evaluate the dose dependent effects of iloprost on skin microcirculation. Iloprost was infused IV starting at 0.0625 ng.kg-1.min-1 and doubling the dose every 15 min up to 2 ng.kg-1.min-1. While tcPo2 at an electrode core temperature of 44 degrees C decreased in both patients and controls, there was a significant dose dependent increase in tcPo2 (37 degrees C) in the controls from 0.25 ng.kg-1.min-1. In the patients the reaction was variable: it was decreased in two and increased in 6, with a maximum either at 0.25-0.5 ng.kg-1.min-1 (n = 3) or at the highest dose (1.0 or 2.0 ng.kg-1.min-1; n = 3). Mean laser Doppler flux in both groups was increased, although the reaction was not consistent in the patients. Density of forefoot skin capillaries was reduced in 3 patients, and in the others the flow velocity was very low. During infusion of iloprost, both an increase in capillary density and blood cell velocity were observed. The effects were of variable intensity and occurred at varying doses, some appeared early and diminished as the dose was increased, and others were found only at 2 ng.kg-1.min-1. Adverse effects were numerous, extending from harmless skin flushing to mental changes and a quickly reversible attack of angina pectoris.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720739 TI - The distribution and origin of substance P immunoreactive nerve fibres in the rat conjunctiva. AB - Indirect immunohistochemistry was used to examine the presence and origin of substance P immunoreactive nerve fibres in the rat conjunctiva. Fluorescent substance P immunoreactive nerve fibres were visualized both in the epithelium and stroma, their number being higher in the stroma where they were associated with blood vessels, the smooth muscle of Muller and the Meibomian glands. Ten to twenty percent of the ganglion cells in the trigeminal ganglion were immunoreactive to substance P, most of them being small in size. Sensory denervation by electrocoagulating the ophthalmic and maxillary branches of the trigeminal nerve caused a complete disappearance of the epithelial fibres and greatly reduced the number of the stromal fibres. These results indicate that the majority of the demonstrated fibres are sensory nerves originating from the trigeminal ganglion. Since sympathectomy had no detectable effect on the number or distribution of substance P immunoreactive nerve fibres, and since some of the fibres remained after sensory denervation it is suggested that at least some of the remaining fibres could be of parasympathetic origin. PMID- 1720740 TI - Transcriptional regulation of murine NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. AB - The cytosolic NADP(+)-dependent methylenetetrahydrofolate dehydrogenase methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is ubiquitously expressed in all mouse tissues and cell lines examined. Northern analyses of the RNA indicated that there is an extensive variation in the levels of mRNA in different tissues. However, the gene is refractory to induction by serum, phorbol esters or growth factors in cultured fibroblasts. The mRNA of the NADP(+)-dependent trifunctional enzyme is stabilized post-transcriptionally by insulin-like growth factor-1. PMID- 1720741 TI - An hsp70 homolog is encoded on the plastid genome of the red alga, Porphyra umbilicalis. AB - A PCR experiment using Porphyra umbilicalis DNA as the template and degenerate oligonucleotides representing conserved regions of hsp70 amino acid sequences generated a 1 kb product that hybridized exclusively to the plastid DNA of this red alga. DNA sequencing of two contiguous EcoRI plastid DNA clones revealed a 620 amino acid open reading frame with 71% identity to the dnaK gene of the cyanobacterium, Synechocystis 6803. Northern hybridization experiments detected a 2.3 kb transcript that is present in control (15 degrees C) cultures and increases approximately 7-fold upon heat shock (75 minutes at 30 degrees C). PMID- 1720742 TI - Structure-function analysis of the human integrin VLA-4 (alpha 4/beta 1). Correlation of proteolytic alpha 4 peptides with alpha 4 epitopes and sites of ligand interaction. AB - The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50 kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions. PMID- 1720743 TI - Interleukin 1 receptor antagonist in human epidermis and cultured keratinocytes. AB - Interleukin 1 (IL-1), present in high amounts in normal human skin without any sign of inflammation, suggests a complex mechanism by which its bioactivity is regulated. The specific receptor antagonist of IL-1 (IL-1ra) was analyzed in human skin, sweat and cultured keratinocytes. Extracts of both skin and cultured keratinocytes blocked the binding of [125I]IL-1 to its receptor whereas sweat did not. The inhibitory activity was cell-associated, was not secreted by cultured keratinocytes, and IL-1ra mRNA was identified in these cells. There was an inverse relationship between the level of IL-1ra and that of IL-1 alpha and beta since extracts of differentiating keratinocytes (DK) and higher IL-1ra levels and expressed more mRNA for IL-1ra than non-differentiated keratinocytes (NDK), whereas NDK contained 4 times more IL-1 alpha and beta proteins than DK. This association of cell differentiation with a shift in agonist/antagonist ratio might be related to important autocrine or paracrine functions of IL-1 in normal and inflamed human skin. PMID- 1720744 TI - Response of vessels to ischaemia. PMID- 1720745 TI - Molecular size of retinal vascular leakage determined by FITC-dextran angiography in patients with posterior uveitis. AB - In order to investigate the selective breakdown of the blood-retinal barrier in posterior uveitis angiograms were performed using fluorescein-conjugated dextrans (FITC-dextrans) of different molecular weights (150-kDa, 20-kDa, and 4-kDa) in two healthy controls and six patients with posterior uveitis, and the results compared with those of conventional fluorescein angiography. The smallest FITC dextran could not penetrate the healthy blood-retinal barrier. Leakage of FITC dextrans of all sizes was seen from swollen optic discs; dextrans of 4-kDa and 20 kDa leaked from areas of macula oedema; and retinal new vessels allowed the passage of 4-kDa molecules only. These results indicate that there is a differential breakdown of the blood-retinal barrier according to molecular size in various clinical situations associated with posterior uveitis. PMID- 1720746 TI - Distribution of epithelial membrane antigen in eccrine poroma. AB - Using immunohistochemical methods, we investigated the distribution of epithelial membrane antigen (EMA) on the normal eccrine gland, eccrine poroma and hidroacanthoma simplex. Granular membrane-associated reaction of EMA was detected on the outer cells of both the intraepidermal and the upper portion of intradermal eccrine ducts, as well as on the luminal surfaces and intercellular canaliculi of eccrine glands. Clear immunolabeling was also present in the tumor cells of eccrine poroma and hidroacanthoma simplex. Thus, it is suggested that the constituent cells of these tumors originate from the outer cells of the intraepidermal and/or the upper portion of the intradermal eccrine ducts. There was no immunolabeling for EMA on the tumor cells of seborrheic keratosis and basal cell carcinoma. Immunohistochemical staining for EMA is a useful tool for the diagnosis of skin appendage tumors. PMID- 1720747 TI - Cortical granule matrix disassembly during exocytosis in sea urchin eggs. AB - Cortical granule exocytosis in sea urchins was studied using hyperosmotic and polymer-containing seawater to halt granule matrix dispersal. Addition of Na2SO4 containing seawater (2.5 osmole/kg) to Strongylocentrotus purpuratus eggs 10 to 40 sec after insemination resulted in arrest of the exocytic wave during propagation. EM examination of these eggs revealed that matrix disassembly occurred in distinct stages. In the earliest stage, granule-plasma membrane fusion had occurred, but the matrix remained completely intact. This early stage was observed in hyperosmotic media, either ionic or nonionic, suggesting that matrix hydration is required for disassembly and exocytic pore widening, but not for membrane fusion. Subsequent stages, in which partially disassembled matrices remained within omega-configured pockets, were captured by activating eggs in 30% dextran in seawater. Stability of these intermediates stages required the presence of Ca2+ and Mg2+; in the absence of divalent cations the matrices completely disassembled and the exocytic pockets flattened. Divalent cations appeared to prevent fragmentation of the matrix lamellae. Late stages of matrix disassembly, in which the lamellae fragmented and formed small particles, were inhibited by media of high ionic strength. Hyperosmolality alone, provided by sucrose, was unable to halt these late stages suggesting that water availability does not play an important role once a critical point in matrix dispersal has been reached. PMID- 1720748 TI - The state of the science. PMID- 1720749 TI - Causes and associations of severe and persistent specific speech and language disorders in children. AB - Eighty-two school-age children with severe and persistent specific speech and language disorders were studied. 71 had specific developmental language disorders, three had structural malformations (cleft palate) and eight had disorders acquired after a period of normal language development, including five with Landau-Kleffner syndrome. The sex ratio was 3.8 boys to one girl. Nearly half had a family history of speech-language disorder, with one in 5.2 affected siblings. Aetiological factors were found in 26 per cent: 11 per cent prenatal, 3 per cent perinatal and 12 per cent postnatal. 21 per cent had had a seizure and 7 per cent had had seizures after the age of eight. 29 per cent were left-handed, 90 per cent were clumsy and 22 per cent first walked after 18 months. The complex origins of specific speech and language disorders are discussed. PMID- 1720750 TI - Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris. AB - Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo. PMID- 1720751 TI - Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates. AB - Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast. PMID- 1720752 TI - [Rate of early abortion after in vitro fertilization and embryo transfer]. AB - The high rate of implantation failures in infertile patients after in vitro fertilization must be regarded as the major problem of the kind of treatment. Usually, no information on the development of the embryo can be obtained for the time between embryo replacement and rising beta-hCG levels. Own studies on the early pregnancy factor (EPF) showed a positive reaction few hours following the contact of a fertilized oocyte with the endometrial surface. Therefore, we used the EPF as a marker for the viability of the embryo in 82 patients after in vitro fertilization and embryo transfer. Within two days after embryo transfer the EPF was positive in 52 (63%) patients and negative in 30 (37%) patients. In these women the embryos may have been lost during handling or may have discontinued further development. Between day 3 and day 12 after transfer the EPF turned to negative values in 35 patients--especially between day 6 and 10. These cases must be regarded as true implantation failures. After day 12 following embryo transfer, rising beta-hCG levels could be measured in 17 women (21%), but only in 12 patients (15%) could a growing embryonic sac be detected by ultrasound. From these figures, we may conclude, that about half of the embryos are lost already during the step of embryo transfer and the other half during implantation. Therefore, more attention should be given to the handling of the embryos to increase the pregnancy rate after in vitro fertilization. PMID- 1720753 TI - Construction of plasmid vectors with a non-antibiotic selection system based on the Escherichia coli thyA+ gene: application to cholera vaccine development. AB - The construction of live oral carriers based on attenuated Salmonella strains as vectors offers a new approach to vaccine development. We have constructed a set of plasmid vectors which have the thyA gene of Escherichia coli (encoding thymidylate synthetase) as the marker for selection and maintenance of plasmid clones. The thyA system offers an alternative to antibiotic-resistance selection markers. It can be easily adapted to a particular host-vector combination since thyA chromosomal mutations can be readily introduced by trimethoprim selection. We also describe the construction of thyA-based plasmids with the Vibrio cholerae rfb genes (encoding O-antigen biosynthesis of the Inaba serotype). These have been found to be useful in the construction of candidate bivalent cholera-typhoid vaccines. PMID- 1720754 TI - Bleomycin resistance as a selectable marker for transformation of the eukaryote, Dictyostelium discoideum. AB - An expression cassette was constructed, which has the bacterial bleomycin (Bm) resistance-encoding gene (ble) fused to the Dictyostelium discoideum actin-6 promoter, with a segment of 3'-flanking DNA from the actin-8-encoding gene placed downstream from the ble gene to serve as a transcription terminator. Plasmid pMUW161, which contains this cassette and the D. discoideum plasmid Ddp2 origin of DNA replication, transformed D. discoideum with high efficiency under Bm selection. Hence, this construct is useful as a dominant selectable marker for D. discoideum. PMID- 1720755 TI - The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6. AB - Lysogens of Shigella flexneri harbouring the temperate bacteriophage, Sf6, have been previously shown to undergo a serotype conversion due to O-acetylation of the O-antigen of the lipopolysaccharide. A partial physical map of the phage genome has been constructed. Analysis of the phage DNA suggests that the phage packages by a headful mechanism and that the mature DNA molecules are terminally redundant. Cloning of the PstI fragments of Sf6 enabled the region encoding the serotype conversion to be localized, showing that this was clearly phage-encoded. The gene was further localized by mutagenesis with Tn5 and the nucleotide sequence of the entire 2693-bp PstI fragment was determined. Two major open reading frames (ORFs) were found capable of encoding proteins of 44.1 and 37.2 kDa. The latter corresponds to the O-antigen acetylase and its gene has been designated oac. The oac gene is capable of converting Sh. flexneri serotypes X, Y, 1a and 4a to 3a, 3b, 1b and 4b, respectively. The Oac protein bears a high degree of homology to the NodX protein of Rhizobium leguminosarum suggesting that it, too, may be a sugar acetylase. The second ORF immediately upstream from oac corresponds to the bacteriophage Sf6 integrase responsible for chromosomal integration and is highly homologous to the integrases of Escherichia coli bacteriophages P4 and phi 80, but less closely related to those of P1, P2, P22, 186 and lambda. PMID- 1720756 TI - Qualitative and quantitative platelet defect with bleeding symptoms as presenting feature of non Hodgkin lymphomas. AB - A young man with bleeding symptoms, mild thrombocytopenia and abundant marrow megakaryocytes was classified as having idiopathic thrombocytopenic purpura. Neither prednisone therapy nor splenectomy modified the clinical picture. Subsequently, a severe defect of platelet aggregation and release reaction was demonstrated. Fifteen months after the onset of bleeding symptoms, fever and hepatomegaly appeared and the diagnosis of T cell non Hodgkin lymphoma was made on the basis of a histologic review of paraffin sections of the spleen. Chemotherapy induced remission of the lymphoma, disappearance of bleeding symptoms and normalization of the platelet count and function. PMID- 1720758 TI - [Recent advances in the research on histamine release]. AB - An increase in inositol 1, 4, 5-trisphosphate (IP3) formation in rat mast cells precedes an elevation in intracellular Ca2+ levels, which triggers the process(es) leading to histamine release. By means of a transmission electron microscope, it was revealed that when permeabilized mast cells were exposed to potassium antimonate, antimonate precipitates in the endoplasmic reticulum (ER) in the form of calcium antimonate, indicating that the ER is the intracellular Ca store in rat mast cells. IP3 at concentrations higher than 0.5 microM preferentially releases Ca2+ from the isolated ER of mast cells. GTP was also effective in releasing Ca2+ from the ER. IP3-induced Ca2+ release was inhibited by pretreatments with cAMP and antiallergic drugs. An increase in the intracellular Ca2+ concentration may lead to an activation of calmodulin, C kinase and cytoskeletal elements in sequence. Furthermore, microtubules may play an important role in the process(es) leading to Ca2+ release from the intracellular Ca store and subsequent histamine release, without affecting IP3 formation. In contrast, microfilaments seem to participate not only in the extrusion but also in the reincorporation of the mast cell granules, having no influence on intracellular Ca2+ release. Substance P (SP) is one of the most effective neuropeptides for releasing histamine from mast cells. Structure activity relationship studies indicate that basicity at the N-terminal and hydrophobicity at the C-terminal are requisite for its histamine releasing activity. SP effectively released Ca2+ from the intracellular Ca store. The site of action of SP on the mast cell surface seems to be the same as that of compound 48/80. Eosinophil major basic protein (MBP) and histone are also effective for releasing histamine. The cDNA sequences of two subclasses of guinea pig MBP have been determined. These proteins may be released at the site of inflammation from the cells activated by the chemical mediators released from mast cells, and consequently, mast cell activation was reinforced. Such cell-to-cell interaction may be the reason for the augmentation of inflammation. PMID- 1720757 TI - [Coupling of muscarinic acetylcholine receptors, m1/m3 and m2/m4, to phosphoinositide metabolism and Ca2+ channels in DNA-transfected NG108-15 cells]. AB - The muscarinic acetylcholine receptor (mAChR) is an integral membrane protein that transduces stimulus to effectors through the activation of guanine nucleotide-binding (G) proteins. Four or more subtypes of mAChR were detected in various tissues, and their primary structures were elucidated by cloning and sequence analysis of complementary DNA. Functional differences between them existed when they were expressed in clonal culture cells. mAChRI (m1) and mAChRIII (m3) preferentially activated phosphoinositide (PI) hydrolysis and opened Ca(2+)-activated K+ channels followed by closure of the M (K+)-currents, while such current activities were rarely evoked by mAChRII (m2)- and mAChRIV (m4)-transformed cells. Although it has been reported that mAChRII and mAChRIV inhibited adenylate cyclase, there was little or no such inhibition by mAChRI and mAChRIII. It is known that heart and neuronal mAChR modulate voltage-sensitive Ca2+ currents, but which species of mAChR subtypes are involved has been poorly understood. Recently we identified that endogenous mAChRIV and exogenous mAChRII expressed in NG108-15 neuroblastoma-glioma hybrid cells, but not mAChRI and mAChRIII, efficiently depressed high-threshold Ca2+ currents in a pertussis toxin sensitive manner. PMID- 1720759 TI - Multicentre combined chemotherapy protocol for large cell advanced non Hodgkin's lymphoma. AB - Between October 1985 and October 1989, 75 previously untreated patients with stage III and IV non Hodgkin's lymphoma, large cell type, were treated with an alternating weekly chemotherapy regimen including the following drugs: week 1: Doxorubicin, vincristine, cyclophosphamide, bleomycin, and intrathecal (i.th.) methotrexate and cytarabine; week 2: Methotrexate with leucovorin rescue; week 3: Doxorubicin, ifosfamide with mesna, etoposide, and i.th. methotrexate and cytarabine; week 4: Methotrexate with leucovorin rescue. Complete responders after three cycles according to this schedule (12 weeks) were given 18 gys cranial irradiation and randomized between one additional cycle or three monthly CHOP (consolidation treatment). Among 66 evaluable patients, 53 achieved a complete remission (CR 80 per cent) and seven a partial remission (11 per cent). There were six failures, and nine early deaths during the initial phase, mostly due to septic problems. Forty-one of the 53 CR patients (77.3 per cent) have remained free of disease with a median follow-up of 15 months (1-49). Eight of the 12 relapses occurred during the first year, the four others at 13, 14, 16 and 38 months respectively. The 2-year survival was 63 per cent for the whole group, and 77 per cent for the CR group. No difference has been observed up until now between the two groups with different consolidation treatment. Therefore, this protocol seems to be able to produce a high rate of complete and durable remission. The analysis of prognostic factors suggests that some high-risk patients should be considered for intensification therapy with the support of autologous bone marrow transplantation. PMID- 1720760 TI - Etoposide, ifosfamide and methotrexate combination chemotherapy in patients with aggressive non-Hodgkin's lymphoma after failure of the LNH 84 regimen. AB - We assessed the efficacy of an etoposide, ifosfamide and methotrexate combination therapy (VIM) in 24 patients failing the LNH 84 protocol. Eight of these patients were refractory to the LNH 84 induction regimen, 10 were partial responders and the six remaining attained complete response after LNH 84 induction but relapsed during consolidation therapy or after completing the whole programme. Twenty three patients were evaluable for response. The VIM regimen provided a 43 per cent complete response rate and an additional 17 per cent partial response rate. The complete response rate was particularly high (67 per cent) in the group of patients who were partial responders to LNH 84 induction treatment. Of the 10 complete responders, five relapsed after 4 to 42 months and five are still alive with no evidence of disease after 27 to 60 months. Overall VIM was well tolerated. Myelotoxicity was the most common side-effect. Infections with fever were observed in 8 per cent of the VIM courses. This study demonstrates that a complete response and a long survival can be obtained in patients after failure of a high-dose doxorubicin containing front-line treatment. PMID- 1720761 TI - Functions and distribution of voltage-gated sodium and potassium channels in mammalian Schwann cells. AB - Recent patch-clamp studies on freshly isolated mammalian Schwann cells suggest that voltage-gated sodium and potassium channels, first demonstrated in cells under culture conditions, are present in vivo. The expression of these channels, at least at the cell body region, appears to be dependent on the myelinogenic and proliferative states of the Schwann cell. Specifically, myelin elaboration is accompanied by a down regulation of functional potassium channel density at the cell body. One possibility to account for this is a progressive regionalization of ion channels on a Schwann cell during myelin formation. In adult myelinating Schwann cells, voltage-gated potassium channels appear to be localized at the paranodal region. Theoretical calculations have been made of activity-dependent potassium accumulations in various compartments of a mature myelinated nerve fibre; the largest potassium accumulation occurs not at the nodal gap but rather at the adjacent 2-4 microns length of periaxonal space at the paranodal junction. Schwann cell potassium channels at the paranode may contribute to ionic regulation during nerve activities. PMID- 1720762 TI - Phorbol-12-myristate-13-acetate (PMA) and inhibitors of protein kinase C alter glial fibrillary acidic protein (GFAP) mRNA levels. AB - Glial fibrillary acidic protein (GFAP) mRNA levels in the human astrocytoma line U-373MG were examined to explore further the effects of agents that regulate protein kinase C. U-373MG cells exhibit a biphasic change in steady-state GFAP mRNA in the presence of the phorbol ester phorbol-12-myristate-13-acetate (PMA). Short-term treatment with PMA results in increased GFAP mRNA, and long-term treatment results in decreased GFAP mRNA. Nuclear run-off experiments demonstrate that the PMA-induced decrease in GFAP mRNA levels is not at the level of GFAP gene transcription. PMA exerts its effect in the presence of protein synthesis inhibitors, demonstrating that de novo protein synthesis is not required for the PMA-induced changes in GFAP mRNA. Staurosporine, a protein kinase C inhibitor, reduces GFAP mRNA expression in a dose-dependent manner; in the presence of PMA the effect is additive. By contrast HA1004, an inhibitor of cAMP-dependent protein kinase, is not inhibitory to GFAP steady-state mRNA. Total protein kinase C activity was determined to be 2,398.8 +/- 94.3 pmol/min/mg protein, with most of the activity in the cytosol. Short-term PMA treatment results in the translocation of the cytosolic protein kinase C activity to the membrane. Long term PMA treatment results in a decrease in total protein kinase C activity indicating that downregulation occurs. These studies demonstrate that in the U 373MG cells, protein kinase C inhibitors and long treatment with PMA result in a decrease in steady-state GFAP mRNA. PMID- 1720763 TI - Tracing transplanted oligodendrocytes during migration and maturation in the shiverer mouse brain. AB - Fragments of neural tissue from normal newborn mouse were stained with Hoechst 33342 dye before transplantation into the newborn shiverer mouse brain. Combination of this technique with immunohistochemistry demonstrated that, after transplantation, these cells are able to survive as long as unstained cells and to myelinate in the shiverer mouse host brain. Stained cells express the normal sequence of differentiation in terms of chronology of differentiation marker expression [04, galactocerebroside (GalC), myelin basic protein (MBP)], as normal cell do in situ. It has thus been possible by this technique to show the migration pathways of transplanted cells and to correlate them with the expression of specific markers: long distance migration along white matter axonal pathways occurs when cells are o4-positive, GalC-negative. By contrast, only GalC positive cells are able to migrate across the grey matter in the absence of radial glia. Finally, it has been possible to propose a migration and differentiation sequence of these cells, suggesting that MBP-positive oligodendrocytes divide after migration in the target zone. PMID- 1720764 TI - Glial immunoreactivity for metallothionein in the rat brain. AB - A series of frozen and vibratome coronal sections of the rat brain were examined by immunocytochemistry for the presence of a cysteine-rich metal binding protein, metallothionein (MT). Astrocytes throughout the brain and brainstem stained positively for MT; neurons and oligodendroglia were unstained. Ependymal cells and tanycyte processes in the hypothalamus were also immunoreactive, along with a narrow zone of immunopositivity along the margins of the area postrema. Gomori positive astrocytes in the hypothalamus, identifiable by toluidine blue staining, metal-containing cytoplasmic granules, represented a subset of MT-positive astrocytes that may be involved in reactions to blood-borne metal compounds that penetrate into circumventricular organs of the brain. PMID- 1720765 TI - Activation of blood coagulation and fibrinolysis in vibration syndrome. AB - The pathophysiology of peripheral circulatory disturbance in patients presenting with vibration syndrome was studied from the viewpoint of blood coagulation. Plasma levels of fibronectin (FN), vitronectin (VN), thrombin-antithrombin III complex (TAT), and alpha 2-plasmin inhibitor-plasmin complex (PIC) were measured in 23 subjects who showed no evidence of vibration-induced white finger [VWF(-) group] and in 24 patients who presented with VWF [VWF(+) group]. In the VWF(-) group, plasma FN concentrations were elevated but plasma TAT and PIC levels were within the normal ranges. In the VWF(+) group, plasma FN concentrations were normal but plasma TAT and PIC levels were significantly elevated. In both groups, plasma VN concentrations were similar to those in normal controls. For purposes of comparison, 32 patients presenting with diabetes mellitus were also studied. They were divided into 2 groups, 13 subjects who showed no evidence of angiopathy [complication(-) group] and 19 patients who presented with angiopathy [complication(+) group]. In the complication(+) group, plasma TAT and PIC concentrations were significantly elevated, as in the VWF(+) group. These results suggest that in vibration syndrome, vibration, cold stimulus, or other factors first injure the vascular endothelium, resulting in a rise in plasma FN, and that in the VWF(+) group, augmentation of coagulation and fibrinolysis induces a state of compensated disseminated intravascular coagulation (DIC). PMID- 1720766 TI - Immunohistologic analysis of the cholesteatoma matrix in children. AB - The immunohistological characteristics of retraction pockets, cholesteatoma matrix and granulomatous tissue were compared in 14 samples from pediatric cholesteatoma. The junction between epidermis and the middle ear mucosa appeared as the most inflammatory area, displaying the characteristics of delayed type hypersensitivity. CD1 + Langerhans cells were observed in all epidermic areas, but expressed class II molecules only in the vicinity of polymorphonuclear infiltrates. Numerous mast cells and IgA producing cells were also observed, suggesting that defenses from the mucosal immune system are summoned and contribute to the pathogenesis of cholesteatoma. PMID- 1720767 TI - Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue. AB - An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a 'direct' fluorescence procedure using FTC-toxin, but not by an 'indirect' procedure using sequential reaction with toxin, anti-toxin and FTC secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the 'direct' and 'indirect' procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the 'indirect' procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a 'direct' procedure using FTC-antiprofilaggrin but only weakly by an 'indirect' double antibody procedure. Insensitivity to 'indirect' procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells. PMID- 1720768 TI - Improved fixation of frozen lympho-haemopoietic tissue sections with hexazotized pararosaniline. AB - In this study, a simple single-step fixation method for frozen tissue sections is introduced using the hexazotized salt of Pararosaniline as preservative agent. Tissue preservation by this method was shown to be superior to the commonly-used fixation with acetone. Fixation with hexazotized Pararosaniline caused a minimal loss of antigenicity as demonstrated using twenty-three monoclonal antibodies directed against lympho-haemopoietic and stromal cells. PMID- 1720769 TI - Digoxigenin as a probe label for in situ hybridization on skeletal tissues. AB - Non-specific staining was encountered using digoxigenin-labelled cDNA probes for in situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described. PMID- 1720770 TI - Multidrug resistance activity in human lymphocytes. AB - The multidrug resistance gene (mdr1) is a member of the recently described ATP binding cassette (ABC) superfamily of transporters. Family members include: (1) the cystic fibrosis transmembrane conductance regulator gene; (2) the hlyB gene of bacteria, and (3) the histocompatibility antigen modifier (HAM) gene. The level of expression of mdr1 correlates with multidrug resistance (MDR), the ability of cells to efflux otherwise toxic doses of several chemotherapeutic agents. MDR activity is also associated with the efflux of cationic lipophilic compounds such as the fluorescent dye rhodamine 123. Recently it was reported that normal lymphocytes efflux rhodamine 123, suggesting that these cells possess MDR-like activity due to the expression of mdr1. In this study, using two-color flow cytometric analysis, we observed that the ability to efflux rhodamine 123 was heterogeneous among human lymphocyte subsets in the order of CD8 greater than CD4 greater than CD2O. Rhodamine 123 efflux and accumulation in lymphocytes was sensitive to the known MDR reversing agents, verapamil and Solutol HS 15. Collectively, these data suggest that an MDR-like transport system is present in normal lymphocytes and may be important for trafficking of molecules involved in lymphocyte function. PMID- 1720771 TI - Ribosome hopping and translational frameshifting are inadequate alternatives to translational attenuation in cat-86 regulation. AB - The induction of cat-86 by chloramphenicol has been proposed to follow the translational attenuation model. In the absence of inducer, the cat-86 gene is transcribed but remains phenotypically unexpressed because the transcripts sequester the ribosome binding site for the cat coding sequence in a stable stem loop structure, preventing translation initiation. The translational attenuation model proposes that the natural inducer, chloramphenicol, stalls a ribosome in the leader region of cat transcripts, which causes localized melting of the downstream stem-loop structure, allowing initiation of translation of the cat-86 coding sequence. Although it is established that ribosome stalling in the cat-86 leader can induce translation of the coding sequence, several subsequent steps predicted by the model remain to be experimentally confirmed. As a consequence, the present evidence for cat-86 regulation can also be explained by two other potential control devices, ribosome hopping and translational frameshifting. Here we describe experiments designed to determine whether the alternatives to translational attenuation regulate cat-86. The results obtained are inconsistent with both competing models and are consistent with predictions made by the translational attenuation model. PMID- 1720772 TI - Regulation and function of the Streptomyces plasmid pSN22 genes involved in pock formation and inviability. AB - pSN22 is an 11-kb multicopy plasmid from Streptomyces nigrifaciens which is being studied in Streptomyces lividans. A segment of about 7 kb of pSN22 contains five genes involved in conjugation. Three of them, traA, traB, and traR, are essential for plasmid transfer and for the mobilization of chromosomal markers (fertility), while the remaining two genes, spdA and spdB, merely enhance the efficiency of plasmid transfer, resulting in the formation of larger pocks. In vitro promoter probing experiments identified a 550-bp BglII-SmaI DNA fragment with promoter activity in both orientations; Northern (RNA blot) hybridization identified corresponding divergent transcripts of 1 and 5.2 kb for traR and the traA-traB spdB operon, respectively. The traR gene product repressed its own transcription and also the transcription of the traA-traB-spdB operon. Plasmids containing a functional traB gene could not "survive" without traR being present in the same cell either in cis or in trans, presumably because unregulated expression of traB is lethal to the host. Plasmids with a functional traA gene but without traR had a low transformation efficiency and inhibited the growth of host cells. PMID- 1720773 TI - Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. AB - The synthesis of nitric oxide (.NO) from L-arginine has been demonstrated in a number of cell types and functions either as a cell signaling agent or as a key component of the cell-mediated immune response. Both constitutive and inducible activities have been described. Herein we report the purification of inducible .NO synthase (EC 1.14.23) from activated murine macrophages using a two-column procedure. Crude 100,000 x g supernatant was passed through a 2'-5'-ADP-Sepharose 4B affinity column followed by a DEAE-Bio-Gel A anion exchange column. The .NO synthase ran as a band of Mr = 130,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration experiments using a Superose 6 HR 10/30 column estimated the native molecular weight to be 260 +/- 30 kDa, indicating that the native enzyme exists as a dimer. Activity was dependent upon L-arginine (Km = 16 +/- 1 microM at 37 degrees C and pH 7.5) and NADPH. Both (6R)-tetrahydro L-biopterin and FAD enhanced activity, whereas Mg2+ and FMN had no effect on activity. Fluorescence studies demonstrated the presence of one bound FAD and one bound FMN per subunit. PMID- 1720774 TI - Multiple cis-acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P. AB - In humans, the H1 RNA, the RNA subunit of RNase P, is synthesized by RNA polymerase III. We have used block replacement mutagenesis to identify the sequences necessary for in vitro transcription of H1 RNA. We find that multiple cis-acting elements located in the H1 RNA 5'-flanking region are necessary for H1 RNA synthesis; no internal sequences are essential. Required cis-acting elements include sequences resembling proximal sequence element, distal sequence element, and TATA motifs. In this respect, the H1 RNA promoter is similar in structure to the promoters of the genes encoding the U6 snRNA, the 7 SK RNA and the MRP RNA. However, our mutational analysis indicates that the H1 promoter is unexpectedly complex, with several additional cis-acting elements spanning nearly 70 base pairs of the H1 RNA gene 5'-flanking sequence. PMID- 1720775 TI - Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1. AB - Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed. PMID- 1720776 TI - Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase. AB - Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits. By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose. Homologous RT subunits rapidly associated to form the respective heterodimers (1 p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed. Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits. In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme. RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66 kDa subunit. PMID- 1720777 TI - Epitope mapping of four monoclonal antibodies recognizing the hexose core domain of Salmonella lipopolysaccharide. AB - Four murine monoclonal antibodies reactive with distinctive regions of the hexose core domain of Salmonella lipopolysaccharide (LPS) were generated and their epitope specificities were delineated. MAST 56 (IgG1) and MAST 50 (IgG3) antibodies elicited by immunizations with Salmonella typhimurium Rb1 and Rb2 mutants, reacted selectively in enzyme immunoassay with the LPS from rough mutants. In contrast, MATy 1 (IgM) and MATy 2 (IgG2b) antibodies raised by an attenuated Salmonella typhi 620 Ty strain were reactive with LPS from both smooth and rough Salmonellae. Immunoblotting analysis showed that MATy 1 distinguished only the bottom bands (naked LPS core) among the heterogeneous LPS populations, whereas MATy 2 gave a ladder pattern (reactive with both naked and O-chain substituted LPS cores). Differential binding specificities of MATy 1 and MATy 2 antibodies to the naked and capped LPS cores were further analyzed utilizing S. typhimurium polysaccharide fractions with different O-chain:core ratios which were obtained after separation by Sephacryl S-200 chromatography. Steric effects on the antibody reactivity by the bulky O-polysaccharide chain were detected. The use of chemically defined native and synthetic saccharides as inhibitors, in combination with the conformation of the Salmonella core oligosaccharide, permitted the definition of antigenic determinants carried in the core domain recognized by each antibody: (i) the branches I and VIII are essential for MATy 1 recognition, (ii) the backbone III-IV-V for MATy 2, (iii) the backbone II-III-IV V for MAST 56, and (iv) the backbone plus the branch III-IV-V-VIII for MAST 50. (formula; see text) PMID- 1720778 TI - Molecular basis of thyroid hormone regulation of myelin basic protein gene expression in rodent brain. AB - Regulation of myelin basic protein (MBP) gene expression by thyroid hormone has been investigated in rodent brain. Quantitation of the 4 major alternatively spliced transcripts by RNase protection assay showed that the individual mRNAs, corresponding to MBP isoforms 21.5, 18.5, 17, and 14 kDa, were decreased from 2- to 17-fold at all ages studied (4-60 days) in hypothyroid animals when compared to euthyroid, but the timing of onset of expression was not altered. MBP mRNA was also reduced in young adult rats thyroidectomized at the age of 5-6 weeks and was restored to normal by thyroxine administration. Nuclear run-off assays showed that the rate of MBP gene transcription is dependent on thyroid state. Co transfection of MBP (-256/+1)-chloramphenicol acetyltransferase chimeric gene with a plasmid expressing thyroid hormone receptor alpha, and in the presence of 3,5,3'-triiodothyronine, into NIH3T3 or NG108-15, increased chloramphenicol acetyltransferase expression 4-fold. Using a footprinting technique and Spodoptera frugiperda 9 (Sf9) nuclear extract infected with baculovirus expressing TR alpha, we have identified a single DNA-binding site (-186/-163) for the receptor. A part of this region contains the AGGACA sequence found in thyroid hormone-responsive elements of other 3,5,3'-triiodothyronine-regulated genes. Our finding of a specific hormone-receptor interaction with the MBP promoter region is the first direct demonstration of a thyroid hormone-responsive element in a brain-specific gene. PMID- 1720779 TI - Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin. AB - The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin. PMID- 1720780 TI - Characterization of Escherichia coli SecA protein binding to a site on its mRNA involved in autoregulation. AB - In order to understand further the autogenous regulation of Escherichia coli secA translation, we have set up a purified system to study the binding of SecA protein to portions of its mRNA. Specific SecA protein-RNA binding was demonstrated by UV cross-linking, filter binding, and gel shift assays. Use of the filter binding assay allowed optimization of binding, which was influenced by Mg2+ and ATP concentrations, and a measurement of the affinity of this interaction. A nested series of RNAs lacking either 5' or 3' portions of geneX secA sequences were used to localize the SecA protein binding site to sequences around the geneX-secA intergenic region. These studies imply that SecA protein directly regulates its own translation by a specific RNA binding activity that presumably blocks translational initiation. PMID- 1720781 TI - Structure of the polyglutamyl side chain posttranslationally added to alpha tubulin. AB - Polyglutamylation, a new posttranslational modification of tubulin identified originally on the acidic alpha variants by Edde et al. (Edde, B., Rossier, J., Le Caer, J. P., Desbruyeres, E., Gros, F., and Denoulet, P. (1990) Science 247, 83 85), consists of the successive addition of glutamyl units to the Glu445. To characterize their linkage mode mouse tubulin was posttranslationally labeled with [3H]glutamate. After digestion of [3H]tubulin with thermolysin, up to eight radioactive peaks were separated on an anion exchange column (DEAE). Combined use of Edman degradation sequencing and mass spectrometry analysis of the first 6 one indicated that they all correspond to the same COOH-terminal sequence 440VEGEGEEEGEE450 bearing one to six glutamyl units on the Glu445. The first glutamyl residue is amide-linked to the gamma-carboxyl group of Glu445, but the additional residues can be linked to the gamma- or alpha-carboxyl groups of the preceding one. All possible linkages for the biglutamylated tubulin peptides (gamma 1 alpha 2, gamma 1 gamma 2) and triglutamylated (gamma 1 alpha 2 alpha 3, gamma 1 alpha 2 gamma 3, gamma 1 alpha 2 gamma 2, gamma 1 gamma 2 alpha 3, gamma 1 gamma 2 gamma 3) were synthesized. These different peptides were successfully separated on a C18 5-micron reverse phase column. We found that the bi- and triglutamylated tubulin peptides behave as the gamma 1 alpha 2 and gamma 1 alpha 2 alpha 3 synthetic peptides, respectively. These results indicate that the second and third glutamyl residues of the polyglutamyl side chain are amide linked to the alpha-carboxyl group of the preceding unit. PMID- 1720782 TI - Rapid detection of proliferating potential in human brain tumors by nucleolar organizer region staining on squash preparations. AB - Rapid detection of the proliferating potential of 37 human brain tumors was attempted using squash preparations stained by a silver colloid technique for argyrophilic protein associated with nucleolar organizer regions (AgNORs). Less than 1 h was required for staining. The mean number of AgNORs in cell nuclei of malignant or recurrent brain tumors (16 cases) including meningeal sarcoma, recurrent meningioma, recurrent craniopharyngioma, anaplastic astrocytoma, glioblastoma multiforme and metastatic brain tumor was 3.18, and the number for benign brain tumors (21 cases) including meningioma, neurinoma, pituitary adenoma, benign astrocytoma, ependymoma, and adenoma of lachrymal gland was 1.85. The former value was significantly greater than the latter value (P less than 0.001). These results indicate that quantitative analysis of AgNORs in brain neoplastic cells, using squash preparations, is useful to differentiate malignant from benign tumors within 1 h. Thus, this method provides rapid and useful information about the proliferative potential of human brain tumors even during operation. PMID- 1720783 TI - Development of serum-free media for the growth of human gastrointestinal adenocarcinoma xenografts as primary tissue cultures. AB - The growth-promoting effect of several hormones and growth factors on eight human colon tumor cell lines (SW 48, SW 403, SW 480, SW 620, SW 948, HT29, LS174T and Caco-2) was studied using seven different chemically defined serum-free media [GF3: Chee's essential medium plus insulin, transferrin and selenium; GF3F: GF3 plus fetuin; GF4: GF3 plus linoleic acid/bovine-serum albumin (BSA); GF5: GF4 plus fetuin, GF5E, GF5 plus EGF; GF5T: GF5 plus triiodothyronine; GF7: GF3 plus EGF, transferrin, insulin, linoleic acid/BSA, oleic acid/BSA and fetuin]. GF5 appears to be the best serum-free medium as it supported continuous growth of all of the colon tumor cell lines. GF5 also supported growth of five of the seven human colon and stomach tumor xenografts as primary tissue cultures. However, the stomach xenograft cells had a very slow growth rate as compared to the colon xenograft cells in the medium. Cells grown in GF5 retained their tumorigenicity in athymic (nude) mice and characteristic cellular morphology. GF7 was the poorest of all of the serum-free media studied as none of the cell lines or xenografts grew in this medium. PMID- 1720784 TI - Inhibition of O6-alkylguanine-DNA alkyltransferase and DNase I activities in vitro by some alkylating substances and antineoplastic agents. AB - The specificities of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase from brain and liver cells of the chick embryo and of DNase I were demonstrated in vitro by their response to substrate DNA pretreated with monofunctional alkylating agents of different O6-guanine alkylating ability and some antineoplastic agents. Treatment of DNA with ethidium bromide, Hoechst 33258, doxorubicin, Fe2+/bleomycin, and suramin resulted in a dose-dependent diminution of alkyltransferase activity (DE50 approximately 5 micrograms/ml, 15 micrograms/ml, 5 micrograms/ml, 5 micrograms/ml, 100 micrograms/ml, respectively). Apart from bleomycin, comparable results were obtained with DNase I. Thermal denaturation of the substrate DNA reduced both alkyltransferase and DNase I activity. No effect was seen with X-irradiation. Cisplatin decreased only DNase I activity. Some topoisomerase II and/or gyrase inhibitors remained without significant effects on the alkyltransferase reaction whereas DNA catabolism by DNase I was diminished in a dose-dependent manner (DE50 between 6.5 and 19 micrograms/ml). PMID- 1720785 TI - Oestrogen-regulated 24-kDa protein--a marker for breast carcinoma in benign breast tissue. AB - A monoclonal antibody (mAb) raised to an oestrogen-induced protein (24-kDa protein) has been suggested as a marker of women at risk of developing breast cancer. Patients with benign breast biopsies who have not developed breast cancer (benign control groups) were compared to women who had malignant breast biopsies. The benign tissue components from each group were studied. Subsequently, women with benign breast biopsies who have not developed malignancy were compared to women with benign breast biopsies who later went on to develop breast cancer. The study was carried out on formalin-fixed paraffin-embedded tissue sections. Comparison between tissue from the benign control groups and from women with breast cancer demonstrated the cytoplasmic staining in apocrine metaplasia of benign controls to be more pronounced. No staining differences were apparent between benign biopsies of patients who have not developed breast cancer and benign biopsies of those who have. We conclude that diminished expression in apocrine metaplasia of mAb to 24-kDa protein may indicate the presence of breast cancer but we have been unable to establish the role of 24-kDa protein as a marker for cancer risk. PMID- 1720786 TI - 5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts. AB - IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5 methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5 azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process. PMID- 1720787 TI - An IGF binding protein is an inhibitor of FGF stimulation. AB - We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation. PMID- 1720789 TI - Thyroid disease in the elderly. AB - Overtreatment of hypothyroidism is particularly hazardous in older patients, who are often maintained on less than half the amount of replacement hormone required by younger patients. With the current sensitive assays for TSH, the goal is to reduce TSH to the normal but not to the suppressed range. For hyperthyroidism, prompt treatment with radioactive iodine is indicated. PMID- 1720788 TI - Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts. AB - We examined the induction, by heat shock, of heat shock transcription factor (HSTF) DNA-binding and hsp 70 gene promoter activities during aging of the IMR-90 human diploid fibroblasts. Cells with population doubling level (PDL) ranging from 15-48 were heat shocked at temperatures of 39, 42, and 45 degrees C for various time periods; the binding of HSTF to its consensus DNA was determined by gel retardation assay and the promoter activity of the human hsp 70 gene was analyzed by transient expression of reporter gene activity. We observed that the induction of HSE-binding activity was inversely related to the PDL of the cells used. Importantly, as cells progress through their life span, a higher temperature and a longer period of heat shock were needed to evoke an optimal increase in HSE-binding activity. A substantial and rapid (within 30 min) increase in HSE-binding activity was observed when PDL 20 cells were heat shocked at 39, 42, or 45 degrees C. However, PDL 35 cells did not respond to 39 degrees C, and PDL 48 cells responded slowly to heat shock at 45 degrees C, but not 39 or 42 degrees C. Experiments on the heat induced increase in hsp 70 promoter driven reporter gene expression provided similar information on the age-dependent decrease in transcriptional activation of hsps. These results were further corroborated by quantitation of the abundance of mRNA of hsp 70. Analysis of the cAMP induced expression of the rat somatostatin promoter driven CAT gene provided evidence that the decrease in transcriptional activation of hsps in aging diploid cells was not a reflection of a generalized dysfunction of signal transduction. We conclude that functional changes in the heat shock response occur before cells lose their capacity to replicate, and we suggest that these changes are likely to have a central role in the expression of the aging phenotype. PMID- 1720790 TI - Dye-ligand affinity partitioning of lactate dehydrogenase isoenzymes. AB - Aqueous two-phase systems consisting of dextran and polyethylene glycol (PEG) were used to study the partition behaviour of isoenzymes of lactate dehydrogenase (LDH; E.C. 1.1.1.27) from rabbit tissues in the presence and absence of a series of triazine dyes covalently coupled to PEG. The variations in the primary structures of LDH1(H4) and LDH5(M4) are reflected by significantly different partition coefficients. A class of dyes exhibiting defined structural elements is able to distinguish between both of these isoenzymes. This may be based on differences in the binding affinity to the catalytic site of the enzyme. The difference in the relative affinities of LDH1 and LDH5 to Procion Blue H-5R, as estimated by affinity partitioning, were corroborated by chromatographic experiments. Affinity partitioning in aqueous two-phase systems can be used to predict and to optimize conditions for the fast and simple chromatographic separation of isoenzymes. PMID- 1720791 TI - The health situation in the Americas. AB - This paper considers the elements that determine the health situation in any country: the specific hazards to health, the health services, and the general determinants which influence both the hazards and the services. The expectation of life--the best single measure of the health situation--is presented for each country of the Americas as representing the resultant of these determinants. The changing pattern of disease in the Americas--the emergence of noninfectious diseases as the major problems--is documented, and the need for epidemiologically oriented health planning is stressed. Data are presented to indicate that medical care is the least significant of the basic triad of public health, and that primary attention must be paid to both disease prevention and living standards. PMID- 1720792 TI - Acoustic chiasm. IV: Eight midbrain decussations of the auditory system in the cat. AB - Conventional retrograde and orthograde axonal transport tract-tracing techniques were used in cats to explore the auditory decussations and commissures in the upper pons and midbrain. In all, 8 decussations differing either in origin or in contralateral termination were found. Three of the 8 decussations (from the dorsal nucleus of the lateral lemniscus to the contralateral dorsal nucleus of the lateral lemniscus, from the dorsal nucleus of the lateral lemniscus to the contralateral inferior colliculus, from the sagulum to the contralateral sagulum) reach their targets via the commissure of Probst. The remaining 5 decussations (from the inferior colliculus to the contralateral inferior colliculus or medial geniculate, from the intermediate nucleus of the lateral lemniscus to the contralateral medial geniculate, from the sagulum to the contralateral inferior colliculus or medial geniculate) reach their targets via the commissure of the inferior colliculus. The results also suggest that the commissure of Probst is not a general avenue for decussating auditory fibers of the lateral lemniscus but is instead a specific avenue only for fibers from the dorsal nucleus of the lateral lemniscus and sagulum. The results also show that, in the cat at least, the dorsal nucleus of the lateral lemniscus does not project beyond the inferior colliculus to either the superior colliculus or medial geniculate--the cells previously reported as doing so are probably those of the immediate neighbors of the dorsal nucleus, the intermediate nucleus of the lateral lemniscus and sagulum. PMID- 1720793 TI - Neurosecretory cells in the honeybee brain and suboesophageal ganglion show FMRFamide-like immunoreactivity. AB - Immunocytochemical analysis of the brain and suboesophageal ganglion of the honeybee Apis mellifera L. was combined with Lucifer Yellow backfilling from the corpora cardiaca and intracellular staining of single neurons. It is shown that more than one third of the cells that display FMRFamide-like immunoreactivity (F LI) project to the corpora cardiaca, suggesting they are neurosecretory. Among the ca. 120 median neurosecretory cells (MNCs) in the pars intercerebralis about 32 show F-LI. The number of immunoreactive MNCs is highly variable and may depend on age and/or diet. Seven of at least 40 lateral neurosecretory cells display F LI. They project through the brain via the medial branch of the bipartite nervus corporis cardiaci II. In the suboesophageal ganglion three types of immunoreactive neurosecretory cells were identified. Together with the median and the lateral neurosecretory cells in the brain these cells project through a single pair of nerves into the corpora cardiaca suggesting that the nervus corporis cardiaci (NCC) of the honeybee is a fusion of NCC I, II, and III described in other insects. PMID- 1720794 TI - Galanin immunoreactivity in the blowfly nervous system: localization and chromatographic analysis. AB - In this study chromatographic, immunochemical, and immunocytochemical methods provide evidence of a galanin-like peptide(s) in an invertebrate, the blowfly Phormia terraenovae. The major portion of the galanin-like immunoreactivity (GAL LI) in fly heads was extractable in acetic acid but not in boiling water, which suggests that the peptide(s) may be highly basic in nature. GAL-LI was present both in the head and body portion of the blowfly in roughly the same amounts. Initial gel filtration data, using a G-50 Sephadex column and a weak phosphate buffer (pH 6.5) as eluent, suggested that a fly GAL-LI peptide(s) from fly heads, eluting as an apparent single peak, was smaller than porcine GAL(1-29) and GAL(1 15). However, concomitant analysis using a G-25 Sephadex column and acetic acid (0.2 M) as eluent, spread the immunoreactive material over a great portion of the chromatogram, although the main portion of the material eluted in the same size range as porcine GAL(1-29). Taken together, the gel filtration data thus suggest that fly GAL-LI peptide(s) may be highly basic but presumably similar in size to vertebrate GAL(1-29). However, the hydrophobic properties of the fly GAL-LI peptide(s) differ from that of porcine GAL as demonstrated by the presence of several immunoreactive components eluting both early as well as late in the chromatogram when using reverse-phase high performance liquid chromatography (HPLC); early peaks may represent highly basic and/or possibly smaller GAL immunoreactive peptide(s), whereas later peaks may represent less basic and possibly elongated forms. Immunocytochemistry indicated that GAL-LI was present in the nervous system of the blowfly. About 160 GAL-immunoreactive neurons were found in the brain and subesophageal ganglion, 26 in the fused thoracic ganglion and 30 in the fused abdominal ganglion. In the brain, GAL-immunoreactive fibers supply specific subdivisions of the central body, optic lobe, superior protocerebrum, and tritocerebrum as well as neuropil in the subesophageal ganglia. In the thoracico-abdominal ganglia, GAL-immunoreactive neuron processes are found inside synaptic neuropil as well as in the neural sheath of the ganglia and several of the dorsal nerve roots. Many of the GAL-immunoreactive neurons react also with an antiserum against porcine galanin message associated peptide, a peptide present in the preprogalanin protein. Immunocytochemical double labeling indicated that some GAL-immunoreactive neurons also reacted with antisera against the molluscan peptides FMRFamide and SCPB, whereas no evidence could be found for colabeling with antisera against tyrosine hydroxylase, substance P and physalaemin.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1720795 TI - Tryptase and histamine release during aspirin-induced respiratory reactions. AB - The involvement of mast cells in the pathogenesis of aspirin (ASA)-induced respiratory reactions was investigated by measuring serum levels of tryptase, a neutral protease that is a specific marker of mast cell activation. ASA challenges were performed in 17 ASA-sensitive patients with asthma and rhinosinusitis, and tryptase and histamine levels were measured in their venous blood samples. In three subjects who experienced moderate to severe respiratory reactions extending to the skin and/or gastrointestinal tract, marked elevations of tryptase levels in postreaction serum samples (peak levels, 51.9 and 40.0 ng/ml) were discovered in two of these three subjects, and a small elevation of tryptase occurred in the serum of the third subject (3.1 ng/ml peak). Plasma histamine levels in postreaction samples were significantly elevated over baseline values in all three subjects (delta mean plasma histamine, 238 pg/ml versus 56 pg/ml for the remaining 14 subjects; p less than 0.04). In the remaining 14 subjects, who experienced similar respiratory reactions without extrapulmonary symptoms during aspirin challenge, changes in tryptase and histamine levels were not observed. PMID- 1720796 TI - Desferal (desferrioxamine)--a novel activator of connective tissue-type mast cells. AB - The effect of Desferal (desferrioxamine), an iron-chelating agent with allergic side effects, was examined on human basophils and rodent mast cells (MCs) in vitro and in the human skin. Even at a high concentration (100 mg/ml), the drug neither induced histamine release (HR) from human basophils nor primed these cells to release higher amounts of histamine when they were activated with f-met peptide or anti-IgE antibodies. In contrast, in all seven subjects studied, intradermal injection of Desferal (0.1 mg/ml to 100 mg/ml) elicited classic wheal and-flare responses. Ingestion of 10 mg of cetirizine, an H1 antagonist, 3 hours before the intracutaneous administration of Desferal, significantly reduced the diameters of both wheal-and-flare reactions, indicating that the drug caused local HR. Desferal also induced HR from rat peritoneal MCs in vitro but had no effect on mouse bone marrow-derived MCs. These results suggest that Desferal has a direct, IgE-independent, stimulatory effect on connective tissue-type MCs. Thus, it may be used as a positive control in skin testing. PMID- 1720797 TI - The ovarian innervation in the dog: a preliminary study for the base for electro acupuncture. AB - The origin of the canine ovarian sensory and sympathetic nerves was studied by applying horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) to the ovarian stroma and into the ovarian bursa. HRP/WGA-HRP positive neurons were found bilaterally in the dorsal root ganglia of T10 to L4 segment with the majority located in T13 to L2. In sympathetic paravertebral ganglia, labeled neurons were distributed bilaterally in ganglia from T11 to L4 with the majorities located in segments T13 to L2. Both distributions show ipsilateral predominance. Labeled prevertebral neurons were mainly located in the aorticorenal ganglion, ovarian ganglia and caudal mesenteric ganglion. No labeled neurons were found in the dorsal motor nucleus of vagus, nodose ganglia or sacral segment from S1 to S3. This study provides the possible morphological basis of electro-acupuncture concerning the somato-visceral reflex of the ovary. PMID- 1720798 TI - The distribution of myelinated nerve fibers in the mature opossum esophagus. AB - Myelination of nerve fibers could be important in establishing normal esophageal peristalsis. We therefore examined the general distribution and the age of appearance of myelinated nerve fibers in the smooth-muscle part of the esophagus of the American opossum. Tissues stained with thionein and Sudan black B were examined by light microscopy. Other tissues were prepared for electron microscopy and examined in the light microscope in Toluidine blue stained sections as well as at electron microscopy. In mature animals (weight greater than 2.0 kg, age greater than or equal to 1 year), myelinated nerve fibers, oriented mainly craniocaudally, were most abundant at the striated muscle-smooth muscle junction, and declined in density distally along the organ. They were nearly absent at the esophagogastric junction. They were more abundant in the stomach just below the esophagogastric junction. The myelinated nerve fibers commonly lay within sheathed fascicles that had the appearance of peripheral nerves, like the shunt fascicles of the stomach and colon. In immature animals myelinated fibers did not appear until a weight of about 1 kg was reached, 50 days after weaning and about 150 days after birth. Since the younger animals are presumably swallowing normally, myelination of the extrinsic nerves is not essential for esophageal motor function. PMID- 1720799 TI - Insulin-like growth factor II (IGF-II) mRNA expression during hepatocarcinogenesis in transgenic mice. AB - Insulin-like growth factor II (IGF-II) mRNA expression is developmentally regulated in liver tissue. We previously observed the reexpression of fetal IGF II mRNAs in human primary liver cancer and in surrounding cirrhotic tissue. In order to determine the steps of liver cancer progression where the activation of IGF-II fetal mRNAs occurs, we analyzed IGF-II mRNA expression during hepatocarinogenesis in transgenic mice carrying an antithrombin III-SV40 early region hybrid gene. The comparative analysis of mRNAs encoding IGF-II and other differentiation-associated proteins, as well as histological analysis, indicate that the reexpression of fetal IGF-II mRNAs takes place in specific steps of liver cancer progression, both in early pretumorous lesions and in well differentiated hepatocellular carcinomas. PMID- 1720800 TI - Surgical treatment of pancreatic cancer. The United States experience. AB - About 28,000 new cases of pancreatic cancer are diagnosed yearly in the United States. The diagnosis is now made up to two months more quickly than just a few years ago, but this has had no impact on survival. In most institutions, 20-25% of patients have resectable lesions. The standard operation is still the Whipple pancreaticoduodenectomy, but many surgeons now use the pylorus preserving modification of that procedure. The operative mortality rate has fallen to less than 5%. The five-year survival rate after a resection for attempted cure is about 9%. Palliation requires cholecysto(docho)jejunostomy and gastrojejunostomy, which is often done prophylactically. The operative mortality rate in patients undergoing palliation is less than 10% (recent UCLA experience), and the average survival is seven months. PMID- 1720801 TI - [Analysis of human papillomavirus type 16 E6/E7 mRNA in cervical cancers and precancerous lesions by means of the polymerase chain reaction with reverse transcriptase reaction]. AB - We analyzed HPV-16 E6/E7 mRNA in human uterine cervical carcinomas and cervical intraepithelial neoplasias (CINs) by polymerase chain reaction (PCR) with reverse transcription (RT-PCR). Simultaneously total RNA and DNA were extracted from 6 cervical carcinomas, 14 CINs and 2 normal cervical tissues by the guanidium isothiocyanate/CsCl method. HPV-16 DNA was detected in 3 cervical carcinomas and 6 CINs. HPV-16 E6/E7 transcripts were detected in all HPV-16 DNA positive cervical carcinomas and CINs. In 2 cervical carcinomas and 5 CINs, 2 spliced E6 mRNA (E6*I and E6*II) and full length E6 mRNA were detected. In one cervical carcinoma and in one CIN, only full length E6-E7 mRNA was detected. Sequence analysis of cloned PCR products showed that both transcripts were generated by splicing out an intron in E6, from nucleotides (nt) 226 to 409 in E6*I and from nt 226 to 526 in E6*II. There was no significance difference in HPV-16 E6/E7 mRNA patterns between cervical carcinomas and CINs. This sensitive RT-PCR technique was available for analysis of HPV-16 mRNA in the small specimens. PMID- 1720802 TI - Recombinant human G-CSF and GM-CSF prime human neutrophils for superoxide production through different signal transduction mechanisms. AB - Recombinant human granulocyte-colony stimulating factor (G-CSF) and recombinant human granulocyte/macrophage-colony stimulating factor (GM-CSF) stimulate neutrophil production from precursors in the marrow and enhance granulocyte functions in vitro. We studied the effects of G-CSF and GM-CSF on neutrophil superoxide production and secretion. G-CSF and GM-CSF alone stimulated neither superoxide production nor secretion, but both agents primed neutrophils for superoxide production stimulated by either N-formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin. Optimal priming occurred with G-CSF at 5.3 ng/ml for 20 minutes and for GM-CSF at 1 ng/ml for 60 minutes. Priming by GM-CSF was more readily inhibited by the tyrosine kinase inhibitor ST638 but was unaffected by staurosporine. Conversely, G-CSF priming was inhibited by staurosporine but not by ST638. Neither protein kinase C translocation nor increased protein kinase C activity, however, were observed after G-CSF/GM-CSF treatment. Priming by G-CSF and GM-CSF was sensitive to pertussis toxin, suggesting the involvement of guanine nucleotide-binding proteins (G-proteins). Neutrophils from three siblings with cyclic neutropenia were studied to observe the effects of G-CSF treatment on neutrophil function in vivo; sibling 1 and sibling 2 were treated with G-CSF for 6 months, but sibling 3 was not in the treatment group. Compared with neutrophils from normal donors, neutrophils from sibling 1 and sibling 2 were primed in vivo for superoxide release stimulated by either ionomycin or FMLP. Superoxide released by neutrophils from sibling 3 was similar to control cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720803 TI - Purification and characterization of the 30,000 dalton native antigen of Mycobacterium tuberculosis and characterization of six monoclonal antibodies reactive with a major epitope of this antigen. AB - The 30,000 dalton native antigen of Mycobacterium tuberculosis is a major constituent of this organism and is secreted into culture medium. We purified this antigen by ammonium sulfate precipitation, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography to yield a single 29 to 30 kd component. The first 20 N-terminal amino acid sequence was determined and found to be identical to that reported for M. bovis alpha-antigen. Immunoelectrophoresis studies demonstrated the purified 30,000 dalton antigen to be immunologically identical with antigen 6 and antigen 85B. The 30,000 dalton native antigen was a potent skin test antigen in sensitized guinea pigs. Six immunoglobulin G1 murine monoclonal antibodies against the 30,000 dalton antigen were generated. By enzyme-linked immunosorbent assay and western immunoblotting, all six monoclonal antibodies reacted with the 30,000 dalton antigen, perhaps with the same epitope. When used with culture filtrates of other mycobacteria, the monoclonal antibodies demonstrated reactivity with M. gordonae and M. kansasii and to a lesser extent with M. avium and M. scrofulaceum. PMID- 1720804 TI - Changes in insulin-like growth factor-binding proteins in bovine mammary secretions associated with pregnancy and parturition. AB - The bovine mammary gland accumulates large quantities of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) during late gestation which are secreted at parturition. The present study was conducted to determine the changes in the profiles of IGFBPs secreted by the mammary gland and in blood during late gestation and early lactation in dairy cows. Ligand blotting of serum and mammary secretions showed that IGFBPs of Mr 25,000, 30,000, 34,000, 42,000, 46,000 and greater than 200,000 were present in both fluids. The binding activity of the 42 46,000 Mr IGFBP predominated in prepartum mammary secretions and colostrum but was reduced postpartum. The binding activities of the 30,000 and 34,000 Mr IGFBPs, relative to other IGFBPs, were increased postpartum. Concentrations of IGF-I and IGF-II in mammary secretions declined from 347.1 and 181.1 nmol/litre 1 week prepartum to 0.7 and 0.3 nmol/litre 1.5 weeks postpartum. The volume of mammary secretions obtained was 0.109 litre and 6.690 litres at 1 week prepartum and 1.5 weeks postpartum respectively. In prepartum serum, the greatest binding activity was at Mr 42-46,000. The activity at this Mr decreased at parturition but was restored postpartum. The binding activities of the 30,000 and 34,000 Mr IGFBPs were increased around parturition. The 25,000 Mr IGFBP had minor activity during all periods. IGF-I concentrations decreased from 10.6 nmol/litres 1 week prepartum to 4.7 nmol/litres 1.5 weeks postpartum but IGF-II concentrations remained constant. In conclusion, IGFBP activity secreted by the mammary gland shifts from primarily Mr 42-46,000 prepartum to Mr 30,000 postpartum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720805 TI - Protein glycation: its role in the changes induced by diabetes in the properties of the serum insulin-like growth factor-I binding proteins. AB - The purpose of this work was to study the effect of diabetes on 125I-labelled insulin-like growth factor (IGF) binding to specific serum binding proteins (IGFBPs) and the possible role of protein glycation in such an effect. Accordingly, ligand blotting and fructosamine assays were performed in serum samples from diabetic and non-diabetic eSS rats as well as in samples of normal rat serum previously incubated with different concentrations of glucose. IGFBPs with molecular weights of 24, 30 and 40 kDa were identified in samples from diabetic and non-diabetic rats. 125I-Labelled IGF-I binding to each of these fractions increased significantly in the serum of diabetic rats. IGF-I binding to IGFBP-40 increased significantly as a function of the degree of glycation of serum proteins. Conversely, the increased binding of IGFBP-24 and IGFBP-30 was related only to the glucose concentration attained at 120 min during the oral glucose tolerance test. Glycation of proteins of normal serum and the binding of labelled IGF-I increased as a function of glucose concentration in the incubation media. In these in-vitro glycated normal sera, only the binding to IGFBP-40 increased significantly; this increase was closely related to the amount of protein glycation. No clear and reproducible changes occurred with the binding of 125I-labelled IGF-I to IGFBP-24 and IGFBP-30 fractions. These results confirm the increase in the binding capacity of IGFBPs reported in diabetic animals. They also show that the increase in IGF-I binding to each IGFBP fraction is regulated by a different mechanism; whereas protein glycation induces changes in IGFBP-40, this mechanism does not affect the binding properties of the other two IGFBPs. The increased binding of IGFBP might affect the availability of free IGF-I, and the consequent alterations in IGF-I-dependent metabolic processes could explain the role of this growth factor in the pathogenesis of chronic complications of diabetes. PMID- 1720806 TI - The insulin-like growth factors and their binding proteins in a case of non-islet cell tumour-associated hypoglycaemia. AB - Non-islet-cell tumours which induce hypoglycaemia are rare. Insulin-like growth factor-II (IGF-II) produced by some tumours is thought to be responsible for the hypoglycaemia and other systemic effects, despite normal or even low serum IGF-II levels. We studied a 44-year-old woman presenting with symptomatic hypoglycaemia associated with a large intraabdominal haemangiopericytoma. The serum IGF-II level was 455 micrograms/l when measured after acid-ethanol extraction (normal range (NR) 450-750 micrograms/l) and 1063 micrograms/l after acid chromatography (normal human serum pool 1068 micrograms/l). Levels of fasting plasma insulin, C peptide, glucose and serum IGF-I levels were low before the operation (less than 2 mU/l (NR less than 2-14), 0.23 nmol/l (NR 0.4-1.2), 3.1 mmol/l, (NR 3.7-5.9) and 0.02 U/ml respectively). After tumour removal, the symptoms resolved rapidly and the patient made a full recovery. Secretion of both insulin and growth hormone was suppressed before the operation in response to a 75 g glucose meal and to an infusion of 100 micrograms GH-releasing hormone (GHRH) respectively in comparison with studies after the operation. Serum IGF-II levels 6 weeks and 12 weeks after the operation fell to 385 micrograms/l (777 micrograms/l; acid chromatography) and 280 micrograms/l (647 micrograms/l; acid chromatography) and serum IGF-I levels increased to 0.35 U/ml and 0.26 U/ml. Serum before the operation and tumour extract contained chiefly a large molecular weight precursor IGF-II (molecular weight 15,000-20,000) which disappeared from the serum after the operation. The IGF-binding proteins (IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-4) were examined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720807 TI - Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility. AB - A murine monoclonal antibody (M31-15) was identified using the penetration inhibiting assay of a human lung adenocarcinoma cell line (MAC10) and remarkably inhibited the phagokinetic tract motility of various cancer cell lines. The antigen, motility-related protein (MRP-1), recognized by M31-15, was 25- and 28 kD proteins, and M31-15 was used to isolate a cDNA clone from a human breast carcinoma cDNA library. Sequence analysis revealed that MRP-1 had strong similarity with a B cell surface antigen (CD37), a melanoma-associated antigen (ME491), the target of an antiproliferative antibody (TAPA-1), a human tumor associated antigen (CO-029), and the Sm23 antigen of the trematode parasite Schistosoma mansoni. PMID- 1720808 TI - CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta. AB - The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells. PMID- 1720809 TI - Role of human decay-accelerating factor in the evasion of Schistosoma mansoni from the complement-mediated killing in vitro. AB - Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that prevents complement (C)-mediated hemolysis by blocking the assembly or accelerating the decay of C3 convertase. Purified DAF is known to incorporate into the membrane of DAF-deficient cells, inhibiting lysis. Since Schistosoma mansoni is a blood dwelling parasite, we investigated whether DAF can be transferred from human erythrocytes to the worm and protect it against C-mediated killing in vitro. We have found that schistosomula (schla) incubated with normal human erythrocytes (N HuE), but not with DAF-deficient erythrocytes, become resistant to C damage in vitro. Protected parasites acquire a 70-kD surface protein which can be immunoprecipitated by anti-DAF antibodies. The acquired resistance is abrogated by treatment of N-HuE-incubated parasites with anti-DAF antibody. These results indicate that, in vitro, N-HuE DAF can be transferred to schla, and suggest its participation in preventing their C-mediated killing. This could represent an important strategy of parasites to evade the host's immune response in vivo. PMID- 1720810 TI - The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1. AB - A skin-associated population of memory T lymphocytes, defined by expression of the cutaneous lymphocyte antigen (CLA), binds selectively and avidly to the vascular lectin endothelial cell-leukocyte adhesion molecule 1 (ELAM-1), an interaction that may be involved in targeting of CLA+ T cells to cutaneous sites of chronic inflammation. Here we present evidence that CLA itself is the (or a) lymphocyte homing receptor for ELAM-1. Antigen isolated with anti-CLA monoclonal antibody HECA-452 from human tonsillar lysates avidly binds ELAM-1 transfected mouse cells. Anti-CLA antibody blocks T lymphocyte binding to ELAM-1 transfectants. HECA-452 and ELAM-1 binding to lymphocytes or to isolated tonsillar HECA-452 antigen is abrogated by neuraminidase treatment implying a prominent role for sialic acid in CLA structure and function. The dominant form of CLA on T cells is immunologically distinct from the major neutrophil ELAM-1 ligand, the sialyl Lewis x (sLex) antigen (NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc), which is absent, weakly expressed, or masked on T cells. However, neuraminidase treatment of CLA+ T cells, but not of CLA- T cells, reveals Lewis x (CD15) structures. In combination with the known requirement for terminal NeuAc alpha 2-3Gal and fucose residues attached to N-acetylglucosamine for ELAM-1 and HECA-452 binding, this finding suggests that CLA may comprise an additionally sialylated or otherwise modified form of sLex. The identification of a lymphocyte homing receptor for skin may permit novel approaches to the diagnosis and therapy of cutaneous and inflammatory disorders. PMID- 1720811 TI - Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. AB - Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo. PMID- 1720812 TI - Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes. AB - In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK) susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function. PMID- 1720813 TI - Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen. AB - The absence of readily manipulable experimental systems to study the cytotoxic T lymphocyte (CTL) response against hepatitis B virus (HBV) antigens has thus far precluded a definitive demonstration of the role played by this response in the pathogenesis of liver cell injury and viral clearance during HBV infection. To circumvent the problem that HBV infection of human cells in vitro for production of stimulator/target systems for CTL analysis is not feasible, a panel of 22 overlapping synthetic peptides covering the entire amino acid sequence of the HBV core (HBcAg) and e (HBeAg) antigens were used to induce and to analyze the HBV nucleocapsid-specific CTL response in nine patients with acute hepatitis B, six patients with chronic active hepatitis B, and eight normal controls. By using this approach, we have identified an HLA-A2-restricted CTL epitope, located within the NH2-terminal region of the HBV core molecule, which is shared with the e antigen and is readily recognized by peripheral blood mononuclear cells from patients with self-limited acute hepatitis B but less efficiently in chronic HBV infection. Our study provides the first direct evidence of HLA class I-restricted T cell cytotoxicity against HBV in humans. Furthermore, the different response in HBV-infected subjects who successfully clear the virus (acute patients) in comparison with patients who do not succeed (chronic patients) suggests a pathogenetic role for this CTL activity in the clearance of HBV infection. PMID- 1720814 TI - New roles for glia. PMID- 1720815 TI - Changes in the distribution of extracellular matrix components accompany early morphogenetic events of mammalian cortical development. AB - As a step in defining the molecular environment for development of the mammalian cerebral cortex, we have used immunohistochemistry to analyze the distribution and remodeling of three major extracellular matrix (ECM) components, fibronectin, chondroitin sulfate proteoglycan (CSPG), and tenascin, during embryonic and early postnatal stages in the mouse. Fibronectin and CSPG are distributed throughout the proliferative zone that initially comprises the thin wall of the telencephalic vesicle, but their distribution changes as newly generated cells form the preplate just beneath the pia. Immunolabeling for CSPG becomes most prominent in the preplate, and fibronectin becomes restricted to that layer. Just after this change occurs, processes of preplate neurons, visualized with antibodies to neurofilaments, become evident within the matrix-rich preplate zone. The association of fibronectin and CSPG with preplate cells persists as cortical plate neurons divide the preplate; both ECM components are now most prominent in the marginal zone and subplate, the layers above and below the cortical plate that are preplate derived. Within the preplate and its derivatives, immunolabeling of fibronectin is punctate and closely associated with radial glial processes, while labeling of CSPG is more intense and diffuse. Labeling of fibronectin and CSPG declines rapidly as the cortical plate begins to differentiate into cortex; labeling for tenascin first appears at this stage in the most mature layers, the marginal zone and subplate, then gradually becomes widespread throughout all of cortex and subcortical white matter. In early postnatal life, tenascin is eliminated from the hollows of the vibrissal barrels in the somatosensory region; it then declines rapidly throughout cortex. The association of both fibronectin and CSPG with preplate cells and the distribution of fibronectin along radial glia during early cortical development suggest that one or both of these transient cell types might produce specific ECM components or induce their local deposition. The spatial and temporal distribution of fibronectin and CSPG suggests a role in defining a destination for migrating neurons that form the cortical plate and in delineating the pathway for early axonal extension. In contrast, the relatively late appearance of tenascin correlates best with the formation of astrocytes and their processes rather than with the establishment of cortical layers or major axonal pathways. These events are well underway before labeling of tenascin is evident. PMID- 1720816 TI - Applications of gallium-67 scintigraphy in the management of patients with malignant lymphoma. PMID- 1720817 TI - Immunohistochemical demonstration of keratin 7 in routinely fixed paraffin embedded human tissues. AB - The immunoreactivity of OV-TL 12/30, a monoclonal anti-keratin 7 antibody (Mab), was investigated on frozen as well as paraffin-embedded human tissues. Its reactivity patterns were compared with another well-characterized monoclonal antibody to keratin 7 (RCK 105), and with broadly cross-reacting monoclonal (OV TL 12/5) as well as polyclonal (pKer) keratin antisera. In frozen sections of normal and malignant human tissues both keratin 7 Mabs gave similar staining patterns. The immunoreactivity for OV-TL 12/30 and the polyclonal antibody (pKer) in tissue sections fixed in 4 per cent formalin or Bouin solution, was completely restored when pretreated with 0.1 per cent pronase, 0.1 per cent trypsin in phosphate-buffered saline (PBS) or with 0.5 per cent pepsin in 0.01 N HCl. Except for loss of immunoreactivity on human normal stomach surface epithelium and glandular mucous cells, Mab OV-TL 12/30 reacted strongly positive with essentially all those formalin- or Bouin-fixed paraffin-embedded tissues that had been shown to stain in non-fixed, frozen sections. In addition to the good correlation in human tissues, a complete correlation between the reactivity on frozen and paraffin-embedded human carcinomas (n = 86) was found as well. While both RCK 105 (anti-keratin 7) and OV-TL 12/5 (anti-keratin 5, 7, 14, 19) did not stain on paraffin-embedded sections, the polyclonal control antiserum (pKer) lost immunoreactivity in some cell types (e.g. mucous cells in compound glands, hepatocytes, pancreatic acinar cells, and proximal and distal convoluted tubules of the kidney). Our study shows that the keratin 7 Mab OV-TL 12/30 is an excellent marker for tumour histopathology since it is reactive in paraffin embedded formalin-fixed human tissues. PMID- 1720818 TI - New monoclonal antibodies reacting with bile ducts: further insights into the pathogenesis of bile ductular proliferation in biliary diseases. AB - We have produced a range of monoclonal antibodies which stain human intrahepatic bile ducts of different sizes. Amongst 26 monoclonal antibodies produced, five clones reacted specifically with bile ducts of different sizes, of which three have been maintained in culture and their viability following freezing and thawing confirmed. Staining patterns varied between normal adult liver tissue, normal fetal liver tissue and a variety of hepatobiliary diseases. The antibodies provide further evidence of the immunological heterogeneity of the human intrahepatic biliary tree and support the hypothesis that proliferating bile ductules are derived from periseptal hepatocytes. The preparation of the antibodies, their staining reactions in normal adult, normal fetal and a variety of liver diseases are described. PMID- 1720819 TI - Cyclical vomiting and hypertension with dermatographism and histamine release. PMID- 1720820 TI - Substance P: a neurogenic mediator of acute cellular inflammation in the dog? AB - Substance P (SP) is a neuropeptide that has recently been implicated in the pathogenesis of neurogenic inflammation. SP has been shown to activate polymorphonuclear leukocytes (PMN) as well as other inflammatory cells. The present study investigated the direct stimulatory and priming effects of SP on canine PMN aggregation and migration. Direct stimulation of cell migration by SP was present at an unphysiologically high concentration of the mediator. However, when micromolar concentrations of SP were added to PMN prior to stimulation with sub-optimal concentrations of leukotriene B4 (LTB4), the cells exhibited enhanced aggregation and migration, i.e. priming, when stimulated with the latter. Since SP has been reported to act via the formyl-Met-Leu-Phe (fMLP) chemotaxin receptor, this mediator was also studied and found not to possess any effects similar to SP. Thus, the results indicate that SP acts as a primer of canine PMN functions in vitro via a receptor different from that for fMLP. Before ascribing SP a mediator role in canine neurogenic inflammation, in vivo studies determining the concentrations of, and responses to SP in inflamed tissue should be performed. PMID- 1720821 TI - Heart rate-dependent alteration of the frequency and coupling interval of ventricular arrhythmias as measured by 24-hour ECG monitoring. AB - Twenty-four hour ECG recordings of 132 patients with frequent (greater than 1000/day) ventricular premature contractions (VPCs) were analyzed using a computerized system, designed to evaluate the relationships between 1) the VPC frequency and heart rate (HR) (VPC-HR relation), 2) the coupling interval (CI) of VPCs and HR (CI-HR relation), and 3) the incidence of ventricular tachycardia (VT) and HR (VT-HR relation). The patterns of the VPC-HR relation included: 1) an increase in VPCs with increasing HR (positive correlation, 43 patients), 2) an increase in VPCs at low HR range and a decrease at high HR range, with increasing HR (bidirectional correlation, 74 patients), 3) a decrease in VPCs with increasing HR (negative correlation, 7 patients) and 4) constant VPCs over all HRs (flat correlation, 8 patients). Patients were divided into 2 broad categories according to whether they had a positive correlation (P group, 43 patients) or the other correlations (non-positive or NP group, 89 patients). Of 132 patients, the CI-HR relation was negative in 129 (98%) and positive in only 3 (2%). Patients with frequent VTs (10 or more events over 24h) were significantly more frequent in the P (9 patients, 21%) than in the NP group (7 patients, 8%, p less than 0.05). However, mean HR, mean CI, total VPC counts and the slope of CI-HR relation were not significantly different between the groups. The VT-HR relation observed in 16 patients with frequent VTs were positive in 9 of the P group and in 2 of the NP group and non-positive in 5 of the NP group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720822 TI - Analysis of perioperative ventricular arrhythmias in valvular heart diseases by Holter ECG recording. AB - Seventy-one consecutive patients undergoing cardiac surgery for acquired valvular diseases were analyzed to determine the incidence of and the predisposing factors to postoperative ventricular arrhythmias. We recorded the Holter ECGs before (pre op), and within 24h (op-day) and 4 to 10 weeks after operation (post-op) and determined the frequency of ventricular arrhythmias and the degree according to the Lown grade. The relationship between the op-day ventricular arrhythmias and clinical, hemodynamic, operative or postoperative variables was examined. The operation included mitral valve replacement or open mitral commissurotomy (49 patients, group M), aortic valve replacement (12 patients, group A) and a combined mitral and aortic operation (10 patients, group A + M). In all groups, the frequency and the degree of ventricular arrhythmias increased at the op-day and decreased at the post-op period approximately to the pre-op level. The frequency and Lown grade of the 3 groups were similar in each of the pre-op, the op-day and the post-op periods. The frequency and Lown grade of the op-day ventricular arrhythmias increased with increases in the arrhythmia frequency and Lown grade at the pre-op period, and in patients with left ventricular (LV) dysfunction at the post-op period, as evidence by an increased LV volume and decreased ejection fraction on echocardiograms. Furthermore, the frequency of ventricular premature contractions in the op-day was significantly less when a cardioplegia solution containing magnesium was used than in the case of a cardioplegia solution without magnesium. The op-day ventricular arrhythmias showed no significant relation to extracorporeal circulation time, aortic cross clamping time, the antiarrhythmic drugs used and the op-day serum levels of K and CK-MB. PMID- 1720823 TI - Effects of calcium channel agonists (BAY K 8644, CGP28392 and YC-170) on 45Ca uptake by rat uterine segments. AB - The characteristics of the stimulating effects of the calcium channel agonists BAY K 8644, CGP28392 and YC-170 on 45Ca uptake by rat uterine segments were investigated. BAY K 8644, CGP28392 and YC-170 caused about 150, 100 and 150% increase, respectively in the 45Ca uptake induced by 20 mM KCl. The ED50 values of BAY K 8644, CGP28392 and YC-170 were 1.8 X 10(-9), 2.5 X 10(-8) and 9.8 X 10( 9) M, respectively. These agonists had little effect on the 45Ca uptake induced by 10(-6) M acetylcholine. They also did not affect the basal 45Ca uptake. Their enhancing effects were blocked by the Ca channel antagonist nitrendipine. We conclude that rat uterine segments have voltage-sensitive Ca channels that are stimulated by Ca channel agonists (BAY K 8644, CGP28392 and YC-170) under depolarizing conditions and that the characteristics of the stimulating effects of CGP28392 and YC-170 on 45Ca uptake by rat uterine segments are qualitatively the same as those of BAY K 8644. PMID- 1720824 TI - Active principle of swine prostate extract: II. Effect of a peptide isolated from swine prostate extract on rat prostate. AB - Previously, we purified a substance from swine prostate extract (PE) that had been reported to have therapeutic effect on benign prostatic hypertrophy. The purified substance (PPE) suppressed 3H-testosterone uptake into the prostate in castrated rats. The present study was carried out to examine the effect of PPE on the weight of accessory sexual organs including the prostate and biochemical parameters in the prostate of normal and/or castrated and testosterone-treated rats. 1) In normal rats, the p.o. administration of PPE daily for a total of 30 days did not affect the prostate weight, but reduced the citric acid content in the prostate. The treatment had little or no influence tissue O2 uptake, aconitase activity or isocitrate dehydrogenase activity in the prostate. 2) In castrated and testosterone-treated rats, the p.o. treatment with PPE for 15 or 30 days reduced the weight of the prostate as well as the total citric acid, DNA and RNA contents in prostatic tissue. However, these biochemical parameters per tissue weight were not obviously affected except for the citric acid content. These findings suggest that PPE is one of the active principals of PE for the therapeutic efficiency on benign prostatic hypertrophy, probably due to its suppressive effect on excessive uptake of androgen by the prostate. PMID- 1720825 TI - Human acidic fibroblast growth factor has trophic effects on cultured neurons from multiple regions of brain and retina. AB - Neurotrophic activities of human recombinant acidic fibroblast growth factor (haFGF) were evaluated on primary cultured neurons from various brain regions and compared with those of CS23, modified human basic fibroblast growth factor. Survival of cultured neurons from embryonic day 16 (E16) rat cortex and substantia nigra was significantly increased by the addition of more than 10 ng/ml haFGF and that from the hippocampus was increased by 100 and 1000 ng/ml. However, enhancement of viability by haFGF was observed only in 1000 ng/ml treated neurons from the striatum, thalamus, colliculus and cerebellum; and it was not observed in septal neurons. Survival of cultured neurons from postnatal day 15 rat on glial feeder layer was significantly increased by the addition of 1000 ng/ml haFGF, except for neurons from the septum. CS23 (10 ng/ml) increased the survival of cultured neurons from all regions mentioned above, and its effects were stronger than those of 1000 ng/ml haFGF. Addition of more than 10 ng/ml haFGF significantly increased the survival of cultured neurons from neonatal day 2 rat retina. Addition of 1000 ng/ml haFGF increased the choline acetyltransferase activity of E16 septal neurons slightly but significantly, but didn't increase the dopamine uptake activity of embryonic day E15 ventral midbrain. These results show that haFGF is effective on limited regions and ages of brain and retina, while CS23 is effective on all regions tested. PMID- 1720826 TI - Hepatitis C virus infection among kidney transplant recipients. AB - The extent of hepatitis C virus (HCV) infection among kidney recipients was investigated in 67 patients by testing for anti-HCV paired serum samples, collected at time of transplantation and during follow-up (average 32 +/- 20 months). Prevalence of anti-HCV at transplant time was 48%, and was related to the time on dialysis and to the amount of blood transfusions. Following transplantation, nine (28%) seropositive patients lost anti-HCV and five (14%), previously seronegative, seroconverted. Anti-HCV was found to be positive in 92% of the patients with chronic liver disease who were on hemodialysis, but in 56% in kidney recipients with chronic hepatitis. Anti-HCV was positive in 50% of patients with resolving hepatitis before transplantation, but only in 21% of those with acute hepatitis following transplantation. This study confirms the high risk of HCV infection among hemodialysis and kidney recipient populations, and also that HCV is closely related with the length of time the patient is on hemodialysis as well as the number of blood units transfused. HCV is the main cause of acute and chronic liver disease in hemodialysis patients and of chronic liver disease in kidney recipients, but does not clearly influence the survival of the allograft nor that of patients. PMID- 1720827 TI - Selective assay of protein kinase C with a specific peptide substrate. AB - Protein kinase C is a family of multifunctional protein serine/threonine kinase and generally accepted to be involved in a wide variety of cellular signal transduction. Biochemical and immunochemical studies as well as sequence analysis of its cDNA clones have revealed the existence of multiple subspecies of this enzyme with obvious tissue-specific expression. Enzymatic properties of type I, II, and III protein kinase C subspecies, which are encoded by gamma-, beta I- and beta II, and alpha-cDNA, respectively, are well characterized. Many proteins and peptides are reported as phosphate acceptors of these protein kinase C subspecies. In this study, it is shown that a synthetic peptide, Gln-Lys-Arg-Pro Ser-Gln-Arg-Ser-Lys-Tyr-Leu, which corresponds to amino acid residues 4-14 of bovine myelin basic protein, is the most specific and convenient substrate for selective assay of protein kinase C among various phosphate acceptor proteins and peptides. This peptide is phosphorylated at Ser-8, but not Ser-11 by protein kinase C subspecies in a manner dependent on Ca2+, phosphatidylserine, and diacylglycerol. This peptide is not phosphorylated by other protein serine/threonine kinases such as cyclic AMP-dependent protein kinase. Thus, it is possible to assay protein kinase C activity in the crude tissue extracts selectively using this peptide as a phosphate acceptor. PMID- 1720828 TI - Effect of the renin response during renin inhibition: oral Ro 42-5892 in normal humans. AB - The effect of the new renin inhibitor Ro 42-5892 was evaluated in healthy volunteers after both intravenous and oral administration. In a preliminary study, 14 subjects received a 10-min infusion of Ro 42-5892 at doses ranging from 0.001 to 1 mg/kg. Plasma renin activity (PRA) and angiotensin (Ang) II levels were maximally suppressed in a dose-dependent manner at the end of the infusion. Plasma active renin concentration increased up to threefold. In a second study, 24 volunteers received placebo or 100, 600, or 1,200 mg of Ro 42-5892 p.o. in a single-blind, randomized fashion. Within 30 min after drug intake, PRA and plasma Ang I and Ang II levels fell to their nadir. Both Ang I and Ang II were measured specifically after extraction on phenylsilylsilica and separation by isocratic HPLC. The degree as well as the duration of inhibition were dose related. The decrease in plasma Ang lasted maximally for 2 h. Active renin increased dose dependently and remained elevated for more than 8 h after the 1,200 mg dose. A theoretical generation rate of Ang I was calculated for individual plasma samples assuming Michaelis-Menten kinetics for competitive inhibition and steady-state conditions. This calculated Ang I generation rate, based on plasma active renin concentrations and drug levels, closely correlated with actually measured Ang I and Ang II levels (r = 0.90, n = 88) over the whole 8 h time period. Thus, a sustained renin inhibition by Ro 42-5892, as indicated by increased plasma active renin levels, induces a much shorter fall in plasma Ang I and II apparently because of a rise in renin secretion. PMID- 1720829 TI - Heart failure augments the cardiovascular and renal effects of neutral endopeptidase inhibition in rats. AB - We compared the cardiovascular and renal actions of the neutral endopeptidase (NEP) inhibitor, SQ 28,603, in normal rats and in rats with healed myocardial infarcts. The infarcted rats were studied in the conscious state 8 weeks after ligation of the left main coronary artery and 4 h after placement of cardiovascular and renal catheters. Infarct size was 39 +/- 1.2% of left ventricle circumference; right ventricle and lung weight to body weight ratios were twice those of normal rats. These postmortem values were shown to be associated with elevated left ventricular end diastolic pressure and high plasma atrial natriuretic peptide (ANP) concentration in separate groups of rats. SQ 28,603 at 100 mumol/kg intravenously (i.v.) caused urine volume and sodium excretion to increase by 79 +/- 11 microliters/min and 8.2 +/- 1.4 microEq/min, respectively, 20 min after injection in infarcted rats; these changes were significantly greater than those in normal rats (12 +/- 5 microliters/min and 1.6 microEq/min, respectively). Thoracic venous pressure decreased by 1.9 +/- 0.4 mm Hg 80 min after SQ 28,603 in infarcted rats and by only 0.1 +/- 0.1 mm Hg in normal rats (p less than 0.05 vs. infarcted rats). SQ 28,603 had no effects on mean arterial pressure (MAP), cardiac output (CO), or glomerular filtration rate (GFR). The observation that NEP inhibition has more pronounced effects in animals with high ambient ANP level than in those with normal ANP is consistent with previous studies in a variety of animal models and supports the concept that NEP inhibition potentiates endogenous ANP. PMID- 1720830 TI - Contribution of spinal dopamine receptors to the hypotensive action of bromocriptine in rats. AB - Bromocriptine, a dopamine (DA) receptor agonist, has been reported to have hypotensive effects in anesthetized and conscious normotensive rats but its mechanism of action is still not fully understood. Therefore, we studied the changes in mean arterial blood pressure (MAP) and heart rate (HR) elicited by an intravenous (i.v.) administration of bromocriptine (150 micrograms/kg), in either pentobarbital-anesthetized or conscious normotensive rats, pretreated with either i.v. (0.3 mg/kg) or intrathecal (i.t.) (93 nmol) domperidone, a DA receptor antagonist that does not cross the blood-brain barrier. In these preparations, i.v. administration of bromocriptine elicited dose-dependent decreases in MAP and rises in HR. The hypotensive effect was antagonized partially by i.t. and fully by i.v. domperidone. However, the latter compound did not modify the tachycardia, which could be blocked by propranolol (0.5 mg/kg i.v.). In rats pretreated with the latter beta-adrenoceptor antagonist, bromocriptine produced only a decrease in blood pressure that was inhibited by i.v. and i.t. domperidone. These results suggest that, in anesthetized and conscious normotensive rats, the hypotension induced by systemic administration of bromocriptine is fully mediated by DA2 dopamine receptors, which are located partly within the spinal cord and partly in the peripheral circulation. PMID- 1720831 TI - The hemodynamic effects of lacidipine in anesthetized dogs: comparison with nitrendipine, amlodipine, verapamil, and diltiazem. AB - The hemodynamic effects of lacidipine in anesthetized, open-chest dogs were compared with those of nitrendipine, amlodipine, verapamil and diltiazem. Lacidipine administered intravenously induced dose-related, long-lasting reductions in systemic and coronary vascular resistance with corresponding increases in aortic flow and coronary blood flow. The hypotensive effect (ED25 for mean blood pressure reduction = 0.006 mg/kg) was still significant 120 min after administration with all doses tested. Nitrendipine was equipotent with lacidipine in reducing the mean blood pressure (ED25 = 0.005 mg/kg), but its effect was shorter acting (significant effect at 120 min only with the highest dose tested). Amlodipine caused a marked and long-lasting hypotension though at higher doses than lacidipine (ED25 = 0.50 mg/kg). Short-lasting hypotensive responses were also detected with verapamil (ED25 = 0.1 mg/kg) and diltiazem (ED25 = 0.12 mg/kg). A reflex increase in heart rate was observed with lacidipine, nitrendipine, and amlodipine, whereas verapamil and diltiazem showed a dose-related bradycardia. No effect on AV conduction was observed with lacidipine and nitrendipine, whereas amlodipine, verapamil, and diltiazem produced second- to third-degree AV block at the highest doses tested. Lacidipine and nitrendipine caused a reflex increase in contractile index at all doses, whereas amlodipine was more similar to verapamil since a marked decrease in contractile index was detected at the highest dose. Diltiazem was practically devoid of negative inotropic effect. PMID- 1720832 TI - Improved kidney function with cilazapril in hypertensive type II diabetics with chronic renal failure. AB - The purpose of this study was to determine efficacy and safety of the angiotensin converting enzyme inhibitor, cilazapril, in the treatment of hypertensive diabetics with renal insufficiency. Fifteen type II diabetics with hypertension and chronic renal insufficiency aged (mean +/- SD) 64 +/- 7 years were studied in a regional clinic and university hospital hypertension unit. The blood pressure was measured biweekly. Urinary collections were done after 2 weeks of placebo and 8 weeks of cilazapril treatment. The blood pressure decreased from 176 +/- 15/105 +/- 9 to 164 +/- 11/95 +/- 9 mm Hg and serum creatinine from 197 +/- 69 to 179 +/ 73 mumol/L. The creatinine clearance rose from 41.6 +/- 11.4 to 47.4 +/- 14.9 ml/min, while protein excretion decreased from 0.8 +/- 1.3 to 0.5 +/- 0.8 g/24 h (p less than 0.05). The blood pressure change was inversely correlated with the creatinine clearance change (r = -0.5, n = 15, p less than 0.05). In these high risk patients, 8 weeks of cilazapril treatment improved both blood pressure control and renal function but renal function improved most in the patients whose blood pressure changed the least. PMID- 1720833 TI - Hemodynamic changes after subselective intracoronary administration of nisoldipine in humans. AB - Hemodynamic changes after the subselective intracoronary administration of 50 micrograms of nisoldipine were analyzed in 24 nonstenotic coronary arteries using a randomized, placebo-controlled, double-blind protocol. The following hemodynamic parameters were studied: (a) epicardial coronary artery diameter, assessed by quantitative angiography; (b) coronary blood flow velocity, measured by an intracoronary Doppler probe; (c) coronary blood flow, calculated from the above parameters; (d) coronary flow velocity reserve, assessed after intracoronary administration of 10 mg of papaverine hydrochloride; and (e) heart rate and arterial blood pressure. Since 3 patients were excluded due to unreliable Doppler signals, a total of 21 patients was eligible for complete analysis (placebo: n = 9; nisoldipine: n = 12). In placebo-treated patients, all studied parameters proved to be very stable on repeat measurement and no significant changes were found. In nisoldipine-treated patients, a significant increase in epicardial diameter (+19%; p = 0.0001) and coronary blood flow (+47%; p = 0.003) was found. The coronary blood flow velocity transiently increased after nisoldipine, with a maximum (+80%) after 2 min and returning to baseline within 10 min. Finally, nisoldipine resulted in a significant decrease in the coronary flow velocity reserve by 20% (p = 0.001). All coronary hemodynamic effects were observed in the absence of changes in heart rate and arterial blood pressure. Therefore, the present data demonstrate that nisoldipine acts as a potent dilator of epicardial as well as resistance vessels in nonstenotic human coronary arteries. PMID- 1720834 TI - Catecholamines and the renin-angiotensin-aldosterone system during treatment with felodipine ER or hydrochlorothiazide in essential hypertension. AB - The neurohumoral responses after 10 mg of felodipine extended release (ER), a new dihydropyridine calcium antagonist, and 25 mg of hydrochlorothiazide (HCTZ) were compared in a randomized, double-blind, crossover trial in 28 mild to moderate hypertensives. Antihypertensive drugs were gradually discontinued. Felodipine ER, 10 mg was given once daily for 2 weeks; after another washout period of 1 week, patients were switched to 25 mg of HCTZ once daily and vice versa. Blood pressure (BP) was measured at baseline, 2.5 h after medication, and after 2 weeks of treatment (24 h postdosing) using an oscillometric device. Felodipine ER and HCTZ both lowered BP effectively. However, felodipine ER was superior in reducing systolic and diastolic BP during the short term and medium term. Treatment with felodipine ER over 2 weeks increased sympathetic outflow as indicated by elevated plasma norepinephrine levels, whereas plasma epinephrine was mainly unaffected, as were plasma renin and aldosterone levels. On the other hand, 25 mg of HCTZ increased plasma renin and aldosterone, but left catecholamines unchanged. Despite persistent increased sympathetic activity, the reduction in BP in this study was more pronounced after felodipine ER as compared to HCTZ. The lack of a difference between heart rates under both medications after 2 weeks of treatment suggests a resetting of the baroreflex by felodipine ER and furthermore that the increased norepinephrine levels may not be clinically relevant, but demonstrate the maintained baroreflex activity. HCTZ, in doses as low as 25 mg, is still capable of stimulating the renin-angiotensin-aldosterone system. PMID- 1720835 TI - Effects of the selective alpha 1-antagonist, doxazosin, on hepatic lipid levels and metabolism in the golden hamster. AB - The effect of the selective alpha 1-antagonist, doxazosin, on lipid metabolism was studied in cholesterol-fed golden hamsters. The hamsters were studied after short-term (1 week) and long-term (6-8 weeks) treatment. Doxazosin was added to the food (0.05-0.1%). Doxazosin lowered, within 1 week, plasma cholesterol and triglyceride concentrations by 12 and 19%, respectively. These effects were slightly larger during long-term treatment (cholesterol by 15% and triglycerides by 27%). Lipoprotein lipase (LPL) activity in postheparin plasma or in adipose tissue or heart was affected by doxazosin. The hepatic triglyceride secretion rate was lowered by 40%. Doxazosin treatment partially prevented the accumulation of cholesterol and triglycerides in the liver. Hepatic cholesterol synthesis was decreased by 25-40%. When determined under optimal conditions, i.e., after prior dephosphorylation, the hepatic microsomal HMG-CoA reductase activity was lowered by 10-25% in the doxazosin-treated animals. However, if HMG-CoA reductase was determined under conditions to prevent phosphorylation and dephosphorylation of the enzyme, representing the in situ expressed activity, the activity in the doxazosin-treated animals was 40% lower than in the controls. These results indicate that the plasma lipid-lowering effect of doxazosin is largely due to its interference with hepatic lipid metabolism and that one of the effects is a lowering of hepatic cholesterol synthesis, probably due to an increase in the phosphorylation grade of HMG-CoA reductase. PMID- 1720836 TI - Baroreflex and atrial natriuretic factor concentration correlate with myocardial infarct size and predict early death in rabbits: implications for drug studies. AB - The severity of myocardial infarction (MI) and its functional consequences are difficult to assess in small animals. We searched for criteria to achieve such an assessment in rabbits 1 week after MI. Thirteen large mongrel rabbits (3-4 kg) were anesthetized with pentobarbitone for ligating a branch of the circumflex coronary artery and 7 rabbits were subject to a sham operation without ligation. All sham-operated rabbits and 12 MI animals survived for 1 week, when blood was obtained for biochemical analyses and the baroreflex was tested. Six animals survived to the third week (survivors) and six died earlier (nonsurvivors). The MI size, measured immediately after death, was 42 +/- 3% of the left ventricular mass in nonsurvivors and 20 +/- 7% in survivors. The plasma atrial natriuretic factor (ANF) concentration was correlated linearly with MI size (r = 0.77) over the whole range of infarct sizes and, like the MI size itself, was associated with the risk of early death (critical limit: 80 pM). Plasma renin activity and catecholamines yielded less prognostic information. The baroreflex control of the heart rate (tested using phenylephrine and nitroprusside) of nonsurvivors was severely impaired and the slopes correlated with MI size (r = 0.90 for phenylephrine and r = 0.67 for nitroprusside). The plasma ANF concentration and the baroreflex both accurately reflected MI size and also correctly classified 11/12 rabbits into survivors and nonsurvivors. An ANF- and baroreflex-based stratification of animals for future studies on therapeutic interventions after MI will reduce the number of animals required by at least 65%, making such studies far more feasible than in the past. PMID- 1720837 TI - VIP-ergic and cholinergic innervations in internal carotid arteries of the cat and rat. AB - Using immunohistochemical methods, choline acetyltransferase and vasoactive intestinal polypeptide immunoreactive (ChAT-I and VIP-I) fine fibers with varicosity-like structures were observed in the rat and cat cerebral arteries. Acetylcholine (ACh) induced dual responses in endothelium-intact internal carotid arteries of the cat; it induced vasodilation at low concentrations and constrictions at high concentrations (greater than 10(-6) M). ACh induced contraction exclusively in endothelium-rubbed preparations. Atropine (10(-7) M) blocked ACh-induced constriction and dilatation. ACh-induced vasodilation was potentiated by M & B 22,948 (2 x 10(-5) M), a selective cyclic GMP phosphodiesterase inhibitor. Vasoconstriction induced by ACh was inhibited by neomycin (3 x 10(-3) M), an inositol phosphate synthesis inhibitor, which did not affect the neuropeptide Y-induced contraction. VIP-induced dilation of the cat internal carotid arteries was not affected by removing the endothelial layer, but was blocked by VIP receptor antagonist ([Ac-Tyr1, D-Phe2]-GRF 1-29 amide) and potentiated by cilostazol (2 x 10(-5) M), a selective cyclic AMP phosphodiesterase inhibitor. These results are consistent with previous findings that cerebral blood vessels receive cholinergic and VIP-ergic innervations, and that ACh-induced endothelium-dependent vasodilation is mediated by cyclic GMP synthesis, and that VIP-induced endothelium-independent vasodilation is mediated by cyclic AMP synthesis. The present study, however, demonstrates for the first time the presence of varicosity-like structure associated with ChAT-I fibers, suggesting the presence of cholinergic nerve terminals and that ACh-induced cerebral vasoconstriction is mediated by phosphatidyl-inositide turnover. PMID- 1720838 TI - Effects of a new nitro compound on the systemic circulatory system in dogs. AB - The present study was undertaken to evaluate the effects of a newly synthesized nitro compound (E-4701) on the systemic circulatory system with special reference to venous return and vascular compliance. Dogs were anesthetized with sodium pentobarbital. After opening the chest, cannulae were inserted into the superior and inferior vena cavae and into the right atrial appendage. The venous flow from the caval veins was redirected to a blood reservoir with an outlet at a constant height. The blood was pumped into the right atrium at a constant flow rate. E 4701 had a hypotensive effect, and also caused a decrease in reservoir blood volume; i.e., a decrease in venous return. Venous return via the superior vena cava was increased, whereas return via the inferior vena cava was decreased. Similar effects on the systemic circulatory system were observed with nitroprusside. The lowest dose of nitroprusside that caused a significant reduction in blood pressure was the same dose as that which caused a decrease in reservoir blood volume. However, a low dose of E-4701 caused a significant reduction in reservoir blood volume without affecting the systemic blood pressure. Arterial and venous compliances were increased by both E-4701 and nitroprusside. Nitroglycerin and isosorbide dinitrate increased venous compliance, but did not affect arterial compliance. The results suggest that E 4701 caused an almost equipotent reduction in blood pressure and venous return by dilating the arterial and venous vascular beds. The capacitance vessels may be more sensitive to E-4701 than the resistance vessels. PMID- 1720839 TI - Effects of isomazole on force of contraction and phosphodiesterase isoenzymes I IV in nonfailing and failing human hearts. AB - The phosphodiesterase (PDE) inhibitor isomazole increased the force of contraction to 278.3 +/- 89.1% (n = 7) of the predrug value in ventricular trabeculae carneae isolated from nonfailing human hearts. This effect can be attributed mainly to a PDE III or a combined PDE III/IV inhibition since at the concentration of the maximal positive inotropic effect of isomazole, PDE III and PDE IV were completely inhibited. In explanted failing human hearts (end-stage myocardial failure, NYHA IV), isomazole increased the force of contraction only marginally to 110.1 +/- 10.7% of the predrug value. The lack of a distinct positive inotropic efficacy of isomazole in failing human hearts could not be explained by an impairment of PDE inhibition since the properties of the PDE I-IV isoenzymes separated by DEAE-Sepharose chromatography and the inhibitory effects of isomazole did not differ in both preparations. The positive inotropic effect of the beta-adrenoceptor agonist isoprenaline was also reduced in failing hearts. However, in the presence of isomazole, the diminished positive inotropic effect of isoprenaline was restored to values obtained with isoprenaline alone in nonfailing hearts. Thus, the decreased effect of inotropic drugs like isoprenaline or isomazole in preparations from failing human heart might be explained mainly by a diminished cAMP formation due to a defect in receptor adenylate cyclase coupling. PMID- 1720840 TI - Pharmacological evaluation of the angiotensin, kinin, and neurokinin receptors on the rabbit vena cava. AB - Angiotensin II, bradykinin, and substance P are powerful vasoconstrictors of venous smooth muscle. In this report, we have characterized the receptors and the cellular mechanisms of these vasoactive peptides on a new isolated smooth muscle preparation, the rabbit vena cava. Receptors were characterized using agonists and antagonists and were found to be of the AT, B2, and NK-1 types. The myotropic responses of the vein to KCl was completely abolished in calcium-free medium; in the presence of nicardipine, nifedipine, and verapamil, three calcium channel antagonists; and of trifluoperazine, a calmodulin antagonist. AT II-, BK-, and SP induced responses were slightly attenuated in calcium-free medium and in the presence of nifedipine and trifluoperazine. Pinacidil inhibited the contractile response of KCl and the three peptides while lidocaine was active against KCl only. Staurosporine and cholera toxin strongly inhibited the contractile responses of the vein to AT II, BK, SP, and KCl, probably by a nonspecific effect. It is concluded that AT II-, BK-, and SP-induced contractions of the rabbit vena cava are mediated by specific receptors and in part by an influx of extracellular Ca2+ through dihydropyridine-insensitive channels. Opening of K+ channels and inhibition of the Ca(2+)-calmodulin complex appear to interfere with the smooth muscle response to the peptides. PMID- 1720841 TI - Effects of new and potent methanesulfonanilide class III antiarrhythmic agents on myocardial refractoriness and contractility in isolated cardiac muscle. AB - The effects of the new and potent methanesulfonanilide class III antiarrhythmic agents (E-4031, UK-66,914, and UK-68,798) on myocardial refractoriness and contractility were compared to those of d-sotalol in ferret isometrically contracting right ventricular papillary muscle preparations. During 1 Hz pacing at 37 degrees C, the four class III agents elicited concentration-dependent increases in ventricular effective refractory period (ERP), with a relative order of potency of UK-68,798 greater than E-4031 greater than UK-66,914 much greater than d-sotalol. EC25 values (effective concentration required to increase ERP 25% above baseline) were (in microM) UK-68,798, 0.018; E-4031, 0.058; UK-66,914, 0.501; and d-sotalol, 43.76. Maximal increases in ERP relative to baseline (% of baseline value) for the class III agents at 37 degrees C (range of 44.5 +/- 4.5 to 63.0 +/- 3.1%) were greater than the maximal increases observed at 27 degrees C (range of 15.0 +/- 3.3 to 31.2 +/- 4.8%), whereas the maximal absolute (ms) increases in ERP above baseline were comparable for the class III agents at both temperatures. Increases in ERP produced by the four class III agents at 37 degrees C were significantly greater at a pacing frequency of 1 Hz (range of 70.0 +/- 7.6 to 102.0 +/- 2.3 ms) than at 3 Hz (range of 18.3 +/- 4.4 to 31.BBB/- 4.8 ms). During a temporary period of hypoxic perfusion at 37 degrees C, increases in ERP produced by the four class III agents were reversed, such that "hypoxic" ERP values approximated pretreatment, baseline values.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720842 TI - Investigation of electrophysiologic mechanisms for the antiarrhythmic actions of R 56865 in cardiac glycoside toxicity. AB - R 56865 is an experimental compound that has been shown to ameliorate the effects of cardiac glycoside toxicity and myocardial ischemia. We evaluated the direct electrophysiological effects of R 56865 and its effects on the electrophysiological sequelae of ouabain toxicity in vivo and in vitro. In normal anesthetized dogs, R 56865 alone at doses of 0.04 to 0.16 mg/kg i.v. had no effect on atrial, AV nodal, or ventricular conduction times and refractoriness, but at doses of 0.64 to 2.5 mg/kg it tended to increase these parameters. In ouabain-pretreated dogs, R 56865 (0.08 to 0.32 mg/kg i.v.) dose-relatedly reduced ouabain-induced ventricular arrhythmias. In normal isolated canine Purkinje fibers, R 56865 (1-10 microM) reduced Vmax at short pacing cycle lengths and decreased the action potential duration at concentrations of 0.1 to 10 microM. R 56865 at concentrations through 10 microM had no significant effect on normal action potentials of canine ventricular muscle and slow response action potentials in guinea pig papillary muscles. In Purkinje fibers exposed to toxic concentrations of ouabain, R 56865 (1 microM) reduced the delayed after depolarization (DAD) amplitude and inhibited triggered activity. R 56865 had no effect on normal automaticity in canine Purkinje fibers at 1 microM, but 10 microM significantly slowed it. R 56865 at 10 microM did not affect isoproterenol enhanced automaticity and only slightly reduced barium-induced abnormal automaticity that occurred at reduced membrane potentials. These results demonstrate that R 56865 reverses cardiac glycoside-induced arrhythmias in anesthetized dogs at doses that do not significantly affect conduction or refractoriness. Suppression of ouabain-induced DAD and triggered activity in isolated Purkinje fibers, at concentrations not affecting normal or abnormal automaticity, may be the mechanism of R 56865's antiarrhythmic actions in vivo. Suppression of DAD does not appear to be associated with blockade of voltage dependent calcium channels, but R 56865 may prevent intracellular sodium overload by limiting excessive sodium entry during ouabain intoxication. PMID- 1720844 TI - Alterations of intracellular calcium homeostasis and myocardial energetics in acute adriamycin-induced heart failure. AB - To elucidate the mechanism of acute contractile failure induced by adriamycin, the intracellular concentrations of free calcium ([Ca2+]i) and energy-related phosphate compounds were determined in isolated ferret hearts. The time-averaged [Ca2+]i was measured at 10 min resolution using fluorine nuclear magnetic resonance (NMR) spectroscopy and the NMR-sensitive Ca2+ indicator 5F-BAPTA. [Ca2+]i significantly increased from a control of 381 +/- 66 nM (mean +/- SEM, N = 5) to 789 +/- 171 nM during 30 min of perfusion with adriamycin (30 mg/L), and remained elevated for at least 30 min after washout. The isovolumic LV pressure decreased to 80.7 +/- 8.9% of control (N = 12, p less than 0.05) and did not recover after washout. Intramyocardial contents of energy-related phosphates were determined by phosphorus NMR spectroscopy in seven other hearts. No significant change in myocardial energy metabolism was observed during adriamycin exposure and after washout; inorganic phosphate did not increase, and phosphocreatine and ATP did not decrease. These results indicate that Ca overload induced by adriamycin is associated with acute contractile failure. Adriamycin has been reported to inhibit Na-Ca exchange and to affect the gating of Ca2+ release channels in sarcoplasmic reticulum. Whatever the cause of the calcium overload, the fact that dysfunction persists as an aftereffect of adriamycin is consistent with the hypothesis that calcium overload, in the absence of ischemia, can leave behind long-lasting contractile dysfunction. PMID- 1720843 TI - Converting enzyme inhibitors and the role of the sulfhydryl group in the potentiation of exo- and endogenous nitrovasodilators. AB - In this study, the effect of bradykinin on coronary flow in the isolated rat heart was significantly potentiated when cysteine or the sulfhydryl-containing converting enzyme inhibitors captopril and zofenoprilat were administered simultaneously. In contrast, the effect of concomitant administration of enalaprilat only slightly increased the effect of bradykinin on coronary flow. In nitrate-tolerant hearts of rats pretreated with isosorbide dinitrate (15 mg daily), the increase in coronary flow by nitroglycerin and bradykinin was significantly less when compared to control hearts. The effect of captopril was not affected by pretreatment. The involvement of endothelium-derived relaxing factor (EDRF) in the effect of captopril was apparent from experiments with L arginine, the precursor of EDRF, and L-NMMA, the "false" precursor of EDRF. L Arginine increased the effect of captopril, whereas L-NMMA showed a competitive antagonism for the effect of captopril on coronary flow in the isolated rat heart. Clinically, the effect of captopril was studied in 10 patients with stable, exercise-induced angina pectoris that had been treated for 3 weeks with slow-release isosorbide dinitrate (20 mg four times daily). At day 7, a baseline exercise test was obtained. Subsequently, patients with chest pain and at least 1 mm ST-segment depression on the ECG during exercise were included. They received on day 14 and 21 either captopril (25 mg) or placebo 1 h before exercise testing in a randomized, double-blind, crossover design. Captopril significantly improved the combined score of maximal ST-segment depression, maximal workload, and time to angina when compared to placebo. No differences in the pressure-rate index at rest and during exercise were seen. These results indicate that the sulfhydryl group of certain angiotensin converting enzyme inhibitors can potentiate their effect on the endogenous nitrovasodilator EDRF. In the clinical situation, this may lead to an improved exercise performance in patients with stable angina pectoris during chronic nitrate treatment, independent of its systemic vascular effects. PMID- 1720845 TI - Influence of tedisamil on the initiation and maintenance of ventricular fibrillation: chemical defibrillation by Ito blockade? AB - We examined the effects of tedisamil on ventricular fibrillation (VF) elicited by regional ischemia and by reperfusion in isolated rat hearts (n = 12/group). During 30 min of ischemia, 0.1, 0.55, and 3 microM tedisamil had no influence on the incidence of VF (an index of VF initiation). However, sustained VF (SVF, defined as that lasting greater than 120 s, an index of VF maintenance) was reduced in a concentration-dependent manner from 73 to 54, 17 (p less than 0.05), and 0% (p less than 0.05), respectively. Tedisamil caused sinus bradycardia but this was not the basis for tedisamil's antiarrhythmic activity since SVF was also inhibited in separate groups of hearts that were paced throughout the study. Tedisamil had no effect on reperfusion-induced VF initiation. However, VF maintenance was, again, inhibited, with SVF incidence reduced from 75% to 40, 20, and 0% (p less than 0.05), respectively, by increasing concentrations of tedisamil. A similar effect was, again, observed in paced hearts. The average cycle length of the electrogram during VF correlated with preceding width of the ventricular complex; both of these variables were concentration-dependently increased by tedisamil and mean values of each correlated inversely with the incidence of SVF. The ratio of SVF cycle length to ventricular tachycardia cycle length was 1:3, and this ratio was conserved in the presence and absence of drug. The data are consistent with a drug-induced increase in the probability of spontaneous termination of multiple wave-front re-entry. PMID- 1720846 TI - Lisinopril combined with atenolol in the treatment of hypertension. Swedish Lisinopril Study Group. AB - This is a double-blind, randomized parallel-group multicenter study comprising 340 hypertensive patients. All were treated with 50 mg of atenolol once daily. If their recumbent diastolic blood pressure was greater than or equal to 95 and less than or equal to 115 mm Hg after a 4-week open run-in period, patients were randomized to receive additionally, 5, 10, or 20 mg of lisinopril once daily or placebo for a further 8-week period in a double-blind fashion. The additional effects on the trough (24-28 h after tablet intake) diastolic blood pressure of placebo and 5 mg of lisinopril were not statistically significant. Lisinopril at 10 and 20 mg reduced the recumbent diastolic blood pressure by 3.2 and 3.3 mm Hg, respectively, more than placebo (p = 0.010-0.011). The recumbent systolic blood pressure and heart rate did not change. The side effect profile of lisinopril was not different from that of placebo and adverse effects were few and mild. PMID- 1720847 TI - Effect of angiotensin converting enzyme inhibition on the menstrual cycle of hypertensive women. AB - Angiotensin II was reported to play a key role in ovulation in rats and it seems also to be involved in the regulation of LH release. Thus, we studied the effect of chronic ACE inhibition on the menstrual cycle, measuring daily plasma estradiol, progesterone, LH and FSH, and renin and prorenin before and during the third month of treatment with enalapril (10 mg b.i.d.) in 10 mild essential hypertensive women. Blood pressure was normalized by treatment. The cyclical changes of steroids and gonadotrophins were unaffected in their temporal relationships and in the magnitude of their variation during the experimental cycle compared with the basal cycle. A synchronization of plasma prorenin with the other hormones was seen both before, as previously reported, and during enalapril treatment. Our data show that peripheral blockade of angiotensin I conversion does not affect the pituitary guidance of the ovarian hormonal response or the ovarian prorenin release during the menstrual cycle. Our data are in agreement with the hypothesis that circulating angiotensin II does not play a key role in the human fertility process and that hydrophilic ACE inhibitors can be safely used in the treatment of hypertensive women of reproductive age. PMID- 1720848 TI - Postsynaptic dopamine DA1- and DA2-receptors in jejunal arteries of rabbits. AB - Changes in the external diameter of rabbit isolated jejunal arteries were measured by a photoelectric device. Both norepinephrine and dopamine produced vasoconstriction; prazosin, but not idazoxan, antagonized these effects. Arteries preconstricted with norepinephrine were dilated by SKF 38393 and LY 171555. SCH 23390 antagonized both compounds, however, in the case of LY 171555 with a lower potency. Sulpiride also interacted with LY 171555. When arteries were preconstricted with PGF2 alpha in the presence of prazosin, idazoxan, and propranolol, SKF 38393 caused vasodilation, while LY 171555 and dopamine were inactive. We conclude that in jejunal arteries both DA1- and DA2-receptors may be present at the vascular smooth muscle. PMID- 1720849 TI - Effect of IgE-stimulated alveolar macrophages on tracheal epithelial bioelectric properties in dogs. AB - To investigate a possible interaction between pulmonary alveolar macrophages (AMs) and airway epithelial cells in patients with allergic conditions, we studied the effect of AMs on bioelectric properties of canine tracheal epithelium under short-circuited conditions in vitro. Mucosal addition of the supernatants from AMs stimulated with monoclonal antidinitrophenyl (DNP) IgE antibody and DNP human serum albumin (DNP-HSA) increased short-circuit current (Isc) of cultured epithelium in a dose-dependent manner. The maximal increase from the baseline value and the EC50 were 10.2 +/- 2.0 microA/cm2 (mean +/- SE, p less than 0.01) and 3 x 10(5) AMs/ml, respectively. This effect was accompanied by the release of prostaglandin E2 and F2 alpha from AMs. In contrast, AMs incubated with anti-DNP IgE antibody alone or DNP-HSA alone had no effect. The AM-induced increase in Isc was attenuated by diphenylamine-2-carboxylate and Cl-free medium but not by amiloride. Pretreatment of AMs with indomethacin or piroxicam inhibited the effect of AMs on epithelial Isc. These results suggest that AMs may stimulate Cl secretion across the airway mucosa through an IgE-dependent release of prostaglandins. PMID- 1720850 TI - Double atrial parasystole showing intermittent trigeminy. AB - Atrial parasystole arising from two different ectopic atrial foci, namely, double atrial parasystole, showed intermittent trigeminy due to 3:1 exit block, in which both the ectopic atrial rates were around the sinus rate. Atrial trigeminy showing positive P waves in leads II and III continued, then intermittent atrial parasystole with negative P waves in leads II and III took over. The double atrial parasystole was considered to be interspersed with reentrant atrial beats. Other possible mechanisms to cause a disruptive influence on the trigeminy of the atrial parasystole with positive P waves in leads II and III are: (a) concealed intraatrial reentry with resetting by the sinus impulse; and (b) delayed capture by the impulse of the third preceding sinus beat, and the subsequent 3:1 exit block. On the other hand, the atrial parasystole with negative P waves in leads II and III tended to show long interectopic intervals, in which some of the parasystolic beats exhibited a coupling to the third preceding sinus beat. This suggested prompt resetting by the sinus impulse and the ensuing 3:1 exit block. PMID- 1720851 TI - Late toxic effects of long-term exposure to lindane in peripheral blood cells in rabbits. I. Function impairment and structural disturbances in leucocytes. AB - Following lindane intoxication in daily doses of 0.1 LD50 during one month (total dose 2.0 LD50) functional changes were noted in granulocytes and structural changes in lymphocytes in peripheral blood of rabbits during 4 months after completion of lindane administration. A significant decrease of the phagocytic activity with a rise in the number of non-phagocytizing granulocytes, quantitative and qualitative changes of nucleoli and lysosomes were found in lymphocytes. The latest effect of lindane is regarded to be a raised number of non-phagocytizing granulocytes 4 months after withdrawal of the pesticide. PMID- 1720852 TI - Late toxic effects of long-term exposure to lindane in peripheral blood cells in rabbits. II. Erythrocyte changes. AB - After lindane administration in daily doses of 0.1 LD50 for one month (total dose 2.0 LD50) changes in the erythrocytes were found persisting for up to 4 months after withdrawal of the pesticide. A significant increase in the number of echinocytes, a greater per cent of reticulocytes, significantly raised level of denatured haemoglobin, and an increased number of erythrocytes of higher sensitivity to oxidating factors in relation to control group was observed. PMID- 1720853 TI - Dexfenfluramine neurotoxicity in brains of non-human primates. AB - Dexfenfluramine, a drug prescribed for appetite suppression, was evaluated in non human primates for its potential to produce toxic effects on brain serotonin (5 HT) neurons. Squirrel monkeys received dexfenfluramine subcutaneously twice daily for four days at doses of 1.25 or 5.00 mg/kg. Two weeks later, a dose-related depletion of 5-HT and 5-hydroxyindoleacetic acid was found, together with a reduced number of 5-HT uptake sites. Morphological studies showed acute pathological changes in 5-HT axons, followed by a persistent decrease in 5-HT axon density. Our findings indicate that dexfenfluramine damages central 5-HT neurons in monkeys and raise concern about the potential neurotoxicity of this drug in man. PMID- 1720854 TI - Pseudomonas cepacia in inpatients with cystic fibrosis. PMID- 1720855 TI - p53 immunostaining in benign breast disease. PMID- 1720856 TI - Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. AB - Hypotension during septic shock, which may reflect increased synthesis of the potent vasodilator nitric oxide (NO), is often refractory to vasoconstrictors. We describe the effects of NO synthase inhibition in two patients with life threatening septic shock in whom conventional therapy had failed to restore blood pressure. NG-monomethyl-L-arginine (L-NMMA) caused dose-dependent increases in blood pressure and systemic vascular resistance in both patients, and a similar effect was observed in the second patient after treatment with NG-nitro-L arginine methyl ester (L-NAME). These findings indicate that NO synthase induction contributes to the pathogenesis of septic shock, and that inhibition of NO synthase may represent a novel therapeutic option. PMID- 1720857 TI - TALC direct recording scales. PMID- 1720858 TI - Suppression of low dose streptozotocin induced diabetes in mice by administration of a nitric oxide synthase inhibitor. AB - Nitric oxide has recently been identified as the primary toxic effector molecule in the lysis of islet cells by inflammatory macrophages. We show here that N nitro-L-arginine-methylester (NAME), an inhibitor of endothelial and macrophage NO synthase partially suppresses diabetes development in the low dose streptozotocin induced diabetes model in C57BL/6J mice. Mean blood glucose levels were lower in the group receiving NAME throughout the observation period of 30d (p less than 0.05-0.001). Similar concentrations of NAME as expected in vivo were tested in vitro in macrophage-islet cell cocultures and were found to partially suppress NO production and islet cell lysis. We conclude that NO synthase activity is a pathogenetic factor in diabetes development. PMID- 1720859 TI - Demonstration of nucleomorph-encoded eukaryotic small subunit ribosomal RNA in cryptomonads. AB - In cryptomonads, unicellular phototrophic flagellates, the plastid(s) is (are) located in a special narrow compartment which is bordered by two membranes; it harbours neither mitochondria nor Golgi dictyosomes but comprises eukaryotic ribosomes and starch grains together with a small organelle called the nucleomorph. The nucleomorph contains DNA and is surrounded by a double membrane with pores. It is thought to be the vestigial nucleus of a phototrophic eukaryotic endosymbiont. Cryptomonads are therefore supposed to represent an intermediate state in the evolution of complex plastids from endosymbionts. We have succeeded in isolating pure nucleomorph fractions, and can thus provide, using pulsed field gel electrophoresis, polymerase chain reaction and sequence analysis, definitive proof for the eukaryotic nature of the symbiont and its phylogenetic origin. PMID- 1720860 TI - Structure and expression of the triose phosphate isomerase (Tpi) gene of Drosophila melanogaster. AB - We report the isolation of the genomic sequence that encodes the enzyme triose phosphate isomerase of Drosophila melanogaster. There is a single copy of the Tpi sequence in the genome of Drosophila, as judged by Southern blots and in situ hybridization to salivary gland chromosomes. The sequence of 3414 nucleotides from the Tpi region was determined. The gene has an intron in the 5' untranslated region of the transcript and a second intron in the coding region at an evolutionarily conserved position. Transcripts initiate at a single site which does not have a TATA box in the usual position. Northern blot analysis of RNA prepared from different developmental stages revealed that Tpi mRNA is present in substantial amounts in oocytes, declines in abundance in early embryos, and begins to increase during mid-embryogenesis. Transcript abundance follows a pattern typical of enzymes involved in intermediate metabolism. A peak is found during third instar followed by a decline during pupal stages and then a second rise near the time of eclosion. PMID- 1720861 TI - Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli. AB - The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced. The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41,281 daltons) and EnvD (104,415 daltons). The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane. The detection of a common transcript for both ORFs indicated the existence of an envCD operon. Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61. Overproduction of EnvC protein appears to be lethal to Escherichia coli. The envD gene, however, could be cloned and expressed at high levels under control of the tac promoter without deleterious effects on the host. PMID- 1720862 TI - Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype. AB - We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon omega failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were FixRed, but on Vigna unguiculata were Fix-. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose. PMID- 1720863 TI - Regulation of replication of plasmid R1: an analysis of the intergenic region between copA and repA. AB - The synthesis of the rate-limiting RepA replication initiator protein of plasmid R1 is negatively controlled by an antisense RNA, CopA. The regulation is posttranscriptional and involves an inhibitory effect on RepA translation mediated by the binding of CopA to its target (CopT) in the leader region of the RepA mRNA. The evolutionary conservation of the intergenic region between the copA gene and the repA reading frame among plasmids related to R1 may be indicative of an important function in this regulation. One possibility is that sequences/structures in this region might be required for the presumed distal effect of CopAQCopT binding. We have performed a mutational analysis of this region, starting with a mutant repA-lacZ fusion plasmid that shows decreased RepA LacZ synthesis compared to a wild-type construct, and have identified five compensatory mutations that increase repA-lacZ expression. Two of these were single base-pair substitutions in the copA promoter leading to a decrease in CopA transcription. The other three mutations increased RepA synthesis in the presence as well as in the absence of functional CopA. Reconstructed plasmids carrying these mutations--in conjunction with the original down-mutation or in an otherwise wild-type background--show the expected increase in copy number. The effect of two of these mutations is consistent with the destabilization of a putative secondary structure which may be responsible for the normally low translation rate of the RepA reading frame. The implications of the types of mutations found in this study, as well as the absence of other classes of mutations, are discussed in terms of alternative possible models of CopA-mediated inhibition of RepA synthesis. PMID- 1720864 TI - Structure of the Hordeum vulgare gene encoding dihydroflavonol-4-reductase and molecular analysis of ant18 mutants blocked in flavonoid synthesis. AB - A full-length cDNA clone encoding barley dihydroflavonol-4-reductase was isolated from a kernel-specific cDNA library by screening with the cDNA of the structural gene (A1) for this enzyme from maize. Subsequently, the gene corresponding to the barley dihydroflavonol-4-reductase cDNA was cloned and sequenced. The gene contains three introns at the same positions as in the Zea mays gene, corresponding to the positions of the first three of the five introns present in the genes of Petunia hybrida and Antirrhinum majus. In vitro transcription and translation of the Hordeum vulgare cDNA clone yielded a protein which converts dihydroquercetin into 2,3-trans-3,4-cis-leucocyanidin with NADPH as cofactor. The protein has a deduced amino acid sequence of 354 residues and a molecular weight of 38,400 daltons. Dihydroflavonol reductases of barley, maize, petunia and snapdragon are highly polymorphic in the NH2- and C-terminal parts of the polypeptide chain while a central region of 324 residues contains 51% identical amino acids. This identity increases to 81% when only the barley and maize enzymes are compared. Recessive mutants in the Ant18 gene tested so far lack dihydroflavonol-4-reductase activity and accumulate small amounts of dihydroquercetin but have retained activity for at least two other enzymes in the flavonoid pathway. In testa-pericarp tissue of mutants ant18-159, ant18-162 and ant18-164, wild-type levels of steady state mRNA for dihydroflavonol reductase have been measured, while mRNA for this enzyme is not transcribed in mutant ant18 161. These data are consistent with the proposal that the Ant18 locus carries the structural gene for dihydroflavonol-4-reductase of barley. PMID- 1720866 TI - Ernst Freese (1925-1990). PMID- 1720865 TI - Analysis of interferon-gamma resistant mutants that are possibly defective in their signaling mechanism. AB - Our previous observations indicated that mutants partially resistant to IFN-gamma cytotoxicity were defective in the induction of indoleamine 2,3-dioxygenase, (IDO). Two mutants highly resistant to IFN-gamma were isolated following a second round of mutagenesis. The resistance to IFN-gamma was inversely correlated with the inducibility of IDO in these mutants. Moreover, several other IFN-gamma responsive genes, including those encoding 2-5A synthetase, GTP cyclohydrolase and HLA-DR alpha, were also differentially altered in their expression upon INF gamma treatment. IFN-gamma receptor gene expression was not changed nor was the binding of the receptor to IFN-gamma. Southern blot analysis failed to reveal any significant abnormality in the IDO gene structure in these mutants. We therefore suggest that these mutants are defective in the IFN-gamma signaling pathway and will be useful in further analysis of the biochemical mechanism of IFN-gamma activated gene expression in target cells. PMID- 1720867 TI - Cytogenetic characterization of radiosensitive mouse mutants. AB - In order to develop mouse models for human mutagen-sensitive syndromes, we carried out cytogenetic characterization of several mouse mutants and MS/Ae mice showing enhanced radiosensitivities. The applied cytogenetic techniques include chromosomal analysis of in vitro cell cultures and lymphocyte cultures as well as in vivo UDS in hepatocytes, induction of micronuclei in polychromatic erythrocytes and translocation induction in spermatogonial stem cells. Among the mutations studied, namely the contrasted allele of steel (Slcon), viable dominant spotting (Wc), wasted (wst), varitint-waddler (Va) and dystonia musculorum (dt) as well as MS/Ae mice, various iso-, hyper- or hypo-sensitive conditions were recorded. Only Va and dt appear to be associated with some deficiency in DNA repair. PMID- 1720868 TI - Adaptive response in irradiated human lymphocytes: radiobiological and genetical aspects. AB - The adaptive response (AR) in human lymphocytes in different experimental protocols was investigated. The AR was found to be present in cells pre-exposed to 3 cGy of X-rays in G0, G1 and S phase as well as with tritiated water (4 muCi/ml) when the 'challenge' dose was given in G2. There was no AR after prior exposure of the cells in S phase to secondary irradiation from 70 GeV protons. The AR was not observed after preliminary X-irradiation of the lymphocytes in G0 and G1 and 'challenge' irradiation in G1. Cells from 6 patients with Down's syndrome were tested. At least 5 of them did not show the AR. The AR is considered to be a phenomenon of the antimutagenic aftereffect. PMID- 1720869 TI - In vitro mutagenesis as a result of 60Co-gamma-ray-induced base damage. AB - We utilized a model system to study the mechanism(s) of mutation resulting from gamma-ray-induced DNA base damage. 60Co-irradiated, uracil-containing M13mp2 DNA was hybridized to normal (non-uracil) linearized double-stranded virus DNA minus the lac reporter region. Only DNA without strand breaks in the reporter region will circularize. This DNA was used as a substrate for a modified T7 DNA polymerase with no residual 3'----5' exonuclease activity (Sequenase 2). The reaction product was transfected into a rec- bacterial host to minimize the occurrence of bypass events in vivo, and mutant progeny were selected. DNA irradiated with 400 or 800 Gy from a 60Co gamma-source gave about a 5-fold increase in the percentage of mutants recovered after synthesis with Sequenase as compared to the recovery of mutants using control DNA. About 20% of the mutants recovered from both irradiated and control templates contained multiple mutations in the target area sequenced. The irradiated samples had an excess of mutations which resulted from changes at pyrimidines. C---T transitions were most common. Mutations at T were mostly (-1) and (-2) frameshifts, particularly at sequences of repeated T's. PMID- 1720871 TI - Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli. AB - Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5' pyrimidine, 3'-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts. PMID- 1720870 TI - Genetic assay of misincorporation. AB - A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13 bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13 lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3' terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper. PMID- 1720872 TI - In vitro effects of L-ascorbic acid (vitamin C) on aryl hydrocarbon hydroxylase activity in hepatic microsomes of mice. AB - When aromatic hydrocarbon (Ah)-responsive and -non-responsive strains of mice were pretreated with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD), vitamin C reduced the microsomal aryl hydrocarbon hydroxylase (AHH) activity. The AHH inhibitors 7,8-benzoflavone (7,8-BF) and 3-methylsulfonyl 3',4,4',5-tetrachlorobiphenyl (3-MSF-3',4,4',5-tetraCB) showed various inhibitory effects depending upon the types of microsomes, whereas vitamin C exhibited inhibition irrespective of the types of microsomes. 7,8-BF and 3-MSF-3',4,4',5 tetraCB as well as vitamin C suppressed the reverse mutation of the Salmonella typhimurium tester strains TA98 and TA100 induced by benzo[a]pyrene. PMID- 1720873 TI - Antimutagenicity of propionic acid bacteria. AB - The antimutagenic effect of dialysed cell extracts of 4 strains of propionic acid bacteria was examined against the mutagenicity of sodium azide in the TA1535 tester strain of Salmonella typhimurium using the Ames test. It was noted that dialysates of 2 strains of Propionibacterium shermanii, P. pentosaceum and P. acnes, significantly reduced sodium azide-induced revertants. The dialysate of propionic acid cocci did not show an antimutagenic effect. The inhibitory activity was enhanced if the mutagen and extract were coincubated for 20 min prior to performing the mutagenicity assay. Antimutagenicity of dialysates from P. shermanii VKM-103 against MNNG and 9-aminoacridine was shown in S. typhimurium strains TA1535 and TA97. The antimutagenic activity was found in the protein fraction of the cell extract of P. shermanii. The proteins of the dialysate of P. shermanii were separated using a Toyopearl gel column into 3 main peaks according to their molecular weights. The antimutagenic activity towards sodium azide was found in the second and the third peaks. We suggest that dialysates of the cells of propionic acid bacteria contain several kinds of antimutagenic substances with different molecular weights. PMID- 1720874 TI - Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes. AB - The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population. PMID- 1720875 TI - An observation on hitherto unknown corticospinal fibers that descend between the tractus corticospinalis lateralis and ventralis in the cat. AB - Following injection of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the pericruciate (sensorimotor) cortex on one side in the cat, we found labeled fibers surrounding the ventral horn of the spinal cord bilaterally, in addition to the crossed and the uncrossed lateral, ventral and dorsal corticospinal tracts (CSTs). These hitherto unknown fibers, which could be traced to the caudal end of the L2 segment, were predominantly contralateral to the WGA HRP injection side. This study indicates the presence of newly recognized CST fibers in the cat. PMID- 1720876 TI - Changes of substance P and somatostatin contents in the gastrointestinal tract of streptozotocin diabetic rats. AB - Substance P and somatostatin contents were measured in the gastrointestinal tract of streptozotocin diabetic rats, 1 month after streptozotocin administration (60 mg/kg), and of age-matched controls with radioimmunoassay. Substance P and somatostatin contents were statistically increased in the extrafundus of the diabetic stomach, but not in the diabetic fundus. Substance P was significantly decreased in the diabetic ileum and caecum. Similarly, somatostatin was decreased in the diabetic caecum. Contrarily, slight increase of somatostatin contents in the diabetic duodenum, jejunum and proximal colon was not statistically significant. PMID- 1720877 TI - Concentration jump studies of intracellularly dialysed Xenopus oocytes show desensitization of kainate receptors. AB - The techniques of intracellular dialysis and 'concentration jump' have been applied to Xenopus oocytes injected with rat brain RNA. Changes in the internal ionic environment of the oocyte through intracellular perfusion via patch pipettes, in whole cell recording mode, led to expected changes in the reversal potential for current induced during application of L-kainic acid. The responses to this amino acid were also studied using the agonist 'concentration jump' technique. Dose-response relationships for L-kainate were similar to those obtained with oocyte superfusion, but the L-kainate-induced currents exhibited desensitization. PMID- 1720878 TI - Evidence for an involvement of eicosanoids in neurokinin3-receptor mediated acetylcholine release from myenteric neurons. AB - The release of acetylcholine (ACh) from myenteric plexus evoked by 5 hydroxytryptamine (5-HT) and senktide (a selective neurokinin3 (NK3) agonist) was depressed by mepacrine, an inhibitor for phospholipase A2 activity. Release of ACh was stimulated by arachidonic acid; this release was partially depressed by nordihydroguaiaretic acid (NDGA), which inhibits lipoxygenase activity. NDGA failed to modify the ACh secretion elicited by 5-HT. Release of ACh evoked by senktide was significantly inhibited by NDGA, suggesting an involvement of eicosanoids in the release of ACh elicited by specific neurokinin receptors in myenteric neurons. PMID- 1720879 TI - Striatal dopamine metabolism increases during long-term haloperidol administration in rats but shows tolerance in response to acute challenge with raclopride. AB - The release and metabolism of dopamine (DA) in the striatum of rats during long term haloperidol administration (32 weeks) was assessed using in vivo microdialysis. Basal levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) were significantly elevated over control values, while basal DA release was not significantly increased. The specific DA D2 receptor antagonist, raclopride (0.5 mg/kg, i.p.), increased DA release and metabolism in control animals, but this effect was profoundly blocked in the haloperidol treated group. These results suggest that chronic haloperidol treatment may induce compensatory increases in basal DA activity even though response to an acute D2 antagonist shows significant tolerance. PMID- 1720880 TI - Identification of myenteric neurons which project to the mucosa of the guinea-pig small intestine. AB - Myenteric neurons which innervate the mucosa of the guinea-pig ileum were characterized by combining retrograde transport of DiI in vitro with immunohistochemistry. Of DiI-labelled myenteric neurons, 43% were immunoreactive for calbindin and substance P, 25% were immunoreactive for calbindin alone, and 18% were immunoreactive for substance P alone. These 3 classes of neurons had Dogiel Type II morphology and are probably sensory neurons. Two classes of probable secretomotor neurons were characterized by immunoreactivity for neuropeptide Y (4%) and vasoactive intestinal peptide (2%). These 5 classes of myenteric neurons represent over 90% of the retrogradely labelled myenteric neurons that project to the mucosa. PMID- 1720881 TI - Role of NK1 tachykinin receptors in thermonociception: effect of (+/-)-CP 96,345, a non-peptide substance P antagonist, on the hot plate test in mice. AB - We have tested the ability of (+/-)-CP 96,345, a novel nonpeptide substance P (SP) antagonist, to block the aversive behaviour induced by intrathecal (i.t.) administration of SP and to induce thermal antinociception in mice. (+/-)-CP 96,345 administered i.t. or i.p. selectively blocked the effect of i.t. SP while leaving the response to i.t. bombesin unaffected. At the same dose proven effective against i.t. SP, (+/-)-CP 96,345 produced thermal analgesia in the hot plate test (52 degrees C). Using isolated organs for bioassay evaluation of activity at tachykinin receptor, (+/-)-CP 96,345 was found to be a potent (pA2 8.11, c.l. 7.9-8.3) and competitive NK1 receptor antagonist while it was devoid of activity at NK2 or NK3 receptors. These findings provide clear indication for the participation of SP, via NK1 receptors, in thermal nociception. PMID- 1720883 TI - Clinical management and diagnostic possibilities in hydatidiform mole with coexistent fetus. AB - The goal of the current review is to present the figures essential for counseling, when hydatidiform mole and normal fetus occur together. Previous reviews of prognosis and risks when mole and fetus are observed together did not adjust for differences in genetic constitution and thus varying risks for gynecologic and obstetric complications. A literature search from 1903 to 1989 revealed 113 reports of pregnancies with mole and fetus in which there appeared to be no major malformations or cytogenetic abnormalities; 87 of those were intended to continue. This group provides the most appropriate risk figures, when mole and karyotypically normal child are detected by first or second trimester prenatal diagnosis. Fifty-two pregnancies (59.8 per cent) proceeded to the 28th week without spontaneous abortion or interruption of pregnancy. None of the children delivered before week 28 survived. Of the pregnancies continuing beyond this time 69.2 per cent of the children survived, 7.7 per cent were live-born with unknown long-term outcome, 17.3 per cent died neonatally, and 5.8 per cent succumbed before delivery. Persistent trophoblastic disease was reported in 19.2 per cent of pregnancies interrupted at diagnosis, as well as in 9.1 per cent of those intended to continue. Due to advances in prenatal diagnosis, clinicians will be confronted with counseling in pregnancies with mole and fetus more often than expected from the literature. Chorionic villus biopsy or amniocentesis can disclose those triploid gestations without possibility of a surviving child. First trimester ultrasound demonstration of a partially cystic placenta and abnormal high se-hCG values should initiate prenatal diagnosis for evaluation of the fetal karyotype, before deciding whether to abort or continue the pregnancy. PMID- 1720882 TI - Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: a model for neurotoxicological studies. AB - The increased desire to use in vitro techniques in neurotoxicology has resulted in the search for clonal cell lines which may be useful for studying disruption by neurotoxicants of various aspects of neuronal physiology and biochemistry. One such cell line is the PC12 cell, a clonal cell line derived from a pheochromocytoma of the rat adrenal medulla. When cultured under normal conditions, PC12 cells resemble adrenal chromaffin cells in morphology, physiology and biochemistry. However, when cultured in the presence of nerve growth factor (NGF) or several other compounds, PC12 cells differentiate to resemble sympathetic neurons morphologically and functionally. Differentiation and the resultant physiological and biochemical changes are some of the most attractive and useful features of this cell line. PC12 cells release, depending on the conditions, dopamine, norepinephrine and acetylcholine and contain Na, K and Ca channels and other membrane receptors, including receptors coupled to G proteins. Moreover, the relative proportion of various subtypes of Ca channels changes during differentiation. Thus, PC12 cells provide an excellent model for studying chemical disruption of processes associated with neuronal differentiation, synthesis, storage and release of neurotransmitters, function and regulation of ion channels and interactions of compounds with membrane bound receptors. The ability of PC12 cells to differentiate in response to NGF and other compounds allows for selective expression of certain channels and proteins and for comparisons of responses in undifferentiated and differentiated cells. The prominent neurotoxicant methylmercury causes potent reductions in uptake of 45Ca and binding of ligands associated with various subpopulations of Ca channels in the PC12 cells, as well as currents carried through putative Ca channels. PMID- 1720884 TI - Coagulation disorders and tumor markers in the diagnosis of pancreatic cancer. AB - Twenty patients (42-80 years old of whom 9 women) affected by instrumentally ascertained pancreatic cancer (7 cases were operated on) were studied. In all of them the following coagulation indices (fibrinopeptide A, FpA; beta thromboglobulin, BTG; platelet factor IV, PF4; fibrinogen degradation products, XDP) and tumor markers (gastrointestinal cancer associated antigen, GICA; tissue polypeptide antigen, TPA; carcinoembryonic antigen, CEA; alpha-fetoprotein, or AFP) were assessed at the time of diagnosis, and 10 and 30 days after diagnosis, to test whether and which of the above parameters are more sensitive for entertaining the underlying affection. In both operated and nonoperated patients FpA was shown to be the most sensitive index. Lesser sensitivity was shown by XDP, GICA, and BTG. AFP proved to be quite useless as its serum levels constantly fell within the normal range. PMID- 1720885 TI - Importance of administration method in high-dose anticancer chemotherapy from toxicological standpoint in rats. AB - We examined death rates in Fisher rats when chemotherapeutic drugs were administered in a prescribed amount by either a single or divided dose. Lower death rates were observed when nimustine hydrochloride (ACNU) was given in a divided dose, or when cyclophosphamide (CY), vindesine sulfate (VDS) or vincristine (VCR) was administered as a single dose. Lower hematological toxicity was observed when ACNU was administered in 2 doses and CY in a single dose. With etoposide, we did not observe any significant difference between the two administration methods. In a combination study with ACNU and VDS, we observed lower toxic death rates when the dose of ACNU was split and VDS was given as a single dose. ACNU-induced death was substantially reduced by syngeneic bone marrow support or granulocyte colony-stimulating factor treatment. Our experiments indicate that the toxicity of the administration of chemotherapeutic agents may be significantly modified by altering the time course of administration. This would hopefully result in reducing the chemotherapy-related mortality associated with high-dose anticancer chemotherapy. PMID- 1720886 TI - Characterization and immunolocalization of an oocyst wall antigen of Cryptosporidium parvum (Protozoa: Apicomplexa). AB - A monoclonal antibody (OW-IGO) raised against purified excysted oocysts of Cryptosporidium parvum reacted in an immunofluorescence assay with the oocyst wall. The corresponding antigen was localized by immunoelectron microscopy in fibrillous material present in the parasitophorous vacuole of developing macrogametes and in the wall of both single and double layered sporulating oocysts. Gold particles were also detected over electron-lucent vesicles of the macrogametes by immunoelectron microscopy. On Western blotting of C. parvum oocyst extracts, major bands at 250 and 40 kDa and several minor components were recognized by Mab OW-IGO. Almost complete abolition of Western blot reactivity occurred after periodate oxidation of oocyst antigen, suggesting that monoclonal antibody OW-IGO reacts with a carbohydrate epitope. Taken together, our results suggest that a fibrillous glycoproteinic material is released in the parasitophorous vacuole from electron-lucent vesicles during gametogenesis, and later condensed in the oocyst wall. PMID- 1720887 TI - Acid phosphatase activity in microfilariae of Setaria labiato-papillosa and comparison with other blood microfilariae of dog and horse origin. AB - Acid phosphatase activity was demonstrated in smears of Setaria labiato-papillosa microfilariae by the naphthol AS-TR-phosphate method. The staining was restricted to 3 distinct sites, corresponding to the excretory pore, the inner body and the anal pore. This staining pattern was compared with those of Dirofilaria immitis, Dirofilaria repens and a Setaria of horse origin. PMID- 1720888 TI - C-reactive protein: a critical review. AB - We have reviewed the literature to determine the value of C-reactive protein (CRP) measurements in the diagnosis and management of a wide range of conditions. CRP levels are of value in 6 clinical situations: (a) monitoring the response to antibiotic treatment in patients with known bacterial infections, (b) in obstetric patients with premature rupture of membranes, a rise in CRP can give early warning of intrauterine infections, (c) differentiation between active disease and infections in patients with systemic lupus and ulcerative colitis where the level of response to active disease has been previously established, (d) as a measure of disease activity and response to disease-modifying drugs in rheumatoid arthritis, (e) early detection of complications in postoperative patients, (f) in differentiating between infection and graft-versus-host-disease in bone marrow transplant patients. CRP levels have been used in an attempt to differentiate between bacterial and viral infections in various clinical situations, however the published literature does not support this role. PMID- 1720889 TI - [Bilateral nodular pulmonary histoplasmosis: cytohistological correlation]. AB - Nodular bilateral pulmonary histoplasmosis: cyto-histological correlation. A case of nodular pulmonary histoplasmosis is reported. A 29 year old man was admitted to hospital with temperature and general weakness following a short stay in a tropical country. Laboratory investigation showed an increased E.S.R. and a routine chest-roentgenogram revealed multiple bilateral nodular lesions confirmed by CT scan. Smears obtained from fine-needle-aspiration biopsy showed the presence of epithelioid cell's clusters with a few giant-cells in a background of inflammatory elements and necrotic debris. The cytological picture was consistent with an inflammatory process with necrotizing granulomatous features. The clinical evolution and the radiological picture progression caused, nevertheless, suspicion of a metastatic tumor. The histological examination of a resected peripheral nodule confirmed the inflammatory nature of the process, revealing the presence of multiple roundish encapsulated conidia 2-4 microns in diameter scattered within a granulomatous and necrotic tissue. The fungi are clearly pinpointed by using special stains like Grocott method. Serological and microbiological investigations are necessary in order to confirm the diagnosis. PMID- 1720890 TI - Gastric syphilis simulating malignant lymphoma. AB - We report a case of syphilis of the stomach, occurring in a 50 year old man, which simulated malignant lymphoma microscopically and had also the endoscopic appearance of a malignancy. To the best of our knowledge this is the first report of gastric syphilis mimicking a lymphoma, and we believe that the recognition of this presentation is of particular importance to correctly set the disorder especially for therapeutic purposes and in order to avoid unnecessary surgery. PMID- 1720891 TI - The inhibitory action of caffeine on calcium currents in isolated intestinal smooth muscle cells. AB - The patch-clamp method has been used to investigate the action of caffeine on the calcium current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum. Caffeine (10 mM) substantially inhibited ICa. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis-(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K+ in the pipette solution, application of caffeine evoked a transient Ca-dependent K+ current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased ICa. Ryanodine had only little effect on ICa, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of ICa. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the ICa inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of ICa inhibition was probably due to a direct blocking action of caffeine on Ca channels. PMID- 1720892 TI - Interleukin-2 lengthens extrajunctional acetylcholine receptor channel open time in mammalian muscle cells. AB - The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000-3,000 units/ml). Cell attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07 +/- 0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system. PMID- 1720893 TI - Childhood pulmonary blastoma: a pleuropulmonary variant of the adult-type pulmonary blastoma. AB - Two fatal childhood cases of the rare pulmonary blastoma are reported. One was associated with a congenital cystic adenomatoid malformation. Both neoplasms extended to involve visceral pleura and were entirely composed of blastemal and mesenchymal elements without recognizable neoplastic epithelial components. The mesenchymal component in both instances consisted of malignant rhabdomyoblasts, undifferentiated mesenchyme, and differentiated, apparently benign, cartilage. Review of the literature suggests that these features may be specific for the childhood forms of pulmonary blastoma. It is further suggested that pulmonary blastoma, malignant mesenchymoma of the lung, and primary pulmonary rhabdomyosarcoma may have a common pathogenetic origin. PMID- 1720894 TI - The influence of semistarvation-induced hyperactivity on hypothalamic serotonin metabolism. AB - Male rats kept in a running wheel developed hyperactivity when food was restricted. Highest activity occurred around noon when food was given. Semistarved sedentary and ad lib fed sedentary and running rats served as controls. Five-hydroxyindole-acetic acid (5-HIAA) in the medial basal hypothalamus was lowest in the sedentary ad lib fed group. Running significantly increased 5-HIAA. Starvation likewise increased 5-HIAA. This effect was further enhanced by hyperactivity. When the circadian rhythm of serotonin (5-HT) and 5 HIAA was studied in the hypothalamus, a minimum of 5-HT as seen in semistarved sedentary and running rats around feeding time (noon). At this time 5-HIAA reached a maximum in the semistarved running rats while semistarved sedentary and ad lib fed rats showed no circadian pattern of 5-HIAA. These data indicate that serotonin turnover in the medial basal hypothalamus is increased as a consequence of semistarvation and hyperactivity. PMID- 1720895 TI - Cerebrospinal fluid calcium, parathyroid hormone, and monoamine and purine metabolites and the blood-brain barrier function in primary hyperparathyroidism. AB - Psychiatric disturbances are common in primary hyperparathyroidism (HPT), but their pathogenesis is essentially unknown. This study deals with cerebrospinal fluid (CSF) calcium homeostasis and its connection with parathyroid hormone (PTH), blood-brain barrier (BBB) function, and central monoamine and purine metabolites in patients with primary HPT. In 22 patients with primary HPT (serum calcium 2.85 +/- 0.21 mmol/l), the CSF concentrations of total and ionized calcium were higher (1.21 +/- 0.08 mmol/l, p less than 0.01, and 1.09 +/- 0.05 mmol/l, p less than 0.001, respectively) than in 11 normocalcemic reference subjects. The values correlated with serum calcium concentration (p less than 0.001) and CSF/serum albumin ratio, a measure of BBB permeability. The latter ratio was elevated in one-third of the patients with HPT, indicating BBB damage. CSF immunoreactive intact PTH was higher in the HPT patients than in the reference group (p less than 0.05), and serum and CSF PTH were positively correlated (p less than 0.05). The CSF levels of the monoamine metabolites 5 hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) were lower, and the level of urate in CSF was higher, in the HPT patients than in the reference subjects, while there were no consistent differences in CSF hypoxanthine or xanthine. CSF 5HIAA correlated inversely with CSF ionized calcium (r = -0.42, p = 0.02). After parathyroid surgery, CSF calcium and urate decreased significantly and CSF monoamine metabolites increased slightly. The decrease in CSF ionized calcium correlated with the alleviation of psychiatric symptoms. The results indicate the importance of increased CSF calcium concentrations in patients with primary HPT and suggest a relation between central calcium regulation and central turnover of monoamines. PMID- 1720896 TI - Evidence for an involvement of 5-hydroxytryptaminergic neurones in the maintenance of operant behaviour by positive reinforcement. AB - The possible involvement of the ascending 5-hydroxytryptaminergic (5HTergic) pathways in the maintenance of operant behaviour by positive reinforcement was examined using a quantitative paradigm based on Herrnstein's (1970) equation which defines a hyperbolic relationship between steady-state response rate and reinforcement frequency in variable-interval schedules. Nine rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei; 12 rats received sham injections. The rats were trained to steady-state in a series of variable-interval schedules of sucrose reinforcement affording a range of reinforcement frequencies. Herrnstein's equation was fitted to the data obtained from each rat and to the averaged data obtained from the two groups. The value of KH (the parameter expressing the reinforcement frequency needed to obtain the half-maximum response rate) was significantly lower in the lesioned group than in the control group; the values of Rmax (the parameter expressing the maximum response rate) did not differ significantly between the two groups. The levels of 5HT and 5-hydroxyindoleacetic acid in the parietal cortex, hippocampus, nucleus accumbens and hypothalamus were markedly reduced in all four regions in the lesioned group, but the levels of noradrenaline and dopamine were not significantly affected. The results indicate that damage to the central 5HTergic pathways resulted in an increase in the "value" of the sucrose reinforcer, without affecting the animals' response capacity. The results are consistent with the suggestion that the 5HTergic pathways may exert some limiting control on the "values" of certain reinforcers. PMID- 1720897 TI - [Acid polypeptides as intensifiers of the formation of irreparable double stranded DNA breaks induced by the gamma irradiation of mammalian cells]. AB - Acid polypeptides, synthetic analogues of a natural modifier of lethal effect of radiation, were shown to inhibit double-strand DNA breaks (DSB) repair, to increase irreparable DSB accumulation and to enhance the formation of structural chromosome rearrangements in gamma-irradiated mammalian cells. The authors discuss the possibility of involvement of proteins, that contain amino acid sequences comparable, in length, with a modifier, into radiation formation of irreparable DSB. PMID- 1720898 TI - Expression of developmentally regulated genes in embryonal carcinoma cells. PMID- 1720899 TI - [Ion channels in cell membranes]. PMID- 1720900 TI - Kawasaki syndrome in 18 children in the west of Scotland. AB - Analysis of the case records of 18 children admitted to the Royal Hospital for Sick Children, Glasgow between June 1981 and May 1990 satisfied the diagnostic criteria of Kawasaki syndrome as published by Japan's Kawasaki disease research committee in September 1984. No aetiological agents were implicated. All children had cardiological work-ups including chest x-ray, ECG, 2-D echocardiography. Two children had gammaglobulin therapy and 16 had treatment with aspirin. All children except one recovered without sequelae. The child with right coronary artery aneurysm has since had successful coronary bypass surgery. PMID- 1720901 TI - Sexual ideology and experience in a Papua New Guinea society. AB - Recent anthropological interest in sexuality has been closely related to symbolic constructions of gender in different societies. Most studies explore the cultural constitution of sexual meanings without addressing how sex is experienced by actors. Material from the Bumbita Arapesh of the East Sepik Province of Papua New Guinea reveals that the local cultural ideology of male sexual domination does not adequately describe actual sexual experiences of Bumbita men and women. Cultural ideology states that men control sexual encounters as a part of their masculine essence, yet in actual marriages women often take the initiative in limiting sexual practice following postpartum prohibitions. Contrary to cultural expectations, men exhibit anxiety concerning sexual relations in a marriage. The public assertion of sexual dominance and suppression of sexual intimacy compensate for a sense of vulnerability accompanying sexual experience. An analytical distinction between "cultural ideology" and "individual experience" helps clarify the climate of sexual relationships in a cultural context. PMID- 1720902 TI - Vasoactive intestinal polypeptide-, neurotensin-, substance P-, gastrin-releasing peptide-, calcitonin-, calcitonin gene related peptide-, and somatostatin-like immunoreactivities in human parathyroid glands. AB - We have found vasoactive intestinal polypeptide (VIP)-, neurotensin-, substance P , gastrin-releasing peptide-, calcitonin-, calcitonin gene related peptide (CGRP 2)-, and somatostatin-like immunoreactivities in extracts of sporadic human parathyroid adenomas (n = 18). The content of CGRP-2, substance P, and somatostatin in adenomas correlated directly with that of parathyroid hormone. In addition, concentrations of VIP versus substance P and somatostatin versus CGRP-2 in adenomas were directly correlated. Neuropeptide content of parathyroid hyperplasias differed from that of adenomas. VIP was detected in only one of seven parathyroid hyperplasias, and neurotensin was undetectable (0/7), whereas substance P was present in six of seven cases and GRP in five of seven hyperplasias. In hyperplasias, content of substance P correlated directly with that of gastrin-releasing peptide. Peroxidase immunohistochemistry localized VIP like immunoreactivity to 20% to 50% of both chief and oxyphilic cells and rare clear cells and capillary endothelium in 11 of 12 adenomas studied. Focal staining was present in glandular epithelium of the rim of adjacent normal parathyroid tissue and in two of three normal parathyroid glands removed with thyroid goiters. This staining was both cytoplasmic and apical membrane. By contrast, in adenomas, neurotensin- and substance P-like positivities were confined to scattered (5% to 10%) oxyphilic cells. Cytoplasmic positivity for parathyroid hormone, noted in 30% to 70% of cells in serial sections, confirmed that these tissues were indeed parathyroid glands. PMID- 1720903 TI - Acute gastric pH changes alter intraluminal but not plasma peptide levels. AB - Gastric acidity is influenced by systemic and local peptide effects. Previous work by others has shown that intraluminally secreted peptides may have a role in local control of gastric acidity; however, the response of these peptides to acute changes in gastric pH is unknown. To determine the effects of acute changes in pH on systemic and intraluminal peptide levels, 14 normal volunteers underwent placement of a nasogastric tube after an overnight fast. Blood and gastric fluid were analyzed on a control day, 2 hours after completion of 24 hours of aluminum magnesium antacid therapy and after 24 hours of H2 blockade. Plasma and acid alcohol-extracted gastric peptide levels were measured with specific radioimmunoassays. Specimens were subdivided into two groups: 28 gastric fluid specimens with a pH less than 4 and 10 specimens with a pH greater than 4. In the patients with a pH greater than 4, the luminal peptides, motilin, neurotensin, pancreatic polypeptide, somatostatin, substance P, and gastrin, were decreased by 50% to 90% and gastrin-releasing peptide was decreased by 36% compared with specimens with a pH less than 4. Conversely, intraluminal vasoactive intestinal polypeptide and calcitonin levels were elevated by 60% and 27%, respectively, in the samples with a pH greater than 4. Intraluminal peptide concentrations are responsive to changes in intragastric pH; however, this response was not seen in plasma peptide levels. PMID- 1720904 TI - Limited proteolysis of vitronectin by plasmin destroys heparin binding activity. AB - Vitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN. Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis. PMID- 1720905 TI - The stimulatory effect of PACAP 38 on amylase release in dispersed rat pancreatic acini. AB - Pituitary adenylate cyclase activating polypeptide 38 (PACAP 38), a novel peptide of the vasoactive intestinal polypeptide (VIP) family, was shown to stimulate enzyme secretion in the dispersed rat pancreatic acini. The dose-response of pancreatic enzyme secretion to PACAP 38 was nearly identical with that to VIP. In the presence of a submaximal dose of PACAP 38 (1 nM), amylase release stimulated by an agonist working via the elevation of intracellular cyclic AMP (VIP, dibutyryl cAMP) was additionally responded, but the amylase release stimulated by an agonist via the elevation of cytosolic free calcium (carbachol, cholecystokinin) was potentiated synergistically. The present data suggest that PACAP 38 is a new candidate for the cAMP-mediated stimulant of pancreatic exocrine secretion. PMID- 1720906 TI - Alpha 2u-globulin nephropathy without nephrocarcinogenesis in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid. AB - Alpha 2u-Globulin (alpha 2u) nephropathy is a male rat-specific condition caused by a diverse group of xenobiotics. Features of this nephropathy include hyaline droplet accumulation in proximal tubules, tubular epithelial necrosis and regeneration, exacerbation of spontaneous renal disease, and induction of renal epithelial tumors. Nephrocarcinogenicity of compounds that cause this nephropathy may be a consequence of increased proximal tubular proliferation resulting from cell injury. These studies document alpha 2u nephropathy without primary renal epithelial tumors in male Wistar rats administered 1 (aminomethyl)cyclohexaneacetic acid (gabapentin), a therapeutic agent with antiepileptic/anticonvulsant properties. In a series of preclinical studies gabapentin was administered to rats at the following doses and durations: 50 and 2000 mg/kg for 2 weeks; 250, 1000, 2000, and 3000 mg/kg for 13 weeks; 250, 1000, and 2000 mg/kg for 52 and 104 weeks. Renal effects were evaluated by biochemical, immunocytochemical, histopathologic, and ultrastructural techniques. Reversible increases in size and distribution of hyaline droplets within proximal tubular epithelium occurred through 1 year of treatment at a severity that was dose dependent. In males given 2000 mg/kg, alpha 2u accumulation, degeneration, and necrosis of the P2 segment and intraluminal cellular casts were seen after 2 days of treatment. In the 2-week study, the size and number of phagolysosomes containing alpha 2u and the renal tissue alpha 2u increased with increasing dose and time. By Day 7, polymorphic crystalline inclusions were abundant in phagolysosomes of 2000 mg/kg males. In subchronic and chronic studies, spontaneous glomerulonephrosis was exacerbated in males given 2000 mg/kg, and, interestingly, no drug-related effect on renal tumor incidence was observed. To the best of our knowledge, this is the first documentation of the absence of nephrocarcinogenic effect in male rats treated for up to 104 weeks with a compound that causes acute and chronic lesions of alpha 2u nephropathy. PMID- 1720907 TI - [The value of prostate-specific antigen]. PMID- 1720908 TI - Digital subtraction cavernosography: method to detect venous leakage. AB - Cavernosography has become an important diagnostic test for detecting venous leakage as a cause of vasculogenic impotence. Digital subtraction cavernosography (DSC) was carried out on 21 patients with a history of venous leakage resulting in impotence. The DSC technique was compared to conventional cineradiography. Major venous leaks were easily identified in 16 patients. DSC was able to detect minor leaks missed by cineradiography in 2 patients. DSC seems to be a reliable technique that is easy to perform. It should be done in conjunction with pharmacologically induced erection. PMID- 1720909 TI - Applications of flow cytometry to artificial insemination: a review. AB - Flow cytometry is a technique in which sub-populations of cells can be analysed and separated according to the staining pattern seen with various fluorescent markers. This review describes some of the ways in which flow cytometry can be applied to the investigation of sperm populations, either as a means of quality control of semen or to examine the characteristics of different sub-populations of sperm within an ejaculate. These methods can replace or augment existing subjective assessments of semen characteristics. Using this technique it is possible to produce aliquots of sexed sperm for insemination or for in vitro fertilisation. An objective assessment can be made of the effects of environmental stress on male physiology by monitoring changes in semen quality. PMID- 1720910 TI - Detailed diagnoses and procedures, National Hospital Discharge Survey, 1989. AB - This report presents statistics on conditions diagnosed and surgical and nonsurgical procedures performed in non-Federal short-stay hospitals. The statistics are based on data collected through the National Hospital Discharge Survey from a national sample of the hospital records of discharged inpatients. Estimates of first-listed diagnoses, all-listed diagnoses, days of care for first listed diagnoses, and all-listed procedures are shown by sex and age of patient and geographic region of hospital. PMID- 1720911 TI - [Sudeck's syndrome of the hip in pregnancy. A case report and review of the literature]. AB - A case algodystrophy of the hip during pregnancy is reported and the literature is reviewed. Mostly symptoms manifest in the last trimenon and sometimes in the postpartum period. Typically only the left hip is involved. The symptomatology shows usually three periods: During a first phase there is a sudden onset of symptoms, while in the second phase symptoms remain stable and after birth the pain disappears in a short time. For diagnosis the clinical signs, radiology, radionuclide scans and MRI are helpful. Therapeutically physiokinesitheraphy and analgesics are recommended. PMID- 1720912 TI - [Fetomaternal transfusion in relation to mode of delivery. Comparison of data from the DFG multicenter study "Rhesus negative" (1965-70) with data from the Freiburg University Gynecologic Clinic (1989)]. AB - Fetomaternal hemorrhage with transfusion of more than 10-25 ml fetal blood into the maternal circulation ("macrotransfusion") is one possible cause of the failure of combined pre- and postpartal anti-D prophylaxis. We analyzed the data from 391 patients who delivered at the UFK Freiburg in 1989. We evaluated the amount of fetomaternal bleeding in different modes of delivery. We observed fetomaternal hemorrhage of clinical relevance in 7.5% of spontaneous delivery, 11.1% of vacuum extraction, 17.7% of cesarean section (p less than 0.05). There was no difference concerning macrotransfusions in the above mentioned modes of delivery. Our data are compared with the data of the DFG multicenter trial "rhesus negative" (1965-79). PMID- 1720913 TI - Which is the best test to evaluate the integrity of sperm plasma membrane? AB - The functional integrity of sperm plasma membrane of 87 semen samples collected from fertile and infertile men was assessed by the hypoosmotic swelling test (HOS). The results were correlated with sperm motility and supravital stains using trypan blue and eosin Y. These data indicate that sperm swelling and staining methods evaluate different properties of spermatozoa. Moreover the present report shows that staining percentages with trypan blue are significantly lower than with eosin Y. PMID- 1720914 TI - Quantitative assessment of human spermatozoa chromatin stainability by flow cytometry: preliminary study. AB - The authors report a human sperm suspension method to assess quantitatively the stainability of the spermatozoa chromatin. Analysis of 15 ejaculates demonstrated the existence of a constant percentage of stained DNA in different ejaculated spermatozoa. The condensed chromatin stainability was then compared to the in vitro decondensed chromatin stainability with the finding of an increased uptake of fluorochrome in relation with the nuclear chromatin decondensation. The quantitative spermatozoa chromatin stainability and its decondensation aptitude may be of importance in sperm physiology. PMID- 1720915 TI - Suggestive evidence for a microanatomical relationship between mast cells and nerve fibres containing substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, and somatostatin in the rat mesentery. AB - A close microanatomical relationship between serotonin-positive mast cells and nerve fibres positive for substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, and somatostatin has been observed in whole mount preparations of rat mesentery by an immunofluorescent double-staining procedure. Peptidergic fibres have been shown either to run in close proximity or come in direct contact with mast cells. This supports earlier morphological and immunohistochemical results suggesting an innervation of mast cells and provides a structural foundation for a series of pharmacological studies which outline the influence of various neuropeptides on mast cell secretory activity. PMID- 1720916 TI - Nigrocaudate and nigroputaminal projections in the monkey. AB - Following injections of tracers into the caudate nucleus, the putamen or into both structures of the same cerebral hemisphere, retrograde cell labeling in the nigral complex of the squirrel monkey has been analyzed. Our goal was to investigate a possible relation of the retrograde neuronal labeling in the substantia nigra pars compacta (SNc) with the histochemical compartmentalization of this structure detected with the aid of the acetylcholinesterase (AChE) technique. Our results confirmed that in the squirrel monkey there was a very precise topography in the nigrostriatal projection system as a whole, and also in the nigrocaudate and nigroputaminal projection systems considered separately. In the cases with injections of two different tracers, we were not able to find any double-labeled nigral cell. Nigrocaudate and nigroputaminal projecting cells generally formed independent groups which at some nigral levels clearly interdigitated, as shown by the group of Parent. However, the presence of nigral cells labeled either by one or another tracer within the same histochemical compartment of the SNc confirmed that the segregation of nigroputaminal and nigrocaudate projecting cells did not explain the AChE compartmentalization of the SNc. Based on our experiments and on the literature we suggest that the histochemical compartmentalization of the SNc could be related, at least in part, to the subdivision of the striatum into AChE-poor striosomes and an AChE-rich extrastriosomal matrix. PMID- 1720917 TI - Increased risk of death and cardiac arrest from encainide and flecainide in patients after non-Q-wave acute myocardial infarction in the Cardiac Arrhythmia Suppression Trial. CAST Investigators. AB - This report examines whether in the Cardiac Arrhythmia Suppression Trial death and cardiac arrest from encainide, flecainide and moricizine during the titration phase and from encainide and flecainide during the follow-up phase were related to presence (Q-wave acute myocardial infarction [Q-AMI]) or absence (non-Q-AMI) of pathologic Q waves. In all, 2,371 patients (70% with Q-AMI, 26% with non-Q AMI, and 4% unknown) entered the titration phase, starting 117 +/- 163 days after index AMI and lasting for an average of 21 days. For the titration phase, no significant differences existed between Q-AMI and non-Q-AMI patients for death and cardiac arrest rate, ventricular premature complex suppression rate, and nonrandomization rate. A total of 1,498 patients entered the follow-up phase of an average of 10 months (starting 129 +/- 158 days after the index AMI), and were randomized to encainide or flecainide, or their matching placebos. In the placebo group, non-Q-AMI patients had a significantly lower rate of death and cardiac arrest than Q-AMI patients (1.0 and 4.6%, respectively; p = 0.04). Encainide and flecainide significantly elevated death and cardiac arrest rate in both non-Q-AMI patients (8.7%, p less than 0.01) and Q-AMI patients (7.8%, p = 0.04). The relative risk for encainide or flecainide over placebo in the non-Q-AMI patients was 8.7, which was significantly higher than 1.7 observed for the Q-AMI patients (p = 0.03). None of the baseline characteristics had any significant interaction with encainide or flecainide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720918 TI - The search for a "cancer stain" in diagnostic surgical pathology. PMID- 1720919 TI - Lack of specificity of monoclonal antibody B72.3 in distinguishing chronic pancreatitis from pancreatic adenocarcinoma. AB - Making the morphologic distinction between chronic pancreatitis and pancreatic adenocarcinoma is a diagnostic challenge in small biopsy specimens and fine needle aspiration samples. It has been suggested that immunohistochemical evaluation for the tumor-associated glycoprotein-72 antigen recognized by the monoclonal antibody B72.3 may be helpful in this setting. Formalin-fixed, routinely processed, paraffin-embedded tissue from 29 known cases of chronic pancreatitis and 31 cases of pancreatic adenocarcinoma were evaluated for reactivity with monoclonal antibody B72.3 using a standard avidin-biotin complex technique. Positive staining was seen in 26 of 31 adenocarcinomas (84%) and in 6 of 29 cases (21%) of chronic pancreatitis. Although monoclonal antibody B72.3 is more commonly reactive with pancreatic adenocarcinoma than with chronic pancreatitis, too many cases of chronic pancreatitis are reactive with this antibody for it to be useful as a diagnostic adjunct. PMID- 1720920 TI - Sinonasal malignant melanoma. A clinicopathologic and immunohistochemical study of 14 cases. AB - The clinical, light microscopic, and immunohistochemical features of 14 sinonasal malignant melanomas were studied to show their diverse morphologic appearance and distinction from therapeutically more amenable neoplasms that occur in this region. The tumors arose in 6 men and 8 women (median age, 70 years). Eleven patients died of disease 7 to 44 months (median, 18 months) after diagnosis. The absolute median survival time was 18.5 months (range, 7 to 44 months). The predominant microscopic appearance was categorized as small blue cell in eight cases, spindle cell in three cases, epithelioid in two cases, and pleomorphic in one case. Eight tumors had multiple patterns. Five sinonasal malignant melanomas had theque-like growth, five had junctional change, and 10 contained at least rare melanin pigment. Fourteen, 13, and 12 sinonasal malignant melanomas were immunoreactive with anti-vimentin, HMB45, and anti-S100 protein antibodies, respectively. One epithelioid tumor positive for vimentin, S100, and HMB45 also contained scattered epithelial membrane antigen-positive and cytokeratin-positive cells, which emphasizes the need for a battery of stains to distinguish sinonasal malignant melanoma from carcinoma. All tumors were negative for leukocyte common antigen, muscle-specific actin, and synaptophysin. Diffuse immunopositivity for vimentin, S100 protein, and HMB45 allows distinction of sinonasal malignant melanomas from histologically similar neoplasms. PMID- 1720921 TI - Immunohistochemical progesterone receptor assay. Measurement by image analysis. AB - To determine the efficiency of image analysis in immunohistochemical progesterone receptor (PgR) measurement, 94 primary breast carcinoma tissue samples were evaluated for PgR by biochemical dextran-coated charcoal assay (DCC) and an immunohistochemical method. Frozen sections immunostained for PgR with a monoclonal antibody (Abbott PgR-ICA, Chicago, IL) and the peroxidase antiperoxidase technique were scored semiquantitatively histologic score by microscopy and quantitatively (percentage nuclear area immunopositivity [PNA] using the CAS 200 image analyzer (Cell Analysis Systems, Elmhurst, IL). There was a positive correlation between dextran-coated charcoal assay and both histologic score (r = 0.82) and PNA (r = 0.69). Selected cutoff points of 60 histologic score and 6.5% PNA based on sensitivity/specificity calculations yielded a predictive value of a negative test of 73% and 80%, respectively, and a positive predictive value of 100% for both; ranges of fmol/mg protein PgR correspond to ranges of histologic score and PNA. The use of an image analyzer to measure PNA in PgR-immunostained sections is a viable alternative to dextran-coated charcoal assay, especially when insufficient fresh tissue is available. PMID- 1720922 TI - Expression of Tn and sialyl-Tn antigens in tumor tissues of the ovary. AB - The expression of sialyl-Tn and Tn antigens in various benign, borderline, and malignant ovarian tumors was examined immunohistochemically using newly developed antibodies specific for sialyl-Tn and Tn antigens. Sialyl-Tn antigen was detected in only one benign tumor, a mucinous adenoma that showed faint cytoplasmic staining in a few cells. However, sialyl-Tn was present in 5 of 12 serous borderline tumors, 10 of 19 mucinous borderline tumors, 10 of 13 serous adenocarcinomas, 15 of 16 mucinous adenocarcinomas, 14 of 15 endometrioid adenocarcinomas, and 7 of 7 clear cell carcinomas of the ovary. The antigen expression was observed throughout the cytoplasm of cancer cells and in the apical cytoplasm and luminal contents of some glands. The incidence and intensity of staining for sialyl-Tn antigen were higher in malignant tumors than in borderline tumors, but these results did not correlate with the histologic classification or differentiation. Coexpression of sialyl-Tn antigen and Tn antigen was observed in two serous adenocarcinomas, six mucinous borderline tumors, five mucinous adenocarcinomas, eight endometrioid, and seven clear cell carcinomas. In no case was Tn antigen expressed without concomitant sialyl-Tn antigen expression. Accumulation of sialyl-Tn antigen seems to be an early event of carcinogenesis of the ovary. PMID- 1720923 TI - Detection of cytomegalovirus in lung allografts. Comparison of histologic and immunohistochemical findings. AB - Although the histologic manifestation of cytomegalovirus (CMV) is usually characteristic intracellular inclusions and cytomegaly, some investigators, using immunohistochemical or in situ hybridization techniques, have demonstrated the presence of histologically occult infections in certain tissues. A series of lung biopsy specimens from pulmonary transplant recipients were studied using a monoclonal antibody (CCH2) to CMV early viral antigen and the results were compared with routine histologic findings. Occult infection could not be demonstrated in any of these cases. These results may reflect the relative sensitivity of the monoclonal antibody used in this study, although other possible factors are discussed. The results suggest that, in lung allograft biopsy specimens, immunohistochemical analysis using monoclonal antibody CCH2 is not likely to increase significantly the yield of positive cases compared with examination of multiple levels of hematoxylin-and-eosin-stained sections. Additional studies are needed to compare the sensitivity of monoclonal antibodies to CMV antigens using a variety of sampling techniques and clinical settings. PMID- 1720925 TI - Platelet thrombospondin and glycoprotein IV abnormalities in patients with essential thrombocythemia: effect of alpha-interferon treatment. AB - Platelet aggregability and some biochemical parameters were evaluated in seven patients with essential thrombocythemia (ET) compared with seven patients with secondary thrombocytosis (ST). Defective platelet aggregation with one or more agonists was seen in five patients with ET whereas aggregation was increased in two other patients. In addition, three patients with ET demonstrated spontaneous platelet aggregation in citrated plasma. This was associated with increased level of thrombospondin (TSP) in the plasma membrane. Interestingly, the presence of a proteolyzed 160 kDa form of TSP was detected in all patients with ET, whereas it was never found in patients with ST. Furthermore, three patients with ET demonstrated increased levels of platelet surface glycoprotein IV (GP IV), the putative receptor for TSP in the plasma membrane. In two of these patients, this correlated with increased surface expression of TSP and spontaneous platelet aggregation. The results suggest a possible link between the increased number of plasma membrane GP IV molecules, the spontaneous expression of TSP on the platelet surface and platelet hyperaggregability in some ET patients. The levels of plasma membrane GP IV and platelet surface-associated TSP tended to be normalized during alpha-interferon treatment, whereas the presence of an altered form of TSP persisted. This last parameter might be of practical usefulness in the characterization of the disease, permitting a clear distinction from ST. PMID- 1720924 TI - Antibody to hepatitis C virus among cardiac surgery patients, homosexual men, and intravenous drug users in Baltimore, Maryland. AB - In order to define the risk factors for infection with hepatitis C virus, the authors determined the prevalence and incidence of antibodies to hepatitis C in three cohorts in Baltimore, Maryland, enrolled in prospective studies of human immunodeficiency virus (HIV-1) infection. Among 500 multi-transfused patients who underwent cardiac surgery in 1985 and 1986, 12 (2.4%) were hepatitis C seropositive before surgery while 19 (3.9%) developed antibodies in the 8-12 months after surgery. The seroprevalence of hepatitis C virus among 225 intravenous drug users followed since 1988 was 85%, which did not vary by HIV-1 status. Longer duration of intravenous drug use was significantly associated with hepatitis C seropositivity. Among 926 homosexual/bisexual men followed since 1984, 15 (1.6%) were hepatitis C seropositive; only intravenous drug use and a history of hepatitis A were marginally associated with hepatitis C in this population. No association was found between hepatitis C virus and HIV-1 or sexual behavior variables in this population. These data suggest that hepatitis C is readily transmitted by blood exposure, but is transmitted inefficiently by sexual means. PMID- 1720926 TI - A cluster of highly polymorphic dinucleotide repeats in intron 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. AB - A cluster of highly polymorphic dinucleotide repeats has been detected in intron 17b of the CFTR gene, 200 bp downstream from the preceding exon. At least 24 alleles, with sizes ranging from 7 to 56 units of a TA repeat, have been identified in a panel of 92 unrelated carriers of cystic fibrosis (CF). The common ones are those with 7, 30, and 31 dinucleotide units, with frequencies of .22, .19, and .12, respectively, among the non-CF chromosomes. Mendelian, codominant segregation of the alleles has been demonstrated in family studies, as expected. A less polymorphic dinucleotide (CA repeat) cluster has also been detected in a region 167 bp downstream from the TA repeat. The length of the CA repeat cluster varies from 11 to 17 dinucleotide units, and it appears to have an inverse relationship to that of the TA repeats. These dinucleotide repeats should be useful in genetic linkage studies, in counseling for CF families with unknown mutations, and in tracing the origins of the various mutant CF alleles. PMID- 1720927 TI - Complete deletion of the proteolipid protein gene (PLP) in a family with X-linked Pelizaeus-Merzbacher disease. AB - Pelizaeus-Merzbacher disease (PMD) is an X-linked neurologic disorder characterized by dysmyelination in the central nervous system. Proteolipid protein (PLP), a major structural protein of myelin, is coded on the X chromosome. It has been postulated that a defect in the PLP gene is responsible for PMD. Different single-nucleotide substitutions have been found in conserved regions of the PLP gene of four unrelated PMD patients. Novel Southern blot patterns suggested a complex rearrangement in a fifth family. Linkage to PLP has been shown in others. We evaluated the PLP locus in a four-generation family with two living males affected with X-linked PMD. Analysis of DNA from the affected males revealed complete absence of a band, with PLP probes encompassing the promoter region, the entire coding region, and the 3' untranslated region and spanning at least 29 kb of genomic DNA. DNA from unaffected relatives gave the expected band pattern. Two obligate and one probable carrier women were hemizygous for the PLP locus by dosage analysis. Although it is unlikely, the previously described point mutations in PLP could represent polymorphisms. The finding of complete deletion of the PLP gene in our family is a stronger argument that mutations in PLP are responsible for X-linked PMD. PMID- 1720928 TI - Immunochemistry of factor VIII:C inhibitor antibodies. AB - Factor VIII:C (FVIII:C) inhibitors are pathologic circulating antibodies that reduce FVIII:C activity. They can arise either as alloantibodies in some congenital hemophiliacs, or as autoantibodies in nonhemophilic patients with acquired inhibitors. The reason for development of such antibodies is not known, but their basic biochemical and clinical characteristics have been reviewed extensively in the past several years. This article surveys recent immunochemical studies to characterize the FVIII:C molecule and antibodies against it. For instance, epitope (antigenic site) mapping investigations conducted to date suggest that binding sites for most inhibitory antibodies can be localized to limited regions of the FVIII:C molecule. Further progress in the understanding of immune responses against FVIII:C is likely to provide a sound basis for the design of new therapeutic approaches to patients suffering the sequelae of FVIII:C inhibitor antibodies. PMID- 1720929 TI - Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity. AB - We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated co-segregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acid androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor. PMID- 1720930 TI - Sclerosing adenosis of the prostate gland. A clinicopathological and immunohistochemical study of 11 cases. AB - Eleven cases of sclerosing adenosis of the prostate gland, a recently reported uncommon pseudoneoplastic lesion with characteristic histological, histochemical, and immunohistochemical features, are described. The well-circumscribed cellular lesions were composed of variably sized and shaped, often compressed, glands and small clusters of epithelial cells embedded in a cellular, often myxoid stroma. Mild cytologic atypia was occasionally present, and one case had moderate cytologic atypia. A distinct basement membrane often surrounded the glands and clusters. Luminal acid mucin was typically present. Keratin-positive basal cells were present in the glands and as spindle cells in the stroma. The basal cells were also immunoreactive for S-100 and muscle-specific actin, suggesting myoepithelial differentiation. Clinical follow-up has shown no evidence of prostatic carcinoma. The available evidence suggests that sclerosing adenosis of the prostate gland is a benign lesion with distinctive features that should enable it to be distinguished from prostatic adenocarcinoma. PMID- 1720931 TI - Spindle cell pseudotumor due to Mycobacterium avium-intracellulare in patients with acquired immunodeficiency syndrome (AIDS). Positive staining of mycobacteria for cytoskeleton filaments. AB - A rare spindle-cell pseudotumor caused by Mycobacterium avium-intracellulare (MAI) that mimics a mesenchymal tumor, was recently reported (7,14). We report on three such pseudotumors in patients with the acquired immunodeficiency syndrome (AIDS), two involving lymph nodes and one involving the bone marrow. In the course of investigating the first-encountered example of this tumor for evidence of smooth-muscle origin of the spindle cells, it was noted that these cells stained positively for desmin by immunoperoxidase techniques (IPX), as did a variety of other cytoskeleton filaments of all sizes. Electron microscopic examination of one of these lesions revealed spindle cells containing lysosomes and large numbers of microorganisms compatible with MAI but no filaments or organelles suggestive of smooth-muscle cells. Further studies revealed that the typical lesions produced by MAI in patients with AIDS, namely aggregates of histiocytes or individual histiocytes laden with organisms, rather than the expansile spindle-cell pseudotumor, also strain strongly for cytoskeleton filaments, as do M. tuberculosis and Mycobacterium leprae. Awareness of the existence of this unusual manifestation of MAI infection in AIDS patients and its desmin positivity can avoid misdiagnosis of a primary or metastatic smooth-muscle neoplasm. The cell of origin appears to be the histiocyte. PMID- 1720932 TI - Development of a palliative care protocol for emergency medical services. PMID- 1720933 TI - Card, poster ready to assist with patient talk. PMID- 1720934 TI - Effect of viruses and interferon on chick embryo otocyst cultures. AB - Chick embryo otocyst organ cultures were subjected to live vesicular stomatitis virus and rubella virus preparations, to interferon (IFN), and to a combination of both virus and IFN, and compared to control untreated otocysts. We observed morphologic and microscopic changes suggestive of individual cell death and delayed organ differentiation in the virus-treated groups, along with an appreciable decrease in size of the otocyst. Low-dose IFN treatment prior to virus inoculation appeared to partially prevent these effects. The addition of IFN alone did not seem to affect the differentiation process. Time-lapse videophotography further confirmed the above findings. This study suggests that the peripheral component of congenital deafness associated with viral infections is likely to be an effect of the virus itself, and not of the IFN. Interferon provides a partial protective effect against the insult from the virus in vitro and does not seem to be toxic to the developing otocyst. PMID- 1720935 TI - Primary culture of strial marginal cells of guinea pig cochlea: growth, morphologic features, and characterization. AB - To further investigate the cellular mechanisms involved in the formation of endolymph, primary cultures of marginal cells of guinea pig were established. Minute explants obtained by mechanical dissociation of stria vascularis were plated on collagen type I precoated impermeable substrate in serum-free, hormone supplemented medium. A confluent layer of epithelial-like cells was obtained within 2 weeks. The cultured cells formed domes, demonstrating that they retain some of their transepithelial properties. Polarization was also suggested by electron microscopic observation of apical microvilli and tight junctions. Immunohistochemical methods revealed that the cultured cells coexpressed cytokeratin and vimentin, demonstrating their epithelial origin, although some degree of dedifferentiation occurred. Thus, a primary culture of marginal cells can be established that may be a suitable model for an in-depth investigation of the function of the marginal cells. PMID- 1720936 TI - Negative control of epithelial cell proliferation by prostatic stroma. AB - The influence of prostatic fibroblasts on the growth of the prostatic carcinoma cell lines PC-3 and LNCaP was examined. In a double layer soft agar system, clonal growth of both cell lines was inhibited by all prostatic fibroblasts tested, irrespective of whether they were derived from malignant or non malignant prostate tissue. Irradiated fibroblasts and quiescent fibroblasts showed similar effects. The finding that growth was also inhibited in the presence of fibroblasts conditioned medium suggests that the observed effects were mediated by a diffusable growth inhibiting factor. Organ-specific production was suggested by the observation that skin fibroblasts stimulated rather than inhibited PC-3 cell growth. These findings indicate a negative control of epithelial cell proliferation by prostatic stroma. PMID- 1720937 TI - Human chromosome 19 confers the CD2/E rosette receptor phenotype in interspecific cell hybrids. AB - Interspecific cell hybrids between Chinese Hamster Ovary (CHO) and phytohaemagglutinin (PHA) stimulated human T lymphocytes were purified by preparative rosetting with sheep red blood cells (SRBC). The hybrid cell clone used in the present study consisted of cells containing a complete set of the 20 CHO chromosomes and one extra human chromosome, No 19. Hybrid cells constitutively expressed high levels of human CD2 surface receptor and formed multilayer rosettes with SRBC and human erythrocytes. In addition to CD2 they produced low levels of a small number of human extracellular proteins. These findings suggest that the factor(s) responsible for CD2 expression are produced by the hybrid and that genes responsible for CD2 expression are located on chromosome 19. However, the present work cannot exclude that material of chromosome 1, where the CD2 gene has been assigned previously, is integrated somewhere in the hybrid karyotype. Further work is needed to clarify this point. PMID- 1720938 TI - Chemotherapy agents and the induction of late lethal defects. AB - Radiation is now known to be capable of producing both lethal and non lethal lesions in a variety of mammalian cells which may not be expressed for several cell divisions after the initial insult. The mechanism by which such an effect occurs is unknown. Because of the possible implications for cancer treatment, if such an effect also occurred following chemotherapy exposure, cells were exposed to various cytotoxic chemotherapy agents with known and well characterised effects on DNA or other areas of cell function or structure. The results indicate that late lethal defects are not detectable after treatment with appropriate ranges of doses of cisplatinum, vincristine, BCNU or adriamycin, but that they are induced by Bleomycin and to a lesser extent by 5-Fluorouracil. Bleomycin is known to cause strand breaks and is regarded as a radiomimetic agent. 5 Fluorouracil may act by preventing efficient and faithful synthesis of DNA, allowing mutations to become integrated into the genome. The occurrence of lethal mutations with both these agents supports previous suggestions that error-prone repair of DNA base sequence abnormalities may be fundamental to the process of late lethal damage production in mammalian cells. The cloning efficiency of cells which survived exposure to Vincristine or BCNU over a wide dose range was found to be significantly increased; this may represent an adaptive response to the drugs. PMID- 1720939 TI - Potentiation of antitumor activity of bleomycin towards solid tumors in mice by Bacillus thuringiensis subsp. israelensis toxin. AB - We previously reported that a 25-kDa Bacillus thuringiensis subsp. israelensis (BTI) toxin is cytotoxic to cultured tumor cells and can also potentiate the cytotoxic effect of some antitumor agents in vitro. In this study, we examined the in vivo effect of BTI toxin on the potentiation of antitumor activity of bleomycin (BLM) against solid tumor-bearing mice. Three tumor cell lines, Ehrlich carcinoma, B16 melanoma and Meth A fibrosarcoma, were employed. It was shown that dose of BTI toxin, ineffective alone, potentiated the antitumor activity of BLM when used in combination. PMID- 1720940 TI - New in vitro line from a human (B) non-Hodgkin lymphoma. AB - An in vitro cell line (HT 58) has been established from a human (B) NHL xenograft. The lymphoma cells in culture retained their lymphoblastic appearance, DNA-content, IgM/lambda monoclonality and many immunophenotypic markers. The clonal chromosomal abnormalities involved the chromosomes 1, 2, 3 and 14. The cells expressed and produced chondroitin sulfate proteoglycans identified with mAbs that were raised against human articular cartilage CSPG. The cells also released IgM into the medium as well as substances that stimulated the proliferation of activated normal peripheral B-cells. PMID- 1720941 TI - [Nephroblastoma in the adult. Apropos of a case]. AB - Adult nephroblastoma is a rare tumor. The authors report a new case observed in a 35 year old woman in whom the diagnosis was made by histopathology. The authors study the particular features of this tumor. Its prognosis appeared to be poorer than that of renal adenocarcinoma. PMID- 1720942 TI - [Prostate-specific antigen. Interpretation of the results in relation to the sampling method]. AB - The authors compare the two prostate specific antigen assay methods most widely used in France. The first method (RIA Baxter) uses an isotope marker (Iodine 125), the other (EIA Biotrol) uses an enzymatic marker (alkaline phosphatase). Prostate specific antigen was assayed by means of these two techniques in two groups of patients: one group of 49 men considered to be free of any prostatic disease, recruited from blood donors; another group of 89 male patients in whom a prostate specific antigen assay was performed prospectively at the first urology outpatients visit. The two prostate specific antigen assay techniques gave different results, but the values obtained by these two methods were not discordant. It is therefore possible to define a coefficient of proportionality of 1.45 regardless of the prostate specific antigen concentration or the urological disease considered (EIA Biotrol x 1.47 = RIA Baxter). PMID- 1720943 TI - [Home therapy in cancer pain--from the viewpoint of the pain clinic]. AB - Recently, the return to everyday life and a meaningful life are the most important goals in the management of cancer pain patients who have not undergone radical therapy. The aim is therefore betterment of QOL (quality of life) of the cancer pain patients who suffer from physical and mental pain, However, one can not expect much from any kind of therapy without adequate relief of physical cancer pain. Accordingly, pain relief methods in a pain clinic are also necessary for home therapy in cancer pain. We should like to show some cases treated in our pain clinic and emphasize the efficacy of our treatment for cancer pain. PMID- 1720944 TI - [Toward effective cancer home therapy--from the nurses point of view]. AB - Home cancer therapy is part of the palliative care intended to enhance the Quality of Life for patients. This treatment in the home should be maintained at the hospital level. The home could be called a "Hospital without walls." Cancer patients, both in the terminal and late stages prefer treatment in their own homes regardless of severity except patients whose cancer is in the early stage. Home treatment primarily relates itself to pain control, TPN, chemotherapy, and symptom control. Our hospital started Home Care Service in 1989, and has since continued work for 18 cancer patients with 122 home visits. Patients in the late stage receiving mostly chemotherapy were treated on an outpatient basis. From August 1990 through July 1991 the patients receiving chemotherapy were 28 in number. Informed consent is essential whenever chemotherapy is involved. Drugs used at home are mostly vescicant or irritant types, in which the route of administration plays a crucial part. But most home cases are given ip treatment. In this process, major efforts have been directed toward flexibility, efficiency, and optimality in providing a support system that responds effectively to multifaceted needs of the community. Clear findings ascertained so far from our experience in this pilot study are as follows. The most urgent needs are 1. Creation of a support system a. A Discharge Planning and Team approach. b. 24 hour support service and emergency care c. A medical and welfare network in the community 2. Education and Training 3. Informed consent 4. Nursing Manual 5. Extensive use of public health insurance, in order to achieve balanced cost sharing. PMID- 1720945 TI - [Clinico-pathological studies on the effects of preoperative hyperthermo chemoradiotherapy of advanced esophageal carcinoma]. AB - We report clinico-pathological studies on the effect of preoperative hyperthermia and chemotherapy combined with radiotherapy (HCR) for progress of the local curability of advanced esophageal carcinoma. The subjects of these studies were 17 patients who underwent subtotal esophagectomy after preoperative irradiation 40 Gy from 1980 to 1989, of which 8 patients had HCR, 6 patients irradiation only (R), 3 patients both irradiation and chemotherapy (CR). The clinical response rate of the patients with R or CR was 33% (PR 3, MR 3, NC 3), and the histological effective (Ef3 or Ef2) rate was 56% (Ef3 1, Ef2 4, Ef1 4). The clinical response rate of the patients with HCR was 88% (PR 7, MR 1), and the histological effective rate was 100% (Ef3 1 Ef2 7). HCR was more effective than R or CR for the local lesion of esophageal carcinoma histopathologically (p less than 0.05). However, the survival rate of patients with HCR was similar to R and CR, respectively. These results suggest that further improvement of the heating methods and the methods of combining hyperthermia with irradiation and chemotherapy is needed. PMID- 1720946 TI - [Clinical pharmacology of anticancer agents--(Part 2). Anticancer antibiotics]. PMID- 1720947 TI - Persistence of cellular and humoral response to synthetic peptides from defined Plasmodium falciparum antigens. AB - The cellular and humoral immune responses to synthetic peptides reproducing the repeat sequences of two major vaccine candidates (circumsporozoite protein and Pfl55/RESA) were investigated in two groups of African subjects according to the length of their stay outside endemic areas. The relation between the lymphoproliferative response and the antibody levels to these antigens was studied. The results confirm the existence of T-cell epitopes within the repeat sequences of the CS protein and the Pfl55/RESA capable of inducing lymphocyte proliferation. Cellular response to all studied peptides was more frequently observed in individuals living in France for less than one year than in individuals living in France for a longer time. T-cell proliferation in the presence of the tetrapeptide and of the octapeptide from the C-terminus repeat of Pfl55/RESA was related, with an immunodominance of the tetrapeptide over the octapeptide. Cellular responses to the CS protein repeat and to the 11-amino-acid peptide from the Pfl55/RESA N-terminus were the longest lasting after termination of exposure. In a given individual, cellular and humoral responses were not related for any peptide studied. PMID- 1720948 TI - Combined Fontana-Masson-mucin staining of Cryptococcus neoformans. AB - Cryptococci react positively with various histochemical stains, including the Fontana-Masson (FM), which stains the cell wall, and mucin stains, such as alcian blue and mucicarmine, which stain the capsule. Combinations of the FM stain with both the alcian blue and mucicarmine stains were performed on paraffin-embedded tissue specimens that were obtained from 15 patients who had culture-proved cryptococcosis. Combined FM-mucicarmine and FM-alcian blue stains were compared with other individual fungal stains. The FM stain, followed by either the mucicarmine or alcian blue stain, distinctively demonstrated both the cell wall and capsule of most organisms. More organisms were recognized in the combined stains than with either stain done individually. No interference between the stains was noted. Combining the FM stain with either of these two mucin stains appears to be helpful for identifying cryptococci. PMID- 1720949 TI - Endoscopic laser recanalization is effective for prevention and treatment of obstruction in sigmoid and rectal cancer. AB - Patients with obstructing cancers are ineligible for preoperative chemotherapy and radiation unless they undergo surgical diversion. Endoscopic laser therapy (ELT) may provide an alternative to colostomy for these patients. We retrospectively reviewed all patients with distal sigmoid and rectal carcinomas who underwent ELT from January 1988 through April 1990. The majority of patients were referred for palliation of advanced disease. Thirty-seven patients underwent 123 ELT sessions (median, 2.5; range, one to 18). In 84% of patients, patency was maintained during a median follow-up of 31.5 weeks (range, one to 123). Morbidity and mortality were 2.5% (3/123) and 5% (1/37), respectively. Sixty-two percent had radiotherapy, chemotherapy, and/or surgery concurrent with ELT. Endoscopic laser therapy can safely and effectively reestablish and maintain luminal patency in patients with obstructing distal cancers. In addition, ELT can enable the administration of preoperative adjuvant radiotherapy and chemotherapy. PMID- 1720950 TI - Metastatic melanoma of the gastrointestinal tract. Results of surgical management. AB - Between 1954 and 1989, 41 patients with melanoma metastatic to the gastrointestinal tract underwent surgical treatment at the Mayo Clinic, Rochester, Minn. The small bowel was most commonly involved (71%), followed by the stomach (27%), large bowel (22%), and esophagus (5%). Gross total excision of all intra-abdominal metastases was performed in 52% of patients. The postoperative mortality was 5% and the median patient survival was 0.8 years, with 1- and 5-year survival rates of 44% and 9%, respectively. Of the patient, tumor, and treatment variables evaluated, patients with small-intestinal metastases had a significantly worse prognosis. Although patients with melanoma metastatic to the bowel have a limited life expectancy, surgical resection of their metastases provides effective palliation. Operative treatment of selected patients with symptomatic melanoma metastatic to the gastrointestinal tract is a worthwhile undertaking. PMID- 1720951 TI - Lucy Wortham James Basic Research Award. Markers of prostatic carcinoma. AB - Markers of human prostatic cancer are important diagnostic aids in the management of this most common tumor among US men. A rapidly expanding body of basic and clinical knowledge about the properties and behavior of these unique organ specific macromolecules bridges the gap between sophisticated immunochemistry and everyday practice. As a consequence, the patient benefits, because greater opportunities for early detection of prostatic cancer provide more freedom in selecting the most effective treatment modalities. Parallel improvement in quantitative assessment of an existing tumor mass allows earlier and more precise monitoring of the positive therapeutic responses and/or disease progression, as well as better overall prognosis. In addition, marker molecules can provide specific targets for antibody-directed therapeutic compounds active against prostatic cancer. This study focused on three such markers: prostatic acid phosphatase, prostate-specific antigen, and a new membrane-associated marker defined by a monoclonal antibody called 7E11-C5. A critical evaluation of clinical usefulness and prospects for future developments are presented. PMID- 1720953 TI - Cytokeratin patterns of human oral mucosae in histiotypic culture. AB - In a three-dimensional culture model, oral epithelial differentiation was investigated ultrastructurally and biochemically for cytokeratin expression. Epithelia from the hard palate, gingiva and alveolar mucosa grown on freely floating collagen lattices populated with fibroblasts from homotypic origins, and fed with medium containing 10% delipidized fetal calf serum for 21 days before analysis, stratified and differentiated to basal cuboidal cells, polyhydral spinous cells and elongated superficial cells. The epithelium of palatal origin had non-nucleated superficial cells resembling orthokeratinized cells. The upper spinous cells had keratohyalin-like granules. The corresponding cells of gingival and alveolar mucosal origins retained their nuclei and had smaller numbers of keratohyalin-like granules. Basal cell keratins (CK 5 and 14) and those of hyperproliferation (CK 6 and 16) were consistently found in all epithelia. Furthermore, simple epithelial keratins (CK 18 and 19) were variably expressed by cells from different oral origins. In epithelial cells from the alveolar mucosa, CK 13 and 19 formed major bands, which correlates with their expression in vivo. In contrast, these polypeptides were either absent or formed minor bands in extracts of gingival and hard palatal cells. Although in small quantities, keratins of terminal differentiation (CK 1, 2, 10 and 11) were detected in gels prepared from palatal epithelia. This expression correlates with the higher morphological differentiation of these cells in this model. The model is of interest for studies of epithelial differentiation, as the differentiation markers of keratinized epithelia (CK 1 and 10) were expressed by cells from palatal origin, and those of non-keratinized epithelia (CK 4, 13 and 19) were prominent in cells from alveolar mucosal origin. PMID- 1720952 TI - Comparison of elemental concentrations in the acinar cells of the human labial salivary gland. AB - Two types of acinar cells were observed in human labial glands by conventional and analytical electron microscopic and light microscopic techniques. The predominant type contained large and prominent secretory granules that were strongly mucicarmine and PAS (with and without diastase) positive. The second type contained small, lacy, secretory granules, and these cells were faintly positive with these stains. The elemental contents of the two types of granules were measured by analytical electron microscopy using digital mapping and spot analysis applied to freeze-dried cryosections prepared from gland slices incubated in vitro under non-stimulated conditions. The large secretory granules had significantly higher Ca, S and Mg concentrations and significantly lower Cl and K concentrations than the small granules. The difference in elemental contents probably reflects differences in the content of secretory macromolecules. Specifically, the S content is thought to reflect the anionic properties of the secretory macromolecules, while the levels of divalent cations are thought to be determined by electroneutrality requirements for macromolecular folding and storage. No differences were found in nuclear or cytoplasmic elemental concentrations between the two cell types. PMID- 1720954 TI - Effect of zinc deficiency on keratins in buccal epithelium of rats. AB - Weanling rats fed a zinc-deficient diet (less than 1 part/10(6)) for 4 weeks develop parakeratotic and hyperplastic buccal epithelium with increased mitotic activity. Normal buccal epithelium contains major keratin polypeptides of 56, 46 and 43 kDa. Four-week zinc-deficient rats lacked the 43 kDa keratin. It appears that the 46 and 43 kDa keratins are related, differing as a result of some post translation modification. A proteolytic cleavage of the 46 kDa keratin to the 43 kDa species is the most likely mechanism. The findings point to a decrease of keratinolytic enzyme activity in the zinc-deficient rats. PMID- 1720955 TI - Transcription in Rhynchosciara americana embryos' early development. AB - Embryonic transcription starts about 6 hr postfertilization and increases during the first 72 hr of development. During this stage rRNA is the most abundant transcript, but some premessenger RNAs are also present. At 96 hr of development poly(A+) mRNA starts to be detected concordant with gastrulation movements. PMID- 1720956 TI - Identification of a novel transcription unit in the human insulin-like growth factor-II gene. AB - The human insulin-like growth factor-II (hIGF-II) gene has until now been thought to be composed of eight exons, including three independent leader exons. In the present study two additional exons, one leader exon and one alternatively used ordinate exon, have been newly identified. They were abundantly expressed in human histiocytoma tissue, generating mRNA species of about 5.0 kb in length. The new leader exon shows significant sequence similarity with the rE1 exon, previously reported to be transcribed only in the rat, and is mapped at nearly the same genomic location as in the rat. On the other hand, sequence similarity with another exon in the corresponding region of the rat genome was also found. It was, however, obvious that the rat sequence would not work as an active exon, since both splice acceptor and donor sites were deviated considerably from the consensus sequences. It has thus become apparent that the complex transcription unit of a single-copy hIGF-II gene comprises at least 10 exons, including four leader exons, one alternative exon and three common protein-coding exons. PMID- 1720957 TI - Endothelial and fibroblastic activation in scleroderma. The myth of the "uninvolved skin". AB - We studied the immunohistochemistry of the skin of scleroderma patients to determine the differences (if any) between clinically "affected" and "nonaffected" areas. We examined paired skin biopsy samples from clinically involved forearm skin ("affected") and clinically uninvolved proximal skin ("nonaffected") taken from 19 patients with diffuse scleroderma and from 15 normal control subjects. We stained the sections with antibodies to endothelial leukocyte-adherence molecule type 1 (ELAM-1; to detect endothelial activation) and to procollagen-1 (PC-1; to detect newly formed, unprocessed collagen). There was increased expression of ELAM-1 and PC-1 in sclerodermatous skin as compared with the controls, but there was no difference between clinically affected and nonaffected skin samples. In 10 of 11 patients whose condition was getting worse, endothelial and fibroblast activation preceded fibrosis. Endothelial and fibroblast activation are more widespread in the skin of scleroderma patients than is evident by inspection on physical examination. What appears to be "normal" skin in diffuse scleroderma is already pathologic, as shown by abnormal endothelial activation and procollagen production. PMID- 1720958 TI - Autoimmunity to human thyroglobulin. Respective epitopic specificity patterns of anti-human thyroglobulin autoantibodies in patients with Sjogren's syndrome and patients with Hashimoto's thyroiditis. AB - We evaluated the epitopic specificity pattern of anti-human thyroglobulin (anti hTg) autoantibodies from patients with primary Sjogren's syndrome (SS). All of the primary SS sera tested contained both IgG and IgM anti-hTg autoantibodies recognizing at least 1 region on hTg; in 65% of the cases, 3 or more regions were recognized. A strong recognition of region II, as is seen in Hashimoto's thyroiditis, was associated with thyroid disorder in primary SS. These results emphasize the importance of region II in autoimmune thyroid disease. PMID- 1720959 TI - Balance of care for the dying between hospitals and the community: perceptions of general practitioners, hospital consultants, community nurses and relatives. AB - A survey was made of the general practitioners, hospital consultants and community nurses who had cared for a random sample of people dying in 1987. Their views and experiences of the balance of care between hospital and the community are reported. All three groups wanted more people to be looked after in their homes rather than in hospital if adequate care could be arranged at home. But they perceived inadequacies in home help and district nursing services and many wanted other community services expanded or introduced. The main shortcomings of the hospital service were seen as inadequate numbers of hospice beds, difficulty obtaining admission for people needing long term care, discharge too early and some over-treatment of people who were dying. There was some evidence from relatives that pain control was better in hospital than at home, and the district nurses also reported that pain was not controlled satisfactorily for patients dying at home as often as it could be. It is concluded that inadequacies in community services may discourage some people from taking on the care of their relatives at home. PMID- 1720961 TI - [Traumatic luxation of the extensor apparatus of the dorsum of the metacarpophalangeal joint of the little finger. Six cases]. AB - Dislocation of the extensor tendon over the MP joint as a result of trauma is a rare and unrecognized injury as our six patients were only operated subsequently. The diagnosis is simple: flexion is possible, while extension is not. The patient is able to hold the extension despie resistance if the dislocation is reduced by artificial extension. X-rays eliminate any possible fracture. Six cases have revealed two different assumptions: either the two tendons (extensor digitorum communis and extensor digiti quinti proprius) are simultaneously dislocated together on the ulnar side, or they are dislocated on either sides of the joint. In the first assumption, the injury occurred ulnar-wise tearing the sagittal band. This case concerns the three older patients. The dislocation was reduced and stabilized by the Michon surgery method. The second assumption concerns the three youngest patients who suffered from a direct axial shock. This separation was reduced by suture of the two tendons on the dorsal base of the joint. The postoperative care was ensured in all cases by a splint attached to the palm, keeping the MP joint in extension and enabling immediate web-fingered reeducation. This therapeutic technique has given satisfactory results as over an average period of six years no recurrence of stiffness has been observed. PMID- 1720960 TI - Biocompatibility of hydroxyapatite-coated hip prostheses. AB - In three patients a mechanically well-fixed Mathys Ceros 80 (Ha) hydroxyapatite coated acetabular component was revised 2, 5 and 13 months after total hip replacement due to component malposition. In each case there was a thin cellular connective tissue membrane between hydroxyapatite and bone, the main cell type being fibroblast with only occasional giant cells. Immunohistological analysis revealed some MHC locus II antigen positive cells that were identified as monocytes. No interleukin-2 receptor positive cells were found. Under clinical cyclic loading conditions there does not seem to be chemical fixation or bony ingrowth into the hydroxyapatite coated prosthesis component. In human lymphocyte cultures, hydroxyapatite (Interpore 200, particle diameters 15-40 microns) did not cause an increase in lymphocyte DNA synthesis as assessed by the 3H-thymidine incorporation method on culture days 1, 3 and 5. As analysed with lymphocyte activation markers, the hydroxyapatite-dependent expression of MHC locus II antigen was modest and differed significantly (P less than 0.05) from that in culture medium only on day 3. Hydroxyapatite induced only a slight interleukin-2 receptor expression that did not differ from culture medium on days 1, 3 and 5. CD4 and CD8 positive lymphocytes as well as monocytes were not seen attached to hydroxyapatite particles during the culture days. Our findings suggest that hydroxyapatite is an immunologically inert implant material. PMID- 1720963 TI - [Necrotizing fasciitis of the upper limb. 12 cases]. AB - Twelve cases of necrotizing fasciitis or streptococcal cellulitis of the upper limb are reported. Four cases presented with a low grade aggressive for and one case was chronic. Seven fulminating cases resulted in two deaths. These different presentations are in fact different stages of the same disease which is a group A beta-hemolytic streptococcal necrotizing infection of the subcutaneous tissue. It is a medical emergency in which surgery is the main treatment. In cases seen early, surgery helps by making an early diagnosis by showing the typical appearance of the subcutaneous tissue and by isolating organisms in wound culture. In fulminant cases, only extensive surgical debridement can control infection. Delayed or incomplete radical excision may lead to disseminated infection. Infection spreading beyond one upper limb worsens the vital prognosis. PMID- 1720962 TI - [Scaphoid-trapezium-trapezoid dislocation. 5 cases]. AB - The authors report 5 cases of scaphoid-trapezium-trapezoid dislocation (STT) which occurred in crush injuries of the hand. A good functional result was achieved by closed reduction and pinning whenever there was no associated soft tissue damage. However, lateral mid carpal osteoarthritis may develop and produce reduction of the abduction of the thumb as well as slight stiffness of the wrist. PMID- 1720964 TI - [Carpal tunnel syndrome. Comparative studies of pre- and postoperative magnetic resonance and electromyography]. AB - A comparative wrist study was conducted in a total of 36 subjects: 20 normal volunteers and 16 patients with diagnosed carpal tunnel syndrome. 1.5 Tesla MR was used with a cylindrical extremity coll. The authors reviewed the anatomy of the wrist in the normal subjects. The accuracy of MR diagnostic criteria for carpal tunnel syndrome was assessed in comparison with EMG results and surgical findings. The authors conclude that MRI has proved to be reliable and useful in the diagnostic work up of carpal tunnel syndrome. Its main indication would be in cases of negative or doubtful EMG. PMID- 1720965 TI - [The neurological forms of thoracic outlet syndrome: the role of the middle scalene]. AB - The authors discuss the pathogenesis of neurological involvement in the thoracic outlet syndrome. They emphasise a factor which has been neglected up until now: the tendon of scalenus medius, around which the primary inferior trunk of the brachial plexus forms a curve with a postero-inferior concavity. An anatomical study reveals this close relation which is only apparent in the horizontal plane. Thirteen clinical cases of pure neurological forms, confirmed by Aran-Duchenne amyotrophy of the hand, illustrate the role of scalenus medius in this nerve lesion. The therapeutic application of this new concept consists of middle scalenectomy and posterior release of the nerve trunks by cervicotomy. PMID- 1720966 TI - [The chronically painful hand. A consecutive series of 60 cases]. AB - This study was based on 60 patients presenting with hand pain refractory to usual treatment. The aim of this study was to assess the pathophysiological and neuropsychological factors involved in a population of patients recruited in a hand surgery unit. Analysis of this series revealed the frequency of desafferentation mechanisms. These symptoms can be treated by means of antidepressants, antiepileptics and peripheral neurostimulation. Anxio-depressive factors were of limited importance in this series, at least in comparison with the population usually examined in a pain treatment centre. However, their presence in a subgroup of patients (26% of cases) and the limitations of symptomatic analgesic treatment justifie a global approach to chronic pain in order to establish the participation of the various factors in each particular case. It is useful to combine aetiological treatments with psychological treatments (antidepressants, relaxation therapy, for example) to increase the patient's tolerance of their persistent painful symptoms. PMID- 1720967 TI - [Digital varices. 12 cases]. AB - Twelve cases of varix of the fingers are reviewed. The mean age was 41 years with a female predominance (75%). A palmar site was predominant (92%) and the ring finger was most frequently involved (50%). No recurrence was observed after a mean follow up of 6 years. PMID- 1720968 TI - [Value of pulp recession in inveterate hook fingers. Techniques/indications]. AB - The aim of this operation is to preserve the sensitivity whenever when a finger has to be amputated. We have reviewed 9 patients operated according to this technique, out of 14 patients for whom an amputation or a shortening arthrodesis was discussed. The results of pulp recession were most encouraging. The incision were located on both sides of the finger and distant from the ungueal matrix. The dorsal skin was elevated in the sub-periosteal plane. Resection of the necessary amount of 2nd phalanx to obtain an extended finger was performed. An osteosynthesis with pins is preferred for a duration of 6 weeks. This operation can be performed for very stiff finger, hooked fingers or dystrophic fingers with a good pulp. In extreme cases of shortening, the nail with its bed ar sacrificed, and the entire pulp is used to cover the distal stump. This principal of pulp recession has been used in two other cases in emergency surgery. The postoperative was marked by finger swelling and some pain of the distal sutured skin. Overall the results were satisfactory and patients were very satisfied to keep their finger, which would otherwise have been doomed. PMID- 1720969 TI - [Reflections on 4 cases of ante-lunar carpal luxations]. AB - Over the past 20 years, 4 cases of ante-lunate carpal dislocation have been treated in the Traumatology and Orthopaedics Department of Besancon. During this same period, 61 cases of retro-lunate dislocation have also been treated. Ante lunate dislocations are very rare lesions as confirmed by a review of the literature. Our cases do not illustrate the mechanisms of such injuries since 3 cases involved patients with multiple injuries Clinical symptoms may be very minor in children in whom such injuries may easily be overlooked. A thorough interpretation of X-rays is necessary for diagnosis in adults. All 4 cases showed a fracture of the lunate in a frontal plane (factor of instability). Such an instable lesion justifies either internal osteosynthesis or percutaneous trans articular pinning when rigid internal fixation is impossible. However, in all cases in which the lesion was treated surgically, the capsular plane was not disrupted. Retrospectively, such a finding contraindicates an open surgical approach. Relationships between such dislocations and isolated fractures of the lunate are discussed. The prognosis is mainly determined by early diagnosis and management rather than the type of treatment itself. PMID- 1720970 TI - [Compression of the deep branch of the ulnar nerve as it exits the pisiform unciform hiatus: report of an anomaly not yet described]. AB - We observed a case of compression of the deep branch of the ulnar nerve distal to the piso-hamate hiatus due to an aberrant fibrous band arising from the hamulus ossi hamatum and ending in the flexor digiti minimi muscle. An anatomical study of eleven fresh cadaver hands revealed that between the piso-hamate hiatus and the palm, the nerve passes through an osteo-fibrous tunnel in which multiple anomalies can occur. The authors think that ulnar nerve neurolysis at the wrist must always extend up to the palm. PMID- 1720971 TI - [Tenosynovial osteochondromatosis of the hand. A case report]. AB - A 14 year old boy presented with swelling of the middle finger. X-rays showed calcifications of the palmar surface of the head of the third metacarpal and over the first and second phalanges. The natural course was followed over a period of two years with a diagnosis of calcifying peritendonitis. Due to the increased volume of the calcification deforming the proximal phalanx, the masses were resected revealing a diagnosis of chondroma. The discovery of free foreign bodies in the synovial sheath of the flexors and histological examination established the diagnosis of tenosynovial osteochondromatosis, which is rarely located in the hand. No recurrence has been observed after 8 years of follow-up. PMID- 1720972 TI - [A rare bone tumor of the hand: a solitary cavernous hemangioma. A case report]. AB - Solitary hemangioma located in the skeleton of the hand is a rare tumor, very different from diffuse angiomatosis or benign, hemangioma, located in the two distal phalanges of the right fourth finger. Due to articular stiffness, pain, and cosmetic problem, the treatment consisted of central ray resection. The pathological examination revealed a true benign skeletal hemangioma with marked bone resorption of the phalangeal skeleton. The soft tissues ant the periosteum were not involved. PMID- 1720973 TI - [Multiple rare tumors of the hand: a case of multifocal reticulohistiocytosis]. AB - The authors report the case of a woman operated for multiple dorsal nodes of the fingers with a lytic appearance on X-rays of the distal joints. The histopathology concluded on multicentric reticulohistiocytosis which is a very rare disease (fewer than 100 cases) affecting the hand (90%), skin and joints, and other organ systems. The prognosis is not good as the disease is disabling to the hand and malignant diseases may occur in 15 to 25% of cases. No treatment, even very aggressive, has been shown to be truly effective. PMID- 1720974 TI - [Incomplete section of a superficial flexor tendon in zone II: successive complications and quadriga syndrome]. AB - Partial section of the flexor tendon may be asymptomatic and heal without sequelae. However, in the absence of primary treatment, they may lead to subsequent complications such as: tendon locking due to incarceration of a partially detached slip of tendon, jump phenomena due to irregularity of the surface or the volume of the tendon, which induces a sudden jerk as the tendon passe through the retinaculum, and lastly, secondary tendon rupture. These complications all occurred in the case reported here, who also presented with syndrome of the quadriga. Locking of the intact deep flexor tendon by the ruptured superficial tendon interfered with the movements of the other fingers. Resection of the ruptured tendon was able to restore complete function. Opinions diverge concerning the need to suture partial sections of the flexor tendons, but the authors agree on the great importance of meticulous surgical exploration and immediate controlled mobilisation, which ensures the best functional results. PMID- 1720975 TI - [Sarcoid synovitis. A case report of localization at the level of the flexor tendons of the fingers]. AB - Sarcoidosis without bone involvement or sarcoid dactylitis, is a very unusual cause of flexor synovitis. Our reported patient initially presented with chronic arthralgia of the knees and ankles. The initial diagnosis of rheumatoid arthritis was incorrect. A surgical flexor synovectomy was performed to release painful compression of the median nerve due to the synovitis. The correct diagnosis was suggested by the histopathological examination showing noncaseating epithelioid granulomas. The diagnosis was confirmed by the association of a negative tuberculin test and raised angiotensin converting enzyme. No recurrence of synovitis occurred after surgical excision and colchicine therapy but arthralgia persisted. PMID- 1720976 TI - [Management of digital ischemia. 5 cases]. AB - Ischaemic fingers, a rare, generally chronic disease, may sometimes be acute, requiring emergency surgical treatment. Five cases are reported: 3 acute and 2 chronic. The 3 cases of acute ischaemia occurred in the context of cardiac arrhythmias in 2 cases and an aneurysm of the ulnar artery in 1 case. Treatment consisted of 2 thrombectomies with microsurgical digital sympathectomy thrombectomies with microsurgical digital sympathectomy and resection of the aneurysm. Complete clinical and functional recovery was obtained in these three cases. The 2 cases of chronic ischaemia were due to diabetes and Buerger's disease. In both cases, medical treatment was followed by thoracic sympathectomy with secondary resection of necrotic tissue as required. In conclusion, the prognosis in the acute cases depends on the rapidity of correction of the arterial obstruction associated with digital sympathectomy. In the case of chronic ischaemia, the clinical course depends on the efficacy of medico-surgical treatment and the severity of the underlying disease. PMID- 1720977 TI - DNA-dependent adenosinetriphosphatase A is the eukaryotic analogue of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication associated ATPases. AB - We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A. All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA dependent ATPase. Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A. Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication. These findings provide strong evidence for the role of ATPase A in DNA replication. These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA dependent ATPase. PMID- 1720979 TI - [Systematic approach to studying proteins from human platelets. Two-dimensional mapping]. AB - Total proteins of human platelets and their membranes were studied by two dimensional electrophoresis using the lysis in sodium dodecyl sulfate solution. Analysis of platelets from 30 donors revealed the presence of 105 repeating fractions on the electrophoregrams. A two-dimensional map of platelet proteins in the molecular mass versus relative electrophoretic mobility plot was constructed. This map made it possible to localize membrane proteins and albumin as well as a protein immunologically related to phenylalanine hydroxylase. Electrophoretic variants of 13 platelet polypeptides were identified. PMID- 1720978 TI - [The Ca2+-activated photoprotein obelin as a calcium transport inducer in proteoliposomes in the membrane of the T-system of skeletal muscles]. AB - The calcium-binding photoprotein obelin extracted and purified from the luminescent hydroid Obelia longissima was used to record the processes of Ca2+ release from proteoliposomes. It has been shown that lecithin proteoliposomes with incorporated rabbit skeletal muscle T-system membranes possess a BAY K-8644 activated permeability which is inhibited by nitrendipine. The Ca(2+)-activated photoprotein obelin is a convenient and perspective tool in studies of fast calcium fluxes. PMID- 1720980 TI - [Isolation and purification of Lpm and alpha-2 M macroglobulins from mink serum]. AB - A procedure for large-scale separation and purification of two mink serum macroglobulins, Lpm and alpha 2M, is described. Individual preparations of each of these macroglobulins were obtained in an immunologically pure state. After precipitation from the serum at 6.5-13% PEG 6000, Lpm and alpha 2M were separated by a pH stepwise gradient elution metal chelate affinity chromatography and purified by chromatographies on Biogel A 1.5 m and DEAE-Trisacryl M. Each of these macroglobulins was tested by counter-immunoelectrophoresis with the corresponding monospecific antiserum. The yields per 100 ml of the source serum were 23-44 mg of Lpm and 7-30 mg of alpha 2M which corresponded to 10-20% of their serum contents. Some of general biochemical properties of mink Lpm and alpha 2M and of all mammalian alpha-macroglobulins are discussed. PMID- 1720981 TI - Developing effective courtroom exhibits: working with a graphics consultant. PMID- 1720982 TI - Myeloid progenitor cell growth characteristics and effect of G-CSF in a patient with congenital cyclic neutropenia. AB - A 17-year-old male with congenital cyclic neutropenia was treated with recombinant human granulocyte colony stimulating factor (G-CSF) administered subcutaneously at 1 to 2 micrograms/kg per day. The peak and nadir counts of neutrophils and the peak counts of monocytes were significantly elevated, and the period of cycling decreased from 3 to 2 weeks. Bone marrow culture studies revealed the following abnormalities in granulocytic progenitor cells (CFU-G): a decrease in the concentrations of G-cluster forming cells, stimulated by a maximal dose of G-CSF, and a tendency of abnormally low responsive growth of the CFU-G to lower concentrations of G-CSF and GM-CSF. Our findings suggest that administration of G-CSF at relatively low doses overcomes or compensates for these abnormalities, though not completely, as fluctuation in the neutrophil counts persisted. PMID- 1720983 TI - [Antifibrotic effects of aldehyde dextran modified superoxide dismutase in experimental silicosis]. AB - The present paper dwells on biomedical study of aldehyde dextran modified superoxide dismutase. Pharmacokinetic data demonstrated that modification of superoxide dismutase increased its half-time. A rat model of experimental silicosis showed that aldehyde dextran modified superoxide dismutase inhibited evolving fibrosis in the lungs. The same dose of native enzyme produced no therapeutic effect. Thus, superoxide dismutase can be considered as a potential agent for treatment of fibrosis due to its modification. PMID- 1720984 TI - [Immunocytochemistry of keratins in the tongue mucosa of rats]. AB - C12 and E2 monoclonal antibodies to keratins stained taste bud cells of foliate papillae as well as the cells of the associated glands. H4 monoclonal antibody to keratin reacted with the surrounding epithelial cells and did not react with the taste bud cells. The results show that the keratin subtype differs between taste bud and surrounding epithelial cells. The similar keratin composition of taste bud and associated gland cells confirm the view on the existence of the same type of cells in these two structures. PMID- 1720985 TI - [Stimulation of proliferative and metabolic processes in the liver after administration of allogeneic hepatocytes]. AB - The interconnection of morpho-functional changes of liver and cellular capacity to synthesize RNA and DNA in condition of the acute liver failure (ALF) and after the administration of the newborn allogenic hepatocytes (NAH) was studied by the autoradiographic method. The administration of NAH prevents, in part, the ischemic cell death and is efficient in the stimulation of the liver regeneration in ALF; it increases metabolic and proliferative activity of hepatocytes as well as of reticulo-endothelial cells. PMID- 1720986 TI - [Immunohistochemical study of the expression of trophoblastic beta-1-globulin in the gastric epithelium of man in stomach cancer]. AB - The expression of specific-pregnancy beta 1-globulin (SP1) was studied in gastric mucosa with chronic gastritis. The expression of SP1 was revealed in focus of intestinal metaplasia, dysplasia as well as in cover epithelial cells. PMID- 1720987 TI - Organ-specific mucin antigens and gastrointestinal carcinogenesis. AB - Organ-specific mucin antigens detected by polyclonal antibodies were first used to characterize abnormal differentiation of gastrointestinal (GI) adenocarcinomas (1,2). These mucin tumour markers can thus be useful as criteria for evaluating the degree of gastric or intestinal differentiation of gastric carcinomas. In addition, some of these mucin antigens are expressed early during GI carcinogenesis where they are associated to premorphological abnormalities (3-5). Consequently, these markers are of importance for studying genetic changes occurring in precancerous mucosa, in which cancer cell transformation can occur. Finally, recent observations have pointed out a putative value of these mucin antigens for screening high risk populations for GI cancers. First of all, we shall define the words: mucin, mucin antigen (or epitopes) and organ-specificity before described abnormal antigen expression during GI carcinogenesis in the cancerous and in the precancerous mucosae and specially concerning their association with premorphological abnormalities. PMID- 1720988 TI - Vital staining of oesophagus in patients with head and neck cancer: still a worthwhile procedure. AB - One hundred three patients with upper aerodigestive cancer were consecutively submitted to upper GI endoscopy with vital staining (Toluidine Blue 1%) of the oesophagus. The aim of the study was not only to confirm the prevalence of synchronous or metachronous tumour but also to verify the usefulness of the vital stain compared to simple endoscopy. Staining was positive in 29 patients (28.1%) for oesophagitis, leukoplakia, Barrett's oesophagus and 3 oesophageal neoplasms (2.9%), two of them unsuspected at endoscopy. We did not observe false positives while 13 cases (13/29-44.8%) were considered normal at endoscopy. Five cases with some endoscopic abnormality of the mucosa did not stain and were considered false negatives. Specificity of the method was 100%, sensibility 85.2%. The recognition of dysplastic changes and neoplasms not suspected at endoscopy should recommend in our opinion the use of vital staining of oesophagus in high-risk groups. PMID- 1720989 TI - Treating carcinoma of the oesophagus. PMID- 1720990 TI - Treating bony metastases. PMID- 1720991 TI - Monoclonal antibody treatment in rheumatoid arthritis: the clinical and immunological effects of a CD7 monoclonal antibody. AB - Six patients with rheumatoid arthritis were treated with a CD7 mouse monoclonal antibody, RFT2, daily for 15 days. Only two patients had a significant improvement in clinical disease activity which lasted 7-14 days. No serious adverse effects occurred although all patients developed antibodies against mouse immunoglobulin. During treatment T-lymphocyte numbers decreased and T-lymphocyte CD7 expression was absent in all but one patient. PMID- 1720992 TI - Characteristics of mast cells in normal bladder, bacterial cystitis and interstitial cystitis. AB - An analysis was made of the numbers and characteristics of mast cells in lateral bladder wall biopsies from 22 patients with interstitial cystitis, 6 with bacterial cystitis and 8 normal controls, using toluidine blue stains and computerised video image analysis techniques. A significantly greater number of mast cells were found within the detrusor muscle in interstitial cystitis than in bacterial cystitis or normal controls. Within the urothelium and submucosa, mast cell numbers were significantly greater than in normal controls in both interstitial and bacterial cystitis. In interstitial cystitis mast cells were significantly larger within the detrusor than in the urothelium/submucosa and they appeared to degranulate predominantly within the superficial layers. Differential staining techniques, using long and short toluidine blue stains, failed to reveal statistically significant evidence of mast cell heterogeneity within the bladder wall in interstitial cystitis. PMID- 1720993 TI - Roles of cyclic GMP and inositol trisphosphate in phototransduction of the molluscan extraocular photoreceptor. AB - The internal messengers mediating the photocurrent of the molluscan extraocular photoreceptor, A-P-1, were examined. In the dark, pressure-injection of cGMP into the A-P-1, voltage-clamped at resting levels, produced a rapid outward current, associated with an increase in conductance. However, the cGMP-induced current and increase in conductance were suppressed by subsequent photostimulation, suggesting hydrolysis of cGMP by light. The steady-state I/V relation for the cGMP-induced current was non-linear. The I/V relation for the instantaneous cGMP induced current, measured 50 ms after the beginning of a voltage step, was linear, and reversed at the membrane potential, -67 mV, which corresponded to the K+ equilibrium potential of A-P-1 in 10 mM K+ normal saline. These findings indicate that the internal cGMP induces a voltage- and time-dependent K+ current. Since the photocurrent results from the suppression of a voltage- and time dependent K+ current similar to above, the photocurrent is considered to be equivalent to the suppression of the cGMP-induced current. Short pressure injection of GDP-beta-S into A-P-1 reduced the subsequent photocurrent. The photocurrent was also suppressed after an external application of Pertussis toxin. On the other hand, the photocurrent was amplified by prior pressure injection of inositol 1,4,5-trisphosphate (IP3). However, a short pressure injection of neomycin into A-P-1 depressed the subsequent photocurrent. These results suggested that the cGMP-induced (dark) current is mediated by cGMP, and that hydrolysis of cGMP by light leads to the photocurrent, then being modified by another messenger, IP3, to be amplified.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1720994 TI - Ultrastructural identification of trigeminal nerve terminals in the pterygopalatine ganglion of rats: an anterograde tracing and immunohistochemical study. AB - Trigeminal nerve terminals in the rat pterygopalatine ganglion (PPG) were ultrastructurally identified using anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L). Electron microscopic immunohistochemistry was used to demonstrate the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) in nerve terminals of the PPG. Adjacent to the rostral part of the PPG an additional minor area was described. Perikarya in this minor rostral part were more spherical and had irregular outlines. Ultrastructurally, the glial enwrapment of the nerve terminals seemed to be more loosely arranged in comparison to that in the major rostral part of the PPG. With PHA-L, numerous labelled nerve fibres and terminals were found in all parts of the PPG. The ultrastructure of these terminals was uniform, many of them showing synaptic contacts. Numerous terminals in the PPG were SP-positive, whereas only a few were CGRP-positive. Fibres stained positive for both neuropeptides. The PPG is shown to be synaptically innervated by sensory fibres arising in the trigeminal ganglion, with the strong suggestion of SP and CGRP acting as neurotransmitters. A modulatory interaction between the autonomic and sensory system, resembling an axon reflex mechanism in the peripheral nervous system is endorsed. PMID- 1720995 TI - Voltage clamp analysis of intact stomatogastric neurons. AB - Two-electrode voltage clamp of intact, identified pyloric neurons of the spiny lobster stomatogastric ganglion reveals two major outward currents. A rapidly inactivating, tetraethylammonium- (TEA) insensitive, 4-aminopyridine- (4AP) sensitive, outward current resembles IA of molluscan neurons; it activates rapidly on depolarizations above rest (e.g. -45 mV), delaying both the axonal sodium and the neuropil-calcium spikes which escape voltage-clamp control. We infer that A-current is distributed both in a space clamped region (on or near the soma) and in a non-space clamped region with access to the generators for sodium and calcium spikes. A calcium-dependent outward current, IO(Ca), activates rapidly at clamp steps above -25 mV and inactivates at depolarizing holding voltages. Increasing depolarization results in an increase in both IO(Ca) and firing rate but a reduction in the amplitude of the sodium spike current. Blockage of IO(Ca) with Cd2+ causes little change in spike firing pattern. These observations are consistent with IO(Ca) being activated primarily in the soma and nearby regions which are under good control with a soma voltage clamp (and distant from the Na(+)-spike trigger zone). While the lack of space clamp limits resolution of charging transients and tail currents, the identification of the major current subgroups can still be readily accomplished, and inferences about the location and function of currents can be made which would not be possible if the cells were space clamped or truncated. PMID- 1720996 TI - Serotonin regulation of neostriatal tachykinins following neonatal 6 hydroxydopamine lesions. AB - In order to determine whether dopamine mediates the effects of serotonin on tachykinin biosynthesis in the neostriatum, serotonin neurotransmission was altered following depletion of dopamine. Neonatal rats received intracisternal injections of saline or the dopamine neurotoxin 6-hydroxydopamine (6HD). This lesion caused significant reductions in the neostriatum of substance P-like immunoreactivity as well as levels of mRNA coding for preprotachykinin (PPT; the prohormone precursor to tachykinins substance P, neurokinin A and related peptides). Two months later, rats were treated for 5-6 days with saline or the serotonin-uptake inhibitor, zimelidine. Zimelidine treatment of unlesioned animals significantly increased PPT mRNA levels in the neostriatum. However, zimelidine treatment failed to increase PPT mRNA content in 6HD-treated animals. By contrast, neostriatal substance P-like immunoreactivity was restored by zimelidine treatment of 6HD-lesioned animals. These results suggest that an intact nigrostriatal pathway may be required for serotonin neurotransmission to alter PPT mRNA levels in the neostriatum. However, neostriatal tachykinins may be regulated by direct serotonin innervation. PMID- 1720997 TI - Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat: distribution and coexistence with putative transmitter agents. AB - Immunoreactivity for the calcium binding protein, calretinin (calretinin-ir), was demonstrated in cell bodies of vagal and glossopharyngeal sensory ganglia (jugular, petrosal, and nodose ganglia) and in associated nerve fibers. In the jugular and petrosal ganglia, many calretinin-ir neurons were also immunoreactive for calcitonin gene-related peptide and substance P. In the nodose ganglion, most of the calretinin-ir neurons lacked these peptides. None of the calretinin-ir neurons in these ganglia were also immunoreactive for tyrosine hydroxylase. PMID- 1720998 TI - Reserpine-insensitive dopamine release in the substantia nigra? AB - Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic pathway, but also from the dendrites in the substantia nigra (SN). Whether the DA release in the SN is sensitive to reserpine treatment, as it is in the stratum, has, however, not been clarified. We have determined the effects of reserpine on the concentrations of DA, serotonin and their metabolites in the SN and in the striatum and as an index of DA release in vivo we have assessed the accumulation of the DA metabolite 3-methoxytyramine (3-MT) following inhibition of monoamine oxidase by pargyline. The effects of reserpine on the concentrations of DA and its metabolites were different in the SN as compared to in the striatum. In the striatum there was a maximal depletion of DA to 2% of controls, but in the SN the DA concentration decreased only to 17% of controls. In the SN, the increases of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were less pronounced than in the striatum. In the striatum reserpine treatment (given 15 h, 3.75 h, or 1.75 h before pargyline) decreased the pargyline-induced 3-MT accumulation to 30% of pargyline-treated controls. However, in the SN no effects of reserpine were observed. The results indicate that DA in the SN partly is situated in a reserpine insensitive pool and that the release of DA might be insensitive to reserpine. These differences between the SN and the striatum could be due to different storage mechanisms. In the striatum DA is stored in classical storage granulas but in SN DA is partly stored in storage granulas and partly in smooth endoplasmatic reticulum. PMID- 1720999 TI - An improved microcomputer program for finding gene- or gene family-specific oligonucleotides suitable as primers for polymerase chain reactions or as probes. AB - We present here an easy-to-use computer program which finds oligonucleotides suitable as primers in polymerase chain reactions (PCR) or as probes for hybridization. In contrast to other programs used for this purpose, the additional advantage of this one is the possibility of directly detecting gene- as well as gene family-specific oligonucleotides. For this purpose, up to 200 different DNA sequences, of maximally 65,000 nucleotides each, can be scanned in a single search to ensure either single or multiple gene binding of the PCR primers or probes. Specific oligonucleotides for genes carrying internal repetitions and for single genes belonging to a set of highly conserved genes can also be detected. Many parameters such as exclusion of simple sequences, which are known to be highly repeated throughout various genomes or regions of stable secondary structures in both primer-primer and primer-template, can be taken into consideration and avoided. Furthermore, the G + C content and the length of the oligonucleotides can be changed in a broad range by the user. PMID- 1721000 TI - Alternated versus syncopated CHOP-PVB: two studies for the treatment of non Hodgkin's lymphoma by the Southwest Oncology Group. AB - Based on a preliminary trial that suggested that CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), and PVB (cisplatinum, vinblastine, bleomycin), are at least partially non-cross-resistant, the Southwest Oncology Group treated patients with unfavorable histology, non-Hodgkin's lymphoma with CHOP and PVB. In the first study, 76 eligible patients were given three courses of CHOP, with complete or partial responders receiving three courses of PVB followed by three further courses of CHOP. Nonresponders after the initial three cycles of CHOP, received six courses of PVB. In the second study, 154 eligible patients were treated with alternating cycles of the two drug regimens. The overall objective antitumor response (CR + PR) was 77% for the first study and 58% for the second. The complete remission rates were 48% and 38%, respectively. The overall survival for both studies is similar. These results are interpreted in terms of the Goldie-Coldman hypothesis. PMID- 1721001 TI - An assessment of alternated versus syncopated CHOP-PVB. PMID- 1721002 TI - [Granulocyte differentiation of human promyelocytic leukemic cells induced by coordinate action of granulocyte colony stimulating factor and retinoic acid]. AB - We analysed the effects of recombinant human G-CSF (rhG-CSF) and retinoic acid (RA) on proliferation and differentiation of HL-60 cells and human acute myeloid leukemic (AML) cells. A synergistic effect on granulocyte differentiation was observed when HL-60 cells and primary cultured acute promyelocyte leukemic cells were cocultured with 10(-8)mol/L RA plus 1:2000 or 1:1000 rhG-CSF. The rhG-CSF plus RA treated cells demonstrated significant increase in the percentage of mature cells. Morphological changes and nitroblue tetrazolium (NBT) reduction activity evidenced more increase than RA treatment alone (P less than 0.001). The results suggest that RA not only inhibits the proliferative action of G-CSF, but also retains and enhances the action of G-CSF to induce differentiation. Therefore, we believe that the combined use of G-CSF with RA may improve the treatment of leukemia. PMID- 1721003 TI - Reduced cardiovascular sympathetic excitatory responses during iloprost infusion in conscious dogs. AB - STUDY OBJECTIVE: The aim was to determine the effects of the stable prostacyclin analogue iloprost (ZK 36374) on systemic haemodynamics and on cardiovascular neural control. DESIGN: The buffering effect was examined of intravenous and intracoronary iloprost infusion on the excitatory sympathetic reflexes elicited from the heart by (1) intracoronary injections of bradykinin and (2) transient coronary artery occlusion. SUBJECTS: 22 conscious mongrel dogs of either sex, weight 20-25 kg, were used. MEASUREMENTS AND MAIN RESULTS: ECG, systemic arterial pressure, left atrial pressure, and left ventricular pressure, and contractility (dP/dt) were continuously monitored for the duration of the experiments. Iloprost infusion reduced left ventricular pressure, mean arterial pressure, and dP/dt without causing significant changes in heart rate. Transient non-hypertensive coronary artery occlusion increased heart rate and depressed contractility. During intravenous iloprost infusion, coronary artery occlusion no longer elicited an increase in heart rate, while left ventricular dP/dt was more drastically reduced. This pattern of response was not substantially modified by beta adrenergic blockade, whereas the blockade of muscarinic receptors with atropine was accompanied by hypotension and a greater reduction of dP/dt. The observation of a reduced pressor response to the intracoronary injections of bradykinin during iloprost administration further indicated a restraining effect of iloprost on the sympathetic reflexes elicited from the heart. CONCLUSIONS: The data suggest the hypothesis that the protective effects on the ischaemic myocardium observed with iloprost infusions may arise not only from its vasodilator and antiplatelet properties, but also from its capacity to blunt excitatory sympathetic reflexes. PMID- 1721004 TI - An in oculo model for quantitation of vascular growth in the rat heart. AB - STUDY OBJECTIVE: The aim was to describe, both qualitatively and quantitatively, vascularisation in an in oculo model that may prove useful for studying neovascularisation in the myocardium. DESIGN: Whole hearts from 13-14 d rat fetuses were implanted into the anterior chamber of host Sprague Dawley rats. Implant growth was monitored and at 3 months postimplantation grafts were recovered. EXPERIMENTAL MATERIAL: Fixed tissue sections were histologically characterised with light and electron microscopy and morphometrically analysed using image analysis. MEASUREMENTS AND MAIN RESULTS: At two weeks, 92% of implanted hearts were beating. At 1 month, well vascularised implants that were beating were slightly larger than those not beating, at 16.0(SD 1.0) v. 12.3(2.0)mm2. By 2 months, beating implants were significantly (p less than 0.05) larger than non-beating implants, at 23.5(3.0) v. 12.4(1.7)mm2. In non-beating implants, the larger the extent of vascularisation, the larger the implant size. Histologically, well vascularised beating implants showed normal adult cardiac myocyte structure, with normal appearing sarcomeres and intercalated discs, but no preferential fibre orientation. The amount of area occupied by muscle and connective tissue varied between implants. Implant vasculature, including arterioles and capillaries, appeared normal. The percentage of vascular area was consistent between grafts. A small percentage of total area was occupied by lymphocytes. CONCLUSIONS: This investigation indicates that sufficient vascularisation is important for growth of grafts, provides for the first time quantitative morphometric data characterising the in oculo cardiac implant model, and reports baseline data that will be useful for comparison to myocardium treated with putative angiogenic factors in the future. PMID- 1721005 TI - A large proportion of afferent neurons innervating the uterine cervix of the cat contain VIP and other neuropeptides. AB - Axonal tracing techniques were used in combination with immunohistochemistry to examine the distribution of neuropeptides in afferent pathways from the uterine cervix of the cat. Primary afferent neurons innervating the uterine cervix were identified by axonal transport of the dye, fast blue, injected into the cervix. Fifteen to twenty-five days after the injection, dorsal root ganglia (L1-S3) were removed and incubated for 48-72 h in culture medium containing colchicine to increase the levels of peptides. Calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), leucine-enkephalin (LENK), somatostatin, substance P and vasoactive intenstinal polypeptide (VIP) were identified by use of indirect immunohistochemical techniques. Eighty-four percent of uterine cervix afferent neurons were identified in the sacral dorsal root ganglia (S1-S3), and 16% in the middle lumbar dorsal root ganglia (L3-L4). In sacral dorsal root ganglia, VIP was present in the highest percentage of dye-labeled cells (71%), CGRP in 42%, and substance P in 18% of the cells. CCK and LENK were present in 13% of the cells. In lumbar dorsal root ganglia, CGRP (51%) was most prominent peptide followed by VIP (34%), substance P (28%), LENK (17%) and CCK (13%). Somatostatin was present in the ganglia but did not occur in dye-labeled neurons. In conclusion, the uterine cervix of the cat receives a prominent VIP- and CGRP-containing afferent innervation. The percentage of neurons containing VIP is three to five times higher than the percentage of these neurons in afferent pathways to other pelvic organs. These observations coupled with the results of physiological studies suggest that VIP is an important transmitter in afferent pathways from the cervix. PMID- 1721006 TI - Localization of neuropeptides in the nervous system of the marine annelid Sabellastarte magnifica. AB - Immunohistochemical studies of the nervous system of Sabellastarte magnifica, a sedentary polychaete, showed the presence of neuropeptide expressing cells and fibers within the double ventral nerve cord. Immunoreactivity to cholecystokinin, neuropeptide Y, enkephalins, substance P, and FMRFamide was found to be present in specific populations of cells, identifiable by their location and by the neuropeptide they expressed. Fibers expressing the various neuropeptides were also observed in particular locations within the nerve cord. This characteristic distribution of the various neuron subgroups and fiber pathways may represent functional circuits within the nervous system of this annelid. PMID- 1721007 TI - Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland. AB - An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation. PMID- 1721008 TI - Aflatoxin B1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and non-tumorous liver tissue. AB - Studies were carried out to test the hypothesis that exposure to aflatoxin B1 (AFB1) is common among individuals with hepatocellular carcinoma (HCC) who are also chronically infected with hepatitis B virus (HBV). Experiments were also carried out to determine whether there is a close association between the presence of AFB1-DNA adducts and the expression of one or more HBV antigens in the tumor or non-tumor regions of the liver. Twenty-seven paired tumor and non tumor liver tissues of HCC patients from Taiwan were analyzed. Monoclonal antibody 6A10, generated against the imidazole ring-opened persistent form of the major N-7 guanine adduct of AFB1, was used for adduct detection by both indirect immunofluorescence and competitive enzyme-linked immunosorbent assay. An avidin biotin complex staining method was used for the detection of HBsAg and HBxAg in liver sections. A total of 8 (30%) HCC samples and 7 (26%) adjacent non-tumor liver tissue samples from Taiwan were positive for AFB1-DNA adducts. For HBsAg, 10 (37%) HCC samples and 22 (81%) adjacent non-tumorous liver samples were positive while 9 (33%) HCC samples and 11 (41%) adjacent non-tumor liver samples were HBxAg-positive. No association with AFB1-DNA adducts was observed for HBsAg and HBxAg. These results suggest that both AFB1 exposure and carrier status of HBsAg/HBxAg may be involved in the induction of HCC in Taiwan. PMID- 1721009 TI - The facilitated effect of retinol on rat hepatocarcinogenesis induced by 3' methyl-4-dimethylaminoazobenzene. AB - To examine the influence of retinol acetate (retinol, known as an inhibitor of tumor promotion) on 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB)-induced hepatocarcinogenesis, rats were fed with a diet containing 0.06% 3'MeDAB for 4 or 7 weeks and then with a normal diet for 21 or 18 weeks. Rats were given retinol (0, 6.25, 12.5 and 25.0 mg/rat, dissolved in DMSO) i.p. every 5 days from the 10th week to the 20th week. As a control, rats were fed a basal diet and given retinol at the same doses as mentioned above. At the 25th week, the incidence of hepatoma (hepatocellular carcinoma and cholangiocarcinoma) of each group was checked. In rats fed diet containing 3'MeDAB for 7 weeks, significant increases in the incidence of hepatoma were seen in retinol-treated groups at various doses. In rats fed 3'MeDAB diet for 4 weeks, all three doses also moderately, though not significantly, increased the incidence of hepatoma. No liver tumor was found in rats fed normal diet followed by treatment with retinol at any dose. Except for slight but detectable elevation of cellular retinoic acid binding protein levels in tumor tissues obtained from rats treated with retinol, no obvious differences in cellular retinol binding protein and gamma-glutamyl transpeptidase in the tumor tissues were observed between retinol-treated and untreated rats. Phytohemagglutinin-induced lymphocyte blastogenesis of the tumor bearing rats with or without retinol treatment showed approximately 50% inhibition compared with that of rats fed normal diet without retinol treatment. These results indicated that the administration of retinol in the early stages of hepatocarcinogenesis enhanced the tumor induction, possibly due to the fixation of malignant transformation of the cells. PMID- 1721010 TI - Urinary excretion of CD23 antigen in normal individuals and patients with chronic lymphocytic leukaemia (CLL) AB - A soluble form of CD23 (sCD23) was found in the urine from 12 normal individuals but was not present in 20 normal sera, suggesting that sCD23 produced by cells in tissues is eliminated in the urine. The sCD23 from urine differed in physicochemical properties from the sCD23 found in supernates from B lymphoblastoid cell lines (B-LCL) and in the sera of patients with B type chronic lymphocytic leukaemia (B-CLL). On SDS-PAGE analysis under reducing conditions urinary sCD23 showed two bands corresponding to molecular weights of 45-60 kD and 28-35 kD indicating that sCD23 may be excreted in combination with another molecule. When subjected to gel filtration in its native state, sCD23 from urine showed a major peak at approximately 150 kD and a minor peak (probably a breakdown product) at 21 kD. Urinary sCD23 was more strongly held by DEAE cellulose and required 0.5 M buffer pH 8.0 for elution, suggesting that it is more anionic than sCD23 from culture supernates. Five MoAbs recognizing different epitopes on sCD23 from B-LCL supernates were tested on urinary sCD23. Four of the MoAbs were reactive but one (EBVCS-1) was not. Urinary sCD23 did not bind to IgE. The level of sCD23 found in normal urine (approximately 0.02-0.05 micrograms/ml) was exceeded in 17 of 24 cases of B-CLL. In one case with a high cell count and a serum concentration of 10 micrograms/ml, the urine contained 80 micrograms/ml sCD23. In another case a high serum sCD23 was not matched by a high urinary level. In this case the gel filtration pattern was closer to that found with urine sCD23 rather than the B-LCL pattern found with sera of other B-CLL patients. PMID- 1721011 TI - Antibodies to mouse laminin in patients with systemic sclerosis (scleroderma) recognize galactosyl (alpha 1-3)-galactose epitopes. AB - Employing radioimmunoinhibition assays with distinct oligosaccharides as inhibitors, this study demonstrates that the epitope recognized on mouse laminin by sera from patients with systemic sclerosis (scleroderma) is a terminal galactosyl (alpha 1-3)-galactose disaccharide. The reaction with this alpha digalactose was further confirmed when the sera were tested in radioimmunoassay (RIA) binding assay and in ELISA with synthetic galactose alpha 1-3 galactose coupled to human serum albumin. The circulating antibody appeared restricted to the IgG class and mostly to the subclass IgG3 and IgG4. Antibodies with the same specificity can be found in patients with American cutaneous leishmaniasis and Chagas disease; however, whilst in these diseases the antibody production is triggered by antigenic determinants present on the surface of the parasites, the events eliciting their appearance in systemic sclerosis are unknown. PMID- 1721012 TI - Antibodies to Mycobacterium tuberculosis-specific epitopes in lepromatous leprosy. AB - Sera from patients with leprosy or tuberculosis and healthy subjects have been analysed for the presence of antibodies to four species-specific mycobacterial epitopes, four different viruses and five autoantigens. Antibodies to the Mycobacterium leprae-specific 35-kD protein and phenolic glycolipid I epitopes were not present in patients with active pulmonary tuberculosis. In contrast, antibody levels to species-specific epitopes of the 38-kD and 14-kD antigens M. tuberculosis were significantly elevated in patients with lepromatous leprosy. Neither of the two antigens is cross-reactive with M. leprae at the B cell level. However, it was considered that cross-reactive helper T cells could recall the response of M. tuberculosis-specific memory B cells, which had been primed through prior self-healing tuberculous infection. As an alternative explanation, the possible role of polyclonal B cell stimulation was considered. This seemed unlikely, however, since: (i) antibody levels to autoantigens, except anti-smooth muscle, were not elevated, and (ii) antibody levels to four distinct viruses, unlike those to all mycobacterial epitopes, showed no correlation with titres, to M. tuberculosis-specific epitopes. PMID- 1721013 TI - Functional characterization of skin-infiltrating lymphocytes in atopic dermatitis. AB - Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In 3H-thymidine incorporation assays, SIL showed proliferation in response to IL-2, IL-3, IL-4, ionomycin (Io) + 12-o-tetradecanoyl-phorbol-13 acetate (TPA) and OKT3 + TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-alpha) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-alpha upon stimulation with mitogens but failed to secrete IFN-gamma. Io in combination with phorbol-ester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-alpha and low amounts of IFN gamma. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-gamma but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2 like' cell-mediated immune response in lesional skin of AD patients. PMID- 1721014 TI - Anti-human thyroid peroxidase and anti-human thyroglobulin antibodies present no cross-reactivity on recombinant peptides. AB - Thyroglobulin (Tg) and thyroid peroxidase (TPO) are two antigens largely recognized by the sera from patients with autoimmune thyroid disease (AITD). Recently, the complete mapping of both antigens was established with rabbit polyclonal antibodies by the use of recombinant proteins expressed in prokaryotic vector. Several investigators have argued for the existence of a cross-reactivity of some hetero- and autologous antibodies versus these two proteins. In the present study, using rabbit polyclonal antibody, mouse polyclonal antibody and autoimmune antibody (aAb), we observed no common epitope on human Tg (hTg) and human TPO (hTPO). PMID- 1721015 TI - Inter-mouse strain differences in the in vivo anti-CD3 induced cytokine release. AB - Triggering of the CD3 molecule by in vivo injection of the hamster anti-murine CD3 monoclonal antibody 145-2C11 in adult BALB/c mice leads to massive although transient T cell activation. High levels of tumour necrosis factor (TNF), interferon-gamma (IFN-gamma), IL-2, IL-3 and IL-6 are released into the circulation 1 to 8 h after a single 10 micrograms 145-2C11 i.v. injection. This release induces an impressive self-limited physical reaction associating hypothermia, hypomotility (as assessed by actimetry), diarrhoea, piloerection and even death when high doses (a single dose of greater than 100 micrograms/mouse injection) are administered. In vivo injection of 145-2C11 to other selected mouse strains, namely NZW, CBA/J and C3H/HeJ, induced both different cytokine release patterns and sickness. 145-2C11 induced significant release of TNF and IL 2 in all four strains. At variance, IFN-gamma was only detected in BALB/c mice sera which, in terms of physical reaction (hypothermia and hypomotility) were the most affected. Higher and long-lasting circulating IL-3/GM-CSF levels were present in CBA/J sera, correlating with a later recovery. These results underline heterogeneity in the in vivo cell activation pattern among different mouse strains, when triggering T lymphocytes via the CD3/Ti molecule as compared to exclusive targeting of monocyte/macrophages by means of lipopolysaccharide. PMID- 1721016 TI - Plasma IGF-I binding proteins in sheep: effect of recombinant growth hormone treatment and nutritional status. AB - In humans the IGF binding proteins (BP) are closely related to metabolic status. In this paper we have examined the influence of controlled feed intake and GH treatment on IGF binding proteins in growing lambs. Analyses were performed on plasma samples from animals maintained on two levels of feed intake (1.75% body weight as lucerne pellets or 3% body weight which is approximately equivalent to an ad libitum intake) either with or without recombinant bovine growth hormone (BST; 0.25 mg/kg body weight/day) administration. Samples used for the analyses reported in this paper were collected at 9.00 hr following 41 d of treatment. Total plasma IGF-I was increased on the higher plane of nutrition (P less than .01) and by BST (P less than .001) but only on high feed intake. IGF is associated with BP of 150 kDa and 40-50 kDa in sheep plasma. 150 kDa bound IGF-I was increased on the higher plane of nutrition (P less than .05) and by BST treatment (P less than .001) but only on the higher feed intake. By contrast no change in 40-50 kDa bound IGF-I was observed with treatment. Unbound IGF-I was also found in sheep plasma (2-5% of total) but demonstrated only minor changes in relation to treatment. Saturation analysis gave estimates of total binding capacity and saturation of the IGF-BP. In ovine plasma the binding capacity of the 150 kDa species is in excess of bound IGF (P less than .001). Saturation did not change with treatment despite the observed differences in 150 kDa bound IGF I. Thus BP(s) contained in the 150 kDa fraction were responsive to treatment. By contrast large differences in saturation of the 40-50 kDa species were observed (P less than .001) despite little treatment dependent change in bound IGF-I. IGF BP(s) in the 40-50 kDa fraction were elevated in the low nutrition group and suppressed on the higher feed intake resulting in near saturation. These data strongly suggest that the IGF BP are modulated according to metabolic status in the sheep. PMID- 1721017 TI - Evaluation of interference by insulin-like growth factor I (IGF-I) binding proteins in a radioimmunoassay for IGF-I in serum from dairy cows. AB - Insulin-like growth factor I (IGF-I) circulates in serum bound to a number of different binding proteins (BPs). With antibodies currently available, BPs must be dissociated and inactivated or removed from serum prior to measurement of IGF I by radioimmunoassay (RIA). Serum samples which spanned a 13-fold range in IGF-I concentration were obtained from lactating dairy cows and used to develop conditions for assay of IGF-I with minimal interference from BPs. Removal of BPs from serum by acid-ethanol extraction resulted in interference in the RIA. Therefore, serum was incubated with 0.1 M glycyl-glycine HCl to inactivate BPs as suggested by Underwood et al. Time, temperature and pH were optimum when serum was incubated for 48 hr at 37 C, pH 3.7. Binding protein inactivation was evaluated by ability of glycyl-glycine incubated serum to reassociate with 125I IGF-I. In addition, BPs isolated by gel filtration of glycyl-glycine incubated serum were tested for interference in the RIA. The concentration of IGF-I in serum where inactivated BPs were removed by acid gel filtration was compared to corresponding glycyl-glycine incubated serum. There was a 1:1 relationship which intersected at zero indicating that total IGF-I could be measured. Therefore, incubation of serum with glycyl-glycine is a reliable method for measuring total IGF-I in serum from dairy cows. PMID- 1721018 TI - Immunological detection of the EGF-like domain of the core proteins of large proteoglycans from human and baboon cartilage. AB - Recent data from the literature have shown that cDNA clones for the carboxyterminal domain of the core protein of large proteoglycan monomers from human cartilage contain an EGF-like domain, which appears to undergo alternative splicing. In the present study we have found that articular proteoglycans from human and baboon separated on agarose flat-bed gels and blotted onto nitrocellulose react with a rabbit antiserum to mouse EGF. In addition both forms of the proteoglycans (band I and band II) seen on these gels are reactive. Reactivity is seen with proteoglycans extracted from human articular cartilage of various ages (fetal, newborn, young and aged) and with proteoglycans extracted from cartilage of thanatophoric dysplasia and homozygous achondroplasia. Reactivity is dependent on prior digestion of the nitrocellulose blot with Chase ABC, suggesting masking of epitope by chondroitin sulfate. Reactivity of the EGF antiserum with cartilage proteoglycan core protein was also demonstrated in an ELISA system with core protein as coating antigen. The reactivity appears to reside in a tryptic peptide generated from Chase/keratanase digested core protein. The immunoreactive species migrates as a 68 KDa species on gradient gels. Immunological detection and quantitative analysis of the EGF-like domain could be useful for analysis of various proteoglycan samples. PMID- 1721019 TI - Clinical usefulness of des-gamma-carboxy prothrombin assay in early diagnosis of hepatocellular carcinoma. AB - Des-gamma-carboxy prothrombin (DCP) was evaluated as a serological marker for hepatocellular carcinoma (HCC), particularly in patients with early HCC. In 1192 patients with various diseases, plasma DCP levels were measured by a newly developed enzyme immunoassay method using an anti-DCP monoclonal antibody. Of the 254 patients with HCC, 143 (56%) had abnormal DCP levels of greater than 0.1 AU/ml. In contrast, elevated DCP levels were rarely observed in patients with chronic hepatitis, liver cirrhosis, metastatic liver cancer, and other malignant tumors. Because no correlation was observed between DCP and alpha-fetoprotein (AFP), the combined measurement of these two complementary markers appears to be useful in the diagnosis of HCC. Since normal levels were observed in 29 of 30 patients (97%) with small liver tumors measuring 2 cm or less in diameter, the diagnostic application of the DCP assay to small liver tumors is limited. However, in patients with tumors larger than 2 cm, the plasma DCP assay may even be more useful than AFP. Among 46 patients with liver cirrhosis or chronic hepatitis who subsequently developed HCC, significantly increased DCP and AFP levels were observed in nine patients (20%) and 14 patients (30%), respectively, when a tumor was detected. When the results of both assays were combined, 19 patients (41%) had elevated levels of one or both markers. Although the plasma DCP assay alone is not sensitive enough to detect early small liver cancers, it could be applied as a complementary tumor marker together with AFP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721020 TI - Acute porphyria with hyperamylasemia. PMID- 1721021 TI - Direct interaction with primed CD4+ CD45R0+ memory T lymphocytes induces expression of endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of vascular endothelial cells. AB - The process of recruitment of leukocytes at sites of inflammation involves direct cell-to-cell interactions between leukocytes and vascular endothelial cells (EC) mediated by various adhesion receptors on leukocytes and their inducible endothelial ligands. In this study we have examined the induction on EC of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) upon their interaction with subpopulations of human T cells. When co-cultured with EC both resting CD4+ T and CD8+ T cells caused a modest increase in the expression of endothelial ICAM-1. Moreover, resting CD4+ but not CD8+ T cells induced expression of ELAM-1 and VCAM-1 on a small fraction of unstimulated EC. Prior activation with phorbol 12-myristate 13-acetate (PMA) significantly increased the ability of T cells to up-regulate endothelial ICAM-1 and also induced the expression of both ELAM-1 and VCAM-1. PMA-primed CD4+ T cells induced both VCAM-1 and ELAM-1 on EC more efficiently than CD8+ T cells. Furthermore, the ability to induce the expression of ELAM-1 and VCAM-1 was confined to the CD4+ CD45R0+ memory/primed subpopulation of T cells. This induction of various endothelial adhesion ligands could also be mediated by antigen-primed CD4+ T cell lines. The CD4+ T cell-mediated induction of adhesion ligands required direct intercellular contact with EC because neither cultures of EC and PMA-primed CD4+ T cells separated by a microporous membrane insert nor the conditioned medium of PMA primed T cells induced expression of ELAM-1 and VCAM-1 on EC. Cyclosporin A significantly inhibited the activation of T cells with PMA but had no effect on the ability of PMA-primed T cells to up-regulate endothelial CAM. Thus, CD4+CD45R0+ T cells via as yet unknown mechanism can significantly enhance the expression of each of the three endothelial adhesion ligands and, thereby, may facilitate the process of recruitment of additional leukocytes to exacerbate inflammation. PMID- 1721022 TI - Homing receptors reexamined: mouse LECAM-1 (MEL-14 antigen) is involved in lymphocyte migration into gut-associated lymphoid tissue. AB - Specific recognition molecules ("homing receptors") on lymphocytes are thought to direct selective entry of cells into different organs. The lectin-related cell adhesion molecule LECAM-1 has previously been supposed to mediate lymphocyte entry into peripheral lymph nodes and, partially, mesenteric nodes but not into Peyer's patches. Here we present evidence that in vivo the molecule is also implicated in homing of mouse lymphocytes to Peyer's patches and may have a more general role as homing receptor for high endothelial venules-bearing lymphoid tissue, but not for most non-lymphoid tissue. PMID- 1721023 TI - Cells producing antibodies specific for myelin basic protein region 70-89 are predominant in cerebrospinal fluid from patients with multiple sclerosis. AB - Cells secreting antibodies against guinea pig myelin and synthetic myelin basic protein (MBP) peptides were evaluated in cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) and a variety of other neurological diseases (OND). The peptides used, reproducing amino acid sequences 1-20, 70-89, 108-126, or 157-166 of MBP, were selected on the basis of their hydrophilic and encephalitogenic properties. Low numbers of cells secreting IgG antibodies against myelin or each of the MBP peptides (about 1 per 50,000) were detected in peripheral blood, with no difference between MS and OND. In CSF, cells secreting IgG antibodies to MBP 70-89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG-secreting cells on average) than in patients with OND (1/2083 IgG-secreting cells on average). The frequencies of cells secreting antibodies against myelin or the three other MBP peptides were similar in MS and OND. Thus, evaluation of B cell immunity at the cellular level indicates that MBP 70-89 is an immunodominant B cell epitope in MS. It is not clear whether this intrathecal anti-MBP 70-89 IgG antibody response has any pathogenetic relevance in MS or is the result of myelin breakdown. PMID- 1721024 TI - Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages. AB - Inbred strains of mice differ considerably in their innate resistance to leishmanial infection. BALB/c mice are highly susceptible to cutaneous leishmaniasis caused by Leishmania major, whereas CBA mice are resistant. We now show that this resistance correlates with the ability of macrophages to synthesize nitric oxide (NO) following activation with interferon-gamma or tumor necrosis factor alpha. Furthermore, the larger amounts of NO generated by resistant macrophages are related to higher levels of NO synthase activity, a difference which is not attributable to the number or the affinity of the receptors for interferon-gamma on these cells. The level of NO synthesis by activated macrophages was also correlated to the resistance in a number of other inbred mouse strains tested; macrophages from the resistant B10.S, C57BL and C3H mice produced significantly higher levels of NO than the macrophages from the susceptible BALB.b and DBA/2 mice. PMID- 1721025 TI - Immunogenicity of multiple antigen peptides (MAP) containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite protein. AB - The immunogenicity of multiple antigen peptides (MAP) constructs containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite (CS) protein was examined in vitro, using a human T cell clone, and in vivo, using four different strains of mice. All the MAP constructs that contained the T cell epitope, (DPNANPNVDPNANPNV), stimulated proliferation and interferon-gamma production by a human T cell clone specific for this epitope which is located in the 5' end of the repeat region of the P. falciparum CS protein. These human T cells did not recognize MAP that contained only the B cell epitope, (NANP)3, which is located in the 3' repeat region. Optimal antibody responses were obtained in mice immunized with MAP containing four copies of tandemly arranged T and B cell epitopes, (TB)4. The murine immune response to the MAP constructs was genetically restricted. Mice of a high responder strain, C57BL, recognized both the 5' and 3' repeat sequences in the MAP as T, as well as B, cell epitopes and developed very high anti-MAP and anti-sporozoite antibody titers. A/J and C3H mice, which were intermediate responders, developed lower antibody titers which varied according to the orientation of the T vs. the B cell epitopes within the MAP constructs. BALB/c mice were nonresponders and did not develop antibodies following immunization with any of the MAP constructs containing the 5' and 3' repeats of the P. falciparum CS protein. PMID- 1721026 TI - Down-regulation by tumor necrosis factor-alpha of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism. AB - Adhesion of human neutrophils to endothelial cells is a crucial step during migration to the extravascular sites of inflammation. A large number of molecules, including the CD44 and LAM-1 antigens, have been described to participate in this process. We have investigated the regulation by human recombinant tumor necrosis factor-alpha (TNF-alpha) of human neutrophil plasma membrane expression of both CD44 and LAM-1 adhesion molecules, as well as that of CD43 sialophorin, which has been involved in adhesion and activation of leukocytes. The expression of these three antigens was down-regulated in neutrophils upon TNF-alpha treatment, as determined by immunofluorescence and immunoprecipitation experiments. However, the expression of other cell surface molecules, such as CD45 or CD11b, was up-regulated. Similar regulatory effects were also observed upon neutrophil treatment with other activating agents such as the chemoattractant peptide formyl-Met-Leu-Phe, the calcium ionophore A23187, or the phorbol ester phorbol 12-myristate 13-acetate. Protease inhibitors virtually abrogated the TNF-alpha-induced down-regulation of CD43 and CD44 expression, but not that of LAM-1, suggesting the involvement of a protease activity in this process. These results underline the role of TNF-alpha on the differential regulation of cell surface expression of neutrophil adhesion molecules, thus implying modifications in the neutrophil adhesive properties. PMID- 1721028 TI - Vascular eicosanoids and platelet-aortic wall interactions in spontaneously hypertensive rats. AB - We studied the aggregation of collagen and ADP-stimulated platelet-rich plasma (PRP) and the formation of thromboxane B2 (TxB2) by collagen-stimulated PRP in spontaneously hypertensive rats (SHR) and in Wistar-Kyoto control rats (WKY). In addition, we evaluated the inhibition of the aggregation of PRP following homologous or heterologous perfusions through isolated aortas, the release of 6 keto-prostaglandin (PG)F1 alpha from these arteries perfused with PRP, and the sensitivity of PRP to the antiaggregatory activity of the stable PGI2 analogue, iloprost, in both SHR and WKY. The lower activities (aggregation induced by ADP and collagen, collagen-stimulated TxB2 production) of SHR platelets, were not accompanied by morphological differences from WKY platelets. These changes were associated with a greater release of arterial 6-keto-PGF1 alpha, with greater platelet antiaggregatory activity of the arterial wall and with higher sensitivity of platelets to iloprost. The lower reactivity of platelets to aggregating agents, and the greater sensitivity to prostacyclin, associated with a greater production of arterial prostacyclin were the major changes observed in SHR animals. These alterations in the SHR vs. normotensive WKY may lead to an enhanced risk of hemorrhage in the hypertensive state. PMID- 1721029 TI - Effects of pentobarbitone on the properties of nicotinic channels of chromaffin cells. AB - We have used the whole cell patch clamp technique to investigate the action of pentobarbitone on the nicotinic channels of bovine chromaffin cells. Application of agonists induced an inward current associated with a large increase in current noise. The noise could be fitted by Lorentzian functions with time constants of 17 +/- 2 ms for 10 microM acetylcholine and 10 +/- 1 ms for 10 microM carbachol. The single channel conductance estimated from the current variance was about 25 pS in each case. Pentobarbitone decreased the time constants in a concentration dependent fashion, but the unit conductances were unaffected. Single channel events were recorded in chromaffin cells held under voltage clamp. Pentobarbitone did not reduce the amplitude of channel openings or the probability of channel opening but reduced the mean channel open time. This reduction was sufficient to account for the decrease in inward current produced by pentobarbitone. PMID- 1721027 TI - The ligand recognized by ELAM-1 on HL60 cells is not carried by N-linked oligosaccharides. AB - The sialyl Lewis-x determinant is a ligand for ELAM-1, a major adhesion molecule for HL60 cells and neutrophils. ELAM-1 expression can selectively be induced on human umbilical vein endothelial cells (HUVEC) by tumor necrosis factor, interleukin 1 and lipopolysaccharide. The determinant sialyl Lewis-x is found on both glycolipids as well as on glycoproteins. Using specific inhibitors of the biosynthesis of N-linked glycosylated glycoproteins, we investigated whether N linked glycans or their modifications are involved in ELAM-1-dependent adhesion of HL60 cells to activated HUVEC. The inhibitors of glycoprotein processing N methyl-deoxynojirimycin, 1-deoxymannojirimycin and swainsonine did not affect ELAM-1-dependent adhesion. Complex-type N-linked glycans are not required for ELAM-1 mediated adhesion, and therefore the ligand for ELAM-1 is most likely a glycolipid, or a glycoprotein carrying O-linked oligosaccharides. PMID- 1721030 TI - Distribution of cholinergic and catecholaminergic nerves in the colon of the rat with aganglionosis. AB - We compared localization and distribution of putative cholinergic fibers by acetylcholinesterase and of adrenergic fibers visualized by the glyoxylic acid technique in the aganglionic segment using whole mount preparations of aganglionosis rat (AGR) and compared them with those of normal littermates. We also attempted simultaneous staining of acetylcholinesterase (AChE) and catecholamine fluorescence (C-F) on the same whole mount preparations to compare the differences in distribution pattern. All AGR used in this study had narrowed segments of the bowel extending from the distal ileum to the anus, and had no ganglion cells in these narrowed segments. In the intermuscular space, normally occupied with myenteric ganglion, of the narrowed distal colon and rectum, various sizes of nerve bundles and fibers reactive for AChE and C-F appeared to make coarse and irregular networks. These thick nerve bundles appeared to ascend to the proximal colon and disappeared in the cecum. In the distal ileum, almost totally absence of AChE positive nerve fibers, but a few fine C-F fibers, probably associated with blood vessels, were observed. By the method of simultaneous staining of AChE and C-F method in the whole mount preparations, the thick nerve bundles in the narrowed segments showed both of AChE positive and C-F positive. However, there were differences in peripheral fine nerve fibers in the segment; especially numerous perivascular C-F positive nerve fibers, but a few AChE positive ones were found. In the upper aganglionic narrowed segments, greatly diminished numbers of AChE positive and C-F positive nerve fibers were found in the circular muscle layer and in the submucosal layer. In the lower aganglionic narrowed segments, there were thick nerve bundles, forming irregular interlaced network. The role of these extrinsic nerve fibers in aganglionic segments is unclear. PMID- 1721032 TI - Three individual regulatory elements of the promoter positively activate the transcription of the murine gene encoding granulocyte colony-stimulating factor. AB - At least three regulatory elements GPE1, GPE2 and GPE3 (G-CSF promoter elements) controlling the gene (G-CSF) encoding granulocyte colony-stimulating factor (G CSF) are indispensable for the constitutive expression of the G-CSF gene in human CHU-2 cells and for its lipopolysaccharide(LPS)-inducible expression in macrophages. The enhancer activities of each regulatory element were examined with or without the SV40 enhancer element placed downstream from the reporter gene. A GPE1 tetramer mediated the constitutive expression in CHU-2 cells, and the LPS-inducible expression in macrophage cell lines, while the GPE2 element was active in CHU-2 and LPS-treated macrophage cell lines only in combination with the SV40 enhancer. A GPE3 tetramer had efficient enhancer activity in CHU-2 cells but not in macrophage cell lines without the SV40 enhancer. In combination with the SV40 enhancer, GPE3 worked as an LPS-inducible enhancer element in macrophage BAM3 cells. Gel retardation assay indicated that the CHU-2 and the macrophage cells contained nuclear factors which specifically bound to each GPE sequence. PMID- 1721031 TI - Ectopic expression of a c-kitW42 minigene in transgenic mice: recapitulation of W phenotypes and evidence for c-kit function in melanoblast progenitors. AB - The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor that is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and adult life, and they result from the partial or complete loss of c-kit function. The W42 allele is a W mutation with severe effects in both the homozygous and the heterozygous states. Previous analysis of the W42 allele identified a missense mutation in an essential amino acid of the c-kitW42 kinase domain that abolishes the in vitro kinase activity of the c-kitW42 protein but does not affect its normal expression. These results suggested that the c-kitW42 allele was a dominant negative mutation within the context of c-kit-mediated signal transduction. To further explore the dominant negative characteristics of the W42 mutation, we have generated transgenic mice in which ectopic expression is driven by the human beta-actin promoter (hAP). Two mouse lines carrying the hAP-c-kitW42 transgene show an effect on pigmentation and the number of tissue mast cells. The patchy coat color pattern of the line 695 mice may reflect variable expression of the transgene in melanoblast progenitors and their descendants and, consequently, is indicative of a function for c-kit in early melanoblasts. Germ cell development and erythropoiesis, however, do not appear to be affected by the transgene. Mice expressing the c-kitW42 transgene therefore recapitulate some of the phenotypes of mice with W mutations. These results are therefore in agreement with the molecular basis of the W42 mutation and the dominant-negative characteristics of the c-kitW42 protein product. PMID- 1721033 TI - The cDNA cloning and RNA distribution of bovine osteopontin. AB - We have isolated and sequenced the bovine cDNA (OPN) counterpart of osteopontin. The cDNA is 1356 nucleotides (nt) in length with an open reading frame of 834 nt, encoding a 278-amino acid (aa) protein. Cell-free transcription and translation of OPN RNA resulted in a major species of approx. 40 kDa in size, in agreement with the predicted size of the deduced aa sequence. Northern analysis of bovine OPN RNA indicated the presence of the message in mineralized, as well as soft tissues. A comparison of the deduced aa sequence among various species indicates both regions of similarity and divergence. One prominent region of dissimilarity in bovine OPN compared to all other species is a 22-aa gap which may represent a loss of a potential Ca(2+)-binding loop. Despite the variability among the species, several regions of conservation are apparent, including a hydrophobic leader sequence, a potential site for Asn-linked glycosylation, a stretch of polyaspartic acid residues, and the cell attachment Arg-Gly-Asp tripeptide. Whether bovine OPN enhances cell attachment is unknown. Furthermore, whether the loss of a potential Ca(2+)-binding loop alters the function of OPN would be interesting to determine. PMID- 1721034 TI - Cloning and sequencing of a jack bean urease-encoding cDNA. AB - A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans. It was necessary to use reverse transcriptase in the cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced. The conceptual translation of the nt sequence gave an 840 aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur. J. Biochem. 175 (1988) 151-165). These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant. PMID- 1721035 TI - Pregnancy-associated plasma protein A interaction with heparin: a critical appraisal. AB - Positive affinity chromatography on heparin-Sepharose has proved a most crucial step in the purification of pregnancy-associated plasma protein A (PAPP-A). In this chromatographic procedure, PAPP-A was purified almost 500-fold from term pregnancy serum. Further purification was achieved by gel filtration and negative immunoaffinity chromatography. Both PAPP-A and free heparin inhibited granulocyte elastase (HGE) activity. Whereas free heparin inhibited only in hypotonic buffers, PAPP-A inhibited HGE in hypertonic buffers also. However, PAPP-A did not inhibit other proteases (trypsin, chymotrypsin, plasmin, fibroblast collagenase) or proteolytic cascades (complement activation). Since heparin was not detected in the purified PAPP-A, the inhibition of HGE was not due to desorbed or leeched heparin ligand. PMID- 1721036 TI - A new staining method for constriction marks in skin. AB - A modified Poley's acid fuchsin-methyl green stain was used to demonstrate ligature marks in tissues taken at autopsy. This stain was compared in 30 cases with haematoxylin-eosin, Mallory's trichrome and Ogata's picro-indigocarmine stains. Using the modified Poley method, it was possible to demonstrate the compression mark in corpses even when post-mortem changes were advanced. The method was found useful in evaluating the cases when the force used was minimal or when the mark was atypical using the usual haematoxylin-eosin method. PMID- 1721037 TI - Diffusion of alcohol upon application of neuroadenolysis of the pituitary gland (NALP). An experimental study using HRP and WGA-HRP in the cat. AB - Diffusion of alcohol in neuroadenolysis of pituitary gland (NALP) was observed by injection of horseradish peroxidase (HRP) or wheat germ agglutinin-HRP conjugates (WGA-HRP) into the pituitary of the cat. After small-amount injection of WGA-HRP into the pituitary, WGA-HRP labeling was observed markedly around the third ventricle and the ventral part of the hypothalamus. While, in large-amount injection of WGA-HRP, it extended to all of the ventricular systems and dipped into the substances of the brain and spinal cord through the ependyma. These results suggest that the main site of action for alcohol injected into the pituitary is probably the hypothalamus. PMID- 1721038 TI - [Isolation and purification of alpha fetal protein utilizing Act-Ultrogel AcA 22 affinity chromatography]. AB - In the present communication, we report a method for the isolation and purification of alpha FP from fetal corpse homogenate in a single-step procedure using Act-Ultrogel AcA 22 affinity chromatogrpahy column. This method proves to be better than the conventional method in simplicity, fastness and validity with recovery, purity and immunoreactivity. The immunoreactivity of the purified alpha FP labelled by the method of Hunter and Greenwood (1962) was 50-60% (B/T%). This was similar to alpha FP standard (WHO). The results indicate that the 125I-alpha FP is suitable for alpha FP radioimmunoassay. PMID- 1721039 TI - New monoclonal antibodies in CD44 and CD58: their use to quantify CD44 and CD58 on normal human erythrocytes and to compare the distribution of CD44 and CD58 in human tissues. AB - The cell-surface glycoproteins CD44 and CD58 are involved in cell adhesion reactions. In this paper 12 monoclonal antibodies in CD44 and two in CD58 are described. Competitive binding assays using CD44 antibodies identified three distinct epitope groups. Antibodies in Group 1 and, with one exception (BRIC 214), antibodies in group 2, but not antibodies in Group 3, recognized epitopes that are sensitive to reduction and to trypsin or chymotrypsin treatment of intact erythrocytes, and so these epitopes probably reside on the N-terminal disulphide-bonded domain of CD44. Antibodies in CD44 did not inhibit the binding of CD58 antibodies to erythrocytes or vice versa. Quantitative binding studies using radioiodinated IgG measured 1888-5592 copies of CD44 and 1772-3290 copies of CD58 on normal erythrocytes. Similar measurements with radioiodinated Fab fragments gave values of 6508-10,450 (CD44) and 3457-7622 (CD58). Immunocytochemical studies indicated that CD44 is much more widely expressed in non-haemopoietic tissues than CD58. Comparison with previously described CD44 antibodies suggests that antibodies in our Group 1 encompass Hermes 2 and that those in Group 2 encompass Hermes 1. All the CD44 antibodies gave weakened reactions with Lu(a-b-) erythrocytes of the In(Lu) type by one or more methods. BRIC 214 and antibodies in epitope Group 3 were used to demonstrate that CD44 on these variant cells gives membrane-bound trypsin and chymotrypsin cleavage fragments of similar molecular weight to those obtained with normal erythrocytes. PMID- 1721040 TI - Expression of the CDw75 (beta-galactoside alpha 2,6-sialyltransferase) antigen on normal blood cells and in B-cell chronic lymphocytic leukaemia. AB - Using monoclonal antibodies (mAb) characterized at the last International Workshop on Human Leucocyte Antigens, we examined the expression of CDw75 antigens (beta-galactoside alpha 2,6-sialyltransferase) on normal peripheral blood cells and on cells from patients with B-cell chronic lymphocytic leukaemia (CLL). The mAb used (HH2, EBU.65, EBU.141 and OKB4) detect different epitopes of CDw75. Normal peripheral blood B cells expressed high levels of CDw75 detectable with HH2, EBU.65 and EBU.141 but did not react with OKB4. Cells from patients with B-cell CLL closely resembled normal B cells. All CDw75 epitopes, including OKB4, were strongly expressed on some Namalwa variant Burkitt lymphoma cell lines. The OKB4 epitope was also present on red cells from all normal donors. The other CDw75 mAb were unreactive with red cells from some normal donors. The CDw75 epitope detected with EBU.65 was present on most CD4+ T cells and on a minority of CD8+ cells. HH2 and EBU.141 stained only small numbers of T lymphocytes. OKB4 did not react with T cells. EBU.65+, CD4+ T cells had low levels of expression of CD45R0, CD29, CD54 and CD58, and had high levels of CD45RA antigen. Phytohaemagglutinin (PHA) activation of cells led to the loss of EBU.65 binding. These results suggest that the CDw75 epitope recognized by the EBU.65 mAb is a marker of native T lymphocytes. On B CLL cells the epitopes detected with HH2, EBU.65 and EBU.141 were destroyed by treatment with neuraminidase. Treatment of B CLL cells and red cells with neuraminidase increased the binding of OKB4, suggesting that this epitope is masked by sialic acid. The results suggest that CDw75 is a sialylated cell-surface antigen expressed in a number of tissue specific isoforms. PMID- 1721041 TI - Intrathymic induction of neonatal tolerance to Mls-1a determinant: clonal deletion and clonal anergy by haematolymphoid cells. AB - Newborn BALB/c (Mls-1b) mice were intravenously injected with either bone marrow cells (BMC) or peritoneal exudate cells (PEC) from Mls semi-allogeneic (BALB/c x AKR)F1 mice. Thymic cells of these mice, obtained 7 days after the injection, were found to be unresponsive to the superantigen Mls-1a, as determined by graft versus-host reactivity. On Day 7, deletion of T cells expressing the V beta 6 element at high levels (V beta 6hi) was observed in thymic cells of mice receiving PEC. In mice given BMC, it took 2 weeks until the proportion of V beta 6hi T cells began to decline, and a longer period was required for complete disappearance of V beta 6hi T cells. These results may indicate that although both BMC and PEC contain cells mediating tolerance, a component(s) of cells responsible for clonal deletion is deficient in BMC. Immunohistological investigation showed that on Day 7 donor type B cells were present in the thymus of mice that received PEC but absent from mice that received BMC, whereas cells expressing donor type class I as well as class II antigens were seen in both recipients. The presence of donor type B cells could be observed 8 weeks after injection of BMC. By this time, the deletion of V beta 6hi T cells was completed. These results indicate, collectively, that the tolerance of both anergy type and deletion type occurs in the naturally developing thymus, and suggest that the presence of B cells in the thymus might be required for clonal deletion. PMID- 1721042 TI - Rapid loss of perforin and serine protease RNA in cytotoxic lymphocytes exposed to sensitive targets. AB - We have previously reported that cytotoxic lymphocytes, when exposed to sensitive target cells, temporarily lose their lytic potential. The mechanism leading to this loss of lytic activity is still unknown but it is reversible and the lytic potency can be recovered when the effector cells are incubated with interleukin-2 (IL-2) for 12-14 hr. In this study, we have investigated the regulation of RNA coding for perforin and for two serine proteases, HSP1 and HSP2, in cytotoxic lymphocytes exposed to sensitive targets. Perforin and the two serine proteases are contained in granules of major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxic lymphocytes, but their exact role in the lytic mechanism is still debated. Here we used four different human cytotoxic lymphocytes (CTL) as effector cells: an MHC-restricted CTL (SG-CTL), a non-MHC restricted CTL (IE6), a natural killer (NK)-like cell line (3.3) and lymphokine activated killer (LAK) cells. In all effector cells we observed a rapid loss of perforin and of serine protease RNAs within 5 min following the addition of sensitive targets. The effector cells recovered the RNA messages as early as 30 min, although the kinetics of recovery was faster with CTL than with NK-like or LAK effector cells. When we exposed the effector cells to resistant targets we did not detect any loss of perforin or serine protease RNAs. Incubation of the effector cells with cycloheximide, prior to the addition of sensitive targets, did not block message loss, indicating that de novo protein synthesis was not required in this process. Cycloheximide treatment, however, inhibited the recovery of perforin and serine protease RNAs. Taken together, our results indicate that the target-mediated loss of lytic activity in cytotoxic lymphocytes may be a consequence of the down-regulation of perforin or of serine protease transcripts, or both. PMID- 1721043 TI - Towards the design of heterovalent anti-malaria vaccines: a hybrid immunogen capable of eliciting immune responses to epitopes of circumsporozoite antigens from two different species of the malaria parasite, Plasmodium. AB - The peptide CS.T3, corresponding to residues 378-398 of the Plasmodium falciparum (Pf) circumsporozoite (CS) protein sequence (except with cysteines 384 and 389 replaced by alanines), has been found to be almost universally recognized by human and mouse T lymphocytes. When colinearly linked to the repetitive B lymphocyte-specific epitope (Asn-Ala-Asn-Pro)n of Pf CS protein, CS.T3 induces T helper activity for an anti-(Asn-Ala-Asn-Pro)n antibody response in mice of different haplotypes. We constructed a double-epitope peptide, CS.T3-R3, by co linearly joining a truncated 18-mer form (IEKKIAKMEKASSVFNVV) of CS.T3 to three tandem repeats (R3) of a B-cell-specific epitope, QGPGAP, of Plasmodium yoelii (Py) CS protein, via a two-glycine spacer. Whereas CS.T3 and R3 did not induce specific antibodies, CS.T3-R3 elicited anti-CS.T3 and anti-R3 antibodies in different mouse strains. Some human anti-Pf sera from malaria-endemic areas contained high-titred anti-CS.T3 antibody IgG, indicating that parasite-derived CS.T3 contains a B-cell determinant which is maintained in the alanine substituted synthetic CS.T3. Antibody absorption experiments showed that CS.T3-R3 contains no new B-cell-specific determinants other than R3 and CS.T3. That the Pf CS protein epitope, CS.T3, supports T-cell help for antibody responses against the Py CS protein repeat epitope, QGPGAP, implies the possible use of CS.T3 in anti-sporozoite multiple-epitope vaccines against different species of Plasmodium. Colinearly linking CS.T3 to R3, via a two-glycine spacer, appears to be a useful model by which different T- and B-cell-specific determinants can be jointed into a heterovalent immunogen while retaining their distinct immunological properties. PMID- 1721044 TI - Tissue-specific expression of the HLA-DRA gene in transgenic mice. AB - Transgenic mice were produced containing a 33 kilobase (kb) DNA fragment encompassing the five exons and all the known regulatory regions of the class II HLA-DRA gene. The transgene displayed regulated expression [constitutive and interferon-gamma (IFN)-gamma induced] of the human products in most mouse tissues. The tissue distribution of the DRA transgene products more closely resembled that of their mouse homologues, the endogenous H-2 Ea products, than the wider distribution of DRA products in humans. This was evident in several tissues (endothelia of small vessels, especially those of glomerular capillaries, Kupffer cells, and epithelial cells lining the gastrointestinal tract), known to differentially express class II molecules in the two species. Thus, the wider human specific pattern of expression requires an exact cis/trans complementation which is incompletely reconstituted in transgenic mice, suggesting that human specific cis-acting elements may have arisen during evolution to direct the expression of class II genes to those anatomical regions which usually lack them in the mouse. The only example of aberrant expression of the DRA gene in the present series of transgenic mice was in the dendritic and/or epithelial cells of the thymic cortex, which displayed greatly reduced DR alpha levels in spite of a normal expression of the endogenous E alpha molecules. PMID- 1721045 TI - Proctor Lecture. Experimental allergic uveitis. Investigations of retinal autoimmunity and the immunopathologic responses evoked. PMID- 1721046 TI - Stability study of Azure B, Eosin Y and commercial Romanowsky Giemsa stock solutions using high performance liquid chromatography. AB - The stability of Azure B and Eosin Y in stock solutions of the individual compounds as well as in mixtures of the two dyes was studied. The purpose of the study of these two essential constituents of the Romanowsky Giemsa stain, commonly used in cytology and histology, was to select a stable mixture as a definitive stock solution. Two specific high performance liquid chromatographic methods were used to monitor qualitative and quantitative changes in solutions. Several parameters influencing the stability of Azure B were examined e.g. the type of counter ion, the presence of Eosin Y and the type of solvent used. The second part focused on the stability of Eosin Y in mixtures with different cationic dyes submitted to high temperatures. In conclusion, an Azure B SCN-Eosin Y acid mixture in dimethylsulphoxide (concentrations 0.75% and 0.12%, respectively) was selected as being the most appropriate composition of a stock solution for the Romanowsky Giemsa stain. PMID- 1721047 TI - Immunogold-silver staining of leucocyte populations in lung sections containing carbon particles requires cautions interpretation. PMID- 1721048 TI - [Incidence and clinical consequences of distant metastases in the area of the jaw/face]. AB - Metastases from primary tumours and distant sites to the maxillofacial region are rare. The establishment of an exact diagnosis is often difficult because of the atypical clinical and roentgenological appearances. Thirty-five cases of distant metastases to oral soft tissues and jaws are presented. The most common primary tumours were renal carcinomas followed by breast cancers. In most cases metastatic lesions lie in the distal segment of the mandible. The possibilities of treatment are very limited. PMID- 1721050 TI - Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase. AB - Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP. The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM). Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template. The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor. MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer. Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested. MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases. Other members of the ipecac class of alkaloids, e.g. emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT. Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested. Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity. Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases. Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents. PMID- 1721049 TI - Electron and proton transport across the plasma membrane. AB - Transplasm membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes contain b cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na+/H+ antiport. In plants part of the proton release can be achieved by activation of the H+ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen. PMID- 1721051 TI - Effect of polyanions on the refolding of human acidic fibroblast growth factor. AB - Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species. PMID- 1721052 TI - Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin. AB - The roles of selected amino acid residues of human 14-kDa beta-galactoside binding lectin were studied by site-directed mutagenesis. Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate beta-galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted. All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin. In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions. It retained asialofetuin binding activity for over a week in the absence of beta-mercaptoethanol, while the wild-type lectin lost it within a day. This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin. Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose. Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation. Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential. PMID- 1721053 TI - Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor. AB - The peripheral nicotinic acetylcholine receptor (nAChR) is phosphorylated on tyrosine residues in vivo and in vitro at a high stoichiometry. We have previously reported that this tyrosine phosphorylation occurs on the beta, gamma, and delta subunits of the receptor and is implicated in both the modulation of the function of the receptor and localization of the receptor at the synapse. The specific tyrosine residue of each subunit which is phosphorylated is now identified. The endogenously phosphorylated nAChR from the electric organ of Torpedo californica was phosphorylated to maximal stoichiometry in vitro exclusively on tyrosine residues as indicated by phosphoamino acid analysis. Two dimensional phosphopeptide maps of thermolysin limit digests of the isolated phosphorylated subunits indicated that each subunit is phosphorylated at a single site. To determine the site of tyrosine phosphorylation of the beta, gamma, and delta subunits, phosphorylated subunits were isolated and digested with trypsin. A single phosphotyrosine containing peptide from each subunit was purified by antiphosphotyrosine antibody affinity chromatography and reverse phase high performance liquid chromatography. The purified phosphopeptides were subjected to sequential Edman degradation and sequence analysis. Comparison of the phosphopeptide sequence data with the deduced amino acid sequence of each subunit indicated that Tyr-355 of beta, Tyr-364 of gamma, and Tyr-372 of delta are the sites of in vitro and in vivo tyrosine phosphorylation of the nAChR. Identification of these sites should facilitate further studies of the role of tyrosine phosphorylation in the regulation of receptor function. PMID- 1721054 TI - Constitutive and inducible nitric oxide synthases incorporate molecular oxygen into both nitric oxide and citrulline. AB - Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO. PMID- 1721055 TI - Structure and function studies of the cGMP-stimulated phosphodiesterase. AB - Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1 methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1721056 TI - Identification of a functional domain of human granulocyte colony-stimulating factor using neutralizing monoclonal antibodies. AB - Human granulocyte colony-stimulating factor (G-CSF) is a hemopoietic growth factor that is being used successfully to treat various forms of neutropenia. To define functionally important regions of G-CSF, we have prepared 37 monoclonal anti-G-CSF antibodies and mapped the regions of G-CSF recognized by different antibody groups. Antibodies recognizing similar epitopes were identified by competition assays, neutralization assays, conformation dependence and cross reactivity with canine G-CSF. Seven of eight neutralizing antibodies fell into two related epitope groups and were conformation-dependent. The eighth was unrelated and conformation-independent. Peptides of G-CSF were generated by chemical or enzymatic digestion and tested for antibody reactivity. One of the neutralizing antibodies (LMM351) recognized a small, disulfide-bonded peptide from the V8 protease digest (residues 34-46). A synthetic peptide (residues 20 58) was recognized by all the neutralizing antibodies, implicating this disulfide bonded loop in receptor binding. The epitopes recognized by nonneutralizing antibodies were found throughout G-CSF. Thus, regions of G-CSF that are not involved in receptor binding have also been defined. A CNBr peptide (residues 1 121) had greatly reduced biological activity, indicating that the COOH terminus is required for receptor binding. We predict that residues 20-46 and the COOH terminus bind to the G-CSF receptor. PMID- 1721057 TI - Transcriptional regulation of p90 with sequence homology to Escherichia coli glycerol-3-phosphate acyltransferase. AB - We have previously isolated cDNA clones for several mRNAs that are dramatically increased in livers of fasted mice refed a high carbohydrate diet. We report here the sequence and regulation of one such mRNA; the 6.8-kilobase mRNA has an open reading frame of 2481 nucleotides, and the coded protein contains 827 amino acid residues (Mr of 90,000) with a 30% identity and an additional 42% similarity in an approximately 300-amino acid stretch to Escherichia coli glycerol-3-phosphate acyltransferase. The p90 mRNA is highly expressed in liver and in adipose tissue. When previously fasted mice were refed a high carbohydrate, fat-free diet, the liver mRNA level for p90 was increased about 20-fold at 8 h. Administration of dibutyryl cAMP at the time of refeeding prevented the increase in the p90 mRNA by 70%. In addition, there was no increase in the p90 mRNA level when previously starved streptozotocin-diabetic mice were refed. In diabetic animals, the p90 mRNA level increased by 2-fold 1 h after insulin injection and reached a maximum of 19-fold after 6 h. The increase in transcription rate of the p90 gene preceded that of steady state mRNA level caused by fasting/refeeding, and cAMP abolished the increase in transcription. Transcription of the p90 gene was not detectable in either fasted or refed streptozotocin-diabetic mice, but increased 4-fold 30 min after insulin administration and further increased up to 8-fold at 2 h. On going protein synthesis was necessary for this increase. PMID- 1721058 TI - A single base pair deletion from the inactive octamer-like motif of the 7S K distal sequence element brings full functionality in vivo. AB - Octamer sequence elements were analyzed for their capacity to induce the 7S K "core" promoter in vivo. The U6 distal sequence element (DSE) which contains a consensus sequence octamer, was able to support efficient 7S K expression in vivo. In contrast, no such function could be attributed to the octamer-like element alone, which is present within the 7S K DSE. However, conversion of this octamer-like element (ATTTaGCAT) to the octamer consensus sequence ATTTGCAT generated a potent DSE, even in the absence of the CACCC box, which constitutes the major functional element of the 7S K DSE. Both the consensus and the octamer like sequences revealed no cooperativity with the CACCC box. Together, these results demonstrate that the octamer-like element of the wild-type 7S K DSE is definitely not functional in vivo. Furthermore, our experiments indicate that in contrast to the RNA polymerase II-transcribed small nuclear RNA genes, in intact cells a single functional DSE motif is necessary and sufficient for maximal transcription by RNA polymerase III of the 7S K RNA gene. PMID- 1721059 TI - Localization of an apolipoprotein A-I epitope critical for activation of lecithin cholesterol acyltransferase. AB - Apolipoprotein (apo) A-I, the major apoprotein of human high density lipoprotein, is a vital cofactor for lecithin-cholesterol acyltransferase (LCAT), the plasma enzyme responsible for esterification of free cholesterol associated with high density lipoprotein. This esterification is an important component of the reverse cholesterol transport process. An immunochemical approach was used to test the hypothesis that a discrete region of apoA-I was important for LCAT activation. Three human apoA-I-specific monoclonal antibodies were found to inhibit LCAT activation in vitro in a manner directly proportional to their ability to bind to apoA-I-proteoliposomes in fluid phase immunoassays. This relationship was not observed with another four apoA-I-specific antibodies that also were able to bind to the apoA-I proteoliposomes. The use of synthetic peptides representing short amino acid sequences of the apoA-I molecule facilitated the identification of discrete but overlapping apoA-I epitopes for those antibodies that interfered with LCAT-mediated cholesterol esterification. These epitopes spanned amino acid residues 95-121 of mature apoA-I. Therefore, this region is most likely involved in the activation of LCAT by apoA-I. PMID- 1721060 TI - Mapping of a contact for the RNA 3' terminus in the largest subunit of RNA polymerase. AB - Stalled elongation complexes of Escherichia coli RNA polymerase were prepared carrying the photo-cross-linkable 8-azido derivative of adenine at the 3' terminus of the nascent RNA chain. Ultraviolet irradiation of such complexes resulted in the cross-linking of radiolabeled RNA exclusively to the beta' subunit of RNA polymerase. The adduct was mapped between Met932 and Trp1020 in the linear sequence of the beta' polypeptide using specific chemical degradation of the cross-linked species. PMID- 1721061 TI - Self-translocation of diphtheria toxin across model membranes. AB - To understand the mechanism of diphtheria toxin membrane translocation, the toxin was entrapped within lipid vesicles, and its low pH-induced translocation across the lipid bilayer was measured. Proteolysis and resistance to guanidinium chloride denaturation were used to demonstrate that the toxin molecules were entrapped. Low pH-induced movement of entrapped toxin to the outer (trans) face of the bilayer was assayed by the binding of external streptavidin to biotin labeled entrapped toxin. Complete translocation was quantified by the amount of protein released into the external medium. Using whole toxin, it was found that the A fragment was efficiently translocated, but the B fragment was not. This was true both in the low temperature (A domain folded) and high temperature (A domain unfolded) toxin conformations previously identified [Jiang J. X., Abrams, F. S., and London, E. (1991) Biochemistry 30, 3857-3864]. Remarkably, even isolated fragment A appeared to self-translocate under some conditions. Toxin-induced translocation may partly result from formation of a nonspecific toxin-induced pore. This idea is supported by the toxin-induced release of fluorescent dextrans coentrapped within the vesicles. However, low pH-induced exposure of entrapped toxin on the outside of the membrane was conformation dependent. Exposure was greatest for the high temperature conformation. This suggests the existence of a more specific translocation process. The nature and relationship of these processes, and their relative roles in translocation in vivo are discussed. PMID- 1721062 TI - Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen. AB - The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically. PMID- 1721063 TI - An adipose tissue-specific beta-adrenergic receptor. Molecular cloning and down regulation in obesity. AB - Clones encoding an atypical beta-adrenergic receptor were isolated from a rat brown adipose tissue cDNA library. This receptor expressed in Chinese hamster ovary (CHO) cells displays a low affinity for beta-adrenergic antagonists and a high affinity for BRL 37344, an agonist that selectively stimulates lipolysis in adipose tissue. The rank order of potency for agonist-mediated increases in intracellular cAMP in transfected cells correlates with that for agonist-mediated stimulation of lipolysis in brown adipocytes. Northern blot analysis demonstrates that this receptor subtype is expressed only in brown and white adipose tissue where it represents the predominant beta-receptor subtype. The amount of atypical beta-adrenergic receptor present in adipose tissue of obese (fa/fa) Zucker rats is reduced by up to 71% as compared with lean (Fa/Fa) control animals. These findings suggest that a change in the expression of this beta-adrenergic receptor subtype may play a role in obesity. PMID- 1721064 TI - Cyclooxygenase gene expression is down-regulated by heparin-binding (acidic fibroblast) growth factor-1 in human endothelial cells. AB - The expression of cyclooxygenase (EC 1.14.99.1, Cox), a rate-limiting enzyme in the biosynthesis of inflammatory prostanoids, is regulated by growth factors and cytokines. We have shown previously that the cytokine interleukin-1, an inhibitor of endothelial growth in vitro and stimulator of prostacyclin production, induces the expression of the 3-kilobase Cox transcript in cultured human umbilical vein endothelial cells (HUVEC). In contrast, the endothelial cell mitogen, heparin binding (acidic fibroblast) growth factor-1 (HBGF-1) inhibits the synthesis of prostacyclin in HUVEC. In this report, we describe the effect of HBGF-1 on Cox mRNA expression in HUVEC. Cells cultured in the presence of HBGF-1 express diminished Cox mRNA levels whereas quiescent cells maintained in serum expressed a 7-fold higher level of the transcript for Cox. Concomitantly, the level of the Cox translation product and prostacyclin synthesis are also reduced by HBGF-1. Further, HBGF-1, in the presence of heparin, down-regulates the levels of the Cox transcript in a dose- and time-dependent manner. The onset of action of HBGF-1 is slow, requiring up to 24 h to depress the level of Cox mRNA. Lastly, the effective dose of HBGF-1 to depress Cox mRNA levels is similar to that required for mitogenesis, suggesting that cell proliferation may be required for the reduction of Cox expression in vitro. Thus, Cox may belong to a class of genes that are reversibly down-regulated during periods of endothelial cell proliferation. PMID- 1721065 TI - Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells. AB - Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells. PMID- 1721066 TI - Rho-dependent transcription termination. Characterization of the requirement for cytidine in the nascent transcript. AB - By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site. PMID- 1721067 TI - Specific ribonuclease activities in spinach chloroplasts promote mRNA maturation and degradation. AB - We have used an in vitro system to characterize ribonuclease activities present in spinach chloroplasts. We show that 3' end maturation of petD mRNA, which encodes subunit IV of the cytochrome b6/f complex, is affected by a 33-kDa protein that binds to a hairpin structure at the 3' end of the mature mRNA. Binding of the 33-kDa protein to the petD hairpin structure decreases the efficiency of 3' end maturation, probably by impeding the progress of the processive 3'-5' exonuclease activity involved in chloroplast mRNA processing. A two-base mutation in the stem of the petD hairpin structure creates a novel recognition site for a ribonuclease which competes with the normal processing exonuclease activity. This mutation results in a very low 3' end processing efficiency for mutant petD transcripts, and instead generates a second processing product that lacks a complete hairpin structure. An endonuclease activity which is biochemically distinct from the previously characterized exonuclease activities has also been identified. This endonuclease activity is EDTA insensitive, and cleaves petD RNA both at the termination codon and at the mature RNA 3' end. Cleavage of petD mRNA at the termination codon leads to rapid degradation of upstream RNA. The possible roles of these ribonuclease activities in chloroplast mRNA decay in vivo are discussed. PMID- 1721068 TI - Crystallins of the octopus lens. Recruitment from detoxification enzymes. AB - The eye lens crystallins of the octopus Octopus dofleini were identified by sequencing abundant proteins and cDNAs. As in squid, the octopus crystallins have subunit molecular masses of 25-30 kDa, are related to mammalian glutathione S transferases (GST), and are encoded in at least six genes. The coding regions and deduced amino acid sequences of four octopus lens cDNAs are 75-80% identical, while their non-coding regions are entirely different. Deduced amino acid sequences show 52-57% similarity with squid GST-like crystallins, but only 20-25% similarity with mammalian GST. These data suggest that the octopus and squid lens GST-like crystallin gene families expanded after divergence of these species. Northern blot hybridization indicated that the four octopus GST-like crystallin genes examined are lens-specific. Lens extracts showed about 40 times less GST activity using 1-chloro-2,4-dinitrobenzene as substrate than liver extracts of the octopus, indicating that the major GST-like crystallins are specialized for a lens structural role. A prominent 59-kDa crystallin polypeptide, previously observed in octopus but not squid and called omega-crystallin (Chiou, S.-H. (1988) FEBS Lett. 241, 261-264), has been identified as an aldehyde dehydrogenase. Since cytoplasmic aldehyde dehydrogenase is a major protein in elephant shrew lenses (eta-crystallin; Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) the octopus aldehyde dehydrogenase crystallin provides the first example of a similar enzyme-crystallin in vertebrates and invertebrates. The use of detoxification stress proteins (GST and aldehyde dehydrogenase) as cephalopod crystallins indicates a common strategy for recruitment of enzyme crystallins during the convergent evolution of vertebrate and invertebrate lenses. For historical reasons we propose that the octopus GST-like crystallins, like those of the squid, are called S-crystallins. PMID- 1721069 TI - Omega conotoxin and prejunctional modulation of the biphasic response of the rat isolated urinary bladder to single pulse electrical field stimulation. AB - 1. Single pulse electrical field stimulation (EFS) produces a biphasic response of muscle strips of the rat isolated urinary bladder consisting of an early and a late contraction which were atropine-resistant and atropine-sensitive, respectively. Repeated application of desensitizing doses of the P2 purinoceptor agonist, alpha, beta-methylene ATP (mATP) inhibited the early response while leaving unaffected the late component. 2. Omega conotoxin (CTX, 0.1 microM) inhibited both the early and the late response either in control conditions or after enhancement by physostigmine (0.1 microM). The effect of CTX was, in both cases, more pronounced on the late than the early response to EFS. CTX (0.1 microM) failed to affect contraction produced by ATP or acetylcholine at concentrations (0.3 mM and 0.5 microM) which produced a response similar to that to EFS. 3. The effect of physostigmine was more intense for the late than the early response and was abolished by atropine. In the presence of CTX, physostigmine enhanced both the early and the late components of the mechanical response to EFS. 4. Nifedipine (0.1-1 microM) reduced to a similar extent both the early and late responses. Bay K 8644 (1 microM) produced a marked enhancement of the response to EFS, which, however, did not have a distinct late peak. In the presence of Bay K 8644, either atropine (3 microM) or tetrodotoxin (1 microM) had minor inhibitory effects indicating the myogenic origin of the response. 5. Neurokinin A (0.1-1 nM) enhanced both the early and late responses to EFS without affecting the contraction produced by exogenous acetylcholine or ATP. A consistent potentiation was evident also in the presence of CTX and for the early response, in the presence of atropine. Clonidine (3 microM) inhibited the response to EFS either in the absence or the presence of physostigmine. The inhibitory effect of clonidine, shown previously to depend upon activation of prejunctional alpha 2-adrenoceptors, was still observed in presence of CTX or atropine. 6. It is concluded that CTX-sensitive voltage dependent calcium channels play a more important role in determining the cholinergic rather than the non-cholinergic, putatively purinergic, component of the biphasic response of the rat bladder to single pulse EFS. The action of CTX is likely to be exerted on N-type rather than L-type (dihydropyridine-sensitive) calcium channels. Prejunctional modulation (enhancement by neurokinin A, inhibition by clonidine) occurs even in the presence of CTX-sensitive channels blockade. PMID- 1721070 TI - Fetuin and alpha-2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes. AB - A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation. PMID- 1721071 TI - Insulin-like growth factor binding protein (IGFBP) inhibits IGF action on human osteosarcoma cells. AB - The influence of a human insulin-like growth factor binding protein, hIGFBP-1, on the action of IGFs on human osteosarcoma cells was examined. hIGFBP-1 was found to block binding of IGFs to their receptors on MG-63 cells and subsequent IGF stimulation of DNA synthesis. Concurrent incubation of hIGFBP-1 with either 125I IGF-I or 125I-IGF-II prevented the binding of both 125I-IGFs to cells in a dose dependent manner. hIGFBP-1 inhibition of IGF binding occurred similarly under both 4 degrees and 37 degrees C conditions. Additionally, hIGFBP-1 facilitated the dissociation of IGFs bound to cells. The inhibitory effect of hIGFBP-1 on IGF 1 mediated 3H-thymidine incorporation into DNA was dose dependent. hIGFBP-1 did not inhibit binding to or stimulation of growth in MG-63 cells by des3-IGF-1, an IGF-I analog with a 100-fold less affinity for hIGFBP-I. This confirmed that hIGFBP-1 competed for IGF receptor binding sites on MG-63. Since hIGFBP-1 did not bind to cells, inhibition of IGF action was indirect, presumably through the formation of extracellular soluble bioinactive IGF-BP complexes. PMID- 1721072 TI - Macrophages express functional receptors for calcitonin-gene-related peptide. AB - The present study was designed to investigate whether non-activated macrophages express calcitonin (CT) or calcitonin-gene-related peptide (CGRP) receptors. To this end, we first analyzed whether CT and CGRP induce a cAMP accumulation in macrophages. Macrophages were treated for 2 min with increasing concentrations of either CT or CGRP in the presence or absence of IBMX. A dose-dependent cAMP accumulation was measured in response to CGRP with a half-maximal effect attained with 1 nM CGRP. CT failed at all doses to induce an accumulation of cAMP. The effects of CT and CGRP on the activation of the Na-H exchanger were next assessed by spectrofluorometry by using the pH-sensitive dye 2,7 biscarboxyethyl-5(6) carboxyfluorescein (BCECF). Steady-state pHi of macrophages in a 7.4, HCO3-free solution (HEPES-buffered) was 7.04 +/- 0.08 (n = 22). pHi recovery following an NH4+/NH3 acid load was inhibited by the removal of Na+ or by the addition of the amiloride analog EIPA; therefore recovery is dependent on Na-H exchange activity. CT had no effect on steady-state pHi but CGRP increased pHi in a dose-dependent fashion (10(-12) to 10(-6) M). The pHi change induced by CGRP was due to the stimulation of the Na-H exchanger as CGRP enhanced the rate of recovery (dpHi/dt) from an acid load from 45.3 to 77.2 microMs-1 (n = 8, P less than 0.002) and was completely blocked by EIPA. These data indicate that CGRP both enhances the activity of the Na-H exchanger and increases intracellular cAMP, thus demonstrating that macrophages express functional CGRP receptors. PMID- 1721073 TI - Multiple pathways for ligand internalization in rat hepatocytes. I: Effects of anoxia, phenylarsine oxide and monensin. AB - It has been suggested that there are multiple pathways for the cellular internalization of insulin. To investigate these pathways we have examined the effects of three perturbations of endocytosis on the insulin internalization process and have compared these effects with those obtained using an asialoglycoprotein, asialofetuin (Afet), and epidermal growth factor (EGF). Freshly isolated hepatocytes were incubated with radiolabeled ligands and internalization measured under conditions of anoxia to deplete cellular ATP, in the presence of phenylarsine oxide (PAO) to inhibit endocytosis, and in the presence of monensin to interfere with endosomal acidification. Afet internalization essentially was blocked by all three treatment processes, while insulin internalization was inhibited approximately 40% in the presence of anoxia, and 54% in the presence of PAO. Monensin exhibited differential effects on internalization of high and low insulin concentrations. The effects of the treatment processes on EGF internalization were intermediate to those seen with Afet and insulin. These results suggest that insulin and EGF utilize routes of internalization exhibiting different energy requirements that may correspond to coated pit, non-coated pit, and fluid-phase internalization pathways. The observations with Afet internalization remain consistent with utilization of the coated pit pathway. PMID- 1721074 TI - Multiple pathways for ligand internalization in rat hepatocytes. II: Effect of hyperosmolarity and contribution of fluid-phase endocytosis. AB - In a companion report (Moss and Ward: J. Cell. Physiol 149:313-318, 1991) evidence was presented for multiple pathways for insulin internalization based on differences between the internalization of insulin and that of two other ligands, asialofetuin (Afet) and epidermal growth factor (EGF), in the presence of several perturbations of endocytosis. In the present study we have explored the characteristics of three internalization pathways and the contribution of each to overall insulin uptake. Freshly isolated hepatocytes were incubated with radiolabeled ligands in the presence of hyperosmolar sucrose, treatment that is thought to inhibit the coated pit pathway of endocytosis. Insulin internalization was decreased approximately 39%, but much greater decreases were observed with Afet (86%) and EGF (62%). Competition between uptake of radiolabeled and unlabeled insulin was observed in hyperosmolar-treated cells, suggestive of endocytosis by a receptor-mediated noncoated-pit pathway. Uptake of radiolabeled insulin that persisted in the presence of hyperosmolarity and high concentrations of unlabeled insulin suggested a third uptake pathway: fluid-phase endocytosis. A rate of fluid-phase endocytosis of 7.2 microL/hr/10(6) cells was determined from the uptake of the fluid-phase marker lucifer yellow. At high insulin concentrations (greater than or equal to 250 ng/ml), fluid-phase endocytosis appears to be the predominant pathway for insulin uptake, but at lower insulin concentrations (physiological) the coated pit and noncoated pit pathways are the primary routes for insulin internalization. PMID- 1721075 TI - Subpopulations of GABAergic neurons containing parvalbumin, calbindin D28k, and cholecystokinin in the rat hippocampus. AB - The possible coexistence of calbindin D28k with parvalbumin and of calbindin D28k with cholecystokinin was studied in nonpyramidal cells of the rat dorsal hippocampal formation. Neighbouring Vibratome sections were immunostained either for calbindin D28k and parvalbumin or for calbindin D28k and cholecystokinin. The cells, halved during sectioning, were identified in both sections immunostained for different antigens. The coexistence of calbindin D28k and parvalbumin in the same neuron was rare throughout the hippocampal formation with the exception of stratum oriens of the CA1 region, where 9.6% of the parvalbumin-immunoreactive cells also contained calbindin D28k. In stratum radiatum of the CA3 region, calbindin D28k and cholecystokinin coexisted in 12.5% and 21.2% of the calbindin D28k and cholecystokinin-immunoreactive cells, respectively. In other regions of the hippocampal formation, the two markers coexisted in less than 5% of the cells of either type. The present results demonstrate that calbindin D28k-, parvalbumin and cholecystokinin-containing nonpyramidal cells represent largely nonoverlapping cell populations and may thus be involved in different inhibitory circuits. PMID- 1721076 TI - Substance P immunoreactivity identifies a projection from the cat's superior colliculus to the principal tectorecipient zone of the lateral posterior nucleus. AB - Cells in the superficial layers of the superior colliculus innervate multiple visual regions within the pulvinar-lateral posterior complex of the cat. To characterize these neurons we have examined their immunocytochemical properties in conjunction with their projection patterns. In the present study, we show that a monoclonal antibody for substance P recognizes a morphologically diverse population of neurons, which can be classified as granular, stellate, angular, and horizontal or nonhorizontal fusiform cell types. These neurons are distributed throughout the superficial layers of the colliculus, with a peak density corresponding to sublayer 2 of the stratum griseum superficiale. Injections of rhodamine latex micropheres into the pulvinar-lateral posterior complex demonstrate that a substantial proportion of these collicular cells (at least 35%) project to this region of the posterior thalamus. The overall population of substance P-containing cells, as well as the immunoreactive projection neurons, is composed of the same proportions of cell classes, with the exception that granular cells were not found to be projection neurons. A distinct wedge of substance P immunoreactivity, consisting of fiber and diffuse extracellular labeling, was discovered in the pulvinar-lateral posterior complex. This staining was demonstrated to be confined entirely within the medial division of the lateral posterior nucleus, which is considered to be the principal tectorecipient zone of the extrageniculate visual thalamus. Lesions of the superior colliculus largely abolished the substance P immunoreactivity in the ipsilateral tectorecipient zone. These results are consistent with the view that substance P plays a role in the functional organization of the principal tectothalamic pathway of the cat's extrageniculate visual system. PMID- 1721077 TI - Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene-related peptide and substance P, and fluoride-resistant acid phosphatase in neonatally capsaicin-treated rats. AB - This study examined the effect of neonatal administration of capsaicin on nociceptive threshold and the distribution of calcitonin gene-related peptide (CGRP), substance P (SP), and fluoride-resistant acid phosphatase (FRAP) in the dorsal horn of the spinal cord during the course of development (10 days to 12 weeks of age) in the rat. As early as 10 days of age, CGRP-like immunoreactivity was reduced in laminae I, II, and V, as well as in the bundles of fibers situated dorsal and ventral to the central canal. However, beginning on or about 6 weeks of age, the density of CGRP-like immunoreactivity in the superficial laminae and in the bundles dorsal and ventral to the central canal increased. Moreover, thick, nonvaricose CGRP-like immunoreactive fibers appeared in laminae III and IV. These recurring fibers were of primary afferent origin as demonstrated by their disappearance after multiple, unilateral rhizotomies. A similar age dependent alteration in the density of FRAP activity was also observed. Although virtually absent at 10 days of age after neonatal administration of capsaicin, the density of FRAP activity increased in lamina II by 8 weeks of age. This activity disappeared after multiple, unilateral rhizotomies, indicating that the FRAP activity that reappeared was of primary afferent origin. Neonatal administration of capsaicin also reduced the density of SP-like immunoreactivity in the dorsal horn as early as 10 days of age, although the density of SP-like immunoreactivity showed some recovery after 6 weeks of age. However, unlike CGRP like immunoreactivity or FRAP activity, the density of SP-like immunoreactivity in capsaicin-treated rats was not detectably altered by multiple, unilateral rhizotomies, indicating that it originated principally from intrinsic dorsal horn neurons. Age-dependent alterations in both thermal and mechanical, but not chemical, nociceptive thresholds were also observed in these same animals. Thus, tail flick latency, hot plate latency, and paw withdrawal threshold were maximally increased at 6 weeks of age, after which time thresholds declined to vehicle-treated values. In contrast, capsaicin-treated animals were uniformly insensitive to ophthalmic administration of capsaicin. The correspondence between developmental alterations in CGRP-like immunoreactivity or FRAP activity and in thermal and mechanical nociceptive thresholds is suggestive of a role of CGRP- or FRAP-containing primary afferents in thermal and mechanical nociception. PMID- 1721078 TI - Development of collateral circulation in a replanted rat hindlimb model. AB - The development of collateral circulation was studied in a replanted rat hindlimb model by use of the hemodynamic techniques of hydrogen washout and laser Doppler capillary perfusion monitoring. Collateral development occurred and provided blood flow whose magnitude increased linearly over time. Twenty-seven percent of preligation blood flow was present at 2 weeks after replantation, 39% at 4 to 5 weeks, and 77% at 8 to 9 weeks by laser Doppler assessment. Laser Doppler correlated more closely with limb survival than did the hydrogen washout technique. Uniform necrosis (eight of eight) occurred after pedicle interruption in the 2-week group. Partial limb survival was noted in five of eight replants at 4 to 5 weeks on transection of the vascular pedicle, while complete survival of all replants (nine of nine) was seen at 8 to 9 weeks. PMID- 1721079 TI - Clinicopathological profile of carcinoma of oesophagus at Aligarh. AB - Forty-seven patients with oesophageal carcinoma were managed in 6 years' time. Average duration of illness was 5.5 months. History of chronic smoking and/or tobacco chewing was present in 80.85% of patients. Carcinoma included squamous cell variety (80.85%) and adenocarcinoma (19.15%). Thirty-one patients were in stage III while 16 patients were in stage II. Surgery included oesophagogastrectomy/oesophagogastrostomy (16 patients), feeding gastrostomy (11 patients), Mousseau-Barbin tube insertion (10 patients), only 10 patients were subjected to palliative radiotherapy. All patients after palliative treatment died within one year whereas 3-year and 5-year survivals after oesophagogastrectomy/oesophagogastrostomy were 68.75% and 31.25% respectively. Local lymph node metastasis adversely affected the 5-year survival rate. PMID- 1721080 TI - Epidermolysis bullosa simplex (Dowling-Meara type) is a genetic disease characterized by an abnormal keratin-filament network involving keratins K5 and K14. AB - The distribution and morphology of tonofilament (TF) clumps were examined by light and electron microscopy in skin samples from a total of 17 patients with the Dowling-Meara (DM) form of epidermolysis bullosa simplex (EBS). TF clumps extending from the basal to the upper-spinous epidermal layer were seen in all lesional skin samples and in the majority of peri-lesional and non-lesional skin samples. TF clumps were also noted in adnexal epithelia, including outer hair root sheaths, sweat ducts, and sebaceous glands. Cultured keratinocytes from two patients also demonstrated characteristic TF clumps. All these epithelial cells have in common their expression of the keratin pair K5 and K14. Post-embedding immunogold electron microscopy using antibodies to K5, K14, and K10 showed similar expressed keratins in DM-EBS skin from four patients compared with normal skin, with K5 and K14 predominantly in the basal cell layer and K10 in the suprabasal layers. The clumped TF in DM-EBS samples were labeled strongly with anti-K5 and K14 antibodies in the basal and suprabasal layers. In contrast, the suprabasal clumps were only slightly reactive with anti-K10 antibodies and labeling was usually restricted to the periphery of the clumps. We conclude that DM-EBS is associated with an intrinsic abnormality of the keratin-filament network involving the K5 and K14 pair that is likely to result in impaired resistance of basal epidermal cells to external shearing forces, leading to the characteristic intraepidermal blisters. DM-EBS may become the first genetic skin disease to be recognized as having a specific keratin abnormality. PMID- 1721081 TI - An immunofluorescence study of the calcium-induced coordinated reorganization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. AB - Indirect immunofluorescence microscopy has been used to investigate the coordinated reorganization of microtubules, microfilaments, and keratin intermediate filaments in cultured human epidermal keratinocytes following a switch from low-Ca++ (0.15 mM) medium to high-Ca++ (1.05 mM) medium. A dramatic reorganization occurs concurrently in the three major cytoskeletal components shortly after the calcium switch. The most prominent features are the alignment of keratin filaments at the plasma membranes of apposed cells, the induction of microfilament rings, the restriction of microtubules to the area within the boundaries of the microfilament rings, and the alignment of actin bundles at cell borders. Additional changes are observed in terminally differentiated cells. This is the first report that describes simultaneous changes in the organization of the three major cytoskeletal components of epidermal keratinocytes. Cytochalasin D and demecolcine (colcemid) studies were performed to determine whether the organization of microtubules, microfilaments, and keratin filaments, as well as the calcium-induced reorganization of these cytoskeletal elements, may be dependent on the existence of structural relationships between them. These studies demonstrate that the disruption of microfilaments results in the formation of a latticelike keratin network, with a close association of actin and keratin being maintained. The formation of keratin filament alignments occurs even in the absence of intact microfilaments. In addition, it was found that the Ca(++)-induced reorganization of microfilaments and keratin filaments is not dependent on an intact microtubule network. Furthermore, the reorganization of actin into concentric rings can be dissociated from changes in the organization of keratin filaments. PMID- 1721082 TI - Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), but not galanin, are autonomic cotransmitters in the porcine pancreas. AB - The neuropeptide galanin has been identified as a potential sympathetic cotransmitter in the canine pancreas. Immunoreactive galanin, also present in nerve fibers of the pig pancreas, was therefore measured in the effluent from isolated perfused pig pancreas with preserved sympathetic (splanchnic) or parasympathetic (vagal) innervation with radioimmunoassays directed against both the N-terminus and the C-terminus of galanin. Electrical vagus stimulation increased the pancreatic exocrine secretion, the secretion of insulin and glucagon, and the release of VIP, but did not influence galanin release. Splanchnic nerve stimulation increased perfusion pressure and glucagon secretion, inhibited insulin secretion, and increased the release of NPY, but galanin release was not affected. We conclude that the pancreatic galanin nerve fibers belong neither to the sympathetic nor to the parasympathetic divisions of the efferent nerve supply to the pig pancreas. PMID- 1721083 TI - Murine eosinophil granulocytes bind the murine macrophage-monocyte specific monoclonal antibody F4/80. AB - Studies designed to identify a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was expressed by eosinophil granulocytes. Eosinophils from several strains of mice stained positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes. PMID- 1721084 TI - Protection against Streptococcus equi infection by monoclonal antibodies against an M-like protein. AB - We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs, 1D10 and 2A6, were shown to be highly protective. It was also demonstrated, by means of a competitive enzyme immunoassay, that these mAbs recognized different epitopes in the preparation. Examination of a dose-response curve for mAbs 1D10 and 2A6 revealed that optimal levels of protection were achieved using 1 mg of either 1D10 or 2A6, or 0.5 mg 1D10 and 0.5 mg 2A6 given together. Immunological reactivity of these mAbs with a preparation of M-like protein showed that the antigens they recognized were comparable in size to some of the antigens recognized by convalescent horse serum antibodies. The role of immunoglobulin isotype in conferring protection is discussed. PMID- 1721085 TI - Classification of acidophilic, neutrotolerant and neutrophilic streptomycetes by nucleotide sequencing of 5S ribosomal RNA. AB - Complete 5S ribosomal RNA sequences were obtained for four acidophilic actinomycetes, seven neutrophilic streptomycetes and a strain of Streptoverticillium baldaccii. All of the organisms contained RNAs belonging to the 120 nucleotide type. An evolutionary tree was generated after combining the test data with results from similar studies on representative Gram-positive bacteria. The acidophilic, neutrotolerant and neutrophilic actinomycetes were recovered in a distinct cluster that was equated with the genus Streptomyces. The sequence data support the view that the genera Chainia, Elytrosporangium, Kitasatoa and Microellobosporia should be considered as synonyms of the genus Streptomyces. The recovery of the Streptoverticillium baldaccii strain on the fringe of the Streptomyces cluster is also consistent with current trends in the taxonomy of these organisms. Further work is needed to determine the taxonomic status of the two streptomycete subgroups that comprised the streptomycete cluster. PMID- 1721086 TI - Effects of methotrexate on RNA and purine synthesis of astrocytes in primary culture. AB - There is increasing evidence to indicate that astrocytes are primary targets for methotrexate (MTX) neurotoxicity. However, the mechanism by which MTX exerts its deleterious effect on astroglial cells is not known. Methotrexate acts by inhibiting dihydrofolate reductase and in other cell systems has been reported to inhibit thymidylate synthesis, purine synthesis or both. To determine the mechanism involved in MTX-induced toxicity to the nervous system, RNA synthesis was studied in two week-old primary astrocyte cultures by measuring [3H]Uridine (Urd) incorporation 24 hours after exposure to varying concentrations of MTX. De novo purine synthesis was also studied by measuring incorporation of [14C]glycine and [14C]formate in cultured astrocytes. The radioactivity level of incorporated Urd in culture decreased to 48%, 53% and 43% after exposure to 1, 10 and 100 microM MTX. Total [14C]glycine incorporation was not affected while incorporation of [14C]formate was almost completely inhibited by MTX. The MTX-induced inhibition of [3H]Urd incorporation was not reversed by concomitant addition of exogenous purine bases (1 and 10 microM adenine, guanine and hypoxanthine) or nucleosides (1 and 10 microM adenosine, guanosine and inosine) to the MTX-treated cultures. On the other hand, addition of formyl-tetrahydrofolate reversed the MTX induced reduction in [3H]Urd incorporation, indicating that the RNA inhibition was due to depletion of folate-dependent substrates for purine synthesis. Our results provide evidence that inhibition of purine and RNA synthesis may be the underlying mechanism involved in MTX-induced injury to the astrocytes, and may be important in the pathogenesis of MTX encephalopathy. PMID- 1721087 TI - Fine structural characteristics of testicular cord formation in the developing rabbit gonad. AB - This paper presents morphological (light- and electron-microscopical) evidence for the role of the mesonephros in contributing cells to the differentiating indifferent gonad and, after sexual differentiation, to the testis. A continuous process is revealed during which segregation of cells occurs from the developing and regressing mesonephros. Additionally, the complementary role of the coelomic epithelium in gonadal ridge and testis formation is demonstrated. The differentiation of testicular cords, their remodelling from a primary reticulum, and the composition and further change of the cellular content during the period after sexual differentiation is described using a computer-aided three dimensional reconstruction system. Apart from these morphogenetic events, cytodifferentiation in the somatic cells of the indifferent gonad and of the early differentiated testis is demonstrated using indirect immunofluorescence in combination with monoclonal antibodies to the intermediate filament proteins keratin 8 and 18 and vimentin. The immunohistochemical results show that different forms of cytodifferentiation coexist among the somatic cells present in the indifferent gonad and in the testis early after sexual differentiation. PMID- 1721088 TI - Immunogold electron microscopic localization of antigenic sites in the outer dense fiber region of rat sperm tail obtained by using antisera to two Sertoli cell secretory proteins. AB - Polyclonal antisera raised against polypeptide components of two rat Sertoli cell secretory proteins, designated protein S70 and S45-S35 heterodimeric protein on the basis of cell origin and estimated molecular weight, were used to identify antigenic sites in 1) rat testis, 2) cultured Sertoli cells, 3) developing spermatids (collected from spermatogenic stage-specific seminiferous tubular segments), and 4) epididymal sperm. Indirect immunofluorescence, immunoperoxidase, and immunogold electron microscopy (single and double labeling) were used. Immunocytochemical techniques have detected antigenic sites in 1) the cytoplasm of Sertoli cells in the intact seminiferous tubule and in culture in the form of a punctuate, granular-like pattern, and 2) the acrosome (but not the Golgi region) and tail of developing spermatids and sperm. In developing spermatids, the principal piece of the tail displays a characteristic apical-to distal immunoreactive banding pattern that correlates both temporally and spatially with the reported multistep assembly of outer dense fibers along the axoneme. The immunoreactivity of the acrosome, connecting piece, and outer dense fibers of the sperm tail was confirmed by immunogold electron microscopy. A precise identification of the component(s) of the outer dense fiber region responsible for the antigenic homology with Sertoli cell secretory proteins is under investigation. PMID- 1721089 TI - Postembedding immunostaining of ultrathin sections realized after their staining with heavy metals. PMID- 1721090 TI - Clinical experience with the triple test for Down's syndrome screening. AB - The introduction of maternal serological screening for chromosome disorders in pregnancy in women aged over 30 years at the estimated date of delivery was monitored in two hospitals. The test used involved measurement of three substances in maternal serum combined with maternal age (the triple test). This is the first report of such screening applied to an unselected antenatal clinic population. Test uptake was high but there was no overall increase in amniocentesis numbers because the increase in younger mothers was compensated for by a decrease in older women who previously, without serological testing, might have gone directly to amniocentesis. It is anticipated that widespread introduction of such testing will lead to improved detection of Down's syndrome as predicted from retrospective studies. PMID- 1721091 TI - Group play therapy. Using an interaction model with delayed regressed behaviors in children. AB - This article gives an overview of individual play therapy and relates the concepts of individual play therapy to group play therapy. A new model of group play therapy, an interactional model, is introduced. The article provides basic guidelines for beginning play therapists. An actual group is described and specific interventions for problems are suggested. PMID- 1721092 TI - SELEXION. Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis. AB - Recently, novel technologies for isolation of nucleic acid molecules with specific biological activities have been reported. In each case, the enrichment process involves repeated rounds of selection from complex mixtures of nucleic acid sequences, followed by polymerase chain reaction (PCR) amplification of ligand sequences that function in the desired manner. Particular variations in experimental conditions can dramatically alter the outcome of these processes. In this study, we use mathematical analysis and computer simulation to predict which variations have the greatest impact and to develop strategies and guidelines for enhanced effectiveness. First, we perform reconstruction tests to demonstrate that a mathematical description based on equilibrium binding is sufficient to explain the high levels of enrichment attained in the laboratory after just a few rounds. Then, we show the expected enrichment for an extensive range of conditions; and, finally, we determine the optimum protein and nucleic acid concentrations to use for maximum enrichment, while also ensuring a high likelihood of recovering even the rare molecule that binds well. The strategies and guidelines for enhanced effectiveness are generally applicable to processes for systematic enrichment of DNA, RNA or peptide ligands and have been implemented in an interactive simulation program for integrated non-linear optimization of enrichment using any target of interest. PMID- 1721093 TI - Clinical pharmacology, metabolism, and tissue distribution of 90Y-labeled monoclonal antibody B72.3 after intraperitoneal administration. AB - B72.3 is a murine monoclonal antibody that recognizes a high-molecular-weight tumor-associated glycoprotein (TAG-72). Nine patients with TAG-72-positive ovarian carcinoma or papillary serous carcinoma of the peritoneum received an intraperitoneal infusion of 2, 4, or 10 mg B72.3 labeled with 0.5-1.2 (mean, 0.8) mCi 90Y. All patients had laparotomy, with multiple tissue and tumor samples removed 3-7 days later. The concentration of the total 90Y label in peritoneal fluid cleared with an extrapolated half-life of 68.6 +/- 4.5 hours. A low molecular-weight 90Y-labeled species of metabolite was identified by high performance liquid chromatography. The concentration of this low-molecular-weight species initially increased in the peritoneal fluid, with a half-life of 0.9 hour, and was rapidly cleared from the peritoneal cavity, with a half-life of 23.1 hours. Both the 90Y-labeled metabolite and the 90Y-labeled B72.3 were absorbed into the plasma, with half-lives of 16 +/- 2.2 hours and 25 +/- 5 hours, respectively. The clearance half-lives for these agents in plasma were 25 +/- 3 hours for the metabolite and 42 +/- 17 hours for B72.3. Approximately 8%-11% of the total injected 90Y label appeared in urine over 72 hours. Most of the label (about 70%) was present as the 90Y-labeled metabolite, but about 30% of the 90Y label in urine appeared identical to the authentic 90Y-labeled B72.3 standard when assayed by chromatography. Tissue distribution studies showed that normal tumor tissue and omentum contained the highest content of 90Y (about 0.017% of the injected dose per gram), followed in descending order by liver, normal lymph nodes, peritoneum, bone, and fascia. The lowest concentrations of 90Y were found in rectus abdominis muscle, bone marrow, and fat. There was substantial heterogeneity in the uptake of the 90Y label into tumor sites among patients and among different sites within the same patient. No correlation could be demonstrated between the TAG-72 content and the amount of 90Y label found in tumor sites. Preliminary radiation dosimetry estimates suggest that the tumor sites received about 82.8 cGy for each millicurie of 90Y administered. Thus, if an adequate total radiation dose can be achieved, 90Y-labeled B72.3 should be therapeutically useful for treating diffuse intraperitoneal disease. PMID- 1721094 TI - Immunological mechanisms in experimental coxsackievirus B3 myocarditis in mice. AB - To address unresolved questions, experimental models of viral myocarditis may be of great value. In this study, immunological mechanisms of myocardial damage in coxsackievirus B3 myocarditis in mice were investigated. The results showed that susceptibility to viral infection is primarily determined by the genetic background of the host, that the severity of myocarditis depends not upon B cells but upon T cells, and that antigen-specific T cells play a pivotal role in the pathogenesis of acute coxsackievirus B3 myocarditis. PMID- 1721095 TI - Mechanisms of cardiac hypertrophy and injury--possible role of protein kinase C activation. AB - To examine the molecular mechanisms by which mechanical stimuli induced cardiac hypertrophy and injury, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos. Marked accumulation of c-fos mRNA followed increases in intracellular Na+ and protein kinase C activation. The accumulation of c-fos mRNA by cardiocyte stretching was suppressed by protein kinase C inhibitors but not by stretch channel blockers. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbolesters stumulated the expression of c-fos. We also examined TGF beta expression in the heart. TGF beta is known to be stimulated by protein kinase C activation, and the mRNA level of TGF beta was increased in in vivo heart by pressure overload. Furthermore, collagen synthesis was stimulated by TGF beta in cultured fibroblasts from hearts. These findings suggest that hemodynamic overload may stimulate cardiac hypertrophy and induce cardiac injury (fibrosis) through protein kinase C activation. PMID- 1721096 TI - [Structure and function of Pseudomonas aeruginosa lipopolysaccharide (endotoxin)]. PMID- 1721097 TI - [Postoperative infections due to Pseudomonas aeruginosa]. PMID- 1721098 TI - [Recent trend of infection caused by Pseudomonas aeruginosa in patients with hematologic disorders]. PMID- 1721099 TI - [The necessity of i.v. human immunoglobulin therapy for Pseudomonas aeruginosa infection]. PMID- 1721100 TI - Analysis of the roles of microvessel endothelial cell random motility and chemotaxis in angiogenesis. AB - The growth of new capillary blood vessels, or angiogenesis, is a prominent component of numerous physiological and pathological conditions. An understanding of the co-ordination of underlying cellular behaviors would be helpful for therapeutic manipulation of the process. A probabilistic mathematical model of angiogenesis is developed based upon specific microvessel endothelial cell (MEC) functions involved in vessel growth. The model focuses on the roles of MEC random motility and chemotaxis, to test the hypothesis that these MEC behaviors are of critical importance in determining capillary growth rate and network structure. Model predictions are computer simulations of microvessel networks, from which questions of interest are examined both qualitatively and quantitatively. Results indicate that a moderate MEC chemotactic response toward an angiogenic stimulus, similar to that measured in vitro in response to acidic fibroblast growth factor, is necessary to provide directed vascular network growth. Persistent random motility alone, with initial budding biased toward the stimulus, does not adequately provide directed network growth. A significant degree of randomness in cell migration direction, however, is required for vessel anastomosis and capillary loop formation, as simulations with an overly strong chemotactic response produce network structures largely absent of these features. The predicted vessel extension rate and network structure in the simulations are quantitatively consistent with experimental observations of angiogenesis in vivo. This suggests that the rate of vessel outgrowth is primarily determined by MEC migration rate, and consequently that quantitative in vitro migration assays might be useful tools for the prescreening of possible angiogenesis activators and inhibitors. Finally, reduction of MEC speed results in substantial inhibition of simulated angiogenesis. Together, these results predict that both random motility and chemotaxis are MEC functions critically involved in determining the rate and morphology of new microvessel network growth. PMID- 1721101 TI - Mayo and the LE cell. PMID- 1721102 TI - Stereospecific effects of a nonpeptidic NK1 selective antagonist, CP-96,345: antinociception in the absence of motor dysfunction. AB - CP-96,345 has been identified as being a highly selective, nonpeptidic agent with subnanomolar affinity for the NK1 receptor. In the present study, we observed that pre but not posttreatment with this agent will produce depression in the second, but not the first phase of the agitation behavior induced by the injection of formalin into a rat's hindpaw. This effect is monotonically dose dependent after intrathecal (10-200 micrograms/10 microliters) or systemic (1-15 mg/kg, ip) administration. Even at the highest dose examined (400 micrograms/10 microliters), there was only a transient motor weakness of the hindpaw. The stereoisomer CP-96,344 has no binding affinity, and has no effect on the formalin response, but shows the same dose profile for motor dysfunction at the highest dose. In contrast, Spantide, a peptidic sP ligand, had only a modest effect upon the formalin response at 1 microgram/10 microliters and produced a prominent, long-lasting motor dysfunction at 4 micrograms/10 microliters. These results provide the first suggestion of sP antagonists having prominent analgesic activity with a significant therapeutic index (analgesic to motor), and emphasizes the probable role of the NK1 class of receptors in the spinal cord in mediating at least one class of nociceptive afferent input. PMID- 1721103 TI - Influence of N omega-nitro-L-arginine methyl on pressor responses elicited by sympathetic nerve stimulation in pithed normotensive and hypertensive rats. AB - The effects of N omega-nitro-L-arginine methyl ester (L-NAME) were examined on pressor responses elicited by sympathetic nerve stimulation (SNS) in the pithed spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats. Frequency-response curves (1-20 Hz) were carried out by using the pithing rod to stimulate the sympathetic chain. SNS produced an increase in the blood pressure of the pithed rats which was dependent on the frequency of the applied stimuli, however, a significantly greater increase was observed for the blood pressure of SH versus WKY rats. Bolus i.v. injections of L-NAME (0.03-1.0 mg/kg) augmented the increase in the blood pressure resulting from SNS. The potentiating effects of L-NAME displayed frequency as well as dose-dependency and the augmentation produced following the administration of L-NAME was greater in magnitude in the SH as compared to WKY rats. These differential effects of L-NAME in SH versus WKY rats suggest that the levels of L-arginine-derived nitric oxide are higher in the SH rats. Such an increase may reflect a compensatory response resulting from the elevated blood pressure of the SH rats. PMID- 1721104 TI - The repertoire of antibodies to a single antigenic determinant. PMID- 1721105 TI - Protective effect of human granulocyte colony stimulating factor (hG-CSF) on Candida infections in normal and immunosuppressed mice. AB - Prophylactic treatment with human granulocyte colony stimulating factor (hG-CSF) affords significant protection against systemic infections caused by C. albicans in cyclophosphamide-treated but not in cortisone-treated mice. Localized candidosis in neutropenic mice does not respond to hG-CSF. Our data show that granulocytes play an important role in the immune defence against deep mycoses, but not against local infections. From our data it is reasonable to assume that prophylactic treatment with hG-CSF may augment the resistance of immunosuppressed patients to deep Candida infection, but it would be of little help against oral candidosis of HIV patients. PMID- 1721106 TI - HIV. Games that viruses play. PMID- 1721107 TI - Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. AB - In a longitudinal study of HIV seropositive patients, there were fluctuations in the specificity of cytotoxic T cells for the virus. This was matched by variability in proviral gag DNA epitope sequences in the lymphocytes of these patients. Some of these viral variants are not recognized by autologous T cells. Accumulation of such mutations in T-cell antigenic targets would provide a mechanism for immune escape. PMID- 1721108 TI - [Cell biology from a medical perspective. VII. Functions of cell compartments: synthesis and degradation of glycoproteins]. PMID- 1721109 TI - [Psychometric speech studies in Alzheimer's dementia with the Aachen aphasia test]. AB - Dementias of the Alzheimer type seem to be frequently accompanied by language disturbances. These may represent a feature which can be of help in distinguishing them from other types of dementias. We used the Aachen aphasia test in 32 patients suffering from Alzheimer dementia according to research criteria, and a mixed sample of 35 patients suffering from other dementias. From these 2 groups 2 subsamples of 21 patients each were gathered which were comparable with regard to age, disease onset, level of education, verbal intelligence and severity of senility. Nevertheless it was possible to distinguish the groups to a certain degree on grounds of psychometric language criteria alone. Alzheimer patients were more severely handicapped communicatively, less dysarthric, produced more automatisms and discretely more phonemic paraphasias with fluent speech which was sometimes paragrammatic. A relatively better level of repetition compared to the Token test and written language was fairly specific. A computer-assisted classification yielded language disturbances similar to Wernicke's aphasia more often than with non-Alzheimer dementias. We found no Alzheimer patients with a Broca's type of language disorder, while amnestic and global types were bound to the level of overall impairment to a certain degree. The significance of these results with regard to the use of psychometric language test in the dementias, particularly Alzheimer's dementia, and to differential diagnostic considerations are reviewed briefly. PMID- 1721110 TI - The involvement of L-type calcium channels in heterosynaptic long-term depression in the hippocampus. AB - The involvement of L-type calcium channels in heterosynaptic long-term depression (LTD) of the stratum radiatum input to area CA1 was studied in rat hippocampal slices. LTD of the radiatum field excitatory postsynaptic potential (EPSP) and population spike, produced by tetanization of the alveus in the presence of picrotoxin, was blocked by the calcium antagonist nimodipine and by a monoclonal antibody to the L-type calcium channel. LTD was produced in the absence of picrotoxin when the L-type calcium channel agonist, BAY-K8644, was applied. This effect was also blocked by nimodipine. These results indicate that L-type calcium channels are involved in heterosynaptic long-term depression. PMID- 1721111 TI - Cytochrome oxidase staining facilitates unequivocal visualization of the primary gustatory area in the fronto-operculo-insular cortex of macaque monkeys. AB - Fronto-opercular and insular cortices of Japanese macaques were histochemically stained for cytochrome oxidase activity. The laminated pattern of enzyme activity differed in the different cytoarchitectonic areas. Area G, the presumed primary gustatory area, as well as area 3 were prominent because the third stripe, corresponding to the termination layers of the specific thalamocortical projection in these areas was very dark and thick in comparison with that from the neighboring areas. This approach provides a convenient histological aid for identification of area G. PMID- 1721112 TI - Calpain activity in a subcellular fraction enriched in partially degraded CNS myelin fragments compared with myelin. AB - Marchi-positive bodies are structures present paranodally in large myelinated nerve fibers. They have morphological and biochemical characteristics closely resembling the partially degraded myelin fragments formed during the early phases of Wallerian degeneration. Levels of calcium-activated neutral proteases (calpains) and their endogenous specific inhibitor calpastatin were measured in highly purified rabbit myelin and a spinal cord subcellular light ('floating') fraction heavily enriched in Marchi-positive bodies. Calpain levels were found to be significantly higher in the floating fraction as compared to myelin. No calpastatin was detectable in either fraction. PMID- 1721113 TI - Lesion-induced establishment of the crossed corticorubral projections in kittens is associated with axonal proliferation and topographic refinement. AB - The aberrant crossed corticorubral projection of the cat, which is very weak compared to the uncrossed one at about 1 month postnatal, becomes pronounced following unilateral lesions of the sensorimotor cortex. In order to determine whether or not terminal proliferation of pre-existing axons underlie this enlargement, the morphological changes of the crossed axons were examined, using the anterograde tracer Phaseolus vulgar- is leukoagglutinin (PHA-L). The crossed corticorubral axons in normal kittens were mostly simple in morphology with infrequent branching and did not often exhibit growth-cone-like axonal endings at 1 month postnatal. Two to 5 days after unilateral lesions of the sensorimotor cortex placed at this age, the axons were as simple as those in normal animals but ended in growth cones more frequently. Seven to 10 days post-lesion, the axons often bore side-branches which ended in growth cones. Two to 3 weeks post lesion axons with sprays of finger-like fine sprouts occurred throughout the projection zone. There was no clear topography for the crossed projection in normal animals, but at 1-2 weeks post-lesion the axons started to show a certain amount of localization in the regions of the red nucleus which corresponded to the densely innervated region on the ipsilateral side. The topography of the crossed projections roughly mirrors that of the ipsilateral projection at about 1 month post-lesion. Thus, the lesions of the sensorimotor cortex induce substantial growth and proliferation of the crossed corticorubral axons. The post lesion changes in axonal morphology and topographic refinement are reminiscent of developmental events. It is likely that the lesions permit the crossed axons, which normally fail to develop, to develop like the uncrossed ones. PMID- 1721114 TI - Non-reciprocal connections between the vestibular nuclei and the cerebellar paramedian lobule, with special reference to the climbing fiber zones: an analysis in the rabbit using the retrograde horseradish peroxidase method. AB - The organization of the secondary vestibular projections onto the cerebellar paramedian lobule (PML) and possible reciprocal corticovestibular connections were investigated by the retrograde horseradish (HRP) technique in the rabbit. Following injections of the tracer into the vestibular nuclear complex (VNC), an ill-defined, sagittal band composed of numerous labelled Purkinje cells was found ipsilaterally throughout the length of the lateral portion of the vermis, the ventral paraflocculus and the flocculus. However, no labelled Purkinje cells were found in the cortex of the PML. The results indicate that zone B, considered to give rise to cerebellar corticovestibular projections, is not present in the rabbit PML. After injections of HRP into the PML, the retrograde labelling pattern in the VNC was analyzed, in relation to the climbing fiber zones identified by retrograde labelling in the inferior olive. No clear-cut correspondence could be found between the vestibular subdivisions and climbing fiber zones in the PML, except that only the interstitial nucleus of the vestibular nerve projects into zone D1 in sublobules e and d. There were no vestibular projections to zone D2. The only salient feature was that cells projecting onto zones C2, C1 and C3 of the PML were arranged rostrocaudally in the inferior vestibular nucleus and the caudal portion of the medial vestibular nucleus. In addition, a topical relationship was found between parts of the VNC and sublobules of the PML. PMID- 1721115 TI - Cerebellar projections of the central cervical nucleus in the rat: an anterograde tracing study. AB - The projection of the spinocerebellar tract arising from the central cervical nucleus was examined by the anterograde transport of wheatgerm agglutinin or cholera toxin subunit B (choleragenoid) conjugated to horseradish peroxidase in the rat. Following bilateral injections of the tracers into the C1-C3 segments, labeled terminals were seen in lobules I-VI, sublobule VIIb, lobule VIII, sublobule IXa + b and the copula pyramids. The labeled terminals were densely distributed in the basal half or basal two thirds of lobules I-III in their apicobasal extent, and the transitional areas between the neighboring lobules. The projection field in the horizontal plane of the lobules was reconstructed from a series of transverse sections through each lobule. In the anterior lobe, labeled terminals were distributed in 3 longitudinal areas, named areas 1, 2 and 3, respectively. These areas were confined in the basal half to the basal two thirds of lobules II and III; area 1 was located in zone A1 of Voogd (within 250 microns of the midline); area 2 was located in zones A1-A2 (about 250 microns lateral to the midline); and area 3 was located in the lateral part of zone A2 to zone B (between 0.5 and 1.0 mm lateral to the midline). In lobule II, another area (area 4) was present lateral to the vermis. In lobule I the 3 longitudinal areas extended over the apicobasal length of the lobule. In sublobule IXa + b and lobule VIII, 4 ill-defined longitudinal areas appeared in the basal two-thirds. The present study confirmed that the projection pattern of the central cervical nucleus is identical in both the rat and the cat. PMID- 1721116 TI - Brainstem mossy fiber projections to lobules VIa, VIb,c, VII and VIII of the cerebellar vermis in the rat. AB - The brainstem mossy-fiber projections to lobules VIa, VIb,c, VII and VIII of the cerebellar vermis were studied by retrograde transport of horseradish peroxidase in the rat. The distribution of labeled cells indicated that these lobules received major projections from topographically different locations of the basilar pontine nuclei and the nucleus reticularis tegmenti pontis. Lobules VIa and VIII received an additional strong projection from the lateral reticular nucleus. Moderate projections were found to reach lobule VIa from the raphe pontis and external cuneate nucleus; lobules VIb,c from the raphe pontis, lateral reticular nucleus, and a group of cells in the lateral tegmentum; lobule VII from the spinal vestibular nucleus and a lateral tegmental cell group; and lobule VIII from the medial and spinal vestibular nuclei, nucleus intercalatus and Roller of the perihypoglossal nuclei, and the main cuneate nucleus. The quantitative and topographical differences in the origin of mossy fibers suggest that these lobules may subserve slightly different functions. PMID- 1721117 TI - Olivocerebellar projection to the cardiovascular zone of rabbit cerebellum. AB - Climbing fiber responses were evoked in the medial vermal cortex of lobule VIIa by stimulation of the contralateral medial accessory olive (MAO) in anesthetized, paralyzed rabbits. Effective stimulating sites were localized in a small medial part of the caudal MAO, at 0.4-1.6 mm rostral from the caudal pole of the MAO (total length of the MAO, 4.2 mm). Stimulation of this MAO area induced depression in renal sympathetic nerve activity and this depressant response disappeared after ablation of lobule VIIa. Following injections of horseradish peroxidase into the small areas of lobule VIb, VIc, VIIa or VIIb, retrogradely labeled cells were found in corresponding small particular regions of the MAO: lobule VIb to the most caudal part, lobule VIc to the next caudal, lobule VIIa to the most rostral within the caudal MAO, and lobule VIIb further rostrally to the intermediate MAO. There was a clear disparity between the medial halves of lobules VI and VII projected from the medial MAO and the lateral halves from the lateral MAO. These results show that climbing fiber projections to lobules VI and VII are topographically organized, and that the medial region of lobule VIIa, related to cardiovascular function, receives climbing fibers from a localized small medial region of the caudal MAO. PMID- 1721118 TI - Corticocortical inputs to the dorsal and ventral aspects of the premotor cortex of macaque monkeys. AB - Recent cytoarchitectonic, histochemical and physiological studies have shown that the lateral part of area 6 (the premotor cortex) of macaque monkeys can be divided into at least two subregions, each of which is considered to play an important role in motor control. One lies in the dorsal aspect of the premotor cortex (PMd) medial to the spur of the arcuate sulcus, and the other in the ventral aspect of the premotor cortex (PMv) lateral to it. Since there is little information on the corticocortical inputs to the PMd, wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into both the PMd and PMv to study corticocortical inputs to these two regions, and the distribution of retrogradely labeled cells was compared. When WGA-HRP was injected into the region immediately lateral to the superior precentral sulcus within the PMd, retrogradely labeled neurons were found in area 6 lying in the mesial wall possibly corresponding to the supplementary motor area (SMA), areas 24 and 23 of the cingulate cortex, rostral region of area 4, and area 5 (area PEa). In contrast, when WGA-HRP was injected into the PMv immediately caudal to the arcuate sulcus and lateral to the spur of the arcuate sulcus, the labeled cells were found in area 7 (areas POa, PF, PFG), area 5 (area PEa), area PFop (secondary somatosensory area), SMA, the cingulate cortex (areas 24), caudal region of area 4 in the rostral bank of the central sulcus, and area 3a. It appears that the differences in the corticocortical inputs contribute to specialization of the PMd and PMv for their differential roles in motor control. PMID- 1721119 TI - Cortical and subcortical connections of the pars compacta of the anterior pretectal nucleus in the rat. AB - The efferent and afferent connections of the dorsal part of the anterior pretectal nucleus, pars compacta (APc), were studied experimentally in the rat by using neurotracers. A restricted number of structures supply afferents to the anterior pretectal nucleus: the visual cortex (areas 17, 18 and 18a), ventral lateral geniculate nucleus and superficial layers of the superior colliculus. Additional afferents have been demonstrated originating from the Darkschewitsch nucleus, periaqueductal gray, zona incerta and anterior cingulate cortex. Efferent fibers are distributed to a sector of the deep mesencephalic nucleus just dorsolateral to the red nucleus, the basilar pontine gray, posterior and olivary pretectal nuclei, superficial layers of the superior colliculus, lateral posterior thalamic nucleus, ventral lateral geniculate nucleus and zona incerta. These anatomical observations indicate that the pars compacta of the anterior pretectal nucleus is closely related to visual centers, suggesting an involvement of this nucleus in visually mediated behavior. PMID- 1721120 TI - Gastroschisis: a case report. PMID- 1721121 TI - Trends in pacemaker use: results of a multicenter registry. PMID- 1721122 TI - Ninety-six episodes of spontaneous ventricular tachycardia in 1 week: success of ramp pacing by a pacer-cardioverter-defibrillator. AB - This case report describes the flexibility and usefulness of a pacer-cardioverter defibrillator for the management of a 63-year-old patient with malignant ventricular tachyarrhythmias. Ninety of 96 episodes of ventricular tachycardia were terminated successfully with ramp pacing in a 1-week period. In those patients who have frequent episodes of ventricular tachycardia that respond to antitachycardia pacing, the multifunction device can add to the patient's comfort and increase acceptance of this type of device. PMID- 1721123 TI - Bipolar atrial triggered pacing to restore normal chronotropic responsiveness in an orthotopic cardiac transplant patient. AB - A not uncommon arrhythmia in cardiac orthotopic transplantation patients is sinus node dysfunction with chronotropic incompetence. This is a result of the surgical procedure that denervates the donor heart while the native sinus node may be normal but isolated in the remnant of the recipient atrial wall that serves as the anastomotic site. We were able to restore "normal sinus node function" in a heart transplant patient utilizing a bipolar single chamber pacemaker programmed to the triggered mode. A single unipolar active fixation lead was positioned in each atria. Both leads were connected to a bipolar AAT pulse generator utilizing a Y adaptator. The native atrium with its innervated intact sinus node effectively drove the donor atrium and thus the heart. PMID- 1721124 TI - Successful percutaneous extraction of a chronic left ventricular pacing lead. AB - This report describes a patient with a chronic endocardial left ventricular pacing lead. To avoid the risk of future embolization, it was felt that the lead should be removed and right ventricular pacing established. The lead was carefully evaluated by transesophageal echocardiography to exclude adherent thrombus. Successful percutaneous lead extraction was accomplished without sequelae, thus avoiding the morbidity of a thoracotomy. PMID- 1721125 TI - Inappropriate shocks and elevation of defibrillation thresholds in a patient with automatic defibrillator patch Silastic erosion and titanium mesh fraying. PMID- 1721126 TI - A case of a pseudomalfunction of a DDD pacemaker. AB - DDD pacemaker pseudomalfunction occurred in a 65-year-old man. This was due to premature ventricular contraction (PVC) response option and cross-talk detection window, which are designed to protect against pacemaker related tachycardia or cross-talk. Pseudomalfunction disappeared by eliminating PVC response option. PMID- 1721127 TI - Repetitive nonsustained monomorphic ventricular tachycardia aborted by methoxamine and sleep "heart rate dependent ventricular tachycardia". AB - Methoxamine, an alpha adrenoreceptor agonist, and sleep abolished repetitive nonsustained monomorphic ventricular tachycardia in a 41-year-old man without detectable underlying heart disease. Detailed pacing studies revealed that the occurrence of ventricular tachycardia was totally dependent on the basic heart rate. Sleep and the alpha adrenoreceptor agonist abolished the ventricular tachycardia by slowing the basic heart rate. Verapamil, propranolol, and disopyramide were able to decrease the upper limit of the tachycardia-initiating heart rate, but none of them were able to increase the lower limit (75/min). Failure to increase the lower limit of the tachycardia-initiating heart rate was a major reason why these conventional antiarrhythmic drugs were unable to suppress his daytime episodes of repetitive ventricular tachycardia. PMID- 1721128 TI - Effects of body constitution and age on maximum pacing rate of activity-modulated rate responsive pacemakers. AB - The pacing rate of activity-modulated pacemakers is triggered by vibrations running through the body. Whether the body constitution predicts maximum pacing rate and may facilitate rate response programming was studied in 16 patients with Activitrax pacemakers. Rate response parameters were programmed to a fixed setting in VVIR/VOOR mode (lower pacing rate 60 ppm, upper pacing rate 125-130 ppm, activity threshold medium/7). Body vibrations were induced by a treadmill exercise test with increasing speed. Maximum pacing rates were measured at the stage of symptom-limited tolerance. Exercise tests with a duration of 7.3 +/- 2.9 minutes resulted in a maximum pacing rate of 98 +/- 22 ppm ranging from 60-122 ppm. Maximum pacing rates did not differ between male (n = 10; 102 +/- 21 ppm) and female (n = 6; 92 +/- 24 ppm). Correlations between maximum pacing rates and body constitutional factors were not significant with r = -0.15 (weight), r = 0.39 (height), r = 0.07 (body surface area), and r = -0.27 (skin-fold thickness). The correlations with body mass index (r = -0.53) and age (r = -0.53) were initially significant, but not after Bonferroni-Simes-Hommel correction. The age dependent relationship may be caused by the shorter exercise duration of older patients indicated by the correlation between exercise duration and maximum pacing rate (r = 0.77), as well as with age (r = -0.73). CONCLUSIONS: body constitution did not modify body vibrations and did not allow prediction of maximum pacing rates; therefore, it is no aid for the programming of rate response parameters. PMID- 1721129 TI - Prehospital transcutaneous cardiac pacing for symptomatic bradycardia. AB - We studied patients with symptomatic bradycardia to determine the importance of presenting hemodynamic status and prehospital transcutaneous cardiac pacing (TCP) upon patient survival. Of 51 patients with witnessed cardiovascular decompensation and initial bradycardia, 27 (53%) received TCP. There were no significant differences between the paced patients and those without TCP for mean times from collapse until cardiopulmonary resuscitation, paramedic arrival and a paceable rhythm, or from paramedic arrival until a paceable rhythm. Overall, emergency department arrival with a palpable pulse (26% in paced vs 13% in nonpaced group; P = 0.20) and survival to hospital discharge (15% in paced vs 0% nonpaced group; P = 0.07) tended to be better for the paced group. No patient without a palpable pulse on paramedic arrival survived to leave the hospital. Of patients with a palpable pulse upon paramedic arrival, survival to hospital discharge was greater for the paced group (80% in paced vs 0% in nonpaced group; P = 0.024). TCP appears to be most beneficial in those patients with bradycardia who have a palpable pulse when first seen. PMID- 1721130 TI - Radiofrequency catheter ablation for Wolff-Parkinson-White syndrome associated with a coronary sinus diverticulum. AB - The case of a patient with Wolff-Parkinson-White syndrome undergoing attempted radiofrequency catheter ablation of a left posterior paraseptal accessory pathway is described. Coronary sinus venography revealed the presence of a large diverticulum attaching near the os. The electrogram recorded from a catheter placed in the narrow neck of the diverticulum revealed a very short atrioventricular time during sinus rhythm. The pathway was easily ablated using radiofrequency energy applied in the neck of the diverticulum, after multiple failed attempts at catheter ablation from the endocardial surface of the mitral annulus. Our report emphasizes the importance of searching for a coronary sinus diverticulum in all patients with posterior accessory pathways undergoing catheter ablation. PMID- 1721131 TI - Association of humps on monophasic action potentials and ST-T alternans in a patient with Romano-Ward syndrome. AB - A case of Romano-Ward syndrome had episodes of torsade de pointes preceded by ST T alternans. ST-T alternans was induced by isoproterenol and abolished by verapamil, lidocaine, mexiletine and MgSO4. A monophasic action potential (MAP) showed humps in MAPs at the right ventricular outflow tract but not at the right ventricular apex in alternate beats. Differences in the MAP duration were noted between the two areas and were associated with ST-T alternans. Atrial pacing abolished both humps and ST-T alternans. These results suggest that humps are a possible reflection of early afterdepolarizations and their appearance is limited to localized regions of the ventricles, which produces regional disparity of repolarization and ST-T alternans. PMID- 1721132 TI - Cardiac electrophysiological experiments in numero, Part I: Concepts and strategies of mathematical and computer models. AB - This article is the first of three articles that review mathematical and computer models of the heart and describe their construction, development, research potential, and clinical utility. This article explains the methodological principles of mathematical and computer simulation of biomedical systems. The strategies of model construction, testing, and application are presented; the advantages and limitations of computer simulation studies are explained, and the basic value of computer simulation for cardiological research and practice is discussed. PMID- 1721133 TI - Hypothesis testing as an approach to the analysis of complex tachycardias--an illustrative case of a preexcitation variant. AB - The correct elucidation of the electrophysiological substrate and mechanism(s) responsible for a complex arrhythmia requires a systematic approach to the analysis of the electrophysiological data. One approach calls for the formulation of a set of hypotheses that could explain the data obtained during the study. The hypotheses are then tested for compatibility with phenomena observed and the one that agrees with the majority of the findings would represent the most tenable explanation. We present the case of a young girl with a wide QRS complex tachycardia and a history of ventricular preexcitation that illustrates this approach. The complexities were resolved only after intraoperative analysis and surgical ablation of a right-sided accessory pathway with decremental properties, and provides further insight into our understanding of the nodoventricular Mahaim fiber. PMID- 1721134 TI - In vivo and in vitro studies of a chronic oxygen saturation sensor. AB - An oxygen saturation sensor, for the purpose of chronically controlling the heart rhythm produced by a pacemaker, should be specific to oxygen saturation and should be minimally affected by the harsh blood environment. For the sensor type we tested we found: (1) one sensor failure in 205.5 canine-months of chronic implantation (n = 11, range 4 to 50 months); (2) hematocrit-induced error of less than 5 percentage points of SvO2 over the range of 50% to 80% SvO2 and 15% to 45% hematocrit; (3) carboxyhemoglobin (HbCO)-induced error of less than 4 percentage points of SvO2 with HbCO up to 20%; (4) a fibrotic sheath-induced error of less than 3 percentage points of SvO2 in the range of 50% to 80% SvO2 due to fibrotic sheath thicknesses up to 0.22 mm; (5) no significant error induced by velocity variations local to the sensor; (6) no significant error due to temperature in the range of 30 degrees to 42 degrees C; and (7) that the sensor could be as close as 0.3mm to the ventricular wall and still only produce an error of 5% SvO2. PMID- 1721135 TI - Tilt table testing for evaluation of neurally-mediated (cardioneurogenic) syncope: rationale and proposed protocols. PMID- 1721136 TI - Retrograde (ventriculoatrial) conduction in congenital complete heart block. AB - Retrograde ventriculoatrial (VA) conduction is documented at the time of dual chamber pacemaker implantation in a 36-year-old patient with congenital complete atrioventricular (AV) block. Programmed ventricular stimulation with stimuli of increasing prematurity demonstrated a lack of decremental conduction via a unidirectional retrograde pathway. Because retrograde VA conduction has been associated with pacemaker mediated endless loop tachycardia, the status of retrograde conduction should be assessed in all patients undergoing dual chamber pacemaker implantation, including those with congenital complete AV block who have previously been considered to have no conductive tissue between atria and ventricles. PMID- 1721137 TI - Congenital myocardial sympathetic dysinnervation (CMSD)--a structural defect of idiopathic long QT syndrome. AB - Concerning the pathogenetic mechanism of idiopathic long QT syndrome (LQTS), the hypothesis of a specific sympathetic imbalance has gained general acceptance, but its validity has never been proven. To test this hypothesis I-123-MIBG, an analogue of norepinephrine and guanethidine, was used to provide scintigraphic display of the efferent cardiac sympathetic innervation. Twelve members of four LQTS families (mean age 38.2 +/- 17.2 years, eight males) and eight healthy volunteers (mean age 48.2 +/- 13.3 years, five males) were studied by means of I 123-MIBG single photon emission computed tomography (SPECT). A quantitative analysis of all scans was performed. All scans of the healthy volunteers show a uniform tracer uptake with sometimes slightly decreased activity in the apex. (1) All patients with QTc greater than 440 msec (n = 5); (2) all, who had suffered from at least one episode of torsade de pointes, ventricular fibrillation (VF) or syncope (n = 5); and (3) all symptomatic patients with QTc prolongation (n = 4) have reduced or abolished (P less than 0.02) MIBG uptakes in the inferior and inferior septal parts of the left ventricle (congenital myocardial sympathetic dysinnervation [CMSD]). Additionally, one female without symptoms or QTc prolongation (LQT) shows an abnormal MIBG SPECT similar to the one of her daughter, who has LQT and symptoms. One male without LQT, who had suffered from VF shows CMSD similar to his father, who has LQT, but no symptoms. All members of the families with normal MIBG SPECTs have neither LQT nor symptoms. In all families CMSD fulfills the criteria of autosomal-dominant inheritance. Normal QTc interval predicted only in 57% normal cardiac sympathetic innervation in the present LQTS families. Therefore, quantitative I-123-MIBG SPECT enables to identify myocardial sympathetic dysinnervation as structural defect in LQTS. CMSD is associated with and without LQT and presents a pattern of autosomal-dominant inheritance. LQT at rest or during exercise was specific (100%), but less sensitive (63%) in the assessment of CMSD than I-123-MIBG SPECT. PMID- 1721138 TI - The initial clinical experience with a third generation cardioverter defibrillator/antitachycardia pacemaker. PMID- 1721139 TI - Defibrillator "twiddler's" syndrome. PMID- 1721140 TI - Performance of implantable cardiac rhythm management devices. PMID- 1721141 TI - Acquisition of hemodynamic data and sensor signals for rate control from standard pacing electrodes. PMID- 1721142 TI - Cardiac pacing in unilateral left superior vena cava: evaluation by digital angiography. AB - In a patient with complete heart block and chronic lymphocytic leukemia a pacemaker lead could not be introduced from either the right or left subclavian vein. Digital subtraction angiography excluded a neoplastic mediastinal mass, demonstrated a unilateral left superior vena cava and defined the best route for lead insertion. PMID- 1721143 TI - Propafenone-induced torsade de pointes: cross-reactivity with quinidine. AB - A 77-year-old female with new onset atrial fibrillation occurring in the absence of structural heart disease developed torsade de pointes during therapy with quinidine bisulfate 500 mg orally every 8 hours. Ten days after quinidine therapy had been discontinued she developed torsade de pointes while receiving propafenone 300 mg orally every 8 hours. This case demonstrates that propafenone may be associated with torsade de pointes and suggests a cross-reactivity between this effect and prior occurrence of torsade de pointes on Class IA antiarrhythmic drug therapy. PMID- 1721144 TI - Effects of transcatheter cardioversion on chronic lone atrial fibrillation. AB - The effectiveness and safety of internal transcatheter cardioversion on chronic lone atrial fibrillation were examined in ten patients resistant to external electrical (400 joules) and pharmacological cardioversion. Transcatheter cardioversion was performed by pulling back the atrioventricular junction catheter just inferior to the site of the His-bundle recording and delivering the shock between a proximal electrode (cathode) and backplate (anode). Transcatheter cardioversion restored sinus rhythm in all of the ten patients. The only complication observed was transient atrioventricular block after the shock and this was treated by temporary pacing. However, atrial fibrillation recurred in five patients at 30, 27, 52, 1, and 6 days, respectively. A second attempt at transcatheter cardioversion was performed in those patients an was successful in three patients. During a follow-up period ranging from 12 to 22 months, eight patients continued in sinus rhythm. Thus, transcatheter cardioversion is considered effective and safe in selected patients with chronic lone atrial fibrillation in whom external cardioversion was unsuccessful. PMID- 1721145 TI - A method for analysis of the local atrial evoked response for determination of atrial capture in permanent pacing systems. AB - We developed a method for detection and analysis of the local atrial evoked response. Eleven patients undergoing permanent dual chamber pacemaker implantations for standard clinical indications were included in the study. Using a pacing system emulator, charge balancing using a variable triphasic stimulus waveform to reduce polarization artifact amplitude was performed first. This could not be completed in one patient because of a poor signal-to-noise ratio. Subsequent analysis of the local atrial evoked response in the remaining ten patients showed the typical signal to be a biphasic waveform with an initial negative deflection followed by a positive deflection nearly equal in amplitude. The mean amplitude of the atrial evoked response was 3.1 +/- 1.4 mV, while the intrinsic P wave amplitude in these same patients averaged 5.6 +/- 3.0 mV. The summed evoked response, a parameter that is directly proportional to the area of the signal, was used to determine atrial pacing threshold. The median atrial pacing threshold determined by the algorithm was 1.00 V. There was no instance of failure to detect loss of capture, nor was loss of capture inaccurately determined when there was still successful atrial pacing. Atrial capture in permanent pacing systems can thus be determined using an algorithm to record and analyze the local atrial evoked response. This method could potentially be useful in the automatic determination of atrial pacing threshold. PMID- 1721146 TI - Aortic leaflet perforation during radiofrequency ablation. AB - A 15-year-old girl underwent successful radiofrequency ablation of an accessory pathway. Following ablation, a new III/VI diastolic murmur was noted. Echocardiography revealed a perforated aortic leaflet, with a small amount of adherent valvular tissue and trivial aortic insufficiency by color Doppler. The patient remains asymptomatic. We are not aware of any similar complication from electrophysiological study, catheter ablation, coronary angiography, or percutaneous transluminal coronary angioplasty. We speculate that the current state of catheter technology contributed significantly to this complication. This case illustrates the need for using care in crossing the valve, continued advances in catheter technology to reduce the incidence of complications, and careful physical examination prior to and following attempts at ablation. PMID- 1721147 TI - Effect of antiarrhythmic drug therapy on the incidence of shocks in patients who receive an implantable cardioverter defibrillator after a single episode of sustained ventricular tachycardia/fibrillation. AB - Seventy-four patients (16 women, 58 men, age 58 +/- 11 years, mean +/- standard deviation) who received an implantable cardioverter defibrillator (ICD) after experiencing a single episode of ventricular tachycardia or ventricular fibrillation were followed to determine if antiarrhythmic drug therapy affects the incidence of ICD discharges. Thirty-three patients (group A) were treated with an antiarrhythmic drug that was either untested or previously demonstrated during electropharmacological testing to be ineffective in suppressing the induction of ventricular tachycardia. Forty-one patients (group B) were not treated with an antiarrhythmic drug. There were no significant differences between the two groups in regards to age, sex, incidence of coronary artery disease, left ventricular function or the type of ICD pulse generator used. During a mean follow-up of 14 months for the entire cohort, 15 patients (46%) in group A and 18 patients (44%) in group B experienced at least one ICD shock. The time to the first appropriate shock (5 +/- 5 months in both groups) and the frequency of ICD shocks (0.3 +/- 0.2/month in group A vs 0.4 +/- 0.5/month in group B) were similar in both groups. The incidence of syncope at the time of ICD discharge was higher in group A than group B patients (31% vs 5%, P less than 0.05). In conclusion, antiarrhythmic drugs that are untested or have failed electropharmacological testing do not appear to reduce the probability of ICD discharge over a short-term (mean 14 months) follow-up in patients who have had only one clinical episode of VT/VF and may increase the risk of syncope during ICD discharge.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721148 TI - Successful catheter fulguration of the His bundle in a postoperative Mustard patient after unsuccessful fulguration of ectopic atrial foci. AB - Incessant supraventricular tachycardia (SVT) resistant to pharmacological agents may cause cardiac dysfunction requiring more aggressive therapy. We present the case of a 12-year-old postoperative Mustard patient who developed biventricular heart failure due to an atrial ectopic tachycardia resistant to amiodarone. Using endocavitary direct current fulguration techniques, catheter ablation of the His bundle was successfully performed after unsuccessful attempts at ablation of the ectopic atrial foci. After placement of a permanent transvenous rate responsive ventricular pacemaker, the patient's clinical status and cardiac function improved. Endocavitary fulguration of the His bundle is technically feasible after the Mustard procedure and should be considered for treatment of selective cases of pharmacologically resistant SVT. PMID- 1721149 TI - One-year follow-up of automatic adaptation of the rate response algorithm of the QT sensing, rate adaptive pacemaker. AB - Optimal functioning of a rate adaptive pacemaker depends upon reliable sensing of the sensor and appropriate programming of the rate of response algorithm. QT sensing pacemakers use data derived from the endocardial electrogram in the programming of the rate response algorithm. In the latest versions of these pacemakers, programming of the rate response algorithm may be performed using either a semiautomatic Fast Learn (FL) procedure or by using the newly developed, fully Automatic Slope Adaptation (ASA) mechanism. We report our experience in a prospective study of 17 patients in the first year postimplantation. ASA was characterized by significant changes only in the values of the slope settings at the lower rate limit (3.7 msec/msec at time 0 to 5.77 msec/msec at 2 weeks, P less than 0.001) during the first 2 weeks after its enablement. Further adaptation between weeks 2 to 4 was observed (5.77 msec/msec to 6.4 msec/msec, P = 0.2) but this was not significant. The slope settings derived using the FL procedure were also checked at 2 and 4 weeks and were reproducible. They were closest in value to the values attained by the automated mechanism at 4 weeks. This suggests that the final value of the slope setting at the lower rate limit using ASA is reached between weeks 2 to 4. Both methods of slope determination result in satisfactory and similar rate response profiles but the time to achieve slope stability will necessarily be slower with ASA. PMID- 1721150 TI - Entrainment of ventricular tachycardia in arrhythmogenic right ventricular tachycardia. AB - In two patients with arrhythmogenic right ventricular dysplasia (ARVD), sustained ventricular tachycardia (VT) was induced by programmed stimulations during serial drug testings. One patient had five and the other had two VT morphologies, and the sites of origin were determined by endocardial catheter mappings. When overdrive pacing was performed, constant fusion in the QRS complex was observed in the two patients. Constant fusion of a different degree was also observed at different paced cycle lengths. Both patients had dilated right ventricles and wall-motion abnormality, and the diagnosis of ARVD was further confirmed by the specimen resected at the site of origin of VT. Therefore, VT in ARVD can be entrained and reentry is the most likely mechanism of such VT. PMID- 1721151 TI - The epidemiology of pacemaker implantations in Fyn county, Denmark. AB - Throughout the last three decades the number of patients with pacemakers has increased. Registers of pacemakers and pacemaker patients have been established to monitor, for example, the battery life expectancy and patient data. We have analyzed the registry for Fyn county, Denmark, which includes data on incidence, prevalence, and mortality rates among patients with pacemakers during the period from 1964 to 1990. The analyses show that the prevalence rate is steadily increasing as a result of a combination of an increasing incidence rate and a stable mortality rate among the pacemaker patients. Based on the development in the first almost 30 years of pacemaker implantations, two trends of the future prevalence of pacemaker patients are projected. Provided constant mortality rates and expected incidence rates of 300 per million person years and 350 per million person years, respectively, the prevalence will reach a plateau of 2.7 per 1,000 persons and 3.1 per 1,000 persons, respectively, within the next 25 years. PMID- 1721152 TI - Patterns of atrioventricular conduction during postexercise recovery in patients with atrial fibrillation and Wolff-Parkinson-White syndrome. AB - The effects of the postexercise recovery phase on the functional anterograde conduction properties of the accessory pathway (AP) were evaluated. Twenty-nine patients with Wolff-Parkinson-White (WPW) syndrome were submitted to supine maximal bicycle exercise testing. In seven patients (group I), in whom sustained atrial fibrillation (AF) could be induced by transesophageal pacing (TP), mean ventricular rate (MVR), the shortest R-R interval (SRR) between preexcited beats, and the observed percentage of preexcited beats were evaluated at rest, after each step of exercise and 2 minutes after the end of exercise. In 22 patients (group II), in whom sustained AF could not be induced, decremental TP was performed to evaluate the shortest atrial cycle length (SCL) with 1:1 conduction over AP at rest, after each step of exercise, and 2 minutes after the end of exercise. In four patients in group I, the protocol was repeated with atropine injected during the last minute of exercise. In 12 patients (three from group I and nine from group II), catecholamine plasma levels were measured at rest, at peak exercise, and during recovery. MVR was 144 +/- 20 beats/min at rest, 186 +/- 21 beats/min at peak exercise (P less than 0.001 vs rest), and 179 +/- 21 beats/min during recovery (P less than 0.001 vs rest; P less than 0.05 vs peak exercise). SRR was 289 +/- 73 msec at rest, 223 +/- 25 msec at peak exercise (P less than 0.05 vs rest), and 227 +/- 29 msec during recovery.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721153 TI - DDIR versus VVIR pacing in patients with paroxysmal atrial tachyarrhythmias. AB - Patients with sinus node dysfunction (SND) in particular those with tachycardia bradycardia syndrome and patients undergoing atrioventricular nodal ablation procedures for refractory paroxysmal atrial tachyarrhythmias (PAT), are candidates for single chamber (VVIR mode) or dual chamber rate responsive (DDIR mode) systems. To evaluate the benefits and disadvantages of each pacing mode we retrospectively analyzed 33 patients with a history of frequent PAT who received a VVIR (22 patients); or a DDDR pacemaker (11 patients) programmed to the DDIR mode. The mean follow-up time was 25 and 18 months, respectively. Preimplant left atrial diameter was significantly smaller in the DDIR group. Chronic atrial fibrillation developed in 54% of the VVIR patients and 27% of the DDIR group, but this difference was not significant. Complications of patients with VVIR pacemakers included new mitral and tricuspid insufficiency, stroke, pacemaker intolerance and aggravated congestive heart failure. Patients with DDIR pacemakers had a lower incidence of symptoms and complications. However, this group received more antiarrhythmic medication, required a closer follow-up, and their pacemakers needed frequent reprogramming. Our findings suggest that VVIR is a poor choice for patients with SND, congestive heart failure, and PAT, and that DDIR may be an acceptable alternative. PMID- 1721154 TI - Bidirectional bundle branch reentry tachycardia associated with Ebstein's anomaly: cured by extensive cryoablation of the right bundle branch. AB - A 30-year-old woman with Ebstein's anomaly presented with a sustained, wide QRS complex tachycardia exhibiting a left bundle branch block morphology. Serial electrophysiological studies revealed right and left bundle branch reentry tachycardias refractory to many conventional antiarrhythmic drugs. Radiofrequency and direct current catheter ablation of the right bundle branch failed to control the tachycardias. The patient subsequently underwent extensive endocardial cryoablation to the right bundle branch resulting in cure of her arrhythmia. PMID- 1721155 TI - Cardiac electrophysiological experiments in numero, Part II: Models of electrophysiological processes. AB - This article is the second part of a three article series reviewing computer simulation models of the heart, in particular of cardiac electrophysiology. The previous section of the review discussed the methodological principles of the construction and application of computer models. This article overviews the development of mathematical and computer modeling studies applied to cardiology. The models are classified according to the physiological processes that were simulated; this article distinguishes models oriented to cardiac mechanics, hemodynamics, and electrophysiology. The electrophysiology models are discussed in more detail and the review classifies them into four main categories: models of cellular processes, models of tissue behavior, models of the ventricular electric field, and models of macroconduction disturbances. In each category, the historical development of the models and their key achievements are described. PMID- 1721156 TI - Adenosine: cardiac electrophysiology. PMID- 1721157 TI - Undersensing as a consequence of lead incompatibility: case report and a plea for universality. AB - Implantation of a single chamber bipolar pulse generator was complicated by transient loss of ventricular sensing, caused by mechanical damage to the unipolar lead connected to the system. A perforation of the insulating sheath was found at the site of the proximal connector block, and undersensing resolved after restoring its integrity with silicone adhesive. The ability to attach an unprotected unipolar lead to a bipolar connector, shared by the Voluntary (VS-1) and International (IS-1) designs, invites the possibility of injury to the insulating sheath by accidental tightening of the proximal screw. There is thus an urgent need for the development and universal adoption of a robust interface standard in lead connector design. PMID- 1721158 TI - Catheter ablation of ventriculoatrial conduction in the treatment of pacemaker mediated tachycardia. PMID- 1721159 TI - The role of diastolic outward current deactivation kinetics on the induction of spiral waves. AB - The mechanism of induced reentry in an initially homogeneous repolarization matrix still remains undefined. In the present study we hypothesized that the slow deactivation rate of the delayed outward current (dIo/dt), which occurs during diastole after complete repolarization, can cause activation failure and facilitate reentry. We modeled the excitation-recovery process using the modified FitzHugh-Nagumo equations in a two-dimensional medium of 128 by 128 cells using the Connection Machine (CM-2), a massively parallel computer that is highly suitable for this class of problem. The model was one cell thick, uniformly excitable, and isotropic. When the rate of Io deactivation was slowed to yield action potential duration (APD) restitution curves similar to experimentally observed arrhythmic ventricular muscle cells ADP restitution curves, premature stimulation (S2) induced nonstationary double spiral waves (Figure 8 reentry). A decrease in dIo/dt increased the radius of the circle around which the tip of the spiral waves rotates and decreased its angular velocity. Wave fronts propagated through areas where the residual diastolic Io was fully inactivated and blocked in areas where its amplitude was high. No such dynamics of wave front propagation could be induced when S2 was applied after the completion of Io deactivation. We conclude that the kinetics of deactivation of the Io during diastole has a profound influence on the dynamics of two-dimensional wave front propagation. The similarities of the APD restitution curve implemented in the computer model with slow deactivation of Io and that observed in our canine model of quinidine induced ventricular tachyarrhythmias suggest that Io deactivation kinetics may play an important role in arrhythmogenesis in the intact ventricle. PMID- 1721160 TI - A massively parallel computer model of propagation through a two-dimensional cardiac syncytium. AB - A computer model of electrical propagation through a two-dimensional (2D) sheet of cardiac tissue has been developed to run on the massively parallel processor Connection Machine (CM-2) computer. The transmembrane ionic currents in each of 16,384 (128 x 128) 100 x 100 microns 2 patches of cardiac tissue are described by modified Beeler-Reuter membrane equations. These equations, along with the parabolic differential equation derived from 2D cable theory, are solved in parallel to study normal and abnormal 2D propagation. The sheet is paced with planar waves at a basic cycle length of 500 msec (control). When a premature ectopic stimulus of sufficient strength and appropriate timing is then applied to a local region of the syncytium, one of two types of reentry is observed: (a) stable figure-of-eight reentry, or (b) unstable but self-sustaining "fibrillation like" reentry. During this fibrillatory activity, action potential durations are 79.8 +/- 36.8 msec (control = 244.9 +/- 0.9 msec) and coupling intervals average 96.7 +/- 31.3 msec (control = 500 +/- 0 msec). We also observed that passive electrotonically-induced depolarization of already refractory tissue extended the refractory period of that tissue, and that the duration of this extension depended on the magnitude of the electrotonic effect. PMID- 1721161 TI - Termination of reentrant propagation by a single stimulus: a model study. AB - A computer model of a ring-shaped one-dimensional cardiac fiber was used to examine responses of reentrant propagation to premature stimuli applied under different degrees of head-tail interaction. Two different types (type I and type II) of termination window (TW) were identified. The type I TW was generated by functional inhomogeneity created by reentrant propagation. The width of the type I TW was proportional to the degree of cellular uncoupling. In contrast, uniform reduction in sodium channel conductance decreased the width of type I TW. The type II TW was generated by electrical alternans created by the head-tail interaction of the reentrant action potential. It was demonstrated that electrical alternans were most significant in medium degree head-tail interaction. For stronger or weaker head-tail interaction, the electrical alternans tended to decrease. The type II TW was located in excitable gaps following reentrant action potentials of short duration. Its size was proportional to the degree of electrical alternans. The type II TW was usually much larger than the type I TW. A premature conditioning stimulus induced alternans and created a type II TW. This response implies that a conditioning stimulus could facilitate greatly the termination of clinical reentrant arrhythmias by programmed electrical stimulation. PMID- 1721162 TI - Effects of procainamide on refractoriness, conduction, and excitable gap in canine atrial reentrant tachycardia. AB - The effects of procainamide were studied in a model of atrial flutter around the tricuspid valve in seven open chest, chloralose-anesthetized dogs (31 +/- 3 kg). A Y-shaped incision in the intercaval area extending to the right atrial appendage was made and five bipolar electrodes were sutured on the atrial epicardium around the tricuspid valve. Reentry tachycardia was induced in the absence and presence of drug by burst pacing. Procainamide (15 mg/kg bolus followed by 0.075 mg/kg/min infusion) produced stable plasma levels (38 +/- 9 microM) during the study. At a pacing cycle length of 200 msec, mean (+/- SD) diastolic threshold at the five sites increased from 1.6 +/- 1.5 to 2.0 +/- 1.7 mA and mean atrial effective refractory period from 125 +/- 9 to 140 +/- 16 msec on drug (P less than 0.05). Procainamide prolonged the cycle length of atrial flutter from 144 +/- 10 to 160 +/- 13 msec and slowed conduction velocity during atrial flutter around the tricuspid valve from 73 +/- 6 to 66 +/- 6 cm/sec (P less than 0.05). A reset response curve was determined by introducing premature stimuli during atrial flutter. Procainamide prolonged effective refractory period during atrial flutter from 101 +/- 13 to 116 +/- 17 msec but did not change the duration of the excitable gap (38 +/- 9 vs 40 +/- 18 msec). Although the reset response curve was predominantly increasing, in six of seven experiments there was present a flat portion at long coupling intervals approaching the atrial flutter cycle length that comprised 23% +/- 10% of the excitable gap.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721163 TI - Afterdepolarizations and triggered activity develop in a select population of cells (M cells) in canine ventricular myocardium: the effects of acetylstrophanthidin and Bay K 8644. AB - Early afterdepolarizations (EADs) are membrane oscillations that interrupt or retard the repolarization phase of the cardiac action potential, whereas delayed afterdepolarizations (DADs) are oscillations that arise after full repolarization. When EADs and DADs are sufficiently large to depolarize the cell membrane to its voltage threshold, they give rise to triggered action potentials, which are believed to underlie some forms of extrasystolic activity and tachyarrhythmias. EAD- and DAD-induced triggered activity have been described and well characterized in isolated Purkinje fibers exposed to a wide variety of drugs, but are rarely seen in syncytial preparations of ventricular myocardium. These results are inconsistent with those of in vivo studies or experiments involving enzymatically dissociated myocytes. In the present study, we used the cardiotonic agent acetylstrophanthidin (AcS) and the calcium channel agonist Bay K 8644 to provide evidence in support of the hypothesis that induction of prominent EADs, DADs, and triggered activity occurs in a select population of cells in ventricular myocardium. The data indicate that EADs, DADs, and triggered activity produced by digitalis and Bay K 8644 are limited to or more readily induced in the deep subepicardial cell layers of the canine ventricle (M cells). Afterdepolarization-induced triggered activity was never observed in the epicardial or endocardial layers. PMID- 1721164 TI - Atrial membranes contain nucleoside diphosphate kinase (NDPK) activity: its role in regulation of muscarinic K+ channels. AB - Guinea pig or rabbit atrial muscarinic K+ channels in cell-free inside-out patches can be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg2+. This ATP-dependent activation is compatible with the existence of a membrane-bound nucleoside diphosphate kinase (NDPK), which directly phosphorylates GK-bound GDP. We show that this ATP dependent activation is also possible in frog atrial cells, and that atrial membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphosphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP dependent activation of muscarinic K+ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction. PMID- 1721165 TI - Proarrhythmic and antiarrhythmic effects of flecainide on nonsustained reentry around the canine atrial tricuspid ring in vitro. AB - The effect of flecainide, 0.3 mg/L and 1.0 mg/L, on inducible nonsustained reentry was studied, in vitro, in the canine tricuspid ring. Nonsustained reentry was engineered by cutting the ring and reconnecting it with an adjustable electronic delay. Delays were used that produced reentry lasting 1-3 beats (group A), 4-10 beats (group B), and 11-25 beats (group C). Reentry was initiated multiple times at each selected delay. A proarrhythmic effect, defined as a significant increase in the duration of reentry, was observed in all 14 trials at the low dose and in two of 15 trials at the high dose in seven experiments. In four more trials a transient proarrhythmic response was seen initially during exposure to the high dose. In five of seven experiments, reentry became sustained after at least one dose of flecainide. Proarrhythmic responses resulted when flecainide increased the tachycardia cycle length more than the effective refractory period and there was less cycle length oscillation after initiation. Antiarrhythmic responses resulted either from a marked increase in effective refractory period at the site of block or production of fixed block. PMID- 1721166 TI - Triggered activity as a possible mechanism for arrhythmias in ventricular hypertrophy. AB - To study the cellular mechanisms of arrhythmias occurring in cardiac hypertrophy, we performed standard microelectrode studies on papillary muscles isolated from control (group N) and hypertrophied ferrets right ventricles. Different stages of hypertrophy, induced by pulmonary banding, were studied: 10-22 days (group H1), 4 6 weeks (H2), and 5 1/2-6 months (H3). During the development of hypertrophy, under beta-adrenergic stimulation, triggered activity (TA) induced by delayed afterdepolarizations appeared in 2 of 5 muscles in group H1 and 8 of 8 in group H2. This arrhythmia was absent in N muscles, as well as in H3, despite a pronounced prolongation of the action potentials at 50% (100 +/- 9.3 msec in group H3 vs 67 +/- 5.7 msec in H2; P less than 0.01) and 90% of repolarization (225 +/- 8.7 in H3 vs 185 +/- 7.4 msec in H2; P less than 0.02). The presence of TA was associated with an increase in the intracellular calcium activity (144 +/- 60 nM in H2 vs 47 +/- 9 nM in N; P less than 0.05). TA properties were as follows. Triggering frequency increased as beta-adrenergic stimulation increased, as pacing cycle length (PCL) decreased, and as duration of the prestimulative pause increased. The duration of salvos of TA increased as duration of the prestimulative pauses increased (NS). The coupling interval of the first triggered beat decreased as PCL decreased (P less than 0.001). The minimal cycle length of salvos of TA was not modified by these parameters. It is concluded that delayed afterdepolarizations-induced TA may occur under beta-adrenergic stimulation during the first stages of ventricular hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721167 TI - The natural history of dual chamber pacing. PMID- 1721168 TI - The mythology of threshold variations as a function of electrode surface area. AB - It has been established that the chronic thresholds of cardiac pacing leads vary as a function of the (spherical) electrode's radius or (geometric) surface area and the thickness of fibrotic encapsulation. Where the radius of the electrode is equal to the thickness of the fibrous capsule (about 0.7 to 1 mm for polished surfaces), threshold should be at a minimum. Where the radius of the electrode is larger or smaller than the thickness of the fibrous capsule, then thresholds should increase since the electric field strength required to stimulate decreases as the square of the distance between the electrode's surface and stimulatable tissue. In addition, it has become (incorrectly) accepted that small electrodes do not sense well. About 8-mm electrodes, therefore, became the "standard" surface area, providing the best tradeoffs between pacing and sensing. Analysis of 18 years of canine data in our laboratory, however, suggest that these relationships may be overemphasized for the surface areas of clinical interest. In fact, new small porous and steroid-eluting electrodes do not have high thresholds, are efficient, and their sensing is excellent. PMID- 1721169 TI - Nonphysiological left heart AV intervals as a result of DDD and AAI "physiological" pacing. AB - DDD and AAI pacemakers are considered physiological, since they preserve atrioventricular (AV) synchrony. Artificial pacing, however, is performed largely from right heart chambers, causing aberrant depolarization pathways. Pacing at the right atrial appendage (RAP) is known to delay left atrial contraction due to interatrial conduction time (IACT), and right ventricular (RV) apical pacing (RVP) delays left ventricular (LV) contraction due to interventricular conduction time (IVCT). These delays may render the left heart AV intervals (LAV) either too short or too long, thus affecting LV systolic function. The purpose of this study was to evaluate the actual LAV intervals during conventional, right heart AAI and DDD pacing. Resulting LAV intervals were compared to programmed AV values during all DDD pacing modalities. Ten patients with DDD and six patients with AAI pacemakers were studied. IACT was measured from the atrial spike to the onset of left P wave, as recorded by an esophageal lead. Systolic time intervals were measured using either a carotid pulse tracing or a densitogram (photoplethysmography). LV function was appraised by measuring rate-corrected LV ejection time (LVETc). IVCT was measured indirectly as the lengthening of LV preejection period (PEP) caused by RV pacing, as compared to normal depolarization pathway. Intrinsic IACT and IVCT were considered zero. Right heart AV intervals (RAV) were measured from surface ECG and LAVs were calculated according to the following equations: Sinus Rhythm: LAV = RAV; Atrial Pace + Ventricular Sense: LAV = RAV - IACT; Atrial Sense + Ventricular Pace: LAV = RAV + IVCT; Sequential AV Pace: LAV = RAV - IACT + IVCT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721170 TI - Applicability of the stimulus-T interval for antitachycardia pacing. AB - At the onset of tachycardia, the refractory period (RP) changes together with the tachycardia termination window. We evaluated dogs with total atrioventricular (AV) block to determine if stimulus-T interval (STI) can be used to adjust the coupling interval(s) of an antitachycardia pacemaker in relation to changes in RP. Endocardial STI was recorded continuously together with six surface ECG leads. Steady-state (greater than 2 min) RP was determined for drive cycle lengths (DCL) 400 msec and 900 msec. The test pulse (TP) coupling interval, with DCL 900 msec, was chosen to be equal to the RP of DCL 400 msec. DCL was then changed to 400 msec until TP captured. STI of DCL of beat before capture was gained was measured. DCL was then changed back to 900 msec and the interval determined when capture was lost. TP was then lengthened by 5 msec and the procedure repeated until TP captured immediately upon changing to DCL 400 msec. RESULTS: The difference between RP at onset of pacing at DCL of 400 msec and RP when capture was achieved with the shortest coupling interval was 35-50 (mean 40) msec. This required 35-90 (mean 62) seconds. The correlation coefficient RP to STI was greater than 0.95. CONCLUSIONS: (1) RP changed by as much as 35-50 msec at the onset of an abrupt increase in rate in a 35-90-second period; and (2) STI enables estimation of RP on a beat-to-beat basis. Capture can therefore be predicted from the previous beat and the coupling interval adjusted accordingly in an antitachycardia pacing mode. PMID- 1721171 TI - Comparison of two antitachycardia pacing modes in supraventricular tachycardia. AB - The comparative efficacy of two different antitachycardia pacing techniques was evaluated in 22 consecutive patients who received the pacemaker Intertach with an atrial electrode for drug refractory, recurrent supraventricular tachycardia (SVT). The Intertach has two consecutive programmable primary and secondary termination modes. The termination programs investigated were adaptive autodecremental burst pacing and adaptive decremental scanning. Atrioventricular nodal reentrant tachycardia was present in 15 patients and atrioventricular reentrant tachycardia due to Wolff-Parkinson-White syndrome in seven patients. The prospective comparison was arranged in a randomized, cross-over study over a period of 12 months. To assess long-term efficacy, diagnostic data of the pacemakers were obtained in intervals of 3 months. In addition, noninvasive programmed stimulation was performed to compare the incidence of pacing-induced atrial fibrillation with both termination programs. During a follow-up of 12 months the overall success rate of autodecremental burst pacing and decremental scanning was 80% and 95%, respectively. Decremental scanning was more effective in 12 patients and less successful in two patients than autodecremental burst pacing. During noninvasive electrophysiological studies, pacing induced atrial fibrillation could be documented in three of ten patients (30%) using autodecremental burst pacing, compared to one of ten patients (10%) using decremental scanning. These data suggest that decremental scanning proved to be more successful in the long-term management of patients with recurrent SVT than autodecremental burst pacing. Furthermore, the occurrence of pacing-induced atrial fibrillation could be documented more frequently with autodecremental burst pacing compared to decremental scanning. PMID- 1721172 TI - Active fixation leads--long-term threshold reduction using a drug-infused ceramic collar. AB - Previous investigators, including our group, have reported the threshold reduction benefits of steroid-releasing leads. To date, all published literature has been for the passive fixation versions. The application of steroids should also enhance the performance of active fixation leads. We have developed and tested an atrial and a ventricular Accufix lead with a dexamethasone acetate releasing, porous ceramic collar (DA DEC). A long-term sheep study has shown a significant reduction in thresholds (THR) when compared to standard Accufix leads without the collar (ACC) for atrial (ATR) and ventricular (VENT) versions (bipolar THR [0.5 msec] at 24 weeks: VENT DA DEC = 0.51 +/- 0.07, VENT ACC = 1.49 +/- 1.03; ATR DA DEC = 1.31 +/- 1.14, ATR ACC = 2.99 +/- 1.31). All other parameters tested, including pacing and sensing impedance as well as polarization overpotential, were similar for the two groups. The Accufix DEC leads therefore have excellent potential for low energy stimulation. PMID- 1721173 TI - Standardized informal exercise testing for programming rate adaptive pacemakers. AB - It is essential that patients with pacemakers capable of rate modulation undergo some form of exercise testing to assure appropriate rate modulation. Informal exercise testing is a reasonable and less expensive alternative to formal treadmill testing. Empiric adjustment of the rate response parameters by assessing the patient's rate response while walking at a self-determined casual and brisk pace has been used. However, no normals exist to determine the appropriate rate response for a "casual" and "brisk" walk. Volunteers were tested with metronome-guided casual and brisk walks in an effort to standardize the informal exercise and determine expected heart rate response for these levels of activity. Results of the metronome-guided rate response in normal volunteers may be useful in determining the appropriate rate response for pacemaker patients when tested in such an informal manner. PMID- 1721174 TI - Determinants of pace-terminable ventricular tachycardia: implications for implantable antitachycardia devices. AB - The next generation of implantable antitachycardia devices incorporate antitachycardia pacing for the treatment of ventricular tachycardia. To evaluate the potential determinants of pace terminability, we analyzed 62 episodes of induced monomorphic ventricular tachycardia. We found that the tachycardia cycle length and cycle length variability are the major determinants of pace terminability. These findings should be considered in the designing of ventricular tachycardia detection and termination algorithms. PMID- 1721175 TI - What's the price to be paid for rate response: AV sequential versus ventricular pacing? AB - The purpose of this study was to compare the effects of atrioventricular (AV) sequential and ventricular pacing at rest and during exercise on parameters of left ventricular performance. Twenty-five patients were studied by means of first pass radionuclide angiography. Pacing rates increased significantly (P less than 0.001) during exercise in both pacing modes, resulting in a significant increase in the cardiac index (P less than 0.001). Pulmonary transit times decreased significantly (P less than 0.001) during exercise in both pacing modes with a significantly shorter pulmonary transit time for AV sequential pacing at rest (P less than 0.01) and during exercise (P less than 0.05), indicating impaired left ventricular function in ventricular pacing. Regional left ventricular wall movement deteriorated significantly during exercise in both pacing modes (P less than 0.02), with a significantly worse performance during ventricular pacing at rest (P less than 0.05) and during exercise (P less than 0.05). Therefore, the price to be paid for rate response is a deterioration of regional wall movement. An additional loss of AV synchrony worsens the situation. It is concluded that rate modulated pacing requires preservation of AV coordination to optimize left ventricular performance. PMID- 1721176 TI - Intrinsic conduction maximizes cardiopulmonary performance in patients with dual chamber pacemakers. AB - Dual chamber pacemaker programmability allows the possibility of atrially-tracked ventricular pacing in patients who would otherwise have intrinsic atrioventricular (AV) conduction. Thirteen patients with permanent AV sequential pacemakers (ages 50-79) were evaluated with paired exercise tests to determine the cardiopulmonary effects of pacemaker induced right ventricular activation compared with normal AV and intraventricular conduction. Peak oxygen uptake (VO2), oxygen pulse (O2P), respiratory rate (RR), and respiratory exchange ratio (RER) were determined using breath-by-breath analysis of expired gases. Patients exercised to fatigue and exercise tests were performed in random sequence. For patients with intrinsic AV conduction (group I, n = 8) the AV delay was programmed to preserve intrinsic conduction during one study; the alternate test used AV delay programming to produce ventricular pacing. Five patients with chronic AV block (group II) acted as a control for the effects of a rate adaptive AV delay compared to a fixed AV delay. Paired t-testing showed a significantly lower peak VO2 (P less than 0.015) and O2P (P less than 0.01) in patients with atrially-tracked ventricular pacing compared to intrinsic conduction. In contrast, group II showed a significant improvement in peak VO2 with rate adaptive AV delay compared to fixed AV delay programming (P less than 0.05). In conclusion, intrinsic conduction should be preserved in patients with dual chamber pacemakers whenever possible. PMID- 1721177 TI - Long-term pacing in heart transplant recipients is usually unnecessary. AB - The indications for and timing of permanent pacing were reviewed in all 17 of 154 adult heart transplant recipients at this center who have had permanent pacemakers implanted. Resting 12-lead ECGs recorded during routine follow-up were examined. A prospective study of pacing requirement was then undertaken. Holter monitoring was performed before and after reprogramming the pacemakers to VVI mode at 50 beats/min. Exercise responses in various pacing modes were then assessed in seven patients with rate responsive pacemakers using a standard Bruce protocol treadmill test. The indication for pacing was sinus node dysfunction in 59% (10/17) and atrioventricular (AV) block in 41% (7/17). The majority of pacemakers were implanted between seven and 21 days after transplantation. There was a progressive reduction in the frequency of pacing on 12-lead ECGs with time after transplantation. There was a progressive reduction in the frequency of pacing on 12-lead ECGs with time after transplantation. Eight of 14 patients with empirically selected programming paced during Holter monitoring. After reprogramming to 50 beats/min VVI mode only three of 14 patients, all with sinus node dysfunction, paced. Rate responsive pacing made no difference to exercise time. The requirement for long-term pacing in cardiac transplant recipients is small (3/154) and is limited to patients with sinus node dysfunction. Rate responsive pacing did not increase exercise tolerance. PMID- 1721178 TI - Relationship between diastolic mitral regurgitation and PQ intervals or cardiac function in patients implanted with DDD pacemakers. AB - Diastolic mitral regurgitation (MR) is known to be induced by prolonging atrioventricular (AV) delay in patients implanted with a DDD pacemaker. We studied the relationship between diastolic MR and PQ intervals on cardiac function in 50 patients (71.3 +/- 11.3 years old: mean +/- SD), who had been implanted with DDD pacemakers. In 19 patients, prior to pacemaker implantation, pulmonary capillary wedge pressure (PCWP) was measured using a Swan-Ganz catheter during AV sequential pacing with an AV delay of 0.165 seconds. Transmitral blood flow was measured with pulsed Doppler echocardiography, while prolonging AV delay stepwise by 0.025 seconds from 0.065 seconds for about 5 minutes each. In nine patients, AV delay could not be prolonged enough due to occurrence of intrinsic AV conduction. In the other 41 patients, diastolic MR was induced by prolonging AV delay. The critical PQ intervals that induced diastolic MR ranged from 0.14 to 0.26 (0.23 +/- 0.03) seconds. Four of five patients whose critical PQ intervals were 0.20 seconds or shorter had heart failure, while 36 patients whose critical PQ intervals were greater than 0.20 seconds were free from signs and symptoms of heart failure. Their PCWPs were 2-27 (7.5 +/- 5.1) mmHg. There was a significant negative correlation between the critical PQ intervals for the appearance of diastolic MR and PCWP during AV sequential pacing, which was performed prior to pacemaker implantation (r = -0.85, P less than 0.001). It is suggested that the appearance of diastolic MR is determined mainly by PQ intervals and cardiac function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721179 TI - A new algorithm for minimizing pacemaker polarization artifact: universally applicable in permanent pacing systems. AB - Polarization artifacts that result from pacing may interfere with analysis of paced evoked responses during, e.g., automatic threshold tracking. We have developed a method for reduction of such artifacts that relies on the introduction of pacing stimuli during the refractory period of unipolar or bipolar paced captured beats after previous identification of a refractory period "template" or baseline. The refractory pacing stimuli cannot capture the heart, and thus any deviation from the template is due to polarization artifact alone. The artifact amplitude is measured and the precharge duration of the triphasic stimulus waveform is changed each time until artifact is minimized, as detected by repeated reversals in the polarity of the polarization artifact. In a series of 11 patients with unipolar and bipolar permanent pacing leads, mean initial artifact before balancing was 1.44 +/- 0.84 mV, which was reduced to 0.44 +/- 0.30 mV after balancing (P = 0.001). Initial precharge duration was 3.2 msec by design; mean final precharge duration was 3.30 +/- 0.34 msec. This algorithm is universally applicable in permanent pacing systems, as it is valid in unipolar and bipolar pacing and it does not require an intrinsic cardiac rhythm. PMID- 1721180 TI - Impact of filtering upon ventricular tachycardia identification by correlation waveform analysis. AB - Signal analysis of digitized waveforms has been postulated as a method for improving sensitivity and specificity of ventricular tachycardia (VT) detection in implantable antitachycardia devices. Such improvement may alleviate the problem of unwarranted delivery of therapy by adding precision to the identification of the pathological VT. Morphological analysis could also allow distinct therapies to be initialized for multiple VTs in the same patient. Correlation waveform analysis (CWA) has been demonstrated to be effective in separating benign rhythms from VT in wideband recordings (1-500 Hz) but the effect of filtering has not been previously examined. Bipolar (1 cm) intraventricular recordings (1-500 Hz) of sinus rhythm (SR) and 25 distinct VTs in 18 patients were analyzed by CWA using a signal-averaged SR template. Passages contained 65.9 +/- 19.8 VT depolarizations (range 45-108). Digital filtering was performed on all data passages with varying passbands. Results for passages with a bandwidth of 1-250 Hz were equivalent to wideband results, i.e., greater than or equal to 92% paired sets of SR and VT were separable at a 95% confidence level. A bandwidth of 1-100 Hz decreased discrimination to 84%. At a bandwidth of 1-80 Hz, 80% of cases were successfully separated, but at 10-80 Hz these results improved to 88%. Bandwidths of 20-80 and 30-80 Hz reduced reliability of CWA performance to 72% and 60%, respectively. Filtering at typical pacemaker/defibrillator passbands produced morphological analysis results equivalent to those yielded at wideband settings. Differences in the range between SR versus VT decreased in filtered recordings but overall detection of VT was not degraded. PMID- 1721181 TI - Early clinical experience with a minute ventilation sensor DDDR pacemaker. AB - The new DDDR pacemaker META DDDR utilizes a minute ventilation sensor based on transthoracic impedance measurements. The sensor determines the metabolic indicated interval, the atrioventricular (AV) delay and the postventricular atrial refractory period (PVARP). The baseline PVARP must be carefully selected to define nonphysiological tachycardias. If a P wave falls within the PVARP the pacemaker will automatically switch to the VVIR mode. This behavior prevents tracking of paroxysmal atrial tachyarrhythmias (PAT). Twenty-eight patients with sinus node dysfunction (n = 20), AV junction ablation (n = 5), complete or intermittent AV block (n = 3); who received a META DDDR pacemaker were studied. The mean age was 65 +/- 13 years. RESULTS: mode switching (reversion) to VVIR was observed in 57% of the patients. Forty-two percent had episodes of mode switching to VVIR during a stress test, four related to PAT, and seven to sinus tachycardia. Fifty percent had episodes of mode switching to VVIR during a 24 hour Holter, four related to PAT, three to retrograde P wave sensing, and two to sinus tachycardia. At the last follow-up, 20 of the 26 patients initially programmed to the DDDR mode remained in the DDDR mode, while five were reprogrammed to the DDD and one to the VVIR mode. Mode switching has a high sensitivity but a low specificity for PAT. It appears to be a useful approach to prevent rapid tracking of atrial tachyarrhythmias. Careful PVARP programming is critical to appropriate reversion behavior, but further modifications of the algorithm are needed to improve its performance. PMID- 1721182 TI - Sensor for right ventricular volumes using the trailing edge voltage of a pulse generator output. AB - Cardiac and thoracic volume (Vol) signals are useful for diagnosis and adaptive rate pacing. Present systems need an AC or pulsed constant current carrier to measure conductivity (proportional to Vol), causing high battery drain and requiring complex detection algorithms or special leads. The aim of this study was to propose a new and simple sensor for cardiac volumes using standard pacing leads and no AC carrier signal. "Constant voltage" pulse generators (PG) deliver a square pulse to the lead via a capacitor (Cap). The signal resulting from the interaction between the PG and the tissues and blood is trapezoidal, with a leading edge voltage determined by PG output and a trailing edge voltage (TEV) dependent on electrode surface and Cap value (device constants), and patient load. The hypothesis that right ventricular (RV) and chest Vol variations could produce load-related TEV changes was tested. Four PGs were connected to bipolar (Bip) leads within containers with 5-80 cc of saline, and TEV was measured with every 5 cc Vol change. Fifteen patients with previously implanted Bip PGs were studied during VOO pacing, scanning the intrinsic cardiac cycle. TEV was noninvasively measured as a function of time from onset of QRS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721183 TI - A new approach to the prevention of endless loop tachycardia in DDD and VVD pacing. AB - Endless loop tachycardia (ELT) is a possible complication in dual chamber pacing; it is usually prevented by programming the atrial refractory period (PVARP) longer than the retrograde ventriculoatrial (VA) conduction interval; this in some patients limits the upper rate. In 15 patients with a DDD (nine patients) or a single-pass lead VDD pacemaker (six patients) and retrograde atrial activation, telemetric recording documented a significant difference in amplitude of antegrade, and retrograde atrial potentials (VDD 1.21 +/- 0.32 mV vs 0.56 +/- 0.23 mV, P = 0.008; DDD 2.7 +/- 1 vs 1.8 +/- 1 mV, P = 0.038; Student's t-test for paired data). In 3/15 patients ELT stopped after programming of atrial sensitivity to a value greater than the retrograde P wave amplitude; in 11/15 patients this occurred at a sensing value lower than or equal to retrograde P wave amplitude with a high pass band filter operating. One patient required PVARP lengthening. Holter monitoring showed no more ELTs. In most patients with a DDD or single-pass lead VDD pacemaker with widely programmable sensing amplitude and Hi/Low bandpass filters, individual programming of atrial channel sensitivity prevents ELT without affecting the PVARP and, consequently, upper rate limit. PMID- 1721184 TI - Pacing threshold spikes months and years after implant. AB - To determine patterns of variation in chronic pacing thresholds, we made 4,942 threshold measurements in 257 patients with 312 leads, at times from implant to 295 months (median 17 months) including 1,053 determinations in 46 children less than 12 years old. Motivation was late sudden death in two single-ventricle pacemaker-dependent children with multiple possible death causes. At stimulus duration 0.5 +/- 0.04 msec, mean of the thresholds, measured 1 month or more after implant, was 1.3 +/- 0.66 volts (V) for endocardial electrodes and 2.8 +/- 1.39 V for epicardially applied electrodes. Highest mean thresholds were in the 6 to 12-year-old age group. In 34 leads studied at implant, again within a month and for at least three years thereafter, time of maximum threshold occurred after one month in 59%, independent of lead type or patient age. Of 107 leads with five or more measurements after 3 months use, gradual increase in threshold continued after 3 months in 24%. An additional 21% had at least one threshold that exceeded the post-three-months individual patient lead mean by three standard deviations. Most striking was the occurrence of transient several-volt increases and decreases in threshold as late as 8 years after lead implantation in at least three children. These temporary changes were detected initially transtelephonically by the vario method of threshold measurement. They occurred during minor illnesses such as summer colds, yet similar illnesses also occurred without threshold elevation. We suggest further study of pacing threshold variations in highly pacemaker-dependent children whose cardiac anatomy makes use of epicardial electrodes necessary. PMID- 1721185 TI - Survival of patients who have received appropriate shocks from their implantable defibrillators. PMID- 1721186 TI - Clinical efficacy of low energy cardioversion in automatic implantable cardioverter defibrillator patients. AB - The inclusion of low energy cardioversion capability into modern implantable antiarrhythmic devices, although an appealing idea, is nevertheless unproven with regard to its potential benefits. Moreover, since occasional reports have surfaced suggesting that ineffective application of low energy shocks may prejudice subsequent arrhythmia reversion, we examined the effectiveness and risks of this feature in a large series of patients performed as part of a US Food and Drug Administration clinical trial performed under an investigational device exemption. A total of 813 induced monomorphic ventricular tachycardias were studied in 244 patients. We found that many of the arrhythmias could be reverted to sinus rhythm with small amounts of energy. Cardioversion energy was less than or equal to 6 joules (J) for 84 (53.2%) and less than or equal to 14 joules in 105 (66.4%) of the 158 patients tested at implant and subsequently remained unchanged through greater than 4 months follow up. The incidence of noncardioversion, acceleration or both occurred in 12.7%, 5.7%, and 13.1%, respectively on a per patient basis. On a per episode basis, nonconversion occurred in 51 (6.3%) and acceleration in 61 (7.5%) of the 813 inductions. There was no correlation between the occurrence of nonconversions and accelerations. The devices were allowed to recycle in the event the arrhythmia was not reverted. The subsequent shock was almost always effective, and in any event, no patient failed to be reverted by the second 30-J rescue shock. Over the entire follow-up period as long as 17 months, there were 17 deaths. Neither the incidence nor the mode of death was correlated with nonconversion or acceleration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721187 TI - Six-year clinical experience with the automatic implantable cardioverter defibrillator. PMID- 1721188 TI - Limitations of the countershock dose response: a study of transthoracic current. AB - Dose response assessment of countershock efficacy has been widely determined with respect to energy but not current. The purpose of this study was to examine the utility of the dose response method in a current-based model of transthoracic defibrillation (pentobarbital anesthetized dogs, n = 8). Ventricular fibrillation induction lasting 15 seconds was separated by 5-minute intervals. Current defibrillation threshold (DFT; the lowest current that successfully defibrillated) was determined by decreasing current on successive trials. Energy DFT equaled the energy value of the corresponding current DFT. Subsequent data were expressed in normalized terms with each DFT assigned a normalized value of 1.00. Three shocks were delivered in random order at each of seven normalized current nodes (total of 21 shocks): 0.55, 0.70, 0.85, 1.00, 1.15, 1.30, and 2.00 x DFT (early testing). Randomization was repeated, and a second set of 21 trials were performed (late testing). Composite plots were made relating normalized current and energy to the percent successful defibrillation. The dose response expressed in normalized energy demonstrated an overall shift to the left compared to current. The difference was significant at every node value below the estimated DFT. Ninety percent of successful trials with respect to current and energy occurred at or above 0.85 DFT and 0.55 DFT, respectively. Significant changes in impedance occurred between early testing (60 +/- 6 ohms) and late testing (47 +/- 5 ohms), n = 8, mean +/- SD. Current, compared to energy, is a more accurate parameter in the dose response assessment of transthoracic defibrillation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721189 TI - Comparison of titanium-mesh and porous disc electrodes for epicardial defibrillation. AB - The object of this study was to compare the effectiveness of chronically implanted porous electrodes with that of smooth mesh titanium electrodes of the same diameter but smaller effective surface area. The criteria used in evaluating the electrodes were (1) acute, subacute, and chronic resistance and (2) acute, subacute, and chronic defibrillation thresholds. Electrode pairs 2.5 cm in diameter were implanted in each of 17 dogs (ten mesh and seven porous). One electrode of each pair was sutured to the right ventricle and one to the left ventricle near the cardiac apex. Defibrillation threshold energy and total resistance were measured at the time of implantation and again 6 and 12 weeks after implantation. The mean initial resistance of the titanium electrodes was 131.7 omega; the mean defibrillation values for the porous electrode implant were 96.9 omega and 7.5 joules, respectively. Three to 6 weeks after implantation, the values for the titanium mesh electrode were 88.9 omega and 12.0 joules, while those for the porous electrode were 59.9 omega and 8.0 joules. In the chronic state, the figures for the titanium mesh electrode were 78.1 omega and 13.0 joules, while those for the porous electrode were 64.3 omega and 8.3 joules. We conclude that defibrillation can be achieved successfully with small epicardial electrodes. The findings suggest that a porous electrode, with its larger effective surface area, has lower electrode/tissue interface resistance in the acute and chronic phases, and, therefore, provides lower defibrillation threshold energy. PMID- 1721190 TI - Implantable cardioverter defibrillator implanted by nonthoracotomy approach: initial clinical experience with the redesigned transvenous lead system. AB - Standard implantation procedure for the implantable cardioverter defibrillator (ICD) has traditionally required a thoracotomy approach. A newly revised nonthoracotomy defibrillator lead system that uses a single transvenous tripolar endocardial lead alone or in combination with a subcutaneous/submuscular patch lead was introduced into clinical trials in September, 1990. Fourteen patients requiring a cardioverter defibrillator for recurrent sustained ventricular tachycardia (eight patients) or aborted sudden cardiac death (six patients) were evaluated for implantation of this lead system. Primary successful lead system implantation was obtained in nine patients. The remaining five patients had unacceptably high defibrillation thresholds (DFTs) for implantation. One of the nine initially successful implants demonstrated unacceptable DFTs and cross-talk inhibition from a permanent pacemaker necessitating removal of the nonthoracotomy lead system and replacement with a conventional lead system via thoracotomy. All remaining primary implanted patients experienced successful conversion of induced ventricular fibrillation prior to hospital discharge. Continued follow-up and greater experience to confirm the durability and efficacy of the nonthoracotomy AICD lead system are needed. PMID- 1721191 TI - Improved patient surveillance and data acquisition with a third generation implantable cardioverter defibrillator. AB - Thirteen patients were implanted with the Telectronics 4210 ATP implantable cardioverter defibrillator (ICD) for ventricular tachycardia or ventricular fibrillation. This device has multiprogrammable antitachycardia pacing, bradycardia pacing, and shock therapies. In addition, there is extensive data logging and ECG snapshot capability for arrhythmia confirmation and response to therapy. These features permit easy retrieval of all detected and treated events, whatever the eventual outcome. In this study, the data logged at predischarge electrophysiological testing was compared to the data recorded in a standard manner. The bulk of the data, however, was derived from long-term follow-up of spontaneous events over a mean period of 203 days (range 154-257). During this period, a total of 6,193 arrhythmia detections were made: 20 were classified as ventricular fibrillation, and 6,173 as ventricular tachycardia. The vast majority of these (93%) terminated spontaneously without ICD intervention (5,738), underscoring the benefit of a standard second confirmation prior to therapy delivery (noncommitted system). There were 394 arrhythmia episodes treated with antitachycardia pacing; of these a total of 8.3% accelerated to either more rapid ventricular tachycardia or ventricular fibrillation (4.3% and 4.0%, respectively). Events were reported in an "episode log" format, listing all arrhythmia detections with time/date annotation; or in a "sense history" format, detailing each episode from start to conclusion. These data demonstrate that this advanced, "tiered" ICD with data recall contributes to better patient management, and permits a more tailored termination prescription for the individual patient. PMID- 1721193 TI - Influence of clinical characteristics and shock occurrence on ICD patient outcome: a multicenter report. The Bilitch Registry Group. AB - Data on 1,281 patients from the Bilitch implantable cardioverter defibrillator (ICD) registry were reviewed to evaluate ICD patient characteristics and survival, and the impact of ICD shock occurrence on outcome. The mean ejection fraction was 34.3%; 78% had coronary disease, 471 patients had at least one shock thought to be appropriate, and 231 patients died. Causes of death included: arrhythmic (41%), nonarrhythmic cardiac (37%), and noncardiac (22%). Cumulative survival from all-cause mortality at 1, 3, and 5 years was 89%, 76%, and 64%; survival from all-cause cardiac death was 93%, 90%, and 76%; survival from arrhythmic death was 96%, 92%, and 87%. Patients who had received a shock had a trend towards a worse long-term prognosis. Shock patients also had a small increase in the prevalence of coronary disease and a somewhat lower ejection fraction than the remainder of the population. PMID- 1721192 TI - When is it safe not to replace an implantable cardioverter defibrillator generator? AB - In most reports on patients receiving implantable cardioverter defibrillators, shocks were received mainly during the first 2 to 3 years. Thus, the question had been raised as to the need for device replacement after 3 or 4 years if no shocks had been received. In order to answer this question, shock experience in 184 patients receiving the implantable cardioverter defibrillator was analyzed. Patients were followed for a mean of 24 +/- 18.7 months. A patient's shock was judged to be appropriate if there was electrocardiographic documentation of sustained ventricular tachyarrhythmia at the time of shock or if it was preceded by sudden onset of presyncopal or syncopal symptoms. The majority of patients had coronary artery disease. In approximately two-thirds of patients, left ventricular ejection fraction was below 40%. One hundred fourteen patients had inducible sustained monomorphic ventricular tachycardia. On follow-up, there were 29 deaths, five of which were sudden. Sixty-eight patients received an appropriate shock during follow-up (37%). Over 90% of these 68 received their first shock within the 2 years after implant. The actuarial risk of receiving an appropriate shock by the fifth year after implant was 69%. Conversely, 31% of patients who survived 5 years had not received an appropriate shock. Hazard analysis indicates that there is a high incidence of first appropriate shock during the year following implant. Subsequently, the incidence dropped to a relatively steady rate with a rise in this rate during the fifth year. This analysis suggested a bimodal distribution of appropriate shocks.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721194 TI - Electroporation of cardiac cell membranes with monophasic or biphasic rectangular pulses. AB - During defibrillation, cardioversion, and electrocution trauma, heart cells are exposed to potential gradients that increase the transmembrane potential (Vm). At sufficiently high Vm, pathological increases in cell permeability can occur. With enzymatically isolated frog heart cells (n = 29) we investigated the voltage and time sufficient for electroporation or cardiac cell membranes with rectangular voltage pulses, particularly with 5-msec monophasic, and 5- or 10-msec biphasic pulses. The rectangular voltage pulse (monophasic 0.1-1.5 V, 0.1-100 msec or symmetric biphasic 0.1-1 V, 0.4-10 msec [total duration]) was applied to the cell membrane using the cell-attached patch clamp technique, and a low voltage pulse train was added so that membrane conductance could be monitored continuously. Step increases in membrane conductance (breakdown) were observed, indicative of electroporation, and occurred with different combinations of pulse amplitude and duration; for example, for monophasic square pulses: (1 V, 0.2 msec) or (0.5 V, 0.5 msec), and for biphasic pulses: (1 V, 0.4 msec total duration) or (0.5 V, 0.8 msec). Using 5- or 10-msec rectangular pulses, breakdown occurred at a voltage around 0.4 V independent of polarity or waveform. The recovery of the permeabilized cell membrane after the voltage pulse was highly variable, in some cases not recovering at all while in other cases recovering after a lapse of seconds to minutes. These results suggest that monophasic and biphasic pulses of approximately 1 V, 0.2-0.4 msec and approximately 0.4 V, 5 msec can permeabilize the heart cell membrane even for minutes, time enough to cause an alteration in the cellular ionic composition leading to depressed or unexcitable tissue, a precursor for cardiac arrhythmia. PMID- 1721195 TI - Ventricular and atrial defibrillation using new transvenous tripolar and bipolar leads with 5 French electrodes and 8 French subcutaneous catheters. AB - This study evaluated the use of new small transvenous atrial and ventricular leads for converting atrial fibrillation (AF) and ventricular fibrillation (VF) in 10 adult male mongrel dogs. Five dogs (group A) received a right atrial "J" (AJ) and right ventricular (RV) active fixation tripolar lead, each consisting of a platinized platinum pacing tip, anode band, and braided defibrillation electrode. The remaining five dogs (group B) received one bipolar RV lead and one tripolar AJ lead. The RV leads were implanted in the right ventricular apex (RVA) and the AJ leads were placed in the atrial appendage. Additionally all dogs received two 8 French subcutaneous defibrillation catheters in the fifth and seventh intercostal spaces. Twenty asymmetric biphasic shocks consisting of five randomized voltage levels were used to convert VF in groups A and B. The bipolar RV lead (group B) had a significantly higher probability of success in converting VF than the tripolar RV lead (group A). In group A defibrillation thresholds for converting AF were obtained using two electrode configurations. No significant difference was observed between the two electrode configurations used to convert AF. Pacing and sensing thresholds were satisfactory for bipolar and tripolar lead configuration. PMID- 1721196 TI - Is defibrillation testing safe? AB - Determination of defibrillation thresholds (DFTs) and implantable cardioverter defibrillator (ICD) testing requires repeated inductions of ventricular fibrillation (VF) and defibrillation attempts using known energy outputs. Little is known about the individual and cumulative effects of repetitive brief episodes of VF and hypoperfusion on cerebral function. The potential clinical utility of quantitative electroencephalographic (QEEG) monitoring during intraoperative ICD testing, by using processed 19-channel EEG (0.5-35 Hz bandwidth), was examined in ten anesthetized patients, five males and five females (mean age 62 +/- 10 years), who underwent ICD implantation and testing. Ischemic QEEG patterns were defined as those with a 3 standard deviation increase (P less than 0.01) in absolute delta (1.5-3.5 Hz) power persisting for greater than or equal to 2.5 minutes. The majority (80%) of the VF episodes (70) were accompanied by QEEG "slowing" (doubling of the pre-VF low frequency delta waves amplitude). All the patients (5/5) experiencing greater than 6 VF episodes showed a statistically significant increase in the low frequency amplitude. In contrast, this EEG abnormality was apparent in only one of five patients experiencing less than 6 VF episodes. These results suggest a cumulative QEEG depression associated with ICD testing. QEEG may provide an objective means for establishing an individualized upper safe limit of DFT testing and the total number of induced VF episodes. PMID- 1721197 TI - Long-term follow-up of patients with nonischemic dilated cardiomyopathy and ventricular tachyarrhythmias treated with implantable cardioverter defibrillators. AB - We analyzed our 10-year cumulative experience of 40 consecutive patients with idiopathic dilated cardiomyopathy and associated ventricular tachyarrhythmias, treated with implantable cardioverter defibrillators. Dilated cardiomyopathy was defined as left ventricular ejection fraction (EF) less than or equal to 50% with no defineable etiology. Patient characteristics included: 24 male, mean age 52 years, mean EF = 33%, New York Heart Association Class I-III, presenting syndrome -cardiac arrest (n = 28), syncope/near syncope (n = 12). At 2.5 years mean follow up, there were 16 deaths: one operative, three sudden, two incessant ventricular tachycardia/ventricular fibrillation (VT/VF), six heart failure, and four noncardiac. The actuarial mortality at 1 and 4 years was 0% and 14% for sudden death, 11% and 34% for cardiac death. The projected mortality was 52% and 78% for same time intervals (P less than 0.01). No useful baseline variable predicted who would or would not receive an ICD shock in follow-up. ICD therapy appears effective in reducing sudden death mortality in this high risk population. PMID- 1721198 TI - Accuracy of rhythm classification using a data log system in implantable cardioverter defibrillators. AB - Because the presence or absence of symptoms alone may be insufficient to correctly diagnose the rhythm for which implantable cardioverter defibrillator therapy is delivered, we hypothesized that the addition of data log information available in Telectronics ATP 4210 may improve the accuracy of rhythm classification. With this system the recorded ventricular electrogram cycle length is reported on a beat-to-beat basis immediately before, during, and after the tachyarrhythmia is detected. Using this information recorded from the data log in 32 separate tachyarrhythmia episodes in 20 patients, we compared the sensitivity, specificity, and predictive accuracy of rhythm classification on the basis of symptoms alone, data log alone, and data log combined with symptoms. While classification based on symptoms alone is highly specific (10/10 episodes), it is insensitive and has an overall predictive accuracy of 53%. By contrast, data log is sensitive (90%) and specific (91%) with better predictive accuracy (94%) than symptoms alone (P = 0.002). The addition of symptoms to information on beat-to-beat cycle length from data log resulted in a slight increase in predictive accuracy. PMID- 1721199 TI - Use of bipolar recordings from patch-patch and rate sensing leads to distinguish ventricular tachycardia from supraventricular rhythms in patients with implantable cardioverter defibrillators. PMID- 1721200 TI - A new approach towards defibrillation electrodes: highly conductive isotropic carbon fibers. AB - A new carbon fiber material was studied for its potential benefit in defibrillation electrodes. Miniaturization of the fiber production can result in small strands with extremely large surface areas. Two carbon fiber patches and a single carbon fiber bundle were used for the purposes of this study, and the results were compared to those obtained with conventional titanium/mesh patch electrodes. Tests performed in a saline filled tank revealed considerably lower resistances through the carbon material when compared to standard mesh electrodes. There was also a higher peak current flow with lower voltage. The carbon fibers exhibited lower impedance for defibrillation and this may be related to low polarization known to occur with carbon materials. This new carbon material may prove to be useful and further research is required. PMID- 1721202 TI - Simultaneous biphasic shocks enhance efficacy of endocardial cardioversion defibrillation in man. PMID- 1721201 TI - Does reception of appropriate shocks from the implantable cardioverter defibrillator affect survival? AB - The implantable cardioverter defibrillator has become an important therapeutic modality for treatment of life-threatening ventricular tachyarrhythmias. Recent reports have suggested that patients who receive appropriate shocks from this device have an inordinately high overall mortality, and questioned the extent of benefit these patients derive from the implant. This report analyzed the survival among 184 patients who received the implantable cardioverter defibrillator to assess survival differences between patients who received appropriate shocks versus those who did not. At a mean follow-up of 24 +/- 18.7 months, 68 patients received an appropriate shock from their device while 116 did not receive an appropriate shock. Overall survival of the entire population was quite similar to those published by others. There was no significant difference between overall survival of patients who received an appropriate shock versus those who did not. However, there was a statistically significant difference in sudden death mortality. The group of patients that received appropriate shocks included all five sudden deaths. This observation suggested that sudden death in this population was likely due to ventricular tachyarrhythmias rather than strictly bradycardia or asystole. The "benefit" of the device to the entire population was also assessed by estimating survival after receipt of the first appropriate shock. Using this approach, an estimated 10% of patients died without receiving an appropriate shock. In other words, ultimately, 90% of patients were expected to benefit from the device. This survival curve, which initiated only after receipt of the first appropriate shock was fairly similar to those estimated from conventional methods. Therefore, survival after receipt of an appropriate shock was comparable to overall survival and there was no significant difference between survival of patients who received appropriate shocks and those who did not. PMID- 1721203 TI - Sustained monomorphic ventricular tachycardia: a single electrocardiographic expression of different patterns of reentry. AB - Sustained monomorphic ventricular tachycardia (SMVT) can be the electrocardiographic expression of a reentrant impulse in the ventricles. In this study we analyzed the different types of reentry that might lead to SMVT. METHODS: The pattern of activation of 73 episodes of SMVT induced in thin sheets of epicardium in 50 Langendorff perfused rabbit hearts were visualized with high resolution epicardial mapping (248 points). RESULTS: Five different patterns of reentry resulting in SMVT were identified: (1) Single-loop reentry around a fixed obstacle (n = 40); (2) Single-loop reentry around a functional arc of conduction block (n = 17); (3) Double-wave reentry around a fixed obstacle (n = 9); (4) Figure-of-eight reentry around two areas of functional block (n = 5); and (5) Multiple synchronized circuits around multiple areas of functional block (n = 2). CONCLUSION: SMVT is a single electrocardiographic expression of different patterns of reentry. Accurate mapping is mandatory to identify the reentrant pathway and the pathophysiological substrate of the arrhythmia. PMID- 1721204 TI - Proarrhythmic response to antiarrhythmic drug as a risk factor for sudden cardiac death in patients with ischemic heart disease. AB - The prognostic significance of arrhythmogenic response to an antiarrhythmic drug was studied. In 782 patients with ischemic heart disease (IHD) and frequent and/or complex ventricular premature beats (VPBs), 1,041 drug tests guided by 24 hour Holter monitoring were conducted. The following drugs were assessed: beta blockers, disopyramide, mexiletine, amiodarone. Proarrhythmia was defined as: (1) greater than 4-fold increase in VPBs, (2) greater than 10-fold increase in repetitive forms, or (3) new occurrence of ventricular tachycardia or ventricular fibrillation (VT/VF). During a follow-up of 1-49 months (mean 22) patients were treated with antiarrhythmic drugs found to be safe in control Holter monitoring. Proarrhythmic effects were observed in 8.4% of patients. No drug was completely free of this type of reaction. In long-term observation, cardiac death and sudden death occurred in 53 and 32 patients, respectively. With actuarial analysis (Kaplan-Meier method, log-rank test) there was a significant difference in cardiac death (P less than 0.01) and sudden death rate (P less than 0.05) of proarrhythmia (+) compared with proarrhythmia (-) patients at 1 year (11% vs 4%, 7% vs 3%) and 3 years (24% vs 11%, 16% vs 7%). Proarrhythmic response was an independent risk factor apart from myocardial infarction, VT/VF, ejection fraction less than 40% and QTc greater than 440 msec. Arrhythmogenic response to antiarrhythmic drugs seems to be an additional predictor of sudden death in IHD. PMID- 1721205 TI - Low energy direct current ablation in patients with the Wolff-Parkinson-White syndrome: clinical outcome according to accessory pathway location. AB - Forty-five patients with the Wolff-Parkinson-White syndrome underwent direct current (DC) ablation using a low energy power source (Cardiac Recorders). Anodal shocks of 10-40 joules were given to either a 6 French quadripolar catheter (Bard), a 7 French bipolar contoured catheter (Bard), or a 7 French deflectable catheter with a 4-mm distal electrode (Mansfield). The indifferent electrode consisted of a large patch that was positioned under the left scapula. There were 26 males and 19 females, with a mean age of 34 years (range 9-67). Accessory pathways were located in the left free wall in 30 patients (67%) and were posteroseptal in 15 patients (33%). The shortest ventriculoatrial interval during mapping (89 +/- 21 msec), the mean cumulative amount of energy per patient (322 +/- 283 joules), and the mean CK-MB rise (45 +/- 30 units, normal 0-30 units) were not significantly different between both groups. Ablation was successful in 29/30 patients (97%) with a left free-wall accessory pathway, and in 13/15 patients (87%) with a posteroseptal accessory pathway. All three patients with failure of ablation had multiple accessory pathways, and two of these patients had Ebstein's anomaly. Patients with left free-wall and posteroseptal accessory pathways, respectively, differed significantly in terms of: total session time (4.1 +/- 1 hours vs 5.3 +/- 1.3, p = 0.0001), total procedure time for ablation (2.6 +/- 0.8 hours vs 3.2 +/- 1.2, P = 0.02), and fluoroscopy time (46 +/- 24 min vs 64 +/- 29, P = 0.006). In 13 patients (29%) with a concealed accessory pathway, these variables were not significantly different from patients with overt preexcitation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721206 TI - Histopathology of monopolar transcatheter radiofrequency ablation at the mitral valve annulus. AB - Although monopolar radiofrequency (RF) catheter ablation is being used to interrupt left-sided accessory pathways in patients with tachyarrhythmia, little is known of the histologic effects from this method of treatment. RF ablation at the mitral valve (MV) annulus was performed in ten dogs to examine the histology of the lesion area. A custom 6 French ablation catheter with a 4 mm distal electrode was positioned beneath the MV adjacent to the annulus. Mean preablation atrial to ventricular electrogram ratio (A/V ratio) was 0.26 +/- 0.17. Thirty +/- 1 watts of RF power were applied for 53 +/- 13 seconds between the distal electrode and a large skin electrode. Nine dogs were sacrificed 6 weeks and one dog 2 days following ablation. Annular lesions were seen in eight of the ten dogs. Lesion volume was 136 +/- 41 mm3 and correlated with the A/V ratio (r2 = 0.74, P = 0.006). Lesions consisted of necrosis of the left ventricle with extension into the atrioventricular groove and left atrium. No injury to the coronary sinus or circumflex artery was observed. A small area of injury was noticed on the mitral leaflet in one dog. Monopolar RF ablation creates lesions at the MV annulus without injury to adjacent vascular structures. PMID- 1721207 TI - Biomagnetic noninvasive localization of accessory pathways in Wolff-Parkinson White syndrome. AB - It was our purpose to assess the clinical relevance of noninvasive magnetocardiographic localization of accessory pathways. Nine patients with Wolff Parkinson-White (WPW) syndrome were studied. For all of them the site of the accessory pathway was known from invasive catheter mapping. A 37-SQUID (superconducting quantum interference device) sensor multichannel system (KRENIKON) was used, allowing synchronous registration with all channels. The site of the electrophysiological activity at the beginning of the delta wave was determined. Magnetic resonance images of the heart were obtained to correlate the biomagnetically localized activity with the anatomy. Magnetocardiographic localization of the bypass tract corresponded with catheter mapping with a spatial difference of 0-5 cm, 1.8 cm on the average, compared to the results obtained by catheter mapping. Thus, magnetocardiography is a promising new method for noninvasive localization of accessory pathways in WPW patients. This may streamline further invasive procedures. PMID- 1721208 TI - Differential response of QTU interval to exercise, isoproterenol, and atrial pacing in patients with congenital long QT syndrome. AB - Sympathetic stimulation is well known to contribute to the genesis of QTU prolongation and ventricular tachyarrhythmias in patients with congenital long QT syndrome. In this study, we performed exercise treadmill testing, isoproterenol infusion (1-2 micrograms/min), and right atrial pacing (cycle length 500 msec) in 11 patients with congenital long QT (LQT) syndrome (LQT group) and in 12 age- and sex-matched controls (control group). The responses of the corrected QT (QTc; Bazett's method) interval and the TU wave complex were evaluated. The QTc interval was prolonged from 482 +/- 63 msec1/2 to 548 +/- 28 msec1/2 by exercise in the LQT group (n = 11; P less than 0.005), and this was associated with fusion of the T waves with enlarged U waves, whereas the QTc interval did not increase with exercise in the control group (n = 12; 402 +/- 19 msec1/2 vs 409 +/- 22 msec1/2). The QTc interval was also prolonged from 466 +/- 50 msec1/2 to 556 +/- 33 msec1/2 by isoproterenol in the LQT group (n = 7; P less than 0.005) in association with morphological changes of the TU wave complex like those seen with exercise, whereas it was only slightly increased from 399 +/- 10 msec1/2 to 436 +/- 13 msec1/2 by isoproterenol in the control group (n = 77; P less than 0.001). However, the QTc interval did not increase with atrial pacing in the LQT group (n = 8; 476 +/- 57 msec1/2 vs 486 +/- 59 msec1/2), whereas it was slightly increased from 400 +/- 21 msec1/2 to 426 +/- 18 msec1/2 by atrial pacing in the control group (n = 8; P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721209 TI - Acute and chronic hemodynamic impact of total right ventricular disarticulation. AB - Right ventricular disarticulation is a radical operation to control ventricular arrhythmias in patients with arrhythmogenic right ventricular dysplasia. This report describes the acute and chronic hemodynamic impact of the procedure based on our experience of five patients with life-threatening arrhythmias unresponsive to medical therapy who have undergone total disarticulation of the right ventricle. Although all patients suffered acute postoperative hemodynamic problems, all survived and returned to an excellent functional class. Right ventricular disarticulation should be considered in patients with drug refractory ventricular tachycardias due to arrhythmogenic right ventricular dysplasia when the arrhythmia either poses a life threat or results in chronic morbidity. PMID- 1721210 TI - Transcoronary atrioventricular nodal modification using microvascular collagen. AB - Surgical and catheter based techniques for atrioventricular (AV) nodal modification have recently been described. Similarly, transcoronary embolization for the treatment of arrhythmias has recently emerged as a potentially useful approach. This report reviews our experience of a novel technique using embolization of the AV node with an inert agent, cross-linked collagen, for the treatment of AV nodal reentrant tachycardia. Three patients with refractory nodal tachycardia received 0.1-0.5 mL cross-linked collagen (2 mg/mL) delivered via a catheter placed within the nodal artery. All developed transient complete AV block with subsequent recovery of conduction. Two patients have had no further tachycardia and were noninducible at restudy. One patient required electrical modification because of recurrent symptoms. One patient sustained a limited posterior infarct due to back-spill of collagen into the distal right coronary artery. This novel technique provides an alternative approach to a cure for AV nodal tachycardia without producing long-term heart block. PMID- 1721211 TI - Lower pacemaker in high degree atrioventricular block is modulated electrotonically by atrial excitations. AB - To see if the lower pacemaker in patients with atrioventricular (AV) block was modulated electrotonically by atrial excitation, we studied R-R intervals in two groups of AV block patients using a phase response curve (PRC). Group I consisted of 20 patients with high degree AV block, including seven patients in whom complete AV block was transiently observed in the course of acute inferior myocardial infarction. Group II consisted of 19 patients with complete AV block. In every patient, PRC was obtained from the continuous electrocardiogram by plotting each R1-R2 interval (response) on the ordinate as a function of the R1 Px interval (phase, x = 1, 2 ...) on the abscissa. In Group I, the R-R interval was prolonged when the P wave fell in the initial half of the R-R interval, and was abbreviated when the P wave fell in the later half of the cycle. In Group II, the fluctuation of the R-R interval was minimum. In ten group I patients, with the improvement of the AV block, PRC became sharper and transition from prolongation to shortening occurred at shorter R-P intervals. We conclude that, in Group I, lower pacemaker was modulated electrotonically by atrial excitations through decreased electrical coupling along the AV node. PMID- 1721212 TI - Multifactorial prediction of arrhythmic events after myocardial infarction. Combination of heart rate variability and left ventricular ejection fraction with other variables. AB - Autonomic dysfunction has recently been shown to identify postinfarction patients at a high risk of arrhythmic events. Therefore, the predictive characteristics of heart rate variability and the left ventricular ejection fraction in combination with other prognostic variables--mean heart rate, late potentials, and ventricular ectopic beat frequency greater than 10/hour (VE10)--were examined in 417 postinfarction patients. The heart rate variability index was the most important factor for the stratification of patients at high risk of arrhythmic events after myocardial infarction and optimum stratification was based on the combination of the heart rate variability index with late potentials or with frequent ventricular ectopic beats. PMID- 1721213 TI - Transesophageal echocardiographic evaluation for mural thrombus following radiofrequency catheter ablation of accessory pathways. AB - BACKGROUND: Catheter ablation of accessory pathways (APs) provides a definitive therapy for patients with Wolff-Parkinson-White Syndrome. The reported incidence of thrombus formation on ablation-induced injuries with direct current shock varies from 0%-20% in animal studies. The purpose of this study was to determine the prevalence of mural thrombus following catheter ablation with radiofrequency current of accessory pathways in humans. METHODS AND RESULTS: Radiofrequency current (30-35 watts) was applied through a catheter electrode placed against the mitral or tricuspid annulus guided by catheter recordings of AP potentials. Transthoracic (TTE) and transesophageal echocardiography (TEE) were performed in 95 of 111 patients, at 18 +/- 6 hours following catheter ablation. After ablation, no thrombus was identified at or near the ablation site in any patient. Two out of 95 patients had a mural thrombus at a remote site that was detected by TEE but not by TTE. No new wall motion abnormality was detected in any patient. No significant regurgitant valvular lesion was found in any patient. CONCLUSION: Intracardiac thrombus was not identified at the site of catheter ablation, possibly owing to the small lesions produced by radiofrequency energy and high blood flow normally present in those areas. However, patients may be at small risk for mural thrombus at a remote site from prolonged placement of catheters. PMID- 1721214 TI - Which factors determine the development of late potentials after first myocardial infarction? A multifactorial analysis. AB - A multifactorial analysis was performed to study the factors that contributed to the occurrence of late potentials on the signal-averaged electrocardiogram in 106 consecutive patients with a first myocardial infarction. Ninety-three (88%) patients received intravenous thrombolytic therapy within 6 hours of symptom onset. Thirty-two (30%) patients had a late potential on the signal-averaged electrocardiogram on day 6, including 17 of 31 (55%) in whom the infarct-related artery was occluded and 15 of 75 (20%) in whom it was patent (P = 0.0004). Twenty three variables were analyzed by a multifactorial stepwise regression analysis. Predictors of a late potential were (1) an occluded infarct-related coronary artery (t = -3.653, P = 0.0004) and (2) the extent of myocardial necrosis as indicated by the peak serum lactate dehydrogenase level (t = 3.094, P = 0.0025). The lower incidence of late potentials when the infarct-related coronary artery was patent was independent of left ventricular ejection fraction and peak enzyme levels after infarction. PMID- 1721215 TI - Diagnosis and surgical treatment of tachycardias in patients with nodoventricular fibers. AB - From May, 1982 to April, 1991 25 patients were examined and 23 had open heart surgery for tachycardia with nodoventricular fibers (NVF) participation. All patients suffered tachycardia for more than 5 years. Most patients had syncope. The patients had: true NVF tachycardia (11), atrioventricular node reentry tachycardia (5), orthodromic atrioventricular tachycardia (AVT) (5), and atrial flutter (1) all with passive propagation through NVF, pseudonodoventricular tachycardia (AVT with slow accessory pathways) two patients. In 18 out of the 23 who had surgery there were no signs of preexcitation or tachycardia events in the follow-up period. PMID- 1721216 TI - Demonstration of right and left atrial dissociation by atrial rapid pacing or extrastimulation during fast-slow (uncommon) form of atrioventricular nodal reentrant tachycardia. AB - Some recent works suggest that extranodal atrial fibers may form part of the reentry circuit in the atrioventricular (AV) nodal reentrant tachycardia (AVNRT). This hypothesis is based on the fact that the perinodal dissection successfully abolished AVNRT while preserving intact AV conduction. Apart from the surgical success, the electrophysiological evidence supporting this hypothesis has not been demonstrated, especially in the uncommon (fast-slow) form of AVNRT. We present some electrophysiological evidence suggesting atrial participation in eight patients with the fast-slow form of AVNRT. During the tachycardia, rapid pacing or extrastimulation was done from the orifice of the coronary sinus (CS) and the right atrium (RA), while recording the electrograms of the CS and the low septal RA. In seven patients, right and left atrial dissociation was demonstrated during pacing from the RA, while in the remaining one this was demonstrated from the CS. The interatrial dissociation will be unlikely if the intranodal reentry circuit connects with the atria via a single upper common pathway. This suggests that the upper turnaround of the reentry circuit involves atrial tissue and that the extranodal accessory pathway with long conduction times may form the ascending limb of the circuit (atrionodal reentry). Alternatively, the reentry circuit is entirely intranodal and two or more connecting pathways are present between the atria and the circuit. PMID- 1721218 TI - The role of catheter ablation techniques in the treatment of classic (type 1) atrial flutter. AB - Several attempts at circuit interruption of type 1 atrial flutter by means of surgical or catheter techniques have been published. We recently reported the results of a series of patients who underwent catheter fulguration of the low septal right atrium, with a mean follow-up of almost 3 years. True electrophysiological success was observed in 7/14 patients (50%). Clinical success, defined as absence of symptoms, was observed in 8 was observed in 8/14 (57%) in this patient population. No serious complications were encountered, but the potential risks of DC shock, and the experience that we gained in right atrial mapping using this approach, led us to reconsider the role of atrial DC ablation in these patients. Additional studies assessing the meaning of fragmented electrograms, and identification of one (or of several) slow conduction areas of the reentrant circuit are ongoing. PMID- 1721217 TI - RR variability and baroreflex sensitivity in patients with ventricular tachycardia associated with normal heart and patients with ischemic heart disease. AB - Recent studies have suggested that disordered autonomic function, particularly the loss of protective vagal reflexes are associated with an increased incidence of arrhythmic deaths following myocardial infarction (MI). Heart rate variability (HRV) and baroreflex sensitivity (BRS) are measures of myocardial autonomic function and predict arrhythmic deaths post-MI. Patients with ventricular tachycardia associated with a "normal heart" frequently have exercise-induced arrhythmia suggesting that the autonomic nervous system is important in the genesis of this form of ventricular tachycardia (VT). This study examines HRV and BRS in patients with VT associated with a "normal heart" and compares these values to patients post-MI with and without evidence of arrhythmia. Twenty patients with VT associated with a "normal heart," 16 patients with MI but without arrhythmia on follow-up, and 11 patients with MI and VT on follow-up were studied. HRV was measured from 24-hour Holter recordings and BRS was measured from plots of change in systolic blood pressure versus change in heart rate following an intravenous injection of 0.4-0.6 mg phenylephrine. HRV was significantly higher in the patients with VT associated with a normal heart (34.2 +/- 10.8 msec) compared to the patients post-MI, without (23.7 +/- 6.7 msec) and with (14.8 +/- 3.8 msec) arrhythmia (F = 9.2, P less than 0.001) and these differences were unaffected by adjustment for age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721219 TI - A comparison of intravenous propafenone and flecainide in the treatment of tachycardias associated with the Wolff-Parkinson-White syndrome. AB - We compared the electrophysiological effects of intravenous propafenone and flecainide on accessory pathway conduction by a randomized crossover study in 16 patients with Wolff-Parkinson-White syndrome. The antegrade refractory period of the pathway increased from 256 +/- 18 msec at baseline to 288 +/- 13 msec on propafenone (P less than 0.05) and to 296 +/- 27 msec on flecainide (P = 0.075). The minimum preexcited RR interval during atrial fibrillation or incremental atrial pacing was prolonged from 225 +/- 37 msec to 262 +/- 22 msec by propafenone (P less than 0.05) and to 301 +/- 31 msec by flecainide (P less than 0.005). The prolongation was significantly greater with flecainide than propafenone (P less than 0.05). Both drugs increased tachycardia cycle length (TCL) from 310 +/- 35 msec to 354 +/- 37 msec (propafenone P less than 0.005) and to 352 +/- 37 msec (flecainide P less than 0.01). Both propafenone and flecainide blocked antegrade conduction in the pathway in five patients. Both drugs rendered atrial fibrillation noninducible in seven patients and orthodromic tachycardia noninducible in five patients. CONCLUSIONS: (1) Flecainide causes a greater prolongation of minimum preexcited RR interval than propafenone; (2) There is no significant difference between propafenone and flecainide on the inducibility of arrhythmias, TCL, or incidence of antegrade conduction block. PMID- 1721220 TI - The mechanism of propafenone-induced slowing of ventricular tachycardia in man as defined by analysis of resetting response patterns. AB - The major finding in this study was that all VTs in patients treated with propafenone had a fully excitable gap. As only well tolerated, uniform VT in patients with chronic coronary artery disease was included for study, this result may not apply for ventricular arrhythmias in other patient populations. This is incompatible with the hypothesis that propafenone slows VT by increasing refractoriness within the VT circuit. Instead, the drug-mediated prolongation in VT cycle length is caused by effects on conduction velocity in fully recovered tissue and/or a change in the barriers of the circuit. PMID- 1721221 TI - Factors associated with recurrence of accessory pathway conduction after radiofrequency catheter ablation. AB - Catheter ablation of 215 accessory pathways (APs) using radiofrequency current (RF) was attempted in 204 consecutive patients. Two hundred twelve of the 215 (99%) APs were successfully ablated. After a minimum of follow-up period of 1 month (mean 8.5 +/- 5.4 months), AP conduction had returned in 17 patients (8%). Recurrence of AP conduction was manifest by atrioventricular (AV) reentrant tachycardia in six patients, palpitations suggestive of AV reentrant tachycardia in five patients, ventricular preexcitation on electrocardiogram in five patients, and inducible AV reentrant tachycardia during a follow-up electrophysiological study in one asymptomatic patient. AP conduction returned as early as 12 hours and as late as 4.7 months, but was evident within 2 months of ablation in 15 of 17 (88%) patients. AP conduction recurred in 12%-14% of anteroseptal, right free-wall, and posteroseptal APs, but only 5% of left free wall APs (P less than 0.01). Retrograde only conducting APs (concealed APs) had recurrence of AP conduction more frequently (16%) than APs that exhibited antegrade conduction (5.5%; P less than 0.01). Failure to record AP potentials from the ablation electrode, reflecting poor AP localization, was a strong predictor for recurrence of AP conduction. AP conduction returned in 19% of 48 APs when AP potentials were not recorded, compared to 5% of 164 APs where AP potentials were recorded from the ablation electrode (P less than 0.01). The time to block of AP conduction from the onset of RF current application was longer in APs with recurrence of conduction (4.9 +/- 6.1 sec vs 2.9 +/- 3.4 sec; P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721222 TI - The effect of age on the electrophysiological and autonomic correlates of sudden death after acute myocardial infarction. AB - To examine the influence of age on the autonomic and electrophysiological correlates of sudden death after myocardial infarction, 223 patients aged less than 60 and 195 patients aged greater than or equal to 60 were followed up for a mena of 790 days. The patients had Holter monitoring and a signal-averaged ECG 5 11 days after infarction. A mean ventricular ectopic beat frequency greater than 10 beats/hour (VE10) was present in 17.0% of young versus 28.2% of old patients (P less than 0.01); a low heart heart variability index in 17.9% of young but in 32.3% of old patients (P less than 0.001) and late potentials in 17.5% but 32% of young and old patients, respectively (P less than 0.01). There was no difference in the incidence of sudden death between young and old patients (3.6% vs 3.1%). However, sudden death accounted for 50%, compared with 24% of all deaths in the young and old groups, respectively (P less than 0.01). Sudden death was more closely associated with low heart rate variability and VE10 in the young than in the older group. The predictive values of a heart rate variability index less than 20 units with VE10 in younger patients were a sensitivity of 50%, a positive predictive accuracy (PPA) of 33% and risk ratio (RR) of 18 (P less than 0.001); these values did not reach significance in older patients (16.7%, 4.3% and 1.4%, respectively.)(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721223 TI - Permanent antitachycardia pacing for chronic atrial tachyarrhythmias in postoperative pediatric patients. PMID- 1721224 TI - Failure of rate responsive ventricular pacing to improve physiological performance in the univentricular heart. AB - The physiological efficacy of single chamber, rate responsive ventricular pacing (VVIR) is unknown for symptomatic patients following the Fontan procedure for univentricular hearts. A total of six postoperative children, ages 6-21 years (mean 13), with symptomatic bradycardia requiring pacing therapy, underwent comparative treadmill exercise testing in randomized fixed rate (VVI) and VVIR pacing modes. In all instances, implanted activity pulse generators (Medtronic Model 8403) were programmed to identical age-appropriate low paced rates during VVI and VVIR modes with the upper rate response at 150 ppm. All studies were performed at least 2 weeks apart. Physiological values of heart rate, blood pressure, work rate (watts), oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were monitored continuously during each test using a 1 minute incremental treadmill protocol. Ventilatory anaerobic threshold (VAT) was calculated from VO2, VCO2, and minute ventilation. The results demonstrated that although there was a significant increase in paced heart rate per minute throughout exercise (P less than 0.01) with VVIR pacing, maximum watts, VO2, and VAT remained unchanged. These findings indicate that in spite of an improved chronotropic response to exercise, children with univentricular hearts following the Fontan procedure continue to demonstrate altered hemodynamics which negate potential benefits of VVIR pacing. PMID- 1721225 TI - Efficacy and safety of oral sotalol in early infancy. AB - Sotalol, a nonselective beta blocking agent with additional Class III activity has been shown to be extremely effective in the treatment of supraventricular tachycardias in adults and children. Little information is available on its use in infants. From August, 1985 to April, 1990, 18 infants, 2 months of age or less, were treated with oral sotalol for supraventricular arrhythmias. Their age ranged from a few hours to 2 months, mean 5 weeks, at the start of treatment. Weights were between 2.58-5 kg, mean 3.9 kg and dosage 2-4 mg/kg/24 hrs given in two equal doses, 12 hourly. Sixteen infants had structurally normal hearts, one had multiple cardiac rhabdomyomas, and one was postoperative Mustard procedure for transposition of the great arteries. Thirteen of 18 infants had reentrant forms of supraventricular tachycardia, six of these had overt preexcitation. Two infants had chaotic atrial tachycardia, two atrial flutter, and one with ectopic atrial tachycardia. Previous antiarrhythmic therapy had been unsuccessful in 12 patients. All infants, except one with chaotic atrial tachycardia, were successfully controlled with sotalol. Ten infants discontinued therapy between the ages of 7 and 18 months as it was felt to be no longer necessary. Mean duration of treatment was 12.8 months. Three had recurrences of their arrhythmia and were again successfully controlled by sotalol. Mild sinus bradycardia occurred in all infants. No other side effects were noted. Sotalol is an effective, safe drug for the treatment of supraventricular tachycardias in early infancy. PMID- 1721226 TI - Steroid-eluting epicardial leads in pediatrics: improved epicardial thresholds in the first year. AB - From 1986 to 1991, we evaluated the clinical use of three new epicardial lead designs incorporating a steroid-eluting electrode. Medtronic models SP2114 (bipolar high profile), 10320 (bipolar low profile), 10295A/4965 (unipolar low profile) steroid epicardial (SE) leads were used on either atrium or ventricle for a total of 21 lead placements in 17 patients. Energy thresholds (T) were calculated and compared with our most recent 16 nonsteroid epicardial (NE) Medtronic model 4951 lead implants for which T was available. SE leads demonstrated no acute T rise and continued T improvements at 1 year of follow-up. We conclude that epicardial application of SE lead technology offers a major improvement in pacing lead function and potential pacemaker longevity over NE leads in pediatric patients in whom endocardial pacing is precluded by size or anatomy. PMID- 1721227 TI - The 1989 World Survey of Cardiac Pacing. PMID- 1721228 TI - Obstructive jaundice. Nonsurgical options for 'surgical' jaundice. AB - The development of nonoperative methods of biliary drainage has altered traditional concepts regarding management of medical and surgical jaundice. Patients with newly diagnosed obstructive jaundice typically are elderly and have an unresectable neoplasm. Because surgical cure is often impossible and operation is usually risky in such patients, decompression of the biliary tree by endoscopic retrograde cholangiopancreatography and endoscopically inserted biliary stents has become an increasingly popular means of palliation. Percutaneous transhepatic cholangiography and surgical bilidigestive bypass remain important alternatives. Selection of optimal management for the individual patient requires an in-depth evaluation by a skilled team consisting of the primary care physician, endoscopist, interventional radiologist, and surgeon. PMID- 1721229 TI - Modification of energy density with inhibitors of carbohydrate and fat digestion. PMID- 1721230 TI - The effect of age, sex, weight and height on the plasma concentrations in healthy subjects of the acidic metabolites of some biogenic monoamines involved in psychiatric and neurological disorders. AB - 1. The plasma concentrations of unconjugated phenylacetic acid and m hydroxyphenylacetic acid are lower in male than in female subjects. 2. The plasma concentrations of unconjugated phenylacetic acid and mandelic acid decrease with increasing weight and height for all subjects combined. The same relationships apply for both males and females but are significant only for males. 3. Homovanillic and vanillylmandelic acid concentrations in plasma increase with age. 4. The importance of using age, sex, weight and height matched groups in studies involving the plasma concentrations of some of the trace amine metabolites in psychiatric disorders has been demonstrated. This is particularly the case for phenylacetic acid, the major metabolite of phenylethylamine which is now thought to be a neuromodulator of catecholaminergic neurotransmission. PMID- 1721231 TI - [Genetical aspects of bronchial asthma]. PMID- 1721232 TI - [Palliative care: therapeutic possibilities for patients at the end of life]. AB - Palliation seeks to provide alleviation of pain and all other physical complaints, together with psychologic and spiritual support. The commonly used symptomatic therapies are undoubtedly fit to significantly improve the patient's quality of life. Subjects of palliative medicine should be included in postgraduate nursing education, covering above all the basic notions listed by the WHO, such as the principles of efficient communication, the pathophysiology of symptoms in advanced cancer, the evaluation and importance of pain, the basic physiologic and spiritual needs of the seriously ill or the dying. It is also mandatory to care about the physiologic needs of the patient's family and to know the physiologic and psychologic reactions in coping with grief. PMID- 1721233 TI - [Neurosurgery in old age. II: CNS tumors--cerebrospinal-vascular diseases--pain surgery--conclusions]. AB - The relative percentage of patients who are admitted to our clinic with benign tumours often susceptible to therapy has increased from 23% (1974) to 55% (1989). In patients over 60 years old, these are above all cranial tumours (most frequently meningiomas), whereas metastases predominate at the spinal cord. 8% of the cranial meningiomas show an apoplectiform course (mostly steal effects). On the other hand, very large cranial meningiomas causing few symptoms are particularly frequent in elderly patients (larger reserve space, reduced tendency to edema). The use of minimally damaging surgical approaches, palliative operations and possibly a cautious radicality adapted to location and extent are especially important for tumour surgery in elderly patients, especially for tumours with a low tendency to proliferate. Our own experience in vascular pathology concern arteriovenous aneurysms and the diagnosis/therapy of spontaneous intracerebral hemorrhage and space-occupying malacia in the region of the cerebrum and the cerebellum. Epidural hematomas and the spinal dural arteriovenous fistulae which often become manifest at an advanced age owing to increasing medullary venous stasis are of particular therapeutic importance in elderly patients. Depending on the situation, direct surgical elimination or treatment by selective embolization may be considered. Typical trigeminal neuralgia is especially frequent in patients over 60 years in neurosurgical treatment of very severe therapy-resistant pain. Whereas the microvascular decompression of the trigeminal roots near the pons (compression by arterial loops) is most important in younger patients, we use the minimally invasive, relatively simple percutaneous thermo-rhizotomy in the Gasserian ganglion with selective functional elimination of the thinner pain fibers in elderly patients. Ablative measures are otherwise possible, above all in nociceptive pain, whereas the stimulation of the lemniscal system is of primary significance in deafferentation pain (e.g. causalgia). Spinal stimulation in inoperable peripheral arterial occlusive disease (leg, pelvis) to improve the microcirculation is of special importance: The prospect of success is greater than 80% in Fontain stage III. We do not apply subarachnoid spinal morphine administration only in malignancies, in contrast to intraventricular morphine administration (right anterior horn). For elderly patients with very severe inoperable lumbar stenosis (mostly spondylarthritis), the dosage-controlled continuous low-dose subarachnoid administration of morphine via a subcutaneously implanted programmable pump with a reservoir is suitable in some cases. These examples show that neurosurgery may be appropriate even in elderly patients. Today, more differentiated and at the same time minimally invasive diagnostic and surgical methods are available. PMID- 1721234 TI - Rabbit serum amyloid protein A: expression and primary structure deduced from cDNA sequences. AB - Serum amyloid A protein (SAA), the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis, is an acute phase plasma apolipoprotein produced by hepatocytes. The primary structure of SAA demonstrates high interspecies homology. Several isoforms exist in individual species, probably with different amyloidogenic potential. The nucleotide sequences of two different rabbit serum amyloid A cDNA clones have been analysed, one (corresponding to SAA1) 569 base pairs (bp) long and the other (corresponding to SAA2) 513 bp long. Their deduced amino acid sequences differ at five amino acid positions, four of which are located in the NH2-terminal region of the protein. The deduced amino acid sequence of SAA2 corresponds to rabbit protein AA previously described except for one amino acid in position 22. Eighteen hours after turpentine stimulation, rabbit SAA mRNA is abundant in liver, while lower levels are present in spleen. None of the other extrahepatic organs studied showed any SAA mRNA expression. A third mRNA species (1.9 kb) hybridizing with a single-stranded RNA probe transcribed from the rabbit SAA cDNA, was identified. SAA1 and SAA2 mRNA were found in approximately equal amounts in turpentine-stimulated rabbit liver, but seem to be coordinately decreased after repeated inflammatory stimulation. PMID- 1721235 TI - Presence of human chromosome 1 with expression of human decay-accelerating factor (DAF) prevents lysis of mouse/human hybrid cells by human complement. AB - Xenogeneic organs transplanted to phylogenetically distant species are subject to rapid destruction mediated by complement. In humans, the complement activation is regulated by several proteins encoded by a series of closely linked genes (RCA locus) located on chromosome 1. The mouse/human hybrid cell line B10 was found to have retained human chromosome 1. FACS analysis confirmed that RCA products such as decay-accelerating factor (DAF) were expressed on the membrane surface of B10 cells. When exposed to human or rabbit complement in the presence of 'naturally occurring' human anti-mouse antibodies these cells were not lysed by human complement but were killed by rabbit complement. This effect could be abrogated by addition of anti-DAF monoclonal antibody (IC6). The results offer potential for genetic manipulation of the human complement regulatory products in animals to overcome xenograft hyperacute rejection. PMID- 1721236 TI - [The course of pregnancy and teratogenicity of antiepileptic agents in 66 patients with epilepsy]. AB - We report on 79 pregnancies in 66 female outpatients with epilepsy. An increase of seizure frequency was significantly more frequent in complex partial seizures than in grand mal seizures and in absences. The reason for these disparities are not clear. In most patients a raised frequency of seizures during pregnancy decreased again after delivery. Carbamazepine was the antiepileptic drug prescribed most frequently followed by valproic acid. The course of the blood levels of carbamazepine and valproic acid was nonuniform during pregnancy. Total concentrations of carbamazepine in cord blood were on average 84.5% of those in maternal blood (n = 22). Valproic acid blood levels were on average 183% of those in maternal blood (n = 15). It is still unclear whether these differences are clinically relevant. During the last weeks of pregnancy we found an increase of the free fraction of carbamazepine and valproic acid. Simultaneously the total protein concentration decreased. Until now these findings are without clinical relevance. The course of labor did not differ from normal population concerning the ratios of spontaneous labor, cesarean section and delivery by forceps. Miscarriage and perinatal mortality were 2.7% each and outnumbered the risk in the general population. In 42.8% of the neonates one to three perinatal complications were observed. The ratio of perinatal complications is not different between patients with monotherapy and combined therapy respectively. There was a tendency to lower values of length, weight and head circumference in the male neonates but not in the female neonates. The risk of minor malformations was 26%, the risk of major malformations was 14% (including one case of suspected malformation) without a discernible correlation with a specific antiepileptic drug. PMID- 1721237 TI - [Cerebral sequelae of stenosing and occlusive diseases of the internal carotid artery. Importance of Doppler transcranial examination. Part 2]. AB - The author reports 57 stenosis or occlusions of the internal carotid artery, 13 dissections and 44 atheromatous lesions. She studies the correlation between intracranial collateralisation detected by transcranial Doppler sonography (TCD) and clinical symptoms as well as lesions seen on cerebral CT-scan. Two types of ischaemic lesions are described: border-zone infarcts, probably haemodynamic in origin and territorial infarcts, probably thromboembolic. TCD lets suppose that strokes in this series have a thromboembolic origin. On the other hand it is possible that collateralisation depending on both anterior and posterior communicating arteries is not sufficient, because such a collateralisation is found above all in the 2 most severe symptomatic groups and in many territorial infarcts. In the atheromatous group, occlusions are often asymptomatic and territorial infarcts less extended, whereas in the dissections group all occlusions determined a territorial infarct, often very important. TCD alone doesn't allow to definitely conclude about pathogeny of ischaemic lesions nor on collateralisation value. The adjunction of CO2 reactivity tests in middle cerebral artery will perhaps give the clues of these problems. PMID- 1721238 TI - [A computer algorithm for diagnostic assessment with DSM-III in the early course of schizophrenic diseases]. AB - The purpose of the computer algorithm described here is the evaluation of diagnostic criteria according to DSM-III for schizophrenia and schizophreniform disorders. It also dates the first time point of the assessment of these diagnoses. The necessary information comes from a semistructured interview, called IRAOS (Interview for the Retrospective Assessment of the Onset of Schizophrenia). With this interview early indicators of a beginning schizophrenia can be evaluated in their chronological order and their type of course. The algorithm was first used in a sample of patients admitted for the first time with a diagnosis of either schizophrenia or paranoid psychosis. One third of these patients fulfills the DSM-III-criterion of a duration of at least six months. The other patients fulfill criterion B of a schizophreniform disorder. To strengthen the validity of a diagnosis including the criteria A up to E successively, the sample is reduced to 70%. The average time point of the first assessment of the diagnosis by the computer algorithm is about 1.5 years before the index admission. Together with the IRAOS the computer algorithm allows an operationalized assessment of the real onset of schizophrenia. PMID- 1721239 TI - [Differentiation of suicide and parasuicide. A study of the last letters of suicidal patients]. AB - The paper presents a content-analytical comparison between "last letters" of suicidals (SUI) and parasuicidals (PARA). So far studies on this topic are mostly performed by "external" parameters, e.g. sociodemographic or anamnestic data. The content-analytical approach provides to describe the presuicidal situation on base of "internal" variables. Compared with the suicidal notes the parasuicidal communication can be evaluated as more ambivalent, emotionally disturbed, and cognitively restricted. The successful reclassification of the notes into the two samples by a stepwise discriminant analysis supports the hypothesis, that both groups are distinct even on base of internal variables. PMID- 1721240 TI - [Diagnostic careers]. AB - Careers of diagnoses: The case history of 80 schizophrenics, 40 borderline personalities and 20 persons with bipolar disorders, who were treated in the Basle University Psychiatric Outpatient Department for at least 5 years, were examined concerning the first diagnosis and the change of the diagnostic evaluation. On one hand the aim was to evaluate on which factors the first diagnosis was based (heredity, anamnesis, previous hospitalizations, actual psychostatus), on the other hand, how these changes of diagnosis could happen (course, new evaluation of psychopathology). It appeared that 60% of the evaluated schizophrenics and 40% of the evaluated patients with bipolar disorders had the same diagnosis at their first consultation as 5 years afterwards, but the same applied only to 12.5% of the patients with borderline personalities. These differences are significant. The average period between first contact and today's definitive diagnosis was 2.2 years for schizophrenics, 7 years for patients with bipolar disorders and 8.8 years for patients with borderline personalities. Also these differences are significant. Furthermore the first diagnosis but also the change of diagnosis and today's diagnosis were evaluated. Thereby it was examined if the original diagnosis and the diagnosis 5 years later can be understood or if they cannot be understood at all or have been made incompletely. The therapy of that time as well as the one of today have been examined and evaluated. PMID- 1721241 TI - Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. AB - Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method. PMID- 1721242 TI - Molecular architecture and electrostatic properties of a bacterial porin. AB - The integral membrane protein porin from Rhodobacter capsulatus consists of three tightly associated 16-stranded beta barrels that give rise to three distinct diffusion channels for small solutes through the outer membrane. The x-ray structure of this porin has revealed details of its shape, the residue distributions within the pore and at the membrane-facing surface, and the location of calcium sites. The electrostatic potential has been calculated and related to function. Moreover, potential calculations were found to predict the Ca2+ sites. PMID- 1721243 TI - Participation of postsynaptic PKC in cerebellar long-term depression in culture. AB - Long-term depression (LTD) in the intact cerebellum is a decrease in the efficacy of the parallel fiber-Purkinje neuron synapse induced by coactivation of climbing fiber and parallel fiber inputs. In cultured Purkinje neurons, a similar depression can be induced by iontophoretic glutamate pulses and Purkinje neuron depolarization. This form of LTD is expressed as a depression of alpha-amino-3 hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA)-mediated current, and its induction is dependent on activation of metabotropic quisqualate receptors. The effect of inhibitors of protein kinase C (PKC) on LTD induction was studied. Inhibitors of PKC blocked LTD induction, while phorbol-12,13-diacetate (PDA), a PKC activator, mimicked LTD. These results suggest that PKC activation is necessary for the induction of cerebellar LTD. PMID- 1721244 TI - Effects of histamine, ethanol, and a detergent on exudation and absorption across guinea pig airway mucosa in vivo. AB - This study examined effects of three substances that cause mucosal provocation (histamine, ethanol, and the detergent dioctylsodium sulphosuccinate (DOSS] on the flux of solutes across airway vascular mucosal barriers in anaesthetised guinea pigs. The inward flux was assessed as absorption of iodine-131 labelled albumin (MW 69,000) from the tracheobronchial surface into the circulation and the outward flux as the exudation of two intravenously administered plasma tracers--125I albumin (MW 69,000) and fluorescein isothiocyanate conjugated (FITC) dextran (MW 70,000)--into the airway. The absorption of technetium-99m labelled DTPA (MW 492) from the tracheobronchial airways was determined in separate experiments. Histamine (5.0 nmol) dissolved in 40 microliters saline and superfused on the tracheobronchial mucosal surface caused significant and similar entry of 125I albumin and FITC dextran into the airway lumen. This dose of histamine did not, however, alter the absorption of small (99mTc DTPA) or large (131I albumin) solutes across the airway mucosa. Ethanol (0.17 mumol), superfused in the same way, also caused significant exudation of the plasma tracers into the airway lumen. In addition, ethanol increased the absorption of 131I albumin without causing change in the disappearance rate of 99mTc DTPA. The detergent, DOSS (0.28 nmol), dissolved in ethanol (0.17 mumol), caused a pronounced increase in exudation and much increased absorption of small and large tracer solutes. Thus three patterns of change in airway mucosal barriers were found. The agents that are toxic to membranes, ethanol and DOSS, caused a bidirectional increase in permeability across the mucosa, whereas histamine caused only an outward exudative flux. The results obtained with histamine are similar to those seen previously with bradykinin, capsaicin, and allergen, suggesting that endogenous inflammatory mediators have a role in mucosal defence, producing entry of plasma exudates into the airway lumen without increasing the mucosal absorption of luminal material. PMID- 1721245 TI - [Prohibition of lindane-containing ectoparasitic agents]. PMID- 1721246 TI - Bradykinin-induced cough reflex markedly increases in patients with cough associated with captopril and enalapril. AB - We studied the effects of angiotensin converting enzyme (ACE) inhibitors on cough responses to bradykinin (BK), substance P (SP) and citric acid in a double blind, random study on 10 hypertensive patients receiving ACE inhibitors. Of these patients, five had reported cough with ACE inhibitors. Cough responses to citric acid were similar between patients with and without cough, and SP up to 10(-5) M did not cause cough in any of the subjects. BK caused cough at 13.4 +/- 1.2 (-log M) in 5 patients with cough associated with ACE inhibitors, but it did not cause cough at concentrations up to 10(-5) M in other 5 patients. One month after the withdrawal of ACE inhibitors, 5 patients were free from cough symptoms, and BK did not cause cough up to 10(-5) M in these patients, except for one who coughed at 10(-9) M, without changes in responses to citric acid. BK caused cough at 14.3 +/- 0.7 (-log M) although BK1-7, a major metabolite of BK by ACE, caused cough at 5.7 +/- 0.7 (-log M) in another 3 patients with cough associated with ACE inhibitor. These results suggest that impaired metabolism of BK induced by ACE inhibitors may relate to the manifestation of cough in hypertensive patients receiving ACE inhibitors. PMID- 1721247 TI - Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. AB - BACKGROUND AND PURPOSE: Accurate and reproducible determination of the size and location of cerebral infarcts is critical for the evaluation of experimental focal cerebral ischemia. The purpose of this study was to compare intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride with immersion of brain tissue in 2,3,5-triphenyltetrazolium chloride to delineate brain infarcts in rats. METHODS: After 6, 24, or 48 hours of ischemia induced by permanent middle cerebral artery occlusion, some rats were perfused with 2,3,5 triphenyltetrazolium chloride; other rats were given an overdose of barbiturates, after which brain sections were immersed in 2,3,5-triphenyltetrazolium chloride. Coronal sections were taken 4, 6, and 8 mm from the frontal pole, and infarct areas in perfused and immersed sections were compared; subsequently, the same sections were stained with hematoxylin and eosin. RESULTS: In rats subjected to 24 or 48 hours of occlusion, areas of infarction were clearly defined with both 2,3,5-triphenyltetrazolium chloride staining techniques, and the infarct sizes correlated well with the results of hematoxylin and eosin staining (r = 0.85 0.94). CONCLUSIONS: These results demonstrate that intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride is an accurate, inexpensive, and efficient staining method to detect infarcted tissue 24 and 48 hours after the onset of ischemia in rats. PMID- 1721248 TI - Analysis of primary HLA-specific cytotoxic T cell response in graft-draining lymph nodes--a transgenic mouse model for in vivo recognition of human MHC antigens. AB - In order to characterize primary anti-HLA cytotoxic T cells and especially those involved in graft rejection, we have utilized a transgenic mouse model. Mice (non transgenic and HLA-transgenic) were grafted with spleen cells originating from H 2-matched transgenic mice expressing HLA-B27 molecules, and cells from graft draining lymph nodes were tested in CML assay to investigate the primary in vivo induced CTL responses. The results showed that HLA-B27 molecules were able to raise strong primary xenogeneic CTL responses. Results from split-well analysis indicated that although recognition of HLA-B27 by primary CTL induced in nontransgenic recipients is predominantly unrestricted by H-2, a small fraction (ranging from 2% to 27%) of the primary in vivo induced CTL is able to recognize HLA-B27 in an H-2-restricted manner. HLA-specific H-2-restricted CTL had never so far been demonstrated in the primary T cell response. Thus the protocol used in our study for the generation of a primary CTL response seems to provide not only a more appropriate representation of cytotoxic T cells sensitized by a graft, but also to be a more sensitive approach than the usually used in vitro mixed lymphocyte culture. PMID- 1721249 TI - Stimulation of distinct T cell subsets in MLR using human macrophage hybridomas differentially expressing class II antigens. AB - Recognition of class II antigens by alloreactive T cells is thought to be the major mechanism by which tissues undergo rejection. However, the specific role of the various class II antigens in the stimulation of these alloreactive cells remains to be elucidated. We have recently generated a series of human monocyte hybridomas that express distinct patterns of class II antigen expression. HLA-DR+ as well as HLA-DR-DP+DQ+ hybrids were capable of promoting T cell proliferation in a unidirectional mixed lymphocyte reaction. T cells stimulated by the HLA-DR+ clone 16.1 were predominantly of the CD4 (helper/inducer) phenotype. In contrast, T cells stimulated by the HLA-DR-DP+DQ+ clone 13 appear to reside in the CD8+ T cell subpopulation. Functional assessment of the T cell blasts generated in these cultures demonstrated a predominant helper T cell effect by those T cells stimulated by the HLA-DR+ clone 16.1, while suppressor cell activity was exhibited by T cells stimulated with the HLA-DR-DP+DQ+ clone 13. These data suggest that there may be a differential role for distinct class II molecules in the stimulation of T cell subpopulations. PMID- 1721250 TI - Interaction between FK506 and clotrimazole in a liver transplant recipient. PMID- 1721251 TI - Improved liver preservation with addition of iloprost to Eurocollins and University of Wisconsin storage solutions. PMID- 1721253 TI - First International Congress on FK 506. August 21-24, 1991, Pittsburgh, PA. PMID- 1721252 TI - Low-potassium UW solution for lung preservation. Comparison with regular UW, LPD, and Euro-Collins solutions. AB - University of Wisconsin solution has been used successfully in clinical kidney and liver preservation. The object of this study was to determine if low potassium UW (LPUW) solution could be applied to pulmonary preservation. Rabbit lungs were stored after hypothermic pulmonary artery (PA) flush with four different solutions (group 1: low-potassium dextran (LPD) solution, group 2: high potassium UW (HPUW) solution, group 3: LPUW solution, group 4: modified Euro Collins (E-C) solution). The lungs were preserved at 10 degrees C for 30 hr and evaluated in an ex vivo ventilation/perfusion apparatus using fresh pooled venous rabbit blood. Mean PA flush pressures (MFP) during harvesting were significantly lower in groups 1 and 3 (8.1 +/- 1.0 mmHg and 7.3 +/- 0.6 mmHg, respectively; mean +/- SEM) than in groups 2 and 4 (15.5 +/- 1.7 mmHg and 12.3 +/- 0.9 mmHg, respectively). Lungs in groups 1 and 3 showed significantly higher PaO2 (103.5 +/ 8.0 mmHg and 89.3 +/- 7.2 mmHg) than groups 2 and 4 (48.3 +/- 7.7 mmHg, 66.7 +/- 4.7 mmHg). Groups 1 and 3 showed significantly lower wet/dry weight (W/D) ratios after reperfusion (6.21 +/- 0.15 and 6.39 +/- 0.23) than groups 2 and 4 (7.70 +/- 0.57 and 7.13 +/- 0.21, respectively). There were no significant differences in MFP, PaO2, PaCO2, mean pulmonary artery pressure, or W/D ratio between groups 1 and 3. These results suggest that LPUW solution may be as beneficial as LPD solution for pulmonary arterial flush and lung preservation. PMID- 1721254 TI - FK 506: historical perspectives. PMID- 1721255 TI - Effective and safe use of FK 506: combination treatment with rapamycin or RS 61443 in experimental organ transplantation. PMID- 1721256 TI - Potential roles of other FK 506-binding proteins in mediating the effects of FK 506. PMID- 1721257 TI - In vivo immunopharmacology of the macrolides FK 506 and rapamycin: toward the era of rational immunosuppressive drug discovery, development, and use. PMID- 1721258 TI - FK 506 assay past and present--characteristics of FK 506 ELISA. PMID- 1721259 TI - Practical aspects of FK 506 analysis (Pittsburgh experience). PMID- 1721260 TI - Monitoring FK 506 concentrations in plasma and whole blood. AB - The FCL provides support for clinical trials of FK 506 in liver transplantation by monitoring drug concentrations in plasma and whole blood. The sensitive EIA method of Tamura et al was adapted for routine use. Extraction of drug from 300 microL plasma or 20 microL whole blood samples was carried out using MeCl. An alternative solid phase (Sep-Pak) extraction method was also tested. Nonlinear temperature-sensitive binding of FK 506 to red cells necessitates plasma separation from whole blood at 37 degrees C. The analytical procedure entails numerous steps and efforts were made to improve the accuracy of the method. Interday CVs of FK 506 in plasma (0.3 to 3.0 ng/mL) and whole blood (4 to 60 ng/mL) ranged up to about 20% during validation testing and are maintained below 30% during routine assay use. However, daily QC samples occasionally yield unexpectedly high FK 506 concentrations. The MeCl and solid phase extraction methods yield FK 506 concentrations that reasonably correlate but the MeCl method produces plasma concentrations that are about 30% lower. FK 506 is appreciably bound in red cells with a nonlinear WBPR of 20 to 50 at low plasma concentrations (0 to 2 ng/mL) and a ratio of about 11 at plasma concentrations above 5 ng/mL. This may complicate conversion from plasma to whole blood for routine therapeutic monitoring. The described procedures for FK 506 are being implemented at various clinical sites in the United States with the FCL providing assistance with a quality assurance program to assure intersite comparability of assays results. PMID- 1721261 TI - Pharmacokinetics of FK 506 in transplant patients. PMID- 1721262 TI - High performance liquid chromatography/mass spectrometry of FK 506 and its metabolites in blood, bile, and urine of liver grafted patients. PMID- 1721263 TI - FK 506: monitoring in plasma or in whole blood? PMID- 1721264 TI - A whole blood FK 506 assay for the IMx analyzer. PMID- 1721265 TI - A combined HPLC-ELISA evaluation of FK 506 in transplant patients. PMID- 1721266 TI - Effect of temperature and hematocrit on plasma concentration of FK 506. PMID- 1721267 TI - Plasma level of FK 506 in newborn goats and infant baboons. PMID- 1721268 TI - Pharmacokinetic study of FK 506 in the rat. PMID- 1721269 TI - Uptake of FK 506 by lymphocytes and erythrocytes. PMID- 1721270 TI - Comparative study of cyclosporine and FK 506 dosage requirements in adult and pediatric orthotopic liver transplant patients. PMID- 1721271 TI - Four-hour versus 24-hour intravenous infusion of FK 506 in liver transplantation. PMID- 1721272 TI - Strategy of FK 506 therapy in liver transplant patients: effect of graft function. PMID- 1721273 TI - Pharmacokinetics of FK 506 during maintenance therapy in liver transplant patients. PMID- 1721275 TI - Evaluation of a novel "intelligent" dosing system for optimizing FK 506 therapy. PMID- 1721274 TI - Pharmacokinetics of cyclosporine and nephrotoxicity in orthotopic liver transplant patients rescued with FK 506. PMID- 1721276 TI - Effects of FK 506 on human hepatic microsomal cytochrome P-450-dependent drug metabolism in vitro. PMID- 1721277 TI - Inhibition of drug metabolism in rat and human liver microsomes by FK 506 and cyclosporine. PMID- 1721278 TI - Nephrotoxic potential of FK 506. PMID- 1721279 TI - Effect of FK 506 on human hepatic cytochromes P-450: interaction with CyA. PMID- 1721280 TI - Interactions of FK 506 and cyclosporine metabolism. PMID- 1721281 TI - Interaction between FK 506 and cyclosporine in dogs. PMID- 1721282 TI - The pharmacodynamics of pentobarbital following FK 506 therapy. PMID- 1721283 TI - Effect of FK 506 chronic administration on bromosulphthalein hepatic excretion in rats. PMID- 1721285 TI - FK 506 modulates D-galactosamine-induced hepatitis in rats. PMID- 1721284 TI - A biochemical and 31P-NMR investigation of the effect of FK 506 and cyclosporine pretreatment on immobilized hepatocytes perifused with ethanol. PMID- 1721286 TI - Inhibition of insulin release by FK 506 and its prevention by rioprostil, a stable prostaglandin E1 analogue. PMID- 1721287 TI - The effects of FK 506, cyclosporine, and rapamycin on liver growth in vitro and in vivo. PMID- 1721288 TI - Antiproliferative effect of FK 506 and cyclosporine on adult human hepatocytes in culture. PMID- 1721290 TI - Cyclosporine and FK 506 induced inhibition of renal epithelial cell proliferation. PMID- 1721289 TI - FK 506 is less cytotoxic than cyclosporine to human and rat hepatocytes in vitro. PMID- 1721291 TI - Growth inhibition of the MOLT-4 human T-leukemia cell line. A comparison of cyclosporine and FK 506. PMID- 1721292 TI - Rapamycin inhibits spontaneous and fibroblast growth factor beta-stimulated proliferation of endothelial cells and fibroblasts. PMID- 1721294 TI - Site of action of cyclosporine and FK 506 in the pathways of communication between the T-lymphocyte antigen receptor and the early activation genes. PMID- 1721293 TI - Immunophilin-ligand complexes as probes of intracellular signaling pathways. PMID- 1721295 TI - Is FKBP involved in the immunosuppressive and/or toxic mechanism of action of FK 506? PMID- 1721296 TI - FK 506 and rapamycin: molecular probes of T-lymphocyte activation. PMID- 1721297 TI - Yeast cyclophilin-related gene encodes a nonessential second peptidyl-prolyl cis trans isomerase associated with the secretory pathway. PMID- 1721298 TI - The FK 506-sensitive nature of the interleukin-2 promoter is derived from a specific array of multiple regulatory elements. PMID- 1721299 TI - Effect of immunosuppressive drugs on cytokine gene transcription studied by message amplification phenotyping (MAPPing) polymerase chain reaction. PMID- 1721300 TI - Up-regulation of gene expression by FK 506. PMID- 1721301 TI - Effect of FK 506 on FK-binding protein and transforming growth factor beta gene expression. PMID- 1721302 TI - Characterization of the human FKBP-12 gene and related pseudogenes. PMID- 1721303 TI - Gene expression of FK 506-binding protein. PMID- 1721304 TI - Thermodynamics of interaction of FK 506-binding protein and its ligands. PMID- 1721305 TI - Purification of a 50- to 58-kDa immunophilin from human spleen and a Jurkat T cell line capable of binding cyclosporine A, FK 506, and rapamycin. PMID- 1721306 TI - Identification of FKBP-related proteins with antibodies of predetermined specificity and isolation by FK 506 affinity chromatography. PMID- 1721308 TI - Mast cell biochemical and functional heterogeneity. PMID- 1721307 TI - In vitro assessment of FK 506 immunosuppressive activity in transplant patients. PMID- 1721309 TI - Antiinflammatory effect of FK 506 on human basophils. PMID- 1721310 TI - Activation of transcription factor NF kappa B in Jurkat cells is inhibited selectively by FK 506 in a signal-dependent manner. PMID- 1721311 TI - The effects of FK 506 on cytokine production are dependent on the mode of cell activation. PMID- 1721312 TI - FK 506, rapamycin, and cyclosporine: effects on IL-4 and IL-10 mRNA levels in a T helper 2 cell line. PMID- 1721313 TI - Kinetics of early T-cell repopulation in the mouse following syngeneic bone marrow transplantation: FK 506 causes a maturational defect of CD4+ CD8- T cells. PMID- 1721314 TI - Effects of FK 506 and cyclosporine on T-cell tolerance: inhibition of a "protective" mechanism that regulates activation-induced apoptosis in developing thymocytes. PMID- 1721315 TI - Comparative in vitro studies on the immunosuppressive activities of mycophenolic acid, bredinin, FK 506, cyclosporine, and rapamycin. PMID- 1721316 TI - Beneficial immunosuppressive effect of combined use of FK 506 and cyclosporine assessed by proliferative responses of cloned human T cells. PMID- 1721317 TI - Combined immunosuppressive effect of FK 506 and other immunosuppressive agents on PHA- and CD3-stimulated human lymphocyte proliferation in vitro. PMID- 1721318 TI - The effect of FK 506 on peripheral blood T-lymphocyte subsets in orthotopic liver transplant patients. PMID- 1721319 TI - Effects of FK 506, mycophenolic acid, and bredinin on OKT-3-, PMA-, and alloantigen-induced activation molecule expression on cultured CD4+ and CD8+ human lymphocytes. PMID- 1721320 TI - Diminished lymphocyte growth from endomyocardial biopsies from cardiac transplant patients on FK 506 immunosuppression. PMID- 1721321 TI - Effect of FK 506 on CD4+ and CD8+ T-cell function in vivo. PMID- 1721322 TI - Effects of FK 506 on in vivo immunity in comparison to cyclosporine. PMID- 1721324 TI - FK 506 favors the generation of memory T cells in vitro. PMID- 1721323 TI - Inhibition of T-cell function by FK 506 and cyclosporine is not accompanied by alterations in intracellular calcium. PMID- 1721325 TI - Immunosuppressive effect of FK 506 on cytotoxic T lymphocytes and delayed-type hypersensitivity in rats. PMID- 1721327 TI - Effect of FK 506 on rat leukocyte chemotaxis. PMID- 1721326 TI - Evaluation of the influence of FK 506, rapamycin, and cyclosporine on processing and presentation of particulate antigen by macrophages: assessment of a drug "carry-over" effect. PMID- 1721328 TI - Categorizing receptor-signaling pathways with FK 506 and rapamycin. PMID- 1721329 TI - Immunosuppressive effects of FK 506 on rat lymphoid organs. PMID- 1721330 TI - Effect of FK 506 on generation of activated macrophages invading rejected skin allografts in rats: discrepancy of in vitro versus in vivo effects. PMID- 1721331 TI - Flow cytometric analysis of lymphocyte populations in FK 506-treated newborn goats. PMID- 1721332 TI - FK 506 and xenogeneic human anti-porcine cellular reactivity. PMID- 1721333 TI - A randomized trial of primary liver transplantation under immunosuppression with FK 506 vs cyclosporine. PMID- 1721334 TI - Use of FK 506 for treatment of chronic rejection after liver transplantation. PMID- 1721335 TI - FK 506 rescue therapy for resistant rejection episodes in liver transplant recipients. PMID- 1721336 TI - FK 506 rescue therapy in liver transplant recipients with drug-resistant rejection. PMID- 1721337 TI - FK 506: reversal of humorally mediated rejection following ABO-incompatible liver transplantation. PMID- 1721338 TI - FK 506 for rescue treatment of acute and chronic rejection in liver allograft recipients. PMID- 1721339 TI - FK 506 rescue therapy in liver transplantation: outcome and complications. PMID- 1721340 TI - FK 506 rescue therapy for refractory acute rejection in five liver recipients. PMID- 1721341 TI - Compassionate use of FK 506 in pediatric liver transplantation: a pilot study. PMID- 1721342 TI - Experience with FK 506 in living related donor liver transplantation. PMID- 1721343 TI - FK 506 versus cyclosporine in pediatric liver transplantation. PMID- 1721344 TI - Liver transplantation of American veterans under FK 506 immunosuppression: a preliminary report. PMID- 1721345 TI - Early experience with FK 506 induction immunosuppression--suggestion for using oral FK 506. PMID- 1721346 TI - The lymphocytotoxic crossmatch in liver transplantation: a clinicopathologic analysis. PMID- 1721347 TI - Correlation of rejection episodes with FK 506 dosage, FK 506 level, and steroids following primary orthotopic liver transplant. PMID- 1721348 TI - Retransplantation of liver: a comparison of FK 506- and cyclosporine-treated patients. PMID- 1721349 TI - Liver retransplantation in adults: overall results and determinant factors affecting the outcome. PMID- 1721350 TI - Changes in quality of life following conversion from CyA to FK 506 in orthotopic liver transplant patients. PMID- 1721351 TI - CMV infection in liver transplantation under cyclosporine or FK 506 immunosuppression. PMID- 1721352 TI - Infectious complications of pediatric liver transplantation under FK 506. PMID- 1721353 TI - The spectrum of aspergillosis in liver transplant patients: comparison of FK 506 and cyclosporine immunosuppression. PMID- 1721354 TI - CMV retinitis and the use of FK 506. PMID- 1721355 TI - Posttransplant lymphoproliferative disorders occurring under primary FK 506 immunosuppression. PMID- 1721357 TI - Interferon therapy of hepatitis following liver transplantation under FK 506 or cyclosporine. PMID- 1721356 TI - Serial monitoring of immunologic function and phenotype of lymphocytes in the blood of transplanted patients randomized to cyclosporine or FK 506. PMID- 1721358 TI - The clinical trial of FK 506 as primary and rescue immunosuppression in adult cardiac transplantation. PMID- 1721359 TI - The clinical trial of FK 506 as primary and rescue immunosuppression in pediatric cardiac transplantation. PMID- 1721360 TI - Quality-of-life advantages of FK 506 vs conventional immunosuppressive drug therapy in cardiac transplantation. PMID- 1721361 TI - FK 506 in clinical kidney transplantation. PMID- 1721362 TI - Renal transplantation under FK 506 in patients with previous loss of renal function due to hemolytic uremic syndrome. PMID- 1721363 TI - Japanese study of FK 506 on kidney transplantation: results of an early phase II study. Japanese FK 506 Study Group. PMID- 1721364 TI - Pediatric renal transplantation under FK 506 immunosuppression. PMID- 1721365 TI - FK 506 conversion of renal allografts failing cyclosporine immunosuppression. PMID- 1721367 TI - Japanese study of FK 506 on kidney transplantation: the benefit of monitoring the whole blood FK 506 concentration. Japanese FK 506 Study Group. PMID- 1721366 TI - FK 506 in a 14-year-old renal allograft recipient with cyclosporine-related liver nephrotoxicity: 1-year follow-up. PMID- 1721368 TI - Transplantation of pediatric en bloc kidneys under FK 506 immunosuppression. PMID- 1721369 TI - Clinical small bowel or small bowel plus liver transplantation under FK 506. PMID- 1721370 TI - Development of renal dysfunction and renal histological lesions in two patients treated with FK 506 for acute rejection following liver transplantation. PMID- 1721371 TI - Toxicity of FK 506 in the cynomolgus monkey: noncorrelation with FK 506 serum levels. PMID- 1721372 TI - Adverse effects associated with the use of FK 506. PMID- 1721373 TI - Efficacy and toxicity of FK 506 for the treatment of resistant rejection in liver transplant patients. PMID- 1721374 TI - Clinicopathological evaluation of kidney transplants in patients given a fixed dose of FK 506. Japanese FK 506 Study Group. PMID- 1721376 TI - FK 506 is a direct glomeruloconstrictor, as determined by electrical resistance pulse sizing (ERPS). PMID- 1721375 TI - Disruption of renal function and gene expression by FK 506 and cyclosporine. PMID- 1721377 TI - Influence of FK 506 on renal blood flow. PMID- 1721378 TI - FK 506 does not affect the glomerular filtration rate and renal plasma flow in the rat. PMID- 1721379 TI - Effect of FK 506 on excretion of urinary enzymes in rats. PMID- 1721380 TI - In vitro effects of cyclosporine and FK 506 on the renal cortex. PMID- 1721381 TI - Short-term FK 506-induced morphological changes in rat kidneys. PMID- 1721382 TI - FK 506 mechanism of nephrotoxicity: stimulatory effect on endothelin secretion by cultured kidney cells and tubular cell toxicity in vitro. PMID- 1721383 TI - In vitro FK 506 kidney tubular cell toxicity. PMID- 1721384 TI - The effects of cyclosporine A, cyclosporine G, and FK 506 upon prostaglandin production in renal mesangial cells in culture. PMID- 1721385 TI - Changes in renal function after liver transplantation under FK 506. PMID- 1721386 TI - A comparison of the renal effects (ERPF, GFR, and FF) of FK 506 and cyclosporine in patients with liver transplantation. AB - 1. The mean "cost" in milliliters per minute of ESLD alone, prior to transplantation, was 35% + 23% (1 SD). In GFR it was 15%. 2. The additional burden of CyA + OLT increases the loss in ERPF an additional 18%; in GFR, it increases loss another 10%. Thus, the total loss in CyA-treated patients was 53% and 25%, respectively. 3. The decrease imposed by FK 506 + OLT on ERPF was only 7%, with no decrease in GFR. 4. Therefore, from the renal point of view, FK 506 would appear to be the superior drug. 5. The large error around mean values underlines the desirability of performing these tests on the individual patient rather than on information from groups, since many values fall near the threshold of the azotemic range (ERPF approximately 175 mL/min). 6. As renal mass was compromised, ie, fall in the ERPF, the GFR increased relatively, ie, the renal filtering membrane became more permeable and the FFs gradually increased. 7. The loss of renal function was significantly less in OLT patients on FK 506 than CyA. However, the greatest loss in expected renal function was due to the basic ESLD itself. PMID- 1721387 TI - Renal function after conversion from cyclosporine to FK 506 in liver transplant patients. PMID- 1721388 TI - Urinary proteins as a marker of drug-induced renal damage following treatment with cyclosporine or FK 506. PMID- 1721389 TI - Association of elevated FK 506 plasma levels with nephrotoxicity in liver-grafted patients. PMID- 1721390 TI - Hemolytic-uremic syndrome in a renal transplant recipient on FK 506 immunosuppression. PMID- 1721391 TI - Effect of FK 506 on glucose metabolism and insulin secretion in normal rats. PMID- 1721392 TI - Mechanisms by which FK 506 affects exocrine pancreas in rats. PMID- 1721393 TI - Effect of FK 506 on function of human islets of Langerhans. PMID- 1721394 TI - Synexin: a target protein for toxic effects of cyclosporine and FK 506 in endocrine cells. PMID- 1721395 TI - New onset of diabetes in FK 506 vs cyclosporine-treated kidney transplant recipients. PMID- 1721396 TI - FK 506-associated diabetes mellitus in the pediatric transplant population is a rare complication. PMID- 1721397 TI - Similar clinical presentation of neurotoxicity following FK 506 and cyclosporine in a liver transplant recipient. PMID- 1721398 TI - Neurologic complications of FK 506. PMID- 1721400 TI - Neuropathologic findings in liver transplantation: a comparative study of cyclosporine and FK 506. PMID- 1721399 TI - Psychiatric morbidity in liver transplant patients. PMID- 1721401 TI - Hypoxic and reoxygenation injury in human and rat hepatocytes--influence of FK 506 and cyclosporine. PMID- 1721402 TI - Effect of FK 506 and cyclosporine on plasma cholesterol levels in rabbits. PMID- 1721403 TI - The effect of FK 506, cyclosporine A, and cyclosporine G on serum 1,25 dihydroxyvitamin D levels. PMID- 1721404 TI - Acute hemolytic anemia in liver and bone marrow transplant patients under FK 506 therapy. PMID- 1721405 TI - A study of the effects of the drug FK 506 on gingival tissues. PMID- 1721406 TI - Pulmonary infiltrates and eosinophilia in an FK 506 liver transplant recipient. PMID- 1721407 TI - Effect of FK 506 on experimental liver carcinogenesis. PMID- 1721408 TI - FK 506 and rapamycin do not affect platelet aggregation or mitochondrial function. PMID- 1721409 TI - In vivo and in vitro effect of FK 506 on rat Leydig cell function. PMID- 1721410 TI - Human islet allotransplantation under FK 506. PMID- 1721411 TI - Long-term survival of islet allografts in diabetic rats treated with FK 506. PMID- 1721412 TI - Intraportal FK 506 improves intrahepatic islet allograft survival. PMID- 1721413 TI - The effect of short-term FK 506 therapy on pancreas transplantation in rats. PMID- 1721414 TI - Prolongation of pancreaticoduodenal allograft survival in rats by treatment with FK 506. PMID- 1721415 TI - Induction of tolerance to islet allografts with preoperative donor spleen cell injection and a short course of FK 506 treatment. PMID- 1721416 TI - FK 506 rescue in chronic graft-versus-host-disease after bone marrow transplantation. PMID- 1721417 TI - Phase II study of FK 506 for allogeneic bone marrow transplantation. PMID- 1721419 TI - The induction of pseudo-graft-versus-host disease following syngeneic bone marrow transplantation using FK 506. PMID- 1721418 TI - The effect of cyclosporine, rapamycin and FK 506 the survival following allogeneic bone marrow transplantation. PMID- 1721420 TI - FK 506 has no short-term effects on endogenous or exogenous myeloid reconstitution in irradiated mice. PMID- 1721421 TI - Prolonged prevention of acute graft-versus-host disease after allogeneic bone marrow transplantation by donor pretreatment using FK 506. PMID- 1721423 TI - Effect of donor pretreatment with FK 506 upon small intestine allotransplantation in rats. PMID- 1721422 TI - Canine total orthotopic small bowel transplantation under FK 506. PMID- 1721424 TI - Lymphocyte traffic and graft-versus-host disease after fully allogeneic small bowel transplantation. PMID- 1721425 TI - Indefinite small bowel allograft survival after limited immunosuppressive therapy is not due to specific hyporesponsiveness. PMID- 1721426 TI - Effect of FK 506 on the survival of rat small bowel allografts. PMID- 1721427 TI - Effect of donor-specific transfusion and FK 506 on small intestine allotransplantation. PMID- 1721428 TI - Effect of FK 506 on growth of transplanted newborn rat intestine. PMID- 1721429 TI - Fulminant graft-versus-host disease after FK 506 treatment in fully allogeneic small bowel transplantation. PMID- 1721430 TI - FK 506 inhibits nitric oxide production by cells infiltrating sponge matrix allografts. PMID- 1721431 TI - Tolerance induction by liver grafting and FK 506 treatment in nonhuman primates. PMID- 1721433 TI - FK 506 inhibits the development of transplant arteriosclerosis. PMID- 1721432 TI - Differential survival of hamster-to-rat liver and cardiac xenografts under FK 506 immunosuppression. PMID- 1721434 TI - Effect of FK 506 on abdominal organ cluster transplantation in pigs. PMID- 1721435 TI - An attempt to induce tolerance to skin grafts in congenic mice with FK 506. PMID- 1721436 TI - Synergistic effect of FK 506 and donor-specific blood transfusion on rat skin but not on composite tissue (limb) allograft survival. PMID- 1721437 TI - Bone marrow cell- and FK 506-induced donor-specific unresponsiveness in rat heart allografts. PMID- 1721438 TI - FK 506 fails to reverse moderate cardiac allograft rejection in a canine heterotopic model. PMID- 1721439 TI - Prolonged cardiac allograft survival in sensitized rats by low-dose FK 506 in combination with splenectomy. PMID- 1721440 TI - Comparative study of FK 506, cyclosporine, and triple regimen immunosuppression with FK 506, cyclosporine, and mizoribine. PMID- 1721441 TI - Immunosuppressive effects of FK 506 in rat lung transplantation. PMID- 1721442 TI - Immunosuppressive effects of FK 506 in canine lung transplantation. PMID- 1721443 TI - FK 506 and cyclosporine: effects and side effects in canine lung transplantation. PMID- 1721444 TI - Dose response to FK 506 in canine lung transplantation. PMID- 1721445 TI - Effects of FK 506 on the biochemical markers in canine lung allograft rejection. PMID- 1721447 TI - FK 506 enhances the beneficial effects of donor-specific blood transfusion on allograft survival in rats. PMID- 1721446 TI - Evidence that FK 506 may abrogate suppressor cell activity induced by blood transfusion. PMID- 1721448 TI - Effects of donor-specific transfusion, cyclosporine A and FK 506 on rat cardiac allograft survival. PMID- 1721449 TI - FK 506: a new therapeutic agent for severe recalcitrant psoriasis. PMID- 1721450 TI - Metabolic effects of FK 506 in patients with severe psoriasis: short-term follow up of seven cases. PMID- 1721451 TI - Efficacy of FK 506 in the treatment of recalcitrant pyoderma gangrenosum. PMID- 1721452 TI - Influence of FK 506 on T lymphocytes, Langerhans' cells and the expression of cytokine receptors and adhesion molecules in psoriatic skin lesions: a preliminary study. PMID- 1721453 TI - Differential effects of FK 506 and cyclosporine on hair regrowth in the DEBR model of alopecia areata. PMID- 1721454 TI - FK 506 treatment of experimental autoimmune uveoretinitis in primates. PMID- 1721455 TI - FK 506 modulates accessory cell adhesion molecule expression and inhibits CD4 lymphocyte adhesion to retinal pigment epithelial cells in vitro: implications for therapy of uveoretinitis. PMID- 1721456 TI - A multicenter clinical open trial of FK 506 in refractory uveitis, including Behcet's disease. Japanese FK 506 Study Group on Refractory Uveitis. PMID- 1721457 TI - Treatment of Cogan's syndrome with FK 506: a case report. PMID- 1721458 TI - The use of FK 506 in new-onset type I diabetes in man. PMID- 1721459 TI - FK 506 in the management of transplant-related nephrotic syndrome and steroid resistant nephrotic syndrome. PMID- 1721460 TI - Effect of FK 506 in the prophylaxis of autoimmune glomerulonephritis in NZB/WF1 mice. PMID- 1721461 TI - Effects of FK 506 on acute experimental allergic encephalomyelitis. PMID- 1721462 TI - The ability of myelin basic protein-sensitised leukocytes to adoptively transfer experimental allergic encephalomyelitis following coculture with FK 506, cyclosporine, or prednisolone. PMID- 1721463 TI - Impact of FK 506 on myocarditis in the enteroviral murine model. PMID- 1721464 TI - Summary of the First International FK 506 Congress: perspectives and prospects. PMID- 1721465 TI - Hepatitis B viral markers in Nigerian patients with primary liver carcinoma. AB - Sera from 65 patients with primary liver carcinoma (PLC) and 69 sex- and age matched controls were examined for Hepatitis B virus markers. Forty two of the patients (65%) and 25 controls (36%) were HBsAg positive. Anti-HBc was demonstrated in over 80% and in 84% of the study groups respectively. Over 70% of hepatitis B infection was anicteric and traditional surgical intervention correlated with prevalence of PLC. The data provide support for the etiological role of HBV in Nigerian patients with PLC; this is the first report that the incidence of PLC is highest in the northern Savannah region of Nigeria. PMID- 1721466 TI - Molecular cloning of the human platelet 12-lipoxygenase. PMID- 1721467 TI - A transgenic mouse model for studying the induction of fetal hemoglobin in the adult. PMID- 1721468 TI - Severity of comorbidity, not type of surgery, affects outcome of prostatectomy. PMID- 1721469 TI - Microfilamentous type VI collagen in the hyalinized stroma of the hypertrophied ligamentum flavum. AB - Thickened ligamenta flava obtained from 14 patients with spinal canal stenosis were examined with special reference to type VI collagen. The characteristic histological finding in the thickened area was rupture or normal elastic fibre meshwork with resultant fibrosis which usually appeared hyaline. Using an immunohistological method, collagen types VI, I and III were found to be present in the hyaline matrix. Ultrastructural study revealed many microfilamentous structures of type VI collagen admixed in loosely packed, banded collagen fibres. With differential salt precipitation of pepsin-extracted collagen the existence of type VI collagen was confirmed by SDS-polyacrylamide gel electrophoresis analysis and Western blotting analysis using anti-type VI collagen antibody. Quantification of type VI collagen in pepsin-extracted crude collagen samples by an inhibition enzyme-linked immunosorbent assay showed an increasing amount of type VI collagen in the thickened ligamenta flava compared to the normal ligaments. Thus, increase of type VI collagen is the main contribution to the thickening of the ligamentum flavum. This may represent an adaptational and reparative process associated with disruption of elastic fibres. PMID- 1721470 TI - Sporadic loss of leucocyte-function-associated antigen-3 (LFA-3) in colorectal carcinomas. AB - Leucocyte-function-associated antigen-3 (LFA-3) is a cell surface glycoprotein involved in T-cell/target cell interaction. The expression of LFA-3 on the cell surface was found to be inevitably associated with the expression of HLA molecules. Loss of LFA-3 or HLA proteins on tumour cells might result in ineffective T-cell/target cell interaction and a failure of immunological tumour surveillance. Immunohistochemistry revealed that LFA-3 is expressed in normal colonic epithelium; however, in a minor fraction of colonic adenomas and in 50.3% of colorectal carcinomas LFA-3 expression was reduced or even absent. To investigate whether the presence or absence of LFA-3 in colorectal carcinomas influences the relapse rate and time of tumour-related death, 149 patients who underwent putatively curative surgery were surveyed for a maximum of 65 months (mean 48 months). In contrast to the prognostic role of tumour stage and grade, the presence versus absence of LFA-3 was not correlated with recurrence or survival. Regarding survival and growth of residual tumour cells after potentially curative resection of the initial tumour burden, we conclude that the status of LFA-3 expression in colorectal carcinoma seems to be irrelevant in vivo. PMID- 1721471 TI - Proliferating cell nuclear antigen expression in central nervous system neoplasms. AB - Proliferating cell nuclear antigen (PCNA) is a cell-cycle-regulated protein, which can be demonstrated in routinely fixed specimens. Studies on various tissues, cell cultures and neoplasms have shown that PCNA labelling index (LI) correlates with flow cytometry, tritiated thymidine LI, bromodeoxyuridine (BrdU) incorporation and Ki67 LI. PCNA LI may have prognostic value in various neoplasms. The present study concerns PCNA immunostaining in a series of neuroglial tumours. We demonstrate that there is a relation between PCNA LI and histological grade, and between PCNA LI and reported thymidine LI, BrdU LI and Ki67 LI. Pleomorphic xanthoastrocytomas and low-grade astrocytomas had the lowest LI, whereas metastases of small cell lung cancer and medulloblastomas had the highest LI. Glioblastomas sometimes showed a certain degree of intratumoral heterogeneity of distribution of immunostained cells. Intratumoral heterogeneity underscores the critical importance of representative sampling of central nervous system neoplasms for kinetic studies. As expected, PCNA LI are somewhat higher than tritiated thymidine LI, BrdU LI and Ki67 LI because PCNA is a marker of G1, S, G2 and M-phases of the cell cycle and not of S-phase only. In addition, because of its long half-life, PCNA may be detected immunohistochemically in cells that have recently left the cell cycle. The immunohistochemical evaluation of PCNA LI is easy to perform on routinely processed material, allowing retrospective studies. PCNA LI may be a useful tool in grading gliomas. However, its prognostic value must be validated by comparing PCNA LI with the follow-up of the neoplasms, and possibly with the responsiveness to anti-proliferative therapy. PMID- 1721472 TI - Expression of interphasic nucleolar organizer regions in normal, dysplastic and neoplastic colorectal mucosa. AB - Silver-binding nucleolar organizer region (Ag-NOR) expression in interphasic nuclei was studied in normal, dysplastic and neoplastic colorectal mucosa at the light microscope level and by means of an image analyser (IBAS II). Both methods showed a progressive increase in the mean number of Ag-NOR sites per nucleus from mild dysplasia to invasive carcinoma. Ag-NOR counts differed significantly in the various classes of lesions (P less than 0.001), except between moderate and severe dysplasia (P greater than 0.05). Severe dysplasia showed a mean number of NORs lower than that for invasive carcinoma though an overlap in the respective frequency distributions was observed. The mean of the variances of the mean dot areas per cell nucleus (pooled variance) also showed a step-wise increase from normal to neoplastic lesions, indicating a greater variability in NOR size as a characteristic of malignant cells. A similar increase was observed in the percentage of nuclear area occupied by Ag-NORs. The mean area per silver-stained dot was also measured in the different classes of lesions by IBAS II. Data obtained showed no significant differences among the values. In conclusion, the wide overlap between the frequency distributions does not allow consideration of the Ag-NOR count alone to be a reliable marker of malignant transformation in a single cell. It appears that the study of Ag-NOR number needs to be evaluated together with dot anisometry in order to be a useful criterion in distinguishing the biological behaviour of neoplastic lesions in colorectal mucosa. PMID- 1721473 TI - Silver-stained nucleolar organizer regions in the normal, hyperplastic and neoplastic endometrium. AB - The numbers of silver-stained nucleolar proteins (AgNORs) were counted in hyperplastic and neoplastic lesions of the endometrium and compared with those of normal proliferative and secretory phase endometrium. In glandular cells in the normal menstrual cycle, the mean number of AgNORs in proliferative phase endometrium (3.8) was significantly higher than that in secretory phase endometrium (2.7, P less than 0.05). The mean number of AgNORs in well differentiated endometrioid type adenocarcinoma (5.5) was significantly higher than that in both complex hyperplasia without cytological atypia (3.6, P less than 0.01), and simple hyperplasia (3.3, P less than 0.01). Mean AgNOR counts in complex hyperplasia with cytological atypia were greater than those in both complex hyperplasia without cytological atypia (P less than 0.05) and simple hyperplasia (P less than 0.05). Thus, complex hyperplasia with cytological atypia appears to be a direct precursor of well-differentiated endometrioid type adenocarcinoma. These findings suggest that the mean numbers of AgNORs are increased in neoplastic changes in the endometrium, and the one-step colloid method for AgNORs may therefore be a simple and useful technique to examine proliferative activity in neoplastic and pre-neoplastic endometrial cells. PMID- 1721474 TI - Co-expression of cytokeratin and vimentin filaments in rete testis and epididymis. An immunohistochemical study. PMID- 1721475 TI - [Activity of elastase-like proteinases and their inhibitors in indigent nearly healthy residents in various biogeochemical conditions of the Chuvash ASSR]. AB - A proteinase-inhibitory balance of blood (elastase-like activity, alpha 1 proteinase inhibitor and alpha 2-macroglobulin activities) was studied in practically healthy inhabitants of the Chuvash ASSR two subregions--Sura river basin and Cubninocivil region, which are distinctly dissimilar in all the biogeochemical parameters involving macro- and microtrace compositions. The higher activity of elastase-like proteinases and decreased content of alpha 1 proteinase inhibitor were detected in practically healthy inhabitants of the river Sura basin, where high incidence of myocardial infarction was found, as compared with those of the Cubninocivil people. The similar alterations in the proteinase-inhibitory balance were observed in blood of experimental animals maintained on a diet containing fresh water from these subregions. The data obtained suggest that there exists causative relationship between biogeochemical parameters and development of imbalance in the proteinase-inhibitor system in practically healthy inhabitants of the river Sura basin. This imbalance is considered as a pathogenetic factor responsible for development of atherosclerosis. PMID- 1721476 TI - [Activity of tissue and plasma kallikrein and level of their precursors in eye tissue structures and media of healthy rabbits]. AB - Activity of tissue and blood plasma kallikreins as well as total content of their inactive precursors were studied in rabbit eye structures and media: iris, ciliary body, cornea, vascular striatum, retina, aqueous humor, tear liquid, lacrimal gland by means of fluorimetric procedure using Z-Phe-Arg-MCA as a substrate. Dissimilar capacity of the trypsin soya bean inhibitor and of aprotinin (basic inhibitor of Kunitz type) to inhibit tissue and blood plasma kallikreins enabled to differentiate the enzymatic activity. Lacrimal gland contained the highest activity of tissue kallikrein which amounted to 70% of total Z-Phe-Arg-MCA-hydrolyzing activity of the homogenate. Total Z-Phe-Arg-MCA amidase activity and activity of individual kallikreins was distinctly lower in all the eye structures and media studied as compared with that of lacrimal gland. Activity of tissue kallikrein was higher than blood serum kallikrein activity in iris, ciliary body, vascular striatum, retina and conjunctiva. The highest content of prekallikreins was found in conjunctiva, aqueous humor, iris and ciliary body. Tissue and blood plasma kallikrein-kinin systems appear to carry out dissimilar functions in eye tissue structures; they are apparently involved in pathogenesis of some eye diseases. PMID- 1721477 TI - [Reactions of rat fetal hepatocytes to hormones in primary cultures grown in selective media with an addition of glucocorticoid and barbiturate]. AB - After growing of fetal rat hepatocytes in the medium containing cortisol and sodium barbital for 4-8 days and subsequent cultivation in the medium without glucocorticoid and barbiturate for 1-4 days insulin retained its ability to stimulate total RNA and protein synthesis, while human growth hormone kept its enhancing action on RNA biosynthesis. However, cortisol did not change protein synthesis and inhibited paradoxically incorporation of 3H-uridine into total RNA, after preincubation of cells in the selective medium. This suggests that prolonged exposure of cultured fetal rat liver cells to cortisol and sodium barbital may cause phenomena similar to those of hormonal and/or enzymatic imprinting. PMID- 1721479 TI - [Anomalous origin of the left coronary artery from the pulmonary artery (Bland White-Garland syndrome) in a 29-year-old male with absolute tachyarrhythmia]. AB - We report on a man without any symptoms until the age of 29 years when a ventricular tachyarrhythmia occurred for the first time. There were electrocardiographic signs of a previous myocardial infarction of the anterior wall, but there had not even been any episode of angina pectoris. The cause was the anomalous origin of the left coronary artery from the pulmonary trunk (Bland White-Garland syndrome). Therapy consisted of ligation of the left coronary artery and the implantation of an aortocoronary vein graft to the left coronary artery in order to reinstall a two-coronary system. Diagnosis is easily made by coronary angiography. At a 3-year follow-up there was an increase of the ejection fraction/cardiac output, a stable sinus rhythm, and an improved physical endurance. PMID- 1721478 TI - New frontiers in ovarian cancer diagnosis and management. AB - Ovarian carcinoma is now the leading cause of death among women. Surgery has reached its limits, and further aggressive surgery will result in an inordinate morbidity and mortality. Ovarian carcinoma is ideally treated by complete surgical removal of the cancer, followed by anti-cancer chemotherapy. Since it is often impossible to remove all of the cancer, adjunctive chemotherapy is playing an increasingly important role in the management of the cancer. New anti-cancer drugs must be found or synthesized, and new combinations of current anti-cancer drugs with mechanisms to protect the bone marrow must be explored. The field of genetics and the identification of the patient at high risk because of a familial history of ovarian cancer must be expanded. The role of tumor markers and oncogenes requires more in-depth study so that these signs can play a greater role in monitoring and identifying the patient with early ovarian cancer. The emerging fields of genetic engineering and biologic response modifiers are opening up new avenues for additional modalities of therapy. The expanding areas of research in cancer are starting to dispel the doom and gloom of the last three decades with a spirit of optimism for the diagnosis and treatment of ovarian cancer, as the new century approaches. PMID- 1721480 TI - Immunohistochemical demonstration of cellular fibronectin and tenascin in human epiretinal membranes. AB - The occurrence of fibronectin, cellular fibronectin and tenascin was studied immunohistochemically in epiretinal membranes from 5 patients with retinal detachment and 3 patients with macular pucker, and in preretinal membranes from 3 patients with proliferative diabetic retinopathy. Positive immunofluorescence for these proteins was seen in all specimens studied. The presence of cellular fibronectin suggests that local production of fibronectin occurs in these membranes. The expression of tenascin, an extracellular matrix glycoprotein, originally found to modulate organosenesis in tendinous and glial tissue, suggests that this glycoprotein also participates in the regulation of cellular growth in these membranes. PMID- 1721481 TI - Effect of dextrans of various molecular weights on mesangial transport in ddY mice pretreated with sheep anti-type IV collagen serum. AB - The authors' previous report concluded that increased mesangial IgA deposition seen in ddY mice pretreated with mesangiotropic anti-type IV collagen serum might be due to a dysfunction of mesangial transport of macromolecules such as polymeric autologous IgA. In the present study we examined the effect of macromolecules upon mesangial transport in similarly treated ddY mice, using intravenously administered FITC-labeled dextrans of various molecular weights as tracers. The intensity of each resulting mesangial dextran deposit inspected directly by immunofluorescence was measured periodically and compared with the deposition of autologous mouse IgA examined using rhodamine-labeled rabbit anti mouse IgA. High-molecular-weight dextran of 2,000 kDa showed prolonged mesangial deposition which paralleled the intensity and distribution of the autologous mouse IgA deposition. In contrast, medium- and low-molecular-weight dextrans of 500 and 150 kDa, respectively, disappeared earlier from the mesangium, unlike the autologous IgA deposition which persisted. Based on these results, it was concluded that the macromolecularity of certain substances and a transport dysfunction of the mesangium may both be major factors in prolonged mesangial deposition. These findings may provide some clues to help clarify the pathogenesis of human IgA nephritis. PMID- 1721482 TI - Two cases of peritoneal serous papillary adenocarcinoma. AB - Two cases of peritoneal papillary carcinoma are reported. The patient in the first case was a 71-year-old woman with symptoms of obstructive ileus. Laparotomy revealed a tumor in the omentum involving the transverse colon, and several small tumors in the peritoneum and pelvic wall. However, no primary site of the tumor was seen in the ovary, pancreas, or gastrointestinal tract. The patient in the second case was a 44-year-old woman with carcinomatous peritonitis. Postmortem examination revealed multiple tumors in the peritoneum, omentum, and pelvic wall. Tumors were also found in the cortex with mild invasion of the underlying parenchyma of the bilateral ovaries, although these lesions were thought to be metastatic. The histologic features of the tumor in both cases were those of tubulopapillary adenocarcinoma containing scattered psammoma bodies. The cells were positive with the PAS-D technique, but negative with alcian blue staining. In both cases, the serum levels of CA-125 were considerably elevated, and the tumor cells showed positivity for CA-125, S-100 protein, cytokeratin and EMA by immunohistochemistry. The present cases were most likely peritoneal serous papillary adenocarcinoma derived from extraovarian peritoneal mesothelium with mullerian potential, being different from the usual type of diffuse malignant mesothelioma. PMID- 1721483 TI - Iloprost and risk of thromboembolism. PMID- 1721484 TI - Reduction of primary posttraumatic adhesion formation with the prostacyclin analog iloprost in a rodent model. AB - Recent evidence suggests that inhibition of postsurgical adhesion formation may be effected by modulation of the activities of inflammatory cells contributing to mesothelial repair. Iloprost, a stable analog of prostacyclin, has been shown to exert vasodilatory, antiinflammatory, fibrinolytic, and antithrombotic influences. To determine whether these properties of iloprost might protect mesothelial surfaces from perioperative damage and hence prevent adhesion formation, we evaluated the effect of iloprost on peritoneal healing in a hamster model for primary pelvic injury. Perioperative iloprost therapy significantly reduced posttraumatic adhesion formation when compared with that in vehicle treated controls. Dose-response studies demonstrate adhesion prevention with doses ranging from 0.04 to 4 mg/kg per 8 hours given subcutaneously over the course of 3 days. These data demonstrate that iloprost is a potent positive modulator of peritoneal healing after pelvic trauma. Further studies to characterize the potential application of iloprost as an adjuvant in reproductive surgery are indicated. PMID- 1721485 TI - C-jun and jun-B oncogene expression during placental development. AB - During embryogenesis, growth and differentiation occur in a sequential, predetermined order suggesting that specific genes are turned on and off in a precise and well-regulated manner. Placental development, which is characterized by massive proliferation and differentiation of multiple cell types, must be similarly regulated. Early response protooncogenes, such as c-jun and jun-B, have been associated with both proliferation and differentiation of different cell types. In this study, using Northern blot analysis, we found that c-jun and jun-B expression occurred in human placentas throughout gestation. Maximal expression of c-jun occurred in early gestation, and maximal expression of jun-B occurred in late gestation. We speculate that peak expression of c-jun in human placenta at early gestation may be related to cytotrophoblastic proliferation and that peak expression of jun-B in late gestation may be related to further terminal differentiation of trophoblastic cells. PMID- 1721486 TI - New monoclonal antibodies identify the glycoprotein carrying the CA 125 epitope. AB - CA 125 is an antigenic determinant located on the surface of ovarian carcinoma cells and elevated in the serum of greater than 90% of patients with carcinoma. The antigen, derived from the ovarian epithelium, has been described as a mucinlike glycoprotein greater than 200 kd. To date little is known of the metabolic regulation or expression of this antigen in either normal or neoplastic tissues. New monoclonal antibodies that we describe here recognize both unique and similar epitopes to OC 125. These reagents may allow for a more complete definition of the structure and expression of the CA 125 complex. These antibodies recognize high-molecular weight (greater than 200 kd) subspecies and a lower-molecular-weight (68 kd) subspecies of the antigen and identify it in the cytoplasm and the extracellular matrix of CA 125-producing cells. PMID- 1721487 TI - Specific binding sites for insulin and insulin-like growth factor I in human endometrial cancer. AB - Insulin and insulin-like growth factor I are known to be mitogenic and therefore may play a role in the development of endometrial cancer. We undertook this study to investigate whether human endometrial cancer tissue has receptors for these substances. Endometrial cancer tissue samples were obtained at hysterectomy from 10 women with endometrial cancer, and control endometrial tissue was collected from normal cycling women undergoing hysterectomy for nonendocrine problems. Binding studies with iodine 125-insulin and [125I]insulin-like growth factor I revealed the presence of specific binding sites for insulin and insulin-like growth factor I in both normal endometrium and endometrial cancer tissue. The percent binding of [125I]insulin in the endometrial cancer tissue (mean +/- SE 2.4% +/- 0.5%/100 micrograms protein) was not significantly different from that in normal endometrium (3.5% +/- 1%/100 micrograms protein). On the contrary, the percent total binding of [125]insulin-like growth factor I in the endometrial cancer (5.3% +/- 1.5%/100 micrograms protein) was significantly (p less than 0.04) higher than that observed in normal endometrium (2.1% +/- 0.4%/100 micrograms protein). There was a significant positive correlation between the histologic grade of the tumor and the insulin-like growth factor I binding (r = 0.865, p less than 0.02). The affinity constants for the high-affinity receptors were similar in the normal and neoplastic endometrium. These results indicate that insulin and insulin-like growth factor I may play a role in the growth and development of endometrial cancer. PMID- 1721488 TI - Image analysis quantitation of immunoreactive retinoblastoma protein in human thyroid neoplasms with a streptavidin-biotin-peroxidase staining technique. AB - Studies investigating the molecular pathogenesis of common thyroid neoplasms have shown altered expression and/or structure of proto-oncogenes, G-proteins, and growth factors. Growth suppressor genes, genomic DNA segments that code for proteins believed to function as growth suppressors, have not been evaluated for a potential role in the pathogenesis of thyroid neoplasms. The retinoblastoma gene (RB1) maps to chromosome 13q14 and encodes a 110 kilodalton variably phosphorylated nuclear protein (Rb) that functions as a growth suppressor in a wide variety of human tissues. The frequent association of Rb protein loss with multiple neoplasms has prompted the authors to apply a specific and rapid immunohistochemical technique using streptavidin-biotin-peroxidase technology evaluated by image analysis that can be used to quantitate the level of immunoreactive Rb protein (iRb) in thyroid neoplasms. In utilizing streptavidin biotin technology for nuclear iRb detection, artifacts that can be associated with the use of avidin such as nonspecific binding at physiologic pH and nonspecific complex formation with cellular components including chromatin are avoided. By this method, positive nuclear staining for iRb in the follicular cells of three follicular adenomas and in CV-1 control cells known to express Rb was demonstrated. Two papillary carcinomas, two medullary carcinomas and a SAOS-2 cell line known to produce a defective form of Rb stained at significantly lower levels (P less than .001). The authors conclude that the streptavidin-biotin peroxidase staining technique evaluated by image analysis is a sensitive and specific detection system for nuclear iRb studies; has significant advantages over previously used techniques; and that thyroid neoplasms may variably express iRb which may, in part, reflect their variable pathogenesis. PMID- 1721489 TI - L and H cells of nodular lymphocyte predominant Hodgkin's disease show immunoglobulin light-chain restriction. AB - The nodular form of lymphocyte predominant Hodgkin's disease (NLPHD) is widely accepted to be a B-cell-derived neoplasm. Despite this consensus, previous studies have not shown monotypic immunoglobulin (Ig) light-chain expression by the putatively malignant L & H cells. We have studied paraffin-embedded tissue from 19 cases of NLPHD for the presence of Ig light and heavy chains and J chain. In addition, frozen tissue, available from one case, was examined with antibodies to light chains. In paraffin material, in each case L & H cells in individual nodules were examined for the presence of Ig light chain restriction. Kappa-light chain restriction was detected in all L & H cells in all nodules in 5/19 cases (26.5%) and this was confirmed in frozen sections available from one case. Of the remaining 14 cases, 13 (68.5%) showed kappa-light-chain restriction in a proportion of nodules and in 1 case light-chain restriction was not observed. Of 14 cases stained with antibodies to Ig heavy chains 13 contained IgG, and in 1 case no heavy chains were demonstrable. Most L & H cells in 15 cases examined contained J chain. Our finding of monotypic L & H cells in NLPHD in 95% of our investigated cases provides strong evidence for the neoplastic nature of L & H cells and supports the hypothesis that NLPHD is a malignant nonHodgkin's B-cell lymphoma. PMID- 1721490 TI - Immunohistochemical demonstration of alphaB-crystallin in hamartomas of tuberous sclerosis. AB - Autopsy material from two individuals with tuberous sclerosis were examined immunohistochemically for the expression of alpha B-crystallin. Positive immunoreactions were detected in the hamartomas of various organs. Abnormal expression of alpha B-crystallin in the hamartomas in conjunction with the alpha B-crystallin gene locus on chromosome 11q22-23 overlapping with one of the candidates of tuberous sclerosis loci suggest that tuberous sclerosis may involve the altered regulation of alpha B-crystallin gene expression. PMID- 1721491 TI - Cytokeratin expression patterns in metastatic transitional cell carcinoma of the urinary tract. An immunohistochemical study comparing local tumor and autologous metastases. AB - The cytokeratin (CK) expression patterns of local, ie, primary or recurrent, high grade-malignant transitional cell carcinomas (TCCs) of the human urinary tract and autologous lymphogenic and hematogenic metastases (n = 33) were compared. Special attention was paid to CK expression in the tumor invasion front and other areas where tumor-stroma interaction occurred to visualize cell populations with a metastatic phenotype. For this purpose, polypeptide-specific monoclonal antibodies to CKs 4, 7, 8, 10, 13, 14, 16, 17, 18, and 19 were used, employing the immunoperoxidase method. Results show that: 1) An increased expression of CK8 and CK18 is seen in the TCC tumor cells at the interface with peritumoral stroma in the tumor invasion front and with intratumoral stroma ('interface phenomenon'). Other than reflecting a quantitative change, this phenomenon might be explained by unmasking of CK8 and CK18 epitopes occurring in these regions. 2) Although in general the expression of CK13 in local TCC is decreased with increase of histopathologic parameters for progression, ie, grade and stage, an extensive proportion of CK13-positive tumor cells still can be found in some TCCs, even in metastases. 3) Morphologically recognizable types of aberrant differentiation in TCC, i.e., pseudosarcomatous or squamous differentiation and marked loss of differentiation, show altered expression of many of the CKs studied. PMID- 1721492 TI - The N terminal region of human tau is present in Alzheimer's disease protein A68 and is incorporated into paired helical filaments. AB - Antibody (Ab) E-1 was raised to the amino terminus (19 to 33 amino acid residues) of human tau. It recognized Alzheimer's disease proteins A68 (MW 60, 64, 68 kd), labeled paired helical filaments, and had no reactivity with tau from rat, mouse, and bovine brains. The results indicate that the N terminus of tau is incorporated in A68 proteins and paired helical filaments and that human tau proteins contain species-specific amino acid sequences. PMID- 1721493 TI - Multinucleated rat alveolar macrophages express functional receptors for calcitonin. AB - The fusion of mononuclear phagocytes occurs spontaneously in vivo and leads to the differentiation of either multinucleated giant cells or osteoclasts in chronic inflammatory sites or in bone, respectively. Although osteoclasts are responsible for resorbing bone, the functional role of giant cells in chronic inflammatory reactions and tumors remains poorly understood. We recently reported that the plasma membrane of multinucleated macrophages is, like that of osteoclasts, enriched in Na-K-adenosinetriphosphatases (ATPases). We also observed that the localization of their Na-K-ATPases is restricted to the nonadherent domain of the plasma membrane of cells both in vivo and in vitro, thus imposing a functional polarity on their organization. By following this observation, we wished to investigate whether these cells also expressed, like osteoclasts, functional receptors for calcitonin (CT). To this end, alveolar macrophages were fused in vitro, and both their structural and functional association with CT was analyzed and compared with those of mononucleated peritoneal and alveolar macrophages. Evidence is presented that multinucleated alveolar macrophages express a high copy number of functional receptors for CT. Our results also indicate that alveolar macrophages, much like peritoneal, express functional receptors for calcitonin gene-related peptide. It is suggested that multinucleated rat alveolar macrophages offer a novel model system to study CT receptors and that calcitonin may control local immune reactions where giant cells differentiate. PMID- 1721494 TI - Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells. AB - To further understand and investigate how ischemia affects the tight junction we have developed a 2-h model of rapidly reversible ATP depletion and cellular injury in confluent LLC-PK1 monolayers. ATP depletion was achieved utilizing substrate-free medium containing 0.1 microM antimycin A (AA). Cellular ATP levels dropped rapidly to less than 5% of control values, but recovery of ATP and cell morphology was possible even after 2 h of exposure to AA. Ruthenium red, an electron-dense marker of tight-junction integrity, was excluded from the tight junctions of control monolayers but penetrated cellular tight junctions during ATP depletion in a duration-dependent manner. Electrical resistance across the monolayers remained unchanged in control monolayers but decreased linearly during ATP depletion to 59% of control values. Transmonolayer movement of [3H]mannitol increased from a control level of 7 to 13.5% during ATP depletion. Recovery of tight-junction integrity was demonstrated by a slowing of [3H]mannitol transfer from the basolateral to the apical medium. The transfer rate in control monolayers was 0.0126%/min. During the initial 120 min of cellular recovery from 2 h of ATP depletion, the transfer rate was 0.0789%/min, but this decreased to 0.0045%/min between 2 and 4 h of recovery. In summary, physiology, biochemical, and morphological evidence indicates that reversible ATP depletion results in rapid opening of cellular tight junctions. After ATP-repletion physiological studies indicate a recovery of tight-junction integrity. PMID- 1721495 TI - Cytokine-induced phagocyte adhesion to human mesangial cells: role of CD11/CD18 integrins and ICAM-1. AB - We examined the actions of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) on neutrophil and monocyte (phagocyte) adhesion to human mesangial cell monolayers (HMC) and assessed the role of phagocyte CD11/CD18 integrin adhesion molecules and HMC intercellular adhesion molecule-1 (ICAM-1) in this process, using subunit specific monoclonal antibodies (MAb). TNF, but not IL-1, provoked rapid (onset less than 1 min) neutrophil and monocyte adhesion to HMC by a phagocyte-directed action. Adhesion was markedly inhibited by MAb against CD18 and CD11b, with lesser or no inhibition being afforded by MAb against CD11a, CD11c, or ICAM-1. In contrast, prolonged exposure of HMC to TNF or IL-1 (1-18 h) increased HMC adhesiveness for phagocytes. These actions were blocked by actinomycin D or cycloheximide and by MAb against HMC ICAM-1 or phagocyte CD18, CD11a, or CD11b, suggesting that cytokines provoked adhesion by inducing HMC ICAM-1 synthesis. In keeping with this interpretation, TNF treatment of HMC was associated with increased ICAM-1 surface expression, as determined by indirect immunofluorescence, and increased ICAM-1 mRNA levels, as determined by Northern blot analysis. The actions of TNF on phagocytes and HMC were additive. HMC injury, as determined by 51Cr release, was only observed when both phagocytes and HMC were activated by TNF. HMC injury was attenuated by anti-CD18 MAb and superoxide dismutase, suggesting that the injury process was, in part, adhesion dependent and mediated by reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721496 TI - RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney. AB - Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present. PMID- 1721497 TI - Amiloride-sensitive sodium channels in rabbit cortical collecting tubule primary cultures. AB - Patch-clamp methodology was applied to principal cell apical membranes of rabbit cortical collecting tubule (CCT) primary cultures grown on collagen supports in the presence of aldosterone (1.5 microM). The most frequently observed channel had a unit conductance of 3-5 pS, nonlinear current-voltage (I-V) relationship, Na permeability (PNa)-to-K permeability (PK) ratio greater than 19:1, and inward current at all applied potentials (Vapp) less than +80 mV (n = 41). Less frequently, an 8- to 10-pS channel with a linear I-V curve, PNa/PK less than 5:1, and inward current at Vapp less than +40 mV was also observed (n = 7). Luminal amiloride (0.75 microM) decreased the open probability (Po) for both of these channels. Mean open time for the high-selectivity Na+ channel was 2.1 +/- 0.5 s and for the low-selectivity Na+ channel was 50 +/- 12 ms. In primary cultures grown without aldosterone the high-selectivity Na+ channel was rarely observed (1 of 32 patches). Lastly, a 26- to 35-pS channel, nonselective for Na+ over K+, was not activated by cytoplasmic Ca2+ or voltage nor inhibited by amiloride (n = 17). We conclude that under specific growth conditions, namely permeable transporting supports and chronic mineralocorticoid hormone exposure, principal cell apical membranes of rabbit CCT primary cultures contain 1) both high-selectivity and low selectivity, amiloride-inhibitable Na+ channels and 2) amiloride-insensitive, nonselective cation channels. PMID- 1721498 TI - Localization and regulation of acid-base secretory currents from individual epithelial cells. AB - The turtle urinary bladder is composed of different epithelial cell types that are suspected to separately produce electrogenic acid and alkali excretion. We measured the electrical currents produced by individual cells, scanning a two dimensional vibrating probe over the luminal surface of the bladder. Acidification (outward current) was produced by the type of epithelial cell rich in carbonic anhydrase (CA cells). The measured currents of these cells quantitatively accounted for the total epithelial acidification current. When alkali secretion was induced by adenosine 3',5'-cyclic monophosphate and acidification was inhibited (by luminal pH 4), we measured inward currents localized to a small number of epithelial cells in four bladders but found no localization in the other seven treated bladders. When alkali secretion was localized and induced without inhibiting acidification, we found both cells producing inward current and cells producing outward current, which demonstrated that the two transport functions can occur simultaneously. We conclude that net acid-base secretion can be determined by regulating the transport rates of separate cells. PMID- 1721499 TI - Effects of angiotensin on renal cortical and papillary blood flows measured by laser-Doppler flowmetry. AB - The effects of angiotensin II (ANG II) or angiotensin III (ANG III) on renal cortical blood flow (CBF) or papillary blood flow (PBF) were investigated in Inactin-anesthetized young rats with the use of laser-Doppler flowmetry. Infusion of equimolar pressor doses of ANG II (300 ng.kg-1.min-1 iv) or ANG III (267 ng.kg 1.min-1) decreased CBF by 31 +/- 2.6% (P less than 0.001) and 20.3 +/- 3.2% (P less than 0.01), respectively but increased PBF by 19 +/- 6.1% (P less than 0.05) and 14.6 +/- 4.4% (P less than 0.05). The ANG II-induced increase in PBF was not prevented by aortic clamping to maintain constant renal perfusion pressure or pretreatment with the prostaglandin synthase inhibitor, indomethacin. The nonpeptide ANG II receptor antagonist, DuP 753 completely abolished the systemic and intrarenal effects of ANG II. After pretreatment with a kallikrein inhibitor, aprotinin, ANG II infusion increased mean arterial pressure but did not affect PBF, suggesting that kinins, but not prostaglandins, modulate the action of systemic ANG II on PBF. We conclude that circulating ANG II induces vasoconstriction in the cortex and also promotes the intrarenal production of kinins, which act to enhance papillary blood flow. PMID- 1721500 TI - Regulation of single calcium channels in cerebral arteries by voltage, serotonin, and dihydropyridines. AB - Unitary currents through Ca channels were measured from cell-attached patches on smooth muscle cells isolated from rabbit cerebral (basilar) arteries. Barium (80 and 10 mM) and calcium (80 and 10 mM) were used as the charge carriers. The dihydropyridine Ca channel agonist BAY R 5417 was used to increase open-state probability (Popen), with 500 nM BAY R 5417 increasing Popen 10-fold at 0 mV. Barium currents through single Ca channels were greater than calcium currents at any voltage, with single-channel conductances negative to -20 mV of 24.6 pS (80 mM barium), 15.1 pS (80 mM calcium), 17.2 pS (10 mM barium), and 5.8 pS (10 mM calcium). The single-channel Popen increased 2.7-fold per 5- to 7-mV membrane depolarization (negative to 0 mV) and was half maximal at +0.4 mV (80 mM calcium) and +13.5 mV (80 mM barium). Ca channels with calcium but not with barium as the charge carrier exhibited pronounced inactivation positive to -20 mV (half time, 112 ms at 0 mV). The dihydropyridine nimodipine (2 nM) inhibited average currents through Ca channels. The cerebral artery constrictor serotonin increased Popen of single Ca channels by as much as 200-fold without an effect on single-channel conductance. A second distinct amplitude of unitary currents was often observed, corresponding to a channel conductance of about one-half the more commonly observed level. The small-conductance-level channel was voltage dependent, did not inactivate over 0.5-s test pulses (with barium), and could be activated by serotonin. PMID- 1721501 TI - Effects of ryanodine and BAY K 8644 on membrane properties and conduction during simulated ischemia. AB - We studied the effect of 1.0 microM ryanodine and 0.1 microM BAY K 8644 (putative modulators of intracellular calcium) on the changes in action potential characteristics, cellular coupling, and longitudinal conduction induced by simulated ischemia (9.0 mM K, 6.5 pH, 0 glucose, 20 mmHg PO2) in superfused guinea pig papillary muscles. Simulated ischemia (SI) depolarized the resting membrane by 5 mV and caused a 28% decrease in action potential upstroke (Vmax), a 65% decrease in action potential duration at 90% (APD90), a 40% increase in internal longitudinal resistance (ri), and a 17% decrease in conduction velocity as compared with the 9-K Tyrode control solution. These changes were reversible and reproducible. The decrease in Vmax induced by SI was greater than that associated with a K(+)-induced change in resting membrane potential (RMP). Ryanodine lessened the SI-induced APD90 shortening by 26%, the decrease in Vmax by 42%, the increase in ri by 33%, and the decrease in conduction velocity by 21%. BAY K 8644 did not alter SI-induced APD90 shortening but augmented the decrease in Vmax by 23%, the increase in ri by 67%, and the decrease in conduction velocity by 59%. Neither ryanodine nor BAY K 8644 altered the SI induced changes in RMP. Our results suggest that changes in intracellular calcium during SI not only influence cellular coupling but also contribute to the apparent non-RMP-dependent component of the change in Vmax and to the change in APD90 induced by SI. PMID- 1721503 TI - Steady state variables in the guarded receptor hypothesis. PMID- 1721502 TI - EDRF-mediated shear-induced dilation opposes myogenic vasoconstriction in small rabbit arteries. AB - In small saline-perfused rabbit mesenteric arteries (diam 221 +/- 4 microns, means +/- SE; n = 48) in situ, the interactions of endothelium-derived relaxing factor (EDRF)-mediated flow-dependent dilation and myogenic constriction were studied. When pump flow was increased two- to fivefold (2.8 +/- 0.1-fold), input perfusion pressure rose by 133 +/- 17%. Vessel diameter first increased passively by 9 +/- 1% and then decreased to or below control values reflecting the vascular myogenic activity. This was followed by a 16 +/- 3% increase in diameter, which was flow dependent, because nonperfused vessels exposed to the same intravascular pressures did not dilate. When the perfusate viscosity was increased with dextran solutions, both the basal diameters and the flow-induced dilator responses were significantly augmented, indicating that the increase in shear stress was the stimulus. The flow-dependent dilation was abolished by inhibition of EDRF with either hemoglobin (10 microM) or NG-nitro-L-arginine (0.3 mM) and also after preincubation with neuraminidase (0.2 U/ml, 30 min), which removes part of the membrane glycocalyx. Thus, myogenic responses in small mesenteric arteries can be effectively opposed by shear-induced release of EDRF. This might be a major mechanism for maintaining adequate tissue perfusion when pressure and shear stress increase simultaneously (e.g., exercise hyperemia, autoregulation) and otherwise myogenic activity would reduce vascular conductivity. PMID- 1721504 TI - Response of muscle protein synthesis to fasting in suckling and weaned rats. AB - Protein synthetic efficiency (KRNA) is low in immature skeletal muscle of suckling rats and increases toward the end of the suckling period. To determine whether immature skeletal muscle is able to further reduce KRNA in response to fasting, suckling (5, 10, and 16 days of age) and weaned (28 days of age) rats were fed, fasted for 10 h, or fasted for 18 h and injected with a flooding dose of L-[4-3H]phenylalanine for measurement of muscle protein synthesis in vivo. In fed rats, fractional rates of protein synthesis (KS) and protein synthetic capacity decreased during the suckling period. KRNA increased toward the end of the suckling period. In 5-day-old rats, fasting for 10 h produced a 50% decline in KS of extensor digitorum longus and plantaris muscles, but KS did not change further after 18 h of fasting. In older suckled and weaned rats, 10 h of fasting decreased KS of extensor digitorum longus and plantaris muscles 30%; after 18 h of fasting, values had declined to 50% of those in fed animals. The reductions in KS in soleus muscles with 10 and 18 h of fasting were similar to those in other muscles at 5 and 10 days but were less than those in other muscles at 16 and 28 days. Changes in KRNA were similar to those for KS in all muscles from all age groups fasted for 10 and 18 h. Protein synthetic capacity decreased approximately 12% after 18 h of fasting, but this effect did not differ between age groups or muscle types.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721505 TI - Use of a low molecular weight preparation of heparin--ORG 10172 (Lomoparan) in the critically ill. PMID- 1721506 TI - Ultrastructural study of the hyaline layer of the starfish embryo, Pisaster ochraceus. AB - The hyaline layer (HL) is a multilayered extracellular matrix (ECM) that coats the external surfaces of sea urchin and starfish embryos. It is thought to protect and lubricate the embryo, stabilize the blastomeres during morphogenesis, and regulate nutrient intake. Ultrastructural studies of chemically fixed embryos have shown the HL to consist of two to four sublayers. However, since chemical fixatives may cause collapse and alter the positions and antigenicity of the extracellular components, fixation methods that exclude chemicals may reveal a picture of the HL closer to what is present in vivo. Freeze substitution, a fixation method whereby tissues are rapidly frozen and dehydrated at low temperatures, has proved useful for fixing material rich in ECM. In this study, embryos of the starfish Pisaster ochraceus were fixed for microscopy using freeze substitution and three chemical methods in order to determine, as accurately as possible, the structure of the HL. Embryos appear to be best preserved by freeze substitution and demonstrate a HL consisting of at least six distinct sublayers. Based on staining with anionic dyes, most sublayers appear to contain glycosaminoglycans. Freeze substituted embryos, which were also stained with monoclonal antibodies raised against their ECM, revealed that some molecules are common to all six sublayers, whereas other molecules may be restricted to specific sublayers. This suggests that each sublayer could have a different function. Additional evidence suggests that microvillus associated bodies, present in other marine invertebrate embryos, may anchor the asteroid HL to the cell surface microvilli. PMID- 1721507 TI - Apical mitochondria-rich cells in the human epididymis: an ultrastructural, enzymohistochemical, and immunohistochemical study. AB - An ultrastructural, enzymohistochemical, and immunohistochemical study of the ductus epididymis in normal men was undertaken to investigate the characteristics of the apical mitochondria-rich cells (AMRCs). These cells, which differ morphologically from the principal cells (PCs), appear in isolation in the caput epididymidis (5.8 +/- 1.7 cells per cross-sectional duct) and only occasionally in the corpus epididymidis. The morphologic appearance of AMRCs varies from slender cells extending from the basement membrane to the lumen to apical cells without apparent contact with the basement membrane. The former display a round pale nucleus located in the middle of the epithelium; the apical cells have a dark nucleus, which, surrounded by a narrow cytoplasmic band, protrudes into the lumen. The cytoplasm of AMRCs is electron-dense and contains numerous mitochondria surrounded by rough endoplasmic reticulum cisternae. In the apical portion, there are lysosomes, vesicles with an electron-dense granule, and vacuoles showing a variable size and content. The stereocilia are shorter and less numerous than those of the PCs. The AMRCs are similar to the PCs in the intensely positive reaction for the enzymatic activity acid phosphatase, as well as in the lack of reaction for alkaline phosphatase and phosphorylase activities. AMRCs differ from PCs in: (1) a more intense reaction to the enzymatic activities ATPase, NADP, and succinic dehydrogenease, (2) a more intense immunostaining by AE1/AE3 and Ks4.62 anti-cytokeratin antibodies, and anti-estradiol receptor protein (D5) antibodies, and (3) a lower staining affinity for epithelial membrane antigen (EMA) antibodies. No positive immunostaining for the anti cytokeratin Ks8.6 antibodies was observed in either AMRCs or PCs. PMID- 1721508 TI - [The cystic fibrosis gene: mutation and the function of CFTR protein]. AB - The spectrum of mutations identified in cystic fibrosis patients includes a major defect (delta F508), found in 70% of CF chromosomes from French patients, and a large number of infrequent mutations. This significant heterogeneity of molecular defects precludes large scale screening for the disease at the time being. The presumptive structure of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), the membrane protein encoded by the CF gene, is briefly recalled. The role this protein is thought to play in the regulation of chloride ion transport across epithelial membranes is discussed, along with the new insights into the molecular mechanisms of CF gained as a result of identification of the CF gene. PMID- 1721509 TI - [Congenital sensory neuropathy with anhidrosis: type IV. Apropos of 2 new cases]. AB - Two new cases of congenital sensory neuropathy (CSN) type IV in brothers aged 10 and 5 years are reported. Features included diffuse lack of response to pain without loss of response to touch, temperature and proprioceptive stimuli. No other neurologic anomalies were found. Both patients had complete anhidrosis. Joint destruction, which was the result of the failure to react to painful stimuli, was the most prominent feature. Nerve biopsy specimens exhibited marked reductions in numbers of amyelinic fibers with normal numbers of myelinic fibers. These two cases of CSN type IV are discussed in the light of previously reported cases and the new classification of congenital sensory neuropathies is reviewed. PMID- 1721510 TI - Perceptions by medical/surgical nurses of hospitalized dialysis patients. AB - The purpose of this study was to determine the perception by medical/surgical nurses of hospitalized dialysis patients. The results, tabulated from a demographic sheet and a 33-item questionnaire, revealed a positive attitude towards end stage renal disease (ESRD) patients by the staff of the facility where the study took place. The factors of age, nursing experience, and education were examined relative to this perception. The majority of the respondents indicated a strong desire to learn more about the needs of ESRD patients. In addition to the results, a valid and reliable tool for measuring nurses' perception of hospitalized dialysis patients was developed. PMID- 1721511 TI - [Active centers of gamma-glutamyltransferase in the aerosol OT reverse micellar system in octane by an inhibitor analysis method]. AB - Regulation of the supramolecular structure and catalytic activity of the heterodimeric enzyme gamma-glutamyltransferase in the system of Aerosol OT reversed micelles in octane was studied. Variation of the hydration degree causes a reversible dissociation of the enzyme to the light and heavy subunits, both possessing the catalytic activity. The subunits were separated on the preparative scale in the reversed micelle system using ultracentrifugation. The active centres of gamma-glutamyltransferase were studied using the enzyme's irreversible inhibitor AT-125 (L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5 isoxazoleacetic acid). It is shown that the separation of the gamma glutamyltransferase subunits results in "opening" of a new active centre in the heavy subunit, whereas in the enzyme's dimeric form this centre is masked and not accessible to the inhibitor's molecule. The kinetic and inhibitor analysis data indicate that the active centres in the light and heavy subunits are similar. PMID- 1721512 TI - Mucin histochemistry by paradoxical concanavalin A staining in early gastric carcinomas. AB - Phenotypic expression of tumor cells was investigated in 33 early gastric carcinomas by mucin histochemistry using paradoxical concanavalin A staining. This staining method had been developed to differentiate 3 classes of mucins located at various sites of the alimentary tract. Twenty-five (76%) tumors contained mixtures of neutral or acid class II mucin and class III mucin, suggesting the origin of multipotential stem cells. The surface mucous cell expression was more dominant than the pyloric gland or intestinal phenotypes in the well-and poorly differentiated adenocarcinomas. The intestinal properties of the tumor cells were noted not only in the well-differentiated but also in the poorly differentiated or signet ring cell carcinomas, not closely being related to the presence of background intestinal metaplasia. Signet ring cell carcinomas revealed a distinct pattern of mucin histochemistry compared with the other types. PMID- 1721513 TI - Experimental study of a new tracheal prosthesis made from collagen-grafted mesh. AB - The efficiency of a new tracheal prosthesis was studied. The prosthesis consists of fine Marlex mesh reinforced with a continuous Teflon spiral, and it is grafted and coated with pig collagen, with the aim of promoting connective tissue infiltration and providing air-tightness during the initial stage of implantation. Complete surgical resection and replacement of a 4-6 tracheal ring segment of the cervical trachea was performed in 13 adult mongrel dogs. Except for two dogs that developed anastomotic insufficiency, the prostheses in all dogs were infiltrated by the surrounding connective tissue, and were completely incorporated by the host trachea at the anastomotic sites. Formation of respiratory epithelium that lined the prosthesis lumen, was seen to varying degrees, and in one dog killed at 4 months after reconstruction, this was confirmed histologically from the upper to the lower anastomosis of the prosthesis. Stenosis of the lumen often occurred, however, due to deformation of the prosthesis and overgrowth of granulation tissue. The authors conclude that this tracheal prosthesis is highly biocompatible, and might be useful for repairing tracheal defects by improving prosthesis processing, especially that used for insertion of stents. PMID- 1721514 TI - Angiopolarity: a new design parameter for cell transplantation devices and its application to degradable systems. AB - The purpose of this study was to obtain directional angiogenesis of small blood vessels and capillaries to an implant made from a resorbable polymer for hepatocyte transplantation. It was intended to mimic the native acinar structure of the liver in form to facilitate replication of the cells and organ growth. The implant device structure was designed for injection to minimize surgical trauma. Hollow microspheres with an open porous wall structure and one large central opening were made from poly(d,1-lactic-co-glycolic acid) (85:15 lactic:glycolic). This polymeric scaffold was seeded with hepatocytes and implanted into the abdominal wall muscle of syngeneic Fisher rats. Specimens explanted up to 56 days postoperatively showed hepatocyte survival and the development of a directional blood supply, a phenomenon known as "angiopolarity." This study should help in addressing the issue as to whether vascular cell implants with posttransplantation organ growth should be attempted. PMID- 1721515 TI - Administration of haptoglobin during cardiopulmonary bypass surgery. AB - A study was undertaken to evaluate hemolysis and subsequent renal damage in 14 patients undergoing cardiopulmonary bypass (CPB) surgery. In all patients, free haptoglobin disappeared completely 30 to 90 minutes into CPB, while free hemoglobin (Hb) levels increased progressively. The NAG index and alpha 1M index also increased progressively, indicating renal tubular injury due to hemolysis (Study 1). An additional 20 patients were monitored intraoperatively for plasma free Hb levels by a newly developed colorimetric method using a haptoglobin coated strip. Free Hb levels during CPB exceeded 30 mg/dl in 14 patients, who were immediately given haptoglobin. This treatment eliminated plasma free Hb within 30 minutes, and effectively prevented hemoglobinuria. Haptoglobin treatment brought significant decreases in the NAG index and alpha 1M index, suggesting a protective effect on renal function (Study 2). PMID- 1721516 TI - Influence of hypertonic volume replacement on the microcirculation in cardiac surgery. AB - We have studied the effects of two types of volume replacement on the microcirculation in an open, controlled study in 45 patients undergoing aorto coronary bypass grafting whose pulmonary capillary wedge pressure (PCWP) was less than 5 mm Hg. Hypertonic saline prepared in hydroxyethylstarch solution (HS-HES, n = 15) and 6% HES 200/0.5 solution (6% HES; n = 15) were infused randomly before operation in order to double the PCWP. Patients not given an infusion served as controls (n = 15). Skin microcirculatory blood flow was investigated by laser Doppler flow (LDF) measured simultaneously at the forearm and forehead before and after cardiopulmonary bypass (CPB). Less HS-HES (3.8 (SD 0.3) ml kg-1) than 6% HES-solution (9.7 (1.5) ml kg-1) was necessary to double baseline PCWP. There were no differences in heart rate and mean arterial pressure (MAP) between the groups. Cardiac index (CI) increased significantly in both volume groups (HS-HES max. +54%; 6% HES max. +30%). Systemic vascular resistance (SVR) decreased after infusion of HS-HES (-30%) and after 6% HES(-19%) and remained almost unchanged in the control group. Plasma viscosity decreased after infusion of HS-HES and increased slightly in control patients (+4%). In comparison with the 6% HES and particularly with the control group, LDF was significantly greater after infusion of HS-HES (forearm +80%; forehead +28%). LDF during CPB and thereafter was always greater than baseline values in the HS-HES group, whereas after bypass LDF was reduced in the 6% HES (-5%) and particularly in the control patients ( 30%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721517 TI - Resistance to atracurium in a patient with an increase in plasma alpha 1 globulins. AB - We observed that a female patient with a poorly differentiated adenocarcinoma of the stomach undergoing gastrectomy was markedly resistant to the action of the neuromuscular blocking drug atracurium. There was no evidence of tumour metastasis and her liver function tests were normal. Electrophoresis of plasma proteins revealed a marked increase in alpha 1 globulin. Alpha 1 acid glycoprotein is an acute phase reactant that is increased in patients with cancer and is present in the alpha 1 globulin electrophoresis pattern. It is likely that the mechanism for the resistance to neuromuscular block in the patient was an increase in drug binding to alpha 1 acid glycoprotein. PMID- 1721518 TI - The transmembrane hyaluronate receptor (CD44): multiple functions, multiple forms. PMID- 1721519 TI - T-cell and B-cell function in lupus. AB - Investigation into the nature of the immunologic abnormalities responsible for autoantibody production continues to be an extremely active area of research in lupus. This paper reviews several areas of current research relevant to our understanding of T-cell and B-cell function in lupus. PMID- 1721520 TI - [An apparently primary lymphoma of the testicle]. AB - A case of apparently primary non-Hodgkin's lymphoma of the testis is described. The patient, in stage IVE at the diagnosis, was treated with left orchiectomy and chemotherapy (MACOP-B). The abdominal CT scan showed a retroperitoneal lymph node involvement with infiltration of the right kidney and liver. The patient died 6 months after onset of the disease due to its progressive course. PMID- 1721521 TI - Management of anorexia-cachexia associated with cancer and HIV infection. AB - The cachexia associated with cancer and AIDS appears to result from a number of processes, most of which impair caloric intake. Although past attempts to reverse anorexia-cachexia have generally been disappointing, several promising new pharmacologic approaches are currently being evaluated, including megestrol acetate, hydrazine sulfate, metoclopramide, and dronabinol. PMID- 1721522 TI - An overview of palliative care in cancer and AIDS. AB - Many patients with cancer or AIDS present with pain and multiple other physical and psychological symptoms. For patients with advanced cancer or AIDS, the symptoms are truly the disease and should be the basis for the therapeutic plan. This requires maintaining an active problemsolving approach, giving particular attention to major clinical problems (eg, pain control), and instituting early treatment of other symptoms. These problems need management throughout the course of disease, not just in the terminal stages. In incurable disease, management priorities must focus on controlling symptoms. PMID- 1721523 TI - Cross-reactivity between streptococcal M surface antigen and human skin. AB - Psoriasis can be triggered by haemolytic streptococcal infections. As M protein is a major pathogenic surface antigen in these streptococci, the cross-reactivity between streptococcal M protein surface antigens and human epidermis was investigated. The conserved component common to the few M proteins investigated consists of an alpha-helical 'coiled-coil' configuration, similar to sub-units of human keratin. The amino acid sequence of protein M6, one of the M proteins that has been fully sequenced, was compared with that of 4721 ubiquitous peptides, by computer-assisted analysis using a protein-sequence data bank. Of all human proteins in the data bank 50-kDa keratin type 1 showed the closest homology with protein M6. Further evaluation revealed that this homology mainly involved the heptapeptide repeat patterns, which form the alpha-helical 'coiled-coil' structure, in both M6 and 50-kDa keratin. Cryostat sections of normal, involved and uninvolved psoriatic skin were studied for cross-reactivity with rabbit antisera raised against 10 different M proteins. All these antisera reacted with the stratum corneum of normal and psoriatic epidermis to a variable extent. Staining of keratinocyte cytoplasm was also observed, but this tended to be more prominent in lesional than in uninvolved and normal skin. Some of the M antisera also stained dendritic cells in the upper dermis as well as endothelium and smooth muscle. These cross-reactivities might be relevant to the pathogenesis of post-streptococcal psoriasis. PMID- 1721524 TI - New approaches to the treatment of follicular lymphoma. AB - The major avenues of clinical research into the treatment of follicular lymphoma, 'more, if so when?', interferon therapy, and antibody therapy, have been presented in the light of present knowledge about the clinical course of the disease. They must be seen within the context of the current philosophical approach to the illness, and the economic climate which prevails, at a time when new drugs, for example fludarabine (Leiby et al, 1987; Reman et al, 1988; Whelan et al, 1991), are showing promise, and differentiating agents are being tested in remission (Cunningham et al, 1985). There can be little doubt that the objective of future research should be to eliminate the disease altogether at the time of initial presentation, since patients entering remission and never having a recurrence have a far greater probability of longevity than those in whom recurrences occur (Lister, 1991). There can also be little doubt that when lymphoma is present and causing symptoms, treatment should be given, since survival is longer for those in whom a response is achieved, at least at presentation, and at first recurrence (Lister, 1991). Since the latter is sadly the reality for the majority, improving treatment at the time of recurrence must also be a priority. Time will tell whether any of the options presently under investigation will be appropriate at all, and if so when. It is certainly the case that some of them will be entirely inappropriate for some patients, because the risk of toxicity will outweigh the potential benefit, especially for the elderly. Further careful identification of prognostic variables may allow for individualization of therapy. It would be comforting to know that the newly found molecular marker of the disease would help us. Its absence may do--but its presence certainly does not, since t(14;18) containing cells may seemingly be present for many years of clinical normality (Price et al, 1991, in press). The challenge to find the right treatment at the right time--or perhaps to identify the 'right patient' for the therapy continues. PMID- 1721525 TI - Primary human acute myeloblastic leukaemia: an analysis of in vitro granulocytic maturation following stimulation with retinoic acid and G-CSF. AB - Acute myeloid leukaemia (AML) is characterized by the inability of myeloid cells to reach terminal maturation. We examined to what degree granulocytic maturation could be achieved by stimulating AML blast cells in an in vitro serum-free system with a combination of granulocyte-colony stimulating factor (G-CSF) and all-trans retinoic acid (RA). Specimens from 41 AML patients were cultured for 7 d and then examined for cytochemistry (myeloperoxidase, Sudan Black, naphthyl-ASD chloracetate esterase, periodic acid Schiff) an nitroblue tetrazolium reduction. The expression of CD11a, CD11b, CD11c, CD15, CD18, CD10, CD24 and B13-3 membrane antigens was also evaluated. Morphological and cytochemical studies were also performed after AML colony culture and culture of normal bone marrow cells (NBM). The comparative analysis of the panel of parameters was indicative of granulocytic maturation although to different degrees. The cells from 25/41 cases showed morphologic maturation (May-Grunwald-Giemsa). A positive correlation was evident between morphological maturation and CD11b expression (11/22 patients) as well as that of CD11c and CD15 (6 patients). Napthyl ASD chloroacetate esterase and PAS stainings also correlated with morphology (in 10/22 and 10/24 patients respectively). Nevertheless, the pattern of granulocytic maturation was remarkably variable among the 41 cases examined. The cells from only a few patients acquired the full spectrum of granulocytic markers. The comparison with normal bone marrow blasts indicates that serum-free culture conditions can, per se, limit granulocytic maturation, but it also confirms the intrinsic inability of AML cells to attain complete maturation in response to two potent granulocytic inducers. PMID- 1721526 TI - Expression of cytoadhesion molecules (CD56, CD54, CD18 and CD29) by myeloma plasma cells. AB - Recently we reported the expression of the human natural killer cell associated antigen CD56 (Leu 19/NKH1) in plasma cells of a majority of multiple myeloma (MM) patients. CD56 is known to be an isoform of the human neural adhesion molecule N CAM which is involved in homotypic adhesive interactions. By immunophenotyping using four CD56 specific monoclonal antibodies and immunoprecipitation analysis we here confirm that the Leu 19 antigen expressed by myeloma plasma cells is identical to N-CAM and corresponds to the 145 kDa isoform. Because of the possible biological role of adhesion molecules on myeloma cells, we compared the expression of N-CAM with the intercellular adhesion molecule 1 (ICAM-1) and the beta 1 and beta 2 integrins. By immunogold-silver staining of cytospin preparations of mononuclear cell suspensions, bone marrow plasma cells of 17 MM patients were analysed. Plasma cells expressed N-CAM (CD56) in 14 patients. ICAM 1 (CD54) in 16 patients, and beta 2 integrins (CD18) in eight patients. beta 1 integrins (CD29) were expressed in all patients. The expression of beta 2 integrins was always very weak while N-CAM, ICAM-1 and the beta 1 integrins showed a moderate to strong positivity. The plasma cells of five haematological normal individuals lacked significant N-CAM expression but were positive for ICAM 1 and both integrin subgroups. One plasma cell leukaemia patient and two out of four end-stage MM patients showed no expression of N-CAM or beta 2 integrins on their circulating plasma cells. Among 11 previously established myeloma cell lines, surface expression of ICAM-1 and the integrins was detected in most cases, while N-CAM was present in only four lines. Most cell lines showed coexpression of the fibronectin receptors (VLA-4 and VLA-5) and the laminin receptor (VLA-6). The collagen receptor (VLA-2) was not expressed. The N-CAM negative cell lines included four cell lines that were derived from plasma cell leukaemia patients. These results indicate that the expression of adhesion molecules is an intrinsic part of the biology of multiple myeloma. PMID- 1721527 TI - Hepatitis C antibody profile and viraemia prevalence in adults with severe haemophilia. AB - Sera from 21 patients who had received large amounts of unheated factor VIII concentrate were tested for antibodies to the hepatitis C virus (HCV) by both commercial (Ortho C100) and 'in house' ELISAs. 'In house' assays utilized recombinant structural (core) or non-structural (replicase) HCV proteins generated by a baculovirus expression system. Antibodies to HCV were detected in 100% of the sera by the core protein based ELISA but in only 62% and 19% by the C100 and replicase based ELISAs, respectively. Hepatitis C viraemia was demonstrated in 90% of the patients by in vitro amplification of the 5' non coding region of the HCV genome. Amplification with primer sets from two other regions of the genome proved less efficient at detecting viraemia. We conclude that the prevalence of hepatitis C infection in haemophiliacs may have been underestimated previously and that almost all HCV-infected patients have evidence of on-going viral replication. PMID- 1721528 TI - Prognostic value of CD34 expression in de novo acute myeloblastic leukaemia. PMID- 1721529 TI - Cell cycle analysis of Krox-20, c-fos, and JE expression in proliferating NIH3T3 fibroblasts. AB - We have shown that early growth response genes which were identified on the basis of their expression during the G0 to G1 transition can also be induced in proliferating fibroblasts. The expression of Krox-20 and c-fos mRNAs increased dramatically upon stimulation of cell populations of increasing density and correlated with the percentage of cells in the G0-G1 stages of the cell cycle. However, fractionation of serum-stimulated cultures into cell cycle stage specific subpopulations using fluorescence-activated cell sorting revealed that the levels of Krox-20 and c-fos mRNAs were equal in all stages of the cell cycle. This result was corroborated by serum and cycloheximide stimulation of stage specific fractions separated by centrifugal elutriation. Expression of the immediate-early gene JE was also induced in all stages of the cell cycle in the elutriated fractions. Thus, although the amount of induced Krox-20, c-fos, and JE mRNA correlated with the number of quiescent cells in a proliferating population, expression of all three genes occurred to the same extent in all stages of the cell cycle at a given culture density. Possible explanations for the density associated increase in induced expression are discussed. We have demonstrated that there are both transcriptional and posttranscriptional components to the stimulated expression of the Krox-20 and c-fos genes in proliferating fibroblasts. Since the increased expression of these genes has the same steady state and transcription rate kinetics as serum response factor-mediated induction, we are currently investigating the role of serum response factor in serum-induced expression in proliferating cells. PMID- 1721530 TI - Visual-field map in the transcallosal sending zone of area 17 in the cat. AB - The representation of the visual field in the part of area 17 containing neurons that project axons across the corpus callosum to the contralateral hemisphere was defined in the cat. Of 1424 sites sampled along 77 electrode tracks, 768 proved to be in the callosal sending zone, which was identified by retrograde transport of horseradish peroxidase that had been deposited in the opposite hemisphere. The results show that the callosal sending zone has a fairly constant width of between 3 and 4 mm at most levels in area 17. However, the representation of the contralateral field at the different elevations of the visual field is not equal in this zone. The zone represents positions within 4 deg of the midline at the 0 deg horizontal meridian, and positions out to 15-deg azimuths in the upper hemifield and out to positions of 25-deg azimuth in the lower hemifield. The shape of the representation is approximately mirror-symmetric about the horizontal meridian, although there is a greater extent in the lower hemifield, which can be accounted for by the greater range of elevations (greater than 60 deg) represented there compared with the upper hemifield (approximately 40 deg). The representation in the sending zone of one hemisphere matches that present in the area 17/18 transition zone, which receives the bulk of transcallosal projections, in the opposite hemisphere. The observations on the sending zone show that callosal connections of area 17 are concerned with a vertical hour glass-shaped region of the visual field centered on the midline. The observations suggest that in addition to interactions between neurons concerned with positions immediately adjacent to the midline, there are positions, especially high and low in the visual field, where interactions can occur between neurons that have receptive fields displaced some distance from the midline. PMID- 1721531 TI - Visual-field map in the callosal recipient zone at the border between areas 17 and 18 in the cat. AB - The representation of the visual field in the callosal fiber recipient zone of area 17 and the adjacent area 17/18 transition zone was determined in the cat. The callosal fiber recipient zone was identified by anterograde transport of tritiated amino acids that had been injected into transcallosal sending zone of the opposite hemisphere. Application of autoradiographic procedures revealed that transcallosal projections are densest in the area 17/18 transition zone, and that their density in area 17 diminishes within 1-2 mm of the transition zone. Of 980 sites sampled in the visual-field mapping part of the study, 507 proved to be in the zone demarcated by transcallosally transported label. In this zone, both ipsilateral- and contralateral-field positions are represented, and the representation of the visual field at the different elevations is not equal. When ipsilateral-field positions are considered, the representation extends to about 4 deg close to the visual axis, and to 15-20 deg at elevations greater than +/- 30 deg, the representation is approximately mirror-symmetric about the horizontal meridian, and the representation is concordant with that of the representation in the area 17 transcallosal sending zone of the opposite hemisphere. PMID- 1721532 TI - Respiratory function in severe gestational proteinuric hypertension: the effects of rapid volume expansion and subsequent vasodilatation with verapamil. AB - OBJECTIVES: (1) To define the baseline respiratory function in untreated severe gestational proteinuric hypertension (GPH) and (2) to assess the effects of volume expansion with dextran (MW = 70,000 Dalton) and subsequent vasodilatation with the calcium antagonist verapamil on the baseline respiratory function in severe GPH. DESIGN: Prospective descriptive study. SETTING: Reproductive Research Unit, Groote Schuur Hospital, Cape Town, South Africa. SUBJECTS: Six women with severe GPH undergoing stabilization and delivery. INTERVENTIONS: Baseline haemodynamic and respiratory function was assessed using invasive monitoring. Patients then underwent volume expansion to a pulmonary capillary wedge pressure of 16 mmHg with dextran-70, followed by vasodilatation with the calcium antagonist verapamil. Haemodynamic and respiratory variables were measured, before and after both the fluid load and the reduction (20%) in the mean arterial pressure. MAIN OUTCOME MEASURES: Mean arterial pressure, heart rate, mean pulmonary arterial pressure, pulmonary capillary wedge pressure, central venous pressure, cardiac index, systemic vascular resistance, pulmonary vascular resistance, respiratory rate, blood gases, alveolar arterial oxygen difference, oxygen availability, oxygen consumption, pulmonary shunt fraction. RESULTS: Baseline oxygen availability/delivery and oxygen consumption indices were consistent with severe tissue ischaemia. Volume loading with 400 +/- 114 ml dextran-70 normalized these variables, and subsequent vasodilatation with verapamil did not reduce these indices below the normal limits for pregnancy. CONCLUSIONS: These data support the theory that some of the complications of severe GPH may follow organ damage due to prolonged tissue ischaemia. They also support the appropriateness of controlled volume expansion in the management of this condition. We suggest, from these data, that the combination of volume expansion and verapamil vasodilatation lowers the blood pressure without compromising the maternal respiratory function. PMID- 1721533 TI - Early fetal hematopoietic development from in vitro differentiated embryonic stem cells. AB - In this report we describe the efficient hematopoietic differentiation of embryonic stem (ES) cells in vitro. When cultured in semisolid medium two of five ES cell lines efficiently generated embryoid bodies (EBs) containing blood islands in which hematopoietic cells from all six myeloid lineages could be detected. Among a variety of growth factors tested, only erythropoietin significantly increased blood island formation. We directly demonstrate the presence of hematopoietic progenitors in the EBs by employing an in vitro precursor assay. Colony-forming cells (CFC) of all myeloid lineages as well as bi and multipotent (CFC-MIX) progenitors were readily identified, and a detailed time-course analysis of their appearance was performed. Despite a high frequency of CFC-MIX in vitro, we did not observe any spleen colony-forming cells (CFU-S) in vivo. We conclude that hematopoietic differentiation of ES cells under these conditions reflects formation of the complete range of blood cells found in the yolk sac of the early fetus. Therefore this system provides a unique model in which to study the earliest events of hematopoietic development in vitro. PMID- 1721534 TI - Downregulation of GMP-140 (CD62 or PADGEM) expression on platelets by N,N dimethyl and N,N,N-trimethyl derivatives of sphingosine. AB - GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721535 TI - Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding. AB - Human immunodeficiency virus 1 reverse transcriptase (RT) purified from virions is composed of a approximately 51,000 Mr polypeptide and a approximately 66,000 Mr polypeptide that are thought to be in heterodimer structure (Chandra et al., 1986; Hansen et al., 1988; Starnes & Cheng, 1989) and are identical except for a 15,000 Mr C-terminal truncation in the smaller species (Di Marzo-Veronese et al., 1986). We prepared individual bacterial-recombinant RTs as the approximately 66,000 Mr polypeptide (p66) or as the approximately 51,000 Mr polypeptide (p51) and then conducted various in vitro protein-protein binding experiments. Analytical ultracentrifugation studies in 0.25 M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1 x 10(4) M-1. p51 failed to dimerize and behaved as a monomer under these conditions. Mixing of the p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 x 10(5) M-1. These results on formation of the p66/p66 homodimer and p66/p51 heterodimer were confirmed by gel filtration analysis using FPLC Superose-12 columns. Binding between p66 and individual p66 segment polypeptides also was observed using an immunoprecipitation assay. Binding between p51 and p66 in this assay was resistant to the presence of approximately 1 M NaCl, suggesting that the binding free energy has a large hydrophobic component. C-Terminal truncation of p66 to yield a 29-kDa polypeptide eliminated binding to p66, and N-terminal truncation of p66 to yield a 15-kDa peptide also eliminated binding to p66. The results indicate that purified individual RT peptides p51 and p66 are capable of binding to form a 1:1 heterodimer and suggest that the central region of p66 is required for this subunit binding; the C-terminal region (15,000 Mr) of p66 appears to be required also, as p51 alone did not dimerize. PMID- 1721536 TI - Drug binding to a DNA BZ molecule: analysis by chemical footprinting. AB - The polymorphism in a DNA 16-mer (designated BZ-II) has been investigated by means of circular dichroism (CD) spectroscopy and chemical footprinting. CD spectra indicate that, in low salt, the oligomer is fully right-handed whereas, in high salt, it possesses a B-Z conformational junction: half of the duplex is right-handed while the other half is left-handed. Treatment of BZ-II with diethyl pyrocarbonate (DEPC) confirms the existence of a left-handed segment of the duplex in high salt: enhanced DEPC scission occurs at the G residues in the alternating CG sequence. The scission patterns of the upper and lower strands in BZ-II by the reactive chemical probe MPE.Fe(II), and the antitumor antibiotics dynemicin and Fe-(II).bleomycin, are different under low salt conditions. The 3' terminal region of both upper and lower strands and the middle region of the upper strand of BZ-II are preferential cleavage sites in low salt. This result suggests that the methylated cytosines or the alternating CG domain in the molecule perturbs the DNA structure. Under high salt conditions, the reactivity of the Z-DNA segment of BZ-II for MPE.Fe(II) and Fe(II).bleomycin is dramatically enhanced, while it is reduced in the case of dynemicin. Excess propidium (PI) eliminates preferential cleavage by each of these chemical probes in high salt conditions. This is due in part to conversion of the BZ-DNA molecule into B-DNA, as is seen by a DEPC modification experiment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721537 TI - Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases. AB - The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116 kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721538 TI - Cyclic GMP-activated channel activity in renal epithelial cells (A6). AB - We studied the effects of guanosine 3',5'-cyclic monophosphate (cGMP) and nitroprusside on ion channels in the apical membrane of confluent A6 cells (a distal nephron cell line) cultured on permeable supports for 10-14 days using patch clamp techniques. In cell-attached patches without any detectable channel activity, activity of a non-selective cation channel with a single-channel conductance of 1 pS was observed after adding nitroprusside. After adding cGMP to the cytosolic surface of inside-out patches with no detectable channel activity, we observed single channel activity similar to the channel observed after adding nitroprusside. These observations imply that nitroprusside activates a non selective cation channel with small single channel conductance (1 pS) via an increase in cGMP which activates the channel. PMID- 1721539 TI - The effect of changes in gramicidin conformation on bilayer lipid properties. AB - The effects of two different gramicidin conformations on lipid phase behaviour and dynamics are compared. Samples of chain-perdeuterated dimyristoylphosphatidylcholine containing gramicidin were first prepared with gramicidin in a state having a circular dichroism spectrum generally identified as corresponding to the non-channel conformation. The effects, on bilayer lipid properties, of gramicidin in this conformation were then determined using deuterium nuclear magnetic resonance measurements of acyl chain orientational order and transverse relaxation times as a function of temperature. These samples were then incubated at 65 degrees C to convert the gramicidin to a state with a circular dichroism spectrum of the type generally identified with the channel conformation. The nuclear magnetic resonance measurements were then repeated. In the gel phase, it was found that transverse relaxation time and chain orientational order of the lipid were insensitive to gramicidin conformation. In the liquid crystalline phase, gramicidin in the channel conformation was found to have a slightly larger effect on transverse relaxation and orientational order than gramicidin in the non-channel conformation. The perturbation of the phase behavior by gramicidin was found to be relatively insensitive to gramicidin conformation. PMID- 1721540 TI - Proteins and peptides bound to long-circulating liposomes. AB - Liposome formulations with prolonged circulation time have recently been developed as a potential sustained-release drug delivery system. Data shown in this report indicate that such formulations can also be used to prolong the circulation time of proteins and peptides by conjugating them to the surface of liposomes. Increase of the circulation halflife ranged from 2- to 150-fold depending on the protein/lipid ratio of the liposomal formulation, liposome size, and the lipid composition of liposomes. Since the proteins/peptides localize on the liposome surface, instead of being entrapped inside the liposomes, they are directly available for binding to its receptor molecules and express the biological activity. This strategy has been successfully applied to two proteins with known fast clearance rate, i.e. asialofetuin and ricin A-chain. The biological activities of both proteins are preserved when they are formulated in liposomes. Incorporation of a peptide, i.e. a-factor of the yeast Saccharomyces cerevisiae, into the liposome membrane also significantly enhanced the circulation time of the peptide. PMID- 1721541 TI - Low levels of the pesticide, delta-hexachlorocyclohexane, lyses human erythrocytes and alters the organization of membrane lipids and proteins as revealed by Raman spectroscopy. AB - We studied the nature of the interaction of delta-hexachlorocyclohexane (delta HCCH), a pesticide having a stereoisomeric structure similar to inositol, with red blood cells. Cell survival data, measured as percent of hemoglobin released by delta-HCCH, show that the cell lysis increases with post exposure time. delta HCCH at 55-60 micrograms/ml causes about 70% cell lysis after 24 h of exposure. The nature of interaction of delta-HCCH with membrane components was evaluated by studying the thermotropic transitions and protein structure of ghosts using Raman spectroscopy. Control ghosts show transitions with onset/completion temperatures 30 degrees C/38 degrees C (high temperature transition) and 3 degrees C/10 degrees C (middle temperature transition) when monitored by the I2935/I2850 ratio. The interaction of delta-HCCH drastically broadens the high temperature transition and shifts it to the temperature range of 10-29 degrees C. The plots of (I2880-90/I2850) vs. temperature show two transitions for control ghosts, one extending from -10 degrees C to 3 degrees C (lower temperature transition) and the other from about 7 degrees C to about 15 degrees C (middle temperature transition). Ghosts lysed with delta-HCCH shows only a single and a very broad transition in the range of about -3 degrees C to about 15 degrees C. These changes in the thermal transition properties suggest that delta-HCCH alters lipid and lipid-protein phases of erythrocyte membranes. The comparison of Raman spectra in the amide I and III regions of erythrocyte ghosts and purified band 3 with several amidated compounds reveals that cytoskeleton proteins contain highly amidated residues (probably glutamine and asparagine). The interaction of delta HCCH with erythrocytes drastically alters the environment of these amidated residues indicating the involvement of cytoskeleton proteins. We conclude that the interaction of delta-HCCH with red blood cells disrupt membrane structure and change the environment of cytoskeleton proteins that could cause cell lysis. PMID- 1721542 TI - Activation of ion transport pathways by changes in cell volume. AB - Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because it's functionally important, it's relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, it's fun to do. PMID- 1721543 TI - The mas oncogene enhances angiotensin-induced [Ca2+]i responses in cells with pre existing angiotensin II receptors. AB - The proposal that the mas oncogene is an angiotensin receptor was evaluated in Xenopus oocytes injected with human and rat mas RNA transcripts, and during transient expression of mas in several cell lines. No evidence of mas-induced angiotensin II (AII) receptors or [Ca2+]i responses was observed in Xenopus oocytes or in most of the transfected cells. However, Cos-1 cells, which showed a small endogenous [Ca2+]i response to AII, exhibited a modest but reproducible enhancement of this response after mas transfection. Such responses were inhibited by [Sar1, Ala8]AII and [Sar1, Ile8]AII, but not by [D-Arg1, D-Pro2, D Trp7,9, Leu11] substance P, an antagonist reported to inhibit mas-induced responses to AII in oocytes. These findings are not compatible with the proposal that the mas oncogene is an angiotensin receptor, but suggest that expression of mas leads to increased responsiveness of the endogenous AII signaling system. PMID- 1721544 TI - Selective stimulation of in situ intermediary metabolism by free calcium in permeabilized rat adipocytes. AB - The hypothesis that ionized calcium [Ca2+]i may stimulate in situ rat adipocyte intermediary metabolism distal to glucose transport was tested. A metabolically active porous adipocyte model was employed in which pathway metabolism is exclusively pore-dependent using glucose 6-phosphate (G6P) as substrate. Cellular [Ca2+]i was, furthermore, directly adjusted to between 0-2.5 microM via the membrane pores. Three metabolic fluxes were examined, (1) glycolysis-Krebs ([6 14C]G6P oxidation), (2) glycolysis to lactate ([U-14C]G6P to [14C]lactate) and (3) pentose pathway ([1-14C]G6P oxidation). Glycolysis-Krebs oxidation was was found to be selectively (33% above basal P less than 0.001) stimulated by 0.625 microM free calcium. In contrast, there was no effect of [Ca2+]i on the other, exclusively cytoplasmic, pathways. The stimulation of glycolysis-Krebs by [Ca2+]i was inhibited by a mitochondrial calcium channel blocker (Ruthenium red) and persisted over a range of ATP/ADP ratios. Separate studies demonstrated that 2-[1 14C]ketoglutarate oxidation was also calcium-stimulated in the porous adipocytes (160% over baseline at 1 microM [Ca2+]i). These studies thus demonstrate that physiologically relevant increments in porous adipocyte [Ca2+]i enhance overall in situ glycolytic-Krebs pathway oxidation by a mechanism which entails mitochondrial calcium uptake. Methodologically, this metabolically active porous adipocyte model presents a novel experimental approach to investigations regarding the effects of ionized calcium on intermediary metabolism beyond glucose transport. PMID- 1721545 TI - Chemotherapy and combined modality treatment in Hodgkin's disease. AB - Several issues in the management of Hodgkin's disease remain to be resolved. These include 1) the most appropriate and effective first-line therapy for advanced disease, 2) the best approach to management of bulky mediastinal disease, 3) the management of uncommon presentations such as subdiaphragmatic disease, and 4) the best approach to salvage therapy for the patient with relapsed or refractory disease. Despite several years of clinical research and several significant advances in the treatment of this disease, the most effective treatment with the minimum amount of acute and delayed toxicity remains to be defined for several subsets of patients. This article reviews recent publications addressing these issues. PMID- 1721547 TI - Usefulness of paradoxical Con A staining for diagnosis of papillary adenocarcinoma of the apocrine anal sac gland in the dog. PMID- 1721546 TI - Lectin staining of peritoneal mesothelial cells in vitro. AB - A survey of lectin-binding specificities present on rodent and human mesothelial cells propagated and maintained in tissue culture was made using fluorescein isothiocynate conjugated (FITC) lectins. Rodent and human cells exhibited cell associated fluorescence following exposure to the FITC-lectins from C. ensiformis, T. vulgaris, A. hypogaea, E. cristagalli and B. simplicifolia, but not with lectins from G. max and D. biflorus. Rodent cells were also positive for FITC-M. pomifera lectin binding. Human, but not rodent, cells were positive for FITC-T. purpureas lectin binding. Exposure of rabbit mesothelial cells in vitro to FITC-lectins that bound to the cell surface resulted in the appearance of discrete loci of putatively intracellular fluorescence. Exposure of cells to ferritin-labelled T. vulgaris lectin at 37 degrees C for as little as 7.5 minutes resulted in the appearance of ferritin-size particles in intracellular vesicles. These results demonstrate 1. the presence of lectin-binding sites in and on peritoneal mesothelial cells from rodents and humans and 2. a possible role of such sites in mediating the entry of lectin-like endogenous molecules into the vacuolar apparatus of these cells. PMID- 1721548 TI - [Cellular resistance to interferon]. AB - The effectiveness of interferon (IFN) action depends not only on the type of IFN but also on the sensitivity of target cells to IFN. Revealing the reasons of different sensitivity of cells to IFN is of significant importance for understanding the mechanisms of cell reaction regulation. The review presents the principle historical stages of study of cell resistance to IFN, some aspects of cell insusceptibility to antiviral and antiproliferative action of various types of IFN are considered. Types of cell resistance to specific IFN effects have been determined. The mechanisms of development of cell resistance and possibilities in correction of cell insusceptibility to IFN are discussed. PMID- 1721549 TI - The expression of c-kit protein during oogenesis and early embryonic development. AB - The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation. PMID- 1721550 TI - Hemodialysis-related leukotriene B4 generation and neutropenia during calcium channel blockade. AB - Cuprophane membranes elict intense blood-surface interactions during clinical hemodialysis. The goal of the present studies was to determine whether calcium channel blockade may alter hemodialysis-related leukotriene B4 (LTB4) generation and neutropenia in 12 patients with end-stage renal failure. The patients were randomized to receive either placebo or nitrendipine for six weeks. Compared with untreated patients, the calcium channel blocker treatment group had significantly lower predialysis plasma LTB4 levels, Nitrendipine reduced both the magnitude of LTB4 generation and of neutropenia during the early phase of hemodialysis, but did not alter the close temporal relation of LTB4 accumulation and neutrophil activation. Therefore, the generation and release of LTB4 by neutrophils may be a calcium-dependent event. Furthermore, these results suggest that activation of neutrophil 5-lipoxygenase may contribute to the alterations in neutrophils of hemodialysis patients. PMID- 1721551 TI - Monoclonal antibodies to the components of the high-molecular-mass aminoacyl-tRNA synthetase complex. AB - Six monoclonal antibodies to the components of the rabbit multienzyme aminoacyl tRNA synthetase complex were generated and characterised. Two, F7 and F31 were directed against arginyl-tRNA synthetase, two, F8 and F25 against glutamyl-tRNA synthetase, and two, F6 and F12 recognised 38 and 43 kDa polypeptides, respectively. All antibodies were species-specific and failed to affect the activity of the respective enzymes. PMID- 1721552 TI - Biological activity of a human monoclonal antibody to Bordetella pertussis lipooligosaccharide. AB - The heterohybridoma cell line HBp2 secreting human monoclonal antibody (hMAb) directed against Bordetella pertussis was generated by fusing SP2/HPT heteromyeloma cells with human spleen lymphocytes, after in vitro stimulation for 6 days. The hybridoma was maintained in culture for more than 1 year with continuous antibody secretion. The hMAb HBp2, an IgM, reacted with untreated and proteinase K-treated B. pertussis outer membrane antigens, whereas the reactivity was lost when the antigen was treated with sodium periodate. Human MAb HBp2 was shown to be specific to B. pertussis LOS by immunoblotting of whole cell extracts after SDS-PAGE. In a dot enzyme immunoassay, HBp2 reacted with all B. pertussis strains and clinical isolates tested except for four atypical variant strains of the LOS B phenotype. Human MAb HBp2 also reacted with a clinical isolate of B. bronchiseptica. No reaction was observed against B. parapertussis and other gram negative species. Together these studies suggested that HBp2 is reactive with carbohydrate epitopes present on the LOS A. Binding assays with live bacteria demonstrated that hMAb HBp2 reacted with cell surface exposed epitopes on B. pertussis but the antibody did not bind significantly to the surface on intact B. bronchiseptica cells. When examined for bactericidal activity in the presence of complement, hMAb HBp2 showed high lytic capability against B. pertussis while no killing was obtained against B. bronchiseptica. These experiments established that LOS A is a target for human bactericidal antibodies. This antigen merits further investigation as a potentially important component in human immunity to B. pertussis infection. PMID- 1721553 TI - Reactive oxygen metabolites cause massive, reversible proteinuria and glomerular sieving defect without apparent ultrastructural abnormality. AB - To identify the specific in vivo renal effect of reactive oxygen species (ROS), hydrogen peroxide (H2O2) was infused directly into the left renal artery in Munich-Wistar rats. H2O2 (5 to 50 mumol over 1 h) induced a dose-dependent increase in urine protein excretion rate in infused kidneys, reaching a maximum at the dose of 35 mumol (on average, a 60-fold increase from baseline). The H2O2 (35 mumol)-induced proteinuria peaked over 1 h and completely normalized by 24 h after the infusion. Electrophoresis revealed that the urine protein is primarily of glomerular origin. Fractional clearances of graded-size neutral dextran of larger molecular radii, an index of glomerular size selectivity, were significantly and substantially elevated immediately but normalized by 24 h after the infusion. GFR and RPF rate remained unchanged throughout the entire time course examined. The H2O2-induced proteinuria was largely prevented by pretreatment with catalase (20 mg, iv) or deferoxamine (30 mg/100 g body wt, iv). Thus, iron-dependent metabolites of hydrogen peroxide appear to be involved in this proteinuria and glomerular size-selective defect. Light and electron microscopy, including determination of anionic site density at lamina rara externa of glomerular capillary wall by polyethyleneimine staining, did not reveal any appreciable abnormality throughout the study period, including at the peak of proteinuria. Thus, ROS can cause massive, reversible proteinuria by inducing a molecular size-selectivity defect of the glomerular capillary wall without apparent ultrastructural abnormalities. The results raise the possibilities: (1) that persistent proteinuria of a variety of renal diseases may reflect persistence of pathogenic ROS acting on glomeruli because the potent proteinuric effect of ROS can be transient (2) that the light and electron microscopy abnormalities in glomeruli of ROS-induced renal injuries reported thus far may have no direct causal linkage to proteinuria; and, finally, (3) ROS induced reversible proteinuria may relate to the mechanism of clinical functional proteinuria, which involves increased oxygen and ROS metabolism, e.g., exercise induced proteinuria. PMID- 1721554 TI - Pathogenesis of chronic active hepatitis B. PMID- 1721555 TI - Congenital hearing loss in Jervell and Lange-Nielsen syndrome. AB - Jervell and Lange-Nielsen syndrome is an autosomal recessive hereditary condition that presents with cardiac abnormalities characterized by a prolonged Q-T electrocardiographic pattern and congenital severe-to-profound auditory deficits. This paper describes the auditory history of twin boys born out of consanguinity and diagnosed with this syndrome. Both infants were products of the neonatal intensive care unit (NICU) and failed initial ABR screening. Diagnostic evaluation demonstrated profound hearing loss and developmental delays for each infant. Because sudden death is a consequence, audiologists are advised to recognize signs and symptoms associated with this syndrome. PMID- 1721556 TI - [The TNM classification adapted to palliative care]. AB - The staging of tumors according to the "TNM" system was developed by P Denoix between 1943 and 1952. The "TNM" system is based on 3 items of data: the clinical aspect of the tumor "T", the regional lymph nodes "N", and the presence or absence of distant metastases. According to the extent of the local, regional and distant sites the TNM system permits definition of tumor stage. These stages allow comparison of the results from different centers to be made and the establishment of treatment protocols. We have taken the principles of the TNM staging of the UICC and the AJCC staging and applied them to "palliative stages" of cancer patients in an attempt to define the profile of the "palliative care patient", and to exchange the results of treatment between cancer centers. PMID- 1721557 TI - Phosphotyrosine-containing proteins in bovine chromaffin cells: effects of insulin-like growth factor I (IGF-I). AB - 1. Antiphosphotyrosine antibodies were used to detect phosphotyrosine-containing proteins in immunoblots of bovine chromaffin cell proteins. 2. Unstimulated cells exhibited two major phosphotyrosine-containing proteins, which had Mr's of 121,000 and 70,000. Insulin-like growth factor I (IGF-I) had little effect on the phosphotyrosine content of these two proteins but greatly increased the phosphotyrosine content of three other proteins of Mr 185,000, 170,000, and 96,000. These proteins were found predominantly in the particulate fraction of cell homogenates. 3. The effects of the IGF-I were time and concentration dependent, with maximal increases in phosphorylation occurring after 1 min of treatment with 10 nM IGF-I. Na3VO4, an inhibitor of phosphotyrosine phosphatases, potentiated the effects of IGF-I. 4. Thus, the IGF-I receptor appears to function as an IGF-I-activated protein tyrosine kinase in chromaffin cells. The tyrosine kinase activity of the IGF-I receptor presumably mediates the effects of IGF-I on chromaffin cell function. PMID- 1721558 TI - Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments. AB - The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5 thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120]. PMID- 1721559 TI - Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides. AB - 3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres. PMID- 1721560 TI - The pattern and timing of cutaneous hair follicle innervation in the rat pup and human fetus. AB - The postnatal development of hair follicle innervation was studied in the rat hindlimb using a silver stain which detects large and medium calibre cutaneous nerve fibres. The pattern and timing of innervation in relation to postnatal changes in follicle growth were studied providing new data on nerve-target interactions in the developing peripheral nervous system. Sensory axons begin to leave the dermal plexus and grow towards follicles at P (postnatal day) 3 but do not start to innervate them until P7 or achieve an adult appearance until P19. The first terminals are circumferential, followed some days later by the appearance of palisade endings. The number of axons innervating a hair follicle increases steadily with age until P19 and there is no evidence of exuberant innervation of follicles during development. Hair follicle density in the rat is maintained during development due to waves of small, vellus follicle growth later in postnatal life as the skin grows. The percentage of follicles innervated however, decreases from the second postnatal week onwards presumably because late developing vellus hairs do not become innervated. Comparative analysis in human fetal abdominal skin using the same silver stain reveals a similar sequence and pattern of innervation to the rat over the period of 22 to 35 weeks EGA (estimated gestational age). Human skin does not, however, undergo the late waves of follicle growth seen in the rat. Follicular density decreases and the percentage of innervated follicles increases in the third trimester of fetal life. PMID- 1721561 TI - Effects of early postnatal receptor damage on dendritic development in gustatory recipient zones of the rostral nucleus of the solitary tract. AB - The rostral gustatory zone of the nucleus of the solitary tract (NST) exhibits extensive anatomical development during the first 3 weeks of postnatal life, and this development requires the presence of intact gustatory receptors during a critical period. We have previously shown that unilateral damage induced to fungiform papillae of the anterior tongue at postnatal day 2 (P2) alters normal migration and ramification of chorda tympani (CT) axons in the rostral NST. In addition to alterations of axonal development, P2 receptor damage decreases the intraneuronal distance between neurons that project axons to the second-order central gustatory relay, located in the caudal parabrachial nucleus (PBN). This observation suggested that P2 receptor damage may alter both axonal development and dendritic development in the rostral gustatory NST. The present study evaluated potential changes in dendritic development of PBN projection neurons following either P2 or P10 receptor damage. Morphological studies were first conducted to quantitatively define somatic characteristics of neurons that project axons to the PBN. Independent experiments used fluorescent labeling combined with subsequent Golgi-impregnation to study dendritic architecture of identified PBN projection neurons. Results confirmed that P2 receptor damage alters dendritic development of PBN projection neurons located in CT terminal fields. Anterior tongue receptor damage at P2 (1) reduces planar length of first- and second-order dendritic branches, (2) reduces the mean number of second-order branches per neuron, and (3) reduces the density of spine processes on second order dendritic branches. A critical period exists for these effects, similar to that reported for axonal development, insofar as P2 receptor damage alters dendritic development of PBN projection neurons, whereas P10 receptor damage does not. Dendrites of identified PBN projection neurons located in regions of the NST that receive primary afferent axons from the glossopharyngeal nerve are not affected by anterior tongue damage at P2. These results show that early postnatal receptor damage influences both pre- and postsynaptic development in the rostral gustatory NST. These anatomical changes are undoubtedly related to alterations in taste-guided behaviors that are observed following P2 receptor damage. PMID- 1721562 TI - Correlation of gangliotetraose gangliosides with neurite forming potential of neuroblastoma cells. AB - Gangliosides of 11 different neuroblastoma cell lines, grown to confluence, were extracted and quantified with respect to: (a) total lipid-bound sialic acid, (b) total gangliotetraose family, and (c) GM1 content. The cultured cells were induced to grow neurites in 3 ways: (a) serum reduction, (b) exogenous ganglioside, and (c) retinoic acid. Neurite outgrowth was quantified in terms of % of cells bearing neurites and average number of neurites per cell. No correlation was observed between neurite outgrowth and total ganglioside concentration, but a reasonably good correlation was observed with respect to neuritogenesis and gangliotetraose content. When exogenous ganglioside was the stimulant the best correlation was with GM1, whereas retinoic acid-stimulated outgrowth was approximately proportional to GD1a content. The 'neurite minus' N1A 103 line, which had the lowest level of GM1, GD1a, and total gangliotetraose gangliosides, showed little if any response to any of the stimuli. PMID- 1721563 TI - Molecular forms of acetylcholinesterase in cerebral cortex and dorsal thalamus of developing rats. AB - Histochemical studies show that primary sensory regions of rat cerebral cortex and dorsal thalamus display transient patterns of intense acetylcholinesterase (AChE) activity during early postnatal development. Sucrose gradient fractionation techniques were used to determine the molecular forms of AChE in developing rat brain at the time of onset (postnatal day 5), during peak expression (days 10-11), and after decline (day 18) of the transient AChE expression. Tissue from auditory and visual regions of cortex and from dorsal thalamus at each age examined contained 10S and 4S forms of AChE, similar to the pattern observed in mature brain. The 10S form was almost totally membrane bound; the 4S form was largely soluble. Hemithalamic lesions reduce both forms of AChE in cortex. These data indicate that transiently expressed AChE does not represent a unique or unusual form of the enzyme. PMID- 1721564 TI - Ontogenesis of the cerebellofugal projection in the rat. AB - Ontogenesis of the cerebellofugal projection was studied in the rat by the tract tracing method with WGA-HRP. The projection, forming a uniform front of compact fibre bundle tipped by growth cones, began entering the brainstem on embryonic day 17 (E17), grew rapidly and orderly with no random extension of fibers, and arrived at the most rostral part of the thalamus already by E18, distributing dense terminals to various brainstem and thalamic nuclei. The course and termination of this projection in prenatal animals was largely similar to normal adult projection although differences were found. Some projections increased postnatally, whereas some projections which were existent in embryos regressed with age and finally disappeared completely. The adult pattern of the projection was attained by 3 weeks of age. It is worth noting that the projections which appeared transiently are similar to those reported as aberrantly regenerated projections in kittens which are born in more mature state than rats and have no such projections at birth. PMID- 1721565 TI - Levels of GH binding activity, IGFBP-1, insulin, blood glucose and cortisol in intensive care patients. AB - OBJECTIVE: To investigate levels of serum GH binding activity, insulin-like growth factor binding protein-1 (IGFBP-1), blood glucose, serum insulin, and cortisol in patients on the Intensive Therapy Unit. DESIGN: Case-control study of severely ill patients admitted to the Intensive Therapy Unit. PATIENTS: Six critically ill patients (51-78 years) who required ventilatory and nutritional support and six healthy age, sex, height and weight matched controls. MEASUREMENTS: Patients and controls were studied for two 24-hour periods; the patients before and after commencing parenteral nutrition, the controls whilst fasted and on a second occasion when fed a diet equal in protein and calories to that of the patients' parenteral nutrition. Samples were taken hourly for measurement of IGFBP-1, blood glucose, serum insulin and cortisol. Growth hormone binding activity was measured at 0 hours. RESULTS: Blood glucose levels were higher in the patients than controls in both the fasted (mean +/- SEM 5.1 +/- 0.5 vs 3.8 +/- 0.2 mmol/l, P = 0.04) and fed states (10.1 +/- 1.6 vs 5.0 +/- 0.1 mmol/l, P = 0.02) and patients' insulin levels were also higher when fed (81.5 +/ 31.6 vs 24.2 +/- 4.8 mU/l, P = 0.046) although there were no significant differences between patients and controls when fasted. IGFBP-1 levels were inversely related to insulin levels in both the patients and controls; mean IGFBP 1 concentrations were higher in fasted patients than in controls (123 +/- 38 vs 52 +/- 9, P = 0.046) but when fed, both groups had similar mean levels. Serum GH binding activity was low in the patients and did not change with feeding. Mean 24 hour cortisol levels were higher in the patients than in controls, whether fasted or fed, and showed no nyctohemeral rhythm. CONCLUSIONS: We have previously reported that critically ill patients have low levels of IGF-I with augmented basal levels of GH. The present results demonstrate that these changes in the GH IGF-I axis are associated with insulin resistance with respect to blood glucose and high levels of IGFBP-1 when patients are fasted. However, when fed, the inverse relationship of IGFBP-1 to insulin is preserved. Patients have low levels of GH binding activity and increased mean cortisol levels. Interventional studies in this patient group with GH and IGF-I must take account of these changes in binding protein and cortisol levels. PMID- 1721566 TI - Autotomy and decreased spinal substance P following peripheral cryogenic nerve lesion. AB - Cryotherapy has been clinically applied to relieve pain by blocking peripheral nerve function. Clinically, analgesia has been successfully achieved but there is suggestion that permanent pain relief may be accompanied by extended motor and sensory deficits. This study was undertaken to determine the effect of a peripheral cryogenic nerve lesion, i.e., of the sciatic nerve, on behavioral effects and substance P content in the dorsal horn of the spinal cord. In rats, the right sciatic nerve was exposed and cryolesioned using one freeze-thaw refreeze cycle. In an alternate group, the right sciatic nerve was cut and a 3-mm region was excised. Animals were allowed to recover 7 or 21 days during which their behavior was assessed. Autotomy, an animal's tendency to attack the nerve injured affected limb, occurred in both the cryolesioned and sectioned groups. They were killed by transcardiac perfusion of fixative and segments L4-S1 were processed for immunocytochemistry. The SP-like immunoreactivity (SPLI) in the right and left dorsal horns was compared and quantitated using a microcomputer imaging device. We utilized a fully automated program to digitize and quantitate the staining of the substantia gelatinosa. There was no significant difference in SPLI in the dorsal horns of the sham-operated controls at either time period. At 7 days the sectioned group demonstrated a 40% decrease in SPLI and 76% decrease at 21 days. In the cryolesioned group, there was a 34% decrease at 7 days and by 21 days there was a 68% decrease in immunoreactivity on the operated side.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721567 TI - Shiverer jimpy double mutant mice. V. Correlation of genotype and myelin proteins. AB - We have reexamined the levels of myelin basic protein (MBP) and proteolipid protein (PLP) in the brains of mice bred to carry both the shi/shi and jp/Y hypomyelination defects. The genotype of each putative double mutant was confirmed by direct DNA analysis: shi/shi by Southern blot analysis, and jp/Y by restriction enzyme analysis of polymerase chain reaction-amplified fragments. MBP and PLP levels were assessed by immunoblotting. All putative double mutants were found to be shi/shi. However, examination of the PLP locus revealed both jp and wild-type genotypes, the latter produced by an expected crossover. Animals proven to be shi/shi*jp/Y had no detectable MBP or PLP; those proven to be shi/shi*+/Y (the crossover) had no MBP but had PLP. These results differ from an earlier report of both MBP and PLP in the brains of presumed shi*jp animals. PMID- 1721568 TI - Regulation of myelin basic protein gene transcription in normal and shiverer mutant mice. AB - We describe the measurement of myelin basic protein gene transcription rate, and of the accumulation of both mature mRNA and total transcripts from the myelin basic protein gene in brains from mice of wild-type and homozygous shiverer genotypes at several ages spanning postnatal development. In wild-type brains the accumulation of total transcripts as well as mature mRNA, and the transcription rate, all follow the same general pattern of rising sharply from a low level at birth to a peak at 20 days, and continuing at a somewhat reduced level into adulthood. Thus a major factor in the developmental regulation of myelin basic protein expression is the control of transcription rate. The shiverer mutation consists of a deletion of the 3' end of the myelin basic protein gene which completely prevents production of mature mRNA and protein, and results in severe dysmyelination and a trembling behavior. In shiverer brains, the transcription rates for the intact 5' end of the gene follow closely those seen in wild-type animals up to the age at which maximal myelination normally occurs. Total myelin basic protein transcripts follow a similar profile but at less than 5% the level seen in wild-type, and, as expected, no mature mRNA is detected. Thus the shiverer deletion does not remove information required for efficient, developmentally regulated transcription, and the low level of myelin basic protein gene transcripts in this mutant must be a result of their reduced stability. A higher than normal myelin basic protein gene transcription rate in older shiverer animals raises interesting questions regarding the regulatory mechanisms controlling myelinogenesis. PMID- 1721569 TI - Alpha-fetoprotein uptake by differentiating neuroretinal structures of the chick embryo. AB - Internalization of exogenous fluoresceinated alpha-fetoprotein (FITC-AFP) was studied at different stages of development on embryonic chick neural retinas maintained, for short periods, in organ cultures. Cellular localization of endogenous, native AFP was carried out by immunohistoperoxidase methods. Cells which specifically internalized exogenous FITC-AFP (neurons and their processes) were precisely those showing positive immunolabeling for the endogenous, native protein. Such a result supports the hypothesis of a predominantly exogenous origin of intracellular neuroretinal AFP. A precise topography and temporal sequence of AFP labeling after internalization in retinal structures is given. AFP uptake was not displayed by undifferentiated cell precursors or germinal cell layers but was apparent in cells with phenotypic characteristics of maturing neurons. Nerve fibers and synaptic layers actively internalized FITC-AFP at specific stages of development. Fully differentiated neurons and processes did not internalize AFP. The possible role of AFP, a carrier of biologically active substances such as fatty acids, in neural retina differentiation is discussed. PMID- 1721570 TI - Variance of interburst intervals in burst suppression. AB - Each EEG performed over a 3 year period at the University of Michigan with a diagnosis of generalized burst-suppression (BS) was reviewed. Ten EEGs from 10 patients with hypoxic-ischemic encephalopathy (HIE-BS) and 21 records from 8 patients with pentobarbital induced burst-suppression for treatment of status epilepticus (SE-BS) were reviewed. For each EEG, the mean duration of 40 interburst intervals (IBIs) as well as their coefficient of variability were calculated. We found that in the SE-BS group the coefficient of variability of IBI duration was highly correlated with the logarithm of mean IBI duration while in the HIE-BS group, there was no significant correlation between these 2 variables. This suggests that the underlying mechanism causing BS is different in the 2 groups and might be related to a uniform and progressive affection of similar brain structures in the SE-BS group and a more patchy and variable pathology in the HIE-BS group. PMID- 1721571 TI - The quantitative extraction and topographic mapping of the abnormal components in the clinical EEG. AB - A method is described which seems to be effective for extracting the abnormal components from the clinical EEG. The approach involves the use of a set a spatial patterns which are common to recorded and 'normal' EEGs and which can account for maximally different proportions of the combined variances in both EEGs. These spatial factors are used to decompose the EEG into orthogonal temporal wave forms which can be judged by the expert electroencephalographer to be abnormal, normal or of artifactual origin. The original EEG is then reconstructed using only the abnormal components and principal component analysis is used to present the spatial topography of the abnormal components. The effectiveness of the method is discussed along with its value for localization of abnormal sources. It is suggested, in conclusion, that the approach described may be optimal for interpretation of the clinical EEG since it allows what is best in terms of quantitative analysis of the EEG to be combined with the best that is available in terms of expert qualitative analysis. PMID- 1721572 TI - Inadequacy of the forehead reference montage for detecting abnormalities of the spinal N13 SEP in cervical cord lesions. AB - Cervical somatosensory evoked potentials (SEPs) recorded using forehead and anterior cervical reference montages were compared in 6 patients whose MRI showed a cervical syrinx. All patients presented with a segmental loss of pain and temperature sensation in upper limbs, but no clinical evidence of dorsal column system dysfunction. Cervical SEPs recorded using the forehead reference montage were normal in all cases, while the N13 potential recorded using an anterior cervical reference was reduced, or absent, in 11 median nerve SEPs out of 12. This discrepancy results from persisting scalp P13-P14 far-field potentials, which were picked up by the forehead, but not by the anterior cervical, reference. It is concluded that the forehead reference montage is inadequate for assessing selectively the spinal N13 potential and should be abandoned for cervical SEP recording. PMID- 1721573 TI - Human brain potential correlates of face encoding into memory. AB - ERPs elicited by photographs of unfamiliar faces were shown to be predictive for their later recognition. We established that these ERP differences were unrelated to fluctuations of attention or other non-specific factors during perceptual processing. Therefore they may be interpreted as manifestations of brain processes that correlate with memory encoding. The scalp topography of this ERP difference was bipolar with greater electrical positivity at frontal and greater negativity at parieto-temporal scalp sites. This topography appears to contrast with the more uniformly positive differences reported for verbal stimuli but is in accord with what might be expected for faces and complex visual stimuli. PMID- 1721574 TI - Auditory attention affects two different areas in the human supratemporal cortex. AB - The effect of selective attention on activity of the right human auditory cortex was studied with a 24-channel planar SQUID-gradiometer. Two conditions were used, favoring either a late attention effect following N100m, or an early effect, overlapping with N100m. In experiment 1 (15 subjects), a randomized tone sequence of 1 and 3 kHz tones was delivered to the left ear with a constant interstimulus interval (ISI) of 405 msec. The subjects' task was to count infrequent longer tones of one of these pitches among shorter standards. An attention effect, called magnetic difference (Md), was found when the responses to the irrelevant standards were subtracted from those to the relevant standards. Md peaked at about 220 msec for the 1 kHz tones and at 195 msec for the 3 kHz tones. The equivalent source of Md was in the supratemporal auditory cortex, about 1 cm anterior to the source of N100m, and in the same location as the source of P200m. In experiment 2 (8 subjects) the paradigm was similar, except that the 1 kHz and 3 kHz tones were led to different ears with a random ISI of 240-300 msec. In this case Md started already at 30-40 msec, adding to the N100m deflection, and the sources of N100m and Md overlapped. Present results show that attention can modify the activity of two different areas in the supratemporal auditory cortex. We interpret both attention effects as alterations of the exogenous evoked response components: the earlier effect as changed activity in neurons underlying N100m to relevant tones and the later effect as a modification of P200m to irrelevant tones. PMID- 1721575 TI - Event-related potentials during arithmetic and mental rotation. AB - In separate studies of arithmetic and mental rotation, similar posterior negative slow waves have been found. This similarity was surprising given the difference in cognitive processing required by these tasks. Furthermore, delayed responses were employed in these studies, so that it was not possible to determine the extent to which the slow wave activity was too late to be associated with processing that was specific to performance of the tasks. This experiment was intended to clarify the task-specific and non-specific nature of the slow wave activity. Subjects performed either an arithmetic or mental rotation task at two levels of difficulty on a random, trial-to-trial basis and gave an immediate response. There were a number of late posterior negativities, each with a different timing and topography, which were sensitive to type of task and/or task difficulty. Some components were associated with early task processing that was synchronized to the stimulus while others, revealed by response-synchronized averaging, were associated with later stages of task processing. There also was post-task activity that was sensitive to the difficulty level of the prior operations. In both tasks, there was a pre-frontal positive wave that persisted over most of the pre-response epoch, evidently related to a process that was active throughout the task. There also was centro-frontal phasic negativity, with a large peak at 380 msec in mental rotation, and a smaller, longer latency peak in arithmetic, apparently related to the complexity of the stimulus. Thus we conclude that arithmetic and mental rotation each elicit task-specific slow wave activity. PMID- 1721576 TI - On the reasons for the delay of P3 latency in healthy elderly subjects. AB - The P3 component reaches its peak later in elderly subjects than in young subjects. The aim of this study was to repeat this finding in the usual auditory oddball task and to look for reasons for the delay by analysing other components and by applying another task, the visual Push/Wait task. It was found in the oddball that the age delay was present for mismatch negativity already but further increased until P3's peak. In the Push/Wait task, the size of the age delay was independent of another delay caused by reduced visual intensity of the stimuli. Further, the age delay had its onset after the occipital P140 component whereas the intensity delay was present before this component. Within the elderly, P3 latencies correlated between the auditory and the visual tasks, and the common factor extracted from both latencies correlated with a test of short term memory span. It is concluded that the age delay of P3 is not due to a delay of perceptual encoding but perhaps due to delayed memory processes in the elderly. As usual, P3 amplitudes were larger and more parietally focussed in the young than in the elderly. Possible differences in motivation might account for this finding. PMID- 1721577 TI - Cortical potentials preceding centrifugal and centripetal self-paced horizontal saccades. AB - Cortical potentials preceding self-paced centrifugal and centripetal saccades were recorded in 15 subjects from F3, Fz, F4, C3, Cz, C4, P3, Pz and P4 versus linked mastoid electrodes. A negative potential starting about 1.0 sec prior to saccade onset, reaching a peak amplitude of 6.8 microV on average, preceded centrifugal saccades. In contrast the negativity preceded centripetal saccades by only 500 msec, and its peak amplitude was smaller (4.6 microV). We conclude that these differences reflect the fact that less 'effort' is needed with centripetal as compared to centrifugal saccades. PMID- 1721578 TI - Two inexpensive electronic circuits for analog display of multichannel EEG signals. AB - Two electronic circuits useful for high-resolution EEG recording systems are described. The first is an analog display driver that formats large numbers of EEG signals for display in a variety of modes on a standard CRT display. The second is an analog demultiplexer circuit that reconstitutes large numbers of analog EEG signals from multiplexed D/A outputs. Each provides a readily expandable, high-resolution EEG display that can easily provide real-time or faster performance and that is much less expensive than computer-based digital display systems with comparable capabilities. PMID- 1721579 TI - Ambulatory EEG cassette recorders for prolonged electroencephalographic monitoring in animals. AB - We describe the use of ambulatory cassette EEG recorders for monitoring in vivo neurophysiologic signals from multiple animals over prolonged periods of time. This technique centers around a simple interface device that attenuates the intracerebral signals to the input range of the recording device and around a common indifferent input for all animals. The resulting analog recordings have the advantage of good signal resolution and rapid review of 24 h of recorded data. PMID- 1721580 TI - Scanning EMG in normal muscle and in neuromuscular disorders. AB - The spatial distribution of motor units in normal subjects and in patients with neurogenic and myogenic conditions was studied. The possibilities and the difficulties in quantifying the records are discussed. Normal values are presented for the brachial biceps and anterior tibial muscles. The results are compared to the previous multielectrode studies. The findings in pathological conditions compared to normals are evaluated. The scanning EMG verified the rearrangement of muscle fibres in abnormal muscles. The most striking finding was the presence of long polyphasic sections in abnormal muscles. However, this parameter did not differentiate neurogenic from myogenic cases. The length of the motor unit cross-section did not differ significantly in the abnormal muscles compared to normal. Thus, the size of motor unit territory does not seem to be a useful parameter to detect pathology. Scanning EMG gives a new dimension to exploring the motor unit characteristics not attainable by conventional methods and provides important information towards a better understanding of concentric needle EMG. Examples are shown from healthy subjects and from patients with neuromuscular diseases, both for the different parameters and special phenomena. PMID- 1721581 TI - Variability of repeated nerve conduction studies. AB - We have determined the variability of repeated measurements of sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) amplitude and motor and sensory conduction velocity (MCV and SCV) and examined the extent to which limb temperature is responsible for the variability. We made 10 serial measurements of SNAP, CMAP, MCV and SCV in each of 3 nerves in a single normal subject. The coefficients of variation for MCV and SCV ranged from 2.0% to 6.7% and the proportion of the variance due to temperature was 0.3-56%. The coefficients of variation were much greater for serial measurements of compound action potential amplitude. We used the standard deviations for serial measurements in each nerve to calculate the number of subjects required to detect a difference of 1/msec between the means of two sets of measurements with a power of 90%. PMID- 1721582 TI - Disturbed modulation of the stretch reflex gain during standing in cerebellar ataxia. AB - The postural control disturbance in patients with spino-cerebellar degeneration (SCD) was evaluated by the displacement of the center of foot pressure (CFP) and the amplitude of the triceps surae muscle H reflex in various standing postures. Twelve patients and 8 age-matched normal subjects (NL) were studied. The CFP, surface electromyograms (EMG) in the lower leg and the soleus H reflex were recorded in all subjects. The CFP displacement area was recorded in 10 patients. The following results were obtained: (1) the range of displacement of CFP from forward to backward leaning as a fraction of foot length was significantly smaller in the patients than in the controls (mean +/- S.D.; SCD = 52.8 +/- 12.7%; NL = 62.3 +/- 9.9%; P less than 0.05%, Student's t test); (2) the ratio of the increase in amplitude of the H reflex to soleus muscle EMG activity when the subjects leaned forward from an upright posture was significantly higher in the patients than in the controls (SCD = 0.41 +/- 0.40; NL = 0.17 +/- 0.15, P less than 0.05); (3) this ratio correlated with the area of CFP displacement during upright standing in the patients (r = 0.75, n = 10, P less than 0.05). These results indicate that the suppression of the stretch reflex observed in normal controls during standing is impaired in patients with SCD. We conclude that a decreased suppression of the stretch reflex is one of the mechanisms responsible for the instability on standing in patients with SCD. PMID- 1721583 TI - Antagonist inhibition during rest and precontraction. AB - The excitability of antagonist soleus motoneurons was tested during fast voluntary contractions of tibialis anterior (TA). Contractions of TA started either from zero or higher levels of tonic contraction of ankle extensors. Results showed that H reflex depression under 3 different conditions: 25% (R25) or 50% (R50) of the maximal isometric dorsiflexion from the resting state and the sequential isometric response (SW) appeared about 20-40 msec prior to the EMG onset of TA. In particular, H reflex depression was clear in the SW response, and the stronger the prior contraction of extensors the greater the H reflex depression. There was a significant difference in the integrated EMG between R25 and SW but none between R50 and SW. The different amounts of inhibition found for dorsiflexion from the resting state and sequential movement is most probably explained by presynaptic inhibition. If presynaptic inhibition were increased throughout the sequential movement, in comparison with the resting state, change in the H reflex gain would occur. PMID- 1721584 TI - The ubiquity of contraction enhanced H reflexes: normative data and use in the diagnosis of radiculopathies. AB - Surface recorded contraction enhanced H reflexes to single stimuli were obtained from muscles supplied by the C5, 6, 7, 8, L3, 4, 5, S1 and 2 nerve roots. For each of 12 such muscles, these reflexes were recorded from 25 control subjects as well as standard unenhanced H reflexes from the gastrocnemius muscles. Amplitude and latency criteria for normality were determined. Thirty-two patients with typical histories and physical findings of radiculopathy secondary to degenerative disease of the spine (C6 nerve root - 9 patients, C7 - 10, C8 - 4, L4 - 4, L5 - 4, S1 - 1) were studied with these H reflexes and the needle electrode. Thirty patients had H reflex abnormalities appropriate to the signs and symptoms. Eighteen had absent H reflexes and 16 had abnormalities to amplitude criteria. None were abnormal by latency criteria. Twelve patients had abnormal needle examinations and all also had abnormal H reflexes. These results indicate that contraction enhanced H reflexes are ubiquitous, readily obtainable and sensitive to the presence of clinically clear-cut radiculopathies. PMID- 1721585 TI - Optimal transcranial magnetic stimulation sites for the assessment of motor function. AB - The optimal placement sites for eliciting motor evoked potentials from the abductor digiti minimi and abductor hallucis muscles by means of transcranial magnetic stimulation were determined using a commercially available circular coli. Fifty volunteers were used for the study. The ability to elicit responses was found to be strongly dependent upon the scalp placement of the stimulator coil. The effects of altering the direction of current flow in the coil were tested on two different sets of volunteers: clockwise in one and counterclockwise in the other. It influenced only the locations of the sites which were optimal for eliciting responses from the abductor hallucis muscles and not those which were optimal for eliciting responses from the abductor digiti minimi muscles. Response latencies were found to be significantly dependent only upon volunteers' heights and not on their sex, age, or weight or the stimulus intensities used to elicit responses. No previous studies have defined the optimal scalp placements for eliciting responses from the lower extremities. This information may have clinical importance for making reliable assessments of patients with significantly impaired motor function. PMID- 1721586 TI - Exploratory mapping of evoked neuromagnetic activity from human peripheral nerve, brachial plexus and spinal cord. AB - Upon conventional median nerve stimulation at the wrist early magnetic fields were recorded using a SQUID magnetometer. At the upper arm, mono- and biphasic compound nerve action fields were detected, depending on the subject's distribution of single fiber conduction velocities. At the upper thorax, brachial plexus fields reversed polarity at the level of Erb's point; their distribution was asymmetric, probably due to volume currents. At the upper lateral neck, fields from proximal plexus, spinal cord (P13m) and the primary somatosensory cortex contralateral to the sensor position were detected. The observed P13m field distribution agrees with the electrophysiological concept of a sagittal segmental dorsal horn generator. PMID- 1721587 TI - Brain excitability and long latency muscular arm responses: non-invasive evaluation in healthy and parkinsonian subjects. AB - Thirty healthy and 35 volunteers affected by Parkinson's disease (PD) were examined. Long latency responses (LLRs) and short latency somatosensory evoked potentials (SEPs) after median nerve stimulation were respectively recorded from forearm flexor muscles, and from 19 scalp electrodes, during relaxation (condition 1), light and maximal muscle contraction (conditions 2 and 3). Linear interpolation of SEPs was performed to produce isopotential colour maps. Latencies and amplitudes of the V1-V2 component in LLR, as well as of parietal, central and frontal scalp SEPs were analysed in the 3 experimental conditions. Highly significant inverse correlation matched the frontal SEP to the LLR V2 component amplitudes, both in healthy and in PD subjects. However, the V2 component--which in the former group was reliably identifiable only in condition 3--was presented in conditions 1 and 2 in a high percentage of PD subjects who also showed an abnormally reduced frontal SEP during complete relaxation. Excitability changes of brain motor areas induced by a sensory input were tested as follows: the motor cortex was transcranially stimulated (TCS) by magnetic pulses with an intensity 10% below (A) or above (B) the threshold for twitch elicitation during complete relaxation of forearm muscles; TCS was randomly preceded (range 14-32 msec) by a shock to the median or ulnar nerve at the elbow with identical characteristics as for LLR elicitation. An initial epoch of 'inhibition' followed by a peak of 'facilitation' of the amplitude of motor responses to TCS was observed when conditioning stimuli to the median nerve preceded TCS by 14-20 and by 24-32 msec, respectively. Contrary to normals, conditioning stimulation of the median nerve did not significantly influence the excitability threshold to TCS in those parkinsonians with depressed frontal N30. PMID- 1721588 TI - Multi-unit activity in sensory fibers is related to intensity of sensation evoked by air-puff stimulation of the glabrous hand in man. AB - An analysis of a multi-unit activity (MUA) recorded from the median nerve at the wrist was undertaken to study intensity coding in the peripheral somatosensory system. Brief air-puff stimuli were applied over the glabrous hand to obtain neural and psychophysical responses. The detection threshold (So) was determined and 6 above-threshold stimulus intensities (So + 0.25 kg/cm2, So + 1.25 kg/cm2, So + 2.50 kg/cm2, So + 3.75 kg/cm2, So + 5.00 kg/cm2, and So + 6.25 kg/cm2) were adopted for magnitude estimation. MUA was recorded for the 6 stimulus intensities and for the threshold (So) level. Response duration, number and frequency of the MUA were evaluated as candidate coding parameters for stimulus intensity. A simple power function with an exponent of 1.03 provided an adequate description of the magnitude estimation function. Similarly, stimulus-response functions of the 3 parameters defining the MUA were well represented by straight lines in double logarithmic plots, with the function of the MUA number having the highest power exponent (0.98) and also the highest correlation coefficient. The results suggest that the total number of MUA, representing spatial recruitment, is the most relevant coding parameter for the subjective magnitude estimation of the stimulus intensity. PMID- 1721589 TI - Sudden unexplained death syndrome--a new manifestation in melioidosis? AB - The indirect haemagglutination (IHA) test using sensitized turkey erythrocytes and the indirect immunofluorescence assay (IgM-IFA) was confirmed to be sensitive in the detection of a recent or current Pseudomonas pseudomallei infection in 19 culture-confirmed Singapore melioidosis patients. All were found to have antibody titres from 4 to 32768 in the IHA test and 10 to 320 in the IgM-IFA test. When these tests were employed on sera from 16 immigrant Thai construction workers who died of sudden unexplained death syndrome (SUDS) and 73 healthy Thai fellow workers, 93.8% and 68.8% of SUDS cases had IHA titre of greater than or equal to 4 and IgM-IFA titre of greater than or equal to 10 respectively, in contrast to 39.7% and 12.3% found among healthy Thai workers. These data indicate that at the time of death, most of the SUDS patients had an active infection with P. pseudomallei, possibly resulting from reactivation of a latent infection. The aetiological role of P. pseudomallei as the major cause of SUDS is discussed. PMID- 1721590 TI - CD34-positive cell proportions in peripheral blood correlate with colony-forming capacity. AB - Blood samples were examined from 25 children with malignancies during hematopoietic recovery following chemotherapy-induced aplasia and from 9 children undergoing tonsillectomy. The proportion of CD34-positive peripheral blood mononuclear cells (PBMNC) evaluated by flow cytometry was compared with the number of colonies (granulocyte-macrophage colony-forming units, CFU-GM; mixed lineage colony-forming units, CFU-GEMM; and erythroid burst-forming units, BFU-E) grown in methylcellulose medium within 2 weeks. A mean of 1387 myeloid colonies (495-4480) per 10(5) PBMNC seeded developed from 13 samples with detectable CD34 populations (between 0.9% and 5.6%), whereas only 152 (9-386, p = 0.002) and 65 (12-137, p = 0.005) colonies were formed from 12 patient and from 9 control samples in which the percentage of CD34-positive cells was too low for analysis. Linear regression analysis revealed that CD34 positivity correlates with colony forming capacity (p = 0.0008, r = 0.782). Flow cytometric evaluation of the CD34 proportions can thus predict the in vitro colony-forming capacity of peripheral blood prior to leukapheresis. PMID- 1721592 TI - [The effects of anti-androgen TZP4238 (17 alpha-acetoxy-6-chloro-2-oxapregna-4,6 diene-3,20-dione) and its related compounds on the in vitro formation of androgen receptor complex]. AB - It was reported that a new steroidal anti-androgen, TZP4238 (17 alpha-acetoxy-6 chloro-2-oxapregna-4,6-diene-3,20-dione), was 10 times stronger than chlormadinone acetate (CMA) as the in vivo anti-androgenic potential. To confirm this result, the effect of TZP4238 and CMA on ventral prostates of intact rats, or castrated ones treated by testosterone, was investigated. Our experiments demonstrated that TZP4238 was 3-5 times stronger than CMA. The effects of this compound on the androgen-receptor complex formation in the human and rat prostates were investigated and compared to the other anti-androgen and related compounds. An aliquot of cytosol or KCl extract from rat or human prostate was incubated with [3H]R1881 in the presence of various tested compounds. By means of dextran coated charcoal assay and sucrose density gradient centrifugation analysis, TZP4238 and TZP4239 showed a stronger inhibitory effect than other compounds except dihydrotestosterone. Judging from all these results, it was estimated that TZP4238 was 2-3 times stronger than CMA. The Scatchard plot analysis revealed that the inhibitory effect of TZP4238 was a competitive type. Between CMA and TZP4238, the difference of in vivo anti-androgenic potential was not identical with that of the inhibitory effect on the androgen-receptor complex formation. PMID- 1721591 TI - Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase. AB - Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands. Major bands had molecular weights of 145, 120, 110, 98, 62, 55 and 42 kD. P145, the most prominent phosphorylated protein, was identified as c-kit product using anti-c-kit-mAb (YB5.B8), suggesting ligand dependent receptor autophosphorylation. Five of six tyrosine phosphorylated bands induced or enhanced by GM-CSF stimulation comigrated with those tyrosine phosphorylated by MGF (138, 120, 76, 55 and 42 kD). P42 was identified, at least in part, as mitogen-activated protein (MAP) kinase. MGF induced tyrosine phosphorylation of a complex of GTPase-activating protein (GAP, 120 kD) and GAP associated proteins (p62/p190) as detected by anti-GAP Ab immunoprecipitation followed by immunoblotting with anti-phosphotyrosine mAb. GM-CSF also stimulated slightly but consistently tyrosine phosphorylation of GAP and p190 but not p62. Both MGF and GM-CSF enhanced Raf-1 phosphorylation and increased Raf-1 associated kinase activity in vitro. Phosphoamino acid analysis revealed Raf-1 phosphorylation by these two growth factors occurred almost exclusively on serine residues. No tyrosine phosphorylation of Raf-1 protein was detected. These data suggest shared and unshared components of signaling pathways of both factors, which may be involved in cell proliferation. PMID- 1721593 TI - Prostaglandin E1 activates a chloride current in Jurkat T lymphocytes via cAMP dependent protein kinase. AB - Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3 phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells. PMID- 1721594 TI - Multifunctional role for fetuin (fetal protein) in lipid transport. AB - Recent studies from this laboratory have shown that fetuin 1) is nearly 50-fold more efficient than albumin in incorporating exogenous fatty acids into cultured cells, (JBC, 265: 5883, 1990), and 2) is associated with a lipoprotein-like particle (FASEB J. 3: 2075-2080, 1989). In the present study, this lipid containing fraction (FLP) was isolated by ultracentrifugation, and its effect on cholesterol efflux from cultured human skin fibroblasts and Hep-G2 cells prelabeled with [14C]cholesterol was investigated. FLP fraction caused a significant efflux of [14C]cholesterol from cells, the same in magnitude as HDL. This effect of fetuin supranatant fraction increased proportionately with concentration and time. Similar results were observed with Hep-G2 cells. This ability to induce efflux of cholesterol was confirmed by a decrease in cholesterol mass of cells after 24 h incubation with FLP. The ultracentrifugal bottom (infranatant) fraction of fetuin (FI) was ineffective in this regard. However, FI was more effective in the incorporation of exogenous fatty acids into cellular triglycerides. These studies suggest that the fetuin molecule is a multifunctional protein (delivery of fatty acids to cells and cholesterol efflux from cells) which may play a role in lipid transport during fetal life. PMID- 1721595 TI - [Presence of anti-alphafetoprotein immunoglobulin G in serum of a patient with hepatocellular carcinoma]. PMID- 1721596 TI - Intrafamilial transmission of hepatitis C virus in Japan. AB - To study the intrafamilial transmission of hepatitis C virus (HCV), 36 family members of 16 patients with anti-HCV (anti-C100-3)-positive chronic liver disease were screened for anti-HCV by an enzyme-linked immunosorbent assay (ELISA). Clusters of anti-HCV-positive individuals were observed in 2 of 16 families (12.5%). Four of 35 family members (11.4%) with no history of blood transfusion were positive for anti-HCV. Two of 17 offspring (11.8%) of anti-HCV-positive females were positive for anti-HCV, while 1 of 5 spouses (20.0%) was positive for anti-HCV. These data suggest that intrafamilial transmission is one of the possible routes of infection for HCV. PMID- 1721597 TI - [Use of the stimulants of nonspecific resistance in the complex treatment of infectious complications in patients with chronic myeloid leukemia]. AB - Effectiveness of three methods of infectious complication therapy in chronic myeloleukemia (CML) patients was studied. Group I patients (n = 20) received antibiotics, group II patients (n = 9) were given antibiotics with gamma globulin, in group III antibiotics combined with individually selected immunomodulators were used. The highest effect was recorded in CML patients who received antibiotics combined with the immunomodulators that has led to normalization of cellular and humoral factors of nonspecific body resistance. PMID- 1721598 TI - Vitamin A deficiency and small intestinal secretory function in the rat. AB - The influence of vitamin A on the functions of the small intestine was examined in rats made vitamin A deficient for 40 days by feeding a special diet after weaning and in pair fed vitamin A deficient rats that were given supplementary vitamin A (240 IU/day) in their drinking water. The basal and stimulated electrogenic secretory and absorptive functions of the jejunum and proximal and distal ileum removed from these rats were examined in vitro using the short circuit current as the index of transport activity. The basal short circuit current in the jejunum and proximal ileum was not significantly different but that of the distal ileum was lower. Electrogenic glucose transfer was not significantly affected by the vitamin deficiency. Cholinergic stimulation using the M1/M2 agonist bethanechol showed a greatly enhanced electrogenic secretion in the jejunum of the deficient rats while secretion stimulated by dibutyryl cyclic adenosine monophosphate was significantly greater in their distal ilea compared with the supplemented group. The vitamin deficiency also disrupted the normal higher/lower hierarchical pattern of transport activity between the proximal and distal ileum. The enhanced secretory activity of the vitamin A deficient small intestine offers a putative explanation for the well known relation between vitamin A deficiency and diarrhoea found in humans. PMID- 1721599 TI - In vivo neural isolation of the canine jejunoileum: temporal adaptation of enteric neuropeptides. AB - This study was designed to assess temporal changes in concentrations of neuromodulatory peptides in plasma and gastrointestinal tissues after in vivo neural isolation of the entire canine jejunoileum. Fasting plasma and transmural biopsy specimens of stomach, duodenum, jejunum, ileum, and colon were obtained from the same dogs before and two, six, and 12 weeks after in situ neural isolation of the entire jejunoileum. Concentrations of vasoactive intestinal peptide, substance P, and neuropeptide Y were determined by quantitative radioimmunoassay. Tissue concentrations of vasoactive intestinal peptide and substance P in the neurally isolated regions increased progressively with time (198% and 217% average maximal increases, respectively), while fasting plasma concentrations changed little. Neuropeptide Y concentrations in plasma and in the jejunoileum were decreased (by 30% to 70%) at two weeks and remained decreased thereafter. Temporal changes in tissue neuropeptide concentrations occur in the neurally isolated jejunum and ileum. These adaptive changes in the neuropeptidergic innervation of the gut may play a role in the alterations in enteric function that occur after extrinsic denervation and after intestinal transplantation. PMID- 1721600 TI - Mucinous neoplasm in the cervix associated with a mucinous neoplasm in the ovary and concurrent bilateral sex cord tumors with annular tubules: immunohistochemical study. AB - The patient described synchronous mucinous tumors of the cervix and ovary and concurrent annular tubules, but without the classical stigmata of Peutz-Jeghers syndrome. The cervical tumor was an invasive mucinous adenocarcinoma with mixed components of minimal deviation and less-well-differentiated endometrioid morphology. The ovarian tumor had the benign appearance of a mucinous adenoma but histologically revealed areas of invasive carcinoma. Immunohistochemical studies of the mucinous neoplasms of the cervix and ovary are discussed. Neither the staining properties of mucin, the pattern of immunostaining for carcinoembryonic antigen, nor any other common markers were helpful in distinguishing the mucinous neoplasms. Positive immunostaining for low-molecular-weight cytokeratin in the filament profile of sex cord tumors with annular tubules was of particular interest since it has not to our knowledge been previously described. PMID- 1721601 TI - [Mechanisms of resistance to methotrexate]. PMID- 1721602 TI - [Membrane defects in paroxysmal nocturnal hemoglobinuria]. PMID- 1721603 TI - [Cellular and molecular mechanisms conditioning the accumulation of resting neoplastic cells in B-lymphocyte chronic lymphatic leukemia]. PMID- 1721604 TI - Changing patterns of male suicide in Scotland. AB - Mortality statistics published annually by the Registrar General Scotland for 1970-1989 are analysed. There has been a recent increase in the suicide rate amongst younger males in Scotland which cannot be explained by changes in the misattribution between suicides (ICD E950-E959) and undetermined deaths (ICD E980 E989). The increase is almost entirely attributable to hanging and the use of motor vehicle exhaust fumes. Analysis of the sex/age/method-specific suicide rates demonstrates that age-specific increases in the male suicide rate are linked to age-specific increases in the use of these two methods. The increased suicide rate involving motor vehicle exhaust fumes can be explained by changes in method availability and acceptability. The increased suicide rate involving hanging may be explained by increased acceptability, possibly flowing from the abolition of judicial hanging in 1965. The increased suicide rate in younger males may reflect a change in the proportion of suicidal attempts resulting in a completed suicide consequent on an age-specific shift to the use of more lethal methods, namely hanging and motor vehicle exhaust fumes. This possibility needs to be evaluated before assessing the influence of other social factors on the suicide rate. PMID- 1721605 TI - Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin. AB - A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active. PMID- 1721606 TI - Prevalence of O and H agglutinins for S. typhi in healthy school children 5-11 years: a cross sectional survey. PMID- 1721607 TI - Role of intravenous immunoglobulin in prevention and treatment of neonatal infection. PMID- 1721608 TI - IgM in the airways of asthma patients. AB - In order to obtain information on the role of IgM in the pathogenesis of asthma, we assayed IgM in the broncho-alveolar lavage fluid (BALF) from 14 non-smoking patients with asthma and 9 non-smoking healthy persons, using an ELISA. The concentrations of IgM in the epithelial lining fluid (ELF) were calculated on the basis of urea in serum and BALF. The concentrations of IgM in ELF after correction for serum IgM, cQIgM, from the patients as a group were higher than those from controls (Mann-Whitney U test, p less than 0.05). The IgM in BALF and serum, analysed by sucrose density gradient centrifugation was pentameric. The transudation of IgM from blood into ELF was compared with that of alpha 2 macroglobulin (A2M), a protein with a similar molecular mass. In 6 patients the cQIgM/cQA2M value was above the upper value of the range for controls suggesting abnormal local production of IgM. The presence of the secretory component (SC) linked to IgM was determined by ELISA. The percentage of SC-IgM in BALF was increased in 5 out of 7 patients tested. The increased local IgM concentrations and its increased local production may suggest a role for local IgM-mediated reactions in the inflammatory events associated with asthma. PMID- 1721609 TI - Allergenic and antigenic cross-reactivities of group IX grass pollen allergens. AB - The allergenic and antigenic cross-reactivities between a major recombinant Poa pratensis (Poa p) IX allergen, rKBG8.3, and its corresponding proteins of different grass pollens were examined. Immunoblotting of the proteins of thirteen different grass pollens using anti-rKBG8.3 antibodies indicated that Poa p IX like proteins are present in ten other grass pollens, albeit in variable amounts and polymorphic forms. These proteins ranged in size from 20 to 88 kDa in different grass pollens. The percent relative binding determined for each grass pollen extract using allergic human sera showed a significant correlation (r = 0.891) with that of anti-rKBG8.3 antiserum. Moreover, there was a strong association (r = 0.901) between the Kentucky bluegrass extract and rKBG8.3 with respect to their inhibition of the binding of human IgE antibodies to allergens in grass pollen extracts. Taken together, these results suggest that the allergenic and antigenic epitopes of the Poa p IX-related proteins in some but not all grass pollens are similar in structure and specificities. It is concluded that the group IX allergens constitute a major family of homologous proteins in several grass pollens. PMID- 1721610 TI - IgG isotype profiles induced in mice by two Trypanosoma cruzi electronegative antigens. AB - In this work we studied the IgG isotypes induced in mice immunized with two Trypanosoma cruzi acidic antigenic fractions (F IV and Eas 4.5) and the level of protection to a later infection with parasites. F IV is a cytosolic antigen from epimastigotes, and Eas 4.5 is an exoantigen released by trypomastigotes. The most relevant epitopes of Eas 4.5 are carbohydrates. A high prevalence of IgG1, low levels of IgG3 and no IgG2 antibodies against F IV and Eas 4.5 were found in sera obtained 2 weeks after the last antigen dose from animals immunized with F IV (group I) or Eas 4.5 (group II). Immunized mice from both groups were infected with trypomastigotes, and the parasitemias detected later on were significantly lower than in control groups (p less than 0.01, group I; p less than 0.001, group II). The amount of IgG2-specific antibodies, which was only detected using epimastigotes as antigen in ELISA, was significantly increased after the infection, but no major changes were seen in the profiles of other isotypes. PMID- 1721611 TI - Effect of a novel antiallergic drug, pemirolast, on activation of rat peritoneal mast cells: inhibition of exocytotic response and membrane phospholipid turnover. AB - The effects of a potent antiallergic drug, pemirolast (TBX), on activation of signal-transducing phospholipase C and A2, were examined in rat peritoneal mast cells. TBX at concentrations between 10(-7) and 10(-5) g/ml effectively inhibited degranulation in response to both antigen and compound 48/80. At the same concentrations the drug markedly suppressed the formation of 1,2-diacylglycerol and phosphatidic acid, and the decrease in phosphatidylinositol, suggesting the prevention of phospholipase C activation. The blockade of phospholipase A2 was suggested by the decrease in agonists-induced arachidonic acid liberation. These results indicate that TBX may exert its inhibitory effects on mast cells, at least in part, by preventing the activation of signal-transducing phospholipases. PMID- 1721612 TI - Effects of PCB (Aroclor 1254) on non-specific immune parameters in rhesus (Macaca mulatta) monkeys. AB - The effects of low level, chronic polychlorinated biphenyl--Aroclor 1254--(PCB) exposure were investigated on non-specific immune parameters in female rhesus (Macaca mulatta) monkeys. Five groups of monkeys were orally administered with PCB at concentrations of 0, 5, 20, 40 or 80 micrograms/kg bw/day. Immunotoxicity testing was initiated after 55 months of exposure. The serum hemolytic complement activity in all PCB treated groups was significantly higher (P less than 0.05) than that in the control group. A statistically significant dose-related increase in natural killer cell activity was evident at the 75:1 effector to target cell ratio. Similarly, a statistically significant dose-related increase was noted for thymosin alpha-1 levels but not for thymosin beta-4 levels. Statistically significant increased interferon levels were noted in the 20 and 80 micrograms/kg groups compared with the control group while the levels in the 40 micrograms/kg group were decreased significantly compared with the control group. The production of tumor necrosis factor by monocytes in the PCB treated groups was not different to that in the control group. The results indicated that long term exposure to PCB modulate several non-specific immune parameters. PMID- 1721613 TI - Differential effects of the immunosuppressive macrolides FK-506 and rapamycin on activation-induced T-cell apoptosis. AB - Activation of certain T-cell lines induces, besides lymphokine production, a suicide process (apoptosis) mediated by fragmentation of the cell's genome. This also occurs intrathymically during negative selection of the T-cell receptor (TcR) repertoire. Cyclosporin A (CsA) has been shown to block activation-driven T cell apoptosis, an effect which may account for the perturbations of TcR repertoire selection caused by this agent in vivo. Recently, the macrolide FK-506 was demonstrated to suppress T-cell activation by inhibiting lymphokine production in a manner apparently similar to CsA. Thus, it seemed important to determine whether FK-506 would also prevent T-cell apoptosis. For the purpose of comparison, we also investigated rapamycin (RAP), a macrolide structurally related to FK-506, but that does not block lymphokine production and antagonizes the immunosuppressive action of FK-506. The DO-11.10 T-cell hybridoma stimulated with ionomycin plus PMA was used as a model system. FK-506 (1.2 nM) totally prevented DNA fragmentation detectable by agarose gel electrophoresis at 16 h of culture. FK-506 still inhibited this phenomenon when added 2 h after the initiation of the cultures but not later. In contrast, concentrations of RAP as high as 1 microM failed to block apoptosis. However, RAP (110 nM) reversed the apoptosis-inhibitory effect of FK-506, even if added 1-2 h after the latter to the cultures. Consistent with this antagonism, RAP also reversed the binding of a radiolabeled derivative of FK-506 in DO-11.10 cells. Therefore, FK-506 interferes with an early event of T-cell activation that leads to apoptosis whereas RAP does not.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721614 TI - Newly established uterine cervical carcinoma cell line with co-amplification of human papillomavirus DNA and c-myc gene. AB - A new human tumor cell line, NCC-c-CX-1 (CX-1), was established from a uterine cervical cancer xenografted in nude mice. This cell line harbored approximately 50 to 100 copies of human papillomavirus (HPV) type 18 DNA per haploid genome, and contained about 16-fold-amplified c-myc gene with rearrangement. These genomic alterations found in CX-1 cells were also present in both primary tumor and xenografted tumor. Histopathologically, original and xenografted tumors were poorly differentiated cancer and were characterized by neuroendocrine features such as positive neuron-specific enolase and chromogranin A by immunohistochemistry and abundant neurosecretory-type granules in the cytoplasm by electron microscopy. However, the established cell line had lost the neuroendocrine features. This cervical cancer cell line may be a useful model for studying cervical carcinogenesis, especially the interaction between HPV and c myc oncogene. PMID- 1721616 TI - Structural features of neurons in whole grafts of the rat inferior colliculus. AB - The inferior colliculus (IC) is a midbrain structure that receives ascending auditory input from brainstem nuclei via the lateral lemniscus, sends efferent fibers to the medial geniculate body of thalamus and receives descending projections from auditory cortex. In the rat, the IC consists of dorsal and external cortices surrounding the central nucleus of IC (CNIC) which is populated by discoid and stellate neurons; the CNIC has a laminar appearance arising from organization of lemniscal fibers and processes of discoid cells. The IC of adult rats was chosen for implantation of whole grafts of E16-17 caudal tectum into unilateral lesion sites. Dendritic and somal architecture of graft neurons was examined 1 to 4.5 months following implantation using rapid Golgi, HRP and Nissl methods. The CNIC of rat is dominated by principal neurons with relatively flattened dendritic fields. In grafts of caudal tectum the most common neuron class observed possesses flattened dendritic arbors which often parallel one another. These neurons also resemble CNIC neurons of host tissue adjacent to the graft border. Spine formations appear on both proximal and distal dendrites of this neural type in both normal and implanted tissues. In addition, comparable somal features of graft neurons include ovoid or fusiform shapes with regular nuclear membranes as found in the normal colliculus. In Golgi stained material fewer stellate class neurons appear as in the normal CNIC, although stellate cell classes are more abundant in the pericentral areas of normal tissue. Both neuron populations are retrogradely labelled in graft and normal IC after HRP injection into the medial geniculate body. These features suggest that the graft core typically consists of prototypic CNIC cells. Other features of neuron and glial cell density vary in graft material which also shows a complex network of vasculature. These results demonstrate that whole grafts of caudal tectum placed into the inferior colliculus can form organized neural architecture similar to the normal CNIC. The somal, dendritic and spine features of these neurons form a potential substrate for connectional and functional properties which establish this preparation as suitable for further investigation as a model for development and recovery of function in the central auditory system. PMID- 1721615 TI - Characterization of high-molecular-mass forms of basic fibroblast growth factor produced by hepatocellular carcinoma cells: possible involvement of basic fibroblast growth factor in hepatocarcinogenesis. AB - Growth factor(s) with a strong mitogenic effect on BALB/c3T3 cells was purified from an extract of C-Li21 cells, a human hepatocellular carcinoma line, by a combination of heparin-affinity chromatography and reversed-phase high performance liquid chromatography (HPLC). Two major peaks of mitogenic activity were obtained by reversed-phase HPLC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the two peaks revealed that one was composed of three proteins with relative molecular masses of 27, 24 and 23 kilodaltons (kD), whereas the other was a single 19-kD protein. Immunoblot analysis showed that all four of these molecules were immunoreactive species of human basic fibroblast growth factor (bFGF). N-Terminal sequence analysis of these molecules revealed that most of them were N-terminally blocked. However, small proportions of the 23 and 19-kD molecules were not blocked, and their respective N-terminal sequences were found to correspond to Gly-40-Gly-27 and Pro29-Phe40 of human bFGF deduced from the cDNA sequence of a human hepatoma cell line, SK-HEP-1. Expression of bFGF in hepatocellular carcinomas was then investigated by RNA blot analysis. All of the examined hepatocellular carcinoma cells expressed bFGF, and the degree of expression was higher in surgically resected hepatocellular carcinomas than in the corresponding adjacent non-cancerous liver tissue. Transcripts of bFGF were not detected in normal liver. These results suggest that C-Li21 cells produce four molecular forms of bFGF, and that bFGF may be involved in hepatocarcinogenesis. Moreover, it appears that bFGF is a potent mitogen toward primary-cultured hepatocytes, and that high-molecular-mass forms of bFGF produced by C-Li21 cells have stronger mitogenic effects on hepatocytes and are more stable under acidic conditions than the low-molecular-mass form, composed of 146 amino acids. PMID- 1721617 TI - Production of the mouse whey acidic protein in transgenic pigs during lactation. AB - The mouse whey acidic protein (WAP) gene was introduced into the genome of pigs and its expression was analyzed in the mammary gland. Mouse WAP was detected in milk of lactating females from five lines at levels between .5 and 1.5 g/liter, thereby representing as much as 2% of the total milk proteins. The corresponding mRNA was expressed in mammary tissue at levels similar to those of pig beta lactoglobulin and beta-casein. The pattern of WAP secretion in three pigs over a period of 6 wk was quantitatively similar to that of pig beta-lactoglobulin. From the eight transgenic pigs analyzed, three successfully completed one lactational period, but five pigs stopped lactating a few days after parturition. Our results show that it is possible to produce large quantities of a foreign protein in milk of pigs over a full lactational period. However, expression of WAP can compromise the mammary gland and render it nonfunctional. PMID- 1721618 TI - Efficacy of dextran therapy in coronary angioplasty. PMID- 1721619 TI - Serotonin, suicide, and aggression: clinical studies. AB - Both suicidal and aggressive, impulsive behaviors have been linked to putative dysregulation in central serotonergic systems. We review data examining the role of serotonin (5-HT) in suicide from postmortem studies and clinical investigations of suicide attempters, including our own preliminary work derived from neuroendocrine challenges with the 5-HT uptake inhibitor clomipramine. Various approaches to the study of 5-HT and aggressive, impulsive behavior, including cerebrospinal fluid studies, investigations of peripheral measures of 5 HT, and neuroendocrine studies utilizing 5-HT probes, are highlighted. Several important caveats, including the challenge of quantifying "suicidality" and "aggression" in reliable and valid ways, should be considered in interpreting the results of clinical studies of 5-HT and suicide and aggression. PMID- 1721620 TI - Insulin-like growth factors: the ovarian connection. AB - A large body of information now supports the existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins. Although much remains to be learned, the emerging consensus would suggest that the intraovarian IGF system is concerned largely with the amplification of gonadotrophin hormonal action for the facilitation of follicular growth and development. Future studies are likely to address the central issue of indispensability and the documentation of a meaningful in vivo role for this and related putative intraovarian regulators. PMID- 1721621 TI - Insulin-like growth factor binding protein-1 and ovarian stimulation. AB - Serum concentrations of insulin-like growth factor binding protein-1 (IGFBP-1) were measured in 42 patients with tubal infertility undergoing two different regimens of ovarian stimulation for in-vitro fertilization. The first group comprised 24 women given a luteinizing hormone-releasing hormone agonist analogue (buserelin, LHRHa) on cycle days 1-4 followed by follicle-stimulating hormone and human menopausal gonadotrophin (LHRHa group). The second group of 18 women received clomiphene citrate and gonadotrophins (CC group). On the day of human chorionic gonadotrophin administration, the sum of follicular diameters (P less than 0.05) and the number of follicles punctured in the LHRHa group (P less than 0.01) were significantly higher than in the CC group. In spite of the greater number of follicles, the oestrogen levels were similar in both groups, but women in the LHRHa group had significantly higher serum IGFBP-1 concentrations during the last 5 days of stimulation. These results suggest that the stimulated preovulatory follicles may contribute to the elevation of serum IGFBP-1. PMID- 1721622 TI - Decompaction and biopsy of late mouse morulae: assessment of in-vitro and in-vivo developmental potential. AB - The present study reports a biopsy technique on decompacted late mouse morulae. Zygotes were collected from hyperstimulated F1 hybrids (C57BL6j females x CBAca males) and cultured in modified Earle's balanced salt solution supplemented with 0.5% bovine serum albumin until the late morula stage (92 h post administration of human chorionic gonadotrophin). Decompaction was obtained by exposure of the embryos to Ca(2+)-Mg(2+)-free phosphate-buffered saline (PBS) or an aqueous solution of ethylene-diaminetetraacetic acid (EDTA) and glycine. Using micromanipulation, a single blastomere was aspirated without removal or softening of the zona. The impact of the different decompaction procedures and the biopsy technique was studied by vital staining, by in-vitro culture up to the early egg cylinder staged and by the recovery of living mice after transfer to pseudopregnant foster mothers. Our investigations revealed no impact of decompaction and biopsy on immediate viability, as assessed by fluorescein diacetate staining. A significant reduction (P less than 0.05) in the number of mouse morulae that reached the early egg-cylinder stage in vitro was observed after the biopsy procedure. We observed that after this microbiopsy technique, successful pregnancies can be obtained but at a lower percentage compared to controls (P less than 0.01). PMID- 1721623 TI - Effects of lysophosphatidylcholine on electrophysiological properties and excitation-contraction coupling in isolated guinea pig ventricular myocytes. AB - Lysophosphoglyceride accumulation in ischemic myocardium has been implicated as a cause of arrhythmias. We examined the effects of lysophosphatidylcholine (LPC) in isolated guinea pig ventricular myocytes. In paced myocytes loaded with the Ca2+ indicator Indo-1-AM and studied at room temperature, 20 microM LPC caused an initial positive inotropic effect followed by spontaneous automaticity, a decline in active cell shortening, and progressive diastolic shortening (contracture) leading to cell death. These changes were accompanied by a progressive increase in cytosolic [Ca2+]i. In patch-clamped myocytes dialyzed internally with high EGTA concentrations, LPC caused membrane depolarization, shortening of the action potential duration, and abnormal automaticity as seen in multicellular preparations. Voltage clamp experiments revealed the appearance of a nonselective leak conductance without significant changes in the delayed rectifier K+ current, inward rectifier K+ current, L-type Ca2+ current, and T-type Ca2+ current. Pretreatment with 20 mM caffeine and [Ca2+]o-free solution did not prevent the leak current. In patch clamped myocytes loaded with 0.1 mM Fura-2 salt, the [Ca2+]i transient induced by either voltage clamps or brief caffeine exposure remained normal until the nonselective leak current developed. The Na(+)-Ca2+ exchange current elicited during caffeine-induced [Ca2+]i transients also did not appear to be altered by LPC. Qualitatively similar results were obtained in myocytes studied at 35 degrees C. The membrane detergent saponin (0.005% wt/wt) mimicked all of the effects of LPC. We conclude that under these experimental conditions the effects of LPC are most compatible with a detergent action causing membrane leakiness with resultant depolarization, [Ca2+]i overload, and contracture. PMID- 1721624 TI - Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis. AB - Cystic fibrosis (CF) is the most common, lethal inherited disorder in the Caucasian population. We have recently reported two African-American patients with nonsense mutations in each CF gene and severe pancreatic disease, but mild pulmonary disease. In order to examine the effect of these nonsense mutations on CF gene expression, bronchial and nasal epithelial cells were obtained from one of these patients (no. 246), a compound heterozygote for nonsense mutations R553X and W1316X; a healthy normal individual; a patient (no. 528) homozygous for the common CF mutation (delta F508); and a CF patient (no. 272) who carries the R553X mutation and a missense mutation, S549N. When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by polymerase chain reaction using primers derived from the CF gene, DNA fragments of the predicted size were observed. However, patient no. 246 with nonsense mutations in each CF gene has no detectable cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA, and therefore should have severely diminished, and possibly absent, CFTR protein. Furthermore, less than 2% of the CFTR transcripts in nasal epithelial cells from patient no. 272 (S549N/R553X) were derived from the gene with the nonsense mutation. We conclude that severe reduction in CFTR mRNA causes CF, but can have different consequences in the lung and pancreas. PMID- 1721625 TI - Glioblastoma expression of vitronectin and the alpha v beta 3 integrin. Adhesion mechanism for transformed glial cells. AB - Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alpha v beta 3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the alpha v beta 3 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain. PMID- 1721626 TI - Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells. AB - Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellular fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribution of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Aggregation responses of neutrophils as well as adhesion responses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-dependent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells. PMID- 1721627 TI - Interferon-alpha restores the deficient expression of the cytoadhesion molecule lymphocyte function antigen-3 by chronic myelogenous leukemia progenitor cells. AB - Hematopoietic cells from the malignant clone in chronic myelogenous leukemia (CML) maintain and expand a proliferative advantage over normal hematopoietic cells within the bone marrow. This advantage is often ameliorated or reversed in vivo by IFN alpha. Based upon earlier studies suggesting decreased adhesiveness of CML progenitor cells, we asked whether CML progenitor cells are deficient in their expression of the cytoadhesion molecule lymphocyte function antigen-3 (LFA 3, CD58) which is normally expressed on hematopoietic progenitors. Progenitor cells from untreated CML patients showed greatly reduced or absent LFA-3 expression, whereas progenitors from patients treated with IFN alpha in vivo or in vitro expressed surface LFA-3 at more normal levels. LFA-3-deficient CML progenitor cells were unable to stimulate normal regulatory proliferative responses in autologous T cells. We hypothesize that IFN alpha-sensitive LFA-3 deficiency reflects a cell surface cytoadhesion defect which may help explain adhesive abnormalities of CML progenitor cells in vitro and clonal proliferation in vivo. PMID- 1721628 TI - Ber-MAC3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen. AB - A new monoclonal antibody Ber-MAC3 is reported. It recognises a formol sensitive epitope of a not yet clustered monocyte/macrophage specific 140 kilodalton glycoprotein that is expressed on the cell surface and in the cytoplasm. In 30 cases of acute and chronic leukaemia, Ber-MAC3 staining was restricted to 15 myeloid leukaemias of M4 and M5 types. The tumour cells of two cases of true histiocytic malignancies were Ber-MAC3 positive, whereas those of all 280 malignancies of lymphocytic origin were negative. The latter included 52 cases of Hodgkin's disease and 41 cases of Ki-1 positive anaplastic large cell lymphomas which had previously been classified as true histiocytic lymphomas. Ber-MAC3 therefore seems to be of considerable value for selective identification of monocytes and macrophages at a certain stage of differentiation and seems to be suitable for diagnosing myelomonocytic or monocytic leukaemia and neoplasms of true histiocytic origin. PMID- 1721629 TI - The spectrum of CAST I and II. PMID- 1721630 TI - Central course of digital axons within the median nerve of Macaca mulatta. AB - The traditional view that axons are not functionally grouped within proximal human nerve is based on the interfascicular dissections of Sunderland ('45). However, microstimulation and microneurography (Schady et al., '83a; Hallin, '90) reveal proximal grouping of cutaneous sensory axons from small areas of skin. In the present studies, conjugates of horseradish peroxidase with wheat germ agglutinin (HRP-WGA) were used to trace the course of digital nerve axons within the median nerve of Macaca mulatta. The electrophysiologic findings were confirmed, suggesting the potential for precise surgical realignment of functionally related axons even after proximal nerve transection. Radial digital nerves were labeled in the thumb (bilateral, 1 animal), the index finger (unilateral, 2 animals), and the middle finger (bilateral, 1 animal). Median nerve cross sections were cut at 1-cm intervals, treated with tetramethyl benzidine to demonstrate HRP-WGA within axons, and compiled to form maps of each digital nerve "territory" within the median nerve. These territories were limited to a single, densely labeled fascicle at the wrist level. They expanded somewhat in the forearm to encompass clusters of labeled axons within a matrix of unlabeled axon profiles. The clusters were more loosely packed in the arm, occupying 1/3 to 1/6 of the nerve cross section at the entrance to the brachial plexus. The three digital nerve territories studied were widely separated at the wrist level. In the proximal arm, there was moderate intermingling of axons from adjacent digits, but those to the middle finger and thumb remained segregated. Territory configuration differed widely overall, but was moderately constant for each digit. The location of territories within the nerve was often strikingly similar from right to left and from animal to animal, with occasional prominent variations reflecting isolated rotation of one nerve. PMID- 1721631 TI - Reduced branching and length of dendrites detected in cervical spinal cord motoneurons of Wobbler mouse, a model for inherited motoneuron disease. AB - The Wobbler mouse (wr) has been proposed as a model for human inherited motoneuron disease (infantile spinal muscular atrophy). The primary defect is thought to be in the motoneurons. Therefore we undertook a survey of the qualitative and quantitative changes occurring in the cervical spinal motoneurons of Wobbler mice during a late stage of the motoneuron disease compared with age- and sex-matched normal phenotype (NFR/wr) littermates. The Rapid Golgi Method was applied. In control and Wobbler mice, four types of neurons were identified according to their dendritic patterns: multipolar, tripolar, bipolar, and unipolar cells. Unipolar cells were observed more often in the Wobbler specimens than the controls and may represent a final stage in the degeneration of other cell types with greater numbers of primary dendrites. Medium (300-999 microns 2) and large (greater than 1,000 microns 2) impregnated neurons (presumably alpha motoneurons) showed strong indications of cell degeneration, including statistically significant reductions in the measurements for dendritic length, distribution, and branching, as well as the number of spines. In contrast, the small (less than 300 microns 2) neurons showed only mild signs of degeneration, including slight reductions in dendritic length, but no significant differences appeared in the distribution and branching of dendrites, or in the number of spines. Instead, a small increase could be detected in the number of primary and secondary dendritic branches emanating from the small neurons, as well as in the number of dendritic spines. These findings suggest that sprouting may occur to a slight extent. Although previous studies document that swelling with subsequent vacuolation of motoneurons is the predominant feature characterizing the Wobbler disease, the mean soma area (microns 2) calculated for the impregnated neurons of the Wobbler specimens showed no significant difference from the controls. It is hypothesized that the advanced signs of the Wobbler motoneuron disease are primarily reflected in the degeneration of the dendrites and spines on the medium and large alpha-motoneurons. The small neurons (presumably a mixed population of gamma-motoneurons, interneurons, and Renshaw cells) possess dendrites and spines that seem to be less affected, and instead show signs of sprouting. PMID- 1721632 TI - Local circuit neurons of macaque monkey striate cortex: III. Neurons of laminae 4B, 4A, and 3B. AB - We continue an investigation of the organization of local circuit neurons (largely inhibitory, GABAergic neurons, with smooth or sparsely spined dendrites) in the primary visual cortex of macaque monkey (Lund, '87: J. Comp. Neurol. 257:60-92; Lund et al., '88: J. Comp. Neurol. 276:1-29). This account covers local circuit neurons of layers 4B, 4A, and 3B; these three layers each receive different intrinsic second-order relays of principal thalamic inputs as well as receiving primary thalamic inputs in the case of two of the three laminae (4A and 3B). The study shows the existence of a number of different local circuit neurons making interlaminar projections between 4B, 4A, and 3B; each provides specific cross links between different combinations of the three laminae. It is known that the functional properties recorded physiologically from layers 4B, 4A, and 3B differ from one another and so these anatomical cross links may allow for correlation between different attributes of visual stimuli, e.g., color or motion, while still enabling separate processing of these different attributes to proceed in each of the three layers and be passed on to extrastriate areas. Whereas no spine-bearing neurons of layers 4B, 4A, or 3B provide "feedback" circuits to layer 4C (the source of their major intrinsic excitatory afferents), some of the local circuit neurons provide precisely structured axon feedback projections to divisions of 4C. The local circuit neurons also project to either lamina 5 or lamina 6, but not both and to superficial layers 3A, 2, and 1. Some local circuit neuron axon projections are of a dimension that would be confined to single functional clusters, e.g., cytochrome-rich "blobs," others reach out far enough to contact nearest neighbor "unlike" functional clusters, and yet others spread far enough to link repeating clusters of single function. PMID- 1721633 TI - Serotoninergic varicosities make synaptic contacts with pleural sensory neurons of Aplysia. AB - Serotonin is a modulatory neurotransmitter that produces many of the cellular changes associated with sensitization of reflexes in Aplysia. These changes have been carefully documented in sensory neurons located in the abdominal ganglion that mediate the gill-siphon withdrawal reflex and in sensory neurons located in the pleural ganglion that mediate the tail-siphon withdrawal reflex. Although serotonin appears to be necessary for sensitization, there is no direct evidence that serotoninergic neurons make synaptic contacts with sensory neurons. In this study, the immunoperoxidase technique was used to label serotonin-immunoreactive neurites surrounding the cell bodies of sensory neurons in the pleural ganglion. Serotonin-immunoreactive neurites had varicosities whose mean short axis diameter was 1.1 +/- 0.6 microns (mean +/- S.D.). The shape of the size distribution was skewed toward larger sizes, however, suggesting that there were multiple subpopulations of varicosities. One subpopulation was that of varicosities located at branch points whose average short axis diameter was larger than normal (1.7 +/- 0.5 microns). Serotonin-immunoreactive varicosities were directly apposed to the sensory neurons without intervening glial cells. In most contacts, serotonin-immunoreactive neurites invaginated into the plasma membranes of the sensory neurons. There were also a few contacts onto spinelike processes, but these were flat rather than invaginated. Serotoninergic neurons whose activity produces changes in the electrophysiological properties of sensory neurons have been identified, but this study provides the first direct evidence for synaptic connections between serotoninergic neurons and sensory neurons in Aplysia. PMID- 1721634 TI - Patient and staff perceptions of caring: review and replication. AB - The present study identified patient (n = 86) and nursing staff (n = 73) perceptions of most and least important caring behaviours. Using a Swedish version of the CARE-Q instrument or a free rating scale, patients ranked items concerned with giving honest and clear information and competent clinical expertise as most important. The nursing staff ranked expressive/affective behaviours as most important. There were significant differences between the two groups in the ranking of 14-30 out of 50 specific behaviours and in 3-5 out of 6 subscales. Patient and staff ratings did not differ appreciably between the methods used, with the exception that staff gave much higher ratings to most items in the free response format. Patients were more discriminating in the importance they assigned to the various items. PMID- 1721635 TI - Distribution of vagal afferent fibers of the guinea pig heart labeled by anterograde transport of conjugated horseradish peroxidase. AB - To determine the distribution of vagal afferent fibers in the heart, wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) or choleragenoid conjugated horseradish peroxidase (B-HRP) was injected into nodose ganglia of guinea pigs. Anterogradely labeled fibers and beaded, terminal-like arborizations were observed in the ascending aorta and aortic arch, the pulmonary trunk and arteries, posterior atrial walls, atrioventricular valves, and ventricles. Control experiments with injection of B-HRP into the cervical vagus nerve indicated that labeled fibers observed in the heart originated from sensory neurons in the nodose ganglia. Neither the density nor distribution of labeling differed between WGA-HRP and B-HRP. Injection of tracer into the left or right nodose ganglion shows that these regions of the heart are bilaterally innervated, although labeling in the left or right posterior atrium was denser after injection into the ipsilateral ganglion. Comparison with a previous study on the distribution of sympathetic afferent fibers in the guinea pig heart suggests that the two afferent systems maintain a complementary, but not mutually exclusive, distribution within the ventricles. Whereas afferents with their source in the spinal ganglia are mainly distributed with coronary arteries on the anterior superior surface of the ventricles, afferent fibers with their source in the nodose ganglia are concentrated within peri-arterial regions of the posterior inferior ventricular epicardium and the posterior septal ventricular endocardium. These differences in distributions of afferent systems could play a role in post infarction autonomic dysfunction and in the symptoms that accompany angina pectoris. PMID- 1721636 TI - Dopamine- and adenosine-3',5'-monophosphate (cAMP)-regulated phosphoprotein of 32 kDa (DARPP-32) in the adrenal gland: immunohistochemical localization. AB - The cellular localization of a dopamine- and adenosine-3',5'-monophosphate (cAMP) regulated phosphoprotein of an apparent molecular weight of 32,000 (DARPP-32) was investigated in mouse, rat, rabbit, guinea pig, cat, monkey (Macaca fascicularis and Marmoset) and human adrenal gland by means of indirect immunofluorescence histochemistry. DARPP-32-like immunoreactivity (-LI) was demonstrated in chromaffin cells in the adrenal medulla of rabbit, guinea-pig, cat, monkey and human, but not in mouse or rat. In the Marmoset monkey, DARPP-32-LI was also observed in the zona glomerulosa of the adrenal cortex. It has been shown that dopamine and dopaminergic agonists inhibit catecholamine release from chromaffin cells and aldosterone secretion from cells in the adrenal cortex. The present results suggest that DARPP-32, an intracellular third messenger for dopamine, may be part of the signal transduction mechanism for dopamine acting on the adrenal gland. PMID- 1721637 TI - The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2. AB - Influenza virus matrix protein-derived peptides were synthesized based on the amino acid motifs for HLA-A2 bound self peptides. Among these peptides a nonamer (amino acids 58 through 66: G I L G F V F T L) was found to be 100 to 1000 times more effective than the commonly used peptide 57-68 (K G I L G F V F T L T V) in sensitizing HLA-A2+ target cells to lysis by influenza virus specific cytotoxic T lymphocytes. The sensitizing activity of the 12-mer 57-68 was not due to contamination with shorter and more active peptides. Intracellular expression of peptide 58-66 (mediated by a stable expression plasmid with DNA coding for this peptide) also sensitized HLA-A2+ cells to lysis. Peptide 58-66 stimulated human PBMC to generate CTL that recognized peptides 58-66 and 57-68 in association with HLA-A2. PMID- 1721638 TI - Reduction of disulfide bonds within lysosomes is a key step in antigen processing. AB - Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2 macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells. PMID- 1721639 TI - Transmembrane signals generated through MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent and independent adhesion in human B cells through a tyrosine kinase-dependent pathway. AB - Transmembrane signals generated following mAb binding to CD19, CD20, CD39, CD40, CD43, Leu-13 Ag, and HLA-D region gene products induced rapid and strong homotypic adhesion in a panel of human B cell lines. Lower levels of adhesion were also observed after engagement of CD21, CD22, and CD23. Adhesion induced by mAb binding to these Ag was identical with respect to the kinetics of adhesion and the morphology of the resulting cellular aggregates, and was distinct from PMA-induced adhesion in both of these properties. Adhesion was not observed in response to mAb binding to MHC class I, CD24, CD38, CD44, CD45RA, or CD72. In contrast to B cell lines, homotypic adhesion was not induced in two pre-B cell lines, in spite of their high level expression of CD19 and HLA-D. Adhesion induced by suboptimal stimulation through these surface Ag or by PMA was mediated primarily through LFA-1 and ICAM-1. However, optimal stimulation through CD19, CD20, CD39, CD40, and HLA-D induced strong homotypic adhesion that was not blocked by anti-LFA-1 mAb. This alternate pathway of adhesion was also observed in LFA-1-deficient cell lines and in the presence of EDTA, suggesting that adhesion was not mediated by integrins. Adhesion in response to engagement of cell-surface Ag was unaffected by H7 or genestein, but was significantly inhibited by staurosporine, and was completely ablated by sphingosine and herbimycin. These studies indicate that engagement of multiple B cell-surface molecules initiates a signal transduction cascade that involves tyrosine kinases but not protein kinase C, and which leads to homotypic adhesion. Furthermore, adhesion was mediated by at least two distinct cell-surface adhesion receptors: LFA-1/ICAM-1 and a heretofore unknown adhesion receptor. PMID- 1721640 TI - The VLA-4/VCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium. AB - Lymphocyte migration to inflammatory sites is an essential factor in the pathogenesis of chronic inflammation. An ensemble of adhesion receptors mediating lymphocyte-endothelial cell recognition and binding are thought to play a crucial role in this process. In the present study, we have explored the molecular basis of lymphocyte adhesion to endothelium in the synovial membrane of patients with rheumatoid arthritis. We established that the very late antigen-4 [VLA-4 (CD49d)] and the vascular cell adhesion molecule-1 (VCAM-1) are important mediators of binding to synovial endothelium of resting and, to a greater extent, of activated T lymphocytes, whereas the leukocyte-function associated antigen-1 [LFA-1 (CD11a/18)]/intercellular adhesion molecule-1 [ICAM-1 (CD54)] pathway is less important in this interaction. In contrast to its prominent role in lymphocyte interaction with endothelium in rheumatoid synovium, the VLA-4/VCAM-1 pathway does not significantly contribute to lymphocyte adhesion to peripheral lymph node high endothelial venule. Thus, the VLA-4/VCAM-1 pathway may be of primary importance in mediating lymphocyte adhesion to inflamed endothelium and in lymphocyte homing to rheumatoid synovium. PMID- 1721641 TI - Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis. AB - Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN. PMID- 1721642 TI - IL-4 regulates c-kit proto-oncogene product expression in human mast and myeloid progenitor cells. AB - The c-kit proto-oncogene encodes the receptor for a novel hemopoietic cytokine, termed stem cell factor (SCF) or mast cell growth factor (MGF) according to its stimulating spectrum. The human receptor for SCF/MGF is expressed in a subset of normal bone marrow progenitor cells, in leukemic myeloid cells, and in mast cells. In the present study, the effects of recombinant human growth regulators (IL-1 through -9, granulocyte-macrophage/granulocyte/macrophage-CSF, IFN, and TNF) on c-kit proto-oncogene product expression were analyzed by indirect immunofluorescence, by using the anti-SCF/MGFR mAb YB5.B8, and Northern blot analyses, by using a c-kit oligonucleotide probe. Of all cytokines tested, IL-4 was found to down-regulate expression of YB5.B8 Ag in the human mast cell line HMC-1 (maximum inhibition, 51.05 +/- 16.36% mean fluorescence intensity of control; p less than 0.02), as well as in primary leukemic myeloid cells. IL-4 was also found to down-regulate expression of YB5.B8 Ag in normal enriched bone marrow progenitor cells. The effects of IL-4 on expression of YB8.B8 Ag in myeloid/mast cell progenitors was dose and time dependent (maximum effects observed on days 2 and/or 4, by using 50 U/ml of rIL-4) and could be neutralized by using anti-IL-4 mAb. Moreover, IL-4 was found to down-regulate expression of c kit mRNA in leukemic myeloid cells as well as in HMC-1 cells. Together, these observations identify IL-4 as a regulator of c-kit proto-oncogene product expression in the human system. The effects of IL-4 on human hemopoietic progenitor cells and mast cells may be mediated in part through regulation of SCF/MGFR expression. PMID- 1721643 TI - The relationship between polymerization of complement component C9 and membrane channel formation. AB - C9 was studied with the objective to clarify the relationship between the process of C9 polymerization and membrane channel formation. Conditions that favor C9 polymerization include low ionic strength and calcium ion in the buffer. Moreover, polymerization is dependent on the concentration of C9. Calcium ion evokes about a threefold increase in the affinity constant for C9 self association, and at 0 degrees C it imparts reversible amphiphilic properties in the molecule. These were discerned by measuring increases in the degree of reversible nonspecific binding of C9 to hydrophobic (tyramine-zymosan) and hydrophilic (arginyl-glutamyl-zymosan) supports as well as to erythrocytes. At 0 degrees C the hydrophilic-to-amphiphilic alteration of C9 is reversible, but upon incubation at 37 degrees C this transition is rendered permanent with the formation of poly(C9). A functional relationship between C9 polymerization and cytolysis was demonstrated by showing that polymerizing C9 can lyse reduced and alkylated erythrocytes. By studying comparative radiolabeling of tyrosine side chains within thrombin-nicked C9 and its polymerized form, it was demonstrated that upon polymerization the membrane-binding site of C9 becomes exposed. It is concluded that the process of circular polymerization of C9 causes a hydrophilic to-amphiphilic transition that is required for membrane perforation and channel formation. PMID- 1721644 TI - Characterization of the anti-inflammatory effect of FK-506 on human mast cells. AB - We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187 induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK 506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP). PMID- 1721645 TI - Serologic reactivities of the 23-kDa integral membrane proteins of schistosomes. AB - The 23-kDa integral membrane proteins of Schistosoma mansoni and Schistosoma japonicum (Sm23 and Sj23) are Ag of some interest in terms of both antiparasite vaccination and immunodiagnosis. We have raised an antiserum against a recombinant fusion protein expressing the extracellular hydrophyllic domain of Sm23 (Sm23HD-pGEX) and used this serum, as well as other antibody reagents reacting with Sm/Sj23, in immunochemical analyses. The immunogenicity and antigenicity of Sm23HD-pGEX, and the surprising lack of cross-reactivity between Sm23 and Sj23 support the hypothesis that Sm/Sj23 are host-like molecules with a very limited number of B cell epitopes that are likely to reside in the extracellular hydrophilic domain. We also present evidence that, unlike the highly immunogenic Sj23, Sm23 is not immunogenic in chronically infected mice. Moreover, we confirm a surface location for Sj23 in adult worms, in S. japonicum. PMID- 1721646 TI - Evaluation of Blalock-Taussig shunts in newborns: value of oblique MRI planes. AB - Eight infants with systemic-pulmonary Blalock-Taussig shunts were evaluated by spin-echo ECG-gated MRI. Contrary to Echocardiography, MRI using coronal oblique projections successfully visualized all palliative shunts entirely in one single plane (including one carried out on a right aberrant subclavian artery). MRI allowed assessment of size, course and patency of the shunt, including pulmonary and subclavian insertion. The proximal portion of the pulmonary and subclavian arteries were also visualized. We conclude that MRI with axial scans completed by coronal oblique planes is a promising, non invasive method for imaging the anatomical features of Blalock-Taussig shunts. PMID- 1721647 TI - The structural basis of protein antigenicity. PMID- 1721648 TI - Chemical and functional analysis of MHC class II-restricted T cell epitopes. AB - Various aspects of antigen degradation and presentation are reviewed, in particular with respect to fragmentation of native vs. denatured proteins, different enzymatic machinery present in different cells and individuals, characterization of epitopes and their persistence on antigen-presenting cells as well as their capacity to interact with different MHC class II molecules. Finally, the structure of antigenic peptides is discussed. PMID- 1721649 TI - Molecular approaches to the study of B cell epitopes. AB - For some years, a major goal of immunochemistry has been to determine the molecular architecture of the antibody-combining site and its cognate surface on the antigen, the antigenic determinant or epitope, and to determine the molecular basis of specificity and affinity. In recent years, the crystal structures of several antigen-antibody complexes have been determined. In addition, recombinant DNA technology is beginning to play an increasingly important role in analysis of protein-protein interaction including the study of antigen structure and its interaction with antibody. The purpose of this review is to briefly present some of the major and common properties of the antigen-antibody interface as it is known today and to demonstrate, using a few selected studies, the efficacy of using site-directed mutagenesis to study the nature of the antigenic surface of protein molecules and its interaction with antibody. PMID- 1721650 TI - Defining antibody-antigen recognition: towards engineered antibodies and epitopes. PMID- 1721651 TI - Binaural properties of single units in the superior olivary complex of the mustached bat. AB - 1. Previous studies of the superior olive of echolocating bats suggest that the lateral superior olive (LSO) retains the same structure and function as in other mammals but that the medial superior olive (MSO) is different in structure and possibly also in function. The present study is an examination of this idea in Pteronotus parnellii, a bat that has a large and well-defined MSO. 2. Using pure tones presented via earphones, we obtained data on frequency tuning for 60 single units and 96 multiunits in LSO and 94 single units and 154 multiunits in MSO. Of these we also obtained binaural response characteristics from 55 single units in LSO and 72 single units in MSO. 3. LSO and MSO each have a complete tonotopic representation, arranged in a sequence similar to that of other mammals studied. However, in both LSO and MSO there is an expanded representation of the frequencies around 60 kHz, the main frequency component of the bat's echolocation call; there is another expanded representation of the range around 90 kHz, the third harmonic of the call. The expansion of these frequency ranges suggests that the functions of LSO and MSO in Pteronotus are related to echolocation behavior. 4. The binaural characteristics of cells in LSO were essentially the same as those seen in other mammals. Most LSO units (93%) were excited by the ipsilateral ear and inhibited by the contralateral ear. The responses of nearly all LSO units were completely suppressed when the sound level at the two ears was equal. 5. The binaural characteristics of cells in MSO were different from those in nonecholocating mammals. Most MSO units (72%) were excited by the contralateral ear but were neither excited nor inhibited by the ipsilateral ear. Of the remaining units, 21% were excited by the contralateral ear and inhibited by the ipsilateral ear, and only 6% were excited by both ears. 6. The temporal discharge patterns of units in MSO differed from the tonic response pattern seen in LSO. Most MSO units had phasic response patterns, with a few spikes at the onset or offset of the stimulus; the response often changed from ON to OFF depending on stimulus frequency. 7. The results support the idea that in evolution LSO has remained unchanged, whereas MSO has undergone adaptation. The function of LSO in Pteronotus seems to be identical to that in other mammals, i.e., analysis of interaural sound level differences to derive azimuthal location. The function of MSO in Pteronotus must be different from that in nonecholocating mammals.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1721652 TI - Metabolic and structural effects of HIV infection in human peripheral blood mononuclear cells can be monitored with 1H NMR spectroscopy. AB - Infection of human peripheral blood lymphocytes by human immunodeficiency virus type 1 (HIV-1) was investigated by means of 1H nuclear magnetic resonance spectroscopy, taking advantage of the presence of signals from fluid lipid domains in the membrane of stimulated lymphocytes. A transient decrease of the lipid methylene signal intensity was observed at the time of HIV internalization, monitoring a general rearrangement of membrane structure associated with virus entry. A similar effect was also observed a few days after infection, when HIV particles are released by infected cells as demonstrated by high reverse transcriptase activity in cell supernatant. Signals arising from choline-based metabolites were also affected by HIV infection, indicating a possible slowing down of phospholipid synthesis. PMID- 1721653 TI - [Color and pulsed Doppler of renal masses. Angiographic and anatomo-pathological correlation]. AB - We report the results of a prospective study about the detection of tumoral neovascularization, the renal vein involvement and the characterization of renal tumors by color and pulsed doppler. Twenty-six renal tumors including 19 renal carcinomas and 7 benign tumors (2 angiomyolipomas, 2 oncocytomas, 2 complex cysts and one adenoma) were prospectively explored. Benign cysts were excluded from the study. The results were correlated to pathologic (24/26) and angiographic datas (24/26). Doppler ultrasonography seems to be an accurate method for the detection of a neovascularization (19 true-positives/19 vascularised tumors, no false positive) but pulsed doppler (no false-negative) is more sensitive than color doppler imaging (3 false-negatives), the results of color doppler were improved by using a new software for the color detection of very low flow. Spectral analysis found 3 different types of doppler curves inside the masses, but all of them were observed in benign and malignant tumors. We did not find any correlation between the size of the tumor and the peak systolic velocity. The specificity of Doppler regarding the renal vein and inferior vena cava involvement seems to be good (no false-positive). But as, in our series, we do not have any case of main renal vein involvement, the sensitivity of this technique cannot be assessed. PMID- 1721654 TI - Influence of immunotherapy on the cellular immunity of unexplained recurrent aborters. AB - Changes in lymphocyte subsets in whole blood were analyzed sequentially by flow cytometry with an automated leukocyte differential system in 15 patients with unexplained recurrent spontaneous abortions, each of whom underwent vaccination(s) with her husband's lymphocytes. Mitogen responses of peripheral blood lymphocytes (PBL) were also examined in these patients. The reactivity of PBL against mitogens revealed no significant change in each patient before and after vaccination(s) with her husband's lymphocytes. The CD4:8 ratio was observed to decrease significantly during 22 and 28 days after the first vaccination with a significant increase in the percentage of T suppressor-cytotoxic (CD8) cells. This change was also observed after the second vaccination. The percentages of other subsets did not change significantly after vaccination(s). In 11 patients out of 15, the pregnancy continued successfully and correlated with a predominance of Ts/c (CD8) over TH/I (CD4) cells in the first trimester. These changes in lymphocyte subsets may indicate the induction of immune enhancing mechanisms and it is suggested that continuation of the predominance of Ts/c cells induced by immunotherapy might be important for the successful maintenance of pregnancy. PMID- 1721655 TI - Histidine decarboxylases from bacteria that colonise the human respiratory tract. AB - We investigated whether production of histamine by bacteria isolated from sputum of patients with infective lung diseases could be attributed to the presence of histidine decarboxylase (HD). Twenty gram-positive and 20 gram-negative organisms were studied for their ability to decarboxylate 14C-histidine in vitro over the pH range 4.5-7.5. Of the bacteria investigated, lysates from the gram-negative species Haemophilus influenzae, H. parainfluenzae, Moraxella (Branhamella) catarrhalis and Pseudomonas aeruginosa liberated 14CO2 and histamine from 14C histidine in the presence of the cofactor pyridoxal phosphate. In contrast, results obtained in the absence of cofactor were similar to those of negative (lysate-free) controls suggesting that the HD enzymes of these species resembled those previously described in other gram-negative bacteria. No HD activity was detected over this pH range in lysates from gram-positive species. This finding correlated with earlier observations that these gram-positive organisms did not produce histamine in vitro. PMID- 1721656 TI - The frequency and management of infectious episodes and sepsis in small cell lung cancer patients receiving intensive chemotherapy with granulocyte-colony stimulating factor. AB - The relation between degree of myelosuppression and episodes of infection was analyzed in 36 patients (92 treatment courses) with small cell lung cancer (SCLC) treated with intensive chemotherapy. The two regimens used were cisplatin (CDDP) + adriamycin (ADR) + cyclophosphamide (CPA) + etoposide (VP-16) + granulocyte colony stimulating factor (G-CSF) and CDDP + teniposide (VM-26) + G-CSF, and they induced grade 3 or 4 leukopenia in 88% of treatment courses and febrile episodes in 60%. In the febrile courses, the mean nadirs of leukocyte and neutrophils (820 +/- 581/mm3, 101 +/- 267/mm3) were significantly longer (P less than 0.01) and the mean durations of grade 3 and 4 leukopenia and neutropenia significantly longer (P less than 0.001) than those of the non-febrile courses. It was noted, however, that febrile episodes appeared frequently in courses having the nadir of leukocytes below 1,000/mm3 (80%) or the nadir of neutrophils below 100/mm3 (74%). The administration of antibiotics was required for about 7 days to patients with febrile episodes. Sepsis was experienced in five courses, in which the neutrophils were all zero. All the patients, however, could be managed by an administration of antibiotics immediately after a febrile episode appeared, without delaying the subsequent chemotherapy except for one patient, who had had a performance status (PS) of 3 prior to chemotherapy. PMID- 1721657 TI - [Aging and exocrine pancreatic function evaluated by the recently standardized secretin test]. AB - The authors studied the relationship between aging and exocrine pancreatic function by the secretin test which was recently standardized by the Japanese Society of Gastroenterology. Pancreatic juice was collected at 10 min intervals for 60 minutes after a bolus intravenous injection of secretin (Secrepan, Eisai Co., Ltd., 100 U/body) through a quadruple-lumen doudenal tube equipped with double balloons. Exocrine pancreatic function was evaluated by three parameters: secretory volume, maximal bicarbonate concentration or bicarbonate output, and enzyme (amylase and lipase) output. Control subjects consisted of 65 outpatients presenting with mild vague abdominal symptoms who fulfilled the following three criteria: 1) good general condition with no known diseases; 2) no abnormality in the liver, bile duct, pancreas, kidney and metabolism judged from blood chemistry, urine and stool analysis, upper GI series, abdominal ultrasonography (US), and endoscopic retrograde cholangiopancreatography (ERCP); 3) alcohol consumption less than 25 g/day. Control subjects were divided into three groups: 15 subjects below 40 years of age (group A), 32 subjects from 40 to 65 years (group B), and 18 subjects of 65 years and above (group C). Nineteen patient with chronic pancreatitis were also studied. The group C showed significantly lower values in secretory volume, bicarbonate output, and enzyme output than group A and B. Enzyme output showed a gradual decrease with aging. However, secretory volume and bicarbonate output showed a gentle convex curve with a peak around age 40 and a rather steep down-slope after late 50s. The degree of the decrease was significantly more marked in volume and bicarbonate output than in enzyme output in group C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721658 TI - [A comparative study of CHOP therapy and COP-BLAM therapy for non-Hodgkin's lymphoma in elderly patients]. AB - Elderly patients (aged greater than or equal to 65 years) with non-Hodgkin's lymphoma were treated either with CHOP or COP-BLAM therapy, and the effectiveness and reverse effects of COP-BLAM therapy were compared with those of CHOP therapy. Thirty-three patients (aged greater than or equal to 65 years) with previously untreated non-Hodgkin's lymphoma were entered either on CHOP or COP-BLAM regimen between September, 1979 and February 1990. To CHOP therapy was performed in 15 patients (median age; 70 years). Eight of them had diffuse large cell type lymphoma (large), five had diffuse medium-sized cell type (medium) and two had diffuse mixed cell type (mixed). As to clinical stage, there were patients in stage II, 4 in stage III and 9 in stage IV in CHOP group. Of 18 patients (median age; 68 years), who were treated with COP-BLAM therapy, 8 had of large lymphoma and 10 medium lymphomas in histopathological classification. In terms of clinical stage, there were 5 patients in stage II, 4 in stage III and 9 in stage IV. CHOP therapy and COP-BLAM therapy were performed according to the method reported by McKelvey et al, and by Laurence et al., respectively, using the full doses of drugs without consideration the age. Complete remission (CR) was achieved in seven (46.7%) of 15 patients treated with CHOP therapy. In this group, five (38.5%) of 13 patients in advanced stages (stage III or IV) entered CR. Of 18 patients subjected to COP-BLAM therapy, 15 (83.3%) achieved CR. Among 13 patients in advanced stage treated with COP-BLAM therapy, CR was achieved in 11 (84.6%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721659 TI - Effects of carbamazepine and ipsapirone on turnovers of monoamines in the rat brain. PMID- 1721660 TI - [The significance of the heparin precipitable fraction in lung diseases]. AB - The authors recently experienced a case of idiopathic interstitial pneumonia (IIP) that exhibited skin ulcers due to increased heparin precipitable fraction (HPF) in plasma. This case prompted us to investigate the occurrence and significance of HPF in interstitial pneumonia (IP). The subjects included patients with IIP (acute exacerbation 6 cases, chronic active stage 12 cases), IP associated with collagen vascular disease (CVD) (9 cases) and granulomatous lung diseases (7 cases). The data indicated that all of the cases with acute exacerbation of IIP exhibited increased plasma HPF values (218-951 mg/dl) compared to those of normal controls (less than 180 mg/dl). In contrast, the values ranged within normal limits in all of the cases with IP associated with CVD. In a companion study, we measured plasma HPF values in patients with lung cancer, bacterial pneumonia and diffuse panbronchiolitis. It was found that 22% of the subjects showed increased plasma HPF values. We also investigated whether there were correlations between plasma HPF and various inflammatory parameters. The data revealed that there were correlations between HPF and ESR, CRP, alpha 1 globulin, alpha 2-globulin, complement (C3) or fibrinogen. However, there was no correlation between HPF and fibronection. These results suggest that plasma HPF is valuable to evaluate the acute exacerbation of IIP, although the elevation of plasma HPF levels is not specific. PMID- 1721661 TI - [Effect of plasmapheresis with substitution by dextran solutions on hemostatic parameters in patients with ischemic heart disease]. AB - Plasmapheresis on a continuous blood flow fractionator to remove 1,200-2,000 ml plasma and substitute for the same values of dextran solutions was employed in the multi-modality treatment of 25 patients with Functional Classes II-IV exertional angina and myocardial infarction. Plasma and thrombocyte hemostatic parameters were examined just before and 24 and 48 hours after plasmapheresis. The analysis of the baseline data revealed that hemostatic changes were directly related to the severity of a disease in the patients and there was a correlation between the increased platelet aggregability and the elevated levels of fibrin monomeric complexes. Plasmapheresis along with substitution of removed plasma for dextran solutions was accompanied by a profound hypocoagulative effect, as evidenced by plasma and thrombocyte hemostatic parameters. In addition, in patients with baseline high values of platelet aggregability plasmapheresis resulted in its decrease, whereas in those with baseline low values, it led to its increase. PMID- 1721662 TI - [Rhythmographic analysis of atrial extrasystole in patients with sick sinus syndrome]. AB - The comparative analysis of 141 electrocardiograms showing premature atrial contraction from 43 patients with the sick sinus syndrome and 19 subjects with normal sinus nodal function revealed new electrocardiographic signs suggesting sinus dysfunction: an increase in the postextrasystolic pause by more than 135% of the preextrasystolic interval; greater full compensatory pause values; extrasystolic appearance of a full compensatory pause with the coupling interval of less than 68% of the presystolic interval; an increase in the sinus cycle followed the postextrasystolic pause by greater than 50 msec. The proposed criteria show a 5.7-fold increase in the informative value of ECG analysis in the diagnosis of the sick sinus syndrome in premature atrial contraction. PMID- 1721663 TI - [Disorders of organic hemodynamics of the liver and their correction in suppurative cholangitis]. AB - The blood flow in the portal vein and hepatic artery was studied by means of ultrasonic Doppler flow measurement to investigate organic and regional hemodynamics of the liver in purulent cholangitis. The blood flow in the portal vein was found to be significantly diminished in patients with acute cholangitis and hepatic failure. Hepatic microcirculation was studied on a experimental model of obstructive jaundice and obstructive purulent cholangitis in rats by polarographic measurement of hydrogen clearance. Considerable reduction of the volume rate of the local blood flow was noted, and the degree of the reduction was related to the severity and duration of the disease. Decompression of the biliary tract by external drainage improved the local blood flow rate which, however, diminished again in prolonged external drainage. The use of pharmacological agents for correction of microcirculation in decompression of the biliary tract led to total and rapid correction of the volume rate of the local blood flow. PMID- 1721664 TI - [Pathophysiology and prevention of retinopathy of prematurity]. AB - In premature infants there is a temporal and causal relation between the change in the erythropoiesis after birth and the manifestation of retinopathy. The physiological substitution of the fetal erythrocytes can be controlled by an analysis of the fetal hemoglobin. In premature infants in which the erythropoiesis has not yet been converted there is an interval of time where there is a chronic hypoxemia of the retina and in which neovascularisation occurs. The oxygen deficit arises because fetal blood - in comparison with adult blood - exhibits a higher affinity to oxygen. If high values of HbF are ascertained in premature infants the chronic hypoxemia of the retina can be avoided by replacing the erythrocytes. In this way, a secondary prevention of the illness is possible. PMID- 1721665 TI - Antigen specificities and clinical distribution of ANCA in kidney diseases. AB - The antigenic specificity and clinical distribution of the antineutrophil cytoplasmic antibodies (ANCA) in kidney diseases have recently been extensively studied. In patients with systemic vasculitis, the great predominance of two major ANCA antigens, proteinase 3 (PR3) and myeloperoxidase (MPO), is now established. PR3 and MPO are colocalized in the azurophilic granules of neutrophils and translocated to the cell surface during activation, and thus are able to interact with autoantibodies after neutrophil preactivation. Furthermore, by comparison of amino acid and DNA sequences, it has been shown that PR3 is identical to myeloblastin, which has been described independently and is involved in the control of growth and differentiation of leukemic cells. Aside from the two major ANCA antigens, a number of neutrophil cytoplasmic antigens recognized by ANCA have been identified, including human leukocyte elastase, lactoferrin, CAP57, and cathepsin G. These rare ANCA specificities occur in a limited number of patients. The variety of ANCA antigen specificities contrasts, however, with the fact that the vast majority of ANCA-positive sera are monospecific for one single ANCA antigen. With regard to clinical distribution, ANCA have major diagnostic significance in the four conditions in which they are frequently detected: Wegener's granulomatosis (WG), Churg and Strauss Syndrome (CSS), microscopic periarteritis (MPA), and necrotic and crescentic glomerulonephritis (NCGN). However, the initial dichotomy between MPO-associated vasculitis (NCGN, MPA) and that associated with anti-PR3 antibodies (WG) appears far from absolute. PMID- 1721666 TI - Antineutrophil cytoplasmic autoantibodies: immunobiological aspects. AB - Antineutrophil cytoplasmic autoantibodies (ANCA) specific for constituents of neutrophil primary granules and monocyte lysosomes have been demonstrated in various vasculitic disorders. The staining pattern in indirect immunofluorescence microscopy using alcohol-fixed neutrophils as substrate allows distinction among 3 types of ANCA: 1) classic anti-neutrophil cytoplasmic antibody (cANCA, formerly known as ACPA); 2) a type with a perinuclear/nuclear staining pattern produced when alcohol-fixed neutrophils are used as substrate (pANCA); and 3) a mixture of both of the above types (xANCA, also described recently as pANCA). Most cANCA are directed against proteinase 3 ("Wegener's autoantigen"). Some pANCA have specificity for myeloperoxidase and are associated with idiopathic crescentic glomerulonephritis ("renal vasculitis") and other systemic vasculitides exhibiting a paucity of immune deposits in blood vessels. In addition to being a useful serological marker, ANCA appear to be directly involved in the pathogenesis of systemic vasculitis. ANCA can activate cytokine-primed granulocytes and monocytes to undergo a respiratory burst and degranulation. This effect leads to vasculitis through the attachment of these cells to the vascular endothelium primed by cytokine-induced expression of adhesion molecules (E-LAM 1) on the endothelium. Thus, the release of toxic oxygen radicals and lytic enzymes is capable of causing vascular damage. In the present paper we report on the main target antigens and on the history, nomenclature, laboratory methods, and etiopathological implication of ANCA. Additional pathophysiological aspects of ANCA and/or autoreactive T cells and immunoregulatory events are also discussed. PMID- 1721667 TI - Distribution of protectin (CD59), a complement membrane attack inhibitor, in normal human tissues. AB - Protectin (CD59) is a recently discovered 18-20 kDa glycoprotein that effectively inhibits lysis by the membrane attack complex of the homologous complement system. This glycoprotein is widely distributed on the membranes of human blood cells (erythrocytes and leukocytes). By using immunofluorescence microscopy, protectin was observed in vascular endothelia throughout the body and in extravascular tissues. Cells expressing protectin were also found in ductal epithelia of pancreatic, biliary, and salivary systems, bronchi, and kidney collecting ducts. Furthermore, protectin was expressed in the epidermis and in the syncytiotrophoblast of placenta. The expression of protectin in endothelia, in various epithelial cells, and in placenta presumably protects autologous tissues and the fetoplacental unit from complement-mediated damage. PMID- 1721668 TI - P-glycoprotein expression is increased in human secretory and gestational endometrium. AB - To determine the expression, distribution, and intracellular localization of the multi-drug resistance gene product P-glycoprotein (Pgp) in the human menstrual cycle and in early gestational endometrium, we retrospectively studied 36 endometrial samples utilizing 3 murine monoclonal antibodies (MAbs), MAb C219, MAb C494, and MAb JSB-1, which recognize spatially distinct cytoplasmic epitopes of Pgp. Formalin-fixed, paraffin-embedded endometrial samples obtained from 36 women of reproductive age with normal menstrual cycles were assigned morphologic menstrual dates: proliferative (N = 10), secretory (N = 19), menstrual (N = 1), and gestational endometrium (N = 6). The cellular localization, staining intensity, and percentage of Pgp immunoreactive cells varied with the phase of the menstrual cycle. Early proliferative endometria revealed no Pgp immunoreactivity for all three MAbs. Mid-proliferative endometria showed weak immunostaining in less than 15% of the glandular epithelia. Late proliferative endometria showed a strong apical paranuclear/Golgi staining pattern. Early secretory endometria showed strong luminal membranous, subnuclear vacuolar membranous, and supranuclear vacuolar membranous immunostaining to all 3 MAbs in greater than 80% of the glandular epithelia. Apical paranuclear/Golgi and membranous staining were present in nonvacuolated mid-secretory glands. Immunoreactivity diminished in the late secretory phase with mild to moderate staining in less than 35% of the endometrial glands. Menstrual endometria showed weak, focal staining. All gestational endometria showed marked cytoplasmic, membranous, and apical/Golgi immunostaining both in the hypersecretory (Arias Stella) endometrial glands as well as in the decidua. In general, the intensity of MAb C494 immunostaining was weaker than that of MAb C219 or JSB-1. These results suggest the following: Pgp expression parallels that of nuclear progesterone receptor expression in the normal human endometrial cycle and early gestational endometrium; Pgp expression corresponds to rising plasma and tissue levels of progesterone as well as to morphologic changes in the endometrial glandular epithelium associated with the marked development of the secretory apparatus; Pgp expression is hormonally regulated and may be involved in uteroplacental transport of substrates important in the implantation process and in early embryo-endometrial interactions; and Pgp may be involved in the transport of progesterone across the uterine epithelium during pregnancy. PMID- 1721669 TI - Immunohistochemical localization of 17 beta-hydroxysteroid dehydrogenase in the human endometrium during the menstrual cycle. AB - Immunoaffinity-purified polyclonal anti-rabbit antibody against human placental 17 beta-hydroxysteroid dehydrogenase (17HSD) was used to localize 17HSD in frozen sections of 21 human endometrial tissue specimens, taken at different stages of the menstrual cycle, and in the human placenta. The presence and distribution of estrogen and progesterone receptors were also analyzed in endometrial specimens using commercial immunohistochemical techniques. In addition, 17HSD was localized by immunoelectron microscopy in the endometrium and the placenta. In the endometrium, immunostaining of 17HSD appeared in the cytoplasm of surface epithelial and gland cells during the early and midluteal phase. During the late luteal phase, it gradually disappeared. No immunostaining was observed in the endometrium during the follicular phase of the menstrual cycle. The changes in staining intensity of 17HSD were associated with changes in the concentration of serum progesterone as judged by radioimmunoassay. An apparent inverse correlation between 17HSD expression and the concentrations of estrogen and progesterone receptors was observed. These results strongly support the concept that progesterone induces an increase in the amount of 17HSD, in the glandular and surface epithelial cells of the human endometrium. In the human term placenta, 17HSD immunostaining was detected exclusively in the cytoplasm of syncytiotrophoblasts. In immunoelectron microscopic studies of the endometrium and placenta, 17HSD staining was observed in the cytoplasm and it was associated with cytoplasmic membranes unrelated to the endoplasmic reticulum. PMID- 1721670 TI - Cytoskeletal gene expression in normal and neoplastic human odontogenic epithelia. AB - In situ and Northern hybridization was carried out to study cytokeratin (Ck) 1, 4, 8, 18, and 19 and vimentin (Vim) gene expression in 13- to 24-week-old human fetal tooth germs, including overlying oral epithelium and odontogenic tumors (N = 6) of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origin. The results were compared with immunocytochemistry using monoclonal antibodies. A relatively strong expression of simple epithelial Ck 19 mRNA, together with low, but significant expression of Ck 8 and 18 mRNAs, was demonstrated in all normal and neoplastic odontogenic epithelia studied. Transcripts for squamous differentiation marker, Ck 4, and for terminal differentiation marker, Ck 1, were detected suprabasally in the fetal oral epithelium, focally in the dental lamina but not in the enamel organ. Ck 4 mRNA was expressed variably in most odontogenic tumors studied, whereas Ck 1 mRNA was detected in one ameloblastoma only. Vim mRNA was not found in the fetal oral epithelia, dental lamina or the enamel organ, but a distinct immunoreactivity with monoclonal antibodies to Vim was seen in the stellate reticulum cells of the enamel organ. The epithelium of most ameloblastomas showed a focal Vim mRNA and polypeptide expression. In addition to Vim, the neoplastic ectomesenchymal cells of ameloblastic fibroma coexpressed low amounts of simple epithelial Cks 8, 18, and 19. The results indicate that the differentiation and cytoskeletal gene expression programs of odontogenic epithelia upon neoplastic transformation are not fully retained. Most ameloblastomas and ameloblastic fibromas show differentiation parameters reminiscent of dental lamina. Ameloblastomas seem to form a heterogenous group of tumors, which may originate from odontogenic epithelial cells at various differentiation levels. The origin of ameloblastic fibroma is more closely related to the tooth germ proper. PMID- 1721671 TI - Enzyme-histochemical method for identification of lymphatic capillaries. AB - Using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining, lymphatic capillaries and blood capillaries were distinguished histochemically on cold glycol methacrylate (JB-4) sections of monkey intestines, on the basis of both enzyme characteristics. The specificity of the 5'-Nase reaction was obtained by inhibiting nonspecific ALPase in the 5'-Nase incubation medium including L tetramisole. This double staining method demonstrated satisfactory isolated visualization of 5'-Nase activity in lymphatic capillaries and of ALPase activity in blood capillaries under light microscopy. The intensity and localization of 5' Nase activity on the walls of lymphatic capillaries were also determined by the lead-based method under transmission electron microscopy. The intense reaction products of 5'-Nase activity were predominantly deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells. PMID- 1721672 TI - [The treatment of large-cell non-Hodgkin's lymphomas with the MACOP-B protocol]. AB - BACKGROUND: To investigate the results of the treatment of large-cell non Hodgkin's lymphomas (LCNHL) with the MACOP-B chemotherapy protocol. METHODS: 20 patients with the following inclusion criteria were treated: LCNHL with a definite majority of large lymphoid cells and absence of previous therapy, HIV infection or severe underlying diseases. RESULTS: Three patients died during therapy and 15 (75%) achieved a complete remission. Actuarial survival after 36 months (0.66) was significantly better (p = 0.05) than that of a comparable historical series of LCNHL treated with CHOP (0.28). Age, stages III-IV, B symptoms, large lymphatic mass (LLM) and bone marrow infiltration did not negatively affect survival. The toxicity of the MACOP-B protocol was high: mucositis (65%), cytopenia (55%), neuropathy (40%), complications of steroid therapy (15%), and mortality directly related with therapy in 15%. Residual masses were found after therapy in 7% (70%) with BD, which were localized in lymphoid areas or in parenchyma (spleen and kidney). Surgical exploration showed that the residual masses were not tumoral in three cases, and in another three magnetic resonance suggested inactive disease. CONCLUSIONS: The MACOP-B protocol is highly effective for the treatment of LCNHL. The essential prognostic factor for survival in these NHL appears to be the cell composition with a great majority of large cells. PMID- 1721673 TI - Endoscopic palliation of malignancies of the upper gastrointestinal tract using Nd:YAG laser: results and survival in 308 treated patients. AB - Three hundred eight patients with obstructing malignant tumors of the gastrointestinal tract were submitted to endoscopic Nd:YAG laser palliation. The number of sessions as well as the number of days and total energy required for initial recanalization of the stenosis were related to tumor length. Luminal patency was obtained in 94% of patients, but improved swallowing was achieved in 74% of patients. The technical success rate did not depend on tumor site, whereas functional positive results are related to the location of the stenosis, with worst results for tumors involving the upper third of the esophagus. Life-table analysis revealed that 1-year survival was 23% when a luminal patency was achieved and 7% when treatment failed. PMID- 1721674 TI - Genetic characterization of the O4 polysaccharide gene cluster from Escherichia coli. AB - The Escherichia coli O4 serotype is among those commonly isolated from urinary tract infections. In order to study the genetics of the O-antigen, the O4 biosynthesis genes from a uropathogenic E. coli have previously been cloned into E. coli K-12. A subclone, GH58, has been identified which reacts with antisera against the O4 serotype. In contrast to the wild-type parental strain, lipopolysaccharide (LPS) from this clone is devoid of rhamnose and does not cross react with O18 antisera. The recombinant plasmid from GH58, pGH58, was used to transform the rfb deletion strain HU1190. The resultant strain agglutinates in O4 antisera, but produces unpolymerized LPS. Escherichia coli K-12 strains HB101 and RC712 containing pGH58 produce polymerized LPS, indicating that the genetic background of the host can influence the LPS encoded by recombinant molecules. A cosmid, pGH84, has been identified which encompasses the entire pGH58 gene sequences and includes an additional 34 kilobases of DNA. HU1190 containing this cosmid agglutinates in O4 antisera and produces a polymerized LPS. By constructing several deletion subclones of pGH84, we have localized the genes necessary for polymerized LPS to a 5.5 kb ClaI-BamHI fragment. P1 transductants that make polymerized and unpolymerized O4 LPS have also been identified. PMID- 1721675 TI - [Positive and negative regulation of replication in hybrid cells]. AB - DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed. PMID- 1721676 TI - [Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies]. AB - To express HIV-1 reverse transcriptase in E. coli a number of genetic constructions containing reverse transcriptase and virus protease nucleotide sequences was obtained. The products of expression were characterized; monoclonal antibodies to reverse transcriptase were produced. The purification of reverse transcriptase was carried out. The substantial proteolysis of reverse transcriptase during purification was shown. The purified preparation is predominantly, an active protein with Mr 57 kDa. Some properties of this protein differed from the reverse transcriptase isolated from HIV. PMID- 1721678 TI - Regulatory disorders. I: Clinical perspectives. PMID- 1721677 TI - [Topography of bacteriophage T7 RNA polymerase using monoclonal antibodies]. AB - Four clones producing monoclonal antibodies inhibiting enzymatic activity were used to localize the functionally important antigenic determinants of T7 RNA polymerase. All antibodies were shown to bind to C-terminal fragment of the protein (residues 589-883). The competition studies showed the specificity of the three antibodies toward one epitope and the fourth antibody to another one. By means of limited cleavage of the RNA polymerase with cyanogen bromide with subsequent electrophoretic separation and immunoblotting the peptides containing antigenic determinants were localized. These are Met861-Ala883 for antibody 4H8 and Met750-Met832 for antibodies 9B2, 3H11 and 2A2. PMID- 1721679 TI - Regulatory disorders. II: Psychophysiologic perspectives. PMID- 1721680 TI - Stress protein synthesis by crayfish CNS tissue in vitro. AB - Some crustacean axons remain functional for months after injury. This unusual property may require stress proteins synthesized by those neurons or provided to them by glial cells. To begin to explore this hypothesis, we examined the conditions that stimulated stress protein synthesis by crayfish CNS tissue in vitro. Incubation for 1-15 h with arsenite or at temperatures about 15 degrees C higher than the acclimation temperature of 20 degrees C induced transient expression of several stress proteins. The heat stress response was blocked by Actinomycin D, suggesting that synthesis of new mRNA was required. In addition, the major crayfish 66 kD stress protein and its mRNA had sequence identities with the 70 kD stress proteins of mammals. Since the crayfish stress response has much in common with that of other organisms, the unique advantages of the crayfish nervous system can be used to study the impact of stress proteins on glial and neuronal function. PMID- 1721682 TI - Neurophysiological mechanisms of aphasia in epilepsy. PMID- 1721681 TI - Calmodulin blockers decrease short-term plasticity of the cholinoreceptors of neurons of the edible snail. AB - A reversible decrease in the rate and depth of the extinction of the reactions of the cholinoreceptive membrane to repeated iontophoretic applications of acetylcholine to the soma by a number of calmodulin blockers was demonstrated in identified RPa3 and LPa3 neurons in the edible snail using the method of recording transmembrane ionic currents: R 24571 (20-50 mumols/liter), trifluoperazine (50-200 mumols/liter), chlorpromazine (20-60 mumols/liter), and prenylamine lactate (30-400 mumols/liter). The results obtained attest to the positive regulation of short-term plasticity of the cholinoreceptors of the neurons in question by calmodulin. PMID- 1721683 TI - The organization of serotonergic projections to cerebral cortex in primates: retrograde transport studies. AB - Retrograde axonal transport and immunocytochemical methods were utilized to determine the origin of serotonergic afferents to selected primary projection and association areas of cerebral cortex in macaque monkeys. After injections of Fast Blue or Diamidino Yellow in primary motor, somatosensory, or visual cortex, retrogradely labeled neurons are found in both the dorsal and median raphe nuclei. The sets of dorsal raphe neurons which innervate these cortical areas differ in their spatial distributions along the rostrocaudal axis of the brainstem; a coarse rostrocaudal topographic relationship is found between these groups of dorsal raphe neurons and their cortical targets. In contrast, neurons in the median raphe which innervate these primary projection areas are not differentially distributed along the rostrocaudal axis. However, in both the median and dorsal raphe nuclei, most neurons projecting to primary visual cortex are situated lateral to the cells which project to motor and somatosensory areas; many of these visually projecting neurons lie among the fascicles of the medial longitudinal fasciculus. For comparison with the serotonergic innervation of primary projection areas, the locations of raphe cells projecting to three areas of association cortex were examined: dorsolateral prefrontal cortex, area 5 and area 7b. Neurons projecting to each of these association areas are found throughout the dorsal and median raphe nuclei. Their distributions are similar to one another; however, more cells projecting to dorsolateral prefrontal cortex are in the rostral part of the dorsal raphe. The dorsal and median raphe neurons projecting to these association areas are intermingled with neurons projecting to motor and somatosensory cortex, but are medial to most of those projecting to visual cortex. Thus, separate cortical areas are innervated by different sets of raphe neurons; these sets partially overlap, yet differ in their rostrocaudal and mediolateral distributions. Ascending serotonergic projections to cerebral cortex form a widely distributed system which exhibits a highly intricate anatomic organization. The present observations support the hypothesis that the dorsal raphe nucleus is comprised of distinct sets of neurons whose output is distributed to multiple, interconnected cortical areas; these serotonergic projections may play a role in the coordination of excitability in functionally related areas of cortex. In contrast, the serotonergic projections arising from the median raphe appear to be more divergent and are likely to have a global influence on cortical activity. Since these individual raphe nuclei have different projection patterns, they are likely to have distinct functional roles. PMID- 1721685 TI - The hypophysiotropic galanin system of the rat brain. AB - The external zone of the rat median eminence contains a large amount of galanin immunoreactive terminals indicating that galanin might function as a hypophysiotropic hormone. The possible sources of these galanin-containing nerve terminals were studied in the male and female rat by means of retrograde labeling in combination with fluorescence immunocytochemistry. Fluoro-Gold was used as retrograde tracer, and it was injected peripherally. Fluoro-Gold does not penetrate the blood-brain barrier, but it is taken up by nerve terminals which project to areas supplied by capillaries that lack the blood-brain barrier. The simultaneous detection of Fluoro-Gold taken up by nerve terminals in the median eminence and the endogenous galanin in thin paraffin sections has revealed that approximately 60% of the hypophysiotropic galanin cells are located in the arcuate nucleus. The remaining portion is located in the parvocellular subdivision of the paraventricular nucleus. Only scattered hypophysiotropic galanin cells are present in the medial preoptic area. PMID- 1721684 TI - PAC 1: an epitope associated with two novel glycoprotein components of isolated postsynaptic densities and a novel cytoskeleton-associated polypeptide. AB - A monoclonal antibody has been raised which recognizes an epitope, PAC 1 (postsynaptic density and cytoskeleton enriched), which is specifically associated with two novel glycoprotein components of forebrain postsynaptic density preparations and a novel neuronal cytoskeletal-associated polypeptide. The monoclonal antibody has been used to study the cellular and subcellular localization of these molecules and for the partial characterization of all three PAC 1 antigens in the rat. The PAC 1 epitope is present on two concanavalin A binding glycoproteins of apparent molecular weights 130,000 (pgp130) and 117,000 (pgp117). Both species are enriched in preparations of rat forebrain postsynaptic densities and to a lesser extent in synaptic membranes. The epitope is also expressed by a polypeptide of 155,000 mol. wt, cp155. This molecule is highly enriched in cytoskeleton rather than membrane preparations. Enzymic removal of N linked carbohydrate lowers the molecular weights of the PAC 1 glycoproteins pgp130 and pgp117 by 11,000 and 14,000 respectively, and suggests that cp155 is not glycosylated. Detergent, alkaline and salt extractions of postsynaptic densities and synaptic membranes indicate that pgp130 and pgp117 are integral membrane glycoproteins and are tightly bound components of postsynaptic density preparations. Immunocytochemical studies of adult rat forebrain show prominent staining of pyramidal cell dendrites and perikarya. There is no evidence of glial staining. Electron microscope studies show staining of microtubules together with punctate deposits of plasma membrane-associated reaction product. Several criteria have been used to show that pgp130 and pgp117 do not correspond to other known neuronal glycoproteins of similar molecular weight. We conclude that the PAC 1 epitope is expressed by two novel synaptic glycoproteins which are very probably integral components of the postsynaptic density and by a novel neuronal cytoskeleton-associated protein. PMID- 1721686 TI - Neurons with access to the general circulation in the central nervous system of the rat: a retrograde tracing study with fluoro-gold. AB - Central nervous system neurons which have access to the general circulation were identified by injecting the retrograde tracer Fluoro-Gold peripherally. Fluoro Gold does not penetrate the blood-brain barrier but is taken up by nerve terminals which project to areas supplied by fenestrated capillaries or to the periphery. Fluoro-Gold-accumulating neurons were present in the following regions or cell groups of the central nervous system: diagonal band of Broca; medial preoptic area; organum vasculosum of the lamina terminalis; subfornical organ; anterior periventricular area; paraventricular nucleus; arcuate nucleus; accessory magnocellular nuclei of the hypothalamus; motor neurons of cranial nerves III-VII, and IX-XII in the brainstem and spinal cord; autonomic ganglionic cells of cranial nerve III (Westphal-Edinger nucleus) in the mesencephalon and the intermediolateral column of the spinal cord; sensory ganglia of the cranial nerve V (mesencephalic trigeminal nucleus); and the C1-C2 and A2 adrenergic cell groups in the medulla. In addition, Fluoro-Gold-accumulating neurons were seen in the sensory ganglia of cranial and spinal nerves. Retrograde labeling with Fluoro Gold can be combined with immunocytochemistry to identify the chemical messengers within Fluoro-Gold-labeled perikarya. Although a large number of neurons are labeled in the central nervous system with Fluoro-Gold when it is administered peripherally, this technique in combination with immunocytochemistry can be a powerful tool to identify selected neuronal systems in the central nervous system. PMID- 1721687 TI - In vivo tonic inhibition of spinal substance P (-like material) release by endogenous opioid(s) acting at delta receptors. AB - Although numerous data support the existence of a presynaptic inhibitory control by opioids of substance P-containing primary afferent fibres entering the dorsal horn of the spinal cord, the exact nature of the opioid receptor involved in this control is still a matter of debate. In the present study, the potential role of delta opioid receptors was investigated by looking for the possible effects of selective delta ligands on the in vivo release of substance P-like material from the whole spinal cord in halothane-anaesthetized rats. Perfusion of the intrathecal space allowed the collection of substance P-like material that was released at a constant rate of approximately 0.65 pg substance P equivalents/min for at least 135 min. The addition of Tyr-D-Thr-Gly-Phe-Leu-Thr (10 microM) or dermenkephalin (10 microM), two selective delta agonists, to the perfusing fluid produced a marked reduction (-50-65%) in substance P-like material outflow which could be prevented by the selective delta antagonist naltrindole (10 microM) but not by naloxone (10 microM), which acts preferentially on mu opioid receptors. Furthermore, naltrindole alone (or the association of this antagonist plus dermenkephalin) enhanced the outflow of substance P-like material (+ 170%) as expected from the blockade of a tonic inhibitory control due to the stimulation of delta receptors by endogenous opioids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721688 TI - Microinjection of neuropeptide Y into the superficial dorsal horn reduces stimulus-evoked release of immunoreactive substance P in the anaesthetized cat. AB - In barbiturate anaesthetized spinal cats, antibody microprobes were used to measure release of immunoreactive substance P in the superficial dorsal horn following electrical stimulation of unmyelinated primary afferents of the ipsilateral tibial nerve. Prior microinjection of neuropeptide Y (0.2-0.6 microliters of 10(-5) mol/l solution) in the region of the substantia gelatinosa reduced the evoked release of immunoreactive substance P for up to 40 min. Microinjection of similar volumes of phosphate-buffered saline at similar sites was without effect. This action of neuropeptide Y could contribute to analgesia, particularly if this neuropeptide is co-released with noradrenaline from axon terminals in the superficial dorsal horn. PMID- 1721689 TI - Effects of neonatal sympathectomy and capsaicin treatment on bone remodeling in rats. AB - Bone metabolism may be influenced by the innervation of skeletal tissues. Neuropeptides such as vasoactive intestinal peptide, from sympathetic nerves, and calcitonin gene-related peptide, from sensory nerves, have been implicated as local modulators of bone metabolism. The effect of neonatal sympathectomy and of capsaicin-induced sensory denervation in rats was studied on the following: (i) the radial bone growth and apposition rate in tibiae (normal growth and modeling) and (ii) the percentage of periosteal surface of the mandible occupied by osteoclasts during induced remodeling. Neonate rats were treated with guanethidine, capsaicin, or appropriate vehicle. At seven weeks, maxillary molars were removed to induce remodeling on the buccal surface of the mandible. Animals were killed four days after surgery. Cross-sectional cortical area, medullary area, and periosteal apposition rate were measured by histomorphometry in ground sections of tibiae. The percentage of periosteal surface at the remodeling site occupied by osteoclasts (stained for acid phosphatase) was measured in frozen, undecalcified sections. There was no significant difference in cortical or medullary area or periosteal apposition rate in tibiae between each drug treatment and its control. However, the mandibular bone surface occupied by osteoclasts was increased 45.5% (P less than or equal to 0.005) in animals treated neonatally with guanethidine compared to controls. In contrast, the mandibular surface occupied by osteoclasts was decreased 21.2% (P less than or equal to 0.04) in animals treated neonatally with capsaicin compared to controls. The alteration of bone remodeling (osteoclast surface) by both treatments indicates that sensory and sympathetic nerves play a role in focal metabolism of bone. PMID- 1721690 TI - Persistence of fluoro-gold following degeneration of labeled motoneurons is due to phagocytosis by microglia and macrophages. AB - When the neural tracer Fluoro-Gold is used to retrogradely label a population of axotomized neurons, cellular labeling can persist in the axotomized nucleus even when Nissl staining indicates that the injured neurons have degenerated. In order to determine the identity of the labeled cells that remain, this study combines retrograde transport of Fluoro-Gold with immunocytochemical methods for identification of specific non-neuronal cell types following peripheral axotomy and Fluoro-Gold labeling of motoneurons in the dorsal motor nucleus of the vagus in neonatal and adult rats. Fourteen days following cervical vagotomy in neonatal rats, Nissl staining revealed a virtually complete loss of vagal motoneurons. Fourteen days after cervical vagotomy in adult rats, vagal motoneuronal loss was not yet extensive but chromatolysis had clearly begun. Injection of Fluoro-Gold into the vagus nerve just prior to the vagotomy led to Fluoro-Gold labeling of remaining vagal motoneurons. In addition, many other small, brightly labeled cells were present in the lesioned vagal nuclei of all rats. Immunofluorescent identification of astrocytes with anti-glial fibrillary acidic protein and microglia and macrophages with OX42 (anti-C3bi complement receptor) and ED1 (anti monocyte/macrophage cytoplasmic antigen) demonstrated that the small, bright Fluoro-Gold-labeled cells were non-neuronal, non-astrocytic phagocytes, including microglia. These results indicate that phagocytic microglia and other macrophages sequester Fluoro-Gold in the axotomized dorsal motor nucleus of the vagus of neonatal and adult rats, leading to persistence of fluorescent cellular labeling following the loss of retrogradely labeled axotomized neurons. PMID- 1721691 TI - The spino-latero-reticular system of the rat: projections from the superficial dorsal horn and structural characterization of marginal neurons involved. AB - The projections of the superficial dorsal horn to the lateral reticular nucleus of the medulla oblongata of the rat, and the morphological types of spinal cord lamina I neurons involved were studied after injecting the retrograde tracer cholera toxin subunit B in the caudal portion of the lateral reticular nucleus. Only injection sites located in the lateral part of the lateral reticular nucleus caused retrograde cell labelling in the superficial dorsal horn (laminae I-III). However, injection sites covering the lateral half of the lateral reticular nucleus and the region intermediate between its lateral border and the ventrocaudal tip of the trigeminal spinal nucleus also labelled cells in the neck of the dorsal horn. In contrast, injection sites confined to the intermediate region gave rise to an almost exclusive cell labelling in laminae I-III. Because the lateral part of the lateral reticular nucleus and the adjoining lateral region are rich in noradrenergic cells, it is suggested that these may be the specific targets of laminae I-III neurons. On the basis of the solid dendritic filling achieved, labelled lamina I cells were classified structurally. Most were fusiform cells (80%) and a minority pyramidal or flattened cells (10% each). Since fusiform cells also project selectively to the parabrachial nuclei, which together with the lateral reticular nucleus have been implicated in respiratory and cardiovascular reflexes, it is suggested that this cell type may convey nociceptive input originating autonomic responses. The pyramidal cells project also in large numbers to the mesencephalic periaqueductal gray which, like the lateral reticular nucleus, exerts descending inhibition on the dorsal horn nociceptive neurons. This suggests that this cell type may activate the spinal midbrain pain modulatory loops centred on both nuclei. PMID- 1721692 TI - Substance P- and calcitonin gene-related peptide immunoreactivity in primary afferent neurons of the cat's knee joint. AB - The distribution of substance P and calcitonin gene-related peptide was determined in primary afferent neurons of the medial and posterior articular nerve of the cat's knee joint. Perikarya of articular afferents were visualized by retrograde labelling with the fluorescent dye Fast Blue which was applied at the transected end of the peripheral nerves. Substance P was found in about 17% of labelled medial articular afferents and in about 16% of labelled posterior articular afferents, respectively, whereas calcitonin gene-related peptide was present in about 35 and 32% of the medial and posterior articular nerve cells, respectively. Taking into account that these neuropeptides are known to be co localized, probably not more than one-third of the joint afferents contain substance P and/or calcitonin gene-related peptide. Quantification of cell diameters revealed that substance P was found only in small- or intermediate sized perikarya (less than 50 microns) indicating that this peptide is predominantly found in unmyelinated neurons. Calcitonin gene-related peptide was present mainly in small- and intermediate- but also in some large-sized neurons (greater than 50 microns) providing evidence that this peptide is found in unmyelinated and to a lesser extent in myelinated neurons. This is consistent with previous studies that show that substance P and calcitonin gene-related peptide are present primarily in unmyelinated and thinly myelinated primary afferents. When the portion of substance P-positive neurons of the medial articular nerve is compared to the number of articular afferents displaying a nociceptive function as determined in earlier electrophysiological studies, it can be calculated that at most 30% of the nociceptive-specific articular afferents contain this neuropeptide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721694 TI - Glutamate immunoreactivity in the rat basilar pons: light and electron microscopy reveals labeled boutons and cells of origin of afferent projections. AB - Immunohistochemical methods that employed a polyclonal antiserum directed against a glutamate-hemocyanin conjugate were utilized to examine the rat basilar pontine nuclei at both light and electron microscopic levels in order to identify putative glutamatergic neural elements. A large number of cells ranging in size from 11 to 32 microns in diameter and present in all subdivisions and at all rostrocaudal levels of the basilar pons exhibited intense glutamate immunoreactivity. Immunoreactive punctate structures, confirmed by electron microscopy to be axon terminals, were homogeneously distributed throughout the pontine neuropil, although a somewhat greater accumulation was apparent medially at mid-levels of the basilar pons and laterally at more caudal levels. Immunolabeled axons were also present throughout the pontine nuclei. In order to demonstrate possible extrinsic sources of glutamate-immunoreactive axon terminals within the pontine gray, injections of wheat germ agglutinin-horseradish peroxidase were made directly into the basilar pons. Tissue was then evaluated for the presence of retrogradely transported wheat germ agglutinin-horseradish peroxidase and the same tissue sections processed for glutamate immunocytochemistry. Following this combined protocol, neuronal somata exhibiting both wheat germ agglutinin-horseradish peroxidase and glutamate immunoperoxidase reaction products were observed within layer Vb of the cerebral cortex, zona incerta, the dentate nucleus of the cerebellum, nucleus paragigantocellularis of the medullary reticular formation, and the dorsal column nuclei. Such double labeled cells were considered to represent glutamatergic neurons that provide axonal projections to the basilar pons. Ultrastructural studies of the pontine nuclei confirmed the presence of glutamate immunogold labeling in dendrites, neuronal somata, axons, and axon terminals. Immunoreactive boutons contained round vesicles and primarily formed asymmetric synapses at various postsynaptic loci which included glutamate-immunolabeled dendritic profiles and somata. These results suggest that glutamatergic basilar pontine neurons form one segment of a multisynaptic pathway involving glutamatergic afferents to the basilar pons, glutamatergic pontocerebellar projection neurons, and the glutamatergic granule cells of the cerebellar cortex. PMID- 1721693 TI - Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine. AB - It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry. PMID- 1721695 TI - [Nature of the reaction of the immunocompetent cells in children with nephrotic syndrome and in their parents with different HLA phenotypes]. AB - The purpose of the study was to analyze the character of the expression of the blood lymphocyte epitopes SD4, SD8 (EBLE SD4, SD8) in a series of the loading in vitro tests in children suffering from the nephrotic syndrome, with different HLA haplotypes. Nine children with the hormone-sensitive nephrotic syndrome (HSNS) and hormone-resistant nephrotic syndrome (HRNS) and 11 parents were examined. Before and after the in-vitro loading with medicamentous agents EBLE SD4, SD8 were determined by flow cytofluorometry, while HLA antigens were tested by the standard micro-lymphocytotoxic method. The studies allowed revealing differences in the responses of EBLE SD8 to the in-vitro loading in children with the HRNS and HSNS. The character of EBLE SD4, SD8 in a child with the NS and its parents may attest to the involvement of those antigens in the pathogenetic component of the given disease. PMID- 1721696 TI - [Study of serum levels of alpha 1-antitrypsin and alpha macroglobulins in children with glomerulonephritis]. AB - To define the clinico-pathogenetic importance of alpha 1-inhibitor of proteinases and alpha 2-macroglobulin of the blood in children with glomerulonephritis, a study was made of the phenotype of alpha 1-inhibitor of proteinases and its concentration in the blood serum of 156 patients with different clinical forms of glomerulonephritis. Overall 1290 practically healthy children were examined as control. The patients suffering from glomerulonephritis did not demonstrate phenotypes responsible for acute deficiency of alpha 1-inhibitor of proteinases (PISS, PISZ). A relationship was established between the amount of alpha 1 inhibitor of proteinases in the blood serum in children with different clinical forms of glomerulonephritis: the patients with the nephrotic form manifested a significant decrease of the inhibitor concentration in the blood serum, whereas in the hematuric form, a significant rise of it was recorded. All the patients suffering from glomerulonephritis showed a significant increase of the content of alpha 2-macroglobulin, particularly in the nephrotic form, which is likely to be determined by the enhanced output of the given protein and its negligible loss with urine in connection with a high molecular weight. PMID- 1721697 TI - [Value of extracorporeal radioimmunoassay in children with nephrotic syndrome]. AB - Extracorporeal radioimmune techniques were employed to study gonadotropic and sex hormones renin and beta 2-microglobulin in patients with nephrotic glomerulonephritis. The hypophyseal and gonadal system showed alterations which were dependent on the disease gravity. The level of renin and beta 2 microglobulin correlated first of all with renal function. PMID- 1721698 TI - [Assessment of the reparative capacity of the small intestine in celiac disease in children according to the DNA and RNA contents in enterocyte nuclei]. AB - In 44 children suffering from celiac disease, cytophotometry was used to measure the content of DNA and RNA in enterocyte nuclei in the jejunum mucosa. The measurements were made after the diagnosis was established, 6-8 months and 1-1.5 years after administration of the agliadin diet. In celiac disease children, there was an increase of the content of DNA and RNA in enterocyte nuclei of the intestinal villi and cryptae and lack of gradient in the distribution of the amount of nucleic acids along the length of the villi, which characterized the hyper-regeneration type of small intestinal mucosa atrophy. The recovery of the crypta/villus gradient accompanied by the normalization of the content of nucleic acids, comparable with the analogous indicators of the unchanged mucosa of the small intestine, allows regarding the measurements of the content of DNA and RNA in the nuclei as an index of reparative capacity of enterocytes in children afflicted with celiac disease. PMID- 1721699 TI - Identity determinants of E. coli tryptophan tRNA. AB - The first base pair of the acceptor stem A1-U72 and the discriminator base G73, as well as the anticodon nucleotides, characterize the tryptophan tRNA in E. coli. To determine the contribution of these nucleotides to the tryptophan acceptor activity, various transcripts of E. coli tryptophan tRNA mutants were constructed. Substitutions of the discriminator base G73, which is conserved within prokaryotic tryptophan tRNAs, impaired aminoacylation with tryptophan. Substitutions of other purine-pyrimidine pairs for A1-U72 revealed that only U72 weakly contributed to recognition by tryptophanyl-tRNA synthetase. The E. coli aspartic acid tRNA transcript introducing the tryptophan anticodon CCA showed almost the same tryptophan charging activity as the tryptophan tRNA transcript possessing a G1-C72 base pair. Only a low activity was detected in the mutant tryptophan tRNA transcript possessing a set of G1-C72 and A73, which is observed in eukaryotic tryptophan tRNAs. These results indicate that the anticodon and G73 are major identity determinants of tryptophan tRNA in E. coli, whereas the A1-U72 base pair is only a weak recognition element. PMID- 1721700 TI - Cloning, characterization and evolution of the BsuFI restriction endonuclease gene of Bacillus subtilis and purification of the enzyme. AB - The restriction endonuclease (R.BsuFI) of Bacillus subtilis recognizes the target DNA sequence 5' CCGG. The R.BsuFI gene was found in close proximity to the cognate M.BsuFI gene, which had previously been characterized (1). Cloning of the R.BsuFI gene in E.coli was only possible with the M.BsuFI Mtase gene present on a compatible plasmid. The cloned R.BsuFI gene was expressed in E. coli and restriction activity was observed in vivo and in vitro. The R.BsuFI gene consists of 1185 bp, coding for a protein of 395 amino acids with a calculated molecular weight of 45.6 kD. The R.BsuFI enzyme was purified to homogeneity following overexpression. It presumably works as a dimer and cleaves the 5' CCGG target sequence between the two cytosines to produce sticky ends with 5' CG overhangs, like the isoschizomers R.MspI and R.HpaII. The relatedness between R.BsuFI and R.MspI is reflected by significant similarities of the amino acid sequences of both enzymes. This is the first case where such similarities have been observed between isoschizomeric restriction endonucleases which belong to 5mC specific R/M systems. This observation suggests that R.BsuFI and R.MspI genes derive from a common ancestor. In spite of such functional and evolutionary relatedness, the R/M systems differ in the arrangement of their R and M genes. In the BsuFI system transcription of the two genes is convergent, whereas divergent transcription occurs in the MspI system. PMID- 1721701 TI - Diversity of a ribonucleoprotein family in tobacco chloroplasts: two new chloroplast ribonucleoproteins and a phylogenetic tree of ten chloroplast RNA binding domains. AB - Two new ribonucleoproteins (RNPs) have been identified from a tobacco chloroplast lysate. These two proteins (cp29A and cp29B) are nuclear-encoded and have a less affinity to single-stranded DNA as compared with three other chloroplast RNPs (cp28, cp31 and cp33) previously isolated. DNA sequencing revealed that both contain two consensus sequence-type homologous RNA-binding domains (CS-RBDs) and a very acidic amino-terminal domain but shorter than that of cp28, cp31 and cp33. Comparison of cp29A and cp29B showed a 19 amino acid insertion in the region separating the two CS-RBDs in cp29B. This insertion results in three tandem repeats of a glycine-rich sequence of 10 amino acids, which is a novel feature in RNPs. The two proteins are encoded by different single nuclear genes and no alternatively spliced transcripts could be identified. We constructed a phylogenetic tree for the ten chloroplast CS-RBDs. These results suggest that there is a sizable RNP family in chloroplasts and the diversity was mainly generated through a series of gene duplications rather than through alternative pre-mRNA splicing. The gene for cp29B contains three introns. The first and second introns interrupt the first CS-RBD and the third intron does the second CS RBD. The position of the first intron site is the same as that in the human hnRNP A1 protein gene. PMID- 1721702 TI - A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution. AB - The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts. PMID- 1721703 TI - Use of single-stranded DNA oligonucleotides in programming ribosomes for translation. AB - Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation. These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin. While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG. However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent. Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA. At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF. We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination. Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis. PMID- 1721704 TI - In vitro self-splicing reactions of the chloroplast group I intron Cr.LSU from Chlamydomonas reinhardtii and in vivo manipulation via gene-replacement. AB - The group I intron from the chloroplast rRNA large subunit of Chlamydomonas reinhardtii (Cr.LSU) undergoes autocatalytic splicing in vitro. Cr.LSU displays a range of reactions typical of other group I introns. Under optimal conditions, the 5' cleavage step proceeds rapidly, but the exon-ligation step is relatively slow, and no pH dependent hydrolysis of the 3' splice site occurs. A requirement for high temperature and high [Mg2+] suggests involvement of additional splicing factors in vivo. The positions of three cyclization sites of the free intron have been mapped; two of these sites represent reactions analogous to 5'-splice site cleavage, whereas the third is an example of G-exchange. Cr.LSU contains an open reading frame (ORF) potentially encoding an 163 amino acid polypeptide. ORF function has been investigated by using chloroplast gene replacement via particle bombardment. We have shown that the ORF can be deleted from Cr.LSU without affecting splicing in vivo and it thus does not encode an essential splicing factor. PMID- 1721705 TI - An STS in a 5' untranslated exon of the human acidic fibroblast growth factor (aFGF) gene. PMID- 1721706 TI - A new DNA marker, D3S740, identifies a MspI polymorphism on chromosome 3p. PMID- 1721707 TI - A new DNA marker, D3S742, identifies a MspI and a TaqI polymorphism on chromosome 3p. PMID- 1721708 TI - A new DNA marker, D3S743, identifies a MspI polymorphism on chromosome 3p. PMID- 1721709 TI - [Penal bone of American mink (Mustela vison Brisson, 1756)]. AB - 30 male minks were examined anatomically, histologically, and radiologically. It was found that penis bone constituted the always-present part of their penis. It was shown, on the grounds of both the X-ray and histological examinations, that there is the connective tissue basis for osteogenesis of penis bone closer ending. PMID- 1721710 TI - Localization of endogenous beta-galactoside-specific lectins by neoglycoproteins, lectin-binding tissue glycoproteins and antibodies and of accessible lectin specific ligands by mammalian lectin in human breast carcinomas. AB - Protein-carbohydrate interactions constitute a system of molecular interaction with relevance to pathologic conditions. Carrier-immobilized carbohydrate structures enable the histochemical investigation of the protein part of this recognitive system. However, thorough systematic studies are inevitably required for standardized application of this relatively novel class of markers. Consequently, serial sections of 21 cases of malignant breast lesion were comparatively analyzed with three different types of probe, specific for beta galactoside-binding lectins. In addition to the chemically lactosylated neoglycoprotein, human lectin-binding glycoproteins, purified by affinity chromatography on resins with an immobilized beta-galactoside-specific lectin, and a lectin-specific antibody were employed to answer the question whether differences occur in their capacity for lectin localization. The patterns of staining were qualitatively similar, the lectin-binding glycoproteins yielding the most intense reaction. Having assured the reliable applicability of the neoglycoprotein, structural alterations of the subterminal carbohydrate residue on the labelled carrier addressed the issue, whether selectivity of binding can be inferred histochemically, allowing rational synthetic tailoring. An N acetylglucosamine residue in beta-1,3-linkage proved to be a less favorable extension than this type of sugar in beta-1,4-linkage or an N-acetylgalactosamine moiety in beta-1,3-linkage. Binding was clearly reduced in cells of normal breast tissue with this probe. In order to gain evidence on the expression of potential carbohydrate ligands for the glyco- and immunohistochemically localized binding activity, a labelled mammalian beta-galactoside-specific lectin was similarly used as histochemical tool. It effectively bound to accessible sites in the sections. The binding pattern was different to that of plant lectins with specificity to beta-galactosides. This result underscores that caution is necessary in the functional interpretation of results of studies with plant, not mammalian lectins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721711 TI - Melioidosis in a patient from Bangladesh. AB - A 54 year old Bangladeshi man presented with a history and chest X-ray appearances suggestive of pulmonary tuberculosis. Following deterioration 4 weeks later, he required ventilation. Although a blood culture isolate was subsequently found to be Pseudomonas pseudomallei, it was initially misidentified and dismissed as a contaminant. Further cultures demonstrated the organism, but the patient died, despite treatment with ceftazidime. The case illustrates the importance of taking a detailed travel history and having a high index of suspicion in patients from South East Asia and the Indian sub-continent, including Bangladesh, where the disease has not previously been considered endemic. PMID- 1721713 TI - Estimating the risk of a fetal autosomal trisomy at mid-trimester using maternal serum alpha-fetoprotein and age: a retrospective study of 142 pregnancies. AB - Risks appropriate for mid-trimester prenatal screening for autosomal trisomies have been estimated from a combination of maternal age and maternal serum (MS) alpha-fetoprotein (AFP) levels at 16-20 weeks gestation. Published data on the frequency of Down's syndrome births relative to maternal age were modified to include the additional age-related frequency of trisomy 18 and trisomy 13 cases to provide an overall risk for an autosomal trisomy at mid-trimester. MSAFP results from a retrospective study of 142 affected (114 trisomy 21, 19 trisomy 18, and 9 trisomy 13) and 113,000 unaffected pregnancies were converted to multiples of the appropriate gestational median (MOM). The AFP levels in the autosomal trisomy pregnancies were found to be significantly reduced at 0.72 MOM of the unaffected pregnancies. Risks (likelihood ratios) were derived from the overlapping log Gaussian distributions for affected and unaffected pregnancies and combined with maternal age risks to give the overall odds of an affected pregnancy. A mid-trimester cut-off risk of 1:280 gave an estimated 37 per cent detection rate for autosomal trisomies in the west of Scotland population for a follow-up (false-positive) rate of 6.6 per cent. These figures compare with a 30 per cent detection and 6.7 per cent false-positive rate if age 35 years and over is used as the sole criterion for selection of at-risk pregnancies. PMID- 1721712 TI - Alpha-fetoprotein and acetylcholinesterase are not predictive of fetal junctional epidermolysis bullosa, Herlitz variant. AB - Junctional epidermolysis bullosa, Herlitz variant (junctional EB-Herlitz) is a lethal autosomal recessive skin disorder currently amenable to prenatal diagnosis only by direct analysis of fetal skin. However, elevated levels of alpha fetoprotein, as well as the presence of acetylcholinesterase in amniotic fluid, have been associated with other severe fetal genodermatoses. Fetal skin samplings were performed in ten pregnancies at risk for fetal junctional EB-Herlitz, with three fetuses affected on the basis of electron microscopic detection of blisters within the lamina lucida and abnormal hemidesmosomes. In neither affected nor unaffected pregnancies were maternal serum or amniotic fluid alpha-fetoprotein levels elevated. Moreover, alpha-fetoprotein levels in both maternal serum and amniotic fluid were not statistically different comparing affected and unaffected fetuses. Acetylcholinesterase was not present in the amniotic fluid samples of the three affected pregnancies. Unlike other severe fetal genodermatoses, neither alpha-fetoprotein nor acetylcholinesterase was predictive of junctional EB Herlitz. PMID- 1721714 TI - Isoelectric focusing pattern of human amniotic fluid alpha-fetoprotein. AB - Isoelectric focusing (IEF) of amniotic fluid alpha-fetoprotein (AFP) in thin layer polyacrylamide gels containing 8 M urea followed by immunoblotting reveals at least nine bands, band I lying next to the cathode. Compared with 298 amniotic fluid samples from normal pregnancies, we found that the density of band V was increased in seven cases of fetal death. In 16 amniotic fluid samples from pregnancies with open neural tube defects (ONTD), band V disappeared or was markedly decreased. In seven cases with elevated AFP and positive acetylcholinesterase (AChE) due to contamination with fetal blood, no difference in pattern was observed compared with samples from normal pregnancies. It is suggested that IEF of AFP and subsequent immunoblotting are an apparently diagnostic test for ONTD and intrauterine fetal death (IUFD). PMID- 1721716 TI - [Hereditary sensory nerve degenerations. Criticisms of classifications]. PMID- 1721715 TI - First-trimester maternal serum alpha-fetoprotein and chorionic gonadotropin in aneuploid pregnancies. AB - First-trimester maternal serum alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) levels were measured in samples from 29 women with cytogenetically abnormal pregnancies and 145 women with cytogenetically normal pregnancies matched for gestational age, race, and sample storage time. All patients had a risk of fetal aneuploidy greater than or equal to that of a mother 35 years of age. AFP was significantly lower in samples from pregnancies affected with trisomy 21 (0.67 MoM; p less than 0.05), while HCG values were no different from those of matched controls. Trisomies 13 and 18 could not be distinguished from matched controls by AFP. However, levels of HCG were significantly lower in such pregnancy samples, with median values of 0.65 MoM in trisomy 13 and 0.32 MoM in trisomy 18 (p less than 0.05). Variations in AFP and HCG levels suggest that expressed differences between autosomal aneuploidies include differences in fetal and placental protein production in the first trimester. PMID- 1721717 TI - [Treatment of relapses and failures in disseminated Hodgkin's disease]. AB - Treatment of disseminated Hodgkin's disease still fails in about 50 percent of the cases. The prognosis is poorer in case of early relapse or when the disease is refractory to first-line therapy from the start. Second- or third-line chemotherapy regimens have given rather disappointing results with a complete remission rate usually around 30 percent and a small number of cures. The indications for radiotherapy in localized lymph node relapses remain to be precisely determined. Based on the theoretical dose-effect concept, high-dose chemotherapy followed by autologous bone marrow transplantation increases the number of prolonged complete remissions in patients who respond to salvage chemotherapy. A wider use of haematopoietic growth factors should reduce the toxicity of this treatment. PMID- 1721718 TI - [Neurobiology of tianeptine. A new pharmaceutic agent]. AB - In ex vivo experiments, tianeptine increased serotonin uptake in the hippocampus and the cortex acutely and after 72 hours following chronic administration for 15 days. This effect results from an increased maximal rate of uptake without changes in the number or affinity to binding sites for I'3H-imipramine or I'3H paroxetin. In addition, tianeptine increased extracellular levels of 5 hydroxyindolacetic acid (5-HIAA) in hippocampus and hypothalamus measured with in vivo voltametry. It can thus be concluded that tianeptine also raises 5 hydrotryptamine (5HT) uptake in vivo. The effects of tianeptine on the serotoninergic system, especially the increase in serotonin uptake, are discussed in relation with its effects on behaviour and the dopaminergic and cholinergic systems. PMID- 1721719 TI - Biochemical pharmacology and analysis of fluoropyrimidines alone and in combination with modulators. AB - After more than three decades since their introduction, fluoropyrimidines, especially FUra, are still a mainstay in the treatment of various solid malignancies. The antitumor effects of fluoropyrimidines are dependent upon metabolic activation. FdUMP, FUTP and FdUTP were identified as the key cytotoxic metabolites that interfere with the proper function of thymidylate synthase and nucleic acids. The relevance of these metabolites is cell-type specific. Recently, fluorouridine diphospho sugars have been detected, but the precise function of this class of metabolites is currently unknown. In mammalian systems fluoropyrimidines and their natural counterparts share the same metabolic pathways since the substrate properties in enzyme-catalyzed reactions are frequently comparable. Ongoing studies indicate that the metabolism and action of fluoropyrimidines exhibit circadian rhythms, which appear to be due to variations in the activity of metabolizing enzymes. Essential for the expanding knowledge of the pathways and effects of fluoropyrimidines has been the constant improvement of analytical methods. These include ligand binding techniques, numerous dedicated HPLC systems and 19F-NMR. Because the overall response rates achieved with fluoropyrimidines are modest, strategies based on biochemical modulation have been devised to enhance their therapeutic index. Biochemical modulators include a wide range of various compounds with different modes of action. In recently completed clinical trials, combinations of FUra with leucovorin, a precursor for 5,10-methylene tetrahydrofolate, or with levamisole, an anthelminthic with immunomodulatory activity, appeared to be superior to FUra alone. At the preclinical level combinations of fluoropyrimidines with, e.g. interferons or L-histidinol were demonstrated to be interesting candidates for further testing. The future therapeutic utility of fluoropyrimidines will depend on both the improvement of combination regimens currently used in the treatment of cancer patients and the judicious clinical implementation of promising experimental modulation strategies. Moreover, novel fluoropyrimidines with superior pharmacological properties may become important as part of or instead of modulation approaches. PMID- 1721720 TI - Maintenance of pBR322-derived plasmids without functional RNAI. AB - pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers. PMID- 1721721 TI - Analysis of the ternary complex of antigen, MHC and T-cell receptor: the influence of the T-cell receptor V beta repertoire on the V beta gene element usage. PMID- 1721722 TI - Detection of P-glycoprotein with JSB-1 monoclonal antibody in B-5 fixed and paraffin-embedded cell lines and tissues. AB - We analyzed the expression of P-glycoprotein (Pgp) by immunohistochemistry using JSB-1 monoclonal antibody (MAb) on paraffin-embedded sections of the multi-drug resistant (MDR) (CHrC5 and CEM-VLB), and sensitive (AuxB1 and CEM) cell lines, and also in normal kidney, colon, adrenal and in kidney and colon carcinomas. After comparing the sensitivity of three different immunohistochemical techniques the peroxidase-antiperoxidase method was found to be the best. We then tested six different fixation methods. The MDR cell lines and human tissues demonstrated the strongest staining with B-5 fixative. Both MDR cell lines, but not the tissues fixed in 1% paraformaldehyde and Zamboni's fixative demonstrated weak staining. No immuno- reactivity could be detected in MDR cell lines and tissues fixed in 10% buffered or nonbuffered formalin or by the AMeX method of tissue processing. The present study clearly shows that the type of fixative is critical for the preservation of Pgp epitope recognized by JSB-1 MAb, and that B-5 fixative is expected to be equally applicable for the detection of Pgp in normal and neoplastic tissues. PMID- 1721723 TI - Biochemical and biophysical analysis of cell-to-cell channels and regulation of gap junctional permeability. PMID- 1721724 TI - An intracrevicular washing method for collection of crevicular contents. AB - Gingival crevicular contents provide a potential source of markers of the destruction of periodontal structures and the disease activity. This communication introduces a new device designed for efficient collection of samples from the critical area of initial tissue break-up at the bottom of a sulcus or a periodontal pocket. The instrument is characterized by two injection needles fitted one within the other so that during sampling the thinner "ejection needle" is at the bottom of the pocket and the "collection needle" at the gingival margin. The washing solution is manually ejected into the crevice and immediately drained through the collection needle into a sample tube by continuous suction. The technique developed provides an easy and useful method for studies of qualitative differences in the crevicular cells and in the chemical components of the crevicular fluid in various clinical situations. Furthermore, the use of the technique can be extended for localized lavage of acute periodontal pockets with appropriate therapeutic solutions. PMID- 1721725 TI - Exposure of commuters to volatile aromatic hydrocarbons from petrol exhaust. AB - Twenty-two volatile aromatic hydrocarbons were determined in the air of an automobile during commuting. Sampling was made on Tenax cartridges and laboratory determinations were carried out using thermal desorption combined with temperature-programmed capillary gas chromatography. Selected hydrocarbons representative of petrol exhaust were determined in the automobile and in an electric commuter train during eight parallel commuter trips. In the automobile, the concentrations of benzene were 35-70 micrograms/m3 and those of total aromatic hydrocarbons 200-400 micrograms/m3. The petrol exhaust levels were 5-10 times higher in the automobile than in the compartment of the commuter train. PMID- 1721726 TI - Prophylaxis of postoperative thromboembolism with combined methods. PMID- 1721727 TI - [Intraprostatic prosthesis: echographic monitoring]. AB - 22 patients affected by BPH have been treated with a spiral urethral prosthesis under local anesthesia with ultrasonic guidance and endoscopic technique. All the patients either refused the surgical procedure or had high operative risk. Insertion guided by ultrasound and endoscopic technique was successful in 19 patients (86%) with a follow-up of 12 months. In our experience the insertion of this prosthesis is a favorable alternative to an indwelling catheter in selected patients. PMID- 1721728 TI - [Transrectal echography in the diagnosis of non-palpable prostatic carcinoma]. AB - 285 affected by BPH have been evaluated by transrectal ultrasound. All the patients had a palpably normal prostate without abnormality suggestive of cancer. 56 patients (19.6%) presented ultrasonic abnormality areas. Under ultrasound guidance, biopsy was done by the transperineal route and biopsy material showed the presence of prostatic carcinoma in 24 patients (8.3%). 6 of these had neoplasm in the non peripheral zone. The study suggests the validity of ultrasound, in particularly transrectal ultrasound, in the identification of non palpable prostatic cancer. In conclusion ultrasound appears very useful in the preoperative evaluation of the patients affected by BPH in order to identify neoplasms non detectable with other methods. PMID- 1721729 TI - [Transrectal echographic micturitional evaluation of bladder neck pathology]. AB - Dynamic transrectal echography is the primary method to evaluate bladder neck diseases during micturition for its safety, noninvasiveness and excellent imaging. In order to find some features for every kind of disease, we have examined 32 patients with BPH, Carcinoma, Marion disease and also evaluated the morphological images after prostate surgery. Enlargement of the prostate gland may restrict the urethral lumen and deviate its intra-prostate way, such that the urethra can't be allocated in one sagittal echographic plane. The third lobe, when it exists, can act like a valve against the urine flow. Prostate carcinoma, at first, can be asymptomatic on urodynamic study. Urethral invasion and its organic stenosis is shown by a very thin, longer and irregular profiles. Bladder neck sclerosis is an unusual disease, but its diagnosis can be easy if a posterior "beak" at internal meatus level, urethral dilatation after stenosis and positive Stop-Test exist. After surgery, one can observe full relaxation of the internal sphincter and the voluntary skeletal sphincter, located at the level of the membranous urethra, providing itself a continence mechanism. PMID- 1721730 TI - Implications of placebo theory for clinical research and practice in pain management. AB - We review three possible theoretical mechanisms for the placebo effect: conditioning, expectancy and endogenous opiates and consider the implications of the first two for clinical research and practice in the area of pain management. Methodological issues in the use of placebos as controls are discussed and include subtractive versus additive expectancy effects, no treatment controls, active placebo controls, the balanced placebo design, between- versus within group designs, triple blind methodology and the double expectancy design. Therapeutically, the possibility of shaping negative placebo responses through placebo sag, overservicing and the use of placebos on their own are explored. Suggestions for using conditioned placebos strategically in conjunction with nonplacebos are made and ways of maximizing the placebo component of nonplacebo treatments are examined. Finally, the importance of investigating the placebo effect in its own right is advocated in order to better understand the long neglected psychological aspects of the therapeutic transaction. PMID- 1721731 TI - Mechanism of ethanol-induced aggregation in whole blood. AB - Effects of ethanol on blood clotting and platelet aggregation have been reported in many models, but its in vitro actions in whole blood, impedance aggregometry have not been reported. We investigated the effect of ethanol in vitro in whole blood and platelet rich plasma of humans and rats, as measured by impedance aggregometry. Ethanol (34 to 170 mM) induced concentration-dependent aggregation in whole blood but not platelet rich plasma. In further studies in rats, aggregation was inhibited by pretreatment of whole blood with the prostacyclin analog iloprost or the enzyme apyrase, which degrades ADP to AMP. Levels of ethanol which produced aggregation in whole blood were also associated with concentration-dependent hemolysis. Based on the requirement for whole blood for ethanol-induced aggregation, the inhibitory effect of apyrase and our observation of hemolysis, and previous studies which have demonstrated the potential contribution of ADP from lysed red blood cells to platelet aggregation, we conclude that ethanol-induced aggregation in whole blood is mediated by erythrocyte lysis and the ADP released from these cells. PMID- 1721732 TI - Cocaine immunotoxicity: abnormal cytokine production in Hispanic drug users. AB - Peripheral blood lymphocytes from 47 Hispanic poly-drug users with a history of cocaine abuse were analyzed for in vitro production of interleukin-1 (IL-1), interleukin-2 (IL-2), gamma-interferon (IFN) and plasma levels of soluble IL-2 receptor (SIL-2R). Cocaine use was confirmed and quantified by analysis of hair and urine samples, and subjects were grouped into 3 based on the extent of cocaine metabolites detected. No significant differences in IL-1 and IFN production were seen between the 3 groups. However, subjects with higher levels of cocaine in hair also showed higher levels of IL-2. In addition, a positive correlation was seen between cocaine concentrations and IL-2 levels. A corresponding negative correlation was seen between cocaine levels and levels of plasma SIL-2R. These findings suggest modulation of the IL-2 network by cocaine in poly-drug users. PMID- 1721733 TI - Neural transplantation--an experimental tool with clinical possibilities. PMID- 1721734 TI - Transplantation of glial cells into the CNS. AB - Glial cell transplantation into the CNS offers an experimental approach to help us unravel the complex interactions that occur between CNS glia, Schwann cells and axons during repair and development. This article reviews recent advances that have been made in our understanding of the nature and potential of CNS repair using this approach, and introduces the idea of using transplantation to address broader issues in glial biology. PMID- 1721735 TI - Genetically modified cells: applications for intracerebral grafting. AB - Grafting cells to the CNS is a useful approach to address fundamental and clinical issues in neurobiology. Recently, a hybrid technique - the genetic modification of cells followed by intracerebral implantation - has emerged, which may potentially enhance the power of CNS grafting. However, several methodological considerations need to be addressed to test the reliability of this new approach. Progress in the gene transfer-grafting technique has implications for expanding the range of issues and problems that may be addressed in both the basic science and clinical arenas. PMID- 1721736 TI - The neuropathology of Alzheimer's disease investigated by transplantation of mouse trisomy 16 hippocampal tissues. AB - This article evaluates the novel application of neural transplantation as a model for studying the neuropathological events associated with Alzheimer's disease and those that have subsequently also been observed in Trisomy 21 (Down syndrome). PMID- 1721737 TI - Identifying and manipulating neuronal stem cells. AB - Fetal brain tissue has been shown to have clear behavioral effects when transplanted into adult lesioned brains. These results have focused attention on the cell types of the embryonic brain. Transplantation experiments using primary cells are beginning to define the plasticity of these cells and the times when they become committed to specific neuronal fates. Growth factors have been defined that regulate the proliferation of these cells in culture. Cell lines have been established that express stem cell properties and that are capable of differentiation when implanted into the developing brain. In this article we review this work on mammalian neuroepithelial stem cells and discuss how these studies might contribute to the therapeutic use of brain transplants. PMID- 1721738 TI - The immune response to intracerebral neural grafts. AB - Neural transplantation offers a potential therapeutic approach to a variety of neurological disorders, most notably those of a degenerative nature. However, the degree of immunological privilege (i.e. isolation from an immune response) in the brain, which is not absolute, may be a significant impediment to the survival of histoincompatible grafts. The nature of this privilege, together with the specific immune events leading to neural graft rejection, are discussed. As a consequence of this immune-mediated rejection, immunosuppression in some form might be necessary to guarantee long-term graft survival. Various strategies are being explored to suppress the immune response to neural grafts, not only for future use in clinical therapies, but also to bring intracerebral allo- and xenotransplantation to the attention of the general neurobiologist. PMID- 1721739 TI - Transplantation to the diseased and damaged retina. AB - Retinas of Royal College of Surgeons (RCS) dystrophic rats undergo a dramatic loss of photoreceptor cells as a result of defective retinal pigment epithelial (RPE) cells. These retinas are therefore a valuable model in the investigation of the role of the RPE on photoreceptor-cell survival and development. Also, rat retinas damaged by excessive light serve as a suitable environment to study survival of transplanted photoreceptor cells. Even though photoreceptor cells are lost in these retinas, a normal inner retinal structure is retained. Both models have recently been used in successful RPE-cell and/or photoreceptor-cell transplantation studies designed to replace defective or lost cells due to retinal disease or damage. These new approaches in the field of retinal transplantation offer unique and novel opportunities for the development of possible therapeutic strategies in human eye disease, and for improving our understanding of the normal relationships between retinal cells. PMID- 1721740 TI - The reconstruction of cerebellar circuits. AB - Repair of adult 'point-to-point' systems by neural grafting is possible only when grafted neurons succeed in synaptically replacing the host's missing neurons, thus re-establishing the anatomical and functional integrity of the impaired circuits. Grafting experiments carried out on the cerebellum of the adult pcd (Purkinje-cell-degeneration) mutant mouse (an animal model of hereditary degenerative ataxia) reveal that embryonic Purkinje cells, by some unknown sorting mechanism, selectively invade the deprived cerebellar cortex. These neurons migrate to their proper domains and, inducing axonal sprouting of specific populations of host neurons, they become integrated synaptically within the pcd cerebellar cortex. However, the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit. PMID- 1721741 TI - Transplants of embryonic motoneurones to adult spinal cord: survival and innervation abilities. AB - One goal of transplantation experiments involving damaged spinal cords is to reconstruct a functional innervation to muscles in the periphery. Embryonic spinal cord grafts have been shown to survive transplantation into adult spinal cord lacking motoneurones. Motoneurones from the graft appear to be able to innervate muscle tissue by being encouraged to grow across a bridge of peripheral nerve. Integration of grafted motoneurones appears to involve their migration from the graft into the host ventral horn, thus replacing depleted host neurones. These results suggest possible strategies of research that might lead to treatments of spinal cord injuries and disorders in which motoneurone loss occurs, such as amyotrophic lateral sclerosis, spinal muscular atrophies and poliomyelitis. PMID- 1721742 TI - The impact of intracerebral retinal transplants on types of behavior exhibited by host rats. AB - Retinae transplanted over the midbrain of newborn rats establish functional connections with host brain centers, which provide a substrate for several distinct visual functions. These responses provide insight into the relationship between anatomy and behavior under normal conditions and after brain injury, as well as into the strategies used by an animal to extract significant information from its visual environment. PMID- 1721743 TI - Transplantation: a new tool in the analysis of the mammalian hypothalamic circadian pacemaker. AB - The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of pacemaker cells that generate circadian rhythmicity in mammals. Transplantation of the nucleus into animals whose own nucleus has been ablated results in the restoration of overt rhythmicity to the arrhythmic host. By using donors and hosts with genetically different circadian characteristics, the unambiguous recognition of the donor rhythm expressed in a transplant recipient is possible. The reappearance of a rhythm indicates that not only has the grafted tissue survived the transplantation procedure, but that pacemaker cells that generate circadian rhythms were included in the graft; this is essential in interpreting results of such transplantation experiments. The restoration of circadian function by neural transplantation has become an important tool for studying the generation and expression of biological rhythms in mammals, and is being used in the investigation of basic questions in this field. PMID- 1721744 TI - Can fetal neural transplants restore function in monkeys with lesion-induced behavioural deficits? AB - Experiments are now being conducted in monkeys to see whether the transplantation of fetal neural tissue, rich in certain neurotransmitter-producing cells, can restore behaviour in animals with movement or learning impairments induced by lesions that have destroyed important neurotransmitter pathways. Transplantation of dopamine neurons in humans may prove to be a useful therapy in Parkinson's disease, in which a severe movement disorder is associated with degeneration of the dopamine system. Transplantation of cholinergic neurones in monkeys can overcome a severe learning impairment induced by lesion of the cholinergic system. Cholinergic transplantation may eventually be of use in a variety of neurodegenerative dementing illnesses. PMID- 1721745 TI - Cholinergic grafts, memory and ageing. AB - Neural grafts rich in cholinergic neurones can survive transplantation to the neocortex or hippocampus in rats. Such grafts have the capacity to ameliorate a variety of functional deficits associated both with explicit lesions that deafferent the neocortex or hippocampus and with natural ageing. The transplantation technique enhances our understanding of the involvement of forebrain cholinergic systems in normal cognitive functions (including memory) and of the role of cholinergic degeneration in the dysfunctions associated with ageing. It is unlikely, however, that these observations will extend to a therapeutic strategy for dementia using neural transplantation, because the human diseases (at least in the case of Alzheimer's disease and multi-infarct dementia) involve widespread degeneration of other populations of cortical neurones that are not so amenable to functional transplantation as the diffuse forebrain cholinergic systems. PMID- 1721746 TI - Prospects of transplantation in human neurodegenerative diseases. AB - Over the past decade experimental data obtained from animals have suggested that restoration or preservation of function through cell transplantation into the CNS might be developed into a useful therapeutic approach in human neurodegenerative disorders. Clinical trials in patients with Parkinson's disease have provided evidence that grafts of fetal dopaminergic neurons can survive and induce functional effects in the human brain, but no treatment based on transplantation is available yet. Initiation of studies of patients with striatal neural grafts in Huntington's disease is supported by findings in animal models, and is motivated by the lack of therapy and the severity of the symptoms in this disorder. Application of cell transplantation to other neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and hereditary ataxia is definitely premature. Further progress can be made only by systematic studies in animals of the scientific issues that can now be defined, but will also require clinical trials in a few well-monitored patients. PMID- 1721747 TI - Ethical issues in brain-cell transplantation. AB - The ethical ramifications of intracranial transplantation are many. While the majority of ethical concerns have focused on the relationship of transplantation of fetal brain tissue to elective abortion, there are other significant issues relating to graft recipients (patients and their families) and to the allocation of public resources for clinical transplantation research. In this article, some of these latter problems will be considered first, followed by a discussion of the constraints derived from the abortion question that are placed on transplantation. PMID- 1721748 TI - Synovial sarcoma: a review and update, with emphasis on the ultrastructural characterization of the nonglandular component. AB - Classic biphasic synovial sarcoma is usually not a problem in identification, whereas the monophasic spindle cell form continues to be a challenge in the differential diagnosis of spindle cell neoplasms. Most synovial sarcomas do not arise from a joint or tendon sheath, and by electron microscopy and immunohistochemistry they differ in several ways from nonneoplastic synovium. The cell of origin of synovial sarcoma is unknown, but certain features are rather consistently observed in the biphasic tumors and are useful in identifying monophasic samples. These features are apparent by immunohistochemistry and electron microscopy, both of which indicate early epithelial differentiation in the nonglandular component of the neoplasm. With immunohistochemistry, some of these cells stain for keratin. By electron microscopy, a gradient of differentiation from unclassifiable spindle cells to fully differentiated epithelial lining cells is demonstrable. A review and illustration of the ultrastructural characteristics in this spectrum of intermediate cells constitute the main emphasis of the article. The cells tend to be oval and polygonal; to be arranged in clusters surrounded by basal lamina or flocculent matrix; to have junctions, including tight junctions, and to form villuslike filopodia, true microvilli, canaliculi, and microlumina. This range of ultrastructural features is usually diagnostic of the nonglandular phase of synovial sarcoma. PMID- 1721749 TI - Malignant mesothelioma of peritoneum. AB - A 57-year-old man with malaise, ascites, and abdominal pain was found to have a peritoneum studied with numerous, small nodular tumor masses. Light microscopy revealed an anaplastic malignant tumor of uncertain differentiation. Mucin stains were negative. Electron microscopy revealed pleomorphic tumor cells with diffusely distributed cytoplasmic tonofilaments and well-developed true desmosomes. No long, thin, branching microvilli were present, yet tumor cells were strongly positive for both callus keratin (polyclonal) and monoclonal cytokeratin (AE1/3) in a diffuse cytoplasmic distribution (a pattern corresponding to the diffuse cytoplasmic tonofilaments). Tumor cells were negative for Leu-M1 and carcinoembryonic antigen. The findings were most consistent with malignant mesothelioma, and additional questioning, after tissue diagnosis, revealed a work history of asbestos exposure. PMID- 1721750 TI - Cytokeratin-containing globular filamentous bodies in renal oncocytoma. AB - Eighty-four cortical neoplasms were studied for cytokeratin and vimentin expression by immunohistochemistry and for intermediate filament aggregates by electron microscopy. Twenty oncocytomas expressed cytokeratin, 16 in a distinctive punctate pattern. These same 16 tumors also contained small globular filamentous bodies (GFB) by electron microscopy. The GFB were characterized by a matrix of intermediate-sized filaments with incorporation of diverse cell organelles such as endoplasmic reticulum, lysosomes, mitochondria, and lipid. The GFB were not within a unit membrane. Although 11 of 64 carcinomas also contained intermediate filament aggregates, only 2 of these solely expressed cytokeratin, and this was restricted to a few cells in small foci. Small GFB were also present in 5 carcinomas by electron microscopy. Three mixed clear and granular cell carcinomas contained only rare cells, whereas 2 sarcomatoid carcinomas, both of rhabdoid cell phenotype, contained numerous GFB that coexpressed vimentin and cytokeratin. Cytokeratin-containing GFB are common in oncocytomas but are uncommon in carcinoma, and, when numerous, may provide a diagnostically useful immunohistochemical feature with which to distinguish oncocytoma from its carcinoma congeners. PMID- 1721752 TI - Transurethral incision of prostate. AB - Transurethral incision of the prostate was performed in 100 males with prostates that measured under 30 g via rectal examination or transrectal ultrasound. Urodynamic measurements including uroflow rate, post-void residual, as well as physical examination have been evaluated in these patients. Eighty-five percent of these patients have been successfully treated with outpatient transurethral incision of the prostate. PMID- 1721751 TI - Oncocytic adrenal cortical adenoma. AB - Follow-up examination of a 61-year-old woman who recently had a papillary transitional cell carcinoma removed from her urinary bladder revealed a 5-cm mass in the left adrenal gland. Clinicopathologic studies showed the tumor to be a heretofore undocumented virilizing oncocytic adrenal cortical adenoma. Ultrastructural examination disclosed that the cytoplasm was filled with mitochondria, many of which had tubulovesicular cristae. The smooth endoplasmic reticulum was well developed, as was the Golgi apparatus. Lysosomes of various types, including lipofuscin pigment, were also evident. Immunohistochemical studies revealed variable immunoreactivity for simple epithelial cell cytokeratins and for synaptophysin (weak staining) in a minority of tumor cells. Two additional cases of oncocytic adrenocortical tumors, presumably adenomas, from other institutions are also discussed. The small size and weight of the tumor, the low mitotic rate, and the absence of necrosis and vascular and capsular invasion portend a good prognosis. The patient is currently free of disease 1 year after extirpation of the tumor. Additional cases and longer follow up times are needed to predict better the behavior of this rare oncocytic adrenal cortical neoplasm. PMID- 1721753 TI - Local hyperthermia for prostate cancer. AB - Hyperthermia for treatment of cancer is known to be beneficial both in itself and in conjunction with other forms of treatment, particularly radiotherapy. We have developed a new machine for local hyperthermia (41-44 degrees C) to the prostate, by transmission of microwaves (915 MHz) via a rectal probe. Patients were treated on an outpatient basis and did not require any anesthesia or sedation. There were no complications. Good results, measured by local control, disappearance of malignancy, relief of obstructive symptoms and pain, have been obtained in a series of 44 cases. More studies with this promising new modality are in progress. PMID- 1721754 TI - Transrectal ultrasound-guided interstitial radiation therapy for localized prostate cancer. AB - The use of interstitial implants for the treatment of low-stage prostate cancer using transrectal ultrasound guidance is evaluated in 80 patients. This outpatient procedure involves the placement of needles through a template and into the prostate. Ultrasound guidance is used to place the needles into a preselected location. The needles are loaded with a radioactive source. In this study Palladium-103 was utilized. This technique allows accurate and complete seeding of the prostate. There was a 50 percent or greater decrease in prostate size in all of the patients who were implanted. Prostate-specific antigen (PSA) levels became normal or decreased by more than 50 percent in 97 percent of the patients. Most patients experienced urethral irritative symptoms which lasted up to five months, but none of the patients experienced rectal symptoms lasting longer than a month. The mean follow-up is 11.8 months which is too brief to ascertain the effectiveness of this therapy. The method appears to be safe and may represent an alternative to external beam irradiation. PMID- 1721755 TI - [Fluorescein iridography in post-thrombotic retinopathy]. AB - Fluorescent iridography evidenced that postthrombotic retinopathy is associated with pathologic changes in the iridal vessels, increase of the circulation time, impaired angioarchitectonics, development of shunts and collaterals, in some cases of new vessels. The degree of the pathologic changes in the iris correlates with the severity of the underlying condition. Extravasal release of fluorescein from the iridal and ciliary vessels evidences an impairment of the permeability of the anterior section of the ++hemato-ophthalmic barrier in postthrombotic retinopathy. PMID- 1721756 TI - [Behavior of intermediate filaments in human epithelial cells during mitosis]. AB - By indirect immunofluorescence microscopy and electron microscopy, we studied the behavior of intermediate filaments during mitosis in three human epithelial cell lines, derived from normal epidermis (PcaSE-1, from a cancer patient), stratified epithelium (CNE, from nasopharyngeal carcinoma) and simple epithelium (SPC-A-1 from lung adenocarcinoma) respectively. CNE cells and SPC-A-1 cells express two different intermediate filament systems; keratin filaments and vimentin filaments, but PcaSE-1 cells only express keratin filaments. The keratin filament system in PcaSE-1 cells remained intact and encircled the developing mitotic spindle as the cells entered mitosis. In contrast, in CNE cells and SPC-A-1 cells, keratin filaments appeared to disassemble into amorphous cytoplasmic bodies during mitosis. However, their vimentin filaments remained morphologically intact throughout mitosis. We propose; (1) The disassembly of keratin filaments in mitotic epithelial cells is more or less associated with the degree of their cell malignancy rather than with the abundance of keratin filaments in interphase. (2) Intermediate filaments may be involved in the positioning and/or centering of the spindle during mitosis. (3) The possible function of vimentin filament system in CNE cells is positioning and orientation of chromosomes. PMID- 1721757 TI - Antimitochondrial and antinuclear antibodies in primary biliary cirrhosis: an update in relation to their biochemical characterization and clinical significance. AB - Antimitochondrial antibodies are found in 85 to 95% of patients with primary biliary cirrhosis. Nine different patterns have been identified but only four are associated with primary biliary cirrhosis. Anti-M2 antibodies are specific for the disease. The M2 antigen was found to be composed of five antigen determinants and related to the E2 component of three multienzyme complexes located within mitochondria. Anti-M4 and M8 antibodies appear invariably associated with anti-M2 and are markers for the "overlap syndrome" between primary biliary cirrhosis and chronic active hepatitis as well as for poor prognosis. Anti-M9 antibodies are preferentially associated with early and/or asymptomatic disease. Antinuclear antibodies are found in 24 to 58% of patients with primary biliary cirrhosis. Six various patterns have been reported. Antibodies directed to the 200 kD polypeptide of the nuclear pore and giving a perinuclear fluorescence are specific for primary biliary cirrhosis. Patients with such antibodies exhibit a less symptomatic disease and lower titers of anti-M2 than those without. PMID- 1721758 TI - Quantitation of tryptase- and chymase-containing mast cells in cutaneous lichen planus. AB - The distribution and density of tryptase- and chymase-positive mast cells in lesional and non-lesional cutaneous lichen planus (LP) was analysed. For this, enzyme-histochemical staining techniques and morphometrical measurements were applied. In non-lesional LP skin, chymase-positive cells (TC mast cells) showed a distribution similar to that found in both non-lesional psoriatic skin and in normal skin. Tryptase-positive cells (reflecting both T and TC mast cells), however, were increased in number in the upper dermis of non-lesional LP skin. In lesional LP skin, there were fewer chymase-positive cells in the upper dermis, whereas there were more tryptase-positive cells. In the upper dermis, no differences in the number of tryptase containing cells were detected between lesional and nonlesional LP skin. In lesions of LP and psoriasis, tryptase positive mast cells are increased but differ in their distribution in the papillary dermis. In psoriatic lesions, tryptase-positive cells are frequently observed in epidermal contact, a feature very rarely seen in LP lesions. The present results suggest that the increased numbers of T mast cells in the upper dermis of nonlesional LP skin may be involved in initiating the LP lesion. It seems unlikely that mast cells could be responsible for the epidermal basal cell damage, though T mast cells do participate in the general inflammatory reaction. PMID- 1721759 TI - Reactivity of HMB-45 monoclonal antibody with sweat-gland tumours of the skin. AB - HMB-45, a monoclonal antibody claimed to be specific for malignant melanoma, has been observed to react with normal eccrine sweat glands and occasionally with normal mammary and bronchial epithelium. In this study we show that HMB-45 also decorates cells in approximately 15% of various sweatgland tumours of the skin. This finding, along with the reported reactivity on mammary carcinomas further outlines the lack of absolute specificity of HMB-45 for cells of the melanocytic lineage. PMID- 1721760 TI - Clinical report and investigation of a patient with localized heat urticaria. AB - Localized heat urticaria is a rare disorder, in which the nature of the mediator is not fully established. We report the case of a 41-year-old woman with the condition, dependent upon mast cell integrity, in which histamine was demonstrated as the dominant, if not sole mediator. Non-sedative antihistamines conferred some therapeutic benefit, but subsequent sequential desensitization has enabled her to lead a full and active life again. PMID- 1721761 TI - Immunohistochemical study of cytokeratin distribution in the collecting duct of the human kidney. AB - In order to assess the expression of different cytokeratins in the collecting duct cells (CDCs) of the human kidney, three consecutive sections were stained with periodic acid-Schiff, CAM 5.2 and AE-1 (CAM 5.2 recognizes cytokeratins #19,18,8 and AE-1 #19,16,15,14,10 of Moll's catalog.), respectively. By comparing these sections, it was found that most CDCs in the inner medulla were both CAM 5.2- and AE-1-positive, whereas in the outer medulla and cortex, 77% of the CDCs were both CAM 5.2- and AE-1-positive, 15% CAM 5.2-positive and AE-1-negative, 8% both CAM 5.2- and AE-1-negative, and 0.4% CAM 5.2-negative and AE-1-positive. Recent studies have shown that most CDCs express low-molecular-weight cytokeratins #7,8,18 and 19 (17, 18, 19, 20). Of these cytokeratins, CAM 5.2 recognizes cytokeratins #8,18,19 and AE-1 recognizes cytokeratin #19. Therefore, most CDCs belong to one of the following three major types; 1. Those positive for cytokeratins #8,18 and 19 (CAM 5.2- and AE-1-positive), 2. Those positive for cytokeratins #8 and 18 and negative for #19 (CAM 5.2-positive and AE-1-negative) and 3. Those negative for cytokeratins #8,18 and 19 (CAM 5.2- and AE-1-negative). A few CAM 5.2-negative and AE-1-positive cells were thought to express high molecular-weight cytokeratins. The significance of these various cytokeratin expressions is discussed. PMID- 1721762 TI - Ceasing of epithelisation and deposit formation of unknown origin on the cornea. AB - The author presents a so far unknown pathological process interrupting permanently the regeneration of the superficially damaged cornea, and its consequences and therapy of the condition as well. The process occurs only in 5.6% of the injured individuals. The occurrence is in no correlation with the quality or extent of the damage. Also it is independent of the form and duration of therapy. The essence of the pathological changes is the slowing of corneal epithelisation within 2-4 days, followed by a complete cessation. After that a thin membrane-like layer develops simultaneously and evenly within 12 days on the area without epithelium, the surface of which is dull, transparent and whitish in colour. Within weeks or months an individually varying thickening of the membrane occurs, but the area does not grow. The surface becomes whitish-grey and is without any epithelium and with no adherence to tear. The deposits are closely and inseparably adherent to their base, their substance is rigid, being brittle only at the margins. The lesion is staining greenish-yellow with Na-fluorescein, and lively blue with toluidine blue. It is staining in small reddish-brown with rose bengal. In vivo the deposits are not measurably influenced by hyaluronidase, trypsin, alpha-chymotrypsin and papain. The microbes play no role in the process. Histological and electron-microscopical examinations suggest the corneal deposit are the product of the necrobiotic process occurring on the corneal surface during regeneration. The specific treatment consists of local application of corticoid-heparin. On the basis of the results of the examinations and literary data the author suggests that the corneal deposition and the similarly rare KCV (keratoconjunctivitis vernalis) plaque formation is the same specific process, i.e. the peculiar manifestation of the atopic state of the organism occurring independently of age. PMID- 1721763 TI - Capsaicin-sensitive bronchopulmonary receptors with dual sensory-efferent function: mode of action of capsaicin antagonists. AB - It has been suggested that capsaicin-sensitive interoceptors subserve dual sensory-efferent function in sense of being sites not only for initiating sensory impulses but also for release of mediators. The efferent response of smooth muscle contraction to capsaicin was analyzed in vitro on the trachea and main bronchi of the guinea-pig. Tetrodotoxin-resistant neurogenic contraction of the trachea evoked by capsaicin was inhibited by pretreatment of the tissue with the mast cell depleting agent of compound 48/80. Pretreatment of the preparation with indomethacin or with antagonists of histamine and 5-HT caused no changes in the responses. Electrical field stimulation of the nerve fibres in the main bronchi induced prolonged capsaicin-sensitive bronchoconstriction. Participation of mast cells and particularly leukotrienes in the responses is suggested. Sensory effect and site of action of capsaicin and its antagonists at the pulmonary receptors were tested in vivo by recording the Bezold-Jarisch reflex in the rat. Ruthenium red (0.5-2 mg/kg i.v.) and resiniferatoxin (0.1 micrograms/kg i.v.) did not evoke the vagal reflex triad of bradycardia, fall in blood pressure and apnoea, but antagonized the effect of capsaicin. The cardiorespiratory reflex triad evoked by stimulation of the regenerative region of the receptors by veratridine was not inhibited by ruthenium red. Furthermore, bradycardia evoked by electrical stimulation of the vagal nerve remained unchanged after pretreatment of the rat with either ruthenium red or resiniferatoxin. It is suggested that capsaicin excites the generator region of the receptors. Ruthenium red and resiniferatoxin antagonize its effect at different sites of the capsaicin receptor coupled cation channel. PMID- 1721764 TI - A hospital palliative care ward for elderly people. AB - In order to improve inpatient facilities for terminal care for elderly people, a special ward has been opened to maximize the quality of remaining life and to achieve 'death with dignity'. The ward is based within a geriatric department and in a District General Hospital. The work of the first year is described. It is considered to have been successful. PMID- 1721765 TI - [A study of crystallization on urolithiasis in vitro]. AB - The formation, growth and aggregation of calcium oxalate crystals were evaluated with a Coulter counter. CG-120 and sodium pentosan polysulfate (SPP) has inhibitory activity on calcium oxalate crystal formation, growth and aggregation in the seeded crystal system and whole urine system. In continuous crystallizer system, they inhibit the nucleation rate. A new system of observing the crystal formation and growth of fragmented stones after extracorporeal shock wave lithotripsy has been developed. CG-120 has an inhibitory effect on their growth. The deposition of calcium oxalate crystals in the rat kidney was induced, and the, the volume and number of crystals were estimated using a Coulter counter. SPP has an inhibitory effect on calcium oxalate crystal growth in vivo. PMID- 1721766 TI - [Activated monocytes involved in allograft rejection of rats: spontaneous plaque forming cell as a new monocyte effector]. AB - Spontaneous plaque-forming cell, SPFC, is a new hemolytic effector of monocytes, which is generated 6 to 7 days after antigen unstimulation or stimulation in man. SPFC is also detected in rats. A significant concomitant response of the SPFC, in number of SPFC at the peak response around the rejection day, and histoincompatibility of allogeneic combinations in skin or heart allograft transplantation of rats was found. Peak number of SPFC (28,850 + 1,343/10(6) on day 6 in H-1 incompatible combination (ACI-Lewis) was higher than that (11,606 + 4,235/10(6)) on day 10 in H-1 compatible combination (F344-Lewis) (p less than 0.05, Student'st-test). The day of peak SPFC response in each experimental group was also concordant with the day of rejection. The OX42 monoclonal antibody against CR3 of leukocyte-adhesion molecules inhibited hemolysis of the SPFC. Since the erythrocyte is a ligand of CR3, it is likely that hemolysis of SPFC is mediated by the CR3 adhesion molecules. In conclusion, SPFC may be an immunologic indicator of allograft rejection in rats. PMID- 1721767 TI - [Experimental studies on the mode of action of cyclosporine and FK506 assessed by proliferation response of human cloned T lymphocytes]. AB - We applied cloned human T lymphocytes established in our laboratory to evaluate the mode of action of Cyclosporine (CsA) and FK506. Phenotypic and functional analysis led us to conclude that HTL403 was a helper T cell clone and HTL805 a cytotoxic one. Susceptibility of HTL-403 to the immunosuppressants demonstrated that alloantigen-driven proliferative response can recover to the rIL2-driven level by the addition of rIL2 at higher concentration of the agents. Although full recovery was not observed in FK506, this finding indicated that FK506 as well as CsA inhibit IL2 secretion from HTL403. FK506 showed remarkable suppressive effect on the proliferative response of HTL-805 even at a considerably low concentration, while CsA suppressed such a response dose dependently. We concluded that FK506 can be used to reverse ongoing acute rejection as well as to prevent acute rejection. PMID- 1721768 TI - [Differential diagnosis of kidney transplant rejection and ciclosporin nephrotoxicity by urine cytology]. AB - To make the differentiation of kidney transplant acute rejection and ciclosporin (CS) nephrotoxicity urine cytology by classical Papanicolaou with immunocytochemical stain has been performed. Increased numbers of renal tubular cells with lymphocytes and monocytes were found in both rejections and CS toxicities. CS toxicities were associated with increased numbers of proximal tubular cells. In immunocytochemical stain, increased numbers of CD25 and CD8 positive cells as well as increased ratio of HLA-DR/cytokeratin positive cells were typically found in rejections. It is concluded that the proposed analysis of urine cytology is a non-invasive and reliable method for daily graft monitoring of acute rejection and CS toxicity. PMID- 1721769 TI - [A study of the simulation model of the lower urinary tract for urodynamics--(the first report)--theoretical evaluation of hydrodynamic model]. AB - From the view point of urodynamics, the lower urinary tract might be able to be simulated by a hydrodynamic model, consisting of a spherical balloon corresponding to the bladder and a circular tube to the urethra. In this hydrodynamic model, the diameter of the tube corresponding to the posterior urethra was assumed to become smaller with increase of the prostatic pressure according to the development of BPH. On this assumption, Bernoulli's equation might be adopted. Because of the turbulent flow in the tube, the velocity of flow was expressed as a function of the invariants, which indicated the pressure in the balloon, the respective lengths and diameters of narrow tube and wide tube, coefficients due to pipe friction, pipe fitting and pipe enlargement. The findings suggested that the smaller the diameter of the narrow tube, the smaller the flow volume showed with the development of BPH. In conclusion the urodynamics in the lower urinary tract could be simulated quantitatively by this model. The study may lead to the development of the urodynamic simulation model for the lower urinary tract. PMID- 1721770 TI - [Significance of prostatic acid phosphatase, gamma-seminoprotein and prostatic specific antigen in the urine. First report: the measurement of PAP, gamma-Sm and PA in the urine of patients with prostatic diseases]. AB - To study the significance of prostatic acid phosphatase (PAP), gamma seminoprotein (gamma-Sm) and prostatic specific antigen (PA) in urine, we have determined the urinary levels of these proteins in women and infants, in patients without prostatic disease, in patients with benign prostatic hypertrophy, and in patients with prostatic adenocarcinoma. Women and infants were found to excrete little PAP (27.9 +/- 4.8 ng/mg) and undetectable levels of gamma-Sm except one case, and undetectable levels of PA in the urine. The excretion of PAP in patients with prostatic carcinoma who were either castrated, or treated with endocrine therapy was lower than the levels in women and infants, or the levels in patients without prostatic diseases, or the levels in patients with BPH. Urinary excretion levels of gamma-Sm and PA were undetectable in the patients with well-controlled prostatic carcinoma. The present study suggests that the determination of PAP, gamma-Sm and PA in the urine of patients with prostatic carcinoma may become a useful tool for monitoring of the primary locus of the carcinoma, but additional assays of urinary PAP, gamma-Sm and PA should be measured at regular intervals to be concluded. PMID- 1721771 TI - [Clinical evaluation of nonseminomatous testicular germ cell tumors]. AB - We treated 26 patients with nonseminomatous germ cell tumors (NSGCT) between January 1976 and March 1989. Histologically, 7 were embryonal carcinoma (27%), 4 were teratoma (15%), 2 were yolk sac tumor (8%), 10 were teratocarcinoma (38%) and 3 were other mixed tumors. As regards staging, 18 belonged to stage I (69%), 1 to stage II A (4%), 1 to stage IIB (4%), 1 to stage IIIA, 2 to stage III B1 (8%) and 3 to stage III B2 (12%). Patients in stage I were treated by orchidectomy with lymphadenectomy and occasionally chemotherapy before 1984, resulting in a 100% 5-year survival. However, after 1985, 5 cases in stage I were treated by orchidectomy alone according to a watch-and-see policy. Two cases among them relapsed within two years and both of them contained immature teratoma elements. Six patients with metastatic tumor were treated with PVB therapy of which response rate was 66.7%. The total 5-year survival rate of patients in stage I, II and III was 100%, 50%, 50%, respectively and that in overall cases was 84.6%. PMID- 1721772 TI - [Pancreatic conservation after autotransplantation in dogs. Value of histologic surface analysis]. AB - A study of an artificial conservation fluid (hyperosmolar, pH = 8, rich in lactobionate and raffinose) was carried out by means of an experimental procedure involving segmental pancreatic autotransplants in dogs. The study covers 14 transplants, seven carried out without conservation and seven with 24-hour conservation at 4 degrees C. The caudal pancreas was removed after splenectomy and either transfused with 250 ml of 4 degrees C Euro-Collins before immediate transplant or with 250 ml of 4 degrees C conservation fluid for 24 hours before the transplant. The caudal pancreas was transplanted onto the right iliac vessels, while an arterio-venous fistula was created on the distal splenic vessels and the pancreatic duct was injected with modified tissucol. At the same time as the transplant, a cephalic pancreatectomy was performed. Laboratory tests included an intra venous glucose tolerance test monitored on days 0 and 28 and blood glucose and serum amylase measured every three days from days 1 to 28. The histological study of the pancreatic tissues 28 days after the transplants involved the light microscopic evaluation of the degree of fibrosis, inflammation of the pancreas, cystosteatonecrosis and peripancreatic inflammation. We used a computerized method to measure the surface area of the islets of Langerhans, as revealed by immunocytochemistry, and the surface area of fibrosis. The blood glucose and the serum amylase analyses from days 1 to 28, and the blood glucose variations during the intra venous glucose tolerance test, showed no differences between the two groups. Standard laboratory parameters were similar in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721773 TI - [Congenital dermoid cysts and fistulae of the nose. Apropos of 19 cases]. AB - Nineteen patients with nasal dermal sinuses were operated over a period of twelve years. 12 patients presented with a fistula and 8 with a cyst. One of them presented with a double sinus ostia. In six patients, intracranial extension was confirmed through a surgical approach. Clinical examination and CT scans determined the choice of surgical approach. All sinuses were removed by two surgical procedures: direct incisions, either vertical midline or horizontal glabellar, and nasofrontal osteotomy via a coronal approach. The main theories of dermoid development are explained and embryogenesis is compared to the operative findings. PMID- 1721774 TI - [Fractures of the orbital floor. Apropos of a homogeneous series of 70 patients]. AB - The authors report their experience based on a homogeneous series of 70 fractures of the orbital floor. Different anatomo-clinical forms were defined in particular fractures of the orbital floor, accompanying an "internal pivoting" of the cheek bone, which by their incarceration mechanism resemble the trap-door fractures. The blow-out fracture associated with the lower orbital margin also raises therapeutic problems. After a clinical and CT study, the authors recommend treatment via the lower orbital approach using silastic implants. The different sequelae are described clinically and are appraised medically and legally. PMID- 1721775 TI - [Giant fibroadenoma of the breast: reconstruction of the breast after resection using the dermal vault technique]. AB - The authors report two cases of giant fibroadenoma of the breast and discuss the diagnostic and therapeutic problems and the value of the "dermal vault" technique. A giant fibroadenoma is a fibroadenoma with a diameter exceeding 6 centimeters. This benign tumor has an inflammatory growth during pregnancy. An exact histological diagnosis is necessary to differentiate GFA from sarcoma phyllodes. The reconstructive concern should not jeopardize the quality of the removal. The "dermal vault" technique seems to meet these requirements: diagnosis and removal are made through a posterior approach and symmetrization of the volumes is easily obtained on clamp. The new location of the NAC should be outlined while bearing in mind that a cutaneous retraction of the upper parts will systematically occur. PMID- 1721776 TI - [Glomus tumor of the sciatic nerve]. AB - Nervous glomus tumor is an exceptional lesion, as only one case has been reported in the literature. A case of sciatic nerve glomus tumor is described. Because of the intra-neural location of the tumor with complete loss of the fascicular structures, the tibial nerve was resected and grafted. Light microscopy clearly demonstrated a glomus tumor; the histological features of these cells are typical of this lesion. PMID- 1721777 TI - [Conchal cartilaginous delta graft in secondary or post-traumatic rhinoplasties]. AB - The authors study several cases of surgical revision in post-traumatic or secondary rhinoplasty. Cartilaginous grafts with temporal fascia are recommended. The removal of ear cartilage is performed via a posterior approach for the concha cartilage associated with a septum or an alar cartilage removal. They describe the delta graft associating a concha cartilage supported by a prop (auricular or septal) to repair a defect of the middle or lower third of the pyramid. PMID- 1721778 TI - [Improving of scars from temporal and frontal face lifts]. AB - In order to prevent a widening of the frontal and temporal area and to avoid reducing the presence of hair in these areas we practice "W" like incisions at the junction of the scalp and skin. We preserve the deep part of the hair follicules and eliminate tension as we suture the wound. We will demonstrate a technique which is simple and quick allowing us to obtain excellent scars and we discuss its physiology. PMID- 1721779 TI - [Contribution of external valves in skin expansion]. AB - The filling period is a delicate phase of skin expansion procedures. Many of these difficulties are due to problems encountered during puncture of the valves when they are buried in the skin (pain, leaks, loss of the valve, inverted valve). The authors report their experience of the use of external filling valves. Based on a series of 68 valves, they describe the implantation technique, indications and their results. PMID- 1721780 TI - [Rapid tissue expansion: value of the measurement of the transcutaneous oxygen pressure and intraprosthetic pressure]. AB - Tissue expansion is often unpleasant for the patient because of its long duration. The authors present a series of 16 rapid expansion prostheses implanted in 10 patients. The expansion was monitored by measuring the percutaneous oxygen partial pressure and intraprosthetic pressure. The expansions were performed without any complications. Monitoring of the expansion by measuring the pcPO2 appears to ensure good results. PMID- 1721781 TI - [The Pers flap in the reconstruction of distal nasal defects]. AB - The in and out flap of Pers, described in 1967, is used for the reconstruction of alar defect. After an anatomical reminder of the blood supplied of the nasolabial region, we mentioned four cases of this flap, with some little modifications of drawing and closing of donor site. The others ways of repear of alar defect are discussed. PMID- 1721782 TI - [Destruction of tattoo by ruby laser]. AB - The originality of tattoo destruction by ruby laser is to selectively treat the tattooed areas without injuring the surrounding normal cells, in order to obtain better healing. Therefore, we selected a red laser (ruby, emitting at 694.3 nm), with very short flashes (100 ns with self Q switched ruby laser). Ruby laser spots of about 1 cm diameter are delivered to on the area to be treated. As the black particles of the tattoo absorb more laser energy than the surrounding pale pink skin (140 Mw/cm2, i.e. 14 j/cm2), we can obtain quite localized destruction and better healing. The beam is focussed on one point of the tattoo with a sighting neon-helium laser. In view of the very short impact, the energy absorbed by the pigmented particles diffuses minimally to adjacent tissues. After the crust falls, carrying away some tattoo pigment on its deeper surface, a pale-pink scar forms, then gradually fades in several months. With thick tattoos, it is necessary to proceed in layers and to plan a course of several treatments about one month apart. Compared with the other methods of tattoo removal (dermabrasion, salt, CO2 laser), ruby laser gives the best cosmetic results, even in keloid prone areas. PMID- 1721783 TI - [Therapeutic attitude in extensive cutaneous lesions in Recklinghausen's disease]. AB - Based on our experience with von Recklinghausen's disease, we present two cases characterized by highly developed skin tumors. One of these cases is unique in the literature. After describing these two cases, we review the different sites of the disease and discuss the limitations of surgery. Particular attention is paid to surgical palliation for patients with end-stage tumors. PMID- 1721784 TI - [Role of neurolysis of the posterior tibial nerve in plantar perforating diseases of diabetic origin]. AB - Perforating ulcers of the foot in diabetics constitute a difficult therapeutic problem. In a series of 17 cases, the authors stress the value of tibial nerve neurolysis associated with periarterial sympathectomy which results in cure of the 15 patients treated within 30 days. Although no electromyographic improvement was observed, perfusion studies and oxymetry demonstrated an improved local vascular situation which promoted healing. PMID- 1721785 TI - [Esthetic surgery and psychological rupture states]. AB - Although the psychological profile of patients requesting cosmetic surgery is often similar, the consequences of surgery can be dramatic in certain cases and result in a true state of rupture. The various forms, depression or aggression, and the conditions of onset are analysed. The four essential predisposing factors are: lack of information, result-satisfaction dichotomy, patient-surgeon divorce, and the responsibility of colleagues who, as a result of their inconsiderable comments, destabilize an already fragile psychological state. PMID- 1721786 TI - [The dry eye]. AB - After reviewing the anatomy and pathophysiology of the lacrimal circuit, the authors describe the diagnostic approach to dry eye syndrome. Two very different entities can be distinguished: acute dryness generally due to a defect of the palpebral lining, for which urgent treatment is required to prevent corneal perforation, and chronic dryness, generally due to a reduction of lacrimal secretion. The aetiological survey must look for a reversible cause, dominated by drugs. Treatment is symptomatic in every case, consisting of the regular prescription of artificial tears. The use of lacrimal plugs to obstruct the lacrimal draining ducts constitute second-line treatment and may improve the functional tolerance. PMID- 1721787 TI - Red cell and plasma concentrations of combined quinine-quinidine and quinine in falciparum malaria. AB - Red cell and plasma quinine-quinidine, and quinine concentrations in children with uncomplicated falciparum malaria who were treated with a combination of quinine/quinidine/cinchonine (combined drug) and quinine alone, respectively, were measured, using the extraction fluorescence method. The cure rates obtained with the high dose regimen of the combined drug (100%) were significantly higher than in the low dose regimen group (37.5%) (p less than 0.05), and the quinine regimen produced a 50% cure rate. Similar mild and transient ECG effects were noted in both the combined drug group and the quinine group. In patients treated with the combined drug, quinine-quinidine concentrations in both red cell and plasma of the high dose regimen group were significantly higher than those in the low dose regimen group (p less than 0.001, p less than 0.001). In quinine-treated patients, red cell quinine concentration in those with RII failure was significantly lower than that in patients with cure or RI failure (p less than 0.05). Both red cell and plasma levels of quinine-quinidine were higher than quinine levels. The red cell:plasma quinine-quinidine concentration ratios rose steadily to the high level from day 3 to day 6, while the ratio of quinine alone fluctuated around the low level and then gradually fell. The evidence suggests that red cell drug concentrations are more closely related to the outcome of treatment than to plasma concentrations and that the combined drug may be very useful for treatment of multi-drug-resistant P. falciparum infections. Further study is needed. PMID- 1721788 TI - Age- and sex-related study of HBV-DNA in HBsAg asymptomatic children from an endemic area (Cameroon). AB - A sero-epidemiological survey was carried out in Cameroon in January 1989 on a sample of 702 children of primary school age. A high HBV endemicity level was observed: 60.3% of the sera were positive to any HBV marker, 23.2% (163 sera) were HBsAg-positive. HBV-DNA positivity was observed in 38/163 (23.3%), thus showing a high level of infectivity among these carriers. Seventy-seven HBsAg positive sera were tested for HBeAg/anti-HBe: 20 (26%) were HBeAg-positive 31 (40%) anti-HBe-positive, and 26 (34%) were negative for both. All sera were anti HD-negative. Twenty-five per cent of HBeAg-positive sera were HBV-DNA-negative. This finding could be explained by a delayed HBeAg/anti-HBe seroconversion phase with fluctuant HBV-DNA. Only one case of HBV-DNA-positive anti-HBe-positive serum was observed. This study showed that HBV-DNA prevalence was significantly higher in boys (31.8%) than in girls (14.1%) (p less than 0.02). This difference was not observed for any HBV marker. We therefore conclude that in boys a prolonged HBV replicative phase might explain the observed high chronicity rate. PMID- 1721789 TI - Birth asphyxia and hypoxic-ischaemic encephalopathy: incidence and severity. AB - Clinically significant birth asphyxia was assessed over a 3-year period in a tertiary referral hospital in Nigeria. The overall incidence was 26.5/1000 live births of whom 12.1/1000 showed severely abnormal features comprising persistent seizures and coma. There was no appreciable difference in incidence for the consecutive years of the study. There was a marked involvement of infants who had suffered intrauterine growth retardation: 51 (30.7%) of these were asphyxiated, whereas only 3% were large for gestational age. The Apgar scoring system seemed not to have compared well with the clinical presentation of hypoxic-ischaemic encephalopathy from birth asphyxia. Much needs to be done to improve health care delivery and reduce the incidence of birth asphyxia. PMID- 1721790 TI - Glycosylated haemoglobin levels in children with protein-energy malnutrition. AB - Fasting blood glucose, serum protein and glycosylated haemoglobin level (HbA1c) were determined in 50 children (aged 1-5 years) suffering from protein-energy malnutrition and in 25 healthy and nutritionally normal children of the same age group. It was observed that HbA1c correlated well with the blood glucose values of the children. It was also observed that they had significantly higher values of HbA1c than the controls, indicating the existence in them of glucose intolerance. Long-term monitoring of the glycaemic status is therefore suggested as a means of assessing any relationship between glycosylated haemoglobin and impaired pancreatic function in such patients. PMID- 1721791 TI - Ambiguous genitalia: medical, socio-cultural and religious factors affecting management in Saudi Arabia. AB - Twenty-eight children with ambiguous genitalia were seen at King Khalid University Hospital over a 6-year period. The incidence of this disorder was 0.4/1000 live births. Of the total, 21 (75%) were Saudis and seven (25%) were non Saudis. The consanguinity rate was 67.9%. Twenty-four (85.7%) were born in hospital and four (14.3%) at home. In only three (10.7%) was the news first broken to the parents by a senior doctor, in 13 (46.4%) by a junior doctor, and in 11 (39.3%) by a nurse. Ambiguous genitalia were observed in 22 (78.6%) at birth and in six (21.4%) were picked up later. Owing to a lack of immediate investigative facilities and for some socio-cultural reasons, 19 of the latter groups were assigned sex without prior investigations. There was an obvious preference to assign male sex. On investigation, 13 (46.4%) had XX chromosomes, 11 (39%) XY and one (3.6%) XO: in three (10.7%), chromosomal results were not available. There were 14 cases (50%) of congenital adrenal hyperplasia, two of 5 alpha reductase deficiency (7.1%), and five of testicular feminization syndrome (17.9%), in addition to others. After investigation, five (17.9%) of the children needed sex reassignment. This was accepted by two and rejected for socio-cultural reasons by three. The opinion of the religious leaders was obtained. Some recommendations on management of these cases are made, based on our local experience. PMID- 1721792 TI - Intestinal lymphangiectasia masquerading as coeliac disease. AB - Intestinal lymphangiectasia (IL) usually presents with either non-specific general or gastro-intestinal symptoms. As IL may mimic other gastro-intestinal disorders, the diagnosis is often delayed. Intestinal lymphangiectasia was diagnosed in three children who were originally treated as cases of coeliac disease. Two were sisters who had been placed on a gluten-free diet, for 3 years in one and 10 years in the other, with no favourable response. The third patient had been tried on various formulae and underwent many investigations for failure to thrive, oedema, abdominal distension and recurrent chest infections. The diagnosis of IL was based on clinical history, physical examination and radiological and histological findings. The three patients were commenced on a medium-chain triglyceride-based diet and vitamins, with satisfactory results. PMID- 1721793 TI - Low birthweight and acute childhood diarrhoea: evidence of their association in an urban settlement of Papua New Guinea. AB - Children under 5 years of age residing in an urban settlement of Papua New Guinea were monitored from May 1987 to July 1988 in an attempt to identify aetiological factors of childhood diarrhoea. Low birthweight was found to be strongly associated with diarrhoea (incidence density ratio (IDR) = 1.60, 95% confidence interval (CI) = 1.26-2.03). The low birthweight effect was noticeable to at least 3 years of age. Greater attention should be paid to reducing the incidence of low birthweight, because such a reduction will not only be of benefit in the control of diarrhoea, but will also alleviate other factors which contribute to infant morbidity and mortality. PMID- 1721794 TI - An Indian family of hereditary pituitary dwarfism. AB - Three girls and one boy out of six siblings born to parents of consanguineous marriage presented with pituitary dwarfism. Their parents were of normal height. All the affected children had features of classical isolated growth hormone deficiency. No hypoglycaemic attacks were noted. Three of them attained puberty at the age of 16 years. PMID- 1721795 TI - Cutis laxa, growth retardation and hip dislocation in a Sudanese child. AB - This case report describes the rare variant of autosomal recessive cutis laxa with bone dystrophy in a Sudanese child. The clinical features include cutis laxa, growth and development retardation, facial dysmorphism, hyperextensible joints, dislocation of the hips and a large umbilical hernia. PMID- 1721796 TI - Priapism in sickle cell anaemia: a case report. AB - Priapism is an unusual and distressing complication of sickle cell anaemia and its management has been varied and generally unsatisfactory. We report priapism in a Libyan boy with sickle cell anaemia, managed successfully by blood transfusion. PMID- 1721797 TI - Prevalence of haemoglobinopathies in school children in Jordan Valley. AB - Blood samples were drawn from 456 healthy children, 6-10 years old, to explore the prevalences of haemoglobinopathies in Northern Jordan Valley. The children were selected by the multi-stage random sampling technique. Complete blood count, haemoglobin electrophoresis and haemoglobin A2 (HbA2) estimations were carried out on all the samples. The prevalences of beta-thalassaemia minor, alpha thalassaemia trait, sickle cell trait, and hereditary elliptocytosis were 15(3.3%), 16(3.5%), 2(0.44%) and 4(0.89%), respectively. PMID- 1721798 TI - Intracranial haemorrhages after Nebo hierochonticus scorpion sting. AB - A 3-year-old boy, who was previously well, developed acute pulmonary oedema, fundal haemorrhages, temporary blindness and deafness following a Nebo hierochonticus scorpion sting. Cranial CT scan 8 days after admission showed bilaterally symmetrical multiple hyperdense areas with intense enhancement in the cerebellar and cerebral hemispheres consistent with multiple haemorrhages. He made a complete clinical recovery with resorption of the retinal haemorrhages 4 weeks after the scorpion sting. Cranial CT scan 8 months later revealed the intracranial lesions to be hypodense with no contrast enhancement, indicating resorption of the haemorrhages. PMID- 1721799 TI - Cardiovascular manifestations of severe scorpion sting in India (review of 34 children). AB - Scorpion sting in children is a hazardous and potentially fatal condition. Of 34 children admitted to hospital in Mahad, Maharashtra State, India following scorpion sting, 14 had hypertension (130/90-170/130 mmHg), five had myocardial failure, acute pulmonary oedema developed in nine, two had tachycardia (110 200/min) and four died. Analysis of data suggests that cardiovascular morbidity and mortality depend upon the time lapse between sting and administration of vasodilators. Current management of human scorpionism consists of early admission to hospital and immediate reduction of raised blood pressure with sublingual nifedipine while peripheral action of venom is antagonized by the post-synaptic alpha blocker prazosin; in addition, digoxin, frusemide, aminophylline and oxygen are administered. The patient is kept under close surveillance in an intensive care unit. Massive life-threatening pulmonary oedema is treated with a sodium nitroprusside drip. We suggest that aggressive medical management directed at the organ system specifically affected by scorpion venom can be effective. PMID- 1721800 TI - Appendicitis presenting with dysuria in a 2-year-old: ultrasound-aided diagnosis- a case report. AB - A 2-year-old Fijian boy presented with a week's history of fever and dysuria. On ultrasound scan of the abdomen, he was found to have an appendicular mass. The role of ultrasound in the diagnosis is emphasized as well as the need for consideration of appendicitis in any young child with abdominal pain. PMID- 1721801 TI - Pulmonary radiological changes in kerosene poisoning in the Asir region of Saudi Arabia. AB - A prospective study of pulmonary radiological changes following accidental kerosene poisoning in 67 children is presented. Abnormalities were seen in 41 patients (61.2%), and these were categorized according to nine patterns. These included varying degrees of perihilar and lung infiltration, pulmonary cystic changes, pleural effusion, empyema, pneumomediastinum and surgical emphysema. The two most common were bilateral perihilar infiltrates with clear lung bases and bilateral perihilar with basal infiltrates. In the majority of cases, these radiological changes resolved completely within 10-12 days with only two needing surgical intervention. PMID- 1721802 TI - Gender and the pattern of transmission of measles infection. A reanalysis of data from the Machakos area, Kenya. AB - Data on measles from the project in the Machakos district, Kenya between 1974 and 1981 have been reanalysed in order to test the impact of sex and cross-sex transmission on severity of infection. In families with several cases, the case fatality rate was high as 11.3% (13/115) during the initial 6 months of the project. In the remaining period, the case fatality rate fell to 2.4% (21/885) (relative risk (RR) = 0.21, 95% confidence interval (CI): 0.11-0.39). During the initial period with high mortality, second cases had 4.74 times higher mortality (95% CI: 1.65-13.66) than index cases and there was no difference in mortality between girls and boys (RR = 0.98). Among secondary cases, though not significant, those infected by someone of the opposite sex had a trend toward a higher risk of dying than those infected by someone of their own sex (RR = 2.44, 95% CI: 0.77-7.78). In families with two children of the same sex, the case fatality rate was 9% compared with 29% in families with a boy and a girl (RR = 3.49; 95% CI: 0.96-12.75). In the subsequent period with low mortality, the difference in mortality between index and secondary cases was less pronounced (RR = 2.32, 95% CI: 1.03-5.25) and girls had significantly higher case fatality than boys (RR = 2.63, 95% CI: 1.09-6.34). There was no difference in this case fatality rate associated with cross-sex transmission of infection (RR = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721803 TI - Aprotinin used in emergency coronary operation after streptokinase treatment. AB - We describe a patient with evolving myocardial infarction who underwent emergency intracoronary thrombolysis followed by immediate coronary artery grafting. Aprotinin was administered intraoperatively to control the potential bleeding problems associated with thrombolysis. Total postoperative blood loss was 260 mL. The case illustrates a further use for aprotinin in cardiac operations when excessive bleeding is anticipated. PMID- 1721804 TI - Manual occluder for use in ophthalmic education. PMID- 1721805 TI - Macular hole formation following laser photocoagulation of choroidal neovascular membranes in a patient with presumed ocular histoplasmosis. PMID- 1721806 TI - A laboratory study of glass ionomer cement as a retrograde root-filling material. AB - This laboratory study investigated the use of various glass ionomer cements for retrograde root filling from the point of view of sealing qualities, ion release and ease of application. The sealing qualities of the material were tested by dye penetration and microscopic and SEM examination. Fluoride and silver ion release tests showed an initial loss of these two ions from the glass ionomer cement. A modified system for mixing and application was developed. Dye penetration did not differ from that of controls using vertically condensed gutta-percha. Glass ionomer cement is possibly a clinical alternative for the sealing of retrograde cavities; however, the silver-reinforced materials may cause tissue irritation from release of silver ions and their corrosion products. PMID- 1721807 TI - Insulin-like growth factors: biochemistry and physiology. PMID- 1721808 TI - Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells. AB - Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10(-5) M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes. PMID- 1721809 TI - Functional expression of the alpha 1 subunit of the AMPA-selective glutamate receptor channel, using a baculovirus system. AB - Using a baculovirus expression vector system, the alpha 1 subunit of the mouse AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-selective glutamate receptor channel was expressed in insect Spdoptera frugiperda cells. Binding studies using [3H]AMPA showed that insect cells infected with the recombinant virus expressed approximately 1.8 x 10(5) binding sites per cell on their surface. The ligand binding characteristics of the receptors expressed in insect cells were examined. The baculovirus-insect cell expression system affords high efficiency expression of the receptor in sufficient amounts to permit structural and functional analyses. PMID- 1721810 TI - Identification of macrophage cell-surface binding sites for cationized bovine serum albumin. AB - Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens. PMID- 1721811 TI - Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. AB - The presence of nitric oxide (NO) in the exhaled air of humans and of anaesthetized rabbits and guinea pigs was demonstrated by chemiluminescence, diazotization and mass spectrometry. This NO is endogenously produced in the lung by an NO synthase, since its generation in guinea pigs and rabbits was inhibited by N omega-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, inhibitors of this enzyme. The effect of the inhibitors was reversed by the precursor of NO synthesis, L-arginine. Since NO is produced by normal vascular endothelium for the physiological regulation of blood flow and pressure and also by activated macrophages to contribute to non-specific immunity, our experiments suggest that NO may play both vascular regulatory and host defence roles in pulmonary physiology and pathophysiology. PMID- 1721812 TI - Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67. AB - Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor. PMID- 1721813 TI - Phosphorylation of nitric oxide synthase by protein kinase A. AB - Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A. PMID- 1721814 TI - [Prospective randomized study on the comparative effect between 10% HES 200/0.5 and 6% HES 200/0.5 in patients with hearing loss]. AB - Two good comparable groups of patients (group 1 treated with 10% HES 200/0.5 + Naftidrofuryl; and group 2 treated with 6% HES 200/0.5 + Naftidrofuryl) with sudden hearing loss were compared with respect to their hearing recovery, hemorheological parameters and intravasal detectable HES fraction. The results showed an overall improvement of the hemorheological features. No difference between the two groups could be found wether in the hearing improvement nor the investigated parameters. We therefore conclude, that HES 6% is a good alternative to HES 10% for hemodilution therapy in cochleo-vestibular disorders. PMID- 1721815 TI - [Drug and non-drug tumor pain therapy in ENT medicine]. AB - The basic of analgesic therapy in patients with advanced cancer of the head and neck is the regular application of analgesics and adjuvant analgesics. Radiotherapy is the treatment of choice in patients suffering from severe pain by bone metastases. Analgesic-resistant pain of dura in patients with tumour infiltration of the skull base is an indication for intraventricularly application of opioids by neurosurgeon. PMID- 1721816 TI - Serotyping of Serratia marcescens: detection of two new O-antigens (O25 and O26). PMID- 1721817 TI - The control of homologous lysis. AB - Complement activation unleashes powerful effector mechanisms against which host cells are protected by homologous restriction factors. These factors are glycolipid-anchored membrane proteins that either induce C3 convertase dissociation (for example decay-accelerating factor) or prevent the full development of the membrane attack complex (for example homologous restriction factor and CD59). In this article Peter Lachmann explores the biology and biochemistry of these important and intriguing molecules. PMID- 1721818 TI - Big MAC attack: complement proteins cause leaky patches. PMID- 1721819 TI - Complement lysis: a hole is a hole. PMID- 1721820 TI - Complement evasion strategies of microorganisms. AB - The success of microorganisms as human pathogens stems partly from their ability to evade recognition and/or avoid destruction by complement and other natural and acquired defense mechanisms. Here, Neil Cooper reviews the various mechanisms that pathogens have evolved to evade the destructive actions of the complement system, with particular emphasis on the many remarkable examples of the duplication of complement-like structural and functional epitopes by microorganisms. Such mimicry not only enables the pathogens to avoid destruction by complement-mediated mechanisms but also, in a number of instances, facilitates infection. PMID- 1721822 TI - CD4-gp120 interactions. AB - The three-dimensional structure of the binding domain of the CD4 molecule has been determined and extensive mutational analyses of the respective binding sites on gp120 and CD4 have been completed. The consequences of gp120-CD4 binding with respect to secondary changes in the virion, or the cell, that may be required for infection or that may interfere with cellular function are current active areas of investigation. PMID- 1721821 TI - Gene expression and cDNA cloning identified a major basic protein constituent of bovine seminal plasma as bovine monocyte-chemoattractant protein-1 (MCP-1). AB - P6 is one of the major basic proteins of bovine seminal plasma. Using cell-free translation of poly(A)+RNA from bovine seminal vesicle tissue and monospecific anti-P6-IgGs, we show that P6 is a secretory product of the seminal vesicles. Immunohistochemical experiments supported this finding. Immunoscreening of a lambda gt11 cDNA library derived from seminal vesicle poly(A)+RNA furnished a number of positive cDNA clones, from which clone pH42 was characterized by sequencing. The partial amino acid sequence of a CNBr-fragment of P6 permitted identification of the reading frame of clone pH42 encoding the precursor protein of P6. The P6 precursor contains a signal peptide of 23 amino acids followed by the mature P6 sequence of 76 amino acid residues. The cDNA sequence of pH42 was 80% homologous with that of the human monocyte-chemoattractant protein-1 (hMCP 1). The respective amino acid sequences for the precursor molecules are 72% identical. Northern analysis of seminal vesicle poly(A)+RNA using pH42 as probe probe identified a 0.9-kb P6 mRNA. Stimulation of P6 mRNA expression by phytohemagglutinin in bovine peripheral mononuclear leukocytes suggests that P6 is identical to bovine MCP-1. PMID- 1721823 TI - Cytokines in clinical cancer therapy. AB - Cytokines have been of much interest in clinical cancer therapy research over the past decade. One important advance during the past year has been the clear demonstration, in large prospective randomized studies, that granulocyte colony stimulating factor and granulocyte-macrophage colony-stimulating factor reduce the neutropenia-related morbidity of cancer therapy. PMID- 1721824 TI - Cyclosporine, FK-506 and other drugs in organ transplantation. AB - As experience of the most effective way to use cyclosporine for immunosuppression in organ transplantation grows, new drugs are emerging, which may improve the potency of future immunosuppressive protocols or at least provide alternative drugs in selected situations. These newer agents include FK506, which is undergoing extensive clinical trials, rapamycin, RS-61443 and deoxyspergualin. The increasing understanding of the mechanism of action of some of these drugs on signal transduction pathways in the T cell should allow the development of drugs with perhaps more specific actions. PMID- 1721825 TI - Macrophages and oxidized low density lipoproteins in the pathogenesis of atherosclerosis. AB - Oxidized low density lipoprotein (LDL) may play an important role in the pathogenesis of atherosclerosis. Recent evidence strongly suggests that oxidized LDL is present in atherosclerotic lesions in vivo: 1) LDL isolated from human and rabbit lesions (but not from normal intima) resembles oxidized LDL in its physical, chemical and immunological properties; 2) Oxidized LDL and/or oxidation specific lipid-protein adducts can be demonstrated in human and rabbit lesions by immunocytochemical techniques; 3) Human and rabbit serum contains autoantibodies against oxidized LDL and oxidation specific lipid-protein adducts; 4) atherosclerotic lesions contain IgG that recognizes oxidized LDL and 5) antioxidant therapy slows the development of atherosclerotic lesions in rabbits. Atherosclerosis in human and rabbit arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase which leads to an enhanced uptake of LDL in macrophages by way of the scavenger receptor(s). The identification of LDL oxidation as one of the key events in the early pathogenesis of atherosclerosis offers an interesting possibility to reduce atherosclerosis by antioxidants, enzyme inhibitors and other compounds that protect LDL against oxidative damage and/or reduce the subsequent harmful effects of oxidized LDL on various cellular functions. PMID- 1721826 TI - Recovery of Ames assay mutagenicity of a polar fraction of a diesel engine particulate extract from capillary gas chromatography. PMID- 1721827 TI - Chromosomal fragile sites. PMID- 1721828 TI - Chiral aspects of drug action at ion channels: a commentary on the stereoselectivity of drug actions at voltage-gated ion channels with particular reference to verapamil actions at the Ca2+ channel. AB - Ion channels may be considered as pharmacological receptors possessing specific drug binding sites with defined structure-activity relationships. Accordingly drug binding to ion channels is stereoselective. Interpretation of this stereoselectivity may be complex because of the existence of differences in affinity and access to different channel states. Such state-dependent interactions may give rise to quantitative and qualitative differences in stereoselectivity. The implications of such differences are reviewed for drug action at Na+, K+ and Ca2+ channels. Detailed attention is paid to the actions of verapamil enantiomers in the cardiovascular system where activities differ in vascular and cardiac tissues because of state-dependent interactions and stereoselective first-oass metabolism. PMID- 1721829 TI - Acquired immunodeficiency syndrome (AIDS)--data as at 1 October 1991. PMID- 1721830 TI - Measles surveillance. Measles in the Caribbean prior to the elimination campaign. PMID- 1721831 TI - Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes. AB - The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells. PMID- 1721832 TI - The identification of tyrosine as a common key residue in unrelated H-2Kd restricted antigenic peptides. AB - We have compared the activity of several Kd- or Ld-restricted antigenic peptides as competitors in a functional competition assay using cytolytic T lymphocyte (CTL) clones. All of four unrelated Kd-restricted peptides tested could compete with each other but not with the Ld-restricted peptide P91A-. 12-24 (P91A). Moreover, the P91A peptide failed to compete with the four Kd-restricted peptides. In contrast, another Ld-restricted peptide [mouse cytomegalovirus (MCMV) pp89 167-176] could clearly compete with both Kd- and Ld-restricted peptides. The comparison of a series of modified MCMV pp89 peptides suggested that distinct structural features allow the interaction of the peptide with the two different MHC class I molecules. We showed previously that the competitor activity of two different Kd-restricted antigenic peptides was reduced substantially upon Ala substitution of the single Tyr residues present in these peptides. We now show a similar effect for two additional Kd-restricted peptides. Our results thus suggest that Tyr may function as an 'anchor' residue for many antigenic peptides that bind to the Kd molecule. Molecular modeling of the presumed antigen-binding site of the Kd molecule revealed the presence of two deep cavities that may be involved in binding peptide amino acid side chains. A model illustrating one possible interaction of a Tyr-containing peptide with the Kd molecule is presented. PMID- 1721833 TI - Failure to detect MHC class II associations of the human immune response induced by repeated malaria infections to the Plasmodium falciparum antigen Pf155/RESA. AB - Available evidence suggests that human T and B cell responses to a major Plasmodium falciparum malaria antigen (Pf155/RESA) in individuals primed by repeated infections are genetically regulated. In the present study we have attempted to establish whether these regulations reflect genetic restrictions imposed on the immune response by class II molecules of the donor's MHC system. T cell activation (proliferation and IFN-gamma release in vitro) and antibody activity (ELISA) were assayed with synthetic peptides corresponding to major Pf155/RESA epitopes. To associate T cell and antibody responses with the donors' MHC class II genotypes, leukocytes from 145 donors living in holo- or hyperendemic regions of Africa (Liberia, Gambia, Madagascar) were used for genomic HLA class II typing of their DRB-DQA and DQB genes by means of restriction fragment length analysis (RFLP). No associations between T cell responses and HLA-DR or -DQ alleles or DRB-DQA-DQB haplotypes were seen among the West Africans even when the donors were divided into high, medium or low responders. This was also true for a small group of HLA class II identical Malagasy donors including three pairs of twins. However, while the T cell responses between the twin pairs varied, those within the pairs were similar. Very similar findings were made with antibodies binding to Pf155/RESA peptides. Our data imply that the impact of MHC class II gene products on specific immune responses to Pf155/RESA epitopes is weak and hard to demonstrate in outbred human populations naturally primed by infection. This may be due to genetic regulations by other, non-HLA class II coded factors superimposed on possible HLA class II restrictions. PMID- 1721834 TI - T cell responses to peptides covering the gag p24 region of HIV-1 occur in HIV-1 seronegative individuals. AB - We demonstrate that peptides (16 amino acids long) covering the sequence of the HIV-1 core protein p24 induce significant proliferation in peripheral blood mononuclear cells (PBMC) of several (greater than 50%) healthy seronegative volunteers as well as seronegative homosexual men. The nature of this response was characterized and compared with those of HIV-infected patients. Several peptides induced responses; however, the most frequent responses in both seropositive and seronegative individuals were noted to the following peptides: 1 and 2 (aa 133-157); 6 and 7 (aa 183-207); 15 (aa 273-287); and 17 and 18 (aa 293 317). The response pattern was related to the disease stage of the patients; seronegative individuals as well as asymptomatic seropositive individuals (CDC II/III) responded to low concentrations of several peptides, but symptomatic patients (CDC IV) only responded to high concentrations of a few peptides. Cell separation studies of PBMC from healthy volunteers showed that the responding cells were CD4+ and expressed the CD45RO differentiation antigen. Furthermore, cord-blood mononuclear cells with less than 5% of CD45RO T cells did not proliferative to any of the peptides. Finally, CD4+ T cell lines specific for both peptides and p24 protein were successfully established from the PBMC of seronegative individuals confirming the data obtained with freshly isolated cells. These studies therefore suggest that the CD4+ cell response to p24 is not strictly disease related, instead, the response may be due to priming of the host with cross-reactive antigens. PMID- 1721835 TI - Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein. AB - CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA. PMID- 1721836 TI - Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor alpha subunit extracellular domain 1-210. AB - The duplicated alpha subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rH alpha 1-210) corresponding to the proposed extracellular domain of the alpha subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human alpha subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rH alpha 1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57 72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's alpha subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells. PMID- 1721837 TI - Cytotoxic CD4+ T cells from a sporozoite-immunized volunteer recognize the Plasmodium falciparum CS protein. AB - The present data provide the first evidence that a protozoan parasite, Plasmodium falciparum, can induce CD4+ cytotoxic T cells in man. The CD4+ cytotoxic T lymphocytes (CTL) were derived from a sporozoite-immunized volunteer who was protected against challenge with P. falciparum sporozoites. These T cells recognize an epitope within the circumsporozoite (CS) protein, an immunodominant sporozoite surface antigen, present also in liver stages of the parasite, which has been investigated as a vaccine candidate. The class II restricted T cell clones specifically lyse autologous B cells pulsed with a synthetic peptide representing a C-terminal sequence of the P. falciparum CS protein. The same peptide, as well as recombinant or native CS protein, also stimulates proliferation and gamma-interferon production by the CD4+ CTL. The CTL epitope, KIQNSLSTEW, is recognized in the context of HLA-DR7 and overlaps both a highly conserved, as well as a polymorphic, region of the P. falciparum CS protein. PMID- 1721838 TI - The nucleotide sequence of a voltage-gated chloride channel from the electric organ of Torpedo californica. AB - The cDNA encoding the voltage-gated chloride channel from the electric organ of Torpedo californica has been isolated and sequenced. The 2.7 kilobase pair cDNA encodes an 810 amino acid polypeptide which is highly homologous at both the DNA (97%) and amino acid (97%) levels to the voltage-gated chloride channel from the electric organ of T. marmorata. The majority of the 24 amino acid differences between the T. californica and T. marmorata voltage-gated chloride channels are clustered in two putative cytoplasmic domains with six differences located between residues 10-92 and 14 differences occurring between residues 576 to 708. Only one amino acid difference occurs in one of the predicted transmembrane domains. The most dramatic difference is an insertion of Asp-Val-Pro-Gly in a large cytoplasmic domain at amino acid residue 627 of the T. californica channel. PMID- 1721839 TI - Sequences involved in brain-specific in vitro transcription from the core promoter of the mouse myelin basic protein gene. AB - In the previous study, we have shown that a short DNA stretch from -35 to -17 in the core promoter of the mouse myelin basic protein (MBP) gene is mainly responsible for brain-specificity in in vitro transcription. In this study, we found from intensive mutation analysis that the TATA-box sequence at -34 is not critical, but sequences downstream from the TATA-box especially around -20 were important for brain-specificity. The existence of a tissue-specific factor for the MBP core promoter different from the TATA-box-binding factor TFIID is implicated. PMID- 1721841 TI - Granulocyte colony-stimulating factor (G-CSF) treatment in a neutropenic leukemia patient with diffuse interstitial pulmonary infiltrates. AB - Adult respiratory distress syndrome (ARDS) in patients suffering from acute leukemia usually occurs during chemotherapy-induced neutropenia. In addition, intensified chemotherapy with high-dose cytosine arabinoside and mediastinal irradiation may contribute to the development of ARDS. This complication is usually refractory to conservative treatment with antibiotics, steroids, and mechanical ventilation. In this report, we describe a 25-year-old patient with acute lymphoblastic leukemia who developed ARDS during the phase of chemotherapy induced neutropenia. Subcutaneous administration of granulocyte colony stimulating factor (G-CSF) at doses of 300-600 micrograms/day led to a prompt increase of peripheral granulocyte counts. With resolution of neutropenia, respiratory function gradually improved, and mechanical ventilatory support was stopped after 2 weeks. From this observation we surmise that the application of G CSF may be an effective therapeutic approach for preventing the fatal outcome of ARDS in leukemia patients with bone marrow aplasia. PMID- 1721840 TI - Early embryonal/fetal lymphopoietic ontogeny and leukemogenesis. PMID- 1721842 TI - Epidermal growth factor-specific protein tyrosine phosphorylation in preimplantation embryo development. AB - We examined whether epidermal growth factor (EGF)-induced preimplantation mouse embryo development and function are mediated by EGF-specific protein tyrosine phosphorylation (PTP). In situ cross-linking and autophosphorylation studies showed that EGF receptor (EGF-R) in Day 4 mouse blastocysts is a protein of approximately 170 kDa that is phosphorylated when exposed to EGF and ATP. Furthermore, EGF induced about a twofold increase in protein tyrosine kinase (PTK) activity in Day 4 blastocysts when incubated in the presence of a peptide substrate with a tyrosine moiety and ATP. RG 50864, a specific inhibitor of EGF dependent PTK, diminished autophosphorylation of the 170-kDa protein and completely blocked PTK activity in the blastocyst induced by EGF. However, this inhibitor did not affect EGF binding to the embryonic cell surface. In contrast, an inactive tyrphostin compound, RG 50862, did not alter EGF-induced PTK activity in the blastocyst. These findings led us to examine the effects of these tyrphostin compounds on preimplantation mouse embryo development and blastocyst hatching in vitro. RG 50864, in a dose-dependent manner, inhibited EGF-dependent development of 2-cell embryos to blastocysts and the number of cells per blastocyst. This inhibitor also antagonized EGF-induced zona-hatching of blastocysts formed from 8-cell embryos in culture. However, the inhibitor was not effective in deterring transforming growth factor-beta 1-induced blastocyst formation. The inactive compound, RG 50862, had no effects on EGF-dependent blastocyst formation or zona-hatching. The data show that the effects of RG 50864 are specific and mediated by inhibition of EGF-specific PTK activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721843 TI - A two-site enzyme-linked immunosorbent assay for inhibin. AB - The investigation of the role of inhibin in the regulation of fertility is hindered by the lack of a routine, specific assay. The pituitary cell bioassay is time-consuming and the existing RIAs, based on either purified bovine 32-kDa inhibin or synthetic alpha-subunit peptides, are not specific for the biologically active inhibin molecules. We have used monoclonal antibodies, one specific for the N-terminal region of the human inhibin alpha chain, and the other raised to a peptide sequence close to the C-terminal of the human beta A inhibin chain, to create a two-site sandwich ELISA specific for alpha beta inhibin molecules. This was used to estimate levels of inhibin in crude bovine and human follicular fluids and fractions concentrated from them. Comparison of the values obtained with the ELISA and those obtained with the pituitary cell bioassay, suggests that the ELISA measures biologically active inhibin. Compared with the peptide-based RIA, the ELISA gave much lower (as little as 100-fold lower) values for the inhibin content of these samples, e.g., bovine follicular fluid 0.375 micrograms/ml (ELISA) compared with 41.0 micrograms/ml (RIA). Such large differences, possibly due to the presence of relatively large amounts of biologically inactive forms of inhibin such as the pro-alpha c or free alpha forms, suggest that those RIAs, which essentially measure the level of alpha inhibin, considerably overestimate the levels of the active forms of inhibin in the samples and that results obtained using these assays may need reinterpretation. PMID- 1721844 TI - Modifications of the mouse zona pellucida during oocyte maturation and egg activation: effects of newborn calf serum and fetuin. AB - A precocious but limited loss of cortical granules (CG) occurs during mouse oocyte maturation both in vivo and in vitro. Although CG loss during maturation in vivo is not associated with changes in the zona pellucida (ZP), a maturation associated conversion of ZP2 to ZP2f occurs during oocyte maturation in vitro in serum-free medium. We now demonstrate that a maturation-associated change of ZP3 to ZP3f, as assessed by a reduction in sperm binding, also occurs during maturation in vitro in serum-free medium, and that both newborn calf serum (NCS) and fetuin, each of which inhibits the ZP2 conversion, also inhibit the ZP3 conversion. The concentration-dependence of the NCS- and fetuin-mediated inhibition of the ZP2 conversion, coupled with the concentration of fetuin present in NCS, is consistent with fetuin being the component present in NCS that is primarily responsible for this inhibition. Although NCS can inhibit the ZP modifications that occur during oocyte maturation in vitro, ionophore treatment of eggs, which results in an extensive release of CGs over a short period of time, overcomes the inhibitory effect of NCS on the ZP2 conversion. Results of these studies suggest a potential regulatory function of serum-derived components in the formation of a fertilizable egg. PMID- 1721845 TI - [Chievitz's organ (the juxtaoral organ): morphological and immunohistochemical studies]. AB - The Chievitz's organ (juxtaoral organ) consisting of a long continuous mass of epithelial cells is located with its medial border by the lateral face of the buccinator muscle. It was successfully identified in 10 out of 40 corpses and histologically examined. Through immunohistological methods using markers like S 100, TPA, PAN, CK-13, NSE and vimentin the epithelial character in the centre of the parenchyma could be demonstrated as well as the marked innervation and vascular supply of the direct surroundings. PMID- 1721846 TI - Identification of antibody epitopes within the CB-11 peptide of type II collagen. I: Detection of antibody binding sites by epitope scanning. AB - Using epitope scanning, the precise location of antibody binding sites on the CB 11 peptide of bovine type II collagen have been identified for the first time. Two hundred and seventy two peptides (8 amino acids in length and overlapping by seven amino acids), representing the complete CB-11 sequence, were synthesised on solid phase supports, in duplicate, and were screened with sera from arthritic and non-arthritic, bovine type II collagen-immunised rats. A total of twenty one different antibody binding sites were identified with no epitope being uniquely recognised by sera from arthritic, as compared to non-arthritic, rats although differences in the relative amount of antibody binding were seen. Individual sera identified between two and thirteen epitopes with one epitope being recognised by all sera. Some of the amino acid sequences, of the CB-11 region of bovine type II collagen, recognised by the rat sera are identical to the sequences in human type II collagen and thus these epitopes may be relevant to autoimmunity to type II collagen in patients with rheumatoid arthritis. PMID- 1721847 TI - Identification of antibody epitopes within the CB-11 peptide of type II collagen. II. Computer modelling studies of peptides and the interpretation of epitope scanning results. AB - Computer modelling techniques were used to investigate the structure of 8-mers from the CB-11 peptide of bovine type II collagen which were recognised by sera from rats which had previously been injected with bovine type II collage. It was discovered that all the hydrophobic peptides recognised by the rat sera were predicted to have collagenous-like secondary structures. The primary structure of the 8-mers which were recognised was also compared against the sequences in the OWL protein sequence database. The combined results of the computer modelling and sequence analysis suggested that the sequence Gly-Pro-Gly-Phe-Pro is a minimal B cell epitope of the CB-11 fragment of bovine type II collagen. PMID- 1721848 TI - Influence of host environment on growth of clonal CD5+B (Lyl+B) cells. AB - Autoimmune NZB mice have increased percentages of CD5+B (Lyl+B) cells in both the spleen and peritoneum. We have previously reported that as NZB mice age they develop a clonal population of hyperdiploid CD5+B cells in the spleen. These cells can readily be transplanted into unirradiated recipients. The growth characteristics of such transplanted hyperdiploid NZB spleen cells were examined in different recipient strains to determine if the immunological status of the host environments affected the growth of the clonal CD5+B cells. Young NZB and NZB.xid recipients (lacking hyperdiploid CD5+B cells) allowed growth and expansion of unpassaged CD5+B cells derived from primary NZB mice. Similarly, (NZBxDBA/2) and (NZBxBALB/c) F1 recipients allowed for expansion of CD5+B cell clones from primary sources. In a separate experiment, T cell-depleted NZB spleen cells containing a hyperdiploid CD5+B cell clone were transferred to SCID mice. The SCID environment supported the growth of the primary clone. None of these recipients normally have elevated CD5+B cells, yet these recipients allowed growth of primary transferred hyperdiploid cells. However, a difference in the ability of these recipient strains in their ability to expand multiply passaged CD5+B cell clones was observed. These results indicate that while hyperdiploid CD5+B cells are difficult to be maintained in culture, they can readily be passaged in vivo. The host environment may provide growth factors or signals for endogenous growth factors. Although the CD5+B clones arise initially in a hyperactive autoimmune environment, a hyperimmune environment is not necessary to support their growth. Transferred CD5+B cells affect the recipient environment and reduce the percentages of normal B cells. PMID- 1721849 TI - IL-4 counteracts anti-mu-induced human B cell proliferation: involvement of a cAMP-dependent inhibitory pathway. AB - In this report we show that IL-4 inhibits DNA synthesis induced by stimulation of human B cells with mitogenic doses of either soluble anti-mu mAb DA44 or phorbol ester. In contrast, earlier steps of anti-mu-induced B cell stimulation, such as RNA synthesis, CD23 expression and IL-6 production, were not inhibited but rather increased in the presence of IL-4. From these results, IL-4 appears therefore to exert two opposite effects on DA44 anti-mu mAb-induced human B cell activation: early steps are stimulated, and later steps inhibited. The results of kinetic analysis were consistent with this model. The inhibitory activity of IL-4 required an active cAMP-dependent pathway since IL-4-mediated inhibition of anti mu-induced B cell proliferation was abolished in the presence of two specific inhibitors of the cAMP pathway (H8 and 2',5'-dideoxyadenosine which are specific for cAMP-dependent protein kinase and adenylate cyclase respectively). Furthermore, IL-4 induced a delayed and prolonged increase in intracellular cAMP concentrations (observed between 4 and 48 hours of culture), and this strongly suggests that the late inhibitory effects of IL-4 is cAMP-dependent. Moreover, this delayed IL-4-mediated cAMP production is probably sufficient to prevent anti mu induced DNA synthesis since addition of the cAMP agonist forskolin on day 1 or 2 of culture also suppresses the anti-mu-mediated B cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721850 TI - LPS and cytokine-induced endothelial cell IL-6 release and ELAM-1 expression; involvement of serum. AB - In this in vitro study, the influence of serum-concentration, heat inactivation of the serum and the origin of the serum on the responsiveness of cultured human umbilical vein endothelial cells (HUVEC) to immunological challenges was investigated. Addition of human serum during stimulation with 1 microgram/ml bacterial lipopolysaccharide (LPS) increased endothelial cell ELAM-1 expression and interleukin (IL)-6 release five to ten-fold. Full endothelial cell responsiveness to LPS required 10 to 50% human serum and was largely abrogated after heating the serum for 30 minutes at 56 degrees C. Addition of newborn or fetal bovine serum instead of human serum, induced even higher IL-6 release and ELAM-1 expression in response to LPS, whilst heat-inactivation of these serum batches only moderately decreased endothelial cell responses. Endothelial cell IL 6 release and ELAM-1 expression after stimulation with IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) were less influenced by heat inactivation of the serum and by omission of serum, whilst responses to PMA remained completely unaffected by such modifications in assay media. Finally, we demonstrated that endothelial cell IL-8 release also and ICAM-1 expression in response to LPS and cytokines were increased by addition of human serum, indicating that the use of serum-free assay media, or the use of media enriched with heat-inactivated (HI) human serum interferes with physiological endothelial cell responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721851 TI - Interferon induction by Lactobacillus bulgaricus and Streptococcus thermophilus in mice. AB - This study investigates the effect of intraperitoneal injection of L. bulgaricus and S. thermophilus on interferon production by Swiss mice. The serum from mice given 5 x 10(7) L. bulgaricus in 0.5 ml saline showed a maximal production of 300 U/ml of alpha/beta interferon activity six hours after injection. Cellular integrity appears to be necessary for stimulation; heat-treated bacteria had little effect, while irradiated-bacteria had a greater effect. TNF was also produced, the sera of mice with high IFN also contained 300 U/ml TNF. Streptococcus thermophilus produced no detectable increase in serum IFN, but the 2'-5' A synthetase activity of peritoneal cells was elevated suggesting that small amounts of interferon were produced. Injection of Streptococcus thermophilus plus Lactobacillus bulgaricus did not change the serum interferon response to L. bulgaricus. These observations suggest that non-pathogenic bacteria such as those used in food processing, can stimulate IFN production in mice. There is some evidence that the bacterial cell walls might be responsible for at least part of this effect. PMID- 1721852 TI - Heterogeneity of gamma-globin chain synthesis in Saudi newborns. AB - Cord blood samples from 655 unselected neonates born to Saudi mothers at King Fahad National Guard Hospital, Riyadh, Saudi Arabia were analysed to determine the levels of gamma-globin chains in Saudis. The percentage of three types of gamma-chains of human fetal hemoglobin (A gamma T, G gamma and A gamma I) was obtained by high-performance liquid chromatographic (HPLC) method. Although the majority of babies (631/655) had normal G gamma values in the range of 58-74%, there were only 69% with normal G gamma/A gamma ratio. The A gamma T chain or HbF Sardinia was present in 28% of the total neonates with a gene frequency of 0.160. The A gamma T values in this group ranged between 11-42%. Eight babies (1.2%) had G gamma levels 45% or less (mean 41 +/- 3%) and in 16 neonates (2.4%), G gamma values were highly elevated (mean 81.4 +/- 2.8%). The frequency of two G gamma globin genes was 0.0061 and 0.0122, respectively, which is comparable with other ethnic or racial groups. The differences in G gamma to A gamma ratio in some Saudi babies could be due to an abnormal arrangement of gamma-globin genes of beta-globin gene cluster which is now being investigated. PMID- 1721853 TI - Expression of CD56/NCAM on hematopoietic malignant cells. A useful marker for acute monocytic and megakaryocytic leukemias. AB - We investigated the expression of CD56 (a neural cell adhesion molecule, NCAM) and CD57 in various hematopoietic and non-hematopoietic malignant cells, using Leu-19 and Leu-7 monoclonal antibodies. Although both molecules are commonly defined as a natural killer cell marker, we found that CD56 was highly expressed on blasts from patients with acute monocytic (4/6) and megakaryocytic (3/3) leukemias. In the latter, FACS two-color analysis revealed that leukemic megakaryoblasts simultaneously expressed CD56 and platelet-related antigens. Among leukemic cell lines, one myelocytic, three monocytic, and two megakaryocytic lines were positive for CD56. On the other hand, except for one large granular lymphocytic leukemia and one multiple myeloma cell line, none of the lymphoid leukemia cell lines or lymphoblasts from patients with acute lymphocytic leukemia (ALL) (0/15), non-Hodgkin's lymphoma (NHL) (0/2), and central nervous system (CNS) leukemia (0/2) reacted with Leu-19 antibody for CD56. The expression of CD56 in leukemia cells was not significantly affected by 12-O-tetradecanoylphorbol-13-acetate (TPA). By contrast, all hematopoietic materials were negative for CD57, while non-hematopoietic neuroblastoma cell lines expressed this molecule (4/5) as well as CD56 (5/5). Cytogenetically, the NCAM gene is located at chromosome 11q23, and chromosome breaks were often observed at this location in various leukemias. Blasts from all five acute non lymphocytic leukemia (ANLL) patients and cell lines with 11q23-proximal chromosomal breaks were positive, while those from one ALL patient with an 11q23 abnormality were negative for CD56, necessitating further studies to clarify the link between the 11q23 abnormality and CD56 expression. PMID- 1721854 TI - Surgical science, general surgery and superspecialization. The development of surgery in Norway in the 20th century. PMID- 1721855 TI - Xenopus c-raf proto-oncogene: cloning and expression during oogenesis and early development. AB - We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis raf protein. raf mRNA is identified as a member of the class of maternal RNAs. It is already relatively abundant at the beginning of oogenesis and is stable at least until the midblastula transition. The RNA is also detected later during embryogenesis in particular in gastrula, neurula, tailbud and feeding tadpole. We have also found the RNA in several adult tissues (skin, testis, stomach, intestine) at different levels. PMID- 1721856 TI - A simplified method for cryopreservation of peripheral blood stem cells at -80 degrees C without rate-controlled freezing. AB - A simplified method was established for cryopreservation of peripheral blood stem cells (PBSCs) using hydroxyethyl starch (HES) and dimethylsulfoxide (DMSO) as a cryoprotective agent at -80 degrees C without rate-controlled freezing. The data indicate that a cryoprotective solution consisting of 6% HES and 5% DMSO produced the highest recovery rates for nucleated cells (92.0 +/- 3.5%), CFU-GM (73.8 +/- 4.1%) and BFU-E (82.2 +/- 6.9%), and the highest trypan blue viability (88.4 +/- 3.6%). For long-term cryopreservation of PBSCs, CFU-GM recovery rates remained almost unchanged during 5-18 months; the mean CFU-GM recovery rate after 18 months of cryopreservation was 70 +/- 11%. When large-scale samples of PBSCs in 100-ml freezing bags were cryopreserved for clinical use, cell and CFU-GM recoveries were similar. Using this method, 10 patients with hematological malignancy received PBSC transplants after marrow-ablative chemotherapy. All demonstrated early engraftment; seven are now alive in unmaintained complete remission for 3.5-15 months after PBSC transplant. This simple and inexpensive method will be useful for the wider application of PBSC transplant as a therapeutic alternative in the treatment of malignant diseases. PMID- 1721857 TI - A novel spinal pathway and other connections to the spinocerebellum in the pigeon. AB - Avian dorsal column nuclei do not project to the cerebellum. Injections of fluorescent tracers into the spinocerebellum of homing pigeons (Columba livia) disclosed a group of neurons located rostral to the dorsal column nuclei which receives spinal primary afferents, as confirmed by double-labeling experiments. Since this group has some similarities to the mammalian group x (location medial to the restiform body, spinal afferents, efferents to the cerebellum), this name was adopted for the pigeon. Further brainstem nuclei projecting to anterior or posterior spinocerebellum and with some relevance to transmission of spinal signals are described. PMID- 1721858 TI - Midbrain periaqueductal gray projections to the dorsomedial medulla in the rabbit. AB - The present study sought to determine the existence of projections from the midbrain periaqueductal gray (PAG) to the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMN) in the rabbit. Fast Blue injections into the NTS/DMN complex revealed a population of retrogradely labeled cells within the ventrolateral PAG. Deposits of wheat germ agglutinin/horseradish peroxidase (WGA/HRP) into the ventrolateral PAG revealed terminal label within the dorsomedial, lateral, ventrolateral, intermediate, and commissural subnuclei of the NTS. Label was also observed within the DMN and a heavy concentration encapsulated this nucleus. These data suggest that the projection from the PAG to the NTS/DMN complex may represent a substrate by which the PAG may influence autonomic and cardiovascular regulation, particularly during emotional arousal. PMID- 1721859 TI - Rat central amygdaloid nucleus projections to the bed nucleus of the stria terminalis. AB - The projections from the central amygdaloid nucleus (Ce) to different subdivisions of the bed nucleus of the stria terminalis (BNST) were investigated using retrograde transport of fluorescent dyes. Iontophoretic injections of either Fast Blue (FB) or bisbenzimide (BB) were applied to the anterior medial, posterior medial, anterior lateral and posterior lateral parts of the bed nucleus of the stria terminalis. The anterior medial BNST receives projections from caudal part of medial Ce (CeM). The posterior medial BNST receives projections specifically from the intermediate subdivision of Ce, though in some cases projections from the ventral subdivision (CeV) of Ce were seen. The anterior lateral BNST receives projections primarily from the caudal lateral Ce (CeL) as well as middle and caudal part of CeM. The posterior lateral BNST receives projection from rostral CeL as well as the CeV and lateral capsular Ce. In general, the results indicate that the major subdivisions of the BNST receive projections from Ce subdivisions having similar connections with diencephalic or brainstem cell groups. Additional evidence is presented suggesting that Ce-BNST projections are part of an extensive system of intrinsic connections linking similar groups of neurons in both the Ce and BNST as well as within Ce. PMID- 1721860 TI - Investigations of origins of serotonergic projection to developing rat visual cortex: a combined retrograde tracing and immunohistochemical study. AB - The present study investigated whether the raphe neurons which give rise to the transient serotonergic fibers in the visual cortex of neonatal rats persist or disappear as the rats mature. Three experiments were performed employing the WGA apoHRP-Au retrograde transport technique in conjunction with 5-HT or WGA-HRP immunohistochemical staining. WGA-apoHRP-Au was injected into the primary visual cortex of all rats 9 days postnatally. In the first experiment, the animals were examined after 2 days; retrogradely labeled cells were observed in the dorsal raphe nucleus (DR), the median raphe nucleus (MR), and in the B9 and B6 cell groups; the majority (82.5%) of the cells was serotonergic. In the second experiment, the examinations took place following a survival time of 8 weeks: virtually all of the original raphe-visual cortical serotonergic neurons were found to the present. In the third experiment, also performed after 8 weeks relabeling the raphe-visual cortical neurons by WGA-HRP, it was found that 37.2% of the raphe neurons which had projected to the neonatal visual cortex no longer possessed such projections. PMID- 1721861 TI - Nontraumatic posterior temporal lobe hemorrhage: clinical computed tomographic correlations. AB - Ten patients with nontraumatic posterior temporal hematomas were analyzed. These hemorrhages were spontaneous (four cases) or hypertensive (six cases). With right posterior temporal hematomas, headache and confusion of sudden onset were the initial common characteristic clinical signs. The absence of prominent lateralizing neurological deficit simulated a diffuse toxic or metabolic encephalopathy. With left-sided hematomas, Wernicke-type aphasia was the initial feature. The 10 hematomas were 1.8 to 2.8 cm in maximal diameter. In these 10 cases, clinical outcome was good, as all patients survived and the hematoma resolved spontaneously. PMID- 1721862 TI - CD7+, CD4-/CD8- acute leukemia with t(11;14)(p15;q11) in a child. AB - A t(11;14)(p15;q11) was the sole chromosome abnormality observed in the malignant cells of a 10-year-old boy with acute leukemia. Morphologically, these cells were classified as L1 by the criteria of the French-American-British Working Group. Cytochemical analysis revealed that the leukemic cells were negative for Sudan Black B, periodic acid Schiff, and esterases, and positive for acid phosphatase. Immunophenotyping disclosed that the cells expressed a very immature antigenic profile [CD34+, CD7+, cytoplasmic CD3+, membrane CD3-, CD4-, and CD8-]. In spite of very intensive chemotherapy, complete remission was never induced, and the child died of progressive disease. The relationship of this case to other reported cases of acute leukemia arising from immature pluripotent hematopoietic cells is discussed. PMID- 1721863 TI - Strenuous exercise: analogous to the acute-phase response? AB - 1. It has been suggested that the physiological consequences of strenuous exercise are analogous to those of the acute-phase response. 2. In 70 male and 20 female competitive distance runners, a marked, but transient, neutrophil leucocytosis occurred immediately after these athletes completed a standard (42 km) marathon race. Concomitant significant increases were noted in the plasma cortisol levels, creatine kinase activity, C-reactive protein level, total protein level and albumin level (P less than 0.01). 3. The plasma fibrinogen, C reactive protein and total protein concentrations were markedly increased both 24 h and 48 h after exercise (P less than 0.01). The serum haptoglobin level was significantly decreased after exercise (P less than 0.01), and increased 48 h later (P less than 0.05). There was no change in the serum iron level, total iron binding capacity, per cent saturation of transferrin and serum ferritin level. 4. A significant increase in interleukin-1-type activity was demonstrated immediately and 24 h after exercise (P less than 0.01). 5. It is concluded that the metabolic sequelae of sustained exercise are similar, but not analogous, to the acute-phase response, and interleukin-1, probably plays a significant role in linking the haematological and immunological changes observed after sustained strenuous exercise. PMID- 1721864 TI - Selective arterial embolization in the treatment of lesions of the musculoskeletal apparatus. AB - The authors describe 42 cases of lesions of the musculoskeletal apparatus (traumatic, pseudoneoplastic or tumorous) in which selective arterial transcatheter percutaneous embolization (SAE) was indicated. In 3 patients SAE was not performed because the angiography had shown it to be too dangerous for the spinal cord. Out of 39 patients 2 were embolized in order to stop unrestrainable hemorrhaging (1 post-traumatic and 1 post-bioptic), 7 in order to reduce intraoperative bleeding, while in 7 cases (aneurysmal bone cyst, angioma of bone) the aim was curative. In the remaining 23 patients SAE was performed for adjuvant (8) or palliative (15) purposes in association with radio-and/or chemotherapy (11). In these last 15 cases the clinical results obtained were good in 67% of the cases, with partial or total regression of pain. Healing was obtained in 100% of the patients treated for curative purposes. PMID- 1721865 TI - [Palliative endoscopic surgical measures in esophageal carcinoma. Indications, technical requirements and results]. PMID- 1721866 TI - Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins. AB - Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA binding proteins is dependent upon chloroplast development, while its mRNA binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants. PMID- 1721867 TI - Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. AB - We have generated transgenic mouse lines carrying and expressing wild-type and 3' modified human tumour necrosis factor (hTNF-alpha, cachectin) transgenes. We show that correct, endotoxin-responsive and macrophage-specific hTNF gene expression can be established in transgenic mice and we present evidence that the 3'-region of the hTNF gene may be involved in macrophage-specific transcription. Transgenic mice carrying 3'-modified hTNF transgenes shows deregulated patterns of expression and interestingly develop chronic inflammatory polyarthritis. Treatment of these arthritic mice with a monoclonal antibody against human TNF completely prevents development of this disease. Our results indicate a direct involvement of TNF in the pathogenesis of arthritis. Transgenic mice which predictably develop arthritis represent a novel genetic model by which the pathogenesis and treatment of this disease in humans may be further investigated. PMID- 1721868 TI - Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences. AB - Antigenic diversity is generated in the wall-less pathogen Mycoplasma hyorhinis by combinatorial expression and phase variation of multiple, size-variant membrane surface lipoproteins (Vlps). The unusual structural basis for Vlp variation was revealed in a cluster of related but divergent vlp genes, vlpA, vlpB and vlpC, which occur as single chromosomal copies. These encode conserved N terminal domains for membrane insertion and lipoprotein processing, but divergent external domains undergoing size variation by loss or gain of repetitive intragenic coding sequences while retaining a motif with distinctive charge distribution. Genetic analysis of phenotypically switched isogenic lineages representing ON or OFF expression states of Vlp products ruled out chromosomal rearrangement or frameshift mutations as mechanisms for Vlp phase variation. However, highly conserved vlp promoter regions contain a tract of contiguous A residues immediately upstream of the -10 box which is subject to frequent mutations altering its length in exact correspondence with the ON and OFF phase states of specific genes. This suggests a mechanism of transcriptional control regulating high frequency phase variation and random combinatorial expression of Vlps. The multiple levels of diversity embodied in the vlp gene cluster represents a novel adaptive capability particularly suited for this class of wall less microbe. PMID- 1721869 TI - Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis. AB - The proto-oncogene c-kit is allelic with the murine white spotting (W) locus and encodes a transmembrane protein tyrosine kinase that is structurally related to the receptors for platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1). Recently the ligand for the c-kit product, stem cell factor (SCF), was identified in both transmembrane and soluble forms. In order to examine the mechanism for receptor activation by SCF and biological properties of the activated c-kit product, we transfected the wild-type human c-kit cDNA into porcine aortic endothelial cells. We found that the receptor was down-regulated and transmitted a mitogenic signal in response to stimulation with soluble SCF. We also demonstrate that SCF induces dimerization of the c-kit product in intact cells, and that dimerization of the receptor is correlated with activation of its kinase. Activation of the c-kit product by SCF was found to induce circular actin reorganization indistinguishable from that mediated by the PDGF beta-receptor in response to PDGF-BB. Furthermore, soluble SCF was a potent chemotactic agent for cells expressing the c-kit product, a property which might be of importance during embryonic development. PMID- 1721870 TI - Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. AB - In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive. PMID- 1721871 TI - Neurons with callosal projections in visual areas of newborn kittens: an analysis of their dendritic phenotype with respect to the fate of the callosal axon and of its target. AB - Combined retrograde transport of Rhodamine-labeled latex beads and intracellular injection of Lucifer Yellow in aldehyde-fixed slices of areas 17 and 18 in kittens indicate that neurons with similar dendritic morphology send axons into the corpus callosum from the 17/18 border and from parts of area 17 destined to become acallosal. At both sites callosally projecting neurons (callosal neurons) include pyramids, spiny stellate cells and star-pyramids; two types of pyramidal neurons can be distinguished on the basis of the complexity of their apical dendrites. At both sites, the dendritic morphology of callosal neurons appears basically unaffected by the ablation at the beginning of the second postnatal week of the contralateral areas 17 and 18 to which they have sent their axon. Thus the dendritic morphology of this type of cortical neuron seems independent of retrograde signals coming from their contralateral target and may instead depend on "programs" intrinsic to the neurons and/or conditions acting locally on their cell bodies, dendrites or initial axon collaterals. PMID- 1721872 TI - Acidic and basic fibroblast growth factors augment growth of fetal brain tissue grafts. AB - The fibroblast growth factor family of peptides (FGF's) are biological regulators which have a diverse array of activities. Among the biological responses reported are inductive effects during early embryogenesis, mitogenic activity on a variety of mesenchymally derived tissues, potent angiogenic activity and neurotrophic activity for both the peripheral and central nervous system. In vitro studies have been performed showing that the FGF's play a regulatory role in the survival and growth of neurons from several regions of the developing rat brain. By using the in vivo model of intraocular transplantation and repeated injections into the anterior chamber, we have been able to observe and follow the survival and growth of small, defined areas of central nervous system (CNS) under the influence of acidic (a) FGF or basic (b) FGF. Acidic FGF significantly enhanced growth of transplanted parietal cortex, embryonic day 17-20 [E17-20], hippocampus [E20] but not spinal cord [E14] when compared to the bovine serum albumin (BSA) vehicle alone. Parietal cortex grafts increased approximately 200% and the hippocampus grafts 100% when stimulated with aFGF. Basic FGF greatly enhanced the growth of intraocularly transplanted parietal cortex (E17-18), hippocampus (E16-17), and spinal cord (E14) by approximately 400%, 100% and 50% respectively when compared to the vehicle alone, and was thus significantly more potent than aFGF at the same concentration. Effects on all areas were seen using concentrations of aFGF down to 25 micrograms/ml and bFGF as low as 2.5 micrograms/ml. Histochemical and immunohistochemical studies carried out on cryostat sectioned grafts suggested either no change or normalization of markers for vascularization, glial and neuronal populations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721873 TI - Patterns of reinnervation of denervated cerebral arteries by sympathetic nerve fibers after unilateral ganglionectomy in rats. AB - In order to clarify the manner in which previously denervated cerebral arteries become reinnervated after unilateral excision of the superior cervical ganglion (SCG), we observed directly the reinnervating sympathetic nerve fibers originating in the contralateral SCG by using anterograde labeling with wheat germ aggulutinin-horseradish peroxidase in rats. The nerve fibers sprouted from the nerve fibers in the contralateral anterior cerebral artery and reinnervated the arterial wall of the anterior cerebral artery of the denervated side as early as one week after ganglionectomy. In addition to this sprouting route, three other reinnervating nerve fiber routes were observed in the circle of Willis of the denervated side two weeks after ganglionectomy: the proximal portion of the internal carotid artery, the route passing between bilateral ethmodial arteries, and the posterior communicating artery. Eight weeks after ganglionectomy, these reinnervating nerve fibers formed a fairly dense plexus in a circular pattern in the circle of Willis. However, the reinnervation could not be observed in the arterial branches derived from the circle of Willis (middle cerebral artery and posterior cerebral artery) even 16 weeks after ganglionectomy. The present results clearly demonstrated the time course, distribution pattern and limitation of the reinnervation from the contralateral SCG following unilateral ganglionectomy. The fact that reinnervation could be observed only in the main cerebral arteries of the circle of Willis, in which the nerve plexus appeared to have a circular pattern, suggests a difference between the qualities of sympathetic innervation controlling the cerebral circulation in these arteries and the other arterial branches related to these differences in reinnervation capacity. PMID- 1721874 TI - Hippocampal neurons transplanted into ischemically lesioned hippocampus: anatomical assessment of survival, maturation and integration. AB - Cerebral ischemia can be caused by many diverse conditions such as cardiac arrest and severe hypotension and is often the cause of secondary brain damage following head injury or infantile birth trauma. The inadequate cerebral blood flow can result in permanent loss of essential brain circuitries and neurological deficits. The CA1 region of the hippocampal formation is the region of the brain that is most often lesioned following transient forebrain ischemia and is associated with impairments of learning and memory. Furthermore, the loss of such a large target area can lead to detrimental post-trauma synaptic reorganization. Since methods are not currently available for the prevention of neuronal loss following cerebral ischemia, a number of anatomical methodologies were utilized to investigate whether transplanted neurons had the potential to afford some measure of repair. The hippocampal CA1 region of the rat brain was lesioned by transient forebrain ischemia and subsequently repopulated with suspensions of fetal hippocampal tissue. The ability of the transplanted neurons to remain viable when placed into a degenerating environment was confirmed by the histological demonstration of 3H-thymidine labelled neurons in the lesioned region. Histological and immunohistochemical techniques showed that the transplanted neurons developed cytological features that were indistinguishable from their normal CA1 counterparts, often showed a remarkable degree of organization, and expressed some of the same neuron specific proteins; specifically calbindin-D28K and parvalbumin. Acetylcholinesterase histochemistry and retrograde axonal transport of Fluorogold demonstrated that some afferent and efferent fibre projections to and from the septal nucleus could be reinstated. The data have shown that the transplanted neurons can demonstrate many of the anatomical properties that are characteristic of the adult cells they have replaced and therefore have great potential for the reconstruction of severe focal lesions due to ischemia. PMID- 1721875 TI - Components of the dynamic response of mammalian muscle spindles that originate in the sensory terminals. AB - One component of the dynamic response of muscle spindles is characterized by a phase lead and frequency dependent sensitivity in response to sinusoidal stretches at frequencies around 1 Hz. Possible mechanisms producing this component, designated the "mid-frequency" dynamics, were investigated by testing the hypotheses that they arise from the mechanical behavior of the intrafusal muscle and alternatively from within the sensory terminals. Destruction of the myofibrillar structure of the intrafusal muscle fibers did not alter the mid frequency dynamics, indicating that they do not arise from viscoelastic properties of the intrafusal muscle. An Arrhenius plot of the temperature dependence of the mid-frequency dynamics yielded an equivalent activation energy of 6.5 Kcal/M in the temperature range 23-42 degrees C and a 3-fold higher activation energy at lower temperatures. These observations are consistent with a dynamic process associated with a membrane-bound biochemical process. The addition of Ca++ and Ca(++)-activated-K+ (K(Ca] channel blockers (ZnCl2, Apamin and TEA) to the bathing solution altered the response dynamics by reducing the mid-frequency phase lead. The results suggest a negative feedback on the membrane potential generated by K+ efflux following a Ca++ influx that opens K(Ca) channels. A quantitative model fit to the experimental data yields a time constant of about 80 ms representing the limiting process associated with activation of the K(Ca) channels in this system. The results indicate that the mechanism underlying the mid-frequency dynamics includes at least two processes: one, not identified in this study, generates the phase lead and another, involving Ca++ and K(Ca) channels, provides a negative feedback that modifies the phase lead. PMID- 1721876 TI - Lateral and medial sub-divisions within the olivocerebellar zones of the paravermal cortex in lobule Vb/c of the cat anterior lobe. AB - The olivocerebellar projection to the c1, c2 and c3 zones in the paravermal cortex of lobule Vb/c has been investigated in the cat using a combined electrophysiological/neuroanatomical tracing technique. The zonal boundaries in the paravermal cortex were located by recording, on the cerebellar surface, climbing fibre field potentials evoked in response to percutaneous stimulation of one or more paws. A small (10-30 nl) injection of WGA-HRP was then made either into the centre or into the medial or lateral geographical half of a chosen zone and the resultant distribution of retrogradely labelled cells within the contralateral inferior olive was plotted. The c1 and c3 zones were each found to consist of two mediolaterally oriented 'sub-zones' which could be distinguished by their olivocerebellar input. The medial part of the c1 zone received climbing fibre input from the rostromedial part of the dorsal accessory olive (DAO) while the lateral part of the c1 zone received climbing fibre input from middle/rostral regions of the medial accessory olive (MAO). Both medial and lateral 'sub-zones' within the c3 zone were found to receive climbing fibre input from the rostral pole of DAO but, whereas there was heavy overlap between the olivary territories projecting to the medial c1 and medial c3 subzones, olivary cells projecting to the lateral part of c3 were located more rostrally within DAO. The c2 zone was found not to be divisible into mediolaterally oriented subzones and to receive climbing fibre input from a region of MAO located rostral and somewhat lateral to the region projecting to the lateral part of the c1 zone. The sub-zonal organisation of the olivocerebellar projection to the c1, c2 and c3 zones is discussed in relation to the functional properties of the different zones. PMID- 1721877 TI - A study of branching in the projection from the inferior olive to the x and lateral c1 zones of the cat cerebellum using a combined electrophysiological and retrograde fluorescent double-labelling technique. AB - The pattern of transverse branching in the olivocerebellar projection to the x zone in the vermis and the lateral c1 zone in the paravermis of the cat anterior lobe was studied using a combined electrophysiological and retrograde double labelling tracer technique. Fluorochrome-tagged latex microspheres were well suited for this purpose. The results show that the region of olive that supplies climbing fibres to the two zones forms a continuous, rostrocaudally directed column about 2.25 mm in length, in a caudo-lateral to rostromedial part of the medial accessory olive (MAO), on average between A-P levels 12.50-10.50. This column may be divided into caudal and rostral halves that project respectively to the x and lateral c1 zones in the apical folia of lobules V/VIa. Partial overlap between these two territories occurs in an intermediate region (A-P levels 12.00 11.00) in middle MAO where olive cells that supply climbing fibres to either x or lateral c1 are intermingled with a smaller population of cells whose axons branch to provide climbing fibres to both zones. Quantitative analysis showed that, when different tracers were injected into each zone in the same animal, double-labeled cells represented only 5-7% of either single-labelled cell population within this area of overlap. It is concluded that, although some transverse branching is present within the olivocerebellar projection to the x and lateral c1 zones in the apical folia of lobule V, such branching is not extensive. PMID- 1721878 TI - Ultrastructure of giant and small thalamic terminals of cortical origin: a study of the projections from the barrel cortex in mice using Phaseolus vulgaris leuco agglutinin (PHA-L). AB - By means of tracing with the lectin Phaseolus-vulgaris leucoagglutinin (PHA-L), we examined in the thalamus of the mouse, the axon terminals of fibers originating in the barrel cortex. Vibratome sections of the brain were subjected to PHA-L immunocytochemistry and processed for light and electron microscopy. We observed small (0.5-0.8 microns in diameter) varicosities of labeled fibers in the nucleus ventrobasalis (VB) and the nucleus posterior (PO) as well as labeled giant terminals (3-5 microns in diameter) in PO. The analysis involved examination of serial sections and computer-aided reconstruction of several terminals. The small varicosities in VB appear to be small axon terminals forming distinct asymmetric synapses with small dendritic profiles. Some labeled terminals are apposed to, but not synaptically related with, the cell bodies of neurons in VB that are retrogradely labeled with PHA-L. The small varicosities seen with the light microscope in PO are terminals forming asymmetric synapses with dendritic shafts. The giant terminals in PO appear as large, vesicle-filled profiles forming part of synaptic glomeruli, i.e. complexes of one corticothalamic terminal engulfing several excrescences of a single dendrite. A giant terminal forms several asymmetric synapses (about 8) with these excrescences, as well as numerous (up to 15) puncta adhaerentia. The glomeruli are enveloped in glial lamellae, and they are often found at the bifurcations of primary dendritic segments. We suggest that the small terminals in VB are in the service of feedback signalling from the barrel cortex to its principal thalamic relay nucleus; the functional importance of this projection may reside in increased spatio-temporal discrimination. We interpret the giant terminals in PO as elements serving feed-forward processing, allowing the barrel cortex to influence, via PO, parts of the motor pathway modulating the animal's ongoing behavior. PMID- 1721879 TI - Pallidotectal projection to the inferior colliculus of the rat. AB - After injection of fluorescent tracer into the inferior colliculus (IC), retrogradely labeled cells were observed not only in the temporoauditory cortex (ACx) and the substantia nigra pars lateralis, but also in the globus pallidus (GP). These labeled GP cells were localized exclusively in the caudal portion of the GP, which has been known to project to the ACx. Employing a retrograde fluorescent double labeling technique, the GP-IC neurons were found to be distributed in a separate manner from the GP-ACx neurons within the caudal GP. The present study provides further anatomical evidence that the caudal GP has a functional role in auditory processing. PMID- 1721880 TI - On the substrate specificity of nitric oxide synthase. AB - Nitric oxide (.NO) synthase (NOS) activity in subcellular fractions from cultured endothelial cells (EC) and lipopolysaccharide-activated J774.2 monocyte/macrophages was investigated by monitoring the .NO-mediated increase in intracellular cyclic GMP in LLC-PK1 pig kidney epithelial cells. The constitutive NOS in EC (NOSc) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOSi) was equally distributed among cytosol and membrane(s). Both the cytosolic NOSc in EC and the membrane-bound NOSi in J774.2 cells were strictly Ca(2+)-dependent, whereas the membrane-bound NOSc in EC and the cytosolic NOSi in J774.2 cells were not. L-Homoarginine and L-arginine-containing small peptides, such as L-arginyl-L-phenylalanine, replaced L-arginine as a substrate for the NOSc in EC and the Ca(2+)-independent NOSi in J774.2 cells, but not the Ca(2+) dependent NOSi. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca(2+)-dependency. PMID- 1721881 TI - Nd:YAG laser versus polidocanol injection for palliation of esophageal malignancy: a prospective, randomized study. AB - Palliation is often the only treatment that can be offered to patients affected by esophageal malignancy. This prospective study was carried out in order to compare two endoscopic palliative treatments: Nd:YAG laser and local injection of 3% polidocanol. We randomized 34 patients with inoperable malignancies to one of the two treatments. After the first course, 88.8% of the patients in the laser group and 81.5% in the polidocanol group were able to swallow a normal oral caloric intake. Only one major complication (esophageal perforation) was observed (polidocanol group) and was successfully treated with endoscopic placement of a prosthesis. We believe that both techniques are safe and effective for the palliation of esophageal malignant strictures but that polidocanol injection is cheap, simple, and more widely available. PMID- 1721882 TI - Small volume India ink injections. PMID- 1721883 TI - [Interferon activates DNA repair genes in UV-irradiated human cells]. AB - Unscheduled DNA synthesis in human fibroblasts pretreated with natural and recombinant interferons was studied by scintillated radiometry. UDS was increased in fibroblasts pretreated 7 days before UV-irradiation. Newly synthesized proteins induced in cells after interferon treatment were compared with heat shock proteins. PMID- 1721885 TI - Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13. AB - Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage. PMID- 1721884 TI - A transposition of the reverse transcriptase gene reveals unexpected structural homology to E. coli DNA polymerase I. AB - The rational design of antiviral agents targeting the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) would greatly benefit from a more intimate knowledge of the structure of RT. Until now, the degree of sequence similarity between RT and E. coli DNA polymerase I (Pol I) has been thought to be confined to several small regions, suggesting little basis for homology molecular modeling. However, we have found that a region in the C terminal of the RT polymerase domain is homologous to a central region of Pol I that lies between the universal polymerase motifs A and C (specifically, helices N-O-P of the Pol I crystal structure); a single transposition closely aligns the RT and Pol I genes, revealing a similar domain structure with 20% residue identity, as well as the possible structural correlates of several RNA-dependent polymerase motifs. The RT from Myxococcus xanthus (a bacterium believed to have diverged from other species 2 billion years ago), if similarly transposed, shows homology to both HIV-1 and E. coli, suggesting the possibility of a very ancient divergence between the RT and Pol I polymerase genes. A second even more significant match to this E. coli region was found in the retroviral ribonuclease H (RNase H) domain, and corresponds precisely to a region that has been aligned by previous investigators with the E. coli RNase H, suggesting that Pol I helices O and P are homologous to helices A and D of the RNase H crystal structure, respectively. These results are consistent with a modular theory of molecular evolution. PMID- 1721886 TI - Null mutation in the stringent starvation protein of Escherichia coli disrupts lytic development of bacteriophage P1. AB - As initial steps toward understanding the regulation and function of the stringent starvation protein (SSP) of Escherichia coli, we have isolated the ssp gene (encoding SSP), defined the operon in which ssp is found, and created insertion-deletion mutations of the ssp gene in recBC, sbc and recD strains by linear DNA transformation. During attempts to move the insertion-deletion structure to other strains by P1 transduction, we found that P1 was unable to form plaques on hosts lacking an intact ssp gene. The delta ssp mutation, however, did not affect transduction of the delta ssp strains and mutant strains were able to support lysogenic P1. When P1 lytic growth was induced, an increase in P1 DNA was detected without lysis or plaque formation. Examination of proteins synthesized in the delta ssp host during induction revealed the absence of P1 late gene products. Also, the apparent continued synthesis of early gene products during late time points was observed in the delta ssp host. The results reported here suggest that the defect in P1 lytic growth brought about by the absence of SSP occurs at the point at which bacteriophage P1 shifts from early to late gene expression. We also report the results of experiments on stable RNA synthesis following amino acid (aa) starvation induced by serine hydroxamate, and experiments on stable RNA synthesis following resupplementation of a limiting aa. These experiments show that SSP is not involved in stable RNA synthesis. Additionally, complementation studies have shown that ssp is identical to the previously described pog gene of E. coli. PMID- 1721887 TI - [Isovolemic hemodilution on non-arteritic anterior optic neuropathy. Initial results of a randomized study]. AB - As there is no generally accepted treatment for non-inflammatory anterior ischemic optic neuropathy, we have started a randomized, controlled clinical trial on isovolemic hemodilution. In this study all patients received a basic treatment of 75 mg of acetylsalicyclic acid daily. Patients were then randomly assigned to either a group without additional therapy or a group with isovolemic hemodilution. Isovolemic hemodilution was carried out over 6 weeks by several blood-letting procedures and replacement of volume by infusion of hydroxyethyl starch solutions (PCV down to 35-32%). So far, 21 patients have fulfilled the inclusion criteria (symptoms less than or equal to 30 days, no medical contraindications, no giant-cell arteritis), and since they matched for age, duration of symptoms, cardiovascular risk factors, they could be observed for at least 3 months. Although only 1 of 10 control patients hat better visual acuity after 3 months; 6 out of 11 hemodiluted patients had better vision after treatment (p = 0.024). These results indicate that isovolemic hemodilution most likely has a beneficial effect on the visual prognosis of patients with anterior ischemic neuropathy. PMID- 1721888 TI - [Conservative therapy of prostate cancer]. AB - Treatment of carcinoma of the prostate with hormones can be carried out as partial or complete androgen deprivation. As primary therapy it may be administered palliatively in advanced carcinomas (almost always metastatic), as adjuvant treatment following radical prostatectomy, as "salvage" treatment in post-irradiation recurrent disease, or secondarily after unsuccessful primary treatment. In the case of primary treatment, androgen deprivation is more effective than chemotherapy (NPCP Protocol 1300). LHRH analogues (of the gosereline acetate type) are equally as effective as orchiectomy (standard therapy), but cause a flare-up of the patient's symptoms within the first two weeks, and are therefore given in combination with an antiandrogen. The use of a pure antiandrogen (of the flutamide type) is equally as effective as the standard therapy, but in contrast to the latter, impotence does not occur. Complete androgen deprivation (LHRH analogues plus pure antiandrogens) is more effective in the case of low-volume metastases--in terms of time-to-progression--than standard therapy. PMID- 1721889 TI - [New substances in therapy of advanced prostate cancer]. PMID- 1721890 TI - Phenotyping of T-cell lymphomas in paraffin sections--which antibodies? AB - Five antibodies, MT1 (CD43), UCHL1 (CD45RO), OPD4, poly-CD3 and beta F1, were assessed for their reactivity with 50 archival cases of T-cell lymphoma in formalin-fixed paraffin-embedded tissue. All cases had been previously characterized as T-cell lymphomas, and the histological types included 14 cases of small cerebriform lymphoma, six cases of angioimmunoblastic lymphadenopathy like T-cell lymphoma, four cases of T-zone lymphoma, five cases of pleomorphic small cell lymphoma, 12 cases of pleomorphic medium and large cell lymphoma, four cases of anaplastic large cell lymphoma, two cases of T-lymphoblastic lymphoma and three cases of enteropathy-associated T-cell lymphoma. UCHL1 and MT1 showed reactivity with the highest percentage of cases (94 and 86% respectively) but lack absolute specificity for T-cells, especially in high-grade lymphomas. Poly CD3 is highly specific for T-cells, and stained neoplastic cells in almost 80% of the cases. beta F1 stained the lowest percentage of cases (40%). UCHL1 and poly CD3 together identified 98% of cases, and this combination is recommended for the diagnosis of T-cell lymphomas in paraffin sections. PMID- 1721891 TI - Granulomatous prostatitis: a clinicopathological study. AB - In a clinicopathological study of granulomatous prostatitis, we have found two distinct histological patterns. Approximately one third of cases consisted of localized, often elongated or stellate lesions, resembling rheumatoid nodules. Where clinical details were available, most of these cases had a history of previous transurethral resection. The remaining cases showed more diffuse involvement of the prostate, with lesions centred on ducts and glands, and were not associated with previous prostatic surgery or systemic illness. Immunohistochemical studies of the associated inflammatory infiltrate showed an apparently random distribution of T- and B-lymphocytes in the former group, while in the latter group there was a concentration of T-cells in and around damaged ducts and glands, suggesting a possible immune-mediated destruction of these structures. PMID- 1721892 TI - Duodenal gangliocytic paraganglioma. Case report with immunohistochemical study on the expression of keratin polypeptides. PMID- 1721893 TI - Inheritance of ribosomal gene activity and level of DNA methylation of individual gene clusters in a three generation family. AB - Ribosomal gene activity and levels of DNA methylation were investigated by cytochemical and immunological methods in the nucleolar organizer regions (NORs) of individually recognised acrocentric chromosomes. Mendelian inheritance of ribosomal gene activity in a three generation family was demonstrated, together with consistent behaviour of individual gene clusters in different carriers, even when environmental conditions were changed. For most chromosomes, an inverse relationship between gene activity and the level of DNA methylation was observed. Exceptions were the two chromosomes 15 and chromosomes 13cp and 22p, all being strongly chromomycin-A3-positive in their short arms. These chromosomes bound to anti-5-MeC antibodies with differential frequencies in the different carriers. The possibility of involvement of repetitive GC-rich DNA in this behaviour is discussed. PMID- 1721894 TI - Fatal hyperammonemia resulting from a C-to-T mutation at a MspI site of the ornithine transcarbamylase gene. AB - Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle in humans and is responsible for lethal neonatal hyperammonemia in males. Partial OTC deficiency also occurs in females and can be responsible for life-threatening hyperammonemic comas in heterozygotes. The cosegregation of the trait with a 5.8-kb abnormal MspI fragment in an affected family led us to hypothesize that this unexpected migration pattern was related to the mutation event in this particular family. Using polymerase chain reaction amplification of the specific mRNA derived from a post-mortem biopsy of the liver, we found that the MspI site located in the seventh exon of the gene was abolished and we finally identified a C-to-T transition at codon 225 of the cDNA, changing a proline to a leucine in the protein. Subsequent digestion of amplified exon 7 using the restriction enzyme MspI allowed direct screening for the mutant genotype during the next pregnancy. The present study supports the view that direct detection of the mutant genotype using either Southern blotting or digestion of amplified exons of the gene can contribute to genetic counselling in noninformative families. Finally, since MspI digestions are routinely performed for restriction fragment length polymorphism-based family studies in OTC deficiency, we suggest that the possible presence of the 5.8-kb abnormal fragment should be investigated on Southern blots of affected individuals. PMID- 1721895 TI - The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2. AB - Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb. PMID- 1721896 TI - The response pattern of human T cells to stimulation following expansion in IL-2. AB - The ability to stimulate and induce responses of T cells is influenced by their age and state of differentiation. We have examined the response upon restimulation of T cells expanded in IL-2. Our results demonstrate that large numbers of T cells, which after activation react by secreting IL-2, can be obtained from small numbers of PBL. In this study T cells were grown in IL-2 and, after 12-14 days, IL-2 was withdrawn in order to deprive them of growth factors. Our findings showed that after resting for 48 hours without IL-2, IL-2-dependent cells reverted to small lymphocytes, ceased to incorporate 3H-TdR, and had no mRNA for activation antigens such as Tac or IL-2. The cells could be reactivated to proliferate by stimulation with a calcium ionophore ionomycin and phorbol dibutyrate (PdB). Cells from insulin dependent diabetes mellitus patients with a defined immunoregulatory defect were then studied. Our results demonstrated that the IL-2 expanded cells evidenced the immunoregulatory defect for IL-2 synthesis that we had initially defined using virgin T cells from peripheral blood. These results demonstrate that studies of immune function can be undertaken in donors from whom limited numbers of peripheral blood lymphocytes are available using primed cells which have been allowed to dedifferentiate. PMID- 1721897 TI - Detection of charges and their distribution on dialysis membranes with cationic/anionic dyes using confocal laser scanning microscopy. AB - Methods for the detection of positive or negative charges on the surface of biomaterials/membranes and inside a membrane are important for the characterisation of such materials. We tested different dyes and optimized staining procedures. Under standardized conditions negatively charged membranes were stained with cationic triarylmethane compounds such as crystal violet and positively charged membranes with the anionic anthraquinone dye anthralan blue B. There was no staining of uncharged cellulose membranes. The applicability of these methods was demonstrated on membranes coated to varying degrees with charged compounds such as heparin, these changes in charge being detectible quantitatively by photometry. The distribution of charges inside a membrane was detected by optical sectioning across the stained (FITC labelled poly-L-lysine) membrane using confocal laser scanning microscopy (LSM). LSM offers a completely new application possibility in biomaterial and biocompatibility research. PMID- 1721898 TI - A rapid method for the cytodiagnosis of multibacillary leprosy. AB - The diagnosis of leprosy is made clinically with histologic and cytologic confirmation. The traditional methods for making the cytologic diagnosis of leprosy take time and training, making them unsuitable for rapid office use. We describe a simple, rapid method for cytologic confirmation of the diagnosis of multibacillary leprosy using slit skin scrapings. PMID- 1721899 TI - [Serotyping of Chlamydia trachomatis isolates]. AB - Antigens can be used in the classification of microorganisms and can give information about the biological behaviour of the infectious agent and the spread of the infection. Starting with the mouse toxicity prevention test, followed by the two-step and then the one-step micro-IFT using polyclonal antibodies, C. trachomatis serovars are now typed with monoclonal antibodies. Worldwide, serovars D, E and F are found with the highest prevalence. No differences have been found between women and men in the prevalence of serovars. In addition, symptomatic and asymptomatic courses of the disease were not correlated with distinct serovars. The persisting technical difficulties of for large-scale typing could be overcome by the use of the polymerase chain reaction followed by treatment with endonucleases. PMID- 1721900 TI - Early detection of stage A prostate carcinoma: combined use of prostate-specific antigen and transrectal ultrasonography. AB - We prospectively studied 103 men who had normal results on digital prostate examinations but had bladder outlet obstruction secondary to prostatic hypertrophy and needed transurethral prostatectomy. All men underwent a preoperative transrectal ultrasonographic examination of the prostate and prostate-specific antigen (PSA) level determination. A total of 30 cancers were ultimately detected, 22 (73%) of which were detected preoperatively by either an abnormal ultrasonogram or elevated PSA levels (or both). Eight of these men were spared transurethral prostatectomy and had definitive treatment based on transrectal biopsy and appropriate staging evaluation. For PSA and ultrasonography combined, the sensitivities and negative predictive values for cancer (92% and 94%, respectively) were superior to the specificities and positive predictive values (71% and 64%, respectively). The combined use of both studies is recommended to rule out cancer in candidates for prostatectomy but not to routinely screen the general male population older than 40 years. PMID- 1721901 TI - Protein synthesis in adult skeletal muscle after tenotomy: responses to fasting and insulin infusion. AB - Muscle growth was established in specific muscles in the hindlimb of adult female rats by tenotomy of the gastrocnemius muscle. Seven days after surgery there was an increase in the wet weight of the soleus (Sol) and plantaris (P) muscles and a decrease in that of the gastrocnemius (G) muscle from the tenotomized limb compared with the respective control muscles from the contralateral limb from the same animal. In all three muscles there was a significant increase in the fractional rate of protein synthesis (ks) in the muscles from the tenotomized limb above the rate of the respective control muscles. In contrast, the extensor digitorum longus (EDL) muscle showed no change in wet weight or ks 7 days after tenotomy of G. Fasting for 12 or 36 h had no significant effect on ks in G, P, or Sol muscles from either the control or tenotomized limbs. In EDL from the control limb, both fasting periods resulted in a significant decrease in ks, although this effect was not seen in the EDL from the tenotomized limbs of the same animals. A subsequent 30-min insulin infusion was similarly ineffectual in G, P, and Sol, with its only effect evident in the EDL from the control limb, where it was sufficient to reverse the decreased ks resulting from the fasting, even though after 36 h fasting the reversal was only partial. PMID- 1721902 TI - Dependency of hypoxic chemotransduction in cat carotid body on voltage-gated calcium channels. AB - The hypothesis that the entry of extracellular calcium ions into some compartment, quite possibly the type I cells, through voltage-gated calcium channels (VGCC) is essential for hypoxic chemotransduction in the cat carotid body was tested using an in situ perfusion technique. The neural output of the carotid body of anesthetized, paralyzed, and artificially ventilated cats in response to perfusions with Krebs-Ringer bicarbonate solution (KRB), calcium-free KRB, KRB containing calcium channel blockers, or KRB containing BAY K 8644 was recorded. Selective perfusion of the carotid body with hypoxic calcium-free KRB significantly decreased carotid chemoreceptor activity, suggesting that extracellular calcium is essential for hypoxic chemotransduction. Selective perfusion of the carotid body with hypoxic KRB containing verapamil (10-100 microM), diltiazem (10-100 microM), or nifedipine (10-100 microM) dose dependently attenuated the increase in chemoreceptor activity produced by hypoxia, suggesting that VGCC need to be activated for hypoxic chemotransduction. The carotid body response to hyperoxic KRB containing the calcium channel agonist BAY K 8644 (10 microM) was 267 +/- 87% of hyperoxic control KRB, suggesting that an enhanced influx of calcium ions through VGCC stimulates carotid chemoreceptor activity. Selective perfusion of the carotid body with severely hypoxic KRB containing BAY K 8644 did not increase chemoreceptor activity above that produced by severe hypoxia alone. This suggests that severe hypoxia increases intracellular calcium in some compartment of the carotid body to achieve stimulatory maximum response and that further increase in intracellular calcium does not produce further elevation of neural activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721903 TI - Lung to blood passage of different-sized molecules during lung inflammation in the rat. AB - The passage of different-sized marker molecules over the lower respiratory tract into the blood circulation during pulmonary inflammation induced by dextran, endotoxin [i.e., lipopolysaccharide from Escherichia coli (LPS)], or ferritin was assessed in the rat. Bovine immunoglobulin G (BIgG, mol wt = 150,000 Da), bovine serum albumin (BSA, mol wt = 67,000 Da), and the nonapeptide 1-deaminocysteine-8 D-arginine vasopressin (dDAVP, mol wt = 1,067 Da) were used as permeability markers after intratracheal instillation. The pathophysiological indexes of a proceeding lung inflammation were increased total cell number, changed leukocyte proportions and increased total protein content obtained in bronchoalveolar lavage, and lung edema formation shown as an increased lung wet-dry weight difference. Intratracheal instillation of dextran induced a moderate neutrophil invasion into the lungs but had no effect on the passage of the different markers over the lungs (BIgG 1.8 +/- 0.6%, BSA 3.5 +/- 1.2%, dDAVP 26.1 +/- 20.7%) compared with control rats instilled with the markers alone (1.8 +/- 0.4%, 4.1 +/ 1.3%, 20.0 +/- 3.8%, respectively). Endotoxin administration resulted in markedly higher lavage cell counts and lung edema concomitantly with an increased lung passage of the markers (3.2 +/- 0.9%, 22.0 +/- 6.1%, 33.3 +/- 12.0%, respectively; P less than 0.01-P less than 0.001). The highest marker passage was obtained when the inflammation was most severe, i.e., after ferritin administration (17.6 +/- 2.3%, 60.0 +/- 6.7%, 41.6 +/- 6.9%, respectively; P less than 0.001), which resulted in markedly elevated lavage cell numbers and protein content as well as edema formation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721904 TI - Multiple effects of BAY K 8644 and nifedipine on isolated diaphragmatic fibers in vitro. AB - The dose-response effects of BAY K 8644 and nifedipine on diaphragmatic contractility were assessed in vitro. Isolated diaphragmatic fibers were obtained from rats and placed in an open-topped channel of a Plexiglas tissue chamber perfused with continuously flowing Krebs solution heated to 37 degrees C. Isometric twitch force, generated in response to 1-Hz supramaximal electrical stimulation (4 times/min), was measured with a highly sensitive photoelectric force transducer. Low doses of BAY K 8644 or nifedipine (10(-7) M) were without effect on twitch tension. For 10(-6) M, twitch tension increased by 10 +/- 1% (P less than 0.005) for both drugs. For 10(-5) M, twitch tension increased by 12 +/- 1% (P less than 0.05), and maximal contractures were observed (BAY K 8644 and nifedipine). Simultaneous drug administration did not reveal mutual antagonism as expected; instead the effects were additive, with twitch tension increasing by 30 +/- 2% (P less than 0.001) for 10(-5) M BAY K 8644 + nifedipine. Both BAY K 8644 and nifedipine altered twitch characteristics. In low-calcium media (0.5 mM) twitch potentiation produced by the two drugs was further enhanced (increasing 60% for 10(-5) M BAY K 8644 or nifedipine). Contractures, by contrast, were abolished. From these results it is difficult to reconcile a unique action of these drugs on calcium channels as is conventionally accepted. PMID- 1721905 TI - Neural regulation of lysozyme secretion from tracheal submucosal glands of ferrets in vivo. AB - To investigate how central and peripheral nerves affect lysozyme secretion from tracheal submucosal glands in ferrets we injected substance P (20 nmol/kg in 200 microliters) intracisternally or intravenously into anesthetized artificially ventilated ferrets. We collected 3-ml samples from a perfused (3 ml/5 min) segment of trachea in situ during 15 min before and 45 min after injection of substance P. Content of lysozyme, a specific marker of tracheal submucosal gland serous cell secretion in ferrets, was measured spectrophotometrically in each sample. Intracisternal substance P increased peak lysozyme output threefold compared with baseline. This increase was abolished completely by cutting both superior laryngeal nerves (SLN) and was partially inhibited by atropine, phentolamine, or propranolol. Intravenous substance P increased peak lysozyme output 10-fold compared with baseline. This increase was partly abolished by cutting both SLN. We concluded that intracisternal substance P stimulated the central nervous system (CNS) and activated cholinergic, adrenergic, and nonadrenergic noncholinergic secretomotor nerves to tracheal glands and that intravenous substance P increased lysozyme secretion both by acting directly on tracheal glands and indirectly on the CNS to activate secretomotor nerves. PMID- 1721906 TI - Influence of antigen on membrane properties of guinea pig bronchial ganglion neurons. AB - The bronchus was isolated from actively sensitized guinea pigs, and the effect of antigen challenge on the excitability of bronchial parasympathetic ganglion neurons was examined with standard intracellular recording techniques. Based on histological examination, we found that mast cells were located near parasympathetic ganglia neurons. Antigen challenge resulted in a loss of mast cell staining and the release of the mast cell-associated mediators, histamine (38 ng/g, approximately 14% of total content) and prostaglandin D2 (PGD2, 118 ng/g wet weight of tissue). Challenging the isolated bronchus with the sensitizing antigen resulted in a transient depolarization (mean 6 mV) of the resting membrane potential of the neurons. Antigen challenge also had a dramatic effect on the accommodative properties of the neurons. Before antigen challenge, two subpopulations of neurons could be differentiated by their response to cathodal current steps: 60% of the cells responded in a "phasic" manner, firing one to six spikes and then accommodated, whereas the balance fired spikes repetitively throughout the current pulse. In phasic firing cells, ovalbumin challenge produced a decrease in accommodation. This was evidenced by a fivefold increase in the number of action potentials elicited during a 500-ms suprathreshold current pulse. The antigen-induced depolarization could be mimicked by histamine, whereas the decrease in accommodation was mimicked by application of PGD2. Leukotriene C4, another mast cell-associated mediator, had no effect on these neuronal properties. These results provide evidence that the immediate hypersensitivity response in guinea pig airways may involve changes in membrane characteristics of bronchial parasympathetic ganglia neurons. PMID- 1721907 TI - Proliferative potential and expression of cell type specific functions in primary mouse colonic epithelial cells. AB - Primary cultures of mouse colonic epithelial cells have been obtained that are typically epithelial by morphology and moreover express keratins and endogenous beta-galactosidase; this latter activity was also demonstrated in the epithelial lining of the mouse colonic mucosa. The proliferative response of the primary colonic epithelial cells to epidermal growth factor, insulin, and the bile acid, deoxycholic acid, has been studied. Using primary cultures maintained at suboptimal growth conditions, which yielded 96 to 100% quiescent cells, epidermal growth factor, insulin, and the bile acid, deoxycholic acid, at concentrations at which it normally occurs in the aqueous phase of human feces, stimulated proliferation as measured by autoradiography. Exposure of the cells to combinations of these factors resulted in additive increases in growth. In conclusion, cells from the normal mouse colon can now be cultured while retaining at least two normal marker functions and moreover respond to some known mitogens and the potential tumor promoter deoxycholic acid. The cells can also be subcultivated while maintaining their epithelial morphology and marker functions for at least 3 passages. PMID- 1721908 TI - Serial cultivation of epithelial cells from human and macaque salivary glands. AB - To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham's F12 and Dulbecco's modified Eagle's media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions conditioned to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression. PMID- 1721909 TI - Regulation of angiogenesis in vitro by collagen metabolism. AB - The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ecstatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by adding L-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix. PMID- 1721910 TI - A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases. AB - Previously we demonstrated that transgenic mice expressing a mutant keratin in the basal layer of their stratified squamous epithelia exhibited a phenotype bearing resemblance to a subclass (Dowling Meara) of a heterogeneous group of human skin disorders known as epidermolysis bullosa simplex (EBS) (Vassar, R., P. A. Coulombe, L. Degenstein, K. Albers, E. Fuchs. 1991. Cell. 64:365-380.). The extent to which subtypes of EBS diseases might be genetically related is unknown, although they all exhibit skin blistering as a consequence of basal cell cytolysis. We have now examined transgenic mice expressing a range of keratin mutants which perturb keratin filament assembly to varying degrees. We have generated phenotypes which include most subtypes of EBS, demonstrating for the first time that at least in mice, these diseases can be generated by different mutations within a single gene. A strong correlation existed between the severity of the disease and the extent to which the keratin filament network was disrupted, implicating perturbations in keratin networks as an essential component of these diseases. Some keratin mutants elicited subtle perturbations, with no signs of the tonofilament clumping typical of Dowling-Meara EBS and our previous transgenic mice. Importantly, basal cell cytolysis still occurred, thereby uncoupling cytolysis from the generation of large, insoluble cytoplasmic protein aggregates. Moreover, cell rupture occurred in a narrowly defined subnuclear zone, and seemed to involve three factors: (a) filament perturbation, (b) the columnar shape of the basal cell, and (c) physical trauma. This work provides the best evidence to date for a structural function of a cytoplasmic intermediate filament network, namely to impart mechanical integrity to the cell in the context of its tissue. PMID- 1721911 TI - Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles. AB - We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1 fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm. PMID- 1721912 TI - Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix. AB - Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1. PMID- 1721913 TI - Prostatic epithelial cells in culture: phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity. AB - The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCI. The enzyme directly solubilized from prostatic cells with Triton X 100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721914 TI - DNA and RNA within the nucleus: how much sequence-specific spatial organization? AB - Spatial organization of various nuclear components is often proposed as a means by which nuclei more efficiently carry out their various tasks. Such functional compartmentalization may involve a sequence-specific packaging and placement of DNA and RNA. Here we review recent insights, allowed primarily by advances in fluorescent in situ hybridization methodology, into the organization of nucleic acids within individual nuclei. PMID- 1721915 TI - Expression of growth-regulated genes in normal and SV40 transformed hamster fibroblasts. AB - Transformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth-regulated genes in SV40-transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1-specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk-ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk-ts13/1%), was also included in these studies. The low serum adapted SV40-transformed cells and the spontaneous tk-ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721916 TI - Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. AB - The disposition, function and fate of centrosomes were analysed in mouse oocytes undergoing in vitro meiotic maturation, using multiple-label fluorescence microscopy. Oocytes fixed at various points during meiotic progression were double labeled with either human centrosome-specific antibody, 5051, and anti tubulin antibodies or 5051 and MPM-2 antibodies in order to evaluate the microtubule nucleation capacity and phosphorylation status of centrosomes during this process. Double labeling with anti-tubulin antibodies revealed two populations of centrosomes that undergo stage-specific changes in number, location and microtubule nucleation capacity in relation to spindle assembly and cytoplasmic events. Specifically, one population was consistently associated with chromatin throughout meiotic maturation whereas a second population of cytoplasmic centrosomes exhibited maximal numbers and nucleation capacity at prometaphase and anaphase of meiosis-I. Quantitative evaluation of cytoplasmic centrosomes indicated increased numbers during the transition from diakinesis to prometaphase and metaphase to anaphase and total disappearance during telophase. Colocalization studies with MPM-2 revealed that centrosomes were always phosphorylated. However, at metaphase of meiosis I and II the microtubule nucleation capacity of centrosomes was diminished. These results suggest the existence of two discrete populations of centrosomes in the mouse oocyte that are coordinately regulated to subserve aspects of microtubule organization relative to both nuclear and cytoplasmic events. PMID- 1721917 TI - Combined effects of extracellular matrix and growth factors on NBT-II rat bladder carcinoma cell dispersion. AB - Using the rat bladder carcinoma cell line NBT-II we showed that collagens but not laminin and fibronectin were able to induce cell scattering. Acidic fibroblast growth factor and transforming growth factor alpha also promoted NBT-II cell dispersion on glass or tissue culture plastic. We have now further analysed the scatter response to these two growth factors in the presence of extracellular matrix molecules. In the presence of growth factors, no peripheral single-cell dispersion occurred on fibronectin and laminin, although time-lapse video analyses revealed intense cell mingling and motility inside the monolayer forming around NBT-II aggregates. Patterns of strings or files of cells protruding from the monolayer were often observed. The presence of a scattering activity in the complex acellular extracellular matrix deposited by NBT-II cells themselves strongly suggested that substratum conditioning was responsible for this effect. On the other hand, the two growth factors accelerated collagen-mediated NBT-II individual cell dispersion and locomotion in a reversible way. As a marker of cell dissociation, we studied desmosome distribution in aggregate cultures: desmosomes were present in aggregates formed in suspension even in the presence of growth factors, whereas internalization occurred after cell-to-substratum contact. On laminin or fibronectin and in the presence of growth factors, peripheral cells inside the halo of NBT-II aggregates did not exhibit desmosome linkages. These observations suggest that scatter effects per se are dependent on the composition of the extracellular matrix. In particular, on a substratum nonpermissive for direct cell translocation, individual cell dispersion can be replaced by en bloc patterns of migration following substratum conditioning by the cells. PMID- 1721918 TI - Role of membrane phosphotyrosine proteins in human spermatozoal function. AB - The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94,000 +/- 3000 and 46,000 +/- 3000 Mr on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94,000 +/- 3000, 46,000 +/- 3000, 25,000 +/- 7000 and 12,000 +/- 2000 Mr, respectively, were labeled with 32P when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher 32P incorporation into 94,000 +/- 3000 Mr proteins and 17% less incorporation into 12,000 +/- 2000 Mr proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94,000 +/- 3000 Mr proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721919 TI - Monoclonal antibodies to Leishmania mexicana promastigote antigens. I. Secreted acid phosphatase and other proteins share epitopes with lipophosphoglycan. AB - The abundant surface glycolipid, lipophosphoglycan (LPG), of Leishmania promastigotes is composed of phosphosaccharide repeating units linked via a phosphosaccharide core to a conserved lyso alkylphosphatidylinositol membrane anchor. It is shown in this paper that monoclonal antibodies (mAbs) directed against LPG also react with an acid phosphatase secreted by L. mexicana promastigotes. Acid phosphatase purified by column chromatography (apparent Mr = 100,000) reacts in immunoblots with the anti-LPG mAb AP3 and another mAb, L3.13, which does not recognize LPG. mAb L3.13 was used to purify the enzyme by affinity chromatography. The resulting glycoprotein has the same molecular weight and binds AP3 on immunoblots. The secreted phosphatase is non-covalently associated with a high molecular weight, galactose-containing glycan or proteoglycan that is recognized by both AP3 and L3.13. In addition to acid phosphatase, other parasite proteins appear to be modified by LPG epitopes. PMID- 1721920 TI - Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells. AB - In order to study the effects of insulin-like growth factor (IGF-I) and insulin like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP 1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells. PMID- 1721921 TI - Evaluation of human sperm morphology using strict criteria after Diff-Quik staining: correlation of morphology with fertilization in vitro. AB - New, very strict criteria were used after Diff-Quik staining for evaluating sperm morphology. The results of morphology scoring were correlated with the fertilization rate in vitro. Semen samples from 64 men participating in an in vitro fertilization programme were used for this study. All men had to have a sperm concentration of greater than or equal to 20 million/ml and a progressive motility of greater than 30%. The morphology evaluation using strict criteria was performed on the same aliquot of semen as that used for in-vitro insemination. If strict criteria showed that normal morphology was less than or equal to 4%, the fertilization rate per oocyte was 23%. If normal morphology was greater than or equal to 11%, 77% fertilization occurred. For proportions of normal morphology between 4 and 11%, the fertilization rate per oocyte was 59% (P less than 0.000001). Among all these morphology groups, classical semen parameters, such as the mean volume, the mean concentration and the mean motility, did not differ significantly, except for the morphology evaluation using WHO criteria. The correlation with fertilization was better for morphology evaluation using strict criteria than for WHO. In conclusion, the method of evaluating sperm morphology based on very strict criteria allows a more accurate prediction of the chance of fertilization in vitro. Further studies should be done to establish the most appropriate cut-off points for severely impaired, intermediate and high fertilization rates. PMID- 1721923 TI - Detection of Helicobacter pylori in stomach tissue by use of a monoclonal antibody. AB - Monoclonal antibodies were produced against an acid glycine extract of Helicobacter pylori ATCC 43504T. One of these appeared to be specific for H. pylori; it recognized all H. pylori isolates by an indirect immunofluorescence assay (IIF) but it did not cross-react with the other strains tested, including different species of the genera Helicobacter, Campylobacter, and Wolinella. Different strains of members of the families Enterobacteriaceae and Pseudomonadaceae or other gram-negative bacteria tested also gave negative reactions. Indirect immunofluorescence assay of antral biopsy specimens identified 54 of 56 infected patients (96.4%), and it may be able to detect nonviable organisms after antibiotic therapy. PMID- 1721922 TI - Molecular relatedness of Staphylococcus epidermidis isolates obtained during a platelet transfusion-associated episode of sepsis. AB - Staphylococcus epidermidis was isolated from the blood of a 25-year-old pregnant woman following the administration of eight units of platelets. She had developed chills and a fever of 41.4 degrees C soon after the transfusions were completed. S. epidermidis was also obtained from all eight platelet units, as well as from the packed-erythrocyte unit associated with the first unit of platelets. The isolation of the same organism from these epidemiologically related sources provided us with the opportunity to phenotypically and genetically characterize the isolates. Several typing methods, including four molecular techniques, were used to increase our chances of finding any differences between the isolates under investigation. Phenotypic analyses demonstrated that S. epidermidis isolates from the patient, platelet units, and erythrocyte unit reacted in exactly the same manner in 15 biochemical tests, exhibited slime production, and had the same antibiotic susceptibility pattern. Genetic analyses, which included plasmid profiles, plasmid cross-hybridization, field inversion gel electrophoresis, and ribotyping, substantiated the relationships between the S. epidermidis isolates from the patient, platelet units, and erythrocyte unit. Eight S. epidermidis control strains unrelated to the case were found to differ significantly from the platelet-related strain. PMID- 1721924 TI - Central projections of the sciatic, saphenous, median, and ulnar nerves of the rat demonstrated by transganglionic transport of choleragenoid-HRP (B-HRP) and wheat germ agglutinin-HRP (WGA-HRP). AB - The central projections of the rat sciatic, saphenous, median, and ulnar nerves were labeled by injecting each nerve with 0.05 mg B-HRP, or 0.5 mg WGA-HRP, or a mixture of both. The B-HRP labeled large dorsal root ganglion cells (30-50 microns) and, correspondingly, 98% of axons labeled in a rootlet were meyelinated; although all sizes of myelinated axons were labeled, a greater proportion fell in the large ranges (2-6.5 microns axon diameter) than in the small ranges (0.5-2 microns). Primary afferents labeled with B-HRP were distributed in laminae I, III, IV, and V of the dorsal horn and extended into the intermediate grey and the ventral horn; Clarke's column and the respective dorsal column nuclei were also densely labeled. Motoneurons of the nerve were densely labeled by B-HRP, including extensive regions of their dendritic trees. In contrast, WGA-HRP labeled small dorsal root ganglion cells (15-25 microns) and in the dorsal rootlets, 84% of the labeled axons were nonmyelinated; the small population of labeled myelinated afferents mainly fell within the smaller ranges (0.5-2.0 microns). Terminal fields of WGA-HRP labeled afferents were restricted to the superficial dorsal horn (laminae I-III), and to limited regions in the dorsal column nuclei. Sciatic nerve projections traced by labeling with B-HRP alone or in combination with WGA-HRP were more extensive than previously described when using either native HRP or WGA-HRP. Afferents to the dorsal horn extended from L1-S1, to Clarke's nucleus from T8-L1, to the ventral horn from L2 L5, and extended throughout the medial and dorsal region of the gracilie nucleus. Motoneurons were found from L4-L6. Using the same tracers, saphenous projections extended in the superficial dorsal horn from caudal L1 to rostral L4, in the deep dorsal horn to mid L4 and along the length of the central part of the gracilie nucleus. The median nerve projected to the internal basilar nucleus from C1-C6, the dorsal horn from C3-T2, Clarke's nucleus from T1-T6, the external cuneate nucleus, and a large central area throughout the length of the cuneate nucleus. Motoneurons were located in dorsolateral and ventrolateral nuclear groups from C4 through C8. The ulnar nerve projections were less extensive but also included the internal basilar nucleus from C1-C6, the medial region of the dorsal horn from C4 T1, Clarke's nucleus from T1-T6, the external cuneate nucleus, and the medial part of the cuneate nucleus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1721925 TI - Central projections from the skin of the hand in squirrel monkeys. AB - Central termination patterns of afferents from the hands of squirrel monkeys were studied after subdermal injections of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) or cholera toxin subunit B conjugated to HRP (BHRP). WGA-HRP more effectively labeled axons terminating in the superficial dorsal horn of the spinal cord, while BHRP more effectively labeled axons terminating in the deeper layers. Injections of both tracers, when restricted to parts of glabrous digits, palm, or dorsal hand, revealed somatotopic patterns in the spinal cord and pars rotunda of the cuneate nucleus that were, in some respects, similar and, in other respects, quite different from those previously reported for macaque monkey (Florence et al., J. Comp. Neurol. 286:48-70, '89). As in macaques, injections in digits 1-5 produced a rostrocaudal sequence of foci of terminations in the cervical spinal cord. However, inputs from the palm were located medial to those from the digits, whereas the palm is represented lateral to the digits in macaque monkeys. Since inputs from the palm is also medial in the dorsal horn in cats (Nyberg and Blomqvist, J. Comp. Neurol. 242:28-39, '85), the condition in squirrel monkeys may be similar to the generalized state. In the cuneate nucleus, single injections in the hand produced dense label in the pars rotunda, and sparse label in the rostral and caudal poles. As in macaque monkeys, inputs from specific parts of the hand related to rostrocaudal clusters of cells that are cytochrome oxidase dense. The representation of the digits differed from macaques in that the digits were represented dorsal to the palm, rather that ventral to the palm as in macaques. Again, comparisons with cats suggest that squirrel monkeys have the more generalized pattern. Finally, inputs from the hair, dorsal surfaces of the digits terminated on the same clusters as the inputs from the glabrous, ventral surfaces, apparently overlapping somewhat. The proximity of these terminations from dorsal and ventral surfaces of the digits may be related to observations that cortical representations of the glabrous surfaces of digits become responsive to dorsal surfaces of the same digits when inputs from glabrous skin are chronically deactivated (e.g., Merzenich et al., Neuroscience 3:33-55, '83). PMID- 1721926 TI - Isolation of monoclonal antibodies monospecific for bovine beta-lactoglobulin. AB - Monoclonal antibodies were generated against purified bovine beta-lactoglobulin A. Six antibodies reactive only with beta-lactoglobulin were selected. At least one of the antibodies appeared to be species monospecific for bovine beta lactoglobulin. The remaining five antibodies recognized proteins in caprine and porcine whey fractions. One monoclonal antibody (59-1) exhibited a distinct 2:1 preference for beta-lactoglobulin B over A at near neutral pH (7.5). These antibodies should prove extremely useful adjuncts in structural studies of bovine beta-lactoglobulin. PMID- 1721927 TI - Granulocyte colony-stimulating factor effects on lymphocytes and immunoglobulin concentrations in periparturient cows. AB - Immunomodulatory effects of recombinant bovine granulocyte colony-stimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony stimulating factor (5 micrograms/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG1, conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bovine granulocyte colony-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to alleviate immunosuppression in periparturient cows. PMID- 1721928 TI - Evaluating acrosome reaction steps with brightfield and differential interference contrast microscopy techniques. AB - Acrosomal integrity of bovine spermatozoa was evaluated using naphthol yellow S erythrosine B stain with bright-field microscopy, .2% glutaraldehyde fixation with Nomarski optics, and live wet smears with Nomarski optics to compare the evaluation techniques. Four ejaculates per two Holstein bulls were enriched through a Percoll gradient, capacitated with heparin, and acrosome-reacted with lysophosphatidylcholine to prepare spermatozoa for in vitro fertilization. Spermatozoa samples were taken after each preparatory step, and percentage of intact acrosomes was evaluated with the stain, .2% glutaraldehyde, and live wet smear. Acrosomal integrity decreased with each preparatory step across all techniques. The decrease in integrity from heparin to lysophosphatidylcholine indicated that all methods detected the acrosome reaction. Technique means across preparation steps showed no differences. Correlations between microscopy techniques were high. The live wet smear used no fixation methods. Both the naphthol yellow S-erythrosine B stain and the .2% glutaraldehyde techniques employed fixation steps that may have introduced artifacts that could influence the data. The live wet smear evaluated by Nomarski optics allowed a comparable evaluation of the acrosome-reacted bovine spermatozoa. Comparing brightfield with differential interference contrast microscopy for detecting the acrosome reaction in bovine spermatozoa showed that both methods were equally accurate. PMID- 1721929 TI - Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm. AB - Semen from four Holstein bulls was evaluated to compare effects of four extender treatments on postthaw semen quality. Extender fractions A and B, either heated whole milk or 20% egg yolk-citrate, were combined to yield the extender treatments 1) milk and milk, 2) milk and egg yolk-citrate, 3) egg yolk-citrate and milk, and 4) egg yolk-citrate and egg yolk-citrate. Semen was evaluated at thawing and after 30, 60, 120, and 180 min of incubation at 38.5 degrees C. Flow cytometry showed that acridine orange-stained sperm were most susceptible to in situ DNA denaturation when fraction A was milk. For sperm stained with rhodamine 123, flow cytometry showed that the proportion with intact mitochondrial membrane potential was lowest of all treatments at thawing but greatest at 180-min incubation with milk and milk extender. Flow cytometry of propidium iodine stained sperm showed greatest proportion of cell membrane intact sperm when fraction A was egg yolk-citrate. Light microscopy showed the lowest proportion of cell membrane intact sperm with milk and milk extender after eosin-aniline blue vital staining. Postthaw motility scores tended to be reduced when both extender fractions were egg yolk-citrate. Results demonstrate differential extender effects on postthaw semen quality and indicate that altering extender composition or sequence of adding extender components may improve postthaw quality of cryopreserved sperm. PMID- 1721930 TI - A pattern of test findings predicting attention problems at school. AB - The aim of this study was to identify different types of neuropsychological test profiles that would predict attention problems at school. Forty-six children with mild developmental disorders, among whom a high frequency of attention deficit disorder (ADD) was expected, were examined just before they started school. The tests were mainly drawn from a new assessment, called NEPSY, and corresponded to the various components of attention. The test profiles were grouped with the aid of a Q-type factor analysis into five subgroups. The test profiles of two of the subgroups were suggestive of attention deficits. The predictions were based on difficulties in tests aimed to evaluate impulse control, sustained attention, and selective attention. As such findings were found in both of the subgroups they were collapsed into one. Half a year later the collapsed subgroups were found to have a higher frequency of attention problems at school than the other three subgroups. PMID- 1721931 TI - Malignant CD5 B cells--biased immunoglobulin variable gene usage and autoantibody production. AB - Malignant CD5 B cells obtained from patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) were analyzed for immunoglobulin variable gene usage, CD5 gene expression and autoantibody production. A statistically significant biased usage of the VH5, VH6 and VKIII immunoglobulin variable gene families was observed. It is important to point out that both VH5 and VH6 are extremely small families which are located at the 3' extremity of the immunoglobulin variable gene locus. We determined that the transcription of the CD5 gene in T cell malignancies, CLL, SLL and a selected group of EBV transformed lines was identical. Autoantibody production was studied in a panel of heterohybridomas obtained by the fusion of CLL cells with mouse myeloma line SP2/0. A large fraction of these heterohybridomas secrete autoantibodies; some were monospecific, some bispecific and some polyspecific. PMID- 1721932 TI - Antigenic mimicry and autoimmune diseases. AB - The finding of cross-reactive autoantibodies or sequence homology does not necessarily mean that this molecular mimicry is biologically meaningful or associated with disease pathogenesis. For example, relatives of persons with putative autoimmune insulin-dependent diabetes [123], and elderly humans [124] have a high incidence of autoantibodies which are generally not associated with autoimmune disease. In addition, natural antibodies to cell constituents [125] may be present in normal sera. These antibodies need to be directed against biologically important domains of host cell proteins in order to mediate autoimmune disease [27]. In spite of extensive homology between two sequences, a cross-reactive immune response may not be generated. The dissimilar amino acids should not be radical substitutions or affect the binding properties of the molecule. For instance, antibodies to synthetic peptides with only one substitution in a 19 amino acid sequence may not bind the whole protein [126]. Despite an identical six amino acid sequence shared by HLA-B27 and an EBV protein, no cross-reactive antibodies to EBV peptides were found in HLA-B27 positive patients with AS or RS. Unless the homology and subsequent crossreactive immune response can recognize a host protein intimately involved in disease pathogenesis, autoimmune disease is unlikely to occur. PMID- 1721933 TI - Anti-Jo-1 autoantibodies and the immunopathogenesis of autoimmune myositis. AB - Polymyositis and dermatomyositis are inflammatory myopathies characterized by proximal muscle weakness and myopathic electromyographic and histological findings. While the causes of myositis are not known, the close association of these disorders with a spectrum of autoantibodies suggests an etiologic and/or pathogenetic role for autoimmune processes. Of particular interest in this regard are antibodies directed against histidyl as well as other tRNA synthetases which are almost uniquely associated with myositis and may define a distinct subset of patients. Recently we isolated the histidyl tRNA synthetase gene which encodes the autoantigen representing the most frequent target of the myositis autoimmune response. The isolation and expression of this gene has allowed us to investigate both the autoreactive epitopes on histidyl-tRNA synthetase and the extent to which these correlate with functional epitopes on the molecule. As described here, the results of these studies as well as other recent data pertaining to the immunopathogenesis of myositis, provide a framework for delineating the mechanisms which render synthetases and other translation-related proteins autoantigenic in myositis, and allow one to examine the significance of such autoimmune responses in the etiology and pathogenesis of inflammatory myopathy. PMID- 1721934 TI - Analysis of prognostic factors in thymectomized patients with myasthenia gravis: correlation between thymic lymphoid cell subsets and postoperative clinical course. AB - We retrospectively examined the postoperative clinical course of 50 consecutive patients with myasthenia gravis who underwent a transsternal extended thymectomy. Twenty-six patients (52%) showed a steady improving postoperative course without any additional immunotherapy (group A). The remaining 24 patients (48%) had an intractable postoperative course, and most of them needed additional immunotherapy (group B). A significant increases in CD3+ cells, HLA-DR+ cells and B-cells and significant decreases in CD1+ cells and CD8+ cells were observed in the thymuses from patients in group B, compared with the findings in group A. In the peripheral blood, the ratio of CD4+ cells to CD8+ cells was significantly increased in group B. Patients who were either under 40 years of age, females, or without thymoma had good clinical courses after the thymectomy alone. PMID- 1721935 TI - Long-term pinealectomy alters hypothalamic serotonin metabolism in the rat. AB - In the present study, the effects of long-term pinealectomy on tryptophan, 5 hydroxytryptamine (5-HT or serotonin), 5-hydroxy-3-indoleacetic acid (5-HIAA), and tryptophan hydroxylase and monoamine oxidase activities were studied in preoptic area-anterior hypothalamus (POA-AH) and in the medial and posterior hypothalamus of the rat. After pinealectomy, 5-HT levels decreased significantly in medial hypothalamus but increased in the POA-AH. The levels of 5-HIAA decreased significantly in the POA-AH and medial hypothalamus. Tryptophan levels remained unchanged while tryptophan hydroxylase activity diminished significantly in POA-AH and medial hypothalamus. Monoamine oxidase activity remained unchanged in the hypothalamic regions. These results suggest that pinealectomy induces differential inhibitory actions on the serotoninergic terminal regions, mainly in anterior and medial hypothalamic areas. PMID- 1721936 TI - Exocrine pancreatic insufficiency-like syndrome in giraffe. AB - Studies were conducted on four giraffe (Giraffa camelopardalis) with diarrhea and three clinically healthy ones. The feces from both healthy and sick animals were examined to determine amylase, lipase and trypsin activity. In the feces of the giraffe with diarrhea a significant decrease of amylase and lipase activity was noted. The trypsin activity remained unchanged. Pancreatic exocrine insufficiency like syndrome was diagnosed on the basis of laboratory investigations and histopathological examinations. The administration of pancreatic enzyme supplements (Pancreatin; Polfa, Poland) had a noticeable effect on the normalization of clinical state and laboratory data of the giraffe with diarrhea. Determination of amylase and lipase activity in the feces may be helpful in determination of the condition of the pancreas in the course of chronic diarrhea in giraffe. PMID- 1721937 TI - [The COP-BLAM III therapy of non-Hodgkin's lymphoma]. AB - COP-BLAM III therapy was given to 18 patients with non-Hodgkin's lymphoma, and the therapeutic effects as well as adverse effects of the treatment were examined. Of the 18 patients 16 had a complete remission (CR) and 2 showed an partial remission (PR) with a total response rate of 100%. In terms of the stage of disease, CR was achieved in all patients in stage III and in 11 of 13 patients in stage IV. Patients with neutrophil counts less than 1,000/microliters were given rhG-CSF (1.5 micrograms/kg/day, sc), which significantly shortened the duration of neutropenia and decreased the number of days with episodes of fever when compared with those not given rhG-CSF, consequently facilitating the treatment without prolonging the dosing intervals. No serious infection was observed. Adverse effects included neutropenia of less than 1,000/microliters in 6 of the 18 patients (33.3%), thrombocytopenia less than 5 x 10(4)/microliters in 3 (16.7%), nausea and vomiting in 8 (44.4%), peripheral neuropathy in 4 (22.2%) and stomatitis in 4 (22.2%). There were no fatalities caused by the treatment. The above findings indicate that COP-BLAM III therapy is capable inducing high frequency of complete remissions in non-Hodgkin's lymphoma and that its combination with G-CSF can improve the results of the therapy and relieve adverse reactions. PMID- 1721938 TI - [Role of membrane molecules in complement system]. PMID- 1721939 TI - [Transport mechanisms across the biological membranes]. PMID- 1721940 TI - 15 parameters of coagulation and fibrinolysis in children with type I diabetes mellitus (onset period). AB - 15 parameters of coagulation and fibrinolysis were investigated in 38 children with type I diabetes mellitus without clinical signs of diabetic angiopathy. Compared to an age matched non diabetic control group spontaneous platelet aggregation was enhanced, plasma levels for factor VIII C, von Willebrand factor, antithrombin III and C-1-inactivator were elevated, alpha-2-macroglobulin was decreased at onset of the disease. During remission (3, 6, 12 months) these changes reverted to normal. Alpha-2-antiplasmin decreased after 12 months. If, during partial remission, diabetic duration was longer than one year an increase of factor VIII C was seen again. In comparison to the controls no significant alterations were found for ristocetin cofactor, fibrinogen, plasminogen and alpha 1-antichymotrypsin. It seems likely that changes in plasmatic coagulation, fibrinolysis and platelet function during the onset period of diabetes mellitus type I are due to metabolic changes and precede diabetic angiopathy. PMID- 1721941 TI - Ring chromosome 22: a case report. AB - A three year old girl with ring chromosome 22 is described. The clinical findings include epicanthus, flat nasal bridge, hypertelorism, long eye-lashes, lymphoedema, hypoplastic toe nails, hydrocephalus and muscular hypotonia. Speech and language development is delayed. At three years the child begins to walk. PMID- 1721942 TI - [The preparation of iron (III) chloride solution from salt with an unknown water content]. AB - The authors have derived an equation of direct relationship between solution density and iron (III) chloride concentration in glacial acetic acid. Using this equation, one may define the concentration of a solution prepared from commercial salt, having determined its density beforehand. PMID- 1721943 TI - [The blood level of cobalamin as an indicator of the functional status of the liver in jaundice of different etiologies]. AB - Blood serum free vitamin B12, bilirubin, alanine aminotransferase levels were measured and thymol test made in 168 patients with viral hepatitis A, 13 with chronic hepatitis, 8 with mechanical jaundice of neoplastic origin, 7 with calculous cholecystitis, and 8 with functional hyperbilirubinemia by the microbiologic methods. Elevated blood serum levels of free vitamin B12, conforming to the disease severity and stage, were revealed in the patients with viral hepatitis A, chronic active hepatitis, and mechanical jaundice of a neoplastic origin with liver involvement. Correlations between cobalaminemia and other functional liver tests were observed. Therefore blood serum vitamin B12 measurements may be used for the assessment of the disease activity and severity of liver parenchyma injury. PMID- 1721944 TI - [A stable reagent for the-single stage determination of inorganic phosphate]. AB - A recipe of a simple reagent for phosphorus detection has been developed, consisting of ammonium molybdate (4 mM), sulfuric acid (0.2 N), and Tween-80 (0.2%). The developing phosphate staining may be registered in 15 min at a wavelength of 350 nm. The product molar extinction is equal to 1.20.10(4) M-1.cm 1, this being close to that of molybdic blue. Phosphate staining is characterized by the stability of results and insensitivity to the presence of a number of substances used in enzymology. The prepared reagent is fit for experiments within a fortnight if stored in the cold. PMID- 1721945 TI - [Indices of lipid metabolism and peroxidation in normal newborn infants]. AB - Studies of lipid metabolism and peroxidation values, carried out in healthy newborns in the town of Irkutsk, have shown that total lipid and triglyceride levels in these newborns over the first ten days of their lives were similar to those in age-matched infants in the European USSR, whereas plasma total cholesterol levels were significantly higher in them as against the newborns in other regions of the country. The studies included measurements of malonic dialdehyde and total antioxidative activity of blood plasma and red cells, of catalase and peroxidase activities, of red cell peroxidation resistance. PMID- 1721946 TI - [Gas chromatographic determination of phospholipid fatty acids and free cholesterol in a single sample]. AB - The results of gas chromatographic measurements of phospholipid fatty acid spectrum and free cholesterol in the blood serum and red cells of diabetics are presented. A deficit of polyunsaturated fatty acids (at the expense of C18:2 and C20:4) in the presence of a high free cholesterol level was found to be a characteristic feature of diabetes mellitus. PMID- 1721947 TI - [The chemiluminescent determination of cholesterol]. AB - A chemiluminescent method has been offered to assay the blood serum total cholesterol. The method is based on coupled enzymatic reactions in two steps: (1) esterified cholesterol hydrolysis by cholesterol esterase in the presence of sodium cholate and (2) cholesterol oxidation by cholesterol oxidase and chemiluminescent assay of the released hydrogen peroxide in peroxidase reaction with luminol and p-iodophenol. Separation of these steps results in a better accuracy of the test as against the one-step procedure. High sensitivity of the method (the detection limit being 1 mM of cholesterol in a luminometer cuvette) permits high dilutions of the serum, this essentially eliminating the admixture effects on the enzymatic reactions. The findings of the suggested method are in good correlation with those of the spectrophotometric assay of cholesterol using the KONE kit. PMID- 1721948 TI - [Determination of apoprotein B using the method of solid-phase immunoenzyme analysis]. AB - Indirect solid-phase enzyme immunoassay was used to measure apoB in human blood serum. ApoB obtained by traditional gel filtration in sepharose 4B and high performance liquid chromatography was used as an antigen. Anti-apoB antibodies were immunochemically identified. Working regimens for the components and conjugates employed were selected. ApoB level in human blood serum, determined by this method, has made up 0.83 +/- 0.1 g/l in men and 0.88 +/- 0.1 g/l in women. PMID- 1721949 TI - [A method of diagnosing congenital adenylosuccinase enzymopathy]. AB - The authors present a simple and convenient method for the diagnosis of a recently described condition, congenital adenyl succinase enzymopathy, that is characterized, among other signs, by the appearance in patients' urine of succinylaminoimidazole carboxamide riboside (SAICRs) and succine adenosine (SA), dephosphorylated (nucleoside) forms of two adenylsuccinase substrates. Both the nucleosides may be sorbed by ion exchanger when the urine is let pass through H+ cationite and then eluated by solved ammonia (earlier this was known only in respect of SAICRs). The new feature of the procedure is detection of the afore said nucleosides in ammonia eluate from its UV spectrum. The value of the ratio between ammonia eluate absorption in 270 and 250 nm (A270/A250) was found a reliable criterion of both SAICRs and SA absence in the urine and therefore of a normal adenyl succinase level (A270/A250 ratio is 0.25-0.45 in this case) and of their presence in the urine and therefore of their presence of adenyl succinase enzymopathy (A270/A250 ratio, above 0.70, typically 0.90-1.0). PMID- 1721950 TI - [Determination of lipids in amniotic fluid using the chemiluminescent method]. PMID- 1721951 TI - [The functional activity of neutrophilic granulocytes in patients with erysipelas]. AB - Dynamic cytochemical studies of neutrophilic granulocyte functional activities in erysipelas patients have revealed that reduced level of cationic proteins and unbalanced activities of the respiratory enzymes help assess the disease severity, and if these disorders persist during the convalescence period and between the recurrences, they indicate a reduction of the nonspecific resistance and help predict the recurrences of the condition. PMID- 1721952 TI - [Anomalous neutrophilic granules in patients with angina and acute respiratory diseases]. AB - Analysis of the informative value of the lysosomal cationic test (LCT) and of the kinetics of neutrophilic nuclear fragmentation helped reveal significant differences in the neutrophil granulocyte morphology and function in the patients with acute bacterial tonsillitis and acute respiratory viral infections. The detected varieties in A granule formation and in cellular ability to lysosomal secretion of cationic proteins allow the employment of LCT in the assessment of the disease severity and in the early differential diagnosis between tonsillitis and acute respiratory diseases. PMID- 1721953 TI - [The effect of molecular oxygen and calcium ions on the membrane permeability of erythrocytes]. AB - Extracellular Ca has shown the highest membrane destroying effect of all the factors studied. Its injurious action augmented when blood samples were exposed to atmospheric oxygen. To improve the diagnostic value of biochemical studies at the expense of red cell membrane stabilization the authors recommend the use of blood citrate plasma as the basic object of enzymic studies. PMID- 1721954 TI - [Blood serum iron: diagnostic significance and study methods (review of the literature)]. PMID- 1721956 TI - [Determination of the erythrocyte deformability index]. AB - The suggested modification of the red cell deformability index consists in estimation of the ratio between the diameters of spots of 0.02 ml of 60% suspension of washed red cells and of 0.2 ml of normal saline, placed onto Filtrak-388 filter. The modification is simple, available, well reproducible, and sensitive. PMID- 1721955 TI - [The state of erythrocyte membranes in diabetes mellitus]. AB - Study of red cell membranes in diabetics has shown that physicochemical shifts in the red cell membrane lipid bilayer, elevated lipid peroxidation, and disordered thiol compound metabolism are among the crucial aspects in the pathogenesis of diabetes mellitus. EPR spectroscopy in complex with other biochemical methods will help monitor the adequacy of therapy. PMID- 1721957 TI - [Evaluation of the intensity of the duodenogastric reflux based on the alkaline phosphatase and trypsin activities in the stomach contents]. AB - Alkaline phosphatase and trypsin activities were measured in the gastric contents of patients with chronic gastritis with visually fixed duodenogastric reflux and without reflux. The authors discuss the advantages of a comparative approach to the enzymatic assessment of duodenogastric reflux presence, intensity, and length. PMID- 1721958 TI - [A comparative evaluation of the results of studies of multi-moment chromatic intubation and cholecystography]. AB - These methods were employed in examinations of 55 patients with various gastrointestinal diseases. The findings of both methods coincided in assessment of gallbladder kinetic function in 36 (66%) patients. In 19 (34%) patients the results did not coincide, in 9 of them multi-moment chromatic duodenal intubation with xylite stimulation has yielded overstated results, and in the remaining 10 patients gallbladder hyperfunction was detected by x-ray examination after egg yolk stimulation. Individual reactions of the body may be responsible for this discrepancy; other explanations are unstable pathologic dysfunction of the gallbladder and the psychoemotional status of patients during the examination. PMID- 1721959 TI - [Development and a clinical test of a method of assessing the interactions between polymorphonuclear leukocytes and monocytes in human blood]. AB - A method for assessment of interactions between blood polymorphonuclear leukocytes and monocytes is described, demonstrating the monocyte ability to stimulate polymorphonuclear leukocyte migration in capillary leukocyte migration test. Clinical trials of the method have shown enhanced monocyte stimulation of the migration of polymorphonuclear leukocytes in psoriasis patients during exacerbations of the disease. The method may be used for assessment of the immune status to detect disorders at the level of intercellular relations. PMID- 1721960 TI - [Determination of the circulating antibodies to cardiomyocyte membrane proteins based on the immunoblotting principle]. AB - Cardiomyocyte membrane proteins (CMP) were isolated from the hearts of subjects dead from injuries or brain tumors by differentiated centrifugation in sucrose density gradient for the determination of anti-CMP antibodies in the blood serum samples of myocarditis patients. CMP preparation reactivity with blood serum antibodies of patients with myocarditis and other myocardial diseases, as well as with systemic lupus erythematosus, was assessed by the immune blotting technique. The findings evidence a high incidence of antibody interaction with CMP with a molecular mass of 67 kD. The method is highly specific and sensitive, its results are fairly well reproducible, and the technique is simple. PMID- 1721961 TI - [A modification of the immunoenzyme method of determining class A immunoglobulins]. AB - The authors suggest a variant of enzyme immunoassay for measuring IgA in the blood and other biologic fluids. This method is based on the use of polystyrene balls, made in the USSR, as the solid phase; the balls are placed in biochemical tubes with a conic bottom. The sensitivity of the method is approximately 40 times superior to the widely known radial diffusion in gel. Use of polystyrene balls presensitized with antispecies antibodies helps cut down the time of the assay. The method is recommended for clinical laboratories that are intended for unsophisticated investigations. PMID- 1721962 TI - [The diagnosis of campylobacteriosis. 1]. PMID- 1721963 TI - [A study of the variability of group A type M29 streptococcus on exposure to biologically active substances]. AB - Biologically active substances spermidine and the compounds contained in the low molecular fraction of cattle blood serum change the phenotype of type 29 group A Streptococcus. Amino acid analysis and enzymatic destruction of cell walls have demonstrated changes of the streptococcal biological characteristics and cell wall structural organization. These changes may result from loss of supervariable component of the M-protein when compounds from cattle blood serum low-molecular fraction are added to culture medium and by unbalanced growth of Streptococcus after spermidine addition to growth medium. PMID- 1721964 TI - [Anaerodisks for the identification of anaerobic microflora]. AB - The authors review the present-day methods for the identification of gram negative anaerobic nonsporogenous bacilli and cocci with the use of anaerodisks. Data are presented on the generic and species composition of obligate anaerobes of the gastric contents, proximal section of the jejunum, and the distal portion of the large intestine. Use of anaerodisks helped identify up to 90% of anaerobic cultures isolated from the gastrointestinal tract of man. PMID- 1721965 TI - [Gas chromatographic analysis of the metabolic products of anaerobic bacteria. 2. Clostridia]. AB - Qualitative and quantitative characteristics of clostridial metabolites (short chain carbonic acids and alcohols) were analyzed in associations of these bacteria with facultative anaerobes. Various bacterial groups were found to vary as regarded the composition and quantitative ratio of the major bacterial metabolites. These data may become the criteria for the indication of pathogenic clostridia in mixed cultures after anaerobic passages for 24-48 h at 37 degrees in a medium with 1% glucose. PMID- 1721966 TI - [A method of determining nitrate reductase in enterobacteria in a solid culture medium]. PMID- 1721967 TI - [Bacterioscopic and bacteriologic study of the vaginal contents of pregnant women]. AB - Comparative analysis of the results of bacterioscopic and bacteriologic studies of vaginal contents of 100 pregnant women suffering from nonspecific inflammatory diseases of the genitals has shown the advantages of the quantitative method of examination as against the bacterioscopic method. This method permits the detection of risk groups among women with Stage II vaginal purity and the identification of the etiologic factor of nonspecific inflammatory diseases of the genitals, thus being conducive to timely etiotropic therapy (bacterial agents). PMID- 1721968 TI - [Evaluation of the coagglutination reaction with urine in generalized forms of meningococcal infection]. AB - The coagglutination test with concentrated urine was tried in 59 patients with validated generalized forms of meningococcal infection and in 23 ones with meningitides and pneumonia of a different etiology to assess the diagnostic value of this test. Positive results were obtained in 64.4% of the test group patient and in 4.3% of the reference patients (in a case with pneumococcal meningitis). The antigen serologic group in the urine coincided with the serologic group of the meningococcus that induced the disease in only 19 (50%) of the 38 patients in whom antigenuria+ was detected. In the rest cases urine samples reacted parallel with 2-7 antisera, in 5 patients with the antisera heterologic towards the serologic group of the meningococcus responsible for the disease. Antigenuria was observed between the second and ninth days of the illness, its peak was recorded on days 4-6. Repeated urine tests are more likely to yield positive results. The authors come to a conclusion that the test is simple and harmless for patients, but the presence of false positive results permits regarding it as but an auxiliary one. PMID- 1721969 TI - [A device for scanning electrophoregrams]. AB - A microprocessor analyzer for electrophoregram scanning on cellulose acetate film, agarose and polyacrylamide gels has been designed. The principal characteristics of the device: scanning time 20 seconds, size of the analyzed plate 140 x 110 mm, scanning length 65-85 mm, maximal number of analyzed fractions 10. Analysis and recording of the results takes up to 1 min. PMID- 1721970 TI - [A flow-type cuvette for spectrophotometry]. PMID- 1721971 TI - Essential hypertension: hemodynamic and therapeutic changes over 20 years. AB - The hemodynamic disturbances in essential hypertension depend on the age of the subjects and the severity of the hypertensive state. In a 20-year follow-up study, we demonstrated a shift in the hemodynamic characteristics from a high output/normal resistance pattern in the early phase toward a low-flow/high resistance pattern in the later period. The arteriovenous oxygen difference increased, particularly during exercise, and the oxygen reserve in venous blood decreased. Treatment with conventional drugs (beta-blockers and/or diuretics) for 20 years with satisfactory control of diastolic blood pressure did not prevent a marked increase in total peripheral resistance and a reduction in stroke index and cardiac index. In recent years, beta-blockers with vasodilating activity have been introduced in the treatment of hypertension (labetalol, acebutolol, dilevalol, celiprolol, and carvedilol). The hemodynamic effects of these compounds clearly differ from the changes induced during acute and chronic conventional beta-blocking treatment. In contrast to the usual beta-blockers, these drugs reduce total peripheral resistance acutely, and reduce cardiac index considerably less; arteriovenous oxygen difference is more normal. In an acute study of carvedilol in 18 patients with essential hypertension, total peripheral resistance was reduced 8% at rest (supine) and 6% during exercise. Exercise heart rate was reduced 12%, but due to an increase in stroke index, the reduction in cardiac index was only 6%-considerably less than what is seen during treatment with conventional beta-blockers. PMID- 1721972 TI - Clinical challenges in hypertension: therapeutic applications of new vasodilating beta-blockers. Symposium proceedings: 13th scientific meeting of the International Society of Hypertension, Montreal, Canada, June 1990. PMID- 1721973 TI - The effects of intravenous carvedilol, a new multiple action vasodilatory beta blocker, in congestive heart failure. AB - Recent studies have shown that beta-blockers may be effective in the management of heart failure. However, negative inotropic effects of these agents may offset the beneficial properties of up-regulation of the beta-receptors and reduction in myocardial oxygen demand. Carvedilol is a new drug which possesses a balanced combination of vasodilatation and beta-blockade. Previous studies have shown that carvedilol may have beneficial effects on left ventricular function in patients with ischemic heart disease. We have performed a preliminary study to address the safety and acute effects of intravenous carvedilol in 17 patients with chronic congestive heart failure secondary to ischemic heart disease. Acute hemodynamic changes were monitored by right heart catheterization and arterial cannulation. Ejection fraction was also monitored by radionuclide ventriculography. Significant reductions in heart rate (79 +/- 14 to 72 +/- 12 beats/min, p less than 0.001) systolic and diastolic blood pressure (137 +/- 20/72 +/- 8 to 119 +/- 19/66 +/- 8 mm Hg, p less than 0.001 and p less than 0.01), systemic vascular resistance (1766 +/- 367 to 1518 +/- 377 dynes/s/cm-5/m2, p less than 0.001) and pulmonary artery wedge pressure (20 +/- 8 to 15 +/- 7 mm Hg, p less than 0.001) were observed. Ejection fraction increased significantly from 24 to 28% (p less than 0.001) but there was little change in cardiac index or stroke volume index. The peak changes occurred at 10 min and the effect on pulmonary wedge pressure was maintained up to 30 min. No adverse effects were noted. The improvements in left ventricular filling pressure and systolic function, and the reduction in sympathetic activity may combine to produce an important therapeutic advantage in congestive heart failure. Further studies with this interesting agent are recommended. PMID- 1721974 TI - The interplay of ischemic and hypertensive pathophysiology on cardiac function. AB - Many patients with systemic hypertension also have concomitant angina pectoris. Hypertension is a risk factor for the development of coronary artery disease as well as an aggravating factor once symptomatic ischemic heart disease develops. It would be prudent to use one agent that reduced the elevated blood pressure and relieved anginal symptoms while providing no negative affects on hemodynamics or other cardiac risk factors. beta-Adrenergic blockers (traditional agents and newer compounds with ancillary properties) and calcium-entry blockers have been shown to be effective as monotherapies in patients with angina. They also have the ability to induce left ventricular hypertrophy regression. There is some evidence that some of these therapies may arrest the development of arteriosclerosis and may have an inhibitory effect on platelet aggregation. What remains controversial is the optimal amount of blood pressure reduction in patients with hypertension coexistent with angina, or whether one specific agent or drug class would have an overall advantage in reducing the risk of morbidity or mortality. PMID- 1721975 TI - Features of the acute hypotensive action of carvedilol and its ameliorating effect on myocardial ischemia. AB - The objective of the present experiment was to investigate the following parameters concerning carvedilol: (a) the time course of acute hypotensive effect, (b) the influence of alpha 1-adrenoceptor blockade on the acute hypotension induced by carvedilol, and (c) the effect of carvedilol on myocardial ischemia. In conscious, catheter-implanted spontaneously hypertensive rats, carvedilol at the doses of 10 and 30 mg/kg, p.o., dose-dependently decreased mean arterial blood pressure (MAP). At 10 mg/kg, MAP was significantly decreased for 16 h, and at 30 mg/kg, it was significantly decreased until 24 h after administration. Thus, carvedilol shows long-lasting antihypertensive effects by a single oral administration. In anesthetized Wistar rats, intravenous administration of carvedilol (0.3 mg/kg) produced an immediate decrease in blood pressure. On the other hand, in phentolamine-pretreated rats whose blood pressure was restored by prostaglandin F2 alpha, the acute hypotension produced by carvedilol was abolished similarly to the case with labetalol (0.6 mg/kg) and prazosin (0.01 mg/kg). The hypotensive effect of either hydralazine (0.2 mg/kg) or nitroprusside Na (0.005 mg/kg) was attenuated by about 20%, whereas the effect of diltiazem (0.2 mg/kg) was not attenuated. These results indicate that alpha 1 adrenoceptor blocking action of carvedilol contributes to a large extent to the acute hypotensive effect of this drug. In anesthetized open-chest dogs, myocardial ischemia was produced by partial occlusion of the left anterior descending coronary artery. Myocardial pH of the ischemic region was decreased by about 0.7 units. Carvedilol at the doses of 0.1 and 0.3 mg/kg, i.v., dose dependently attenuated the decrease in myocardial pH accompanied with decreases in MAP and heart rate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721976 TI - Comparison of the effects of carvedilol, propranolol, and verapamil on in vitro platelet function in healthy volunteers. AB - The effects of the two beta-blockers carvedilol and propranolol and the calcium antagonist verapamil on platelet aggregation induced by ADP, epinephrine, or collagen was examined in the serum of eight healthy volunteers. The inhibitory potential of the three drugs on the arachidonic acid-prostaglandin pathway was measured by the assessment of the formation of the stable metabolite thromboxane B2. Furthermore, the influence of drugs on intracellular cAMP levels was measured in platelets aggregated with ADP, epinephrine, or collagen. All three drugs were able to inhibit platelet aggregation induced by a threshold concentration of ADP, epinephrine (10 microM), or collagen (1 micrograms/ml) in a concentration dependent manner, preventing aggregation completely in the high micromolar range. Propranolol is 1.6 and 2.3 times more active than verapamil and carvedilol in inhibiting ADP-induced platelet aggregation. Although the two beta-blockers were marginally more potent than verapamil in inhibiting platelet aggregation induced by collagen, this was not statistically significant. Both propranolol and verapamil were slightly more active than carvedilol in inhibiting epinephrine induced platelet aggregation, a trend consistent with the IC50 values. Intracellular cAMP levels decreased in platelets aggregated with ADP, epinephrine, and collagen. The inhibition of platelet aggregation with the three drugs resulted in a return of intracellular cAMP levels to basal values. Carvedilol and propranolol were of similar potency and around 1.5-3 times as potent as verapamil for all agonists. The formation of thromboxane B2, an end product of the arachidonic acid pathway, could be completely inhibited by the three drugs. Propranolol was 2.4 and 2.1 times more potent than carvedilol and verapamil when platelets were aggregated with ADP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721977 TI - A comparative study of carvedilol, slow-release nifedipine, and atenolol in the management of essential hypertension. AB - Carvedilol is a novel treatment for hypertension, having a balanced pharmacology of vasodilation and beta-receptor blockade. We present here the results of a three-way, multicenter, comparative study on the use of carvedilol, slow-release nifedipine, and atenolol in the management of essential hypertension. A total of 311 patients was entered into the study, of which 293 were randomized to one of the three treatment regimens. Full data are available on 255 patients. Systolic and diastolic blood pressure measurements, in both sitting and standing positions, were taken, together with the heart rate. There was no consistently significant difference between treatments with respect to blood pressure control. Differences in heart rate were more pronounced, with the reduction due to carvedilol being generally intermediate between nifedipine and atenolol. Further studies of carvedilol in hypertension, as well as other indications, are warranted. PMID- 1721978 TI - Hypertension and coronary artery disease: a therapeutic challenge. AB - The coexistence of the syndromes of essential hypertension and coronary artery disease (CAD) poses a major but common therapeutic challenge. High blood pressure is one of the most potent risk factors for the early development of CAD. Conversely, the presence of CAD significantly worsens the predictive prognosis associated with high blood pressure. Moreover, metabolic risk factors for the acceleration of both syndromes are similar, particularly with regard to abnormalities of the blood lipid profile, carbohydrate intolerance, and obesity. It is clinically crucial, therefore, to direct drug therapy not only at the immediate alleviation of the symptoms and signs of each syndrome but also to control the cardiac and vascular risk factors common to both syndromes. Carvedilol is a third-generation vasodilating beta-adrenoceptor antagonist with advantageous ancillary pharmacologic properties for the treatment of the patient with high blood pressure complicated by CAD. The immediate advantages of the drug in the treatment of both syndromes are distinct. In the patient with high blood pressure, carvedilol controls the pressure throughout the 24 h of the day and suppresses the increase associated with exercise. In the patient with CAD, the drug is efficacious in relieving anginal pain and electrocardiographic signs of myocardial ischemia. By reducing blood pressure and heart rate and retarding their increases during exercise, the drug exhibits a potent ability to reduce left ventricular work, wall stress, myocardial oxygen consumption, and left ventricular myocardial ischemia. In the patient in whom both syndromes coexist, carvedilol affords a remedy for both.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721979 TI - Effects of carvedilol on serum lipids in patients with essential hypertension. AB - The purpose of this study was to investigate the effect of carvedilol on serum lipids in patients with mild-to-moderate essential hypertension. Twenty-one patients with blood pressure greater than or equal to 160/95 mm Hg after a 4-week placebo run-in period were initially given 10 mg of carvedilol once daily. The dose was increased to 20 mg after 4 weeks if the target blood pressure was not achieved. The duration of treatment was 12 weeks. After 12 weeks of administration, blood pressure and the pulse rate (PR) declined significantly (blood pressure from 173/105 to 142/91 mm Hg, p less than 0.001; PR from 74 to 67 beats/min, p less than 0.001); however, serum lipids [total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein (HDL), HDL2, and HDL3], lipoprotein fraction (alpha, pre-beta, and beta), apoprotein fraction (A I, A-II, CII, CIII, and E), and atherogenic index [(total cholesterol - HDL cholesterol) divided by HDL cholesterol] were not altered significantly. There were no side effects reported during the trial. From these results, it can be concluded that carvedilol has no adverse effect on the coronary risk profile as reflected by lipid measurements, and is an efficacious, safe, well-tolerated antihypertensive drug in patients with mild-to-moderate hypertension. PMID- 1721981 TI - The kidney in hypertension: treatment strategies. AB - As in any patient with hypertension, the first goal in the hypertensive patient with renal disease is to control the hypertension. The second goal is to reduce overall cardiovascular risk, which includes an increased likelihood of coronary events. The third goal is to minimize the likelihood that renal disease will progress, or minimize the rate of progression. There is substantial evidence to indicate that control of hypertension, however achieved, will slow the rate of progression of renal injury and destruction. Studies in animal models have indicated that angiotensin-converting enzyme inhibitors are more consistent in preventing progressive renal injury, and perhaps have a more sustained action than standard agents employed hitherto. In the patient with a clear indication for beta-adrenergic blockade, such as a prior coronary event or angina pectoris, a beta-adrenergic blocking agent should be employed. Preliminary, but intriguing, evidence is available to suggest that a beta-adrenergic blocking agent that incorporates a vasodilator action may enjoy special benefits in terms of achieving the three goals. In the case of renal injury, completion of the studies required to prove this point will improve our ability to deal with the problem of hypertension in the patient with renal disease. PMID- 1721980 TI - A multicenter comparison of carvedilol with hydrochlorothiazide in the treatment of mild-to-moderate essential hypertension. AB - The efficacy and safety of carvedilol, a beta-blocker with vasodilatory properties, were compared with that of hydrochlorothiazide (HCTZ) both at a once daily dose of 25-50 mg in a double-blind, randomized, parallel-group, multicenter study. Following a single-blind placebo phase of 3-6 weeks, 201 eligible patients (aged 27-88 years) were randomized to receive 8 weeks of treatment with either carvedilol or HCTZ, 25 mg doubling to 50 mg at week 4 if the respone was inadequate. Sitting and standing blood pressure and heart rate were recorded 2 h after the first dose and 24 h postdose at weeks 4 and 8. The analysis included 179 patients (11 having withdrawn, including 5 for adverse events, and 11 excluded for protocol violations). There were no statistically or clinically significant differences between treatment groups. Eighty-six percent of patients in the carvedilol group and 88% in the HCTZ group had an 8-week sitting diastolic blood pressure less than or equal to 90 mm Hg or decreased by greater than or equal to 10 mm Hg. Safety profiles were similar for both agents, with a tendency to lower uric acid and total cholesterol levels with carvedilol. Carvedilol and HCTZ at doses compared in this study have similar efficacy and tolerability, with laboratory evidence to suggest a more favorable metabolic profile for carvedilol. PMID- 1721982 TI - Pharmacokinetics and efficacy of carvedilol in chronic hemodialysis patients with hypertension. AB - The efficacy, safety, and pharmacokinetics of carvedilol were investigated in an open trial performed on six patients with hypertension and chronic renal failure requiring hemodialysis. The plasma level of unchanged carvedilol after a single dose of 10 mg reached a peak 1-5 h after administration both on days with and without hemodialysis. The drug was gradually metabolized thereafter and had almost disappeared from the plasma after 24 h. Blood pressure was lowered by carvedilol both on days with and without hemodialysis. No carvedilol passed through the dialysis membrane. During the 4-week administration period of carvedilol at 10 mg/day, assessment of plasma samples taken just prior to early morning administration demonstrated no drug accumulation. Blood pressure was well controlled during the administration period. Tolerance to the antihypertensive effect was not observed. Heart rate was not significantly changed at any time. There were no side effects in any of the patients during the trial, and laboratory parameters remained unchanged. These results indicate that carvedilol is a safe and effective antihypertensive agent for use in patients on chronic hemodialysis. PMID- 1721984 TI - Arteriosclerosis obliterans of the lower limbs as a model of peripheral vascular disease with hypertension. AB - In patients with arteriosclerosis obliterans of the lower limbs, hypertension is often characterized by a disproportionate increase in systolic pressure whereas diastolic blood pressure remains normal or even low. This hemodynamic pattern is associated with a reduction in systemic and forearm arterial compliance, whereas cardiac output and peripheral vascular resistance remains within the normal range in the absence of complications. The reduced arterial compliance is influenced not only by structural modifications of the arterial wall but also by changes in vasomotor tone. Whereas increased sodium intake and beta-blockade by propranolol reduce arterial compliance, nitrate derivatives increase arterial compliance with a resulting selective decrease in systolic pressure. PMID- 1721983 TI - Clinical efficacy of carvedilol in severe hypertension. AB - In an open clinical study, the efficacy and safety of carvedilol was investigated in 26 severely hypertensive patients controlled inadequately on a diuretic [diastolic blood pressure (DBP) greater than 120 mm Hg at first visit and greater than 110 mm Hg following more than 1 week administration of a diuretic]. Following diuretic treatment all patients were initially administered 5 mg of carvedilol once daily. The dose was gradually increased to 10 mg and 20 mg until DBP was reduced below 100 mm Hg or until it was reduced by at least 10 mm Hg. Antihypertensive activity of carvedilol (5 mg) was sufficient in only three cases, but after 4 weeks (inpatients) or 8 weeks (outpatients) administration of carvedilol (10 mg or 20 mg), DBP/systolic blood pressure was significantly reduced from 176 +/- 6/117 +/- 3 to 145 +/- 3/94 +/- 2 mm Hg (p less than 0.001) in all patients. Overall, a sufficient antihypertensive effect was observed in 80% of the patients. Heart rate was significantly decreased from 76 +/- 2 to 67 +/- 2 beats/min, but no patient experienced bradycardia. Carvedilol was generally well tolerated. These findings suggest that 10-20 mg of carvedilol once daily, in combination with a diuretic, is an effective and safe treatment for patients with severe hypertension. PMID- 1721985 TI - Antihypertensive treatment in concomitant peripheral vascular disease: current experience and the potential of carvedilol. AB - The relative importance of hypertension as a risk factor for peripheral vascular disease is of the same order as coronary artery disease. The design of drug studies in occlusive vascular disease presents several problems. First, investigations must be placebo-controlled and crossover in design. Second, since these patients are very much at risk from other vascular occlusions, length of treatment phase is critical. Third, drug doses are also critical--probably best chosen by titration to similar antihypertensive effect. Fourth, patients must be trained in treadmill procedure. Fifth, measurements of limb blood flow must be accompanied where possible by "functional" assessment, e.g., claudication distance. With respect to the specific problem of low perfusion pressure distal to the blockage of peripheral vasculature, resting blood flow may remain normal, implying compensatory reduction in tone of arteriolar resistance vessels. Thus, regional circulation distal to blockage is sensitive to changes in perfusion pressure. There is the risk of "steal" with vasodilator agents; however, conflict exists in the literature over effects of beta-blockers in this situation. In view of its peripheral hemodynamic profile, the theoretical possibilities with the beta-blocker/vasodilator carvedilol in patients with hypertension and peripheral vascular disease seem extremely rewarding, but remain to be borne out in practice. PMID- 1721986 TI - Congestive heart failure: pathophysiology and management with special reference to systemic hypertension. AB - The recognition and management of heart failure is based on the knowledge of the underlying disease and precipitating factors. The underlying causes are all cardiovascular whereas precipitating factors comprise both cardiac and a variety of noncardiac factors. The influence of hypertension on the development of heart failure is complex. Increased ventricular systolic pressure raises myocardial oxygen demand, resulting in ischemic heart syndromes and arrhythmias. Also, systemic hypertension leads to hypertrophy, resulting in systolic and diastolic function abnormalities. Generally, heart failure is controlled by treating the underlying cause, by removal of precipitating factors, and by treatment of failure itself. Heart failure therapy involves general measures, and pharmacological and surgical therapy. The pharmacological treatment involves the use of diuretics, vasodilators, and positive inotropic agents. In patients with heart failure and hypertension, arterial and mixed type vasodilators are the drugs of choice. Positive inotropic agents have to be used with care because of the potential induction or aggravation of myocardial ischemia. Interest in beta adrenergic-blocking agents, especially those with ancillary properties similar to vasodilators, has recently surged and will continue to provoke more and more clinical research in an attempt to unravel the complexities of these cardiovascular diseases and their therapies. PMID- 1721987 TI - Quantitation of urinary alpha 2u-globulin and albumin by reverse-phase high performance liquid chromatography. AB - A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the quantitation of alpha 2u-globulin, the major urinary protein excreted by adult male rats, and albumin has been developed. Total urinary proteins, isolated by a simple Sephadex G-25 gel filtration step, are separated and quantitated by reverse-phase HPLC on a C4 Macrosphere 300 column. The proteins are separated and eluted with a two-step gradient of acetonitrile in aqueous trifluoroacetic acid. Detection limits of 9 and 25 micrograms/mL of urine were established for albumin and alpha 2u-globulin, respectively. Quantitation of urinary excretion of the two proteins in young adult male and female rats and aging male rats showed that values obtained with this method compared favorably with values from previously developed immunological techniques. To quantitate total urinary protein excretion, we modified the Bradford protein assay to use rat urinary protein as standard. Given the established importance of alpha 2u globulin in the development of male rat-specific nephrotoxicity and nephrocarcinogenicity, these methods should be useful for studying the renal handling of this protein under normal and nephrotoxic conditions. PMID- 1721988 TI - [The modification of the toxicity produced by chemotherapy in testicular cancer by adapting its intensity to prognostic groups]. AB - BACKGROUND: The reduction of iatrogenesis is fundamental in the treatment of germ cell testicular tumors (GTT) because of the high incidence of cures achieved. On the other hand, the tumoral mass and the serum concentration of the beta fraction of the gonadotropin hormone (CGH) and of alphafetoprotein allow the differentiation of 2 clear prognostic groups; those for which the intensity of chemotherapy may be adapted to reduce its collateral effects and improve the results. METHODS: In the Oncology Department of the Hospital de la Santa Creu i Sant Pau 23 patients with GTT of good prognosis were treated between 1984-1990. These patients were given the combination of etoposide-cisplatin (EP) over the same period. Twenty patients with a bad prognosis received the alternative scheme of bleomycin-vincristine-methotrexate-cisplatin/etoposide-cisplatin- phosphamide (BOMP/EPI). RESULTS: In comparison to the classical treatment with cisplatin vinblastine-bleomycin (PVB) the EP association demonstrated less iatrogenesis except in regards to the formation of granulocytopenia which was higher. The BOM/EPI combination conditioned greater hematological toxicity during the acute phase and the first observations suggested a diminution of chronic iatrogenesis. CONCLUSIONS: These results indicate that chemotherapy in testicular cancer may be adapted to the aggressiveness of the with the aim to thereby reduce global toxicity. PMID- 1721989 TI - [Pulmonary Kaposi's sarcoma in a heterosexual parenteral drug addict patient with the acquired immunodeficiency syndrome]. AB - A case of Kaposi syndrome is described in a 28-year-old heterosexual male with acquired immunodeficiency syndrome. The disease began clinically with pulmonary disease, without mucocutaneous lesions. This form of presentation is extremely infrequent and has not been described in non-homosexual subjects. Clinical manifestations were fever, cough and dyspnea. Thoracic radiography observed a perihilar interstitial pattern which evolved to a bilateral nodular pattern with perihilar adenopathy. There was endobronchial disease, however pulmonary biopsy was required for diagnosis. Complete tumoral remission was achieved with adriamycin, bleomycin and vincristine. PMID- 1721990 TI - [Cytomegalovirus ileocolitis in patients with HIV infection is a treatable cause of severe abdominal problems]. PMID- 1721991 TI - Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection. AB - In situ hybridization histochemistry was used to study changes in mRNAs coding neuropeptides such as alpha-calcitonin gene-related peptide (CGRP), beta-CGRP, cholecystokinin (CCK) and galanin, in rat facial motoneurons following axotomy of the facial nerve. In control rats, 38%, 55% and 7% of the facial motoneurons expressed alpha-CGRP, beta-CGRP and CCK mRNAs, respectively. No galanin mRNA containing motoneurons were observed in these animals. The levels of mRNA for alpha-CGRP, CCK and galanin were increased while the beta-CGRP mRNA level was decreased after axotomy. The levels of mRNAs for these peptides returned to the control values by 2-4 weeks after nerve crush, whereas nerve resection had more prolonged effects. Within 3-4 weeks after injury, nerve resection had greater effects on beta-CGRP, CCK and galanin mRNAs than did nerve crush. Thus, there appear to be differences in the regulation of mRNA expression of these peptides in axotomized motoneurons. PMID- 1721992 TI - Characterization of Xenopus laevis proenkephalin gene. AB - Enkephalins are opiate peptides found in a variety of tissues including brain and pituitary. In brain, they function as neurotransmitters, neuromodulators and neurohormones. Recent studies show that proenkephalin mRNA is expressed early in development both in mammals and the amphibian, suggesting that enkephalins may play a unique role in embryogenesis. In order to characterize factors which regulate the onset and patterning of expression of this gene in adult and developing frog embryos, the proenkephalin A gene was cloned from Xenopus laevis. The clones have been characterized by DNA sequencing and restriction endonuclease mapping. The gene is made up of three exons which span approximately 12 kb. Exon I encodes the 5' untranslated region of the mRNA. Exon II contains the signal peptide and the N terminus of the mature protein. Biologically active opioid peptides are generated from exon III. Comparison to mammalian proenkephalin genomic sequence indicated that nucleotide sequences of the 5' flanking region, noncoding exon I and exon II were not well conserved but exon III was highly conserved. Primer extension and RNase protection assay analyses of the RNA transcripts revealed two major 5' ends. The putative TATA box, CAAT box, CRE and Pit 1 elements have been identified on this gene by sequence homology to published consensus sequences. To assay for sequences that could potentially regulate Xenopus proenkephalin expression, we transfected constructs that contained upstream genomic sequences linked to the CAT reporter gene into various eukaryotic cell lines. The expression of the fusion gene constructs were detected and could be induced 10- to 30-fold upon treatment with forskolin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721993 TI - A synaptic vesicle specific GTP-binding protein from ray electric organ. AB - A cDNA encoding a synaptic vesicle associated GTP-binding protein was identified by screening a lambda gt11 expression library derived from the electric lobe of Discopyge ommata with polyclonal antibodies recognizing vesicle-specific proteins of Mr 25,000. Nucleotide sequence analysis defines an open reading frame of 218 amino acids. The protein belongs to the ras superfamily and shares about 75% amino acid identity with smg-25A, B and C identified in bovine brain and rab3A characterized in rat brain. Northern blot analysis revealed a 4.5 kb transcript present only in neural tissues, the highest level of expression being observed in electric lobe. Western blot analysis of total tissue homogenates derived from D. ommata detected the protein in electric organ, forebrain and to a lesser extent in electric lobe and spinal cord. No immunoreactivity was detected in non neuronal tissues. Blotting of subcellular fractions derived from electric ray electric organ revealed that the GTP-binding protein co-purifies with synaptic vesicles. The neural specific expression and the localization to synaptic vesicles suggest a role of this protein in synaptic vesicle trafficking and targeting. PMID- 1721994 TI - Primary demyelination induced by exposure to tellurium alters mRNA levels for nerve growth factor receptor, SCIP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin proteolipid protein in rat sciatic nerve. AB - Weanling rats fed a diet containing tellurium develop a peripheral neuropathy characterized by a highly synchronous primary demyelination; this demyelination is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis. Expression of mRNA for the major structural proteins of PNS myelin, myelin basic protein and P0, is coordinately down regulated during the demyelinating phase and then up-regulated during the remyelinating phase (Toews et al., J. Neurosci. Res., 26 (1990) 501-507). We now report tellurium-induced alterations in gene expression for several proteins which are not major structural components of myelin in the peripheral nervous system. Expression of mRNA for nerve growth factor receptor in sciatic nerve was very low in control animals, but was markedly up-regulated after 3-5 days of exposure to tellurium, a time corresponding to the beginning of demyelination. Levels remained elevated during the subsequent period of remyelination. Expression of mRNA for SCIP (a presumptive transcription factor) was also up regulated in sciatic nerve following tellurium exposure, with a time course similar to that for nerve growth factor receptor. When examined as a fraction of total RNA, steady-state mRNA levels for 2',3'-cyclic nucleotide 3' phosphodiesterase and the myelin proteolipid protein were decreased during the demyelinating phase; however, this decrease could be largely accounted for by increased levels of total RNA. When analyzed on a 'per nerve' basis, steady-state mRNA levels for these two proteins were actually increased about 2-fold by 9 days after beginning tellurium exposure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1721995 TI - Conservation of antigenic epitopes of the inhibitory glycine receptor in rodent and goldfish CNS. AB - Monoclonal antibodies against the inhibitory glycine receptor of rat spinal cord were used to identify corresponding receptor polypeptides in goldfish CNS. Both Western blot analysis and quantitative receptor immunoassays revealed crossreacting antigens in goldfish brain membranes. A polypeptide of 46 kDa molecular weight is immunologically related to the 48 kDa alpha subunit of the mammalian receptor. Similarly, a large receptor-associated protein of 93 kDa is present both in goldfish and mammals. Throughout the goldfish CNS, glycine displaceable [3H]strychnine binding codistributes with the alpha subunit protein as determined immunologically. Glycine receptor contents were highest in goldfish medulla oblongata, medium in optic tectum and mesencephalon, whereas little or no receptor was detected in cerebellum, olfactory bulb, and spinal cord. Immunohistochemistry confirmed that the alpha subunit antigen and the 93 kDa protein were located in the plasma membrane of neurons and concentrated in small clusters found on the soma and dendrites. These data indicate that immunological properties and cellular distribution of glycine receptors are conserved from fish to mammals. PMID- 1721996 TI - Effects of background music on the remembering of filmed events. AB - The use of background music within films provides a naturalistic setting in which to investigate certain issues of schematic processing. Here, the relative placement of music was manipulated such that music either accompanied a scene's outcome, and thereby accentuated its affective meaning, or foreshadowed the same scene, and thereby created expectancies about the future course of events. In addition, background music was either congruent or incongruent with the affect of an episode's outcome. When subjects were later asked to recall the series of filmed episodes, results showed that expectancy violations arising from mood incongruent relations led to better memory in the foreshadowing condition, while mood-congruent relations led to better performance in the accompanying condition. Results from a recognition task further revealed that scenes unavailable for recall could be recognized when cued by background music. These overall findings are discussed in terms of selective-attending processes that are differentially directed as a function of background music. PMID- 1721997 TI - [A method for the quantitative determination of reticulocytes in induced reticulocytosis]. AB - A technique accelerating and simplifying the determination of reticulocyte counts in animals with reticulocytosis has been suggested. The results are correctly evaluated without microscopy. The technique can be also used for the identification of patients with hemolytic anemias, especially in field conditions. An application for the technique developed has been submitted and positive approval has been obtained on November 20, 1987. PMID- 1721998 TI - [Results of treatment of paralytic ileus caused by diffuse intra-abdominal metastasis with motilin. A pilot study of 25 patients]. AB - In a pilot-study 25 patients presenting with a paralytic ileus due to diffuse intraabdominal metastases were treated with motilin. There were 16 male (64%) and nine female (36%) patients. Gastric cancer was the most frequent cause (40%), followed by pancreatic (36%), and colorectal (20%) cancer. 36% had received a postoperative chemotherapy before commencing the motilin-scheme. In 92% motilin therapy was started within 48 hours after the diagnosis of paralytic ileus. There were no serious side-effects of motilin therapy. In approximately 80% the pretherapeutic state was improved. Thus, it seems worthwhile to investigate the influence of motilin on paralytic ileus in incurable cancer patients in a prospective controlled trial. PMID- 1721999 TI - [Laboratory chemical inflammation parameters in chronic inflammatory bowel diseases]. PMID- 1722000 TI - Neoplastic meningitis. AB - Neoplastic meningitis appears to be increasing in frequency with improvements in the treatment of many cancers. It is most often recognized in patients with leukemia, breast cancer, lymphomas, and small-cell cancer of the lung, although it may be seen with virtually any malignancy. Treatment should include intrathecal chemotherapy, radiation therapy to symptomatic areas of the CNS, and optimal therapy of the systemic cancer. New efforts are underway to decrease the toxicity and improve the efficacy of antineoplastic therapy for this devastating complication of cancer. PMID- 1722001 TI - Nerve plexus metastases. AB - Metastatic plexopathy is often a disabling accompaniment of advanced systemic cancer and may involve any of the peripheral nerve plexus. Brachial plexopathy most commonly occurs in carcinoma of the breast and lung; lumbosacral plexopathy is most common with colorectal and gynecologic tumors, sarcomas, and lymphomas. Regardless of the location, carcinomatous plexopathy typically is associated with severe unrelenting pain as the cardinal clinical feature. Later, weakness and focal sensory disturbances occur in the distribution of plexuses involvement. Epidural tumor involvement frequently (in more than 50% of patients) coexists with either plexopathy. In previously treated patients, the main differential diagnostic consideration is radiation-induced plexopathy. Treatment of metastatic plexopathy is palliative and includes radiotherapy to the tumor mass and chemotherapy. In selected patients, subtotal surgical resection of the tumor may be warranted. The response to therapy is modest and generally short lived. Efforts should be made to provide adequate pain control, to maximize remaining neurologic function, and to prevent complications of immobility produced by the neuromuscular dysfunction. PMID- 1722002 TI - Interactions of agonists with M2 and M4 muscarinic receptor subtypes mediating cyclic AMP inhibition. AB - In this study the similarities and differences between the M2 and M4 subtypes in their recognition of agonists were explored. A CHO-K1 cell line transfected with the human m2 receptor was used as a homogeneous M2 tissue for comparison with two putative M4 systems (rat striatum and the N1E-115 mouse neuroblastoma cell line). The equilibrium binding dissociation constants and intrinsic efficacies for seven muscarinic agonists were determined for their stimulation of cyclic AMP inhibition via the M2 and M4 receptors. Partial receptor occlusion with propylbenzilylcholine mustard was used to determine binding constants for the more efficacious drugs and the reference agonist oxotremorine-M. The binding dissociation constants and relative efficacies for other agonists were then determined in reference to oxotremorine-M by a null method. For the M2 receptor the agonist binding dissociation constants ranged in potency from oxotremorine (1.5 microM) to bethanechol (171 microM), whereas relative efficacies varied from that of muscarine (relative efficacy = 0.9) to the value for McN-A343 (relative efficacy = 0.04). In general, most agonists bound with similar potencies to M2 and M4 receptors (Kd values within a factor of 2-3). However, oxotremorine bound to the N1E-115 and striatal M4 receptors about 3-fold and 10-fold less potently, respectively, than it did to the M2 receptor. Another exception was pilocarpine, which bound to the N1E-115 receptor (1.9 microM) with 8-fold and 12-fold higher potency than to the CHO-K1 M2 receptor and the striatal M4 receptor, respectively. Despite the low affinity of bethanechol for the M2 receptor, it was an efficacious agonist (maximal response equivalent to that of oxotremorine-M; relative efficacy = 0.6) at this subtype, whereas it was a partial agonist (60%) with lesser efficacy in the clonal M4 system. In contrast, McN-A343 and arecoline were significantly more efficacious at the two M4 receptors than they were at the M2 receptor. The M4 system in the rat striatum displayed some similarity to the N1E-115 M4 system, with regard to the efficacy ranking for certain agonists (arecoline greater than bethanechol greater than McN-A343 greater than or equal to pilocarpine). This rank order was different from the ranking of these four agonists in the M2 system, indicating that these two M4 receptors are more similar to each other in efficacy ranking than they are to the M2 receptor. However, the rat striatal and N1E-115 M4 receptor differed in their binding of oxotremorine and pilocarpine, indicating that these two M4 systems were not pharmacologically identical.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722003 TI - Changes in gene expression and DNA methylation in adrenocortical cells senescing in culture. AB - Recent experiments in cultured bovine adrenocortical cells show that the previously observed phenotypic switching of CYP17 (steroid 17 alpha-hydroxylase) expression is preceded at a much earlier time by changes in methylation in the CYP17 5' flanking region. Two CpG sites that are methylated in the adrenal cortex in vivo were observed to undergo rapid demethylation when adrenocortical cells were placed in culture. Two adjacent CpG sites that are also methylated in vivo did not demethylate; these two sites are completely nonmethylated in fibroblasts. All CpG sites downstream, in the promoter or coding region, are always methylated in all tissues and in bovine adrenocortical cells even after many population doublings in culture. In contrast to the specific and rapid demethylation of sites in CYP17, satellite I shows a slower and apparently random loss of methylation that extends over the entire replicative life span. These changes in methylation provide examples of genetic instability in cells that undergo senescence in culture. Future experiments will focus on the relationship of these events to the phenotypic switching process. PMID- 1722004 TI - Protein synthesis and the components of protein synthetic machinery during cellular aging. AB - The slowing down of protein synthesis is a change widely observed during the aging of organisms. It has also been claimed that a decline in the rate of protein synthesis occurs during cellular aging. However, the evidence in favour of this view is not clear-cut, and reliable estimates of rates of protein synthesis during cellular aging have yet to be made. Studies on various components of the protein synthetic machinery during cellular aging have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Detailed studies on the structure and function of ribosomes, tRNA isoacceptor profiles, activities of aminoacyl-tRNA synthetases, levels and activities of initiation factors, rates of protein elongation, and the accuracy of protein synthesis will be needed before the molecular mechanisms of the regulation of protein synthesis during cellular aging can be understood. PMID- 1722005 TI - Cellular ageing related proteins secreted by human fibroblasts. AB - Fibroblast secreted proteins participate in the formation of extracellular matrix. Extracellular matrix affects growth factor action, mediates cell adhesion and supports cell growth. Structural and quantitative characteristics of secreted proteins are modified in a similar manner, during both in vivo and in vitro cellular ageing. Such ageing related modifications may either be directly controlled by primary ageing causes, or evolve from a reformation of the extracellular matrix induced by a few ageing defects in key proteins such as fibronectin. They may result in the further inhibition of cell adhesion, cell stimulation by growth factors and, eventually, of cell proliferative ability. PMID- 1722006 TI - Actin cytoskeletal network in aging and cancer. AB - The cytoskeleton is being recognized as an important modulator of metabolic functions of the cell. The actin cytoskeletal network, in particular, is involved in events regulating cell proliferation and differentiation. The state of actin in a variety of cell types is regulated by signals arising from the cell surface through a wide spectrum of interactions. In this review, we explore the role of actin cytoskeletal network in a series of events which are known to influence cell proliferation and differentiation. These include interaction of actin network with extracellular matrix proteins, cell surface membranes, second messengers, cytoplasmic enzymes and the nucleus. Because of the involvement of the actin network in such diverse interactions, we propose that alterations in the actin cytoskeletal function may be an important aspect of generalized decrease in cellular functions associated with aging. Preliminary data indicate that alterations in the cytoskeletal network do occur in cells obtained from older individuals. Alterations in actin state are also reported during malignant transformation of cells in culture, and in naturally occurring tumors. Taken together, the existing data seem to suggest that changes in the actin cytoskeletal network may be a part of the aging process as well as malignant transformation. Therefore, the study of the actin cytoskeletal network and its regulation has the potential to yield important information regarding cellular senescence and neoplastic transformation. PMID- 1722007 TI - Involvement of microtubules in modifications associated with cellular aging. AB - Microtubules are ubiquitous cellular components involved in the control of cell structure and functions, such as cell division, regulation of shape and polarity, intracellular transport, etc. Consequently, any alteration affecting them in structure or function has a good chance of affecting the cell and generally leads to cell dysfunctions. This has been shown for instance, after treatment with microtubule-interacting drugs. Cellular aging is also characterized by the appearance of various cell dysfunctions, but the possible involvement of the microtubules in the aging process, although a rather tempting hypothesis, has not yet been extensively investigated. In this paper, I will first rapidly review the different components that build, organize and control the microtubules in normal cells, independently of the aging process. I will then consider the possible involvement of the microtubules in the aging process, more particularly in models of cells aging in vitro and in aging neuronal cells, which have been the most extensively investigated. There is some evidence for alterations in the microtubule organization both in cells aging in vitro and in the aging brain. But the interpretation of these data awaits further experiments, taking into account the latest progress in tubulin genetics and in microtubule biochemistry. Microtubules could also represent one of the cellular targets affected after signal transduction and could thus be involved in the resulting cellular responses. This hypothesis will be discussed, as it offers new insights into the regulation of microtubule organization, dynamics and functions in normal cells, which will be worthwhile to investigate during the aging process. PMID- 1722008 TI - Changes in the cell surface of human diploid fibroblasts during cellular aging. AB - The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG 3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG 3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging. PMID- 1722009 TI - Oxidants and antioxidants in proliferative senescence. AB - In terms of the amount of experimental research it has generated the free radical theory of ageing is one of the most popular hypotheses to explain this ubiquitous phenomenon. From the theory two postulates were derived: either cellular defence mechanisms against free radical-dependent oxidants deteriorate during ageing of cells, or essential, unrepairable damages are imparted to the cell by oxidants regardless of the activity of antioxidant defence systems. The many reports dealing with a putative breakdown in antioxidant defence systems failed to positively support this postulate. However, a minor depletion in cellular glutathione by exposure to a model lipophilic peroxide led to a significant decrement in DNA and protein synthesis. In other words, the glutathione redox cycle is intrinsically fallible with respect to defending the cellular DNA replication system against this model lipophilic peroxide. Interestingly, after ageing in culture cells a partial uncoupling of the NADPH-producing and consuming systems tends to take place. Experiments involving the addition of antioxidants to the culture medium have failed to significantly extend the lifespan of cultured diploid somatic cells. The level of antioxidants appears to be a modulator rather than a primary determinant of cellular ageing in culture. Several lines of evidence suggest that DNA damages accumulate during ageing of the organism, but no oxidant-related DNA damage has been pinpointed in the cultured cell system. Human mutants with defects in antioxidant enzymes have not shown conclusive signs of accelerated ageing. Cells from patients with Werner's syndrome (progeria of the adult), on the other hand, do not suffer from a defect in their antioxidant defence system, nor do they accumulate more than normal amounts of autofluorescent products resulting from lipid peroxidation. The recent finding that Werner's syndrome constitutes a mutator phenotype may prompt the comparison of oxidant- and ageing-related mutation spectra in order to investigate a mutational theory of ageing as a new derivative from the free radical hypothesis. PMID- 1722010 TI - Homoeostatic imbalance during cellular ageing: altered responsiveness. AB - The inability of normal cells to maintain themselves for ever is a reflection of homoeostatic imbalance and a progressive failure of maintenance. Ageing cells respond less to growth stimulants whereas they show increased sensitivity to toxic agents including antibiotics, phorbol esters, radiation and other physical stresses. No major quantitative and qualitative defects in the receptor systems have been detected that could explain the reasons for altered responsiveness during ageing. Random metabolic defects in the processes involved in maintaining homoeostasis may be critical for causing homoeostatic imbalance, cellular ageing and death. PMID- 1722011 TI - Ageing in the chick lens: in vitro studies. AB - In principle, ageing may be due to the interaction of several factors, including the accumulation of random changes both genomic and non-genomic, secondary changes in a tissue contingent upon the changing function of other tissues, and programmed non-random changes in the tissue-specific expression of various genes. The use of a single tissue comprising one cell type only, in which the major gene products are well defined, in which there is a well attested series of developmental and age-related changes in cell properties and gene expression and which can be studied and compared in vivo and in vitro, offers advantages for investigation of these questions. The vertebrate eye lens possesses these advantages. The crystallins (proteins expressed at super-abundant levels in the lens) are well characterised. The lens epithelial cells (LEC) grow readily and can differentiate into the lens fibre cells in vitro, and, finally, such terminally differentiated cells may also be derived, by a process of transdifferentiation, from neural retina cells (NRC) in vitro. Thus the effect on ageing changes of the tissue of origin may also be studied. This article reviews our previous studies on long-term changes in growth potential, differentiation capacity and crystallin expression of chick lens cells in ageing cultures, their overall similarity to events in vivo and the effect on ageing changes of genotypes affecting the growth rate. It presents new information on these genetic aspects, and on crystallin expression in long-term ageing cultures of transdifferentiated neural retina, and compares the behaviour of ageing chick lens cells with that reported for mammals. PMID- 1722012 TI - Cellular ageing of Alzheimer's disease and Down syndrome cells in culture. AB - In Alzheimer's disease, the typical clinical symptoms and the pathological findings are restricted to the nervous system. Nevertheless, like in some other neurologic-metabolic disorders, several alterations are found in peripheral tissues. The aim of this study was to examine whether cellular properties which can be studied in vitro on skin fibroblast cultures obtained from Alzheimer's disease patients differ from those of age-matched controls. Down syndrome patients were also included, since the same neuropathological findings are present in nearly 100% of Down syndrome patients. Since Alzheimer's disease is an age-related disorder, we examined the growth characteristics of skin fibroblast cultures. The in vitro senescence of cultured fibroblasts is widely accepted as a model for in vivo ageing. Normal growth properties were found. We can conclude that there is no premature ageing in Alzheimer's disease nor in Down syndrome and that the abnormalities found in peripheral tissues are related to the disease itself. The beta amyloid precursor protein (beta APP) has been shown to have adhesive interactions. We therefore investigated several parameters of adhesion in the skin fibroblast cultures: adhesion to a fibronectin coat, adhesion to extracellular matrix of Alzheimer's disease cultures and semi-quantification of adhesion-related molecules (beta 1-integrin, cell surface proteoglycans, extracellular matrix proteoglycans, extracellular matrix fibronectin). No significant difference was found in the parameters examined. PMID- 1722013 TI - Differentiation of primary and secondary fibroblasts in cell culture systems. AB - As a function of the advancing development of Valo chicken, C3H mice, BN rats, and man in the embryonic, juvenile, adolescent, and senescent phases, stem cells and fibroblasts in the connective tissues of skin and lung differentiate along an 11-stage differentiation sequence in five compartments of the fibroblast stem cell system, when studied in primary ex vivo-in vitro systems. In the fibroblast stem cell system, three stem cells develop in the stem cell compartment along the cell lineage S1-S2-S3, three mitotic fibroblasts (MF) differentiate along the sequence MF I-MF II-MF III in the fibroblast progenitor compartment, three postmitotic fibroblasts (PMF) proceed in the fibroblast maturing compartment along the row PMF IV-PMF V-PMF VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. After a species- and tissue-specific period of high metabolic activity, PMF VI either dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment. The reiterated appearance of the 11 cell types in primary stem cell and fibroblast populations and the reiterated age-related changes in the cell type composition of the primary stem cell and fibroblast populations make it very likely that stem cell, mitotic and postmitotic fibroblast equivalents exist in vivo and that age-related changes of the frequencies of the stem cell and fibroblast equivalents result from the progressing differentiation of stem cell, mitotic, and postmitotic fibroblast equivalents along the 11 stage differentiation sequence in the fibroblast equivalent stem cell system in vivo. Secondary fibroblast populations derived from connective tissue of prenatal and postnatal skin of Valo chicken, C3H mice, BN rats, and man, including the normal embryonic human lung fibroblast cell line WI38, were also found to develop along a terminal stem cell sequence. Thus, secondary fibroblast populations in vitro constitute a representative material for studies of general and special issues of cell biology, such as terminal differentiation, aging, apoptosis, and transformation, as long as stem cell system-specific concepts and methods are employed in such investigations. PMID- 1722014 TI - Protein markers for cellular mortality and immortality. AB - Fixed mortality of normal somatic cells is a well-established fact though the mechanism underlying this universal phenomenon remains unknown. Use of immortal cells in conjunction with their normal mortal counterparts has delineated the dominant genetic nature of the senescent phenotype over immortalization. Although the involvement of proteins in determining the entry/exit/arrest of cells in the cell cycle is evident from the literature, none of them has been confirmed for its role in senescence-associated irreversible cell cycle exit/arrest. The identification of true mortality markers might be possible by selecting a system of natural and conditional aging achieved by the fusion of mortal and spontaneously immortalized cells of the same origin. We report here a few such protein markers which might serve as useful handles to tease out the molecular events determining mortality/immortality of cultured cells. PMID- 1722015 TI - Senescence and the accumulation of abnormal proteins. AB - Mammalian cells can produce abnormal proteins in a number of different ways. These include random errors during protein synthesis, spontaneous or metabolite induced modifications of amino acid sidechains and changes in polypeptide folding. The evidence that such alterations occur in proteins during growth and senescence is discussed. An important function controlling the accumulation of abnormal proteins is the rate at which they are hydrolysed by proteases. Modified proteins are much better protease substrates than their normal parent molecules, but in spite of this sensitivity to proteolysis they accumulate during ageing. This indicates a drop during senescence in the activity of those proteases degrading abnormal polypeptides. Ways in which abnormal proteins could inhibit cell growth and how these inhibitions may be negated during the immortalisation of diploid cells are discussed. PMID- 1722016 TI - Negative growth effectors and cellular senescence. AB - Current studies suggest a genetic program governs the lifespan of each organism. Using cellular senescence as a model system, components of this program for aging have been sought. Human diploid fibroblasts, upon reaching senescence, express active inhibitors of DNA synthesis. It is believed that such inhibitors could be members of a new family of negative growth effectors involved in the pathway to senescence. Factors capable of inhibiting DNA synthesis in a similar manner have also been identified from human quiescent fibroblasts and liver cells as well as from quiescent rodent liver cells. The relationship of these inhibitors to previously identified negative growth effectors and aging are discussed. PMID- 1722017 TI - Telomere loss: mitotic clock or genetic time bomb? AB - The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization. PMID- 1722018 TI - DNA methylation and cellular ageing. AB - Methylated cytosine (m5C) in DNA appears to be an important modulator of the expression of some genes. There are several lines of evidence that gradual loss of m5C is relevant to in vitro cellular ageing: m5C loss occurs during cell culture; m5C loss is detectable at an early stage of culture; m5C loss appears to be related to cell division not just duration in culture; the rate of m5C loss appears to be related to in vitro lifespan of the cell strain in question; and the total loss of m5C during an in vitro lifespan is significant by comparison with induced-changes in m5C levels which effect cell growth, or cause cell-death in culture. Progressive loss of m5C in dividing cells may thus produce the multi step cell division "clock" which underlies the Hayflick phenomenon. PMID- 1722019 TI - A re-examination of the effects of ionizing radiation on lifespan and transformation of human diploid fibroblasts. AB - Human diploid fibroblasts, strain MRC-5, were sequentially irradiated with 60Co gamma rays at intervals during their in vitro lifespan. The results indicate that 3 or 6 doses of 1 Gy can increase lifespan, and the same was true for cells treated with 3 doses of 3 Gy. Higher doses (5 x 3 Gy) did reduce growth potential, suggesting either that mid-late passage cells become more sensitive to radiation, or that doses beyond a given threshold reduce population lifespan by multiple cellular hits. The life extension induced by gamma rays might be due to an induced hypermethylation of DNA. Alternatively, oxygen radicals produced by irradiation might trigger an adaptive stress response which would remove damaged macromolecules and thereby increase the cells' growth potential. Whichever explanation is correct, the results show that the human fibroblast system is not appropriate for the study of the well known effect of ionizing radiation in shortening the lifespan of experimental animals. Contrary to earlier published results, populations of cells treated with cumulative doses of 15 Gy or 18 Gy and held for nearly 3 months after they had reached senescence (Phase III), produced no foci of transformed cells. PMID- 1722020 TI - Molecular genetic approaches to the study of cellular senescence. AB - Normal cells in culture exhibit limited division potential, which is used as a model for cellular aging. In contrast, tumor-derived, carcinogen- or virus transformed cells are capable of dividing indefinitely (immortal). Fusion of normal with immortal human cells yielded hybrids having limited life span, indicating that cellular senescence is a dominant phenotype and that immortality is recessive. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. In order to identify the chromosomes and genes involved in growth regulation, that had been modified in immortal cells, we used the technique of microcell fusion to introduce either a normal human chromosome 11 or 4 into cell lines representative of the different complementation groups. Chromosome 11 had no effect on the in vitro life span of the different immortal human tumor lines. However, when a normal human chromosome 4 was introduced into cell lines assigned to complementation group B, the cells lost the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. These results suggest that a gene(s) on human chromosome 4 has been modified in immortal cell lines assigned to complementation group B, to allow escape from senescence. They also provide evidence for a genetic basis for cellular aging. PMID- 1722021 TI - Somatic mutations and cellular aging: two-dimensional DNA typing of rat fibroblast clones. AB - Aging may be explained, to some extent, as a stochastic process of macromolecular damage. The rate of such a process should then determine longevity and be genetically controlled, as can be derived from the species specificity of maximum lifespan. The genome of the somatic cell is a major candidate to study for loss of DNA sequence integrity during aging. Unfortunately, a lack of adequate techniques has thus far hampered progress in testing the aging genome for changes in its DNA sequence content. Here we discuss recently developed sophisticated technology for studying spontaneous somatic mutations in relation to aging. More specifically, we describe the use of a novel two-dimensional DNA typing technique for the analysis of fibroblast clones derived from primary cultures established from skin biopsies of rats of different ages. Preliminary data are presented indicating the occurrence of DNA sequence changes in mini- and microsatellite regions of the rat genome at an average frequency of 2.7 x 10(-3) per analyzed DNA fragment. Age-related variations in the somatic mutation frequency of these genomic regions were not observed. PMID- 1722023 TI - Cellular ageing. PMID- 1722022 TI - Genetic basis of limited cell proliferation. AB - Knowledge about the changes that occur as cells traverse their replicative lifespans grows apace, as evidenced by the articles in this issue. Controversy over the interpretation of this knowledge continues, however, and is indeed fuelled by new discoveries (e.g., see Cristofalo, 1990; Holliday, 1990; Smith, 1990). This paper makes a brief commentary on the problems of cellular ageing, with particular emphasis on the unfolding picture of the genetic control of ageing and longevity which derives from evolutionary theory (Kirkwood and Rose, 1991). The case is argued for a synthetic view which recognizes that the immediate causes of limited cell proliferation probably involve some form of active genetic control, but that the ultimate reason for cell ageing is found in evolutionary theories which suggest that the ageing process is not actively programmed and that senescence may be due to the accumulation of damage. PMID- 1722024 TI - Aging under glass. PMID- 1722025 TI - Chromatin reorganization during senescence of proliferating cells. AB - It was previously proposed (Macieira-Coelho, 1979) that aging of proliferating cells is the result of genome reorganization taking place during the division cycle. This hypothesis was investigated and a reorganization could indeed be ascertained in the different hierarchical orders of DNA structure; a correlation was found between changes in chromatin organization and the impairment of cell cycle-related events. Indeed, like the latter, the reorganization of chromatin structure is characterized by a succession of subtle changes through the cell population life span, and a final short stage with abrupt events. The final events seem to concern mainly the organization of heterochromatin. The reorganization in the genome is accompanied by structural changes in the cellular scaffold and an evolution of cell morphology. The remodeling occurring in the cell through serial divisions seems to take place in such a way as to decrease the probability of further reorganizations, tending to a limit. The decline of the proliferative activity seems to be the result of the tendency to reach this limit. PMID- 1722026 TI - Cystic fibrosis. The mutant protein responds. PMID- 1722027 TI - Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation. AB - Cystic fibrosis is associated with a defect in epithelial chloride ion transport which is caused by mutations in a membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator). Heterologous expression of CFTR produces cyclicAMP-sensitive Cl(-)-channel activity. Deletion of phenylalanine at amino acid position 508 in CFTR (delta F508 CFTR) is the most common mutation in cystic fibrosis. It has been proposed that this mutation prevents glycoprotein maturation and its transport to its normal cellular location. We have expressed both CFTR and delta F508 CFTR in Vero cells using recombinant vaccinia virus. Although far less delta F508 CFTR reached the plasma membrane than normal CFTR, sufficient delta F508 CFTR was expressed at the plasma membrane to permit functional analysis. delta F508 CFTR expression induced a reduced activity of the cAMP-activated Cl- channel, with conductance, anion selectivity and open-time kinetics similar to those of CFTR, but with much greater closed times, resulting in a large decrease of open probability. The delta F508 mutation thus seems to have two major consequences, an abnormal translocation of the CFTR protein which limits membrane insertion, and an abnormal function in mediating Cl- transport. PMID- 1722028 TI - Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF. AB - A key event in the response of cells to proliferative signals is the rapid, transient induction of the c-fos proto-oncogene, which is mediated through the serum response element (SRE) in the fos promoter. Genomic footprinting and transfection experiments suggest that this activation occurs through a ternary complex that includes the serum response factor (SRF) and the ternary complex factor p62. Interaction of p62TCF with the SRF-SRE binary complex requires a CAGGA tract immediately upstream of the SRE. Proteins of the ets proto-oncogene family bind to similar sequences and we have found that a member of this family, Elk-1, forms SRF-dependent ternary complexes with the SRE. Elk-1 and p62TCF have the same DNA sequence requirements and antibodies against Elk-1 block the binding of both proteins. Furthermore, we show that like p62TCF, Elk-1 forms complexes with the yeast SRF-homologue MCM1 but not with yeast ARG80. But ARG80 mutants that convey interaction with p62TCF can also form complexes with Elk-1. The similarity, or even identity, between Elk-1 and p62TCF suggests a novel regulatory role for Ets proteins that is effected through interaction with other proteins, such as SRF. Furthermore, the possible involvement of an Ets protein in the control of c-fos has interesting implications for proto-oncogene cooperation in cellular growth control. PMID- 1722029 TI - [Stevens-Johnson syndrome as a complication in tuberculosis treatment with thioacetazon]. PMID- 1722030 TI - Barrier functions of the leptomeninges: a study of normal meninges and meningiomas in tissue culture. AB - The anatomical arrangement of the pia mater suggests that it may act as a regulatory interface between cerebrospinal fluid and the surface of the brain and between arterioles within the brain and the surrounding neural tissue. However, the functional aspects of such a barrier are difficult to evaluate in vivo. In the present study, the enzymic content and endocytotic capacities of normal leptomeningeal cells in situ and meningioma cells in confluent tissue culture are examined in relation to barrier functions of meningeal cells. Growth of cells in culture was obtained from human fetal and newborn rat leptomeninges and from 9/13 meningiomas. But, in only two meningiomas were the cultured cells characterized as meningeal in origin by using the strict criteria of desmosomes identified by immunocytochemistry or by electron microscopy. These two tumours had high (8 8.7%) Ki-67 labelling indices. Glutamine synthetase activity is present in normal meninges and in meningioma cells in culture; this enzyme together with catechol-O methyltransferase could play a role in limiting the diffusion of neurotransmitters into brain tissue. A steady rate of endocytosis of carbon particles and fluorescent latex beads, 0.2-1 microns in diameter, was observed in cultured meningioma cells. Such endocytosis was inhibited by cytochalasin B indicating the active participation of intracellular microfilaments. Similar endocytosis has been observed in normal leptomeninges in vivo. The results of this study suggest that meningioma cells in culture reflect the barrier functions of the pia mater and may be used as a model to further investigate the functions of the pia mater. PMID- 1722031 TI - Femoxetine blocks the morphine-induced increase in 5-HT metabolism, as measured by in vivo voltammetry in the nucleus raphe magnus of freely-moving rats. AB - Tricyclic antidepressants, when administered acutely, are known to potentiate morphine-induced antinociception. Systemic administration of morphine has been shown to increase the metabolism of serotonin (5-HT) at the level of the nucleus raphe magnus, as measured by in vivo electrochemistry, in freely-moving rats. Using a similar electrochemical detection of 5-hydroxyindole (peak "3") in the nucleus raphe magnus, the present study investigated the effect of the specific 5 HT uptake inhibitor, femoxetine, on peak 3 and on changes in the metabolism of 5 HT, induced by morphine. Acutely administered femoxetine (40 mg/kg i.p.) induced a significant decrease in peak 3 and completely abolished the effect of morphine (10 mg/kg i.p.) on the metabolism of 5-HT. These data do not support the contention that potentiation of morphine-induced analgesia, by tricyclic depressants results from an interaction between the tricyclic antidepressants and the morphine-induced increase in metabolism of 5-HT, at the level of the nucleus raphe magnus. PMID- 1722032 TI - Involvement of substance P in hyperalgesia induced by intrathecal galanin. AB - Previously we have demonstrated that an intrathecal injection of galanin (GAL) decreases the nociceptive threshold for mechanical stimulation without effect on thermal nociceptive responses. The present experiments were conducted to determine whether substance P (SP) would be involved in such a decrease in the nociceptive threshold produced by GAL. An intrathecal injection of anti-SP monoclonal antibody inhibited the nociceptive threshold-decreasing effect of intrathecal GAL (0.1 nmol/rat). This antibody significantly suppressed the contractile action of SP (3 nM) on the longitudinal muscle and that of neurokinin A (3 nM) to a lesser degree. Binding of [125I]Tyr8-SP to this antibody was inhibited by SP in a concentration-dependent manner in the range 0.1-33 nM without suppression by GAL at a concentration of 3300 nM. In addition, an intrathecal injection of the anti-SP monoclonal antibody increased the nociceptive threshold for mechanical stimulation in carrageenin-inflamed rats without effect on thermal nociceptive behaviors. The capsaicin (0.5 microM) evoked release of immunoreactive SP from dorsal-half slices of the spinal cord was increased by galanin (1 microM, but not 0.1 microM) without effects on basal release. An intrathecal injection of GAL did not produce aversive responses (biting, licking and scratching) at doses of 0.1 and 1 nmol/rat. GAL (0.1 nmol/rat) did not affect biting/licking behaviors evoked by SP (1 nmol/rat), but inhibited SP-evoked scratching behavior. These results suggest that the nociceptive threshold-decreasing action of intrathecal GAL is at least in part mediated by SP, and that GAL may act on primary afferent terminals to increase the release of SP evoked by stimulation. PMID- 1722033 TI - [Work places can be much more hazardous than smoking or passive smoking]. PMID- 1722034 TI - Medical and social factors as predictors of outcome in infant tracheostomy. AB - We examined the relative impact of infant tracheostomy in comparison to associated medical and social factors, on developmental outcome as part of a cross-sectional follow-up of 32 children. These children had no mental retardation, physical handicap, or severe neurological problems, but had a history of long-term tracheostomy in infancy, ranging from 3 to 146 months duration. Medical factors evaluated included prematurity, neurological status, severity of illness, and number of weeks hospitalized. Social factors included parental education and occupation. Outcome measures included IQ, language quotient, growth parameters, and behavioral competence. Correlation analyses, stepwise multiple regression analyses, and t-tests were used. Early medical illnesses were significant predictors of cognitive, language, and growth outcome. Severity of medical complications at birth and the presence of any neurological problem predicted 49% of the variance in IQ at follow-up. Social class was the only variable to predict behavioral outcome, accounting for 28% of the variance. For children without confounding medical conditions, tracheostomy had a negative impact on overall language and auditory comprehension. Once children with confounding medical risk factors were removed from the sample, children with history of infant tracheostomy exhibited significantly lower overall mean language scores (106 versus 120), and lower mean language comprehension scores (104 versus 119) than a matched comparison group. PMID- 1722035 TI - Longitudinal serum IgG response to Pseudomonas cepacia surface antigens in cystic fibrosis. AB - In cystic fibrosis (CF), serum antibody against surface antigens of Pseudomonas aeruginosa is detected only after colonization. Since pulmonary acquisition of P. cepacia usually follows colonization with P. aeruginosa and since P. aeruginosa colonized patients with CF have demonstrable antibody against outer membrane proteins of P. cepacia, it appears that acquisition of the latter organism occurs in the presence of specific serum antibody. To test this hypothesis, serum obtained from six P. aeruginosa-colonized patients 4 and 2 years prior to and 3 months and 2 years after P. cepacia colonization were assayed for total and specific IgG to P. cepacia outer membrane components. Four patients demonstrated 6-fold or greater increases in specific IgG titers to whole outer membranes following colonization. By immunoblot, all patients had demonstrable serum IgG against the 27- and 36-kDa outer membrane proteins of P. cepacia 4 and 2 years prior to colonization. Immunoblots after P. cepacia acquisition demonstrated an intensification of the 28- and 36-kDa bands and the appearance of antibody to a very low molecular weight compound which was not hydrolyzed by proteinase K and was present in purified LPS. These observations suggest that low serum titers of antibody against two P. cepacia outer membrane proteins are present in patients with CF prior to P. cepacia colonization, and that these antibodies fail to protect for intrinsic or extrinsic reasons. PMID- 1722036 TI - Changing attitudes towards narcotic use in cancer pain management in Japan. AB - Morphine consumption for medical purposes in Japan showed a 17-fold increase between 1979 and 1989, due to increased use in cancer pain management. This increase is a reflection of the improving attitude of the health care professionals and health policy makers towards narcotics use. The WHO Cancer Pain Relief Programme has ultimately become the basis for a national cancer pain relief programme. The Ministry of Health and Welfare amended the Narcotics and Psychotropics Control Law in 1990, to improve accessibility of morphine preparations to cancer patients with pain, and edited four manuals for palliative care, that include guidelines on cancer pain relief, and legislative management of narcotics use in hospital, clinic and pharmacy. PMID- 1722037 TI - Effective integration of pain management into comprehensive cancer care. AB - Pain management is an integral component of comprehensive cancer care. The combined goals of optimal comfort and optimal function require a working understanding of how pain therapy interacts with cancer and cancer therapy. The two main aspects of cancer which affect pain management are the cancer's treatability and its non-pain pathophysiology. Cancer treatability determines the importance of pain management and the appropriateness of invasive pain-blocking procedures. Cancer non-pain pathophysiology often hinders pain control by preventing oral administration of medications, narrowing a patient's therapeutic window for opioid analgesics, limiting psychological therapies, and interfering with invasive pain relieving procedures. Cancer therapy can impair or enhance pain therapy and vice versa. Cancer therapy can impair pain therapy by its production of adverse effects or by its direct causation of pain. Cancer therapy can enhance pain therapy by reducing the amount of cancer, by including drugs which act as coanalgesics, and by providing intravenous access devices for parenteral opioid administration. Pain therapy can impair cancer therapy by augmenting or complicating cancer therapy's adverse effects. Pain therapy can enhance cancer therapy by improving organ function and patient performance status permitting previously limited or contraindicated cancer therapies to be given. Five case studies are presented to illustrate how effective integration of pain management into comprehensive cancer care is mandatory for optimal care of cancer patients and their families. PMID- 1722038 TI - [Language development and play behavior of retarded children]. AB - The relationship of symbolic play behavior, general cognitive functioning and language in 108 mentally retarded children was studied. Correlations between play and language measures were found with mental age partialed out. Clinical implications for planning language facilitation programs with mentally retarded children are discussed. PMID- 1722039 TI - [Diagnosis of humidifier lung--comparison of various serologic procedures]. AB - Humidifier lung is a form of exogenous-allergic alveolitis caused by microbial growth in humidifiers and air conditioners. It was the aim of the present study to employ and test the ELISA method as an alternative to antibody determination. 134 employees in a large printhouse equipped with air conditioning plant were examined by us. Specific IgG antibodies against contaminated humidifier fluid were determined by means of a solid-phase radioimmunoassay (protein A RAST) that we had developed further. Alternatively we examined a commercially available ELISA method (Pharmacia IgG-RAST 40; enzyme: beta-galactosidase) and an assay based on protein A peroxidase. The influence of different test conditions was studied. All the methods examined proved suitable for determining the specific IgG antibodies. The commercial beta-galactosidase assay could be adapted to application on microtitre plates in a slightly modified form. In the peroxidase assay it is recommended to use very low serum and enzyme concentrations on account of its high sensitivity. Examination of all the 134 serum samples yielded a high correlation between the results of these two non-radioactive methods and those obtained with the protein-A RAST. PMID- 1722040 TI - [Isolation of species-specific Mycobacterium bovis antigen and testing of its cutaneous tuberculin activity]. AB - Polyclonal rabbit antibodies specific to a species-specific M. bovis antigen were used to isolate a species-specific antigen from the PPD. The preparation obtained was active and possessed a higher specificity than the PPD in skin reactions in sensitized guinea pigs. PMID- 1722041 TI - Pulmonary blastoma with germ cell (yolk sac) differentiation: report of two cases. AB - Pulmonary blastoma is a rare lung neoplasm of disputed histogenesis and variable biologic behavior. Typical cases contain both epithelial and mesenchymal tissues, and a variety of patterns of differentiation have been described. While expression of oncofetal antigens in these tumors has been noted rarely, a coexisting component of germ cell tumor has not been reported previously. We describe the clinical and pathologic features of two cases of pulmonary blastoma having alpha-fetoprotein production and histologic areas of yolk sac tumor. We also report the finding of immunohistochemical staining of fetal lung tissue for alpha-fetoprotein. PMID- 1722042 TI - Evaluation of histologic, morphometric, and immunohistochemical criteria in the differential diagnosis of small cell carcinomas of the cervix with particular reference to human papillomavirus types 16 and 18. AB - Clinicopathologic analyses including immunohistochemical, morphometric, virologic, and DNA ploidy studies were performed on seven cases of small cell (undifferentiated) carcinoma (SCC) and 13 cases of small cell squamous carcinoma (SCSC) of the uterine cervix in an attempt to evaluate which criteria are the most useful in identifying aggressive cervical carcinomas composed of small cells. Highly malignant behavior was found to correlate most closely with the histologic pattern of the tumor. Diffuse infiltration by round to spindle-shaped cells with hyperchromatic nuclei similar to small cell carcinoma in other organs correlated with a high frequency of lymph node metastasis and tumor recurrence. In contrast, tumors with well-defined nests similar to large cell nonkeratinizing squamous cell carcinoma were associated with low rates of lymph node metastasis and recurrence. Although there were trends in the distribution of neuroendocrine and cytokeratin immunohistochemical markers, frequency of detection of HPV 16 and 18 DNA sequences, and ploidy patterns, these features showed considerable overlap and none assisted in consistently separating these two types of neoplasms. Consideration of several features, however, could assist in the differential diagnosis. Women with SCC tended to be younger (mean age 36 yr) compared to women with SCSC (mean age 50 yr). A squamous intraepithelial lesion, i.e., cervical intraepithelial neoplasia, was present in association with 60% of SCSC but was not found in any case of SCC. Tumors positive for keratin and negative for neuroendocrine markers were invariably SCSC, whereas those negative for keratin and positive for neuroendocrine markers were always SCC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722043 TI - Spindle cell squamous carcinoma of the thyroid: an unusual anaplastic tumor associated with tall cell papillary cancer. AB - Five cases of spindle cell squamous carcinoma of the thyroid associated with tall cell papillary carcinoma were examined. The unusual spindle cell squamous carcinoma in these cases resembles those previously described in other sites, including the breast and oropharynx. Three patients demonstrated concurrent spindle cell squamous anaplastic carcinoma and tall cell papillary carcinoma at initial diagnosis, whereas two patients developed concurrent tumor morphologies subsequent to initial diagnoses of only papillary carcinoma. The mean age of the patients was 77 yr, with a female:male ratio of 4:1. Thyroglobulin immunoreactivity was demonstrated in the tall cell papillary component of each case but was absent in both the squamous and spindle cell components. Low molecular weight cytokeratin immunoreactivity was strongly and diffusely present in the tall cell papillary and squamous components, whereas the spindle cell areas demonstrated only focal weak to negative reactivity. Only the squamous cell component demonstrated consistent immunoreactivity with high-molecular-weight keratin antibodies. These unusual tumors have not been previously described in the thyroid, and the association of spindle cell squamous anaplastic carcinoma with tall cell papillary carcinoma in five independent cases may indicate a particular relationship. PMID- 1722044 TI - Immunohistochemical distinction of classic and chondroid chordomas. AB - Chondroid chordomas are cartilage-rich neoplasms, most often located in the spheno-occipital region, that have a better prognosis than classic chordomas. The immunohistochemical features of 19 classic and chondroid chordomas were studied retrospectively using avidin-biotin-complex (ABC) immunoperoxidase histochemistry on formalin-fixed, paraffin-embedded tissue. Of the 19 tumors, all located in the spheno-occipital region, 5 exhibited predominantly chondroid morphologic features. The 14 classic chordomas showed the following pattern of antigen expression (percent of tumors positive): epithelial membrane antigen (EMA) 100%, AE 1/3 (a "cocktail" of monoclonal antibodies directed against low and high molecular weight epidermal cytokeratins) 100%, DP keratin (DPK) 100%, vimentin 100%, S100 86%, neuron specific enolase (NSE) 100%, carcinoembryonic antigen (CEA) 57%, and HMB-45 (an anti-melanoma-associated antibody) 57%. The five chondroid chordomas exhibited the following pattern: EMA 0%, AE 1/3 0%, DPK 0%, vimentin 100%, S100 100%, NSE 100%, CEA 0%, and HMB-45 0%. The focal, weak HMB-45 positivity (performed on the index case because of a clinical concern of metastatic melanoma) seen in 57% of the classic chordomas is a previously unreported finding. This finding suggests either that classic chordomas are capable of HMB-45 expression or that this antibody has broader reactivity than previously recognized. The lack of cytokeratin, EMA, and CEA expression by the chondroid chordomas is similar to chondrosarcomas as reported in the literature and dissimilar to the classic chordoma group. These immunohistochemical findings suggest that chondroid chordomas may more validly be classified as low grade chondrosarcomas. PMID- 1722045 TI - A single tryptic fragment of colicin E1 can form an ion channel: stoichiometry confirms kinetics. AB - The molecularity of the ion channel formed by peptide fragments of colicin has taken on particular significance since the length of the active peptide has been shown to be less than 90 amino acids and the lumen size at least 8 A. Cell survival experiments show that killing by colicin obeys single-hit statistics, and ion leakage rates from phospholipid vesicles are first order in colicin concentration. However, interpretation in molecular terms is generally complicated by the requirement of large numbers of colicin molecules per cell or vesicle. We have measured the discharge of potential across membranes of small phospholipid vesicles by following the changes in binding of potential sensitive spin labeled phosphonium ions as a function of the number of colicin fragments added. Because of the sensitivity of the method, it was possible to reliably investigate the effect of colicin in a range where there was no more than 0.2 colicins per vesicle. The quantitative results of these experiments yield a direct molecular stoichiometry and demonstrate that one C-terminal fragment of the colicin molecule per one vesicle is sufficient to induce a rapid ion flux in these vesicles. In addition, the experiments confirm earlier findings that the colicin fragments do not migrate from one vesicle to another at pH 4.5. Similar results are obtained with large unilamellar vesicles. PMID- 1722046 TI - Isolation and mass spectrometric identification of five metabolites of FK-506, a novel macrolide immunosuppressive agent, from human plasma. PMID- 1722047 TI - Naloxone blocks conditioned place preference induced by substance P and [pGlu6] SP(6-11). AB - The effect of prior treatment with the opioid receptor (opioceptor) antagonist naloxone on conditioned place preference produced by the neurotachykinin substance P (SP) and its C-terminal hexapeptide analog [pGlu6]-SP(6-11) (SPC) was investigated in rats. Place conditioning was assessed using a circular open field partitioned into four quadrants that were equally preferred by the rats prior to drug treatment. On three successive days, rats received an intraperitoneal (i.p.) injection of naloxone-HCl (1 mg/kg) or vehicle 15 min before an i.p. injection of either 37 nmol/kg SP, equimolar dosed SPC or corresponding diluent vehicle. After injection the rats were placed into their assigned treatment corral for 15 min. During the test for conditioned corral preference (CCP), when provided a choice between the four quadrants, rats injected with SP or SPC spent more time in the treatment corral compared to vehicle controls, indicative of a positive reinforcing action of these peptides. The pre-treatment with naloxone blocked the positive reinforcing effects of both SP and SPC; when injected alone, naloxone did not influence the preference behavior. Gross locomotor activity was affected by neither treatment. Thus, the positive reinforcing effects of SP and SPC may be mediated via interactions with the endogenous opioid system(s). PMID- 1722048 TI - Effects of positive end-expiratory pressure (PEEP) ventilation on the exocrine pancreas in minipigs. AB - The effect of positive end-expiratory pressure (PEEP) ventilation on the pancreas was studied in 22 anesthetized minipigs. Five pigs were treated with intermittent positive pressure ventilation (IPPV) for 21 h (Group I) and six pigs were treated with a PEEP of 15 cm H2O for 21 h (Group II). We next explored the influence of PEEP ventilation while stimulating the pancreas with Ceruletide, a synthetic cholecystokinin (CCK) analog, at 2.3 micrograms/kg per h.i.v. for 3 h. Ventilation with IPPV for 4 h (n = 5, group III) was compared with PEEP of 15 cm H2O for 4 h (n = 6, group IV). Changes in serum-lipase were observed in all groups. The average lipase level rose from 12.6 U/l to 67 U/l group I and from 15.1 U/l to 129.2 U/l in group II (P = 0.025 for group I vs group II). In group IV (PEEP and Ceruletide), the mean lipase level rose about six times more than in group III (IPPV and Ceruletide). The difference was significant (P less than 0.00005). By three-factor ANOVA analysis, effects due to the drug (P less than 0.000006) and to PEEP (P less than 0.038) as well as a threefold interaction could be demonstrated, i.e., using Ceruletide a greater PEEP effect on lipase than without the drug (P less than 0.023) took place. There were no significant histological changes of the pancreas in groups I and III. In group II (21 h PEEP), vacuolization of acinar cells was evident; on the ultrastructural level, indication was given that these vacuoles derived both from the Golgi apparatus and from fusion of individual zymogen granules. In group IV (PEEP and Ceruletide), the focal appearance of fatty-tissue necrosis and acinar cells as well as hemorrhage of the gland was observed. We conclude that PEEP ventilation impairs the function of the pancreas and produces even more deleterious effects when the gland is stimulated. These effects should be considered when PEEP is used in clinical practice in the treatment of acute pancreatitis. PMID- 1722049 TI - [A new application of plastination in bone histology]. AB - The plastination techniques originally developed for macroscopy can be modified for the preparation of plastinated sections for microscopy. Particularly good penetration of the specimen during preparation of the histological section is obtained, when the techniques described for freeze substitution, degreasing and forced impregnation with resin are employed. The different types of polymers are compared to determine the particular advantages and disadvantages of each. The sectioning technique and the use of histological stains are described for both undecalcified and decalcified bones. The modified Spalteholz technique with decalcification of sections and second plastination procedure for the preparation of transparent decalcified bone sections is described. This makes it possible to produce plane parallel standardized sections for morphometric examination of the vascular structure of the bone. The possible uses of the plastination for the histological examination procedures currently in use, such as fluorescence microscopy, microangiography and microradiography, are shown. The relative values of different microangiographic techniques have been determined for the first time by means of further developments of the Spalteholz and plastination techniques, because they have provided the possibility of standardizing sections. Thus, the plastination technique appears to be especially useful for the examination of microscopic specimens. PMID- 1722050 TI - [A case of fentiuram poisoning]. PMID- 1722051 TI - PCR-synthesized single-stranded DNA: a useful tool for 'hyb' and 'HAP' standardization for construction of subtraction libraries. PMID- 1722052 TI - Steroid receptor profile and receptor stability in subfractions of human prostatic tissues. Critical aspects on microassays. AB - Androgen (AR), progesterone (PR), and estrogen (ER) receptor contents in cytosol and salt-extractable nuclear subcompartments from 6 normal, 39 benign hyperplastic (BPH), and 7 malignant prostatic tissue specimens were analyzed by radioligand-binding assay techniques. In addition, the temperature stability of AR and PR was measured in another three BPH specimens. Five punch-needle biopsy samples from prostate cancers were also analyzed for AR and PR content. All receptor data were calculated from saturation analyses. The highest AR content was found in the cytosol and nucleic from malignant prostatic tissues. The highest PR concentrations were found in BPH cytosol, whereas nuclei of all types of tissues were negative with regard to this receptor. Markedly lower concentrations of ER were found in cytosol and nuclei from BPH as compared with malignant and normal tissues. PR was the most temperature-stable receptor; a marked receptor loss at room temperature was not registered until after 12 h. AR was stable for 4-5 h in cytosol and for 8-9 h in nuclei. Needle-biopsy specimens from prostate cancer showed highly variable and confusing results for AR and PR content, indicating that microassay studies using biochemical techniques on small tissue samples are unreliable and should not be recommended. PMID- 1722053 TI - Microassays for androgen and progesterone receptor quantitation as compared with standard saturation analyses in human prostatic tissues. AB - Simultaneous measurement of androgen and progesterone receptor content in cytosol and salt extractable nuclear subcompartments of benign hyperplastic prostatic tissue was carried out with various microassay techniques and compared to the results from analyses on bulky tissue from the same tissue specimens. The microassays were carried out as modified saturation analyses or as single concentration assays at various degrees of dilution with tris-EDTA-glycerol (TEG) buffer. Tissue samples for the standard assay weighed between 1.76 and 3.22 g, whereas the microassay samples weighted between 0.14 and 0.47 g. When considering the results of the standard assay as the "true" value, the microassays on the same tissue samples tended to underestimate both the androgen and progesterone receptor contents. Data from the microassays showed a wide variation of the androgen and progesterone receptor content in cytosol and nuclei. With the standard assay technique no detectable amount of progesterone receptor was found in the nuclei, whereas the microassays often indicated false-positive progesterone receptor content in this subcompartment. Therefore, the measurements of steroid receptors using biochemical microassays in prostatic tissue are unreliable and not suitable for clinical use, at least with the techniques available today. Reports in the literature based on such assays should therefore be interpreted with great caution. PMID- 1722054 TI - Androgen receptor assays in specimens of prostatic tissue obtained by transurethral resection and transvesical adenomectomy. AB - The main goal of this study was to ascertain whether routine transurethral resection (TUR) of prostate may provide useful material for the evaluation of androgen receptor (AR) status. At the same time, either intracellular distribution of binding affinity and capacity of receptor molecules were particularly taken into account. Based on our previous findings in breast and endometrial cancer, we suggest that a "functional" receptor status may correspond to the presence of type I (high affinity, low capacity) AR in both soluble and nuclear fractions. However, the precise significance of type II (lower affinity, higher capacity) binding sites remains to be clarified. Ten samples of large prostatic adenomas, obtained by transvesical adenomectomy (TVA), were compared with ten parallel specimens obtained by an in vitro TUR, whereby a pure cutting current was used. The AR assay was carried out with a standard competition method using tritiated mibolerone as the radioligand and Scatchard analysis for data processing. No significant difference between the TUR and TVA groups emerged concerning type I AR content of soluble, nuclear or soluble together with nuclear fractions; this was also true when the results were expressed either as fmol/ml homogenate or as fmol/mg DNA. Similarly, concentrations of type II AR in TVA and TUR samples did not differ significantly in either cell compartment, although they were widely scattered, especially in the soluble fraction. In the light of our findings, it is suggested that TUR specimens represent suitable material for receptor studies, provided that only cutting current is employed and that the use of coagulation current, to control bleeding from the prostatic bed, is confined to the final step of the TUR procedure. PMID- 1722056 TI - 31P MR spectroscopy and 1H MR imaging of the human prostate using a transrectal probe. AB - 1H magnetic resonance imaging and 31P magnetic resonance spectroscopy of the human prostate using transrectal surface coils are discussed. 1H MR images were characterized by a high sensitivity, revealing many details in the prostate. Localized 31P spectra acquired during the same investigation showed phosphorous metabolites, which may help differentiate between benign prostatic hyperplasia and prostate carcinoma. An endoscopic transmit-receive radio frequency (RF) antenna is also described which can be used with very low RF power. PMID- 1722057 TI - [The classification of cryosurgical resections of the liver in reoperated patients]. AB - The authors present a classification of cryosurgical operations in resection of the liver in reoperated patients. The basis of this classification is different combination of usual and cryosurgical (cryoresection and cryodestruction) methods, which allow to increase the radicality of the operations. Ten kinds of cryointerventions (5 radical and 5 palliative) were established. The classification proposed allows the operative volume for resection of the liver in reoperated patients to be correctly planned. PMID- 1722055 TI - The evaluation of markers of prostatic function. AB - The concentrations of three secretory proteins of the human prostate, including prostatic secretory protein of 94 amino acid residues (PSP94), prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP), were measured by enzyme linked immunosorbent assay (ELISA) in semen from a collective of patients suffering from various inflammatory diseases of the genital tract. In addition, levels of the conventional markers citrate, glucosidase and fructose were determined. As compared with semen from men exhibiting no inflammatory condition, only levels of glucosidase in cases of epididymitis and concentrations of PSP94 in the collective suffering from prostatitis showed significant reductions. The changes in the secretion of PSA, PAP, fructose and citrate in the semen of patients with inflammation of genital tract tissue were not significant at the 95% range of confidence. PMID- 1722058 TI - [Catalytic properties of neuraminidase of non-cholera vibrios]. AB - Main catalytic properties of commercially available neuraminidase preparations from noncholeric vibrios were studied. The enzymatic activity was measured using a simple resorcinol procedure. Optimal conditions for neuraminidase effect: pH 5.5-6.0 and buffer composition, were characterized. Affinity of the enzyme to various substrates was studied using 10 natural and synthetic sialoconjugates. Km values were studied for fetuin, ovomucin and transferrin used as optimal substrates. When influence of meta ions, detergents, complexes and other compounds was studied, activation of neuraminidase was found in presence of bivalent metal ions, especially of Ca2+, while chelate-forming complexes and heavy metal salts inhibited the enzyme. These results may be used in studies of the neuraminidase action mechanism and regulation of its activity. PMID- 1722059 TI - [Interleukin-1 formation during inhibition of proteolytic enzyme activity]. AB - Contrycal and phosphomethylsulfonyl fluoride prevented a decrease in activity of interleukin-1 (IL) in 3-days-old culture of mononuclear cells from human peripheral blood stimulated with lipopolysaccharide. These protease inhibitors did not affect per se the IL formation as distinct from soy bean trypsin inhibitor which developed immunostimulating and pyrogenic activities of IL in the cell culture. Use of contrycal enabled to increase content of IL in human and rabbit whole blood culture stimulated by lipopolysaccharide as well as to reinforce the rate of rabbit fever caused by exogenous pyrogen. PMID- 1722060 TI - [Status of the kallikrein-kinin system, proteinase inhibitors and nonspecific resistance factors in patients with chronic kidney failure on hemodialysis as well as in kidney transplant recipients]. AB - A state of the kallikrein-kinin system, activity of proteolysis inhibitors were studied simultaneously with the functional activity of neutrophils and content of lysozyme in blood serum of 21 patients with chronic kidney insufficiency. Two types of alterations in the kallikrein-kinin system were found in 13 patients maintained on hemodialysis: in four patients content of kallikrein was increased 8-fold as compared with normal level with a decrease in content of prekallikrein, while in nine patients activity of kallikrein was similar to control values but content of prekallikrein was still further decreased. Content of alpha 1 proteinase inhibitor (PI) was distinctly decreased (2-2.5-fold) in these patients, however, the decrease of the inhibitor was not observed in four patients; activity of alpha 2-macroglobulin tended to decrease. The ratio of active neutrophils and content of lysozyme were increased in blood serum of the majority of the patients. Hemodialysis, activation of the kallikrein-kinin system and stimulation of neutrophils appear to be responsible for a decrease in PI activity. The decrease in the PI activity and stimulation of the kallikrein-kinin system suggest that impairments in regulation of proteolysis could be corrected by means of exogenous proteinase inhibitors. In crisis of allogenic kidney rejection activities of PI and prekallikrein were decreased. Drastic, uneven alterations of the patterns studied were detected in pyo-inflammatory complications not related to the rejection crisis. PMID- 1722061 TI - [Methods of studying alpha 2-antiplasmin activity]. AB - Methods of alpha 2-antiplasmin (AP) study are reviewed. Estimation of the AP concentration, content of the plasmin-AP complex and of functional AP activity are discussed. PMID- 1722062 TI - [The time characteristics of the chronotropic and anti-arrhythmia effects of kordaron]. PMID- 1722063 TI - Group A streptococcal antigens and vaccine potential. AB - Attempts over the past seventy years to produce an effective vaccine to protect humans against group A streptococcal infections and their immunologically mediated sequelae (acute rheumatic fever and post-streptococcal glomerulonephritis) have been frustrated by two basic problems, first, the ability of the highly protective cell-surface M proteins to elicit potentially harmful host reactions and second, the existence of a large number of distinct serovars of M proteins and the fact that human immunity to group A streptococcal infections is predominantly M serovar-specific. In recent years, progress towards overcoming these problems has been greatly facilitated by an increased understanding of the structural and immunological properties of protective group A streptococcal antigens, which has emerged from molecular biology studies. This article reviews these studies and discusses the potential for developing an effective group A streptococcal vaccine. PMID- 1722064 TI - Detection of stable epitopes on formaldehyde-detoxified Pasteurella multocida toxin by monoclonal antibodies. AB - Progressive atrophic rhinitis in pigs can be prevented by vaccination of pregnant sows with formaldehyde-detoxified preparations of either crude extract of toxigenic Pasteurella multocida or purified P. multocida toxin (PMT). The protective value of a vaccine is expected to be related to its content of detoxified immunogenic PMT, but previously described methods for detection of PMT cannot be used for the quantification of formaldehyde-detoxified PMT. In contrast to the epitopes on PMT recognized by previously produced anti-PMT monoclonal antibodies (mAbs), two of the epitopes recognized by mAbs developed in this study showed a significant stability after treatment with formaldehyde under conditions relevant for production of vaccines. When analysed in a sandwich ELISA based on these two mAbs, the titre of a preparation of PMT, detoxified by treatment with 1% (w/v) formaldehyde for 48 h at 20 degrees C, decreased by less than 30% when compared to the titre of native PMT. A close relationship between the amount of formaldehyde-treated PMT in a vaccine determined by the sandwich ELISA and its immunogenic properties in mice was observed. PMID- 1722065 TI - Structural relationships between hepatitis B surface antigen in human plasma and dimers from recombinant vaccine: a monoclonal antibody study. AB - Ten monoclonal antibodies were obtained from mice immunized with a yeast recombinant hepatitis B vaccine. They were selected at an early stage for their ability to bind to native surface antigen particles (HBsAg) in human plasma. All antibodies recognized conformational epitopes which were destroyed completely or almost completely by reduction of disulphide bridges. They were divided into five epitope groups by their competition for binding to recombinant S protein, though epitopes within each group are not identical. Recombinant S protein migrated on SDS-PAGE in the absence of reducing agents as a mixture of monomers and dimers/oligomers. Sucrose gradient analysis suggests that all these forms are co aggregated into HBsAg-like particles. On Western blots, all ten antibodies either bound only to dimers/oligomers or strongly preferred them over monomers. The results suggest that, of the antibodies produced in response to recombinant vaccine in mice, most of those which bind strongly to 'native' HBsAg particles in human plasma recognize surface structures created by interaction between two subunits. PMID- 1722066 TI - [Methods of the demonstration of the lymphatic vessels]. AB - The methodological tools for the study of the lymphatic vascular system are reviewed. The use of intraparenchymal and retrograde injection techniques often results in poorly reliable specimens, whereas direct injections allow an accurate topographical study of superficial and deep lymphatic vessels. The results obtained by the following methods are discussed: 1) Gerota's method (modified by Ottaviani, 1954) which can be carried out by glass needles and syringes and a mass of Prussian blue; 2) polymers injection as Geon, Rhodopas AX, "Mercox" methyl-methacrylate and Neoprene 842 A. Especially the last yields three dimensional casts which are matchless in elegance and definition of the relationship between lymphatic and blood vessels; 3) three-dimensional models from serial ultrathin sections, which represent a fundamental tool in order to go through the processes of transendothelial transport; 4) in vivo cinematography documents the lymph pulsed flow, the contractile activity of superficial lymphatic collectors and the play of their valves. PMID- 1722067 TI - On the role of the peptide galanin in regulation of growth hormone secretion. AB - The effects of the peptide galanin on growth hormone secretion were studied in vitro using cultured rat and human anterior pituitary cells, and in vivo by iv administration of galanin in both rats and humans. Galanin in concentrations from 10 nmol/l to 1 mumol/l did not alter basal GH release, but slightly inhibited GHRH-stimulated GH release from cultured rat anterior pituitary cells. Galanin (1 mumol/l) did not significantly change basal or GHRH-stimulated GH secretion from cultured human anterior pituitary cells. In contrast, iv injection of 1 microgram (300 pmol) galanin to rats induced an increase in plasma GH that was reproducible at repetitive injections. The galanin-induced GH release in rats was of a lower magnitude than the increase in plasma GH after iv injections of GHRH, and was seen with a 5-15 min delay in comparison to iv administered GHRH. In man, iv infusions of galanin (40 pmol.kg-1.min-1.(40 min)) also caused a significant increase in plasma GH, but it occurred 25-30 min after the beginning of the infusion. These results suggest an indirect action of galanin on GH release in both rats and humans, i.e. galanin does not directly affect the somatotropes. In agreement with a central action, no binding sites for galanin could be demonstrated in the rat anterior pituitary by autoradiography. Since galanin did not affect somatostatin release from fragments of rat mediobasal hypothalamus, the stimulatory effects of galanin on GH release are most likely mediated via a stimulatory effect on GHRH neurons. PMID- 1722068 TI - Immunocytochemical and ultrastructural studies of hyaline inclusions in sporadic motor neuron disease. AB - We investigated hyaline inclusion bodies (HI) immunocytochemically and ultrastructurally in six cases of sporadic motor neuron disease (MND). All HI contained large amounts of ubiquitin and some HI were stained at the core or the center with anti-neurofilament antibody, with the surrounding halo unstained. No HI were stained with antibodies raised against cytoskeletal proteins such as high molecular weight microtubule-associated proteins and phosphorylated tau. Ultrastructurally, HI were chiefly composed of filaments measuring about 20 nm in diameter thicker than neurofilaments, and contained fine granules and frequently one or more of four characteristic profiles, i.e., small electron-dense materials resembling Bunina bodies, bundles of tubular filaments measuring approximately 20 nm in diameter, large electron-dense cores, and focal accumulations of randomly arranged neurofilaments. Hyaline inclusions can be regarded as one of the characteristic markers for sporadic MND as well as familial amyotrophic lateral sclerosis. Hyaline inclusions have a markedly heterogeneous ultrastructure and, therefore, differences in immunoreactivity with antineurofilament antibodies are not unexpected. PMID- 1722069 TI - Nucleolar organizer regions in soft tissue tumours. AB - The diagnostic perspectives of the combination of silver NOR staining method with TV image analysis were tested in 138 soft tissue tumours. In addition to counting the dots per cell, we also measured the summed areas of dots within the nucleus. The so called "malignancy factor" (MF), i.e. the number of dots multiplied by summed areas of dots in one nucleus, was calculated. Tumours were divided into four groups: 1) benign, 2) benign with diagnostic difficulty, 3) intermediate malignant and 4) malignant. The number of dots, the summed area of dots and the MF were separately evaluated by the "t" test. Groups 2 and 3 were not distinguishable at all (p. - 0.788-0.863), while the others showed significant differences in the former values (p less than 3.5 x 10(-6) - 10(-6). When the number of dots was considered in itself, some overlaps occurred between the fourth and the other groups. With the determination of MF, however, no overlap was encountered at all. MF seems to be superior to traditional dot counting when malignant, intermediate and benign soft tissue tumours are to be distinguished. PMID- 1722071 TI - Solubilization of the NMDA receptor ion channel complex from rat brain. PMID- 1722070 TI - Structure and expression of inhibitory glycine receptors. PMID- 1722072 TI - Transmitter-activated ion channels as the target of chemical agents. AB - The transmitter-activated ion channels are known to be important target sites of a variety of therapeutic and toxic agents. The GABA-activated chloride channel has been shown to be modulated by general anesthetics, alcohols, and the pyrethroid, cyclodiene and lindane insecticides. The general anesthetics halothane, enflurane and isoflurane greatly augmented the GABA-activated current before desensitization took place, and suppressed it after desensitization at clinically relevant concentrations equivalent to 1-2 minimum alveolar concentrations. The stimulating effect appears to be a mechanism of general anesthesia. It seems that general anesthetics have a specific affinity for the GABA receptor-channel complex. Ethanol also augmented the GABA-activated peak chloride current with little or no effect on the desensitized sustained current. Longer chain alcohols n-butanol, n-hexanol, n-octanol, and n-decanol also exerted the same type of effect, with the potency and efficacy increasing with lengthening of the carbon chain. The GABA receptor-channel complex has also been shown to be an important target site of certain insecticides. The type II pyrethroids deltamethrin and fenvalerate augmented the GABA-activated peak chloride current when applied concurrently with GABA, but the effect was diminished as the pyrethroids were applied for long periods of time prior to GABA application. The latter effect might explain the controversy in the literature regarding the pyrethroid action on the GABA system. The type I pyrethroid allethrin suppressed the GABA-activated peak chloride current when co-applied with GABA. Both types of pyrethroids suppressed the N-methyl-d-aspartate-induced current. Lindane and the cyclodienes dieldrin, endrin, heptachlor-epoxide, and isobenzan suppressed the GABA-activated chloride current. These effects can account for the convulsant action of lindane and the cyclodienes. PMID- 1722073 TI - Chemical kinetic investigations of the channel-opening process of neurotransmitter receptors. PMID- 1722074 TI - Initiation and pattern of angiogenesis in wound healing in the rat. AB - The object of this study was to examine the initiation and pattern of capillary growth associated with wound healing. Collagen sponges were implanted subcutaneously in the hind limbs of adult male rats to stimulate the formation of granulation tissue. Blood vessels of the hind limbs of euthanized rats were perfused with Mercox (an acrylic monomer) via the abdominal aorta at selected periods of time following sponge implantation. When the perfusate was completely cured, the sponge and parajacent tissues were excised and subsequently macerated by alternating immersion in 40% KOH and distilled water. Cast replicas of the vascular lumina were coated with gold and imaged by scanning electron microscopy. At 6 hr, punctate depressions at the periphery of the replicas of vein and venule lumina were noted. The depressions represented sites of leukocyte margination. By 24 hr, the depressions increased numerically, indicating a great increase in the sites of leukocyte margination. The number of these depressions decreased by 48 hr. Concomitantly, the depressions representing endothelial cell nuclei became more pronounced, indicating nuclear hypertrophy of these cells. In addition, capillary bud formation was initiated. At 72 hr, capillary buds were quite apparent and arose solely from venules. Between 7 and 14 days, replicas of capillary lumina were longer and formed an elaborate network, presumably by end to-end, side-to-side, and end-to-side anastomoses. The network was formed circumferential to the sponge and then capillary sprouts entered the sponge's interstitial spaces. PMID- 1722075 TI - Trigeminal facial pain: a model of peptides and monoamines in intracerebral cerebrospinal fluid. AB - A biochemical model of chronic trigeminal facial pain with elevated substance P (SP) and co-dysfunctional dopamine (DA), norepinephrine (NE) and purinergic systems is proposed. The serotonergic system is hypoactive as judged by low 5 hydroxyindoleacetic acid (5HIM). In distinction, intracerebral opioids may not be dysfunctional in facial pain as measured by normal levels of beta endorphin (BE). The neuropeptides somatostatin (SOM), cholecystokinin (CCK), met and leu enkephalin (MENK, LENK) have very small picogram concentrations in these pain patients, but no definite conclusion can be reached on their role in trigeminal pain, alone or with monoamines, because of the small numbers, both sample size and concentrations. Interpretive obstacles to such human neurochemical studies suggest that future work might move to human clinical trials comodulating SP down, inhibitory peptides (SOM, CCK) up, and enhancing monoamine systems. PMID- 1722076 TI - Bone cement and wound healing. An experimental study in the rat. AB - The local effect of methyl methacrylate on wound healing was studied in rats using viscose cellulose sponges. A 7 mm diameter cylindrical section was removed from the sponges, filled with freshly prepared bone cement or left empty in controls. Two sponges were implanted subcutaneously into each rat. The concentrations of DNA, RNA-ribose, nitrogen, hydroxyproline, uronic acids and hexosamines of the sponges were determined 5, 10 and 21 days postoperatively. The concentrations of collagen and glycosaminoglycans were similar in experimental and control samples and only small differences were detected in the amounts of DNA. It was concluded that methyl methacrylate bone cement did not have any practical harmful effects on wound healing. PMID- 1722077 TI - Synergistic inhibition of human immunodeficiency virus type 1 replication in vitro by two-drug and three-drug combinations of 3'-azido-3'-deoxythymidine, phosphonoformate, and 2',3'-dideoxythymidine. AB - The effects of 3'-azido-3'-deoxythymidine (AZT), phosphonoformate (PFA), and 2',3'-dideoxythymidine (ddT) and their combination on human immunodeficiency type 1 (HIV-1) replication were studied by measuring the HIV-1 p24 antigen expression and reverse transcriptase (RT) release in HIV-1-infected MT4 cells in vitro. RT activity was also measured in a cell-free system by using poly(rA)-oligo(dT) as the primer-template, and cell growth inhibition was measured in noninfected MT4 cells. The interactions of these two- and three-drug combinations were evaluated by the combination index (CI) method and isobologram techniques. The 50% effective concentrations (EC50s) of AZT, PFA, and ddT were 0.014 to 0.005, 9.4 to 8.8, and 8.4 to 2.5 microM, respectively, for p24 enzyme-linked immunosorbent assays (ELISAs) and 0.005 to 0.0034, 1.43 to 1.37, and 2.87 to 2.83 microM, respectively, for RT activity in vitro; for RT activity in the cell-free system, the EC50s were 0.00019 to 0.00024, 0.012 to 0.02, and 0.00074 to 0.0005 microM, for AZT-5'-triphosphate, PFA, and ddT-5'-triphosphate, respectively. AZT in combination with PFA (1:200) or ddT (1:5) as well as the combination of these three drugs (1:200:5) synergistically inhibited HIV-1 replication and RT activity in the cell-free system over a wide range of drug concentrations, with the CIs ranging from 0.5 to 0.09, in which CIs of less than 1, 1, and greater than 1 indicate synergism, additive effect, and antagonism, respectively. Three- and two drug combinations of AZT, PFA, and ddT showed similar degrees of synergism against HIV-1 replication in p24 assays and RT release assays, whereas the combination of AZT and ddT was found to be the most selective in terms of its anti-HIV-1 effect versus cytotoxicity. Dose reduction indices calculated from both HIV-1 replication inhibition, as measured by p24 ELISA and by RT activity in the cell-free system, indicated that two- and three-drug combinations at high effect levels and the selected combination ratios allow 2- to 240-fold dose reduction over the single drug alone in terms of their anti-HIV-1 effects. The three-drug combination showed the highest dose reduction index. These finding suggest that increased efficacy and reduced toxicity may be achieved in AIDS therapy by using AZT, PFA, and ddT in two- or three-drug combinations. PMID- 1722078 TI - A DNA sequence upstream of the tet(O) gene is required for full expression of tetracycline resistance. AB - The DNA sequences upstream of the tet(O) and tet(M) open reading frames (ORFs) (ca. 300 bp) were found to share a higher degree of homology than those of the tet(O) and tet(M) ORFs themselves. A transcription initiation site for tet(O) was located by primer extension analysis. Campylobacter coli was found to use a promoter sequence different from that used by Escherichia coli. The sequence upstream of tet(O) was shown to be required in cis for high-level resistance to tetracycline. PMID- 1722079 TI - Selective anabolism of 6-methoxypurine arabinoside in varicella-zoster virus infected cells. AB - 6-Methoxypurine arabinoside (ara-M) is a highly selective inhibitor of varicella zoster virus (VZV). It belongs to a class of purine arabinosides whose anti-VZV activity in vitro correlates with substrate utilization by the VZV-encoded thymidine kinase (TK) (D. R. Averett, G. W. Koszalka, J. A. Fyfe, G. B. Roberts, D. J. M. Purifoy, and T. A. Krenitsky, Antimicrob Agents Chemother. 35:851-857, 1991). In this study, the mechanism of action of ara-M was explored. VZV-infected human fibroblasts selectively accumulated ara-M and its phosphorylated metabolites, whereas in uninfected fibroblasts or in those infected with a TK deficient strain of VZV, there was virtually no cellular uptake of ara-M. The major intracellular metabolite of ara-M in VZV-infected cells was identified as the triphosphate of adenine arabinoside (ara-ATP). Appreciable levels of ara-ADP, ara-AMP, and ara-MMP were also detected. However, di- or triphosphorylated forms of ara-M were not detected. Moreover, in VZV-infected cells, the concentrations of ara-ATP which accumulated in the presence of ara-M were up to eightfold higher than those generated with ara-A itself. In contrast, in uninfected cells, the levels of ara-ATP which accumulated in the presence of ara-M were barely detectable. Clearly, Ara-M activation was dependent on the activity of the virus encoded TK, while ara-A anabolism resulted primarily from the activity of host cell enzymes. Therefore, ara-M selectively generates the DNA polymerase inhibitor ara-ATP in the VZV-infected cell. PMID- 1722081 TI - [Current status of transcatheter arterial embolization in urology]. AB - We report our experience of 155 transcatheter arterial embolization (TAE) procedures performed over a period spanning 12 months. The changes relative to the therapeutic approach in renal tumors are discussed. Palliative TAE has increased in comparison to presurgical TAE and the range of possibilities in benign renal pathological conditions has been extended (trauma, hemorrhage, hypertension, etc.). The morbidity ascribable to this technique continues to be low. New embolization materials (ethanol, fine particles, etc.) that are more effective and produce less side effects have become available. To date, TAE continues to be a highly effective therapeutic modality with specific indications and scant morbidity. PMID- 1722080 TI - Seasonal and geographical variation of organochlorine residues in birds from northwest Mexico. AB - Eight species of birds (129 individuals) were collected from three agricultural areas with long histories of pesticide use in northwestern Mexico. Plucked carcasses were analyzed for organochlorine (OC) pesticides and polychlorobiphenyls (PCBs). DDE was found in all of the samples and at higher levels than other OCs. Mean (geometric) DDE concentrations varied from 0.04 (microgram/g) ppm in mourning doves (Zenaida macroura) to 5.05 ppm in double crested cormorants (Phalacrocorax auritus). Hexachlorocyclohexane (HCH) was detected in 95% of the samples, but at lower levels than DDE. Hexachlorobenzene (HCB) residues were detected more frequently in birds from Mexicali (62%, p less than 0.05) than in those from Yaqui and Culiacan. HCH and HCB concentrations were significantly higher in birds from Mexicali during the winter than in the summer (p less than 0.05), indicating accumulation of these compounds during that period. Other OCs such as DDT, DDD, dieldrin, oxychlordane, heptachlor expoxide, endosulfan, and endrin were found at lower levels and less frequently. PCBs (quantitated as Aroclor 1260) were found mostly in cattle egrets (Bubulcus ibis) and cormorants at the three locations. Overall, concentrations of OCs were higher for Mexicali than for Yaqui and Culiacan (p less than 0.01). In a few cases, DDE levels were above those that might adversely affect birds. PMID- 1722082 TI - [Endo-urologic surgery of urothelial tumors of the upper urinary tract]. AB - The endourological approach for utothelial tumors of the upper urinary tract (UUT) is a controversial issue of which there is little experience to date. However, the data reported in the series of other authors as well as our own series support its utilization. Of a total of 137 patients suspected of having UUT tumor, 66 patients underwent endourological management: 56 by ureteroscopy (URS) and 10 by percutaneous nephroscopy (PN). The presence of tumor was discarded in 26 patients, 10 underwent open surgery to treat the tumor, and the remaining 30 patients were primarily treated by URS (20), PN (9), or combined treatment (1). Overall, 30 of 111 patients (27%) were treated by endoscopy; 28 attempted cure and 2 were palliative procedures. Twenty-six of these 30 patients had a previous history of urothelial tumor, 7 had a single kidney, and in 5 patients the tumor had presented following cystectomy. Except for the T2 tumor submitted to palliative treatment and one case with diffuse carcinoma in situ, all tumors were TA-1, 20 were G1, 8 were G2, and were G3. Seven of the 30 patients had died after a mean follow-up of 28.4 months (range 3-117 months: 2 immediately postoperatively from pathological conditions unrelated to the operation (acute CVA, biliary sepsis), 1 from conditions unrelated to the urinary tract or tumor, 2 from disseminated bladder urothelial tumor, and 1 from disseminated primary adenocarcinoma of unknown origin. Currently, 23 patients (76.6%) are alive; of these, 7 (23.3%) have had tumor recurrence: 2 required treatment by nephroureterectomy but the remaining 5 patients were also treated endourologically with success. The progression index was 7% (2/28). Analysis of prognostic factors revealed a close correlation between the histologic grade of malignancy, malignant urinary cytology, and the frequency of tumor recurrence. Tumor recurrence was observed to be 60% in those with a positive cytology and only 17.6% in those with a negative cytology. G3 tumors recurred 50% of the time, G2 37.5%, and Go-1 22%. The frequency of tumor recurrence was also different in patients who had received adjuvant topical BCG or MMC therapy (20%) in comparison to those who received no adjuvant therapy (40% recurrence). On the other hand, no significant difference was observed relative to the technique utilized to treat the tumor: 3/12 (25%) of those who underwent electroresection or electrocoagulation and 6/16 (37.5%) of those submitted to Nd:YAG laser.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722083 TI - [Direct percutaneous access to the ureter: percutaneous ureterostomy]. AB - Ureteral occlusion with proximal ureteral urinary diversion is usually required in patients with ureteral fistulae secondary to malignant disease. Palliative endourologic management as described with the percutaneous ureterostomy is a viable alternative in treating ureteral fistulae in this patient population. PMID- 1722084 TI - [Trigonocervicoprostatotomy]. AB - Endoscopic trigonocervicoprostatotomy incision has become a minimally aggressive alternative to surgical treatment of early benign prostatic hypertrophy (BPH). The present article analyzes its indications, advantages and disadvantages. We describe the technique utilized and report on the results achieved in 146 patients (mean age 62.7 years, mean follow-up 15.7 months). Clinically, the results were completely satisfactory in 78.1% of the cases, symptomatology improved in 15.7%, and 4.8% warranted a second procedure. The incidence of retrograde ejaculation was observed to be only 20.5%. With regard to the urodynamics, the preoperative mean maximum flow rate of 9 ml/sec. increased to almost 20 ml/sec. postoperatively. The results of urethral evaluation support the hypothesis of Turner-Warwick which ascribes the obstructive symptomatology in these patients to cervical dysfunction. PMID- 1722085 TI - [Intraprostatic prostheses]. AB - We evaluated the usefulness of the intraprostatic spiral prosthesis as an alternative to surgical treatment of benign prostatic hyperplasia (BPH) (40 cases), cancer of the prostate (7 cases), and cervico-prostatic sclerosis (2 cases). These 49 prostheses were placed in elderly patients (mean age 79 years) who had a long indwelling urethral catheter (mean 6.2 months) and were at high risk for surgery (ASA IV 83.6%). In 17% of the cases, placement of the prosthesis permitted performing curative surgery subsequently, once patient general condition had improved. Overall, good results were achieved in 63% (53 patients with BPH) of the cases who tolerated the long indwelling prosthesis well (mean 22 months). The results were assessed according to the quality of micturition (normal in 74.3%), correct placement of the stent (80%), no post-micturition residual volume (88.5%), and a flow rate of 6-12 ml/sec. (60%). Poor results (9 cases) owing to migration of the stent (7 cases) and/or complications: perforation of urethra (1 case), secondary stricture (1 case) and partial calcification of the prosthesis (1 case). The advantages and disadvantages of other currently utilized stents (Prostakath, Variospire, Wall-Stent, and Double Pezzer) are discussed. PMID- 1722086 TI - [Treatment of benign prostatic hypertrophy using transurethral thermotherapy. Initial experience]. AB - One hundred patients with benign prostatic hypertrophy have been treated from July 22, 1990 to April 3, 1991 (86 had been treated between July 22, 1990 and February 27, 1991) at the Department of Urology of Clinica La Luz. Measurement of residual urine with a catheter, maximum flow and index, and nocturnal frequency and urgency were determined in all patients. Patient subjective evaluation post thermotherapy used a score from 0 to 10. Patients with improvement of 4 parameters were classed as excellent, with 3 as good, 2 fair, and 1 or 0 as poor. Of the overall patient group, 62 were evaluable. Of these, excellent or good results were achieved in 39 out of 62 patients (63%), fair in 8 patients (13%), and poor in 15 (24%). Of these 62 patients, if we exclude those with a catheter before treatment, high residual volume, middle lobe, had undergone previous urethral endoscopic surgery, or with prostates larger than 45 mm. longitudinal diameter on the ultrasound, we are left with a group of 25 patients. In this patient group excellent and good results were achieved in 21 of 25 patients (84%) and fair in 16%. We had no poor results.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722087 TI - [Development of an instrument for endoscopic liquefaction of the prostate]. AB - Although the concept of prostate liquefaction had been proposed by Wickham 15 years ago, its clinical application has been delayed by various technical problems. The authors report on the investigative procedures that have been undertaken to design and develop the ELSA (Endoscopic Liquidiser and Surgical Aspirator) prototype which has been utilized in the clinical setting with promising results. PMID- 1722088 TI - [Immunologic sperm detection]. AB - Proof of prostatic acid phosphatase with the enzyme-immuno-assay (EIA) is specific and as sensitive as the common phosphatase reaction. The EIA for prostatic-specific-antigen (PSA) is prostate specific as well, but less sensitive than the phosphatase assay. In a review of 30 own cases no indications for nonspecifity of the immuno assay were obtained. A positive EIA-test in absence of spermatozoa can be explained by either azoospermia or a prolonged persistence of prostatic acid phosphatase in comparison with spermatozoa. PMID- 1722089 TI - Topological mapping of antigenic sites on the Rift Valley fever virus envelope glycoproteins using monoclonal antibodies. AB - A panel of 17 monoclonal antibodies (MAbs) to the G1 and G2 envelope glycoproteins of Rift Valley fever (RVF) virus were used to analyze the topography and functional properties of the viral antigenic sites. Four heterogeneous antigenic regions which may be interlinked were identified on the G1 protein and four distinct domains on the G2 protein by competitive binding assays. Comparison of the biological activities and epitope specificities of the MAbs against G1 showed that the antigenic domains I, II, and IV were involved in virus neutralization and haemagglutination at different potencies. For both the G1 and G2 proteins, determinants mapping to domain G1 Ia and G2 Ia were associated with very strong neutralization independent of complement (C'), suggesting that they represent biologically important areas. Domain G2 II was involved in haemagglutination and weak C' dependent neutralization while the other two G2 regions had no haemagglutination function and neutralized to a low level only in the presence of C'. Epitopes Ia and IIb on G1 and Ia and IIa on G2 were also associated with protection of mice against virulent RVFV infection, indicating that both envelope glycoproteins play an important role in RVF viral infection and pathogenesis. PMID- 1722090 TI - Preparation and characterization of a neutralizing monoclonal antibody directed to VP4 of rotavirus strain K8 which has unique VP4 neutralization epitopes. AB - For selecting the neutralizing monoclonal antibodies (N-MAbs) directed to VP4 of rotavirus strain K8, which has unique VP4 neutralization epitopes, we prepared several reassortant viruses by mixed infection of two different strains K8 (serotype 1) and P (serotype 3) in vitro: three reassortant clones having VP4 of K8 and VP7 of P and four clones having VP4 of P and VP7 of K8. By using these reassortants in screening hybridomas, a N-MAb (K8-2C12) directed to strain K8 specific VP4 was obtained. The MAb K8-2C12 neutralized only K8 when tested against numerous strains of different serotypes, while in enzyme-linked immunosorbent assay this MAb reacted also with simian rotavirus SA11 (serotype 3), bovine rotavirus NCDV (serotype 6), and human rotavirus (HRV) strain 69M (serotype 8). Neutralization-resistant mutants of K8 were selected by the K8-2C12 antibody and VP4 amino acid sequences of the mutants were determined. Single amino acid substitution was detected in the three mutant clones at position 394, which is included in the major cross-reactive neutralization region identified in other rotaviruses. PMID- 1722091 TI - Cytokine enhancement of simian immunodeficiency virus (SIV/mac) from a chronically infected cloned T-cell line (HuT-78). AB - Simian immunodeficiency viruses (SIV) are a family of primate lentiviruses similar to human immunodeficiency viruses (HIV) in their genetic sequence and pathogenesis. However, host-derived cofactors which may determine the extent of viral replication are not clearly defined for SIV or HIV infections. A HuT-78 cell line chronically infected with SIV/mac strain 251, was biologically cloned and characterized for the ability to produce infectious viral particles, viral structural protein profile, cellular antigen surface phenotype and tested to determine the effects of recombinant cytokines on SIV replication. Reverse transcriptase (RT) assay was used to measure the replication of SIV/mac in response to various concentrations of recombinant cytokines (1-1000 units/ml). We report that tumor necrosis factor-alpha (rTNF-alpha), gamma-interferon (rIFN gamma), interleukin 2 (rIL-2), and granulocyte-macrophage colony stimulating factor (rGM-CSF) induced approximately a 2 to 3 fold increase in virus RT activity compared with untreated SIV-infected HuT-78 cells. In contrast, viral replication was not enhanced or minimally enhanced by interleukin 1 (rIL-1), interleukin 3 (rIL-3), or interleukin 4 (rIL-4) at similar dosages. Furthermore, SIV replication in response to rTNF-alpha and rIFN-gamma occurred in a dose dependent fashion. These data suggest that SIV-infected T-lymphocyte lines are responsive to particular cytokines resulting in increased virus production. PMID- 1722092 TI - Pancreatic adenocarcinoma responding to cisplatin based cytotoxic chemotherapy. PMID- 1722093 TI - Effect of exogenous insulin treatment on fetal growth and maternal alpha fetoprotein levels in pregnant mice. AB - The effect of exogenous insulin on the maternal serum level of alpha-fetoprotein and fetal weight was studied. Insulin was given either as one subcutaneous injection on the 13th day of pregnancy (pulse treatment) or as subcutaneous injections each day throughout pregnancy (long-term treatment). Neither of the treatment procedures resulted in a significant alteration in the average fetal weight. However, pulse treatment on the 13th day of pregnancy resulted in elevated maternal serum levels of alpha-fetoprotein and in a greater number of fetuses and larger fetal mass per animal as examined on the 18th day of pregnancy. By contrast, long-term treatment with insulin from the first day of pregnancy resulted in a lower level of maternal serum alpha-fetoprotein and in significantly lower fetal mass per animal. It is concluded that insulin treatment as such, and especially the mode of treatment, plays a crucial role for fetal growth and synthesis of alpha-fetoprotein. PMID- 1722094 TI - Alpha- but not beta-receptor blocking agents inhibit the antiarrhythmic effect of iloprost on ouabain-induced arrhythmia in guinea-pigs. AB - The effects of iloprost, prazosin and propranolol were tested on ouabain-induced arrhythmia in guinea-pigs. Each drug used alone showed an antiarrhythmic effect. In a second step, iloprost was given in combination with drugs blocking alpha- and beta-adrenergic receptors. Propranolol and iloprost caused a statistically significant and comparable increase of the threshold dose of ouabain for the onset of arrhythmia (OA), the occurrence of premature ventricular beats (PVB), ventricular flutter (VF) and ventricular fibrillation (FIB). The effect of a combination of iloprost and propranolol was comparable to the effect of each drug administered alone. Prazosin enhanced the threshold dose of ouabain for OA and PVB in a statistically significant manner. The effect of a combination of iloprost and prazosin was nearly the same for OA and PVB compared to the single effect of these drugs. The threshold dose of ouabain was decreased for VF and FIB when a combination of iloprost and prazosin was given, compared to iloprost used alone. These results support the assumption that the adrenergic nervous system is involved in the antiarrhythmic effect of iloprost. PMID- 1722095 TI - Effects of Bay K 8644 and nifedipine on locomotor activity and striatal homovanillic acid concentration in acutely ethanol-treated rats. AB - The possible involvement of voltage sensitive calcium channels (VSCCs) in both locomotor activity and striatal homovanillic acid (HVA) alterations in acutely low (0.5 g/kg) and high (2 g/kg) dose ethanol-treated rats was investigated. Both doses of ethanol produced inhibitory effects on both locomotor activity and striatal HVA levels. Bay K 8644 significantly potentiated and nifedipine reversed the locomotor inhibitory effect of low-dose ethanol, whereas both drugs did not alter locomotor activity and striatal HVA levels, when used alone. On the other hand, both Bay K 8644 and nifedipine significantly reversed the decrease in striatal HVA levels induced by the low and high doses of ethanol. PMID- 1722096 TI - Counseling and care for the pregnancy complicated by gastroschisis. AB - Twenty-one consecutive cases of fetal and neonatal gastroschisis were retrospectively reviewed. There was 100% survival if major nonintestinal malformations did not coexist; however, 28.6% of these patients had other major malformations and 66% of them died. There were significantly fewer small for gestational age infants if the defect was diagnosed prenatally (20% versus 75%, p less than 0.003). There was a 60% cesarean delivery rate in prenatally diagnosed infants and 0% if diagnosis occurred at delivery (p less than 0.01). PMID- 1722097 TI - Role of interferon in lethality and lymphoid atrophy induced by Coxsackievirus B3 infection in mice. AB - To assess the importance of interferon (IFN) in the pathology of coxsackievirus B3 (CVB-3) infection, we evaluated both mortality rate and lymphoid involution in young adult BALB/C mice infected with lethal doses of the virus and treated either with anti-IFN antibody or with murine IFN-alpha/beta. Administration of antibody to IFN caused a profound worsening of the pathology and an increase in the mortality rate in infected animals. Treatment with murine IFN exerted a significant ameliorative effect on lethality when administered concomitantly with or soon after virus infection. The extent of this protection was correlated with the plasma levels of exogenous or endogenous IFN at 6 h postinfection, whereas no correlation with IFN titers was found later. The effects of IFN apparently were not directly mediated by antiviral effects, because at the times studied, no relation was found between IFN levels and virus titers, at least in the plasma of the infected animals. Lymphoid atrophy, assessed by measuring spleen weight, was only partially reversed by early IFN treatment. These data suggest that IFN production is critical during the early phases of infection, whereas it does not seem to play a significant protective role at later stages. PMID- 1722098 TI - Induction of immune response to bovine herpesvirus-1 with anti-idiotypic antibodies. AB - Previously, we prepared rabbit anti-idiotypic (anti-Id) antibodies against murine monoclonal antibodies (MAbs) specific for the major bovine herpesvirus-1 (BHV-1) envelope glycoproteins. Glycoprotein III (gIII) contains neutralization epitopes and may be the virus attachment protein. Anti-Id antibodies to a neutralizing MAb that reacts with gIII were purified by sequential immunoaffinity chromatography. Immune responses to the purified anti-Id reagent and BHV-1 were compared in mice. Both groups of mice produced BHV-1-specific neutralizing antibodies. However, lymphocyte proliferative responses and interferon and interleukin-2 production were specific for the respective immunizing antigens. These results suggest that the anti-Id reagent may bear an internal image of a B-cell-stimulating epitope of glycoprotein gIII; however, this epitope does not stimulate a virus-specific cellular immune response in mice. PMID- 1722099 TI - In vitro T-cell proliferative response to the flavivirus, west Nile. AB - West Nile virus (WNV)-specific murine T-cell proliferation in vitro was investigated in terms of conditions that optimize antigen-specific responses and reduce background proliferation. The responder populations consisted of splenocytes from WNV-primed mice enriched for L3T4+ T cells. Ia+ antigen presenting cells (APC) were derived from splenocytes of WNV-primed or naive mice. Antigen was a lysate prepared from WNV-infected Vero cells at 12 h postinfection. Strong virus-specific proliferative responses were observed when antigen-pulsed APC were cocultured with responders at a 1:1 ratio. Substantial nonspecific proliferation occurred when culture medium supplemented with 5% fetal bovine serum (FBS) was used, whereas with 1% normal mouse serum a higher degree of antigen specificity was evident, although the magnitude of the responses was lower. The best separation between antigen-specific and background proliferation was obtained by using an exogenous source of T-cell growth factors to amplify for 2 days the proliferation of L3T4+ cells triggered by an initial 3 days of culture with antigen-pulsed APC. This investigation has defined optimal conditions for investigating the stimulation of WNV-primed L3T4+ T-cell proliferation in response to the presentation of viral gene products by Ia+ APC. This assay should permit detailed analysis of the efficiency of various APC populations and identification of viral antigens that stimulate the proliferation of Class II MHC restricted T cells. PMID- 1722101 TI - The effects of exercise on dose and dose distribution of inhaled automotive pollutants. AB - The purpose of this study was to determine how changes in ventilation rate and in the entry route of air pollutants into the respiratory tract (nose versus mouth breathing) affected the respiratory tract uptake and penetration of inhaled gaseous and particulate pollutants associated with automobile emissions. Experiments were performed with female beagle dogs exposed while standing at rest or while exercising on a treadmill at 5 km/hour and a 7.5 percent grade. Dogs were exposed to nitrogen dioxide at concentrations of 1 and 5 parts per million (ppm), to formaldehyde at 2 and 10 ppm, and to an aerosol of ammonium nitrate particles (0.3 micron mass median aerodynamic diameter) at 1 mg/m3. Total respiratory system uptake and effects on breath time, expired tidal volume, fractional expiration time, minute ventilation, respiratory gas exchange, ventilation equivalents for oxygen and carbon dioxide, and dynamic pulmonary resistance and compliance were measured in exercising and resting dogs exposed for two hours to 5 ppm nitrogen dioxide and 10 ppm formaldehyde in combination with 1 mg/m3 of ammonium nitrate particles. Regional penetration of pollutants through oral and nasal airways and pollutant uptake in the lung were measured in a separate group of six tracheostomized dogs standing at rest while being exposed to nitrogen dioxide, formaldehyde, and ammonium nitrate particles. Hypercapnic stimulation was used to modify ventilation rates in the tracheostomized dogs while pollutant penetration and uptake were measured. Dogs exposed to 5 ppm of nitrogen dioxide at rest tended to breathe more rapidly (p less than 0.05) and more shallowly (a nonsignificant trend) than dogs exposed to purified air. The changes observed were similar in direction, but of smaller magnitude, to changes observed when the same dogs were exposed during exercise to ozone at 0.6 ppm in a separate study. Rapid-shallow breathing was not observed when the dogs were exposed during exercise to 5 ppm nitrogen dioxide. Dogs exposed to a mixture of 10 ppm formaldehyde and 1 mg/m3 ammonium nitrate particles during exercise showed a shift to larger tidal volume breathing, but the response was much less pronounced than the slow-deep breathing pattern response observed in a separate study of dogs exposed to 10 ppm formaldehyde alone. The total respiratory system uptake of formaldehyde from the formaldehyde and ammonium nitrate mixture was larger than that measured for 10 ppm of formaldehyde alone in another exercise and exposure study.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722100 TI - Anti-idiotypic antibodies against anti-CD4 antibodies MT151 and OKT4A. AB - Anti-idiotypic antibodies (Ab2) binding to the antigen-combining site of other antibodies may functionally and even structurally mimic antigen. Ab2 to antibodies directed against the lymphocyte CD4 receptor for human immunodeficiency virus type 1 (HIV-1) may mimic the receptor and therefore inhibit viral infectivity. We have produced Ab2 against monoclonal anti-CD4 receptor antibodies (Ab1). The Ab1 strongly inhibit HIV-1 binding to the receptor. Six monoclonal rat Ab2 and two polyclonal rabbit Ab2 were produced against the Ab1 MT151 and nine monoclonal Ab2 against the Ab1 OKT4A. These Ab2 bound only to Ab1 and not to a panel of nine unrelated murine monoclonal antibodies (MAbs). The Ab2 completely inhibited the binding of the homologous Ab1 to CD4-positive target cells, and recombinant soluble CD4 inhibited binding of Ab2 to Ab1. Thus, the Ab2 seemed to mimic the Ab1-binding site of the CD4 receptor, although the results of inhibition assays did not exclude steric hindrance of antibody-combining sites. However, none of the 17 Ab2 bound to gp120 of HIV-1 envelope or inhibited syncytia formation between cells infected and uninfected with HIV-1. These results suggest that the Ab2 do not mimic the HIV-1 binding site of the CD4 receptor. They further suggest that the Ab1 may not bind within the virus-binding site of the CD4 receptor. PMID- 1722102 TI - Quantification of alphafetoprotein (AFP) endocytosis by PHA-activated peripheral blood mononuclear cells (PBMC) from HIV-infected individuals. A useful test of predictive value for the progression of AIDS. AB - Serum alphafetoprotein (AFP) is actively taken up, through receptor-mediated endocytosis, by many embryo-fetal cells during ontogenic development but also by neoplastic cells, as well as by normal peripheral T lymphocytes after mitogenic activation. We have previously shown that the ability to internalize AFP is impaired in mitogen-activated T cells from several groups of (HIV+)-seropositive individuals and that this expression roughly correlates with the progression of the disease. It is not clear whether this impaired AFP-endocytosis results from an HIV-mediated inhibition of AFP-receptor expression or if it is the consequence of a target signal transduction impairment due to HIV-infection. In the present work we have explored both possibilities by studying AFP-endocytosis in peripheral blood mononuclear cells (PBMC) from seropositive (HIV+)-asymptomatic heterosexual individuals and in in vitro HIV-infected PBMC from healthy donors, either quiescent or stimulated with phytohemagglutin (PHA). Our results suggest that this novel abnormality of T-cells associated with HIV-infection reflects an unusual proliferative response of PBMC to mitogenic stimuli. PMID- 1722103 TI - Elevated in vitro translation of a 25-kDa protein in renal cell carcinoma. AB - As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma. PMID- 1722104 TI - Flow cytometric analysis of lipid droplet formation in cells of the human monocytic cell line, U937. AB - Active uptake of a free fatty acid, oleate, and its incorporation into triacylglycerol and phospholipid in the human monocytic cell line, U937, was identified using 14C-labelled oleate. Excess triacylglycerol accumulates in the form of lipid droplets in the cytoplasm. The extent of lipid droplet formation in each cell can be assessed in the absence of any staining by 90 degrees light scatter intensity, one of the basic parameters of flow cytometry. PMID- 1722105 TI - The DNA-dependent and RNA-dependent DNA polymerase activities of the reverse transcriptases of human immunodeficiency viruses types 1 and 2. AB - We have constructed a series of plasmids which, when introduced into Escherichia coli, induce the overexpression of soluble wild-type and mutated forms of the reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). These proteins were analyzed previously for their RNA-dependent DNA polymerase (RDDP) and ribonuclease H (RNase H) activities. In the present study we assayed the different mutant RTs for their DNA-dependent DNA polymerase (DDDP) activity, employing an in situ polyacrylamide gel activity assay. The results indicate that both the RDDP and DDDP catalytic functions of HIV-1 RT mutants are affected similarly by mutations suggesting a high degree of overlap between the catalytic domains involved in both activities. Contrariwise, many of the HIV-2 RT mutants display no correlation between these two DNA polymerase activities, that is, the DDDP activity was not affected by the mutations introduced in the native enzyme in contrast to the RDDP activity. We were thus able to generate mutants of HIV-2 RT that unlike the wild-type RT, are capable of transcribing only DNA and not RNA. The disparity in mutational catalytic relations between the two HIV-related RTs may reflect a possible difference in the structure and folding properties of the two proteins. PMID- 1722106 TI - Search for epitopic sites of antibodies to germ cell alkaline phosphatase. AB - This study comprises a search for epitopic sites of the germ cell alkaline phosphatase by the use of polyclonal and monoclonal antibodies. The aim of this study was to define epitopes of the germ cell alkaline phosphatase molecule by synthetic peptides representing the known sequence of the molecule. The amino acid sequences of the epitopes to which antisera react were not known. We used overlapping peptides covering the whole 532 amino acid germ cell alkaline phosphatase molecule, including the signal 19 amino acid peptide. A polyclonal, and three monoclonal, antibodies known to react equally well with placental alkaline phosphatase isozyme (PLAP), but which differ in their reactivities with the germ cell alkaline phosphatase isozyme, were used. We have revealed potential epitopic sites of the germ cell alkaline phosphatase molecule using first polyclonal anti-PLAP, followed by monoclonal antibodies for the mapping. We could also show that the antibodies exhibited higher reactivities with such sites if they were combined in a linear synthesis. This indicates that the germ cell alkaline phosphatase molecule has discontinuous epitopes. PMID- 1722107 TI - Muramyl tripeptide phosphatidylethanolamine encapsulated in liposomes stimulates monocyte production of tumor necrosis factor and interleukin-1 in vitro. AB - Muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic lipophilic analogue of muramyl dipeptide (MDP), can be incorporated into the lipid membrane of liposomes. Liposomes containing MTP-PE (L-MTP-PE) stimulated monocytes to selectively kill tumors, but not normal cells in vitro. Furthermore, the activation of monocyte tumoricidal function was demonstrated following the i.v. infusion of L-MTP-PE in a phase I trial with cancer patients. The purpose of this study was to determine the mechanism by which L-MTP-PE activates monocytes. Monocyte tumoricidal function is linked to both interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, normal human monocytes were incubated for various times with L-MTP-PE, empty liposomes, or medium in the presence or absence of gamma interferon (IFN-gamma). The supernatants were removed and assayed for TNF and IL-1 using the L929 and D10.G4.1 assays, respectively. TNF was detected after a 4 hr incubation with L-MTP-PE but not with empty liposomes or medium. TNF secretion peaked at 8 hr and was sustained for up to 72 hr. A 4 fold increase in TNF mRNA levels was demonstrated after 8 hr. An increased level of IL-1 beta mRNA was detected after a 4 hr incubation, but only low level IL-1 secretion was detected in monocytes incubated with L-MTP-PE. Adherent monocytes were frozen and thawed to release intracellular IL-1. Intracellular IL-1 was significantly increased in monocytes incubated with L-MTP-PE. Intracellular IL-1 levels peaked by 8 hr and decreased by 72 hr. Activators were then assayed in the presence or absence of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722108 TI - Interleukin-4 activates ion channels in B lymphocytes. AB - The lymphokine interleukin-4 (IL-4) has been shown to induce dramatic changes in the physiology of resting B cells. We have applied the patch clamp technique in the cell attached and inside/out configurations to resting and IL-4-treated B cells to determine whether specific ion conductances result as a consequence of IL-4 action. We report here that two distinct ion channel events occur in B lymphocytes after treatment with IL-4, (i) induction of an inward rectifying K+ channel that is not observed in untreated cells, and (ii) activation of a large conductance anion channel that is normally silent in non-treated cells in the cell attached patch configuration. These data present the first evidence of a direct effect by IL-4 on ion channels and we suggest roles for these two ionic conductances in IL-4-induced B cell activation. PMID- 1722109 TI - [The histopathological diagnosis of Hirschsprung's disease. Our experience over 18 years]. AB - Having had several cases of difficult interpretation from the anatomopathological point of view, we make a review of all intestinal biopsies carried out in 54 patients for diagnosis of Hirschsprung's disease and above all of the suction rectal biopsies. The result has been seven cases with false diagnosis that represents 22 per 100 of the series. We analyse the possible causes that can lead to false positives and negatives results with the suction rectal biopsy, like the height where the biopsy has been taken, the age of the patients (71 per 100 of false results were in children under one month) and finally other diagnosis like hyperganglionisme. PMID- 1722110 TI - Anatomical mapping of Merkel cells in normal human adult epidermis. AB - The distribution of Merkel cells (MCs) in normal human skin and mucosa was studied using the mouse monoclonal antibody Troma-1, reacting specifically with component 8 of the Moll cytokeratin catalogue. The specificity of this antibody for MCs in human skin was assessed by double indirect immunofluorescence (IIF) and immunoelectron microscopy. Two-hundred and thirty 6-mm punch biopsies were obtained from 44 different sites from six human cadavers within 48 h post-mortem. IIF was performed with Troma-1 on EDTA-split epithelial sheets and the MCs were counted and the mean values per mm2 calculated for each site. Regions with greater than 50 MC/mm2 were the lips, hard palate, palms, finger pads, proximal nail fold, and dorsum of the feet. Three different patterns were observed in the epidermis or mucosa: MCs grouped in clumps, linear and arciform arrangements, and scattered MCs. In the hair follicles grouped MCs were observed in the bulb and scattered MCs were seen in the outer root sheath. PMID- 1722111 TI - Substance P antagonist and immune hypersensitivity reactions. PMID- 1722112 TI - Heparin alters the expression of different forms of immunoglobulin mu heavy chains and their associated proteins by pre-B cell lines and normal Ly-1 (CD5+) B cells. AB - Studies presented here show that heparin alters immunoglobulin expression by murine pre-B cell lines and normal Ly-1 (CD5+) B cells. Previous studies have shown that pre-B cell lines 70Z/3 and NFS-5.3 express mu heavy chains in the cytoplasm and a small amount on the cell surface. Both these cytoplasmic and surface mu are disulfide-linked to omega (lambda 5) surrogate light chains and are noncovalently associated with iota (Vpre-B) variable region-like proteins. We show that culturing 70Z/3 with heparin reduces the amount of the membrane-form mu (micron) on the cell surface. Culturing NFS-5.3 with heparin similarly decreases the membrane-form mu; however, it increases the surface level of a pentameric mu molecule containing secreted-form mu (microS) heavy chains, disulfide-linked omega (lambda 5) chains, and noncovalently associated proteins. Culturing peritoneal B cells with heparin also increases the production of the secreted form microS, detectable in this case by the secretion of classical pentameric IgM. Similarly, injecting heparin intraperitoneally increases IgM secretion by peritoneal Ly-1 B cells. Thus heparin could influence pre-B cell and B cell differentiation and function. PMID- 1722113 TI - Esophageal endoprosthesis therapy. AB - Currently various methods for the treatment of obstructing esophageal lesions are used in the endoscopy setting. The placement of an endoscopic esophageal endoprosthesis (stent) offers a palliative form of therapy for esophageal cancer designed to enhance the patient's quality of life. The gastroenterology nurse or associate must be familiar with the various types of prostheses available, methods of insertion and patient care needs related to esophageal stent placement in order to provide optimal patient care. This article offers a review of esophageal endoprosthesis therapy. The various types of endoprostheses utilized in the United States, insertion techniques, advantages and disadvantages of stent placement and implications for patient care will be discussed. PMID- 1722114 TI - Laser bronchoscopy, indications and possible complications. AB - Laser bronchoscopy can be a very effective way to relieve the agony associated with the sensation of asphyxia due to central airway obstruction. In selected patients and when performed by skilled hands the results can indeed be dramatic. The procedure is not curative in the case of malignant central airway obstruction since only the endotracheal or endobronchial portion of the tumor can be treated. It can be curative in a selected group of patients with primarily benign conditions. There can be complications that could be fatal. One must always weigh the risks involved. PMID- 1722115 TI - Mechanosensitive ion channels as reporters of bilayer expansion. A theoretical model. AB - Various amphipathic compounds have been found to activate mechanosensitive (MS) ion channels in the bacterium Escherichia coli. These results were interpreted qualitatively in terms of the bilayer couple hypothesis. Here we present a mathematical model that describes the results quantitatively. According to the model, the uneven partitioning of amphipaths between the monolayers of the cell membrane causes one monolayer to be compressed and the other expanded. Because the open probability (Po) of the E. coli channels increased independently of which monolayer the amphipaths partitioned into, the model suggests that Po of the MS channels is determined by the monolayer having higher tension. We derived a relation between Po and amphipath concentration. The kinetics of Po variation after exposure of the cell membrane to the amphipaths was calculated based on this relation. The results fit satisfactorily the experimental data obtained with the cationic amphipath chlorpromazine and with the anionic amphipath trinitrophenol. Experiments which should further test the predictions following from the model are discussed. PMID- 1722116 TI - Effects of pipette geometry on the time course of solution change in patch clamp experiments. AB - The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand. PMID- 1722117 TI - Pacemaker activity of the rabbit sinoatrial node. A comparison of mathematical models. AB - In the past decade, three mathematical models describing the pacemaker activity of the rabbit sinoatrial node have been developed: the Bristow-Clark model, the Irisawa-Noma model, and the Noble-Noble model. In a comparative study it is demonstrated that these models, as well as subsequent modifications, all have several drawbacks. A more accurate model, describing the pacemaker activity of a single pacemaker cell isolated from the rabbit sinoatrial node, was constructed. Model equations, including equations for the T-type calcium current, are based on experimental data from voltage clamp experiments on single cells that were published during the last few years. In contrast to the other models, only a small amount of background current contributes to the overall electrical charge flow. The action potential parameters of the model cell, its responses to voltage clamp steps and its current-voltage relationships have been computed. The model is used to discuss the relative contribution of membrane current components to the slow diastolic depolarization phase of the action potential. PMID- 1722118 TI - Hydrogen ion currents in rat alveolar epithelial cells. AB - Alveolar epithelial cells isolated from rats and maintained in primary culture were studied using the whole-cell configuration of the "patch-clamp" technique. After other ionic conductances were eliminated by replacing permeant ions with N methyl-D-glucamine methanesulfonate, large voltage-activated hydrogen-selective currents were observed. Like H+ currents in snail neurons and axolotl oocytes, those in alveolar epithelium are activated by depolarization, deactivate upon repolarization, and are inhibited by Cd2+ and Zn2+. Activation of H+ currents is slower in alveolar epithelium than in other tissues, and often has a sigmoid time course. Activation occurs at more positive potentials when external pH is decreased. Saturation of the currents suggests that diffusion limitation may occur; increasing the pipette buffer concentration from 5 to 120 mM at a constant pH of 5.5 increased the maximum current density from 8.7 to 27.3 pA/pF, indicating that the current amplitude can be limited in 5 mM buffer solutions by the rate at which buffer molecules can supply H+ to the membrane. These data indicate that voltage-dependent H+ currents exist in mammalian cells. PMID- 1722119 TI - Connexin32 gap junction channels in stably transfected cells: unitary conductance. AB - Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class. PMID- 1722120 TI - Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties. AB - Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (gj) to transjunctional voltages (Vjs) of either polarity, with the ratio of minimal to maximal gj (gmin/gmax) being approximately 0.1 at the highest Vjs. Steady-state gj values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which gj was reduced by 50%, was approximately 25-30 mV; A, the Boltzmann parameter describing voltage dependence, was approximately 0.06 (corresponding to an energy difference between states of approximately 1 kCal/mol and to approximately 2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (tau greater than 5 s at 20-40 mV, tau = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (alpha) was approximately 30% lower than that of the closing rate constant (beta) over the Vj range 0-70 mV; at higher voltages, voltage sensitivity of alpha and beta saturated. The kinetic response of gj to a paradigm in which gj was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state. PMID- 1722121 TI - Use of microphotohemolysis to distinguish differences in erythrocyte treatments. AB - A new microhemolytic assay was used to determine if this assay could distinguish between normal erythrocytes and those which had been experimentally altered. Solutions of erythrocytes (4% hematocrit in buffer with fluorescein isothiocyanate tagged to 150,000 MW dextran, FITC-DEX) were placed in a hemacytometer and epi-illumination with a Leitz fluorescent microscope was used to activate the fluorochrome. The resultant hemolysis that occurred only in the discrete area of activation was dependent on the total light energy used for activation (45-180 J/cm2). It was quantitated by an analysis of the amount of light transmittance through that area. The presence of glucose in the buffers decreased the rate of hemolysis and increased the time to reach 50% of the maximal response (T50). Erythrocytes treated with diamide had up to a fourfold increase in the rate of hemolysis and a 48% decrease in the T50, while chlorpromazine produced a 51% decrease in the T50 but had no effect on the rate of hemolysis. Gluteraldehyde produced a graded suppression of the hemolysis. These results demonstrate that the microphotohemolytic assay can be used with energy response curves to provide a relatively quick, quantitative determination of altered erythrocytes. PMID- 1722122 TI - Quantitation of erythrocyte photohemolysis by light microscopy. AB - In a solution of erythrocytes and a photo-active compound, light activation of the compound produces hemolysis of the cells. This study describes a new assay in which focused light through a microscope is used to induce a circumscribed hemolysis of erythrocytes that have been mixed with fluorescein isothiocyanate dextran 150,000 (FITC-DEX) and placed in a hemacytometer. The hemolytic response was monitored by detecting transmitted light intensity in the area (1.8 x 10(-4) cm2) of activation. Only cells within the specific microscopic field of activation hemolyze. This allows for multiple sites of activation within one sample. The hemolytic response was dependent on the concentration of FITC-DEX (0.5-4 mg/ml) and on light intensity (53-210 J/cm2) but not on small changes in hematocrit (3%-5%) or on the presence of platelets and leukocytes. Rabbit erythrocytes, however, were almost twice as sensitive as those from guinea pigs. Since the photohemolytic response will depend on the composition and strength of the erythrocyte membrane and presence of oxidant defense mechanisms, we suggest that this assay could be used to detect drug- or disease-induced changes in the red blood cell membrane. PMID- 1722123 TI - Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis. AB - Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples. PMID- 1722124 TI - Application of photodiode array detection and fast atom bombardment mass spectrometry for the identification of the arginine residue in neuropeptides. AB - Chemical derivatization by phenylglyoxal (PGX) was applied to the identification of arginine in the neuropeptides dynorphin A (1-6) and substance P. The obtained products were separated on a short reversed phase C18 column and analysed on-line with the photodiode array UV technique. The selective attachment of a chromogenic molecule into the arginine residue resulted in significant change in the absorbance spectra around 250 nm, depending on the number of PGX molecules attracted. Further analysis employed fast atom bombardment mass spectrometry (FAB MS) and C-terminal sequencing for detailed verification of the derivatives formed during modification with PGX. The results clearly demonstrated that the photodiode array technique, when combined with chemical modification of certain amino acids, provides new possibilities for the analysis of peptide structures. PMID- 1722125 TI - Specific high performance liquid chromatographic determination of the molecular weight and concentration of hyaluronic acid in complex mixtures by labelled hyaluronate binding proteins. AB - A simple High performance liquid chromatographic (HPLC) method for the specific determination of the molecular weight and concentration of hyaluronic acid (HA) in complex mixtures has been developed. Hyaluronate-binding proteins isolated from bovine cartilage labelled by 125I or fluoresceinisothiocyanate were used as specific markers. The specific binding affinities of the markers were compared and were found to have association constants of 1.6 x 10(7) M-1 and 1.2 x 10(7) M 1 respectively. The HA levels and molecular weight distributions can be easily determined in the range 10-500 ng/mL in complex mixtures by the use of markers, molecular sieving HPLC columns and appropriate detectors. It has been demonstrated clearly that the method is useful for the highly specific determination of the parameters in complex biological samples such as serum and synovial fluids and is recommended for clinical applications. PMID- 1722126 TI - Aprotinin and cardiac surgery. PMID- 1722127 TI - Palliation of malignant dysphagia with laser therapy: predictability of results. AB - This study reports the results of 189 patients treated by laser therapy for malignant dysphagia. Ninety-one per cent of patients derived benefit from treatment, but the long-term survival rate was poor, with only 12 per cent of patients surviving for 6 months. Survival was not influenced by either tumour length, site or histological type. Patients with adenocarcinomas initially showed improved swallowing after laser treatment compared with those with squamous tumours, but this difference had disappeared by 2 months. The results of laser treatment were not influenced by either tumour length or site. PMID- 1722128 TI - Talc as a pleural sclerosant. PMID- 1722129 TI - Binding sites for [3H][Sar9, Met(O2)11]substance P in rat brain and guinea pig ileum. AB - [3H][Sar9,Met(O2)11]substance P (SP) with high specific activity (32 Ci/mmol) was used to study neurokinin-1 (NK-1) binding sites on rat brain and smooth muscle membranes of the guinea pig ileum. The specific binding of [3H][Sar9,Met(O2)11]SP was shown to be saturable, reversible and increased in parallel with the protein concentration. Scatchard analyses of equilibrium binding experiments revealed that [3H][Sar9,Met(O2)11]SP binds to a class of non-interacting binding sites in rat brain membranes (Kd = 2 nM, Bmax = 56 fmol/mg of protein) and ileum muscle membranes (Kd = 2 nM, Bmax = 194 fmol/mg of protein). Competition of [3H][Sar9,Met(O2)11]SP, 125I-BH[Sar9,Met(O2)11]SP and 125I-BH.SP with different tachykinin-related peptides gave the following rank order of potencies: SP greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than N Ac[Arg6,Sar9,Met(O2)11]SP(6-11) greater than neurokinin A (NKA) greater than or equal to eledoisin greater than or equal to neurokinin B (NKB) greater than [MePhe7]NKB (4-10) greater than [beta-Ala8]NKA(4-10). A very similar pattern was observed on ileum muscle membranes. [3H][Sar9,Met(O2)11]SP was found to be highly selective for NK-1 binding sites in rat brain and in the intestinal tissue. Binding showed good correlation with the biological activity of tachykinins and related peptides. From these data it can be suggested that (a) the NK-1 receptor characterized in the central nervous system is identical to the one in the periphery, (b) the NK-1 binding site of the muscle membranes appears to be similar to the contractile receptor of the guinea pig ileum and (c) the functional site mediating relaxation of the dog carotid artery is similar to the contractile receptor of the guinea pig ileum. PMID- 1722130 TI - Identification and ultrastructural localization of a calretinin-like calcium binding protein (protein 10) in the guinea pig and rat inner ear. AB - Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, at Mr = 29 k. Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons. PMID- 1722131 TI - An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia. AB - Changes in MAP2 and clathrin immunoreactivity were studied in gerbil hippocampus after transient cerebral ischemia. MAP2 immunoreactivity decreased significantly by 1 h in the subiculum-CA1 and CA2 areas which correspond to reactive change, while no decrease was observed in CA1 until day 4. Before the initiation of delayed neuronal death, MAP2 immunoreactivity was not changed in CA1. On the other hand clathrin immunoreactivity increased in the pyramidal cell layer of CA1 by 3 h after ischemia and remained high for 2 days. Clathrin immunoreactivity in the pyramidal cell layer of CA1 diminished after delayed neuronal death. The transient change of clathrin was noted especially in CA1 in the period prior to delayed neuronal death. These results imply an abnormal change in clathrin turnover after ischemia, which may participate in the pathogenesis of delayed neuronal death. PMID- 1722132 TI - Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain. AB - The cellular distribution of insulin-like growth factor I (IGF-I) immunoreactivity was examined in the rat brain from embryonic day 15 to maturity. IGF-I immunoreactivity was found in the perikarya of neurons distributed along the entire extension of the neuronal tube in all the embryonic ages studied (E15, E17, E19 and E21). In E21 animals, the majority of immunoreactive neurons was located in the olfactory bulb, cerebral cortex, hippocampus, striatum, diencephalon, mesencephalic colliculi, trigeminal nuclei, trigeminal ganglion and in motoneurons of the brainstem. In 10- and 20-day-old rats, in addition to the above areas, IGF-I immunoreactivity was also observed in capillary walls, ependymal cells, choroid plexus, glial cells and most fiber paths. In postnatal ages, immunoreactivity in neuronal somas was mainly restricted to the cell nuclei. However, IGF-I immunoreactivity in the neuronal cytoplasm was observed in 20-day-old rats treated with colchicine while fiber paths and neuronal cell nuclei were negative in these animals. In the telencephalon of 20-day-old rats injected with colchicine, the most intense immunoreactive neurons were observed in the olfactory bulb, cerebral cortex, tenia tecta, hippocampus, islands of Calleja, septal nuclei, striatum, endopyriform nucleus and amygdala. Most diencephalic nuclei, the substantia nigra, the mesencephalic colliculi, Purkinje cells in the cerebellar cortex and several nuclei in mesencephalon, pons and medulla oblongata were also immunoreactive. In adult rats injected with colchicine, IGF-I immunoreactivity was located in the same areas as in 20-day-old rats. The number of immunoreactive cells and the intensity of the staining was reduced in adult rats as compared to that found in young postnatal animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722133 TI - Involvement of non-selective cationic channels in the generation of pacemaker depolarizations and firing behaviour in cultured frog melanotrophs. AB - The firing patterns of cultured frog melanotrophs were studied using the patch clamp technique. In the cell-attached mode, unitary currents were frequently observed as well as biphasic waveforms which were attributed to action potentials 'leaking' through the patch membrane. An inwardly rectifying single-unit current was observed with pipette solutions containing either 100 mM K+ or 100 mM Na+. Under both conditions, these channels displayed an identical I/V relationship, yielding a unitary conductance of 110 pS. The channel opening time was extremely long (50-3000 ms) and single-channel currents showed typical relaxations, which triggered bursts of action currents. In the whole-cell configuration large (2-12 mV) fluctuations in the membrane voltage of current-clamped cells frequently occurred. The deflections appeared to result from single-channel currents. Depolarizing 'events' often led to the discharge of action potentials. Taken together, our data provide evidence for the existence of high-conductance cationic channels in frog pars intermedia cells. These channels may, at least in some cases, be responsible for the generation of pacemaker depolarizations, thereby regulating firing behaviour. It is concluded, that the current traversing a single channel can seriously affect the membrane potential and excitability of frog melanotrophs. PMID- 1722134 TI - Intrinsic connections in cat visual cortex: a combined anterograde and retrograde tracing study. AB - Area 18 of cat visual cortex was examined for intrinsic axons following small, columnar injections of an anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Locally projecting axons radiated from the injection site and branched to form 10-15 discrete, approximately circular patches 500-750 microns in diameter consisting of many bouton-studded terminal arborizations. Labeled fibers and boutons ramified densely in layers I, II/II, V, and VI, and were noticeably less dense in layer IV. Afferent and efferent pathways originating from the same cortical columns were studied by injecting a mixture of PHA-L and wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Between 10 and 15 patches of cells retrogradely labeled by WGA-HRP surrounded each injection site. Within a patch, labeled cells were found in all layers and included both pyramidal and non-pyramidal cells. The distribution of PHA-L labeling was similar to that obtained when PHA-L was injected alone. Most often, the labeled patches resulting from injections of such mixtures contained both anterograde and retrograde labeling. However, patches consisting of retrograde labeling alone and of anterograde labeling alone were also observed, indicating that the local connections linking neighboring cortical columns were not always reciprocal. PMID- 1722135 TI - Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PC12 cells. AB - The conductance properties of neuronal nicotinic acetylcholine receptors (neuronal nAChR channels) in PC12 cells were studied using single channel and whole cell gigaohm seal voltage clamp techniques. Acetylcholine receptor agonists were applied using external pipettes. Neuronal nAChR channels were selective for monovalent cations, and were impermeable to anions. Based on measurements of shifts in reversal potential with changes in external Na and K ion concentrations and evaluation of the GHK constant field relation, PK/PNa was 1.45 (ignoring Ca permeability). PCa/PK was 1.75 based on the shift in reversal potential seen when raising the external Ca concentration from 2 to 50 mM (holding the external Na ion concentration constant), and determining the best fit to the extended GHK constant field relation using ionic activities. This value is larger than that determined for the muscle-type nAChR channel. Evaluation of the extended GHK current equations using these permeability coefficients indicated that at subthreshold voltages approximately 5% of total current through neuronal nAChR channels will be carried by Ca ions. Thus, these channels may be important mediators of activity-dependent Ca influx in the nervous system. PMID- 1722136 TI - Home hospice care. AB - Home hospice care, which helps patients with terminal illnesses remain at home, is becoming more common and accepted in the United States. The Medicare hospice benefit reimburses hospices for the care of elderly patients. The goal of hospices is to help the patient and family remain in control of the dying process as much as possible. Palliative care rather than the extension of life is emphasized. Hospice techniques for controlling symptoms such as pain, nausea, and vomiting are discussed in this article. PMID- 1722137 TI - Palliative care. The nurse's role in helping families through the transition of "fading away". AB - Oncology nurses are finding themselves increasingly involved in providing palliative care. A foundational tenet of palliative care is that the entire family, not just the patient, is the unit of care. Although nursing literature on care of the family is growing, nursing approaches tend to be described in general terms. A recent study of families caring for a member with advanced cancer generated a theoretic scheme which indicated that the families' experience could be conceptualized as a transition: the transition of fading away. This theoretic scheme served as the foundation for identifying nursing approaches which could be helpful to families engaged in the transition. This paper expands on the nursing approaches so that nurses may be better prepared to help families through the transition of fading away. PMID- 1722138 TI - In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain. AB - The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab')2 or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab')2-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins. PMID- 1722140 TI - RNA-protein interactions. PMID- 1722139 TI - Search for the critical characteristics of phenotypically different B cell lines, Burkitt lymphoma cells and lymphoblastoid cell lines, which determine differences in their functional interaction with allogeneic lymphocytes. AB - Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity ot lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation. PMID- 1722141 TI - Percutaneous transhepatic biliary drainage for malignant biliary obstruction: a report of two cases with five year survival. AB - We report two cases of malignant biliary obstruction in whom percutaneous transhepatic biliary drainage has contributed to patient survival of more than 5 years. To our knowledge, this represents the first such case report in the literature. Both patients suffered biliary obstruction from poorly differentiated adenocarcinoma, in all likelihood from the head of the pancreas. Morbidity has been low with only two episodes of significant sepsis and one episode of GI haemorrhage in one patient and both patients were able to enjoy an excellent quality of life. These cases demonstrate that percutaneous biliary drainage has a place not only in the short term palliation of patients with malignant biliary obstruction, but in the long term as well. PMID- 1722142 TI - The cell biology of antigen processing. AB - T lymphocytes recognize antigen only after a series of intracellular events known as antigen processing. The result of antigen processing is the production of short segments of the primary peptide sequence bound to a polypeptide-binding groove on major histocompatibility complex (MHC) molecules. Antigen originates from one of two sites: intracellular or extracellular. There are two corresponding pathways for antigen processing and two corresponding classes of MHC molecule. Analysis of each pathway has demonstrated that their separation is not purely anatomical, but is maintained by molecular interactions with other molecules. Antigen processing has been shown to regulate the overall immune response, but the mechanisms involved remain obscure. PMID- 1722143 TI - [Treatment strategy and results in primary inoperable rectal cancer]. AB - The authors report their experience with laparotomy in patients with rectal carcinoma to identify those patients with locally non-resectable primary cancer of the rectum who may be treated initially by high-dose radiotherapy. The goal of this so-called "staging laparotomy" is to assess mobility and tumor size by means of bimanual palpation, to stage the abdominal cavity and to create total fecal diversion by performing an endcolostomy in order to condition these patients for maximum tolerance during the protracted radiotherapy course (greater than 50 Gy/5 6 weeks). The formation of an endcolostomy seems to avoid severe morbidity and even mortality of high-dose radiotherapy without delay of further surgery. Twenty two patients with locally advanced rectal carcinoma were treated in this way. The tumor was resectable in 18 of the 20 patients who underwent relaparotomy after high-dose radiotherapy. Six out of the 13 patients with a radical resection died with evidence of disease during the follow-up (2 with local disease). One patient died with no evidence of disease after 5 years and 6 patients are still alive without disease after an average of 37 months (15-67 months). PMID- 1722144 TI - [Developmental rehabilitation. A new concept in aid to the handicapped]. PMID- 1722145 TI - Inflammation, cytokines and nutrition. PMID- 1722146 TI - Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida. AB - The third lipoamide dehydrogenase structural gene of Pseudomonas putida, lpd3, was isolated from a library of P. putida PpG2 DNA cloned in Escherichia coli TB1. The nucleotide sequence of lpd3 and its flanking regions indicate that lpd3 is not part of an operon, which is unique for a prokaryotic lipoamide dehydrogenase. An open reading frame was found 207 bases upstream from the start of transcription, but is encoded on the strand opposite lpd3. There is no evidence of an open reading frame immediately downstream from lpd3. The coding region of lpd3 consists of 1401 bp, providing for 466 amino acids plus a stop codon with a G/C content of 62.4%. The transcriptional start site was located 33-bp upstream from the start of translation. The third lipoamide dehydrogenase (LPD-3) shares amino acid identity with the other two lipoamide dehydrogenases of P. putida, 45% with that of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes, and 45.9% with the lipoamide dehydrogenase of the branched-chain oxoacid complex. LPD-3 is more closely related to eukaryotic lipoamide dehydrogenases since it has 53.6% amino acid sequence identity with pig and human lipoamide dehydrogenases and 51.1% identity with yeast lipoamide dehydrogenase. LPD-3 was not produced in wild-type P. putida PpG2 under a variety of growth conditions. However, LPD-3 was produced in P. putida PpG2 carrying pSP14, a pKT240-based clone with the entire lpd3 gene plus 104 bases of the leader. The only demonstrated role of LPD-3 in P. putida is as a substitute for lipoamide dehydrogenase of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes when the latter is inactive or missing. PMID- 1722147 TI - Recognition of the folding consensus in RNA secondary structures by the topological-filtering method. AB - Functionally homologous RNA sequences can substantially diverge in their primary sequences but it can be reasonably assumed that they are related in their higher degree structures. The problem to find such structures and simultaneously satisfy as far as possible the free-energy-minimization criterion, is considered here in two aspects. Firstly a quantitative measure of the folding consensus among secondary structures is defined, translating each structure into a linear representation and using the correlation theorem to compare them. Secondly an algorithm for the parallel search for secondary structures according to the free energy-minimization criterion, but with a filtering action on the basis of the folding consensus measure is presented. The method is tested on groups of RNA sequences different in origin and in functions, for which proposals of homologous secondary structures based on experimental data exist. A comparison of the results with a blank consisting of a search on the basis of the free energy minimization alone is always performed. In these tests the method shows its ability in obtaining, from different sequences, secondary structures characterized by a high-folding consensus measure also when lower free energy but not homologous structures are possible. Two applications are also shown. The first demonstrates the transfer of experimental data available for one sequence, to a functionally related and therefore homologous one. The second application is the possibility of using a topological probe in the search for precise structural motifs. PMID- 1722148 TI - Two lipid-anchored cAMP-binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A. AB - We show that the yeast, Saccharomyces cerevisiae, contains two cAMP-binding proteins in addition to the well-characterized regulatory (R) subunit of cytoplasmic cAMP-dependent protein kinase (PKA). We provide evidence that they comprise a new type of cAMP receptor, membrane-anchored by covalently attached lipid structures. They are genetically not related to the cytoplasmic R subunit. The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross-react with the two membrane-bound cAMP binding proteins. In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity-labeled proteins. Although they differ from one another in molecular mass and subcellular location, peptide maps of the cAMP-binding domains resemble each other and both proteins are membrane-anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane. Both anchors can be metabolically labeled by Etn, myo-Ins and fatty acids. In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man. After cleavage of the anchor with glycosylphosphatidylinositol-specific phospholipase C from trypanosomes, the solubilized cAMP-binding protein from plasma membranes reacts with antibodies which specifically recognize the cross-reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors. Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates. The plasma membrane cAMP receptor is, in addition, modified by an N-glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass. PMID- 1722149 TI - Expression of CYP2D6 in developing human liver. AB - The CYP2D6 protein is a polymorphic isoenzyme involved in the biotransformation of several drugs including the probe drug dextromethorphan. The rise in the protein concentration, immunochemically determined with a specific antibody, was shown to occur within the first week following birth, whatever the gestational age at birth. In fetuses, the concentration of hepatic CYP2D6 protein was very low or undetectable in 70% of samples tested. In the remaining 30%, its concentration was comparable to that of newborns aged 1-7 days. This early rise was associated with spontaneous abortion in 70% of positive samples, whereas in fetuses with an intermediate CYP2D6 protein concentration, 80% were from induced abortions. The rise in CYP2D6 protein was associated with the developmental onset of dextromethorphan O-demethylation, but not N-demethylation, even if activity was lower in fetal than in neonatal and in adult liver microsomes. Lastly, the CYP2D6 RNA is detectable earlier than the protein and exhibits a peak of hepatic accumulation in newborns, before declining in adulthood. A positive correlation between RNA accumulation and protein concentration can be demonstrated only in the adult. This suggest that regulation is primarily at the transcriptional level, but cannot rule out the participation of post-transcriptional events in the regulation process throughout ontogenesis. PMID- 1722150 TI - Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase. AB - Placental (PLAP) and germ cell (GCAP) alkaline phosphatases were probed immunologically with a library of 18 murine monoclonal antibodies reacting with different conformational epitopes on PLAP. Three main antigenic domains (I, II and III) were mapped by antibody competition experiments and the relative binding of the antibodies to site-directed PLAP mutants. Relative affinities of each of the antibodies for the wild type (wt) GCAP were 2-3-fold lower than the values found for wt PLAP. Relative affinity was determined for a series of PLAP mutants, in which one, two or three amino acids were substituted for the corresponding wt GCAP residues by site-directed mutagenesis. Substitutions at residues 15, 38, 67, 241 or 254 induced a major decrease in affinity (6-10-fold) primarily for those antibodies reacting within domain I, whereas changes at positions 84 and 297 led to a 2-3-fold enhancement of affinities as measured with antibodies reacting within the three domains. Arg209 was found to constitute the only difference between the S and F allelic phenotypes of PLAP and to structure the epitope for the F/S allotype-discriminating antibodies. Arg241 was found to constitute the epitope for the antibody 17E3 that discriminates between PLAP and GCAP. Mutagenesis at position 68 or 133 had little effect on the overall reactivity with the antibody panel. Substitution in wt PLAP of Glu429 for Gly429 or even for His429 (found at this position in tissue-nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies. The conformational change accompanying mutagenesis of Glu429 in PLAP, is important in view of the recent identification of Gly429 as the major determinant of the unique GCAP inhibition by the uncompetitive inhibitor L-Leu. Relative affinity values determined for the rare L-Leu sensitive heterodimeric FD and SD PLAP phenotypes, suggested that the reactivity pattern of the D homodimer with the antibody panel, would resemble more closely that of wt GCAP than wt PLAP. Our data suggest that the uncompetitive inhibition of GCAP by L-Leu is due to an enzymatically critical conformational change in a loop region proximal to the active site of the enzyme, induced by substitution of a single amino acid residue. PMID- 1722151 TI - Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria. AB - Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation. PMID- 1722152 TI - Complete primary structure of porcine tenascin. Detection of tenascin transcripts in adult submaxillary glands. AB - Tenascin is an extracellular matrix protein that is postulated to modulate tissue differentiation and cell migration during development. cDNA clones for tenascin were isolated from a cDNA library of adult porcine submaxillary glands. Three forms of tenascin clones were observed which varied with the number (8-10) of fibronectin type III (FN-III) domains. A major form consists of the N-terminal domain involved in the hexamer formation of tenascin subunits, 14 epidermal growth-factor-like domains, nine FN-III domains, and the fibrinogen-like domain. A minor form with ten FN-III domains has never been described. Another striking feature is the lack of an RGD sequence that has been implicated to be crucial for cell adhesion, whereas RGD is present in both chicken and human tenascin sequences. In the adult, tenascin is expressed in very restricted tissues such as brain and chicken gizzard. A survey of tenascin transcripts in various adult rat normal tissues, including brain, revealed that the transcripts were detected only in submaxillary glands where tenascin expression has never been reported. PMID- 1722153 TI - Changing attitudes in the treatment of benign prostatic hyperplasia: the role of 5 alpha-reductase inhibition. Proceedings of two symposia: Nice, France, November 11, 1989 and Amsterdam, The Netherlands, June 14, 1990. PMID- 1722154 TI - Benign prostatic hyperplasia: a population-based study. AB - To evaluate the incidence and outcome of initial surgery for benign prostatic hyperplasia (BPH) and to clarify the natural occurrence and progression of such urologic diseases, two studies have been conducted in a free-living population in Rochester, MN. The first followed 330 men who had not been diagnosed with prostate or bladder cancer at the time of prostatectomy. All surgery subjects were area residents and between 46 and 95 years of age (mean age 70 years). Among the operated subjects, 14 (4.2%) had serious intraoperative complications, 32 (9.7%) were rehospitalized for urologic complications within 30 days after surgery, and 13 (3.9%) experienced other serious complications in that same time period. Blood transfusions within 30 days of surgery were necessary in 45 patients (14%). The risk of reoperation within 6 years of the initial surgery was calculated at 15.1% (95% CI: 9.7, 20.6). On the basis of age- and sex-specific mortality statistics for Rochester, short- and long-term postoperative mortality was not statistically significantly different from that expected. Results of the second study are not yet available. This population-based evaluation of the natural history of urologic disease is expected to clarify the relative utility of various treatment options and provide a useful perspective on the management of BPH. PMID- 1722155 TI - Descriptive analysis of a series of operations for prostatic adenomas in inhabitants of Lyon, France, in 1988. Urological College of Lyon. AB - A retrospective, population-based study was conducted in Lyon, France, to elucidate the benefits and risks of a treatment for prostatic adenoma (benign prostatic hyperplasia). Case records were reviewed for all prostatectomies performed on patients in Lyon in 1988 for benign prostatic hyperplasia. Data were obtained from all records of public and private hospitals. Of 408 procedures, 312 involved endourethral resection (transurethral resection) and 96 open surgery. The mean weight of resected tissue was 20.3 +/- 0.9 g after endourethral resection and 71.7 +/- 6.4 g after open surgery. About 20% of the resections took place in a university hospital center, 43% in a not-for-profit private hospital, and 37% in a private clinic; there were eight deaths in the first 3 postoperative months, and 11 patients required hospitalization for urologic complications. Length of hospital stay uniquely correlated with age and type of surgery. PMID- 1722156 TI - The natural history and course of untreated benign prostatic hyperplasia. AB - An understanding of the natural history or developmental growth and clinicopathologic evolution of benign prostatic hyperplasia (BPH) is important in assessing prognosis, providing adequate treatment, and evaluating the potential usefulness of newer therapeutic agents. Currently, the general view is that BPH is basically a progressive disease characterized by different growth rates in different individuals. However, the reason for possible fluctuations in growth rate, or even that of spontaneous regression in some individuals as the result of unknown endogenous factors in the host, remains to be determined. PMID- 1722157 TI - Pathogenesis of benign prostatic hyperplasia. AB - The pathogenesis of benign prostatic hyperplasia (BPH) remains largely unresolved. Three major theories have evolved over the years, each emphasizing a possible causative mechanism. The first theory, the dihydrotestosterone hypothesis, is based on the failure of BPH to develop in men castrated prior to puberty. The second, the embryonic reawakening theory, assumes a reawakening of the embryonic induction potential of prostatic stroma. The third, or stem cell theory, postulates the development of BPH through an increase in the number of stem cells or through an abnormal increase in clonal expansion of amplifying or transit cells. These mechanisms may act in concert. PMID- 1722158 TI - Epidemiology of benign prostatic hyperplasia: present knowledge and studies needed. AB - Despite being such a common condition, morphologically prevalent in 88% of autopsies of old (greater than 80 years) men, little epidemiologic research has been undertaken on benign prostatic hyperplasia (BPH). The prevalence of BPH, and probably the incidence, increases with age although the belief that BPH is a direct consequence of aging per se still awaits proof. Many observers have concluded that the age association reflects age-related hormonal changes, although this also requires proof. BPH is a very common condition in aging men: 3 in every 10 may ultimately require surgery for this condition if current estimates of prevalence are correct. Despite its common occurrence, little is known with any degree of certainty about risk factors for BPH apart from being male, being old, and having had a pair of functioning testicles since puberty. There are good reasons why the epidemiology of BPH has remained poorly understood although the application of more epidemiologic thought could pay great dividends, particularly if prostate screening programs could be exploited maximally. PMID- 1722159 TI - Is there a relationship between benign prostatic hyperplasia and prostatic cancer? AB - No definite relationship can be demonstrated between benign prostatic hyperplasia (BPH) and prostatic cancer on the basis of various anatomic and morphologic features. However, interesting recent observations suggest a link between BPH and prostate cancer. Clinically, the simultaneous presence of benign and malignant disease is observed quite often, and this coexistence is bound to increase with the age of patients. Possible intermediate forms between BPH and early cancer have been investigated. Two premalignant lesions have been identified: atypical adenomatous hyperplasia and prostatic intraepithelial neoplasia. PMID- 1722160 TI - Benign prostatic hyperplasia: symptoms and objective interpretation. AB - Considerable new knowledge about benign prostatic hyperplasia has been gained over the past two decades, particularly with regard to its natural history, hydrodynamic changes in the lower urinary tract, and the symptomatic and urodynamic results of treatment. A survey of the literature has been undertaken with special attention to the results of interventional studies and suggestions for future research. PMID- 1722161 TI - Symptomatology and diagnosis of benign prostatic hyperplasia. AB - The symptoms of benign prostatic hyperplasia are well known. Diagnosis rests on digital palpation of the enlarged prostate. Objective parameters are laboratory tests, imaging techniques, endoscopy, and urodynamic investigations. Laboratory tests include urinalysis and serum creatinine measurement. Markers are not reliable and the determination of acid phosphatase and prostate-specific antigen is not recommended routinely for patients with prostatism. Imaging techniques are usually restricted to a complete echographic investigation of the entire urinary tract. Endoscopic manipulations are seldom necessary. Uroflow measurements are customarily performed, but the need for a complete urodynamic investigation is still open to debate. PMID- 1722162 TI - Patient reports of symptoms and quality of life following prostate surgery. AB - In the United States, prostate surgery, most often transurethral resection of the prostate, is a common treatment for men with benign prostatic hyperplasia. For at least 80% of patients, this elective surgery reduces everyday symptoms associated with urination, and reduces the risk of episodes of acute retention, so that the quality of life is improved. However, there is considerable variability in the way symptoms affect patients. The potential value of surgery cannot be based solely on a symptom profile, but must include information about the patient's own response to those symptoms. PMID- 1722163 TI - Imaging of the prostate gland. AB - The advent of ultrasonography and the development of intrarectal techniques have made possible semiquantitative prostatic volumetrics for more accurate preoperative assessment of benign prostatic hyperplasia (BPH) as well as earlier diagnosis of prostatic carcinoma (PC). Computed tomography has increased the scope of prostatic imaging by including regional lymph nodes as well. Magnetic resonance imaging (MRI) has enabled viewing of the internal architecture of the prostate gland, thus allowing precise diagnosis of BPH and recognition of carcinoma. MRI is also the most exact method of staging PC. PMID- 1722164 TI - Benign prostatic hyperplasia: surgical versus nonsurgical treatment. A critical review. AB - With the advent of new treatments for benign prostatic hyperplasia (BPH), the urologist must objectively assess the results of new and traditional therapeutic options. This requires three sets of data-not all of which may be readily available-including a precise knowledge of the natural history of BPH; an objective assessment of the results of surgery, both open and transurethral; and an accurate estimate of objective and subjective results of medical treatment based on randomized studies with a placebo arm. The two types of medical treatment available are hormonal and neuropharmacologic manipulation. Hormonal treatment, using agents such as luteinizing hormone-releasing hormone agonists or antiandrogens, reduces the volume of the epithelial component of the prostate, but urodynamic improvement does not always parallel volumetric reduction. Neuropharmacologic treatment using alpha-blockers alone or in combination with anticholinergic agents has no effect on prostatic volume, but reduces the tone of the muscular component. PMID- 1722165 TI - Changing approaches in the treatment of benign prostatic hyperplasia. AB - A large number of new options for the management of benign prostatic hyperplasia (BPH) have become available, including several classes of drugs that are under investigation. Various principles of management include hyperthermia, balloon dilatation, the introduction of spirals to keep the prostate open, and exploitation of the endocrine dependence of BPH through androgen withdrawal, using 5 alpha-reductase and aromatase inhibition. The efficacy of these alternative forms of treatment will become better defined during the next few years. Whatever the result, it is likely that the indication for surgery will decrease. Therapy must also take into account the sexual activity and expectations of the patient, an area that until recently has been largely neglected. PMID- 1722166 TI - Steroid hormones and the pathogenesis of benign prostatic hyperplasia. AB - The pathogenesis of benign prostatic hyperplasia (BPH) is still poorly understood: there is, however, general acceptance that the condition is not premalignant and that it has an etiology distinct from that of cancer. Interest now focuses on the biochemistry of the target prostate cells and the propensity of the gland for uncontrolled growth. Dihydrotestosterone (DHT) is the active intracellular androgen formed from testosterone by 5 alpha-reductase. DHT concentrations appear a little higher in BPH tissue than in normal tissue, and there is no doubt that DHT-receptor complex modulates gene expression. Current studies suggest that DHT is essential but not sufficient for proliferation, and that other regulatory factors, including peptide growth factors, are prerequisite. The growth responsiveness of prostate tissue to androgens may be dependent on the balance between epithelial and stromal tissues, with biologic processes in the epithelium indirectly controlled by androgen-dependent mediators of stromal origin. PMID- 1722167 TI - 5 alpha-metabolism in finasteride-treated subjects and male pseudohermaphrodites with inherited 5 alpha-reductase deficiency. A review. AB - Male pseudohermaphrodites (MPHs) with inherited 5 alpha-reductase deficiency and decreased dihydrotestosterone production have a global defect in 5 alpha metabolism affecting both C19 androgen metabolism and C21 steroid metabolism. However, the decreased 5 alpha-reduction of testosterone to dihydrotestosterone is the only impaired steroid conversion to have clinical consequences, e.g., ambiguous genitalia, impaired prostate differentiation and development, and decreased facial and body hair. The 5 alpha-steroid metabolite profile in the MPHs was compared with that of men with benign prostatic hyperplasia who were administered varying doses of the 5 alpha-reductase inhibitor finasteride. Finasteride was found to be a potent inhibitor of both C19 androgen and C21 5 alpha-steroid metabolism affecting both hepatic and peripheral 5 alpha metabolism. The 5 alpha-steroid metabolite profile was strikingly similar to that of MPHs with inherited 5 alpha-reductase deficiency. The data suggest that a 5 alpha-reductase gene codes for an enzyme with affinity for multiple steroid substrates. PMID- 1722168 TI - Hormonal effects of a 5 alpha-reductase inhibitor (finasteride) on hormonal levels in normal men and in patients with benign prostatic hyperplasia. AB - Finasteride is a potent competitive 5 alpha-reductase inhibitor, active at a dose as low as 1 mg/day. After a single dose, the effects on 5 alpha-reductase last as long as 7 days. Both hepatic and target tissue 5 alpha-reductase are inhibited. Plasma testosterone and estradiol are unaffected and luteinizing hormone levels do not change. During chronic treatment with finasteride 5 mg/day, the effects on 5 alpha-reductase are maintained. Since the only significant effect of chronic finasteride therapy appears to be 5 alpha-reductase inhibition, and testosterone or estradiol levels are not affected, neither libido nor potency is lost. Testosterone is the active androgen at the muscular level; therefore, muscular catabolism is not expected. PMID- 1722169 TI - No pharmacokinetic interaction between iloprost and digoxin. PMID- 1722170 TI - Bilateral projections from the parabigeminal nucleus to the superior colliculus in monkey. AB - We examined the distribution of labeled neurons in the parabigeminal nucleus of the monkey following injections of retrograde fluorescent tracers into the superior colliculus. The extent of the visual field representation included in the injection site was assessed from the location of labeled cells in striate cortex. The results suggest a rough topographic organization of the parabigeminal nucleus, with the lower quadrant represented anteriorly and the upper quadrant posteriorly. We also found bilateral projections from the parabigeminal nucleus to both superior colliculi, but the crossed projection appeared to terminate only in that part of the colliculus where the vertical meridian is represented. Parabigeminal cells with a crossed projection were larger than those projecting to the ipsilateral colliculus. The results suggest that the organization of the monkey's parabigemino-tectal system is fundamentally similar to that of many other vertebrates. PMID- 1722171 TI - Auditory corticocortical interconnections in the cat: evidence for parallel and hierarchical arrangement of the auditory cortical areas. AB - The origin and laminar arrangement of the homolateral and callosal projections to the anterior (AAF), primary (AI), posterior (PAF) and secondary (AII) auditory cortical areas were studied in the cat by means of electrophysiological recording and WGA-HRP tracing techniques. The transcallosal projections to AAF, AI, PAF and AII were principally homotypic since the major source of input was their corresponding area in the contralateral cortex. Heterotypic transcallosal projections to AAF and AI were seen, originating from the contralateral AI and AAF, respectively. PAF received heterotypic commissural projections from the opposite ventroposterior auditory cortical field (VPAF). Heterotypic callosal inputs to AII were rare, originating from AAF and AI. The neurons of origin of the transcallosal connections were located mainly in layers II and III (70-92%), and less frequently in deep layers (V and VI, 8-30%). Single unit recordings provided evidence that both homotypic and heterotypic transcallosal projections connect corresponding frequency regions of the two hemispheres. The regional distribution of the anterogradely labeled terminals indicated that the homotypic and heterotypic auditory transcallosal projections are reciprocal. The present data suggest that the transcallosal auditory interconnections are segregated in 3 major parallel components (AAF-AI, PAF-VPAF and AII), maintaining a segregation between parallel functional channels already established for the thalamocortical auditory interconnections. For the intrahemispheric connections, the analysis of the retrograde tracing data revealed that AAF and AI receive projections from the homolateral cortical areas PAF, VPAF and AII, whose neurons of origin were located mainly in their deep (V and VI) cortical layers. The reciprocal interconnections between the homolateral AAF and AI did not show a preferential laminar arrangement since the neurons of origin were distributed almost evenly in both superficial (II and III) and deep (V and VI) cortical layers. On the contrary, PAF received inputs from the homolateral cortical fields AAF, AI, AII and VPAF, originating predominantly from their superficial (II and III) layers. The homolateral projections reaching AII originated mainly from the superficial layers of AAF and AI, but from the deep layers of VPAF and PAF. The laminar distribution of anterogradely labeled terminal fields, when they were dense enough for a confident identification, was systematically related to the laminar arrangement of neurons of origin of the reciprocal projection: a projection originating from deep layers was associated with a reciprocal projection terminating mainly in layer IV, whereas a projection originating from superficial layers was associated with a reciprocal projection terminating predominantly outside layer IV.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722173 TI - Convulsions and wet-dog shakes produced by systemic or intrahippocampal administration of ruthenium red in the rat. AB - In this work we have studied in the rat the behavioral effects of the intraperitoneal (i.p.) and intrahippocampal (i.h.) administration of ruthenium red (RuR), an inorganic dye which has been shown to inhibit neurotransmitter release in synaptosomes. The i.p. injection induced initially flaccid paralysis and subsequently generalized tonic-clonic convulsions. It contrast, unilateral RuR microinjection into the CA1 area of the hippocampus produced complex seizure behavior and wet-dog shakes (WDS). The i.p. administration of the serotonin receptor antagonist ketanserin markedly inhibited the WDS induced by i.h. RuR. In contrast, the i.h. injection of ketanserin and of the gamma-aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol(THIP) and baclofen together with RuR did not affect the frequency of WDS nor the seizure behavior. However, the i.h. injection of the GABA uptake blocker nipecotic acid, simultaneously with RuR, increased the frequency of WDS. The release of [3H]GABA, measured in synaptosomes of different cerebral structures of the rats injected i.p. with RuR, and in slices of the CA1 area after i.h. injection of the dye, was not affected. Histological observations of the injected area showed a specific and intense staining of the somas of the CA1 pyramidal neurons. It is concluded that the convulsant action induced by i.h. RuR microinjection is probably the result of an increased excitability of these CA1 neurons, which is independent of any action on GABA release. PMID- 1722172 TI - Chemoarchitectonic organization of the cat primary auditory cortex. AB - Acetylcholinesterase (AChE) activity, demonstrated histochemically, defines an area of cortex on the middle ectosylvian gyrus that appears to correspond to the cytoarchitectonically defined area 41 and the physiologically defined primary auditory area (AI). In this area there are high levels of AChE in layers III, IV and VI while in the surrounding areas there are comparatively low levels of enzyme in these layers. The monoclonal antibody CAT 301, which was raised against a cell surface proteoglycan, also defines this area. There are high levels of CAT 301 immunoreactivity in cell bodies and the neuropil of layer III and an absence of very large immunoreactive neurons in layer V. Furthermore there are higher levels of the calcium binding protein, parvalbumin and the metabolic enzyme, cytochrome oxidase, in layers III and IV of AI, than in most of the surrounding cortex. By contrast the distribution of the calcium binding protein, calbindin and the distribution of myelinated fibers are similar in area 41 and the surrounding areas. PMID- 1722174 TI - Phenotyping of peripheral blood hemopoietic progenitor cells--in vitro cultures using CD34-/CD33-immunomagnetic purging. AB - In contrast to many detailed studies on the antigenic profile of hemopoietic progenitor cells from human bone marrow, sparse information, so far, has been gathered with regard to the antigen expression of hemopoietic progenitors present in peripheral blood. Previous studies by multiparameter flow-cytometry have revealed substantial differences of the coexpression of the CD33-, CD19-, and CD74- antigens, respectively, on CD34-positive cells from blood versus those from bone marrow, respectively. Immunomagnetic purging with monoclonal antibodies detecting the CD34-, and the CD33- antigen, respectively, has been used to further characterize the expression of these antigens on day 8 and d-14 granulocyte/macrophage and erythroid colonies as grown from circulating progenitor cells. Purging with CD34 monoclonal antibody abrogated all colony formation, whereas purging with CD33 antibody led to differential inhibition of the various progenitors. Purging bone marrow cells with CD34 antibody, an inhibition of only about 25% was observed with regard to erythroid colonies, whereas an inhibition of about 85% was observed for CFU-GM. These findings reinforce the view that circulating progenitor cells represent relatively immature stages of differentiation, when compared to bone marrow progenitors. Particularly, d-8 erythroid colonies from blood do not represent the equivalent of the genuine CFU-E as described from bone marrow, but they seem to be early stages of BFU-E development. PMID- 1722175 TI - [The correction of pancreatic enzyme secretion depending on the properties of the duodenal contents]. AB - Substrates both specific and unspecific for pancreatic enzymes abolish and modify the inhibition of pancreatic enzymes secretion induced by their intraduodenal administration. Enterosorbents and gastric mucus, depending on the strength of their sorption properties, abolish or decrease the inhibition of secretion of pancreatic enzymes induced by intraduodenal administration of pancreatic enzymes. The complicated mechanism of correction of pancreatic enzymes secretion seems to depend on duodenal chyme properties. PMID- 1722176 TI - Transvaginal color Doppler imaging for hemodynamic assessment of reproductive tract tumors. AB - Transvaginal color Doppler flow imaging was carried out on 68 Japanese women (normal, 10; uterine myoma, 21; cervical carcinoma, 7; endometrial carcinoma, 10; benign ovarian tumor, 12; ovarian carcinoma, 8). Blood flow velocity waveforms were evaluated by calculation of the resistance index (RI). In 6 patients with cervical carcinoma neovascularization was evident within the cervix. In all patients with endometrial carcinoma such signs were present adjacent to and/or within the endometrium. These findings were absent in normal women and in those with myomata. There was a significant difference between the RI (0.510 +/- 0.097) in patients with cervical carcinoma and in normal women (0.881 +/- 0.048) in the ascending branch. In endometrial carcinoma the RI (0.535 +/- 0.158) was significantly lower in the arcuate artery compared to the normal uterus (0.768 +/ 0.075) and patients with uterine myoma (0.679 +/- 0.131), respectively. There was no area of neovascularization in the normal ovaries. Neovascularization was confirmed in four patients with a benign ovarian tumor and in all patients with an ovarian carcinoma. A significantly lower RI was obtained in cases of ovarian carcinoma (0.503 +/- 0.122) than in patients with benign ovarian tumors (0.888 +/ 0.216). Transvaginal color Doppler imaging and pulsed Doppler analysis may be useful diagnostic tools to differentiate benign and malignant tumors. PMID- 1722177 TI - Transcription of sequences upstream of the rat prolactin gene suggests the existence of a second promoter. AB - The transcription of the rat prolactin gene domain has been examined using a modified Southern blot procedure. Cloned genomic DNAs were resolved by electrophoresis in agarose, transferred to nitrocellulose, and probed with radiolabeled RNA that had been synthesized in vitro by nuclei isolated from pituitary tumor cells. Data presented in this paper illustrate that single copy genomic sequences located within 7.3 kb upstream of exon 1 are transcribed. Single copy or low copy number DNA sequences that reside greater than 7.3 kb upstream of exon 1, or downstream of exon 5 were not transcribed at detectable levels. These data suggest that a second promoter may exist upstream of the rat prolactin gene and that this second promoter may be active in pituitary cells. PMID- 1722178 TI - Role of chloride ions in progesterone production by chicken granulosa cells. AB - The importance of chloride ions in luteinizing hormone (LH)-stimulated progesterone production by chicken granulosa cells from the two largest preovulatory follicles was investigated in vitro. Reduction of the extracellular chloride concentration from 147.8 mM to 2.8 mM, by substitution with equimolar concentrations of non-permeant glutamate and aspartate, inhibited the ability of LH to stimulate progesterone production and cAMP accumulation during a 4 h incubation. LH-stimulated granulosa cell progesterone production was also suppressed in a concentration-dependent manner by the chloride channel blockers 4 acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS; 10(-8)-5 x 10(-5) M) or 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS; 10(-8)-5 x 10(-5) M). The inhibitory effect was observed within 30 min of the addition of the blockers and was irreversible. DIDS appeared to act at a site(s) proximal to the generation of cAMP, since concentrations of DIDS (10(-8)-10(-6) M) which inhibited LH- and human chorionic gonadotropin-stimulated progesterone production, did not affect progesterone production stimulated by dibutyryl cAMP, 8-bromo cAMP or forskolin. In addition, concentrations of DIDS (10(-8)-10(-6) M) which attenuated LH-stimulated progesterone production also reduced the accumulation of extracellular cAMP. These studies suggest that chloride ions may play an important role in the stimulatory action of LH on chicken granulosa cell progesterone production. PMID- 1722179 TI - Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture. AB - The effects of dibutyryl cyclic AMP [Bu)2cAMP) and phorbol ester (TPA), in the absence or presence of follicle-stimulating hormone (FSH) and/or testosterone, on the development of tight junctions by immature rat Sertoli cells (Sc) were investigated in vitro using the two-compartment culture system. The tight junction status was evaluated by repeated measurements of transepithelial electrical resistance (TER). Untreated cell monolayers developed stable TER of approximately 120 omega cm2 during 3 days of culture. Continuous presence of FSH (200 ng/ml) from day 1 onward significantly increased the TER up to approximately 300 omega cm2 after a transient (24-36 h) delay. The initial delay was prolonged to 3-4 days by the addition of 1-methyl-3-isobutylxanthine (MIX) (0.2 mM), whereas the subsequent increase of TER was significantly potentiated by the concomitant presence of testosterone (10 microM). Cholera toxin (CHT; 10 ng/ml) and forskolin (FR; 50 microM) mimicked these FSH effects. (Bu)2cAMP, at concentrations which maximally stimulated immunoactive inhibin secretion (100-500 microM), inhibited the initial TER increase and significantly decreased the TER level when added on days 1 and 5 of culture, respectively. In contrast, low concentrations of (Bu)2cAMP (4-20 microM) consistently stimulated the TER development, mimicking the stimulatory phase of FSH action. TPA (100 nM) alone had no effect on TER development, but potentiated the stimulatory effect of testosterone in a manner similar to FSH, CHT, FR or low concentrations of (Bu)2cAMP. These results demonstrate, for the first time, a concentration dependent, dual effect of exogenous cAMP on the Sc function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722180 TI - Twelve of fourteen surface epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor. AB - On the surface of the free (receptor-unbound) form of hCG, we have previously identified 14 topographically distinct epitopes (Schwarz et al. (1986) Endocrinology 118, 189-197; Berger et al. (1990) J. Endocrinol. 125, 301-309). Here we report that only two of them, i.e. the (adjacent) beta 3 and beta 5 epitopes, can be recognized by 125iodine-labeled monoclonal antibodies when hCG was specifically bound to the rat testis hCG receptor. The exclusive accessibility of precisely these two surface epitopes indicates that hCG assumes a defined rather than a stochastic orientation in its receptor-bound state. The inaccessibility of 12 of 14 epitopes is consistent with the idea that the 341 residues long extracellular domain of the recently cloned hCG receptor (MacFarland et al. (1989) Science 245, 494-499) is the ligand binding domain. It is proposed that the extracellular domain is folded in a way that a cavity is formed large enough to accommodate hCG. Thereby, a considerable portion of the total surface of hCG is covered, as reflected by the masking of most of its epitopes. PMID- 1722181 TI - Effects of ageing on nicotine-induced contraction and substance P-like materials release in guinea-pig bronchus. AB - 1. Effects of ageing on nicotine-induced contraction and release of substance P like materials in the bronchial preparations from guinea-pigs of different ages were studied. 2. The pD2 value (potency) of nicotine decreased with age from 10 to 100 weeks. The pD2 value of substance P did not change with age suggesting that substance P receptor mechanisms do not alter with age. 3. The amount of substance P-like materials released by nicotine (10(-4) M) decreased with age from 10 to 100 weeks, supporting our previous findings that nicotine contracts the guinea-pig bronchus through the release of substance P-like materials. 4. These results suggest that the age-related decrease in the pD2 value (potency) of nicotine is due to the reduction in the amount of substance P-like materials released by nicotine. PMID- 1722182 TI - Diastolic tension and contraction amplitude in calcium-loaded rat ventricular myocardium are differently affected by drugs. AB - 1. To obtain a measure of drug effects on myocardial function during diastole, the following experimental protocol was designed: rapid electrical stimulation (5 Hz) at high Ca02+ caused an elevated diastolic tension, which could be subjected to drug-induced alterations. 2. Antiarrhythmic drugs (quinidine, propafenone, procainamide, mexiletine) were able to lower diastolic force without appreciably decreasing contraction amplitude. Calcium antagonists (nifedipine, verapamil) lowered both parameters in parallel. 3. Veratridine and Bay K 8644 both enhanced diastolic tension, but only Bay K 8644 concomitantly elevated contraction amplitude. 4. These findings may be explained when taking into account differential actions of sodium- and calcium channel modulating drugs, respectively, on cellular Ca2+ movements. In quantitative terms, the non-linear dependence of myocardial force on Cai2+ also had to be considered. PMID- 1722183 TI - The dual effect of BAY K 8644 on excitation-contraction coupling in gastric smooth muscle. AB - 1. BAY K 8644 at concentrations of 10(-10)-10(-6) M had a stimulant effect on the spontaneous electrical and contractile activity of smooth muscle preparations from cat and guinea pig stomach. 2. Nifedipine (10(-6) M) antagonized the BAY K 8644-induced spike potentials and the related phasic contractions. 3. Neither the excitatory nor the inhibitory effect of BAY K 8644 was significantly influenced by atropine (10(-7) M), phentolamine (10(-7) M), propranolol (10(-7) M) or TTX (10(-6) M). 4. TEA (10(-3) M) abolished the inhibitory effect of BAY K 8644 on the spike generation and increased the amplitude of the phasic contractions. PMID- 1722184 TI - Substance P and its fragments affect Ca2+/calmodulin-dependent synaptosomal membrane protein phosphorylation from rat cerebral cortex. AB - 1. We have used synaptosomal membranes to study the influence of substance P and its fragments and analogues of its C-terminal fragment on Ca2+/calmodulin dependent synapsin I endogenous phosphorylation. 2. SP1-11, SP1-4, [Tyr8]SP6-11 and [pGlu6, Tyr8]SP6-11 at 10(-3) M greatly inhibited synapsin I phosphorylation. 3. SP6-11 at all investigated concentrations and SP1-11, SP1-4, [Tyr8]SP6-11, [pGlu6, Tyr8]SP6-11 at 10(-4) and 10(-5) M were ineffective. 4. The results indicate that SP1-11 and its N-terminal fragment and analogues of its C-terminal fragment act on the phosphorylation of specific synaptic protein (synapsin I) and therefore may influence the release of neurotransmitters, membrane conductance and potentiation or inhibition of other signalling systems. PMID- 1722185 TI - [Positional control of the distribution of spontaneous sister chromatid exchanges along mouse chromosomes]. AB - The use of a new method having combined C-band staining and differential staining of sister chromatids allowed to determine a pattern of distribution of spontaneous sister chromatid exchanges (SCE) along cytologically marked chromosomes 1, 2 and 6 of house mouse. All chromosomes displayed the same pattern of SCE distribution: SCEs are most frequent in the middle part of the chromosome arm and rather rare near the centromere and the telomere. It has been suggested that this pattern of distribution is positional, rather chromatin-specific. The chromosome 1 carrying paracentric inversion with breakpoints in the middle part of the arm and just near the telomere has the same pattern of SCE distribution as normal chromosome 1. Double insertion of homogeneously staining regions in the middle part of the chromosome 1 produces increase in the SCE number per chromosome proportional to the physical length of the insertion. In contrast to meiotic recombination, interference between SCEs is not detected. No evidence for existence of the hot-spots of SCE on the junctions between C-positive and C negative regions, as well as between G-bands and R-bands, has been produced. PMID- 1722186 TI - [Detection of chromosomal abnormalities using cordocentesis]. AB - Four cases of cytogenetic prenatal diagnosis of fetuses with chromosomal aberrations are presented: (1) the Patau syndrome; (2) and (4) the Down syndrome; (3) the Klinefelter syndrome. Cordocentesis has been shown to be expedient for rapid and accurate determination of fetus karyotype. Indicative for cytogenetic examination were ultrasonic data, maternal age, the values of AFP, HGG and nonconjugated estreol in maternal serum. Comparison of ultrasonic examination of fetuses with the data on abortus autotopsia was undertaken. The results demonstrate importance of ultrasonic, cytogenetic, biochemical and morphological research in prenatal malformation diagnosis. PMID- 1722187 TI - [Magnetocardiography. Biomagnetic localization of impulse development and transmission of the heart]. AB - Magnetocardiography is a non-invasive biomagnetic technique for measuring magnetic fields produced at the surface of the body when the heart is stimulated to beat. The measurement is contact-free and is independent of tissue resistance. For the first time, magnetocardiography employing multi-channel systems permits the accurate, non-invasive localization of accessory conduction pathways and ectopic ventricular activity. PMID- 1722189 TI - Induction of the acrosome reaction in sperm by exposure to low temperature increases their rate of fusion with zona-free hamster oocytes. AB - The fusion rate of human sperm with zona-free hamster ova was investigated after induction of the acrosome reaction by exposure to a low temperature (4 degrees C). Sperm were collected from 14 patients, and selected by the 'swim-up' method. The sperm were incubated for 24 h at either room temperature (control group) or at 4 degrees C (low temperature group), followed by additional incubation at 37 degrees C for 3 h. The mean sperm penetration rate, number of swollen sperm heads as well as the number of sperm attached to the oocyte increased significantly after exposing sperm to low temperature. The sperm penetration rate showed a significant correlation (Spearman test, r = 0.572, n = 28, P less than 0.0035) with the acrosome reaction in the low temperature group. These results were associated with an increase in the rate of penetration of hamster ova observed in this study, presumably due to the increase in induction of the acrosome reaction by low temperature. Incubation of sperm at low temperature might be useful in the evaluation of so-called false negative results in the zona-free hamster test. PMID- 1722188 TI - [Systematics, differentiation, and detection of bacterial infections-- the family Mycobacteriaceae]. AB - Comparative 16S rRNA sequencing allows to infer natural relationships among bacteria, to characterize and identify microorganisms at a molecular level and to develop DNA probes specific at any desired taxonomic level (e.g. family, genus, species). Probes targeted at ribosomal RNA are suitable for in situ hybridization of whole, intact bacterial cells as well as in polymerase-chain-reaction techniques for sensitive detection and identification of bacteria. Comparative 16S rRNA sequencing provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. Certain growth characteristics, i.e. thermotolerance and growth rate correspond to natural relationships among the mycobacteria. However, the phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotochromogenic and nonphotochromogenic mycobacteria. The use of oligonucleotides targeted at highly or semi-conserved regions within the 16S rRNA molecule allows a universal procedure for amplification and rapid sequence determination of 16S rDNA-gene fragments from any virtually bacterial organism. This method of amplification of 16S rDNA-gene fragments was used to identify a novel, uncultured pathogen and opens new perspectives for other infectious diseases of unknown cause. PMID- 1722190 TI - Evaluation of the acrosome reaction using monoclonal antibodies against different acrosomal antigens--comparison with the triple stain technique. AB - The acrosome reaction of human sperm was evaluated in vitro with the aid of there monoclonal antibodies TuS-1, TuS-19 and TuS-20, which react with antigens in the anterior part of the acrosome. Data were compared with results obtained using the triple stain technique. Seminal smears were performed prior to, and following incubation for 5 and 24 h in Hams-F10 containing 3% human serum albumin. Using the triple stain technique, 15.2 +/- 7.1% of sperm exhibited acrosome activation after 5 h and 16.8 +/- 8.4% after incubation for 24 h. The corresponding values obtained using antibody TuS-1 were 12.9 +/- 5.8 and 13.2 +/- 2.2%, with TuS-19 10.1 +/- 3.8 and 10.8 +/- 1.4% and with TuS-20, 9.0 +/- 3.4 and 10.4 +/- 2.9%. When the same sperm smears were stained firstly with rose Bengal, and secondly with TuS-1 both methods stained the same cells and the same region of the cells, thus indicating that both methods stain the same substrate. Staining of the acrosome with monoclonal antibodies gives clearer results than the triple stain technique, and could be used in preference in future studies dealing with diagnosis of the acrosome reaction in vitro. PMID- 1722191 TI - Effect of isoprinosine on IL-2, IFN-gamma and IL-4 production in vivo and in vitro. AB - The effects of an immunopotentiating drug, isoprinosine, on the splenocytes of BALB/c mice to produce cytokines were investigated. Isoprinosine enhanced IL-2 production, upregulating the expression of IL-2 receptor in vitro. It also significantly increased the IFN-gamma secretion and decreased the IL-4 production in vivo. The significance of these findings in terms of immune regulation is discussed. PMID- 1722192 TI - FK-506 prevents diabetes in diabetes-prone BB/Wor rats. AB - The effect of the immunosuppressant FK-506 on the development of diabetes in BB/Wor rats was investigated. Using a treatment schedule (25 micrograms i.m. from day 27 to 120), not associated with detectable general ill effects, this drug was found to completely inhibit the appearance hyperglycemia and to reduce the histological signs of pancreatic insulitis. The treatment was also able to reduce the percentages of Ia+ T-lymphocytes and to block the appearance of detectable serum levels of gamma interferon (IFN). PMID- 1722193 TI - Immunotoxin with mistletoe lectin I A-chain and ricin A-chain directed against CD5 antigen of human T-lymphocytes; comparison of efficiency and specificity. AB - Monoclonal anti-CD5 antibody was coupled to the enzymatically active subunit of plant toxin [either mistletoe lectin I (ML) or ricin]. The obtained conjugates proved to be selectively toxic to CD5-bearing target cells. The immunotoxin prepared from ML A-chain (MLA) was as toxic as native ML and approximately 80 fold more active than the corresponding conjugate with ricin A-chain (RTA). The comparative studies of the structural properties of isolated MLA and RTA were carried out using intrinsic fluorescence spectroscopy. The results showed similar properties for both proteins. No antigenic cross-reactivity against both toxins was detected when using polyclonal antibodies. The results suggest that MLA antibody conjugates may be potential candidates for therapeutical use. PMID- 1722194 TI - The mechanism of action of a synthetic immunomodulator, 3,6-bis(2 piperidinoethoxy)acridine trihydrochloride (CL 246,738), in natural killer cell activation in animals. AB - CL 246,738 is a low molecular weight, synthetic immunomodulator. The present study was done to determine the interaction among interferon (IFN), macrophages, and natural killer (NK) cells in mice following oral administration of CL 246,738. Splenic NK activity as evidenced by lysis of YAC-1 lymphoma cells in vitro was found to be augmented by the compound not only in normal mice, but also in immunodeficient beige and nude mice. Lytic activity remained elevated from one to seven days after a single treatment and the peak activation varied depending on the source of NK cells. NK cell activity associated with the peritoneal exudate cell population peaked at day 1 and returned to normal by day 2, whereas NK cell activity of peripheral blood lymphocytes peaked at day 3 and remained significantly elevated until day 7. Liver associated NK activity peaked at day 4 and remained significantly elevated at day 7 after treatment with CL 246,738. Lung associated NK activity was elevated by day 1 after treatment, peaked at day 4 and returned to normal by day 7 after drug administration. The drug was also effective in inducing IFN in all mouse strains tested. When these drug-treated mice were given antibody to IFN-(alpha + beta) but not to IFN-(beta), both IFN levels and NK cell activity decreased, suggesting the importance of IFN-(alpha) in this system. Furthermore, mice that had received carrageenan prior to, but not after CL 246,738 administration showed reduced serum IFN titers as well as decreased NK cell activity, indicating that macrophages played an intermediate role in immune enhancement by the drug.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722195 TI - Genetic control of the expression of two biological activities of an antitumor polysaccharide, lentinan. AB - In order to make clear whether the expression of biological activities and antitumor polysaccharides are under genetic control, the responses of mice to lentinan, a beta-1,6;1,3-glucan, in the induction of several acute phase proteins (APPs) and T-cell-mediated vascular dilation and hemorrhage (VDH) were investigated. Twenty inbred strains of mice were divided into two groups according to their phenotypes in the induction of APPs when they were administered lentinan i.p. at a dose of 10 mg/kg; sensitive strains showed a marked increase in levels of APPs and resistant strains showed as low a level of APPs as non-treated control mice. No sex-related differences and no relation with H-2 halotypes were found in the responses. Only low-level responses were observed in F1 hybrid mice obtained by crosses between a sensitive and a resistant strain, indicating that the low APP response to lentinan is dominant. The N2 progeny between the F1 and a high responder segregated into high and low responders at a ratio of almost 1:1. These results suggest that a single major gene on an autosome is responsible for the induction of APPs. The induction of VDH also depended on the strains of mice. However, the strain distribution pattern of the VDH phenotype was distinct from that of the APP phenotype, indicating that the VDH-controlling gene was different from the APP-controlling gene. Further analyses with F1 hybrid and backcross progeny mice suggested that the high VDH response was dominant, and that the phenotype was determined by a single major gene. PMID- 1722196 TI - How can the aromatic side-chains modulate the conductance of the gramicidin channel? A new approach using non-coded amino acids. AB - In order to elucidate the role of the aromatic side-chains in the mechanism of transduction of monovalent cations through the channel of linear gramicidin, two series of analogues containing non-coded aromatic amino acids were synthesized. In the first series, the four tryptophans were replaced by either four L-3-(8 quinolyl)alanyl or four L-3-(4-quinolyl)alanyl residues and single channel conductance measurements showed that these substitutions led to a strong lowering of the channel conductance, which is attributed to a modification of the orientation of the aromatic side-chains due to an increase of their hydrophobicity. In the second series, the analogues contained both tryptophyl and naphthylalanyl residues in various amounts and positions. The single channel conductance data indicated that the conductance was mainly governed by the number of polar residues (Trp) and not by their positions. The conformational consequences of these results are discussed together with their influence on the energy profile of the gramicidin channel. PMID- 1722197 TI - Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor. AB - The neuropeptide galanin (1-29) binds with high affinity to hypothalamic receptors (KD approximately 0.9 nM) and regulates feeding behavior. The N terminal fragments (1-16), (1-16)NH2 are high affinity (KD approximately 6 nM) full agonists in vivo and in vitro. L-Ala substitutions show that amino acid residues Gly1, Trp2, Asn5, Tyr9, and Gly12 are important for the high affinity binding of galanin (1-16). Shortening the fragment (1-16) to galanin (1-7) causes a gradual drop of affinity: galanin (1-15), (1-14), and (1-13) have submicromolar KD values and galanin (1-12) has KD approximately 3 microM. Cyclic analogs of galanin (1-12) of different ring size were synthesized by condensing Gly1 and Gly12 without or with spacer groups. These analogs, independent of ring size, had a lower affinity than the linear galanin (1-12). Derivatization of the N-terminus of galanin (1-29), (1-16), and (1-12) all resulted in a large drop of affinity for the receptors, suggesting again the importance of the free N-terminal Gly. PMID- 1722198 TI - Cellular and molecular biology of neuronal intermediate filaments. PMID- 1722199 TI - Reflex responses of laryngeal and pharyngeal submucosal glands in dogs. AB - In dogs tracheal secretion is enhanced reflexly and by locally acting mediators such as substance P (SP). To evaluate the role of these mechanisms on submucosal gland secretion in the larynx (L) and pharynx (Ph), we compared the effects of mechanical stimulation of intrapulmonary irritant receptors and stimulation of pulmonary C-fiber receptors by capsaicin (20 micrograms/kg iv) with the response produced by intravenous SP. In six alpha-chloralose-anesthetized, paralyzed, and artificially ventilated dogs, submucosal gland secretion was monitored by analyzing the areas covered by hillocks of liquid and calculating the volume of secreted liquid (microliter) in the L and Ph. Mechanical stimulation of the carina increased both the number of hillocks and the volume of secreted liquid in the L. Excitation of pulmonary C-fiber receptors also increased the number of hillocks, and total volume of secreted liquid was elevated from 1.9 +/- 0.5 to 8.3 +/- 1.4 microliters (P less than 0.01). These responses were significantly reduced by prior cervical vagotomy and intravenous administration of atropine. Neither stimulation of irritant receptors nor stimulation of pulmonary C-fiber receptors caused discernible effects on Ph submucosal gland secretion. However, intravenous SP increased the number of Ph hillocks and elevated the volume of secreted Ph liquid from 1.0 +/- 0.6 to 10.2 +/- 1 microliters (P less than 0.01); similar responses to intravenous SP were observed in the L. Prior intravenous administration of atropine methylnitrate or bilateral vagotomy did not alter Ph or L secretory responses to intravenous SP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722200 TI - Cyclic AMP antagonist Rp-cAMPS inhibits amylase exocytosis from saponin permeabilized parotid acini. AB - Rp-cAMPS, the Rp-diastereomer of adenosine 3',5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken together with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini. PMID- 1722201 TI - CSK: a protein-tyrosine kinase involved in regulation of src family kinases. AB - The functions of src family protein-tyrosine kinases are thought to be regulated negatively by the phosphorylation of highly conserved tyrosine residues close to their carboxyl termini. Recently we have purified and cloned a protein-tyrosine kinase (designated as CSK) that can specifically phosphorylate the negative regulatory site of p60c-src. To elucidate the relationship between CSK and other types of src family kinases, we investigated the tissue distribution of CSK and examined whether CSK could phosphorylate the negative regulatory sites of src family kinases other than p60c-src. Western blot analysis indicated that CSK was enriched at the highest level in lymphoid tissues in which the expression of p60c src is considerably lower than those of other types of src family kinases. CSK phosphorylated p56lyn and p59fyn, which are known to be expressed in lymphoid tissues at a relatively high level. The putative regulatory site, tyrosine 508, was found to be essential for phosphorylation in p56lyn, and the kinase activities of these src family kinases were repressed by phosphorylation with CSK. These findings raise the possibility that CSK might act as a universal regulator for src family kinases. PMID- 1722202 TI - Human immunodeficiency virus reverse transcriptase displays a partially processive 3' to 5' endonuclease activity. AB - We have examined the ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV-RT) using a natural sequence 83-nucleotide-long RNA template to which was hybridized a DNA oligomer. This substrate configuration allowed for the simultaneous electrophoretic resolution of 5'-, 3'-, and internally derived RNase H cleavage products. Assays performed in the presence of excess challenger RNA to sequester the RT permitted the analysis of products resulting from a single round of binding of RT to substrate. Substrate cleavage was highly sensitive to ionic strength, showing greatest activity at low KCl concentrations. The increase in cleavage correlated with an increase in the half life of the enzyme on the RNA-DNA hybrid from approximately 31 s to 6.2 min at 80 and 5 mM KCl, respectively. Internally derived cleavage products generated in challenged reactions were primarily 2-9 nucleotides in length. These lengths indicate that the products were generated by an endo- rather than an exonuclease activity. The directionality and processivity of the endonuclease were also determined by examination of cleavage products from challenged reactions. Although the lengths of 5'-derived products markedly decreased with time, no change in the size distribution of 3'-derived products was observed, indicating that cleavage proceeded processively in the 3' to 5' direction. The 5'-derived products were shortened more in reactions performed under conditions allowing multiple versus single enzyme-binding events, suggesting that the endonuclease action of a single enzyme is not processive enough to generate the maximum possible amount of cleavage on each substrate. Therefore, HIV-RT displays a partially processive 3' to 5' endonuclease activity. PMID- 1722203 TI - Calcium homeostasis in Trypanosoma brucei. Identification of a pH-sensitive non mitochondrial calcium pool. AB - The objective of this study was to characterize mechanisms which maintain intracellular calcium homeostasis in bloodstream forms of Trypanosoma brucei. The identification of homeostatic pathways is required to understand signal transduction in these organisms. The fluorescent probes Fura-2, 2',7' bis(carboxyethyl)-5(6)-carboxyfluorescein, and bisoxonal were used to measure intracellular calcium ([Ca2+]i), intracellular pH (pHi), and membrane potential, respectively. Homeostatic pathways maintained [Ca2+]i at 98 +/- 12 nM in the presence of 1.8 mM extracellular calcium despite a steady leak of calcium into the cell. The addition of 2.7 microM nigericin acidified the cytosol, depolarized the plasma membrane, and induced an approximate 3-fold increase in [Ca2+]i. The rise in [Ca2+]i could not be induced with valinomycin or gramicidin D under conditions where membrane depolarization occurred. By contrast, the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, elevated [Ca2+]i in a manner that was not additive with nigericin. Changes in pHi appeared to regulate [Ca2+]i since: 1) stepwise addition of K+ to nigericin-treated cells generated and incremental increase in pHi and concomitant decrease in [Ca]i; 2) addition of serum to nigericin-treated cells allowed simultaneous recovery of pHi and [Ca2+]i; and 3) addition of NH4Cl to untreated cells resulted in a biphasic change in pHi with corresponding biphasic change in [Ca2+]i. The rise in [Ca2+]i was derived from an intracellular pool which was not dependent on functional cytochrome oxidase, mitochondrial alternative oxidase, or the F0F1-ATPase. These data demonstrate that large quantities of calcium are reversibly stored in a non mitochondrial pH-sensitive intracellular pool in T. brucei. We conclude that changes in pHi can serve to trigger calcium signals in these organisms. PMID- 1722204 TI - Plasticity of Escherichia coli porin channels. Dependence of their conductance on strain and lipid environment. AB - The conductance properties of three members of the porin family which form channels across the outer membrane of Gram-negative bacteria were compared. With their endogenous lipopolysaccharide (LPS) bound, the closely related porins F and C from Escherichia coli reveal significantly different conductance steps and closing potentials, with values of 0.82 nS (nanosiemens) and 89 mV for F-type channels, and 0.49 nS and 158 mV for C-type pores (1 M NaCl), respectively. On the basis of their closing potentials, the two channel types can be distinguished unequivocally. If reconstituted in asolectin and extraneous LPS, porin C forms F type in addition to C-type channels. Substitution of asolectin by mitochondrial lipids yields the native C-type pores only. Both channel types can be induced to assume the mutually other channel configuration by variation of ionic strength. A multiplicity of channel subtypes is observed by variation of the pH of the medium. The three channels within a trimer are, however, consistently of the same type. Since structural studies have revealed a single channel per monomer, the several conductance steps observed are likely to reflect distinct configurations of the same channel. Best channel recoveries were observed if endogenous LPS remained associated to porin during purification. Significant yields could nevertheless be obtained also if LPS was removed from porin and replaced with various precursors or chemically synthesized analogues. As function requires the presence of glycolipids, yet crystallization is perturbed by heterodisperse endogenous LPS, the smallest monodisperse analogues yielding good channel recovery were determined. The minimal synthetic moiety is a monoglucosaminetetraacyl compound. The characteristics of porin B from E. coli BE are shown to be indistinguishable from those of porin F. The conductance properties of this porin, refolded from random coil configuration, are indistinguishable from those exhibited by native protein. The formation of channels is thus encoded by the sequence of the mature polypeptide alone. PMID- 1722205 TI - Characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene. AB - To identify the transcription regulatory elements of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, DNA fragments located in the 5' upstream region were fused with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into various cell lines to test for promoter activity. The results of these studies suggested that there were at least two positive and one negative cisacting elements involved in CFTR transcription initiation. One of them was a proximal, positive element delimited by the 5' deletion constructs -226 base parts upstream of the transcription start site. This minimal promoter sequence (-226 to +98) alone seemed to be sufficient to direct cell-specific CAT expression. The sequences immediately upstream of -227, on the other hand, appeared to contain a negative regulatory element; inclusion of this sequence with the proximal element (e.g. a construct containing sequences -345 to +98) rendered the CFTR promoter inactive. This negative regulatory element could also suppress the activity of a heterologous promoter. In addition, the DNA transfection study suggested the existence of another positive regulatory element outside the CFTR promoter region examined, as the inability of this region (e.g. -658 to +98) to function in a CAT assay could be overcome by the presence of a viral enhancer element. PMID- 1722206 TI - An induced mRNA secondary structure enhances repZ translation in plasmid ColIb P9. AB - Translation of the repZ gene encoding a DNA replication initiation protein of plasmid ColIb-P9 depends on not only the translation of a transcribed leader sequence (repY) but also the specific intergenic base pairing within RepZ mRNA between two short complementary sequences located in the repY and inc gene regions. In addition, repZ translation can be negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. Here we present evidence indicating that a stable secondary structure of RepZ mRNA, designated as structure III, sequesters one of the complementary sequences and the ribosome binding site, thereby preventing repZ translation. When site-directed mutagenesis was used to destabilize structure III without changing the ribosome-binding site, a significant level of repZ expression was observed even in the absence of repY translation. Under these conditions, however, repZ expression could be substantially reduced by additional mutations that directly diminished the intergenic base pairing at the mRNA level between the two complementary sequences. These results indicate that repY translation is essential for the disruption of structure III by inducing the formation of a new secondary structure through the intergenic base pairing, and more importantly, that this new structure enhances repZ translation. We also found that the site of repY translation termination played a critical role in the intramolecular conformational alteration of structure III. PMID- 1722207 TI - A single gene encodes the catalytic "A" subunit of the bovine vacuolar H(+) ATPase. AB - We have previously demonstrated that the 73-kDa (A) subunit of the bovine coated vesicle (H+)-ATPase possesses a nucleotide binding site required for catalytic activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638). Here we report the cDNA sequence of the coding region of the bovine brain A subunit. Comparison of the deduced amino acid sequence with those previously reported for the A subunits of vacuolar ATPases from lower eukaryotes, plants, and archaebacteria reveals significant homology, especially in sequences implicated in nucleotide binding. The message encoding the bovine brain A subunit is relatively large, approximately 4.6 kilobases; Northern blotting of RNA isolated from rat brain and human brain tumor cells reveals a message of similar size. Northern analysis of several bovine tissues indicates that only one message for this subunit is expressed. Southern blot analysis of bovine genomic DNA indicates that the bovine A subunit is encoded by a single gene. PMID- 1722208 TI - Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. AB - The intergenic region between the mouse alpha-myosin heavy chain (MHC) and beta MHC genes was analyzed in terms of its ability to drive gene expression in transgenic mice. Earlier, we reported that the entire intergenic region was sufficient to direct expression of the bacterial chloramphenicol acetyl transferase reporter gene in a tissue-specific and developmental stage-specific manner. Additional transgenic lines have been generated which include two deletions. The first deletion, alpha-3, which lacks the distal 2.5 kilobase pairs of the upstream region, is competent to direct tissue- and developmental-specific expression of the transgene. A larger deletion, in which only 138 base pairs upstream of the transcriptional start site remain, shows no chloramphenicol acetyltransferase activity in either muscle or non-muscle tissue. Tissue surveys of transgene expression indicated low levels of activity in the lung, and analyses via the polymerase chain reaction confirmed the presence of the endogenous alpha-MHC gene transcripts in this tissue. Subsequently, an alpha-MHC gene-specific riboprobe was used to detect the cognate transcripts in lung sections by in situ hybridization. The data show that, in the lung, the transcripts are localized to the thick intimal wall of the veins and venules. PMID- 1722209 TI - Purification of p100, a protein antigenically related to the signal transducing G proteins Gt and Gi. Evidence for an adaptin-like protein. AB - A 100-kDa protein, termed p100, cross-reacts with antisera raised against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha subunit of the retinal G protein Gt. p100 is abundantly expressed in liver and, on subcellular fractionation of rat liver homogenates, is distributed between the cytosolic and microsome fractions (Traub, L. M., Evans, W. H., and Sagi Eisenberg, R. (1990) Biochem. J. 272, 453-458; Udrisar, D., and Rodbell, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6321-6325). We have now purified p100 to near-homogeneity from rat liver microsomes. The protein was purified approximately 500-fold by ATP extraction followed by a series of four chromatographic steps. Similar to partially purified p100, on two-dimensional electrophoresis, the final preparation contained a major series of five immunoreactive 100-kDa charge isoforms. Partial amino terminus amino acid sequencing of the purified protein revealed that p100 is a previously unidentified protein. Further analysis of the soluble form of p100 showed the protein migrated with an apparent molecular weight of approximately 110,000 on gel filtration, indicating that the soluble protein occurs as a monomeric polypeptide. The soluble form of p100 was also partially purified from rat liver cytosol and amino acid sequencing yielded the same amino-terminal sequence as obtained from the microsome-associated form. The amino-terminal sequence of p100 exhibits significant similarity to the deduced amino-terminal amino acid sequences of both alpha- and gamma-adaptins. Using the amino-terminal sequence from p100, we have raised antipeptide polyclonal antisera. The antisera reacted specifically with the purified 100-kDa protein on immunoblots. With the purified protein and specific antisera now available, it will be possible to explore the physiological role of p100. PMID- 1722210 TI - Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain. AB - A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed. PMID- 1722211 TI - Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis. AB - Rat pancreatitis-associated protein (PAP) is an additional protein appearing in pancreatic juice after induction of prancreatic inflammation. Its messenger RNA was cloned and sequenced from pancreas. The deduced amino acid sequence revealed that PAP was synthetized as a preprotein with, in its mature form, a predicted molecular weight of 16,630. A search in protein data bases revealed a marked homology with the carbohydrate binding region of animal lectins; no hemagglutination activity could be shown for PAP, but the protein induced extensive bacterial aggregation. In healthy rats, the very low level of PAP expression in pancreas could be increased up to 4-fold by physiological stimuli such as chronic hormonal or cholinergic stimulation of pancreatic secretion and adaptation of rats to a carbohydrate-rich diet. By contrast, induction of acute experimental pancreatitis by retrograde injection of sodium taurocholate resulted in dramatic overexpression. Pancreatic concentration of PAP mRNA increased more than 300 x within 12 h whereas concentrations of mRNAs encoding major secretory proteins such as amylase decreased. PAP overexpression persisted during the 2 days of the acute phase and then returned to the control level during pancreatic recovery. PAP mRNA could not be evidenced in liver, stomach, salivary glands, brain, kidney, or testis. Its pattern of expression during severe pancreatic aggression suggests that it might be a stress protein involved in the control of bacterial proliferation. PMID- 1722212 TI - Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone. AB - Tartrate-resistant acid phosphatase (TRAP) is a characteristic constituent of osteoclasts and some mononuclear preosteoclasts and, therefore, used as a histochemical and biochemical marker for osteoclasts and bone resorption. We now report the isolation of a 1397-base pair (bp) full-length TRAP/tartrate-resistant acid ATPase (TrATPase) cDNA clone from a neonatal rat calvaria lambda gt11 cDNA library. The cDNA clone consists of a 92-bp untranslated 5'-flank, an open reading frame of 981 bp and a 324-bp untranslated 3'-poly(A)-containing region. The deduced protein sequence of 327 amino acids contains a putative cleavable signal sequence of 21 amino acids. The mature polypeptide of 306 amino acids has a calculated Mr of 34,350 Da and a pI of 9.18, and it contains two potential N glycosylation sites and the lysosomal targeting sequence DKRFQ. At the protein level, the sequence displays 89-94% homology to TRAP enzymes from human placenta, beef spleen, and uteroferrin and identity to the N terminus of purified rat bone TRAP/TrATPase. An N-terminal amino acid segment is strikingly homologous to the corresponding region in lysosomal and prostatic acid phosphatases. The cDNA recognized a 1.5-kilobase mRNA in long bones and calvaria, and in vitro translation using, as template, mRNA transcribed from the full-length insert yielded an immunoprecipitated product of 34 kDa. In neonatal rats, TRAP/TrATPase mRNA was highly expressed in skeletal tissues, with much lower (less than 10%) levels detected in spleen, thymus, liver, skin, brain, kidney, brain, lung, and heart. In situ hybridization demonstrated specific labeling of osteoclasts at endostal surfaces and bone trabeculae of long bones. Thus, despite the apparent similarity of this osteoclastic TRAP/TrATPase with type 5, tartrate-resistant and purple, acid phosphatases expressed in other mammalian tissues, this gene appears to be preferentially expressed at skeletal sites. PMID- 1722214 TI - Identification of protein-binding sequences mediating constitutive and 12-O tetradecanoylphorbol-13-acetate-induced VL30 transcription in cultured mouse and human keratinocytes. AB - A retrovirus-like 30S (VL30) gene induced in mouse epidermis after a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) was used as a model gene to define mechanisms of transcriptional regulation in keratinocytes. Sequences important for TPA and epidermal growth factor-induced transcription were found to be separated from eacho other within the long terminal repeat. Deletion mapping of the long terminal repeat region and linking short sequences to a heterologous promoter made it possible to identify a 28-base pair VL30 TPA responsive element. VL30 TPA-responsive element mediated both basal and TPA induced transcription in the mouse keratinocyte cell line Balb/MK and in normal human keratinocytes. Gel-retardation and transient transfection experiments indicated that two nuclear factors (VLX and VLY) bind independently to the VL30 TPA-responsive element in juxtaposed positions and that the binding sites collaborate functionally in constitutive and TPA-induced transcription. The sequence involved in VLY binding shows no homology to previously identified binding motifs. Two different sequences involved in mediating TPA-induced transcription of the urokinase plasminogen activator and of the c-jun gene, respectively, competed for proteins with affinity toward the VLX binding site. No competition was found with sequences containing the consensus AP-1 binding site. PMID- 1722213 TI - The pro-alpha 1(V) collagen chain. Complete primary structure, distribution of expression, and comparison with the pro-alpha 1(XI) collagen chain. AB - We have isolated overlapping cDNA clones from human and hamster libraries which comprise the entire coding sequences for the prepro-alpha 1(V) collagen chains of both species. The translated polypeptide has a signal peptide of 36 amino acids, a central triple helical domain of 338 uninterrupted Gly-X-Y triplets, and 266 amino acids which comprise the C-telopeptide and propeptide. The N-propeptide and telopeptide are comprised of 522 residues in humans and 524 residues in hamsters. The cDNA-derived pro-alpha 1(V) amino acid sequences exhibit a variety of structural features characteristic of fibrillar collagens. Pro-alpha 1(V) is found to be unique among fibrillar collagen chains, however, in lacking potential cross-linking lysyl residues in either telopeptide, and in possessing potential N asparaginyl-linked carbohydrate attachment sites in its N-propeptide. Of particular interest is the strong homology found between the pro-alpha 1(V) and pro-alpha 1(XI) collagen chains in most domains, with the notable exception of a subdomain in the globular region of the N-propeptide. RNase protection analysis of RNA with a variety of pro-alpha 1(V) cDNA-derived riboprobes indicates a broad distribution of expression of the pro-alpha 1(V) chain in tissues and suggests that transcripts encoding the pro-alpha 1(V) chain and the putative pro-alpha 1'(V) chain are not products of the same gene. PMID- 1722215 TI - Responses of porcine gastric and duodenal smooth muscle to VIP. AB - 1. Mechanical activity was recorded in isolated muscle preparations from circular and longitudinal layers of gastric fundus, corpus and antrum and from the duodenum of pigs, using conventional organ bath technique. Rectangular current pulses were applied to the muscle strips for electrical field stimulation (EFS). 2. Fundic and circular corpus preparations developed a marked spontaneous tonic activity. Vasoactive intestinal polypeptide (VIP, 10(-9)-10(-7) mol l-1) inhibited this spontaneous activity. This inhibitory effect was not affected by application of tetrodotoxin (TTX) showing its myogenic nature. 3. Pretreatment of fundic and circular corpus preparations with VIP reduced the excitatory responses to substance P, bombesin, serotonin and histamine, but it had no effect on the acetylcholine (ACh)-induced tonic and phasic activity. 4. Longitudinal duodenal preparations showed purely phasic activity which was almost insensitive to VIP. In circular duodenal preparations particularly strong spontaneous tonic contractions were observed which could be inhibited by VIP. 5. Circular duodenal preparations excised 3-5 cm postpyloric had a spontaneous tone which could reach up to 80% of the maximum contractions induced by 10(-4) mol l-1 ACh. These preparations were chosen for further pharmacological studies and for experiments with EFS. VIP was the most powerful substance for the inhibition of spontaneous tone, followed by serotonin, PGE2 and bradykinin. This type of preparation exhibited particularly strong inhibitory effects to EFS; even single stimuli could induce near maximum relaxation. The inhibition induced by EFS was unaffected by treatment with ATP, guanethidine, atropine, methysergide and apamin. TTX completely abolished the EFS-induced relaxation, showing its neurogenic nature. 6. Porcine circular duodenum is a good model for studying the transmitter system of the non-adrenergic, non-cholinergic (NANC) innervation. The results are consistent with the assumption that VIP is the transmitter in this system, although the very slow time-course of the VIP-induced inhibition in comparison with the EFS-induced inhibition is not consistent with this notion. PMID- 1722217 TI - A common autoepitope near the carboxyl terminus of the 60-kD Ro ribonucleoprotein: sequence similarity with a viral protein. AB - The Ro ribonucleoprotein is composed of hY RNA and a 60.7-kD peptide that is antigenic for autoantibodies produced by many patients with systemic lupus erythematosus or Sjogren's syndrome and mothers of newborns with complete congenital heart block. A major immunoreactive fragment (13 kD) of the 60-kD Ro is bound by 28 of 45 (62%) of the anti-Ro sera tested. Amino acid sequence analysis localizes this fragment to the carboxyl end of the 60-kD Ro peptide. All possible overlapping octapeptides of this 13-kD peptide of 60-kD Ro have been assessed for antigenicity. Sera that bind the 13-kD peptide fragment in immunoblot generally also bind the octapeptides of Ro spanning the sequence AIALREYRKKMDIPA (P less than 0.01). Inhibition studies with synthetic peptides and purified Ro have established specificity for reference serum antibody binding to an antigenic octapeptide, EYRKKMDI, from this region. The closely related sequence EYRKKLMD is found in the nucleocapsid protein of vesicular stomatitis virus and may portend an immunologic link to this or a related viral antigen. These results also demonstrate that despite fine specificity variation between human sera, there are recurring patterns of anti-Ro binding shared by some patients who have precipitating anti-Ro autoantibodies. PMID- 1722218 TI - A review of 254 ectopic pregnancies in a teaching hospital in the Trent Region, 1977-1990. AB - A total of 254 cases of ectopic pregnancy were reviewed in a teaching hospital in Sheffield, in three defined periods: I, 1977-9; II, 1985-7 and III, 1988-90. A previous history of infertility was noted in 37% of cases. Overall, the presenting symptoms, clinical, laboratory, operative as well as histological findings, are in broad agreement with other series. The incidence increased steadily from 8.6 per 1000 total births in period I to 16.5 per 1000 total births in period III. A number of changes noted in recent years include: (1) the diagnosis of ectopic pregnancy was made significantly (P less than 0.05) earlier; (2) a significantly (P less than 0.05) greater proportion of ectopic pregnancies had an association with the following factors: previous tubal surgery, the diagnosis established with ultrasonography, laparotomy preceded by laparoscopy and treatment by conservative surgery; and (3) a significantly (P less than 0.05) smaller proportion of ectopic pregnancies had the diagnosis based on pelvic tenderness or pelvic mass. During the period 1988-90 a total of 126 laparoscopies were performed for suspected ectopic pregnancy, of which 82 (65%) were confirmed to have ectopic pregnancy and 44 (35%) were thought to have no evidence of ectopic pregnancy on laparoscopy. However, two of the latter cases were subsequently found to have an ectopic pregnancy within 2 weeks. The clinical implications of these findings are discussed. PMID- 1722216 TI - Systemic lupus erythematosus: RNA-protein autoantigens, models of disease heterogeneity, and theories of etiology. PMID- 1722219 TI - Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti E7 prevalence in cervical cancer patients. AB - Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18 positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Muller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa. PMID- 1722220 TI - 43-kilodalton glycoprotein from Paracoccidioides brasiliensis: immunochemical reactions with sera from patients with paracoccidioidomycosis, histoplasmosis, or Jorge Lobo's disease. AB - Sera from patients with paracoccidioidomycosis (PCM), histoplasmosis (HP), or Jorge Lobo's disease (JL) were titrated against purified gp43 from Paracoccidioides brasiliensis by using both enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IPP) reactions with 125I-labeled antigens. In IPP, PCM sera and other sera could be distinguished on the basis of serum titers, whereas in ELISA, 53% of the HP sera and 29% of the JL sera reacted similarly to the PCM sera. To investigate the possible role of the carbohydrate epitopes in these reactions, we compared the reactivities of sera from several patients with native and deglycosylated gp43. Competition experiments were carried out with monosaccharides as inhibitors. The results suggest that greater than 85% of the reactions of the PCM sera with gp43 involved peptide epitopes. Cross-reactions with HP and JL sera in ELISA were predominantly attributed to periodate-sensitive carbohydrate epitopes containing galactosyl residues. HP and JL sera which reacted strongly with gp43 in ELISA were only weakly reactive or did not react in IPP with labeled antigens in solution. Moreover, ELISA reactions could be significantly inhibited either by monosaccharides or by periodate treatment. Apparently, carbohydrate epitopes in gp43 are more accessible to the antibodies when the molecule is bound to a plastic substrate than when it is in solution. Structural changes in the gp43 antigen arising by N deglycosylation abolish reactivity with PCM sera and support the existence of conformational peptide epitopes. PMID- 1722221 TI - Reproducibility of a scoring system for gram stain diagnosis of bacterial vaginosis. AB - A total of 225 pairs of duplicate Gram-stained slides from three hospitals in Jakarta were evaluated independently by a local (University of Indonesia, Jakarta) and a referral (University of Washington, Seattle) laboratory by the new scoring criteria proposed by Nugent et al. The correlation coefficients of the duplicate Gram stain scores ranged from 0.65 to 0.83. The kappa statistics for the bacterial vaginosis category (no, score of 0 to 6; yes, score of 7 to 10) ranged from 0.62 to 0.77. These findings confirm the good to excellent interobserver reliability of the new scoring system and the importance of slide preparation. PMID- 1722222 TI - Distribution of acetylcholinesterase in the hippocampal region of the mouse: II. Subiculum and hippocampus. AB - The distribution of acetylcholinesterase (AChE) was examined in the subiculum and hippocampus of the adult mouse (Mus musculus domesticus). A distinctly stratified AChE pattern was observed in both areas and was compared in detail with cytoarchitectural fields and layers. In the subiculum, the lateral plexiform layer was lightly stained superficially and moderately stained at depth, where it abutted the lateral, moderately stained cell layer. Medially, a moderately stained deep plexiform layer separated the darkly stained superficial plexiform layer from the equally AChE-intense cell layer. At depth, the subicular cell layer was delimited by a band of very high AChE activity. In regio superior of the hippocampus, AChE-intense bands delimited the moderately stained strata moleculare, radiatum, and oriens toward the subjacent layers. In the stratum pyramidale, precipitate insinuated between the cell bodies gave a dark appearance to the deep part of the layer. The homologous strata of regio inferior appeared darker, but the relative staining intensities corresponded largely to those in regio superior. AChE activity in the layer of mossy fibers was almost absent septally but increased gradually to very high levels temporally. The AChE staining pattern, in conjunction with cytochemical and morphological evidence, strongly suggests a division of the pyramidal cell layer of the mouse and rat into superficial and deep substrata and discourages the definition of a prosubiculum in rodents. A comparative analysis of the AChE pattern reveals that: 1) in the subiculum, differences between species are observed within a generalized pattern of medial darkly staining and lateral lightly staining portions; 2) in the hippocampus, a conservation of the AChE pattern is seen in strata associated with intrinsic hippocampal connection; while 3) numerous interspecific differences are found in the stratum moleculare. PMID- 1722223 TI - Lysosomal activity in developing cat alpha-motor axons under normal conditions and during retrograde axonal transport of horseradish peroxidase. AB - The occurrence of acid phosphatase (AcPase)-positive bodies, i.e., lysosomes, in lumbosacral alpha-motor axons of kittens, 0-16 weeks of age, was analyzed by light and electron cytochemical methods under normal conditions and after intramuscular injection of horseradish peroxidase (HRP). Axonal lysosomes were rare early postnatally. In 3-week-old animals, a few AcPase-positive bodies appeared in the axoplasm at some nodes of Ranvier in the peripheral nervous system (PNS) and internodally in the intrafunicular motor axon parts within the central nervous system (CNS). From 6 weeks postnatally, a nodal concentration of AcPase-positive bodies was also noted in the CNS. The number of AcPase-positive bodies continued to increase gradually in the course of neuronal maturation. In 16-week-old animals, axonal AcPase activity was still at considerably lower levels than at adult stages. At all ages, acid hydrolase-containing organelles were most commonly found at ventral root nodes. After injection of HRP in the medial gastrocnemius muscle, accumulations of AcPase-positive bodies were seen in the axoplasm at some PNS nodes of the HRP-injected sides of kittens aged 8, 12, and 16 weeks. Incubation for demonstration of both HRP and AcPase activity showed that some organelles at HRP-transporting nodes contained both types of reaction product. The nodal AcPase activity in the intrafunicular, CNS parts of alpha motor axons of the HRP-exposed sides did not differ from that of the contralateral, uninjected sides. In view of our previous observations in alpha motor neurons of adult cats in which a lysosome-mediated degradation of axonally transported materials may take place at PNS nodes of Ranvier, the present study illuminates possible differences in the ability to interfere with axonal transport between developing and mature neurons. The infrequent presence of lysosomes in developing alpha-motor axons and the implied disability of their nodal regions to interfere with axonally transported constituents in a way similar to that seen in adult animals may be of significance in that trophic and chemical signals can pass unhindered between the periphery and perikaryon. However, this could also have negative consequences for the vulnerable immature neuron in that various materials retrieved at the axon terminals outside the CNS are permitted a more-or-less free access to the perikaryon. PMID- 1722224 TI - Distribution and morphology of human cone photoreceptors stained with anti-blue opsin. AB - Primate cones maximally sensitive to short wavelength light (blue cones) have been previously identified by using indirect methods. We stained 7 wholemounted human retinas obtained from 6 female donors, using an affinity purified antibody to a 19 amino acid peptide sequence at the N-terminus of blue opsin (Lerea et al., '89: Neuron 3:367-376), standard PAP immunocytochemistry, and controls. Cones were counted where all outer segments could be traced to inner segments and were measured where cells were well aligned vertically. We find that: (1) 7% of cones within 4 mm of the foveal center are labeled by antiblue opsin; (2) compared to neighboring red/green cones, blue cone inner segments are 10% taller, have a larger cross-sectional diameter near the junction with the outer segment, and a smaller diameter near the external limiting membrane, resulting in a more cylindrical shape, (3) foveal blue cones are sparse, irregularly spaced, and missing in a zone about 100 microns (0.35 degrees) in diameter near the site of peak cone density, (4) the highest densities of blue cones (greater than 2,000 cells/mm2) are found in a ring at 0.1-0.3 mm eccentricity, and (5) the shortest distances between neighboring cones are between blue and red/green cones, and the blue and red/green mosaics are statistically independent. These findings are consistent with psychophysical reports of foveal tritanopia and maximum sensitivity to blue light at 1 degree eccentricity. Blue cone spacing may limit resolution of the blue channel out to 20-30 degrees eccentricity. The blue and red/green mosaics appear to be formed by separate processes. PMID- 1722225 TI - Olivary morphology and olivocerebellar topography in adult lurcher mutant mice. AB - In adult lurcher mice, in which virtually all cerebellar Purkinje cells have degenerated as a direct consequence of mutant gene action, the inferior olivary complex suffers a severe retrograde transneuronal atrophy. Our analysis indicates a 63% cell loss in the lurcher inferior olive, homogeneously distributed between the medial and dorsal accessory, and principal olivary subdivisions. Olivary neurons are reduced in cross-sectional area by 30% in lurcher mice, compared to normal controls. All olivary subdivisions morphologically identifiable in normal mice are also found in the lurcher inferior olive. Analysis of olivocerebellar topography by retrograde transport of lectin-conjugated horseradish peroxidase and fluorogold, in both single and double labeling paradigms, reveals no abnormalities in the general organization of this highly ordered projection. This stability may be based on the initial establishment of the topographic pattern in late embryogenesis or early postnatal periods, prior to the onset of lurcher Purkinje cell degeneration, or, alternatively, the lurcher gene may not alter critical afferent and target characteristics at stages when the topographic relationship is being established. Once established, the olivocerebellar projection is apparently not dependent on the Purkinje cell for long-term maintenance of its general topographic organization. PMID- 1722226 TI - Pathology of infection caused by Dermatophilus-like organisms in porcine tonsils. AB - The authors investigated the occurrence of Dermatophilus-like organisms in sulphur granules of porcine tonsils. Light and electron microscopic studies, together with histochemical examination, were carried out to elucidate the mode of growth of the organism in the tonsils, the interaction between the organisms and host cells, and the nature of the radiating clubs around the organisms. Sulphur granules were found in about 15 and 70 per cent of market pigs and breeding pigs, respectively. Of the pigs having tonsillar granules, Dermatophilus like organisms were observed in about 70 per cent of market pigs, and in nearly all breeding pigs. The organisms invaded tonsillar crypts to produce lesions resembling actinomycotic abscesses up to 5 mm in diameter. Dermatophilus-like organisms were demonstrated in various morphological forms ranging from filamentous to tuber-shaped or coccoid bodies. In the lesion, the bacterial cells adjacent to the host cell reaction showed distinct degenerative changes forming thick amorphous masses on the surface of the bacterial cells. The amorphous masses seemed to be derived from the bacterial cells but showed histochemical components different from those of the bacterial cells. These masses had numerous protrusions forming clubs. Phagocytic neutrophils close to the amorphous masses were presumed to play a role in deposition of the club material. Macrophages also appeared to participate in the inflammation leading to a granulomatous lesion. These findings suggested that the clubs might be formed by an interaction between the organisms and host cell reaction. PMID- 1722227 TI - Crusted scabies in a patient with chronic graft-versus-host disease. AB - We describe a 20-year-old man with chronic graft-versus-host disease and progressive cutaneous changes. His skin became more lichenified despite therapy with azathioprine, prednisone, and cyclosporine. Although it was initially thought that lichenoid graft-versus-host disease had developed, it was subsequently discovered that the patient had crusted (Norwegian) scabies. PMID- 1722228 TI - Reactivity of monoclonal antibody OKM5 with sebaceous carcinoma. AB - Two cases of sebaceous carcinoma (SC), as well as epithelial tumours, melanoma, and lymphoma, were examined using immunoperoxidase and a panel of monoclonal antibodies on cryostat sections. The results showed that, whereas all SC cells in both cases reacted strongly with monoclonal antibody OKM5, other tumour cells (except juvenile xanthogranuloma cells) did not. The pagetoid cells within the epidermis of SC also reacted with OKM5 antibody. Although the nature of the phenomenon merits further study, this reactivity, or cross-reactivity, might possibly aid diagnosis of SC. PMID- 1722229 TI - A pedunculated follicular hamartoma: a case showing a central trichofolliculoma like tumor with multiple trichogenic tumors. AB - We report a rare case with a pedunculated nodule on the nasal septum, which seems a kind of follicular hamartoma with a pedunculated appearance. Our case was a 77 year-old Japanese male with an 11 x 11 x 10 mm sized, skin-to-pink colored, pedunculated nodule on the center of his nasal septum. Histopathological study revealed the nodule to be a well-formed dilated hair follicle at the central pore of the tumor, where keratin debris and vellus hairs were present. In addition, epithelial cells forming long thin strands or immature follicle-like structures had proliferated multifocally from the epidermis of the wall of the central follicle to the peripheral epidermis. One distinctive feature was the formation of a primary germinal bud with thick concentric collagen fibers and sparse elastic fibers. Although a combined tumor of trichofolliculoma with highly-graded desmoplasia, multiple trichogenic trichoblastomas, and perifollicular fibromas was suggested, calling it a pedunculated follicular hamartoma seemed more useful because the pathological features were basically similar to reported follicular hamartomas and the clinical feature was so distinctive. PMID- 1722231 TI - A high frequency of detection of Helicobacter pylori in whitish exudate of gastric ulcer. AB - Helicobacter pylori has been implicated in the pathogenesis of chronic gastritis and peptic ulcer. However, no report to date has been made on the presence of H. pylori in the whitish exudate of peptic ulcers. We examined the biopsy specimens of 52 patients (42 men and 10 women, aged 29-78, not taking steroids or nonsteroid antiinflammatory drugs), and evaluated the frequency of detecting the organism in the whitish exudate of gastric ulcers in comparison with that in the ulcer border, gastric fundic gland, and pyloric gland using microaerobic culture and acridine-orange stain. H. pylori was detected in high rates: 38 (73%) of 52 cases by culture, and 50 cases (96%) by staining of the whitish exudate, 42 (81%) of 52 cases by culture, and 46 cases (88%) by staining of the ulcer border. The frequency for the other regions were 81% by culture and 90% by staining of the fundic gland, and 81 and 90% of the pyloric gland, respectively. Of all sites studied by the staining method, H. pylori was detected at the highest frequency in the whitish exudate. The fact that many H. pylori live in the whitish exudate of gastric ulcer, suggests a causal relationship between H. pylori and gastric ulcer. PMID- 1722230 TI - Galanin. PMID- 1722232 TI - Pancreatic enlargement in alcoholic pancreatitis: prevalence and natural history. AB - We determined the prevalence and natural history of pancreatic enlargement by abdominal ultrasonography or computed tomography in 72 patients with alcoholic pancreatitis. Pancreatic enlargement was observed in 54 patients (75%); it was diffuse in 28 (52%) and focal in 26 (48%). The focal enlargement was frequently cystic (50%), while the diffuse enlargement was only occasionally cystic (7%). Sequential imaging of the pancreas in 29 patients demonstrated partial to total resolution of pancreatic enlargement in greater than 50% during 6 months of follow-up. Determination of serum amylase and p-isoamylase activity was neither sensitive nor specific for pancreatic enlargement in alcoholic pancreatitis. PMID- 1722233 TI - [Study on fluctuation of several parameters in varicella]. AB - Specific immunological responses (varicella-specific IgG and IgM) and various parameters (interferon, C3, C4, platelet count, erythrocyte count, erythrocyte CR1) in varicella infection were determined over the course of the disease (days) and their relationships to severity and immunological significance evaluated. Defining the day varicella appeared as disease day 0, IgG appeared on the disease day 4 and IgG on day 5. The preceding disease days were dominated by non-specific immunological mechanisms. Interferon appearance preceded that of these antibodies but did not correlate with severity. In serious cases, C3 and C4 increased in the acute stage while platelet count declined. Erythrocyte count decreased in severe cases after disease day 5. Three patients with lower erythrocyte CR1 activity values remained critical. These findings suggested that determining platelet count is expedient in determining severity in the early stage and that the complement pathway is a major component of early immunological response. PMID- 1722234 TI - [Study of the increasing effect of therapeutic efficacy of amikacin in combination with human granulocyte-colony stimulating factor (G-CSF) against experimental urinary tract infection in diabetic mice]. AB - Clinically, prophylactic effects of Granulocyte-colony stimulating factor (G-CSF) on granulocytopenia originating from the use of anticancer agents are thought possible, and clinical studies are being performed. Recently the application of G CSF against infectious diseases has been considered, and we reported the prophylactic effect of G-CSF against experimental urinary tract infection induced by Pseudomonas aeruginosa in diabetic mice treated Streptozotocin, a compromised host model. In this report, we investigated the therapeutic effect of G-CSF alone and in combination with Amikacin (20 mg/kg) against experimental urinary tract infection induced by Pseudomonas aeruginosa in diabetic mice. In diabetic mice, the therapeutic administration of G-CSF alone and Amikacin alone did not produce an increase of 7 day survival rate and decrease of incidence of infection. But, combinational administration of these increased the 7 day survival rate and yielded a lower incidence of infection. Thus, the therapeutic efficacy of Amikacin was improved by a combinational use of G-CSF. This result suggests that the synergy of bactericidal effect of neutrophils accelerated by G-CSF with Amikacin, and suggests the meaning and the efficacy of G-SCF as an additional therapy with antibiotics for infectious diseases. PMID- 1722235 TI - [Pathophysiology and therapy of chronic hepatitis]. PMID- 1722236 TI - [Diagnosis and management of infections complicated with hematological diseases]. PMID- 1722237 TI - Calcifying and keratinizing ameloblastoma of the maxilla. AB - A case is described of ameloblastoma of maxilla presenting with numerous calcified keratin pearls. The significance of cellular variation in relation to the behavioural potential of the ameloblastoma in general is briefly discussed. PMID- 1722238 TI - Net charge transport during sodium-dependent calcium extrusion in isolated salamander rod outer segments. AB - The light-sensitive current and the current associated with the extrusion of internal Ca2+ in exchange for external Na+ have been recorded from detached rod outer segments from the salamander retina by the use of the whole-cell voltage clamp technique. No significant current-carrying mechanisms are present in the outer segment membrane apart from the light-sensitive conductance and the Na:Ca,K exchange, and exchange currents can therefore be recorded directly without the use of subtraction procedures or pharmacological blockers. The charge moved by the exchange was studied by loading outer segments with a known amount of calcium and then recording the exchange current on return to a Na(+)-containing solution. Calcium is not sequestered to any significant extent in a slowly exchanging internal store, as the charge recovered is unaffected if admission of the Na(+) containing solution is delayed for 40 s. The number of charges flowing into the cell in exchange for each Ca2+ ion extruded was found not to deviate significantly from one over a wide range of ionic conditions and membrane potentials. These results show that the stoichiometry of the exchange is fixed over a wide range of conditions, and that the size of the inward exchange current is therefore directly proportional to the rate of Ca2+ efflux through the carrier. PMID- 1722239 TI - Optical measurements of Na-Ca-K exchange currents in intact outer segments isolated from bovine retinal rods. AB - The properties of Na-Ca-K exchange current through the plasma membrane of intact rod outer segments (ROS) isolated from bovine retinas were studied with the optical probe neutral red. Small cellular organelles such as bovine ROS do not offer an adequate collecting area to measure Na-Ca-K exchange currents with electrophysiological techniques. This study demonstrates that Na-Ca-K exchange current in bovine ROS can be measured with the dye neutral red and dual wavelength spectrophotometry. The binding of neutral red is sensitive to transport of cations across the plasma membrane of ROS by the effect of the translocated cations on the surface potential of the intracellular disk membranes (1985. J. Membr. Biol. 88: 249-262). Electrogenic Na+ fluxes through the ROS plasma membrane were measured with a resolution of 10(5) Na+ ions/ROS per s, equivalent to a current of approximately 0.01 pA; maximal electrogenic Na-Ca-K exchange flux in bovine ROS was equivalent to a maximal exchange current of 1-2 pA. Electrogenic Na+ fluxes were identified as Na-Ca-K exchange current based on a comparison between electrogenic Na+ flux and Na(+)-stimulated Ca2+ release with respect to flux rate, Na+ dependence, and ion selectivity. Neutral red monitored the net entry of a single positive charge carried by Na+ for each Ca2+ ion released (i.e., monitored the Na-Ca-K exchange current). Na-Ca-K exchange in the plasma membrane of bovine ROS had the following properties: (a) Inward Na-Ca-K exchange current required internal Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 0.9 microM), whereas outward Na-Ca-K exchange current required both external Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 1.1 microM) and external K+. (b) Inward Na-Ca-K exchange current depended in a sigmoidal manner on the external Na+ concentration, identical to Na(+)-stimulated Ca2+ release measured with Ca(2+)-indicating dyes. (c) The neutral red method was modified to measure Ca(2+)-activated K+ fluxes (half-maximal stimulation at 2.7 microM free Ca2+) via the Na-Ca-K exchanger in support of the notion that the rod Na-Ca exchanger is in effect a Na-Ca-K exchanger. (d) Competitive interactions between Ca2+ and Na+ ions on the exchanger protein are described. PMID- 1722240 TI - Light and dark active phosphodiesterase regulation in salamander rods. AB - We studied the activation of 3',5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) by using a cell-permeant enzyme inhibitor. Rods of Ambystoma tigrinum held in a suction electrode were jumped into a stream of 3 isobutyl-1-methylxanthine (IBMX), 0.01-1 mM. Initial transient light-sensitive currents fit the notion that dark and light-activated forms of PDE contributed independently to metabolic activity and were equivalently inhibited by IBMX (apparent Ki 30 microns). Inhibition developed within 50 ms, producing a step decrease of enzyme velocity, which could be offset by activation with flashes or steps of light. The dark PDE activity was equivalent to light activation of enzyme by 1,000 isomerization rod-1s-1, sufficient to hydrolyze the free cGMP pool (1/e) in 0.6 s. Steady light activated PDE in linear proportion to isomerization rate, the range from darkness to current saturation amounting to a 10-fold increase. The conditions for simultaneous onset of inhibitor and illumination to produce no net change of membrane current defined the apparent lifetime of light-activated PDE, TPDE* = 0.9 s, which was independent of both background illumination and current over the range 0-3 x 10(5) isomerization s-1, from 50 to 0 pA. Adaptation was a function of current rather than isomerization: jumps with different proportions of IBMX concentration to steady light intensity produced equal currents, and followed the same course of adaptation in maintained light, despite a 10-fold difference of illumination. Judged from the delay between IBMX- and light-induced currents, the dominant feedback regulatory site comes after PDE on the signal path. The dark active PDE affects the hydrolytic flux and cytoplasmic diffusion of cGMP, as well as the proportional range of the cGMP activity signal in response to light. PMID- 1722241 TI - Axonal sprouting and changes in fibre types after running-induced muscle damage. AB - We have recently observed increase in Type I fibres in mouse soleus--but not extensor digitorum longus--muscles as a result of repeated muscle damage induced by voluntary wheel running. The most likely mechanism underlying the changes in fibre type composition is a redistribution of motor units with axonal sprouting and formation of new synapses. To test this hypothesis we exercised mice on a motor-driven treadmill once (3 x 3 h with 30 min rest periods in between, 14 m min-1, slope 6 degrees) or repeatedly (8-10 times at intervals of 3-5 days) and quantified axonal sprouting after staining with zinc iodide-osmium. In the contralateral solei, muscle damage and fibre type changes were evaluated with standard histochemical techniques. Significant numbers of damaged muscle fibers were found 0-15 days after a single exercise as compared to unexercised control animals (range 0.0-0.3% of the fibres in sedentary, n = 5, vs 2.1-14.8% in exercised muscles, n = 10) and repeated damage occurred in repeatedly exercised animals. In muscles of sedentary animals 3.8 +/- 1.4% SD of the examined endplates (n = 880, 5 muscles) had nodal or terminal sprouts. The incidence of sprouting was significantly elevated 3-21 days after a single exercise (7.5 +/- 1.8%, n = 2855, 12 muscles, P less than 0.01 signed-rank test), and more so after repeated running (12.0 +/- 2.5%, n = 1505, 6 muscles, P less than 0.01). Fibre type distributions were not different from controls 3 weeks after a single running episode, but after the 6-7 weeks of repeated running a significant increase in undifferentiated fibres at the cost of Type II fibres was found (9.7 +/- 3.4% versus 1.0 +/- 0.5% in sedentary controls, P less than 0.05, t-test); undifferentiated fibres express both Type I and Type II myofibrillar ATPase and are considered as fibres in the process of changing their types. These observations strongly support the assumption that sprouting and formation of new synapses--followed by motor unit enlargement and redistribution--occur as a result of muscle damage. PMID- 1722242 TI - Basket cells in the monkey fascia dentata: a Golgi/electron microscopic study. AB - This study describes non-granule cells in the fascia dentata of rhesus monkeys and baboons. Their cell bodies are located in the molecular layer and at the hilar border of the granular layer. They are called basket cells since their axons give rise to collaterals that branch in the close vicinity of the parent cell body and form symmetric synapses with dendrites and cell bodies of granule cells. These neurons are further classified with regard to the shape and location of their cell bodies and the orientation of their dendrites. Basket cells in the molecular layer are mainly bipolar with dendrites oriented perpendicular to the granular layer. These dendrites are densely innervated by presynaptic boutons forming asymmetric synapses. We have rarely observed molecular layer basket cells with dendrites traversing the granular layer and invading the hilus. We thus conclude that these cells are mainly activated by extrinsic afferents terminating in the molecular layer. Basket cells at the hilar border display pyramidal, fusiform or multipolar cell bodies that give rise to apical dendrites traversing the molecular layer and basal dendrites invading the hilar region. Large boutons establish asymmetric synapses with identified basal dendrites of these neurons. The dendrites of all types of basket cell are smooth, i.e. they had few or no spines. Many of them display varicosities. Cell counts in Cresyl Violet-stained sections revealed a ratio of basket cells to granule cells of 1:500. Essentially, the types of basket cell in the monkey fascia dentata are similar to those described previously for the rat. This contrasts sharply to our recent findings for pyramidal neurons and granule cells of the monkey hippocampus which showed an increased complexity and variability when compared with rodents. These data do not support the hypothesis that only local circuit neurons evolve in phylogeny. PMID- 1722243 TI - Desensitization and resensitization rates of glutamate-activated channels may regulate motoneuron excitability. AB - 1. Single-channel properties of desensitizing glutamate-activated channels were analyzed in outside-out patch-clamp recordings from a motoneuron-enriched cell fraction from embryonic chick. A piezo-driven device was used to achieve fast solution exchange at the electrode tip, resulting in maximum activation within 2 ms. 2. Quisqualate/AMPA receptors, with a 13-pS conductance, desensitized rapidly; the desensitization rate depended on agonist concentration but not on membrane potential. When quisqualate was applied slowly, the quisqualate activated channels desensitized without prior channel opening, indicating desensitization from the closed state. After a 10-ms refractory period, resensitization of all channels required up to 300 ms; resensitization rate did not depend on the duration of the preceding quisqualate application. 3. At agonist concentrations less than or equal to 1 mM, kainate receptors, with a 20 pS conductance, did not desensitize. At kainate concentrations greater than or equal to 1 mM, though, kainate receptors desensitized to a low steady-state conductance within approximately 200 ms. Resensitization of all channels required as long as 3 s, which could render kainate receptors inexcitable during high frequency activation. 4. Desensitization rates of whole-cell currents were similar to those observed in outside-out mode. Glutamate- and quisqualate activated responses were similar, suggesting that the rapidly desensitizing quisqualate-sensitive receptor type may dominate the kinetics of whole-cell excitatory postsynaptic currents (EPSCs) in this preparation. 5. It may be concluded that the efficacy of glutamate-mediated synaptic transmission is modulated by differences in the rates of desensitization and resensitization. PMID- 1722244 TI - Corticostriatal transformations in the primate somatosensory system. Projections from physiologically mapped body-part representations. AB - 1. The basal ganglia of primates receive somatosensory input carried largely by corticostriatal fibers. To determine whether map-transformations occur in this corticostriatal system, we investigated how electrophysiologically defined regions of the primary somatosensory cortex (SI) project to the striatum in the squirrel monkey (Saimiri sciureus). Receptive fields in the hand, mouth, and foot representations of cortical areas 3a, 3b, and 1 were mapped by multiunit recording; and small volumes of distinguishable anterograde tracers were injected into different body-part representations in single SI areas. 2. Analysis of labeled projections established that at least four types of systematic remapping occur in the primate corticostriatal system. 1) An area of cortex representing a single body part sends fibers that diverge to innervate multiple regions in the putamen, forming branching, patchy fields that are densest in the lateral putamen. The fields do not form elongated cylindrical forms; rather, they are nearly as extended mediolaterally as they are rostrocaudally. 2) Cortical regions representing hand, mouth, and foot send globally somatotopic, nonoverlapping projections to the putamen, but regions with closely related representations (such as those of the thumb and 5th finger in area 3b) send convergent, overlapping corticostriatal projections. The overlap is fairly precise in the caudal putamen, but in the rostral putamen the densest zones of the projections do not overlap. 3) Regions representing homologous body parts in different SI cortical areas send projections that converge in the putamen. This was true of paired projections from areas 3a and 3b, and from areas 3b and 1. Thus corticostriatal inputs representing distinct somatosensory submodalities can project to the same local regions within the striatum. Convergence is not always complete, however: in the rostral putamen of two cases comparing projections from areas 3a and 1, the densest zones of the projections did not overlap. 4) All projections from SI avoid striosomes and innervate discrete zones within the matrix. 3. These experiments demonstrate that the somatosensory representations of the body are reorganized as they are projected from SI to the somatosensory sector of the primate putamen. This remapping suggests that the striatal representation of the body may be functionally distinct from that of each area of SI. The patchy projections may provide a basis for redistribution of somatosensory information to discrete output systems in the basal ganglia. Transformations in the corticostriatal system could thus be designed for modulating different movement-related programs. PMID- 1722245 TI - A characterization of glycinergic receptors present in cultured rat medullary neurons. AB - 1. Whole-cell current responses to bath application of glycine, beta-alanine, and taurine were studied in medullary neurons cultured from embryonic rats. 2. Two current components were seen in the responses to bath application of agonist, one component that desensitized and another that did not. 3. The two current components have different dose-response characteristics, with the nondesensitizing component being activated more effectively at lower concentrations than the desensitizing component and also reaching its peak at lower concentrations. The agonist concentrations producing half-maximal responses are 26 +/- 4 (SE, n = 6) and 69 +/- 17 (n = 7) microM for the nondesensitizing and desensitizing components, respectively, for glycine; 54 +/- 7 (n = 9) and 127 +/- 37 (n = 7) microM for beta-alanine; and 153 +/- 24 (n = 9) 443 +/- 99 (n = 3) microM for taurine. Thus, for each component, the order of potency is glycine greater than beta-alanine greater than taurine. 4. When total responses to glycine, beta-alanine, and taurine are compared in the same cells, taurine and beta-alanine are less potent agonists than glycine, with relative potencies of 1:0.4:0.1 for glycine-beta-alanine-taurine. 5. The desensitizing component is more sensitive to strychnine than the nondesensitizing one. The strychnine concentrations that block 50% of the response to a control dose of agonist are 15 and 500 nM for the desensitizing and nondesensitizing components, respectively, for glycine; 60 nM and 1 microM for beta-alanine; and 18 and 500 nM for taurine. 6. The complete occlusion between the responses to glycine and beta-alanine or glycine and taurine suggests that these agonists activate the same receptors. 7. The two current components may be manifestations of one receptor population with complicated kinetics or two independent receptor populations. PMID- 1722246 TI - Inhibition of 5-HT3 receptor-mediated ion current by divalent metal cations in NCB-20 neuroblastoma cells. AB - 1. The effect of micromolar concentrations of divalent metal cations on ion current activated by 5-hydroxytryptamine (5-HT) was investigated in NCB-20 neuroblastoma cells by the use of the whole-cell, patch-clamp technique. 2. Ion current activated by 5-HT in these cells was mimicked by 5-HT3 receptor agonists, blocked by nanomolar concentrations of selective 5-HT3 receptor antagonists and reversed polarity at approximately 0 mV. These properties indicate that this current is carried primarily if not exclusively by the nonspecific cation channel activated by the 5-HT3 receptor. 3. The Group IIb metal cations Cd2+ and Zn2+ and the Group Ib cation Cu2+ inhibited 5-HT-activated current with inhibition increasing in a concentration-dependent manner over micromolar concentrations of the ions. The order of potency of the ions for inhibiting 5-HT-activated current was Zn2+ (IC50 = 20 microM) greater than or equal to Cu2+ (IC50 = 25 microM) greater than Cd2+ (IC50 = 75 microM) at -50 mV. The other divalent metal cations tested (Ba2+, Co2+, Mg2+, Mn2+, and Ni2+) produced little or no inhibition of 5 HT-activated current at concentrations up to 200 microM. 4. Inhibition of 5-HT activated current by Cd2+ and Zn2+ was dependent on membrane potential with the Kd increasing e-fold per 72 and 52 mV, respectively. Inhibition by Cu2+ was much less voltage dependent with the Kd increasing e-fold per 233 mV. 5. Inhibition by all three cations decreased with increasing concentration of agonist over a range of 5-HT concentrations from 1 to 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722247 TI - Occult regional lymph node metastases from breast carcinoma: immunohistological detection with antibodies CAM 5.2 and NCRC-11. AB - Ninety-eight consecutive patients with primary operable breast cancer and an initial diagnosis of no regional lymph node metastases as assessed by conventional light microscopy were studied. Immunohistological staining of routine lymph node sections was assessed using two monoclonal antibodies: CAM 5.2 (Becton Dickinson) with specificity for low molecular weight cytokeratin, and NCRC-11 (CRC Laboratories, Nottingham) with specificity for epithelial mucin antigen. Positive staining for occult metastases was seen in nine patients with CAM 5.2 and in eight of these nine with NCRC-11. At a follow-up out to 14 years, there was no difference in overall survival, in recurrence-free survival, or in frequency of or time to presentation of local or regional recurrences between occult metastasis-positive and occult metastasis-negative patients. This study concludes that while immunohistological staining of routine lymph node sections increases the diagnostic yield of metastases, it is not to be recommended as this increase is of no useful clinical value. PMID- 1722248 TI - The significance of cerebrovascular amyloid in the aetiology of superficial (lobar) cerebral haemorrhage and its incidence in the elderly population. AB - Cerebrovascular amyloid deposition (CVAD), caused by deposition of the beta/A4 protein, has been previously identified as a cause of cerebral haemorrhage, yet its prevalence is uncertain. The presence of vascular amyloid was studied in brains of 169 patients by immunohistochemical and Congo red staining. Fifty patients had cerebral haemorrhage (CH), 56 had cerebral infarction (CI), and 63 had neither haemorrhage nor infarction (control group). CVAD was found in 38 per cent of the CH group, 25 per cent of the CI group, and 32 per cent of the control group. The incidence of CVAD increased with age in each group. Immunohistochemical staining with an antibody to beta/A4 protein was more sensitive than Congo red staining the demonstrating the extent of vascular amyloid. Within the CH group, CVAD was present in the vessels at the site of haemorrhage in 6/8 (75 per cent) of pure superficial (lobar) cerebral haemorrhages. While amyloid was detected in vessels in the brain of 10/37 (27 per cent) of pure deep cerebral haemorrhages, none was present in vessels at the site of haemorrhage. CVAD is a common pathological finding in the elderly and has a significant association with pure superficial (lobar) cerebral haemorrhages. PMID- 1722249 TI - Patterns of cytokeratin expression in human gingival epithelia. AB - Specimens of human gingiva were collected from teenage and adult subjects and frozen sections were stained with an extensive panel of monoclonal antibodies with defined specificities for individual cytokeratins. The results indicated different and distinctive patterns of keratin expression by the oral gingival, oral sulcular and the junctional epithelia. It was observed that epithelium with staining characteristics of sulcular epithelium extended over the gingival crest onto the oral surface of the gingiva. Junctional epithelium showed the unusual pattern of co-expression of keratins typical of the stratifying and of the simple epithelial phenotypes. The patterns of gingival keratin expression are compared with those of other mucosal epithelia. The findings are discussed in relation to mechanisms that may determine or influence the junctional epithelial phenotype. PMID- 1722250 TI - Immunolocalization of proteins specific for adhaerens junctions in human gingival epithelial cells grown on differently processed titanium surfaces. AB - The localization of desmoplakins 1 and 2 (DP 1&2), components of desmosomes, vinculin, and actin, was studied in gingival epithelial cells grown on cell culture glass and on titanium plates with various surface topography. The results showed that epithelial cells attached and spread more readily on smooth than on rough, sandblasted titanium surfaces. Moreover, the cells appeared to develop more granular DP 1&2 immunoreactivity at their ventral surfaces when grown on smooth or etched titanium as compared to glass. In cells grown on sandblasted titanium surfaces, DP 1&2-specific immunoreactivity was primarily located at cell cell contacts. Cells grown on smooth titanium surfaces harbored a fine network of actin filaments with apparent cell-to-cell organization. Vinculin was confined to cell-cell contact areas. No vinculin-containing focal adhesions could be detected, suggesting that the cells adhere either by means of close contacts, extracellular matrix contacts, or by means of hemidesmosomes. The findings suggest that smooth of finely grooved titanium surfaces could be optimal in maintaining the adhesion and specialized phenotype of gingival epithelial cells. PMID- 1722251 TI - A culture of one: case study of play therapy with an abused child. AB - This article presents a case study of play therapy with an emotionally disturbed abused child. The article discusses the intuitive and individualized approach to the world of the abused child (conceptualized as a "cultured of one") in which the nurse therapist's role is to develop a trusting relationship with the child. Distance between the nurse's world and that of the abused child can be bridged by learning the child's symbolic language through play and by sharing and enlarging the child's symbolic vocabulary. PMID- 1722252 TI - Effects of K+ channel blockers and cromakalim (BRL 34915) on the mechanical activity of guinea pig detrusor smooth muscle. AB - There is strong evidence that cromakalim (BRL 34915) relaxes smooth muscle by opening cell membrane K+ channels. The aim of this study was to use relatively selective K+ channel blockers to investigate 1) the K+ channel type(s) opened by cromakalim in guinea pig detrusor and 2) the role of different K+ channel types in the control of basal tension. Cromakalim produced a concentration-related relaxation (IC50 = 0.50 +/- 0.03 microM, n = 42) of 15 mM K(+)-evoked mechanical activity. The ATP-sensitive K+ channel blocker glyburide (0.3-3 microM) antagonized the effects of cromakalim in an apparently competitive manner (pA2 = 6.76). Charybdotoxin and iberiatoxin (3-30 nM), blockers of the large conductance, Ca(++)-activated K+ channel, appeared to functionally antagonize cromakalim. Apamin (1 microM) and leiurotoxin I (0.3 microM), blockers of the small conductance, Ca(++)-activated K+ channel, and noxiustoxin (0.3 microM), a blocker of squid axon delayed rectifer K+ channels, all failed to antagonize cromakalim. Cumulative administration of charybdotoxin and iberiatoxin produced marked, concentration-related stimulation of mechanical activity per se whereas glyburide, noxiustoxin, apamin and leiurotoxin I had no effect. Apamin and leiurotoxin I did stimulate mechanical activity to a small extent when administered noncumulatively, however. The results suggest that cromakalim opens ATP-sensitive K+ channels in detrusor and suggest that cromakalim does not open CA(++)-activated K+ channels and noxiustoxin-sensitive, delayed rectifier K+ channels. The marked stimulatory effects of charybdotoxin and iberiatoxin per se suggest an important role for large conductance, Ca(++)-activated K+ channels in the control of basal tension and, presumably, membrane potential in detrusor smooth muscle cells. PMID- 1722253 TI - Response of extrapyramidal and limbic neuropeptides to fenfluramine administration: comparison with methamphetamine. AB - The responses of extrapyramidal and limbic neuropeptide and striatal dopamine and serotonin systems were evaluated after treatment with fenfluramine in rats. After multiple administrations of fenfluramine, its active metabolite, norfenfluramine, and methamphetamine (METH), striatal neurotensin (NT) content was similarly increased to approximately 200% of control. In contrast, nigral NT levels were unaltered by fenfluramine, intermediately increased by norfenfluramine (148% of control) and maximally increased by METH (267% of control). Striatal and nigral substance P (SP) and dynorphin A (Dyn) systems were unaltered by fenfluramine, whereas norfenfluramine caused an intermediate increase in striatal Dyn content but did not significantly alter striatal SP or nigral SP and Dyn levels. However, METH significantly elevated striatal and nigral Dyn and SP concentrations to 280 to 425% (Dyn) and 140% (SP) of control. For the most part, the response of the limbic peptides was similar to that seen in the striatum with a couple of notable differences. Further investigation of the striatal NT system showed that the increases induced by fenfluramine were completely blocked by the D1 antagonist, SCH 23390, and the noncompetitive N-methyl-D-aspartate antagonist, MK801. Depletion of 5-hydroxytryptamine with pretreatment by parachloroamphetamine did not alter the response of the striatal NT system to fenfluramine. The present results demonstrate common and unique features in the response of peptide systems to fenfluramine and methamphetamine, which might explain some of the similarities and differences between these two drugs. PMID- 1722254 TI - Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts. AB - Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2 +/- 0.2 pA (mean +/- SE, n = 21) at +/- 40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg2+ as well as a reduction in pH. Detection of single-channel events was done at 0.5, 1 and 2 M KCl. The single-channel amplitude in 0.15 M KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with a PK/PCl = 32 (n = 6). Treatment with alkaline pH (8.3) increased the current and anion permeability (PK/PCl = 16, n = 6). The membrane becomes completely cation selective when low concentrations (12 microM) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 microM), propranolol (70 microM) and quinine (0.6 mM) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies. PMID- 1722255 TI - Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation. AB - The fadR gene of Escherichia coli encodes a protein that acts as a negative regulator (repressor) of the inducible beta-oxidation pathway. We report that the FadR protein also functions as a positive transcriptional activator of the fabA gene, which encodes the enzyme introducing the double bond of the unsaturated fatty acids of E. coli. PMID- 1722256 TI - Crystallization and preliminary X-ray diffraction studies of dialkylglycine decarboxylase, a decarboxylating transaminase. AB - The pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (E.C. 4.1.1.64) has been crystallized by vapor diffusion from a 15% polyethyleneglycol solution with sodium pyruvate as coprecipitant. The space group of the crystals is either P6(2)22 or the enantiomorph, P6(4)22, with one subunit of 46,500 Da per asymmetric unit. The unit cell has dimensions a = b = 152.7 A, c = 86.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and a solvent content of approximately 61%. diffraction extends to 2.3 A resolution. PMID- 1722257 TI - Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli. AB - Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A. PMID- 1722258 TI - [Augmentation of activities of peripheral granulocytes and of resistance against bacterial infection after administration of G-CSF into mice]. AB - We evaluated the metabolic capability of murine peripheral granulocytes after administration of recombinant human granulocyte colony-stimulating factor (rhG CSF) by quantitative flow cytometric assay for H2O2-dependent oxidative product formation. Intraperitoneal administration of a daily dose of 10 micrograms of rhG CSF for 5 days induced doubling of the leukocyte population. Differential counting of peripheral leukocytes and scattergram by flow cytometry showed an increased mature granulocyte population. After stimulation with phorbol myristate acetate, the granulocytes of the rhG-CSF-administered mice demonstrated some hyperresponsive population and an increased H2O2 production. The hyperresponsive population showed H2O2 production 4-6 times higher than did normal cells. Granulocytes from the G-CSF-treated mice revealed an augmented phagocytic activity and an increased expression of Mac-1 molecules. Moreover, mice treated with G-CSF showed an enhanced resistance against intravenous infection with a lethal dose of E. coli. Granulocytes showing such markedly increased oxidative metabolism may be a significant component of the host defence to various infective organisms. PMID- 1722259 TI - [DNA analysis and gene diagnosis of cystic fibrosis]. AB - Cystic fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, affecting approximately 1 in 2,000 newborns but the actual estimate varies with the geographic location. The incidence of CF in non Caucasian populations is low. Intensive efforts using genetic linkage information ultimately led to the cloning of the CF gene prior to the identification of the gene product or its function. The gene encodes what is believed to be a transmembrane protein, which has been named the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR contains two nucleotide-binding folds (NBF) which show homology to numerous transport proteins with the greatest homology to the P-glycoproteins that are encoded by the multiple drug-resistance loci. A three- base deletion resulting in the loss of phenylalanine residue (delta F508) in the tenth exon of the CFTR gene is the mutation occurring on the majority of CF chromosomes. The overall frequency of delta F508 in the present mutant CF gene pool is about 70%, but the study populations are not equally represented: there is marked variation in the population of delta F508 among different geographic populations. Recently, numerous additional, less common mutations have been found. Some mutations occur on 2-5% of the CF chromosomes. Many of these are rare 'private' mutations, occurring in individual families of all racial and ethnic backgrounds. By contrast over 80% of Western European CF mutations have been identified. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but further improvements are needed in DNA-based genetic screening for CF carrier status. PMID- 1722260 TI - [Immunohistochemical analysis of adenomatoid tumor of the uterine corpus- comparison with mesothelioma]. AB - Immunohistochemical study was carried out in a case of adenomatoid tumor of the uterine corpus. The patient was a 35-year-old female. The tumor showed classical histochemical and immunohistochemical findings of mesothelioma, i.e., presence of hyaluronic acid on the cellular surface and cytokeratin in the cytoplasm. In addition, the tumor showed positive reaction to anti-vimentin. Furthermore, absence of Ber-Ep4 supports mesothelial origin of this tumor. EMA reaction was reported only in one case in the literature. The result was negative as in our case. Therefore, it was suggested that at least some of the adenomatoid tumor were negative for EMA as in malignant mesothelioma, although this tumor was benign. Therefore, it was suggested that loss of EMA in mesothelial tumor was not always related to anaplastic change. PMID- 1722261 TI - A case of Landau-Kleffner syndrome: speechaudiometry and clinical efficacy of treatment. PMID- 1722262 TI - Mesocortical neurotensin/dopamine system: is it involved in the pathogenesis of schizophrenia? PMID- 1722263 TI - Locus coeruleus and norepinephrine in Parkinson's disease. PMID- 1722264 TI - [Voiding disturbance in elderly males examined by prostate mass screening. Gunma Urological Oncology Study Group]. AB - Information about voiding disturbance was obtained from 34,140 elderly males in Gunma Aged Club Association (Q group) through a questionnaire and from 8,129 males by prostate mass screening for prostate disease (MS group) as to: (1) the characteristics of the subjects examined by the mass screening, (2) the relationship between the voiding disturbance and aging and (3) the frequency of voiding disturbance in the normal elderly males. In MS group, the percentage of young subjects, especially less than 60 years old, who complained voiding disturbance was higher than the expected percentage on the assumption that it increases with aging. The percentage of voiding disturbance in these young subjects was also higher than that of males in the same decade in Q group. In their past and present history, the percentage of benign prostatic hypertrophy (BPH) in these young subjects was higher than the expected percentage on the assumption that the incidence of BPH increases with aging. Judging from these results, it was estimated that they were not satisfied with the medical care by which they were treated or were being treated, and they received this mass screening to obtain consultation for their complains. Loss of the force was related with the prostate size estimated by digital rectal examination but not with aging. However, nocturia was well related with aging but not with the prostate size. Hesitancy, strain and dribbling were not apparently related with either the prostate size or aging. It was demonstrated that frequency of nocturia was 0 to 1 in less than 60 years old normal males, 0 to 2 in 60 to 79 years old ones and 0 to 3 in over 80 years old ones. The other symptoms studied in each decade were also analyzed and discussed. PMID- 1722265 TI - Urinary protein excretion in children from families with Balkan nephropathy. AB - We studied the urinary excretion of total protein and five other proteins in urine samples from a total of 831 children and adolescents. The characteristics of the children were: (1.) they resided in areas where Balkan nephropathy (BEN) is endemic and also had family members suffering from BEN; (2.) they resided in areas where BEN is endemic, but the families had no members suffering from the disease; (3.) they lived in nonendemic settlements; (4.) they lived in the city of Nis. Urinary excretion of total protein (TP), albumin (ALB), alpha 2 macroglobulin (alpha 2m), transferrin (TF), IgG, and beta 2-microglobulin (beta 2m) were measured. This study showed that urinary excretion of beta 2m and albumin in children from endemic settlements and from families affected with Balkan nephropathy were not different from the control rural settlements. However, children residing in the city of Nis had significantly increased urinary beta 2m and albumin excretion, over 1.5 to 2.6 times the excretion in the other three groups, although excretion of either protein remained within accepted normal ranges in all groups. The increased excretion of beta 2m and albumin in children from the city of Nis could probably be related to the different growth conditions and/or the effect of toxic environmental factors. These data could serve as a reference base for future comparative studies of urinary protein excretion in children as well as in BEN populations. PMID- 1722266 TI - [Histological classification of uveal melanoma]. AB - Cytological classification of choroidal malignant melanomas recommended by WHO and based on the Callender classification (spindle A and B, mixed and epithelioid) is presented. Prognosis according to the histological types is discussed. PMID- 1722267 TI - Recording of voltage and Ca(2+)-dependent currents in Xenopus oocytes using an intracellular perfusion method. AB - We describe a method for internal perfusion of Xenopus laevis oocytes that allows control of the composition of intracellular and extracellular solutions, including the possibility of sequential introduction of different substances inside and outside the cell. Using this method, it was possible to record Ca2+ dependent Cl- current and to inhibit it by intracellular perfusion of EGTA containing solution. With a high BA2+ solution at the external surface of the perfused oocyte, Ba2+ currents through voltage-dependent Ca2+ channels were observed in native and in cardiac RNA-injected oocytes. Finally, a delayed rectifier K+ current was recorded and blocked by internally perfused Cs+ in oocytes injected with mRNA of a cloned (MBK1) K+ channel. The method is expected to be useful for the study of function and modulation of ion channels and transporters in the oocyte, which is an important and widely used model system. PMID- 1722268 TI - A method for the determination of projection areas of GABA immunoreactive neurons in the invertebrate nervous system. AB - Axonal transport of metallic salts (nickel or cobalt chloride) has been widely used for the anatomical mapping of neural pathways. We show here that when nickel is introduced into GABAergic neurons it completely eliminates GABA immunolabelling. We have used this property to determine the axonal projections of GABAergic neurons in the stomatogastric system of Crustacea. For example, following nickel backfills from either cut axons or from terminals, GABA immunostaining labels only those GABA-immunoreactive neurons which had not been retrogradely labelled with nickel and hence did not project in the cut nerve or to the neuropile uptake site. By comparing such immunolabelled preparations with those not pretreated with nickel the projection patterns of all the GABA immunoreactive neurons in a given system can be revealed. This effect of nickel appears to be selective for GABA immunostaining, insofar as it does not interfere with the immunodetection of either the peptide proctolin or a FMRFamide-like peptide. This method may prove to be a useful tool for analyzing GABAergic neuronal pathways in the nervous systems of invertebrates. PMID- 1722269 TI - Sequential double labelling with different fluorescent dyes coupled to dextran amines as a tool to estimate the accuracy of tracer application and of regeneration. AB - We present a technique to estimate the accuracy of a given application procedure for neuronal tracers. In a second series of animals we used this technique for the estimation of successful regeneration of peripheral nerves. Dextran amine coupled to rhodamine was applied to the cut trochlar nerve in Xenopus tadpoles. To assess the accuracy of tracer application, experiments were done in which a second dye, dextran amine coupled to fluorescein, was applied after 1 day proximal to the first dye. More then 90% of all trochlear motoneurons were doubly labelled after this procedure. Their total numbers were not significantly different from numbers obtained after single labelling with HRP in a comparable age group. To assess success of regeneration after 5 and 8 days, the second application of fluorescein dextran amine was distal to the first application side. Statistically significant differences suggest incomplete regeneration of many neurons. After 42 days the numbers of singly and doubly labelled motoneurons was in the same proportion as before regeneration. This suggests that about 90% of the surviving motoneurons had successfully regenerated back to the periphery. PMID- 1722270 TI - Predicting location of continuous epitopes in proteins from their primary structures. PMID- 1722271 TI - Immunoelectron microscopy and image processing for epitope mapping. PMID- 1722272 TI - Characterization of antigenic structures by mapping on resin-bound epitope analogs. PMID- 1722273 TI - Epitope mapping of allergens for rapid localization of continuous allergenic determinants. PMID- 1722274 TI - Selection of T cell epitopes and vaccine engineering. PMID- 1722275 TI - In situ hybridization and immunodetection techniques for simultaneous localization of messenger RNAs and protein epitopes in tissue sections and cultured cells. PMID- 1722276 TI - Design of simplified ribonuclease P RNA by phylogenetic comparison. PMID- 1722277 TI - Identification of an immunoreactive non-proteinaleous component in Borrelia burgdorferi. AB - Investigations of immunoblots using Borrelia burgdorferi antigen demonstrated that a band, migrating faster than the bromophenol blue front in sodium dodecyl sulfate-gel electrophoresis, reacted strongly with sera containing anti-Borrelia burgdorferi antibodies preferentially of the IgG class. Extraction of this antigenic component and chemical analyses showed that the substance was composed mainly of fatty acids and carbohydrates. Typical structures of classical lipooolysaccharides such as 3-deoxy-D-manno-2-octulosonic acid, hydroxy fatty acids or lipid A could not be detected. PMID- 1722278 TI - The role of free radical scavengers, inhibitors of prostaglandin synthesis, and hypomethylating agents in reactivation of latent herpes simplex virus. AB - Reactivation of herpes simplex virus (HSV) was assumed to be dependent on prostaglandin synthesis [Kurane et al. (1984) J Gen Virol 65:1665-1674]. Since free radicals are generated in the course of prostaglandin synthesis and inhibitors of prostaglandin synthesis are free radical scavengers, the question arose whether free radicals are most important in viral reactivation. Therefore, the influence of five different inhibitors of prostaglandin synthesis was compared with the activity of two combinations of classical radical scavengers. Since the efficiency of both groups of compounds was similar, the role of free radicals in viral reactivation was strongly assumed, whereas the role of prostaglandins has to be revisited. All drugs tested were effective in the early phase after explantation of the latently infected ganglia and could be omitted in the later stages of co-cultivation. As free radicals are suspected of activating nuclear factors, it was of interest to confirm the enhancement of HSV reactivation by DNA hypomethylating drugs. 5-Azazytidine proved to be more effective than dimethylsulfoxide (DMSO). The effect of combinations of DMSO with inhibitors of prostaglandin synthesis was dependent on the mode of action of the inhibitors. PMID- 1722279 TI - Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts. AB - The mutagenicity spectra of the organic extracts of both airborne particulate matter and diesel and gasoline soot particles were determined using a battery of 9 bacterial strains of different genetic specificity. The assays with crude extracts and with fractionated acidic, neutral and basic components revealed striking differences in the patterns of mutagenic responses produced by each of the complex mixtures investigated. The mutagenicity of air particulate matter was shown to depend mainly on direct-acting acidic and neutral compounds, with a lesser contribution of basic promutagens which required exogenous metabolic activation by liver S9. The assays with a diesel soot extract indicated the prevailing contribution of direct-acting acidic and neutral compounds, and suggested an important role also for nitro derivatives other than nitropyrenes. The gasoline exhaust was characterized by powerful promutagenic compounds, belonging to either the acidic, neutral or basic fractions. The implications of these results are discussed with respect to the contribution of engine exhausts to air pollution, and the possible use of mutagenicity spectra in the analysis of environmental complex mixtures. PMID- 1722280 TI - Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1 (3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells. AB - The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino) 1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay. PMID- 1722281 TI - Mutagenicity of some heterocyclic amines in Salmonella typhimurium with metabolic activation by human red blood cell cytosol. AB - Purified human red blood cell cytosol was used to activate the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4 dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3 b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) into mutagenic intermediate(s) in the Salmonella test. The liquid preincubation method in the presence of strain TA98 was utilized. In order to understand the mechanism involved in this metabolic activation, some modulators were incorporated in the medium. The results suggest that an oxygenated hemoprotein, probably oxyhemoglobin, is involved in the activation into genotoxic intermediate(s). PMID- 1722282 TI - Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[a]pyrene diol epoxides in CHO cells. AB - The initiation of carcinogenesis by carcinogens such as 7r,8t-dihydroxy-9,10t-oxy 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7r,8t-dihydroxy-9,10c-oxy 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37 degrees C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in greater than 20% survival, and were found to increase linearly with dose. In the DNA-repair deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25 fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10(6) survivors at a dose that resulted in one adduct per 10(6) base pairs) with our previous studies of BPDE-1, we found that BPDE-II was 4-5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts. PMID- 1722283 TI - Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[a]pyrene. AB - Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[a]pyrene, 9r,10t-dihydroxy-7c,8c-oxy 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10 tetrahydrobenzo[a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D0's, the DNA-repair-deficient cell lines were found to be 4-5-fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells. PMID- 1722284 TI - Localization of beta pre-protachykinin mRNA in nodose ganglion. AB - The neurons which synthesize tachykinins in the capsaicin-sensitive afferent nerves of the respiratory tract are largely localized to the nodose ganglia. Using a radiolabelled antisense cRNA probe constructed from cDNA for the major precursor of substance P and neurokinin A (beta-preprotachykinin: beta-PPT), we have localized specific mRNA for this peptide in neurons of the nodose ganglion of rat using in situ hybridization. 26% of neurons gave a positive hybridization signal, which was in agreement with the same proportion of cell bodies showing substance P-like immunoreactivity. The specificity of the hybridization was confirmed by the absence of labelling using RNase pre-treatment and a sense probe having the same sequence as beta-PPT mRNA. This approach may now allow the investigation of factors which regulate synthesis of tachykinins at a gene transcriptional level. PMID- 1722285 TI - Studies of reflexogenic effects of capsaicin and neuropeptides on neural afferents in the dog parietal pericardium. AB - Stimulation of neural afferents in the parietal pericardium of anaesthetized, open-chest dogs by local application of capsaicin (0.1-100 micrograms) consistently induced dose-related pressor effects and tachycardia, whereas the application (0.1-1 microgram) of neuropeptides substance P (SP), neurokinin A (NKA), neurokinin B (NKB) or calcitonin gene-related peptide (CGRP) had no cardiovascular effect. Capsaicin-induced reflex responses were not affected by vagotomy, but were abolished by bilateral sectioning of the upper thoracic (T1 T4) white rami communicantes and stellectomy. Capsaicin-induced reflex tachycardia could also be abolished by a beta-adrenoceptor blockade with propranolol (0.5 mg/kg, IV), while ganglionic blockade with pentolinium (0.5 mg/kg, IV) eliminated both the tachycardia and pressor effects. Intravenous treatment with the cyclo-oxygenase inhibitors, indomethacin (5 mg/kg) or aspirin (100 mg/kg) had no effect on reflex pressor and heart rate responses to pericardial capsaicin. Also local treatment of the pericardium with either indomethacin (1 microgram/ml) or dual cyclooxygenase/lipoxygenase inhibitor, BW755C (10 micrograms/ml) failed to affect the responses to capsaicin. We conclude that (i) capsaicin-sensitive afferents which are present in the dog pericardium have a spinal origin and can initiate sympathetically-mediated reflex cardiovascular changes; (ii) the reflexogenic action of capsaicin on pericardial afferents does not depend on local production of eicosanoids; (iii) neuropeptides appear to be without reflexogenic effects on neural afferents in the dog parietal pericardium. PMID- 1722286 TI - Isocratic reverse-phase HPLC separation and RIA used in the analysis of neuropeptides in brain tissue. AB - A reverse-phase, high-performance liquid chromatographic (HPLC) method was employed to separate and characterise five neuropeptides from complex mixtures, with important advantages over methods employed earlier using combined HPLC-RIA studies. Peptides were separated using 0.5M pyridine-0.5M formic acid buffer, pH 4, containing propan-l-ol 14% (met-enkephalin, leu-enkephalin, neurotensin) or 20% (CCK-8-S, substance P) at a flow rate of 1.0 ml/min. Isocratic conditions, and volatile solvents, resulted in a highly reproducible method, producing samples in a form designed for subsequent RIA. The application and importance of the procedure is demonstrated by comparison of the measurements of apparent peptide levels in crude brain extracts with those of authentic peptides as determined after HPLC purification. PMID- 1722287 TI - Central hypoxaemia in rats provokes neurological defects similar to those seen in experimental diabetes mellitus: evidence for a partial role of endoneurial hypoxia in diabetic neuropathy. AB - Endoneurial hypoxia has been put forward as a factor contributing to diabetic neuropathy. The aim of this study was to determine whether alterations in motor nerve conduction velocity, Na+/K(+)-ATPase activity and substance P content of nerve and skin tissue, characteristic of the diabetic rat, could develop in non diabetic animals subjected to a central hypoxaemia for five weeks. Compared to normoxic controls, five weeks of central hypoxaemia caused a fall in motor nerve conduction velocity of 30% (P less than 0.01), a decrease in sciatic nerve substance P content (68%; P less than 0.001) combined with elevated substance P content per unit area foot skin (44%; P less than 0.01). This pattern of change is qualitatively similar to that seen in diabetic rats. The Na+/K(+)-ATPase activity, however, was unaltered by the hypoxic environment. These findings support strongly a partial role for hypoxia in the pathogenesis of diabetic neuropathy. PMID- 1722288 TI - Substance P-evoked release of GABA from isolated spinal cord of the newborn rat. AB - Isolated spinal cords of newborn rats were perfused with artificial cerebrospinal fluid and the effects of substance P and its analogs on the release of endogenous GABA were examined. Application of substance P evoked a dose-dependent release of GABA from spinal cords. The threshold concentration of substance P for induction of a significant increase in the GABA release was 3 microM. The substance P evoked GABA release was neither blocked by removal of Ca2+ from perfusion medium nor by tetrodotoxin. In contrast, the GABA release evoked by high K+ (90 mM) was abolished in Ca(2+)-free medium, and the GABA release evoked by veratridine (5 microM) was suppressed by tetrodotoxin (1 microM). A GABA uptake inhibitor, cis-4 hydroxynipecotic acid, markedly augmented the GABA release induced by high K+, but not that induced by substance P or veratridine. These results suggest the possibility that a carrier-mediated mechanism might be involved in the GABA release induced by substance P, as well as by veratridine, in the newborn rat spinal cord. Two N-terminal fragments of substance P, substance P free acid and substance P1-10 amide, as well as [D-Arg1,D-Trp7,9,Leu11]substance P (spantide), evoked an increase in the GABA release, whereas substance P1-6, and a C-terminal fragment, substance P5-11 were inactive. Somatostatin and compound 48/80 also evoked a GABA release, which was independent of external Ca2+ and resistant to tetrodotoxin. [D-Pro4,D-Trp7,9,10]substance P4-11 (10-15 microM) inhibited the GABA release evoked by substance P, somatostatin and compound 48/80.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722289 TI - Substance P-evoked release of acetylcholine from isolated spinal cord of the newborn rat. AB - Isolated spinal cords of newborn rats were perfused with artificial cerebrospinal fluid and the release of endogenous acetylcholine was measured using high performance liquid chromatography with an electrochemical detection system. Application of high-K+ (90 mM) medium evoked about an eight-fold increase in the acetylcholine release, and the K(+)-evoked release was Ca2+ dependent. Veratridine (20 microM) also evoked about a four-fold increase in the acetylcholine release, and this increase was suppressed by 0.2 microM tetrodotoxin. Application of substance P at 0.3-3 microM evoked a concentration dependent release of acetylcholine. The substance P-evoked acetylcholine release was Ca2+ dependent and abolished by tetrodotoxin. Neurokinin A, neurokinin B, acetyl-Arg6-septide and senktide (3 microM each) also evoked a release of acetylcholine. Electrophysiological experiments using isolated spinal cords of newborn rats showed that bath application of substance P induced a depolarization of motoneurons, which was enhanced by edrophonium. This enhancement of substance P-induced depolarization by edrophonium disappeared in a low-Ca2+ medium or in the presence of atropine and dihydro-beta-erythroidine. In the presence of edrophonium and dihydro-beta-erythroidine, substance P induced an inhibition of monosynaptic reflex, and this inhibition was abolished by atropine. These results suggest that substance P and other tachykinins induce a release of acetylcholine from the newborn rat spinal cord by exciting cholinergic neurons. PMID- 1722290 TI - The effect of selective serotonergic neurotoxin treatment on tachykinin levels in the rat ventral spinal cord. AB - The levels of 5-hydroxytryptamine and tachykinin neuropeptides substance P, neurokinin A, neurokinin B and neuropeptide K were measured in the spinal cord of rats treated by intraventricular injection of the selective serotonergic neurotoxin 5,7-dihydroxytryptamine. The spinal cord levels of 5-hydroxytryptamine as measured by high performance liquid chromatography with electrochemical detection decreased by more than 90% in the ventral and dorsal cord compared to controls. The levels of substance P as measured by radioimmunoassay were significantly reduced (66%, P less than 0.01) in the ventral lumbar cord only. In this region, neurokinin A, neurokinin B and neuropeptide K levels were determined by combined high performance liquid chromatography and radioimmunoassay. The neurotoxin treatment also caused a significant reduction of neurokinin A (72% reduction, P less than 0.01) and a non-significant reduction of neuropeptide K, but virtually no change in the neurokinin B level. Immunohistochemical studies of the ventral lumbar cord of sham-operated animals showed immunoreactivity for 5 hydroxytryptamine as well as for substance P and neurokinin A in nerve fibres around motor neurons. In neurotoxin-treated rats this region was devoid of immunohistochemically detectable substance P- and neurokinin A-positive fibres and showed very sparse or no 5-hydroxytryptamine immunoreactivity. We conclude that among the tachykinins both neurokinin A and substance P, but probably not neurokinin B, co-exist with 5-hydroxytryptamine in nerve terminals in the rat ventral spinal cord. PMID- 1722291 TI - A novel epitope expressed on the surface of developing and mature astrocytes. AB - Plasmalemmal fractions from cultured astrocytes have been used as the immunogen in generating a monoclonal antibody, termed 8C10, which binds to the surface of cultured astrocytes of the rat. 8C10 immunoreactivity is present on the membrane surface of cultured type 1 astrocytes, type 2 astrocytes, oligodendrocytes, meningeal cells, and 02A progenitor cells, and it persists after aldehyde fixation. The antibody also stains aldehyde-fixed central nervous system, in a pattern which suggests that the plasma membranes of fine astrocytic processes in adult neuropil express the epitope. Astrocytic perikarya and processes in white matter are also stained, but there is no immunoreactivity present in neuronal processes or perikarya. Astrocytic processes in developing cerebellar cortex are stained at postnatal ages when some of these processes are guiding the migration of neuronal perikarya. PMID- 1722292 TI - Mapping of the canine lumbosacral spinal cord neurons by Nauta method at the end of the early phase of paraplegia induced by ischemia and reperfusion. AB - The Nauta impregnation method was used to map the neuronal changes in the canine lumbosacral segments following ischemia and reperfusion. The early perikaryal changes ensuing during the first phase after 30 min of thoracic aorta cross clamping alone or followed by 30 min of reperfusion were mapped. During the second phase (one to six postischemic reperfusion days) the dendritic, preterminal and synaptic degeneration developed. The influence of 30 min cross clamping immediately followed by perfusion fixation is characterized by the occurrence of flocculent argyrophilic clusters in the cytoplasm of middle-sized and large neurons of L3-S1 segments. Declamping of the thoracic aorta followed by 30 min of reperfusion basically modifies the susceptibility of lumbosacral neurons to Nauta impregnation promoting somatic and dendritic argyrophilia mainly of small (less than 15 microns) neurons, localized mostly in the fifth, sixth and seventh layers, respectively. This early appearing somatic and dendritic argyrophilia is not abolished by a pretreatment of sections with acetone in which cholesterol and its esters are highly soluble, or chloroform-methanol which extracts total lipid. After 24 h of reperfusion the somatic and dendritic argyrophilia is lost but the first signs of drop-like degeneration are detected in all but three superficial dorsal horn layers. At the end of the third reperfusion day, an atypical form of bouton degeneration was found, consisting of massive occurrence of enlarged (greater than 4 microns) boutons encircled by a clear halo. Laminar distribution of enlarged degenerating boutons coincides with laminar quantitative distribution of small argyrophilic neurons detected 30 min after reperfusion. The basic orientation of the many terminal fibres attached to enlarged boutons suggests that they belong to the axons localized mainly in the lateral and anterior columns. Despite a dense argyrophilic network pervading the gray matter of lumbosacral segments only pale shadows of middle-sized and large neurons were found at the end of the sixth reperfusion day and neither somatic nor vessel wall argyrophilia could be detected. All animals surviving one, three and six days postoperatively suffered from fully developed paraplegia. PMID- 1722293 TI - Topographic distribution of the neurons of the central complex (centre median parafascicular complex) and of other thalamic neurons projecting to the striatum in macaques. AB - The distribution of the neurons of the central complex (or "centre median parafascicular complex") and of other thalamic regions projecting to the striatum was studied using a cartographic technique based on ventricular landmarks. The brain of a macaque was used as a reference for the cytoarchitectonic study of the complex. Three parts were isolated: the pars parafascicularis (or medial part), the pars media (or middle part) and the pars paralateralis (or lateral part). Wheat germ agglutinin conjugated to horseradish peroxidase was stereotaxically injected into either the sensorimotor or the associative territory of the striatum (i.e. the striatal space occupied by the axonal endings coming from either the sensorimotor or the associative cortex) of four macaques. Neurons projecting to the sensorimotor territory of the striatum were found to be located within the pars media (middle part) of the central complex while neurons projecting to the associative territory of the striatum were located within the pars parafascicularis. In all experimental cases, labelled neurons were scarce or absent in the pars paralateralis (or lateral part). Outside the central complex, neurons projecting to the sensorimotor territory of the striatum were scattered within the lateral part of the lateral mass, in the intralaminar nuclei and in the posterior part of the internal lamina. Neurons projecting to the associative territory of the striatum were observed mainly in the paraventricular region, dorsal to the rostral part of the lateral mass, and in the dorsolateral part of the nucleus oralis medialis. Our three-dimensional analysis of the clusters of the central complex cells projecting to the two striatal territories justifies the partitioning of the central complex into three parts. The pars media (or middle part), which projects to the sensorimotor territory of the striatum, receives selectively pallidal afferent axons. It belongs to the Nauta-Mehler loop, a closed loop linking the central complex to the basal ganglia. The pars parafascicularis, which projects to the associative territory of the striatum, seems more related to oculomotor neuronal systems. The pars paralateralis (or lateral part) appears to have very little, if any, relation with the striatum. PMID- 1722294 TI - Transmitter synthesis increases in substantia nigra neurons of the aged mouse. AB - Striatal dopamine concentrations are relatively well maintained with age despite extensive death of the nigrostriatal neurons whose terminals contain the dopamine. Counts of nigrostriatal dopaminergic neurons in C57BL mice identified using immunocytochemistry, Fluoro-Gold retrograde axonal transport and Nissl staining were combined with measures of striatal dopamine and DOPA after saline, pargyline or NSD-1015 treatment. On average, 68% of the dopaminergic nigrostriatal neurons died between ages 8 and 104 weeks and there was a 3-fold increase in dopamine synthesis per average neuron in the aged mice. Increased transmitter synthesis by surviving neurons may serve to compensate brain function in old age. PMID- 1722295 TI - Decreased spinal cord content of calcitonin gene-related peptide in the spontaneously hypertensive rat. AB - Calcitonin gene-related peptide (CGRP), produced by alternative processing of the primary transcript of the calcitonin gene, is a potent vasodilator. We have shown that dietary calcium deficiency accompanied by decreased serum ionized calcium significantly decreases the neuronal content of CGRP in laminae I and II of the dorsal horn of the spinal cord in the growing rat. The spontaneously hypertensive rat (SHR) is characterized by decreased serum ionized calcium levels and is thought to most closely resemble human essential hypertension. To determine if the neuronal content of CGRP is decreased in the SHR compared to the Wistar-Kyoto (WKY) parent strain, CGRP was localized immunocytochemically in the dorsal horn of the spinal cord. The density of immunocytochemical staining was quantitated by computer-assisted image processing of laminae I and II of the upper thoracic spinal cord of 12-14 week old male SHR (n = 4) and WKY (n = 4) normotensive, control rats. The SHR had significantly decreased neuronal CGRP content compared to the WKY rats (107 +/- 5 vs 121 +/- 6 arbitrary units, P less than 0.01). In contrast, the neuronal density of substance P (SP), which frequently co-exists with CGRP in this neuronal population, was not different between the two groups (SHR, 91 +/- 6 (n = 4) vs WKY, 88 +/- 3 arbitrary units (n = 4)). PMID- 1722296 TI - Quisqualate-sensitive glutamate receptors of the locust Schistocerca gregaria are antagonised by intracellularly applied philanthotoxin and spermine. AB - The effects of intracellularly and extracellularly applied synthetic analogues of delta-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor. PMID- 1722297 TI - Projections from serotonin- and substance P-like immunoreactive neurons in the midbrain periaqueductal gray onto the nucleus reticularis gigantocellularis pars alpha in the rat. AB - Serotonin- and substance P-like immunoreactive (5HT-LI and SP-LI) neurons in the midbrain periaqueductal gray (PAG) of the rat were observed to send their axons to the nucleus reticularis gigantocellularis pars alpha (Rgc alpha) by the retrograde horseradish peroxidase (HRP)-tracing method combined with the 5HT- or SP-immunohistochemical technique. These 5HT- or SP-LI PAG neurons were distributed mainly in the ventrolateral subdivision and ventral portion of the medial subdivision at the middle and caudal levels of the PAG, and additionally in the nucleus raphe dorsalis (DR). PMID- 1722298 TI - Tumor necrosis factor reduces the ACh-induced outward current in identified Aplysia neurons. AB - Effects of extracellularly applied recombinant human tumor necrosis factor (rhTNF) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhTNF (200-500 U/ml) reduced the ACh-induced current in the neurons without affecting the holding current and resting membrane conductance. The suppressing effect of rhTNF on the current was completely reversible. Inhibition by rhTNF was non-competitive. Heat inactivated rhTNF was without effect. Our results suggest that the immunomodulator TNF can act on the ACh receptor in the nervous system. PMID- 1722299 TI - Changes in lectin, GAP-43 and neuropeptide staining in the rat superficial dorsal horn following experimental peripheral neuropathy. AB - The density and distribution of reactivity for two lectins (soybean agglutinin (SBA) and RL-29), growth associated protein-43 (GAP-43) and the neuropeptides substance P and calcitonin gene-related peptide were analyzed in the spinal cord dorsal horn of rats with an experimental peripheral neuropathy. Twenty-eight days postsurgery, the density of label for RL-29 and GAP-43 was increased in laminae I and II on the experimental compared to the control side. In contrast, the density of neuropeptide label was decreased in the same region. Furthermore, on the experimental side, the distribution of both SBA and RL-29 reactivity was increased, extending into lamina III. We hypothesize that the increases in density and distribution of reactivity for the lectins and GAP-43, as well as the decreases in neuropeptide reactivity, reflect injury-induced regenerative changes in primary afferent terminals. PMID- 1722300 TI - Relative enrichment of the lighter 59 kDa form of glutamic acid decarboxylase in nerve endings: an immunoblotting study in pituitary neurointermediate lobe. AB - Previous studies established that glutamate decarboxylase (GAD) is present in the brain of higher vertebrates in two forms composed of the homodimeric association of two subunits 59 and 63 kDa, respectively, that were found to be structurally related. We have performed quantitative comparative immunoblotting of whole brain and pituitary neurointermediate lobe (NIL) extracts. While GAD in the brain is present in cell bodies and nerve endings, it is known to be contained in the NIL exclusively in nerve endings that belong to a relatively long gamma-aminobutyric acid (GABA)ergic pathway of central origin. The relative immunolabelling ratio of the 59 kDa to the 63 kDa subunit was at least 10-fold higher in the NIL when compared to whole brain extracts. Accordingly we suggest that the GAD composed of the 59 kDa subunits, which appears to be greatly enriched in GABAergic nerve endings, might represent the form of GAD on which short term activity regulation is exerted in relation to neuronal activity. It might result from the post translational processing of the GAD formed from the 63 kDa subunits during the axonal transport. PMID- 1722301 TI - An implantable tumor-window chamber model for the study of photodynamic therapy. AB - In order to study both the anti-tumor effects and early vascular events in photodynamic therapy, a useful animal model has been developed. A window chamber is surgically placed on the dorsum of the Fischer-344 rat, and 500-microns fragments of the rat mammary adenocarcinoma 13672 are placed under direct vision into the subcutaneous tissue. Implantation of the chamber has been successfully completed in more than 50 rats. The operative procedure is straightforward and is accomplished in less than 1 hour. Using tumor fragments, tumor viability has been 60%. We have demonstrated obvious and reproducible neovascularization occurring as soon as 1 day after implantation. The application of this system to an experimental protocol comparing the photosensitizers dihematoporphyrin ether (DHE) and chloraluminum sulfonated phthalocyanine (CASP) has yielded important information on early vascular events resulting from photodynamic therapy. PMID- 1722302 TI - [Acute phase proteins in monoclonal gammapathies]. AB - IL-6 is now recognized as a growth factor for plasma cells as well as a C Reactive Proteine inducer. This prompted a reappraisal of acute phase reactants in monoclonal gammapathies. Eight acute phase proteins were assayed in patients with multiple myeloma (n = 51), MGUS (n = 17) and Waldenstrom's macroglobulinemia (n = 5). The CRP level was above 10 mg/l in 27% of all myeloma patients, in 39% of patients with active myeloma, in 4 of 5 patients with Waldenstrom's macroglobulinemia and in none of the MGUS patients. Fibrinogen, alpha-1 antitrypsin and orosomucoid levels were significantly higher in the myeloma group than in the MGUS group. Differences were not significant for haptoglobin, ceruleoplasmin, transferrin, and alpha-2-macroglobulin. Serial assays in 22 myeloma patients showed that CRP levels were correlated with disease activity. A biologic inflammatory syndrome, defined as a significant variation in two or more acute phase reactants, was demonstrated in 41% of myeloma patients, 18% of MGUS patients, and 60% of Waldenstrom's macroglobulinemia patients. Active disease was significantly more common among myeloma patients with biologic evidence of inflammation, as compared with myeloma patients without biologic inflammation. These data suggest that similarly to IL-6 acute phase reactants are markers for disease activity in multiple myeloma. PMID- 1722303 TI - [Prevention of thrombosis in orthopedic surgery]. AB - The high incidence of thromboembolic complications in orthopedic surgery necessitates an effective thromboprophylactic regime. Current methods of prophylaxis are discussed, and principles of thromboprophylaxis with low molecular weight heparin are presented. PMID- 1722304 TI - Evolution of tRNAs and tRNA genes in Acholeplasma laidlawii. AB - The genes for 22 tRNA species from Acholeplasma laidawii, belonging to the class Mollicutes (Mycoplasmas), have been cloned and sequenced. Sixteen genes are organized in 3 clusters consisting of eleven, three and two tRNA genes, respectively, and the other 6 genes exist as a single gene. The arrangement of tRNA genes in the 11-gene, the 3-gene and the 2-gene clusters reveals extensive similarity to several parts of the 21-tRNA or 16-tRNA gene cluster in Bacillus subtilis. The 11-gene cluster is also similar to the tRNA gene clusters found in other mycoplasma species, the 9-tRNA gene cluster in M.capricolum and in M.mycoides, and the 10-tRNA gene cluster in Spiroplasma meliferm. The results suggest that the tRNA genes in mycoplasmas have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to mycoplasmas and B.subtilis. The anticodon sequences including base modifications of 15 tRNA species from A.laidlawii were determined. The anticodon composition and codon recognition patterns of A.laidlawii resemble those of Bacillus subtilis rather than those of other mycoplasma species. PMID- 1722305 TI - Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco ringspot virus and its binding to polyamines. AB - Several mutants for the minus strands of the self-cleaving domain of the satellite RNA of tobacco ringspot virus have been synthesized by joining chemically synthesized oligoribonucleotides with RNA ligase. Kinetic properties of the enzyme strands (50 nucleotides) against substrates (15-mer and 18-mer) were investigated. Structural properties of the unpaired part in the cleavage region were estimated from mutagenesis. The catalytic domain alone was proved to be responsible for the rejoining reaction of cleaved substrates. It was also found that the ribozyme could be divided into two strands without loss of activity. Effects of concentration of magnesium ion and polyamines on the cleavage reaction for the two-stranded ribozyme are also reported. PMID- 1722306 TI - MspI RFLP in the human fumarylacetoacetate hydrolase (FAH) gene. PMID- 1722307 TI - TaqI RFLP at the c-kit oncogene locus (KIT). PMID- 1722308 TI - MspI RFLP of FSHB on chromosome 11p. PMID- 1722309 TI - PCR-based detection of two MspI polymorphic sites at D18S8. PMID- 1722310 TI - The impact of granulocyte colony-stimulating factor on quality of life in patients with severe chronic neutropenia. AB - Quality of life (QOL) was assessed in 10 patients receiving treatment with recombinant methionyl human granulocyte colony-stimulating factor (r-met-GCSF). The Ferrans & Powers Quality of Life Index (Cancer II) was administered at three different time points to patients with severe chronic neutropenia. Mean overall QOL increased from time 1 to time 2 from a score of 23.72 to a score of 35.93 (p = 0.006). Overall QOL was maintained throughout the study with a mean score at time 3 of 36.96 (p = 0.002). Measures on all QOL subscales, including health and functioning and socioeconomic, improved significantly. Daily therapy with r-met GCSF significantly improves the QOL of patients with severe chronic neutropenia. PMID- 1722311 TI - [Clinical experience with FK 506]. AB - FK 506 is a superior immunosuppressive agent that should improve patient survival after the commonly performed transplant procedures, make feasible transplantations that have been previously impractical, allow immune intervention for serious autoimmune diseases, and create a better spin-off understanding of basic biologic processes including signal transduction. PMID- 1722312 TI - [LTB4-metabolism and histamine liberation by granulocytes in the disease pattern of cystic fibrosis (CF)]. AB - We determined the generation and liberation of LTB4 in peripheral granulocytes and the histamine release from basophils in patients suffering from cystic fibrosis (CF, median 17.2 years of age, n = 12). We compared the data with an age matched group of healthy donors (n = 12). All patients suffered from an exacerbation of a chronic pulmonary infection caused by P. aeruginosa. Peripheral granulocytes were stimulated at different days before, during and after antiinfectious treatment with Ca-Ionophore, opsonized zymosan and arachidonic acid. The granulocytes from patients with CF as compared to the control group showed an increased omega-Oxidation of the synthesized LTB4 into 20-COOH- and 20 OH-LTB4 after stimulation with Ca-Ionophore and opsonized zymosan (Ca-Ionophore: ratio of LTB4 versus omega-oxidated products (CF): 0.77 +/- 0.007 mean +/- S.E.M., n = 12, control group: 1.07 +/- 0.1, n = 12, p less than 0.01). Stimulation of the cells with Ca-Ionophore combined with arachidonic acid led to a significantly increased formation of lipoxygenase products in the patient group. No significant differences in the basophil counts were determined between both populations. However, the absolute histamine content per basophil was elevated in the CF group (2.4 +/- 0.3 versus 1.6 +/- 0.2 mean +/- S.E.M., n = 12/12, p less than 0.04). Stimulation of basophils with Ca-Ionophore and anti-IgE leads to a significant higher release of histamine per basophil in CF patients (Ca-Ionophore: 2.6 +/- 0.2 versus 1.3 +/- 0.8 pg/basophil, mean +/- S.E.M., p less than 0.05). These data indicate that basophils in CF may have a greater potential to release mediators. During the antiinfectious treatment a normalization of the altered pattern was observed. Within the CF-groups a strong correlation between the release of LTB4, its metabolites, the histamine release per basophil, the total histamine content and clinical (e.g. pO2 FEV1) and laboratory findings (e.g. IgE and IgG levels, CRP) was established. Our data suggest that the inflammatory process in patients with CF is associated with an alteration of the lipoxygenase pathway and histamine releasability of granulocyte subpopulations which correlates with the clinical signs of inflammation. PMID- 1722313 TI - Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation. AB - Senescent human diploid fibroblasts (HDF) contain no detectable cdc2 mRNA or p34cdc2 protein. Similarly, young quiescent HDF have only low levels of cdc2 mRNA and protein. After serum stimulation, quiescent HDF accumulate increasing amounts of cdc2 mRNA and protein and go through DNA synthesis and mitosis. In contrast, serum-stimulated senescent HDF fail to accumulate detectable amounts of cdc2 mRNA and protein and fail to enter S phase. Mitosis is likewise deficient in senescent cells even when they have been induced to synthesize DNA by simian virus 40 large tumor antigen. Since p34cdc2 or its homologues appear to be required for DNA synthesis and mitosis in eukaryotes, a lack of these molecules in serum stimulated senescent HDF could be an important reason for their inability to enter S phase or mitosis. Nuclear microinjection of cdc2 DNA into senescent HDF causes rounding up of the cells but no induction of DNA synthesis. Since cyclins A and B are important cofactors of the protein kinase activity of p34cdc2 or its homologues, we analyzed expression of these genes in serum-stimulated senescent HDF and determined that they contain little or no cycA or cycB mRNA. These deficiencies may be relevant to the lack of DNA synthesis and mitosis in senescent HDF. PMID- 1722314 TI - Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis. AB - Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO. PMID- 1722315 TI - De novo chromosome formation in rodent cells. AB - A hybrid cell line was produced by the fusion of an EC3/7 mouse cell with a Chinese hamster ovary cell. The EC3/7 cell carries a dicentric chromosome with a functional marker centromere. This marker centromere contains human, lambda, and bacterial vector DNA sequences and a dominant selectable gene (aminoglycoside 3' phosphotransferase type II; neo). In the hybrid, the marker centromere separated from the dicentric chromosome and formed a full-sized chromosome (lambda neo). The newly formed chromosome is stable, even under nonselective culture conditions. This functional chromosome, which is the result of an amplification process, is composed of seven large, different-sized amplicons. Each amplicon contains multiple copies of human, lambda, neo, and mouse telomeric DNA sequences. Individual amplicons are separated from each other by mouse major satellite DNA sequences. The marker centromere was localized to a terminal amplicon by anticentromere immunostaining. The number of amplicons in the newly formed chromosome is remarkably consistent. This finding suggests that the length of the newly formed chromosome is highly constrained. PMID- 1722316 TI - In vitro selection of lymphocytic choriomeningitis virus escape mutants by cytotoxic T lymphocytes. AB - Cytotoxic T lymphocyte (CTL)-mediated cytolysis is induced via the interaction of the specific T-cell antigen receptor and the peptidic viral antigen associated with the major histocompatibility complex class I antigen. Here we demonstrate in vitro that lymphocytic choriomeningitis virus (LCMV) can escape the cytotoxic activity of LCMV-specific cloned CTLs by single amino acid changes within the recognized T-cell epitope defined by residues 275-289 of the LCMV glycoprotein [LCMV-GP-(275-289)]. LCMV-infected fibroblasts at a multiplicity of infection of 10(-3) exposed to virus-specific CTL at an effector-to-target cell ratio of 4:1 4 hr after infection was optimal for virus mutant selection. The selections were carried out with three LCMV-GP-(275-289)-specific CTL clones expressing T-cell antigen receptors containing the identical variable gene segments V alpha 4 and V beta 10 but different junctional regions; selection was also possible with LCMV GP-(275-289)-specific cytotoxic polyclonal T cells. The most common escape mutation was an amino acid change of asparagine (AAT) to aspartic acid (GAT) at position 280; an additional mutation was glycine (GGT) to aspartic acid (GAT) at position 282. The results presented show that relevant point mutations within the T-cell epitope of LCMV-GP-(275-289) occur frequently and that they are selectable in vitro by CTLs. PMID- 1722317 TI - Completely functional double-barreled chloride channel expressed from a single Torpedo cDNA. AB - We have performed an electrophysiological analysis of the recently cloned Torpedo marmorata Cl- channel. Functional expression of Cl- channels in oocytes of Xenopus laevis previously injected with cRNA yielded an outward-rectifying current activated by hyperpolarization. Replacement of Cl- with other anions significantly reduced or inhibited the current. Single-channel recordings from cell-attached patches exhibited burst-like Cl- channel activity with rapid fluctuations between three equally spaced substates (0 pS, 9 pS, and 18 pS). The properties of the cloned Cl- channel were almost identical to those of the reconstituted native T. californica Cl- channel and were in full agreement with the predictions of the double-barreled channel model [Hanke, W. & Miller, C. (1983) J. Gen. Physiol. 82, 25-45]. Our results imply that the cloned cDNA codes for the completely functional Torpedo electroplax Cl- channel. PMID- 1722318 TI - Response to erythropoietin in erythroid subclones of the factor-dependent cell line 32D is determined by translocation of the erythropoietin receptor to the cell surface. AB - Regulation of the expression of the erythropoietin (Epo) receptor (EpoR) gene is under the control of transcriptional regulatory factor GATA-1. GATA-1 is expressed widely among the nonerythroid, factor-dependent subclones of the interleukin 3-dependent mouse cell line 32D. Consequently, to determine whether GATA-1 and EpoR gene expression are linked even in nonerythroid cells, we have studied the correlation of GATA-1 expression with expression and function of EpoR in these cell lines. EpoR mRNA (by RNase protection analysis) and EpoR protein (by specific antibody immunoprecipitation of metabolically labeled EpoR protein) were detectable not only in 32D and 32D Epo (an Epo-dependent subclone) but also in 32D GM, a subclone dependent for growth on granulocyte/macrophage colony stimulating factor. EpoR mRNA also was detectable by PCR in 32D G, a subclone dependent for growth on granulocyte colony-stimulating factor. However, only 32D Epo cells bound 125I-labeled Epo and expressed EpoR protein on the cell surface, as determined by immunoprecipitation of surface-labeled proteins. These results indicate that, in these factor-dependent cell lines, the major regulatory step determining the erythroid-specific response to Epo is the efficiency of EpoR protein translocation to the cell surface. Mechanisms that could affect lineage specific translocation are the presence of a chaperone protein, erythroid specific editing of EpoR mRNA, or altered processing of the EpoR protein to the cell surface. In this model, lineage-restricted responses to growth factors such as Epo are determined not by expression of the genes for growth factor receptors but, rather, by appropriate processing of the receptor protein. PMID- 1722319 TI - Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family. AB - CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function. PMID- 1722320 TI - Rapid de novo generation of defective interfering RNA by cucumber necrosis virus mutants that do not express the 20-kDa nonstructural protein. AB - It is generally believed that serial passage at high multiplicity of infection (moi) is required for the generation of defective interfering (DI) particles. High levels of DI RNAs are found associated with persistent infections initiated with laboratory cultures of cucumber necrosis virus (CNV). Two synthetic CNV transcripts that were derived through site-directed mutagenesis of a highly infectious CNV cDNA clone and that do not express the CNV 20-kDa nonstructural protein were found to generate high levels of symptom-attenuating DI RNAs de novo without serial high-moi passage in transcript-inoculated plants. Such de novo generation of DI RNAs did not occur in infections initiated with wild-type transcript until at least eight serial high-moi passages. The observation that a CNV nonstructural protein mutant rapidly generates DI RNA de novo may provide insight into mechanisms that underly DI particle formation in RNA viruses in general. PMID- 1722321 TI - Cloning of the cocaine-sensitive bovine dopamine transporter. AB - A cDNA encoding the dopamine transporter from bovine brain substantia nigra was identified on the basis of its structural homology to other, recently cloned, neurotransmitter transporters. The sequence of the 693-amino acid protein is quite similar to those of the rat gamma-aminobutyric acid, human norepinephrine, and rat serotonin transporters. Dopamine transporter mRNA was detected by in situ hybridization in the substantia nigra but not in the locus coeruleus, raphe, caudate, or other brain areas. [3H]Dopamine accumulation in tissue culture cells transfected with the cDNA was inhibited by amphetamine, cocaine, and specific inhibitors of dopamine transport, including GBR12909. PMID- 1722322 TI - Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene. AB - The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase delta was localized to chromosome 19. PMID- 1722324 TI - Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. AB - We have recently described a nonnucleoside compound that specifically inhibits the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS. This compound, nevirapine (BI-RG-587), interacts with highly conserved tyrosine residues at positions 181 and 188 in the reverse transcriptase to inhibit the recombinant enzyme and virus replication in cell culture with 50% inhibitory concentrations in the 40 nM range. HIV-1 variants resistant to nevirapine emerged with passage in cell culture in the presence of drug. This resistant phenotype was stable with continued passage in the absence of drug. These mutants had a substitution of cysteine for the tyrosine at position 181. Introduction of this mutation into the recombinant enzyme increased the inhibitory concentration of nevirapine 100-fold. Substitution of cysteine for tyrosine at residue 181 into the wild-type viral genome conferred a similar reduction in susceptibility to nevirapine. Mutants were also resistant to a tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione derivative and two 6-phenylthiouracil derivatives but retained their sensitivity to the other reverse transcriptase inhibitors, 3'-azido-3'-deoxythymidine and foscarnet. PMID- 1722323 TI - Spectrum of mutations in aspartylglucosaminuria. AB - Aspartylglucosaminuria (AGU) is an inherited lysosomal storage disorder caused by the deficiency of aspartylglucosaminidase. We have earlier reported a single missense mutation (Cys163----Ser) to be responsible for 98% of the AGU alleles in the isolated Finnish population, which contains about 90% of the reported AGU patients. Here we describe the spectrum of 10 AGU mutations found in unrelated patients of non-Finnish origin. Since 11 out of 12 AGU patients were homozygotes, consanguinity has to be a common denominator in most AGU families. The mutations were distributed over the entire coding region of the aspartylglucosaminidase cDNA, except in the carboxyl-terminal 17-kDa subunit in which they were clustered within a 46-amino acid region. Based on the character of the mutations, most of them are prone to affect the folding and stability and not to directly affect the active site of the aspartylglucosaminidase enzyme. PMID- 1722325 TI - An optimal viral peptide recognized by CD8+ T cells binds very tightly to the restricting class I major histocompatibility complex protein on intact cells but not to the purified class I protein. AB - CD8+ cytotoxic T lymphocytes recognize cell surface complexes formed by class I major histocompatibility complex (MHC-I) glycoproteins and antigenic peptides. We have identified a peptide nonamer (termed IV9) derived from the human immunodeficiency virus that is over a millionfold more active (at subpicomolar concentrations) than peptide analogues longer or shorter by one or two amino acid residues. Although IV9 does not detectably bind to isolated MHC-I molecules as measured by equilibrium dialysis, we quantitated its specific binding in unaltered form to MHC-I on intact cells. Less than 1% of cell surface MHC-I forms complexes with IV9, which suffices to trigger maximal cytotoxic T-lymphocyte activity. By contrast, a peptide dodecamer that includes the IV9 sequence and is active at micromolar concentrations does not bind to MHC-I on intact cells, raising the possibility that this longer peptide undergoes processing. Using stoichiometrically iodinated IV9 to obviate the ambiguities associated with trace labeling methods, we measured the dissociation kinetics of purified peptide/MHC-I complexes isolated by affinity chromatography and found these complexes to be exceedingly stable (t1/2 = 200-600 hr). PMID- 1722326 TI - Lineage commitment of hemopoietic progenitor cells in developing blast cell colonies: influence of colony-stimulating factors. AB - In clonal cultures of normal mouse marrow cells, combination of granulocyte, granulocyte-macrophage, or multipotential colony-stimulating factor (G-CSF, GM CSF, or multi-CSF, respectively) with stem cell factor (SCF) did not alter the number of blast colonies stimulated to develop compared with SCF alone but induced an up to 25-fold increase in their mean cell content and an up to 6-fold increase in their mean progenitor cell content. Costimulation of blast colony formation by SCF plus G-CSF did not change the relative frequency of progenitor cells of different types within the colonies compared with colonies stimulated by SCF alone. However, combination of GM-CSF or multi-CSF with SCF significantly increased the relative frequency of granulocytic progenitors and, for multi-CSF, also of eosinophil progenitor cells. These changes in the relative frequencies of progenitor cells committed to the various lineages support the hypothesis that hemopoietic regulators have some ability to induce selective lineage commitment in the progeny of multipotential cells. PMID- 1722327 TI - Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation. AB - The original proposal that substance P is involved in the regulation of nociceptive information at the first sensory synapse in the spinal cord has been substantiated by a wide range of evidence, but definitive support has been lacking, due primarily to the lack of evidence that a specific nociceptive response in the dorsal horn can be blocked by a substance P antagonist. Here, we present evidence that CP-96,345, a specific substance P (NK-1) receptor antagonist, selectively blocks a slow, prolonged excitatory postsynaptic potential following noxious cutaneous stimulation or a train of intense electrical stimuli to sensory nerves but does not affect the response to innocuous input or the brief response to single electrical stimuli to C fibers. These results indicate the specific involvement of substance P in the mediation of a prolonged after-excitation to noxious stimulation. This may have important implications for the etiology and treatment of chronic pain and for plastic changes in nociceptive pathways. PMID- 1722328 TI - Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase. AB - The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine. PMID- 1722330 TI - Cytotactin binding: inhibition of stimulated proliferation and intracellular alkalinization in fibroblasts. AB - Cytotactin is an extracellular matrix protein that is dynamically and transiently expressed in a place-dependent fashion during development by glial cells, fibroblasts, and several other cell types. In the present study, the effects of cytotactin on cell proliferation were examined in fibroblastic cells in culture. NIH 3T3 mouse cells plated on tissue culture substrata in the presence of soluble cytotactin remained rounded for longer periods than untreated control cells, similar to their response to cytotactin-coated substrates. These rounding effects could be prevented by pretreatment of the cells with nocodazole, a microtubule disrupting agent. Cytotactin inhibited the proliferation of fibroblasts in culture in a dose- and time-dependent manner, and this inhibition occurred even after nocodazole treatment. In addition, the presence of cytotactin inhibited proliferation stimulated by growth factors or tumor promoter. These effects on cell growth were accompanied by an early inhibition of the intracellular alkalinization that normally occurs upon mitogenic stimulation by a number of growth-promoting agents. Together these observations suggest that cytotactin is an endogenous cell surface modulatory protein and provide a possible mechanism whereby cytotactin may contribute to pattern formation during development, regeneration, tumorigenesis, and wound healing. PMID- 1722329 TI - Prohormone processing in Xenopus oocytes: characterization of cleavage signals and cleavage enzymes. AB - In this study, we characterize the sequences required for the cleavage of prohormones in Xenopus oocytes. We demonstrate that the yeast alpha-factor and the Aplysia egg-laying hormone (ELH) precursors are not cleaved in oocytes following simple pairs of basic residues, such as Lys-Arg, but that the ELH precursor is cleaved following the consensus sequence Arg-Xaa-(Lys/Arg)-Arg. This motif is conserved among precursors that are cleaved in virtually all mammalian cell types. Mutations that generate this sequence in the alpha-factor prohormone also result in efficient processing within oocytes. Cleavage at this consensus sequence may be due to the action of the Xenopus homologues of mammalian furin. PMID- 1722331 TI - Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. AB - We have analyzed the expression of the cytokine leukemia inhibitory factor (LIF) during embryogenesis and in tissues of neonatal and adult mice. The site of the most abundant LIF expression is the uterine endometrial glands, specifically on day 4 of pregnancy. Analysis of LIF expression in pseudopregnant mice and in females undergoing delayed implantation showed that it is under maternal control and that its expression coincides with blastocyst formation and always precedes implantation. These results suggest that a principal function of LIF in vivo may be to regulate the growth and to initiate implantation of blastocysts. PMID- 1722332 TI - Characterization of a protein that regulates the DNA-binding activity of NF-AT, the nuclear factor of activated T cells. AB - NF-AT is a T-cell-restricted activity that regulates interleukin 2 expression by binding to positions -285 to -254 of the interleukin 2 gene. Prior studies have shown that NF-AT is found only in T cells that are appropriately stimulated with "two signals" and is sensitive to the immunosuppressive drugs cyclosporin A and FK506. However, NF-AT has yet to be characterized biochemically. Here we show that the activity of NF-AT in electrophoretic gel mobility-shift assays is greatly enhanced by a distinct protein with an apparent molecular mass of 28 kDa. This modulator has been enriched from the flow-through of NF-AT affinity columns and enhances by severalfold the activity of the factor that elutes from these columns. The modulator has a predominantly nuclear location, but unlike NF-AT, the modulator is found in nuclear extracts of many cell types, even unstimulated T cells and T cells immunosuppressed with cyclosporin A or FK506. This modulator protein may regulate NF-AT binding activity posttranscriptionally, and it should prove useful in the isolation and biochemical characterization of NF-AT. PMID- 1722333 TI - Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide. AB - Galanin (Gal), a 29-amino acid C-terminally amidated neuropeptide, is widely distributed throughout the central and peripheral nervous system. The primary structures of rat and bovine Gals were derived from the cDNA sequences of their precursors. To elucidate the structure of human Gal (hGal), we extracted 280 postmortem pituitaries in trifluoroacetic acid and purified hGal binding activity, by three successive HPLC steps, to homogeneity based on a radioreceptor assay. The primary structure of hGal was determined by automatic Edman degradation to be Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu- Gly-Pro-His-Ala Val-Gly-Asn-His-Arg-Ser-Phe-Ser-Asp-Lys-Asn-Gly-Leu-Thr- Ser-COOH. The structure was confirmed by plasma desorption time-of-flight mass spectrometry, revealing a mass of 3156.1. Compared to the 29-residue porcine, rat, and bovine Gals, hGal uniquely comprises 30 amino acids possessing an additional nonamidated serine residue as C terminus. The nonamidated carboxylic group at the C terminus was proven by synthesis of amidated and nonamidated hGal and by mass spectrometry after selective methylation of all free carboxylic groups. Synthetic hGal possesses full biological activity on isolated rat fundus muscle strips. PMID- 1722334 TI - Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes. AB - Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucleases. The biological roles of these enzymes in metazoans have not been tested. We have cloned the human cDNA (APE) that encodes the main nuclear AP endonuclease. The predicted Ape protein, which contains likely nuclear transport signals, is a member of a family of DNA repair enzymes that includes two bacterial AP endonucleases (ExoA protein of Streptococcus pneumoniae and exonuclease III of Escherichia coli) and Rrp1 protein of Drosophila melanogaster. Purified Ape protein lacks the 3'-exonuclease activity against undamaged DNA that is found in the bacterial and Drosophila enzymes, but the lack of obvious amino acid changes to account for this difference suggests that the various enzyme functions evolved by fine tuning a conserved active site. Expression of the active human enzyme in AP endonuclease-deficient E. coli conferred significant resistance to killing by the DNA-alkylating agent methyl methanesulfonate. The APE cDNA provides a molecular tool for analyzing the role of this central enzyme in maintaining genetic stability in humans. PMID- 1722335 TI - Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex-peptide complexes. AB - Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) class II gene-linked autoimmune demyelinating disease of the central nervous system. To develop therapies that will specifically inactivate only the autoantigen-reactive T cells, mice were treated with soluble MHC class II molecules that had been complexed with encephalitogenic peptides. Intravenous injections of 300 micrograms of complexes consisting of encephalitogenic peptide 91-103 of myelin basic protein plus I-As protein on day 0, 4, and 7 were effective in preventing experimental allergic encephalomyelitis. Similarly, administration of 45 micrograms of I-As protein complexed to peptide 139-151 from proteolipoprotein on day 1, 4, and 7 prevented mortality and significantly reduced paralysis induced by immunization with the encephalitogenic proteolipoprotein peptide. Histological examination of sections of animal brains revealed that treatment with I-As protein plus myelin basic protein 91-103 peptide prevents the development of inflammatory lesions characteristic of experimental allergic encephalomyelitis. Thus, treatment with MHC-self-peptide complexes could serve as a highly specific therapeutic modality in treating autoimmune disease when the putative autoantigen and the MHC restricting elements are known. PMID- 1722336 TI - A gene expression screen. AB - A gene expression screen identifies mRNAs that differ in abundance between two mRNA mixtures by a subtractive hybridization method. The two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for polymerase chain reaction (PCR) amplification. The multiple cDNA fragments isolated from any given gene can be treated as alleles in a genetic screen. Probability analysis of the frequency with which multiple alleles are found provides an estimation of the total number of up- and down-regulated genes. We have applied this method to genes that are differentially expressed in amphibian tadpole tail tissue in the first 24 hr after thyroid hormone treatment, which ultimately induces tail resorption. We estimate that there are about 30 up regulated genes; 16 have been isolated. PMID- 1722337 TI - Expression cloning of a cDNA encoding the bovine histamine H1 receptor. AB - A functional cDNA clone for the histamine H1 receptor was isolated from a cDNA library of bovine adrenal medulla by a combination of molecular cloning in an expression vector and electrophysiological assay in Xenopus oocytes. The H1 receptor cDNA encodes a protein of 491 amino acids (Mr 55,954) with seven putative transmembrane domains, illustrating the similarity to other receptors that couple with guanine nucleotide-binding regulatory proteins (G protein coupled receptors). The sequence homology between the H1 and H2 receptors is not higher than that between the histamine H1 and m1-muscarinic receptors. The cloned receptor protein expressed in COS-7 cells bound specifically to [3H]mepyramine, an H1 receptor antagonist, and this binding was displaced by H1 receptor antagonists and histamine with affinities comparable with those in membranes of bovine adrenal medulla. H1 receptor mRNA was shown to be expressed in brain and in peripheral tissues, including lung, small intestine, and adrenal medulla. This investigation discloses the molecular nature of the H1 receptor--a receptor that mediates diverse neuronal and peripheral actions of histamine and that may be of therapeutic importance in allergy. PMID- 1722338 TI - A developmental switch in B lymphopoiesis. AB - B and T lymphocytes are generated from hematopoietic stem cells during both fetal and adult life. A critical unresolved issue is whether the differentiation pathways in lymphopoiesis are the same in fetal and adult animals or whether they differ, similar to the hemoglobin switch in erythropoiesis. We report here that a developmental switch occurs in B lymphopoiesis. We isolated "pro-B" cells (i.e., cells that have initiated, but not completed, heavy-chain gene rearrangement) from fetal and adult sources and investigated their B-cell progeny generated both in vitro and in vivo. Most of the cells from fetal liver, but few from adult bone marrow, expressed CD5. Further, fetal pro-B cells failed to generate cells expressing high levels of IgD in severe combined immunodeficiency mice, whereas adult pro-B cells gave rise to CD5-B cells bearing IgD at levels comparable to the bulk of cells in the spleen of adult mice. Thus, all committed B progenitors in fetal liver of day 16 gestation mice give rise to phenotypically distinct progeny when compared to cells at a comparable differentiation stage in the bone marrow of adult animals. We conclude that the cohort of B-lineage progenitors in early fetal development is committed to a differentiation pathway distinct from that seen in the adult. PMID- 1722339 TI - Regional brain glucose metabolism: correlations to biochemical measures and anxiety in patients with schizophrenia. AB - Regional brain glucose metabolism in 20 patients with schizophrenia (DSM-III) was investigated by positron emission tomography (PET) with uniformly labeled 11C glucose as the tracer. Monoamine metabolites were analyzed in cerebrospinal fluid (CSF) and serum, and prolactin was analyzed in serum. Intensity of anxiety was rated directly after the PET study. Ten healthy volunteers served as controls. In the patients, weak positive and negative relationships were found between homovanillic acid in CSF and prolactin in serum, respectively, and regional metabolic rates. In all subjects, positive correlations were found between the level of anxiety and the regional glucose metabolism. In the controls, positive correlations were found between anxiety and the frontal/parietal ratios of the left hemisphere, whereas anxiety scores of the patients correlated negatively to relative metabolic rates of the right medial frontal cortex and the left thalamus. These observations may indicate alterations in the neuronal systems participating in the initiation of anxiety and arousal in schizophrenia. PMID- 1722340 TI - Nurse fails to follow "doctor's policy": death results. Case in point: Hartman v. Riverside Methodist Hosp. (577 N.E.2d 112--OH (1989)). PMID- 1722341 TI - Serotonergic interhemispheric asymmetry: neurochemical and pharmaco-EEG evidence. AB - 1. Postmortem neurochemical investigations revealed interhemispheric asymmetry in the mediofrontal region of human brain. Significantly higher right hemisphere serotonin metabolite (5HIAA) content as well as increased maximal imipramine binding (IB) were found in the right hemisphere than in the left side. 2. IB did not show a gender difference in the mediofrontal area. However, women had higher IB in the right orbital frontal cortex than did men. 3. In vivo pharmaco-EEG results tend to support the postmortem neurochemical data. Intravenous chlorimipramine resulted in an asymmetric topographic distribution of the P300 auditory evoked potential, peak amplitudes were shifted to the right hemisphere. PMID- 1722342 TI - Effects of acute administration of SCH 23390 on dopamine and serotonin turnover in major dopaminergic areas and mesencephalic raphe nuclei--comparison with ritanserin. AB - 1. The effects of acute administration of SCH 23390 (0.05 and 0.25 mg/kg s.c.), a dopamine D-1 receptor antagonist having also a moderate serotonin-S2 (5-HT-2) receptor blocking activity, and ritanserin (0.5 mg/kg), a specific 5-HT-2 antagonist, on dopamine (DA) and serotonin (5-HT) turnover were investigated in dopaminergic (nucleus caudatus, nucleus accumbens, substantia nigra, A10 area) and serotonergic (nucleus raphe dorsalis and nucleus raphe medialis) rat brain nuclei. 2. Acute SCH 23390 (both doses) increased the metabolism of DA and tended to augment the rate of DA synthesis (accumulation of DOPA after inhibition of aromatic acid decarboxylase) in the nucleus accumbens, but not in the nucleus caudatus. In addition, SCH 23390 had a moderate effect on DA metabolism in substantia nigra. SCH 23390 did not alter the turnover of 5-HT in any of the nuclei studied. 3. Acute administration of ritanserin did not modify 5-HT or DA turnover in any of the nuclei studied. 4. In conclusion, these results suggest that acute SCH 23390 administration preferentially activates the mesolimbic DA system. The lack of effect of ritanserin on DA or 5-HT turnover in nigrostriatal and mesolimbic DAergic areas suggests that under basal conditions the blockade of 5-HT2 receptors do not change monoamine metabolism in these areas. The role of 5 HT-2 blockade in the actions of SCH 23390 on DA turnover appears thus to be of a minor importance. PMID- 1722343 TI - The response of the pineal melatonin biosynthesis to the selective MAO-A inhibitor, clorgyline, in young and middle-aged rats. AB - 1. Clorgyline increased pineal melatonin and N-acetylserotonin (NAS) and decreased 5-hydroxyindoleacetic acid (5-HIAA) content in 3 and 12 months of age male Sprague-Dawley rats kept under 12:12 h light: dark schedule. Exposure to light for 24 h before clorgyline administration resulted in additional elevation of NAS and melatonin. NAS and melatonin levels after clorgyline injections were significantly higher while 5-HIAA levels were significantly lower in young than in middle-aged rats. 2. The 5-HIAA/5-HT ratio (index of monoamine oxidase activity) was higher in middle-aged than in young rats suggesting the lesser degree of clorgyline-induced inhibition of MAO-A in old than in young rats. 3. It is suggested that melatonin response to a single dose of the selective MAO-A inhibitor might be used for the assessment of the aging changes of the rat (and human) pineals. PMID- 1722344 TI - A rapid method for the estimation of prostaglandin E2 in intestinal tissues using fluorescence derivatization. AB - A sensitive spectrofluorimetric method is described to determine small quantities of prostaglandin E2 in complex biological systems as intestinal tissues. The method is based on a solid phase extraction combined with a coupling with a fluorescent marker and measuring the derivatization product by fluorescence densitometry. After mixing the tissue with an ice-cold perchloric acid solution, adjusting the pH, centrifugation and filtration steps, the prostaglandins are retained on a solid phase extraction C18 disposable column. They are eluted with diethylether, derivatized with 4-bromomethyl-7-methoxy-coumarin using potassium carbonate as condensating agent and finally analysed using fluorescence densitometry on silica gel TLC plates. Applying this method, amounts down to 5 ng (per gram wet tissue) could be measured in intestinal tissues, the s.e.m. for replicated total analysis being less than 15%. The foregoing method is applied for the determination of PGE2 released in the intestinal wall under the influence of laxatives. PMID- 1722345 TI - [Combination therapies with anti-retroviral agents in HIV and AIDS infections]. PMID- 1722346 TI - Toxicity of chlorpyrifos in Nubian goats. AB - A single oral administration of chlorpyrifos to Nubian goats at 1200, 600 and 300 mg/kg caused nervous signs and death within 15 minutes to 2 days of treatment. An oral dose of the compound at 150 mg/kg was toxic, but not fatal to goats. Animals given chlorpyrifos orally at daily doses of 300, 150 and 75 mg/kg showed signs of toxicity and died within 2 to 7 days of treatment. Pathological, biochemical and haematological changes are described. PMID- 1722347 TI - Neuropeptides in temporomandibular joints with rheumatoid arthritis: a clinical study. AB - There is evidence that neuropeptides play a role in the development of arthritis. Synovial fluid from arthritic temporomandibular joints in patients with rheumatoid arthritis was therefore investigated for presence of the neuropeptides calcitonin gene-related peptide, substance P, neurokinin A and neuropeptide Y. All four peptides were found in the synovial fluid above plasma level, but calcitonin gene-related peptide showed the highest concentration and substance P the lowest. PMID- 1722348 TI - Antibodies to recombinant and synthetic peptides derived from the hepatitis C virus genome in long-term-studied patients with posttransfusion hepatitis C. AB - Eight of 13 Swedish patients (62%), studied prospectively, who developed posttransfusion non-A, non-B hepatitis (PT-NANBH) had earlier been found to seroconvert for antibodies to hepatitis C virus (anti-HCV) c100-3 in the first generation anti-HCV enzyme-linked immunosorbent assay 1-18 (mean, 8) weeks after onset of hepatitis. By using a second-generation test utilizing antigens encoded by the core NS3 and NS4 region of HCV, a further four patients non-reactive to c100-3 (NS4) were found to seroconvert. Thus 12 of 13 (92%) Swedish patients with PT-NANBH were shown to have HCV infection. In addition, the serologic reactivity for several individual synthetic peptides and/or recombinant HCV proteins was studied in seven anti-HCV c100-3 seroconverts studied long-term after onset of acute PT-HCV infection. No special patterns were found that could differentiate patients who recovered from those who developed chronic HCV infection. It was concluded that the addition of new recombinant antigens derived from the core and NS3 region to c100-3 (NS4) both improved the sensitivity of the anti-HCV test and shortened the window phase to seroconversion. PMID- 1722349 TI - Studies of isolated parietal and enterochromaffin-like cells from the rat. AB - Rat gastric mucosal cells isolated by enzyme dispersion were separated by elutriation centrifugation. The amount of histamine and the number of enterochromaffin-like (ECL) cells and parietal cells were determined in the crude mucosal cells and the various elutriation fractions. The mucosal cells contained 2.6% ECL and 20% parietal cells. Elutriation centrifugation resulted in good separation of parietal cells and ECL cells. Most of the ECL cells were elutriated in the small cell fractions. Scattered ECL cells were also present in the fraction enriched with parietal cells. Histamine and carbacholine stimulated aminopyrine uptake in a concentration-dependent manner with about the same efficacy, 5.6 times the base-line value. When combined with the phosphodiesterase inhibitor isobutyl methylxanthine, the maximal histamine stimulation was increased to 16.8 times the base-line value, and the sensitivity increased about 10-fold. Gastrin at high and unphysiologic concentrations stimulated only faintly the aminopyrine uptake in parietal cells and the histamine release from ECL cells. PMID- 1722350 TI - Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1 methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis. PMID- 1722351 TI - Long-term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR deficient rabbits. AB - Familial hypercholesterolemia (FH) is an inherited disorder in humans that is caused by a deficiency of low density lipoprotein receptors (LDLRs). An animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit, was used to develop an approach for liver-directed gene therapy based on transplantation of autologous hepatocytes that were genetically corrected ex vivo with recombinant retroviruses. Animals transplanted with LDLR-transduced autologous hepatocytes demonstrated a 30 to 50 percent decrease in total serum cholesterol that persisted for the duration of the experiment (122 days). Recombinant-derived LDLR RNA was harvested from tissues with no diminution for up to 6.5 months after transplantation. PMID- 1722352 TI - Reverse transcriptase encoded by a human transposable element. AB - L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition. A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases. The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity. This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D. Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements. PMID- 1722353 TI - [Surgical treatment of bleeding pyloroduodenal ulcers]. PMID- 1722354 TI - Confocal microscopy of genome exposure in normal, cancer, and reverse-transformed cells. AB - Genome exposure studies were carried out on malignant CHO-K1 and C6 rat glioma cells and their respective, phenotypically normal counterparts (reverse transformed CHO-K1, and both reverse-transformed C6 glioma and normal rat fibroblasts). Cells were subjected to the nick-translation technique previously developed to make visible the exposed (i.e., DNase I-sensitive) nuclear DNA, and examined by both epifluorescence and confocal microscopy. The confocal microscopy, by permitting examination of sections throughout the nucleus, made possible clearer identification of the regions of exposed and sequestered DNA in the cells studied. A peripheral shell of exposed DNA with some discontinuities was displayed in the great majority of the cells with normal phenotype, but in none of the cancer cells. Both types of cells displayed regions of exposed DNA in the nuclear interior, particularly surrounding the nucleoli. In accordance with previous theoretical proposals we postulate: the peripheral nuclear shell of exposed DNA contains differentiation-specific genes that include the specific growth-control genes and that are functional in normal cells but not in cancer; the exposed genes surrounding the nucleoli may represent housekeeping genes active in both normal and cancer cells; and the DNase I-resistant DNA in the interior of the nucleus we postulate to consist for the most part of genes specific to alternative differentiation states and to be sequestered and inactive. Previous differences in evaluation of roles of peripheral and internal DNA sensitivity to DNAse I hydrolysis appear to be reconciled by this formulation. Identification of exposed DNA may be useful in cancer diagnosis. PMID- 1722355 TI - Esophageal intubation for malignant fistulas. AB - Between April 1978 and December 1989 at the Endoscopy Division of the National Cancer Institute of Milan, 140 patients were intubated for esophageal neoplasms; 19 of these subjects underwent endoscopic intubation for malignant fistulas complicated by pneumonia and/or mediastinitis. The prostheses were tolerated well and enabled the restoration of oral nutrition. The mean survival was 4.7 months (range, 0.5-17 months). No major complications occurred. Tube dislodgement was observed in 2 cases (10.5%). Two patients died of causes that were not related to the procedure. PMID- 1722356 TI - Percutaneous stenting for malignant biliary stenosis. AB - Percutaneous stenting for malignant biliary stenosis is quite beneficial to patients with unresectable or recurrent disease, tremendously improving the quality of their lives. Percutaneous transhepatic biliary drainage (PTBD) was attempted in 92 patients with obstructive jaundice during the period between January 1986 and July 1989. Implantation of an endoprosthesis was performed in 14 cases (15.2%) and succeeded in 12 (85.7%). When a guide wire could not be passed distally across the stricture site, percutaneous transhepatic cholangioscopy (PTCS) through the dilated PTBD fistula was carried out to enable its passage. PTCS is also valuable in the preoperative diagnosis of obstructive jaundice. The patients who are not candidates for surgery are suitable for this procedure. A Miller double-mushroom stent is used as the endoprosthesis in the majority of cases. One patient with recurrent hepatoma has lived at home with this stent for greater than 3 years due to repeated transarterial embolization and chemotherapy and does not need to wash or change the stent. PMID- 1722357 TI - Behavior of alpha 2u-globulin accumulating in kidneys of male rats treated with d limonene: kidney-type alpha 2u-globulin in the urine as a marker of d-limonene nephropathy. AB - Effects of d-limonene on alpha 2u-globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d-limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type alpha 2u-globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum alpha 2u-globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating alpha 2u globulin levels were not affected by the d-limonene administration. While the molecular weight of the major alpha 2u-globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type-alpha 2u-globulin was observed in the urine from treated rats. The results were indicative of elimination of alpha 2u globulin from the kidney to urine, the appearance of kidney-type-alpha 2u globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the alpha 2u-globulin (kidney type) in the urine might be used as an indicator of chemically induced alpha 2u globulin nephropathy. PMID- 1722359 TI - Inductive events in the neural tube. PMID- 1722358 TI - Prion dimers: a deadly duo? PMID- 1722360 TI - Molecular analysis of neuronal physiology by gene transfer into neurons with herpes simplex virus vectors. AB - A genetic analysis of mammalian neuronal physiology might now be possible due to the development of defective herpes simplex virus vectors, which allow gene transfer directly into mature neurons, in culture or in the adult brain. Genetically altered proteins that play critical roles in neuronal physiology, including those responsible for the generation of action potentials, synthesis and release of neurotransmitters, and signal transduction enzymes, can now be stably expressed in neurons. The effect of such altered proteins on neuronal physiology can therefore be examined, using the tools of modern neuroscience. Genetic manipulation is biochemically specific and stable, and can be targeted both to a particular cell type and to a particular subregion of the cell to yield insights into the molecular basis for specific brain functions. PMID- 1722361 TI - The cognitive basis of a biological disorder: autism. AB - This article summarizes recent evidence indicating that individuals suffering from autism have a specific problem in understanding intentions and beliefs. We propose that this problem arises because they are incapable of forming a special kind of mental representation. A single cognitive deficit defines what is common to all autistic individuals. In contrast there is a wide range of proposals for the biological origins of the disorder. PMID- 1722362 TI - Quantal analysis and synaptic efficacy in the CNS. AB - Quantal analysis of synaptic transmission at connections between neurons in the CNS has provided insights concerning the structural constraints on transmitter release and postsynaptic responsiveness. However, it has proven difficult in many cases to resolve the size and variability of a single quantum or to distinguish clear peaks in amplitude histograms of evoked responses, due in part to the superposition of background instrumental and biological noise. These limitations raise questions about recent attempts to use direct or indirect methods of quantal analysis in order to distinguish between pre- and postsynaptic loci of the modifications underlying long-term potentiation, particularly since the interpretations are model-dependent and the statistical treatments and experimental techniques employed incorporate simplifying assumptions not yet proven. PMID- 1722363 TI - Neural models of stereoscopic vision. AB - Human stereopsis remains an enigma: how does the brain match features between the left and right eye images and compute disparity between these matched features? Developments in computational neuroscience and machine vision have led to several models of human stereopsis that provide insight into possible mechanisms underlying this phenomenon. These models, reviewed in this paper, adopt one of three general strategies. One class of models employs cooperative interactions, whereby a unique solution to the matching problem emerges from excitatory and inhibitory interactions among binocular neural elements. A second class of models implements matching and disparity computation serially over multiple spatial scales. A third class relies on local, non-interacting computations performed in parallel to overcome speed limitations inherent in the other models. Considered together, these theoretical developments offer fresh insights concerning the actual neural concomitants of binocular stereopsis. PMID- 1722364 TI - Autoimmunity to glutamic acid decarboxylase (GAD) in Stiff-Man syndrome and insulin-dependent diabetes mellitus. AB - Stiff-Man syndrome (SMS) is a disorder of the CNS, characterized by rigidity of the body musculature, which has been hypothesized to result from an impairment of GABAergic neurotransmission. GABA is the main inhibitory neurotransmitter of the brain. It is also a putative signal molecule in the pancreas, where it is produced by beta cells (insulin-secreting cells)--the autoimmune target in insulin-dependent diabetes mellitus (IDDM). Autoantibodies to the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) have been found in SMS and in IDDM. This review summarizes evidence suggesting that SMS may be an autoimmune disease and discusses the possible significance of the autoimmune response to GAD in SMS and IDDM. PMID- 1722365 TI - Glycine receptors: heterogeneous and widespread in the mammalian brain. AB - The amino acid glycine is an established inhibitory neurotransmitter in the spinal cord and brain stem. Its postsynaptic receptor has been purified, and cDNAs of the receptor subunits have been cloned and sequenced. Recent molecular studies indicate considerable diversity of the inhibitory glycine receptor (GlyR); this diversity stems from both the existence of several alpha-subunit genes encoding isoforms of distinct pharmacology, and alternative splicing of their primary transcripts. In situ hybridization studies reveal a widespread expression of GlyRs throughout the mammalian CNS, suggesting that glycinergic neurotransmission may be implicated in many higher brain functions. PMID- 1722366 TI - Non-synaptic mechanisms of Ca(2+)-mediated injury in CNS white matter. AB - Clinical deficits after injury to the CNS are due, in large part, to dysfunction of white matter (myelinated fiber tracts), including descending and ascending tracts in the spinal cord. A crucial set of questions, in the search for strategies that will preserve or restore function after CNS injury, centers on the pathophysiology of, and mechanisms underlying recovery of conduction in, CNS white matter. These questions are relevant both to spinal cord injury, and to brain infarction, which frequently affects white matter. PMID- 1722367 TI - Sexual differentiation of monoaminergic neurons--genetic or epigenetic? AB - It is currently believed that sexual differentiation of the brain is mediated entirely by the epigenetic action of gonadal steroids during a critical period of development. Ingrid Reisert and Christoph Pilgrim review sexual dimorphisms of monoaminergic systems, which also appear to be generated by sex steroids. However, there are a number of observations that are not explainable by the 'androgen theory of sexual differentiation'. Results obtained from cultures of embryonic rat brain tissue appear to indicate that dopaminergic neurons may develop morphological and functional sex differences in the absence of sex steroids. Hormone-independent and -dependent developmental processes may affect diencephalic and mesencephalic dopaminergic neurons in a regionally diverse fashion. Factors other than sex steroids need to be examined. It is possible that some sexual dimorphisms in the nervous system may develop under primary genetic control. PMID- 1722368 TI - Miltenberger class IX of the MNS blood group system. AB - Mi.IX is a new phenotype in the Miltenberger series of the MNS blood group system with a frequency of 0.43% in Denmark. Mi.IX red cells are Mur+ but do not express any of the other established Miltenberger determinants. They react with a new antibody, anti-DANE, which defines a determinant present on Mi.IX cells but not on cells of other Miltenberger phenotypes. Four Mi.IX propositi have been found. Their families show that MiIX is inherited with a MS complex (lod score 3.69 at theta = 0.00) which produces a trypsin-resistant M antigen. DANE has been allotted the ISBT number 002032 (MNS32). Serological and immunochemical studies with human and monoclonal antibodies to various determinants on glycophorin A (GPA) suggest that Mi.IX is associated with an aberrant GPA molecule that lacks the trypsin cleavage site at amino-acid residue 39, retains the chymotrypsin cleavage site at residue 34 and has an apparent Mr of about 1,000 less than normal GPA. It is proposed that this Mi.IX molecule has an amino acid and possibly also a glycosylation change in the region of amino-acid residues 35-39. PMID- 1722369 TI - Nomenclature for factors of the HLA system, 1990. PMID- 1722370 TI - Misperceptions and inadequate pain management in cancer patients. AB - This article examines misperceptions and barriers to adequate pain relief in cancer patients. Healthcare professionals have gaps in their knowledge of opioid drugs as well as misconceptions concerning tolerance, physical dependence, and addiction that often lead to the underprescribing of these agents. The pervasiveness of the "say no to drugs" message in our society and the fear of addiction on the part of patients and their families creates yet another barrier to the legitimate use of opioids to treat cancer pain. Legal and regulatory documents filled with arbitrary and ill-defined labels meant to promote the legitimate use of these drugs and curtail their misuse may instead intimidate healthcare professionals and negatively influence prescribing habits. Increased educational efforts for pharmacists and other healthcare professionals as well as the development of clinical role models and state cancer pain initiatives are cited as means to break down these barriers in order to achieve adequate pain relief for all cancer patients. PMID- 1722371 TI - Phenytoin-tizanidine interaction. PMID- 1722372 TI - [A comparison of the take of embryonic amygdala and visual cortex transplants in the rat brain in connection with the possible compensation of functional disorders]. AB - Transplantation of embryonic (E17-18) visual cortex and amygdala was performed into corresponding damaged areas of the adult rat brain. It was shown in Nissl and Golgi preparations (by comparing qualitative and quantitative findings) that 2-6 months after operation the grafts were successful in case of putting them into the corresponding brain areas (cortex to cortex, or amygdala to amygdala). Graft's integration resulted in a selective increase of dendrite length and ramification towards the area of graft-host interface both in amygdala and visual cortex grafts. In case of inadequate graft-host integration the stratification of the grafted visual cortex could be observed. The structural reorganization of grafted neurones is compared with physiological and behavioural findings during the recovery processes in damaged brain. PMID- 1722373 TI - [Serum acute phase proteins for determining disease activity of ulcerative colitis and Crohn disease]. AB - We studied the activity assessment of ulcerative colitis and Crohn's disease by 5 acute phase reactants: C-reactive protein (CRP), alpha 1-acid glycoprotein, alpha 1 antitrypsin, haptoglobin and fibrinogen. From a large register of patients with inflammatory bowel disease (IBD) we chose randomly 91 patients: 61 with ulcerative colitis and 30 with Crohn's disease. As a reference point in the disease activity assessment we used standard clinical indices. Statistical analysis was performed by non-parametric methods: the Kruskal-Wallis and Fisher's exact test. The disease activity assessment in patients with ulcerative colitis by the index according to Powell-Tuck indicated that the patients with active disease (N = 19) had significantly higher levels of all acute phase proteins mentioned above except fibrinogen (alpha less than 0.05 to 0.001) than patients in remission (N = 42). Analysis of the same data by Fisher's exact test indicated that there had been a probability for all the proteins measured to be higher than the normal values, particullary CRP (p less than 10(-8) and the other somewhat less. In patients with Crohn's disease, the disease activity assessment was performed by 2 indices. According to "The Crohn's Disease Activity Index" (CDAI), only alpha-1 acid glycoprotein and haptoglobin (alpha less than 0.05) were higher in patients with active disease (N = 4) than in patients with remission (N = 26).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722374 TI - Basophil histamine release in atopic patients after in vitro provocation with thiopental, Diprivan and chlormethiazole. AB - The degree of histamine release induced by three different anesthetic drugs was studied in vitro using basophil leukocytes from atopic patients (n = 11) and controls (n = 14). In all, eight dilutions (1/2 to 10(-5)) of Diprivan and its solvent Intralipid, thiopental and chlormethiazole in aqueous solution, were used. Histamine was released in four controls with weak dilutions (1/2 to 10(-5)) of Diprivan (n = 2) and thiopental (n = 2). The reaction with thiopental was greater than that with Diprivan. Five of the atopic subjects released histamine with one or more drug: thiopental and Diprivan four times each, Intralipid twice, and chlormethiazole once. Histamine release was greater in these patients than in controls, and occurred with dilutions ranging from 1/2 to 10(-2), except for one case. It is concluded that atopic patients release histamine with hypnotic anesthetic drugs more easily than normal subjects. In the clinical setting, where blood concentration of the drugs studied is equivalent to a dilution of less than 10(-3), they do not release much histamine. They may be used in atopic patients if the drugs are injected slowly. PMID- 1722375 TI - Effects of 1.5% glycine solution with and without 1% ethanol on the fluid balance in elderly men. AB - Ten male patients scheduled for transurethral prostatic resection (aged 57-79) were given irrigating fluid by intravenous infusion at 50 ml.min-1 over 20 min. Each patient was subjected to two infusions: 1.5% glycine in water on one occasion, and the same solution but with 1% ethanol added on the other. Urine and blood samples were collected at regular intervals for up to 2 h after infusion, and the changes in the distribution of water and electrolytes between fluid compartments were calculated. Transient prickling skin sensations were frequently reported effects of the infusions. Two patients experienced visual disturbances. There were no changes in the blood ammonia and plasma vasopressin levels. During the infusions, the estimated blood volume and the total plasma sodium and potassium content increased. The solutions produced osmotic diuresis with increased urinary excretion of water and electrolytes. After ending the fluid administration, blood volume was rapidly restored. Over the following 120 min the irrigant water was redistributed intracellularly or removed by urinary excretion. The addition of ethanol did not alter the overall effects of glycine solution on the fluid balance. PMID- 1722376 TI - Ventricular dysrhythmias following an alfentanil anesthetic in a patient on reserpine for hypertension. AB - The case is presented of a hypertensive patient on reserpine, who developed ventricular irritability after administration of alfentanil, a new ultra-short acting narcotic used in anesthesia. The dysrhythmias resolved after cessation of the offending agent. There are no reported cases of interaction of reserpine and alfentanil in the literature. PMID- 1722377 TI - Tissue kallikrein stimulates mucociliary activity in the rabbit maxillary sinus. AB - The in vivo effect of tissue kallikrein on the mucociliary activity in the rabbit maxillary sinus was investigated administering the substance (0.1-5 mU/kg) via a. maxillaris and recording the response with a non-invasive photoelectric technique. Tissue kallikrein accelerated mucociliary activity, with a maximum response for the dose 5 mU/kg (33.7 +/- 13.4% from basal levels, n = 5). The effect had a latency of abut 1 min, with a peak within 2-3 min after the beginning of the administration. The response to tissue kallikrein displayed tachyphylaxis with a second dose producing a weaker response. Pretreatment with the protease inhibitor aprotinin (10,000 KIU bolus/kg) inhibited the action of tissue kallikrein. Tissue kallikrein probably stimulates mucociliary activity by producing lysylbradykinin from kininogen. Bradykinin has in an earlier study been shown to stimulate mucociliary activity. PMID- 1722378 TI - Cytoarchitecture of cochlear nucleus in the chinchilla. AB - The morphology of the cochlear nucleus in the normal, adult chinchilla, as demonstrated by Nissl staining, was examined. The cytoarchitecture was determined from sections viewed at the light microscope level. The chinchilla cochlear nucleus was found to possess most of the features reported in other mammalian cochlear nuclei. It could easily be divided into dorsal and ventral components due to an intervening layer of granule cells, and most cell types previously reported in mammals were also found in the chinchilla cochlear nucleus. A distinct distribution pattern of cell types exists within each part. PMID- 1722379 TI - Changes in the testicular acid and alkaline phosphatase activities at different durations following prostatectomy: a correlative study with spermatogenesis. AB - The purpose of this investigation was to make a correlative study between spermatogenesis and testicular acid and alkaline phosphatase activities in mature prostatectomized animals at different post-operative periods. The results demonstrate that there was a significant augmentation in the activity of testicular acid and alkaline phosphatases subsequent to 14 and 21 days of prostatectomy. A parallel quantitative study of spermatogenesis at stage VII of the seminiferous cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7sd), revealed that there was a significant reduction in the number of step 7 spermatids after 14 and 21 days. No change was observed in the above testicular enzymes and spermatogenesis after 7 days of prostatectomy. Therefore, it is concluded that prostatectomy can alter the above testicular enzyme activities and spermatogenesis in chronic prostatectomized state. PMID- 1722380 TI - Effect of lorglumide (CR-1409) on pancreatic secretory and trophic response to caerulein in newborn rats. AB - The authors investigated whether lorglumide a specific CCK-receptor antagonist affects the pancreatic actions of caerulein in female newborn Wistar rats. Pancreatic secretory response (expressed as the decrease in specific trypsin activity in the pancreas) was studied in 11-day-old rats following acute administration of saline (control), caerulein (0.3, 1, or 3 micrograms/kg s.c.) either without or with lorglumide (10 mg/kg s.c.). Lorglumide was given 15 min before caerulein. In chronic studies rats were treated 3x/day for 10 days from the day of birth (Day 1) with caerulein and lorglumide as above. On Day 11 the rats were decapitated and exsanguinated, their pancreas removed and analyzed. Acute administration of caerulein induced a dose-dependent depletion of specific trypsin activity from the pancreas and this was antagonized by lorglumide. Chronic treatment with each dose of the peptide increased total pancreatic trypsin content. Besides, the 3 micrograms/kg dose caused to increase pancreatic protein, DNA, and amylase content and to increase plasma corticosterone level. Chronic administration of lorglumide did not influence normal pancreatic growth, while it strongly inhibited the increase in trypsin content evoked by caerulein. However, lorglumide, given alone or in combination with caerulein, induced a significant increase in pancreatic amylase content without affecting plasma corticosterone level. PMID- 1722381 TI - Immunocytochemical localization of interferons in human trophoblast populations. AB - It is known that Interferon (IFN) is present in normal body fluids and tissues during pregnancy. Using an immunohistochemical technique and a panel of monoclonal antibodies we have localized IFN-alpha, -beta and -gamma directly on formalin-fixed paraffin-embedded normal human placentae at different stages of pregnancy and in the hydatidiform mole. The results show that IFNs is mostly localized in villous syncytiotrophoblast and in extravillous interstitial trophoblast. No reactivity was observed in villous cytotrophoblast or in cytotrophoblast cell columns. The most intense staining was observed for IFN alpha and -beta, while IFN-gamma was rather weak. There is then a gradual diminution in IFN reactivity with increasing gestation age being almost imperceptible at term. These results suggest that IFN may deploy antiviral, immunomodulator and differentiation activities during normal human pregnancy. PMID- 1722382 TI - Calcium determination in primary hyperparathyroidism. AB - Calcium is the most closely controlled substance in the blood. The biologic variation of total calcium is approximately 2% and of the biologically active free (ionized, ionic) calcium only 1%. Thus, the monitoring of calcium in blood requires analytic procedures of high precision and accuracy. For patients with asymptomatic primary hyperparathyroidism, calcium monitoring involves the measurement of total calcium and free calcium. This review first considers the measurement of total calcium and then free calcium. PMID- 1722383 TI - Immunoassays for parathyroid hormone 1-84 in the diagnosis of hyperparathyroidism. AB - The two most frequent causes for hypercalcemia are primary hyperparathyroidism and hypercalcemia associated with malignancy. Elevated or inappropriately high PTH serum levels are the hallmark of hyperparathyroidism. Sensitive immunometric assays for the secreted, biologically active, intact parathyroid hormone molecule, PTH-(1-84), employ two populations of region-specific antibodies, take advantage of saturation kinetics rather than competitive binding, and have many technical advantages over conventional radioimmunoassay. Approximately 90% of patients with primary hyperparathyroidism have elevated serum levels of PTH-(1 84) by immunometric assay; the remainder have inappropriately elevated values of PTH for the serum calcium concentration. Clinical correlation studies comparing measurements of PTH using antisera that recognize the carboxyl, midregion, or amino terminus of PTH with PTH levels determined by immunometric assays demonstrate elevated values in equivalent numbers of hyperparathyroid individuals. Immunometric assays for PTH-(1-84) have their greatest value in separating patients with hyperparathyroidism from those with hypercalcemia of malignancy. In earlier studies using region-specific antisera, there was virtually always an overlap of serum PTH levels in hyperparathyroidism and hypercalcemia associated with malignancy. In contrast, analysis of results using PTH-(1-84) immunometric assays in several hundred reported patients shows a complete separation of PTH values. Clinical judgment, combined with measurement of PTH in the setting of hypercalcemia, can lead to the diagnosis of hyperparathyroidism with confidence in essentially all patients. PMID- 1722384 TI - MR findings in infantile Refsum disease: case report of two family members. PMID- 1722385 TI - Infantile Refsum disease. PMID- 1722386 TI - Confirmation of the assignment of the vitronectin (VNRA) and fibronectin (FNRA) receptor alpha-subunits. AB - The receptors for the extracellular structural components fibronectin and vitronectin each consist of two subunits called alpha and beta. Using human rodent somatic cell hybrids and cDNA probes corresponding to the alpha-subunits, we have confirmed the mapping of the fibronectin receptor (FNRA) to chromosome 12 and the vitronectin receptor (VNRA) to chromosome 2. PMID- 1722387 TI - Effects of gangliosides on the expression of autoimmune demyelination in the peripheral nervous system. AB - To test whether gangliosides (GA) might exert neuritogenic effects in vivo, experimental allergic neuritis (EAN) was studied clinically, neuropathologically, and immunologically in Lewis rats immunized with bovine peripheral nerve, P2 myelin protein, P2 myelin protein plus two different doses of GA, P2 with galactocerebroside (GC), and GA alone, each emulsified in adjuvant. All except the GA-treated group developed signs of EAN between days 11 and 14 after the injection. Rats immunized with P2 alone were the most severely affected. Rats given P2 plus GA and those given P2 plus GC displayed a significantly lower clinical score. Histological analysis revealed a comparable degree of inflammation of the peripheral nervous system and demyelination in the spinal nerve roots of bovine peripheral nerve- and P2-immunized rats. The P2 plus GA and P2 plus GC groups revealed similar degrees of pathology in the spinal nerve roots but the latter group stood apart from the rest in that it showed widespread peripheral nervous system changes extending distally into the sciatic nerve. Serological analysis demonstrated that P2 and GC, but not GA, elicited antibody (IgG) responses, but there was no correlation between antibody titer and clinical or histological involvement. The present data fail to support an enhancing role for gangliosides in the expression of EAN and, by extrapolation, in the Guillain Barre syndrome, for which EAN serves as the laboratory model, and in which suggestions have been made that antibodies to GA may have pathogenetic significance. PMID- 1722388 TI - Anti-tumor necrosis factor therapy abrogates autoimmune demyelination. AB - To define a role for the cytokine tumor necrosis factor (TNF) in immune-mediated demyelination, the effect of anti-TNF antibody was investigated with a form of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice induced by the adoptive transfer of myelin basic protein-(MBP)-sensitized T lymphocytes, an animal model of the human disease multiple sclerosis (MS). In three separate experiments, no mouse sensitized for EAE and then treated with anti-TNF by intraperitoneal injection developed signs of central nervous system (CNS) disease. Examination of CNS tissue from anti-TNF-treated animals showed no pathological changes. CNS tissue from control animals demonstrated extensive inflammatory cell infiltration and demyelination. To test whether anti-TNF therapy was inhibitory to encephalitogenic cells, preincubation of MBP-sensitized T lymphocytes with anti-TNF in vitro prior to injection into recipient mice was performed, and resulted in no diminution of their ability to transfer EAE. In addition, spleen cells from anti-TNF-treated mice were capable of serial transfer of EAE, similar to spleen cells from control animals. However, spleen cells from anti-TNF-treated mice did not produce TNF on stimulation with MBP or concanavalin A. This study showed that anti-TNF antibody can inhibit effectively the development of EAE by interfering with the effector, rather than the induction, phase of the disease. Anticytokine therapy may have important applications in the development of new therapeutic strategies for MS. PMID- 1722389 TI - [Determination of nasal transepithelial potential difference (DDPTE) in cystic fibrosis. Analysis of a simplified measurement technique]. AB - Measurements of nasal transepithelial potential differences (TEPD) were performed in 77 patients in order to assess a routine simplified method of recording. TEPD assays were performed in 34 patients with cystic fibrosis aged 1 month to 25 years, in 22 children with another chronic respiratory illness and in 21 subjects without any bronchopulmonary impairment. In the cystic fibrosis group TEPD values (mean +/- SD) were significantly higher (-49.077 +/- 9.38 mV) than in patients with chronic respiratory illnesses (-20.590 +/- 5.011 mV) or in subjects without bronchopulmonary impairment (-19.857 +/- 5.033 mV) (p less than 0.0001). Measurements could not be performed in 10 patients due to major nasal inflammation. The excellent specificity (100%) and sensitivity (93%) of the method confirm its diagnostic value. It may be used from the neonatal period and may represent an alternative to the sweat test, especially in dubious cases. PMID- 1722390 TI - Rice protein 16KD--a major allergen in rice grain extract. AB - The allergenic activity of Rice protein 16 KD (RP16KD) isolated from water soluble rice proteins was examined by radioallergosorbent test (RAST), RAST inhibition and histamine release assay. All of the 31 sera which showed positive RAST values for rice grain extract were positive for RP16KD RAST. Furthermore, there was a significant correlation (r = 0.56, p less than 0.01) between these RAST values. PR16KD effectively inhibited IgE binding to the rice grain extract disc in RAST inhibition assays using 4 sera with positive RAST values for both antigens. In 17 subjects with positive RAST values for rice grain extract, a significant positive correlation (r = 0.53, p less than 0.05) was found between the maximum percent histamine releases from their leukocytes by rice grain extract and RP16KD. These data strongly suggest that RP16KD is one of the major allergens of rice grain. PMID- 1722391 TI - Electrophysiological responses to non-electrolytes in lingual nerve of rat and in lingual epithelia of dog. AB - Epithelial and neural mechanisms underlying the trigeminal chemoreception of non electrolytes were investigated in whole-nerve recordings from lingual nerve and in Ussing-chamber studies of isolated lingual epithelia. The non-electrolytes included menthol, amyl acetate, phenethyl alcohol, toluene, methanol, ethanol, propanol, butanol, hexanol and octanol. They produced different lingual nerve responses: methanol and ethanol only increased ongoing activity; longer-chain alcohols initially increased but then suppressed activity below baseline; phenethyl alcohol and toluene only suppressed activity. Their threshold concentrations for lingual nerve responses, with the exception of menthol, were proportional to the octanol:water partition coefficients of the stimuli. The threshold concentration for menthol was significantly lower than predicted by this coefficient. Calculation of the free energy of transfer from the threshold concentrations for the n-alcohols suggests that they undergo partition into a hydrophobic environment such as is found in lipid bilayers. Lanthanum chloride, which inhibited lingual nerve responses to hydrophilic compounds, presumably by blocking their diffusion across tight junctions, did not inhibit responses to these non-electrolytes. At high concentrations, hexanol acted as an anaesthetic in that the lingual nerve no longer responded to thermal and chemical stimuli whereas ethanol, which only increased lingual nerve activity, did not inhibit those responses. Epithelial transport, as indicated by the short-circuit current (Isc) measured across tongues bathed in symmetrical solutions of Krebs-Henseleit buffer, was reversibly inhibited by ethanol, hexanol, octanol, phenyl ethanol and menthol. The stimulus concentration necessary to inhibit 50% of the Isc decreased with increasing octanol:water partition coefficient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722392 TI - Haemangiopericytoma--Queensland Radium Institute experience and a review of the literature. AB - We report the presenting clinical characteristics, management, relapse patterns and survival of 17 patients with haemangiopericytoma treated at the Queensland Radium Institute, Australia from 1962 to 1989. Twelve patients were referred at the time of first diagnosis and were treated with curative intent. Three patients were treated with palliative intent when referred following initial diagnosis, and the remaining two patients were referred at the time of relapse. Disease was metastatic at presentation in 4 patients. Radiotherapy was used as a component of primary treatment of disease in 11 patients, in both patients referred for management of local relapse of haemangiopericytoma, and for palliation of metastatic disease developing in 3 patients. One patient received chemotherapy as part of initial treatment. Nine patients have died with survival from first treatment ranging from 3 to 139 months. All 8 surviving patients remain free of disease at 6 to 94 months from first treatment. PMID- 1722393 TI - Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase. AB - Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396 400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3 kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo. PMID- 1722394 TI - Prostaglandin-concentration-dependent desensitization of adenylate cyclase in human erythroleukaemia (HEL) cells is abolished by pertussis toxin and enhanced by induction by dimethyl sulphoxide. AB - Prostaglandin-regulated cyclic AMP metabolism in human erythroleukaemia (HEL) cells was similar to that previously described in platelets [Ashby (1989) Mol. Pharmacol. 36, 866-873], displaying prostaglandin-concentration-dependent desensitization that could be explained by the presence of separate stimulatory and inhibitory prostaglandin receptors. Pertussis toxin abolished prostaglandin concentration-dependent desensitization, indicating that the process is mediated through a pertussis toxin-sensitive GTP-binding protein. Treatment of HEL cells for 4 days with the inducer dimethyl sulphoxide enhanced prostaglandin concentration-dependent desensitization, but did not alter the initial rate of cyclic AMP synthesis or the amount of Gi2 alpha measured by immunoblotting, suggesting that the inhibitory receptor was selectively induced by changing the cells to a more platelet-like form. PMID- 1722395 TI - Eponemycin, a novel antibiotic, is a highly powerful angiogenesis inhibitor. AB - Eponemycin, a novel antibiotic, was examined as to its anti-angiogenic activity in an in vivo assay system involving chorioallantoic membranes (CAMs) of growing chick embryos. Eponemycin powerfully inhibited angiogenesis in the CAMs. This powerful inhibition was dose-dependent, the inhibitory activity becoming detectable at a dose of 7.5 fmol/egg and the ID50 value being 250 fmol/egg, suggesting that eponemycin exhibits more potent anti-angiogenic activity than Ch 55, a synthetic retinoid, which had been the strongest angiogenesis inhibitor identified so far. To determine which event(s) in the angiogenesis process was affected by eponemycin, experiments were conducted using systems involving cultured vascular endothelial cells. Eponemycin effectively inhibited both the proliferation and migration of endothelial cells, indicating that the antibiotic affected these two important events during angiogenesis, resulting in effective inhibition of angiogenesis. These results strongly suggest that eponemycin could be a promising candidate as an angiogenesis inhibitor for the control of aberrant angiogenesis occurring in different diseases such as tumor development and diabetic retinopathy. PMID- 1722396 TI - Growth signal erythropoietin activates the same tyrosine kinases as interleukin 3, but activates only one tyrosine kinase as differentiation signal. AB - By Western blotting with anti-phosphotyrosine-specific antibody, we demonstrated that both erythropoietin (Epo) and interleukin 3 (IL3) induce rapid and transient tyrosine phosphorylation of a common set of proteins of 45, 55, 69, 87, 90, 95 and 160 KDa as a growth signal in Epo- and IL3-dependent FD-M6 cells. In contrast, only two proteins of 87 and 90 KDa were transiently phosphorylated in Epo-induced erythroid differentiation of SKT6 cells. Furthermore, no tyrosine phosphorylation was observed in dimethyl sulfoxide-induced differentiation of SKT6 cells. Taken together with other observations, these results indicate that Epo, IL3 and GM-CSF activate the same tyrosine protein kinases as growth signal and that Epo-induced differentiation signal uses only a part of the tyrosine kinase pathway. PMID- 1722397 TI - Diabetes and glucose transporter gene expression in rat small intestine. AB - The expressions of Na(+)-dependent glucose transporter (SGLT1) and five facilitative glucose transporter genes (GLUT1-5) in the small intestine of streptozotocin (STZ)-induced diabetic rats were examined by RNA blotting analysis. The transcripts of SGLT1 mRNA gave bands of 4.5 Kilobases (Kb) and 2.8Kb (very faint band). The levels of SGLT1 mRNA were significantly increased in 30- and 60-day STZ rats, but not changed in acute diabetic rats (2- to 10- day STZ rats). The GLUT2 mRNA levels changed in parallel with the D-galactose transport activity, being increased about 4-fold in 5-day STZ rats. The transcripts of GLUT5 mRNA gave three bands of 5.1Kb, 2.8Kb and 2.OKb, whose levels were significantly reduced in 30- and 60-day STZ rats. These results suggest that the facilitative glucose transporter (GLUT2), in addition to the Na(+)-dependent glucose transporter (SGLT1), may play an important role in intestinal glucose transport in diabetic rats. PMID- 1722398 TI - Identification and NH2-terminal amino acid sequence of three insulin-like growth factor-binding proteins in porcine serum. AB - Three distinct species of IGFBP in porcine serum were identified by NH2-terminal amino acid sequence analysis. The IGFBPs identified include pIGFBP-2 (34 kDa), three isoforms of pIGFBP-3 (43, 40 and 30 kDa) and two isoforms of pIGFBP-4 (30 and 26 kDa). The three isoforms of pIGFBP-3 were found to have a common NH2 terminal amino acid sequence, as were the two isoforms of pIGFBP-4. These results indicate that porcine serum contains a truncated form of IGFBP-3 and two forms of pIGFBP-4, similar to those previously isolated from human and rat serum. Furthermore, the presence of a truncated form(s) of the GH-dependent IGFBP-3 in porcine serum suggests that elucidating its origin and function may be important in understanding how IGFBPs affect the somatogenic actions of GH. PMID- 1722399 TI - Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin. AB - We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC. PMID- 1722400 TI - Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Le(a) and sialosyl-Le(x), and sulfated glycans modulate this binding. AB - GMP-140 (CD62; PADGEM) is a member of the selectin family expressed highly at the surface of platelets and endothelial cells by agonists such as thrombin or phorbol esters. Previous studies indicate that the lectin domain of GMP-140 recognizes sialosyl-Le(x) (SLex) and to a lesser extent Le(x) (Polley MJ, et al., Proc Natl Acad Sci USA 88:6224, 1991). We now report that GMP-140 binds to sialosyl Lea (SLea) and to SLex, and that degree of binding to SLea is greater than that to SLex under our experimental conditions. Binding of activated platelets to SLea or SLex was inhibited to various degrees in the presence of sulfated glycans, suggesting that sulfated glycans induce conformational change in the lectin domain of GMP-140 and modulates its binding affinity to SLea and SLex. PMID- 1722401 TI - Effect of NCDC, a protease inhibitor, on histamine release from rat peritoneal mast cells induced by anti-IgE. AB - NCDC dose-dependently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE. Moreover, NCDC inhibited Ca(2+)-mobilization from intracellular Ca(2+)-stores as well as histamine release in mast cells activated by anti IgE, the effect on both of these phenomena being closely correlated. Anti IgE induced a rapid increase in IP3 production from phosphoinositides in mast cells, with its production in 15 sec, followed to baseline levels within 1 min. Anti-IgE stimulated PLC activity on mast cells membrane preparation. NCDC dose dependently inhibited the generation of IP3. These results suggest that the inhibitory effect of NCDC on the release of histamine induced by anti-IgE is due to, in part at least, the inhibition of PI-specific PLC and that the inhibitory effects of NCDC are involved in intracellular calcium store. PMID- 1722402 TI - Purification and characterization of a human NO synthase. AB - A NO synthase (NOS, EC 1.14.23) was isolated from human cerebellum by two sequential chromatography steps, that is affinity chromatography on 2'5'ADP sepharose and size exclusion chromatography on Superose 6. Human NOS migrated as a single band of 160 kDa on SDS/PAGE. The enzyme was Ca2+/calmodulin-regulated and NADPH/tetrahydrobiopterin (BH4)-dependent, which are characteristics of a type I NOS previously isolated from rat cerebellum. Antisera raised against purified rat cerebellar NOS crossreacted specifically with a 160 kDa protein in crude supernatant fraction of human cerebellum and purified human NOS but not in crude supernatant fraction of the temporal lobe. These findings provide evidence that nitrinergic signal transduction through conversion of L-arginine to L citrulline and NO does also occur in humans and NO may function as a neurotransmitter in the human central nervous system. PMID- 1722403 TI - Enhanced lipolysis in 3T3-L1 adipocytes following prolonged exposure to tolbutamide. AB - The effect of tolbutamide on lipolysis was examined in 3T3-L1 adipocytes. Whereas lipolysis was reversibly inhibited by tolbutamide, prolonged treatment with this agent dose-dependently increased both basal and isoproterenol-stimulated lipolysis in washed adipocytes. The latter effect of tolbutamide was not accompanied with altered cAMP levels in the cells and was abolished in the presence of cycloheximide. Moreover, the lipolytic responses induced by isobutylmethylxanthine, forskolin and dibutyryl cAMP were also augmented by prolonged treatment of adipocytes with tolbutamide. Thus, it appears that development of enhanced lipolysis in 3T3-L1 adipocytes following prolonged exposure to tolbutamide requires continuous protein synthesis and probably involves a step distal to cAMP production. PMID- 1722404 TI - Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes. AB - We report the cloning and the deduced amino acid sequence of cDNAs encoding both the human serotonin 5-HT2 and 5-HT1C receptors. The human 5-HT2 and 5-HT1C receptors shared 87% and 90% amino acid homology, respectively, with their rat counterparts. The most divergent regions of the 5-HT2 receptor between human and rat were the N-terminal extracellular domain (75% homology) and the C-terminal intracellular domain (67% homology between amino acids 426-474). The greatest variability between the human and rat 5-HT1C receptors were at the N-terminal extracellular domain (78% homology) and the third cytoplasmic loop (71% homology). The availability of the cloned human 5-HT2 and 5-HT1C receptors will help facilitate the further understanding of the molecular pharmacology and physiology of these receptors. PMID- 1722405 TI - Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase. AB - Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase. PMID- 1722406 TI - Dexamethasone prevents the growth inhibitory effects of recombinant tumor necrosis factor in a rat hepatoma cell line Reuber-RC-3: an association with the changes in the messenger RNA levels for multidrug resistance gene. AB - Two days exposure to recombinant tumor necrosis factor (rTNF-alpha) produced a dose-dependent reduction in (methyl-3H) thymidine incorporation in RC-3 cells (ID50 = 25 units/ml). Prolonged treatment with rTNF-alpha further resulted in a significant reduction in colony formation (ID50 = 200 units/ml), which was reversed upon removal of the agent. Interferon levels were undetectable in the supernatants of the rTNF-alpha treated cells. Simultaneous exposure to dexamethasone prevented the growth inhibition in rTNF-alpha-treated RC-3 cells. Significant dose-dependent increase in the steady state levels of the mRNA for multidrug resistance (MDR1) gene was observed after rTNF-alpha treatment while simultaneous exposure to dexamethasone produced a substantial reduction in the mRNA levels for MDR1 gene. These data suggest that growth inhibitory effects of TNF are regulated by dexamethasone and are associated with changes in MDR1 mRNA levels in hepatoma-derived cells. PMID- 1722407 TI - Human PAF receptor gene expression: induction during HL-60 cell differentiation. AB - Platelet-activating factor is a potent lipid mediator of inflammation and immune regulation. Its numerous biological activities are mediated through specific receptors on the plasma membranes of responsive cells. The expression of such receptors may be modulated by various agents, including those responsible for cell differentiation. Here, we demonstrate that differentiation of the human promyelocytic leukemia cell line HL-60 by 1 alpha,25(OH)2 vitamin D3 towards the macrophage phenotype is associated with induction of PAF receptor gene expression: PAF receptor mRNA accumulation correlates with the induction and development of specific PAF responsiveness as assayed by [Ca2+]i fluxes. Our studies suggest that PAF responsiveness parallels macrophage differentiation and that PAF receptor expression can be regulated at the transcriptional level. PMID- 1722408 TI - Constitutive expression of c-jun and jun-B in cell lines infected with human T lymphotropic virus types I and II. AB - To better understand the transcriptional regulation of human T-lymphotropic viruses, expression of the nuclear proto-oncogenes, jun-B and c-jun were examined in cell lines infected with HTLV-I/II. Constitutive high levels of jun-B and c jun expression were observed in HTLV-I (MT-2, Hut-102, IR, FS, SP) and HTLV-II infected cell lines (Mo-T, PAN). In contrast, the uninfected cell lines (Jurkat, Hut-78) expressed only basal levels of jun. This expression of jun was not dependent upon IL-2, as both IL-2 dependent (IR, FS, SP, and Pan) and IL-2 independent (MT-2, Hut-102, Mo-T) cell lines constitutively expressed transcripts for jun-B and c-jun. These data demonstrate that deregulated expression of nuclear protooncogenes such as jun may lead to cellular proliferation and the protein products of these nuclear oncogenes may potentially serve as transcriptional activators of HTLV-LTR by complexing with other nuclear proteins. PMID- 1722409 TI - Loss of murine tumor thymidine kinase activity in vivo following 5-fluorouracil (FUra) treatment by incorporation of FUra into RNA. AB - The effects of 5-fluorouracil (FUra) treatment on thymidine kinase (TKase) activity were examined in vivo in CD8F1 mice bearing first generation CD8F1 mouse mammary tumors. TKase activity was not affected by low dose FUra25 (25 mg/kg), a dose which substantially inhibited thymidylate synthase (TSase), but was severely inhibited 24 hr following treatment with FUra100, a weekly maximally tolerated dose, as judged by activity measurements and labeling of DNA with [3H]thymidine. The amount of (FU)RNA was increased markedly with increasing FUra dose from 0.4 nmol/mg DNA at FUra25 to 2.2 nmol/mg DNA at FUra100. At FUra100, TKase activity gradually declined over 24 hr to less than 10% of the control value, remained low for a further 48 hr, and then was gradually restored to control levels by 168 hr. The loss of TKase activity followed the incorporation of FUra into RNA which peaked at 4-5 hr. TKase activity was not restored by removal of endogenous inhibitors but was restored by treatment with uridine. TKase activity was not inhibited by therapeutic levels of methotrexate (300 mg/kg). TKase from murine colon 38 carcinoma was also severely inhibited, but the activity from colon 26 was only partially (50%) inhibited. Ornithine decarboxylase was also inhibited by FUra100 treatment in the CD8F1 tumor. These results demonstrate that certain short-lived, proliferation-related enzymes are affected by FUra doses higher than those required for TSase inhibition, and this effect appears to correlate with incorporation of FUra into RNA. Thus, in some tumors high doses of FUra can inhibit salvage as well as de novo synthesis of thymidylate providing an increased block of DNA synthesis and increased therapeutic advantage. PMID- 1722410 TI - Enhancement of etoposide and methotrexate sensitivity by indomethacin in vitro. AB - The possibility of increasing the activity of etoposide (VP-16) by combining this anti-cancer agent with indomethacin (Indo) was investigated by treating murine and human cultured tumor cells with a combination of Indo and VP-16 and quantitating VP-16 cytotoxicity by the [3H]thymidine incorporation assay. Non toxic concentrations of Indo were found to enhance the sensitivity to VP-16 in cultured Lewis lung carcinoma (LLC), YAC-1, P815, CCRF-CEM and K562 cells which were all relatively sensitive to VP-16. With the LLC, the Indo effect was dose dependent and near maximal at an Indo concentration of 0.5 micrograms/ml. Indo also increased the response of LLC cells to methotrexate, but not to bleomycin. Ibuprofen was less effective than Indo in enhancing VP-16 sensitivity in LLC cells. The enhanced sensitivity of VP-16 by Indo was not reversed by the prostaglandins PGE2 and PGD2, the analogs carbocyclic thromboxane A2 and carba prostacyclin or conditioned medium removed after 24 h or 48 h of culture from near confluent LLC cell monolayers. This finding suggests that Indo is not augmenting VP-16 cytotoxicity by inhibiting cyclo-oxygenase activity and prostaglandin production. The lipoxygenase inhibitor, eicosatetraynoic acid (ETYA), was also ineffective in reversing the Indo augmentation of VP-16 sensitivity. This finding indicates that Indo is not acting by inhibiting cyclo oxygenase and converting larger amounts of arachidonic acid to lipoxygenase products, such as leukotrienes, that could then interact with VP-16 to increase its sensitivity. In other studies, Indo was found to significantly increase the steady state accumulation of [3H]-VP-16 in all five cell lines studied. With the LLC cells, this increased steady state was achieved within 15 min after the addition of Indo to these cells and this enhanced VP-16 uptake was not reversed by the addition of prostaglandin E2 or prostaglandin D2. Thus, taken together, these studies indicate that Indo most likely enhances the cytotoxicity of VP-16 by increasing the cellular accumulation of VP-16. This newly identified function of Indo may be of potential clinical significance in the treatment of cancers in man. PMID- 1722411 TI - Heat shock protects cultured neurons from glutamate toxicity. AB - Expression of heat shock proteins (HSPs) occurs in brain after ischemia and status epilepticus. We report that induction of the heat shock response in cortical cultures protects neurons from glutamate-induced excitotoxicity. Cultures heated to 42.2 degrees C for 20 min showed an overall decrease in protein synthesis but an increase in the synthesis of approximately 72 and approximately 85 kd proteins and in the levels of HSP70 mRNA. Heat shock inhibited excitotoxicity in cells exposed to glutamate at 3 or 24 hr following heat exposure, but not when the interval between heat and glutamate exposure was shortened to 15 min or lengthened to 48 hr. Protection due to heat shock required new protein synthesis, since it did not occur when protein or RNA synthesis inhibitors were added. By ameliorating excitotoxic processes, HSPs may attenuate brain injury in certain pathologic conditions. PMID- 1722412 TI - Single-channel currents underlying glycinergic inhibitory postsynaptic responses in spinal neurons. AB - Single-channel properties of glycine receptors have been characterized so far only in cultured neurons. To characterize the glycine receptor channels in situ, we applied the patch-clamp technique to spinal neurons in slice preparations. Glycine-gated, single-channel currents were recorded in outside-out patches excised from spinal neurons. In the falling phase of glycinergic inhibitory synaptic currents, single-channel currents were resolved as discrete steps. In both cases, the glycine-gated channels showed similar multiple conductance levels. These results suggest that the receptor channel properties are indistinguishable in the synaptic and extrasynaptic sites. We conclude that multiple conductance states of a receptor channel are the native feature of the glycine receptor in situ. PMID- 1722413 TI - Down-regulation of myelin-associated glycoprotein on Schwann cells by interferon gamma and tumor necrosis factor-alpha affects neurite outgrowth. AB - To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF-alpha) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-gamma and TNF-alpha synergistically inhibited the neurite outgrowth promoting properties of the Schwann cells by specifically down-regulating myelin associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAg inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by which inflammatory cytokines interfere with Schwann cell-neuron interactions. PMID- 1722414 TI - Mediator secretion from human skin mast cells provoked by immunological and non immunological stimulation. AB - Mast cells of the human skin not only release mediators following immunological activation, but may also be stimulated to release histamine by the neuropeptides substance P, vasoactive intestinal polypeptide and somatostatin or by other basic secretagogues such as morphine, poly-L-lysine and compound 48/80. Release of histamine under these conditions is rapid and accompanied by minimal generation of the eicosanoids, prostaglandin (PG)D2 and leukotriene (LT)C4. Transient elevations of intracellular calcium are associated with mediator secretion induced by both stimuli, that induced by anti-IgE being derived from extracellular sources through channels in the plasma membrane while that stimulated by neuropeptides is mobilized intracellularly. Similarly, elevations of intracellular cyclic adenosine monophosphate (AMP) induced by anti-IgE occur only in the presence of extracellular calcium whereas with substance P elevations are apparent even in the absence of extracellular calcium. With the latter stimulus, histamine release is complete before the peak cyclic AMP is achieved. Histamine release stimulated by both secretagogues is unaffected by sodium cromoglycate or nedocromil sodium but is reduced by both salbutamol and isobutylmethylxanthine. Despite these biochemical and temporal differences, degranulation induced by both secretagogues proceeds by compound exocytosis which is indistinguishable under the electron microscope. PMID- 1722415 TI - Role of cytoskeletons on Ca2+ release from the intracellular Ca store of rat peritoneal mast cells. AB - Cytochalasin D, colchicine or vinblastine effectively inhibited both histamine release and 45Ca uptake induced by compound 48/80 in rat mast cells. The inhibitory effects of cytochalasin D or colchicine on histamine release were exerted more remarkably when permeabilized mast cells were stimulated with either Ca2+ or inositol-1,4,5-trisphosphate (IP3). Since colchicine, vinblastine or cytochalasin D were not effective in inhibiting IP3 formation, it was assumed that microtubules or microfilaments may not participate in the initial stages of the membrane events leading to histamine release. By contrast, in Ca2+ release from the intracellular Ca store both colchicine and vinblastine (but not cytochalasin D) were effective in inhibiting Ca2+ mobilization, indicating that microtubules, rather than microfilaments, are intimately related to Ca2+ release from the endoplasmic reticulum (ER). By means of a fluorescence microscope, it was revealed that colchicine decreased the fluorescence intensity of FITC-labeled anti-tubulin antibody in the mast cells, while the amount of tubulin polymer in mast cells increased after exposure to compound 48/80. The findings indicate that colchicine simply suppressed polymerization of tubulin, while the rearrangement of microtubules so as to increase the polymerization took place after exposure to compound 48/80. Using an electron microscope in combination with potassium antimonate technique, Ca-antimonate dots were clearly observed in a cluster on the surface of the ER and a distinct connection between the ER and microtubules was also observed. It was concluded that microtubules play an important role in the processes leading to Ca2+ release from the intracellular Ca store and in subsequent histamine release. PMID- 1722416 TI - Histamine-releasing autoantibodies in chronic urticaria. AB - Circulating histamine-releasing factors have been identified in the serum and plasma of chronic-urticaria patients by in vivo skin testing and in vitro histamine release from heterologous mixed leukocytes. Quantitative mast cell studies of serum skin test biopsies and electron microscopy indicate that the serum factors release histamine by mast cell degranulation. Peripheral blood basophils and total cellular blood histamine are reduced in chronic-urticaria patients suggesting that the circulating serum factors cause sustained degranulation. Histamine-releasing activity has been identified by skin testing in ultrafiltered serum fractions less than 30 kDa and greater than 100 kDa. In vitro histamine-releasing activity was confined to ultrafiltered serum fractions greater than 100 kDa and was present in IgG purified from some chronic-urticaria sera by protein G affinity chromatography. The dose-response relationship and kinetics of histamine release in vitro were similar to those of anti-human IgE. 'Desensitisation' of basophils by prior incubation with anti-IgE in the absence of calcium and competitive inhibition studies with myeloma IgE serum indicated that histamine-releasing autoantibodies in chronic-urticaria sera and purified IgG have the properties of anti-IgE. Plasma exchange in 4 patients with active chronic urticaria refractory to antihistamine therapy showing in vivo and in vitro histamine-releasing activity was followed by temporary remission of disease activity in 2 of them. It is possible that chronic urticaria is an autoimmune disease. PMID- 1722417 TI - Immunodetection of the ligand-activated receptor for epidermal growth factor. AB - Many receptors for cellular growth factors are known to be protein tyrosine kinases which become activated upon ligand binding at their extracellular domain. We describe here a method to detect the activation state of Epidermal Growth Factor receptor (EGFr) with a monoclonal antibody (mAb74). This antibody was found to preferentially recognize the ligand-activated EGFr as detected by immunoprecipitation, Western blotting and immunocytochemical techniques. mAb74 did not recognize other tyrosine-phosphorylated proteins and was not inhibited by phosphotyrosine, suggesting that it is recognizing an epitope specific for the ligand-activated EGF receptor. The reactivity of mAb74 towards EGFr was closely correlated with the EGF-dependent tyrosine phosphorylation of endogenous substrates. This antibody allows one to detect the activated EGF receptor in vitro or in vivo even in a complex mixture of other tyrosine kinases and substrates. PMID- 1722418 TI - Platinum-based chemotherapy followed by radiation therapy of locally advanced nasopharyngeal cancer. A retrospective analysis of 39 cases. AB - A retrospective analysis was performed of 39 patients with locally advanced nasopharyngeal cancer treated with combined chemotherapy and radiation therapy during the last five years at our departments. There were 26 men and 13 women with median age 55 (24-75) years. Histology was squamous cell carcinoma in 6 patients and undifferentiated carcinoma in the remaining 33 patients. Induction chemotherapy consisted of either regimen A (cisplatin 100 mg/m2 day 1, 5-FU 1,000 mg/m2 days 2-6 as continuous infusion, bleomycin 15 mg days 15 and 29 i.m., mitomycin 4 mg/m2 day 22 and hydroxyurea 1,000 mg/m2 daily days 23-27) or regimen B (carboplatin 300 mg/m2 day 1, 5-FU 1,000 mg/m2 days 1-5 as continuous infusion and methotrexate 1.2 g/m2 day 14 with leucovorin rescue). After completion of induction chemotherapy 13 patients (33%) had complete remission (CR) and 19 (49%) partial remission (PR). The CR rate was increased after radiation therapy to 72%. Survival rates were 88% at 12 and 78% at 24 months. Median time to progression was 29.5 months. In conclusion, induction chemotherapy with a platinum-based regimen followed by radiation therapy achieved a high rate of local control. If the treatment also prolongs survival must, however, be studied by randomized trials. PMID- 1722419 TI - The relation between maternal alcohol consumption and child development: the epidemiological evidence. PMID- 1722420 TI - Study shows PSA better than DRE in screening for prostate cancer. PMID- 1722421 TI - Nomenclature for factors of the HLA system, 1990. WHO Committee for Factors of the HLA System. PMID- 1722422 TI - Regulatory volume decrease in small intestinal crypts is inhibited by K+ and Cl- channel blockers. AB - Total crypt volume has been estimated by analysis of photographic images of intact viable crypts isolated from guinea-pig small intestine. Exposing these crypts to a hypotonic medium, led to transient swelling followed by regulatory volume decrease (RVD) in 12-20 min. RVD was blocked by inhibitors of K+ and Cl- conductance, suggesting that it occurs by activation of K+ and Cl- permeability pathways and loss of these ions. PMID- 1722423 TI - Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies. AB - Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another. PMID- 1722424 TI - How does ppGpp affect translational accuracy in the stringent response? AB - With an in vitro poly(Phe) synthesis system we have tested recent models concerning translational accuracy in the stringent response during aminoacid starvation. We have found that cognate, deacylated tRNA of very high concentrations is unable to block the A-site. No influence of EF-Tu.ppGpp on ribosomal proofreading has been found. Alternative mechanisms to keep translational errors low by the stringent response are discussed. PMID- 1722425 TI - Structure and evolution of ribonuclease P RNA. AB - Eubacterial RNase P contains a catalytic RNA that cleaves 5' leader sequences from precursor tRNAs. We review the current understanding of RNase P RNA structure and evolution, from the perspective of phylogenetic comparative analysis. PMID- 1722426 TI - Aprotinin prevents bleeding and has effects on platelets and fibrinolysis. PMID- 1722427 TI - Neutrophil chemotactic activity of PAF, histamine and neuromediators in bronchial asthma. AB - Human blood polymorphonuclear neutrophils (PMN) are thought to be involved in the pathogenesis of asthma through their recruitment into the bronchoalveolar lumen and the lung by local release of chemotactic factors. Therefore chemotactic activities of several mediators (PAF, histamine and three neuropeptides substance P, VIP and a somatostatin analog) were compared on blood PMN from both healthy subjects (HS) and asthmatic patients (AP). The maximal response to PAF was significantly different (P less than 0.05) with cells from both groups. Moreover activity for the HS peaked at 10(-6) M, whereas the AP showed peak chemotactic activity at 10(-8) M. Histamine had no chemoattractant effect on PMN. Substance P did not induce PMN locomotion, whereas VIP induced a chemotactic response in a dose-dependent manner, particularly with cells from HS as compared to those from AP. BIM 23014 (a somatostatin analog) exhibited chemotactic activity which was also more pronounced with PMN from HS as compared to those from AP. Our findings showed that blood PMN could be involved in asthma through their heightened locomotor reactions to mediators which are known to be released locally by activated cells in bronchoalveolar lumen. PMID- 1722428 TI - The role of electrolytes in early stages of cell proliferation. PMID- 1722429 TI - [Professor and mentor in surgery]. AB - The surgical monitor supervises students, teaches them various skills and evaluates their progress. In the course of overseeing the students' clinical practice, the monitor helps them apply the theoretical concepts learned in class to the reality of life on the ward. If the experience is not properly planned, the goals of the clinical practicum, post-operative care, the student's personal goals and those of the monitor can become confused and intertwined. The author discusses the socialization process every student must go through to forge her own professional identity. Students must also adapt to the hospital "sub culture", which can be dramatically different from nursing school, and they must learn to think critically and analytically. The author, himself a monitor, explains how the students' progress is evaluated. The monitor presents himself as a confident, independent role model, equal to others on the health care team. The article concludes by pointing out that the Quebec Ministry of Education wants students to become integrated into the future job market; the new nursing focus is educating people who can be independent generalists. To sum up: the monitor is a coach, critic, consultant, supervisor, and advisor--all rolled into one. PMID- 1722430 TI - Basal cells in the mouse olfactory epithelium after axotomy: immunohistochemical and electron-microscopic studies. AB - The olfactory epithelium of mice after axotomy was investigated to clarify the stem cells of olfactory cells by double immunostaining using antikeratin (MA903) and anti-bromodeoxyuridine (BrdU) antibodies and by conventional electron microscopy. When a single dose of BrdU was given to mice 9 days after axotomy, immunostaining for BrdU was found in the globose basal cells which were negative for MA903, but not in the basal cells proper which were positive for MA903. The BrdU-immunoreactive cells increased 3- to 6-fold over the number of these cells in the controls, indicating active cell proliferation. At other postoperative days (4 and 14 days), fewer BrdU-immunoreactive cells were found. Furthermore, three pulses of BrdU resulted in numerous BrdU-immunolabelings in the globose basal cells and a few in the basal cells proper. There was no detectable difference in the number of labeled basal cells proper in operated and unoperated mice. In the electron micrographs 9 days after axotomy, the basal cells proper, flat-shaped in unoperated mice, appeared cylindrical or pyramidal in shape and the globose basal cells often lay between the basal cells proper. In unoperated controls, the globose basal cells were located above the flat-shaped basal cells proper. The results suggest that the stem cells of the olfactory cells are globose basal cells and not basal cells proper, and that the shape of basal cells proper changes in relation to the active proliferation of stem cells. PMID- 1722431 TI - Generation of monoclonal antibodies detecting specific epitopes in olfactory and respiratory epithelia. AB - Two panels of monoclonal antibodies have been generated, each panel having a distinct specificity for antigens located in the ciliary zone of either the olfactory or respiratory epithelium of rats. Tissue specificity was confirmed in enzyme-linked immunosorbent assays on membrane fractions from various tissues. During ontogeny, the expression of olfactory-specific antigens preceeds that of respiratory-specific antigens; this observation correlates with differences in the genesis of the respective cilia type and confirms that different molecular entities are recognized. A spatial segregation of immunoreactivity in the chemosensory epithelium was observed for one of the olfactory-specific monoclonal antibodies; negative zones were located in the dorsal recess of the nasal cavity and on the tips of the turbinates. Olfactory-specific antibodies reacted with distinct polypeptide bands on Western blots from olfactory ciliary preparations. PMID- 1722432 TI - Target-specific innervation by autonomic and sensory nerve fibers in hairy fetal skin transplanted into the anterior eye chamber of adult rat. AB - Pieces of hairy skin tissue of fetal rat were transplanted into the anterior eye chamber of adult rats. The ability of autonomic and sensory nerve fibers from the host iris to innervate the grafted skin tissue was immunohistochemically and enzyme-histochemically examined using antisera against tyrosine hydroxylase (TH), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), and a reaction medium for acetylcholinesterase (AchE). The grafted tissue was successfully implanted and connected with the host iris. Epidermis, dermis, subcutaneous tissue, hairs, hair follicles, sebaceous glands, and piloerector muscles developed in the graft. Two weeks after transplantation, TH-, SP-, and CGRP-immunoreactive fibers were observed in association with the blood vessels in the graft. Four weeks after transplantation, TH-immunoreactive fibers were distributed in the piloerector muscles, whereas SP- and CGRP immunoreactive fibers were present around the hair follicles. VIP-immunoreactive and AchE-positive fibers were restricted to the host iris at all survival times. These results suggest that the outgrowth of autonomic and sensory nerve fibers from the host iris show target specificity for the grafted skin tissue. PMID- 1722433 TI - Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat. AB - The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non noradrenergic (putative cholinergic) neurons, and (2) the types of peptide containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed "baskets" surrounding individual GAL negative somata. PMID- 1722434 TI - Effects of atrial septostomy in patients with terminal cor pulmonale due to pulmonary vascular disease. AB - Fourteen patients with pulmonary vascular disease, either primary pulmonary hypertension or the result of cardiac defects, underwent balloon atrial septostomy (BAS) over the period of July 1981 to June 1988 because of symptoms of syncope, fatigue, right heart failure, and cardiac arrest. Ages ranged from 4 mo to 50 yr. Two moribund patients died within 24 h of the septostomy due to severe hypoxemia and unrelieved low cardiac output; three others died 2 wk to 35 mo later; the remaining 9 patients have improved symptomatically and are alive 11 to 96 mo after septostomy. One received a heart and lung transplant 19 mo later. We conclude that, in patients with symptomatic cor pulmonale secondary to pulmonary vascular disease, atrial septostomy can improve symptoms and may serve as a palliative bridge to heart and/or lung transplantation. PMID- 1722436 TI - Goldmann tonometry and fluorescein solution: a way to avoid contact lens staining. AB - Topical instillation of fluorescein during standard Goldmann tonometry may cause subsequent contact lens staining. We evaluated a method of Goldmann tonometry that uses only a small amount of fluorescein. In this experimental technique, the fluorescein is applied only to the tip of the applanation cone with fluorescein coated tissue paper. Thirty patients had their intraocular pressures (IOPs) determined by this method. The IOP measurements were then repeated on the same eyes after instillation of fluorescein directly onto their eyes. In addition, 20 soft contact lens wearers had their IOPs determined by the experimental method, with contact lenses fitted immediately after the measurements. There was no statistically significant difference between the IOPs measured by the standard technique and by the experimental method. In addition, no fluorescein staining was detected in the 20 soft contact lenses examined. Our results suggest that the proposed method is accurate and useful in determining IOP prior to soft contact lens fitting. PMID- 1722435 TI - Fibroblasts are critical determinants in prostatic cancer growth and dissemination. AB - Fibroblasts are important contributors to both benign and malignant growth of prostate epithelial cells in vivo. In the human prostate cancer model that we have established, we can grow human LNCaP tumors reproducibly in athymic mice by coinoculating the animals with human LNCaP epithelial cells plus fibroblasts derived from either the prostate or bone; human lung, normal rat kidney, and embryonic mouse fibroblasts were inactive. We have delivered conditioned medium isolated from competent fibroblasts directly to sites where the tumor cells were injected and found that the conditioned medium alone confers tumorigenicity. Further studies of the mechanism of fibroblast-epithelial interaction have indicated that close metabolic cooperation between fibroblast and epithelial cells, involving the production of growth factors by the epithelial cells and the production of extracellular matrices and growth factors by the fibroblasts (assayed in vitro), is important in promoting prostate tumor growth in vivo. We have also investigated the possible in vivo interaction between extracellular matrix proteins such as laminin, collagens, heparan sulfate proteoglycans and Matrigel and prostate epithelial cells. Selective extracellular-matrix components were found to confer tumorigenicity to the prostate epithelial cells. Moreover, extracellular-matrix components were observed to induce cancer cell differentiation and alter permanently the morphology, gene expression and tumorigenic potential of the cancer epithelial cells. PMID- 1722437 TI - Ultrasensitive, specific, two-antibody immunoradiometric assay that detects free alpha subunits of glycoprotein hormones in blood of nonpregnant humans. AB - This noncompetitive, sensitive, immunoradiometric assay of the free alpha subunit of human pituitary glycoprotein hormones is based on two monoclonal antibodies and an avidin-biotin separation system. The affinity of the first antibody, mouse anti-alpha subunit covalently conjugated to biotin, is 3.8 x 10(11) L/mol. The second antibody, radiolabeled with 125I, has an affinity of 5.4 x 10(11) L/mol. A polystyrene ball coated with avidin serves as the separation system. Tests of "purified" immunochemical-grade intact human glycoprotein hormones yielded cross reactions of approximately 2% in the assay. Sephadex G-100 column chromatography showed that this "cross-reaction" was caused by contamination of the various hormone preparations with free alpha subunit. When the intact glycoprotein hormones were further purified with specific anti-alpha monoclonal antibody, their reaction in the alpha subunit assay was undetectable (less than 0.01%). Interassay CV averaged 3.5%, and intra-assay CV averaged 7.5% at low concentrations of subunit. The detection limit of the assay (0.01 micrograms/L) is adequate to detect free alpha subunit in the blood of normal humans. Mean (SD) concentrations of free alpha subunit in normal humans were as follows: eugonadal men = 437 (35) ng/L; postmenopausal women = 1231 (40) ng/L; eugonadal women, follicular phase = 1061 (40) ng/L; eugonadal luteal phase = 780 (45) ng/L. PMID- 1722439 TI - Discrepancies between methods for amylase in cases of macroamylasemia. PMID- 1722438 TI - Bile acids and conjugates identified in metabolic disorders by fast atom bombardment and tandem mass spectrometry. AB - From a study of the collision-activated fragmentation of bile acids, a qualitative analytical method based on negative ion fast atom bombardment tandem mass spectrometry has been developed. The times for sample preparation and analyses are short. Both free and conjugated bile acids are detected as they occur in biological fluids, without derivatization. For identifying bile acids and conjugates, the method offers better specificity and sensitivity than does the fast atom bombardment mass spectrometric technique alone. Specific scan modes have been developed for the selective detection of taurine conjugates, delta 4 unsaturated taurine conjugates, delta 4-3-keto free acids and their glycine conjugates, free acids and glycine conjugates bearing a hydroxyl group at the C 12 position, sulfates of glycine and taurine conjugates, and a C29 dicarboxylic bile acid, specific for generalized peroxisomal disorders. Applications of this technique demonstrate its potential usefulness, principally in the diagnosis of several peroxisomal disorders. PMID- 1722440 TI - Effect of stress on serotonin, norepinephrine, epinephrine and corticosterone contents in the soft-shelled turtle. AB - 1. Adult soft-shelled turtles were exposed to hyperosmotic and dehydration stresses. 2. Acute treatment for 0.5, 1 or 2 h with sodium chloride (3.6%, single intramuscular injection, 2 mL volume) caused depletion of pineal serotonin contents followed by elevation of norepinephrine and epinephrine levels. In addition, it depleted corticosterone and norepinephrine from the adrenal gland. 3. The serotonin level also decreased with a concomitant increase of 5 hydroxyindoleacetic acid but without any discernible change in catecholamine content after chronic treatment with sodium chloride (3.6%, 0.5 mL daily for 7 days). 4. Dehydration for 7 days brought about depletion of serotonin and epinephrine levels and elevation of norepinephrine level. 5. The findings suggest that hyperosmotic stress has a definite influence on pineal-paraphyseal serotonin, 5-hydroxyindoleacetic acid, norepinephrine and epinephrine concentrations, and adrenal corticosterone and norepinephrine contents in Lissemys turtles. Dehydration stress also modulates pineal-paraphyseal serotonin, norepinephrine and epinephrine concentrations. PMID- 1722441 TI - T cells bearing gamma/delta T cell receptor and their expression of activation antigen in peripheral blood from patients with Sjogren's syndrome. AB - T cells bearing gamma/delta T cell receptor (gamma/delta + T cells) and their expression of activation antigen (HLA-DR) or the marker of natural killer (NK) cells (CD56), were examined in the peripheral blood lymphocytes (PBL) from twenty two patients with Sjogren's syndrome (SS) by three-color flowcytometry to elucidate possible pathological roles of the T cell subset in SS. The frequency of gamma/delta + T cells in PBL was not elevated in SS patients, while that of gamma/delta - T cells, which are T cells bearing the alpha/beta T cell receptor (alpha/beta + T cells), was significantly low in the patients, as compared with 22 healthy controls. We found that the proportions of activated cells (HLA-DR+) in both the gamma/delta + and alpha/beta+T cell subsets were significantly higher in the patients than in the controls. The proportions of HLA-DR+ cells in cells in both patients and controls. Furthermore, the frequency of activated cells in both T cell subsets correlated with the duration of disease in SS patients. However, no difference was found in the percentages of total CD56+ cells, CD56+CD3- cells (true NK cells), CD56+CD3+T cells, CD56+gamma/delta+T cells, or CD56-gamma/delta+T cells between the patients and controls. The above results indicate that immunologic activation in SS patients is progressive and involves both alpha/beta+ and gamma/delta+ T cell subsets. PMID- 1722442 TI - Polyarthritis, mononeuritis multiplex and eczematous ulcerative skin rash in a patient with myelodysplastic syndrome and peripheral large granular lymphocytosis. AB - A patient with polyarthritis, peripheral mononeuritis multiplex with spatial and temporal fluctuation, and eczematous, ulcerative skin rash in the lower extremities was found to have myelodysplastic syndrome (MDS) in the bone marrow and concomitant large granular lymphocytosis in the peripheral blood. Histochemical study showed that cells with large granular lymphocyte markers (CD2+, 11b+, 16+, 57+, HLA-DR+) had infiltrated into the skin and around the nerve fibers. Both the bone marrow dyscrasia and rheumatic manifestations of this patient improved significantly after prednisolone therapy. The unusual rheumatologic manifestations of this patient appear to derive from a delicate balance between MDS and large granular lymphocytosis. PMID- 1722443 TI - Islet amyloid polypeptide: production by an osteoblast cell line and possible role as a paracrine regulator of osteoclast function in man. AB - 1. The recently discovered peptide islet amyloid polypeptide shows considerable sequence homology with calcitonin-gene-related peptide, itself an alternative product of the calcitonin gene. The possibility that islet amyloid polypeptide might affect calcium homoeostasis and bone cell function was investigated. 2. Islet amyloid polypeptide messenger RNA was found to be expressed by human HTb 96 osteoblast-like cells in culture, and islet amyloid polypeptide immunoreactivity was present in the cell culture medium. 3. Infusion of islet amyloid polypeptide (150 pmol min-1 kg-1) caused a fall in serum calcium and phosphate concentrations in five patients with Paget's disease of the bone. This was similar to that caused by infusion of calcitonin (50 pmol min-1 kg-1). 4. These findings raise the possibility that islet amyloid polypeptide may act as a local factor within bone, produced by osteoblasts and regulating osteoclast function. The possibility of an action of islet amyloid polypeptide on the renal handling of calcium seems unlikely but is not totally excluded. PMID- 1722444 TI - Computational implications of NMDA receptor channels. AB - We have summarized the quantitative relations developed so far for the description of NMDA receptor function. One of the most important gaps in our knowledge relates to desensitization. A full quantitative treatment of computational uses of NMDA receptor channels must await a formalization of this process and also a more detailed examination of the occupation of closed states of the receptor whose binding sites are occupied. As this information becomes available and the role of NMDA receptors in the function of brain circuits is further explored, we should be able to define accurately this second computational mode. PMID- 1722445 TI - Inhibition studies on liver arylformamidases of rainbow trout and cattle. AB - The effects of L-tryptophan, L-kynurenine, 3-hydroxy-L-kynurenine, ascorbate, some amino acids, 5-hydroxy-L-tryptophan, anthranilate, sodium bisulfite, EDTA, divalent ions and ionic strength on purified liver arylformamidases of rainbow trout and cattle were investigated. PMID- 1722446 TI - The interpretations of fox possession: illness as metaphor. AB - Although fox possession is a rare phenomenon in contemporary Japan, it is a matter of concern in psychiatry and in folk healing. The case study presented here deals with the social process of role transformation, from client to shaman, as a way of self healing. The discussion then assumes that fox possession renders multiple interpretations and argues that it is, in fact, a metaphorical representation in much the same way that biomedical interpretation is. The argument also proposes that an etiological (causal) understanding of fox possession is less productive than a metaphorical one. PMID- 1722447 TI - Pseudohorn associated with basal cell carcinoma. AB - Cutaneous horns usually represent compacted keratin arising from an underlying pathologic process and are important to dermatologists because they may indicate an underlying malignancy. The differential diagnosis of cutaneous horns includes pseudohorns, which have the morphologic appearance of a cutaneous horn but consist entirely of benign or malignant tumor. We describe a second type of pseudohorn consisting of hair, dried serum, and inflammatory exudate that was associated with an underlying basal cell carcinoma. PMID- 1722448 TI - Bivariate flow karyotyping of human chromosomes: evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence, and relative chromosomal DNA content. AB - The total variation of chromosome peak positions, in bivariate distributions of Hoechst 33258 and chromomycin A3 fluorescence of 19 healthy individuals, was compared with the experimental variation, determined from 23 bivariate distributions of chromosomes prepared separately from a single cell lineage. The experimental variation in Hoechst and chromomycin fluorescence and the relative chromosomal DNA content were determined from experiments performed over several days. The additional variance contributed by time was the same as the daily variance. The accuracy by which the relative chromosomal DNA content can be calculated from bivariate peak positions was investigated. A least squares method was used to fit the distributions of relative DNA content, obtained, respectively, from mono- and bivariate flow analyses of chromosomes from the same cell lineage. In general the DNA contents match quite well, but for a few chromosomes a difference was found, statistically discernible at the 5% level. The average relative chromosomal DNA content of the chromosomes from the 19 normal individuals, calculated from bivariate peak positions, showed a linear relation with the estimates published by other investigators. PMID- 1722449 TI - An evaluation of myelomeres and segmentation of the chick embryo spinal cord. AB - We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2 day embryos with colchicine does produce exaggerated myelomeres, in confirmation of Kallen (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites. PMID- 1722450 TI - Transcription factor AP-2 is tissue-specific in Xenopus and is closely related or identical to keratin transcription factor 1 (KTF-1). AB - This paper identifies a new, developmental role for transcription factor AP-2 in the activation of amphibian embryonic epidermal keratin gene expression. Keratin transcription factor KTF-1 is shown by several criteria to be identical or closely related to AP-2. KTF-1/AP-2 is shown to be tissue-specific from its first transcription in Xenopus embryos, and restricted to a small number of adult tissues, including skin. Epidermis-specific keratin transcription closely follows specification of the embryonic ectoderm in Xenopus, and is subject to regulation by growth factors and embryonic induction. We further show that in mouse basal keratinocytes, a KTF-1/AP-2-like factor is present and binds to a DNA sequence previously shown to be important in the regulation of the keratin K14 gene, which is actively expressed in these cells. Thus, the study of AP-2 and its role in the regulation of keratin gene transcription should enhance our understanding of both amphibian embryonic development and mammalian skin differentiation. PMID- 1722451 TI - The diagnostic importance of alpha-fetoprotein. PMID- 1722452 TI - A study of alpha-fetoprotein in primary liver cancer in Tanzania. AB - Alpha-fetoprotein (AFP) was detected, by Ouchterlony immunodiffusion technique, in 81.5% of patients with histologically confirmed diagnosis of hepatocellular carcinoma. The test gave negative results with 35 cases of acute viral hepatitis, 7 haemochromatosis, 6 micronodular cirrhosis and 2 cholangiocellular carcinoma. Curiously, one patient with postnecrotic cirrhosis, a well recognized sequela of viral hepatitis, whose liver cell regeneration also showed "atypical changes", was AFP positive. AFP was not detected in sera from the general population which comprised 1029 male blood donors, 144 antenatal and 106 maternity cases. The only exception was the case of a woman who aborted a 5-month old foetus. A follow-up serum sample taken 3 months later was, however, negative for AFP. The frequency of hepatitis B surface antigen (HBsAg) detection in patients with hepatocellular carcinoma (25.9%) was 4 to 5 times higher than that in the general population. This strong association between HBsAg and primary liver cancer in countries where liver tumours are often AFP secretors suggests a role for hepatitis B virus, not only in the aetiology of the cancer, but also in the reactivation of the gene encoding this foetal protein. PMID- 1722453 TI - Maternal levels of alpha-fetoprotein in African women. AB - In a prospective and cross-sectional study, 147 serum samples from normal antenatal indigenous Kenyan women were analysed for alpha-fetoprotein (AFP) concentrations between 8 and 28 weeks gestation. There was progressive rise in AFP levels with gestation, most rapid between 12 and 24 weeks. There were wide variations in AFP concentrations at every gestation but no correlation was established with maternal age or parity. All the eleven women with very high AFP values had subsequent complications, including 2 abortions, 1 APH, 7 PET and one pair of twins. The need for more studies on the value of AFP in monitoring of pregnancy in the African set up is emphasised. PMID- 1722454 TI - Primary structure of a barley-grain aspartic proteinase. A plant aspartic proteinase resembling mammalian cathepsin D. AB - Two enzymatically active heterodimeric forms of an aspartic proteinase, a putative 32 kDa + 16 kDa precursor form and a putative 29 kDa + 11 kDa mature form, are present in resting barley grains (Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J. & Mikola, L., 1990, in the press). The cDNA corresponding to this enzyme has been cloned and sequenced. The full-length 1863 bp cDNA sequence codes for an open reading frame of 508 amino acids. The open reading frame consists of a 66-amino acid preprosequence and a 442-amino acid mature protein. Comparison of the N-terminal amino acid sequences of the enzyme subunits with the sequence of the cDNA clone indicates that the heterodimeric enzyme is translated as a proenzyme which is processed into two subunits. The localisation of the experimentally determined N-terminal amino acid sequences of all four subunits (32 kDa + 16 kDa and 29 kDa + 11 kDa) in the same transcript, as well as the detection of only one 2.0-kb mRNA on Northern blots from resting seeds, clearly indicates that the larger (32 kDa + 16 kDa) enzyme is an intermediate precursor form of the smaller (29 kDa + 11 kDa) enzyme. The processing pattern of the barley enzyme, which is the first sequenced plant aspartic proteinase, differs from that of all other known aspartic proteinases. The barley enzyme is highly similar to mammalian and yeast aspartic proteinases, especially to human and porcine cathepsin D. This similarity is clearly dispersed over two regions, separated by a dissimilar, barley-specific region of 104 amino acids. PMID- 1722455 TI - Structural characterization of mating pheromone precursors of the ciliate protozoan Euplotes raikovi. High conservation of pre and pro regions versus high variability of secreted regions. AB - The precursors of Euplotes raikovi pheromones Er-2 and Er-10 have been structurally characterized from the sequences of their coding regions that were amplified and cloned using the polymerase chain reaction and oligonucleotide primers corresponding to conserved sequences of the gene for pheromone Er-1. The predicted amino acid sequences contain 75 residues distributed through three domains: signal peptide, pro segment and mature pheromone. Despite the conservation of the overall length, there is variation in the size of the pro segments and of the mature pheromones. The comparison of the sequences shows a gradient of identity from the amino to the carboxyl terminus; the signal sequences are identical (with greater than or equal to 95% identity in the nucleotide sequences), the pro segments more variable and the mature pheromones quite diverse. The processing site of the pro pheromones, to produce the mature forms, is apparently characterized by the unusual Xaa-Asp sequence. PMID- 1722456 TI - Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA. AB - Phospholipase B has not yet been well defined. The most important points about this enzyme are its relationships with lysophospholipase and phospholipase A1. As reported [Saito, K., Sugatani, J. & Okumura, T. (1991) Methods Enzymol. 197, 446 456], Penicillium notatum phospholipase B is a glycoprotein with a molecular mass of 95 kDa and intrinsic lysophospholipase and phospholipase B activities; however, by endogenous proteolytic modification, its phospholipase B activity is lost almost completely, whereas its lysophospholipase activity remains unchanged. A cDNA library of P. notatum was screened by hybridization with two synthetic oligodeoxyribonucleotide probes, which corresponds to two different pentapeptides of the enzyme. A hybridization-positive clone, pPLB18, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence was quite different from that found previously. Therefore, we rescreened the cDNA library with a Sau3AI fragment derived from pPLB18 and isolated a new clone, pPLB15. Comparison of the nucleotide sequences of pPLB15 and pPLB18 revealed that pPLB18 contained an insertion sequence of 53 bp. Consequently, the reading frame was open downstream for 603 amino acid residues. From the assigned sequence, it was deduced that the limited proteolysis occurred between Leu175 and Asp176; eight cysteine residues and 16 potential N-glycosylation sites were also found. No amino acid sequence similarity was found with other proteins, including other phospholipases. PMID- 1722457 TI - Post-transcriptional regulation of the transferrin receptor and 4F2 antigen heavy chain mRNA during growth activation of spleen cells. AB - The expression of several cell surface proteins including the transferrin receptor and 4F2 antigen is induced when quiescent cells are activated and proliferate. We have studied this induction in mouse spleen cells after stimulation with 4 beta-phorbol 12-myristate 13-acetate (PMA), the Ca(2+) ionophore, ionomycin and recombinant interleukin-2 (IL-2). The 4F2 antigen heavy chain and transferrin receptor mRNA were barely detectable in resting cells, but increased 60-fold within 4 h of growth stimulation. The corresponding proteins became measurable at the cell surface after 6 h, prior to the S phase. In run-on transcription assays the transferrin receptor gene was transcribed to almost the same extent in resting and growth-stimulated cell populations and the 4F2 antigen heavy chain gene was induced fivefold. This suggests that post-transcriptional control mechanisms are mainly responsible for the accumulation of the respective mRNA at the onset of cell proliferation. In the case of the transferrin receptor, the induction correlated with an activation of the mRNA-binding iron-regulatory factor which is known to increase the stability of the cytoplasmic transferrin receptor mRNA. PMID- 1722458 TI - Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls. AB - Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Gotz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N [14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water phase and the lipid-phase. Incubation of intact mitochondria with N ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle. PMID- 1722459 TI - Expression of the wild-type and mutated vacuolar storage protein phaseolin in Xenopus oocytes reveals relationships between assembly and intracellular transport. AB - The role played by subunit assembly in the intracellular transport of the bean storage protein phaseolin, a soluble trimeric glycoprotein, was investigated using Xenopus oocytes injected with RNA. We show that phaseolin assembly is dependent upon the level of synthesis of the protein and is required for intracellular transport out of the endoplasmic reticulum. We also show that a fraction of the assembled phaseolin is permanently retained in a post-endoplasmic reticulum compartment. Deletion of the C-terminal alpha-helical domain fully prevents in vivo assembly but not endoplasmic reticulum retention. This indicates that this domain is necessary for trimerization but not for interactions of unassembled subunits with endoplasmic reticulum components. The truncated phaseolin has high in vivo stability. The potential implications of these findings on the possibility to improve the nutritional value of phaseolin through genetic engineering are discussed. PMID- 1722460 TI - Concentration of azithromycin in human prostatic tissue. AB - Prostatic tissue was obtained from 36 patients at two study locations and assayed for azithromycin by HPLC or bioassay. The mean concentration of azithromycin in human prostatic tissue (2.54 micrograms/ml) 14 h after 500 mg oral dosing (two 250 mg doses 12 h apart) was much greater than plasma concentrations (less than or equal to 0.1 micrograms/ml). Azithromycin was slowly eliminated from prostatic tissue (half-life 60 h) and a mean concentration of 0.62 micrograms/ml remained 137 h after dosing. PMID- 1722461 TI - A rapid change in phosphorylation on tyrosine accompanies fertilization of sea urchin eggs. AB - Alterations in protein phosphorylation, particularly phosphorylation on tyrosine, frequently accompany cell change and are important agents in the cascades initiated by extracellular signals. This paper examines whether the activation of the sea urchin egg at fertilization involves an early and rapid phosphotyrosine response. Using an anti-phosphotyrosine antibody and a rapid sampling technique, we find a very early increase in the phosphorylation on tyrosine of two proteins of approximately 91 kDa and 138 kDa. A similar phosphorylation occurs after activation of the eggs by the calcium ionophore, ionomycin, suggesting the stimulation of a Ca(2+)-sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization. PMID- 1722462 TI - Epidermal growth factor (EGF) decreased endothelin-2 (ET-2) production in human renal adenocarcinoma cells. AB - Production of immunoreactive (ir-) endothelin-2 (ET-2) in renal adenocarcinoma cells, ACHN, was reduced by transforming growth factor-beta, basic fibroblast growth factor, transforming growth factor-alpha and, most strikingly, by epidermal growth factor (EGF). These growth factors did not show such inhibitory effects on the secretion of ir-ET-1 in ET-1-producing cells, indicating that the production of ET-2 and ET-1 is regulated differently by the growth factors. EGF specifically reduced the secretion of not only ir-ET-2 but also ir-big ET-2 with only a small decrease in total protein synthesis. Northern blot analysis indicated that EGF controls the ET-2-production at the transcription levels of ET 2 gene. PMID- 1722463 TI - Functional characterization of RK5, a voltage-gated K+ channel cloned from the rat cardiovascular system. AB - A voltage-sensitive K+ channel previously cloned from rat heart designated RK5 (rat Kv4.2) (Roberds and Tamkun, 1991, Proc. Natl. Acad. Sci. USA 88, 1798-1802) was functionally characterized in the Xenopus oocyte expression system. RK5 is a homolog of the Drosophila Shal K+ channel, activates with a rise time of 2.8 ms, has a midpoint for activation of -1 mV and rapidly inactivates with time constants of 15 and 60 ms. RK5 is sensitive to 4-AP, IC50 = 5 mM, and is insensitive to TEA and dendrotoxins. The voltage dependence and kinetics of the RK5 induced currents suggest this channel contributes to the Ito current in heart. PMID- 1722464 TI - Transcript hairpin structures are not required for RNA polymerase pausing in the gene encoding the E. coli RNase P RNA, M1 RNA. AB - Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E. coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P. These pauses are immediately downstream of 2 phylogenetically conserved stem-loop structures in the RNA. In the present work, single-base changes which disrupted Watson-Crick base-pairing in the hairpins were introduced into rnpB. Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half-lives. A mutation which disrupted the second hairpin increased the pause half-life 2-fold. The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event. PMID- 1722465 TI - Relationship between DNA methylation and cell proliferation in Trypanosoma cruzi. AB - 5-Azacytidine treatment of T. cruzi epimastigotes in culture induces active cell proliferation. This effect was detected as an increase in the cell number and [3H methyl]thymidine incorporation into DNA. 5-Azacytidine does not alter other metabolic parameters. We have previously demonstrated that 5-azacytidine induces DNA hypomethylation in T. cruzi. Accordingly, we suggest that this chemical modification may be related to the control of T. cruzi cell division. PMID- 1722466 TI - Effects of the membrane potential upon the Ca(2+)- and cumene hydroperoxide induced permeabilization of the inner mitochondrial membrane. AB - A protonophore-induced delta psi decrease in a 180-140 mV range causes an increase in the lag-period of Ca(2+)-induced mitochondrial permeabilization but has little effect on the cumene hydroperoxide-induced permeability transition of mitochondria. Suppression of the non-specific permeability induction seems to be mediated by an increase in [ADP] in the mitochondrial matrix. A further decrease in delta psi leads to additional suppression of the non-specific permeability as a result of a partial ruthenium red-sensitive efflux of the previously accumulated Ca2+. On the other hand, complete dissipation of delta psi causes immediate induction of the non-specific permeability. It is concluded that only complete dissipation of delta psi caused by H+ leakages may act as a trigger for non-specific permeability induction. PMID- 1722467 TI - PGE2 regulates cholecystokinin-octapeptide (CCK-8)-stimulated Cl- conductance in isolated zymogen granules from rat pancreas. AB - In this study we have examined the effects of prostaglandin E2 (PGE2), the cyclooxygenase inhibitor, indomethacin, and a protein kinase A inhibitor (PKA-I) on the Cl- conductance in isolated zymogen granules (ZG) from cholecystokinin octapeptide (CCK-8) pre-stimulated pancreatic acini. The Cl- conductance in isolated ZG from CCK-8 pre-stimulated rat pancreatic acini increases with increasing CCK-8 concentrations and decreases at supramaximal CCK-8 concentrations. The basal and CCK-8-stimulated Cl- conductance in ZG is inhibited by pretreatment of acini with PGE2 (10(-6) M). This PGE2-induced inhibition is abolished in the presence of PKA-I (20 U/ml). Furthermore, pretreatment of acini with indomethacin (10(-5) M) or PKA-I (20 U/ml) abolishes the decrease in the CL- conductance at supramaximal CCK-8 concentrations (10(-9) M). We conclude that the inhibition of the CL- conductance in isolated ZG at high CCK-8 concentrations is mediated by an enhanced production of PGE2, and that PGE2 operates by stimulating adenylate cyclase (AC) with a consequent rise in cAMP and activation of PKA. PMID- 1722468 TI - Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle. AB - We isolated a new calcium-binding protein from porcine cardiac muscle by calcium dependent hydrophobic and dye-affinity chromatography. It showed an apparent molecular weight of 11,000 on SDS-PAGE. Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein. PMID- 1722469 TI - [The psychomotor development of infants in the 1st year of life within the family]. PMID- 1722470 TI - A microspectrophotometric study of DNA ploidy patterns of colorectal adenomas. AB - Eighty-four adenomas (31 mild, 25 moderate, 28 severe atypia), 32 invasive carcinomas and 15 samples of normal mucosa were collected form polypectomy and surgically resected specimens, and their DNA ploidy patterns were examined by microspectrophotometry. The frequency of polyploidy and aneuploidy were 7% and 0% in normal mucosa, 13% and 0% in mild atypia, 20% and 44% in moderate atypia, 32% and 22% in severe atypia and 31% and 53% in invasive carcinomas, respectively. Furthermore, these abnormal ploidy patterns were found frequently in moderate atypia accompanying severe atypia and mild atypia accompanying moderate atypia. These findings suggest that adenomas with moderate atypia might be the early stage of histologically overt cancer. The thin-section method (4 microns or 7 microns) was useful for measuring regional DNA ploidy patterns of colorectal adenoma. PMID- 1722471 TI - Successful treatment of a case of hepatocellular carcinoma with tumor necrosis factor and local hyperthermia. AB - A case of unresectable hepatocellular carcinoma which responded favorably to combined therapy with tumor necrosis factor (TNF) and local hyperthermia is reported. A 58-year-old man was admitted to our hospital in June 1988 for treatment of hepatocellular carcinoma affecting S4 and S8. After three sessions of transcatheter arterial embolization (TAE) therapy, the serum alpha 1 fetoprotein level decreased, and a reduction in the size of the lesions was also noted. Thereafter, the patient received local hyperthermia once a week (60 minutes of irradiation from a Thermotron-RF8 at 1,100W), but the alpha 1 fetoprotein level increased again in February 1989. On examination, enlargement of the S8 lesion and a new nodule in S7 were recognized. Since TAE was contraindicated due to liver dysfunction, human recombinant TNF (1 x 10(6)U) was given by intravenous infusion together with local hyperthermia once a week. Eight sessions of the combined therapy reduced the serum alpha 1-fetoprotein level markedly (7,512.0 to 2,782.0 pg/ml) and after eighteen sessions, 58.1% regression of tumor size (partial response) on computed tomography scans was observed. This anecdotal case supports previous experimental evidence suggesting that TNF plus hyperthermia may be effective for treating unresectable hepatocellular carcinoma. PMID- 1722472 TI - An application of recombinant human granulocyte colony-stimulating factor (rhG CSF) in a case of hepatocellular carcinoma combined with liver cirrhosis in which leukopenia developed after chemoembolization. AB - A 63-year-old Japanese woman who was being treated for liver cirrhosis was diagnosed as having hepatocellular carcinoma in the caudate lobe of the liver. Transcatheter hepatic arterial chemoembolization was performed for this lesion, but severe neutropenia occurred. To restore white blood cell (WBC) counts, recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered (250 micrograms per day during 10 days, intravenously). Subsequently, WBC counts recovered immediately without side effects. This suggests that rhG-CSF could be useful for the treatment of neutropenia after chemoembolization, even in cirrhotic patients. PMID- 1722473 TI - Tagging the genome of the murine leukemia retrovirus SL3-3 by a bacterial lac operator sequence. AB - The bacterial lactose operator (lacO) was introduced into the PstI site of the long terminal repeat of the SL3-3 murine leukemia virus, generating a virus, SL3 3lacO, that can replicate in NIH3T3 cell cultures. DNA sequences harboring the lacO sequence might be recovered by molecular cloning in Escherichia coli lac+ lacZ+ using bacteriophage lambda or plasmid vectors. The high copy numbers of the lacO sequence titrate out the lac repressor, leading to the induction of the lac operon in the host. We show here that the lacO and the proviral sequences are carried stably together in the genomes of SL3-3lacO-infected cell cultures and in viral particles. This system is designed to facilitate studies on the provirus and the site of viral integration. PMID- 1722474 TI - cDNA encoding murine FK506-binding protein (FKBP): nucleotide and deduced amino acid sequence. AB - A FKBP cDNA encoding murine FK506 binding protein (FKBP) has been cloned, and its complete nucleotide sequence has been determined. The open reading frame within the 1556-bp cDNA segment encodes an 108 amino acid (aa) protein that differs from the human FKBP by three aa and from the bovine FKBP by five aa. Molecular modeling of the protein places the aa substitutions at positions not directly involved in drug binding or interaction with the potential drug target protein, calcineurin A. PMID- 1722475 TI - Genes for insulin-like growth factors I and II are expressed in senescent rat tissues. AB - Insulin-like growth factor IGF-I and IGF-II gene expression was measured for the first time in rat liver, brain and heart during the later stages of life, i.e., young adulthood (6 months) to senescence (25 months). IGF-I mRNA was detected in all three tissues at all ages. Its relative level decreased the most in the liver with advancing age. IGF-II mRNA was detected in the brain and heart but not the liver at all ages. The level of IGF-II mRNA decreased only slightly in the brain from young adulthood to senescence. PMID- 1722476 TI - Soluble forms of isobutylmethylxanthine enhance the ocular hypotension induced by catecholamines. AB - Isobutylmethylxanthine (IBMX) suspended in 0.5% hydroxypropyl methylcellulose has been shown to enhance the reduction in intraocular pressure (IOP) induced by epinephrine and norepinephrine in rabbits and beagles. In the present study, the effects of two soluble forms of IBMX, i.e. the ethylenediamine salt (IBMX-ED) and IBMX bound to cyclodextrin in saline (IBMX-CD), were studied in rabbits. When used alone, 1% IBMX-ED did not affect the IOP, but the hypotensive response to 0.1% epinephrine was enhanced dose-dependently by combination of the catecholamine with IBMX-ED. Although 1% IBMX-CD applied alone also failed to influence the IOP, the hypotensive response to epinephrine, dipivalyl epinephrine and norepinephrine was enhanced by combination of these substances with IBMX-CD. Soluble IBMX-ED as well as soluble IBMX-CD and the IBMX suspension enhanced, to a similar extent and in a dose-dependent manner, the IOP reduction induced by catecholamines. IBMX-CD, which has a neutral pH, may be a suitable form of IBMX for future long-term experiments. PMID- 1722477 TI - Monoclonal antibody to the corneal endothelium: partial characterization of the antigen and its expression in fetal and adult rabbits. AB - Monoclonal antibodies (mAbs) were developed against corneal endothelial cells (CECs) using cultured bovine CECs as immunogens by the mouse hybridoma technique. One of these mAbs, KP14D10 (IgM type), reacted specifically with CECs in human, bovine and rabbit ocular tissues as judged by immunohistochemical methods. This mAb also reacted with some epithelial cells of human esophageal and gastric glands but not with those of bovine and rabbit origin. The molecular weight of the antigen was determined to be 60,000 Da by the immunoblotting method. This antigenicity was apparently decreased by treatment with hyaluronidase, trypsin, and pronase. In the immunohistochemical study of rabbit fetal corneas, the immunofluorescence appeared specifically in the CECs after the 15th day of gestation and its intensity became stronger as gestation advanced. These results suggest that this antibody would be a valuable tool in the further analysis of corneal development and of divese pathological alterations of CECs. PMID- 1722478 TI - [Proposed normal values for alpha fetoprotein in maternal serum for the detection of neural tube closure defects and Down syndrome. Preliminary study]. AB - To establish a normal range of alpha fetoprotein in maternal serum (AFP sm) in the population at the "20 de Noviembre" Hospital in Mexico City, there were studied 46 patients with a normal pregnancy confirmed with ultrasonography between 16 to 18 week of gestation. The 97 determinations of AFPsm were made by radioimmunoanalysis. The multiples of the median (MoM) were 2.16 al 16th weeks, 2.33 a 17th week 2.43 al 18th week of pregnancy. Now the methods to determine abnormalities in the product and the genetics studies are proposed only to the patients considered high risk (older than 35 years old, previous malformed son or family history of those abnormalities). AFPsm and the establishment of normal range allow us to amplify the study to every pregnant woman and to determine those malformations in the pregnant women of low risk which represents about 90 95% of DCTN and about 80% of SD. PMID- 1722479 TI - [Diagnosis of ovum viability: comparative analysis between ultrasound and chorionic gonadotropin hormone]. AB - In a retrospective study carried out in the Hospital de Gineco-Obstetricia del Centro Medico Leon, Gto., Instituto Mexicano del Seguro Social, 61 patients were studied in order to compare the sensitivity and specificity values and the correlation coefficient between the hormonal assays (Human Chorionic Gonadotropin, HCG) and the ultrasound scanning. The qualitative concentrations of HCG had a sensitivity of 37.5% and a specificity of 100%. The levels of subunit beta of HCG had sensitivity of 25% and specificity of 100%. The whole correlation coefficient of the hormonal method (HCG) was R = 0.51 (P less than 0.01). The ultrasound monitoring had a sensitivity of 855 and specificity of 100%, with a correlation coefficient R = 0.88 (P less than 0.01). It was concluded that ultrasound scanning has a better sensitivity and higher correlation than human chorionic gonadotropin assays in the diagnosis of ovum vitality. PMID- 1722480 TI - Detection of semen and blood stains using polilight as a light source. AB - Photoluminescence spectra of dry untreated semen have been measured and a suggested method for rapid detection of untreated semen stains is derived from these measurements. The method is presented in the form of a flow chart to cover most crime scene situations. The absorption spectrum of dry untreated blood has also been measured and a suggested method for enhancement and photography of blood stains is derived from this measurement. The method is presented in the form of a flow chart. Both methods are based on the use of a high intensity light source such as the Polilight. PMID- 1722481 TI - [The image of the heart. The origin of the heart symbol--1: the heart-shaped leaf in antiquity, in early Christianity and other cultures]. PMID- 1722482 TI - Non-parallel secretion of pancreatic amylase and trypsinogen following hepatectomy in rats. AB - To explore the changes in exocrine pancreatic function in the stages of active regeneration and recovery after hepatectomy, we measured the caerulein-stimulated amylase and trypsinogen output in anesthetized rats 4 days and 8 days after about 70% hepatectomy. Both 4 days and 8 days after hepatectomy, the amylase output was significantly greater than in the control groups. On the other hand, 4 days after hepatectomy, the trypsinogen output was almost the same as in the control groups, and 8 days after hepatectomy, it was significantly lower than in the control groups. Thus, the secretion of amylase and trypsinogen 8 days after hepatectomy was not parallel. The amylase content of the pancreas was significantly larger after hepatectomy than in the control groups, but the trypsinogen content was significantly smaller. These results indicate a non-parallel secretion of pancreatic digestive enzymes, as well as differences in the functions of the acinar cells after partial hepatectomy, and the important role of amylase in glucose metabolism after hepatectomy. PMID- 1722484 TI - Duodenal diversion of percutaneous biliary drain through a percutaneous endoscopic gastrostomy: report of a case. AB - Occasionally, percutaneous biliary drainage is the only possible form of treatment in a patient with a malignant obstruction at the porta hepatis. We report on a case of gallbladder carcinoma with a complete block at the porta hepatis, which was palliated with a percutaneous biliary drain. Enteral reinfusion of bile was accomplished through a duodenal tube placed through a percutaneous endoscopic gastrostomy. PMID- 1722483 TI - Abnormal prothrombin (DES-gamma-carboxy prothrombin) in hepatocellular carcinoma. AB - Des-gamma-carboxy prothrombin (DCP), a protein induced by vitamin K absence or antagonist-II (PIVKA-II) was measured by an enzyme immunoassay (E-1023) using anti-DCP monoclonal antibody in 92 patients with various hepatobiliary diseases. Thirty-six of the 38 patients (94.7%) with hepatocellular carcinoma (HCC) had abnormal DCP levels greater than 0.1 arbitrary unit (AU)/ml, but only 18 of the 35 patients (51.4%) had AFP greater than 100 ng/ml (suspicious levels for HCC). There was no correlation between plasma or serum DCP and serum alpha-fetoprotein (AFP) levels. Serum alpha fetoprotein was elevated (above 20 ng/ml) in 23 of the 35 patients (65.7%), and DCP was elevated in all of the remaining 12 patients with normal AFP. DCP levels returned to normal levels following curative hepatic resection or orthotopic liver transplantation for HCC. DCP is a useful tumor marker in the diagnosis and postoperative monitoring of patients with HCC. PMID- 1722485 TI - Patterns of DNA methylation are indistinguishable in different individuals over a wide range of human DNA sequences. AB - Patterns of DNA methylation at 5'-CCGG-3' and 5'-GCGC-3' sequences were determined in about 570 kb, equivalent to about 0.02% of the human genome, by using HpaII and HhaI restriction endonucleases, respectively, and randomly selected cosmid clones of human DNA as hybridization probes. Many of these human DNA sequences were of the repetitive type. The DNAs from human lymphocytes, from a mixture of all blood cells or from several established human cell lines (HeLa, KB, 293, or DEV) were included in these analyses. In the segments of the human genome investigated, the patterns of DNA methylation were characterized by often completely or partly methylated 5'-CCGG-3' or by partly methylated 5'-GCGC-3' sequences. Even among individuals of different genetic origins (East-Asian or Caucasian), these patterns of DNA methylation proved indistinguishable by the method applied. The cytokine-dependent stimulation of human lymphocytes to replicate in culture did not affect the stability of these patterns. In the same DNA sequences from several human cell lines, much lower levels of DNA methylation were observed. In human cell lines some of the investigated sequences were unmethylated. The results presented lend credence to the notion that the human genome exhibits highly cell type-specific patterns of DNA methylation which are often indistinguishable among different individuals even of different genetic backgrounds. PMID- 1722486 TI - AUR Memorial Award 1991. Immunogenicity of gadolinium-based contrast agents for magnetic resonance imaging. Induction and characterization of antibodies in animals. AB - To evaluate the immunogenic potential of gadolinium-based magnetic resonance imaging (MRI) contrast agents, Sprague-Dawley rats were sensitized with gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) dimeglumine and with Gd DTPA covalently linked to either human serum albumin, dextran, or polylysine. IgG antibodies directed against Gd-DTPA were detected in immune sera by an enzyme linked immunosorbent assay (ELISA), and were confirmed by competitive inhibition of antibody binding using free Gd-DTPA dimeglumine. Antiserum induced by immunization with human serum albumin-(Gd-DTPA) was characterized by a monophasic competition curve with 50% inhibition (IC50) = 5.5 x 10(-4) M when Gd-DTPA dimeglumine was used as both the well-coating and the displacing agent in a competition ELISA. Antiserum induced by Gd-DTPA dimeglumine alone was characterized by a biphasic competition curve with IC50 = 6.5 x 10(-7) M and 7.9 x 10(-4) M. Antisera obtained after exposure to either dextran-(Gd-DTPA) or polylysine-(Gd-DTPA) were of insufficient titer for characterization. The detection of antibodies specific for Gd-DTPA suggests in vivo protein binding with formation of hapten-carrier conjugates. This hypothesis is supported by increased relaxivity values observed when Gd-DTPA dimeglumine is incubated in serum rather than in water. Gd-DTPA dimeglumine and albumin-(Gd-DTPA) are immunogenic in rats under idealized experimental conditions. Additional studies will be necessary to determine the potential for immunologic response in humans to gadolinium chelates under conditions of exposure inherent in clinical use. PMID- 1722487 TI - Central projections of auditory-nerve fibers of differing spontaneous rate. I. Anteroventral cochlear nucleus. AB - Auditory nerve fibers have been subdivided into three functional groups (Liberman, M.C. [1978] J. Acoust. Soc. Am. 63:442-455) differing in acoustic sensitivity and spontaneous discharge rate (SR). Using intracellular injection of horseradish peroxidase, the present study analyzes the projections of these three neuronal subclasses to the various subdivisions of the anteroventral cochlear nucleus (AVCN) and to the different cell types found therein. The average number of swellings and number of cells contacted decreased from low- to medium- to high SR groups. However, these differences in terminal elaboration were not evenly distributed throughout the AVCN. The small cell cap was almost exclusively innervated by low- and medium-SR fibers, i.e., those with the highest acoustic thresholds. Within anterior AVCN, spherical-cell innervation was seen from all SR groups, whereas almost all multipolar cell innervation was from low- and medium SR fibers. In the posterior AVCN, multipolar-cell innervation was equally likely from all SR groups, whereas globular cells were preferentially contacted by high SR fibers. These SR-based trends in cochlear nucleus innervation help explain some of the known physiological properties of cell-types in each subdivision. They also suggest that additional physiological study of the small cell cap may be key in elucidating the functional significance of the low-SR population. PMID- 1722488 TI - Morphological taxonomy of the neurons of the primate striatum. AB - A quantitative taxonomy of primate striatal neurons was elaborated on the basis of the morphology of Golgi-impregnated neurons. Dendritic arborizations were reconstructed from serial sections and digitized in three dimensions by means of a video computer system. Topological, metrical, and geometrical parameters were measured for each neuron. Groups of neurons were isolated by using uni- and multidimensional statistical tests. A neuronal species was defined as a group of neurons characterized quantitatively by a series of nonredundant parameters, differing statistically from other groups, and appearing as a separate cluster in principal component analysis. Four neuronal species were isolated: (1) the spiny neuronal species (96% of striatal neurons) characterized by spine-free proximal dendrites (up to 31 microns) and spine-laden distal dendrites, which are more numerous, shorter, and less spiny in the human than in the monkey, (2) the leptodendritic neuronal species (2%) characterized by a small number of long, thick, smooth, and sparsely ramified dendrites, (3) the spidery neuronal species (1%) characterized by very thick dendritic stems and a large number of varicose recurrent distal processes, and (4) the microneuronal species (1%) characterized by numerous short, thin, and beaded axonlike processes. All striatal neurons give off a local axonal arborization. The size and shape of cell bodies were analyzed quantitatively in Golgi material and in materials treated for Nissl-staining, immunohistochemical demonstration of parvalbumin and histochemical demonstration of acetylcholinesterase. Only three types were distinguishable: small, round cell bodies corresponding to either spiny neurons or microneurons, medium-size elongated cell bodies, which were parvalbumin-immunoreactive and corresponded to leptodendritic neurons, and large round cell bodies, which were acetylcholinesterase-positive and corresponded to spidery neurons. Thorough analysis of previously elaborated classifications revealed that spidery neurons do not exist in rats and cats and that large cholinergic neurons in these species correspond to leptodendritic neurons. From this, it can be assumed that the dendritic domain of striatal cholinergic neurons is considerably smaller in primates than in other species. Computer simulations based on both the frequency of each neuronal species and their three-dimensional dendritic morphology revealed that the striatum consists of two intertwined dendritic lattices: a fine grain lattice (300-600 microns) formed by the dendritic arborizations of spiny, spidery, and microneurons, and a large-grain lattice (1,200 microns) formed by the dendritic arborizations of leptodendritic neurons. This suggests that cortical information can be processed in the striatum through two different systems: a fine-grain system that would conserve the precision of the cortical input, and a large-grain system that would blur it. PMID- 1722489 TI - Distribution of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) cells and fibers in the monkey amygdaloid complex. AB - The NADPH-d histochemical method stains a selective population of neurons in the central nervous system. Although the functional significance of the enzyme in these cells is unknown, it has nonetheless proved to be a useful marker. In the present study we describe the distribution of NADPH-d-positive cells and fibers in the amygdaloid complex of the Macaca fascicularis monkey. NADPH-d-positive neurons were distributed throughout the amygdaloid complex. Based on the intensity of the reaction product, three different types of NADPH-d-positive cells were described: type 1 cells, the most intensely stained, varied in morphology and were most commonly found in the accessory basal, basal, and lateral nuclei and in the nucleus of the lateral olfactory tract; type 2 cells, the most common NADPH-d-positive cells, were more lightly stained, were generally stellate in shape, and were found in the lateral, basal, and accessory basal nuclei; type 3 cells were very lightly stained, oval or round in shape, and mostly found in the medial, anterior cortical, and paralaminar nuclei. NADPH-d staining was also associated with axonal fiber plexuses in various regions of the amygdala. The highest densities of stained fibers were found in the lateral nucleus, the parvicellular portion of the accessory basal nucleus, and the anterior amygdaloid area. The lowest densities of NADPH-d-positive fiber staining were found in the amygdalohippocampal area, in the lateral part of the central nucleus, and in the intercalated nuclei. In addition to the neuronal and fiber staining, a diffuse, blue neuropil staining was also observed, most commonly in the anterior cortical nucleus, the medial nucleus, the intercalated nuclei, and especially in the amygdalohippocampal area. The distribution of NADPH-d staining often respected nuclear boundaries within the amygdala and was particularly helpful in clarifying the borders of the amygdalohippocampal area. PMID- 1722490 TI - GABA-synthesizing neurons in the medulla: their relationship to serotonin containing and spinally projecting neurons in the rat. AB - GABA-synthesizing neurons were identified in the medulla of the rat by peroxidase antiperoxidase (PAP) immunohistochemistry for glutamic acid decarboxylase (GAD). Using diaminobenzidine (DAB) either alone or intensified with silver, a relatively large number of GAD-immunoreactive neurons were evident within the reticular formation, raphe nuclei and vestibular nuclei. In all these areas, profuse GAD-immunoreactive varicosities appeared to contact the soma and dendrites of both non-GABA and GABA neurons. These observations suggest that GABA neurons may act as interneurons or local projection neurons within the medulla and accordingly exert a potent inhibitory and/or disinhibitory control on bulbar projection neurons. Within the ventral reticular formation (pars alpha and ventralis of the gigantocellular reticular field) and raphe magnus, large numbers of prominent GAD-immunoreactive neurons resembled in size and morphology and overlapped in distribution the serotonin-immunoreactive neurons of the same regions. However, by sequential double immunostaining utilizing DAB as a chromogen for serotonin (5-HT) and benzidine dihydrochloride (BDHC) for GAD, it was found that GAD-containing neurons were distinct from 5-HT-containing neurons. Following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord and combined processing for WGA-HRP (using tetramethylbenzidine [TMB] with cobalt) and immunohistochemistry (with DAB), a contingent of spinally projecting neurons were found to contain GAD. The GAD-immunoreactive reticulo- and raphe-spinal neurons were most frequent within the pars alpha and ventralis of the gigantocellular reticular fields and the raphe magnus, where they were approximately equal in number to the coexistent, but distinct 5-HT spinally projecting neurons. GABA neurons of the medulla may thus contribute directly to the bulbar inhibitory influence upon spinal sensory and motor systems. PMID- 1722491 TI - Spinal distribution of ascending lamina I axons anterogradely labeled with Phaseolus vulgaris leucoagglutinin (PHA-L) in the cat. AB - The location of the ascending axons of spinal lamina I cells was studied in cats that received injections of Phaseolus vulgaris leucoagglutinin (PHA-L) in the superficial dorsal horn of the cervical or lumbosacral enlargement. Lamina I axons that could be ascribed to the spinothalamic tract (STT) were of particular interest. The cases were divided into three sets: in seven optimal cases the injections were restricted to lamina I; in ten nominal cases the injections involved laminae I-II or laminae I-III and occasionally lamina IV; and in eight mixed cases laminae I-V were injected. Since ipsilateral propriospinal and bilateral supraspinal axons originate from laminae I and V, but only ipsilateral propriospinal axons from laminae II-IV, this categorization facilitated a comparative analysis. Ascending axons labeled immunohistochemically with avidin/Texas Red were observed in oblique transverse sections from the C1, C3/4, T6, T12, and L3/4 levels. Incidental axonal labeling occurred in the ipsilateral dorsal columns because of passing primary afferent fiber uptake and, in nominal and mixed cases with involvement of laminae III-IV, in the superficial dorsolateral funiculus at the location of the spinocervical tract. Ipsilateral ascending lamina I axons in optimal cases were located in Lissauer's tract and in the white matter adjacent to the dorsal horn. Since these appeared to terminate in lamina I, and few remained at C1, they were ascribed to propriospinal projections. Contralateral ascending lamina I axons in optimal and nominal cases were distributed throughout the dorsal and ventral portions of the lateral funiculus (LF), but, despite considerable variability between animals in their location and dispersion, they were consistently concentrated in the middle of the LF (i.e., at the level of the central canal). This concentration was observed in a slightly more ventral location at C1, and a similar but weaker concentration of lamina I axons was located slightly more dorsally in C1 on the ipsilateral side. These supraspinal lamina I projections were ascribed to the spinomesencephalic tract (SMT) and to the STT. In mixed cases, additional ascending axons ascribed to lamina V cells were labeled in the ventrolateral and ventral funiculi. Many labeled axons were found in this region following a large injection of biocytin into lumbosacral laminae V-VIII in a supplementary case. These results thus together support previous descriptions of a dorsoventral distribution of STT axons according to laminar origin, but they contradict recent reports that lamina I axons ascend in the dorsolateral funiculus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722492 TI - Immunoadjuvant activity of oral Lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infections. AB - Lactobacilli, often used as effectors of host functions, could play an important role in maintaining human health by controlling other intestinal microorganisms capable of producing harmful effects. Using an experimental model, we studied the effect of different oral doses of Lactobacillus casei on the secretory IgA response and the protective capacity of the microorganism in preventing intestinal infections. The optimization of the protective dose of Lb. casei by previous feeding and the use of the lactobacillus as an immunological way to control enteric infections were investigated. We found that conventional mice were protected against infection with Salmonella typhimurium and Escherichia coli by previous feeding for 2 consecutive days with a daily Lb. casei dose of 1.2 x 10(9) cfu/mouse. Previous feeding for 7 d proved less effective, and feeding for 5 d afforded no protection at all. We were also able to demonstrate that the protective effect of Lb. casei against Sal. typhimurium and Esch. coli was connected mainly with the high level of IgA antipathogen antibodies present in intestinal secretions. beta-Glucuronidase (EC 3.2.1.31) and beta-galactosidase (EC 3.2.1.23) activities, measured both in the intestinal fluid and histological samples, showed a marked increase in intestinal inflammatory response on day 5 of feeding. These results show that Lb. casei plays an important role in the prevention of enteric infections, a low dose being enough for protection against intestinal infections by increasing IgA secretion into the intestinal lumen, thus providing adequate defences for the mucosal surface. A previously administered dose of this magnitude could therefore be used as an oral adjuvant in preventing enteric infections. PMID- 1722493 TI - Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA. AB - Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems. PMID- 1722494 TI - Human group II phospholipase A2. Characterization of monoclonal antibodies and immunochemical quantitation of the protein in synovial fluid. AB - Murine monoclonal and rabbit polyclonal antibodies were generated against human group II phospholipase A2 (PLA2) in order to study the role of this enzyme in inflammatory disease, the source of its synthesis, and the interaction of PLA2 with its substrate. Monoclonal antibody PLA187 exhibits potent inhibitory activity toward human PLA2 using autoclaved E. coli membranes as the substrate. Three other monoclonal antibodies (PLA184, PLA185, and PLA186) also inhibit enzyme activity, but with about 50-fold less potency. Based on the results of double-antibody competition experiments and enzyme inhibition profiles, PLA184 and PLA185 appear to recognize the same epitope. Monoclonal antibody PLA186 recognizes an epitope which is spatially distinct from that recognized by PLA184/185. The results also suggest that the epitope recognized by PLA187 may overlap with both epitopes recognized by PLA186 and PLA184/185. A double-antibody sandwich ELISA was developed using a combination of PLA185 and rabbit polyclonal antibody against PLA2. The ELISA provides a sensitive and quantitative method for monitoring specifically group II PLA2 in various biological sources, independent of factors which may affect enzyme activity. We have utilized this assay to quantitate PLA2 levels in synovial fluid from the joints of individuals with rheumatoid arthritis as well as from non-arthritic joints. Our results indicate that elevated levels of group II PLA2 in synovial fluid are not necessarily associated with arthritis. PMID- 1722495 TI - BLT esterase activity as an alternative to chromium release in cytotoxic T cell assays. AB - Granules released by cytotoxic T cells (CTL), during recognition and killing of target cells, contain granule enzyme A. This serine protease has an esterase activity, which is easily measured using the substrate benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). BLT activity, routinely used as an assay for granule release, provides an alternative to the standard chromium release assay as a measure of CTL-mediated killing. The two methods were highly comparable when either exogenous synthetic peptide or endogenously produced epitopes were used as targets and human CTL clones acted as effectors. The advantages of the BLT assay are that it uses inexpensive non-radioactive reagents, the assay can be run over any period between 4 and 30 h and can be performed with as few as 10(4) CTLs if synthetic peptide epitopes are used. PMID- 1722496 TI - Evaluation of blood lead level among population in Riyadh, Saudi Arabia. AB - Air pollution is a national problem, that recognizes no geographical or political boundaries. Most of the atmospheric lead is emitted from two main sources, motor vehicles and industrial sources, such as metal melting, coal and oil combustion, iron and steel production. The aim of the present work is to measure the blood lead level among residents near and far from the high ways, to evaluate the effect of motor vehicle emission on the environmental pollution along the high ways in Riyadh City. PMID- 1722497 TI - Idiotypic cascades associated with the CD4-HIV gp120 interaction: principles for idiotype-based vaccines. AB - Idiotypes (Id) are antigenic determinants expressed on the variable (V) region of the immunoglobulin molecule. Id-bearing antibodies, or Ab-1, are produced upon stimulation with a given antigen. Ab-1 may elicit the production of anti idiotypic antibodies (anti-Id) or Ab-2. The anti-Id also expresses Id determinants and may in turn elicit the production of anti-anti-Id or Ab-3. The production of Ab-1, Ab-2, and Ab-3 responses resulting from stimulation with the antigen is representative of components within an Id cascade. The existence of this Id cascade is the basis for the development of Id based strategies for controlling the immune response to infectious agents and tumors. In this review we will focus on several aspects regarding the Id cascades that may be operational during the immune response to the human immunodeficiency virus (HIV). In light of several studies which suggest the existence of Id-anti-Id interactions operating during the course of HIV infection, we will discuss the potential applications of Id based strategies in manipulating the immune response to HIV. PMID- 1722498 TI - HIV-neutralizing antibodies: epitope identification and significance for future vaccine. PMID- 1722499 TI - Peptide component vaccine engineering: targeting the AIDS virus. AB - Most of the successful vaccines developed to date induce protective immunity resembling that produced by natural infection. HIV infection does not induce protective immunity. Thus, previously successful approaches based on live- or killed-virus preparations may not yield an effective and safe AIDS vaccine and many feel that a more highly engineered vaccine will be required. Synthetic peptides represent extremely powerful tools for vaccine research and construct optimization. The theory and practice of vaccine engineering using synthetic peptide components is reviewed with special emphasis on progress towards development of a vaccine for AIDS. PMID- 1722500 TI - Biologically active transcripts of a large satellite RNA from arabis mosaic nepovirus and the importance of 5' end sequences for its replication. AB - Synthetic transcripts of a satellite RNA associated with a lilac isolate of arabis mosaic nepovirus (ArMV) were made from cDNA clones. Transcripts having either six (M1R) or 29 (M3R) extra nucleotides at their 5' ends replicated in the presence of ArMV genomic RNA in manually inoculated Chenopodium quinoa plants, even though M1R also differs from the native sequence at nucleotide position 2. Transcript 12R, which has 11 guanosyl residues and 27 other nucleotides not present in the natural satellite RNA at its 5' end, and also lacks the two 5' terminal nucleotides (UA), replicated inefficiently, both in transformed tobacco plants and in plants that had been manually inoculated. Transcripts from another construct (M2R) lacking eight 5'-terminal bases of the native sequence did not multiply in plants. Each of these transcripts directed the in vitro synthesis of a protein (Mr 39K) encoded by satellite RNA, although 12R was the least efficient message. Analysis of the 5'-terminal sequences in progeny RNA from M1R showed that the non-native bases were removed and the second nucleotide corrected, suggesting that VPg plus a few initial 5'-terminal bases might serve as a primer for plus-strand synthesis of this satellite RNA. When M1R was inoculated with genomic RNAs from ArMV of ash or ivy, the transcripts replicated and were encapsidated. However, when the same amounts of M1R were inoculated with genomic RNAs of ArMV from hop or sugar-beet, progeny of the transcripts were not detected either in virions or in plants. Less surprisingly, this RNA transcript did not multiply in the presence of dogwood mosaic, strawberry latent ringspot, grapevine fanleaf or cherry leaf roll nepoviruses. PMID- 1722501 TI - Monoclonal antibodies to three structural proteins of avian infectious bronchitis virus: characterization of epitopes and antigenic differentiation of Australian strains. AB - Ten monoclonal antibodies (MAbs) directed against three structural proteins of infectious bronchitis viruses (IBV), the peplomer (S), membrane (M) and nucleocapsid (N) proteins, were characterized and used to determine the antigenic relationship between Australian IBV strains. One MAb (MAb 5) was directed against an epitope on the S1 subunit of the peplomer, another (MAb 2) against an epitope on the M glycoprotein and eight MAbs (MAbs 1, 7, 9, 16, 24, 26, 27 and 51) were directed against seven non-overlapping epitopes on the N protein. None of the MAbs neutralized infectivity or inhibited haemagglutination of the virus. Conservation of the nine epitopes detected by these MAbs was determined in 13 serotypes of Australian IBV strains. Only epitope 5 on the S1 subunit of the peplomer was conserved in all strains. Epitope 2 on the M protein showed a high degree of conservation although this epitope was absent from four strains. None of the eight epitopes on the N proteins was conserved in all IBV strains but four epitopes (1, 16, 24 and 27) showed a high degree of conservation. Epitope 9 on the N protein was present only in IBV strains of one serotype whereas epitope 7 on the N protein distinguished vaccine viruses of serotype B from other IBV strains. The presence or absence of nine epitopes on three structural proteins differentiated IBV strains into five antigenic groups. PMID- 1722502 TI - Bovine immunodeficiency virus: immunochemical characterization and serological survey. AB - Bovine immunodeficiency virus (BIV) was purified by isodensity centrifugation; viral activities were monitored in gradient fractions using the reverse transcriptase assay and a p26-specific monoclonal antibody ELISA. In the coincident peak fractions (density about 1.17 g/ml) proteins with Mr values of 26K, 17K, 53K, 14K and 100K (with decreasing intensity) were detected by Western blotting using serum of a calf after experimental BIV infection. When 957 randomly collected cattle sera from The Netherlands were tested by indirect immunofluorescence and confirmed using Western blot and/or radioimmunoprecipitation, 1.4% appeared seropositive. Thus BIV infection is not uncommon in one European cattle population. PMID- 1722503 TI - Proliferative responses of T cells primed against human rhinovirus to other rhinovirus serotypes. AB - Lymphocytes from mice immunized with human rhinovirus (HRV) serotypes 1A or 15 proliferated in vitro in response to HRV and the activated cells were shown to be helper T (Th) cells. Lymphocytes from mice primed with HRV-1A responded to seven of eight heterologous virus serotypes, the responses to other minor cell receptor group viruses being greater than to those belonging to the major cell receptor group. A similar bias was seen with cells from mice primed with HRV-15 in that they responded preferentially to other major receptor group viruses. This pattern of cross-serotype recognition was shown to be similar in three inbred mouse strains and was not dependent upon the major histocompatibility complex haplotype. These results have revealed that there are determinants within the viral proteins of a number of serotypes of HRV that are recognized by Th cells primed against a single HRV serotype. Thus, at the level of Th cell recognition of HRV, a cross-serotype reactivity is seen which is not reflected in the B cell antibody response to virus, which is generally highly serotype-specific. PMID- 1722504 TI - Evidence that human cytomegalovirus assembly protein shares antigenic sites with an uninfected cell membrane protein. AB - Immunological abnormalities of an autoimmune nature often develop during acute primary human cytomegalovirus (HCMV) infection. IgM antibodies reacting with the membrane of uninfected human embryonic fibroblasts can be detected in most patients undergoing a primary HCMV infection. In this work, we have found that there is a common antigenic epitope shared by a cell membrane component of Mr 60K (mp60), which is recognized by IgM in sera from patients with primary HCMV infection, and a linear determinant in the C-terminal half of the HCMV assembly protein of Mr 38K (vp38), which is known to be one of the most IgM-reactive antigens of HCMV. While vp38 seems to contain other specific IgM-reactive regions, IgM reactivity to mp60 is due exclusively to this shared epitope. Furthermore, mp60 is found abundantly on the surface of human red blood cells, a possible explanation for the pathogenesis of the haemolytic anaemia that may appear during primary HCMV infection. PMID- 1722505 TI - Detection of B19 parvovirus in human fetal tissues by electron microscopy. AB - We present the electron microscopy observations on samples from 38 pregnancies that were investigated for B19 parvovirus infection. Thirty-four had resulted in fetal loss thought to be due to a virus infection and 22 of the 38 were positive for B19 parvovirus in one or more of the tissues. Twenty-one placentas and 75 fetal tissue samples were examined. Fresh samples were investigated by immune electron microscopy while formalin-fixed tissues were examined as thin sections and by negative staining of tissue extracts with direct electron microscopy. Electron microscopy was more sensitive on fresh than on fixed samples. The ultrastructural observations on thin sections of fixed tissues yielded new information locating B19 parvovirus particles in both nucleus and cytoplasm of infected fetal cells. The diagnostic results of the range of electron microscopy assays were compared with those of two hybridization methods. The fresh samples yielded comparable results from electron microscopy and hybridisation assays but on formalin-fixed materials hybridisation was more sensitive. PMID- 1722506 TI - A developmental handshake: neuronal control of ionic currents and their control of neuronal differentiation. PMID- 1722507 TI - Development of ion channels in early embryos. PMID- 1722508 TI - Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain. AB - Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division. PMID- 1722509 TI - Homoarginine labeling is suitable for determination of protein absorption in miniature pigs. AB - Intestinal proteolysis and absorption of dietary protein was followed using a chemical label of the lysine side-chain. Protein-bound lysine was transformed into homoarginine (HA), an amino acid that is not used for protein synthesis. Disappearance in the intestinal tract of HA originating from labeled casein and soybean isolate and appearance in peripheral blood was followed in four miniature pigs fitted with permanent T-cannulas at the proximal jejunum and ileum. Less than 0.2% of HA appeared in the digesta at the ileum when up to 13.5 mmol of HA was infused either into the jejunum or jugular vein. Several factors suggest that HA in fact traces the exogenous (dietary) protein: 1) the quick postprandial appearance of HA in plasma; 2) the high (greater than 97%) oro-ileal absorption of HA-labeled casein and soybean isolate; 3) the similarity of chymotryptic proteolysis in vitro of guanidinated and native casein; 4) the fact that only trace amounts of the marker reenter the intestinal lumen from the blood. Therefore HA-label is suitable for the differentiation of exogenous and endogenous protein in the chyme and thus the measurement of oro-ileal protein absorption and irreversible loss of endogenous protein in the chyme of the small intestine. PMID- 1722510 TI - Sputum Gram stain controls. PMID- 1722511 TI - Mechanical and chemical root preparation in vitro: efficiency of plaque and calculus removal. AB - The aim of this investigation was to compare the effectiveness of several different methods of root instrumentation by measuring and comparing the amount of residual stained material following treatment. Also, 2 different methods of quantitating residual stained material were compared. A total of 90 periodontally involved teeth were extracted and randomly assigned to 1 of 8 treatment groups or to the untreated control group. Experimental treatments consisted of one or a combination of the following: Columbia 13-14 curet, P-10 ultrasonic instrument, diamond-coated P-10 ultrasonic instrument, or antiformin/citric acid chemical treatment. Selected samples were examined using light microscopy in order to determine the amount of cementum removed during root instrumentation. Residual stained material was quantitated using a method of grid-square analysis as well as by the use of photographs and a digitizing tablet. Following instrumentation, it was noted in histologic sections that the complete removal of cementum was rare, although all of the cementum was removed by some experimental treatments in some areas. All mechanical methods of root instrumentation were found to be essentially equal in effectiveness with respect to the removal of plaque and calculus. Chemical debridement alone was found to be ineffective. It was further noted that the grid-square method of analysis produced measurements that were 2 to 8 times higher than measurements produced by the digitizing tablet. PMID- 1722512 TI - Gold treatment of rheumatoid arthritis decreases synovial expression of the endothelial leukocyte adhesion receptor ELAM-1. AB - Leukocyte adhesion receptors on endothelial cells play an important role in the evolution of synovitis. We studied sequential synovial biopsies at Weeks 0, 2 and 12 in 11 patients with rheumatoid arthritis beginning parenteral gold therapy either alone or combined with 120 mg intramuscular methylprednisolone acetate at Weeks 0, 4 and 8 of treatment. Expression of endothelial leukocyte adhesion molecule 1 (ELAM-1) decreased on synovial blood vessels after both 2 and 12 weeks treatment (p less than 0.05), while the overall vascularity of the synovium did not change. Neutrophil numbers within the synovial membrane also decreased although this did not reach statistical significance. In contrast, there was no significant change in numbers or subset distribution of T cells or in Class II MHC expression by synovial lining cells, mononuclear cells or endothelial cells. Our results suggest that one of the early effects of intramuscular gold and glucocorticoid therapy may be a downregulation of the acute inflammatory process associated with the endothelial expression of a neutrophil adhesion receptor and the subsequent recruitment of neutrophils into the joint. PMID- 1722513 TI - Channel reconstitution in liposomes and planar bilayers with HPLC-purified MIP26 of bovine lens. AB - The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than +/- 20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60-80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (approximately 120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 to 7 sec. Cs+ did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs+ occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na+ solutions (zero K+). These data indicate that MIP26 channels are not K(+)-selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels. PMID- 1722514 TI - Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers. AB - Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl2 buffers (cis, 100 mM/trans, 25 mM), single channel conductances at -40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (PNa:PBa:PCl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.23:0.04:1 and 0.05:0.03:1, respectively. Both channels exhibited characteristic voltage dependent bursting activities. Open probability for type I channels had a maximum of approximately 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from the cis side, while it blocked type II channels from either side. PMID- 1722516 TI - [Transvesical prostatectomy for benign prostatic hyperplasia--a 364 cases analysis]. AB - During a 16 year period from January 1972 to December 1988, transvesical prostatectomies were performed on 364 patients with benign prostatic hyperplasia in the Department of Urology, Kaohsiung Medical College Hospital. The average operational procedure time was 116.1 minutes and the average amount of tissue resected was 38.3 grams. The most common complications were wound infections (6.8%), stress incontinence (1.92%), urethral stricture (4.12%) and impotence (0.27%). In this series, epididymitis and/or orchitis was noted in 4.3% of those who underwent vasectomies and 1.8% of those who did not; thus, epididymitis cannot be prevented by a prophylactic vasectomy. There was one postoperative death (mortality rate-0.27%). The present study found that 6.86% of the transvesical prostatectomy patients will require longer hospitalization and greater amounts of antibiotics due to post-operative wound infection; therefore the use of transvesical prostatectomy will decline and only be recommended for fit patients with others genitourinary diseases such as bladder diverticulum or giant bladder calculus. PMID- 1722515 TI - Ca(2+)-dependent chloride conductance in Necturus taste cells. AB - This report describes the occurrence and localization of a Ca(2+)-dependent chloride conductance in taste cells of Necturus maculosus. Lingual epithelium from Necturus was removed with blunt dissection and mounted in a modified Ussing chamber which allowed individual taste cells to be impaled with intracellular micropipettes. Solutions in the mucosal and serosal chambers could be changed independently and the properties of apical and basolateral membranes tested separately. Action potentials in taste cells, elicited by brief depolarizing current pulses passed through the intracellular recording microelectrode, provided an accurate description of whether voltage-dependent conductances had been blocked or unmasked by the experimental conditions. We found that Ca2+ influx during the action potential triggers a prolonged depolarization due to Ca(2+)-dependent conductance changes, particularly in the presence of TEA to block repolarizing K+ currents. This afterdepolarization could last up to 7 sec and is due, in part, to a Ca(2+)-dependent Cl- conductance. Other Ca(2+) dependent channels such as Ca(2+)-dependent K+ channels or nonselective cation channels may also contribute to the afterpotential. Calcium-dependent conductance channels were situated on apical and basolateral membranes of the taste cells. We speculate that Ca(2+)-dependent Cl- channels may play a role in discriminating chloride salts from salts of other anions and may help shape receptor cell responses elicited by taste stimuli. PMID- 1722517 TI - Clinical study on coupling interval of ventricular premature contraction in patients with organic heart disease. AB - The characteristics of the coupling interval (CI) of ventricular premature contraction (VPC) were studied in 100 patients with frequent VPCs using 24 h ambulatory ECG recording. All R-R intervals were registered on computer to determine the mean value of CIs (Mean CI) and the standard deviation of CIs (SD CI). We compared the Mean CI and the SD-CI between idiopathic VPCs and VPCs with organic heart disease (OHD). In addition, we evaluated the efficacy of disopyramide (DP) and mexiletine (MX) and we examined the relationship between the efficacy of these drugs and the characteristics of CI. The Mean CI of VPCs with OHD was longer than that of idiopathic VPCs (530 vs. 494 msec, p less than 0.05). The SD-CI of VPCs with OHD was larger than that of idiopathic VPCs (54.1 vs. 39.3 msec, p less than 0.01). In all treated cases, the drug efficacy was not different between DP (11/18, 61%) and MX (9/19, 47%). However when we isolated cases of OHD, we found a tendency that DP (9/12, 75%) was more effective than MX (7/16, 44%). In cases where DP was administered, the Mean CI of VPCs was not different between effective and ineffective cases, while in cases where MX was administered, the Mean CI of ineffective cases had a tendency to be longer than that of effective cases (552 vs. 506 msec, p less than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722518 TI - [Autoantibodies related to interferon therapy in chronic hepatitis B may affect the therapeutic response to interferon]. AB - We have evaluated the problem of autoantibodies such as antinuclear antibodies (ANA), smooth muscle antibody (SMA) and antibodies to thyroid microsomal (TMA) and to thyroglobulin (TGA) related to interferon therapy in 27 patients with chronic hepatitis B. Anti-interferon antibody was also studied by Western blot method. Eight patients had ANA and 2 had SMA during interferon therapy. However, 6 of the 8 patients were ANA positive and one of the 2 was SMA positive prior to interferon treatment. No patients developed TMA, TGA or anti-interferon antibody. Eight (29.6%) of the 27 patients had clearance of both DNA polymerase and HBeAg and persistent normalization in alanine aminotransferase levels with interferon therapy. Seven of the 8 responders developed none of the autoantibodies related to interferon therapy. These results suggest that the presence of ANA or SMA during treatment may affect the therapeutic response to interferon. PMID- 1722519 TI - [Treatment of chronic diseases of the small intestine]. AB - Multimodality and differentiated treatment of small-intestinal diseases is to combine methods of etiological action with pathogenetic treatment of the main clinical syndromes: chronic diarrhea, malabsorption syndrome, hypercatabolic exudative enteropathy. Each nosological form should be treated specifically. Pathogenetic treatment involves diet therapy, chemotherapeutic correction of metabolic processes (vitamin administration, recovery of normal protein and lipid metabolism, water and electrolyte balance, anemia), management of chronic diarrhea. Treatment regimens are specified for gluten enteropathies, total variable immunodeficiency, Whipple disease, small-intestinal diverticulosis, Crohn's disease, amyloidoses, intestinal lymphoma and retroperitoneal lymph nodes. Clinical experience justifies the above methods as highly effective. PMID- 1722520 TI - [A method of determining middle molecules]. AB - The authors suggest a method for the detection of 'medium molecules' to be used for rapid diagnosis of poisoning. They present their arguments in favor of a higher specificity of this method as against other rapid methods for 'medium molecule' detection in biologic fluids of the body. The authors' data evidence a high correlation between the results of the suggested method and gel filtration technique. The procedure may be used for the rapid diagnosis of peptidemia severity in the patients with grave endogenic intoxication and in cases when endotoxicosis is not the underlying condition. PMID- 1722521 TI - [The significance of middle-molecular peptides in the blood in acute forms of ischemic heart disease]. AB - Estimation and analysis of correlations permit disclosing principally new mechanisms in assessment of the homeostasis. The authors have derived curvilinear correlations between medium-molecular peptides, immune complexes, and lipids in coronary patients with the acute forms of the condition. Close relationships (high correlations) between homeostasis parameters were revealed, that were found more manifest in the patients with unstable angina pectoris. The immune complexes and lipids are regarded as the principal signs, and the medium-molecular peptides are functionally related to these. A lower correlation between the parameters and change of the argument/functional relationships in the patients with myocardial infarction are characteristic of essential imbalance of the homeostasis system. PMID- 1722522 TI - [Lipid peroxidation and the antioxidant activity of the sputum in chronic obstructive bronchitis]. AB - A method for measuring lipid peroxidation (LPO) products and antioxidant activity of the sputum in patients with chronic obstructive bronchitis is suggested. Both measurements are based on the reaction of LPO products with thiobarbituric acid. PMID- 1722523 TI - [The use of a fatty emulsion for determining blood lipids in parenteral nutrition]. AB - The author has investigated the possibility of using as the reference agent in estimation of the resorption rate from vascular bed of fatty emulsions the very agent that is introduced or the oil, one of the ingredients of this agent. Fatty emulsions for parenteral nutrition intralipid (Sweden), lipofundin-C (Finland), venolipid (Japan), lipidin and lipidin-2 (USSR), and specially purified sunflower oil were used to plot the calibration curve. PMID- 1722524 TI - [The use of biochemical indices in the prognosis and diagnosis of ischemic heart disease]. AB - The authors review the experience gained in search for the prognostic and diagnostic criteria of coronary heart disease and its various forms. They recommend using the index of red blood cell fatty acid unsaturation as the characteristic, indicating liability to coronary disease; lysosomal beta glucosidase activity for assessment of the severity of myocardial injury; simultaneous measurements of hydroxyproline and uric acid for estimation of the completeness of cicatrization and of the myocardial injury severity; and the fatty acid unsaturation index and hydroxyproline level for the assessment of the efficacy of rehabilitation measures. PMID- 1722525 TI - [A method of determining glucose oxidase-immobilized glucose]. AB - A method for manual measurement of glucose in biologic fluids has been developed, making use of glucose oxidase immobilized on a carbamide derivative of microcrystal cellulose; two variants are suggested: a rapid and a routine one. The method is characterized by a high analytical reliability, its results are in high correlation with the results of measurements by Beckman glucose analyzer (r = 0.92, p less than 0.001). The method is economic (glucose oxidase reagent may be used for more than 300 times), easily available, and is 3 to 6 times more rapid than the method with soluble glucose oxidase. It is particularly convenient for urgent laboratory diagnosis. PMID- 1722526 TI - [Determination of thrombocyte aggregation by a modification of K. Wu and J. Hoak's method]. AB - The authors review the results of estimating platelet spontaneous aggregation by Wu and Hoak's method and by its modifications. They describe a modification of the procedure that essentially improves the accuracy of investigation, is sufficiently sensitive, simple, and rapid. Its informative value was confirmed in experiments with spontaneous platelet aggregation in rabbits with cholesterolemia. PMID- 1722527 TI - [Methods of isolating intact thrombocytes from blood]. AB - A combined method of platelet isolation in albumin density gradient followed by gel filtration through a column packed with Sepharose 2B appears to be the best for the isolation of intact platelets free from plasma proteins, fit for research purposes; for clinical studies a more available technique for intact platelet isolation in albumin density gradient is recommended. PMID- 1722528 TI - [The cytochemical characteristics of neutrophil leukocytes in lung cancer before and during antineoplastic chemotherapy]. AB - A number of cytochemical characteristics and the NBT test were studied in neutrophilic granulocytes of 194 patients with diffuse pulmonary carcinoma (Stages III-IV), 31 patients with chronic nonspecific pulmonary diseases, and 20 normal subjects. Changes in the neutrophilic morphology and function were revealed in lung cancer patients, presenting as elevated alkaline phosphatase activity, reduced myeloperoxidase activity and lipid and glycogen levels, increased endogenous activation of the neutrophils in the NBT test, and decreased reaction activity in zymosan stimulation. Antitumor chemotherapy involved a lowering of the cationic protein level, as well of the acid phosphatase activity, and elevation of glycogen content. Stimulated NBT test was highly sensitive to cytostatic therapy. Tumor dissemination and morphologic variant contributed to changes in the neutrophilic morphology and function. PMID- 1722529 TI - [Laboratory diagnosis of secondary hyperparathyroidism (review of the literature)]. PMID- 1722530 TI - [The effect of smoking on the composition of the peripheral blood in normal subjects]. AB - The peripheral blood erythrocytic and leukocytic status was studied in 60 healthy young tobacco-smokers and in 30 non-smokers. The smokers were divided into two subgroups, each with 30 members: those smoking for not more than 5 years and those smoking for 6 to 10 years. A trend to inhibition of erythro- and leukocytopoiesis was detected in Subgroup 1 tobacco smokers: reticulocyte maturation rate was reduced, as was bone marrow production and the level of circulating red cells, macrocyte count was increased and planocytosis was likely to develop, leukocyte counts were decreased at the expense of the neutrophils, eosinophils, and monocytes; basophil count was growing. Subgroup 2 tobacco smokers presented with normalization of erythro- and leukocytopoiesis: reticulocyte maturation rate was growing, as was bone marrow production and the count of circulating red cells, erythrocytogram normalized, leukocyte count was increasing at the expense of the neutrophils, eosinophils, and lymphocytes; basophil count has decreased. The detected changes in the peripheral blood erythro- and leukocytic composition, related to the duration of tobacco-smoking, appear to reflect different phases of tobacco smoke toxic product effects on the bone marrow and the formation of the defense, adaptive, allergic, and immunologic reactions of the body in conditions of prolonged tobacco antigenemia. PMID- 1722531 TI - [Determination of the reactive luminol-dependent chemiluminescence of neutrophils]. AB - Reactive luminol-dependent neutrophilic chemiluminescence was estimated in 25 children aged 4 to 14. No essential differences in spontaneous and induced chemiluminescence of venous and capillary blood samples diluted 1:100 were detected. Therefore both methods may be used. PMID- 1722532 TI - [Automation of hematologic studies in a clinical-diagnostic laboratory in a multi profile hospital]. AB - The authors share their experience gained with automation of hematologic investigations at a clinical diagnosis laboratory. A CDL information system, basing on personal computers, has been created, functioning in complex with hematologic autoanalyzers. The reports on the laboratory's activity are made automatically, as is the laboratory register and processing of applications for tests. PMID- 1722533 TI - [Immunologic indices in chronic pancreatitis and in its combination with other diseases of the digestive system]. AB - Analysis of the immune system status of 200 patients with chronic pancreatitis has shown more marked immune shifts in the patients with the primary condition, who presented with high levels of immune complexes and autosensitization to pancreatic tissue. When pancreatitis was associated with intestinal abnormalities, T lymphocyte function was depressed, as was nonspecific defense of the body. In cases when chronic pancreatitis resulted from diseases of the bile duct, stomach, or duodenum, the findings evidenced high blood serum levels of immunoglobulins A, G, and M, and significant bacterial sensitization. These results may be useful in choosing immunocorrecting therapy. PMID- 1722534 TI - [Identification of lymphocyte subpopulations using monoclonal antibodies]. AB - Presents the tube variant of the indirect immunofluorescence method with IKO monoclonal antibodies, that permits studies of the immune status of humans (with 4-5 monoclonal antibodies) without sophisticated equipment or unavailable reagents; the method is simple and rapid. PMID- 1722535 TI - [The effect of heterophilic antibodies on the result of the passive hemagglutination reaction in intestinal infections]. AB - When commercial erythrocytic diagnostic agents, prepared from sheep red cells, are used in the passive hemagglutination test with blood sera from patients with intestinal infections, the possibility of nonspecific reactions with heterophilic antibodies of human body should be borne in mind. To completely eliminate these antibody effects on the results of the test, titration of the tested serum should be started from 1:64, 1:80, or 1:100, depending on what titration scale is used. PMID- 1722536 TI - [A rapid indication of Helicobacter pylori in patients with gastroduodenal pathology]. AB - Rapid detection of H. pylori in samples from gastroduodenal patients is based on the use of standard ingredients made in the USSR: disks with urea from kits and indicator strips to show pH values of the Rifan type, within the pH range 5.8 7.4. This method helps detect pH changes emerging in H. pylori urea disintegration in the liquid and gaseous (air) phases. The sensitivity, specificity, and rate of the test are not inferior to those of the commercial urease test. PMID- 1722537 TI - [Determination of the cytotoxic activity of Vibrio cholerae]. AB - Cytotoxic activities of broth culture supernatants of 39 V. cholerae strains 01 and non 01 were studied in L-929 cell cultures. The examined strains differed by the cytotoxic factor production, which factors were identified (with the use of anti-cytolysin serum) as cytolysin. The strains capable of choleric enterotoxin secretion did not produce cytolysin. PMID- 1722538 TI - [The activity of antiseptics and disinfectants against different species of non fermenting gram negative bacteria]. AB - The efficacies of 7 antiseptics and disinfectants, routinely used in clinical surgery, in respect of six nonfermenting gram-negative bacterial species were under study. The microorganism strains were isolated from various objects in a surgical hospital. The detected differences in the activities of the tested agents towards different microorganism species point to the necessity of a differentiated approach to the choice of antiseptics and disinfectants with due consideration for their effects on nonfermenting bacteria. PMID- 1722539 TI - [Characteristics of the biochemical properties of pseudomonads isolated from environmental objects]. AB - Biochemical characteristics of 117 Pseudomonas cultures isolated from water bodies and washings off the hospital environment objects were under study, including the strains from patients' wound surfaces. Analysis of the strains' characteristics permitted referring 83.8 +/- 3.7% of them to typical ones as regards their biochemistry. 45.9 +/- 7.5% of these were P. putida, 21.4 +/- 4.8% were P. aeruginosa, 10.2 +/- 1.01% P. fluorescens strains, and the shares of P. cepacia and P. stutzeri strains were 11.2% each. 16.2% of the strains were found atypical because of lysine decarboxylase and growth at 42 degrees C; they were conditionally referred to P. putida. One third of these cultures were isolated in hospital. The detected differences may be helpful in identification of Pseudomonas. PMID- 1722540 TI - [A rapid method of determining the anti-lysozyme activity of microorganisms]. AB - Rapid methods for anti-lysozyme activity measurements, developed by the author, permit reducing the time of investigation to 24 and 4 hrs. Both the modifications are based on the routine technique and are not inferior to it in sensitivity and specificity. PMID- 1722541 TI - [The 20th anniversary of the quality control system in Finnish laboratories]. PMID- 1722542 TI - [An apparatus for spraying chromatographic plates and paper]. PMID- 1722543 TI - [The cytologic diagnosis of papillary syringadenoma]. PMID- 1722544 TI - [Determination of anticonvulsants using high performance liquid chromatography]. PMID- 1722545 TI - [A method of recording shifts in the leukocyte formula]. PMID- 1722546 TI - [An apparatus for automated control of exposure in microphotography using a photo flash]. AB - The authors suggest a device for automated exposure in micro-photographing with the use of the Elektronika B5-22 flash light. This pulsed light source is optimal as regards its spectral composition. The gist of the method is as follows: the flash photographic element, built in a light-proof case, disposed on the microscope ocular, permits governing the light pulse length. PMID- 1722547 TI - [Equipment for microphotography using the Zenit camera]. PMID- 1722548 TI - [Determination of the antioxidant properties of the blood and their diagnostic significance in the elderly]. AB - Blood antioxidant system parameters were examined in elderly subjects. The authors have developed methods for measurements of catalase, superoxide dismutase, and lipid peroxidation products. They introduce a new factor 'F' that is supposed to characterize the blood antioxidant system; this factor is based on the values of catalase and superoxide dismutase activities and the intensity of lipid peroxidation. The authors come to a conclusion that the blood antioxidant and oxidant systems may be more accurately described with the use of this new factor F. In case of an abdominal tumor whole blood catalase level is elevated and superoxide dismutase activity significantly reduced. Factor F values were found extremely low before death, therefore this factor may be considered an important criterion of a critical state. The blood antioxidant parameters of patients with diabetes mellitus and essential hypertension did not much differ from those of age-matched healthy subjects. PMID- 1722549 TI - Effects of recombinant human granulocyte and granulocyte-macrophage colony stimulating factors on neutrophil function following autologous bone marrow transplantation. AB - Functional activity of peripheral blood neutrophils was assessed in eight patients at 4, 6, 8, 10 and 12 weeks following autologous bone marrow transplantation (ABMT). Functions studied included superoxide generation (O2-) intracellular killing of Staphylococcus aureus, phagocytosis and killing of Candida albicans. Neutrophils were tested following in vitro preincubation with 300 pM granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.2 nM granulocyte colony-stimulating factor (G-CSF) or buffered solution (diluent) as control. Our data indicate that during the early period (weeks 4-6) following ABMT most of the patients exhibited diminished neutrophil oxidative metabolism, defective phagocytosis and killing of C. albicans and reduced capacity to kill S. aureus. In some patients a gradual increase in the functional activity of neutrophils occurred with time. Both GM-CSF and G-CSF induced in vitro amplification of (a) O2- production in response to fmet-leu-phe (FMLP) (b) phagocytosis and killing of C. albicans and (c) killing of S. aureus. This study suggests that GM-CSF and G-CSF may enhance the depressed functional activity of neutrophils following ABMT. PMID- 1722550 TI - Basis for natural resistance to methotrexate in human acute non-lymphocytic leukemia. AB - The basis of intrinsic resistance of blasts from patients with acute non lymphocytic leukemia (ANLL) to methotrexate was studied. MTX polyglutamate formation was measured in blast cells from 19 patients with ANLL and in 7 pediatric patients with acute lymphocytic leukemia (ALL), after in vitro incubation for 24 h with 3H-methotrexate. There was no significant differences seen in the total amount of MTX plus polyglutamates measured between ANLL and ALL blasts, indicating that transport defects do not account for intrinsic MTX resistance in ANLL. However, there were significant differences between the amounts of long chain MTX polyglutamates found in ANLL cells as compared to ALL cells. Most, but not all, ANLL blasts were unable to form long chain polyglutamates. In as much as the level of MTX polyglutamates found in blast cells after MTX administration allows for retention of this drug, this property may explain, at least in part, the refractoriness of most patients with ANLL to methotrexate. PMID- 1722551 TI - Human respiratory mucosa in a nonadhesive stationary organ culture system. AB - Fragments of human adenoid tissue were transferred to a nonadhesive, stationary organ culture system. The culture period was 40 days. In culture, beating cilia could be observed at the surface of the fragments. Light microscopy, scanning electron microscopy, and transmission electron microscopy showed that the tissue fragments were covered by a multilayered, pseudostratified, ciliated epithelium. Beneath the epithelium was a basement membrane. At the start of the culture period, the central parts of the fragments were dominated by lymphocytes. These lymphocytes gradually disappeared and were replaced by a collagen-containing stroma with scattered fibroblasts. The tissue fragments can be used as an organ culture model for normal respiratory mucosa. PMID- 1722552 TI - Region-specific regulation of preproenkephalin mRNA in cultured astrocytes. AB - Regulation of preproenkephalin (PPE) mRNA was examined in astrocytes cultured from several regions of the neonatal rat brain. Astrocytes from these regions expressed differing levels of PPE mRNA, with higher levels in astrocytes from the hypothalamus followed by frontal cortex and striatum. Further, PPE mRNA was regulated differently in hypothalamic than in striatal glia. Treatment of striatal astrocytes with the beta-adrenergic agonist, isoproterenol, or with agents which directly increased intracellular cAMP (forskolin or 8-bromo-cAMP) elevated levels of PPE mRNA. By contrast, none of these treatments altered levels of PPE mRNA in hypothalamic astrocytes despite increasing cAMP levels 60-fold. These observations indicate that there is striking regional heterogeneity in the expression and regulation of PPE mRNA by astrocytes, suggesting that proenkephalin or its derived peptides help to mediate region-specific brain functions. PMID- 1722553 TI - Molecular analysis of the responder satellite DNA in Drosophila melanogaster: DNA bending, nucleosome structure, and Rsp-binding proteins. AB - The Responder (Rsp) locus of Drosophila melanogaster, the target locus of segregation distortion, is a satellite DNA array. This repeat array imparts some fitness advantage to the chromosomes bearing it. In this paper, we report the following three related molecular properties of this satellite repeat: (1) Sequence-directed curvature--On a polyacrylamide gel, Rsp-containing fragments migrate slower than would be predicted on the basis of their physical sizes. The extent of migration retardation correlates with the size and position of the Rsp sequence in a DNA fragment, suggesting that Rsp DNA is bent. The bending is shown to be affected by a DNA-binding drug (Hoechst 33258). (2) Nucleosome structure- Nucleosomes associated with Rsp repeats have an unusual spacing pattern. Instead of being spaced at approximately 190-bp intervals as is the bulk chromatin, they are separated at approximately 240-bp intervals, roughly the size of a dimeric Rsp repeat. The nucleosomal structure in the Rsp region is preferentially disrupted by Hoechst 33258, whereas the bulk chromatin appears to be insensitive to the drug. (3) Rsp-DNA binding proteins--Gel mobility-shift assays using nuclear extracts from pupae and end-labeled Rsp repeat demonstrate the presence of three distinct DNA-protein complexes. Competition assays suggest that these complexes are specific to the Rsp sequence, and two of these nucleoprotein complexes seem to be influenced by the presence of Hoechst 33258. The observed complexes are formed by nonhistone proteins of somatic origin and may be related to the normal functions of Rsp, rather than to the germ-line segregation distortion activities. PMID- 1722554 TI - The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells. AB - The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non-piliated variants of the same strains (greater than 10x). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (greater than 30x) compared with the non-piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells. PMID- 1722555 TI - Cloning and expression of the rfe-rff gene cluster of Escherichia coli. AB - We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants. PMID- 1722556 TI - Structure of two retrons of Escherichia coli and their common chromosomal insertion site. AB - It has been shown that certain strains of myxobacteria and of Escherichia coli have a genetic element encoding a reverse transcriptase (RT). This element, called a 'retron', produces a covalently linked RNA-DNA compound (msDNA-RNA). Here, I report the complete nucleotide sequence of retron EC-86, the retron in E. coli B, together with its flanking regions. Retron EC-86 contains genes for msDNA RNA (msd, and msr), a gene for RT (ret) and a gene for an open reading frame whose function is unknown. The upstream junction is composed of the sequence GCGCGCGC, but there are no direct or inverted repeats at the retron-host junctions. It is also shown that another retron of E. coli, EC-67, which was isolated originally from the clinical strain CL1 and was later found to be present also in a clinical E. coli isolate from Brazil, is inserted at the same chromosomal site as retron EC-86. Retron EC-67 contains only msd, msr, and ret. I suggest that these two retrons were independently inserted into the same site of their host strains via a novel mechanism of integration. PMID- 1722557 TI - Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D. AB - We report the cloning and mapping of the entire rfb gene cluster of a group C2 Salmonella strain. Comparison with the rfb region of group B strain LT2 and group D strain Ty2 reveals an 11.8 kb central region of limited similarity flanked by regions of high similarity. The genes from the central region confer a group C2 O antigen structure on a Salmonella LT2 partial delete strain. The significance of this region in relation to function and evolutionary origin is discussed. We also report evidence for the existence of an O-antigen chain-length determinant in Escherichia coli K12 and propose a model for a possible mechanism by which a preferred chain length is determined. PMID- 1722558 TI - The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing of plasmid-free segregants. AB - The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence. PMID- 1722559 TI - Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain. AB - The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterica serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopolysaccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed. PMID- 1722560 TI - The sugar-specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate-specific porins. AB - Escherichia coli K-12 strain PS1-28-37 carries the multicopy plasmid pPSO28-37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid bilayer membrane demonstrated that ScrY formed ion permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose-dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto-oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli is discussed with respect to the kinetics of sugar movement through the channel. PMID- 1722561 TI - Genetic mapping of starch- and lambda-receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis. AB - Cysteine mutagenesis was used to test the proximity of 16 residues to protein ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes. PMID- 1722562 TI - Differential inhibition and potentiation by cell-permeant analogues of cyclic AMP and cyclic GMP and NO-containing compounds of exocytosis in human neutrophils. AB - The chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu Phe), complement C5a and platelet-activating factor (PAF), induce beta glucuronidase release and aggregation and an increase in cytosolic Ca2+ [Ca2+]i in human neutrophils. We studied the roles of cAMP and cGMP in neutrophil avtivation, using their cell-permeant analogues, N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) and N2,2'-O-dibutyryl guanosine 3':5'-cyclic monophosphate (Bt2cGMP) and the NO-containing compounds, sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and its prodrug, molsidomine (SIN-10). Bt2cAMP, Bt2cGMP, SIN-1 and SIN-10 but not SNP inhibited exocytosis induced by fMet-Leu-Phe. Superoxide dismutase potentiated the inhibitory effect of SIN-1. Bt2cGMP and SNP potentiated C5a-induced beta-glucuronidase release, Bt2cAMP, KCN, SIN-1 and SIN-10 being ineffective. KCN partially reversed the stimulatory effect of SNP, and in the presence of superoxide dismutase, SIN-1 potentiated C5a induced exocytosis. PAF-induced beta-glucuronidase release was not affected by Bt2cAMP, Bt2cGMP, SNP and SIN-1. Bt2cGMP was more effective than Bt2cAMP to inhibit aggregation and the increase in [Ca2+]i induced by fMet-Leu-Phe at submaximally effective concentrations. C5a-induced rises in [Ca2+]i were not affected by Bt2cAMP and Bt2cGMP. Bt2cAMP but not Bt2cGMP inhibited the effect of PAF at submaximally effective concentrations on [Ca2+]i. Our data suggest (I) that Bt2cGMP and Bt2cAMP differentially modulate neutrophil activation, that (II) NO-containing compounds partially mimic the effects of Bt2cGMP on exocytosis and that (III) cGMP plays an inhibitory role in fMet-Leu-Phe- and a stimulatory role in C5a-induced beta-glucuronidase release. PMID- 1722563 TI - Inhibition of tachykinin-induced hypotension in dogs by CP-96,345, a selective blocker of NK-1 receptors. AB - The effects of substance P, neurokinin A, neurokinin B, [Sar9, Met(O2)11] substance P, [Nle10]-neurokinin A (4-10) and senktide (succinyl-[Asp6, MePhe8] substance P (6-11)) on blood pressure and heart rate were studied in anesthetized dogs. Dose-dependent decreases in blood pressure and increases in heart rate were caused by each peptide except senktide. The latter elicited weak hypotensive or hypertensive responses at high doses. The order or potency was as follows: [Sar9, Met(O2)11]-substance P greater than or equal to substance P greater than neurokinin A greater than neurokinin B greater than [Nle10]-neurokinin A (4-10) much greater than senktide. CP-96,345, [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2 methoxyphenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine] a selective NK-1 tachykinin receptor blocker, inhibited substance P-induced hypotension in a dose related manner. Responses to each of the other peptides were inhibited by CP 96,345, 1.0 mg/kg (excluding senktide against which CP-96,345 was not tested). CP 96,344 (1.0 mg/kg i.v.) the 2R-3R enantiomer of CP-96,345 which does not block NK 1 receptors, had no effect on substance P-induced hypotension. We conclude that tachykinin-induced hypotension in dogs is mediated by NK-1 tachykinin receptors. PMID- 1722564 TI - Comparison of two non-cross-resistant combinations (ABVP/LOPP) with COPP plus bleomycin in the treatment of advanced Hodgkin's disease. AB - Eighty patients with advanced Hodgkin's disease were randomized either to treatment with combination of doxorubicin, bleomycin, vinblastine, and prednisone (ABVP), alternating with lomustine, vincristine, procarbazine, and prednisone (LOPP)--Group A, or to combination of cyclophosphamide, vincristine, procarbazine, prednisone, and low dose of bleomycin (COPP-Bleo)--Group B. Thirty nine out of 41 patients (95%) in Group A achieved complete remission (CR) as compared to 25 CR in 39 patients (64%) in Group B. Patients with systemic symptoms, bulky disease, and nodular sclerosis achieved significantly more CR after treatment with ABVP/LOPP regimen than with COPP-Bleo regimen. Ninety percent of patients are alive in Group A (median observation time 97+ months) as compared to 58% in Group B (median observation time 97+ months). Ninety-two percent of complete responders are in CR in Group A as compared to 53% of complete responders in Group B. These differences between both groups are significant. More serious (WHO grade III and IV) myelosuppression as well as stomatitis and alopecia were observed in Group A. Gastrointestinal toxicity and neurotoxicity was more frequent in Group A. No patient died due to toxicity in Group A as compared to one patient in Group B. Non-cross-resistant alternating regimen ABVP/LOPP was more effective in the treatment of advanced Hodgkin's disease than the COPP-Bleo regimen, especially for patients with advanced Stage IVB Hodgkin's disease. PMID- 1722565 TI - Tumor markers in patients undergoing hemodialysis or kidney transplantation. AB - The following tumor markers, AFP, CEA, CA 19-9, CA-125 and CA 15-3 were studied in 50 healthy volunteers (group A), in 23 patients on chronic hemodialysis (group B) and in 30 successfully transplanted individuals (group C) who did not present any clinical symptoms or signs of neoplasia. The levels of AFP, CEA and CA 15-3 were significantly higher in group B when compared to groups A and C. The levels of CA 19-9 and CA-125 did not differ significantly among the three groups. Transplanted individuals (group C) presented significantly lower levels of CEA and AFP and higher levels of CA 15-3 when compared to group B patients. The levels of all markers were not influenced by sex or time on dialysis. It is concluded that: (1) CA 19-9 and CA-125 can be considered as reliable tumor markers in patients undergoing hemodialysis or kidney transplantation. (2) The elevation of CEA and AFP levels in hemodialysis and their decline to normal levels found in the group of successfully transplanted individuals, suggest a possible active role of functioning renal tissue in their clearance. (3) The etiology of CA 15.3 elevation following successful kidney transplantation remains obscure and requires further evaluation. PMID- 1722566 TI - Autosomal recessive polycystic kidney in rats. AB - We evaluated the characteristics of renal lesions in rat autosomal recessive polycystic kidney (ARPK). In rat ARPK, small cysts appeared primarily in the medulla 2 months after birth and gradually extended to the cortex, forming large cysts involving the entire layer after 8 months. By immunofluorescence microscopy, type IV collagen was more strongly stained in the epithelial basement membrane of the rat ARPK than in the normal rat tubular basement membrane (TBM). Electron microscopy demonstrated a marked thickening, slight splitting and lamination of the TBM in the ARPK. As peroxidase-labeled lectins, dolichos biflorus very strongly stained the cyst epithelium whereas lens culinaris did not. These findings indicate that cysts in rat ARPK originate in the collecting duct. PMID- 1722567 TI - [AIDS in the world]. PMID- 1722568 TI - [Possibility of the combined use of tumor markers in endometrial carcinoma]. AB - The Authors have studied the haematic levels of CA 125, CA 19-9, CA 50, CEA, TPA, alfa-feto-proteina e CA 15-3 in 24 women with endometrial carcinoma and in 28 healthy women. The results show that these markers are not useful for the screening of endometrial carcinoma. PMID- 1722569 TI - Cell types expressing the Wilms' tumour gene (WT1) in Wilms' tumours: implications for tumour histogenesis. AB - Wilms' tumour (nephroblastoma), a childhood embryonal kidney tumour, is believed to arise from malignant transformation of abnormally persistent metanephric blastemal cells. At a histological level, tumours show a remarkable mimicry of the normal nephrogenic pathway. There is histological and epidemiological evidence for at least two pathogenetic groupings within Wilms' tumour which may reflect different timings of the tumorigenic insult in this pathway and/or involvement of different genes. Tumorigenesis is thought to result from loss of function of a so-called tumour-suppressor gene which has an essential role in control of normal genitourinary development. Such a candidate, Wilms' tumour gene (WT1) mapping to chromosome 11p13, has been isolated and is known to be mutated in some tumours. We have examined the cell types expressing this gene in 32 Wilms' tumours and in nephroblastomatosis by in situ mRNA hybridization. Our results show that WT1 is expressed only in neoplastic structures whose normal counterparts also express the gene and that abnormally persistent high levels of expression are common in both these lesions. Thus, WT1 expression is a good marker for tumour differentiation and reveals how the normal pattern of differentiation is disrupted in Wilms' tumours. We postulate that mutation of the WT1 gene at the 11p13 locus results in Wilms' tumours associated with intralobar nephrogenic rests, which frequently show stromal-predominant histology. We have used our results and ideas to reinterpret current theories on tumour histogenesis and propose a model which explains how patterns of epithelial differentiation are disrupted in Wilms' tumour and how malignant stroma can result from mutation in WT1. PMID- 1722570 TI - High-affinity binding and activation of a truncated FGF receptor by both aFGF and bFGF. AB - We recently reported the cloning and overexpression of full-length forms of human fibroblast growth factor (FGF) receptors, bek and flg. These receptors contain three immunoglobulin (Ig)-like domains and an unusual acidic motif in the extracellular region, a single transmembrane segment and a protein tyrosine kinase cytoplasmic domain containing a 14 amino acid insert. Each of the related full-length gene products interacts at high affinity with both acidic FGF and basic FGF. We now report the isolation of cDNA clones encoding two variant forms of human bek. One variant form encodes a potentially secreted bek protein containing only the first Ig-like domain and acidic motif, whereas the other variant includes all of the full-length bek protein except the first Ig-like domain and acidic motif. Overexpression of the latter bek form in NIH3T3 cells has been used to demonstrate that the N-terminal Ig-like domain and acidic region are not required for binding or activation of bek by aFGF or bFGF. PMID- 1722571 TI - Coexpression of the stem cell factor and the c-kit genes in small-cell lung cancer. AB - Stem cell factor (SCF) is a pluripotent growth factor which is suggested to play an important role in proliferation and differentiation in various types of fetal and adult tissues as the ligand of the c-kit proto-oncogene product. However, very little is known about expression of the SCF gene in human malignancies. We analysed DNA and RNA extracted from 28 cell lines and 16 fresh tumor specimens of lung cancer as well as 24 cancer cell lines of various origin for SCF expression. Now we report that the SCF gene is expressed in a wide variety of human cancers including lung cancer, in marked contrast to c-kit, which is expressed in very few types of cancers. As a consequence, coexpression of both the ligand and the receptor is seen only in small-cell lung cancer, suggesting possible involvement of autocrine stimulation via this ligand-receptor system in the pathogenesis of this aggressive cancer. In addition, this study revealed that the human SCF gene is transcribed into two major forms of alternatively spliced mRNAs with different molar ratio in fetal, adult and malignant tissues. PMID- 1722572 TI - Ras oncogenes amplify lymphokine (interleukin 3, granulocyte-macrophage colony stimulating factor) induction by calcium ionophore. AB - Interleukin 3 (IL-3) expression in PB-3c mastocytes is transiently induced in vitro by treatment with the drug A23187, a calcium ionophore, or constitutively following ras-dependent transformation in vivo. While the mechanism of oncogenically induced IL-3 expression is not clear, A23187-mediated lymphokine mRNA accumulation is primarily the result of calcium-dependent mRNA stabilization. We investigated whether the expression of various ras alleles influenced IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA induction by A23187. It was found that activated forms of ras potentiated ionophore-mediated lymphokine mRNA accumulation. This enhancement involves a post transcriptional mechanism, as ionophore-induced lymphokine mRNAs are significantly more stable in ras oncogene-expressing lines than in the control line. We propose that one way by which ras genes exert their oncogenic potential is by extending the half-life of short-lived growth factor mRNAs. PMID- 1722573 TI - A proposed conceptual model of human behavior and its implications for design of warnings. AB - A model of human behavior is proposed that hierarchically describes levels of operator performance. Judgement-based performance occurs at the top level, and the lower levels correspond to knowledge-, rule-, and skill-based performance. Different forms of information denoted as values, symbols, signs, and signals are used at each of these levels of performance, allowing the effectiveness of different warning applications to be inferred. To be effective, warning information must be presented in the form appropriate for the operator's level of performance. Values therefore are appropriate when performance is at the judgement-based level. Explicit verbal information (symbols) is most likely to be effective when directed toward changing behavior from a knowledge- to a rule based level, as when recommending actions in novel situations or to a judgement based level, when goal priorities need to be changed. Signs are likely to be effective when performance is at a rule-based level, while signals are best for guiding needed transitions from a skill- to a rule-based level. Warning information should be carefully matched to the level of performance at which errors are taking place to be most effective and avoid information overload. To attain this goal, task analysis that focuses on cognitive activity is of essence. This includes measuring users' knowledge and documenting the flow of information during task performance. PMID- 1722574 TI - [The X chromosome, fetal hemoglobin and sickle cell anemia]. PMID- 1722575 TI - Identifying infants at risk: North Carolina's High-Priority Infant Program. AB - Recent federal legislation (e.g., PL 99-457, Part H, and amendments to the MHC block grant and Medicaid legislation) has focused attention on infants at risk for health and developmental problems. The ability to plan services for these infants would be strengthened by data that could be used for program accountability and evaluation. Using nurses based in local health departments, North Carolina's High-Priority Infant program currently identifies and follows infants with biologic (e.g., very low birth weight), environmental (e.g., psychosocial problems), and established (e.g., Down syndrome) risk conditions. Data from this program were collected to analyze specific features of its implementation, a necessary precursor to evaluating outcome. PMID- 1722576 TI - Comparison of transabdominal and transcervical CVS and amniocentesis: sampling success and risk. AB - A total of 2931 women randomized to either transabdominal CVS, transcervical CVS, or amniocentesis were studied. Unless intended or unintended abortion had occurred, they had completed up to 28 weeks of pregnancy. No significant difference was seen between total fetal loss in the transabdominal CVS group and the amniocentesis group (6.5 and 6.8 per cent, respectively, SE difference = 0.92 per cent, p = 0.01). The total fetal loss in the transcervical CVS group was 10.1 per cent. After pooling our data with data from the Canadian randomized study and the American non-randomized study, the difference in risk between transcervical CVS and amniocentesis was 1.8 per cent (SE difference = 0.64 per cent, p = 0.8). When the number of failed procedures and those cases evaluated as unfeasible for the assigned method--for anatomical reasons--are compared, the overall sampling efficacy is poorer transcervically than transabdominally. PMID- 1722577 TI - Chorionic villus sampling: analysis of fetal losses to delivery, placental pathology, and cervical microbiology. AB - A population of 1639 patients were seen for chorionic villus sampling (CVS). Embryonic death was identified at ultrasound in 5.3 per cent of patients. The number of patients undergoing CVS was 1551, with 1416 transcervical procedures and 135 transabdominal procedures. The most common indication for CVS was advanced maternal age. Spontaneous pregnancy losses identified by increased risk of pregnancy loss with increasing aspiration attempts. The total fetal loss for this population was 5.4 per cent with the pregnancy loss estimated due to procedure being 1.2 per cent. Analysis of placentae from patients having CVS and amniocentesis showed no differences. Microbiological assessment prior to CVS was similar to previous publications. PMID- 1722578 TI - Chorionic villus cDNA library displays expression of butyrylcholinesterase: putative genetic disposition for ecological danger. AB - Gene expression in chorionic villi may be particularly vulnerable to environmental exposure to poisonous substances. To reveal villus gene products which are thus subject to poisoning, molecular cloning was employed. A single sample of apparently normal chorionic villi (approximately 40 mg, from 9 weeks' gestation) was microscopically dissected to ensure purity of fetal tissue. Total RNA was extracted by isothiocyanate and directly employed for reverse transcription. A chorionic villus cDNA library was constructed from this preparation in the phage vector lambda gt10 and contained 60,000 independent recombinants. In the present study, this cDNA library was screened with labelled cDNA probes encoding human butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE). Nine BCHEcDNA clones were isolated out of 1.6 x 10(6) phages (5.7 x 10( 6) of screened recombinants) and exhibited similar restriction patterns to those observed for BCHEcDNA from other human tissues. In contrast, no ACHEcDNA clones could be found in 4.0 x 10(6) screened phages (less than 2.5 x 10(-6) of recombinants). These findings demonstrate efficient transcription (similar to fetal brain levels) from the BCHE gene but not from the ACHE gene in chorionic villi, and support the notion that BCHE is involved in chorionic villus growth and development. PMID- 1722579 TI - First-trimester screening for fetal chromosomal abnormalities. Preliminary results. Dutch Working Party on Prenatal Diagnosis. AB - We have started a multicentre trial to study the possibilities of first-trimester maternal serum screening for fetal chromosomal abnormalities. Maternal blood samples were obtained before 13 weeks of gestation. We present the preliminary results of the first 950 patients on alpha-fetoprotein (AFP). Results on cancer antigen 125 (CA 125) in Down's syndrome and normal pregnancies are also presented. We conclude that the results on AFP are promising and that CA 125 might be predictive for fetal Down's syndrome. PMID- 1722580 TI - Alpha-fetoprotein in fetal serum, amniotic fluid, and maternal serum. AB - In order to gain more insight into the association between alpha-fetoprotein (AFP) and fetal chromosomal disorders, especially Down's syndrome, we measured AFP in fetal serum, amniotic fluid, and maternal serum at cordocentesis. We compared the concentration and gradient of AFP in these three compartments. Our data confirm earlier findings on second-trimester fetal serum AFP concentration. The results indicate that low maternal serum AFP in pregnancies with fetal chromosomal disorders could result from an impaired fetal kidney function as well as from impaired membrane or placental passage of AFP, rather than from reduced fetal AFP production. PMID- 1722581 TI - Homeless families in Hackney. AB - A retrospective study of medical records in Hackney and Tower Hamlets showed that the health of infants and school-children in temporary accommodation was impaired. Perhaps the most striking result was the high proportion of low birthweight in all four groups (10-25%). Of particular concern is the finding that 25% of babies living in Bed and Breakfast (B&B) hotels were born with a weight less than 2,500 g. Amongst other findings was that a third of the infants 'born and bred' in B&B accommodation and their Tower Hamlets controls had been below the 10th centile at some time in their lives. The immunisation rates, however, were good. In the study of school-children, the school medical officer found that 30% of the children in B&B hotels were considered not to be in normal health compared with 20% of Finsbury Park residents. Ten percent of the children in B&B were considered not to have normal development and 47% of these had been referred for further investigation compared with 25% of the local residents. In contrast with the infants, the immunisation rates were poor. While it is not possible to infer any causal relationship from this study, it must be questioned whether these high risk children should be living in temporary accommodation for prolonged periods of time. PMID- 1722582 TI - Tissue characterization by color-Doppler. PMID- 1722583 TI - [Echography and computerized tomography of the abdomen in Whipple's disease]. PMID- 1722584 TI - Evaluation and management of pain in children with rheumatic diseases. AB - The evaluation and management of pain in children with rheumatic diseases are still in the early stages of empirical development. Nevertheless, with the advent of the PPQ, systematic research efforts are now underway to develop the reliability and validity of the PPQ's measurement characteristics for pediatric rheumatic diseases. With these developments, the inclusion of pediatric pain measurement as an essential outcome variable in controlled clinical trials should be advocated. The major points to be advocated by professionals who care for these children are (1) Children can accurately report their pain when age appropriate measures are used; (2) pharmacologic treatment is not a sufficient condition for adequate pain management; (3) cognitive-behavior therapy techniques can facilitate children's coping with "breakthrough" recurrent musculoskeletal pain with no known side effects; and (4) adequate pain control should be viewed as a quality assurance issue, considered as another indicator of the adequacy of pediatric health care. PMID- 1722585 TI - [Treatment with octreotide (SMS 201-995) in a case of intestinal carcinoid tumor]. AB - Over the past few years the usefulness of some somatostatin's analogues in the treatment of intestinal tract endocrine tumors has been demonstrated. Notwithstanding, the results obtained are variable. The case of a carcinoid tumor with a hepatic metastasis is presented and its clinical as well as its biochemical and its morphological results are evaluated after treatment with octreotide over a seven months period. It is important to highlight the great clinical improvement obtained at the beginning of treatment. Treatment was not effective in the control of tumor progression. After the injection of such a drug, a decrease in serotonin and 5-hydroxy-indoleacetic acid serum levels was observed as well as a reduction in the urinary metabolite. It is concluded that octreotide is very useful for the symptomatic treatment of carcinoid syndrome. PMID- 1722586 TI - [Prenatal screening for trisomy 21 in maternal blood. Focus]. PMID- 1722587 TI - [Rheology and gravidic hypertension]. AB - Gestational hypertension is the development of hypertension and proteinuria after the 20th week of gestation. The most common causes of increased peripheral resistance are the vasoconstriction and hemoconcentration with plasma volume contraction. Additional rheological parameters are an elevated red blood cell aggregation and impaired erythrocyte deformability. Preeclamptic patients showed a significantly low cardiac output and central venous pressure than normal pregnant women. It has already been shown by the studies by Hytten and Paintin (1963) and also by the subsequent studies by Garn et al. (1981), Murphy et al. (1986) that a strong correlation exists between newborn weight and plasma volume. Other authors (Gallery et al. (1979/1981)) show the possibility that plasma volume contraction plays an even larger role than vasoconstriction in the fetal growth retardation that often accompanies maternal hypertension. This possibility is supported by the finding that hypertension and perinatal complications can be reduced in some pregnant women by the admission of oncotic solutions (i.e. hydroxyethyl-starch) that expand plasma volume. Volume expansion with hydroxyethyl-starch appears to be of therapeutic benefit for hypertensive patients and patients with fetal growth retardation with low cardiac output. PMID- 1722588 TI - Carcinoma of the prostate. Serum tumour markers. AB - Health examination - screening: Tumour markers in serum are of minor value when used for detection of the disease in health screening protocols. Reflection of the course of disease: Tumour markers are of great value for monitoring the response to a given therapy and for early detection of relapse. In recent years, measurements of PSA has replaced PAP for this purpose. Prognostic indicator: Tumour markers in serum may also be used to indicate the biological activity of the tumour. Also for this purpose PSA appears to be a more reliable marker than PAP. General recommendation: For newly detected cases of prostatic carcinoma determination of a limited number of prognostic markers is recommended. If no treatment is instituted all these determinations may be repeated at an interval of about one year. Once treatment has been instituted assay of serum markers may be restricted to PSA alone and may be carried out at about six-monthly intervals. In protocolled clinical trials the intervals may be shortened. PMID- 1722589 TI - [Hepatitis C virus, 1991]. PMID- 1722590 TI - [Organ-preserving principle in the surgical treatment of primary multiple cancer of the large intestine]. AB - This paper presents the analysis of 193 surgical cases of multifocal cancer of the colon. The choice of the radical surgery in synchronous tumors depended on the involved portions of the colon, distance between the lesions, cancer dissemination and general condition of the patient. Out of radical interventions, extensive colon resections (atypical and subtotal) made up 18% only (16 of 89 patients). In metachronous cancer, colon resection (n-67) was performed with due consideration of the second tumor site and lymph outflow from the affected zone (39 typical radical operations and 28 resections with removal of interintestinal anastomosis). Postoperative lethality in synchronous cancer reached 11.2%, in metachronous 10.4%, 5-year survival was 59.3% and 57.7%, respectively. The latter figures appeared only a little lower that in solitary colon cancer (63%). PMID- 1722591 TI - Gene silencing in mammalian cells by uptake of 5-methyl deoxycytidine-5' triphosphate. AB - Chinese hamster ovary (CHO) cells were subjected to electroporation in the presence of 5-methyl deoxycytidine-triphosphate. This treatment increases by 10 to 100-fold the frequency of cells lacking thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, or adenine phosphoribosyltransferase. The inactivation of the genes coding for these enzymes is thought to occur following the direct incorporation of the methylated nucleotide triphosphate into DNA. The enzyme deficient clones were stable, but almost all were reactivated at high frequency by the demethylating agent 5-azacytidine, to produce derivatives with enzyme activity. The results indicate that there is a direct relationship between DNA methylation and gene silencing. PMID- 1722592 TI - Gene silencing in mammalian cells by direct incorporation of electroporated 5 methyl-2'-deoxycytidine 5'-triphosphate. AB - DNA methylation is an important process contributing to transcriptional regulation in animal and plant cells. We observed that electroporation of Chinese hamster V-79 cells in the presence of millimolar concentrations of 5-methyl-2' deoxycytidine 5'-triphosphate (5mdCTP) led to high-level direct incorporation of this nucleotide into DNA polymer. Under optimum conditions, approximately 2 x 10(8) molecules of 5 mdCTP were calculated to have been incorporated into each unit genome (6 pg of DNA). Since a diploid mammalian genome contains approximately 1.2-1.5 x 10(9) cytosines, this level of 5 mdCTP incorporation corresponds to substitution of up to 16.6% of total cytosines by 5 methylcytosine, or about 100-150 new methylated cytosines per average gene. The pattern of genomic methylation produced under these conditions differed from that produced physiologically in that 5mdCTP was substituted into DNA at random cytosines, superimposing a novel methylation pattern upon that derived from the normal enzyme-driven postreplicational process. This novel pattern of methylation showed no preference for CpG dinucleotides, but was nevertheless found capable of silencing HPRT gene expression and producing a condition of resistance to 6 thioguanine. The epigenetic nature of this gene silencing event was shown by the very high level of reexpression of HPRT mRNA following exposure of HPRT cells to the demethylating agent 5-azadeoxycytidine. PMID- 1722593 TI - Immunocytochemical evidence for methamphetamine-induced serotonergic axon loss in the rat brain. AB - Central serotonin (5-HT) axons were visualized by immunocytochemistry to assess both acute and long-lasting changes in innervation density following methamphetamine administration to rats. Two morphologically distinct subtypes of 5-HT axons (fine and beaded) were differentially affected by d-methamphetamine (d MA); the density of fine serotonergic axons was selectively decreased both 4 hours and 2 weeks after administration of d-MA. Acute depletion of 5-HT from fine axons, but not from beaded axons, was observed in the brains of all rats treated 4 hours previously with either a 100 mg/kg or 15 mg/kg dose of d-MA. Persistent loss of 5-HT axons was observed in 30% of rats treated 1 or 2 weeks previously with doses of d-MA which produce long-term deficits in biochemical markers for 5 HT. In the fraction of animals that exhibited denervation, fine serotonergic fibers were selectively ablated by d-MA, but beaded serotonergic fibers were spared. Thus, d-MA is similar to other amphetamine derivatives (e.g., p chloroamphetamine, 3,4-methylenedioxyamphetamine) in that it acts selectively upon a morphologically distinct class of 5-HT axons but differs in that it produces long-lasting axon loss in only a fraction of animals. These data provide morphologic evidence of 5-HT axon loss following methamphetamine administration and further confirm the differential vulnerability of a particular morphological subtype of serotonergic axons to the neurotoxic effects of substituted amphetamines. PMID- 1722594 TI - LTP changes the waveform of synaptic responses. PMID- 1722595 TI - Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen. AB - The nephritogenic antigen that induces antiglomerular basement membrane antibody induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic. The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the alpha 3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen. PMID- 1722596 TI - Neutralization of CRPV infectivity by monoclonal antibodies that identify conformational epitopes on intact virions. AB - Monoclonal antibodies were generated against cottontail rabbit papillomavirus (CRPV) and tested for neutralization of CRPV-induced papillomas on domestic NZW rabbits. Intact CRPV was semi-purified on CsCl gradients and used to immunize BALB/c mice. Hybridomas were prepared from a fusion with lymph node cells, and supernatants from growing hybridomas were analyzed by enzyme-linked immunosorbent assay (ELISA) for reactivity to both intact and disrupted CRPV virion antigen. Supernatants from 22 cultures were initially selected that were responsive to CRPV. Ten were reactive to intact CRPV alone, 4 were reactive only to disrupted CRPV, and 8 were reactive to both intact and disrupted CRPV virion antigen. None of these supernatants contained antibodies which recognized epitopes on CRPV capsid proteins (L1 and L2) that were separated on Western blots. Five hybridomas which produced antibodies that bound to intact CRPV, and did not react to intact HPV-11 or BPV-1 were selected and tested for antibody-mediated neutralization of CRPV infectivity. All five monoclonal antibodies were neutralizing, and identified epitopes on intact CRPV virions which were non-linear and conformational in nature. The five neutralizing monoclonal antibodies appeared to recognize a similar epitope or epitope cluster on the intact CRPV virion as determined by competition ELISA. PMID- 1722597 TI - Cyclic variations in the permeability of the cell wall of Saccharomyces cerevisiae. AB - To study cell-cycle-related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cells of increasing size. The subpopulations represented different stages of the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulations became enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MATa cells from alpha-factor induced G1-arrest. These cultures grew synchronously for at least two generations. The cell wall porosity increased sharply in these cultures, shortly before buds became visible and was maximal during the initial stages of bud growth. It decreased in cells which had completed nuclear migration and before abscission of the bud had occurred. The porosity reached its lowest value during abscission and in unbudded cells. We examined the incorporation of mannoproteins into the wall during the cell cycle. SDS-extractable mannoproteins were incorporated continuously. However, the incorporation of glucanase-extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration. This coincided with a rapid decrease in cell wall porosity, indicating that glucanase-extractable mannoproteins might contribute to this decrease. PMID- 1722598 TI - [The structural-functional organization of the auditory cortex in rats]. AB - Using axonal transport of horseradish peroxidase and electrophysiological mapping technique, studies have been made on structural and functional organization of the auditory cortex in rats. After the injection of HRP to peripheral parts of the auditory cortex, mainly initial neurones were found in the dorsal and median parts of the geniculate body. Electrophysiological experiments revealed the localization of neurones with widespread frequency-threshold curves and high thresholds at characteristic frequency. On the basis of the data obtained, it is suggested that similar to other mammals, rats have the areas of secondary fields of the auditory cortex which surround central coniocortex. PMID- 1722599 TI - Breast reconstruction in women with Poland's syndrome. AB - The authors point to the considerable variability in the development of the mammary gland in Poland's syndrome cases. Using computed tomography they draw attention to some other anatomical deviations of the deeper-seated structures of the affected side of the chest. Finally, they discuss various techniques of breast reconstruction for hypoplasia or aplasia. PMID- 1722600 TI - Transferred triage to a level I trauma center in a mass catastrophe of patients; many of them with burns. AB - Three airplanes were involved in an airshow accident at the Ramstein military airbase on 28th of August 1988 causing immediate 45 dead and approximate 400 injured people. As a Level I Trauma facility we received 47 patients in different state of distress direct from airfield within an hour. The disaster plan was activated and sufficient personal and infrastructure could be mobilized. PMID- 1722601 TI - Local anaesthesia in cosmetic surgery. AB - The author reports on his experience with a new form of local anaesthesia in cosmetic surgery, namely a cream for local application produced by the Swedish firm ASTRA under the name of EMLA. It is a mixture of prilocaine and lidocaine. The effect sets in 45 min after application. The technique and indication range in cosmetic surgery are described. PMID- 1722602 TI - Use of the free jejunal loop to replace cervical oesophagus in an experiment. AB - The paper presents the results of experimental studies exploring the possibility to replace the cervical oesophagus with a free intestinal loop. The experiments were carried out in dogs and cervical oesophagus replacement was performed using a jejunal loop. Nutrition was provided by mesenteric vessels anastomosed by means of a microsurgical operative technique. The intestinal segment was subsequently used to replace the resected segment of the cervical oesophagus. The results of our experiments indicate this new operative technique can be used in clinical practice. PMID- 1722603 TI - Paraffinoma of the male breast. AB - A case of paraffinoma of hte male breast is reported following injections of paraffin in a male transsexual. PMID- 1722604 TI - Mental and social problems of families with handicapped child. AB - Studies were carried out in a series of 100 mothers of children with facial clefts who were treated up to the age of 13 to 15 years at the Department for Plastic Surgery. Clefts were used as a model situation for studies into the response of parents--and especially of mothers--to the birth of a child with an inborn malformation. The main results showed: that three quarters of mothers designated the birth of a handicapped child as a mental shock followed by a variety of neurotic symptoms. A certain proportion of mothers suffered from reactive depression which was not diagnosed and therefore not treated. It was confirmed that the poor mental condition of the mother in the predominant part of the series examined persisted up to the adolescence of the affected child and exerted marked negative effects on the mental prosperity of the child. This resulted in a latent or manifest parental attitude of rejection and in the development of mental handicap in their children (impaired self-esteem, impaired sexual identification etc.). The stress situation was increased by factors including manifestation of stigmatization by the familial environment and by the collective of children which had an adverse effect approximately on about three quarters of mothers and children. The discussed forms of medical care and counselling could in the post partum period and later help to reduce the mental stress experienced by the parents, as well as promote the somatic and mental development of the child. PMID- 1722605 TI - Combined dermatoplasty in deep burns in the region of flexion of the large joints in children. AB - In 13 children with deep burns in the region of functionally important anatomical flexions, the wounds were covered with rotation skin flaps from the vicinity. The purpose of the method used is the prevention of post-burn contractures, which are particularly expressed in the process of the child's growth. After observation of the operated patients over 3 years, the results of operation justify the conclusion that it is purposeful to utilize skin resources from the vicinity of the wound surface to achieve more rapid and more effective restoration of anatomical integrity and function in deep burns on flexions of the body. PMID- 1722606 TI - Meningiomas differentiating to arachnoid trabecular cells: a proposal for histological subtype "arachnoid trabecular cell meningioma". AB - Three cases of meningiomas which had abundant small vacuoles in the tumor tissue are reported. By electron microscopy, the tumor cells exhibited long and thin processes, the tips of which were united by desmosomes. The tumor tissue was revealed to have wide extracellular spaces which corresponded to the vacuoles observed by light microscopy. In previous literature, various terms have been used when referring to this meningioma, such as microcystic meningioma or vacuolated meningioma. Since the ultrastructure of the tumor showed similarity to that of normal arachnoid trabecular cells, we propose to call the tumor "arachnoid trabecular cell meningioma" denoting its morphological nature clearly. PMID- 1722607 TI - Reexamination of granulovacuolar degeneration. AB - Granulovacuolar degeneration (GVD) in the hippocampal pyramidal neurons of Alzheimer-type dementia was examined. Immunohistochemical examinations showed that the majority of centrally located granules were positive for ubiquitin. Based on electron microscopic observations, morphogenesis of GVD is considered to be as follows. Slight-to-moderate amounts of electron-dense material appear in the cytoplasm at the early stage, and are then surrounded and demarcated by a two layered membrane (probably from smooth endoplasmic reticulum). Following this some inner material is digested forming floccular and liquid-like materials, while undigested material remains as coarse electron-dense granules. Specifically, granulovacuoles are considered to be an age-related special type of autophagosome. Analytical electron microscopy disclosed that the granules in GVD contained some aluminum. PMID- 1722609 TI - Membranous lipodystrophy (Nasu-Hakola disease) with thalamic degeneration: report of an autopsied case. AB - An autopsied case of membranous lipodystrophy (Nasu-Hakola disease, NHD) with thalamic degeneration was reported. A 34-year-old Japanese man was diagnosed as having NHD by bone biopsy prior to the onset of clinical symptoms. His maternal grandfather and paternal grandmother are cousins, but this family history is negative for NHD. He developed frontal lobe syndrome at the age of 35 with progressive dementia, and died of acute renal failure at the age of 46. Gross inspection of the brain detected atrophy and softening of the cerebral white matter, predominantly in the frontal lobe. Microscopically, numerous spheroids, predominant fibrillary gliosis with less prominent demyelination "dissociation glio-myelinique" and scanty sudanophilic lipid droplets were observed, indicating the sclerosing type of NHD. An unusual patholgoical finding in this case was selective involvement of the thalamic nuclei with preservation of the other gray matter except for focal cortical necrosis. The topography of the affected thalamic nuclei is similar to that of systemic thalamus degeneration. An association with thalamic degeneration in NHD has not been previously reported. The present case suggests that NHD also affects the thalamus. PMID- 1722608 TI - Immunohistochemical study of microtubule-associated protein 2 and ubiquitin in chronically aluminum-intoxicated rabbit brain. AB - Experimental neurofibrillary change was produced in rabbit brain by daily subcutaneous aluminum tartrate injection for 40 days. The production of experimental neurofibrillary changes was confirmed by immunostaining with antibodies against neurofilament triplet proteins and the brain tissue was studied immunohistochemically with antibodies against microtubule-associated protein (MAP) 2 and ubiquitin. The hippocampal neurons of the chronically aluminum-intoxicated rabbit brain showed diminished staining of dendrites by anti MAP2 antibody. The length of anti-MAP2-positive dendrites in hippocampus was significantly shorter than that of the control brain. In the cortex somata of a subset of pyramidal neurons were intensively stained by anti-MAP2 antibody, while the MAP2 immunoreactivity of distal dendrites was diminished. The immunostaining by anti-ubiquitin antibody revealed the positive staining of the neurons bearing experimental neurofibrillary changes in the lower brain stem nuclei. It is speculated that MAP2 dislocation and ubiquitination are accompanying phenomena of the production of experimental neurofibrillary changes in chronically aluminum intoxicated rabbit brains. PMID- 1722610 TI - Insulin-like growth factor binding protein control secretion and mechanisms of action. PMID- 1722611 TI - Regulation and actions of insulin-like growth factor binding protein-3. PMID- 1722612 TI - Regulation of gene expression of rat insulin-like growth factor binding proteins 1 and 2. PMID- 1722613 TI - Hormonal regulation of insulin-like growth factor binding protein-1 expression in the rat. PMID- 1722614 TI - Cerebrospinal IGF binding proteins: isolation and characterization. PMID- 1722615 TI - The effect of quantity and nutritional quality of dietary proteins on plasma concentration of insulin-like growth factor binding proteins (IGFBP) and the saturability of IGFBP with endogenous IGF-I. PMID- 1722616 TI - Presence of insulin-like growth factors and their binding proteins in rat milk. PMID- 1722617 TI - Characterization of the biological activity of IGF I analogs with reduced affinity for IGF receptors and binding proteins. PMID- 1722618 TI - Insulin-like growth factors and their receptors in muscle development. AB - Several proteins involved in IGF action are expressed in C2 cells and their abundance was found to vary as a function of development. IGF-I and II mRNA levels rose 10 and 25-fold, respectively, during differentiation, and were accompanied by an increase in growth factor secretion. The accumulation of IGF-II in conditioned culture medium was much greater than that of IGF-I. There was also an increase in the number of IGF-I receptors and IGF-II/CIMPR on the cell surface during differentiation. The sustained rise in IGF-II/CIMPR expression appeared to be a consequence of a similar increase in its mRNA abundance. The mechanisms responsible for the transient increment in IGF-I receptor number were not assessed, although it is likely that the decline in IGF-I receptor content after 72 hours in differentiation medium was a consequence of down-regulation by the IGF-II that accumulated in the medium (35). In contrast to the 13-fold rise in IGF-II/CIMPR mRNA levels, transcript levels for the CDMPR remained constant during C2 cell development, enzymatic activities of two lysosomal enzymes did not change, and only a small increment was detected at a single time point in the expression of several lysosomal enzyme mRNAs. In addition, during C2 muscle differentiation, a novel IGF binding protein was induced. These results demonstrate modulation of several components of IGF signaling pathways in differentiating myoblasts, and argue for a local role for IGFs in muscle development. PMID- 1722619 TI - Insulin-like growth factor receptors in testicular vascular tissue from normal and diabetic rats. AB - Testicular blood vessels contain IGF-I and IGF-II/M6P receptors. Binding to these receptors was altered following treatment with streptozotocin to induce diabetes. Intensity of labelling and size of receptors were examined using SDS-gel electrophoresis and autoradiography. The IGF-I and IGF-II/M6P receptor of the diabetic rat testicular microvessels appear to have a lower molecular weight as compared to controls. Macro- and microvascular tissues from diabetic rats apparently contain more IGF-I receptors than normal Sprague-Dawley rats. Using immunohistochemical techniques, the IGF-II/M6P receptor appears to dissociate easier from diabetic rat testicular arteries than from control animal blood vessels. M6P appears to increase both IGF-I and IGF-II binding to the rat IGF II/M6P receptor, at least as visualized using affinity crosslinking analysis. Whether these differences in the IGF receptors are involved in the development of diabetic vascular disease is not yet known. PMID- 1722620 TI - Insulin-like growth factor II: complexity of biosynthesis and receptor binding. AB - Insulin-like growth factor II (IGF-II) belongs to the insulin family of peptides and acts as a growth factor in many fetal tissues and tumors. The gene expression of IGF-II is initiated at three different promoters which gives rise to multiple transcripts. In a human rhabdomyosarcoma cell line IN 157 IGF-II mRNAs of 6.0-kb, 4.8-kb, and 4.2-kb are present. Fractionation of cellular extracts on sucrose gradients and Northern blot analysis showed that only the 4.8-kb mRNA was associated with polysomes, whereas the other transcripts cosedimented with monosomal particles. This suggests that only the 4.8-kb mRNA is translated to IGF II. The cell line secretes two forms of immunoreactive and bioactive IGF-II to the medium of molecular size 10 kd and 7.5 kd which may be involved in autocrine control of cell growth. IGF-II binds to two receptors on the surface of many cell types: the IGF-I receptor and the mannose-6-phosphate (Man-6-P)/IGF-II receptor. There is consensus that the cellular effects of IGF-II are mediated by the IGF-I receptor via activation of its intrinsic tyrosine kinase. The Man-6-P/IGF-II receptor is involved in endocytosis of lysosomal enzymes and IGF-II. In selected cell types, however, Man-6-P induces cellular responses. We have studied rat brain neuronal precursor cells where Man-6-P acted as a mitogen suggesting that phosphomannosylated proteins may act as growth factors via the Man-6-P/IGF-II receptor. In conclusion, the gene expression and mechanism of action of IGF-II is very complex suggesting that its biological actions can be regulated at different levels including the transcription, translation, posttranslational processing, receptor binding and intracellular signalling. PMID- 1722621 TI - Expression of IGF-II, the IGF-II/mannose-6-phosphate receptor and IGFBP-2 during rat embryogenesis. PMID- 1722622 TI - Development of a specific radioimmuno assay for E domain containing forms of insulin-like growth factor II. PMID- 1722623 TI - Insulin-like growth factor binding proteins in the nervous system. PMID- 1722624 TI - Insulin-like growth factor I: a possible modulator of intercellular communication in the brain. PMID- 1722625 TI - Electrophysiological properties of human neutrophils. PMID- 1722626 TI - Anti-cytoplasmic antibodies in Wegener's granulomatosis are directed against proteinase 3. PMID- 1722627 TI - [Antiandrogen therapy of benign prostatic hyperplasia--review of the agents evaluation of the clinical results]. AB - Various non-surgical therapeutic modalities such as balloon dilation of the prostatic urethra, hyperthermia of the prostate and medication with antiandrogens and/or adrenergic blockade have been attempted for the patients with benign prostatic hyperplasia (BPH) especially in an early stage or in a poor operative risk. The observation that androgen deprivation induces shrinkage of the hyperplastic prostate represents the basis for the treatment of BPH with antiandrogen. Although several antiandrogens are now in clinical use in our country, there still remain problems to be solved. We reviewed the mechanism of action and the clinical results of antiandrogens in the treatment of BPH. The improvement following antiandrogen therapy occurred among the patients with symptomatic BPH, in 50-80% subjectively and in 40-50% objectively. The therapy appeared to be more effective in an early stage of the disease. However, the limitation of the duration of the effects and unfavorable side effects should also be noticed. The progestational agents such as gestonorone caproate, chlormadinone acetate and allylestrenol suppress more or less sexual function by interference of the pituitary-gonadal axis. Besides, coincidental prostate cancer must be excluded since antiandrogen therapy might hinder the natural course of the cancer. PMID- 1722628 TI - [Clinical results and problems of anti-androgen therapy of benign prostatic hypertrophy]. AB - We evaluated the effect of anti-androgen therapy for benign prostatic hypertrophy. Patients showed a significant reduction in the prostatic weight measured by means of transrectal ultrasonography after 3 to 4 months of treatment. However, there were no patients who showed any symptomatic improvement despite a reduction in the prostatic weight. They had prostatic stones more frequently than the group who showed symptomatic improvement properly. We summarized some problems of anti-androgen therapy for benign prostatic hypertrophy. PMID- 1722629 TI - [Clinical experience of local hyperthermia for benign prostatic hyperplasia]. AB - A total of 20 patients with benign prostatic hyperplasia underwent transrectal local hyperthermia. For heating of the prostate gland, the PROSTATHERMER (Biodan Medical System, Israel) was used. Patients were treated twice weekly, for 1 hour, with 6 sessions on an outpatient basis. Four of the 20 patients who had acute toxicity such as urethral irritability due to urethral thermoprobe could not tolerate the treatment. In the majority of the patients who were completely treated, a significant decrease in frequency of nocturia, decrease in post-void residual urine capacity and increase in urine flow rate were observed. No significant change in prostate volume was noted. With a mean follow-up of 6 months, only 1 patient required subsequent prostatic resection. These findings indicate that local hyperthermia applied by this method is effective in the treatment of benign prostatic hyperplasia and that improvement of the thermometry system is needed. PMID- 1722630 TI - [Transrectal hyperthermia for benign prostatic hyperplasia]. AB - Transrectal hyperthermia was performed on 30 patients with benign prostatic hyperplasia twice a week for a total of ten times with the temperature of the prostatic tissue set at 43.0 degrees C. In our in vitro experiment using an agar phantom, the highest temperature was observed at approx. 1.5 cm from the point where the 915-MHz microwave was generated. Our histopathological study of the prostatic tissue, resected at open surgery after three days of hyperthermia, indicated that the effect of hyperthermia first occurred in the interstitial tissues, and then extended to the epithelial cells. Subjective symptoms and objective findings were evaluated. In almost all cases, improvement was observed in subjective symptoms after completion of the treatment. The residual urine volume improved significantly. Also, significant improvement was observed in our urodynamics study. In 16 out of 30 cases (53%), both subjective symptoms and objective findings were still improved after six months. PMID- 1722631 TI - [Transrectal hyperthermia for benign prostatic hyperplasia]. AB - The PRIMUS system was designed as a dedicated microwave hyperthermia system for treatment of benign prostatic hyperplasia (BPH). Completed cases were 36 patients. Hyperthermia was subjectively effective in 31 patients (86%), but no appreciable changes were noted with regard to the size of the prostate. Side effects occurred in 10 of the evaluable 49 cases (20%). However, no severe side effects were recognized except in one case. The effective ratio revealed 25 of 36 cases (69%). Therefore, transrectal prostatic hyperthermia would be a useful method especially in cases of BPH with severe complications. PMID- 1722632 TI - [Clinical evaluation of balloon dilatation in patients with benign prostatic hyperplasia]. AB - Balloon dilatation of the urethra was performed on 38 patients (aged 61 to 89 years) with benign prostatic hyperplasia. Of the 38 patients, 34 (89.5%) achieved improvement of symptoms including urinary retention and difficulty on urination and no recurrence of symptoms was observed during a follow-up period. The maximum flow rate remarkably increased after the treatment in the patients and there was no increase of residual urine volume in 17 patients followed up for 6 to 12 months. The balloon dilatation of the prostatic urethra proved to be useful for patients with benign prostatic hyperplasia. PMID- 1722633 TI - [Transurethral balloon dilatation of the prostate: initial results, indication and complication]. AB - Transurethral balloon dilatation therapy was performed on 40 patients with benign prostatic hypertrophy (BPH) under local anesthesia. During the procedure, urinary urgency occurred in 80% of the patients. After prostatic dilatation, macrohematuria was observed in almost all patients. The longest follow-up period after dilatation was now 22 months, and the average was 9.5 months. After treatment, residual urine volume decreased, and average flow rate and maximal flow rate improved from 5.7 and 10.4 ml/sec to 8.2 and 15.6 ml/sec, respectively. Overall clinical efficacy was 67.5%. Urethral dilatation therapy was thought to be an effective and non-invasive therapy for BPH. PMID- 1722634 TI - [Treatment of benign prostatic hypertrophy using balloon dilation]. AB - Balloon dilation of the prostatic urethra was performed for the management of benign prostatic hypertrophy. The patients selected were mainly high risk patients who were poor surgical candidates for transurethral resection of the prostate. Ten patients between 61 and 87 years old with a mean age of 77.8 years were treated. Of the 10 patients 6 had urinary retention. The procedures were performed under spinal anesthesia, using 75 Fr dilation balloon for 10 minutes twice at 3 atmospheres. In a couple of months after treatments, 5 patients showed improvement in both uroflowmetry and clinical symptoms. Three patients revealed improvement of clinical symptoms only and 2 were in vain. One year after treatment, 3 out of 6 patients had persistent improvement. Balloon dilation of prostatic urethra was safe and showed promising effectiveness for high risk patients. PMID- 1722635 TI - [Clinical study of a metallic prostatic stent]. AB - Treatment using a metallic spiral was attempted on 22 patients. They had urinary retention in 16 cases and dysuria in 6. In 21 of them, the spiral was successfully placed under transrectal ultrasound control. In all 21 patients, voiding was possible immediately after placement of the spiral. As for urodynamic study, urine volume was 100-230 ml (mean: 172 ml), maximum flow rate was 13-21 ml/sec (mean: 17.3 ml/sec), and average flow rate was 4-12 ml/sec (mean: 8.2 ml/sec). Residual urine volume was less than 30 ml in 20 patients and 200 ml in one. As for complications, proximal migration of the spiral was observed in 6 patients. In 4 of them, the new spiral was placed. Perineal discomfort was seen in 3 patients, in 2 the spiral was removed. Pyuria associated with bacterial infection did not continue after the treatment in patients having a catheter. Severe urge incontinence and encrustation were never seen. The above findings suggest that, though a longterm study has not done, this treatment could be an effective treatment particularly for the elderly patients with general complications and/or who require removal of an indwelling catheter for clinical or social reasons. PMID- 1722636 TI - [Clinical evaluation of serum basic fetoprotein in patients with urogenital malignancies and renal transplantation]. AB - The serum basic fetoprotein (BFP) in patients with urogenital diseases was measured by enzyme immunoassay (EIA). The positive range of serum BFP was defined to be 75 ng/ml or more. In benign cases except for renal transplantation, the positive rate of serum BFP was 11.1% (5/45), and relatively high (21.4%, 3/14) in benign prostatic hypertrophy. In cases of urogenital cancers before treatment, the positive rate of serum BFP was 29.1% (16/55), and increased with the progression of clinical stage. Eleven of the patients with positive serum BFP before treatment were re-examined after treatment, and all of them exhibited a marked decrease of the titer of serum BFP. In seventeen renal transplant patients, the positive rate of serum BFP was 100% (8/8) in acute rejection, 66.7% (2/3) in chronic rejection and 0% (0/6) in rejection-free condition. We conclude that serum BFP is a clinically beneficial marker for renal transplant rejections and urogenital malignancies. PMID- 1722637 TI - [Clinical study of urinary fungal infection: survey of patient background and fungal strains from the urine]. AB - Clinical features of urinary fungal infections were analyzed in 152 patients in whom fungi were cultured from urine and mycologically identified between February, 1989 and June, 1990. The average age was 66 years old, and approximately 70% of the patients were 60 years old or older. Systemic and urinary tract underlying diseases were observed in 145 patients (95.4%) and 70 patients (46.1%) had urinary indwelling catheters. Previous use of antimicrobial agents before isolation of urinary fungi was recorded in 103 patients (67.8%), the 2nd and 3rd generation cephems and new quinolones being most frequent. A mixture of fungi and bacteria was observed in 68 patients (44.7%). Gram-positive cocci were isolated from 50% of them. Of 173 strains of fungi, C. glabrata was the most frequent (31.2%), followed by C. albicans (27.8%), C. tropicalis (17.3%) and T. beigelii (15.0%). All strains of C. glabrata, C. albicans, C. tropicalis and T. beigelii were highly sensitive to flucytosine (5-FC), miconazole (MCZ) and amphotericin B (AMPH), except for 3 strains which were resistant to 5-FC. The consecutiveness was confirmed by repeated culture in 59.4% of the patients. Fungi in the urine, however, spontaneously disappeared in the other patients. PMID- 1722638 TI - [Cefixime concentration in human prostatic tissue following 3-days of administration]. AB - The penetration of Cefixime (CFIX) into the prostatic tissue and the serum was examined in 54 patients with benign prostatic hypertrophy treated with transurethral resection of the prostate. CFIX was administered orally in a dose of 200 mg 2 times daily for 3 days preoperatively. The blood samples were taken at the time of the tissue sampling. The patients were divided into 2 groups. In group 1 (16 patients), the tissue sampling was done about 17 hours after the final drug administration. The mean concentration of CFIX was 0.83 +/- 0.49 micrograms/g in the prostatic tissue and 0.84 +/- 0.63 micrograms/ml in the serum. In the prostatic tissue, CFIX was detected in only 4 patients, in the other 12 patients, CFIX was not detected. In group 2 (38 patients), tissue sampling was done 5.5 hours after the final drug administration. The mean concentration of CFIX was 1.08 +/- 0.47 micrograms/g in the prostatic tissue and 3.18 +/- 1.28 micrograms/ml in the serum. PMID- 1722639 TI - Acupuncture to the skin induces release of substance P and calcitonin gene related peptide from peripheral terminals of primary sensory neurons in the rat. AB - We immunohistochemically examined the short term effects of electro-acupuncture (E-acupuncture) to the skin on substance P (SP)- and calcitonin gene-related peptide (CGRP) containing primary sensory neurons in the rat. Immunoreactivity to SP and CGRP in these neurons at the treatment site decreased after 30 min of E acupuncture. These results suggest that E-acupuncture induces release of SP and CGRP from peripheral terminals of primary sensory neurons. PMID- 1722640 TI - Effects of ryo-kan-kyomi-sin-ge-nin-to extract on degranulation of and histamine release from rat mast cells. AB - The effects of Ryo-kan-kyomi-sin-ge-nin-to (RKSG)) extract, a medicinal agent traditionally used in China and Japan for treatment of asthma, on the degranulation of and histamine release from rat mast cells were studied. At a concentration of 5 mg/ml RKSG, degranulation of mast cells stimulated either by antigen (DNP-Ascaris) or compound 48/80 was markedly suppressed. At a concentration of 1-5 mg/ml RKSG, histamine release from mast cells due to application of either antigen or compound 48/80 was inhibited in a dose-dependent fashion. These results suggest that RKSG may be useful for the treatment of type I allergy-related diseases. PMID- 1722641 TI - Steady-state effects of arginine vasopressin on force and pHi of isolated mesenteric resistance arteries from rats. AB - Control of intracellular pH (pHi) in rat intact resistance arteries has been assessed during activation with arginine vasopressin (AVP) or depolarization with a high potassium concentration. Isometric force of isolated arteries was measured simultaneously with pHi using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Under control conditions, pHi was unchanged during AVP-induced force development but fell during potassium activation. After inhibition of Na(+)-HCO3- cotransport, AVP-induced force development was associated with a fall in pHi. After inhibition of Na(+)-H+ exchange pHi was unchanged during activation with AVP. In the absence of bicarbonate, inhibition of Na(+)-H+ exchange caused an exaggerated fall in pHi during activation with AVP. When AVP was added to depolarized vessels, a further force development and an increase in pHi was seen. This increase in pHi was not affected by amiloride but disappeared after inhibition of Na(+)-HCO3- cotransport by 4,4'-diisothiocyanostilbene-2,2' disulfonic acid or sodium-free conditions. These data suggest that AVP, but not depolarization, changes the characteristics of the Na(+)-HCO3- exchange and the Na(+)-H+ exchange so that these transport systems extrude the acid load associated with the force development more efficiently. We suggest that the importance of the effect of vasoconstrictor hormones on the characteristics of acid extrusion from vascular smooth muscle cells (VSMC) in situ lies in their ability to maintain pHi at resting levels during the metabolic load associated with contraction in the tonically active VSMC. PMID- 1722642 TI - Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons. AB - [3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+) sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions. PMID- 1722643 TI - Two outward K+ currents in bovine pigmented ciliary epithelial cells: IK(Ca) and IK(V). AB - Pigmented ciliary epithelial cells were studied using the whole cell voltage clamp technique. Depolarizing steps from a holding potential of -80 mV resulted in a small initial inward current followed by a large outward current. Prolonged depolarizing voltage steps revealed inactivating and noninactivating components of outward current. Outward current was sensitive to the level of Ca2+ in the pipette and was increased by the calcium ionophore A23187; it was blocked by tetraethylammonium (TEA+), quinine, and 4-aminopyridine (4-AP). 4-AP blocked 70% of the outward current with a Ki of 7 x 10(-5) M, and part of the remaining current was abolished by Ni2+. Ni2+ caused a reduction in outward current by blocking IK(Ca) indirectly via decreasing Ca2+ entry through T-type Ca2+ channels. Separating Ni(2+)-sensitive from -insensitive outward conductance gives components that correspond notionally to IK(Ca) and IK(V), respectively. On this basis IK(Ca) represents approximately 28% of K+ outward current. Charybdotoxin blocked 26% of the outward conductance at very depolarized voltage steps as calculated from the slope of the current-voltage curve in this region. It is concluded that there are two major components to the outward current: IK(V), an inactivating voltage-sensitive K+ current, and IK(Ca), which is dependent on the entry of Ca2+ through T-type Ca2+ channels and comprises approximately a quarter of the total K+ outward current under the conditions described. Because of their relative voltage-activation properties, IK(Ca) will be the more important in terms of K+ transport and the secretion of aqueous humor by the ciliary epithelium. PMID- 1722644 TI - Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems. AB - The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [3H]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) approximately 4-diphenylacetoxy-N methylpiperidine methbromide (4-DAMP) (1.6) much greater than pirenzepine (136) greater than 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (Ki) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed Ki values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh elicited and Gi-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation. PMID- 1722645 TI - Nitric oxide: mediator of NANC hyperpolarization of opossum esophageal smooth muscle. AB - Activation of intrinsic nonadrenergic noncholinergic (NANC) esophageal nerves during peristalsis or by electrical field stimulation (EFS) in vitro produces a hyperpolarization followed by a depolarization of the circular smooth muscle of the opossum esophagus. N omega-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, and nitric oxide (NO) were used to test the hypothesis that NO or a NO-containing compound is a mediator of this NANC nerve-induced hyperpolarization of circular esophageal smooth muscle. The transmembrane potential difference of esophageal circular smooth muscle cells was recorded with glass microelectrodes. Nerve-mediated membrane responses were evoked by single electrical pulses of 0.5 ms duration and 50 V amplitude. L-NNA abolished the initial hyperpolarization and reduced the amplitude of and the time to maximal depolarization. L-Arginine (1 mM), the substrate for NO synthase, antagonized the effect of L-NNA. Exogenous NO produced hyperpolarization of the smooth muscle membrane potential and attenuated the amplitudes of EFS-induced hyperpolarization and depolarization. The effect of NO was blocked neither by L-NNA nor by tetrodotoxin (1 microM). The data support the hypothesis that NO or a NO containing compound mediates NANC nerve-induced responses of the esophageal smooth muscle membrane. PMID- 1722646 TI - A capacitative Ca2+ influx is required for sustained fluid secretion in sublingual mucous acini. AB - The Ca2+ dependence of muscarinic-induced fluid and electrolyte secretion was studied using rat sublingual mucous gland preparations. During stimulation, secretions from vascularly perfused glands were totally inhibited when perfused with a Ca(2+)-free medium. Fluid secretion correlated with sustained losses of 42K+ and 36Cl- content and sustained increases in 22Na+ content and the intracellular free Ca2+ concentration ([Ca2+]i) in fura-2-loaded acini. The magnitudes of the initial agonist-induced changes in Na+, K+, and Cl- content and [Ca2+]i were unaltered in a Ca(2+)-free medium, whereas extracellular Ca2+ removal resulted in the recovery of these ions during the sustained phase to pre stimulation levels. The recovery of Cl- content induced by Ca2+ depletion was totally blocked in the presence of bumetanide, an inhibitor of Na(+)-K(+)-2Cl- cotransport, while 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion exchange, did not influence Cl- recovery in a HCO(3-) containing solution (25 mM NaHCO3, 5% CO2). The stimulated increase in [Ca2+]i was not inhibited by the addition of voltage-activated Ca2+ channel blockers (D 888, nifedipine, and diltiazem) or in a Na(+)-free medium. Studies using the quench of fura-2 by Mn2+ as an index of Ca2+ influx and thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, indicate that a capacitative Ca2+ entry pathway mediates Ca2+ entry during stimulation. The above data demonstrate that Ca2+ uptake, which is dependent on the refill status of the agonist-sensitive intracellular Ca2+ pool, is a prerequisite for sustained muscarinic-induced fluid and electrolyte secretion in the rat sublingual mucous gland. PMID- 1722647 TI - Carbachol acts through protein kinase C to modulate cholecystokinin receptors on pancreatic acini. AB - Cholecystokinin (CCK) and cholinergic agonists are both major stimulants of pancreatic enzyme secretion and both utilize a common calcium-phosphoinositide mediated receptor coupling system. In this study we investigated the modulation of pancreatic acinar CCK receptors by the muscarinic agonist carbachol (CCh) and investigated the intracellular mechanisms involved in the modulation. Acini were isolated from rat pancreas and dispersed in N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid-Ringer solution. Preincubation with 0.1 mM carbachol for 60 min reduced the CCK octapeptide (CCK-8; 100 pM)-stimulated amylase release by 43 +/- 5%. Binding of 125I-Bolton-Hunter-labeled CCK-8 (125I-BH-CCK-8) revealed two classes of CCK receptors, a high affinity with a dissociation constant (Kd) of 20 pM and a low affinity with a Kd of 2.3 nM. Pretreatment with 100 microM CCh decreased total binding by 35 +/- 6%, affecting the binding capacity of the high affinity site, without change in the maximal binding capacity of the low-affinity site and no change in the Kd of either site. Preincubation of acini with 12-O tetradecanoylphorbol 12,13-acetate (TPA, 1 microM), an activator of protein kinase C (PKC), decreased subsequent CCK-8-stimulated amylase release, and total binding of 125I-BH-CCK-8 to a similar extent as with pretreatment with CCh. The inhibitory effect of TPA or CCh on CCK-8-stimulated amylase release was reversed by simultaneous preincubation with H-7, an inhibitor of PKC. Pretreatment of acini with the calcium ionophore A23187, vasoactive intestinal peptide, or 8 bromoadenosine 3',5'-cyclic monophosphate had no effect on 125I-BH-CCK-8 binding. After CCh or TPA preincubation, CCK-8-stimulated production of [3H]inositol phosphates was inhibited by at least 49%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722648 TI - On the activation of outwardly rectifying anion channels in excised patches. AB - Previous studies have shown that outwardly rectifying anion channels can be activated in excised patches by exposure to protein kinases or large depolarizing voltage pulses and by raising the bath temperature to 37 degrees C. These maneuvers presumably induce some conformational change in the channel or a regulatory molecule. However, the mechanisms underlying stimulation have not been defined. We have tested several procedures known to influence the structure and solubility of proteins for their effects on activation of anion channels in excised patches. Spontaneous and voltage-induced activation of outward rectifiers was enhanced by increasing the ionic strength or the pH of the bath solution. These maneuvers had no effect on the activity of calcium-activated cation nonselective channels. Activation of outward rectifiers depended on the anion used and followed the (inverse) Hofmeister series consistent with a salting-in process. Divalent cations also enhanced activation with relative potencies Ba greater than Ca greater than Mg but at a much lower concentration (4 meq/l). Exposing patches from T84 cells to purified catalytic subunit of adenosine 3',5' cyclic monophosphate-dependent kinase had no effect on the outward rectifier but did activate the low-conductance Cl channel. The results indicate that the outward rectifier is labile and raise the possibility that nonspecific physical mechanisms may contribute to its activation in excised patches by kinases and other stimuli. PMID- 1722649 TI - Release of tachykinins by histamine, methacholine, PAF, LTD4, and substance P from guinea pig lungs. AB - The release of substance P- and neurokinin A-like immunoreactivities (SP-LI and NKA-LI) after tracheal infusion of histamine, methacholine, leukotriene D4, and platelet-activating factor was measured in isolated guinea pig lungs superfused through the trachea. Infusion of each of these agonists was associated with a significant (P less than 0.05) increase in the recovery of both SP-LI and NKA-LI from lung perfusates compared with preinfusion baseline recoveries of these peptides. After infusion of bronchoactive mediators, approximately 4-15 times more NKA-LI than SP-LI was recovered from the lung superfusate. Coincident with the release of neuropeptides, mediator infusion was accompanied by an increase in airway opening pressure (Pao). Addition to the perfusate of the neutral endopeptidase inhibitor thiorphan, 1 microM increased the change in Pao induced by histamine (10(-8) mol, P less than 0.005) and methacholine (10(-8) mol, P less than 0.02) and increased the recovery of NKA-LI (P less than 0.05 for histamine and methacholine). Addition of isoproterenol to the perfusion buffer reduced, but did not abolish, either the Pao response or the increased recovery of NKA-LI (P less than 0.05) observed after histamine infusion. We conclude that bronchoactive agonists have the capacity to release both SP-LI and NKA-LI, and we speculate that NKA contributes to the bronchomotor response observed in response to histamine or methacholine. PMID- 1722650 TI - [Anterior resection of the rectum and local recurrence]. PMID- 1722651 TI - Single ion channels, medicine, and measurement science. PMID- 1722652 TI - On-line coupling of supercritical fluid extraction with multidimensional microcolumn liquid chromatography/gas chromatography. AB - An on-line multidimensional supercritical fluid extraction/microcolumn liquid chromatography/capillary gas chromatography system (SFE/LC/GC) has been developed and applied to the quantitative determination of trace levels (parts per billion) of chlorpyrifos insecticide in grass field samples. This system provides all the advantages of an on-line multidimensional system, including increased resolving power, high sensitivity, quantitation, precision, and automation potential. Off line analysis of the grass extracts by GC with an electron capture detector yielded a complex chromatogram from which it was difficult to quantitate the chlorpyrifos, but analysis of the extract by LC/GC yielded a simple chromatogram from which chlorpyrifos could be quantitated. On-line SFE/LC/GC resulted in reduced sample preparation with the grass extract being deposited directly on the LC microcolumn via an impactor interface, followed by the LC/GC separation. The reproducibility of the on-line SFE/LC/GC procedure was studied and found to yield a relative standard deviation of 10.8% for the determination of chlorpyrifos insecticide in grass field samples at a concentration of 160 ng/g. Using this method, the entire analysis including extraction, clean-up, and gas chromatography required less than 0.1 mL of organic solvent. PMID- 1722653 TI - Ion channel sensors for glutamic acid. AB - Coulometric biosensors using glutamate receptor (GluR) ion channel protein as a signal-amplifying sensory element that exploit the glutamate-triggered Na+ ion current through bilayer lipid membranes have been fabricated. The formation of stable planar bilayer lipid membranes was achieved by applying the folding method across a small circular aperture bored through a thin polyimide film. The multichannel type sensing membranes, formed across an aperture of ca. 120 microns diameter, contained more than 10 GluR proteins and showed L-glutamate-triggered response as a composite of individual single-channel currents. The single-channel type sensing membranes, formed across an aperture of ca. 20 microns diameter, contained a sufficiently small number of GluR proteins so that the response was observed as a series of single-channel pulse currents. Dependence of the integrated channel current on the glutamate concentration was examined. A sharp concentration dependence of up to ca. 1.5 x 10(-7) M and 3 x 10(-6) M for the multichannel and single-channel type sensors, respectively, was observed. A high selectivity for L-glutamate compared with D-glutamate for inducing the channel current was observed. A detection limit as low as ca. 3 x 10(-8) M was attained for the multichannel type sensor. This remarkable sensitivity is discussed in terms of the potential use of GluR ion channel protein for a new type of sensing system. PMID- 1722654 TI - Identification of Mycoplasma gallisepticum by use of monoclonal antibody in a rapid slide agglutination test. AB - Monoclonal antibody (MAb) against Mycoplasma gallisepticum strain PG31 was produced in BALB/c mice. The MAb (designated M9) was of IgG3 isotype and reacted with an epitope in M gallisepticum antigens with molecular weights of 35, 90, 95, and 98 kilodaltons (kDa). The M9 reacted with M gallisepticum antigens in the dot blot ELISA and in western blot assays. It agglutinated M gallisepticum strains PG31, F, R, S6, A5969, and 9 field isolates from various sources. A coagglutination assay, using Staphylococcus aureus (Cowan strain 1), was developed to enhance the agglutination of some weakly agglutinating M gallisepticum isolates. The M9 did not react with M synoviae, M iowae, M meleagridis, M gallinarum, or M gallinaceum in any of the aforementioned assays. This MAb may be useful in facilitating laboratory diagnosis of M gallisepticum infections. PMID- 1722656 TI - [Artificial nutritional approach in the palliative treatment of esophageal cancer]. AB - Authors, after to linger over necessity of artificial nutritional approach in patients with inoperable esophageal cancer, emphasize the various moments and condition where is right to operate. In conclusion, they wish the increase of early diagnosis, one and only to guarantee appraisable results. PMID- 1722655 TI - Effects of a highly concentrated hypertonic saline-dextran volume expander on cardiopulmonary function in anesthetized normovolemic horses. AB - Conventional fluid resuscitation is unsatisfactory in a small percentage of equine emergency surgical cases because the large volumes of fluids required cannot be given rapidly enough to adequately stabilize the horse. In anesthetized horses, the volume expansion and cardiopulmonary effects of a small volume of highly concentrated hypertonic saline-dextran solution were evaluated as an alternative initial fluid choice. Seven halothane-anesthetized, laterally recumbent, spontaneously ventilating, normovolemic horses were treated with a 25% NaCl-24% dextran 70 solution (HSD) at a dosage of 1.0 ml/kg of body weight, IV, infused over 10 minutes, and the effects were measured for 120 minutes after infusion. Plasma volume expansion was rapid and significant (from 36.6 +/- 4.6 ml/kg to 44.9 +/- 4.8 ml/kg), and remained significantly expanded for the duration of the experiment. Packed cell volume, total blood hemoglobin, and plasma protein concentrations significantly decreased, confirming rapid and sustained volume expansion with hemodilution. Cardiac index and stroke index immediately increased and remained high for the entire study (from 69.6 +/- 15.3 ml/min/kg to 106.6 +/- 28.4 ml/min/kg, and from 1.88 +/- 0.49 ml/beat/kg to 2.50 +/- 0.72 ml/beat/kg, respectively). Systemic vascular resistance significantly decreased immediately after HSD infusion and remained decreased for the duration of the study (from 1.41 +/- 0.45 mm of Hg/ml/min/kg to 0.88 +/- 0.22 mm of Hg/ml/min/kg). Arterial and venous blood oxygen content decreased significantly because of hemodilution, but actual oxygen transport transiently increased at the 10-minute measurement before returning toward baseline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722657 TI - [Pseudotumoral lipid pneumopathies. Apropos of six cases]. AB - In cases of localised pneumonia due to oil aspiration, the diagnosis may be difficult and is often assessed by thoracotomy. Six cases of lung paraffinoma are reported. The lesion, localised in the lower lobe in five patients out of six, was discovered on screening chest x-rays. In two cases, two lesions were observed in the same patient. All six patients underwent lung resection. Diagnosis was made on histologic examination showing foreign body reactions against oil. Oil aspiration was due to oily nose drops in one patient and to the use of paraffin oil on a tracheostomy in two others. The preoperative diagnosis may be suspected on bronchial lavage CT scan and MRI. When the diagnosis is strongly suspected, thoracotomy can be avoided as in some cases withdrawal of the medication can be followed by progressive resolution of the radiological signs. PMID- 1722658 TI - Effects of flutamide and hydroxy-flutamide on the growth of human benign prostatic hyperplasia cells in primary culture: a preliminary report. AB - Tissues from human benign prostatic hyperplasia [BPH] were collected from twelve patients undergoing routine transurethral resection of the prostate to relieve urine out-flow obstruction. Viable epithelial organoids were obtained after enzymatic digestion of the tissue. Primary cultures of epithelium were successfully maintained on collagen gel for up to 21 days. Immunocytochemical staining revealed that there was no expression of either desmin or vimentin in these cells; however, the anticytokeratin antibodies LP-34 (cytokeratins 4, 5, 6, 10, 13, 16, 17 and 18), LE-61 (cytokeratin 18) and CAM 5.2 (cytokeratins 7 and 8) all showed positive responses, indicating the epithelial nature of the cells. Cell growth was significantly increased in the presence of 3 x 10(-10) M testosterone propionate [TP] in the culture medium. The presence of the non steroidal anti-androgens, Flutamide and Hydroxy-Flutamide [Flu-OH], in the concentration range 1.0-0.001 micrograms per ml of medium inhibited the growth in the presence of androgens in a dose-dependent manner. The anti-androgens failed to affect cell growth in the absence of TP. In view of these preliminary findings, it is postulated that the antiandrogens might be acting either by displacing the androgen from its receptor or alternately by inhibiting the activity of prostatic 5 alpha-reductase. PMID- 1722659 TI - Improved treatment of disseminated B16f10 melanoma in mice with anticancer drugs in combination with L-histidinol. AB - An artificial hematogenous-metastasis model, in which B16f10 melanoma cells injected into the tail veins of C57/BL mice arrest in the lungs and proliferate as discrete pulmonary foci, was employed to examine effects of L-histidinol on the capacity of a number of conventional antineoplastic agents to manage disseminated disease. Treatment responses were evaluated by determining both the number of lung foci and/or by evaluating animal survival. L-Histidinol, on its own, was found to have a significant and dose-dependent capacity to reduce the number of lung foci and to extend survival of animals bearing disseminated B16f10 melanoma. L-Histidinol enhanced the ability of bis-chloroethylnitrosourea, 5 fluorouracil, and 1-beta-D-arabinofuranosulcytosine to reduce the number of lung foci. The latter combinations also gave marked improvements in survival, whether administered 1 or 7 days after the intravenous injection of tumor cells. PMID- 1722660 TI - Selective usage of TCR V beta in tumor-specific CTL lines isolated from ovarian tumor-associated lymphocytes. AB - We have developed a series of six CTL lines exhibiting preferential killing of autologous tumour cells from tumour-associated lymphocytes (TALs) infiltrating malignant ovarian ascites. Five out of six CTL lines showed increased percentages of V beta 8.1+ cells, four showed increased percentages of V beta 5.3+ cells and one showed increased percentages of V beta 6.7+ cells. On the contrary, fresh isolated TALs or autologous PBMCs, when stimulated by OKT3 mAb or other TAL cultures with nonspecific cytolytic function, did not show preferential usage of any of these families. Most important, V beta 8.1+ and V beta 6.7+ T cells within the CTL-TALs mediated cytoxicity against autologous targets. This represents the first demonstration of preferential usage of TCR V beta in ovarian tumor-specific CTLs and suggests possible association between tumor surveillance and TCR V beta genes. PMID- 1722661 TI - Plasmids of Pseudomonas cepacia strains of diverse origins. AB - Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains. PMID- 1722662 TI - Phylogenetic characterization and in situ localization of the bacterial symbiont of shipworms (Teredinidae: Bivalvia) by using 16S rRNA sequence analysis and oligodeoxynucleotide probe hybridization. AB - It has been proposed that a bacterium isolated from the gills of shipworms (teredinid mollusks) is, by virtue of its ability both to degrade cellulose and to fix dinitrogen, the symbiont that enables these mollusks to utilize wood as their principal food source. The phylogenetic affiliation of four of these bacteria isolated from wood-boring bivalve mollusks was determined by 16S rRNA sequence analysis by using the reverse transcriptase method with six oligodeoxynucleotide primers. The four bacterial strains tested had indistinguishable 16S rRNA sequences, supporting the previous conclusion, based on phenotypic characterization, that these isolates represent a single species. Evolutionary distance matrix analysis of the RNA sequence indicated that the bacterial symbiont falls within the gamma-3 subdivision of the Proteobacteria and is distinct from other known bacterial genera. In situ localization of the bacterial symbiont in tissue sections of the shipworm Lyrodus pedicellatus was determined by using a 16S rRNA-directed oligodeoxynucleotide hybridization probe specific for the bacterium isolated from shipworm gill tissue. Fluorescence microscopy showed that the specific probe bound to L. pedicellatus tissue at sites coincident with the location of symbiont cells and that it did not bind to other host tissues. This technique provided direct visual evidence that the cellulolytic, nitrogen-fixing bacterial isolates were the symbionts observed within the gill of L. pedicellatus. PMID- 1722663 TI - Distribution, clearance, and mortality of environmental pseudomonads in mice upon intranasal exposure. AB - When introduced intranasally, P. cepacia AC1100 (approximately 10(8) CFU/animal) and P. aeruginosa AC869 (approximately 10(3) CFU/animal) were readily cleared from the mouse. However, a approximately 10(7)-CFU dose of AC869 persisted for 14 days. Strain AC869 had a 50% lethal dose of 2.7 x 10(7) CFU. Slight morbidity occurred in animals treated with approximately 10(7) CFU of AC869 or approximately 10(8) CFU of AC1100. PMID- 1722664 TI - Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity. AB - Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N nitrosoguanidine together with selective enrichment on the glucose analog 2 deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested. PMID- 1722665 TI - Impact of endocervical specimen quality on apparent prevalence of Chlamydia trachomatis infections diagnosed using an enzyme-linked immunosorbent assay method. AB - Duplicate endocervical swabs were collected from 1824 patients for detection of Chlamydia trachomatis. Specimen pairs were combined into 400 microL of 0.9% saline solution. After vortexing, a 40-microL sample was smeared and stained with Papanicolaou's method for detection of endocervical and/or metaplastic (E-M) cells. The remaining specimen was tested for C trachomatis antigen with the use of an enzyme-linked immunosorbent assay (ELISA) procedure (Chlamydiazyme, Abbott Laboratories, North Chicago, Ill). Chlamydia trachomatis antigen was detected and confirmed (with the use of a blocking antibody [Abbott Laboratories]) in only 16 (1.7%) of 918 specimens that lacked detectable E-M cells, but it was detected significantly more frequently not only in 88 (13.3%) of 661 specimens that contained detectable E-M cells but also in 32 (13.1%) of 245 specimens that contained too many red blood cells to analyze microscopically. Of the initially positive ELISA results, none of 37 were falsely positive from specimens that contained 11 or more E-M cells, but significantly more (six [27.3%] of 22) were falsely positive from specimens that lacked detectable E-M cells. Variations in specimen quality had a significant impact on the incidence of both true-positive and false-positive ELISA results and could significantly influence understanding of the prevalence of chlamydial infections in women. PMID- 1722666 TI - Immunochemical analysis of high molecular-weight human salivary mucins (MG1) using monoclonal antibodies. AB - Using four Mabs with different specificities for salivary mucins, an ELISA has been developed in which human whole saliva, glandular salivas, salivary protein fractions and purified, high molecular-weight, mucin fractions (MG1) isolated from human submandibular and sublingual glandular tissues have been immunochemically analysed. All four Mabs reacted with MG1s. Three of them reacted with the purified, low molecular-weight salivary mucins (MG2). None was reactive with parotid saliva. MG1 preparations isolated from submandibular and sublingual glandular tissues of one and the same individual displayed different patterns of reactivity with these Mabs, indicating that they differ immunochemically. Analysis of the MG1s in salivas derived from individual salivary glands showed differences in immunochemical composition. These results indicate that the MG1 fraction in human whole saliva consists of several immunochemically different species. PMID- 1722667 TI - Immunological relevance of malonic dialdehyde (MDA): IV. Further evidences about the epitope recognized by antibodies obtained from rabbits immunized with MDA modified lysozyme. AB - Reactions of MDA with primary amino groups produce inter- or intra-molecular 1 amino-3-imino-propene (AIP) bridges, leading to structural modifications of biological molecules. In this work, applying electrophoresis followed by transfer onto nitrocellulose membranes, we observed that serum of a rabbit immunized with MDA-modified lysozyme (ML) reacts not only with ML and native lysozyme (L), but also with MDA-modified ribonuclease, cytochrome c or polylysine (MR, MC and MP respectively), while it does not react with native ribonuclease, cytochrome c or polylysine (R, C and P respectively). These results confirm previous ones indicating that sera of rabbits immunized with ML contain antibodies reacting specifically with epitopes containing AIP bridges. PMID- 1722668 TI - Quantification of ATP-producing and consuming processes in quiescent pig spleen lymphocytes. AB - ATP production in quiescent pig spleen lymphocytes was estimated on the basis of their coupled respiration. ATP-consuming processes were assessed from the effects of inhibitors of protein synthesis, proteolysis, RNA synthesis, Na+ K(+)-ATPase and Ca(2+)-ATPase on respiration. About 95% of the total ATP consumption could be assigned to specific processes. More than 50% of the ATP produced appear to be consumed by the cation transport ATPases. PMID- 1722669 TI - Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture. AB - We observed previously in vitro that the cytotoxicity of bleomycin (BLM), an anticancer drug in current use, was greatly potentiated by exposing cultured cells to appropriately chosen electric pulses. We then showed in vivo, on tumor bearing mice, that the same electric pulses also potentiated the antitumoral activity of BLM. In the present work, we demonstrate on DC-3F cells in vitro, that this potentiation is closely related to cell electropermeabilization and the consequent direct internalization of BLM molecules in the cytosol. The survival response curve (SRC) of the electropermeabilized (EP) cells exposed to BLM (plotted as logarithm of survival versus external drug concentration) shows a linear pattern usual for the SRCs of intact cells exposed to current cytotoxic drugs, though in the nanomolar range of concentrations. We have succeeded in determining the relation between BLM cytotoxicity on EP cells and the number of electroloaded BLM molecules per cell (that is the average number, per cell, of BLM molecules internalized into the cytosol). We conclude that (1) BLM molecules possess very intense cytotoxic activity which in non-EP cells is drastically limited by the intact plasma membrane; and (2) in these intact cells, the plasma membrane is responsible for the unusual upward concave curvature of the SRC resulting from exposure to BLM. PMID- 1722670 TI - Sialic acid removal modulates the myocardial and vascular activity of calcium channel ligands. AB - Selective removal of sialic acid from isolated guinea pig left atrial strips and rabbit thoracic aortic ring segments was performed by neuraminidase prepared from Clostridium perfringens and was controlled electron microscopically. Preincubation of these organs (2 units/mL; 2 hr) resulted in enzyme mediated hydrolysis of total tissue sialic acid; 55.2% for atria and 60.9% for aorta. Contractile force of atria and arterial diameter of thoracic aorta were measured isometrically and isotonically by means of a force displacement transducer. Pretreatment of both organs with neuraminidase (2 units/mL; 2 hr) in a carbogen saturated organ bath caused a moderate left-hand shift of the cumulative concentration response curves for the dihydropyridine type calcium antagonist nisoldipine, the phenylalkylamine derivative gallopamil and the benzothiazepine diltiazem. EC50 values were significantly lower (P less than 0.05), particularly in the atrial muscle, when compared to untreated preparations. There was no effect of neuraminidase on the negative inotropic and vasodilator potency of the calcium channel modulator fendiline. Conversely, neuraminidase induced a right hand shift in the concentration response curves shown by the pure calcium agonist (-)-S-Bay K 8644 leading to significantly higher EC50 values in both organs. Similarly, the contractile potency of calcium chloride (atria) and potassium chloride (aorta) was attenuated upon neuraminidase treatment. From the results obtained it is concluded that sialic acid removal may modulate the action of calcium channel ligands through an inhibitory effect on transmembrane calcium fluxes and/or by decreasing the external calcium availability. Whether the present results suggest a functional role for sialic acid in the regulation of calcium channels warrants further investigation. PMID- 1722671 TI - Formation of molecular iodine during oxidation of iodide by the peroxidase/H2O2 system. Implications for antithyroid therapy. AB - The first step in the biogenesis of thyroid hormones is the oxidation of iodides taken up by the thyroid gland. Oxidation of I- by the H2O2/peroxidase system leads to the formation of iodinium ions I+ which bond to thyroglobulin by electrophilic substitution. However, it is not clear whether I- is transformed directly to I+ or whether it passes through a molecular iodine intermediate. This latter possibility is indicated by the oxidation potentials of the reactions. I2 can be detected in vitro from the formation of I3- ions, although this has yet to be confirmed in vivo. The present study was designed to determine, albeit indirectly, whether this reaction occurs in vivo. If I2 is produced, it may form charge transfer complexes with numerous drugs. We also investigated the action of various drugs on lactoperoxidase and assessed their antithyroid activity in the rat by assay of plasma levels of T3, T4, and TSH. We found a good correlation between the value of Kc, the formation constant of the complex of the drug with molecular iodine, and the antithyroid activity in vivo. This correlation was observed in four different classes of compound. The possibility that molecular iodine is produced in the thyroid gland has implications for antithyroid therapy. PMID- 1722672 TI - Carbonyl reduction of metyrapone in human liver. AB - Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase. PMID- 1722673 TI - [Antigenic structure of the foot-and-mouth virus. VI. Functional segments of the immunodominant region of the VP1 protein of foot-and-mouth virus strains O1K and A22]. AB - B- and T-epitopes have been localized within the protective fragments of VP1 protein, viz., 136-152 of the O1K strain and 135-159 of the A22 strain of the foot-and-mouth disease virus (FMDV). Antibodies eliciting after immunization of various animals with the 135-159 A22 peptide are directed to different sites of the peptide. Immunogenicity of fragments of the 135-159 A22 peptide on mice correlates with their activity on T-cells of the same animals and protective activity on guinea pigs. The investigations were carried out using synthetic fragments of the 136-152-O1K and 135-159-A22 peptides. PMID- 1722674 TI - [Alkaline phosphatase in reverse micelles of surfactants in organic solvent]. AB - A dimeric enzyme (alkaline phosphatase from calf intestinal mucosa) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. The dependence of the enzyme's activity on the hydration degree (on the size of micelles) is a curve with two optima corresponding to the hydration degrees [H2O]/[AOT] = 17 and 25; when the inner cavity radii of reversed micelles are equal to the size of the enzyme's monomer (Mr = 70 000) and of the dimer (Mr = 140 000). Ultracentrifugation experiments showed that a reversible dissociation of the enzyme into subunits takes place as a result of the change of the hydration degree; the first and second maxima corresponding to the functioning of the monomeric and dimeric forms of the enzyme, respectively. PMID- 1722675 TI - [Conformational analysis of tachykinins. I. N-terminal fragments of substance P, physalemin, hylambatin, and uperolein]. AB - Theoretical conformational analysis of N-terminal fragments of the title peptides has been carried out using the potential energy calculations. The number of conformational states for each fragment is very limited, and they are easily interconverted. Since these fragments cannot form alpha-helises, it is unlikely that upon binding of tachikinins to their receptors, their N-terminal fragments could overcome the hydrophobic barrier of the cell membrane's lipid belayer. PMID- 1722676 TI - Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1. AB - We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection. PMID- 1722677 TI - Similarities of viral proteins to toxins that interact with monovalent cation channels. PMID- 1722678 TI - Merkel cell carcinoma of the head and neck associated with Bowen's disease. AB - The Merkel cell carcinoma occurs primarily in the skin of the head and neck, and develops in the dermis with a trabecular growth pattern. Immunohistochemistry reveals positive staining for neuron-specific enolase, neurofilaments, cytokeratin and chromogranin A. Electron microscopically, the tumor cells contain dense-core granules, spinous cytoplasmic processes, desmosomes, zonulae adherentes and paranuclear filament aggregates besides frequent mitoses, focal necroses and lymphocyte and plasma cell infiltrates. The Merkel cell carcinoma is often co-existent with other malignancies such as squamous cell carcinoma or, as in the present study, with Bowen's disease. The definite diagnosis of the Merkel cell carcinoma can be effected only by electron microscopic examination of the tumor. PMID- 1722679 TI - Immunoreactive nerve fibers in the nasal mucosa. An experimental study on neuropeptides Y, calcitonin gene-related peptide and galanin. AB - The presence of immunoreactive nervous fibers in the respiratory nasal mucosa of rats and guinea pigs was studied by means of a modified peroxidase antiperoxidase technique for whole mounting. The fibers with neuropeptide Y (NPY) always appeared in the walls of blood vessels, while the fibers immunoreactive to calcitonin gene-related peptide (CGRP) were found in nerve tracts near the vessels and the acini of seromucous glands as thick networks located in the subepithelial layers. Immunoreactivity (IR) for galanin was found in the mucosa studied. The findings after surgical and chemical denervation of the trigeminal and superior cervical ganglia may support the theory that the fibers with NPY are of a sympathetic nature with the superior cervical ganglion their site of origin, while the CGRP-IR fibers may have a sensory nature. PMID- 1722680 TI - Differential immunohistochemical detection of cytokeratins and vimentin in the surgically removed human endolymphatic duct and sac. AB - Immunohistochemical detection of intermediate filament proteins and different subgroups of cytokeratins (Cks) was used to characterize the epithelium of the surgically removed adult human endolymphatic duct (ED) and sac (ES). The epithelium of the ED and ES demonstrated immunostaining for Cks 7, 8, 14, 17, 18 and 19, a pattern typical of so-called "complex" or "mixed" epithelia. This is a remarkable finding, since this pattern differs strikingly from previously reported data on the adult human cochlea and vestibular labyrinth that demonstrated a Ck pattern typical of "simple" (or single-layered) epithelia. Furthermore, the epithelium of the ED and ES demonstrated co-expression of Cks and vimentin. The present data indicate that the epithelium of the ED and ES exhibits another type of epithelial differentiation and demonstrates a higher degree of complexity than the other epithelia in the adult human inner ear. PMID- 1722681 TI - Acitretin decreases tumor cell-induced angiogenesis. AB - The effects of acitretin and etretinate on angiogenesis induced in Balb/c mice by intradermal injection of keratinocyte tumor cell lines were evaluated. It was shown that both retinoids are capable of inhibiting angiogenesis evoked by a human epidermoid carcinoma cell line (A431). Acitretin, but not etretinate, inhibited also angiogenesis induced by the spontaneously transformed murine keratinocyte cell line Pam 212 and by the established tumorigenic SKv cell line harboring the HPV16 genome. We suggest that inhibition of blood vessel formation may be one of the mechanisms responsible for the anticancerogenic effect of retinoids. PMID- 1722682 TI - Serum-free culture of enriched murine haemopoietic stem cells. I: Effect of haemopoietic growth factors on proliferation. AB - Using a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (termed the FACS-BM population), and a serum-free culture system, the requirements for development of multipotential cell have been investigated and compared to previous results using serum containing cultures. In both serum-free and serum supplemented cultures interleukin-3 (IL-3) was a potent colony stimulating factor, although it was more effective in serum free conditions. However, colony stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage-colony stimulating factor (M-CSF) was markedly reduced in the absence of serum. Significantly, the ability of interleukin-1 (IL-1) and granulocyte-colony stimulating factor (G-CSF) to synergise with these two growth factors was retained in serum-free conditions, indicating that these growth factors act directly on the FACS-BM without serum co factors. Furthermore synergistic interactions between IL-3 plus IL-1, and IL-3 plus M-CSF were only manifest in serum-free conditions. The significance of these results in relation to the ability of these growth factors to act directly on multipotential cells is discussed. PMID- 1722683 TI - cDNA cloning and expression of a human FGF receptor which binds acidic and basic FGF. AB - We have isolated and characterized a cDNA clone, phFGFR, encoding a human fibroblast growth factor (FGF) receptor. phFGFR contains an open reading frame which encodes an 820 amino acid polypeptide with three immunoglobulin-like domains in the extracellular part and an intracellular split tyrosine kinase domain. Transient expression in COS-1 cells and immunoprecipitation using an antiserum raised against a C-terminal peptide, gave rise to two components, representing mature (130 kDa) and precursor (115 kDa) forms of the phFGFR encoded polypeptide, which was denoted hFGFR-1. Crosslinking of iodinated acidic FGF (aFGF) and basic FGF (bFGF) to transiently expressing COS-1 cells revealed a major band of 95 kDa, which was competed for by both aFGF and bFGF. From Scatchard analyses, the Kd:s for binding of aFGF and bFGF to hFGFR-1 were estimated to 25 pM and 41 pM, respectively. Thus, phFGFR encodes a human FGF receptor with high affinity for both aFGF and bFGF. PMID- 1722684 TI - Thyrotropin inhibits while insulin, epidermal growth factor and tetradecanoyl phorbol acetate stimulate insulin-like growth factor binding protein secretion from sheep thyroid cells. AB - Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP 2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function. PMID- 1722685 TI - IGF-I and IGF-binding proteins: stimulatory and inhibitory factors secreted by human prostatic adenocarcinoma cells. AB - Deregulation of growth observed in malignant cell cultures has been assumed to be the result of increased secretion by these cells of autocrine growth factors, as well as the decreased sensitivity of these cells to inhibitory molecules which are diffused from normal or transformed cells. Our results show that PC-3 cells secreted into the medium, factors having stimulatory and inhibitory activities. We found an IGF-like molecule in medium conditioned by PC-3 cells. Its concentration was less than 1 ng/ml of conditioned medium. We demonstrated that PC-3 cells have receptors for IGF-I and are stimulated by this growth factor. However, the dose response curve shows that 1 ng/ml of IGF-I is not sufficient to indicate autocrine growth regulation by IGF of prostatic carcinoma cells. IGF binding proteins of 90,000, 45,000, 34,000 and 28,000 molecular weight were also secreted by PC-3 cells. It is noteworthy that the secreted proteins which had the greatest inhibitory effect on chick embryo fibroblast growth also has the strongest IGF-binding activity. The probability that the IGF-binding protein secreted by PC-3 cells inhibited serum stimulation of DNA synthesis by preventing stimulation induced by IGF present in the serum is discussed. It is of interest that these IGF-binding proteins inhibited chick embryo fibroblast proliferation but did not inhibit PC-3 cells. This is in agreement with the assumption that IGF present in the medium is not an autocrine growth factor for these cells. PMID- 1722686 TI - Acidic fibroblast growth factor overexpression in corneal epithelial wound healing. AB - aFGF expression was studied in normal and regenerating cornea of adult rats. aFGF mRNA and proteins were expressed mainly in corneal epithelium but not in stroma. After burning of the epithelium by iodine vapours, the intact epithelial cells migrated to cover the wounded area during the first 4 days and then divided to reconstitute a normal multilayered epithelium 6 days after injury. aFGF mRNA localized by in situ hybridization on regenerating epithelium showed a peak between 6 hr and 2 days after denudation, decreasing to basal levels 6 days later. This induction of aFGF mRNA preceded the increased amount of aFGF peptides, as assessed by indirect immunofluorescence staining. Thus aFGF overexpression is clearly correlated with active migration in epithelial wound healing. PMID- 1722687 TI - [Mouse type II cytokeratin genes CK-4 and CK-8 mapped on chromosome 15]. AB - Human cytokeratin cDNA fragment was used as a probe and hybridized to mouse hamster hybrids DNA. The Southern hybridization analysis showed that the mouse type II cytokeratin genes CK-4 and CK-8 located on chromosome 15. PMID- 1722688 TI - Chemoattractant(s) in culture supernatants of HTLV-I-Infected T-cell lines. AB - Supernatants obtained from four HTLV-I transformed cell lines (MT2, MT4, C91/PL, and 81-66/45) induced in vitro migration of monocytes, polymorphonuclear leukocytes (PMN), and lymphocytes. The MT2, C91/PL, and 81-66/45 cell lines expressed both lymphotoxin (LT) and tumor necrosis factor (TNF-alpha) mRNA transcripts, and had TNF biological activity. In contrast, the MT4 cells did not express LT mRNA, had low levels of TNF-alpha transcript, and no TNF activity in the supernatant. Anti-TNF-alpha MAb, which blocks the chemotactic activity of recombinant TNF-alpha, had no inhibitory effect on the induction of migration by the MT2 and MT4 supernatants. Hence, no correlation was evident between TNF and chemotactic activity in supernatants of different HTLV-I-infected cell lines. Upon fractionation on Sephadex G50, the monocyte chemoattractant(s) eluted with two peaks in the 8-12 kD region, a size compatible with the chemotactic cytokines IL-8 and monocyte chemotactic protein (MCP). However, anti-IL-8 and anti-MCP antibodies did not have any effect, and Northern blot analysis showed that HTLV-I transformed cell lines did not express mRNA transcripts of either IL-8 and MCP. These results demonstrate that HTLV-I transformed T-cell lines produce chemoattractant(s) active on PMN and monocytes, distinct from LT, TNF-alpha, IL 8, and MCP. Production of chemoattractants may play a role in the pathogenesis of diseases associated with HTLV-I infection. PMID- 1722689 TI - Platelet preservation during cardiopulmonary bypass with iloprost and Duraflo-II heparin-coated surfaces. AB - To test the hypothesis that temporary platelet inhibition during cardiopulmonary bypass (CPB) with surface heparinized systems may result in platelet preservation, nine Yorkshire pigs were placed on CPB for 3 hours. Platelet labeling was done in all pigs with Indium-111 tropolone. CPB was instituted with a roller pump, a hollow fiber membrane oxygenator (Bentley CM-50 [Baxter-Bentley Laboratories, Irvine, CA]), and an arterial filter. The extracorporeal perfusion systems were surface-coated with the Duraflo-II heparin complex. Group A pigs (n = 5) were systemically heparinized (activated coagulation time longer than 400 sec). Group B pigs (n = 4) were placed on CPB without systematic heparinization, but have received the stable prostacyclin-analog Iloprost (ZK36374) at 1 ng/kg/min i.v. from 30 min before and during CPB. Platelet counts declined in group A pigs at 5 min, 1 hr, 2 hr, and 3 hr of CPB to 79.8% (mean), 66.5%, 71.3%, and 69.0% of pre-CPB values, respectively (p less than 0.05). In group B pigs, mean platelet count during CPB was higher than 90% of control value. Percentage of injected radioactivity detected in the oxygenator was 2.82% in group A pigs versus 0.73% in group B pigs (p = 0.0541). Surface heparinization with the Duraflo II heparin coating complex in combination with Iloprost-induced temporary platelet inhibition resulted in platelet count preservation during CPB in the pig model. PMID- 1722690 TI - Peritoneal fluid kinetics during CAPD measured with intraperitoneal dextran 70. AB - Simultaneous measurement of transcapillary ultrafiltration (TCUF), lymphatic absorption rate (LAR), and intraperitoneal volume (IPV) was performed by means of intraperitoneally-administered polydisperse dextran 70 in nine CAPD patients during a 4 hr dialysis dwell (glucose 1.36%). The recovery of dextran was 88 +/- 1%. LAR, calculated as the amount of dextran lost, divided by the dialysate dextran concentration, was 1.30 +/- 0.12 ml/min. The time course of TCUF could be described as a hyperbola. Therefore, the application of the Lineweaver-Burke plot made it possible to calculate TCUFmax (median 641 mL) and its half-time (t50: median 211 min). delta IPV4h, calculated as the difference between the Lineweaver Burke adjusted TCUF4h and LA4h, was correlated with measured delta IPV4h after drainage (r = 0.89, p less than 0.001). The latter was dependent upon LAR (r = 0.71) and effective peritoneal surface area, as represented by mass transfer area coefficients (MTC) of low molecular weight solutes (creatinine r = -0.76, glucose r = -0.81). High MTC values of these solutes were exponentially related to a short t50 (creatinine r = -0.76). LAR was correlated with the MTC of intraperitoneally administered inulin (r = 0.83). Dextran 70 had no measurable effect on solute transport. It is concluded that dextran 70 is a useful marker for the measurement of peritoneal fluid kinetics, even during CAPD with a low glucose concentration in the dialysate. PMID- 1722691 TI - General anaesthetics induce only histamine release selectively from human mast cells. AB - We have examined the in vitro effects of increasing concentrations of propofol (5 70 micrograms ml-1), ketamine (10(-6)-10(-3) mol litre-1) and thiopentone (10(-5) 8 x 10(-4) mol litre-1) on the release of preformed histamine and de novo synthesized mediators (peptide leukotriene C4 (LTC4) or prostaglandin D2 (PGD2] from human basophils and mast cells isolated from lung parenchyma and skin tissue and from heart fragments. Propofol, ketamine and thiopentone failed to induce the release of histamine and de novo synthesis of LTC4 from basophils. Propofol induced histamine release from lung (mean 8.6 (SEM 1.6)%) and skin mast cells (3.8 (1.5)%), but not from heart mast cells. Ketamine caused release of histamine from lung (6.2 (0.9)%) and skin mast cells (2.5 (1.5)%). Thiopentone caused a small amount of histamine release from lung mast cells (3.1 (1.2)%). Propofol, ketamine and thiopentone did not induce de novo synthesis of PGD2 and LTC4 from lung and skin mast cells. These results demonstrate that general anaesthetics induce only histamine release selectively from human mast cells. PMID- 1722693 TI - Role of fixed parenchyma cells in blastema formation of the planarian Dugesia japonica. AB - Observations of fixed parenchyma cells using electron microscopy were carried out in an attempt to understand the morphogenetic process of blastema formation in regenerating planarians. Fixed parenchyma cells could be found throughout one-day blastemata. In the mid-blastema region where migrating regenerative cells build up a compact cell aggregate, long and slender cytoplasmic processes of the fixed parenchyma cells were seen occupying spaces among regenerative cells. A characteristic feature of such processes was orderly arranged microtubules. Ruthenium red staining revealed thickened portions of cell coats on these processes and occasional formation of gap junctions between the cytoplasmic process of the fixed parenchyma cell and the regenerative cell undergoing migration. Colchicine treatment (M/1,000) caused detachment of the cytoplasmic processes from the regenerative cells. Microtubules within such processes became depolymerized. As a result, directional migration of regenerative cells was inhibited by colchicine treatment. To determine the extracellular site of fibronectin, immunoelectron microscopy was performed in one-day blastema. Immunogold labeling was detected at the surface area of fixed parenchyma cells and regenerative cells. In particular the reactivity was conspicuous at the cytoplasmic process of the fixed parenchyma cells. These observations suggest that the cytoplasmic processes of fixed parenchyma cell are related to directional movement of regenerative cells by providing a contact guidance system. The biological implications of this system are discussed in relation to the extracellular matrix components. PMID- 1722692 TI - A comparison of the cardiovascular effects of phenylpropanolamine and phenylephrine containing proprietary cold remedies. AB - 1. The cardiovascular effects of the proprietary cold remedies, Mu-cron and Boots Cold Relief tablets were compared with 'placebo' Boots Pain Relief tablets in a double-blind study involving 16 healthy volunteers. Measurements (impedance cardiography, forearm plethysmography) were made over 4 h after oral drug administration. 2. Two Mu-cron tablets (containing phenylpropanolamine [(1R,2S)- plus (1S,2R)-norephedrine] 50 mg) increased blood pressure (maximal effect 18 +/- 1/8 +/- 1 mm Hg (mean +/- s.e. mean), P less than 0.001), stroke volume (4.9 +/- 0.8 ml m-2, P less than 0.05), total peripheral resistance (243 +/- 27 dyn s cm-5 m2, P less than 0.001) and forearm vascular resistance (1.3 +/- 0.3 mm Hg ml-1 min, P less than 0.01) and reduced the ratio of pre-ejection period to ventricular ejection time (-0.031 +/- 0.003, P less than 0.05) and forearm blood flow (-2.6 +/- 0.5 ml min-1, P less than 0.05) but did not affect heart rate or cardiac index. 3. Two Boots Cold Relief tablets (containing phenylephrine 10 mg and caffeine 60 mg) caused a small and short-lived increase in total peripheral resistance but did not have consistent effects on other measurements. Two Boots Pain Relief tablets (containing caffeine 60 mg) did not have important cardiovascular effects. 4. The cardiovascular effects of phenylpropanolamine, including vasoconstriction and an increase in cardiac performance, are consistent with its alpha- and beta 1-adrenoceptor agonist action. While it may help the symptoms of rhinitis, its use in patients with heart disease or hypertension is hazardous.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722694 TI - Embryonic expression of beta-actin-lacZ hybrid gene injected into the fertilized ovum of the domestic fowl. AB - An experiment was carried out to investigate the expression of cloned DNA injected into the germinal disc of the chick fertilized ovum. The beta-actin-lacZ hybrid gene, MiwZ, was injected, in the closed circular form, into the cytoplasm of the germinal disc at the single-cell stage. The embryos were cultured in vitro, then in recipient eggshells up to day 4 of incubation. The survival rate of the embryos at day 4 was 42% (55/130), and the rate of embryos expressing MiwZ was 64% (35/55). Twenty-two embryos expressed the MiwZ in both embryonic and extraembryonic tissues, while the remainder expressed the MiwZ in only extraembryonic tissues. Mosaic expression was observed in most of the embryos expressing MiwZ in embryonic tissues. Expression throughout all tissues of the embryo including blood cells occurred in one case. In this case, the injected DNA was assumed to have integrated at an earlier stage. The results indicate that it is now possible to investigate the promoter activities of introduced exogenous genes as well as the effect of introduced genes on embryogenesis in early chick embryos. This technique may also facilitate the production of transgenic chicks. PMID- 1722695 TI - Characterization of cytokeratin patterns in the developing human tongue. AB - The characterization of cytokeratin (CK) in adult oral mucosa and developing teeth have been well documented in human. Cytokeratin distribution in developing oral mucosa has not yet been described. The aim of this study was to identify the expression of CK in human fetal tongue (week 10 to week 23) and to correlate the results with morphological maturation. Simple epithelial CK are expressed in all cell layers during the early stages, essentially in peridermal cells. From the 14th week, CK 18 is present only in the taste buds, making this polypeptide a reliable marker for this sensory organ. CK 4 and 13 are expressed from the 10th to the 23rd week by both ventral and dorsal lingual epithelia. Terminal differentiation keratins (CK 1, 2 and 10-11) can only be detected immunohistochemically at the 14th week in some cells on the external surface of some papillae. The number of these papillae and positive cells increase at the 19th and 23rd weeks. The terminal differentiation markers are expressed several weeks earlier than the formation of a well-distinguished keratinized layer. PMID- 1722696 TI - Dengue virus-induced helper cytokine has two polypeptide chains which bear different determinants. AB - Dengue type 2 virus (DV) induces generation of a T cell helper cytokine (HF) in mouse spleen which enhances the antigen-specific antibody plaque forming cell count in syngeneic mice. The present study was undertaken to investigate the molecular structure of HF. It was observed that the activity of HF was abrogated by treatment with reducing agents such as glutathione, ouabain or dithiothreitol (DTT) which cleave disulphide bonds, separating the polypeptide chains. The two polypeptide chains could be purified by high performance liquid chromatography of DTT treated HF. The individual chains had no helper activity, but it could be restored by mixing the two. One chain of the HF bonded to the DV-antigen coupled immunosorbent column and the other to the anti-I-Ak antibody coupled column. Thus, DV-induced HF is a disulphide bonded double chain structure, one chain having antigen and the other having I-A determinants; the presence of both chains is essential for helper activity. PMID- 1722697 TI - Superiority of second over first generation chemotherapy in a randomized trial for stage III-IV intermediate and high-grade non-Hodgkin's lymphoma (NHL): the 1980-1985 EORTC trial. The EORTC Lymphoma Group. AB - A first-generation CHOP-like cyclic combination chemotherapy (CT) regimen using cyclophosphamide 600 mg/m2 IV d1, hydroxorubicin (doxorubicin) 50 mg/m2 IV d1, VM26 60 mg/m2 IV d1, and prednisone 40 mg/m2 PO d1-5 (CHVmP) was compared to a second-generation combination wherein vincristine 1.4 mg/m2 IV and bleomycin 6 mg/m2 IM/IV were added at mid-interval (d15) to the former drugs (CHVmP + VB) in the treatment of intermediate- and high-grade malignant NHL. From April 1980 to January 1986, 141 eligible patients with stage III-IV unfavorable histologies (except T lymphoblastic NHL) entered this EORTC randomized trial. In both arms adjuvant radiotherapy (30 Gy) was given in instances of bulky or residual disease. In all patient subsets the outcome favored the second-generation regimen. The difference was even greater in patients with Diffuse Large Cell Lymphoma (DLCL). At 5 years, overall survival was 53% with CHVmP + VB versus 29% (p = 0.002). The advantage was due to a higher complete remission (CR) rate (80% versus 50%, p = 0.01). Indeed, once CR was achieved the relapse-free survival (RFS) was not significantly influenced (59% versus 49%). No significant additional toxicity could be attributed to vincristine and bleomycin. This study demonstrates a clear benefit for intermediate- and high-risk malignant NHL and particularly DLCL from intercalating non-myelotoxic drugs at mid-cycle intervals, without adverse effects. PMID- 1722698 TI - Palliative oncological management: do we need specialization? PMID- 1722699 TI - Derivatized dextran inhibition of smooth muscle cell proliferation. AB - Proliferation of vascular smooth muscle cells is postulated to be one of the key events in the pathogenesis of atherosclerosis or during the development of focal glomerular sclerosis. Several studies have suggested that the antiproliferative effects of heparin appear to be regulated by different structural determinants. Our experiments show that dextrans substituted with carboxylic and benzylamide sulphonate groups markedly inhibit the growth of smooth muscle cells in vitro. Studies on the structure-function relationships of these products to their effect on rat aorta smooth muscle cells are reported. The antiproliferative capacity is similar to that of heparin. PMID- 1722700 TI - ADAPTU: animated dynamics analysis program at Tubingen University. AB - Molecular dynamics calculations are being used more and more in computational chemistry to investigate inter- and intra-molecular properties, as well as to determine structures. Visualization of a calculated trajectory is needed to allow a detailed analysis of the huge amount of data generated by such calculations. In addition to animating trajectories, ADAPTU was written to permit diagram generation in two and three dimensions for a detailed analysis, the extraction and listing of properties of a selected conformation and the visualization of the development of constraints in a restrained dynamics. The displayed trajectory, as well as the three-dimensional (3D) diagrams can be scaled, translated and rotated, and displayed in cross stereo representation. Our graphics device is a E&S PS300 system, which is connected to a host by Ethernet or by an asynchronous line. PMID- 1722701 TI - A leucine zipper-like motif may mediate HIV reverse transcriptase subunit binding. PMID- 1722702 TI - Great gating in Kiev. Symposium on Molecular Neurobiology sponsored by the National Academy of Sciences (USA), and the Academy of Sciences (USSR), Kiev, USSR, May 21-25, 1991. PMID- 1722703 TI - Invertebrates: witnesses to the evolution of neuroreceptors and ion channels in the nervous system. Invertebrate molecular neurobiology: key to evolution of neuroreceptors and ionic channels. A Jacques Monod Conference sponsored by the Life Science Department of the Centre National de la Recherche Scientifique, Aussois, France, April 15-19, 1991. PMID- 1722704 TI - Treatment of iatrogenic choriovitreal neovascularisation in sickle cell disease. AB - The effect of scatter photocoagulation on the perfusion of iatrogenic choriovitreal neovascularisation (CVN) has been assessed by a randomised trial in 35 CVN lesions in 18 eyes with proliferative sickle retinopathy. No difference in size or vascularity of CVN lesions was apparent between the nine treated and nine control eyes over a median follow-up of 42 months. Scatter photocoagulation by the stated protocol was not effective in the treatment of CVN. PMID- 1722705 TI - Cell surface expression of ICAM-1 (CD54) and LFA-3 (CD58), two adhesion molecules, is up-regulated on bone marrow leukemic blasts after in vivo administration of high-dose recombinant interleukin-2. AB - High-dose recombinant interleukin-2 (rIL-2) therapy can induce long-term remission in patients with melanoma and renal and colon cancer. More recently, in vivo IL-2 therapy was shown to induce complete or partial remission in some cases of relapsed chemotherapy-resistant acute myeloid leukemia. We have investigated the phenotypic modifications of bone marrow cells obtained from five patients with acute myeloid leukemia in relapse receiving high-dose i.v. rIL-2. We found that, in three of five patients, IL-2 could induce, in vivo, an increase in the expression of CD54/ICAM-1 and to a lesser extent of CD58/LFA-3 on bone marrow leukemic blasts. This demonstrates that rIL-2 modifies directly or indirectly the expression of the cell surface molecules of the tumor cells themselves. Upregulation of such adhesion molecules could account for the enhancement of cell interactions between the tumor and effector cells such as T, natural killer, and phagocytic cells as well as being indicators of differentiation signaling. PMID- 1722706 TI - [Frequency and differential diagnosis of cribriform structures of the prostate]. AB - Systematic investigations of the prostate in 450 autopsies of individuals who had lived to the age of 40 to 80 years as well as in 500 prostate biopsies and 480 so called adenomectomies of the prostate led to detection of cribriform hyperplasia in 30 postmortems (7%), 6 biopsies (1.2%), and 5 adenomectomies (1%). The average age of all hyperplasia cases amounted to 69 years. Cribriform hyperplasia was recordable with and without cellular atypia. Most of the foci, single and multiple up to 4, were not larger than 25 mm2. Statistical calculations revealed good correlations with adenomatous and microglandular hyperplasia as well as with dysplasia of the prostate. A significant relationship was found to exist between cribriform hyperplasia and cellular atypia as well as with prostate carcinoma. Concomitantly recorded were 315 carcinomas, with 81 of these being of the pluriform type and with 68 of the latter (84%) exhibiting cribriform structures. Problems relating to differential diagnosis were found to result from the presence of both benign and malignant structures and are discussed in some detail. PMID- 1722707 TI - Pachyonychia congenita. Immunohistologic findings. AB - Pachyonychia congenita (PC) is a very rare hereditary disorder of keratinization. Immunohistological findings has so far been lacking. Reported in this paper is a case of Jadassohn-Lewandowsky type PC in a woman aged 18 years on which immunohistological investigations could be performed. Several monoclonal antibodies to filaggrin and keratin were used to stain tissue sections of lesional plantar skin, with a view to studying impairment of epidermal differentiation. While staining patterns comparable to those of normal skin were exhibited by anti-filaggrin and some antikeratins (RPN 1161, A51-B/H4), substantially altered immunostaining was recordable from other anti-keratins. Only superficial vital keratinocytes were stained by RKSE 60 against keratin 10 and K 8.12 against keratins 13 and 16. The authors, in other words, obtained information on expression of keratin 10, normally occurring in all suprabasal keratinocytes, as well as of the basal proliferation keratin 16 in the uppermost vital cell positions of PC lesion. The above results are likely to suggest impairment of keratin expression in cases of PC. PMID- 1722708 TI - Mast cell fixation and staining in image analysis. AB - Modern image analysers automatically perform densitometric measurements and elaborate digital images. Elaboration however is subject to operator interpretation and often eliminates precious information from the areas of interest. For this reason, it was appropriate to find a staining method which would overcome this drawback and, in the case of mast cell histochemistry, limit staining to granule content. The following current staining techniques were tested: Toluidine Blue in buffered solution (solut. a) and in 0.003% alcoholic solution (solut. b) and alcoholic Astra Blue, pH 0.2 Densitometric analysis was performed on both 5 microns and semithin sections of mouse tongue fixed in Isotonic formaldehyde-acetic acid (IFAA). Digital images were obtained using 630 nm and 546 nm wavelengths for Toluidine Blue and 610 nm for Astra Blue. Direct comparison between the two Toluidine Blue solutions revealed that more pixels were captured by the 5 microns sections stained with solut. a, whilst the opposite occurred in semithin sections. Both dyes introduced a certain amount of error due to the orthochromatic component of the nucleus and cytoplasmic basophily, which had to be eliminated through image elaboration. Because of its subjective nature, this operation may in turn lead to further errors. The choice of Astra Blue as an alternative to Toluidine Blue in densitometric analysis of mastocytes is based on its property to restrict staining to the granules of mast cells. A comparison between Astra Blue and the two Toluidine Blue solutions showed that, at all transmission levels, preparations stained with Astra Blue captured more pixels than those stained with Toluidine Blue. Consequently our results suggest that the most suitable technique for densitometric image analysis is fixation of mast cells in IFAA followed with Astra Blue. PMID- 1722709 TI - Hepatoid gastric adenocarcinoma. A histological and immunohistochemical study of a case. AB - A case of gastric adenocarcinoma with patterns resembling those of hepatocellular carcinoma is reported. The hepatoid component of the tumour was characterized by discrete masses, nests and broad bands of large polyhedral cells with central nuclei, prominent nucleoli, and abundant eosinophilic cytoplasm; single-nucleus giant cells were frequently noted. Varying numbers of tumour cells stained immunohistochemically for alpha-fetoprotein (AFP), alpha-1-antitrypsin (AAT), and albumin. Thus, in this hepatoid gastric adenocarcinoma tumour cells demonstrated both morphologic and immunohistochemical features of partial differentiation in hepatocellular carcinoma. Careful histological examination in conjunction with the immunohistochemical demonstration of AFP and other serum proteins can provide an useful contribution to the diagnosis of this rare histological type of gastric carcinoma. PMID- 1722710 TI - [Biomonitoring of the ecotoxicological situation in reserves by demonstrating organochlorine pesticides in fish tissues]. AB - The experience of sampling of fish tissues for chlororganic pesticides (COP) content or its metabolites is generalized. The greatest quantity of these substances concentrate in brain and liver tissues and in brain tissues the pesticides accumulate during all the period of ontogenesis, but in liver - only during last months of active feeding period. According to the difference in pesticide content in fish tissues in spring and autumn we can localize the source of COP contamination of water fauna and terrestrial ecosystems of different protected areas possessing water bodies. It has been demonstrated on example of white fishes sensitive to COP and inhabiting the northern regions of Hallarctic as well as on sazan - a representative of ichthyofauna of the river Vakhsh in a deserted reserve the "Tiger's Walley". PMID- 1722711 TI - [The inhibition of transcription in Bacillus subtilis during repression by a secreted enzyme of its own synthesis]. PMID- 1722712 TI - [The antigenic characteristics of the capsid proteins in strains of the potato virus Y]. AB - The antigenic properties of capsid proteins of potato virus Y (PVY) strains have been studied, the most wide antigenic specificity of necrotic group strains has been marked. Several antigen-active strains of necrotic group strains (PVY-N Far East, PVY-N Leningrad, PVY-N Moscow) and common group (PVY-O-3 Moscow, PVY-O Far East) have been revealed. Antisera against these strains reacted with any PVY strain. Virus specific surface epitope, corresponding to position 198-208 of polypeptide chain has been located, which is a group-specific epitope of potyviruses, identified in the Far East. PMID- 1722713 TI - Differential inhibition of IL-1 beta activities and receptor binding by monoclonal antibodies mapping within a discrete region of the protein. AB - The importance of the region in position 148-192 for the biological activities and receptor-binding capacity of the human IL-1 beta protein has been assessed by the use of mAbs. Four mAbs have been used, which recognize different epitopes within the 148-192 region. None of the mAbs could inhibit binding of IL-1 beta to IL-1RI (expressed on T cells and fibroblasts), suggesting that the 148-192 region does not contain IL-1RI binding sites. Conversely, mAbs Vhp20 (recognizing the fragment 166-169) and BRhD2 (directed to an epitope in the sequence 177-186) recognize sites partially involved in binding to IL-1RII (expressed on B cells, macrophages, and PMN). Only mAbs BRhD2 and FIB 1 (which recognizes an epitope in the sequence 174-186) can inhibit IL-1 beta-induced thymocyte proliferation, whereas all four can inhibit the adjuvant capacity of IL-1 beta in vivo. It is concluded that the region 148-192 encompasses domains important for T cell activation but not for binding to the IL-1RI on T cells, others involved in immunostimulation in vivo, and others important for binding to IL-1RII, although not directly involved in it. PMID- 1722714 TI - [Angiogenesis: a crucial element in tumor development]. PMID- 1722715 TI - Prospects for the use of synthetic antigens in immunodiagnosis. AB - Methods for the localization and prediction of protein antigenic determinants are described, and the diagnostic potential of synthetic peptide antigens in cases of HIV, hepatitis B, and influenza is demonstrated. Attention is concentrated on the principles governing the creation and application of artificial diagnostic preparations. PMID- 1722716 TI - Effect of conjugation on the biodistribution of 111In-labelled anti-PAP and anti PSA monoclonal antibodies examined in nude mice with PC-82 human tumor xenografts. AB - The effect of conjugation on the biodistribution of 111In-labelled antibodies was studied in nude mice carrying human prostatic cancer xenografts (PC-82). Two monoclonal antibodies and their fragments raised against human prostate-specific acid phosphatase (PAP) and prostate-specific antigen (PSA) were used. We used the cyclic anhydride of DTPA (CA-DTPA) as a chelating agent, or, alternatively, 1-(p aminobenzyl)diethylenetriaminepentaacetic acid (NH2-Bz-DTPA) was attached as a linker to the carbohydrate components of the parent molecules. The conjugation method, the amount of circulating antigen and the size of the antibody component affected the blood clearance of the labelled derivatives. F(ab')2 fragments displayed a faster blood clearance than the corresponding derivatives of intact IgG1s. Aminobenzyl derivatives of anti-PAP-IgG1 showed a faster blood clearance than the corresponding CA-DTPA derivatives, but, in the case of derivatives of anti-PSA-IgG1, this was less clear, possibly due to the high PSA concentrations in the mouse sera. All the derivatives studied accumulated in the liver independently of the size of the antibody derivative, most probably due to the formation of antigen-antibody complexes. All CA-DTPA derivatives showed a higher kidney accumulation than the corresponding aminobenzyl derivatives. CA-DTPA-anti PAP-F(ab')2 fragments showed a higher kidney uptake than the corresponding anti PSA-F(ab')2 derivatives, since a large fraction of the latter are complexed with circulating antigen, thereby slowing down its reabsorption by the kidney. In addition, the lower kidney accumulation for anti-PSA-F(ab')2 fragments might be, at least partly, due to the electronegative charge of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722717 TI - Usefulness of peritoneal fluid amylase levels in the differential diagnosis of peritonitis in peritoneal dialysis patients. AB - Peritonitis continues to be a major cause of morbidity in peritoneal dialysis patients despite recent technological advances (Y systems) that have reduced peritonitis rates to much more acceptable levels. Most of the time when a peritoneal dialysis patient presents with peritonitis, it is infectious in origin. However, these patients occasionally develop other intra-abdominal pathology that requires more intensive medical care or, at times, surgical intervention. To help in the early differential diagnosis of the cause of peritonitis in these patients, peritoneal fluid amylase levels were prospectively obtained from 50 patients presenting to the hospital with peritonitis. Thirty nine of them had typical infectious peritonitis, and their mean peritoneal fluid amylase level was 11.1 (range, 0 to 90). Six patients had pancreatitis and a mean peritoneal fluid amylase level of 550 U/L (range, 100 to 1,140 U/L). Five patients were found to have other intra-abdominal pathology, and their mean peritoneal fluid amylase level was 816 U/L (range, 142 to 1,746 U/L). In patients who did not respond to initial therapy, sequential peritoneal fluid amylase levels did not increase in patients with typical infectious peritonitis whereas it did increase in patients with other intra-abdominal pathology. In conclusion, it was found that peritoneal fluid amylase levels were helpful in the differential diagnosis of peritonitis in these patients. An elevated level (greater than 100 U/L) differentiated those patients with other intra-abdominal causes of peritonitis from those with typical infectious peritonitis. PMID- 1722718 TI - Are genetically hypertensive rats deficient in nerve growth factor? AB - We have shown previously that sympathetic ganglia from genetically hypertensive Otago Wistar (GH) rats contain fewer neurons than those of normotensive animals and are heavily innervated by substance P-containing axons. In adult GH rats treated over days 1-7 of postnatal life with 10 micrograms/rat/day of nerve growth factor (NGF) s.c., ganglion cell numbers were similar to those of normotensive rats. By contrast, NGF treatment of neonatal normotensive animals did not affect ganglion cell numbers. In NGF-treated GH animals, the numbers of substance P-positive axons were substantially reduced relative to age-matched controls. We conclude that the abnormalities seen in ganglia of GH rats may involve a perinatal deficiency of a nerve growth factor. PMID- 1722719 TI - High density cAMP-gated channels at the ciliary membrane in the olfactory receptor cell. AB - Spatial distribution of the cAMP-gated channel was investigated in amphibian olfactory receptor cells. Low doses of cAMP applied to the cytoplasmic side of a membrane patch excised from cilia produced single channel activity of unitary conductance 28pS. Variance analysis showed that the ciliary membrane contained 920 cAMP gated-channels/microns2 in the newt and 2400 channels/microns2 in the toad. In contrast, the membrane of the dendrite and cell body contained only 2 cAMP-gated channels/microns2 (newt) and 6 channels/microns2 (toad). Thus, there is a high density of cAMP-gated channels in the cilia where olfactory transduction is thought to take place. PMID- 1722720 TI - Olfactory bulb DA receptors may be located on terminals of the olfactory nerve. AB - The glomerular layer of the olfactory bulb contains a substantial population of dopaminergic neurons. We determined the quantity and location of D1 and D2 dopamine receptors which are the presumed targets of these neurons. Binding of the D1 selective ligand [3H]SCH23390 was slightly above background and was distributed through all layers of the bulb except the olfactory nerve layer. In contrast there were relatively high levels of [3H]spiperone binding to D2 DA receptors in the glomerular and olfactory nerve layers. The presence of relatively high concentrations of D2 DA receptors in both the nerve layer and glomerular layer suggests the novel hypothesis that these receptors may be localized on terminals of the olfactory nerve. PMID- 1722721 TI - Characterization of myelin basic protein charge isomers from adult mouse brain. AB - ADULT mouse MBP charge isomers (C1 or component 1, C2 or component 2, etc.) were purified from an acid soluble brain protein fraction by cation exchange chromatography. They were characterized by their elution profiles, their migration rates in alkaline-urea gels and by SDS-PAGE. Mouse C1 and C2 were both 14 kD in size, while C3, C4 and C5 consisted of the 18.5, 17 and 14 kD isoforms. Comparison of mouse and human MBP charge isomers showed that they were similar in that they both showed extensive charge heterogeneity, but different in that mouse MBP charge isomers were more cationic than their human counterparts. Finally, a possible explanation for the presence of the 14 kD MBP isoform in mouse myelin was suggested. PMID- 1722722 TI - Chromaffin allografts into arachnoid of spinal cord reduce basal pain responses in rats. AB - In this present study, behavioral responses to a subcutaneous formalin test for pain are evaluated in rats that previously received an allograft of adrenal chromaffin tissue into arachnoid of the dorsal spinal cord and in control animals. In the group of rats with grafts, a significant basal analgesia, reversed by the opioid antagonist naloxone, is found. These findings suggest that the grafts secrete some substance that reduces the response to painful stimulation and whose action is blocked by naloxone. PMID- 1722723 TI - The transcription control region of the rat alpha-fetoprotein gene. DNA sequence and homology studies. AB - The alpha-fetoprotein (AFP) gene, an important system for studying developmental and tissue-specific gene expression, is regulated mostly through the control of transcription. The promoter and cis-acting DNA elements which regulate the rat gene lie within a 7 kbp region upstream of the cap site. We have determined the sequence of this entire region. It contains several repetitive elements and a species-specific distribution of DNA methylation sites. We aligned our rat AFP sequence with fragmentary mouse and human AFP sequences to define blocks of highly conserved sequence, which we then analyzed for homology to known transcription regulatory sequences. Our analysis demonstrates that the regulatory region of the rat AFP gene is unusually complex. PMID- 1722724 TI - Cloning and characterization of a cDNA encoding the bovine insulin-like growth factor binding protein 1 (bIGFBP-1). AB - This communication reports the complete amino acid sequence (263 amino acids) for the insulin-like growth factor binding protein 1 in the bovine (bIGFBP-1). It is 70% homologous with the equivalent IGFBP-1 in human and rat. PMID- 1722725 TI - Flight activity of insecticide resistant and susceptible Anopheles stephensi mosquitoes in actograph chambers lined with malathion, gamma HCH or dieldrin. AB - The activity and resting behaviour of resistant and susceptible Anopheles stephensi Liston were recorded in acoustic actograph chambers lined with residual deposits of malathion, dieldrin or gamma HCH. In gamma HCH-treated flight chambers, SS and RS mosquitoes became active only after picking up lethal doses of insecticide, which explains why few SS and RS mosquitoes survive release into gamma HCH-treated experimental huts. Similar results were obtained in flight chambers treated with dieldrin; however, more mosquitoes would be expected to survive dieldrin under field conditions because resistance to this insecticide is greater than to gamma HCH. Mosquitoes in contact with malathion showed a three phase activity pattern: an initial active phase, an inactive phase, and hyper activity/convulsions. Initial activity or irritability was especially pronounced in SS and RS but absent in RR mosquitoes. Whether or not irritability would protect RS mosquitoes from malathion would probably depend on the ratio of sprayed to unsprayed surfaces in treated huts. PMID- 1722726 TI - Variation in fluorescein-labelled lectin staining of salivary glands in the Anopheles gambiae complex. PMID- 1722727 TI - Base-line susceptibility of the oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), to five insectides. AB - Susceptibility to five insecticides was assessed on seven colonies of the oriental latrine fly, Chrysomya megacephala (Fabricius), originating from Japan, Hong Kong, Philippines, Indonesia and Papua New Guinea, and on C. saffranea (Bigot) from PNG. All the colonies were susceptible to fenitrothion, diazinon, dichlorvos, permethrin and gamma-HCH, although the Jakarta, Manila and Tokyo colonies of C.megacephala were least susceptible (most tolerant) to diazinon, dichlorvos and permethrin. Feral forms of both Chrysomya species were 4-5-fold more susceptible to permethrin at the LD50 level than were all six colonies of the synanthropic form of C.megacephala. PMID- 1722728 TI - Behaviour and fitness of gamma HCH/dieldrin resistant and susceptible female Anopheles gambiae and An.stephensi mosquitoes in the absence of insecticide. AB - The effects of gamma HCH/dieldrin resistance genes on various fitness components of mosquito larvae and adult females in the absence of insecticide were investigated in backcrossed strains of Anopheles gambiae Giles and An.stephensi Liston. Among larvae, heterozygotes (RS) developed slightly but significantly faster than homozygotes for resistance (RR) or susceptibility (SS). The lifetime fecundity of RR females in population cages was only half to two-thirds that of SS and RS females despite similar longevities; several reasons were identified: RR gravid females were less responsive to oviposition-site stimuli, their spontaneous activity--as measured in an acoustic actograph--was only half that of SS or RS females, and RR females produced fewer eggs per unit bloodmeal. When inseminated females were recorded in LD 12:12, RR were again less active than SS or RS. When the lighting was switched to a regime simulating full-moonlight, the activity pattern of SS and RS changed and they flew for longer periods. In contrast, the activity of RR females was the same in LD 12:12 as in 'moonlight'. In a test simulation of potential predation, RR mosquitoes took to flight least readily. All component tests on adult females therefore point to RR as being the least fit of the three genotypes. The behavioural tests suggest that resistance has raised the response threshold of RR females to diverse stimuli. A possible physiological mechanism underlying RR behaviour is that a change in the cyclodiene receptor on the chloride channels has increased their permeability to chloride ions, causing hyper-inhibition of the nervous system. PMID- 1722729 TI - Activity and mating competitiveness of gamma HCH/dieldrin resistant and susceptible male and virgin female Anopheles gambiae and An.stephensi mosquitoes, with assessment of an insecticide-rotation strategy. AB - The effects of gamma HCH/dieldrin resistance genes on flight activity and mating competitiveness were investigated in males from backcrossed strains of Anopheles gambiae Giles and An.stephensi Liston. Activity of males and virgin females of both species, as recorded in an acoustic actograph, occurred mainly at dusk (the E peak). The activity pattern of An.gambiae males was not affected by resistance genes; in mating competition and predator avoidance experiments, however, RR males were less successful than RS males which were less successful than SS males. The activity pattern of An.stephensi differed from An.gambiae in that the E peaks of RR males and females in a gradual dusk regime were out of synchrony with those of SS and RS, the E peaks of RR occurring slightly later. Thus, RR males and females tended to mate assortatively in mate competition experiments. When a sudden dusk regime was substituted for the gradual dusk regime, activity of RR An.stephensi became synchronized with SS and RS activity, but in mating competition experiments RR still tended to mate assortatively. Estimates of male competitiveness, together with previously-obtained estimates of female fitness, were included in population genetics models. Computer simulations showed that the frequency of resistance in populations of An.gambiae and An.stephensi should decrease in the absence of insecticide at a rate comparable with known field reversions. PMID- 1722730 TI - Recovery of Triatoma infestans populations after insecticide application: an experimental field study. AB - The capacity of populations of Triatoma infestans (Klug) (Hemiptera: Reduviidae) to survive and recover was assessed after application of insecticide (gamma-HCH at a rate of 0.5 g a.i./m2) at different seasons. T. infestans populations were maintained in experimental chicken houses under natural climatic conditions in a region of Argentina endemic for Chagas disease transmitted by these bugs. Based on previous studies of T. infestans populations in these habitats, each experimental group was set up with a total of 626 T. infestans, comprising 390 eggs, 204 nymphs of particular stages, fourteen male and eighteen female adults. The chicken houses were dismantled and rebuilt at monthly intervals to study the vector population changes over a period of 33 months. When the insecticide was applied during winter, spring or summer, populations of T. infestans recovered to untreated or precontrol levels during the next reproductive season (i.e. during the hot season, October-March). In contrast, populations treated during autumn (March) remained at very low densities for 2 years and then increased rapidly to surpass the untreated populations. All populations of the bugs fell to very low numbers (sometimes less than twenty individuals) after gamma-HCH applications, but none was driven to extinction. In all cases, the density of surviving populations was independent of their density before treatment. The fact that all treated populations recovered within 1-3 years, to at least the density of untreated populations, shows the high reproductive potential of T. infestans to recover from very low population densities. Moreover, the additive effect of climatic-induced mortality and insecticide-induced mortality is only apparent when insecticides are applied just before the onset of the cold winter months during which reproductive rates are at their lowest. PMID- 1722731 TI - Changes in selected biochemical parameters in the brain of the fish, Anguilla anguilla (L.), exposed to lindane. PMID- 1722732 TI - Effect of lindane on the blood of a freshwater fish. PMID- 1722733 TI - Acute lethal toxicity of some pesticides to Brachionus calyciflorus and Brachionus plicatilis. PMID- 1722734 TI - Acceptor activity of affinity-immobilized dextransucrase from Streptococcus sanguis ATCC 10558. PMID- 1722735 TI - [Did the care of patients with tumor improve based on the recommendations of the World Health Organization?]. PMID- 1722736 TI - Effects of ACTH and angiotensin II on cytosolic calcium in cultured adrenal glomerulosa cells. Role of cAMP production in the ACTH effect. AB - We have used microspectrofluorometry and video imaging techniques in order to study and compare the changes in intracellular calcium concentrations [( Ca2+]i) of individual Fura-2 loaded glomerulosa cells cultured for three days and stimulated either with angiotensin II (AT), K+, or adrenocorticotropin (ACTH). As previously demonstrated for freshly isolated cells, K+ ion induces an immediate increase in [Ca2+]i, although AT induces a biphasic response, characterized by an initial transient spike, followed by a sustained plateau. In this study, we demonstrate, for the first time, that ACTH is able to induce a [Ca2+]i increase in cultured glomerulosa cells from rat and bovine sources. Moreover, it is clear that the pattern of [Ca2+]i increase elicited by ACTH is different from that observed with AT. In most cases, addition of ACTH leads to a slow increase in [Ca2+]i after a long latency period ranging from 10-15 min, which could be correlated to cAMP time-production. The present results show that: (a) in the absence of extracellular Ca2+, ACTH does not increase [Ca2+]i; (b) the response develops slowly and cases immediately after [Ca2+]e depletion or addition of calcium channel blockers, such as nifedipine or omega-conotoxin; (c) the addition of the calcium channel agonist Bay K 8644 enhances the ACTH response; (d) the cAMP analog, 8-Br-cAMP, induces an increase in [Ca2+]i similar to that observed with ACTH, which is also dependent of the presence of calcium in the extracellular medium; (e) time-production of ACTH-induced cAMP follows quite well the increase in [Ca2+]i; (f) Bay K 8644 also enhances the 8-Br-cAMP induced increase in [Ca2+]i; and (g) ACTH-induced Cai response is inhibited by the specific protein kinase A blocker, HA1004. These observations, combined with previous results obtained on the effects of ACTH on calcium currents and action potentials, suggest that the [Ca2+]i increase induced by ACTH results from a calcium influx through dihydropyridine and omega-conotoxin sensitive calcium channels, which need to be phosphorylated by cAMP for full activation. The use of video-imaging techniques has allowed us to examine the spatial distribution of changes in [Ca2+]i in single cells. The ability to simultaneously record images of a number of cells confirm the heterogeneity of cellular responses, and corroborate results obtained through photocounting only. Our results indicate that ACTH initially increases [Ca2+]i locally beneath the cell membrane and throughout the cell thereafter, whereas angiotensin II elicits a more prominent effect in certain regions of the cell and eventually extends to the entire cell surface. PMID- 1722737 TI - Cytokinin increases intracellular Ca2+ in Funaria: detection with Indo-1. AB - Changes in intracellular [Ca2+] ([Ca2+]i) after cytokinin-treatment in protonema cells of the moss Funaria hygrometrica have been measured using the pentapotassium salt of Indo-1. The extent of dye loading strongly depended on lowering the pH of the incubation medium to 5.0. Exposing dye-loaded cells briefly with Mn2+ did not quench fluorescence suggesting that the source of fluorescence is from the cytoplasm and not from the cell wall. Indo-1 remains responsive to changes in [Ca2+]i in Funaria cells. The [Ca2+]i in quiescent cells (with and without extracellular Ca2+) is 250 nM, which is within the range of reported [Ca2+]i of other plant cells. Treatment of cells with extracellular cytokinin in 4 mM Ca2+ induced a three-fold increase in [Ca2+]i to 750 nM in target caulonema cells. This increase was not observed in Ca(2+)-free medium. These target cells respond to cytokinin treatment by an asymmetrical division, while non-target chloronema cells do not divide. Cytokinin appears to increase [Ca2+]i by extracellular Ca2+ uptake. However, non-target chloronema cells and tip cells also respond to cytokinin treatment by increasing [Ca2+]i. The differential physiological response of these cell types to hormonal stimulation must lie further down the signal transduction chain. PMID- 1722738 TI - Roles of potassium and chloride ions in cAMP-mediated amylase exocytosis from rat parotid acini. AB - The roles of potassium and chloride ions in cAMP-mediated amylase exocytosis were studied using intact and saponin-permeabilized parotid acini. Cyclic AMP-evoked amylase release from saponin-permeabilized parotid acini decreased markedly when KCl in the incubation medium was isoosmotically replaced by K-glutamate, NaCl, Na isothionate, or mannitol. Quinidine and barium, K+ channel blockers, clearly inhibited amylase release from the permeabilized acini, but not from intact ones. The chloride channel blocker DPC (diphenylamine-2-carboxylate) also inhibited amylase release, while DIDS (4,4'-diisothiocyanostilben-2,2'-disulfonate) or bumetanide had little effect, if any, on the exocytosis. Hyperosmolarity with mannitol markedly reduced amylase release from permeabilized acini. These results suggest that potassium and chloride ions play important roles in cAMP-mediated amylase exocytosis, and that these ions act on secretory granules inside the acinar cells. PMID- 1722739 TI - Ontogeny of insulin-like growth factor I, its receptor, and its binding proteins in the rat hypothalamus. AB - A role for the insulin-like growth factors (IGFs) in brain growth and differentiation has recently been suggested. In previous studies on fetal hypothalamic cells we found a trophic influence of IGF-I on in vitro survival and differentiation of both neurons and glia. We have now investigated the expression of IGF-I, its receptor and its binding proteins in the rat hypothalamus to determine whether endogenous IGF-I might serve as a trophic factor during development of this brain area. Both IGF-I receptors and IGF-I binding proteins showed marked developmental stage-dependent variations. Thus, IGF-I receptors as measured by both binding and cross-linking techniques, were highest during fetal life and steadily decreased thereafter to reach low adult levels. Changes in receptor numbers rather than in its affinity constant accounted for the differences seen in binding activity during development. In addition, we found 3 different IGF-I binding proteins (IGFBPs) of apparent Mr of 24, 29 and 32 kDa respectively, whose levels also showed a specific developmental pattern. Highest levels of the 29 and 32 kDA IGFBPs were found in fetal and early postnatal life, whereas levels of the 24 kDa form were highest in young adults. Changes in the concentration of IGFBPs rather than in their affinities for IGF-I accounted for the different binding capacities found. Using a specific IGF-I radioimmunoassay we found that IGF-I-like immunoreactivity (IGF-I-li) levels had no direct correlation with developmental stage. IGF-I-li levels oscillated with no apparent trend throughout development of the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722740 TI - Unilateral olfactory deprivation: effects on succinate dehydrogenase histochemistry and [3H]leucine incorporation in the olfactory mucosa. AB - Surgically closing one external naris reduces airflow through one half of the nasal cavity, decreasing the access of odors to the receptor sheet. In rats, unilateral naris occlusion performed near birth results in large reductions in the size of the olfactory bulb, the primary central relay, when examined 30 days later. Previous research has demonstrated that there is a rapid reduction in [3H]2-deoxyglucose (2-DG) and [3H]leucine uptake in the bulb within hours after naris closure. The present study examined whether similar rapid changes could be observed in the sensory periphery. Pups occluded on P1 and examined on P3 with succinate dehydrogenase histochemistry exhibited reduced staining on the closed side of the nasal cavity, suggesting occlusion results in reductions in mucosal metabolism. Larger differences in staining were observed in pups examined at P6. [3H]Leucine incorporation was quite similar on both sides of the nasal septum as late as 30 days post occlusion, suggesting less dramatic changes in protein synthesis. The results suggest that naris closure does indeed have rapid effects on mucosal function, but indicate that the changes are different than those observed in the bulb. PMID- 1722741 TI - Agarose gel electrophoresis of glucose-6-phosphate-dehydrogenase isoenzymes. PMID- 1722742 TI - A 64 kDa membrane antigen is a recurrent epitope for natural autoantibodies in patients with Graves' thyroid and ophthalmic diseases. AB - OBJECTIVE: We have explored the recently described 64 kDa extraocular muscle antigen that is associated with autoantibodies in the serum of patients with severe Grave's ophthalmopathy. The localization of the antigen and the specificity of autoantibodies for both eye muscle antigens and ophthalmopathy patients were investigated. DESIGN: Western blotting and immunoprecipitation of metabolically labelled antigen from eye muscle and control tissues with sera from ophthalmopathy, Graves' without ophthalmopathy, and normals were used. PATIENTS: Sera from normals (n = 9), patients with recent onset Graves' ophthalmopathy (n = 23), and patients with Graves' disease without ophthalmopathy (n = 8) were utilized. MEASUREMENTS: Immunoblots using detergent phase separated (amphiphilic) antigen preparations from fetal eye muscle, skeletal muscle and control tissues were quantitated. Metabolically labelled eye muscle and skeletal muscle antigens were immunoprecipitated using patient and control IgG. RESULTS: In the eye muscle detergent phase, immunoreactivity around 64 kDa was detected in 30% of the patients with ophthalmopathy (n = 23) as well as 38% of patients with Graves' disease and no ophthalmopathy (n = 8) and in 30% of normal sera (n = 9). There was significantly more of this anti-64 kDa reactivity in sera from the ophthalmopathy patients compared with the normals (P less than 0.01). 64 kDa reactivity to detergent phase antigens prepared from human thyroid, skeletal muscle, brain, and liver was also observed with these positive sera indicating the polyreactivity of the IgG interactions to conserved antigens in this region. CONCLUSIONS: We conclude that IgG antibodies binding to a recurrent 64 kDa antigen are present in many normal human sera, with increased concentrations detectable in sera from Graves' ophthalmopathy patients. Such 'specificity crossover' with similar molecular weight transmembrane antigens is likely to be caused by natural autoantibodies reacting with recurrent autoepitopes rather than a factor aetiological in the disease process. PMID- 1722743 TI - Current topics in viral hepatitis. PMID- 1722744 TI - Expression of intercellular adhesion molecule 1 (ICAM-1) on human articular cartilage chondrocytes. AB - Monoclonal antibodies have been used to demonstrate the induction of intercellular adhesion molecule 1 (ICAM-1) on chondrocytes in human articular cartilage. ICAM-1 was found not to be constitutively expressed but could be induced by exogenous interleukin 1 alpha(IL1- alpha) at concentrations ranging from 0.01 to 20 ng/ml during in vitro culture. Maximum expression was observed with 2-5ng/ml. In time-course experiments ICAM-1 was not expressed after 4h in culture with IL1 alpha. Expression was induced by 16h and was sustained for a minimum of 6 days in the continued presence of the cytokine. The endothelial leukocyte adhesion molecule (ELAM-1) was not expressed on chondrocytes and was not induced by IL1-alpha. PMID- 1722745 TI - [New aspects of the pathogenesis and treatment of infection and septic shock]. PMID- 1722746 TI - Association of RNA with the B and C snurposomes of Xenopus oocyte nuclei. AB - We studied the time course of [3H]-uridine incorporation into the B and C snurposomes of Xenopus oocyte nuclei. B snurposomes constitute most of the non nucleolar granules in the 1-4 micron size range; they contain the five splicing small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) plus a variety of associated proteins. The organelles referred to as spheres consist of a C snurposome with one or more B snurposomes on its surface. C snurposomes can exist independently of Bs and many are smaller than the structures usually classified as spheres. C snurposomes contain the trimethylguanosine moiety characteristic of snRNAs, as well as the Sm epitope found on several small nuclear ribonucleoproteins (snRNPs), but it is not known which snRNA(s) they contain. When oocytes are incubated with [3H]uridine, all of the nucleoli and chromosome loops label strongly and rapidly. By contrast, labelled RNA appears slowly in the B snurposomes and then only in a fraction of them. After a 24 h incubation, about half of the Bs are labelled, and half are unlabelled or weakly labelled. This observation suggests that there are "mature" and "immature" B snurposomes, and that only the latter acquire newly synthesized RNA. The nature of this RNA is unknown, but it probably includes the splicing snRNAs. B snurposomes on the surface of Cs also constitute a heterogeneous population, some becoming labelled and some remaining unlabelled during a 24 h incubation. An analysis of the label in "doublets" (one B and one C snurposome) suggests that RNA may pass from the Bs to the Cs. PMID- 1722747 TI - Antibodies directed against a meiosis-specific, chromatin-associated protein identify conserved meiotic epitopes. AB - The molecular mechanisms by which meiotic events are regulated are at present unknown. To approach this problem, we have exploited the natural synchrony of Lilium meiocytes to compare the nuclear protein profiles of a variety of stages of meiosis. This approach has facilitated the identification of a number of nuclear proteins that appear and disappear in a stage-specific fashion. Here we report the presence of an abundant nuclear protein that first appears during premeiotic interphase, a period during which the irreversible commitment to meiosis occurs. Antibodies directed against this protein demonstrate its meiosis specificity as well as conservation of the epitope(s) in both mono- and dicotyledonous plant species. Chromatin fractionation studies indicate that this protein, which we have termed meiotin-1, is associated with strings of nucleosomes. Implications for meiotic chromatin packaging and chromosome structure are discussed. PMID- 1722748 TI - Gastric carcinoma metastatic to the bone marrow: immunoperoxidase identification of KMO-1 antigen in MGG-destained aspirate. AB - A case is presented that illustrates the application of the immunoperoxidase technique to the May-Grunwald-Giemsa (MGG)-destained bone marrow aspirate. The cytologic findings in a MGG-stained smear of the bone marrow suggested a metastatic epithelial tumor. Subsequently, a positive reaction to KMO-1, a monoclonal antibody raised against a colon carcinoma cell line, was demonstrated in tumor cells in the MGG-destained smear sample as well as in the paraffin embedded section of the primary gastric cancer. The demonstration of the cancer associated antigen in the MGG-destained material may be useful in establishing the diagnosis of metastatic tumor in the bone marrow. PMID- 1722749 TI - Some famous persons with visual problems as shown on postage stamps. AB - A number of persons important in all fields of human endeavor became blind or were born blind. The reason for the loss of vision varies a great deal, but many of them continued a productive life and contributed to the welfare and advancement of mankind. It is also surprising how many famous people lost one eye or lost nearly all vision in one eye. These were not only soldiers and warriors exposed to accidental traumas, but also writers, scientists and even physicians. PMID- 1722750 TI - [Determination of probiont structure]. PMID- 1722751 TI - An outdoor artificial stream system designed for ecotoxicological studies. AB - Six outdoor artificial streams were designed to simulate natural stream environments. Their primary intended use is to define acceptable threshold toxicity concentrations for chemicals and effluents in the aquatic environment and so provide data that can be used to define water quality criteria more accurately than is possible on the basis of data obtained solely from laboratory tests. Each stream was divided into pool and riffle sections that were colonized by communities of periphyton and invertebrates. They were operated as partly flow through, partly recirculating systems. The design and construction of the streams are described in detail, and brief descriptions are given of methods of establishing aquatic communities, measuring water quality parameters, and treating the streams with chemicals and effluents. The performance of the streams in environmental research is illustrated by reference to typical data for primary production, for invertebrate diversity and abundance, and for chemical concentrations measured during an experiment of two weeks duration. PMID- 1722752 TI - The effect of valproic acid on 5-hydroxytryptamine and 5-hydroxyindoleacetic acid concentration in hippocampal dialysates in vivo. AB - The effect of valproic acid (100, 200 and 400 mg/kg i.p.) on extracellular 5 hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) has been studied using intracerebral microdialysis of the ventral hippocampus. Valproate caused a dose-dependent increase in dialysate 5-HT, but in contrast no effect on 5-HIAA level was observed. These findings are considered with regard to a possible role of 5-HT in mediating the anticonvulsant action of valproate. PMID- 1722753 TI - Reduction in brain serotonin markers by alpha-ethyltryptamine (Monase). AB - alpha-Ethyltryptamine (Monase) is both an inhibitor of monoamine oxidase and a monoamine releasing agent. To determine whether alpha-ethyltryptamine induces serotonergic deficits similar to that of other monoamine releasing agents such as 3,4-methyl-enedioxymethamphetamine (MDMA) and para-chloroamphetamine (PCA), rats were given multiple doses (8 x 30 mg/kg s.c.) of alpha-ethyltryptamine acetate. Serotonin and 5-hydroxyindole acetic acid (5-HIAA) levels and the number of 5-HT uptake sites (determined from [3H]paroxetine binding) were significantly decreased in frontal cortex samples at one week killing. A significant decrease in 5-HT and 5-HIAA levels was also observed in rat hippocampal samples. The results provide evidence that alpha-ethyltryptamine may induce serotonin neurotoxicity similar to that of MDMA and PCA. PMID- 1722754 TI - Muscarinic receptor agonist-mediated modulation of neuronal activity in rat cerebral cortex. AB - Multiple cortical neuronal responses were elicited by the iontophoretic application of muscarinic receptor agonists and antagonists in the rat cerebral sensorimotor cortex in vivo. (1) The muscarinic receptor agonist, oxotremorine-M induced a biphasic effect on spontaneous firing. This was evident as an early brief increase in the firing rate over the spontaneous discharge followed by secondary inhibition of spontaneous activity. The excitation could be blocked by the muscarinic receptor non-selective antagonist atropine and by both the M1 receptor antagonist pirenzepine and the M2 receptor antagonists gallamine or methoctramine. Oxotremorine-M inhibition of spontaneous activity was not affected by the M1 receptor antagonist pirenzepine, while evaluation of its sensitivity to gallamine and methoctramine was not possible since these two M2 receptor antagonists also depressed spontaneous activity, unlike pirenzepine. Of the other two muscarinic receptor agonists, oxotremorine had inconsistent and weak excitatory effects whilst McN-A-343 had only weak excitatory or inhibitory effects on spontaneous activity. (2) Oxotremorine-M, oxotremorine and McN-A-343 had a depressant action on neuronal discharges evoked by glutamate or acetylcholine. A depressant effect of oxotremorine-M was also demonstrated on the early excitation evoked by subsequent applications of oxotremorine-M itself. Of the three muscarinic receptor agonists tested, oxotremorine-M was the most potent in evoking a long-term depression of evoked discharges, lasting from several minutes (greater than 5 min) to as long as 40 min. Oxotremorine-M-induced depression of evoked responses was most sensitive to the M2 receptor antagonists, whereas oxotremorine-induced depression was more sensitive to the M1 receptor antagonist pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722755 TI - Microdialysis studies on 3,4-methylenedioxyamphetamine and structurally related analogues. AB - The acute effect of 3,4-methylenedioxyamphetamine (MDA) and three structural analogues on the extracellular concentrations of dopamine (DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum was studied using in vivo microdialysis in awake, freely moving rats. MDA significantly (P less than 0.001) increased and decreased the extracellular concentrations of DA and its metabolites, respectively, following i.p. administration. Similarly, acute administration of the N-methyl (MDMA) and N ethyl (MDE) derivatives of MDA significantly (P less than 0.05) increased the concentration of DA in the striatum. The alpha-ethyl homologue of MDMA, MBDB, increased the extracellular concentration of DA but significantly (P less than 0.05) less than MDA, MDMA, or MDE. The rank order of potency for these amphetamine derivatives to increase the extracellular concentration of DA was: MDA greater than MDMA greater than MDE greater than MBDB greater than vehicle. The increase in extracellular DA concentration following a single administration of these compounds was negatively correlated with the level of serotonin (5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the contralateral striatum measured 7 days following drug administration. Thus, extending the alkyl group on the nitrogen or alpha-carbon of MDA reduces the ability of these compounds both to increase acutely the extracellular concentration of DA and to produce long-lasting depletions of 5-HT in the brain. PMID- 1722756 TI - Bronchial smooth muscle responses evoked by toluene diisocyanate are inhibited by ruthenium red and by indomethacin. AB - We have investigated the ability of ruthenium red, an inorganic dye with Ca2+ entry-blocking properties and a selective antagonist of capsaicin, and of indomethacin, a cyclooxygenase inhibitor, to inhibit bronchial smooth muscle responses evoked by toluene diisocyanate in guinea pigs. Previous exposure of isolated guinea pig bronchi to ruthenium red significantly decreased the response produced by toluene diisocyanate. Further, the response to toluene diisocyanate was significantly decreased by pretreatment with indomethacin. These findings provide evidence that toluene diisocyanate-induced contractions of guinea pig bronchi are produced indirectly by generation of a prostanoid that activates capsaicin-sensitive afferents via a ruthenium red-sensitive mechanism. PMID- 1722757 TI - Rostrocaudal and lateromedial density distributions of superior colliculus neurons projecting in the predorsal bundle and to the spinal cord: a retrograde HRP study in the cat. AB - Efferent neurons of the cat superior colliculus (SC) which project in the predorsal bundle (PDB) and to the spinal cord (PDB neurons) form a major pathway by which the SC controls the changes of the direction of gaze in response to stimuli of visual and other modalities. Knowledge of rostrocaudal and lateromedial density distributions of different groups of PDB neurons within the SC is necessary to analyse their relationships with the topography of sensory and motor maps. Density gradients may also bear on the efficacy of connections originating from topographically different collicular regions. In the present study, large injections of HRP/WGA-HRP were made in the C1 segment of the spinal cord and in the pontobulbar tegmentum. Judged by several morphological criteria, axons of passage, including those not subjected to a direct mechanical damage, were participating in the uptake of tracers. Therefore, labeled SC neurons corresponded to the nearly total populations of contralaterally projecting tectospinal neurons (TSNs) and neurons projecting in the PDB, respectively. Subtraction of the TSN density map from that of the whole PDB population was used to infer the distribution of tectal neurons terminating in the rhombencephalic tegmentum (TRhN). This subtotal labeling method proved useful in resolving the contradictions between the earlier HRP studies on the TSN and TRhN topography. The following density distributions were obtained for different groups of PDB neurons: 1) The mean TSN density is more than two times higher in the lateral half of the SC, representing the lower visual field. In this region the density remains constant from rostral to caudal, i.e., from the representation of vertical meridian to large contralateral azimuths. In the medial half, the average density decreases from rostral to caudal. Consequently, TSNs do not show the caudalward increment predicted by the higher efficacy of caudal stimulation points in eliciting head movements. 2) The distribution of PDB neurons is symmetrical with respect to the representation of the horizontal meridian. It is close to homogeneous at all azimuths of the retinotopic map and within the zone limited by small (10-15 degrees) upward and downward elevations.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722758 TI - Topographical mapping of the thalamocortical projections in rodents and comparison with that in primates. AB - The general topographical organization of the thalamo-cortical projection of two rodents, the Siberian hamster (Phodopus sungorus) and the Guinea pig (Cavia aperta) was investigated with the HRP-method and compared with that of the new world primate marmoset (Cal-lithrix jacchus) as shown in a companion study by Brysch et al. (1990). HRP was injected into various regions of the cortex in different animals and hemispheres, and plots were made of the retrogradely stained thalamic projection neurons. The thalamocortical projection is virtually identical in both rodent species. It is topological throughout in that nearby cortical injections label nearby, though overlapping cell groups in the thalamus. Cortical injections in a rostro-caudal progression labelled thalamic projection zones on top of each other, layered like tiles on a roof or fish scales, beginning in the rostromedial and ending in the caudo-dorsal thalamus. The progression vector of thalamic zones projecting successively from more rostral to more caudal cortical zones is twisted and turns from a predominantly mediolateral direction in the anterior thalamus to an essentially ventro-dorsal direction in the posterior thalamus In the marmoset, the thalamo-cortical topography follows the same topological rule, with the exception of the lateral geniculate body which is translocated latero-ventrally and separated from the rest of the thalamus as in all primates. This suggests a general thalamo-cortical mapping rule common to all mammals which can be related to gradients and timing of cell birth in the thalamus. It is proposed that this mapping rule is the consequence of successive appositions of neurons in the medio-ventral thalamus during ontogenetic development. PMID- 1722759 TI - Initial stages of retinofugal axon development in the hamster: evidence for two distinct modes of growth. AB - In order to characterize differences in growth patterns of axons as they elongate toward their targets and during the initial stages of terminal arbor formation within the targets, we examined the primary visual system of fetal and newborn hamsters using three morphological methods: the Cajal-deCastro reduced silver method, the rapid Golgi technique, and anterograde transport of HRP. Axons emerge from the retina between the 10th and 11th embryonic days (E10-E11). The front of retinal axons crosses the chiasm, extends over the primitive dorsal nucleus of the lateral geniculate body (LGBd) by E13, and advances to the back of the superior colliculus (SC) by E13.5-E14. The rate of axon growth during this advance is nearly 2 mm/day. Collateral sprouts appear on axons around E15.5. In the LGBd and SC, these sprouts arise from multiple sites along the parent axons. Only one or a few of the sprouts continue to grow and branch, while others are eliminated. The net rate of axon collateral advance in this second phase is an order of magnitude slower than during the stage of axon elongation. Thus, formation of CNS projections may involve two qualitatively distinct modes of axon growth. The arborization mode contrasts with the elongation mode by the presence of branching, a lack of fasciculation and a slower average rate of extension. The stereotypic direct advance of axons during elongation also differs from the remodelling which occurs during arborization. The delay between axon arrival at targets and onset of arborization could be a reflection of axons "waiting" for a maturational change to occur in the retina or in targets. Arborization in the LGBd and SC is initiated around the same time, implicating the former possibility. However, a slower differentiation of retinal arbors in the SC, in addition to morphological differences of arbors in the two structures, suggests that alterations in substrate factors also play a critical role in triggering the early stages of arbor formation. PMID- 1722760 TI - Two populations of glial cells from fish optic nerve/tract with distinct electrophysiological properties. AB - The electrophysiological properties of the two major glial cell types in cultures from the regenerating goldfish optic nerve/tract were studied with patch-clamp techniques. Spindle-shaped cells express myelin proteins. These oligodendrocyte like cells possess outwardly rectifying currents, do not show glutamate activated currents and are rarely electrically coupled to neighboring cells. Cells of epitheloid morphology probably represent astrocytes. They are GFAP-positive and do not exhibit myelin proteins. These cells have glutamate activated currents, display a linear current to voltage relationship and are extensively electrically coupled thus displaying properties similar to mammalian astrocytes. PMID- 1722762 TI - L-tryptophan-induced eosinophilia-myalgia syndrome. II. Partial correction of abnormal tryptophan metabolism by pyridoxine. AB - Abnormal metabolism of tryptophan is one of the possible aetiological factors in the L-tryptophan-induced eosinophilia-myalgia syndrome (EMS). We studied the plasma levels of tryptophan and serotonin and the urinary excretion of kynurenine and 5-hydroxyindoleacetic acid after oral intake of L-tryptophan in 1 subject with EMS and 2 healthy subjects. The test was repeated with concomitant administration of pyridoxine. In the patient there were elevated levels of plasma tryptophan during the loading and increased elimination of kynurenine in the urine both during and after the L-tryptophan test. During pyridoxine administration tryptophan levels and kynurenine elimination were much reduced, and kynurenine elimination was similar to that of controls. This study (i) confirms that an abnormal metabolism of L-tryptophan occurs in EMS patients and (ii) shows that this can be corrected by pyridoxine. PMID- 1722761 TI - Nigrotectal projections in the primate Galago crassicaudatus. AB - The pattern of the nigrotectal projection in Galago crassicaudatus was determined using retrograde and anterograde transport methods. These experiments revealed that pars reticulata and pars lateralis of the substantia nigra project to all layers of the ipsilateral and contralateral superior colliculus, except to layer I. The nigrotectal projection is not homogeneous, but is concentrated in particular collicular layers and sublayers, and the intensity and laminar distribution of the projection varies along the rostral-caudal dimension of the superior colliculus. The ipsilateral and contralateral nigrotectal projections are generally similar, except that a tier of dense label which is prominent in the ventral part of much of the ipsilateral layer IV is not obvious contralaterally; moreover, the contralateral projection is much sparser than the ipsilateral. Deposits of tracers at different medial-lateral locations within the substantia nigra did not result in different laminar patterns of anterogradely transported label in the superior colliculus. Based on the known connections and functions of the collicular layers and sublayers, the pattern and distribution of the nigrotectal projection suggests that the substantia nigra may use this pathway to gain access to particular components of vision- and visuomotor-related networks. PMID- 1722763 TI - Pancreatic secretion of zinc and copper in normal subjects and in patients with chronic pancreatitis. AB - Pancreatic secretion of zinc and copper in duodenal juice were measured in 7 healthy persons and in 9 patients with chronic pancreatitis. Stimulation with cholecystokinin and secretin increased secretion of zinc in healthy persons but not in patients. Copper secretion was not influenced. In patients with chronic pancreatitis, the correlations between zinc secretion, and amylase and trypsin secretion were significant while in healthy subjects they were not. Possibly pancreatic zinc secretion in the duodenal juice might be used as a measure of exogenic pancreatic function, and determination of zinc in duodenal juice may replace enzyme determinations in the diagnosis of chronic pancreatitis. PMID- 1722765 TI - Modulation of nicotinic acetylcholine receptors by intracellular calcium and cyclic AMP in Lymnaea stagnalis neurones. AB - Acetylcholine (ACh)-receptor ion channels were investigated under the modulatory action of calcium and cyclic AMP in completely isolated Lymnaea stagnalis neurones using the noise analysis technique. Elevation of the intracellular Ca2+ concentration in dialyzed neurones produced a reduction in the amplitude of ACh induced current accompanied by slight decrease in the mean channel open time and a simultaneous 1.5-fold increase in mean channel conductance. Direct introduction of cyclic AMP into neurones or elevation of intracellular cyclic AMP level by application of serotonin or forskolin produced 20-40% reduction in ACh-induced conductance without significant effect on the measured parameters of the ion channels. The inhibitory effects of calcium and cyclic AMP appear to be independent. Our findings indicate that reduction in ACh induced conductance under calcium and cyclic AMP modulation results from an alteration in the channel gating mechanism. Since the efficiency of ion transfer is independent of cyclic AMP, and it even rises with the elevation of calcium concentration, the inhibition of ACh responses may be accounted for by a decrease in the rate constant for channel opening, so that channels activated by acetylcholine remain in a closed state over longer intervals. PMID- 1722764 TI - Overexpression of class I major histocompatibility complex accompanies insulitis in the non-obese diabetic mouse and is prevented by anti-interferon-gamma antibody. AB - Overexpression of class I major histocompatibility complex (MHC) proteins on pancreatic islet cells is a characteristic of autoimmune Type 1 (insulin dependent) diabetes mellitus in humans and in animal models. Studies of post mortem pancreases from humans with Type 1 diabetes suggest that overexpression of class I MHC proteins may precede mononuclear cell infiltration of the islets (insulitis). Pancreatic histology from the earliest stages of human Type 1 diabetes is rarely available. We have used the non-obese diabetic mouse, given cyclophosphamide to accelerate Beta-cell destruction, to investigate the temporal relationship between the overexpression of class I MHC protein and mRNA and other pathological changes associated with Beta-cell destruction. Prior to cyclophosphamide, immunoperoxidase staining showed that expression of class I MHC proteins was greater on islet cells and infiltrating inflammatory cells of the non-obese diabetic mouse than on islet cells of other mouse strains, whereas staining on exocrine cells was similar. On day three after cyclophosphamide administration, when insulitis had regressed, islet class I MHC protein expression had diminished. A dramatic increase in class I MHC protein expression occurred between days seven and nine, concomitant with reinfiltration of the islets by mononuclear cells; overexpression was seen both on islet cells and on surrounding exocrine cells, but only in the presence of mononuclear cell infiltration. By day 21, class I MHC protein overexpression was again confined to the islets, the exocrine pancreas being free of infiltration. Class I mRNA also increased dramatically by day eight but had virtually returned to normal by day 12.2+ effected by cytokines secreted by activated immuno-inflammatory cells. Class I MHC overexpression should enhance targeting of cytotoxic T cells to Beta cells bearing autoantigen. PMID- 1722766 TI - Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherichia coli K-12. AB - The rfb genes of Vibrio cholerae O1 (Ogawa serotype) were subcloned into a derivative of pBR322. This plasmid was transformed into several Escherichia coli K-12 mutant strains which produce an incomplete lipopolysaccharide (LPS)-core oligosaccharide region. The data indicate that the V. cholerae O-antigen is assembled onto the E. coli LPS and that at least two glucoses are needed in the core in order to achieve a high level of production. These data are consistent with the reported presence of glucose in the V. cholerae LPS-core-oligosaccharide region. PMID- 1722767 TI - Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O antigen of an E. coli O2: K1 strain. AB - The rfb gene cluster which determines the biosynthesis of the O2 O antigen has been cloned from an Escherichia coli O2: K1 strain isolated from a case of septicaemia in chickens. The region required for expression of the O antigen in E. coli K-12 was localised to a 10.7 to 14.15-kb segment which was shown to be chromosomal in origin with a close linkage to the gnd and his genetic loci. PMID- 1722768 TI - Improved vectors for transcriptional signal screening in corynebacteria. AB - New promoter-probe and terminator-probe shuttle vectors for Escherichia coli and corynebacteria were constructed. These vectors, working with the uidA gene of E. coli as reporter gene, are very useful for the cloning and subsequent analysis of transcriptional regulatory signals. The beta-glucuronidase encoding activity of uidA can be easily detected on agar plates containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc). In the terminator probe vector pUT2, uidA is expressed from a promoter of the Brevibacterium lactofermentum cryptic plasmid pBL1. Multiple cloning sites (MCS) located immediately upstream of uidA allow introduction and selection of terminators or regulatory signals. In the promoter-probe vector pUT3, transcription readthrough from vector promoters is prevented by a terminator of B. lactofermentum isolated using pUT2. We have successfully used pUT2 and pUT3 to isolate several terminators and promoter regions active in B. lactofermentum and shown that the E. coli strong terminator cartridge omega appears less efficient in corynebacteria. PMID- 1722769 TI - [Uterine cervix cancer. Clinical stage III. Combined radiotherapy and chemotherapy treatment]. AB - 55 patients with stage III carcinoma of the uterine cervix were entered into a prospective randomized study to evaluate the possible radiation-potentiating properties of bleomycin. Group A received classical radiation treatment with telecobalt-therapy 50 Gy/25 fractions plus 32 Gy/4 fractions (Cathetron). The other two groups received 15 mg of bleomycin by continue infusion two time of week during 5 week, groups B before, and group C after, irradiation. The morbidity was minimal. The initial response was complete in 49 cases and partial in 6 cases. At 2 years there were 26 recurrences, 22 (88.8%), locoregional recurrences and 4 distant metastasis, 3 in the group of bleomycin treatment. The probability of actuarial survival was 62.1%, 30.1% and 35.6% respectively to groups A, B and C. Addition of bleomycin to radiotherapy failed to increase the recurrence-free survival. PMID- 1722770 TI - The study of variation in the human genome. AB - Regions of the genome showing high evolutionary stability are often conserved as a result of functional constraints. Conversely, more variable regions are likely to represent DNA with no functional or structural importance. However, as in the case of immunologically important regions, sequence divergence does not always indicate lack of functional importance. There is thus a wealth of information from both a functional and an evolutionary point of view that comes from studies of DNA sequence variation, a neglected aspect of the genome endeavor. Naturally, one cannot sequence hundreds of individuals in full, but a useful compromise is to use less expensive methods and to limit the more expensive types of analysis to an appropriately chosen sample of loci. The sample could be determined after careful consideration of categories of DNA segments with respect to individual variation. The study of such categories of DNA variation patterns can help in the understanding of the role of each gene and vice versa. One other important application requiring a study of DNA variation in different human populations is forensic DNA typing. This study requires a knowledge of allele frequencies in different human populations. Evidence of a match between two DNA samples is meaningless if the approximate population frequency of the DNA pattern is not known. It has been suggested (E. Lander) that one use the highest frequency for the most common allele as a baseline frequency estimate. Obviously, systems in which this is employed require an extensive analysis of population-specific allele frequencies. In general, the best way of studying interindividual variation when detecting or describing new polymorphisms is to include interethnic variation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722771 TI - Study of total serum amylase, its salivary and pancreatic fraction and the pancreatic to salivary amylase ratio in testicular tumours. AB - Serum levels of total amylase, its pancreatic fraction (P), salivary fraction (S), and the ratio of pancreatic to salivary fraction (P/S) were determined in 52 cases of histologically proved testicular germ cell tumours and 33 healthy controls. Total serum amylase remained unchanged, but the salivary fraction had a lower mean value. P/S ratio and the pancreatic fraction were significantly elevated in both seminomatous and non- seminomatous tumours. The ratio was more frequently raised in non-seminomatous (100%) as compared to seminomatous (66.67%) tumors. Following treatment there was no appreciable fall in P/S ratio in non seminomatous tumors whereas in seminomas there was a slight increase in the ratio. The pancreatic fraction showed a transient fall in seminomatous but not in non-seminomatous tumors following treatment. The pancreatic fraction of amylase and the P/S ratio may help in the diagnosis of testicular germ cell tumor but does not appear to be of use in assessment of prognosis or monitoring the course of the disease during treatment. PMID- 1722772 TI - Intrathymic presentation by dendritic cells and macrophages: their role in selecting T cells with specificity for internal and external nominal antigen. AB - The present study focused on the question of whether intrathymic dendritic cells and macrophages (DC/M phi) are involved in the processes of T-cell repertoire selection and establishment of tolerance towards nominal antigen. Proliferation of thymocytes (TC) was determined under limiting dilution (LD) conditions after depletion of DC/M phi and after reconstitution of TC, which were depleted of cells expressing major histocompatibility complex (MHC) class II antigens, with thymic DC/M phi. Trinitrophenyl (TNP) [coupled to ovalbumin (OVA)] was used as an internal antigen in prenatally trinitrobenzenesulphonic acid (TNBS)-treated mice and as an external antigen in prenatally untreated mice. Intrathymic DC/M phi were clearly involved in selecting the repertoire of T cells specific for external antigen: they presented the antigen and initiated proliferation of thymic T cells, which were depleted of MHC class II antigen-expressing cells. But they were not the only cells to present nominal antigen in the thymic environment. Intrathymic DC/M phi could also deliver negative signals. This became apparent when evaluating presentation of TNP in prenatally TNBS-treated mice. Thymus-derived DC/M phi from prenatally TNBS-treated mice could not initiate proliferation of TC in response to TNP-OVA. Instead, when prenatally TNBS-treated mice received an antigenic challenge [TNP-sheep red blood cells (SRBC)], thymic DC/M phi inhibited proliferation of cortisone-resistant TC from untreated and prenatally TNBS-treated mice. This can be explained by assuming that in the process of establishing tolerance, intrathymic DC/M phi may exert cytotoxic/cytostatic activity. PMID- 1722773 TI - Specificity of the immune response to the group B polysaccharide of Neisseria meningitidis. AB - A panel of monoclonal antibodies (mAb) and polyclonal sera of murine, human and equine origin, of IgM isotype and with specificity for Neisseria meningitidis group B polysaccharide, an alpha(2----8)-linked homopolymer of sialic acid, were examined for their antigenic and biological specificities. The nature of the antigenic determinants on B polysaccharide was investigated using a series of N acyl derivatives of B polysaccharide, two sialic acid polymers containing alpha(2 ---9)-linkages and a series of polynucleotides. The panel of antibodies recognized an array of unrelated antigenic determinants on the B polysaccharide, despite its structural simplicity, and all but one were highly effective in an in vitro bactericidal assay and/or in an in vivo murine passive protection model. There was no evidence that B polysaccharide induced antibody capable of blocking biological activity (blocking antibody). PMID- 1722774 TI - Expression of Epstein-Barr virus nuclear antigens in anti-IgM-stimulated B cells following recombinant vaccinia infection and their recognition by human cytotoxic T cells. AB - Cytotoxic T lymphocytes (CTL) recognizing Epstein-Barr virus (EBV) nuclear antigens (EBNA) are an important host defence mechanism in restricting the proliferation of EBV-infected B cells. Previously, B-type lymphoblastoid cell lines (LCL) infected with vaccinia recombinants encoding for the EBNA proteins have been used to identify A-type-specific CTL epitopes. However, to localize the CTL epitopes encoded by both A- and B-type transformants, B-type LCL are an inappropriate host for vaccinia. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-IgM (mu-chain specific)-stimulated human B cells allowed vaccinia virus to replicate more efficiently than either phytohaemagglutinin-stimulated lymphocytes (PHA blasts) or CTL and expressed EBNA proteins following recombinant vaccinia infection. Furthermore, the presentation and recognition of target epitopes expressed on vaccinia-infected anti-mu-stimulated B cell blasts were comparable to that on similarly infected LCL. Anti-mu-stimulated B cells were used to define the CTL epitopes recognized by a panel of CTL clones from an EBV-immune donor. Using recombinant vaccinia-infected anti-mu-stimulated B cells, the CTL response from this donor was mapped to the EBNA6 protein. Most importantly, in vitro stimulation of unfractionated mononuclear cells with vaccinia-infected anti-mu B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 localized the epitope for the majority of the EBNA6 specific CTL clones to the sequence EENLLDFVRFM, apparently in association with HLA-B44. This work clearly demonstrates that anti-mu-stimulated B cells not only provide an efficient model for localizing the CTL epitope(s) but also raises the possibility of reactivating a memory T-cell response to any gene product expressed by recombinant vaccinia. PMID- 1722775 TI - Deficiency of complement decay-accelerating factor (DAF, CD55) in non-Hodgkin's lymphoma. AB - We have assessed levels of surface-expressed complement regulatory proteins, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) on cells from patients with hematological malignancies. Neither malignant cells nor unaffected nucleated blood cells from the patients lacked MCP. On the other hand, complete deficiency of DAF was found in 2/10 of non-Hodgkin's lymphoma (NHL), while none of the 38 patients with acute nonlymphocytic leukemia (ANLL) (14 cases), chronic myelogenous leukemia (CML) (6 cases), acute lymphocytic leukemia (ALL) (12 cases) and chronic lymphocytic leukemia (CLL) (6 cases) lacked DAF. The two patients with DAF-negative NHL had no history of paroxysmal nocturnal hemoglobinuria (PNH), and their peripheral blood cells were DAF-positive. One DAF-negative NHL exhibited T cell markers and the other those of B cell. In both cases, treatment of the DAF-negative lymphoma cells with antibody against MCP (M177) followed by Mg(2+)-EGTA-serum resulted in efficient deposition of homologous C3. These results infer that some NHL specifically lack DAF and, through treatment with M177, are targeted by homologous C3. PMID- 1722776 TI - Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen. AB - The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance. PMID- 1722777 TI - Detection of antigen in the coelomocytes of the earthworm, Eisenia foetida (Annelida). AB - Earthworms, Eisenia foetida, are able to respond to antigenic stimulation by the formation of the antigen-binding molecules by coelomocytes--the effector cells of annelids' defence reactions. The ability to react with gold-labelled antigen was detected in agranular coelomocytes by electron microscopy. Furthermore, flow cytometry analysis used for quantitative evaluation of antigen binding showed significant increase of both antigen-binding cells and the amount of antigen bound per cell after stimulation. The antigen binding was inhibited by preincubation of cells with several similar proteins, although the most potent inhibitor was the immunizing antigen. PMID- 1722778 TI - The influence of FK-506 on the thymus and spleen in normal C3H/He mice: flow cytometric analysis of lymphocyte subpopulations. AB - Although FK-506 has been widely investigated as a potent suppressor of organ allograft rejection in animals, little is known about the effect of FK-506 on normal animals. In this study, we investigated the influence of FK-506 on the thymus and spleen in normal C3H/He mice at the age of 5 weeks. Mice were given dosages of FK-506 varying between 0.3 and 30 mg/kg/day intramuscularly for 5 days. After treatment with FK-506, the total number of lymphocytes in the thymus was significantly decreased in a dose-dependent manner; however, no change was evident in the total number of lymphocytes in the spleen. FK-506 also influenced the immunophenotypes in thymocytes, but not in splenocytes. The absolute number of CD4+ CD8- or CD4- CD8+ thymocytes decreased significantly in the 0.3 mg/kg/day treated group. CD4+ CD8-, but not CD4-CD8+ thymocytes decreased further in the 1 mg/kg/day-treated group. Neither subpopulation decreased any further with continuing administration. On the other hand, the number of CD4+ CD8+ thymocytes did not decrease significantly in the 0.3 mg/kg/day-treated group, whereas it did decrease significantly in the 1 mg/kg/day-treated group, decreased further with the increase in dosage of FK-506, and decreased markedly in the 30 mg/kg/day group, The number of CD4-CD8-thymocytes did not show any change, even in the high dosage groups. These results indicate that FK-506 influences the subpopulations of thymocytes according to its dosage. At a lower dosage, it affects mature CD4+ CD8- or CD4- CD8+ cells, but not immature CD4+ CD8+ and CD4-CD8- cells.2 PMID- 1722779 TI - [Accumulation of two different hydroxyethyl starch preparations in the placenta after hemodilution in patients with fetal intrauterine growth retardation or pregnancy hypertension]. AB - In a prospective clinical study the safety of two hydroxyethylstarch preparations (HES steril 10%, Fresenius AG, Oberursel = HES-A; Haemufusin, Kabi-Pfrimmer, Erlangen = HES-B) were assessed. In 60 patients with fetal growth retardation and/or gestational hypertension, hematocrit, aPTT, factor VIIIR: Ag, fibrinogen, uric acid, cord blood hemoglobin, hematocrit, pH-value and the fetal/maternal hydroxyethylstarch concentration before and after eight (HES-B) or nine (HES-A) days of treatment were monitored. 500 ml HES-A (n = 36) or HES-B (n = 24) together with the same volume electrolyte solution, were infused daily. Both substances lowered significantly the maternal and fetal hematocrit. Histopathological changes of placenta (trophoblast cells and stroma) taking place after the infusion of HES-A or HES-B were depicted by light microscopy. Administration of HES-A or HES-B was associated with lower values of factor VIIIR: Ag and a prolongation of aPTT, but only HES-B demonstrated a significant effect (31% vs. 12%, p less than 0.01). We observed in 4 (16.7%) cases severe uterine bleeding complications and one woman (4.2%) with abruptio placentae in the group HES-B. Light microscopy shows vacuoled trophoblast and stroma cells after HES infusions. The marked vacuolisation of the placenta after HES-B is due to differences in the physicochemical characteristics of HES-A and HES-B. For this reason, we prefer to administer HES-A in the dilution treatment of patients with placental insufficiency. PMID- 1722780 TI - Alpha-2-macroglobulin-kallikrein complex: a temperature-sensitive mediator in contact-system-induced inflammation with a potential role in late and delayed hypersensitivity responses. AB - Maximal complexing of alpha 2-macroglobulin (alpha 2M) and kallikrein (KK) occurs at a temperature of 22-24 rather than at 37 degrees C. The protease expressivity of the complex is also maximal at 22-24 degrees C. alpha 2M-KK complex, sustained permeability changes in guinea pig skin. These findings suggest that the complex, rather than free KK, could play a role in the kinin release reported in some late phase reactions, some instances of delayed-type hypersensitivity and some cold induced reactions. PMID- 1722781 TI - Change in sensitivity to lysophosphatidylserine of mouse bone marrow-derived mast cells during cultivation with fibroblasts. AB - Lysophosphatidylserine (lysoPS) is known to enhance IgE-mediated activation of rodent connective tissue mast cells (CTMCs). In the present study, we investigated the effect of lysoPS on degranulation of interleukin-3-dependent mouse bone marrow-derived mucosal mast cells (BMMCs) and of their CTMC-like differentiated cells. In the absence of lysoPS, BMMCs released approximately 20% of their histamine when sensitized with anti-dinitrophenyl (DNP) IgE and challenged with DNP-conjugated antigen. When stimulated in the presence of lysoPS, no appreciable enhancement was observed. On the other hand, histamine release from BMMCs, which had differentiated to CTMC-like cells by co-culture with 3T3 fibroblasts, was enhanced 2- to 3-fold by the addition of lysoPS. The maximum potentiation was observed at 5 x 10(-6) M lysoPS. These results suggest that mast cells might acquire their dependence on exogenous lysoPS during differentiation from mucosal mast cells to CTMC-like cells. PMID- 1722782 TI - Characteristics of histamine secretion from mast cells stimulated with sodium orthovanadate and other vanadium compounds. AB - Sodium orthovanadate was found to be an effective histamine liberator from serosal mast cells of the rat and mouse. The release process was slow, non cytotoxic and strongly dependent on pH and extracellular calcium. The effect was highly tissue and species specific and human basophil leucocytes, human lung mast cells and tissue mast cells of the rat and guinea pig were only weakly responsive or essentially unreactive. Other oxyanions of vanadium with the metal in the (+V) oxidation state also evoked histamine release from rat peritoneal mast cells but neither vanadyl sulphate (+IV oxidation state) nor the analogous orthophosphate anion were effective secretagogues. On the basis of these results, the possible mechanism of action of vanadate is discussed. PMID- 1722783 TI - Release of mediators from human gastric mucosa and blood in adverse reactions to benzoate. AB - A study was carried out on 29 patients to investigate the amount of histamine liberation and release of platelet-activating factor and 6-keto-prostaglandin F1 alpha from gastric mucosa and whole blood or mononuclear cells by sodium benzoate. The patients suffered from asthma (10), atopic dermatitis (7) and chronic urticaria (4). 8 patients with unrelated, non-immunologic diseases served as controls. In the oral provocation test (OPT) 3 patients experienced a recurrence of their original disease, whilst 1 asthmatic patient reacted with abdominal disorder. The release of histamine and prostaglandin from mucosa was significantly increased by sodium benzoate in comparison to the spontaneous release observed in patients. The mucosa of the control persons did not react to sodium benzoate. Furthermore, there was a significant difference in prostaglandin release between patients with positive OPT and the control persons. No difference could be found between patients with negative OPT and those with positive OPT. Additionally, in the mediator release from whole blood or mononuclear cells there was no obvious difference apparent. These results suggest a possible involvement of prostacyclin and histamine in adverse reactions to benzoate. Due to the sensitivity of the method, a mediator release from mucosa can already be demonstrated in a preclinical state of the pseudoallergic reaction in the absence of clinical symptoms. PMID- 1722784 TI - Cefminox sodium penetration into prostatic tissue with and without inflammation. AB - The concentrations of cefminox sodium (CMNX) in serum and prostatic tissue were determined in 36 cases of prostatic hyperplasia with and without inflammation. Mean ratios of CMNX in tissue over concentration in serum were 0.11 +/- 0.07 for patients with inflammation and 0.09 +/- 0.06 for those without inflammation. There was no significant difference between the two groups. These data suggest that CMNX penetration into prostatic tissue is not influenced by the presence of inflammation. PMID- 1722785 TI - [Reduction of postoperative blood loss and donor blood use in heart surgery with aprotinin: experience with various dosages]. AB - The effect of high dose aprotinin was evaluated in a prospective study on 100 patients undergoing cardiopulmonary bypass. Special attention was made on postoperative blood loss and transfusions of bank blood postoperatively. In the first part of the study, after induction of anesthesia, a loading dose of 2,000,000 kallikrein-inhibiting-unit (KIU) = 280 mg aprotinin was given intravenously over a 30-min period. Immediately afterward, a continuous infusion of 500,000 KIU/h was started and maintained until skin closure. Another 2,000,000 KIU was added to the priming volume of the heart-lung machine. A control group of 50 patients was randomized with similar indication for surgery and past cardiac history. The total loss from the thoracic drains was significantly reduced in the aprotinin group as compared with the loss in the control group (490 +/- 265 ml versus 1045 +/- 380 ml). In a separate group of risk patients (redo-operations, infective endocarditis) the total blood loss was even more significant reduced in the aprotinin group (690 +/- 195 ml versus 1585 +/- 290 ml). Patients of the aprotinin group received markedly less bank blood postoperatively (350 +/- 100 ml versus 900 +/- 240 ml without aprotinin). Part II of the study (36 patients) consisted of lower dosage (2,000,000 KIU intravenously during induction of anesthesia only or 2,000,000 KIU in the priming volume of the heart-lung machine only). Patients who received aprotinin in the heart-lung machine only showed no significant difference regarding blood loss and blood requirement to patients with high dose aprotinin. It appears possible that aprotinin reduces the activation of the coagulation during cardiopulmonary bypass and preserves platelet function without affecting platelet consumption during the extracorporeal circulation. The results of our study demonstrate that high dose aprotinin markedly reduces blood loss as well as homologous blood requirement in the early postoperative course of cardiosurgical patients. Similar effects due to reduced aprotinin dose have been observed in patients receiving aprotinin in the extracorporeal circulation only. PMID- 1722786 TI - Galanin-like immunoreactivity is increased in the brain of estradiol- and methyltestosterone-treated eels. AB - A galanin-like peptidergic system was demonstrated in the brain of Anguilla. A group of immunoreactive perikarya was located in the nucleus preopticus periventricularis close to the recessus preopticus. Galaninergic fibers occurred in various brain areas. Galanin identified in mammalian pituitary cells was undetectable in fish adenohypophysial cells. Estradiol increased the immunostaining of the rostral perikarya and brain fibers in both male and female European eels kept in fresh water and in female American eels in sea water. Methyltestosterone, an aromatizable androgen, increased galanin immunoreactivity in rostral perikarya and brain fibers of male European eels and female American eels. The cross-sectional area of these perikarya increased significantly after both treatments whereas cell bodies of the posteroventral hypothalamus were slightly affected. Dihydrotestosterone showed no clear effect. Fibers close to the corticotropes were sometime increased, but galanin synthesis was not induced in pituitary cells. In contrast, estradiol induced galanin synthesis in rat pituitary cells, but had a still controversed effect on hypothalamic galanin. A putative influence of galanin on the pituitary-gonadal axis is discussed as gonadal hormones diversely affect gonadotropes and gonosomatic indices in Anguilla. PMID- 1722787 TI - Immunohistochemical characterization of nurse cells in normal human thymus. AB - Lymphoepithelial complexes known as thymic "nurse" cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35 beta H11 and 34 beta E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B1)], class II histocompatibility antigens (HLA-DR), and thymosin alpha 1. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed. PMID- 1722788 TI - Expression of cytokeratin polypeptides during development of the rat inner ear. AB - The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells. PMID- 1722789 TI - Use of pesticides in practice--good news and a warning. PMID- 1722790 TI - The origin and development of benign prostatic hyperplasia. An age-dependent process. AB - Although the exact cause of benign prostatic hyperplasia (BPH) is not well defined, it is thought to occur as the result of epithelial-stromal interactions in the appropriate hormonal milieu. Benign prostatic hyperplasia originates in the periurethral and transition zones of the prostate in a microscopic (histologically identifiable) state as early as the third decade of life. With advancing age and the presence of androgens, approximately 50% of microscopic BPH will develop into macroscopic (palpably enlarged prostate) BPH. However, clinically significant BPH necessitating treatment will develop in only 50% of men with an enlarged prostate gland. In the United States, the estimated risk of a 50-year-old man undergoing a prostatectomy in his lifetime is approximately 25% to 40%. If left untreated, a significant number of symptomatic patients will remain stable or improve without adverse sequelae. PMID- 1722791 TI - The pathophysiology of benign prostatic hyperplasia. AB - Although benign prostatic hyperplasia (BPH) is one of the most common disease processes affecting the aging male, surprisingly little is known about its pathophysiology. Cause-and-effect relationships have not been established, despite intense research efforts in the last four or five decades aimed at elucidating the underlying etiology of prostatic growth in older men. Previously held notions that the clinical symptoms of BPH (prostatism) are due simply to a mass-related increase in urethral resistance are too simplistic. It is now clear that a significant portion of the symptoms are due to obstruction-induced detrusor dysfunction. Moreover, obstruction may induce a variety of neural alterations in the bladder and prostate that contribute to symptomatology. Undoubtedly, the constellation of cellular pathologies that give rise to the symptoms of BPH will be far more complex than we currently realize. Only by unraveling these complexities, however, will we be able successfully to design alternative strategies to treat, and possibly prevent BPH. PMID- 1722792 TI - One-year experience in the treatment of benign prostatic hyperplasia with finasteride. The MK-906 (Finasteride) Study Group. AB - Finasteride (MK-906) is a 5 alpha-reductase inhibitor that reduces circulating dihydrotestosterone (DHT) levels without lowering testosterone levels. The goal of this study was to evaluate the effects of long-term (12 months) treatment with finasteride in 67 men with benign prostatic hyperplasia to determine if previously reported short-term efficacy was maintained with chronic therapy. Treatment with 10 mg of finasteride resulted in a 78% to 80% reduction in DHT levels (P less than 0.001) levels, and a small but significant increase in testosterone levels (P less than 0.05) that were maintained over the 12-month period. Significant reduction in prostate volume was observed after 6 months of treatment and maintained at month 12 (P less than 0.05). In patients with baseline maximum flow rates less than or equal to 15 ml/second, maximum urinary flow significantly increased by a mean of 4 ml/second (P less than 0.01), with at least a 3 ml/second improvement observed in up to 70% of the patients at month 12. These results indicate that finasteride can reduce prostate size and improve maximum urinary flow with no loss of efficacy after 1 year of treatment. It is concluded that finasteride has the potential to be an effective chronic therapy for benign prostatic hyperplasia. PMID- 1722793 TI - Response of prostate volume, prostate-specific antigen, and testosterone to flutamide in men with benign prostatic hyperplasia. AB - Patients diagnosed as having benign prostatic hyperplasia (BPH) had determination of prostate volume (PV), prostate-specific antigen (PSA), and serum testosterone before consideration for entry into a double-blind, randomized trial of flutamide (750 mg/day for 6 months). The mean PSA level for these patients (N = 43) was 7.6 ng/ml (range: 1.0 to 45.7), and the mean PV was 76.8 cm3 (range: 24 to 198). Linear regression analysis demonstrated a strong correlation between the two (r = 0.876, P less than 0.05). Every 10 cm3 of prostate volume accounted for 1.02 ng/ml of PSA in the serum. Twenty-two patients (11 treated with flutamide, 11 with a placebo) agreed to enter the study. Prostate volume decreased by 35% and PSA by 65% (P less than 0.001) within 6 months. These changes occurred despite a 58.3% increase in serum testosterone levels (P less than 0.01). Patients treated with a placebo experienced no significant changes. Side effects were minimal, and flutamide was well tolerated. These data suggest that androgen deprivation therapy with flutamide may be an effective and safe treatment for BPH. PMID- 1722794 TI - LHRH agonists. A nonsurgical treatment for benign prostatic hyperplasia. AB - Luteinizing hormone-releasing hormone (LHRH) agonist, when administered in a continuous, nonpulsatile manner, causes desensitization of the LHRH receptor complex on the gonadotroph cells in the anterior pituitary gland. Biosynthesis and secretion of luteinizing hormone cease, and testicular androgenic production is inhibited. When used in this capacity, LHRH agonists can be an effective treatment for benign prostatic hyperplasia. After 4 to 6 months of therapy, prostatic volume decreases by 25% to 30%, voiding symptoms improve significantly in approximately 25% to 33% of patients, and the peak urinary flow rate increases substantially (more than 15 ml/second) in approximately 25% to 33% of patients. During the first month of treatment, serum luteinizing hormone, follicle stimulating hormone, testosterone, dihydrotestosterone, 17 beta-estradiol, and prostate-specific antigen decline to low values and remain low throughout treatment. Prostatic 5 alpha-reductase activity and androgen receptor content also decrease with treatment. Side effects are significant: impotence, decreased libido, and hot flushes are the most common. Because the effect of LHRH agonist therapy on the serum testosterone concentration is reversible, treatment of benign prostatic hyperplasia with an LHRH agonist must be considered life-long therapy. Thus, this therapy should be reserved for patients who are impotent or who are poor surgical risks. PMID- 1722795 TI - The emerging role of alpha antagonists in the therapy of benign prostatic hyperplasia. AB - The rationale for using alpha blockade to treat benign prostatic hyperplasia (BPH) is based on the physiology and pharmacology of prostate smooth muscle. Approximately 20% of the area density of the prostate adenoma is smooth muscle. In vitro isometric tension studies have demonstrated that the contractile properties of the human prostate adenoma are mediated primarily by alpha 1 adrenoceptors. Alpha blockers presumably decrease the resistance along the prostatic urethra by relaxing the smooth muscle component of the prostate. Over the past 14 years, at least 16 clinical trials have confirmed the efficacy of alpha blockade in the treatment of BPH. The primary advantage of terazosin over all other commercially available alpha blockers is that its longer half-life allows for a once-daily dosage regimen. Two Phase II studies conducted in the United States, a multicenter dose titration randomized withdrawal study and the author's personal experience with terazosin, are summarized in this report. Overall, the peak urinary flow rate increased 50% and the mean urinary flow rate increased 46% following terazosin therapy. The mean obstructive and irritative scores improved 67% and 35%, respectively. The adverse reactions occurring with an incidence greater than 5% included headache (10%), asthenia (7%), and dizziness (14%). All adverse events were reversible on termination of therapy. The preliminary experiences with alpha blockers for the treatment of BPH has been very encouraging. Yet, the definitive role of alpha blockade in BPH awaits the reporting of multicenter, randomized placebo-controlled studies. PMID- 1722796 TI - Rationale for using aromatase inhibitors to manage benign prostatic hyperplasia. Experimental studies. AB - Today, human benign prostatic hyperplasia (BPH) is considered primarily to be a disease of the stroma, in which estrogens are thought to play a considerable causative or permissive role. The growing incidence of BPH with increasing age coincides with a shift in the androgen:estrogen ratio in favor of estrogens, not only in terms of serum hormone values, but also in the prostate itself. Furthermore, evidence has been provided for a preferential accumulation of estrogens in the stroma of human hyperplastic tissue, and the presence of an estrogen receptor satisfying the classical criteria of high affinity and low capacity has been demonstrated. Also, animal studies have emphasized the potential role of estrogens in the pathogenesis of BPH. Experimentally, stimulation of the stroma, particularly of smooth muscle, can be induced by aromatizable substrates, such as androstenedione, in the prostates of beagles and cynomolgus monkeys. These effects can be antagonized by aromatase inhibitors, such as atamestane. In addition, the increase in intraprostatic estrogen concentrations and immunohistochemically detectable estrogen receptor content induced by androstenedione in intact dogs is completely reversed by simultaneous treatment with atamestane. In conclusion, clinical data, as well as that from animal models, emphasize an important role for estrogens in the development of BPH. Estrogen deprivation might, therefore, represent a useful treatment for human BPH. PMID- 1722797 TI - Atamestane, a new aromatase inhibitor for the management of benign prostatic hyperplasia. AB - Atamestane is a new, competitive, and irreversible inhibitor of estrogen biosynthesis. Its pharmacologic action has been evaluated in mice, rats, rabbits, dogs, monkeys, and humans. In rats, atamestane leads to a decrease of pregnant mare serum gonadotropin-stimulated ovarian estrogen production, and inhibits androstenedione-induced estrogenic effects such as uterine growth and abortion. In all species tested, atamestane lacks other intrinsic hormonal or antihormonal activities, and shows no inhibition of other cytochrome P450-dependent enzymes of adrenal steroidogenesis. However, it inhibits estrogen-related negative feedback. The extent and consequences of the induced counterregulation of the pituitary hypothalamic axis show major sex- and species-specific differences. Atamestane is highly effective in inhibiting estrogen-induced hyperplastic changes in the fibromuscular stroma of the prostate in androstenedione-treated dogs and monkeys. In male volunteers and patients with benign prostatic hyperplasia (BPH), atamestane induces an expected reduction of serum (and BPH tissue) estrogen concentrations without significant changes in androgen levels. In conclusion, all available results indicate that atamestane is a selective (no inhibition of adrenal function), pure (no endocrine side effects), and highly effective steroidal aromatase inhibitor, with an excellent safety profile. Based on the discussion of its clinical potential, atamestane seems to be a promising compound for the management of BPH. PMID- 1722798 TI - Transurethral incision of the prostate and bladder neck. AB - Transurethral incision of the prostate (TUIP) is compared to transurethral resection of the prostate (TURP) by reviewing nonrandomized, matched, and randomized studies. These studies indicate that incision of the prostate and bladder neck relieves outflow urinary obstruction, as does TURP. The incision is relatively easier to learn and perform, and requires shorter operative time compared to TURP. The incidence of retrograde ejaculation is lower after incision than after TURP--16% versus 63%, on average. Transurethral incision of the prostate has a potential for reduced costs due to reduced operative time, shortened hospital stay, and the potential for local anesthesia. PMID- 1722799 TI - A permanent, epithelializing stent for the treatment of benign prostatic hyperplasia. Preliminary results. AB - Currently, there is much enthusiasm in the urologic community for the development of alternative treatments to transurethral prostatectomy for management of benign prostatic hyperplasia (BPH). At the Mayo Clinic, the role of a permanently implanted intraurethral stent (UroLume Wallstent) is being examined. It is a biocompatible prosthesis made from a "super" alloy that is woven into a tubular mesh. It is both flexible and self-expanding with no elastic recoil; when fully expanded, it has a large internal diameter of 42 Fr (1.4 cm). Twelve patients (mean age = 67 years; range = 62 to 77 years) with obstructive BPH have been treated with this stent. After 3 months, the decrease in the total symptom score was 65% (mean preoperative score, 13.9 +/- 5.2; mean postoperative score, 4.8 +/- 3.7, P less than 0.001), whereas the increase in peak urinary flow rate was 99% (mean preoperative value, 10.1 +/- 3.3 ml/second; mean postoperative value, 20.1 +/- 6.2 ml/second; P less than 0.001). The postvoid residual urine volume decreased by 76% (mean preoperative value, 133 +/- 68 ml; mean postoperative value, 32 +/- 38 ml, P less than 0.001). There has been no difficulty with infection, encrustation, stent erosion, stent migration, incontinence, or potency. Eight patients (67%), however, did have irritative voiding symptoms after stent placement. These untoward effects subsided markedly during the follow up period. No patient has required either pain or antispasmodic medications, and as of this time, it has not been necessary to remove any stent because of side effects. These results suggest that the intraurethral stent may be a viable treatment option for patients with BPH.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722800 TI - Local microwave hyperthermia as a treatment alternative for benign prostatic hyperplasia. AB - It appears that the technology for local microwave application of heat to the prostate for the management of benign prostatic hyperplasia has arrived. There are a number of issues to be resolved in the coming years that will determine the role this modality will play in the overall management of men with benign prostatic hyperplasia. These issues include: transurethral versus transrectal route, hyperthermia (42 degrees C to 44 degrees C) versus thermotherapy (greater than 45 degrees C), and a proper assessment as to whether the technique is really efficacious, given the known placebo response in all studies currently available. The results with the transrectal route appear to improve patients' symptoms objectively and subjectively, without causing irreversible tissue effects. Thus, its action has been likened to alpha blockade. But, it appears that the transrectal approach is relatively inefficient because of a significant loss in microwave power with rectal cooling. A probe placed transurethrally can accurately and easily deliver the intended power to the center of the prostate, where theoretically it has its greatest effect on both the dynamic and static components of outlet obstruction. Currently, the transurethral devices described by Sapozink and Devonec will produce histologic necrosis. The theoretical value of combining urethral heating with cooling is that it will allow treatments of greater power deeper in the prostate adenoma, but the greatest advantage over transurethral heating without cooling may be in the ability to effect a response in a single session. Finally, the placebo response is a well known phenomenon seen in all drug trials conducted for the management of benign prostatic hyperplasia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722801 TI - Stretch-activated ion channels in guinea pig outer hair cells. AB - Two types of stretch-activated (SA) ion channels have been found in the lateral wall of isolated outer hair cells (OHC) from the guinea pig cochlea. One type had a reversal potential of -12 mV and was non-selective to cations, passing Ca2+ as well as monovalent ions. The channel had a conductance of 38-50 pS and the amplitude of the current through the open SA channel was independent of suction. The probability of the channel being open increased with applied suction and was voltage dependent with the maximum probability occurring at pipette potentials of -40 to -60 mV. The second type of SA channel had a conductance of approximately 150 pS and a reversal potential of approximately -50 mV. The ionic selectivity of this channel has not yet been determined, but it is probably K+ selective. OHCs have been shown to undergo a slow change in length in response to acoustic stimulation directed at the lateral wall of the OHC. The SA channels reported here could affect the motile response by altering the membrane potential or by allowing the entry of free Ca2+ which could lead to a change in OHC length through the interaction of actin and myosin. SA channels could also play an important role in regulating the osmotic pressure of OHC thereby influencing its electro-mechanical response. PMID- 1722802 TI - Outer membrane permeability of Acinetobacter calcoaceticus and its implication in antibiotic resistance. AB - In order to understand high and broad antibiotic resistance of Acinetobacter calcoaceticus, the outer membrane permeability was studied. The permeability coefficients of zwitterionic cephalosporins in the intact cell outer membrane were 0.14-1.12 x 10(-4) cm3/min/mg protein. These values were two to seven times lower than the permeability coefficients of the same beta-lactams in the outer membrane of Pseudomonas aeruginosa. The diffusion rates of carbapenems and zwitterionic cephalosporins into liposomes containing purified outer membrane appeared to be about 1-3%, that of the Escherichia coli B outer membrane. These results indicate that the outer membrane of A. calcoaceticus acts as a substantial barrier against the penetration of these antibiotics. About 80 times of the A. calcoaceticus outer membrane was needed in the liposome compared with E. coli B to show the same extent of saccharide permeability. Two minor outer membrane proteins (less than 5% of total outer membrane protein) were identified to be the porins. These results suggest that one of the causes for the high antibiotic resistance of A. calcoaceticus is attributable to the presence of a small number of small-sized porins. PMID- 1722803 TI - Genetic linkage of lung cancer-associated MspI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P450IA1 gene. AB - Individuals with high genetic risk of lung cancer had previously been identified by MspI polymorphisms of the cytochrome P450IA1 gene. In the present study we analyzed the structures of individual P450IA1 genes by PCR direct sequencing of genomic DNA of each genotype raised by the MspI polymorphisms, which were ascribed to a single point mutation in the 3'-flanking region. We then found a novel point mutation in the coding region of the gene which results in the substitution of Ile for Val at residue 462 in the heme binding region. We further analyzed the genetic association between this amino acid replacement and MspI polymorphisms in the general population, using a new method to detect polymorphisms not recognized by restriction enzymes. The results showed that there are at least two forms of human P450IA1 protein with different primary structures and that one of the forms is closely linked with the lung cancer susceptible genotype of MspI polymorphisms. Thus MspI polymorphisms, which are associated with increased risk of lung cancer, are linked to at least one amino acid substitution, which gives an important clue, at the molecular level, toward elucidation of increased susceptibility to lung cancer. PMID- 1722804 TI - Tamm-Horsfall-Protein excretion as a marker of ascending limb transport indicates early renal tubular damage in diabetes mellitus type I. AB - Tamm-Horsfall Protein (THP) is a 95 kD glycoprotein which is secreted in the thick ascending loop of Henle (TALH) of the kidney. After renal tubular damage the secretion of THP is reduced. In diabetes mellitus (DM), TALH has not been studied. To differentiate between glomerular (albumin), proximal tubular microglobulinuria (alpha 1-microglobulin), and TALH function (THP), we investigated 65 patients 4-61 years of age. In well-controlled DM, mean hemoglobin A1 equalled 7.4% and proximal tubular parameters indicated reversible damage early after onset. THP excretion (per 24 hrs or per day) was significantly elevated in DM duration of greater than ten years, suggesting enhanced TALH ion transport (glomerular hyperfiltration). THP secretion decreased in DM duration of greater than 15 years despite normal albumin excretion. Thus, renal THP excretion indicates early medullary dysfunction (TALH) in DM type I. PMID- 1722805 TI - Influence of glycemic control and hypertension on urinary microprotein excretion in non-insulin-dependent diabetes mellitus. AB - In order to evaluate the influence of glycemic control and hypertension on the development of diabetic nephropathy, we measured urinary excretion of albumin (AER) and other microproteins in non-insulin-dependent diabetes mellitus (NIDDM), and reexamined the 103 patients who had had AER less than 300 micrograms/min at the initial study 12-18 months later. AER in the patients with HbA1c greater than or equal to 7.5% increased significantly in both the normoalbuminuric (AER less than 30 micrograms/min) and microalbuminuric (30-300 micrograms/min) groups, whereas no significant change in AER was observed in the patients with HbA1c less than 7.5%. In the microalbuminuric group, AER in both hypertensive and normotensive patients increased significantly. In this group, the change in AER correlated positively with the change in alpha 1-microglobulin (alpha 1M). These results indicate that glycemic control has a greater influence on the development of nephropathy in its early stage than hypertension and that alpha 1M is as a good predictor of nephropathy as albumin. PMID- 1722806 TI - Expression of intermediate filament in endometrial glands changes with the onset of pregnancy and in endometriosis. AB - Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermediate filament proteins, cytokeratin and vimentin, in human endometrium and first trimester decidua and in ectopic endometrium from women with endometriosis. Stromal elements, including vascular endothelial cells, were consistently vimentin-positive and cytokeratin-negative. Surface and glandular epithelial cells of human endometrium co-expressed vimentin and cytokeratin during all stages of the menstrual cycle, but failed to express vimentin after the onset of pregnancy. This suggests that intermediate filaments, and especially vimentin, may have a role to play in the proliferation and/or differentiation of the endometrial glands during decidualization. Ectopic endometrium showed a staining pattern similar to normal endometrium. PMID- 1722807 TI - Characterisation of two new monoclonal antibodies directed against rat microglia. AB - With the aid of cultured rat microglial cells as immunogen, we raised two monoclonal antibodies, designated murine clone (MUC) 101 and 102, which recognised subsets of resident microglial cells in the normal central nervous system and cells of the mononuclear phagocyte system in peripheral organs. These antibodies were characterised by immunoperoxidase immunocytochemistry, immunoelectron microscopy, and immunoblotting. The immunostained cells were identified as microglial cells by double-immunofluorescence labelling with the B4 isolectin from Griffonia simplicifolia, an established microglial cell marker. Under normal conditions, both antibodies labeled resident microglia but with different distribution patterns. Under pathological conditions, e.g., after facial nerve transection, they labeled activated, perineuronal microglia in the operated facial nucleus. Immunoelectron microscopy demonstrated a membrane localisation of the antigen recognised by MUC 102. In peripheral organs, MUC 101 and 102 reacted with different cell populations of the mononuclear phagocyte system, particularly in thymus, spleen, and peripheral lymph node. Western blot experiments showed that MUC 101 recognised two proteins of 116 and 95 kD in fractions obtained from operated facial nucleus while MUC 102 reacted with two proteins of 62 and 70 kD molecular weight. These immunocytochemical results 1) confirm the antigenic similarity between microglia and cells of the monocyte macrophage cell lineage, and 2) indicate that considerable antigen heterogeneity might exist among resident microglia. MUC 101 and 102 could thus become useful for studying the function of microglial cells both under normal and pathological conditions. PMID- 1722808 TI - Ultrastructure and synaptic organization of axon terminals from brainstem structures to the mediodorsal thalamic nucleus of the rat. AB - The ultrastructural characteristics and synaptic organization of afferent terminals from the brainstem to the mediodorsal thalamic nucleus (MD) of the rat have been studied with the electron microscope, by means of anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Labeled fibers were seen predominantly in the lateral portion of MD after the injections of WGA-HRP into the substantia nigra pars reticulata (SNr), the superior colliculus (SC), and the dorsal tegmental region (DT). The boutons arising from the SC were relatively small (less than 1.5 microns in diameter), formed asymmetric synaptic contacts with small dendrites and dendritic spines, and contained round synaptic vesicles. The axon terminals from the DT were mostly large boutons (2-4.5 microns) with asymmetric synaptic specializations and round vesicles. These boutons and their postsynaptic targets formed synaptic glomeruli that were entirely or partially ensheathed by glial lamellae. The ultrastructural features are almost identical to those of boutons in the medial and central segments of MD that were previously shown to originate from the basal amygdaloid nucleus and the piriform cortex. The boutons from the SNr had a wide range in size, but the majority were medium-sized to large (1.5-4 microns). The nigral boutons established symmetric synaptic contacts with dendritic shafts and occasionally with somata, and contained pleomorphic vesicles. However, like the DT terminals, they participated in glomerular formations. The nigral terminals closely resemble previously described terminals in the medial part of MD from the ventral pallidum, except that the nigral terminals formed en passant and axosomatic synapses as well as axodendritic synapses. A combined immunohistochemistry and WGA-HRP tracing study revealed that the nigral inputs were immunoreactive for glutamic acid decarboxylase and the axon terminals from the DT were immunoreactive for choline acetyltransferase. In a separate study, the colliculothalamic fibers have been shown to take up and transport the transmitter specific tracer [3H]-D-aspartate, and are therefore putatively glutamatergic and/or aspartatergic. Taken together with this, the present results suggest that the collicular afferents are excitatory and glutamatergic and/or aspartatergic, that the inputs from the DT are also excitatory and cholinergic, while the nigral inputs are inhibitory and GABAergic. PMID- 1722809 TI - Densimetric determination of carbohydrate content in glycoproteins. AB - Carbohydrates play important roles in activity, stability and pharmacokinetics of glycoproteins and the degree of glycosylation varies with proteins. In this communication, a simple method of determining the carbohydrate content was developed, which consists of measuring the density increments of a glycoprotein and its non-glycosylated counterpart, and then dividing the difference between the two values by the density increment of carbohydrates. The density increment was relatively constant for various sugars except for sialic acid, and hence assumed to be 0.39. Thus, we obtained carbohydrate contents of 38, 28, 8 and 7% for Chinese hamster ovary cell-expressed erythropoietin (EPO), stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and platelet-derived growth factor (PDGF), respectively. These values are in close agreement with those determined by other methods. PMID- 1722810 TI - A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine. AB - A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide. PMID- 1722811 TI - Inhibitory effect of tranilast on substance P-induced plasma extravasation in rat skin. AB - The effect of tranilast, an inhibitor for IgE-mediated mediator release from mast cells, on plasma extravasation induced by the intradermal injection of substance P in rats was examined. Tranilast (100 mg/kg, intraperitoneally) decreased plasma extravasation induced by substance P (10(-7)-10(-5) M). Tranilast decreased plasma extravasation induced by the amino-terminal peptide substance P1-9 (10(-6) 10(-4) M), which is active for rat mast cells, but not by the carboxy-terminal peptide substance P6-11 (10(-6)-10(-4) M), which is inactive for the mast cells. Therefore, tranilast prevents substance P-induced plasma extravasation most likely by inhibiting mast cell degranulation. PMID- 1722812 TI - Differential stimulatory and inhibitory effects of interleukin 4 on granulocytic and monocytic colony formation in human bone marrow cultures. AB - Both stimulatory and inhibitory effects of interleukin (IL) 4 on myelopoiesis have been described. In this paper we further define the specificity of these effects. IL-4 was added to cultures of bone marrow cells in which colony formation was stimulated with several colony-stimulating factors (CSFs): monocyte CSF (M-CSF), granulocyte CSF (G-CSF), IL-3, IL-5 and conditioned medium from phytohemagglutinin-stimulated peripheral blood cells (PHA-CM). Inhibition of monocytic colony formation by IL-4 was observed in cultures that were stimulated with IL-3 or with PHA-CM, similar to the inhibition in M-CSF- or GM-CSF stimulated cultures. The enhancement of granulocytic colony formation by IL-4 was restricted to G-CSF-induced colony growth. No enhancement was observed in cultures that were stimulated with IL-5 or with PHA-CM from which the G-CSF was neutralized by anti-G-CSF antibodies. Both the inhibiting and enhancing effects of IL-4 were preserved in cultures that were stimulated with concentrations of CSF that exceeded more than tenfold the plateau concentrations. Enhancement of granulocytic and inhibition of monocytic colony formation by IL-4 occurred simultaneously in cultures stimulated with PHA-CM or with a combination of G-CSF and M-CSF. In summary, we show that the inhibiting effect of IL-4 on monocytic colony formation is independent of the growth factor used, whereas the stimulatory effect of IL-4 on granulocytic colony formation is restricted to G CSF-induced cultures. Simultaneous occurrence of both effects results in preferential growth of granulocytes. PMID- 1722813 TI - Clinical and hemodynamic results after Fontan operation. AB - Twenty-six patients, ranging in age from 2 to 14 years (mean, 6 years and 8 months), with tricuspid atresia or other complex cyanotic cardiac diseases underwent a modified Fontan operation between 1980 and 1990. In 13 patients, palliative operations had been previously performed. There were 7 mortalities within 1 month after the operation. Seventeen of the 19 survivors were followed up for 5-122 months (mean, 27) with no late deaths. Fifteen patients were in the New York Heart Association functional class 1. Two patients required reoperations at 2 and 57 months postoperatively: the former for atrio-ventricular valvular regurgitation, and the latter for late-developing conduit obstruction and residual atrial right to left shunting. Fourteen patients underwent cardiac catheterization after 1-57 months (mean, 9.8) postoperatively. Two patients had a residual right to left shunt at the atrial level, while the oxygen saturation in the remaining 12 patients was above 90%. Thus, the modified Fontan operation offers an alternate surgical approach for patients with complex congenital heart disease for whom no other palliative procedure can be expected to obtain significant improvement. It can be performed with good clinical and hemodynamic results in selected patients. PMID- 1722814 TI - Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry. AB - Thermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m1, m2, m3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 degrees C, respectively. After cooling to 5 degrees C and reheating, peaks denoted fr, mr and pr were observed at 23, 73 and 94 degrees C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 degrees C and extending to just above 40 degrees C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 degrees C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 degrees C and is characterized by peaks m1, m2 and n. Peak m3 at 75-76 degrees C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 degrees C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 degrees C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 degrees C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 degrees C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 degrees C caused by the remelting of ribosomal RNA. Peak pr at 94 degrees C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not. PMID- 1722815 TI - Regulation of levels of purine biosynthetic enzymes in Bacillus subtilis: effects of changing purine nucleotide pools. AB - The genes encoding the enzymes of IMP biosynthesis in Bacillus subtilis constitute the pur operon, whereas the genes encoding GMP biosynthetic enzymes, guaA (GMP synthetase) and guaB (IMP dehydrogenase), and the purA gene encoding adenylosuccinate (sAMP) synthetase all occur as single units. The purB gene encodes an enzyme involved in both IMP and AMP biosynthesis and is located in the pur operon. The levels of purine biosynthetic enzymes (except for GMP synthetase) were repressed in cells grown in the presence of purine compounds. Transcription of the pur operon is regulated negatively by adenine and guanine compounds. Our results suggest that ATP and guanine (or hypoxanthine) act as low molecular mass repressors. The level of IMP dehydrogenase was repressed by guanosine, but not in the presence of adenine, and was negatively correlated with the GTP/ATP pools ratio. The level of sAMP synthetase was repressed by adenine and increased by guanosine, and was positively correlated with the GTP/ATP pools ratio. It appears that the mode of regulating purine biosynthetic enzyme levels coincides with the cellular need for the individual enzymes. PMID- 1722816 TI - Acute phase reactants in neonatal bacterial infection. AB - The C-reactive protein (CRP) level was evaluated in 142 infants requiring investigation for suspected infection. After excluding two neonates because of incomplete data, there remained 140 neonates, of whom 16 had septicemia. Fifteen of 16 had increased CRP levels. The CRP value was not elevated in any baby (n = 5) who had positive blood cultures for Staphylococcus epidermidis, all of whom had an uneventful clinical course. The CRP level was elevated in all six babies with meconium-aspiration syndrome, but was normal in five infants whose viral cultures were positive. Ninety-nine percent of uninfected babies had normal CRP values. Overall, CRP was a valuable test for diagnostic confirmation of bacterial infection. Elevated CRP level was always accompanied by at least one abnormality in the other tests performed. Although the study was not intended to predict clinical onset of bacterial disease, our results suggest that the CRP level, because of a high negative predictive value, may be useful in ruling out bacterial infection. PMID- 1722817 TI - Distribution of type I phytochrome (phyA) RNA1 and RNA2 in etiolated pea seedlings. AB - Four-day-old etiolated pea seedlings were divided into 11 parts along the axis, from which poly(A)+ RNA and DNA were extracted. Using a slot-blot hybridization assay, the abundance of pea type I phytochrome (phyA) poly(A)+ RNA was measured in each portion of the etiolated pea seedling. For the quantification of the phyA RNA, pea phyA RNA synthesized in vitro was used as an RNA standard. The hook region contained the highest abundance of phyA RNA (approximately 0.3 ng phyA RNA per microgram DNA) in the etiolated seedling. Two mRNAs of different length (the shorter designated as RNA1 and the longer RNA2) are produced in detectable amounts from single pea phyA in the etiolated seedling; the ratio of the abundance of phyA RNA1 to phyA RNA2 was determined in each of the 11 parts by a primer extension assay. The abundance of phyA RNA1 in the plumule and hook regions was 3-5-fold higher than that of RNA2, whereas the ratio of their abundance was approximately unity in other regions. A time course study of the abundance of both RNAs was carried out during the imbibition of seeds and indicated that the accumulation of phyA RNA1 occurred more rapidly in the cotyledons than that of RNA2 during the first day of imbibition, whereas the accumulation of phyA RNA2 increased rapidly during the second day and became as high as that of phyA RNA1 by the third day. PMID- 1722818 TI - Expression of early genes in light-induced chloroplast differentiation of cultured plant cells. AB - The objective of this study was to identify genes, preferentially of the plastid, which are rapidly expressed during the initial phase of blue-light-induced chloroplast development in suspension-cultured cells (Chenopodium rubrum L.) and to analyse the encoded proteins. A cDNA library (lambda gt 10) was constructed using total RNA from plastid preparations of dark-grown cells exposed to blue light for 3, 6 and 12 h. By differential screening, at least three clones were identified which correspond to rapidly light-induced plastid genes. For these and a number of nuclear genes represented by other clones, a temporary accumulation of the specific mRNA was observed between 12 and 48 h of blue-light exposure. With regard to their nucleotide sequence and derived amino-acid sequence they seem to represent a novel group of genes distinctive in structure and encoded product. PMID- 1722819 TI - Antagonistic blue- and red-light regulation of cab-gene expression during photosynthetic adaptation in Scenedesmus obliquus. AB - During adaptation of the photosynthetic apparatus of the green alga Scenedesmus obliquus to various light qualities, the accumulation of chlorophylls and pigment protein complexes (with specific consideration of chlorophyll a/b-binding (Cab) proteins) and cab-gene expression were determined. The fluence rate dependences for chlorophyll accumulation and cab-gene expression were very different. Very low fluence rates of violet (404 nm), blue (461 nm) and red (650 nm) light below the photosynthetic threshold, i.e. between 10(-3) and 10(-1) mumol m-2 s-1, inhibited all of these reactions in cells grown under heterotrophic conditions. At elevated fluence rates (above 1 mumol m-2 s-1), red light retained its negative regulation, whereas blue light stimulated pigment accumulation. Under autotrophic conditions the pattern was more complex, because chlorophyll accumulation was unaffected by light below the photosynthetic threshold. However, the expression of cab-genes was inhibited by red light but stimulated by blue light. Cells adapted to fluence rates, which ensured photosynthetic energy supply (above 1 mumol m-2 s-1), showed an increase in chlorophyll accumulation, blue light being more effective than red light. The results confirm and extend our previous discovery of two antagonistically acting photoreceptors in Scenedesmus which mediate and coordinate the complex functional and structural changes associated with photosynthetic adaptation. One of these receptor pigments is a blue-light receptor with positive action; the other is a violet-red-light receptor which can operate far below the photosynthetic threshold and exerts a negative regulation. PMID- 1722820 TI - Kinetic properties of the glycine receptor main- and sub-conductance states of mouse spinal cord neurones in culture. AB - 1. The kinetic properties of the two most frequent conductance states of glycine receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using the outside-out patch clamp technique. At low concentrations of glycine (0.5, 1 and 2 microM), single-channel currents were recorded with two predominant amplitudes corresponding to a dominant or main-conductance state of about 42 pS and a sub-conductance state of about 27 pS. Both conductance states opened singly and in bursts of several openings. Total current evoked and single channel opening frequency increased as glycine concentration was increased from 0.5 to 2 microM. 2. For both conductance states mean open times were increased and open time frequency histograms were shifted to longer times as glycine concentration was increased from 0.5 to 2 microM. For both conductance states, three exponential components were required to fit best open time frequency distribution histograms at all glycine concentrations (0.5, 1 and 2 microM). The time constants of the exponential components for each conductance state were not significantly different across concentration, suggesting that the main- and sub conductance states of the channel each opened into at least three open states. For the main-conductance state, the time constants were 1.09 +/- 0.09, 4.06 +/- 0.26 and 9.79 +/- 0.30 ms. For the sub-conductance state, the time constants were 0.55 +/- 0.04, 2.64 +/- 0.35 and 8.57 +/- 1.08 ms. The increase in long open times with concentration was due primarily to a shift in relative frequency of occurrence of openings from the shortest to the two longest open states. 3. Closed time frequency distributions of closures between main-conductance state openings, closures between sub-conductance state openings and closures between both main- and sub-conductance state openings were fitted with multiple exponential components, suggesting that the channel had several closed states. The two shortest time constants (0.16 +/- 0.01 and 1.26 +/- 0.13 ms) did not vary significantly with concentration (0.5-2 microM) or method of analysis. The longer time constant varied with concentration. 4. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time. For both conductances states, mean burst durations were increased and burst duration frequency histograms were shifted to longer times as glycine concentration was increased from 0.5 to 2 miroM. Burst duration frequency histograms contained four exponential components for the main-conductance state and three exponential components for the sub-conductance state.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722822 TI - Videodisks help patients choose therapy. PMID- 1722821 TI - Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium. AB - 1. Intracellular voltage recordings using conventional and double-barrelled chloride-selective microelectrodes have been used to identify several transport mechanisms at the apical and basolateral membranes of the isolated bovine retinal pigment epithelium (RPE)-choroid preparation. Intracellular recordings were obtained from two cell populations, melanotic (pigmented) and amelanotic (non pigmented). The electrical properties of these two populations are practically identical. For melanotic cells the average apical resting membrane potential (VA) is -61 +/- 2 mV (mean +/- S.E.M., n = 49 cells, thirty-three eyes). For these cells the ratio of apical to basolateral membrane resistance (a) was 0.22 +/- 0.02. The mean transepithelial voltage and resistance were 6 +/- 1 mV and 138 +/- 7 omega cm2, respectively. 2. The apical membrane, which faces the distal retina, contains a Ba(2+)-inhibitable K+ conductance and a ouabain-inhibitable, electrogenic Na(+)-K+ pump. In addition it contains a bumetanide-sensitive mechanism, the putative Na(+)-K(+)-Cl- cotransporter. The basolateral membrane contains a DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid)-inhibitable chloride channel. The relative conductances of the apical and basolateral membranes to K+ and Cl- are TK approximately 0.9 and TCl approximately 0.7, respectively. 3. The ouabain-induced fast phase of apical membrane depolarization (0-30 s) was used to calculate the equivalent resistances of the apical (RA) and basolateral (RB) cell membranes, as well as the paracellular or shunt resistance (RS). They are: 3190 +/- 400, 17920 +/- 2730 and 2550 +/- 200 omega (mean +/- S.E.M., n = 9 tissues), respectively. From these data the equivalent electromotive forces (EMF) at the apical (EA) and basolateral (EB) membranes were also calculated. They are: -69 +/- 5.0 and -24 +/- 5.0 mV, respectively. 4. Intracellular Cl- activity (aiCl) was measured using double-barreled ion selective microelectrodes. In the steady state aiCl = 61 +/- 4.0 mM and the Nernst potential ECl = -13.5 +/- 1.5 mV (mean +/- S.E.M., n = 4). 5. In the intact eye or in retina, RPE-choroid preparations it has been shown that the transition between light and dark alters the K+ concentration in the extracellular (or subretinal) space between the photoreceptors and the apical membrane of the RPE. These light-induced changes in subretinal [K+]o were qualitatively simulated in vitro by altering apical K+ between 5 and 2 mM. This produced a sequence of voltage changes at the apical and basolateral membranes that had three operationally distinct phases. Phase 1 is generated by the combination of an apical membrane K+ diffusion potential and inhibition of the electrogenic Na(+)-K+ pump.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722823 TI - Optimism expressed over prostate drug. PMID- 1722824 TI - [Diagnosis of arrhythmia by Holter ECG]. PMID- 1722825 TI - [Arrhythmia--progress in diagnostic methods--body surface mapping]. PMID- 1722826 TI - [Supraventricular premature contraction]. PMID- 1722827 TI - [Premature ventricular contraction]. PMID- 1722828 TI - [Parasystole]. PMID- 1722829 TI - [The effects and pharmacokinetics of rhG-CSF on the treatment of neutropenia in patients with renal failure]. AB - rhG-CSF (recombinant human granulocyte colony stimulating factor) promotes production and release of neutrophil from bone marrow, and it enhances neutrophil function. In this study, the pharmacokinetics, effects on neutrophil and immune functions and efficacy and safety of rhG-CSF were studied in patients with end stage renal failure (CRF). To 9 patients with CRF; 2 patients on conservative therapy and 7 patients under regular hemodialysis, 50 micrograms/m2 rhG-CSF were administered intravenously under the schedule of single or 2 week consecutive injection. In single injection study, serial changes in plasma rhG-CSF concentration and peripheral blood cell count were examined following the administration. In consecutive injection study, plasma rhG-CSF concentration, anti-rhG-CSF antibody, peripheral blood cell counts, blood chemistry and coagulation factors, and neutrophil and immune functions were examined. As the results, 1) Half life of rhG-CSF, 2.87 +/- 0.65 hr, was about 2 times longer than that in healthy subjects, and it was not affected by hemodialysis treatment. 2) Marked increase in leukocyte and neutrophil counts and mild increase in lymphocyte count were observed during single and consecutive administration of rhG-CSF. There was no significant change in other leukocyte differentiations, RBC, or platelet count. 3) Neutrophil alkaline phosphatase score increased significantly during single and consecutive administration, and other neutrophil function also improved in several patients with impaired neutrophil function. 4) Slight bone pain and increase in serum alkaline phosphatase were observed in about a half of patients during consecutive injection study. Neither antibody nor accumulation of rhG-CSF was noted.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722830 TI - [Infection of hepatitis C virus in patients with chronic renal failure undergoing hemodialysis therapy and staff members]. AB - The prevalence of antibody to hepatitis C virus (anti-HCV) was determined in 564 patients and 145 staff members of nine hemodialysis (HD) units in Nagano Prefecture using an enzyme-linked immunosorbent assay based on the C 100 HCV antigen (the first generation anti-HCV assay). And also serum HBV markers were tested in these subjects. One hundred patients (18%) were anti-C100 HCV positive, indicating that this figure represents a much higher prevalence than that (0.9%) among general population in the same geographical area. Out of 141 patients without history of blood transfusion, 17 (12%) were positive for anti-C 100 HCV, suggesting that blood-transfusions-unrelated acquisition of HCV infection can occur. Anti-HCV prevalence correlated with both the blood units transfused and the duration of HD treatment. There was a significant difference in the prevalence of anti-C 100 HCV in individual dialysis units ranging from 0% to 53%. In the dialysis unit with prevalence of 53%, approximately half of the anti-HCV positive patients were found to have chronic liver disease. The prevalence of hepatitis B virus (HBV) markers among HD patients, on the other hand, was 36% (202/564). Fifty one (51%) of 100 anti-C 100 HCV positive patients and 151 (33%) of 464 anti-C 100 HCV negative patients were positive for HBV markers, with significant difference in HBV infection rate between the 2 groups. The prevalence of chronic liver disease, defined as abnormal serum transaminase levels for more than 6 months was significantly higher in anti-HCV positive patients than in anti HCV negative ones (39% vs 10%, p less than 0.05), suggesting that HCV infection may contribute to chronic liver disease in HD patients. Among 145 staff members, only 3 (2%) were positive for anti-HCV, whereas 25 (17%) were positive for hepatitis B core antibody (anti-HBc), indicating prior HBV infection. With applying the second generation anti-HCV assay, which can detect antibodies to both capsid and nonstructural products of HCV gene, anti-HCV prevalence increased by two times in HD patients, but didn't change in HD staff members.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722831 TI - [Inhibitory effects of rhIL-1 beta pretreatment on bleomycin-induced pneumonitis in mice]. AB - We studied the effects of recombinant human interleukin-1 beta (rhIL-1 beta) pretreatment on bleomycin (BLM)-induced pneumonitis in mice. Lung injury was dose dependently induced by BLM (50 mg/kg, 100 mg/kg, 150 mg/kg i.v.). The mice were pretreated with IL-1 (1 microgram/mouse i.p.) at 0.5, 6, 12 or 24 hours before the administration of BLM. Wet lung weight, lung weight-to-body weight ratio and bronchoalveolar lavage cell findings were analyzed with respect to time, and lung specimens on day 28 after administration of BLM were histopathologically examined. When mice were pretreated with IL-1 at 0.5 hr or 6 hr before the administration of BLM, changes in all the parameters were significantly suppressed. The results indicate that IL-1 pretreatment protects mice from BLM induced pneumonitis and its effects are time-dependent. PMID- 1722832 TI - [Autosomal dominant (adult type) polycystic kidney disease]. PMID- 1722833 TI - [VAB-6 chemotherapy of advanced testicular cancers--its efficacy and limitation]. AB - Fifteen patients with advanced testicular germ cell tumor were treated by VAB-6 chemotherapy after radical orchiectomy. CR was achieved in two (13.3%) and PR in nine (60.0%). The one, three and five year survival rates were 92.2, 64.2 and 51.4%, respectively. Nine patients (60.0%) remain with no evidence of disease (NED) after combined therapy (chemotherapy and surgery) with a median follow up of 4.3 months. We performed resection of the residual tumors and examined histologically. Out of 11 cases, four (36.3%) were with necrosis/fibrosis, two (18.2%) teratoma and five (45.5%) cancer. All of the six cases with non-cancerous tissues remain in NED. However, only two of the five cases with cancerous tissues remain in NED. We studied the NED rate based on four prognostic factors, (1) bulky abdominal diseases, (2) advanced lung diseases, (3) other metasteses, and (4) choriocarcinoma. In our analysis, advanced lung diseases and choriocarcinoma were poor prognostic factors. We should treat these poor prognostic cases initially with VP-16, ifosfamide, high doses cisplatin etc, which were used as salvaged drugs. PMID- 1722834 TI - [Treatment of newly diagnosed stage D2 prostatic carcinoma with hormonal therapy alone, or chemotherapy agents in combination with hormones]. AB - From June 1984 to March 1988, patients with newly diagnosed stage D2 prostate cancer were treated with protocol 1. This comprised oral hormonal agents either diethylstilbestrol diphosphate (Honvan: 300 mg/day) or estramustine phosphate (Estracyt: 560 mg/day), or chlormadinone acetate (Prostal: 100 mg/day), plus intravenous cyclophosphamide (CPM, 0.5-1 g/m2) every 3-4 weeks. From May 1988, protocol 2 was used in a randomized study of castration alone versus castration plus intravenous methotrexate (MTX, 20 mg/m2) every 2 weeks. Forty-nine of 53 patients who underwent the two protocols were evaluable for the response. The response rates according to the NPCP criteria were 92% (11/12) for Honvan, 100% (9/9) for Estracyt, 78% (7/9) for Prostal and castration plus MTX, and 80% (8/10) for castration alone. There were no significant differences among these treatments. The median response duration and survival time (months) were 16 and 44, respectively, for Honvan, 19 and 37 for Estracyt, 12 and 43 for Prostal, 11 and 15 for castration plus MTX, and 13 and 13 for castration alone. The short survival times of the castration alone and castration plus MTX groups were due to a short follow-up period. There were no statistical differences among the oral hormonal agent plus CPM groups. However, the 2-year survival rate (Kaplan-Meier method) was higher in the CPM and MTX groups than in the castration alone group. Survival was longer in the good performance status (P.S.) group than the poor P.S. group (p less than 0.05 by Wilcoxon test) and in the responders than the non responders (p less than 0.01). Side effects were not excessive in the chemotherapy groups and patient compliance was good. PMID- 1722836 TI - [Pulmonary thromboembolism]. PMID- 1722835 TI - [Biologically active helevin-based compositions in the treatment of pleural empyema]. AB - The article analyses the method of topical open treatment of postoperative pyothorax in 17 patients with the use of biologically active helevin-based compositions. The control group was made up of 11 patients with a similar suppurative process who received topical treatment by the same method but with the use of ointments with antibiotics and antiseptics on a fat base. With the use of biologically active compositions (BAC) on a helevin-base, the empyema cavity was completely freed from the pyonecrotic masses and its walls filled with granulation tissue on day 7 of treatment. In the control group the cavity was completely cleansed only on day 19 of treatment. It is shown that BAC possesses a high antibacterial effect not only due to the antibacterial components but because it is based on the laws of physical antiseptics. PMID- 1722837 TI - [Determination of histamine and serotonin in whole blood using the method of high performance liquid chromatography]. AB - The authors have developed a method for measuring histamine and serotonin in whole blood, based on reverse-phase high-performance liquid chromatography of 5 dimethylaminonaphthyl sulfonic (DNS) amine derivatives with fluorometric detection. Chromatography in a mini-column packed with polyamide was employed for the isolation and purification of DNS-histamine and serotonin. The lowest detectable level of histamine is 2-2.5 ng/ml of blood, that of serotonin 1-1.5 ng/ml. The method was tried with whole blood samples from children suffering from various allergic conditions. Whole blood histamine and serotonin concentration were found particularly elevated in the patients with gastrointestinal allergies. PMID- 1722838 TI - [Chemiluminescence in the peroxidase oxidation of luminol with hydrogen peroxide in various media]. AB - The effects of culture media of various compositions on chemiluminescence developing in peroxidase oxidation of luminol with hydrogen peroxide were under study. The findings evidence that the presence of carbonate and bicarbonate ions in the medium results in a two-staged chemiluminescence kinetics and in more intensive chemiluminescence in the peroxidase-luminol-hydrogen peroxide system. This fact has brought the authors to a conclusion that carbonate and bicarbonate containing media are more effective for the detection of low peroxidase concentrations by the chemiluminescence technique. PMID- 1722839 TI - [The concentration of glucose in the amniotic fluid in a high risk pregnancy group]. AB - Amniotic fluid glucose levels were measured in 125 pregnant women included in high-risk group in respect of perinatal pathology. In the reference group the measurements have demonstrated an agreement between glucose concentrations in the amniotic fluid and gestation term and an inverse relationship between pregnancy progress and glucose level, whereas in diabetics this relationship was direct. Fetal developmental defects were associated with elevated glucose content in the amniotic fluid, concomitant placental abnormalities were conducive to reduction of these levels, and no correlations between this parameter and pregnancy terms were observed. PMID- 1722840 TI - [Carbohydrate metabolism enzymes in children with extrahepatic portal hypertension]. AB - Glycogen and protein concentrations and the activities of liver glycogen metabolic enzymes were measured in 22 children aged 4 to 15, suffering from extrahepatic portal hypertension. Glucose-6-phosphatase, amylo-1,6-glucosidase, fructose-1,6-diphosphatase, phosphorylases alpha and beta, phosphoglucomutase, and phosphohexose isomerase levels were analyzed. Liver biopsy specimens obtained by surgical marginal biopsy were used in the study. No or drastic reduction of phosphorylase alpha activity and reduction of glycogen concentration and glucose phosphatase activity were found characteristic of extrahepatic hypertension. Analysis of correlations of the findings has demonstrated a medium correlation in 4 cases and a strong correlation between the findings in 1 case, the possibility being estimated as 0.95-0.99. The highest number of correlations was observed with phosphorylase alpha and glucose-6-phosphatase (3 correlations). Liver blood stream impairments result in injury to one of its main biochemical functions, i.e., the maintenance of blood glucose homeostasis, this leading to reduction of the adaptation potential of the body; this should be borne in mind when planning therapeutic measures for patients with extrahepatic hypertension. PMID- 1722841 TI - [Bioluminescent determination of NAD- and NADP-dependent lymphocytic glutamate dehydrogenases]. AB - A sensitive bioluminescent rapid method for estimating the activity of pyridine nucleotide-(phosphate)-dependent glutamate dehydrogenases (NAD(P)-GDH) of human peripheral blood lymphocytes is suggested. 10000 cells is sufficient per analysis, up to 100 measurements may be made within 3 hrs. Normal subjects and subjects often suffering from acute respiratory diseases were examined for the aforesaid parameter. The findings evidence the diagnostic significance of NAD(P) GDH for the detection of subjects at risk of developing acute respiratory diseases. PMID- 1722842 TI - [Determination of plasminogen in blood using the method of inhibitor type immunoenzyme analysis]. PMID- 1722843 TI - [Determination of the enzyme activity of the fibrinolytic system using fibrinogen conjugated with peroxidase]. AB - The authors suggest a method for enzymic fibrinolytic activity measurements, based on the detection in the solution of degradation products of fibrin, prelabeled with peroxidase. The method is highly sensitive, permits the detection in solution of as little as 0.01 MU per ml of plasminogen activator. As the basic method, it can be employed for measurements of plasminogen activator, plasminogen activator inhibitors, and plasmin inhibitors in biologic fluids, blood plasma including. Since the method is simple and the majority of its steps may be automated, it can be used in clinical laboratories. PMID- 1722844 TI - [The surface charge of erythrocytes in children with chronic hepatitis]. AB - Surface charge of the peripheral blood red cells was studied with the device for cellular phoresis in 36 schoolchildren suffering from chronic hepatitis and 20 healthy children. The surface charge was found regularly reduced when the disease exacerbated, this permitting a judgement on the activity of the hepatic process. The developed method for rapid diagnosis of chronic hepatitis is recommended to be used during the outpatient stage of examinations and for the assessment of membranotropic therapy efficacy. PMID- 1722845 TI - [Monitoring the chemiluminescence of leukocytes in suppurative surgical infections]. AB - The authors suggest that analysis of chemiluminescence of patient's blood leukocytes be used for monitoring the status of surgical patients. Chemiluminescence value on the 15th min starting from the onset of phagocytosis may reflect the neutrophil activation. Monitoring of the status of patients with purulent surgical diseases has shown that a sluggish course of the inflammatory processes is related to inadequate functional activity of the leukocytes, and temporary activation of the neutrophils is recorded in 83% of cases when the patient's status improves: either initially in case of an acute course, or in late periods if the disease course is protracted. PMID- 1722846 TI - [Detection of the oligoclonal fractions of immunoglobulins in the cerebrospinal fluid and its significance]. PMID- 1722847 TI - [Isoforms of creatine kinase isoenzymes in the blood in myocardial infarct (review of the literature)]. PMID- 1722848 TI - [Evaluation of the alternative approach to complement activation using C3 dependent adhesion]. AB - The authors suggest a method of functional probing of the complement alternative cascade, the jist of it being analysis of the neutrophil adhesion to sephadex granules treated with serum or another complement-containing substrate. The advantages of the method are analyzed, and the range of its application suggested. PMID- 1722849 TI - [Erythrocytic immunoreagents for diagnosing autoimmune disorders of the pancreas]. AB - Amidol sensitization of formalin-treated sheep red blood cells was found to be the optimal method for the preparation of an erythrocytic diagnostic agent from the summary antigen of the pancreas. Organ specificity of serum antibodies detection with the use of the developed diagnostic agent was provided by preliminary adsorption of the tested sera with rhesus-positive human red cells, group IV (AB). Use of this diagnostic agent in the neutralization test permitted a highly specific detection of the pancreatic antigen. Antigen-specific lymphocytes detectable in the indirect rosette-formation test with the erythrocytic diagnostic agent from the pancreatic summary antigen, based on chicken red cells were found highly specific. PMID- 1722850 TI - [The informativeness of immunoenzyme analysis in cancer of the gastrointestinal tract]. AB - Carcinoma-associated antigen (CA) 19-9, carcinofetal and mucinoid antigens are the optimal combination of markers for the diagnosis and monitoring of the course of gastric, colonic, and rectal carcinomas. CA-125 and alpha-fetal protein antigen are but fourth-stage markers for carcinomas of these sites. Neuron specific antigen may be used for the differential diagnosis of pancreatic diseases. The findings evidence no strict specificity of the markers used in the study. PMID- 1722851 TI - [The spleno-cytotoxic test--an indicator of toxemia in oncologic patients]. AB - Biologic integral methods for indication of blood toxicity, the migration activity of intact donor leukocytes in agar, and the splenocytotoxic test were used in radiotoxemia assessment in 62 oncologic patients with radiation reactions and in studies on the efficacy of detoxifying therapy with polyvisoline, a new blood substitute, in 25 subjects. Both the methods for studies of the blood serum toxicity were found sufficiently informative and unsophisticated, but the splenocytotoxic test proved to be more sensitive, simple, and rapid. PMID- 1722852 TI - [The use of the serum reference sample containing the antinuclear factor (homogeneous) to determine the working dilution of diagnostic fluorescing immunoglobulins against human IgG(H)]. AB - A previously developed serum reference sample containing the antinuclear factor (homogeneous) in a standard dilution 1:40 was used to estimate the working dilution of commercial lots of diagnostic fluorescing immunoglobulins against human IgG (H) (IGF). Use of a working dilution of IGF in the diagnostic titer helps optimize the immunofluorescence test and save IGF. PMID- 1722853 TI - [The use of sectional polystyrene plates in the set-up of lanthanide immunofluorescence analysis]. AB - The authors have examined the possibility of using sectional polystyrene plates, made in this country, in time-resolved fluoroimmunoassay (tr-FIA) with Venezuelan equine encephalomyelitis and tick-borne encephalitis arboviruses, and with influenza A virus. The plates presensitized with specific antibodies were found fit for the detection of the antigens of the above viruses. These plates are not recommended for the detection of influenza A virus-specific proteins adsorbed directly onto the microplate surface. PMID- 1722854 TI - [An improvement in the accuracy of determining the titer of staphylococcal alpha antitoxin]. AB - The modification of the titration technique involves addition (in microvolumes) of various solvent quantities to equal blood serum volumes, followed by levelling of liquid volumes, and then addition of equal toxin doses. Serum dilutions: No. 1 - 1: 0.5 of toxin working dose, etc., provide a registration scale with 1 U interval. This modification makes the method more accurate and simple. PMID- 1722855 TI - [DNAase activity, a method of differentiating hospital strains of Salmonella typhimurium]. AB - Measurements of DNAse activities in Salmonella typhimurium strains of various origins have demonstrated that hospital strains are characterized by the highest DNAse activity. This activity correlates with the presence of R-plasmids in the cells. High DNAse activity of hospital strains is a stable marker, and therefore may be used for intraspecies differentiation of S. typhimurium. PMID- 1722856 TI - [Preparation of dried semiproduct in the manufacture of medicinal forms of bacteriophages]. AB - The authors have found the optimal regimen for dehydration by spraying the Staphylococcus and Shigella bacteriophage semiproducts. The semiproduct quality answers the requirements to such reagents. The spraying method is recommended for bacteriophage preservation in preparing some dosage forms (suppositoria, ointments). PMID- 1722857 TI - [A micromethod of determining the sensitivity of anaerobic bacteria to antibacterial preparations]. AB - An economic rapid 'cassette' micromethod for estimating the sensitivity of obligate anaerobes to antibiotics has been developed, applicable at clinical laboratories. The method permits simultaneous testing of a microorganism with a wide range of antibiotics and assessment of the minimal inhibiting concentration of the agent, making use of the minimized volume of the inoculum. The technique has been tried with 176 anaerobe strains isolated from maxillofacial purulent foci from 76 patients; sensitivities to 42 antibiotics and antiseptics have been tested. Parallel inoculations were carried out, as was a comparative study with the traditional serial dilutions in solid nutrient medium. The suggested method is available and rapid; the results are ready within 1-3 days. PMID- 1722858 TI - [An immunoenzyme test system for detecting rickettsiae of the tick-borne spotted fever group]. AB - An enzyme immunoassay (EIA) test system has been developed for the detection of tick spotty fever (TSF) group Rickettsia. A long scheme of immunization in an isogenic system on rabbits has been used with an adjuvant without bacterial components, and based on aluminum hydroxide. This EIA test system detects whole soluble R. akari, R. sibirica, and R. conorii antigens and is at least 10-20 times more sensitive than the indirect hemagglutination test. Treatment in a temperature gradient permits the detection of the corpuscular antigen. A considerable vector infection rate has been revealed in individual examinations of ticks in foci of tick rickettsiasis, whereas such studies carried out in nonendemic areas brought negative results. PMID- 1722859 TI - [Preparation of an erythrocytic diagnostic agent based on staphylococcal proteinase]. AB - Various methods for sheep red cell sensitization for the preparation of erythrocytic diagnostic agent from staphylococcal proteinase were under study. Sensitization with chromium chloride yielded the best results as regarded the sensitivity of the agent, optimal sensitizing dose, and sensitin consumption. The resultant erythrocytic diagnostic agent retained about 10% of the initial enzymic solution activity. The authors have demonstrated a high specificity of the erythrocytic diagnostic agent from staphylococcal proteinase, that may be used for the serologic diagnosis of staphylococcal infection and for proteinase indication in various staphylococcal preparations. PMID- 1722860 TI - [The International Federation of Clinical Chemistry (IFCC). Methods of the IFCC for measuring the catalytic concentration of enzymes]. PMID- 1722861 TI - Pristanic acid and phytanic acid in plasma from patients with a single peroxisomal enzyme deficiency. PMID- 1722862 TI - [A meta-analysis of controlled studies of the treatment of chronic hepatitis due to the hepatitis B virus]. AB - BACKGROUND: Since the results obtained in controlled studies of antiviral treatment of chronic hepatitis by the hepatitis B virus are frequently insignificant the authors have undertaken a combined study of the different controlled trials with the meta-analysis technique. METHODS: The results of 7 controlled trials of treatment with adenosin arabinoside (AAR) and 18 published studies on therapy of a control group with interferon were studied. Meta-analysis was performed by two methods: Mantel expressing the results in odds ratio (OR) and Cochram-Dersimonian-Laird which consider the results in differences of risk (RD). RESULTS: The meta-analysis of AAR treatment shows a mean difference of response of 11% with no homogeneity among the different studies published. This lack of homogeneity may be due to the different selection criteria of the patients which determine very variable degrees of response. The controlled studies with interferon showed a mean difference of response of 20% with homogeneity of effects. No differences were observed in the degrees of response according to whether recombinant lymphoblastoid interferon was used or if the dose was of 5 or 10 megaunits per day. Non were differences observed in degrees of response with respect to the time in which the loss of viral DNA was evaluated. CONCLUSIONS: Following analysis of the results it was concluded that interferon is clearly effective in the treatment of chronic hepatitis B and based on published studies adenosin arabinoside demonstrate less efficacy. PMID- 1722863 TI - [Interferon in chronic hepatitis]. PMID- 1722864 TI - Histochemical staining of nerve endings as an aid to free muscle transplantation. AB - Histochemical staining techniques that identify intact motor nerve fascicles are available to aid free muscle transplantation. Cholinesterase activity of myelinated axons can be identified by Karnovsky and Roots's technique. Axon viability can be assessed based on the presence of axoplasmic enzyme activity. By reacting serial sections for cholinesterase activity and carbonic anhydrase activity, which labels sensory axons, an accurate cross-sectional map of regenerating or functional sensory and motor nerve fibers can be constructed. Resolving the motor and sensory identities of fascicles in a mixed peripheral nerve should lead to more precise coaptation of recipient motor fibers to the motor nerve of the transferred muscle and enhance reinnervation. PMID- 1722865 TI - Renal magnesium wasting associated with therapeutic agents. AB - Renal magnesium wasting is an important cause of hypomagnesemia observed in hospitalized patients. The purpose of this review is to present an index case of symptomatic hypomagnesemia associated with renal magnesium wasting during capreomycin therapy, and to survey the available literature regarding the various therapeutic agents associated with the causation of this syndrome. Finally, we have considered the pathophysiologic mechanisms that may contribute to the development of the multiple electrolyte abnormalities observed in these patients, and have outlined the current strategies to treat this syndrome. PMID- 1722866 TI - [HCV infection among cirrhotic patients as a risk factor for generation of hepatocellular carcinoma: preliminary report]. PMID- 1722867 TI - RNA-mediated transfer of cellular immunity to a synthetic env antigen of the human immunodeficiency virus (HIV-1). AB - A sheep was immunized with a synthetic peptide corresponding to amino acid residues 586-606 of the precursor envelope protein GP-160 of the HIV-1 including a conserved epitope region of the GP-41 transmembrane protein in the mature viral particles, referred to as SM 284 HIV-1 [1]. It is demonstrated that immune RNA extracted from the lymphoid organs of the immunized animal (SM 284 HIV-1 I-RNA) was able to transfer immune cellular reactivity to SM 284 HIV-1 in vitro to human and rabbit lymphocytes and in vivo to Cebus apella monkeys. The transfer was detected by the leukocyte adherence inhibition test (LAI) as an indicator of cellular reactivity. One of the most relevant results was the demonstration that SM 284 HIV-1 I-RNA was able to induce cellular immunological memory in vivo in monkeys. These results may be relevant to delineate a new alternative for immunomodulation against HIV infection. PMID- 1722868 TI - Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes. AB - The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the beta subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of 32P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the beta-subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn2+ (Ka = 1.0 mM) was much better than Mg2+ (Ka = 6.3 mM) in supporting pp43 phosphorylation. Insulin stimulated phosphorylation of pp43 (t1/2 = 3.6 min) proceeded at a much slower rate compared to that of the beta-subunit of IR (t1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4:1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR beta-subunit suggest that pp43 was not derived from IR beta-subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase. PMID- 1722869 TI - Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary. AB - Two affinity-purified polyclonal antibodies have been generated that differentially recognize two mol wt (Mr) variants of prostaglandin H synthase (PGS) in the rat ovary: antibody-2 recognized PGS of 72,000 Mr (PGS72), and antibody-3 recognized PGS of 69,000 Mr (PGS69). Immunoblot analyses showed that PGS72 was rapidly induced by LH in granulosa cells of preovulatory (PO) follicles and was associated with the increased production of prostaglandins (PGs) obligatory for ovulation. PGS72 was low (negligible) in other ovarian tissues, including PO follicles, corpora lutea, and interstitium. In contrast, PGS69 was constitutively present in small antral and PO follicles (primarily in thecal cells), was unaffected by LH, and was found at higher levels in corpora lutea throughout pregnancy and in the ovarian interstitium. PGS69 (but not PGS72) was also detected by immunoblots in rat adrenal glands, heart, uterus, and kidney. Immunofluorescent localization of PGS72 and PGS69 to ovarian tissue sections confirmed the cell-specific distribution of PGS observed by immunoblot analyses of cell extracts. Immunofluorescent detection of PGS72 required methanol fixation, whereas PGS69 was also observed with paraformaldehyde fixation and Triton X-100 permeabilization, further suggesting biochemical differences in these molecules. Immunoreactive PGS69 in PO follicles, thecal cells, and granulosa cells was associated with low amounts of indomethacin-sensitive production of PGs by these tissues in vitro, which was unaffected by inhibitors of transcription or translation. In contrast, stimulation of PGs in PO follicles by LH in vitro correlated with the marked induction of PGS72, but not PGS69, and was sensitive to both transcriptional and translational inhibitors. Collectively, these studies provide the first evidence that the rat ovary contains two immunologically distinct forms and Mr variants of PGS, each of which is selectively regulated by hormones, localized to specific cell types, differentially sensitive to inhibitors of transcription/translation, and differentially solubilized for immunocytochemical localization. PMID- 1722870 TI - Regulation of alpha 2-macroglobulin by luteinizing hormone and prolactin during cell differentiation in the rat ovary. AB - Alph alpha 2-macroglobulin (alpha 2M), a protease inhibitor which also binds growth factors and cytokines, is temporally expressed in association with remodelling phenomena in the ovary: ovulation and luteinization. Specific hormonal, cellular, subcellular, and molecular events regulating alpha 2M mRNA and protein have been analyzed during follicular growth, ovulation, and luteinization using complementary in vivo and in vitro models. Data demonstrate that alpha 2M mRNA and protein are synthesized in thecal cells of developing follicles in response to low levels of LH. Conversely, alpha 2M mRNA and protein are only synthesized by granulosa cells of follicles that have been stimulated to luteinize either in vivo by the LH surge or in vitro by FSH and testosterone and are also exposed to PRL. The obligatory requirement for PRL is specific; associated with increased numbers of PRL-binding sites; mediated by time dependent appearance of alpha 2M in the endoplasmic reticulum (12 h), Golgi apparatus (24 h), and secretion vesicles (48 h); and involves in part increased transcription of the alpha 2M gene. PMID- 1722871 TI - Differential regulation of gene expression by estrogen in estrogen growth independent and -dependent MCF-7 human breast cancer cell sublines. AB - We have examined the ability of estradiol (E2) to regulate the expression of three mRNAs [for pS2, progesterone receptor (PR), and estrogen receptor (ER)], known to be under E2 regulation in the parental E2 growth-responsive MCF-7 cells, in an E2 growth-independent MCF-7 K3), previously isolated from the parental estrogen-dependent MCF-7 K1 human breast cancer cells after long term growth in vitro in the absence of estrogen, acquired estrogen-independent growth in vitro as well as the ability to form tumors in nude mice in vivo without estrogen. We find that the content of pS2 mRNA and the transcription rate of the pS2 gene, while being markedly increased by E2 in MCF-7 K1 cells, are no longer stimulated by E2 in this subline, although protein kinase activators tremendously increase (greater than 10-fold) pS2 mRNA in both K1 and K3 cells. In fact, basal pS2 mRNA levels are elevated 2.8 +/- 0.4-fold in MCF-7 K3 cells, and E2 evokes a concentration-dependent suppression of the pS2 mRNA level. In contrast, PR mRNA in the K3 subline, as in the parental K1 cells, is still up-regulated by E2, and ER mRAN content and the ER mRNA transcription rate are still down-regulated by E2 and show normal E2 dose-response relationships, implying that the ER in this subline is functional. These results demonstrate that the progression to estrogen independent growth in K3 cells is accompanied by a change in the regulation of some estrogen-induced genes by estrogen. While PR and ER retain normal patterns of regulation by E2, the pS2 gene in the estrogen growth-independent K3 subline is differentially affected and is no longer stimulated by E2. Our data suggest that this altered regulation of the pS2 gene is probably not caused by a defect of the ER or ER regulation in this subline. PMID- 1722872 TI - Different rates of age-related loss for four murine monoaminergic neuronal populations. AB - The age-related loss of locus coeruleus (LC) noradrenergic neurons, substantia nigra compacta (SNc) dopaminergic neurons, dopaminergic retinal amacrine (rAm) neurons and raphe serotonergic neurons, identified using antibodies against tyrosine hydroxylase (TH) and serotonin (5HT) was investigated in C57B1 mice aged 8 to 104 weeks. The neuronal somata were counted and their locations three dimensionally reconstructed from serial sections alternately immunoreacted or Nissl stained. Nonlinear estimation analysis showed that decaying exponential equations best fitted the plots of neuronal numbers versus age and each subtype was lost according to different exponential constants of -0.015, -0.013, -0.004 and -0.001 for LC TH+, SNc TH+, rAm TH+ and raphe 5HT+ neurons, respectively. Neurons were lost from all different subregions within the nuclei or the retinae. Counts of immediately adjacent TH-immunoreacted and Nissl-stained sections through the LC at different ages indicate that the neuronal loss was due to neuronal death rather than loss of TH immunoreactivity. The markedly different rates of age-related neuronal loss for the four monoaminergic subtypes offer a model to study the underlying molecular and cellular mechanisms. PMID- 1722873 TI - A novel species-specific RNA related to alternatively spliced amyloid precursor protein mRNAs. AB - Using an S1 nuclease protection assay, we have identified a novel "variant" Amyloid Precursor Protein (APP) RNA in human brain which is 3-6-fold more abundant than APP-770, but less abundant than APP-751 or APP-695. This variant, referred to as amyloid precursor-related protein 365 (APRP-365), is not detected in mouse and rat brain RNAs. A 1.6 kilo-basepair cDNA clone corresponding to this variant APP RNA predicts the existence of a 365 amino acid protein that is similar to the amino-terminal end of APP-770 but lacks the beta-amyloid peptide and any hydrophobic transmembrane spanning domains. In a modified polymerase chain reaction (PCR), we used amplification of reverse transcribed mRNA to confirm and extend our S1 observations. Together, the features of APRP-365 suggest that the human variant is a soluble protein containing a Kunitz protease inhibitor domain. PMID- 1722874 TI - Quantitative measurement of alternatively spliced amyloid precursor protein mRNA expression in Alzheimer's disease and normal brain by S1 nuclease protection analysis. AB - We have used an S1 nuclease protection strategy to measure alternatively spliced amyloid precursor protein (APP) mRNAs associated with Alzheimer's disease (AD) to determine whether the expression of either one or more of the transcripts correlate with observed amyloid plaque pathology. Comparison of AD with normal cortex reveals that increasing plaque density parallels an increase in the fraction of APP-695 and a corresponding decrease in APP-770 and 751 mRNA fractions. A specific increase of APP-695, the protease inhibitor-lacking APP RNA form, in those brain regions most involved with amyloid plaque formation, suggests that an imbalance in the protease inhibitor is potentially significant in the disease. These data are consistent with cellular/tissue region-specific regulation of alternative splicing accounting for AD-related changes in the expression of APP mRNA forms. PMID- 1722875 TI - Superinduction of cytotoxic interferon-beta in glioma cells. AB - The possibility for new interferon therapy was investigated using the effect of endogenous human interferon-beta (HuIFN-beta) on various culture cell lines. Cell lines were exposed to superinduction agents (poly I: poly C, cycloheximide, and actinomycin D) and the production of endogenous interferon analyzed. Quantitative determination of HuIFN-beta and messenger ribonucleic acid (mRNA) showed HuIFN beta was induced in all of five glioma cell lines, one of two melanoma cell lines, and all of three lung carcinoma cell lines as well as fibroblasts. Northern blot analysis showed HuIFN-beta mRNA induced in glioma cells was identical to that from fibroblasts. Endogenous HuIFN-beta induced from glioma cells had a cytostatic or cytocidal effect against various human glioma cell lines, even those resistant to fibroblast-derived HuIFN-beta. These results show it may be possible to use the induction of excess endogenous cytotoxic HuIFN-beta in human glioma tissue itself. PMID- 1722876 TI - Melatonin secretion in normal pressure hydrocephalus after cerebral aneurysm rupture--investigation before and after ventriculoperitoneal shunt. AB - Melatonin (MLT) secretion was examined in six normal pressure hydrocephalus (NPH) patients before and after ventriculoperitoneal (VP) shunt surgery. Ten healthy subjects were used as controls. Venous blood samples were taken daily at 2 p.m., 8 p.m., 2 a.m., and 8 a.m. Radioimmunoassay of MLT used a new specific antiserum and separation method achieving low cross-reactivity and high-efficiency MLT separation. Plasma levels in the control group at 2 p.m. and 2 a.m. were significantly different, showing diurnal rhythm (DR). The patients' MLT levels before VP shunt were significantly lower than control levels and the DR was absent. Postoperatively, the values were significantly different from preoperative values only at 2 a.m., but the DR reappeared. Thus, in NPH, VP shunt surgery improved the melatonin DR, probably through normalization of the dilated third ventricle. PMID- 1722877 TI - Spontaneous intracerebral hemorrhage in cases of severe angiospasm following ruptured aneurysm--a pathological study. AB - Eleven patients with a history of moderate or severe angiospasm following ruptured cerebral aneurysm developed spontaneous intracerebral hemorrhage between 31 and 111 months after aneurysm surgery. In all cases, hemorrhage occurred in the ipsilateral hemisphere to the original aneurysm. In nine patients, the hematoma was surgically evacuated and bleeding perforating arteries were resected for histological examination. Computed tomographic scans showed the hematomas to be unusually extended compared to those after hypertensive intracerebral hemorrhage. The histological examination showed various degenerative changes in the elastic lamina and media of the perforating arteries, even though most patients were young and normotensive. These findings suggest that patients who have suffered severe cerebral angiospasm may have a higher risk for subsequent development of intracerebral hemorrhage than those without prior angiospasm. PMID- 1722878 TI - Investigation of normal pressure hydrocephalus by 123I-IMP SPECT. AB - We evaluated N-isopropyl-p-[123I]iodoamphetamine (123I-IMP) single photon emission computed tomography (SPECT) as a method for identifying normal pressure hydrocephalic (NPH) patients eligible for shunting procedures. 123I-IMP SPECT scans were taken before and after cerebrospinal fluid (CSF) taps in NPH cases. Post-subarachnoid hemorrhagic (SAH) patients showed apparent frontal blood flow reduction but non-SAH cases did not. The frontal blood flow increased in comparison with the temporal flow after CSF tapping in SAH cases who benefited most from shunting. Cerebral blood flow study before and after CSF removal is a potential method for classifying NPH patients likely to benefit from the shunting operation. PMID- 1722880 TI - Ruptured distal anterior cerebral artery aneurysms presenting as acute subdural hematoma--report of three cases. AB - The authors report three cases of distal anterior cerebral artery aneurysm presenting as acute subdural hematoma (SDH). Two patients were comatose on admission and died of massive SDH. One patient underwent aneurysmal neck clipping in the chronic stage and returned to normal daily life. A convexity SDH continuous with a wedge-shaped interhemispheric SDH was the characteristic computed tomographic appearance in all cases. There was no accompanying subarachnoid or intracerebral hemorrhage in one case (pure SDH). These cases are 9.4% of 32 ruptured distal ACA aneurysms treated in our institute in the last 14 years, a higher incidence than reported previously. PMID- 1722879 TI - Clinicopathological study of bacterial intracranial aneurysms. AB - The authors report the clinicopathological findings in six cases of bacterial intracranial aneurysms. All patients received appropriate high-dose antibiotics, and four were treated surgically. One patient with multiple aneurysms of the main trunks died of disseminated intravascular coagulation. Autopsy disclosed no apparent aneurysm or inflammatory cell infiltration, but a partially interrupted internal elastic lamina and thickened intima were disclosed at the angiographical aneurysm sites. These findings suggest that 1) appropriate high-dose antibiotics are effective against inoperable bacterial aneurysms in the main trunks, 2) new aneurysms may be formed in patients with cyanotic congenital heart disease, because bacterial emboli can directly reach the cerebral circulation and reimplant on the fragile arterial walls after vasculitis. Histological examination of aneurysmal walls revealed inflammatory cell infiltration after resolution of clinical endocarditis. This suggests that both appropriate high dose antibiotic therapy and surgery should be considered in patients with distal bacterial aneurysms. PMID- 1722881 TI - Treatment of metastatic brain tumor from esophageal carcinoma--report of four cases. AB - Metastasis to the brain from esophageal carcinoma is rare. Recently we had four cases, all treated by tumor removal. Three received postoperative whole or local brain irradiation. Anticancer pellets were implanted in two. The postoperative 1 year survival rate was 37.5%, which suggests the prognosis is not worse than for other metastatic brain tumors. PMID- 1722882 TI - Cerebral arteriovenous malformation causing benign intracranial hypertension- case report. AB - A 32-year-old male with a large arteriovenous malformation (AVM) in the right temporoparietal area presented with features of benign intracranial hypertension. This association is rare. The pathogenesis is believed to be due to cerebral venous hypertension. The excision of the AVM eliminated the intracranial hypertension. PMID- 1722883 TI - Peduncular hallucinosis associated with ruptured basilar-superior cerebellar artery aneurysm--case report. AB - A 65-year-old female developed peduncular hallucinosis 3 days after rupture of a basilar-superior cerebellar artery aneurysm. There were no neurological deficits except slight anisocoria when she first complained of hallucinations. Vasospasm of the perforating arteries to the upper brainstem, rather than direct brainstem damage caused by the bleeding, was probably the cause. Peduncular hallucinosis is possibly the only localizing sign of ruptured upper posterior circulation aneurysm. PMID- 1722884 TI - Hypothalamic histiocytosis X with diabetes insipidus and Korsakoff's syndrome- case report. AB - A 54-year-old female presented with apparent isolated hypothalamic histiocytosis X associated with diabetes insipidus and Korsakoff's syndrome. Computed tomographic and magnetic resonance imaging demonstrated a single hypothalamic mass. A craniotomy for biopsy found granulation tissue of unknown cause. Further investigation discovered genital bleeding before admission. Biopsy of the cervix uteri revealed histiocytosis X. Further studies showed the disease was restricted to the hypothalamus and the endometrium of the cervix uteri. Low-dose irradiation led to partial regression of the hypothalamic mass and improvement of Korsakoff's syndrome. Even when a diagnosis of isolated hypothalamic histiocytosis X is confirmed, the possibility of another histiocytosis X lesion in an unexpected region must be considered. PMID- 1722885 TI - Wound healing following stab injury on rat cerebral cortex. AB - We examined how wound healing was initiated and completed. Stab injury was made over the right parietal cortex with 2.5 mm depth and 4 mm length. Either 3 days, 7 days or 1 month after this operation, operated rats were perfused and fixed with 4% paraformaldehyde. The brains were removed, embedded in paraffin, cut coronally at a level of caudate-putamen complex and thin-sliced into 6 microns thick sections with a microtome. The sections were stained immunohistochemically for detection of glial fibrillary acidic protein (GFAP), and co-stained for myelination with Woelcke's staining method. Sections were also stained immunohistochemically for Laminin after pretreatment by pepsin. Furthermore, the sections were stained either haematoxylin-eosin staining, Laidlaw's Reticulum staining for evaluation of reticulin and phosphotungusten acid haematoxylin (PTAH) staining for delination of collagen. We, first, confirmed astrocytic proliferation induced by the stab injury. Then, astrocytes can be seen crowded around the injured site 7 days after injury. Both Laminin and Reticulum stain show the so-called neovascularization around the stab wound 7 days after injury when astrocytes proliferated most vigorously as mentioned above. PTAH stain showed collagenous fibre 1 month after injury when astrocytes congregated along the wound site, and Laminin fibres were localized to the injury site. Reticulin fibres disappeared. In conclusion, it takes more than a month for the wound site to regain the steady state. PMID- 1722886 TI - Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat. AB - The organization of connections between the amygdala, prefrontal cortex and striatum was studied using anterograde and retrograde tract tracing techniques in the rat. The anterograde transport of Phaseolus vulgaris leucoagglutinin and wheat germ agglutinin conjugated to horseradish peroxidase was used to examine the striatal projections of the prefrontal cortex. These studies revealed that the prelimbic area of the medial prefrontal cortex projects mainly to the medial part of the striatum, whereas the dorsal agranular insular area of the lateral prefrontal cortex projects mainly to the ventrolateral part of the striatum. The organization of amygdaloid projections to the prefrontal cortex and its associated portions of the striatum was investigated using the fluorescence retrograde tract tracing technique. Different color fluorescent dyes, True Blue and Diamidino Yellow, were injected into the prefrontal cortex and striatum. These studies demonstrated that medial portions of the basolateral nucleus, and adjacent portions of the lateral, basomedial and amygdalo-hippocampal nuclei, project to both the medial prefrontal cortex and its associated medial striatal region. The rostral pole and lateral portions of the basolateral nucleus project to both the lateral prefrontal cortex and its associated lateral striatal region. Many neurons in the basolateral amygdaloid nucleus, and to a lesser extent other amygdaloid nuclei, were double-labeled in these experiments, indicating that these cells send collaterals to both the prefrontal cortex and striatum. These findings indicate that discrete areas of the amygdala, and in some cases individual amygdaloid neurons, can modulate information processing in the first two links of distinct cortico-striato-pallidal systems arising in the medial and lateral prefrontal cortex. PMID- 1722887 TI - Spinal distribution and collateral projections of rat spinomesencephalic tract cells. AB - The distribution of cells belonging to the rat spinomesencephalic tract was studied by means of the retrograde transport of fluorescent dyes. Bilateral midbrain injections of cytoplasmic and nuclear tracers were made in order to evaluate the location of ipsilateral, contralateral, or bilaterally projecting cells. Spinal neurons with ascending projections to midbrain and descending propriospinal projections were identified by midbrain and spinal injections of different cytoplasmic labels. The locations of spinomesencephalic tract cells included seven regions of the spinal gray matter: marginal zone, lateral neck of the dorsal horn, nucleus proprius, the region around the central canal, the lateral cervical and spinal nuclei and the ventral horn. Cells projecting to the ipsilateral or contralateral midbrain had similar distributions and were frequently found in clusters with overlapping dendritic fields. Approximately 75% of spinomesencephalic cells projected to the contralateral midbrain. The largest contribution to the spinomesencephalic tract cell population was found in cervical cord segments 1-4. Cells with bilateral projections accounted for nearly 2% of all labeled cells, whereas 5% had both ascending and descending projections. Spinomesencephalic cells were found to have varying dendritic fields and morphology, e.g. fusiform, pyramidal, round/oval, and multipolar. The results of the present study lend further support to the view that the spinomesencephalic tract is a multi-component pathway with varied origins and projection targets. PMID- 1722888 TI - Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia. AB - Recent evidence has suggested that cAMP plays a role as a second messenger in the decrease in nociceptive threshold (or hyperalgesia) produced by agents acting on primary afferent terminals. In support of this hypothesis we report that intradermal injection of a direct activator of adenyl cyclase, forskolin, produces a dose-dependent hyperalgesia in the rat. The duration of this hyperalgesia was prolonged by the phosphodiesterase inhibitors, isobutylmethylxanthine and rolipram. Forskolin hyperalgesia was antagonized by the Rp isomer of cyclic adenosine-3'5'-monophosphothioate, an analog of cAMP that prevents the phosphorylation of the cAMP protein kinase. The Rp isomer of cyclic adenosine-3'5'-monophosphothioate also inhibited the hyperalgesia induced by a membrane-permeable analogue of cAMP, 8-bromocyclic adenosine monophosphate, as well as the hyperalgesia induced by agents that are presumed to act directly on primary afferent nociceptors: prostaglandin E2, prostaglandin I2, (8R,15S) dihydroxyicosa(5E-9,11,13Z)tetraenoic acid; and the adenosine A2-agonist 2 phenylaminoadenosine. Although the cAMP second messenger system contributes to primary afferent hyperalgesia, we found no evidence for a contribution of protein kinase C. Thus, hyperalgesia induced by prostaglandin E2, prostacyclin (prostaglandin I2), (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid, the adenosine A2-agonist 2-phenylaminoadenosine, 8-bromocyclic adenosine monophosphate and the direct activator of adenyl cyclase, forskolin, were not significantly attenuated by the selective inhibition of protein kinase C by the 19-31 fragment of protein kinase C. Two other inhibitors of protein kinase C, sphingosine and staurosporine, also failed to attenuate prostaglandin E2-induced hyperalgesia. PMID- 1722889 TI - Sprouting of cholinergic axons does not occur in the cerebral cortex after nucleus basalis lesions. AB - Different doses of the excitotoxin quisqualate were used to make lesions in the caudal part of the ferret nucleus basalis, i.e. the part that projects to the visual cortex. The higher doses of the excitotoxin destroyed all nerve growth factor receptor-immunoreactive cells in the caudal nucleus basalis and gave rise to up to 75% loss of acetylcholinesterase-containing axons in the visual cortex. In sections stained for Nissl substance there was generalized tissue damage around the injection sites and extensive loss of all neuron types in areas surrounding the caudal nucleus basalis. Lower doses of the excitotoxin damaged only a proportion of the nerve growth factor receptor-immunoreactive neurons in the caudal nucleus basalis and produced a much lower depletion of acetylcholinesterase-positive fibres in the visual cortex. The only damage seen in sections stained for Nissl substance was a loss of magnocellular neurons in the vicinity of the injection sites. A quantitative morphological approach was used to show that either one week or three months after the lesions there was a linear correlation between the proportion of acetylcholinesterase-positive axons lost in the visual cortex and the proportion of nerve growth factor receptor immunoreactive cells that had disappeared from the caudal nucleus basalis. Since the correlation lines for the short-term (one week) survival and the long-term (three months) survival experiments coincided, this indicated that no collateral sprouting of cholinergic axons had occurred in the visual cortex of the long-term survival animals regardless of size of the lesion in the nucleus basalis. PMID- 1722890 TI - Topographical organization of amygdaloid projections to the caudatoputamen, nucleus accumbens, and related striatal-like areas of the rat brain. AB - The topographical organization of amygdaloid projections to the caudatoputamen, nucleus accumbens, and lateral portions of the bed nucleus of the stria terminalis and central amygdaloid nucleus was investigated, in the rat, using the retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase. Although the caudatoputamen and nucleus accumbens are the principal components of the striatum, there is evidence that lateral portions of the bed nucleus of the stria terminalis and central amygdaloid nucleus may be striatal-like structures. The basolateral nucleus was the main source of amygdaloid fibers to all of these structures. In many instances labeled areas of the basolateral nucleus were continuous with labeled areas in the adjacent lateral and basomedial nuclei. Amygdaloid neurons projecting to the striatum and striatal-like areas exhibited an overlapping topographical organization. In general, the medial-to-lateral coordinate in the striatum corresponds to the medial-to-lateral coordinate in the basolateral nucleus. There was also a partial reversed sagittal topography in that the caudal caudatoputamen receives its principal projection from the rostral basolateral nucleus. However, the rostral basolateral nucleus had a stronger projection to the rostral caudatoputamen and lateral nucleus accumbens than the caudal basolateral nucleus. The principal striatal projection of the caudal basolateral nucleus was to the medial nucleus accumbens. Amygdaloid labeling produced by injections into the medial nucleus accumbens was very similar to that seen with injections into the lateral portions of the bed nucleus of the stria terminalis and central amygdaloid nucleus. The retrograde amygdaloid labeling seen in this investigation, when compared to labeling seen with cortical injections in previous studies, suggests that specific amygdaloid domains project to particular cortical areas as well as to the principal striatal targets of the same areas. PMID- 1722891 TI - Release of pro-thyrotropin-releasing hormone connecting peptides PS4 and PS5 from perifused rat hypothalamic slices. AB - Thyrotropin-releasing hormone prohormone contains five copies of the thyrotropin releasing hormone progenitor sequence Gln-His-Pro-Gly, each flanked by pairs of basic amino acids and separated by intervening sequences (connecting peptides). Using a perifusion system for rat hypothalamic slices, we have studied the ionic mechanisms underlying the release of two connecting peptides originating from the thyrotropin-releasing hormone precursor: prepro-thyrotropin-releasing hormone (160-169) (Ps4) and prepro-thyrotropin-releasing hormone-(178-199) (Ps5). Quantification of these two peptides in the effluent fluid was performed using sensitive and highly specific radioimmunoassay procedures. Reverse phase high performance liquid chromatography analysis of the effluent perifusate showed that released peptides co-eluted with synthetic Ps4 and Ps5. The secretion of Ps4 and Ps5 was stimulated by depolarizing agents such as (i) high potassium concentrations, (ii) ouabain, an Na+/K(+)-ATPase inhibitor, and (iii) veratridine, a stimulator of voltage-operated Na+ channels. The response to potassium (70 mM) was not affected by the specific Na+ channel blocker tetrodotoxin. The K+ channel blocker tetraethylammonium did not modify K(+) evoked release of Ps4 and Ps5. These data suggest that voltage-operated Na+ channels are not involved in the stimulatory effect of high K+ on the release of Ps4 and Ps5. The lack of effect of picrotoxin, a Cl- channel blocker, on the secretion of the connecting peptides indicates that chloride ions play a minor role in the release process. In contrast, deprivation of Ca2+ in the perifusion medium suppressed K(+)-evoked release of the two peptides, indicating that voltage-operated Ca2+ channels are implicated in the release process. Taken together, the present results show that non-thyrotropin-releasing hormone peptides originating from the thyrotropin-releasing hormone precursor are secreted by mediobasal hypothalamic fragments. The release of these peptides is stimulated by depolarization through a calcium-dependent process. These data indicate that Ps4 and Ps5 may be released at the level of the median eminence into the portal circulation, suggesting that these peptides may play a role in the control of anterior pituitary cells. PMID- 1722892 TI - Effects of eye-enucleation on substance P-immunoreactive fibers of some retinorecipient nuclei of the rat in relation to their origin from the superior colliculus. AB - We have previously shown that retinal deafferentation causes a decrease in immunoreactive dendrites of substance P-positive neurons of the superficial superior colliculus of the rat. Since some retinorecipient thalamic and pretectal nuclei are putative targets for substance P-containing cells of the superior colliculus, the present study attempted to ascertain whether substance P immunoreactive fibers in these nuclei are also affected by retinal denervation. We found that unilateral eye removal produced a progressive increase in fibrous substance P immunoreactivity in the nucleus of the optic tract, lateral posterior nucleus, and lateral geniculate nucleus of the side contralateral to the enucleation. On the other hand, unilateral lesions to the superficial layers of the superior colliculus produced a dramatic reduction in substance P immunoreactivity in the ipsilateral nucleus of the optic tract, lateral posterior nucleus, and dorsal and ventral lateral geniculate nuclei. In bilaterally enucleated animals, unilateral lesion to the superior colliculus produced, as expected, loss of immunoreactive fibers only in the lateral posterior nucleus and the retinorecipient nuclei ipsilateral to the lesion. These results suggest that transneuronal changes in the distribution of substance P in collicular neurons observed after enucleation could be reflected in their projections to the other primary visual centers and to the lateral posterior nucleus. PMID- 1722893 TI - Convergence of synaptic inputs from the striatum and the globus pallidus onto identified nigrocollicular cells in the rat: a double anterograde labelling study. AB - Two major sources of afferent synaptic inputs to projection neurons in the rat substantia nigra reticulata are the striatum and the globus pallidus. In order to understand better the functional relationships between these two afferents in the control of the activity of nigrofugal neurons, experiments have been performed to test the possibility that single nigrofugal cells receive convergent synaptic inputs from the striatum and the globus pallidus. To address this question we have used two different approaches. First, we have developed a double anterograde labelling technique suitable for both light and electron microscopy and combined this procedure with the retrograde transport of lectin-conjugated horseradish peroxidase in order to retrogradely label the nigrocollicular cells. Second, we have combined the anterograde transport of Phaseolus vulgaris-leucoagglutinin from the globus pallidus and immunocytochemistry for DARPP-32 as a marker for the striatal terminals, with the retrograde transport of lectin-conjugated horseradish peroxidase from the superior colliculus. In the double anterograde labelling experiment, biocytin was injected in the striatum, Phaseolus vulgaris leucoagglutinin in the globus pallidus and lectin-conjugated horseradish peroxidase in the superior colliculus. Following these injections, rich plexuses of biocytin- and Phaseolus vulgaris-leucoagglutinin-labelled terminals were found in the ventral two-thirds of the substantia nigra. The biocytin-positive terminals (striatonigral) were generally small and formed rich plexuses without any apparent neuronal association whereas the Phaseolus vulgaris-leucoagglutinin labelled terminals (pallidonigral) were much larger and formed baskets around the perikarya of retrogradely and non retrogradely labelled cells in the substantia nigra reticulata. In areas of the substantia nigra reticulata where the fields of biocytin- and Phaseolus vulgaris-leucoagglutinin-labelled terminals overlapped, the perikarya and the proximal dendrites of retrogradely and non retrogradely labelled cells were found to be apposed by numerous Phaseolus vulgaris leucoagglutinin-immunoreactive pallidonigral terminals and a few biocytin labelled striatonigral terminals. In the sections prepared for electron microscopy, the biocytin was localized using 3,3'-diaminobenzidine tetrahydrochloride whereas Phaseolus vulgaris-leucoagglutinin was localized using benzidine dihydrochloride. It was thus possible to distinguish the biocytin- from the Phaseolus vulgaris-leucoagglutinin-labelled terminals in the electron microscope by the texture of the reaction product associated with them.4+ Examination of 231 biocytin-labelled (striatonigral) terminals and 105 Phaseolus vulgaris-leucoagglutinin-immunoreactive (pallidronigral) terminals revealed that the striatonigral terminals were generally small, contained few mitochondria and formed symmetric synapses predominantly with the distal dendrites (77%) and far less frequently with the perikarya (3%) of substantia nigra reticulata cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1722894 TI - Dopamine regulates the electrical activity of frog melanotrophs through a G protein-mediated mechanism. AB - Recently we have demonstrated that dopamine inhibits action potentials in cultured frog melanotrophs through D2 receptor-mediated activation of hyperpolarizing potassium current and reduction of calcium and sodium currents. Herein, the respective roles of G proteins, guanosine-5'-triphosphate and adenosine-3':5'-cyclic-monophosphate in dopamine-induced electrical responses were investigated using the whole-cell patch-clamp technique. Pretreatment of melanotrophs with pertussis toxin (1 microgram/ml) abolished the hyperpolarization and arrest of action potentials evoked by dopamine (1 microM) in 77% of the cells studied. Addition of guanosine-5'-O-(2-thiodiphosphate) (500 microM) to the intracellular solution did not alter the effects of a first exposure to dopamine, but completely blocked the response of cultured melanotrophs to subsequent pulses of dopamine. In cells which were dialysed with guanosine-5'-O-(3-thiotriphosphate) (100 microM) dopamine caused a sustained hyperpolarization and an irreversible inhibition of spikes. Voltage-clamp recordings with electrodes containing guanosine-5'-O-(3-thiotriphosphate), showed that the increase of potassium current and decrease of calcium and sodium currents caused by dopamine were irreversible. These effects were not modified when the pipette contained, in addition to guanosine-5'-O-(3-thiotriphosphate), a high concentration of adenosine-3':5'-cyclic-monophosphate (100 microM) together with the inhibitor of phosphodiesterases 3-isobutyl-1-methylxanthine (100 microM). It is concluded that, in cultured frog melanotrophs, a pertussis toxin sensitive G protein is implicated in the coupling of dopamine D2 receptors to activation of potassium channels and inhibition of calcium and sodium channels. Our results also indicate that the G protein-mediated signal transduction does not involve the adenylate cyclase system. PMID- 1722895 TI - Enhancement of spinothalamic neuron responses to chemical and mechanical stimuli following combined micro-iontophoretic application of N-methyl-D-aspartic acid and substance P. AB - A role for sensitization of nociceptors in the generation of primary hyperalgesia is well documented. More recent work has begun to define a role of an increased excitability of neurons within the spinal cord in the generation of secondary hyperalgesia. The present study demonstrates increased responses of primate spinothalamic neurons following co-administration of N-methyl-D-aspartic acid (NMDA) and substance P (SP) by micro-iontophoresis. Wide dynamic range and high threshold STT neurons in laminae I-VI showed an increased frequency of discharges following application of NMDA which was characterized by a slow onset to peak discharge rate and a slow return to background levels of discharge. Combined application of NMDA with SP resulted in an enhancement of responses to NMDA that often long outlasted the administration of SP. This increase in response of the cells to NMDA was not produced by repeated application of NMDA alone or following combined application of NMDA with an SP analog. NMDA responses were reduced or prevented in all cases by co-application of an NMDA-receptor antagonist. Finally, long-lasting potentiation of NMDA responses by SP was paralleled by enhanced responses to mechanical stimulation of skin. It is proposed that a mechanism involving the combined synaptic release of excitatory amino acids and peptides leads to secondary hyperalgesia. PMID- 1722896 TI - [Evaluation and prognosis of fetal arrhythmia]. AB - From January 1986 to August 1990, a fetal arrhythmia was diagnosed in 97 pregnant women referred to our unit. Atrial or ventricular extrasystoles were the most frequent rhythm disturbance encountered (71%). They were always well tolerated and all disappeared during the perinatal period. Tachycardia was found in 16 fetuses; 6 had a supraventricular reentrant tachycardia, 6 an atrial chaotic tachycardia, 1 an ectopic atrial tachycardia and the remaining 3 an atrial flutter. A congenital heart malformation was present in 4 fetuses. The arrhythmia induced hydrops fetalis in 25% of cases. One hydropic fetus died in utero and another needed premature delivery. Bradycardia was diagnosed in 12 cases, 3 had benign atrial blocked extrasystoles, 3 others sinus bradycardia due to fetal distress and 6 atrio-ventricular block. Atrio-ventricular block was associated with congenital malformation in 4 cases (66%). All these fetuses were hydropic and died. The 2 fetuses without cardiac malformation tolerated well their bradycardia during fetal life. Fetal arrhythmia is not rare, but in most cases is benign. Sustained tachycardia requires prompt treatment, because when hydrops fetalis appears the prognosis is worse. The major prognostic factor for atrio ventricular block is the association with a cardiac malformation. PMID- 1722897 TI - Zidovudine and other reverse transcriptase inhibitors in the management of human immunodeficiency virus-related disease. AB - Licensed in 1987, zidovudine remains the only medication with proved efficacy for the treatment of disease caused by the human immunodeficiency virus (HIV). New information on the pharmacology (adults and children), effects of kidney and liver dysfunction on the disposition of the drug, and drug-drug interactions have improved the way we use and monitor this agent. The serious toxicity associated with zidovudine has led researchers to develop safer dosage regimens. Also, recognition that zidovudine slows but does not halt progression of disease has increased the search for effective alternatives. The best-studied agents are didanosine (2',3'-dideoxyinosine, ddl), zalcitabine (2',3'-dideoxycytidine, ddC), and foscarnet. PMID- 1722898 TI - Assays for angiogenesis: a review. AB - Accurate, reliable quantitation of the neovascular (angiogenic) response, both in vitro and in vivo, is an essential requirement for the study of new blood vessel growth. Over many years, ingenious ways have been developed for measuring this process, and they have contributed much to our present understanding of the vasculogenesis and angiogenesis that accompany normal embryonic development, lactation and wound healing, as well as tumor growth and a variety of other disease states ranging from diabetic retinopathy to autoimmune vasculitis. In this review we describe and evaluate the methodology and specific features of some of the most frequently used of these assays. PMID- 1722899 TI - Differential HIV replication and HIV-induced interferon production in mononuclear phagocytes: relationship to cell maturation. AB - We have investigated the replication of human immunodeficiency virus (HIV) and HIV-induced interferon (IFN) production in human mononuclear phagocytes at 2 different stages of in vitro maturation. Blood monocytes and monocyte-derived macrophages from 6 healthy, HIV-seronegative donors were challenged with HIV1IIIB and HIV2ROD. Freshly separated monocytes produced IFN when inoculated with both HIV types. In these cultures, an inverse correlation was observed between the amount of IFN production and the rate of HIV replication. In contrast to the monocytes, 5-day-old monocyte-derived macrophages did not produce IFN when challenged with HIV, but a significant replication of HIV1IIIB and HIV2ROD was found in all cultures. PMID- 1722900 TI - A multi-arterial clamping method with arterial hypotension does not bring about complete brain ischemia in dogs. AB - The completeness of brain ischemia with a multi-arterial clamping method in dogs was examined using EEG, evoked potentials (EPs) and vessel staining with Evans blue. EEG was monitored by bipolar parietal lead. EPs stimulating electrodes were inserted into the first thoracic (T1) epidural space and recording electrodes into the C2 epidural space, brain stem and cerebral cortex. EPs were measured at 30 s intervals with 50 measurements each time using 3.0 mA current of 100 microseconds duration. In dogs in which brain ischemia was brought about by ventricular fibrillation (VF group, n = 5) EEG disappeared within 40 s in all dogs and the amplitudes of EPs at the C2 spinal cord, brain stem and cerebral cortex after 10 min ischemia were 57%, 0% and 0%, respectively. In the dogs in which a multi-arterial clamping method (clamping internal thoracic arteries, brachiocephalic trunk and left subclavian artery while lowering systolic arterial pressure (AP) below 50 Torr) was used (AC group, n = 5) EEG was still recognizable at 5 min in 2 dogs and the amplitudes of EPs at the C2, brain stem and cerebral cortex at 10 min ischemia were 103%, 53% and 0%, respectively. Stainings with Evans blue were observed in all soft tissue at and below thoracic level, entire intervertebral venous plexus, venous sinuses of cranial dura mater and spinal cord below the lower part of cervical region. Bright red fluorescence by Evans blue was observed microscopically in the vessels of the spinal cord, brain stem and cerebrum (1 dog only). In conclusion a multi-arterial clamping method with arterial hypotension brings about only incomplete brain ischemia. PMID- 1722901 TI - [Interferon]. PMID- 1722902 TI - Nucleolar organizer regions in cardiac lesions induced by doxorubicin. AB - The use of the argyrophilic (Ag) staining technique for nucleolar organizer regions (NORs) revealed nuclear changes in myocytes of the left atrium of 10 rats treated twice a week for 6 weeks with doxorubicin (1 mg/kg body weight) iv and sacrificed after 6 weeks without treatment. The changes were easily detected qualitatively and further assessed by quantification. Cardiac myocytes of doxorubicin-treated rats had larger nuclei and/or a larger quantity of AgNORs that were either dispersed in a number of small dots or clustered in rounded, rod shaped, or tortuous large structures. AgNOR alterations may reflect a defect of nucleolar association leading to an impairment of protein synthesis that could be involved in doxorubicin cardiotoxicity. PMID- 1722903 TI - Hyaline droplet accumulation in rodent kidney proximal tubules: an association with histiocytic sarcoma. AB - Since recognition during the last decade that certain renal carcinogens can initially cause an accumulation of hyaline (protein) droplets in proximal tubules of male rats, it has become appropriate to establish whether this phenomenon of protein overload can also occur in rodent kidneys unrelated to chemical treatment. Kidney tissue from a number of selected rodent studies held in the National Toxicology Program (NTP) or Food and Drug Administration (FDA) archives were evaluated for hyaline droplet accumulation in proximal tubules. The survey concentrated on rats and mice of both sexes bearing hematopoietic tumors, as our preliminary observations had suggested this direction of study. The tissues of 101 Sprague-Dawley, 25 Osborne-Mendel, and 70 Fischer 344 rats and 96 B6C3F1 mice were examined. These animals provided an assortment of tumors including histiocytic sarcoma, lymphocytic lymphoma, mononuclear cell leukemia, and sarcoma. Hyaline droplet accumulation, primarily involving the P2 segment of proximal tubules, was diagnosed in 96% of rats with histiocytic sarcoma (74/77 cases in Sprague-Dawley, 17/18 in Osborne-Mendels, 7/7 in Fischers) and in 55% of B6C3F1 mice with histiocytic sarcoma (18/33 cases). There appeared to be a qualitative correlation between hyaline droplet accumulation and degree of tumor burden. Thus, in cases negative for hyaline droplets, the tumor was often confined to a single location, while increasing involvement of proximal segments beyond P2 occurred with more extensive multi-organ dissemination of the tumor. By immunohistochemistry on 11 cases of rat and 8 cases of mouse histiocytic sarcoma, the protein in hyaline droplets was identified as lysozyme, a known major secretory product of monocytes and macrophages. The hyaline droplets were negative for alpha 1-antitrypsin, alpha 2u-globulin, rat or mouse immunoglobulin, and albumin. More sparsely scattered droplets and granules present in proximal tubules of Fischer rats with mononuclear cell leukemia were negative for lysozyme but positive for either iron or lipofuscin pigment. The study establishes a clear association between renal tubule hyaline droplet and lysozyme accumulation in rats and mice with histiocytic sarcoma. Hyaline droplets secondary to neoplasia should be distinguished from chemically-induced hyaline droplet nephropathy in the male rat involving alpha 2u-globulin. PMID- 1722904 TI - [Transitory carotid ischemic attacks: clinical and pathogenic aspects]. AB - The aim of this study is to describe the clinical characteristics and the pathological mechanisms of carotid transient ischemic attacks (TIA) in 117 patients which were hospitalised for such symptoms. Our results show a male predominance, except for age group under 40 and over 79 years. The principal cardiovascular index and risk factors are: arterial hypertension, smoking, hyperlipidemia, vascular intermittent claudication and hematocrit greater than 46%. Amongst our patients, 17% with hemispherical and mixed TIAs had a cerebral infarction proved by CT-Scan, the recent aspect and localisation of which were compatible with symptoms. The atherosclerotic causes are more frequently associated with mixed and retinal TIAs than hemispheric TIAs. This fact may be attributed to a larger proportion of stenotic atherosclerotic lesions by mixed TIAs than hemispheric ones. The cardiac embolic pathogenic mechanism is responsible for 11% of TIAs if considered individually; of 5% if associated with carotid atherosclerosis. PMID- 1722905 TI - 7th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS), 21st Central European Neurological Symposium (CNS 21), 147th meeting of the Swiss Neurological Society. May 22-25, 1991, St. Gallen. Abstracts. PMID- 1722906 TI - [Psychometric differentiation of schizophrenic and schizoaffective psychoses]. AB - The investigation aims at cognitive deficits of schizoaffective disorders, at the differentiation of the schizomanic and the schizodepressive subtypes, and at the cognitive differentiation of schizophrenic and schizoaffective disorders. Schizoaffective patients show no cognitive deficits in comparison to a nonclinical sample. There are no cognitive differences between the schizomanic and the schizodepressive subtype, but significant differences between schizophrenic and schizoaffective patients in test-scores concerning concentration and abstract reasoning. No differences in test-scores concerning verbal abilities are found. PMID- 1722907 TI - [Reliability and validity of the Zurich Questionnaire of Coping with Illness]. AB - The coping behavior of three groups of patients (N = 585) was assessed by a 45 item-questionnaire, a revised version of the Zurich Coping Questionnaire (ZKV) published earlier. The following three coping patterns resulted, established by factor analysis: ZKV-1: Depressive coping (Cronbach-alpha = .86), ZKV-2: Self encouragement/distraction (.81) and ZKV-3: Problem tackling (.60). Retest stability for the two scales 1 and 3 was high (r greater than .74, 12-month interval). Concurrent and prognostic validity was assessed. The results indicate that the ZKV is a useful instrument for coping research, especially for the prognostic rating of the subsequent coping behavior. PMID- 1722908 TI - Laboratory use of hirudin. PMID- 1722909 TI - [Commitment. Symbolic and real constraints]. PMID- 1722910 TI - [Introduction to the lecture of Jacques Lacan]. PMID- 1722911 TI - [The lie of the image]. PMID- 1722912 TI - Surrogate methods to diagnose gonococcal and chlamydial cervicitis: comparison of leukocyte esterase dipstick, endocervical gram stain, and culture. AB - This study compared leukocyte esterase dipsticks (LED) and endocervical Gram stains (EGS) as surrogates for culture diagnosis of gonococcal and chlamydial cervicitis in 495 STD clinic patients. Overall, gonorrhea prevalence was 15.7%; chlamydia prevalence (in the subgroup that was tested) was 17.8%. In diagnosing gonorrhea, LED and EGS performed similarly, with sensitivities of 68% and 76%, respectively, and identical specificities of 44%. In diagnosing gonococcal or chlamydial cervicitis, LED and EGS sensitivities fell to 48% and 47%, respectively, whereas specificities increased to 55% and 75%. These data suggest that, although both tests are imperfect surrogates for gonococcal and chlamydial culture, LED sacrifices little in sensitivity compared with EGS. Because LED does not require ancillary supplies, equipment, electricity, or trained personnel, its use may be feasible when Gram-stain diagnosis is impossible. Modifications of LED technology and specimen preparation should be sought to improve LED performance. PMID- 1722913 TI - [Differential cytokeratin expression in cultivated smooth muscle cells of primary and re-stenosed plaque tissue?]. PMID- 1722914 TI - [Hemorheologic and clinical in vitro and in vivo studies of the effectiveness of isovolemic hemodilution using Gelafusal and Infukoll M40 in patients with peripheral arterial occlusive disease]. PMID- 1722915 TI - [Neurogenic malregulation of microcirculation as a cause of chronic pain]. PMID- 1722916 TI - Endoscopic palliation of tracheobronchial malignancies. PMID- 1722917 TI - Vitronectin: a new molecular connection in haemostasis. PMID- 1722918 TI - Interaction study between Org 10172, a low molecular weight heparinoid, and acetylsalicylic acid in healthy male volunteers. AB - Potential interactions between Org 10172 (Lomoparan, i.v. bolus injection of 3,250 anti-Xa units followed by 750 units twice daily s.c. for 8 days) and acetylsalicylic acid (ASA) (500 mg orally 14 and 2 h before i.v. Org 10172 administration) were studied in eight healthy male volunteers using an open, randomised three-way cross-over design. Except for moderate bruising at venepuncture and s.c. injection sites which were equally distributed over all three treatments (Org 10172 alone, ASA alone, Org 10172 and ASA combined), no side effects were observed. The effects of the separate drugs on several haemostatic parameters were as expected, although the prolongations in bleeding time after ASA were highly variable and tended to be somewhat more pronounced after the combination (p greater than 0.05). Org 10172 did not influence the inhibiting effects of ASA on platelet function nor the functional recovery afterwards, as evaluated by thromboxane A2 (TXA2) generation and collagen-induced platelet aggregation. Furthermore, no important interactions were observed with regard to the coagulation tests and plasma anti-Xa activity. Although this study did not entirely exclude small interactions between Org 10172 and ASA in this relatively small group of subjects, these effects are probably without clinical significance. PMID- 1722919 TI - A new HLA-DR2-related specificity (DR2LUM) in South African populations detected using polymerase chain reaction and sequence-specific oligonucleotide typing. PMID- 1722920 TI - [Endosonography of the prostate]. AB - The role of transrectal prostate ultrasonography (TRUS) for screening and early detection is controversial due to its low sensitivity and specificity. The digital rectal examination (DRE) in combination with PSA still remains useful in screening for prostate cancer. Less than 4% of patients with normal DRE and normal PSA have prostate cancer found on random biopsies. We recommend TRUS for exact determination of prostate volume, in patients with suspicious DRE findings, in combination with the Biopty gun for ultrasound-guided biopsies, and in staging before radical prostatectomy. PMID- 1722921 TI - [Diagnosis of localized prostate cancer: screening and preoperative staging]. AB - In 712 patients, mapping of the prostate by six systematic ultrasound-guided core biopsies was performed without major side effects using the "biopyt gun". The histologic findings provided data on patients with normal and those with abnormal prostates on digital rectal examination (DRE). Only 3 of 72 (4%) nonurologic patients with normal prostate-specific antigen (PSA; less than 4 ng/ml) had prostate cancer. In patients with firm prostates on DRE and normal PSA, 13 out of 101 (13%) had prostate cancer. In patients in whom PSA was greater than or equal to 4 ng/ml, 92 of 158 (58%) had prostate cancer. In patients with clinical stage B or C and PSA less than 4 ng/ml, 20/56 (36%) had prostate cancer, compared to 155 of 187 (83%) patients with PSA greater than or equal to 4 ng/ml. Transrectal ultrasound (TRUS) seemed not to be useful in screening for prostate cancer, due to its low specificity of 54%, although in patients with clinical stage B or C TRUS identified 157/175 (90%) patients with prostate cancer. For staging prostate cancer we compared in 103 men with pelvic lymph node dissection the value of digital rectal examination, computerized tomography (CT), magnetic resonance imaging (MRI), PAS, TRUS, and random systematic biopsy for identification of lymph-node-positive patients before radical prostatectomy. CT had a sensitivity of only 7% and a specificity of 96% in detecting lymph nodes, whereas MRI had a sensitivity of 50% and a specificity of 100%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722922 TI - [Incidental prostate cancer: volume, location and degree of differentiation of the tumor in the radical prostatectomy specimen and value of subclassification to stage A1 and A2]. AB - Morphometric analysis was performed on 22 radical prostatectomy specimens of clinical stage A1 and 22 specimens of stage A2 prostate cancers. Of 44 stage A cancers (86%), 38 arose in the transition zone of the prostate, while only 6 were peripheral zone tumors. The subclassification into stages A1 and A2 based on the percentage of cancer in the transurethral resection specimen was not able reliably to separate patients with high-volume stage A cancer from those with low volume stage A cancer. The same was true when the patients were subclassified according to the criteria of the TNM system (TNM 1987). However, all cases (n = 6) with Gleason grade 4 elements in the TUR chips had relatively high-volume residual TUR cancer (greater than or equal to 1.7 cm3) in the radical specimen. Unsuspected cancers unrelated to the incidental prostate cancer were found in 73% of the specimens. The vast majority (87%) were peripheral zone cancers. Eight unsuspected cancers were larger than the Stage A cancer, but only 2 of the 8 were larger than 1 cm3. Our data suggest that the subclassification of stage A into stages A1 and A2 or the subclassification according to the TNM criteria (TNM 1987) does not reliably separate patients who are at risk of cancer progression. Further diagnostic procedures are necessary in these patients. Post-TUR serum PSA levels (Yang) provided valuable additional information in this series. Post-TUR PSA levels increased with increasing residual cancer volume in the prostate. Below a post-TUR PSA of 1 ng/ml, total residual cancer volume was less than 0.4 cm3 in 7 of 8 cases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722923 TI - [Radical prostatectomy and adjuvant endocrine treatment of prostatic cancer with lymphatic metastasis?]. AB - The limits of curability of prostate cancer still have not been exactly defined. Data derived of randomized, retrospective comparative studies of patients with positive lymph nodes suggest an advantage in overall survival and cancer mortality if such tumors are treated by means of radical prostatectomy with immediate adjuvant endocrine therapy. An analysis of such publications, however, shows that the more favourable results are based on the unequal distribution of important prognostic factors. Several publications agree that adjuvant endocrine treatment in N+ disease leads to a prolongation of time to progression which is clinically and statistically significant. Up to now, however, a significant prolongation of survival has not been shown with early endocrine treatment. Patients have a choice between an initial short period of time until progression occurs if endocrine treatment is delayed. During this time they will be sexually potent. On the other hand, for the price of loss of potency and libido an initial longer period of time free of progression can be expected. It is unclear at this moment whether it makes sense to carry out a radical prostatectomy for palliative reasons. To come to a proper decision it is necessary to compare the risk of the untreated primary tumour and the risk of the radical prostatectomy in this situation. This comparison is very difficult and depends on factors which are not ready for comparison at this moment. Local progression under endocrine treatment is relatively rare and can usually be controlled by conservative means (TUR, radiotherapy). At this moment there are insufficient arguments to carry out palliative radical prostatectomy as a routine in lymph node positive patients. PMID- 1722924 TI - A histomorphologic and immunohistochemical study of chordoma in twenty ferrets (Mustela putorius furo). AB - The histomorphologic and immunohistochemical features of chordoma in 20 ferrets were evaluated. The mean age was 3.4 years, and, in the cases for which sex was known, females (n = 10) outnumbered males (n = 5) two to one. All 20 tumors occurred on the tip of the tail. Nineteen of 20 tumors (95%) were composed of three tissue components, often arranged concentrically with lobules of physaliferous cells at the periphery, trabecular bone in the center, and cartilage in between. The bone often contained marrow and hematopoietic cells. One tumor lacked chondromatous or osseous tissue. Immunohistochemical results were consistent with previous studies of chordoma. All 20 tumors (100%) were positive for keratin and vimentin intermediate filaments; 15 (75%) were positive for S-100 protein; and 17 (85%) were positive for neuron specific enolase. This neoplasm shares morphologic and immunohistochemical features with "classic," as well as chondroid chordoma, of human beings, making it a potential animal model. PMID- 1722925 TI - Cytokine modulation of the interaction between bluetongue virus and endothelial cells in vitro. AB - An in vitro model was developed to examine the interaction between endothelial cells and the host inflammatory response in bluetongue virus (BTV) infections. Whole cell enzyme-linked immunosorbent assays, a tritiated thymidine uptake assay, and a colorimetric assay of mitochondrial function were used to assess how four cytokines (interleukin-1, interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) affect endothelial cell metabolism and susceptibility to BTV infection. Concurrent alterations in major histocompatibility complex (MHC) antigen expression were also examined. BTV infection suppressed target cell mitochondrial function and DNA synthesis and enhanced MHC class I expression. Interferon-gamma and tumor necrosis factor alpha suppressed viral antigen expression and were synergistic early in the infection. Interferon gamma enhanced MHC class I and induced MHC class II antigen expression in both BTV infected and uninfected endothelial cells. The other cytokines had minimal effect on endothelial cell surface antigen expression, although interleukin-1 (IL-1) did inhibit cell growth. Infected endothelial cell cultures produced interferon at 20 hours and 40 hours after infection. Electron microscopic analysis confirmed previous findings in other cell lines regarding BTV morphogenesis in endothelial cells, the putative target cell population in vivo. PMID- 1722926 TI - Bronchiolar metaplasia and Ulex europaeus agglutinin I (UEA-I) affinity in Mycoplasma hyopneumoniae-infected lungs of six pigs. PMID- 1722927 TI - Bacterial histiocytic colitis in a lowland gorilla (Gorilla gorilla gorilla). PMID- 1722928 TI - Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen. AB - Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs. PMID- 1722929 TI - Inhibition of human immunodeficiency virus replication in a human T cell line by antisense RNA expressed in the cell. AB - The effect of the expression of antisense RNA against the human immunodeficiency virus (HIV) genome in a human T-cell line CEM on HIV replication was investigated. A 2.7 kilobase (kb) fragment of the HIV genome, including tat and a part of rev and env, was cloned into the retroviral vector WB in the antisense orientation under the SV40 or H-2K promoter. CEM cells transduced with this antisense gene via recombinant retrovirus expressed the RNAs of three different molecular sizes containing the antisense construct. CEM cells and these transduced cells were infected with HIV. HIV replication was evaluated 4-10 days later by an immunofluorescence assay and by determining the reverse transcriptase activity in the culture supernatant. The results indicate that although the recombinant retrovirus WB strongly enhanced the HIV replication in CEM cells, the expression of antisense RNA in the cells was highly effective in impeding the replication of HIV. The inhibitory effect was especially high in CEM cells transduced with the antisense gene under the control of SV40 promoter. In this case, HIV antigen-positive cells and reverse transcriptase activity in the culture supernatant of transduced cells were reduced to 30-50% and 5-10% of those in CEM cells and in the CEM cells transduced with WB, respectively. PMID- 1722930 TI - Identification of viral proteins encoded by two DNA fragments of herpesvirus of turkeys (HVT). AB - Herpesvirus of turkeys (HVT) vaccine is used worldwide to immunize chickens against Marek's disease (MD). Polyclonal antiserum directed against one virus cross-reacts with proteins of the other, while only 5% homology at the DNA level was demonstrated between the two viruses. A partial library of HVT DNA fragments ranging from 1.5 to 13.5 kbp in size was constructed in pBR 322. Under stringent conditions of hybridization (low salt concentration, 10% dextran sulfate at 68 degrees C), two of the cloned HVT DNA fragments (13.5 and 11.0 kbp) hybridized to three MDV DNA fragments: BamHI-C, D, and G. The 13.5 kbp fragment detected two transcripts of 1.8 and 3.0 kb in RNA extracted from HVT-infected chicken embryo fibroblasts, while the 11.0 kbp fragment detected three transcripts of 1.6, 2.0, and 3.0 kb in the extracted RNA. Using hybrid selection, specific RNA was isolated by hybridization with the two cloned HVT DNA fragments and then was translated in vitro using rabbit reticulocyte lysate, and the resulting proteins were analyzed by NaDodSO4-PAGE. The RNA selected by the two HVT DNA fragments coded for a 30 kD protein and for several smaller proteins 10-20 kD in size, while the RNA selected by the 11.0 kb HVT DNA fragment was translated into a 40 kD protein. Immunoprecipitation of these in vitro synthesized proteins with hyperimmune anti-HVT chicken serum showed that they were of viral origin. PMID- 1722931 TI - National Hospital Discharge Survey: annual summary, 1988. 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, conditions diagnosed, and surgical and nonsurgical procedures performed, and by geographic region, bed size, and ownership of hospitals that provided inpatient care. Measurements of hospital utilization are given by frequency, rate, percent, and average length of stay. PMID- 1722932 TI - [The diagnosis of histiocytic lymphomas]. AB - The histogenesis, classification, terminology of histiocytic lymphomas is analyzed. The possibilities are shown of their diagnosis evaluating lectin histochemistry data which is of major importance for the prognosis of the clinical course of the disease. PMID- 1722933 TI - [Structural changes in the stomach, duodenum and pancreas in ischemic heart disease]. AB - The authors carried out a morphological, histochemical and morphometric study of the gastric mucosa, duodenum and pancreas in 22 cadavers of patients who suffered of chronic ischemic heart disease. It was established that patients with chronic ischemic heart disease develop dystrophic, atrophic, sclerotic and compensatory adaptative changes that may lead to marked structural-functional reorganization of the tissues. The morphological basis of trophic disorders are pronounced disturbances of the intramural microcirculatory bed. Changes were revealed of the morphology of the insular apparatus of the pancreas evidencing inclusion of the endocrinous component in the pathogenesis of chronic ischemic heart disease. PMID- 1722934 TI - [New, in Austria registered specialty drugs. Neupogen (G-CSF)]. PMID- 1722935 TI - [Effect of barucainide on left ventricular ejection fraction in patients with ventricular cardiac arrhythmias]. AB - We studied the effect of barucainide, an investigational class lb antiarrhythmic drug, on ventricular arrhythmias and left-ventricular ejection fraction in 10 patients with frequent and complex ventricular arrhythmias (Lown grade 4a/4b). The study was conducted as a single-blind and placebo-controlled trial. With placebo, mean frequency of ventricular arrhythmias was 6238 VPB/24 h, 510 couplets/24 h, and 24 salvos/24 h. Mean left-ventricular ejection fraction was 37.6%, ranging from 18% to 58%. Therapy with barucainide (300-400 mg/day) resulted in a significant reduction of ventricular arrhythmias in 7 of 10 patients; in one patient barucainide had a clear proarrhythmic effect. Over all, left-ventricular ejection fraction (37.6% +/- 12% with placebo vs 36.1% +/- 11% with barucainide) was not significantly altered. In one patient, however, it was depressed by more than 5%; one patient complained of shortness of breath during exercise. None of the four patients with an initial ejection fraction below 35% showed a drop of ejection fraction during therapy with barucainide. The only main adverse effect was a small, but significant (p less than 0.005) rise of serum kreatinine (1.13 +/- 0.26 vs 1.39 +/- 0.38 mg%) in all patients. We conclude that barucainide has a good antiarrhythmic effect and is usually well tolerated in patients with markedly depressed left-ventricular function. The mechanism causing the rise of serum-kreatinin, however, needs to be clarified in further studies. PMID- 1722936 TI - Bovine herpesvirus-1 (infectious bovine rhinotracheitis virus)-based viral vector which expresses foot-and-mouth disease epitopes. AB - A recombinant infectious bovine rhinotracheitis virus (IBRV) vector has been constructed to express bovine growth hormone signal sequence plus a foot-and mouth disease virus [FMDV (O1K)] capsid protein (VP1) epitope as the N-terminal sequence of an IBRV glycoprotein gIII fusion protein on the surface of virus infected cells and on the surface of virus particles. Sequences encoding the first 38 amino acids of IBRV gIII were deleted from the recombinant to avoid redundant glycoprotein signal sequences, but IBRV gIII epitopes detected by anti gIII monoclonal antibodies were retained. Phenotypes were confirmed by in situ immunostaining of virus plaques with anti-FMDV peptide sera, by immunogold staining of permeabilized- and non-permeabilized infected cells, and by virus neutralization experiments with anti-FMDV peptide sera. Vaccination with the IBRV FMDV recombinant induced protective levels of anti-FMDV antibodies in calves and protected them from challenge with virulent IBRV. PMID- 1722937 TI - Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted. AB - The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV). Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals. Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle. Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg. Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure. In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle. Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes. PMID- 1722938 TI - Effects of the nitric oxide synthase inhibitor NG-nitro-L-arginine on the erectile response to cavernous nerve stimulation in the rabbit. AB - Using a rabbit model, the involvement of the L-arginine/nitric oxide pathway in penile erection was investigated. The mean basal intracavernous pressure was 21 cm H2O. Cavernous nerve stimulation (4-8 V, 20-30 Hz) increased the pressure to approximately 130 cm H2O. This response was highly reproducible and usually associated with full penile erection. The pressure increase could be quantified in terms of: (1) the slope of the initial, ascending part of the pressure increase; (2) delta P, which was defined as the maximal pressure obtained by the stimulation minus the basal pressure before the stimulation; (3) T90, which was defined as the time to reach 90 per cent of delta P. Intrapenile administration of the L-arginine/nitric oxide synthesis inhibitor NG-nitro-L-arginine had no effect on systemic arterial blood pressure. However, NG-nitro-L-arginine (0.22 and 2.19 mg), administered via the same route, abolished the erectile response induced by cavernous nerve stimulation; T90 increased and slope and delta P decreased significantly. NG-nitro-D-arginine (2.19), on the other hand, had no inhibitory effect. L-arginine (21.07 mg), given either directly or after NG-nitro L-arginine had no consistent effect on the functional response to cavernous nerve stimulation. The results suggest that pharmacologically induced effects on intracavernous pressure in the rabbit can be described quantitatively, and that this model may be useful to study the mechanisms controlling penile erection in vivo. The pronounced inhibitory action of NG-nitro-L-arginine demonstrates the important role of the arginine/nitric oxide pathway in mediating relaxation of penile smooth muscles necessary for erection. PMID- 1722939 TI - Parotid secretion of fluid, amylase and kallikrein during reflex stimulation under normal conditions and after acute administration of autonomic blocking agents in man. AB - The purpose of this work was to study the effect of graded mechanical and gustatory stimulation on the secretion of the acinar products fluid and amylase and the ductal product kallikrein from the human parotid gland (n = 9). The involvement of parasympathetic and sympathetic nerves in the salivary reflexes was subsequently examined using receptor blocking agents (n = 4). Chewing elevated the secretion of all products as compared to rest (P less than 0.013). When increasing the length of the chewing object, secretion of fluid (P less than 0.013), but not enzymes, further increased. The shift from mechanical to gustatory stimulation with 0.5% citric acid enhanced significantly the secretion of amylase and kallikrein (P less than 0.009), while application of 5.0% citric acid increased the secretion of both acinar products (P less than 0.009) more than kallikrein. A differentiated reflex control of salivation both with regard to input and output was thereby indicated. The muscarinic-cholinergic antagonist oxyphencyclimin reduced median fluid secretion between 54 and 76% depending on the stimuli. During citric acid stimulation, but not during chewing, fluid secretion was reduced about 40% by the beta 1-adrenergic antagonist metoprolol, and about 20% by the alpha 1-adrenergic antagonist prazosin. Median amylase secretion was reduced 30% during chewing and 75% during gustatory stimulation by metoprolol. It was concluded that the masticatory-salivary reflex mainly activated parasympathetic pathways producing saliva of low protein content.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722940 TI - Resiniferatoxin-, capsaicin- and CGRP-evoked porcine coronary vasodilatation is independent of EDRF mechanisms but antagonized by CGRP(8-37). AB - In the present study the effects of activation of capsaicin-sensitive C-fibre afferents by resiniferatoxin and capsaicin as well as the effects of the co stored peptides calcitonin gene-related peptide substance P and neurokinin A on porcine coronary vascular tone in vitro was investigated. Resiniferatoxin, capsaicin, calcitonin gene-related peptide and neurokinin A all evoked a sustained, concentration-dependent vasodilatation of potassium (60 mM) precontracted arteries. Substance P also caused vasodilatation of the precontracted arteries but this effect was transient and tachyphylaxis developed rapidly upon repeated administration. Incubation with the calcitonin gene-related peptide fragment (8-37) did not influence the vascular tone per se but markedly attenuated the dilatory effect of calcitonin gene-related peptide and totally abolished the vasodilatation induced by resiniferatoxin and capsaicin while leaving the effect of neurokinin A and substance P unaltered. Incubation with methylene blue, an inhibitor of endothelium-derived relaxing factor mechanisms, which completely blocked the substance P-evoked vasodilatation, as well as substance P-tachyphylaxis, did not influence the vasodilator response to resiniferatoxin, capsaicin or calcitonin gene-related peptide. The neurokinin A evoked vasodilatation was most likely mediated through activation of neurokinin 1 receptors since it remained unchanged in the presence of the neurokinin 2 receptor antagonist dactinomycin and (Nle10)-neurokinin A (4-10), which selectively activates neurokinin 2-receptors, had only a minor dilatory effect on the precontracted arteries.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722941 TI - [Dimethyl sulfoxide in the treatment of interstitial cystitis]. AB - The paper presents the results obtained with endovesical dimethylsulphoxide in the treatment of interstitial cystitis in 30 women. Up to 80% patients showed clinical improvement with an average of 10 installations. Volume of maximal vesical capacity was increased in 24 patients (80%), the increase being greater than 100 cc in 10 cases. Presently, 24 (80%) patients remain under treatment, 14 with one instillation monthly and 10 once every six months. Six patients are fully asymptomatic without treatment after an average symptoms-free interval of 32 months. Since this is directly related to a decrease both in pain and miction frequency, the increase in vesical capacity appears to be a good improvement index. Dimethylsulphoxide in neither a healing nor a definite therapy but it seems to be effective in the management of this unknown disease. PMID- 1722942 TI - Neuroendocrine responses to L-tryptophan as an index of brain serotonin function: effect of weight loss. PMID- 1722943 TI - Melatonin interaction with the benzodiazepine-GABA receptor complex in the CNS. PMID- 1722944 TI - Effects of tryptophan 2,3-dioxygenase inhibitors in the rat. PMID- 1722945 TI - Acute effects of meals on brain tryptophan and serotonin in humans. PMID- 1722947 TI - Plasma free 5HT, plasma 5HIAA and whole blood 5HT in the rat. PMID- 1722946 TI - Implications of interferon-induced tryptophan catabolism in cancer, auto-immune diseases and AIDS. AB - Tryptophan (Trp) is an indispensable amino acid required for biosynthesis of proteins, serotonin and niacin. Indoleamine 2,3-dioxygenase (IDO) is induced by infections, viruses, lipopolysaccharides, or interferons (IFNs) and this results in significant catabolism of Trp along the kynurenine (Kyn) pathway. Intracellular growth of Toxoplasma gondii and Chlamydia psittaci in human fibroblasts in vitro is inhibited by IFN-gamma and this inhibition is negated by extra Trp in the medium. Similarly, growth of a number of human cell lines in vitro is inhibited by IFN-gamma and addition of extra Trp restores growth. Thus, in some in vitro systems, antiproliferative effects of IFN-gamma are mediated by induced depletion of Trp. We find that cancer patients given Type I or Type II IFNs can induce IDO which results in decreased serum Trp levels (20-50% of pretreatment) and increased urinary metabolites of the Kyn pathway (5 to 500 fold of pretreatment). We speculate that in vivo antineoplastic effects of IFNs and clinical side effects are mediated, at least in part, by a general or localized depletion of Trp. In view of reported increases of IFNs in autoimmune diseases and our earlier findings of elevated urinary Trp metabolites in autoimmune diseases, it seems likely that systemic or local depletion of Trp occurs in autoimmune diseases and may relate to degeneration, wasting and other symptoms in such diseases. We find high levels of IDO in cells isolated from synovia of arthritic joints. IFNs are also elevated in human immunodeficiency virus (HIV) patients and increasing IFN levels are associated with a worsening prognosis. We propose that IDO is induced chronically by HIV infection, is further increased by opportunistic infections, and that this chronic loss of Trp initiates mechanisms responsible for the cachexia, dementia, diarrhea and possibly immunosuppression of AIDS patients. In these symptoms, AIDS resembles classical pellagra due to dietary deficiency of Trp and niacin. In preliminary studies, others report low levels of Trp and serotonin, and elevated levels of Kyn and quinolinic acid in AIDS patients. The implications of these data in cancer, autoimmune diseases and AIDS are discussed. PMID- 1722948 TI - Effects of clomipramine on extracellular serotonin in the rat frontal cortex. PMID- 1722949 TI - Tryptophan metabolism in mice infected with Schistosoma mansoni. PMID- 1722950 TI - Induction of indoleamine 2,3-dioxygenase in human cells in vitro. AB - IFN-gamma is the most effective principle responsible for IDO induction in human cells in vitro. Lymphocyte factors support the effect of other IFN species. Induction of IDO and influence of various factors on IDO is correlated to pteridine synthesis. Cooperative effects of several mediators may account for in vivo observations concerning enhanced excretion of neopterin and tryptophan metabolites in certain disease states. PMID- 1722951 TI - Tryptophan metabolism in healthy subjects: influence of pyridoxine after single or repeated administrations. PMID- 1722952 TI - Increased L-tryptophan, 5-hydroxyindoleacetic acid, 3-hydroxykynurenine and quinolinic acid concentrations in cerebral cortex following systemic endotoxin administration. PMID- 1722953 TI - Effects of 5-hydroxytryptophan on eating behavior and adherence to dietary prescriptions in obese adult subjects. PMID- 1722954 TI - Brain indole metabolism assessed using in vivo dialysis. PMID- 1722955 TI - Plasma free 5HT and platelet 5HT in depression: case-control studies and the effect of antidepressant therapy. PMID- 1722956 TI - Tryptophan and its metabolites in a family with Hartnup disease. PMID- 1722957 TI - Effects of profound insulin-induced hypoglycemia on quinolinic acid in hippocampus and plasma. PMID- 1722958 TI - Heterogeneity of the CD8 lymphocytes in healthy and HIV 1 infected subjects. AB - CD8 lymphocytes have been subdivided on the basis of Leu2 antigen cell surface density and coexpression of Leu4 and Leu11 antigens in two categories: Leu2bright and Leu2dim. Some CD8 lymphocyte phenotypes present in small percentages in some subjects do not come under these subsets. Flow cytometry analysis moreover shows that the distribution of fluorescence intensity tends to aggregate in most subjects in three distinct spots, here called Leu2HD, ID, and LD. The first, defined by fluorescence levels higher than 450, corresponds to the area of the Leu2+ Leu4+ lymphocytes that express the Leu7 antigen. These cells are all Leu11- and reach remarkably higher percentages in subjects with HIV 1 infection. They are totally included in the area of the Leu2bright cells, but their lowest level of fluorescence is higher. Our data seem to indicate a greater phenotypic homogeneity for these cells. The Leu2LD lymphocytes included in fluorescence levels lower than 175 are all Leu4-Leu11+ and 30% coexpress the Leu7 antigen. They are included in the fluorescence area of the Leud2dim cells, but reach medially lower fluorescence levels. The subset named LeuID has a mean fluorescence ranging between 175 and 450 and includes cells of the Leu4+ 11- and Leu4-11+ phenotypes. Moreover, HIV positive subjects exhibit very low percentages of Leu4+ 11+ and Leu4-11- cells. It is interesting to note that the Leu7 antigen density on the CD8 cell surface is highest for the Leu2HD lymphocytes and diminishes proportionally to the Leu2 antigen density in the Leu2ID and Leu2LD lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1722960 TI - Staining with Ziehl's fuchsin of semithin sections mounted on slides. PMID- 1722959 TI - CD8 lymphocytes during Epstein Barr virus (EBV) infection: A CD29 positive population is expanded in acute infectious mononucleosis. AB - The expression of phenotypic markers on CD4 and CD8 lymphocytes during the acute and convalescent phases of Epstein Barr virus (EBV) induced infectious mononucleosis was examined by two colour flow cytometry. Activated CD8 cells constitute the major population increased during acute infectious mononucleosis; in this phase we observed a preferential expansion of the CD8 CD29+ compared to the CD8 CD45RA+ cells. Serum soluble CD8 levels were also raised during the acute phase and a correlation with CD8 CD38+ and CD8 CD29+ cell numbers was found. The convalescent phase of infectious mononucleosis was characterized by a progressive return of CD8 subset and of soluble CD8 to baseline normal values. These results demonstrate that acute EBV infection induces the expansion of a CD8 subset with peculiar surface antigenic profile. PMID- 1722961 TI - [Locoregional treatment of carcinoma of the penis]. AB - Locoregional treatment of penile carcinoma continues to be a controversy between those who advocate early treatment of lymph nodes and those who advocate delaying treatment. The present study reviews 81 cases of penile carcinoma and describes our approach to treatment of the lymph nodes. A study undertaken to compare the different treatment modalities revealed that at 71 months mean follow-up 64% of the patients submitted to inguinal lymphadenectomy were alive and disease free versus 33% of those submitted to radiotherapy. Statistical analyses comparing the survival rates revealed that the likelihood of survival at 5 years was 100% for those patients submitted to prophylactic lymphadenectomy versus 51% for those submitted to therapeutic lymphadenectomy. We can conclude that since nodal metastasis represents a worse prognosis in these patients, it is advisable to perform prophylactic or therapeutic lymphadenectomy early and, furthermore, regional treatment by radiotherapy appears to be of little use. PMID- 1722962 TI - [Prostate-specific antigen as a prostatic tumor marker. Analysis of results in 206 patients]. AB - The results of prostatic specific antigen (PSA) determinations in 206 patients studied as a part of routine prostatic evaluation are presented. In 109 cases histologic evaluation was performed and a clinico-histologic correlation was made in all the 109 patients. We found 57 cases with benign prostatic hyperplasia (BPH), 36 cases with prostatic adenocarcinoma (PADC), 15 patients with prostatitis; and 1 patient with a leiomyoma of the prostate. When the PSA value is between 0-10 ng/ml no differential diagnosis between BPH and PADC can be made because there are no significant differences in these values. But with results above 15 ng/ml the diagnosis of PADC is more evident. Patients with permanent vesical catheter because of acute retention presented high values of PSA and it is possible to misinterpret the results and make an incorrect diagnosis of PADC. PSA is a very useful procedure in the pre and post-operative control of patients with radical prostatectomy. We consider it to be a useful tool in the diagnosis and treatment of cancer of the prostate and used together with the other diagnostic tests available. PMID- 1722963 TI - [Endoscopic cervicotomy in post-prostatectomy sclerosis of the bladder neck]. AB - We treated 23 patients with bladder neck sclerosis following treatment of prostatic adenoma by TUR (20 patients, 87%) and adenomectomy (3 patients, 14%). All patients entered a protocol for bilateral longitudinal bladder neck incision (cervicotomy) and injection of orgotein in the area of incision. Of these, 21 patients were evaluable; 18 (85%) had no recurrence, symptoms remained unchanged without ring in 2 (10%) and there was 1 (5%) recurrence. The results achieved by this simple technique combined with local antiinflammatory therapy make it the treatment of choice for this pathological condition. PMID- 1722964 TI - [Spontaneous subcapsular and perirenal hematoma in a patient undergoing periodic hemodialysis]. AB - Spontaneous renal subcapsular hematoma in patients undergoing hemodialysis is a very uncommon condition that is attributed to acquired cystic disease of the kidneys and other causes. We report on a patient undergoing hemodialysis due to chronic renal failure who developed right spontaneous perirenal subcapsular hematoma ascribable to no underlying pathology. Treatment was by nephrectomy. PMID- 1722965 TI - Neurogenic component of different models of acute inflammation in the rat knee joint. AB - This study was performed to investigate whether different models of acute joint inflammation showed a neurogenic component and to establish whether this is mediated through sensory afferent or sympathetic efferent nerve fibres. Intra articular injection of 2% carrageenan, 20 micrograms substance P, 1% formalin, and 2% urate all produced an inflammatory response. Prior surgical denervation of the joint significantly inhibited this response in the carrageenan and formalin models, but not the others. Pretreatment of the joint with 1% capsaicin (about one week previously) significantly reduced the inflammatory response in all models except formalin. In animals pretreated long term with reserpine (to deplete sympathetic nerve endings of their neurotransmitters) significant reductions occurred in the inflammatory responses to substance P and urate. Intraarticular injection of compound 48/80 produced a marked inflammatory response, which was only significantly reduced by capsaicin pretreatment. These results suggest that both the formalin and carrageenan models of inflammation depend to some extent on the integrity of the sensory innervation of the joint, and thus have a neurogenically mediated component to the inflammatory process they generate. In these models there seems to be little contribution from sympathetic efferent fibres. Each model of inflammation showed a different pattern of response to the pretreatments, suggesting that the mediators of the inflammatory process may differ in each case. PMID- 1722967 TI - A double blind, randomised, multicentre comparison of two doses of intravenous iloprost in the treatment of Raynaud's phenomenon secondary to connective tissue diseases. AB - OBJECTIVE: To compare low (0.5 ng/kg/min) and standard dose (2 ng/kg/min) iloprost (a stable carbacyclin analogue of prostacyclin) in patients with Raynaud's phenomenon secondary to connective tissue disorders. DESIGN: Double blind, random allocation, three six hour infusions on consecutive days. Follow up period eight weeks. SETTING: Rheumatology units, five teaching hospitals. PATIENTS: 55 Patients with Raynaud's phenomenon (greater than seven attacks per week), 32 secondary to well documented classical progressive systemic sclerosis (American Rheumatism Association criteria), 11 CREST syndrome, 5 mixed connective tissue disease, 1 rheumatoid arthritis, 1 Sjogren's syndrome, 1 childhood dermatomyositis, and 4 abnormal nailfold capillaroscopy and antibody profiles but no definite diagnosis. INTERVENTIONS: All other treatment for Raynaud's phenomenon was discontinued two weeks before entry. 28 Patients were randomly allocated to receive the low dose, 27 the standard dose. Differing dilutions allowed infusion rates to be started at 10 ml/h with increments of 10 ml/h every 15 minutes until infusion rates reached 0.5 ng/kg/min and 2 ng/kg/min respectively. MAIN OUTCOME MEASURE(s)--Reduction in frequency, duration, and severity of attacks of Raynaud's phenomenon. Assessment of ulcer and ischaemic lesion healing. RESULTS: Both dosage regimens were equally effective in reducing severity, frequency, and duration of Raynaud's attacks. Ulcer healing occurred to similar degree in both treatment groups (standard dose 44%, low dose 39%). Low dose was associated with significantly fewer side effects. CONCLUSIONS: Both dosage regimens reduce severity of Raynaud's phenomenon and encourage ulcer healing. Low dose was associated with fewer side effects and was better tolerated by the patients. PMID- 1722966 TI - Cross reaction of antibodies to a glycine/alanine repeat sequence of Epstein-Barr virus nuclear antigen-1 with collagen, cytokeratin, and actin. AB - P62 is a synthetic peptide which corresponds to the glycine/alanine repeat sequence of Epstein-Barr virus nuclear antigen-1. It is the main epitope recognised by anti-rheumatoid arthritis nuclear antigen antibodies. It was shown previously that anti-P62 antibodies were raised fourfold in patients with rheumatoid arthritis compared with controls. To examine the possibility that this increase was due to cross reactive autoantibodies binding to P62, anti-P62 antibodies from serum samples taken from 10 patients with rheumatoid arthritis and five healthy controls were purified by affinity chromatography. Immunoglobulin G anti-P62 antibodies purified from four of 10 serum samples from patients with rheumatoid arthritis also reacted with human epidermal keratin, denatured collagen type II and actin, but not with influenza antigens, as determined by enzyme linked immunosorbent assay (ELISA). Anti-P62 antibodies in serum samples from healthy controls and patients with rheumatoid arthritis reacted with epidermal keratin by immunoblotting. It is suggested that antibodies to the glycine/alanine repeat sequence of Epstein-Barr nuclear antigen-1 recognise homologous epitopes on keratin, actin, and collagen. It is also possible that molecular mimicry between a major epitope on the Epstein-Barr virus and several autoantigens might contribute to the breakdown of tolerance and autoimmunity in patients with rheumatoid arthritis. PMID- 1722968 TI - Function and distribution of acetyl- and butyrylcholinesterase in canine tracheal smooth muscle. AB - The total cholinesterase activity in canine tracheal smooth muscle was found to consist of butyrylcholinesterase and acetylcholinesterase in a ratio of 3:1. Most of the acetyl- and butyrylcholinesterase sites were distributed on the muscle surface; the remaining hydrolytic sites were associated with internal structures. Intracellular acetylcholinesterase staining was associated with the perinuclear envelope, sarcoplasmic reticulum and Golgi apparatus. Intracellular butyrylcholinesterase was associated with the perinuclear envelope, sarcoplasmic reticulum and the contractile filaments. Inhibition of acetylcholinesterase by the selective agent 1,5,bis(allyl-dimethylammoniumphenyl)-pentane-3-one dibromide (BW 284C51) led to a parallel leftward shift in the concentration-response curve for bath-applied acetylcholine. A similar shift was observed in the frequency response curve for neurally released acetylcholine. Inhibition of butyrylcholinesterase by the selective agent tetraisopropyl-pyrophosphoramide potentiated the response to bath-applied and neurally released acetylcholine; the potentiation was limited to acetylcholine concentrations greater than or equal to 1 microM and frequencies greater than or equal to 10 Hz. It is concluded that both acetyl- and butyrylcholinesterase participate in the hydrolysis of acetylcholine in canine tracheal smooth muscle. The role of acetylcholinesterase is evident over the entire range of concentrations (1 nM to 100 microM) and frequencies (1 to 90 Hz) examined, whereas the role of butyrylcholinesterase is confined to the higher end of the concentration and frequency ranges used. PMID- 1722969 TI - [Effects of single ethanol administration on liver regeneration in rats]. AB - The effects of a single ethanol administration on liver weight gain, RNA synthesis, DNA synthesis, and polyamine metabolism as parameters of liver regeneration in rat liver after partial hepatectomy were studied. Ethanol, given 1 hr before partial hepatectomy at the dose of 3 g/kg, inhibited RNA synthesis 4 hr after partial hepatectomy and DNA synthesis 24 hr after partial hepatectomy in the liver, but did not affect liver weight gain. Further, we studied the effects of a single ethanol administration on polyamine metabolism in order to elucidate the mechanism of the inhibition of liver regeneration by ethanol. Ethanol inhibited the increase in the putrescine level in the liver 4 and 8 hr after surgery, which was a metabolic product of ornithine decarboxylase. But spermidine and spermidine levels in the liver were not affected by ethanol administration. Hepatic spermidine N1-acetyl transferase activity increased by ethanol administration 6 hr after partial hepatectomy in compensation for the suppression of putrescine. Judging from these results, a single dose of ethanol inhibited liver regeneration polyamine metabolism after partial hepatectomy in rats. PMID- 1722970 TI - Developmental changes in serotonergic neurons by maternal ethanol consumption in the rat offspring. AB - The brain levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5 HIAA), the synthesis rate of 5-HT and 5-HIAA, and the elimination rate of 5-HIAA in the rat offspring brain exposed to ethanol were examined. Ethanol was administered as a drinking water (group L) and in combination with 4 g/kg. p.o. of ethanol (group H) to the pregnant mothers during days 3 to 21 of gestation. There was no difference in brain 5-HT levels between control and groups L and H at 2, 3, 4 and 6-7 weeks of age. A significant decrease in brain 5-HIAA levels was observed at 3 and 6-7 weeks of age in group L and group H, respectively. In the pups of group H, the 5-HT synthesis rate and the elimination rate of 5-HIAA reduced at 4 and 6-7 weeks of age in comparison with the respective control pups. On the other hand, in the pups of group L, the 5-HT synthesis rate increased, and the 5-HIAA synthesis rate reduced at 3, 4 and 6-7 weeks of age. These results suggest that differential exposure to ethanol, such as group L and H, in the CNS during developmental period induces a differential change in the activity of serotonergic system. PMID- 1722971 TI - The influence of endothelium on the action of PGF2 alpha and some dihydropyridine type calcium antagonists in porcine basilar arteries. AB - Vascular endothelium modulates the effect of various vasoconstricting mediators as well as the affinity of dihydropyridine-type calcium entry blockers. To further investigate this influence, vasoconstriction by PGF2 alpha as opposed to KCl and the affinity of nitrendipine and some related 3-ester side-chain derivatives were determined in isolated porcine basilar arteries in the presence and in the absence of intact endothelium, as well as in the presence of methylene blue. Treatment with methylene blue or mechanical endothelial damage increased the contractile work of basilar arteries stimulated by PGF2 alpha and reduced the affinity of the dihydropyridines in such precontracted vessels. Both experimental conditions resulted in nearly the same effect. In addition, the degree of intact endothelium, as determined by substance-P-induced vasodilation, significantly correlated with the corresponding efficacy of all dihydropyridines examined. In contrast, KCl-mediated contractions remained unchanged. It is suggested that the endothelium (probably due to the production and release of endothelium-derived vasorelaxing factors, such as EDRF and/or prostacyclin) may attenuate PGF2 alpha induced transmembrane calcium influx through receptor operated calcium channels, whereas potential operated calcium channels seems to be unaffected. PMID- 1722972 TI - Characterization of the maize Globulin-2 gene and analysis of two null alleles. AB - The most abundant proteins present in maize (Zea mays L.) embryos are saline soluble globulins. A Mr 45,000 globulin component, designated GLB2, is encoded by the Glb2 gene. A cDNA clone corresponding to Glb2 was used as radiolabeled probe to examine the expression of Glb2 in developing embryos and other maize tissues. Glb2 transcripts accumulate during embryo development and are not detectable in germinating kernels. Glb2 transcripts are found only in the developing embryo, and not in endosperm, seedling, or unfertilized ears. Analysis of globulin profiles in embryos homozygous for either a previously described null allele, Glb 2-0, or a novel null allele, Glb2-N1, revealed that these embryos lack not only the GLB2 protein but also globulins of lower molecular mass which may represent processed forms of GLB2. Southern blot analysis of DNA from Glb2-0/0 and Glb2 N1/N1 plants in which a Glb2-specific clone is used as probe indicates that the two null alleles are genetically distinct. PMID- 1722973 TI - Chromosome assignments of the genes for glucocorticoid receptor, myelin basic protein, leukocyte common antigen, and TRPM2 in the rat. AB - We have utilized rat-mouse somatic cell hybrids to make chromosomal assignments for the glucocorticoid receptor (GR), myelin basic protein (MBP), leukocyte common antigen (LCA), and testosterone-repressed prostate message-2 (TRPM2) genes in the rat. The genes for GR and MBP both map on chromosome 18 of the rat, which corresponds to the mapping of both genes on chromosome 18 of the mouse. The gene for LCA maps on chromosome 13, which is where C4b-binding protein beta-chain (C4BPB), coagulation factor V (F5), and renin have previously been assigned. This linkage group appears to be homologous to a substantial portion of mouse chromosome 1 and human chromosome 1q. Finally, the TRPM2 gene has been assigned to rat chromosome 15. PMID- 1722974 TI - Genetics of isocitrate dehydrogenase in Anopheles stephensi. PMID- 1722975 TI - Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. AB - Cancers have a formidable capacity to develop resistance to a large and diverse array of chemical, biologic, and physical anti-neoplastic agents. This can be largely traced to the instability of the tumor cell genome, and the resultant ability of tumor cell populations to generate phenotypic variants rapidly. It is therefore argued that anti-cancer strategies should be directed at eliminating those genetically stable normal diploid cells that are required for the progressive growth of tumors. Microvascular endothelial cells comprising the tumor vasculature represent such a normal cell target. Moreover, specificity for tumor associated vasculature by anti-cancer agents may be achieved by virtue of the fact that many of the endothelial cells that comprise these blood vessels are in an immature, cycling, and 'activated' state, in contrast to the endothelial cells associated with normal tissue and organ blood vessels. PMID- 1722976 TI - RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes. AB - We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes. PMID- 1722977 TI - Resistance to LDL oxidative modifications of an N-terminal apolipoprotein B epitope. AB - The immunoreactivity of human apolipoprotein B (apo B) towards 5 monoclonal antibodies was studied by enzyme immunoassay in native and in vitro oxidized low density lipoproteins (LDL). LDL oxidative modifications were obtained by incubation with either copper ions or an association of lipoxygenase and phospholipase A2. The monoclonal antibodies used in the inhibition analysis were directed to epitopes located in the amino-terminal region (1D1), in the middle part (2D8, L7, 4G3) and in the carboxy-terminal region (L3) of the apo B molecule. The results demonstrated that the immuno-reactivity of 1D1 epitope was little affected by LDL oxidation with copper ions or lipoxygenase plus phospholipase A2, whereas the immunoreactivity of the other epitopes were markedly decreased by these LDL modifications. Immunoreactivity changes were more important in L3 and L7 epitopes than in 2D8 and 4G3 epitopes. Since it is known that L3 and L7 epitopes are located in apo B domains rich in lipid-associated peptides whereas 1D1 is in a domain poor in such peptides, these results suggest a relationship between the lipid environment of an apo B epitope and its susceptibility to alteration by LDL oxidation. PMID- 1722978 TI - Bombesin receptor antagonists. 2. Analogues based on substance P antagonists. AB - Although bombesin (BN) and substance P share only the C-terminal dipeptide amide, some substance P receptor antagonists are also weak bombesin receptor antagonists. In order to increase the selectivity of the antagonism for the BN receptor, a series of hybrid peptides were synthesized by the solid-phase methodology, and screened on 3T3 fibroblasts for binding and mitogenic activity. The analogues inhibiting BN-induced thymidine incorporation were further tested for peripheral (amylase release and urinary bladder contraction) and central activity (grooming behaviour). PMID- 1722979 TI - Effect of drugs on histamine release. AB - The effects of histamine release in the pathogenesis of bronchial obstruction in asthma, and the action of several pharmacological agents on this release--both by prevention of this mediator's secretion and its antagonism, is briefly reviewed. It is concluded that some drugs can prevent either the histamine secretion or antagonize its effects, partially or totally, but some results must be carefully interpreted in what the clinical practice is concerned, as its practical application needs further investigation. PMID- 1722980 TI - A disorganized increase of galanin-like immunoreactivity in cerebral cortex of Alzheimer-type dementia brains. AB - Galanin (GAL), a neuropeptide which has been reported to inhibit the evoked release of acetylcholine, is widely distributed in the mammalian central nervous system. In the present study, GAL-like immunoreactivity (LI) was measured in the cerebral cortex of autopsy brains of patients with the Alzheimer-type dementia and controls using an enzyme immunoassay technique. GAL-LI tended to be increased in the ATD brains and the difference was statistically significant in the frontal cortex. Moreover, although significant correlations of GAL-LI between regions of the cerebral cortex were found in the control brains, no such correlations were found in the ATD brains. These findings suggest a disorganized increase in GAL-LI in the cortex of ATD brains. PMID- 1722981 TI - Evolution of the myelin integral membrane proteins of the central nervous system. AB - The predominant integral membrane protein of the CNS myelin of amphibia, reptiles, birds and mammals is proteolipid protein (PLP) and P0, the main glycoprotein in PNS myelin. Alternative splicing of the transcripts of the single genes of PLP and myelin basic protein (MBP) is the underlying mechanism by which the isoforms of the two main proteins of the myelin membrane arise. DM20 is an isoform of PLP in mammalian, avian and reptilian myelin. It does not occur in the CNS myelin of amphibia. DM20 lacks an extended hydrophilic sequence exposed on the extracytoplasmic surface of the lipid bilayer as a result of the usage of a cryptic donor splice site within exon III. We report about comparative studies on PLP and its DM20 isoform on the protein and DNA level of frog, chicken, rat CNS and the P0-related IP proteins of the CNS of trout. Chemical cleavage at tryptophan residues with N-chlorosuccinimide yields identical patterns of PLP peptides which refers to a high conservation between amphibia, birds and mammals and is totally different from the cleavage pattern of hydrophobic myelin proteins IP-1 and IP-2 of trout CNS and that of P0 of rat PNS. The N-terminal 19 amino acid residues of IP-1 of trout CNS- and P0 of frog PNS myelin were sequenced and proved to be homologous on one hand with the P0 analogue of CNS of the shark, a cartilage fish, and on the other hand with P0 protein of PNS of birds and mammals. The complete amino-acid sequence of chicken CNS PLP was derived from its cDNA. Coding and noncoding segments of the PLP gene of frog were sequenced: there is a high degree of conservation between amphibian and mammalian PLP within the hydrophobic domains. Numerous mutations were found within the part of exon III encoding the hydrophilic domain. Base exchanges within the putative splice site in exon III explain the absence of DM20 in the protein pattern of amphibia CNS myelin. This result is being discussed in view of the membrane organization and the function of PLP. PMID- 1722982 TI - Pancreatic islet-cell epitope recognized by an anti-sulphatide monoclonal antibody. AB - Insulin-dependent (Type 1) diabetes mellitus is recognized as an autoimmune disease and islet-cell antibody (ICA) is present in the majority of patients at diagnosis. ICA labels both beta and alpha cells and is believed to be directed against a glycolipid. In this study we examine the presence of sulphatide (3' sulphogalactosylceramide) or closely related structures (sulpholactosylceramide and seminolipid) in islet cells by means of a monoclonal antibody, Sulph I. Histological examination of pancreatic tissue from Lewis and BB rats, and BALB/c and NOD mice showed a pronounced labelling of the islets of Langerhans with Sulph I. No staining of the exocrine pancreatic tissue, the heart, the liver, the adrenals, the thymus, the spleen or lymph nodes was seen, but staining of some tubular cells and glomerular cells in the kidney as well as of myelin in nerve cells was found. Cytological examination of isolated Lewis islet cells and their cell subpopulations, separated using a fluorescence-activated cell sorter (FACS), showed positive surface labelling of 97.3 +/- 2.2% (SD) of the beta cells and 84.4 +/- 3.0% of the non-beta cells. Thus, the epitope on the glycolipid sulphatide or closely related structures is--with the exception of neural and certain kidney tissue--specifically present in islet cells. Furthermore, the staining pattern of the antibody used, Sulph I, was equivalent to that of ICA. PMID- 1722983 TI - Voltage-sensitive Na+ channels: motifs, modes and modulation. AB - Much recent progress has been made in understanding the structural organization and functional properties of voltage-dependent Na+ channels, in particular in the areas of activation, ion conductance, and inactivation. At the same time, however, electrophysiological studies have revealed new, more complex functional properties in the form of at least two gating modes and the existence of as yet unidentified modulatory factors. PMID- 1722984 TI - Mammalian phosphorylating ion-motive ATPases. AB - The pumps discussed in this review are three members of the phosphorylating class of ion transport ATPases. They are the Na(+)-K(+)-, Ca(2+)- and H(+)-K(+) ATPases. Recent work on their topology, possible transport mechanisms, ion binding sites and role of the different subunits found for the Na(+)-K(+)- and H(+)-K(+)-ATPases is presented, with a suggestion of a unifying 10-membrane segment model for the catalytic subunit of this class of enzyme. PMID- 1722985 TI - Assembly of macromolecular pores by immune defense systems. AB - Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability. PMID- 1722986 TI - Membrane permeability. PMID- 1722987 TI - Insulin-like growth factor binding proteins: structural and molecular relationships. PMID- 1722988 TI - Oral iloprost in healthy volunteers. AB - Iloprost is a potent chemically stable PGI2-mimetic. Therapeutic efficacy was shown after i.v. infusion treatment in several states of peripheral vascular disease. For out-patient therapy an oral dosage form should be developed. Based upon dissolution profiles and in vivo data of a pig model, three different film coated pellet formulations were selected for pharmacokinetic characterization in nine healthy volunteers. In the first part of the study groups of three test subjects were treated with increasing dosages (150-300 micrograms) of iloprost. At 300 micrograms flush and headache led to the discontinuation of those titration. All formulations exhibited dose-dependent serum level profiles. The cross-over characterization in all test subjects showed that one formulation, which exhibited a modified in vitro dissolution of 60% of the dose within 1 h in pH 7.4 phosphate buffer, was optimal from the pharmacokinetic profile. After oral administration of this formulation the bioavailable dose fraction was highest and half-maximal serum levels lasted for 2.4 h (mean); therapeutic serum levels were maintained for 2.1-5.0 h. This formulation was chosen for further investigation to imitate therapeutic serum level profiles as obtained after i.v. infusion for 4 6 h with a once-a-day dosage form. PMID- 1722989 TI - Effect of high-dose corticosteroids on the incidence of deep vein thrombosis after total hip replacement. AB - Fifty patients receiving uncemented total hip prostheses were examined by venography of the legs on the 2nd postoperative day. The patients were randomly divided into two groups, a non-steroid group (n = 26) and a steroid group (n = 24). Both groups received dextran thrombo-prophylaxis. The patients in the steroid group were treated with high-dose corticosteroids. The incidence of deep vein thrombosis (DVT) was 38% (19/50). No patients had clinical signs or symptoms of DVT. All thrombi were located distally in the leg. DVT was bilateral in nine patients, in the operated leg in three, and in the non-operated leg in seven. The administration of high-dose corticosteroids did not influence the incidence or pattern of DVT. All patients were followed up clinically and plethysmographically up to 12 months after surgery. Distally located asymptomatic DVT were not given specific treatment. The postoperative course was uneventful except for one patient in each group who developed clinically, apparent DVT more than 3 weeks after operation, although the initial venographic studies were normal. PMID- 1722990 TI - Treatment of systemic sclerosis. AB - The last few years have brought a resurgence in interest in managing systemic sclerosis. In the past year, there have been reports of small series of patients and several well-designed, double-blind, controlled trials. Treatment has been directed at a variety of potential pathogenic mechanisms. Stanazol, iloprost, plasmapheresis, thyroxine, and calcitonin were felt to have positive effects on vascular aspects of disease, ie, Raynaud's phenomenon. Immune-mediated treatment using antithymocyte and antilymphocyte globulin, cyclosporine, and methotrexate were encouraging in a small number of patients, but controlled studies of plasma exchange, extracorporeal phototherapy, and 5-fluorouracil were not very exciting. Changing fibroblast function was used with some success in some patients with D penicillamine and interferon gamma, but not ketotifen. A very dramatic improvement in the survival of renal crisis occurred with the use of angiotensin converting enzyme inhibitor. Hopefully, this improvement in survival will also occur in the other visceral abnormalities. All this activity in the therapy of systemic sclerosis will certainly lead to improvement in the overall management of disease. PMID- 1722991 TI - Usefulness of antiplatelet drugs in the management of heparin-associated thrombocytopenia and thrombosis. AB - Heparin-associated thrombocytopenia and thrombosis is a severe complication of systemic heparin therapy. Its treatment is mainly based upon discontinuation of heparin therapy. However in some patients requiring emergency cardiac or vascular surgery, reexposure to heparin may be unavoidable. We report the management of two such patients by use of antiplatelet drugs for a vascular procedure. In the two cases, a combination of iloprost, a stable prostacyclin analogue (1 to 2 ng/kg/mn) with aspirin and dipyridamole was shown to inhibit ex vivo the heparin induced platelet aggregation. These antiplatelet agents were continued during the perioperative period. A successful vascular procedure was achieved with full heparinization without subsequent thrombocytopenia or thrombotic or hemorrhagic complications. This experience supports the hypothesis that heparin can be readministered early to patients with heparin-associated thrombocytopenia and thrombosis, provided antiplatelet therapy is given. PMID- 1722992 TI - Platelet activation and alpha granule secretion in type IIB von Willebrand's disease. AB - Type IIB von Willebrand disease is characterized by enhanced ristocetin-induced platelet aggregation, spontaneous platelet aggregation, thrombocytopenia and the absence of the largest plasma von Willebrand factor (vWf) multimers. The absence of the largest plasma vWf multimers is related to their enhanced binding to platelets. The abnormal affinity of the IIB von Willebrand factor to platelets results in thrombocytopenia, but the mechanism is not known. We have studied the platelets from three patients with type IIB von Willebrand disease and have found evidence of platelet activation and alpha granule secretion as defined by increased amounts of von Willebrand factor, fibrinogen and the alpha granule protein PADGEM/GMP-140 on the surface of these platelets. The degree of thrombocytopenia appears to be directly related to the number of platelets with fibrinogen bound to the surface. PADGEM/GMP-140, an alpha granule membrane protein, fuses with the platelet plasma membrane after activation and is a site on platelets which binds to neutrophils or monocytes. This alpha granule protein may play an additional role in platelet clearance and thrombocytopenia in type IIB von Willebrand disease. This may, in part, explain the absence of thromboembolic phenomena despite the presence of activated platelets in patients with type IIB von Willebrand disease. PMID- 1722993 TI - Postnatal transition of gamma-globin gene expression in normal Japanese population. AB - Temporal course of postnatal changes in the gamma isoform composition of human fetal haemoglobin (HbF) was studied in 259 cord, 272 infantile and 216 adult Japanese blood samples. Reversed phase high-performance liquid chromatography was used for determination of the three isoforms of the gamma chain (A gamma T, A gamma I and G gamma), and the adult samples, usually with less than 1% of Hb F, were enriched for Hb F by an alkali denaturation-salting out procedure. The results show that the G gamma gene expression, kept at a higher and strictly controlled level in the neonatal period, undergoes a gradual change during 1 year, beginning 3-4 months after birth, to the adult stage which is characterized by a generally lower and loosely-controlled expression of the gene. Further change seems to occur after 1 year, settling to the final adult level. The A gamma T gene frequency is estimated as 0.139 in the present Japanese adult population, and is essentially identical to the value for the newborns in the same population (0.141). PMID- 1722994 TI - The successful treatment of two cases of severe aplastic anaemia with granulocyte colony stimulating factor and cyclosporine A. PMID- 1722995 TI - Adult T-cell leukaemia in patients with OKT4 epitope deficiency. PMID- 1722996 TI - Determination of the axial ratio of globular proteins in aqueous solution using viscometric measurements. AB - The paper presents the results of viscosity determinations on aqueous solutions of several globular proteins in a wide range of concentrations. On the basis of these measurements a general formula connecting the relative viscosity with concentration and axial ratio of the dissolved proteins was established. By applying the formula the axial ratios of bovine gamma-globulin and horse albumin molecules were calculated. PMID- 1722997 TI - Effect of dietary tryptophan on muscle, liver and whole-body protein synthesis in weaned piglets: relationship to plasma insulin. AB - Two experiments were carried out with piglets, 3-5 kg live weight, to evaluate the effects of feeding a tryptophan (TRP)-deficient diet for 2 weeks on protein synthesis rates measured in vivo 2 h after a meal. In the first experiment on twenty piglets fed on 250 g protein/kg diets, TRP deficiency (0.77 g/16 g nitrogen) as compared with adequacy (1.17 g/16 g N) significantly decreased feed intake, growth performance and fractional protein synthesis rates (FSR), without variation of RNA in longissimus dorsi (LD) and with parallel increases in RNA in semitendinosus (ST) muscle and liver. In the second experiment thirty-two piglets were tube-fed deficient and adequate diets at the two feeding levels (LF) previously achieved. Both TRP and LF significantly increased growth performance and FSR, but not RNA, in LD and ST muscle, with a trend to a synergy between the two factors (TRP x LF interaction). In another muscle, trapezius (TR), the same interaction was only apparent in RNA content. Among the three muscles it was in LD that FSR was the most responsive to dietary TRP (significant muscle x TRP interaction). In the liver the TRP x LF interaction on FSR and not RNA was the major significant effect, indicating that higher TRP and higher LF were both required to get the maximum protein synthesis rate. At 30 min after a meal the same significant interaction effect was shown on plasma glucose, whilst the higher LF increased plasma insulin with both diets. After a further 30 min the appearance of a similar significant effect of the TRP x LF interaction on plasma insulin resulted from its abatement when the deficient diet had been fed at high LF. These results suggest that dietary TRP deficiency decreased muscle and liver protein synthesis rates in relation to a decrease in the post-prandial release of insulin following a decreased rate of nutrient absorption. PMID- 1722998 TI - A DR7 specific monoclonal antibody TAL13.1, raised against a transfectant detects IL-4 upregulated antigen on peripheral B-lymphocytes. AB - A monoclonal antibody TAL13.1 was raised against mouse L cells transfected with the human HLA-DRB1*0701 gene. This antibody was found to be polymorphic recognizing a determinant expressed by the DR7, DRB1*0701 and DRB1*0702 gene products. Four polymorphic sites unique to this specificity have been identified within the DR beta 1 domain. These are residues 11-14, 25, 30 and 71-74, one or a combination of which is postulated as being responsible for conferring the specificity of the antibody. In Western blot analysis TAL13.1 was found to react with the DR alpha beta dimer, but not with the free alpha or beta chains. However, in flow cytometry it failed to bind a DR alpha/DQ beta mixed pair transfectant confirming that it recognizes an epitope on the DR beta not the DR alpha chain. Although TAL13.1, a low affinity antibody is negative or only weakly positive on resting peripheral blood lymphocytes (PBLs), we have demonstrated that by interleukin-4 (IL-4) stimulation we can up-regulate the levels of antigen already present and gain a level of binding comparable to that found on B lymphoid cell lines (B-LCLs) where it has been found to be a valuable reagent in their characterization. PMID- 1722999 TI - Adhesion and growth of cultured human endothelial cells on perfluorosulphonate: role of vitronectin and fibronectin in cell attachment. AB - The suitability of neutralized perfluorosulphonic acid (Nafion) as a surface for the attachment and growth of human cells was investigated in tissue culture. Nafion was equivalent to tissue culture polystyrene (TCP), and markedly better than polytetrafluoroethylene (Teflon), for the attachment and growth of human umbilical artery endothelial (HUAE) cells. The attachment and growth of HUAE cells on Fn-coated Nafion was equivalent to that on Fn-coated TCP. The contribution to the attachment and spreading of HUAE cells that is due to adsorption of serum fibronectin (Fn) or vitronectin (Vn) on to the Nafion or TCP was directly tested by selective removal of Fn or Vn from the serum before addition to the culture medium. HUAE cells seeded on to Nafion or TCP in medium depleted of Vn failed to attach and spread on to these surfaces, as measured after 4 or 24 h of culture. HUAE cells seeded in medium depleted of Fn, but containing Vn, attached and spread on to Nafion, albeit to a decreased extent as compared to that in intact serum when measured after 4 h of culture, and there was no effect of depletion of Fn when measured after 24 h of culture. HUAE cells seeded on to TCP in medium depleted of Fn became attached and spread during 4 h of culture. Our results show that Nafion is a suitable polymeric surface for the attachment and growth of human cells, including endothelial cells. For HUAE cells, adsorption on to the surface of an adhesive glycoprotein, such as Vn or Fn, is an essential step for attachment and spreading of the cells onto the Nafion surface. PMID- 1723000 TI - Enzymatic degradation and immunogenic properties of derivatized dextrans. AB - Dextran T40 was modified with carboxylic, benzylamide and benzylamine-sulphonated groups (samples 1-10). The polymers were incubated with dextranase and the decrease of molecular weight was determined by high performance size exclusion chromatography. It was shown that the higher the substitution of dextrans, the lower their degradability. Modification of dextran with benzylamine and benzylamine-sulphonated groups appeared to hinder the formation of the enzyme substrate complex more than the same quantity of carboxylic groups. The immunogenicity of one of the modified dextrans, containing 54% of carboxylic groups and 19.5% of benzylamine-sulphonated groups, was determined after subcutaneous and intravenous administration into Balb/c mice. The antibody titre was very low even if administered in complete Freund's adjuvant and did not depend on the injected dose. On the average, the titres of antibodies were lower by five orders of magnitude compared to bovine gamma globulin. PMID- 1723001 TI - An autoantibody cross-reactive to hepatitis C virus core and a host nuclear antigen. AB - GOR, an epitope borne by the amino acid sequence, GRRGQKAKSNPNRPL, is recognized by anti-GOR antibodies specifically found in patients with non-A, non-B hepatitis (NANBH). The epitope is not coded for by the hepatitis C virus (HCV), the presumed causative agent for NANBH, but by a single copy gene of the host. Anti GOR antibodies, distinct from anti-HCV (c100-3) antibodies, were revealed to have dual specificities; they target both the presumed core gene product of HCV and a host component. This cross recognition is probably derived from homologous regions between the GOR epitope and a viral epitope on the core protein in HCV. It is therefore suggested that anti-GOR is an autoantibody induced by HCV infection. This may explain the autoimmune disease like aspect of NANBH pathogenesis. PMID- 1723002 TI - Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies. AB - Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125 I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg. PMID- 1723003 TI - Antisense oligonucleotide derivatives as gene-targeted drugs. AB - The strategies and problems involved in designing oligonucleotide derivatives as gene-targeted drugs are discussed. Experiments with isolated and cellular nucleic acids, studies with infected cell cultures, and preliminary animal tests all demonstrate that various derivatives of complementary oligonucleotides (antisense oligonucleotide derivatives) can act as extremely specific and potent inhibitors of gene expression. The design and synthesis of more stable oligonucleotide analogues that can enter mammalian cells and efficiently affect preselected nucleic acids will result in the development of a new generation of drugs, including those with antiviral and anticancer properties. PMID- 1723004 TI - Nucleolar activation during artificial demethylation of DNA in cultured pig embryonic kidney cells. AB - The level of nucleolar activity after incubation of pig embryonic kidney cells with an inhibitor of enzymatic methylation of DNA, 5-azacytidine, which decreased DNA methylation from (3.0 +/- 0.2) to (1.0 +/- 0.3) mol% of 5-methylcytosine in total cytosine, was studied. Addition of 5-azacytidine to the culture medium for 12 or 24 h followed by incubation of the cells in fresh 5-azacytidine-free culture medium for 48-72 h led to the activation of rRNA synthesis. This was evident from the increased level of incorporation of [3H]uridine recorded by biochemical techniques and also from the increase in the intensity with which the nucleoli were stained with silver nitrate and from the changes in their ultrastructure. When the cells were maintained in the presence of sodium butyrate, an inhibitor of histone deacetylases, rRNA synthesis was activated in stationary phase cultures, but the inhibitor had no marked effect on the nucleolar activity of cells which had been preincubated with 5-azacytidine. The results obtained are discussed from the standpoint of the mechanisms associated with the regulation of the transcription of ribosomal genes. PMID- 1723005 TI - Epitope mapping of the low-molecular-mass subunits of reverse transcriptase in human immunodeficiency virus type 1 by monoclonal antibodies. AB - With the aid of monoclonal antibodies to the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1), low-molecular-mass subunits (p29, p32, and p40) were identified in HIV-1 RT purified from HIV (HTLV-IIIB) virions by isoelectric focusing. Epitope mapping with synthetic polypeptides from various regions of the pol gene suggests that the low-molecular-mass subunits result from N-terminal cleavage of the p51 subunit. The subunits could be separated only by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting. They could not be separated on chromatographic columns, suggesting that the subunits are complexed or conformationally arranged in such a way that their separation on the basis of molecular mass is not possible. The molecular mass of the active enzyme eluted from a chromatographic column (Sephacryl S-300) loaded with a mixture of the subunits was estimated to be 100 kDa. PMID- 1723006 TI - The immunogenicity of an antigen-polyelectrolyte complex mixture is higher than that of a general complex carrying a number of antigen molecules. PMID- 1723007 TI - Effects of He-Ne laser irradiation on chromatin properties and synthesis of nucleic acids in human peripheral blood lymphocytes. AB - Irradiation of human peripheral blood lymphocytes with an He-Ne laser (at 632.8 nm) at doses between 28 and 112 J m-2 caused changes in the chromatin during the first 6 h after exposure that were similar to those found after stimulation of the lymphocytes by phytohaemagglutinin (PHA), i.e. it increased chromatin accessibility to the low-molecular-mass ligand acridine orange (AO) and increased incorporation of the labelled RNA precursor [14C]uridine into the cells. The curves of AO-chromatin binding and RNA synthesis after either He-Ne irradiation with an He-Ne laser (56 J m-2) or PHA treatment were multipeak in nature. For the first 6 h after stimulation the curves for the two treatments were similar. After 7 h, the rate of RNA synthesis in laser-irradiated lymphocytes dropped to the control level, whereas in the PHA-stimulated cells [14C]uridine incorporation increased substantially. Unlike the case with PHA, treatment with an He-Ne laser did not induce resumption of DNA synthesis in lymphocytes. Lymphocytes irradiated by laser in the presence of interleukin-2 (IL-2) retained the level of labelled thymidine incorporation characteristic of intact cells cultivated in the presence of IL-2. On the other hand, irradiation by an He-Ne laser produced a potentiating action on the response of peripheral blood lymphocytes to PHA, with thymidine incorporation being stimulated. This effect may explain the mechanism of wound healing by an He-Ne laser radiation: chromatin activation in the cells of wounds and ulcers makes these cells more responsive to the natural stimulators present in tissues. PMID- 1723008 TI - Covalent binding of bleomycin to concanavalin A and immunoglobulin G enhances the ability of the bleomycin-Fe(II) complex to destroy the erythrocyte membrane. AB - The antibiotic bleomycin was examined as a possible component of hybrid molecules composed of an address fragment and a generator of reactive oxygen species. The bleomycin-Fe(II) complex was found to destroy the erythrocyte membrane by generating reactive oxygen. The ability of antioxidants to slow down haemolysis points to a free-radical mechanism for this process. The protective effects of catalase and superoxide dismutase indicate that hydrogen peroxide and the superoxide radical formed on autoxidation of the complex are essential for membrane damage. Haemolytic activity is also exhibited by bleomycin-Fe(III) reduced in the NADPH-cytochrome P450 reductase reaction. The covalent binding of bleomycin to such address molecules as concanavalin A and antierythrocyte immunoglobulin G enhances the ability of the bleomycin-Fe(II) complex to destroy the plasma membrane of erythrocytes. PMID- 1723009 TI - Characteristics of the macromolecular components of the extracellular matrix in human hyaline cartilage at different stages of ontogenesis. AB - The composition of the collagen and proteoglycan components of the extracellular matrix in human rib cartilage under normal conditions at different stages of ontogenesis (from 7 weeks of intrauterine development to 60 years of age) has been analysed. Polyacrylamide gel electrophoresis of collagen CNBr-peptides has shown the presence of type I collagen in embryonal cartilage and a gradual decrease in the quantity of this component relative to type II collagen with increasing age. Analysis of reducible and mature collagen cross-links revealed traces of lysylpyridinoline in addition to hydroxylysylpyridinoline in human rib cartilage. The increase in the content of mature cross-links during ontogenesis was accompanied by a decrease in the content of dihydroxylysinonorleucine. Electrophoretic analysis of proteoglycan monomers revealed four fractions of different mobility and the ratio of these fractions altered in the course of ontogenesis. An increase in the glucosamine/galactosamine ratio in the core proteins was observed with increase in age of the donors. During electrophoretic analysis of the link protein fraction a protein of molecular mass 200 kDa was found. This protein first appeared after 9 weeks of intrauterine development and was present in the rib cartilage at all subsequent stages of embryogenesis. This protein has been identified as tenascin by immunoblotting. PMID- 1723010 TI - Inhibition of contractile activity of rat cardiomyocytes in culture by alpha- and gamma-endorphins. PMID- 1723011 TI - Coagulation changes and the influence of the early perfusate in the course of orthotopic liver transplantation (OLT) when aprotinin is used intra-operatively. AB - The changes in relevant haemostatic parameters during the course of ten orthotopic liver transplantation were studied when aprotinin was given intra operatively. Increases of tissue-type (P = 0.008) and urokinase-type (P = 0.009) plasminogen activators during the anhepatic phase could be correlated with hyperfibrinolysis. Thrombin-antithrombin III complexes (TAT) increased after revascularization of the liver graft (P = 0.003). Parallel studies in the perfusate showed that TAT concentrations were 350% and protease inhibitor activities (antithrombin III, protein C) only 52% of the systemic circulation before reperfusion, suggesting that thrombin activation together with protease inhibitor consumption occurs during graft liver reperfusion. The relatively smaller increases in profibrinolytic parameters and a lower blood loss when compared with other groups may be explained by aprotinin administration in our patients. PMID- 1723012 TI - Modulation of lipopolysaccharide-induced cytotoxic factor and interferon production by moxibustion in mice. AB - Pretreatment of mice with moxibustion (Mox) modulated lipopolysaccharide (LPS) induced endogenous cytotoxic factor (CF) and interferon (IFN) production in serum. CF was measured by the L929 cytotoxicity test and IFN by the cytopathic effect microassay on L929 cells with vesicular stomatitis virus. Significant inhibition of CF activity was observed when Mox and LPS were applied simultaneously. Its potentiation was maximal, about 9 times the control level, when treatment intervals between Mox and LPS were 24-72 hours, and declined thereafter. Mox treatment modified LPS-induced IFN production with a similar biphasic pattern but the onset of modification was delayed. This is the first report of modulation of cytokine production by Mox treatment. PMID- 1723013 TI - Two year experience with transrectal prostate ultrasound. AB - During the last two and a half years, transrectal prostate ultrasound has been used extensively at our institution. Three hundred and twenty patients have been evaluated in a double blind fashion as part of a study comparing digital rectal examination and transrectal prostatic ultrasound. Prostate cancer was detected in twenty three patients (7.2%); 13 had palpable nodules (4.1%) and 10 had non palpable nodules (3.1%). Of the 23 patients, 19 had clinically localized (Stage B) prostate cancer. Clinical and pathologic staging correlated in 15 patients (79%). This compares favorably to clinical staging accuracy of 55% in patients prior to utilization of transrectal prostatic ultrasound. PMID- 1723014 TI - Granulocyte colony-stimulating factor and its receptor. AB - Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein of Mr of about 20,000, which stimulates proliferation and differentiation of progenitor cells of neutrophils. Recent clinical application of G-CSF has proven that this hormone is effective in treatment of patients suffering from neutropenia. In the last few years, the biochemical and molecular nature of the G-CSF receptor has been characterized. The G-CSF receptor is a glycoprotein of Mr 100-130,000, and is expressed on the cell surface of various myeloid cells. A homodimer of this polypeptide can bind G-CSF with a high affinity, and transduce G-CSF-triggered growth signals into cells. Its extracellular domain contains a sequence of about 200 amino acids which can be found in various cytokine receptors. In addition, it contains an immunoglobulin-like domain and three fibronectin type III domains. The overall structure of the beta-chain (gp130) of the interleukin 6 receptor was found to be very similar to that of the G-CSF receptor. PMID- 1723015 TI - The effect of basic fibroblast growth factor on the neovascularisation process: skin flap survival and staged flap transfers. AB - Forty-two male Sprague-Dawley rats were utilised to determine whether the angiogenic property of basic fibroblast growth factor (bFGF) could be applied to improve the survival of the ischaemic portion of a random skin flap and to accelerate the process of staged flap transfer. In the ischaemic flap model, bFGF enhanced the development of vascular connections between the bed and the flap and prevented marginally perfused areas from undergoing necrosis. No effect was observed in the staged reconstruction model using the same dosage of bFGF. A speculative explanation is given for the differential effect of BFGF in these two models. The application of angiogenic factors may improve the survival of the random portion of skin flaps. Further investigations are needed to determine whether exogenously applied angiogenic factors can have a beneficial effect in staged flap reconstructions. PMID- 1723016 TI - Prefabrication of skin flaps using vein grafts: an experimental study in rabbits. AB - Prefabrication provides a new method for creating donor sites which are not limited by natural vascular territories. There are several methods for prefabrication, and these include implantation of greater omentum, blood vessels or muscle flaps. Based on the concept that an arterio-venous (A-V) shunt results in sufficient neovascularisation to support a free flap, we used a rabbit model to investigate the characteristics of these flaps. Prefabrication of an abdominal wall donor site was performed using the left epigastric vein in 20 male New Zealand white rabbits. An 8 x 10 cm skin flap was elevated 10 days after prefabrication, either as an island or a free flap. Survival of the skin flaps exceeded 93% and was independent of position of the vascular pedicle, direction of blood flow, or nature of the flap (island or free flap). Angiograms showed a very rich neovascularisation within the prefabricated flap. PMID- 1723017 TI - Experience with the intraprostatic spiral. AB - Thirty-six patients have been followed up after insertion of an indwelling intraurethral device for prostatic obstruction. Follow-up exceeded 1 year in some cases. The main problem was dislocation of the spiral not only in the early stages but also after several months. Endoscopic assessment of the obstruction seems important during the pre-operative investigations. The ideal indication is prostatic obstruction in patients who are unsuitable for surgery and who need relief of acute retention for a limited time only. In selected patients the prostatic spiral has a place in the treatment of obstructive prostatism. PMID- 1723018 TI - Ultrastructural localization of galanin immunoreactivity in the rat median eminence. AB - The aim of this study was to examine, by use of a pre-embedding immunoperoxidase technique, the ultrastructural localization of galanin immunoreactivity in the external layer of the rat median eminence. Galanin immunoreactivity was only observed in axonal profiles. Immunoreactive fibers were found in contact with the following non-immunoreactive structures: (1) axonal profiles that contain dense granular vesicles and clear vesicles, (2) axonal profiles that contain predominantly clear vesicles, (3) glial cell bodies, and (4) processes of tanycytes. Labeled terminals were also observed in the proximity of the perivascular space of the portal vessels. The results suggest possible interactions between galanin-immunoreactive terminals and other terminals containing peptide and/or other transmitters in the external layer of the median eminence. PMID- 1723019 TI - Staining of glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic nerve constriction injury. AB - The change in staining density of glial fibrillary acidic protein (GFAP) was analyzed in rats that sustained a chronic constriction injury produced by sutures tied loosely around one sciatic nerve. This injury model of peripheral neuropathy resulted in a behavioral hyperalgesia evidenced by a decrease in mean foot withdrawal latency to radiant heat. Increased GFAP immunostaining was observed in the gray matter of the spinal cord ipsilateral to the lesion and specific to spinal segments in which the sciatic nerve is distributed. Elevated GFAP staining density was attributed primarily to hypertrophy of astrocytes rather than their proliferation or migration since counts of astrocyte profiles demonstrated no significant difference when comparing the lesioned to the control side. The magnitude of the increase in GFAP staining correlated with the degree of hyperalgesia. Thus, these data suggest that astrocytes participate in the sequelae occurring in the dorsal horn following constriction injury of a peripheral nerve. PMID- 1723020 TI - Synaptology and origin of somatostatin fibers in the rat lateral septal area: convergent somatostatinergic and hippocampal inputs of somatospiny neurons. AB - This study deals with the synaptology, morphologically identified postsynaptic targets, and origin of somatostatin (SOM) fibers in the rat lateral septal area (LSA) with special reference to those forming pericellular baskets. Septal vibratome sections were immunostained for SOM-14 in 3 experimental groups: control animals, rats subjected to a chronic transection of the ascending afferents to the septum, and animals with acute fimbria-fornix lesion. Light microscopy revealed that the SOM-immunoreactive fibers form pericellular baskets predominantly in the intermediate and ventral parts of the caudal half of the LSA. Electron microscopic analysis showed that the somatospiny neurons are postsynaptic targets of these pericellular baskets. Eight days after a unilateral cut placed at the ventral border of the septum, virtually all SOM-immunoreactive axon terminals disappeared from the ipsilateral intermediate and ventral LSA, and they were substantially reduced in the dorsal LSA. However, in these rats SOM positive neurons could be observed in the LSA on the lesioned, but not on the contralateral side. Furthermore, on the lesion side of the anterior periventricular hypothalamus an increase was detected both in the number and the intensity of immunostaining of SOM-positive neurons. Thirty-six h following a unilateral transection of the fimbria-fornix, the SOM-immunoreactive axon terminals in the LSA remained intact; only immunonegative degenerated hippocamposeptal boutons were detected forming synaptic contacts with somatospiny neurons. Axosomatic synapses of SOM-positive boutons regularly appeared at the neck of somatic spines which were postsynaptic to degenerated hippocamposeptal fibers. The results indicate that the septal SOM fibers are of multiple origin. Those forming pericellular baskets in the LSA originate in ventral extraseptal, probably periventricular hypothalamic areas. SOM fibers scattered in the dorsal LSA are most likely processes of local SOM neurons. The accumulation of immunoreactive SOM in some cells of the undercut septum is a sign of axonal lesion, indicating that these neurons project outside the septum. The SOM innervation of somatospiny neurons which also receive hippocampal input and have been reported to contain gamma-aminobutyric acid (GABA) may be a morphological substrate of the SOM-related disinhibition in the LSA. PMID- 1723021 TI - Decreased norepinephrine release in anterior hypothalamus of NaCl-sensitive spontaneously hypertensive rats during high NaCl intake. AB - We previously demonstrated that dietary NaCl supplementation reduces endogenous norepinephrine stores and turnover in the anterior hypothalamic area (AHA) of male NaCl sensitive spontaneously hypertensive rats (SHR-S) but not in NaCl resistant control rats and have implicated this mechanism in the pathogenesis of NaCl sensitive hypertension. In the current study, we tested directly the hypothesis that dietary NaCl supplementation decreases the release of norepinephrine from nerve terminals in the AHA of SHR-S using the push-pull perfusion technique. Conscious, freely moving SHR-S and control Wistar-Kyoto (WKY) rats were studied after 2-3 weeks of 8% or 1% NaCl feeding. In the 1% NaCl fed SHR-S, 3-methoxy-4-hydroxyphenylglycol (MOPEG, the major metabolite of norepinephrine in brain) levels averaged 272 +/- 32 pg/10 min; norepinephrine levels, 17 +/- 2 pg/10 min; in the 8% NaCl fed SHR-S, MOPEG levels averaged 72 +/ 7 pg/10 min; norepinephrine levels were 6 +/- 1 pg/10 min. There was a positive linear correlation (r = 0.777; P less than 0.01) between MOPEG and norepinephrine levels in AHA perfusates, indicating that perfusate MOPEG levels provide a useful index of norepinephrine release from AHA nerve terminals. In contrast, MOPEG levels in AHA perfusates were not affected by dietary NaCl intake in control WKY, and in control posterior hypothalamic perfusates, were not affected by dietary NaCl intake in SHR-S.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723022 TI - Effects of colchicine on retrogradely-transported WGA-HRP. AB - The effects of colchicine treatment on retrogradely-transported WGA-HRP were examined in the cat. The ventroposterolateral nucleus of the thalamus was bilaterally injected with WGA-HRP followed 2 days later by a unilateral injection of colchicine into the dorsal column nuclei (DCN). The cats were sacrificed by perfusion 24 h later. Retrogradely-transported WGA-HRP within DCN neurons was visualized in coronal, vibratome-cut sections of the medulla using the procedure of Rye and collaborators (J. Histochem. Cytochem., 32 (1984) 1145-1153). On the uninjected side, as expected, the reaction product filled numerous neuronal perikarya and their dendrites. In contrast, on the colchicine-treated side, the reaction product was restricted to dendrites; little labeling was observed within perikarya. These findings appear to reflect colchicine's effects on the translocation of lysosomes from neuronal perikarya to their dendrites are important for the interpretation of data from experiments using colchicine to enhance perikaryal immunohistochemical staining. PMID- 1723023 TI - The effect of a new immunosuppressive agent, FK-506, on xenogeneic neural transplantation in rodents. AB - This study examines the effect of a novel immunosuppressive agent FK-506 (FK) on the survivability of embryonic (E14) rat ventral mesencephalic tissue after intracerebral grafting to the lateral ventricle of adult mice. The recipient mice were given FK in doses of 10 mg/kg or 1 mg/kg for 2 weeks postgrafting, at which time they were sacrificed and histologically processed except for one group of animals on the high dose (10 mg/kg). In this group most animals died from side effects of the drug during the following days. Only the mice receiving the high dose of FK displayed healthy grafts without signs of rejection. PMID- 1723024 TI - Cyclic AMP modulates sensory-neural communication at the vestibular end organ. AB - Adenosine 3':5'-cyclic phosphate (cAMP) is a second messenger that plays an important role in mediating neuronal interactions in many systems. A possible role for cAMP in sensorineural communication at the vestibular end organ was studied. The putative roles for cAMP action investigated here were: the ability of cAMP to act as the second messenger for the efferent transmitter, acetylcholine, and the possible involvement of cAMP in modulating spontaneous or mechanically-evoked afferent nerve firing. Levels of cAMP were increased pharmacologically with forskolin, 3-isobutyl-1-methyl xanthine (IBMX) and dibutyryl cAMP. Changes in multiunit afferent nerve firing measured from the ampullar nerve of the semicircular canal, and the transepithelial potential measured across the neuroepithelium of the semicircular canal were recorded. At selected doses, all drugs produced a similar increase in spontaneous multiunit afferent nerve firing with a concomitant decrease in the transepithelial potential. Mechanically-evoked hair cell activity and the response to exogenously applied acetylcholine were unaffected by these drugs. We are suggesting that the excitatory aspects of the acetylcholine response are not mediated via a cAMP dependent mechanism. However, cAMP does play an important role in modulating spontaneous afferent nerve firing in the semicircular canal. The finding that spontaneous afferent nerve firing can be biochemically modulated without altering mechanically-induced afferent firing is novel and deserves further investigation. PMID- 1723025 TI - Axonal mapping of the giant peptidergic neurons VD1 and RPD2 located in the CNS of the pond snail Lymnaea stagnalis, with particular reference to the innervation of the auricle of the heart. AB - VD1 and RPD2 are two giant neuropeptidergic neurons located respectively in the visceral and right parietal ganglion of the central nervous system (CNS) of the pond snail Lymnaea stagnalis. They are the most prominent representatives of a system of neurons expressing a gene that is similar to the gene expressed in R15 of Aplysia californica. Both neuronal systems are involved in the regulation of cardio-respiratory phenomena. In the present study the axonal branches of VD1 and RPD2 were mapped using immunocytochemical and tracer studies. To this end the alpha 1-antiserum (directed to one of the VD1/RPD2 neuropeptides) was used in combination with Lucifer yellow (LY) and Ni-lys tracers. In whole mount preparations of the CNS, immunostained axons of VD1 and RPD2 were observed to run to the pleural, cerebral and pedal ganglia and in several nerves. Upon LY injection of VD1 thin axon branches were observed in the internal right parietal nerve. These run to the skin in the mantle area near the pneumostome and osphradium. The skin of the lips appeared to receive a similar innervation via the lip nerves. Thick LY filled axons of VD1 and RPD2 were observed in the intestinal nerve. They could be traced to the heart region. The pericardial branch of the intestinal nerve innervates the pericardium and heart (Ni-lys tracing). Immunocytochemically, using the alpha 1-antiserum, it was demonstrated that this nerve branch carries the axons of VD1 and RPD2 to the venous side of the auricle, where they enter the pericardial cavity and ramify in the auricle (but not in the ventricle).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723026 TI - Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons. AB - The mechanisms regulating the expression and localization of excitatory amino acid (EAA) neurotransmitter receptors in neurons of the developing mammalian brain, and roles for these receptors in the plasticity and degeneration of neural circuits are not well understood. We previously isolated and characterized a 71 kDa glutamate binding protein (GBP) from rat brain, and have recently obtained evidence that this GBP is a component of a functional N-methyl-D-aspartate (NMDA) receptor-ion channel complex. We have now used antibodies to this putative NMDA receptor protein to examine its expression and localization, and consequences of its activation in cultured embryonic (18 day) rat hippocampal neurons. Immunocytochemistry and Western blots using monoclonal antibodies to the GBP demonstrated an increase in GBP-positive neurons and their staining intensity with time in culture. GBP was localized to the somata and dendrites of pyramidal like neurons and was sparse or absent in the axons. The expression and compartmentalization of GBP occurred in isolated neurons indicating that direct cell interactions were not required for these processes. Cell surface staining for GBP occurred in patches on the soma and dendrites. The developmental expression of GBP immunoreactivity closely paralleled the expression of sensitivity to NMDA neurotoxicity. There was a direct relationship between GBP immunoreactivity and neuronal vulnerability to glutamate-induced degeneration; vulnerable neurons stained heavily whereas resistant neurons showed either low levels of staining or no staining. Finally, a GBP antiserum greatly reduced NMDA neurotoxicity (but not kainate neurotoxicity). Taken together, these findings demonstrate the expression of presumptive NMDA receptors within a subpopulation of embryonic hippocampal neurons, and their segregation to the soma and dentrites of pyramidal neurons. This spatial distribution of glutamate receptors among and within neurons is likely to play important roles in regulating the structure of neural circuitry during development, and may also be an important determinant of selective neuronal vulnerability in pathological conditions. PMID- 1723027 TI - Aortic dissection presenting as acute pancreatitis: CT diagnosis. AB - A 42-year-old male developed epigastric pain and elevation of serum amylase of 2045 U/L. A contrast-enhanced abdominal CT disclosed inflammatory changes involving the pancreas and peripancreatic tissues and findings indicative of aortic dissection. The possibility of aortic dissection should be considered in the management of patients with acute pancreatitis. PMID- 1723028 TI - X-ray microanalysis of cAMP-induced ion transport in cystic fibrosis fibroblasts. AB - cAMP-induced ion transport in normal and cystic fibrosis (CF) fibroblasts was investigated by X-ray microanalysis. Stimulation with cAMP causes an increase in cellular Na content and a decrease in cellular Cl and K content. No significant difference in response between CF and normal cells was noted. In this respect, fibroblasts differ from epithelial cells, where cAMP-induced Cl- efflux blocked in CF patients. Isoproterenol produced similar changes in Na and K content as cAMP, but did not effect Cl content. PMID- 1723029 TI - Novobiocin and butyrate synergistically enhanced cytokeratin assembly and acetate uptake of human liver cells. PMID- 1723031 TI - Prevention of cerebral stroke by arotinolol in salt-loaded SHRSP. AB - The preventive effects of long-term treatment with arotinolol on the development of cerebral stroke were examined in SHRSP fed a high salt diet. Arotinolol (4.87 mg/kg per day for 20 weeks) prevented cerebral lesions, reduced signs of stroke and delayed early mortality but did not alter blood pressure from control SHRSP, when the administration of the drug was started before the onset of hypertension. At dosage levels similar to arotinolol, both pindolol and labetalol were less effective in preventing cerebral lesions despite lower blood pressure. Propranolol produced no detectable effect on blood pressure or frequency of cerebral lesions. Furthermore, arotinolol (4.27 mg/kg per day) markedly inhibited the development of stroke without blood pressure reduction, when the administration was started after the onset of severe hypertension. These results suggest that arotinolol is more effective in preventing cerebral stroke than pindolol, labetalol and propranolol, and that factors other than blood pressure reduction may be involved in this preventive effect. PMID- 1723030 TI - Conserved beta-tubulin binding domain for the microtubule-associated motors underlying sperm motility and fast axonal transport. AB - An antiserum against tubulin, NS20, has been previously shown to inhibit anterograde and retrograde axonal transport by 50% in vivo and in vitro. We report here that Protein A purified NS20 antibodies also attenuate sperm motility by 50% in demembranated sea urchin sperm. This inhibition is absorbed out by preincubating the NS20 antibodies with a biochemically purified porcine microtubule preparation, with recombinant Trypanosoma beta- (but not alpha-) tubulin and most specifically, with a 37 amino acid (a.a.) synthetic peptide corresponding to a domain near (but not including) the porcine beta-tubulin C terminus. Furthermore, addition of this beta-tubulin peptide alone is sufficient to attenuate motility by 50% in demembranated sperm, indicating that this critical 37a.a. NS20 antigen is a motor binding domain. Together, the results suggest that at least two phenotypically distinct forms of microtubule-based motility, axonal transport and flagellar beating, are homologous at the fundamental level of the microtubule domains (the beta-tubulin peptide and we suggest a distinct but similarly located alpha-tubulin domain) mediating the attachment of tubulin-associated motors. PMID- 1723032 TI - Nine mutations in the cystic fibrosis (CF) gene account for 80% of the CF chromosomes in French patients. AB - Thirteen mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been screened in a French sample of 185 cystic fibrosis (CF) patients, together with their respective associated RFLP haplotypes at the linked D7S23 locus (XV2C and KM19 markers). The respective frequencies of the mutations showed that 9 of them account for 80% of the CF chromosomes. Implications for prenatal diagnosis and heterozygote detection are defined and discussed. The well known great excess of RFLP B marker within CF chromosomes is partially explained by two already characterized mutations highly associated with haplotype B: delta F508 and G542X. Similarly, the excess of haplotype D within CF chromosomes is partially explained by the association between delta I507 and this haplotype. These results may suggest the existence of two still untested or uncharacterized mutations, whose frequencies could be near 1%, one which would be associated with haplotype B and a second which would be associated with haplotype D. The possible cause of the specific association between most of the main different CF mutations and the RFLP haplotype B is discussed. PMID- 1723033 TI - Transperineal ultrasound examination in the evaluation of prostatic size. AB - Eighty patients with benign prostatic hyperplasia underwent transperineal ultrasonographic evaluation of prostatic size and calculation of prostatic volume. In 10 patients transabdominal prostatic ultrasonography was also performed and the findings compared with those of transperineal examination. The estimated prostatic volume measured by transperineal ultrasonography was compared to the actual weight of the surgically removed gland. The correlation coefficient was 0.89 (P less than 0.0001). The transperineal and transabdominal ultrasonographic estimated volumes were also compared. The correlation coefficient was 0.92 (P less than 0.0001). No pain or discomfort were reported by the patients. Transperineal ultrasonography appears to be an accurate, fast and easy method to evaluate prostatic volume. PMID- 1723034 TI - Criteria for use of filgrastim (granulocyte colony-stimulating factor). PMID- 1723035 TI - CD5-T lymphocytes in renal transplant recipients. AB - The CD3+CD5--subpopulation of T cells has been shown to be increased in patients following allogeneic bone marrow transplantation, and a statistical association has been found with graft-versus-host disease (GVHD). We studied this population in renal transplant recipients. There was no correlation with rejection episodes but we found an increase in this CD3+CD5--population in patients on cyclosporin, and we suggest that these cells may be involved in the mechanism of action of this drug. In patients on azathioprine the absolute number of CD3+CD5- lymphocytes is reduced, along with other lymphoid subpopulations. PMID- 1723036 TI - Mineral metabolism in the developing turkey embryo--I. The effects of developmental age and shell-less culture on trace element contents of selected tissues. AB - 1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days. The embryos incubated ex ovo exhibited a progressive decline in the rate of growth and were hypocalcemic and hypoproteinemic compared to their in ovo counterparts from day 18 to day 26 of incubation. 2. The ratio of the concentrations of alpha-fetoprotein and albumin (AFP/A) in serum was determined for both groups of embryos. The AFP/A ratio may be useful as a biochemical index to stage avian embryonic development. Using this index it was concluded that ex ovo embryos exhibited a progressive developmental retardation compared to in ovo embryos. 3. Significant differences were observed in serum trace element concentrations for embryos incubated in ovo vs ex ovo. Most notably, serum copper concentration was significantly lower in ex ovo embryos on days 18 and 22 of incubation and significantly higher on day 26 of incubation compared to serum from embryos incubated in ovo. 4. Livers from embryos incubated ex ovo exhibited significant differences trace element levels compared to those incubated in ovo. By day 26 of incubation the concentration and total amount of zinc and iron were markedly elevated, whereas copper was greatly reduced in the livers of embryos incubated ex ovo compared to the corresponding in ovo levels. 5. Hearts from embryos incubated ex ovo contained less zinc and copper and more iron by day 26 of incubation than those from embryos incubated in ovo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723037 TI - Genotype-treatment interaction between amantadine and chlorpromazine in the mouse. AB - 1. Repeated administration of amantadine prior to chlorpromazine to two different strains of mice altered both locomotor activity, concentrations of brain biogenic amines and selected major metabolites as a function of mouse strain. 2. Amantadine antagonized chlorpromazine effect on motility which was associated with increases in whole brain levels of homovanillic acid in the CDF-1 but not C57BL/6 mice. 3. Conversely, the treatment with amantadine prior to chlorpromazine reduced whole brain normetanephrine and 5-hydroxyindoleacetic acid levels from respective controls in the C57BL/6 and CDF-1 mice, respectively. 4. The results suggest that genetic factors underly differential alteration of brain dopamine and serotonin which may underly the mechanism of amantadine efficacy in neuroleptic-induced extrapyramidal disorders and to the variable responses to amantadine therapy. PMID- 1723038 TI - Stimulation of frog ciliated cells in culture by acetylcholine and substance P. AB - 1. Ciliary beat frequency in epithelial outgrowths from cultured explants of Rana pipiens palate changed markedly from second to second. 2. Acetylcholine (10(-8) to 10(-3) M) and substance P (1.35 x 10(-7) to 1.35 x 10(-5) M) increased and stabilized ciliary beat frequency. The effect of acetylcholine and part of the effect of substance P were blocked by atropine (10(-4) M). 3. Acetylcholine appears to act directly and substance P both directly and indirectly through the release of acetylcholine. PMID- 1723039 TI - Real time recognition of complete atrioventricular blocks. AB - Common heart arrhythmia monitors are limited to the electrical activity of the ventricles. Severe cardiac malfunctions exhibit typical defects of atrioventricular conduction which often can be efficiently treated with medicine or electrotherapy. The method described here enables the detection of complete atrioventricular blocks and may be used for the improvement of arrhythmia monitoring. PMID- 1723040 TI - Collagenase production at the border of granulation tissue in a healing wound: macrophage and mesenchymal collagenase production in vivo. AB - We demonstrated the cells producing collagenase and the time course of collagenase-production at early stages of wound healing, using histology and two immunohistochemical procedures on cross sections of rat skin harvested 0, 3, 5, 7 and 12 days after full-thickness incisions. A monospecific rabbit polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to demonstrate collagenase production. Specificity of this reaction was confirmed by blocking the reaction with excess homogeneously purified antigen. Macrophages were simultaneously labelled using a mouse anti-rat monoclonal antibody recognizing exclusively mature macrophages. Intracellular collagenase was not reliably detectable at day 0, but was prominent at days 3 and 5 and thereafter declined. Double labeling technique showed occasional macrophages producing collagenase in the developing granulation tissue, but most cells labeled as macrophages were negative for collagenase. Most activity was found in fibroblasts adjacent to granulation tissue elements. Since the granulation tissue parallels revascularization in a dendritic pattern, a cross section at three days typically shows an annulus of collagenase-positive cells surrounding a branch of the active granulation tissue. At days 5, 7 and 12 after wounding the pattern of collagenase expression became indistinct as more tissue was involved in the granulation process. However, double-labelling for macrophages and collagenase showed the dichotomy between collagenase expression and presence of macrophages to persist. The finding that collagenase is produced in connective tissue adjacent to granulation tissue suggests an inductive process, possibly due to diffusion of cytokines produced by granulation tissue elements. PMID- 1723041 TI - Nucleoli, nucleolar chromosomes and ribosomal genes in the human spermatocyte. AB - The formation and development of nucleoli and their connections with the nucleolar chromosomes were studied in human spermatocytes using electron microscopy, silver staining of nucleolus organizer regions (NORs), high resolution autoradiography and in situ hybridization in order to localize rRNA genes and their transcription in the different stages of meiotic prophase I. At leptotene, new nucleoli were formed, consisting of a fibrillar centre surrounded by a cap of dense fibrillar component. Following [3H]uridine uptake, label was found only over the dense fibrillar component. In situ hybridization revealed rDNA mainly in the dense fibrillar component and in the chromatin. During zygotene, nucleoli increased in size. The fibrillar centre was connected with the secondary constriction region of the nucleolar bivalent and was partially surrounded by dense fibrillar component. This shell of dense fibrillar component merged into a fibrillo-granular mesh that extended away from the fibrillar centre. Autoradiography following [3H]uridine uptake again showed the label overlaying the dense fibrillar component and the proximal part of the fibrillo granular strands. With in situ hybridization in both the light and electron microscope, signal was mainly found in the dense fibrillar component. A small quantity of label was observed in the peripheral region of the fibrillar centre and in the adjacent chromatin. From early to late pachytene segregation of nucleolar components occurred, with a reduction in the dense fibrillar component that formed a narrow rim around the fibrillar centre with small extensions along the granular component. [3H]uridine incorporation progressively decreased. In situ hybridization showed signal located mainly in the dense fibrillar component and in the chromatin corresponding to the condensed short arm of the nucleolar bivalent. Our results indicate that the majority of rDNA is located and transcribed in the dense fibrillar component; only a small amount is present in the peripheral part of the fibrillar centre and may be transcribed there. Moreover, from leptotene to zygotene, rDNA unravels from the nucleolar chromosome into the nucleolar dense fibrillar component. From zygotene to late pachytene a progressive return to the condensed acrocentric short arm is observed. PMID- 1723042 TI - Excitatory amino acid transmitters in epilepsy. AB - For the majority of human epilepsy syndromes, the molecular and cellular basis for the epileptic activity remains largely conjectural. The principal hypotheses currently concern: defects in membrane ionic conductances or transport mechanisms; defects in gamma-aminobutyric acid (GABA)-mediated inhibitory processes; and enhanced or abnormal excitatory synaptic action. Substantial evidence exists in humans and animals for acquired abnormalities in excitatory amino acid neurotransmission that may participate in the abnormal patterns of neuronal discharge, and this could provide the morphological basis for a recurrent excitatory pathway sustaining seizure discharges in temporal lobe epilepsy. In practice, two approaches appear significant in the suppression of seizures. One is to act postsynaptically on receptors to decrease the excitation induced by glutamate, and the other is to decrease synaptic release of glutamate and aspartate. Agents acting upon adenosine or GABAB receptors decrease glutamate release in vitro but do not have significant anticonvulsant activity, probably because of their predominant actions at other sites. Lamotrigine blocks stimulated release of glutamate and shows anticonvulsant activity in a wide range of animal models. PMID- 1723044 TI - New reduced peptide bond substance P agonists and antagonists: effects on smooth muscle contraction. AB - Following the recent discovery of a new substance P (SP) competitive pancreatic acini cell receptor antagonist containing a reduced peptide bond in place of the C-terminal peptide bond, a new series of full chain and short chain (heptapeptide and hexapeptide) substance P analogues have been prepared in which one of the C terminal-region peptide bonds has been replaced by CH2NH or CH2O groups. They were compared for their ability to recognize NK1 and/or NK2 tachykinin receptor binding sites on guinea pig ileum and rat duodenum smooth muscle preparations, respectively. It was found that all full sequence SP pseudopeptides were agonists with much reduced bioactivity in both tested systems and, in addition, [Gly9 psi(CH2NH)Leu10,Leu11]SP was found to be a relatively selective agonist for NK1 binding sites. Substitution of leucine at position 11 of SP heptapseudopeptides with phenylalanine generated a pseudopeptide with weak agonist activity when Gln at position 5 was replaced by D-Phe, or antagonists when this residue was replaced by D-Nal or D-Cpa. [Leu10 psi(CH2NH)Leu11]SP-(6-11) with Gln at position 6 substituted by D-Phe was a relatively stronger antagonist in both assay systems. These results suggest that, as with several other peptide systems of late, manipulation of the peptide bonds in SP can produce receptor antagonists which in some cases approach the potency of the classic spantide series and, furthermore, that the approach might be used to induce NK receptor specificity in both agonist and antagonist analogs. PMID- 1723043 TI - Biochemical markers associated with the stages of promotion and progression during hepatocarcinogenesis in the rat. AB - Specific biochemical changes occurring during hepatocarcinogenesis have been sought by many investigators. The development of multistage models for hepatocarcinogenesis in the rodent has renewed interest in such marker alterations in preneoplastic as well as neoplastic hepatocytes. Preneoplastic altered hepatic foci (AHF) exhibit specific histomorphologic changes as viewed with tinctorial stains and show a variety of biochemical changes as evidenced by enzyme and immunohistochemistry and by other histochemical markers. During the reversible stage of promotion when AHF are scored by multiple markers, the distribution of markers within these lesions differs with the use of different promoting agents. One interpretation of this finding is that each promoting agent stimulates the replication of a set of initiated cells exhibiting the phenotypic characteristics of a specific programmed phenotype. The same markers score AHF during the stage of progression, but many AHF in this stage are phenotypically heterogeneous, exhibiting in tissue sections a "focus-in-focus" pattern of marker alteration. These latter changes can be correlated with the appearance of karyotypic alterations in preneoplastic hepatocytes. On the other hand, it has been difficult to demonstrate the activation, either mutational or transcriptional, of proto-oncogenes until this stage of progression in rat hepatocarcinogenesis. Thus, a study of biochemical and molecular markers during the stages of hepatocarcinogenesis may lead to a better understanding of potential mechanisms involved in the development of neoplasia through the stages of initiation, promotion, and progression. PMID- 1723045 TI - NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype. AB - The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi. PMID- 1723046 TI - The grasping response in rats: interaction between gamma-type endorphins and haloperidol. AB - gamma-Type endorphins mimic neuroleptics in inducing a grasping response in rats. It was studied whether the haloperidol-induced grasping response was altered after blockade of gamma-type endorphin activity in the rat brain. To achieve this blockade rats were injected i.c.v. with gamma-endorphin antiserum or with a monoclonal anti-idiotype desenkephalin-gamma-endorphin antibody, which may bio inactivate the gamma-type endorphins or block the putative receptors for gamma type endorphins, respectively. The results showed that both treatments attenuated the haloperidol-induced grasping response, particularly 3 h after haloperidol treatment. The influence of these antibodies appeared to be specific, since other sera were without effect. Thus there may be an interaction between the endogenous gamma-type endorphin activity and the haloperidol-induced grasping response. PMID- 1723047 TI - Effect of extracellular calcium concentration on potency of muscarinic agonists at M1 and M2 receptors in rabbit vas deferens. AB - The M1 potency (decrease in neurogenic response) of carbachol, oxotremorine, muscarine, arecaidine propargly ester (APE) and 4-(4-chlorophenylcarbamoyloxy)-2 butynyltrimethylammonium iodide (4-Cl-McN-A-343) in rabbit field-stimulated vas deferens (prostatic segments) increased up to 15-fold when calcium was lowered (3.5-1.0 nM). The M2 receptor-mediated increase in twitch height in response to carbachol and the M1 affinity of pirenzepine were independent of calcium concentration (2.5 and 1.0 mM). Thus, prostatic segments of rabbit vas deferens can be used successfully at 1.0 mM calcium to determine M1 potencies of agonists and M1 affinities of antagonists without counteracting stimulation of M2 receptors. PMID- 1723048 TI - Changes of serotonin and catecholamines are related to pharmacokinetic alterations of clomipramine in rat brain. AB - When rats received a single i.p. injection of clomipramine (20 mg/kg), clomipramine and desmethylclomipramine were rapidly distributed into the brain and their concentrations were markedly higher in the brain than in the serum, while the concentration of the metabolite in the brain was much lower than that of clomipramine. Chronic administration of clomipramine gradually increased the concentrations of the metabolite in both serum and brain, but did not significantly change those of clomipramine. The levels of serotonin (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites in the brain were compared with the concentrations of clomipramine and desmethylclomipramine at different times. The levels of 5-HT, NE, DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brain did not appear to be affected by changes in the concentrations of the drug and metabolite. The level of 5-hydroxyindole acetic acid (5-HIAA) was reduced following clomipramine injection and the reduced level was maintained during chronic treatment. Chronic treatment for more than 7 days increased the 3-methoxy-4-hydroxyphenylglycol (MHPG) level with no alteration of NE level. This elevation appeared to be induced by desmethylclomipramine. PMID- 1723049 TI - C-terminal neuropeptide Y fragments are mast cell-dependent vasodepressor agents. AB - Neuropeptide Y (NPY) is a well-established vasopressor agent present in sympathetic perivascular nerves. Recently, it was found that high doses of the peptide cause a biphasic pressor-depressor response upon intravenous administration. We now report that C-terminal NPY fragments (NPY-(18-36) and NPY (22-36] given intravenously to conscious or pithed (areflexive) male Sprague Dawley rats mimic the depressor component of the NPY-(1-36) response while displaying very low pressor activity. Additionally, we have found that the depressor component is blocked by the histamine H1-antagonist, mepyramine. Since the fragment, NPY-(22-36), was equipotent with NPY in inducing histamine release from isolated peritoneal mast cells, we conclude that short C-terminal NPY fragments, like NPY itself, act on mast cells to initiate histamine-mediated cardiovascular actions. Such actions may conceivably be accounted for by the abundance of positively charged amino acid residues in the C-terminus. Moreover, these fragments have little affinity for vascular NPY receptors, as indicated by their poor ability to displace iodinated NPY or peptide YY (PYY) from specific binding sites on vascular smooth muscle cells derived from rat aorta. In conclusion, we propose that short C-terminal NPY fragments, which contain several positively charged amino acid residues, retain the ability of NPY to release histamine from rat mast cells while being essentially devoid of direct vascular motor activity. PMID- 1723050 TI - Neurokinins produce selective venoconstriction via NK-3 receptors in the rat mesenteric vascular bed. AB - The vasoactive properties of the neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and some selective analogues were assessed in the arterial and venous mesenteric beds of the rat. Although both sides of the mesenteric vasculature displayed endothelium-dependent relaxation in response to acetylcholine (ACh) or bradykinin (BK) (1 and 10 nmol), SP and the selective NK-1 analogue, [Sar9,Met(O2)11]SP were inactive. Of the three selective neurokinin agonists used, [Sar9,Met(O2)11]SP (NK-1), [beta-Ala8]NKA-(4-10) (NK-2) and [MePhe7]NKB (NK-3), only the latter induced a dose-dependent pressor effect in the venous mesenteric vasculature. Injections of SP and the selective NK-1 and NK 2 analogues at high doses (10 nmol), did not change the perfusion pressure in the mesenteric bed even when the mesenteric vasculature was treated with methylene blue (50 microM) to inhibit the effects of endothelium-derived relaxing factor (EDRF) or with NG-nitro-L-arginine (L-NNA) (20 microM) to inhibit the formation of EDRF or with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate] (CHAPS 20 mM, 30 s) to remove the endothelial layer. In contrast, the vasoconstrictor effects of noradrenaline (NA), angiotensin II (ATII), NKB and [MePhe7]NKB on the venous side of the circulation were enhanced following treatment with L-NNA, methylene blue or CHAPS. The present results suggest that neurokinins act on the rat mesenteric bed by increasing the perfusion pressure of the venous vasculature via activation of NK-3 receptors. Neurokinins are inactive on the arterial mesenteric vasculature.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723051 TI - Tributyltin stimulates reactive oxygen formation in toadfish macrophages. PMID- 1723052 TI - Expression pattern of a hematopoietic proteoglycan core protein gene during human hematopoiesis. AB - Expression of the hematopoietic proteoglycan core protein (HpPG) gene was examined in normal peripheral blood, normal bone marrow, and leukemic peripheral blood leukocytes samples to assess the expression pattern of the HpPG gene in these cells and to ascertain points of regulation of this gene during hematopoiesis. In situ hybridization to normal bone marrow and peripheral blood leukocytes demonstrated that the gene was expressed in the promyelocytes at a approximately two fold greater level than in the segmented neutrophils and the expression decreased as the granulocytes matured. The ratio of expression in the other leukocytes to expression in the segmented neutrophils were as follows: eosinophils/basophils approximately 7; monocytes approximately 2; lymphocytes less than 1. Expression of the HpPG gene during myeloblast differentiation was assessed by Northern blot analysis of acute myelogenous leukemia (AML) RNA samples. The expression of this gene, when compared to the levels in HL-60 cells, was approximately ten fold lower in the poorly differentiated blast cells obtained from three AML patients classified M"0". Conversely, the expression in the more differentiated blast cells obtained from 10 of 11 AML patients classified as M1 and M2 were at levels similar to the levels in HL-60 cells. The expression level found in eight lymphoid leukemias was approximately ten fold or more lower than in HL-60 cells. Gene copy number determination confirmed that the HpPG gene is present in one copy per haploid genome. Thus the HpPG gene's expression pattern denotes a single copy gene being differentially expressed during hematopoiesis with initial regulation occurring very early in this developmental process and an additional up-regulatory event occurring during granule genesis. PMID- 1723053 TI - The distribution of J1/tenascin and its transcript during the development of the avian cornea. AB - J1/tenascin is a glycoprotein that is associated with mesenchymal cell motility and proliferation in a variety of embryonic systems. Immunohistochemistry and Western blotting were used to determine if J1/tenascin is present in developing eye tissues, especially in the developing cornea at the times when the neural crest-derived precursors of the corneal endothelium and keratocytes invade the corneal anlage. Anti-J1/tenascin staining was found in both the primary and secondary corneal stromata, Descemet's membrane, the lens capsule, and in the vitreous body. The distribution of J1/tenascin appears to be limited to a subset of eye matrix that is stained by anti-fibronectin. The cellular origin of J1/tenascin was determined by in situ hybridization. J1/tenascin transcript was detected in the developing corneal endothelium, in corneal fibroblasts just before and during their migration, in the ciliary body, and in the lens. This supports the notions that migrating cells synthesize J1/tenascin as they migrate through matrices rich in collagen and fibronectin, and that some epithelia are also a source of J1/tenascin. PMID- 1723054 TI - Cytokeratin in early hamster embryogenesis and parthenogenesis: reorganization during mitosis and association with clusters of interchromatinlike granules. AB - In vivo obtained golden hamster embryos were used to study, by immunofluorescence and immunoelectron microscopy, the main cytokeratin pattern rearrangements during completion of meiosis and the first cleavage division. Our results point to three major re-organization steps: (1) diffuse immunofluorescent cytokeratin spots characteristic of recently ovulated oocytes rearrange into large cortical patches interconnected by fibrils in one-cell embryos; (2) during mitosis a homogeneous cytokeratin spotty pattern reappears; (3) in two-cell embryos cortical and perinuclear cytokeratin fibrillar networks become prominent. Parthenogenotic oocytes were able to mimic the major cytokeratin patterns observed until the first embryonic mitosis, supporting the concept of a maternally established common response to activation. Despite the lack of fibrillar immunofluorescent reactivity during mitosis, electron microscopy demonstrates persistence of 10 nm filament meshworks. These cytokeratin meshworks often associate with clusters of interchromatinlike granules, which persist in the cytoplasm for a short period after nuclear envelope reassembly. PMID- 1723055 TI - Detection and characterization of chimeric yeast artificial-chromosome clones. AB - Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA. PMID- 1723056 TI - A de novo cystic fibrosis mutation: CGA (Arg) to TGA (stop) at codon 851 of the CFTR gene. PMID- 1723057 TI - Keratin expression in normal vulva, non-neoplastic epithelial disorders, vulvar intraepithelial neoplasia, and invasive squamous cell carcinoma. AB - The expression of AE1, AE2, AE3, and CAM 5.2 antikeratin monoclonal antibodies was investigated in 68 vulvar specimens by an avidin-biotin complex (ABC) method. They included normal vulva (NV, 10), non-neoplastic epithelial disorders (NNED, 31), vulvar intraepithelial neoplasia (VIN, 17), and squamous cell carcinoma (SCC, 10). AE1 weakly stained the basal cell layer in NV, exhibited a uniform suprabasal stain in hyperplasia, failed to stain dysplastic areas in VIN I-II, and was patchy and disorganized in VIN III and SCC. AE2 stained the upper third of the epithelium in NV, NNED, and VIN I-II, but it failed to stain VIN III basaloid and SCC; VIN III bowenoid was focally positive. AE3 offered little information, because it stained all lesions; VIN III and SCC, however, exhibited a patchy and disorganized stain. CAM 5.2 was expressed in only half of the cases of VIN III basaloid and in one case each of VIN I-II and SCC. We conclude that keratin expression varies according to the degree of dysplasia; AE1 may serve to separate certain cases of NNED from VIN I-II; AE2 and CAM 5.2 are useful to differentiate both histologic types of VIN III. PMID- 1723058 TI - Apathetic hyperthyroidism in middle age. AB - Apathetic hyperthyroidism was first described in the medical literature by Lahey in 1931. It is a form of hyperthyroidism found principally in the elderly population. In this disorder the usual hyperkinetic presentation of thyrotoxicosis is replaced by apathy and inactivity, often leading to an erroneous psychiatric diagnosis. Although there is a paucity of literature on apathetic hyperthyroidism, it has been described in the elderly and as an extremely rare complication of hyperthyroid disorder in children. It was described only rarely in middle age. The following case highlights the diagnostic and therapeutic dilemmas encountered in a middle-aged patient who presented with dementia and apathetic hyperthyroidism. PMID- 1723059 TI - Experimental epilepsy in vitro: neuromodulating activity of anti-brain autoantibodies from rats exposed to electroconvulsive shock. AB - Neuromodulating activity of anti-brain autoantibodies obtained from electroshocked (ECS) rats was tested on the neurons of isolated suboesophageal ganglion of the snail Helix pomatia. In 16 out of 18 spontaneously active (pacemaker) neurons, ECS IgG containing anti-brain autoantibodies induced short lasting epileptiform discharges and membrane depolarization. Membrane input resistance and time constant decreased, while membrane capacitance increased after addition of ECS IgG. Amplitude of evoked action potential (AP) decreased, whereas AP duration, rise time and fall time slightly increased. Thus, anti neural autoantibody-positive IgG from rats with experimental epilepsy, but not autoantibody-negative IgG from control rats, significantly affected the bioelectrical properties of the isolated snail neurons. These results suggest that anti-neural autoantibodies present in epileptic animals are capable of influencing in vivo the function of the brain neurons. PMID- 1723060 TI - Differential appearance of autoantibodies to human brain S100 protein, neuron specific enolase and myelin basic protein in psychiatric patients. AB - Sera from psychiatric patients (32 with senile dementia, 56 with Alzheimer's disease, 189 with schizophrenia, 117 with manic-depressive psychoses, 52 with other nonorganic psychoses, 44 with paranoid state, 58 with neurotic depression and 78 with alcoholic syndrome), normal subjects (112 blood donors) and 43 hospitalized elderly patients with chronic cardiac failures without senile syndrome were examined by means of an enzyme-linked immunosorbent assay (ELISA) for the presence of autoantibodies to human brain S100 protein, neuron specific enolase (NSE) and myelin basic protein (MBP). These varied antibrain autoantibodies occurred at different frequencies. The highest incidence of anti S100 and anti-NSE antibodies was in Alzheimer's disease and senile dementia, than in manic-depressive and other nonorganic psychoses, and the lowest in paranoid state, neurotic depression, schizophrenia and alcoholic syndrome. The frequency of anti-S100 autoantibodies was higher than that of anti-NSE. Autoantibodies reacting with MBP were revealed in a very small number of psychiatric patients. In healthy individuals and control cardiac patients, the incidence of antibrain autoantibodies was low. These results suggest a differential correlation between antibrain autoantibodies and psychiatric diseases. PMID- 1723061 TI - T cell can recognize the allospecificities formed by the substitution of amino acids associated with HLA-Bw4/Bw6 public epitopes. AB - Our previous studies clearly showed that HLA-B35 and HLA-Bw53 differed only by the amino acids associated with HLA-Bw4/Bw6 epitopes, in that the former possessed Bw6 and the latter Bw4 epitope. It remains to be known whether T cell can discriminate HLA-B35 from HLA-Bw53, although the difference between these HLA antigens is discriminated by monospecific human alloantisera. To investigate allorecognition of these HLA antigens by T cells, anti-HLA-Bw53 cytotoxic T lymphocytes (CTLs) were generated. Anti-HLA-Bw53 cytotoxic T lymphocytes (CTLs) were generated. Anti-HLA-Bw53 bulk CTLs from an individual with HLA-B35 clearly discriminated HLA-Bw53 from HLA-B35. On the other hand, anti-HLA-Bw53 bulk CTLs from an individual without HLA-B35 revealed weak cross-reactivity with HLA-B35 and HLA-B51. The additional studies using HLA-Bw53 or HLA-B35-specific CTL clones showed that some but not all of the CTL clones definitively distinguish the difference between HLA-Bw53 and HLA-B35. Thus, the allospecificities formed by HLA-Bw4/Bw6 epitopes were discriminated by allogeneic T cells. The present study demonstrated that HLA-Bw4/Bw6 public epitopes play an important role in allorecognition of T cells. PMID- 1723062 TI - The impact of naturally occurring DR3 microvariants, DRw17 and DRw18, on T-cell allorecognition. AB - The limited amino acid sequence differences between the DR3 microvariants, DRw17 and DRw18, are found in the second variable region of the DR beta chain (residues 26 and 28) as well as in framework residues 47 and 86. Using selected responder/stimulator combinations, alloproliferative T-lymphocyte clones (TLC) were generated which recognize either a supertypic DR3-related determinant(s) or only those T-cell recognition determinants created by the four amino acids which differ between DRw17 and DRw18. Results indicate that the microvariation creates potent T-cell recognition determinants while leaving the DR3-related determinant(s) unaffected. Several TLC were generated which recognize the DRw18 molecule strongly and the DRw52c molecule weakly reflecting the sequence similarity between these molecules. In addition, one TLC was generated which recognizes DRw18 and DRw14,Dw9 but not DRw14,Dw16 molecules, a result not predicted by linear amino acid sequence comparisons. The intricate and sometimes unpredictable allorecognition patterns observed demonstrate that the molecular context of a specific amino acid sequence is as important as the actual sequence in forming a T-cell recognition site and, thus, in shaping the immune response profile of a given allele. PMID- 1723063 TI - Fine specificity of the alloantiserum MSD-51: epitope mapping of HLA-DRw53 determinants. AB - HLA-DRw53 is a supertypic specificity expressed by HLA-DR4-, HLA-DR7-, and HLA DR9-positive cells. In the present study, the fine specificity of an HLA-DRw53 specific alloantiserum (MSD-51) was analyzed in serology and by the isoelectric focusing technique. In serology, MSD-51 recognized HLA-DRw53-positive cells with the exception of cells expressing the HLA-DR7/DRw53/DQw9 haplotype. The immunoprecipitation studies and the use of the IgM-reducing agent dithiothreitol revealed that MSD-51 consisted of at least two antibodies: (1) an IgM antibody which reacted with the HLA-DRB4 gene product of HLA-DRw53-positive cells, except HLA-DR7/DRw53/DQw9-expressing cells, and (2) at least one IgG antibody which recognized a linear sequence or conformational structure formed by positions 67 to 70 on the HLA-DRB1 gene product of HLA-DR4- and HLA-DR9-positive cells. These findings demonstrate the complexity of the supertypic HLA-DRw53 antigen analyzed with a serologically well-defined HLA-DRw53-specific alloantiserum. PMID- 1723064 TI - HLA-DR and -DQ allelic sequences in multiple sclerosis patients are identical to those found in the general population. AB - The HLA-DR2/Dw2 haplotype is associated with multiple sclerosis (MS) in the North American Caucasian population. HLA-DRB, -DQA, and -DQB N-terminal domain sequences derived from amplified cDNA in a series of North American Caucasian MS patients were examined to determine if unique or rare class II alleles could be found. In addition, class II allelic sequences were analyzed from clinically discordant, HLA-genoidentical siblings from a multiplex MS family. All alleles observed, whether from HLA-DR2/Dw2 positive or negative individuals, were identical to those most commonly expressed in the general population. These data demonstrate that, if HLA class II truly confers susceptibility to MS, commonly expressed alleles are involved. PMID- 1723065 TI - T-cell clones identify three distinct epitopes associated with HLA-Dw14. AB - Alloreactive T-cell clones were used to study allodeterminants associated with the HLA-DR1, -DR4, and -DRw14 allelic families. Three clones derived by priming against the DR4,Dw14 alloantigen were tested against a panel of HLA-D homozygous B-cell lines and homozygous and heterozygous peripheral blood lymphocytes. Each clone was blocked by monoclonal antibodies specific for HLA-DR, but not HLA-DQ or -DP, molecules, and each showed a unique pattern of allorecognition when tested against the cell panel. Clone 14B appeared to recognize a specific sequence, termed L67-A74, comprised of amino acids in the third hypervariable region of the alpha-helix of the DR beta 1 molecule, and expressed on certain DR1-, DR4-, and DRw14-positive cells. Clone EMO25 recognized the same L67-A74 sequence, but only when expressed on DR4-positive cells, suggesting a role for residues in the first and second hypervariable regions of DR4-positive DR beta 1 molecules in T-cell recognition. Clone EM036 also recognized the L67-A74 sequence, but only when expressed on DR4,Dw14.1-positive cells, implicating residues at positions 57 and 86 of the alpha-helix in T-cell recognition. These results demonstrate the range of specific T-cell responses that are possible against alloepitopes expressed by a single class II allele (Dw14), and are an indication of the diverse regions of the class II molecule that can contribute to allorecognition sites. PMID- 1723066 TI - Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation. AB - CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells. PMID- 1723067 TI - Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase. AB - The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane-associated glycoprotein. Western blot analysis on disulfide bond reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS-polyacrylamide gels. Silver staining of the purified antigen on SDS-polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N-linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N-terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast-mediated bone resorption. PMID- 1723068 TI - Molecular epidemiology of Yersinia enterocolitica O:3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes. AB - Yersinia enterocolitica is a major enteric pathogen associated with a wide variety of clinical and immunologic manifestations, including transfusion associated disease, from which there is a high mortality. Although previously rare in the United States, in the late 1980s Y. enterocolitica O:3 emerged as the predominant serotype in the United States, as it has been in Canada, Europe, and Japan. Epidemiologic investigation of this serogroup has been hampered by the limited availability of a phage typing system and the fact that Y. enterocolitica harbors few plasmids that are useful as strain markers. We therefore analyzed whole-cell DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to study a group of 61 (50 human, 11 porcine) Y. enterocolitica isolates. Initially, 20 different restriction enzymes were used: NciI appeared to give the best discrimination of hybridization banding patterns (ribotypes) within Y. enterocolitica O:3. Ribotyping distinguished seven clones among all the study isolates and four clones within Y. enterocolitica O:3 (53 isolates studied) and clearly differentiated Y. enterocolitica O:3 from Y. enterocolitica O:9; O:1,2,3; O:20; and O:5,27. Most serogroup O:3 isolates belonged to two clones, ribotypes I and II, including 23 of 24 Y. enterocolitica O:3 (13 human, 11 porcine chitterling) isolates recovered from a recent outbreak of Y. enterocolitica in children in Atlanta associated with chitterling preparation and 3 transfusion associated O:3 isolates from the United States. Y. enterocolitica O:3 ribotypes I and II were also isolated in Japan, ribotypes II and IV were isolated in Belgium, and ribotype I was isolated in Canada. Ribotype patterns I and II corresponded to phage types 9b and 8, respectively. Ribotyping was able to distinguish individual strains of Y. enterocolitica O:3, but suggests that a limited number of clones have disseminated within the United States and globally. The finding of identical ribotype patterns in chitterling and human specimens from the Atlanta outbreak supports epidemiologic evidence that swine were the source of infection and major reservoir for Y. enterocolitica O:3. PMID- 1723069 TI - Restriction fragment length polymorphisms in rRNA operons for subtyping Shigella sonnei. AB - Shigella sonnei is the most frequent cause of shigellosis in the United States. Epidemiologic studies of this organism have been hampered by the lack of adequate typing procedures. Ribosomal DNA analysis (ribotyping), a method which analyzes restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has recently been shown to be useful for microbial species identification and subtyping. To determine whether ribotyping could be used to distinguish between S. sonnei isolates, we conducted Southern hybridization studies on isolates from 16 different geographic locations and from four recent outbreaks. S. sonnei genomic DNA fragments generated following digestion with SalI hybridized with Escherichia coli 16S and 23S rRNAs to produce six distinct patterns; strains with patterns 1, 2, and 3 were each further subdivided into two additional patterns by using PvuII, SmaI, and SstI, respectively. Epidemiologically related strains had identical patterns. Ribotyping appears to be a useful tool for epidemiologic studies of shigellosis caused by S. sonnei. PMID- 1723070 TI - Anti-Salmonella lipopolysaccharide monoclonal antibodies: characterization of Salmonella BO-, CO-, DO-, and EO-specific clones and their diagnostic usefulness. AB - To facilitate the identification and serotyping of Salmonella species, we established a wide variety of murine monoclonal antibodies (MAbs) that were reactive with the lipopolysaccharides (LPSs) of Salmonella serogroups B to E. An effective approach for generating LPS-reactive hybridomas was used; this required immunization of mice with LPS-coated bacteria. To screen for diagnostically useful MAbs, the MAbs were tested by enzyme-linked immunosorbent assay against a set of purified LPSs from smooth and rough Salmonella strains. At least four major groups of antibody specificities were identified: Salmonella (i) BO specific, (ii) CO specific, (iii) DO specific, and (iv) EO specific. For a more detailed epitope analysis, a panel of eight different serogroup-specific MAbs which were shown to bind the O-antigenic polysaccharide chains, yielding characteristic ladder patterns in Western blots (immunoblots) against the LPS of Salmonella serogroups B to E, were selected. The availability of various chemically defined LPS structures and Salmonella O-antigen glycoconjugates permitted the definition of O-antigenic polysaccharide epitopes recognized by each MAb that serologically corresponded to factors O3, O4, O5, O6, O7, O8, O9, and O10 on the basis of the Kauffmann-White scheme for Salmonella classification. The diagnostic accuracy of these immunochemically defined O-specific MAbs for Salmonella serotyping was demonstrated by correct identification of all 167 salmonellae (including 72 serotypes from serogroups B to E) among the 294 bacterial strains in a slide agglutination test. No false-positive reactions were detected. PMID- 1723071 TI - Evaluation of acridinium-ester-labeled DNA probes for identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture. AB - The detectability of mycobacteria in culture by the use of nonisotopic, chemiluminescent DNA probes for Mycobacterium tuberculosis and the M. avium-M. intracellulare complex (MAC) was evaluated and compared with that by the use of 125I-labeled DNA probes for the same mycobacteria. In the assay, rRNA-directed DNA probes labeled with acridinium ester (AE-DNA probes) were used. Unhybridized probes were chemically degraded, and the esterified acridinium on the hybridized probes was hydrolyzed by the addition of alkaline hydrogen peroxide solution, resulting in the production of visible light which was measured with a luminometer. The detection limits of the AE-DNA probes were almost the same as those of the 125I-labeled DNA probes. A total of 107 clinical isolates of mycobacteria (47 isolates of M. tuberculosis, 36 MAC, and 24 atypical mycobacteria other than MAC) were tested. The sensitivity and specificity of the AE-DNA probes for M. tuberculosis were 100% both for the conventional method and with the 125I-labeled DNA probe. The sensitivity and specificity of the AE-DNA probes for MAC were 97.2 and 100%, respectively, for the conventional method and were both 100% with the 125I-labeled DNA probes. Because the procedure is simple, reliable, rapid (it can be completed within an hour), and safe (it does not use radioisotopes), it can easily be performed in any clinical laboratory. PMID- 1723072 TI - Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals. AB - We designed a polymerase chain reaction (PCR) for amplifying the Helicobacter pylori gene encoding 16S rRNA. Primers for the specific detection of H. pylori were designed for areas of the 16S rRNA gene in which there is the least sequence homology between H. pylori and its closest relatives. The specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from the campylobacters H. cinaedi, H. mustelae, and Wolinella succinogenes, which are the closest relatives of H. pylori, as determined by 16S rRNA sequencing. Serial dilution experiments revealed the detection of as little as 0.1 pg of DNA by PCR and 0.01 pg by nested PCR. H. pylori DNA was detected successfully in clinical paraffin-embedded and fresh gastric biopsy specimens from patients positive for the bacterium and also in fecal suspensions seeded with the organism. The DNA from the nonculturable coccoid form of H. pylori was also identified by the primers. Universal primers designed for highly conserved areas on the 16S rRNA gene enabled large amplification products to be produced for direct sequencing analysis. Gastric bacteria resembling H. pylori have been isolated from animals. DNA of these animal gastric bacteria amplified with H. pylori-specific primers yielded PCR products identical to those from human isolates of H. pylori, as confirmed by the use of a 20-base radiolabelled probe complementary to an internal sequence flanked by the H. pylori-specific primers. The results of PCR amplification and partial 16S rRNA gene sequence analysis strongly support the contention that the gastric organisms previously recovered from a pig, a baboon, and rhesus monkeys are H. pylori. PMID- 1723073 TI - Immunoblot studies to analyze antibody to the Rickettsia typhi group antigen in sera from patients with acute febrile cerebrovasculitis. AB - In 1986, an unusual syndrome of acute febrile cerebrovasculitis in the Piedmont Region of Virginia was reported. All patients had encephalopathy and prior exposure to both a sylvan environment and flea-infested animals. The initial serological studies suggested a rickettsial origin, corroborating clinical, epidemiological, and histopathological findings. Sera from four of five patients were subsequently studied by immunoblotting. Unabsorbed and absorbed sera were tested with electrophoresed and electroblotted Rickettsia typhi, Legionella bozemanii, and Proteus vulgaris OX19 antigens. The unabsorbed sera reacted with all three antigens. The P. vulgaris- and L. bozemanii-absorbed sera reacted with R. typhi only and without significantly less intensity. In contrast, the reactivity of R. typhi-absorbed sera was significantly lower with all three antigens. These results indicate that these patients had specific antibodies to a typhus group antigen. Although our findings suggest that a rickettsia of the typhus group may have caused this syndrome, no definitive diagnosis could be achieved because a rickettsial organism was not isolated. PMID- 1723074 TI - Evaluation of hepatitis C virus kits. AB - Hepatitis C virus (HCV) antibodies were detected in 85.9% of the samples by commercial enzyme immunoassay (EIA) kits. Most EIA-positive samples were reactive (80%) by the RIBA HCV test (Ortho Diagnostics). Samples with optical density values greater than or equal to 2.0 were mostly reactive (87%) by RIBA HCV test, in contrast to those with values less than or equal to 1.0 (6.6%). Samples which were indeterminate by the RIBA HCV test were positive (88.4%) by HCV neutralization EIA (Abbott Laboratories), along with 29.4% of samples which were nonreactive by the RIBA HCV test. PMID- 1723075 TI - Homotypic and heterotypic serological responses to rotavirus neutralization epitopes in immunologically naive and experienced animals. AB - Gnotobiotic or specific-pathogen-free animals with no previous exposure to rotavirus were vaccinated with strain UK, serotype G6. The highest serological response was to homologous virus; significant but lower responses occurred to viruses with either VP4 or VP7 related to that of vaccine virus; responses to other viruses were of low titer or infrequent. Adult cows vaccinated with UK virus produced increased titers of antibody to all rotavirus serotypes. The increases in titer to homologous virus and to other natural and reassortant viruses sharing VP7 with the vaccine virus were significantly higher than those to all other viruses. These results suggest the presence of common epitopes which are not well recognized in primary infections. PMID- 1723076 TI - Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences. AB - Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA. PMID- 1723078 TI - Monoclonal antibodies to the fusion protein of bovine respiratory syncytial virus. AB - Five monoclonal antibodies specific for bovine respiratory syncytial virus were characterized by Western immunoblotting, radioimmunoprecipitation, and epitope mapping assays. The monoclonal antibodies were found to be specific for the fusion protein, and there were at least two antigen binding sites, one of which was neutralizing. PMID- 1723077 TI - Human cytomegalovirus structural proteins: immune reaction against pp150 synthetic peptides. AB - In the present study, several peptides of the major structural antigen (pp150) of human cytomegalovirus (CMV) have been chemically synthesized and tested by a modified slot blotting procedure for their ability to bind CMV-specific immunoglobulin G (IgG) and IgM present in human sera. The sequences of the peptides were deduced on the basis of either (i) their presence in a fusion protein already known to be frequently recognized by human antibody or (ii) their high content of hydrophilic amino acids as deduced from the published nucleotide sequence. An important IgM-binding epitope was found to be located in the last 38 amino acids at the carboxy terminus of the molecule. This region reacts with anti CMV IgM present in the great majority (83.3%) of IgM-positive human sera, and adsorption experiments have shown that IgM titers to the entire pp150 decrease 25 to 50% in most sera previously absorbed with this region. The overall results obtained endorse the continued synthesis of other sequences in order to define a group of peptides sensitive and specific enough to replace the virus and infected cells as an antigenic substrate in the serological evaluation of anti-CMV antibody. PMID- 1723079 TI - Gold blot for detection of immunoglobulin M (IgM)- and IgG-specific antibodies for rapid serodiagnosis of melioidosis. AB - Gold blot tests for rapid serodiagnosis of melioidosis were developed and evaluated with sera from 40 melioidosis patients and 159 normal controls. The sensitivity and specificity were 87.5 and 88%, respectively, for the immunoglobulin M (IgM) test and 100 and 91%, respectively, for the protein A test for IgG. Combination of the IgM gold blot and protein A gold blot yielded 97.5% sensitivity and 94.3% specificity. The tests were rapid and simple. PMID- 1723080 TI - Pigmented purpuric eruptions: immunopathologic studies supportive of a common immunophenotype. AB - Traditionally, the pigmented purpuric eruptions (PPE) have been subdivided into four histologic categories, based upon variations in histologic pattern and clinical morphologies. In recent years, it has become apparent that the lesions behave similarly, and this family of eruptions has become grouped under the term PPE. The pathogenesis of these eruptions is largely unknown. In this study, we examined 13 examples of the histologic and clinical subtypes of the PPE with a panel of hematolymphoid immunophenotypic markers, in hopes of better understanding the pathogenesis of these curious eruptions. All lesions examined showed a predominantly T cell infiltrate composed of a mixture of OPD4 (CD4) positive and OPD4 negative cells. B cells were rare. Macrophages did not comprise a significant component of the infiltrate. The staining pattern was relatively constant, independent of the histologic pattern (i.e., Schamberg's vs. Gougerot Blum, etc) of the PPE. Our results suggest that PPE are a group of related eruptions which may have a common pathogenesis. It seems likely that the vascular damage and erythrocyte extravasation are secondary to a localized cell-mediated immunologic event. PMID- 1723081 TI - Immunoperoxidase technique modified by counterstain with azure B as a diagnostic aid in evaluating heavily pigmented melanocytic neoplasms. AB - Heavily-pigmented melanocytic neoplasms are difficult to evaluate on routine hematoxylin and eosin stained slides because pigmented melanocytes are difficult to distinguish from the numerous melanophages that are usually seen in the background of these lesions. Immunoperoxidase staining for S100 protein or HMB-45 antibody using diaminobenzidine (DAB) as chromogen, which forms a brown product, does not adequately distinguish melanocytes from melanophages. We modified this technique by replacing hematoxylin as the counterstain with azure B, which stains melanin green-blue. Thus, positive melanocytes appear brown while melanin granules in their cytoplasm are green-blue. However, negative melanophages only stain green-blue. This technique is useful in evaluating heavily pigmented melanocytic lesions such as malignant melanomas, melanosis of regressing malignant melanoma, residual malignant melanoma in areas of granulation tissue with melanophages, blue nevi, pigmented spindle cell variant of Spitz's nevi and combined nevi. PMID- 1723082 TI - Calcium uptake in rat parotid gland secretory granules. AB - 45Ca2+ uptake in isolated rat parotid secretory granules was examined in the presence of oxalate. Uptake of calcium was dependent on time, with the maximum occurring at 15 min. The uptake of calcium was dependent on adenosine-5' triphosphate (ATP), and substitution of ATP with beta, gamma-methylene-ATP did not stimulate calcium uptake. Enzyme marker analysis indicated that mitochondria accounted for no greater than 3.0 +/- 0.2% of the observed ATP-dependent calcium uptake. Calcium uptake was blocked by the ATPase inhibitors tributyltin, IC50 = 12.2 +/- 0.6 nmol/L and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS), IC50 = 3.0 +/- 0.3 mumol/L. These results indicate that in the parotid secretory granule there is a calcium uptake mechanism that is dependent on the hydrolysis of ATP and is suppressed by two inhibitors of granule ATPase. PMID- 1723083 TI - Inhibition of NK cell generation by Corynebacterium parvum. AB - Treatment of mice with Corynebacterium parvum (Cp) resulted in a substantial decrease of splenic NK activity associated with a reduced number of LGL. Cp also inhibited in vitro augmentation of NK cytotoxicity by IFN or IL-2 as well as generation of LAK activity. Localization experiments by using radiolabelled LGL indicated that the lower number of LGL in the spleen was not attributable to a Cp induced alteration of LGL homing. Finally Cp was found to affect the ability of bone marrow cells to reconstitute NK activity in lethally irradiated mice, indicating that it can interfere with development of NK cells from bone marrow progenitors. PMID- 1723084 TI - Comparison of the in vitro and biophysical effects of cyclosporine A, FK-506, and mycophenolic acid on human peripheral blood lymphocytes. AB - The immunosuppressive drugs FK-506 and mycophenolic acid (MPA) have recently been described, but their mode(s) of action are not well understood. We have compared them to cyclosporine A (CsA) in several assays. We have shown that CsA (1 microgram/ml), MPA (0.1 microgram/ml), and FK-506 (0.5 microgram/ml) all induce a state of unresponsiveness to anti-CD3 stimulation as measured by [3H]-thymidine uptake. This suggests that the target of these drugs may be present only after mitogenic stimulation. These drugs also cause a hyperpolarization of the plasma membrane of lymphocytes. This effect is blocked by quinine or verapamil. All three immunosuppressors only slightly modulate the increase in intracellular Ca++ caused by Con-A or by anti-CD3 stimulation but do not affect Ca++ levels alone. They also decrease expression of IL-2 receptors on alpha CD3-stimulated lymphocytes. Similarities in their modes of action, as measured by these biophysical and cell biological tests, indicate the possibility that these three drugs will show similarities in their clinical performance. PMID- 1723085 TI - The interferon family stimulates the secretions of prolactin and interleukin-6 by the pituitary gland in vitro. AB - The effects of interferon-alpha, interferon-beta 1 and interferon-gamma on the secretions of prolactin (PRL) and interleukin-6 by primary cultured rat anterior pituitary cells were examined. These three interferons caused dose-dependent increases in PRL secretion within 30 min, and dose-dependent stimulation of interleukin-6 were weaker than the effects of interleukin-1 and tumor necrosis factor-alpha. These results suggest that interferons regulate PRL secretion from the pituitary gland, and that there may be a pathway in which interferons stimulate PRL secretion through interleukin-6 release. PMID- 1723086 TI - Combined chemotherapy with bleomycin, adriamycin, and platinum in advanced thyroid cancer. AB - Twenty-two advanced consecutive thyroid cancer patients with varying histologies were treated with the so called BAP regime which consisted of bleomycin (B) 30 mg a day for three days, adriamycin (A) 60 mg/m2 iv in day 5, and cisplatinum (P) 60 to mg/m2 iv in day 5. Patients with progressive, symptomatic recurrent or disseminated disease unresponsive to hormonal and/or isotopic treatment were eligible. Nine patients had an objective response: two long-lasting complete and seven partial responses were observed out of 21 evaluable patients. Stable disease was observed in four additional patients. The median duration of response was 12 months (range, 6-29). The total series experienced a median survival of 11 months (range, 1 to 57), with 2 patients actually disease free. Several histologic types of thyroid carcinoma responded, but the best responses were observed in medullary and anaplastic giant-cell carcinomas. Toxicity was reversible in all but one patient. Of the patients failing on BAP chemotherapy three responded to a four drug second line combination containing vincristine, fluorouracil, BCNU and methotrexate. BAP regime can achieve reasonable palliation, and probably increases survival, in poor-prognosis thyroid cancers. PMID- 1723088 TI - Interferon stimulates the expression of 2',5'-oligoadenylate synthetase and MHC class I antigens in insulin-producing cells. AB - The pathogenesis of type 1 diabetes involves autoimmune processes directed against the pancreatic beta-cells. The etiology is not known, but circumstantial evidence suggests a connection between virus infection and development of the disease. Therefore, because the interferon-(IFN) dependent 2',5'-oligoadenylate (2-5A) synthetase system constitutes an important part of the nonspecific immune defense against viral infections, the activity of the enzyme was examined in islets of Langerhans, RIN cells, and GH3 cells. First, the 2-5A synthetase was expressed constitutively in all cell types and, second, all cells were sensitive to stimulation with IFN-alpha. The 2-5A synthetase activity induced by 1,000 U/ml of IFN-alpha increased by 400% in pancreatic islets and by more than 1000% in GH3 and RIN cells. However, the IFN-alpha concentration needed to induce half-maximal 2-5A synthetase activity was nearly the same in the three cell types (i.e., ranging from 59 to 66 U/ml IFN-alpha). The 2-5A synthetase present in islets and RIN cells was highly sensitive to poly (I:C). In pancreatic islets and RIN cells, the 2-5A synthetase enzyme generated dimers and trimers of 2',5'-oligoadenylates. Furthermore, exposure of RIN cells to IFN-alpha showed an increase in MHC class I expression already at 5 U/ml and maximal expression at about 200 U/ml IFN-alpha. The examined endocrine cells express the 2-5A synthetase enzyme as well as MHC class I antigen constitutively, but also by stimulation with IFN in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723087 TI - Ontogeny of angiotensinogen mRNA and angiotensin II receptors in rat brain and liver. AB - The renin-angiotensin-system (RAS) is active in fetal and neonatal life. This study was undertaken to examine the ontogenic regulation of angiotensinogen (AT) gene expression and angiotensin II (A II) receptors in liver and brain. AT gene expression was studied in fetal, neonatal, adult and aged rats, using slot blot hybridization to quantify AT mRNA levels. During fetal life (gestational days 15 20), AT mRNA was more abundant in brain than in liver. Soon after birth, brain AT mRNA levels increased to a concentration 3 fold above fetal levels. In contrast, liver AT mRNA abundance increased 30-fold within 12 h of birth. Aging (3-20 months) resulted in a gradual decrease in AT mRNA in both the brain and liver. Liver A II receptors in the neonate were 2-fold higher than in the fetus, but returned to fetal levels by 8 weeks of age. In the brain, A II receptor abundance increased to a level 75% above fetal levels in 7 days old animals, but returned to fetal levels by 14 days of age. These studies suggest than in the fetus, the liver is not the primary source of AT but that unknown factors at parturition result in a dramatic increase in liver AT mRNA. In contrast, the more modest increases in brain AT mRNA parallel the gradual maturation of the CNS. In both tissues, further aging resulted in a gradual decrease in AT mRNA, reflecting either increased sensitivity to feedback downregulation by A II or age related increases in other extrahepatic sites of AT synthesis. Age related changes were also found in the A II receptor in both the liver and brain. PMID- 1723089 TI - [Surgical therapy of HCG-producing mediastinal tumor accompanied with intrapulmonary metastasis]. AB - The patient is a 31-year-old man who was suffering from hyperthyroidism and left hemothorax. His serum HCG level was extremely elevated and chest X-ray showed a mass shadow of anterior mediastinum and bilateral multiple intrapulmonary metastasis. Our clinical diagnosis was primary HCG-producing germ cell carcinoma of mediastinum and immediately administered CDDP and VP-16. Chemotherapy was effective and tumor extirpation was carried out for mediastinum and lungs by median sternotomy. All of the resected specimen showed no cancer cells and 4 years have passed with no evidence of recurrence. Aggressive surgical approach is indicated for mediastinal germ cell carcinoma accompanied with intrapulmonary metastasis when chemotherapy is effective. PMID- 1723090 TI - [Follow-up studies on hepatoma developing from liver cirrhosis]. PMID- 1723091 TI - [A case of acquired systemic anhidrosis and the relationship to autoimmune diseases]. PMID- 1723092 TI - Glutamate-activated channels in adult rat ventral spinal cord cells. AB - 1. Currents in response to rapid application of glutamate and its agonists were studied in cells dissociated from the ventral spinal cord of adult rats. 2. Glutamate activated an inward current that desensitized in less than 15 ms. 3. Responses to quisqualate and to DL-alpha-amino-3-hydroxy-5-methyl isoxeazolepropionic acid (AMPA) also desensitized with time constants ranging from 7 to 18 ms in whole cell configuration and from 3.4 to 4.3 ms in outside-out configuration. Desensitization rate was independent of membrane potential. Single channel conductance was 12 pS. 4. Currents in response to N-methyl-D-aspartate activation also desensitized; the time constants ranged from 15 to 50 ms. Single channel conductance was 23 pS. 5. Kainate responses did not desensitize appreciably. Single-channel conductance was 17 pS. 6. These data obtained from adult cells are similar to values reported for cultured embryonic and neonatal neurons, indicating minimal postnatal changes in these aspects of glutamate receptors. PMID- 1723093 TI - Relationship of intrinsic connections to forelimb movement representations in monkey motor cortex: a correlative anatomic and physiological study. AB - 1. Intracortical microstimulation (ICMS) and horseradish peroxidase (HRP) histochemistry were combined to examine the relationship between intrinsic connections and intracortical microstimulation sites eliciting evoked movements in the forelimb representation of adult macaque monkey motor cortex. 2. The distribution of sites from which stimulation-evoked movements about individual forelimb joints were elicited under anesthesia varied considerably among animals. Identical movements could often be elicited from multiple, noncontiguous sites. 3. After single, small extracellular HRP injections at sites from which thumb movement was evoked, small groups of retrogradely labeled cells and dense patches of axon terminations were found scattered across a wide area of the forelimb representation. Terminal patches were discontinuous and arose from horizontal, intracortical axons. 4. Correlating the HRP labeling with the physiologically defined movement maps revealed a profuse set of intrinsic, bidirectional connections that connect digit representations and representations of movements about the wrist, elbow, and shoulder. 5. HRP injections placed in the forelimb representation close to the physiologically defined face representation resulted in virtually no retrogradely labeled cells or terminal fiber labeling that crossed into the face representation. A patch of anterolaterally placed label that was present may be the dissociated rostrolateral arm area of other authors. 6. Taken together, these data suggest that extensive, horizontally oriented, intrinsic axon collaterals provide inputs to many different forelimb movement representations and may be recruited during complex movements to coordinate the activity of motor cortical zones whose predominant output is to forelimb muscle groups acting synchronously. PMID- 1723094 TI - Computer simulations of N-methyl-D-aspartate receptor-induced membrane properties in a neuron model. AB - 1. To evaluate the role of N-methyl-D-aspartate (NMDA) receptors in simulations of the lamprey spinal locomotor network, we developed a computer-simulated electrical model of a neuron that contains NMDA channels in addition to voltage gated Na+, K+, and Ca2+ channels and Ca(2+)-activated K+ channels [K(Ca) channels]. 2. The voltage dependence of the Mg2+ block of the Na(+)-K+ current flow through the NMDA channel was modeled according to a scheme of open-channel block. To account for the regulation of K(Ca) channels by NMDA and membrane voltage, we modeled two separate Ca2+ pools that had different voltage dependencies and dynamics. 3. Pacemaker-like membrane potential oscillations could be elicited in the model neuron, which resembled those observed experimentally in the presence of bath-applied NMDA and tetrodotoxin. The effect of changing different channel parameters were tested to determine under which conditions such membrane potential oscillations could occur. 4. The oscillation amplitude was determined by the potential levels at which the NMDA channels and voltage-dependent K+ channels, respectively, were activated. The oscillation frequency and the relative durations of the de- and hyperpolarized phases of the oscillations were determined by the balance between the depolarizing (NMDA channels) and hyperpolarizing [K(Ca) channels] currents. 5. Simulated alterations of the Mg2+ concentration and the K+ conductance as well as injection of constant current caused changes of the oscillations corresponding to those observed experimentally. The de- and hyperpolarizing phases could be reset by brief current pulses. 6. We conclude that the present model can account for the effects of bath-applied NMDA on spinal neurons. This permits an incorporation of NMDA receptor-mediated properties in simulation models of the lamprey locomotor network. PMID- 1723095 TI - Quinolones and fenbufen interact with GABAA receptor in dissociated hippocampal cells of rat. AB - 1. Interaction of quinolone antibiotics and the anti-inflammatory agent fenbufen with the gamma-aminobutyric acid-A (GABAA) receptor-chloride channel complex in pyramidal neurons freshly dissociated from the hippocampal CA1 region of the rats was investigated in whole-cell mode, using the patch-clamp technique under voltage-clamp conditions. 2. Quinolones in clinical doses had no effects on the GABA-gated Cl- current (ICl) but slightly suppressed the response at concentrations greater than 10(-5) M. A metabolite of fenbufen, 4-biphenylacetic acid (BPA), also had little effect on the GABA response at therapeutic concentrations. 3. Coadministration of one of quinolones and BPA suppressed the GABA-gated ICl with increase in each of them in a concentration-dependent manner, and there was a parallel shift of the concentration-response curve for GABA to the right but with no effect on the maximum response, thereby indicating a competitive antagonism. The inhibitory potency of antibiotics in combination with BPA was in the order of norfloxacin much greater than enoxacin greater than cyprofloxacin greater than pipemidic acid much greater than ofloxacin greater than cinoxacin = piromidic acid = nalidixic acid = 0. 4. Norfloxacin and BPA, administered simultaneously, also strongly suppressed pentobarbital sodium (PB) gated ICl, but they did not act on benzodiazepine (BZP) receptors. 5. Both GABA- and PB-induced ICls reversed at the Cl- equilibrium potential (ECl). In the presence of BPA, the quinolone-induced inhibition of GABA-gated ICls showed no voltage dependence. 6. It was concluded that, in the presence of an anti inflammatory agent, the quinolone antibiotics decrease the affinity of GABAA receptors, the result being induction of epileptogenic neurotoxicities. PMID- 1723096 TI - Comparison of two screening methods, modified Hb H preparation and the osmotic fragility test, for alpha-thalassemic traits on the basis of gene mapping. AB - We evaluated 61 patients with two screening tests for alpha-thalassemia traits on the basis of endonuclease gene mapping. Comparing these two methods--the osmotic fragility test of the red cell and modified hemoglobin H inclusion staining for the sensitivity--we found that the latter was much superior to the former with 100% sensitivity in detecting heterozygous alpha-1 thalassemia and it was also specific as a confirmatory test for thalassemia traits. Red cell indices are still the basic screening tool and can be used together with modified Hb H inclusion staining. The osmotic fragility test was not better than the red cell indices and was not confirmatory. Besides the MCV, RBC, and discrimination functions, we found that RBC distribution width-standard deviation (RDW-SD) was consistently low in heterozygous alpha-1 thalassemia but not in heterozygous alpha-2 thalassemia. None of the above tests was shown to be really helpful in screening in the latter situation. We conclude that the modified Hb H inclusion staining is superior to the osmotic fragility test in screening of alpha-1 thalassemia. PMID- 1723097 TI - New method for the determination of pancreatic amylase evaluated. AB - We have carried out a multicenter evaluation of a new reagent carrier for Reflotron, specific for pancreatic amylase where the salivary isoenzyme is inhibited by two specific monoclonal antibodies. This new procedure combines easy handling with low imprecision (median CV less than 3%) in control material, serum, heparinized blood, and plasma) and close correlation (r = 0.991 to 0.999) with established manual and automated methods. The same close correlation was found with values obtained from either venous or capillary finger-stick blood. Salivary amylase up to 54 kU/L (37 degrees C) was inhibited to about 97%. Endogenous interference by hemoglobin, bilirubin, triglycerides, cholesterol, or hematocrit was found to be negligible within a wide range of interferent concentrations. Out of a panel of 28 commonly used drugs it was shown that only two (ascorbic acid and paracetamol), and then only at toxic concentrations, caused a deviation in amylase activity of greater than 10%. From the results of this study we conclude that this new method is suitable for highly precise and accurate measurements of pancreatic amylase in emergency and routine laboratories. PMID- 1723098 TI - Inhibitory effect of levocabastine on experimental allergic conjunctivitis in guinea pigs. AB - The effect of levocabastine on allergic and histamine (Hi)-induced conjunctivitis in guinea pigs was compared with those of cromolyn sodium and amlexanox. Levocabastine inhibited both antigen- and Hi-induced conjunctivitis. Amlexanox had no effect on Hi-induced conjunctivitis; however, the drug elicited a significant inhibition of allergic conjunctivitis. On the other hand, the effect of cromolyn sodium was almost negligible in both cases. Levocabastine moderately inhibited Hi release from guinea pig conjunctiva induced by antigen-antibody reactions. Amlexanox also caused a significant inhibitory effect, whereas cromolyn sodium was not effective in suppressing Hi release induced by antigen challenge. Furthermore, levocabastine prevented an increase in the vascular permeability elicited by both Hi and antigen instillation. PMID- 1723099 TI - The effect of dihydropyridine calcium channel agents on 5-HT metabolism in the CNS of the rat. AB - The effects of dihydropyridines on the levels of 5-hydroxytryptamine (5-HT) and 5 hydroxy-3-indole acetic acid (5-HIAA) in the spinal cord and various brain regions of the rat have been studied. Nimodipine, nitrendipine and nifedipine (10 mg kg-1), nisoldipine (5 mg kg-1), and BAY K8644 (0.2 and 2 mg kg-1) were administered i.p. 1 h before killing. The administration of nifedipine and nitrendipine increased 5-HT turnover in all of the areas studied except for the spinal cord. Nisoldipine increased 5-HT turnover in midbrain, hippocampus and cortex, while the effect of nimodipine was restricted to midbrain. BAY K8644 at 2 mg kg-1 produced the same effects as nifedipine and nitrendipine; however, at low doses (0.2 mg kg-1), this compound increased 5-HT turnover only in midbrain and medulla oblongata. These results indicate that both dihydropyridine calcium channel agonist and antagonists are able to activate the 5-HT-ergic system in the central nervous system of the rat in-vivo. Therefore, it seems likely that such effects could be due to indirect actions or to interactions of the compounds with receptors other than the voltage-sensitive calcium channels. PMID- 1723100 TI - Transcatheter hepatic arterial therapy for symptomatic liver malignancy. AB - Transcatheter hepatic arterial chemoembolization was performed in ten patients with symptomatic unresectable liver malignancy. Nine patients experienced control of symptoms for 52-100% of the duration of their survival, although one patient died 10 days after the procedure. No objective evidence of decrease in tumour size was seen at review but three cases showed selective decrease in tumour vasculature or tumour necrosis. PMID- 1723101 TI - Anion channel forming activity from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense. AB - The plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense secretes an anion channel forming activity (CFA) into the culture field. The CFA inserts spontaneously into planar lipid membranes when culture fluid of this species is added to the aqueous phase of the bilayer chamber. The channels formed are highly anion selective. The conductance decreases for larger anions (Cl- greater than SCN- greater than SO2-(4] and is practically zero for gluconate. The channels show a unique voltage dependence: (i) The single-channel conductance increases linearly with voltage up to 200 mV saturating at 250 mV with 25 +/- 1 pS (300 mM KCl). The channel is closed at negative voltage relative to the side of insertion (diode-type I-V curve). (ii) The average number of open channels also increases with voltage. The Poisson distribution of channel numbers indicates independent opening of the channels. Channel activity can be abolished by protease treatment of the planar bilayer. The channels can be blocked by indanyloxyacetic acid (IAA 94) and by pH greater than 10. The CFA was purified yielding one major band on the SDS gel with a relative molecular mass of 65,000. The putative involvement of the CFA in the toxicity of this plant pathogen is discussed and compared to other toxins like colicins and to the diptheria toxin group. PMID- 1723102 TI - Ionic basis of methacholine-induced shrinkage of dissociated eccrine clear cells. AB - The goal of the present study was to elucidate the ionic mechanisms by which cholinergic stimulation induces cell shrinkage in eccrine clear cells. Dissociated Rhesus monkey eccrine sweat clear cells were prepared by collagenase digestion of freshly isolated secretory coils and immobilized on a glass slide in a perfusion chamber at 30 degrees C. The cell was visualized by light microscopy with differential interference contract (DIC) and was recorded with a video system (15,000 x total magnification). The cell volume was calculated from the maximal cross section of the cell. Methacholine (MCh)-induced cell shrinkage, which was as much as 30% of resting cell volume, was dose dependent and pharmacologically specific. MCh-induced cell shrinkage was persistent in some cells but tended to partially wane with time in others. MCh-induced cell shrinkage was dependent on the chemical potential gradient for KCl, i.e., increasing [K] in the bath ([K]o) from 5 to 120 mM caused MCh to induce cell swelling, whereas removing [Cl]0 at 120 mM K partially restored the MCh-induced cell shrinkage. The interpolated null [K]o (medium [K] where the cell volume did not change by MCh) of 71 mM agreed with the predicted [K]o,null. MCh-induced cell shrinkage was inhibited completely by 1 mM quinidine (K-channel blocker) and partially by 1 mM diphenylamine-2-carboxylic acid (DPC, a Cl-channel blocker), but not by 0.1 mM ouabain or 0.1 mM bumetanide, suggesting that MCh-induced cell shrinkage may be due to activation of both K and Cl channels with the resultant net KCl efflux down the chemical potential gradient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723103 TI - Chloride channels in the nuclear membrane. AB - Chloride-selective ion channels were measured from isolated rat liver nuclei. Single ion channel currents were recorded in both "nuclear-attached" and in excised patches in the inside-out configuration of the patch-clamp technique. Two types of chloride conductance were defined, a large conductance (150 pS; iCl,N) channel with complex kinetics and multiple substates, and a second smaller conductance (58 pS;ICln) channel sensitive to block by ATP. The channels were inhibited by pharmacological agents known to block chloride channels and were insensitive to internal and external changes in calcium and magnesium. Presumably the channels reside in the external membrane of the nuclear double membrane and may mediate charge balance in the release and uptake of calcium from the perinuclear space. PMID- 1723104 TI - Patch clamping VDAC in liposomes containing whole mitochondrial membranes. AB - Whole mitochondrial membranes isolated from Neurospora crassa were reconstituted into liposomes and patch clamped. Clear activity characteristic of the mitochondrial channel VDAC was found, namely: open state conductance of 650 pS (in 150 mM KCl, 1 mM CaCl2, 20 mM HEPES, pH 7.2), voltage-dependent closure at both positive and negative potentials, change in conductance upon channel closure of about 450 pS in response to negative and positive potentials, and increased voltage dependence in the presence of Konig's polyanion. This is the first clear demonstration of VDAC single channels using the patch-clamp technique, even though others used this method before to study whole mitochondrial membranes and liposomes containing mitochondrial proteins. We also found one other channel with a conductance change of about 120 pS. PMID- 1723105 TI - Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts. AB - Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30 50 microM) of indo-1 and with high concentrations (3.5-4.5 mM) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca2+]i) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases. aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin 2 overloaded cells, a significant initial rise in [Ca2+]i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells. PMID- 1723106 TI - Nucleotide sequence of a DNA region comprising the gene for elongation factor 1 alpha (EF-1 alpha) from the ultrathermophilic archaeote Pyrococcus woesei: phylogenetic implications. AB - The gene encoding elongation factor 1 alpha (EF-1 alpha, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeote Pyrococcus woesei was localized within a Bg/II fragment of chromosomal DNA. Sequence analysis showed that the EF 1 alpha gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNAser (GGA). The three genes follow each other immediately in the order EF-1 alpha.S10.tRNA(ser) after a putative promoter located 55 bp upstream of the EF-1 alpha gene. Alignment of the derived EF-1 alpha sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed that Pyrococcus EF-1 alpha is highly homologous (89% identity) to Thermococcus celer EF-1 alpha, both being strikingly more similar to eukaryotic EF-1 alpha than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony--although not evolutionary parsimony--support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of the Pyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation of Pyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins. PMID- 1723107 TI - Kinetics of rapid RNA evolution in vitro. AB - A rapidly acquired partial resistance to the replicase antagonist, ethidium bromide (EB), seen by Spiegelman and coresearchers in Q beta RNA variants competitively replicating under defined conditions in vitro, reflected existence of a pool of mutant RNA molecules, preadapted to EB, and their cross-propagation from the pre-EB optimum species, MDV-1, and from other kindred variants, some of which remained undetected, according to this quantitative analysis of midivariant RNA replication kinetics. DNAlike features of their evolution, such as the cloning of variants from an MDV-1 subtype and a compliance with the fundamental theorem of natural selection, resulted from the suppression, both real and apparent, of intrinsic RNA heterogeneity through sampling and detection methods, and also by the ascendency of self-propagation over cross-propagation with advancement of a superior variant. The deficit in mean polymer fitness, compared with optimum levels, determines the lower limit of this heterogeneity. Stability conditions for frequency equilibrium and strategies for counteracting viral drug resistance have been considered. PMID- 1723108 TI - Allergic and inflammatory aspects of chronic rhinosinusitis. AB - The pathophysiology of rhinosinusitis is complex and poorly understood. Although it is recognized that obstruction of the sinus ostia which is surgically correctable contributes to recurrent bacterial sinusitis, allergic and nonallergic inflammation may contribute to or mimic infectious rhinosinusitis. Those aspects of rhinosinusitis which are not necessarily surgically correctable require consideration and may affect surgical prognosis. This article focuses on those considerations. PMID- 1723109 TI - Endoscopic diagnosis, medical treatment and a working classification for chronic sinusitis. PMID- 1723110 TI - New trends in diagnosis and treatment of arrhythmias. Proceedings of an international symposium, Montreux, Switzerland, November 8-9, 1990. PMID- 1723111 TI - Mechanisms of ventricular arrhythmias: a perspective. AB - The most important ventricular arrhythmias, the ventricular tachycardias (VTs) and ventricular fibrillations (VFs), are thought to underlie the majority of cases of sudden cardiac death. In ischemic heart disease, they can be divided into several pathophysiological entities: (a) arrhythmias occurring during the acute reversible phase of ischemia, (b) arrhythmias taking place during reperfusion of acutely ischemic myocardium, (c) arrhythmias occurring 24-72 h after acute infarction, and (d) arrhythmias associated with chronic infarction. In all three settings, the mechanisms sustaining ventricular arrhythmias need to be distinguished from initiating mechanisms. With the exception of the 24-72-h stage, these arrhythmias are sustained by circus movement with reentry: the electrophysiological determinants of circus movements at a cellular level and, consequently, the appearance of the circulating wave fronts, differ according to the ischemic phase. In acute ischemia, multiple circulating waves, with somewhat large diameters, change their vortexes from beat to beat. In chronic infarction, the location of the stable circuits with elongated central zones of block are closely related to myocardial fiber architecture and probably to scar tissue. These differences indicate that (a) in acute ischemia, the conduction disturbances are mainly determined by the development of inexcitability at the level of cardiac membranes; and (b) in chronic infarction, the site of conduction block and the pivoting points of the wave fronts are determined by impairment of electrical cell-to-cell coupling. In contrast to the mechanisms sustaining VT and VF, the initiating mechanisms are less well defined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723112 TI - Classification of arrhythmias. AB - In view of the broad spectrum of arrhythmias and their considerable spontaneous variability, there is a need for a classification of arrhythmias as a basis for scientific and clinical decision making. From the clinical point of view, a classification should consider (a) hemodynamic consequences, (b) prognostic significance of arrhythmias, and (c) should allow assessment of efficacy of antiarrhythmic treatment. Hemodynamic consequences of tachycardias are related to the degree of heart rate: The shorter the RR interval the shorter the diastolic filling period, resulting in a decrease of the stroke volume and--above a critical heart rate--in a decrease of the cardiac output. The critical heart rate, on the other hand, is essentially related to the functional status of the heart: The more pronounced the cardiac impairment, the lower the critical heart rate. A second factor favoring hemodynamic impairment due to arrhythmias is the loss of the sequence of atrioventricular contractions. Despite the clinical relevance of hemodynamic consequences of arrhythmias, there is no accepted classification taking these aspects into account. In the past, more interest was directed toward the prognostic significance of arrhythmias. In 1971, Lown and Wolf published a classification of ventricular arrhythmias, assigning risk to advanced grades. This proposal, as those from others, took into account arrhythmias of ventricular origin only. The major concern about the Lown classification, however, relates to the consequences of maximal grading: a patient is assigned to a grade depending on the highest ranking. Thus, a person can only be in one grade, leaving all other arrhythmias grouped below as well as the true frequency of ventricular arrhythmia obscure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723113 TI - Hemodynamic consequences of arrhythmias. AB - Hemodynamic effects during arrhythmias may be caused by underlying pathology (i.e., infarction) as well as disturbance of rate or conduction pattern. In all arrhythmias, compensatory mechanisms tend to restore normal hemodynamics, and with good left ventricular function this can be achieved despite wide disturbance of rhythm. Hemodynamic effects of ectopic beats can result in dramatic fall of stroke volume and reduction in cardiac output, which is greater for ventricular than for atrial ectopics. Prolonged tachycardias are also tolerated up to far higher rates (180/min) if they are atrial, not ventricular, in origin. Mean blood pressure is often maintained even when systolic pressure and cardiac output are reduced. Even in healthy young subjects it is possible for cardiac ischemia to be induced by excessive heart rates. Some of the most deleterious effects are produced by simultaneous atrial and ventricular contraction, which results in continued suppression of cardiac output, both during tachycardias and at normal heart rates. Such situations are often highly symptomatic. Few measurements are available during external chest compression, and these suggest only marginal improvement in hemodynamics, with low pressures and output. PMID- 1723114 TI - Diagnostic approach to cardiac arrhythmias. AB - The diagnostic approach to cardiac arrhythmias should be logical and starts with the clinical history, which provides two types of information: (a) the presence of symptoms, and (b) the clinical context, including the presence of an underlying heart disease. Clinical history and examination are helpful in the choice of pertinent invasive or noninvasive tests. The tolerance of the arrhythmia is not helpful in determining the type of arrhythmia because ventricular tachycardia, for example, may be well tolerated or even asymptomatic. The electrocardiogram (ECG) in sinus rhythm may be suggestive of the origin or etiology of arrhythmia as the presence, for example, of the Wolff-Parkinson-White pattern. An essential step in the diagnostic approach to arrhythmia is the ECG documentation. Ambulatory Holter monitoring, radiotelemetry, intermittent recorders, exercise testing, and electrophysiological testing will help in this endeavor. The latter is particularly useful in paroxysmal circus movement tachycardias. Once the tachycardia is recorded, a number of clues, including the regularity of the RR interval and the width of the QRS complex, may facilitate the diagnosis. In tachycardias with wide QRS complexes, preexcitation has to be excluded. The first step is then to look for atrioventricular dissociation, which is diagnostic of ventricular tachycardia. Other diagnostic clues (QRS duration, axis deviation, QRS morphology) may be useful. In case of difficulty because of preexisting bundle branch block or aberrancy, esophageal, right atrial, or His bundle recordings are indicated. If the tachycardia is not well tolerated, prompt termination with electrical DC shock should be performed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723115 TI - Antiarrhythmic treatment of atrial arrhythmias. AB - Atrial premature beats seldom require an antiarrhythmic treatment; reassurance and suppression of coffee, alcohol, and tobacco generally suffice. Acute atrial fibrillation is best treated by electrical cardioversion if it induces acute cardiovascular decompensation. If it is not poorly tolerated, the arrhythmia may be treated with digitalis at doses sufficient to keep the ventricular response rate at 70-90/min. This therapy may restore sinus rhythm, but conversion to sinus rhythm often requires the combined use of digitalis with a beta-blocker or class I antiarrhythmic drug (quinidine, disopyramide, procainamide, propafenone, or flecainide). Digitalis must be avoided in the presence of a preexcitation, and class IA agents, which facilitate atrioventricular (AV) nodal transport, must never be used without digitalis. Chemical cardioversion may also be achieved by i.v. amiodarone. Long-term prevention of recurrences after cardioversion or in the presence of recurrent paroxysmal atrial fibrillation requires digitalis combined with a class I agent, or a beta-blocker, preferably sotalol. Amiodarone is also very efficacious. Special mention should be made of atrial fibrillations of vagal or sympathetic origin, which are best treated by amiodarone, or beta blockade (nadolol), respectively. In the presence of chronic established atrial fibrillation, digitalis in combination with a beta-blocking agent or a calcium antagonist, such as verapamil or diltiazem, may be useful to slow the ventricular response rate. If successful control cannot be obtained, catheter ablation of the AV node with implantation of a rate-responsive pacemaker must be contemplated. The therapeutic approach in patients with chronic atrial fibrillation, whether or not associated, is similar to atrial flutter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723116 TI - Antiarrhythmic treatment of atrioventricular tachycardias. AB - Atrioventricular (AV) tachycardia includes both AV nodal reentrant tachycardia (AVNRT) and AV reentrant tachycardia (AVRT) using an accessory pathway. The treatment of the acute attack is different from the long-term treatment of both AVNRT and AVRT. Verapamil and adenosine, by prolonging the refractory period of the AV node, are highly effective in terminating acute attacks of AVNRT and orthodromic AVRT. Conversion to sinus rhythm is achieved in approximately 90% of the episodes of tachycardias with both agents given intravenously. The initial dose of verapamil is 0.075-0.1 mg/kg and a subsequent bolus of 5 mg can be given to a maximal dose of 15-20 mg. The initial dose of adenosine is 3 or 6 mg, but doses of 9 or 12 mg can be administered if smaller dosages have been unsuccessful. Other agents producing lengthening of the refractory period of the accessory pathway in AVRT or of the fast pathway in AVNRT often terminate reentry tachycardia. Such agents are class IC antiarrhythmic drugs such as flecainide or propafenone and class IA drugs such as procainamide. In patients with accessory pathways and antidromic tachycardia or atrial fibrillation conducting via an accessory pathway, treatment with verapamil or digoxin should be avoided because these agents may enhance the conduction properties of the accessory pathway, thereby leading to an increase of the ventricular rate or even to ventricular fibrillation. Prevention of AVNRT episodes can be obtained with various antiarrhythmic drugs. Digoxin alone or in combination with beta-blockers is effective in approximately 50% of the cases and especially when the combination proved to be successful during electrophysiological testing.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723117 TI - Clinical pharmacology and beta-blocking efficacy of propafenone. AB - Propafenone, like encainide and flecainide, is an antiarrhythmic drug characterized by slow kinetics of interaction with cardiac sodium channels. In addition, it shares structural similarities with propranolol and has been shown to exert consistent beta-blocking action in vitro. On the other hand, the extent of beta-blockade noted after oral administration of propafenone in vivo has been reported to range from undetectable to clinically significant. Although the explanation for this discrepancy was initially unclear, recent work has convincingly demonstrated that the degree of beta-blockade during propafenone therapy reflects genetically determined variations in the metabolism of the parent drug, which has greater beta-blocking potency than do its metabolites. At lower dosages, beta-blockade is significantly greater and more likely to be clinically noticeable in the small percentage of patients who have poor metabolism and in whom deficient 5-hydroxylation is associated with higher plasma propafenone concentrations. Because this metabolic pathway is saturable, at the usual highest clinical dosage (900 mg/24 h) plasma propafenone concentration rises disproportionately in patients with normal 5-hydroxylation, and more comparable degrees of beta-blockade are noted in both metabolizer types. Beta blockade during propafenone therapy, which can contribute both to the genesis of side effects and to the suppression of arrhythmias, is a potentially important drug effect that can largely be predicted by genetic variations in the metabolism of the drug. PMID- 1723118 TI - Negative inotropic effects of antiarrhythmic drugs: a clinical point of view. AB - From a clinical point of view, the negative inotropic effects of antiarrhythmic drugs lead to the following questions: Do antiarrhythmic drugs induce or worsen congestive heart failure (CHF)? Which patients are at increased risk of developing CHF with antiarrhythmic drugs? Which antiarrhythmic drugs are most likely to induce CHF clinically? The present review of the recent literature demonstrates that antiarrhythmic drugs may induce or worsen CHF in a small number of patients. This is true for all antiarrhythmic drugs, but only disopyramide (and flecainide) are antiarrhythmic drugs with a relevant rate of clinical CHF. Patients with a history of heart failure, a low left ventricular ejection fraction, and cardiomyopathy are at increased risk of developing CHF with antiarrhythmic drugs, especially if the drugs are administered intravenously and in high doses. PMID- 1723119 TI - Combination of antiarrhythmic drugs. AB - Antiarrhythmic treatment with single agents is often ineffective and can be limited by dose-dependent side effects. Therefore, combinations of antiarrhythmic drugs in smaller and well-tolerated doses are advocated in cases refractory to single antiarrhythmic drugs. Basically, substances belonging to the same electrophysiologic class should not be combined. However, drugs of different subsets of class I may be combined. Agents that have pharmacokinetic interactions, such as quinidine and amiodarone, should not be given together because this combination may be associated with a considerable proarrhythmic effect. A combination of beta-adrenoreceptor blockers with class I antiarrhythmic drugs may be effective, mainly in cases in which the arrhythmia is dependent on adrenergic stimulation. The combination of class III and IB substances can be useful in some cases, from the electrophysiological and clinical point of view. Among the successful combinations of this type are amiodarone and mexiletine, sotalol and mexiletine, or sotalol and tocainide. In 34 patients, the reduction of ventricular premature beats by sotalol alone was 28%, and by sotalol plus mexiletine or tocainide was 79%. Complex ventricular arrhythmias were suppressed by sotalol alone by less than 40% and by sotalol plus mexiletine or tocainide by more than 80%. There was no difference in the effectiveness of mexiletine and tocainide (both of them being class IB drugs) in this combination. However, mexiletine was associated with fewer adverse effects than was tocainide. In patients refractory to amiodarone alone or to a combination with mexiletine, the combined treatment with amiodarone and class IC drugs such as flecainide and encainide prolongs the cycle length of ventricular tachycardia, but does not suppress induction of ventricular tachycardia during programmed stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723120 TI - Effect of congestive heart failure treatment on incidence and prognosis of ventricular tachyarrhythmias. AB - The prognosis for patients with congestive heart failure (CHF) is poor, with a mortality exceeding 50% within 5 years from diagnosis. This poor prognosis remains despite improved pharmacological therapy. Because the prevalence of sudden death among these patients is high, reported to exceed 40%, the prognostic importance of ventricular tachyarrhythmias has attracted much interest. Long-term electrocardiographic monitoring of patients with CHF reveals a high prevalence of ventricular premature beats, which in many patients occur frequently or are complex according to Lown criteria. Ventricular tachycardia (three or more consecutive beats) has been recorded in 40% or more of the patient population. Whether the occurrence and/or severity of ventricular tachyarrhythmia detected on Holter electrocardiograms relates to the subsequent prognosis is, however, debated. The occurrence of ventricular tachyarrhythmia may just be an expression of severely compromised left ventricular function, which, in turn, decides the subsequent outcome of the disease. Besides myocardial injury, patients with CHF have many factors that may contribute to the high prevalence of ventricular arrhythmias. Among these are elevated levels of plasma norepinephrine. Angiotensin II may increase the sensitivity to sympathetic nervous system arousal but also promotes renal loss of potassium and magnesium. Treatment with digitalis and diuretic drugs may provoke arrhythmias as well. Heart failure therapy may, however, also improve ventricular arrhythmias. Accordingly, it has been demonstrated that captopril therapy significantly reduces ventricular prematurity, compared with digitalis. In contrast, however, enalapril improvement of mortality was due to a reduction of progressive heart failure, with no difference seen in the incidence of sudden cardiac death (the CONSENSUS study).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723121 TI - Antiarrhythmic therapy for ventricular arrhythmias. AB - Treatment of ventricular arrhythmias has received great attention during the past 20 years. However, results of recent trials with class I antiarrhythmic drugs in patients after myocardial infarction have raised many questions about the risk benefit ratio of antiarrhythmic therapy, at least in asymptomatic subjects. Theoretically, the only two reasons to treat ventricular arrhythmias are (a) the presence of symptoms related to the arrhythmia, and (b) the presence of an increased risk of sudden death. The prognostic significance of a ventricular arrhythmia depends on the type of underlying cardiac disease, on the extent of left ventricular dysfunction, on arrhythmia-related symptoms, and on specific characteristics of the ventricular arrhythmia itself. All these factors should be assessed to allow an adequate selection of patients who really need antiarrhythmic therapy (including nonpharmacological modes of treatment), and to allow the identification of patients for whom antiarrhythmic therapy is clearly unnecessary. Such a risk stratification strategy is essential, because many if not all antiarrhythmic agents have potentially serious adverse effects such as proarrhythmic or negative inotropic effects. PMID- 1723122 TI - Safety and toxicity of antiarrhythmic drug therapy: benefit versus risk. AB - Although antiarrhythmic drugs remain the first and most frequently used approach to therapy for arrhythmias, there is growing concern about their safety. It has long been recognized that this class of drug is associated with frequent side effects, especially in patients with extensive underlying heart disease. However, the recent report from the Cardiac Arrhythmia Suppression Trial (CAST) has pointed out that serious toxicity may occur even in patients with less serious heart disease. There are two major reasons for antiarrhythmic therapy. First is for relief of symptoms documented to be the result of arrhythmia. Although there are few studies showing that the antiarrhythmic drugs are effective for this indication, clinical experience does confirm that this is the case. The second indication is to prevent sudden cardiac death. Although antiarrhythmic drugs are of benefit for preventing recurrent arrhythmias in those patients who have already experienced a sustained ventricular tachyarrhythmia, there are, as yet, no data that they are effective for preventing such arrhythmias in patients thought to be at high risk; for example, postinfarction patients or those with a cardiomyopathy and who have nonsustained ventricular tachycardia. Unfortunately, therapy with antiarrhythmic drugs is associated with substantial risks. Although the majority of the side effects are not serious but only "nuisance" complaints, there are more serious toxic reactions that are often idiosyncratic. Organ toxicity may occur with some of these drugs. However, the most serious problems are cardiac side effects including conduction abnormalities, worsening of congestive heart failure, and aggravation of arrhythmia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723123 TI - Clinical pharmacology of antiarrhythmic drugs: variability of metabolism and dose requirements. AB - The clinical use of the presently available antiarrhythmic agents is rendered difficult by narrow therapeutic ranges, highly variable drug elimination kinetics, and complex dose-concentration-effect relations. Because of their electrophysiological mechanisms of action, these drugs may produce proarrhythmic effects, conduction disturbances, and myocardial depression even at the assumed optimum therapeutic serum levels. Several antiarrhythmic drugs, such as procainamide, quinidine, propafenone, encainide, and amiodarone, are transformed to pharmacologically active metabolites, which may contribute to their antiarrhythmic and toxic effects. In addition to marked genetic differences in hepatic metabolism rates (polymorphic hydroxylation and acetylation), some drugs, e.g., quinidine, propafenone, and amiodarone, produce an inhibition of hepatic metabolizing enzymes and thus cause practically relevant drug interactions. A careful dosage adaptation in individual patients is therefore required to achieve maximum therapeutic benefit and to reduce the risk of unwanted side effects. Serum drug concentration measurements may contribute to an improved use of antiarrhythmic agents, but monitoring alone does not replace the electrophysiological assessment of therapeutic efficacy. PMID- 1723124 TI - Effect of antiarrhythmic therapy on mortality after myocardial infarction. AB - In an attempt to improve survival of patients with coronary artery disease and high-grade ventricular ectopic activity, several studies using different antiarrhythmic drugs were undertaken. A meta-analysis of all randomized controlled trials using type I antiarrhythmic agents showed that the treatment effect was much more likely to be adverse than beneficial. In contrast to these studies, the pooled results of major secondary prevention trials using beta blocking agents could demonstrate a significant reduction in the sudden death rate by an average of 24% during observation periods of 9-36 months. In the beta blocker trials, however, patients with contraindications for this type of drug, such as overt congestive heart failure or chronic obstructive lung disease, were excluded. In these patients a type III antiarrhythmic drug, such as amiodarone, may have a place, and in fact, the Basel Antiarrhythmic Study of Infarct Survival, a prospective, controlled, randomized trial using low-dose amiodarone as an antiarrhythmic agent, could demonstrate a 60% reduction in sudden death rate and a 74% reduction in arrhythmic events incidence during the first year after myocardial infarction. Therefore, in patients with repetitive ventricular ectopic activity after myocardial infarction and adequate left ventricular function, a therapeutic attempt with beta-blockers without intrinsic sympathomimetic activity seems advisable. Beside beta-adrenergic blockade, low dose amiodarone is an alternative, especially in patients with impaired left ventricular function or other contraindications for beta-blockers. PMID- 1723125 TI - New approaches to risk stratification after myocardial infarction. AB - Parameters to assess the presence of electrical instability after myocardial infarction include spontaneous ventricular arrhythmias, late potentials, and programmed ventricular stimulation. The accuracy of the long-term electrocardiogram in correctly identifying high-risk patients has been questioned because spontaneous ventricular arrhythmias also occur in a large proportion of patients who do not develop ventricular tachycardia or sudden death during follow up (false-positive results). In addition, many patients died suddenly without having these markers. Late potentials, although showing a good correlation to subsequent occurrence of sustained ventricular tachyarrhythmia or sudden death, are also burdened by the problem of a great number of false-positive results. Programmed ventricular stimulation (such as late potentials) assesses the presence of an arrhythmogenic substrate. An abnormal finding such as inducibility of ventricular tachyarrhythmia is predictive of subsequent occurrence of ventricular tachyarrhythmias. Combining these approaches, additionally including a low ejection fraction, subgroups of patients at very high risk of sudden death or sustained ventricular tachyarrhythmia can be identified. Noninvasive procedures (such as Holter monitoring or recording of late potentials) are desirable for screening purposes, whereas it would be acceptable to use more aggressive invasive techniques in certain subsets of patients. A step-like approach using noninvasive recording of late ventricular potentials as the initial step would allow the preselection of patients for further evaluation by invasive electrophysiological techniques. PMID- 1723126 TI - Nonpharmacological therapy for refractory arrhythmias. AB - Because the great majority of clinically relevant arrhythmias are based on the principle of reentry, the understanding of the basic principle of this mechanism leads to alternative nonpharmacological therapy for such arrhythmias. The reentry circuit might be interrupted definitively by surgical or catheter ablative procedures, such as DC-energy shock delivery or radiofrequency coagulation, which will both lead to complete prevention of the tachycardia. On the other hand, electrical stimulation may influence the circus movement by wavefront collision and thus interrupt the tachycardia after it started. Antitachycardia pacemakers work on this principle. One major impact of antiarrhythmic treatment is the prevention of sudden death. The implantable defibrillator is currently the only treatment that really proves to be reliable in aborting sudden death. It is therefore the "gold standard" of antiarrhythmic treatment in life-threatening arrhythmias. PMID- 1723127 TI - Predisposing factors for ventricular arrhythmias. AB - The majority of ventricular arrhythmias that affect humans are reentrant. Three components are then of relevance: the substrate, trigger, and facilitatory factors. The substrate comprises an area of slow conduction and unidirectional block. The anisotropic conduction of ventricular muscle is a contributory factor but, on its own, is unlikely to support a pathological arrhythmia. Ischemia and infarction are important but are not the exclusive disease mechanisms for creating patchy electrical conditions that will support reentry. Trigger factors are not well understood, but the perturbing influence of ectopic beats is important. Ectopic beat frequency correlates with risk, but variable coupling may be a more effective trigger, operating by "scanning" the cardiac cycle. Facilitatory factors act when both substrate and trigger are present and include autonomic tone, electrolyte imbalance, and biochemical influences. In the future, they may be important therapeutic targets. Substrate, trigger, and facilitators- all are interdependent. Separating their individual contribution is difficult and may be inappropriate. Sometimes the substrate is created by the trigger or facilitator and the facilitator may be the actual trigger. These issues raise exciting prospects for arrhythmia control but should not overshadow the fact that minimizing or preventing disease of ventricular muscle is the optimal clinical goal. PMID- 1723128 TI - Surgical treatment of tachyarrhythmias. AB - Tachyarrhythmia surgery should be divided into two separate groups: supraventricular and ventricular. Supraventricular tachyarrhythmias (SVT): The first surgical cure of the Wolff-Parkinson-White syndrome (WPW) in 1968 led to a better understanding of the pathophysiology and anatomy of this syndrome. WPW should now be classified by its anatomical location as defined by the preoperative and intraoperative mapping. At present, there are two surgical approaches for WPW, endocardial or epicardial. Improvement of the surgical results has broadened the indications for surgery of WPW, making it the most commonly performed operation for SVT. Surgical treatment is briefly discussed for AV nodal reentrant tachycardia, ectopic (focal) atrial tachycardia, atrial flutter, and atrial fibrillation. Ventricular tachyarrhythmias (VT): Different types of direct operations have been applied to the treatment of VT in ischemic heart disease. Because of the fairly high mortality and recurrence rate of these major operations in patients with poor ventricular function, there is now a marked increase in the use of implantable cardioverter-defibrillators as an indirect surgical approach. PMID- 1723129 TI - Future trends in antiarrhythmic therapy. AB - It is difficult to define what the trends will be in antiarrhythmic therapy in the coming years, particularly at a time when rhythmology has been somewhat destabilized by the unexpected results of large therapeutic trials on sudden death prevention. Schematically, drugs that do have established antiarrhythmic properties do not provide any benefit, and they can even make the patients' situation worse. On the other hand, beta-blockers, which do not satisfy the admitted rules of antiarrhythmic efficacy, are indeed capable of preventing death, and they are so doing in subgroups with heart failure, a classic contraindication. Finally, drugs such as calcium antagonists, which supposedly address the ischemic mechanism of death, do not seem to provide any benefit in this kind of trial, whereas drugs such as amiodarone have such a complex action that their probably favorable effect is not easy to explain. This uncomfortable situation probably relates to the fact that the various modes of reasoning concerning the mechanisms of arrhythmias and the effects of drugs have been too straightforward, not to say simplistic. Routinely observed arrhythmias may not have the same determinants as the arrhythmias finally responsible for death, particularly if the role of adrenergic stimulation is taken into account to explain the difference in severity. Routinely used antiarrhythmic drugs may well be effective on the former and innocuous for the patients, and at the same time ineffective on the latter and detrimental to the more severely diseased patients. Our modes of evaluation of the real effects of the drugs certainly have to be revisited, as well as the important parameters to be taken into account in the evaluation of arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723130 TI - Identification of CD34+ cells in normal and pathological bone marrow biopsies by QBEND10 monoclonal antibody. AB - Monoclonal antibody QBEND10 is reactive with the CD34 antigen in aldehyde-fixed, decalcified, paraffin-embedded bone marrow biopsies. In normal bone marrow it stained endothelial cells lining arterioles and capillaries, sinusoidal (littoral) cells and 0.89% of all haemopoietic cells. QBEND10+ mononuclear cells were seen as isolated, randomly distributed mononuclear cells in normal and regenerating bone marrows. Conversely, QBEND10+ cells were increased and present in aggregates of three or more cells in 6/8 cases of acute leukemia; in two cases of CD34-negative leukemia and in two patients after complete remission no aggregates were seen. QBEND10 immunohistochemistry may therefore be useful for diagnosis and follow-up of myeloid leukemias. In addition, increased numbers of CD34+ cells arranged in clusters were seen in 4/9 cases of refractory anemia with excess blasts (RAEB), 1 case of chronic myelomonocytic leukemia, 3/3 cases of RAEB in transformation, and in 3/7 cases of chronic myelogenous leukemia: in all these cases, CD34 staining of the bone biopsy may have prognostic value. QBEND10+ endothelial cells were significantly increased in all the pathological conditions examined (1.43% of all nucleated cells versus 0.80% in normal bone marrow; p = 0.0063), but especially in myeloid leukemias and in two fibrotic syndromes examined. PMID- 1723131 TI - Long survival of leukemic mice by repeated combination treatment of cyclophosphamide and recombinant human granulocyte colony-stimulating factor. AB - The therapeutic and hematological effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) in combination with cyclophosphamide (CY) were investigated in a murine myeloid leukemia model. Ten daily administrations of rhG CSF following CY prolonged the survival time of leukemic mice more than either agent alone. Hematological examination indicated that this effect was attributable to suppression with rhG-CSF of the leukemic repopulation after CY injection. In addition, rhG-CSF accelerated recovery from CY-induced neutropenia. Based on these hematological changes, a treatment regimen was established consisting of a single injection of CY on day 1 and daily injections of rhG-CSF on days 2-6; this combination treatment was given to the leukemic mice for up to four cycles, with a pause of one day between each cycle. The leukemic mice completed each cycle of treatment with few failures, and it resulted in a long survival time for the leukemic mice. The mean survival time of the mice receiving four cycles of treatment was 47 days, 30 days longer than that of the untreated mice. Hematological examination performed at the end of each cycle showed that the leukemic cell population was controlled at a level equal to or below the pre treatment level, and peripheral blood neutrophils were maintained at a level equal to or above the normal level. These results indicate the possible effectiveness of combining rhG-CSF with chemotherapeutic drugs in controlling leukemic cell growth, and the effectiveness of rhG-CSF in enhancing neutrophil recovery after chemotherapy. However, it was found that the leukemic cells became resistant to treatment with rhG-CSF after four cycles of combination treatment, suggesting that great care should be taken in the clinical application of rhG CSF, even when the growth of acute myelogenous leukemia cells is not apparently stimulated by it. PMID- 1723132 TI - Neurofibromatosis and increased risk of leukaemia--is the G-CSF gene involved? PMID- 1723133 TI - [Individual risk-related after-care in colorectal cancer?]. AB - Efficacy of the regular follow-up program and influence on survival rate following treatment of recurrence were evaluated. 556 follow-up records of patients after resection of colorectal cancer were analysed. The primary drop-out rate was 12.4%. Recurrences were found in 26.6% (n = 128). 53.1% of recurrences were symptomatic at diagnosis of recurrence. Curative resection of recurrence was only performed in 19.5%. 46.1% were given palliative and 34.4 no specific oncologic treatment. We define efficacy as the rate of curative asymptomatic recurrence. This was 3.5% of all patients. From the curative resection of recurrence only 6 patients were free of recurrence longer than 2 years. No second resection of recurrence was possible. Different treatment of recurrence did influence the survival rate (p = 0.09). There was no difference in prognosis for asymptomatic and symptomatic recurrences (p greater than 0.8). In order to increase the efficacy of follow-up for colorectal cancer we are introducing a new concept based on individual risk factors. PMID- 1723134 TI - 5-Azacytidine induction of a cellular heat shock protein and expression of the major immediate early protein of human cytomegalovirus in Vero cells. AB - Human cytomegalovirus can support an abortive infection in Vero cells and only some immediate early events are expressed in a low proportion of the cell population. It has been shown that cellular factors partially remove blocks in cells abortively or latently infected by HCMV, whereas 5-azacytidine is known to act on gene expression and cell differentiation. In this work we present evidence that the treatment of HCMV infected Vero cells with 5-azacytidine induces the expression of the major immediate early protein (68K) of Human cytomegalovirus by enhancing a 72K cellular heat shock protein. PMID- 1723135 TI - Capsule-like structures in Clostridium difficile strains. AB - Fourteen strains of Clostridium difficile, previously characterized by SDS-PAGE, were examined for the presence of surface structures. None of the strains were fimbriated but, when grown in the presence of glucose, all exhibited a thin ruthenium red-positive layer. Two strains, belonging to different electrophoretic groups, were also observed after treatment with homologous and heterologous antisera. The strain belonging to the electrophoretic group 2, usually associated with the disease, showed a 30-80nm thick capsule in ultrathin sections. The strains belonging to group 5, mainly observed in isolates from healthy children, exhibited a thinner polysaccharide layer (10-20 nm). No stabilization was observed when these strains were treated with heterologous antisera. This capsule like material did not seem to be associated with adhesive properties of C. difficile strains. PMID- 1723136 TI - Comparative ability of various detergents to extract proteins of Borrelia burgdorferi and Borrelia hermsii. AB - Borrelia burgdorferi and Borrelia hermsii were treated with the following detergents: sodium dodecyl sulphate (SDS), N-lauryl sarcosine (Sarkosyl) and Triton X-100, and the soluble and insoluble fractions obtained after each detergent treatment were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Sarkosyl as well as SDS solubilized all Borrelia proteins, whereas Triton X-100 treatment selectively solubilized the majority of other borrelial proteins, leaving two proteins of 41 kDa and 66 kDa in Borrelia burgdorferi and one protein of 39 kDa in Borrelia hermsii associated with the Triton X-100 insoluble fraction. PMID- 1723137 TI - Acute phase reactants in leprosy. AB - Serum levels of C-reactive protein (CRP) and plasma levels of fibronectin (Fn) were studied in 74 untreated leprosy patients. CRP was detected by latex agglutination in 25.6% of the patients. A significant increase in Fn levels was seen in all the groups of leprosy patients, as compared to the controls. PMID- 1723138 TI - [Activity of glucose-6-phosphate dehydrogenase and level of nucleic acids in Escherichia coli during various rates of growth]. AB - Nucleic acids content and activity of glucose-6-phosphate dehydrogenase were studied at various growth rates. The activity of glucose-6-phosphate dehydrogenase was shown to correlate with the growth rate and with the DNA content in Escherichia coli. PMID- 1723139 TI - Dissociation of hydroxylase and lyase activities by site-directed mutagenesis of the rat P45017 alpha. AB - Site-directed mutagenesis of the arginine-rich region within the putative active site of the rat testicular P45017 alpha (17 alpha-hydroxylase cytochrome P-450, the product of P450XVII gene) was performed to identify specific amino acids that contribute to either the hydroxylase or lyase activities catalyzed by this enzyme. The conversion of Arg346 to alanine differentially abolished lyase activity without affecting hydroxylase activity, resulting in an accumulation of the 17 alpha-hydroxylated intermediate, and partial lyase activity was recovered by conversion of this mutant to Lys346. Similar results were obtained with the conversion of Arg357 to alanine, although this mutant also diminished hydroxylase activity, and full lyase and hydroxylase activities were recovered with a lysine in this position. Major reductions in hydroxylase activity were apparent with the conversion of Arg363 to Ala, and this inhibition was reversed by a lysine at position 363. In contrast, differential effects were not observed with the mutants Arg361 Ala, Arg361 Lys, or Tyr334Phe. Both mutations at the Arg361 position resulted in a total loss of hydroxylase and lyase activities, and mutation at the Tyr334 position had no effect on either activity. The identification of specific amino acids that are essential for either the hydroxylase or lyase reaction indicates that the steroid substrate-protein interaction changes during the course of the two consecutive reactions and reveals the potential for separation of the two activities by chemical and biological modulators within the active site region of the P45017 alpha. PMID- 1723140 TI - Isolation and characterization of transcripts induced by androgen withdrawal and apoptotic cell death in the rat ventral prostate. AB - A variety of stimuli have been identified which initiate transcription-dependent programmed cell death (apoptosis) in specific target cells. Since the withdrawal of androgens induces regression and apoptosis in rat ventral prostate (RVP) epithelial cells, and it is known that the androgen receptor is a transcriptional regulator, we used subtraction cDNA cloning to isolate differentially expressed transcripts from the RVP of androgen ablated rats. In addition to sulfated glycoprotein-2 and glutathione S-transferase (GST), which had been previously described, several other transcripts were found to be elevated 3- to 8-fold in the regressing RVP. DNA sequencing revealed that two of these cDNA clones encode matrix carboxyglutamic acid and gamma-actin, respectively. A third cDNA contained novel sequence information and was named RVP.1. The RVP.1 transcript is expressed at very low levels in the RVP and epididymis of normal adult rats (less than 0.01% of the total mRNA) and is undetectable in other tissues, such as kidney, liver, and muscle. RVP.1 encodes a putative 280-amino acid protein, which shares no significant homology with previously described protein functional domains. We examined the expression of these transcripts in serum-starved NIH 3T3 cells to determine whether any of them are elevated in cells that are growth arrested. It was found that only GST mRNA levels are increased under these conditions. These data may suggest that induction of some genes, such as RVP.1, could be associated with apoptosis, whereas other transcripts, such as GST, may be up-regulated in response to altered rates of cellular metabolism. PMID- 1723141 TI - Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. AB - The pituitary gonadotropins FSH and LH are key hormones for regulating gametogenesis and steroidogenesis in the ovary and testis. The cell surface receptors that mediate the biological activities of these hormones are thought to be expressed in a cell-specific fashion in the ovary and are regulated as animals progress through the reproductive cycle. Using cloned receptor cDNAs, we have examined the expression and hormonal regulation of the ovarian FSH and LH receptor mRNAs in the rat. A quantitative reverse transcription-polymerase chain reaction amplification scheme was used to measure relative levels of the FSH and LH receptor mRNAs, while in situ hybridization was used to localize FSH and LH receptor transcripts. In immature animals, low levels of FSH receptor mRNA are observed in the granulosa cells of small follicles, while low levels of LH receptor mRNA are found in the thecal cells of these same follicles. After stimulation with PMSG, levels of both mRNAs increase, and the LH receptor mRNA is localized in both the granulosa and thecal cells of large follicles. Further treatment of PMSG-primed animals with hCG results in down-regulation, particularly of the LH receptor mRNA in granulosa cells. In adult animals, LH receptor mRNA levels change dramatically during the estrous cycle, particularly after the preovulatory LH surge. FSH receptor mRNA levels show a similar pattern of change, but the FSH receptor mRNA is of lower abundance and is not as highly regulated as the LH receptor mRNA. FSH receptor mRNA is confined to the granulosa cells of healthy developing follicles, whereas LH receptor mRNA is localized predominantly to thecal cells of small follicles on estrous morning, then appears in the granulosa cells of growing follicles by diestrous morning. LH receptor mRNA is also found in interstitial tissues and corpora lutea throughout much of the estrous cycle. Our results indicate that the gonadotropin receptor genes are regulated in a complex fashion during the recruitment, maturation, and ovulation of the ovarian follicle. PMID- 1723142 TI - Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing. AB - Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP. PMID- 1723143 TI - Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells. AB - Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2 day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1 treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo. PMID- 1723144 TI - Cloning and functional characterization of a novel mas-related gene, modulating intracellular angiotensin II actions. AB - The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family. A new mas-related gene (mrg) was cloned in a human genomic library at low stringency using the mas cDNA as probe. Mrg codes for a seven-hydrophobic-segment receptor that is 35% identical to the mas product and 29% identical to the rat thoracic aorta gene product. Mrg mRNA was not detected in several rat and human adult tissues that normally express the angiotensin II (AII) receptor, and transfections of COS and CHO cells with the mrg gene did not modify the number of AII binding sites. These results indicate that mrg and the human AII receptor genes are not identical. However, injection of mrg mRNA into Xenopus oocytes markedly increased the electrophysiological response to angiotensin peptides, indicating some functional similarities with the mas product. The reduction of the response after defolliculation of the oocyte, together with the full agonist effect of Sar1IIe8AII and the partial agonist effect of Sar1Ala8AII, seem to indicate that mrg interacts with the signaling pathways of the endogenous Xenopus angiotensin receptor to potentiate the response to AII. PMID- 1723145 TI - Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect. AB - In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5 isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723146 TI - The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc. AB - Retinol-binding protein (RBP) is a major secretory product of the porcine conceptus. Using an oligonucleotide probe corresponding to a highly conserved region of all known mammalian RBP, we have isolated an apparently full-length cDNA clone for porcine conceptus RBP from a cDNA library constructed from pig conceptuses collected between days 13-17 of pregnancy. The cDNA was 937 base pairs in length and coded for a protein whose inferred amino-terminal sequence was identical to that reported for both porcine conceptus RBP and porcine serum RBP. Its length was consistent with the size (approximately 1 kilobase) of the RBP message in porcine conceptuses. Porcine conceptus RBP and human serum RBP share 91% amino acid sequence identity. The inferred differences in sequence were evenly distributed throughout the length of the polypeptide. RBP mRNA was detectable within the trophoblast of day 11 porcine conceptuses by in situ hybridization with a 618-basepair 35S-labeled probe corresponding to the 3' end of porcine RBP. Silver grain density was distributed relatively uniformly over the trophoblast and the inner cell mass. Western blot analysis of conceptus culture medium demonstrated that the conceptuses of cattle (on day 19) and sheep (on day 15) as well as pigs secrete RBP during early pregnancy. Secretion of large quantities of RBP by the trophoblast of preimplantation pig conceptuses suggests important roles for vitamin A and RBP near the time of conceptus elongation. PMID- 1723147 TI - Stage-specific expression of plasmodial proteins containing an antigenic marker of the intraerythrocytic cisternae. AB - A monoclonal antibody, LWLI, recognized 3 proteins of 45, 50 and 102 kDa in Plasmodium falciparum-infected erythrocytes. The 45- and 50-kDa proteins were parasite-encoded and displayed markedly different peptide maps, indicating that they were distinct plasmodial polypeptides with a common antigenic epitope rather than differentially processed forms of a primary translational product. The 45 kDa protein was present throughout intraerythrocytic growth, while the 50-kDa molecule was not detected earlier than 11 h in the life cycle. The 102-kDa protein was only expressed in trophozoite- and schizont-infected red cells: its structural relationship to the 45- and 50-kDa proteins, if any, remains undefined. By indirect immunofluorescence and immunoelectron microscopy, LWLI bound to flattened intraerythrocytic cisternae exported into the erythrocyte cytoplasm. The results support the theory that proteins recognized by the antibody were concentrated in these compartments and their common antigenic epitope may serve as a marker for the cisternae. Stage-specific expression of LWLI reactive proteins implicates developmental regulation of cisternal functions during asexual parasite development. PMID- 1723148 TI - Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion. AB - We have previously shown that only a single 19-kDa fragment of the Plasmodium falciparum major merozoite surface protein (MSP1) is carried with an invading merozoite into the infected red cell. This fragment (MSP1(19] is derived from the C-terminal membrane-bound end of a major product, MSP1(42), of the primary stage of MSP1 proteolytic processing. Using a monoclonal antibody mapped to an epitope within the N-terminal region of MSP1(42), we have shown that a soluble 33-kDa polypeptide (MSP1(33) corresponding to the N-terminal region of MSP1(42) is shed into culture supernatants during merozoite release and erythrocyte invasion. These observations provide further evidence that the secondary processing of MSP1(42) involves a highly site-specific proteolytic activity. PMID- 1723149 TI - Identification of a common Plasmodium epitope (CPE) recognised by a pan-specific inhibitory monoclonal antibody. AB - A Plasmodium falciparum genomic expression library was screened with a monoclonal antibody produced from mice infected with Plasmodium yoelii. Eleven unique clones were isolated all of which contained the sequence NKND, IKND or KKND. This sequence was confirmed as the epitope of M26-32 by testing a series of overlapping peptides and the allowable substitutions determined by testing the binding of M26-32 to peptides containing all possible single amino acid replacements of NKND. Potential epitopes of M26-32 occur in many plasmodial proteins and this is consistent with the large number of proteins recognised in these parasites by Western blotting. Since this monoclonal antibody shows marked in vitro inhibition of P. falciparum growth, these data suggest that an anti malarial vaccine may be produced by targeting such common plasmodial epitopes without necessarily identifying the corresponding antigens. PMID- 1723150 TI - Patients infected with Leishmania donovani chagasi can have antibodies that recognize heat shock and acidic ribosomal proteins of Trypanosoma cruzi. PMID- 1723151 TI - Mutations affecting local anesthetic block of the nicotinic acetylcholine receptor ion channel. PMID- 1723154 TI - Study of regional cerebral blood flow in experimental head injury: changes following cerebral contusion and during spreading depression. AB - Changes in regional cerebral blood flow (rCBF) following fluid-percussion brain injury (cerebral contusion) were studied in rats using the autoradiographic method. The direct current potential was monitored to identify spreading depression (SD). The rCBF was measured during SD and 2, 4, and 24 hours after injury. rCBF was almost nil in the contused area and decreased considerably in the cortices of the injured side for 4 hours after insult, then recovered by 24 hours. Focal relative rCBF increase occurred in the parietal cortex during SD, and was probably hyperperfusion due to SD. However, the rCBF did not increase over the sham-operated control. The injury probably caused hypoperfusion within 4 hours of insult and abolished the vascular response to SD. PMID- 1723152 TI - Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus. AB - The effects of three different opioid agonists on contractions and [3H] acetylcholine (ACh) release evoked by 5-hydroxytryptamine3 (5-HT3) and neurokinin 3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu (mu)-opioid receptor agonist (D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAMGO; 1 nM-100 nM) and the selective kappa (kappa)-opioid receptor agonist U50488 (10 nM-1 microM) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC50 estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM-51 nM. The selective delta (delta)-opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 microM). 3H-overflow from LMMP preparations preincubated with [3H]-choline was measured as an indicator of [3H] ACh release. DAMGO (1 nM-100 nM) and U50488 (10 nM-1 microM) inhibited the increases in release of [3H]-ACh evoked by 5-HT (10 microM) and by senktide (10 nM) in a concentration-dependent manner. IC50 estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [3H]-ACh release and lay in the range 6 nM-23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723155 TI - Direct calorimetry using Swan-Ganz catheter for evaluation of general metabolic expenditure in acute cerebrovascular disease--comparison between direct Fick method and indirect calorimetry technique. AB - Oxygen consumption calculated by the direct Fick method using a Swan-Ganz catheter (D-VO2) and indirect calorimetry using a metabolic computer (ID-VO2), carbon dioxide production calculated by the latter method, and respiratory quotient were determined pre- and postoperatively in 12 patients with acute hypertensive intracerebral hemorrhage and eight patients with acute ruptured intracranial aneurysm. The mean D-VO2 value was slightly lower than the mean ID VO2 value, but had a significantly positive correlation. The regression curve was very close to the line of identity. The total metabolic expenditure can be calculated from D-VO2 and daily urinary nitrogen excretion. Direct calorimetry using a Swan-Ganz catheter is a simple method to evaluate metabolic expenditure in acute hemorrhagic cardiovascular disease. PMID- 1723153 TI - Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations. AB - The present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the beta-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydro ouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors. To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I-III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I-III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored. These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1723156 TI - Pharmacokinetic study of selective continuous internal carotid CDDP infusion in malignant brain tumors. AB - Cis-diamminedichloroplatinum (CDDP) was administered by selective continuous internal carotid infusion to nine patients with malignant brain tumors, including five glioblastomas, one mixed glioma, and three metastatic tumors. CDDP was infused through a catheter in the internal carotid artery at 100 mg/hr in all cases, except one glioblastoma case in which the lower rate of 10 mg/hr was used. The results of CDDP concentration measurements were: 1) CDDP in the blood peaked at termination of CDDP infusion and then decreased slowly, 2) CDDP infiltrated intratumoral cysts and accumulated there, 3) CDDP in the cerebrospinal fluid peaked 6-18 hours after infusion, and 4) the tumor/plasma CDDP ratio varied from 2 to 8. The size of tumors decreased moderately in three of the nine cases, but no complete response was achieved. The histological changes due to CDDP were observed in the tumor tissue and were absent in the normal brain parenchyma. PMID- 1723157 TI - Intraoperative radiation therapy for malignant glioma. AB - Intraoperative radiation therapy (IORT) was used as part of the initial therapy for malignant glioma in 32 of 73 patients with histologically verified anaplastic astrocytoma (grade III astrocytoma) and glioblastoma multiforme. The initial treatment for all cases was subtotal or total tumor resection combined with external irradiation and chemotherapy. IORT was performed 1 week after tumor resection, with doses of 10-50 Gy (mean 26.7 Gy) in one session. Fourteen of 32 cases had IORT two times because of tumor recurrence. The IORT patients had survival rates at 24 and 36 months after initial treatment of 57.1 and 33.5% (median survival 26.2 months). The other 41 patients had 23.6 and 13.1% survivals (median survival 20.7 months), which were significantly lower (p less than 0.01). Tumor recurrence within the original lesion site was suspected because of clinical condition, computed tomography, and magnetic resonance imaging studies in 65.6% of the IORT group (21 cases) 12 months after initial treatment. Twenty cases of death in the IORT group, including five autopsy cases, demonstrated regional tumor recurrence with a high incidence of intraventricular tumor invasion. The authors consider IORT is beneficial for selected malignant glioma patients, including tumor recurrence, because of prolonged survival. PMID- 1723158 TI - Effects of encephalo-duro-arterio-synangiosis on childhood moyamoya patients- swift disappearance of ischemic attacks and maintenance of mental capacity. AB - The effect of encephalo-duro-arterio-synangiosis (EDAS) upon chronic cerebrovascular ischemia in 65 pediatric moyamoya patients was evaluated by the postoperative interval before complete disappearance of ischemic attacks and changes in pre- and postoperative intelligence (IQ) or development quotients (DQ). The ischemic attacks disappeared after a mean period of 239 days, in three fourths of patients within a year and in about one-fourth within the second year. This was very fast compared with the natural course of the disease. There was no significant difference in DQ/IQ before and after the operation. The mentally normal (IQ/DQ greater than or equal to 86) population in the postoperative patients was greater than in the natural course of the disease, although fewer in the preoperative group. This shows that EDAS delayed or prevented the deterioration in mental capacity usually present but often overlooked in the natural course of pediatric moyamoya disease. PMID- 1723159 TI - Foramen magnum decompression for syringomyelia associated with basilar impression and Chiari I malformation--report of three cases. AB - Anterior or posterior decompression of the foramen magnum was performed in three patients with syringomyelia associated with basilar impression and Chiari I malformation. The operative results were evaluated using the pre- and postoperative magnetic resonance (MR) images. Two patients with combined anterior and posterior cervicomedullary compression due to basilar impression and tonsillar descent received suboccipital craniectomy, upper cervical laminectomy, and dural plasty without any intradural manipulations via the posterior approach. One patient with prominent anterior cervicomedullary compression due to basilar impression and a sharp clivoaxial angle was operated on by the transoral anterior approach. Postoperatively, all patients showed a sustained shrinkage of the syrinx and rounding of the flattened cerebellar tonsils. Two patients showed upward movement of the herniated tonsils. All patients had improved symptoms during 2-4 years follow-up. Treatment of syringomyelia associated with basilar impression and Chiari I malformation requires more efficient decompressive procedures at the foramen magnum based on neurological and MR findings. PMID- 1723160 TI - Coexistence of intracranial and spinal meningiomas--report of two cases. AB - The authors report two rare cases of multiple meningiomas in both the intracranial and spinal regions. A 64-year-old female presented with a right sphenoidal ridge meningioma and a cervical extramedullary meningioma. Tumor histology was transitional and vacuolated types, respectively. The tumors were removed successfully in two stages, craniotomy then laminectomy 3 months later. A 62-year-old female presented with a right sphenoidal ridge meningioma (meningotheliomatous type) which was totally removed. An extramedullary spinal meningioma became symptomatic 33 months later, which was also removed totally. The meningiomas in the first case had different subtypes, but immunohistochemical characteristics including microcyst formation were similar. The second case had meningiomas of the same subtype with similar characteristics, but different fibrous septum development. Multiple meningiomas, even in different compartments of the central nervous system, may have common characteristics. PMID- 1723161 TI - Subarachnoid dissemination of pineal germinoma 9 years after radiation therapy without local relapse--case report. AB - A 22-year-old female developed intracranial and spinal subarachnoid metastases 9 years after radiation therapy for a pineal germinoma. Computed tomographic scans showed no evidence of local recurrence. Cerebrospinal axis irradiation achieved total remission. Delayed subarachnoid dissemination may be caused by germinoma cells remaining dormant in the subarachnoid space, outside the radiation field. PMID- 1723162 TI - Intracranial and spinal germinomas occurring four years after spinal cord germinoma--case report. AB - Intracranial and thoracic tumors developed in a 31-year-old male 4 years after irradiation therapy for a cervical tumor. He received irradiation for the pineal, suprasellar, and thoracic tumors. All tumors disappeared by neuroradiological imaging after treatment. The previous and present tumors were probably germinomas, possibly with multifocal origins. PMID- 1723163 TI - Subependymoma of the lateral ventricle--case report. AB - A 56-year-old male presented with mild gait disturbance and short-term memory disturbance. Computed tomographic scans revealed an isodense mass with a large cyst in the left lateral ventricle, extending to the right. The tumor was removed totally via the left frontal transcortical approach. Light microscope examination found clusters of isomorphic cells separated by a dense fibrillar matrix. No ependymal rosettes or blepharoplasts were found. Some cluster cells had positive immunoperoxidase staining for glial fibrillary acidic protein and S-100 protein. Electron microscope observation found tumor cells with gap junctions and zonula adherens resembling the junctional complexes of normal ependymal cells, many microvilli and cilia, and long processes containing abundant glial fibrils. Such "transitional cells" may be important in establishing the origin of subependymoma. PMID- 1723164 TI - Pituitary prolactinoma associated with polycystic ovary--case report. AB - A 25-year-old female presented with pituitary prolactinoma associated with polycystic ovarian disease and amenorrhea. After trans-sphenoidal adenomectomy, the serum prolactin level returned to normal. Postoperative ultrasonography revealed resolution of the polycystic ovary. Regular menses recommenced 2 months after surgery. Our experience suggests that pituitary prolactinoma may be a cause of polycystic ovarian disease. PMID- 1723165 TI - Fenestration of the intracranial internal carotid artery--case report. AB - A rare case of fenestration of the intracranial internal carotid artery (ICA) was identified by angiography in a 51-year-old female with suspected subarachnoid hemorrhage. Only two similar cases have been reported previously. Fenestration tends to develop on the right side and near the bifurcation of the ophthalmic artery. Congenital factors at the 4 mm embryonic stage may be involved in the fenestration of the intracranial ICA. PMID- 1723166 TI - Anticoagulant-related intracerebral hemorrhage in patients with prosthetic heart valves--report of two cases. AB - Two cases of intracerebral hemorrhage in patients with prosthetic heart valves receiving anticoagulant therapy without preceding embolic cerebral infarction are reported. Phytonadione and fresh frozen plasma were immediately given, and the intracerebral hematoma evacuated successfully. In one case, intractable bleeding occurred perioperatively until the thrombotest value reached 40% or more. This patient later developed fatal massive multiple intracerebral hemorrhages. PMID- 1723167 TI - Influence of extracellular matrix on the proliferation and differentiation of glioma cells in culture. AB - The influence of extracellular matrix on the proliferation and differentiation of glioma cells in vitro was investigated by culturing glioma cells in collagen gel and on collagen and laminin films. Rat C-6 glioma cells extended thin cytoplasmic processes, proliferated, and differentiated in collagen gel and on collagen film. The cells were stellate with multiple thin processes like the astrocyte in vivo. The intracellular content of cyclic adenosine-monophosphate in rat C-6 glioma cells on collagen film increased approximately four fold over the control level. In laminin film culture, rat C-6 glioma cells extended thin cytoplasmic processes with many knotty structures and proliferated. These findings confirm that extracellular matrix induces the differentiation of glioma cells. PMID- 1723168 TI - Serological analysis of tumor antigens in malignant glioma patients. AB - Sera from 27 malignant glioma patients were tested for antibodies to surface antigens of cultured human glioma cells using enzyme-linked immunosorbent assay. Average antibody titer for glioma cell lines was 0.558 +/- 0.123, which was significantly higher than in the normal control group (0.165 +/- 0.082). Surprisingly, average antibody titer for autologous glioma cells was low (0.207 +/- 0.154) in these patients. The results suggest that various surface antigens in glioma cells include specific autologous antigens, antigens associated with gliomas, and common antigens present on cultured normal and malignant cells. These analyses are important in the evaluation of monoclonal antibodies and explanation of escape mechanisms. PMID- 1723169 TI - Cerebrospinal fluid placental alkaline phosphatase in the intracranial germinomas: results of enzyme antigen immunoassay. AB - The authors investigated the placental alkaline phosphatase (PALP) activity in cerebrospinal fluid (CSF) by enzyme-antigen immunoassay using polyclonal antibody as a marker for intracranial germinomas in 17 patients with germ cell tumors and 20 with other disorders. The detection limit of PALP activity was 0.072 optical density units equivalent to 5.9 ng/ml. Five of nine germinomas demonstrated high CSF PALP activities before treatment. These high PALP activities became undetectable following radiation therapy. The other tumors were small or had no CSF contact. CSF PALP activity is a useful tumor marker for pure germinomas. PMID- 1723170 TI - AVDO2 and EEG as indicators for treatment of post-traumatic brain swelling- experimental study. AB - A cat model of compression ischemia using epidural balloon inflation investigated: 1) the relationship between postischemic cerebral blood flow (CBF) and metabolism and brain swelling, 2) the use of arteriovenous oxygen difference (AVDO2) and electroencephalographic (EEG) frequency band analysis for monitoring CBF and cerebral metabolism, and 3) indications for selecting the therapy. Global ischemia was induced by brain compression, followed by rapid decompression, and AVDO2, CBF, cerebral metabolic rate for oxygen, and EEG were monitored. The animals were divided into delayed brain swelling and no swelling groups. The severity of compression ischemia influenced the recovery of CBF and cerebral metabolism. The AVDO2 and EEG reflected the CBF and cerebral metabolism. These parameters are useful in selecting the therapy for focal brain injury. PMID- 1723171 TI - Intracranial pressure responses during hyperbaric oxygen therapy. AB - The responses of intracranial pressure (ICP) to hyperbaric oxygen (HBO) therapy and arterial gas pressures were investigated. ICP was measured through a ventricular or spinal drainage catheter in patients with brain tumor or cerebrovascular disease. Changes in ICP, heart rate (HR), arterial blood pressure (ABP), and transcutaneous partial pressure of carbon dioxide (PtcCO2) or oxygen (PtcO2) were recorded continuously during air or 100% O2 breathing at 1 and 2.5 atmospheres absolute (ATA). HR and PtcCO2 decreased and mean ABP was unchanged during HBO inhalation. ICP was reduced at the beginning and tended to increase gradually during HBO inhalation. The change from air to O2 without altering respiratory frequency and volume caused a gradual increase of ICP and PtcCO2 with a transient ICP reduction in an artificially respirated patient. Intentionally reduced respiration to maintain PtcCO2 at the value at 2.5 ATA with air caused the ICP to return to near the value at 2.5 ATA with air even during HBO inhalation. These findings suggest that reduced ICP is initially due to direct cerebral vasoconstriction caused by hyperoxia and is maintained mainly by induced hypocapnia during HBO inhalation. Care is required when giving HBO therapy to patients with a high ICP and/or who are respirated artificially. PMID- 1723172 TI - Aneurysm of the lenticulostriate artery--report of four cases. AB - Four rare cases of aneurysm of the lenticulostriate artery (LSA) are presented. LSA aneurysms were located at the origin in three patients and distally in one. Two cases were of multiple aneurysms, one was associated with hypertensive intracerebral hematoma (putaminal hemorrhage), and the other with moyamoya disease. Two patients were successfully treated by microsurgical procedures. The occurrence of LSA aneurysm suggests that aneurysm formation and growth are accentuated by hemodynamic alteration and stress. PMID- 1723173 TI - Brain metastasis of testicular tumor with massive hemorrhage--report of two cases. AB - The authors report two cases of brain metastasis from testicular tumor with massive, sudden intratumoral hemorrhage. In both cases, the hemorrhage occurred during the 1st admission day and carried a high risk of fatality. Early, aggressive surgical removal is advisable before general deterioration. Postoperative chemotherapy with an agent different from the one applied to primary lesion is also recommended because of drug tolerance. PMID- 1723175 TI - Intradural-extramedullary spinal cavernous angioma--case report. AB - An extremely rare case of intradural-extramedullary spinal cavernous angioma in a 65-year-old male is reported. The clinical presentation was recurrent episodes of subarachnoid hemorrhage (SAH). Magnetic resonance images disclosed the tumor at the 1st thoracic vertebra, without gadolinium enhancement. The tumor was tightly adhered to the cord surface and successfully removed microsurgically. The previous two reports of this unusual tumor also showed repeated SAH and the tumor attachment to the spinal cord or nerve roots. Intradural-extramedullary cavernous angioma possibly originates in the surface of the spinal cord or nerve root and then extends extraphytically. PMID- 1723174 TI - Pediatric orbital eosinophilic granuloma with intra- and extracranial extension- case report. AB - A rare case of eosinophilic granuloma of the orbit in a 3-year-old boy presented as right upper eyelid swelling and proptosis. Computed tomographic scanning revealed a soft-tissue dense mass in the lateral wall of the right orbit. Magnetic resonance (MR) imaging demonstrated tumor extension into the orbit, anterior and middle cranial fossae, and extracranial region. The tumor was completely removed. Histological diagnosis was eosinophilic granuloma. No evidence of recurrence was found 14 months later. MR imaging is useful for diagnosis of the lesion, and particularly for surgical management. PMID- 1723176 TI - Moyamoya disease developing from unilateral moyamoya disease--case report. AB - The authors report a case of unilateral moyamoya disease which developed into moyamoya disease 3 years later. Unilateral moyamoya disease is generally defined as moyamoya disease, but the exact relationship is unknown. In this case, occlusive changes developed in the stenotic carotid fork, and in a similar portion contralaterally which was intact. Follow-up 4-vessel angiography is strongly recommended even for unilateral moyamoya disease. PMID- 1723177 TI - The effect of intrauterine alcohol exposition in various durations on early cognitive development. AB - Fifty-three children exposed to alcohol of various duration were examined at 18 to 19 months of age. No significant difference was found in developmental outcome between non-exposed (n = 56) and during the I trimester exposed children (n = 21). Exposure until the III trimester (n = 19) or continuous exposure (n = 13) caused significantly lower scores in language and total mental assessment. The procentual numbers of children with possible developmental risk (failure or pass at acceptance limit) in various developmental fields grew with increasing duration of intrauterine alcohol exposure. Failures in gross or fine motor items differed significantly between the non-exposed and the two long exposed children but in language items only between the non-exposed and the children exposed until the III trimester. A high number of exposed children scored just the limit values indicating a milder form of developmental delay. In comparison with the developmental results of one year of age an increase in delay in cognitive development was seen. The incidences of other teratogenic effects (pre- or postnatal growth retardation, facial dysmorphia) of fetal alcohol exposure were highest in the continuous exposed group. Five (38%) of the continuous exposed children had typical fetal alcohol syndrome and only 1 (5%) of the children exposed until the III trimester. PMID- 1723178 TI - Levels of dynorphin peptides, substance P and CGRP in the spinal cord after subchronic administration of morphine in the rat. AB - Rats were rendered dependent on morphine by repeated injections of morphine, in increasing doses for 14 days and sacrificed. Levels of peptides in the dorsal spinal cord and dorsal root ganglia were analyzed in rats decapitated 2 hr, 24 hr (acute abstinent) or 7 days (late abstinent) respectively, after the last injection of drug. Dynorphin A was significantly decreased in rats abstinent for 24 hr, while dynorphin B remained unaffected. Substance P and CGRP, both putative transmitters in nociceptive primary afferent neurones, and partly existing together in the same neurone, were affected differently. Significantly less substance P but unchanged levels of CGRP were detected in rats abstinent for 24 hr, while on the other hand, CGRP but not levels of substance P, were increased 2 hr after the final injection. In dorsal root ganglia, levels of substance P were lower at 2 hr, while levels of CGRP were unaffected. In late (7 days) abstinence, no effect of opiate on any peptide was detected. PMID- 1723179 TI - Two mental calculation systems: a case study of severe acalculia with preserved approximation. AB - We report the case of an aphasic and acalculic patient with selective preservation of approximation abilities. The patient's deficit was so severe that he judged 2 + 2 = 5 to be correct, illustrating a radical impairment in exact calculation. However, he easily rejected grossly false additions such as 2 + 2 = 9, therefore demonstrating a preserved knowledge of the approximate result. The dissociation between impaired exact processing and preserved approximation was identified in several numerical tasks: solving and verifying arithmetical operations, number reading, short-term memory, number comparison, parity judgement, and number knowledge. We suggest the existence of two distinct number processing routes in the normal subject. One route permits exact number representation, memory and calculation using symbolic notation. The other route allows for approximate computations using an analog representation of quantities. PMID- 1723180 TI - An investigation of naming errors following semantic and phonemic cueing. AB - This study investigated the types of verbal errors produced by aphasic patients following phonemic and semantic cueing. Twenty-eight aphasic patients--10 Broca's, 10 Wernicke's and 8 conduction aphasics--served as subjects. Semantic and phonemic cues were administered on object and action confrontation-naming tasks. When subjects did not respond correctly to phonemic cueing, a significantly greater number of phonemic errors were produced, with a concurrent decline in related words and extended circumlocutions. When subjects failed to respond to semantic cueing on the action task, there was an increase in a number of error categories. PMID- 1723181 TI - Sexual dimorphism in accessory olfactory bulb mitral cells: a quantitative Golgi study. AB - The purpose of the present study was to identify the existence of sexual dimorphism in the dendritic field of accessory olfactory bulb mitral cells in rats and to investigate the effects of male orchidectomy and female androgenization on the day of birth upon this dendritic field. The rapid Golgi method was used to conduct a quantitative study of various characteristics of the dendritic field of accessory olfactory bulb mitral cells. The results indicated greater values for males than females for the following characteristics: (i) somatic area; (ii) degree of branching in the dendritic field; (iii) total dendritic length; and (iv) dendritic density around the neuronal soma. Orchidectomy of males, as well as androgenization of females, on the day of birth inverted these differences. PMID- 1723182 TI - Neurogenic and non-neurogenic inflammation in the rat paw following chemical sympathectomy. AB - Rats with chemical sympathectomy, induced either at neonatal age (long-term sympathectomy) or in adult animals (short-term sympathectomy) by guanethidine or by 6-hydroxydopamine, were used to determine the contribution of sympathetic noradrenergic fibres to afferent neuron-mediated responses and to non-neurogenic inflammation in the rat. Following long-term sympathectomy with 6-hydroxydopamine there was a 66% depletion of noradrenaline in the paw skin. This was accompanied by a 20-53% increase in the levels of sensory neuropeptides in the paw skin and sciatic nerve. A hypersensitivity towards heat stimuli was observed in the tail immersion test. Neither neurogenic plasma protein extravasation following antidromic nerve stimulation or upon local mustard oil application nor the development of the non-neurogenic carrageenan oedema and its susceptibility towards indomethacin were impaired. Neonatal guanethidine sympathectomy caused an 86% depletion of noradrenaline in the paw skin and neurogenic plasma protein extravasation upon antidromic nerve stimulation was impaired. Sensory neuropeptides were unchanged in the skin after neonatal guanethidine and only calcitonin gene-related peptide content was increased in the spinal cord and sciatic nerves. The other observations (i.e. the sensitivity towards heat stimuli, the neurogenic mustard oil inflammation and the non-neurogenic carrageenan oedema) were similar to those observed after neonatal 6 hydroxydopamine treatment. Following the short-term treatment protocol of 6 hydroxydopamine, an 82% depletion of noradrenaline in the skin was accompanied by an increase in calcitonin gene-related peptide content, whereas after adult guanethidine (60% depletion of noradrenaline) levels of sensory neuropeptides were unchanged. Neurogenic plasma protein extravasation was found to be unimpaired after either type of short-term chemical sympathectomy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723183 TI - [Early recognition of partial learning disorders]. AB - The long-term outcome of learning disabilities (lower school performance, higher rates of unemployment and unskilled jobs, secondary psychic disorders and juvenile delinquency) requires their early recognition even at pre-school age. The concept of learning disabilities should be adapted to the concept of specific developmental disorders (ICD-10, F80-F83). The diagnosis of specific developmental disorders at pre-school age shows many difficulties due to the drawbacks of the available psychological tests. A highly economic and reliable battery of tests is presented as an alternative, which enables us to diagnose general intelligence and specific developmental disorders. The diagnostic procedure was standardised on 657 children of four and five years of age. PMID- 1723184 TI - [Developmental delay in students starting school and possibilities for detection in preschool screening by the public health office]. AB - There is an incomplete degree of school maturity in approximately ten per cent of our school beginners because of developmental delays which may have genetic or psychosocial causes or may be caused by an infantile cerebral dysfunction. A consequence of these developmental delays are the partial disturbances of performance frequently resulting in failure at school. An early registration of the partial disturbances of performance for example at pre-school medical check ups is important for the prognosis of these children. The examination scheme of the Public Health office Schweinfurt is presented. PMID- 1723185 TI - [Early rehabilitation in the former East Germany]. AB - This is an account of the early rehabilitation training and care afforded to handicapped infants and babies in special groups in creches in the city of Magdeburg. The patient groups are described, as well as the nature and execution of the rehabilitation work and the requirements in respect of space and personnel. The legislation that was valid in the former German Democratic Republic is also described. The authors' ideas in respect of future work in rehabilitation and medical paedagogy are presented. PMID- 1723186 TI - Electroretinogram b-wave implicit time and b/a wave ratio as a function of intensity in central retinal vein occlusion. AB - The ability of electroretinogram (ERG) b-wave implicit time and b/a wave ratio to predict iris neovascular response was analyzed as a function of stimulus intensity over a 3.6 log unit intensity range in 39 patients with central retinal vein occlusion (CRVO). Predictive power for CRVO patients was evaluated using ROC area at intensities of 1.23, 1.83, 2.43, and 3.03 effective log quanta/rod, where reliable data for both parameters were obtainable from most patients. The relative predictive power of b-wave implicit time and b/a wave ratio were shown to vary with stimulus intensity. The predictive power of b-wave implicit time, as measured by ROC area, declined to below significance at high intensity (above 1.83 log quanta/rod), while b/a wave ratio performed best at middle intensities (1.83 and 2.43 log quanta/rod) and not as well at high and low intensities. Further analysis of statistical behavior of both ERG parameters was obtained from the t statistic. Insight into the mechanism influencing predictive power of b wave implicit time was derived from measurements on normal adults and CRVO patients with response data taken at high intensities. These results suggest that an optimal stimulus intensity range can be found for these ERG parameters in the evaluation of CRVO. PMID- 1723187 TI - Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels. AB - In the heart, the guanosine 5'-triphosphate (GTP)-binding protein Gs is activated by hormone binding to beta-adrenergic receptors and stimulates the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A. Additionally, Gs can modulate cardiac Ca channels directly in cell-free systems. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (ICa) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the ICa density increased from 12 to 40 microA/cm2 following application of 1 microM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1 microM ISP enhanced ICa only from 9 to 17 microA/cm2 after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPs (the Rp-isomer of adenosine 3',5'-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on ICa. Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of ICa, which probably reflects the direct stimulation of Ca channels by Gs under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 microM cAMP, bath application of 20-40 microM 3-isobutyl-1-methylxanthine (IBMX) enhanced the ICa density to about 41 microA/cm2 indicating saturation of the cAMP pathway. Under this condition, 1 microM ISP was without significant effect on ICa. This result may suggest that direct Gs stimulation is rather ineffective on Ca channels after maximal cAMP dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the Gs pathway in intact myocytes. Third, simultaneous application of 1 microM ISP and 40 microM IBMX enhanced ICa up to densities of around 75 microA/cm2 during cell dialysis with 100 microM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that Gs is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of Gs may act to prime L-type Ca channels for cAMP dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes. PMID- 1723188 TI - Analyses by the intention-to-treat principle in randomized trials and databases. PMID- 1723189 TI - A right atrial mass in the presence of a permanent pacemaker electrode in a patient with polycythemia vera. AB - A case of a huge right atrial mass that developed 2 years after a permanent pacemaker implantation is described. The patient had a history of polycythemia vera, which is known to present a high tendency towards the development of thrombosis. In light of this fact, we suggest that in similar cases a full echocardiography follow-up should be performed, and long-term anticoagulant therapy should be considered in selected cases. PMID- 1723190 TI - The etiology of syncope in pacemaker patients. AB - A total of 46 patients with syncopal episodes after VVI pacemaker implantation were studied. Of these, 92% had one to three syncopal episodes and 8% more than three. All underwent a thorough clinical examination, which included chest X ray, echocardiogram, neurological exam, and the following protocol: 24-hour Holter monitoring, EEG, blood pressure (BP) measurement in three positions, Doppler exam of the carotid vessels, fasting blood glucose, and head-up tilt table test (60 minutes, 60 degrees). Holter monitoring showed exit block in two patients (4.3%) and failed sensing in one (2.1%). In two patients there was unilateral slowing on EEG. Orthostatic hypotension was found in four patients (8.6%), and hypoglycemia in three insulin-dependent diabetics. An occlusive atherosclerotic plaque in the carotid artery was found in three patients (6.5%). Syncope was induced in 17 patients (36.9%) by the tilt table test, after a mean standing time of 47 +/- 11 minutes. The mean resting systolic BP of these patients was 140 +/- 24 mmHg, and fell to a mean level of 56 +/- 8 mmHg (mean systolic BP drop was 79 +/- 8 mmHg). Sixteen of these 17 patients with positive tilt table were being paced at the time of syncope and one had a spontaneous heart rate of 73 beats/min. In 14 cases (30.4%) the cause of syncopal episodes after this extensive workup remained unexplained. These results indicate that pacemaker dysfunction is not a major cause of syncopal episodes in pacemaker patients and that these are most often due to vasovagal syncope. Long-term follow-up is warranted to determine the prognostic significance of various types of syncope in pacemaker patients. PMID- 1723191 TI - The effect of exercise on the atrial electrogram voltage in young patients. AB - Atrial electrogram sensing is an important function in active individuals with permanently implanted bipolar dual chamber pacing systems. We undertook to determine the effect of vigorous exercise on the atrial electrogram size in 11 children and young adults (average age 12 years). Using a telemetry signal through a handheld programming wand, nine tracings were completely and clearly recorded for analysis. Six patients had tined/passive fixation atrial leads and three patients had screw-in/active fixation lead systems. All leads were bipolar. The atrial electrogram size for each patient was measured at rest and at each minute of exercise. The atrial electrogram size decreased with exercise from a mean of 5.08 mV to 3.44 mV (range 0.9-4.25 mV) (P = 0.002). The 1.64 mV mean decrease represented a 33.8% reduction (range 19%-56%) (P less than 0.001). There was no difference in the change in atrial electrogram size between the two lead types. Treadmill exercise testing with telemetric data of atrial electrograms showed a decrease in atrial electrogram size produced by exercise and may be helpful in determining appropriate atrial sensitivity settings in selected individuals. Because of the documented decrease in atrial electrogram size produced by exercise, we recommend obtaining maximal atrial electrograms at the time of implant and use of pacing systems that allow maximal flexibility in atrial sensing especially in athletically active individuals. PMID- 1723192 TI - Benefits of smaller electrode surface area (4 mm2) on steroid-eluting leads. AB - The purpose was to test whether a reduction of pacemaker electrode surface area below 8 mm2 improves leads that elute steroid from the electrode tip to the surrounding myocardium. A standard-sized 8 mm2 lead with 1 mg dexamethasone was implanted in 12 patients and a lead with 4 mm2 electrode surface area and 0.5 mg dexamethasone in ten patients. Pacing threshold, impedance, and sensing threshold were measured at implantation and after 1, 4, and 12 weeks. Pacing thresholds were similar for both groups and were always less than or equal to 0.8 V at 0.5 msec pulse duration in all patients. Impedance was significantly higher (P less than 0.05) for the 4 mm2 lead (implantation: 726 +/- 119 ohms; 1 week: 596 +/- 71 ohms; 4 weeks: 624 +/- 68 ohms; 12 weeks: 643 +/- 56 ohms) than for the 8 mm2 lead (implantation: 422 +/- 43 ohms; 1 week: 402 +/- 48 ohms; 4 weeks: 439 +/- 57 ohms; 12 weeks: 449 +/- 61 ohms). R wave amplitudes did not differ between both groups; no sensing failure occurred at 5 mV sensitivity. Compared to the 8 mm2 lead the reduction of surface area to 4 mm2 did not influence pacing threshold, but resulted in a higher pacing impedance. The amount of pacing energy was lower in the smaller-sized electrode. For clinical impact, low pacing threshold and high impedance leads are the condition to implant pulse generators with smaller battery capacity. PMID- 1723194 TI - The influence of elevated 50 Hz electric and magnetic fields on implanted cardiac pacemakers: the role of the lead configuration and programming of the sensitivity. AB - The influence of the electromagnetic interference (EMI) on performance of 15 implanted cardiac pacemakers (12 generator models) was tested during exposure at a high voltage substation. All patients had an adequate spontaneous heart rate during the study. Tests were performed in the ventricular inhibited mode with unipolar sensing in all pacemakers and repeated with bipolar sensing in four pacemakers. The sensitivity was set to a regular, functionally proper level and then to the highest available level. Exposure was done to moderate (1.2-1.7 kV/m) and strong (7.0-8.0 kV/m) electric fields, which correspond to the immediate vicinity of 110 and 400 kV power lines, respectively. In moderate electric fields the output was inhibited in one pacemaker at regular sensitivity (1.7-3.0 mV) and in five pacemakers at the highest sensitivity (0.5-1.25 mV). In strong electric fields the output was inhibited in five pacemakers at regular sensitivity and several pacemakers converted to noise reversion mode at the highest sensitivity. In bipolar mode only one of four pacemakers at high sensitivity (0.5-1.0 mV) was inhibited in the strongest electric field, whereas all four did so in the unipolar mode. One pacemaker with unipolar sensitivity at 0.5 mV was interfered by 63 microT magnetic field. The results confirm that the programmed sensitivity level and the lead configuration markedly influence pacemakers' vulnerability to EMI. Bipolar sensing mode is rather safe in the presence of EMI, which is encountered in public environments. The programmable features of today's pacemakers permit individualized, less stringent safety measures to avoid electromagnetic hazards. PMID- 1723193 TI - Catheter ablation of the atrioventricular junction using a helical microwave antenna: a novel means of coupling energy to the endocardium. AB - Catheter ablation with either direct current defibrillator discharges or radiofrequency energy produces tissue injury via current flow from an electrode into the adjacent myocardium. In order to affect tissue at a distance, excessive power density may be produced at the electrode-tissue interface with the possibility of explosive gas formation or coagulum formation. A novel microwave catheter was developed with a helical antenna distally. This coil, although not in direct contact with the endocardium, radiates an electromagnetic field into the tissue that, in turn, causes thermal injury. The utility of this system for ablation was assessed in six dogs. The antenna catheter was introduced percutaneously and positioned so as to record the largest His electrogram. Microwave power (50 watts at 2,450 MHz) was applied for 114 +/- 118 seconds. Complete AV block was produced in all six animals with 1.8 +/- 1.2 applications. There was no ventricular ectopy or change in blood pressure during microwave ablation. One dog died 6 days after ablation. The remaining five dogs had persistent, complete AV block during 6 weeks of follow-up. Pathological analysis at 6 weeks revealed a large (mean 2.8 x 4.7 mm) fibrovascular scar in the region of the AV junction. Percutaneous microwave ablation of the endocardium appears feasible. By radiating an electromagnetic field without direct contact, this system can produce large lesions without being limited by desiccation of tissue and impedance rise. PMID- 1723195 TI - Atrioventricular electrotonic interaction in complete atrioventricular block: an experimental model using radiofrequency energy in the rabbit heart. AB - Provided the conditions for electrotonic transmission exist, an automatic focus surrounded by a block zone may be externally modulated. The atrioventricular (AV) electrotonic interaction was studied in 16 perfused rabbit hearts with supra Hisian complete AV block induced using low radiofrequency energy doses (2.5 watts; 10 seconds). In nine experiments the sinus node was preserved (group I), whereas in seven it was removed maintaining an AV nodal rhythm (group II). The V V (ventricular cycle length) and V-A (coupling of the intervening atrial beat) in both groups, and also the A-A (atrial cycle length) and A-V (coupling of the intervening ventricular beat) intervals in group II, were measured beat by beat after current delivery. The phase response curves V-V versus V-A, and A-A versus A-V showed AV interaction in five experiments from group I, and in four from group II, as follows: (1) accelerating phase response curve, characterized by a pacemaker acceleration (V-V or A-A abbreviation) at a critical V-A or A-V coupling interval; maximum acceleration could be progressively (phase response curve without rapid cross-over) or briskly (phase response curve with rapid crossover) reached; from this point onwards acceleration decreased with a further increase in V-A or A-V coupling interval (acceleration slope). (2) Biphasic phase response curve, characterized by initial delaying and late accelerating phases. Maximum acceleration and the acceleration slope were both smaller in accelerating phase response curves without rapid cross-over. On reverting complete block in two experiments, a progressive increase in maximum acceleration and acceleration slope was observed. CONCLUSIONS: (1) AV interaction in complete AV block can be manifested as accelerating or biphasic phase response curves; (2) transition from electrotonic interaction to conduction seems to be a continuum. PMID- 1723196 TI - Lack of physiological adaptation of the atrioventricular interval to heart rate in patients chronically paced in the AAIR mode. AB - Seventeen consecutive patients, aged 56 +/- 12, were chronically paced in the AAIR mode for a symptomatic sinus node disease with atrial chronotropic incompetence defined by a peak exercise heart rate (HR) less than 75% of the maximal predicted heart rate (MPHR) mean = 65 +/- 10%). Sensors used were activity sensing (n = 7), minute ventilation (n = 6), or respiratory rate (n = 4). Basic pacing rate was programmed at 71 +/- 5 beats/min and the maximal sensor rate at approximately 85% MPHR (143 +/- 10); other sensor parameters were programmed individually. Six months after implant, two standardized and symptom limited exercise tests were performed in random order, AAI and AAIR modes, respectively. AAIR pacing significantly improved peak exercise HR (139 +/- 14 vs 112 +/- 30 beats/min; P less than 0.01), maximal sustained workload (132 +/- 42 vs 110 +/- 38 watts; P less than 0.02), and total exercise duration (724 +/- 299 vs 594 +/- 245 sec; p less than 0.02) compared to the AAI mode. In all 17 patients, HR was continuously sensor driven in the AAIR mode, making it possible to precisely study the adaptation of the stimulus-R interval and of the stimulus R:RR ratio during exercise. Six patients normally adapted with a progressive shortening. Six others did not adapt at all without any variation of interval. Five patients paradoxically increased their stimulus-R interval (286 +/- 10 msec at peak E vs 220 +/- 19 msec at rest) and their stimulus-R:RR ratio (67 +/- 20% vs 29 +/- 4%), producing P waves occurring immediately after, or even within the R wave of the preceding cycle; two patients complained of severe exercise related symptoms corresponding to the so-called "AAIR pacemaker syndrome." The principal factors involved in the nonadaptation of AV interval to HR were related to the patient (organic heart disease, with the particular problem of the denervated heart; the bradytachy syndrome; and the use of drugs, especially beta blockers and Class I antiarrhythmic drugs) or to the pacemaker ("overstimulation" phenomenon). These observations constitute an additional argument for wider indications of implanting DDDR units in these patients. PMID- 1723197 TI - Radiation-induced effects in multiprogrammable pacemakers and implantable defibrillators. AB - Twenty-three multiprogrammable pacemakers and four implantable cardioverter defibrillators (ICDs) containing either complementary metal-oxide semiconductor (CMOS) or CMOS/Bipolar integrated circuit (IC) technology were exposed to 6-MV photon and 18-MeV electron radiation at various dose levels. Of the 17 pacemakers exposed to photon radiation eight failed before 50 Gy, whereas four of the six pacemakers exposed to electron radiation failed before 70 Gy. Photon scatter doses were well tolerated. For the ICDs detection and charging time increased with accumulated radiation dose, the charging time increased catastrophically at less than 50 total pulses delivered when compared with the charging time of six implanted ICDs. Sensitivity and output energy delivered by the ICD pulse were constant during the test. It was found that devices using the shorter channel length IC technology (i.e., 3 microns CMOS) were per se harder to ionizing radiation than the devices using larger channel length IC technologies (i.e., either 8 microns CMOS or combined 5 microM CMOS/20 V Bipolar). In fact, none of the devices based on 3 microns CMOS IC technology failed before 76 Gy, which is above the highest dose level (70 Gy) normally used in radiation oncology treatments. PMID- 1723198 TI - Direct current shock ablation: quantitative assessment of proarrhythmic effects. AB - Catheter ablation using direct current (DC) shock has proved invaluable in the management of a variety of tachycardias. However, sporadic reports of fatal arrhythmias following ablation have raised the question of the proarrhythmic potential of DC shock ablation. The present study was undertaken in 45 patients to assess prospectively any proarrhythmia related to DC shock ablation, using matched pre- and postablation Holter monitors and programmed electrical stimulation (PES). Nineteen of these patients had Holter monitors for three successive postablation days to observe trends. There was unmatched data in 11 additional patients. All 56 patients provided prospective follow-up for clinical events. There was no immediate sustained VT/VF at the time of the ablation. Four patients had sustained VT in the first 72 hours after ablation; three episodes were similar to the preablation clinical arrhythmias; one patient had torsades de pointes interrupting bradycardia. Twelve patients met Holter, PES, or clinical criteria for proarrhythmia; none were treated on the basis of these findings. On Holter monitoring, there were significant increases in VPCs/hour and couplets/hour in patients undergoing atrial or atrioventricular junctional ablations; and an increase in couplets after accessory pathway ablations. Increases in these categories were not significant for VT patients; nor were increases in episodes of VT/hour or atrial arrhythmias significant in any group. Patients were followed for 44 +/- 33 months, with an actuarial survival of 95% at 1 year, 88% at 3 years, and 85% at 4 years. There were six deaths during follow up. Two patients had sudden death: one at 2 months had early evidence of proarrhythmia; the other at 32 months may have represented later myocardia deterioration. One patient died of heart failure at 77 months; and there were three noncardiac deaths. DC shock ablation in humans is much less proarrhythmic than in dogs. The low incidence of clinical proarrhythmic events during prolonged follow-up after discharge resulted in low sensitivity, specificity, and positive predictive values for Holter and PES, although the negative predictive values of these tests were greater than 90%. Only one of 12 patients who met criteria for proarrhythmia in the days immediately following ablation had subsequent clinical events consistent with proarrhythmia. These results may be useful as standards for comparison with results of radiofrequency or other ablation modalities. PMID- 1723199 TI - Cardiac electrophysiological experiments in numero, Part III: Simulation of arrhythmias and pacing. AB - This paper is the third and final part of a series of articles reviewing mathematical and computer models of the electrophysiological processes. This section reviews the arrhythmia simulation and discusses models of arrhythmogenic processes, fibrillation and defibrillation, and of heart-pacemaker interaction. The models of arrhythmogenesis are classified into three main sections: models of reentry and vortex reentry, models of myocardial electrotonic interactions, and models of macroreentrant supraventricular tachycardias. This final part of the review discusses the future potential of mathematical and computer models of different cardiac processes. PMID- 1723200 TI - Influence of time of sampling onset on parameters used for activation time determination in computerized intraoperative mapping. AB - The purpose of this work is to determine the sensitivity of the estimated time of peaks and maximum slopes, commonly used in activation time computations, to the instant at which sampling is initiated. Based on complex and quickly changing waveforms, 471 monopolar (MP) and bipolar (BP) epicardial responses in man were selected. These were decimated from 10 kHz to simulate sampling at frequencies ranging from 200 Hz to 2,000 Hz. The peak and maximum absolute slope for BP and the minimum slope for MP were computed repeatedly starting at successive 100 microseconds intervals extending throughout the sampling period and compared with these parameters computed from the waveform sampled at 10 kHz. Slopes were estimated using each of four different algorithms. The average greatest shift (AGS) due to variations in sampling onset ranged from 11.2 +/- 3.5 (200 Hz) to 0.3 +/- 0.2 msec (2,000 Hz). For bipolar algorithms, the peak performed better than the slope algorithms (AGS: 5.9 +/- 3.3 to 0.3 +/- 1.0 msec). For MP algorithms, 2 point linear, and 3 and 5 point Lagrange slope estimates performed similarly (AGS: 5.6 +/- 3.3 to 0.3 +/- 0.2 msec); a 5 point least square fit algorithm performed poorly. Sampling MP and BP electrograms below 500 and 400, respectively, often caused maximum shifts greater than 4 msec. Thus, the resolution of the peak and estimated slope is not limited to the sampling period, variations in initiation of sampling can cause significant outliers especially at low sampling rates, and MP electrograms should be sampled faster than BP electrograms for comparable accuracy. PMID- 1723201 TI - Significance of QRS alternans during narrow QRS tachycardias. PMID- 1723202 TI - [Plasma renin activity in women with hyperthyroidism]. AB - Serum renin activity was measured in 35 women aged 18-57 (mean age = 37.3) with Graves-Basedow hyperthyreosis, before and after the thyrostatic treatment. Serum renin activity was significantly higher in yet untreated patients as compared to the healthy controls, but after treatment it did not differ from that of the controls. The results seem to confirm a causative relation between the thyroid hormones and the renin-angiotensin-aldosterone system. PMID- 1723204 TI - Antiangiogenic effect of heparin and other sulphated glycosaminoglycans in the chick embryo chorioallantoic membrane. AB - The effect of heparin and other sulphated glycosaminoglycans on normal capillary growth was studied in the chick embryo chorioallantoic membrane, with or without hydrocortisone. Contrary to previous reports, heparin was shown to have an antiangiogenic effect in itself, and an additive effect was obtained when it was combined with hydrocortisone. Heparan sulphate also had an antiangiogenic effect in the chorioallantoic membrane, while keratan sulphate, dermatan sulphate or chondroitin sulphate had no such effect. Copper ions, added in small amounts, did not influence the antiangiogenic effect of heparin, and nor did iron, zinc and magnesium ions, and EDTA. PMID- 1723203 TI - Clinical immunology. PMID- 1723205 TI - [Production of immune interferon in experimental tuberculosis and antituberculosis vaccination in mice]. AB - The serum content of interferon (IF) and its correlation with diverse immunologic parameters were studied in tuberculosis-resistant mice. In primary contamination the IF level was much higher in B10 mice highly sensitive to tuberculosis infection in all periods of the study than that in highly resistant A/Sn mice. In the vaccinated mice which were later infected this tendency remained but IF levels were 1.5--2 times higher than in primary contamination. Comparison of the IF level and the studied resistance parameters in the resistant mice revealed an inverse correlation. If the immunologic parameters in resistant A/Sn mice are higher than those in B10 mice the latter have significantly higher serum IF levels. PMID- 1723206 TI - Critical issues in defining the role of serotonin in psychiatric disorders. PMID- 1723207 TI - Effect of the lysosomotropic drug suramin on islet lysosomal enzyme activities and the insulin-secretory response induced by various secretagogues. AB - The trypanocidal drug suramin is known to concentrate in lysosomes and to depress the activity of different lysosomal enzymes. We have previously shown that suramin can inhibit the activity of the islet lysosomal enzyme acid amyloglucosidase, a glycogenolytic glucose-producing hydrolase, which seems to be involved in certain insulin-secretory processes. In the present investigation we studied the pH dependency and dose-response effects of suramin on islet lysosomal enzyme activities as well as the effect of suramin treatment on the insulin secretory response to various secretagogues in mice. It was found that two injections of suramin (0.18 mmol/kg) to normal NMRI mice at -24 and -2 h induced a moderate depression of the activities of islet acid amyloglucosidase (-22%) and acid phosphatase (-13%), whereas no effect was recorded for the activities of acid alpha-glucosidase, N-acetyl-beta-D-glucosaminidase and the non-lysosomal enzyme neutral alpha-glucosidase. Direct addition of different concentrations of suramin to islet homogenates showed that the drug was a potent inhibitor of acid amyloglucosidase and acid alpha-glucosidase at pH 4.0. At pH 5.0, suramin induced a large increase in acid alpha-glucosidase activity, whereas acid amyloglucosidase and acid phosphatase were inhibited. Suramin-injected mice showed a reduced insulin-secretory response to the sulphonylurea drug glibenclamide (-45%), whereas the insulin response to the cholinergic agonist carbachol or the phosphodiesterase inhibitor IBMX (1-isobutyl-3-methylxanthine) was unaffected. It is concluded that suramin inhibits islet acid amyloglucosidase activity in vivo and in vitro, whereas its effect on acid alpha-glucosidase is complex and pH dependent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723208 TI - Australian Halobacteria and their retinal-protein ion pumps. AB - Halophiles collected in Western Australia have been found to be examples of extremely halophilic rod-shaped archaebacteria, members of the genus Halobacterium. Most of them contain retinal proteins, and these proteins differ from one another and also from both bacteriorhodopsin (bR) and halorhodopsin [and sensory rhodopsins (sR)] isolated from Halobacterium salinarium (halobium), as revealed by their peptide maps and amino acid sequences. However, these retinal proteins still have the ability to pump protons or chloride ions in the light. These new ion pumps, designated archaerhodopsins (aR) [Mukohata et al. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345], are almost identical in terms of their molecular sizes and transient photochemical properties to the ion pumps identified previously. Differences are found in the: (1) apparent extinction coefficient of dark/light-adapted aR-2; (2) titration profiles at acidic pH of the absorption spectra of all aRs; and (3) circular dichroism spectra, which are influenced by the coexistent isoprenoid bacterioruberin. The amino acid sequences of two proton pumps from the Australian halobacteria, namely aR and aR-2, are approximately 90% homologous and both sequences are about 60% homologous with that of bR. Hydropathy plots suggest that these pumps also have a seven-helical structure similar to that of bR. The amino acid residues are highly conserved in the helical regions, in particular in the case of helices C and G (91 and 84%, respectively), among the three proton pumps. PMID- 1723209 TI - Disposition of an antineoplastic sesquiterpene lactone, [3H]-plenolin, in BDF1 mice. AB - The pharmacokinetics of a radiolabelled analog of helenalin, [3H]-plenolin ([3H] 11,13-dihydrohelenalin), was determined in BDF1 mice following intravenous, intraperitoneal, and oral administration. A two-compartment pharmacokinetic model predicted that the maximum terminal (beta) half-life of [3H]-plenolin was 57.3 hours. Urinary excretion accounted for 40.3% to 64.4% of the administered radioactivity, while fecal excretion accounted for 9.3% to 39.7%. The fecal excretion data also suggested that [3H]-plenolin was secreted in the bile. Following intraperitoneal administration of [3H]-plenolin, no radioactivity was sequestered in the major organs. However, radioactivity was sustained in the carcass and skin for 24 days. [3H]-Plenolin was rapidly taken up by murine tumor cells and human fibroblasts. The drug did not significantly associate with DNA, RNA, or protein of P388 leukemia or human fibroblast cells. PMID- 1723210 TI - [The structure and function of RNA replicon]. PMID- 1723211 TI - [Arterial hypertension, left ventricular hypertrophy and arrhythmias]. AB - Arterial hypertension is the most common cause of chronic pressure overload of the left ventricle. Electrocardiographic and echocardiographic signs of left ventricular hypertrophy in hypertensive patients are associated with an increased cardiovascular mortality and incidence of sudden death habitually due to ventricular arrhythmias. The significance of a normal increase in systolic blood pressure during exercise in persons without evident resting hypertension is uncertain. M-mode and 2D echocardiography, 24-hour continuous ambulatory electrocardiographic (Holter), exercise testing and 24-hour ambulatory blood pressure monitoring (ABPM) were performed on 22 normotensive patients (group I); 25 normotensives with exaggerated blood pressure response to exercise (greater than 220 mmHg) (group II) and 33 hypertensive patients (group III). None was taking cardioactive drugs. Left ventricular hypertrophy (LVH) was found on one patient of group I (4.5%), 13 of group II (52%) and 20 of group III (61%). Left ventricular mass index (LVMI) was linearly correlated with maximum exercise blood pressure (group I: r2 = 0.518, p less than 0.0002; group II: r2 = 0.098, NS; group III: r2 = 0.407, p less than 0.0001) with 24-hour systolic pressure overload (ABPM) (group I: r2 = 0.848, p less than 0.0001; group II: r2 = 0.705, p less than 0.0001; group III: r2 = 0.839, p less than 0.0001) and 24-hour diastolic pressure overload (ABPM) (group I: r2 = 0.612, p less than 0.0001; group II: r2 = 0.815, p less than 0.0001; group III: r2 = 0.807, p less than 0.0001) within each group but not between different groups. The hypertensive subjects (group III) had a higher average heart rate (p less than 0.0001) more supraventricular premature (p less than 0.0001) and ventricular premature (p less than 0.0001) beats than the normotensive (group I) and normotensive patients with abnormal increases in systolic blood pressure response to exercise (group II) (p less than 0.0001) (NS) and (p less than 0.0002), respectively. LVMI was linearly correlated with ventricular premature beats (group I: r2 = 0.072, NS; group II: r2 = 0.823, p less than 0.0001; group III: r2 = 0.691, p less than 0.0001). Frequent and complex ventricular arrhythmias were more common in patients with LVH normotensives or hypertensives than without LVI (p less than 0.0001) and the age increases their severity. We conclude that normotensives with hypertensive response to exercise have similar incidence of LVI; if those patients develop sustained hypertension, LVI was previous to arterial hypertension. There are two types of hypertrophy: secondary hypertrophy is linked to the high afterload and vasoconstriction typical in hypertension.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1723212 TI - [In situ and invasive cancer of the gallbladder: nucleolar organizer regions]. AB - We studied samples from gallbladders of 10 subjects with invasive and 10 with in situ carcinoma of this organ. The number of nucleolar organizer regions, as revealed by colloidal silver staining, was at least 4 times larger in cancer samples than in control ones. Mean number of nucleolar organizer regions for invasive lesions was higher than for in situ ones (11.6 +/- 2.3 vs 9.4 +/- 1.3, p less than 0.04). The study of cellular markers may be helpful in establishing the preneoplastic nature of gallbladder lesions. PMID- 1723213 TI - [Neoplasms of Vater's papilla: study of nucleolar organizer regions]. AB - We analyzed the number and aspect of nucleolar organizer regions in 11 specimens from ampullary carcinoma, by means of silver colloidal staining. The number of regions was higher than that found in intestinal villi but similar to that in cripti. The size of the regions was larger than any of those mentioned above. These elements may contribute to evaluate endoscopic samples in the diagnosis of ampullary lesions. PMID- 1723214 TI - [Progeny in women with fetal alcohol syndrome]. AB - Among children referred to our genetic clinics for mental or growth retardation we identified 8 of their mothers with the fetal alcohol syndrome. This was complete in 5 and partial in 3. All of their alcoholic mothers had died. Most of the patients were unwed mothers with mental retardation and no elementary education. One of them was also alcoholic and her third offspring had the syndrome. The etiology of this syndrome and possible preventive measures are discussed. PMID- 1723215 TI - Bacteria and viruses induce production of interferon in the cerebrospinal fluid of children with acute meningitis: a study of 57 cases and review. AB - The CSF of 57 infants and children with bacterial or enterovirus meningitis was analyzed for the presence of interferon (IFN). CSF was collected when the diagnosis of meningitis was made; a bacterium or enterovirus was isolated in all cases. IFN was detectable in CSF in 24% of cases of bacterial meningitis and in 75% of cases of viral meningitis. Titers of IFN were generally lower in cases of bacterial meningitis. Neither the presence of IFN nor the level of IFN titers correlated with the patient's age or number of white blood cells or mononuclear cells in the CSF. Coxsackievirus induced production of IFN more consistently and in higher titers than did echovirus. None of 35 control patients had detectable IFN in CSF. A literature review and our data indicate that the presence of IFN in CSF suggests infection of the CNS but does not differentiate bacterial from viral infection. The finding of IFN in the CSF of children with bacterial meningitis supports evidence that bacteria and other nonviral microorganisms induce IFN production. The protective role of IFN in nonviral infections deserves further investigation. PMID- 1723216 TI - [Visceral leishmaniasis associated with Hodgkin's disease: diagnostic difficulties]. AB - A case of Hodgkin's disease associated from the start with visceral leishmaniasis in the absence of antitumoral treatment shows that leishmaniasis is a severe opportunistic infection in endemic areas and can be masked by the tumoral syndrome of an underlying pathology. Conversely, patients with visceral leishmaniasis must be investigated for a cause of immunosuppression with, in particular, biopsy of accessible lymph nodes. The exceptionally favourable course of this particular case deserved to be high-lighted. PMID- 1723217 TI - [Treatment of human papillomavirus infection of the uterine cervix with gel interferon]. AB - Treatment of uterine cervix infection caused by human papillomavirus still is an enigma. Some drugs have been tested with cure rates between 60 to 70%. Most of such agents are substances which produce a strong epithelial desquamation and have shown strictly toxic side effects. The use of interferon in this infection has been studied, but different authors disagree in their therapeutic findings. A randomized, double blind trial was conducted to compare interferon to placebo. Preliminary results are presented in this article. 47 patients were followed so far. 18 of them were evaluated and the results are shown in this article. PMID- 1723219 TI - [Enoxacin in the treatment of asymptomatic bacteriuria in patients with prostatic adenoma]. AB - Fifty patients suffering from prostatic adenoma with asymptomatic bacteriuria, were admitted to an open non comparative trial. Enoxacin was administered at the daily dosage of 300 mg every 12 hours for 10 days. Three cycles of treatment were performed during three consecutive months. Treatment efficacy was established by assessing patient symptoms related to the infection such as pollakiuria , nocturia, decreased flow rate, stranguria, daily temperature. Cultural tests were also performed. All observations were collected at baseline and at the end of each cycle of therapy. Cure was obtained in 43 patients (87, 75%), 1 patient (2,04%) relapsed, 5 patients (10.2%) withdrew because of inefficacy of treatment and 1 patient died of heart failure. No side effects were observed. These results suggest that enoxacin may be successfully used in the treatment of asymptomatic bacteriuria. PMID- 1723218 TI - [Influence of diet composition on the late course of acute pancreatitis. Experimental study in rats]. AB - Acute pancreatitis was induced with sodium taurocholate 1% in two lots of rats fed during 21 days with diets that differed in lipid composition. Serum amylase, pancreatic tissue enzymes (trypsinogen, chymotrypsinogen and amylase), pancreatic tissue nucleotides (RNA and DNA) and biopsies for histological study were collected in normal pair fed animals, and in the experimental lots 1, 4, 7 and 15 days after AP was induced. ANOVA and Student t-test were used for the comparison of biochemical data (p less than 0.05). They showed that acute pancreatitis aggravated progressively until the fourth day independently of the regimen. On the 15th day, the histological and biochemical parameters reached normal values. The authors concluded that high lipidic diet was not the main factor responsible for progressive injury of the pancreas. PMID- 1723220 TI - [Urological and sexual evaluation of treatment of benign prostatic disease using Pygeum africanum at high doses]. AB - This clinical study has been designed to evaluate the efficacy of an extract of Pygeum Africanum (Tadenan) (Roussel-Pharma) in patients suffering from prostatic hypertrophy or chronic prostatitis. The drug has been administrated to 18 patients, for 60 days, as double of standard dosage (200 mg/die per os, instead of 100 mg/die). Because of the high frequency of association of sexual disorders with those two pathologies, we have extended the study also to sexual disorders selecting patients suffering from prostatic hypertrophy or chronic prostatitis and, simultaneously, from sexual disturbances. No side effects have been observed during the treatment. The urinary disturbances have been evaluated by anamnesis and prostatic transrectal echography; sexual disorders have been evaluated by anamnesis and nocturnal penile tumescence and rigidity (NPTR) monitoring. Furthermore, dosage of serum levels of the hormones LH, FSH, Prolactin, 17 beta Estradiol and Testosterone has been performed before and after therapy. Pygeum Africanum extract administration improved all the urinary parameters we investigated; prostatic echography relieved reduction of peri-urethral edema. Also an improvement of sexual behaviour has been obtained; but we have not found significant differences between serum hormonal levels before and after therapy, as well as for NPTR. PMID- 1723221 TI - [The treatment of viral hepatitis]. PMID- 1723222 TI - Reliability of questionnaire responses as compared with interview in the elderly: views of the outcome of transurethral resection of the prostate. AB - Three hundred and eighty-eight men undergoing transurethral resection of the prostate for benign prostatic hypertrophy completed a presurgical questionnaire and three follow-up questionnaires 3, 6 and 12 months after surgery. The questionnaires covered details of prostatic symptoms, general health, and expectations and results of surgery. At each follow-up point 40 randomly selected patients were interviewed by two female research assistants. The response rate to the questionnaires was over 90% at each follow-up point while that for the interviews was lower at around 80%. We examine the reliability of the postal questionnaires in assessing health status by comparing questionnaire and interview responses, with a view to the wider employment of such a method in the follow-up of surgical patients. In general, and as reported elsewhere, responses to questions on easily defined topics are highly comparable between questionnaire and interview. Responses to more subjective questions are moderately reliable, but with a tendency for postal questionnaires to underestimate a patient's health problems. It is difficult to assess the reliability of the questionnaires with regard to questions of an intimate nature since such questions caused embarrassment during interview with consequent incomplete responses. PMID- 1723223 TI - Heat-shock proteins and pathogenesis of bacterial infections. PMID- 1723225 TI - NG-monomethyl-L-arginine increases platelet deposition on damaged endothelium in vivo. A scanning electron microscopic study. AB - There is increasing evidence that nitric oxide (NO) synthetized in vascular endothelium and in platelets by NO synthase influences vascular tone, down regulates platelet function and platelet-vessel wall interaction both in vitro and in vivo. We investigated the effect of a NO synthase inhibitor, NG-mono methyl-L-arginine (L-NMMA, 100 mg/kg iv) on platelet-endothelial cell interaction in rabbit arteries ex vivo using scanning electron microscope (SEM). The effect of L-NMMA was examined on intact endothelium and on that damaged by arterial constriction. The infusion of L-NMMA increased systemic blood pressure and decreased carotid blood flow, however, it did not change the appearance of an intact endothelium and did not result in platelet activation on intact endothelial cells. In contrast, SEM of endothelial areas damaged by constriction showed extensive platelet adhesion and aggregation on subendothelium. These morphological changes were not detected in control animals with intact or damaged by arterial constriction endothelium. These results show that under physiological conditions, the inhibition of NO synthase alone does not result in platelet activation in vivo. However, when combined with endothelial injury it may lead to platelet activation and thrombosis. PMID- 1723226 TI - Alteration of the stromal architecture and depletion of keratan sulphate proteoglycans in oedematous human corneas: histological, immunochemical and X-ray diffraction evidence. AB - The structure and content of the extracellular stromal matrix of several oedematous human corneas was investigated using electron microscopy, X-ray diffraction and biochemical techniques. Electron microscopy revealed the presence of wavy lamellae and various sized collagen-free 'lakes' within the stroma of the oedematous corneas, with their posterior sections containing by far the largest 'lakes'. The existence of 'lakes' was supported by the equatorial X-ray diffraction evidence. Staining the oedematous corneas with Cuprolinic blue prior to electron microscopical and meridional X-ray diffraction studies demonstrated a loss of stromal proteoglycans normally associated with collagen. Immunochemical evidence demonstrated reduced levels of antigenic keratan sulphate in the oedematous corneas while biochemical techniques revealed constant chondroitin sulphate levels in the same corneas. PMID- 1723224 TI - Parasite heat-shock proteins and host responses: the balance between protection and immunopathology. PMID- 1723227 TI - Gap junction protein tissue distribution and abundance in the adult brain in Drosophila. AB - The distribution of gap junction (GJ) protein in Drosophila tissues and developmental stages was determined by probing immuno-blots with an anti Drosophila GJ protein antibody (R2AP18). All tissues and developmental stages examined contained 18, 24 or 72 kD GJ protein. GJ protein was notably abundant in immuno-blots of homogenates of adult brain tissue. This was confirmed by the direct visualization of GJs in thin sections of adult brain by electron microscopy. GJs were particularly large and numerous between glial cells in the optic lobes and peripheral glial sheath. R2AP18 reactivity was used to identify GJ protein in immunoblots of cell fractions from isolated adult heads. The final GJ-enriched pellets, derived by extracting crude membrane fractions with urea and N-lauroyl sarcosine, contained GJs with reduced profile widths (13-15 nm vs 16-18 nm for native GJs) and which, unlike native GJs in the crude membrane fractions, were immuno-labelled by R2AP18. Immuno-blots of the urea-sarcosine extracted GJ pellets and supernatant contained higher molecular weight R2AP18 immuno-reactive proteins in addition to the 18 kD form which was present in the tissue homogenate and crude membrane fractions. The results confirm previous observations that urea sarcosine causes alterations in GJ structure and suggest that urea-sarcosine treatment exposes antigenic determinant(s) which are unavailable for R2AP18 binding in non-extracted native GJs. The abundance of GJs in the adult brain and the relatively simple R2AP18 staining patterns in immuno-blots of GJ-enriched fractions from isolated adult heads suggest that this tissue will be useful for further biochemical and molecular studies of GJs in Drosophila. PMID- 1723228 TI - Mechanisms of lindane-induced hepatotoxicity: alterations of respiratory activity and sinusoidal glutathione efflux in the isolated perfused rat liver. AB - 1. Lindane (25-60 mg/kg) at 24 h after dosage induced a dose-dependent increase in oxygen consumption by perfused rat livers, an effect not observed at early times (2-6 h) after administration. About 60% of the increase in liver oxygen uptake is suppressed by the antioxidant, desferrioxamine, indicating enhanced free radical activity induced by the insecticide. 2. The hepatic content of total GSH equivalents (GSH + 2GSSG) decreased 4 h after lindane treatment (60 mg/kg), together with significant diminution in net and fractional rates of sinusoidal GSH efflux, that returned to control values 24 h after treatment. 3. These data indicate that lindane resulted in marked changes in hepatic oxidative capacity and glutathione metabolism, which condition the production of oxidative stress in the liver at different times of intoxication. PMID- 1723229 TI - Effect of phenobarbital and 3-methylcholanthrene on the early oxidative stress component induced by lindane in rat liver. AB - 1. Lindane administered to untreated rats or rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) increased liver lipid peroxidation, of the same magnitude in all groups. 2. PB pretreatment produced a 50% increase in lipid peroxidation (TBAR) by liver homogenates and microsomes, an effect accompanied by increases in cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH oxidase and microsomal superoxide anion production, MC pretreatment resulted in increases in liver cytochrome P-450 and NADPH oxidase only. 3. Pretreatment of rats with PB, but not MC or lindane, gave increases in glutathione peroxidase and reductase. 4. Pretreatment with PB, but not MC, increased liver GSH. Lindane decreased liver GSH to the same extent as PB plus lindane. 5. Biliary GSH, GSSG and bile flow were decreased by lindane to similar extents in all groups. 6. Lindane induced periportal necrosis with haemorrhagic foci in all groups. 7. Data presented indicate that the early lipid peroxidative response of liver to lindane was unchanged by PB- or MC-stimulated hepatic microsomal enzyme induction. PMID- 1723230 TI - [Therapy of HIV-associated Kaposi's sarcoma]. PMID- 1723231 TI - [Plasma nucleic acids and their possible pathophysiologic significance in systemic lupus erythematosus]. AB - The discussion of the possible pathophysiological role of plasma and/or serum nucleic acids from patients suffering from systemic lupus erythematosus is nearly identical with the still unsolved question regarding the antigen, i.e., the possible autoantigen-inducing antibodies against native double-strand DNA (dsDNA). This question is of special interest, since native dsDNA per se is not immunogenic. As repeatedly demonstrated, circulating antibodies of the isotype IgG against dsDNA can be correlated with the disease activity and, particularly, with renal involvement. Antibodies against native dsDNA were isolated from affected organs from SLE patients. The analysis of circulating immune complexes revealed dsDNA, as well as antibodies against dsDNA as complex components. DNA anti-dsDNA serum immune complexes could also be demonstrated in patients with a so-called seronegative SLE, i.e., in patients not showing any free antibodies against native dsDNA in their sera. Furthermore, the involvement of antibodies against native dsDNA in pathogenic mechanisms of SLE patients becomes particularly evident from animal models. Regarding the induction of dsDNA antibodies, it is of special interest that in animal models such as the MLR/lpr mouse, anti-dsDNA antibodies are rather antigen-selected than they are a consequence of a "random" polyclonal B cell stimulation. Likewise, by the demonstration of somatic mutations of clonal human IgG-anti-dsDNA antibodies from SLE patients it has recently been possible to prove that these autoantibodies are also most probably antigen-selected.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723232 TI - von Willebrand factor antigen as an acute phase reactant and marker of endothelial cell injury in connective tissue diseases: a comparison with CRP, rheumatoid factor, and erythrocyte sedimentation rate. AB - Plasma von Willebrand factor antigen concentrations in 125 patients with connective tissues diseases were compared to the levels of the acute phase reactant C-reactive protein, with rheumatoid factor and with erythrocyte sedimentation rate. There was a positive correlation between von Willebrand factor antigen and C-reactive protein, but no correlation with rheumatoid factor or erythrocyte sedimentation rate. There were, however, a significant number of instances of high C-reactive protein with low von Willebrand factor antigen, and also low C-reactive protein with high von Willebrand factor antigen. Although von Willebrand factor antigen may be regarded as an acute phase reactant, high levels in the absence of other indicators of an acute phase response may be suggestive of damage/injury to the endothelium. PMID- 1723233 TI - Computerized angiographic study of the vascular supply of the pectoralis major muscle. AB - The pectoralis major muscle or musculocutaneous flap is well suited to repair immediately wide defects following surgical removal of carcinomas or traumas of the cervicofacial and thoracic regions. Microsurgery has recently suggested exciting and successful solutions for the same purposes, but we think it is still important to reassess and perfect the flaps already known in order to achieve a better cost-benefit ratio. We examined the intramuscular vascular anatomy of 22 pectoralis major muscles using an image analyzer and a computerized measuring system to quantify the essential features objectively. The data show the segmentation of the pectoralis major muscle into two subunits, each provided with its own vascular supply. Slight anatomical differences and the presence of a well developed intramuscular vascular supply makes the pectoralis major muscular and musculocutaneous flap a useful and safe procedure. PMID- 1723234 TI - Successful healing of a burn injury covering 100% of TBSA and 96% IIIrd degree with inhalation injury. AB - The authors describe successful healing of a burn injury covering 100% of TBSA with 96% full-thickness skin loss and inhalation injury. The patient was admitted to the burn department of our hospital on September the 4th 1987. He smoothly overcame the shock stage with help of fluid replacement and application of alkaline drugs in large quantities. Early escharectomy and repeated micrografting were performed. The treatment is discussed. PMID- 1723235 TI - Multiple symmetric lipomatosis. AB - Multiple symmetric lipomatosis (MSL) is a rare disorder only mentioned in about 200 cases in the medical literature. It manifests as massive lipomatous deposits in specific areas of the body. The cause is unknown, although there frequently is a history of alcoholism. Surgical lipectomy has so far been the choice of treatment. We present a review of the disease and report one case successfully treated with liposuction. PMID- 1723236 TI - Extensive corium grafts. AB - Even in our times of revolutionary advances in the technology and operative techniques in plastic surgery, the corium is a material with multiple applications and can be found in the patient at varying thickness. The aim of the article is to show that its indication to reinforce flaccid musculature or to cover muscular defects of the abdominal wall is fully justified even in a time of routine use of plastic nets. PMID- 1723237 TI - Bilateral breast reconstruction after mastectomy. AB - The authors discuss the development of breast reconstruction following mastectomy in Czechoslovakia and present their first case of bilateral reconstruction employing the technique of the TRAM flap. In addition, they explore the possibility of eliminating high-risk parenchyma during with contralateral reconstruction. PMID- 1723238 TI - Craniofacial morphology in unilateral cleft lip and palate in adults. AB - X-ray measurements were used for studies of craniofacial morphology in unilateral (right-sided) cleft lip and palate in 58 adult males operated upon with the same technique. The results obtained showed a slightly smaller neurocranium without marked changes of the cranial base. Basic facial skeletal deviations included a shortening of maxillary depth, reduction of the upper face height, widening of some maxillary dimensions (interorbital and of nasal cavity), retroinclination of upper incisors and alveolar process and mandibular changes resulting from growth deficiency (they consisted of shortening of the body and ramus, elongation of anterior mandibular height, obtuse gonial angle, acute chin angle, steeper slope of the body, retroinclination of incisors and retrognathia). Described changes caused an impairment of sagittal and vertical jaw relations, anterior crossbite, flattening of the face and a limitation of its anterior growth rotation. There was also a displacement of the whole maxilla backwards and a reduction of the height and of the thickness of the upper lip (increasing retrocheilia). The elongation of the lower face and thus of the whole face was produced by the increase of the anterior height of the mandible, impaired overjet and by maxillary dentoalveolar retroinclination. A slight transversal flattening of the face (greater retrusion in the centre than in the zygomatic regions) was present as well. PMID- 1723239 TI - The influence of external factors on the development of primary palatal cleft. AB - The study was designed to assess the effect of heavy metal fallouts in industrial areas on the development of primary palatal cleft in Northern Moravia and Silesia over a period of five years (1985-1990). The study involved a total of 110 children; fallout levels in their places of residence were determined. Heavy metal fallout levels were measured in the ashes of needles and forest soil in the nearest possible locality to the place of birth of the child. The data obtained show a striking correlation between increases in cadmium and cobalt levels and the development of primary palatal cleft. PMID- 1723240 TI - Comparison of non-stereo polaroids and slides in detection of diabetic retinopathy. AB - Fundus photography plays an important role in detection and assessment of diabetic retinopathy. This study compared the detectability of diabetic retinopathy lesions on Polaroid prints and Ektachrome slides obtained with a non mydriatic camera. The number of lesions detected with Ektachrome slides was higher compared to Polaroids. The method of viewing the Ektachrome slides was also shown to be important. Slides when projected onto a white screen revealed a higher number of lesions compared to the same slide viewed through a macroscope. If a non-mydriatic camera is used to screen diabetic retinopathy, judgement from a projected Ectachrome slide is recommended. PMID- 1723241 TI - [Treatment of malignant stenosis of the cervical esophagus]. PMID- 1723242 TI - Immunotherapeutics of multiple sclerosis. AB - Immunosuppressive therapy may act specifically by inhibiting a certain stage of the immune response, or, by contrast, by inducing an overall inhibition of the immune system. Low dosage, a specific immunosuppressive therapeutic protocols with corticosteroids or azathioprine are used routinely, although there is no evidence of their effects. There are, however, unquestionable data on the numerous (and serious) side effects. High dosage, a specific immunosuppression appears to be better tolerated (probably due to the brevity of the treatment) and to have certain immunodepressant effects within the CNS. ACTH and methylprednisolone are recommended for short-term relapse therapy. Specific immunosuppressive treatment will no doubt be part of future autoimmune disease therapy, but for the time being is no more than theoretically stimulating experimentation (anti-idiotypic monoclonal antibodies, specific T cell suppressor factor, T cell "vaccination"). Important multicentric studies on cyclosporin-A and copolymer 1 are almost complete on the numerous side effects as regard to cyclosporin-A. Clinical trials with alpha or beta-interferons are still in progress and, so far at least, these drugs appear to be characterized by the absence of important side effects. PMID- 1723243 TI - Electrophysiologic characteristics of basal forebrain neurons in vitro. AB - Our data show that different cell types recorded in vitro can be identified by their intrinsic membrane properties. One type of neuron, namely S-AHP cells, have the ability to fire single action potentials in a rhythmic fashion following sufficient membrane depolarization. The rate is apparently controlled by several voltage-dependent conductances. S-AHP cells are normally quiescent at their resting potentials but will discharge once threshold is reached (-55 to -60 mV). Importantly, S-AHP (or F-AHP) cells will not convert into burst-firing neurons merely with changes in membrane potential. On the other hand, burst-firing cells have the ability to switch to a repetitive-firing pattern following membrane depolarization. All of these data provide a first step in an understanding of the firing rates of basal forebrain neurons, however, our results must be consolidated with existing in vivo studies for a more general understanding of basal forebrain function. Comparing our data to an in vivo preparation of the MS/nDB with synaptic afferents surgically removed may be one approach to correlating in vitro and in vivo studies. Vinogradova et al. (1980) used single unit recording techniques in unanesthetized chronic rabbits and compared the firing rates of cells before and after deafferentation. These authors reported a preservation of burst-firing neurons (25% of the cells) after deafferentation but with a significant reduction in the mean frequency of bursts. In addition a higher percentage of regularly firing cells also occurred following deafferentation (Vinogradova et al., 1980). It is interesting to speculate that these regularly firing cells may correspond to S-AHP cells in our in vitro studies, and some of the burst-firing units may correspond to the burst-firing cells we record in slices. Nevertheless, the in vivo data strongly suggests that endogenous regular spiking as well as rhythmic burst capabilities are present in some MS/nDB cells, however, the firing rates of most MS/nDB neurons are strongly influenced by synaptic afferents (see also Vinogradova et al., 1980; 1987). The endogenous activity in vivo can be explained, in part, by the intrinsic properties elucidated in our in vitro studies. How the synaptic afferents control MS/nDB circuitry and integrative output is premature to speculate without a more thorough understanding of the synaptic mechanisms involved. It is possible that future in vitro studies will help define these mechanisms and again contribute to an understanding of basal forebrain function. PMID- 1723244 TI - Substance P excites cultured cholinergic neurons in the basal forebrain. PMID- 1723245 TI - The pharmacology of basal forebrain involvement in cognition. PMID- 1723246 TI - [Choroidal neovascularization, experimental and clinical study]. AB - As a special lecture at the 95th annual Congress of the Japanese Ophthalmological Society in 1991, we presented experimental studies on choroidal neovascularization (ChNV), and clinical studies on senile disciform macular degeneration (exudative age-related macular degeneration). We produced experimentally ChNV on monkey eyes using intense photocoagulation with krypton laser. We showed the retinal pigment epithelium (RPE) played a heavy role as inducer or inhibitor for ChNV at different stages of development or involution of experimental ChNV. Senile disciform macular degeneration is becoming a leading cause of blindness in the elderly in Japan. We examined 473 eyes in 398 cases of this disease during the past 5 years. Nineteen percent were bilaterally affected, males were affected 3 times prevalent than in female, and average age was 67 in years. Predisposing signs were degeneration or atrophy of RPE, hard or soft drusen, and serous detachment of RPE in the macula. In early stage, serous retinal detachment stage appeared and showed good outcome by laser treatment. Subretinal hematoma form showed next better outcome in acute onset and acute course. Advanced form of disciform lesion showed worse outcome. A form (subretinal cystic form) associated with large serous RPE detachment showed the worst outcome and scarcely indicated for laser treatment. We describe clinical features of each form and stages of the disease and clinical course. Early detection, early correct diagnosis and early laser treatment must be essential for prevention of blindness due to this disease. PMID- 1723247 TI - Characterization of the 5q- breakpoint in an acute nonlymphocytic leukemia patient using pulsed-field gel electrophoresis. AB - Multiple genes of hematopoietic importance have been localized to the long arm of chromosome 5 including granulocytemacrophage colony stimulating factor (GM-CSF) and interleukins (IL) 3, 4 and 5 to 5q23-31, colony stimulating factor 1 (CSF1) to 5q33.1 and its receptor (c-fms) to 5q33.3. The genes coding for platelet derived growth factor receptor (PDGFR) and acidic fibroblast growth factor (FGFA) have been localized to 5q31-32 and 5q31.3-33.2, respectively. These genes fall in the region of chromosome 5 which is deleted in the 5q- refractory anemia syndrome (5q-RA) and acute nonlymphocytic leukemia (ANLL). We have characterized this region in a 5q- patient with therapy-related ANLL (t-ANLL) by pulsed-field gel electrophoresis and Southern blotting analysis utilizing DNA probes for PDGFR, c fms, and FGFA. A single 300 kbp M1uI restriction fragment was detected in the patient using a PDGFR probe as compared to a 200 kbp fragment in normal controls. BssHII digestions also showed restriction fragment length difference. Similar data for both M1uI and BssHII digestions were also obtained when c-fms was used as a probe. Southern blotting analysis of EcoRI-digested DNA showed that each of the PDGFR, c-fms, and FGFA alleles were deleted. These results suggested that one chromosome 5 has a large deletion involving PDGFR, c-fms and FGFA, which is consistent with the cytogenetic analysis of the patient. In contrast, the other chromosome 5, which appeared normal cytogenetically, may have a smaller deletion (or alteration) in proximity to but not involving any of these 3 genes. PMID- 1723248 TI - Automated nonisotopic assay for protein-tyrosine kinase and protein-tyrosine phosphatase activities. AB - A sensitive, automated, and nonisotopic assay for protein-tyrosine kinases and phosphatases has been developed. The assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology. The assay is specific for phosphotyrosine residues, can be performed faster, and is at least 100-fold more sensitive than the current standard filter type radioassay. Myelin basic protein and a synthetic peptide corresponding to the autophosphorylation site of p56lck performed equally well in the detection of p56lck kinase activity. Myelin basic protein phosphorylated on tyrosine residues by p56lck was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity. The assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein-tyrosine kinases and phosphatases. PMID- 1723249 TI - Eosin Y staining of proteins in polyacrylamide gels. AB - A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins. PMID- 1723250 TI - An improved method for isolating RNA from porcine adipose tissue. AB - In the present study, we describe a method that we developed to isolate total RNA from porcine adipose tissue. This method entails homogenizing porcine adipose tissue in 10 ml of 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Sarcosyl, 0.1 M beta-mercaptoethanol, pH 7.0, and then performing two CHCl3 extractions to remove lipid before following the procedure described by P. Chomczynski and N. Sacchi (1987, Anal. Biochem. 162, 156-159). This modification improved the yield of RNA approximately threefold (yield was 88 +/- 7 micrograms total RNA/g of tissue) without affecting RNA quality. PMID- 1723251 TI - Purification of RNA using an anti-RNA monoclonal antibody. AB - Studies of the synthesis and modification of RNA employ many types of in vitro reactions. Often, the RNA product must be concentrated or purified away from other reaction components such as salts, unincorporated nucleotides, protein, or DNA. Here I describe an immunological approach suitable for the isolation of RNA from in vitro reactions. A variety of RNAs of differing size and nucleotide sequence were immunoprecipitated with a monoclonal antibody specific for RNA. RNA binding took place in seconds with nearly quantitative recoveries. Immunoprecipitation was more efficient than ethanol precipitation in removing unincorporated nucleotides. Proteins which do not bind to RNA remained soluble. The immunoprecipitated RNA sample was solubilized directly with a buffered solution suitable for gel electrophoresis under denaturing conditions. Thus, RNAs can be rapidly concentrated for electrophoresis in a single step. Antibody-RNA binding was reversible under nondenaturing conditions in the presence of excess rRNA. This procedure serves as a novel means of purifying RNA and RNA-binding proteins from in vitro reactions. PMID- 1723252 TI - Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology. AB - Nonradioactive detection methods for DNA, RNA, and protein analysis have been the subject of research for several years. In this paper the application of the digoxigenin nucleic acid labeling system, in combination with the new alkaline phosphatase substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl -1,2-dioxetane, to the special requirements of the analysis of transgenic plants is described. Earlier detection systems lacked the required ultrasensitive limits of detection necessary because of the large genomes found in plant cells. Routine detection of single-copy genes from transgenic plant species requires the detection of bands of picograms of specific DNA, which is easily achieved by employing the AMPPD substrate. Optimal conditions of genomic Southern analysis have been successfully adapted for Northern blotting techniques. Detection of foreign proteins in transgenic plants has proven difficult because of the very small amounts of detectable specific protein. Until now, utilization of biotinylated antibodies in combination with a streptavidin-alkaline phosphatase conjugate has been the most sensitive procedure. By introducing the AMPPD substrate, a further significant enhancement of sensitivity leading to detectable signals in the picogram range can be obtained. PMID- 1723253 TI - The transverse tubular system of the hypertrophic myocardium: morphology and morphometry in spontaneous hypertensive rats (SHR). AB - We reported previously on a modified Golgi stain that, in conjunction with high voltage electron microscope stereoscopy, gives striking views of the elaborate network of the transverse tubular system (T system) in rat myocardium. In this report we used the same techniques to study three-dimensional arrangements of the T system in the left ventricular myocardium of spontaneous hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). High voltage electron microscope stereoscopy revealed distinctive morphological characteristics of the T system, such as undulating running, short dead-end branches, and labyrinth-like tubular aggregates in the hypertrophic myocardium of SHR. Quantitative analysis of the SHR T system indicated a surface area greater than that of WKY. These findings may support the hypothesis that making an additional T system membrane will compensate for the smaller surface-to-volume ratio. However, the normal regulatory mechanism required to maintain the surface-to-volume ratio does not function properly in SHR, resulting in morphological abnormalities and functional disturbances of the myocardium. PMID- 1723254 TI - Association of MspI restriction fragment length polymorphisms with transferrin in horses. PMID- 1723255 TI - A MspI restriction fragment length polymorphism at the ovine locus for glucagon. PMID- 1723256 TI - Topography, extent, and clinical relevance of neurochemical deficits in dementia of Lewy body type, Parkinson's disease, and Alzheimer's disease. AB - Cholinergic and monoaminergic (dopaminergic and serotonergic) activities have been examined in postmortem brain tissue in senile dementia of Lewy body type, Parkinson's disease, and Alzheimer's disease. Quantitative data suggest that although extrapyramidal symptoms relate to striatal levels of dopamine, cognitive impairment is most closely associated with cholinergic (but not monoaminergic) deficits in temporal and archicortical areas. Hallucinations, which are most frequent in Lewy body dementia, appear to be related to an extensive cholinergic deficit in temporal neocortex and the resulting imbalance between decreased cholinergic and relatively preserved serotonergic activities. Topographic analyses such as these including consideration of quantitative "threshold" effects, may be relevant to the future anatomic focus of neurochemical investigations in dementia and to the development of appropriate experimental models. PMID- 1723257 TI - Analysis of monoamines in the cerebrospinal fluid of Chinese patients with Alzheimer's disease. AB - We have established HPLC assay conditions that could measure the levels of 14 monoamines and their metabolites simultaneously. Monoamine levels in cerebrospinal fluids of 12 Chinese patients with Alzheimer's disease were compared with those in samples from patients with benign prostate hyperplasia. Of the 14 monoamines and metabolites, only three were found to be present in all samples. Although the levels of 5-hydroxy-3-indoleacetic acid (HIAA) and homovanillic acid (HVA) in cerebrospinal fluid of patients with Alzheimer's disease were lower, and the levels of 3-methoxy-4-hydroxyphenylglycol (MHPG) higher, as compared to control patients, no significant differences were found between these two groups. PMID- 1723258 TI - Exogenous nerve growth factor reverses age-related structural changes in neocortical neurons in the aging rat. A quantitative Golgi study. AB - The role of chronic exogenous intracerebroventricular administration of nerve growth factor (NGF) on the morphology of layer V pyramidal cell dendrites in aging rats was quantified using Golgi impregnations. Both dendritic branching and dendritic spines from the basilar tree of randomly selected pyramidal neurons of the frontal cortex were evaluated in young control (4-month-old) Fischer 344 rats, in old controls (24-month-old), and in 24-month-old rats administered NGF for 4 weeks. Sholl analysis of basilar dendritic trees showed that neuronal branching in older rats was significantly greater than that in young rats (probably due to compensatory dendritic hypertrophy). The extent of dendritic material in aged rats receiving NGF, however, was identical to that in young rats, that is, the dendritic tree had regressed in size. Dendritic spine response to NGF treatment depended on the region of the dendritic tree sampled. Normal aging resulted in spine loss. However, NGF treatment restored dendritic spine densities to those seen in young controls on terminal tip segments ("plastic" regions). Internal branch segments ("nonplastic" regions) showed no response to NGF. As dendritic spines are thought to represent the neuroanatomic basis of learning and memory, results suggest that NGF can influence the morphology of cortical neurons (probably indirectly via the basal forebrain projections) and therefore may play an efficacious role in the treatment of geriatric cognitive dysfunction and even perhaps in Alzheimer's disease. PMID- 1723259 TI - Targeted killing of squamous carcinoma cells by a monoclonal antibody-peplomycin conjugate which recognizes the EGF receptor. AB - We determined in vitro the antitumor activity of a conjugate prepared by binding a monoclonal antibody (B4G7), which recognizes the human epidermal growth factor (EGF) receptor, with peplomycin (PEP), which is an antitumor agent effective against squamous cell carcinoma. This B4G7-PEP conjugate was prepared by coupling of B4G7 and carboxymethylpeplomycin active ester. The conjugate killed A431 cells of squamous cell carcinoma which overexpress EGF receptors at lower concentrations than PEP alone. On the basis of its IC50, the conjugate was six times more potent than PEP alone. A simple mixture of B4G7 and PEP was as effective as PEP alone in cytotoxicity. The addition of ten times the amounts of B4G7 to this conjugate decreased its cytotoxicity. When other squamous cell carcinoma cell lines with different levels of EGF receptors (NA, Ca9-22, TE-1, TE 8) were treated with the conjugate, cells were killed dose-dependently and the cytotoxicity was dependent on the number of EGF receptors. When each squamous cell carcinoma cell line was treated with a control conjugate prepared by combining PEP with mouse IgG instead of B4G7, no cytotoxicity was observed. These results indicate that B4G7-PEP will be a useful weapon in multidisciplinary treatment which utilizes the EGF receptor, as these receptors are detected in a higher incidence in squamous cell carcinoma. PMID- 1723260 TI - Heterogeneity of keratin expression and actin distribution in benign and malignant mammary diseases. AB - Immunoreactivity of monoclonal anti-cytokeratin KL1, PKK1, K8.12 and anti-actin antibodies in 101 cases of diseased human breast lesions showed irregular keratin distribution in luminal cells of terminal ductal-lobular unit and basal layer cells of the interlobular and main duct. Actin staining was confined to myoepithelial cells. Benign lesions showed great heterogeneity in luminal cells of the terminal ductal-lobular units. Breast carcinoma showed a reduced staining for keratins, heterogeneity of keratin expression was found in solid tubular carcinoma, and actin was usually absent: however, papillo-ductal or comedo type had actin positive myoepithelial cells around carcinoma foci. PMID- 1723261 TI - The relation of argyrophilic proteins of nucleolar organizer regions (AgNORs) to the proportions of Ki-67 or DNA polymerase alpha-reacting cells in non-Hodgkin's lymphomas. AB - In order to examine the relationship between argyrophilic proteins of nucleolar organizer regions (AgNORs) and the proliferation activity of cells, we investigated lymph nodes obtained from 25 untreated non-Hodgkin's lymphoma (NHL) patients. Two monoclonal antibodies (MoAb) (Ki-67 antibody and anti-DNA polymerase alpha antibody) were used for evaluating cell proliferation activity. A linear relation between the mean number of agNORs per nucleus and the proportion of NHL cells reacting with Ki-67 MoAb was observed (r = 0.48, P less than 0.05). A similar relation between AgNORs and DNA polymerase alpha MoAb was also observed (r = 0.51, P less than 0.01). From these data, it was confirmed that AgNORs reflect the proliferation activity of NHL cells. We conclude that the AgNOR staining procedure is one of the simplest and most reliable methods for analyzing cell proliferation potential. PMID- 1723262 TI - Influence of suramin alone or in combination with DHT and PDGF on the cell proliferation of benign and malignant human prostatic tissues in organ cultures. AB - We studied the suramin-induced influence on the cell proliferation of 16 benign and 6 malignant lesions of the human prostate maintained in vitro as organ cultures. The cell proliferation was assessed by nuclear labeling with tritiated thymidine autoradiography. We also studied the dihydrotestosterone (DHT)-and platelet-derived growth factor (PDGF)-induced modulation of suramin influence on such prostate organ culture cell proliferation. Our results indicate that more than half of the benign prostatic tissues showed cell proliferation which was modulated by DHT and/or PDGF, while none of the six carcinomas responded to such hormonal stimulation. Suramin alone inhibited the cell proliferation of only 19% of the prostate organ culture under study, while in combination with DHT and/or PDGF this inhibition level reached 48%. However, we occasionally observed that S alone or in combination with DHT and/or PDGF was also able to stimulate prostate cell proliferation. We think that organ cultures of human prostatic tissues might represent a helpful pre-clinical tool to study the anti-tumoral influence of suramin, which is a new antineoplastic generative compound. PMID- 1723263 TI - Ki-67 as a marker for cell cycle regulation by interferon. AB - The effects of interferon (IFN) on the expression of the nuclear antigen Ki-67 were studied in the two IFN-sensitive tumour cell lines Daudi and 251 MG, known to be arrested in the cell cycle in separate stages. The GO/G1-arrested Burkitt's lymphoma cell line Daudi displayed an increasing fraction of Ki-67 negative cells with time, concomitant with an increasing proportion of growth arrested cells. A small fraction of Ki-67 positive cells were found mainly arrested in G2/M. In contrast, no effect on Ki-67 expression was seen in IFN-resistant Namalwa cells, nor in the sensitive glioma cell line 251 MG, which is blocked in the S phase of the cell cycle. Agents blocking the cells in other phases of the cycle did not affect Ki-67 expression. However, after serum deprivation, no Ki-67 expression was found in the glioma cell line, while restimulation initiated expression after 12 hours as cells entered the S phase. We conclude that the Ki-67 antigen was not down regulated in all cells inhibited by IFN and thus does not seem to be useful to monitor clinical effects of IFN treatment. PMID- 1723264 TI - Immunomodulatory activity of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent anti-HIV nucleotide analogue, on in vivo murine models. AB - In order to evaluate the influence of antiviral nucleoside analogues upon the natural immune system, we investigated the immunomodulatory activity of 9-(2 phosphonylmethoxyethyl)adenine (PMEA), a nucleotide analogue with potent anti-HIV and anti-herpes activity, in a murine system. C57BL/6 mice were inoculated intraperitoneally with 10, 25 and 50 mg PMEA/kg. Mononuclear cells were isolated from their spleens, and some natural immune functions were evaluated. The results show that PMEA significantly increases the levels of natural killer (NK)-cell cytotoxicity. We also found that alpha/beta IFN production was substantially increased in PMEA-treated mice, while both IL-1 and IL-2 production was decreased. Thus, PMEA can increase some natural immunity functions, such as NK activity and IFN production. These results suggest that PMEA might be active in vivo against HIV and herpes viruses both as an immunomodulator and as an antiviral compound. PMID- 1723265 TI - On the pharmacological phenocopying of memory mutations in Drosophila: alkylxanthines accelerate memory decay. AB - Theophylline and 3-isobutyl-1-methylxanthine, two cyclic nucleotide phosphodiesterase inhibitors, when fed to wild-type Drosophila adults, cause the rapid decay of learning index after training in a shock-odor learning paradigm. The drugs practically do not affect the olfactory acuity of flies, hence they influence the learning/memory process itself. The time courses of memory decay resemble those of the memory mutants rutabaga and amnesiac and, to a lesser extent, dunce2 and dunceM11. Theophylline further deteriorates the learning performance of dunceM11. Biochemical characterization of the inhibition of the two major phosphodiesterase isoenzymes in Drosophila by theophylline predicts only a slight inhibition of these enzymes in vivo, in accordance with the unchanged level of cAMP in wild-type fly heads during drug feeding. 8 Phenyltheophylline, an adenosine receptor antagonist in mammals, slightly retards memory decay in the wild-type. It is suggested that alkylxanthines induce memory decay in Drosophila by interfering with cAMP dynamics at more than one point of its metabolism. PMID- 1723266 TI - A simple method for determination of intramolecular amino acid sequence of unpurified protein. Application to human serum protein adsorbed by silica particles. AB - Silica particles adsorbed several kinds of human serum proteins, especially 23 kDa molecular weight protein. After SDS-PAGE of adsorbed serum proteins, gel pieces containing 23 kDa protein was cut out and set in slot of stacking gel in second SDS-PAGE following overlay of Staphylococcus aureus V8 protease. After electrophoresis, gel was subjected to electroblotting onto polyvinylidene difluoride membrane. Both bands of dye-stained 23 kDa and the peptide were cut out from membrane and analyzed for amino acid sequence. Obtained sequences agreed well with amino terminal and intramolecular sequences of human HDL apolipoprotein, A-I. PMID- 1723267 TI - [Localization of an antigenic determinant of recombinant interleukin-2, recognized by monoclonal antibody 13B1]. AB - We suggest a simple approach to localization of antigenic determinants for the monoclonal antibody 13B1 raised against recombinant human interleukin-2. The approach is based on the limited trypsin proteolysis, peptide separation by the O'Farrell method and identification of the peptides, interacting with monoclonal antibodies, and comparison of the charge and length of these peptides with the corresponding values of theoretically possible peptides. PMID- 1723268 TI - [Use of synthetic carriers and adjuvants for increasing the immunogenicity of a synthetic peptide from the CS-protein of Plasmodium falciparum]. AB - In order to increase immunogenicity of the peptide (NANP)3, we have prepared a large set of fully synthetic constructions based on the peptide, glycopeptide adjuvant GMDP and some synthetic carriers. Immunogenicity of these constructions was tested on mice (line C57B1/6) responding to the peptide polymer (NANP)40 without carrier and on mice (line BALB/c) not responding to this antigen. Immunogenic constructions based on synthetic polytuftsin induced as high titres of anti-(NANP)3 antibodies as the standard conjugate KLH--(NANP)3. The chimeric peptide consisting of (NANP)3 and tuftsin dimer induced anti-(NANP)3 antibodies in both lines of mice as well. The GMDP covalent attachment to the immunogenic constructions increased the antipeptide antibodies titre. The results are discussed in terms of an approach to synthetic vaccines. PMID- 1723269 TI - [Artificial oligomerization of enzymes--a new way of regulating their catalytic activity in reversed micellar systems]. AB - Comparative studies were carried out in the catalytic activity regulation of native alpha-chymotrypsin and its artificially produced hexameric form as an example of non-dissociating oligomeric enzyme (covalently cross-linked by means of succinimidyl-3-(2-pyridylthiopropionate] in the Aerosol OT reversed micelles in octane. Native (monomeric) alpha-chymotrypsin exhibits maximal catalytic activity in the reversed micelles at the hydration degree w0 = 10, when the radius of the micelle inner cavity is equal to the radius of the alpha chymotrypsin globule. For the alpha-chymotrypsin hexamer, optimum is observed at w0 = 45, with the inner micellar cavity radius (r = 68 A) being approximately equal to the radius of the sphere surrounding the octahedral combination of the six monomeric alpha-chymotrypsin molecules (r = 61 A). Thus, construction of the corresponding oligomeric structures is made easy, with the optimal catalytic activity in a preset range of the hydration degrees. PMID- 1723270 TI - [Synthesis of RNA using T7 RNA polymerase and immobilized DNA in a stream type reactor]. AB - The DNA ligase-induced assembly of synthetic oligodeoxyribonucleotides on polymer supports was used to obtain immobilized DNA, containing the T7 RNA polymerase promoter and coding for a 14-membered oligoribonucleotide. The obtained template can carry out RNA synthesis in a flowing-type reactor. Sepharose 4B and Toyopearl HW-55 were used as supports. PMID- 1723271 TI - [Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2' fluoronucleoside and arabinofuranosylcytosine]. AB - A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes. PMID- 1723272 TI - Immunoblot analysis to demonstrate antigenic variability of clinical isolated. Pseudomonas pseudomallei. AB - Pseudomonas pseudomallei (Ps.ps.) is the causative organism of melioidosis, and is widely distributed in Southeast Asia and Northern Australia. Clinical manifestations range from subclinical infection to fulminant septicemia. To demonstrate the antigenic variability of Ps.ps., 62 clinical isolates from 31 blood, 13 sputum, 9 pus, 3 urine and 6 body fluid culture specimens were studied by SDS-PAGE and immunoblotting. In SDS-PAGE, there were approximately 20 antigenic components with molecular weights ranging from 14 to 66 kilodaltons (KD) which suggested that there was antigenic variability among these 62 clinical isolates of Ps.ps. Attempts to correlate immunoblot profiles with clinical illness or sources of specimens were not successful but 6 common antigens were identified with molecular weight of 17.5, 21, 33, 34, 40 and 45 KD, respectively. Among these antigens, the 45 KD component was recognised by all patients' sera. Thus, the 45 KD protein antigen may be useful for the future approach in immunodiagnosis of melioidosis. PMID- 1723273 TI - Identification of the main epitope on human cytochrome P450 IID6 recognized by anti-liver kidney microsome antibody. AB - Antibodies present in the sera of a group of children with autoimmune hepatitis react with human cytochrome P450 IID6. cDNA constructions of various fragments of human P450 IID6 were made and expressed and the resulting peptides were tested in immunoblot with patients' sera. These allowed identification of at least two antigenic sites on the P450 molecule. The main one, recognized by all sera tested, is located between amino acids 239 and 271. Synthesis of three peptides covering this area of the molecule allowed identification of a sequence of three amino acids (tyrosine-tryptophane-asparagine) located at position 261-263 that constitutes the essential part of the epitope. A protein sequence data-base search revealed homologies between this region of human P450 and proteins from Salmonella typhimurium, from human T lymphotropic virus types 1 and 2 and Herpes simplex virus type 1. PMID- 1723274 TI - Repetitive P68-autoantigen specific epitopes recognized by human anti-(U1) small nuclear ribonucleoprotein autoantibodies. AB - The major target of anti-(U1)snRNP autoantibodies, a serological marker of patients with mixed connective tissue disease and related rheumatic disorders, is a 68 kDa protein (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein particles. With recombinant p68 fusion proteins, multiple autoepitopes have been identified, and one of these has been mapped to the pentamer sequence ERKRR, which is located within antigenic domain A in the amino terminal half of p68. The lysine residue (K) of this epitope can be replaced by isoleucine without loss of autoantibody binding. Here we have investigated whether other variants of this epitope are present on the p68 autoantigen and if these are recognized by anti-p68 autoantibodies. We identified four related motifs in the carboxy-terminal half of the p68-protein, and three of these (all containing glutamic acid instead of lysine (ERERR] mapped to the previously characterized autoantigenic domains C and D. Immunoreaction of anti-ERKRR autoantibodies, affinity-purified from domain A with recombinant fusion proteins containing either domain C or domain D of p68, revealed that anti-ERKRR autoantibodies cross-react with the ERERR-motifs. This finding, which was confirmed by competitive inhibition-ELISA with solid-phase coupled domain A-, C- and D-fusion proteins and ERKRR-containing synthetic peptides as competitors, suggests that a subset of patient autoantibodies is directed against repetitive structures on a single snRNP component. PMID- 1723275 TI - ACTH and angiotensin II regulation of insulin-like growth factor-I and its binding proteins in cultured bovine adrenal cells. AB - Insulin-like growth factor-I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of ACTH and angiotensin II (AII), as well as the secretion of IGF-I and its binding proteins (IGFBPs). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days of treatment with AII (1 microM) or ACTH (10 nM) the number of stained cells increased by 5- and 14-fold respectively. In all cases the staining was specific, since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I into the medium, evaluated by a specific radioimmunoassay, was increased two- and sevenfold by AII and ACTH respectively. Using the method of Western ligand blotting, the major form of IGFBP secreted by control adrenal cells was found to be a 38-42 kDa doublet protein. Two minor forms with apparent molecular weights of 28-31 kDa and 24 kDa have also been identified. Following acid-ethanol extraction of the conditioned medium, all the IGFBPs were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent, AII pretreatment increased the 38-42 kDa IGFBP by several fold, decreased the 28-31 kDa IGFBP and had no effect on the 24 kDa IGFBP. In conclusion, these results demonstrate (i) that bovine adrenal cells contain IGF-I-like immunoreactive material, (ii) that the stimulatory effects of ACTH and AII on IGF I secretion by bovine adrenal cells are due mainly to an increase in the number of IGF-I-producing cells and (iii) that ACTH and AII modulate the secretion of IGFBP by adrenal cells. Although the roles of IGFBPs have not been defined in adrenal cells, they are capable of modulating the biological action of IGFs in other cell cultures. Regulation of both IGF-I and its binding proteins by the two specific hormones ACTH and AII suggests important roles for these binding proteins in modulating the action of IGF-I in bovine adrenal cell function. PMID- 1723276 TI - Blocking antibodies to inhalant allergens and asthma. AB - The mechanisms of specific immunotherapy are not still established. Among the lot of immunological changes, induced by immunotherapy, the increase of specific IgG 1, and then of IgG 4 antibodies, during the first months is well demonstrated. The skin-tests, the histamine-release and the human basophil degranulation are significantly decreased after incubation of allergen with the serum of desensitized patients. This antigen neutralizing capacity (blocking antibodies) disappeared when IgG 4 were suppressed from the serum by the mean of activated columns linked either with allergen, or protein A or antihuman IgG4 antibodies. Conversely, the total amount of serum blocking activity was found using pure IgG 4. However, the role of IgG 4 antibodies remains a subject of controversy. Since a clear correlation has yet to be established with symptom scores. PMID- 1723277 TI - [Hodgkin's disease during pregnancy. Study of late effects in the newborn infants]. AB - An analysis of the possible late effects in 15 children of mothers with Hodgkin Disease, who were given combined chemotherapy during pregnancy, including five who were in the first trimester of development, was conducted. None of the newborns were found to have congenital abnormalities during birth. The 15 children, ranging in ages between 4 to 17, are alive, showing both normal physical and psychomotor development, as well as normal laboratory and cytogenetic results. The thirteen mothers who were in complete remission as a result of the chemotherapy used at recommended dosages and at accepted intervals, are alive and without any evidence of the illness, considering them as cured. Based on these results, pregnancy should not be considered as a contraindication for the adequate treatment of Hodgkin's disease, since those fetuses who received cytotoxic agents in utero, did not show a greater incidence of congenital malformations and have not shown any evidence of side-effects due to the chemotherapy. PMID- 1723279 TI - Scanning electron microscopic study of capillary change in bleomycin-induced pulmonary fibrosis. AB - The architectural changes which occur in the capillaries are difficult to illustrate without a three-dimensional tool, such as scanning electron microscopy. Therefore, a scanning electron microscopic study was occasionally undertaken to show the capillary changes of lung fibrosis. Fibrosis was induced in twenty rats by an intratracheal injection of bleomycin. After 30 days the rats were sacrificed, and light microscopy and scanning electron microscopy were performed. The vascular trees of both lungs were cast with methacrylate. Light microscopically, the pulmonary fibrosis was patchy and inflammatory cell infiltration was rather sparse. Scanning electron microscopically, the intercapillary spaces became wider; and some capillaries revealed large irregular dilatation. The pleural and alveolar capillaries were variably dilated. The pleural capillary diameter was increased (P = 0.06), and the capillary plexus diameter was decreased (P = 0.00). Distance between the capillary branches of the pleural surface was increased (P = 0.06). The appearance of irregularly shaped capillaries, an increase in diameter with variable dilatation of alveolar capillary rings and a decrease in branching between the capillaries, resulting in a loss of surface area are the main scanning electron microscopic findings of the remodeling which occurs pulmonary capillaries in bleomycin-induced pulmonary fibrosis. PMID- 1723278 TI - Requirements for in vitro growth of human thymocytes. AB - Since it is difficult to study human thymocyte maturation in vitro, we have developed an in vitro thymocyte culture system which has allowed us to select the optimal growth conditions for thymocyte subpopulations. Three thymocyte subpopulations (CD3-CD1-, CD1+CD3-, and CD3+CD1-) were isolated by a single step percoll density gradient centrifugation and indirect panning procedure using anti CD1 and anti-CD3 monoclonal antibodies, and their purity was checked by flow cytometry. The combination of concanavalin A (Con A), tetradecanoylphorbol acetate (TPA), and IL-2 was shown to be the most reliable stimulus for the proliferation of CD3-CD1- thymocytes for up to 15 days in a culture system in vitro. Flow cytometric analysis for the phenotypic change of CD3-CD1- thymocytes revealed a steady increase of CD3 antigen after a 3-day cultivation, whereas there was no change in CD1 antigen intensity. A combination of Con A and IL-2 was both sufficient and necessary to induce growth of CD3+CD1- thymocytes. The major population of immature cortical thymocytes (CD3-CD1+ or CD3+CD1+), which are considered to be the most unresponsive dead-end cells, could not be maintained or stimulated with any combination used in this experiment, even in the presence of thymic accessory cells. PMID- 1723280 TI - Low number of functionally active B lymphocytes in the peripheral blood of HIV-1 seropositive individuals with low p24-specific serum antibody titers. AB - The in vitro synthesis of HIV-1, p24-, reverse transcriptase (RT)- and gp120 specific immunoglobulin (Ig) G by unstimulated peripheral blood mononuclear cells (PBMC) from 38 asymptomatic and 10 symptomatic HIV-1-seropositive individuals was analysed. In the majority of these individuals, spontaneous production of HIV-1- and gp120-specific IgG from PBMC cultures was demonstrated. In addition, in the majority of the PBMC cultures from individuals with high serum antibody titers to p24, spontaneous production of p24-specific IgG was shown. In contrast, no p24 specific IgG production was detected in PBMC cultures from seropositive individuals with low or no serum antibody titers to p24. A similar relationship between low or absent RT-specific serum antibody titers and the absence of in vitro RT-specific IgG synthesis was not demonstrated. Furthermore, it was shown that the number of p24-specific B lymphocytes in circulation, as calculated by a spot enzyme-linked immunosorbent assay, were significantly lower in individuals with low serum antibody titers to p24. These results suggest that the decline in p24-specific serum antibodies observed during progression towards AIDS is not merely a reflection of the clearance via immune complexes, but may also be attributable, at least in part, to a reduction of p24-specific antibody-producing active B lymphocytes. PMID- 1723281 TI - The effect of methamphetamine on serotonin and its metabolite in the suprachiasmatic nucleus: a microdialysis study. AB - The suprachiasmatic nucleus (SCN) has been identified as a major circadian pacemaker. Methamphetamine has been shown to modify the behavior of circadian rhythms. We detected extracellular serotonin (5-HT) and its metabolite 5 hydroxyindoleacetic acid (5-HIAA) in the SCN in freely moving rats, using a microdialysis method, to investigate biochemical effects of methamphetamine in the SCN. Methamphetamine infusion into the SCN dose-dependently increased extracellular 5-HT and decreased extracellular 5-HIAA. PMID- 1723282 TI - A comparative immunocytochemical and immunochemical analysis of glycoproteins synthesized in the bovine subcommissural organ. AB - To extend our previous immunochemical investigations in the chick embryo (Karoumi et al., 1990 b), we raised antibodies in the rabbit against crude extracts of the subcommissural organ (SCO) of the bovine. The antiserum labeled A99 was absorbed by crude brain extracts and its specificity was tested by different techniques. Comparison of crude SCO and cerebral hemispheres supernatants after immunoblotting allow to identify specific 98, 60, 52, 42, 38, and 32 kDa polypeptides in the SCO profile. Immunoaffinity chromatography on A99 immunoadsorbent of crude SCO, cerebral hemispheres (CH) and classical ependyma (CE) supernatants was followed by electrophoretical analysis and electrotransfer. Concanavalin A (Con A) and wheat germ agglutinin (WGA) labeling procedures demonstrated the presence of numerous glycopeptides specific of crude SCO supernatants and having an apparent molecular weight ranging from 240 to 50 kDa. In the CH-eluted fraction, 50 and 52 kDa glycopeptides were revealed by ConA and WGA, whereas in the CE-immunopurified fraction no band was visualized. The similarity of the chick embryo and bovine electrophoretic pattern corresponding to the SCO eluted fractions speaks in favour of a high degree of conservation of the SCO secretory material and an evolutionary stability of the antigens recognized by A99IgG. PMID- 1723283 TI - The outcome of extremely low birthweight infants. AB - During the years 1978-89, all surviving extremely low birthweight infants (BW less than 1000 g, ELBWI) in the region of Southern Finland were admitted to the Children's Hospital, University of Helsinki and followed up to six years. The number of liveborn ELBWI increased from 30 to 50/year during the first and last third of the follow-up. During the same twelve year period, the number of the surviving infants increased from 8 to 25/year, with the number and proportion of infants with birthweights of less than 800 g and with gestational ages of less than 27 weeks increasing from 3 to 15/year. Despite of the greater proportion of smaller infants the proportion of infants without intraventricular hemorrhage increased from 50 to 85%. The proportion of children with normal neurodevelopment at two years increased from 40-70% during the first five years of the study, to 63-84% during the last three years of the study. The proportion of children with major disabilities decreased from 28 to 8%. The factors associated with poor neurodevelopment were sepsis, year of birth, intraventricular hemorrhage, and birthweight. The neurological status at one year was a valid predictor of the outcome: at four years 94% of the infants were assessed and normal remained normal as neurologically abnormal remained abnormal or slightly abnormal. The neurologically normal ELBWI were tested at six years: visuomotor coordination was immature in 50%, emotional immaturity was found in 25% and delay of language development in 13%. In our unit increased survival of ELBWI infants has not been associated with an increase in the number of ELBWI infants with handicaps.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723284 TI - Antisense RNA inhibition of hematopoietic growth factor production. AB - Vectors that generate antisense RNA targeted to granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA sequences were constructed using a strong viral promoter and a T cell-specific control element from the human CD2 gene. Stably transfected lymphoid clones expressing antisense RNA were tested for their ability to synthesize GM-CSF in response to stimulation with phorbol 12-myristate 13-acetate (TPA) and ionomycin. At early time points (4 and 8 hr) following stimulation, mean GM-CSF production by clones expressing antisense RNA was 10% the mean of control clones (p less than 0.001). Analysis of mean log data for 15 antisense clones demonstrated that GM-CSF production remained depressed at 12 and 24 hr time points, averaging 37% of that of the control clones (p less than 0.01). We conclude that antisense inhibition of growth factor production may be an effective strategy to investigate the role of specific growth factors in hematopoiesis in vivo in transgenic mice. PMID- 1723285 TI - Constitutive production of granulocyte colony-stimulating factor by hybrids of a SV40-transformed mouse macrophage and a renal adenocarcinoma cell line. AB - Mouse macrophage BAM3 cells produced colony-stimulating factors (CSFs) after stimulation with bacterial lipopolysaccharide (LPS). By assaying the CSF using various interleukin 3-dependent cell lines, it was shown that most of the CSFs produced by BAM3 cells were granulocyte CSF (G-CSF). The granulocyte-macrophage CSF (GM-CSF) gene was also expressed in BAM3 cells after stimulation with LPS. When BAM3 cells were fused with the mouse renal adenocarcinoma cell line RAG which does not produce G-CSF, two of four hybrid cell lines constitutively produced large quantities of G-CSF. About 300 bp of the promoter region of mouse G-CSF chromosomal gene was inserted upstream of the Escherichia coli chloramphenicol acetyltransferase gene, and introduced into BAM3, RAG and hybrid cells. The G-CSF promoter was activated by stimulation with LPS, in BAM3 cells, but was inert in RAG cells. On the other hand, there was significant constitutive CAT activity in the hybrid cells. PMID- 1723286 TI - High affinity receptors to acidic and basic fibroblast growth factor (FGF) are detected mainly in adult brain membrane preparations but not in liver, kidney, intestine, lung or stomach. AB - We have previously shown that only adult brain contained a detectable amount of high affinity receptors for basic Fibroblast growth factor (bFGF) whereas adult liver, kidney, lung, intestine or stomach showed only low affinity binding sites. We now have studied and compared the distribution of the receptors for acidic Fibroblast growth factor (aFGF) with that of bFGF receptors in the same tissues. Membrane binding of 125I-aFGF was time dependent, reversible and displaced by an excess of unlabeled aFGF. Scatchard analyses of binding data obtained with all tissue membrane preparations revealed the presence of at least one class of low affinity/high capacity interaction sites characterized by apparent Kd values ranging from 3.9 to 6.9 x 10(-8) M. Interestingly and as for bFGF, high affinity receptors for aFGF could be detected only in adult brain membranes. Cross-linking and Scatchard analyses indicate that this family of interaction was characterized by four molecular species of 175, 125, 95 and 70 kDa and by an apparent Kd value of 1.8 x 10(-10) M. Moreover, cross-competition binding assay revealed that these brain high affinity receptors were common for both acidic and basic FGF. These results suggest that these growth factors may share identical functions mediated by the same receptors highly expressed in the brain. Using a cDNA probe for the Bek form of FGF receptors, we were able to show that all the tissues studied expressed this mRNA (4.5 kb transcript) but probably not in sufficient amounts to account for the number of high affinity receptors that we detected only in the brain. PMID- 1723287 TI - Image analysis cytology for DNA determination in breast and prostate cancer. AB - Nuclear DNA distribution in fine-needle specimens from 112 breast carcinomas and 45 prostatic tumours was studied. The distributions were described statistically with five separate descriptors, namely mean deviation from 2C, percentage of cells exceeding 2.25C and 4.5C respectively, DNA index and entropy (a mathematical measure of degree of scatter of the DNA content of the nuclei). It was shown that information about DNA index only ('diploid' versus 'aneuploid') is too limited and the term 'diploid' even incorrect and misleading for description of human cancer cell populations. Merely an addition of the percentage of cells exceeding 2.25C allowed separation of a majority of the carcinoma specimens from specimens taken from normal tissue. Moreover, in carcinoma specimens 96% of the patients had between 1 and 100% of the cancer nuclei cell population exceeding 4.5C in contrast to all normal specimens, where this percentage was 0. Entropy was clearly correlated with percentage of Ki-67-positive cells, indicating that it contains information on both scatter of nuclear DNA content and proliferation. Plotting of the scatter (entropy) versus DNA index allowed separation of greater than 97% of the carcinoma specimens from specimens taken from normal individuals. With the use of all five descriptors of DNA distribution it was possible to separate all breast cancer specimens and all moderately and poorly differentiated prostate cancer specimens from normal specimens. The discrimination between well differentiated prostate cancer and hyperplasia constituted a special problem in that it was not possible to confirm the diagnosis well-differentiated prostate cancer by objective measurements in 8% of the patients, even when all five descriptors were used for the analysis. None of the verified carcinoma specimens had a DNA distribution that was truly 'diploid' in the sense of the distributions of cancer nuclei and normal cell nuclei from the actual organ being quite similar. PMID- 1723288 TI - The role of maintenance therapy in the treatment of large-cell non-Hodgkin's lymphoma. AB - Eighty-one patients with large-cell non-Hodgkin's lymphoma achieving complete restaging verified remission after induction chemotherapy (CHOP-Bleo or m-BACOD) were randomized to the following 3 arms: 1. No further treatment (observation). 2. Early consolidation therapy with 6 courses of CVP (cyclophosphamide, vincristine, prednisone) given monthly. 3. Maintenance therapy with cyclophosphamide and prednisone given every 6 weeks for 2 years. The relapse-free survival was better in the maintenance and consolidation arms than in the observation arm. The additional therapy given after the initial complete remission produced lasting disease control in a considerable number of patients and with acceptable toxicity. The authors feel that patients with large-cell lymphoma do not need more aggressive and toxic initial management because the use of maintenance therapy can increase the number of patients remaining in complete remission by more conventional, less toxic chemotherapy. PMID- 1723290 TI - Epidemiological stratification of malaria in the region of the Americas. PMID- 1723289 TI - [ProMACE-MOPP vs. ProMACE-CytaBOM polychemotherapies in the treatment of large cell and unclassifiable non-Hodgkin's lymphomas]. AB - The results of two distinct and successive clinical pilot studies investigating feasibility of aggressive chemotherapy in large cell and unclassified non Hodgkin's lymphomas are reported. In the first study (1986-87) the ProMACE-MOPP chemotherapy (P-M), a 2nd generation regimen, was administered to 10 patients, whereas in the second study (1987-88) the ProMACE-CytaBOM schedule (P-C), a 3rd generation regimen, was administered to 13 patients. The clinical and prognostic features of the two groups of patients were quite comparable, the only difference being the different follow-up times (median: 43 vs 26 months). The number of complete remissions, freedom from relapse and overall survival were slightly better in the P-M group, with-out a statistically significant difference, despite the longer follow-up time. Hematological toxicity was higher in the P-M study, while gastrointestinal (mucositis, hepatic transitory damage) and neurological toxicity (peripheral neuritis) were somewhat lower. In conclusion, the P-M regimen is worth considering for the treatment of high grade malignant lymphomas, although it was not recently designed and is not widely used at present. PMID- 1723291 TI - Use of low molecular mass RNA profiles to identify lactic acid bacteria and related organisms associated with foods. AB - Fourteen strains of lactic acid bacteria and species of Brochothrix, Carnobacterium, Enterococcus, Erysipelothrix, Kurthia and Listeria were examined using low molecular mass RNA (5S rRNA and tRNA) profiles. These profiles were developed on denaturing polyacrylamide gels. Gel strengths between 9 and 14% were tested to improve resolution of distinct bands for densitometrical analysis. Profiles generated on 12% gels proved to be the best for scanning. Scans of class 2 tRNAs by densitometry showed a characteristic profile for each genus. In the case of Lactobacillus each species studied gave a unique profile. The technique of low molecular mass RNA profiling may provide a useful means for identifying different bacteria from ecosystems such as meats. PMID- 1723292 TI - Outgrowth of stable class I major histocompatibility complex-expressing subsets from immunogenic variants of a murine mammary carcinoma: association with a differentially staining region on chromosome 9. AB - We have examined interactions among intratumor subpopulations during the rejection of immunogenic variants of a murine mammary carcinoma (SPI) and in the outgrowth of tumorigenic "revertant" subsets. Analysis of subclones isolated during the early phase of rejection of one immunogenic variant revealed extensive cellular heterogeneity of tumor-forming ability and class I major histocompatibility complex (MHC) expression. Two main categories of subclones were identified. One set expressed high levels of class I MHC (MHCH) and grew poorly or not at all in normal syngeneic mice. The second set of clones expressed generally low levels of class I MHC (MHCL) and exhibited progressive growth in vivo, similar to the parent tumor. The steady-state mRNA levels for class I MHC and beta 2-microglobulin were constitutively elevated in MHCH clones compared to MHCL clones or the parent tumor. However, in vivo tumorigenic outgrowths from immunogenic variants always expressed the MHCH phenotype. A cytogenetic analysis was carried out to determine the clonal origin and lineage relationship of in vivo selected tumor outgrowths. Surprisingly, tumor outgrowths from mixtures of karyotypically distinct MHCH and MHCL subclones were derived from one lineage within the MHCH subset, despite the fact that MHCH subclones exhibited slower growth in vivo than MHCL subsets when analyzed individually. These results suggest that in polyclonal populations the various subsets sometimes interact in a way that overrides the influence of immunogenic and MHC phenotypes of individual subclones. PMID- 1723293 TI - Neuroimmune modulation of lymphocyte function--I. Substance P enhances immunoglobulin synthesis in lipopolysaccharide activated murine splenic B cell cultures. AB - Murine B cells have been shown to possess substance P (SP) receptors, but their functional and biological significance remains unresolved. While previous studies have suggested that SP can induce B cells to secrete Ig, the effect could be indirect since mixed cultures were used. In order to assess directly the ability of SP to trigger normal B cells, we have studied the effects of this neuropeptide on purified splenic B cells in vitro. Although an activation, e.g. lipopolysaccharide (LPS), was required, the functionality of the B cell SP receptors was clearly shown by the ability of subnanomolar concentrations of this neuropeptide to augment antibody secretion in a dose-dependent fashion. Specifically, IgM and IgG levels, determined by an isotype-specific sandwich ELISA, were greatly enhanced at 10(-10) M SP by as much as 500 and 572% respectively, while IgA levels were only modestly affected. Even picomolar concentrations of SP could significantly increase IgM levels. This observed enhancement of Ig production was SP specific since B cells co-cultured in the presence of excess SP antagonist were reduced to basal LPS-stimulated Ig levels. Furthermore, this synergistic stimulation by SP and LPS upon normal B cells could not be attributed to SP-induced cell proliferation since stimulatory concentrations of SP were not mitogenic and at high concentrations could inhibit cell proliferation. Rather, it was observed that the increased IgM and IgG secretion was in part attributable to a greater number of B cells secreting antibodies as demonstrated with an ELISPOT assay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723294 TI - Growth factor requirements for the stimulation of germinal center B cells: evidence for an IL-2-dependent pathway of development. AB - Germinal center (GC) B cells readily undergo apoptosis, a tendency which can be suppressed in vitro by immobilized anti-Ig; mAb to CD40 and soluble CD23 (in synergy with IL-1 alpha) also effect rescue of GC cells from programmed cell death. In the present study, the signals which stimulate rescued GC populations to DNA synthesis have been examined and compared to those established for the activation of follicular mantle (FM) B cells. On co-culture with anti-Ig, optimal responses in FM B cells can be achieved with a combination of IL-4 and CD40 antibody; these activities also provided a modest stimulus to GC cells but, for this population, anti-Ig was ineffective at augmenting the response further. Stimulations of GC B cells were enhanced, however, when performed on a support of primary fetal lung fibroblasts; a major influence of stroma was to promote, by direct cell-cell contact, the CD40-dependent survival of GC B cells. FM B cells were relatively independent of such stromal support. In marked contrast to FM cells, GC B cells were found to respond by enhanced DNA synthesis to IL-2 even when quite low concentrations of the factor were present (IC50 = 2 U/ml). Stimulation of GC cells via this pathway was augmented almost 2-fold on the inclusion of anti-Ig whereas neither fibroblasts, IL-4, nor CD40 antibody made any additional contribution to the IL-2-dependent response. The requirements found for stimulating GC cells in vitro are discussed with reference to the signals that this population may encounter in appropriate microenvironments in vivo: the variety of options apparently available could reflect changing priorities at different stages of a developing GC response. PMID- 1723295 TI - Extension of a minimal T cell determinant allows relaxation of the requirement for particular residues within the determinant. AB - The determinant recognized by a class II restricted helper T cell clone raised against a peptide corresponding to the C-terminal 24 residues of the heavy chain of influenza virus hemagglutinin (HA) was examined in detail. The sequence 309VKQNTLKL316 was identified as the minimal determinant for T cell activation but its stimulatory capacity was augmented by extension at either end. Sets of peptide analogs, in which each residue within the minimal determinant was replaced in turn by every one of the other naturally occurring amino acids, revealed either an absolute requirement for the native residue or a very limited degree of replaceability, at seven of the eight positions. Only the N-terminal residue 309V could be replaced with almost any other amino acid without loss of reactivity; in fact, substitution at this position with residues containing bulky side groups enhanced the response. The reactivity of the clone with analogs of the longer peptide 307KYVKQNTLKL316, which induces maximal levels of stimulation, revealed a very different pattern of replaceability for certain residues; in particular, the requirement for a lysine at position 310 was no longer apparent. This study presents a complete analysis of the importance of each individual residue to the integrity of a T cell determinant and provides evidence that the critical requirement for a particular amino acid at a given location may be overridden by N-terminal extension of the minimal determinant. These findings indicate that, within different homologs of the native sequence, particular residues may assume quite different roles. PMID- 1723296 TI - Dual function of recombinant human CD58: inhibition of T cell adhesion and activation via the CD2 pathway. AB - To produce large quantities of recombinant CD58 (rCD58) glycoproteins for biochemical and functional studies, a cDNA clone containing the phosphatidylinositol-linked form of human CD58 was expressed in insect cells using the baculovirus system. Gel filtration showed rCD58 to form soluble oligomeric aggregates which were functionally, antigenically, and biochemically similar to their natural counterpart. Sequence analysis of the amino- and carboxy terminal ends of released rCD58 protein revealed that the 28 amino acid signal peptide was accurately removed. In contrast, the hydrophobic C-terminal peptide was not removed. rCD58 binds to its natural ligand CD2 with a dissociation constant Kd = 5 x 10(-8) M, which is equivalent to the affinity of physiological T cell adhesion mediated by the membrane bound CD2-CD58 receptor-ligand pair. Rosette formation of human T lymphocytes with sheep and human erythrocytes was completely abrogated. In addition, the mixed lymphocyte reaction was significantly inhibited by rCD58. Moreover, cytotoxicity of human NK clones (CD2+CD3-) was inhibited by rCD58 similar to inhibition by CD58 mAbs. In contrast, rCD58 synergized with mitogenic CD2R mAbs in T cell triggering. These data demonstrate that rCD58 might serve as a biological immunomodulator which influences T cell adhesion and activation. PMID- 1723297 TI - Molecular motions of polysaccharides in the solid state: dextran, pullulan and amylose. AB - Dextran, pullulan and amylose have been investigated by differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical and dielectric spectroscopy over a wide range of temperatures and frequencies. No melting or glass transition is seen below the range of thermal degradation (about 300 degrees C) for either amylose or pullulan; only dextran shows a Tg at 223 degrees C (delta cp = 0.40 J/g deg). The viscoelastic spectrum of the 'dry' polysaccharides is characterized by a low temperature relaxation that occurs at 94, -73 and -59 degrees C, at 1 kHz, (activation energy 32, 39 and 52 kJ/mol) in dextran, pullulan and amylose respectively and is assigned to small entity local motions of the polysaccharide backbone. Absorbed water strongly modifies the relaxation spectrum, inducing a new relaxation below room temperature and dissipation regions associated with water loss above room temperature. The former appears at temperatures higher than the relaxation characteristic of the dry polymer and moves to lower temperature with increasing water content. In normal 'room humidity' conditions (about 10% absorbed water) the water-induced relaxation, attributed to the motion of complex polymer-water relaxing units, is the only observable feature in the dynamic mechanical and dielectric spectrum below room temperature. PMID- 1723298 TI - [Preparation and use of monoclonal antibodies against seal alkaline phosphatase]. AB - Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively. PMID- 1723299 TI - [Interaction of rabbit IgG with anti-IgG: localization of epitopes and absence of immunoreactivity of the pFc'-fragment]. AB - To localize essential epitopes of rabbit IgG, a series of proteolytic IgG fragments obtained by papain (Fab, Fc) or pepsin (pFc', F(ab')2) proteolysis have been prepared and their interaction with sheep antibodies against rabbit IgG has been studied. The data obtained suggest that essential immunoreactive epitopes of rabbit IgG are located in the CH2 domain and hinge region. This finding is in line with the results obtained by computing the antigenic sites of immunoglobulins. However, the deviation from the computed antigenic structure was deduced from the complete lack of immunoreactivity of the pFc fragment, it being a dimer of the terminal CH3 domain of the Fc fragment. The hinge region comparable in size with the dimensions of the epitope reveals high affinity binding to anti-IgG, thus testifying to the localization of the expressed epitope or its essential part in the hinge region. Proteolytic cleavage of this region leads to a significant decrease in the binding of the IgG fragment to anti-IgG. In addition to the CH2 domain and hinge region, a relatively low interaction of the antigen-binding antibody fragments with anti-IgG was found. PMID- 1723300 TI - Memory effects of clomipramine treatment: relationship to CSF monoamine metabolites and drug concentrations in plasma. AB - Performance on tasks tapping automatic and voluntary aspects of memory, attention, and motor speed was examined in 14 patients with major depressive disorder, before and after 3 weeks of treatment with clomipramine (150 mg/day), a potent serotonin and noradrenaline uptake blocker with anticholinergic side effects. Performance on tasks requiring frontal functions improved or did not change, whereas verbal learning and retention, where hippocampal functioning is critical, were impaired. The latter tasks were negatively related to cerebrospinal fluid (CSF) 5-HIAA levels and plasma concentration of clomipramine. The results provide further support for the regulatory role of monoaminergic systems in cognition. Furthermore, we found the automatic-voluntary capacity distinction less heuristically useful. Physiological mechanisms regulating different aspects of cognition and memory appeared to be more closely related to the type of task used than to its capacity-demanding properties. PMID- 1723301 TI - IgM natural autoantibodies against bromelain-treated mouse red blood cells recognise carbonic anhydrase. AB - Carbonic anhydrase (CA) from mouse erythrocyte membranes is recognised as an autoantigen in Western blotting experiments with FUB 1, a murine IgM monoclonal antibody that binds both phosphatidylcholine and bromelain-treated mouse red blood cells (BrMRBC). Serum from mice stimulated with lipopolysaccharide (LPS serum) also recognises CA. From SDS-PAGE, and blotting experiments with whole mouse erythrocytes, we found two closely spaced glycoprotein bands in the 30 kD region that reacted with both FUB 1 and LPS-serum. One of the molecular weight markers, bovine carbonic anhydrase which is of a molecular weight of about 30 kD, electrophoresed in the same 30 kD region also reacted with these antibodies. Carbonic anhydrases from a range of mammalian species were found to be crossreactive with FUB 1 and LPS-serum by Western blotting, whereas human glycophorin A and human asialoglycophorin were not recognised by the antibodies. FUB 1 specifically recognises both native and denatured bovine carbonic anhydrase in ELISA assays. The serological identity of the determinants of CA and BrMRBC was confirmed by specific absorption of both FUB 1 and LPS-serum with BrMRBC and normal mouse erythrocytes. We propose that a native autoantigenic epitope on erythrocytes may be revealed by the proteolytic action of bromelain and that this determinant is associated, at least in part, with carbonic anhydrase. PMID- 1723302 TI - Monoclonal antibodies to different neo-epitopes on fibrinogen and fibrin degradation products. AB - The measurement of fibrin or fibrinogen degradation products is widely used in clinical practice for the diagnosis and follow up of coagulolytic disturbances. Recently D-dimer assays have become very popular owing to their direct application to plasma. However, in some clinical situations there is a need to differentiate fibrin from fibrinogen degradation products. These are still routinely measured by conventional assays on serum. We tried to develop various monoclonal antibodies specific for the neo-epitopes unmasked during the degradation of fibrin or fibrinogen. Fifteen mice hybridomas producing the expected antibodies were obtained and ten were extensively characterized. They could be classified in three reactivity classes: D and D-dimer, D-dimer and early fibrinogen or fibrin degradation products. These monoclonal antibodies were used to develop latex slide assays and ELISA techniques. Two types of assays were obtained; those which were specific for fibrin-related products and those evaluating the totality of fibrin or fibrinogen degradation products. Assays discriminating the fibrinogen split products from those derived from fibrin, and performed directly on citrated plasma can be proposed. They provide complementary information in clinical states such as DIC, pulmonary embolism, leukaemias, thrombolysis, etc. PMID- 1723303 TI - Immunochemical evidence for intramolecular interaction of the carboxy terminal A alpha-appendages of plasma fibrinogen. AB - A monoclonal antibody (Mab), 45J, which reacts with intact fibrinogen, has been employed to demonstrate the interaction of the carboxy terminal regions of the A alpha-chain in non-denatured plasma fibrinogen. The 45J Mab recognizes an epitope in the mid section of the carboxy terminal end of the A alpha chain. The epitope is destroyed by plasmin and trypsin digestion. The 45J Mab and a horseradish peroxidase conjugate of the 45J Mab (45J-HRP) were used in an ELISA to demonstrate that the antibody could recognize two copies of the same epitope on purified fibrinogen or denatured plasma fibrinogen. Fibrinogen in non-denatured plasma could not be detected by this single antibody ELISA. This immunochemical study demonstrates that only one copy of the epitope on the C-terminal protuberance of the A alpha-chain is exposed in non-denatured plasma. However, once the plasma fibrinogen has been denatured, as in the purification process, both copies of the epitope are available for antibody binding. This finding suggests that in plasma there is an intramolecular interaction between the carboxy terminal ends of the fibrinogen A alpha-chains which can be destroyed by denaturation. PMID- 1723304 TI - The effects of some plasma proteins on fibrin network structure. AB - Pronounced differences are found between characteristics of networks developed in plasma and those developed in pure fibrinogen solution. Networks in plasma have thicker fibres, are more permeable and have lower tensile strength. In this investigation the role of some plasma proteins as determinants of network structure under physiological conditions of clotting has been examined in an attempt to account for the differences in network structure in plasma and fibrinogen solution. The effect of physiological concentrations of antithrombin III, fibronectin, albumin, alpha globulin and gamma globulin on fibrin network structure was examined using mass-length ratio (muT) from turbidity, bulk network permeability (tau) and kinetics of network development. It was found that differences in fibrin network structure developed in plasma and pure fibrinogen solution could not be accounted for by alterations induced in network properties by albumin, gamma globulin, alpha globulin, fibronectin and antithrombin III. It is concluded that the final network structure is determined by the kinetics of fibrin fibre growth and is highly responsive to the presence of plasma proteins. PMID- 1723305 TI - Multicolour immuno-staining of fibrinogen polypeptide chains for identification of their derivatives in electrophoregrams. AB - A novel electrophoretic procedure enabling multiple, direct immunoprobing of electrophoregrams without depending on Western blotting is described, and applied to the identification of the derivatives formed in the early stages of clot stabilization. Multicolour immunostainings for positive identification of cross linked chains in partially stabilized fibrin clots indicated that the early products of alpha-chain cross-linking by factor XIII are largely hybrids of co cross-linking of alpha- and gamma-chains rather than alpha-chain polymers suggested from previous studies employing non-specific staining of electrophoregrams. Furthermore, plasma-fibrinogen dimers were found to contain cross-linked alpha-chains with an electrophoretic mobility very near that of gamma-gamma-dyads. A similar product is produced by tissue-transglutaminase, but not by factor XIII. PMID- 1723306 TI - Atherosclerotic plaque growth: presence of stimulatory fibrin degradation products. AB - Focal smooth muscle cell proliferation is widely perceived as a key event in the formation of stenosing atherosclerotic lesions, but the stimuli for this remain uncertain. Soluble extracts of human aortic intima from proliferative gelatinous and transitional lesions, as well as surface encrusted thrombi, have been shown by us to be mitogenic for the chick chorioallantoic membrane (CAM). They have also been shown to stimulate increase in vascularity of the CAM. When active samples were passed through anti-albumin and anti-whole-serum affinity columns, mitogenic activity in the unabsorbed, fibrin related antigen fraction remained close to the original whole extract level. In contrast, when the unabsorbed fractions from anti-whole-serum columns were passed through an antifibrinogen affinity columns, the activity was reduced to insignificant levels. Similarly, whole extracts lost activity after passing through an antifibrinogen column. This has been taken one stage further by dividing the unabsorbed fraction from an anti whole-serum column into two equal volumes and passing one half through an antifibrinogen fragment D affinity column, and the other through an antifibrinogen fragment E affinity column. The activity of the unabsorbed fraction from the fragment D column remained the same, but that from the fragment E column was significantly reduced. Most of the fibrin degradation products (FbDP) in lesion extracts are derived from fibrin, not fibrinogen, and clotting out fibrinogen and fragment X with thrombin did not remove the activity. Whole extracts of atherosclerotic lesions clotted on the CAM surface as has previously been shown with plasma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723307 TI - Factors relevant to stimulatory activity of fibrin degradation products in vivo. AB - Extracts of atherosclerotic lesions contain a range of fibrin degradation products (FbDP), similar fragments have been detected in extracts from human and mouse healing skin wounds and from the invasive edge of human breast carcinomas, which are all proliferating systems. We have previously shown that FbDP stimulate cell proliferation including angiogenesis in the chick chorioallantoic membrane (CAM), and sought to characterize further the active components. Fibrin prepared from platelet-rich and platelet-free plasma, and purified Kabi fibrinogen, was treated with plasmin, and the digests were all active. FbDP from platelet-rich plasma clots also increased vascularity of the CAM. Prior removal of fibronectin from plasma by gelatin-Sepharose affinity chromatography did not affect proliferative activity. Current studies showed that long digests of fibrin, in which the only major band detectable is fibrin fragment E are active. Commercial fibrinogen derived fragment E, itself inactive on the CAM, becomes active after exposure to thrombin cleavage of fibrinopeptides. Recently fragment E has been isolated from shorter digests, by simple filtration through a Millipore 0.2 microns centrifuge filter. It displayed similar activity to the fragment E obtained from long digests. Fragment E in plaque extracts has been shown consistently to lack fibrinopeptide A indicating it is of fibrin origin. PMID- 1723308 TI - Fibrin degradation products generation and fibrinopeptide A release in normal plasma incubated with thrombolytic agents: proposed mechanisms. AB - Clinical data have shown that the evaluation of fibrin degradation products (FbDP) does not reflect the efficiency of thrombolytic therapy in vivo. In this study, we found that the addition of plasminogen activators to normal plasma resulted in generation of FbDP and release of fibrinopeptide A (FpA) as shown by ELISA and HPLC. This FpA release was concomitant with fibrinogen degradation, and was not inhibited by thrombin inhibition or by prothrombin depletion in plasma. Thus, the increase in FpA did not result from coagulation activation and may result from the plasmin-induced release of FpA from fibrinogen degradation product E1. The generation of cross-linked FbDP after tPA addition occurred in normal plasma as well as in factor-XIII-deficient plasma and quickly reached a plateau. It was not inhibited by hirudin. Therefore FbDP in these plasmas probably derived from the plasmin degradation of cellular transglutaminase cross linked fibrin/fibrinogen derivatives present in plasma. PMID- 1723309 TI - Endothelial cell growth: biology and pharmacology in relation to angiogenesis. AB - The vascular system is lined by a monolayer of endothelial cells which proliferate very slowly under normal conditions. The formation of new capillary vessels is associated with some physiological circumstances and several pathological conditions. Angiogenesis requires migration, differentiation and proliferation of endothelial cells. The mechanism of tube formation is still poorly understood. Tumour growth is angiogenesis-dependent and angiogenesis is directly or indirectly induced by the tumour. Induction of angiogenesis is an important step in carcinogenesis and in metastatic development. Angiogenesis is induced during the transition from hyperplasia to neoplasia. Numerous angiogenic factors have been identified, most are mitogenic for endothelial cells and some are only responsible for tube formation. However, it is difficult to recognize which factor is the most important in vivo. Since angiogenesis is necessary for tumour growth, any natural or synthetic antiangiogenic compound may have an antineoplastic potential. Inhibition of tumour angiogenesis under the control of a tumour suppression gene could play an important role. Pharmacological compounds, such as heparin, heparin fragments and corticosteroids, have been shown to be antiangiogenic substances. More recently two new inhibitors of capillary endothelial cell proliferation and/or angiogenesis have been described: they are a cartilage-derived inhibitor and platelet factor 4. PMID- 1723310 TI - In vivo and in vitro studies of angiogenin--a potent angiogenic factor. AB - Angiogenin is a potent blood-vessel-inducing polypeptide with a molecular weight of 14,000 that has a unique ribonucleolytic activity. First isolated from the conditioned medium of tumour cells, angiogenin has since been purified from normal plasma, which suggested that its propensity to induce neovascularization should be strictly controlled. Modulation of that activity might involve interaction of angiogenin with cell-surface receptors and extracellular matrix of endothelial cells, tight-binding inhibition of both its ribonucleolytic activity and cell binding property by ribonuclease inhibitor, as well as the overall influence of divalent copper, a modulator of angiogenesis. PMID- 1723311 TI - Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging. AB - An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy. PMID- 1723313 TI - Self-association of bovine pancreatic trypsin inhibitor: specific or nonspecific? PMID- 1723312 TI - Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles. AB - We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin. PMID- 1723314 TI - Expression of receptors for extracellular matrix proteins in human endothelial cells. AB - The interaction with matrix components of the basal membrane is an important factor in the control of vascular endothelial cell function in both normal and pathological conditions. In the present work we define integrin receptors in vascular endothelial cells and analyze whether their expression is affected by endothelial cell activators. Using in vitro adhesion tests we show that human endothelial cells (HEC) can attach and spread on substrates coated with several matrix components including fibronectin, laminin, collagen type IV, fibrinogen and vitronectin. Using specific antibodies we detect integrin receptor complexes of the beta 1 and beta 3 families that can support adhesion of HEC to the above matrix proteins. These are the alpha 2/beta 1 collagen receptor, the alpha 3/beta 1 receptor for fibronectin, collagens and laminin, the alpha 5/beta 1 fibronectin receptor, the alpha 6/beta 1 laminin receptor and the alpha V/beta 3 receptor for vitronectin and fibrinogen. When HEC are exposed to a combination of tumor necrosis factor alpha (TNF alpha) and immune interferon (IFN-gamma) the amount of the alpha V/beta 3 vitronectin receptor at the cell surface was decreased by a factor of 50-70%, while the beta 1 integrin complexes were not affected. HEC cells thus express receptors for several matrix components and inflammatory mediators such as TNF alpha and IFN-gamma can selectively alter the expression of some of them. PMID- 1723315 TI - Stromal fibroblastic and hematopoietic progenitors in patients with graft-versus host disease (GVHD). AB - We cultured bone marrow cells from patients receiving bone marrow transplantation (BMT) to assay bone marrow fibroblast colony-forming cells (CFU-F) and hematopoietic progenitors (CFU-mix, CFU-C, BFU-E and CFU-E), and compared the mean values obtained from patients with and without graft-versus-host disease (GVHD). The value of CFU-F colonies from 15 patients was always less in patients with acute GVHD Grade II,III than in those with Grade 0,I over the period 30 to 110 days after BMT. The CFU-F colonies from patients with Grade 0,I consisted of approximately the same number of small and large colonies, whereas virtually all CFU-F colonies from patients with Grade II,III were small. Fibroblasts collected from bone marrow cells cultured for 2-3 weeks were incubated with IL-1 (50 U/ml) for 24 h. The concentrations of G-CSF in the culture supernatants from patients with Grade II,III were higher than in those with Grade 0,I. The results of assays of hematopoietic progenitors from 48 patients showed that the number of hematopoietic progenitors decreased as the severity of acute GVHD increased. These results suggest that the myelosuppression seen in GVHD may be associated with reduced numbers of CFU-F in bone marrow. PMID- 1723316 TI - Lack of effect of perindopril on plasma and adrenal corticosteroids in the guinea pig during the estrous cycle and under contraceptive treatment. AB - We studied changes in cortisol, aldosterone, progesterone, estrogens and cholesterol in cyclic female guinea-pigs and in animals under contraceptive, treated or not with an inhibitor of angiotensin converting enzyme (ACE): perindopril. Perindopril decreased ACE by 80% without affecting steroid profiles. Peak value for plasma progesterone occurred at meta-estrus and diestrus. It disappeared under contraceptive treatment. The very low levels of estrogens in the female guinea pig remained unchanged in all cases. Plasma cortisol concentrations were higher at pro-estrus and estrus whereas plasma aldosterone concentrations remained constant during the estrous cycle and under contraceptive treatment. Furthermore, aldosterone did not change under perindopril treatment despite the decrease of the activity of ACE. The contraceptive treatment decreased plasma cholesterol levels. Under perindopril treatment, this drop was amplified. No change was detected in adrenal steroid concentrations, except for progesterone which decreased under contraceptive treatment. PMID- 1723317 TI - Effects of the nature of dietary proteins, lecithin and methionine on rat plasma lipids. AB - In order to study the complex interrelationships between on the one hand, dietary proteins, lecithin and methionine and, on the other, blood cholesterol and triglyceride levels, groups of .10 rats were fed for six weeks with diets only differing by the nature of proteins which comprised 10% of the diet. These diets were composed of egg white, heated soybean flour, casein, heated soybean flour supplemented with 1% methionine, or with 4% of the lipids replaced by soybean lecithin, heated 5-day germinated soybean supplemented or not with 1% methionine and casein with 4% of the lipids replaced by lecithin. Egg white caused no change in blood cholesterol compared to heated soybean meal (0.955 +/- 0.18 vs 0.83 +/- 0.14), but caused a significant increase in blood cholesterol levels compared to casein (0.955 +/- 0.18 vs 0.81 +/- 0.11 g/l). No effect was found on blood triglycerides levels. Lecithin caused no change when it partially replaced lipids in the soybean or casein diets thought it increased triglycerides with the casein diet (1.16 +/- 0.31 vs 0.84 +/- 0.24 g/l). Supplementation of the heated soybean diet with 1% methionine led to an increase in blood triglycerides (0.85 +/- 0.26 vs 1.18 +/- 0.34 g/l). After germination, this effect disappeared. The effects of the type of protein, partial replacement of dietary lipids by lecithin and supplementation with methionine are discussed. PMID- 1723318 TI - Lactate steady state velocity and distance-exhaustion time relationship in running. AB - The relationship between distance and exhaustion time is linear for running exercise at constant velocity lasting 5 to 45 minutes. The slope of this relationship has the dimension of a velocity (VCRIT) which can be sustained during a long time. The individual VCRIT have been studied in 8 runners by measuring exhaustion time for 4 to 5 constant-velocity running exercises performed to exhaustion. The velocity correspond to a lactate steady state (VCHASSAIN) has been estimated according to a two-step protocol proposed by Chassain for exercises on a cycle ergometer. The running velocity corresponding to maximal aerobic metabolism (VLEGER) was estimated by means of the track test proposed by Leger and Boucher. VCRIT was very well correlated (r greater than 0.97) and almost equal to VCHASSAIN. VLEGER was also very well correlated with VCHASSAIN and VCRIT. PMID- 1723319 TI - Decrease in tetanic tension elicited by beta-adrenergic stimulation. AB - The effect of beta-adrenergic stimulation on tetanic tension (TT), maximal rate of rise of tension (+TT) and phospholamban (PHL) phosphorylation were studied in the perfused rat heart. 3 x 10(-8) M isoproterenol perfused at different [Ca2+]o 0.25, 1.35 and 3.85 mM, significantly decreased TT while increased +TT and PHL phosphorylation at the three [Ca2+]o studied. Regression lines of the relationship between +TT and TT from individual data obtained at each [Ca2+]o in the presence and in the absence of isoproterenol, show that for the same level of +TT, TT is lower in the presence of isoproterenol, i.e. at high levels of PHL phosphorylation. The slopes of the lines were 0.137 s and 0.427 s (P less than 0.05) in the presence and absence of isoproterenol respectively. The decrease in TT produced by the beta-agonist can be attributed to its relaxant action prevailing over its inotropic effect and may represent the mechanical expression of the enhanced phosphorylation of phospholamban. PMID- 1723320 TI - Hypertensive factor in different models of experimental hypertension. AB - A hypertensive factor (HF), isolated from rat erythrocytes, has been shown to stimulate in vitro calcium uptake in aortic rings and to elevate blood pressure when injected into normotensive rats. In the present study, we investigated tissue responsiveness to HF in spontaneously hypertensive rats (SHR), Wistar Kyoto rats (WKY), 2-kidney, 1-clip renovascular hypertensive rats and uninephrectomized rats that were given water or saline to drink or that were treated with DOCA and given water or saline to drink (DOCA-salt). Tissue responsiveness was determined by incubating aortic rings from the rats in the different groups with a constant amount of HF and measuring "lanthanum-resistant" calcium uptake. Tissue sensitivity to HF was greater in SHR than in WKY. In contrast, tissue sensitivity to HF was not enhanced in 2-kidney, 1-clip renovascular and DOCA-salt hypertensive rats relative to their appropriate controls. These results suggest that the increased tissue responsiveness to HF found in SHR is not universally associated with elevated blood pressure; increased tissue sensitivity seems to be a specific characteristic of genetic hypertension. PMID- 1723321 TI - [Central temperature during chronic exposure to a warm climate]. AB - Rectal temperature was determined at rest in 603 healthy melanoderm african men, in their natural environment. The ambient temperatures were noted between 20 and 38 degrees C on the morning and 23 to 46 degrees C on the afternoon, according to the season and place. On the morning results a significant positive correlation was found between rectal and ambient temperatures. On the afternoon results the situation was more complex: there was a positive correlation only with comfortable and warm ambient temperatures (23-35 degrees C), but not with higher temperatures (36-46 degrees C). Furthermore 28 young sportmen performed a submaximal 30 min exercise test on a cycloergometer at 2 ambient temperatures (comfortable and hot) and the difference in rectal temperature was appreciated between the beginning and the end of each activity. Results showed the same increase in the body temperature during the 2 tests. These results suggest that human thermoregulation remains effective in the subtropical area but can be adapted to the climatic conditions in permanent residents. Then the internal temperature is modified by the increasing ambient temperature, but there is a superior limit of this deep body temperature: when it is overpassed, a strong corrective mechanism is applied. PMID- 1723322 TI - In-vivo and in-vitro studies on the effects of chronic dexamethasone treatment on cardiovascular responses to sympathetic stimulation. AB - Rats treated with dexamethasone, 1.5 mg/kg s.c. weekly for 3 weeks exhibited significantly greater increases in mean arterial pressure than their controls, following either sympathetic nerve stimulation or noradrenaline administration. The atria from dexamethasone-treated rats showed greater chronotropic activity in response to noradrenaline but not to field stimulation, whereas the force of contraction was significantly less than that of the controls after either field or noradrenaline stimulation. Isolated rat tail artery preparations from dexamethasone-treated rats were found to be twice more sensitive to noradrenaline than the controls. Prazosin antagonised the noradrenaline-induced pressor response to the same extent in control and dexamethasone-treated rats. Dexamethasone treatment did not significantly increase the sensitivity to KCl or the angiotensin-potentiated pressor response to noradrenaline. This study shows that dexamethasone treatment increases postsynaptic sensitivity of the cardiovascular system to noradrenaline in rats. PMID- 1723323 TI - [Effects of beta-adrenergic receptor blockade on hyperlactacidemia induced by exercise of different intensity]. AB - The effects of beta-adrenergic blockade on the exercise-induced hyperlactatemia (Lap) have been studied in 31 adult male subjects [age: 25 +/- 1 years; body weight: 69 +/- 1 kg; VO2max: 54 +/- 1 ml O2.kg-1.min-1 (mean values +/- SEM)] randomly divided in 3 groups. All exercises were performed on a 10% inclined treadmill. In group 1 (n = 11), the subjects were walking during 20 minutes at 5 km.h-1 (55.6 +/- 1.4% VO2max). In group 2 (n = 10), they were running during 9 minutes at 8 km.h-1 (79.4 + 1.5% VO2max). The subjects of the third group (n = 10) were submitted to a 4 minutes run at 9.5 km.h-1 92 +/- 1.6% VO2max). These exercises were performed 1 hour after ingestion of a placebo or a single dose of 40 mg propranolol, in a double-blind randomized order. Blood samples were drawn at regular time intervals from an antecubital vein. Exercise tachycardia was reduced by about 20% (P less than 0.001) by propranolol in each group. Lap was significantly reduced by 15% by propranolol (P less than 0.005) at the lowest exercise intensity (55.6% VO2max), remained unchanged at 79.4% VO2max and was significantly enhanced by 16% during the recovery period following the run at 92% VO2max. These results clearly showed that the effects of acute beta-adrenergic blockade on Lap depend on exercise intensity. PMID- 1723324 TI - Evaluation of the stimulatory capacity of procaine on Na transport through frog skin. AB - Procaine has different effects on various ionic conductive pathways through the frog skin. We investigated the season and temperature dependence of the stimulation by mucosal procaine, of the Na-conductive pathway. For this stimulation, we found higher half-maximal saturation constants (KNa) in winter animals (6.38 +/- 0.8 mmol/l), than in summer ones (4.03 +/- 0.7 mmol/l). Summer frogs kept for 2 weeks at 4 degrees C, reacted like winter frogs (6.24 +/- 0.8 mmol/l). However, the maximal sodium currents (INa max) did not depend on temperature adaptation. Procaine-induced increased of KNa is associated with an increase of INa. The effects of procaine associated with BIG (benzoylimidazole-2 guanidine) were non-additive, while with vasopressin they were additive. A biphasic, dose-dependent response was recorded after procaine application to the inner surface. Vasopressin counteracted the serosal procaine-induced inhibition of the Na-transport. PMID- 1723325 TI - Postprandial modifications of plasma secretin levels during pancreatic secretion in dogs. AB - In dogs with a direct pancreatic fistula, a duodenal cannula and a catheter in a saphenous vein, plasma secretin levels, changes in the flow, bicarbonate and chloride concentrations of the exocrine pancreatic secretion as well as in the pH of intraduodenal content have been studied 12 hours after the ingestion of a standard diet. Under these conditions the pancreatic secretion showed a biphasic response with a maximum flow and bicarbonate concentration during the 0-4 and 8 12 h postprandial periods. This coincided with a marked decrease of pH in the intraduodenal content, with values close to 4.5. On the other hand, during the first postprandial hour, plasma secretin values increased from basal ones (218.66 +/- 27 pg/ml) to 448.94 +/- 66 pg/ml, remaining elevated for four hours after the meal ingestion. However, no increase occurred between 8-12 h when intraduodenal pH reached values below 4.5. This study indicated that: 1) plasma secretin levels increased significantly (P less than 0.05) after the ingestion of a standard solid diet, and 2) determinants liberating secretin were not only the presence of a duodenal pH below 4.5, but probably the presence of some macronutrients from the meal. PMID- 1723326 TI - Sustained changes in blood alpha amino nitrogen compartmentation during recovery from cafeteria feeding in rats. AB - We have previously reported that blood urea and blood cell amino acids levels are reduced in rats obese by feeding a palatable cafeteria diet. In order to distinguish whether these changes result from the altered diet, or from the obesity per se, we have studied cafeteria fed rats after returning to standard diet. As in previous studies, obesity induced by cafeteria feeding (for 90 days) was maintained when the cafeteria diet was removed and rats were fed standard diet only. After removal of the cafeteria diet, blood urea levels of 24 h starved obese rats were lower (23%) than those of starved control rats. Blood cell amino acid levels of obese were lower than control ones from day 50 onwards, during and after cafeteria feeding (21% lower on day 100 of life), and thus coincided with divergence of body weights; these differences were maintained despite removal of cafeteria diet. The effects of starvation on plasma amino acid levels were more marked in obese than control rats, during and after cafeteria feeding. Thus the effects on blood amino acids and urea levels in cafeteria diet induced obese rats are related to the obese status rather than to the diet composition. PMID- 1723327 TI - Interference of lung distension with the cardiovascular response to chemoreceptor stimulation in anaesthetized rats. AB - Experiments were performed to examine the role of the stimulation of pulmonary stretch receptors in cardiovascular response to peripheral chemoreceptor stimulation in spontaneously breathing anaesthetized rats. The effects of continuous positive tracheal pressure (0.1 to 0.4 kPa) were examined in normal rats and in rats pretreated with almitrine bismesylate, a potent stimulator of the arterial chemoreceptors. In the untreated group, an increase of a maximum 4 +/- 0.7 beats.min-1 in heart rate was found during testing at pressure of 0.2 and 0.3 kPa when interference with augmented breath was avoided. These pressures at the tracheal level had no effect on systemic blood pressure, so that baroreflex influences can be discounted. During strong almitrine-induced chemostimulation, a prolonged bradycardia developed with the long-lasting hyperventilation. The same continuous positive pressures were unable to overcome the chemoreflex bradycardia. No changes in heart rate were observed under these conditions, whatever the potentiation of the mecanoreceptors for distension in the presence of alveolar hypocapnia. It was concluded that the stretching of lung mecanoreceptors on the cardiac control is of little importance in anaesthetized rats and that bradycardia generally dominates during strong stimulation of the chemoreceptors even when lung distension is artificially increased. PMID- 1723328 TI - Dialysis of plasma soluble lipofuscins in patients with end-stage renal failure. AB - Fluorescence spectrophotometry demonstrates that the levels of plasma soluble lipofuscins (SL) in patients with end-stage renal failure, undergoing continuous ambulatory peritoneal dialysis (CAPD) or haemodialysis (HD), remain significantly higher than in normal subjects. Plasma samples from these patients show the presence of SL generated from 3-hydroxy-anthranilic acid [excitation (ex) at approximately 324 nm and emission (em) at approximately 413 nm] and of other SL generated from dopa, catecholamines, 3-hydroxykynurenine and from structurally related precursors (ex at approximately 345 nm, em at approximately 445 nm). These precursors form the melanin components, which are approximately 3 wt % of SL. The fluorescence of SL appears to originate mainly from the melanin components. Peaks and shoulders at these wavelengths are found in the spectra of all dialysates. Based on intensity measurements at 413 nm and 445 nm, the weekly clearance rates with HD are in general greater than those with CAPD. The saponified cellulose ester membrane used in HD passes only lower-molecular-weight SL and/or components of SL. After HD, the greatest reductions in plasma intensities are found at approximately 324 nm and approximately 413 nm. The clearance rates (l/week) are always greater at 413 nm [means HD: 18.47 +/- 4.44 standard deviation (SD), n = 8; CAPD: 12.50 +/- 2.47, n = 4] than at 445 nm (HD: 10.94 +/- 3.86; CAPD: 7.95 +/- 1.75) both with HD and CAPD. In CAPD, the membrane also permits the passage of large amounts of albumin and other high-molecular weight substances.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723329 TI - [Effects of C-terminal fragment of substance P-CP 5-11 on the activity of neurons of the dorsal raphe nucleus]. AB - The experiments on Wistar rats showed that microinjection of C-terminal fragment of substance P-CP5-11 (1 microgram) into one of the antinociceptive system structure--dorsal raphe nucleus, caused a prolonged (24 hours of observation) analgetic effect by the hot plate test. Neuronal activity of dorsal raphe nucleus simultaneously enhanced. The CP5-11 antinociceptive activity was higher than the CP1-11 one. The conclusion is that CP1-11 and in particular its C-terminal fragment CP5-11 play a role in activation of antinociceptive system. PMID- 1723330 TI - [Immunogenesis and axoplasmic transport in Wistar rats]. AB - Immunization of Wistar rats with thymus dependent antigens (sheep red blood cells SRBC) is accompanied by a reliable increase in the synthesis of RNA and proteins in thalamic cerebral cortex and spinal marrow (48 hrs after antigen injection) and also in an increase in the intensity of rapid axoplasmic transport (RAT) along motor fibers of sciatic nerve (5,48,72 hrs following the beginning of immunization). There was a consecutive augmentation in AFC number in mesenteric and partly in inguinal lymph nodes (96 hrs after SRBC injection). Thus, time dependence between immunogenesis and axoplasmic transport in experimental animals (Wistar rats) was determined for the first time. It identifies another, previously unstudied, channel in interactions of immune and nervous systems. PMID- 1723331 TI - The effects of semantic and phonemic prestimulation cues on picture naming in aphasia. AB - In this study of auditory prestimulation cues, picture naming performances under phonemic and semantic conditions were compared to picture naming performance under a neutral condition. Twenty aphasic subjects named 324 pictures (108 pictures x 3 conditions) each. Responses were scored using a coding system adapted by the investigator from classification systems used by Williams and Canter (1982) and Kohn and Goodglass (1985). Results indicated that naming accuracy was facilitated by phonemic and semantic cues. An examination of the distribution of errors under the three conditions revealed systematic effects of phonemic and semantic cues on the frequency of occurrence of specific error types. Increases in semantic paraphasia proportion scores and decreases in unrelated word error proportion scores were associated with the semantic condition, while increases in phonemic paraphasia proportion scores were associated with the phonemic condition. The finding that naming performance of aphasic adults varies as a function of the type of information provided by the cue is discussed in relation to cascade visual confrontation naming models. PMID- 1723332 TI - Writing with the right hemisphere. AB - We studied writing abilities in a strongly right-handed man following a massive stroke that resulted in virtually complete destruction of the language-dominant left hemisphere. Writing was characterized by sensitivity to lexical-semantic variables (i.e., word frequency, imageability, and part of speech), semantic errors in writing to dictation and written naming, total inability to use the nonlexical phonological spelling route, and agrammatism in spontaneous writing. The reliance on a lexical-semantic strategy in spelling, semantic errors, and impaired phonology and syntax were all highly consistent with the general characteristics of right hemisphere language, as revealed by studies of split brain patients and adults with dominant hemispherectomy. In addition, this pattern of writing closely resembled the syndrome of deep agraphia. These observations provide strong support for the hypothesis that deep agraphia reflects right hemisphere writing. PMID- 1723333 TI - There is an entity called agrammatic aphasia. AB - Agrammatism as a phenomenon of neuropsychological relevance has been recently attacked--from conceptual and empirical angles. This article examines the facts, as they emerge from three recent experimental studies that have concluded that agrammatism does not exist (Miceli et al., 1989; Martin et al., 1989, Badecker et al., in press), and draws the opposite conclusion: that agrammatism is of interest to students of language and that patients belonging in this clinical category also reveal uniform patterns of aberrant behavior that are of great linguistic and psycholinguistic relevance. PMID- 1723334 TI - Grammatical class effects in word production: finding the locus of the deficit. PMID- 1723335 TI - Morphological details of primate axons and dendrites revealed by extracellular injection of biocytin: an economic and reliable alternative to PHA-L. AB - The objective of this study was to determine if biocytin would reliably label details of distant axons and dendrites when injected extracellularly in primates. Biocytin (2.5-5%) was injected iontophoretically or by pressure into several areas of the visual and somatosensory systems of macaque monkeys, squirrel monkeys, tree shrews and galagos. After survival times that ranged from 9 h to 2 weeks, fine details of anterogradely filled axons and/or retrogradely filled dendrites were reliably revealed with an avidin-biotin-HRP complex (ABC solution) that was enhanced with heavy metals. Biocytin labeling was successfully combined with choline acetyltransferase (ChAT) or cytochrome oxidase (CO) histochemistry to reveal double-labeled cells. Our results show that biocytin is a versatile, easy-to-use label that completely fills cell processes both anterogradely and retrogradely in several primate species. PMID- 1723336 TI - Colchicine-induced deafferentation of the hippocampus selectively disrupts cholinergic rhythmical slow wave activity. AB - It has been proposed that hippocampal rhythmical slow wave activity (RSA or theta rhythm) induced by sensory stimulation (atropine-sensitive theta) is generated by the cholinergic septo-hippocampal system. Although ablations of the septum or its projections to the hippocampus disrupt hippocampal RSA, such non-selective lesions damage both cholinergic and non-cholinergic septo-hippocampal inputs. The present study assesses the effects of a selective septal neurotoxic lesion on hippocampal electrical activity. Colchicine, which has been reported to be selectively toxic to cholinergic neurons in the medial septum, was injected into the right lateral ventricle, and electrodes were implanted bilaterally into the dorsal hippocampus of female Sprague-Dawley rats. Hippocampal electrical activity was recorded 10-14 days later from the ipsilateral (colchicine-treated) and contralateral (control) hemispheres during locomotor activity or immobility. RSA ranging from 6.3 to 8.7 Hz was evoked in both hippocampi during mobility. Following i.p. administration of an anesthetic dose of urethane, hippocampal RSA at a frequency of 4 Hz could be elicited in the control hemisphere (n = 12) of all animals by pinching the tail. RSA was absent in 6 of 9 animals in the colchicine-treated hemisphere. RSA from control and treated hemispheres persisting after urethane administration was abolished by 5 mg/kg of scopolamine, thus verifying its cholinergic nature. A decrease in the number of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum and a depletion of acetylcholinesterase (AChE)-staining in the hippocampus were evident in the hemisphere ipsilateral to colchicine administration. These data support the septal pacemaker hypothesis of hippocampal theta-rhythm and further demonstrate the neurotoxic effect of colchicine on septo-hippocampal cholinergic neurons by the induction of a functional alteration. The selective disruption of cholinergic neurons in the medial septum by colchicine provides a means to dissociate the contribution of septal cholinergic and non-cholinergic components to hippocampal electrical activity. PMID- 1723337 TI - Ethanol enhances GABAA receptor-activated chloride currents in chick cerebral cortical neurons. AB - Primary cultures of cerebral cortical neurons were prepared from 7- to 8-day-old chick embryos. The effect of ethanol on GABA-activated membrane current was examined using whole-cell voltage-clamp recording in cells maintained for 3-25 days in vitro. In approximately 60% of neurons examined ethanol caused a potentiation of the membrane current elicited by GABA. The threshold concentration of ethanol was 1 mM, and the potentiating effect of ethanol on GABA activated currents was maximal at 10 mM. In many cells higher concentration (40 50 mM) of ethanol inhibited GABA-activated currents. These effects of ethanol were all reversible. PMID- 1723338 TI - A new population of neurons with crossed axons in the lamprey spinal cord. AB - Neurons with contralateral, rostrally and caudally projecting axons were studied in whole mounts of lamprey spinal cord using retrograde labelling techniques with fluorescent dextran-amines, cobalt-lysine or horseradish peroxidase. A previously unknown large population (180-300 cells per hemisegment) of small (less than 25 microns) cells with contralateral projecting axons is described. Their axons extend over less than 5 segments rostrally or caudally. The number of these cells per segment was relatively constant in the rostral half of the spinal cord, but increased significantly in the caudal half. In comparison, medium-sized cells with contralateral axons corresponding to previously identified premotor interneurons were far less numerous (14-21 per hemisegment) and their axons extended more than 5 segments. Contralaterally projecting edge cells (intraspinal stretch receptor neurons) with principal rostral or caudal axons plus short collaterals in the other direction were distributed throughout the length of the spinal cord, whereas large and giant cells with a varied morphology were found in the caudal half. PMID- 1723339 TI - Separate signals mediate hypoglossal motor neuron response to axonal injury. AB - Nerve transection causes decreased choline acetyltransferase (ChAT) expression and appearance of nerve growth factor receptor (NGFr) in hypoglossal motor neurons. Topical application of vincristine to the hypoglossal nerve blocks axonal transport of WGA for more than one week and causes loss of ChAT but no appearance of NGFr. These results indicate that loss of ChAT is related to interruption of axonal transport, but another signal induces de novo expression of NGFr. PMID- 1723340 TI - Effect of adrenalectomy and dexamethasone on neuropeptide content of dorsal root ganglia in the rat. AB - Neuropeptides, including substance P (SP), calcitonin gene-related peptide (CGRP) and somatostatin (SS) in dorsal root ganglia (DRG) may play a role in neurogenic inflammation and pain transmission. Adrenal corticosteroids regulate neuropeptide synthesis in some areas of the CNS and may modulate neurogenic inflammation and sensory perception. We have investigated the effects of adrenalectomy and dexamethasone (0.2 mg/kg/day) treatment on neuropeptide content of rat cervical DRG using specific and sensitive radioimmunoassays. In control animals, a differential distribution of neuropeptide was found; SP and CGRP content increased from C4 to C7 in contrast to SS content, which decreased from C4 to C7. Ten days following adrenalectomy, the mean SS content of cervical DRG decreased significantly to 79.6 +/- 4.5% of sham-operated controls. In contrast, SP and CGRP content increased significantly 10 days after adrenalectomy to 134.6 +/- 6.9% and 132.0 +/- 11.6% of sham-operated controls, respectively. The effects of adrenalectomy on CGRP and SS were reversed by administration of dexamethasone. These results suggest that glucocorticoids affect the neuropeptide content of DRG in the adult rat. PMID- 1723341 TI - Somatotopic organization of the dorsal column nuclei in the rat: transganglionic labelling with B-HRP and WGA-HRP. AB - To analyze the patterns of cutaneous primary afferent fibers projecting to the dorsal column nuclei in the rat, horseradish peroxidase (HRP)-based tracers were injected intracutaneously into a number of discrete regions of the forelimbs and hindlimbs. Three-4 days following the HRP injections, the rats were perfused transcardially; 60 microns transverse sections were cut, and the HRP was reacted using the tetramethyl benzidine method. Comparisons were made of projections following injections with choleragenoid-conjugated horseradish peroxidase (B-HRP) or with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). B HRP and WGA-HRP produced similar patterns of labelling, but B-HRP produced greater intensity of labelling and slightly larger projection areas. In the cuneate nucleus (CN), HRP labelling of primary afferents from small, delimited regions, e.g., from a portion of the skin of a single digit, appeared to be precisely restricted in rostrocaudally oriented columns, with little or no overlap (in the mediolateral and dorsoventral plane) into adjacent regions. With respect to rostrocaudal organization, a region in the CN containing a dense population of cutaneous primary afferent fibers appeared to be similar to the middle, or cluster, region in cats and in raccoons and the pars rotunda in primates. Projection patterns were very consistent from rat to rat, but their somatotopic organization differed from that suggested by electrophysiological studies: cutaneous afferents from forelimb digit 1 projected near the ventral border of the CN; those from digit 5 projected dorsomedially to those from digit 1; the projections from the remaining digits formed a crescent between the projections from digits 1 and 5. In the gracile nucleus, the organization of cutaneous afferent projections from hindlimb digits was more variable and complex than that found in the CN. PMID- 1723342 TI - Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells. AB - This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UC11. Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture. Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor. Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNF alpha) or interleukin-1 beta (IL1 beta), but not interleukin-2 or interleukin-6, decreased the density of SP receptors. This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation. The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines. The decrease in SP receptor density was readily reversible on washout of the cytokines. The EC50 for TNF alpha was approximately 0.5 ng/ml, the EC50 for IL1 beta was approximately 0.1 ng/ml. Radioligand binding studies with [125I]TNF alpha indicated the presence of a low density of high affinity binding sites for this ligand: Kd = 2.5 +/- 0.6 ng/ml, Bmax = 14.8 +/- 2.7 fmol bound/mg protein (assuming trimeric form of ligand bound). The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors. PMID- 1723343 TI - Detection of circulating histamine releasing autoantibodies with functional properties of anti-IgE in chronic urticaria. AB - Circulating histamine releasing factor(s) have been demonstrated previously in chronic urticaria by an immediate weal-and-flare response to intradermal autologous serum injection. We have studied 25 chronic urticaria patients by in vivo skin testing with autologous sera and an in vitro histamine release assay using mixed leukocytes of healthy donors, to define the nature and functional properties of the serum factor(s). Twenty showed a weal response to autologous serum (mean +/- s.e.m., 37.3 +/- 6.8 mm3). Weal formation was confined to ultrafiltered serum fractions greater than 100 kD in nine of nine patients. There was no response in 10 healthy controls of five patients with symptomatic dermographism. Fourteen chronic urticaria sera elicited histamine release greater than 10% (mean +/- s.e.m., 44.3% +/- 6.7) above basal levels from leukocytes of at least one of seven healthy donors. This in vitro response was also confined to ultrafiltered serum fractions greater than 100 kD in seven of seven sera and was present in IgG fractions of six of seven chronic urticaria sera that showed histamine releasing activity. Functional studies indicated that this histamine releasing autoantibody had the properties of anti-IgE: chronic urticaria sera 'desensitized' basophil leukocytes to subsequent challenge with other chronic urticaria sera and to goat anti-human IgE antibody; human myeloma IgE inhibited histamine release from leukocytes in response to chronic urticaria sera; removal of surface-bound IgE by lactic acid 'stripping' reduced histamine release in response to chronic urticaria sera and anti-IgE and subsequent passive sensitization with IgE myeloma serum partially restored it. Stainable peripheral blood basophils/mm3 in chronic urticaria patients were significantly reduced (mean +/- s.e.m, 7.9 +/- 2.0) when compared to healthy controls (39.6 +/- 4.4), P less than 0.001. These results suggest that histamine releasing autoantibodies are important in the pathogenesis of chronic urticaria by stimulating or facilitating degranulation of basophils and cutaneous mast cells through cross linking cell surface IgE receptors. PMID- 1723344 TI - Tumour necrosis factor stimulates human skin mast cells to release histamine and tryptase. AB - Besides its effects on tumour cells, tumour necrosis factor (TNF) also acts on a variety of other cells, thus enhancing inflammatory and immune processes. In view of the prominent role of the mast cell in such processes, the aim of the present study was to assess the effects of recombinant TNF-alpha on human mast cells. Mast cells from the infant foreskin obtained during circumcision were dispersed by an enzymatic technique using collagenase and hyaluronidase. Cells thus obtained were pooled, washed and separated by Percoll gradient centrifugation. Mast cells, with a purity of 70-90% were incubated for 60 min with 10(-11) to 10( 7) M rTNF-alpha. Histamine and tryptase levels were assessed in the cell supernatant by spectrofluorometry and radioimmunoassay (RIA) respectively. A concentration dependent release of histamine was observed, which reached a maximum of 11.5 +/- 2.2 nmol/10(6) cells at 10(-8) M rTNF. Release of tryptase was also concentration dependent and reached a maximum of 293 +/- 105 mU/10(6) cells (10(-8) rTNF). rTNF-alpha thus appears to be a direct stimulus for mast cells to degranulate and to release both histamine and tryptase. PMID- 1723345 TI - Larval exoantigens from ascarid nematodes are potent inducers of histamine release from human blood basophils. AB - Histamine release (HR) from washed human blood cells after challenge with the excretory-secretory antigens (ES) of the parasitic nematodes Toxocara canis and Ascaris suum was studied, employing a recently developed microfibre-based assay combined with hyperosmolar release media for maximal sensitivity. Blood samples were obtained from 30 patients suspected of parasite infection and 11 healthy volunteers serving as controls. Specific antibodies of IgE, IgG1 and IgG4 subclasses were determined by ELISA. Virtually no HR could be provoked by ES in the 11 controls. In contrast, HR was seen in 16 patients after challenge with T. canis ES and in 11 patients with A. suum ES. In the majority of these, HR was detectable after challenge with ES protein concentrations of less than 1 ng/ml, and the maximal HR obtained with ES was greater than that seen with optimal concentrations of anti-IgE. The HR after ES antigen challenge correlated with the amount of specific IgE in patient plasma, and coincided with the presence of specific IgG1 and IgG4. PMID- 1723346 TI - The actions of intermediate and long-chain n-alkanols on unitary NMDA currents in hippocampal neurons. AB - The actions of the n-alkanols butanol, pentanol, and octanol on unitary currents passing through N-methyl-D-aspartate (NMDA) ion channels have been studied in cultured CA1 hippocampal neurons. The cell-attached patch clamp method, with L homocysteic acid included in the patch pipette, was used to record single channel NMDA currents at the cell resting potential or for hyperpolarizing patch potentials. With the n-alkanols added to the bath solution, the mean open times for the NMDA channel were diminished and the channel conductance was unchanged. A decrease in mean open time to about 70% of control value was found with butanol (3 mM), pentanol (1 mM), and octanol (0.02 mM). In addition the n-alkanols had small effects to decrease the frequency of channel openings and to increase the amplitude of the unitary currents. The effects of the alcohols on intracellular calcium levels, during NMDA applications, were also measured using the fluorescent dye FURA II. PMID- 1723347 TI - cis-dominance of rat atrial natriuretic factor gene regulatory sequences in transgenic mice. AB - We have produced transgenic mice that express the prokaryotic marker protein chloramphenicol acetyltransferase under the control of regulatory sequences derived from the rat atrial natriuretic factor gene. The transgene, which contains 2.4 kilobases of the rat atrial natriuretic factor gene regulatory region, was found to direct 4000-fold more chloramphenicol acetyltransferase expression in adult atria than in ventricles. Low-level activity was also detected in the hypothalamus, demonstrating that these sequences contain the signals necessary for cardiac and central nervous system expression of the hormone atrial natriuretic factor. Developmental analyses showed early, high level transgene expression in fetal atrial and ventricular tissues but marked reduction of ventricular transgene expression following birth. Further, the developmental expression patterns of the endogenous murine atrial natriuretic factor gene and rat transgene were found to be quite distinct. Although both the rat and mouse atrial natriuretic factor genes are activated early in embryogenesis, perinatal ventricular expression appears to differ in these two rodent species. The transgene is expressed in a pattern analogous to the neonatal rat rather than the endogenous murine gene. These studies demonstrate that the cis-acting signals required for correct tissue specificity and developmental regulation of the rat atrial natriuretic factor gene are encoded in this 2.4 kilobase fragment and that these sequences act in a dominant fashion. PMID- 1723348 TI - Arachidonic acid metabolites regulate the secretion of atrial natriuretic peptide in cultured rat atrial cardiocytes. AB - Prostaglandins F2 alpha and E2 increase release of immunoreactive (irANP) in primary cultures of rat atrial cardiocytes. This effect is independent of cell density in the cultures and does not appear to operate through a cAMP-dependent mechanism. Studies that probed the PGF2 alpha effect with a number of different pharmacological antagonists suggest that it is tied to a calmodulin-dependent step. This latter effect does not appear to be related to increased calcium entry through voltage-gated channels in the plasma membrane nor to mobilization of ryanodine-sensitive intracellular calcium pools. Inhibitors of the lipoxygenase pathway, a second avenue of arachidonate metabolism, resulted in a decrease in irANP release from cultured atrial or ventricular cardiocytes. Leukotriene C4, a lipoxygenase product, had a modest effect to promote irANP release over a 24-h period. However, pretreatment of anesthetized rats with nordihydroguarietic acid, a lipoxygenase inhibitor, had no effect on stretch-dependent release of irANP from the heart in vivo. These findings suggest that the prostaglandins represent the more important group of arachidonate metabolites in regulating irANP release physiologically. PMID- 1723349 TI - Biosynthesis, secretion, and receptor selectivity of human brain natriuretic peptide. AB - Biosynthesis, secretion and receptor selectivity of human brain natriuretic peptide (hBNP) were studied. The BNP mRNA level in the ventricle was approximately 40% of that in the atrium and, taking tissue weight into account, the total amount of BNP mRNA in the ventricle was about twofold greater than the total amount in the atrium. The plasma BNP-like immuno-reactivity (-LI) level in normal subjects was 0.90 +/- 0.07 fmol/mL, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with congestive heart failure, with a progressive rise in proportion to its severity. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root, and the difference in the plasma BNP-LI level between the CS and the aorta (Ao), delta (CS-Ao)BNP, increased with the severity of congestive heart failure. In addition, the difference in the BNP-LI level between the anterior inverventricular vein (AIV) draining the ventricle and the aorta (delta (AIV-Ao)BNP) was comparable to delta (CS-Ao) BNP, indicating that BNP is secreted predominantly from the ventricle. Binding ability to human clearance receptors (C receptors) and cyclic GMP (cGMP) production of hBNP were investigated and compared with those of ANP. hBNP bound to human C receptors very weakly (about 7%), but exerted cGMP production similar to ANP in cultured human mesangial cells and bovine endothelial cells. In conclusion, hBNP is a novel cardiac hormone mainly synthesized in and secreted from the ventricle and plays physiological and pathophysiological roles in the dual cardiac natriuretic peptide system. PMID- 1723350 TI - Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma viride. AB - Antagonistic activity of the bacterium Pseudomonas cepacia against Trichoderma viride was greatly influenced by nutritional and environmental conditions. Xylose and trehalose strongly enhanced the antifungal activity of P. cepacia, whereas mannitol and glucose had little effect. The carbon sources that enhanced the antagonistic activity also inhibited sporulation of T. viride. Antagonism of P. cepacia was enhanced by ammonium nitrogen; however, with nitrite or nitrate there was only a little antagonism. The antagonism of P. cepacia was optimal at pH 5.0. Although P. cepacia showed maximum antagonism against T. viride at 37 degrees C, the antagonism was fairly good at temperatures as low as 18 degrees C, indicating that there is a broad range of temperature for the antifungal activity of P. cepacia. PMID- 1723351 TI - Experience with chronic peritoneal dialysis in infants. AB - We describe our experience with chronic peritoneal dialysis in 17 infants. Fourteen boys and 3 girls, aged 2.7-22.5 months, were on continuous ambulatory peritoneal dialysis (CAPD) for 215.1 months with 5 infants on continuous cycling peritoneal dialysis (CCPD) for 71.2 months subsequently. Analysis of growth and biochemical data on 16 CAPD and 5 CCPD patients showed poor linear growth despite improved weight gain, high incidence of developmental delay, tendency to lower plasma albumin, phosphate and sodium and increased plasma cholesterol and triglyceride levels. Overall chronic peritoneal dialysis proved to be a possible and effective means of dialysis in infants. PMID- 1723352 TI - Naturally occurring free phosphotyrosine in human liver. AB - We report an endogenous tyrosine-driven phosphorylating activity in human liver extracts. The detection is achieved after selective enrichment of soluble components present in a post-mitochondrial supernatant fraction (PMS) while measuring in vitro kinase activity. A putative functional role is inferred from the competence displayed by exogenous free target amino acids when added to the reaction. We demonstrate that exogenous tyrosine is specifically phosphorylated. In view of the close association between protein phosphorylation and cell function, our observations broader the scope of interpretation for the pivotal role phosphoamino acids might have in cell metabolism. PMID- 1723353 TI - Endogenous opioid peptides and blood pressure regulation during controlled, stepwise hemorrhagic hypotension. AB - In the present study, the role of the endogenous opioid peptide systems in the regulation of blood pressure during standardized, stepwise hemorrhagic hypotension was investigated in anesthetized rats. Central as well as peripheral administration of naloxone resulted in an increase in the bleeding volumes required to reduce blood pressure. Bleeding volumes also increased after the peripheral injection of naloxone methobromide, an analog of naloxone that does not readily cross the blood-brain barrier. Following central administration of antisera against beta- and alpha-endorphin and dynorphin A(1-13), the amount of blood that had to be withdrawn to induce hypotension was elevated. In rats treated with an antiserum against [Met5] enkephalin or gamma-endorphin, bleeding volumes did not differ from those of rats treated with control serum. These data indicate that activation of central and possibly also of peripheral opiate receptors plays a role in the control of blood pressure during blood loss. Dynorphin A(1-13), beta- and alpha-endorphin, or closely related peptides might be the endogenous ligands for the receptors that are blocked by naloxone. PMID- 1723354 TI - Histamine release during intestinal ischemia-reperfusion: role of iron ions and hydrogen peroxide. AB - Reactive oxygen intermediates (ROI) play a major role in the mucosal damage developing during the reperfusion period following intestinal ischemia. We have shown previously that histamine (H) release is related to the ROI generated by xanthine oxidase during intestinal ischemia-reperfusion. The present study sought to determine the possible chain of events leading to H liberation. The artery supplying a segment of the ileum was occluded for 2 hr in 51 anesthetized dogs, and plasma levels of H were determined radioenzymatically in the venous effluent. Catalase was applied to scavenge hydrogen peroxide; dimethylsulfoxide and mannitol were used as hydroxyl radical scavengers; the role of catalytically active iron was assessed by using desferrioxamine. Pretreatment with either catalase or desferrioxamine, but not with dimethyl sulfoxide or mannitol, was effective in reducing the postocclusive H release. The results provide further in vivo evidence that ROI are causative agents in H liberation during reperfusion of the ischemic gut. Hydrogen peroxide can interact with catalytically active iron and generate highly reactive oxidants, which in turn are responsible for H release. The exact nature of these oxidants is still uncertain. PMID- 1723355 TI - Administration of the calcium agonist BAY K 8644 in endotoxic shock. AB - Septic shock is characterized by a decreased vascular tone and a depressed myocardial function. An impairment in cellular calcium availability has been incriminated in both phenomenons. The effects of BAY K 8644, a dihydropyridine derivative agent acting as a slow calcium-channel activator, were studied and compared to those of norepinephrine (NE) on an experimental endotoxin shock model in the dog. Thirty minutes after intravenous administration of E. coli endotoxin (3 mg/kg), fluid therapy with normal saline was initiated to restore and maintain pulmonary artery capillary wedge pressure at baseline level. In the first part of the study (8 dogs), the effects of increasing doses of 1, 2, 4, and 8 mcg/kg/min of BAY K 8644 were evaluated. BAY K 8644 administration resulted in an increase in mean arterial pressure (MAP) (65 +/- 16 to 116 +/- 24 mmHg, P less than 0.001), systemic vascular resistance (SVR) (1,451 +/- 526 to 2,632 +/- 804 dynes.sec.cm-5, P less than 0.01), and left ventricular stroke work (LVSW) (0.08 +/- 0.04 to 0.16 +/- 0.07 g.m/kg, P less than 0.05), without change in mean pulmonary artery pressure, cardiac output, O2 transport, or O2 consumption (VO2). In the second part of the study (10 dogs), BAY K 8644 and NE were administered in a randomized order at increasing doses to achieve an identical increase in MAP. For a 20 mmHg increase in MAP, BAY K 8644 increased more SVR than NE (1,284 +/- 299 to 1,717 +/- 551 vs. 1,415 +/- 312 to 1,470 +/- 603 dynes.sec.cm-5, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723356 TI - Absence of increased urinary excretion of adenosine-deaminase-binding protein by patients with chronic renal tubular malfunction. AB - The urinary excretion of adenosine-deaminase-binding protein, a constituent of the brush border of proximal renal tubule cells, has been investigated in 39 patients with disorders associated with malfunction of the renal tubules, and its excretion has been compared with that of two low molecular mass plasma proteins and an enzyme derived from renal tubular cells. None of the 36 patients with disorders associated with chronic renal tubular malfunction were found to be excreting significantly increased quantities of adenosine-deaminase-binding protein but 30 had increased excretion of retinol-binding protein, alpha 1 microglobulin, or N-acetyl-beta-D-glucosaminidase. Measurement of urinary adenosine-deaminase-binding protein may be useful in the assessment of acute renal tubular injuries but it is not of value in the detection of chronic renal tubular disorders. PMID- 1723357 TI - A sensitive fluorescence assay for quantitation of fludarabine and metabolites in biological fluids. AB - Rapid and quantitative dephosphorylation of the new anticancer nucleotide analogue fludarabine phosphate to its nucleoside 9-beta-D-arabinofuranosyl-2 fluoroadenine (F-ara-A) renders this metabolite the target for pharmacologic investigations. At clinically effective doses of fludarabine phosphate (18-30 mg/m2 per day) comprehensive pharmacokinetic analysis of F-ara-A has been limited by the sensitivity of UV based HPLC assays. To address this problem we developed a sensitive test based on the condensation of F-ara-A with chloroacetaldehyde to form the fluorescent derivative, arabinosyl-1,N6-etheno-isoguanine. Combined with a solid-phase extraction step prior to derivatization and separation of the reaction products by reverse-phase HPLC, this assay had a quantitation limit of 2 pmol F-ara-A per ml plasma. Slightly modified, the system was also applicable to urine specimens, with a quantitation limit of 1 nmol F-ara-A per ml urine. PMID- 1723358 TI - Antibodies to dietary antigens in rheumatoid arthritis--possible molecular mimicry mechanism. AB - Antibodies in serum from some patients with rheumatoid arthritis, recognize bovine albumin present in the milk, as determined by immunoprecipitation analysis from 125I-milk extracts. This antigen was also immunoprecipitated from bovine sera. These and ELISA studies showed that BSA is preferentially recognized over other proteins present in the milk. Panel studies demonstrated that although the average reactivity for BSA was high, only one third of the sera tested displayed a reactivity above the mean. The possibility of a molecular mimicry mechanism in RA between this food antigen and other human antigens was investigated. A sequence alignment analysis showed that the residues 141-157 of bovine albumin significantly differed from the corresponding fragment of human albumin, but were highly homologous with human collagen type I, C1q and vitamin D binding protein. In support of the immunogenicity of this fragment, we found that representative RA sera displayed a specific reactivity for a synthetic peptide containing the BSA residues responsible for the homology. Furthermore, most of the epitopes recognized on BSA by the RA sera seem to be conformationally dependent as heat denaturation or reduction followed by alkylation lead to a diminished recognition. PMID- 1723359 TI - In vitro stability of human atrial natriuretic peptide (h-ANP). PMID- 1723360 TI - Selective decontamination of the digestive tract. Theoretical and practical treatment recommendations. PMID- 1723361 TI - 5-HT3 receptor antagonists. An overview of their present status and future potential in cancer therapy-induced emesis. AB - The serotonin (5-hydroxytryptamine, 5-HT) antagonists, which bind at the type 3 receptor (5-HT3 receptor), have been evaluated in several preclinical models and found to be effective in alleviating cancer therapy-related emesis. The antiemetic efficacy of ondansetron (GRF-38032F, odanserin), granisetron (BRL 43694), tropisetron (ICS-205930), MDL-72222 and MDL-73147EF, batanopride (BMY 25801-01) and several others is at various stages of investigation. Ondansetron is currently marketed in several countries and the same will soon be true for granisetron. At this stage it is not yet possible to evaluate the comparative efficacy of each of these compounds, although recent preclinical data reveal some differences in the affinity of these compounds, for other receptors. Side effects related to these agents have been minor, consisting mainly of slight headaches; possible rises in liver enzymes related to some compounds need further evaluation. Future studies will need to determine the exact role of 5-HT3 antagonists, although their cost may confine their use to patients at high risk for side effects from metoclopramide. PMID- 1723363 TI - Therapeutic dilemmas in external ocular diseases. AB - Three ocular conditions continue to pose therapeutic dilemmas for the practising clinician. Acanthamoeba keratitis, which presents with ocular pain, redness, tearing, photophobia and lid oedema, should be considered in any chronic, progressive corneal ulceration that is unresponsive to conventional treatment. Although the best treatment for this infection has yet to be defined, surgery should be reserved for those patients with progressive destructive disease or corneal penetration. Topical antibiotics and oral ketoconazole may be beneficial, as may surgical debridement in conjunction with topical antibiotic-antifungal combinations. However, since more than two-thirds of reported cases involve contact lens wearers, patients should be instructed as to the importance of regular lens care regimens. Giant papillary conjunctivitis occurs more frequently in soft contact lens wearers than in those wearing hard lenses, but may also occur in association with ocular prostheses, cataract surgery and corneal transplants. Symptoms of increased lens awareness, mucus accumulation, itching and blurred vision occur. Stopping use of contact lenses usually improves or eliminates these irritating effects, but is not always practical. Thus, resolution or improvement of symptoms while the patient continues to wear contact lenses is desirable, making lens hygiene essential in treatment. Pharmacological treatment includes the use of topical corticosteroids and agents that stabilise mast cells, such as cromolyn sodium. The dry eye syndrome can occur alone or as a part of Sjogren's syndrome. The irritation, redness, and other symptoms associated with ocular dryness are usually treated by preparations of either mucomimetics, polyvinyl alcohol or cellulose derivatives, which provide moisture and prevent evaporation from the surface of the eye. PMID- 1723364 TI - Drug treatment of colorectal cancer. Current status. AB - Drug therapy is most often used in colorectal cancer for palliation of metastatic disease. Current data also support the use of adjuvant chemotherapy following complete surgical resection in patients with locoregional lymph node metastases. The agent most widely used in the treatment of colorectal cancer is the antimetabolite fluorouracil (5-fluorouracil; 5-FU). This fluoridated pyrimidine has been available for over 30 years, yet to date no other single agent has proven to be more efficacious. Controversy exists about the most desirable schedule for administration of fluorouracil. Efforts have been made to improve upon its therapeutic index and efficacy by using the concept of biomodulation, in which chemicals which are not themselves active antineoplastic agents against colorectal cancer are administered with fluorouracil in an attempt to enhance the sensitivity of the cancer cell to fluorouracil. Biomodulation agents currently in use in clinical practice include leucovorin (calcium folinate), methotrexate, and interferon-alpha. Other biomodulation strategies are currently under investigation. Adding putatively active antineoplastic agents to fluorouracil to form combination chemotherapy regimens has not yielded convincingly superior results to treatment with fluorouracil alone, and the toxicities of many of these combination regimens have been formidable. Secondary therapies following failure of fluorouracil-based regimens have been similarly disappointing. Current areas of investigation into the chemotherapy of colorectal cancer include development of new agents, locoregional administration of chemotherapy, and manipulation of intrinsic drug resistance mechanisms of the cancer cells. PMID- 1723365 TI - Treatment and prophylaxis of Pneumocystis carinii pneumonia in AIDS patients. AB - Pneumocystis carinii pneumonia (PCP) is seen in people with a defect in cell mediated immunity. Today the most common cause for this is the Acquired Immunodeficiency Syndrome (AIDS). There have been some remarkable advances recently in the development of new drug regimens to combat this otherwise fatal infection. Although cotrimoxazole (trimethoprim-sulfamethoxazole) is still the drug of first choice it cannot be tolerated by a significant proportion of patients, and therapies such as pentamidine (pentamidine-isethionate) [intravenous or nebulised], dapsone-trimethoprim, eflornithine (DFMO; difluoromethylornithine), trimetrexate, and clindamycin-primaquine are finding therapeutic niches. The major advantage in these other agents is not improved efficacy but different toxicity profiles, enabling therapy to be most appropriately tailored to individual patients' conditions. Although the majority of patients should now survive an attack of PCP, relapses will occur if prophylaxis is not used. There is also the capacity to predict accurately which patients are at risk for this pneumonia and prevent it through the use of chemoprophylaxis. These advances in the treatment and prevention of PCP, together with anti-retroviral therapy, mean that this is an area of AIDS management that has resulted in improved long term survival. PMID- 1723366 TI - Ivermectin. A review of its antifilarial activity, pharmacokinetic properties and clinical efficacy in onchocerciasis. AB - Ivermectin, a derivative of avermectin B, is an orally effective microfilaricidal agent. It is the current drug of choice for treating patients infected with the nematode Onchocerca volvulus, which is a major cause of blindness in inhabitants of some tropical areas. Ivermectin is administered orally as a single dose of 150 micrograms/kg given annually. Skin and ocular microfilarial counts are dramatically reduced after the first dose, with some evidence for a resulting decrease in transmission of infection by the blackfly vector. With the exception of rare serious reactions such as severe systemic postural hypotension, ivermectin is generally well tolerated. The drug has the clear advantages of ease of administration and better tolerability compared with diethylcarbamazine and suramin, agents previously used to treat onchocerciasis. Thus, ivermectin is suitable for inclusion in mass treatment programmes and is the best therapeutic option presently available to combat onchocerciasis. As such it provides hope for many thousands of people at risk of becoming blind, and represents a major contribution to tropical medicine. PMID- 1723362 TI - Drug antioxidant effects. A basis for drug selection? AB - A free radical is any species capable of independent existence that contains one or more unpaired electrons. Free radical reactions have been implicated in the pathology of more than 50 human diseases. Radicals and other reactive oxygen species are formed constantly in the human body, both by deliberate synthesis (e.g. by activated phagocytes) and by chemical side-reactions. They are removed by enzymic and nonenzymic antioxidant defence systems. Oxidative stress, occurring when antioxidant defences are inadequate, can damage lipids, proteins, carbohydrates and DNA. A few clinical conditions are caused by oxidative stress, but more often the stress results from the disease. Sometimes it then makes a significant contribution to the disease pathology, and sometimes it does not. Several antioxidants are available for therapeutic use. They include molecules naturally present in the body [superoxide dismutase (SOD), alpha-tocopherol, glutathione and its precursors, ascorbic acid, adenosine, lactoferrin and carotenoids] as well as synthetic antioxidants [such as thiols, ebselen (PZ51), xanthine oxidase inhibitors, inhibitors of phagocyte function, iron ion chelators and probucol]. The therapeutic efficacy of SOD, alpha-tocopherol and ascorbic acid in the treatment of human disease is generally unimpressive to date although dietary deficiencies of the last two molecules should certainly be avoided. Xanthine oxidase inhibitors may be of limited relevance as antioxidants for human use. Exciting preliminary results with probucol (antiatherosclerosis), ebselen (anti-inflammatory), and iron ion chelators (in thalassaemia, leukaemia, malaria, stroke, traumatic brain injury and haemorrhagic shock) need to be confirmed by controlled clinical trials. Clinical testing of N-acetylcysteine in HIV-1 positive subjects may also be merited. A few drugs already in clinical use may have some antioxidant properties, but this ability is not widespread and drug derived radicals may occasionally cause significant damage. PMID- 1723367 TI - Naftifine. A review of its antimicrobial activity and therapeutic use in superficial dermatomycoses. AB - Naftifine is an allylamine derivative for topical administration with a mechanism of action distinct from that of other classes of antifungal agents. It inhibits squalene epoxidase and may have certain anti-inflammatory properties, but its precise mechanism of action is as yet unclear. In vitro, naftifine has potent fungistatic and fungicidal activity against dermatophytes. This correlates well with its clinical and mycological activity in patients with dermatophytoses. There is improvement in clinical symptoms and overall therapeutic success after a 2- to 5-week course of therapy in a high percentage of patients (usually over 80%) with tinea cruris or corporis, and in a slightly smaller percentage of those with tinea pedis. Naftifine is moderately active in vitro against moulds, but is generally less active against yeasts, including Candida albicans. However, it has proved reasonably effective in the treatment of patients with cutaneous candidiasis, although further studies are necessary to establish its place in therapy for this indication. In view of its good local tolerability, absence of systemic adverse effects, novel mechanism of action and effectiveness with once daily application, naftifine offers a useful addition to available pharmaceutical options in patients with dermatomycoses. PMID- 1723368 TI - Doxacurium. A review of its pharmacology and clinical potential in anaesthesia. AB - Doxacurium, a benzylisoquinolinium diester, provides nondepolarising neuromuscular blockade (and thus surgical relaxation), without vagolytic or sympathomimetic effects. At equipotent doses, the duration of action of doxacurium is similar to that of the long-acting neuromuscular blocker pancuronium. Supplemental doses of doxacurium administered at approximately 25% recovery reliably increase the depth and duration of neuromuscular blockade without accumulation. Although patients will recover spontaneously from doxacurium neuromuscular blockade, pharmacological reversal with neostigmine is recommended in line with accepted clinical practice, and permits acceleration of recovery when necessary. Doxacurium may also be used to facilitate endotracheal intubation. However, even with large doses onset of action is delayed, suggesting more rapidly acting neuromuscular blocking agents will be preferred when rapid sequence induction and intubation is required. Doxacurium has been successfully used in children, the elderly, and in patients with renal failure, hepatic failure, or cardiovascular disease. The absence of clinically significant effects on cardiovascular parameters and relative lack of histamine release observed in clinical trials indicates that doxacurium is suitable for use in high-risk patient groups. Thus, while there are only a limited number of studies comparing doxacurium with other nondepolarising neuromuscular blockers, particularly other 'cardiostable' agents, its ability to produce sustained effective muscle relaxation without significantly affecting the cardiovascular system or inducing histamine release would seem to make it a preferred agent for surgery of longer duration, particularly in high-risk patient groups. PMID- 1723370 TI - Do calcium antagonists have a place in the treatment of mood disorders? PMID- 1723369 TI - Idarubicin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in the chemotherapy of cancer. AB - Idarubicin is a 4-demethoxy-anthracycline analogue of daunorubicin which, when administered intravenously in combination with cytarabine, has therapeutic efficacy superior to that of standard induction and salvage treatment regimens in acute myelogenous leukaemia. Idarubicin alone or in combination regimens has also been effective in limited studies in patients with other acute leukaemias, advanced breast cancer, multiple myeloma and non-Hodgkin's lymphoma. Idarubicin is more lipophilic than its parent drug daunorubicin. It intercalates DNA, induces DNA strand breaks and delays cell cycle progression. Leucopenia is often dose-limiting in patients with solid tumours treated with idarubicin, and most other adverse effects are similar in incidence and severity to those experienced with other anthracycline cytotoxic agents, except for alopecia which appears to occur with a reduced incidence and severity. Cardiotoxic effects have been reported with idarubicin as with other anthracyclines, but animal data and preliminary clinical findings suggest the possibility of reduced cardiotoxicity with idarubicin--a potentially important advantage if confirmed on further study. A maximum cumulative dose of idarubicin beyond which the incidence of cardiotoxicity rapidly increases has not been determined. Thus, intravenous idarubicin is a useful alternative to other anthracyclines (particularly in combination with cytarabine) in the treatment of acute myelogenous leukaemia, and there is some evidence that it is less cardiotoxic than other anthracycline drugs. Further studies are required to establish the use of intravenous idarubicin as a replacement for other intravenous anthracyclines, especially its effect on response duration and survival, and to confirm the evidence of reduced cardiotoxic effects. The role of idarubicin as consolidation and maintenance therapy for various malignancies requires further investigation, as does the potential use of oral idarubicin which is currently under development. PMID- 1723371 TI - The use of antidepressants in the treatment of chronic pain. A review of the current evidence. AB - In the last 30 years antidepressant drugs have been used increasingly in the treatment of patients with chronic pain. This article reviews the results of some 40 placebo-controlled studies. It is difficult to make comparisons between the various studies because they often differ in terms of pain conditions, patient selection, antidepressant drug used, dosages, trial design, etc. However, in spite of this heterogeneity and other methodological problems it is clear that a wide range of pain conditions are responsive to antidepressant drug treatment, in particular: headache, migraine, facial pain, neurogenic pain, fibrositis, and probably arthritis and rheumatoid arthritis. More data need to be gathered in cancer pain, and in other conditions such as low back pain for which no, or very limited, effect has been shown. The beneficial effects of antidepressant drugs is in most cases of a mild to moderate degree, some time lag is necessary before it is completely manifest, and it tends to persist over time if drug treatment is continued in the long term. Strong evidence of efficacy is not evident for all the antidepressants, and there are probably significant differences in this respect between various drugs. The effect of a drug on pain does not seem necessarily to be related to its effect on mood. Further studies are needed to clarify this topic, and it will be necessary to examine specific pain conditions, compare different antidepressants, with reference to each other and to placebo, further investigate the role of drug plasma concentrations and control for the presence of concomitant psychiatric disturbances and for organic lesions responsible for the pain symptomatology. PMID- 1723372 TI - The use of interferon-alpha in virus infections. AB - The interferons (IFN) act too slowly to arrest acute viral infections, but interferon-alpha (IFN alpha) preparations have proved useful in some chronic infections and will clearly be used increasingly in these in the future. In the preparations derived from human leucocytes or cultured B lymphoblastoid cells, which are in routine clinical use, mixtures of a number of distinct subtypes of human IFN alpha have been identified. There are also 3 slightly different versions of the same single subtype, IFN alpha-2, made by recombinant DNA procedures in bacteria. IFN alpha preparations are injected intramuscularly or subcutaneously. Dose-related side effects are common but usually tolerable, but prolonged treatment may cause increasing fatigue and depression. Some patients form neutralising antibodies which block the effects of the IFN; these appear to be relatively more common after recombinant IFN alpha-2 than after IFN derived from human cells. Given intranasally, IFN alpha can prevent a subsequent experimental rhinovirus infection, or the spread of natural colds within a family. Repeated administration progressively damages the nasal mucosa, so that long term prophylaxis is not possible. IFN alpha has proved useful in patients with papillomavirus warts of the larynx, ano-genital region (condyloma acuminata) and skin (common warts). Treatment regimens remain to be optimised and are likely to include surgery or other treatments. IFN alpha and zidovudine (azidothymidine) synergistically inhibit the growth of HIV in vitro, and combination are on trial in patients with early AIDS. Very large doses of IFN alpha are effective against Kaposi's sarcoma in some AIDS patients. In chronic hepatitis B, continuing virus replication may lead to cirrhosis or primary liver cancer. Earlier clinical trials with IFN alpha gave inconclusive results, but recent large studies have confirmed that 25 to 40% of patients obtain benefit; this probably results from both the antiviral and the immunomodulatory effects of IFN alpha. In patients with chronic hepatitis C, the biochemical markers usually improve rapidly during IFN alpha administration, but relapse if treatment is stopped after only a few months; to increase the chances of sustained cure, the treatment period is now being prolonged. PMID- 1723373 TI - Treatment of Wilms' tumour. Current recommendations. AB - Wilms' tumour (nephroblastoma, renal embryoma) is the fifth most common paediatric malignancy, arising from the embryonal tissue of kidneys and first formally described by Max Wilms in his classic 1899 monograph. Until the early part of this century, Wilms' tumour was associated with a less than 20% survival rate. The current survival rate exceeds 80%, primarily due to large multi institutional trials such as the National Wilms' Tumor Study (NWTS). These studies have refined and defined the roles of surgery, chemotherapy, and radiation in treating Wilms' tumour, based on staging and histology. The dramatic improvement in the prognosis for children with Wilms' tumour, especially over the past 20 years, represents a landmark achievement in the history of paediatric oncology. Specific treatment recommendations are based on the current National Wilms' Tumor Study IV schema. Stages I and II favourable histology patients do not receive radiotherapy, but are treated postoperatively with 'pulsed' or 'conventional' dactinomycin and vincristine; stage III favourable histology requires postoperative abdominal radiotherapy followed by triple agent, 'conventional' or 'pulsed' chemotherapy (dactinomycin, doxorubicin and vincristine). Patients with stage IV favourable histology, stages II to IV anaplastic, clear cell or rhabdoid histology, are treated similarly with aggressive triple-agent chemotherapy, with the addition of radiotherapy to selected sites. Recurrent and adult Wilms' tumours have poor prognoses and are treated with aggressive surgery, radiotherapy and chemotherapy. PMID- 1723374 TI - Cyclophosphamide toxicity. Characterising and avoiding the problem. AB - Cyclophosphamide, an orally active alkylating agent, is widely used to treat a variety of malignant and nonmalignant disorders. Although it has some tumour selectivity, it also possesses a wide spectrum of toxicities. The requirement of metabolic activation before cyclophosphamide exerts either its therapeutic or toxic effects is well established, but has not led to effective counter-measures. Clinically, damage to the bladder (haemorrhagic cystitis), immunosuppression (when not desired) and alopecia are the most significant toxicities associated with cyclophosphamide. Cardiotoxicity is also a possibility when very high doses are given. Preventing these toxicities has focused on modifications of the treatment regimens and, in the case of haemorrhagic cystitis, the administration of a drug which is excreted in the urine where it inactivates the bladder-toxic species. As treatment regimens for cancer become more effective in prolonging a patient's life, and as cyclophosphamide receives increasing use for nonmalignant disorders, the potential for cyclophosphamide-induced cancers, particularly in the bladder, must be recognised. Although the toxicities associated with cyclophosphamide are serious, this agent remains a highly effective drug in many situations. Research on the pathways which play an important role in activating this drug may improve our ability to target particular diseases and decrease unwanted side effects. PMID- 1723375 TI - Practical treatment recommendations for pharmacotherapy of Behcet's syndrome. AB - Behcet's syndrome is a disease of unknown aetiology classified among the vasculitides. It runs a course of exacerbations and remissions which gradually abate with time. Eye disease, the most frequent cause of serious morbidity, may lead to blindness in 20% of those affected. The syndrome may occasionally be fatal due to vasculitis leading to arterial occlusion, ruptured arterial aneurysms or pulmonary vasculitis, or involvement of the central nervous system. Immunosuppressive drugs have been shown to be moderately successful in inducing and maintaining remissions. Azathioprine at a dose of 2.5 mg/kg/day has been shown to control the progression of existing, and the development of new, eye disease. Cyclosporin A is also beneficial in controlling active eye disease and although it has a more rapid action than azathioprine, its toxicity limits its long term use. Colchicine, although widely prescribed, has been shown in a controlled trial to be effective only in reducing the development of erythema nodosum and arthralgia. Systemic corticosteroids, once widely used, are now reserved only for the most severe cases of inflammatory eye disease and vasculitis, where they are frequently used as intravenous pulse therapy. Local mydriatics are used to prevent synechiae. Local treatment with corticosteroids, sometimes in conjunction with antibiotics, control oral and genital ulcers which may also be controlled by immunosuppressives, which are reserved for the most severe cases. Thrombophlebitis usually only requires antiplatelet agents, whereas arteritis is treated conventionally with a combination of corticosteroids and immunosuppressive drugs, usually cyclophosphamide. PMID- 1723376 TI - Granisetron. A review of its pharmacological properties and therapeutic use as an antiemetic. AB - Granisetron (BRL 43694) is a highly selective 5-HT3 receptor antagonist which possesses significant antiemetic activity, likely mediated through antagonism of 5-HT3 receptors on abdominal vagal afferents and possibly in or near the chemoreceptor trigger zone. Clinical trials in cancer patients demonstrate that, compared with placebo, granisetron significantly reduces the incidence of nausea and vomiting for 24 hours after administration of high-dose cisplatin. In large comparative trials, 70% of patients who received granisetron prior to cisplatin or other chemotherapy experienced complete inhibition of vomiting with little or no nausea for 24 hours after antineoplastic administration; these results were similar to those obtained with high-dose metoclopramide plus dexamethasone, and superior to a combination of chlorpromazine plus dexamethasone, or prochlorperazine plus dexamethasone, or methylprednisolone monotherapy. The most frequently reported adverse event associated with granisetron administration is headache which occurs in about 10 to 15% of patients while constipation, somnolence, diarrhoea and minor transient changes in blood pressure have been reported less frequently. Extrapyramidal effects, which can occur with high-dose metoclopramide and may be a limiting factor in its use, have not been noted with granisetron administration. Thus, granisetron is an effective, well tolerated and easily administered agent for the prophylaxis of nausea and vomiting induced by cancer chemotherapy which appears to be devoid of extrapyramidal side effects associated with metoclopramide. As a member of a new class of drugs, the selective 5-HT3 receptor antagonists, granisetron provides the medical oncologist with a new, potentially more acceptable antiemetic therapy. PMID- 1723378 TI - Colfosceril palmitate. A review of the therapeutic efficacy and clinical tolerability of a synthetic surfactant preparation (Exosurf Neonatal) in neonatal respiratory distress syndrome. AB - Colfosceril palmitate (dipalmitoylphosphatidylcholine) is the primary surface active agent of natural lung surfactant and the major constituent of exogenous surface replacement preparations. Exogenous surfactants derived from either natural (i.e. animal and human) or synthetic sources are indicated for the prophylaxis and treatment of neonatal respiratory distress syndrome. One of the synthetic surfactants, Exosurf Neonatal, is the focus of this review. This preparation is composed of colfosceril palmitate plus cetyl alcohol and tyloxapol, which facilitate rapid spreading and adsorption of the surface-active agent at the air-alveolar interface. For review purposes, this preparation is referred to only as colfosceril palmitate. Comparative trials with air placebo have shown that colfosceril palmitate improves clinical outcome in infants weighing greater than 700g at birth by reducing mortality and increasing the number of infants who survive without bronchopulmonary dysplasia. It also reduces the number of deaths from respiratory distress syndrome and decreases the incidence of air leak events such as pulmonary interstitial emphysema and pneumothorax. Although colfosceril palmitate itself is very well tolerated and does not increase the incidence of most complications of prematurity or of respiratory distress syndrome, its use is associated with a higher incidence of apnoea of prematurity and pulmonary haemorrhage compared with air placebo, possibly because of earlier extubation of surfactant-treated infants following an improved clinical course and decreased pulmonary vascular resistance secondary to improved ventilation, respectively. Colfosceril palmitate thus has an established efficacy in the prophylaxis and treatment of premature infants with respiratory distress syndrome. Ongoing trials may identify whether prophylactic or rescue administration of the surfactant preparation is the preferred approach and whether different dosage regimens or different administration techniques impart greater therapeutic efficacy. Importantly, it also remains to be determined whether any of the available surfactant preparations, including Exosurf Neonatal, will provide distinct therapeutic advantages over the others. PMID- 1723381 TI - Pulp revascularization in reimplanted immature monkey incisors--predictability and the effect of antibiotic systemic prophylaxis. AB - In 32 monkeys 105 immature maxillary incisors were extracted and reimplanted either immediately or after 30 or 60 min wet or dry storage. Of the monkeys, 17 (group I) did not receive and 15 (group II) received prophylactic treatment with 4 mg/kg doxycycline before extraction and 2 mg/kg for 5 d after reimplantation. The observation time varied from 6 to 8 weeks. After being histologically processed, the material was evaluated with respect to the amount of vital tissue and presence of micro-organisms in the pulpal lumen. A comparison revealed no difference in the results between the groups. The results were therefore pooled and statistically analysed with respect to the significance of apical foramen width, extra-alveolar time, wet or dry storage and presence of micro-organisms in the pulpal lumen for the occurrence of complete pulp revascularization (CPR). The overall frequency of CPR was 18%. Log-linear analyses (SAS, 1985) of the material as a whole or of separate parameters consistently revealed a relationship between presence of micro-organisms and absence of CPR (P = 0.0001). A higher frequency of CPR and a lower frequency of micro-organisms (P = 0.05) was found only for the group of immediately reimplanted teeth. The presence of micro-organisms could be explained for 61 teeth. In 27 of these, blood clots containing bacteria in the apical portion of pulpal lumen indicated contamination during the extra-alveolar time, while in 34, the micro-organisms originated from plaque covered mechanical damage in the cervical part of the root surface. PMID- 1723380 TI - Cellular but not humoral antibacterial activity of earthworms is inhibited by Aroclor 1254. AB - Earthworms, Eisenia fetida andrei and Lumbricus terrestris, exposed to Aroclor 1254, followed by infestation with Aeromonas hydrophila, elicited two types of responses. First, in E. fetida, there was no change in the LD50 nor in the in vitro antibacterial growth capacity of cell-free coelomic fluid. Thus, Aroclor exerts no influence on antibacterial proteins nor on the chloragogue cells responsible for their release. Second, in L. terrestris, both a high LD50 value and no antibacterial activity indicate that A. hydrophila was not pathogenic. The 10(4) times higher sensitivity of exposed L. terrestris suggests that Aroclor inhibits leukocyte activity since E. fetida eliminates nonpathogenic bacteria by a cellular mechanism. PMID- 1723379 TI - Salmeterol xinafoate. A review of its pharmacological properties and therapeutic potential in reversible obstructive airways disease. AB - Salmeterol xinafoate, like salbutamol (albuterol), is a saligenin derivative, and a selective beta 2-adrenoceptor agonist. It produces bronchodilation for at least 12 hours following inhalation of a single 50 micrograms dose. Salmeterol is intended for regular twice-daily treatment of reversible airways obstruction and not for immediate symptomatic relief, and when used in this manner, 50 micrograms twice daily is more effective than salbutamol 200 micrograms or terbutaline 500 micrograms administered 4 times daily, or individually titrated oral doses of theophylline in improving objective and subjective criteria of efficacy in patients with mild to moderate asthma. Salmeterol 100 micrograms inhaled twice daily may provide better control than the lower dose in patients with severe asthma. The long duration of effect of salmeterol makes it particularly suitable for treating patients with nocturnal asthma in whom it improves sleep quality. The place of salmeterol, like that of other beta 2-adrenoceptor agonists used regularly in the treatment of asthma, is being debated. Patients in need of regular beta 2-agonist therapy should also be regarded as candidates for inhaled corticosteroids to counteract underlying inflammation. Thus, salmeterol may be particularly useful in patients requiring regular treatment with beta 2-agonists for nocturnal asthma and results of trials in progress involving large numbers of patients are awaited with interest. PMID- 1723382 TI - Effect of topical application of doxycycline on pulp revascularization and periodontal healing in reimplanted monkey incisors. AB - Maxillary incisors in 47 monkeys, 54 in the experimental group (I) and 117 in the control group (II), were extracted and reimplanted, either immediately or after 30 or 60 min wet or dry storage. Incisors in the experimental group I were additionally kept 5 min in a suspension of 1 mg doxycycline in 20 ml physiologic saline, freshly prepared for each of the 15 animals before reimplantation. The observation time varied from 6 to 8 weeks. The teeth were removed in tissue blocks, histologically processed and evaluated for occurrence of complete pulp revascularization (CPR), presence of the micro-organisms in the pulpal lumen and ankylosis or inflammatory root resorption. Then the results were statistically evaluated, using log-linear analyses and chi-square tests (SAS, 1985) for the comparisons between group I and group II. These analyses revealed that topical application of doxycycline increased the frequency of complete pulp revascularization (P less than 0.002) and decreased the frequency of micro organisms in the pulpal lumen (P less than 0.001). Furthermore, the frequencies of ankylosis (P less than 0.05) and inflammatory root resorption (P less than 0.001) were also decreased compared with the control group of teeth. It was concluded that the effect of topical treatment with doxycycline was most probably exerted on the micro-organisms that contaminated root surface during the extra alveolar time; contamination of necrotic pulp tissue from the mechanical damage in the cervical part of the root surface was not affected. PMID- 1723383 TI - Prostates, pates, and pimples. The potential medical uses of steroid 5 alpha reductase inhibitors. AB - The steroid 5 alpha-reductase enzyme is responsible for the formation of DHT from testosterone. DHT has been the major androgen implicated in the pathogenesis of benign prostatic hyperplasia, male pattern baldness, acne, and idiopathic female hirsutism. Although specific inhibitors of 5 alpha-reductase are not yet generally available for human use, it is expected that they will become available within the next several years. Based on biochemical, histologic, and anatomic information from animals given 5 alpha-reductase inhibitors, preliminary data on their use in humans, and knowledge gained from men with the inherited 5 alpha reductase deficiency, it is expected that these 5 alpha-reductase inhibitors may have a major role in the medical management of benign prostatic hyperplasia. In addition, it is possible that these compounds will hold promise for the prevention of male pattern baldness and for the treatment of resistant acne and idiopathic hirsutism. PMID- 1723384 TI - HCV infection, hepatic HLA display and composition of the mononuclear cell inflammatory infiltrate in chronic alcoholic liver disease. AB - Viral infection may play a role in alcoholic liver disease with histological features of chronic active hepatitis (CAH). Human leucocyte antigen (HLA) hepatocellular display is supposed to allow HLA-restricted T-lymphocyte cytotoxicity in chronic viral hepatitis. We studied the presence of serum anti hepatitis C virus (HCV) antibodies, the hepatic HLA display and the composition of the mononuclear cell infiltrate in 16 patients with alcoholic liver disease and histological features of CAH and in 11 patients with alcohol-related degenerative changes. All patients were negative for hepatitis B virus (HBV) markers. Anti-HCV were tested by microplate ELISA. Class I HLA A, B, class II HLA DR, lymphocytes pan T, T helper/inducer, T suppressor/cytotoxic, B, and K NK cells were stained on liver cryostat sections by monoclonal antibodies and double indirect immunoperoxidase. Anti-HCV were present in all the patients with features of CAH and absent in those with only degenerative changes. In livers with features of CAH the mononuclear cell infiltrate consisted largely of T lymphocytes with marked prevalence of suppressor/cytotoxic cells in periportal and lobular areas. K NK cells were rare. Class I HLA, diffusely displayed on bile duct epithelium and on sinusoidal cells, also appeared on liver cells in the areas of periportal and lobular necrosis, namely on the hepatocytes in close contact with suppressor/cytotoxic T cells. In livers with only degenerative changes class I HLA were diffusely displayed on bile duct epithelium and on sinusoidal cells but absent on the hepatocytes. In all the specimens HLA DR antigens were expressed on sinusoidal and inflammatory cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723385 TI - Lipoproteins of human peripheral lymph. Apolipoprotein AI-containing lipoprotein with alpha-2 electrophoretic mobility. AB - Evidence from diverse sources has implicated a central role of apolipoprotein AI (apo AI), the most abundant protein of plasma high-density lipoproteins, in the transport of cholesterol from peripheral tissues to the liver (reverse cholesterol transport). Particles containing only apo AI appear to be more effective as cholesterol acceptors in tissue culture than do particles which also contain apo AII. The apo AI-containing lipoproteins of plasma have been extensively studied, but there is less information on those in tissue fluids, to which most peripheral cells are exposed. In the present study the heterogeneity of apo AI-containing particles in human peripheral lymph, collected from the dorsum of the foot, has been examined by starch block electrophoresis, exclusion chromatography and immunoelectrophoresis. The apo AI-containing particles of lymph were found to be more variable in both electrophoretic mobility and size than those of plasma from the same subjects. Of particular interest was a subpopulation which migrated on electrophoresis with the same mobility as alpha-2 macroglobulin. This fraction accounted for approximately 7% (range: 4-12%; n = 5) of lymph apo AI, contained no immunodetectable apo AII, and by exclusion chromatography was composed of particles the size of, or smaller than, albumin. Such physicochemical properties suggest that these alpha-2 migrating particles may function as the principal primary acceptors of cell cholesterol in the extracellular matrix of human peripheral tissues. By isoelectric focusing, lymph apo AI was found to contain a higher proportion of more negatively charged isoforms than the apo AI of plasma. PMID- 1723377 TI - Ofloxacin. A reappraisal of its antimicrobial activity, pharmacology and therapeutic use. AB - Ofloxacin is a fluoroquinolone whose primary mechanism of action is inhibition of bacterial DNA gyrase. In vitro it has a broad spectrum of activity against aerobic Gram-negative and Gram-positive bacteria, although it is poorly active against anaerobes. Ofloxacin, unlike most other broad spectrum antibacterial drugs, can be administered orally as well as intravenously. Penetration into body tissues and fluids is highly efficient. Clinical trials with orally and intravenously administered ofloxacin have confirmed its potential for use in a wide range of infections, where it has generally proved as effective as standard treatments. Ofloxacin in well tolerated, and in comparison with other available fluoroquinolones is less likely to cause clinically relevant drug interactions. Ofloxacin thus offers a valuable oral treatment (with an option for intravenous administration if necessary) for use in a wide range of clinical infections, but with a particular advantage in more severe or chronic infections when recourse to parenteral broad spectrum agents would normally be required, thereby providing cost savings and additionally allowing outpatient treatment. PMID- 1723386 TI - Follitropin receptor down-regulation involves a cAMP-dependent post transcriptional decrease of receptor mRNA expression. AB - The regulation by FSH (follitropin; follicle-stimulating hormone) of FSH receptor mRNA and protein (FSH binding) was studied using cultured Sertoli cells isolated from 21-day-old rats. FSH induced a dose-dependent and almost complete down regulation of receptor mRNA at 4 h after addition of the hormone. At subsequent time points (16 h and later) the FSH receptor mRNA levels had returned close to control values. The effect of FSH was mimicked by dibutyryl cyclic AMP (dbcAMP) and forskolin, and the phosphodiesterase inhibitor methyl-isobutylxanthine (MIX) prolonged the FSH action. These findings indicate that the effect of FSH on its receptor mRNA was mediated by cAMP. A down-regulatory effect of FSH and dbcAMP on FSH receptor mRNA was also observed in the presence of the protein synthesis inhibitor cycloheximide, suggesting a direct effect of FSH/dbcAMP on the expression of the FSH receptor gene. Transcriptional run-on experiments revealed that FSH did not inhibit initiation of the FSH receptor gene; hence a post transcriptional mechanism is involved. Binding of 125I-FSH to the cultured Sertoli cells was rapidly (4 h) decreased when the cells were incubated with FSH or FSH in combination with MIX. This effect can be explained by ligand-induced receptor sequestration. In contrast, incubation of Sertoli cells with dbcAMP had no effect on binding of 125I-FSH after 4 h, but resulted in a 60% loss of FSH binding sites after 24 h, probably caused by decreased mRNA expression. In conclusion, FSH receptor down-regulation in Sertoli cells is effected not only by the well-documented ligand-induced loss of receptors from the plasma membrane, but also involves a cAMP-mediated decrease of FSH receptor mRNA through a post transcriptional mechanism. PMID- 1723387 TI - Relationship between structure and biological and protective activities of pertussis toxin. AB - The relationship between mouse-protective activities against aerosol and intracerebral (i.c.) challenge with virulent B. pertussis organisms and pertussis toxin (PT)-neutralizing activities against ADP-ribosylation (ADPR), CHO cell clustering (CC), leukocytosis-promoting (LP) and islet-activating (IA) activities of anti-PT antibody, was investigated using anti-PT mouse monoclonal antibodies (MAbs): ten anti-S1, four anti-S23, one anti-S2, two anti-S3, and three anti-S4 MAbs. All the anti-S1 MAbs neutralized ADPR activity of PT. Among them, six MAbs: 1B7, 1D7, 3F11, 10D6, 8G4 and E1E, showed mouse-protection in either an aerosol or i.c. challenge system. These protective MAbs neutralized both LP and IA activities but showed little or no neutralization against CC activity except for 1B7. All anti-S2 and/or S3 MAbs showed higher CC-neutralizing activity and protected the mice against the aerosol challenge, but very little against the i.c. challenge. The mouse-protection was not necessarily parallel to either PT neutralizing activity or CC-neutralizing activity. The protective MAbs against S1, S2 and S3 decreased the number of bacteria and the amount of pertussis toxin in the lungs of mice challenged with the aerosol. These results suggest that the role of the anti-PT antibody in mouse-protection is not only neutralization of PT but also prevention of bacterial growth in the respiratory tract. Four anti-S1 and two anti-S4 MAbs showed neither neutralization nor protection. Competitive ELISA using the biotinylated MAbs suggested that four anti-S1 MAbs: 1B7, 1D7, 3F11 and 10D6, which showed the highest and almost complete mouse-protection, may recognize the same or closely related epitope(s) or areas on PT. The foregoing results suggested that prediction of the protection of MAbs only by the neutralizing activity against any one of the PT activities must be difficult and that competition ELISA may be helpful for such predictions. PMID- 1723388 TI - Weissenbacher-Zweymuller syndrome: long-term follow-up of growth and psychomotor development. AB - A child with the distinguishing characteristics of Weissenbacher-Zweymuller syndrome (WZS), a rare syndrome characterized by multiple skeletal and radiological abnormalities, dwarfism and developmental delays, was followed from birth to eight years. Follow-up showed that the radiographic anomalies eventually disappeared, and that height, motor, cognitive and language development returned to normal by eight years of age. The child's normal development at school age supports the theory that WZS is a dysmaturational, rather than dysplastic, syndrome. Diagnosis of the syndrome at birth is essential to ensure proper management of the child and counselling for the parents. PMID- 1723389 TI - Regulation of gene expression by interferons. AB - IFNs are a family of proteins produced by various cells following stimulation by biological or synthetic inducers. The interaction with the membrane receptor is followed by the activation of the expression of particular genes that are responsible for their antiviral, antiproliferative and immunomodulatory properties. Initiation of transcription is an early and critical event in the control of eukaryotic gene expression. In this review we will briefly discuss the mechanisms exploited by IFNs to control at transcriptional level the expression of inducible genes. In particular we will focus on some characteristics if cis acting DNA elements that are located upstream from the initiation site for RNA transcription and of nuclear trans-acting factors that are required for modulation of gene expression by IFNs. PMID- 1723390 TI - Expression of micF involved in porin synthesis in Escherichia coli: two distinct cis-acting elements respectively regulate micF expression positively and negatively. AB - micF RNA, whose sequence is highly complementary to a 5'-portion of ompF mRNA, has been implicated in the osmoregulation and thermoregulation of the ompF porin gene in Escherichia coli. To define and characterize cis-acting regulatory regions upstream of the micF promoter, a series of deletions of the micF promoter fused to the lacZ gene were constructed. Two distinct regions, which function differently, were identified as cis-acting regulatory elements, namely, one responsible for OmpR-dependent activation and the other for OmpR-independent repression of micF expression. The former contains the OmpR-binding site, which simultaneously regulates both the genes, micF and ompC, in response to the medium osmolarity. The latter may be involved in an unknown regulatory process of micF expression. PMID- 1723391 TI - Retrotransposon Gypsy and genetic instability in Drosophila (review). AB - The laboratory mutator strain (MS) has properties which can be characterized as genetic instability. It exhibits the high level of gypsy autonomous transposition in somatic and germ cells. This paper summarizes all the data concerning this system and gypsy itself that has been obtained in our works during the last years. PMID- 1723392 TI - Cytokeratin intermediate filament pattern and human papillomavirus type in uterine cervical biopsies with different histological diagnosis. AB - The cytokeratin pattern and the presence of human papillomavirus (HPV) were analyzed in 53 uterine cervical biopsies. The biopsies were histologically characterized and the diagnosis ranged from normal through dysplasia to carcinoma. The cytokeratins were identified by their immunological reactivity with the monoclonal antibodies AE1 and AE3. The tissue was typed for the presence of HPV types 11, 16 and 18. We have previously shown that there was no correlation between the expression of cytokeratins No. 14, 15, 16 and 19 (K14, K15, K16 and K19) and the histological diagnosis of cervical biopsies. The present study shows that the cytokeratin pattern cannot be correlated to HPV infection of the cervical tissue either. PMID- 1723393 TI - Glucose stimulates and potentiates islet amyloid polypeptide secretion by the B cell. AB - Islet amyloid polypeptide (IAPP) has been shown to be actively secreted by the pancreatic B-cell along with insulin. To determine whether the modulation of B cell IAPP secretion is similar to that of insulin, we assessed IAPP release in response to glucose at 4 different concentrations (1.67, 5.5, 8.8 and 16.7 mM) and to non-glucose secretagogues at different glucose concentrations in a neonatal rat islet monolayer culture preparation. Glucose alone stimulated IAPP and insulin secretion in a dose dependent fashion with maximal release for both peptides occurring at 8.8 mM. B-cell secretion of IAPP in response to arginine, isobutylmethylxanthine or both together was potentiated by increasing glucose concentrations from 1.67 to 16.7 mM. This same pattern of glucose potentiation was observed for insulin secretion. The data indicate that the pattern of peptide responses of cultured neonatal B-cells to glucose is similar for both IAPP and insulin release. Furthermore, the data suggest that glucose is capable of potentiating B-cell secretion of both IAPP and insulin. PMID- 1723394 TI - Anabolic actions of a mixed beta-adrenergic agonist on nitrogen retention and protein turnover. AB - Subcutaneous administration of a mixed beta-agonist induced increases in muscle (+13%) and heart (+17%) weights, which were accompanied by a reduction in back ( 13%) and perirenal (-27%) fat stores in young male rats, while no changes in liver were observed. The values of nitrogen retention (mg/day) were significantly higher in the beta-agonist treated animals (+16%) as well as those of muscle DNA content (+8%). On the other hand, no statistically significant changes in muscle protein synthetic activity (g protein synthetized/g RNA) were detected (control: 13.4 vs treated: 14.6), while muscle proteolytic activity was decreased (-9%) in those rats administered with the repartitioning agent. In this context, it is suggested that the anabolic effect of metaproterenol should be attributed to a reduction in muscle protein degradation rather than to changes in protein synthesis. PMID- 1723395 TI - Evaluation of RNA rich fraction of Litmosoides carinii in induction of protection in infected rats. AB - The RNA rich fraction of adult L. carinii worms was evaluated in evoking a protective response in infected rats. The RNA immunization was seen to be effective in limiting the microfilaraemia in peripheral blood as well as the adult worm burden. The antibodies to both RNA antigen and adult worm antigen were high in this group of animals at the peak of infection. The RNA immunization was seen to evoke hyperresponsiveness in lymphocytes to mitogens like adult worm antigen, PHA and Con A. PMID- 1723396 TI - Use of non-verbal construing and metaphor in psychotherapy. AB - This article begins by looking at the way we try to construe or make sense of the world in which we live. How in the very nature of such sense-making there is the potential for sowing the seeds of self-deception by trying to fit our (relatively simplified) bipolar constructs to a complex unitary universe. Non-verbal and verbal construing are compared. Verbal construing enables us to reach an even more sophisticated level of self-deception, via myths created by limiting metaphors--such as "man-the-machine." The institutions which we create and the people who are invested in them tend to solidify in-group myths, often with the help of metaphor. It is argued that personal, constructivist, and eclectic approaches to psychotherapy can be envisaged as enterprises to begin the process of demythologising clients in the safety of the therapeutic setting. In therapy, clients can experiment with empowering themselves in reconstruing the problem areas of their lives and actively testing out these new constructions against personal experience. Clients can also learn to explore the meaning behind symptoms, i.e., to give cognitive form to feeling with the help of their intuitive sensitivity. PMID- 1723397 TI - Pain management and yoga. AB - The use of yoga and yoga related techniques in pain management is reviewed and discussed. Self-awareness, relaxation, approaches which use respiration, increased self-understanding and self-acceptance, changed context of pain, increased control, life style improvements, group and social support proved beneficial. The use of yoga in pain management has its transpersonal and philosophical dimensions. Independence and self-confidence of suffering people may be protected in this way. PMID- 1723398 TI - Smoking cessation in a single session: an update. AB - An approach to helping people cease smoking in a single, 50-minute session is outlined. A number of elements including direct suggestions to satisfy patients' expectations, metaphors, indirect commands, and motivation-enhancing statements are used, these being combined and sequenced in a pattern considered to be appropriate to the patient. Within elements, possible alternatives are outlined. PMID- 1723399 TI - Do doctors and patients agree? Views of the outcome of transurethral resection of the prostate. AB - In an attempt to establish the extent to which patients and doctors agree on the outcome of health care, the pre- and postoperative states of health of 388 men undergoing transurethral resection of the prostate for benign disease were studied. Generally, high levels of concordance (greater than 70%) were obtained. The strongest agreement was for clearly defined events, such as episodes of acute retention (95%); the weakest agreement occurred over the most subjective symptoms, prognostic expectations, and ambiguous terms (around 60%). The level of agreement was not associated with any characteristics of the patient, surgeon, or treatment. PMID- 1723400 TI - Feasibility and effectiveness of en bloc resection of the esophagus for esophageal cancer. Results of a prospective study. AB - En bloc resection of the esophagus was attempted by right thoracotomy, laparotomy, and left cervicotomy in 82 patients suffering from an esophageal cancer. Tumors were classified by the depth of wall penetration and node involvement: 21 tumors penetrated at the most into but not through the muscle (W1), and 61 invaded the full thickness of the wall (W2). Twenty-five were associated with normal lymph nodes (N0), 26 with metastatic thoracic nodes only (N1), and 31 with metastatic extrathoracic nodes (N2). Digestive continuity was restored by gastric pull-up in all cases except one in which the transverse colon was used. Thirty day and hospital mortality were 0 and 2.4% (2/82) respectively. Posterior mediastinectomy was feasible in 65 patients but in seven of them an unsuspected metastatic spread was detected during the second step of the operation. It was not feasible in 17 patients owing to the involvement of adjacent mediastinal organs. It was feasible in all W1 tumors and in 72% of W2. Feasibility did not significantly depend on the tumor location or length. Of the 24 palliative operations, 17 were carried out for N2 tumors. After potentially curative mediastinectomy (N = 58), three year survival was 38% and after palliative operation (N = 24), 18 months survival was 11% only. After mediastinectomy, survival dropped as the node involvement and the depth of wall penetration increased (W1: 66%, W2: 28%, N0: 58%, N1: 32% at three years and N2: 17% at 2 years). Overall survival in group N2 was not significantly different from that achieved in palliative cases and no patient classified W2 N2 was alive at 18 months.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723401 TI - Biologically localized firefly luciferase: a tool to study cellular processes. PMID- 1723402 TI - Mitomycin and bleomycin. PMID- 1723403 TI - Dunaimycins, a new complex of spiroketal 24-membered macrolides with immunosuppressive activity. III. Immunosuppressive activities of dunaimycins. AB - The immunosuppressive effects of the dunaimycins, a new complex of spiroketal 24 membered macrolides, were compared to cyclosporin A, ascomycin, and rapamycin. Each dunaimycin was a potent inhibitor of the mitogenic response observed in mixed murine splenocyte or human leukocyte cultures, and like immunosuppressive drugs these compounds were relatively less potent inhibitors of the constitutive proliferation of murine EL4 thymoma cells. Dunaimycin D4S showed no selectivity in inhibiting the mitogenic response of spleen cells to concanavalin A, pokeweed mitogen, lipopolysaccharide, or phytohemagglutinin. Cyclosporin A and ascomycin did not inhibit interleukin 2 dependent proliferation, whereas the dunaimycins and rapamycin blocked the uptake of [3H]thymidine in mixed cultures supplemented with exogenous interleukin 2. In addition, dunaimycin D4S had no apparent affinity for cyclosporin A or FK-506 immunophilins. Although the dunaimycins inhibited the activity of Na+, K(+)-ATPase, inhibition of this enzyme appeared insufficient to explain the biological activity of these new macrolides. Over a narrow concentration range, dunaimycin D4S showed in vivo immunosuppressive activity in the murine popliteal lymph node hyperplasia model. PMID- 1723404 TI - Development of resistance to ceftazidime and co-amoxiclav in Pseudomonas pseudomallei. PMID- 1723405 TI - Exercise training and responsiveness of isolated coronary arteries. AB - Exercise is associated with release of catecholamines and vasoactive intestinal polypeptides. Recurrent exposure to catecholamines modifies the sensitivity of adrenoceptors. To test the hypothesis that exercise training may affect the sensitivity of the epicardial coronary arteries, we performed studies on isolated coronary arteries from male dogs capable of running on a treadmill. The animals were separated randomly into two groups: sedentary and exercise training. After 11 wk, rings of left circumflex and left anterior descending coronary arteries were studied in vitro. Contractions to alpha 1-adrenergic agonists (norepinephrine and phenylephrine) were not affected by exercise training. During contractions with prostaglandin F2 alpha, endothelium-dependent relaxations to alpha 2-adrenergic agonists (norepinephrine and UK 14304) were not reduced significantly by exercise training. The concentration-relaxation curves to beta adrenergic agonists (norepinephrine, isoproterenol, and epinephrine) were shifted to the right after training. The concentration-response curves to vasoactive intestinal polypeptide, but not that to substance P, were shifted to the right in rings with endothelium from exercise-trained animals. These findings demonstrate a decrease in responsiveness of canine vascular smooth muscle to beta-adrenergic agonists and to vasoactive intestinal polypeptide after exercise training. PMID- 1723406 TI - Intravascular anti-IgE challenge in perfused lungs: mediator release and vascular pressor response. AB - Intravascular application of goat anti-rabbit immunoglobulin E (IgE) was used to stimulate parenchymal mast cells in situ in perfused rabbit lungs. Sustained pulmonary arterial pressure rise was evoked in the absence of lung vascular permeability increase and lung edema formation. Early prostaglandin (PG) D2 and histamine release into the perfusate was documented, accompanied by more sustained liberation of cysteinyl leukotrienes (LT), LTB4, and PGI2. The quantities of these inflammatory mediators displayed the following order: histamine greater than cysteinyl-LT greater than PGI2 greater than LTB4 greater than PGD2. Pressor response and inflammatory mediator release revealed corresponding bell-shaped dose dependencies. Cyclooxygenase inhibition (acetylsalicylic acid) suppressed prostanoid generation, increased LT release, and did not substantially affect pressor response and histamine liberation. BW755 C, a cyclo- and lipoxygenase inhibitor, blocked the release of cysteinyl-LT and markedly reduced the liberation of the other inflammatory mediators as well as the pressor response. The H1-antagonist clemastine caused a moderate reduction of the anti-IgE-provoked pressure rise. We conclude that intravascular anti-IgE challenge in intact lungs provokes the release of an inflammatory mediator profile compatible with in situ lung parenchymal mast cell activation. Pulmonary hypertension represents the predominant vascular response, presumably mediated by cysteinyl-LT and, to a minor extent, histamine liberation. PMID- 1723407 TI - Chemotherapy for advanced testicular cancer. AB - Testis cancer has become one of the most curable of all solid malignancies. More than 95% of patients should be cured with appropriate treatment and should have few long-term treatment-related side effects. Current chemotherapy for advanced testis cancer has resulted from an orderly sequence of chemotherapy trials that serves as a model for cancer chemotherapy development. Recent studies have defined the least therapy required to minimize toxicity while maintaining a high cure rate in "low-risk" patients. Further improvements are necessary in "high risk" patients; evaluation of new regimens by means of randomized trials is essential. PMID- 1723409 TI - Isolation and characterization of 1,200 kDa peptide of alpha-connectin. AB - When rabbit skeletal muscle myofibrils were kept for 12 h at 4 degrees C, alpha connectin was partially degraded and 1,200 kDa peptide was newly formed [Takahashi, K. & Takai, H. (1988) Abst. 80th Jpn. Soc. Zootech. Sci. p-102]. The latter was isolated together with remaining alpha-connectin. Ultracentrifugation of the mixture at low ionic strength resulted in sedimentation of alpha connectin, leaving the 1,200 kDa peptide in the supernatant. Physicochemical properties of the isolated 1,200 kDa peptide were investigated: UV absorption spectra, circular dichroism spectra, amino acid composition, and molecular size and shape. Polyclonal antibodies against the 1,200 kDa peptide [PcAb(1200)] bound the Z line and I-band. The position of the stripe in the I band near the N1 line due to the binding of PcAb(1200) moved both away from the Z lines and from the A band as sarcomeres were elongated. Therefore, it is considered that the 1,200 kDa portion of alpha-connectin is elastic. PMID- 1723408 TI - Serum markers in germ cell neoplasms. AB - Innovations in the treatment of testicular cancer, including surveillance of clinical stage I patients and curative chemotherapy for disseminated disease, have increased the need for sensitive ways to stage and monitor patients, both during and after therapy. Serum tumor markers, in combination with radiographic studies, have significantly improved our ability to evaluate and treat patients with seminomas and NSGCT. Elevated AFP and BHCG levels provide prognostic information at diagnosis, indicate persistent disease following orchiectomy or RPLND, and signal a recurrence after chemotherapy. Significantly delayed clearance of markers during chemotherapy often indicates persistent disease. Serum markers help define the duration of therapy, thus minimizing the substantial toxicities often associated with curative chemotherapy. Despite these advances, areas of concern remain. A small percentage of patients with NSGCT and the majority of patients with seminoma have undetectable levels of AFP and BHCG. The search for additional sensitive and specific serum markers in these cases has not been wholly successful. LDH, PLAP, and BFP occasionally serve as useful markers in seminoma but suffer lack of specificity. In addition, normal postoperative or postchemotherapy serum marker levels do not always ensure complete remission. This is difficult clinically when residual masses persist following therapy. Resection is always required to rule out persistent disease. The next decade may reveal additional useful serum tumor markers and potentially new imaging techniques incorporating antimarker antibodies to differentiate necrotic tissue from active disease. PMID- 1723410 TI - Genetic and molecular properties of human and rat renin-binding proteins with reference to the function of the leucine zipper motif. AB - The presence of a leucine zipper motif was recognized in the deduced amino acid sequences of human and rat renin-binding proteins (RnBPs) on cloning and sequence analysis of the RnBP cDNAs. The in vitro synthesized RnBPs, with the respective cDNAs, formed heterodimers with porcine renin and homodimers. On comparison of these properties with those of porcine RnBP, the leucine zipper motif was suggested to be a functional domain common to animal RnBPs. In addition to the motif, a hydrophobic domain adjacent to the motif and 10 cysteine residues were also well conserved in the three RnBPs. Moreover, about 85% of their amino acid sequences were identical. The RnBP mRNAs were expressed in the kidneys as the same size of 1.5-kb and the genes are suggested to exist as single copies in the genomes. Despite the high similarities in genetic and molecular properties, the molecular weights of human and rat RnBPs were 43,000, which is 1,000 larger than that of porcine RnBP. The immunoreactivities of human and rat RnBPs toward anti porcine RnBP antiserum were 88 and 8% that of porcine RnBP, respectively, and the affinities of the two RnBPs for porcine renin were remarkably less than that of porcine RnBP. Moreover, the human and rat RnBP homodimers were partly dissociated under the conditions under which porcine RnBP existed as a dimer. These results indicate distinct differences in the molecular properties among the three RnBPs, in spite of their being highly similar structurally and functionally. PMID- 1723411 TI - Alpha-macroglobulin-like plasma inactivator for Vibrio vulnificus metalloprotease. AB - The metalloprotease produced by Vibrio vulnificus (VVP) is known to be quickly inactivated by plasma proteins which belong to the class of alpha-macroglobulins in vitro at a molar ratio of 1:1. But the in vivo potential of the inactivators has not been studied. Macroalbumin (MA), a member of alpha-macroglobulins in guinea pig plasma, was found to inactivate VVP by means of physical entrapment in vitro. In vivo actions of VVP, permeability-enhancing and hemorrhagic actions, were greatly augmented by simultaneous injection of the antibody against MA, which had no effect on in vitro proteolytic action toward azocasein. The interstitial-tissue space in the normal guinea pig skin contains a negligible amount of MA. However, sufficient MA was present in the extravascular fluid collected after the intradermal injection of VVP. Besides, in the extravascular fluid, VVP formed a complex with MA and no inactivator other than MA was found. These results indicate that plasma MA leaked from the vascular system owing to the permeability-enhancing and hemorrhagic actions of VVP, resulting in inactivation of VVP in situ. PMID- 1723412 TI - Calcium transport sensitive to ruthenium red in cytochrome oxidase vesicles reconstituted with mitochondrial proteins. AB - We describe a calcium transport that is sensitive to ruthenium red in liposomes reconstituted with mitochondrial extracts. This system is able to build an internally negative membrane potential, which allows the electrogenic influx of Ca2+ and Sr2+. Proteins with molecular weights higher than 35 kDa were incorporated to the vesicles, and enhanced the accumulation of the cation in an energy-dependent fashion. PMID- 1723413 TI - Renal hyaline droplets in tumour-bearing female Wistar rats. AB - Renal hyaline droplets were defined by histochemical and ultrastructural methods in eight female Wistar rats in a carcinogenesis bioassay. All eight rats had neoplasms of varied type (five histiocytic sarcoma, one phaeochromocytoma, one rhabdomyosarcoma, one leiomyosarcoma). Renal hyaline droplets were not seen in female rats without tumours and, although in this study, rats with tumours did not all have hyaline droplets, the source of the protein is likely to be the neoplasm. Presence of hyaline droplets may be useful as a confirmatory criterion in tumour diagnosis. PMID- 1723414 TI - Lectin histochemistry of foamy cells in non-nervous tissues of feline sphingomyelinosis. AB - Foamy cells in non-nervous tissues from a female Siamese cat with sphingomyelinosis were examined by lectin histochemistry. Many foamy cells, so called Niemann-Pick cells, were found in the non-nervous tissues, such as liver, spleen, lung, kidney, adrenal gland, lymph node and tonsil. These cells were positive for Concanavalia ensiformis agglutinin, Ricinus communis agglutinin-I and wheat germ agglutinin. Storage materials in the foamy cells were different from those in affected cells of the nervous tissues. This study suggests that lectin histochemistry might be helpful in the diagnosis of sphingomyelinosis. PMID- 1723415 TI - Isolation and characterization of monoclonal antibody directed against bovine alpha s2-casein. AB - A monoclonal antibody 62-1A of isotype IgM, directed against bovine alpha s2 casein, was isolated and characterized. Monoclonal antibody 62-1A recognized bovine alpha s2-11P- and alpha s2-9P-caseins by indirect solid phase radioimmunoassay and Western blot analysis. Crossreactivity toward native genetic variants (A, B, and C) of alpha s1-casein was similar but lower (approximately 60 to 70%) than that for alpha s2-11P-casein). Little or no crossreactivity was observed for other bovine milk or serum proteins. Antibody affinity for alpha s2 11P-casein was 1.3 x 10(9)/M. As little as 25 ng/ml (.5 ng/well) of alpha s2-11P casein was detected by solid phase radioimmunoassay. PMID- 1723416 TI - Isolation and phagocytic properties of neutrophils and other phagocytes from nonmastitic bovine milk. AB - A technique for the separation of neutrophils from macrophages-epithelial cells in samples of nonmastitic bovine milk with low cell counts has been developed. The procedure is based on centrifugation in a discontinuous metrizamide gradient and is rapid, taking less than 40 min. The recovery of the neutrophils is about 30% and their viability about 90%. The isolated neutrophils showed an appreciable unstimulated luminol- and lucigenin-dependent chemiluminescence, which was due to NADPH oxidase rather than to xanthine oxidase. The neutrophils had a higher rate of ingestion of C3-opsonized particles than macrophages-epithelial cells, whereas no significant differences in phagocytosis of IgG-opsonized yeast or unopsonized yeast were detected between the two cell populations. The macrophages-epithelial cells produced no luminol-dependent chemiluminescence and induced considerably lower activity in the lucigenin-dependent system than neutrophils, indicating that these cells contain no myeloperoxidase. Analyses of the activity of the neutrophils in response to C3-opsonized yeast particles showed that the luminol dependent chemiluminescence of cells isolated from residual milk increased significantly over the lactation period. Moreover, a tendency to a higher phagocytosis and chemiluminescence of neutrophils isolated from residual milk than from stripping milk was indicated. PMID- 1723417 TI - Comparative acute toxicities of selected pesticides to Anguilla anguilla. AB - The acute toxicities (24, 48, 72 and 96 hr) of eight pesticides to Anguilla anguilla were determined. The organochlorine pesticide, endosulfan was the most toxic, with LC50 values in the range of 0.042 to 0.041 mg/L. Endosulfan was followed in order of decreasing toxicity by diazinon, fenitrothion, chlorpyrifos, lindane, methidathion, trichlorfon and methylparathion. When fishes were exposed to the pesticides tested they exhibited signs of restlessness, erratic swimming, convulsions and difficulty in respiration. This response was more persistent in fishes exposed to organophosphorus pesticides. PMID- 1723418 TI - [Neurons in the visual cortex of Microtus brandti]. AB - Neurons were described in the visual Cortex of Microtus brandti, a Mongolian harmful rodent living in day-activity. We find following types of neurons in our Golgi-material: 1. spiny neurons: pyramidal and stellate neurons. 2. smooth or sparsely spined neurons: smooth, large neurons, sparsely spined small neurons with descending axons, sparsely spined neurons with ascending axons. Double bouquet-, chandelier and neuroglioforme cells are not impregnated. There are no bipolare neurons (Martinotti cells) among the neurons with ascending axons. The small, sparsely spined neurons are not only in lamina IV - like in other species but they can also be found in laminae II to IV. Their distribution of spines on the distal parts of dendrites seems to be characteristical for rodents. The lamination of the visual cortex of Microtus brandti is the same like in the rat. All cells are of large size in relation to the body mass of the animal. PMID- 1723419 TI - Cytoarchitectonic pattern of the hypothalamus in the catfish, Clarias batrachus (Linn.). AB - The hypothalamus of the catfish, Clarias batrachus has been investigated to reveal the organization of various parvocellular (aldehyde fuchsin-negative) nuclear complexes and to suggest homologies. The hypothalamus of C. batrachus can be divided into the preoptic area, postoptic area, tuberal area and the laterally placed inferior lobes. Application of cytoarchitectonic criteria permits the delineation of 9 distinct nuclear complexes in the preoptic area extending along the periventricular margin of the preoptic recess. The postoptic area is represented by only two nuclear groups, the nucleus postopticus lateralis and the nucleus of the horizontal commissure. The tuberal area consists of as many as 14 nuclei, some are divisible into subnuclei, arranged in an elaborate pattern. The nucleus lateralis tuberis is located in the mid-ventral margin of the tuberal area and revealed two subdivisions, the pars anterior and pars posterior. At the mid-tuberal level, the nucleus anterior tuberis is well-developed and revealed dorsal, ventral and lateral subdivisions. The paraventricular organ is encircled by nucleus of the paraventricular organ. The conspicuous lateral recesses are flanked dorsally by nucleus recessus lateralis superior and ventrally by nucleus recessus lateral by nucleus recessus lateralis superior and ventrally by nucleus recessus lateralis inferior. In the caudal tuberal area, in close proximity of the pituitary, the nucleus arcuatus hypothalamicus is described for the first time. The recessus posterioris is encircled by nucleus recessus posterioris. The inferior lobes are massive and each consists of 7 well-defined nuclear complexes. The nucleus lobi inferioris centralis is represented by giant-sized cells clustered in the center of the lobes. The periventricular nucleus of the inferior lobes occupies the area surrounding the recesses of the inferior lobes. The hypothalamus shows 4 circumventricular organs. The organum vasculosum laminae terminalis is situated along the ventral wall of the preoptic recess. While the paraventricular organ is located in the dorsal wall of the third ventricle, the posterior recess organ is located in the postero-ventral wall of the third ventricle. The saccus vasculosus is small, capsule-like and located caudally in between the two mamillary nuclei. PMID- 1723420 TI - The connections of the anterior pallium in Pleurodeles waltl and Triturus carnifex: an HRP study. AB - In order to provide cues about the evolution of the telencephalon in tetrapods, the connections of the anterior pallium were studied in two adult Urodeles, Pleurodeles waltl and Triturus carnifex, by means of the HRP-tracing method. The staining of HRP-immunopositive cell bodies indicates that the pallial regions studied receive afferent projections from the main olfactory bulb and are reciprocally interconnected by intrapallial associative fiber systems. In the ventral hemispheric wall, HRP-immunoreactive perikarya are observed in the pars medialis of the amygdala and in the rostral and caudal striatum. Triturus exhibits a more complex pattern of pallial afferents, including interhemispheric connections and thalamic ascending projections that were not discovered in Pleurodeles. HRP-immunopositive fibers are observed in the dorsal and medial walls of the telencephalon, from the rostral part to the foraminal level. In Triturus, the dorsal fibers extend to the caudal part of the hemisphere. Another group of labelled fibers extends, throughout the lateral and ventral walls, to the most caudal part of the telencephalon, and, through the stria medullaris and the habenular commissure, crosses over to the controlateral hemisphere. These results allow us to specify the basic pattern of the pallial connections in Urodeles and to compare them with data previously obtained in other Amphibians. PMID- 1723421 TI - A histochemical study of bone remodeling during experimental apical periodontitis in rats. AB - An apical periodontitis experimental model was produced by means of opening the pulp chamber of the mandibular first molar in Wistar strain rats. In particular, the behavior of bone tissue in the vicinity of the root apex was investigated histochemically, ultrastructurally, and quantitatively. In addition, in order to demonstrate the effects of prostaglandin on the formation process of apical periodontitis, we examined the effects of indomethacin on bone remodeling during experimental apical periodontitis. These experiments suggested that prostaglandin may stimulate osteoclastic bone resorption and that the relationship between bone resorption and formation in apical periodontitis is a coupling phenomenon. PMID- 1723422 TI - Clonal cells from embryonic retinal cell lines express qualitative electrophysiological differences. AB - Cells from the embryonic quail retina were immortalized with the v-mil oncogene and cloned by limiting dilution. Their phenotype was examined using the whole cell patch clamp method. Three membrane currents, IK(IR), INa and IK, were found at different frequencies within a sample of 170 cells drawn from a large clone. Nearly all combinations of these three markers were found and the frequency of combinations showed that the markers assorted independently. Examination of clones of less than 10 cells showed that heterogeneity originates with a high probability within clones, arguing that chromosomal mutation, for example, is unlikely to account for phenotypic diversity. A possible explanation is that phenotypic differences between cells might reflect the local exchange of instructive signals. If so, then the genes for the three phenotypic markers are controlled independently. PMID- 1723423 TI - A monoclonal antibody against HSV type 1 ribonucleotide reductase cross-reacts with the P0 protein of peripheral nerve myelin. AB - An epitope on peripheral nerve myelin was detected by the use of a mouse monoclonal antibody directed against the 38 kDa subunit of herpes simplex virus (HSV) type 1 ribonucleotide reductase. Immunohistochemistry showed reactivity solely in PNS myelin. In nerve roots there was a sharp border in transitional zones to the negative CNS myelin. The immunoreactivity was found in rat, guinea pig, bovine and human peripheral nerves. Western blot analysis of peripheral nerve myelin as well as purified P0 revealed a distinctly stained band corresponding to a molecular weight of approximately 29 kDa. The present finding of a shared antigenic determinant between HSV ribonucleotide reductase and peripheral nerve P0 may be of pathogenetic relevance in virus induced demyelinating diseases in the peripheral nervous system. PMID- 1723424 TI - Phase II study of cyclophosphamide (C), epirubicin (E), oncovin (O), prednisone (P), methotrexate (M) + leucovorin and bleomycin (B) (CEOP-MB) in intermediate and high grade non-Hodgkin lymphomas. AB - Thirty-five patients with non-Hodgkin lymphoma of intermediate or high-grade histology were treated with cyclophosphamide, epirubicin, vincristine, prednisone, methotrexate plus leucovorin and bleomycin (CEOP-MB). The complete response rate was 66%. The relapse rate of these complete responders was 39%. After a median follow-up of 3 years the median duration of complete response was 23 months and the median survival of the complete responders 52 months. The median survival of the entire group was 35.5 months. The toxicity was acceptable with no cases of congestive heart failure and no toxic deaths. Epirubicin appears to have a better therapeutic index than doxorubicin in aggressive treatment protocols in advanced non-Hodgkin lymphomas. PMID- 1723425 TI - Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo. AB - Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs. PMID- 1723426 TI - Salivary gland antigens of Ixodes dammini are glycoproteins that have interspecies cross-reactivity. AB - Serum from rabbits and rats exposed to Ixodes dammini adults and larvae, respectively, contain antibodies to a large number of antigens in salivary gland homogenates from adult ticks that cross react with the antigens of a closely related species, Ixodes scapularis, and significantly with Dermacentor variabilis antigens. The salivary gland antigens of I. dammini are glycoproteins composed of both N- and O-linked carbohydrate chains. The antigenic determinants reside in both the polypeptide and carbohydrate chains. PMID- 1723427 TI - Toward defining the function of the cystic fibrosis gene product. PMID- 1723428 TI - Outcome of mid-trimester pregnancies complicated by oligohydramnios. Quantitative evaluation of the prognostic value of maternal serum alpha-fetoprotein level. PMID- 1723429 TI - Area and volume measurement of posterior fossa structures in MRI. AB - This study was undertaken to determine the extent to which area measures of posterior fossa structures can be used confidently to represent structure volumes. MRI scans were obtained from three groups: fragile X males, males with other developmental disabilities, and males with normal IQ. The areas of the midbrain, pons, medulla, cerebellar vermis, and fourth ventricle were measured in midsagittal sections. Volumes of midbrain, pons, medulla, cerebellum, fourth ventricle, and third ventricle were obtained by measuring these structures in contiguous axial slices. In addition, the largest axial area for each structure was identified. Analysis revealed that midsagittal area measures for pons, medulla, and fourth ventricle were significantly correlated with structure volumes, and that all of the largest axial area measures were significantly correlated with structure volumes. However, only the midsagittal area measure of fourth ventricle and the largest axial area measures of fourth ventricle and cerebellum were correlated with volume measures with an r of .80 of greater. Results of this study suggest that area measures may not accurately represent three-dimensional structure size. PMID- 1723430 TI - Structural organization of male-specific visual neurons in calliphorid optic lobes. AB - The superiority of male flies over female flies in locating and intercepting small rapidly moving targets has been ascribed to differences in their visual systems. In males, this sexual dimorphism is externally expressed by an area of high visual acuity called the acute zone. Selective cobalt uptake reveals 12 types of male-specific visual interneurons in the male lobula, the axons of which terminate in neuropil supplying premotor descending neurons to neck and flight motor circuits. The dendritic fields of the individual male-specific neurons can be extrapolated out into visual space to demonstrate that each is assigned a discrete area of the visual panorama. The dendritic fields of 10 of the 12 male specific neurons subtend areas of the retina associated with the male acute zone. The functional significance of male-specific neurons is discussed with respect to their putative receptive field and a model circuit for target location by male flies. PMID- 1723431 TI - The functional organization of male-specific visual neurons in flies. AB - Intracellular recording and Lucifer yellow dye filling of male fleshflies, Sarcophaga bullata, have revealed male-specific neurons in the lobula, the axons of which project to the origin of premotor channels supplying flight motor neurons. Dendrites of male-specific neurons visit areas of the retinotopic mosaic supplied by the retina's acute zone, which is used by males to keep the image of a conspecific female centered during aerial pursuit. Only males engage in high speed aerobatic chases, and male-specific neurons are suspected to underlie this behavior. Physiological determination of receptive fields of male-specific neurons substantiates the fields predicted from anatomical studies and demonstrates that they subtend the acute zone. Male-specific neurons respond in a manner predicted on theoretical grounds from observations of tracking behavior. Such properties include directional selectivity to visual motion and higher sensitivity to motion of small images than to wide-field motion. The present account substantiates and extends neuroanatomical evidence that predicts that male-specific lobula neurons comprise a distinct circuit mediating conspecific tracking. PMID- 1723432 TI - Descending pathways connecting the male-specific visual system of flies to the neck and flight motor. AB - During sexual pursuit, male flies Sarcophaga bullata, stabilize the image of a pursued target on the dorso-frontal acute zone of their compound eyes. By retinotopic projection, this region is represented in the upper frontal part of the lobula where it is sampled by ensembles of male-specific motion- and flicker sensitive interneurons. Intracellular recordings of descending neurons, followed by biocytin injection, demonstrate that male-specific neurons are dye-coupled to specific descending neurons and that the response characteristics of these descending neurons closely resemble those of male-specific lobula neurons. Such descending neurons are biocytin-coupled in the thoracic ganglia, revealing their connections with ipsilateral frontal nerve motor neurons supplying muscles that move the head and with contralateral basalar muscle motor neurons that control wing beat amplitude. Recordings from neck muscle motor neurons demonstrate that although they respond to movement of panoramic motion, they also selectively respond to movement of small targets presented to the male-specific acute zone. The present results are discussed with respect to anatomical and physiological studies of sex-specific interneurons and with respect to sex-specific visual behavior. The present study, and those of the two preceding papers, provide a revision of Land and Collett's hypothetical circuit underlying target localization and motor control in males pursuing females. PMID- 1723433 TI - [Cell-associated complement regulatory proteins and their relation to disease processes]. AB - C3-deposition is a key step for activation of the complement system, which involves C9-mediated immunocytolysis, immunoadherence, C3 receptor-mediated phagocytosis, NK potentiation, anaphylatoxin release, and amplification of C3 activation. Foreign material is eliminated even in the preimmune stage by these complement functions. Self cells, on the other hand, must circumvent the C3 attack, so that they express complement regulatory proteins, namely C3b/C4b receptor (CR1, CD35), decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46). We herein review the properties of these regulatory proteins and discuss the relationships between disease processes and the aberrance of these regulatory proteins. PMID- 1723434 TI - [Expression of CD56 antigen on acute nonlymphocytic leukemia]. AB - CD56 antigen (detected by NKH-1) is distributed on NK cells, monocytes, and ectodermal neural cells. In this study, the blasts of 29.2% of 27 patients with acute nonlymphocytic leukemia (ANLL) expressed CD56 antigen, but not CD16, CD2, or CD3 antigen. Leukemic cells isolated from 3 patients with CD56-positive ANLL did not have NK activity. There were no significant differences between CD56 positive and CD56-negative ANLL in CD13-positive cases, CD33-positive cases, and HLA-DR-positive cases. These results suggest that CD56-positive ANLL could be so called mixed-lineage leukemia (lymphoid-associated antigen in ANLL). PMID- 1723435 TI - [Transurethral microwave thermotherapy of benign prostatic hypertrophy]. AB - Transurethral microwave thermotherapy using Prostatron was performed in 31 patients with benign prostatic hypertrophy, and the clinical effectiveness was evaluated by analyzing the subjective and objective responses following the treatment. The 22F balloon catheter to be placed in the prostatic urethra incorporates the microwave antenna, a cooling system and a fiberoptic thermosensor which allow an effective delivery of microwave energy to the center of the prostate, while preserving the mucosa and periurethral tissue. The maximum urethral temperature during the treatment ranged from 43.3 to 45.5 degrees C (44.7 +/- 0.96 degrees C: mean +/- S.D.) and the average power output was 27.4 Watt. The treatment was performed in a single session of an hour on the outpatient basis. In one patient who could not be relieved of the indwelling catheter underwent a transurethral resection, and the histological effect of thermotherapy on the resected specimen was examined. In the prostatic tissue, heat-induced necrotic change of the interstitial tissue as well as degenerative change of the acinar epithelium were remarkable, whereas the urethral mucosa was well preserved. In the remaining 30 patients, the clinical effects were evaluated 8 weeks after the treatment by a score scale for subjective symptoms, residual urine and maximum urinary flow rate, which was compared with the pretreatment score. Improvement of both subjective symptoms and objective findings was observed in 13 subjects (43.3%), that of subjective symptoms only in 14 cases, and that of objective findings only in 2 cases, resulting in a notable improvement in total 29 cases (97.7%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723437 TI - [Differential diagnosis of extrasystole and transient disorders of intraventricular conduction]. PMID- 1723436 TI - [Autologous blood transfusion aided with erythropoietin in transurethral resection of the prostate]. AB - Erythropoietin (EPO) with an established clinical efficacy in renal anemia has in recent years become applied as an aid to autologous blood transfusion in surgical patients. This report describes our experience with autotransfusion along with the use of EPO in transurethral resection of the prostate (TUR-P), indicating its usefulness. Ten patients with benign prostatic hypertrophy aged 60 to 74 years received 3000 units of EPO, with an iron preparation, nine times beginning 3 weeks prior to operation. Autologous blood of 300 ml was collected from the patient each at 2 and 1 week before operation and was used at TUR-P. Five other patients who underwent TUR-P with the same volume of autotransfusion accompanied by preparative medication with the iron alone served as controls. In the EPO treated group (mean age, 68.3 years) the mean value for hemoglobin concentration (Hb) was 14.0 +/- 1.6 g/dl on the day of operation, which showed a recovery rate of 94.9 +/- 5.4% (Hb recovery rate) as against the pre-EPO treatment value (mean: 14.8 +/- 1.3 g/dl). This Hb recovery rate was significantly greater (p less than 0.001) when compared to 82.2 +/- 2.5% in the control group (mean age, 68.2 years). Of the EPO treated patients, those in their sixties (n = 6; mean age, 66.3 years) exhibited a significantly higher Hb recovery rate (98.3 +/- 3.5%) than the rate (89.9 +/- 3.0%) for patients in their seventies (N = 4; mean age, 71.3 years) (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723438 TI - [Differential diagnosis of extrasystole and transient disorders of intraventricular conduction (solution of the clinical problem)]. PMID- 1723439 TI - [Elaboration of a quantitative method of evaluation of disease severity in patients with ventricular disorders of cardiac rhythm]. AB - Mathematical approaches to the evaluation of the severity of the pathological process were used to develop an objective quantitative method to assess the severity of ventricular arrhythmias. The method is highly informative and reliable. It may be useful in the control of the efficacy of antiarrhythmic therapy and in the application of a differential approach to choice of therapy strategy for the patients. PMID- 1723440 TI - [Isolated right ventricle after coronary perfusion as a model for the study of ischemic and reperfusion-induced arrhythmia in rats]. AB - A catheter through which perfusion was performed with oxygenated saline (2.1 ml/min) was introduced into the right coronary artery ostium of the rat right ventricle that had been isolated during cardioplegia. Super perfusion (12 ml/min) was simultaneously made. Termination of the perfusion caused arrhythmias at minutes 6 to 28 of ischemia. The highest likelihood of occurrence of such arrhythmias was observed on minutes 16-20 (premature beats being seen in 86% of the experiments, extrastimulus-induced tachycardias in 75%, spontaneous tachycardias in 25%). Reperfusion was made at 3, 5, 7, 10, 13, 15, 20, 30 and 60 min following ischemia (n = 7 in each case). The occurrence of reperfusion arrhythmias is likely to be related to the duration of ischemia with the highest likelihood of 20 minutes after ischemia (tachycardia and fibrillation were observed in 100 and 71%, respectively). PMID- 1723441 TI - [Anesthesiologic aspects of palliative surgery and the correction of congenital vascular anomalies in infants]. PMID- 1723442 TI - Developmental profile of patients with maple syrup urine disease. AB - The progress of nine maple syrup urine disease patients (eight classical, one possible variant) was reviewed to look for similarities in developmental patterns. A consistent developmental profile of stronger Verbal than Performance IQ and lower than familially expected IQ was seen. Younger age at diagnosis was associated with a milder neonatal course. Those who were asymptomatic or suffered mild complications in the newborn period have higher IQs than children experiencing moderate or severe complications. Children with asymptomatic or mild neonatal course also required less special education services. Overall, a picture of ubiquitous motor, visual-analytic and learning deficits is seen. PMID- 1723444 TI - Dihydropyridines and their future in hypertension. A symposium. Basel, Switzerland, February 13, 1991. PMID- 1723443 TI - A closer look at acetyl and pentafluoropropionyl derivatives for quantitative analysis of morphine and codeine by gas chromatography/mass spectrometry. AB - PFPA and acetic anhydride derivatives of morphine and codeine were evaluated with respect to stability, chromatography, potential for analytical interferences by other opiates, and suitability of major fragment ions for analysis by GC/MS with deuterated internal standards and selected ion monitoring (SIM). The PFPA derivatives showed acceptable stability and could be analyzed without interference from other opiates, but the codeine derivative had relatively poor chromatography and its mass spectrum had only two ions suitable for SIM. The acetic anhydride derivatives were stable and chromatographed well, but diacetyl hydromorphone enol, a minor product of derivatization of hydromorphone, interfered with analysis of morphine. 3-Monoacetylmorphine, a minor product of derivatization of morphine, prevented use of the abundant m/z 285 ion of derivatized D3-codeine as a qualifying ion in quantitative assays. The acetic anhydride derivative of morphine cannot be distinguished from the corresponding derivative of the heroin metabolite 6-monoacetylmorphine. PMID- 1723445 TI - Calcium antagonists and tissue protection. AB - The tissue-protective effect of the calcium antagonists is a complex phenomenon that needs to be considered at the cellular as well as the organ level, and with respect to both the vasculature and myocardium. As far as the vasculature and the myocardium are concerned, hypertension is a major risk factor. The dihydropyridine-based calcium antagonists are protective under these circumstances, not only because of their blood pressure-lowering effect but also because of their ability to slow plaque formation and possibly to provide some protection against oxyradical-induced injury. With regard to the myocardium, tissue protection in the presence of hypertension involves not only a reduction in hypertrophy, but also a reduction in ischemia-induced injury and the incidence of damage due to lipid peroxidation. PMID- 1723446 TI - Nitrendipine and atenolol in essential hypertension in young and middle-aged patients: effect on serum lipids and left ventricular mass. AB - This study was designed to compare the antihypertensive efficacy of nitrendipine and atenolol in young and middle-aged patients with mild or moderate essential hypertension and to assess treatment effects on plasma lipids and potential changes in left ventricular mass (LVM). After 2 weeks off medication and a 4-week placebo phase, patients who met the inclusion criteria [sitting diastolic blood pressure (DBP) 95 to 114 mm Hg, age below 50 years] entered a 12-week dose adjustment and maintenance period with nitrendipine or atenolol. Serum lipids were determined before and after therapy. At the same time, LVM was evaluated echocardiographically (M mode). Twenty-two patients completed the double-blind, randomized study. After 12 weeks on nitrendipine, the systolic blood pressure (SBP) and DBP were reduced (p less than 0.005 and p less than 0.001, respectively). No significant changes in heart rate were observed. There were no changes in the lipid profile, and LVM was reduced from 93.7 to 23.4 to 82.4 +/- 22.6 g/m2 of body surface (p less than 0.05). On atenolol the SBP and DBP were reduced (p less than 0.001 and p less than 0.001, respectively). The expected reduction in heart rate was significant (p less than 0.05). Total cholesterol and LDL cholesterol increased by 11% (p less than 0.05) and 12.3% (p less than 0.01), respectively. HDL cholesterol showed a small reduction. Tryglycerides increased by 22% (n.s.). LVM did not change. In conclusion, nitrendipine and atenolol showed comparable antihypertensive efficacy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723447 TI - Influence of therapy on silent ischemia and ventricular arrhythmias in hypertensive patients. AB - To assess whether therapy with hydrochlorothiazide (HCTZ) or the calcium antagonist nitrendipine influences silent ischemia or arrhythmias, we studied 10 asymptomatic hypertensive male patients with positive Tl-201 scintigraphy in a double-blind, crossover protocol. Blood pressure (BP) and 48-h Holter monitoring were obtained after 2 weeks of placebo and 8 weeks each of HCTZ and nitrendipine therapy. Ischemia was defined as greater than 1 mm ST-segment depression lasting greater than 1 min and was quantified by the number of episodes, duration, and area under the curve (AUC). The mean number of PVCs per hour and the number of episodes of ventricular tachycardia (greater than 3 beats) were also assessed. Diastolic BP was significantly reduced by both HCTZ and nitrendipine (98 +/- 6 vs. 90 +/- 6 vs. 88 +/- 7 mm Hg, respectively, p less than 0.05), but systolic BP was unchanged for either drug. The number of ischemic episodes was reduced by nitrendipine, from 2.4 +/- 3 to 0.8 +/- 2, (p less than 0.05) but not by HCTZ (2.4 +/- 3 to 1.5 +/- 3, p = NS). The duration of ischemia (37 +/- 43 vs. 5 +/- 9 min, p less than 0.05) as well as the AUC (41 +/- 45 vs. 7 +/- 14 mm/min, p less than 0.05) were reduced only by nitrendipine. The number of PVCs rose with HCTZ therapy, from 19 +/- 34 to 69 +/- 88 (p less than 0.05) and was unchanged by nitrendipine (19 +/- 34 vs. 19 +/- 40, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723448 TI - Cerebral blood flow is not altered by treatment with nitrendipine in patients with mild to moderate hypertension. AB - We evaluated the effects of nitrendipine, a calcium antagonist of the dihydropyridine group, on cerebral blood flow (CBF) in nine patients with mild to moderate hypertension. The CBF was determined under placebo, 2 h after the first dose (short term) and after 4 weeks (long term) of regular nitrendipine treatment. The CBF was determined using the xenon-133 method. All patients were on placebo for 2-4 weeks before nitrendipine administration. The mean age was 48 years (range of 35-67 years). Blood pressure fell from 169 +/- 19/110 +/- 16 to 147 +/- 12/94 +/- 11 mm Hg after the first dose and to 140 +/- 12/92 +/- 9 mm Hg after long-term administration (p less than 0.01). The corresponding CBF did not change significantly from 37.5 +/- 4.8 ml/100 g/min before to 38.7 +/- 4.5 ml/100 g/min 2 h after the first dose and 38.1 +/- 6.0 ml/100 g/min after long-term administration of nitrendipine (p = 0.3 and p = 0.8, respectively). In conclusion, nitrendipine did not alter the CBF of mildly to moderately hypertensive patients. Particularly, the CBF was maintained during short-term and long-term administration despite significant blood pressure reduction. PMID- 1723449 TI - Renal protection with the calcium antagonists. AB - In addition to their role as highly potent antihypertensive drugs, calcium antagonists may also play an important future role in the area of tissue protection and preservation. Calcium antagonists exert favorable effects on renal hemodynamics related to their reversal of renal vasoconstrictors. Calcium antagonists are also capable of blocking intracellular calcium overload induced by various types of ischemic or toxic stimuli. Features such as these may be of substantial value in ameliorating acute renal insufficiency secondary to renal ischemia, iodinated radiographic contrast agents, or the administration of various nephrotoxic drugs. The latter includes agents such as the aminoglycoside antibiotics, cyclosporine A, and the cancer chemotherapeutic agent cisplatin. Recent prospective, controlled studies from our group indicate that calcium antagonists protected against postischemic acute renal failure in the setting of cadaveric renal transplantation. Moreover, in a prospective, randomized, controlled clinical trial, we were able to demonstrate that the prophylactic use of nitrendipine reduced the decrease in GFR in patients receiving radiographic contrast agents. Calcium antagonists may also play a beneficial role in preventing progressive renal disease. Data from a number of studies conducted in experimental animals, as well as information from clinical trials, support such a view. Although the mechanisms of action of calcium antagonists in the setting of chronic renal failure are not yet fully established, their beneficial effects may be related to protective actions such as the reduction in renal hypertrophy, modulation of mesangial cell uptake of macromolecules, changes in permselectivity of the glomerulus, and a decreased free radical formation. These various aspects will be the topic in this review. PMID- 1723450 TI - The future of calcium channel blocker therapy in diabetes mellitus. AB - Diabetes mellitus is associated with significant morbidity and mortality caused by the micro- and macro-vascular complications that all too frequently develop during the lifetime of the diabetic patient. In attempts to treat the complications of diabetes, several different treatment strategies have been investigated. The role of tight blood glucose control in the treatment of diabetic vascular complications has recently been challenged, as the existing data in support of this mode of therapy are currently inconclusive. Perhaps more effective in preventing many of the vascular complications is the rigorous treatment of hypertension that frequently accompanies diabetes mellitus. Epidemiological studies have demonstrated that the presence of hypertension significantly contributes to the development and progression of diabetic nephropathy, retinopathy, cardiovascular disease, and possibly neuropathy. Preliminary clinical studies demonstrate that the progression of diabetic renal disease can be slowed by vigorous antihypertensive therapy. Among the various antihypertensive agents used to treat the hypertension associated with diabetes mellitus, calcium channel blockers are emerging as one of the agents of first choice. This is because of their very low side effect profile and their absence of detrimental effects on serum lipid levels and glucose tolerance. Calcium channel blockers may be of additional potential benefit to the diabetic patient by slowing the progression of atherosclerosis, reversing the intracellular calcium defects that may contribute to the pathogenesis of diabetic cardiomyopathy, and protecting against the progression of chronic renal disease. PMID- 1723451 TI - Treatment of mild hypertension. AB - The mortality of males and females increases two-fold with increasing diastolic blood pressure (DBP) from 83 to 102 mm Hg. There is a corresponding significant increase in cardiovascular morbidity. While there is a general consensus about the need for drug treatment for those patients with DBP of 100 mm Hg or more, there is a controversy as to the justification of pharmacotherapy in the DBP range of 90-100 mm Hg. The arguments against drug treatment of patients with sustained mild hypertension with DBP less than 100 mm Hg rest on results of the large scale clinical trials, which have shown only modest benefit, not commensurate with the risks and inconveniences of pharmacotherapy. Critical appraisal of trials conducted in the past decade suggest a good efficacy of treatment of mild hypertension. Furthermore, recent studies indicate that hypertension is not a symptomless well-being. The inconveniences and side effects of treatment could be greatly reduced with lower doses and selective application of the most appropriate drugs. In a randomized, double-blind, parallel-group study of 80 patients with mild hypertension, 10-20 mg of nitrendipine once a day was an efficacious blood pressure lowering treatment with no significant difference in the incidence of side effects compared to a placebo-treated control group. PMID- 1723452 TI - Metabolic neutrality in nitrendipine therapy. AB - There is evidence that hypertensive patients frequently have other metabolic disorders, such as hyperlipidemia and diabetes mellitus. It is also known that the reduction in high blood pressure alone, disregarding the other cardiovascular risk factors, is unable to reduce mortality to the level of the general population. Moreover, the occurrence of metabolic side effects with some antihypertensive drugs deserves particular attention in the treatment of hypertension. Calcium antagonists seem to be devoid of untoward metabolic effects. In particular, several studies have shown that nitrendipine does not deteriorate glucose tolerance. We have evaluated the effects of nitrendipine on insulin response to i.v. glucose load: no change was observed after 2 months of treatment in both serum insulin levels and glucose percent removal rate in comparison to pretreatment values. No unfavorable change was detectable in the studies aimed at investigating the effects of nitrendipine on lipid metabolism parameters. We observed a 22% increase of the percent removal rate of a lipid emulsion (Intralipid) after nitrendipine (3.11 +/- 1.0 vs. 3.80 +/- 1.0%/min, p less than 0.03). This finding suggests a favorable effect of nitrendipine on triglyceride catabolism, possibly mediated by an interference with lipoprotein lipase activity. The metabolic neutrality of nitrendipine, therefore, leads to considering the usefulness of this drug in an antihypertensive treatment that should not disregard the global risk profile. PMID- 1723453 TI - Treatment of isolated systolic hypertension in the elderly. AB - Isolated systolic hypertension affects between 10 and 20% of the elderly population and carries a substantial risk of cardiovascular complications. As no prospective, randomized trials have produced scientific evidence of a treatment benefit in elderly patients with isolated systolic hypertension, opinion on when and how to treat this condition differs among expert committees as well as among individual doctors. This article reviews the present treatment policies in patients with isolated systolic hypertension. It describes the ongoing intervention studies that have been designed to examine the hypothesis that antihypertensive treatment confers a benefit to elderly patients with isolated systolic hypertension in terms of a reduced morbidity and mortality. The Syst-Eur trial, which was recently initiated by the European Working Party on High Blood Pressure in the Elderly, is described in greater detail. PMID- 1723454 TI - Nitrendipine efficacy and safety in patients with mild and moderate essential hypertension. AB - The antihypertensive effect of nitrendipine was examined in 29 outpatients with a mild or moderate hypertension and type II diabetes or a dyslipidemic condition. The drug was administered for 90 days at a daily dose of 10 to 40 mg. Following a washout period, the blood pressure (measured by a Dinamap device) was 181/99 mm Hg supine and 172/104 mm Hg standing. Nitrendipine caused a reduction in both pressures and after 90 days their values were 148/74 and 143/80 mm Hg, respectively. Heart rate was not affected by the drug, which also caused no variation in blood pressure, total and HDL cholesterol, and triglycerides. In more than 20% of the cases, treatment was associated with headache and flushing, which did not necessitate discontinuation of treatment. Thus, nitrendipine is an effective antihypertensive agent and causes no untoward metabolic effects. PMID- 1723455 TI - Low-dose nitrendipine in mild hypertension: a double-blind, placebo-controlled, comparative study. AB - The objective of this study was to determine whether 10 mg of nitrendipine once daily is adequate for treatment of mild hypertension. The study was a randomized, double-blind, multicenter, and placebo-controlled group comparison over 8 weeks (a 2-week placebo-controlled washout phase followed by a 6-week treatment phase) with measurement of blood pressure, routine laboratory tests, compliance monitoring, and recording of side effects every 2 weeks. The study subjects were 141 outpatients with mild arterial hypertension and were given one 10-mg tablet of nitrendipine or placebo once daily. The end point used was reduction in diastolic blood pressure (DBP) by at least 10 mm Hg or to less than or equal to 90 mm Hg; the responder rate at the end of the treatment phase was determined. After 6 weeks of treatment, the mean reduction in DBP was 11.8 mm Hg under nitrendipine compared with 5 mm Hg under placebo. The responder rates were 73% (53 of 73 patients) in the nitrendipine group and 26% (18 of 68 patients) in the placebo group. This difference is statistically significant. Side effects were reported by a total of 14 patients (6 in the nitrendipine group, 8 in the placebo group). There was one dropout in each group. No changes in laboratory parameters were observed. In conclusion, nitrendipine is suitable for monotherapy of mild arterial hypertension in the dosage of 10 mg once daily used in this study. PMID- 1723456 TI - Circadian rhythm of blood pressure and calcium entry blockade: a chronobiologic study with nitrendipine in essential hypertension. AB - In order to evaluate the chronobiologic effect of nitrendipine, 27 patients with mild-to-moderate arterial essential hypertension were studied. After randomized administration of 20 mg of nitrendipine and placebo, systolic and diastolic blood pressure (SBP and DBP, respectively) and heart rate (HR) were measured for 24 h using an automatic noninvasive device. The data of the time series were statistically analyzed by single and mean cosinor methods (obtaining mesor, amplitude, and acrophase) and by ANOVA and Student's paired t test. Nitrendipine significantly reduced the SBP and DBP mesors without affecting the HR mesor, and reduced the SBP and DBP amplitudes while increasing the HR amplitude. After placebo, group circadian rhythms were observed for SBP, DBP, and HR and maintained after nitrendipine. In conclusion, a single-dose administration of nitrendipine is effective in lowering blood pressure. The increased HR amplitude is probably due to a tachycardic reaction. The preservation of the SBP, DBP, and HR group circadian rhythms agrees with the lack of interference of the drug with the neurohormonal mechanisms. PMID- 1723457 TI - Effectiveness of nitrendipine, 20 mg once daily in the management of hypertension in general practice. AB - The effectiveness of nitrendipine, given as a single 20 mg tablet in the morning, was evaluated in general practice in 6,058 hypertensive patients. They filled in a questionnaire on their activities and previous antihypertensive treatment, if any. Visits were planned after 2, 6, and 12 weeks. Then, patients and general practitioners gave their assessment of the treatment. Eighty-four percent completed the 12-week study while receiving 20 mg of nitrendipine once daily. Adverse events were observed in 26% of the patients, mainly during the first 2 weeks, where flushing and peripheral edema occurred in 9 and 7% of the patients, respectively. Both led to withdrawal of 4% of the included patients over 3 months. A supine diastolic blood pressure below 90 mm Hg was achieved in 65% of the patients, irrespective of age, sex, activity, smoking habits, and presence of diabetes or previous antihypertensive therapy. In conclusion, this large-scale study further established the effectiveness of nitrendipine as monotherapy given once daily in most hypertensive patients. Eight patients in 10 felt they had benefited from the treatment. The investigators were satisfied with the results in 66% of the patients. They considered that the main advantage of nitrendipine was ease of use. PMID- 1723458 TI - Antihypertensive monotherapy with nitrendipine in general practice. AB - Efficacy and tolerability of antihypertensive monotherapy with the calcium antagonist nitrendipine were investigated in a 6-month open trial in 495 patients with mild to moderate essential hypertension from 101 practicing internists and general practitioners. Previous antihypertensive therapy (57.4%) was stopped for 1 week and therapy then started with nitrendipine, 20 mg once daily. Sixty-one patients discontinued therapy prematurely because of unwanted effects, mostly characteristic with dihydropyridines (headaches, flushing, and ankle edema), and 23 patients because of insufficient efficacy. In 75% of the remaining 411 patients, the goal blood pressure was achieved by nitrendipine monotherapy (10 mg in 17.6%, 20 mg in 73.3%, and 20 mg b.i.d. in 8%) and diastolic blood pressure was between 90 and 95 mm Hg in another 6%. The reduction in blood pressure did not result in changes of heart rate or weight. Nitrendipine was effective in patients of all age groups but patients older than 60 years of age showed a significantly greater fall in systolic pressure than middle-aged or young patients. At the end of the study, 15 patients still reported side effects. Nitrendipine appears to be well suited for first-line therapy of mild to moderate essential hypertension. PMID- 1723459 TI - Antiperoxidative actions of calcium antagonists and atherogenesis. AB - Recent experimental and clinical studies suggest that structurally disparate calcium channel blockers retard the progression of atherosclerosis. However, mechanisms of action by which calcium blockers exert their antiatherosclerotic effects have not been completely elucidated. Formation of atherosclerotic lesions involves cells (macrophages, endothelial cells, and platelets) not expressing voltage-dependent (L-type) calcium channels, the major drug receptors for calcium channel blockers. Therefore, it is possible that these drugs act by non-L-type channel mechanisms. Recent reports indicate that nifedipine, verapamil, and diltiazem exert antiperoxidative effects on membrane lipids. It has been suggested that antiperoxidants such as probucol and butylated hydroxytoluene (BHT) exert antiatherosclerotic effects by preventing oxidation of low-density lipoprotein (LDL), a modification thought to confer atherogenic properties to the lipoprotein. Therefore, the beneficial effects of calcium channel blockers might be related to their antiperoxidative activity. PMID- 1723460 TI - A comparative study of the effects of nitrendipine and enalapril in essential hypertension. AB - In this study, we compared the effects of nitrendipine (20-40 mg daily) and enalapril (20-40 mg daily) in 44 patients with mild to moderate essential hypertension. After a 4-week placebo period, the patients entered a double-blind, crossover study of 16 weeks, divided by a second 4-week placebo period. Sitting and standing blood pressures (standard mercurymeter) were measured every 2 weeks. Ten patients dropped out, so 34 patients were evaluable. Two patients dropped out because of surgery, one patient was withdrawn because of accelerating hypertension, and seven patients discontinued because of side effects (two on placebo, four on enalapril, and one on nitrendipine). Sitting blood pressures decreased from 172 +/- 3/107 +/- 1 to 159 +/- 3/94 +/- 1 mm Hg on nitrendipine (p less than 0.001) and to 157 +/- 4/96 +/- 2 mm Hg on enalapril (p less than 0.001). The heart rate did not change. Both compounds had no significant effect on serum lipids and on renal function. With regard to side effects, flushing occurred in 10 patients on nitrendipine and in 3 on enalapril (p less than 0.05); cough was noted in 3 patients on enalapril. When using a diastolic pressure less than 95 mm Hg as a response, 72% responded on nitrendipine and 64% on enalapril (n.s.). In conclusion, nitrendipine and enalapril, given as monotherapy, were equally effective antihypertensive agents in this group of patients with uncomplicated, moderate, essential hypertension. The use of either of the tested agents seems to be more limited by its specific side effects than the lack of antihypertensive efficacy. PMID- 1723461 TI - A double-blind, randomized, comparative study of nitrendipine and enalapril in elderly hypertensive patients. AB - We conducted a randomized, double-blind, double-placebo study in two groups of elderly hypertensive patients aged over 60 years to compare nitrendipine to enalapril, given once daily over 4 months, as monotherapy with 20 mg tablets. Clinic blood pressure was measured monthly and 24 h ambulatory monitoring was obtained at day 0 and day 120. Fifty-two patients entered the study (22 in the nitrendipine group, 30 in the enalapril group); 4 patients in both groups were dropped from the study because they withdrew their consent to participate. Three patients under nitrendipine and five patients under enalapril were not included in the analysis of data because of insufficient follow-up. The two groups were comparable on final analysis. The clinical blood pressure was similarly reduced in both groups at 24 h postdose (172/97 to 157/85 mm Hg on nitrendipine vs. 177/98 to 154/86 mm Hg on enalapril). Seventy percent of the patients had their blood pressure normalized at the end of the study in the two groups. With ambulatory blood pressure recording, no 24 h blood pressure significant differences were found on day 120 when we calculated the mean of systolic blood pressure (SBP) or diastolic blood pressure (DBP) (137 +/- 12/82 +/- 9 mm Hg on nitrendipine vs. 136 +/- 16/82 +/- 11 mm Hg on enalapril), or for daytime or nighttime periods. The pulse pressure or the mean of the five highest SBP values was also similar in nitrendipine and enalapril patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723462 TI - Tolerance to nitrendipine in patients with arterial hypertension accompanying chronic renal failure. AB - To test the tolerance to nitrendipine in arterial hypertension secondary to chronic renal parenchymatous disease, a group of 10 patients so diagnosed and adequately controlled with a diuretic alone or with a beta-blocker were switched to nitrendipine at a dose to insure similarly adequate control of blood pressure. Patients were followed for 1 year at monthly intervals during which blood pressure measurement and the determination of a biochemical profile were performed. During the follow-up, nitrendipine adequately controlled blood pressure and a significant fall was observed in hematocrit (p less than 0.05) and serum uric acid (p less than 0.01) values. Meanwhile, the glomerular filtration rate, determined by the creatinine clearance, did not exhibit significant changes nor did the 24-h urinary excretion of proteins. The tolerance of the drug was adequate, with two patients presenting minimal ankle edema. These results seem to indicate that nitrendipine can be safely used in patients with arterial hypertension secondary to chronic renal parenchymatous disease. PMID- 1723463 TI - Arteriosclerosis and antihypertensive response to calcium antagonists in end stage renal failure. AB - The relationship between the presence of arterial calcinosis and the antihypertensive response to calcium blockers was studied in 40 hypertensive patients with end-stage renal failure (ESRF) on chronic hemodialysis, before and during 16 weeks after administration of nitrendipine in monotherapy. In a double blind, placebo-controlled, randomized study, nitrendipine reduced systolic blood pressure regardless of the presence or absence of arterial calcifications. The antihypertensive effect was significantly more pronounced in subjects with aortic calcium deposits in comparison with patients without clinical signs of arteriosclerosis (p less than 0.01). The diastolic blood pressure was significantly reduced only in patients with aortic calcifications, and remained unchanged in subjects with noncalcified aorta. The aortic pulse wave velocity decreased significantly in patients with aortic calcifications (p less than 0.001), but remained unaffected in patients with noncalcified vessels. Multivariate regression analysis showed that the antihypertensive action of nitrendipine was correlated to the presence of aortic calcium deposits independent of age or baseline blood pressure levels. The results of the present study indicate that an overt arteriosclerosis as demonstrated by the presence of aortic calcifications on abdominal radiographs is a good indication for the use of dihydropyridines in patients with ESRF. PMID- 1723464 TI - Cardiovascular side effects after renal allograft rejection therapy with Orthoclone: prevention with nitrendipine. AB - Orthoclone (OKT-3), a monoclonal antibody, is an effective immunosuppressant in organ graft recipients. One of the reported side effects is serious pulmonary edema, heart failure, hyperdynamia, and elevation of blood pressure. It should be assessed whether patients treated with OKT-3 benefit from antihypertensive therapy with a calcium channel blocker before and during the allograft rejection therapy to prevent from cardiovascular side effects. To assess a preventive cardiovascular effect of therapy with nitrendipine before and during the OKT-3 rejection therapy, the patients studied (n = 28) were randomly allocated to two study groups. Group a without nitrendipine comprised 15 patients, and group b with 2 x 10 mg of nitrendipine daily comprised 13 patients. In study group a (without nitrendipine therapy), in 8 of 15 patients, there was a short-lasting increase in blood pressure during 3 h after the first injection of OKT-3 by 20.0 +/- 12.8 (systolic)/10.1 +/- 6.7 (diastolic) mm Hg. Whereas this initial rise of blood pressure on the first day was accompanied by an increase in heart rate by 24.1 +/- 10.8 beats/min, the longer-lasting increase in blood pressure at day 2 was not associated with significant changes in heart rate. In group b (patients receiving 2 x 10 mg of nitrendipine before OKT-3 therapy was started and during the whole treatment course), in 3 of 13 patients, a short increase in blood pressure (13.7 +/- 2.9/7.8 +/- 5.1 mm Hg) was recorded 5 h after the first dose of OKT-3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723465 TI - Influence of the calcium antagonist nitrendipine on the hepatotoxic effect of cyclosporine A. AB - Calcium antagonists have a protective effect on different forms of nephrotoxicity due to cyclosporine A (CsA). The objective of the present study was to examine the influence of a calcium antagonist on the liver function in CsA-treated patients after kidney transplants. Different quantitative liver function tests were performed in six patients before and 3 months after administration of the calcium antagonist nitrendipine. Indocyanine green clearance (ICG-Cl) as a marker for hepatic blood flow and excretion showed a significant increase under nitrendipine. In addition, there was an improvement of the galactose elimination capacity. Although nitrendipine and lidocaine are both metabolized by the cytochrome P system, there was no reduction in lidocaine clearance. These results suggest an improvement of liver function under nitrendipine. Whether these findings alone are the expression of an improved liver blood flow or whether there is an additional hepatoprotective characteristic of the calcium antagonist nitrendipine cannot be determined. PMID- 1723466 TI - Long-term (3 years) sustained antihypertensive and metabolic actions of nitrendipine in severe, complicated, and resistant hypertension. AB - The antihypertensive efficacy of once-daily nitrendipine was studied in 18 patients with severe, resistant, refractory, and complicated hypertension. The dose range was 20-120 mg/day adjusted weekly for a total treatment period of 3 years. Nitrendipine produced a significant reduction in blood pressure compared to pretreatment baseline values with no significant effects on heart rate. Renal function was preserved and there was an increase in urine flow, urinary excretions of Na+, kallikrein, and prostaglandin E2, and plasma renin. Some patients experienced known calcium antagonist side effects but the drug was otherwise well tolerated. PMID- 1723467 TI - Effects of nifedipine and nitrendipine on insulin secretion in obese patients. AB - Data on the influence of calcium antagonists on glucose tolerance and insulin release in humans are conflicting. The present double-blind, double-dummy, controlled trial was designed to investigate the effect of a short-term (7 days) treatment with nitrendipine, 20 mg b.i.d.; nitrendipine, 20 mg once daily; or placebo on blood glucose and plasma insulin and C-peptide response to an intravenous glucose load in mildly or transiently hypertensive nondiabetic obese patients. No statistically significant differences were found in fasting glucose, insulin, and C-peptide, or in the glucose disappearance rate and in any of the parameters for insulin and C-peptide response after i.v. glucose, between the three groups of patients. However, a slight decrease in early insulin response to glucose was observed in the nifedipine and the nitrendipine groups. This study confirms that calcium antagonists have no clinically relevant effect on glucose homeostasis even if a slight alteration of insulin release after glucose load cannot be ruled out. PMID- 1723468 TI - Nitrendipine once or twice daily in hypertensive non-insulin-dependent diabetes mellitus. AB - Twenty (12 male, 8 female) hypertensive patients were recruited into the study; their mean (+/- SD) sitting blood pressure (BP) was 166 +/- 13/101 +/- 4 mm Hg, age 59.6 +/- 5.8 years, weight 83.4 +/- 14.2 kg, and glycosylated hemoglobin (HbA1) 9.9 +/- 2.6%. After a run-in period of 4 weeks, patients were randomized to receive nitrendipine once or twice daily, with 15 patients completing the 12 week study period. Prior to treatment with nitrendipine, the patients subsequently treated on a twice-daily regimen had a higher sitting systolic BP (SBP) of 173 +/- 15 mm Hg compared to the once-daily patient group at 159 +/- 7 mm Hg (p less than 0.05). There were no differences between the two treatment groups in weight or diabetes control. Once-daily nitrendipine effectively reduced the sitting SBP and diastolic BP (DBP), i.e., SBP fell from 159 +/- 7 to 147 +/- 12 mm Hg (p less than 0.05) and DBP fell from 100 +/- 4 to 86.3 +/- 8 mm Hg (p less than 0.001). Nonsignificant reductions in weight from 81.5 +/- 11.3 to 80.9 +/- 11.2 kg and HbA1 from 9.8 +/- 3.3 to 8.4 +/- 1.3% were also seen. Similarly, with twice-daily nitrendipine, a significant (p less than 0.01) fall in sitting systolic BP from 173 +/- 15 to 148 +/- 9 mm Hg and DBP 103 +/- 4 to 85 +/- 8 mm Hg was also achieved. Little or no change was observed in weight or glycemic control in this patient group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723469 TI - Effect of nitrendipine in mild or moderate essential hypertensive subjects with type II diabetes. AB - The antihypertensive effect of nitrendipine, 20 mg once daily was studied in 30 patients with mild or moderate hypertension and type II diabetes mellitus under metabolic control. Blood pressure was measured at the end of a 15-day washout period from previous antihypertensive treatment and on the 15th, 30th, 60th, and 90th day of treatment. Systolic blood pressure and diastolic blood pressure were significantly reduced by nitrendipine. The reduction involved lying and orthostatic blood pressure and was maintained throughout the treatment period. Heart rate, fasting and postprandial glycemia, and lipid profile were not significantly affected by nitrendipine. It is concluded that this drug effectively lowers blood pressure in diabetic hypertensive subjects with no alteration of lipid and glucose metabolism. PMID- 1723470 TI - Tau-ubiquitin protein conjugates in a human cell line. AB - The microtubule-associated protein tau, and the cytoplasmic protein ubiquitin, are constituents of pathological neurofibrillary tangles found in Alzheimer's disease. In order to see if there is any physiological relationship between these proteins in a functioning human system, human neuroblastoma (LAN-5) cells were grown in vitro and differentiated to a neuronal phenotype. Cell extracts were analyzed by SDS-PAGE, immunoblot, and immunoprecipitation techniques. The colocalization of ubiquitin and tau immunoreactivity was noted in 12- and 35-kDa bands, predominantly located in a cell membrane fraction. The bands were also isolated by immunoprecipitation with the Alz-50 antibody and then identified with a ubiquitin antiserum. These findings show a relationship between tau and ubiquitin in a human neural cell line. This interaction suggests that tau may normally be degraded by an ubiquitin-dependent mechanism and alterations in it may contribute to the formation of neuro-fibrillary pathology. PMID- 1723471 TI - Microbial pathogenesis and tyrosine dephosphorylation: surprising 'bedfellows'. AB - The importance of parasite-directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with the normal function of protein tyrosine phosphatase in eukaryotic cells. PMID- 1723472 TI - Site-directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study. AB - The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel-forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water-filled pore with an approximate diameter of 9.3 +/- 0.4 A. PMID- 1723473 TI - Antibodies to metallothionein. PMID- 1723474 TI - Epitope mapping of metallothionein antibodies. PMID- 1723475 TI - Quantification and identification of metallothioneins by gel electrophoresis and silver staining. PMID- 1723476 TI - Quantification of metallothionein by silver saturation. PMID- 1723477 TI - Linker insertion mutagenesis as probe of structure-function relationships. PMID- 1723478 TI - Nucleic acid sequence localization by electron microscopic in situ hybridization. PMID- 1723479 TI - The use of autoantibodies in the study of nuclear and chromosomal organization. PMID- 1723480 TI - Distribution of chromosomal proteins in polytene chromosomes of Drosophila. PMID- 1723481 TI - Fluorescent detection of nuclear RNA and DNA: implications for genome organization. PMID- 1723482 TI - Imaging of leukocytes within the rat brain cortex in vivo. AB - Confocal laser scanning microscopy was used in a rat closed cranial window preparation in order to study rhodamin 6G-labeled leukocytes within the brain cortex in vivo. Leukocytes were visualized up to 150 microns beneath the rat brain surface in noninvasive optical sections. In pial venules, leukocytes were seen flowing with the blood stream, rolling along or sticking to the endothelium, and migrating through the vessel wall. Within cerebral capillaries, leukocyte flux, velocities, and leukocyte plugging were measured. After additional intravenous administration of fluorescein, the plasma, leukocytes, and erythrocytes were visualized simultaneously. Based on stacks of optical sections of fluorescein-labeled capillaries, the individual capillaries were localized within the three-dimensional microvascular network. The usefulness of this technique was illustrated in a feasibility study in which leukocyte sticking to the vascular walls of venules, leukocyte extravasation, and intracapillary leukocyte plugging were monitored in a model of global cerebral ischemia. PMID- 1723483 TI - Methylene blue stains some vascular smooth muscle cells in rabbit coronary arteries and arterioles. PMID- 1723484 TI - Monoclonal antibody to a structure expressed on a subpopulation of rat CD8 T cell subsets. AB - We have developed a monoclonal antibody, RTS-1, which can divide a rat CD8 (+) peripheral T cell population into two functionally distinct subsets. The cell surface structure defined by this antibody is a glycoprotein with a molecular weight of 220 kDa found to be a high molecular isoform of rat CD45 antigen. CD4 (+) T cells were not stained by RTS-1 antibody. The cytotoxic T cell-enriched population did not express RTS-1 epitope on the cell surface. CD8 (+) spleen cells as well as RTS-1(+)CD8(+)T cells exhibited strong inhibition on mitogen induced immunoglobulin G production by rat B cells. Furthermore, RTS-1 antibody, but not the control antibody, abolished CD8(+)T cell-mediated inhibition of immunoglobulin G production by rat B cells. These data suggest that RTS-1 antibody recognizes a unique determinant of rat CD45 antigen that is expressed on a fraction of CD8(+) cells. PMID- 1723485 TI - Hormonal therapy for hepatocellular carcinoma. AB - Hepatocellular carcinoma is not only the leading cause of male cancer death in Taiwan, but also one of the most common cancers in the world. The survival of hepatocellular carcinoma patients is very low, mainly due to the lack of effective treatments. Radiation and chemotherapies in general are not satisfactory: surgery itself is the most effective treatment for hepatocellular carcinoma but only on small resectable tumors. The overall prognosis is still poor. Previously, we have found that the level of glucocorticoid receptor and its mRNA in hepatocellular carcinoma was significantly higher than that of the adjacent liver tissue. This correlated well with the elevated serum alpha fetoprotein levels in patients with hepatocellular carcinoma. Recently, a female hormone, progesterone, has been found to inhibit the expression of alpha fetoprotein in hepatoma cells. In addition, progesterone has been used to treat a few hepatocellular carcinoma patients with promising responses. These results together with our hypothesis that the expression of alpha-fetoprotein is regulated by glucocorticoid receptor complex in proliferating hepatocellular carcinoma cells lead to the conclusion that steroid hormones and/or their antagonists may interfere with the function of glucocorticoid receptors in tumors, consequently regulate tumor growth. The potential of hormonal therapy for treatment of hepatocellular carcinoma is worthy of further investigation. PMID- 1723486 TI - Effect of culture conditions on PLP and MAG gene expression in rat glioma C6 cells. AB - The effects of culture conditions on the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells was studied. Early passage (40-46) cells had higher steady-state level of PLP- and MAG-specific mRNA than late (100) passage cells when grown in defined (serum-free) medium. The PLP gene expression was increased whereas the MAG gene expression was reduced in the presence of 10% fetal calf serum in either passage. The level of both PLP- and MAG-specific messages was also directly related to the cell density indicating cell contact-induced stimulation of the gene expression. Furthermore, the cells apparently secrete factors into the medium, which upregulate the gene expression in autocrine fashion. The results also indicate a dissimilarity of regulatory mechanisms involved in the expression of the PLP and MAG genes. PMID- 1723487 TI - Surface layers of bacteria. AB - Since bacteria are so small, microscopy has traditionally been used to study them as individual cells. To this end, electron microscopy has been a most powerful tool for studying bacterial surfaces; the viewing of macromolecular arrangements of some surfaces is now possible. This review compares older conventional electron-microscopic methods with new cryotechniques currently available and the results each has produced. Emphasis is not placed on the methodology but, rather, on the importance of the results in terms of our perception of the makeup and function of bacterial surfaces and their interaction with the surrounding environment. PMID- 1723488 TI - Avian myeloblastosis virus reverse transcriptase inhibition by nalidixic acid. AB - Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on the avian myeloblastosis virus reverse transcriptase activity. The drug inhibited the DNA synthesis reaction catalyzed by the viral enzyme in the presence of different template-primers. The inhibitory effect by nalidixic acid was higher with polyriboadenylic acid than with polyribocytidylic acid as a synthetic template. With activated DNA as a template nalidixic acid preferentially inhibited the TMP incorporation when compared with the dAMP incorporation. Both these results showed the importance of the presence of adenine in the templates for a more efficient inhibition by nalidixic acid. The inhibition for this drug was also shown in the presence of Mn2+ instead of Mg2+ as the divalent cation, and with a 2'-fluorinated analogue of polyriboadenylic acid as the template. Kinetic data showed a non-competitive inhibition by nalidixic acid in relation to polyriboadenylic acid and to TTP in the reaction catalyzed by reverse transcriptase. PMID- 1723489 TI - Expression and differential glycosylation of human sex hormone-binding globulin by mammalian cell lines. AB - The human sex hormone-binding globulin (SHBG) gene is responsible for the production of plasma SHBG by the liver and androgen-binding protein in the testis. Cell-specific glycosylation events during synthesis may account for minor differences in the biochemical properties of SHBG and androgen-binding protein, and we have, therefore, expressed a human SHBG cDNA in chinese hamster ovary (CHO) cells and a mouse hepatoma cell line (BW-1), and compared the products to SHBG in serum. The SHBG produced in this way is a homodimer of subunits that exhibit size microheterogeneity similar to SHBG in human serum, and its affinity for 5 alpha-dihydrotestosterone (Kd = 0.6 nM) and other steroids is essentially identical to that of natural SHBG. When medium from transfected CHO and BW-1 cells was subjected to Concanavalin-A (Con-A) chromatography, the relative amounts of SHBG retained by Con-A were 74% and 86%, respectively. In addition, when SHBG produced by CHO cells was separated into two fractions by Con-A chromatography and analyzed by polyacrylamide gel electrophoresis, SHBG that did not interact with Con-A migrated with a slightly larger apparent mol wt than that of SHBG that binds Con-A; this can be explained by the presence of triantennary, rather than biantennary, N-linked oligosaccharide chains. These data also demonstrate that the subunit microheterogeneity associated with plasma SHBG reflects differences in glycosylation during synthesis, which appear to be cell type specific. PMID- 1723491 TI - Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection. AB - The development of technology to increase the sensitivity and speed of detection of bacterial pathogens in samples is important for diagnosis and monitoring of illness. We have developed a sensitive and rapid method for the detection of bacteria, using Escherichia coli as a model, which combines transcription-based target amplification with a bead-based sandwich hybridization assay using rare earth metal chelate labelled probes and time-resolved fluorescence detection. Using these methods as little as 100 copies (0.00016 attomoles) of purified native Escherichia coli rRNA or just one bacterial cell in a spiked sample could be detected. These results demonstrate that amplification of rRNA by transcription-based amplification and detection by time-resolved fluorescence provide a sensitive technology for the direct detection of micro-organisms without the requirement for prior cultivation. PMID- 1723490 TI - Use of antibodies against the P36 protein of Mycoplasma hyopneumoniae for the identification of M. hyopneumoniae strains. AB - Mycoplasma hyopneumoniae, the principal aetiological agent of porcine enzootic pneumonia, synthesizes a 36 kDa protein (P36) which is an early and strong immunogenic factor in experimentally and naturally infected swine. Polyclonal antibodies were made against the recombinant P36 protein in rabbits and used for the identification of M. hyopneumoniae by the immunoblot technique. The proteins from the M. hyopneumoniae reference strains and from 13 M. hyopneumoniae field strains isolated from naturally infected pigs in Switzerland, Hungary, France and Canada were analysed by the immunoblot technique using anti-P36 antibodies. All 13 field strains and the three reference J strains of M. hyopneumoniae, received from different collections and laboratories, exhibited a strong reaction with a protein of 36 kDa indicating that the P36 protein is a common M. hyopneumoniae antigen. None of the different porcine Mycoplasma species including M. flocculare, M. hyorhinis, M. hyosynoviae, A. axanthum, A. laidlawii and A. granularum showed any reaction on the immunoblot with the anti-P36 antibodies. In addition, we have found no reaction with anti-P36 antibodies using 47 different Mycoplasma or Acholeplasma species isolated from human, mice, rat, poultry, ruminant, dog and cat. In conclusion we have shown that P36 is a protein that is a common antigen of M. hyopneumoniae strains and is not found in other Mycoplasma or Acholeplasma species tested. Because of its high specificity, P36 protein, or antibodies made against this protein can be used for the identification of M. hyopneumoniae strains. PMID- 1723492 TI - Carcinogenic ranking of aromatic amines and nitro compounds. AB - A scheme is proposed for ranking the carcinogenicity of aromatic amines and nitro compounds based on both qualitative (weight of evidence) and quantitative (carcinogenic potency, i.e. the TD50 value) factors. The scheme has been drawn up specifically with a view to linking with workplace hygiene controls. Other essential features are that a reliable database exists for the TD50 values for many compounds and that the scheme is capable of usage by non-toxicologists. Validation of the scheme using 38 aromatic amines or nitro compounds indicates that the main objectives have been met. Extension to different chemical classes should be possible but has not been attempted in this work. An example of a potential hygiene control scheme for use alongside the carcinogenicity ranking is described. PMID- 1723493 TI - Genotoxicity of beryllium, gallium and antimony in short-term assays. AB - The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BeCl2, Be(NO3)2, GaCl3, Ga(NO3)3, SbCl3, SbCl5, and an oxide, Sb2O3, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl3 and Sb2O3, and 2 beryllium compounds, BeCl2 and Be(NO3)2, induced SCEs significantly. Sb2O3, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses. PMID- 1723494 TI - High mutability in rye (Secale cereale L.). AB - Analysis of the rye cultivar Ailes of several descents derived from crosses between plants carrying specific genotypes and/or chromosome constitutions resulted in the detection of high chromosome (2.05 x 10(-2)) and gene (9.3 x 10( 3)) mutation frequencies. The existence of a transposon system responsible for this instability is suggested. PMID- 1723495 TI - Absence of mutagenicity of the antitumor drug 3-nitrobenzothiazolo[3,2 a]quinolinium chloride (NBQ) in the germ cells of Drosophila melanogaster males. AB - The antitumor drug, 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ) was tested for genotoxicity with the sex-linked recessive lethal test by feeding Drosophila melanogaster males. Although toxic to adults, the drug tested negative at the concentrations studied. PMID- 1723496 TI - Growth of Chinese hamster ovary (CHO) cells in the presence of MMC in low-serum medium. AB - Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10% fetal bovine serum (FBS) were adapted to 0.5% FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10(-7) M and 10(-8) M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5% serum proliferate, though at a slower rate than cells in 10% serum. Treatment with MMC revealed that at 10(-7) M, MMC is cytotoxic to cells to both the media; at 10(-8) M, MMC is non-cytotoxic to cells in both media. PMID- 1723497 TI - Micronucleus formation in cultured fetal liver blood cells using mitomycin C. AB - Mouse fetal-liver blood cells were cultured and used to investigate micronucleus formation after exposure to mitomycin C (MMC). The isolated fetal cells were incubated in a medium supplemented with erythropoietin (EPO), and the frequency of micronuclei formation was detected in polychromatic erythrocytes (PCE). The effects of four variables were investigated: (1) MMC exposure dose, (2) MMC exposure time, (3) incubation time, and (4) EPO concentration. PCE were formed by proliferation and differentiation of the erythroid cells in culture. Micronucleated PCE (MNPCE) were observed in a dose-dependent manner after exposing the cultured cells with up to 1.0 microgram/ml MMC. The optimum time of MMC exposure and post-exposure incubation was 3 h and 48 h, respectively, and the optimum EPO concentration was 0.25 U/ml. Mouse fetal-liver PCE are sensitive primordial cell targets that can be obtained in relatively large numbers from a single pregnant animal. The procedure is relatively simple and potentially useful in detecting mutagens and carcinogens capable of causing chromosomal damage. PMID- 1723498 TI - Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA100. A comparative study. AB - The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2 propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system. PMID- 1723499 TI - Induction of sperm abnormalities in mice by norfloxacin. AB - Norfloxacin was tested in the mouse sperm morphology test. Data obtained suggest that norfloxacin may have 2 different effects on sperm development: a stimulating effect on spermatogenesis and a possible mutagenic effect that results in an increase in sperm abnormalities. The first effect might be caused by a hormonal action. A dose-response relationship was not observed in sperm morphology changes. Consequently norfloxacin cannot with certainty be judged a positive inducer of abnormal sperm, but further studies are essential to clarify the obtained results. PMID- 1723500 TI - Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli. AB - The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed. PMID- 1723501 TI - The influence of the nucleotide excision-repair system on mutagenesis in Salmonella typhimurium LT2 after exposure to low doses of monofunctional alkylating agents. AB - The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and N-ethyl-N nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS less than ENU less than ENNG less than MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of all the alkylating agents used, the nucleotide excision repair-proficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision repair enzymes and constitutive O6-methylguanine-DNA methyltransferase, is discussed. PMID- 1723502 TI - In vivo exposure of human lymphocytes to technetium-99m in nuclear medicine patients does not induce detectable genetic effects. AB - Recent reports have demonstrated that exposure of nuclear medicine patients to thallium-201 does not result in a detectable increase in mutation at the hprt locus in human lymphocytes. In an effort to study further the potential genetic effects of medical exposures to low dose radiation, we have examined chromosome aberrations and mutations in peripheral blood lymphocytes from nuclear medicine patients exposed to clinical doses of technetium-99m. Our results show that there is no exposure-related increase in chromosomal damage; furthermore, the data do not confirm earlier reports of exposure-related increases in mutations induced by technetium-99m. PMID- 1723503 TI - Chromosomal effects of theophylline measured in mouse marrow cells in vivo. AB - Doses of 62.5, 125 and 250 mg/kg of theophylline were administered to male B6C3F1 mice by intraperitoneal injection. Chromosome aberrations were scored in first division metaphases of marrow cells 18 and 36 h post-treatment and sister chromatid exchanges were quantified in second-division metaphases at 24 h. A modest but statistically significant increase in the number of SCEs occurred, but chromosome aberrations were not significantly different from controls following treatment with any level of the drug at either time period. PMID- 1723505 TI - Stimulation of bacterial mutagenicity by inhibitors of mammalian cell multidrug resistance. PMID- 1723504 TI - In vitro studies on genotoxicity and cytotoxicity of the anticancer drugs cisplatin and cofplaton, a caffeine-8-ether plus cisplatinum compound. AB - The cytotoxic and genotoxic properties of the newly designed anticancer drug 'cofplaton' were investigated. Since cofplaton is a cisplatin (CDDP) plus caffeine compound, the widely applied anticancer drug CDDP alone or in combination with caffeine was studied in parallel. As measured by the MTT test the cytotoxicity of the two drugs was comparable, but cofplaton exhibited significantly fewer genotoxic side effects than CDDP in the chromosome aberration test as well as in the SCE assay. First results from animal studies indicate that cofplaton exerts antitumor activity comparable to CDDP. Because of its relatively low genotoxicity, cofplaton seems to be a promising drug in human anticancer therapy. PMID- 1723506 TI - The induction of specific-locus mutations with N-propyl-N-nitrosourea in stem cell spermatogonia of mice. AB - A specific-locus test to determine the effect of N-propyl-N-nitrosourea (PNU) on the stem-cell spermatogonia of mice has been performed. Male wild-type mice (C3H/He) were treated with an intraperitoneal injection of 200 mg/kg [corrected] of PNU. Eight weeks after the injections, the males were mated with tester stock females (PW), homozygous for 6 visible recessive genes. Twelve mutants among 8605 offspring were observed. The mutation frequency with PNU was calculated to be 23.2 x 10(-5)/locus/gamete, showing a significant difference from that of the non treated control. The mutations were all heritable and half of them were viable in homozygous condition. The mutation frequency with PNU was about one-third of that with N-ethyl-N-nitrosourea, a highly potent mutagen for mouse stem-cell spermatogonia. PMID- 1723507 TI - African trypanosomes express an immunogenic protein with a repeating epitope of 24 amino acids. AB - Infection with intracellular protozoan parasites such as Plasmodium, Leishmania and Trypanosoma cruzi induces a strong antibody response against proteins containing tandem repeats, suggesting that these repetitive epitopes may camouflage vulnerable parasite antigens from a 'protective' immune response. We tested this theory by immunoscreening a cDNA expression library of African trypanosomes, extracellular parasites that evade their hosts' immune response by antigenic variation, and found that the most frequently detected trypanosome protein contains more than 40 tandem copies of a 24-amino acid repeat with a consensus sequence of A-M-E-D-E-L-D-S-L-R-A-L-N-E-Q-Y-E-A-L-Q-R-T-N-A (net charge = -4). This protein is encoded on an mRNA of more than 20 kb and has slight sequence similarities with cytoskeletal, intermediate filament proteins in other organisms. Thus, protozoan proteins with tandemly repeating epitopes do not exist solely to divert the humoral immune response; they have other specific physiological functions for the parasites and affect the overall parasite-host interaction in unknown and perhaps different ways. PMID- 1723508 TI - Electrophysiological characterization of diazepam binding inhibitor (DBI) on GABAA receptors. AB - The gamma-aminobutyric acid (GABAA) receptor complex is a hetero-oligomeric protein which contains an integral chloride channel and several modulatory domains. The ligands of benzodiazepine recognition sites can up- or down-regulate the activity of the GABAA receptor. The effects of DBI (diazepam binding inhibitor) on GABAA receptors have been studied in cultured mammalian central neurons. Experiments performed with patch-clamp techniques, as well as with conventional intracellular microelectrodes, have revealed a reversible reduction of GABA-induced responses by micromolar concentrations of DBI. This effect was prevented by Ro 15-1788 (flumazenil), a selective benzodiazepine receptor antagonist. From these data, DBI is capable of reducing the activity of the GABAA receptor complex by specifically interacting with the benzodiazepine recognition site. The idea of DBI being a negative allosteric modulator of GABAA receptor channels is in agreement with biochemical, as well as behavioral, pharmacology data. PMID- 1723509 TI - Alcohol, the chloride ionophore and endogenous ligands for benzodiazepine receptors. AB - Considerable evidence suggests that at least some of the effects of ethanol are mediated by an action on the GABAA receptor chloride channel complex. More speculative is the suggestion that ethanol might interact with endogenous ligands for the benzodiazepine receptor on the complex. This paper considers the evidence for such interactions. PMID- 1723510 TI - Behavioral syndrome induced by allylnitrile, crotononitrile or 2-pentenenitrile in rats. AB - A single oral administration of allylnitrile, crotononitrile or 2-pentenenitrile in rats induced behavioral abnormalities, such as head-twitching, head weaving, hindlimb abduction, backward pedaling and pivoting. The head-twitching, which was most consistently observed, was suppressed by serotonin (5-HT) antagonists, cyproheptadine or methysergide or by the 5-HT depleter, dl-p-chlorophenylalanine but was accentuated by the 5-HT releaser, dl-p-chloroamphetamine. The results suggest that the 5-HT system is involved in producing the behavioral abnormalities. To discover the effects of allylnitrile, crotononitrile and 2 pentenenitrile on the metabolism of 5-HT and dopamine, 6 areas of the brain of the rat were examined on days 1, 6, 15 and 30 after injection. Each of the nitriles caused significant increases in the level of 5-HT and 5 hydroxyindoleacetic acid (5-HIAA) and in the ratio of 5-HIAA/5-HT, one day after injection. The increase in 5-HIAA was most remarkable, suggesting an enhancement of the serotonergic system. The three nitriles had no effect on the metabolism of dopamine, over a period of 30 days. PMID- 1723511 TI - (+) 3-[3-hydroxyphenyl-N-(1-propyl) piperidine] selectively differentiates effects of sigma ligands on neurochemical pathways modulated by sigma receptors: evidence for subtypes, in vivo. AB - The effects of sigma ligands, (+)3PPP 3-[3-hydroxyphenyl-N(1-propyl) piperidine] and (-)butaclamol, were evaluated in vivo on the metabolism of dopamine (DA) and in the striatum release of adrenocorticotrophic hormone (ACTH) and prolactin in the rat and changes in levels of cyclic guanosine monophosphate (cGMP) in the cerebellum of the mouse and compared with the effects of (+)NANM (N-allyl normetazocine, SKF 10,047) and (+)pentazocine. Both (+)3PPP and (-) butaclamol decreased the release of prolactin and did not affect the metabolism of DA. N Allyl-normetazocine and (+)pentazocine increased release of prolactin and have been shown previously to increase the metabolism of DA. All four ligands increased release of ACTH; however, only the increases caused by (+)NANM and (+)pentazocine were reversed by pretreatment with CPP, a N-methyl-D-aspartate (NMDA) receptor antagonist. (+)Pentazocine and (+)NANM inhibited the NMDA receptor-mediated changes in levels of cGMP in the cerebellum of the mouse, while (+)3PPP and (-)butaclamol did not attenuate the response to NMDA. In addition to further confirming a functional interaction between sigma receptors and NMDA receptors, these studies divide the observed effects of putative sigma ligands into two groups, characterized by benzomorphan compounds and non-benzomorphan compounds, suggesting the possibility of subtypes at sigma receptor in vivo. PMID- 1723512 TI - Dissimilar responses of adult thalamic monoaminergic and somatosensory afferent fibers to implantation of thalamic fetal cells. AB - It is generally accepted that transplanted fetal neurons can, after several weeks to months, establish connections with the host CNS. Host afferent systems seem, however, to show different types of responses to the presence of grafted fetal neurons. The present study is a preliminary step to identify mechanisms involved in the reactions of adult axons to transplanted fetal neurons. The right ventrobasal thalamus of adult rats was depleted of neurons by in-situ injection of kainic acid and cell suspensions from homotopic thalamic embryonic primordia which were injected into the lesioned area. After various post-implantation delays, ranging from five to 30 days, two types of experiments were performed: (i) noradrenaline and serotonin immunohistochemistry with specific antibodies on alternate sections; and (ii) anterograde tracing using wheat germ agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei and the principal sensory trigeminal nucleus. Five days after transplantation, host monoaminergic fibers (either noradrenergic or serotoninergic) had already grown into the transplants. Ingrowing fibers were thin and poorly varicose, exhibiting endings morphologically similar to the growth cones observed during axogenesis. Seven days after grafting, growth cones were no longer visible and monoaminergic fibers exhibited either normal-sized or very large varicosities. Large varicosities progressively decreased in number and, after three weeks, the fibers displayed a normal adult morphology, forming a dense network all over the transplants. In contrast, host somatosensory afferents, labeled by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, did not grow into the transplants. Intermingling of somatosensory afferents and transplanted cells was observed only after 10 days, when grafted neurons extended outside the original transplantation site into the neuron-depleted area containing the somatosensory afferents. The present results demonstrate that adult monoaminergic and somatosensory afferents, when deprived of their usual target, do not react in a similar way to the addition of fetal neurons. It is proposed that adult monaminergic fibers have the ability to regain morphological (and probably functional) immature forms which were considered to be restricted to the period of axogenesis or to lesion-induced regeneration. In contrast, fetal transplants do not seem to induce, by themselves, a similar alteration of genetic expression in adult somatosensory neurons. It has been proposed that "diffuse" and "point-to-point" axonal systems may be differentiated in the CNS on anatomical bases. The present results add to the identification of two different systems by demonstrating that, in the thalamus, they present dissimilar responses to the implantation of fetal cells. PMID- 1723513 TI - Plasticity in crossed and uncrossed tuberomammillary-striatal projections in relation to recovery from behavioral asymmetries induced by hemivibrissotomy. AB - The influence of unilateral removal of the adult rat's vibrissae on crossed and uncrossed tuberomammillary-striatal projections was examined with the horseradish peroxidase tract tracing technique. Striatal afferents from the caudal magnocellular and the postmammillary caudal magnocellular subnuclei of the tuberomammillary nucleus were investigated. Between four and 20 days after clipping the vibrissae on one side, more retrogradely labeled cells were found in the crossed and uncrossed projections from both subnuclei to the caudate-putamen of the hemisphere opposite to vibrissae removal (i.e. the sensory deprived hemisphere) than in the projections to the caudate-putamen on the vibrissae clipped side. In contrast, between one and three days after clipping the vibrissae no asymmetries in cell-labeling in these projections were found. This time-course of changes in the tuberomammillary-striatal projections is similar to the time-course of behavioral recovery reported after unilateral removal of vibrissae. Thus, the neural changes in these projections appeared at the point in time when rats recovered from behavioral asymmetries induced by vibrissae removal. PMID- 1723514 TI - Similarities and differences in the action of resiniferatoxin and capsaicin on central and peripheral endings of primary sensory neurons. AB - We have compared the ability of capsaicin and resiniferatoxin, a natural diterpene present in the latex of plants of the Euphorbia family to excite and desensitize capsaicin-sensitive primary afferents in a variety of models. Both capsaicin and resiniferatoxin inhibited the twitch contractions of the rat isolated vas deferens and prevented, in a concentration-related manner, the effect of a subsequent challenge with 1 microM capsaicin (desensitization). Resiniferatoxin was 1000-10,000 times more potent than capsaicin in both cases. The time course of action of resiniferatoxin was much slower than that of capsaicin, suggesting a slower penetration rate in the tissue. The action of resiniferatoxin was blocked by Ruthenium Red, a proposed antagonist at the cation channel coupled to the capsaicin receptor. Both capsaicin and resiniferatoxin produced a contraction of the rat isolated urinary bladder. Resiniferatoxin was about as potent as capsaicin in this assay although it was 500-1000 times more potent than capsaicin in desensitizing the primary afferents to a subsequent challenge with capsaicin itself. Resiniferatoxin did not affect motility in the isolated vasa deferentia or urinary bladder from capsaicin-pretreated rats. After topical application onto the rat urinary bladder both resiniferatoxin (10 nM) and capsaicin (10 microM) increased bladder capacity as assessed in a volume-evoked micturition reflex model in rats without affecting micturition contraction. Intrarterial injection of resiniferatoxin or capsaicin in the ear of anesthetized rabbits evoked a systemic depressor reflex due to activation of paravascular nociceptors, resiniferatoxin being about three times more potent than capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723515 TI - Neuropeptide changes in a primate model (Cebus apella) for tardive dyskinesia. AB - Tardive dyskinesia has been connected with regional reductions of GABA functions in the basal ganglia. In view of the possibility that peptides are involved in neuroleptic-induced dyskinesias substance P and dynorphin A levels were measured in the basal ganglia of the Cebus apella model for tardive dyskinesia. In addition, regional glutamate decarboxylase activities, dopamine, homovanillic acid and dihydroxyphenylacetic acid levels were monitored. A significant dyskinesia-related decrease in glutamate decarboxylase activity was found in the subthalamic nucleus, the medial segment of globus pallidus and the rostral part of substantia nigra in accordance with earlier findings. Cebus monkeys with an intact GABA system (neuroleptic-treated controls without dyskinesia) showed increased levels of substance P and homovanillic acid in the caudate nucleus. The changes were confined to the caudal part of the body of the caudate and the nucleus accumbens. On the other hand, the dyskinetic monkeys, with a defective GABA system, did not demonstrate a similar substance P rise in the caudate or nucleus accumbens, but showed a depression of homovanillic acid levels in the caudal part of the body of the caudate nucleus. Dynorphin A, dopamine and dihydroxyphenylacetic acid showed no dyskinesia-related changes. In conclusion, the difference in glutamate decarboxylase activity between animals developing dyskinetic symptoms vs those who did not, was reflected by regional changes in substance P and homovanillic acid levels. PMID- 1723516 TI - [Ergometric test in the diagnostic and prognostic evaluation of arrhythmia]. AB - One thousand three hundred patient's stress tests were analyzed to value arrhythmia outline and its relationship with anamnesis and clinical data. The patients were divided into two groups: group A, with no arrhythmias at rest, and group B with arrhythmias at rest. All classic nosographic arrhythmias were considered. In some teenager patients with arrhythmias at rest and no organic cardiopathy, anomalies disappeared during stress, showing the benignity of the phenomenon. Supraventricular stress induced arrhythmias has 1.2% of incidence in normal subjects, but 81% in heart disease patients. Supraventricular tachycardia was induced in 6 patients. Ventricular stress induced arrhythmias are found in 4.9%. Two cases of sudden death occurred in our groups. PMID- 1723517 TI - [AIDS in the world]. PMID- 1723518 TI - A method for identifying megakaryocytic and erythroid cells by a combined immunocytochemical and cytochemical stain. AB - The effectiveness of immunoperoxidase staining for platelet glycoprotein IIb/IIIa coupled with cytochemical staining for hemoglobin has been evaluated as a dual staining technique for identifying lineage-specific characteristics of the megakaryocytic and erythroid series in cells of the same hemopoietic tissue preparation. A sequence of staining was established which produced a circumferential brown stain around cells of the megakaryocytic series, and a diffuse yellow to orange stain in the cytoplasm of cells of the erythroid series. Each staining reaction possessed a high degree of specificity and sensitivity which enabled the combined stain to provide a convenient means for establishing the megakaryocytic and erythroid nature of individual cells in hematological specimens where classification cannot be achieved with certainty on morphological grounds. PMID- 1723519 TI - Viral-like characteristics of particle aggregates in a lymphoblastic cell line. AB - A lymphoblastic cell line (K45) established from a child with acute lymphoblastic leukemia (ALL) is characterized by a profusion of intracytoplasmic particle aggregates (PA) similar in morphology to those occurring in fresh childhood ALL cells. The PA in K45 cells were examined for morphology and capacity for nucleic acid synthesis to test the hypothesis that they are identical to those in fresh ALL cells, and also to identify characteristics which might distinguish PA from cell organelles and which might determine if they are viral-like. In contrast to cell organelles, the PA in both K45 and ALL cells were found to be characterized by a localized thickening of the particle wall. Furthermore, autoradiography of K45 cells showed uptake by PA of 3H uridine rather than 3H thymidine indicating an RNA composition. The presence of PA in profusion was associated with, but preceded necrotic death of K45 cells. These combined features suggest that the PA in K45 and in ALL cells are identical, that they are distinct from cell organelles, are not formed as a consequence of the initiation of cell death and that, while their exact nature remains unknown, a viral origin cannot be excluded. PMID- 1723520 TI - Intravascular large cell lymphoma: diagnosis on renal biopsy. AB - The case is reported of a woman aged 60 yrs who presented with systemic symptoms and who was found to have proteinuria of 3.5 g per day. A renal biopsy revealed numerous neoplastic cells filling many of the glomerular capillary lumina. Immunoperoxidase stains revealed that the phenotype of the malignant cells was LCA+, L26+, MB2+, UCHL1-, CD43-, CAM5.2- and S100-, indicating that they were of lymphoid origin and B-cell lineage. The diagnosis of intravascular large cell lymphoma was therefore made. Remission was induced by chemotherapy with CAVP (cyclophosphamide, adriamycin, vincristine and prednisone). A subsequent relapse was treated with cyclophosphamide, VP16 and prednisone, and again remission occurred. This is the first case known to the authors in which the diagnosis of intravascular large cell lymphoma was made on renal biopsy. We confirm the experience of others that chemotherapy with regimens utilized in other varieties of large cell lymphoma may also be appropriate for this unusual neoplasm. PMID- 1723521 TI - Dimethyl suberimidate as an effective crosslinker for antibody-enzyme conjugation. AB - Dimethyl suberimidate (DMS), a bifunctional reagent was used for the first time to crosslink the alpha-feto protein monoclonal antibodies (AFPMAb) to horse radish peroxidase (HRP). Three batches of conjugates were prepared, purified by Sephadex gel chromatography and evaluated for their immunological reactivity. The Rz values obtained for AFPMAb-HRP conjugate were 0.39 to 1.36. Under optimised conditions the ELISA results showed the optical density of 1.9. The iso-electric focusing for the conjugate revealed different degrees of crosslinking between antibodies and HRP. It was evident that isoperoxidase-C was involved in the crosslinking process. From the dot ELISA, as low as 25 pg of AFP in the test samples could be detected with AFPMab-HRP conjugate. The conjugate prepared by DMS was stable at 0 degrees C for more than 10 months. PMID- 1723522 TI - Bleomycin A2 and B2 purification by flash chromatography for chemical and biochemical studies. AB - The clinically used formulation of the anticancer antibiotic, Blenoxane, is a mixture of bleomycin congeners. A new approach to separating the major A2 and B2 congeners has been developed utilizing the flash chromatography technique. A 5-6 inch column of fine mesh silica gel with a solvent system of 1% ammonium formate:methanol (2:3) was used. Low air pressure was applied to the column to increase the flow rate such that separation was complete in approximately 20 min. Reverse phase size exclusion gravity chromatography with Sephadex G-15 column bedding was an effective, rapid procedure for removal of the 1% ammonium formate, the lowest percentage practical for separating the bleomycins. This separation approach does not damage the antibiotics, as demonstrated by NMR spectroscopy, thin layer chromatography, and DNA cleaving activity. Although not as useful for detection of trace amounts of the drug in biological systems as some of the known HPLC methods, this method is excellent for separating large quantities of the drug (8-32 mg) in order to obtain congeners pure enough for synthetic, biochemical, and biophysical studies. PMID- 1723523 TI - Survival and B-cell function of neonatal pig pancreatic islet-like cell clusters in an extracellular matrix. AB - Islet-like cell clusters (ICCs), formed from single cells of pig pancreas in suspension culture, were embedded in an extracellular matrix. It was recently reported that nicotinamide prevented dissolution of the extracellular matrix by the ICCs. In this experiment, various conditions for embedded culture of ICCs in an extracellular matrix were studied, in an attempt to maintain the function as well as the extracted insulin content of culture specimens. The ICCs in the matrix were refed with RPMI 1640, containing 10 mM nicotinamide, 10% fetal bovine serum (FBS), 11 mM D-glucose and with or without 0.1 mM 3-isobutyl-1 methylxanthine (IBMX) or 1.0 micrograms/ml caerulein. A comparison between the different culture media showed that embedded ICCs, maintained in RPMI 1640 with caerulein, in the presence of nicotinamide, had higher insulin content accumulation than when maintained in medium containing nicotinamide alone, but had impaired glucose-stimulated insulin secretion. In the medium containing IBMX and nicotinamide, embedded ICCs showed higher insulin accumulation but lower insulin content, compared to ICCs maintained in the presence of caerulein, and also showed impaired glucose-stimulated insulin release. Thus, the effect of nicotinamide on the survival and function of B-cells is amplified by the presence of caerulein or IBMX. PMID- 1723524 TI - Influence of exogenous application of pancreatic extracts on endogenous pancreatic enzyme secretion. AB - The existence of negative feedback inhibition of human pancreatic enzyme secretion by proteases is controversially discussed. We have recently demonstrated that jejunal application of porcine pancreatic extracts, in a dose commonly used to treat digestive insufficiency, stimulated rather than inhibited, human pancreatic enzyme secretion. We have now studied the influence of duodenal application of high concentrations of either pure trypsin or porcine pancreatic extracts with trypsin-equivalent activity, on human pancreatic enzyme secretion. Twenty-three male volunteers were intubated with a gastric tube and a two-lumen jejunal tube to collect secretions separately via the first and third tubes and to perfuse either pure trypsin or porcine pancreatic extracts distal to the pylorus via the second tube. PEG-4.000 was continuously perfused via the second tube to correct for losses of volume. Volunteers received PEG alone during the first hour, phenylalanine during the second, PEG alone again during the third, and either phenylalanine together with trypsin or porcine pancreatic extracts during the fourth h. Activities of lipase, amylase, and chymotrypsin were measured in 15-min fractions. In addition, human lipase secretion was measured with an enzyme immunoassay, which does not crossreact with porcine lipase. Plasma cholecystokinin (CCK) was measured using a sensitive bioassay, which utilizes amylase release by isolated rat pancreatic acini. Perfusion of the duodenum with phenylalanine caused a statistically significant stimulation of enzyme secretion. This stimulation could be inhibited by high concentrations of pure trypsin. In contrast, application of porcine pancreatic extracts, which contained the equivalent activity of trypsin, caused further increases of lipase secretion when compared to phenylalanine alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723525 TI - Alterations of amylase secretion in the chronically reserpinized rat: an acetylcholine-mediated phenomenon. AB - Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired secretion, and decreased pancreatic content of amylase. However, the mechanisms of altered acinar pancreatic function in both CF patients and reserpine-treated rats are still unknown. The present study was designed to investigate the secretory response of the rat pancreas after reserpine treatment. Rats were given reserpine (1 mg kg-1 day-1 ip) or vehicle, and killed 2, 4, or 7 days later. To distinguish between specific effects of reserpine and those related to secondary malnutrition caused by the drug, secretory response of a group of pair-fed animals to reserpine was also investigated. During reserpine treatment, body weight and food intake were significantly reduced from the 2nd day on. Amylase release from dispersed pancreatic acini, prepared from control pair-fed and from reserpine-treated rats were used to evaluate functional secretory capacity. After 7 days, both chronic reserpine and pair-feeding significantly decreased pancreatic amylase concentration to 75 and 50% of controls, respectively. Reserpine caused desensitization of the pancreatic acini secretory response to carbamylcholine, without altering maximal amylase release. Desensitization occurred gradually and reached a maximum after 7 days. Secretory responses to caerulein and to the phorbol ester 12.0-tetradecanoyl phorbol-13 acetate (TPA) were not altered. These findings indicate that desensitization of amylase release after reserpine was specific to this muscarinic agonist. It may result from increased endogenous acetylcholine release in the course of treatment. PMID- 1723526 TI - Dorsal hippocampal lesion abolishes the response to FG-7142 in the rat. AB - The effects of the anxiogenic beta-carboline FG-7142 (15 mg/kg IP) on exploratory locomotor activity were assessed in rats with sham or ibotenic acid (IA) lesions of the dorsal or ventral hippocampus. FG-7142 reduced exploratory activity similarly in control animals as well as in those with IA lesions of the ventral hippocampus. In contrast, FG-7142 had no effect on rats with dorsal hippocampal lesions. The results suggest that the dorsal hippocampus plays a unique role in FG-7142-mediated attenuation of locomotor exploration. Other studies suggest that serotonergic systems may mediate these properties of FG-7142. PMID- 1723528 TI - TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells. AB - The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells. PMID- 1723527 TI - Pharmacological profile of a potential anxiolytic: AP159, a new benzothieno pyridine derivative. AB - AP159 [N-cyclohexyl-1,2,3,4-tetrahydrobenzo(b)thieno(2,3c)pyridine]-3- carboamide,hydrochloride) showed clear anti-conflict activity in rats in the absence of effects on muscle relaxation, potentiation of anesthesia (in mice) or anticonvulsant activity (in mice). This anti-conflict effect was antagonized by treatment with Ro15-1788. By contrast with the deficits produced by diazepam, AP159 did not impair passive avoidance. The latter drug also improved scopolamine induced amnesia in the same task. AP159 did not inhibit 3H-flunitrazepam binding, but potently inhibited 3H-8OH-DPAT binding. This compound increased serotonin and 5HIAA content of the midbrain raphe nuclei and of the amygdala centralis. AP159 has been shown to be a novel non-BZP anxiolytic agent with no side effects in laboratory animals; it could be a clinically effective anxiolytic agent. PMID- 1723529 TI - A study of the shape of dose-response curves for acute lethality at low response: a "megadaphnia study". AB - Dose-response curves were developed for the immobilization response in Daphnia magna to four toxicants. The purpose of this work was to study the effect of the form of the model and the number of concentration levels used on the estimates of typical low-dose effective concentrations (1%, 5%, 10%). The generalized four parameter logistic model was used as the reference. When using 12 concentration levels, one of the logistic family two- or three-parameter models was shown reliably to represent each of these various sets of dose-response data, and to provide adequate estimates of EC01 and EC05, as well as EC10 and EC50. For two of the toxicants, an asymmetric model was required. When reducing the number of concentrations to five, the EC10 and EC50 were well estimated by the probit model, with acceptable results at the EC05 level. PMID- 1723530 TI - [Macroamylasemia and acquired immunodeficiency syndrome]. PMID- 1723531 TI - [Round table discussion. Amniocentesis before 15 weeks of amenorrhea. Limits of the method]. PMID- 1723532 TI - Sustainability of forestomach hyperplasia in rats treated with ethyl acrylate for 13 weeks and regression after cessation of dosing. AB - In a chronic study conducted by the National Toxicology Program (NTP), gavage administration of 100 or 200 mg ethyl acrylate (EA)/kg/day, 5 days/week, to F344 rats and B6C3F1 mice resulted in a significant dose-dependent increase in the incidence of squamous cell papillomas and carcinomas of the forestomach of both sexes of rats and mice. No increase in the incidence of tumors was observed at any other site in these rats. Chemically-induced cell proliferation is currently thought to play a role in the development and progression of chemically-induced neoplasia. Therefore, a stop-study was initiated where 100 or 200 mg EA/kg (in corn oil) was administered daily, 5 days/week, for 13 weeks. Rats sacrificed at the end of the treatment regimen had severe epithelial hyperplasia of the forestomach. No lesions were observed in the glandular stomach or liver of EA treated rats. Forestomach hyperplasia induced by EA included upward and downward cell proliferation. However, forestomachs of rats treated for 13 weeks and sacrificed 8 weeks after the last EA dose exhibited a significant decline in the incidence and severity of forestomach mucosal hyperplasia. Histopathologic evaluation of forestomachs of EA-treated rats (13 weeks) which were allowed a 19 month-recovery (with no exposure to EA) showed further decline in the incidence and severity of mucosal cell hyperplasia. These results indicate that gavage administration of EA to rats results in extensive and sustained forestomach mucosal hyperplasia. The sustainability of forestomach hyperplasia is apparently dependent on the continued exposure of rats to ethyl acrylate, and regressed after cessation of dosing. Furthermore, although enough post-treatment time was allowed for tumors to develop after cessation of EA administration, forestomachs exhibited a nearly complete recovery with no increased incidence of papillomas or carcinomas. It, therefore, remains to be determined what duration of exposure or other factors are critical for reversibility or progression of EA-induced forestomach mucosal hyperplasia to neoplasia. PMID- 1723533 TI - Morphologic and immunohistochemical characterization of Leydig cell tumor variants in Wistar rats. AB - During a routine long-term drug safety study, lasting approximately 2 1/2 yr, male Wistar rats, treated with a prolactin-inhibiting compound, developed an excess of Leydig cell tumors (LCTs). Most tumors were typical for the rat but a small number showed an unusual variation and some appeared malignant. The variation consisted of glandular and/or tubular structures within the tumor mass which occasionally anastomosed and contained an eosinophilic periodic-acid Schiff (PAS) positive material. In a few of these variants, malignant features such as cellular atypia, capsular, and lymphatic invasion and necrosis were seen. No metastases were detected. Detailed morphological and immunohistochemical investigations were conducted in order to establish the cell of origin of these variants. Glandular/tubular structures were found to stain with varying intensity for vimentin and cytokeratin, but were always negative for beta-tubulin. The results indicated that the cell of origin of these LCT variants was indeed the Leydig cell and that glandular and/or tubular structures within LCTs represented a form of Leydig cell metaplasia. PMID- 1723534 TI - Tumor-associated trypsin inhibitor in pancreatic diseases. AB - We have evaluated tumor-associated trypsin inhibitor (TATI) as a marker for pancreatic and hepatic cancer. Of the patients studied 52 had pancreatic cancer, 30 primary liver cancer, 32 chronic pancreatitis, 25 biliary tract inflammatory disease, and 28 liver cirrhosis. A considerable number of falsely elevated values were observed in benign biliary diseases and in chronic relapsing pancreatitis. In pancreatic cancer the sensitivity of TATI was 63% while that of CEA was 40% and of CA19-9 77%. TATI is a marker of pancreatic disease but it does not differentiate between pancreatitis and pancreatic cancer. In liver cancer TATI and AFP has similar sensitivity and specificity. PMID- 1723535 TI - Cytoskeletal and contractile proteins in detrusor smooth muscle from bladders with outlet obstruction--a comparative study in rat and man. AB - Detrusor biopsies were obtained from patients with urinary outlet obstruction due to prostatic enlargement and from age-matched control patients. The relative amounts of actin and myosin and their isoforms, as well as desmin and filamin were determined and compared with corresponding results from bladders from control rats and rats with 10 days of experimental outlet obstruction of the urinary bladder. In the human control detrusor the actin/myosin ratio was similar to that in the control rat. The isoform distribution of the myosin heavy chains differed between man and rat. In the biopsies from the patients with outlet obstruction and in the obstructed rat bladders the actin/myosin ratio was increased. A change in the myosin heavy chain distribution in the obstructed bladders was observed for both species. The filamin/actin ratio increased significantly in the obstructed rat bladders and tended to increase in the obstructed human bladders. Desmin was the dominating intermediate filament protein. The desmin/actin ratio increased in obstructed bladders in man and in rat. PMID- 1723536 TI - Failure of perivascular sympathectomy to remove adrenergic nerves from peripheral vessels of the rabbit ear skin. AB - A perivascular sympathectomy was performed at the base of the ear artery in 11 New Zealand white rabbits. Two days later three were killed, and the central nerve was cut in the remaining eight. The contralateral ear served as a control. Specimens were taken from the distal parts of both ears two days later and the glyoxylic acid-induced fluorescence method was used to show the adrenergic nerves. The arteries of the normal, control ear were surrounded by a dense plexus of fluorescent adrenergic nerves, which were abolished by perivascular sympathectomy only in the segment from which the adventitia had been removed. The adrenergic innervation was normal proximal to the site of perivascular sympathectomy, but there was a short segment of the central vessel distally in which it was diminished. No changes in adrenergic innervation were found in the distal third of the rabbit ear. PMID- 1723537 TI - The cure of leukemia: a model for the role of humoral growth factors in cancer treatment. PMID- 1723538 TI - Decreased responsiveness of platelets to a stable prostacyclin analogue in patients with Crohn's disease. Reversal by n-3 polyunsaturated fatty acids. PMID- 1723539 TI - [The 1991 Nobel Prize in physiology and medicine--small cellular parts--great progress]. PMID- 1723540 TI - [Man as a symbolic animal--symptoms as symbolic interaction]. AB - This is an article about views of human nature. In western medicine man is conceived mainly as a biological being, a mammal. Philosophical-ontological questions about man's nature are answered implicitly in clinical practice. They are not, however, reflected explicitly. In this article it is argued that symptoms are primarily human experience. To understand these experiences and their expressions, it is necessary to understand man as an "animal symbolicum", a term introduced by the German philosopher Ernst Cassirer. Language is the most specific expression of man's symbolic activities, and symptoms are expressed verbally. Speech is part of social interaction--we, as human beings, have "the power of speech". This way of thinking is illustrated by clinical examples, and an alternative medical practice, homeopathy, is analyzed as a special means of symbolic interaction. PMID- 1723541 TI - Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression. AB - 3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC 2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies. PMID- 1723542 TI - Expression of CD31 epitopes on human lymphocytes: CD31 monoclonal antibodies differentiate between naive (CD45RA+) and memory (CD45RA-) CD4-positive T cells. AB - The CD31 antigen, a member of the immunoglobulin superfamily with a possible cell adhesion function, is expressed on approximately 50% of peripheral blood lymphoid cells at relatively low intensity (10-20% of the level on monocytes). In the accompanying paper we showed that a mAb, 5A2.G5, which identifies a glycosylation dependent epitope of the CD31 antigen, bound to fewer lymphocytes than two other CD31 mAb, B2B1 and 2BD4, although the 3 antibodies bound equally well to monocytes. We have now analyzed the pattern of expression of epitopes of the CD31 antigen on lymphoid cell subpopulations using two-color immunofluorescence and flow cytometry. Large granular lymphocytes (CD16+), CD8-positive T cells and B cells (SMIg+) were mostly CD31-positive as indicated by the binding of mAb B2B1 and 2BD4. Single populations displaying some overlap with the negative control were obtained in each case. In contrast, CD4-positive T cells fell into two discrete populations with respect to CD31 antigen expression. mAb 5A2.G5 displayed weaker binding to all lymphoid cell types, indicating that the pattern of glycosylation of the CD31 antigen differs between lymphocytes (of all types) and cells of the myeloid lineages. The heterogeneity of CD31 antigen expression by CD4-positive cells was further examined by dual-labelling of purified CD4 cells with mAb B2B1 and CD45RA or CD29 mAb which identify naive and memory T cells respectively. The CD31 antigen was found to be preferentially expressed by the CD45RA-positive, naive cell population. PMID- 1723543 TI - Inhibition of cell proliferation with an HLA-A-specific monoclonal antibody. AB - A new mouse IgG2A monoclonal antibody--JD 12--is described, which was selected for its ability to inhibit peripheral blood mononuclear cell (PBMC) proliferation. Serologic, immunoprecipitation and immunoelectrophoretic studies showed JD12 to be monomorphically reactive with HLA-A molecules with an affinity of 10(9) M-1. JD12 was capable of inhibiting growth in resting or stimulated PBMC with an ID50 of 300 ng/ml in a time-dependent and dose-dependent manner without cytotoxicity. IgG, F(ab)2 and F(ab) were active. Growth stimulation by phorbol ester was not inhibited, suggesting that JD12 action occurred prior to protein kinase C activation. DNA and RNA synthesis were both slowed with a resulting G0/G1 block. Analysis of separated PBMC components showed that adherent cells displayed increased activity (clumping and RNA synthesis) upon JD12 binding. In addition, the inhibitory activity of JD12 could be transferred to new cultures by cell-free, IgG-free supernatant from JD12-treated PBMC, suggesting that the antiproliferative effects could be mediated, at least in part, in an indirect manner through an inhibitory factor. Therefore, potent, noncytotoxic inhibition of cell proliferation, DNA and RNA synthesis via MHC molecules can be mediated specifically through effects initiated by binding to HLA-A molecules alone. PMID- 1723544 TI - Road transport impact on the environmental health. PMID- 1723545 TI - Risk assessment/risk management of motor vehicle emissions. PMID- 1723546 TI - Study of respirable suspended particulates and polycyclic aromatic hydrocarbons in indoor and outdoor air. AB - To study the concentration change of respirable suspended particulate (RSP) in indoor and outdoor air environment, a new portable sampler (AND-sampler) was made. The sampler could collect particles above 10 microns, 2 to 10 microns, and below 2 microns in aerodynamic diameter separately. The relationship between the airborne particulate concentration in indoor and outdoor air environment varied with the aerodynamic diameter of the particles. The concentration of RSP in indoor air environment increased in proportion to that in outdoor environment. Polycyclic aromatic hydrocarbons (PAH) concentration in airborne particulate also varies with the aerodynamic diameter of the particles. Fine particles below 2 microns in aerodynamic diameter contains high concentration of PAH. The PAH concentration in indoor air environment increases in proportion to that in outdoor air. PMID- 1723547 TI - Mast cells in the chick digestive tract. I. Development. AB - Mast cells in the digestive tract of the developing chick embryo were studied with histochemistry and electron microscopy (EM). The cells appeared in the esophagus, proventriculus and small intestine on about the 13th day of incubation, whereas mast cells in the tongue appeared earlier. The staining properties and ultrastructure of the mast cells varied with development. In 13- and 15-day embryos, mast cells showed a pale metachromasia with toluidine blue, and stained blue with Alcian Blue-Safranin O (AB-S). In the 18-day embryo, mast cells stained a deep purple with toluidine blue. Stained with AB-S, most of the mast cell granules stained blue, but some red granules were also seen in a few cells. In the newly hatched chick, the cells stained a strong reddish purple with toluidine blue. Stained with AB-S, a few cells contained only blue or red granules, but most contained both. Observations with the EM revealed that the internal structure of the granules varied with the stage of embryonic development. The basis for the changes in staining properties and ultrastructure of the mast cell in the developing chick embryo were discussed. PMID- 1723548 TI - Mast cells in the chick digestive tract. II. Fixation, distribution, histochemistry and ultrastructure. AB - Mast cells in the chick digestive tract were investigated with different fixatives, histochemical methods and transmission electron microscopy. Carnoy's fluid was the best fixative for the chick mast cell. Fixation in formalin reduced the number of mast cells and dissolved the granules. Mast cells stained reddish purple with toluidine blue, and bright blue with Alcian blue-periodic acid schiff (AB-PAS). Staining with Alcian blue-safranin O (AB-S) resulted in some cells only containing blue or red granules, and others containing both blue and red granules in the same cell. The result of AB-S staining was obviously affected by fixation. The ultrastructure of chick mast cells was described. Internal large granules had a variable morphology; some higher or lower electron density with a homogeneous appearance; some were finely granular; some had a mottled appearance, and a few showed a reticular structure. The distribution of mast cells varied. The proventriculus was the most abundant in mast cells among the organs examined. Only one type of mast cell was found in the chick digestive tract. The fixation, staining and the ultrastructure of chick mast cell granules were discussed. PMID- 1723549 TI - Effect of dimethylsulfoxide-hydroxyl radical scavenger on cerulein-induced acute pancreatitis in rats. AB - Acute oedematous pancreatitis was induced in Wistar male rats by intravenous (i.v.) infusion of cerulein at the rate of 5.10(-6)g.kg-1.h-1 for 3 or 6 hrs. The effects of dimethylsulfoxide (DMSO)-hydroxyl radical scavenger in this model of disease were examined. DMSO was injected i.v., 0.75 g.kg-1, at 0 and 3 hrs during cerulein infusion. Treatment with this agent exerted a protective effect on the rat pancreas in the cerulein-induced acute pancreatitis. This effect is expressed by inhibition of lipid peroxidation in pancreatic tissue, less oedema formation, diminution of hyperlipasemia and a significant reduction in acinar cell vacuolisation. Our data suggest that besides the hydroxyl radical, other oxidants (secondary?) may cause cytotoxicity in this model of disease. We suggest that phagocytic inflammatory cells provide such oxidants. PMID- 1723550 TI - Outline of clinical studies on recombinant human granulocyte colony stimulating factor (KRN 8601) in Japan. AB - In phase I studies of KRN 8601, single administration and six-day consecutive administration studies were conducted in healthy male adults using intravenous drip infusion and subcutaneous administration. Safety and tolerance to KRN 8601 were confirmed and a dose-related increase of neutrophil counts by KRN 8601 was observed. In an early phase II study, the safety and tolerance to KRN 8601 in cases of neutropenia following cancer chemotherapy were shown at doses of 25-800 micrograms/m2 and an improvement of neutropenia was seen at doses of more than 50 micrograms/m2. In a phase II study, the optimal dose was investigated for subcutaneous administration and intravenous drip infusion in cases of neutropenia induced by chemotherapy for malignant lymphomas. The optimal dose was 75 micrograms/body (about 50 micrograms/m2) for subcutaneous administration and 100 200 micrograms/m2 for intravenous drip infusion. In a phase III study, a double blind prospective randomized trial comparing KRN 8601 with an inactive placebo against malignant lymphomas was performed and the inhibitory, improvement and recovery-promoting effects of KRN 8601 (75 micrograms/body, sc) on neutropenia were demonstrated. PMID- 1723551 TI - Leptospirosis, renal failure and raised amylase levels. PMID- 1723552 TI - The basic defect in cystic fibrosis. AB - Recent evidence strongly suggests that the cystic fibrosis gene product (CFTR) is a Cl- channel. Its properties, however, differ from those of a 30-50 pS outwardly rectifying channel previously implicated as defective in cystic fibrosis. It is still uncertain whether the pleiotropic effects of the CF defect, such as increased airway Na+ absorption and mucus sulfation, are secondary to reduced Cl- conductance, or reflect additional functions of CFTR. PMID- 1723553 TI - Microscopy-based techniques. AB - Microscopic evaluation of specimens for detection of STD is a key element in the diagnosis and timely management, particularly of symptomatic patients. It is rapid and inexpensive, and can be used effectively with minimal resources. When cultures are available, it should be used in conjunction with them, since culture results are not available at the time the patient is seen. PMID- 1723554 TI - The current use of interferons, interleukin-2 and tumor necrosis factor in renal cell cancer. AB - Cytokines such as interferons, interleukin-2 and tumor necrosis factor have been widely tested in the treatment of advanced renal cell cancer. However, the rates of objective remissions (PR and CR) are disappointing and rarely exceed 20% overall. Until now, a definitive cure of a patient with renal cell cancer treated with cytokines has not been reported in the literature. The combination of interferon-alpha and interleukin-2 in low-dose regimens seems to offer the best achievable results with the lowest morbidity of the patients in renal cell cancer. Since optimal treatment regimens are still not defined, treatment with these substances should only be carried out in prospective trials. PMID- 1723555 TI - Squamous cell metaplasia in the human lung: molecular characteristics of epithelial stratification. AB - Squamous cell metaplasia (SCM) is a frequent epithelial alteration of the human tracheobronchial mucosa. This review pays particular attention to the fact that SCM can mimic esophageal, and in some instances even skin-type differentiation, showing striking similarities not only in morphology but also in terms of gene expression. Therefore, characterization of this dynamic process lends insight into the process of stratification, squamous cell formation, and "keratinization" in a pathologically relevant in vivo situation in man. First, the concept of metaplasia is presented with certain historical viewpoints on histogenesis. Then, the morphological characteristics of normal bronchial epithelium are compared with the altered phenotype of cells in SCM. These changes are described as a disturbance of the finely tuned balance of differentiation and proliferation through the action of a variety of extrinsic and intrinsic factors. Molecular aspects of altered cell/cell and cell/extracellular matrix interactions in stratified compared with single-layered epithelia are discussed with reference to SCM in the lung. Intracellular organizational and compositional changes are then summarized with special emphasis on the differential distribution of the cytokeratin (CK) polypeptides. Finally, the still unresolved problems of the histogenetic relationships between normal bronchial mucosa, SCM, and pulmonary neoplasms are addressed. As these questions remain open, examples for detection of well defined "markers" are provided that may be employed as objective criteria for determining clinically important cellular differentiation features. PMID- 1723556 TI - Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies. Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in human lymphoid tissues. AB - During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the alpha-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15,000 to 20,000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11,000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages. PMID- 1723557 TI - Facial nerve paralysis associated with Kawasaki disease. AB - Kawasaki disease is a multisystem disorder with varying clinical expression. This is a report on one case of Kawasaki disease which during its clinical course developed facial nerve palsy and spontaneous recovery without specific treatment. It is hoped that this report will serve to remind physicians of the association of facial nerve paralysis with Kawasaki disease. PMID- 1723558 TI - Patients' perception of itch induced by histamine, compound 48/80 and wool fibres in atopic dermatitis. AB - Itch and flare responses were investigated in 32 patients with atopic dermatitis (AD) and in 32 healthy controls. Itch was induced chemically by intradermal injections of histamine (1, 3.3, 10 and 100 micrograms/ml) and compound 48/80 (10 micrograms/ml) into non-lesional skin and mechanically by wearing a woollen sweater. Continuous recording of itch intensity allowed the calculation of itch duration (ID), maximal itch intensity (Imax) and a "total itch index" (Tii). The itch responses were significantly increased in AD patients compared with controls for wool fibres and one of the histamine concentrations (10 micrograms/ml), but not for the remaining three histamine concentrations or compound 48/80. Conversely, the flare response was significantly smaller in AD patients than in controls for the two strongest histamine solutions and compound 48/80. Significant dose-response relationships were found between histamine concentration and each of ID, Imax, Tii and flare in both patients and controls. The slope of the flare-regression line was significantly steeper in controls than in AD patients, whereas the slopes of the itch-regression lines did not differ significantly between the two groups, i.e. their ability to discriminate between weak and strong histamine concentrations did not differ significantly. No increased skin mast cell releasability in vivo to compound 48/80 was shown in AD patients compared with controls. The itch and flare responses of AD patients did not correlate significantly with clinical itch intensity, eczema score or serum IgE level. PMID- 1723559 TI - Beta-carotene in prevention of cutaneous carcinogenesis. AB - Beta-carotene, administered orally to mice, caused a decrease in angiogenesis evoked by HPV-transformed tumorigenic cell lines (SKv-t, HeLa). It did not affect angiogenesis induced by the non-tumorigenic SKv (not-t) cell line, and increased lymphocyte-induced immune angiogenesis. We suggest that the anti-cancerogenic effect of beta-carotene may be due, at least in part, to its inhibitory effect on formation of new blood vessels within the tumour mass. PMID- 1723560 TI - Identification of two maturation-related, wheat-germ-lectin-binding proteins on the surface of mouse sperm. AB - Epididymal maturation is essential for mammalian sperm to develop fertility. Wheat germ agglutinin (WGA), a lectin which specifically recognizes N-acetyl glucosamine and sialic acid, was used to investigate membrane characteristics of epididymal sperm in the mouse. Histochemical and cytochemical localization revealed that WGA-binding sites were increased as sperm became mature. The binding sites were mainly localized in the plasma membrane of the anterior acrosome and tails. On Western blots of NP-40 extracts, two WGA-binding glycoproteins, GP-49 and GP-83, were identified on the sperm recovered from both corpus and cauda epididymidis. GP-83 was removed from the sperm surface by centrifugation and resuspension in phosphate-buffered saline (PBS) three times. GP-49 was resistant to centrifuging at 400 g for 5 min up to seven times and the treatment with 1M NaCl in PBS. These observations suggest that GP-49 is very likely an intrinsic membrane protein of the sperm, whereas GP-83 is an extrinsic protein. PMID- 1723561 TI - [Recurrences of epithelial tumors of the head and neck: review of the literature and a critical analysis of the problem]. AB - Recurrence is a tumor renewal in a previously treated field or surgical wound after a disease-free period. The present work investigates loco-regional recurrences. In the first part of the work the most frequent recurrence sites, types and their characteristics (i.e. infiltrating, exophytic, ulcerating) are studied along with the problems in clinical and pathological diagnosis, new diagnostic imaging (CT and MR) and the UICC classification system. The second part of the work is dedicated to examining classical treatments (surgery, radiotherapy, chemotherapy and radiochemotherapy associations) as well new therapies (i.e. cryosurgery, hyperthermia, immunotherapy, photodynamic therapy). A review of the literature is made for indications, results and complications. An extensive study of the cases of the Head and Neck Group of the University of Turin during the 1983-1990 period is also reported. This experience is based on 312 cases: 68 (oropharynx and larynx) treated by surgery, 51 (neck nodes and stomal recurrences) treated by radiotherapy and hyperthermia, 35 (neck nodes) by hyperthermia alone, 168 (all sites) by chemotherapy and 20 by immunotherapy. In the third part of this work supportive therapies and practical problems are reported. In conclusion a therapeutic approach is proposed in relation to the site of the recurrence and previous treatments. PMID- 1723562 TI - Histochemical and immunohistochemical analyses of primary carcinoma of the liver. AB - Hematoxylin and eosin (H-E) stained liver sections of 47 autopsy cases of hepatic malignancies were examined. There were 43 cases of hepatocellular carcinoma (subtypes of 30 trabecular, 7 solid, 5 pseudoglandular, and one scirrhous carcinoma), 3 of cholangiocellular carcinoma and one of mixed carcinoma. After immunohistochemical staining, benign hepatocytes reacted positively with anti epithelial membrane antigen (EMA). Hepatocellular carcinoma cells reacted more weakly than benign hepatocytes. It was noted that the microtubular structure, which could not be demonstrated even by alcian blue or cationic ferric hydroxide colloid stabilized with cacodylate (Fe-CaC), was clearly detected with anti-EMA. The EMA-positive microtubular structures may indicate terminal cholangiolar differentiation. Based on EMA, seven more cases formerly classified as hepatocellular carcinoma by H-E were reclassified as mixed carcinoma, totaling eight (17.0%). The histologic classification of "mixed carcinoma" has been 1.5 to 2.0% of primary liver cancers in Japan, but we suggest there may be more cases of "mixed carcinoma" identified in the future. In conclusion, we emphasize that EMA staining is useful for more accurate classification of hepatic tumors. PMID- 1723563 TI - Connections of the insular cortex in kittens: an anatomical demonstration with wheatgerm agglutinin conjugated to horseradish peroxidase technique. AB - The postnatal development of the afferent and efferent connections of the feline insular cortex was investigated using wheat germ agglutinin conjugated to horseradish peroxidase method. The overall pattern of connections was found to be substantially the same as that observed in adults. In the cortex, anterograde and retrograde labeling was found in the presylvian sulcus, cingulate gyrus, cruciate sulcus, medial prefrontal area, lateral suprasylvian cortex, and posterior rhinal sulcus. Subcortical regions containing label included the lateralis medialis suprageniculate nuclear complex, ventral medial thalamic nucleus, claustrum, lateral amygdaloid nucleus, hypothalamus, and raphe nuclei. In addition, axonal labeling was seen in the striatum, superior colliculus, and pontine nuclei. In contrast, the insular associational projections in kittens differed markedly from those found in adults. In the early postnatal period (1-14 days), dense terminal labeling was found in the cortical layers I, V and VI as well as in the underlying white matter, whereas only moderate to sparse labeling was observed in layers II, III and IV. By four-weeks, an adult-like distribution of terminals was present in each cortical areas: labeling was found mainly in layers I, II, III and IV, with less in layers V and VI. The present results suggest that the basic framework of the insular connections is formed prenatally and that the fine tuning of the axonal terminals and the formation of synapses occurs mainly during the first four postnatal weeks. PMID- 1723564 TI - The regulation of antibody responses by mini-cytokines. AB - The presence of adrenocorticotropic hormone (ACTH) and substance P (SP) receptors on leukocytes is suggestive that these cells can respond to these ligands. To address this possibility, we have investigated the consequences of ACTH and SP stimulation of B cells. As a result, enhanced immunoglobulin synthesis mimicking an IL-4-like mechanism was noted. Importantly, this stimulation could be induced at ligand concentrations at or near the kD for their receptors. Herein these effects by ACTH and SP were described using B cell lymphoma cell lines and normal B cells. PMID- 1723565 TI - LN-10, a brain derived cDNA clone: studies related to CNS development. PMID- 1723566 TI - Macromolecular changes in the aging brain. PMID- 1723567 TI - Reactive sprouting (pruning effect) is altered in the brain of rats perinatally exposed to morphine. PMID- 1723568 TI - Virus-host cell interactions in human immunodeficiency virus infections. AB - The isolate HTLV-IIIB is used by a large number of investigators for a variety of studies. The data shown here emphasize the uniqueness of this isolate with respect to certain biological behaviors, in particular, the ability to infect cells other than T lymphocytes and mononuclear phagocytes, and the ability to infect chimpanzee T cells. Data also presented indicates that HTLV-IIIB has a greater binding affinity for the CD4 receptor molecule, which may help to explain its observed biological uniqueness. Caution in generalizing from findings based on studies with HTLV-IIIB alone is recommended. In addition, evidence demonstrating CD4-mediated entry of HIV-1 into normal human monocyte/macrophages is included. PMID- 1723569 TI - Human phosphoribosylpyrophosphate synthetase (PRS) 2: an independent active, X chromosome-linked PRS isoform. PMID- 1723570 TI - Pyridine nucleotide metabolism: purine and pyrimidine interconnections. PMID- 1723571 TI - Applications of PRPP metabolism in human erythrocytes. PMID- 1723573 TI - The effect of pyrroline-5-carboxylate on R5P and PRPP generation in mouse liver in vivo. PMID- 1723572 TI - Incorporation of purine ribonucleotides into nucleic acids of rat liver after castration. PMID- 1723574 TI - Adhesion proteins: in vitro and in vivo studies of leukocyte-endothelial interactions. PMID- 1723575 TI - Osteoarthritis. PMID- 1723576 TI - Adhesion-promoting receptors on phagocytes. AB - In order for leukocytes to migrate to sites of inflammation they must first adhere to the vascular endothelium. This initial adhesion, however, must be rapidly broken to allow diapedesis. Recent studies have shown that the CD18 family of leukocyte integrins plays a pivotal role in the transient adhesions needed for diapedesis. The means by which CD18 molecules mediate transient adhesion and the respective roles of CD18 molecules and other adhesion molecules in inflammation are discussed. PMID- 1723577 TI - Quantitative determination of trucking industry workers' exposures to diesel exhaust particles. AB - As part of a case-control mortality study of trucking industry workers, exposures to diesel aerosol were measured among the four major presumably exposed job groups (road drivers, local drivers, dock workers, and mechanics) in the industry. Eight industrial hygiene surveys were conducted during both warm and cold weather at eight U.S. terminals and truck repair shops. A single-stage personal impactor was used to sample submicrometer-sized diesel particles on quartz fiber filters. Laboratory and field studies demonstrated that the elemental carbon content of the particles is a useful and practical marker of exposure to vehicular diesel exhaust. A thermal-optical analysis technique was used to determine the concentration of elemental carbon in the filter samples. Overall geometric mean exposures to submicrometer-sized elemental carbon ranged from 3.8 micrograms/m3 in road (long distance) drivers (N = 72) to 13.8 micrograms/m3 in dock workers (N = 75). Geometric mean background area concentrations, measured in the same cities where workers were sampled, were 2.5 micrograms/m3 on major highways (N = 21) and 1.1 micrograms/m3 in residential areas (N = 23). A factorial analysis of variance indicated that exposures in two job groups, dock workers (particularly those exposed primarily via diesel forklift trucks, introduced relatively recently) and mechanics (working in poorly ventilated shops during cold weather), were significantly higher than background concentrations and were significantly higher than the exposures in the local and road drivers. The exposures of the truck drivers could not be distinguished from background highway concentrations but were significantly higher than background concentrations in residential areas. PMID- 1723578 TI - Compatibility of dexamethasone sodium phosphate with hydromorphone hydrochloride or diphenhydramine hydrochloride. AB - The stability and compatibility of dexamethasone sodium phosphate and hydromorphone hydrochloride or diphenhydramine hydrochloride at various concentrations at room temperature was studied. Solutions containing equal volumes in the following ranges of concentrations were prepared: dexamethasone sodium phosphate 0-10 mg/mL, diphenhydramine hydrochloride 0-50 mg/mL, and hydromorphone hydrochloride 0-40 mg/mL. Samples of each combination were analyzed immediately after mixing and at 7 and 24 hours using a stability-indicating high performance liquid chromatographic assay. The pH of each solution was measured, and each combination was visually inspected. Precipitation occurred in solutions containing dexamethasone and hydromorphone hydrochloride or diphenhydramine hydrochloride when equal volumes of the most concentrated solutions were mixed. Some of the combinations at lower concentrations were visually compatible, and more than 90% of the initial concentrations of both drugs (i.e., dexamethasone hydromorphone and dexamethasone-diphenhydramine) remained in these compatible solutions. Dexamethasone is visually compatible with diphenhydramine or hydromorphone but only within specific concentration ranges. In visually compatible solutions, both drug combinations are stable for up to 24 hours at room temperature. PMID- 1723579 TI - [Cervical seminoma. Presentation of a case]. AB - A germinal cells tumor in a male manifested as left supraclavicular mass, without other localisation, is presented. The pathologic anatomy corroborate the diagnosis of seminoma. Very few cases of a germinal cell tumor in the supraclavicular fossa, with a normal testicular exploration, have been reported. PMID- 1723580 TI - Serious complications with dextran-70 despite hapten prophylaxis. Is it best avoided prior to delivery? AB - Dextran is used clinically for plasma volume expansion, improvement of blood flow and thromboprophylaxis, but has been associated with untoward side effects. Immunoprophylaxis with dextran I (hapten), before the infusion of dextran-70, has reduced the incidence of serious dextran-induced anaphylactoid reactions. We report three cases of severe reactions occurring during anaesthesia in spite of immunoprophylaxis. One patient given dextran-70 before Caesarean section had a mild reaction but gave birth to a child with serious brain damage. One patient with an extremely high titre of dextran-reactive antibodies died from myocardial infarction and another patient recovered without sequelae. From our experience we conclude that dextran-induced anaphylactoid reactions are still a serious problem despite immunoprophylaxis. Dextran-70 should be avoided during pregnancy and should not be given during Caesarean section before delivery of the child. Even in the presence of immunological prophylaxis, vigilant observation of the patient is essential and resuscitation equipment must be available when starting a dextran infusion. PMID- 1723581 TI - [Confabulatory paraphrenia, taxonomy and psychopathology]. AB - A 60 year old woman has elaborated for the last 20 years a delusional system, which refers to an illegitimate birth but which also claims that she has published works of well-known French Writers and that she has been for some time in concentration camp. There is probably a relationship to what happened to her during infancy (she spent the 13 first years of life in foster homes and institution). To some extent she justifies Bruno Bettelheim's thesis relative to a similarity of experiences of concentration camp and deprived infancy. Such a delusional system can be classified under the krapelinian heading of paraphrenia fantastica. Its does not seem to have been included in the DSM-III and DSM-III-R. PMID- 1723582 TI - [Massive pulmonary embolism disclosing thrombocytopenia induced by low molecular weight heparin. Therapeutic success of prostacyclin]. AB - The authors report a case of massive pulmonary embolism revealing thrombocytopenia induced by a low molecular weight heparin (LMWH) initially proposed for the treatment of superficial phlebitis. The diagnosis was confirmed by in vitro aggregation tests and a fall in the platelet count when the LMWH was reintroduced. The outcome was clinically, angiographically and hematologically satisfactory in response to in situ treatment with prostaglandin, subsequently replaced by Vitamin K antagonists. PMID- 1723583 TI - Screening for carcinoma of the prostate with prostate specific antigen. AB - A free screening clinic for cancer of the prostate was held in Madison County, New York, in September 1990, in conjunction with Prostate Cancer Awareness Week, a program of the Prostate Cancer Educational Council. Serum prostate specific antigen and digital rectal examination were used to screen 565 men. The two tests were equally effective in identifying patients with carcinoma. Of 118 patients with one or both tests positive, 54 were biopsied. Carcinoma was found in 20 of these. Four carcinomas were found in patients with prostate specific antigen (PSA) greater than four ng per ml with negative rectal examination. The costs for adding PSA to the protocol appeared reasonable in terms of the number of carcinomas identified. PMID- 1723584 TI - Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone. AB - Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22 degrees C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity (P less than or equal to 0.05), with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation (P less than or equal to 0.05) were detected for inactivations up to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation. PMID- 1723585 TI - Propidium iodide as an indicator of Giardia cyst viability. AB - The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants. PMID- 1723586 TI - Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes. AB - Specific DNA probes based on variable regions V1 and V3 of 16S rRNA of lactic acid bacteria were designed. These probes were used in hybridization experiments with variable regions amplified by using the polymerase chain reaction. In this way, a rapid and sensitive method was developed for the identification and classification of Lactococcus and Leuconostoc species. PMID- 1723587 TI - [Mechanism of actinomycin biosynthesis]. PMID- 1723588 TI - Acetonema longum gen. nov. sp. nov., an H2/CO2 acetogenic bacterium from the termite, Pterotermes occidentis. AB - A previously undescribed, H2-oxidizing CO2-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic. Gram-negative, endospore-forming motile rods which measured 0.30-0.40 x 6-60 microm. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G + C in their DNA. Optimum conditions for growth on H2 + CO2 were at 30-33 degrees C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H2 + 2 CO2----CH3COOH + 2 H2O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related, to but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites. PMID- 1723589 TI - Involvement of afferent nerves in pulpal blood-flow reactions in response to clinical and experimental procedures in the cat. AB - A unilateral resection of the mandibular nerve (n = 20) was made 10-14 days before investigation of the contribution of afferent nerves in vasodilator reactions in the dental pulp. Lower canine teeth were subjected to various stimuli and pulp blood-flow responses monitored by laser Doppler flowmetry. An absence of response to bipolar electrical (5 impulses, 50 microA, 5 ms, 2 Hz) stimulation on the tooth surface was used to demonstrate a successful chronic nerve lesion. Local application of capsaicin (10(-4) M) in a deep dentinal cavity induced a long-lasting increase in pulpal blood flow in control teeth only. Bradykinin (10(-3) M) induced significantly larger responses in control than in denervated teeth (58.3 +/- 9.8% and 24.5 +/- 4.9%, respectively, p less than 0.005, n = 8); in addition, the onset was slower and the duration of the response significantly (60%) shorter than in control teeth. Intermittent grinding of surface dentine instantly increased flow in control teeth by 53.0 +/- 12.5% (n = 12) whereas in denervated teeth the response was delayed and significantly (70%) smaller. Deeper preparation produced responses of similar magnitude in control and denervated teeth (69 and 50%, respectively) but the onset was delayed in denervated teeth. Low-intensity ultrasonic stimulation caused vasodilation in intact teeth (38% increase) but had no effect in denervated teeth. This effect was abolished after local anaesthetic (mepivacaine) injection. Sympathectomy (n = 3) did not influence stimulation-induced blood-flow responses in the dental pulp.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723591 TI - Immunoaffinity purification of human alpha-fetoprotein (AFP) using monoclonal antibodies. AB - A two-step immunoaffinity isolation procedure for human alpha-fetoprotein (AFP) was developed resulting in highly purified AFP at high yield. A monoclonal antibody immunoadsorbent was used in the first step. Elution of AFP was carried out at alkaline pH by a solution of 0.5 mol/l ammonia containing 0.5 mol/l sodium chloride. To remove impurities caused by the first step, an anti-mouse immunoglobulin antibody immunoadsorbent was applied in the second step. PMID- 1723590 TI - Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte-macrophage protein recognized by mouse monoclonal antibody MOMA-2. AB - These studies were made as a first step in elucidating unknown functions of enamel proteins in odontogenesis. The cytoplasm of ameloblasts and the proteins in dentine matrix before mineralization were stained with this monoclonal antibody. SDS-PAGE and Coomassie blue staining of proteins extracted from enamel showed several protein bands. Immunoblotting revealed that proteins recognized by this antibody were situated between 20-30 kDa. These results indicate that enamel proteins, presumably amelogenins, have an epitope resembling monocyte-macrophage protein. The findings suggest that epithelial-mesenchymal interaction in odontogenesis and preosteoblast-preosteoclast interaction in osteogenesis may be similar. PMID- 1723592 TI - Modification of low density lipoproteins by sodium hypochlorite. AB - Human low density lipoproteins (LDL) were incubated with increasing amounts of sodium hypochlorite. A decrease of the number of free amino groups on the LDL surface starts only upon addition of 30-40 moles NaOCl per mole apoB, whereas all detectable SH groups are oxidized after addition of nearly 17-20 moles NaOCl. All hypochlorite-modified LDL samples have a higher electronegative surface charge compared with native LDL as revealed by agarose gel electrophoresis and partition of LDL in an aqueous polyethylene glycol/dextran two-phase system. The more NaOCl is used to alter LDL, the higher is the electrical surface charge. Changes in surface charge are found already at low NaOCl concentrations where no decrease of amino groups is detected. It is assumed that changes in surface charge are caused by the formation of monochloramines and especially at low degrees of modification by a further unknown contribution. An effect on the primary structure of apoB or peroxidation-like changes in NaOCl-altered LDL could not be found under our experimental conditions. The results are discussed with respect to such modifications under in vivo conditions by hypochlorous acid generated in stimulated phagocytosing cells. PMID- 1723593 TI - The use of macromolecular protease inhibitors to study liposome-cell interactions. AB - A method is introduced that quantitates the release of a high molecular weight compound from small unilamellar vesicles. The technique is based on the liposomal encapsulation of trypsin inhibitor from bovine lung. Because liposomes are impermeable to all components of the test system (inhibitor, enzyme, macromolecular substrate), only the free inhibitor released from disturbed liposomes is measured. Using this technique, the exit of the macromolecular solute from liposomes induced by erythrocyte ghosts was studied. The results were compared with the leakage of a low molecular weight fluorescent marker. They indicate that, especially during the first hours of incubation, ghost-induced release of solutes is dependent on their size. PMID- 1723594 TI - Properties of an immunoglobulin binding factor from human seminal plasma. AB - A soluble factor (IBF) in human seminal plasma that binds serum immunoglobulins (Ig) of various species was purified to homogeneity by ammonium sulfate precipitation, preparative isoelectrofocusing, and gel filtration chromatography. The purified IBF interacted weakly with Fc and F(ab')2 fragments and not with Fab. It interacted with anti-Leu 11b and polyclonal anti-Fc gamma RIII antibodies, but not with other anti-Fc gamma R antibodies (32.2, IV.3 and 3G8). IBF is probably a non-glycosylated protein with isoelectric point ranging from 5.1 to 5.8. The estimated Mr of the purified native IBF is 27 kD, determined by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) under non reducing condition. In its native form, IBF did not bind Ig or interact with anti Fc gamma R antibodies. Following SDS-PAGE under reducing condition, IBF migrated as a single protein with an estimated Mr of 16 kD and interacted with Ig of various species and with anti-Leu 11b antibodies. When carboxymethylated, however, IBF did not bind IgG. The present results suggest that free sulfhydryl groups of IBF is required for Ig binding. PMID- 1723595 TI - Identification of two calcineurin alpha isoforms in bovine brain by two different monoclonal antibodies. AB - Calcineurin (calcium- and calmodulin-stimulated phosphatase) alpha subunit purified from bovine brain was found to be composed of two polypeptides, 61 KDa (alpha 1) and 59 KDa (alpha 2). The two peptides were separated and extracted from polyacrylamide gel. The immuno-peptide mapping of the purified peptides by partial proteolysis showed that the 59-KDa polypeptide was not a degradative product of the 61-KDa polypeptide. The interaction of the enzyme with two monoclonal antibodies, Vj6 and Vd3, raised against bovine brain calcineurin revealed that the 61-KDa polypeptide was recognized by both Vj6 and Vd3, whereas the 59-KDa one was recognized only by Vj6. These results indicate that there are at least two isoforms of calcineurin alpha subunits in bovine brain. PMID- 1723596 TI - Changes in the carbohydrate metabolism in the selected tissues of Mus booduga gray after BHC treatment. AB - Adult mice, Mus booduga were fed orally with bennzenehexachloride (BHC) at a dose of 50 mg/kg body weight every day for 1, 5 and 15 days. Significant decrease in the pyruvate content was observed at all periods of treatment. In support of this increase in lactate content and lactate dehydrogenase (LDH) activity was noticed in all the three tissues. Enzymes of TCA cycle namely isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) were inhibited suggesting abnormality in mitochondrial oxidative metabolism as a consequence of BHC toxicity. PMID- 1723597 TI - Potentiation of the analgesic effects of tryptophan by allopurinol in rats. AB - The administration to rats of tryptophan (CAS 73-22-3) in high dosage causes a significant increase in pain threshold values. The analgesic effects of tryptophan are potentiated by allopurinol (CAS 315-30-0). The analgesic effects shown by tryptophan injection are associated with increased levels of serotonin and 5-hydroxyindoleacetic acid in some areas of the brain. The combined allopurinol and tryptophan treatment elevates the serotonin levels furtherly when they are compared with the values observed in animals receiving tryptophan only. The analgesia caused by tryptophan administration has been attributed to an increased activity of the serotoninergic system involved in the control of pain transmission. PMID- 1723598 TI - Effect of an immunostimulatory substance of Klebsiella pneumoniae on inflammatory responses of human granulocytes, basophils and platelets. AB - RU 41740 (Biostim), a biological response modifier of bacterial origin obtained from the 01.K2 strain of K. pneumoniae, was studied with regard to its effect to modulate the chemiluminescence response, phagocytosis and leukotriene formation from polymorphonuclear leukocytes (PMNs), the histamine release from basophils and the aggregation of human platelets. For histamine release human basophils were analyzed with either the calcium ionophore A23187 or hemolysin producing bacteria. RU 41740 reduced the E. coli K12 pANN5211 induced histamine release whereas the calcium ionophore A23187 induced histamine release was not affected. The studies with regard to adherence and phagocytosis showed a significant increase in phagocytosis for S. aureus in the presence of RU 41740. With regard to the chemiluminescence response of human granulocytes, donor cells were divided into cells with a high and a low response. The chemiluminescence response of high responder cells towards a potent stimulus, e.g. the calcium ionophore A23187, was decreased by RU 41740 whereas weak stimuli (e.g. E. coli K12) were not affected. In contrast to these results the chemiluminescence response of low responder cells to a weak stimulus e.g. E. coli K12 was increased by RU 41740 and the potent stimuli were not affected. RU 41740 also modulates the eicosanoid synthesis of human polymorphonuclear granulocytes. For the calcium ionophore A23187 induced leukotriene production an increase of LTB4 and a decrease of LTC4 by RU 41740 was observed. Furthermore, preincubation of platelets with various concentrations of RU 41740 for 5 min shows an inhibition of platelet aggregation, when an additional stimulation with the calcium ionophore A23187 was carried out. PMID- 1723599 TI - Conformational changes of recombinant human granulocyte-colony stimulating factor induced by pH and guanidine hydrochloride. AB - Fluorescence and circular dichroism were used to follow the pH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going from pH 7 to 4, with a midtransition pH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as the pH was changed from 6 to 2.5, with a midtransition pH of 4.5. Near UV circular dichroic spectra also showed changes between pH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at five pH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at all pH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable between pH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on the pH used. These results are consistent with the pH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF. PMID- 1723600 TI - Immunostained serotonergic fibers are decreased in selected brain regions of alcohol-preferring rats. AB - The levels of serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA) are decreased in the hippocampus, nucleus accumbens, and cortex of selectively bred alcohol-preferring (P) rats, compared with the alcohol-nonpreferring (NP) rats. In this study, we have confirmed these findings by immunocytochemistry and quantitative image analysis which indicates that there is a reduction of 5-HT immunostained fibers in several brain areas of alcohol-naive P rats. Three major areas possibly related to alcohol drinking, hippocampus, accumbens, and cortex, were examined. Pathways to these areas were also examined. The 5-HT fiber bundles had the same pattern in P and NP rats. However, in the terminal regions of the ventral hippocampus, the amount of 5-HT fibers was reduced in P rats as compared with NP rats. The 5-HT fibers in the hilus and CA4 of the dentate gyrus were also significantly decreased in the P rats. No differences in fiber density were seen in the anterior nucleus accumbens, but a significant decrease was seen in the middle medial and posterior accumbens of P rats. In the cortical regions examined, decreases in 5-HT fibers were observed in the posterior cingulum and anterior frontal cortex, but not in the insular frontal cortex of P rats. These observations indicate that there are quantitative decreases in 5-HT innervations or that the 5-HT in some 5-HT fibers is reduced to a level undetectable by immunocytochemistry in the brains of P rats when compared with that of NP rats. PMID- 1723601 TI - Opposite effects of naltrexone on ETOH intake by Syracuse high and low avoidance rats. AB - Rats which were selectively bred for good (Syracuse High Avoidance: SHA) and poor (Syracuse Low Avoidance: SLA) shuttle-box avoidance learning were used to assess the effects of naltrexone on ethanol ingestion. Male rats from both strains were offered a free choice of water and ethanol (10%, v/v) for two 8-day periods between which was inserted a 4-day period of forced ethanol consumption. The net ethanol consumption and ethanol preference ratio were significantly greater in control SHA rats than in control SLA rats in the first choice period, but they did not differ in the forced and the second choice periods. Chronic naltrexone administration from an implanted 30-mg pellet showed bidirectional effects, i.e., suppression of ethanol consumption in SLA animals and enhancement in SHA rats. PMID- 1723602 TI - Effect of alcohol on spleen cells and their functions in C57BL/6 mice. AB - Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4+ cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67-90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons. PMID- 1723603 TI - Up-regulation of substance P binding sites in the vagus nerve projection area of the cat brainstem after nodosectomy. A quantitative autoradiographic study. AB - Substance P (SP) regulates visceral functions in the nucleus of the solitary tract (NST) area. High affinity SP binding sites labelled with [3H]SP or [125I]SP show a heterogeneous distribution in the cat medulla with high densities in the rostral and dorso-caudal parts of both the median subnucleus of NST and the dorsal motor nucleus (DMN). We previously observed a significant loss of SP immunoreactivity in the vagal area of the cat after an ipsilateral nodosectomy. It was thus important to study the correlated plasticity of SP binding in the context of the regulation of receptor function. Whichever labelled ligand was used, a unilateral nodose excision was followed by an ipsilateral increase in SP binding in the NST (200%) and the DMN (300%) after 30 days of survival. This increase was region-specific and did not match exactly the decrease in SP immunoreactivity following nodosectomy. This SP receptor density up-regulation could be due to long-term deprivation of SP afferent fibres in the NST and partly in the DMN. In the latter the increase of SP receptors occurred in both the cytoplasm of large neurons and the neuropile and did not affect the glia. The up regulation phenomenon seems to be specific for SP receptors in the cat (at least in the DMN) and may constitute a reactive mechanism against the injury of axotomy of DMN neurons. PMID- 1723604 TI - Distal arthrogryposis, specific facial dysmorphism and psychomotor retardation: a recognizable entity in surviving patients with the fetal akinesia deformation sequence. AB - Two non-related patients, a boy and a girl, are described suffering from distal arthrogryposis and facial dysmorphism consisting of flat face, hypertelorism and telecanthus, small mouth with thin, downturned upper lip, micrognathia, cleft palate and simple, low-set, posteriorly angulated ears. Feeble fetal movements (case 2), polyhydramnion (case 1) and lung hypoplasia (case 2) were present. On follow-up, both children were severely developmentally retarded. These findings are consistent with the Fetal A/hypokinesia Deformation Sequence (FADS). Survival beyond the neonatal period in this heterogeneous condition seems to be rare. Clinical descriptions of infants with FADS surviving the neonatal period are most important in order to delineate clinically recognizable entities; it may help to disclose pathogenetic basic mechanisms. PMID- 1723605 TI - Structure of a psoralen derivative of a monosubstituted 18-crown-6 ether. AB - N-[(2,5,9-Trimethyl-7-oxo-7H-furo[3,2-g]-[1] benzopyran-3-yl)methyl] 1,4,7,10,13,16-hexaoxacyclooctadecane- 2,3-dicarboximide, C29H35NO11, Mr = 573.60, monoclinic, P21, alpha = 18.877 (9), b = 6.888 (6), c = 24.488 (10) angstrom, beta = 119.90 (4) degrees, V = 2760.2 angstrom3, Z = 4, Dx = 1.380 Mg m 3, lambda (Cu K a) = 1.54178 angstrom, mu = 0.85 mm-1, F(000) = 1216, T = 170 K, R = 0.065, wR = 0.057 for 3720 observed reflections. There are two molecules (I and II) in the asymmetric unit and the 18-crown-6 part of molecule II is disordered. The psoralen moieties of the two molecules are nearly centrosymmetrically related. PMID- 1723606 TI - Acceleration of purine synthesis in mouse liver by glycogenolytic hormones. AB - Administration (ip) into fed mice of glucagon, epinephrine, vasopressin, oxytocin, angiotensin II, and dibutyryl cyclic AMP (dbcAMP) resulted in a rapid (within 2.5 to 15 min) elevation of PRPP content (two- to threefold) and in acceleration of the rate of de novo purine synthesis (twofold). Inhibition of the epinephrine-stimulated glycogenolysis by 2,5-anhydromannitol diminished markedly the acceleration effect of the hormone on the rate of purine synthesis. Administration of the hormones caused a rapid rise in the liver content of glucose 6-phosphate (G6P) by 15-70% but did not increase the ribose 5-phosphate (R5P) content. Liver ATP content was not affected. The hormones did not cause direct activation of PRPP synthetase, as gauged by the specific activity of the enzyme, its Km for substrates R5P and ATP, and its sensitivity to inhibition by ADP and GDP. The hormones did not increase the liver content of the enzyme activators Pi and Mg2+. The results suggest that the glycogenolytic hormones accelerate purine synthesis by a metabolic mechanism associated with the enhancement of glycogenolysis. PRPP synthesis is probably enhanced by the glycogenolysis-induced alterations in the cellular content of some metabolites other than R5P. PMID- 1723607 TI - Pyrimidine nucleotide synthesis in the rat kidney in early diabetes. AB - Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de novo and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative Ka and Km values for PPRibP and the possibility of metabolite channeling. PMID- 1723608 TI - Treatment of good-risk disseminated non-seminomatous germ cell tumours: the less bleomycin, the more cisplatin? PMID- 1723609 TI - RNA synthesis in isolated rat osteoclasts: inhibitory effect of calcitonin. AB - The metabolism of RNA has not been studied in the osteoclast (OC) because these bone-resorbing cells are only available in small numbers and cultures are always contaminated with other cells. Using two single-cell assay techniques, tritiated uridine (3H-UdR) autoradiography and gallocyanin quantitative cytophotometry, we have examined RNA synthesis in OCs isolated from neonatal rats. Oligo-nuclear OCs showed greater nuclear uptake of 3H-UdR than cells with many nuclei, and the variance of nuclear labeling within polykarya was greater in the latter, possibly because they contain nuclei of various ages. Salmon calcitonin (sCT) was a potent (ED50 approximately 5 x 10(-12) M) and rapid (40% reduction in 2 h, 75% reduction in 6 h) inhibitor of 3H-UdR uptake, and also reduced cytochemical total cellular RNA by 22% within 4 h. Forskolin (10(-5) M) inhibited nuclear uptake of 3H-UdR, suggesting that the sCT response may be mediated by cyclic AMP. Following a short (30 min) exposure to sCT, there was a progressive decline in labeling, followed by complete recovery by 4.5 h, a response possibly related to the phenomenon of calcitonin-induced persistent activation of adenylate cyclase. Inhibition of OC RNA synthesis may be an important component of its anti-resorptive action. PMID- 1723610 TI - Bone mass and comparative rates of bone resorption and formation of fibular autografts: comparison of vascular and nonvascular grafts in dogs. AB - The metabolic fate of whole grafts that were either vascularized or nonvascularized were compared. This study was designed to quantify and correlate changes in bone resorption, formation, and mass in orthotopic, stably fixed, weight-bearing autografts. The grafts were 8-cm segments of the fibula that were internally fixed. Fibula segments subjected to sham operations, nonvascularized autografts, and vascularized autografts were studied in 16 dogs at three months after surgery. Three months prior to surgery the dogs were labeled repeatedly over two months with 3H-tetracycline and 3H-proline. Metabolic turnover of whole grafts was evaluated by quantifying loss of 3H-tetracycline for measuring postoperative resorption of bone mineral and loss of 3H-collagen for resorption of bone collagen. Net changes in bone dry weight, calcium, and collagen per whole grafts were obtained to determine differential changes in the mineral and matrix mass. The difference in change between bone resorption and bone mass was used to determine the amount of new bone formation that had replaced the resorbed bone. Vascularized autografts lost more mass (12%), and had more bone resorption (40%) and more bone formation (28%) than sham operated and unoperated fibulas. Nonvascularized grafts lost much more bone mass (48%) because resorption was large (61%) and formation was relatively small (13%). More new bone was formed in vascularized autografts than in nonvascularized autografts. During the incorporation of bone grafts, resorption is an early and rapid process, whereas formation is a late and slow process.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723611 TI - Chronic effects of cadmium on kidney, liver, testis, and fertility of male rats. AB - Male Wistar rats (n:20), at 5 wk of age, were given cadmium in drinking water (10 mg/L water) for 52 wk; 8 males and 20 female rats, as controls, were given tap water. At the end of 28 and 40 wk, some of the cadmium-treated males and control group male rats were sacrificed for the histopathological examination of testis, kidney, and liver. At the end of 56 wk, histopathological examinations were performed in the same way. Liver, kidney, and testis cadmium levels were also determined by atomic absorption spectrophotometry. All the cadmium-treated male rats showed pathological testicular alterations, and liver and kidney damage after chronic exposure. Cadmium levels were found to be highest in the kidney (1.009 +/- 0.034 microgram/g wet tissue in the infertile group). At the end of the 52-wk period, reproductive capacity of the cadmium-treated rats was investigated and was found to be lost in 39.89% of the animals. PMID- 1723612 TI - Interactions of dietary lead with fish oil and antioxidant in chicks. AB - Two experiments were conducted with day-old broiler chicks reared to 18 or 19 d of age. The objectives were: (1) to examine the effects of the antioxidant ethoxyquin (EQ) on peroxidation in feeds containing fish oil (FO) or lead (Pb), and (2) to determine whether systemic effects of Pb, which are attributed to tissue peroxidation, can be reversed by dietary EQ. Experiment 1 was a 2 x 2 factorial arrangement with the factors being 4% dietary cottonseed oil (CSO) vs FO and dietary Pb as lead acetate trihydrate (0 vs 1000 ppm). Feed was mixed 1 d prior to initiation of the experiment and stored at 4 degrees C until it was placed in the feeders. Experiment 2 was a 2 x 2 x 2 factorial arrangement with the factors being 3.5% dietary oil (CSO vs FO), dietary Pb (0 vs 1000 ppm), and EQ (0 vs 75 ppm). Feed was mixed 1 d prior to initiation of the experiment and held at room temperature thereafter. Growth depression by FO and Pb was less pronounced in Experiment 1 than in Experiment 2. In Experiment 2, FO and Pb increased the concentration of feed peroxide, and the increases were prevented by EQ. The growth depression by FO was completely reversed by EQ. EQ reversal of Pb induced growth depression, although substantial, was not complete. The FO diet without Pb had a peroxide content (12.4 meq/kg feed) similar to the CSO + Pb diet (12.3 meq/kg feed); however, growth was not similar (407 vs 213 g body weight at 19 d, respectively). The results suggest that the toxic effects of Pb are mediated by peroxidative alterations both in the feed and in tissues. The ability of EQ to reverse significantly Pb effects on growth suggests a systemic action of this antioxidant. PMID- 1723613 TI - The effect of in ovo boron supplementation on bone mineralization of the vitamin D-deficient chicken embryo. AB - It has been hypothesized that boron (B) is an essential element for animals, but its action will vary greatly depending on the nutriture of the organism. One of the nutrients implicated as having an interaction with boron is cholecalciferol (Vit D3). This study was carried out to determine if such an interaction exists. The study was conducted utilizing vitamin D-deficient chicken embryos that were injected through the shell at 8 d of embryogenesis with carrier (NaCl and/or acetone), B (0.5 mg), B + Vit D3 (0.5 mg and 0.3 microgram, respectively), or Vit D3 (0.3 or 1.5 micrograms). The in ovo concomitant administration of boron and vitamin D enhanced (p less than 0.05) the hatchability of the vitamin D-deficient embryos. Furthermore, boron and/or vitamin D3 increased (p less than 0.05) the percent of bone ash and decreased (p less than 0.05) the exaggerated height of the proliferative zone of the epiphyseal growth plate normally observed in vitamin D deficiency, suggesting a more rapid bone formation. The results provide further evidence supporting the hypothesis that boron plays a role in bone mineralization through an interaction with vitamin D. PMID- 1723614 TI - The effect of silicon (Si) on lipid parameters in blood serum and arterial wall. AB - The influence of silicon treatment on the levels of lipid parameters of blood serum and aortic wall was studied in rats. The concentrations of total cholesterol, phospholipids, triglycerides, HDL-cholesterol, LDL-cholesterol, and HDL-phospholipids were measured in sera of rats receiving per os a soluble, inorganic silicon compound--sodium metasilicate nonahydrate (Na2SiO3.9H2O)- dissolved in the drinking water. In the aortic tissue levels of total cholesterol, triglycerides and phospholipids were estimated. An increase in HDL cholesterol and HDL-phospholipid concentrations, with a simultaneous decrease of LDL-cholesterol and triglyceride levels, was observed in the sera of the tested group. The levels of total cholesterol and phospholipids in the sera, as well as the concentrations of lipids in the aortic walls, showed no significant differences. The results obtained could provide evidence for the existence of an additional mechanism of silicon antiatheromatous action, concerning the modification of activity of enzymatic systems involved in lipids metabolism. PMID- 1723615 TI - Effect of dimethyl sulfoxide on enlarged hearts of copper-deficient rats. AB - The purpose of this study was to examine, by transmission electron microscopy (TEM), the nature of the protective effect of dimethyl sulfoxide (DMSO) on hearts of copper-deficient (CuD) rats. Male, weanling Sprague-Dawley rats were fed, in a two-way design, CuD (0.45 micrograms/g) or copper-sufficient (CuS, 5.4 micrograms/g) diets with or without 5% DMSO in their drinking water. After 28 d, CuD rats showed typical signs of copper deficiency, including reduced liver and heart Cu, enlarged hearts, and anemia. DMSO-treated, CuD rats had lower heart weights and higher hematocrits than CuD rats. DMSO enhanced organ Cu concentrations in CuS, but not in CuD rats. TEM of CuD hearts showed myofibrillar distortion and enlarged, vacuolated mitochondria with fragmented cristae; morphometric measurements indicated an enhanced mitochondrial/myofibrillar ratio (mito/myo), but an increase of both mitochondrial and myofibrillar mass relative to CuS hearts. Compared to CuD hearts, DMSO-treated CuD hearts showed better mitochondrial morphology and myofibrillar organization, as well as a greater mito/myo, but lower mitochondrial and myofibrillar masses. Its function as a hydroxyl radical scavenger indicates that DMSO could protect CuD hearts, in particular their mitochondria, against oxidative damage. However, because measurements of thiobarbituric acid reactive substances were not consistent with this theory, other metabolic mechanisms, direct and indirect, must be examined. PMID- 1723616 TI - Determination of nonheme iron using inductively coupled plasma-atomic emission spectrometry. AB - A technique for the rapid and accurate estimation of nonheme iron using inductively coupled plasma-atomic emission spectrometry is described. Yttrium was used as an internal standard. An external calibration method was used. The standards were prepared in a matrix composed of 2.5N HCl in 10% (w/v) trichloroacetic acid. The supernatant and coagulum fractions of liver nonheme iron were separated by the method of Drysdale and Ramsay with minor modification. The data determined by this procedure was compared and found to be agreement with data determined by the method of Hallgren. To evaluate the iron status of rats, hemoglobin and liver nonheme iron were determined. Hemoglobin and all of the nonheme iron fractions of the rats fed an iron-deficient diet were significantly lower than those of the rats fed an iron-sufficient diet. The blood content in the liver was estimated to be 80 microL/g from the blood iron concentration, and the difference between total and nonheme iron concentration in liver. PMID- 1723617 TI - The influence of selenium and vitamin E on the enhanced respiratory burst reaction in smokers. AB - The respiratory burst reaction (RBR) of neutrophilic granulocytes of the peripheral blood was estimated by means of the luminol reaction in 10 smokers and in 10 nonsmokers. Compared to the nonsmokers, the RBR of smokers' granulocytes showed a significantly higher rate of RBR. RBR consists of two enzymatic systems, i.e., NADPH-oxidase generating superoxide anions and myeloperoxidase, generating hypochlorous acid. Furthermore the superoxide anion may undergo dismutation to oxygen and peroxide. Thus, since the RBR may cause an oxidative stress, the smokers were supplemented for 10 d with antioxidants, i.e., 200 micrograms L-Se methionine and 1000 mg vitamin E/d. After 10 d of supplementation with the antioxidants, the RBR of the smokers was significantly decreased by 20-75 percent. Since the oxidative stress associated with RBR may cause autodigestive reactions in the lungs of smokers, it may be beneficial for smokers to use relatively high doses of such antioxidants in order to hamper the pathological processes associated with smoking. PMID- 1723618 TI - Aluminum decreases the zinc concentration of soft tissues and bones of rats fed a low calcium-magnesium diet. AB - The relationship between magnesium (Mg) and zinc (Zn) in soft tissues and bone of rats was studied after administration of unbalanced mineral diets. Minerals and metals in soft tissues and bone were determined using inductively coupled plasma emission spectrometry (ICP). There were significant positive correlations between serum Zn and Mg levels, between serum Zn and Zn content of soft tissues and bone, and between serum Mg levels and Zn content of bone and soft tissues in rats fed unbalanced mineral diets. A significant positive correlation was also found between Zn and Mg content in the lumbar spine and femoral bone of rats. It appears that altered bone mineralization induced by unbalanced mineral diets leads to mobilization of Mg and Zn from rat bones in similar ways. PMID- 1723619 TI - Effect of delayed gastric emptying on fluoride absorption in the rat. AB - The rate and site of fluoride (F) absorption were compared in fasted 350 g male rats given 50 micrograms F (as NaF) in either water or a 7.5% pectin solution. Absorption was measured at intervals up to 2 h following gastric intubation. Gastric emptying was measured by inclusion of 14C-PEG in the F solution. The extent of gastric F absorption was derived from rates of gastric emptying (14C PEG loss) and F loss. Pectin markedly slowed gastric emptying, but by 2 h, more than 90% of the solution had passed into the small intestine in both groups, and F absorption exceeded 90% in both groups. The rate of F absorption was initially much slower in the pectin group than in the group given F in water, and plasma F concentration increased more slowly and reached a lower maximum value. Absorption from the stomach was greater in the pectin group, but still accounted for only approx 25% of total gastrointestinal absorption. The reduced rate of F absorption and slower rise in plasma F concentration accompanying delayed gastric emptying indicate that passage of F into the small intestine is the major factor in rapid F absorption. PMID- 1723620 TI - Serum zinc and copper in myocardial infarction with particular reference to prognosis. AB - Serum copper and zinc estimations in humans were made to find their diagnostic and prognostic value in cases of myocardial infarction. Following infarction, there was an increase in serum copper levels from the first 24 h up to the 7th day, with gradual decline that did not reach the normal value up to the 14th day. The serum zinc levels declined in the first 24 h until the 4th day and increased to the normal value on the 14th day. It is concluded that, for diagnosis of myocardial infarction, serum zinc levels are more useful during the first week and copper levels in the second week after the onset of infarction. PMID- 1723621 TI - Octamer-dependent regulation of the kFGF gene in embryonal carcinoma and embryonic stem cells. AB - Expression of kFGF, which belongs to the family of fibroblast growth factor genes, is restricted to undifferentiated embryonal carcinoma and embryonic stem cells. Stem cell specific expression of kFGF is controlled by a distally localized enhancer, conferring both positive and negative regulation to the kFGF and tk promoters. This enhancer contains a consensus octamer binding sequence that controls positive regulation in EC and ES cells. The octamer sequence binds Oct1 and Oct4 in nuclear extracts from undifferentiated EC cells, while only Oct1 is bound in nuclear extracts from RA differentiated cells. These results suggest that the kFGF gene is a target for positive regulation by Oct4 and implicate Oct4 as target for regulation by the retinoic acid receptors. PMID- 1723622 TI - A common disinfectant used in condom processing inhibits endonuclease digestion of sperm DNA. AB - DNA recovered from a condom found at a crime scene appeared undigestible with restriction enzymes, preventing characterization by Southern blot and polymorphic probe hybridization. Several chemical substances used in the processing and treatment of condoms were tested for inhibitory action of restriction enzymes. In particular dibenzalkonium chloride appeared to promote enzyme inhibition at very low concentrations. The effectiveness of treatments to restore cleavage of sample DNA in the presence of such contaminants is discussed. PMID- 1723623 TI - International eye banking and the Eye Bank Association of America (EBAA). PMID- 1723624 TI - [Regeneration of peripheral nerves and intracerebral transplantation]. AB - Axoplasmic flow is essential to the regeneration of peripheral nerves. We observed a mean of 12 mm/day for the slow axoplasmic flow and a mean of 410mm/day for the fast axoplasmic flow. In the process of regeneration of peripheral nerves, however, slow transport increased to 14.7mm/day and fast transport to 572mm/day on day 7. We reviewed the relevant literature on the axoplasmic flow and described the topics in this report. Some central nerves may show poor regeneration but it has been confirmed that nerve cells grow and survive by intracerebral nerve transplantation, and this technique has been applied to the treatment of Parkinson's disease. Further development can be expected for the regeneration of central nerves through transplantation. PMID- 1723625 TI - [Tissue repair of uterine cervix--cell-biological properties of normal uterine cervical epithelia of transformation zone in vitro]. AB - The objective of this study is to culture the epithelia of the transformation zone of the uterine cervix for long term and evaluate their biological characteristics, such as morphology, growth behavior, alkaline phosphatase activity and heterotransplantability. The epithelia of transformation zone of 15 cases of myoma uteri were cut into 1 x 1 x 1 mm fragments and placed directly on the cover glass. The explants were cultured at 37 degrees C in 5% CO2 and 95% air. In vitro outgrowth of squamous cells (squamous cell outgrowth pattern) was observed in 44, that of columnar cells (columnar cell outgrowth pattern) was observed 49, a mixture of squamous and columnar cell outgrowth patterns was 52 out of 198 explants of transformation zone. The squamous cells were polygonal in shape and showed a pavement-like cell arrangement. The glandular cells grew in whorled fashion. Along the margins of the outgrowth of glandular cells, two types of cells were seen after 2 weeks of culture. One type contained secretory vacuoles of glandular cell, and the other type contained a large number of tonofilaments of squamous metaplastic cells. These phenomena suggested that biological characteristics of the cells in vivo can well be retained in vitro for a relative long term (about 6 weeks). PMID- 1723626 TI - Plasma desorption mass spectrometry of phosphopeptides: an investigation to determine the feasibility of quantifying the degree of phosphorylation. AB - The intact ion yields of phosphotyrosine, phosphothreonine and mono- and diphosphoserine residue-containing peptides have been compared with the non phosphorylated sequences using plasma desorption mass spectrometry. Equimolar mixtures of the phosphorylated (Mp) and non-phosphorylated peptides (M) were also analysed. The positive mass spectra of these mixtures show a higher intensity of the [M + H]+ compared with the [Mp + H]+. In the negative mass spectrum, the bias towards the [M - H]- compared with the [Mp - H]- was reduced, but the spectra generally did not accurately reflect the stoichiometry. PMID- 1723627 TI - Epitope mapping of allergens and antigens of timothy pollen extract. AB - For the identification of allergens in crude timothy pollen extract, 2D immunoblotting was performed with subsequent use of patients' IgE. Only about 15 of more than 70 detectable components with molecular weights of 55, 38, 35, and 32 kD showed IgE-reactivity. The 55 and 35 kD allergens were identified as glycoproteins by a glycan detection kit. To investigate the protein structure of the allergens, proteins of the pollen extract were cleaved by CNBr and endoproteinases. The cleavage products were identified by patients' IgE and monoclonal antibodies (moab) raised against timothy pollen proteins. While the use of endoproteinases resulted in a complete loss of some allergens, CNBr produced smaller IgE-reactive proteins, e.g. the 38 kD component was split into a 32 kD component and the original 32 kD protein was partially cleaved into IgE reactive fragments of 30, 21, and 15 kD. In order to investigate the IgE-binding epitopes we intend to determine the amino acid sequence of the cleavage products. PMID- 1723628 TI - Identification of human blood plasma and serum proteins in two-dimensional gels by use of protein A-Sepharose: application to alpha 1-microglobulin. AB - The relative position identification of some of the proteins in human blood plasma and serum after separation by two-dimensional electrophoresis (2-DE) in gel slabs have been studied. Antibodies specific to the protein to be identified were first immobilized on Protein A-Sepharose. Serum or plasma samples devoid of this protein were obtained following adsorption and subsequently the protein itself was eluted. The preparations were examined by 2-DE, silverstaining and blotting with parallel analysis of the purified proteins. Position identification in 2-DE gels of the spots of alpha 1-microglobulin (= protein HC) is shown as an example. The technique has general applicability not only for identification of the positions in 2-DE gels of serum and plasma proteins but also for proteins in other fluids, tissues and organs from humans and other species. PMID- 1723629 TI - [Modifications of the enzymatic content and morphology of the exocrine pancreas of rats with experimental hepatic cirrhosis induced by thioacetamide]. AB - In order to assess the experimental level of enzymatic content in exocrine pancreas of cirrhotic rats, cirrhosis was induced with administration of thioacetamide (50 mg/kg) during ten weeks to male Wistar rats with an initial average weight of 350 g. Contents of amylase, lipase and trypsinogen were determined in pancreatic tissues and amylase, lipase and biliary salts in duodenal juice obtained by cannulation and perfusion with physiologic serum. A higher presence of trypsinogen and amylase was detected in pancreatic tissues, and of lipase in the duodenal juice, with a trend, although insignificant, towards a decrease in biliary salts among the cirrhotic group. No changes were observed in the morphologic study. The hypothesis that a deficit of biliary salts in experimental cirrhosis could be responsible of the enzymatic increase in the pancreatic tissue and, in particular, of the selective excretion of lipase, is discussed. PMID- 1723630 TI - [Toxic syndrome and myalgia as manifestations of Whipple's disease]. PMID- 1723631 TI - Antibodies against central nervous system tissue (anti-CNS) detected by ELISA and western blotting: marker antibodies for neuropsychiatric manifestations in connective tissue diseases. AB - Organ specific antibodies against epitopes of the central nervous system (CNS) tissue were detected by ELISA and Western blotting (WB) in sera from patients with ANA positive collagen disorders using a 100,000 g supernatant from beef or rat brain. The corresponding CNS-antigens consist of six major determinants at molecular weights 29, 48, 56, 68 kD and six minor determinants at 130, 110, 86, 60, 38, 34 kD. All except the 38 kD polypeptide were organ specific. Forty-six of 91 patients with ANA positive collagen disorders reacted with at least one of these determinants; 43 of them had cerebral symptoms in contrast to only three of the 43 anti-CNS negative patients. Sera from patients with other disorders did not react with these epitopes. We conclude that anti-CNS antibodies detected by Western blotting may be marker for neuropsychiatric manifestations in patients with collagen disorders. PMID- 1723632 TI - Structure-function studies of S-antigen: use of proteases to reveal a dominant uveitogenic site. AB - Retinal S-antigen induced experimental autoimmune uveitis (EAU) is a severe, predominantly T-cell mediated inflammatory disease of the uveal tract and retina of the eye. Pretreatment of LEW rats with the monoclonal antibody, MAbS2.4.C5, which defines an epitope in S-antigen, has been shown to effectively inhibit the subsequent induction of EAU with S-antigen. Using synthetic peptides and cyanogen bromide fragments of S-antigen we found the binding site of MAbS2.4.C5 to be located at the carboxy terminus of the molecule corresponding to amino acid positions 375 to 380. Limited Staphylococcus aureus V8 protease digestion yielded several polypeptide fragments including one large 43 kD fragment which retained antibody binding to a variety of both polyclonal and monoclonal antibodies which identify epitopes that span the length of the S-antigen. This treatment, however, completely destroys the MAbS2.4.C5 binding site and dramatically reduces uveitopathogenicity. Limited trypsin and papain digestion, on the other hand, had little effect on pathogenicity or on MAbS2.4.C5 binding to S-antigen or its peptide fragments. These results indicate that the carboxy-terminus of S-antigen plays a predominant role in the pathogenesis of EAU. PMID- 1723633 TI - The alpha-galactosyl epitope on human normal and autoimmune thyroid cells. AB - As much as 1% of circulating IgG in man (the natural anti-Gal antibody) is directed against the alpha-galactosyl epitope, with the structure Gal alpha 1--- 3Gal beta 1----4GlcNAc-R. The alpha-galactosyl epitope is abundantly expressed on cells of nonprimate mammals, prosimians, and New World monkeys. Its expression is diminished in Old World monkeys, apes, and humans. It has been previously suggested that interaction between anti-Gal and aberrantly expressed alpha galactosyl epitopes on thyroid cells may contribute to the initiation of autoimmune thyroid disorders. To study this possibility, alpha-galactosyl epitope expression on thyroid cell membranes of normal individuals and patients with Graves' disease was assessed by a sensitive radioimmunoassay. alpha-Gal-actosyl epitopes were found both on normal and diseased thyroid cells. Whereas the concentration of these epitopes on Graves' disease thyroid membranes was somewhat higher than that observed in normal glands, the difference was not significant. The activity of the enzyme, alpha 1-3-galactosyltransferase, which synthesizes the alpha-galactosyl epitope, was higher in microsomal fractions obtained from some patients as compared with healthy controls, but not significantly different. In view of the abundance of anti-Gal antibody in the circulation, it is argued that, under physiologic conditions, the interaction of this antibody with alpha galactosyl epitopes does not elicit pathologic effects. However, aberrant expression of the alpha-galactosyl epitope may result in effective anti-Gal binding to thyroid cells (e.g., rearrangement of this structure on the cell membrane or its increased expression).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723634 TI - DNA ploidy studies of benign and malignant tumours: comparison of flow cytometry and image analysis techniques using two types of cytological specimen. AB - DNA ploidy studies were carried out on Feulgen stained smears and cytocentrifuge preparations from 35 malignant tumours and four benign neoplasms using the CAS image analyser. The smears were prepared from scrapings from fresh tumour tissue whereas the cytocentrifuge preparations were prepared from single nuclear suspensions from paraffin-embedded cell blocks from the same tumour. Histograms obtained by image analysis of the tumour scrapes were compared with those obtained on the cytocentrifuge preparations. Concordant results were obtained in four benign tumours (100%) and 32 malignant tumours (91%). The results obtained by image analysis were also compared with results obtained by flow cytometry of the tumour tissue. Discordant results were obtained for three malignant tumours. Possible reasons for the discrepancy include sampling error, tumour heterogeneity and selective loss of cell populations during processing. PMID- 1723635 TI - The molecular mechanism of nondegranulative release of biogenic amines. AB - According to current teaching biogenic amines are released by exocytosis, i.e. by evacuation of amine storing vesicles or granules into the extracellular space. The release of transmitter amines is quantal, i.e. occurs in packs of transmitter molecules. These packs are assumed to be identical with vesicle contents, in other words, the smallest releasable quantum equals the amine content of one vesicle. However, there are experimental observations which do not fit in with this version of an exocytotic release theory. Observed quantitative discrepancies could be explained if the release mechanism allowed a fractional release of transmitter amine from several vesicles instead of the total evacuation of a few. The lack of adequate knowledge about the mechanisms of storage of biogenic amines within the vesicles has up til now rendered it difficult to envisage the machinery behind a fractional release of the amine content of a vesicle. In extensive in-vitro studies we have found that the matrices of amine storing granules (i.e. from mast cells, chromaffin cells and nerve terminals) show the properties of weak cation exchanger materials, carboxyl groups serving as amine binding ionic sites. When exposed to cations like sodium and potassium ions, the amines are released from their storage sites according to kinetics characteristic of weak cation exchangers. In vivo, amine release from cat adrenals on splanchnic nerve stimulation also occurs according to ion exchange kinetics. Histamine release from mast cells is considered to occur as the result of degranulation, i.e. the expulsion of histamine storing granules to the extracellular space, a typical example of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723636 TI - Hormonal manipulation of prostatic cancer. PMID- 1723637 TI - Air pollution: II--Road traffic and modern industry. PMID- 1723638 TI - Hypothalamic degradation of galanin(1-29) and galanin(1-16): identification and characterization of the peptidolytic products. AB - The degradation of the neuropeptide galanin(1-29) and its fully active synthetic N-terminal fragment galanin(1-16) in hypothalamic tissue, where these peptides potently affect feeding behaviour, is studied. Galanin(1-29) had a half-life of 100 min while galanin(1-16) had a half-life of 28 min when incubated with a hypothalamic membrane preparation. The putative sites of peptidolytic cleavage of the active N-terminal fragment galanin(1-16) were determined as being between amino acids Leu4 and Asn5, between Asn5 and Ser6, and between His14 and Ala15, respectively. The synthetic analogs of galanin(1-16) where Leu4, Asn5 or Ser6 was substituted by Ala were all more stable to peptidolysis; [Ala4]galanin(1-16) had a half-life of 55 min. Cleavage of the galanin(1-16) between His14-Ala15 yields a ligand-galanin(1-14) which binds to the receptor with high affinity (KD approximately 10(7) M), while cleavage at amino acid residues Leu4, Asn5 and Ser6 results in inactive peptide fragments with affinities for the galanin receptor below 10(-4) M. The enzyme(s) responsible for degradation of galanin were identified as endopeptidase(s), which were partially inhibited by bacitracin (1 mg/ml) by up to 50%, but not significantly by EDTA (1 mM), phosphoramidon (1 microM), phenylmethylsulfonyl fluoride, (100 microM) or aprotinin (10 micrograms/ml). PMID- 1723639 TI - Basic fibroblast growth factor promotes in vivo cerebral angiogenesis in chronic forebrain ischemia. AB - This experiment was designed to determine if intraventricular administration of basic fibroblast growth factor (bFGF) could promote cerebral angiogenesis in a model of mild chronic forebrain ischemia. Wistar rats underwent bilateral carotid artery ligation. Animals received intraventricular injections of bFGF every 4 days for 28 days. Basic fibroblast growth factor caused a significant dose dependent increase in capillary density compared to ischemic controls in all regions examined. These results support the hypothesis that chronic intraventricular administration of bFGF induces in vivo cerebral angiogenesis. PMID- 1723640 TI - Calcitonin gene-related peptide in monkey spinal cord and medulla oblongata. AB - The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers and cell bodies was studied in the spinal cord and the medulla oblongata of the grey monkey (Macaca fascicularis) using peroxidase-antiperoxidase (PAP) immunohistochemistry. At all levels of the spinal cord many CGRP-IR motoneurons and fibers were seen in the motor nuclei. In the medulla, CGRP-IR cell bodies were encountered in nucleus raphe obscurus, nucleus raphe pallidus and nucleus raphe magnus, nucleus reticularis lateralis as well as in the area dorsal to the inferior olive. Bulbar motoneurons were much more intensely stained than spinal cord motoneurons, indicating higher levels of CGRP-like immunoreactivity (LI) at the medullary level. The concentration of CGRP-LI measured by radioimmunoassay showed higher levels in the dorsal quadrants as compared to the ventral quadrants, but the dorsal/ventral ratio was lower than has previously been reported from the rat. The present results demonstrate that using the PAP technique CGRP-LI can be visualized in a larger number of spinal cord motoneurons of the monkey than earlier revealed by immunofluorescence. Moreover, the finding supports the view that the CGRP-IR nerve endings in the spinal motor nuclei originate from cell bodies in the medullary raphe nuclei. PMID- 1723641 TI - In vivo brain incorporation of [1-14C]arachidonate in awake rats, with or without cholinergic stimulation, following unilateral lesioning of nucleus basalis magnocellularis. AB - Regional brain incorporation of a radiolabeled unsaturated fatty acid, [1 14C]arachidonic acid (14C-AA), was measured in awake rats following unilateral lesioning of the nucleus basalis magnocellularis (NBM). Right-sided lesions were produced in 3-month-old, male rats by stereotaxic injection of 10 micrograms ibotenic acid. Two weeks after lesioning, rats were subjected to one of two protocols: (1) 5 min intravenous infusion of 14C-AA (170 microCi/kg); or (2) i.p. injection of arecoline (5 mg/kg), a cholinergic agonist, followed by 5 min intravenous infusion of 14C-AA. All animals were killed 15 min postinfusion. Brains were frozen and sectioned for quantitative autoradiography or were stained for acetylcholinesterase (AChE). Animals with unilateral NBM lesions displayed reduced AChE staining in prefrontal, frontal and parietal cortices of the lesioned side, but there was no right-left difference in incorporation of 14C-AA without cholinergic stimulation. Arecoline administration increased 14C-AA incorporation into the prefrontal and frontal cortices ipsilateral to the NBM lesion as compared to the contralateral side and the increase was most prominent in deeper cortical layers such as layers IV and V. Right-left differences in incorporation were not apparent in parietal, temporal, or occipital cortices, where reduction of AChE activity was minimal or absent, nor in subcortical structures. The results suggest that the intravenous 14C-AA technique combined with cholinergic stimulation can be used to detect compensatory regulation of phospholipid-coupled signal transduction caused by a deficit in cholinergic input into the cerebral cortex. PMID- 1723642 TI - A carbohydrate epitope defined by monoclonal antibody VC1.1 is found on N-CAM and other cell adhesion molecules. AB - VC1.1 is a monoclonal antibody that stains the surfaces of neuronal subsets in the brain. Previous work showed that VC1.1 recognizes 3 polypeptide bands with molecular weights of 95-105 kDa, 140 kDa and 170 kDa and two high molecular weight proteoglycans with weights of approximately 680 and 650-700 kDa. The heterogeneity and molecular weight range of these bands suggested that VC1.1 might recognize a carbohydrate moiety associated with a family of cell surface molecules. It had been previously demonstrated that a separate monoclonal antibody, HNK-1 also recognized a cell surface associated epitope characterized as a sulfate- and glucuronic acid-containing N-linked carbohydrate. This epitope has been shown to be present on members of the N-CAM adhesion molecule family. In this report, we demonstrate that VC1.1 recognizes an N-linked carbohydrate group that is attached to myelin-associated glycoprotein and N-CAM. Immunocytochemical and biochemical comparisons of VC1.1 and HNK-1 staining in rat and cat brain indicate that these two antibodies probably recognize overlapping, or identical carbohydrate epitopes. PMID- 1723643 TI - Non-dopaminergic projections from the substantia nigra pars lateralis to the inferior colliculus in the rat. AB - The substantia nigra pars lateralis (SNI) of the rat was found, by the anterograde and retrograde tracing methods, to send projection fibers to the peripheral shell region surrounding the central nucleus of the inferior colliculus (IC), bilaterally with a clear-cut ipsilateral dominance. SNI neurons sending their axons to the IC were distributed throughout the entire rostrocaudal extent of the SNI. None of these SNI neurons showed tyrosine hydroxylase-like immunoreactivity. PMID- 1723644 TI - Substance P and NMDA receptors mediate a slow nociceptive ventral root potential in neonatal rat spinal cord. AB - Substance P and glutamate actions have separately been implicated in the generation of nociceptive-related slow ventral root potentials (slow VRPs). We report that slow VRPs are dependent on both substance P and NMDA receptor mediated neurotransmission. Slow VRPs of 10-40 s duration were evoked by electrically stimulating a lumbar dorsal root and recorded at the corresponding ipsilateral ventral root in spinal cords isolated from 1- to 5-day-old rats; the monosynaptic reflex was also recorded. The NMDA receptor antagonist APV (5-20 microM) and the substance P antagonist spantide (10-20 microM) both reversibly depressed the slow VRP without affecting the monosynaptic reflex; spantide and APV applied together nearly abolished the slow VRP. The quisqualate-kainate receptor antagonist CNQX (1-5 microM) reduced the monosynaptic reflex and an early component of the slow VRP. A slow VRP could be elicited by brief (0.1-1.0 s) focal applications of either substance P (2-20 microM) or NMDA (10 microM), and also by CGRP (2-20 microM). Substance P-evoked and NMDA-evoked responses were blocked by their respective antagonists spantide and APV. Each was also cross sensitive to the other antagonist. Both excitatory amino acids, acting on an NMDA receptor, and substance P, acting on a tachykinin receptor, thus appear to be involved in generating this slow potential. Both NMDA and tachykinin receptors are necessary to generate a full response. PMID- 1723645 TI - Effects of 5-hydroxytryptophan on extracellular serotonin in the spinal cord of rats with experimental allergic encephalomyelitis. AB - Serotonin (5-HT) and the serotonin metabolite, 5-hydroxyindoleacetic acid (5 HIAA) were collected by in vivo dialysis in the lumbar spinal cord of control rats and rats with hindlimb paralysis induced by experimental allergic encephalomyelitis (EAE). Both 5-HT and 5-HIAA were significantly decreased in baseline samples from EAE rats compared to controls. This decrease in extracellular 5-HT and 5-HIAA in the EAE rats was accompanied by marked morphological changes in spinal cord axons and axon terminal plexuses that were stained for 5-HT-like immunoreactivity. The 5-HT precursor, 5-hydroxytryptophan (5-HTP)-increased 5-HT and 5-HIAA levels in dialysate samples from both control and EAE animals. However, the 5-HTP-induced increase in extracellular 5-HT was significantly greater in the EAE rats than in the controls, despite a lower baseline 5-HT level in the EAE animals. In contrast to 5-HT, both baseline and post-5-HTP levels of 5-HIAA were significantly higher in control animals than in EAE animals. The decreased extracellular 5-HT and 5-HIAA in baseline samples from the EAE rats compared to controls is probably a consequence of the damage to descending 5-HT axons and axon terminals that occurs during the disease. The larger increase in extracellular 5-HT in EAE animals after precursor injection may reflect both decreased 5-HT reuptake from the extracellular space by damaged 5-HT terminals and disruption of the blood-brain barrier that allows entry into the central nervous system of 5-HT that was synthesized from 5-HTP in the periphery. PMID- 1723646 TI - Differential distribution of GABA- and serotonin-containing afferents on an identified central neuron. AB - The distributions of gamma-aminobutyric acid (GABA)- and serotonin (5-HT) containing terminals impinging on the surface of the Mauthner (M-) cell were studied at the light microscopic level using double immunofluorescent labeling and were compared with that of the glycine receptor. The latter was visualized indirectly, using a monoclonal mouse antibody which recognizes its 93-kDa associated protein. This neuron has two large principal dendrites: one extending ventrorostrally (ventral dendrite) and the other dorsolaterally (lateral dendrite). There are also two other classes of smaller processes: one that projects ventrally (small ventral dendrites) and one penetrating in the axon cap (cap dendrites), a peculiar neuropil surrounding the initial segment of the M cell axon. A cellular regionalization of these afferent systems was found: GABA boutons, labeled for glutamic acid decarboxylase (GAD), were localized preferentially on the lateral dendrite while 5-HT-filled endings predominated on the ventral one. The density of these two classes of inputs was comparable in the other areas of the M-cell: less of their terminals were in contact with the soma outside the axon cap, and more numerous boutons, which presented either GABA or 5 HT immunoreactivities, were apposed to the small ventral dendrites. This preferential pattern of innervation differed with the ubiquitous presence of glycine receptor clusters on the M-cell membrane. Finally no evidence of a colocalization of GABA and 5-HT in afferent endings was detected at any portion of the M-cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723647 TI - [Electrochemotherapy, a new antitumor treatment: first clinical trial]. AB - Electrochemotherapy (ECT) is a new antitumor treatment which consists in delivering electric pulses to the tumor some minutes after an intravenous injection of bleomycin. We report here the first clinical trial of ECT, applied to patients with permeation nodules of head and neck squamous carcinomas. ECT was well tolerated by patients, no serious incident occurred and a clear antitumor efficiency was found. PMID- 1723648 TI - Production and characterization of new monoclonal antibodies to human osteoclasts. AB - Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts. PMID- 1723649 TI - Regulation of plasminogen activator and plasminogen activator inhibitor production by growth factors and cytokines in rat calvarial cells. AB - Fetal rat osteoblast-enriched calvarial cells were used to study the effects of various growth factors and cytokines on plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities and the possible relationship of these effects to bone resorption. Confluent cultures were exposed to various factors under serum-free conditions, and levels of PA and PAI activities were examined in both conditioned medium (CM) and cell layer using the 125I-fibrin plate assay, fibrin zymogram, and reverse fibrin zymogram. According to the 125I fibrin plate assay or zymogram, incubation of cells with acidic fibroblast growth factor (aFGF), basic FGF (bFGF), epidermal growth factor (EGF), and platelet derived growth factor (PDGF) elevated the PA activity in the CM as well as in the cell layer extract. Incubation with interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and insulin-like growth factor I (IGF-I) produced no change in PA activity in either CM or cell layer. Addition of transforming growth factor beta (TGF beta) to calvarial cells resulted in nearly undetectable PA activity in CM with the fibrin plate assay but increased PA activity on the fibrin zymogram after PAI was separated from PA by SDS-PAGE. A reverse fibrin zymogram indicated that PAI activity was greatly enhanced in TGF beta-treated CM. TGF beta treatment also increased PA activity in the cell layer of calvarial cells. Treatment of calvarial cells with bFGF and PDGF slightly increased PAI secretion into medium. This increase, however, was not as dramatic as the increase of PA induced by these two agents. IL-1 alpha and TNF alpha did not change PAI concentration in CM. No detectable PAI activity was found in the cell layer in control and treated groups. The PA found in the CM and cell layer of rat calvarial cells was the urokinase type; the PAI stimulated by TGF beta was the endothelial cell type, PAI-1. The regulation of PA activity by growth factors and cytokines did not correlate with their resorption-stimulating activities. Thus, PA secreted by osteoblasts may not be the only factor involved in the initiation of bone resorption. Delineation of the function of PA and PAI in the physiology of bone tissue awaits further studies. PMID- 1723650 TI - The matrix of endochondral bone differs from the matrix of intramembranous bone. AB - Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induce cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM. PMID- 1723652 TI - Allergy to thiopentone. PMID- 1723651 TI - Nonspecific histamine-releasing properties of general anesthetic drugs. PMID- 1723653 TI - Anaphylactoid reactions to narcotic analgesics. PMID- 1723654 TI - Anaphylactoid reactions to radiocontrast material. PMID- 1723655 TI - Anaphylactoid reactions to intravenous solutions used for volume substitution. PMID- 1723656 TI - The value of mammography in different ethnic groups in Israel--analysis of mortality reduction and costs using CAN*TROL. AB - Routine follow-up using screening mammography is currently considered the best method available for the early detection of breast cancer. The potential impact of such an activity is highly dependent on the size of the population suitable for and attending the screening program, and on the basic disease rates in this population. A computer-based model for designing cancer control strategies (CAN*TROL) was used. The screening scenario involved annual mammography of females 50 to 70 years of age in the two major Jewish ethnic groups in Israel. Two outcome parameters were studied: change in mortality rate and costs. Using the same scenario, a greater reduction in mortality rates was achieved in the whole screening period among European-American born females (-20%) than among Asian-African born females (-14.7%). Cost showed different trends over time in the two groups, but totaled about $130,000,000 in each of the screening groups. The total cost of saving one case of death from breast cancer was much less for European-Americans ($316,000) than for Asian-Africans ($1,944,000). These results reflect the higher risk for breast cancer among European-American females as well as the difference in age structure of these two groups. The planning of every screening program should include an effort to estimate the real impact on mortality and the cost-effectiveness in the specific population involved. PMID- 1723657 TI - A dipstick, dot-ELISA assay for the rapid and early detection of bladder cancer. AB - A mouse monoclonal antibody (CK1K10) specific for keratin was developed. Enzyme immunoassays (EIAs) were performed to detect keratin in urine samples using CK1K10 and anti-mouse IgG. A high degree of sensitivity and specificity was obtained in the detection of bladder cancer patients. A fast dot-ELISA was developed in which urine is applied onto nitrocellulose (NC) sheets or dipsticks. Dipsticks coated with urine samples can be stored at room temperature for more than 5 days and at -20 degrees C for more than 3 months without loss of efficiency. A high positive correlation was found between the fast dot-ELISA and other EIAs. The assay does not require sophisticated equipment nor highly trained technical staff and can be performed in less than 30 min. The keratin dot-ELISA could be a reliable screening test for the early detection of bladder cancer. PMID- 1723658 TI - The potassium channel MBK1 (Kv1.1) is expressed in the mouse retina. AB - 1. The neurons of the retina have electrical properties that are different from those of most of the other neurons of the central nervous system. To identify the voltage-gated ion channels found in the retina, we screened mouse retinal cDNA libraries with oligonucleotide probes homologous to the mammalian K+ channel MBK1 (Kv1.1) and ligated two partial clones to produce a full-length clone with no significant differences from MBK1. 2. Expression of MBK1 mRNA was determined by RNAse protection. MBK1 mRNA was detected in retinal RNA and was also detected in brain, liver, and heart RNAs. 3. We transcribed the full-length clone, injected it into oocytes of Xenopus laevis, and measured the membrane currents 2 to 6 days later. Depolarization from a holding voltage of -90mV induced a slowly activated outward current with a peak value as large as 20 microA. The current inactivated very slowly with a single exponential time course [mean time constant, 6.5 +/- 0.4 sec (SEM) for activation voltage of -10mV]. 4. The outward current was reduced to half-maximal by 0.42 mM tetraethylammonium, 1.1 mM 4-aminopyridine, and 3.2 mM Ba2+ but was not significantly attenuated by Co2+ (1 mM). 5. The reversal potential (measured with tail currents) changed by 53mV per decade change of [K+] from 1 to 77 mM. 6. The voltage for half-maximal activation of the conductance was -26.6mV (+/- 1.7mV), and the voltage required for an e-fold increase in conductance was 6.9mV (+/- 0.5mV). 7. Thus, the mRNA for MBK1 found in the mouse retina causes the expression of a voltage-dependent K+ current which has properties suitable for may retinal neurons. PMID- 1723659 TI - Developmental expression of the glial fibrillary acidic protein (GFAP) gene in the mouse retina. AB - 1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2. In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from "dedifferentiation" of Muller cells as a consequence of disruption of normal neuron-glia interactions. 3. We have carried out immunocytochemical and in situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development. 4. Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10. 5. Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of approximately 160-200 microns per day. 6. An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops. 7. Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development. PMID- 1723660 TI - Monoclonal antibody with specificity to a conserved epitope in the C-terminal domain of histone H1 variants. AB - A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1(0) in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1(0), and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression. PMID- 1723661 TI - Isolation and genetic study of p-fluoro-DL-phenylalanine-resistant mutants overproducing beta-phenethyl-alcohol in Saccharomyces cerevisiae. AB - p-Fluoro-DL-phenylalanine (PFP)-resistant mutants which produce a large amount of beta-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae. Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine. The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased. Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, try1-pfp, caused both PFP resistance and beta-phenethyl-alcohol overproduction. This was supported by molecular genetic studies with cloned tyr1-pfp DNA. PMID- 1723662 TI - A mutated ARO4 gene for feedback-resistant DAHP synthase which causes both o fluoro-DL-phenylalanine resistance and beta-phenethyl-alcohol overproduction in Saccharomyces cerevisiae. AB - o-Fluoro-DL-phenylalanine (OFP)-resistant mutants which overproduce beta phenethyl-alcohol were isolated from a laboratory strain of Saccharomyces cerevisiae. Cells of one of the mutants accumulated tyrosine and phenylalanine 1.5-3 fold more than did wild-type cells. Its 3-deoxy-D-arabino-hepturosonate-7 phosphate (DAHP) synthase (EC 4.1.2.15), encoded by ARO4, was free from feedback inhibition by tyrosine. Genetic analysis revealed that the mutation was controlled by a single dominant gene, ARO4-OFP, encoding feedback-resistant DAHP synthase by tyrosine, and that this gene caused both the OFP resistance and beta phenethyl-alcohol overproduction. This was supported by molecular genetic studies using cloned ARO4 both from the wild-type and its mutant strain. PMID- 1723663 TI - The group IIB intron from the green alga Scenedesmus obliquus mitochondrion: molecular characterization of the in vitro splicing products. AB - In the presence of high molar salt concentrations, the mitochondrial group IIB intron (rI1) from the green alga Scenedesmus obliquus is capable of splicing in vitro. After establishing the optimal conditions for RNA processing the in vitro splicing products were unequivocally identified in self-splicing experiments by Northern hybridization analysis employing 3'end-labelled RNAs or exon- and/or intron-specific probes. Finally, two trans-esterification products were identified by sequencing of the spliced RNA. From our data we conclude that the processing of group II introns from both algal and yeast mitochondria is preceded by identical consecutive trans-esterification steps. The predicted secondary and tertiary structure of intron rI1 of S. obliquus contains all the motifs necessary for optimal self-splicing and which are characteristic of other group IIB introns from different species. PMID- 1723664 TI - Lack of a functional plastid tRNA(Cys) gene is associated with loss of photosynthesis in a lineage of parasitic plants. AB - We recently reported that the gene for chloroplast tRNA(Cys)(GCA) is a pseudogene in the plastid DNA of Epifagus virginiana, a non-photosynthetic parasitic flowering plant in the family Orobanchaceae. Since this is the only tRNA(Cys) gene in the plastid genome, and since Epifagus appears to possess a functional plastid translational apparatus, it seems probable that nuclear-encoded tRNAs are imported into plastids to effect translation. In this study we have surveyed species closely related to Epifagus to establish how widespread the loss of this tRNA gene has been. We find that Conopholis americana, another non-photosynthetic parasite, lacks the gene altogether, but that seven closely-related photosynthetic plants (both parasitic and free-living) maintain an intact chloroplast tRNA(Cys) gene. Thus, the tRNA(Cys) gene appears to have become non functional at the same time that photosynthetic ability was lost. This may be because the levels of putatively imported tRNAs are sufficient to meet the demands of plastid gene expression under nonphotosynthetic conditions only. PMID- 1723665 TI - [Microwave--a new technique in histological fixation and staining]. PMID- 1723666 TI - An animal investigation into the effects of tympanostomy on middle ear mucociliary function and cilia. AB - The effect of tympanostomy on guinea-pig middle ear mucosa with particular reference to mucociliary function and cilia was investigated. 13 guinea-pigs were used; 5 acted as the control group with no surgical intervention whilst the other 8 had perforations made in one tympanic membrane. 6 weeks later an attempt was made to measure mucociliary function in all the ears, but this was unsuccessful. However, it would appear that fashioning a perforation in the tympanic membrane causes no histological changes in this animal model. PMID- 1723667 TI - Absorption of cytokines via oropharyngeal-associated lymphoid tissues. Does an unorthodox route improve the therapeutic index of interferon? PMID- 1723668 TI - The expression of sub-population markers on B cells: a re-evaluation using high sensitivity fluorescence flow cytometry. AB - A number of markers which have been proposed to identify B cell subsets have been reassessed on human B cells, using an immunofluorescence technique optimized for sensitivity and an analytical mode which yields histograms showing the distribution of fluorescence on B cells. The results show that CD38, CD22, CD23, FMC6, and anti-IgM react with all blood B cells, albeit with a broad and complex distribution of fluorescence. CD5, CD9, CD10, CD43, and IgD can be regarded as subset markers since they give clearly bimodal distributions of fluorescence intensity. CD5 staining showed at least three populations, with a small number (3 5 per cent) of cells brightly stained and a population of variable size staining weakly. No clearly defined populations were seen with CD45R0, although staining was slightly above background. An antibody against the LAM-1 molecule reacted with all blood B cells. Expression of the IL-2 receptor p55 chain (CD25) was clearly bimodal, whereas the p75 chain was essentially negative on B cells. The relationship between subsets in blood and subsets in tissue, and between subsets identified by different markers in blood, is discussed. PMID- 1723669 TI - Kinetics of the ontogenic and reversible hemoglobin switching in the mouflon (Ovis musimon) and sheep x mouflon hybrid. AB - 1. Hemoglobin (Hb) switching in the perinatal life of wild mouflon (Ovis musimon) was characterized by the replacement of Hb F by 60% levels of Hb C, and subsequently of Hb C by Hb B. 2. The recently discovered Hb M variant was not replaced by Hb C; thus, Hb BM heterozygote newborns synthesized 30% Hb C at the expense of Hb B. 3. Hybrid B mouflon x B sheep synthesized only 5% Hb C at birth but were able to produce 30% Hb C in adult life following induced anemia. 4. Adult BB and BM mouflons, after the same extent of induced anemia, synthesized HB C levels similar to those produced at birth. The results indicate a mouflon beta globin gene cluster arrangement similar to those of sheep and goat, the beta C gene having an intermediate expression. Results also suggest a selective disadvantage in hybrid animals. PMID- 1723670 TI - Evolutionary trends of neurofilament proteins in fish. AB - 1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution. PMID- 1723671 TI - Evolution of the "titin epitope" in neurofilament proteins. AB - 1. The specificity of a monoclonal antibody raised to human titin was characterized. The antibody reacts with an epitope which is common to titin and the high mol. wt subunits NF-H and NF-M of mammalian neurofilaments. 2. Mapping of the epitope indicated that it is located in the carboxyterminal extension of NF-H and NF-M, and that its reactivity does not depend on the phosphorylation state of the molecule. 3. A comparative study on neurofilament protein of lower vertebrates revealed that this epitope has been conserved during vertebrate evolution. PMID- 1723672 TI - Epidermal growth factor messenger RNA production in human lacrimal gland. AB - Experimental models and clinical investigations have suggested that epidermal growth factor (EGF) may have a role in corneal wound healing. It has been identified as a normal component of human tears. Rabbit and mouse lacrimal glands have recently been shown to synthesize EGF messenger RNA (mRNA). The purpose of the present study was to determine whether the human lacrimal gland synthesizes EGF mRNA. Total cellular RNA was isolated from pathologic specimens of normal human lacrimal glands from two individuals. Reverse transcriptase was used to generate complementary DNA (cDNA) using a human EGF-specific mRNA primer. Amplification of EGF-related cDNA sequences was performed with the polymerase chain reaction (PCR) and human EGF-derived up- and downstream primers. The PCR products from both lacrimal glands contained an amplified product of the expected length of approximately 410 base pairs. The PCR-generated fragment was verified as an EGF-related amplification product with Southern blotting using a synthetic oligonucleotide probe derived from the mature coding sequence of EGF. These results conclusively demonstrate that the human lacrimal gland synthesizes EGF and suggest that the lacrimal gland could have a regulatory role in maintaining the ocular surface and possibly regulating corneal wound healing through the secretion of EGF. PMID- 1723673 TI - High-dose aprotinin (trasylol) in reducing bleeding and protecting lung function in potential bleeders undergoing cardiopulmonary bypass. AB - Intraoperative high-dose aprotinin during cardiopulmonary bypass was used to investigate if high-risk bleeders could be changed to bleed normally or less as well as see if aprotinin could preserve lung function. Eleven matched controls were compared with eleven aprotinin patients taking warfarin or aspirin preoperatively. The mean (+/- SEM) 12-h and 24-h postoperative amount of bleeding, volume of blood product transfusion and hemoglobin reduction in the aprotinin group were 328 +/- 45 ml, 418 +/- 63 ml, 341 +/- 99 ml and 1.8 +/- 0.5 g% respectively, which were significantly lower than the respective values of 716 +/- 86 ml (P less than 0.01), 1,029 +/- 115 ml (P less than 0.01), 985 +/- 294 ml (P less than 0.05) and 4.1 +/- 0.4 g% (P less than 0.02) in the controls. There was a 65% blood-saving effect by aprotinin in this study. The hypercapnea rate was 45% in the treated patients, and 82% (P less than 0.05) in the controls reflecting better preservation of pulmonary diffusion function which is clinically important following major surgery. PMID- 1723674 TI - Serum SP1, hPL and beta-hCG levels in trophoblastic diseases. AB - Serum SP1 (pregnancy specific beta 1 glycoprotein), hPL (human placental lactogen) and beta-hCG (beta-human chorionic gonadotropin) in patients with choriocarcinoma, invasive mole, and hydati-diform mole were determined by radioimmunoassay (RIA), and compared with those in normal males, non-pregnant women and normal pregnant women in order to evaluate the clinical significance of SP1, hPL and beta-hCG determinations. Serum SP1 levels at the time of admission were highest in hydatidiform mole (5.1 +/- 0.6 micrograms/L) and lowest in choriocarcinoma (0.5 +/- 0.3 micrograms/L). Serum hPL levels were 68.2 +/- 9.7 ng/L in hydatidiform mole and 26.4 +/- 8.3 ng/L in choriocarcinoma. Serum SP1 and hPL levels in trophoblastic diseases were lower than in normal pregnancies (SP1 11.5 +/- 5.1 micrograms/L, hPL 216.8 +/- 48.1 ng/L). SP1/beta-hCG ratios were less than 1.5 in 4/43 (9.3%) cases of hydatidiform mole and 17/19 (89.5%) cases of invasive mole and choriocarcinoma. The beta-hCG/hPL ratios were below 15 in 35/43 (81.4%) cases of hydatidiform mole and 4/19 (21.1%) malignant trophoblastic diseases. The prognosis after operation and chemotherapy was favourable if patient's SP1 and beta-hCG levels gradually decreased. PMID- 1723675 TI - Towards a quantitative grading of bladder tumors. AB - The histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated. Three hundred thirty-three images were digitized from 111 bladder tissue sections (4 microns thick, Feulgen stained), using the ICAS microscope-camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity. The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as "in between grade 1 and grade 3" and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population. The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields-of-view are chosen for analysis. PMID- 1723676 TI - An improved Hedley method for preparation of paraffin-embedded tissues for flow cytometric analysis of ploidy and S-phase. AB - A modification of the Hedley-method for flow cytometric DNA analysis of paraffin embedded tissues is presented. Dewaxed and dehydrated tissue from paraffin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was advantageous, for example, for the analysis of core biopsies, and all measured samples showed extremely low frequencies of clumped cell nuclei. This made is easier to detect polyploid nuclei and even rare nuclei of high ploidy could be identified. S-phase analyses were more precise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high-polyploid nuclei found in various diploid, tetraploid, and aneuploid human myosarcomas of the uterus. PMID- 1723677 TI - [Value of anti-human heterogeneous lens culinaris agglutinin reactive AFP monoclonal antibody in the diagnosis of primary hepatocellular carcinoma (PHC)]. AB - In this paper, we report on the preparation and application of anti-human heterogeneous AFP-R-LCA monoclonal antibodies (VG5, VD12, VB5, VA8, VD1). These McAbs were more sensitive and specific for AFP-R-LCA than the anti-AFP polyclonal antibody routinely used. The twin site (a and b) sandwich ELISA method so established was used to test the serum samples of 69 PHC patients, 67 patients with benign liver diseases, 30 pregnant women and 30 normal controls. The results showed that this twin site sandwich ELISA method gave a false positive reaction in only 2.1% and was 81.2% (56/69) positive reaction, giving a positive reaction to 5-100 ng/ml. It was positive in PHC patients with AFP levels less than 400 ng/ml. This method, being simple, accurate and reliable, is valuable in the differential diagnosis of PHC from benign liver diseases. PMID- 1723678 TI - [Gastrointestinal carcinoid tumor--an immunohistochemical and histochemical study]. AB - Fourteen gastrointestinal carcinoid tumors were studied by immunohistochemical and histochemical methods. Neuro-specific enolase (NSE) was detected in all 14 cases with patterns of homogeneously moderate to strongly diffuse cytoplasmic stain. 12 of 14 neoplasms (85.7%) were positive for Grimelius technic. 5 (35.7%) were positive for Masson-Fontana stain. The results indicate that the immunohistochemical demonstration of NSE is a marker more sensitive than the silver stain for gastrointestinal carcinoid tumors. Distribution and significance of silver stain are discussed. PMID- 1723679 TI - Nuclear translocation of fibroblast growth factor during Xenopus mesoderm induction. AB - Mesoderm induction, the earliest inductive cell-cell interaction in vertebrate embryogenesis, is thought to be mediated by polypeptide growth factors including fibroblast growth factor (FGF). Here we present an immunocytochemical analysis of FGF during mesoderm induction in Xenopus laevis. Antibodies to both basic and acidic FGF were immunoreactive with oocytes and early embryos. Immunostaining was predominantly intracellular and was concentrated in the marginal zone and vegetal pole throughout cleavage and blastula stages. In addition, basic FGF (bFGF) antibodies showed intense nuclear staining in these regions, at and following the mid-blastula transition, when embryonic transcription begins. Acidic FGF (aFGF) also appeared in some nuclei at these stages. Taken together the evidence suggests that FGF is prepositioned in mesoderm-forming regions and is actively involved in mesoderm induction in vivo. PMID- 1723680 TI - FGFR-4, a new member of the fibroblast growth factor receptor family, expressed in the definitive endoderm and skeletal muscle lineages of the mouse. AB - We have used the polymerase chain reaction to clone from fetal cerebellar RNA a novel member of the fibroblast growth factor receptor family, FGFR-4. cDNAs encoding a full-length receptor were isolated and RNA expression examined in adult and fetal tissues by RNA blot analysis. Transcripts were detected in adult lung, liver and kidney and in fetal RNAs from 11.5 to 16.5 days post coitum (p.c.). In situ hybridization was performed to examine developmental expression. FGFR-4 RNA was expressed in definitive endoderm of the developing gut and extraembryonic endoderm of the yolk-sac from 8.5 to 14.5 days p.c. At early somite stages, FGFR-4 was also expressed in the myotomal component of the somite, and by 14.5 days p.c. in the myotomally derived skeletal muscle. No expression was seen at any stage in cardiac muscle. Several endodermal derivatives, the liver, lung and pancreas, expressed FGFR-4 at 14.5 days p.c. In addition, FGFR-4 RNA was detected in the adrenal cortex, collecting tubules of the kidney and condensing cartilage at this time. These results suggest that FGFR-4 is likely to have diverse roles in development, which may include regulation of definitive endoderm and skeletal muscle lineages. PMID- 1723681 TI - Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. AB - Recent studies have shown that the dominant white spotting (W) locus encodes the proto-oncogene c-kit, a member of the tyrosine kinase receptor family. One symptom of mice bearing mutation within this gene is sterility due to developmental failure of the primordial germ cells during early embryogenesis. To elucidate the role of the c-kit in gametogenesis, we used an anti-c-kit monoclonal antibody, ACK2, as an antagonistic blocker for c-kit function to interfere with the development of male and female germ cells during postnatal life. ACK2 enabled us to detect the expression of c-kit in the gonadal tissue and also to determine the functional status of c-kit, which is expressed on the surface of a particular cell lineage. Consistent with our immunohistochemical findings, the intravenous injection of ACK2 into adult mice caused a depletion in the differentiating type A spermatogonia from the testis during 24-36 h, while the undifferentiated type A spermatogonia were basically unaffected. Intraperitoneal injections of ACK2 into prepuberal mice could completely block the mitosis of mature (differentiating) type A spermatogonia, but not the mitosis of the gonocytes and primitive type A spermatogonia, or the meiosis of spermatocytes. Our results indicate that the survival and/or proliferation of the differentiating type A spermatogonia requires c-kit, but the primitive (undifferentiated) type A spermatogonia or spermatogenic stem cells are independent from c-kit. Moreover, the antibody administration had no significant effect on oocyte maturation despite its intense expression of c-kit. PMID- 1723682 TI - The 'Sicilian Gambit'. A new approach to the classification of antiarrhythmic drugs based on their actions on arrhythmogenic mechanisms. The Task Force of the Working Group on Arrhythmias of the European Society of Cardiology. AB - The Queen's Gambit is an opening move in chess that provides a variety of aggressive options to the player electing it. This report represents a similar gambit (the 'Sicilian Gambit') on the part of a group of basic and clinical investigators who met in Taormina, Sicily, to consider the classification of antiarrhythmic drugs. Paramount to their considerations were (1) dissatisfaction with the options offered by existing classification systems for inspiring and directing research, development and therapy, (2) the disarray in the field of antiarrhythmic drug development and testing in this post-CAST era, and (3) the desire to provide an operational framework for consideration of antiarrhythmic drugs that will both encourage advancement and have the plasticity to grow as a result of the advances that occur. The multifaceted approach suggested is, like the title of the manuscript, a gambit. It is an opening rather than a compendium, and is intended to challenge thought and investigation rather than to resolve issues. The manuscript incorporates first, a discussion of the shortcomings of the present system for drug classification; second, a review of the molecular targets on which drugs act (including channels and receptors); third, a consideration of the mechanisms responsible for arrhythmias, including the identification of 'vulnerable parameters' that might be most accessible to drug effect; and finally, clinical considerations with respect to antiarrhythmic drugs. Information relating to the various levels of information is correlated across categories (i.e., clinical arrhythmias, cellular mechanisms and molecular targets), and a 'spread sheet' approach to antiarrhythmic action is presented that considers each drug as a unit, with similarities to and dissimilarities from other drugs being highlighted. A complete reference list for this work would require as many pages as the text itself. For this reason, referencing is selective and incomplete. It is designed, in fact, to provide sufficient background information to give the interested reader a starting frame of reference, rather than to recognize the complete body of literature that is the basis for this paper. PMID- 1723683 TI - Effect of FK-506 on heart allograft survival in the highly sensitized recipient rats as compared with ciclosporin and 15-deoxyspergualin. AB - The effect of FK-506 on allograft survival in unsensitized recipients has been established in several animal experimental models. In this present study, we investigated the immunosuppressive capacity of FK-506, in comparison with ciclosporin (Cs) and 15-deoxyspergualin (DSG), on heart allograft in presensitized rats. Heart grafts from male ACI rats were heterotopically transplanted to male LEW rats. All recipient rats were sensitized with donor-type whole blood admixed with immunoadjuvant (adjuvant complete Freund) 7 days prior to transplantation. FK-506, Cs and DSG were administered from day 0 to day 14 posttransplantation. The results showed that both FK-506 and Cs significantly prolonged heart allograft survival of presensitized recipients in a dose dependent manner. The minimum effective dose in the treatment was less for FK-506 than for Cs. However, DSG showed almost no effect of prolongation in this experiment. PMID- 1723684 TI - Prophylactic administration of urinary trypsin inhibitor prevents postoperative hyperamylasemia after R2 gastrectomy in patients with gastric cancer. A prospective randomized trial. AB - The purpose of this prospective randomized study was to determine whether prophylactic administration of urinary trypsin inhibitor prevents postoperative damage to the pancreas caused by R2 gastrectomy in patients with gastric cancer, by analyzing related enzyme activities. Among the 22 patients who underwent distal partial gastrectomy together with R2 lymphadenectomy, 12 were given the drug for 3 days postoperatively, and 10 no therapeutic agent. These groups were otherwise comparable. Postoperatively, the control patients had significantly higher levels of total amylase activity in the serum and P-type amylase when compared to the preoperative data. These amylase activities almost remained at the preoperative level in those given the drug. When the ratio of increase in enzyme activities (postoperative value divided by preoperative value) were compared, on the 7th postoperative day, total amylase activity in the serum and P type amylase were significantly different between the two groups (p less than 0.05). This difference was also evident when comparing total amylase activity in the urine (p less than 0.05). These findings indicate that prophylactic administration of urinary trypsin inhibitor would aid in preventing postoperative hyperamylasemia caused by R2 gastrectomy in patients with gastric cancer. PMID- 1723685 TI - Uptake of immunoreactive leukocyte elastase-inhibitor complexes in macrophage like cells in abscesses. AB - Leukocyte elastase was demonstrated immunohistochemically not only in PMN leukocytes in subcutaneous abscesses but also in scattered macrophage-like cells which in addition contained immunoreactive alpha 1-proteinase inhibitor (alpha 1PI) and alpha 2-macroglobulin (alpha 2M). These cells were located mainly in the marginal region of the abscess. It is concluded that the intracellular deposits in the macrophage-like cells represent phagocytized elastase and/or elastase alpha 1PI and elastase-alpha 2M complexes. A contributing, local production of the inhibitor is, however, not excluded by the present findings. PMID- 1723686 TI - The presence of an M4 subtype muscarinic receptor in the bovine adrenal medulla revealed by mRNA and receptor binding analyses. AB - The muscarinic receptor subtype present in the bovine adrenal medulla was characterized. Hybridization of RNA to highly specific m1-m5 muscarinic receptor cDNA probes detected the presence of only m4 subtype mRNA in this tissue. Muscarinic receptor binding studies using the non-selective ligand [3H]N-methyl scopolamine showed a single class of binding sites with a maximum density of 19.8 fmol/mg protein and a dissociation constant (KD) of 220 pM in the adrenal medulla, while the M1 selective ligand [3H]telenzepine did not bind detectably. Competition of specific antagonists with [3H]N-methyl-scopolamine for binding to the membranes produced a rank order of potencies with a profile that fitted either the cloned m3 or m4 receptor. In further comparative studies, the adrenal gland of the rat showed the presence of m4 subtype mRNA in addition to the m3 subtype. PMID- 1723687 TI - Substance P-induced diacylglycerol formation in rat parotid acinar cells. AB - The mechanisms underlying the ability of substance P, to stimulate the sn-1,2 diacylglycerol (DAG) formation were studied using rat parotid acinar cells. During a 60 s stimulation, 1 microM substance P caused a rapid rise in DAG accumulation at 5 s, whereas a low (0.1 microM) concentration of agonist did not. During long term stimulation for 30 min, DAG accumulation induced by 1 microM substance P reached near maximal levels at 5 min and remained elevated for at least 20 min. In contrast, DAG formation induced by 0.1 microM substance P exhibited a peak at 5 min, gradually declined and returned to near basal levels at 30 min. Furthermore, DAG accumulation in response to substance P at 5 and 20 min increased in a dose-dependent manner. The breakdown of both [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2) stimulated by 1 microM substance P significantly increased from 5 to 20 min and returned to basal levels by 30 min; however, the breakdown of [32P]PIP2 was greater than that of [32P]PIP. At a low concentration of substance P, [32P]PIP2 breakdown reached maximal levels at 5 min followed by a progressive decrease and returned to basal levels at 30 min, whereas the breakdown of [32P]PIP reached maximal levels at 5 min and returned to near basal levels at 10 min. Both concentrations of substance P caused some [32P]phosphatidylinositol breakdown at 5 min. Changes in [3H]inositol trisphosphate induced by substance P were similar to those in [32P]PIP2. In addition, substance P (1 microM) did not stimulate the release of [3H]choline or [3H]ethanolamine metabolites into the medium. Substance P-induced DAG formation was not inhibited by staurosporine, a protein kinase C inhibitor. These results suggest that DAG formation caused by substance P is closely associated with the hydrolysis of phosphatidylinositides but not that of phosphatidylcholine or phosphatidylethanolamine, and is not regulated by protein kinase C-dependent mechanism(s). PMID- 1723688 TI - Species differences in the effects of substance P on inositol trisphosphate accumulation and cyclic AMP formation, and on contraction in isolated iris sphincter of the mammalian eye: differences in receptor density. AB - The effects of substance P (SP) on inositol trisphosphate (IP3) accumulation, myosin light chain (MLC) phosphorylation, cAMP formation and contraction were studied in iris sphincter smooth muscle of different mammalian species. SP receptor density was also examined in membrane fractions from this tissue. The data obtained can be summarized as follows. (1) In the iris sphincters of rabbit, bovine and pig, SP receptors are coupled to the phospholipase C system, whereas in dog, cat and human these receptors are coupled to the adenylate cyclase system. (2) In those species which employ the phospholipase C system, SP induced IP3 accumulation, MLC phosphorylation and contraction in a dose-dependent manner; in contrast, in those species in which SP induced the formation of cAMP we found the neuropeptide to cause muscle relaxation. The findings on cAMP formation in intact tissue were confirmed in iris sphincter membranes. Both the effect of SP on IP3 accumulation in rabbit and bovine sphincters and its effect on cAMP formation in the dog were blocked by the SP antagonist, (D-Pro2, D-Trp7, 9)-SP. (3) The density of SP receptors in rabbit, bovine and dog were found to be 227, 110.9 and 13.6 fmol mg-1 protein, respectively, and the Kd values were 1.9, 1.8 and 1.3 nM, respectively. (4) Of the neuropeptides investigated SP, neurokinin A and neurokinin B had significant stimulatory effects on IP3 accumulation and on contraction in the rabbit iris sphincter; however, neither neurokinin Y nor the calcitonin gene-related peptide (CGRP) had any effect on these responses. In addition, none of the neuropeptides studied had any effect on IP3 or on contraction in the dog iris sphincter. While it is possible that SP may have dual actions, with the predominant action dependent on the species, the data presented could suggest the presence of two SP receptor subtypes, one coupled to phospholipase C and the other to adenylate cyclase. The results of this investigation indicate major species differences in biochemical and functional responsiveness to SP and in SP receptor density in the iris sphincter of the mammalian eye, and support a modulatory role for the neuropeptide in muscle response in this tissue. PMID- 1723689 TI - Pigment epithelial cell changes precede vascular transformations in the dystrophic rat retina. AB - In the Royal College of Surgeons rat with inherited retinal dystrophy, vascularization of the retinal pigment epithelium (RPE) is preceded by migration and proliferation of Muller cell processes in the subretinal space where they contact the RPE. Later, RPE cells envelope subretinal vessels which have lost their perivascular Muller cell sheath. To characterize RPE cell changes and interactions in relation to glial and vascular transformations in retinal dystrophy, we used immunocytochemical techniques and antibodies against cytokeratin (CK) and glial fibrillary acidic protein (GFAP). Prior to the proliferation of Muller cell processes in the dystrophic retina, CK filaments in RPE cells formed a circumferential meshwork with intense cytoplasmic and perinuclear labeling as in control RPE cells. Following entry of Muller cell processes into the membranous debris zone and formation of RPE-Muller cell contact, RPE cells became pleomorphic and extended prominent apical processes in the debris zone. Some CK-reactive RPE cells detached from Bruch's membrane and migrated into the debris zone. Electron microscopic study showed extensive areas of close RPE-Muller cell contact at this time. Obvious junctional specializations of the plasma membranes were not seen but prominent tubulo-vesicular profiles occupied the cytoplasm of altered RPE and Muller cell processes. Following RPE vascularization, hypertrophic CK-positive cells surrounded blood vessels and accompanied them into the inner retina. Electron microscopic analysis showed that RPE-associated vessels were fenestrated and devoid of their perivascular glial sheath. Apparent proliferation of RPE cells and redistribution of CK filaments were observed. Our study shows that RPE cell alterations accompany Muller cell and vascular changes which result in altered RPE-Muller cell and RPE-endothelial cell relationships in the dystrophic rat retina. The altered relationships among RPE, Muller and endothelial cells may result in increased cellular interaction and promote proliferation and transformation of all three cells types in diseased retinas. PMID- 1723690 TI - A WGA-HRP study of the fiber arrangement in the cat optic radiation: a demonstration via three-dimensional reconstruction. AB - The fiber arrangement of the optic radiation was investigated in fourteen adult cats. The retinotopies of the lateral geniculate nucleus (LGN) were first identified electrophysiologically, and thereafter, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was iontophoretically injected into defined positions of the LGN. These corresponded to the central (medial LGN), horizontal peripheral (lateral LGN), dorsal (rostral LGN), and ventral (caudal LGN) retina. Geniculocortical fibers from the each position of the LGN and corticogeniculate fibers projecting to these positions were always labeled reciprocally. Labeled terminals were found massively in layer IV with some extending to the lower part of layer III, but layers VI and I also contained substantial numbers. Although most of the labeled neurons were localized in layer VI, some neurons were labeled in layer V and transsynaptically in layer IV. Labeled fibers were superimposed in three-dimensionally reconstructed maps of the white matter for the easy understanding of the pathways connecting the LGN and the visual cortex. They were localized in certain zones in the white matter without wide dispersion; however, we did not obtain any findings which suggested clearly different populations of geniculocortical and corticogeniculate fibers. In agreement with previous studies, fibers from the rostral LGN and the caudal LGN projected to the striate cortex in a regular order, rostrocaudally, and fibers from the medial LGN and the lateral LGN projected to the striate cortex inversely (i.e. lateromedially). This inverse projection resulted because fibers from the lateral LGN traversed fibers from the medial LGN in a lateromedial direction; however, there was only partial crossing of these two pathways. The distribution of geniculocortical fibers together with corticogeniculate fibers formed topographic zones arrayed mediolaterally in the white matter. Thus, fibers of the medial LGN were positioned in the intermediate zone, and fibers of the rostral LGN and the lateral LGN were positioned in the rostral and caudal parts of the lateral zone, respectively. Fibers of the caudal LGN were found in the medial zone. This fiber arrangement displayed a rough centroperipheral retinotopy in that fibers representing the central area were placed between fibers representing the peripheral retina. Finally, this fiber arrangement was compared with that of the optic nerve and optic tract. PMID- 1723691 TI - Cholinergic stimulation of substantia nigra: abolition of carbachol-induced eating by unilateral 6-hydroxydopamine lesion of nigrostriatal dopamine neurones. AB - Microinjection of cholinergic agonists into the substantia nigra is known to elicit increases in eating, drinking and sexual behaviour under appropriate circumstances. It has been suggested that these effects are dependent on stimulation of nigrostriatal dopamine-containing neurones in the substantia nigra pars compacta, but no direct evidence has confirmed this. The present experiment was therefore undertaken to determine whether unilateral lesions of nigrostriatal dopamine neurones made by 6-hydroxydopamine would attenuate or abolish eating in satiated rats elicited by intranigral microinjection of the muscarinic agonist carbachol. Two groups of rats were tested: a 6-hydroxydopamine- and a sham-lesion group. Before lesions were made intranigral microinjection of 0.5 microgram/0.5 microliter carbachol stimulated significantly more eating than control microinjections in both groups. After 6-hydroxydopamine lesions, microinjection of carbachol elicited no more eating than vehicle alone. Rats given sham lesions (ascorbate-saline vehicle only) showed increased feeding to intra-nigral carbachol before and after sham-lesioning. Post-mortem analysis by HPLC was used to determine the concentration of dopamine, DOPAC, HVA, serotonin and 5-HIAA in the lesioned and non-lesioned hemispheres of both 6-hydroxydopamine- and sham lesioned rats. In caudate-putamen there were significant reductions in the concentration of DA (to 50.03% of the level in control sides), DOPAC (to 49.34%) and HVA (to 63.98%) in the 6-hydroxydopamine-lesioned but not sham-lesioned rats. The concentration of dopamine, DOPAC and HVA were not affected in the nucleus accumbens. The turnover of dopamine (assessed by calculating the ratio of dopamine to DOPAC) in the caudate-putamen but not nucleus accumbens was also altered by the 6-hydroxydopamine lesions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723692 TI - Superficial tectal neurons projecting to the dorsolateral pontine nucleus in the rabbit. AB - After WGA-HRP injections in the pontine grey involving the dorsolateral pontine nucleus, a great number of labeled cells were found in the superficial layers of the ipsilateral superior colliculus. The majority of these cells were located in the stratum griseum superficiale (SGS). Few labeled cells were found in the stratum opticum, and the stratum zonale (SZ) showed no labeled cells. Labeled cells in the SGS formed a rather homogeneous population as most of them had fusiform somata with an upper dendritic process which runs vertically to reach the SZ. These cells were mainly located in the middle third of the SGS, forming a sublamina in this layer. These results demonstrate the participation of the superficial tectal layers in the ipsilateral descending pathway of the superior colliculus, allowing visual information to reach precerebellar stations at the dorsolateral pontine nucleus. PMID- 1723695 TI - [Changes in the hormonal content of the blood during chronic electrostimulation of the hypothalamus and amygdaloid complex]. AB - The changes of corticotropin, thyrotropin in hypophysis, triiodothyronine, thyroxin in thyroid gland, adrenaline, norepinephrine, hydrocortisone, 11-OKS in adrenal glands and pancreas, caused by prolonged electrostimulation of hypothalamic ventro-median nucleus, lateral nucleus of amygdala and the combination of the latter with the SP injection, were studied in chronic experiments on rabbits. The electrostimulation increased the hormone contents in the blood, whereas glucagon contents as well as norepinephrine contents was decreasing in stimulation of hypothalamus and amygdala, resp. The SP injection against the background of electrostimulation of the amygdala resulted in the hormone status standardisation. PMID- 1723694 TI - Hepatitis C virus infection in homosexual men: a seroepidemiological study in gay clubs in north-east Italy. AB - Seroprevalence of Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV antibodies was studied in a group of 259 apparently healthy homosexual men of the Veneto Region (Italy). Subjects were recruited between 1987 and 1989 from homosexual men's clubs. Seropositivity was evaluated in relation to main risk factors associated with the lifestyle and sexual behaviours of this population. Serological evaluation revealed an overall prevalence of HCV infection of 18.9% in the study population as a whole, but on breaking the samples down into three subgroups according to optical density (O.D.) values and to the year of sera collection, different seroprevalences were observed. Prevalence of anti-HCV was higher in 1987 and steadily decreased in 1988 and 1989; 4.1% of subjects gave positive results at O.D. greater than 2.0, while 6.2% were positive at O.D. between 0.8 and 2.0 and 9.6% at O.D. between cut-off and 0.8. Anti-HCV positivity was not correlated with HIV nor HBV positivity. No correlation was found between HCV seropositivity and either the type of anogenital intercourse or sexual promiscuity, but the prevalence increased (p = n.s.), as observed for HIV (p less than 0.05) and HBV (p = n.s.), with the number of intercourses per month. Epidemiological and preventive aspects arising from the investigation are discussed herein. PMID- 1723696 TI - [TSH receptor antibody in hyperthyroid patients due to Graves' disease treated with antithyroid drugs]. AB - As an immunologic process, TSH receptor antibody (TRab) may be synthesized by the lymphocytes within the thyroid gland. Two techniques were devised to express this TRap activity: a) thyroid stimulating immunoglobulin (TSI) expressed as thyroid c AMP synthesis and b) TSH binding inhibiting immunoglobulin (TBII) expressed as TSH displacement. In general, serum TSI and TBII activity correlated well with each other in hyperthyroid patients. Measurement of TSI and TBII is useful to assess improvement of immunologic abnormality induced by antithyroid drugs. The data so far gathered indicated that TRab disappears from the blood in about 70 80% of hyperthyroid patients in response to antithyroid drugs. However, this does not indicate that intrathyroidal TRab synthesis has ceased, since thyroglobulin is still secreted supernormally and T3 fails to suppress the thyroid in some patients. On the other hand, normalization of thyroglobulin and absence of TRab in the blood indicated complete normalization in the patients as evidenced by positive T3 suppressibility. By using a number of new drugs, further efforts must be made to completely normalize immunologic abnormality in 100% of patients. Recurrence of hyperthyroidism of Graves' disease is generally associated with re appearance of TRab in the blood in most of the recurrent patients after discontinuation of antithyroid drugs. PMID- 1723693 TI - Swine and cattle enterotoxigenic Escherichia coli-mediated diarrhea. Development of therapies based on inhibition of bacteria-host interactions. AB - Enterotoxigenic Escherichia coli (ETEC) frequently occurs in diarrheal disease afflicting domestic animals. In this paper is summarized the research carried out over the last decade on the two important determinants of virulence that plays a role in the development of the infection, namely the colonizing ability of the small intestine mediated by specific fimbrial adhesins acting as lectins and the production of enterotoxins. Recent progress in knowledge of the phenomenon led to alternative strategies of prevention and cure of enteric infection. Since bacterial recognition of mucosa surface receptors in an initial event in colonization, several approaches based on the competitive inhibition of ETEC adhesion have been developed. This review examines the following approaches: competitive colonization with non pathogenic strains, design of adhesin or toxin vaccines, receptor analog therapy and methods for in vivo suppression of virulence factors. PMID- 1723697 TI - Characterisation of anti-peptide CFTR antibodies. PMID- 1723698 TI - Evidence for a pH-dependent anion channel in sheep heart mitochondria. PMID- 1723699 TI - An intracellular anion channel from rat brain microsomes which is also permeable to cations. PMID- 1723700 TI - Partial denaturation and renaturation of beta-lactoglobulin at air-water interfaces. PMID- 1723701 TI - A comparison of the rat anti-proteinases alphamacrofetoprotein and decidualisation associated protein. PMID- 1723702 TI - Protein factors involved in the expression of the alpha-fetoprotein gene. PMID- 1723703 TI - Purine de novo synthesis and enzymes at the inosinic branch point in human lymphocytes. PMID- 1723704 TI - A transfected human fucosyltransferase cDNA determines biosynthesis of oligosaccharide ligand(s) for endothelial-leukocyte adhesion molecule I. PMID- 1723705 TI - Relevant factors in the identification of hearing loss. AB - This study examined factors which may affect early identification of hearing loss. The medical records of 123 children with educationally significant hearing impairment were examined. Information about each child's degree and type of hearing loss, etiology, referral source, birth and medical history, additional handicaps, age of suspicion of loss, mode of identification, age of identification, and age at which aided was entered into a database for further analysis. The age range for identification was 7 weeks to 10 yr, with a median age of 2.1 yr. Children with a greater degree of hearing loss, an additional handicap, additional medical conditions, or an etiology strongly associated with hearing loss were identified earlier than those without these factors. Unexpectedly, children with a history of middle ear dysfunction were identified no later than those without, and children with a positive family history of hearing loss were identified later than those with a negative family history. These results agree with other studies which show that, in general, children are identified and habilitated at a later age than that recommended by both the American Speech-Language-Hearing Association Committee and the Joint Committee on Infant Hearing. PMID- 1723706 TI - [Late discovery of drug-related hepatotoxicity]. PMID- 1723707 TI - [Moxisylyte-induced cytolytic hepatitis?]. PMID- 1723708 TI - [Moxisylyte: affecting the liver. A case of readministration]. PMID- 1723709 TI - Structure and transcription of the singed locus of Drosophila melanogaster. AB - Developmental and genetic studies of the singed gene of Drosophila melanogaster indicate that the gene has a role in somatic cells during the formation of adult bristles and hairs, and in the female germline during oogenesis. During metamorphosis a single 3.6-kilobase (kb) RNA is made, and this RNA is also present in adults and early embryos. Early embryos and adult females have additional 3.3- and 3.0-kb RNAs. The RNAs differ only in the length of the 3' untranslated region and a single gene product of 57 kilodaltons is predicted. Analysis of RNA from females lacking ovaries suggests that the 3.3- and 3.0-kb RNAs are made only in ovaries. The absence of the 3.3- and 3.0-kb RNAs in pupae and the time course of their appearance in adult females after eclosion suggests that transcription of singed in the ovary is from middle to late stages of oogenesis. Analysis of RNA in embryos from the reciprocal crosses between wild type and singed-3 showed that all three RNAs are maternally inherited with very little zygotic transcription in embryos. The mutation singed-3 appears to separate the two requirements for singed function as it has an extreme effect upon bristle development, but does not obviously affect oogenesis. In singed-3, there is a deletion at the 5' end of the gene, but the coding region is intact. Transcription in singed-3 is from a cryptic promoter in the upstream flanking sequences which is sufficiently active during oogenesis for fertility, but less active than the wild-type promoter during metamorphosis. The role of the single singed gene product may be in the asymmetric organization and/or movement of cytoplasmic components. PMID- 1723710 TI - [Developmental language disorder in children of severely dysfunctional families]. AB - Developmental language disorder (DLD) is the most common type of language disorder and involves difficulty in comprehending oral language (receptive type) or in expressing verbal language (expressive type). The relationship between DLD encountered in general practice and severe family dysfunction was evaluated. Severely dysfunctional families are characterized by chronic and persistent conflicts, absence of closeness and lack of trust between parents, bad relations between parents and children, and lack of parental support. 20 children, 3.6% of a study population of 550 children aged 3-5 years, were found to be suffering from DLD. The proportion of boys with this disorder from severely dysfunctional families was considerably higher than from control families (73.5% vs 9.5%, p less than 0.0001). In girls, the excess of those from severely dysfunctional families was smaller, and the difference was not significant (20.3% vs 6.6%, p greater than 0.3). Mean years of education of fathers of children with DLD was significantly less than that of fathers of control families (p = 0.04), and less than that of mothers of children with DLD (not significant, but control sample small). It was also less than that of mothers of control families (p = 0.02). In control families there was no difference between the education of fathers and of mothers. The excess of cases of DLD in dysfunctional families was significantly greater when there were more than 4 children in the families (p = 0.01). There was a predominance of the expressive as compared to the receptive type of DLD. Early detection of severely dysfunctional families and of children with DLD may promote early intervention and increase effectiveness of treatment. PMID- 1723711 TI - [The diagnosis of prostatic carcinoma]. AB - Thanks to the combination of high-resolution transrectal ultrasound and the "Biopty-Gun", it has now become possible for the first time to obtain multiple punch biopsies of the prostate, painlessly and with few complications. "Mapping" of the prostate by 6 randomized punch biopsies permits an evaluation of the sensitivity and specificity of such screening procedures as digital rectal examination (DRE), prostate-specific antigen (PSA) and transrectal ultrasound (TRUS). In addition, randomized biopsies provide reliable information about prostatic carcinomas with a significant volume. The indications and efficacy of the various diagnostic methods are discussed in detail. PMID- 1723712 TI - [Allergic enteropathy. Intestinally-mediated fungus allergy]. AB - This article reports on a 53-year-old woman suffering from recurrent diarrhea since 1980 who, in November 1988, was admitted to the Medical Department of the University of Erlangen-Nuremberg with four to eight loose to liquid stools a day. Since January 1988, in addition to abdominal symptoms, including meteorism and recurrent abdominal pain, intermittent bouts of fever of up to 40 degrees C lasting six to eight hours and frequently occurring in the evening, had been noted. The patient's symptoms improved at weekends and during the holidays. Since 1977, she had been employed at a cheese counter, selling cheese. Noteworthy findings were an eosinophilia of 6% and, with the prick skin test, weakly positive reactions induced by two moulds with no increased total IgE and negative IgE RAST for diverse foods and moulds. An in vitro histamine-releasing test performed on colonic mucosal particles obtained at colonoscopy, a sensitization of the mucosa to moulds was demonstrated, and the patient was then given the mast cell stabilizing agent DNCG. This treatment cleared the symptoms, and she put on weight. In vitro tests on colonic biopsy material repeated in 1989 revealed a significant reduction in the release of histamine in response to the anti allergic treatment. PMID- 1723713 TI - [The structure and function of botulinum type C neurotoxin]. AB - The structure gene for botulinum type C neurotoxin was cloned from the toxigenic bacteriophage obtained from Clostridium botulinum type C, and the whole nucleotide sequence was determined. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The signal peptide was not found after the first methionine residue. Upstream of the ATG codon, sequences predicted as a Shine-Dalgarno and a promoter were found. When the deduced amino acid sequence of type C toxin was compared with those of type A and D botulinum toxins and tetanus toxin, type C toxin shared about 52% identity with type D toxin, but shared only about 33% identity with type A and tetanus toxins. The structure and function of type C toxin were estimated from the results of epitope map with monoclonal antibodies and DNA thermal stability map. PMID- 1723714 TI - D12S17 (YNH15) and D12S25 (CRI-L809) are the same polymorphic locus. PMID- 1723715 TI - Characterization of the adherence of human monocytes to cytokine-stimulated human macrovascular endothelial cells. AB - At sites of inflammation, interactions between monocytes and vascular endothelium play an important role in the margination and extravasation of monocytes. The aim of this study was to investigate the relative contributions of the CD11/CD18 family of leucocyte adhesion molecules on monocytes and ICAM-1 and ELAM-1 molecules on endothelial cells (EC) to the binding of monocytes to EC stimulated with recombinant interleukin-1 alpha (rIL-1 alpha), rIL-6, recombinant tumour necrosis factor-alpha (rTFN-alpha) or recombinant interferon-gamma (rIFN-gamma). The adhesiveness of EC for monocytes increased 1.8-2.3-fold after incubation of monolayers of venous or arterial EC with rIL-1 alpha or rTNF-alpha for 4 hr, and 1.6-2.0-fold after stimulation of both types of EC with rIL-1 alpha, rTNF-alpha or rIFN-gamma for 24 hr. Incubation with rIL-6 was without effect. The monoclonal antibodies (mAb) against CD11a, b, c and CD18 on monocytes did not inhibit the increase in the number of monocytes bound to rIL-1 alpha-, rTNF-alpha-, or rIFN gamma-stimulated EC. However, mAb against ELAM-1 expressed on the surface of 4 hr rIL-1 alpha-stimulated EC slightly inhibited (15-21%) the enhanced monocyte binding. ICAM-1, which exhibited marked expression on 24 hr rIL-1 alpha-, rTNF alpha- or rIFN-gamma-stimulated EC, did not contribute to the enhanced monocyte binding. The percentage of EC-bound monocytes which had stretched out over the surface of cytokine-stimulated venous or arterial EC was significantly increased compared to the percentage found for non-stimulated EC. It was observed that mild fixation of EC as well as treatment of EC with cytochalasin B or mAb against ICAM 1 did not affect the number of monocytes that were bound to EC, but considerably reduced the percentage of EC-bound monocytes with a stretched morphology. It is concluded that the binding of monocytes to cytokine-stimulated EC is dependent on the type of cytokine and the duration of cytokine stimulation. The increase in the binding of monocytes to cytokine-stimulated EC occurred as a result of CD11/CD18- and ICAM-1-independent factors. The subsequent morphological changes, i.e. stretching of monocytes over the surface of EC, required viable EC and ICAM 1. PMID- 1723716 TI - Human complement regulatory proteins expressed on mouse A9 cells containing a human chromosome 1. AB - The structural genes of human complement regulatory proteins are clustered on chromosome 1 at position q3.2. Human chromosome 1 was transferred into a mouse fibroblast cell line, A9 [designated as A9(neo-1)], and the surface expression of its gene products participating in complement regulation, namely C3b/C4b receptor (CR1, CD35), decay-accelerating factor (DAF, CD55), membrane co-factor protein (MCP, CD46) and C3d/EB virus receptor (CR2, CD21), were assessed using respective monoclonal antibodies by flow cytometry. CR1 became positive within 7 days of culture. MCP appeared in a small population of cells by Day 3 and, together with DAF, began to increase on Day 7. CR2 appeared on Day 14. The order of the expression was CR1 greater than DAF = MCP greater than CR2. On Day 42, however, all became negative except for MCP, which was markedly diminished. These human regulatory proteins were specifically associated with the presence of human chromosome 1, since none of them were expressed on human chromosome 12 transferred A9 cells [A9(neo-12)]. Intact A9 and A9(neo-12) cells activated human complement via the alternative pathway. The activation of this pathway was suppressed in the A9(neo-1) cells that expressed CR1, DAF and MCP. Slight protective activity was still observed in the 42-day cultured A9(neo-1) cells expressing only trace MCP. These results suggest that human complement regulators, expressed via the transferred human chromosome 1, can protect heterologous cells from complement, overcoming their ability to activate the human alternative pathway. PMID- 1723717 TI - Examination of chlamydial glycolipid with monoclonal antibodies: cellular distribution and epitope binding. AB - A chlamydial glycolipid antigen (GLXA) is shed into the medium of C. trachomatis infected cell cultures. This study screened monoclonal antibodies (mAb), prepared in different laboratories by immunization with embryonated egg propagated elementary bodies (EB), for their ability to bind with infected cells and to react with purified GLXA isolated from supernatants of infected McCoy cells. The fluorescent antibody (FA) staining pattern exhibited by a number of mAb indicated that they bound antigen present within the inclusion and at the inner membrane surface of infected cells; the observed pattern differs significantly from the distribution seen when anti-lipopolysaccharide (LPS) (mAb) were used. The staining pattern observed by immunofluorescence was confirmed and extended by ultrastructure studies of immunogold-labelled, infected human endometrial gland epithelial cells (HEGEC) and a human endometrial carcinoma-derived cell line (RL95-2). Additionally, the immunoelectron microscope studies revealed binding within the inclusion and on reticulate bodies, within the cell cytoplasm and at the surface of infected cells. The specificity of the reactive mAb, examined by molecular shift chromatography and isolated, affinity-purified GLXA, indicated that two mAb of the IgG isotype recognized an antigen which had been purified from tissue culture supernatants by affinity chromatography using an IgM mAb. The results suggest that GLXA is an important determinant whose role and function during in vitro and in vivo infections deserves further analyses. PMID- 1723718 TI - A traditional Chinese herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-yoei-to) augments the production of granulocyte-macrophage colony stimulating factor from human peripheral blood mononuclear cells in vitro. AB - Ren-shen-yang-rong-tang (Japanese name: Ninjin-yoei-to, NYT) is a traditional Chinese herbal medicine. Leukocytosis and elevated levels of colony-stimulating factor (CSF) in peripheral blood were found previously after the administration of this compound to mice. In this study, human peripheral blood mononuclear cells (PBMC) were cultured in the presence of NYT in vitro, and the levels of granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) in the supernatant of cultured PBMC were measured using a sensitive enzyme-linked immunosorbent assays. NYT significantly (P less than 0.01) augmented GM-CSF production but not G-CSF production by PBMC in vitro. PMID- 1723719 TI - Dimensional oscillation. A fast variation of energy embedding gives good results with the AMBER potential energy function. AB - The structure of the AMBER potential energy surface of the cyclic tetrapeptide cyclotetrasarcosyl is analyzed as a function of the dimensionality of coordinate space. It is found that the number of local energy minima decreases as the dimensionality of the space increases until some limit at which point equipotential subspaces appear. The applicability of energy embedding methods to finding global energy minima in this type of energy-conformation space is explored. Dimensional oscillation, a computationally fast variant of energy embedding is introduced and found to sample conformation space widely and to do a good job of finding global and near-global energy minima. PMID- 1723720 TI - Synthesis and biological activity of 2-phenylethyl ester analogues of C-terminal heptapeptide of cholecystokinin modified in Trp 30 region. AB - We have tried to evaluate the significance of the tryptophan side chain residue and of the surrounding peptide bonds in the antagonist activity of cholecystokinin analogues lacking the C-terminal amide function and having a D tryptophan. In order to perform this study, analogues of the C-terminal heptapeptide of cholecystokinin were synthesized by replacing the C-terminal phenylalanine residue with 2-phenylethyl alcohol and by either replacing the tryptophan residue with an alanine, a norleucine and a phenylalanine residue, or introducing a "reduced peptide bond" in the tryptophan 30 region. Most of these compounds were able to reproduce only part of the response of cholecystokinin in stimulating amylase release from rat pancreatic acini, as was already observed for 2-phenylethyl ester analogues of CCK. These results point out the key role of tryptophan 30 in the biological response of cholecystokinin. PMID- 1723721 TI - Simultaneous immunohistochemical demonstration of vasoactive intestinal polypeptide and its receptor in human colon. AB - The present study reports an immunohistochemical approach for localizing the immunoreactivity of vasoactive intestinal polypeptide (VIP) receptor in the human colon, by using a monoclonal antibody which recognizes the VIP-receptor of a human colonic adenocarcinoma cell line. Simultaneous demonstration of immunoreactive VIP-receptor of a human colonic adenocarcinoma cell line. Simultaneous demonstration of immunoreactive VIP-receptor and VIP was achieved by a double-labelling procedure employing immunogold silver staining for VIP receptor, and a biotinylated secondary antibody followed by streptavidin-Texas Red, to visualize VIP. The immunoreactive VIP receptor was found at two locations receiving dense VIP innervation: myenteric ganglia and mucosal epithelium. Epithelial cells displayed intense labelling at the basolateral membrane, which confirmed earlier binding studies on fractionated membranes. A small number of enteroendocrine cells was also recognized by the VIP-receptor antibody. Smooth muscle and cells of the immune system were not stained by the monoclonal antibody, indicating that it recognized an epitope not common to VIP-receptors of all locations. Thus, the immunohistochemical approach of VIP-receptor localization differs from autoradiography in (a) precise cellular localization, (b) possibility of simultaneous demonstration of receptor and ligand immunoreactivity, and (c) selectivity to a certain receptor population which, however, is presently not fully characterized. PMID- 1723722 TI - Post embedding immunoelectron microscopy of human breast cancer: a comparison of three acrylic resins. AB - The suitability of three acrylic resins for the immunoelectron microscopical localization of cell surface and cytoskeletal antigens in surgically excised, immersion fixed human breast cancer, using an immunogold system, has been assessed. Good localization of milk fat globule membrane was achieved with LR White, LR Gold and Lowicryl K11M, although the embedding schedule for LR White had to be modified. The best results were achieved with Lowicryl K11M. Only scanty labelling of actin and cytokeratin was seen in LR White embedded tissue, whereas there was clear localization in LR Gold and Lowicryl K11M embedded samples. Tubulin and alpha-actinin was detected at low level in tissues in the low temperature embedding resins, but not in LR White embedded samples. The morphology of the latter was poorer, and there was greater variability in ultrastructure and labelling. Of the two low temperature embedding resins, Lowicryl K11M gave slightly better results. However, the advantages could be outweighed by the problem incurred in achieving the low temperatures, and by poorer handling properties than LR Gold. PMID- 1723723 TI - Detection of different developmental stages of malaria parasites by non radioactive DNA in situ hybridization. AB - A highly sensitive non-radioactive DNA in situ hybridization procedure is described that enables detection and unequivocal identification of various developmental stages of human and rodent malaria parasites. Using biotinylated species-specific DNA probes, erythrocytic parasites can be specifically stained in blood smears. Similarly exoerythrocytic stages can be visualized in cell culture and in sections of paraffin-embedded liver. In blood smears, the hybridization procedure provides a rapid detection of (low) parasitemia and species-determination for experienced microscopists at 100 to 400x magnification. Moreover, the procedure can be applied even after previous Giemsa staining of the preparation, enabling revision of patient smears which were difficult to read after routine Giemsa staining. PMID- 1723724 TI - Blockade of the antigen-antibody reaction using benzil condensation with the guanidyl residue of arginine. AB - Benzil blockade of the guanidyl group of arginine was tried on sections of paraffin-embedded tissue fixed in two different fixatives, in an attempt to evaluate the relevance of this amino acid to the reaction of several proteins with their corresponding antibodies. The two fixatives were 10% formaldehyde, and Bouin's fluid without acetic acid. Both polyclonal and monoclonal antibodies against proteins or peptides (lysozyme, adrenocorticotropic hormone, growth hormone, placental lactogen, and prolactin) were used on human biopsies or material from autopsies. The blockade was effective when monoclonal antibodies were used, whereas no effect or only a small decrease of the intensity of the reaction was observed with polyclonal antibodies. The least definitive result was obtained with prolactin, where a complete blockade was never achieved with monoclonal antibodies. Calcitonin, a peptide that does not contain arginine, was used as a control not susceptible to benzil blockade; no blockade of immunostaining was observed. PMID- 1723725 TI - Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure. AB - In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5-30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results. PMID- 1723726 TI - Mucosubstance histochemistry of pleomorphic adenoma of parotid and submandibular salivary glands of man: light and electron microscopy. AB - Lumina and adluminal cells in human salivary gland pleomorphic adenomas were found to contain neutral, carboxylated, and occasionally sulphated glycoproteins. A variable component of luminal contents and secretory granules did not appear to contain glycoprotein and possibly consisted of protein. Glycosaminoglycans, which appeared to be hyaluronic acid and chondroitin sulphate, were demonstrated rarely in lumina, often between epithelial cells, and forming the matrix of myxoid tissue and, together with collagen, chondroid tissue. No differences were seen between tumours from parotid glands and those from submandibular glands. Glycoproteins demonstrated in the epithelium are similar to those of intercalary ducts of parotid and submandibular glands, and may represent a primitive form of salivary secretion. Glycosaminoglycans secreted intercellularly by epithelial cells cause their increasing separation to form myxoid or chondroid tissue. This stromalization extends to lumina to produce a loss of epithelium. Pleomorphic adenoma appears to be a manifest example of variable derepression of the genotype. PMID- 1723727 TI - Glycopeptide-albumin derivative: it preparation and histochemical ligand properties. AB - Carrier-immobilized mono- or disaccharides and other carbohydrate structures, derived by custom-made chemical synthesis, have already proven to be valuable ligands for localizing carbohydrate-binding proteins in tissue sections. Defined purified glycopeptides, as components of neoglycoproteins, offer the possibility of increasing their structural complexity and, thereby, their receptor selectivity. To test the feasibility of this approach, the glycopeptide man6 glcNAc2-asparagine derived from ovalbumin was purified after pronase digestion. It was coupled to bovine serum albumin as carrier protein with the homobifunctional linking agent bis-(sulphosuccinimidyl)suberate to yield the diglycosylated concanavalin A-reactive product. Following biotinylation, it was used to detect mannose-specific binding sites in fixed cells of seven human leukemia or lymphoma lines and in fixed, paraffin-embedded sections of human breast cancer. In comparison to chemically mannosylated bovine serum albumin with ten sites of glycosylation or to ovalbumin, this derivative produced a similar pattern of reaction with a quantitatively lower extent of staining in most cases. Remarkably, the presence of potential endogenous ligands for the detected receptor sites was ascertained using the plant lectin concanavalin A. Thus, the conjugation of a purified, deliberately selected glycopeptide to a suitable carrier produces a histochemical tool for detecting glycopeptide-specific binding sites. PMID- 1723728 TI - Blocking of nucleophilic amino groups with carbonyldiimidazole. PMID- 1723729 TI - Antibodies to synthetic platelet-activating factor (1-O-alkyl-2-O-acetyl-sn glycero-3-phosphocholine) analogues with substituents at the sn-2 position. AB - We obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentadecyl)-2-O-acetyl-sn glycero-3- phosphocholine(acetyl-CPGPC), 1-O-(15'-carboxypentadecyl)-2-O-N,N dimethylcarbamoyl-sn-glycero-3 - phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O (15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-pho sphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl much greater than dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethylcarbamoyl greater than acetyl much greater than butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC BSA; and butylcarbamoyl greater than dimethylcarbamoyl greater than acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these antibodies. Anti dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG inhibited a PAF induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG. PMID- 1723730 TI - Sialylation processes in mitochondria: evidence for two distinct sialyltransferases located in the outer membrane. AB - Previous studies have shown that purified mitochondrial outer membrane is able to catalyze the transfer of sialic acid from CMP-Neu5Ac to an exogenous asialoglycoprotein acceptor, asialofetuin. Considering the heterogeneity of the glycan chains borne by this glycoprotein, an investigation of mitochondrial sialyltransferase activities was undertaken. Our data provide evidence for the existence of two distinct sialyltransferases in purified mitochondrial outer membranes. The use of different acceptor substrates, the temperature dependence of these enzymes, and their different sensitivity towards a sulfhydryl reagent, p CMB, allowed us to discriminate between a galactoside alpha(2-3) sialyltransferase and a galactoside alpha(2-6) sialyltransferase presumably involved in the sialylation of O- and N-glycan chains of glycoprotein, respectively. These results are discussed in terms of mitochondrial autonomy for post-translational events. PMID- 1723731 TI - Simultaneous separation and determination of hydrocarbons and organochlorine compounds by using a two-step microcolumn. AB - The simultaneous separation and determination of a mixture of hydrocarbons and organochlorine compounds was successfully carried out by using sorption chromatography on a two-step microcolumn of silica and aluminium oxide for their fractionation, and a dual detector system. In addition to the separation and identification of hydrocarbons and heterocompounds containing nitrogen, oxygen and sulphur atoms, separation and identification of chlorinated hydrocarbons (dichlorobenzenes, p-chlorotoluene, hexachlorobutadiene, 1,2,4-trichlorobenzene and 2-chloronaphthalene), pesticides (chlorpicrin, aldrin, lindane, alpha- and beta-benzene hexachloride (BHC), endrin, dieldrin, endosulphan, methoxychlor) and herbicides (propanil, dichlobenil, trifluralin, difolatan) were achieved in mixtures containing polychlorinated biphenyl, strobane and chlordane. PMID- 1723732 TI - Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography. AB - A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography-mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies. PMID- 1723733 TI - Optimization of oxidation of glycoproteins: an assay for predicting coupling to hydrazide chromatographic supports. AB - A rapid, simple assay for aldehydes generated by oxidation of saccharide units in glycoproteins, using dyes containing hydrazide functionalities, is described. The assay is used, in conjunction with tests of biological activity, to predict oxidation conditions that will result in a maximum of active protein coupled to a hydrazide chromatographic support. Glycoproteins are labeled with Lucifer Yellow CH or Texas Red Hydrazide, and the extent of labeling is determined. Using the assay, it is shown that the efficiency of coupling to Affi-Prep Hydrazide is proportional to oxidation. PMID- 1723734 TI - Hepatitis C antibody testing: problems associated with non-specific binding. AB - The prevalence of antibodies to hepatitis C virus (anti-HCV) was measured in a number of groups known to be at increased risk of blood-borne viral infections, using an enzyme-linked immunosorbent assay (EIA) based on a nonstructural peptide generated by recombinant DNA technology. The assay was repeatably reactive in 75.6% of men with haemophilia, 61.9% of intravenous drug users, 34.1% of homosexual men who were regular attenders at a gay sauna and 30.8% of prisoners. A lower reactivity was detected in sera collected from female prostitutes (10.4%), patients undergoing maintenance haemodialysis (5.9%), or renal transplantation (6.9%) and patients attending a sexually transmitted diseases clinic (6.2%). We also measured reactivity among inmates of a large institution for the mentally handicapped in which hepatitis B is known to be endemic, and in panels of sera which had been stored for 25-35 years. The test was positive in 41.1% of mentally handicapped patients with Down's syndrome and 7% of subjects with other forms of mental retardation. Similarly some 23% and 20% of sera collected in 1954 and 1964 from patients with a variety of illnesses were found to be reactive. As most diagnostic assays suffer from some degree of non specificity and confirmatory tests for the anti-HCV assay were not initially available in Australia, we analysed the distribution of optical density (OD) values in the different groups, in an attempt to obtain an insight into the specificity of the results being obtained. Whereas the ODs of sera collected from patients with haemophilia and IVDU had a bimodal pattern, with two well separated sets of results on either side of the cut-off.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723735 TI - Immunoaffinity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals. AB - The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies. The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine. PMID- 1723736 TI - A study of the effect of chemical inactivants on the epitopes of Rift Valley fever virus glycoproteins using monoclonal antibodies. AB - A panel of 23 monoclonal antibodies (mAbs) was used to study the effect of formalin, beta propriolactone (BPL) and binary ethylenimine (BEI) on the epitopes of the Rift Valley fever virus glycoproteins. After the initial inactivation period BEI had very little adverse affect on the epitopes whereas BPL significantly altered seven and formalin partially changed the conformation or accessibility of most of the epitopes. The epitopes of all of the inactivated antigens showed a reduced activity against the specific mAbs over a six month storage period. PMID- 1723737 TI - Age-related changes in galanin-immunoreactive cells of the rat medial septal area. AB - Age-related changes in the cholinergic cells have been reported in the rat medial septal area. The neuropeptide galanin is colocalized with acetylcholine in the majority of the medial septal neurons. To assess possible age-related changes in the galanin-containing septal cells, we have examined, with immunohistochemical methods, the distribution pattern, density, and morphological features of galanin containing cells in the rat medial septal nucleus (MS) and the nucleus of the diagonal band of Broca (DBB) in 1, 3-6, 9-12, 16-18, 24-27, and 28-30 month-old rats. A morphometric computerized analysis was also performed. In addition, the intensity of the immunolabelling was measured by densitometry. Galanin-like immunoreactivity (galanin-LI) was present in both the MS and the DBB. Our results clearly indicate a progressive age-related decrease in the number of galanin positive cells throughout the MS-DBB complex. Our quantitative study revealed a significant loss of galanin-positive cells in the MS-DBB complex of 16-18 (50.4%), 24-27 (52.3%), and 28-30 (52.4%) month-old rats compared to 3-6 month old animals. A non-significant reduction (28.6%) in galanin-LI cell number was observed in 3-6 month-old rats compared to 1 month-old animals. The morphometric analysis demonstrated a significant reduction (18%) in the surface of galanin positive cells remaining in the 28-30 month-old group. Furthermore, a significant decrease in the immunolabelling intensity was consistently observed in animals of 16 month-old and older. To determine whether changes in galanin-positive cells were associated with cholinergic changes, the number of cells stained for acetylcholinesterase (AChE) was estimated in 3-6, 9-12, 16-18, and 24-27 month old rats. There was a 43% decrease in the number of AChE-positive cells and a 71% loss of galanin-positive cells in 24-27 month-old rats compared to 3-6 month-old. The galanin-cell loss in the medial septal area was therefore associated with a parallel, although smaller, cholinergic septal cell loss. PMID- 1723738 TI - Application of alkaline unwinding to analysis of strand breaks induced by bleomycin in hamster lung DNA in vivo. AB - Strand breaks in hamster lung DNA were analyzed following in vivo exposure to bleomycin. An alkaline unwinding procedure involving separation of single-strand from double-strand DNA by hydroxylapatite chromatography followed by fluorescence detection of DNA with bisbenzimide was adapted for these studies. Procedures were developed that allowed preparation of DNA from lungs of control animals with a relatively low amount of single-strand DNA relative to double-strand DNA. Time dependence of unwinding was demonstrated using samples that were damaged deliberately by brief probe sonication. To verify that residual bleomycin remaining in lungs at the time of sacrifice did not cause strand breaks during sample preparation, bleomycin was added to minced lung in vitro. Under these conditions, the ratio of single-strand to double-strand DNA was not increased significantly. Substantial strand breaks were produced in vivo at 15 min and 1 h following intratracheal instillation of bleomycin into hamsters, as evidenced by a 5-6-fold increase in the ratio of single-strand to double-strand DNA relative to controls. The DNA damage appeared to be repaired within 1 day following exposure. PMID- 1723739 TI - Bacterial endotoxin: molecular relationships between structure and activity. AB - The significance of endotoxins in bacterial infection and their role as bacterial surface antigens (O antigens) have stimulated investigations into their chemical nature and the mechanisms of their biologic action during the last few decades. This article summarizes some of the recent results and emphasizes structure activity relationships. PMID- 1723740 TI - Immunotherapy with antibodies to core lipopolysaccharide: a critical appraisal. AB - The unknowns persisting in the understanding of the mode of action of anti-core lipopolysaccharide antibodies are discussed, and a study of two anti-lipid A monoclonal antibodies is reviewed. This article also critically analyzes the results of the recent clinical trials with monoclonal antibodies. PMID- 1723741 TI - Assessing the viability of Mycobacterium leprae by the fluorescein diacetate/ethidium bromide staining technique. AB - In the present study we have evaluated the Fluorescein Diacetate/Ethidium Bromide (FDA/EB) staining technique to assess the viability of Mycobacterium leprae obtained from biopsies of leprosy patients under different periods of treatment. Bacillary suspensions were obtained from skin punch biopsies and stained with the FDA/EB solution. The average percentage of green cells seen which were deemed to be viable were: 67.2% of green cells in patients without previous treatment; 45.6% in patients with 1 to 6 months of treatment; 25.9% for patients with 7 to 12 months of treatment and 10.5% in patients with 13 to 24 months of treatment. All the patients studied were on multidrug therapy. The differences obtained in the percentages of green cells in the different groups of patients were statistically significant as determined by the Wilcoxon's test. The decrease in the percentage of green cells observed with increasing periods of treatment suggests that the FDA/EB technique correlates with the actual viability of M. leprae. The application of this technique in the routine procedures performed with Hansen's disease patients could be very useful for monitoring the effectiveness of treatment in leprosy patients. PMID- 1723742 TI - Significance of neither green nor red NGR bacilli in FDA-EB staining. AB - This Study deals with determination of viability by FDA-EB method. It has been observed that some of the bacilli do not take any colour in FDA-EB preparations. These can be called "neither green nor red" (NGR) bacilli. These non-staining bacilli should be taken into account when reporting viability by FDA-EB method. PMID- 1723743 TI - [Evaluation of DNA-probe assay for the clinical diagnosis of Mycoplasma pneumoniae infections]. AB - DNA probe-assay using the Gen-Probe kit was carried out to detect Mycoplasma pneumoniae infections. Fifteen children visited Ota General Hospital complaining of dry cough and high grade fever. Throat swabs of the patients were examined to detect M. pneumoniae ribosomal RNA by Gen-Prove kit. Five out of 15 patients were positive for DNA probe assay of M. pneumoniae. Clinical and laboratory data including serological examinations were compatible with M. pneumoniae infection in these cases. Following the improvement of clinical symptoms and signs by receiving erythromycin or minocycline, the positivity for DNA probe assay turned to negative. Among the ten patients, who were negative for DNA probe assay, 2 cases were suspected of M. pneumoniae infection on the basis of clinical and laboratory findings. One patient had already taken antibiotics. Therefore, in these two patients, there was a possibility that the bacterial numbers were too small to be detected by DNA-probe assay. The data described above support that DNA-probe assay is useful for the diagnosis of M. pneumoniae infections in the early stage. DNA-probe assay is also valuable to follow up the clinical course of the patients. PMID- 1723744 TI - [Molecular mimicry and autoimmunity]. PMID- 1723745 TI - Rat anterior pituitary neuropeptides following chronic prolactin manipulation: a combined radioimmunoassay and mRNA study. AB - Prolactin secretion is highly regulable, and the possibility exists that there are local intrapituitary factors controlling prolactin secretion. Recently, the neuropeptides vasoactive intestinal peptide (VIP), galanin and substance P (SP) have been co-localized to the lactotroph in the female rat. We investigated the effects of alterations in prolactin status in vivo on pituitary and hypothalamic expression of these peptides by specific radioimmunoassays and mRNA analysis. In the anterior pituitary, following haloperidol treatment, the contents of both VIP and galanin were suppressed to below detectable levels. Similarly, after bromocriptine treatment, the content of VIP was decreased to below the detection limit of the assay while galanin (14.2 +/- 1.3 vs control 21.0 +/- 2.1 fmol/mg, P less than 0.05) also showed a significant reduction. The levels of VIP mRNA and galanin mRNA in these groups showed the same qualitative change as their respective peptides. Concurrent treatment with high-dose oestrogen modified the VIP peptide response to bromocriptine (1368.7 +/- 149.2 vs bromocriptine 843.4 +/ 82.7 fmol/mg, P less than 0.05) but not to haloperidol. Oestrogen-induced decreases in galanin content were not influenced by either treatment. The pituitary content of SP showed a fall after oestrogen treatment (1.1 +/- 0.01 vs control 6.4 +/- 0.8 fmol/mg, P less than 0.05) which was not significantly altered by either bromocriptine or haloperidol. Likewise, SP mRNA levels in the pituitary were decreased by 90% following oestrogen treatment. Hypothalamic expression of these peptides did not change with any of the treatments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723746 TI - Most of the circulating insulin-like growth factors-I and -II are present in the 150 kDa complex during human pregnancy. AB - The aim of this study was to assess the molecular size distribution of insulin like growth factors (IGFs) complexed to IGF-binding proteins (IGFBPs) in serum of non-pregnant and pregnant women. Sera were fractionated on a size-exclusion column at pH 7.4 to resolve different IGF-IGFBP complexes from unbound IGFs. Each fraction was further chromatographed on a size-exclusion column under acid conditions to dissociate IGFs from binding proteins prior to measurement of the IGF content of the complexes. The IGF-containing fractions from the acid column were assayed specifically for IGF-I and IGF-II. Serum pooled from pregnant women contained more IGF-I and IGF-II than serum from non-pregnant women. In both groups most of the IGF-I and IGF-II was found in a large (150 kDa) complex in serum and the remainder was present in complexes eluting in the 40-50 kDa region at pH 7.4. Some free IGF-I was also detected. Serum fractions collected by size exclusion chromatography at pH 7.4 were also analysed by Western-ligand blotting to characterize the IGFBPs in the two main IGFBP size classes. In serum pooled from non-pregnant women, the 150 kDa IGF-IGFBP complexes contained a 40-50 kDa IGFBP doublet following Western-ligand blot analysis. This IGFBP co-migrated with a pure IGFBP-3 standard. IGFBPs of 34, 28 and 24 kDa all contributed to the 40-50 kDa IGF-IGFBP complexes of the pH 7.4 chromatograph.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723747 TI - Labeling of developing vascular endothelium after injections of rhodamine-dextran into blastomeres of Xenopus laevis. AB - The goal of this work has been to label endothelial cells with fluorescent marker and to record their behavior during angiogenesis in vivo. Single blastomeres in 16-128-cell-stage embryos of pigment-deficient Xenopus laevis were injected intracellularly with 5% tetramethyl-rhodamine dextran. Subsequently, the embryos and tadpoles were examined with an epifluorescence microscope, a silicon intensified target (SIT) camera, and video recordings. Clones that would include endothelium could be selected as early as stages 33-36 on the basis of heavy labeling in the ventral mesodermal core of the tail. Strands of fluorescent cells and early vessels appeared in the tail at stages 39-41. Subsequently, groups of endothelial cells were followed in case histories in the tail and in the aortic arches and gills of tadpoles. Two main results were that the patterns of fluorescent endothelial cells were stable in established arteries, veins, and capillaries for at least 2-12 days, and that labeled endothelial cells migrated distally in elongating sprouts. In addition, it was inferred that endothelium was derived from multiple blastomeres, probably in the ventral vegetal regions. Only small fractions of total endothelium were labeled from any single blastomere. None of the early blastomeres produced exclusive clones of vascular endothelium; other labeled cell types in various clones included muscle fibers, lymphatics, mesodermal stellate cells, blood cells, gut, proctodeum, and some epidermis, in addition to endothelial cells. The method of intracellular marking of blastomeres is recognized as a direct approach for charting lineage and fate tables in embryos of Xenopus and other species. The present study extends the period of observation in vivo for up to 2 weeks in the growing tadpole and focuses on endothelial cells during angiogenesis. Even though fluorescent dextran was apparently packaged in vesicles and metabolized, individual cells and small groups could be identified and followed with time. This method provides excellent opportunities for addressing problems in vascular development in the living animal. PMID- 1723748 TI - Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction. AB - Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential. PMID- 1723749 TI - Progressive supranuclear palsy as the sole manifestation of systemic Whipple's disease treated with pefloxacine. PMID- 1723750 TI - Pseudotumour cerebri and chronic benzene hexachloride (lindane) exposure. PMID- 1723751 TI - Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity. AB - Zinc-positive boutons, originating in the medial cortex of lizards, exhibit glutamate immunoreactivity. This finding supports the presumed homology between lizard zinc-positive boutons and the hippocampal mossy fibres of mammals, which are also glutamate-immunoreactive and zinc-positive. Zinc-positive boutons of lizards contain a chelatable pool of zinc located in the hippocampal mossy fibres of mammals. These synaptic systems also contain glutamate, which indicates a possible simultaneous action of zinc and glutamate during synaptic transmission. PMID- 1723752 TI - Fibrinolysis and angiogenesis in wound healing. AB - The healing wound offers a clear example of the sequence of events in chronic inflammation leading to repair. Although angiogenesis has an obvious and essential role in this process, it has been little studied. For an angiogenic factor to seem relevant, it would have to be shown to precede the peak of increased vascularity. To define this peak, the vessel content of simple, incised mouse wounds was estimated using morphometry of histological sections, and found to rise to a maximum at days 5 and 6. Total angiogenic activity of aqueous extracts was found to reach a peak at day 3. The detection of such activity on the chick chorioallantoic membrane is very dependent on the preparation technique and the choice of proteinase inhibitors. Previous in vitro work by us using purified material has shown fibrin degradation products to be effective in stimulating angiogenesis. Fibrin degradation products are prominent on immunoblotting from day 3, when macrophages are plentiful, with a similar band pattern to human granulation tissue. PMID- 1723753 TI - Immunohistochemical positivity for neuron-specific enolase and Leu-7 in malignant mesotheliomas. AB - The discovery of immunostaining for neuron-specific enolase (NSE) and Leu-7 in a small cell mesothelioma prompted us to study some putative immunohistochemical markers of neuroendocrine differentiation in malignant mesotheliomas and to examine any diagnostically important immunohistochemical distinctions or similarities between malignant mesothelioma and other histologically similar lung tumours. Most mesotheliomas were positive for NSE (96 per cent) and Leu-7 (70 per cent) and positivity for these two markers was also found in small cell carcinomas (NSE 25 per cent, Leu-7 81 per cent) and adenocarcinomas (NSE 28 per cent, Leu-7 28 per cent) but carcinosarcomas were positive for only NSE (44 per cent). Chromogranin A positivity was found only in occasional small cell carcinomas (6 per cent) and adenocarcinomas (6 per cent). No tumour was positive for bombesin. The high incidence of NSE and Leu-7 positivity in mesotheliomas is an important original observation because it guards against the unjustified exclusion of mesothelioma from a differential diagnosis on the basis of positivity for these two markers. PMID- 1723754 TI - Effect of tranilast on endothelin-induced bronchoconstriction in guinea pigs. AB - Tranilast, an anti-asthmatic drug with anti-allergic properties, inhibited endothelin (ET)-induced asthmatic like respiratory obstruction in guinea pigs when administered orally at 200 mg/kg 1 h prior to the intravenous injection of ET. ET also caused a bronchoconstriction detected as an increase in inflation pressure in Konzett-Rossler apparatus. Tranilast (200 mg/kg, p.o. 1 h before ET) inhibited ET-induced increased inflation pressure. ET-induced bronchoconstriction detected as an increase in inflation pressure was clearly inhibited by the administration of indomethacin (used as a reference drug) at doses of 1 and 5 mg/kg 30 min prior to onset of the reaction. In addition to the above in vivo experiments, tranilast at concentrations between 10(-5) and 10(-4) g/ml inhibited ET-induced contraction of isolated guinea pig tracheal muscle, whereas indomethacin did not affect this in vitro response. In order to elucidate the inhibitory mechanism of tranilast on ET-induced bronchoconstriction, the effects in Ca(2+)-free medium and on ET-induced histamine and prostaglandin F2 alpha release from tracheal muscle were investigated. Consequently, tranilast did not affect ET-induced contraction of guinea pig tracheal muscle in Ca(2+)-free Tyrode's solution and ET induced neither histamine nor PGF2 alpha release. These results suggest that tranilast inhibits ET-induced bronchoconstriction by inhibiting the ET-induced calcium influx into tracheal muscle. PMID- 1723755 TI - [Current topics on glutamate receptors]. AB - Glutamate (Glu) receptors are classified into two major categories in the mammalian central nervous system: inotropic receptors linked to ion channels and metabotropic receptors linked to phosphatidylinositol (PI) metabolism. Classification of the inotropic Glu receptors is based on the differential sensitivity to excitement by N-methyl-D-aspartic acid (NMDA), DL-alpha-amino-3 hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainic acid (KA). The NMDA sensitive subclass is supposed to be a receptor ionophore complex consisting of at least four different subcomponents, including an NMDA recognition site, a glycine (Gly) recognition site, a polyamine recognition site and a cation channel. The NMDA site is radiolabeled by both Glu and competitive antagonists, such as (+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and DL-(E) 2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653). The Gly domain, which is labeled by both [3H]Gly and [3H]5,7-dichlorokynurenic acid, is sensitive to D serine but insensitive to strychnine, and this domain seems to be absolutely required for an activation of the NMDA channel by agonists. The ionophore domain is identified by radiolabeled non-competitive NMDA antagonists that gain access to the binding sites within the channel only when it is gated by agonists. The opening of an NMDA channel is allosterically potentiated by Gly and several polyamines. In contrast, an activation of the NMDA channel is blocked by both H+ and divalent cations such as Mg2+ and Zn2+. [3H]AMPA binding displays pharmacological profiles of the AMPA-sensitive subclass with a rank order of agonistic potencies of quisqualic acid (QA) greater than or equal to AMPA greater than Glu greater than KA, which is apparently different from that found for the KA-sensitive subclass (domoic acid greater than or equal to KA greater than QA greater than Glu). In contrast, several quinoxaline derivatives competitively antagonize neuronal responses mediated not only by the AMPA receptor but also by the KA receptor. The metabotropic Glu receptors, which stimulate PI metabolism through an activation of the guanosine triphosphate-binding proteins, are activated by Glu, QA and trans-1-amino-cyclopentyl-1,3-di-carboxylic acid (ACPD). Responses mediated by the metabotropic receptors are competitively blocked by 2 amino-3-phosphonopropionic acid. Three or four cloned complementary deoxyribonucleic acids (cDNAs) encoding inotropic Glu receptors are isolated from a rat brain cDNA library. Pharmacological and electrophysiological properties of receptor-ion channels encoded by a transfection of these cDNAs are similar to those observed with the AMPA receptor as well as the KA receptor, but not with the NMDA receptor. PMID- 1723756 TI - Comparative study between modified Freyer's prostatectomy, classical Freyer's prostatectomy and Millin's prostatectomy. AB - Classical Freyer's method was modified in which, prostatic fossa was packed after the enucleation of the gland for hemostatic purpose; and the pack and suprapubic drain were brought out laterally. The pack was removed after 48-72 hours postoperatively and catheter was introduced per urethrum for early closure of suprapubic ostomy. Fifty such cases of Modified Freyer's prostatectomy were compared with cases of classical Freyer's prostatectomy (n = 25) and Millin's prostatectomy (n = 25). The duration of surgery and requirements for perioperative blood transfusion were found to be less with our method. PMID- 1723757 TI - Studies on monoclonal anti-idiotypic and anti-isotypic antibodies against leukemia and myeloma: III. Analysis of monoclonal antibodies recognizing the antigenic epitopes by use of ELISA additivity test and microcomputer grouping programme. AB - ELISA double antibodies additivity test was employed to identify the epitopes which can be recognized by monoclonal antibodies (McAbs) against IgM of a patient with B chronic lymphocytic leukemia (B-CLL). The computer grouping programme analysis showed that 4 anti-isotype McAbs could be divided into two groups and 10 anti-idiotype McAbs could be divided into four groups. The result was consistent with that of the indirect ELISA, ELISA sandwich as well as ELISA inhibition test. The above findings suggested that there are at least 6 distinct IgM epitopes which can specifically react with 14 McAbs. Our study indicated that the combination of the ELISA additivity test and the computer grouping programme analysis provides a helpful tool for researchers into the relationship between the structure and function of antigens. PMID- 1723758 TI - The effect of anti-AFP-F(ab)'2 and adriamycin conjugate on transplantable tumors in mice. AB - Efficacy of the conjugate anti-AFP-F(ab)'2 with adriamycin, anti-AFP-F(ab)'2-adr, against transplantable tumors was studied in mice. The results showed that LD50 of anti-AFP-F(ab)'2-adr in mice was 7.43 mg/kg and that anti-AFP-F(ab)'2-adr in a dose of 1/10-1/5 LD50 could inhibit the growth of transplanted hepatocellular carcinoma. In addition, anti-AFP-F(ab)'2-adr with local application of AFP could markedly inhibit the growth of transplanted Ehrlich ascites cancer. From the above it is suggested that anti-AFP-F(ab)'2-adr can be used to treat hepatic carcinoma with satisfactory results. PMID- 1723759 TI - Peritrichous flagellation in Plesiomonas shigelloides strains. AB - In total, 131 strains of Plesiomonas shigelloides isolated from various sources were tested for peritrichous flagella by a flagella staining method. When incubated on a solid medium for 18 hr at 25 C, peritrichous flagella were demonstrated in 89 (68%) of them. With an electron microscope, the peritrichous flagella were clearly distinguished from the lophotrichous ones by their wavelength. PMID- 1723760 TI - Age-dependent hyperresponsiveness of spontaneously hypertensive rats to the pressor effects of intravenous neuropeptide Y (NPY): role of mode of peptide administration and plasma NPY-like immunoreactivity. AB - The effects of various doses of intravenously (i.v.) infused (5-min duration, 0.1 3.2 nmol/kg/min) or bolus-injected (0.1-3.2 nmol/kg) porcine and/or rat/human neuropeptide Y (NPY) on mean arterial pressure (MAP), heart rate (HR), and plasma concentrations of porcine NPY-like immunoreactivity (pNPYir) were examined in conscious, unrestrained spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats of various ages. When administered as an infusion to 12- to 17-week-old SHR, WKY, and SD rats, porcine NPY (pNPY) was more potent in increasing MAP in SHR than in either WKY or SD rats. Infusions of rat/human NPY instead of pNPY resulted in similar increases in potency in 12- to 17-week-old SHR as compared with WKY. This potency-associated hyperresponsiveness to infused pNPY was also observed when 36- to 41-week-old and 6-week-old SHR and WKY were examined, but infused NPY induced similar HR reductions in age-matched rats regardless of rat strain. Furthermore, doses of infused pNPY that elicited significantly greater pressor responses in SHR and WKY (0.32 nmol/kg/min in 12- to 17-week-old rats and 1.0 nmol/kg/min in 6-week-old rats) resulted in essentially identical plasma pNPYir concentrations in the two rat strains. In contrast, hyperresponsiveness to the MAP effects of bolus injections of pNPY in 12- to 17-week-old SHR was manifested as an increase in efficacy rather than potency, was associated with significantly smaller reductions in HR in SHR than in WKY and occurred at plasma pNPYir concentrations that were significantly larger than those required for infusion-associated hyperresponsiveness. These results are consistent with the hypothesis that NPY is an important contributor to the development and maintenance of essential hypertension. PMID- 1723761 TI - Systemic hypoxia activates a coronary vasoconstrictor reflex response that is blocked by prazosin. AB - We assessed the presence of an alpha 1-adrenoceptor-mediated coronary vasoconstrictor reflex response during acute systemic hypoxia in eight chloralose anesthetized dogs. We avoided local vasodilator responses to myocardial and coronary hypoxia and to circulating factors by perfusing the left common coronary artery at constant pressure with normoxic blood while the dogs were ventilated with 5% O2-95% N2. Left ventricular afterload was held constant by withdrawing arterial blood during hypoxia-induced peripheral vasoconstriction. Left ventricular (LV) preload, as indicated by left atrial pressure, was unchanged. beta-adrenoceptor-mediated coronary dilation and positive chronotropic and inotropic responses to hypoxia were blocked by propranolol. Para-sympathetic mediated coronary dilation and bradycardia were blocked by atropine. Under these conditions, systemic hypoxia caused a 19.7 +/- 2.1% decrease in left common coronary blood flow. Blockade of left coronary alpha 1-adrenoceptors with prazosin prevented coronary vasoconstriction during repeated systemic hypoxia. In four other similarly prepared dogs, repeated systemic hypoxia without alpha 1 adrenoceptor blockade reproducibly reduced left coronary blood flow 16.3 +/- 3.5 and 15.7 +/- 3.1%, respectively. The results of this investigation provide the first evidence of a coronary vasoconstrictor reflex response to systemic hypoxia. This response is mediated by alpha 1-adrenoceptors. PMID- 1723762 TI - Endothelium-derived nitric oxide as a mediator of acetylcholine-induced coronary vasodilation in dogs. AB - To investigate the role of endothelium-derived nitric oxide (NO) in regulation of the tonus of the coronary artery system in vivo, the effect of NG-mono-methyl-L arginine (L-NMMA), a specific inhibitor of the synthesis of NO from L-arginine, on acetylcholine (ACh)-induced coronary vasodilation was examined in open-chest, anesthetized dogs. At baseline, the infusion of ACh and adenosine into the left circumflex coronary artery (LCX) increased the LCX blood flow. Continuous infusion of L-NMMA (2 mumol/min, for 20 min) into LCX significantly attenuated ACh-induced increase in the LCX flow: percentage increases in the LCX flow by ACh (10 and 100 ng/kg) before L-NMMA were 42.0 +/- 8.7 and 137.3 +/- 12.5%, respectively, whereas those after L-NMMA were 14.0 +/- 4.9 (p less than 0.02) and 88.9 +/- 10.9% (p less than 0.005), respectively. L-NMMA showed no effect on adenosine-induced increase in the LCX flow. After the L-NMMA infusion was terminated, the effect of ACh partially returned to the baseline level. In other dogs, L-arginine (3 mg/min, for 20 min) was administered just after the L-NMMA infusion, which completely reversed the inhibitory effect of L-NMMA on ACh induced vasodilation. Endothelium-derived NO is a mediator of ACh-induced vasodilation of the coronary resistance vessels in dogs. PMID- 1723763 TI - Participation of endothelium-derived relaxing factor and role of cyclic GMP in inhibitory effects of endothelium on contractile responses elicited by alpha adrenoceptor agonists in rat aorta. AB - The participation of NO production and the role of cyclic GMP in inhibitory function of endothelium were investigated in rat aortic rings exposed to alpha adrenoceptor agonists. Both endothelium and 8-Br cyclic GMP (in endothelium denuded rings) depressed more markedly not only maximal contractions but also equipotent contractions elicited by two partial agonists (indanidine and B-HT 920) than responses to the full agonist phenylephrine. The influence of endothelium on maximal responses to the three agonists was abolished by both the nitric oxide (NO)-synthase inhibitor NG-nitro-L-arginine methylester (L-NAME, 30 microM) and by the guanylate cyclase inhibitor methylene blue (methylene blue, 0.3 and 1 microM). Both endothelium and 8-Br cyclic GMP (in endothelium-denuded rings) increased the EC50 value of phenylephrine. This effect was more pronounced in the case of endothelium (10-fold), however, than in the case of 8-Br cyclic GMP (fourfold at 30 microM), and the rightward shift produced by endothelium remained significant (twofold) in the presence of L-NAME or methylene blue. In addition, the effect of 8-Br cyclic GMP on phenylephrine-induced contractions was considerably enhanced in the presence of endothelium or after partial alkylation of receptors by phenoxybenzamine in endothelium-denuded rings. These results indicate that the L-arginine-NO-cyclic GMP pathway accounts for most of the inhibitory influence of endothelium on alpha-adrenergic responses in aortic rings. They indicate differential effects of cyclic GMP depending on the agonist and show that 8-Br cyclic GMP does not impair the basal inhibitory effect of endothelium on aortic contraction to alpha-adrenergic agonists. PMID- 1723765 TI - Cardiac electrophysiologic and inotropic actions of new and potent methanesulfonanilide class III antiarrhythmic agents in anesthetized dogs. AB - The effects of cumulative intravenous (i.v.) administration of potent and selective methanesulfonanilide class III antiarrhythmic agents on cardiac electrophysiologic and hemodynamic parameters were compared with those of D sotalol in chloralose-anesthetized dogs. The new class III agents tested were E 4031 [1-(2-(6-methyl-2-pyridyl)ethyl)-(4-methanesulfonamidobenzoyl)pipe ridine]; UK-66,914 [N-(4-(1-hydroxy-2-(4-(4-pyridinyl)-1-piperazinyl)ethyl)phenyl) methanesulfonamide], and UK-68,798 [1-(4-methanesulfonamidophenoxy)-2-(N- (4 methanesulfonamidophenethyl)-N-methylamino)ethane]. The class III agents produced significant and dose-dependent increases in ventricular refractoriness, with effective doses required to increase ventricular relative refractory period 20 ms above baseline (ED20, micrograms/kg i.v., with 95% confidence limits) of 5.2 (4.2 6.6) for UK-68,798, 17 (13-23) for E-4031, 75 (58-99) for UK-66,914, and 3,700 (2,600-5,800) for D-sotalol. Significant increases in the electrocardiographic QT and QTc intervals paralleled the increases in ventricular refractoriness for the four class III agents. Significant increases in left ventricular (LV) + dP/dt also paralleled increases in ventricular refractoriness and QT intervals for E 4031 (10-1,000 micrograms/kg i.v.), UK-66,914 (100-1,000 micrograms/kg i.v.), and UK-68,798 (30-1,000 micrograms/kg i.v.), but not for D-sotalol. No concomitant alterations in LV-dP/dt were observed for the new and potent methanesulfonanilide class III agents, resulting in significant increases in the ratio of LV + dP/dt/ dP/dt for E-4031, UK-66,914, and UK-68,798. Potent and selective methanesulfonanilide class III agents therefore may augment cardiac contractility in addition to prolonging ventricular refractoriness. PMID- 1723764 TI - Discrepancies between inotropic responses and beta-adrenoceptor characteristics after global ischemia in isolated hearts. AB - The influence of global ischemia on cardiac beta-adrenoceptors was studied in rat and guinea pig Langendorff hearts (LH), both by functional and binding experiments using the specific beta-adrenoceptor ligand (-)-[125I] iodocyanopindolol. Neither ischemia (30 or 60 min) nor postischemic reperfusion caused any change in beta-adrenoceptor density, affinity or in the beta 1/beta 2 ratio in LH of normal rats or in LH of rats pretreated with reserpine or 6 hydroxydopamine (6-OHDA), or in guinea pig LH, whereas perfusion of rat LH with 10(-5) M isoprenaline (15 min) caused the expected decrease in beta-adrenoceptor density. After ischemia, isoprenaline was no longer able to influence beta adrenoceptor density, suggesting that the internalization mechanism is impaired. In functional studies, perfusion of the rat LH with 10(-5) M isoprenaline (15 min) shifted the concentration-response curve for isoprenaline to the right. Thirty-minute global ischemia virtually abolished the inotropic but not the chronotropic response to isoprenaline. Ischemia did not impair the inotropic response to ouabain or to calcium, indicating that the contractile apparatus itself was still largely intact. Our results suggest that the contractile failure after ischemia is not caused by a decrease in beta-adrenoceptor density or by a defect in the contractile apparatus but by an impaired second-messenger system. PMID- 1723766 TI - G proteins subserve relaxations mediated by adenosine receptors in human coronary artery. AB - The coupling of the human coronary adenosine receptor to a G protein was investigated in vitro. Hearts were obtained from accidental death victims and the left anterior descending coronary artery (LAD) was taken for experimentation. Cholera toxin (CT) and pertussis toxin (PT) ADP-ribosylated proteins with Mr of 45, 49 (CT), and 41 (PT) kDa. Both processes were sensitive to GTP gamma S. In LAD rings contracted with KCl, adenosine (ADO) and its analogs 5'-N ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CAD) produced concentration-dependent relaxation. These concentration-response curves were shifted to the right significantly in the presence of the competitive ADO receptor antagonist, 8-phenyltheophylline (8-PT), indicating the involvement of ADO receptors. Treatment with NaF/AlCl3, which uncouples G protein-mediated responses, caused significant attenuation of the relaxation responses to ADO, NECA, and CAD. When the rings were incubated with CT, there was an attenuation of the relaxations produced by ADO, CAD, NECA, and isoproterenol (ISOP). Incubation with PT resulted in significant inhibition of the relaxations induced by ADO, NECA, and CAD. The results provide evidence for the presence of CT- and PT sensitive G protein(s) subserving the relaxing adenosine receptors in human coronary artery. PMID- 1723767 TI - Hypothyroidism renders protection against lethal ventricular arrhythmias in a conscious canine model of sudden death. AB - The protective effect of hypothyroidism against lethal ventricular tachyarrhythmias (VT) in the subacute phase of experimental myocardial infarction (MI) was investigated in 10 thyroidectomized dogs using a conscious model of sudden coronary death. Four weeks after surgical ablation of the thyroid, and having established biochemical hypothyroidism, anterior MI was produced by 120 min of occlusion-reperfusion of the left anterior descending coronary artery. In the subacute phase of MI, the inducibility of VT was investigated using programmed ventricular stimulation (PVS), and the effects on spontaneous development of ventricular fibrillation (VF) were studied by production of posterolateral ischemia at a site remote from the area of the previous infarction. Ischemia was produced by the passage of anodal direct current through a silver wire electrode implanted in the left circumflex coronary (LCX) artery. The results were compared to those from a cohort of 20 existing euthyroid controls that had undergone an identical experimental protocol. No differences were found in heart rate and other electrocardiographic parameters such as the PR, QRS, and QT (paced at 2.5 Hz) and the QTc interval between the hypo- and euthyroid groups. During PVS in the subacute phase of anterior MI, the measured threshold voltage and ventricular refractory periods were similar in both groups. The incidence of inducibility of VT was 100% in the euthyroid animals compared to 60% in the hypothyroid dogs, suggesting an antiarrhythmic effect of hypothyroidism. The incidence of sustained vs. nonsustained VT was similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723768 TI - On the mechanism by which doxorubicin abolishes the oscillatory events induced by Ca overload in single cardiac myocytes. AB - The mechanism by which doxorubicin (DOXO) modifies the voltage and current changes induced by Ca overload was investigated in isolated guinea pig ventricular myocytes. In the presence of norepinephrine (NE 0.1 microM), drive induces an oscillatory potential (Vos) superimposed on a prolonged depolarization (Vex): DOXO (10-50 microM) decreases or abolishes Vos but exaggerates Vex. In high [Ca]o (5.4-8.1 mM), drive induces Vos and Vex, and DOXO has the same effects as in NE. Trains of voltage-clamp steps induce Ios and Iex (the currents underlying Vos and Vex, respectively): DOXO decreases or abolishes Ios but increases Iex both in NE and high [Ca]o. DOXO does not decrease the slow inward current Isi. Caffeine (5 mM) abolishes Vos and Ios and increases Vex and Iex (as DOXO does), and adding DOXO slightly increased Vex and Iex. Ni (2 mM) (which blocks Na-Ca exchange) decreases or abolishes DOXO-induced Vex and Iex. In papillary muscles, reduced [Na]o or high [Ca]o increases force, but DOXO has little inotropic effect. Reduced [Na]o and high [Ca]o also increase force less in the presence of DOXO. We conclude that DOXO abolishes Vos and Ios but not by blocking the adrenergic receptor, decreasing Isi, or inhibiting Na-Ca exchange. Instead, DOXO may act by impairing uptake and release of Ca by the sarcoplasmic reticulum membrane. PMID- 1723769 TI - SK&F 86002 inhibits tumor necrosis factor formation and improves survival in endotoxemic rats. AB - We evaluated the effects of SK&F 86002, a dual 5-lipoxygenase/cyclooxygenase inhibitor, on the responses to 30 mg/kg Escherichia coli endotoxin (bacterial lipopolysaccharides, LPS) in conscious male Sprague-Dawley rats. Injection of LPS increased serum tumor necrosis factor (TNF alpha) activity from 20 U/ml (baseline) to 1,066 +/- 430 and 2,825 +/- 1,155 U/ml (n = 9) at 30 min and 1 h after administration of LPS, respectively (p less than 0.01 as compared with the vehicle control group). This dose of LPS reduced the survival rate to 9% at 48 h (mean survival time 9.7 +/- 2.7 h), increased heart rate (HR) to 494 +/- 18 beats/min, increased the hematocrit to 56 +/- 2 vol%, reduced the circulating platelet count at 6 h to 40% of the initial value, and produced leukopenia of 30% of the initial value at 1 h, with recovery to the initial value at 6 h. Administration of 30 mg/kg SK&F 86002 (intragastric, i.g.) 1 h before injection of LPS reduced serum TNF alpha concentrations to 92 +/- 58 and 184 +/- 117 U/ml at 30 min and 1 h (p less than 0.05), respectively, and also reduced the hemoconcentration. The survival rate was improved to 60% (mean survival time 38.1 +/- 4.3 h; p less than 0.05), although there was no effect on the hemodynamic responses to LPS. SK&F 86002 significantly reduced thrombocytopenia at 30 min and 1 h (p less than 0.05), but not at 3 and 6 h, and had no effect on changes in white blood cell (WBC) count.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723770 TI - Reduction in myocardial ischemic/reperfusion injury and neutrophil accumulation after therapeutic administration of streptokinase. AB - The benefit of thrombolytic agents to reduce myocardial infarct size, improve left ventricular (LV) function, and prolong survival in human subjects is generally recognized, although the precise mechanism is poorly defined. This study was designed to evaluate the cardioprotective effects of streptokinase (SK) in rats, a species less responsive to plasminogen activators, using a model of mechanical occlusion and release of the left coronary artery. Myocardial injury and polymorphonuclear leukocyte (PMN) infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the LV free wall (LVFW). After coronary artery occlusion for 0.5 h and reperfusion for 24 h (myocardial ischemia, MI/R), CPK specific activity decreased from 7.0 +/- 0.3 U/mg protein in the sham + vehicle group to 5.6 +/- 0.5 U/mg protein in the MI/R + vehicle group (n = 19, p less than 0.01), while MPO activity increased from 0.14 +/- 0.03 U/g tissue in the sham + vehicle group to 2.8 +/- 0.7 U/g in the MI/R + vehicle group (p less than 0.001). Administration of SK (100,000 IU/kg + 50,000 IU/kg/h for 2 h beginning 15 min before coronary artery reperfusion) reduced the loss of CPK specific activity from reperfused myocardium (6.8 +/- 0.5 U/mg protein, n = 23, p less than 0.05 as compared with the MI/R + vehicle group) and attenuated the increase in MPO activity (1.3 +/- 0.4 U/g tissue, p less than 0.05 as compared with the MI/R + vehicle group). This dose of SK did not change plasma fibrinogen concentration, slightly reduced plasminogen activity (i.e., 20% from control value), and markedly reduced alpha 2-antiplasmin activity (i.e., 60% from control values). A lower dose of SK (i.e., 10,000 IU/kg + 5,000 IU/kg/h for 2 h) did not reduce myocardial injury, did not attenuate the increase in MPO activity, and had no effect on the measured hemostatic parameters. Survival in all MI/R groups ranged from 62 to 66%, and there were no differences in survival between any of the groups (p greater than 0.05). In a model of arachidonic acid-induced rat hindpaw inflammation, SK had no effect on the increase in MPO activity, suggesting that the increase in myocardial MPO activity was not due to a direct effect on inflammatory cell accumulation. In in vitro studies, SK (1-1,000 U/ml) did not scavenge superoxide anion produced by purine (10 mM) and xanthine oxidase (10 mU/ml), nor did it reduce superoxide release, beta-glucuronidase release, or neutrophil aggregation of rabbit peritoneal neutrophils activated with fMLP.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1723771 TI - Reduction in myocardial hemorrhage and the extent of necrosis by gallopamil (D600) in dogs with coronary artery reperfusion. AB - To determine whether gallopamil (D600), a methoxy derivative of verapamil, administered immediately before coronary artery reperfusion reduces the extent of myocardial hemorrhage and necrosis, the left anterior descending coronary artery was occluded for 3 h and reperfused for 3 h in anesthetized, open-chest dogs. To quantify the extent of the hypoperfused zone (HZ), 99mTc-labeled albumin microspheres were injected into the left atrium 1 min after occlusion. Five minutes before reperfusion, dogs were randomly assigned to a control group or a gallopamil-treated group that immediately after assignment received 0.08 mg/kg gallopamil followed by a continuous infusion of 0.2 mg/kg/h for 3 h. Three hours after reperfusion, the left ventricle was cut into slices for triphenyltetrazolium chloride staining and autoradiography. There were no differences in the extent of the HZ between the two groups. Gallopamil significantly reduced the extent of myocardial necrosis from 81.3 +/- 4.2% (n = 8) of the HZ in the control to 46.1 +/- 13.1% (n = 9, p less than 0.05) in the treated group. The extent of gross hemorrhage was significantly smaller in the gallopamil-treated group (1.3 +/- 0.9% of the left ventricle or 3.1 +/- 1.8% of the HZ, p less than 0.01) as compared with the control group (6.2 +/- 1.4% of the left ventricle or 20.0 +/- 4.6% of the HZ). Thus, gallopamil administered immediately before coronary artery reperfusion limited infarct size and reduced the extent of myocardial hemorrhage. PMID- 1723772 TI - Lack of negative inotropic effects of the new calcium antagonist Ro 40-5967 in patients with stable angina pectoris. AB - We screened the antiischemic, hemodynamic, and inotropic effects of different dosages of the new calcium channel blocker Ro 40-5967 in 65 patients with stable effort-induced angina pectoris. In a double-blind way, patients were randomized to recieve a single oral dose of 50, 100, or 200 mg Ro 40-5967 or placebo, given as a drinking solution. Left ventricular ejection fraction (LVEF), blood pressure (BP), and heart rate (HR) were measured at rest and during a supine bicycle exercise test on day 0 (baseline) and 2 h after drug intake on day 1. Twenty-four hours later, the bicycle exercise test was repeated. Ro 40-5967 improved exercise duration and resting LVEF. After 200 mg, exercise time increased significantly from 8.4 +/- 0.8 min (mean +/- SEM) to 9.6 +/- 0.7 min (p = 0.018), and LVEF at rest increased from 54.5 +/- 2.2 to 58.1 +/- 2.6% (p = 0.045). Time to 0.1 mV ST segment depression increased significantly from 4.3 +/- 0.8 to 5.5 +/- 0.9 min in the 100-mg group (p = 0.013) and from 4.3 +/- 1.3 to 5.4 +/- 1.5 min in the 200 mg group (p = 0.027). Maximum ST-segment depression decreased significantly at all dose levels (p = 0.01), with the maximum decrease noted in the 200-mg group (from 0.21 +/- 0.03 to 0.15 +/- 0.02 mV, p = 0.004). BP, HR, and rate-pressure product did not change significantly at rest or at maximum exercise. A single dose of Ro 40-5967 has antiischemic properties in patients with stable angina pectoris, with maximum effects obtained after 200 mg. No signs of negative inotropy were noted, and the drug was well tolerated. PMID- 1723773 TI - Impact of diagnosis and treatment of hypertension on quality of life: a double blind, randomized, placebo-controlled, cross-over study of betaxolol. AB - In an experimental study, 150 general practitioners studied 468 apparently healthy subjects whose blood pressure (BP) level was unknown or had not been measured for 1 year. The study lasted for 10 weeks. If BP was greater than 95 mm Hg on the first two visits, subjects were randomized into two experimental groups for 8 weeks, with cross-over from betaxolol to placebo and vice versa after 4 weeks. Quality of life was measured at visits 1, 3 (2 weeks), 5 (6 weeks), and 7 (10 weeks) in five ways: well-being, physical state, sexual functioning, sleep, and cognitive acuity. BP level appeared to be effectively controlled by betaxolol as compared with placebo. The results show that no effects on quality of life could be detected by labeling subjects as hypertensives. Equally, almost no effects of active treatment could be established. Learning effects on the two cognitive acuity measurements used were attenuated by betaxolol, however. Apparently, in a carefully controlled study, the effects of increased medical attention and care outweight the potentially negative effects of labeling and treatment. PMID- 1723774 TI - Prevention of cyclosporine A-induced vascular toxicity by pentoxifylline. AB - To determine whether an in vivo treatment with pentoxifylline (PTX) can prevent the vascular toxicity of cyclosporine A (Cx), three groups of rats were studied in parallel. The first group received daily injections of Cx (20 mg/kg intramuscularly) and pentoxifylline (80 mg/kg intraperitoneally) for 7 days, the second group was treated with Cx only, and the third group served as control (vehicle treatment). Cx serum levels were similar in groups 1 and 2. In thoracic aortic rings isolated from Cx group (group 2), the concentration-response curves to phenylephrine were potentiated: there was a significant leftward shift (p less than 0.001 vs. control) in the EC50 values and an increase in the maximal responses (p less than 0.05). After mechanical removal of the endothelium or inhibition of endothelium-derived relaxing factor formation (incubation with NG monomethyl-L-arginine, L-NMMA), this enhanced responsiveness to phenylephrine persisted. In preparations from the same group (group 2), the endothelium dependent relaxations to acetylcholine (ACh) were decreased whereas the endothelium-independent relaxations to nitroprusside (NTP 0.01-10 nM) and forskolin (1 nM) were slightly attenuated but without changes in the maximal response. In the group cotreated with Cx and PTX (group 1), the responses to ACh, NTP, and forskolin were not different from controls whereas the greater responsiveness to phenylephrine was only partially attenuated. In vivo cotreatment with PTX may prevent the endothelial dysfunction and the functional changes in smooth muscle cells induced by Cx. PMID- 1723775 TI - Electrophysiological effects of CD-349, a dihydropyridine-type calcium antagonist, on goat cardiac Purkinje fibers. AB - We examined the calcium antagonistic action of CD-349, a dihydropyridine derivative, on goat cardiac Purkinje fibers using the two-microelectrode voltage clamp method. CD-349 at a concentration of 10(-5) M shortened the action potential duration without changing the maximum rise in the action potential (Vmax) in goat Purkinje fibers. CD-349 at 3 x 10(-7) to 3 x 10(-6) M inhibited the slow inward current (Isi) in a concentration-dependent manner. At the holding potential of -55 mV, CD-349 exerted a tonic block of Isi, and, furthermore, it exerted a use-dependent block at a frequency range of 1 Hz, but it did not exert a use-dependent block at 0.5 and 0.2 Hz. This may be because CD-349 delayed the recovery process from the inactivation of Isi. The amplitude of the block of Isi was larger at the holding potential of -45 mV than at -55 mV. The inactivation curve of Isi shifted toward a negative potential in the presence of CD-349. Nifedipine also exerted a tonic block of Isi. The onset of the action of nifedipine was quicker than that of CD-349 or nitrendipine. A use-dependent block at 1 Hz and delay of the recovery process from inactivation was also observed with nifedipine. The inactivation curve shifted toward the negative potential with nifedipine. On washout of the drugs, the effects of CD-349 or nitrendipine were not readily reversed compared with those of nifedipine. CD-349 had no effect on either inward rectifying (IK1) or delayed outward potassium (IK) or hyperpolarization-activated inward (If) currents. These observations suggest that, in cardiac tissues, CD-349 selectively inhibits the calcium current, presumably by acting on the inactivated channel. PMID- 1723776 TI - Comparison of three intracellular markers for combined electrophysiological, morphological and immunohistochemical analyses. AB - Hypothalamic paraventricular and supraoptic neurons were recorded intracellularly in coronal slices and injected with Lucifer yellow, ethidium bromide or biocytin. Electrical properties, morphological staining and neurophysin immunohistochemistry were compared among the 3 markers. Lucifer yellow electrodes had a high resistance and frequently blocked during experiments. Neurons recorded with Lucifer yellow electrodes had low input resistances and low-amplitude, broad spikes. Lucifer yellow labeling in whole mount was highly fluorescent, revealing distal dendrites and axons. Of cells injected with Lucifer yellow, 64% were recovered but were faint after immunohistochemical processing. Recordings with ethidium bromide electrodes were similar to controls, although electrode blockage sometimes occurred. Only somata and proximal dendrites of ethidium bromide-filled neurons were visible in whole-mount. Forty percent of cells injected with ethidium bromide were recovered after immunohistochemical processing; these were invariably faint. Recordings with biocytin-filled electrodes were similar to control recordings. Biocytin-filled, HRP-labeled cells showed distal dendrites and often dendritic spines and axons in 50-75-microns sections. Seventy percent of biocytin-injected cells labeled with fluorescent markers were recovered and remained strongly labeled after immunohistochemical processing. Biocytin had the best electrical and staining properties for combined electrophysiological and anatomical studies. PMID- 1723777 TI - A procedure for the simultaneous visualization of two anterograde and different retrograde fluorescent tracers. Application to the study of the afferent-efferent organization of thalamic anterior intralaminar nuclei. AB - The present report describes a method for the simultaneous visualization, in the same structure, of two different sets of afferent pathways and the neurons of origin of some efferent projections. This method has been applied in the cat for studying, in the thalamic anterior intralaminar nuclei, the topographical relationships of afferent arising from the spinal cord and deep cerebellar nuclei with neurons projecting to different cortical areas. Spino- and cerebello thalamic terminals were anterogradely labeled by injections of the fluorescent dyes fast blue (FB) and 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) in the spinal cord and cerebellum. Thalamo-cortical neurons were retrogradely labeled by injections of fluorescent tracers in the precruciate and anterior suprasylvian cortices. The findings show that spinal and cerebellar afferent fibers and the cells of origin of intralaminocortical projections are organized in a clear modular manner and indicate that the method used here is suitable for analyzing simultaneously, in light microscopy, multiple input-output interrelationships of a single structure. PMID- 1723778 TI - A fast staining method for CNS slices. AB - We describe a staining technique for quick observation of the microanatomy of the vertebrate CNS. Successive soaking of the sections in tannic acid and ferric chloride produces a black colour in the gray matter. The procedure takes 5 min and is useful for identifying electrode penetrations, localization of lesions or teaching neuroanatomy. PMID- 1723779 TI - Gastric tumors. PMID- 1723780 TI - Prediction of alpha helices and T cell-presented sequences in proteins with algorithms based on strip-of-helix hydrophobicity index. AB - Recurrent aliphatic hydrophobic amino acids which occur in the sequence of a protein or a peptide at positions which form an axial, hydrophobic strip when the sequence is coiled as an alpha helix might stabilize coiling against hydrophobic surfaces. That effect can lead to helix formation against hydrophobic cores of nascent proteins or excised T cell-presented peptides and to protease protection and scavenging for presentation by MHC molecules. Such consensus sequences of recurrent hydrophobicity creating a scavenger "S" site might overlap to varying degrees the T cell-presented "T" epitope which actually sits in the antigen binding site of a MHC molecules, as long as a cleavage "C" site does not fall between them when they are relatively separated. Cooperatively among the residues in an axial, hydrophobic strip to stabilize helix formation is reflected in the SOHHI, which is the mean hydrophobicity of residues in such potential strips. Algorithms based on the SOHHI, with additional considerations related to length and caps, lead to sensitive and efficient predictions of structural helices and of T cell-presented epitopes. In experimental tests of these ideas, the SOHHI was found to correlate to helical coiling of amphiphilic peptides in the presence of lipid vesicles. These principles lead to hypotheses to alter the potency and range of MHC restriction of peptide vaccines or to decrease the immunogenicity of therapeutic proteins. PMID- 1723781 TI - Systematic mutational analyses of protein-protein interfaces. PMID- 1723782 TI - Production and purification of antibodies against rat liver P450 enzymes. PMID- 1723783 TI - Quantitation of related gene products by nuclear run-on and northern blot analysis. PMID- 1723784 TI - Intratypic variations in neutralizable epitopes among herpes simplex virus type 2 isolates. AB - Intratypic variation among 94 isolates of herpes simplex virus type 2 (HSV-2) was investigated using 4 different monoclonal antibodies (MAbs). By neutralization test, these MAbs appeared to be directed to at least 2 distinct epitopes on the viral glycoprotein D (gD), i.e., 6G6.G9 and 6E8.F11 which did not require complement (C-MAb) and gD-105 and gD-110 whose neutralizing activities could be enhanced by complement (C+MAb). The C-MAb pairs each separately could detect significant intratypic variations among the isolates. Whether these variations also existed in the gD epitope(s) recognized by C+MAbs remains to be elucidated. The results suggested that intratypic variation occurred on at least one of the neutralizable (thus related to protective immunity) epitopes on gD of HSV-2. PMID- 1723785 TI - Protease inhibitors prevent the development of human rotavirus-induced diarrhea in suckling mice. AB - Oral inoculation of human rotavirus MO strain (serotype 3) into 5-day-old BALB/c mice caused gastroenteritis characterized by diarrhea (90% on the average, on day 2). Using this animal model, preventive effect of antiviral agents on the development of rotavirus-induced diarrhea was examined. The infectivity of human rotavirus was enhanced by treatment with protease in vitro. A cysteine protease inhibitor, E-64-c, was given orally at 12 hr and 24 hr after MO infection. Oral administration of 0.3 mg of E-64-c decreased the diarrhea ratio to 17.5% on day 2 and to 10% on day 3. Oral administration of 0.15 mg of cysteine protease inhibitor, ovocystatin, completely prevented the diarrhea on day 2. Serine protease inhibitor, aprotinin (0.15 mg x 2), also prevented the diarrhea on day 2 to 14.3%. These protease inhibitors were nontoxic in vitro and to suckling mice. The histopathological changes in the small intestine due to infection recovered 2 days after MO infection in mice treated with E-64-c and ovocystatin. These results suggest that protease inhibitors are protective agents for human rotavirus infection by inhibiting proteases required for viral replication. PMID- 1723786 TI - Keratinolytic and keratinophilic fungi in the soils of Papua New Guinea. AB - A study was made of soil samples collected during an expedition to the Highlands of Papua New Guinea. Fungi were isolated from the samples by the method of hair baiting (To-Ka-Va). Of the 33 species isolated, about half showed keratinolytic activity. Such activity is previously unreported for Mucor hiemalis f. hiemalis, Myrothecium roridum, Paecilomyces carneus, P. marquandii, Penicillium brevicompactum, Rhinocladiella mansonii and Verticillium lecanii. The species most active keratinolytically were Chrysosporium an. Arthroderma cuniculi, C. an. A. curreyi, C. indicum, Myceliophthora vellerea and Trichophyton ajelloi. The spectrum of fungi with keratinolytic activity isolated from the different sites differed considerably according to the frequency of use by man, heaviest use being correlated with greatest activity. The pH of the soil (varying from 5.8 7.5) had little influence on the type of such fungi isolated. PMID- 1723787 TI - Estrogen induces ultrastructural changes in progesterone receptor-containing GABA neurons of the primate hypothalamus. AB - Estrogen affects gonadotrophin levels and sex behavior in monkeys. This action could be via inhibitory GABA-ergic neurons in the hypothalamus. We tested for direct estrogen actions on such neurons. Seven days after ovariectomy (OVX) or OVX + estrogen treatment (10 mg estradiol valerate in 1 ml sesame oil s.c. on the day of OVX), light- and electron-microscopic double immunostaining procedures were used for simultaneous visualization of immunoreactivity for progesterone receptors (PR) and glutamic acid decarboxylase (GAD), and to detect ultrastructural changes in PR-containing neurons in the arcuate and ventromedial hypothalamic nuclei of colchicine- and noncolchicine-treated African green monkeys (Cercopithecus aethiops). Immunoreactivity for PR was found only in cell nuclei, and estrogen treatment enhanced the intensity of the immunostaining: in estrogen-treated monkeys in the arcuate nucleus 62%, while in the ventromedial nucleus 42% of the neurons contained PR-immunoreactive nuclei. All of the PR containing neurons were immunopositive for GAD in colchicine-pretreated monkeys. OVX induced whorl body formation, while estrogen treatment of OVX animals resulted in a large number of nematosomes. While all of the whorl bodies and the majority of nematosomes were observed in PR-immunopositive GAD neurons, nematosomes were also found in non-PR-containing GAD-immunoreactive cells. PMID- 1723788 TI - Preproenkephalin A gene expression in rat pineal. AB - Preproenkephalin A (ppEnk) mRNA has been identified in extracts of rat pineal gland using oligonucleotides complementary to nonoverlapping regions of this mRNA. The mRNA has an apparent molecular size of 1.4 kb, equivalent to that found in rat brain. Slot-blot analysis, used to determine the relative amount of ppEnk mRNA in several tissues, demonstrated that the pineal contains 70% of the concentration of ppEnk mRNA found in the hypothalamus. Using a highly specific radioimmunoassay, rat pineals were demonstrated to contain approximately 3.5 pg methionine-enkephalin (Met-Enk) per gland. Following enzymatic digestion to release cryptic Met-Enk from larger molecular forms, pineals contained approximately 160 pg Met-Enk per gland. Gel filtration analysis of pineal extracts demonstrated that the peak of the cryptic Met-Enk has a molecular weight of 32 kDa (presumably proenkephalin). These results suggest that in rat pineal ppEnk gene products may contribute to pineal functioning. PMID- 1723789 TI - Species differences in the distribution and coexistence ratio of serotonin and substance P in the monkey, cat, rat and chick spinal cord. AB - Serotoninergic raphe-spinal motor neuron projections exhibit wide species differences in both innervation pattern and coexistence of serotonin and substance P. The coexistence ratios vary widely ranging from more than 80% (rat) to less than 1% (chick). Serotonin and substance P positive fibers are also unevenly distributed in the ventral horn of different species: dense clusters of serotonin and substance P positive fibers were preferentially located in the motor neuron pools of extensor muscles of the hip joint (chick) as well as antigravity muscles of the forelimb (cat and rat). PMID- 1723790 TI - Argiotoxin636 inhibits NMDA-activated ion channels expressed in Xenopus oocytes. AB - Argiotoxin636, a component of the spider venom of argiope species, was chemically synthesized together with a number of derivatives in order to analyse their blocking activity on mammalian glutamate receptors. Xenopus laevis oocytes injected with rat brain mRNA served as assay system. The results showed that argiotoxin636 had a higher affinity for N-methyl-D-aspartate (NMDA) than for kainate receptors, blocking the corresponding ion channels in a voltage-dependent manner. Modifications of the polyamine tail or the terminal arginine residue strongly reduced the blocking potency. The iodinated monohydroxyl phenylderivatives, however, retained their NMDA-selective binding and could serve as non-competitive antagonists for radioligand binding assays aiding in the biochemical isolation of glutamate receptors. PMID- 1723791 TI - Inhibition of T-type calcium currents by dihydropyridines in mouse embryonic dorsal root ganglion neurons. AB - The effects of dihydropyridines (DHPs) normally considered to be specific for L type calcium channels were studied on the T-type Ca channel current of acutely isolated dorsal root ganglion (DRG) neurons taken from 13-day-old (E13) mouse embryos. Potent but reversible inhibitory effects of the DHP nicardipine were found in the micromolar range. For example, 5 microM nicardipine suppressed 93 +/ 5% of T-type currents. In comparison, other classical DHPs such as nifedipine, PN 200-110 and nitrendipine had only weak effects (less than 20% inhibition) at the same concentration. The inhibition by nicardipine was found slightly to be voltage dependent and the drug induced a leftward shift in the steady-state inactivation. The DHP agonist (-)-Bay K 8644, which dramatically increased the L type current, weakly decreased T-type Ca currents (17 +/- 8% at 5 microM). In conclusion, neuronal T-type Ca channels may be potential targets for some dihydropyridines. This property is not only a feature of the central nervous system (J. Physiol., 412 (1989) 181-195) and can be extended to peripheral neurons. PMID- 1723792 TI - Abnormal production of interleukin-1 by microglia from trisomy 16 mice. AB - The production of interleukin-1 (IL-1) was examined in cultured CNS microglia obtained from trisomy 16 (Ts16) fetal mouse brain, a model system for studies relevant to Down syndrome (DS). When compared to microglia from their normal littermates, Ts16 microglia produced significantly higher levels of IL-1 activity both before and following stimulation with lipopolysaccharide (LPS). IL-1 release was stimulated by alpha/beta interferon (IFN) in the normal but not Ts16 microglial cultures. The overall level of IL-1 production in normal littermates, however, was still less than that seen in Ts16. Thus, microglia from Ts16 mice may function in an inappropriate manner and, if this abnormality occurs in vivo, may have wide ranging effects on a developing nervous system. PMID- 1723793 TI - Histochemistry and electron-microscopic observation in two cases of granular dystrophy of the cornea. AB - We report on 2 cases of granular dystrophy (Groenouw type I). We studied the nature and the ultrastructure of the typical stromal deposits by means of histochemical methods and of electron-microscopic examination. These deposits did not stain with Alcian blue, periodic acid-Schiff, Mallory, van Gieson, von Kossa or Congo red so that they are not constituted of proteoglycans, collagen, calcium or amyloid. They probably contain a proteic material. At the electron-microscopic level we observed electron-dense material which was made up of filaments immersed into an amorphous matrix. PMID- 1723794 TI - Interdigitating cells in the peritumoral infiltrate of laryngeal carcinomas: an immunocytochemical and ultrastructural study. AB - Interdigitating cells (IDCs) have been found in the peritumoral infiltrate of 18 patients with squamous cell carcinoma of the larynx. These cells have a dendritic shape and are characterized by the expression of S-100 protein and CD1a antigens. By electron microscopy, these cells are seen to establish intimate contacts with the apposed lymphocytes, which sometimes show signs of functional activation and proliferation. These findings indicate that IDCs may play a role in setting up a T-cell immune reaction against neoplastic cells, which may influence the biological behaviour and/or local growth of the tumour. Moreover, monocytes and cells with intermediate features between monocytes and IDCs are also found in the peritumoral infiltrate, thus suggesting that IDCs differentiate in situ from monocytic precursors, possibly under the influence of either tumour-derived factors or the local lymphoid microenvironment. PMID- 1723795 TI - T-lymphoblastic lymphoma arising in the small intestine. AB - The authors report the clinical, pathological and immunological features of a case of T-lymphoblastic lymphoma presenting with protein-losing enteropathy. There was extensive multifocal involvement of the duodenum, jejunum and ileum. The mediastinum was not enlarged; the peripheral blood picture and bilateral bone marrow trephine biopsies were unremarkable. The tumor cells were positive for terminal deoxynucleotide transferase, CD3, CD2, CD7 and CD10; they were negative for CD1, CD5, CD4, CD8 and HLA-DR. The immunophenotype was that of an immature thymic T-cell. Monocytic and B-cell markers were negative. Despite initial dose reduction in chemotherapy, the patient still developed massive intestinal hemorrhage and succumbed 2 wks after treatment. Postmortem examination confirmed absence of thymic involvement. The overall picture strongly suggests a primary intestinal origin of this T-lymphoblastic lymphoma which contradicts the conventional wisdom that T-lymphoblastic lymphoma arises in the thymus from primitive cortical lymphocytes before rapidly disseminating. PMID- 1723796 TI - Cloning and sequence analysis of the common alpha-subunit complementary deoxyribonucleic acid of turkey pituitary glycoprotein hormones. AB - Two cDNA clones of nearly full length that encode the turkey pituitary common alpha-subunit glycoprotein hormone have been isolated from a pituitary cDNA library and their nucleotide sequences have been determined. The longer alpha subunit clone was 777 bp in length. It contained 88 bp of the 5'-untranslated region (UTR), an open reading frame of 360 bp (that encodes the turkey alpha subunit 24 amino acid leader polypeptide fragment and 96 amino acid apoprotein), and a 312-bp 3'-UTR followed by a 17-bp poly A tract. When the nucleotide sequence of the turkey alpha-subunit was compared with the sequence of the chicken alpha-subunit clone, the coding region was greater than 98% homologous, but was only 69 to 76% homologous when compared with mammalian alpha-subunit sequences. Northern blot analysis showed an approximate 800 bp processed transcript that hybridized to the labeled turkey alpha-subunit cDNA. There was a greater than fourfold up-regulation of the steady-state levels of turkey alpha subunit transcription when intact cultured pituitaries were treated with chicken gonadotropin-releasing hormone I. PMID- 1723797 TI - Acute effects of 3,4-methylenedioxymethamphetamine (MDMA) on monoamines in rat caudate. AB - Extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT) were assayed in the caudate of freely moving rats using microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-EC) to detect changes in their release. Dialysates were assayed at 20-minute intervals for four hours after an intraperitoneal (IP) injection of MDMA (10 mg/kg). In a separate study to determine MDMA effects on total caudate levels of the above neurochemicals, animals were injected IP with MDMA (10 mg/kg) and then sacrificed at 20, 60, 120 and 180 minutes after treatment. Brains were quickly removed, and caudate nuclei were dissected for neurochemical analysis using HPLC-EC. MDMA elicited an amphetamine-like increase in DA release, followed by an increase in DA content. DOPAC and HVA were both reduced in homogenate. 5-HT release was also increased, followed by a drop in caudate homogenate levels by three hours. DA extracellular content was 686% of control at 80 minutes; caudate homogenate levels were 122% at 120 minutes. 5-HT extracellular release was 123% at 20 minutes, then decreased thereafter. It is concluded that the acute effect of MDMA on caudate is at least as great on the DA as it is on the 5-HT system. PMID- 1723798 TI - Region-selective reduction of brain serotonin turnover rate and serotonin agonist induced behavior in mice treated with clonazepam. AB - Evidence supports a complex interaction between benzodiazepines and the central serotonergic system. This study attempts to correlate biochemical changes in the serotonin (5HT) system induced by clonazepam (CLON) with the behavioural response to a 5HT agonist. The acute administration of CLON to mice produced a time dependent decrease in 5HT turnover rate in the raphe area (dorsal and medial raphe nuclei) and modified the serotonergic syndrome induced by 5-methoxy-N,N, dimethyltryptamine (DMT). One hour after CLON administration, a dose-dependent increase in 5HT concentration was found in the raphe area, while 5 hydroxyindoleacetic acid (5HIAA) levels remained stable, leading to an increase in 5HT/5HIAA ratio, indicative of reduced 5HT turnover rate. No significant changes were detected in the frontal cortex of CLON-treated mice. After 4 days of CLON treatment, the 5HT turnover rate was still decreased in the raphe area and unchanged in the frontal cortex. Acute CLON administration produced dose dependent alterations in locomotor activity, not observed after subchronic administration. Lateral head weaving, a motor manifestation of the serotonergic syndrome produced by DMT, was less intense in CLON-treated animals. The modifications in the 5HT system induced by CLON are region selective, suggesting differences in the receptors implicated in the interaction. Altered synaptic availability of 5HT as a result of CLON administration may be responsible for the differential response to DMT in control and CLON-treated mice. PMID- 1723799 TI - Vasoactive intestinal polypeptide (VIP) potentiates the behavioral effect of substance P intrathecal administration. AB - Intrathecal (IT) injection of 20 micrograms substance P (SP) induced a behavioral syndrome consisting of scratching and biting the flanks (83% and 57%, respectively, of 48 rats), and distress-like vocalization (42% of 26 rats tested) in response to a previously innocuous tactile stimulus with a von Frey fiber (allodynia). These behavioral events following SP were of short latency (1-2 min) and duration (around 10 min). Injection IT of 5 micrograms, but not 1 microgram, of vasoactive intestinal polypeptide (VIP), concurrently with SP, significantly increased the frequency of both scratching and biting bouts over that produced by SP alone. VIP IT alone (1 or 5 micrograms) did not stimulate scratching-biting, but induced allodynia in a significant proportion of rats. PMID- 1723800 TI - Muscimol injections into the median raphe nucleus increase serum ACTH and corticosterone concentrations via a nonserotonergic mechanism. AB - Midbrain raphe serotonin (5-HT) neurons can influence the pituitary-adrenal axis. The midbrain raphe nuclei also contain a number of non-5-HT neurons, including gamma-aminobutyric acid (GABA) interneurons which can modulate 5-HT neuronal activity. We investigated the effects of intraraphe injections of the GABAA agonist, muscimol, on serum adrenocorticotropin hormone (ACTH) and corticosterone concentrations. Rats were infused with muscimol (0, 25, 50, and 100 ng in 0.5 microliters saline) into the median raphe nucleus (MR). The animals were killed 30 min later, and trunk blood was collected for measurement of serum concentrations of ACTH and corticosterone by radioimmunoassay. Muscimol dose dependently increased plasma concentrations of these two pituitary-adrenal hormones. In order to determine the role of MR 5-HT neurons in these effects, separate groups of implanted animals were infused with either the serotonergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) or ascorbic acid vehicle into the MR. Two weeks later, the animals were infused with muscimol (100 ng in 0.5 microliters) and sacrificed as above. Treatment with 5,7-DHT, which markedly reduced hippocampal concentrations of 5-HT (-83%) and 5-HIAA (-73%), did not block intra-MR muscimol-induced elevations in ACTH and corticosterone. Thus, 5-HT neurons within the MR apparently do not mediate the increased activity of the pituitary-adrenal axis produced by stimulation of MR GABAA receptors. PMID- 1723801 TI - The neurochemical and stimulatory effects of putative metabolites of 3,4 methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in rats. AB - Rats were injected SC with a dose of 10 mg/kg (as base) of 3,4 methylenedioxyamphetamine (MDA), or 3,4-methylenedioxymethamphetamine (MDMA), 4 hydroxy-3-methoxyamphetamine, alpha-methyldopamine and alpha methylnorepinephrine, metabolites of MDA, and alpha-methylepinephrine, a putative metabolite of MDMA, twice daily for either 5 or 7 consecutive doses. The rats were killed 24 h after the last injection and monoamines in discrete brain regions were assayed. MDA, MDMA, 4-hydroxy-3-methoxyamphetamine and alpha methyldopamine, but not alpha-methylepinephrine, decreased the concentration of serotonin (5-HT) in the frontal cortex. MDA and MDMA, but not 4-hydroxy-3 methoxyamphetamine, alpha-methyldopamine and alpha-methylepinephrine, also decreased the concentration of 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortexes. In stimulatory studies, MDA and MDMA, but not their metabolites except alpha-methylepinephrine, which increased activity at 15 and 30 min, increased locomotor activity from 15 to 180 min following the drug administration. PMID- 1723802 TI - The effects of repeated administration of MDMA on the expression of sexual behavior in the male rat. AB - 3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") is a potent neurotoxin which preferentially produces 5-HT nerve terminal degeneration in the CNS in both rodents and primates. Timely research on the behavioral effects of acute and long term treatment of MDMA is critical due to the neuropathological effects of MDMA and its abuse liability. Presently, there are no published reports that have systematically examined the effects of acute or chronic treatment of MDMA on animal sexual behavior. Accordingly, the effects of repeated systemic administration of MDMA on a variety of parameters of male sexual behavior in sexually vigorous male rats were studied. Treatment consisted of subcutaneous injections of MDMA (40 mg/kg) or saline (1 ml/kg) every 12 hours for 4 consecutive days. In addition, neurochemical assessments of brain 5-HT and 5-HIAA depletion following repeated MDMA treatment were also conducted using reverse phase liquid chromatography. The results of this study revealed that repeated systemic administration of MDMA to sexually vigorous male rats produced a transient disruption of the expression of male copulatory behavior. In addition, in MDMA-treated males that did display copulatory behavior, both the ejaculation latency and postejaculatory interval were dramatically lengthened when compared to saline injected controls. Surprisingly, one week after the first behavioral test, copulatory behavior in MDMA treated rats appeared unaffected despite a marked depletion of 5-HT and 5-HIAA content in the striatum, and hippocampus. PMID- 1723803 TI - [The standardization of colloid osmotic pressure of blood substitutes with a perfluorocarbon base]. AB - Besides a number of other characteristic features the colloidal osmotic pressure of blood substitutes is of importance, since it regulates the physiological balance between the intra- and extravascular fluid content. The colloidal osmotic pressure in blood is determined predominantly by the albumin fraction. The entropy increase on diluting albumin which, can't pass semipermeable vascular walls of the intravascular substances are used in blood substitutes to maintain the colloidal osmotic pressure. Their selection and appropriate are of great importance for the efficiency of blood substitutes. PMID- 1723804 TI - Profibrinolytic and anticoagulant properties of the pentosan polysulphate derivative bego 0391. PMID- 1723805 TI - Neurogenic function of the diabetic rat bladder: alteration by calcium channel effectors. AB - The in vitro effects of a calcium channel antagonist (nifedipine) and agonist (BAY K8644) on the neurogenic responses of the streptozotocin-induced diabetic rat bladder were investigated. The bladder body and bladder base were studied separately. There were no significant differences in neurogenic responses in diabetic bladder body compared to control body, but the diabetic bladder base demonstrated an increased contractile response at each frequency compared to control base. The rate of contractile response was similar in controls and diabetics but was significantly different between body and base. Although declining with time, contractile responses in the diabetic bladder body and base were increased from control in the absence of extracellular calcium. Differences were found in effects upon maximum responses between diabetic and control tissues treated with nifedipine and BAY K8644. BAY K8644 did not completely reverse the effect of nifedipine on the neurogenic responses in the diabetic bladder body. Effects of diabetes on the bladder body and base are associated with changes in calcium channel activity of bladder smooth muscle. PMID- 1723806 TI - On the superposition of currents from ion channels. AB - We derive a number of statistical properties of the superposition of several independent channels contributing to a patch-clamp recording. Failure of these properties indicates dependence of the channels and may suggest the nature of interactions. We show how properties such as dwell-time distributions of the individual channels may be determined from those of the superposition in the case that the channels are independent. PMID- 1723807 TI - Adaptive processing techniques based on hidden Markov models for characterizing very small channel currents buried in noise and deterministic interferences. AB - Techniques for characterizing very small single-channel currents buried in background noise are described and tested on simulated data to give confidence when applied to real data. Single channel currents are represented as a discrete time, finite-state, homogeneous, Markov process, and the noise that obscures the signal is assumed to be white and Gaussian. The various signal model parameters, such as the Markov state levels and transition probabilities, are unknown. In addition to white Gaussian noise, the signal can be corrupted by deterministic interferences of known form but unknown parameters, such as the sinusoidal disturbance stemming from AC interference and a drift of the base line owing to a slow development of liquid-junction potentials. To characterize the signal buried in such stochastic and deterministic interferences, the problem is first formulated in the framework of a Hidden Markov Model and then the Expectation Maximization algorithm is applied to obtain the maximum likelihood estimates of the model parameters (state levels, transition probabilities), signals, and the parameters of the deterministic disturbances. Using fictitious channel currents embedded in the idealized noise, we first show that the signal processing technique is capable of characterizing the signal characteristics quite accurately even when the amplitude of currents is as small as 5-10 fA. The statistics of the signal estimated from the processing technique include the amplitude, mean open and closed duration, open-time and closed-time histograms, probability of dwell-time and the transition probability matrix. With a periodic interference composed, for example, of 50 Hz and 100 Hz components, or a linear drift of the baseline added to the segment containing channel currents and white noise, the parameters of the deterministic interference, such as the amplitude and phase of the sinusoidal wave, or the rate of linear drift, as well as all the relevant statistics of the signal, are accurately estimated with the algorithm we propose. Also, if the frequencies of the periodic interference are unknown, they can be accurately estimated. Finally, we provide a technique by which channel currents originating from the sum of two or more independent single channels are decomposed so that each process can be separately characterized. This process is also formulated as a Hidden Markov Model problem and solved by applying the Expectation Maximization algorithm. The scheme relies on the fact that the transition matrix of the summed Markov process can be construed as a tensor product of the transition matrices of individual processes. PMID- 1723808 TI - Relationship of liking of one's given names to self-esteem and social desirability. AB - 59 men and 108 women university students rated their first, middle, and last names on seven-point Likert scales. Also, they responded to the Coopersmith Self esteem Inventory and the Crowne-Marlowe Social Desirability Scale. Analysis indicated significant sex differences only on the self-esteem measure. Both men and women who scored higher in self-esteem liked their first, middle, and last names better. Persons who had stronger preferences for their own first or last names tended to score higher on social desirability. PMID- 1723809 TI - RNA structure and NMR spectroscopy. PMID- 1723810 TI - Stress increases the secretory product of rat nasal mucosa goblet cells. AB - The effect of stress on the secretory activity of rat nasal mucosa goblet cells was examined by quantitative histochemistry. Mucosa was prepared from the nasal septum of male rats with or without electric shock applied to the tail. Mucosa was fixed with formaldehyde and stained with alcian blue. The epithelial layer was further dissected, and examined microscopically. The percentage of stained area was estimated with the aid of a microscope connected to a computerized video system. The stained area, which corresponded to the secretory granules of mucosal goblet cells, was greater in the electrically shocked group than the control group. This result raises the possibility that stress enhances the secretory activity of goblet cells in nasal mucosa. PMID- 1723812 TI - A patient weighted symptom score system in the evaluation of uncomplicated benign prostatic hyperplasia. AB - A new scoring system is presented for use in patients with uncomplicated benign prostatic hyperplasia. The system is based on the patients' information on the magnitude of twelve symptoms related to bladder and voiding function (symptom score), and three questions related to sexual function. Furthermore the patients are asked to grade the impact of these symptoms on their daily lives (bother score). Multiplication of the two scores related to bladder and voiding function gives the total score. The score related to sexual function is used only for comparing the situation before and after treatment. The new patient weighted total score may assist in creating a solid base for the indication for and evaluation of surgical and non-surgical treatment of uncomplicated benign prostatic hyperplasia. PMID- 1723811 TI - Effect of gold therapy on CD5+ B-cells and TCR gamma delta+ T-cells in patients with rheumatoid arthritis. AB - Circulating CD5+ B-cell levels in 15 patients with rheumatoid arthritis (RA) not receiving remittive therapy was significantly increased when compared to 17 normal controls (mean +/- SE: RA, 19.7 +/- 2.85%; controls, 11.6 +/- 1.67%; P less than 0.02). In contrast, 24 patients with RA receiving gold sodium thiomalate therapy (GST) had similar CD5+ B-cell levels (11.88 +/- 1.65) when compared to controls and significantly reduced levels when compared to the RA group not receiving remittive agents (P less than 0.01). Furthermore, TCR gamma delta+ T-cell levels were also assessed in these patients groups. These values were not significantly different between any of the groups (controls, 4.46 +/- 1.36%; GST, 6.88 +/- 1.73%; RA, 2.73 +/- 0.55%), although 42% of the GST treated group had gamma delta+ T-cell levels higher than the entire untreated RA group. No correlation was observed between the levels of TCR gamma delta + T-cells and CD5+ B-cells in any of these groups. These results suggested that therapy does influence the level of CD5+ B-cells and gamma delta+ T-cells in these patients. PMID- 1723813 TI - Indications for prostatectomy in the treatment of benign hyperplasia of the prostate. AB - Six hundred and forty patients with benign hyperplasia of the prostate (BPH) were admitted to our department the last 20 years before we started to perform transurethral resection of the prostate (TURP). Sixty per cent of these patients had various degrees of impairment of the renal function, 45% had an abnormal electrocardiogram. Three hundred and ninety-four patients (61.6%) underwent open prostatectomy, four of these died of urinary tract infection postoperatively and another four of severely impaired renal function. Four hundred and thirty-six patients with BPH have been treated at our department during the 5 years since we started to do TURP in 1985. Sixteen per cent had reduced renal function and 25% abnormal electrocardiogram. Three hundred and eighty-seven patients (89%) underwent prostatic surgery, 324 of them (74.3%) TURP. Two patients died to cerebral complications after TURP. Our conclusion is that if patients with BPH are operated at an early stage, their kidneys will not deteriorate and they will not develop severe heart diseases. PMID- 1723814 TI - Efficacy of alfuzosine (an alpha 1-adrenoreceptor blocking drug) in benign hyperplasia of the prostate. AB - Thirty-three patients with symptoms of outflow obstruction due to BPH were randomized into a double-blind study for 8 weeks treatment with alfuzosine 2.5 mg x 3 or placebo. Alfuzosine blood concentrations were analysed. The residual volume was significantly reduced in the alfuzosine group, from 98 to 43 cc (p less than 0.02) and the average urinary flow as measured by timed micturition was significantly improved (p = 0.023). Subjective symptoms were reduced in both groups. However, no statistically significant differences between the groups were recorded. Side-effects were limited and without clinical importance. PMID- 1723815 TI - Evaluation of prostate-specific antigen and prostatic acid phosphatase in untreated prostatic carcinoma and benign prostatic hyperplasia. AB - Prostate specific acid phosphatase (PAP) (Abbott, solid-phase enzyme immunoassay) and prostate specific antigen (PSA) (Hybritech, immunoradiometric assay) were determined in 162 newly diagnosed prostatic carcinoma patients, 187 patients with benign prostatic hyperplasia (BPH) and 127 controls. The upper limit of normal in controls for PAP was 2.2 micrograms/l and for PSA 5.0 micrograms/l. In the BPH group PAP was raised in 21%, for PSA in 41%. When the cut-off level of PSA was raised to 10.0 micrograms/l, 20% of BPH patients had an increased level. PSA was superior to PAP for the detection of prostatic cancer in all stages. Of the 162 patients with prostatic carcinoma, 88 had localised diseases and 74 had metastatic spread. PSA and PAP levels increased with each advancing clinical stage. PAP was elevated in 35% of the patients with cancer confined to the prostate. PSA in 69%. (PSA level 10.0 micrograms/l: 57%). In those patients with metastatic spread PAP was elevated in 77% compared with 96% for PSA. (PSA level 10.0 micrograms/l: 92%). The combined use of PSA and PAP does not give a greater accuracy in the screening of prostate cancer when compared with the sole use of PSA. PAP was elevated in only 4 patients when PSA was normal. In the BPH group there was no proven effect of micturition, frequency or residual urine on the SPA level. However, in this group infection may cause a rise in the PSA level. PMID- 1723816 TI - Experimental evaluation of heparin-coated cardiopulmonary bypass equipment with low systemic heparinization and high-dose aprotinin. AB - Cardiopulmonary bypass (n = 8 calves) with heparin-coated perfusion equipment, low-dose systemic heparinization (activated clotting time: ACT greater than 180 s) and high-dose aprotinin administration was evaluated in comparison to standard perfusion equipment with full-dose systemic heparinization (ACT greater than 480). All animals were perfused for 6 hours and similar values were observed for blood gases and mixed venous oxygen saturation in both groups. The heparin doses given in the study group before and during the 6 hours of perfusion totalized 14660 +/- 2553 IU as compared to 60833 +/- 5137 IU for the control group. No protamin was given in the study group whereas an equivalent of 27000 +/- 5805 IU was necessary to reverse heparin in the control group. There was no difference for prebypass hematocrit versus postbypass hematocrit in the two groups. Prebypass plasma hemoglobine was 8.4 +/- 2.1 mumol/L in the study group versus 10.0 +/- 3.8 mumol/L in the control group. After mixing with the priming volume, plasma hemoglobine was 8.6 +/- 2.5 mumol/L in the study group versus 6.7 +/- 1.6 mumol/L in the control group. The highest value was observed in the study group after 2 hours of perfusion (8.2 +/- 2.1 mumol/L) versus 5 hours of perfusion in the control group (7.4 +/- 3.6 mumol/L). Prebypass LDH levels of 1610 +/- 150 IU in the study group versus 1740 +/- 210 IU in the control group moved to 1870 +/- 200 IU in the study group at 24 hours after perfusion versus 2650 +/- 400 IU in the control group at 24 hours and decreased thereafter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723817 TI - Establishment of epithelial cell lines from human and mouse thymus immortalized by the 12S adenoviral E1a gene product. AB - To understand the role of thymic epithelial cells in the development of immature thymocytes, the establishment and cloning of thymic epithelial cell lines must be required. In the present study, we established human and mouse thymic epithelial cell lines through the immortalization by the transfection of cDNA sequences of adenoviral E1a 12S mRNA. This procedure resulted in the isolation of five stable cell lines (one human cell line and four mouse cell lines). These cell lines were positive in cytokeratin demonstrated by immunohistochemistry. Electron microscopic study revealed that they had bundles of tonofilaments and desmosome like tight junctions. These findings indicate that the cell lines immortalized by E1a gene have retained the properties of epithelial cells. MHC class II antigens were not expressed on these cell lines. When interferon-gamma was added to the cultured epithelial cell lines, MHC class II antigens were induced in their cytoplasm and on their surface membrane, demonstrated by immunohistochemical and immunofluorescent studies. It is suggested that these stable cell lines from human and mouse thymus might serve a good tool for the further study of thymocytes differentiation and of unknown cytokines derived from thymus epithelium. PMID- 1723819 TI - The first lipocalin with enzymatic activity. PMID- 1723818 TI - Use of monoclonal antibodies to identify thymopoietin in cultured human thymic epithelial cells. AB - Murine monoclonal antibodies (mAbs) were developed to discriminate thymopoietin, a human thymic hormone, and thysplenin, a closely related molecule found in spleen. Three of these recognized both native and synthetic thymopoietin as well as thysplenin. Together they define two non-overlapping epitopes which withstand sodium dodecyl sulfate denaturation and can be detected by western blotting. We used these three mAbs to demonstrate the production of thymopoietin by cultured thymic epithelial cells for up to several weeks. Three additional mAbs were selective for thysplenin. Highly specific mAbs will be useful for characterizing further these physiologically distinct polypeptides. PMID- 1723820 TI - [A monoclonal antibody study of the receptor area of the Venezuelan encephalitis virus]. AB - The receptor region for virus-cell interaction in Venezuelan equine encephalomyelitis (VEE) and Eastern equine encephalomyelitis (EEE) viruses was studied using a panel of 17 monoclonal antibodies (MCA). They were able to block agglutination of goose erythrocytes. The dominant role of glycoprotein E2 in the formation of viral receptor for EEE and VEE viruses was demonstrated. Competitive radioimmunoassay identified three antigenic sites in this region. These sites were also responsible for virus neutralization. MCAs to these sites protected outbred mice against lethal infection. The presence of a highly conservative region in VEE (site E2-3) and EEE (site E2a) which produced cross-reacting antibodies blocking hemagglutination of Western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya, and Pixuna viruses was established. A hypothesis is suggested concerning the existence of similar regions for the entire alphavirus genus, and the role of this region in virus-cell interaction. PMID- 1723821 TI - [The biological and immunochemical properties of monoclonal antibodies to the Venezuelan equine encephalomyelitis virus]. AB - Five strains of hybrid cells producing highly active monoclonal antibodies (MCA) to Venezuelan equine encephalomyelitis (VEE) virus were generated. VEEC6 MCA had high virus-neutralizing and hemagglutinating activities attesting to their direction to the "critical" site of E2 glycoprotein. MCA VEEA5 directed to the same glycoprotein did not overlap spatially with the previous one. MCA VEEB5 and VEEA4 interacted with conformational virus determinants taking part in hemagglutination. PMID- 1723823 TI - [The interferon system in juvenile rheumatoid arthritis]. PMID- 1723822 TI - [Monoclonal antibodies to the Machupo virus: their isolation and preliminary characteristics]. AB - Six monoclonal antibody-producing hybridoma cell lines were generated by fusion of NS-1 myeloma cells with BALB/c immune splenocytes. Monoclonal antibodies (MCA) specific to Machupo virus NP protein were used to study cross-reactivity between pathogenic and nonpathogenic arenaviruses. It was shown that 3140 MCA cross reacted in IFA with Lassa, Tacaribe, and Tamiami arenaviruses whereas 3101 MCA reacted with Machupo virus alone. It was assumed that these 3101 MCA could be used for differentiation of Machupo virus in IFA. PMID- 1723824 TI - [Specific brain antigens as the indicators of the blood-brain barrier permeability in Alzheimer's disease]. AB - A study was made of the hypothesis of blood-brain barrier dysfunction in Alzheimer's disease (AD) and of the role it plays in the disease pathogenesis. Use was made of the enzyme immunoassay test systems to detect neurospecific proteins in the blood serum within the following sensitivity range: gliofibrillar acid protein 10 ng/ml, alpha 2-glycoprotein of the brain 0.9 ng/ml, alpha 1- and alpha 2-specific brain globulins 50 pg/ml. Gliofibrillar acid protein and alpha 2 glycoprotein of the brain appeared the most sensitive markers of the process. Demonstration of these antigens in the blood serum in a concentration exceeding donor's ones suggests the impairment of the blood-brain integrity in active passage of brain proteins to the blood. A direct relationship is shown between the level of gliofibrillar acid protein in the blood serum and the severity of the clinical picture of AD. PMID- 1723825 TI - The role of haematopoietic growth factors in bone marrow transplantation. AB - Bone marrow transplantation (BMT) is being increasingly used for treating a large number of diseases. The recent availability of haematopoietic growth factors (HGFs) for therapeutic purposes has offered a powerful tool in order to overcome one of the most important BMT-related complications, namely severe myelosuppression. This article, besides an overview on the main results achieved with the clinical use of HGFs for haematopoietic reconstitution after BMT, also deals with some innovative applications of these molecules in BMT setting, as their use for peripheral stem cell transplantation and bone marrow failure after BMT. PMID- 1723826 TI - Albumin, HES 120 and dextran 70 as adjuvants to red blood cell concentrates: a study on colloid osmotic pressure changes in vitro. AB - An in vitro model of surgical bleeding was developed to simulate continuous blood loss and replacement therapy with plasma substitutes and red cell concentrates. The model was used to determine the lowest colloid concentration in vitro for each of the plasma substitutes that sustains colloid osmotic pressure above 2.4 kPa (or 18 mmHg) when used up to the recommended maximal total dose. Plasma, supernatant separated from red cell concentrates and dextran 70, hydroxyethyl starch 120 or albumin were mixed to create dilutions imitating plasma composition in the course of clinical blood loss and replacement therapy. The relative volume of each component was calculated according to the model when the bleeding was equal to multiples of 10% of blood volume up to a blood loss of 120%. Our measurements indicate that the colloid concentrations of 5.0% for albumin, 4.0% for hydroxyethyl starch 120 and 3.5% for dextran 70 preserve colloid osmotic pressure above 2.4 kPa. PMID- 1723827 TI - The dominant form of the pigmentary orthochromatic leukodystrophy. AB - The present report documents a family with three cases in two successive generations of pigmentary orthochromatic leukodystrophy (POLD). The clinical features of these cases and histochemical and ultrastructural investigations of two of the brains from successive generations are discussed. A review of the familial cases of POLD reported in the literature is also presented. Transmission of these cases was by a dominant inheritance. Onset of the clinical symptoms occurred at 42 to 54 years of age; duration of the disease was from 2-11 years, and death occurred at 45 to 57 years of age. Clinical manifestations of all three cases were severe headaches; bilateral pyramidal, pseudobulbar, cerebellar, and frontal release signs; gait disturbances; euphoria, or apathy; epileptic seizures; and dementia. The neuropathological pattern consists of slight cerebral atrophy, brownish discoloration of the cerebral white matter with demyelination and severe gliosis, sparing the sub-cortical U fibers; presence in the macrophages of lipid pigment granules that are sudanophilic, non metachromatic, and PAS and iron positive. The electron microscopic pattern of the lipid pigment in the macrophages is that of ceroid: electron-dense, membrane-bound intracytoplasmic lysosomes with curvilinear and/or fingerprint profiles. PMID- 1723828 TI - Argyrophilic ubiquitinated cytoplasmic inclusions of Leu-7-positive glial cells in olivopontocerebellar atrophy (multiple system atrophy). AB - We described cytoplasmic inclusions in glial cells in 18 patients with olivopontocerebellar atrophy (OPCA) (multiple system atrophy, MSA). These glial inclusions showed intense argyrophilia with modified Bielschowsky's and Bodian's silver impregnation techniques, and were observed in the pons, cerebellar white matter, midbrain, medulla oblongata and basal ganglia, as well as cerebral white matter and spinal cord. None of the 54 control cases had glial argyrophilic inclusions. Immunohistochemically, these inclusions were intensely labeled by anti-ubiquitin antibody. Some of them reacted with an antibody to Rosenthal fiber (RF) protein. The cytoplasm of ubiquitinated inclusion-bearing glial cells was immunostained by anti-Leu-7 antibody, but not by anti-GFAP antibody. Ultrastructurally, the glial inclusions were composed primarily of approximately 24- to 40-nm fibrils, which were coated with osmiophilic granular material along their length in longitudinal section. These fibrils appeared as annuli in cross section. Often, a central granule approximately 5 nm in diameter was seen in the lucent lumen of a cross-sectioned fibril. The granule-coated fibrils were not seen in the glial filament-containing astrocytes. Electron microscopic examination of silver-impregnated specimens revealed that the granule-coated fibrils had strong affinity for silver. Immunoelectron microscopy using the indirect immunoperoxidase techniques with antibodies to ubiquitin and RF protein revealed that the electron-dense reaction products respective to both were located on constituents of glial inclusions. Our observation that Leu-7-positive glial cells, mainly oligodendroglial cells, had argyrophilic ubiquitinated inclusions may be of significance for the evaluation of the pathology of OPCA(MSA). PMID- 1723829 TI - Immunohistochemistry of primitive neuroectodermal tumors in infants with special emphasis on cytokeratin expression. AB - Eleven primitive neuroectodermal tumor (PNET) biopsies from infants under the age of 3 years were studied for the presence of various differentiation markers for neuroectodermal stem cells. Special emphasis was placed on the expression of cytokeratin proteins. The tumor cells expressed different cytokeratin proteins (CK8, CK13, CK18, CK19, KL1, AE1/AE3, MNF16) in 3 of 11 cases. These cases were furthermore characterized by a strong expression of glial fibrillary acidic protein, S-100 protein and vimentin. Vimentin and cytokeratin proteins were co expressed; cross-reactivity between these two intermediate filaments could be excluded by immunoblotting. It is noteworthy that the three positive tumors were all from infants in their 1st year. We assume that PNETs in early infancy are characterized by a particularly wide range of differentiation patterns. The presence of cytokeratin proteins in these cases seems to be associated with the expression of vimentin and must be regarded as an indicator of an early developmental stage of the tumor cells. PMID- 1723830 TI - Immunohistochemical characterization of primitive neuroectodermal tumors and their possible relationship to the stepwise ontogenetic development of the central nervous system. 1. Ontogenetic studies. AB - Aim of the present study was to establish different immunohistochemical staining patterns for a subsequent comparison with those of primitive neuroectodermal (PNET) subsets, i.e. PNET-NOS (not otherwise specified) or PNET with focal neuronal, astrocytic or ependymal differentiation, to relate neoplastic to embryonal development. Tissue of the developing central nervous system, with special emphasis on the stepwise development of the rhombencephalon, the cerebellar and the retinal anlage, from 20 different human embryos and fetuses ranging from 3 to 30 weeks of gestational age (GA) was examined. Six neuronal markers, synaptophysin, chromogranin A, neuron-specific enolase (NSE), neurofilament protein (NFP; 160 kDa, 200 kDa, 70 and 200 kDa) and six other markers, glial fibrillary acidic protein (GFAP), S-100 protein, vimentin, myoglobin, desmin, cytokeratin, were assessed immunohistochemically. GFAP and S 100 protein appeared at the 6th week of GA in primitive glial cells of the cerebellar anlage, brain stem, rhombencephalon, and developing spinal cord, together with--as first neuronal marker--chromogranin A, then NFP (70 and 200 kDa, and 160 kDa) from the 8th week onward. NSE started in the 11th week and synaptophysin not earlier than the 16th week of GA. Interestingly, the differentiation of the retinal anlage started rather late with NSE positivity beginning from the 16th week and positive reactions to synaptophysin and NFPs only from the 25th and chromogranin A from the 28th week of GA onward. PMID- 1723831 TI - The multidrug-resistance gene MDR1 is expressed in human glial tumors. AB - The most consistantly reported alteration of multidrug-resistant carcinoma cells is the overexpression of a membrane glycoprotein, termed P-glycoprotein. In this study we examined whether the strong intrinsic chemotherapy resistance of glial tumors might be related to the expression of the MDR1 gene which codes for P glycoprotein. Fourteen glial tumors were examined immunohistochemically using the monoclonal antibody C219. In addition, RNA samples of 11 of these tumors were analysed using a sensitive Northern blot assay. P-glycoprotein is expressed in all 14 glial tumors; the number of stained tumor cells, however, varied considerably ranging from 0.3% to 15%. There was no correlation between the number of MDR1-positive cells and the histological malignancy. Varying amounts of MDR1 mRNA were detectable in 7 from 11 examined tumors. The results of our study show that the MDR1 gene is expressed in human glial tumors and suggest that the multidrug transporter may contribute to the clinical non-responsiveness of these tumors to chemotherapy. PMID- 1723832 TI - Demonstration of amyloid beta-protein in a 32-year-old man with progressive dementia. AB - We report the immunolocalization of extensive amyloid beta-protein in senile plaques, cerebrovascular amyloid deposits, neurofibrillary tangles and preamyloid in a 32-year-old man with progressive dementia not due to trisomy 21 or trauma. These amyloid deposits were non-reactive to antibodies directed against scrapie amyloid. Our data indicate that the presence of amyloid beta-protein is not limited to normal aging, Alzheimer's disease and related disorders but is also found in younger individuals with progressive dementia. PMID- 1723833 TI - Regulation of serum insulin-like growth factors and their binding proteins in Ecuadorean patients with growth hormone receptor dysfunction (growth hormone insensitivity). AB - Regulation of serum insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and growth hormone (GH) binding protein (GHBP) has been investigated in Ecuadorean patients with GH receptor dysfunction (GHRD) and in their heterozygous relatives (parents). Serum IGF-I and IGF-II levels were measured by radioimmunoassay (RIA). IGFBPs were identified by Western ligand blotting of serum samples, following separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and relative quantification was performed with a scanning laser densitometer. Serum GHBP levels were measured with a ligand-mediated immunofunctional assay (LIFA) using a monoclonal antibody raised against the GHBP. These values were then compared with values obtained from normal, sex matched Ecuadorean controls. Serum IGF-I, IGF-II, IGFBP-3 and GHBP concentrations were markedly reduced and serum IGFBP-2 values increased in the patients with GHRD. Serum IGF-I and IGF-II values were positively correlated in the patients with GHRD, but were not related to the age of the patient. IGFBP-2 and IGFBP-3 concentrations were inversely correlated in the patients with GHRD. When analysed by age, however, IGFBP-2 was related inversely and IGFBP-3 related positively to the age of the patient. The serum IGF-II levels of the male heterozygotes were significantly reduced when compared with sex-matched controls, but considerable overlap with normal values was found. No other biochemical indices were significantly altered in either male or female heterozygotes. Thus, although GHRD is characterized by dramatic reductions in serum levels of GHBP, IGF-I, IGF-II and IGFBP-3, none of these assays provides a reliable biochemical marker for heterozygosity. PMID- 1723834 TI - Effects of exogenous insulin-like growth factor I on insulin-like growth factor binding proteins in a case of growth hormone insensitivity (Laron-type). AB - The electrophoretic profiles of serum insulin-like growth factor binding proteins (IGFBPs) from an 18-year-old patient with growth hormone (GH) insensitivity were analysed by Western ligand blotting before and after administration of recombinant human insulin-like growth factor I (IGF-I). Under basal conditions, the profile was the same as that observed in all cases of GH deficiency or insensitivity, that is, low IGFBP-3 (seen as two bands of 41.5 and 38.5 kDa) and enhanced IGFBP-2 and, to a lesser extent, IGFBP-1. Subcutaneous injection of IGF I, 40 micrograms/kg, provoked an increase in serum IGF-I levels to close to the lower limits of the normal range and a small increase in IGFBP-3, suggesting that IGF-I has a direct effect on the synthesis of this GH/IGF-I-dependent IGFBP. Moderate increases were also observed in IGFBP-2 and IGFBP-4. Repeated doses of IGF-I over 7 days had no further effect on these changes, which persisted for a few days after the last injection. PMID- 1723835 TI - Impact of increased growth hormone secretion on carbohydrate metabolism in adolescents with diabetes. AB - Growth hormone (GH) and fasting insulin concentrations rise during puberty in normal subjects. Any increase in GH secretion in adolescents with insulin dependent diabetes mellitus (IDDM) might be expected to lead to further insulin resistance and metabolic disturbance. Despite the high incidence of delayed growth in IDDM, the relationship between GH, insulin-like growth factor I (IGF-I) and IGF binding protein 1 (IGFBP-1) has not been clearly established. Twenty-six adolescents with IDDM and 34 healthy siblings underwent measurement of their overnight GH secretory profiles (20.00-08.00 hours, 15-minute sampling). The diabetic subjects were studied either on their normal insulin regimen (n = 15) or during a euglycaemic clamp (n = 26). A second clamp study was undertaken (n = 7) with addition of pirenzepine to suppress GH secretion. GH profiles in the diabetic subjects were characterized by increases in both pulse amplitude and baseline GH concentrations. Deconvolution analysis also revealed an increase in the frequency of GH secretory episodes. In the subjects with diabetes, a direct link between the dawn rise in insulin requirements, increased concentrations of beta-hydroxybutyrate and the elevated concentrations of GH was established. These abnormalities were reversed by the suppression of GH pulse amplitude following pirenzepine. Serum IGF-I concentrations and IGF-I bioactivity in the diabetic subjects were low and were positively correlated with mean GH concentrations. In conclusion, well controlled adolescents with IDDM show persisting abnormalities of GH, beta-hydroxybutyrate and IGF-I despite normoglycaemia. The role of inappropriate insulin delivery in the development of these abnormalities is discussed. PMID- 1723836 TI - Clinical spectrum of the syndrome of growth hormone insensitivity. Advisory Committee on Growth Hormone Insensitivity. AB - A survey of 46 patients with suspected growth hormone (GH) insensitivity is presented. Clinical details and plasma samples from these patients were analysed centrally. A diagnostic score based on basal levels of GH and insulin-like growth factor I (IGF-I), IGF-I response to GH (0.1 U/kg daily for 4 days) and height SDS was determined for all patients to identify those with GH insensitivity. A total of 29 such patients was identified (14 males and 15 females; age range, 3.2-22.6 years). Their mean height SDS was -6.0 (range, -8.9 to -2.6), mean weight SDS was -2.9 (range, -5.2 to 1.2). Pubertal development was delayed in three out of three boys and two out of six girls of pubertal age. Their basal GH level was 32.4 mU/l (range, 0.8-158.2 mU/l) and mean basal IGF-I level was 33.6 ng/ml (range, 20-97 ng/ml). None of the 29 patients showed a significant serum IGF-I response to injections of GH. PMID- 1723837 TI - Chlamydia infections in Kawasaki disease. PMID- 1723838 TI - Ultrastructural cytochemistry of eosinophilic inclusions in the cells of hyperplastic nodules and hepatomas in mouse liver. AB - Intracisternal inclusions in the cells of 48 hyperplastic nodules and hepatomas in mouse liver were examined by electron microscopy to determine the precise compositions of the inclusions in relation to their ultrastructure, and some preliminary attempts at isolation and chemical analysis of the inclusions were performed. We classified the inclusions into two types. One was mainly of larger size, consisting of a single electron-lucent core and a granular cortical zone of high electron density. The other type was smaller, with a number of tiny, electron-lucent areas crowded into the central area instead of a single core. The cortical material of the inclusions was digested by pepsin treatment of thin sections, whereas the core and the electron-lucent areas within the cortical zone were not extracted. On the other hand, in materials treated with ethanol before post-osmication, only the core and electron-lucent areas within the cortical zone were partially extracted. The ultrastructure of the isolated inclusions was very similar to that of inclusions in situ. The chemical composition of the isolated fractions was estimated to be 60% protein and 35% lipid. Electrophoretically, the protein of this fraction showed a single band. We conclude that the cortical substance is proteinaceous in nature, probably consisting of a single protein or a group of proteins with identical electrophoretic mobility, whereas the core is composed of lipid. The possibility that the inclusions are due to an impairment in the mechanism of intracellular lipoprotein transport is discussed. PMID- 1723839 TI - Solid adenosquamous carcinoma of the liver. A case report and review of the literature. AB - This is a report of a fatal case of a primary and solid adenosquamous carcinoma (ASC) of the liver in a 58-year-old Japanese woman. There was no association with biliary cysts. Histochemistry and immunohistochemistry support the contention that the neoplasm arose from squamous metaplasia of a mucus-secreting adenocarcinoma (MSA) of intrahepatic biliary duct epithelium. PMID- 1723840 TI - Fluorescent antibody titres after recovery from visceral leishmaniasis. PMID- 1723841 TI - [Combination chemotherapy with ifosfamide (or cyclophosphamide), adriamycin, cis platinum and peplomycin (IAPP) for hormonally resistant metastatic prostatic cancer]. AB - From January, 1986 to April, 1990, combination chemotherapy with ifosfamide (or cyclophosphamide), adriamycin, cis-platinum and peplomycin was performed in 15 patients with hormonally resistant metastatic adenocarcinoma. Three patients had partial response (PR) and 9 remained objectively stable (ST). The median response duration of PR + ST (12) was 5.7 months (range 2.8 to 18.0+). Three patients progressed while on this therapy. Of 8 patients with prior treatment of chemotherapy or chemo-hormonal therapy, 6 achieved an objective response (2 PR, 4 ST). Severe toxicities occurred in 2 patients. One died of lung fibrosis induced by peplomycin and the other received urinary diversion for persistent hemorrhagic cystitis. These results compare favorably with previous reports of chemotherapy treatment of metastatic prostatic cancer patients who failed on hormonal manipulation. However, careful treatment is needed for lung fibrosis and hemorrhagic cystitis. PMID- 1723842 TI - [BEP (bleomycin, etoposide, cisplatinum) chemotherapy as an induction therapy of advanced extragonadal germ cell tumor]. AB - Five patients with advanced extragonadal germ cell tumor (EGGCT) were treated with cisplatinum + vinblastine + bleomycin (BVP) or cisplatinum + etoposide + bleomycin (BEP) chemotherapy as an induction therapy. All patients had high levels of tumor markers and multiple distant metastasis in poor nutritious conditions. Two patients given BVP therapy died 6 and 9 months after the chemotherapy. By contrast, three patients given BEP therapy achieved complete remission and were surviving longer than one year without any evidences of disease. Granulocytopenic fever was seen soon after the first course of BEP therapy. Prophylactic granulocyte-colony stimulating factor (G-CSF) treatment, enabled two of them to receive full dose chemotherapy without any fever attacks. In conclusion, BEP chemotherapy is effective to EGGCT as well as far advanced testicular tumors and G-CSF treatment is useful for achievement of induction chemotherapy to advanced germ cell tumors. PMID- 1723843 TI - [Effect of KT-611 (Naftopidil) on the contraction of human prostatic tissue and its use in benign prostatic obstruction]. AB - A new alpha 1-adrenergic receptor antagonist, KT-611 (naftopidil) antagonized dose dependently the contraction induced by agonists, noradrenaline and phenylephrine in the human prostatic tissue. KT-611 at a dosage of once or twice a day was evaluated for its effects on 49 patients with benign prostatic hypertrophy. The drug improved subjective and objective symptoms significantly. The residual urine was reduced in volume and percentage significantly. The average and maximum flow rates increased significantly. The optimal dosage was presumed to be in the range of 25 to 75 mg once a day or 25 to 100 mg twice a day. Adverse reactions and abnormal laboratory findings were all slight. KT-611 was concluded to be useful in the treatment of patients with benign prostatic hypertrophy. PMID- 1723844 TI - [A case of squamous cell carcinoma of the urinary bladder effectively responsive to combination chemotherapy]. AB - A case of invasive pure squamous cell carcinoma of the urinary bladder effectively responsive to combination chemotherapy in a 79-year-old female is reported. Clinical staging of the tumor was T3b. We used combination chemotherapy with methotrexate, peplomycin and cis-platinum (MBD regimen) before radical cystectomy. Remarkable regression of the tumor was identified by computed tomographic scan after one course of chemotherapy and the surgical specimen revealed no viable tumors. The patient has been very active in her daily life without evidence of local recurrence or metastasis for more than one year. Squamous cell carcinoma of the bladder generally is regarded as having a poor prognosis with 5-year survival rates ranging from 7.4 to 26% and effective chemotherapy has not yet been established. Our experience suggests that the cure rate of pure squamous cell carcinoma may be improved markedly by the use of MBD regimen. PMID- 1723845 TI - Protective effect of liposome-entrapped superoxide dismutase and catalase on bleomycin-induced lung injury in rats. I. Antioxidant enzyme activities and lipid peroxidation. AB - The influence of liposome-entrapped catalase and/or superoxide dismutase on bleomycin-induced rat lung injury was studied. Liposome-entrapped catalase and/or superoxide dismutase increase antioxidant enzyme activities in the lung tissue of bleomycin-treated rats. The level of lipid peroxidation products (malondialdehyde, conjugated dienes, lipid hydroperoxides) was significantly lower in liposome-entrapped catalase and/or superoxide dismutase supplemented rats with bleomycin-injured lungs. It is suggested that liposomes are good vectors for drugs in the treatment of bleomycin-induced lung fibrosis. PMID- 1723847 TI - Etiologic complexities of diaphragmatic defects: right diaphragmatic hernia, pulmonary hypoplasia/agenesis, and hydrocephalus in sibs. AB - Here we review the complexities of diaphragmatic defects and describe sibs with small, right diaphragmatic defects with pulmonary hypoplasia/agenesis and hydrocephalus. Despite a poor initial prognosis, the propositus has progressed remarkably well. Antenatal sonographic study detected hydrocephalus but not the diaphragmatic defect in the sib of the propositus. Because diaphragmatic defects are most commonly found in association with other anomalies and may occur in association with chromosome anomalies careful workup of all affected infants is crucial for accurate genetic counseling. PMID- 1723848 TI - Immunohistochemical study on the developing optic nerves in human embryos and fetuses. AB - For the purpose of investigating the time of development and myelination of the optic nerve, we applied immunohistochemical stains to the optic nerves of 26 human embryos and fetuses aged 6-39 weeks. The minimum diameter of the optic nerve's cross section increased with the development of embryos; 0.15 mm at the 9th week, 0.4 mm at the 11th week, 1.5 mm at the 24th week, and 2.1 mm at the 32nd week. With regard to myelin basic protein (MBP), the localization could not be observed at 28 gestational weeks. The MBP was proved to be positive in oligodendrocytes of the optic nerve at the age of the 32nd gestational week. The localization of glial fibrillary acidic protein (GFAP) was proved partially in astrocytes around the optic nerve in the case of 24 gestational weeks. The remarkable localization of GFAP-positive astrocytes inside the nerve fibers was proved at the 27th gestational week. The histological appearance of GFAP-positive cells advanced to that of MBP-positive ones after 8 weeks interval. The degree of MBP staining was less marked than that of GFAP in examined fetal cases. These results suggest that astrocytes and oligodendrocytes correlate to the developmental growth and myelination of the optic nerves during the fetal development. PMID- 1723849 TI - Facioscapulohumeral muscular dystrophy: muscle fiber type analysis with particular reference to small angular fibers. AB - Muscle biopsies from 14 patients with facioscapulohumeral muscular dystrophy (FSHD) aged from 5 to 45 years were studied histochemically with fiber type analysis, focusing on small angular fibers (SAF) to clarify their significance. There were no duration-related or age-dependent histopathological differences between child and adult patients. Variations in fiber size and SAF were observed in all, myonecrosis with occasional phagocytosis in 10 and regenerating fibers in 12 biopsies. Cellular responses including inflammatory cell infiltration (7 biopsies) and connective tissue proliferation (8 biopsies), and fiber architectural changes (9 biopsies) were additional common findings. Although SAF are also commonly seen in patients with Kugelberg-Welander disease and amyotrophic lateral sclerosis, in FSHD they were mostly type 2C fibers which frequently exhibit alkaline phosphatase-positive activity. Therefore SAF in FSHD are mostly the products of a regeneration rather than denervation process. PMID- 1723850 TI - Human granulocyte colony-stimulating factor: its basic aspects and clinical applications. AB - Human granulocyte colony-stimulating factor (G-CSF), a 20 KD glycoprotein consisting of 174 amino acids, is one of the physiological regulators mainly produced by endothelial cells, monocytes-macrophages and fibroblasts, under various stimulations such as infections. The molecule specifically and markedly stimulates the production of neutrophils associated with an expansion of the hematopoietic stem cells/progenitor cells and elevates the functional activities of neutrophils. Under favor of the unique biological activities, recombinant human G-CSF (rhG-CSF) has now become an epoch-making agent for the purposes of reduction of bacterial/fungal infections, particularly in neutropenias of various causes, increased dose intensity of chemotherapeutic drugs in cancers, and solving various problems in hematopoietic stem cell transplantation. PMID- 1723851 TI - Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains. AB - An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723852 TI - Intermediate filament--like proteins in the fibrous sheath of the mouse sperm flagellum. PMID- 1723853 TI - Characterization of a non-tyrosine kinase FGF-binding protein. PMID- 1723854 TI - Fibroblast growth factors in normal and malignant melanocytes. PMID- 1723855 TI - The role of fibroblast growth factor in eye lens development. AB - In this review we have presented evidence that FGF plays an important role in inducing events in lens morphogenesis and growth. Our studies show that FGF stimulates lens epithelial cells in explants to proliferate, migrate, and differentiate into fibers at low, medium, and high concentrations, respectively. This has some important implications for understanding the behavior of lens cells in the eye. The fact that aFGF is detected in the equatorial region of the lens where cells are actively proliferating, possibly migrating, and differentiating into fibers suggests that these events may be under autocrine control in vivo, at least to some extent. Because FGF is also present in the ciliary and iridial region of retina and in the vitreous, paracrine control may also be involved. Cell proliferation, fiber differentiation, and (possibly) cell migration occur in characteristic spatial patterns that are related to distinct compartments of the lens. We suggest that cells in the germinative zone receive only a low level of FGF stimulation arising from the cells themselves and possibly also from the ciliary and iridial regions of the retina but, whatever the source, this is only sufficient to stimulate proliferation. Lens epithelial cells that migrate or are displaced into the transitional zone below the lens equator receive some FGF from these sources but in addition receive a strong stimulus from the high level of FGF in the vitreous; thus, fiber differentiation is induced. Cells at the junction between these two zones may receive an intermediate level of FGF stimulation, sufficient to induce cell migration. In essence, we are proposing that, in the eye, FGF acts as a lens morphogen in the sense that different levels of FGF stimulation elicit different lens cell responses. Hence its characteristic distribution in the eye establishes lens polarity and growth patterns. Since FGF has an inductive effect on lens cells from mature age animals, we also propose that this specific distribution of FGF in the eye is also important for maintenance of a normal lens throughout life. Finally the synergistic effects of insulin/IGF on the FGF-induced responses highlight the importance of considering the distribution of members of the insulin/IGF family of molecules in vivo. Mechanisms that control levels of both the FGF and insulin/IGF families of factors in the eye are probably of crucial importance in the formation and maintenance of a normal lens. PMID- 1723856 TI - Differential localization and possible functions of aFGF and bFGF in the central and peripheral nervous systems. AB - We investigated the relative distribution of acidic and basic FGF (aFGF and bFGF) in the nervous system of the rat, using a combination of biological, biochemical, immunochemical, and immunohistochemical methods that can differentiate unambiguously between aFGF and bFGF. We found that different regions of the nervous system contained varying levels of aFGF and bFGF. In the central nervous system, bFGF was present nearly exclusively in astrocytes. Most neurons did not contain detectable amounts of bFGF immunoreactivity, with the notable exception of pyramidal cells in hippocampal area CA2. Interestingly, bFGF immunoreactivity was localized to the nucleus of both CA2 neurons and astrocytes. Astrocytes in vitro were also found to express bFGF, whereas cortical neurons in culture did not contain detectable amounts of bFGF. Transection of the optic nerve led to an approximately twofold increase of bFGF in the distal stump, which is consistent with the observation that bFGF is expressed by astrocytes. Transection of rat and chicken sciatic nerve resulted in a rapid and complete disappearance of aFGF from the distal nerve stump, suggesting that aFGF is present in axons projecting through the sciatic nerve. We observed, in agreement with this notion, that cultured sensory neurons contain reasonably high levels of FGF-like bioactivity. Similar levels of activity were found in developing sciatic nerve, suggesting that neuronal aFGF might be involved in regulating the development of the peripheral nervous system. PMID- 1723857 TI - A role for fibroblast growth factor in oligodendrocyte development. AB - The differentiation of oligodendrocyte-type 2 astrocyte (O-2A) glial progenitor cells into myelin-forming oligodendrocytes is influenced by several polypeptide growth factors. Exposure of O-2A progenitors to basic fibroblast growth factor (bFGF) promotes their sustained proliferation, blocks their differentiation, and maintains both a high level of platelet-derived growth factor (PDGF) alpha receptors and PDGF sensitivity. Exposure to PDGF, in contrast, promotes only a limited number of cell divisions prior to their differentiation and triggers progenitor cell migration. In the continued presence of bFGF the cells have a stellate morphology with short processes. Upon addition of PDGF these stellate cells become bipolar with long processes, and on removal of PDGF their morphology reverts back to stellate. Thus the phenotype of O-2A progenitor cells in response to these growth factors is plastic. Our studies suggest that bFGF (or a related ligand) in the CNS may sensitize O-2A progenitors to PDGF and thereby initiate their ability to migrate into white matter tracts prior to the onset of myelination. PMID- 1723858 TI - aFGF content increases with malignancy in human chondrosarcoma and bladder cancer. PMID- 1723859 TI - Adult brain but not kidney, liver, lung, intestine, and stomach membrane preparations contain detectable amounts of high-affinity receptors to acidic and basic growth factors. PMID- 1723860 TI - Characterization of the FGFR-3 gene and its gene product. PMID- 1723861 TI - Acidic fibroblast growth factor, heart development, and capillary angiogenesis. PMID- 1723862 TI - FGF mediation of coronary angiogenesis. PMID- 1723863 TI - Acidic and basic fibroblast growth factors are present in glioblastoma multiforme and normal brain. PMID- 1723864 TI - Acidic fibroblast growth factor in normal, injured, and jimpy mutant developing mouse brain. PMID- 1723865 TI - Structure-function studies of acidic fibroblast growth factor. PMID- 1723866 TI - Structural modifications of acidic fibroblast growth factor alter activity, stability, and heparin dependence. PMID- 1723867 TI - Characterization of monoclonal antibodies cross-reacting with myocardial and retinal sodium-calcium exchange proteins. PMID- 1723868 TI - Development of a 16S rRNA-based oligomer probe specific for Listeria monocytogenes. AB - Crude rRNA was isolated from Listeria monocytogenes, L. innocua, and L. ivanovii and sequenced by a reverse transcriptase method. Only two sequence regions were found to differ for L. monocytogenes versus L. innocua or L. ivanovii. Two oligonucleotide probes (RL-1 and RL-2) complementary to these two regions of rRNA of L. monocytogenes were synthesized. The RL-1 probe had one base while the RL-2 probe had two bases which differed for L. monocytogenes versus L. innocua and L. ivanovii. Use of a dried gel hybridization in place of Northern (RNA) hybridization or dot blot hybridization indicated that the RL-2 probe hybridized with all 36 strains of L. monocytogenes tested but not with 6 other Listeria species and 11 other bacteria tested. The RL-2 probe is specific for L. monocytogenes, while the RL-1 probe showed some cross-reactions with other Listeria species. An alkaline phosphatase-labeled RL-2 probe could be used in a dot blot hybridization test and gave good results, but a 32P-labeled RL-2 probe was more sensitive than the nonradioactive probe and the 32P-labeled probe was useable for up to 2 months, even though the 32P was highly degenerated. PMID- 1723869 TI - [Use a of DNA probe to detect cellular immunity against intracellular parasitism]. AB - By using a specific, repetitive DNA probe, we have been able to detect picograms of P. berghei DNA. With this probe we have determined that: a) P. berghei, inoculated into Norway Brown rats, reaches its peak of proliferation in the liver 44 h after infection; b) gamma interferon inhibits in a dose-dependent fashion the development of liver exoerythrocytic forms (EEF) in vivo and in vitro, and; c) endogenous gamma interferon inhibits the development of EEF in hosts immunized with irradiated sporozoites. Related with and derived from these findings, we have found that, in order to obtain an effective immunity against malaria in experimental animal models, effector mechanisms mediated by T cells are required. This is substantiated by the following facts: a) immune hosts inoculated with monoclonal antibodies against gamma interferon reversed their immunity against a sporozoite challenge; b) This immunity was also reversed when the animals were depleted from their CD8 positive cytotoxic T cells. Therefore, sterile immunity against this parasite requires not only the presence of antibodies but also the inhibition of the EEF by gamma interferon with participation of CD8 positive T cells. PMID- 1723870 TI - Histopathogenesis of inflammatory linear verrucose epidermal naevus: histochemistry, immunohistochemistry and ultrastructure. AB - Skin lesions of three patients with inflammatory linear verrucose epidermal naevus (ILVEN) were examined. Histologically, orthokeratosis and parakeratosis were alternately seen in the acanthotic epidermis. By N-(7-dimethylamino-4-methyl 3-coumarinyl)maleimide staining, the horny cells in the parakeratotic epidermis showed a cytoplasmic SH pattern and a weak membranous SS pattern. The orthokeratotic epidermis revealed an increased involucrin expression, whereas the parakeratotic epidermis showed almost no involucrin expression. Ultrastructurally, in the parakeratotic epidermis, the living keratinocytes had prominent Golgi apparatuses and vesicles in the cytoplasm. In the intercellular spaces in the upper spinous layer through to the lower horny layer, an electron dense, homogeneous substance was deposited. The cytoplasm of the horny cells was filled with keratin filaments and contained remnants of nucleus and cytoplasmic membrane structures, and some lipid droplets. The marginal band formation was incomplete. Most of these ultrastructural abnormalities were not found in the orthokeratotic epidermis. There are both similarities and differences in histopathogenesis of the parakeratotic epidermis between ILVEN and psoriasis. A unique finding was the lack of involucrin expression in the ILVEN parakeratotic epidermis. PMID- 1723871 TI - No significant change of glycoconjugates exists in the epidermis of familial benign chronic pemphigus. PMID- 1723872 TI - Construction of stable, single-copy luciferase gene fusions in Escherichia coli. AB - A ColE1-based plasmid for transcriptional gene fusions was constructed that contains both the promoterless luxAB genes of Vibrio harveyi and a tet marker within the inverted repeats of a left end-truncated Tn5 element. Introduction of this plasmid into an Escherichia coli strain containing a plasmid (pTF421) that over-produces ColE1 RNA1 (and thus inhibits replication of the ColE1 plasmid) allowed selection for cells that had a single copy of the luxAB operon transposed into the chromosome beginning 5 days post-transformation. The long latent period necessary for Tn5 transposition is analogous to that found in other systems, where transposition frequencies and mutation rates increase in a time-dependent manner when selected for upon prolonged incubation on Petri dishes under bacteriostatic conditions. PMID- 1723873 TI - Effects of chicken anemia agent on lymphokine production and lymphocyte transformation in experimentally infected chickens. AB - One-day-old chicks with no maternal antibodies to chicken anemia agent (CAA) were inoculated intramuscularly with CAA grown in MDCC-MSB1 cells. A control group of birds from the same source was inoculated intramuscularly with a lysate from uninfected MSB1 cells. Birds were killed at 8, 15, 22, 29, and 43 days postinoculation (PI), and the spleens were removed. Spleen cells were dispersed and stimulated with various concentrations of Concanavalin A (Con A), and lymphocyte transformation responses were determined. Supernatants from Con A stimulated cultures were assayed for T-cell growth factor (TCGF) and interferon. Decreased lymphocyte transformation and TCGF production were demonstrated at 8 and 15 days PI. This was followed by a stimulation in activities before a return to control levels at 43 days PI. Interferon levels were elevated 8 days after infection. This was followed by a significant decrease in activity compared with controls at 15, 22, and 29 days PI, and a return to control levels by 43 days PI. The results suggest that CAA infection in young chickens can produce a dramatic decrease in immune competence, which, although transitory, is likely to seriously compromise the ability of birds to mount a successful immune response to invading pathogens. PMID- 1723874 TI - The intra-operative use of trasylol (aprotinin) in liver transplantation. AB - Aprotinin has been reported to reduce blood loss in difficult cases requiring cardiopulmonary bypass surgery and more recently in liver transplantation. Over a 9-month period we compared the effects of an intra-operative infusion of aprotinin on transfusion requirements and coagulation profiles in 12 patients undergoing liver transplantation for end-stage cirrhosis with an equal number of consecutive transplants in patients with similar pathology who did not receive aprotinin. Transfusion of blood and blood products was reduced to one-third in the aprotinin-treated group. Operative time was also significantly reduced, as was ICU stay post-operatively. Aprotinin profoundly inhibits fibrinolysis and this is likely to be the major effect by which blood loss is reduced. Thromboelastography revealed severe fibrinolytic changes in the anhepatic stage in 4 of 6 controlled patients; this accelerated in 3 following reperfusion of the new graft. By contrast, only 1 patient of 12 in the aprotinin-treated group showed fibrinolytic activity in the anhepatic period, and none showed evidence of fibrinolysis following reperfusion of the new graft. PMID- 1723875 TI - The development and repertoire of B-1 cells (CD5 B cells). AB - A small subset of mouse and human B cells produces much of the serum immunoglobulin, including many common autoreactive antibodies, and accounts for most cases of B-cell chronic lymphocytic leukemia. An exciting recent conference* focused on the development, repertoire and lineage classification of these cells. The meeting was convened for a discussion of 'CD5 B cells' but ended with a discussion of 'B-1 cells'. PMID- 1723876 TI - Shared epitopes among HLA class II alleles: gene conversion, common ancestry and balancing selection. AB - The extent and pattern of HLA class II sequence polymorphism raise a variety of evolutionary questions, notably those concerning the genetic mechanisms for generating diversity, the rate of change and the nature of the selection pressure maintaining this variation. Phylogenetic analysis of primate MHC class II sequences suggests that the allelic lineages are ancient, having diverged long before separation of the hominoid species. For the beta-chain loci, however, considerable allelic diversification within these lineages has occurred after speciation. The striking patchwork pattern of polymorphism with different alleles containing common sequence motifs can be accounted for by common ancestry, by gene conversion or by convergent evolution, depending on the location of the shared epitope. PMID- 1723877 TI - Maintenance of immunological memory: a role for CD5+ B cells? AB - How memory is retained is an immunological mystery. One possibility, argued here by Fons UytdeHaag and colleagues, is that memory is imprinted in the somatically mutated Ig expressed by certain CD5+ B cells. The theory proposes that the Ig expressed by this self-renewing population acts as surrogate antigen, selecting and stimulating emerging antigen-specific lymphocytes. PMID- 1723878 TI - Naturally-occurring peptide antigens derived from the MHC class-I-restricted processing pathway. AB - The extraction of naturally-processed peptides from MHC class I glycoproteins has paved the way for a major advance in the understanding of the antigen processing pathway that ultimately induces cytotoxic T-cell responses. Here, Olaf Rotzschke and Kirsten Falk review these new developments and discuss their findings in terms of a novel hypothesis of MHC class-I-restricted processing. PMID- 1723879 TI - Viral homologies with myelin basic protein. PMID- 1723880 TI - Locomotor sites mapped with low current stimulation in intact and kainic acid damaged hypothalamus of anesthetized rats. AB - To determine whether local neurons mediated the locomotor effects of electrical stimulation of the lateral hypothalamus, kainic acid injections (0.5-1.25 micrograms), intended to destroy neural somata as opposed to fibers of passage, were made unilaterally in the tuberal-posterior hypothalamus of 22 rats. The area of lesion and its contralateral homolog were mapped for locomotor stepping sites in Nembutal-anesthetized rats mounted in a stereotaxic apparatus such that locomotor stepping rotated a wheel. Stimulation (25 and 50 microA, 50 Hz, 0.5-ms cathodal pulses, 10-s trains) was delivered through 50-80 microns glass pipettes filled with 2 M saline. Contralateral to the lesion, locomotor stepping sites were common in the perifornical lateral and medial hypothalamus and less dense in the zona incerta. On the side of the kainic-acid lesion, locomotor sites were generally absent in the central part of the damaged area. If they did appear within the area of lesion, they tended to be near the border with intact tissue. In a few cases, locomotor stepping sites were found centrally located in the lesion amidst widespread loss of somata. In four rats, additional maps of anterior locomotor regions in the preoptic area ipsilateral to the lesion suggested that their descending fibers were largely spared by the kainic lesions. Local neurons appear to be major contributors to the locomotion elicited by electrical stimulation of the lateral hypothalamus, but fibers of passage may also participate. PMID- 1723881 TI - Plasma viraemia as a marker of viral replication in HIV-infected individuals. AB - Two hundred and twenty-eight blood samples from 154 HIV-seropositive subjects were investigated for the presence of HIV in peripheral blood mononuclear cells (PBMC) and plasma (without prior ultracentrifugation or filtration), using normal PBMC as the target for replication. HIV was recovered from PBMC and plasma in 80.5 and 19.5% of the patients, respectively. Plasma viraemia was significantly associated with clinical manifestations of HIV infection, indicating that HIV replication increased as disease progressed. This was confirmed by the statistically significant correlations between plasma viraemia and low CD4 cell counts (P less than 0.01) and low anti-p24 antibody titers (P less than 0.01). On patient follow-up, detection of HIV in plasma was transient. p24 antigenaemia was only detected in 13.6% of cases and was also associated with advanced clinical stages of the disease. When HIV RNA detection by polymerase chain reaction (PCR) was compared with plasma viraemia, HIV RNA was detected in plasma in all symptomatic cases and in 53.8% (seven out of 13) of asymptomatic patients [Centers for Disease Control (CDC) stages II and III], confirming that PCR was far more sensitive than plasma culture. These results indicate that cell-free virus production is associated with the clinical stage of HIV infection and may serve as a marker for disease progression. Detection of HIV RNA by PCR appears to be the most sensitive method to detect viraemia. PMID- 1723882 TI - Structure of diethyl 1,2,3,3b,4a,5,6,7,8,8a,8b,9-dodecahydro-1,5-dioxo-4,8,9 metheno-4H-cycl openta [1,2-a:4,3-a']dipentalene-4,10-dicarboxylate. AB - C24H24O6, Mr = 408.45, triclinic, P1, a = 10.276 (2), b = 13.000 (2), c = 7.782 (1) A, alpha = 98.06 (1), beta = 97.63 (1), gamma = 84.43 (1) degree, V = 1017 A3, Z = 2, Dx = 1.33 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.89 cm-1, F(000) = 432, T = 296 K, R = 0.048 for 3321 unique reflections with Fo2 greater than 3 sigma(Fo2). Both cyclopentenones are canted in the endo direction (6.0 and 7.9 degrees), the one syn to the ethoxy groups less so for steric reasons. When the ester groups are disregarded, the polyquinane portion of the molecule is seen to possess a non-crystallographic twofold axis. The overall molecular geometry is reconcilable with the absence of any significant alteration in the density maximum of the C--C pi bonds at this level of downward pyramidalization. PMID- 1723883 TI - Zinc deficiency, ethanol, and myocardial ischemia affect lipoperoxidation in rats. AB - The production of oxygen free radicals can be stimulated by excess iron, cadmium, nickel, and the like. Inversely, copper, zinc, and selenium inhibit production, either via their own action or via antiradical metalloenzymes. The study involved determining the effect of zinc deficiency combined with chronic ethanol administration on the status of blood and tissue free radicals, as well as on cardiac function in isolated, perfused rats' hearts. Animals were fed a basic diet containing residual zinc at 0.2-0.3 ppm. Following a zinc deficiency lasting 5 wk, which during the last 4 wk was accompanied by chronic ethanol administration, hearts were submitted to ischemia for 30 min in vitro, followed by reperfusion. Biochemical analyses (zinc, superoxide dismutase, malondialdehyde, conjugated dienes, and so on) were performed in the blood and in the homogenates of different organs. The experimental zinc deficiency caused a slight decrease of superoxide dismutase activity, accompanied by increased production of peroxidated lipids. Ethanol administration appeared to increase the levels of peroxidated lipids in the heart. Finally, the combination of zinc deficiency and ethanol administration had very harmful effects, especially on lipid peroxidation and contractile function of the isolated, perfused heart in preischemic conditions. PMID- 1723884 TI - The distribution and half-life for retention of vanadium in the organs of normal and diabetic rats orally fed vanadium(IV) and vanadium(V). AB - The concentration of vanadium in organs of diabetic rats that had been fed vanadium, either as V(IV) or V(V), in their drinking water has been determined. The kidney was found to have the highest concentration, about 185 nmol/g wet tissue. This averages about three times higher than for the liver or spleen, for which concentrations were comparable. The lung, blood plasma, and blood cells tended to have the lowest accumulations of vanadium. A time-course study indicated that the half-life for elimination of vanadium from the bodies of vanadium-fed rats is about 12 d. PMID- 1723885 TI - A reevaluation of the Fe(II), Ca(II), Zn(II), and proton formation constants of 4,7-diphenyl-1,10-phenanthrolinedisulfonate. AB - The compound 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid (BPDS) has been found to be very useful in studying Fe uptake by plants, because it forms a large charged complex that is not absorbed. The quantity of BPDS bound to metals in hydroponic solutions can be estimated from calculations using formation constants of BPDS complexes. These formation constants were used in an earlier experiment to predict the availability of Cu to corn plants. In the experiment, bioassays indicated that Cu was not as phytoavailable in the BPDS-added solutions as predicted by chemical equilibrium calculations. To determine sources of error in this prediction, metal and proton BPDS formation constants were reevaluated at 25 degrees C and 0.10M ionic strength. The CaBPDS formation constant was determined by direct measurement of CaBPDS3 formation and was shown to be approximately 1.0; a value much less than that reported before. Formation constants for the HBPDS, H(BPDS)2, and H(BPDS)3 beta 1, beta 2, and beta 3 complexes were, respectively, 5.05 +/- 0.044, 7.44 +/- 0.019, and 9.33 +/- 0.28. The BPDS sulfonic acid group pKs were less than 1.0, not 2.8 as has been reported. The FeBPDS3 complex determined by ligand competition with EDTA (ethylenediaminetetraacetate) was 20.24 +/- 0.08. Copper and Zn constants were determined using the method of corresponding solutions. The CuBPDS, CuBPDS2, and CuBPDS3 beta 1, beta 2, and beta 3 constants were, respectively, 9.76 +/- 0.08, 15.9, and 20.9. The ZnBPDS, ZnBPDS2, and ZnBPDS3 beta 1, beta 2, and beta 3 constants were, respectively, 6.43 +/- 0.07, 10.7 +/- 5.4, and 17.3 +/- 0.8. Results indicated that BPDS affinity to metals was similar to that of its parent compound, phenanthroline, and that errors in published formation constants caused erroneous predictions of Cu phytoavailability used in an earlier experiment. PMID- 1723886 TI - Effect of selenium compounds and thiols on human mammary tumor cells. AB - The effect on cell viability and growth rate of sodium selenite, selenocystine, sodium selenate, and selenomethionine at selenium concentrations of 6.25 and 12.5 uM was studied in vitro on cells of the human mammary tumor cell line HTB123/DU4475. Selenite and selenocystine affected both cell viability and growth rate of the tumor cells at these selenium concentrations. Selenite and selenocystine decreased intracellular glutathione concentrations, but did not affect tumor cell glutathione peroxidase activity. After six days of exposure to either selenate or selenomethionine, the viability of tumor cells remained stable, but cell growth, as measured by numbers of cells, was retarded. Neither selenate nor selenomethionine produced changes in concentrations of intracellular glutathione. The toxic effect of selenite on tumor cells was enhanced by addition of 0.25 mM glutathione to the growth medium. Preincubation of the tumor cells with 62.5 uM buthionine sulfoximine decreased cellular glutathione to 15% of controls at 24 h and enhanced the toxicity of selenite toward the tumor cells. Glutathione, 2-mercaptoethanol, and L-cysteine were all toxic to the tumor cells in a dose-dependent manner. PMID- 1723887 TI - Temporal changes in tissue glutathione in response to chemical form, dose, and duration of selenium treatment. Relevance to cancer chemoprevention by selenium. AB - Selenium has been reported to affect glutathione (GSH) concentrations in short term animal-feeding experiments. Given the central role that this tripeptide plays in maintaining cellular homeostasis, it was hypothesized that perturbations in glutathione metabolism induced by selenium might account for its cancer chemopreventive activity. In the present study, four experiments were conducted in which the effect of acute, short-, or long-term exposure to selenium was assessed. Selenium was provided as either sodium selenite or D,L selenomethionine. Selenite was observed to induce a biphasic response in total liver GSH. Injected selenium caused an acute reduction in GSH, whereas short-term feeding (up to 8 wk) increased both total GSH and oxidized glutathione (GSSH), an effect that gradually diminished in magnitude with prolonged feeding. Our data suggest that such changes are unlikely to account for the chemopreventive activity of selenium for the following reasons: Perturbations in glutathione metabolism occurred only at doses of selenite that approached toxicity. These doses are higher than what would be required for producing cancer chemoprevention. The transient nature of these changes also contrasts with the need for a continuous supplementation of selenite in suppression of tumorigenesis. Furthermore, selenomethionine was found to have little activity in altering glutathione metabolism, even though it compares favorably with selenite as a cancer chemopreventive agent. Nonetheless, these findings do not discount the possibility that sulfhydryl compounds, such as glutathione, might be used to modify the toxicity and/or enhance the cancer prophylactic activity of selenium compounds. PMID- 1723888 TI - A spectrophotometric study of the VO(2+)-glutathione interactions. AB - The interaction of the vanadyl (IV) cation with reduced glutathione (GSH) has been investigated by electronic absorption spectroscopy, at different metal-to ligand ratios and pH values. The interaction depends strongly on the initial VO2+/GSH ratio. Starting with a tenfold GSH excess, coordination takes place through the two carboxylate groups of the ligand, generating (at pH = 7) a blue 1:2 VO2+/GSH complex; this stoichiometry could be confirmed by photometric titration experiments. Higher GSH concentrations produce a violet complex, which can also be obtained by addition of GSH to the blue species. Some measurements with the three component amino acids of GSH, as well as results obtained from the VO3-/GSH system, allowed a wider insight into the characteristics of this violet complex, in which the cation interacts with S and N atoms of the peptide. PMID- 1723889 TI - Glucocorticoid effects on zinc transport into colostrum and milk of lactating cows. AB - Colostrum Zn concentrations were measured in eight randomly selected Holstein dairy cows. Overall mean Zn concentrations were highest within 12 h postpartum (257 +/- 14 microM, mean +/- SEM), fell to 141 +/- 8 microM by 24 h, and then declined at a linear rate of 30 microM/d during the following 48 h. Zn concentrations at 3 d (82 +/- 5 microM) were not different from 150-d milk samples (72 +/- microM). In a second experiment, 32 early-gestation cows were blocked by stage of lactation into four groups in a randomized block design and injected with 0, 15, 30, or 45 mg of dexamethasone. Milk and blood samples were collected at 0, 12, and 24 h after injection and analyzed for Zn, and for fat, protein, and lactose in milk. Cows administered 0 and 15 mg of dexamethasone showed no difference in milk Zn concentrations compared to pretreatment measurements; however, milk Zn concentrations in cows administered 30- and 45-mg doses increased significantly. Plasma cortisol decreased in the dexamethasone treated cows. Plasma Zn and milk fat, protein, and lactose did not change. These data indicate that glucocorticoids can mediate Zn uptake and transport by the mammary glands of lactating cows and suggest that the high Zn concentration in colostrum could be a result of the preparturient surge of cortisol. PMID- 1723890 TI - Cytotoxicity of zinc chloride in mice in vivo. AB - Intraperitoneal administration of zinc chloride (ZnCl2) to Swiss albino mice in vivo induced a significant (p less than or equal to 0.05) increase in the frequencies of chromosomal aberrations of the bone-marrow cells at all concentrations used following acute (7.5, 10, 15 mg/kg body weight) and chronic (2.0, 3.0 mg/kg body wt) treatment. The degree of clastogenicity was directly proportional to the concentrations (p less than or equal to 0.05, trend test) and indirectly to the period of treatment (p less than or equal to 0.05, ANOVA test). It induced a dose-dependent, statistically significant increase (Mann-Whitney U statistics, Student's t-test) in sperm-head abnormalities. The data designate ZnCl2 as a potent clastogen and as a toxic chemical at the concentrations used. PMID- 1723891 TI - Clinical effect of recombinant human granulocyte colony-stimulating factor (rhG CSF) on various types of neutropenia including cyclic neutropenia. AB - Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was investigated for its clinical efficacy in the treatment of various types of neutropenia (3 cases with idiopathic neutropenia of suspected drug induction, 5 cases with idiopathic neutropenia of other origin, and 2 cases with cyclic neutropenia). Treatment with glycosylated rhG-CSF produced in the Chinese Hamster Ovary cells at dose levels of 2-5 micrograms/kg/day caused rapid increases of neutrophil counts associated with an improvement of the infection. In cyclic neutropenia patients, marked reduction in the duration of the neutropenic period was observed with rhG-CSF administration started before the period. Intercurrent stomatitis, which occurred in 1 patient, was markedly milder as compared to a previous episode which occurred during an untreated neutropenic period. The treatment of rhG-CSF was well tolerated and no adverse events were observed, nor was there any detectable anti-rhG-CSF antibody in any patients studied; hence the clinical use of rhG-CSF is considered to be safe. These results suggest beneficial effects of rhG-CSF on the recovery of neutrophil counts in cyclic and other types of idiopathic neutropenias, as well as for the treatment of neutropenia-associated infection. PMID- 1723892 TI - Autocrine versus lymphocyte-dependent mechanisms for macrophage activation. PMID- 1723893 TI - CSF chromogranin A-like immunoreactivity in schizophrenia. Assessment of clinical and biochemical relationships. AB - Chromogranin A (CgA) is co-released with catecholamines and peptides and has a wide distribution in the brain. Chromogranin A provides a measure of tonic arousal. CSF CgA-like immunoreactivity (CgA-LI) was studied in 42 drug-free male schizophrenic patients. 33 of these patients were first studied during chronic haloperidol maintenance treatment. Withdrawal from haloperidol maintenance treatment was associated with a significant increase in CSF CgA-LI, particularly in the patients who did not relapse. Contrary to expectation CSF CgA-LI was higher in drug-free patients who slept longer the night before the lumbar puncture. Significant relationships were observed between CSF CgA-LI and CSF homovanillic acid, acetylcholinesterase, neuropeptide Y-LI and 5-hydroxy-indole acetic acid, but not with CSF norepinephrine or 3-methoxy-4-hydroxyphenylglycol. Ventricular brain ratios correlated negatively with CSF CgA-LI levels. PMID- 1723895 TI - Confocal microscopy of the lizard motor nerve terminals. AB - Confocal imaging was performed on the ceratomandibularis nerve muscle preparation of the lizard Anolis carolinensis, using 4-Di-2-ASP as a fluorescent probe. The imaging system consisted of a Sarastro Phoibos 1000 (Molecular Dynamics) scanning system and a Zeiss Universal microscope. The data were analyzed using the VANIS set of programs on a Silicon Graphics Personal Iris computer. A three dimensional reconstruction of the nerve terminals was performed using look-through and depth coded projections. The volume of the nerve terminal was estimated in 15 neuromuscular junctions and found to be 84,835 microns 3 (+/- 8558 SEM). The largest diameter in each one of the 178 individual boutons was estimated from the look-through projections of 17 nerve terminals in 9 preparations. It was found to be 4.71 microns (+/- 0.08 SEM). The diameter perpendicular to the largest diameter in the same projection at 0 degrees was 3.3 microns (+/- 0.054 SEM). Thus it seems that the synaptic boutons of the ceratomandibularis are suitable for combined optical and electrophysiological recordings. PMID- 1723894 TI - A comparison of the adsorption of three adhesive proteins to biomaterial surfaces. AB - The adsorption of three cell adhesive proteins with known thrombogenic activity [fibrinogen (FGN), fibronectin (FN), and vitronectin (VN)] was quantified from mono-component protein solutions, from a quaternary-component protein solution, and from plasma and diluted plasma in order to compare their potential for adsorption to polymeric substrates from solutions of varying complexity. The surfaces studied included polyethylene (PE), silicone rubber (SR), Teflon-FEP (FEP), and two polyetherurethanes: one with a poly(tetramethylene oxide) soft segment (PTMO-PU) and one with a poly(ethylene oxide) soft segment (PEO-PU). The adsorption of these proteins from single-component solutions followed the Freundlich isotherm and the adhesive proteins showed similar trends in Freundlich parameters for surfaces of similar surface wettability. Adsorption from a quaternary-component solution composed of physiological molar ratios of the three proteins and human serum albumin (HSA) revealed a significant enrichment of adsorbed vitronectin as determined from ratios of the adsorbed surface fraction of each protein to its respective bulk fraction. The other proteins' adsorption was enriched to a lesser extent in the decreasing order of FGN greater than FN greater than HSA for all surfaces. The relative enrichment of VN from plasma was also high as compared with its bulk concentration, whereas the enrichment of FGN, FN, and HSA was much lower and of approximately the same magnitude. Compared with the three other proteins, VN showed a resistance to displacement from the polymer substrates as either the plasma concentration was increased or the length of contact with plasma and diluted plasma was increased. PMID- 1723896 TI - Women and children. PMID- 1723897 TI - A tissue inhibitor of metalloproteinases and alpha-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown. AB - Immature female Sprague-Dawley rats were primed with 20 IU eCG at 28 days of age and treated with 10 IU hCG 48 h later. Ovulation followed at 12-14 h. Ovaries were extracted at various times after hCG by use of Triton X-100 and 10 mM CaCl2 (Triton extract) followed by heating to 60 degrees C for 6 min with 0.1 M CaCl2 in 50 mM Tris/0.15 M NaCl, pH 7.5 (heat extract). These extracts were tested for their ability to inhibit tissue metalloproteinases by use of the small uterine metalloproteinase (UMP) of the rat. The ovary contains three plasma-derived inhibitors (alpha 1-macroglobulin [alpha 1 M], alpha 2-macroglobulin [alpha 2 M], and alpha 1 inhibitor3 [alpha 1I3]) and one tissue-derived inhibitor of the tissue inhibitor of metalloproteinases (TIMP) family. alpha 1 I3 was shown to inhibit UMP and rat collagenase. The concentration of the tissue inhibitor rose 5 fold from 0.6 micrograms UMP blocked per gram wet tissue in ovaries not primed with eCG to 3.2 micrograms UMP blocked at 8 h after hCG. Over this same time interval, the sum of alpha 1M + alpha 2M per gram of ovary rose 7-fold from 3.2 to 22.4 micrograms UMP inhibited and alpha 1I3 rose 2-fold from 4.4 to 10.7 micrograms UMP inhibited. The increases in the tissue inhibitor are interpreted to be due to increased synthesis by the tissue, whereas the changes in alpha macroglobulins are postulated to be due to increased vascularity and increased permeability of the vessels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723898 TI - An antigenic determinant common to human chorionic somatomammotropin and human growth hormone revealed by limited proteolysis of immune complexes. AB - An epitope of human chorionic somatomammotropin for one of the monoclonal antibodies raised against the whole antigen has been identified. We compared the release of peptides from limited proteolysis of the antigen in the presence and absence of the related antibody. Using enzymes of different specificity, we could determine the amino acid sequence that can be considered at least inclusive of the epitope. The monoclonal antibody selected is 100% cross-reactive with human growth hormone, so the antigenic determinant identified is shared by the two protein hormones. PMID- 1723899 TI - Intercalation binding of 6-substituted naphthothiopheneamides to DNA: enthalpy and entropy components. AB - N-(3-dimethylaminopropyl)naphtho[2,1-b]thiophene-4-carboxamide and the 6 substituted methoxy, methyl, fluoro, chloro, bromo, trifluoromethyl, and cyano derivatives have been shown to bind to DNA via intercalation with binding constants in the 35-900 X 10(3) range at 25 degrees C, pH 7, and [Na+] = 0.019M. Both electron-donating and -withdrawing substituents enhance intercalation binding, but the binding affinity is most enhanced by the cyano substituent. Calorimetric titrations for calf thymus DNA differ dramatically from those reported for ethidium [Hopkins et al. (1990) Biopolymers Vol. 29, pp. 449-459]. Apparent enthalpy parameters (delta HB) for intercalation are constant only at low coverage of sites and become much more positive as saturation is approached. In the plateau region, delta HB values for the parent and the cyano-, fluoro-, chloro-, and bromo-substituted compounds are nearly the same (approximately -5.9 kcal/mol). For the methyl- (-6.8 kcal/mol) and methoxy- (-7.5 kcal/mol) substituted compounds, the delta HB values are more exothermic than that for the unsubstituted compound, whereas delta HB for the trifluoromethyl compound is approximately 1 kcal/mol less exothermic. The corresponding delta SB values, corrected for mixing effects, are in the 7-15-cal/deg/mol range and are approximately linearly related to delta HB if the cyano derivative is excluded. PMID- 1723900 TI - Influence of high cholesterol feeding on the pattern and progression of experimental cerebral ischemia. AB - The aim of our research was to study if cholesterol feeding might affect the ischemic changes in the vessels surrounding infarction foci in Sephadex G-75 induced cerebral ischemia model (SG-75). One hundred-twenty-four rabbits were divided as follows: group I was given standard food for 5 weeks; group II: as group I and then injected with SG-75; group III: standard food plus 1% cholesterol for 5 weeks; and group IV: as group III and then injected with SG-75. Rabbits were sacrificed 3 h, 6 h and 2, 5 and 7 days after ischemia had occurred. Vessels surrounding infarction foci (SIF) were identified by using a 6% carbon perfusion. Samples were examined by light microscopy and transmission electron microscopy (TEM). The occurrence of hemorrhagic infarction (HI) showed a clear time/course increase in group II whereas a decrease after 2 days in group IV was observed. The rate of HI was 40% and 20% in group II and IV, respectively. SIF vessels showed red blood cells leakage in group II, whereas multiple platelet thrombi appeared in group IV. This phenomenon caused a more extensive ischemic damage, when compared to group II. By making use of a widely employed model of high cholesterol diet and of a more physiological model of cerebral ischemia devised by us, we have provided the evidence that the hypercholesterolemia induced changes in the SIF vessels strongly affect the pattern and progression of cerebral ischemia. PMID- 1723901 TI - [Effects of cycloheximide on protein, RNA and DNA synthesis in cultures of CHO cells and human diploid fibroblasts]. AB - In CHO cell line and primary human diploid fibroblasts culture an incorporation of protein, RNA and DNA biosyntheses precursors was investigated under different conditions of inhibition of translation by cycloheximide (CHM). Both CHO and human fibroblasts transitory treatment by CHM in the serumfree medium resulted in inhibition of protein and DNA syntheses during S-period while RNA synthesis increased up to 130% (CHM concentration from 0.003 to 2 Mg/ml), as well as in Go- an incorporation of 3H-U increased to 200% (CHM concentration-100 Mg/ml). Long term treatment (48 hours) in the serum-free medium resulted in decreased uptake of 3H-T and 3H-L during first 6 hours of experiment, while incorporation of 3H-U increased to 160%. By 16-th hour of treatment characters of protein, RNA and DNA syntheses came back to control levels. PMID- 1723902 TI - [Study of chemically modified hemoglobin as an artificial oxygen carrier in the model of hemorrhagic shock in dogs]. AB - Hemodynamic and gas-transporting properties of the chemically modified hemoglobin solution have been studied on the model of hemorrhagic shock in dog. It has been shown that the polymerized hemoglobin solution exerts the hemodynamic action just as the plasma substitute "polyglucin" does. However, in contrast to the latter, polyhemoglobin circulating in the vascular bed for a prolonged period of time increases the blood oxygen capacity and oxygen delivery to tissues with the resultant increase in body total oxygen taking-up. PMID- 1723903 TI - [Immunochemical identification of onco-ovarian acid-soluble alpha-2-globulin]. AB - Antisera were obtained by rabbits immunization with pooled extract from ovarian carcinoma and its metastases into the ome tum. Using standard test-system antigen, precipitating in agar, was identified in the tissue of 12 out of 24 primary ovarian carcinomas and in 8 out of 20 metastases. It was not revealed in adult healthy internal organs tissues and in fetal tissues, except embryonal large intestine, where it was determined in trace amounts in isolated samples. Using immunodiffusion method it wasn't determined in blood serum of healthy people and patients, pregnant women and neonates. Immunochemical identification using standard test-systems showed that this antigen is not identical with already known carcino-embryonic antigen, placental, reactive and onco-ovarian proteins. It presents acid-soluble alpha-2-globulin (ASAG-2) with MW 55 kD determined by gel-filtration and 55 kD determined by electrophoresis in 10% polyacrylamide gel with dodecylsulfate under reducing conditions. Physico chemical and antigenic properties of ASAG-2, as we think, give the opportunity to present it as a new onco-ovarian antigen, which differs from the known proteins. PMID- 1723904 TI - [Role of pain component in the organization of chemosensory taste reaction]. AB - The investigation of algoinductor (histaminergic) and peptidergic relations in peripheral pain reaction of taste was performed by using of different histamine liberators (applied on tongue). By fluorescence-histochemical methods it was shown that histamine in the apical portion of papilla is derived from cells of taste buds and in the basal zone--from connective tissue cells (including mast cells). It was established in behavior trials on peptidergic system that consumption of taste solutions became changed. It was suggested that histaminergic structures together with SP-containing fibers ensure food controlling in oral cavity. PMID- 1723905 TI - Fibroblast growth factors: from genes to clinical applications. PMID- 1723906 TI - Placental transport of lindane during early and late stages of gestation in rats. PMID- 1723907 TI - Toxicity of beta- and gamma-hexachlorocyclohexane in rats of different ages. PMID- 1723908 TI - Residues of organochlorine pesticides in milk gland secretion of cows in perinatal period. PMID- 1723909 TI - Toxicity testing of aromatic hydrocarbons utilizing a measure of their impact on the membrane integrity of the green alga Selenastrum capricornutum. PMID- 1723910 TI - Toxicity and bioconcentration of hexachlorocyclohexane (HCH) in an air-breathing catfish, Saccobranchus fossilis (Bloch). PMID- 1723911 TI - Fibrinogen and fibrin formation and its role in fibrinolysis. PMID- 1723912 TI - Fibrinogen-fibrin: preparation and use of monoclonal antibodies as diagnostics. PMID- 1723913 TI - Hematopoietic colony-stimulating factors. AB - In summary, hematopoietic growth factors have been discovered, biochemically characterized, cloned, produced by recombinant DNA technology, and put into clinical use in a period of 25 years. We are approaching a greater understanding of the cellular anatomy and molecular mechanisms that regulate production of the CSFs, the ways in which the CSFs interact with their cell surface receptors and trigger their biological effects, the nature of these receptors themselves and their mechanisms of signal transduction, and the effects of the CSFs in vitro and in vivo on hematopoietic progenitor cells and mature leukocytes. However, many questions remain. What is the mechanism that couples growth-factor binding to the triggering of cellular proliferation? How do multi-CSF and GM-CSF cross-compete at the level of the cell-surface receptor, and yet show no primary amino acid sequence homology? What are the mechanisms that regulate the tissue expression profile of multi-CSF compared to the genetically similar growth factor GM-CSF? And, what are the optimal dosages, schedules of administration, and combinations of CSFs optimal for each of several conditions of marrow failure? These are but a few of the questions that continue to occupy much current research interest. PMID- 1723914 TI - Substance P modulates the time course of nicotinic but not muscarinic catecholamine secretion from perfused adrenal glands of rat. AB - 1. Substance P (SP) and acetylcholine (ACh) are contained within the splanchnic nerve terminals in the adrenal gland and can be released in response to stress. In the rat, the release of aCh brings about secretion of catecholamines (CA) by acting on nicotinic and muscarinic receptors on the adrenal chromaffin cells. 2. In the present study, we have used a rat isolated adrenal gland preparation to investigate the effects of SP, perfused at different concentrations, on CA secretion evoked by 10(-5) M nicotine and 10(-4) M muscarine. 3. In the first 10 min stimulation period (S1), in the absence of SP, nicotine (10(-5) M) evoked substantial and equal secretion of noradrenaline (NA) and adrenaline (Ad). In a second 10 min stimulation period (S2), carried out 18 min after S1, the nicotinic response was desensitized. In contrast, the muscarinic response, which preferentially evoked Ad secretion in S1 (Ad/NA: 8.7/1), was well maintained in S2. 4. SP present in S1 had no effect on desensitization of the subsequent nicotinic response in S2. 5. At low concentrations (10(-7)-10(-10) M), SP changed the time course of nicotine-induced CA secretion during S1 by enhancing CA secretion in the first 4 min and inhibiting CA secretion thereafter. The maximal effect occurred at 10(-9) M SP. 6. At a higher concentration (10(-5) M), SP inhibited total nicotinic CA secretion throughout S1 and produced a biphasic secretion of CA (depressed in the presence of SP and enhanced after wash out of SP). Pre-exposure of adrenal glands to SP (10-' to 10- M) for 10min produced marked inhibition of the nicotine-induced CA secretion. 7. In contrast to the effect of SP on the nicotinic response, SP from 10- to 10-SM had no effect on muscarinic CA secretion. 8. This difference in sensitivity of the nicotinic and muscarinic responses to SP points to a diversity of mechanisms available for control of adrenal catecholamine secretion. In addition to the ability of SP to increase or decrease the total amount of adrenal CA secretion, dependent on the concentration of SP, the present study shows that SP can change the time-course of nicotinic CA secretion. These results with the rat adrenal gland perfused in vitro suggests both a quantitative and temporal role for SP as a novel modulator of adrenal CA secretion. PMID- 1723915 TI - Nitric oxide synthase in cultured endocardial cells of the pig. AB - 1. Endocardial cells release factors which regulate myocardial contractility and guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels. One of these factors is indistinguishable from endothelium-derived relaxing factor (EDRF). 2. The effluent from pig heart endocardial cells cultured on microcarrier beads caused the relaxation of a pig coronary artery ring denuded of endothelium. This relaxation was enhanced by a combination of superoxide dismutase and catalase and was attenuated by haemoglobin, which binds nitric oxide (NO), and by inhibitors of NO synthase, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine. 3. A Ca(2+)-, L-arginine- and NADPH-dependent enzyme activity which generated NO was detected by a specific spectrophotometric assay in cytosol prepared from endocardial cells. The formation of NO was inhibited in a concentration-dependent manner by L-NMMA (but not D-NMMA) and this could be partially reversed upon addition of excess L-arginine. 4. Like endothelial cells from the blood vessels, the endocardial cells possess the ability to synthesize NO, which may act to regulate myocardial contractility. PMID- 1723916 TI - Prostacyclin activates tachykinin release from capsaicin-sensitive afferents in guinea-pig bronchi through a ruthenium red-sensitive pathway. AB - 1. We have investigated the ability of prostacyclin (PGI2) to contract guinea-pig isolated bronchi and the possible involvement of capsaicin-sensitive primary afferents in the response to PGI2. 2. PGI2 (0.1-100 microM) produced concentration-dependent contractions of the guinea-pig isolated bronchi. In vitro capsaicin desensitization (10 microM for 30 min followed by washing) significantly reduced the PGI2-induced contraction at all concentrations tested. A capsaicin-resistant component of contraction (40-60% of the overall response) was also evident. 3. Ruthenium red (3 microM), an inorganic dye which acts as a selective functional antagonist of capsaicin, significantly decreased PGI2 induced contractions, without affecting the response to substance P, neurokinin A or acetylcholine. 4. MEN 10, 207, (Tyr5, D-Trp6,8,9, Arg10)-neurokinin A (4-10) (3 microM), a selective antagonist of NK2-tachykinin receptors, significantly decreased PGI2-induced contractions and neurokinin A-induced contractions, without affecting the response to acetylcholine. 5. The effect of ruthenium red and MEN 10,207 on the one hand, and that of ruthenium red and capsaicin on the other was non additive. 6. These results indicate that PGI2-induced contraction of the guinea-pig isolated bronchi involves two distinct mechanisms, one of which involves transmitter (tachykinins) release from peripheral endings of capsaicin sensitive primary afferents. In as much as PGI2-activation of primary afferents is sensitive to ruthenium red, we suggest that PGI2 shares a common mechanism of tachykinin release with that activated by capsaicin. PMID- 1723918 TI - Sulfated glycoprotein-2 is increased in rat hippocampus following entorhinal cortex lesioning. AB - Thios study showed responses of sulfated glycoprotein-2 (SGP-2) in the rat hippocampus after deafferenting lesion. SGP-2 is a plasma protein that also occurs in many peripheral tissues. In some circumstances, elevations of SGP-2 mRNA are associated with cell degeneration and responses to injury. This study used entorhinal cortex lesions (ECL) to partially deafferent the hippocampus by damaging the perforant path and to induce synaptic remodeling. SGP-2 mRNA is increased in hippocampal astrocytes after ECL. Western blot analysis of soluble hippocampal proteins identified 3 major forms of rat SGP-2 protein: a precursor (61 kDa) and 2 reduced subunits at 39.5 and 35 kDa. These forms increased at 4 days post ECL ipsilaterally to the lesion. By immunocytochemistry (ICC), SGP-2 showed an increased immunoreactivity on the lesioned side by 2 days post ECL that continued through 14 days post ECL. Besides immunopositive astrocytes, punctate immunochemical reaction products occurred among the degenerating fibers of the perforant path. We conclude that changes of SGP-2 protein in the hippocampus after ECL occur roughly in parallel with increases of SGP-2 mRNA. The punctate immuno-deposits could represent secreted SGP-2 and may be useful as a marker for degenerating pathways. PMID- 1723917 TI - Coronary vasodilatation induced by endotoxin in the rabbit isolated perfused heart is nitric oxide-dependent and inhibited by dexamethasone. AB - The coronary vasoconstriction induced by the thromboxane mimetic U46619 (9, 11 dideoxy methanoepoxy 9 alpha, 11 alpha prostaglandin F2 alpha, 3-30 nM) was significantly attenuated in hearts obtained from rabbits treated with endotoxin (lipopolysaccharide, LPS, 200 micrograms kg-1, i.v.) 4 h before isolation of the heart. Under these conditions the vasoconstriction induced by two inhibitors of nitric oxide (NO) synthase, NG-monomethyl-L-arginine (L-NMMA) and N-iminoethyl-L ornithine (L-NIO) (1-100 microM for each) was significantly enhanced when compared to that induced in hearts from control animals. Both the decreased response to U46619 and the increased response to inhibitors of NO synthase were significantly attenuated by administration of dexamethasone (4 mg kg-1, i.v.) 90 min before treatment with LPS. These data are consistent with the induction, by LPS, of an NO synthase, and the inhibition of this induction by dexamethasone. The enhanced NO synthesis contributes to the haemodynamic changes known to occur in endotoxin shock. PMID- 1723919 TI - Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. III. Alterations following single and continuous beta, beta'-iminodipropionitrile administrations. AB - beta,beta'-Iminodipropionitrile (IDPN) administration produces giant neurofilament-filled axonal swellings in the first proximal internodes of large myelinated sensory and motor fibers without any accompanying axonal degeneration. In the present study, we asked whether proximal giant axonal swellings are sufficient to elicit aberrant neurofilament (NF) phosphorylation in neuronal perikarya. Rats were given a single intraperitoneal (i.p.) injection of IDPN (2 g/kg) followed by IDPN (0.1%) in the drinking water (continuous IDPN exposure) or tap water (single IDPN exposure) for two days to 7 weeks. Immunoreactivity to phosphorylated NF (pNF) epitopes (using monoclonal antibodies 6-17 and 7-05) was observed in L4 and L5 dorsal root ganglia (DRG) neurons beginning between one and 5 days, corresponding to the development of proximal giant axonal swellings. Quantitation of DRG neurons demonstrated maximal numbers of immunoreactive cell bodies to pNF epitopes (46-51%) by one week. The number of immunostained DRG cells was maintained in animals given continuous IDPN exposure, but declined significantly (P less than 0.001) in rats given a single injection of IDPN to 26 +/- 0.80% and 6 +/- 0.04% at 3 and 5 weeks, respectively. Ventral and dorsal root fibers, which undergo axonal atrophy distal to axonal swellings, showed intense immunoreactivity to pNF epitopes and a marked reduction or a complete lack of immunostaining to antibody 2-135 (directed against non-phosphorylated NF epitopes); pretreatment with alkaline phosphatase reversed this staining pattern. In a separate study, a similar alkaline phosphatase-sensitive lack of staining to antibody 2-135 was also observed in atrophic motor fibers in the DRG 4 weeks following nerve crush. It is suggested that aberrant NF phosphorylation in DRG neuronal cell bodies from IDPN-treated rats arises secondarily to an alteration in a retrogradely transported 'trophic' signal(s) to the neuron due to the presence of giant axonal swellings. Furthermore, pNFs in atrophic axons may correspond to stationary or slowly moving NFs in the axoplasm. PMID- 1723920 TI - Enkephalins, substance P and acetylcholine microinjected into the nucleus ambiguus elicit vagal bradycardia in rats. AB - Little is known about putative transmitters in the nucleus ambiguus (NA) mediating parasympathetic control of the heart, although Met-enkephalin (m-ENK), Leu-enkephalin (l-ENK), substance P (SP) and acetylcholine (Ach) have been detected in the cell bodies and fibers of this nucleus. The effects of these substances on arterial pressure (AP) and heart rate (HR) were studied by microinjecting them (4-20 nl) into the NA. Experiments were done in 26 spinal (high cervical) rats that were anesthetized with urethane and artificially ventilated. L-Glutamate (GLU) was microinjected into the right NA to identify the location of cell bodies from which decreases in HR and AP could be elicited. m ENK, l-ENK, SP or Ach was then microinjected into these sites. Microinjection of 1 nmol of GLU elicited significant decreases in HR (-72.2 +/- 9.7 bpm, n = 15) which were not accompanied by significant decreases in mean AP. Microinjection of m-ENK (15-200 pmol; n = 7), l-ENK (15-200 pmol; n = 6), SP (0.9-15 pmol; n = 7) and Ach (2.0-20 pmol; n = 7) into the NA decreased HR in a dose-dependent manner but did not affect AP. The magnitudes of HR responses to m-ENK, l-ENK, SP and Ach were smaller but of longer duration than the changes in HR to microinjection of GLU. These results suggest a physiological role for GLU, enkephalins, SP and Ach in the vagal control of HR mediated by the NA. PMID- 1723921 TI - Does insulin-like growth factor I (IGF-1) trigger the cell body reaction in the rat sciatic nerve? AB - Regeneration was measured after the infliction of a crush lesion on rat sciatic nerves which 4 days earlier had been subjected to a distal conditioning transection. Such nerves exhibited an increased outgrowth of nerve fibers as compared to nerves subjected to a single crush lesion. This increased outgrowth could be prevented, if the nerve was locally perfused around the site of the transection during the 4 days conditioning interval, with cycloheximide, actinomycin D and vinblastine, inhibitors of protein-, RNA-synthesis and retrograde axonal transport, respectively. The inhibitory effect of cycloheximide could be overcome by simultaneous perfusion with insulin-like growth factor I (IGF-1). The results suggest that proteins including IGF-1 which are synthesised locally around a nerve lesion and then transported retrogradely could trigger regenerative events in the neuronal cell body. PMID- 1723922 TI - Immunohistochemical localization of myelin-associated glycoprotein isoforms during the development in the mouse brain. AB - The developmental changes in localization of myelin-associated glycoprotein (MAG) isoforms in the mouse brain were demonstrated by an immunohistochemical method using antisera specific to two MAG isoforms. The antiserum to the large isoform of MAG (L-MAG) stained the myelin sheaths and the cytoplasm of oligodendroglia in the active myelinating stage in the mouse central nervous system. However, the antiserum to the small isoform of MAG (S-MAG) stained only myelin sheaths in the adult stage. These findings suggest that L-MAG plays an important role in active myelination. PMID- 1723923 TI - Differential alterations of second messenger systems and cerebral glucose use following excitotoxic lesion of rat cerebral cortex. AB - Quantitative autoradiography of [3H]forskolin and [3H]phorbol 12,13 dibutyrate (PDBu) binding was examined 21 days after unilateral lesioning of the rat visual cortex using ibotenic acid. In the same animals, the functional deficit was assessed using quantitative [14C]-2-deoxyglucose autoradiography. [3H]Forskolin binding was significantly reduced in each layer of the lesioned visual cortex by at least 40% compared to the contralateral hemisphere. Significant reductions in [3H]forskolin binding were observed in the superior colliculus (-15%) and dorsal lateral geniculate body (-12%) ipsilateral to the lesioned cortex. [3H]PDBu binding was significantly reduced in the lesioned visual cortex (layers V-VI) by 34%, compared with the control hemisphere. There were no significant alterations in [3H]DPBu binding in any other brain regions. Following ibotenate-induced lesioning of the visual cortex, glucose use was significantly reduced throughout the lesioned cortex by at least 25% with minor alterations in glucose use in the ipsilateral dorsal lateral geniculate body and superior colliculus. The present study highlights the relative robustness of [3H]PDBu binding compared to [3H]forskolin binding after excitotoxic damage to the cerebral cortex and suggests that [3H]forskolin binding sites are present on cortical efferent fibres. PMID- 1723924 TI - Novel inward rectifier blocked by Cd2+ in crayfish muscle. AB - The characteristics of a voltage- and time-dependent inward rectifying current were examined with voltage clamp techniques in crayfish muscle. The inward current, carried by K+, was activated by hyperpolarization. Although this inward current increased with the extracellular K+ concentration [( K+]o), the voltage dependence of the underlying conductance was independent of [K+]o. The current was unaffected by Cs+ and Ba2+, but was blocked by low concentrations of Cd2+. Therefore, this inward rectifier is different than previously described ones. PMID- 1723925 TI - Normal rate of Schwann cell proliferation in the MBP-deficient shiverer mouse during Wallerian degeneration. AB - Myelin basic protein (MBP) processed by macrophages was reported to promote Schwann cell division during Wallerian degeneration. In the present study we have shown that there was no difference in the rate of Schwann cell proliferation between shiverer mice, which totally lack MBP, and control mice after nerve transection. Furthermore, addition of autologous peritoneal macrophages in cultures led to enhanced thymidine uptake by Schwann cells, irrespective of the presence or absence of MBP. These results suggest that myelin components other than MBP play a role in Schwann cell proliferation induced by macrophages. PMID- 1723926 TI - Modulation of cortical in vivo acetylcholine release by the basal nuclear complex: role of the pontomesencephalic tegmental area. AB - Acetylcholine (ACh) release in vivo from rat cortices was determined by microdialysis either after injection of drugs into the basal nuclear complex (NBM) or after electrolytic lesion of the pontomesencephalic tegmental nucleus (PPT). Scopolamine (SCOP) (5-10 micrograms) increased and oxotremorine (10 micrograms) reduced cortical ACh release, indicating that an inhibitory mechanism operates within the area. The gamma-aminobutyric acid (GABA)ergic antagonist, picrotoxin (2.5 micrograms), by disinhibiting the cholinergic basocortical neurons, induced an increase that was not affected by SCOP. Acute lesion of the cholinergic PPT efferents to NBM raised cortical basal release. Thus, ACh released from the PPT terminals apparently modulates the function of basocortical neurons mainly through a polysynaptic link via GABAergic neurons. PMID- 1723927 TI - Immunocytochemical characterization of the suprachiasmatic nucleus and the intergeniculate leaflet in the diurnal ground squirrel, Spermophilus lateralis. AB - The suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) are retinorecipient structures that play important roles in the expression of circadian rhythmicity. We examined these two structures in a diurnal ground squirrel, Spermophilus lateralis, using immunohistochemical techniques, and cholera toxin-bound horseradish peroxidase. A number of immunoreactive substances are distributed within the ground squirrel SCN in a pattern similar to that reported in many other mammals. These include vasopressin, vasoactive intestinal polypeptide, serotonin, neuropeptide Y (NPY), and glial fibrillary acidic protein. The squirrel SCN differs from that of most other species examined to date in two respects. First, a dense cluster of cells containing immunoreactive L enkephalin (L-ENK-IR) is observed in the center of the SCN. Second, there is a contralateral, but no ipsilateral, projection from the retina to the SCN. In the lateral geniculate region there is a substantial region that contains NPY immunoreactive cells and receives a bilateral retinal projection. This region is assumed to be homologous with the IGL described in other mammals. Cells containing L-ENK-IR are distributed throughout the LGN in groups that overlap, but which have a distinctly different distribution than the more extensive groups of NPY-IR cells. PMID- 1723928 TI - Cranial nerve growth in birds is preceded by cholinesterase expression during neural crest cell migration and the formation of an HNK-1 scaffold. AB - The expression of the neural crest cell (NCC) markers acetylcholinesterase (AChE) and the HNK-1-epitope is compared from the emigration of cephalic NCC until the formation of the cranial nerves V-X in chicken and quail hindbrain. We show that NCC transiently express acetylcholinesterase (AChE) activity during their emigration; NCC migrate into butyrylcholinesterase (BChE)-positive areas of the cranial mesenchyme. Along these migratory tracks that foreshadow the course of later projecting cranial nerves, BChE increases strongly in cells that may represent immature Schwann cells. Both AChE and BChE, but not HNK-1, are expressed in the ectodermal placodes. In NCC, HNK-1 is expressed strongly only when they approach their destination sites. Their intense expression of HNK-1 then leads to the establishment of tunnel-shaped HNK-1 matrices, within which G4 positive cranial neurites begin to extend. We conclude that AChE and HNK-1 expression in cephalic NCC serve different functions, since AChE is related to their migration, and HNK-1 to their aggregation and the formation of an extracellular neurite scaffold. PMID- 1723929 TI - Morphological analysis of neovascularization at early stages of rat splenic autografts in comparison with tumor angiogenesis. AB - This study was undertaken to reveal the neovascularization at early stages of splenic autografts three-dimensionally, to illustrate the differences between it and tumor angiogenesis, and to establish its origin. Early vascular formation after transplantation of the rat spleen or Walker tumor into the major omentum was examined by using a video macroscope, vascular casting methods and the organ culture technique. A complex vascular network layer (vascular cortex) was first formed beneath the capsule of an autograft; later, vascular buds grew from this network toward the necrotic center. They anastomosed and changed into a form resembling withered twigs (vascular medulla). Tumor angiogenesis did not present such morphological features and was characterized by capillary loop formation with a columnar vertex resembling an "inverted V". This fundamental structure did not change throughout angiogenesis except for dilation and irregularity of vascular diameter. The organ culture technique demonstrated that the preliminary vasculature was formed in splenic autografts by regeneration of preexisting vessels in the graft and not by invading capillaries. Transmission electron microscopy showed that the cells present had characteristics of sinus endothelial cells. These results suggest that preexisting sinus endothelial cells rearrange themselves after devascularization and reconstruct a new vasculature that anastomoses with the penetrating capillaries. This mechanism establishes vascular circulation at an early stage, and accelerates regeneration of the splenic autograft before complete necrosis. PMID- 1723930 TI - A cytochemical and immunocytochemical study of DNA distribution in spermatid nuclei of mouse, rabbit, and bull. AB - DNA distribution in mouse, rabbit and bull spermatids was analyzed by electron microscopy, after using a Feulgen-like HCl-osmium ammine procedure, and after immunocytochemistry with anti-DNA antibodies. In addition, nucleic acids were visualized with the intercalating dye ethidium bromide and phosphotungstic acid. The parts of DNA displaying a beta helix configuration (possibly A-T rich parts) were identified by epifluorescence microscopy after staining with Hoechst 33258. In all 3 species, young spermatid nuclei were seen to have large areas poor in DNA, as well as DNA-rich areas, which were mostly concentrated into a peripheral layer close to the acrosome and into one or several masses, displaying species specific locations. These DNA-rich areas were stained with Hoechst 33258. Elongating spermatid nucleic contained homogeneously distributed DNA, and this was evident following both immunocytochemistry and nucleic acid histochemistry in all 3 species. However, the distribution appeared more heterogeneous after the Feulgen-like procedure, and was accompanied by a disappearance of Hoechst fluorescence. In fully elongated spermatids, all nuclear areas stained with Hoechst 33258, while the 3 other techniques labeled either all or species specific parts of the condensed chromatin. The reasons for these variable reactions are discussed in terms of technique specificities, DNA configuration and nucleoprotein moiety replacements. PMID- 1723931 TI - Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture. AB - Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall. PMID- 1723932 TI - Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis. AB - The expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of alpha-type cytokeratins in the alpha-keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with alpha- and beta-type keratin filament bundles in the beta-keratin-rich layers of epidermis known as the beta stratum. PMID- 1723933 TI - Expression of carbohydrate epitopes L2/HNK-1 and L3 in the larva and imago of Drosophila melanogaster and Calliphora vicina. AB - The carbohydrate epitopes L2/HNK-1 and L3 belong to two overlapping families of adhesion molecules in the vertebrate, and probably the invertebrate nervous systems. To investigate their pattern of expression during the development of insects, cryosections of late third instar larvae and imagoes of Drosophila melanogaster and Calliphora vicina were studied by indirect immunofluorescence using several monoclonal antibodies to the L2/HNK-1 and one monoclonal antibody to the L3 epitope. Each monoclonal antibody to the L2/HNK-1 epitope showed a different immunohistological staining pattern, which differed from that of the L3 monoclonal antibody. In both insect species the immunohistological staining patterns for the two carbohydrate epitopes were similar at the two developmental stages, with immunoreactivity not confined to the nervous system. In larvae, immunoreactivities of the monoclonal antibodies L2.334 and L3.492 were predominantly associated with the extracellular matrix as indicated by co localization with laminin, particularly in the imaginal discs, while L2.349 revealed a more cell surface-associated distribution. In imagoes, immunoreactivities were detectable in most organs studied. PMID- 1723934 TI - Dissociated cell culture of rat cerebral cortical neurons in serum-free, conditioned media: GABA-immunopositive neurons. AB - The gamma-aminobutyric acid (GABA)ergic properties of embryonic (E15d) rat cortical neurons were studied in dissociated serum-free culture by immunohistochemical methods. GABA-like immunoreactivity was found in a subpopulation of neurons from the first day onwards. The number of GABA-positive neurons reached mature values (10.5-12.6%) within the first week, while their morphological differentiation was not found to be fully completed until the 11th day of culture and was characterized by several discrete developmental stages. First, GABA-positive neurons gained their mature complement of neurites at 3 days in vitro (DIV). Three days later somal maturation became evident, followed at least by the maturation of the neuritic arbor. Double-labelling studies revealed the coexpression of GABA and tyrosine hydroxylase within the same cells. The similarities of relative number, morphology, time course of development and biochemistry of cultured GABAergic neurons compared with those in situ suggest that the applied culture system is a useful model to investigate several aspects of GABAergic neurotransmission at the cellular level. PMID- 1723935 TI - A transitory population of substance P-like immunoreactive neurones in the developing cerebral cortex of the mouse. AB - Immunocytochemical methods were used to investigate the developmental expression of substance P (SP) in mouse cerebral cortex. SP-like-immunoreactive cells were first detected at postnatal day 0 (P0), their numbers being notably increased by P2. Immunopositive cells were especially abundant in layer VIb and in the subjacent future white matter, although they were also present in layer V. Between P5 and P8 the number of SP-like-immunoreactive cells gradually decreased, being almost completely absent by P12. At these stages cells were only observed in the deepest cortical layers. From P16 onwards, the adult pattern of SP-like immunoreactivity emerged with a few immunopositive cells scattered throughout the cortical layers. The present data show a transitory population of SP-like immunoreactive cells present in the mouse cerebral cortex during the first postnatal week. On the basis of close correlations of SP-like expression with the distribution or transitory populations and the timing of cell death in rodents, we propose that most of the SP-like-immunoreactive cells reported here would probably disappear by cell death. PMID- 1723936 TI - Normal development and the effects of early rhizotomy on spinal systems in the rat. AB - The normal postnatal development of 4 spinal systems was examined in the dorsal horn of the rat spinal cord using histochemical and immunocytochemical techniques. We used thiamine monophosphatase (TMPase), a marker for dorsal root ganglion cells and their projections, a tachykinin, substance P (SP), which is provided by both dorsal root and intrinsic systems, and two markers for descending systems, serotonin (5-HT) and the synthesizing enzyme for noradrenalin, dopamine B-hydroxylase (DBH). The responses of each of these systems to unilateral dorsal lumbosacral rhizotomy on postnatal day 5 was then examined and quantified using image analysis methods to determine whether the extent of plasticity of spinal systems is different after a neonatal lesion than after a comparable lesion made in the adult. Each system differs in development, distribution, and in response to rhizotomy. TMPase is present in the dorsal horn on the day of birth (DPN0) and reaches adult levels of density by 5 days postnatal (DPN5). SP reaction product is present in a distribution similar to the adult in the dorsal horn on DPN0 and reaches adult levels of density by the second postnatal week. 5-HT is present in the dorsal horn on DPN0, shows a laminar distribution at DPN5, and acquires the adult distribution and density at the end of the second week. DBH is present in the dorsal horn on DPN0, acquires the adult distribution at DPN5 and adult levels of density at the end of the second postnatal week. Unilateral lumbosacral rhizotomy in 5 day old rats completely and permanently abolishes TMPase in the dorsal horn by 4 days postoperatively (4DPO). SP is decreased by 4 DPO (9 DPN) but recovers almost completely by 30 DPO. 5-HT is increased by 10 DPO and remains elevated thereafter. DBH is not changed postoperatively. There is shrinkage of lamina I and II by 10 DPO but the recovery of SP and the increase in density of 5-HT staining is proportionally greater than the extent of shrinkage. Therefore, shrinkage contributes to but does not entirely account for either the apparent recovery of SP staining or the increase in density of 5-HT staining. The responses of the TMPase, 5-HT and DBH systems to neonatal rhizotomy are very similar to the response to rhizotomy in adults and there is therefore no evidence for greater plasticity of these systems after neonatal rhizotomy than after adult rhizotomy. The SP systems show more rapid depletion and a greater and more rapid recovery than after adult deafferentation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723937 TI - [A case of schwannomatosis--clinical, pathological and biochemical studies]. AB - A case of schwannomatosis is described, including clinical, pathological and biochemical features. A 16-year-old male patient was admitted because of multiple subcutaneous tumors without family history. No cafe au lait spots were found. Magnetic resonance images (MRI) revealed multiple tumors of cranial and spinal nerves. The tumors in the scalp, right forearm and left spinal nerve at the level of C5 were excised out surgically. The pathological study of all tumor specimens showed a typical appearance of schwannoma with Antoni A and B tissues but not that of neurofibroma. Electrophoretic study of the extract from the tumor detected a basic protein at a molecular weight of 18.5 KD, which has been reported to be a tumor marker protein of benign schwannoma. From these findings, this patient had the features of schwannomatosis, clearly distinguished from those of neurofibromatosis. PMID- 1723938 TI - Temporal pattern of skeletal muscle changes in lambs fed cimaterol. AB - The objectives of this study were to compare the efficacy of 3-week vs 6-week dietary administration of the beta-adrenergic agonist cimaterol on skeletal muscle growth, and to measure the changes in muscle nucleic acid and protein concentration and content to provide evidence regarding the mechanism(s) by which cimaterol stimulates muscle hypertrophy in growing ruminants. Two groups of 12 Dorset or Dorset-Finn cross ram lambs weighing 36 kg or 33 kg were assigned to treatment intervals of 3 or 6 weeks, respectively. Lambs within each weight group were randomly assigned to receive 0 or 10 ppm cimaterol in a complete mixed diet fed ad libitum. Initial live weights and treatment periods were chosen to achieve similar slaughter weights. Cimaterol increased the mass of three hind leg muscles 30% and 25% on average (both P less than .001) with 3- and 6-week administration, respectively, resulting in identical average muscle weights of treated lambs at both treatment intervals. The mean mass of these 3 muscles, expressed as a percentage of body weight, was increased 18.6% (P less than .001) at both treatment intervals. RNA concentration and content of the semitendinosus muscle were increased 24.8% (P less than .01) and 84.6% (P less than .001), respectively, after 3 weeks of treatment, but neither was significantly different from controls after 6 weeks. DNA concentration in the muscle was reduced 42% (P less than .05) with 3-week cimaterol administration, and was 25% less than controls (P greater than .05) in lambs fed cimaterol for 6 weeks. Total DNA content of the semitendinosus was unchanged at either treatment interval.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1723939 TI - [The jockey mobile genetic element codes a DNA polymerase similar to retroviral reverse transcriptase]. PMID- 1723940 TI - In vivo formation of codeinone and morphinone from codeine. Isolation and identification from guinea pig bile. AB - Codeinone (CO) and morphinone (MO) were isolated and identified in the bile of guinea pigs given sc injections of codeine. Authentic CO was synthesized and characterized by the NMR and mass spectra of its 2-mercaptoethanol (ME) adduct. This material was then used as the standard to identify the CO-ME adduct in the bile of codeine-treated animals. The MO-ME adduct was also identified in the bile with authentic materials prepared earlier. The results of our investigations indicated that 10.5 and 2.7% dose of CO and MO, respectively, were produced for 6 hr after the codeine was given. The metabolites were separated by preparative HPLC on a reverse phase column packed with C18 gel using a 10 mM sodium phosphate buffer, pH 6.8/CH3CN, 1:1 (v/v) as an eluate. For the further purification of metabolites, we used another reverse phase column with the same mobile phase. A structural elucidation of the ME adduct of metabolites was then performed by fast atom bombardment mass spectroscopy and 400 MHz fourier transform-NMR spectrometric analysis, and identified as (8S)-(2 hydroxyethylthio)dihydrocodeinone and (8S)-(2-hydroxyethylthio)dihydromorphinone, respectively. PMID- 1723941 TI - Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. AB - We have adapted a non-radioactive technique to detect localized mRNAs in whole mount Xenopus embryos. Synthetic antisense RNA transcribed in the presence of digoxygenin-UTP is used as a probe and is detected via an anti-digoxygenin antibody. We show that localized mRNAs can be detected from late gastrula to tadpole stages and that high as well as low abundance RNAs can be detected. The method was tested on muscle actin and alpha-globin RNAs, whose localization has previously been characterized. In addition, we used the method to determine the distribution of XA-1 RNA, an anterior ectoderm-specific RNA, which we show is expressed in the periphery of the cement gland as well as in the region of the hatching gland. The sequence of an XA-1 cDNA predicts a protein rich in proline and histidine. PMID- 1723942 TI - Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro. AB - The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Here we studied the expression of tenascin along developing peripheral nerves in chick embryos and tested its activity as a substrate for cultured neurons. Motor axons grow out through the tenascin-rich, anterior part of the sclerotome. Shortly after, tenascin surrounds axon fascicles of ventral roots. At the limb levels, outgrowing axons accumulate in the tenascin containing girdle region forming a plexus. In the limb, tenascin first appears in bracket-like structures that surround the precartilage cell condensations of the femur and humerus, respectively. These regions coincide with the channels along which axons first grow in from the girdle plexus to form the limb nerves. Later, the major tenascin staining is associated with the cartilage and tendon primordia, and not with the limb nerves. We used tenascin as a substrate for cultured neural explants and single cells in order to test for its function in neurite outgrowth. Dissociated embryonic neurons of various types attached to mixed polylysine/tenascin substrates and sprouted rapidly after a lag of several hours. Outgrowth was inhibited and neurites were detached by anti-tenascin antibodies. On substrates coated with tenascin alone, neurite outgrowth was achieved from 3 day spinal cord explants. Whereas growth cones were well spread and rapidly moving, the neurites were poorly attached, straight and rarely branched. We speculate that in vivo tenascin allows axonal outgrowth, but inhibits branching and supports fasciculation of newly formed axons. PMID- 1723943 TI - Differential cytokeratin gene expression reveals early dorsal-ventral regionalization in chick mesoderm. AB - The induction and spatial patterning of early mesoderm are known to be critical events in the establishment of the vertebrate body plan. However, it has been difficult to define precisely the steps by which mesoderm is initially subdivided into functionally discrete regions. Here we present evidence for a sharply defined distinction between presumptive dorsal and presumptive ventral regions in early chick mesoderm. Northern blot and in situ hybridization analyses reveal that transcripts corresponding to CKse1, a cytokeratin gene expressed during early development, are present at high levels in the presumptive ventral mesoderm, but are greatly reduced or undetectable in the future dorsal region of mesoderm, where the formation of axial structures occurs later in development. This distinction is present even while the mesoderm layer is being formed, and persists during the extensive cellular movements and tissue remodelling associated with morphogenesis. These results point to an early step in which two fundamentally distinct states are established along the presumptive dorsal ventral axis in the mesoderm, and suggest that determination in this germ layer occurs in a hierarchical manner, rather than by direct specification of individual types of histological differentiation. The differential expression of CKse1 represents the earliest molecular index of dorsoventral regionalization detected thus far in the mesoderm. PMID- 1723944 TI - Primary neurons that express the L2/HNK-1 carbohydrate during early development in the zebrafish. AB - In zebrafish, many nerve pathways in both the CNS and periphery are pioneered by a small and relatively simple set of 'primary' neurons that arise in the early embryo. We now have used monoclonal antibodies to show that, as they develop, primary neurons of several functional classes express on their surfaces the L2/HNK-1 tetrasaccharide that is associated with a variety of cell surface adhesion molecules. We have studied the early labeling patterns of these neurons, as well as some non-neural cells, and found that the time of onset and intensity of immunolabeling vary specifically according to cell type. The first neuronal expression is by Rohon-Beard and trigeminal ganglion neurons, both of which are primary sensory neurons that mediate touch sensitivity. These cells express the epitope very strongly on their growth cones and axons, permitting study of their development unobscured by labeling in other cells. Both types initiate axogenesis at the same early time, and appear to be the first neurons in the embryo to do so. Their peripheral neurites display similar branching patterns and have similar distinctive growth cone morphologies. Their central axons grow at the same rate along the same longitudinal fiber pathway, but in opposite directions, and where they meet they appear to fasciculate with one another. The similarities suggest that Rohon-Beard and trigeminal ganglion neurons, despite their different positions, share a common program of early development. Immunolabeling is also specifically present on a region of the brain surface where the newly arriving trigeminal sensory axons will enter the brain. Further, the trigeminal expression of the antigen persists in growth cones during the time that they contact an individually identified central target neuron, the Mauthner cell, which also expresses the epitope. These findings provide descriptive evidence for possible roles of L2/HNK-1 immunoreactive molecules in axonal growth and synaptogenesis. PMID- 1723945 TI - A point mutation in the proteolipid protein gene of the 'shaking pup' interrupts oligodendrocyte development. AB - The differentiation of the oligodendrocyte from its bipotential progenitor culminates in the production of the myelin-specific proteins and the elaboration of membrane processes that ensheath the axon. Mutations in proteolipid protein (PLP) and its alternatively spliced isoform DM-20, the major protein constituents of central nervous system myelin, are characterized by a significant reduction in the number of mature oligodendrocytes, resulting in severe hypomyelination, tremor and early death. The canine shaking pup carries such a mutation, a single base change that substitutes a proline for a histidine near the first transmembrane region of PLP and DM-20. This mutation hinders oligodendrocyte differentiation, as evidence by a splicing pattern at the PLP locus characteristic of immature oligodendrocytes. The spliced transcript expressed earliest in development, DM-20, continues to be overexpressed in shaking pup oligodendrocytes. The disruption of the normal maturation schedule in these X linked dysmyelinating disorders suggests that PLP or DM-20 plays a fundamental role in oligodendrocyte development. We propose that, while the more abundant PLP is the primary structural component of myelin, DM-20 may be critical to oligodendrocyte maturation. PMID- 1723946 TI - Morphological and radiochemical evidence for the metabolism of exogenous proteins by the preimplantation sheep blastocyst. AB - The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical. The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined. The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues. After culture, the cellular morphology of the explants was well preserved. Fresh and 24 h cultured tissues were incubated with horse-radish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast. Comparison of the uptake of [3H]dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis. During culture of explants of blastocyst with 125I labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins. That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with beta-lactamase labelled with [14C]leucine. A major product (Mr approximately 17 x 10(3) present in the medium was likely to have been ovine trophoblast protein-1. It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis. Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation. PMID- 1723947 TI - Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies. AB - In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells. PMID- 1723948 TI - In situ hybridization analysis of TGF beta 3 RNA expression during mouse development: comparative studies with TGF beta 1 and beta 2. AB - To date, three closely-related TGF beta genes have been found in the mouse; TGF beta 1, TGF beta 2 and TGF beta 3. Previous experiments have indicated that TGF beta 1 and TGF beta 2 may play important roles during mouse embryogenesis. The present study now reports the distribution of transcripts of TGF beta 3 in comparison to the other two genes and reveals overlapping but distinct patterns of RNA expression. TGF beta 3 RNA is expressed in a diverse array of tissues including perichondrium, bone, intervertebral discs, mesenteries, pleura, heart, lung, palate, and amnion, as well as in central nervous system (CNS) structures such as the meninges, choroid plexus and the olfactory bulbs. Furthermore, in several organ systems, TGF beta 3 transcripts are expressed during periods of active morphogenesis suggesting that the protein may be an important factor for the growth and differentiation of many embryonic tissues. PMID- 1723949 TI - Structure and expression pattern of the murine Hox-3.2 gene. AB - The murine homeobox-containing gene Hox-3.2 is the most 5' member of the Hox-3 complex on chromosome 15 isolated to date. Conceptual translation of the longest ORF gives a protein of 260 amino acids lacking the conserved hexapeptide found in most homeobox genes. Northern analysis detects three transcripts of 1.5, 1.9 and 3.2 kb in day 9 to 15 p.c. embryos. As early as day 8.5 p.c., transcripts can be detected in the posterior part of the embryo by in situ hybridization. At this developmental stage no or only very weak expression is visible in the neural plate. At day 10.5 Hox-3.2 is detected in the ventral part of the neural tube with a sharp anterior boundary at the level of the third thoracic prevertebra. This anterior boundary remains at day 12.5 and day 14.5. In contrast to Hox-3.1, Hox-3.2 is not expressed in the dorsal horns containing the sensory neurons at day 14.5 p.c. Hox-3.2 transcripts are also detected in the posterior prevertebrae, the hindlimb buds and the cortex of the developing kidney. Unlike Hox-1.4 and Hox-1.3 and their paralogs, Hox-3.2, -2.5 and -4.4 (5.2) show strikingly different anterior boundaries of expression in the CNS and prevertebrae. PMID- 1723950 TI - Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. AB - Several mouse genes designated 'Pax genes' contain a highly conserved DNA sequence homologous to the paired box of Drosophila. Here we describe the isolation of Pax8, a novel paired box containing clone from an 8.5 day p.c. mouse embryo cDNA library. An open reading frame of 457 amino acids (aa) contains the 128 aa paired domain near the amino terminus. Another conserved region present in some other paired box genes, the octapeptide Tyr-Ser-Ile-Asn-Gly-Leu-Leu-Gly, is located 43 aa C-terminal to the paired domain. Using an interspecies backcross system, we have mapped the Pax8 gene within the proximal portion of mouse chromosome 2 in a close linkage to the surf locus. Several developmental mutations are located in this region. In situ hybridization was used to determine the pattern of Pax8 expression during mouse embryogenesis. Pax8 is expressed transiently between 11.5 and 12.5 days of gestation along the rostrocaudal axis extending from the myelencephalon throughout the length of the neural tube, predominantly in two parallel regions on either side of the basal plate. We also detected Pax8 expression in the developing thyroid gland beginning at 10.5 days of gestation, during the thyroid evagination. In the mesonephros and metanephros the expression of Pax8 was localized to the mesenchymal condensations, which are induced by the nephric duct and ureter, respectively. These condensations develop to functional units, the nephrons, of the kidney. These data are consistent with a role for Pax8 in the induction of kidney epithelium. The embryonic expression pattern of Pax8 is compared with that of Pax2, another recently described paired box gene expressed in the developing excretory system. PMID- 1723951 TI - Effects of phosphodiesterase inhibition on the excitability of hippocampal pyramidal neurons in vitro. AB - Extracellular field potentials were evoked in the CA1 pyramidal cell layer of the isolated rat hippocampus by electrical stimulation of the stratum radiatum. Of the three phosphodiesterase (PDE) inhibitors, 3-isobutyl-1-methylxanthine (IBMX), zardaverine and rolipram, only the adenosine receptor antagonist, IBMX, increased the amplitudes of the extracellular excitatory postsynaptic potential (EPSP) and population spike (PS). The beta-adrenoceptor agonist, isoproterenol, also facilitated these potentials and became more potent in the presence of zardaverine or rolipram. The results suggest that PDE blockade increases the excitability of pyramidal neurones only after preceding stimulation of beta adrenoceptors. PMID- 1723952 TI - Regulation of cardiovascular sympathetic neurons by substance P and gamma aminobutyric acid in the rat spinal cord. AB - The spinal regulation of cardiovascular sympathetic preganglionic neurons by substance P (SP) and gamma-aminobutyric acid (GABA) was investigated in conscious rats. Intrathecal injection at the T-9 spinal level of bicuculline, a GABAA receptor antagonist, evoked increases in mean arterial pressure (MAP) and heart rate (HR) which were maximal at 5.0 and 0.5 nmol, respectively. Phaclofen, a GABAB receptor antagonist, produced no cardiovascular changes up to 2 mumol while 10 mumol evoked a rise in MAP and HR. Muscimol, a GABAA receptor agonist, produced a decrease in MAP which was maximal at 5.0 nmol and had no effect on HR. Baclofen, a GABAB receptor agonist, was without cardiovascular effects up to 5.0 nmol, while 50 and 100 nmol evoked a fall in MAP and HR. The pressor response to SP (16.25 nmol, T-9) was antagonised by 0.5-50 nmol muscimol or baclofen in a dose-related manner and the pressor response to SP was still inhibited by 40 nmol GABA in capsaicin-treated animals. However, when SP was injected at T-2, the rise in both MAP and HR was blocked by 50 nmol baclofen. Similarly, 50 nmol muscimol blocked the rise in both MAP and HR induced by 15 nmol thyrotropin-releasing hormone. In contrast, 50 nmol glycine failed to alter the cardiovascular response to SP co-injected either at T-9 or T-2. Baclofen was found to reduce significantly the basal release of epinephrine when injected at the T-9 level. These results provide pharmacological evidence for a possible tonic GABAergic inhibitory input onto cardiovascular sympathetic preganglionic neurons mediated by GABAA and GABAB receptors. PMID- 1723953 TI - Functional characterization of the muscarinic receptor in rat lungs. AB - The effects of various muscarinic antagonists on antigen- and acetylcholine induced bronchoconstriction were studied. In isolated and ventilated lungs of naive rats, the pA2 values with respect to acetylcholine-induced bronchoconstriction were 9.01 (atropine), 8.39 (ipratropium bromide), 7.39 (pirenzepine), 5.94 (AF-DX 116, a M2-selective muscarinic antagonist), 6.91 (UH AH 37, a novel muscarinic antagonist) and 9.37 (4-DAMP: 4-diphenylacetoxy-N methylpiperidine methobromide). Except for ipratropium bromide, the slopes of the Schild plots were not significantly different from unity. None of the drugs were potent or effective in inhibiting bronchoconstriction or histamine release evoked by antigen challenge in actively sensitized rats. However, in vivo, in anesthetized spontaneously breathing rats, vagotomy and atropine (1 mg/kg) did reduce antigen-induced bronchoconstriction. It is concluded that functional muscarinic receptors in isolated rat lungs are probably of the M3 receptor subtype. With respect to antigen-induced bronchoconstriction and mediator release in a denervated model such as the isolated lung, they are of little, if any, importance. In vivo, vagotomy and atropine reduced antigen-induced bronchoconstriction, probably by blockade of a vagal reflex which is thought to play a role in antigen-evoked bronchoconstriction. PMID- 1723954 TI - Exchange of copper between serum albumin and bleomycin. AB - 1. The present in vitro study shows that bleomycin is able to take up copper from albumin, which functions as a copper transport protein in plasma. 2. Calculations, based on plasma concentrations of Cu(II)-albumin and bleomycin, suggest that only a small fraction of bleomycin (less than 3%) may directly accept copper from Cu(II)-albumin. PMID- 1723955 TI - [Determining the character of extrasystole by the results of functional tests]. PMID- 1723956 TI - Plexiform fibrohistiocytic tumour: clinicopathological, immunohistochemical and ultrastructural analysis in favour of a myofibroblastic lesion. AB - Plexiform fibrohistiocytic tumour is a recently described, seemingly benign neoplasm of superficial soft tissue which is poorly recognized and the differentiation pattern of which remains obscure. Fourteen new cases are presented here. These presented predominantly in the upper limb of infants and children, although the age-range was wide. A morphological spectrum depending on the relative proportions of the spindle cellular and nodular histiocyte-like components was evident. Immunohistochemical analysis revealed positivity of tumour cells in both components for smooth muscle actin, suggestive of myofibroblastic differentiation, as was borne out ultrastructurally in two cases. In addition, a minority of the histiocyte-like cells were also CD68 positive but negative for leucocyte common antigen, HLA-DR, Mac387 and lysozyme. In view of the ultrastructural and other immunohistochemical results, this is regarded as further evidence that the CD68 epitope recognized by KP-1 is not confined to cells of monocyte/macrophage or myeloid lineage. Plexiform fibrohistiocytic tumour appears to be a clinicopathologically distinctive myofibroblastic neoplasm which may warrant reclassification in due course. PMID- 1723957 TI - Expression of ICAM-1, VCAM-1 and ELAM-1 in angiofollicular lymph node hyperplasia (Castleman's disease): evidence for dysplasia of follicular dendritic reticulum cells. AB - The inducible adhesion molecules mediate important functions in the lymphoid tissues. We have investigated the expression of intercellular adhesion molecule 1 (ICAM-1), endothelial leucocyte adhesion molecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1), and platelet endothelial cell adhesion molecule (PECAM/CD31), using immunocytochemistry on cryostat sections of five lymph nodes from patients with Castleman's disease of the hyaline-vascular type. All five cases were characterized by marked hyperplasia of follicular dendritic reticulum cells, which were extensively present even in the mantle zone. Hyperplastic follicular dendritic reticulum cells showed marked expression of VCAM-1, and weak expression of ICAM-1. In two cases, several dysplastic giant cells with aberrant, polyploid nuclei showed aberrant expression of ELAM-1, an endothelium-restricted molecule. Dysplastic giant cells were positive with DRC-1 (an antibody to dendritic reticulum cells), VCAM-1 and occasionally ICAM-1, were negative for the endothelial cell markers factor VIII-related antigen and CD31 and were non proliferating (Kl-67-). Cells positive for ICAM-1 or VCAM-1 were rare in the interfollicular areas. In all cases vascular hyperplasia was prominent, but endothelial cells were poorly activated in terms of expression of inducible adhesion molecules and of HLA-DR antigens. The possibility that dysplastic follicular dendritic reticulum cells have a pathogenetic role in Castleman's disease is discussed. PMID- 1723958 TI - Morphology and immunohistochemistry of carcinoma in situ adjacent to testicular germ cell tumours in adults and children: implications for histogenesis. AB - Observations differ on the pre-invasive malignant lesions associated with the various categories of testicular germ cell tumours. Such lesions have been found to be similar in appearance and are assumed to be composed of multipotent cells, or conversely a distinctive pre-invasive stage has been reported in association with each form of germ cell neoplasm. This study was undertaken to see whether distinctive morphological and immunohistochemical features of carcinoma in situ adjacent to various categories of germ cell tumours could be established. Carcinoma in situ adjacent to seminomas, teratomas and mixed germ cell tumours in 18 adults was indistinguishable morphologically. Placental alkaline phosphatase was demonstrated immunohistochemically but vimentin and low molecular weight cytokeratins were uniformly absent in these abnormal germ cells from all three groups. These findings support the concept of a multipotent pre-invasive malignant cell for both seminoma and teratoma in the adult. Carcinoma in situ was not seen adjacent to 15 spermatocytic seminomas, nor was placental alkaline phosphatase demonstrated in tubules adjacent to these tumours. These negative findings are additional evidence that spermatocytic seminoma differs from classical seminoma in its histogenesis. Carcinoma in situ, as defined morphologically and immunohistochemically in adults, was not identified adjacent to yolk sac tumours and differentiated teratomas in 20 prepubertal testes. The possibility that pre-invasive malignancy in children may not resemble that in adults must be considered when assessing the malignant potential of cryptorchid testes on biopsies taken during orchidopexy. PMID- 1723959 TI - Polymorphous low-grade (terminal duct) adenocarcinoma of the parotid gland. PMID- 1723960 TI - Metastasizing phyllodes tumour with malignant fibrous histiocytoma-like areas. PMID- 1723961 TI - Cell adhesion and focusing of inflammatory responses. PMID- 1723962 TI - [Determination of pancreatic blood flow at the early stage of acute haemorrhagic necrotizing pancreatitis induced by sodium taurocholate in rats]. AB - Acute haemorrhagic necrotizing pancreatitis was induced by injecting 5% sodium taurocholate (Na-Tc) directly into the common biliopancreatic duct in rats. In control group 0.9% NaCl was used. The activity of serum lipase and amylase distinctly increased at 3 h and went up to the maximum at 12 h after injection of Na-Tc. The pancreatic blood flow and tissue perfusion per gram increased apparently at 1 h and decreased at 12 h after injection of Na-Tc by using the fractional indicator distribution technique with 86RbCl. The results demonstrated that the early stage of acute haemorrhagic necrotizing pancreatitis induced by Na Tc in rats was still a primary inflammatory response. PMID- 1723963 TI - [Immunohistochemical observation on keratin filaments of cultured tumor cells by ABC staining]. AB - Avidin-Biotin Peroxidase complex technique, ABV staining, was employed by using monoclonal anti-keratin antibody HK2 in this study. The organization and dynamics of keratins in both interphase and mitotic T56 and HeLa cells were analysed. We also observed the effects of microtubule (MT) and microfilament (MF) inhibitors, colchicine and cytochalasin B, on the organization of keratin filaments in T56 and HeLa cells. The results showed that a significant alteration in the structural organization and distribution of keratin filaments occurred during mitosis, and an extensive rearrangement of keratin networks of the two cell lines was induced in interphase after the MT and MF were disrupted by combined treatment with the two drugs, colchicine and cytochalasin B; the keratin networks turned into a star-like lattice rapidly within 1-2h. Neither colchicine nor cytochalasin B alone elicited significant organizational change in the keratin networks of the two cell lines. PMID- 1723964 TI - Biological microemulsions: Part III--The formation characteristics and transport properties of saffola-aerosol OT-hexylamine-water system. AB - The results of formation, phase behaviour and physical properties of biological microemulsions prepared from saffola/AOT/hexylamine/water in presence of different additives, viz. cholesterol, crown ether, urea and brine, are presented. It has been found that the additives and temperature have striking effects; mono-, bi- and triphasic solutions interchanging proportions among themselves. The conduction of microemulsion at different [Water/AOT] ratios (w = 9,10,14,18,20,39 and 45) has shown conspicuous dependence on temperature with a significant degree of percolation, whereas the dependence of viscosity on temperature has shown normal declining trend with temperature. A maximum in viscosity with respect to its variation with amount of water has been observed. The Walden product (lambda eta) has evidenced noncompensation of ion transport by conduction with the viscosity of the medium. The activation energies evaluated for conduction (delta E*cond) and viscosity (delta E*vis) are systematic except at [Water/AOT] ratio, w = 20. The additives cholesterol, crown ether and their mixture have shown a decreasing effect on the delta E*cond for percolation, whereas delta E*vis has increased in their presence. The bicontinuous microemulsion has the prospect for use as liquid membrane. PMID- 1723965 TI - Intraductal hepatocellular carcinoma with normal liver--case report. AB - Hepatocellular carcinoma commonly invades the portal vein but is rarely seen in the bile duct. When seen, a minor intraductal component usually accompanies a prominent hepatic involvement. We report a case of hepatocellular carcinoma that entirely involved the common bile duct, the hepatic involvement was undiscernible at operation or ultrasonography. The patient had obstructive jaundice both at first presentation and at recurrence. The liver was normal at both explorations. The elevated AFP levels returned to normal after second resection. The diagnosis was confirmed by electron microscopy. PMID- 1723966 TI - CD-13 ('gp150'; aminopeptidase-N): co-expression on endothelial and haemopoietic cells with conservation of functional activity. AB - This report details experimental results which show the presence of enzymic aminopeptidase-N-like activity on endothelial cells, concomitant with cell surface expression previously detected by both ELISA and indirect immunofluorescence. This activity, detected using selected chromogenic substrates in 'functional' assays, is shown to be due (at least in part) to a molecule previously termed 'gp 150' and recognized by MoAb belonging to CD-13, since such antibodies can be shown to inhibit this activity. Activity, as detected on endothelial cells, is similar to that observed on various haemopoietic cells. These assays not only provide a functional basis to cell surface gp150 (CD-13) co expression on haemopoietic and endothelial cells but have also been used to help define structural epitopes present on aminopeptidase-N/gp150, previously analysed using radiolabelled antibodies in competitive binding assays. Appraisal of these new data suggests that the number of distinct antibody binding sites on this molecule is greater than that previously demonstrated. This study is therefore an important first step in investigating the potential involvement of this selective peptidase molecule in the control of haemopoietic cell growth and differentiation and in haemostatic mechanisms. PMID- 1723967 TI - Low affinity binding of mouse immunoglobulin to human CD5+ B cells. AB - Polyreactive immunoglobulin (Ig) secreted by CD5-bearing B cells has the capacity to bind a broad range of self and foreign antigens. Flow cytometric analysis was used to detect low-affinity binding of mouse Ig molecules to the surface of CD5 bearing B cells from patients with chronic lymphocytic leukaemia (CLL). Mouse Ig isotypes G, A, and M, and IgG subclasses G1, G2a, and G3 bound to the B cell surface via a mechanism not involving the antigen-binding site of the mouse Ig molecule. Fab and F(ab')2 fragments of mouse Ig associated with the CLL cells in a similar manner to intact Ig, indicating that Fc receptor interactions were not involved. Dissociation of the mouse Ig from the B cell surface by three washing steps distinguished this lower-affinity binding from high-affinity binding which occurs through the antigen-binding site of a mouse monoclonal antibody (MoAb) when it recognizes a specific cell surface epitope. Blocking studies suggest that the low-affinity binding occurred via surface IgM and surface IgD (sIgM/sIgD) on the CD5-bearing B cells. The results are consistent with the expression of polyreactive Ig on the surface of CD5-bearing B cells in CLL. PMID- 1723968 TI - Palliative treatment of esophageal carcinoma using esophageal dilation and prosthesis. AB - Esophageal cancer is incurable in most patients. Tumor anatomy must be carefully defined using radiographic and endoscopic techniques. These techniques can also provide useful information to plan palliative treatment. The goals of palliation must be explicitly discussed and defined with the patient and family. Palliative manipulation is best done by a physician with experience in the procedures, after consideration of all available options to ensure effective palliation with minimal risk of complications. Esophageal dilation is an integral part of most palliative treatment programs, either as sole or adjunctive therapy. Dilation can maintain luminal patency in most patients and can be performed easily, effectively, and safely in an outpatient setting. An esophageal prosthesis can further alleviate symptoms in patients in whom more conventional palliative techniques are unsuccessful. Because prosthesis placement is associated with a relatively high rate of complications, it should be reserved for patients with advanced refractory disease or tracheo-esophageal fistula, for whom no other palliative alternatives exist. PMID- 1723969 TI - Palliative treatment of esophageal carcinoma using laser and tumor probe therapy. AB - Laser therapy and BICAP tumor probe therapy offer effective and safe means of palliating obstructive esophageal cancer. Future developments may allow more precise application of these techniques, based on more sensitive means of identifying abnormal tumor tissue. The development of endoscopic ultrasonography and the use of tissue sensitizing agents (such as hematoporphyrin derivatives) may allow more accurate means of differentiating normal from abnormal tissue. This may lead to more precise and extensive tumor destruction, less injury to normal tissue, and less potential for complication. As the use of both of these modalities becomes more widespread, their role in the treatment of esophageal cancer will continue to grow. PMID- 1723970 TI - Surgical treatment of esophageal carcinoma. AB - The view of surgical treatment of esophageal carcinoma is pessimistic. This is so because (1) most patients present late in the disease and are beyond the benefits of surgical therapy for cure, (2) resection is associated with a high operative mortality, and (3) the poor 5-year survival rates for surgical resection cited in the literature are poor, although these rates may include procedures done for palliation and not for survival. Early disease is potentially curable. With a better understanding of the biology of the tumor and with improved staging techniques, patients who are able to undergo an en bloc resection designed for cure according to the classic principles of surgical oncology are selected. Five year survival rates for curative en bloc resections range from 40% to 50%. For patients unable to undergo a curative resection, esophagectomy with esophagogastrostomy offers the best palliation. Transhiatal esophagectomy performed through an abdominal and cervical incision is an appropriate palliative procedure for tumors located in the lower third and cervical portions of the esophagus. The standard esophagectomy using a right thoracotomy is the preferred technique for a palliative resection of tumors located in the upper and midthoracic esophagus. Unresectable tumors may be palliated by intubation and, in some specific situations, surgical bypass. PMID- 1723971 TI - Monozygotic twins with sickle cell anemia and discordant clinical courses: clinical and laboratory studies. AB - We describe a rare set of monozygotic twins with coexistent sickle cell anemia and alpha-/alpha alpha thalassemia who have asynchronous painful crises of different frequency and severity. Studies include measurements of cell deformability and other hemorheologic tests, cell density distribution, the percentage of irreversibly sickled cells, adherence of red cells to endothelial cells, membrane heme and membrane free iron, calcium containing internal vesicles and serum antioxidants. Results of these studies, including estimates of organ damage (bone, spleen, retina), were similar except for an increase in red cell membrane free iron in the patient with more frequent and severe painful crises. The study supports the concept that non-inherited factors are important contributors to the frequency and severity of painful crises in sickle cell anemia. PMID- 1723972 TI - Retroviral transfer of a human fetal globin gene carrying the -202 G gamma beta (+)-HPFH mutation into the human erythroleukemia line, KMOE. AB - The presence of point mutations at position -202 relative to the mRNA Cap site of both human fetal gamma-globin genes is linked with elevated fetal globin levels in adults. The question addressed in this study is whether the -202 mutation affects gamma-globin gene expression in the same manner as the -117 hereditary persistence of fetal hemoglobin (HPFH) A gamma-globin mutation. The -117 mutation was found to cause over-expression and confer inducibility of a retrovirally transferred gamma-globin gene in cytosine arabinoside (araC)-treated KMOE cells in an earlier study. In this study, fetal globin genes driven by either the normal G gamma or -202 HPFH G gamma-globin promoter were retrovirally transferred into human erythroid KMOE cells. The -202 HPFH mutation did not cause over expression or confer inducibility of the transferred gamma-globin gene in araC treated KMOE cells. Thus, the -202 HPFH mutation affects gamma-globin gene expression by a different mechanism than the -117 HPFH mutation. Furthermore, this study provides evidence against a general increasing of gamma-globin gene expression as might be expected from the -202 mutation altering binding of a ubiquitous factor such as Sp1. PMID- 1723973 TI - Expression of fibronectin receptor (integrin) in the uterus of rats in relation to the estrous cycle. AB - The expression and localization of fibronectin receptor (integrin), fibronectin, laminin and collagen type IV in the endometrium of the rat uterus during each period of the estrous cycle were investigated by immunofluorescent microscopy. Fibronectin receptor was observed at the epithelial cells of the endometrium and at vascular endothelial cells. At proestrus, when epithelial cells actively migrate, fibronectin receptor was observed at the basal and lateral epithelial cell surfaces. During estrus, fibronectin receptor had begun to disappear, little fibronectin receptor was observed at metestrus or diestrus. No prominent changes in the localization of fibronectin (seen at the vascular endothelial cells and in the stroma) or of laminin and collagen type IV (seen at the muscles and at the basement membranes of the epithelial and vascular endothelial cells) were observed in relation to the estrous cycle. Thus, uterine epithelial cells, like epithelial cells of the healing cornea, increase their expression of fibronectin receptor during active migration, probably facilitating their attachment to stromal fibronectin. This fibronectin-fibronectin receptor mechanism may underlie epithelial repair, whether the defect results from physiological processes or from an insult. PMID- 1723974 TI - Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs. AB - Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion. PMID- 1723975 TI - Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study. AB - Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather large capacity for PGH synthesis and the present results may provide a basis for a better understanding of both normal physiological functions as well as intestinal disease states involving disorders of prostaglandin synthesis. PMID- 1723976 TI - Topology of chromogranins in secretory granules of endocrine cells. AB - Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra granular topology are of special interest with respect to their putative functions. Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities. Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide- and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules. PMID- 1723977 TI - Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation. AB - Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells. PMID- 1723978 TI - Immunocytochemical localization of a galanin-like peptidergic system in the brain and pituitary of some teleost fish. AB - Immunostaining of brain and pituitary sections of teleost fishes (eels, salmonidae, cyprinidae, gourami, sculpin, mullet) with anti porcine galanin (GAL) revealed the presence of immunoreactive (ir) perikarya and a rich network of fibers. Ir-perikarya were located rostrodorsally to the recessus preopticus, and in the posterior tuberal hypothalamus. Ir-fibers were abundant in basal telencephalon and around diencephalic ventricular recesses but never contacted their lumen. Furthermore, they were observed in basal hypothalamus, brainstem and ventral medulla. Ir-fibers passed along corticotropic (ACTH), gonadotropic cells and somatotropes (GH cells) in eel and trout pars distalis, but rarely ended in caudal neurohypophysis. In goldfish pituitary ir-fibers occurred in neural digitations and among different cell types which however did not contain a GAL like peptide. The relation GAL fibers/GH cells appeared more evident in species with a high growth rate. The other species showed a similar distribution of brain GAL. In eels and trout, ir-perikarya were not observed in areas containing somatostatin, GH- and ACTH-releasing factor, and ACTH-like perikarya, suggesting that GAL did not coexist with these peptides. The widespread distribution of a GAL-like peptide in teleost brain suggests that it could play a role of neurotransmitter and/or neuromodulator and regulate the secretion of adenohypophysial hormone(s). PMID- 1723979 TI - Ontogeny of enhancing factor in mouse intestines and skin. AB - Enhancing Factor (EF) is a 14 kDa protein isolated from mouse small intestines, which enhances the binding of 125I-EGF to A431 cells. This observation as well as our earlier in vitro studies have indicated that EF is a modulator of EGF. In adult mice, localization of EF by immunohistochemistry shows it is present predominantly in the Paneth cells of the small intestines and to a lesser extent in the stomach and colon. This study of the ontogeny of EF shows that the appearance of the protein coincides with the appearance of mature Paneth cells. In new born mouse skin EF is localized in the hair follicles in the first hair cycle from day 2 to day 8. It is however absent in the adult skin. Thus EF is associated with tissues which have a high growth rate. PMID- 1723980 TI - Shared dilemmas of research and practice in developmental and behavioral pediatrics: introduction to the special issue on methodology. AB - Research in developmental and behavioral pediatrics is characterized by special methodological problems involving measurement, design, and analysis related to research questions, populations, and nature of interventions in this field. To sensitize researchers and practitioners to these problems and to enhance the quality of research and critical consumption of research reports, this special issue provides a summary of basic methodological issues in developmental and behavioral pediatrics. Advances in this field will require the kind of critical observation and decision making that are shared by researchers and practitioners and are illustrated by contributions to this issue. PMID- 1723981 TI - A guide to critical reading of the literature in behavioral and developmental pediatrics. AB - The field of behavioral-developmental pediatrics covers a wide range of topics, problems, and conditions. A variety of professionals including pediatricians, psychologists, child psychiatrists, social workers, nurses, and educators are involved in research and clinical care. Each of these disciplines uses a variety of methodologies, each of which has its advantages and disadvantages. Different categories of research and methods of critiquing the literature in behavioral and developmental pediatrics are described. PMID- 1723982 TI - The meaning of measures: pitfalls in behavioral and developmental research. AB - This article considers a number of issues encountered in the selection and use of measures in research in the area of behavioral and developmental pediatrics. In addition to considering basic psychometric principles, it is argued that problems inherent in the developmental process, the lack of correspondence between study and standardization samples, and methodological factors affecting measurement may threaten the validity of individual measures and bias research outcome. A number of recommendations are made to counter the effects of these common measurement problems that indirectly result from the absence of a "gold standard" for measuring behavior and development. PMID- 1723983 TI - Methodological considerations in pediatric behavioral research: measurement. AB - Measurement is the critical building block underlying all science. Adequate measures should demonstrate reliability, validity, and sensitivity. Special problems associated with the selection and development of measures used in behavioral pediatric research are reviewed. These include measurement specificity, construct complexity, the illusive "gold standard," construct independence, theoretical clarity, timing of measurements, and developmental considerations. The advantages and limitations of a variety of behavioral assessment strategies are described: questionnaires, interviews, behavioral observations, permanent products, biological measures, and provider ratings. Programmatic measurement research not only improves the science of behavioral pediatrics but also has widespread clinical application. PMID- 1723984 TI - Data analysis techniques in behavioral pediatrics: some practical advice. AB - A review of the last three volumes of the Journal of Developmental and Behavioral Pediatrics revealed that 27% of the articles used correlations, 17% chi 2, 16% t tests, 15% analysis of variance or covariance, and 13% involved regression analysis. Multivariate techniques, discriminant or factor analysis, logistic regression, and sensitivity/specificity were used in less than 5% of articles; the average number of statistical techniques per article was 2.5. These statistical techniques are described in general, conceptual terms in regard to appropriate and inappropriate usage. Clinical examples are provided. Familiarity with these techniques allows the reader "statistical access" to almost 90% of the articles in behavioral pediatrics and other areas of the medical literature. PMID- 1723985 TI - Adjustment for cofactors in pediatric research. AB - Behavioral and developmental pediatric research often seeks to form causal inferences from associations between variables obtained in nonrandomized studies. To do this it is necessary to distinguish the effects of the independent variable of interest from other factors with which it is correlated. We review statistical adjustment procedures for assessing the effects of the independent variable after controlling for other variables, called cofactors. We present guidelines for cofactor adjustment for different patterns of causal relationships occurring in the developmental pediatric literature. We also review procedures for selecting a subset of cofactors from a larger array of candidate variables. Finally, we examine common methodological complications related to cofactor adjustment, including the presence of measurement error in the independent variable and/or the cofactors. PMID- 1723986 TI - Design issues in a randomized clinical trial of a behavioral intervention: insights from the Infant Health and Development Program. PMID- 1723987 TI - Electron-microscopic analysis of synaptic input from the perigeniculate nucleus to the A-laminae of the lateral geniculate nucleus in cats. AB - The perigeniculate nucleus of carnivores is thought to be a part of the thalamic reticular nucleus related to visual centers of the thalamus. Physiological studies show that perigeniculate neurons, which are primarily GABAergic, provide feedback inhibition onto neurons in the lateral geniculate nucleus. However, little is known about the anatomical organization of this feedback pathway. To address this, we used two complementary tracing methods to label perigeniculate axons for electron microscopic study in the geniculate A-laminae: intracellular injection of horseradish peroxidase (HRP) to fill an individual perigeniculate cell and its axon; and anterograde transport of Phaseolus vulgaris leucoagglutinin to label a population of perigeniculate axons. Labeled perigeniculate terminals display features of F1 terminals in the geniculate neuropil: they are small, contain dark mitochondria, and form symmetric synaptic contacts. We found that most of the perigeniculate terminals (greater than 90%) contact geniculate cell dendrites in regions that also receive a rich innervation from terminals deriving from visual cortex (e.g., "cortico-recipient" dendrites). The remainder of the perigeniculate synapses (10%) contacted dendrites in regions that also received direct retinal input (e.g., "retino-recipient" dendrites). Serial reconstruction of segments of dendrites postsynaptic to perigeniculate terminals suggests that these terminals contact both classes of relay cell in the A-laminae (X and Y), although our preliminary conclusion is that an individual perigeniculate cell contacts only one class. Finally, our quantitative comparison between labeled perigeniculate terminals and unlabeled F1 terminals indicates that these perigeniculate terminals form a distinct subset of F1 terminals. We quantitatively compared the labeled perigeniculate terminals to unlabeled F1 terminals. Although the parameters of the perigeniculate terminals fell entirely within the range of those for the unlabeled F1 terminals, as populations, we found consistent differences between these two groups. We thus conclude that, as populations, other sources of F1 terminals are morphologically distinct from perigeniculate terminals and innervate different targets. PMID- 1723988 TI - Human olfactory epithelium in normal aging, Alzheimer's disease, and other neurodegenerative disorders. AB - By use of immunohistochemistry, we characterized the molecular phenotype of human olfactory epithelial (OE) cells and assessed the nature of the dystrophic olfactory neurites described initially in Alzheimer's disease (AD). Keratin 8 was present in all classes of OE cells. Sustentacular cells lacked other cell type specific polypeptides and were distinguished from neurons and basal cells because the latter two classes of OE cells expressed neural cell adhesion molecules (N CAMs) and microtubule associated proteins (MAPs), i.e., MAP5. Basal cells expressed nerve growth factor receptors (NGFRs), which distinguished them from olfactory neurons. Unlike their perikarya, olfactory axons expressed vimentin and GAP-43, but not peripherin or neurofilament (NF) proteins. Olfactory nerves were distinguished from other axons because the latter were positive for all three NF subunits and peripherin, in addition to vimentin and GAP-43. Dystrophic neurites in the OE were GAP-43 positive, but they also expressed proteins that were not detected in normal olfactory nerves (i.e., synaptophysin, MAP2, tau, peripherin, NF proteins). Further, rare NF positive olfactory neurons gave rise to NF positive dystrophic neurites. These neurites were present in all 11 AD cases, 11 of 14 subjects with other neurodegenerative diseases, and 6 of 8 neurologically normal adult controls, but no dystrophic neurites were seen in 9 fetal and neonatal cases. We conclude that the molecular phenotype of different human OE cells is distinct and that dystrophic olfactory neurites occur very frequently in neurologically normal adults. The relevance of these neurites to aging or specific disease processes remains speculative. PMID- 1723990 TI - Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction. AB - Afferent pathways from the urinary bladder were examined with axonal tracing techniques in normal female Wistar rats and in those with partial urethral ligation. Following injection of wheat germ agglutinin-horseradish peroxidase (HRP) into the bladder wall, HRP was detected in lumbosacral dorsal root ganglion cells and in afferent projections to the L6-S1 spinal cord at sites in laminae I, II, V-VII, and X known to receive visceral afferent input. Partial urethral ligation (6 weeks) produced a sixfold increase in bladder weight and altered the morphology of bladder afferent pathways. Changes included an increase in the average cross-sectional area of labelled neuronal profiles in L6 and S1 dorsal root ganglia in obstructed (766 +/- 378 microns 2, P less than 0.001) compared to control rats (528 +/- 189 mu 2). The cross-sectional area of the largest profiles also increased by approximately 40%. The mean number of labelled dorsal root ganglion cell profiles was similar in ligated (837 +/- 198) and control (883 +/- 352) groups. When compared to control animals the obstructed animals exhibited a 60% increase in the area of the labelled afferent terminal field in the intermediolateral region of the L6-S1 spinal cord. This increased labelling was even more remarkable given that the volume of tracer per bladder weight injected into the hypertrophied bladder was 87% less than controls. These results provide evidence that bladder afferents project to regions of the spinal cord known to regulate micturition and that these afferents can undergo morphological alterations and/or changes in axoplasmic transport in response to urethral ligation. Changes may occur in response to increased target organ mass, increased neural activity, or alterations in the levels or activity of neurotrophic factors. PMID- 1723989 TI - Acoustic chiasm. III: Nature, distribution, and sources of afferents to the lateral superior olive in the cat. AB - The outcomes of seven experiments are reported, each directed to the nature and sources of the excitation and inhibition impinging on the lateral superior olive (LSO) in cats. In the first experiment, we used conventional 14C 2-DG methods to determine the specificity, precision, and extent of symmetry in the stimulation reaching LSO from the ipsilateral and contralateral ears. In Experiment 2, we sought the presence of GABA and glycine receptors in LSO using conventional, in vitro receptor-binding methods. On the basis of these results, we used in vitro high-affinity uptake methods in Experiment 3 to seek evidence that some of the terminals as well as the receptors in LSO are glycinergic. In Experiment 4, we used immunocytochemical methods to show that the somata known to supply the contralateral projections to LSO, and their terminals in LSO, are each immunoreactive with an antibody directed to a glycine-protein conjugate. In Experiment 5, we made use of a glycinergic neuron's avidity for transporting glycine retrogradely to label the likely sources of the glycinergic terminals in LSO. In Experiment 6, we used immunocytochemical methods to show that the spherical and globular cells of the ventral cochlear nucleus and terminals in LSO and in MTB are glutamatergic and/or aspartergic. In Experiment 7, we used receptor binding methods to determine whether the glutamate/aspartate receptors in LSO are probably of the kainate or of the quisqualate type. The results of the several experiments suggest that probably glutamate-quisqualate synapses mediate LSO's ipsilaterally driven excitatory responses and glycinergic synapses mediate its contralaterally driven inhibitory responses. The two types of input appear to be well matched in LSO's medial and middle limbs with glycinergic terminals mostly perisomatic and glutamatergic terminals mostly peridendritic. However, LSO's low frequency lateral limb appears to be somewhat different; it receives less stimulation from the contralateral ear. Instead, LSO's lateral limb may receive some of its glycinergic input directly from the ipsilateral ventral cochlear nucleus and/or indirectly via the juxtaposed lateral nucleus of the trapezoid body. PMID- 1723991 TI - Connectional studies of the primate lateral geniculate nucleus: distribution of axons arising from the thalamic reticular nucleus of Galago crassicaudatus. AB - Anterograde and retrograde transport methods have been used to explore the interconnections between the thalamic reticular nucleus (TRN) and the dorsal lateral geniculate nucleus of Galago crassicaudatus. We first defined the region of the TRN, which is connected to the lateral geniculate nucleus, by examining the distribution of geniculo-TRN axons, cortico-TRN axons arising from area 17, and the location of TRN-geniculate neurons. Following an intraocular injection of 3H-proline/3 H-leucine, trans-synaptically transported protein is present bilaterally within the lateral portion of the caudal TRN. This same caudal and lateral region is also targeted by cortico-TRN axons and contains neurons which project upon the lateral geniculate nucleus. Light microscopic anterograde transport methods were used to analyze the distribution of TRN-geniculate axons. Our data reveal that all layers and interlaminar zones of the dorsal lateral geniculate nucleus contain TRN axons. Electron microscopic-autoradiographic data support and extend our light microscopic findings by revealing labeled TRN terminals within all geniculate layers. These TRN profiles are the same size throughout the geniculate and exhibit morphological characteristics similar to F1 terminals described by others. That is, they possess predominantly pleomorphic vesicles, a dark cytoplasmic matrix, dark mitochondria, and symmetrical synaptic contacts. Two additional features of TRN terminals have been observed in some profiles. These include dense-core vesicles and a dense, punctate cytoplasmic matrix, which is sometimes associated with the postsynaptic specialization. In addition to their morphology and size, the postsynaptic targets of TRN terminals are similar within the three sets (parvi-, magno-, and koniocellular) of geniculate layers. TRN profiles terminate upon dendrites of all sizes and somata. These findings suggest that the TRN modulates the retino-geniculocortical pathway and that this modulation is occurring in all three streams. PMID- 1723992 TI - Thalamic processing of vibrissal information in the rat. I. Afferent input to the medial ventral posterior and posterior nuclei. AB - Retrograde tracing with true blue (TB) and diamidino yellow (DY) and anterograde tracing with either wheatgerm agglutinin-conjugated horseradish peroxidase (WGA HRP) or Phaseolus vulgaris leucoagglutinin (PHA-L) were employed to investigate the projections from trigeminal nucleus principalis (PrV) and trigeminal subnucleus interpolaris (SpI) to their targets in the medial ventral posterior (VPM) and posterior (POm) nuclei of the thalamus. Many more cells in both PrV and SpI were labeled by tracer injections into VPM than into POm. Only a very small number of double-labeled neurons were observed in either PrV or SpI. However, a significantly higher percentage of SpI cells projected to POm or to both POm and VPM than was the case for PrV. Anterograde tracing with WGA-HRP showed that the projections from both PrV and SpI to VPM were much denser than those from the same nuclei to POm. Small injections of PHA-L into either PrV or SpI produced a focus of fairly dense labeling in VPM and much more diffuse terminal labeling in POm. These anatomical data provide evidence for two separate trigeminothalamic pathways, one originating from PrV and the second originating from SpI. Both of these pathways converge and diverge at the thalamic level. That is, information from the PrV pathway and from the SpI pathway are both provided to VPM in a morphologically restricted fashion and to POm in a morphologically widespread fashion. PMID- 1723993 TI - Thalamic processing of vibrissal information in the rat: II. Morphological and functional properties of medial ventral posterior nucleus and posterior nucleus neurons. AB - Extracellular recording, intracellular recording, intracellular horseradish peroxidase injection, and receptive field mapping techniques were employed to evaluate the physiological and morphological properties of medial ventral posterior nucleus (VPM) and posterior nucleus (POm) neurons in normal adult rats. Overall, we physiologically characterized 148 VPM and 121 POm neurons. Over 82% of the VPM cells were excited only by deflection of one or more mystacial vibrissae, 10% were activated by displacement of guard hairs, and the remainder were either excited by indentation of the skin or were unresponsive. Less than 40% of the POm cells were activated by vibrissa deflection, 18% were excited by displacement of guard hairs, and another 17% were unresponsive. Most of the rest of the POm cells were excited by stimulation of skin, mucosa, or activation of muscle-related afferents. Small percentages of POm cells responded only to noxious stimulation, were classified as having a wide dynamic range, or were inhibited by peripheral stimulation. Electrical stimulation of either PrV or SpI activated most neurons in both VPM and POm. This excitation was almost invariably followed by a long-lasting hyperpolarization which was generally strong enough to prevent responses to either electrical stimuli delivered in the brainstem or mechanical stimulation of the periphery. The receptive fields of vibrissa sensitive cells in POm were generally much larger than those of cells in VPM. Data obtained with extracellular recording indicated that VPM and POm cells responded to an average of 1.4 and 4.0 vibrissae, respectively. Intracellular recording from smaller samples of VPM and POm cells demonstrated the existence of inputs that were insufficient to produce spikes from the cell, but did yield epsp's. When both sub- and suprathreshold excitation were considered, the average number of vibrissa in the receptive field of a VPM cell was 2.7 and the value for POm cells became 7.8. HRP-filled neurons recovered in POm (N = 20) generally had much larger dendritic arbors than neurons in VPM (N = 31). For the former cells, the size of the dendritic tree was significantly correlated with the number of vibrissa to which the cell responded; for the latter neurons, it was not. PMID- 1723994 TI - Somatotopic organization of hindlimb skin sensory inputs to the dorsal horn of hatchling chicks (Gallus g. domesticus). AB - The somatotopic organization of skin sensory nerve projections to the lumbosacral dorsal horn of hatchling chickens was determined with the aid of transganglionic transport of horseradish peroxidase (HRP) processed with tetramethylbenzidine histochemistry. A total of eight hindlimb nerves were studied, five of which were purely cutaneous. When combined, the innervation fields of these nerves covered most of the hindlimb surface, allowing a nearly complete somatotopic map of the hindlimb to be generated. This report describes a novel pattern of cutaneous nerve projections to the dorsal horn. Unlike other vertebrates, cutaneous nerves of chickens formed two separate, somatotopically organized projections across the mediolateral axis of the dorsal horn; when serially reconstructed and superimposed, these projections produced two nonoverlapping somatotopic maps of the skin surface lying side by side. Each of these separate maps was nearly identical to the other in overall topology. These two separate maps appear to represent distinct modalities of sensory information, as projections composing the medial map were preferentially labeled by choleragenoid-HRP, whereas those composing the lateral map were preferentially labeled by wheat germ agglutinin HRP. In mammals, these HRP ligands selectively label the central projections of myelinated and unmyelinated cutaneous afferents, respectively. The present study, therefore, strongly supports the cytoarchitectonic findings of Brinkman and Martin (Brain Res. 56:43-62, '73) that lamina III lies medial, rather than ventral, to lamina II in the chicken dorsal horn. Further, the present studies also suggest that laminae II and III of chickens are homologous to the homonymous laminae in the dorsal horn of mammals. PMID- 1723995 TI - A serial section electron microscope study of an identified Ia afferent collateral in the cat spinal cord. AB - Serial section electron microscopy has been used to examine a horseradish peroxidase (HRP)-labelled group Ia afferent collateral from its entry point in the grey matter to its termination in Clarke's column of the cat spinal cord. A wide range of geometries and myelination patterns were identified along the collateral, including 1) nodes specialized to exhibit a single synaptic bouton, 2) nodes specialized to exhibit two or more synaptic boutons connected by fine, unmyelinated lengths of the collateral, 3) terminal heminodes, along which boutons were separated by unmyelinated branches, and 4) complex arrangements along which myelinated and unmyelinated branches gave rise to one or more boutons. Thirty-six synaptic boutons of varied shape and size were exhibited by this collateral. Previous studies have shown that the geometry, branching, and myelination pattern of an axon play an important role in determining the amplitude and duration of an action potential propagating along that axon. In turn, the amplitude and duration of a presynaptic action potential influence the efficacy of transmitter release. The varied axonal geometries and myelination patterns observed in the present study provide further evidence in support of our previous proposal that there may be considerable nonuniformity in the efficacy of synaptic transmission among release sites arising from the same primary afferent fiber. PMID- 1723996 TI - Developmental expression of neural cell adhesion molecules in the mouse neocortex and olfactory bulb. AB - Polyclonal antibodies to N-CAM and L1 and monoclonal antibodies to epitopes of N CAM (designated 12F11, 8A2, and 12F8) were used to investigate the spatial and temporal distribution of these neural cell adhesion molecules during the development of mouse cortex and olfactory bulb. The aim of the study was to correlate developmental events such as cell migration, dendritic and axonal outgrowth, and synaptogenesis with the appearance and disappearance of specific molecules involved in cell-cell interactions. Western transfer studies indicated that 12F8 antibody recognized polysialic acid found on embryonic N-CAM; 8A2 antibody primarily recognized the 140 kD component of N-CAM while the 12F11 antibody recognized the 180 and the 140 kD forms. The study demonstrates a high degree of cell surface molecular specialization of different compartments in developing neocortex and olfactory bulb. L1 is found on a variety of unmyelinated fiber tracts including thalamocortical fibers, olfactory nerve, and inner plexiform layer of the olfactory bulb. In contrast, N-CAM epitope recognized by 12F11 antibody is present on olfactory nerve fibers but appears later and is much weaker than L1 on thalamocortical fibers and is absent from the olfactory lobe inner plexiform layer. Dendritic regions are best labeled by 12F8 antibody; the epitope becomes faint in adult cortex but remains strongly expressed in olfactory bulb. This study reveals that widespread N-CAM expression in the central nervous system is constituted by a diversity of local expression of different molecular forms of N-CAM; their different anatomical distributions suggest they may each have unique roles. PMID- 1723997 TI - Central projections of auditory nerve fibers in the barn owl. AB - The central projections of the auditory nerve were examined in the barn owl. Each auditory nerve fiber enters the brain and divides to terminate in both the cochlear nucleus angularis and the cochlear nucleus magnocellularis. This division parallels a functional division into intensity and time coding in the auditory system. The lateral branch of the auditory nerve innervates the nucleus angularis and gives rise to a major and a minor terminal field. The terminals range in size and shape from small boutons to large irregular boutons with thorn like appendages. The medial branch of the auditory nerve conveys phase information to the cells of the nucleus magnocellularis via large axosomatic endings or end bulbs of Held. Each medial branch divides to form 3-6 end bulbs along the rostrocaudal orientation of a single tonotopic band, and each magnocellular neuron receives 1-4 end bulbs. The end bulb envelops the postsynaptic cell body and forms large numbers of synapses. The auditory nerve profiles contain round clear vesicles and form punctate asymmetric synapses on both somatic spines and the cell body. PMID- 1723998 TI - Fine structure of the ventral lateral nucleus (VL) of the Macaca mulatta thalamus: cell types and synaptology. AB - Ultrastructure of the major cerebellar territory of the monkey thalamus, or VL as delineated in sagittal maps by Ilinsky and Kultas-Ilinsky (J. Comp. Neurol. 262:331-364, '87), was analyzed by using neuroanatomical tracing, immunocytochemical, and quantitative morphometric techniques. The VL nucleus contains nerve cells of two types. Multipolar neurons (PN) retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from the precentral gyrus display a tufted branching pattern of the proximal dendrites and have a range of soma areas from 200 to 1,000 microns2 (mean 535.2 microns2, SD = 159.5). Small glutamic acid decarboxylase (GAD) immunoreactive cells (LCN) exhibit sizes from 65 to 210 microns2 (mean 122.5 microns2, SD = 32.8) and remain unlabeled after cortical injections. The two cell types can be further distinguished by ultrastructural features. Unlike PN, LCN display little perikaryal cytoplasm, a small irregularly shaped nucleolus, and synaptic vesicles in proximal dendrites. The ratio of PN to LCN is 3:1. The LCN dendrites establish synaptic contacts on PN somata and all levels of dendritic arbor either singly or as a part of complex synaptic arrangements. They are also presynaptic to other LCN dendrites. Terminals known as LR type, i.e., large boutons containing round vesicles, are the most conspicuous in the neuropil. They form asymmetric contacts on somata and proximal dendrites of PN as well as on distal dendrites of LCN. The areas of these boutons range from 0.7 to 12 microns2 and the appositional length on PN dendrites ranges from 1.1 to 14 microns. All LR boutons except the largest ones become anterogradely labeled from large WGA-HRP injections in the deep cerebellar nuclei. These boutons are also encountered as part of triads and glomeruli, but very infrequently since the latter complex synaptic arrangements are rare. The most numerous axon terminals in the neuropil are the SR type, i.e., small terminals (mean area 0.42 micron2) containing round vesicles. The SR boutons become anterogradely labeled after WGA-HRP injections in the precentral gyrus. They form distinct asymmetric contacts predominantly on distal PN and LCN dendrites; however, their domain partially overlaps that of LR boutons at intermediate levels of PN dendrites. The SR boutons are components of serial synapses with LCN dendrites which, in turn, contact somata and all levels of dendritic arbors of PN. They also participate in complex arrangements that consist of sequences of LCN dendrites, serial synapses, and occasional boutons with symmetric contacts.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1723999 TI - Morphology of intracellularly labeled canine intracardiac ganglion cells. AB - The purpose of this study was to determine the morphological organization and structure-function correlation of mammalian intracardiac ganglion cells. Conventional intracellular microelectrode techniques were applied to the tissue whole mount preparation of canine intracardiac ganglia. Forty neurons were intracellularly recorded and labeled by means of horseradish peroxidase iontophoresis. Cell morphology was quantitatively analyzed by light microscopy and camera lucida technique. Somata were elongated (mean 62 x 40 microns) and had 2-12 primary dendrites restricted within the ganglion. Almost half of the neurons had either a short axon that was traced only within the ganglion or no axon distinguishable. These neurons may have perhaps been intraganglionically active neurons. The other cells had a long axon that either coursed out of the ganglion to peripheral cardiac tissue or exited the ganglion via interganglionic nerve to innervate more remote cardiac tissue or cells in other intracardiac ganglia. Interaction between neurons was suggested by the close proximity of processes from different neurons. Previously defined electrophysiological cell types (R-, S , and N-cells), which were significantly different in their passive and active membrane properties, had different morphological features of the somata but not the axonal or dendritic processes. Intraganglionic or long axon neurons were not associated with a particular electrophysiological cell type. These findings provide the possibility of ganglionic modulation of vagal efferent activity in mammalian heart and also provide some morphological basis for the electrophysiological cell types. PMID- 1724000 TI - Application of interferons in the control of infectious diseases of cattle. AB - Recovery from infection involves a number of complex interactions between specific cells of the immune system. Many of these interactions are mediated by cytokines, which can activate these cells to kill or reduce the replication rate of the pathogen. Availability of large quantities of recombinant cytokines has provided the opportunity to investigate the mechanism or mechanisms of action of each cytokine in vitro and in vivo. In the present review, we describe the application of interferons to reduce morbidity and mortality of cattle suffering from bovine respiratory disease and mastitis. The potential application of interferons in disease modulation as well as the impediments to their use are discussed. PMID- 1724001 TI - Immunobiology of hematopoietic colony-stimulating factors: potential application to disease prevention in the bovine. AB - Colony-stimulating factors are a family of glycoproteins instrumental in regulation of hematopoiesis and inflammation. Clinical effects of various colony stimulating factors have been reported in murine and human hosts. This review summarizes findings from some clinical trial evaluations of macrophage colony stimulating factor, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-6, and interleukin-7 administration to other species. These factors stimulate clonal expansion of progenitor cells in the bone marrow, induce differentiation of various cell lineages to a mature phenotype, and, in some cases, enhance the effector activities of immune cells. Each colony stimulating factor has distinct lineages of bone marrow cells upon which they act, although there is some overlap in lineage activity and synergy between colony-stimulating factors. The close relationship in biological activity among different colony-stimulating factors is also reflected at the genomic level at which genes for some hematopoietic growth factors have been mapped to a region of human chromosome 5. Recently, colony-stimulating factor administration to cattle and its potential application to disease control in bovine preventive medicine programs has been investigated. Data from recent hematological, immunological, and intramammary bacterial (Staphylococcus aureus and Klebsiella pneumoniae) challenge studies in dairy cows are reviewed. These studies, with limited numbers of cows, found that rate of new infections, as well as duration and severity of infection, were reduced by pretreatment of cows with granulocyte-colony stimulating factor. The dose-dependent hematological and immunomodulatory effects of granulocyte colony-stimulating factor administration may explain reduced severity and incidence of mastitis in dairy cows given granulocyte colony stimulating factor. PMID- 1724002 TI - Logistic models describing effects of temperature and humidity on residual effectiveness of chlorpyrifos and cyfluthrin formulations against German cockroaches (Dictyoptera: Blattellidae). AB - Multifactored logistic models were developed for chlorpyrifos and cyfluthrin formulations based on mortality data from laboratory studies with the German cockroach, Blattella germanica (L.). Insecticides were applied to stainless steel surfaces and aged at three different temperatures (23, 30, and 37 degrees C) and two levels of relative humidity (40 and 70%). After the insecticides dried, the treated panels were placed opposite plywood panels to simulate a crack and crevice application. At appropriate aging times, treated panels were removed from environmental chambers for bioassay. The combined effects of high temperature, high humidity, and aging of residues caused the greatest decline in cockroach mortality for chlorpyrifos. Increasing temperature and aging of residues resulted in decreased cockroach mortality for cyfluthrin formulations; however, mortality was greater than 87% for all formulations through 84 d. Information from this study can be incorporated into integrated pest management programs for German cockroaches. PMID- 1724003 TI - Monoclonal antibody OKB19, reactive with a B-lymphoid differentiation antigen (CD19), binding to basal layer keratinocytes of normal human skin. AB - Monoclonal antibody (moAB) OKB19 reacts with CD19 antigen, which is the broadest lineage-specific surface marker on B-lymphocytes. In frozen tissue sections, using an immunohistochemical technique, the OKB19-positive cells in the basal layer were sharply demarcated from the negative suprabasal layers. In normal hair follicles, the OKB19 reactivity was also confined to one layer of the dermal side of the outer root sheath. However, this reactivity gradually disappeared in the lower areas. The inner surface of the lumina in the eccrine duct was weakly stained with OKB19. The basal keratinocytes were also stained with OKB19 in the lesional epidermis of the various dermatoses examined in this study, when the basal keratinocytes remained unaffected. Even in the hyperproliferative state of psoriasis, the OKB19 reactivity was confined to the basal layer. Several kinds of tumor cells derived from the skin were not stained with OKB19. No labeling was seen even in the basaloid cells of basal cell carcinoma, which are morphologically similar to basal keratinocytes. B4 and Leu-12, other monoclonal antibodies reacting with CD19, did not recognize any keratinocytes in the normal human skin. MoAB OKB19, therefore, reacts with an antigen present on basal keratinocytes and provides a probe for the isolation of the basal keratinocyte subpopulation. Thus, this antibody should be useful in studies of not only B lymphocyte differentiation, but also normal and aberrant differentiation of the epidermal keratinocytes. PMID- 1724004 TI - Immuno-electronmicroscopic studies on the gamma-aminobutyric acid and glycine receptor in the intermediolateral nucleus of the thoracic spinal cord of rats and guinea pigs. AB - Gamma-aminobutyric acid (GABA) and glycine are known as major inhibitory neurotransmitters in the intermediolateral nucleus of the spinal cord. Distribution and density of GABA immunoreactive axon varicosities and glycine receptor immunoreactive dendrites and somata in the intermediolateral nucleus were examined by immuno-electronmicroscopy. GABA immunoreaction was observed in the axon varicosities of axo-dendritic and axo-somatic synapses. Glycine receptor immunoreaction was seen in association with the postsynaptic membrane of dendrites and soma. GABA immunoreactive axon varicosities were larger (1.01 +/- 0.49 x 1.20 +/- 0.38 microns) than axon varicosities presynaptic to glycine receptors (0.72 +/- 0.22 x 0.98 +/- 0.33 microns). The density of GABA immunoreactive axon varicosities was 3.65/100 microns 2 and that of glycine receptor immunoreactive synapses was 4.78/100 microns 2. A subpopulation of GABA immunoreactive axons (42%) made synaptic contact with glycine receptor immunoreactive dendrites or soma, indicating the coexistence of GABA and glycine. PMID- 1724005 TI - Long-acting alpha 1-adrenoceptive sympathomimetic agent suppresses sympathetic outflow to muscles in humans. AB - To clarify the effect of a long-acting selective alpha 1-adrenoceptive sympathomimetic agent on sympathetic outflow to muscles (muscle sympathetic activity, MSA) in humans, 5 mg of midodrine hydrochloride was injected intravenously in ten healthy male subjects aged 19-25. Spontaneous MSA was significantly suppressed as well as plasma norepinephrine level without changing heart rate, or systemic blood pressure. The suppression continued for more than 90 min, and this is supposed to be due to negative feedback action on the baroreflex loop. The absence of significant changes in heart rate or systemic blood pressure in healthy subjects suggests that hemodynamic homeostasis is maintained by reducing the MSA in response to a long-acting sympathomimetic agent, like midodrine hydrochloride, which constricts peripheral vessels directly. PMID- 1724006 TI - Efferent connections of lobule IX of the posterior cerebellar cortex in the rabbit--some functional considerations. AB - The Purkinje cell projection from the cardiovascular region of sublobule b of the uvula (medial area of zone A) has been investigated using anterograde tracing methods in the rabbit. The importance of the integrity of the identified pathways in mediating the cardiovascular responses from the uvula has been studied in subsequent lesioning experiments. Wheat germ agglutinin-conjugated horseradish peroxidase or tritiated amino acids were microinjected into sublobule IXb. This resulted in anterogradely labelled Purkinje cell axons in both the inferior and superior cerebellar peduncle. In agreement with previous studies in rabbit we also found labelled fibres at the level of the fastigial nucleus and vestibular complex. However, the labelled fibres we observed in the parabrachial nucleus have not been reported in previous studies except in the prosimian primate. Projections from IXb showed terminal-like patterns of label in the ventromedial region of the caudal fastigial nucleus, the dorsal areas of the superior and inferior vestibular nuclei and in the medial and lateral divisions of the parabrachial nucleus. Labelled fibres were also seen coursing in the lateral vestibular nucleus. Lesioning experiments have revealed that the integrity of the superior cerebellar peduncle is essential for the expression of the cardiovascular responses (bradycardia and depressor response) elicited from the uvula in the anaesthetized rabbit. In contrast, the pattern of cardiovascular response evoked in a decerebrate rabbit (tachycardia and pressor response) was abolished when the inferior cerebellar peduncle was lesioned. PMID- 1724007 TI - Cellular alterations of parotid gland of rats with acute pancreatitis induced by cerulein. AB - To explore the cellular and subcellular alterations of the parotid gland during acute pancreatitis, we examined the redistribution of lysosomal enzyme, cathepsin B, along with the discharge of the LDH and cathepsin B from parotid acini of rats with acute pancreatitis induced by a supramaximal dose of cerulein (5 micrograms/kg/h for 3.5 h). Both the serum amylase level and parotid-gland amylase content were increased significantly in rats with acute pancreatitis. The dry-/wet-wt ratio ratio was significantly lower than in the control. In vitro studies showed that discharge of LDH from the parotid acini and leakage of cathepsin B from lysosomes in the acini were significantly higher than in the control. In addition, there was redistribution of the cathepsin B (shifting from the lysosomal pellet to the zymogen pellet) in the parotid gland. These results indicate that in acute pancreatitis there is edema and accumulation of amylase in the parotid glands, along with increased cellular and lysosomal fragility. Thus, there seems to be a close relationship between the exocrine pancreas and the parotid glands. Gut hormones, such as cerulein, also appear to play an important role in the pathophysiology of the parotid glands. PMID- 1724008 TI - The resorption of FITC-dextran 10,000 from the peritoneum in different modifications of bile-induced acute pancreatitis and in bacterial peritonitis. AB - The resorption from the peritoneum of fluorescein-isothiocyanate-conjugated (FITC) dextran with a mol wt of 10,000 was studied after 6, 15, and 24 h in rats with (1) only laparotomy (LC), (2) bacterial peritonitis (BP), (3) bile-induced acute pancreatitis (AP), (4) acute pancreatitis induced with contaminated bile (AIP), and (5) cerulein administration during acute pancreatitis (CAP). Animals in the AIP and CAP groups had a significantly higher mortality rate at 24 h and higher hematocrit at 6 h, indicating severe disease in these animals. At 6 and 15 h, all groups displayed similar peritoneal resorption. After 24 h, all groups with active inflammation showed significantly higher resorption than laparotomy controls. We conclude that peritoneal resorption as defined is independent of the severity and mode of induction of acute experimental pancreatitis and that it is the same as in bacterial peritonitis. PMID- 1724009 TI - Characterization of secretory responses in exocrine pancreas of genetically obese Zucker rats. AB - Secretory responses of the exocrine pancreas in the obese Zucker rat with an inherited genetic disorder were compared with those in lean control rats. Amylase concentration in pancreatic acinar cells of obese rats was reduced to 62% of the control value, whereas trypsinogen concentration was increased to 269%. The activity ratio of amylase to trypsinogen in the pancreatic acini of obese rats was about one-fourth of that in controls. The activity ratio of amylase to trypsinogen in pancreatic juice released by stimulation with varying concentrations of CCK8 or carbachol in the obese rats was also reduced to one fourth of that in control rats. The concentration of the secretagogs inducing half-maximal secretory responses (EC50) in the obese rats was almost identical to that in the controls. The downward shift of the dose-response relation for these secretagogs in the acini of obese rats was analogous to that in streptozotocin induced diabetic rats, cold-exposed rats, or lactating rats, as demonstrated previously. The present results may be explained by the changes in enzyme content and secretory responses found in pancreatic acini of obese Zucker rats, which would be attributable mainly to congenital disturbance in "insulo-acinar axis." PMID- 1724010 TI - [An experimental study on compression neuropathy--changes of the neurofilament and axonal tubulin content detected by enzymatic antibody technique]. AB - Changes of axonal transport were investigated by enzymatic antibody technique in an experimental model of compression neuropathy. Neurofilament and tubulin were selected as marker protein and avidin-biotin-peroxidase complex method was used to stain specimens. Compression was applied on the sciatic nerves of the mongrel dogs by the KEIO compression clamps with compression force of 15, 30, and 50 grams. Staining was performed at 1, 2, 3, 4, 6, 8 weeks after the onset of the compression. Electrophysiologic and histologic examination were also studied. As a result, neurofilament and axonal tubulin content decreased in the site distal to the compression clamps. The degree of the changes was correlated with the duration and force of the compression. The decrease of axonal tubulin content closely corresponded with the decrease of neurofilament content. In conclusion, axonal transport was impaired in the peripheral nerve even under a low compression force. PMID- 1724011 TI - The head and neck surgeon--activist or moralist? PMID- 1724012 TI - Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica. AB - Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa. PMID- 1724013 TI - Phenotypic abnormalities in CD8+ T lymphocyte subsets in patients with rheumatoid arthritis. AB - We analyzed the cell surface phenotype of CD8+ cells in both peripheral blood and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Utilizing the monoclonal antibodies anti-CD45RA, anti-CD29 and anti-S6F1-, one can define both suppressor effector (CD45RA+CD29-S6F1-) and killer effector (CD45RA-CD29+S6F1+) cells within the CD8 population. In patients with OA, normal proportions of CD8+CD45RA+, CD8+CD29+ and CD8+S6F1+ cells were found in both peripheral blood and SF. The peripheral blood of patients with RA, in contrast, showed a decreased percentage of CD8+CD45RA+ cells (13.4 +/- 2.6) (p less than 0.05), but a normal percentage of CD8+CD29+ and CD8+S6F1+ cells. In the SF of patients with RA, we observed a more dramatic decrease in CD8+CD45RA+ suppressor effector cells (6.4 +/- 5.0) (p less than 0.001), a significant increase in killer effector cells as measured by both CD8 + CD29+ (35.5 +/- 9.9) (p less than 0.001) and CD8 + S6F1+ cells (28.2 +/- 11.4) (p less than 0.01). These changes may contribute to the immunologic abnormalities previously noted in this disease and may provide some insight into the pathophysiologic mechanisms of RA. PMID- 1724014 TI - Solubilization and functional reconstitution of a chloride channel from Torpedo californica electroplax. AB - Chloride channels were detergent-extracted from Torpedo electroplax plasma membrane vesicles and reconstituted into liposomes by rapid detergent removal and a freeze-thaw-sonication procedure. Concentrative uptake of 36Cl-, driven by a Cl gradient was used to determine conductance properties of reconstituted channels. Chloride flux assayed by this method is strongly selective for Cl- over cations, is blocked by SCN-, inactivated by treatment with DIDS, and exhibits an anion selectivity sequence Cl- greater than Br- greater than F- greater than SO4(2-), as does the voltage-gated Cl- channel from Torpedo observed in planar lipid bilayers. The channels are localized to the noninnervated face of the electrocyte, and a novel trapped-volume method is used to estimate a channel density on the order of 500 pmol/mg protein. An initial fractionation of the membrane extract by anion exchange chromatography yields fivefold enrichment of the channel activity. PMID- 1724015 TI - Chloride channels on epithelial cells cultured from human fetal epididymis. AB - Using single-channel recording techniques, we have detected two types of outwardly rectifying chloride channel on epithelial cells cultured from human fetal epididymis. A small-conductance channel (2.8-5.0 pS) was spontaneously active in 29% of cell-attached patches but rapidly disappeared on patch excision. This channel often occurred in clusters and exhibited slow kinetics with open and closed times of the order of tens or hundreds of msec; an open-state probability that was essentially independent of voltage; and a very low permeability to bicarbonate relative to chloride. Exposing epididymal cells to either forskolin (3 microM) or adrenaline (1 microM) activated this channel (up to 350-fold), suggesting that it may be involved in cyclic AMP-mediated anion secretion by the male reproductive tract. The large-conductance channel (14 to 29 pS) was never detected in cell-attached patches but could be activated by depolarization (40 mV) in 3% of excised, inside-out patches. Once activated, opening of this 'large' channel was voltage independent, and it had a relatively high permeability to both gluconate (Pgluconate/Pchloride = 0.24) and bicarbonate (Pbicarbonate/Pchloride = 0.4). The proportion of excised patches that contained this channel was increased 2.5-fold by prior stimulation of the epididymal cells; however, because the channel was never observed in cell-attached patches its physiological role must remain uncertain. PMID- 1724016 TI - A subpopulation of bone marrow-derived macrophage-like cells shares a unique ion channel pattern with microglia. AB - Rat microglia share a number of antigenic, functional, and morphological similarities with macrophages from other tissues, but are characterized by a distinctly different pattern of ion channels in the cellular membrane (Kettenmann et al., J Neurosci Res 26:278-287, 1990). Macrophages typically express outward and inward K+ currents. In contrast, microglia lack outward currents and only show inwardly rectifying K+ currents, regardless of the isolation or cultivation method employed for microglia. In this study we demonstrate that a subpopulation of bone marrow-derived macrophage-like cells possesses inward rectifier K+ currents, but no outward currents and thus with regard to the electrophysiological characteristics closely resembles microglia. A second population of bone marrow-derived macrophage-like cells shows the usual channel pattern described for other body macrophages. Our results strengthen the hypothesis that in the bone marrow distinct pools of precursor cells exist, possibly reflecting an early differential lineage determination for body and brain macrophages, i.e., microglia. PMID- 1724017 TI - Differentiation and heterogeneity in T-antigen immortalized precursor cell lines from mouse cerebellum. AB - Recently, various techniques have been developed to transfer oncogenes into brain cells in order to generate immortalized neural cell lines. It is of interest to establish how well such cell lines reflect their cellular origin. Here we report the characterization of sixteen cell lines from mouse cerebellum and, as a control, six cell lines from skin. Lines were established by immortalizing postnatal primary cell cultures with a retrovirus carrying a modified temperature sensitive variant of SV40 large T antigen. The cell lines reflect many properties of the cell type from which they were derived. All of the sixteen cerebellar lines expressed one or more markers of the neural precursor cells, namely, nestin and epitopes for NG2 and A2B5. In contrast, none of the six skin lines expressed neural precursor markers. Both types of cell lines expressed vimentin and fibronectin. Differentiation occurred in some of the cerebellar lines and was enhanced in defined medium. A small percentage of cerebellar cells, usually less than 5%, was positive for a marker of differentiation, e.g., glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), or L1. Expression of GFAP colocalized with that of nestin at varying levels of intensity, indicating a gradual replacement of nestin by GFAP in the cytoskeleton. Both the cells positive for precursor markers and those positive for differentiation markers tended to be located in clusters, suggesting that stochastic processes or cell cell interactions are important for the determination of the fate of cells within a clonal cell line in vitro. The degree of differentiation seemed to correlate with a shift from serum-containing to defined medium, but not with a shift from the permissive to the nonpermissive temperature for T antigen expression. The immortalization approach described here thus allows the establishment of cell lines which are "captured" in the precursor state of the developing mouse neuroepithelium. PMID- 1724018 TI - [Colour endoscopy and magnifying colour endoscopy]. PMID- 1724019 TI - Acute phase proteins and infectious complications after surgery for esophageal cancer. AB - Severe septic complications are the major cause of operative mortality in patients with esophageal cancer. We examined the levels of acute phase proteins together with infection-related complications after surgery in a large number of patients with esophageal cancer and compared them with a group of patients with gastric cancer and healthy controls. Elevations in alpha 1-antitrypsin, alpha 1 acidglycoprotein, haptoglobin and ceruloplasmin were evident in patients with esophageal cancer, being more predominant when compared to the findings in patients with gastric cancer. Although the mean levels of alpha 2-macroglobulin were not significantly elevated in either patients with esophageal cancer or those with gastric cancer, the average level immediately prior to surgery was higher in esophageal cancer patients with postoperative septic complications than in those without any such problems. Preoperative radiation therapy and total parenteral nutrition did not significantly alter the levels of acute phase proteins. It would thus appear that the elevation in alpha 2-macroglobulin is associated with the occurrence of infectious complications following surgery in patients with esophageal cancer. PMID- 1724020 TI - Binding inhibitors restore furosemide potency in tubule fluid containing albumin. AB - We have previously suggested that albumin in tubule fluid at concentrations found in the nephrotic syndrome (NS) binds furosemide, thereby diminishing diuretic effect. This mechanism may contribute to diuretic resistance in NS. If this hypothesis is correct, displacement of albumin from furosemide should restore diuretic response in tubule fluid containing albumin. To test this supposition, in vivo loop microperfusion was performed in rats using perfusates containing 6 microM furosemide in the presence or absence of 3.8 microM albumin, or furosemide and albumin to which 12 microM warfarin or 5.4 mM sulfisoxazole had been added. These drugs are inhibitors of albumin-furosemide binding in plasma. Albumin in the perfusate impaired furosemide effect on loop chloride reabsorption (1248 +/- 59 vs. 886 +/- 65 pEq/min; P less than 0.05). Addition of warfarin or sulfisoxazole to perfusate containing albumin normalized furosemide's effect. Neither drug affected furosemide response in the absence of albumin. Dansylsarcosine, a probe that binds albumin at a different site than furosemide, failed to normalize furosemide response in albumin perfusates. These data suggest that albumin in tubule fluid reduces diuretic response through a diminution in the free furosemide concentration. In as much as this mechanism contributes to diuretic resistance observed clinically in NS, displacement of furosemide from albumin binding sites may be a therapeutic strategy warranting study. PMID- 1724021 TI - Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization. AB - We used the technique of in situ hybridization to determine if cells expressing PDGF B-chain mRNA can be detected in a model of mesangial proliferative nephritis in the rat induced with antibody directed against the Thy 1 antigen present on the mesangial cell membrane. The method involved hybridization with a digoxigenin labeled cRNA probe for the murine PDGF B-chain followed by detection with an anti digoxigenin-alkaline phosphatase conjugate and subsequent colorimetric reaction. In normal rats (N = 4), the majority of glomeruli (74%) were negative for PDGF B chain mRNA, whereas 65% of glomeruli from rats with mesangial proliferative nephritis (N = 4) had segmental or diffuse staining for PDGF B-chain mRNA in a mesangial pattern. The difference, as measured using a semiquantitative scale, was significant (mean scores 0.4 +/- 0.2 vs. 1.9 +/- 0.2; scale 0 to 3+; P less than 0.001). The increase in PDGF B-chain mRNA positive cells localized to areas of hypercellularity and was associated with a significant increase in cells positive for PDGF B-chain by immunostaining with a specific monoclonal antibody (0.8 +/- 0.1 vs. 1.7 +/- 0.4, scale 0 to 3+, normal vs. diseased rats, P less than 0.005). Complement depletion, which prevents the mesangial cell proliferation, also prevented the increase in cells expressing PDGF B-chain mRNA and protein. Thus, this method of in situ hybridization can successfully detect cells expressing PDGF mRNA in active glomerulonephritis, and may be useful for detecting cells expressing genes for other growth factors and cytokines in both human and experimental models of glomerular injury. PMID- 1724022 TI - [Changes in hormonal homeostasis and development of disorders of the cardiovascular system in patients with prostatic cancer on estrogen therapy]. AB - Cardiac pains related to estrogen therapy for prostatic cancer (PC) emerged in 53% of treated patients with ischemic heart disease (IHD). The pain complaints were associated with impairment of coronary circulation in 48% of cases. This clinical condition is attributed to elevated STH levels and a trend to hypercorticism. In hypertensive PC patients estrogens provoked more frequent and severe headaches which occurred at initial stages of the treatment in 23% and after 1-year administration of hormones in 44% of patients. Hypertensive reactions may be caused by aldosterone and prolactin hyperproduction. Observation of the therapist and endocrinologist can help to prevent complications in IHD patients with PC. PMID- 1724023 TI - [Clinical-biochemical parallels in various methods of proteinase inhibitor kontrykal administration to patients with nonspecific lung diseases]. AB - Changes in clinical manifestations, activity of kinin blood system, serum alpha 1 proteinase inhibitor and proteinase-inhibitory system of bronchial lavage were evaluated in patients with acute pulmonary abscess in the presence of chronic bronchitis and lingering pneumonia treated locally (intrapulmonary and endobronchial administration) and conventionally with antibacterial and antiproteolytic drugs. Forty patients with acute pulmonary abscess and chronic bronchitis and 49 with the abscess and pneumonia received multimodality treatment including intrapulmonary and endobronchial introduction of antibiotics and contrykal. Thirty-five abscess patients with chronic bronchitis and 52 such with lingering pneumonia received intravenous droplet contrykal in identical day dose. Clinico-biochemical follow-up results were indicative of local contrykal advantages as the patients stayed in hospital for a shorter period, did not develop complications and chronic forms of nonspecific pulmonary diseases. PMID- 1724024 TI - [Ways of improving treatment of pulmonary hemorrhage]. PMID- 1724025 TI - [The effect of hyperbaric oxygenation on glycosylated proteins in diabetes mellitus]. AB - Hyperbaric oxygenation (HBO) effects on the levels of circulating glycosylated proteins with various life spans in the blood stream (glycosylated hemoglobin (GH) and serum glycosylated fructose amine (SF) were studied in diabetes mellitus patients not suffering from vascular complications but developing various microangiopathies. Measurements of blood glycosylated proteins and mean daily glycemia carried out over the course of HBO treatment in the diabetics without microangiopathies evidenced an essential reduction of blood GH, SF, and glucose, reaching, in some cases, the normal values. Patients with vascular involvement (lower limb angiopathy, nephropathy, retinopathy) exposed to HBO developed but a trend to reduction of blood levels of glycosylated proteins and glucose. These results indicate a positive therapeutic effect of HBO courses on the status of diabetics with slightly manifest microvascular involvement. In cases with grave vascular involvement exposure to HBO appears inefficient. PMID- 1724026 TI - [Blood cell catecholamines during different methods of postoperative analgesia]. AB - Blood cell catecholamine levels were measured in the patients over the course of the postoperative period administered various types of analgesia, the standard mode with narcotic analgetics and the method suggested by the authors, consisting in blocking of the afferent nervous painful pulsation via the anterior abdominal wall. The blocking is carried out with a micro-irrigator placed in the muscular layer of the anterior abdominal wall during surgery. Procaine solution was employed for anesthesia. Postoperative analgesia efficacy was assessed from blood catecholamine levels. Analysis has shown the efficacy of the suggested analgesia mode. PMID- 1724027 TI - [Determination of the superoxide dismutase activity in puncture biopsy specimens of the liver in chronic liver diseases]. AB - Analysis of liver biopsy specimens from patients with chronic active hepatitis has shown reduced superoxide dismutase (SOD) and catalase activities. More profound changes were revealed in the patients with fibrosis, cirrhosis, and primary biliary cirrhosis. Suppressed activities of antioxidative enzymes and dissociation of their systemic interaction were found to be related to the pathologic process severity. Measurement of SOD activity in a biopsy specimen appears to be clinically valuable and may be used for the assessment of liver antioxidative defense in patients with liver conditions. PMID- 1724028 TI - [Mathematical evaluation of methods of determining the fibrinolytic activity of whole blood]. AB - Mathematical analysis of the adequacy of laboratory tests for estimation of blood fibrinolytic activity, routinely used in practical obstetrics, has shown that any of the known methods may be used in healthy nonpregnant and pregnant women for the diagnosis of fibrinolysis system abnormalities. To diagnose such conditions in women with pathologic pregnancy two laboratory tests should be employed with due consideration for their impact. PMID- 1724029 TI - [The evaluation of lyophilized plasma as a reference standard for the prothrombin complex]. AB - Analysis of a number of parameters of a lyophilized donor plasma reference sample to be used for the assessment of the prothrombin complex factors has shown the stability of this sample over the course of 14 months (a follow-up period). The sample may be used as a reference one in estimation of plasma prothrombin complex factor activities, and of PPSB activity. The reference sample activity should be estimated from the fresh donor plasma native pool, whose procoagulant activity is conventionally estimated as 100% or 1 U/ml. Use of a lyophilized reference sample helps obtain objective results compatible within this country, particularly so if all biologic reagents included in the test systems are standardized. PMID- 1724030 TI - [A graphic method of determining plasminogen]. AB - The author suggests a graphic method for plasminogen estimation. The euglobulin fraction lysis is recorded in the hemocoagulographer cuvette, this essentially improving the accuracy of studies. PMID- 1724031 TI - [A sensitive method of diagnosing hypercoagulation of the blood]. AB - A method for the diagnosis of blood hypercoagulation is suggested, based on recalcification principle in conditions of low contact activation of the coagulation system. A specific feature of the method is employment of lesser amounts of ionized calcium than is necessary for balanced recalcification of citrate blood, promoting recovery of the initial concentration of ionized calcium. Use of this method in examinations of 232 patients has demonstrated its higher diagnostic potential in the detection of blood hypercoagulation as against balanced recalcification technique. PMID- 1724032 TI - [The cytologic diagnosis of histiocytosis X based on bronchoalveolar lavage data]. PMID- 1724033 TI - [The cytologic diagnosis of synovial sarcomas]. AB - Specific features of the cytologic picture were studied and the criteria of the cytologic verification of the tumor type and genetic appurtenance defined in cytologic studies of puncture biopsy specimens, removed tumor impressions, scrapings off, and histologic sections in the patients with malignant tumors of the synovial tissue. Analysis of the results of histologic studies has demonstrated the potentialities of the puncture cytologic method in the combined preoperative diagnosis of synovial sarcomas. PMID- 1724034 TI - [The diagnosis of male infertility based on the lysozyme level in sperm]. AB - Spermal samples of 60 men were examined, 40 of these men suffering from sterility. A significant reduction of the share of mobile spermatozoa in sterile patients was parallelled by a reduced spermal lysozyme level, as against that in healthy controls. In other words, a relationship between spermal lysozyme level and spermatozoa mobility was revealed. The detected regular reduction of spermal lysozyme level permits using this parameter as a diagnostic test. PMID- 1724035 TI - [The diagnosis of mushroom poisoning (review of the literature)]. PMID- 1724036 TI - [The release of biologically active substances from blood leukocytes in allergic diseases]. AB - Elevated spontaneous liberation of biologically active substances from the peripheral blood leukocytes was revealed in 42% of children suffering from various allergic conditions. This liberation was particularly marked during the disease exacerbation and in grave forms of allergies in the patients with elevated serum IgG levels and in those with bronchial hyperreactivity. Liberation of biologically active substances was more often recorded in schoolchildren than in preschool children. Membrane stabilizers (intal and zaditen) were more effective in the patients with elevated liberation of biologically active substances. The authors have analyzed the conditions of tests for spontaneous liberation of biologically active substances and some characteristics of these biologically active substances. PMID- 1724037 TI - [Indices of the humoral response to respiratory syncytial virus infection in children with acute bronchitis]. AB - Examinations of 30 children with acute bronchitis have revealed a respiratory syncytial (RS) viral infection in 18. A reduced response to RS virus was seen in patients with bronchitis complicated by obstruction, in contrast to that in cases without obstruction. Humoral response to this virus was reduced and delayed in younger children as against that in elder ones. Even high levels of specific IgG did not result in vitro neutralization of RS virus. PMID- 1724038 TI - [Antibody-dependent cytotoxic method of determining the number of natural killer cells in the peripheral blood]. AB - A method for assays of human peripheral blood natural killers, making use of monoclonal antibodies, has been developed. The method is unsophisticated, reagent saving, does not involve the use of special equipment. Comparison of the results of the cytotoxic method 'with those of routine tests has shown a good correlation of the results. PMID- 1724039 TI - [Experience in preserving the viability of Pseudomonas aeruginosa in the laboratory]. AB - Experiments with 39 type collection strains and 25 newly isolated strains of P. aeruginosa have demonstrated the possibility of preserving the culture viability for a long time in sterile tap water at 6-8 degrees C. The initial morphologic, cultural, biochemical, and antigenic characteristics of the cultures are unchanged in sealed ampules for up to 1 year and in glass tubes corked with rubber corks for up to 8 months. PMID- 1724040 TI - [The serologic diagnosis of group B salmonelloses]. AB - Antibacterial sera activities towards Citrobacter O22 and Salmonella typhimurium were tested with O22, O1, O4, O5, O12 erythrocytic diagnostic agents. Cross activities of O22, O1, O4, O5, O12, and Hi antigens were tested with the same and Hi erythrocytic diagnostic agents in the antibody neutralization test. The findings have confirmed the identity or very close relationship between the tested O antigens. Screening for antigens of the excretions from patients with S. typhimurium and Citrobacter O22 infections has shown that indication of Hi antigen may be considered as the differentiating criterion between these infections. PMID- 1724041 TI - [A rapid method of bacteriocin typing of microorganisms]. AB - The technique of making paper disks with Klebsiella bacteriocins is described, as is the method for improving the stability of bacteriocin preparations. The authors have developed a method for bacterial bacteriocin typing, facilitating the work and taking but 4-5 hrs. The results of klebocin typing of Klebsiella-237 strains with the use of the developed technique are presented. PMID- 1724042 TI - [Increase in the sensitivity of the indirect hemagglutination reaction using polyethylene glycol]. AB - The authors have examined the potentiality of polyethylene glycol (PEG) to improve the sensitivity of indirect hemagglutination test (IHAT) in studies of postvaccinal immunity to measles, diphtheria, and tetanus. Parallel studies of 170 blood sera in routine IHAT and in IHAT modification with PEG have demonstrated a marked (more than 6-fold) elevation of the test sensitivity (t = 7.11; p less than 0.001). PMID- 1724043 TI - [Serotyping of Proteus using a rapid system]. AB - A rapid method for Proteus serologic typing is suggested, based on Soviet commercial diagnostic adsorbed type sera. The rapid system involves replacing two staged typing with one-staged, making use of polyvalent sera constructed by uniting 8-9 type sera according to the suggested schemes. O- and H-antigens are types in accordance with special tables. Fifteen O-antigens and 4 H-antigens, whose total incidence among Proteus clinical strains is under 2 percent, cannot be typed with the use of this system. The suggested rapid system is suggested as a rapid simple method for typing Proteus clinical strains in intricate epidemiologic situations. PMID- 1724044 TI - [The diagnostic value of studying blood serum albumin]. PMID- 1724045 TI - [The use of the aggregate hemagglutination reaction and immunoenzyme analysis in determining staphylococcal enterotoxin type D]. AB - Type D purified staphylococcal enterotoxin and anti-enterotoxic serum to it were prepared. IgG isolated from the anti-enterotoxic serum by the affinity chromatography method was used to improve the specificity and sensitivity of the aggregate hemagglutination test (AHAT) and enzyme immunoassay (EIA). The developed AHAT and EIA variants intended for the indication of type D staphylococcal enterotoxin are characterized by a high resolution ability and strict specificity. The suggested methods are simple and may be used for the rapid diagnosis of staphylococcal toxin infections. PMID- 1724046 TI - [A method of determining the content of antibiotics in tears and liquid media of the eye]. AB - The authors have developed a simple highly accurate method for measuring antibiotic concentrations in liquid media of the eye. It is based on the ability of antibacterial drugs to diffuse in agar medium. The studies may be carried out at any bacteriologic laboratory. PMID- 1724047 TI - [Determination of the degree of seeding by Helicobacter pylori in the gastric mucosa]. PMID- 1724048 TI - [A standard tampon for quantitative studies of mixed anaerobic-aerobic microflora]. AB - A tampon made of the new spongy polyvinyl-formal AQUIPOR 3500/40 is suggested for quantitative studies of mixed anaerobic-aerobic microflora. Comparison of the adsorption characteristics, the preservation of the tested pathologic material during the collection and transportation of the material, and the potential antibacterial characteristics of the suggested tampon and the traditional cotton wool tampons has shown the advantages of the new tampon, that permits standardization of the investigation by the quantitative bacteriologic method and preserves both aerobic and anaerobic microorganisms viable. This tampon may be used in studies of purulent wound and periodontal pocket microflora. PMID- 1724049 TI - [The state of hemostasis in obliterating diseases of the vessels of the lower limbs]. PMID- 1724050 TI - [The histodyl test in studies of gastric secretion]. AB - The results of the histodyl test used in studies of gastric acid production in patients with peptic ulcer evidence that this test helps obtain additional information on the mechanisms of hyperacid acid production and on the ratio of the digestion nervous reflex and humoral phases. The test objectivises pH metry data, is reliable, and may be useful in practical gastroenterology. PMID- 1724051 TI - [Determination of the optimal conditions for the storage of polystyrene plates sensitized with a commercial Rickettsia prowazekii antigen]. PMID- 1724052 TI - [Determination of triglycerides in a high density lipoprotein fraction]. PMID- 1724053 TI - [Determination of the content of fructose amino acids in the urine of patients with nephrolithiasis]. PMID- 1724054 TI - [A culture medium for the primary differentiation of Enterobacteriaceae]. PMID- 1724055 TI - A triple staining procedure to evaluate phagocytic role of differentiated astrocytes. AB - To determine whether phagocytic activity is affected by a viral infection known to induce astrocyte differentiation, a triple procedure (PAP labeling for GFAP, PAS reaction for added baker's yeast cells and hematoxylin for nuclear staining of the whole monolayer) was applied to Junin virus-inoculated cultures, as well as matched controls. The three-step staining simplified yeast cell count for subsequent statistical analysis, which discerned preferential uptake by differentiated rather than immature astrocytes. Accordingly, greater cell maturation induced by Junin virus was concomitant with early enhancement of phagocytic activity. PMID- 1724056 TI - Hepatitis C virus screening: UK Blood Transfusion Service on the threshold. AB - The implications of testing all blood donations in the UK for antibody to hepatitis C virus (HCV) are considered. Although the risks of serious liver disease arising from transfusion-transmitted HCV are relatively low in the UK, the cost of such screening will be high in terms of financial outlay and lost donations. In the UK, at least, screening of all blood donations for anti-HCV is unlikely to be as cost effective as screening for HBsAg or anti-HIV. PMID- 1724057 TI - Intranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen. AB - The plasmid pBRD026, which directs expression of the B subunit of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3' end of the gene. An oligonucleotide linker containing restriction sites for BglII and SpeI was inserted at the SpeI site at the 3' end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly-Pro-Gly-Pro which we propose acts as a 'hinge' between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetella pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice. PMID- 1724058 TI - Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O antigen specificity. AB - The gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O-antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O-antigen biosynthesis were generated from separate cosmid clones by deletion with Clal. These plasmids expressed O-antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These plasmids also mediated O-antigen expression in an E. coli K12 rfb-delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O antigen biosynthesis to be defined to a maximum of 11 kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O-antigen unit. Analysis of plasmid-encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM-2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes. PMID- 1724059 TI - The cystic fibrosis heterozygote--advantage in surviving cholera? AB - Cystic fibrosis (CF) is the most common fatal genetic disorder of caucasians. While it has been hypothesized that there is a CF heterozygote advantage which allowed the gene to achieve such high prevalence, the nature of that advantage remains a mystery. The recent identification and sequencing of the CF gene has increased the probability that the CF heterozygote advantage will be discovered. In this hypothesis we review the information which is known about the selection of the CF mutation and its cellular consequences, and present evidence which suggests that resistance to cholera may have been the environmental factor which selected CF heterozygotes over their 'normal' homozygote cohort. Future lines of experimentation and possible clinical applicability to therapy of secretory diarrhea are presented. PMID- 1724060 TI - [Kinetics of work of ion channels operated by nicotinic cholinoreceptors in the membrane of mollusk neurons]. AB - The patch-clamp technique in cell-attached configuration was applied to investigate suberyldicholine-induced currents in Planorbarius corneus neurons. The single channels currents registered as inward ones at hyperpolarization of the patches were due to chloride ions efflux from the cell. Suberyldicholine (5 mumol/l) was added into the patch electrode. Usually there were more than one channel in the membrane fragment under the clamp control. The open and closed times, bursts and clusters' lengths were measured and histograms of single channel parameters were obtained. Histograms were approximated by a sum of exponentials. Single exponentials were fitted to open times; to = 27 +/- 3 ms (n = 21). Histograms of closed times were fitted by three exponentials: tc1 = 9.5 +/ 1.0 ms (n = 21)--short gaps; tc2 = 171 +/- 33 ms (n = 19)--intermediate gaps: tc3 = 5.2 +/- 1.0 s (n = 21)--long gaps. The openings were grouped in bursts: tb2 = 203 +/- 23 ms (n = 10) and in clusters of bursts: tk3 = 1.5 +/- 0.5 s (n = 9). There were some evidences that the single burst was the result of opening, closing and reopening of just the same channel. A kinetic model with one open and three nonconducting states is plausible for single-channel data. Two long-lasting closed times may be associated with desensitization states of the receptor channel complex. Estimates of the kinetic model parameters were obtained, that may be useful in the further study of a mode of action for cholinergic drugs in Planorbarius corneus neurons. PMID- 1724061 TI - Brief fasting decreases protein synthesis in the brain of adult rats. AB - The influence of starvation on protein synthesis in the adult rat brain was studied in vivo by an intravenous injection of a flooding dose of unlabeled valine including a tracer dose of L-[3,4(n)-3H]valine. Brief starvation (24 hours) induced a 20% decline in fractional and absolute rates of brain protein synthesis. This decline resulted from a 20% decrease in the efficiency of protein synthesis (microgram protein synthesized per day per microgram RNA) whereas the capacity for protein synthesis (microgram RNA per mg protein) was maintained. Prolonged starvation (5 days) was marked by no further significant changes in the fractional rate, absolute rate and efficiency of protein synthesis, whereas the capacity for protein synthesis decreased slightly. The relative contribution of brain to whole-body protein synthesis increased during fasting, and neither the protein nor the RNA brain content did change during the experiment. These results clearly indicate that brain proteins are spared in response to brief and prolonged food deprivation, and that brain protein synthesis is very sensitive to short-term fasting. PMID- 1724062 TI - Proteolipid protein and DM-20 are synthesized by Schwann cells, present in myelin membrane, but they are not fatty acylated. AB - Proteolipid protein (PLP) and DM-20 were intensely labeled after immunoprecipitation of total cellular proteins and myelin proteins labeled with [35S]methionine in nerve slices. These results provided evidence that PLP and DM 20 are incorporated into the myelin membrane following their synthesis in Schwann cells. In contrast, PLP and DM-20 were not fatty acylated after incubation of the nerve slices with [3H]palmitic acid, however, P0 glycoprotein and 24 kDa protein were heavily fatty acylated. The lack of fatty acylation of PLP and DM-20 in the peripheral nervous system suggests that fatty acyltransferase responsible for their acylation is absent or non-functional in the peripheral nervous system. PMID- 1724063 TI - Characterization of microcarrier cultures of neurons and astrocytes from cerebral cortex and cerebellum. AB - In the present investigation a method is described for culturing cerebellar granule cells (glutamatergic neurons), cerebral cortical neurons (GABAergic neurons) and cortical astrocytes on Cytodex 3 microcarriers. It was possible to obtain a high yield of attached neurons and astrocytes on the microcarriers and the cell specific characteristics such as the ability to release neurotransmitter (neurons) and a high activity of glutamine synthetase (astrocytes) were preserved. This system, allowing mixtures of neurons and astrocytes at any given ratio to be produced, may constitute an attractive model system by which the interaction between neurons and astrocytes with regard to exchange of neurotransmitter precursors as well as other compounds may be studied. PMID- 1724064 TI - [Electrocardiographic changes in hypertensive emergencies. Their incidence and possible pathogenetic mechanisms]. AB - The aim of the study was to analyse electrocardiographic alterations in 30 patients with slight to moderate essential arterial hypertension during the course of hypertensive attacks (DAP greater than 115 mmHg). Standard hematochemical tests were performed in basal conditions, together with 24-h ECG monitoring and an echocardiogram to measure the left ventricular mass index. Echographic monitoring was carried out during hypertensive attacks and for 2 h after the return to basal pressure values. In basal conditions patients showed slight hypopotassemia (23%), left ventricular echographic involvement (57%), left ventricular hypertrophy with or without systolic strain (43%), and ventricular extrasystole (VE) classified as Lown's 1st and 2nd class (17%). During the course of hypertensive attacks, there was a significant increase in systolic strain, the appearance of anterolateral subendocardial ischemia (10%), left anterior hemiblock (3%), lateral subepicardial ischemia (3%), and a marked increase in VE (67%) which were complex in 40% of cases (Lown's classes 3, 4 and 5). A significant correlation was found between the left ventricular mass index and VE/h. The authors stress the multifactorial pathogenesis of echographic alterations and underline left ventricular involvement, acute hemodynamic strain and consequent alterations of coronary perfusion, hypopotassemia, and increased levels of circulating catecholamines. PMID- 1724066 TI - Neurons in the substantia gelatinosa rolandi (lamina II) project to the caudal ventrolateral reticular formation of the medulla oblongata in the rat. AB - Following injections of cholera toxin subunit B in the caudal ventrolateral reticular formation, in an area between the lateral reticular nucleus and the ventrocaudal tip of the spinal trigeminal nucleus, pars caudalis, large numbers of retrogradely labelled cells occurred in lamina II, amounting to 28% and 21% of all labelled neurons in cervical and lumbar enlargements, respectively. Most lamina II cells presented dendritic arbors distributed as narrow radial sheets, resembling the central cells of Ramon y Cajal. It is suggested that the ventrolateral bulbar reticular formation is the only significant supraspinal target of lamina II tract neurons. PMID- 1724065 TI - Prostaglandins sensitize nociceptors in cell culture. AB - Using the whole cell patch clamp technique, a population of nociceptors were identified, by virtue of their small size and capsaicin responsivity. Response to capsaicin was increased following treatment with the hyperalgesic prostaglandins, PGE2 and PGI2. Treatment of the cells with the cyclic adenosine monophosphate (cAMP) analogues, 8 bromo cAMP and dibutyryl cAMP, also resulted in an increase in the capsaicin-induced currents. The effects of the cAMP analogues were greater than that produced by prostaglandin treatment. We conclude that PGE2 and PGI2 act directly on nociceptors, with cAMP as second messenger, to sensitize them to noxious stimulation. PMID- 1724067 TI - Presence of nitric oxide synthase activity in the neurons of the rat embryonal cerebrum. AB - Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor (EDRF) in blood vessels, has been reported to be formed in adult brain tissue, and the possible physiological significance of NO in neuron functions has been suggested. The present study was undertaken to demonstrate the biochemical evidence of the presence of NO synthase activity in intact neurons. In this report, we will demonstrate first that NO synthase is also present in fetal brain where neurons can easily be isolated from and can be cultured in vitro and then we will present the evidence that the cultured neurons are responsible for NO production. We will also show that the distribution pattern of NO synthase in adult brain detected by our biochemical method is well consistent with the immunohistochemical data reported so far. NO synthase activity, which was measured as the formation of [3H]citrulline from [3H]arginine in the presence of Ca2+ and reduced nicotinamide adenine dinucleotide phosphate (NADPH), existed in the homogenate of adult rat cerebrum, olfactory bulb and cerebellum. The cerebellum showed the strongest NO synthase activity and the cerebrum showed the least but significant activity. It was also revealed that NO synthase activity also existed in the homogenate of cerebra of rat embryos, the activity, however, was about half of that in adult cerebrum. Furthermore, the homogenate of the cultured neurons prepared from the rat embryonal cerebra has also shown NO synthase activity. These results suggest that NO has some important roles in the development of the neuron system. PMID- 1724068 TI - Novel substance P antagonist, CP-96,345, blocks responses of cat spinal dorsal horn neurons to noxious cutaneous stimulation and to substance P. AB - Responses of dorsal horn neurons to iontophoretic application of substance P (80 120 nA), and to noxious thermal and noxious mechanical stimulations of the receptive field in the hind limb were tested in adult cats before and after the administration of the specific, non-peptide, NK-1 receptor antagonist CP-96,345 (0.5 mg/kg, i.v.). CP-96,345 inhibited the response of the neurones to substance P and also the response of these substance P-sensitive neurones to noxious thermal stimulation. The response of the substance P-insensitive neurones to noxious heat stimulations were, however, unaffected by CP-96,345. The effect of CP-96,345 on the response of neurones to noxious mechanical stimulation was variable. The results confirm the role of substance P in thermal nociception. PMID- 1724069 TI - Production of nerve growth factor in rat skeletal muscle. AB - Production of the nerve growth factor (NGF) was confirmed by Northern blot hybridization using a specific probe of synthetic cDNA. In normal rat skeletal muscle, this probe clearly showed a band equivalent to 1.3 kilobases (kb) of messenger RNA (mRNA) of NGF of male mouse submaxillary gland. By denervation, the density of the bands derived from muscles increased by a factor of more than 3 at 4 and 6 days later compared to the control. The synthesis of mRNA of NGF in muscle was also confirmed following tetrodotoxin (TTX) blockade of sciatic nerve without denervation. PMID- 1724070 TI - Acute effects of tetrahydroaminoacridine on beta-adrenoceptor-linked cyclic AMP accumulation in brain of young and middle-aged rats. AB - The effects of acute treatment with 1,2,3,4-tetrahydro-9-aminoacridine (THA), a 4 aminopyridine derivative clinically effective in Alzheimer's disease, on beta adrenoceptor-linked cyclic AMP accumulation have been investigated in cortical and hippocampal structures of young and middle-aged rats. In a first series of experiments, pretreatment with 2.5 mg/kg THA decreased basal cyclic AMP accumulation. When a phosphodiesterase inhibitor was added to the preparation, THA again decreased cyclic AMP levels in young rats, but failed to significantly modify cyclic AMP accumulation in middle-aged animals. Finally, in isoprenaline stimulated conditions, acute treatment with tacrine was able to diminish cyclic AMP accumulation in every group of rats. It is suggested that the neurochemical action of THA in mammalian brain is more complex than earlier has been anticipated and may involve an action on beta-adrenoceptors. PMID- 1724071 TI - Cholinergic nigrotectal projections in the rat. AB - In order to verify a possible target site of cholinergic neurons in the substantia nigra of the rat, a retrograde fiber tracing method was combined with choline acetyltransferase immunohistochemistry. After injection of WGA-HRP into the superior colliculus, approximately 12% of retrogradely labeled cells in the substantia nigra were found to express choline acetyltransferase immunoreactivity. These double labeled cells were located in the dorsal and lateral portions of the substantia nigra pars reticulata at its middle and caudal levels. PMID- 1724072 TI - Diurnal changes in pineal extracellular indoles of freely moving rats. AB - We observed 24 h changes of 'extracellular' indoleamines in pineal microdialysates taken from freely moving rats under a 12 h light/12 h dark cycle. Pineal indoleamines in microdialysates were analyzed by high-performance liquid chromatography with electrochemical detection. Consequently, they exhibited marked diurnal variations. The diurnal level in the extracellular 5-HT, higher for the light and lower for the dark, was parallel to that in 5 hydroxyindoleacetic acid and diametrically opposite to that in N-acetylserotonin (NAS). The release of 5-HT into the extracellular space transiently increased for the first 2 h after the dark onset and then became lower during the dark. PMID- 1724074 TI - [Clinical aspects of viral meningitis in children]. PMID- 1724073 TI - Effects of hydroxyethyl starch, nimodipine, and propylene glycol on cochlear blood flow. AB - A primary goal of pharmacologic treatment for otopathologies of vascular origin is to elevate cochlear blood flow (CoBF), thus facilitating the transport of oxygen and nutrients without compromising perfusion pressure in other tissues. In the present investigation, significant increases in CoBF were measured during intra-arterial infusion of the plasma expanding agent, hydroxyethyline starch (HES), and the vasodilator nimodipine, in anesthetized adult male guinea pigs. There were no changes in systemic blood pressure during the infusion of HES or nimodipine. Intra-arterial infusion of propylene glycol (PG), which is used as a nonaqueous solvent, produced inconsistent CoBF effects accompanied by initial decreases in systemic blood pressure with subsequent increases. It is concluded that nimodipine and HES are very promising agents for inducing increases in CoBF, whereas PG produced inconsistent effects on CoBF while elevating blood pressure, thus compromising its potential usefulness in the treatment of otopathologies. PMID- 1724075 TI - Effects of tachykinins on myenteric neurones of the guinea-pig gastric corpus: involvement of NK-3 receptors. AB - Responses of gastric myenteric neurones evoked by the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) were investigated using conventional intracellular recording methods. Application of the tachykinins caused a long lasting depolarization of the membrane potential which was associated with increased spike discharge and augmented excitability of the cells. The responses slowly desensitized. Additionally, cross desensitization occurred between SP, NKA and NKB. Both the NK-1 receptor agonist [Sar9,MetO2(11)]SP and the NK-2 receptor agonist [beta-Ala8]NKA(4-10) had no effect on the electrical properties of the neurones. Only the NK-3 receptor agonist [MePhe7]NKB mimicked the excitatory response observed during SP, NKA and NKB applications. [MePhe7]NKB-induced desensitization abolished the response to SP, NKA and NKB. However, long lasting applications of [Sar9,MetO2(11)]SP or [beta-Ala8]NKA(4-10) had no effect on the SP, NKA or NKB responses. The excitatory effect of SP, NKA and NKB remained unchanged during application of the tachykinin analogues [D-Arg1,D-Trp7,9,Leu11]SP and [Tyr5,D-Trp6,8,9,Arg10]NKA(4 10). The results indicate that SP, NKA and NKB act as excitatory neuromodulators within the enteric nervous system of the stomach. The effects of SP, NKA and NKB appeared to be mediated by activation of NK-3 receptors. PMID- 1724076 TI - Indolizinsulphones. A class of blockers with dual but discriminative effects on L type Ca2+ channel activity and excitation-contraction coupling in skeletal muscle. AB - The alpha 1 subunit of the L-type Ca2+ channel plays a dual role in skeletal muscle. It is essential both for L-type Ca2+ channel activity and for the functioning of the voltage-sensor structure that is situated in the triads as a key element for excitation-contraction coupling. This paper shows, with mouse muscle cells in primary culture, that indolizinsulphone SR33557 which has its binding site on the alpha 1 subunit blocks both L-type Ca2+ channel activity and contraction as the more classical 1,4-dihydropyridine blockers. However, unlike other Ca2+ channel blockers, it can pharmacologically discriminate between the two different roles of the alpha 1 subunit. SR33557 inhibition of both contractile and L-type Ca2+ channel activities is very voltage dependent and increases at depolarized potentials. Complete blockade of contraction was observed at low SR33557 concentrations (K0.5 = 20 nM) and was associated with only minor L-type Ca2+ channel blockade (30%). The remaining and major part of the L-type Ca2+ channel activity (70%) was blocked at much higher SR33557 concentrations (K0.5 = 0.6 microM). The results indicate that SR33557 has a much higher affinity for the alpha 1 subunit inserted into the voltage-sensor structure. They also suggest that the voltage-sensor structure, which probably includes most of the total T-tubule alpha 1 subunit, has intrinsic (but relatively small) Ca2+ channel activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724077 TI - Intracellular calcium does not directly modulate cardiac pacemaker (if) channels. AB - A study on the cardiac pacemaker current if in inside-out macro-patches of sino atrial (SA) node cells has recently demonstrated that if channels are directly activated by intracellular cAMP. Using the same preparation, here we investigate the possibility that internal Ca2+ ions play a role in the modulation of if channels. Our results indicate that Ca2+ ions do not have a direct activating effect on if. PMID- 1724079 TI - NK-1 receptors are the only class of tachykinin receptors found on mouse cortical astrocytes. AB - Extending our previous studies, our results indicate that cultured cortical astrocytes from the mouse possess only NK-1 receptors coupled to phospholipase C. An excellent correlation was found in the potency of tachykinins and selective analogs at inhibiting 125I-BHSP binding and at stimulating phospholipase C activity, their rank order being that of NK-1 receptors. No binding sites could be found with ligands of NK-2 or NK-3 receptors. No additive effect could be shown with NK-2 or NK-3 agonists when phospholipase C activity was estimated with high concentrations of NK-1 agonists. C- or N-terminal SP fragments did not modify SP- or [Pro9]SP-evoked responses. PMID- 1724078 TI - Comparison of prolonged in vivo inhibitory activity of several potent bombesin (BN) antagonists on BN-stimulated amylase secretion in the rat. AB - New BN analogues designed to be competitive receptor antagonists at the BN/gastrin releasing peptide receptor(s) can exhibit diverse properties ranging from full antagonist, partial agonist or weak agonist activity, depending on the assay system and animal species employed. Here we evaluate the following 3 antagonists which have the most potent receptor affinities in several in vitro assay systems and are representative of 3 main classes of BN antagonists for their in vivo effects on pancreatic amylase secretion in the rat: [D Cpa6,Phe14,psi 13-14]BN(6-14), [D-Phe6]BN(6-13) propylamide, and [D-Phe6]BN(6-13) methyl ester. After injection in the rat, the methyl ester was clearly the most potent antagonist and completely inhibited BN-stimulated amylase release at the 20 nmol/kg (IV bolus) for about 2 h. In contrast, the propylamide analogue at the 200 nmol/kg (IV bolus) dose produced incomplete inhibition of amylase release. Inhibition was transient and lasted for only about 1 h, possibly reflecting the significant agonist activity of this latter peptide in the rat pancreatic amylase secretion test in vitro. The psi-analogue, while being the longest acting analogue, was also incapable of lowering amylase to basal level at 50 times the BN dose, suggesting that it is a mixed agonist-antagonist in vivo as was also previously shown in vitro in the rat. PMID- 1724080 TI - In vitro enhancement of natural cytotoxicity by atrial natriuretic peptide fragment 4-28. AB - The presence of atrial natriuretic peptide (ANP) binding sites in the thymic cortex, medulla, and splenic white pulp suggests that this peptide may have immunoregulatory activity. We examined the effect of ANP on human natural killer (NK) cell activity. ANP significantly augmented NK cell cytotoxicity after twenty four hours of incubation but had no effect on NK activity after short-term incubations of one hour. In addition, atrial natriuretic peptide did not effect the expression of natural killer or T cell surface markers. This study demonstrates that atrial natriuretic fragment 4-28 enhances natural killer cell activity. PMID- 1724081 TI - Isolation and characterization of galanin from sheep brain. AB - Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26. PMID- 1724082 TI - Effects of proteolytic enzyme inhibitors on the nasal absorption of vasopressin and an analogue. AB - Proteolytic enzyme inhibitors were examined as absorption enhancers for the nasal delivery of vasopressin (AVP) and desmopressin (1-d-8-DAVP) in rats. Aprotinin, soybean trypsin inhibitor, and camostat mesilate were used as enzyme inhibitors. The nasal absorption of AVP and 1-d-8-DAVP was evaluated by measuring its antidiuretic effect. Nasal administration of AVP (0.005 IU/kg) or 1-d-8-DAVP alone (2.5 ng/kg) produced a small antidiuretic effect. Coadministration with aprotinin (1000 and 10000 KIU/kg) or soybean trypsin inhibitor (1.25 and 6.25 mM) did not change the antidiuretic effect. However, coadministration with camostat mesilate (1 to 50 mM) significantly increased the antidiuretic effect and, thus, the nasal absorption of AVP and 1-d-8-DAVP. The activities of aminopeptidase, cathepsin-B, and trypsin in the nasal mucosal tissue of rats were 7 nmol/min/mg protein, 0.7 nmol/min/mg protein, and 4.6 pmol/min/mg protein, respectively. Aprotinin and soybean trypsin inhibitor inhibited only the trypsin activity, whereas camostat mesilate inhibited aminopeptidase and trypsin activities. Aprotinin (MW 6500) and soybean trypsin inhibitor (MW 8000), with relatively high molecular weights, may not permeate into the nasal mucosal tissue. In contrast, camostat mesilate is slowly absorbed (8%/hr) and could inhibit the proteolytic activity in the nasal mucosa, resulting in enhanced nasal absorption of AVP and 1 d-8-DAVP. PMID- 1724083 TI - [The morphological characteristics of the parotid and submandibular salivary glands following neonatal thymectomy]. AB - The time course of structural and functional changes in the parotid and submaxillary salivary glands after neonatal thymectomy was considered. A combination of various methods including histological, histochemical, cytophotometric ones and transmission electron microscopy, was employed. They showed that neonatal thymectomy resulted in an increase in secretory activity of the salivary glands and was followed by hemolymphodynamic disorders and lymphoplasmocytic infiltration. PMID- 1724084 TI - P-glycoprotein expression in gastroesophageal adenocarcinomas, their metastases, and surrounding mucosa: a mapping study. AB - Previously, we identified P-glycoprotein in primary gastroesophageal adenocarcinomas and in adjacent mucosa. This study is a further investigation of P-glycoprotein expression in adenocarcinomas and benign mucosa. Sixteen resection specimens were studied (seven for gastric adenocarcinoma, seven for esophageal adenocarcinoma, one for adenocarcinoma at the gastroesophageal junction, and one for severe dysplasia in Barrett's esophagus). Multiple samples of tumor and mucosa were submitted according to a specimen diagram. Lymph node and distant metastases were studied when available. P-glycoprotein expression was identified in paraffin-embedded tissues by immunohistochemistry using monoclonal antibody C219 and was scored as the percentage of cells stained. P-glycoprotein was identified in six of 16 resection specimens. Intratumoral variability of C219 score was noted in three resections. No increase in expression was identified in lymph node or distant metastases as compared with primary tumors or in the invasive margin of the tumor as compared to the center. For every case in which tumors expressed P-glycoprotein, it was also diffusely present in all types of benign gastric and Barrett's mucosa, both adjacent to and distant from (up to 8 cm) the tumor. We also studied biopsies from 10 patients with Barrett's esophagus who did not have carcinoma. P-glycoprotein was only focally present in one of the 10 biopsies. Mucosa expressing P-glycoproteins may be the substrate from which a P-glycoprotein positive tumor arises. PMID- 1724085 TI - Small lymphocytic lymphomas with predominant splenomegaly: a comparison of immunophenotypes with cases of predominant lymphadenopathy. AB - In this study, we compared small lymphocytic lymphomas with predominant lymphadenopathy with those with predominant splenomegaly and found differences in morphology and immunophenotype as well as clinical features. Cases with lymphadenopathy were characterized by widespread disease, CLL type morphology with proliferation centers, and a CD5, CD11c, CD23, CD43 positive, CD45Ro negative immunophenotype. Cases with predominant splenomegaly had more localized disease, a mantle zone pattern or a diffuse growth pattern without proliferation centers, and a CD5, CD11c, CD23, CD43 negative, and sometimes CD45Ro positive immunophenotype. CD45Ro (UCHL1) positivity and alkaline phosphatase staining were associated with a mantle zone growth pattern. Comparison with other small lymphocytic lymphoma subtypes indicated that each has its own specific immunophenotype. PMID- 1724086 TI - A comparison between bromodeoxyuridine and 3H thymidine labeling in human breast tumors. AB - The fraction of tumor cells in the DNA synthesis (S) phase of the cell cycle is a strong prognostic indicator in human breast cancer. Most studies measuring S phase in primary tumors have used either in vitro labeling with 3H thymidine or DNA flow cytometry. The former involves a cumbersome and time-consuming radioactive assay, while the latter is heavily dependent on the quality of the preparation and the effectiveness of the algorithms used. In this study, in vivo or in vitro labeling with bromodeoxyuridine (BrdUrd) was compared with in vitro labeling using 3H thymidine in a series of 33 human breast tumors. Thymidine labeling showed strong correlation with both in vitro (r = 0.96) and in vivo (r = 0.83) BrdUrd incorporation. The reliability between observers was higher for BrdUrd counting (r = 0.94) than for thymidine counting (r = 0.87). The mean labeling index of 15 tumors labeled in vivo with BrdUrd was 5.9% compared to 4.7% for 18 tumors labeled in vitro (p = 0.34). There was poor correlation between flow cytometric and microscopic analysis of BrdUrd incorporation (r = 0.37). We conclude that microscopic analysis of in vivo or in vitro BrdUrd incorporation is a rapid and reliable method to estimate breast tumor proliferation. PMID- 1724087 TI - Myeloperoxidase: a specific marker for myeloid cells in paraffin sections. AB - Immunohistochemical detection of intracellular myeloperoxidase, a major constituent of primary granules of neutrophilic myeloid cells, was determined in paraffin sections of 161 specimens using a rabbit polyclonal antibody to human myeloperoxidase and an indirect immunoperoxidase technique. In normal tissues and in a variety of myeloproliferative disorders, myeloid cells of both neutrophilic and eosinophilic types, at all stages of maturation, exhibited strong cytoplasmic reactivity for myeloperoxidase. Myeloperoxidase was readily detected in myeloblasts and immature myeloid cells of acute myelogenous leukemia, progranulocytic leukemia, monomyelocytic leukemia, erythroleukemia, myeloblastomas, and other hematopoietic disorders. Erythroid precursors, megakaryocytes. other hematopoietic disorders. Erythroid precursors, megakaryocytes, lymphoid cells, mast cells, and plasma cells were nonreactive. Cells of monocytic derivation revealed variable reactivity and were typically weakly positive or nonreactive. In a few specimens, rare histiocytes were reactive, some possibly due to phagocytosed material. Cells comprising the infiltrate of a spectrum of lymphoid malignancies, e.q., lymphoblastic lymphoma or leukemia, chronic lymphocytic leukemia, hairy cell leukemia, non-Hodgkin's lymphomas of T- or B-cell type, and Hodgkin's disease, were nonreactive, as were the non-neoplastic tissues present in these specimens, except for occasional cells of myeloid derivation. Myeloperoxidase was not observed in the neoplastic cells of a wide variety of epithelial tumors and sarcomas, or in the contiguous non-neoplastic tissues. Immunoreactivity for myeloperoxidase was well preserved following fixation in a variety of fixatives, including Zenker's-acetic acid solution (employed for processing bone marrow biopsies), B5 solution, and formalin. Immunohistochemical detection of myeloperoxidase represents a sensitive and highly specific technique for identification of mature and immature myeloid cells in paraffin-embedded tissue. PMID- 1724088 TI - Co-expression of four intermediate filament subclasses in childhood glial neoplasms. AB - Immunohistochemical analysis of intermediate filament (IF) proteins was performed on frozen sections of 16 childhood glial tumors using a library of 10 antigen specific IF protein directed monoclonal antibodies (MoABs) and a four-step biotin streptavidin-alkaline phosphatase conjugated antigen detection immunocytochemical technique. Human glial fibrillary acidic protein (GFAP) and vimentin were expressed in all brain tumors. High molecular weight (200 kDa) neurofilament (NF H) protein was expressed in 15 out of 16 tumors; medium molecular weight (160 kDa) neurofilament (NF-M) in seven out of 16 tumors; and low molecular weight (68 kDa) neurofilament (NF-L) in five out of 16. Positive acidic keratin reactivity was found in five out of 16 tumors using MoAB AE1. Expression of a keratin pair was detected with MoAB AE2 in five out of 16 tumors. A second keratin pair in 14 out of 16 glial tumors was demonstrated with MoAB AE3. Immunostaining with AE5 defined the expression of another basic keratin (64 kDa) in nine out of 16 glial tumors. Finally, in 14 out of 16 astrocytomas an individual 51 kDa acidic keratin (detected with MoAB AE8) was expressed. Glial tumor cells contain cell lineage specific and nonspecific IF proteins in the following IF pattern: AE3+, AE8+, GFAP+, vimentin+, and NF-H+. The heterogenous composition of these cytoskeletal IF proteins in childhood glial tumors may reflect a direct stage dependent correlation with their neoplastic transformation. PMID- 1724090 TI - Effects of mild acidosis on mechanical restitution and postextrasystolic potentiation on mammalian cardiac muscle. PMID- 1724089 TI - Primitive multipotential primary sarcoma of bone: a case report and immunohistochemical study. AB - A tumor initially presenting as a round cell sarcoma in the proximal tibia of a 42-yr-old male disseminated to involve the femur, multiple tarsal bones, and the lungs. In addition to round cell areas resembling lymphoma, other areas of the tumor produced osteoid matrix and still other areas suggested epithelial differentiation. Immunohistochemical staining revealed the presence of epithelial markers in areas of epithelial differentiation, leukocyte markers in areas of lymphomatous differentiation, and neither marker in areas of osteosarcomatous differentiation. The finding of epithelial elements in a primary bone tumor is rare, and this report is one of the first confirming their presence using immunohistochemical markers. The findings are consonant for neither epithelial nor lymphoid neoplasms and in all probability represent multipotential differentiation in a primitive mesenchymal tumor. PMID- 1724091 TI - Transesophageal pacing and pharmacologic interventions in cardiac arrhythmias. PMID- 1724092 TI - Changes in limbic evoked potentials produced by the atypical benzodiazepine Ro 5 4864. PMID- 1724093 TI - Morphological and ultrastructural studies of Mycoplasma flocculare and Mycoplasma hyopneumoniae in vitro. AB - Cells of Mycoplasma flocculare were found to vary in size and shape, especially in the later phases of growth, whereas those of Mycoplasma hyopneumoniae were fairly uniform irrespective of growth phases. Filamentous cells were present in cultures of M flocculare in the stationary and declining phases, but were never found in cultures of M hyopneumoniae. The filamentous and bizarre forms observed when mycoplasmas were suspended in phosphotungstic acid probably result from the action of the hypotonic solution. The surface of all cells was covered by a fuzzy coat consisting of fine hairs or bristles. An electron-lucent region was usually seen in cells negatively stained after centrifugation, but was only occasionally seen in cells negatively stained directly from the medium. Intracytoplasmic membranes were present in sectioned cells. No attachment organelle was found in cells of either species. PMID- 1724094 TI - Central effects of tachykinin peptide on tracheal secretion. AB - Tachykinin peptides acting on structures located on the ventral surface of the medulla can increase cholinergic outflow to the tracheal smooth muscles and augment respiratory motor output. In the experiments reported here (performed in anesthetized, paralyzed and artificially ventilated dogs), we examined the effects of tachykinin peptides substance P on secretion from submucosal glands. Changes in secretion were measured in an exposed section of tantalum-coated tracheal epithelium. Substances P was administered intracisternally or applied topically on the intermediate area of the ventral surface of medulla (VMS). Intracisternal infusion and the local medullary administration of tachykinin peptide caused a significant increase in tracheal submucosal gland secretion. Atropine given intravenously prevented the secretory changes induced by central action of tachykinins. In addition, prior application of 2% lidocaine to the medullary surface blocked the responses caused by substance P locally applied on the VMS. These findings suggest that substances P acting centrally can tracheal fluid secretion mainly via cholinergic mechanisms, and that the ventral surface of the medulla is one of the site of these action. PMID- 1724095 TI - [Nucleic acid levels in milk and blood of healthy cows and those with mastitis]. AB - Investigations were carried out in 30 ncb breed cows divided into two groups. First group was consisted with cows in good health. In second group there were cows ill with mastitis. Number of somatic cells in milk was evaluated by California Mastitis Test and nucleic acids content in milk and blood by spectrophotometric method. Nucleic acids content in group of healthy cows amounted 273 micrograms/100 ml in milk and 0.114 g/100 ml in blood. In this group correlation factor concerning nucleic acids content in milk and blood amounted r = +0.57. In group ill cows nucleic acids content in milk and blood amounted respectively 500 micrograms/100 ml and 0.140 g/100 ml, and value of correlation factor r = -0.09 was noted. Differences in nucleic acids content in milk and blood between both groups healthy and ill cows correspond to values 227 micrograms/100 ml and 0.026 g/100 ml and were statistically significant by p less than 0.01. Above results allow to formulate the following conclusions: 1. Statistically high correlation between quality of somatic cells and nucleic acids content in milk exists--it is confirmation well known fact. 2. The rise of nucleic acids content in blood of cows can be an index of udder inflammation in cows. PMID- 1724096 TI - Platelet-derived growth factors and heparin-binding (fibroblast) growth factors in the synovial tissue pathology of rheumatoid arthritis. AB - The pathology of rheumatoid arthritis (RA) is characterized by massive tumor like hyperplasia of synovial connective tissues. Fibroblast like cells and microvascular endothelial cells are the predominant cell types present in this invasive tissue, particularly at sites of bone erosions. Identification of growth factors or cytokines that drive this process is an important goal of current research. Here we review evidence that platelet-derived growth factor (PDGF)-like and heparin-binding fibroblast growth factor (HBGF)-like polypeptides play a significant role in this process. For example, messenger RNA transcripts for PDGF A, PDGF-B, HBGF-1, and HBGF-2 are present in RA synovial tissue specimens, and immunoreactive PDGF-like and HBGF-1- and -2-like polypeptides are present in RA synovia. Levels of expression are significantly higher in RA synovia than in osteoarthritis (OA) synovia, and their expression correlates with the extent and intensity of mononuclear cell infiltration. Similarly, PDGF-receptor expression is elevated in RA synovia compared with OA synovia. High levels of tyrosine phosphorylation and Fos and Myc expression are also characteristic of RA synovia and occur in cells after PDGF- and HBGF-receptor interaction. These and other observations strongly support the view that PDGF-like and HBGF-like factors are involved in stimulating the proliferative and invasive phenotype of RA synovial connective tissue cells. PMID- 1724097 TI - [Is there a correlation between improved blood fluidity and neurological symptoms in cerebral infarct?]. PMID- 1724098 TI - [Hemorheologic therapy of asystematic vertigo]. AB - Hemorheological Treatment in Vertigo. Depending on data of hemorheology disturbances in risk factors (1.2.3.) and carotid artery arterosclerosis- progression in vertigo of non-vestibular origin (4.) this study evaluates treatment effects by basis, i.e. correction of risk factors only, versus additional hemorheological treatment (Lowering hct., HAES, Pentoxifylline) Patients: N = 88 fe. 51 m 37 age 25-86 mean 65.1. RESULTS: ++ base gr. 35% hemorh. 62% + base gr. 40% hem. 38% no effect base gr. 25% hemorh none. PMID- 1724099 TI - [Effect of hydroxyethyl starch and dextran on metabolism of extremity muscles in patients with arterial occlusive disease]. PMID- 1724101 TI - The dependence of activation of platelets by a plasminogen activator on the evolution of thrombin activity. AB - Both augmentation of thrombin activity and activation of platelets have been reported to accompany administration of plasminogen activators in vivo. To determine whether the platelet activation is a consequence or a cause of the procoagulant effects, we assessed the effects of t-PA on spontaneous activation and aggregation of platelets and on clotting in recalcified human whole blood. Spontaneous activation of platelets occurred in the stirred samples 8.9 +/- 2 minutes (n = 5) after recalcification. Aggregation and clotting followed immediately afterward. Activation, aggregation and clotting were accelerated in a dose-dependent manner by 3 minutes of preincubation with t-PA (2-30 micrograms/ml) before recalcification. The procoagulant effect of t-PA (5 micrograms/ml) was abolished by concomitant incubation with hirudin (0.5 nM) or aprotinin (200 KIU/ml) consistent with the hypothesis of plasmin-mediated evolution of thrombin being responsible for the procoagulant effect. However, platelets could be activated independently by other agonists (collagen, 3 micrograms/ml; and ADP, 25 microM) in the presence of hirudin. Despite the procoagulant effect of t-PA, aggregation to collagen (2-5 micrograms/ml) and PAF (0.9 microM) was diminished in samples incubated with t-PA for 30 minutes (37 degrees C). Fibrinogen degradation products elaborated during this interval (25.6 micrograms/ml; n = 3) were responsible for this anti-aggregatory effect. The results indicate that platelet activation in recalcified whole blood depends on procoagulant effects of t-PA. PMID- 1724100 TI - Immune regulation of Epstein-Barr virus (EBV): EBV nuclear antigen as a target for EBV-specific T cell lysis. PMID- 1724102 TI - The role of protein C as an inhibitor of blood clotting during extracorporeal circulation. AB - The plasma levels of protein C, AT III, the perioperative administration of fresh frozen plasma (FFP) and AT III concentrate were compared under the use of various drugs in a randomized, prospective double-blind study in 40 patients in whom an aortocoronary bypass operation was carried out. We formed four groups of ten patients: group A served as a control group, group B received a prostacyclin (PGI2) infusion of 10 or 20 ng/kg/min, group C high-dose aprotinin substitution, and group D was treated with a combination of prostacyclin and aprotinin. After an initial short-term rise in the inhibitors protein C and AT III, there was a fall in all groups in the further course of extracorporeal circulation. The initial preoperative values were reached again on the morning of the first postoperative day. This indicates a raised turnover and in association with this a raised rate of elimination of these factors caused by an increased thrombin activation during the extracorporeal circulation which cannot be prevented by the usual heparinization. Whereas prostacyclin had no effect on our results mediated by thrombocytic mechanisms, use of aprotinin led to a significant saving in the requirement for perioperative fresh frozen plasma and AT III substitution therapy. A clinical advantage of prostacyclin and aprotinin combination was not observed. In view of our results (individual analyses were mainly in the normal range), we consider that AT III and fresh frozen plasma should not be substituted routinely during or after extracorporeal circulation. PMID- 1724104 TI - Report on the nomenclature of te IGF binding proteins. PMID- 1724103 TI - [Dynamics of RNA synthesis and transport into the intact and regenerating rat liver in the presence of sublethal doses of actinomycin D]. AB - The time-dependent characteristic of RNA synthesis has been studied against the effect of sublethal doses of actinomycine D in cells with different proliferative activity, connection of synthesized RNA with RNP-particles of nuclei and its transport into the ribosomes and cytoplasmic postribosomal fraction. Against a background of actinomycine D sublethal doses (150 and 300 micrograms per 100 g body weight) from 0.5 to 4% of RNA is synthesized in comparison with the original level. The level of specific radioactivity of RNA depends on the original functional state of the liver. The newly synthesized RNA is found in the nuclei RNP-particles and transported into the cytoplasm. It is localized in ribosomes (2 3% in intact and about 6% in regenerating liver from the original level) and in the postribosomal fraction (8-10% and 16-18% in intact and regenerating liver, respectively. It is supposed that RNA synthesized against the effect of sublethal doses of actinomycine D takes part in conservation and restoration of realization of genetic information system activity, that is observed 12 hours after the antibiotic injection. PMID- 1724105 TI - Acridine orange staining in early diagnosis of mycotic keratitis. AB - We have assessed the comparative sensitivities of acridine orange staining and 10% potassium hydroxide wet mount in the examination of corneal scrapings from one hundred and sixty-one clinically suspected cases of mycotic keratitis. The sensitivity of acridine orange was found to be 76% as compared to that of potassium hydroxide (65%). The predictive value for culture result was also seen to be higher. Although both the values were not statistically significant (P greater than 0.05), acridine orange staining was a rapid and simple technique with an added advantage of detecting inflammatory cells in corneal scrapings. PMID- 1724106 TI - Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro. AB - Recently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin-3 (IL-3) dependent multipotent hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP-mix cell lines A4, C2GM, and 15S were cocultured with periosteum-free 17-day old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI-3b-conditioned medium (an unpurified IL-3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate-resistant acid phosphatase activity and confirmed by 45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI-3b-conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP-mix cell lines therefore offers a model system with many possibilities for the study of osteoclastogenesis and its regulation. PMID- 1724107 TI - Role of colony-stimulating factors in osteoclast development. AB - Effects of various colony-stimulating factors (CSFs) [interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), and granulocyte CSF (G-CSF)] on osteoclast-like cell formation were examined in two different culture systems: the one-step mouse marrow culture system and the two-step coculture system of mouse primary osteoblastic cells with the bone marrow cells collected from the colonies that formed in the methylcellulose in the presence of the CSFs. In the one-step mouse marrow cultures, none of the CSFs stimulated the formation of tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) positive multinucleated cells (MNCs). Furthermore, the CSFs other than G-CSF inhibited in a dose-dependent manner the TRAP-positive MNC formation induced by 1 alpha-25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. In contrast, when marrow cells were first cultured in semisolid methylcellulose in the presence of a CSF and the recovered marrow cells from the semisolid cultures were subsequently cocultured with primary osteoblastic cells in the presence of 1 alpha,25-(OH)2D3, numerous TRAP-positive MNCs were formed. [125I]salmon calcitonin specifically bound to TRAP-positive cells formed in this two-step culture system. Over 90% of the TRAP positive mononuclear cells and MNCs accumulated [125I]calcitonin. M-CSF was the most potent in inducing TRAP-positive MNCs, followed by GM-CSF, IL-3, and G-CSF in that order. No TRAP-positive cells were formed in the absence of either osteoblastic cells or 1 alpha,25-(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724108 TI - Genotypic and phenotypic changes of a mouse lymphoma during long-term cultivation. AB - NK/Ly mouse lymphoma ascites cells were explanted and established in culture. The cells grew in monolayer and showed fibroblast-like morphology. The original NK/Ly cells contained one large metacentric marker chromosome, while the in vitro growing cells had two metacentrics in the early passages. These so-called bi armed chromosomes were growing in number, up to seven, along with the number of subcultures. No tumour "take" was observed when the cells were given into mice intraperitoneally. However, a solid tumour developed following injection of cells into the leg muscle. The histological picture of this solid tumour was anaplastic sarcoma. It is concluded that the accumulation of bi-armed chromosomes, which were formed either by anaphase bridges, or by centric fusion of telocentric chromosomes, led to a profound alteration of lymphoma resulting a phenotype of anaplastic sarcoma. PMID- 1724109 TI - Some aspects of purine nucleotide metabolism in human lymphocytes: nucleotide content in human lymphoblastoid lines transfected with HIV-1. PMID- 1724110 TI - New aspects of purine nucleotide metabolism: formation of ADP from inorganic phosphate during degradation of AMP. PMID- 1724111 TI - Derivation of new drugs for therapy of AIDS. PMID- 1724112 TI - Analysis of neocortex in three males with the fragile X syndrome. AB - Fragile X [fraX] syndrome is a common hereditary disorder associated with a fragile site marker at Xq27.3 which clinically presents as a form of mental retardation (MR). Postmortem investigation of 3 fraX positive males with mild to moderate MR did not document any gross neuropathological changes. Golgi analysis of neocortical dendritic spine morphology extended our previous observations of immature, long, tortuous spines in one adult case of fraX (Rudelli, et al., Acta Neuropathologica 67:289-295, 1985) to 2 new cases. Evidence for similar dendritic spine abnormalities was found, although Golgi analysis was less than optimal because of incomplete dendritic stain impregnation. Neocortical intra-layer cell density was also investigated in all 3 cases. Cresyl violet stained neurons were counted in 10 randomly selected fields in neocortical layers II-VI of cingulate and temporal association areas (Brodmann's areas 23 and 38). Neuron counts in fraX and control neocortex showed no significant differences. Thus, abnormal dendritic spine morphology with preservation of neuronal density appears to characterize the neocortex in individuals with this common form of mental retardation. PMID- 1724113 TI - Syndrome of developmental retardation, facial and skeletal anomalies, and hyperphosphatasia in two sisters: nosology and genetics of the Coffin-Siris syndrome. AB - We report on 2 sisters, 3 and 6 years old, with a possible new syndrome consisting of developmental retardation, facial and skeletal anomalies, and hyperphosphatasia. This disorder closely resembles the Coffin-Siris syndrome (McKusick number 135900). We describe the difficulties in achieving a diagnosis. A major diagnostic clue was the radiological recognition of hypoplasia/aplasia of the terminal phalanx of the 5th finger. Minor facial anomalies and mental retardation alone had not led to the proper diagnosis. Still, several diagnostic possibilities remain. For unknown reasons both children have an increased level of serum alkaline phosphatase activity. PMID- 1724114 TI - [Hormonal changes and biologically active substances in alcoholic women in the reproductive age]. AB - The levels of hormones and some bioactive substances were measured in women of a reproductive age, suffering from alcoholism, with different stages and duration of this condition. Studies of the serotonin-reactive, cholinergic, and cholino reactive systems have shown a discordant activity of the neuromediator systems: reduced activity of the serotonin-reactive and elevated tone of the cholinergic systems. The findings evidence that chronic alcohol poisoning is associated with activation of the hypothalamohypophyseal system followed by depression of this activity with the progress of alcoholism. Alcoholism involves disordered functioning of the ovaries, thyroid, and adrenals; these disorders may augment due to direct toxic effect of alcohol and its metabolites on the endocrine glands. All this eventuates in menstrual dysfunction and infertility. PMID- 1724115 TI - Measures of maternal alcohol use as predictors of development in early childhood. AB - The effect of prenatal alcohol exposure on growth, dysmorphology, and cognitive development at 6 years was examined in children whose mothers had completed a self-administered questionnaire during pregnancy. Drinking patterns prior to pregnancy recognition and indications of problem drinking (IPD) were assessed. Heavier alcohol intake was associated with slower growth in height and head circumference and increased dysmorphology, as evidenced by facial features associated with fetal alcohol syndrome (FAS) and the prevalence of probable/possible fetal alcohol effects (FAE). Indications of problem drinking predicted facial features associated with FAS and cognitive deficits (i.e., lower WPPSI Verbal IQ scores and lower scores on a test of receptive language function, the Token Test). Effects of alcohol consumption on head circumference and of indications of problem drinking on Verbal IQ and Token Test scores remained significant, even after excluding children born to mothers having drinkers (over seven drinks a day) and children with probable/possible FAE. Verbal IQ was an average of 7.1 points (95% confidence interval = 0.01, 14.25) lower among children born to mothers having more than one indication of problem drinking than it was among those born to women having fewer indications; Token Test scores were 4.3 points lower (95% confidence interval = 1.38, 7.24). Although the confidence intervals for these estimates are broad in this small, heterogeneous sample, their magnitude, if confirmed, is significant given that the population standard deviation for Verbal IQ is 15, and that for the Token Test is 5. PMID- 1724116 TI - Oral allergy syndrome: the effect of astemizole. AB - The effect of treatment with astemizole (Hismanal) on symptoms elicited by ingestion of hazelnuts in birch pollen-allergic patients (the oral allergy syndrome) was investigated. Thirty patients with a well-documented allergy to silver birch, experiencing symptoms when ingesting hazelnuts, were included in the study. All had a positive skin prick test (SPT) to birch, whereas 29 and 27, respectively, showed a positive RAST and basophil histamine release test (HR) to birch. In contrast, only 15 patients had a positive SPT to hazelnut, 13 had a positive RAST, whereas 24 had a positive HR. After two oral provocations with hazelnuts the patients were randomized to receive either 10 mg of astemizole or placebo daily for 2 weeks in a double blind protocol followed by two oral provocations. Treatment with astemizole significantly reduced the symptoms compared with placebo (P = 0.004); however, without completely abolishing the symptoms. PMID- 1724117 TI - Regulation of histamine release from human basophil leucocytes: role of H1, H2 and H3 receptors. AB - A novel class of histamine receptors (H3), controlling histamine synthesis and release, was described in rat and human brain and peripheral nerve endings. The present study was undertaken to evaluate whether H3 receptors contribute to the regulation of histamine release from human basophils. Basophil leucocytes were incubated with a H3 antagonist (thioperamide; concentrations ranging from 1 nM to 10 microM) or with a H3 ((R)alpha methyl-histamine; concentrations ranging from 1 to 100 mM), and subsequently were stimulated with optimal doses of anti-IgE and formyl-methionyl-leucyl-phenyl-alanine (f-met peptide). No significant modifications of histamine release were observed after incubation either with the H3 agonist or with the H3 antagonist. By contrast, a H2 antagonist (cimetidine; concentrations ranging from 1 to 100 microM) exerted a dose-dependent enhancing effect on anti-IgE- and, to a lesser extent, on f-met peptide-induced histamine release. A H1 antihistamine (chlorpheniramine; concentrations ranging from 100 nM to 1 microM), at the highest concentration employed, displayed an inhibitory activity on IgE-dependent and IgE-independent histamine release. Exogenous histamine was shown to exert a dose-dependent inhibitory effect on two-staged anti-IgE-induced histamine release. Taken as a whole, these results suggest that H3 receptors are not involved in the regulation of histamine release from human basophils; by contrast, H2 receptors participate in controlling histamine release from human basophils, as previously demonstrated by other authors. PMID- 1724118 TI - Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. AB - Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei. PMID- 1724119 TI - Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates. AB - Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724120 TI - Microwave-accelerated fixation and lacZ activity staining of testicular cells in transgenic mice. AB - Microwave energy has been used in conjunction with glutaraldehyde to rapidly fix testicular samples of transgenic mice (whole tubules, individual cells, and cryosections) as a preparation for histochemical bacterial beta-galactosidase activity staining. The results demonstrate that the microwave-enhanced aldehyde fixation step is a convenient and simple adaptation for routine analyses, with almost no artifactual consequences or gross distortions in morphology at the microscopic level. The entire procedure (from sacrificing the animal to microscopic observation of the blue spermatogenic cells) can be completed in 1 h. PMID- 1724121 TI - [Correction of disorders of tissue perfusion in surgical patients in critical conditions and their evaluation by changes in the indices of the acid-soluble fractions of blood plasma]. AB - Rheopolyglucin infusion and vasodilation therapy with droperidol have been performed in 16 critically ill surgical patients with low O2 tissue utilization with the aim in view to assess tissue perfusion disturbances and normalize peripheral blood flow. Basic central hemodynamic parameters and oxygen transport system parameters have been determined. Using gel filtration, acid-soluble blood plasma fractions have been studied spectrophotometrically. Despite the fact that rheopolyglucin infusion caused an increase in oxygen flow index (OFI) at the expense of cardiac index (CI) growth, O2 extraction remained practically unchanged. Vasodilation therapy, with slight changes of CI and OFI, caused an increase in O2 consumption and arterio venous difference, as well as a reduction of mixed venous blood pO2, this accompanied by an increase in acid-soluble blood plasma fractions, especially second fraction at 255 nm. A direct correlation has been established between basic oxygen transport system parameters, characterizing O2 tissue utilization and changes of acid-soluble blood plasma fractions parameters. The changes in acid-soluble blood plasma fractions parameters may serve one of the criteria characterizing the efficacy of procedures aimed at the improvement of tissue perfusion. PMID- 1724122 TI - Prophylactic and therapeutic effects of phosphonoformate against feline leukemia virus in vitro. AB - Phosphonoformate (PFA), a noncompetitive inhibitor of reverse transcriptase (RT), inhibited feline leukemia virus (FeLV) infection of 2 feline cell lines and inhibited progeny virus RT activity in a chronically FeLV-infected cell line. Feline leukemia virus infection of 3201 cells, an FeLV-negative lymphoma cell line, was inhibited by greater than 70% at a concentration of only 1 microM PFA and by greater than 90% at concentrations of 64 to 256 microM PFA, as evidenced by RT activity. However, FeLV antigen expression by 3201 cells remained relatively constant over noncytotoxic concentrations of PFA. Because the persistence of viral antigen expression with concomitant suppression of RT activity appears to be unique and because 3201 cells express small amounts of an endogenous retrovirus (RD-114) and contain endogenous FeLV proviral sequences, a possible role of endogenous retroviruses acting as helper viruses was suggested. Feline leukemia virus infection of 81C cells, a sarcoma-positive, leukemia negative fibroblast cell line, was inhibited by greater than 50% at a concentration of 64 microM PFA and by greater than 98% at concentrations of 256 to 512 microM PFA, as indicated by suppression of focus formation. The feline lymphoid cell line FL-74 is a large producer of FeLV. When FL-74 cells were cultured in the presence of 256 microM PFA, virus production (virus budding and viral antigen) was not affected, but progeny virus lost RT activity and infectivity. Direct addition of PFA (256 microM) to FeLV also reduced RT activity and infectivity. These data indicate that PFA can directly and rapidly inactivate retrovirus independent of cellular processing, presumably by inhibiting RT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724123 TI - The concept of neuroglia. PMID- 1724124 TI - Beta 1-integrin-mediated neuronal responses to extracellular matrix proteins. AB - Immunologic and biochemical studies demonstrate that beta 1-class integrin heterodimers mediate attachment and neurite outgrowth by primary neurons and neuronal cell lines in response to neurite-promoting ECM molecules. Individual cells are likely to express several alpha beta 1 integrins, each possessing unique ligand specificities. In some cases, a cell may express two beta 1 integrins that recognize different binding domains in a single ECM ligand, as appears to be the case for the alpha 3 beta 1 and alpha 1 beta 1 integrins on PC12 cells. Thus, the responses of a neuron to even a single ligand may be regulated by the combined action of several receptors. Finally, preliminary studies suggest that neurons can regulate the ligand specificity of individual integrin heterodimers (e.g., alpha 1 beta 1). Given the tremendous diversity in the extracellular matrix surrounding them, it is not surprising that neurons possess intrinsic factors that dictate if and how they respond to a particular ECM component. PMID- 1724126 TI - Myelin and demyelination. PMID- 1724125 TI - Posttranscriptional regulation of myelin protein gene expression. AB - Regulation of myelin protein gene expression occurs at many different levels including transcription, mRNA translocation, translation, and posttranslational modification of myelin proteins prior to their assembly into the membrane. Translocation of myelin basic protein (MBP) mRNAs into oligodendrocyte processes was observed in vivo and in primary cultures, but no such translocation was observed for the mRNAs encoding the proteolipid protein (PLP) or myelin associated glycoprotein. More than 99% of the mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase (CNP) remained associated with cell bodies. In the jimpy mutant, MBP mRNA translocation appeared to be impaired, but translocation occurred normally in quaking brains in vivo. We have found that steroids, such as glucocorticoids, stimulate the translation of MBP and PLP mRNAs in cell-free systems and inhibit the translation of CNP mRNA. This pattern of regulation is consistent with compositional changes noted in myelin during development. We have localized a nine nucleotide segment within the 5'-untranslated region of the MBP mRNA that is involved in the action of steroids on translation of this mRNA. We have also determined that the protein synthetic step modulated by the steroids is chain initiation, enhancing the rate at which new ribosomal subunits bind to the MBP mRNAs. PMID- 1724127 TI - Glial electrophysiology and transport. PMID- 1724128 TI - Five electrophysiological properties of glial cells. PMID- 1724129 TI - Membrane-associated sodium channels and cytoplasmic precursors in glial cells. Immunocytochemical, electrophysiological, and pharmacological studies. PMID- 1724130 TI - A molecular approach to myelination using recombinant retroviruses. PMID- 1724131 TI - Defining critical regions in the promoter for myelin basic protein gene transcription. PMID- 1724132 TI - Adhesion of primary Schwann cells to HNK-1 reactive glycosphingolipids. Cellular specificity. PMID- 1724133 TI - Components of astrocytic extracellular matrix are regulated by contact with axons. PMID- 1724134 TI - Anion conductance blocked by divalent cations in cultured rat astrocytes. PMID- 1724135 TI - Single ion channel currents from vesicles in teased rat spinal roots. PMID- 1724136 TI - Properties of ion channels in cultured adult human Schwann cells. PMID- 1724137 TI - Capsaicin-induced c-fos in peripheral nervous system glial cells. PMID- 1724138 TI - Physiological properties of oligodendrocytes during development. AB - The electrical properties of oligodendrocytes during their development in cell culture were analyzed by combining two techniques: cell identification with cell type and stage-specific antibodies and the patch-clamp technique. The transition from the bipotential precursor cell, which can still develop into astrocytes and oligodendrocytes, into an oligodendrocyte results in a marked change in the ion channel pattern. During this developmental transition, voltage-activated Na+ and several types of K+ currents disappear, whereas a comparatively passive, inwardly rectifying K+ current becomes dominant. GABAA receptor-mediated Cl- currents and a pH-activated Na+ current are down-regulated at this transition but are still present at all developmental stages. In contrast, electrical coupling develops only in oligodendrocytes. This change in the channel repertoire could reflect the transition of a cell in a mobile, mitotic, plastic state (the glial precursor) to a more differentiated specialized state (the oligodendrocyte). PMID- 1724139 TI - [Surgery for hyperthyroidism. Results and current trends]. AB - From 1974 to 1990, the same surgeon operated 96 cases of hyperthyroidism: 72 cases of Basedow's disease, and 24 basedoid goiters. The preliminary preparation with antithyroid agents, sedation, Lugol and beta-blockers is particularly important, as is rehydration, high-dose sedation and beta-blockers, if required, during the postoperative period. There are few complications: 1 hematoma, 1 permanent unilateral laryngeal paralysis, 1 permanent hypocalcemia, 5 transient psychical and/or cardiac disorders, 1 malignant exophthalmia. There was no thyrotoxic crisis. Long-term follow-up allows nothing 4 recurrences as well as hypothyroidism in 46% of all cases. The size of the thyroid remains left in place is closely related to hypothyroidism. One case of vesiculo-papillary carcinoma is noted among the 24 cases of basedoid goiters (4.5%), which justifies total thyroidectomy in such cases. There also was one papillary carcinoma among the 72 patients with Basedow's disease (1.3%). PMID- 1724140 TI - [Ultrastructural study of pemphigus foliaceus and pemphigus vulgaris antigens. Apropos of 2 cases]. PMID- 1724141 TI - [Disseminated spiked hyperkeratosis]. PMID- 1724142 TI - Dementia with argyrophilic grains. PMID- 1724143 TI - [Evaluation of the efficacy of temephos in the campaign against dracunculosis]. AB - Efficacy of water source treatment with temephos 200 EC had been tested with three methods after treatment of a natural reservoir in a temporary river bed. 1. Residual pesticide in water treatment was measured with a biological test based on the pesticide toxicity on mosquito larvae from a laboratory stock for which the standard LD50 for the considered pesticide is stable and accurately known. After pesticide treatment, samples of the treated source water were taken. 30 larvae were placed in 200 ml of the treated water either pure or diluted in untreated water (ten dilutions tested). The mortality rate of larvae after 24 hours was observed and marked on a probit scale. The LD50 was read directly from the curve. The corresponding dilution of the water sample that contained one standard LD50 was expressed in mg.l-1, using the value of the standard LD50. This method was found reproducible and sensitive (up to 0.001 p.p.m.), depending on the value of the standard LD50. 2. Lethality of cyclopides was measured by numeration of survivals in 10 liters samples and compared to the natural population of a close untreated water reservoir in the higher part of the same river. Results showed that half-life of temephos was shorter than 3 days and the concentration corresponding to the LD50 for cyclopides was reached a week after treatment. Two or three weeks after treatment the cyclopide population was significant and further pesticide application was needed. 3. Decrease of incidence of dracunculiasis was compiled by weekly examination of the population during the season of transmission. Authors observed an incidence of 37% during the 86/87 season.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724144 TI - Autoradiographic localization of octylonium bromide binding sites in the rat gastrointestinal tract. AB - The anatomical localization of the binding sites of the spasmolytic drug octylonium bromide (OB) in the rat gastrointestinal tract was analyzed by use of light microscope autoradiography. The drug was visualized after in vitro incubation of frozen sections of the gastrointestinal tract with a 10 nM concentration of 14C-OB and after in vivo injection into the ascending, transverse, descending and sigmoidal portions of the colon. In vitro experiments demonstrated the specific accumulation of 14C-OB within the colonic and rectal smooth muscle. In contrast, no specific binding of the radiolabeled drug was noticeable in the stomach or in the small intestine. In vivo intracolonic injection of 14C-OB showed a significant accumulation of the drug in the colonic musculature 2 min after administration. The predominant localization of 14C-OB in the colonic and rectal musculature could explain its effectiveness in suppressing the amplitude and frequency of colonic contractions and in controlling the irritable bowel syndrome. PMID- 1724145 TI - Microvascular, flow and O2 consumption responses of ischemic myocardium to prazosin. AB - The effects of selective alpha 1-adrenoceptor blockade with prazosin were investigated in ischemic rabbit heart. Using labeled microspheres, regional coronary blood flow was measured before and 1 hr after occlusion of the circumflex coronary artery in controls and animals given 1 mg/kg of prazosin 10 min after ligation. Small artery and vein O2 saturations were obtained microspectrophotometrically from hearts of anesthetized rabbits. Fluorescein isothiocyanate-dextran (150 mg/kg) was administered to mark the perfused microvessels and alkaline phosphatase stain was used to locate the total microvasculature. Prazosin depressed the arterial blood pressure. Coronary flow and O2 consumption (10.9 +/- 4.5 and 6.1 +/- 1.8 ml O2/min/100 g, nonoccluded and occluded) were significantly lower in the occluded region in the control group, while O2 extraction (4.9 +/- 1.4 and 6.8 +/- 2.6 ml O2/100 ml) was locally elevated. The results of occlusion in the prazosin-treated group were similar for flow, O2 consumption (9.9 +/- 4.5 and 6.7 +/- 5.3 ml O2/min/100 g, nonoccluded and occluded) and O2 extraction (6.6 +/- 2.3 and 8.2 +/- 2.3 ml O2/100 ml). The per cent of perfused microvessels was not significantly altered by occlusion in the control group. In the prazosin group, the percentage of occluded region subendocardial capillaries and arterioles perfused was significantly greater than in the control group, e.g., 65 +/- 9% perfused capillaries/mm2 in control occluded subendocardium vs 78 +/- 9% perfused capillaries/mm2 in prazosin-treated occluded subendocardium. Thus, the slight increase in microvessel utilization with prazosin was not sufficient to improve the flow or the O2 consumption in the ischemic myocardium. PMID- 1724146 TI - The neurons of origin of non-cortical afferent connections to the oculomotor nucleus. A retrograde labeling study in the rabbit. AB - The cellular origin of the brainstem projections to the oculomotor nucleus in the rabbit has been investigated by using free (HRP) and lectin-conjugated horseradish peroxidase (WGA-HRP). Following injections of these tracers into the somatic oculomotor nucleus (OMC), retrogradely labeled cells have been observed in numerous brainstem structures. In particular, bilateral labeling has been found in the four main subdivisions of the vestibular complex, predominantly in the superior and medial vestibular nuclei and the interstitial nucleus of Cajal, while ipsilateral labeling was found in the rostral interstitial nucleus of the medial longitudinal fascicle (Ri-MLF), the Darkschewitsch and the praepositus nuclei. Neurons labeled only contralaterally have been identified in the following structures: mesencephalic reticular formation dorsolateral to the red nucleus, abducens internuclear neurons, group Y, several areas of the lateral and medial regions of the pontine and medullary reticular formation, ventral region of the lateral cerebellar nucleus and caudal anterior interpositus nucleus. This study provides also information regarding differential projections of some centers to rostral and caudal portions of the OMC. Thus, the rostral one-third appears to receive predominant afferents from the superior and medial vestibular nuclei, while the caudal two-thirds receive afferents from all the four vestibular nuclei. Finally, the group Y sends afferents to the middle and caudal, but not to the rostral OMC. PMID- 1724147 TI - Cortico-cortical projections from the prefrontal cortex to the superior temporal sulcal area (STs) in the monkey studied by means of HRP method. AB - Cortico-cortical connections from the prefrontal cortex to the superior temporal sulcal cortex (STs area) were studied in the monkey by means of retrograde axonal transport of horseradish peroxidase (HRP). After injections of 0.15-0.6 microliter of 50% HRP into the STs area, labeled cells were found in various cortical regions. In the prefrontal-STs projections, main features of topographic correlation were revealed; the posterior part of the STs area receives fibers from the superior frontal convexity (areas dorsal to the principal sulcus) and areas 8 and 6, whereas the anterior part of the STs area receives fibers from the inferior frontal convexity (areas ventral to the principal sulcus) and the frontal pole (area FD). The principal sulcus sends fibers to the entire STs area except for its ventral wall of the posterior part. A small cortical area adjacent to the inferior ramus of the arcuate sulcus (area 45 of ref. 41) sends fibers to the entire STs area. In addition, the orbitofrontal cortex projects mainly to the rostral part of the STs area, and the parahippocampal gyrus (areas TF and TH) projects to the deeper part of the entire STs area. PMID- 1724148 TI - [The content of DNA, RNA and total protein in different skeletal muscles, in the diaphragm, in the right and left heart ventricles and in the musculature of the esophagus, the stomach and the urinary bladder of normally developed piglets and splayleg piglets of different body weights]. AB - Studies were conducted into concentrations of RNA, DNA, and total protein in various skeletal muscles, diaphragm, right and left ventricles as well as in musculature of the oesophagus, stomach, and urinary bladder of 25 normally developed piglets and 24 sprayleg piglets of differentiated body weight (B.W.). DNA concentration was found to be at its highest in piglets of lowest B.W. The Protein: DNA-quotient increased along with growing B.W. The average value of this quotient in the anterior tibial muscle and brachial triceps muscle of sprayleg piglets was lower than that in normally developed piglets. Average values of the RNA:DNA quotients in the brachial triceps muscle, anterior section of the long dorsal muscle, left ventricle, and stomach muscles were also lower in sprayleg piglets. In newborn piglets, DNA concentrations in ventricles as well as in the muscles of the stomach and oesophagus were higher than those recordable from skeletal muscles. Causes of incapability of newborn piglets to stand on their own feet are discussed in some detail. PMID- 1724149 TI - [Structural changes in the lymph node sinuses in acute blood loss]. AB - By means of light and scanning electron microscopy sinuses of the popliteal lymph nodes have been studied in 50 Wistar male rats. After modelling of an acute hemorrhage in the animals (in 15, 120 min and 1 day) volume of the medullary and marginal sinuses increases essentially, while blood stream intensity decreases in peripheral tissues. Amount of fenestrae in the sinusal lining decreases, that is, perhaps, caused by edema of cytoplasm in the lining cells and accompanied with formation of numerous cytoplasmic protrusions into the sinusal lumen. In 3 days the blood stream level in the peripheral tissues restores, structural organization of the littoral cells in the lymphatic sinuses normalizes. The data obtained demonstrate that during the first day after the acute hemorrhage decrease of hematolymphatic exchange intensity in lymph nodes and enhancement of transitory lymph flow in them take place. PMID- 1724150 TI - [Perivascular lymphoid nodules]. AB - As a result of investigation of lymphoid formations in the walls of the human tracheobronchial tree and the pulmonary stroma by means of morphological and morphometrical techniques, certain regularity in arrangement of the perivascular lymphoid noduli has been stated along the course of the lympho- and hemomicrocirculatory bed links. The periarterial lymphoid noduli of the tracheobronchial tree have certain morphological similarity and some differences, when comparing them with usual lymphoid noduli. PMID- 1724151 TI - Presence of the HNK-1 epitope (3-sulfoglucuronic acid) on oligosaccharides from squid skin chondroitin proteoglycans and degradation of proteoglycans by proteolytic enzymes. AB - Using competitive ELISA it was found that chondroitin proteoglycans from the skin of squid, which is a primitive organism, are reactive with the HNK-1 monoclonal antibody and that all HNK-1 epitopes of the proteoglycans were present only on their oligosaccharides. Although of the presence of appreciable amounts of glycine, serine and glutamic acid and the absence of hydroxyproline, the proteoglycans were degraded by collagenase and other proteolytic enzymes. PMID- 1724152 TI - Characterization of mRNAs and coding potential of the PET54 gene from Saccharomyces cerevisiae. AB - The nuclear PET54 gene in yeast controls expression of two mitochondrial genes: COX1 at the level of pre-mRNA splicing and COX3 at the level of mRNA translation. Two size classes (1.6 and 1.1 kb) of transcripts that contain the PET54 coding region are produced in vivo. Relative to the majority of yeast mRNAs analyzed so far, the 5' untranslated leader region of the 1.6 kb transcript is unusually long (254 bases), while that for the major 1.1 kb transcript is unusually short (1 base). The majority of each class of PET54 mRNA was associated with polysomes in vivo. The possibility that two polypeptides are produced in vivo from the 1.1 kb PET54 mRNA was raised by the work of Sedman et al. [J. Virol. 64: 453-457, 1990], which showed that translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' end of the mRNA. However, two sensitive assays for production of a second polypeptide, which is predicted to be 22 kD, were employed and no second polypeptide was detected. Furthermore, a nonsense mutation introduced near the beginning of the PET54 open reading frame abolished both COX1 and COX3 gene expression. These results indicate that the PET54 gene encodes predominantly a single functional polypeptide that is employed for expression of both the COX1 and COX3 genes of mitochondrial DNA. PMID- 1724153 TI - Metalloproteases of Serratia liquefaciens: degradation of purified human serum proteins. AB - Two representative strains of Serratia liquefaciens, SL 5 (serotype O5:H1) and SL 11 (serotype O1:H1), produced proteases characterized by molecular weights of 52.5 kilodaltons and isoelectric points of 6.2; both enzymes were inhibited by 50 mM EDTA. As demonstrated with SDS-PAGE electrophoresis, the two metalloproteases attacked the following purified human serum proteins: complement components C3, C4, C5, C6, C7, C8, and C9, transferrin, alpha 1-antitrypsin, alpha 2 macroglobulin, fibronectin, type III fibrinogen, immunoglobulin G (heavy chains), and IgM (heavy chains). However, C1q, IgA, haptoglobin, and C-reactive protein were refractory. PMID- 1724154 TI - Surface-exposed antigenic determinants in outer membranes of wild Yersinia pestis isolates. AB - Immunogenic surface exposed envelope proteins of Yersinia pestis strains were investigated with SDS-PAGE and immunoblots with antisera of immunized guinea pigs and convalescent patients. The sarkosyl-insoluble outer membrane proteins (OMPs) of three human isolates and one laboratory strain of Y. pestis grown in different rich media to exponential or stationary phase, and cultivated at 28 degrees C or 37 degrees C, were assayed for the presence of immunogenic peptides. Seven guinea pig sera immunized with one of the four Y. pestis strains and four human sera from plague-infected patients indicated that at least four outer membrane proteins with molecular weights of 45 KDal, 42 KDal, 21 KDal and 16.5 KDal were strongly recognized by at least one of them. All but one of the guinea-pig serum and one human serum recognized the 45 KDal protein. The 42 KDal was identified only in outer membrane isolated from Y. pestis cells grown at 28 degrees C. All immunogenic OMPs were found in the four strains investigated. Labelling of intact Y. pestis cells with Iodogen and 131I further demonstrated the surface-exposed location of the immunogenic OMPs. PMID- 1724155 TI - Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle. AB - We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel. PMID- 1724156 TI - alpha 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but not in the C-terminal inter-domain region. AB - alpha 2-Macroglobulin is cleaved by human immunodeficiency virus-1 protease. The cleavage site is the Phe684-Tyr685 bond in the "bait region", an exposed part of alpha 2-macroglobulin, creating the "F-form". The methylamine derivative of alpha 2-macroglobulin is also cleaved at the same bond. The homologous chicken ovomacroglobulin does not form an F-form structure with the protease, although, F form generation by other enzymes is known. This is possibly due to the lack of a suitable cleavage sequence in the corresponding region of ovomacroglobulin. In human alpha 2-macroglobulin, the interdomain segment between the main part of the molecule and the receptor-binding C-terminal domain is not cleaved by the HIV protease although typical cleavage sequences occur. In AIDS, therefore, HIV protease from infected cells in unlikely to interfere with receptor-binding of alpha 2-macroglobulin. PMID- 1724157 TI - [Evaluation of the efficacy of the information and health education campaigns in the area of AIDS. IV. Knowledge among students of secondary schools in Lombardy and Abruzzi]. PMID- 1724158 TI - [Experience with a pertussis vaccination program. A preliminary study of a local health unit serving 50,000 inhabitants]. PMID- 1724159 TI - [Significance of enteric phages as indicators of the efficacy of a purification plant]. PMID- 1724160 TI - [Prevalence of dental caries in adolescents 14-18 years of age. I. A transversal study in a school community of the lower Latium]. PMID- 1724161 TI - [A comparative study of DNA probe, direct immunofluorescence and cell culture in the detection of Chlamydia trachomatis in genital infections]. PMID- 1724162 TI - Why and how to counteract caesarean section epidemic. PMID- 1724163 TI - [Campylobacter infections]. PMID- 1724164 TI - [Medical responsibility and managerial responsibility in the local health unit concern]. PMID- 1724165 TI - A direct comparison of pharmacologic effects of retinoids on skin cells in vitro and in vivo. AB - The purpose of these studies was to directly compare the pharmacologic effects of retinoids on cutaneous cells in vitro and in vivo. Previously, it was demonstrated that all-trans-retinoic acid stimulates the proliferation of growth arrested human keratinocytes and fibroblasts in culture. In the present studies, all-trans-retinoic acid was compared to three other retinoids--13-cis-retinoic acid, P-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl-1-p ropenyl] benzoic acid (TTNPB) and M-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1-p ropenyl] benzoic acid (meta-carboxy-TTNPB]--for growth stimulation using a cultured human squamous epithelial cell line (UM-SCC-1) and human dermal fibroblasts. These four retinoids were also evaluated for their effects on reduction of horn-filled utriculi when topically applied to the skin of rhino mice. All-trans-retinoic acid stimulated proliferation of both fibroblasts and epithelial cells over concentrations ranging from 0.01 to 1.0 micrograms/ml. In fibroblasts, 13-cis-retinoic acid was less potent than all trans-retinoic acid, whereas, in epithelial cells these two retinoids were equipotent. In contrast, TTNPB was more potent than all-trans-retinoic acid at stimulating the growth of both fibroblasts and epithelial cells. The analog, meta carboxy-TTNPB was essentially inactive as a growth stimulator of both cell types. In the rhino mouse utriculus reduction model, the rank order of potency for the retinoids was the same as that for in vitro cell growth stimulation (TTNPB greater than all-trans-retinoic acid greater than 13-cis-retinoic acid). Meta carboxy-TTNPB was inactive at reducing utriculi at a dose of 5,000 times the ED50 of TTNPB.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724167 TI - Identification of a conserved epitope in class I alcohol dehydrogenase isoenzymes using monoclonal antibodies. AB - Two monoclonal antibodies raised against native horse alcohol dehydrogenase (HADH) bind preferentially to the enzyme attached to solid supports and recognize the denatured and carboxymethylated HADH subunits. Both antibodies cross-react with the human class I isoenzymes but do not recognize the class III ADH isoenzyme. Protease digestion, electrophoresis and HPLC have been used to identify the linear epitope which is contained in the sequence Pro344-Glu357 of the HADH subunit. PMID- 1724166 TI - Clinical and biochemical effects of an oral leukotriene biosynthesis inhibitor (MK886) in psoriasis. AB - Antipsoriatic agents have been shown to decrease skin levels of arachidonic acid and its metabolites including 12-monohydroxy-eicosatetranoic acid (12-HETE), and leukotriene B4 (LTB4). In addition, specific systemic and topical lipoxygenase inhibitors have been reported to be effective in the treatment of psoriasis. The objective of this study was to investigate the effect of a potent oral leukotriene biosynthesis inhibitor (MK886) in patients with chronic plaque psoriasis. Clinical response together with the changes of LTB4 levels in lesional skin biopsy specimens, and urinary leukotriene E4 (LTE4) excretion were evaluated. In addition, markers of inflammation, proliferation and keratinization were studied immunohistochemically. No change in clinical scores or lesional LTB4 levels were observed with a 10 1/3-day course of MK886. A statistically significant reduction in urinary LTE4 excretion was observed: mean LTE4 (ng/h) were 5.14 before treatment and 1.51 on day 11 with MK886; and 7.55 before treatment and 6.57 on day 11 with placebo treatment. Epidermal accumulation of polymorphonuclear leukocytes (PMN) tended to diminish in the MK886 treatment group. These results indicate that although a reduction (greater than 70%) in urinary LTE4 excretion was found, and a slight decrease of epidermal PMN accumulation was observed, no correlative changes in clinical scores or LTB4 levels in skin lesion were found with a short course of MK886. PMID- 1724168 TI - [Cancer of the movable tongue and mouth floor. Development aspects]. AB - A series of 145 patients with tongue and floor of mouth neoplasms is presented. Age, sex, TNM classification, lymph node presence or absence at the time of first visit and management protocols have been studied in each case. Management protocols have been divided into primitive tumor treatment (iridium and/or surgery) and lymph node treatment (radical surgery and radiotherapy). Results have been assessed considering tumoral stage and management scheme. PMID- 1724169 TI - A new developmental screening test. The Denver II. AB - To maximize the potential of a child with a developmental delay, early detection and intervention is essential (Frankenburg and Thornton, 1989). The nurse practitioner who includes a developmental screening with the child's general assessment may detect a developmental delay during a routine office visit. The purpose of this article is to provide an overview of the Denver II, a new developmental screening test. PMID- 1724170 TI - The role of palliative surgery in hospice care. PMID- 1724171 TI - Selenium in Kashin-Beck disease areas. AB - In this article, the duplication portion technique was used to determine the daily intakes of selenium and ten other elements in the 24-h total diets collected in the typical Kashin-Beck endemic areas, i.e., Shanxi Province and Inner Mongolia Autonomous of China. The contents of Ca, Mg, Cu, Fe, Zn, Mn, Al, Sr, Ba, and P in freeze-dried samples were determined by ICP-AES. Se was determined by differential pulse catalytic polarography. The average Se contents in total diets of Shanxi Kashin-Beck endemic and nonendemic areas were 0.009 and 0.021 micrograms/g (dry weight), respectively (P less than 0.001), corresponding to the daily intakes for Se of 4.6 and 10.5 micrograms. After the Se-supplemented fertilizer was applied (225 g of Na2SeO3/ha), the average Se content in total diets of Kashin-Beck disease area was increased to 0.0336 micrograms/g, which corresponded to the average daily intake for Se of 16.8 micrograms. In Inner Mongolia Kashin-Beck endemic and nonendemic areas, the average Se contents in total diets were 0.006 and 0.017 micrograms/g, respectively (p less than 0.001), corresponding to the average daily intakes for Se of 3 and 8.5 micrograms. The contents of other ten elements in total diets in endemic and nonendemic areas were reported and compared. PMID- 1724172 TI - Effect of time and sex on tissue selenium concentrations in chicks fed practical diets supplemented with sodium selenite or calcium selenite. AB - An experiment was conducted with 384 1-d-old male and female broiler-chicks. The basal corn-soybean meal diet (.07 ppm Se DM basis) was supplemented with 0, .1, .2, or .3 ppm added Se as either sodium selenite (Na2SeO3) or calcium selenite (CaSeO3), and fed for 1, 3, or 5 wk. There was no effect of Se source or level on feed intake or gain, but males consumed more (P less than .01) feed than females. There was no effect (P greater than .10) of sex or Se source on plasma, liver, or kidney Se concentration. The Se concentration of all tissues increased (P less than .01) with time and increasing dietary Se concentration. Based on multiple regression slope ratios of liver, kidney, and plasma Se concentrations, Se from CaSeO3 was as available (103%) as Se from Na2SeO3. PMID- 1724173 TI - In vivo nephrotoxicity induced in mice by chromium(VI). Involvement of glutathione and chromium(V). AB - The role of glutathione (GSH) and chromium (V) in chromium (VI)-induced nephrotoxicity in mice was investigated at 24 h after K2Cr(VI)2O7 ip injection. Nephrotoxicity was assessed by measurements of relative kidney weight and serum urea nitrogen. Cr(VI) nephrotoxicity was accompanied by decreased renal GSH and glutathione reductase (GSSG-R) levels. Pretreatment with buthionine sulfoximine, an inhibitor of GSH biosynthesis, enhanced Cr(VI)-induced nephrotoxicity, and remarkably diminished kidney GSH and GSSG-R levels. In contrast, pretreatment with glutathione methyl ester, a GSH-supplying agent, prevented Cr(VI) from exerting a harmful effect on mouse kidney and restored kidney GSH level. Administration of a Cr(V) compound, K3Cr(V)O8, induced much higher toxicity in mouse kidney than Cr(VI), but it failed to diminish renal GSH level. Another Cr(V) compound, Cr(V)-GSH complex, and Cr(III) nitrate did not cause a nephrotoxic effect in mice. The mechanism of Cr(VI)-induced nephrotoxicity was explained using GSH and Cr(V). PMID- 1724174 TI - Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes. AB - The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes (PMN) using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range 10-500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide. PMID- 1724175 TI - Serum and parotid saliva testosterone, calcium, magnesium, and zinc levels in males, with and without periodontitis. AB - Fourteen male patients with periodontitis and 10 patients free of periodontitis were included in the study. The concentrations of testosterone (T), calcium (Ca), magnesium (Mg), and zinc (Zn) were measured in serum and parotid saliva. Patients with periodontitis had increased Ca and decreased Zn serum levels, and they had decreased Ca and increased T levels in parotid saliva. Furthermore, there was a low correlation between parotid saliva T and Mg levels in the patients with periodontitis (r = 0.61, n = 14, t = 2.663, p less than 0.005), and there is an inverse relationship between serum and parotid saliva Mg levels (r = - 0.58, n = 14, t = 2.468, p less than 0.05). PMID- 1724176 TI - Effect of automobile exhaust on the distribution of trace elements and its modulation following Fe, Cu, and Zn supplementation. AB - The effect of automobile exhaust on the distribution of trace elements with special reference to Pb and its modulation following Cu, Zn, and Fe supplementation, in mouse organs, has been studied using Energy Dispersive X-ray Fluorescence technique. Seven elements, namely K, Fe, Cu, Zn, Br, Rb, and Pb, were detected in all the organs. The maximum concentration of Pb was found in lungs followed by that in liver and kidney. The effect of automobile exhaust was found to be significant on the concentrations of Fe and Pb; their concentrations were found to increase in all the organs. However, the concentrations of Cu and Zn were found to be decreased significantly in the liver. In the animals given Fe, Cu, or Zn supplementation along with motor exhaust, the percentage change in the concentration of Pb in lungs was decreased, and that of Fe was increased significantly. In kidney, no significant change was observed for the animals given Cu and Zn, whereas for animals given Fe, the level of Pb decreased significantly. In liver, the reduction in the level of Zn in the exhaust-exposed animals was made up and the level of Pb was reduced following Zn supplementation. These results clearly indicate that Fe and Zn play an important role in Pb metabolism and tend to lower the absorption of Pb. The effect of Fe is more pronounced than that of Zn, whereas the effect of Cu seems to be insignificant. PMID- 1724177 TI - Iron, copper, and zinc status in rats fed supplemental nickel. AB - Literature data concerning the effect of increasing dietary Ni concentrations on Fe, Cu, and Zn status in rats are sparse and, in part, controversial. Therefore, the effects of the addition of either 0, 3, 50, or 100 mg Ni/kg diet on Fe, Cu, and Zn status of rats were investigated in two separate experiments. Purified diets were used that were composed according to the established nutrient requirements of rats. Ni in kidney was increased with increasing Ni intakes. Dietary Ni did not significantly influence Fe concentrations in plasma, liver, kidney, femur, and spleen. Likewise, the addition of Ni to the diet did not alter Cu status. Zn concentrations in femur were significantly decreased after feeding the diets with 100 mg Ni/kg. However, Zn in plasma, liver, kidney, and spleen was not affected. It is concluded that variations in dietary Ni concentrations have no major impact on Fe, Cu, and Zn status in rats. PMID- 1724178 TI - Dietary fluoride, unlike bromide or iodide, counteracts phosphorus-induced nephrocalcinosis in female rats. AB - Supplemental dietary F has been shown to counteract P-induced nephrocalcinosis in female rats. In order to obtain information as to the specificity of this F effect, the effect of other halogens, namely Br and I, on P-induced nephrocalcinosis was studied in weanling female rats. Supplemental dietary Br (5.24 mmol/kg of diet) and I (1.43 mmol/kg of diet) did not influence P-induced nephrocalcinosis, whereas F at equimolar dietary concentrations had marked antinephrocalcinogenic activity. The halogens were added to the diets in the form of KBr, KI, and NaF; the diets were balanced for the kations with Cl salts. The addition of KI to the diet to a concentration of 5.24 mmol/kg caused pronounced growth retardation, decreased feed intake, hepatomegaly, and signs of lethargy. It is concluded that the protective effect of dietary F against P-induced nephrocalcinosis does not extend to other halogens. PMID- 1724180 TI - [The incidence of viral and microbial antigens and the serum interferon titer in certain forms of rheumatism]. AB - The presence of some viral and inframicrobial antigens in peripheral leukocytes was investigated by the indirect immunofluorescence technique in 120 patients with different forms of rheumatism and 50 clinically healthy controls. Mycoplasma pneumoniae and type 3 para-influenza virus were detected most frequently. The determination of serum interferon titer revealed a rise of this product in rheumatic patients. PMID- 1724179 TI - Aluminum effect on the activity of superoxide dismutase and of other antioxygenic enzymes in vitro. AB - The effect of Al on superoxide dismutase (SOD) and on other antioxygenic enzymes: horseradish peroxidase, catalase, and glutathione peroxidase, has been investigated in vitro. In the case of SOD, the effect of metal chelators (EDTA and deferoxamine) and a possible synergistic effect with iron salts have also been tested using the pyrogallol assay. There is no significant inhibitory effect of Al on the activity of any of the above-mentioned enzymes. Noticeable increases in SOD activity were observed when metal chelators were added to the medium, but not when high concentrations of Al were present too, in the case of deferoxamine (DFO). The former fact seems to be a consequence of the chelation of transition metal ions that catalyze pyrogallol autoxidation by a mechanism not inhibitable by SOD, interfering in its action, which may account for part of the DFO antioxidant effect observed in vivo. The latter phenomenon could be owing to a saturation of the chelating capacity of DFO by an excess of Al present in the medium, which should bring the system back to the interfering conditions explained above. It can be concluded that Al, either in the presence or in the absence of iron salts, does not inhibit SOD activity in vitro. Moreover, no significant binding of Al to SOD was demonstrated, and the amounts of its metal constituents, Cu and Zn, were not affected by preincubation of the enzyme with Al. The effect of the different compounds tested on the rate of autoxidation of the indicating scavenger, pyrogallol, and a suitable hypothesis on their role in the oxidation process are also discussed. PMID- 1724181 TI - [Prevalence of antibody to hepatitis C virus in hospital personnel]. AB - Prevalence of antibody to hepatitis C virus (anti-HCV) has been widely investigated in many categories; however no data are available on hospital personnel. The aim of our study was to investigate whether hospital personnel are at risk for HCV infection. METHODS: sera collected during a prospective study on HBV infection in hospital workers done in our institution in 1985 were analyzed for the ELISA test for anti-HCV from Ortho Diagnostic System. Sera were stored at -20 degrees C and were never defrosted until tested. A population of a consecutive series of healthy volunteer blood donors was used as a control group. RESULTS: the anti-HCV prevalence was higher in hospital personnel, than in blood donors (4.5 versus 1.1, p less than 0.001, Odds Ratio 4.5, Confidence Limits 2.9 7.2). CONCLUSION: although anti-HCV is not an "ideal" test for epidemiological purposes, our study suggests that hospital personnel is at high risk for HCV infection. PMID- 1724182 TI - Microwave assisted staining of nerve and muscle biopsy tissue. AB - This paper reports a technique using microwaves to assist penetration of stains into biopsy sections of muscle and peripheral nerve. The technique results in more consistent and reliable staining of tissue sections for examination by light microscopy. PMID- 1724183 TI - Basic blue 75: a new stain for erythroblasts. AB - C.I. basic blue 75 in an aqueous alkaline solution stains the nuclei of mature and immature erythroblasts bright red. Simultaneously, the stain colors the cytoplasm of erythroblasts blue in immature cells and purple in mature cells. Colors of the type described were not found in other normal and abnormal hematopoietic cells. PMID- 1724184 TI - Staining of nuclei in fungi by ethidium bromide. AB - Ethidium bromide, (0.1% solution in ethanol-water, 1:3, v/v) was used to stain nuclei in mycelia and spores of different fungi. Nuclei looked bright brick red under green excitation. This method is very efficient, specific, reproducible and cost-effective. PMID- 1724185 TI - The cystine-stabilized alpha-helix: a common structural motif of ion-channel blocking neurotoxic peptides. AB - Neurotoxic peptides from venoms of scorpions and honey bees exhibit a consensus pattern in the two disulfide bridgings related to the sequence portions Cys-X-Cys and Cys-X-X-X-Cys. A revised three-dimensional structure of charybdotoxin, as determined by two-dimensional nmr spectroscopy, confirms that the consensus cystine dislocation generates in all these toxins a common structural element, i.e., the cystine-stabilized alpha-helical (CSH) motif, which may be correlated with their common ion channel blocking activity. PMID- 1724186 TI - Interleukin 4 drives phytohemagglutinin-activated T cells through several cell cycles: no synergism between interleukin 2 and interleukin 4. AB - Cell kinetic studies of T cells stimulated with the interleukin 2 (Il-2), Il-4, or both lymphokines were performed with conventional [3H]thymidine incorporation and with the bivariate BrdU/Hoechst technique. Il-2 and Il-4 are able to drive phytohemagglutinin-activated T cells through more than one cell cycle. Neither synergistic nor inhibitory effect on T-cell proliferation was seen for the stimulation with both Il-2 and Il-4 as compared with the effect of Il-2 alone. The quantitative data of the cell cycle distribution of phytohemagglutinin activated T cells suggest that the population of Il-4-responsive cells is at least an overlapping population, if not a real subset of the population of the Il 2-responsive cells. PMID- 1724187 TI - Insulin modulation of acute-phase protein production in a human hepatoma cell line. AB - Insulin is widely used as a growth factor in hepatocyte culture but its effect on the production of acute-phase proteins has not been studied. By measuring four positive (fibrinogen, alpha 1-antitrypsin, alpha 1-acid glycoprotein, and alpha 1 antichymotrypsin) and four negative (albumin, prealbumin, transferrin, and retinol binding protein) acute-phase proteins produced by the Hep G2 hepatoma cell line, we have shown that insulin is an important modulator of acute-phase protein production. Our data show that insulin is able to inhibit the synthesis of prealbumin, transferrin, and fibrinogen. The results also show a complex interaction between insulin, interleukin 6, and glucocorticoids because insulin is able to inhibit the dexamethasone induction of alpha 1-antichymotrypsin, and in the presence of interleukin 6, dexamethasone is able to regulate the production of fibrinogen and prealbumin. The regulatory role of insulin in fibrinogen production was confirmed by pulse chase labeling followed by immunoprecipitation and fluorography. PMID- 1724188 TI - Semliki Forest virus envelope proteins function as proton channels. AB - It has been shown that isolated nucleocapsids of Semliki Forest virus (SFV) contract upon low pH exposure (Soederlund et al., 1972). This contraction of the nucleocapsids has been used as an indicator to demonstrate that the spike proteins of SFV can translocate protons into the interior of the virus particle upon low pH (5.8) exposure. Spikeless virus particles obtained after bromelain digestion, which were used as a control, did not translocate protons. This implies that the ectodomain of the spike plays a crucial role for the proton translocation. PMID- 1724189 TI - Surface heterogeneity of bovine sperm revealed by aqueous two-phase partition. AB - Ejaculated, bovine sperm have been subjected to multiple partition in aqueous two phase systems. This partition, carried out in a countercurrent fashion, reveals heterogeneity of the sperm population with respect to surface properties. The sperm, when partitioned in phase systems that detect non-change associated surface properties (change-insensitive) are largely distributed as two distinct populations. In charge-sensitive phase systems (which principally detect cell surface molecules carrying charge) the sperm do not show any obvious surface heterogeneity. Considerable heterogeneity is revealed in affinity-ligand phase systems containing palmitic acid coupled to one of the phase components poly(ethylene glycol). There is a difference in surface heterogeneity between sperm which have been washed in buffer or left unwashed, direct from the ejaculate. This is indicative of weak adsorption of proteins to the sperm surface in seminal fluid. These results show that bovine ejaculated sperm is a heterogeneous cell population having unequal distributions of a number of different surface molecules. PMID- 1724190 TI - [Current status of radiotherapy in treatment of esophageal cancer]. AB - An improvement of the quality of life by alleviating pain, improving swallowing and increasing weight is achieved in about 80% of patients with esophageal cancer. With survival rates of 41%, 21% and 11% after 1, 2 and 5 years, respectively the curative potential of radiotherapy is considered low. Survival is significantly better in patients with small tumors than with large tumors. Radiochemotherapy must still be considered an experimental modality. Afterloading may be helpful for palliation. Postoperative and postradiation results are relatively positive due to improved local control and patient selection. PMID- 1724191 TI - [Prevention of thrombosis with low molecular weight heparin and hydroxyethyl starch in general surgery]. AB - In an open, randomized, controlled and prospective study we compared the efficacy of LMWH with unfractionated heparin (UFH) and the effects of both heparin preparations on haemostatic and fibrinolytic parameters. We also stratified subgroups which received HES intra- and postoperatively. The study groups were well matched with regard to baseline characteristics. With LMWH 4 patients (4.1%) displayed a positive fibrinogen uptake test (1 patient with HES). With UFH 5 patients (5%) demonstrated a positive uptake (3 patients with HES). Antifactor Xa levels were significant higher in the LMWH groups; all other fibrinolytic and haemostatic factors, bleeding and local wound complications did not differ. These data suggest that LMWH in equally effective and safe as UFH in general surgery. HES may have and additive effect. PMID- 1724192 TI - [Perioperative chemotherapy of stomach cancer]. AB - Surgery is the treatment of choice in early stages of gastric carcinoma. Systemic chemotherapy (CTx) is indicated in unresectable and metastatic disease. New studies have shown that CTx perioperatively improved median survival times compared with surgery alone in locally advanced, technically inoperable tumors. Moreover CTx induced resectability in 47-80% of the patients. Therefore, younger patients in these stages of disease should be treated with effective CTx, such as EAP, in order to induce objective remissions so that residual tumor masses can be surgically removed. PMID- 1724193 TI - [Ductal pancreatic cancer]. AB - In a prospective study of 587 patients tumor resection was possible in 138 (23.5%). Only 91 (65.4%) had been classified as R0-resections. Since 1982 subtotal duodenopancreatectomy has been the preferred method for treating pancreatic head cancer. Operative mortality for the whole series was 6%, for subtotal duodenopancreatectomy only 2%. The 5-year-survival rate for all patients was 2.9% with a mean survival of 4 months. For tumor resection it was 11%, for R0 resection 16%. However, in stages I and II the 5-year-survival was 46% and in stage III with a solitary lymph node metastasis, 30%. PMID- 1724194 TI - [Is there an advantage to neo- or adjuvant chemotherapy in pancreatic cancer?]. AB - In view of the lack of representative clinical studies indication and potential value of neo-/adjuvant chemotherapy of pancreatic cancer has still to be discussed on the basis of palliative treatment results, prognostic and pathohistological data, theoretical considerations and experimental data. A critical analysis, however, indicates that the widespread therapeutical nihilism at least with regard to adjuvant trials should be obsolete today. On the contrary, adjuvant chemotherapeutic trials should be recommended, alone or in combination with radiation or immunotherapy. PMID- 1724195 TI - [Laser and afterloading therapy: esophageal cancer]. AB - In the palliative treatment of malignant esophageal stenosis endoscopic laser therapy and intracavitary radiation currently represent the best alternative. The results achieved with elimination of dysphagia, duration of the complaint-free interval, survival period, and complications are presented from a group of 167 patients. Referring to the quality of life, an evolving tendency towards the use of laser therapy instead of endoscopic intubation is reported. PMID- 1724196 TI - [Endoscopic neodymium YAG laser therapy of colorectal adenomas and cancers]. AB - Endoscopic Nd:YAG laser therapy (lambda = 1.06 microns; 80-100 W) of colorectal neoplasia is indicated in: 1. Premalignant, sessile adenoma. After snare resection for histologic estimation of dignity laser ablation is curative when it is benign. 2. Obstructed colorectal cancer can be recanalized preoperatively to perform orthograde bowel lavage, total colonoscopy and elective primary tumour resection. 3. Inoperable colorectal cancer can be irradiated palliatively by the laser to arrest bleeding, to avoid obstruction and to reduce protein and electrolyte secretion. PMID- 1724197 TI - [Esophageal tube in inoperable esophageal and cardia cancer: indications and technique]. AB - Endoscopic tube implantation remains still one of the alternatives in palliation of inoperable patients with malignant stenosis of the esophagus and cardia. Although LASER-treatment has less complications, patients in poor general condition, patients with tracheo-esophageal fistulas, patients with external compression of the esophageal lumen, are clear indications for tube implantation. The right choice of palliative technique should depend on the individual situation of the patient. PMID- 1724198 TI - [After care as a supportive measure in palliative surgery--pain therapy]. AB - Nearly 50% of all patients with cancer will suffer from chronic pain. Aside from specific anticancer treatment these patients need an adequate analgetic drug therapy. By using the WHO analgesic ladder correctly more than 85% of cancer pain can be treated effectively. Nevertheless, there are several reasons for an unsatisfactory management of cancer pain and in practice we often make simple but relevant mistakes in dealing with analgetic drugs, especially with opioids. Non analgetic treatment of cancer pain e.g. neurolytic or neurosurgical blocks are relatively unknown. An adequate and satisfactory care for patients with cancer pain is based on an individual comprehensive approach, including analgetic treatment, non-analgetic techniques and last but not least the consideration of psychological factors that influence and determine the severity of pain. PMID- 1724199 TI - [Monitoring and management of the central nervous system in treatment of tumor cachexia]. AB - After palliative surgery artificial nutrition is indicated primarily in patients receiving postoperative chemotherapy or radiotherapy. The techniques of ambulatory enteral or parenteral nutrition have been standardized during the past decade. They can be used with minimal risk even in patients with advanced cancer. An improvement of the nutritional status can be expected in most of the patients. Further clinical trials are needed to determine whether the patients' quality of life improves. PMID- 1724200 TI - [Collaboration in tumor after care with the established physician]. AB - The organization of cancer followup, the investigations and subsequent treatment are the responsibility of only those doctors directly involved. The followup work consists of three types: diagnostic after curative treatment, therapeutic with adjuvant chemotherapy and palliative. The cooperation of hospital doctors and practitioners has proved effective. The patient's data are recorded in the accompanying documentation. The follow-up system has met with high patient compliance (94%). The drop-out rate is 6%. Twenty percent of patients with colorectal cancer who underwent curative surgery had pathologic findings. One half of them could be treated therapeutically or palliatively. PMID- 1724201 TI - [Surgical oncology--oncologic surgery]. AB - Oncologic surgery still is the fundamental principle of cancer therapy. This is only useful when leading to a complete tumor extirpation (so-called R0 resection). Under these conditions an improvement of prognosis can be achieved. Unfortunately this aim can only be reached in some 50-70% of our patients. For all the other patients non-surgical therapy in surgery has to be secured by oncologic surgery. A most interesting development takes at present the preoperative chemotherapy or radiation therapy respectively. The aim is down staging of the tumor to make a final R0-resection possible after all. PMID- 1724202 TI - [Modern cancer therapy--an interdisciplinary task]. AB - The diagnosis of cancer and the treatment of cancer patients are initially an interdisciplinary entity. Progress has been achieved through oncological multimodal cooperation between various forms of organisations. The ensuing problems confirm the following two main points. (1) The continuing delay in diagnosis of cancer still prevents most patients from benefitting from the implementation of optimal therapeutic possibilities and interdisciplinary cooperation. (2) What the surgeon does not achieve or neglects during the primary operation cannot be compensated for through conservative oncological measures. PMID- 1724203 TI - [Sudeck's disease after radius fracture]. AB - Clinical and radiological features of Sudeck's dystrophy following radius fracture are described. Peripheral and endogenous factors are pointed out. Therapy consists in preventing and alleviating pain by primary immobilisation combined with gentle physical therapy of the non-involved parts of both upper extremities. Calcitonin, non-steroid antirheumatic and anxiolytic drugs should be administered simultaneously. The physician's care and tolerance of the mentally upset and labile patient are essential. Physiotherapy plays a major role in the long-standing rehabilitation process. PMID- 1724204 TI - [Are anti-thyroid drugs and iodine preparations for surgical preparation in Basedow's disease still current today?]. AB - From 1979 to 1989, in 1938 hyperthyroid patients with different preoperative treatment, thyreotoxic crisis did no longer occur. In a prospective study, 28 patients with graves' disease were operated in severe hyperthyroidism (max. fT3 37.3 pmol/l, normal range 4.0-9.0; max. fT4 12.6 ng/dl, normal range 0.7-1.9) premedicated only by betablockers: the man of fT3 sank significantly within 1 hour after operation and returned to normal range after 8 hours. The fT4 sank significantly within the 1st postop. day and reached its normal value on the 4th postop. day. No rise of fT3 or fT4 was ever observed, nor was there any clinical sign of thyreotoxicosis. Therefore in hyperthyroidism, a routine preoperative treatment with antithyroid drugs or iodine is not necessary. PMID- 1724205 TI - Phosphatidylinositol-glycan linked proteins of the erythrocyte membrane. AB - The human erythrocyte bears a number of proteins anchored to the outer membrane surface via a phosphatidylinositol-glycan linkage. This class of proteins includes several complement regulatory proteins (including decay-accelerating factor, CD59 antigen (protectin), and C8 binding protein) as well as several enzymes and at least one protein important in cell-cell interaction. In addition, a number of blood group antigens have been identified to reside on proteins with phosphatidylinositol anchors. One blood group (Cromer) resides on DAF. Study of variants in this blood group system has led to interesting information about the function and expression of this protein. Several other blood groups, such as JMH and Holley/Gregory, appear to reside on as yet unidentified phosphatidylinositol linked proteins. In paroxysmal nocturnal haemoglobinuria, a variable proportion of red cells fail to express or express weakly all phosphatidylinositol-linked proteins. The origin of this deficiency is now being worked out. In addition, individuals with inherited deficiency of DAF or CD59 (protectin) have been identified. Only the latter deficiency leads to a PNH-like syndrome. PMID- 1724206 TI - Allotypes and other epitopes of immunoglobulins. PMID- 1724207 TI - [The prevention of an excitation-conduction block during acute myocardial ischemia: is there a role for prostacyclin or for histamine?]. AB - We have investigated (multivariate Cox's model) the relative risk of stable excitation-conduction block (ECB) in right ventricular myocardial strips (2 x 5 x 1 mm) from 26 female guinea-pigs, bathed in a 2-compartment chamber (3 ml) on the anterior side of which modified Tyrode's solution (K+ 12 mM, HCO3- 9 mM, pH 7 +/- 0.05, pO2 80 +/- 10 mmHg and absence of glucose) is supraperfused (stimulation rate: 450 ms; wire in the posterior compartment), thus enabling simulation of electrophysiologic changes seen during acute myocardial ischemia. Using glass microelectrodes, action potential amplitude (APA), durations (APD50 and 90%), resting membrane potential (RMP) and upstroke velocity (Vmax) are investigated. The hypothesis was tested of prostacyclin and histamine involvement in the genesis of ischemia-induced ECB in this model. Either a weak prostacyclin stimulator (cicletanine 10(-5) M, IPSEN, Paris, F, in DMSO 1:100; n = 16) or a potent prostacyclin generation blocker (indomethacin 10(-5) M, Sigma, in DMSO 1:100; n = 10) and either DMSO alone (1:100; n = 16) or a specific histaminergic H1 receptor antagonist (terfenadine 10(-5) M, Sigma, in DMSO 1:100; n = 10) were supraperfused using a randomization scheme. Each animal was used twice and either a first or a second occlusion (supraperfusing the modified Tyrode's solution for 30 min) period was performed and the randomized substances were supraperfused, thus enabling obtention of n = 52 experiments for analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724208 TI - [Diagnosis of Chlamydia trachomatis by culture and Macchiavella's staining technique]. AB - The author modified and tested staining according to Macchiavelli for detection of C. trachomatis in tissue cultures. As compared with IF staining by the monoclonal antibody the author found a high sensitivity (95.7%) and specificity (99.0%) of this method. Giemsa staining evaluated in passing light has a lower sensitivity (85.5%) and lower specificity (94.1%). The advantage of Macchiavelli staining is also the low price of the chemicals used. Imunofluorescent staining with the monoclonal antibody remains also now the reference method and is most reliable. For some materials which are obtained with difficulty (e.g. punctates etc.) the author recommends to use the IF technique. PMID- 1724209 TI - Class 1 heparin binding growth factor promotes the differentiation but not the survival of ciliary neurones in vivo. AB - Molecules isolated from target tissues of various neuronal populations have been shown to enable the survival of those innervating neurones. A molecule derived from bovine heart which can support the survival of parasympathetic neurones of the chick embryo ciliary ganglion in vitro has been sequenced and identified as a member of the class 1 heparin binding growth factors (HBGF-1). When injected into developing chick embryos during the period of naturally occurring cell death in the ciliary ganglion, HBGF-1 failed to rescue those neurones which normally die. There was, however, a premature increase in the activity of choline acetyltransferase within the ganglion and a shift to the right in the size distribution of the neurones. We conclude that HBGF-1 may promote the differentiation of some subpopulation of neurones of the ciliary ganglion of the chick embryo but does not act in vivo as a survival molecule. PMID- 1724210 TI - Alterations of visual cortical connections in cats following early removal of retinal input. AB - The major afferent pathways to the visual cortex were studied in cats enucleated at postnatal days 0, 15, 30 and 60 using the retrograde tracer wheat germ agglutin conjugated horseradish peroxidase (WGA-HRP). Following enucleation at days 0 and 15, the lateral gyrus was shrunken and cortical thickness was decreased, but the thickness of layer I was increased relative to normal adult cats. The laminar portion of the lateral geniculate complex occupied only half its normal volume, but the pathway from the intralaminar nuclei to visual cortex was doubled in volume. The ipsilateral and contralateral claustrum and ipsilateral medial septal nucleus also provided increased input to visual cortex. The changes in these projection patterns were not as dramatic in the cats enucleated at days 30 and 60. The callosal pathway between areas 17 and 18 was also significantly altered in cats enucleated on the day of birth and at day 15. Callosal cells were found in the infragranular layers throughout area 18. The number of supragranular callosal cells in the medial half of area 18 and in area 17 was reduced to approximately one third the normal adult number, but the mediolateral extent of this zone was not reduced. In the infragranular layers, the callosal cell number was approximately twice that in adult cats, and they occupied at least twice their normal mediolateral extent. Following bilateral enucleation at days 30 and 60, the cell numbers and mediolateral extent of the callosal cell zones approximated those in normal adult cats. Taken together, these results indicate that early deafferantiation of a cortical area can alter the thalamic, extrathalamic and callosal connections of that area. PMID- 1724211 TI - Development of projections of primary afferent fibers from the hindlimb to the gracile nucleus: a WGA-HRP study in the rat. AB - The projection of primary afferent fibers to the gracile nucleus was studied during development. Injections of wheat germ agglutinin-horseradish peroxidase were made into the hindlimb of fetal, postnatal and adult rats. In most cases the sections were alternately stained for wheat germ agglutinin-horseradish peroxidase including counter stain with Neutral red and for acetylcholinesterase. At embryonic day 17 labelled fibers could be traced to the mid-cervical spinal cord but not further rostrally. At embryonic days E18 and E19 labelled fibers penetrate the rostral pole of the nucleus, which does not happen more caudally. At embryonic day E21 the caudal-most pole of the gracile nucleus still is not penetrated by labelled fibers. From postnatal day 1 onwards labelled fibers are found throughout the entire rostrocaudal extent of the gracile nucleus. These results suggest that primary afferent fibers from the hindlimb first grow to the rostral pole of the gracile nucleus and penetrate the rostral pole immediately upon their arrival. During further development more caudal parts of the gracile nucleus are gradually penetrated in a rostrocaudal fashion by primary afferent fibers of the hindlimb. PMID- 1724212 TI - Cell death in the developing trigeminal nuclear complex of the rat. AB - The time course and distribution of cell death in the trigeminal nuclear complex of the rat has been examined with the aid of sections stained for Nissl substance and succinic dehydrogenase activity. Pyknotic figure counts in the principal trigeminal nucleus and in each of the three spinal trigeminal subnuclei revealed that cell death commences at E19, in the region of the junction between the principal nucleus and the subnucleus oralis, close to the site of entry of trigeminal afferents into the brainstem. Cell death subsequently spreads rostrally and caudally into the rest of the principal and spinal trigeminal nuclei. Cell death ceases simultaneously, at about P10, in all parts of the trigeminal nuclear complex examined. Neurons could not be reliably distinguished from glial cells in prenatal animals, but data for neuronal numbers postnatally indicate that much of this cell death is indeed due to loss of neurons. The data suggest that, in the trigeminal nuclear complex, only half the number of neurons produced survive to maturity. These findings are of significance for those investigators using this system in studies of plasticity. PMID- 1724213 TI - Diurnal and postural variation in serum alpha 1-microglobulin in normal individuals. PMID- 1724214 TI - High-performance liquid chromatographic determination of bilirubin distribution on serum proteins. AB - We describe a high-performance liquid chromatographic method for the determination of bilirubin distribution on serum proteins. The procedure is based on both size-exclusion and adsorptive properties of the column packing material LiChrosorb Diol. The elution of all sample constituents is accomplished by means of an aqueous mobile phase containing phosphate buffered solution of adult serum, supplemented by a small amount of bilirubin. The different distribution of bilirubin on albumin, beta-lipoprotein, and as free (loosely bound) pigment fraction is exemplified by assaying normal adult and infant sera. PMID- 1724215 TI - Short- and long-term effects of ammonium chloride on RNA, DNA and protein concentrations in muscle and serum in the rainbow trout, Oncorhynchus mykiss. AB - 1. Effects of ammonium-induced acidosis on white muscle RNA, DNA and protein concentrations were examined in the rainbow trout, Oncorhynchus mykiss. Serum protein concentrations were also determined. 2. A single administration of ammonium chloride at a dose of sublethal level caused no changes in these parameters after 3 and 6 hr. 3. Multiple administrations at a lesser dose caused significant decreases in RNA and serum protein concentrations 48 and 72 hr after the first administration. 4. DNA and muscle protein concentrations remained unchanged throughout the experimental period. 5. Significant decreases in RNA-DNA ratios and RNA-muscle protein ratios were primarily attributed to decreases in RNA concentrations. PMID- 1724216 TI - Morphological aspects of human lens capsules. A comparative LM, SEM and TEM examination. AB - Lens capsules of patients of advanced age, obtained after extracapsular cataract surgery, were carefully prepared for a combined LM, TEM and SEM investigation, after preliminary washing and mounting onto a holder in a buffer solution. After pre-fixation with GA, samples were postfixed for LM/TEM and OsO4/K4Fe)CN)6 and stained with toluidine-blue/basic fuchsin for LM and with uranyl acetate/lead citrate for TEM; for SEM the GA-pre-fixed samples were post-fixed by the Tannin Arginine-OsO4 non-coating technique. At LM-level discrimination between healthy and degenerating cells was possible after toluidine staining. At SEM-level protrusion of the cell nucleus and fibrillation and blebbing of the cell membrane as the result of capsular degeneration could be observed with the TAO-method. At TEM-level protrusion of the cell nucleus, degeneration of the cytoplasm, ballooning of the mitochondria, the presence of microfilaments and the occurrence of vacuoles were visible as the result of capsular degeneration on cataract formation. PMID- 1724217 TI - Control of translation in adenovirus-infected cells. AB - The initiation of protein synthesis in adenovirus-infected cells is regulated during the late phase in two ways, which may be related. The overall translation rate is maintained by a small viral RNA, VA RNAI, which prevents the phosphorylation of initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. In addition, the relative efficiency of translation of host cell and viral mRNA populations is regulated in the infected cell during the late phase such that viral mRNAs are selectively utilized. Three viral elements have been implicated in this process: the 5' leader present on most late viral mRNAs; the late protein, 100K; and VA RNA. This article reviews the mechanisms underlying these translational control phenomena. PMID- 1724218 TI - Regulation of viral and cellular RNA turnover in cells infected by eukaryotic viruses including HIV-1. AB - The following reviews the role of mRNA stability in the regulation of both viral and cellular gene expression in virus-infected cells. Indeed, several eukaryotic viruses, including the human immunodeficiency virus, HIV-1, regulate cellular protein synthesis via such control mechanisms. The following systems will be discussed: (i) the degradation of viral and cellular mRNAs in cells infected by herpes simplex virus (HSV) and advances made using the HSV virion host shutoff mutant; (ii) the degradation of viral and cellular mRNA and ribosomal RNA in cells infected by vaccinia virus and the possible role of the oligoadenylate synthetase-RNase L pathways; (iii) the turnover of RNAs in cells infected by encephalomyocarditis virus, reovirus, and La Crosse virus; and finally (iv) recent studies from our laboratory on the degradation of cellular mRNAs in cells infected by HIV-1. PMID- 1724219 TI - Boundaries as distance regulators in personal relationships. AB - Received wisdom suggests that boundaries are, or should be, important in intimate relationships. In this essay, we focus primarily upon the beliefs and phenomenology relating to a variety of boundaries, and provide a discussion of some conceptual issues, in order to understand better the development, facilitation, and maintenance of, as well as restraints upon, intimacy. Although we attend mainly to dyadic relationships, we believe that our observations and suggestions have application to larger groups. PMID- 1724220 TI - Muddles, bewilderment, and practice theory. AB - "Useful distinctions in conceptual schemes lead to explanatory or descriptive metaphors that have a clear form. Muddles, on the other hand, are created when useful distinctions that could be drawn are not[,] or when an unnecessary distinction is drawn" (5, p. 71; italics omitted), or when when a useful distinction is minimized or blurred. The field of family therapy (particularly its theories), as a whole, has various muddles of each kind. The purpose of this essay is to describe some of these muddles (in the field and its theories) and to suggest that the distinctions (a) between descriptions of doing therapy that exclude the therapist and descriptions of doing therapy that include the therapist (1, 5-9, 11), (b) between explanation and description, and (c) between ideology and grounded theory, are useful, and that in de-radicalizing and minimizing these distinctions, these differences creates mayhem and muddle--a veritable fog of non-clarity. PMID- 1724221 TI - [Effect of various types of vagotomy on gastric secretion of histamine in dogs]. AB - The chronic experiments on dogs with fundal fistulas have revealed that the partial stomach denervation (selective proximal, selective distal and subcardial vagotomies) decreases the gastric secretory responses caused by submaximal and maximal doses of histamine. Selective and extragastral vagotomies do not change the histamine gastric secretion in dogs, stimulated by the maximal histamine dose. PMID- 1724222 TI - [Contribution of motor transport powered by diesel engines to air pollution by sulphur compounds]. PMID- 1724223 TI - [Accumulation and metabolism of organochlorine pesticides in the rat liver]. PMID- 1724224 TI - [Oxycardiotocography. A new procedure for measuring fetal oxygen saturation sub partu. Introduction to the method]. PMID- 1724225 TI - [Supportive measures for cytostatic therapy of gynecologic cancers]. PMID- 1724226 TI - [Adjuvant chemotherapy in radically operated cervix cancer]. PMID- 1724227 TI - [Is supravesical urinary diversion in terminal gynecologic cancer indicated?]. PMID- 1724228 TI - [Therapy of tubal pregnancy with prostaglandin]. PMID- 1724229 TI - Gestational choriocarcinoma with haemorrhagic pleural effusion. AB - A case of gestational choriocarcinoma with haemorrhagic pleural effusion is described. The importance of detection of human chorionic gonadotrophin in pleural fluid is highlighted. PMID- 1724230 TI - Differential transcriptional control of the H-2K and H-2D loci of the major histocompatibility complex in fibrosarcoma cells. AB - In this study we demonstrate a differential transcription of H-2K and H-2D class I genes in two different tumor cell clones; one is highly metastatic (IE-7) and the other is not metastatic (IC-9), both derived from the same fibrosarcoma, T 10, induced in an (H-2b x H-2k)F1 mouse. The expression of the two parental H-2K alleles is transcriptionally suppressed in both of these clones. In addition the IC-9 clone does not transcribe also the H-2Dk allele. Our data rule out the possibility that this suppression results from enhanced RNA degradation, impaired polyadenylation, DNA rearrangement, or changes in DNA methylation within these genes. Interferons (IFN) are known to enhance MHC expression by acting on a consensus IFN responsive element present in the promoter region of MHC genes. However, IFN-gamma, which is the most potent IFN in this respect, failed to activate the expression of the silent MHC genes in our cells. This finding may reflect a defect within the promoter region of these genes or changes in their chromatin structure. PMID- 1724231 TI - Effects of feeding low protein diet with and without leucine supplementation on protein status of lactating females and their pups. AB - Female rats were fed low protein diet (10% casein) either as such or supplemented with 3% leucine during pregnancy and lactation. Changes in litter size and the survival rate, growth and protein status of the pups were noted. The milk yield and hepatic and mammary gland protein status of the mothers were also studied. Feeding low protein diet reduced litter size, increased their mortality and resulted in poor growth of the pups. It also resulted in poor hepatic and mammary gland protein status of the mothers, as well as reduced their milk yield. On adding 3% leucine to 10% casein in the diet, the changes observed in the low protein group, did not alter in any manner. PMID- 1724232 TI - Atresia of the left atrioventricular valve with patency of the aorta: anatomico functional analysis of 23 patients. AB - We analyzed the findings in 23 patients with atresia of the left atrioventricular valve and a patent aorta seen in the period from January 1980 to July 1989. Having divided the cases according to the anatomical findings, we made a subsequent analysis of the clinical and surgical results with the aim of establishing the management most likely to diminish risks, still high, in treatment of this complex anomaly. From the anatomical viewpoint, three variants were observed. In the first, made up of 15 cases, there was absence of the left atrioventricular connexion. The characteristic finding in the second group, with five cases, was an imperforate left atrioventricular valve in the setting of concordant atrioventricular connexions. The third group, of these cases, was dominated by the presence of isomerism of the atrial appendages, both appendages being of left morphology in one case, and of right morphology in the other two. Further anatomical variation was then found in each group. Nine of the 15 with absence of the left atrioventricular connexion had the right atrium connected to a dominant left ventricle in presence of a rudimentary and incomplete right ventricle associated with discordant ventriculo-arterial connexions, all of them being in usual atrial arrangement and three with pulmonary stenosis. The remaining six in this first variant had the right atrium connected to a dominant right ventricle. In the five patients with imperforate left atrioventricular valves, two had discordant and three had concordant ventriculo-arterial connexions. In the three cases with isomerism, two had absence of the left atrioventricular connexion, with a dominant right ventricle. The last patient had an imperforate left atrioventricular valve and a discordant ventriculo-arterial connexion. From the functional viewpoint, there were 14 patients (10 with absence of an atrioventricular connexion, four with imperforate atrioventricular valve) with congestive heart failure and nine patients (five from the first, one from second, and three from the third variant) with hypoxia. Long-term follow-up (median 16.4 months--varying from 1 to 41 months--in the group with congestion and 27.7 months--varying from 12 to 57 months--in those with hypoxia) showed favorable clinical evolution in 11 (91%). We conclude that an anatomico functional division can point towards the most appropriate management in this complex anomaly. PMID- 1724233 TI - Evaluation of disopyramide and mexiletine used alone and in combination for ventricular arrhythmias in patients with and without overt heart disease. AB - The efficacies and side effects of disopyramide and mexiletine used alone and in combination were assessed in 29 patients with chronic ventricular arrhythmias. In combination therapy, one half or two thirds of the conventional doses of each drug were administered. Each patient underwent Holter electrocardiographic monitoring during 4 different periods: baseline, disopyramide alone, mexiletine alone and combination of the two drugs. The mean baseline number of ventricular premature complex per hour was 783 +/- 521 (mean +/- SD), which was significantly reduced with all three therapies. Disopyramide alone significantly reduced the ventricular premature complex frequency in patients with organic heart disease (P less than 0.05), but did not significantly reduce the ventricular premature complex frequency in patients with no apparent heart disease. In contrast, mexiletine alone significantly decreased the ventricular premature complex frequency in no apparent heart disease patients (P less than 0.05), but did not significantly reduce the ventricular premature complex frequency in organic heart disease patients. With disopyramide alone, patients having a significant reduction in ventricular premature complexes (greater than or equal to 83% reduction in ventricular premature complexes) or elimination of ventricular tachycardias tended to be more frequently found in organic heart disease than in no apparent heart disease. The opposite was observed with mexiletine alone. QTc interval with disopyramide alone was significantly prolonged, and the prematurity index of ventricular premature complexes was significantly lowered as compared to mexiletine alone or combination therapy (P less than 0.01 for disopyramide versus mexiletine; P less than 0.05 for disopyramide versus combination therapy). During combination therapy, no patients withdrew from the study due to side effects. However, 3 patients receiving single drug therapy withdrew from the study due to severe side effects. Consequently, disopyramide is suggested to be more effective on ventricular premature complexes in organic heart disease than in no apparent heart disease patients, whereas the opposite was true for mexiletine. A combination of disopyramide and mexiletine in smaller doses may provide almost the same or enhanced antiarrhythmic effects, no aggravation of electrocardiographical parameters and less incidence of side effects when compared to the conventional dose of each drug alone. PMID- 1724234 TI - Substance P-induced histamine release from rat peritoneal mast cells and its inhibition by antiallergic agents and calmodulin inhibitors. AB - Substance P-induced histamine release and Ca2+ release from the intracellular Ca store of rat peritoneal mast cells were inhibited by both antiallergic drugs and microtubule inhibiting agents. It was found that in the case of antiallergic compounds, histamine release inhibition may be intimately related to the inhibition of Ca2+ release from the intracellular store in which the microtubules play an important role. When mast cells were pretreated with either theophylline or dibutyryl cAMP, the inhibition of histamine release was closely related to the inhibition of Ca2+ release from the intracellular Ca store. Calmodulin inhibitors were also effective in inhibiting histamine release from mast cells induced by substance P. The inhibitory potencies of calmodulin inhibitors on histamine release from mast cells were closely correlated with those exerted on calmodulin activity. PMID- 1724235 TI - Benign cystic vs. solid lesions of the parotid gland in HIV patients. AB - In this study of 13 patients with cystic lesions of the parotid gland, 9 patients were known to be antibody positive for the human immunodeficiency virus (HIV) and 4 were subsequently tested to be positive. All patients had computed tomographic (CT) confirmation of parotid gland cysts. Five patients had fluid aspirates showing high amylase levels. All cystic lesions had lymphoepithelial features and lymphoid histology similar to those seen in HIV infection. This study includes a review of 148 HIV patients reported in the literature, as well as our experience. Of all the reported cases, when gross pathology suggested cystic lesions, the incidence of malignancy was close to 1%. The incidence of malignancy for a solid mass, however, was close to 40%. We propose a nonsurgical management protocol which includes CT scan and needle aspiration with tissue for cytology and fluid for amylase level if possible. Watchful observation is advised for cystic pathology. PMID- 1724236 TI - Collagen-associated sulphated proteoglycans. Ultrastructure after formaldehyde cetylpyridinium chloride fixation. AB - In the course of an ultrastructural cytochemical study of intracellular sulphated proteoglycans involving the addition of cetylpyridinium chloride in the primary aldehyde fixative, a remarkable ultrastructural preservation of the collagen associated sulphated proteoglycans was observed. Together with the preservation of their localization among the collagen fibrils (with, for some of them, a 50 nm periodic association with d-bands) and of their native elongated shape, previously observed under similar technical conditions, these stick-shaped and chondroitinase ABC-sensitive proteoglycans exhibited a typical pattern with several dense longitudinal parallel tracks (periodicity: 3-4 nm) not described as yet. Readily observable without high iron diamine-staining, the morphology of these cetylpyridinium chloride-precipitated and collagen-associated polyanions was particularly enhanced after incubation in the diamine solution which ascertained their sulphate content. Such a common ultrastructural organization with parallel tracks for both intracellular (i.e., in eosinophilic polymorphonuclear cells and Kurloff cells) and extracellular CPC-precipitated sulphated proteoglycans could correspond to intrinsic properties of the complexed molecules and could be related to 'double track' proteoglycans observed under other technical conditions in basement membranes. PMID- 1724237 TI - Observer variation in quantification of immunocytochemistry by image analysis. AB - This paper reports the findings of a study designed to examine observer variation as a source of inaccuracy inherent in the use of computer-assisted image analysis to measure areas of stained tissue. The rat pituitary immunostained for prolactin and galanin was used as an example to estimate patterns of immunoreactivity exhibited by different cell types. Six observers, with differing experience, selected grey level threshold values on 40 fields of images of stained tissue making three repeats of each field. The 40 fields consisted of 20 serial pairs of colocalized fields, one immunostained for prolactin, the other for galanin. The 20 pairs consisted of four pairs from each of five animals. Analysis of observer variation in the selection of threshold values showed large differences in the within- and between-observer variation. Analysis of the components of variance in the estimation of the ratios of stained tissues showed that the major source of variation was the within-observer component. An additional experiment using two observers, where half of the images were compared to the original microscope images before setting threshold levels, showed that the opportunity to make a comparison did not reduce observer variation. It is suggested that any study which uses semi-automatic methods to segment regions of a digital image can benefit from an analysis of this kind so that the sources of variation can be determined to enable maximum discriminating power in future studies. PMID- 1724238 TI - Comparison of the effects of four hyperplastic agents on hamster cheek pouch mucosa. AB - Hyperplasia in the hamster cheek pouch was examined using 4 different hyperplastic agents: oil of turpentine 50% v/v in liquid paraffin; vitamin A palmitate 10% w/v in liquid paraffin; 12-O-tetradeconyl-phorbol-13-acetate 16nM in acetone; and ethylphenylpropiolate 0.04mM in acetone. Acetone, paraffin and untreated control groups were also examined. Cheek pouches were painted 3 times a week for up to 4 weeks with each solution. Samples were removed and prepared for light microscopy 24 hours after 2 weeks of painting and 24 hours, 6, 12 and 18 weeks after 4 weeks of painting. Hyperplasia was produced by application of turpentine, vitamin A and TPA after 2 weeks of application. Further increases in epithelial width occurred after 4 weeks of painting in the turpentine and vitamin A groups but a decrease was seen in the TPA group. Six weeks (vitamin A and TPA groups) or 12 weeks (turpentine group) after the completion of treatment the epithelium had a normal histological appearance. No differences between the control or EPP treated cheek pouch mucosa could be detected. Turpentine and vitamin A can be used as models of reversible hyperplasia in the hamster cheek pouch. PMID- 1724239 TI - [The expression of various cytokeratins by epithelial cells of calcified odontogenic cysts]. AB - The calcifying odontogenic cyst (COC) is a rare lesion without specific clinical characteristics. Its diagnosis is essentially histopathological showing the presence of ghost cells associated with narrow squamous epithelium, the basal strata of which consist of clearly delineated cells differentiating in areas of the stellate reticulum in a similar way to ameloblastoma. The results of ultrastructural observations of the ghost cells as well as histochemical and immunohistochemical studies suggest that they are the sites of abnormal keratinisation. The aim of this study of one COC mas to locate the stages in epithelial maturation associated with the formation of ghost cells. Six monoclonal antibodies were used; three with a wide spectrum (KL1, AE3, AE1), two with a narrow spectrum against high molecular weight cytokeratins (AE2, AE8) and one against vimentin (M725). Histopathological examination of the COC revealed three different types of cells in the epithelial lining and the epithelial islands; small basal cubic cells surrounding larger ones placed centrally or suprabasally; balloon shaped cells or flattened ghost cells rolled up on themselves to resemble keratinising pearls, or cornifying cells. The ghost cells and cornifying cells had an altered distribution of their cytokeratins demonstrated by the absence of staining of antibodies against cytokeratins. The differentiated cells adjacent to them showed cytokeratins typical of squamous epithelium rather than those associated with the process of keratinization. The coexistence of cornifying and ghost cells testifies to the great potential of odontogenic epithelium to form numerous epithelial islands. PMID- 1724240 TI - Extracellular matrix and intermediate filaments in the first stages and repair of experimental gingivitis in man. AB - By the indirect immunoperoxidase labelling procedure the expressions of type I and III collagens, laminin and fibronectin and of KL1 cytokeratin and vimentin were examined in the first stages and repair of experimental gingivitis in young subjects. A two month longitudinal study was performed using the tissue from buccal marginal gingival biopsies of four subjects taken sequentially at five specific times: before and during plaque accumulation, and during plaque elimination. The sites examined microscopically were the coronal half of the junctional epithelium and the underlining infiltrated connective tissue fraction. No clinical change could be observed during the study period. Histological examination showed reversible cellular changes during the accumulation of plaque. There were increases in vascularization and cellularity and loss of collagen. They recovered 56 days after plaque elimination their baseline level. Electron microscopic examination showed myofibroblastic aspects in some fibroblasts. The changes in the expression of laminin, fibronectin and KL1 in the J.E. might be due to a proliferation rate enhancement, and suggest an adaptation to the alterations brought about by the inflammatory process. They also reinforce the hypothesis that this epithelium resembles a developmental tissue. Type I collagen demonstrated the "collagen loss-repair" cycle shown in connective tissue by the histological study. The rise in type III collagen and the vimentin fall, both at the initial stage, suggest that these protein profiles may yield information for clinical research purposes during the very early inflammatory process. The variations in fibronectin indicate its key role in the early inflammatory and repair processes. Finally, the variations in matrix and cytoskeletal proteins variations, as well as the morphological modifications observed, were nearly all reversible. PMID- 1724241 TI - Regulators of angiogenesis: stimulators, inhibitors, and extracellular matrix. PMID- 1724242 TI - Release of basic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: a trivial phenomenon or a novel secretion mechanism? AB - Basic fibroblast growth factor (bFGF), a potent angiogenesis inducer, lacks a signal sequence. Therefore, it has been proposed that bFGF is primarily released from dead or damaged cells. Other proteins devoid of secretion signals, interleukin 1 beta (IL-1 beta) and the muscle lectin L-14, have been shown to be released via exocytosis, a novel secretion pathway independent of the "classic" endoplasmic reticulum-Golgi route. In the light of these findings and of our own recent results, we discuss evidence that bFGF can be released from single, uninjured cells and mediate functions in an autocrine manner. As is the case for IL-1 beta and L-14, externalization of bFGF may occur via exocytosis, a pathway utilized during development and differentiation. PMID- 1724243 TI - Platelet-derived endothelial cell growth factor. AB - Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45 kDa single chain polypeptide which stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo. Analysis of a full length PD-ECGF cDNA revealed an open reading frame coding for 482 amino acids without homology to other known proteins. No signal sequence was observed, and analysis of the biosynthesis and processing of PD-ECGF in a thyroid carcinoma cell line revealed that PD-ECGF is released only very slowly. PD-ECGF becomes covalently associated with nucleotide triphosphates (e.g., ATP) in vivo, as well as in vitro. The physiological significance of this posttranslational modification remains to be elucidated. The tissue distribution and target cell specificity of PD-ECGF suggest roles in angiogenesis (e.g., during wound healing and in the developing placenta), as well as in the maintenance of the integrity of the endothelial cell lining of large vessels. PMID- 1724244 TI - Endothelial cell regulation by transforming growth factor-beta. AB - Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-beta. Interrelationships between these effects have shed some light on the mechanism of action of TGF-beta and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-beta. In addition, TGF beta and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-beta has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-beta under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-beta, thus further implicating TGF-beta in the maintenance of the quiescent, differentiated aggregation of EC as found in vascular structures in vivo. While more information is needed to define what, if any role TGF-beta plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-beta are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF beta primes them for differentiation and/or is critical for the maintenance of a differentiated state. PMID- 1724245 TI - A metalloproteinase inhibitor as an inhibitor of neovascularization. AB - Metalloproteinases and their endogenous inhibitors are key components of an enzyme system which is important in a number of fundamental biochemical and cellular processes. Our recent work has focused on the role of a particular metalloproteinase, collagenase, and the role of an endogenous inhibitor of this enzyme in the control of neovascularization. The proteolytic degradation of extracellular matrix components by capillary endothelial cells (EC) has been shown to be one of the key prerequisites of the angiogenic process. As part of a study of the effect(s) of the inhibition of collagenase on neovascularization, we have recently reported the purification, characterization and partial NH2 terminal sequence of a cartilage-derived inhibitor (CDI) of angiogenesis in vivo and in vitro. Evidence is presented which suggests that one means of controlling deregulated vascular growth characteristic of a number of "angiogenic diseases" may be at the level of the control of metalloproteinase activity. PMID- 1724246 TI - Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis. AB - Capillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future. PMID- 1724247 TI - Detection of cytokine-induced protein gamma-immune protein-10 (gamma-IP10) in atypical melanocytic proliferations. AB - gamma-Immune protein-10 (gamma-IP10) is a cytokine whose expression has been shown to be induced by interferon-gamma. It is a member of a group of closely related cytokines (e.g., interleukin 8 and platelet factor 4) with chemotactic properties. gamma-IP10 has been detected in keratinocytes, lymphocytes, monocytes, and endothelial cells in immunologically mediated processes, such as positive tuberculin skin tests, and in growth-activated keratinocytes, such as in psoriasis. Keratinocytes in normal epidermis do not produce gamma-IP10. We tested the hypothesis that keratinocytes adjacent to dysplastic nevi and melanomas would produce gamma-IP10, perhaps as part of an immune response to a tumor, and that this response would not be seen in ordinary melanocytic nevi. We used an affinity purified, polyclonal rabbit anti-gamma-IP10 antibody to examine 10 nevi with moderate to severe histologic dysplasia, one superficial spreading melanoma, and 10 compound melanocytic nevi with no features of dysplasia. As predicted, keratinocytes surrounding all of the cytologically atypical melanocytic lesions displayed strong staining with gamma-IP10. There was no staining of keratinocytes adjacent to ordinary melanocytic nevi. The observed keratinocyte staining with gamma-IP10 may be related to a host immune response to antigenically abnormal cells. PMID- 1724248 TI - Substance P immunoreactivity in rosacea. PMID- 1724249 TI - The pathogenesis of Sweet's syndrome. PMID- 1724250 TI - Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope. AB - The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens. PMID- 1724251 TI - Histochemical detection of burn-induced lipid peroxidation in sebaceous glands of rat skin. AB - Increased lipid peroxide levels both in skin and serum have been reported after cutaneous thermal injury. However, it still remains unclear where lipid peroxides are produced at the site of burned skin. In the present study, a histochemical method using cold Schiff's reagent was applied in order to detect the localization of lipid peroxide. Schiff positivity was detected in sebaceous glands, and the extent of positivity seemed to correlate with the serum lipid peroxide levels. These results may suggest that lipid peroxides produced in sebaceous glands after thermal injury enter the blood stream and are partially responsible for the elevated serum lipid peroxide levels. PMID- 1724252 TI - An electron microscopic study of experimentally-induced comedo and effects of vitamin A acid on comedo formation. AB - The mechanisms of comedogenesis and comedolysis were investigated at the ultrastructural level after applying oleic acid (OA) only, or oleic acid together with vitamin A acid (VAA), to rabbit ears daily for two weeks. 1) In the follicular epithelium of the experimentally-induced comedo (EIC) by OA, several ultrastructural changes similar to those in human comedo were observed. EIC in rabbit ears appeared to be induced both by the hyperkeratinization of the follicular epithelium and by the delayed desquamation of horny cells due to the persistence of intercellular binding apparatus. 2) VAA strongly inhibited the formation of EIC. In the follicular epithelium, two different types of changes, non-cohesive hyperkeratinization and inhibition of keratinization, were observed. These represent the cell injury and recovery stages, respectively. VAA induced the disturbance of follicular epithelial keratinization and reduction of intercellular bindings between horny cells. These effects might prevent the cohesion and accumulation of horny cells and inhibit EIC formation. 3) The number of Odland bodies (Ods) showed an inverse correlation with the cohesion of horny layer. These findings support the theory that Ods have a desquamating function as extracellular lysosomes. The change in Ods would also contribute to both EIC formation by OA and the inhibition by VAA. In addition, VAA caused a characteristic increase in Ods with lamellar structures. It is suggested that Ods with lamellar structures have a desquamating function. PMID- 1724253 TI - An immunohistochemical study of mixed tumor of the skin. AB - An asymptomatic tumor developed on the upper lip of a 63-year-old man. Histologically, the tumor contained glandular and cystic structures forming many branching lumina, and many scattered single cells in an abundant mucoid to chondroid stroma. The tumor was diagnosed as mixed tumor of the skin. Histochemically, the cells composing the tubular structures contained neutral mucopolysaccharides and the stroma, acid mucopolysaccharides. Immunohistochemically, the cells of the glandular and cystic structures showed epithelial and sweat gland differentiation (EMA-, CEA-, BRST-1- and BRST-2 positive), while the cells scattered in the stroma showed a tendency toward myoepithelial differentiation (S-100 protein- and vimentin-positive). PMID- 1724254 TI - The ATPase-staining of Langerhans cells in previously stored skin tissues. AB - The ATPase-staining of Langerhans cells is usually done on fresh, unfixed biopsy specimens. In the present study, we attempted to stain these cells in previously stored tissues of guinea pig skin. The ATPase activity disappeared after freezing or preservation in formalin or ethanol. In contrast, tissue specimens which had been stored in physiological saline at 4 degrees C stained as well as fresh cells even after 14 days. PMID- 1724255 TI - Mind the theory/practice gap in nursing. AB - The theory/practice gap is a recurrent theme in the nursing literature. Numerous suggestions have been made about how this gap may be narrowed or even closed. However, there appears to have been little attempt to date to examine the ways in which research conducted into the hidden curriculum, and philosophical analysis of the concepts involved, might inform our understanding of the reasons for the existence of the theory/practice gap. This paper attempts to utilize some of the contributions from these areas of research, and as a result will argue that, while narrowing of the theory/practice gap may be a realistic goal, any attempt to close it completely will be doomed to failure. PMID- 1724256 TI - [The structure of the superior colliculus in the microphthalmic mouse strain 944]. AB - The structure of the superior Colliculus (CS) in congenitally blind mice was compared with that in healthy control mice. Thus Nissl staining and degeneration experiments were done resulting in differences in the Stratum opticum (SO) of the CS between microphthalmic and control mice. In microphthalmic mice the volume of SO decreases in 14% and packing density of neurons increases in 20% in comparison with controls. Terminal degeneration after lesion of the ipsilateral visual cortex is seen in the surface laminae only. This connection seems independent of visual signals. PMID- 1724257 TI - Divergence and collateral axon branching in subsystems of visual cortical projections from the cat lateral posterior nucleus. AB - The projections from the lateral (LPl), intermediate (LPi) and medial (LPm) subdivisions of the cat lateral posterior nucleus (n. LP) to visual areas 17, the posteomedial (PMLS) and posterolateral (PLLS) lateral suprasylvian and anterior ectosylvian (AEV) were studied using the retrograde labeling technique following concomitant injections of fluorescent dyes (Fast blue, Nuclear yellow, Evans blue and Rhodamine beta-isothiocyanate) into the different cortical loci. The results showed a medial-lateral topographical reversal of the visual n. LP-cortical connections: The ventral portion of LPl projects to area 17 whereas more dorsolateral regions of LPl and lateral LPi provide input to PMLS. Cells in medial LPi project mainly to the PLLS cortex and AEV receives afferents from the LPm. Areas of overlap were identified within the ventral LPl which projects to both area 17 and PMLS and within the LPi/LPm border region at the origin of connections to both PLLS and AEV. Furthermore, some single neurons within the areas of overlap were found to be double-labeled indicating divergent projections to their respective cortical targets via collateral axon branching. The data show that divergence and axonal branching are common features of the different n.LP visual cortical subsystems and support the notion of the existence of families of thalamo-cortical systems which are distinct in their connection patterns and underlying functional properties. PMID- 1724258 TI - A comparative study of the transganglionic transport of cholera toxin-horseradish peroxidase (CT-HRP) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the trigeminal system of the guinea pig. AB - A comparative study of cholera toxin (CT) and wheat germ agglutinin (WGA) conjugated with horseradish peroxidase (HRP) was made on trigeminal central projections of the lower incisor gingiva afferent neurons in the guinea pig. Considerably more CT-HRP-labeled endings were observed in the trigeminal sensory nuclear complex (TSNC) and in the cervical spinal cord (C1-C8). The substantia gelatinosa (lamina II) of both the caudal nucleus of the TSNC and C1-C2 was the only area where WGA-HRP labeled more terminals. CT-HRP-labeled fibers and endings were traced up to C7-C8, whereas with WGA-HRP were rare caudal to C5. A comparison of the two methods currently in use, i.e. the 2-step glutaraldehyde and sodium periodate, showed that the latter yields conjugates which are more sensitive as neuroanatomical tracers. PMID- 1724259 TI - Variations in time of serum pancreatic enzyme levels in chronic pancreatitis and clinical course of the disease. AB - Thirty-four patients with chronic calcified pancreatitis were evaluated clinically and biochemically (at a time when painful relapses were not present) every 9 mo for 3 yr; 25 of them were also studied at 4 and 9 yr. Serum elastase 1, trypsin, lipase, and amylase in the same sera were measured at each visit; levels on entry and variations during the study were compared with the clinical and functional data of the patients. On entry, low levels of elastase-1 were found in 11.7% of the patients, high levels in 41.1%; in contrast, high levels of trypsin and lipase were found in only a small number of patients (5.8 and 11.7%, respectively), whereas low levels were present in a substantial number (47.8 and 32.3% for trypsin and lipase, respectively). Over time, we found a significant (p = 0.000002) reduction in elastase-1 levels. Such reduction was not found for trypsin, lipase, or amylase. The reduction of serum elastase-1 was significantly (p less than 0.003) more frequent in patients presenting a reduction in painful relapses than in patients with a stable or increased attach rate; this association was weaker (p less than 0.05) for lipase and trypsin, and absent for amylase. No correlation was found between circulating enzymes and either alcohol consumption or age of patients. In patients with severe exocrine impairment, low levels of elastase were found in only 20% of the cases, whereas trypsin and lipase were reduced in 73.3 and 53.3% of the cases, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724260 TI - The patterns of beta-cell regeneration in untreated diabetic and insulin-treated diabetic Syrian hamsters after streptozotocin treatment. AB - Our previous study showed that Syrian hamsters recover from streptozotocin (SZ) induced diabetes spontaneously and that insulin therapy adversely affects diabetes. The recovery and lack of recovery was associated with beta-cell regeneration and lack of regeneration. In this study, we examined the patterns of beta-cell regeneration in hamsters by using an immunohistochemical autoradiographical method. We used tritiated thymidine to examine the DNA synthesis in islet cells and evaluated the number of labeled cells that were or were not immunoreactive with antiinsulin, antiglucagon, and anatisomatostatin. The results showed that, in untreated control hamsters, beta-cell regeneration took place both from preexisting beta cells and from undifferentiated cells (labeled cells unstained with any of the three antibodies) in equal proportions. In the SZ-treated hamsters, however, most regenerating cells seemed to derive from undifferentiated cells within the islets. Insulin therapy inhibited beta cell regeneration from both the preexisting cells, but more so from the undifferentiated cells. The number of extrainsular islet cell foci was the same in all treatment groups and paralleled the number of islets in each animal during the entire experiment. We concluded the following about diabetic hamsters: 1. beta-Cell regeneration occurs primarily from undifferentiated cells within the islet; 2. Exogenous insulin inhibits beta-cell differentiation from the precursor cells; and 3. The response of intrainsular and extrainsular islet cells to SZ and insulin therapy is similar and the number of extrainsular islet cell foci per pancreas parallel the changes in the number of islets. PMID- 1724261 TI - [Experimental studies on morphological changes of microvascular architecture following the free gingival autograft on denuded alveolar bone]. AB - The purpose of the present studies was to examine the healing process following the free gingival autograft placed on the recipient bed either with or without periosteum in 54 adult mongrel dogs with healthy periodontium. A recipient bed was prepared on denuded alveolar bone in a definite portion of the attached gingiva of the right maxillary canine tooth, and the graft was taken from the attached gingiva of the left maxillary canine and transplanted in the recipient bed. Morphological changes were observed by means of vascular corrosion casts on the postoperatively 3rd, 5th, 14th, 21st, 28th, 42nd, 56th and 84th day. The healing process following the free gingival autograft on the denuded alveolar bone showed that this graft survived in its margin by recirculation from the cut margin of the recipient bed, and in its center the necrotic tissue changed to granulation tissue, which gradually cicatrized. This was different from the healing process following the free gingival autograft on periosteum, in which the graft was survived entirely by recirculation from the vascular plexus of the periosteum on the recipient bed. This may help to restore the function proper. PMID- 1724262 TI - Advanced nasopharyngeal carcinoma: radiation treatment and chemotherapy approaches, and life table survival results. AB - Induction combination chemotherapy and radiotherapy was tried in 50 patients with T3-T4 nasopharyngeal carcinoma. The results were compared to another matching group of 50 patients given radical radiotherapy. The study showed preliminary improvement of local control rates of the tumour in patients treated by combination chemotherapy with radiation. The actuarial method of survival computation was applied, also the variance and standard error of the four years survival rates for both treatment groups was computed. Comparison of the survival rates showed no significant difference between group I (received combined chemo and radiotherapy) and group II (Radiation therapy) throughout the observation period. PMID- 1724263 TI - Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6 dideoxyhexose abequose. AB - The rfb region of Yersinia pseudotuberculosis serogroup IIA has been cloned and expression of O antigen in Escherichia coli K12 was demonstrated. Transposon mutagenesis analysis confined the DNA region required for O antigen expression to a 19.3 kb fragment, and the O antigen expressed was visualized by SDS-PAGE and silver staining. Southern hybridization analysis demonstrated significant levels of similarity between the Yersinia rfb region and the 3,6-dideoxyhexose pathway genes rfbF and rfbG, previously isolated from Salmonella enterica LT2, but no similarity to the abequose synthase gene rfbJ of the same strain or the paratose synthase gene rfbS isolated from S. enterica Ty2. The evolutionary relationship between the abequose biosynthetic genes of the two species of Salmonella and Yersinia is discussed. PMID- 1724264 TI - Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions. AB - Monoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs. PMID- 1724265 TI - Identification of single bacterial cells using digoxigenin-labelled, rRNA targeted oligonucleotides. AB - Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5' end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gram-negative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence. PMID- 1724266 TI - Insulin-like growth factors I and II and their binding proteins in human milk: effect of heat treatment on IGF and IGF binding protein stability. AB - Insulin-like growth factors (IGF-I and -II) are peptide growth factors that contribute to the growth-promoting properties of human milk. IGFs in extra cellular fluids are associated with high-affinity binding proteins (IGFBPs). In this study, IGF-I and -II, and IGFBPs in human milk were characterized, and the stability of the IGFs and IGFBPs to heat treatment was investigated. IGF-I and II were quantified by radioimmunoassays of acid-chromatographed samples, and IGFBPs were characterized using Western ligand blotting. The concentration (mean +/- SD) of IGF-I in human milk was 1.5 +/- 0.5 micrograms/L, compared to 2.7 +/- 0.7 micrograms/L for IGF-II. Heat treatment did not significantly affect either IGF-I or -II content. Human milk contains a single, nonglycosylated, IGFBP, with an apparent Mr of 31 k, which was immunoprecititable by an antibody to IGFBP-2. Stability of the IGFBP to heat treatment was assessed and was not significantly affected by heat treatment. Therefore, both IGF-I and -II, and the IGFBP in human milk appear to be stable under heat treatment conditions routinely used for processing banked human milk. PMID- 1724267 TI - Cell adhesion properties of myelin-associated glycoprotein in L cell fibroblasts. AB - Myelin-associated glycoprotein (MAG) is a cell surface molecule expressed by oligodendrocytes and Schwann cells. In order to determine whether MAG expression can confer adhesive properties to cells which normally do not aggregate in suspension, the cDNA encoding the long form of MAG (L-MAG) was introduced into L cell fibroblasts by retroviral infection. Clonal L cell lines expressing MAG were then subjected to a cell aggregation assay. Our results indicate that L-MAG can function as an intercellular adhesion molecule in a heterologous cell system. A critical threshold value of L-MAG expression was required for cell aggregation to occur. The adhesive properties of these cells were specific to MAG, since monoclonal antibodies directed against its extracellular domain inhibited aggregation. Furthermore, the adhesion was found to be calcium- and temperature independent. Cell sorting experiments demonstrated that L-MAG-expressing cells bind in a heterotypic fashion to parental L cell fibroblasts. These results suggest that L-MAG can function as a heterotypic cell adhesion molecule recognizing a cell surface molecule(s) expressed by L cells. PMID- 1724268 TI - Identification of transcriptionally regulated genes after sciatic nerve injury. AB - Mammalian peripheral nerve fibres can regenerate after injury. In an attempt toward a better understanding of the underlying molecular events, we have isolated novel and known rat cDNA sequences, the expression of which are regulated during sciatic nerve regeneration. For this purpose, cDNA libraries were constructed from either the nerve segment distal to the crush site or the corresponding contralateral uninjured nerve of the same animals. These libraries were screened by differential hybridization and several transcriptionally repressed and induced sequences were isolated. Out of 2,000 cDNA clones screened from the distal library, 11 sequences were found to be induced in the distal nerve segment. This set of induced cDNAs included the rat homolog of vimentin, 28 S and 18 S ribosomal RNA species, and two novel sequences. Of 5,000 screened colonies of the contralateral library, 30 colonies contained sequences that were repressed in the distal segment after nerve crush. They were identified as myelin basic protein, myelin P0, alpha-globin, cytochrome oxidase subunit 1, creatine kinase (muscle type, M) and collagen type I. In addition, five novel sequences were found that were dramatically repressed after sciatic nerve crush. Representative clones were tested by northern blot analysis to study their time course of transcriptional regulation during nerve regeneration. The observed patterns suggest that the regeneration phenomenon shows complex gene regulation in which the nonneuronal cells of the distal segment play an important role. Further characterization of the isolated regulated known and unknown sequences will increase our understanding of the molecular events associated with neuronal regeneration. PMID- 1724269 TI - Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor. AB - A primary culture system of nearly pure neuronal cells from 14-day-old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum-free medium, and borated polylysine-coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron-specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth-associated protein GAP-43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bromodeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron-specific enolase. Recombinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-beta 1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE-positive neurons, probably belonging to the non-motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE-positive. PMID- 1724270 TI - Acidic fibroblast growth factor (aFGF) is expressed in the neuronal and glial spinal cord cells of adult mice. AB - Fibroblast growth factors (FGFs) are known to be synthesized in the central nervous system (CNS) and to act on CNS cells in vitro, but less is known about their synthesis, expression, and role in vivo. In this work, using specific anti acidic fibroblast growth factor (aFGF) antibodies, we have shown for the first time, by immunohistochemistry, that aFGF is expressed in spinal cord cells of young adult normal mice. This expression is predominant in the cell nucleus. Using immunohistochemical double staining procedures, we identified the cell type expressing aFGF as neurons, astrocytes, and oligodendrocytes, but for each type, cells were not all positively immunostained. PMID- 1724271 TI - [An experimental study on healing process of replanted mature permanent teeth. Changes of periodontal vascular network]. AB - This study was conducted to clarify both changes of periodontal vascular architecture and concomitant remodelling phenomena associated with hard tissues in the healing process of replanted teeth of first premolar. Utilizing both vascular corrosive resin casts method, scanning electron microscopy, and histological examinations, 45 matured mongrel dogs were used for this study. The results were as follows: 1. 4 days after operation: Newly formed vascular networks with a exceedingly irregular course were observed on lower two thirds of the alveolar wall. They derived from comparatively less damaged periodontal vascular components. No vascular networks were observed surrounding in the crevicular area of the replanted tooth where the periodontal membrane tissue was thoroughly damaged when tooth was extracted. 2. 1 week after operation: Newly formed periodontal vascular networks with a slightly irregular course were observed over the entire alveolar bone surface. 3. 2 weeks after operation: Formation of Sharpey's fibers occurred. The surrounding alveolar bone was remodelled and rearrangement of periodontal vascular architecture was observed. Also, several Howship's lacunae were observed on the root surface where characteristic capillary loops with glomerular-like appearance penetrated into these lacunae. 4. 3 weeks after operation: Root resorption was advanced and capillary loops with glomerular-like appearance were extensively distributed in association with each lacunae. On the other hand, the less space where periodontal membrane vasculature occupied, the more space was occupied by osteoid tissue. 5. 4 weeks after operation: Blood vessels within the periodontal space were reduced in number and the osteoid tissue showed bony fusion adjacent to the extensively resorbing surface of dentin. 6. 12 weeks after operation: Functional arrangement of Sharpey's fibers was completed. Restoration of Howship's lacunae on the root surface and two layered arrangements of vascular network within the periodontal space were observed. Newly formed periodontal vascular architecture showed a fine meshwork pattern, which was somewhat different from that of the control (noreplantation) group. 7. 20 weeks after operation: Increased number of capillary loops was observed with leakage of methacrylate resin through the weakened endothelial linings of capillaries in one case. It is supposed that this leakage through capillaries is correlated with the inflammatory root resorption that occurs clinically in marginal periodontitis. Also in some cases, periodontal capillary network showed secondary occlusal traumatic changes. Above results indicated that periodontal vascular architecture varied depending upon the reactions of periodontium following tooth replantation and the prognosis of replanted tooth was deeply associated with repair of periodontal vascular network. PMID- 1724272 TI - Neonatal seizures and a 2-year neurological outcome. AB - A study of 57 infants with neonatal seizures admitted to the Special Care Baby Unit of the Jos University Teaching Hospital over a 3-year period showed perinatal asphyxia and hypoglycaemia as the principal aetiologic factors in 47 and 19 per cent of the cases, respectively. Seizures were commoner in preterm infants, and among them outcome was also poorer. As regards aetiological factors, outcome was poorest with perinatal asphyxia; with a mean (SD) mental age of 72.5 (9.1) weeks at a chronological age of 24 months. Outcome in infants with seizures and coma was most favourably predicted by the absence of abnormal neurological signs, and the way the infant was feeding at 7-10 days. All infants who were clinically and neurologically normal and taking more than half their estimated requirements by mouth at 7 days were not handicapped. The overall incidence was 7.5/1000 live births. The mortality (19.3 per cent) was closely related to the aetiology. In view of the fact that the associated adverse perinatal events are largely preventable, improved prenatal and perinatal health care delivery should lead to a decline in the frequency of neonatal seizures. PMID- 1724273 TI - [Comparative study of the effectiveness of Bonnecor and ethacizine in various disorders of cardiac rhythm]. AB - The data obtained on 68 patients suffering from cardiac arrhythmia treated with bonnecor or ethacizine demonstrate that bonnecor corrects ventricular extrasystole including high-grade arrhythmia in 80.4% of the patients while ethacizine in 84.6%. Acute tests with the drugs can effectively prognosticate the outcomes of bonnecor or ethacizine long-term treatment. The drugs eliminate delta wave and stimulation-induced arrhythmias in 90% and 44-56% of patients with WPW syndrome, respectively. Bonnecor is less effective in nodal tachycardia. Both the drugs noticeably suppress conductivity. PMID- 1724274 TI - [Association of I and II class HLA antigens with Cushing's syndrome]. AB - Incidence of class I and II HLA antigens was studied in 69 patients suffering from Icenko--Cushing syndrome. Positive association was reported for antigens DRw53, DQw3, DR4, DR5, negative for antigen DR2. Antigen combinations DR5, DR7 and DR4, DR5 proved more prevalent. The risk to develop the disease rose 2-3-fold in case of HLA-DR5 phenotype occurrence with antigen DR4 or DR7. Association with DRw53 and DQw3 antigens showing wide specificity seemed most significant. PMID- 1724275 TI - Peptidergic innervation of mesenteric lymphatics in guinea pigs: an immunocytochemical and pharmacological study. AB - By immunocytochemistry, substance P immunoreactive (SP-IR) and vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibers were examined in guinea pig mesenteric lymph collectors. The immunoreactive nerve fibers, located in the adventitia of lymphatics, were few and were irregularly distributed along the vessel wall. These fibers appeared to be more numerous and more evenly distributed along the corresponding artery and vein walls within the same area. SP immunoreactivity in the vascular nerves was depleted in guinea pigs injected with capsaicin but was unaffected by the injection of 6-hydroxydopamine. By contrast, VIP-IR nerve fibers were unaffected by both treatments. It is concluded that SP-IR nerve fibers in the lymphatics are likely to be of sensory origin and that VIP containing nerves in the lymph collectors are distinct from SP containing and noradrenergic nerves. It is also suggested that lymph collectors possess a complex although limited innervation pattern not only of autonomic nerve fibers containing classic neurotransmitters but also of peptidergic nerve fibers of a different origin with a vasomotor and/or sensory action. PMID- 1724276 TI - [Surgical therapy of proximal bile duct cancer]. AB - During the past 13 years a total of 60 patients (33 male, 27 female, median age 64.8 years) were operated upon and 21 of these patients underwent resection with a resectability rate of 35%. The remaining 39 patients had a palliative procedure. In 7 patients some form of bypass procedure was performed. 25 patients underwent some form of drainage procedure and in 7 patients only an explorative laparotomy was undertaken. Patients having resection surgery had a postoperative complication rate of 29% and there were 2 postoperative deaths (9.5%). The complication rate in the palliation group was 38%. The mean survival time in patients operated on with surgical resection was 34.1 months, palliative procedures 4.8 months and in patients with nonresectable tumors 3.6 months. In the resection group (n = 21) curative resection (= R0-resection) was performed in 14 patients, whereas in 7 patients there was a histologically invasion of the bile duct (= R1-resection). The mean survival time in the R0-group was 45.7 months and 11.8 months in the R1-group (Breslow p less than 0.0098, Mantel-Cox p less than 0.0070). We conclude that radical surgical resection offers the best possibility of prolonged survival with a good quality of life in patients with hilar cancer. PMID- 1724277 TI - K1 killer toxin, a pore-forming protein from yeast. AB - K1 killer toxin is a secreted, pore-forming protein that kills sensitive yeast cells. The heterodimeric toxin is processed from a precursor in the Golgi, and has allowed identification of the KEX2- and KEX1-encoded proteases. The toxin binds to a glucan receptor on the cell wall of target yeast, and mutational analysis implicates both the alpha- and beta-toxin subunits in receptor binding. Toxin-resistant mutants with altered cell-wall glucans have helped to outline a pathway of assembly of these polysaccharides. Patch-clamp technology has demonstrated the nature of the lethal channel in toxin-treated plasma membranes. The hydrophobic alpha-subunit-encoding region is the site of all mutations affecting channel formation. Immunity to the toxin is conferred by the toxin precursor, and immunity mutations map to the region encoding the alpha subunit. The precursor probably competes with the toxin to prevent channel formation in toxin-producing cells, but the basis of this remains unknown. This toxin/immunity system has a domain structure that differs from that of other characterized toxins and has no known homologues. PMID- 1724278 TI - The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor. AB - A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule. PMID- 1724279 TI - Analysis of Haemophilus influenzae type b lipooligosaccharide-synthesis genes that assemble or expose a 2-keto-3-deoxyoctulosonic acid epitope. AB - We recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2-keto-deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb Pstl-BamHl fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5 K, 5.1 K and 5.5 K. Only the 5.5 K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl-BamHl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis. PMID- 1724280 TI - Analysis of the topology of the cytochrome d terminal oxidase complex of Escherichia coli by alkaline phosphatase fusions. AB - The cytochrome d complex of Escherichia coli is a heterodimer located in the bacterial cytoplasmic membrane, where it functions as a terminal oxidase of the aerobic respiratory chain. The topology of each of the two subunits of the cytochrome d complex was analysed by the genetic method involving alkaline phosphatase gene fusions. These fusions were generated by both an in vivo method using the transposon TnphoA and an in vitro method of construction. A total of 48 unique fusions were isolated and the whole-cell alkaline phosphatase-specific activities were determined. Data from these fusions, in combination with information from other studies, provide the basis for two-dimensional models for each of the two subunits, defining the way in which the subunits fold in the inner membrane of E. coli. PMID- 1724281 TI - [Antibiotics of an aromatic nature from Pseudomonas cepacia]. AB - A raw antibiotic has been isolated from culture fluid of strain Pseudomonas cepacia 5798. It was methylated and separated into individual components using the column and thin-layer chromatography. The isolated substances possessed antimicrobial activity; two of them were studied more in detail by the UV-, IR- and PMR-spectroscopy methods. Results of the study of physicochemical properties of methyl derivatives of antibiotics from P. cepacia permit supposing that the latter are aromatic substances with nitrogen atoms in the side chain. PMID- 1724282 TI - [The N-terminal peptide of the hexon of human adenovirus type 2: its chemical synthesis and antigenic properties]. AB - Peptides corresponding to the N-terminal section of protein of a hexone of the 2nd type human adenovirus have been synthesized. Being conjugated with a carrier of BSA they induced formation of antibodies which reacted both with peptides and with viral proteins. Sera to native proteins were also bound to synthetic peptides. Immunogenicity strengthening in peptides conjugated with synthetic polyelectrolytes is shown. The N-terminal section of hexon is supposed to participate in formation of an antigenic determinant possessing group specificity. PMID- 1724283 TI - Emerging themes in the history of medicine. AB - The critical themes that have emerged in medical history over the last 25 years reflect the development of a "new social history of medicine." The earlier focus on the evolution of medical knowledge has been replaced by work that emphasizes the nature of the social responses to disease, their sociocultural meanings and historical epidemiology. Because medical historiography reflects debates and questions in contemporary medical culture, the AIDS epidemic is likely to have an important impact on the study of medical history in the immediate future. PMID- 1724284 TI - New AIDS drugs. PMID- 1724285 TI - Antigenic structure of histone H1(0). AB - In order to study the antigenic structure of histone H1(0) the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti H1(0) antiserum. The C-terminal fragments 99-193 (obtained following acetic acid hydrolysis) and 107-193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1-30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1-22 and 1-28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1(0) are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose. PMID- 1724286 TI - Insulin-like growth factor receptor gene expression in the rat ovary: divergent regulation of distinct receptor species. AB - The intraovarian insulin-like growth factor (IGF) system constitutes a triad composed of ligands, receptors, and binding proteins. Although conventional radioligand receptor assays have documented the presence of specific receptors for insulin and insulin-like peptides in some rat somatic ovarian cell types, the exact cellular localization and hormonal regulation of the receptors in question remain matters of inquiry. To reevaluate the very presence, cellular localization, and hormonal regulation of the IGF receptor gene family in the rat ovary, solution hybridization/RNase protection assays were used wherein ovarian total RNA (20 micrograms) from immature (21-23 days old) rats was hybridized with 32P-labeled type I IGF receptor, type II IGF/mannose-6-phosphate receptor, and insulin receptor riboprobes. Single protected fragments 261 (type I IGF receptor), 500 (type II IGF/mannose-6-phosphate receptor), and 478 (insulin receptor) bases long were evident in whole ovary, granulosa, and theca interstitial cells. Hypophysectomy of immature rats led to significant (P less than 0.05) albeit variable decrements in the relative (densitometrically quantified) ovarian abundance of transcripts corresponding to the type I IGF (but not insulin or type II IGF/mannose-6-phosphate) receptor. Treatment of immature hypophysectomized rats with FSH (10 micrograms/rat.day x 2.5 days) resulted in a significant (P less than 0.05) increase (4-fold) in transcripts corresponding to the type I IGF receptor in both whole ovarian material and freshly isolated granulosa cells. Similar (3.7-fold) increments (P less than 0.05) were noted after treatment with a diethylstilbestrol-containing sc silastic implant applied for a total of 5 days.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724287 TI - The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. AB - Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter. PMID- 1724288 TI - Inhibitors of phosphodiesterase III block stimulation of Xenopus laevis oocyte ribosomal S6 kinase activity by insulin-like growth factor-I. AB - Insulin-like growth factor-I (IGF-I) stimulated Xenopus laevis oocyte ribosomal S6 kinase activity 5- to 10-fold, with an apparent EC50 of 0.8 +/- 0.1 nM after 90 min of hormone treatment. IGF-I-stimulated enzyme activity was inhibited by treatment of oocytes with nonselective phosphodiesterase (PDE) inhibitors, with apparent IC50 values of 2 +/- 1 microM papaverine, 20 +/- 2 microM isobutylmethylxanthine, and 128 +/- 16 microM theophylline. Type III PDE inhibitors also inhibited IGF-I-stimulated S6 kinase activity with IC50 values of 9.7 +/- 0.3 microM Cl-930 and 84 +/- 23 microM imazodan (Cl-914). These drugs apparently affected an intracellular molecular event leading to activation of S6 kinase, since Cl-930 prevented IGF-I-stimulation of S6 kinase, but had no direct inhibitory effect when added to the S6 kinase enzyme assay mixture. While hormone stimulated S6 kinase activity was inhibited by isobutylmethylxanthine (nonselective PDE inhibitor) and Cl-930 (PDE III inhibitor), Ro 20, 1724 and rolipram (PDE IV inhibitors) and dipyridamole (PDE V inhibitor) had no significant effect on activated enzyme levels. The time course for IGF-I stimulation of oocyte S6 kinase displayed a small early peak of activity approximately 0.15-0.4 time required for 50% of cell population to display white spots (GVBD50) and a second major increase in activity at 0.6-0.7 GVBD50 that was sustained until meiotic maturation was complete. The second wave of enzyme activation was inhibited by Cl-930, but the early increase was not.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724289 TI - Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. AB - The formation of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, aromatase cytochrome P450 (P450AROM; the product of the CYP19 gene). Previous studies have demonstrated that aromatase activity in human adipose and ovarian granulosa cells is subject to complex multifactorial regulation and that changes in activity are correlated with changes in the levels of mRNA encoding P450AROM. We have previously isolated the human CYP19 gene. Two unique untranslated first exons (exons I.1 and I.2) have been identified in mRNA specific for P450AROM in human placenta. Although the proportion of transcripts encoding exon I.2 is very small, genomic clones encoding the sequences of both exons I.1 and I.2 have recently been isolated. The corpus luteum of human ovary differs in that promoters I.1 and I.2 are completely inactive. Sequence analysis of the DNA immediately 5' of exon II (which contains the start site of translation) demonstrates the presence of a TATAA sequence beginning 149 basepairs 5' of the ATG initiation codon identified in placental exon II. Using a combination of primer extension and S1 nuclease protection analysis, it appears that the initiation site of ovarian P450AROM transcripts aligns 26 basepairs down stream of the sequence TATAA. It appears, therefore, that the expression of P450AROM-specific mRNA in corpus luteum is regulated by an additional promoter (promoter II), which is located just 5' of exon II. Consistent with these observations, Northern analysis of poly(A)+ RNA isolated from placenta and corpus luteum demonstrates that the major promoter of placental P450AROM is promoter I.1, while the major promoter in the corpus luteum is promoter II.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724290 TI - Direct measurement of calpastatin subtypes by sandwich enzyme immunoassay using monoclonal antibodies. AB - Six stable hybridoma cell lines secreting monoclonal antibodies to human calpastatin were established. All monoclonal antibodies belong to the IgG1 subclass and recognized different epitopes on calpastatin. At least two groups were distinguished; the first group was specific for muscle-type (M-) calpastatin and the second group recognized not only M-calpastatin but also erythrocyte-type (E-) calpastatin. The inhibitory effect of all monoclonal antibodies on calpastatin activity was relatively low even at high concentrations of antibodies. Enzyme immunoassay systems were developed for direct determination of calpastatin subtypes in human cells requiring no other sample treatment than the disruption of the cells. The assay methods were, in principle, based on the sandwich enzyme immunoassay using epitope-specific monoclonal antibodies. The enzyme immunoassay system for M-calpastatin was specific for M-calpastatin and could not detect E-calpastatin. The enzyme immunoassay system for total calpastatin detected not only M-calpastatin but also E-calpastatin. The sensitivity of these assay systems was 10 pmol l-1 of calpastatins. Antigenicity of calpastatins was found to be unchanged in the presence of EDTA and haemoglobin. Good reproducibilities of within-and between-assay series and excellent recovery of exogenous calpastatins from cell lysates were observed. From these results, it seems that our newly developed subtype-specific enzyme immunoassay systems for calpastatins are useful in biochemical studies and clinical testing for determination of calpastatin subtypes. PMID- 1724291 TI - Experience with serotyping rotavirus strains by reverse transcription and two step polymerase chain reaction with generic and type-specific primers. AB - Six VP7 serotypes or G types (G1-G4, G8 and G9) occur in group A human rotaviruses. Gouvea et al. recently reported a novel G-typing method based on reverse transcription (RT)-polymerase chain reaction (PCR) amplification of the VP7 gene with type-specific primers [Gouvea, V. et al. (1990). Journal of Clinical Microbiology 28, 276-82]. When we followed their protocol, 40 (89%) of 45 faecal rotavirus specimens were typed into G1-G4 and G9. The five specimens that were untypeable by the RT-PCR method contained three G2 and two G4 rotavirus specimens which were identified by an ELISA using G type-specific monoclonal antibodies. On the other hand, the RT-PCR typing assay was able to determine the G type of the seven isolates that were untypeable by the ELISA. Of 33 faecal rotaviruses that were typed by both assays, 100% agreement of the result was observed. In addition, when applied to some animal rotaviruses, the RT-PCR method identified G3 feline and canine rotavirus strains. We conclude that further refinement of the RT-PCR assay is desirable in order to more readily type G2 and G4 strains, although this assay displayed an applicability to epidemiologic studies comparable with ELISAs. PMID- 1724292 TI - Transport complexes associated with slow axonal flow. AB - Cytoskeletal proteins--neurofilament polypeptides, tubulin and actin--are transported along axons by slow transport. How or in what form they are transported is not known. One hypothesis is that they are assembled into the cytoskeleton at the cell body and transported as intact polymers down the axon. However, recent radiolabeling and photobleaching studies have shown that tubulin and actin exist in both a mobile phase and a stationary phase in the axon. Consequently, it is more likely that cytoskeletal proteins move along the axon in some form of transport complex and are assembled into a cytoskeleton which is stationary. In this overview we discuss these topics and consider the evidence for the existence of transport complexes associated with slow axonal flow. Such evidence includes the slow transport of particulate complexes containing tubulin and neurofilament polypeptides along reconstituted microtubules in vitro, and the coordinate slow transport of actin with actin-binding proteins in vivo. PMID- 1724293 TI - Ionic transporting systems of skeletal muscle in relation with innervation and their involvement in myotonic diseases. AB - Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na+, Ca2+, and K+ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na+, K+) ATPase in myotonic disorders. PMID- 1724295 TI - Intraoperative monitoring of the somatosensory evoked potentials and cerebral blood flow during aneurysm surgery--safety evaluation for temporary vascular occlusion. AB - Somatosensory evoked potentials (SEPs) and cerebral blood flow (CBF) were monitored during temporary vascular occlusion in 67 aneurysm operations to evaluate the usefulness of SEP as an indicator for cerebral ischemia. The SEP N20 component completely disappeared during temporary vascular occlusion in 24 cases, 23 of which demonstrated complete recovery following recirculation and had no postoperative neurological sequelae. The only one with postoperative sequelae demonstrated rapid, within 1 minute, loss of N20 followed by no recovery. Another eight cases showed prolongation of central conduction time during vascular occlusion and had no postoperative sequelae. The SEP N20 attenuation reflected the CBF reduction in the middle cerebral artery (MCA) territory during MCA occlusion. However, CBF changes in the internal carotid artery (ICA) territory during ICA occlusion greatly varied. No detectable changes in SEP were found during anterior cerebral artery occlusion for anterior communicating artery aneurysms. This study indicates that intraoperative SEP monitoring is useful to detect ischemia in the MCA territory and that rapid disappearance of the N20 component is a danger signal. PMID- 1724294 TI - Antiproliferative effect of trapidil on PDGF-associated growth of human glioma cell lines in vitro. AB - The effects of trapidil on platelet-derived growth factor (PDGF)-associated growth of glioblastoma cells were studied. The assessment using PDGF-dependent rat lung endothelium cells revealed secretion of a PDGF-like factor from SF-126 cell line but not from SF-188. Human recombinant PDGF stimulated proliferation of both these glioblastoma cell lines. The anti-PDGF monoclonal antibody inhibited the growth of SF-126 more than SF-188. The results suggest the presence of an autocrine growth mechanism in SF-126 cells mediated by PDGF. The growth of both SF-126 and SF-188 cells was suppressed by trapidil, a specific PDGF antagonist, at 10 and 50 micrograms/ml, respectively. The proliferative response to exogenous PDGF and the antagonistic effect of trapidil were greater in the SF-126 cell line. In addition, trapidil markedly reduced production of prostaglandin E2 in both glioblastoma cell lines. This anti-proliferative effect on malignant glioma cells suggests that trapidil might be a new therapeutic agent for malignant gliomas. PMID- 1724296 TI - Congenital hydrocephalus mimicking Dandy-Walker syndrome induced by 6 aminonicotinamide injection in pregnant rat. AB - Fetal hydrocephalus was induced by single intraperitoneal injection of 8 mg/kg 6 aminonicotinamide (6-AN), a niacinamide antagonist, in Sprague-Dawley rat on day 13 of gestation. Materials for histological examination were obtained by uterotomy 1, 2, 4, and 8 days after injection, and untreated fetuses of the same ages were used as controls. Macrocephalus was clear at day 17 (4 days after injection), when cerebral dysgenesis was suggested by bromodeoxyuridine immunohistochemical study. The entire ventricular system was dilated, including the aqueduct and foramen of Monro, and hypoplasia of the cerebellum was also observed. On day 21, macrocephalus was remarkable, and considerable hypoplasia of the choroid plexus and cerebellum and agenesis of the corpus callosum were recognized. These results indicate that this experimental hydrocephalic model associated with various central nervous system anomalies mimics human Dandy Walker syndrome, suggesting the pathogenesis of Dandy-Walker syndrome to be a feature of systemic metabolic deficits. PMID- 1724297 TI - Giant intracranial aneurysms--magnetic resonance imaging follow-up and clinical symptoms. AB - Twenty-four intracranial aneurysms over 20 mm in diameter were studied with magnetic resonance (MR) imaging. MR imaging follow-up of eight cases revealed induced thrombus with homogeneous intensity and decreased size even after complete intraluminal thrombosis. Most cases demonstrated homogeneous intensity thrombus in contrast to the heterogeneous intensity of spontaneous thrombus. The clinical symptoms could not be explained retrospectively by the thrombus characteristics. Perianeurysmal high intensity, indicating cerebral edema, was detected in one case presenting with a rapid increase in size. MR imaging is useful for following these pathological intra- and perianeurysmal changes. PMID- 1724298 TI - Post-irradiation vasculopathy of intracranial major arteries in children--report of two cases. AB - We report two rare cases of post-irradiation vasculopathy of intracranial major arteries in children. A 13-year-old girl suffered from transient right hemiparesis 1 year after irradiation for suprasellar germinoma. Left carotid angiograms revealed marked stenoses of the intracranial internal carotid, middle cerebral, and anterior cerebral arteries, which were previously normal, and moyamoya vessels. A 2.5-year-old girl underwent internal irradiation with 198Au colloid for cystic craniopharyngioma. At the age of 10 years, she suddenly became unconscious after vomiting. Computed tomographic scans showed a right frontal intracerebral hematoma. Right carotid angiograms disclosed complete obstruction of the intracranial internal carotid, middle cerebral, and anterior cerebral arteries and moyamoya vessels, previously not present. The danger of radiation therapy causing occlusive vasculopathy in small and major cerebral arteries in children is emphasized. To prevent permanent ischemic neurological deficits, vasculopathy should be treated either medically or surgically as early as possible. PMID- 1724299 TI - Cerebral alveolar hydatid cyst--case report. AB - A rare case of cerebral alveolar hydatid disease in a 41-year-old female is presented. The larval mass was subtotally removed and the presence of alveolar hydatid cysts established histologically. Postoperatively, slight mental disturbance persisted but paresis did not develop. PMID- 1724300 TI - Aspergillus mycotic aneurysm--case report. AB - A 61-year-old female developed subarachnoid hemorrhage after trans-sphenoidal surgery for Rathke's cleft cyst. Neuroradiological examination revealed a large aneurysm at the C1 portion of the right internal carotid artery. Autopsy revealed marked proliferation of aspergillus hyphae in the wall of the aneurysm. A review of previously reported cases of fungal aneurysm proposes two developmental processes. Aneurysms secondary to fungal meningitis tend to be large in size and located in the major cerebral artery trunk, but aneurysms following fungal sepsis tend to be small and in peripheral branches. The former aneurysms are probably caused by fungus invasion into the intracranium, usually from the paranasal sinus, and the latter may be due to fungal emboli like bacterial emboli in bacterial endocarditis. Ruptured fungal aneurysms are difficult to treat, so fungal meningitis or sepsis must be eradicated before an aneurysm develops. PMID- 1724301 TI - Excision and end-to-end anastomosis of a fusiform aneurysm of the distal posterior inferior cerebellar artery associated with ischemia--case report. AB - A 35-year-old male with a sudden onset of severe vertigo and vomiting had a fusiform aneurysm of the distal portion of the left posterior inferior cerebellar artery. The symptoms were caused by cerebellar infarction probably due to an embolism from the aneurysm. The aneurysm was excised and the artery reconstructed by end-to-end anastomosis with an excellent outcome. Histological examinations showed mural thrombus but no wall dissection. PMID- 1724302 TI - Brain metastasis of thyroid papillary carcinoma--case report. AB - A rare case of brain metastasis of thyroid papillary carcinoma is reported. The cerebral metastasis was surgically treated without irradiation despite the presence of a spinal metastasis. No recurrence was demonstrated by computed tomography 1 year postoperatively. We suggest that surgery is indicated for a brain metastasis of thyroid papillary carcinoma even if other metastatic lesions are present. PMID- 1724303 TI - Magnetic resonance imaging of fat embolism syndrome--case report. AB - The authors describe a case of fat embolism syndrome in a 20-year-old male. Magnetic resonance (MR) imaging demonstrated multiple small cerebral infarcts suggesting this is the cause of the associated cerebral dysfunction. He underwent chemotherapy and hyperbaric oxygen therapy and recovered rapidly. MR imaging can provide prognostic indications in fat embolism syndrome. PMID- 1724304 TI - Distribution and morphology of substance P-immunoreactive structures in the olfactory bulb and olfactory peduncle of the common marmoset (Callithrix jacchus), a primate species. AB - The present study describes the morphology and distribution of substance P immunoreactive (SP-ir) elements in the olfactory bulb (OB) and olfactory peduncle (OP) of the common marmoset (Callithrix jacchus), a primate species. SP-ir neurons are very abundant in the OB and belong to two types. External tufted cells are present in the glomerular layer (GL), whereas granule cells are found in the deeper layers, especially in the granule cell layer (GRL), but also scattered in the OP. SP-ir fibers, putatively of central origin, were identified in the OP. They ascend into the bulbar layers. The SP-chemoarchitecture of the marmoset OB and OP does not differ more from rat, guinea pig and cat, than the SP chemoarchitecture of these species varies among one another. PMID- 1724305 TI - Synapses formed by ectopic corticofugal axons: an electron microscopic study of crossed corticorubral projections in kittens. AB - The corticorubral projections in newborn kittens are bilateral, while the projections are unilateral in adults. We addressed the question whether or not the crossed corticorubral projection in kitten forms synaptic contacts in the red nucleus. The neurons in the sensorimotor cortex of the kitten were labeled by Phaseolus vulgaris-leucoagglutinin or biocytin. Electron microscopic observations revealed that corticofugal axons form synapses both in the contralateral and the ipsilateral red nucleus; most of them were on small postsynaptic profiles, possibly dendritic spines or distal dendrites. PMID- 1724306 TI - Cholinergic projections from the globus pallidus to the lateral amygdaloid nucleus in the cat. AB - Direct projections from the globus pallidus (GP) to the lateral amygdaloid nucleus (A1) were found in the cat by the anterograde and retrograde tracing methods. Pallido-amygdaloid fibers originated mainly from the caudal half of the GP and distributed throughout the entire extent of the A1; most densely in the lateral part of the A1. Choline acetyltransferase immunohistochemistry in combination with retrograde fluorescent tracing indicated that most pallido amygdaloid neurons were cholinergic. PMID- 1724307 TI - Vasoactive intestinal polypeptide in dorsal root terminals of the rat spinal cord is regulated by the axoplasmic transport in the peripheral nerve. AB - Vasoactive intestinal polypeptide (VIP) immunoreactivity in the upper spinal dorsal horn is markedly increased after transection, crush or vinblastine treatment of the ipsilateral, segmentally related peripheral nerve. After regeneration of the peripheral nerve, VIP disappears from the upper dorsal horn. Transection-induced VIP increase is abolished by rhizotomy. It is concluded that the expression of VIP is restricted by factor(s) carried by retrograde axoplasmic transport to dorsal root ganglion cells. PMID- 1724308 TI - Ultrastructural evidence for an olfactory-autonomic pathway through the rat central amygdaloid nucleus. AB - The innervation of medullary projection neurons in the central amygdaloid nucleus (Ce) by afferents from the ventral taenia tecta (VTT) was investigated using combined lesion-induced axonal degeneration and retrograde transport of horseradish peroxidase-conjugated wheat germ agglutinin (HRP-WGA). Injections of HRP-WGA into the nucleus tractus solitarii resulted in retrograde labeling of neurons in the medial Ce. Ultrastructurally HRP-WGA reaction product was identifiable in the perikarya and proximal dendrites of Ce neurons. Degenerating terminals, probably due to damage at the HRP-WGA injection site, were few and confined to the ventral Ce. Electrolytic coagulation of the VTT resulted in approximately 9% of terminals in medial Ce showing signs of degeneration at 5 days post-lesion. Of the terminals sampled, slightly more than 40% were in contact with the dendrites of retrogradely labeled neurons. Where evident, these terminals formed exclusively symmetrical synaptic contacts. These data provide evidence for an oligosynaptic olfactory-autonomic pathway in the rat that may mediate olfactory influences on gastric and cardiovascular aspects of autonomic function. PMID- 1724309 TI - Long-term survival of severed crayfish giant axons is not associated with an incorporation of glial nuclei into axoplasm. AB - Glial nuclei have been reported to be incorporated into the axoplasm of surviving distal stumps (anucleate axons) weeks to months after lesioning abdominal motor axons in rock lobsters. We have not observed this phenomenon in crayfish medial giant axons (MGAs) which also survive for weeks to months after lesioning. Glial nuclei were not observed within MGAs perfused with a physiological intracellular saline. However, incorporation of glial nuclei was observed after MGAs were perfused with intracellular salines containing Fast green. From these and previously published data, we confirm that glial incorporation into axoplasm can occur, but we suggest that is is not a common mechanism used by crustaceans to provide for long-term survival of anucleate axons. PMID- 1724310 TI - Potentiation of a behavioural response in mice by spinal coadministration of substance P and excitatory amino acid agonists. AB - The functional interaction in the spinal cord between substance P and excitatory amino acid agonists was investigated. Behavioural responses were scored in mice after intrathecal administration of excitatory amino acid agonists and substance P, given separately or in combination. A strong potentiation of the effect was seen when substance P was coadministered with N-methyl-D-aspartate (NMDA), alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) or kainic acid (KA). The potentiation was blocked by the corresponding antagonists: the selective NMDA receptor antagonist (+/-)-3- (2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the substance P analog, [D-Arg1,D-Trp7,9,Leu11]-substance P (Spantide). These findings indicate a functional interaction between substance P and glutamate in the dorsal horn of the spinal cord, compatible with the hypothesis that corelease of substance P and glutamate from primary afferent neurons may enhance nociception. PMID- 1724311 TI - Retrograde axonal transport of the GTP-binding protein Gi alpha: a potential neurotrophic intra-axonal messenger. AB - When a neuronal target is to provide information to the nucleus of the neurone innervating it, it faces the problem of getting its message up the long length of axon separating the cell body from the site of receptor activation at the terminal. The retrograde axonal transport of the neurotrophic molecule, nerve growth factor (NGF), provided one possible mechanism for this information transfer in the sympathetic nervous system. However, some neurotrophic molecules are not retrogradely transported, indicating the message is carried back by a different mechanism. In this paper, we examined such a novel mechanism mediated by the retrograde axonal transport of the alpha subunit of the second messenger protein, Gi. It is proposed that some non-transported neurotrophic molecules may produce a stable second messenger that is itself transported to the nucleus to convey the target derived information for survival. PMID- 1724312 TI - Asymmetric increase in substance P immunoreactivity in the rat and guinea pig substantia nigra after unilateral neocortical ablation. AB - Substance P was visualized in the rat and the guinea pig basal ganglia, mesencephalon and spinal cord after surgical unilateral cortical ablation. An increased density in substance P-like immunoreactive patterns in the substantia nigra was observed ipsilaterally to the lesioned hemisphere. These results, together with previous observations in cases of Alzheimer's disease presenting with asymmetric cortical atrophy, suggest an influence of the corticostriatal pathway on substance P-like immunoreactivity in these subcortical structures. Finally, these experiments proved to be a reliable animal model for the study of the effects of neocortical lesions on the neuropeptidergic innervation of certain subcortical regions. PMID- 1724313 TI - In vitro denervation of frog skeletal muscle: expression of several conductance classes of nicotinic receptors. AB - The question arose as to whether the altered kinetic properties of the nicotinic acetylcholine receptor-ion channel complex (AChR) observed after muscle denervation could be due to post-translational modifications. Single AChR properties of in vitro denervated skeletal muscle were studied using the cell attached patch-clamp technique. Patch-clamp recordings from junctional regions of fibers daily treated with cycloheximide, a protein synthesis inhibitor, and phenylmethylsulfonyl fluoride, a protease inhibitor, revealed single conductance class of acetylcholine (ACh)-activated currents after acute dissociation and several conductance classes 3-14 days after muscle denervation. alpha Bungarotoxin blocked all conductance states induced by ACh. Biochemical assays carried out on the same experimental conditions disclosed that chronic treatment of the fibers with cycloheximide inhibited the incorporation of [3H]leucine into precipitable protein. The present findings provided evidence that the altered functional properties of nicotinic receptors after muscle denervation may occur without new transcription. PMID- 1724314 TI - Laser treatment of subfoveolar choroidal neovascular membranes. AB - We prospectively studied the safety and efficacy of subfoveolar laser treatment of choroidal neovascular membranes (CNVM) in 29 eyes. With one to four treatments, all CNVMs were successfully eradicated. Central visual acuity improved in 31% and stabilized in 69%. Vision did not deteriorate in any of the eyes. In appropriately selected eyes, subfoveolar treatment appears safe and effective in halting the exudative response and minimizing scotoma size, thus facilitating the use of low-vision aids. PMID- 1724315 TI - Positive effect of granulocyte-colony stimulating factor on erythropoiesis in humans. AB - We studied the effects of granulocyte-colony stimulating factor (G-CSF) on human erythropoiesis in vivo. Changes in the peripheral blood were analyzed in 9 subjects; 3 healthy volunteers, 3 patients with pancytopenia and hypersplenism and 3 patients with chronic renal failure and severe anemia being treated with hemodialysis. We monitored erythropoiesis according to the number of highly fluorescent cells (HFC) present in the peripheral blood after staining with Auramine 0. These cells are relatively immature reticulocytes that contain much RNA. All subjects received recombinant human G-CSF at a daily dose of 100 micrograms/m2 administered intravenously for 3 to 5 days. Plasma erythropoietin was measured in patients receiving dialysis before and several times after its administration. The number of HFC increased significantly in all subjects. In 1 of 3 patients receiving dialysis, the plasma erythropoietin increased transiently but insignificantly. These findings show that G-CSF affects human erythropoiesis in vivo, but that the mechanism of its effect did not involve an increase in plasma erythropoietin. PMID- 1724317 TI - Correlations between symbolic motor performance and achievement in school for children with minimal brain dysfunction. AB - Significant correlations were found between grades for reading, writing, and calculating and each child's individual mean reaction time for symbolic motor performance tasks with letters and numbers as stimuli for 240 elementary school boys with minimal brain dysfunction and in first to fourth classes in Prague (Czechoslovakia) and Skopje (Yugoslavia). For age(class)-matched groups of 382 healthy boys the correlations disappeared by the age of ten years. Similar results were noted for groups of boys in both cities. PMID- 1724316 TI - [Receptor status (ER and PgR) determined with histochemical and biochemical methods in breast carcinoma]. AB - Recently, a method similar to ER.ICA has been proposed for the progesterone receptor (PgR) using two monoclonal antibodies, JZB39 and KD68, specific for human PgR and characterized by a molecular weight of 95 and 120 Kd, respectively. A series of 73 breast cancer patients was studied with regards to ER and PgR using both immunocytochemical (ICA) and biochemical (DCC) assays. Results showed no substantial differences between the two methods when considering common clinical-pathological parameters. Overall agreement between ICA and DCC methods was found: 79% for PgR and 78% for ER. A slight quantitative correlation was also observed between the "score values" of the ICA method and the Fmol content of ER and PgR using the Brave-Pearson test (r = 0.49 for PgR; r = 0.43 for ER). Specificity of PgR.ICA method was 77% for PgR and 72% for ER; sensitivity was 82% and 83%, respectively. The ICA method is a reliable technique to assess PgR presence as well as ER. Further studies are necessary to evaluate the prognostic role of nuclear PgR. PMID- 1724318 TI - Leu 7 monoclonal antibody in diagnostic histopathology: its lack of neuroendocrine specificity. PMID- 1724319 TI - Juvenile intracortical adamantinoma of the tibia with predominant osteofibrous dysplasia-like features. AB - In view of the still disputed relationship between adult adamantinoma and osteofibrous dysplasia in children, a unique case of adamantinoma, indicating a direct relationship between the two lesions, is presented with a review of the literature. The patient was a six-year-old boy who complained of pain and swelling in the left lower leg. Roentgenographs showed a loculate osteolysis surrounded by sclerosis within the cortex of the tibial shaft that would be typical of osteofibrous dysplasia. Although an osteofibrous-dysplastic component predominated histologically, some small islands of epithelial cells were scattered throughout the lesion. Immunohistochemically, the tumor cells of these epithelial islands gave a constant positive reaction for cytokeratin as well as vimentin, while the stromal cells in the osteofibrous dysplasia-like lesion were positive for vimentin only. This type of lesion is recorded in the Bone Tumor Registry of Westphalia at a rate of 8.3% for osteofibrous dysplasia, and of 25% for adamantinoma. A review of the literature, yielding reports with remarkable uniformity on 14 cases beyond the present one, suggests the existence of a separate clinicopathologic entity to be called juvenile intracortical adamantinoma with predominant osteofibrous dysplasia-like features, and which might be a regressing form of adamantinoma specific in childhood. PMID- 1724320 TI - Immunohistochemistry on needle biopsies of childhood malignancies. AB - Ultrasound-guided percutaneous needle biopsy proved to be a reliable and safe method to obtain material for histopathological and immunohistochemical diagnosis prior to treatment in childhood malignancies. A principal tumour identification could be obtained by a combined morphological and phenotypic examination of 38 small-sized tumour biopsy specimens using a fairly limited panel of immunological reagents, including antibodies to leucocyte common antigen (CD 45), certain B- and T-cell markers, various intermediate filaments (cytokeratin, desmin and vimentin), and neuroblastoma cells (UJ 167.11, A2B5, and UJ 13A; the latter recognizes NCAM). Five undifferentiated neuroblastomas were all positive with the neuroblastoma antibodies but negative for the other markers, including vimentin. The negative reactivity for desmin and vimentin was the major immunohistochemical distinction between neuroblastomas and rhabdomyosarcomas. In addition, limited reactivity with the neuroblastoma antibodies was seen in blastematous parts of Wilms' tumour, duct-like structures in a hepatoblastoma, and in tumour cells in a few undifferentiated myelo- and lympho-proliferative lesions. This study shows the importance of a combined evaluation of morphology and the pattern of immunoreactivity employing multiple markers. PMID- 1724321 TI - Histochemical localisation of acid phosphatase and non-specific esterase in the midguts of two species of tick, Boophilus microplus and Rhipicephalus appendiculatus, as determined by light microscopy. AB - Serial sections of glycol methacrylate-embedded and frozen midguts of Boophilus microplus and Rhipicephalus appendiculatus were studied histochemically by light microscopy. The use of the naphthol AS-TR phosphate technique combined with glycol methacrylate embedding enabled the precise localisation of lysosomal enzyme activity, despite the ubiquity of haematin granules in tick midgut epithelia. The presence of acid phosphatase and non-specific esterase activity in the same cells was observed in all of the various developmental stages and feeding phases of the ticks. The pattern of appearance of these cells paralleled the reported level of protease activity in the midgut lumen. The cells were found to be solitary, particularly during the slow digestive phases, and appeared to move into the lumen, where they eventually disintegrated. The cells therefore appear to function as holocrine secretory cells. This is the first report indicating the presence of such secretory cells in the midgut of unfed ticks. The disintegration of these cells in the lumen suggests that lumenal digestion may be more important than hitherto realised. PMID- 1724322 TI - Unique features of the trophoblast interferons. AB - The trophoblast interferons (IFN) are Type I IFN with about 50% amino acid sequence identity to the leukocyte IFN (IFN-alpha). They are the major secretory products of the trophoblast of ruminant ungulate species during pregnancy in the period immediately preceding attachment and implantation when they have been implicated in the phenomenon known as maternal recognition of pregnancy. The trophoblast IFN have antiviral and antiproliferative activities typical of other Type I IFN, but unlike IFN-alpha, -beta and -omega are poorly responsive to viral induction and have a highly restricted pattern of expression. Nevertheless, a recombinant bovine IFN-alpha can mimic many of the properties of the trophoblast IFN and has been used pharmacologically to improve pregnancy success in sheep. It still remains unclear, however, whether the trophoblast IFN have unique biological properties or whether they are unusual merely by virtue of the location, magnitude and temporal nature of their expression at a critical time during pregnancy. PMID- 1724324 TI - [Treatment with iloprost of critical ischemia of the lower limbs associated with cardiac insufficiency. Study of the interaction with pharmacokinetics of digoxin]. AB - Twelve patients with critical ischaemia of the lower limbs were treated with iloprost. The purpose of this study was to investigate for a possible iloprost digoxin interaction and to evaluate the clinical benefit provided by short- or long-term iloprost therapy. The pharmacokinetics of digoxin were studied before and during iloprost treatment. Under iloprost the absorption of digoxin was delayed by about one hour, but the area under the plasma digoxin concentration curve remained unmodified. In 11 of our 12 patients the clinical effect of iloprost was satisfactory both immediately and after 6 months. Pain vanished in 6 patients and diminished in 6 patients. All skin ulcers were healed. In most cases this improvement persisted beyond the study period: 2 patients treated at the beginning of the study and who are still followed up have remained improved after 2 1/2 years. Two patients with pain relapse received iloprost in repeated 10 days' courses with successful results. The treatment was relatively well tolerated (headaches, flushing, abdominal pain). Thus, iloprost can avoid amputation in severe arteritis unsuitable for revascularization and for which there is no effective treatment. Patients under digoxin may continue to take this drug in the same doses during treatment with iloprost. PMID- 1724323 TI - [Serum antibodies to hepatitis C virus: analysis of risk factors of seropositivity in a population of blood donors in metropolitan France]. AB - Risk factors were analyzed in a group of 117 blood donors seropositive for hepatitis C virus antibody. One risk factor, at least, was found in 63 (53.8%) subjects. Frequently encountered risk factors were 1) travels in developing countries (31/117); 2) blood transfusions (20/117); 3) health care works (10/117); 4) intravenous drug addiction (8/117); 5) homosexual or multiple heterosexual contacts (3/67). Our study emphasizes the high percutaneous transmission of hepatitis C in contrast with the low sexual transmission. No risk factor could be found in 54 (46.2%) of the 117 seropositive subjects: the route of transmission in these cases is an intriguing issue which certainly deserves further epidemiological investigations. PMID- 1724325 TI - [Incidence and prognostic significance of ventricular extrasystole in pilots of combat aircraft and civilian planes]. AB - It is a real challenge, for the doctor internist-cardiologist, who deals everyday, with problems of arrhythmia, especially in pilots of combat aircraft, to discover those rare cases of premature ventricular contractions (PVC) where exists great probability that PVC were caused by ischemic heart disease. Appearance of complex PVC in aircrew is seldom. Isolated appearance of single, unifocal PVC in young pilots without any symptoms and in absence of organic changes or functional heart disorder, should be considered as normal medical finding. PMID- 1724326 TI - [Determination of correlations between the main pharmacokinetic parameters and physiopathological factors of flecainide in the elderly]. AB - The purpose of the study was to evaluate the correlation between pharmacokinetic and physiopathological parameters of flecainide in elderly population. Pharmacokinetics of flecainide was determined in seventeen patients, aged more than 60 years, with clinically documented ventricular extrasystoles. Patients received 1.5 mg/kg flecainide either by intravenous administration or by slow perfusion. In elderly population the elimination half-life, the mean residence time and the volume of distribution of flecainide are increased and the plasmatic clearance is reduced. All of these parameters show an important interindividual variability. The relationships between pharmacokinetic and physiopathological parameters were evaluated with multivariate analysis. The correlation obtained (89.86% for total clearance, 64.64% for volume of distribution, 42.59% and 31.12% for elimination and distribution half-lives) provide a relatively good determination of pharmacokinetic parameters of flecainide in aged patients. PMID- 1724327 TI - [Treatment with iloprost of critical ischemia of the lower limbs associated with cardiac insufficiency. Study of interaction with the pharmacokinetics of digoxin]. AB - We have treated with intravenous iloprost twelve patients suffering from cardiac insufficiency compensated under oral digoxin (NYHA class II) associated with severe limb ischaemia due to arterial insufficiency. Our aim was to study its possible interaction on digoxin levels and to evaluate the long-term efficacy of iloprost. Although iloprost slowed the digoxin absorption by approximately one hour, we found no clinically significant difference between the digoxin pharmacokinetic data before and during treatment by iloprost. Moreover, 11 out of the 12 patients had a good clinical fate after the treatment, which persisted at 6 months. The pain disappeared in 4 and diminished in 7; and all skin ulcers healed. This improvement has lasted up to two and a half years in two patients. The clinical tolerance of iloprost was acceptable despite frequent headache and flushing associated with hypotension and nausea. We conclude that iloprost seems to be a very promising treatment of severe limb ischaemia when no intervention on the proximal arteries is possible. The patients on digoxin can continue their treatment without dose alteration while starting on iloprost. PMID- 1724328 TI - Neher and Sakmann win Nobel Prize for patch-clamp work. PMID- 1724329 TI - Balloon dilation of the prostate. Critical evaluation. AB - We describe the results of prostatic balloon dilation in 7 patients with a follow up of 9 months. Symptom scores dramatically fell in 5 patients (success rate 71%), which is confirmed by a significant improvement in peak flow in 4 of them. Two patients failed to improve and needed a subsequent intervention which revealed a stage A2 prostatic carcinoma in one. During subsequent open prostatectomy, a condition analogous to dilation with the Deisting dilator was seen. We conclude that the indications and results obtained with the balloon dilator are similar to those with the Deisting dilator. PMID- 1724330 TI - Ion channels and excitatory transmission in the smooth muscle of the urinary bladder. AB - The bladder of most non-human species receives dual purinergic and cholinergic excitatory innervation. Activation of P2x-purinoceptors depolarizes the cells, increases the spike frequency and causes contraction. Addition of agonists rapidly activates non-selective cation channels, which underly the excitatory junction potentials seen on simulation of the intrinsic nerves. In all species studied including humans the detrusor possesses well developed post-junctional purinergic mechanisms, and interspecies differences here cannot account for the large difference in the potency of the purinergic innervation seen. Muscarinic receptor activation causes contraction through pharmacomechanical coupling, using inositol trisphosphate as a second messenger. Increase in intracellular calcium results in outward currents through Ca activated K channels. In most species little depolarization or increase in spike frequency is seen, although in some, a slowly developing inward current mechanism is activated, which might account for the small delayed muscarinic depolarization seen following the purinergic excitatory junction potential. PMID- 1724331 TI - Calcium-activated cation channel in rat portal vein myocytes. AB - A rapid transient rise in the free cytosolic Ca2+ concentration is one of the earliest events in smooth muscle cell activation and is involved in the opening of several classes of Ca2(+)-sensitive ion channels such as K+ channels and Cl- channels. In portal vein smooth muscle cells, in addition to the Ca(2+)-dependent Cl-current, single-channel activities were recorded in response to external application of 10 mM caffeine, in the absence of EGTA in the pipette solution. The conductance of this novel type of channel was around 200 pS for membrane potentials ranging between -100 to +60 mV. The single-channel activities were also induced by external application of noradrenaline (10(-5) M) or acetylcholine (10(-5) M), by Ca2+ entry through voltage-dependent Ca(2+)-channels, and by intracellular application of ryanodine (10(-5) M). The caffeine-activated single channel currents disappeared when 10 mM EGTA were added to the pipette solution or after replacement of external Ca2+ with Ba2+. These results show that these channels are Ca(2+)-dependent. Alteration of the Cl- equilibrium potential did not produce any change in the reversal potential of the caffeine-activated single channel current, indicating that it was not carried by Cl- ions. The value of the reversal potential was about + 10 mV, irrespective of the CsCl-, KCl- or NaCl containing solutions used to fill the pipette. Caffeine activated single-channel currents when cells were bathed in 90 mM Ba2+ plus 1 mM Ca(2+)- or 91 mM Ca(2+) containing solutions, showing that divalent cations permeate the channels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724332 TI - Potassium channel activation, hyperpolarization, and vascular relaxation. AB - 1) Numerous compounds and changes in physical state functions shift the membrane potential of vascular smooth muscle to more negative values. The consequence is a vasodilatation because Ca2+ channels are closed. K+ channel opening frequently causes the hyperpolarization. 2) Acidification of the blood substitute solution, a fall in O2 partial pressure, and an increase in blood flow dilate arterial vessels. Acidosis is associated with a rise in K+ permeability and a simultaneous fall in Na+ permeability. Prostacyclin has a 20-30% share, and EDHF a 70-80% share in hypoxic vasodilatation. Experiments with iloprost (PGI2 analogue) confirmed the K+ channel opening properties of this drug. A voltage-dependent K+ channel and a Ca(2+)-activated K+ channel, via the influence of cA-PK or cG-PK, are responsible for the hyperpolarization with iloprost and with oxygen deficiency. 3) With 23Na+ nuclear magnetic resonance techniques, it has been demonstrated that with flow-dependent vasodilatation, proteoheparan sulphate integrated in the membrane of endothelial cells possibly served as a "flow sensor". With an external strain, such a compound can go from a randomly coiled state to an oriented state. Based on these viscoelastic properties, heparan sulphate proteoglycan is present as a random coil under "no flow" conditions and as an unfurled filament structure with increasing flow. This conformational change produces additional anionic binding sites to which Na+ ions of the blood are bound. A membrane hyperpolarization could be directly initiated by this Na+ binding via the protein fraction within the macromolecule or via a change in zeta potential. Therefore, these ions can trigger the signal transduction for a vasodilatory vessel reaction. Decrease in flow is followed by a structural change of the macromolecule towards coil conformation, a release of Na+ ions and, thus, an interruption of the signal chain. 4) Cicletanine, aqueous garlic extract, and ajoene cause a concentration-dependent membrane hyperpolarization and are potent vasodilators. A cicletanine concentration, which is attained by the dosage given to patients, is sufficient to produce these effects. Under noradrenaline, the cicletanine effect is amplified. Aqueous garlic extract and ajoene exert a hyperpolarizing and vasodilating influence even in a concentration which may occur in the extracellular space by the administration of a single garlic clove. 5) The stationary activation curve "developed force vs. membrane potential" satisfactorily explains the effects of K+ channel openers. The tight electromechanical coupling expressed by this curve comprises a 50% vasorelaxation for a 2.5 mV hyperpolarization. In the linear part of the curve, the coupling ratio is 5.1 mV/g.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1724333 TI - Decrease in memory (CDw29-high) cerebrospinal fluid cells from acute MS patients. AB - The CD45R (2H4) and CDw29 (4B4) molecules are probably involved in the regulation of immunological memory: CD45R functionally defines naive or virgin cells (prior to activation by exposure to antigen), while CDw29 characterizes previously activated (memory) cells. By means of two-colour fluorescence analysis, we studied CD4 and CD8, CD45R and CD4 and CD8 CDw29 cells from the cerebrospinal fluid (CSF) of 27 acute (MSa), and 10 stable (MSs) multiple sclerosis (MS) patients. We also compared 17 patients affected with various non-inflammatory (OND) diseases of the central nervous system (CNS). The most striking finding was the increased percentage of CD4+CD29-, CD4+CD45R- and CD8+CD45R- cells and the decreased percentage of CD4-CDw29+ and CD8- CDw29+ subsets in MSa patients compared to OND and MSs populations. These data suggest a decrease in memory cells (CDw29+) during the acute phase of MS. The modulation of CDw29 receptor could play a role in the genesis of acute MS attacks. PMID- 1724334 TI - Antibody to reverse transcriptase of human retroviruses in multiple sclerosis. AB - HTLV-1, HIV-1 and HIV-2 western blot analysis of sera from patients with multiple sclerosis (MS), from patients with other neurological diseases and from blood donors, revealed a rather frequent cross-reactivity with retroviral proteins in the MS group, though no patient was positive with the corresponding specific ELISA serology. Statistical analysis revealed a significant difference between the MS group and the two control groups for HIV-1 and HIV-2 reverse transcriptase fragments and for HTLV-1 p24. The general significance of these observations is discussed in the light of a retroviral hypothesis for the aetiology of MS. It is suggested that, if a retrovirus is present in MS patients, it does not necessarily belong to the HTLV sub-family and could as well be a lentivirus, like Visna virus, the causative agent of a demyelinating disease in sheep which is one -natural--model for MS. PMID- 1724335 TI - Deposition of beta/A4 protein along neuronal plasma membranes in diffuse senile plaques. AB - The origin of the extracellular beta-amyloid protein (beta/A4) found in senile plaques and the cellular mechanisms responsible for its deposition in cerebral tissues are still an unresolved issue in Alzheimer's disease. In this study we analyzed in detail the distribution of various epitopes of beta/A4 in relation to local cellular elements in diffuse plaques of the hippocampal region. We also correlated our findings with the presence and distribution of non-beta/A4 epitopes of the amyloid precursor protein (APP) and with synaptophysin immunoreactivity in the cortical neuropil. Discontinuous beta/A4-immunoreactive deposits were found along dendrites, and around the soma of neurons included in the plaques. Furthermore, increased synaptophysin reactivity with slightly dilated synaptophysin-immunolabeled presynaptic terminals were found in diffuse plaques. APP epitopes could not be found in diffuse plaques. However, some of the APP antibodies, mainly those to the C-terminal portion of APP, and antibodies to beta/A4 recognized clusters of flat vesicular profiles (0.6-1.4 micron in width and 2-3 microns in length) in the neuropil of cortical areas where plaques had developed. Our findings are compatible with a neuronal origin of beta/A4 in diffuse plaques and with a primary release of beta/A4 at synaptic sites along the immunostained neurites. They also suggest that diffuse plaques might be preceded by minute lesions of the neuropil where beta/A4 is not yet released from the precursor molecule. PMID- 1724337 TI - Measures of serotonin metabolism in anorexia nervosa. AB - Thirty-five consecutive attenders at a clinic specializing in anorexia nervosa were studied. All conformed to a DSM-III-R diagnosis for anorexia nervosa. In addition, 3 cases suffered from major depressive disorder and 9 from dysthymia. Blood from all patients was analysed for monoamine oxidase, serotonin (5-HT), 5 hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), tryptophan and platelet paroxetine binding. Findings showed that blood 5-HT was higher than normal in all patient groups, and was highest in those having affective disorder with anorexia nervosa. However, of the patients with anorexia nervosa alone, a subgroup having greatest weight loss had blood 5-HT levels significantly below all other groups. Lack of significant changes in other parameters compared with normal subjects points to the possibility of abnormal 5-HT storage or release. PMID- 1724336 TI - Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study. AB - Anesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P less than or equal to 0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress. PMID- 1724338 TI - Cerebrospinal fluid variables among alcoholics lack seasonal variation. AB - Seasonal influences on indices of serotonergic function, including cerebrospinal fluid (CSF) concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), have been reported in psychiatric patients and healthy volunteers. We examined seasonal differences in CSF concentrations of 5-HIAA among 135 alcoholics admitted to a research ward who had a lumbar puncture. No significant seasonal differences were found for either CSF concentrations of 5-HIAA or CSF concentrations of other monoamine metabolites or peptides. The possible explanations for these negative findings are discussed. PMID- 1724339 TI - Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers. AB - Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proenhancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picomole, respectively. The proenhancer strategy was effective in assays for beta-D galactosidase, beta-D-glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement. PMID- 1724340 TI - Effects of a combined estrogen-gestagen regimen on serum levels of the carboxy terminal propeptide of human type I procollagen in osteoporosis. AB - Estrogen stimulates osteoblastic collagen production in vitro, but whether the same stimulation takes place in vivo is still unknown. To test the stimulatory effects of a combined estrogen-gestagen regimen in vivo we monitored serum levels of the carboxy-terminal propeptide of human type I procollagen (S-PICP) in a group of 12 osteoporotic women over a 150 week treatment period. Spinal bone mineral content (BMC) increased to a maximum of 5% over pretreatment values around week 90. Serum alkaline phosphatase (S-AP) and serum bone gla protein (S BGP) both fell from initial values of 220 U/liter and 39 ng/ml, respectively, to 146 U/liter (p less than 0.01) and 27.2 ng/ml (NS) around week 60 and remained reduced over the remaining treatment period. S-PICP also fell from 117 to 68 micrograms/liter at week 60 and 70 micrograms/ml at week 150 (P less than 0.01). This is equal to a reduction to 32 +/- 10% pretreatment levels. The reduction in S-PICP was not significantly different from that of the other two markers of bone formation (S-AP and S-BGP). Thus, provided the metabolic clearance of PICP remains unaltered after hormone replacement therapy, no major stimulation of osteoblastic collagen type I synthesis was demonstrable during estrogen-gestagen treatment in this population of osteoporotic women. The changes in bone markers seen in this study are therefore consistent with an estrogen-mediated reduction in the frequency of remodeling activation. Because of the reduction in bone turnover and methodologic limitations of bone marker assays, however, smaller increases in the amount of bone formed per activation could remain undetectable. PMID- 1724341 TI - Effects of acid and basic fibroblast growth factor and heparin on resorption of cultured fetal rat long bones. AB - We tested acid and basic fibroblast growth factor (aFGF and bFGF), members of the heparin binding FGF family, for their ability to stimulate bone resorption as measured by the release of previously incorporated 45Ca from cultured fetal rat long bones in the presence and absence of heparin. Purified low-molecular-weight heparin (LMW heparin) at 5-125 micrograms/ml had no direct stimulatory effect. There was little effect from aFGF (10(-11)-10(-8) M) alone, but increased resorption was observed in the presence of LMW heparin. With bFGF, increased bone resorption was observed at 10(-9) M but not at 10(-8) M. The stimulatory effects of aFGF and bFGF in the presence of LMW heparin were not blocked by the addition of indomethacin (10(-6) M), which blocks prostaglandin production, or hydroxyurea (10(-3) M), which blocks DNA synthesis. However, pretreatment with aphidicolin (3 x 10(-5) M), a potent inhibitor of DNA synthesis, blocked the effect of acid FGF and diminished the effect of bFGF. These results indicate that both aFGF and bFGF can stimulate bone resorption by a prostaglandin-independent mechanism, particularly in the presence of heparin. The activation of FGF-mediated bone resorption by heparin could play a role in producing the osteoporosis that has been described with heparin therapy and mastocytosis. PMID- 1724342 TI - Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: a role for prostacyclin in adaptive bone remodeling? AB - Cyclic mechanical loading sufficient to engender strains of physiologic magnitude applied to recently excised canine cancellous bone cores in vitro increased the release of prostaglandin E (PGE) and prostacyclin (PGI2, measured as its breakdown product 6-keto-PGF1 alpha), during a 15 minute loading period in which PG levels were measured in perfusing medium at 5 minute intervals. Peak production occurred in the 0-5 minute sample. Mean levels preload compared to during load were PGE, 2.66 and 3.67 ng/ml (p less than 0.002); and 6-keto-PGF1 alpha, 543 and 868 pg/ml (p less than 0.007). The elevated levels then declined to preload levels during the loading period. However, the 5-10 minute but not the 10-15 minute samples still contained levels greater than preload values. A second 15 minute period of load, 1 h following the end of the first, produced smaller increases in the levels of release that were statistically significant only for the first 0-5 minute sample during load (preload compared to load mean values, PGE, 1.09-1.66 ng/ml, p less than 0.02; 6-keto-PGF1 alpha, 401-558 pg/ml, p less than 0.04). Immunolocalization revealed PGE and 6-keto-PGF1 alpha in lining cells and 6-keto-PGF1 alpha but not PGE in osteocytes. Addition to the medium of 1 microM PGE2, approximating the concentration produced by loading, had no significant effect on the specific activity of the extractable RNA fraction labeled with [3H]uridine, whereas 1 microM PGI2 produced an increase similar to that seen previously with loading.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724343 TI - Neurones with epileptiform discharge in the central nervous system and chronic pain. Experimental and clinical investigations. AB - Epileptiform discharge was recorded from neurons in the thalamic nuclei of chronic pain patients during stereotactic surgery. Hyperactive neurons showed regular firing of 3-5 trains of epileptic-like group discharges with a frequency of 4 to 5 Hz. As described by Lombard et al. (1979), we operated on the dorsal root unilaterally, sectioning C5 to Thl in male Wistar rats. One to three months after the operation, hyperactive neurons were examined in the contra-lateral thalamic nuclei (VP, zona incerta), and lemniscus medialis. The firing patterns and distribution of hyperactive neurons in these animals was very similar to those of humans. The hyperactive neuron was unaffected by electrical stimulation of the nucleus raphe dorsalis (NRD) and locus ceruleus (LC). Administration of phenytoin and diazepam reduced the firing. However, no effect was seen with valproic acid. During spreading depression of the sensorimotor cortex, a remarkable reduction was seen on the firing of thalamic hyperactive neurons. This suggested that hyperactive neurons of the thalamic nuclei received facilitory effects from the sensorimotor cortex with little influence from adrenergic or serotoenergic systems. PMID- 1724344 TI - Application of anticytokeratin antibodies to colon carcinomas. AB - Mouse monoclonal anticytokeratin antibodies (BA 16; BA 17; CO 8.2; C18.2; C 10; LE 41) were used for examination of 5 patients with large bowel carcinoma ("left colon") using the indirect immunoperoxidase technique. For each case cryostat sections were examined from three localizations: primary tumour of large bowel, mucosa in the vicinity of the tumour, and mucosa of distant part from the primary tumour. The antibodies BA 16 and BA 17 produced strongly positive results. Antibody C 18.2 seems to be less suitable for detection of cytokeratin expression in this part of large bowel. The obtained results indicate that no significant differences were found in expression of cytokeratins in the three localizations by histologically well or moderately differentiated adenocarcinoma. PMID- 1724345 TI - [Relation of prolactin with nodular hyperplasia and carcinoma of the prostate]. AB - The relationship between prolactin in NH and prostate carcinoma has several aspects: under normal conditions, prolactin enhances androgens output, increasing the Leydig cell susceptibility to LH. At the prostate level, it increases testosterone binding to the prostate epithelium and stimulates the conversion of testosterone to dihydrotestosterone. On its own, it decreases prostatic protein degradation and increases the DNA synthesis index. With regard to Nodular Hyperplasia, once its hormone dependency is rated, it also appears to be stimulated by high levels of prolactin. The NH cell cultures with human prostate increase their growth, cell division, as well as the DNA synthesis. The aspects of the prolactin-prostate carcinoma interaction involve considering this hormone as a possible carcinogenic agent, emphasizing the existence of high plasma levels of this hormone in individuals with prostate carcinoma, where any androgen excess is considered a major factor at the genesis of this tumour, and prolactin increases indirectly its testicular synthesis. Also, prolactin itself stimulates the growth and development of carcinomatous human cell lines, increasing its effect in the presence of testosterone. For this reason, controlling this hormone levels becomes an element to have into consideration in the treatment and follow up of these patients; also, the reevaluation of any drug therapy that, besides the initial goal pursued, does not maintain baseline prolactin levels. The association of anti-prolactin drugs has to be understood as a co-adjuvant therapy when there is androgenic deprivation, never as a single treatment. PMID- 1724346 TI - [Cancer of the prostate: current diagnostic aspects]. AB - Presentation of the initial results from a program for early diagnosis of prostate cancer, implemented a year ago at the Urology Unit of the Miguel Servet Hospital with the collaboration of the Region's specialists. All patients attending the Urology services, regardless the pathology, are evaluated when rectal examination is suspicious or the plasma PSA levels are higher than 4 ng/ml. Assessment is made through transrectal ultrasound scanning, with random or ultrasound-directed prostatic biopsy depending on the findings. A total of 83 prostatic biopsies have been analyzed n patients thus selected, presence of prostatic carcinoma becoming apparent in 52 (62.6%), 19 of which have undergone radical prostatectomy. The association suspected rectal examination/increased PSA has produced the higher percentages of diagnostic precision (80%) clearly improving those of rectal examination and PSA alone. The methods for local and nodular staging are analyzed, considering the systematic use of laparoscopic lymphadenectomy and biopsy of seminal vesicles highly useful for higher diagnostic precision in these patients. The diagnostic relevance of prognostic factors in advanced cancer is analyzed, this analysis being mandatory to evaluate the different therapeutic. PMID- 1724347 TI - [PSA in the staging and monitoring of cancer of the prostate]. AB - The prostatic surface antigen (PSA), exclusively secreted by the prostatic epithelial cells, can be raised in the sera of patients with various prostatic pathologies. Serum levels of this marker depend on many factors (prostatic manipulation, associated inflammation, volume of benign node hyperplasia, volume and grading of tumour differentiation), all of which will have to be taken into account when interpreting any specific level. The usefulness of this antigen has a distinctive role in the suspected diagnosis, staging and monitoring following treatment of prostate cancer. PSA has shown to be more effective than other markers in the diagnosis of prostate cancer. Following the review of 106 patients with prostate cancer in various clinical stages, PSA was higher in 76.5% and PAP only in 49%. There is also a correlation between PSA and the extension of the disease. Of 43 patients surgically (stages B, C, D1) or radiologically (stages D2) staged, increases PSA has been found in 25% B stages, 77.7% C stages and 88.4% D stages. With regard to post-treatment monitoring, PSA is highly useful to determine its effectiveness, detect any residual illness and predict a recurrence or progression. PMID- 1724348 TI - Interleukin-1-induced cartilage degradation is independent of substance P level in rabbit knees. AB - The effect of SP on recombinant human IL-1 alpha-induced cartilage degradation in rabbit knees was investigated. Co-injection of 10 ng IL-1 and 1 micrograms SP did not increase cartilage degradation above the level observed after injection of 10 ng IL-1 alone. Co-injection of 100 ng IL-1 and 150 micrograms Spantide, a potent SP antagonist, did not reduce the level of cartilage degradation. Infusion of 100 ng SP/day for two weeks did not induce cartilage degradation. These observations suggest that cartilage degradation induced by IL-1 is independent of SP concentrations in rabbit knees. PMID- 1724349 TI - Effect of capsaicin on carrageenan-induced inflammation in rat pleurisy and exudate substance P level. AB - Injection of 2.5 mg of lambda-carrageenan into the rat pleural cavity resulted in a time-dependent increase in pleural exudate substance P (SP) levels up to 24 hr. Synergistic increases in the exudate formation were observed when a sub-optimal quantity of carrageenan was injected with SP. Pre-treatment of rats with capsaicin at 50 and 100 mg/kg s.c. daily for one week prior to the induction of pleurisy blocked the increase in exudate volume and SP levels when compared to that normally detected after carrageenan injection. These results suggest that inhibition of SP production may improve inflammatory conditions. PMID- 1724350 TI - Effects of indomethacin, triamcinolone, and dexamethasone on recombinant human interleukin-1-induced substance P and prostaglandin E2 levels in rabbit knee joints. AB - Intra-articular (i.a.) injection of recombinant human interleukin-1 alpha (rHuIL 1 alpha) in rabbit knees induced both an inflammation, as determined by increases in leukocytes in the joint fluid, and cartilage degradation, as measured by loss of proteoglycan. Substance P (SP) and prostaglandin E2 (PGE2) levels in the joint lavage are also elevated. Treatment with 5 mg indomethacin/kg, p.o., b.i.d., 2 mg triamcinolone i.a., and 10 mg dexamethasone/kg, p.o., reduced synovial lavage leukocyte counts, as well as PGE2 and SP lavage concentrations induced with IL-1 injection. However, none of the treatments inhibited rHuIL-1-induced proteoglycan loss. PMID- 1724351 TI - Effects of inhaled or intravenous thiorphan on substance P-induced airway obstruction. AB - Male Hartley guinea pigs were treated with the enkephalinase inhibitor thiorphan by inhaled or intravenous administration routes and potentiation of substance P induced airway obstruction examined. Inhaled thiorphan was several thousand times more potent than intravenous thiorphan. The biologic half-life was less than 5 min for aerosolized thiorphan and greater than 20 min after intravenous administration. The very high potency of aerosolized thiorphan may be a result of its direct targeting to enkephalinase concentrated within the airway epithelium. The short duration of action of inhaled thiorphan may reflect its rapid diffusion from the airway surface. PMID- 1724352 TI - Adhesion molecules in the pathogenesis of asthma. AB - Using a primate antigen inhalation model the mechanisms involved in the induction of airway hyperresponsiveness were studied. Antigen inhalation induced a prolonged airway eosinophilia. Chronic airway eosinophilia, intratracheal instillation of eosinophil major basic protein, repeated antigen inhalation, and airway epithelial desquamation induced, or were associated with, an increase in airway responsiveness. Using monoclonal antibodies to intercellular adhesion molecule-1 (ICAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1), the contributions of these cell adhesion molecules to antigen-induced airway hyperresponsiveness were studied. ICAM-1 partially mediated eosinophil adhesion to endothelium in vitro, was upregulated on inflamed endothelium and epithelium in vitro and in vivo, and contributed to repeated antigen-induced airway eosinophilia and hyperresponsiveness. ELAM-1 was upregulated on inflamed endothelium (but not epithelium) in vitro and in vivo, but did not contribute to the repeated antigen-induced airway eosinophilia and hyperresponsiveness. These results indicate that antagonism of ICAM-1, but not ELAM-1, may provide a novel therapeutic approach to reducing the airway inflammation and hyperresponsiveness commonly found in asthmatics. PMID- 1724353 TI - sCD14 prevents endotoxin inducible oxidative burst response of human monocytes. AB - Luminol-enhanced chemiluminescence (LECL) was used to determine the effect of soluble CD14 (sCD14) on the endotoxin inducible generation of reactive oxygen species in human monocytes. LPS is unable to activate monocytes under serum free conditions, but LECL responses were measured after pretreatment of LPS stock solution with serum, according to Wright et al., who described a LPS-binding protein (LBP), necessary for mediating LPS binding to the receptor CD14 on monocyte surfaces. In normal human serum a soluble form of CD14 (sCD14) exists, from which nothing is known about its possible function. sCD14 reduces the endotoxin inducible monocyte activation in our in vitro model in a dose dependent manner (5-30 micrograms/ml) suggesting an immunomodulatory function. Therefore it seems to be a new candidate for a therapeutic concept in endotoxic shock prevention. PMID- 1724354 TI - [Epitope analysis of CD68 antigens]. AB - Different epitopes of CD68 antigen are detectable by three new generated monoclonal antibodies (MABs) BL-M68/1-3 (all mouse IgM). The MAB BL-M68/3 reacts with an epitope, which is stable also in formalin-fixed paraffin-embedded tissues. In immunohistochemical blocking experiments using the own biotinylated MABs and six CD68 MABs from the IV. Workshop on Human Leucocyte Differentiation Antigens two new epitopes could be defined. The number of known CD68 epitopes increases at least up to seven. PMID- 1724355 TI - [Our experience in treating endometrioid ovarian carcinoma]. AB - The authors describe 12 women with endometrioid ovarian carcinoma at the age of 28 to 74 years. Endometrioid carcinoma is accompanied by endometrial carcinoma in 4 women. Five patients are at I stage, 5 patients--at II stage and 1 patient--at III and IV stage. All women are operated and have total hysterectomy with bilateral adnexectomy and 7 women have omectomy and 5 are without omectomy. All are treated by postoperative telegammatherapy: 8 patients-at the region of the pelvis 1--woman-at the pelvis and para-aortal lymph chains, 2 patients--with band irradiation of the whole abdomen and 1 woman is irradiated palliatively. 7 out of 8 patients treated radically and followed over 5 years, are clinically healthy and only 1 is dead and she been at III stage. In the last case there has been mesonephroid component and it has caused probably more malignant course of the disease. An inference is made that the complex treatment of endometrioid carcinoma of the ovary--radical operation and postoperative irradiation, gives good results. PMID- 1724356 TI - Childhood Ki-1 lymphoma: presentation as a buttock mass. AB - Ki-1 lymphoma is a rare, large-cell anaplastic non-Hodgkin's lymphoma that most commonly affects older children and young adults. Presentation usually occurs as a localized infiltration of the skin and lymph nodes. We report an unusual case of childhood Ki-1 lymphoma that presented as a buttock mass in an eight-year-old girl, Pathologic evaluation revealed the characteristic lymphoma cells expressing Ki-1 antigen (CD-30), HLA-DR, interleukin 2 (CD-25), T-cell gene rearrangement, and the cytogenetic karyotype t(2;5). The patient is in complete remission following treatment with combination chemotherapy. This report broadens the clinical spectrum associated with Ki-1 lymphomas and illustrates the importance of combining routine pathologic examination with other specialized diagnostic techniques in the evaluation of childhood soft-tissue masses. PMID- 1724357 TI - Development and migration of Purkinje cells in the mouse cerebellar primordium. AB - The mode of Purkinje cell migration in the mouse cerebellar primordium was examined immunohistochemically, by marking Purkinje cells with anti-spot 35 antibody and labeling them with 5'-bromodeoxyuridine. The cells migrated radially from the neuroepithelium of the fourth ventricle towards the cortical surface between the 13th and 17th days (E13-E17) of gestation. Regional differences in the migratory process were evident: the final settlement of the Purkinje cells proceeded earlier in the lateral and posterior parts of the primordium, exhibiting latero-medial and posteroventral-anterodorsal diminishing sequences. To elucidate the factors involved in the migration, the arrangement of radial glial fibers, and expression of the cell adhesion molecule, tenascin, were examined immunohistochemically with the monoclonal antibody 1D11, a marker for both immature and mature astroglia, and an anti-tenascin antibody. At E14, 1D11 immunopositive fibers were seen to extend from the ventricle to the pial surface, and the cell bodies of immature glia migrated after E15 towards the cortex, shortening the radial processes whose end-feet were attached to the pia mater. Tenascin, which possesses a neuron-glial adhesiveness, was also expressed on the radial fibers during the migration of the Purkinje cells. The fibers were closely apposed to the migratory Purkinje cells, and their arrangement and orientation accorded with the migratory direction of the Purkinje cells. Further, changes in the molecular species of antigens detected by both the 1D11 and anti-tenascin antibodies were observed by immunoblotting analysis during the course of cerebellar development. These findings suggest that the arrangement of radial glia and expression of adhesion molecules may be involved in the control and guidance of Purkinje cell migration. PMID- 1724359 TI - Anatomical relationships between sensory afferent arborizations in the cricket cercal system. AB - The anatomical relationships between sensory afferents within a topographic map in the cricket cercal sensory system were studied using a computer-based reconstruction system developed in our laboratory. Individual identified mechanosensory afferents were characterized physiologically, stained with cobalt, silver intensified, and reconstructed in three dimensions. All reconstructions were scaled to a common standard. The results indicate that there is very little variability in the position or extent of the terminal arborization of identified mechanosensory afferents. The topographic map was divided relatively equally into four regions representing each of the four classes of afferents studied. These regions were discrete but not completely segregated. Approximately 30% of the topographic map contained regions of overlap between two or more classes or afferents. PMID- 1724360 TI - Immunoregulatory role of gamma delta T cells. AB - While the major population of T lymphocytes express T cell receptor (TCR) alpha beta-chains and recognize peptide antigens in association with either Major Histocompatibility Complex class I or class II molecules, a consensus view does not exist concerning either the nature of the antigen recognized or the nature of the restriction element utilized by the minor population of T cells which express TCR gamma delta-chains. We have identified a unique subpopulation of gamma delta T cells which uniformly express the C gamma 4, V delta 6 TCR and which produce a number of cytokines in the absence of exogenous stimulation. Adaption of these cell lines to serum-free culture conditions resulted in a cessation of cytokine production which could then be induced by the addition of extracellular matrix (ECM)-proteins to the culture. The response to the ECM-proteins could be completely inhibited by an antibody to the murine vitronectin receptor (VNR). However, engagement of the VNR by its ligand was not sufficient for the induction of cytokine production as anti-TCR antibodies inhibited the response to ECM proteins and gamma delta TCR loss mutants failed to respond. Collectively, these data demonstrate that not only is coexpression of the VNR and the gamma delta TCR required for the induction of cytokine production by this subpopulation of T cells, but that the TCR must also be engaged by its ligand, most likely a cell surface autoantigen expressed by the T cells themselves. PMID- 1724358 TI - Descending brainstem projections of the pedunculopontine tegmental nucleus in the rat. AB - Descending brainstem projections from the pedunculopontine tegmental nucleus (PPN) were studied in the rat by use of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde tracer lectin-conjugated horseradish peroxidase (HRP-WGA). Results of these experiments demonstrated prominent bilateral projections to the pontomedullary reticular nuclei, but direct connections to the motor and sensory nuclei of the cranial nerves could not be ascertained. The PPN fibers terminated mainly in the pontine reticular nuclei oralis and caudalis and in ventromedial portions (pars alpha and pars ventralis) of the gigantocellular reticular nucleus. A smaller number of labeled fibers distributed to more dorsal regions of the gigantocellular nucleus, lateral para gigantocellular, ventral reticular nucleus of the medulla and lateral reticular nucleus. Although a significant number of PHA-L labeled fibers was seen in two cases in the contralateral medial portion of the facial nucleus, and all cases exhibited a sparse predominantly ipsilateral projection to the lateral facial motor neurons, the retrograde tracing experiments have revealed that these facial afferents originated in the nuclei surrounding the PPN. The results are discussed in the context of PPN involvement in motor functions. It is suggested that the PPN may participate in a complex network involved in the orienting reflex. PMID- 1724361 TI - Prospects for immunotherapy directed to the T cell receptor in human autoimmune disease. PMID- 1724362 TI - The potential of restricted T cell recognition of myelin basic protein epitopes in the therapy of multiple sclerosis. PMID- 1724363 TI - T cell receptor gene rearrangements in the human response to myelin basic protein. PMID- 1724364 TI - Specific immunoregulation of the induction and effector stages of relapsing EAE via neuroantigen-specific tolerance induction. AB - The effects of neuroantigen-specific tolerance on the induction and effector stages of relapsing experimental autoimmune encephalomyelitis (R-EAE) were examined. The incidence of clinical and histologic signs of active MSCH-induced R EAE, and accompanying neuroantigen-specific DTH responses, were dramatically reduced in SJL/J mice tolerized via the i.v. injection of syngeneic splenocytes coupled with MSCH, PLP, or encephalitogenic PLP peptides 7-14 days before priming. MBP-specific tolerance was not effective in preventing active R-EAE. In contrast to MSCH-induced active R-EAE, treatment of recipient mice with splenocytes coupled with MBP and the encephalitogenic MBP 84-104 peptide, but not with PLP, suppressed of clinical signs of adoptive R-EAE mediated by MBP-specific effector T cells in a dose-dependent manner. Neuroantigen-coupled splenocytes were also efficient in treating established disease as tolerization of SJL/J mice after the first incidence of clinical disease significantly reduced the incidence and severity of subsequent paralytic relapses. Antigen-specific tolerance thus provides a powerful approach for the prevention and/or treatment of autoimmune disease. PMID- 1724365 TI - [Reduction of per- and postoperative blood loss with aprotinin (Trasylol) during extracorporeal circulation]. AB - Aprotinin is a pharmacological agent which, when given in high doses during cardiopulmonary bypass (CPB), seems to reduce postoperative blood loss significantly and thereby reduces the need for blood transfusion. This study was undertaken to confirm these claims and to show that there was also decreased peroperative bleeding and a shorter operation time. The immediate postoperative clinical course was also assessed. The study was a prospective, randomised double blind trial versus placebo in 60 coronary patients undergoing at least 2 aorto coronary bypass grafts for the first time within a 3 month period. During surgery after stopping the CPB the blood loss recorded by aspiration was 49 +/- 61 ml in the aprotinin group and 90 +/- 84 ml in the placebo group (p less than 0.05). The quality of haemostasis in the operated area evaluated independently by the anaesthetist was judged to be excellent in 30 patients in the aprotinin group compared with only 19 in the placebo group (p less than 0.001). The time between coming off CPB and skin closure was significantly shorter in the aprotinin group (42 +/- 10 min versus 49 +/- 12 min) and the dose of protamine injected at the end of the operation was 19 +/- 38 mg in the aprotinin group compared to 43 +/- 46 mg in the placebo group (p less than 0.05). The blood loss recorded over 48 hours in the intensive care unit was nearly three times less in the aprotinin group (380 +/- 125 ml) than with placebo (852 +/- 523 ml).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724367 TI - Nasal decongestants in the treatment of acute otitis media. PMID- 1724366 TI - [The diagnostic value of a new whole blood histamine release test using paper disc-coupled antigens--results compared with intracutaneous test, RAST, and eye test]. AB - We have developed a novel histamine release test (HR) using whole blood and antigen-coupled RAST paper discs, for the screening of allergens rather in a short time with a small amount of blood. Histamine was determined by a RIA kit. In order to evaluate the diagnostic value, the results of the test were compared with those of RAST, intracutaneous tests, and eye tests in 45 allergic patients. HR correlates better with RAST than intracutaneous test in almost all antigens. The closest positive correlation with the other tests was seen in mite allergen, followed by pollen, foods and mould spores in that order. When the HR was positive for house dust, 100% of the patients were also positive for the eye test, which is reported to closely correlate with bronchial provocation tests. HR seemed to be a useful test not only for the screening but also for the determination of pathogenic allergens. PMID- 1724368 TI - [Synthesis and immunochemical properties of oligopeptides--fragments of capsid proteins of the hepatitis A virus]. AB - The hepatitis A virus (HAV) capsid protein VP1, VP2 and VP3 are exposed at the virion surface and should therefore contain antigenic determinants. Algorithms for hydrophilicity, antigenicity and flexibility were used to predict probable antigenic sites. Synthesis of 7- to 23-membered overlapping peptides from seven sites, viz., 1-11, 1-17, 2-33, 11-25, 73-82, 76-86, 98-109, 98-112, 102-107, 102 108, 108-127, 113-123, 118-140, 276-298 from VP1, 42-62 from VP2, 76-85 from VP3, and 1-23 from VP4, was performed by various solid-phase methods. Free peptides and their conjugates with different carriers were used for immunization and study of antigenicity. The peptides did not interact with antibodies to the hepatitis A virus, whereas their conjugates did not induce the formation of anti-HAV antibodies. PMID- 1724369 TI - Surface modification of enzymes for therapeutic use: monomethoxypoly (ethylene glycol) derivatization of ribonuclease. AB - Bovine pancreatic ribonuclease A (RNase) was modified at various extent at the lysine residues by monomethoxypoly(ethylene glycol) (MPEG) activated as active ester. For pharmacokinetic experiments a radioactive adduct was also prepared with tritiated amino acid as spacer between polymer and protein. The modification reduced only slightly the RNase catalytic activity and Km towards the substrate cytidine-2',3'-cyclic monophosphate. On the other hand extensively modified MPEG RNase samples, showed significant decrease in activity towards ribonucleic acid. The polymer modification did not change the pH activity profile, increased the stability to proteolytic digestion, while the behaviour towards denaturants and heat was not modified. The native and MPEG-RNase administered IV, IM and SC to rats, showed impressive differences in pharmacokinetics: the half-life of the modified enzyme, evaluated in blood by radioactivity, was increased of 40-50 folds with respect to the native form. PMID- 1724370 TI - The E5 oncoprotein target: a 16-kDa channel-forming protein with diverse functions. PMID- 1724371 TI - v-Ha-ras-induced mouse skin papillomas exhibit aberrant expression of keratin K13 as do their 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate induced analogues. AB - Introduction of the v-Ha-ras gene into primary epidermal keratinocytes, followed by grafting of these cells to animals, leads to the formation of benign epidermal tumors that resemble papillomas induced chemically by a two-stage carcinogenesis protocol. In this study, we investigated v-Ha-ras-induced papillomas for aberrant expression of type I keratin K13, previously described in 7,12 dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13- acetate (DMBA/TPA) induced mouse epidermal tumors. Papillomas produced from three independent infection series were removed 3 wk after grafting concomitant with control grafts originating from mock-, neo-, and v-fos-infected primary keratinocytes. Combined analysis of the grafts by western blotting of extracted keratins and immunofluorescence studies of frozen sections with a K13-monospecific antibody revealed K13 expression in all v-Ha-ras-induced papillomas and absence of this keratin in all control grafts. K13-positive cells in papillomas were restricted to the suprabasal cell layers of the lesions and, at this stage of papilloma development, occurred as foci of varying extensions. Analysis of genomic DNA from v-Ha-ras-induced papillomas for the methylation state of a CpG dinucleotide in the distant promoter region of the K13 gene revealed the occurrence of unmethylated DNA copies that were generated at the expense of methylated DNA copies ubiquitously present in normal epidermis. The ratio of unmethylated to methylated DNA copies correlated with the extent of suprabasal K13 protein expression. Thus, all features of aberrant K13 expression previously described in DMBA/TPA-induced papillomas were shared by v-Ha-ras-induced papillomas. PMID- 1724372 TI - Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium. AB - Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)-beta 1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol-bis(beta aminoethyl ether) N,N,N',N',-tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot analysis of expression of TPA-inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds. PMID- 1724373 TI - Macromolecular prodrugs. IXX. Kinetics of hydrolysis of benzyl dextran carbonate ester conjugates in aqueous buffer solutions and human plasma. AB - Benzyl carbonate esters of dextran with varying degrees of substitution have been synthesized. The kinetics of the hydrolytic cleavage of the carbonate ester bond in aqueous solution within the pH range 0.44-10.46 (37 degrees C) has been investigated. The degradation reactions followed pseudo-first-order kinetics and a rate expression encompassing hydrogen ion-, hydroxide ion- and water-catalyzed hydrolysis of the dextran conjugates was derived. No influence of the degree of substitution on the reaction rates was observed. In alkaline solution a slightly enhanced lability of trifluorethyl benzyl carbonate ester compared to the benzyl dextran carbonate esters was observed, indicating a lack of any significant intramolecular catalytic effect in the hydrolysis of the dextran esters. Almost identical rates of liberation of benzyl alcohol were found in 80% human plasma and aqueous buffer of pH 7.4, indicating the lack of enzyme-mediated cleavage of the dextran carbonate ester bond. PMID- 1724374 TI - Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors. AB - Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159 170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized. PMID- 1724375 TI - An experimental test of a model for repeated Ca2+ spikes in osteoblastic cells. AB - A model for cytosolic Ca2+ spikes is presented that incorporates continual influx of Ca2+, uptake into an intracellular compartment, and Ca(2+)-induced Ca2+ release from the compartment. Two versions are used. In one, release is controlled by explicit thresholds, while in the other, release is a continuous function of cytosolic and compartmental [Ca2+]. Some model predictions are as follows. Starting with low Ca2+ influx and no spikes: (1) induction of spiking when Ca2+ influx is increased. Starting with spikes: (2) increase in magnitude and decrease in frequency when influx is reduced; (3) inhibition of spiking if influx is greatly reduced; (4) decrease in the root-mean-square value when influx is increased; and (5) elimination of spiking if influx is greatly increased. Since there is good evidence that hyperpolarizing spikes reflect cytosolic Ca2+ spikes, we used electrophysiological measurements to test the model. Each model prediction was confirmed by experiments in which Ca2+ influx was manipulated. However, the original spike activity tended to return within 5-30 min, indicating a cellular resetting process. PMID- 1724376 TI - Peroxisomal disorders. AB - The concept that there are human disease states that are associated with abnormal peroxisomal function is of recent origin. This is due in part to the relatively recent discovery of the organelle itself by de Duve in 1983, and to the earlier belief that it was a vestigial structure in mammals. The recognition that the organelle is significant in mammals was ushered in by Paul Lazarow's observation that rat peroxisomes catalyze the beta-oxidation of fatty acids. By 1981, more than 40 enzymes had been localized to the peroxisome, and the number continues to grow. Respect for the physiological role of the peroxisome in man has been heightened by our recent recognition that peroxisome malfunction causes profound disturbances. The Zellweger cerebro-hepato-renal syndrome represents the most serious peroxisomal disease. It is associated with malfunction of virtually every organ, and children with the disease usually do not survive beyond the 4th month. Application of newly developed diagnostic techniques has shown that the clinical spectrum and frequency of peroxisomal disorders are greater than had been realized. Eleven separate peroxisomal disorders have now been identified. Our laboratory alone has identified more than 2000 patients. Disturbances of very long chain fatty acid and ether phospholipid metabolism are present in 9 of the 11 peroxisomal disorders. In this presentation, we will provide an overview of the peroxisomal disorders, with emphasis on disturbances of fatty acid and ether lipid metabolism. PMID- 1724377 TI - A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA. AB - To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein RNA complexes on DEAE-Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine rich sequence at positions 136-144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113-120; they are suggested to be involved in interaction with signal recognition particle RNA. PMID- 1724378 TI - Improved detection of in situ hybridization by laser scanning confocal microscopy. AB - Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques. PMID- 1724379 TI - Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head. AB - ZP3, a glycoprotein of the murine zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining, when incubated with rabbit antiinhibitor antiserum (anti-SVI). The percentage of SVI-treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti-SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti-SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface, in the absence of zona glycoproteins, is sufficient to induce the acrosome reaction. PMID- 1724380 TI - The clinical significance of serum CD8 antigen levels in adult patients with Hodgkin's disease. AB - Increased suppressor T-cell activity has been observed in patients with Hodgkin's disease. In order to evaluate the clinical significance of soluble CD8 antigen (sCD8), which is released from CD8+ suppressor/cytotoxic T-lymphocytes, we determined sCD8 levels in the sera of 82 consecutive patients with newly diagnosed untreated Hodgkin's lymphoma who were entered into prospective trials of the German Hodgkin's Disease Study Group. sCD8 levels were significantly higher (p less than 0.01) in stage IV (781 U/ml, n = 19) than in stages I-IIIB (443 U/ml; n = 63). Patients with B-symptoms (n = 36) had slightly higher levels (611 U/ml) than patients without (n = 46) systemic symptoms (447 U/ml; p = 0.08). In 77 patients evaluable for response, the complete remission (CR) rate of patients with sCD8 less than 750 U/ml was higher (54/60 or 90%) than that of patients with sCD8 greater than 750 U/ml 11/17 or 65%; p = 0.01). The time to treatment failure was significantly longer in patients with sCD8 less than 750 U/ml (p = 0.008), even among the group with stages IIIB/IV only (p = 0.04). Our data suggest that the pretreatment levels of sCD8 in adult patients with Hodgkin's lymphoma have prognostic relevance, and that they should be determined especially in patients with advanced disease. Increased understanding of the role of sCD8 may shed light on the pathogenesis of Hodgkin's disease. PMID- 1724382 TI - Signal transduction by the SRC family of tyrosine protein kinases in hemopoietic cells. PMID- 1724381 TI - Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24. AB - Human mast cell growth factor (MGF) complementary DNAs (cDNAs) were cloned from HeLa cells using the polymerase chain reaction with oligonucleotides corresponding to murine and human MGF sequences. Sequencing of the cloned human MGF polymerase chain reaction products revealed two types of cDNA: a full length form corresponding in size to the murine cDNA, and an alternately spliced clone with a deletion of the sixth exon of the gene. Since membrane-bound MGF is predicted to be proteolytically cleaved within the sequences encoded by exon 6 to generate a soluble protein, this alternately spliced cDNA would likely encode a noncleavable, membrane-bound form of MGF. No difference in biological activity on human bone marrow cells was observed with recombinant, soluble forms of both types of human MGF protein. Our previous localization of the murine MGF gene to the Sl locus on chromosome 10 suggested (via conserved linkage groups) that the human MGF gene would be located on human chromosome 12. Therefore, rodent-human somatic cell hybrids with or without an entire human chromosome 12 and hybrids retaining partial 12 were tested by Southern blot analysis and used to show the presence of the human Mgf locus at chromosome region 12q. Chromosomal in situ hybridization localized the gene to 12q22-q24 in the region predicted by the comparative mapping of the murine Mgf/Sl locus. PMID- 1724384 TI - Invasion of visual cortex by the auditory system in the naturally blind mole rat. AB - Previously we have shown that the dorsal lateral geniculate body (LGB), which is strictly visual in sighted mammals, receives a strong auditory input in the naturally blind mole rat (Spalax ehrenbergi). Here we show with the 2 deoxyglucose technique and with single-unit recordings that in this species the initially non-degenerated visual cortex, as defined by its connection with LGB, is also activated by the auditory modality. These findings suggest that cross modal compensation may occur as a natural consequence of the degeneration of a sense organ. PMID- 1724383 TI - Cryopreserved primate bone marrow cells can be used for retroviral-mediated gene transfer. AB - Retroviral-mediated gene transfer into Rhesus monkey bone marrow cells, which were cryopreserved, stored, and then transduced at the time of thawing, was studied for potential application in gene therapy protocols. Albumin density gradient fractionation was used to define subpopulations of cryopreserved cells transduced by a murine retroviral vector. The transfer of the bacterial neomycin phosphotransferase gene into Rhesus monkey bone marrow that was cryopreserved and thawed was found to be preferential in a light-density population enriched for granulocyte-macrophage colony-forming units (CFU-GM). These results are similar to results obtained with freshly harvested bone marrow in which this population is enriched for CD34 antigen-positive cells, as well as for cells that were undergoing cell division. This method may be useful in enriching for transduced precursors in future gene transfer experiments in primates. PMID- 1724385 TI - Substance P immunoreactivity in the superficial laminae of the hamster olfactory bulb. AB - The direct application of colchicine to the hamster olfactory bulb has engendered the appearance of a broader distribution of substance P-like immunoreactivity (SP LI) than has been previously reported. In the glomerular layer, both external tufted and periglomerular cells were strongly immunopositive. These two classes could be identified by somal size and the presence and branching pattern of intraglomerular dendrites. SP-LI neurons were also identified in the external plexiform layer and many were similar to middle tufted cells. These findings demonstrate that substance P expression is not uniquely associated with classes of projection neurons, but is also found in periglomerular cells, a class of inhibitory local circuit neurons. PMID- 1724386 TI - Biocytin: intracellular staining, dye-coupling and immunocytochemistry in carp retina. AB - Correlation of electrophysiological and morphological, including ultrastructural, characteristics of neurones is important for understanding the functional organization of neuronal systems. Further correlation with neurotransmitter content is essential for determining the neurochemical(s) used by a given neurone for propagating its signal. The two main neuronal markers presently available (lucifer yellow and horseradish peroxidase) are not satisfactory for correlating all three aspects. We have devised a new simple procedure whereby retinal interneurones can be labelled with biocytin by positive ionophoresis of an unbuffered solution. Biocytin readily crosses gap junctions thus revealing extensive networks of coupled cells. In the case of H1 horizontal cells, which are known to be GABAergic, the neurotransmitter can also be demonstrated by superimposed immunocytochemistry. PMID- 1724387 TI - Expression of mRNA for Gs alpha and Gi2 alpha in primary cultured mouse cerebral cortical neurons. AB - Developmental changes of mRNAs for alpha-subunits of GTP binding protein (G protein) such as Gs alpha and Gi2 alpha during neuronal development were examined in both primary cultured cerebral cortical neurons and cerebral cortices obtained from age-matched mice. The expression of mRNAs for both G-protein alpha-subunits in primary cultured neurons showed a development pattern similar to that in the cerebral cortex in vivo. Both mRNAs were expressed at an early stage of neuronal development, and the expression patterns of mRNA for both G-protein alpha subunits were found to be only slightly changed during development. These results suggest that the G-protein appears at an early stage of neuronal development and may play an important role therein. PMID- 1724388 TI - Lesions of inferior temporal area TE in infant monkeys alter cortico-amygdalar projections. AB - When inferior temporal area TE is removed bilaterally in infant monkeys, the normally transient projection from area TEO to the lateral basal nucleus of the amygdala is maintained, and the normally limited projection from area TEO to the dorsal part of the lateral nucleus of the amygdala expands to invade the terminal space in the lateral nucleus that is normally occupied by terminals from area TE. The maintenance and sprouting of these projections from area TEO could play a role in the permanent preservation of visual memory ability in monkeys that have received bilateral removal of area TE in infancy. PMID- 1724389 TI - Effects of recombinant human granulocyte colony-stimulating factor on neutrophil function in normal rats. AB - We studied the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on neutrophil functions in vitro using neutrophils isolated from the venous blood of normal rats. FMLP-induced superoxide anion (O2-) release, phagocytosis, and FMLP-induced chemotaxis were evaluated. These functions were significantly enhanced by rG-CSF treatment. In addition to performing neutrophil function assays, we evaluated FMLP binding to rat neutrophils after rG-CSF treatment. FMLP specific binding was not changed by rG-CSF treatment. In addition, we intravenously injected rG-CSF (10 micrograms/kg) or control vehicle into rats for 7 consecutive days, and evaluated the functions of neutrophils isolated from venous blood at 6 h after the final injection. The neutrophil count in the peripheral blood of rG-CSF-treated rats was increased significantly compared with that in control rats. FMLP-induced O2- release, phagocytosis, FMLP induced chemotaxis and spontaneous migration of rG-CSF-treated neutrophils were significantly enhanced in comparison with those in control rats. These findings demonstrate that rG-CSF not only increases neutrophil counts in peripheral blood, but that it also enhances neutrophil functions, both in vitro and in vivo. PMID- 1724390 TI - In vivo effects of recombinant human granulocyte colony-stimulating factor on normal neutrophil function and membrane effector molecule expression. AB - Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is now undergoing clinical trials. We investigated the effects of rhG-CSF on the function of neutrophils in vivo in healthy volunteers. rhG-CSF (0.5 micrograms/kg) was injected subcutaneously for 6 consecutive days. The number of neutrophils in peripheral blood decreased transiently within an hour, and thereafter increased 2-10-fold compared to the control 6 to 8 h after injection. The circulating neutrophils remaining during this early neutropenic period showed increases in such functions as random motility, chemotaxis, phagocytosis and superoxide anion production. On the other hand, the function of neutrophils which increased 6 to 8 h after rhG-CSF injection was normal. No decrease of neutrophil function was observed following the use of rhG-CSF. CD33-positive cells increased 3 days after rhG-CSF administration. CD11a (LFA-1) expression on the membranes circulating neutrophils decreased 6 h after rhG-CSF administration. This phenomenon suggested that neutrophils adhered to the reticuloendothelial system during neutropenia, and that there was an influx of CD11a-negative mature cells into the circulatory pool thereafter. All our findings suggest that rhG-CSF enhances the function of normal neutrophils in vivo, and that it is effective against microbial infection very soon after administration. PMID- 1724391 TI - Epitope localization of monoclonal antibodies against factor VIII light chain which inhibit complex formation by factor VIII with von Willebrand factor. AB - We obtained three clones of monoclonal antibodies against factor VIII by immunization with purified human factor VIII. The anti-factor VIII procoagulant activity of these antibodies ranged from 2 to 53 Bethesda units/mg of IgG. According to an immunoblotting study, all antibodies reacted with the 80 kDa light chain but not with 72 kDa peptides derived from thrombin digestion of factor VIII. We attempted to localize the antigenic epitopes of these antibodies by a competitive blocking assay using synthetic peptides and recombinant fragments of the amino-terminal region of factor VIII light chain. In the former assay, a 50 microM peptide containing the fifteen amino acid residues from the Val1670-Glu1684 completely inhibited the binding of the three monoclonal antibodies to immobilized factor VIII. In the latter experiment, 13 reactive recombinant peptides were obtained. Sequences of these peptides revealed fourteen overlapped amino acid residues from Glu1675 to Pro1688. All three antibodies at a final concentration of around 10 micrograms/ml completely inhibited the binding of 125I-labelled factor VIII to immobilized von Willebrand factor (vWF). We conclude that ten amino acid residues, 1675EDFDIYDEDE1684 in the factor VIII light chain are important for complex formation with vWF. PMID- 1724392 TI - Changes in polypeptide-specific factor VIII antigen levels during whole blood clotting. AB - The time-course of factor VIII (FVIII) proteolysis during whole blood coagulation was monitored using polypeptide-specific enzyme-linked immunosorbent assays (ELISAs) developed with monoclonal antibodies to FVIII. Such assays of whole blood from normal subjects revealed that, within 30 min after coagulation had started, levels of the amino-terminal region of FVIII light chain and in the middle region of FVIII heavy chain had decreased to baseline in parallel with factor VIII procoagulant activity, but that levels of the 70 kDa thrombin digest decreased more slowly, with 75 U/dl of FVIII antigen (FVIII:Ag) remaining even after 2 h. Similar analysis of the blood of a patient with congenital hypoprothrombinemia indicated a lag phase of 20 min before proteolysis started. No significant change was observed until 1 h after calcium chloride was added to prothrombin-depleted plasma. On the other hand, in the presence of tissue thromboplastin, levels of FVIII:Ag in all ELISAs decreased rapidly. These results indicate a requirement for thrombin generation in the proteolysis of FVIII during the process of whole blood clotting. PMID- 1724393 TI - [Post-traumatic microangiopathies]. PMID- 1724394 TI - [Reparative effect of nucleic acid preparations in experimental stomach ulcer]. PMID- 1724395 TI - [Mediators and their antagonists in shock therapy]. AB - The list of shock mediators currently comprises more than 150 candidates. A careful analysis using the criteria of Koch-Dale together with decision trees for exclusion of bias revealed that only histamine, C5a, beta-endorphin, tumor necrosis factor (TNF) thromboxane B2, platelet-activating factor (PAF), and oxygen free radicals are shown to be causally associated with shock symptoms. Although experimental studies with inhibitors of these mediators were convincing, there is still a lack of evidence under clinical conditions (exception histamine: anaphylactic shock). Combinations of antagonists against different causal mediators are the most promising future approaches. PMID- 1724396 TI - [Percutaneous transhepatic cholangioscopy for gallstone therapy and tumor diagnosis]. AB - Using percutaneous-transhepatic cholangioscopy with a guided small calibre, cholangioscope (3.5 mm) in patients with obstructive jaundice it is possible to perform biopsies under visual control or to look for the exact intraductal extension of a tumor with only little stain on the patient. The access may be via a puncture of the right or the left hepatic duct. Since 1987 we have used this method 61 times in 45 patients. Some 32 patients suffered from malignancy of the bile duct. Of 13 patients with benign obstruction we performed a bougienage in 8. The other 5 patients were treated by stone extraction. PMID- 1724397 TI - Substance P: immunocytochemical localization and biological role in hamster gonads during ontogenesis. AB - The cellular localization of substance P immunoreactivity was demonstrated at light microscopy level in hamster gonads during foetal and postnatal development. Selective immunostaining was observed in both the foetal and the adult generation of Leydig cells, but only in the luteal and interstitial cells of the adult ovary. To date there have been few studies dealing with the potential role of substance P in the control of gonadal functions. To examine whether it exerts an effect on gonadal steroidogenesis, the gonads of newborn, prepuberal and adult hamsters were cultured in vitro in the presence of substance P (10(-7) M), HCG (5 IU/ml) or substance P + HCG. Production of the sex steroid hormones testosterone, oestradiol-17 beta and progesterone was measured by radioimmunoassay. Substance P was observed to have a modulatory effect on steroid production by foetal, immature and mature gonads of both sexes. PMID- 1724399 TI - Reverse glycobiology: the LEC-CAMs and their carbohydrate ligands. PMID- 1724398 TI - Current concepts in the treatment of genitourinary tract disorders in the older individual. AB - Genitourinary problems, including neurogenic dysfunction, impotence, prostatism, urinary tract infections, and prostate cancer, are common in the elderly, and most of the symptoms can be alleviated through pharmacological management. Patients with neurogenic dysfunction who present with symptoms such as incontinence and urinary retention can be appropriately managed with bladder and sphincter relaxants or stimulants. Anticholinergic agents in the form of oxybutynin, flavoxate, and propantheline are effective bladder relaxants, and phenoxybenzamine, prazosin, and terazosin are commonly used as sphincter relaxants. Bethanechol chloride is the agent most commonly used to stimulate bladder contraction, but physicians should be careful when prescribing it for elderly patients with cardiovascular problems. Organic and psychogenic causes of impotence usually overlap, and oral agents have limited use in the treatment process. The use of yohimbine has increased recently, but its value and rate of success remains questionable. Testosterone is being used widely to treat impotence, but it is only helpful to patients with hypogonadism and should be used with discretion in the elderly, who have a high incidence of prostate cancer. Vasoactive intracavernous pharmacotherapy, on the other hand, is a recently discovered alternative to testosterone with promising results. Although the treatment of choice for benign prostatic hypertrophy is surgery, there have been important pharmacological advances in treating this disorder. alpha Adrenergic antagonists and anti-androgenic agents have been found to relieve the symptoms of prostatic enlargement. The use of chemotherapeutic and antibiotic agents to treat and suppress acute and chronic urinary tract infections is reviewed; these are second only to pulmonary infections as the most frequent cause of febrile episodes in patients over the age of 65. Lower urinary tract infections can be treated with almost any antibacterial agent. Upper urinary tract infections require full genitourinary evaluation and appropriate antibiotics should be used according to the urine culture sensitivity studies. With the advent of new hormonal agents, more choices are now available for the management of prostate cancer, which is the second most common malignancy in men. Diethylstilbestrol (stilboestrol), an oral estrogen, remains a commonly used agent to achieve castrate levels of androgens in advanced prostatic carcinoma. Agonist analogues, such as goserelin and leuprorelin, of gonadotrophin-releasing hormone (GnRH) [luteinising hormone-releasing hormone (LHRH); or gonadorelin] achieve the same results as diethylstilbestrol but without the cardiovascular side effects. Antiandrogens are also being used in combination with GnRH agonists to produce complete androgen blockage, with mixed results. PMID- 1724401 TI - Nucleolar changes in human embryo during the pre-implantation stage. Activation of ribosomal genes during the nucleologenesis. AB - We studied the structural and functional organization of human embryo nucleoli during the pre-implantation stage from spare embryos obtained by in vitro fertilization. In human embryo nucleoli, structural modifications occur during the first cleavages. They lead to the constitution of a reticulated nucleolus from an initial structure called primary nucleolus. They appear during the third cleavage and correspond to the nucleogenesis. Autoradiography shows no transcription in the primary nucleolus. Transcription of rDNA starts on the periphery of the initial structure after the 4-cell stage. It corresponds to the beginning of the embryonic gene expression. The entire nucleolus will be progressively concerned by this transcription during the reticulation. Silver staining at the ultrastructural level shows that Ag-NOR proteins are missing in the primary nucleolus. In the beginning of nucleogenesis, Ag-NOR proteins are first located on the nucleolar periphery. Their following distribution corresponds to the structures containing rDNA in the nucleolus. Nucleologenesis in the human embryo during the pre-implantation stage first requires an association of rDNA with a primary structure, then an activation of ribosomal genes. PMID- 1724400 TI - An ultrahigh-sulphur keratin gene of the human hair cuticle is located at 11q13 and cross-hybridizes with sequences at 11p15. AB - A human hair cuticle ultrahigh-sulphur keratin Q (UHSK) gene (KRN1) has been mapped by Southern analysis of a somatic cell hybrid panel and by in situ hybridization. A probe containing the coding region of this gene mapped to 11pter greater than 11q21 using the hybrid cell panel and on in situ hybridization mapped to two regions on chromosome 11: the distal part of 11p15, most likely 11p15.5, and the distal part of 11q13, most likely 11q13.5. A probe from the 3' noncoding region of KRN1 mapped to 11q13.5 indicating that this was the map location of the cloned gene. The sequence of 11p15.5 is termed KRN1-like (KRN1L). The results reveal that the cuticle UHSK gene family is clustered in the human genome. PMID- 1724402 TI - Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays. AB - Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis. PMID- 1724403 TI - Chemical activation of nociceptive peripheral neurones. AB - In inflammation, non-neuronal cells produce a variety of chemical mediators that act on nociceptive neurones. Ultimately, the discharge of these neurones is controlled by the activity of membrane ion channels. Some chemical mediators (e.g. ATP, protons, 5-hydroxytryptamine) act on receptors that are linked directly to ion channels. Other mediators (e.g. bradykinin) act indirectly through receptors linked to second messenger systems and in this way modulate the activity of ion channels and either activate or sensitize the neurones. The eicosanoids, which are produced by a variety of cell types, have important intra- and inter-cellular roles in nociception. The interactions between neurones and non-neuronal cells are likely to be complex as some types of non-neuronal cells express receptors for sensory neuropeptides (substance P). Recent studies also suggest that cytokines and growth factors can have long term effects on nociceptive neurone function. PMID- 1724404 TI - Opioid-responsive and opioid-non-responsive pain in cancer. AB - Cancer pain in general responds in a predictable way to analgesic drugs and drug therapy is the mainstay of treatment, successfully controlling pain in 70-90% of patients. The two major problem areas are pain associated with nerve damage, and 'incident' (movement-related) bone pain. Nerve damage pain tends not to respond well to morphine or other opioids. The difficulty with severe incident pain is that if the dose of opioid is titrated sufficiently to relieve the pain on weight bearing or on movement and is then given regularly at this level, it is too much for the patient at rest. The patient may then experience excessive side-effects at rest, but still have pain on movement. Other examples of pain which may be resistant to treatment with opioid analgesics are bladder and rectal tenesmus, pancreatic pain, and pain associated with decubitus ulcers or other superficial ulcers subjected to pressure or shearing forces. Management of non-opioid responsive pain may include a variety of treatments involving adjuvant analgesic drugs and non-drug measures. PMID- 1724405 TI - Immunology of postviral fatigue syndrome. AB - Postviral fatigue syndrome is associated with persistent infection by a virus. The patient with the condition has failed to eliminate the virus in the usual time. There is little evidence of a deficient immune response by the patient as the explanation for the viral persistence, and it must be assumed that most of the explanation lies in down-regulation of virus expression in infected cells. The general symptomatology of postinfectious syndromes may be mediated by cytokines liberated as part of the infection. Part of the syndrome may also be due to local effects of virus infection in muscles or the central nervous system (CNS). PMID- 1724406 TI - Somatostatin-like immunoreactivity in the region of the prepacemaker nucleus in weakly electric knifefish, Eigenmannia: a quantitative analysis. AB - The diencephalic prepacemaker nucleus (PPn) is a bilateral cluster of neurons that controls frequency modulations of the otherwise very regular electric organ discharge cycle in weakly electric knifefish, Eigenmannia. Ultrastructural evidence had suggested that the action of excitatory and inhibitory synapses contacting PPn neurons is modulated by neuropeptides. In this investigation, we examined the distribution of somatostatin-like immunoreactive (S-IR) structures in the region of the PPn. In transverse sections, we found 5 bilateral S-IR structures in the region of the PPn: a 'dorsohorizontal stripe'; a 'lateral cell group'; a 'diagonal stripe'; a 'tubercular stripe'; and a 'hypothalamic stripe'. The dorsohorizontal stripe, consisting of S-IR fibers, terminals, and roughly 200 cell bodies unilaterally, stretches from the edge of the third ventricle at the thalamic dorsal-posterior nucleus and the central posterior nucleus approximately 400 microns laterally to the PPn. S-IR cell bodies show a distinct pattern of distribution within this stripe. Almost half of the somata are located within 50 microns of the ventricle. These ventricular cells are small and often densely clustered. Laterally, towards the PPn, the number of labelled cells decreases, whereas their size gradually increases. The lateral cell group consists of roughly 20 somata in the medial region of the subelectrosensorius nucleus. Fibers of the diagonal stripe travel from the hypothalamus dorsalis to the PPn. Fibers of the tubercular stripe originate from S-IR cell bodies in the medial zone of the periventricular nucleus of the posterior tuberculum and merge with the diagonal stripe. Fibers of the hypothalamic stripe connect the hypothalamus lateralis and the diagonal stripe. The density of immunolabelling in the hypothalamic stripe is significantly higher in mature than in immature females. PMID- 1724407 TI - Cytomegalovirus infection of the developing brain alters catecholamine and indoleamine metabolism. AB - Tissue concentrations of noradrenaline (NA), serotonin (5-HT), dopamine (DA) and selected metabolites were measured in the spinal cord, cerebellum, cerebral cortex and caudate-putamen of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. MCMV-infected animals exhibited transient signs of neurological impairment, including apparent hypertonicity of hindlimb extensors and abnormal gait, beginning on days 14-16 and continuing for 3-5 days. At the onset of neurological impairment, tissue concentrations of NA were significantly reduced in the spinal cord (20%), cerebellum (32%) and cerebral cortex (40%) of infected animals. Levels of 5-HT were significantly increased in the caudate-putamen (50%), while 5 hydroxyindoleacetic acid (5-HIAA) was increased in both the spinal cord (94%) and caudate-putamen (65%). The ratio of 5-HIAA/5-HT, which is frequently used as an estimate of turnover of 5-HT, was significantly increased in the spinal cord (90%) at the onset of neurological impairment. In the caudate-putamen of MCMV infected animals, there were significant increases in the tissue levels of DA (37%), homovanillic acid (HVA, 41%) and 3,4-dihydroxyphenylacetic acid (DOPAC, 34%). All neurochemical parameters were normal in the MCMV-infected animals by postnatal day 70, approximately 50 days after the resolution of neurological signs. These results indicate transient alterations in monoamine metabolism in the developing nervous system during the pathogenesis of cytomegalovirus-induced movement and postural disorders. PMID- 1724408 TI - Facilitatory role of calcitonin gene-related peptide (CGRP) on excitation induced by substance P (SP) and noxious stimuli in rat spinal dorsal horn neurons. An iontophoretic study in vivo. AB - The effects of iontophoretically applied calcitonin gene-related peptide (CGRP) and/or substance P (SP) onto physiologically identified neurons in the rat lumbar dorsal horn were studied. The consistent excitatory effects of SP found on the spontaneous and the noxious evoked activity were significantly potentiated by CGRP application. This peptide facilitated the noxious evoked activity and did not affect the spontaneous activity of the neurons. The responses to non-noxious stimuli were unaffected in all cases. PMID- 1724409 TI - Glutamate uptake in primary cultures of biliary epithelial cells from normal rat liver. AB - Biliary epithelial cells (BEC) were isolated from normal rat liver with high purity (greater than 95%) as revealed by morphological criteria as well as staining for gamma-glutamyl transferase and cytokeratin 19. During cultivation for 96 hr flattening of the cells and a loss of microvilli was apparent, while the cytokeratin 19-positive phenotype was maintained. The BEC contained a sodium dependent as well as a sodium-independent uptake system for glutamate with high capacity. Both activities increased transiently during cultivation peaking after 72 and 48 hr, respectively. After 72 hr, apparent kinetic constants could be calculated for the sodium dependent (Km = 13.6 mM; Vmax = 388 nmoles/min/mg protein) and for the sodium-independent system. (Km = 10.8 mM; Vmax = 132 nmoles/min/mg protein). The transient increase of both transport systems was suppressed by dexamethasone. The sodium-dependence showed a threshold concentration of about 35 mM sodium. Inhibition by kainate was much less potent for BEC than for hepatocytes. These data indicate that BEC contain transport systems for glutamate different from those in hepatocytes and which may be involved in the intrahepatic reabsorption of glutamate from bile. PMID- 1724410 TI - Microcarrier-attached rat hepatocytes as a xenobiotic-metabolizing system in cocultures. AB - A method for the primary culture of rat liver cells on collagen-coated dextran microcarriers is described. Ethoxycoumarin deethylase (EOD) activity 24 hr after inoculation was comparable for liver cells cultured on microcarriers and on collagen-coated dishes. Cells were cultured on microcarriers for up to 48 hr and retained 25% of the initial EOD-activity that was seen in freshly isolated liver cells. Microcarrier-attached hepatocytes were cocultured with BALB/c 3T3 cells to study the metabolism-mediated cytotoxicity of cyclophosphamide (CPA). In the absence of hepatocytes, growth of 3T3 cells was not affected by CPA at concentrations up to 3600 microM. In coculture with hepatocytes, cytotoxicity of CPA was expressed in a time- and concentration-dependent manner. At high concentrations, CPA slightly depressed the EOD-activity of hepatocytes. Our results indicate that cocultivation of microcarrier-attached rat liver cells with target cells represents a valuable approach to the study of the metabolism mediated toxicity of xenobiotics in vitro. PMID- 1724411 TI - Surgical treatment and long-term neurodevelopmental outcome for infants with idiopathic aqueductal stenosis. AB - Evaluation of in utero shunting for fetal ventriculomegaly requires an analysis of the ex utero treatment of a comparison population of infants with idiopathic aqueductal stenosis (IAS). In this study, 14 neonates with IAS were followed with detailed developmental assessment profiles for 18 months or longer. Using magnetic resonance imaging, the preoperative and postoperative frontal cortical mantle widths (FCMW) were determined for each patient. Four of 14 children demonstrated normal outcomes while 5 of 14 children showed abnormal outcomes. The remaining 5 children demonstrated minimally impaired outcomes. No child with a postoperative FCMW below 30 mm showed normal development on any scale, while all children with abnormal development demonstrated a postoperative FCMW of 21 mm or less. In conclusion, the prognosis for normal long-term neurodevelopment in infants with IAS, despite prompt ex utero treatment, is guarded; the majority of such children will show developmental delays of varying degrees. The FCMW serves as a reasonable prognostic indicator in this patient population. PMID- 1724413 TI - [Selection of methods for HIV screening of donors' sera and blood products]. AB - Blood and blood products are very important agents of HIV transmission. In this paper, 11,251 samples from 10,422 donors' sera and 150 blood products were screened for anti-HIV by ELISA, PA, IF and WB. The results were all negative. The donors lived in the 10 municipalities and counties of Sichuan Province. We also performed a prospective investigation to learn if these four methods were suitable to different blood products. We found that positive or weak positive reactions of ten occurred when gamma-globulins were detected by IF and ELISA, but the results were negative when gamma-globulins were tested by PA and WB. As a screening assay for anti-HIV, PA is most suitable for gamma-globulin. WB assay is the optimal method for anti-HIV testing in terms of both specificity and sensitivity. However, because the necessary reagents are very expensive and the procedure is time-consuming, we suggest that the WB assay may be used as a confirmatory test for anti-HIV assay in countries and regions where its general use is cost-prohibitive. PMID- 1724412 TI - Cytokeratin expression in a congenital multipotential primitive neuroectodermal tumor. AB - A case of an uncommon congenital primitive neuroectodermal cerebellar tumor (PNET) in a 5-month-old child is reported. After subtotal surgical resection, the residual tumor did not respond to radiation and chemotherapy. Histologically, the tumor was composed of small, round, undifferentiated cells and several other patterns like astrocytomatous, oligodendrogliomatous, and ependymomatous structures. Immunostaining was positive for most of the cells for vimentin and S 100, fewer were positive for glial fibrillary acid protein (GFAP) and neuron specific enolase, and only a few for synaptophysin. Surprisingly, the tumor showed strong expression of several monoclonal cytokeratins (CK) with different molecular weights, together with epithelial membrane antigen. Furthermore, we found a coexpression of the tumor cells for CK and vimentin, while CK-GFAP and CK S 100 were negative. Ultrastructurally, intracytoplasmic intermediate filaments could be observed corresponding to immunohistochemical CK expression. The very strong CK and vimentin expression in this case was interpreted as a sign of the embryonic nature of the tumor. PMID- 1724414 TI - [Bioassay for plasma cholecystokinin bioactivity]. AB - A sensitive and specific bioassay for the measurement of plasma cholecystokinin (CCK) is reported. CCK was extracted from plasma by SEP-PAK cartridges and the extracts were measured for CCK levels based on their ability to stimulate amylase release from dispersed rat pancreatic acini. Plasma CCK levels as low as 0.86 pmol/L (CCK-8 equivalent) were detectable. Gastrin in physiological concentration did not affect the bioassay. The amylase release stimulated by plasma extracts was abolished by Bt2-cGMP, a specific CCK receptor antagonist, indicating that there might be no secretin or VIP in plasma extracts. With this bioassay, fasting and postprandial plasma CCK levels of 11 normal volunteers were determined. The fasting level of CCK was 0.95 +/- 0.22 pmol/L (mean +/- SE). It increased to 2.98 +/- 0.67 pmol/L 15 minutes after feeding and remained significantly higher than the fasting level during a 60-minute period. PMID- 1724415 TI - Eosinophilic granuloma of bone and biochemical demonstration of 49-kDa CD1a molecule expression by Langerhans-cell histiocytosis. AB - Histiocytic cells infiltrating the lesions in eosinophilic granuloma of bone as well as in cutaneous histiocytosis X were studied using a murine monoclonal antibody (MA) produced with proliferating cells from an eosinophilic granuloma of bone. This MA reacts with Langerhans cells (LC) of normal human skin or mucous membranes and with proliferating cells of eosinophilic granuloma of bone and skin lesions of Letter-Siwe disease, as shown by immunohistochemistry and immunogold labelling. As other murine MA's obtained after immunization with human cortical thymocytes, this MA immunoprecipitates the 49-kDa CD1a antigen found on human LC and thymic-cell surfaces but not its breakdown product after treatment with trypsin, as demonstrated by analysis of immunoelectron labelling, cytofluorometry and gel electrophoresis. This first production of a CD1a MA from an eosinophilic granuloma supports the concept of Langerhans-cell histiocytosis. PMID- 1724416 TI - 'Partial D' women with anti-D alloimmunization in pregnancy. AB - We report five women with Rh 'partial D' who were found to have anti-D during pregnancy. Two patients were category IVa, their red cells typing as Go(a+); one was category VI; and two had a 'partial Du' which could not be categorized. The maternal anti-D concentration increased during four of the eight pregnancies studied, but none reached significant levels. Three of six D-positive cord blood samples had a positive direct antiglobulin test (DAGT). Although one baby became jaundiced, no treatment was required. The policy in the Oxford Region for the management of patients with a weak expression of D is outlined. PMID- 1724417 TI - Anti-kinetochore staining for single laser, bivariate flow sorting of Indian muntjac chromosomes. AB - Flow cytometric chromosome sorting typically relies upon dual-laser, bivariate analysis after staining with two different base pair-specific dyes for resolution of chromosomes with similar DNA content. The availability of FITC-conjugated antibodies offers the possibility of single-laser bivariate analysis when combined with propidium iodide (PI) DNA staining, but requires exploitable antigenic differences between chromosomes of interest. A technique was developed for indirect immunofluorescent anti-kinetochore staining of Indian muntjac chromosomes in suspension. Primary antibody binding within permeabilized whole cells minimized centrifugation-induced loss of chromosomal integrity. Subsequent FITC-conjugated second antibody binding was not affected by concurrent PI counterstaining. Anti-kinetochore staining facilitated resolution of chromosomes No. 2 and X, which formed a doublet peak upon univariate DNA content analysis, as well as recognition of the Y2 peak which was indistinguishable from debris by univariate analysis. The technique allowed greater than 90% purification of each Indian muntjac chromosome. PMID- 1724418 TI - Tenascin Mr 220,000 isoform expression correlates with corneal cell migration. AB - The three isoforms of chicken tenascin, an extracellular matrix glycoprotein, are generated by alternatively spliced fibronectin type III domains. The resulting proteins migrate as bands of Mr 220,000 (ten220), Mr 200,000 (ten200) and Mr 190,000 (ten190) on SDS-PAGE. We describe here two monoclonal antibodies, one specific for ten220 (mAb T17) and another that recognizes all isoforms (mAb T16). These were used to examine the differential expression of isoforms during development. Most impressive is the close correlation between ten220 expression and cell migration in the embryonic cornea. Initially (stage 18), ten190/200 can be detected within the corneal epithelium and along the basement membranes of the lens and sclera. Ten220 appears within the primary stroma immediately prior to the invasion by neural-crest-derived cells. This expression is maintained during the subsequent migration of fibroblasts from the conjunctiva into the primary stroma. With the completion of migration and the marked increase in matrix synthesis by corneal fibroblasts, ten220 disappears. Ten190/200 remains in the region adjoining the endothelium, the Bowman's membrane and the adjacent stroma. The cell-migration-associated isoform is isolated from extracts of embryonic tissues as a homohexamer. Low molecular weight forms appeared absent but a new tenascin band of Mr 210,000 could be detected in brain extracts which may be a new isoform. We conclude that the synthesis of tenascin isoforms is under tight developmental control and speculate that a function of the additional domains is to facilitate cell migration. PMID- 1724419 TI - Nerve-dependent and -independent tenascin expression in the developing chick limb bud. AB - The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target. PMID- 1724420 TI - Expressing antisense P0 RNA in Schwann cells perturbs myelination. AB - Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense P0 RNA under the control of the human beta-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of P0 antisense RNA. Among the impaired cells, the majority had segregated and ensheathed individual axon but had not differentiated further. They did not express P0 but did express myelin- associated glycoprotein and galactocerebroside. A minority of partially inhibited Schwann cells were also observed that elaborated thin myelin sheaths containing variable numbers of compacted and noncompacted lamellae. These data indicate that restricting the level of P0 expression inhibits spiralling of the Schwann cell membrane and subsequent compaction. PMID- 1724421 TI - The extracellular matrix of lip wounds in fetal, neonatal and adult mice. AB - Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth. PMID- 1724422 TI - A two-dimensional gel database of rat liver proteins useful in gene regulation and drug effects studies. AB - A standard two-dimensional (2-D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2-D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol lowering drugs and a high-cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2-D gel system (the Iso-Dalt system), it can be directly related to an expanding body of work in other laboratories. PMID- 1724423 TI - Discrepant results of different tests for antibodies to hepatitis C in children with malignant diseases and in patients on renal replacement therapy. AB - The serostatus of hepatitis C in pediatric oncological patients and in individuals on renal replacement therapy was tested for circulating antibodies to the c100-3 recombinant antigen of hepatitis C. Upon prescreening, 12 of 82 patients in the pediatric oncological group, 6 of 108 renal transplant recipients, and 17 of 150 patients on chronic intermittent hemodialysis were repeatedly positive. Further testing of these 35 sera by supplemental test assays revealed conflicting data, mostly in the pediatric oncological group and in renal transplant recipients. Only in 10 sera were identical results obtained, suggesting that positive test results in some groups at risk have only a low predictive value. PMID- 1724424 TI - Hypothermia augments non-cholinergic neuronal bronchoconstriction in pithed guinea-pigs. AB - Electrical stimulation at C4-C7 in the spinal canal of pithed guinea-pigs injected with atropine, d-tubocurarine and pentolinium caused frequency-dependent bronchoconstriction. Such non-cholinergic responses to electrical stimulation, unlike responses to substance P, were abolished by pretreatment with capsaicin but not by mepyramine or propranolol. Bronchoconstrictor responses to electrical stimulation were inversely related to rectal temperature (between 30-40 degrees C) whereas responses to substance P increased with increasing temperature over the same range. Ouabain (i.v.) augmented responses to electrical stimulation at 35-37 degrees C but depressed those at 30-32 degrees C. Both morphine and the alpha 2-adrenoceptor agonist B-HT920 (i.v.) inhibited non-cholinergic-mediated bronchoconstrictor responses at 30-32 degrees C. These results stress the importance of adequate control of body temperature in this preparation. Lowered body temperature may increase neuronal output of neuropeptides whilst depressing bronchial smooth muscle sensitivity. The data support previous conclusions regarding the role of Na+/K+ activated ATPase in temperature-induced changes in sensitivity to bronchoconstrictor stimuli. PMID- 1724425 TI - Sodium pump activity and contractile effect of ouabain in human placental veins. AB - The aim of the present study was to determine the number and affinity of [3H]ouabain binding sites, the sodium pump activity and the mechanisms involved in the contractile effects of ouabain in human placental veins. Scatchard analysis suggested the existence of a single population of binding sites with a KD of 196.7 nM and a Bmax of 1606 fmol/mg protein. The sodium pump activity was determined from the 86Rb+ uptake, which was reduced concentration dependently by ouabain (10(-8)-10(-4) M), and from the K+ (7.5 mM)-induced relaxation in veins preincubated in a K(+)-free medium and precontracted with PGF2 alpha (10(-6) M), which was also blocked by the glycoside (10(-6) M). Ouabain (10(-7)-10(-4) M) induced concentration-dependent contractions, which were not modified by either nifedipine or Bay K 8644 (10(-7) and 10(-6) M). Ca2+ omission from the medium or amiloride (10(-4) M) inhibited these contractions, whereas monensin (10(-6) M) potentiated them. These data indicate that human placental veins possess sodium pump activity and that its inhibition by ouabain induces potent contractions mainly mediated by Ca2+ entry through the Na(+)-Ca2+ exchange system. PMID- 1724426 TI - European guest lecture. Subfoveal new vessels in age related macular degeneration. PMID- 1724427 TI - Reproductive death of Chinese hamster V79 cells after exposure to chemical inhibitors of DNA synthesis. AB - 1. The results of this study have contributed to the definition of three categories of chemical inhibitors of DNA replication in mammalian cells. 2. Inhibitors of replicon cluster initiation [4-nitroquinoline-N-oxide (4-NQO), etoposide (VP-16), teniposide (VM-26), amsacrine (m-AMSA), N-methyl-N'-nitro-N nitrozoguanidine (MNNG), cis-Pt(II)diammine dichloride (cis-PDD)], which needed similar doses to produce a slow and persistent (up to 4 hr) inhibition of DNA synthesis, followed by significant cell killing. 3. Inhibitors of DNA replication by indirect action [3-aminobenzamide [correction of 3-aminobezamide] (3-AB), cycloheximide (CHX), puromycin (PRC), bisbenzimide Hoechst No. 33258 (H-33258]), that showed reduced cytotoxic effects, and caused a slow (60 min) and reversible inhibition of DNA synthesis. 4. Inhibitors of formation and/or polymerization of deoxyribonucleotides [5-aminouracil (5-AU), bisbenzimide Hoechst No. 33342 (H 33342)], which induced a fast (20 min) and reversible suppression of DNA replication, associated with limited cell killing. PMID- 1724428 TI - Cell-cell adhesion molecules. PMID- 1724429 TI - Role of the cytoskeleton in nucleocytoplasmic RNA and protein distributions. AB - Establishment and maintenance of correct partitioning of proteins and RNA molecules between nucleus and cytoplasm in a sine qua non of the viability of eukaryotic cells. Cytoskeletal elements play several roles in such partitioning: controlling the diffusion of proteins within the main cell compartments; presenting transportable macromolecular ligands to receptor sites within the pore complexes; maintaining the structure and dynamics of the pore complexes themselves. The solid-state transport machinery which moves mRNA molecules between particular sites in nucleus and cytoplasm is dependent on actin and other fibrils, and the migration of other major RNA types might show similar dependence. These various aspects of macromolecule partitioning illustrate one way in which the cytoskeleton is fundamental to the eukaryotic state. PMID- 1724430 TI - Keratin genes, epidermal differentiation and animal models for the study of human skin diseases. AB - The examples shown here illustrate the power of gene targeting to the epidermis as a means of developing animal models for the study of human skin diseases. In this short review, I have focused on contributions which stem predominantly from my own laboratory [31, 32, 34, 37, 47]. However, other laboratories have also contributed heavily to the development of this technology [28-30, 35, 36]. The opportunities for these models are vast: there are a myriad of human skin diseases which have been characterized extensively at a biological level, but whose aetiology is presently unknown. A combined knowledge of (a) the biochemistry of epidermal differentiation; (b) epidermal-specific gene expression; and (c) transgenic mouse technology has provided the foundation for these and future studies in this area. PMID- 1724431 TI - Association of pp60c-src with the cytoskeleton upon platelet activation. PMID- 1724432 TI - Use of the polymerase chain reaction to amplify and clone alpha-chains of bovine collagen IV. PMID- 1724433 TI - LDV: a novel cell adhesion motif recognized by the integrin alpha 4 beta 1. PMID- 1724434 TI - Indirect measurement of betaine--homocysteine methyltransferase activity using 1H nmr spectroscopy. PMID- 1724435 TI - Prostaglandin F2 alpha stimulates cAMP phosphodiesterase via protein kinase C in cultured human granulosa cells. AB - To investigate the involvement of cyclic AMP phosphodiesterase in the antigonadotrophic actions of prostaglandin F2 alpha (PGF2 alpha), human granulosa cells were cultured in serum-supplemented medium for 72 h, treated for 30 min with cloprostenol or phorbol myristate acetate (PMA) and then exposed to human chorionic gonadotrophin (hCG) +/- isobutyl-methylxanthine (IBMX) for a further 4 h. In the absence of IBMX, cloprostenol and PMA inhibited hCG-stimulated progesterone production. However, in the presence of 0.5 mM IBMX, inhibition of phosphodiesterase prevented these antigonadotrophic effects. Phosphodiesterase activity was assessed by a novel direct assay performed on intact cells after 3 days of culture. PGF2 alpha, cloprostenol and 4 beta-PMA all enhanced cAMP degradation whilst an inactive phorbol ester (4 alpha-PMA) had no effect. Down regulation of protein kinase C by 4 beta-PMA pre-treatment prevented the subsequent stimulation of phosphodiesterase activity by both cloprostenol and 4 beta-PMA. We conclude that the antigonadotrophic actions of PGF2 alpha in cultured human granulosa cells involve a stimulation of cAMP phosphodiesterase mediated via protein kinase C. PMID- 1724436 TI - Hodgkin's disease: controversies and challenges for the future. PMID- 1724437 TI - Fifteen-year experience in Hodgkin's disease: role of combined modality treatment and splenectomy in the incidence of secondary acute leukemia. AB - BACKGROUND: Following irradiation alone, secondary acute leukemia is extremely uncommon; following chemotherapy alone, the risk is increased, but not as much as when continued maintenance chemotherapy or combined modality treatments are used. PATIENTS: The risk of secondary acute nonlymphocytic leukemia (ANLL) was assessed in 552 patients with Hodgkin's disease (HD), who were diagnosed at the Hematology Institute of Bologna from 1970 through 1984 and followed-up through July, 1990. Median follow-up time was 12 years. RESULTS: ANLL developed in 14 of 328 patients treated with the combined modality of extended-field radiotherapy (RT) plus chemotherapy (CT). All ANLL was observed in patients who had received the mechlorethamine-vincristine-procarbazione-prednisone (MOPP) regimen. No ANLL was documented among 115 patients treated with RT alone, nor among the 109 given CT alone. Leukemia, which developed 34-184 months after diagnosis of HD, was always preceded by a preleukemic phase and was fatal (after 1-12 months) to 13 patients. The karyotype of the leukemia cells was studied in 11 of the 14 patients and was always abnormal. CONCLUSIONS: A Cox's Linear Logistic Model that was performed did not demonstrate that the treatment categories, age, sex, and splenectomy were prognostic factors. Because all ANLL was observed in patients who were treated with extended-field RT plus MOPP and who had undergone splenectomies at diagnosis of HD, further research with larger numbers of patients and longer follow-up should be pursued. Thus with such additional data, clinicians could come to appreciate fully the statistical significance of our interesting observations. PMID- 1724438 TI - Angiogenesis under normal and pathological conditions. AB - Angiogenesis, i.e. the generation of new blood capillaries, occurs in utero (during embryonal and fetal development) and in both physiological and pathological situations during extrauterine life. Several angiogenic factors have now been isolated, including angiogenin, acidic and basic fibroblast growth factors, and alpha and beta transforming growth factors. Their amino acid sequences have been determined and their genes cloned. Other factors await complete characterisation. An account is given of techniques used in the investigation of angiogenesis, both in vivo (transparent chambers; corneal micropockets; implantation on chick chorioallantoic membrane; employment of polymers for the sustained release of angiogenesis factors) and in vitro (cloning and long-term culture of capillary endothelial cells). The angiogenesis induced by solid tumours differs from other forms in that it is not self-limited and continues indefinitely until eradication of the tumour or death of the host. Anti angiogenic factors have also been identified, particularly a new class of nonglucocorticoid steroids. Their employment in tumour therapy is a possibility, since neoplastic expansion is essentially dependent on angiogenesis. PMID- 1724439 TI - Flow cytometry to estimate circulating hematopoietic progenitors for autologous transplantation: comparative analysis of different CD34 monoclonal antibodies. AB - We report that the 2 fluorescein (FITC) conjugated CD34 monoclonal antibodies available to date, namely FITC-8G12 and FITC-QBEND10, exhibit different capabilities of detecting circulating hematopoietic progenitors (CHP) as cells expressing the CD34 antigen (CD34+) by direct immunofluorescence flow cytometry. Mean fluorescence intensity conferred by FITC-QBEND10 to CD34+ CHP is 2.8-4.3 times lower than that conferred by FITC-8G12. By indirect immunofluorescence, native unconjugated QBEND10 and 8G12 antibodies detect CD34+ CHP in a comparable manner, thus indicating that the decrease in QBEND10 affinity is due to FITC conjugation. Utilization of FITC-QBEND10 instead of FICT-8G12 to estimate infrequent (usually less than or equal to 5%) CD34+ CHP for clinical decision making in autologous transplantation of CHP exposes clinicians to the risk of either overestimating (false positive) or underestimating (false negative) these cells in peripheral blood. Recently published guidelines for large-scale collection of CHP for autologous transplantation in cancer patients were based on data obtained with FITC-8G12. The same guidelines cannot be considered valid if FITC-QBEND10 is employed. We recommend FITC-8G12 as the optimal reagent to date for standardizing results of autologous CHP transplantation in cancer therapy. PMID- 1724440 TI - Quantitation of HBF gamma-chain types by HPLC in patients with myelodysplastic syndrome. AB - High levels of HbF were found in patients with myelodysplastic syndrome (MDS), as well as a possible switching of the ratio of the gamma chains from the adult to the newborn type in 25% of our patients. These abnormalities in general were not present in the parents. The possibility of having thalassemia or other hemoglobinopathies was excluded. The fetal hemoglobin percentage was quantitated by alkali denaturation of the hemolysate, and the gamma chain content was determined in blood hemolysate by HPLC following HbF isolation. A 7/3 ratio of fetal Gy/Ay was found in three patients. Since the survival of MDS patients with high HbF levels was longer than that of patients with low levels of HbF, this finding may be used as a potential prognostic factor. PMID- 1724441 TI - [Immunohistochemical findings of epiretinal membranes after silicone oil injection]. AB - During vitreoretinal surgery, 23 epiretinal membranes from eyes treated with silicone oil were removed. They were examined by immunohistochemical methods and compared with 15 membranes from eyes affected by proliferative vitreoretinopathy (PVR) and not treated with silicone oil and with 4 membranes from eyes with intermediate uveitis. PVR membranes from eyes treated with silicone oil showed a high level of macrophages and a strong expression of HLA-DR. Additionally, lymphocytes were found in PVR membranes, a finding that has not been described before. Similar changes were seen in proliferations removed from eyes affected by uveitis, but these membranes were found to have smaller amounts of extracellular substance. In contrast to this, most cells in the PVR membranes from eyes not treated with silicone oil react with vimentin, GFAP and cytokeratin. In none of these membranes were we able to find T-lymphocytes. It is not possible to say whether or not the different findings are attributable ot the silicone oil. PMID- 1724442 TI - Immunohistochemical demonstration of mycobacterial antigens in intracranial tuberculoma. AB - Mycobacterial antigens have been demonstrated immunohistochemically in the paraffin sections of 10 intracranial tuberculous granulomas and the results were compared with the detection of acid fast bacilli by conventional Ziehl-Neelsen method. In none of the 10 specimens, acid fast bacilli were demonstrated while mycobacterial antigens were characterised as diffusely staining granular brownish pink material within the cytoplasm of giant cells and macrophages. In 14 specimens of granulomatous lesions due to non-tuberculous aetiology, immunohistochemical stains were negative for mycobacterial antigen. Thus demonstration of mycobacterial antigen will be not only useful in establishing mycobacterial aetiology of a caseating intracranial granuloma but also can be used as an alternative method to the conventional Ziehl-Neelsen method. PMID- 1724443 TI - Significance of mucin stain in uterine cervical cancers. AB - Histopathological examination of 140 cases of cervical cancer was supplemented with staining for the presence of mucin. Secretion of mucin was detected in 27 cases. Fourteen cases of large cell non-keratinizing (LCNK) squamous cell carcinoma were reclassified, 12 as squamous cell carcinoma with mucin secretion and two as adeno-squamous carcinoma. One case of undifferentiated carcinoma was labelled as adenocarcinoma on the basis of mucin stain. None of the keratinizing squamous cell carcinomas were positive for mucin. The results indicate that mucin expression should be looked for even in those carcinomas where no obvious gland formation is seen. PMID- 1724444 TI - Production of monoclonal antibodies specific for ganglioside GD3. AB - Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3(NeuAc-NeuAc), GD3(NeuAc-NeuGc), GD3(NeuGc-NeuAc), and GD3(NeuGc NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc) but not with GD3(NeuGc-NeuAc) or GD3(NeuGc NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal beta 1-3GalNAc, NeuAc alpha 2-3Gal beta 1-3GalNAc, or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAc alpha 2-8NeuAc alpha 2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha 2-8Sia alpha 2-3Gal beta 1-4Glc residue of ganglioside GD3. PMID- 1724445 TI - Synthesis and cell attachment activity of bioactive oligopeptides: RGD, RGDS, RGDV, and RGDT. AB - Tetrapeptides, Arg-Gly-Asp-Ser (RGDS), Arg-Gly-Asp-Val (RGDV), and Arg-Gly-Asp Thr (RGDT), present in the cell-attachment domain of fibronectin, vitronectin, and collagen, respectively, were synthesized by using an improved liquid-phase procedure. Bioactivities of RGD and RGDX (X = S, V, and T) as cell recognition determinants were investigated by two methods to evaluate interactions of these oligopeptides with L-929 fibroblast cells originating in mouse epithelia. In the first method, these oligopeptides were immobilized to ethylene-acrylic acid copolymer (EAA) film, and the cell-attachment activity of the immobilized film was measured. In the second method, interaction of oligopeptides with the cells was evaluated by measuring the cell inhibition caused by oligopeptides. It was found that RGD and RGDX exhibit remarkable cell-attachment activity, and the activity of oligopeptides depends on X. PMID- 1724446 TI - Engineering T lymphocyte antigen specificity. AB - Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review. PMID- 1724447 TI - Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages. AB - The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation. PMID- 1724448 TI - Changes in the abundance and distribution of actin and associated proteins during terminal differentiation of human epidermal keratinocytes. AB - We have examined the abundance and distribution of actin and several actin associated proteins in human epidermal keratinocytes before and after initiation of terminal differentiation. Keratinocytes were placed in suspension in methylcellulose for 1 h or 24 h and then extracted for immunoblotting. At 24 h, when the proportion of cells expressing the terminal differentiation marker, involucrin, had increased approximately 3-fold, there were marked decreases in the levels of vinculin, talin, filamin and gelsolin. The level of actin was unchanged and the level of alpha-actinin decreased only slightly. To complement the immunoblot analysis, we also examined the distribution of each protein in basal (involucrin-negative) and suprabasal (involucrin-positive) cells in stratified colonies, using confocal microscopy. Gelsolin, filamin, vinculin, talin, alpha-actinin and filamentous actin were all less abundant in suprabasal cells than in basal cells. There were also differences in the distribution of all the proteins in the basal compared to the suprabasal layers. In addition to the changes associated with terminal differentiation, there was variation in the distribution of focal contacts and stress fibres and in gelsolin levels between basal cells at the periphery of colonies and those in the centre. These results are discussed in the context of the known association of the actin cytoskeleton with receptors of the integrin family, the loss of integrins that occurs during keratinocyte terminal differentiation, and the possible role of the cytoskeleton in signalling between integrins and the nucleus. PMID- 1724449 TI - Differences in adhesion to tissue culture plastic of clonally related transformed and control sublines from an epithelial cell strain. AB - Clonally related sublines of the NAL1A lung epithelial cell strain were used in a comparison of the mechanism of attachment and the morphology of control and transformed epithelial cells. The initial attachment and spreading of the control cells on tissue culture plastic was shown to be dependent upon adsorption of serum vitronectin to the substratum. The alpha v subunit of the vitronectin receptor was detected in both the control and transformed cells by immunoprecipitation and immunoblot methods. The spontaneously transformed cells differed from the control cells in that, whereas attachment to tissue culture plastic could occur by binding to adsorbed vitronectin, the transformed cells could also become attached, with time, by a vitronectin-independent mechanism. Attachment by this vitronectin-independent reaction was inhibited by the protein synthesis inhibitor cycloheximide and also by the microtubule-disrupting drugs demicolcemid and nocodazole. The morphologies of attached control and transformed cells cultured on tissue culture plastic were disrupted by treatment with cytochalasin B, demicolcemid or nocodazole, indicating that the shape of these cultured epithelial cells is dependent upon the microtubule system as well as the actin filaments. These results show one important difference between the control and transformed cells, in that the transformed cells can attach to tissue culture plastic by a vitronectin-independent mechanism that involves new protein synthesis by the cell. Another interesting difference is that this vitronectin independent attachment of the transformed cells was sensitive to inhibition by microtubule-disrupting agents. On the other hand, the attachment of either transformed or control cells to fibronectin- or vitronectin-coated surfaces was not affected by microtubule-disrupting agents. PMID- 1724450 TI - High molecular weight tau: preferential localization in the peripheral nervous system. AB - Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 x 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system. PMID- 1724451 TI - Multiple families of proteins in the secretory granules of Paramecium tetraurelia: immunological characterization and immunocytochemical localization of trichocyst proteins. AB - Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14-25 x 10(3)), acidic (pI 4.4-5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb A1-3 and B5-5) showed a relatively wide range of pI values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group II proteins (defined by mAb B4-4 and B3-5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electron-dense striations with 55 nm intervals. However, these regions were not accessible to antibody B4-4 in ctmx. Group III proteins (defined by mAb B7-4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups IV are two subunits of heterodimers. Proteins of groups III and IV were localized in the core of ctmx, but were distributed uniformly in xtmx. Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx. PMID- 1724453 TI - Relationship between naturally occurring human antibodies to casein and autologous antiidiotypic antibodies: implications for the network theory. AB - Previous studies on human autologous antiidiotypes have been based largely upon analyses of autoimmune disease. We have previously described polyclonal, naturally occurring human autoantibodies directed against antibodies with specificity toward bovine casein in the sera of IgA-deficient humans. In order to define this system more exactly we have not produced two murine monoclonal antibodies directed against bovine milk kappa-casein to use as clonal tools to identify specific antiidiotypes in these human sera. Kappa-casein is an important part of the casein micelle in milk and cheese; in addition to being an important immunogen for man, kappa-casein is known to have conserved amino acid sequence and two antigenic epitopes. Data presented here show that the serum of up to 74% of IgA-deficient and 10% of normal humans have specific autologous antiidiotypes in their serum which bind to monoclonal antibodies directed to bovine kappa casein. These human antibodies [intact or F(ab)'2] can be blocked from binding to the monoclonal anti-kappa-caseins by pure bovine kappa-casein or the kappa-casein peptide fragment. In contrast to previous studies in autoimmune disease, serum levels of the autoantiidiotypes were directly proportional to the level of IgG antibody to bovine kappa-casein. These observations suggest that continual exposure to a ubiquitous dietary antigen may produce an antigen driven system in which stimulation of both Ab1 and Ab2 occurs in concert. PMID- 1724452 TI - Role of ion channels in lymphocytes. AB - Ion channels, and ion fluxes in general, appear to regulate a wide variety of processes important to lymphocyte function in normal and disease states. These include resting ionic homeostasis and the more complex signaling events involved in activation, proliferation, cytotoxic function, and volume regulation. The wider application of patch-clamp and microfluorimetry techniques to lymphocytes has helped to clarify some issues and raised many more. It seems likely that rapid progress will be made in our understanding of these areas through a combination of immunological, biochemical, and electrophysiological approaches. PMID- 1724454 TI - Clinical and structural characteristics of periodontal tissues in young and old dogs. AB - The aim of this study was to examine some clinical and structural features of healthy periodontal tissues in young and old beagle dogs. The material consisted of 10 beagle dogs; group I (1-year old) and group II (8-9 years of age). All animals belonged to the same beagle dog colony and had been carefully monitored from birth. A given day was termed day 0 on which the teeth of all 10 dogs were scaled and polished and a 6-week period of enhanced plaque control was initiated. On day 42, clinical examinations were performed and biopsies obtained from the right mandibular 4th (4P) and 3rd (3P) premolar regions. The biopsies were prepared for histometric and morphometric analyses. Clinically, the lower premolars of the old but not the young dogs showed signs of marked wear. In the old dogs, the free gingival unit had a more curved and bulky appearance than in the young animals and in the old dogs, the free gingiva was consistently separated from the attached gingiva by a gingival groove. The histometrical dimensions of the free marginal gingiva and the width of the coronal portion of the periodontal ligament did not differ between the 2 groups of dogs. The apical cells of the junctional epithelium (aJE) in the young dogs were consistently located at the cemento-enamel junction (CEJ), whereas in the old dogs, aJE was consistently located apical to the CEJ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724455 TI - Facing visual illiteracy in South African health education: a pilot study. AB - Medical and health educators in South Africa are facing many problems. These are caused by the diverse communication characteristics and socioeconomic, as well as educational, differences of its communities. Visual literacy is often ignored when audiovisual media are used in medical and health education. Consequently, communication and education fail because some target groups, e.g. illiterate patients from rural areas, are unable to interpret visual messages correctly. This study determined to what extent illiterate female patients were able to interpret instructional illustrations on breast feeding. Findings indicate that visual literacy is a key factor in effective medical and health education. There was a distinct variation in the visual literacy skills of this particular target group. The implication is that extreme care must be taken when visual materials are designed for health education in South Africa. PMID- 1724456 TI - Study of the role of airborne lead in food chain of crops grown around a main highway in Egypt. AB - The present study was conducted to clarify the fate of airborne lead derived from petrol and its role in the food chain of tomato cultivated in lead-contaminated soil. Settled dust was sampled at 4 stations at 7, 75, 150 and 500 meters from Alexandria/Cairo highway, and analysed for lead, sulfate and nitrate. Tomato fruits--and plants--were harvested at the sampling sites and analysed for lead. Settled dust and lead content steadily decreased by distance from the highway, indicating that traffic derived lead aggregates with other pollutants in particles and settle at distance of a few hundred meters. The lead tends to be evenly distributed in aggregates of different particle sizes, while the sulfate and nitrate tend to concentrate in medium-sized aggregates. The lead in plants of different stages of growth correlated with that in the dust, and was the highest in fruit and roots. Lead contamination of the plant had an impact on tomato crop reduction. The levels of lead in tomato fruits were 17.7 ppm in the samples collected at 7-500 meters from the highway, while the legal limit for lead in food is 1 ppm. PMID- 1724457 TI - Intracellular recordings from nonspiking interneurons in a semiintact, tethered walking insect. AB - Nonspiking interneurons were investigated in a tethered, walking insect, Carausius morosus, that was able to freely perform walking movements. Experiments were carried out with animals walking on a lightweight, double-wheel treadmill. Although the animal was opened dorsally, the walking system was left intact. Intracellular recordings were obtained from the dorsal posterior neuropil of the mesothoracic ganglion. Nonspiking interneurons, in which modulations of the membrane potential were correlated with the walking rhythm, were described physiologically and stained with Lucifer Yellow. Interneurons are demonstrated in which membrane potential oscillations mirror the leg position or show correlation with the motoneuronal activity of the protractor and retractor coxae muscles during walking. Other interneurons showed distinct hyperpolarizations at certain important trigger points in the step cycle, for example, at the extreme posterior position. Through electrical stimulation of single, nonspiking interneurons during walking, the motoneuronal activity in two antagonistic muscles--protractor and retractor coxae--could be reversed and even the movement of the ipsilateral leg could be influenced. The nonspiking interneurons described appear to be important premotor elements involved in walking. They receive, integrate, and process information from different leg proprioceptors and drive groups of leg motoneurons during walking. PMID- 1724458 TI - Tamoxifen's effects on the zebra finch song system are estrogenic, not antiestrogenic. AB - Antiestrogens fail to block the masculine ontogeny of the zebra finch song system that is hypothesized to occur as a result of early estrogen action. Moreover, they hypermasculinize the male, and masculinize the female song systems. In experiment 1, we assessed whether these antiestrogenic effects might mimic estrogenic actions. Zebra finch chicks received one of two treatments. They were given estradiol benzoate (EB) or vehicle daily for the first 20 days after hatching and sacrificed at 60 days of age, or they received EB or vehicle for the first 25 days after hatching, at which time they were sacrificed. In the day 60 group, certain attributes of the song system were hypermasculinized in males and masculinized in females by EB, when compared with controls. In the day 25 group, males treated with EB were partially demasculinized, while the females were partially masculinized. In experiment 2, we assessed whether simultaneous treatment with tamoxifen was capable of antagonizing the effects of EB obtained in experiment 1 (day 60 group). Sixty-day-old females, previously treated with both EB and tamoxifen for the first 20 days after hatching, had more masculine song regions than females treated with either EB alone or tamoxifen alone. In males, the effects of the combined treatment of EB and tamoxifen over those produced by tamoxifen alone were not as dramatic as in the female. These results are similar to those obtained in systems where tamoxifen is purely estrogenic and suggest that in the song system, tamoxifen acts as an estrogen, not an antiestrogen. PMID- 1724459 TI - A deceptive staining artifact in ultramicrotomed polymer sections. PMID- 1724460 TI - Attenuated nocturnal rise in pineal and serum melatonin in a genetically cardiomyopathic Syrian hamster with a deficient calcium pump. AB - Day and nighttime melatonin production in the pineal gland was compared in normal and cardiomyopathic (polydystrophic) adult male Syrian hamsters. These strains of hamsters were selected for comparison because the cardiomyopathetic hamster displays a deficient transmembrane Ca(2+)-pump in a number of tissues, and intracellular CA2+ concentrations ([Ca2+]i) play a central role in the nocturnal increase in pineal melatonin synthesis. Daytime levels of all constituents measured, i.e., pineal N-acetyltransferase (NAT) activity, pineal and serum melatonin levels, and pineal 5-hydroxytryptophan (5-HTP), serotonin, and 5 hydroxyindole acetic acid (5-HIAA) contents, were comparable in control and dystrophic hamsters. In contrast, the nighttime rises in pineal NAT activity and pineal and serum melatonin levels were significantly attenuated in the dystrophic hamsters. By comparison, the pineal contents of 5-HTP, serotonin, and 5-HIAA were essentially the same in both groups of hamsters with both pineal serotonin and 5 HIAA values exhibiting the usual nighttime drop. It is presumed that the alterations in nocturnal melatonin production in the pineal gland of the cardiomyopathic hamster may relate to a generalized deficiency in the Ca(2+)-pump in pinealocyte plasma membranes, which leads to unusually high [Ca2+]i, causing a depression of NAT activity; this leads to the commensurate decline in pineal and serum melatonin levels. Harderian gland NAT activity and melatonin levels were essentially similar in the two groups of animals, although NAT activity was slightly depressed in the dystrophic hamsters killed during the day. The reduced amounts of intrascapular brown fat in the cardiomyopathic hamster is speculated to be a result of the diminished amount of melatonin produced in these animals. PMID- 1724461 TI - Pineal serotonin is resistant to depletion by serotonergic neurotoxins in rats. AB - Administration of 3,4-methylenedioxymethamphetamine (MDMA) or para chloroamphetamine (pCA) to adult rats is neurotoxic to serotonin (5HT) nerve terminals and cell bodies. MDMA (10 mg/kg) reduces 5HT levels in the frontal cortex, medial basal hypothalamus, and striatum acutely and 17 days after a series of multiple injections. The acute reductions occur within 1-2 hr after injection of doses greater than 3 mg/kg. A single injection of pCA reduces 5HT levels in the above mentioned brain regions as well as in the brain stem. However, none of these treatments are able to alter 5HT levels in the pineal gland. It appears that the pineal does not contain the 5HT reuptake system that is thought to be necessary for the neurotoxicity of MDMA and pCA. PMID- 1724462 TI - Positional information determines the anatomy and synaptic specificity of cockroach filiform hair afferents using independent mechanisms. AB - Mutant first instar cockroaches (Periplaneta americana) with supernumerary filiform hair sensilla on their cerci were used to study the effects of cell body position on axonal morphology and synaptic connections. The wild-type cercus has two hairs, one lateral (L) and the other medial (M), each with an underlying sensory neuron. Silver-intensified cobalt fills show that the supernumerary lateral neuron (SIN) in the mutant has the same shape of arborization as L, and electrophysiological recording shows that it forms synaptic connections with the same subset of giant interneurons (GIs) as L in the terminal ganglion: GI3 and GI6. The supernumerary medial neuron (SuM) has the same axonal morphology as M and synapses with the same GIs as does M: ipsilateral GIs 1 and 2 and contralateral GIs 1, 2, 3, 5 and 6. In 0.1% of approximately 8000 animals screened, a supernumerary hair arose on the cercal midline (C hair). The C neuron sends its axon to the CNS in the same branch of the cercal nerve as the L and SIN, and has a similar arborization. However, the C neuron forms synapses with the same GIs as do M and SuM. Electron microscopy of horseradish peroxidase injected neurons was used to confirm that the C afferent forms a monosynaptic connection to GI2. It was concluded that the position of the sensory neuron cell body does control its axonal morphology and synaptic connectivity, but that these characteristics are produced by independent mechanisms. PMID- 1724463 TI - Lack of expression of alpha or omega interferons by the horse conceptus. AB - Horse conceptuses were collected on Days 13, 15, 20 and 25 after ovulation. Whole conceptuses (Days 13 and 15) or extra-embryonic membranes (Days 20 and 25) were homogenized and poly-adenylated RNA (poly A RNA) was isolated by binding to oligo (dT)-cellulose. Poly A RNA (1 microgram/well) was separated by size on a denaturing 1% agarose gel and blotted onto nitrocellulose filters (northern blotting). DNA probes were prepared from plasmids containing equine alpha 1, omega 1 and omega 2 interferons and human beta actin. The presence of messenger RNA (mRNA) was detected by specific hybridization of 32P-cDNA probes labelled by random priming. After hybridization at 37 degrees C, filters were washed at room temperature and 56 degrees C, and bands of 32P-cDNA:mRNA heteroduplexes were identified by autoradiography on Kodak XAR-5 radiographic film. The presence of bands on autoradiographs of Southern blots of horse DNA indicated that hybridization and probe conditions were adequate to support hybridization. The actin probe hybridized to all mRNA tested on northern blots, indicating that the mRNA was of high aquality. No hybridization was seen on northern blots using any of the interferon probes. These results indicate that poly A RNA with a high degree of homology to alpha or omega interferons does not accumulate in the horse conceptus. PMID- 1724464 TI - Plasmin, plasminogen activators and inhibitor in human osteoarthritic cartilage. AB - Osteoarthritis (OA) is characterized by a progressive erosion of cartilage, which is mediated by the protease degradation of the extracellular matrix components. Plasmin, plasminogen activators (PA) and the inhibitor of plasminogen activator (PAI) are thought to play an important role in the regulation of the OA pathophysiology process. Our study determined the activity of plasmin and PA in OA and normal cartilage. Moreover, the presence and the content of each form of PA, uPA and tPA, as well as the inhibitor PAI-1, were determined using immunohistological techniques and ELISA. Our studies were carried out on fresh cartilage, cultured tissue explants and chondrocytes. OA cartilage demonstrates about 5 times more plasmin activity than the controls (p less than 0.001). Moreover, a statistically significant correlation was found between the plasmin activity and the free collagenolytic form in OA specimens showing severe lesions (r = 0.50; p less than 0.05). Our study revealed that PA content and activity increase in OA cartilage following culture explant experiments. Immunohistochemical studies showed the presence of both uPA and tPA forms in OA cartilage lesions only. Protein determinations revealed uPA as the predominant form. PAI-1 was significantly decreased (p less than 0.04) in OA, and was located mainly extracellularly. Chondrocyte cultures showed the ability to synthesize both forms of PA and PAI-1. Our study demonstrated an increased level of plasmin activity in OA cartilage. This is likely related to increased PA activity, in which the urokinase type appeared to be predominant in OA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724465 TI - Levels of serum keratan sulfate rise rapidly and remain elevated following anterior cruciate ligament transection in the dog. AB - The levels of serum keratan sulfate (KS) were measured in 9 dogs who underwent anterior cruciate ligament transection. They rose in some animals as early as 7 days after surgery, long before any decrease in the articular cartilage content of KS containing proteoglycans could be measured; and they reached their maximum in most dogs by Day 21 and remained elevated for at least 13 weeks. In contrast, the serum levels of the KS epitope showed no increase in sham operated control animals. Analysis of synovial fluid suggested an increase in the catabolism of cartilage proteoglycans in the operated joint contributed significantly to the increased levels of serum KS. PMID- 1724466 TI - Fibrous myopathy from butorphanol injections. AB - We describe an unusual, and probably underrecognized manifestation of intramuscular narcotic abuse. Fibrous myopathy is a deforming, fibrous infiltration of the muscles used for injection over a prolonged period. It is usually associated with pentazocine or meperidine injections, and to our knowledge, this is the first report of butorphanol causing the entity. PMID- 1724467 TI - In vitro antiviral activity of a peptide-nucleic acid solution against the human immunodeficiency virus and influenza A virus. AB - A peptide-nucleic acid solution which had previously been reported to show in vivo efficacy in several viral infections (i.e. influenza, hepatitis, mumps, encephalitis, etc) was tested in three independent laboratories, including the US National Institutes of Health by specific in vitro methods for HIV and Influenza A. The results of these studies demonstrated significant anti-viral activity of the peptide-nucleic acid solution against the Human Immunodeficiency Virus (HIV) and the Influenza A virus. PMID- 1724468 TI - Maturation and aging of the axonal cytoskeleton: biochemical analysis of transported tubulin. AB - Changes in solubility and axonal transport of tubulin during maturation and aging have been investigated using sciatic motor fibers of rats at 4, 7, 14, 30, and 80 weeks of age. One to six weeks after injection of L-[35S]methionine into the spinal cord, labeled cytoskeletal proteins in consecutive segments of the sciatic nerve and the ventral roots were fractionated into soluble and insoluble forms by extraction in 1% Triton at low temperature. In 4-week-old rats, the two forms of tubulin were transported coordinately in a single wave with the average rate of 2 mm/day. At 7 weeks of age, two components in tubulin transport were observed to develop, possibly reflecting the maturation of the axonal cytoskeleton. The slower main component (1.5 mm/day) contained most of the insoluble form together with the neurofilament proteins and the faster component (3 mm/day) was enriched in the soluble form. Though significantly different in composition, the two components correspond to slow component a (SCa) and slow component b (SCb) originally defined in the optic system. A progressive decrease in transport rates of both SCa and SCb was observed with rats at 14, 30, and 80 weeks of age. In addition, there was a large decrease in the proportion of insoluble tubulin during the course of transport in animals older than 30 weeks. This loss of the insoluble form seems to be accounted for partly by the proteolytic degradation of the severely retarded SCa proteins. Changes in axonal transport of tubulin may thus reflect age-related changes in dynamics and turnover of the axonal cytoskeleton. PMID- 1724469 TI - Corticospinal neurons exhibit a novel pattern of cytoskeletal gene expression after injury. AB - We examined changes in the expression of major cytoskeletal protein mRNAs in adult hamster corticospinal neurons after axotomy. While a number of studies had determined that peripheral neurons exhibit major alterations in cytoskeletal gene expression after axotomy, no previous studies had addressed the question of whether or not intrinsic mammalian CNS neurons, which do not have the ability to successfully regenerate axons after injury, alter their expression of tubulin and neurofilament genes after injury. In the present study we used in situ hybridization methods to examine this issue. 35S-labeled cDNA probes for the low molecular weight neurofilament protein (NF-L) mRNA and an alpha-tubulin mRNA species (M alpha 1) were used for in situ hybridizations of sections of the sensorimotor cortex obtained 2, 7, and 14 days after unilateral axotomy of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed dramatic decreases in both alpha-tubulin and NF-L mRNA levels within axotomized neurons in layer Vb of the sensorimotor cortex. Tubulin mRNA levels were decreased as early as 2 days after injury whereas NF-L mRNA levels were not decreased until later times. Ribosomal RNA (rRNA) levels in axotomized corticospinal neurons were also examined using in situ hybridization with a 35S-labeled rDNA probe. These studies showed only a slight decrease in rRNA levels in corticospinal neurons at 14 days after axotomy. Immunoblotting experiments of total protein from corticospinal axons in the medulla were performed to assess whether the axonal composition immediately proximal to the injury site reflected changes in cell body gene expression. Both alpha-tubulin and NF-L levels were found to decrease in corticospinal axons by 28 days after injury. These findings, to our knowledge, are the first to demonstrate that a class of mammalian CNS neurons have an intrinsically different cytoskeletal response to axonal injury than do PNS neurons. The failure to upregulate tubulin gene expression following injury may contribute to the ineffective regenerative response of these long-tract CNS neurons. PMID- 1724470 TI - Tyrosine phosphorylation and immunodetection of vinculin in growth cone particle (GCP) fraction and in GCP-cytoskeletal subfractions. AB - The growth cone, the motile tip of developing neuronal processes, is considered responsible for the exact guidance of axons and synaptogenesis. High activity of tyrosine kinases in growth cones may contribute to the functions of growth cones. Our previous work revealed that vinculin is one of the endogenous substrates for intrinsic tyrosine kinases in the growth cone particle (GCP) fraction isolated from fetal rat brain. In the present study, we examined tyrosine phosphorylation and immunoblot analysis of vinculin in various fractions from fetal rat brains and adult synaptosomal fraction. Tyrosine phosphorylation of vinculin in the GCP fraction was more prominent than in any other fraction from fetal brain or synaptosomes from adult. Compared to other fractions, however, the enrichment of vinculin in the GCP fraction was not observed. Tyrosine phosphorylation of vinculin in the fraction was inhibited by genistein, a specific tyrosine kinase inhibitor. Although vinculin was also phosphorylated by protein kinase C in the GCP fraction, it incorporated a much smaller amount of 32P than MARCKS protein or GAP-43. The cytoskeletal subfraction from the GCP fraction contained a considerable amount of vinculin and it was one of the major substrates for tyrosine kinases in the GCP cytoskeleton. The membrane skeleton from the GCP fraction contained a low amount of vinculin but showed high kinase activity that phosphorylated vinculin. Taken together, our results suggest that tyrosine phosphorylation of vinculin contributes to the cytoskeletal organization of growth cones. PMID- 1724471 TI - Hypothyroidism selectively reduces the rate and amount of transport for specific SCb proteins in the hyt/hyt mouse optic nerve. AB - Thyroid hormone significantly affects molecular and neuroanatomical properties of the developing nervous system. Altered connectivity in hypothyroidism may reflect reductions in process growth, alterations in process maintenance, or changes in synaptogenesis or synaptic maintenance. These events are dependent on microtubules, neurofilaments, microfilaments, and associated molecular components. Reductions in delivery of microtubules and neurofilaments to the distal axon by slow component a (SCa) of axonal transport may contribute to the neuroanatomical abnormalities of hypothyroidism (Stein et al., J Neurosci Res 28:121-133, 1991). However, hypothyroidism might also affect the axon and synaptic connections by altering slow component b (SCb), which includes actin microfilaments and proteins that contribute to synaptic function, i.e., clathrin, HSC70 (clathrin uncoating ATPase), spectrin, and calmodulin. To determine the effect of hypothyroidism on SCb proteins, slow axonal transport was analyzed in optic nerves of hyt/hyt hypothyroid mice, which have severe primary hypothyroidism, and euthyroid control mice. Clathrin, spectrin, HSC70, and actin showed significant reductions in transport velocity in hyt/hyt optic nerves relative to euthyroid nerves, but the transport rate for calmodulin was less affected. However, the amount of calmodulin was significantly elevated in hyt/hyt nerve over euthyroid nerves. Hypothyroidism selectively reduces transport of SCb proteins, which are thought to play significant roles in synaptic function and in the growth cone. The effects of hypothyroidism on microtubules and neurofilaments combined with actions on SCb suggest that changes in neuronal function associated with reduced thyroid hormone during development and maturity (i.e., alterations in neuronal connectivity, nerve conduction, and synaptic function) may be mediated in part by effects on slow axonal transport. PMID- 1724472 TI - Sequence of the rabbit neurofilament protein L. AB - In the course of screening a rabbit brain cDNA library with a probe for the H neurofilament protein, we identified a neurofilament L-cDNA. Its nucleotide sequence is 88% identical to that of human, indicating that L is highly conserved among species. The similarities between the sequences of L from rabbit and mouse suggest that the species-specific accumulation of neurofilaments that occurs in rabbit during aluminum intoxication is not a consequence of the primary structure of L. PMID- 1724473 TI - A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF-M and NF-H. AB - We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C-terminal "tail" sequences of the two high molecular weight neurofilament subunits, NF-M and NF-H. A series of recombinant fusion proteins containing selected NF-M and NF-H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS-PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF-M and NF-H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF-M and NF-H sequence. These antibody studies showed that the extreme C-termini of NF-M and NF-H are immunologically absolutely distinct from one another and also indicate that the extreme C-terminus of NF-M is immunologically much more conserved than the analogous region of NF-H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C-terminus of NF-M. We also show that the unusual immunological properties of the bovine NF-M tail we have previously observed do not extend to the extreme C-terminal region, which appears immunologically no different from the analogous region of other NF-M molecules. The peculiarities of bovine NF-M could be explained by the presence of a KSP motif that resembles the NF-H KSP prototype. PMID- 1724474 TI - Skull metastasis of primary hepatoma--case report. AB - Skull metastasis of hepatoma is rare. A case of a 56-year-old man with painless masses over the skull is presented in this paper. The largest mass in the occipital area measured 10 x 15 cm2; skull radiographs and computed tomography (CT) of the brain revealed several skull tumors with epidural extensions. The largest mass was almost totally excised, and metastatic hepatoma was diagnosed by a histological examination and supported by abdominal ultrasound findings. Literature in regard to hepatoma with skull metastasis is also reviewed. PMID- 1724475 TI - [A case of Schonlein-Henoch purpura complicated with protein-losing enteropathy and hyperamylasemia]. PMID- 1724476 TI - Malignant lymphomas in HIV-seropositive patients. Frequency, features, and prognosis. Report on 31 cases. AB - In a random HIV-seropositive population, malignant lymphomas were diagnosed in 31 patients, of whom 24 (77%) had non-Hodgkin lymphoma (NHL) and 7 (23%) Hodgkin lymphoma (HL). The prevalence of NHL among AIDS patients was 8% (23/279 cases), with a prevalence of 17% among autopsied patients (16/96 cases). No patient with HL had AIDS at the time of diagnosis. In 7 of 23 AIDS patients with NHL (30%) the diagnosis was made only post mortem; among these were all 5 patients with primary CNS NHL. Median survival from the time of diagnosis was 1 month for patients with NHL and 3 months for those with HL. In individual patients, survival for several years may be possible with chemotherapy. Certain patients with NHL appear to benefit from intensive chemotherapy with a combination of methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOPB protocol). Appropriate, therapeutic strategies taking into account the patients' individual conditions, including the overall prognosis, urgently requires development. Metastatic CNS involvement, which was the primary cause of death in 5 of 11 patients with NHL (45%) receiving chemotherapy, represents a serious limitation to successful treatment. PMID- 1724477 TI - Alpha-lipoic acid is an effective inhibitor of human immuno-deficiency virus (HIV 1) replication. AB - Alpha-lipoic acid, a naturally occurring disulfide-compound that acts as a cellular coenzyme, inhibits replication of HIV-1 in cultured lymphoid T-cells. Alpha-lipoic acid was added 16 hours after infection of the T-cell lines Jurkat, SupT1 and Molt-4 with HTLV IIIB and HIV-1 Wal (a wild type HIV-1 isolate). We observed a dose dependent inhibition of HIV-1-replication in CPE (Cytopathic effect) formation, reverse transcriptase activity and plaque formation on CD4 transformed HeLa-cells. An over 90% reduction of reverse transcriptase activity could be achieved with 70 micrograms alpha-lipoic acid/ml, a complete reduction of plaque-forming units at concentrations of greater than or equal to 35 micrograms alpha-lipoic acid/ml. An augmentation of the antiviral activity was seen by combination of zidovudine and low dose of alpha-lipoic acid (7 micrograms/ml). Trypan blue staining revealed no toxic effects of alpha-lipoic acids on peripheral blood mono-nuclear cells and T-cell lines even in concentrations of greater than or equal to 70 micrograms/ml. Therefore, we propose the inclusion of alpha-lipoic acid into chemotherapy trials in combination with zidovudine. PMID- 1724478 TI - [Blood serum inorganic phosphorus: diagnostic significance and method of determination (review of the literature)]. PMID- 1724479 TI - [The role of the clinical laboratory in the evaluation of the tubular function of the kidneys]. AB - Renal tubules contribute to maintenance of the body equilibrium by regulating urinary excretion of electrolytes and low molecular mass components. Many transport mechanisms for regulation of concentrations of various components of the tubular fluid are situated in the renal tubules. Studies on the specification and localization of subcellular mechanisms functioning in the renal tubules is a progressive borderline trend of investigation. A new approach has been used to solve the problems of clinical nephrology and maintenance of the body balance. Processes of calcium, sodium, and monosaccharide reabsorption in the tubular fluid are described in brief. PMID- 1724480 TI - [Change in the flow line of erythrocytes during microelectrophoresis in a magnetic field in normal and pathologic conditions]. PMID- 1724481 TI - [Coagulation properties of the thrombocytes in macro-focal myocardial infarct]. AB - Platelet coagulative and fibrinolytic activities were studied in 37 patients with large myocardial infarction. Plasma samples rich for platelets (with the standard platelet level) and with low platelet counts were analyzed. A lowered coagulative activity of the platelets was revealed in such patients, parallelled by loss of these cells' ability to enhance fibrinolysis. These shifts are particularly marked in the first days of the illness. PMID- 1724482 TI - [The determination of tissue thromboplastin activity in cells]. AB - A method for estimation of thromboplastin activity in blood and tissue cells has been developed, experimentally and theoretically substantiated. Blood plasma free from blood coagulation contact phase factors is used as a substrate for the detection of thromboplastic activities of blood and tissue cells. PMID- 1724483 TI - [Cytologic diagnosis of malignant tumors of the peripheral nervous system]. AB - Histologic and cytologic parallels in 9 cases with malignant schwannomas were correlated to findings in preparations of verified fibrosarcomas and muscular sarcomas, characterized by a similar cytomorphologic picture, in order to define the cytologic differential diagnostic criteria of nerve tissue malignant tumors. Preoperative and suboperative specimens were under study. Analysis of the results evidences that in the majority of cases malignant schwannoma cytogram can be correctly verified if the level of tumor cell differentiation and the respective cytomorphologic shifts be taken into consideration, this being of paramount importance both for the choice of the treatment strategy and for the prognosis. PMID- 1724484 TI - [Presymptomatic and prenatal laboratory diagnosis of hereditary diseases using the DNA analysis method (review of the literature)]. PMID- 1724485 TI - [Cytomorphologic characteristics of poorly differentiated forms of lung cancer]. AB - Lung cancer was diagnosed in 462 of the 738 patients with pulmonary diseases examined by x-ray surgical and radiocontrast methods. Non-differentiated lung cancer was detected in 43 of these, highly differentiated squamous-cell glandular carcinoma in 14, and poorly-differentiated squamous-cell or glandular cancer in the rest 405 (88%) patients. Differential-diagnostic cytomorphologic features of poorly-differentiated squamous-cell and glandular pulmonary carcinoma are presented. PMID- 1724486 TI - [The development of the scientific ideas of Prof. A. Ia. Al'tgauzen in the works of his collaborators (on the 100th anniversary of the birth of Prof. A. Ia. Al'tgauzen and the 60th anniversary of the founding of the Chair of Clinical Laboratory Diagnosis]. PMID- 1724487 TI - [A modification of the leukocyte migration inhibition test in vivo]. AB - Estimation of the degree of leukocyte migration inhibition in the oral cavity, following exposure to allergen solution, is a test widely used to assess a subject's allergy to drugs. To simplify this test and make it more effective, the authors suggest placing the samples in plate wells and drying them there; optically transparent polystyrene plates for enzyme immunoassay are employed. Effective adsorption of the cells on the carrier and the possibility of analyzing the samples with the help of multiple magnification permit a reliable detection of the migration inhibition. The method was tried in examinations of workers engaged in the manufacture of an x-ray contrast agent triambrin. Reduced migration levels were detected in 8 of the 12 examinees. PMID- 1724488 TI - [The polybrene test in the diagnosis of autoimmune hemolytic anemia]. AB - The efficacies of Coombs' test and the polybrene test, heretofore not used in this country, for the diagnosis of autoimmune hemolytic anemias are compared in examinations of 27 donors and 62 patients with this condition. The results evidence a high efficacy of the polybrene test. The test is simple and available for wide use at clinical laboratories. PMID- 1724489 TI - [The immunoturbidimetric determination of class G immunoglobulin using the FP-900 analyzer]. AB - The immunoturbidimetric micromethod for measuring the blood serum IgG has been adapted to the FP-900 analytic system. Monospecific sera to IgG (H) manufactured by several institutions in this country were used in the study. The volumes of antiserum solvents were selected and the analytical reliability of the suggested micromethod assessed. Programs for introduction of the parameters for IgG measurements with FP-900 analyzer are presented. PMID- 1724490 TI - [The effect of sodium nucleinate on the proliferative and specific activity of hybridoma cells in vitro]. AB - Sodium nucleinate, an officinal preparation (Na PHK), added to the growth medium in concentrations of 10 to 50 micrograms/ml enhanced the growth of hybridoma cells in vitro. Na PHK presence in the cultivation medium in the above concentrations enhanced the specific antibody-producing activity of hybridomas. PMID- 1724491 TI - [A method of electrophoretic differentiation of the genera of glucose nonfermenting gram-negative bacteria]. AB - Glucose nonfermenting gram-negative bacteria were electrophoretically typed for the extracellular protein spectra. The method permits differentiation on the generic level the cultures of such bacteria, characterized by atypical biochemistry, within 10 hours. PMID- 1724492 TI - [Characteristics of the anaerobic bacterial spectrum in children with periodontitis]. AB - Analysis of the bacterial spectrum of 25 children with periodontitis has revealed that obligate anaerobes make up 65% of the bacteria. In 96% of cases anaerobic microorganisms are isolated in associations. Bacterial associations are analyzed. PMID- 1724493 TI - [The use of reference sera in clinical diagnostic laboratory practice]. PMID- 1724494 TI - [A rapid response laboratory in a pediatric clinic]. AB - Laboratory studies are an essential aspect in the management of children with grave diseases, helping to plan the therapeutic measures and to identify the disease. The most acute syndromes in pediatric emergency care are: coma, convulsions, dehydration, metabolic disequilibrium, hypovolemic or anaphylactic shock, a grave infection, chemical or drug poisoning. The laboratory tests that should be available within few minutes are blood cell count, blood and gas analysis, sodium, potassium, calcium, glucose measurements. The results of total proteins, serum creatinine and urea measurements, bleeding tests, analysis of blood smear, sedimentation rate, ALT, AST, osmolality, urinary electrolytes, creatinine and cerebrospinal fluid examinations should be available within sixty min. New accurate and rapid techniques and instruments facilitate the diagnostic and therapeutic approach to pediatric emergency. PMID- 1724495 TI - [Evaluation of the economic indices of the work of clinical diagnostic laboratories (methodologic recommendations)]. PMID- 1724496 TI - [The use of detergents in the polarographic determination of superoxide dismutase and catalase activities]. AB - A number of detergents were screened to choose the most suitable one for polarographic measurement of superoxide dismutase and catalase activity. Triton X 100 and triton X-305 nonionic detergents proved to be the best of all the tested ionic and nonionic detergents. The sensitivity of the method with these tritons is close to the sensitivity of the original method and, in contrast to it, does not involve addition of blood plasma. Effects of a number of substances, used in studies of biological samples, on activity measurements by the developed methods were under study. Superoxide dismutase activities were measured in tissues of hepatomas with different growth rates. PMID- 1724497 TI - [A study of acid nuclease activity]. AB - A method for serial measurements of blood serum acid DNAse (3.1.4.5) and acid RNAse (3.1.4.23). The authors recommend measurements of acid nucleases at clinical biochemistry laboratories and use of these parameters for the prediction of outcomes of balneotherapy and other treatment modalities. PMID- 1724498 TI - [Determination of alpha-amylase activity using a new chromogenic substrate]. AB - alpha-Amylase activity was measured with the use of a new chromogenic insoluble substrate, Testamyl, developed and manufactured by Kemotex, Tallinn, Estonia. Reproducibility trials of the new method have demonstrated a low coefficient of variations within a lot (4.4%) and among the lots (3.5%). alpha-Amylase activity values obtained with the use of Testamyl and by other technique are in good correlation. Routine laboratory equipment is employed in the test; this permits use of Testamyl kits for measurements of alpha-amylase activities in the blood serum and urinary samples both at laboratories of specialized treatment and diagnosis institutions and at clinical laboratories. The method is convenient for rapid diagnosis. PMID- 1724499 TI - [Acid-soluble fractions of the blood in patients with nonspecific diseases of the respiratory organs]. AB - A method permitting the differentiation between the types of pulmonary inflammatory processes in patients with nonspecific diseases of the respiratory organs by the parameters of blood plasma acid-soluble fraction is suggested. If the inflammation is of an infectious origin (pneumonia, chronic bronchitis), the ratio of low-molecular to high-molecular fractions of the blood plasma acid soluble fraction is 4.2 +/- 1.8. In allergic inflammations (bronchial asthma) this ratio is 9.0 +/- 5.8. The results of this test were in line with clinical laboratory and x-ray findings in 86.7% of the examinees. In complex with other methods of examination, the described technique may be used for the differential diagnosis of allergic and infectious inflammations in patients with respiratory diseases. PMID- 1724500 TI - [The determination of aldosterone, thromboxane A2 and prostacyclin in biological fluids]. AB - The authors' findings have lead them to a conclusion that urinary prostacyclin may be measured directly without preliminary extraction. Double freezing defrosting of the samples did not change urinary thromboxane A2 level. Prolonged (up to 8 months) storage did not tell on urinary thromboxane A2 and blood plasma aldosterone levels. PMID- 1724501 TI - [H2O2-induced blood serum biochemiluminescence]. AB - Significant differences in the intensities of H2O2-induced biochemiluminescence of venous and capillary blood sera were revealed, as was a direct relationship between this parameter and hemoglobin level in the samples; this permits a conclusion on the improper employment of H2O2-induced biochemiluminescence of blood serum in diagnostic studies. PMID- 1724502 TI - [Cyclic nucleotides in malignant neoplasms of the colon]. PMID- 1724503 TI - [Determination of the blood thiol disulfide balance using the coulometric titration method]. PMID- 1724504 TI - [The use of light diffusion in the determination of microalbuminuria]. PMID- 1724505 TI - [The immunoenzyme analysis of the specific alpha2-glycoprotein in the brain in patients with neuropsychic diseases]. AB - The authors have developed an enzyme immunoassay test system for measurements of the specific cerebral alpha 2-glycoprotein (alpha 2-GP), whose sensitivity limit is 0.8 ng/ml. Blood serum alpha 2-GP level is 7 ng/ml, cerebrospinal fluid level is 12.8 ng/ml in health; detection of alpha 2-GP in these biological fluids in concentrations surpassing such values indicates impairment of the hematoencephalic barrier permeability and active release of the cerebral proteins into the blood. The hematoencephalic barrier is most frequently disrupted in the brain-blood direction in purulent meningitis, encephalitis, open craniocerebral injuries; such impairments may be detected with the use of alpha 2-GP measurements. A direct correlation between the blood serum alpha 2-GP level and the severity of the disease clinical course has been revealed. PMID- 1724506 TI - [Gamma-glutamyl transpeptidase: methodologic approaches to its determination and its diagnostic value (review of the literature)]. PMID- 1724507 TI - [The blood coagulation status and the immunochemical and clinical indices in patients with active rheumatism during treatment with nerobol]. AB - Blood coagulation, immunobiochemical, and clinical characteristics were studied in patients with rheumatic fever running a protracted course treated by combined therapy including nerobol. To assess the therapeutic efficacy of nerobol, the patients were divided into 2 groups: Group 1 (30 patients) were administered combined antirheumatic therapy, Group 2 (30 patients) were administered the same + nerobol in a daily dose of 15 mg for 3-4 weeks. The findings evidence a positive effect of the drug on the status of patients in whom rheumatic fever runs a protracted course: blood procoagulative activity is reduced and anticoagulative activity elevated, blood immunobiochemical and clinical characteristics normalized. PMID- 1724508 TI - [The use of the kallikrein-kininogen system indices in the prognosis of the development of hemorrhagic erysipelas]. AB - A number of the kallikrein-kinin system parameters (kallikrein, prekallikrein, total arginine esterase activity, alpha 1 protease inhibitor, and alpha 2 macroglobulin) were measured in 59 patients with erythematous erysipelas and in 51 ones with hemorrhagic erysipelas over the course of the disease. Marked activation of the blood kallikrein-kinin system was seen in all the patients during the initial period of the disease, manifesting by elevated levels of kallikrein, total arginine esterase activity, alpha 1 protease inhibitor, alpha 2 macroglobulin, and a lowered prekallikrein concentration. In erythematous erysipelas the peak of activation was recorded in the first days of the disease, whereas in hemorrhagic condition it was observed during the second week of erysipelatous inflammation. Different patterns of changes in the kallikrein-kinin system over the course of the disease permit using one of its parameters, kallikrein activity, for the prediction of the development of local hemorrhagic syndrome in erysipelas patients already during the earliest (prehemorrhagic) stage of the condition. PMID- 1724509 TI - [Hematologic indices in participants in the liquidation of the consequences of the accident at the Chernobyl Nuclear Power Station 3 years following their work in Chernobyl]. AB - Blood analysis with the use of automated analyzer H-1 (Technicon), carried out in 31 subjects who were engaged in liquidation of the power plant accident aftereffects 3 years following their work at the station, has revealed elevated red cell counts, elevated counts of peripheral blood large nondifferentiated cells, dose-dependent increase of neutrophil count, reduced counts of lymphocytes and basophils, enlarged nuclear size in mononuclears and reduced optic density of these cells. PMID- 1724510 TI - [The nitroblue tetrazolium reduction test with mononuclear phagocytes]. PMID- 1724511 TI - [The function of neutrophils in dermatoses]. AB - A total of 102 patients were examined, 32 of these with true eczema, 38 with exudative mycosis of the soles, and 32 with eczema etiologically related to a fungal infection. Analysis of the immune and biochemical reactions in the examinees has shown a marked reduction of adenyl nucleotides in the leukocytic suspension and neutrophils of patients with a mycotic infection as against those with true eczema. The lowest creatine phosphate levels were detected in the leukocytic suspension and neutrophils of the patients suffering from eczema etiologically related to mycosis and exudative mycosis of the soles. These results give grounds to search for effective corrective therapy. PMID- 1724512 TI - [The determination of the heparin-dependent monocyte activity in rheumatoid arthritis]. AB - Heparin elevates the level of NBT-positive monocytes in donors and patients with osteoarthrosis deformans and rheumatoid arthritis. Patient's rheumatoid arthritis cells were found the most sensitive to heparin. Excessive heparin-dependent activity of monocytes in this case reflects the activity of the rheumatoid process, the NBT test with heparin being more sensitive in the diagnosis of disease activity than measurement of red cell sedimentation rate or of blood C reactive protein, gamma- and alpha 2-globulin levels. PMID- 1724513 TI - [A cytochemical study of the blood cells in the newborn]. AB - The cytochemical parameters (acid and alkaline phosphatases, peroxidase, and glycogen) of the peripheral blood leukocytes were studied in 58 clinically full term newborns delivered by cesarean section and in 22 healthy newborns delivered spontaneously. Statistically significant deviations of a number of blood biochemical characteristics were detected in the newborns delivered by cesarean section in the course of the early neonatal period (on days 1, 5, 10 of life), this pointing to endogenously predetermined instability of the body of the risk group babies. The authors regard the cytochemical analysis of the blood cells as a highly sensitive method for the detection of disordered adaptation of the newborns. PMID- 1724514 TI - [Tissue typing using a Soviet automated luminescent microscope-photometer]. AB - Automated fluorescent microscope developed by the authors permits photometry of microquantities of cellular suspensions in scanning standard 60-well plates with a flat bottom. Automated and semiautomated modes of operation are possible. Fluorescent stains and schemes of staining the examined cells that yield stable results in the lymphocytotoxic test have been selected. Comparative analysis of the efficacies of histocompatibility antigens detection by the routine and the fluorescent technique has shown a number of advantages of the fluorescent method. Specific features of fluorescent stains used for staining live cells are described. PMID- 1724515 TI - [The use of Soviet antisera for determining immunoglobulins in the ICS-11 system (USA)]. PMID- 1724516 TI - [A highly sensitive immunoenzyme analysis using electrochemical detection]. AB - The authors have developed a method for improving the sensitivity of electrochemical enzyme immunoassay, based on binding the enzymatic reaction volatile component to a complexone, followed by destruction of the complex in a flow-type amperometric detector. The lowest limit of the measured levels of blood serum antibodies and antigens is 10(-11)-10(-12) M. The method was tried in enzyme immunoassay of the blood serum IgE in patients with asthma and chronic obstructive bronchitis. Reduction of IgE level after therapy was found to be a regular feature; the authors claim that it may be considered as one of the criteria indicating the treatment efficacy. PMID- 1724517 TI - [A method of indirect immunofluorescence for diagnosing chlamydia infections]. AB - The diagnostic value of indirect immunofluorescence in the detection of antichlamydial IgG antibodies in patients with chronic chlamydial salpingitis is insufficient. The condition may be diagnosed if antichlamydial IgG antibodies are detectable in the serum diluted at least 1:128. Examinations of paired sera are of no value in chronic chlamydial salpingitis. PMID- 1724518 TI - [Serotypes of Pseudomonas aeruginosa strains isolated in obstetric institutions in Azerbaijan]. AB - Examinations of Pseudomonas aeruginosa strains isolated from clinical material from newborns and puerperae and from the environmental objects (washings off, water) have shown that 45.45% of these strains belonged to serotype 04, 24.55% to serotype 02, and 16.38% to other serotypes. Serotype 04 was the most incident in all the tested samples, it was detectable in newborns' biotopes and on environmental objects. Serotype 02 strains ranked second in incidence; they were detected in clinical material from the newborns but not on environmental objects. PMID- 1724519 TI - [A method of rapid indication of an antigen]. AB - A simple and sensitive method for rapid detection of an antigen is offered. 0.025 ml of antiserum is placed on the slide, 0.025 ml of clarified extract containing the antigen added, and then 0.025 ml of intact 20.10(6) suspension of Kowan 1 Staphylococcus strain is added. When antigen-antibody complex is forming, staphylococcal cell aggregation starts. A staphylococcal reagent, manufactured by the Pasteur Research Institute of Epidemiology and Microbiology in Leningrad, may be employed, this making the method more available. The indication sensitivity is 0.2-1.25 mln cells per ml, this value depending on the bacterial species. PMID- 1724520 TI - [New selective agents for the isolation of Pseudomonas aeruginosa]. AB - Two new selective media were prepared for the isolation of P. aeruginosa cultures from clinical material: RChZh medium with ramrod inhibitor and Zh medium including a selective complex (hydrazine sulfate, gramurin, ethonium, N cetylpyridine chloride). The media are characterized by marked selective characteristics, simple to use, contain no unavailable reagents, inexpensive, and easy to prepare. PMID- 1724521 TI - [The results of a comparative study of different series of commercial casein carbon agar]. AB - Comparative assessment of the diagnostic properties of 30 lots of commercial casein-carbon agar used in practical bacteriology for the diagnosis of pertussis has shown essential differences in the efficacies of various lots of this medium. Only 11 of the 30 tested lots provided the formation of B. pertussis colonies in the periods established by the instruction. Addition of antibiotics to the medium reduced its diagnostic characteristics still more. PMID- 1724522 TI - In vitro and in vivo effects of R023-6152 on heart mitochondrial calcium and energy metabolism. AB - Previous studies have shown that Ca2+ channel antagonists in all chemical classes can inhibit Na(+)-induced CA2+ release from mitochondria. The effects of R023 6152, a new thiazepinone Ca2+ channel antagonist, on isolated rabbit heart mitochondrial Ca2+ transport and respiratory activity were compared with those of diltiazem. Heart mitochondria were also isolated and assayed from dogs treated in vivo with either R023-6152 or diltiazem. The results indicate that R023-6152 produces half-maximal inhibition of Na(+)-induced Ca2+ release from isolated mitochondria at relatively the same concentrations (10-30 microM) as diltiazem but also produces inhibition of mitochondrial Ca2+ uptake and state 3 respiration at concentrations (25-100 microM), at which diltiazem has no effect. The greater lipophilicity of R023-6152 in gaining access to and inhibiting the phosphate transporter in the mitochondrial membrane as compared with that of diltiazem may explain these results. Heart mitochondria isolated from dogs treated with diltiazem and R023-6152 exhibited lower rates of state 3 respiration as compared with controls. We suggest that this may result from a reduction in transsarcolemmal Ca2+ flux causing a down-regulation in mitochondrial dehydrogenase activity and not from any direct intracellular effects of the two drugs. PMID- 1724523 TI - Angiotensin converting enzyme inhibition in heart, kidney, and serum studied ex vivo after administration of zofenopril, captopril, and lisinopril. AB - Angiotensin converting enzyme inhibition in heart, kidney, and serum were studied ex vivo after oral administration of lisinopril (10 mg/kg), zofenopril (10 mg/kg), and captopril (30 mg/kg) to rats to study the time course, degree, and sites of inhibition of ACE by a quantitative in vitro autoradiography and enzymatic assay. ACE activity in all regions of the heart, kidney, and serum was markedly reduced 4 h after administration of lisinopril and zofenopril and only partially recovered toward control levels at 24 h. After captopril treatment, ACE activity was partially inhibited in heart, kidney, and serum at 1 h and fully recovered toward control levels in most regions at 24 h. These results suggest that these inhibitors reduce ACE in all regions of the heart and kidney without regional selective inhibition. Lisinopril and zofenopril at these doses produced longer-lasting ACE inhibition than captopril. ACE recovery after ACE inhibitor treatment in serum was faster than in heart or kidney. PMID- 1724524 TI - Blood flow regulation during acute regional ischemia in feline hearts: importance of postjunctional alpha 1- and alpha 2-adrenoceptors. AB - Influence of postjunctional alpha 1- and subsequent alpha 2-adrenergic antagonism on myocardial blood flow was measured in a group of anesthetized cats with acute occlusion of the left anterior descending coronary artery (LAD) and a control group (n = 10 for both). The relatively selective postjunctional alpha 1 (doxazosin) and alpha 2-adrenergic (SK&F 104078) antagonists were applied after beta-adrenergic blockade (propranolol). Regional myocardial blood flow was obtained with radiolabeled microspheres. Major hemodynamic determinants for perfusion were kept constant both within and between groups by right atrial pacing and aortic obstruction. Mean coronary resistance in nonischemic myocardium was permanently lower in the occlusion group as compared with controls (p less than 0.01). Subsequent alpha 2-adrenergic antagonism reduced mean coronary resistance in controls only (p less than 0.05). Cardiac output (CO) and dP/dt was reduced in LAD-occluded hearts after alpha 2-adrenergic blockade (p less than 0.01, p less than 0.05). The study demonstrates the significance of postjunctional alpha 2-adrenergic-mediated vasoconstriction in well-perfused myocardium of control hearts, whereas such vasoconstriction was deteriorated in LAD-occluded hearts. A role for myocardial alpha 2-adrenoceptors for maintenance of global cardiac function in acute regional ischemia was also indicated. PMID- 1724525 TI - Combination therapy with lovastatin and guar gum versus lovastatin and cholestyramine in treatment of hypercholesterolemia. AB - Sixty-two patients (34 men and 28 women) aged 19-64 years, half of whom had familial hypercholesterolemia, treated initially for 18 weeks with lovastatin alone were randomly allocated either to lovastatin (L) and cholestyramine (16 g/day) or lovastatin and guar gum (L + GG 20 g/day) treatment for 18 additional weeks to compare the hypocholesterolemic effects of these two combination therapies. The patients were selected for this study from a larger study of patients (n = 120) with severe hypercholesterolemia [serum total cholesterol (serum Chol) 6.5-16.3 mM before treatment], and only those patients in whom serum Chol after lovastatin alone (dose 80 mg/day) remained greater than or equal to 5.2 mM were eligible for evaluation of combination therapies. Serum Chol decreased from 10.6 +/- 1.6 to 5.9 +/- 1.3 mM (mean +/- SD) (p less than 0.001) and low-density lipoprotein cholesterol (LDL Chol) from 8.5 +/- 1.8 to 4.1 +/- 1 mM (p less than 0.001) in patients treated with L + GG (values before the beginning of lovastatin and at the end of the combination therapy). The respective changes were from 10.9 +/- 2.2 to 5.5 +/- 1.2 mM (p less than 0.001) and from 8.7 +/- 2.3 to 3.5 +/- 1.2 mM (p less than 0.001) in patients treated with lovastatin and cholestyramine (L + C). At the end of the study, both serum Chol (p less than 0.005) and LDL Chol (p less than 0.01) were significantly lower with L + C than with L + GG.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724526 TI - In vivo inhibition of platelet aggregation and occlusive coronary thrombus formation by a new calcium antagonist (R023-6152). AB - Reports have shown that all three major classes of calcium antagonists can inhibit platelet aggregation in vitro. In this study, we compared the platelet antiaggregatory effects of R023-6152, a thiazepinone-type calcium antagonist, with diltiazem in an in vivo canine model of spontaneous coronary thrombosis. Plasma serotonin (5-HT) levels measured in the coronary sinus were used as an index of in vivo platelet aggregation and coronary flow measured by a Doppler flow probe. Untreated controls developed total coronary occlusion in 62 +/- 18 min after the current used to initiate thrombus formation was discontinued. Control 5-HT levels peaked at 213 +/- 63 ng/ml just before occlusion. Dogs receiving intravenous (i.v.) R023-6152 (200 micrograms/kg) or diltiazem (50 micrograms/kg) immediately after the current was discontinued exhibited no significant elevations in 5-HT values (12.3 +/- 1.4 and 1.84 +/- 0.42 ng/ml for R023-6152 and diltiazem, respectively) or development of coronary occlusions in the next 3 h. Small, transient decreases in arterial pressure (8-10 mm Hg) and changes in contractility occurred during infusion of both drugs. Gravimetric determinations of thrombus weights showed significantly smaller thrombi in the drug-treated animals as compared with controls. The results indicate that both R023-6152 and diltiazem are effective in suppressing in vivo platelet aggregation associated with occlusive coronary thrombus formation. PMID- 1724527 TI - Effects of some inorganic divalent cations and protein kinase C inhibitors on endothelium-dependent relaxation in rat isolated aorta and mesenteric arteries. AB - The effects of nitrendipine and several metal ions possessing Ca2+ antagonistic activity were examined on acetylcholine (ACh) and histamine-induced endothelium dependent relaxations in norepinephrine (NE)-precontracted rat aortic rings and perfused mesenteric arteries. Nitrendipine (1 nM) profoundly attenuated ACh and histamine-induced relaxations of perfused mesenteric arteries but was ineffective against either agonist in aorta. The transition metal ions Co2+, Mn2+, and Ni2+, but not the nontransition ions (Cd2+, Sn2+, and Zn2+), markedly inhibited ACh and histamine relaxations in the aorta, whereas all metal ions antagonized KCl contractions. At the highest concentration devoid of effect on arterial perfusion pressure, none of the transition metal ions altered endothelium-dependent relaxations in the mesenteric arteries. Endothelium-independent relaxations induced by sodium nitroprusside (SNP) were attenuated by Mn2+ but not by Co2+ or Ni2+. Calmidazolium or W-7 inhibited ACh- and histamine-induced relaxations in both aorta and mesenteric arteries, whereas staurosporine and H-7 were ineffective against aortic relaxations; in mesenteric arteries, staurosporine but not H-7 attenuated both endothelium-dependent and -independent relaxations. We conclude (a) that the transition metal ions most likely inhibit endothelium derived relaxing factor (EDRF) (NO) release in the aorta through endothelial receptor-operated Ca2+ channels; (b) that the effects of nitrendipine (shared by nifedipine) in mesenteric arteries result from an interaction with a site that may have structural similarities with, but is distinct from, the L-type Ca2+ channel; and (c) that the inhibitory effects of the calmodulin antagonists may reflect an action on endothelial NO synthase. PMID- 1724528 TI - CAS 936, a novel syndnonimine with direct vasodilating and nitric oxide-donating properties: effects on isolated blood vessels. AB - Organic nitrates and sydnonimines exert their vasorelaxant activity by a common mechanism of action, i.e., release of nitric oxide (NO) and stimulation of the soluble guanylate cyclase of vascular smooth muscle cells. We wished to investigate the vasodilating activity of the novel sydnonimine CAS 936 in guinea pig isolated pulmonary arteries without endothelium. CAS 936 had no effect on contractions induced by norepinephrine (NE) or by the PGF2 alpha-analogue U46 619, but induced a longlasting relaxation of potassium depolarized arteries and of A23 187-contracted vessels. This effect was concentration-dependent (IC50 approximately 16 microM). Oxyhemoglobin and methylene blue had no inhibitory effect on CAS 936, whereas they inhibited the relaxations induced by SIN-1, a sydnonimine which acts by releasing NO. These results suggest that the vasodilating activity of CAS 936 is not related to NO. On the other hand, in vivo metabolites of CAS 936 inhibited NE- and U46 619-induced contractions. Oxyhemoglobin inhibited this effect. Therefore, we conclude that the CAS 936 molecule possesses a vasodilating activity of its own, whereas the metabolites may function as NO donors. The primary target of the intrinsic vasodilating activity of CAS 936 is very likely the vascular smooth muscle cell membrane. To determine which mechanism of action (NO unrelated or NO related) contributes mainly to the in vivo effects of CAS 936, studies of the metabolic fate of CAS 936 may be crucial. PMID- 1724529 TI - ULFS-49 causes bradycardia without decreasing right ventricular systolic and diastolic performance. AB - The effects of ULFS-49, a new calcium channel blocker, on right ventricular (RV) systolic and diastolic performance were evaluated in nine anesthetized, closed chest dogs by load-insensitive indexes. ULFS-49 (0.3 mg/kg) decreased heart rate (HR) from 76 +/- 25 to 47 +/- 11 beats/min (p less than 0.01) and cardiac output (CO) from 1.89 +/- 0.62 to 1.42 +/- 0.72 L/min (p less than 0.01), as RV free wall end-diastolic area increased from 486 +/- 126 to 581 +/- 45 mm2 (p less than 0.01) and RV end-diastolic volume increased from 66.6 +/- 26.4 to 85.3 +/- 28.5 ml (p less than 0.05). Pacing at 100 beats/min ablated these hemodynamic and dimensional changes. RV free wall contractility was assessed by the slope and midrange intercept values of the relation between RV end-systolic pressure (Pes) and end-systolic free wall area (Aes) and between RV free wall segmental work (SW) and end-diastolic area (Aed). RV free wall stiffness was measured by exponential fit of the RV end-diastolic pressure (Ped)-Aed points during caval occlusion. With pacing at 100 beats/min, the slope of the Pes-Aes relationship was unchanged by ULFS-49 (0.52 +/- 0.29 vs. 0.60 +/- 0.35 mm Hg/mm2) as was the midrange intercept (382.3 +/- 114.7 vs. 387.1 +/- 121.5 mm2). After administration of ULFS-49, the slope of the SW-Aed relation increased from 31.8 +/- 14.4 to 37.3 +/- 17.7 mm Hg.mm2 (p less than 0.05) without changing the midrange intercept (410.5 +/- 108.1 mm2 vs. 413.0 +/- 107.0 mm2). Similarly, neither the position nor curvature of the Ped-Aed relation was changed by ULFS 49. These data demonstrate that ULFS-49 causes significant bradycardia and increases the size of the right ventricle without directly depressing RV free wall systolic or diastolic performance. PMID- 1724530 TI - Skeletal muscle blood flow and O2 uptake during intravenous nicotine with and without hypertension. AB - The mechanism by which nicotine causes peripheral vasoconstriction and its relationship to the increased risk of peripheral vascular disease in smokers are unknown. To study the peripheral vascular effects of nicotine, we measured hemodynamic responses and oxygen consumption of the in situ gracilis muscle during intravenous (i.v.) nicotine infusions in anesthetized dogs. Nicotine 36.0 micrograms/kg/min increased gracilis artery pressure (Pga) 91 +/- 17% and muscle vascular resistance (MVR) 96 +/- 18% whereas muscle blood flow (MBF) and oxygen consumption (MVO2) were unchanged from baseline. In dogs with extracorporeal controlled normotension during nicotine infusion, however, Pga was held at baseline levels but similar increases in MVR were observed (95 +/- 11%) as flows decreased 52 +/- 9%. Oxygen consumption decreased in direct proportion (53 +/- 5%) to MBF, indicating complete impairment of oxygen extraction (A-VO2). Thus impaired oxygen extraction was masked in dogs in which Pga was allowed to increase because of sustained pressure-dependent flows. Phenoxybenzamine block of muscle alpha-adrenoceptors increased MBF and MVO2 in both normotensive and hypertensive dogs. Combined alpha- and beta-blockade effectively neutralized all sympathoadrenal responses in the muscle. All the above results occurred regardless of innervation. Plasma levels of norepinephrine (NE) and epinephrine (EPI) increased greater than 1,000% during nicotine infusion. Apparently, these levels were high enough to (a) override dilator effects of plasma EPI and (b) cause vasoconstriction in muscle independent of nerve supply. Infusion of nicotine in the gracilis artery had no effect on muscle hemodynamics. Nicotine induced increase in plasma catecholamines resulted in a powerful constriction of both resistance and oxygen-exchange vessels of skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724531 TI - Effects of nifedipine on global myocardial ischemic injury in hearts from diabetic and age-matched control rats. AB - The purpose of this study was to examine the protective activity of a low concentration of nifedipine (3 x 10(-8) M) against global myocardial ischemic injury in isolated perfused hearts from streptozotocin (STZ) diabetic rats (DR) as compared with control rats (CR). Hearts were subjected to 45-min global ischemia followed by 45-min reperfusion. During ischemia, the period of time until onset of the ischemic contracture was significantly longer in hearts from DR than in hearts from CR (20.3 +/- 1.0 and 15.5 +/- 0.5 min, p less than 0.05). The degree of the ischemic contracture was similar in both types of hearts. During reperfusion, a significantly smaller recovery of left ventricular pressure (LVP) was observed as was a tendency for postischemic coronary flow (CF) to be lower in hearts from DR than in those from CR. After pretreatment with nifedipine, the time of onset of the ischemic contracture was significantly more delayed in hearts from DR than in those from CR: from 20.3 +/- 1.0 to 28.1 +/- 1.2 min (p less than 0.05) and from 15.5 +/- 0.6 to 18.1 +/- 0.9 min (p less than 0.05), respectively. In addition, the degree of the ischemic contracture was reduced (45.3%) in hearts from DR but not in those from CR. During reperfusion, the CF was increased only in hearts from DR. No beneficial effects on functional recovery of LVP were observed in either type of hearts, although recovery tended to be better in diabetic hearts. Nifedipine in a low concentration appears to be more effective against myocardial ischemic injury in diabetes, resulting in an improvement in postischemic CF.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724532 TI - Hypotensive and bradycardic effects elicited by spinal dopamine receptor stimulation: effects of D1 and D2 receptor agonists and antagonists. AB - In anesthetized rats, intrathecal (i.t.) administration, at the upper thoracic level of the spinal cord of fenoldopam (a selective dopamine D1-receptor agonist) or quinpirole (a selective D2-receptor agonist) decreased blood pressure (BP) and heart rate (HR) in a dose-dependent manner. Apomorphine, a nonselective DA receptor agonist, produced similar effects. Apomorphine-induced hypotension was competitively antagonized by either SCH 23390 or remoxipride, selective D1- and D2-receptor antagonists, respectively, but only remoxipride antagonized the bradycardia. Furthermore, SCH 23390 antagonized the hypotensive effect of fenoldopam but did not change that induced by quinpirole. Remoxipride antagonized the hypotensive effect of quinpirole but did not alter the hypotensive effect of fenoldopam. Quinpirole-induced bradycardia was antagonized only by remoxipride. Bradycardia elicited by fenoldopam did not appear to be generated by dopamine receptor stimulation, as suggested by the lack of blocking effects of SCH 23390 and remoxipride. Data obtained with fenoldopam were corroborated with use of SK&F 38393, another dopamine D1-receptor agonist. We conclude that hypotensive effects of i.t.-administered DA receptor agonists appear to result from activation of spinal D1- and D2-receptors whereas bradycardia is related only to activation of spinal D2-receptors. PMID- 1724533 TI - Characterization and modulation of antigen-induced effects in isolated rat heart. AB - The response to antigen (trinitro-phenyl-haptenized ovalbumin) and the modulatory role of several antiallergic drugs was studied in isolated hearts from actively sensitized rats. Antigen induced a triphasic effect on coronary flow (CF) and left ventricular pressure (LVP) characterized by short-term increase (0-1.5 min = phase 1) and a severe decrease (1.5-7.5 min = phase 2) followed by a less pronounced long-lasting decrease (7.5- greater than 20 min = phase 3). The first phase was accompanied with a substantial release of 5-hydroxytryptamine (5-HT), histamine, and leukotrienes measured in cardiac effluents. The histamine2 (H2) receptor antagonist cimetidine (60 microM) reversed the antigen-induced increase in CF to a decrease. In contrast, H1-receptor blockade by mepyramine (6 microM) had no effect. Methysergide (10 microM) and ketotifen (0.1 microM) evoked a mild suppression during all three phases. Indomethacin (10 microM) was almost inactive while tolfenamic acid (1 microM) was slightly active in this respect during phase 2. Addition of the 5-lipoxygenase inhibitor AA 861 (1 microM) resulted in complete suppression of the antigen-induced decrease in CF. The leukotriene antagonist FPL 55712 (5 and 50 nM) evoked a dose-dependent suppression with respect to the anaphylactic phases 2 and 3. A similar reduction was obtained with sodium cromoglycate (1 mM). AA 861, FPL 55712, and sodium cromoglycate also suppressed the antigen-induced decrease in LVP. The antigen-induced histamine release was not affected by the aforementioned drugs. Our results provide evidence that H2-receptor blockade during cardiac anaphylaxis enhances coronary constriction and may be detrimental in this condition. On the other hand, leukotriene antagonists and 5-lipoxygenase inhibitors may exert beneficial effects during cardiac anaphylaxis. Further experiments in this area are needed to clarify the precise role of mast cell-generated mediators in cardiac anaphylaxis possibly leading to new therapeutic approaches in this life threatening disorder. PMID- 1724534 TI - Differences between rat and guinea pig aorta in postreceptor mechanisms of alpha 1-adrenoceptors. AB - The relative importance of different postreceptor mechanisms associated with alpha 1-adrenoceptor activation in guinea pig aorta and rat aorta was compared. In both tissues, a concentration-dependent correlation was observed between contractile responses produced by high concentrations of norepinephrine (NE) and inositol-1-phosphate (IP1) accumulation. Blockade of Ca2+ entry through voltage dependent membrane channels by nifedipine had no inhibitory action on NE-induced contractile responses in guinea pig aorta, but significantly inhibited NE-induced contractile responses in rat aorta. Nifedipine had no major effect on NE-induced IP1 accumulation in either tissue. A medium with no Ca2+ inhibited NE-induced contractile responses and IP1 accumulation in guinea pig aorta, but had a more marked effect on contractile responses and IP1 accumulation in rat aorta. The combination of a high concentration of nifedipine and a medium with no Ca2+ almost completely inhibited NE-induced contractile responses and IP1 accumulation in both tissues. Exposure to EGTA also virtually completely inhibited NE-induced contractile response and IP1 accumulation in both tissues. Significant 45Ca2+ entry was stimulated in rat aorta by NE, and this entry was completely blocked by nifedipine. No significant 45Ca2+ entry was stimulated by NE in guinea pig aorta. We conclude that the most important postreceptor event linked to alpha 1 adrenoceptor stimulation in guinea pig aorta is activation of the phosphatidylinositol pathway, whereas in rat aorta Ca2+ entry through voltage dependent membrane channels is as important as activation of the phosphatidylinositol pathway. We also conclude that activation of the phosphatidylinositol pathway in both tissues is critically dependent on the presence of a small amount of Ca2+, which may enter from the external medium. PMID- 1724535 TI - Effects of nitroglycerin and nitroprusside on vascular capacitance of anesthetized ganglion-blocked dogs. AB - To determine whether changes in vascular capacitance induced by nitroglycerin (NTG) and nitroprusside were due to changes in compliance or unstressed vascular volume, doses producing similar reductions in arterial pressure (Psa) were studied on separate days in six dogs anesthetized and ventilated with pentobarbital after splenectomy during ganglion blockade with hexamethonium. Mean circulatory filling pressure (Pmcf) was determined during transient circulatory arrest induced by acetylcholine at baseline blood volumes and after increases of 5 and 10 ml/kg. Central blood volumes (CBVs, pulmonary artery to aortic root) were determined from transit times, and separately measured cardiac output (CO) was estimated by thermodilution (right atrium to pulmonary artery). NTG and nitroprusside produced similar reductions in Psa and Pmcf without significantly altering right atrial pressure (Pra), pressure gradient for venous return, or CO. Total vascular compliance was not altered, but total vascular capacitance was increased on an average of 4.0 +/- 1.4 ml/kg after NTG and 3.0 +/- 1.3 ml/kg after nitroprusside by increases in unstressed volume. Both drugs caused a variable reduction in CBV, averaging 2 ml/kg. Thus, both drugs produced a large increase in peripheral venous capacitance by increasing unstressed vascular volume without altering total vascular compliance. PMID- 1724536 TI - Differences in infarct size with lidocaine as compared with bretylium tosylate in acute myocardial ischemia and reperfusion in pigs. AB - The effects of the antiarrhythmic drugs lidocaine and bretylium tosylate on myocardial necrosis were studied in anesthetized pigs subjected to 60-min coronary occlusion followed by 3-h reperfusion. Group A (n = 7) received lidocaine (average dose +/- SD = 1,828 +/- 515 mg) before and during coronary occlusion and after reperfusion; the other series (group B, n = 7) received bretylium tosylate (3,457 +/- 1,323 mg). Infarct size was 16.3 +/- 14.7% in the lidocaine group as compared with 68.6 +/- 12.6% (p less than 0.01) in the bretylium group. In vitro release of superoxide anion from porcine granulocytes was studied using the lucigenin-dependent chemiluminescence technique. Lidocaine significantly reduced the peak chemiluminescence response to zymosan from 3.34 +/ 0.44 without lidocaine to 2.23 +/- 0.46 (p less than 0.01) and 1.06 +/- 0.17 mV (p less than 0.001), with lidocaine concentrations of 20 and 200 micrograms/ml, respectively. Bretylium had no effect on the chemiluminescence response. Adherence of porcine granulocytes to plastic was also reduced from 332 +/- 32 cells/mm2 (no lidocaine) to 247 +/- 35 and 206 +/- 26 cells/mm2 with lidocaine concentrations of 20 and 200 micrograms/ml, respectively (p less than 0.001). Bretylium had no significant effect. Eight additional bretylium-treated pigs received either rabbit antiporcine granulocyte serum (group C, n = 4) to reduce circulating granulocytes or nonimmune serum (group D, n = 4). Infract size in the granulocyte-depleted pigs was 26.6 +/- 5.6% as compared with 51.4 +/- 5.5% in pigs that received nonimmune serum (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724537 TI - Mechanism of hypoxia-induced alteration in cerebral arteriolar tone in rats. AB - We studied the changes in pial arteriolar diameter during hypoxia and 60 and 120 min after restoration of normoxia, using the closed cranial window technique in artificially ventilated and normocapnic rats. Pial arteriolar diameter increased 16 +/- 4% during hypoxia (PaO2 less than 25 mm Hg, lasting 10 min). Although blood pressure (BP), heart rate (HR), and blood gases returned to prehypoxic level when measured at 60 and 120 min after hypoxia, pial arterial diameter decreased significantly (13 +/- 5 and 16 +/- 6% below control levels, respectively). This posthypoxic vasoconstriction was reversed by treatment of the brain surface with L-660,711 (10(-5) M), a specific leukotriene D4 receptor antagonist. These data suggest that leukotrienes may be involved in delayed cerebral vasoconstriction that follows a brief period of hypoxia. PMID- 1724538 TI - Comparative cardiac effects of KT-362 and verapamil in isolated heart- correlation to calcium channel current depression. AB - Direct cardiac effects of KT-362 (5-[3 [[-2-(3,4-dimethoxyphenyl)-ethyl]amino]-1 oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate), a drug that may inhibit intracellular calcium mobilization as well as extracellular calcium influx was compared to verapamil. Guinea pig hearts (n = 19) were used to examine the changes in atrial rate, atrioventricular conduction time (AVCT), coronary flow, myocardial oxygen consumption (MVO2), and isovolumetric left ventricular pressure (LVP). Both drugs concentration-dependently and reversibly decreased atrial rate, contractility, and MVO2; AVCT increased during spontaneous rhythm. The increases in AVCT and the incidence of AV dissociation were accentuated during cardiac pacing. Verapamil significantly increased coronary flow, while KT 362 did not. Median effective concentration (EC50) was about 25 times lower for verapamil in depressing LVP and about three times lower in depressing atrial rate and AV conduction. The changes in calcium channel current in voltage-clamped single canine Purkinje cells (n = 6) were also examined. Verapamil (0.3 microM) and KT-362 (7 microM) decreased peak Ca2+ channel current at maximum activation (+10 mV) by 38.1 +/- 8% and 28.6 +/- 6%, respectively, without shifting the current-voltage relationship. This study indicates that verapamil is more potent than KT-362 in depressing contractile function, heart rate, and AV conduction in isolated hearts and calcium current in isolated cardiac Purkinje cells. Moreover, there was a much greater difference between the EC50 for verapamil and that for KT-362 for the depression of indices of contractility (23-30-fold) than for the depression of sinoatrial and atrioventricular nodal function (2.5-4-fold). PMID- 1724539 TI - Implication of the central nervous system in the systemic and regional hemodynamics of two centrally acting hypotensive drugs, flesinoxan and clonidine, in the rat. AB - The systemic and regional hemodynamic effects of the selective 5-HT1A receptor agonist flesinoxan (3-300 micrograms/kg, i.v., in cumulative doses) were investigated in normotensive anesthetized and pithed rats using a pulsed Doppler system and were compared to those of the alpha 2-adrenoceptor agonist clonidine. In anesthetized rats, flesinoxan and clonidine induced dose-dependent decreases in blood pressure and heart rate. Total peripheral resistance and hindquarters vascular resistance dose-dependently decreased after flesinoxan administration whereas cardiac output remained unchanged. Clonidine dose-dependently decreased cardiac output and did not change total peripheral resistance. These results indicate that the decrease in blood pressure induced by flesinoxan is due to a reduction in total peripheral and hindquarters vascular resistance. In contrast, clonidine decreased blood pressure by reducing cardiac output. In the pithed rat, the systemic and regional hemodynamic effects of flesinoxan were abolished whereas those of clonidine were reversed. These results provide evidence for the participation of the central nervous system in the systemic and regional hemodynamic effects of flesinoxan. However, direct administration into the central nervous system remains to be performed in order to strengthen this conclusion. PMID- 1724540 TI - The acute hemodynamic, hormonal, and pharmacokinetic properties of oral spirapril in patients with moderate to severe heart failure. AB - The acute hemodynamic, hormonal, and pharmacokinetic responses to the oral angiotensin-converting enzyme (ACE) inhibitor spirapril were studied in 15 patients with moderate to severe congestive heart failure in a baseline controlled dose-ranging study. Doses of 0.3, 1.0, 1.5, 3.125, and 6.25 mg were investigated for 24 h in three groups of five patients each. All doses demonstrated a significant reduction in serum ACE, even after 24 h. Significant reductions in mean arterial pressure, systemic vascular resistance, and pulmonary capillary wedge pressure were observed with doses greater than 1.0 mg spirapril. Maximal significant hemodynamic effects occurred approximately 4-6 h after drug administration. The plasma concentrations of spirapril and its metabolite spiraprilate were dose-dependent. After administration of spirapril, the quick rise to the peak level of spiraprilate suggests rapid metabolism of spirapril into spiraprilate and a slow elimination of this metabolite. No severe hypotension or other serious side effects occurred in the patients studied. The results indicate that spirapril may be expected to be an effective drug in the treatment of congestive heart failure. From our findings we conclude that 1.5 mg spirapril is an optimal starting dose in patients with moderate to severe congestive heart failure. PMID- 1724541 TI - Age-dependent vasodilation of the skin microcirculation by atrial natriuretic factor. AB - The microcirculatory responses to systemically administered alpha-human atrial natriuretic factor [99-126)hANF) (ANF) were investigated in the skin of eight young (18-25 years) and eight elderly (71-84 years) healthy volunteers in a double-blind, randomized, placebo-controlled study. Laser-Doppler flux (LDF), transcutaneous oxygen pressure (TcPO2), and skin temperatures on the finger (Td) and on the cheek (Tc) were measured during 60 min of i.v. ANF administration at a low rate (0.25 micrograms/min), resulting in plasma ANF concentrations in the upper normal range, and at a high rate (2.0 micrograms/min), inducing a 17- to 23 fold increase in plasma ANF. ANF infusion did not induce consistent changes in TcPO2 or Td at either rate. Placebo-corrected LDF increased significantly following low-rate ANF infusion in the elderly but remained unchanged in the young (mean +/- SD) +22 +/- 21% (p less than 0.05) and +7 +/- 32% (p greater than 0.1), respectively; following high-rate ANF infusion in LDF the changes were greater, with +28 +/- 56% and +19 +/- 42%, respectively. Because of the large SD the increases were no longer significant. During high-rate ANF infusion Tc remained unchanged in the young, but in the elderly Tc decreased--1.1 +/- 1.3% (p less than 0.05), which is probably due to the fall in blood pressure, which was more pronounced in the elderly than in the young: mean arterial blood pressure (MAP) decreased -9 +/- 3% (p less than 0.05) in the elderly and just -6 +/- 3% (p less than 0.05) in the young, both corrected for placebo changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724542 TI - Hemodynamic and morphological effects of quinapril during genetic hypertension development. AB - The relative contributions of the hemodynamic and morphological (vascular and cardiac) modifications induced by long-term administration of an angiotensin I converting enzyme inhibitor, quinapril, to the drug's long-lasting preventive effects vis-a-vis genetic hypertension development (GHD) have been investigated in young spontaneously hypertensive rats (SHRs). Two groups of SHRs were given quinapril (10 mg/kg/day) or distilled water from 5 to 20 weeks of age. The drug was then stopped, but observations continued for another 7 weeks. At selected times systemic and regional hemodynamic parameters as well as cardiac and vascular morphological effects were investigated. During the treatment period, quinapril partially opposed GHD and limited the early rise in total peripheral and regional vascular resistances observed in control animals. Quinapril's partial preventive effect vis-a-vis GHD persisted, but faded after treatment withdrawal. From a morphological point of view, quinapril strongly opposed aortic wall hypertrophy as evidenced by significant reductions in media thickness and wall to lumen ratio and by a significant increase in aortic nuclear density. Quinapril also limited vascular fibrosis development. At the cardiac level, quinapril reduced heart weight to body weight ratio and opposed myocardial hypertrophy and cardiac collagen synthesis. All these vascular and cardiac morphological changes were delayed (starting after 9-15 weeks of treatment) as compared to quinapril's hemodynamic effects. Finally, the drug's vascular and cardiac antihypertrophic properties persisted after treatment withdrawal. In conclusion, our data indicate that the early systemic and regional hemodynamic effects of quinapril initiate its antihypertensive action, but the drug-induced delayed and prolonged vascular morphological changes later take over and may be partly responsible for quinapril's residual blood pressure lowering effects after treatment withdrawal. PMID- 1724543 TI - Enalapril, an inhibitor of angiotensin converting enzyme, increases the junctional conductance in isolated heart cell pairs. AB - The effect of enalapril and angiotensin II on junctional conductance (gj) of isolated rat heart cell pairs was investigated. It was found that enalapril (1 micrograms/ml) increases gj by 106 +/- 3.1% (SEM) (n = 20) within 4 min. The effect of enalapril on gj was not suppressed by propranolol (10(-6) M) or by a cAMP-dependent protein kinase inhibitor. Angiotensin II (1 micrograms/ml) reduced gj by 55%. These observations might indicate that an intrinsic renin-angiotensin system in heart is involved in the control of gj in cardiac muscle. PMID- 1724544 TI - Single incubation double esterase cytochemical reaction using a single coupling reagent. AB - Most methods used in double esterase cytochemistry for the diagnosis and classification of acute myeloid leukaemias require double incubation and staining, using separate coupling reagents. We evaluated a method by Swirsky on our normal and abnormal blood and bone marrow smears where only a single incubation and the use of a single coupling reagent is required. Its short incubation period and its strong positive reaction for butyrate esterase in demonstrating cells of monocytic lineage gives it an advantage over the conventional double incubation technique. PMID- 1724545 TI - Surgical rehabilitation. PMID- 1724546 TI - Fluorescein diacetate and ethidium bromide staining to determine the viability of Mycobacterium smegmatis and Escherichia coli. AB - The ability of the fluorescein diacetate and ethidium bromide fluorescent staining method to assess the percentage of viable bacterial cells in suspension was compared with the plate counting method. Mycobacterium smegmatis and Escherichia coli bacterial cell suspensions were incubated at 60 degrees C. At different time intervals samples were taken and the percentage of viable cells in each sample was assessed by the fluorescent staining method and compared with the plate counting method. The fluorescent staining method showed a positive correlation with the plate counting method. However, the viable counts by the plate counting method were lower than the staining method when incubated at 60 degrees C, indicating a lag period in the decay of enzymes after bacterial death. Hence, the fluorescent staining technique can be used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli. PMID- 1724547 TI - The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins. AB - The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L2 whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha-L fucosidase prevented binding of only UEAI to the two proteins while beta galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [3H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [3H]galactose did. PMID- 1724548 TI - Molecular cloning and sequence analysis of a Rickettsia tsutsugamushi 22 kDa antigen containing B- and T-cell epitopes. AB - The identification of Rickettsia tsutsugamushi T-cell epitopes is necessary for the characterization of the protective immune response (of which the T-cell response is essential) against scrub typhus rickettsiae. A T-helper cell line derived from R. tsutsugamushi (Karp strain) immune mice reacted with rickettsial protein antigens eluted from the 18-35 kDa region of polyacrylamide gels. Within this region is a 22 kDa protein which is reactive with immune serum. The gene encoding the 22 kDa scrub typhus antigen (sta22) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the sta22 gene revealed a potential open reading frame (ORF) in the sta22 sequence encoding a 22 kDa protein. A recombinant 22 kDa protein synthesized in E. coli maxicells was reactive with anti-rickettsial antibodies. The codon usage of the adenine and thymine rich sta22 sequence was similar to other previously sequenced R. tsutsugamushi genes. Computer analysis of the deduced amino acid sequence suggested that the Sta22 protein has several amphipathic regions which may be potential T-cell epitopes. The recombinant Sta22 protein eluted from polyacrylamide gels induced a strong proliferative response from the scrub typhus rickettsiae reactive T-cell line. Recognition of the R. tsutsugamushi Sta22 polypeptide by both cellular and humoral immune mechanisms implicates this antigen as one of potential importance in vaccine development. PMID- 1724549 TI - Molecular determinants of retrovirus epidemiology: the double frameshift hypothesis. PMID- 1724550 TI - Protective effect of human granulocyte colony-stimulating factor (hG-CSF) on Cryptococcus and Aspergillus infections in normal and immunosuppressed mice. AB - Prophylactic treatment with human granulocyte colony-stimulating factor (hG-CSF) affords significant protection against systemic aspergillosis or pulmonary aspergillosis in neutropenic (cyclophosphamide-treated) mice but not in cortisone treated animals. Cryptococcosis does not respond to hG-CSF therapy. Our data show that granulocytes play an important role in the immune defense against aspergillosis, but not against cryptococcosis. Combined treatment using hG-CSF and conventional antimycotics shows a significant beneficial effect in systemic or pulmonary aspergillosis. PMID- 1724551 TI - Clinical isolates of yeast produce a gliotoxin-like substance. AB - Candida infections are major causes of morbidity in compromised human hosts, but our understanding of the virulence of Candida remains incomplete. The possibility that toxic fungal metabolites belonging to the chemical class epipolythiodioxopiperzine (ETP), which are reported to possess immunomodulating and antiphagocytic properties may be produced by Candida species was investigated. Reversed phase HPLC analysis of flash evaporated chloroform extracts of 7 day cultures of clinical Candida isolates grown in Minimal Essential Medium (MEM) with 5% fetal calf serum revealed the presence of a compound which eluted at the same time as the ETP, gliotoxin. Of 50 strains of yeast tested, 32 produced this gliotoxin-like material. This material was tested for other properties of ETP type toxins including the presence of mercaptans (Ellman reaction), ultraviolet absorbance spectrum and antibacterial activity against Micrococcus lutea. These tests revealed gliotoxin-like material from yeast cultures to be similar to commercially supplied gliotoxin. This represents the first report of the presence of ETP-like compounds in yeast and raises the possibility that ETP's may contribute to the virulence of the organism. PMID- 1724552 TI - Degradation products of myelin-oligodendrocyte-associated proteins in a light CNS subcellular fraction. AB - The presence of degradation products of the myelin/oligodendrocyte glycoprotein (MOG) and a new myelin/oligodendrocyte associated protein, FD1, defined by a monoclonal antibody was established in a subfraction (the floating fraction, or FF) of adult rabbit CNS. The histochemical distribution of FD1 was determined by indirect immunofluorescence using conventional and confocal microscopy. FD1 was found to be present in oligodendrocytes, and at the outer rim of CNS myelin sheaths. Strong antibody reactivity was noted at nodes of Ranvier, as well as in regions with a high nodal density. No staining of compact myelin was seen. In the PNS, inner and outer cytoplasmic compartments of the Schwann cells as well as their cell bodies were stained, with no staining of compact myelin. The FF has previously been shown to be highly enriched in Marchi-positive bodies. These structures are situated paranodally in the CNS of myelinated nerve fibers, and their presence has been interpreted as reflections of myelin breakdown and turnover occurring in association with myelin sheath segments situated close to nodes at Ranvier in adult, normal vertebrate CNS. The present findings extend previous observations of partially degraded myelin-associated proteins in the FF, and give further results indicating that Marchi-positive bodies are aspects of intermediate stages in myelin catabolism. PMID- 1724553 TI - Effect of guanidinoethanesulfonic acid on brain monoamines in the mouse. AB - Guanidinoethanesulfonic acid (GES) is known to induce convulsive seizures when administered intracisternally to rabbits and cats. The effects of GES on behavior, electroencephalographic recording and brain monoamine levels were examined in mice. When GES (900 nmol) was intraventricularly injected into mice, focal clonic movements of the face, vibrissae and ears together with twitching of the limbs were observed 0.5-1 min after the injection. Hypersensitivity was observed up to 7 min after the injection, after which the mice behaved normally. GES also induced sporadic spike discharges on electrocorticogram. The levels of 5 hydroxytryptamine (5-HT) of the GES-injected mice were lower than those of the saline-injected mice in the hippocampus, diencephalon, pons-medulla oblongata and cerebellum 5 min after the injection. No changes in the norepinephrine or dopamine levels were found after the GES injection. The level of 5 hydroxyindoleacetic acid increased in the striatum and cerebellum 5 min after the GES injection. These results suggest that GES-induced convulsive activities enhance the serotonergic neuroactivity in order to suppress the convulsions. PMID- 1724554 TI - Effect of 2-guanidinoethanol on levels of monoamines and their metabolites in the rat brain. AB - The contents of monoamines and their metabolites in rat brains 3 hours after the intracerebroventricular injection of 6 mumol of 2-guanidino-ethanol (GEt) were measured by HPLC. GEt which is a configurational analogue of 4-aminobutanoic acid (GABA) induced severe running fits and tonic-clonic convulsions as well as epileptic discharges. In GEt-administered rats, dopamine (DA) decreased in the cortex, hippocampus and hypothalamus. 3,4-Dihydroxyphenylacetic acid (DOPAC) increased to about the same level in all brain regions, therefore the distribution of DOPAC appeared to be homogeneous in the brain. The homovanillic acid levels also increased in the striatum and hippocampus. No significant change in the norepinephrine contents was observed in any region. The turnover ratio of DA increased significantly except in the striatum. Serotonin levels increased in the hypothalamus and midbrain by GEt administration, though 5-hydroxyindoleacetic acid levels showed no change in any of the brain regions. These data suggest that the activity of dopaminergic and serotonergic neurons are increased by GEt. PMID- 1724555 TI - Serotonin-positive fibers within the spinal motor nucleus of the newborn rat, with special reference to co-localization of substance P. AB - Developmental changes of serotonin-positive fibers with special reference to co localization with substance P was examined immunohistochemically in the ventral horn of rat lumbar spinal cord. Only about 20% of serotonin-positive fibers co labelled with substance P on postnatal day (P) 0. The ratio of co-localization gradually increased, and reached the adult value by P28 (60-70%). Enkephalin positive fibers were not co-localized with serotonin at any age examined. Although the densities of serotonin, substance P and enkephalin per unit area of the ventral horn gradually increased with development up to P28, the density of serotonin in the adult was decreased compared to P28 animals. PMID- 1724556 TI - Evidence for acute tolerance to the behavioral effects of lindane: concomitant changes in regional monoamine status. AB - The effects of the organochlorine insecticide lindane on plus-maze ((+)-maze) behavior of rats and on regional monoamine status were studied at two time points, 30 min and 24 hr post-dosing. Animals were given lindane, 20 mg/kg, 30 min before (L1/2 group), or 40 mg/kg, 24 hr before (L24 group), experimental time; these schedules allowed the study of animals with equivalent brain concentration of lindane at two different time points after administration. The (+)-maze results indicated a reduction in the number of entries into the arms of the maze 30 min after administration of lindane that was not present 24 hr later, suggesting the development of an acute tolerance to the behavioral effects of the chemical. In a parallel group of animals, concentrations of noradrenaline (NA), serotonin (5HT), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by HPLC in seven brain regions. Significant decreases in NA concentrations were found in L1/2 animals in hippocampus and cerebral cortex and also in the L24 group in the latter region. 5HT/5HIAA ratios increased in several brain regions in the L24 rats but not in the L1/2 rats, thus showing an inverse relationship with behavioral effects. DOPAC/DA and HVA/DA ratios in hypothalamus and cerebral cortex were, by contrast, increased in both groups of treated animals. In conclusion, it appears that an acute tolerance can develop to the behavioral effects of lindane, and that there are modifications produced in central monoaminergic systems by lindane that show brain region and and treatment schedule specificity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724557 TI - Gene probe for P0 messenger RNA used to index acrylamide toxic neuropathy in rats. AB - Cumulative exposure to the neurotoxicant acrylamide produces axonal damage in the distal ends of both central (CNS) and peripheral (PNS) nerve fibers and subsequent hind-limb paralysis. The messenger RNA which codes for the PNS myelin glycoprotein P0 (P0-mRNA) was used to monitor this toxic neuropathy in Sprague Dawley rats prior to, concurrent with, and subsequent to, ultrastructurally and immunocytochemically defined nerve damage. Rats were dosed every other day with acrylamide (50 mg/kg, IP) and sampled intermittently throughout a 4 week exposure period. Slot blot and Northern gel analyses of the proximal and distal sciatic nerve were used to determine a quantitated measure of P0-mRNA. Twenty-four hours after the first treatment, in the absence of ultrastructural damage, P0-mRNA increased 55% over control levels in the distal sciatic nerve. After 12 treatments, and concomitant with the appearance of spinal cord and PNS neuropathic damage and hindlimb dysfunction, P0-mRNA decreased 45% below control levels. Levels of P0-mRNA from rats exposed to 12 treatments of acrylamide but allowed to recover for 40 days, returned to 79% of control values to reflect the regeneration and remyelination occurring in the distal sciatic nerve. In spite of these fluctuations in levels of P0-mRNA, immunocytochemical staining of P0 protein in plastic sections of the distal sciatic nerve was present throughout all sample times. These results suggest that changes in neural specific mRNAs are sensitive to neurotoxic damage and can be used to monitor the pathogenesis of nerve degeneration. PMID- 1724558 TI - Possible excitatory and inhibitory feedback to the superior colliculus: a combined retrograde and immunocytochemical study in the prepositus hypoglossi nucleus of the guinea pig. AB - We have investigated in the guinea pig the precise localization and the immunoreactivity of the neurones in the prepositus hypoglossi nucleus involved in a direct ascending projection onto the superior colliculus. The projecting neurones were characterized by a retrograde tracer (WGA-ApoHRP coupled to gold particles), injected in the intermediate and deep layers of the superior colliculus. After revealing gold particles, the sections were then treated using an antibody either against GABA or against glutamate, thus allowing identification of gold-filled-immunoreactive neurones. The retrogradely labelled cells were exclusively distributed on the contralateral side, and preferentially in the caudal two thirds of the prepositus hypoglossi nucleus, in its ventral and ventrolateral division. In addition, about 23% of these projecting neurones appear immunopositive when the sections are treated with a GABA antibody and around 27% are immunopositive to glutamate. Furthermore, these two classes of GABA-like or glutamate-like projecting neurones are intermingled within the prepositus hypoglossi nucleus. We conclude, in spite of a probable underestimation of these two populations, that the ascending projection is formed by an excitatory pathway that probably involves glutamate as well as an inhibitory pathway mediated by GABA. Thus we cannot consider this feedback as exclusively inhibitory as was suggested in theoretical models of the oculomotor system. PMID- 1724559 TI - Glutamate receptor agonist-induced inward currents in spinal dorsal horn neurons dissociated from the adult rats. AB - Inward currents to glutamate receptor agonists, quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA) were examined in spinal dorsal horn neurons by whole-cell voltage-clamp techniques after acute dissociation. Neurons were dissociated from the superficial dorsal horn (laminae I/II) of the adult rat (8 16 weeks old) spinal cords by enzymatic and mechanical treatment. The KA-induced current was sustained during KA application, while the QA- and NMDA-induced currents were attenuated. The NMDA response was augmented dose-dependently by addition of glycine (10(-7)-5 X 10(-6) M) and became obscure in the absence of glycine. The NMDA current was depressed by D-2-amino-5-phosphonovaleric acid (APV). Analyses of dose-response curves of these inward currents indicate that both the QA and KA currents were competitively blocked by 6-cyano-7 nitroquinoxaline-2,3-dione (CNQX), while the NMDA current was blocked non competitively. PMID- 1724560 TI - Origin of genetically encoded protein synthesis: a model based on selection for RNA peptidation. AB - The difficulty in explaining the origin of genetic coding centres on the need to identify selective advantages that could account for the synthesis of peptidyl tRNA, the essential intermediate in genetically programmed translation. It is resolved by a recognition of the functional advantages derivable from the post transcriptional addition of peptide cofactors to RNA apo-catalysts. This enables the formulation of a theory for the origin of the genetic encoding of protein synthesis by RNA. PMID- 1724561 TI - [Carcinoid of the larynx]. AB - The case of carcinoid of the larynx is presented. Attention is focused on the difficulties with establishing the diagnosis. The principles of treatment this kind of neoplasm are reported. PMID- 1724562 TI - Regional variation in oral mucosal drug absorption: permeability and degree of keratinization in hamster oral cavity. AB - The regional permeability of oral mucosa to salicylic acid was investigated in vivo in hamsters along with histological variations, especially the degree of keratinization. Histological sections from six regions, i.e., sublingual mucosa, buccal mucosa, dorsum of tongue, ventral surface of tongue, labial mucosa, and cheek pouch mucosa, were prepared to assess the degree of keratinization. The area under the plasma concentration-time curve of salicylic acid following the administration of salicylic acid to the oral mucosa with a film dosage form and the thickness of stratum corneum of each site were in inverse proportion to each other, suggesting that the stratum corneum layer represents the principle barrier to drug absorption. PMID- 1724563 TI - Calcium antagonistic properties of the sesquiterpene T-cadinol: a comparison with nimodipine in the isolated rat aorta. AB - (+)-T-Cadinol is a sesquiterpene with smooth muscle relaxing properties. In the isolated rat aorta, T-cadinol relaxed contractions induced by 60 mM K+ in a concentration-dependent fashion. The dihydropyridine calcium antagonist nimodipine was approximately 4,000 times more potent than T-cadinol. While both drugs nearly abolished the K(+)-induced contractions, they only partially relaxed contractions induced by phenylephrine. The relaxation induced by T-cadinol and nimodipine in K(+)-contracted aortic rings, was completely reversed by the calcium channel activator Bay K8644. In aortic preparations partially depolarized by 20 mM K+, Bay K8644 induced a concentration-dependent contraction. Nimodipine shifted the Bay K8644 concentration-response curve to the right in a parallel manner, consistent with a competitive mode of inhibition. T-cadinol at concentrations less than 10(-3.5) M also produced a right-ward shift of the Bay K8644 concentration-response curve with a maintained maximum response. However, the highest T-cadinol concentration used 10(-3.5 M) significantly reduced the maximum response. In conclusion, although T-cadinol and nimodipine display marked structural differences, their pharmacological profiles of action have several features in common, suggesting that T-cadinol is a calcium antagonist, possibly interacting with the dihydropyridine binding sites on the calcium channels. PMID- 1724564 TI - [An evaluation of the toxic gas pollution in an open-pit mine]. AB - The present work aims at giving a complete characteristic of the toxic chemical factor in the working environment of a pit "Kremikovtsi", for evaluating the occupational risk for the engaged workers. The basic pollution sources of the pit are the eliminating toxic gases during work from different types of machines and heavy-freight vans. The already measured concentrations of carbon monoxide, carbon dioxide, nitric oxides, hydrocarbons, aldehydes and lead, manganese, ferrous and mercury aerosols in all working places of the pit are under MAC. The calculated total index of toxicity for all working places surpasses up to 3 times the unit and is rated. 1st degree--unfavourable conditions of work concerning the toxic chemical factor. PMID- 1724565 TI - Effects of chronic electroconvulsive shock on interstitial concentrations of dopamine in the nucleus accumbens. AB - There is accumulating evidence that chronic electroconvulsive shock (ECS) can increase the functional output of central dopaminergic systems. The present experiments investigated the effects of acute and chronic ECS on interstitial concentrations of dopamine (DA) in the nucleus accumbens (NAC) using in vivo microdialysis in awake freely moving rats. ECS (150 V, 0.75 s) increased interstitial concentrations of DA, DOPAC and HVA to approximately 130% of baseline values. The magnitude of the ECS-induced increase in DA was not affected by chronic ECS. In contrast, the response of the DA metabolites was attenuated in the chronic ECS group. Chronic ECS did not influence apomorphine (25 micrograms/kg, SC)-induced decreases in extracellular concentrations of DA or its metabolites in the NAC, thus providing no support for the hypothesis that chronic ECS produces subsensitivity of DA autoreceptors. d-Amphetamine (1.5 mg/kg SC) induced increases in extracellular DA were significantly prolonged in the NAC of the chronic ECS group. In accordance with previous reports, the locomotor stimulant effects of d-amphetamine were also enhanced in the chronic ECS group. These data provide further evidence that chronic ECS can increase certain behavioral and neurochemical indices of meso-accumbens DA function. PMID- 1724566 TI - PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas. AB - Despite studies indicating the presence of specific pancreatic acinar receptors for PACAP-38, a peptide that was recently isolated from ovine hypothalamus, the actions of the new peptide on pancreatic enzyme secretion have not been examined. The present study demonstrates that in terms of cAMP production and amylase release from dispersed acini from rat pancreatic acini, PACAP-38 and an N terminal fragment, PACAP-27, have the same potency and efficacy as vasoactive intestinal peptide (VIP). As with VIP, these actions are potentiated by adding an inhibitor of cyclic nucleotide phosphodiesterase, and combination of PACAP-38 with bombesin, CCK-8, carbachol or the calcium ionophore A23187 results in 2-fold augmentation of the secretory actions of these agents. Inhibition of PACAP-38 induced cAMP production and amylase release by two VIP-receptor antagonists indicates that the secretory effects of PACAP-38 are mediated by interaction with VIP receptors. PACAP-38, a new brain-gut peptide, may be a physiological modulator of pancreatic enzyme secretion. PMID- 1724567 TI - Segmental distribution of colonic neuropeptides in Hirschsprung's disease. AB - Despite continued research, the pathophysiologic mechanism responsible for functional obstruction in the aganglionic segment of bowel in Hirschsprung's disease remains controversial. Narrowing of the affected segment is thought by many investigators to be the result of loss of intrinsic inhibitory innervation. For this hypothesis to be consistent, inhibitory neuropeptides should be present in the dilating, transitional segment of bowel. In order to quantitate reported changes in peptidergic nerve staining in Hirschsprung's disease, we measured concentrations of five neuropeptides (vasoactive intestinal peptide, peptide histidine-methionine, met5-enkephalin, substance P and bombesin-like immunoreactivity) by radioimmunoassay in the affected segments of bowel from six patients with Hirschsprung's disease. Tissue extracts were prepared using gut obtained at surgery from the: (1) constricted, aganglionic segment, (2) dilating, aganglionic transitional segment and (3) dilated, proximal ganglionic segment. Concentrations of vasoactive intestinal peptide, peptide histidine-methionine, substance P and met5-enkephalin were significantly reduced in both the muscularis externa and the mucosal-submucosal layers from the constricted aganglionic segment. By contrast, concentrations of the candidate inhibitory neuropeptides, vasoactive intestinal peptide and peptide histidine-methionine, were minimally reduced in the dilating, aganglionic transitional segment. These results are consistent with the hypothesis that constriction of the aganglionic segment is due to loss of intrinsic inhibitory innervation. Concentrations of bombesin-like immunoreactivity were similar in the three segments of human gut, suggesting the presence of this immunoreactive neuropeptide in extrinsic nerve fibers. PMID- 1724568 TI - A human monoclonal antibody against the CD4-binding site of HIV1 gp120 exhibits potent, broadly neutralizing activity. AB - A human monoclonal antibody (HuMAb) against HIV1, 1125H, was isolated from an asymptomatic, seropositive haemophiliac. This antibody was specific for gp120, and its binding to gp120 was inhibited by soluble CD4, indicating that its epitope was in or near the CD4-binding site. 1125H antibody recognized a variety of divergent HIV1 strains, including most laboratory strains tested as well as some early passage isolates. Commensurate with its specificity and high apparent affinity, 1125H exhibited potent neutralizing activity against IIIB, MN, RF and SF-2 strains. The epitope recognized by 1125H was destroyed by reduction of disulphide bonds, but not by removal of N-linked sugars. Thus, the epitope was conformationally determined and did not involve carbohydrate. Data from radioimmunoprecipitation/SDS-PAGE analysis of proteolytically cleaved viral lysate further indicated that the epitope of 1125H was not affected by cleavage at the V3 loop of gp120, provided that gp120 disulphide bonds remained intact. The potential use of HuMAb 1125H in passive immunotherapy against HIV is discussed as well as the importance of including its epitope in an AIDS vaccine. PMID- 1724569 TI - Citrus psorosis is probably caused by a bipartite ssRNA virus. AB - Isolate 90-1-1 Concordia (Argentina) of the citrus psorosis agent was graft transmitted to citrus and mechanically transmitted to Chenopodium quinoa, which was used as a local lesion assay host. Infected citrus and C. quinoa plant lesions were used as starting materials for the purification of the psorosis associated agent. In extracts partially purified by differential centrifugation, infectivity was abolished by RNase treatment, even in 0.3 M NaCl, indicating that ssRNA is required for biological activity. The total loss of infectivity produced by proteinase K treatment and the decline in infectivity caused by phenol extraction indicated that protein may be essential for infectivity. When partially purified extracts were subjected to sucrose density gradient centrifugation, infectivity on C. quinoa from certain 2-fraction combinations was higher than expected, compared to the infectivity of the individual fractions. Therefore, infectivity was not associated with a single component but with the combination of at least two components which were distinguishable on sedimentation. The infectious material was present in the top and bottom zones of a sucrose gradient, which on further purification by a second gradient and agarose gel electrophoresis, revealed the presence of a 50-kDa protein. This protein was absent in comparable gradient fractions from healthy plants, and therefore most likely represented the capsid protein of both the top and bottom sucrose gradient zone components. Taken together, these results led to the conclusion that the citrus-psorosis-associated virus (CPsAV) is a multipartite virus, containing ssRNA and a 50-kDa coat protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724570 TI - Antigenic analysis of Plasmodium yoelii liver stages by fluorescence antibody assays. AB - Little is known about the immune response against liver stage antigens which were first described for Plasmodium falciparum. In order to provide a basis for experimental studies, we analysed antigenically the liver stages of Plasmodium yoelii using sera of restricted specificity. Several distinct fluorescence patterns could be described in maturing liver forms. One pattern was identified as corresponding to antigens specific to the liver phase which are also species specific. Another pattern corresponds to sporozoite surface antigens which were predominant in liver trophozoites. Trophozoite-like liver forms were detected at least 7 days after the injection of irradiated sporozoites suggesting that parasites may persist and contribute to the immunity induced by this procedure. PMID- 1724571 TI - Treatment of experimental alveolar echinococcosis: comparative study of mebendazole, isoprinosine and a mebendazole isoprinosine association. AB - A comparative biochemical study of the effects of mebendazole and Isoprinosine used separately or associated was conducted in six-month old gerbils infected three months ago by Echinococcus multilocularis metacestodes. Mebendazole treatment induced a decrease of glucose (81%) whereas Isoprinosine increased the glucose uptake in the parasite. The combination of the two drugs led to a decrease of about 40% of the glucose concentration. The specific activities of alkaline and acid phosphatases were also modified. Isoprinosine seemed more effective when used alone than associated with mebendazole with the chosen procedure of treatment. PMID- 1724572 TI - [Expression of HLA class II antigens on human monocytes and its relation to antigen-presenting function]. PMID- 1724573 TI - [Granule membrane protein 140--a new adhesive protein molecule]. PMID- 1724574 TI - Fluorescent probes as tools to assess the receptor for the urokinase-type plasminogen activator on tumor cells. AB - Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients. PMID- 1724575 TI - [3H]paroxetine binding and serotonin content of rat and rabbit cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region. AB - The high-affinity binding of [3H]paroxetine to membranes was measured in different regions of the rat and rabbit brain: cingulate, frontal, parietal, piriform, entorhinal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum (rat) or caudate nucleus and putamen (rabbit); ventral mesencephalic tegmentum; and midbrain raphe nuclei region. The tissue concentrations of serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxy-l-tryptophan (5-HTP) were also determined by high-performance liquid chromatography (HPLC) in the same brain samples. The regional density of [3H]paroxetine binding varied in both species; the highest values (Bmax) were found in the midbrain raphe region and ventral mesencephalic tegmentum. The cortical values ranged from moderate to low, with a significantly higher density in the cingulate cortex of the rat compared with rabbit. In the rat, there was also a higher density in the ventral than dorsal hippocampus, and the caudal than rostral neostriatum. In the rabbit, the hippocampal and neostriatal values were generally lower and more uniform. In both species, there was an excellent correlation between regional 5-HT levels and specific [3H]paroxetine binding (r = 0.87 in the rat and 0.96 in the rabbit). Considering the available quantitative data on the number of 5-HT nerve cell bodies and axon terminals in different regions of the rat brain, it appears likely that the high amount of [3H]paroxetine binding in the midbrain raphe region and ventral mesencephalic tegmentum reflects the presence of 5-HT uptake sites on 5-HT nerve cell bodies and dendrites as well as axon terminals. In other brain regions, the heterogeneous distribution of [3H]paroxetine binding parallels that of the number of 5-HT axon terminals, emphasizing the potential usefulness of this radioligand as a marker of 5-HT innervation density. PMID- 1724576 TI - On the role of calcium in the acute release of tissue-type plasminogen activator and von Willebrand factor from the rat perfused hindleg region. AB - The involvement of calcium in the release of tissue-type plasminogen activator (t PA) and von Willebrand Factor (vWF) from vascular endothelial cells was studied ex vivo using a rat hindleg perfusion system. By adding either platelet activating factor or bradykinin to the perfusing Tyrode solution, a rapid release of t-PA and vWF was induced. Extracellular calcium was required for the acute release of both glycoproteins as this release was totally abolished in the presence of EGTA. The calcium ionophore A-23187 induced (Ca-dependently) the release of both proteins, suggesting that Ca-influx was also sufficient to induce release. The absence of an effect of the calcium L-type channel blockers, verapamil and diltiazem, and of the calcium channel agonist BAY K-8644, suggested that endothelial voltage-operated calcium channels were not involved in release. Trifluoperazine, a calmodulin antagonist, significantly inhibited the induced release of t-PA and vWF, while the "intracellular calcium antagonist" TMB-8 had no effect. Lanthanum chloride (200 microM) inhibited the induced release of t-PA but not that of vWF. Our results suggest that Ca2+ influx is essential for the release of t-PA and vWF from the perfused rat hindleg. PMID- 1724577 TI - Hemostasis in patients undergoing extracorporeal circulation: the effect of aprotinin (Trasylol). AB - The administration of aprotinin during extracorporeal circulation (ECC) reduces blood loss. To explore the mechanism of this effect, a placebo-controlled double blind study was performed in 20 patients (10 were administered with a high dose of aprotinin, 10 with placebo) undergoing a primary, elective operation of coronary artery bypass grafting (CABG) with ECC. Biological tests were performed at 4 different time points during the operation. A marked reduction in the placebo group of ristocetin-induced platelet agglutination (binding of von Willebrand factor [vWF] to platelet glycoprotein [GP] Ib) was shown during ECC and at the end of surgery, but not in the aprotinin group. This abnormality is not related to the hydrolysis of vWF or GP Ib, since washed platelets were resuspended in pooled normal plasma which provided a constant amount of vWF in this test and since the plasma concentration of the fragment of GP Ib (glycocalicin) did not correlate with this abnormality. Despite a high concentration of heparin (5-7 IU/ml) in patient's plasma during bypass, activation of blood coagulation in both groups was evidenced by an increase in ATm (thrombin-modified antithrombin III) level. The level of ATm in the placebo group reached a maximum at the end of ECC during rewarming, while in the aprotinin group, ATm level at this time point was significantly lower than in the control group. In comparison to the placebo group, the generation of the fibrin degradation products (DDE complexes) was inhibited by aprotinin during ECC, but the level of DDE complexes in the aprotinin group was slightly elevated after ECC, although much less than in the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724578 TI - Induction of CD14 antigen on the surface of U937 cells by an interleukin-6 autocrine mechanism after culture with formalin-killed gram-negative bacteria. AB - In order to better understand the regulation of CD14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116-54. Various cytokines such as interleukin-1 beta, interleukin-2, interleukin-6, interferon-gamma and tumor-necrosis factor-alpha were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116-54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116-54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116-54 also induced strong expression of CD14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6. PMID- 1724579 TI - Antiport mediated rounding and endocytosis are enhanced by sulphate. AB - Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway. PMID- 1724580 TI - [Heparin-bound growth factors produced by the established RH-PA cell line]. AB - Two types of heparin-bound growth factors (HBGF) are isolated from serum-free culture media of RH-PA cells, which are effective producers of urokinase type plasminogen activator, and from a lysate of these cells. According to affinity to heparin, molecular weight and cells proliferation stimulation, these HBGF are similar to basic and acidic fibroblast growth factors. It is shown that the obtained preparations, produced by RH-PA cells at 2 pg/ml concentration and more, exert mitogenic effect towards RH-PA and NIH 3T3 cells. This effect is additive to the bovine serum factors. It is established that urokinase at the 0.5-25 micrograms/ml concentration also exerts mitogenic effect. The addition of HBGF in concentration of more than 0.05 micrograms/ml significantly stimulates urokinase production. It is suggested that both the factors--HBGF and urokinase--may be elements of the common mechanism of autocrine regulation of the RH-PA cell line proliferation and urokinase production. PMID- 1724581 TI - Crossprotective immunity between the Florida and a Zimbabwe stock of Anaplasma marginale. AB - Cattle immunised by infection with the Florida stock of Anaplasma marginale were protected against subsequent homologous challenge, as demonstrated by complete prevention of microscopically detectable parasitaemia. Identically immunised cattle were partially protected against challenge with the Norton, Zimbabwe stock of A. marginale as determined by the significant prolongation of the prepatent period, a significantly lower peak parasitaemia, and a significantly lower percentage drop in haemoglobin concentration when compared to non-immunised calves challenged identically. The partial protection induced by live Florida stock immunisation demonstrates that while protection-inducing epitopes are shared between the two stocks, induction of complete immunity against a Zimbabwe stock may require alternative presentation of Florida stock epitopes or inclusion of additional Zimbabwe stock epitopes in the immunogen. PMID- 1724582 TI - Computer predictions of antigenic domains in herpes simplex virus types 1 and 2 glycoprotein D as compared with experimentally proven domains. AB - The primary amino-acid sequence of the glycoprotein D (gD) of herpes simplex virus types 1 and 2 (HSV-1, HSV-2) was analyzed by computer programs that provided values for hydrophilicity, surface probability, flexibility, and antigenicity, as well as the secondary structure conformation. Putative antigenic domains with a high hydrophilicity, surface probability, and antigenicity index were determined and compared with the reported antigenic domains in HSV-1 and HSV 2 gD protein based on experimental data. The major experimentally proven antigenic domains were detected by the computer analyses. Additional putative antigenic domains with potential for the synthesis of antigenic viral peptides were determined. PMID- 1724583 TI - [The strain-specific diagnosis of influenza by using lanthanide immunofluorescence analysis based on monoclonal antibodies to the hemagglutinin of the influenza A virus]. AB - Nine monoclonal antibodies (MCA) to hemagglutinin of influenza A/Taiwan/1/86 (H1N1) virus and 5 MCA to influenza A/Mississippi/1/85 (H3N2) virus were generated and characterized. The MCA were used for the development of diagnostic test systems on the basis of time-resolved fluoroimmunoassay. The same MCA were used as primary and detecting antibodies in the test system specific for HA of the H1 serosubtype, whereas in the test system specific for influenza A serosubtype H3 virus MCA of different epitope appurtenance were used as primary and secondary antibodies. The sensitivity of the test system for HA of serosubtype H1 was found to be 10 ng/ml and that for serosubtype H3 5 nh/ml. The developed test systems were tried on the clinical material collected during the epidemic periods of 1983-1989. PMID- 1724584 TI - [The comparative characteristics of the interferon-inducing capacity of 2 preparations related to aromatic hydrocarbons]. AB - Aromatic hydrocarbons are rightly considered to belong to most active synthetic interferon inducers among low molecular compounds. A comparative evaluation of L 1 (acridanon) and amixin (fluorenon) showed L-1 to have more marked interferon inducing properties. Both compounds differed not only in the dynamics and levels of interferon synthesis in different organs which suggests the possibility of their employment in different diseases, but also in the efficacy of the modes of application. L-1 induced IF synthesis most actively after subcutaneous inoculation, amixin after oral administration. PMID- 1724585 TI - [The possibility of the restoration of interferon formation by biologically active thymus factors in experimentally induced immunodepression]. AB - Interferon production was investigated in mice of CBA line with experimentally induced immunosuppression. With all types of treatment (irradiation, administration of hydrocortisone, cyclophosphane, ALS) the capacity of host cells for interferon production was shown to be reduced. Administration of biologically active thymus factor, thymostimulin, to experimental animals resulted in significant restoration of alpha/beta and gamma interferon production. PMID- 1724587 TI - [Cellular and molecular mechanisms in heart failure]. AB - In patients with heart failure there are distinct functional abnormalities in the myocytes themselves. This review deals with the deteriorations in the myocardial energy metabolism and the recently found alterations in the neurohumoral and hormonal signal transduction and signal realization within the cardiac cells. Beside the reduction in the volume of mitochondria in the overloaded myocardium the energy starvation is also reflected by a decrease in the content of high energy phosphates. Studies on nonfailing and failing human ventricular myocardium identified significant alterations in the neurohumoral regulation of the heart including the fluxes and the transport processes of Ca2+ as well as the beta adrenoceptors, G-proteins, cAMP levels and cAMP-mediated processes. Recent data on the existence of auto-antibodies against the ADP/ATP translocator of the mitochondrial membrane and of stimulatory acting autoantibodies against i) the L type calcium channel and ii) the beta 1-adrenoceptor, respectively, in patients with dilated cardiomyopathy, may open a new view in the etiology of heart failure and for consequences in the therapeutic concept of these diseases. PMID- 1724586 TI - [The incidence of the contamination of immunoglobulin preparations with the hepatitis B virus surface antigen]. PMID- 1724588 TI - [Pain therapy in patients with gynecologic tumors. Analysis of ambulatory pain management]. AB - In a retrospective study efficacy and side-effects of long-term pain management of 58 patients with gynaecological cancer were analysed. Pathophysiological changes of the underlying tumor determined the choice of pain therapy. In more than 90% sufficient analgesia was obtained with tolerable side-effects. When patients became unable to swallow oral medication or showed intolerable side effects, continuous subcutaneous or epidural opioid administration by means of lightweight disposable infusors proofed to be an efficient alternative in pain therapy. PMID- 1724589 TI - [Changes in the cytoarchitectonics of the field 4 of human cerebral cortex in perinatal developmental defects]. AB - Knowledge of the morphological characteristics is essential for the understanding of the pathogenesis and finding of effective treatment for children with perinatal brain disorders (cerebral palsy). The authors studied histological sections (Nissl-stained) of field 4 of the cerebral cortex from 20 patients aged 3 to 14 years. Demonstrating the signs of nonspecific changes in the nervous tissue and revealing the damage of development, the results of the work can be used for interpreting the data of a quantitative image analysis of the cortex cytoarchitectonics. PMID- 1724591 TI - Aeroallergen analyses and their clinical relevance. I. Immunochemical quantification of allergens by RAST-inhibition, Mab-ELISA, basophil histamine release, and counter current immuno electrophoresis. AB - The aim was to compare IgE and IgG4 RAST-inhibition assay (RI), monoclonal antibody ELISA (Mab-ELISA), counter current immuno electrophoresis (CCIE) and histamine release from basophil leukocytes (HR) for allergen quantification with special reference to aeroallergen detection. As components of indoor aeroallergens, cat, dog, and Derm. pter. allergen extracts were selected for the experiments. To evaluate unspecific interference, these allergens were compared mutually and with Cladosporium herbarum. Allergen extracts in varying dilutions were mixed with crushed glass fibre filter materials, eluted, recovered by centrifugation, and allergen concentration quantified by the assays. Equal sensitivity was found for both IgE- and IgG4-RI assaying cat allergen (in the range 5-50 SQ-U/ml) and dog allergen (in the range 10(2)-10(3) SQ-U/ml). The IgG4 RI assaying Derm. pter. was more sensitive (50 SQ-U/ml) than IgE-RI (2*10(3) SQ U/ml). The ranges of allergen detection limits for the Mab-ELISA were equal for cat and Derm. pter. (10-10(2) SQ-U/ml). The range of allergen detection limits for CCIE, assaying dog were 10(4)-10(5) SQ-U/ml. The ranges of allergen detection limits for HR were equal for cat and Derm. pter. (10-10(2) SQ-U/ml), and 10(2) 10(3) SQ-U/ML for dog. Because of cross-reactivity, a minor degree of interference was observed in the IgE-RI and the HR test for the highest concentration of cat and dog allergens. PMID- 1724590 TI - Inhibition of bone resorption by a monoclonal antibody that reacts with a 150 kD membrane protein in chicken osteoclasts. AB - Bone resorption is a multistep process that includes the maturation of osteoclast precursors, the special attachment of fully differentiated osteoclasts to mineralized bone surface, and the dissolution of inorganic mineral, as well as the breakdown of organic matrix. We have produced a large panel of monoclonal antibodies directed against chicken osteoclasts to obtain specific probes for studying the function of osteoclasts. One of our antibodies, K20, inhibited bone resorption of isolated osteoclasts almost completely. Several pieces of evidence suggested that the antigen detected by this antibody was located in the plasma membrane of the osteoclast. In western blot analysis K20 antibody specifically recognized a 150 kD protein in the medullary bone microsome fraction under reducing and nonreducing conditions. In addition to osteoclasts and some bone and bone marrow mononuclear cells, a positive immunoreaction was seen in the kidney tubules. These data suggest that monoclonal antibody K20 reacts with an osteoclast surface antigen that is functionally important in bone resorption. PMID- 1724592 TI - Suppressive effect of loratadine on allergen-induced histamine release in the nose. AB - It has been speculated whether the recently developed non-sedating antihistamines may possess other properties than merely being antagonists at the H1-receptors. To investigate this suggestion 12 patients with strictly seasonal allergic rhinitis participated in a double-blind placebo controlled randomized cross-over study outside the pollen season. At steady state levels of 10 mg loratadine, a new non-sedating antihistamine, the patients were challenged with methacholine. This was followed by a nasal challenge with increasing doses of allergen. 24 h later the patients were rechallenged nasally with the same methacholine dose as the day before. The volume of the methacholine-induced nasal secretion was measured and the response to allergen was determined by scoring technique. In returned nasal lavage fluid the levels of histamine and TAME-esterase activity were measured. It was found that loratadine significantly reduced the immediate allergic nasal symptoms compared with placebo (P less than 0.01). Loratadine also reduced the allergen-induced release of histamine into the nasal cavity after the strongest allergen dose, from 9.6 +/- 1.5 (mean +/- SEM) to 6.4 +/- 1.4 ng/ml (P less than 0.05). A similar decrease in the TAME-esterase activity after treatment with loratadine was observed. The TAME-esterase activity decreased from 11.6 *10(3) +/- 2.47 *10(3) to 5.60 *10(3) +/- 1.45 *10(3) CMP (P less than 0.05). There were no significant changes between the active and placebo treatments regarding the methacholine-induced secretory response. This was true for the initial methacholine challenge as well as the secretory response 24 h later.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724593 TI - Indications for gastric bypass in palliative operations for pancreatic carcinoma. AB - This review was undertaken to determine whether there are specific factors which predict the development of gastric outlet obstruction (GOO) in patients with pancreatic carcinoma. One hundred forty-two patients with biopsy proven pancreatic carcinoma had palliative operations of whom 74 had gastric bypass (GB). Of the 68 who did not, four died after biliary bypass. The 64 patients who remained at risk for GOO are the subject of this report. Seven of those patients developed GOO in the postoperative period and were compared with the 57 who did not. No significant difference was found between the two groups when they were compared on the basis of 20 historic, laboratory, and operative finding criteria. These data indicate that accurate prediction of subsequent GOO is not possible based on available objective data. Because GB creation does not increase operative blood loss, operative time, postoperative stay, or postoperative morbidity, and because prediction of need is difficult, prophylactic GB should be applied very liberally. PMID- 1724594 TI - Catabolite inactivation, cyclic AMP and protein phosphorylation in the methylotrophic yeast Hansenula polymorpha. AB - The inactivation of the peroxisomal enzyme alcohol oxidase and the cytoplasmic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase was found to occur after addition of glucose to methanol-grown cells of the yeast Hansenula polymorpha. The concentration of cyclic AMP increased nearly twofold within 3 min under the same conditions. In crude extracts of H. polymorpha about 20 proteins are phosphorylated by cyclic AMP dependent protein kinases, among them also fructose-1,6-bisphosphatase. No phosphorylation of the alcohol oxidase protein could be detected. From this fact, it was concluded that the inactivation of the peroxisomal alcohol oxidase is independent of cyclic AMP-dependent protein phosphorylation. PMID- 1724595 TI - Analysis of the promoter region in the rRNA operon from Mycobacterium bovis BCG. AB - At least two transcriptional initiation sites were observed in rRNA operon of Mycobacterium bovis BCG at approximately -187 and -265. The former transcriptional signal was recognized by Escherichia coli RNA polymerase, whereas the later was not. PMID- 1724596 TI - Immunoaffinity purification of glucose/xylose isomerase from Streptomyces. AB - A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes. PMID- 1724597 TI - Antikeratin 14 monoclonal antibody staining in psoriasis and seborrhoeic keratosis: immunofluorescence and two colour FACS studies. AB - A monoclonal antibody (ES3A) was raised against a mouse graft-versus-host reaction (GVHR) model. This antibody was against basal cell cytoplasm and reacted with an acidic (pI 6.2) 50 kDa keratin of human epidermis. However, ES3A reacted with several lower layers of epidermal cells in psoriasis and seborrhoeic keratosis. Acanthotic seborrhoeic keratosis showed varying patterns even in a single lesion. If combined with FACS analysis, ES3A-positive cells could be quantified. Normal skin showed 28%, while psoriasis and seborrhoeic keratosis showed 44% and 51%, respectively. ES3A-positive compartments of the acanthotic type of seborrhoeic keratosis were larger than those of the hyperkeratotic type. ES3A may be suitable for quantification of germinative or proliferative cells. PMID- 1724598 TI - Alterations in sphingomyelin and fatty acids in human benign prostatic hyperplasia and prostatic cancer. AB - The present experiments were conducted to examine and characterize the lipid composition of benign prostatic hyperplasia (BPH) and prostatic cancer tissues (CAP). Lipids were extracted from these tissues and analyzed by thin layer chromatography (TLC) and gas liquid chromatography (GLC). The protein profiles of these tissues were analyzed by SDS-polyacrylamide gel electrophoresis. The results of these studies demonstrate that the principal lipids of BPH and CAP tissues are phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, fatty acids and cholesterol. The sphingomyelin level is significantly higher in CAP tissues compared to BPH tissues. The differences in the enzymatic activities responsible for the biosynthesis and degradation of sphingomyelin appear to explain at least partially the alteration in sphingomyelin level between BPH and CAP tissues. The major fatty acids of phosphatidyl choline and phosphatidyl ethanolamine were palmitic (16:0), stearic (18:0), oleic (18:1) and arachidonic (20:4) acid. The arachidonic acid level was significantly decreased in CAP tissues compared to BPH tissues. SDS polyacrylamide gel electrophoresis revealed several protein differences between BPH and CAP tissues. The most significant difference was the decrease in 12 kDa protein in CAP tissues compared to BPH. The present data, therefore, suggests that there are significant alterations in sphingomyelin, fatty acids and protein profiles between BPH and CAP tissues. PMID- 1724599 TI - In vitro perfusion and incubation of rat pancreatic lobules in a kinetic system. AB - The use of pancreatic lobule preparations is one of the well-established approaches to study stimulus-secretion coupling in the exocrine pancreas in vitro. We have developed a kinetic system for the perfusion and intermittent incubation of rat pancreatic lobules. This model allows repeated hormone stimulation for up to 3 h while permitting rapid changes of the cellular environment with no accumulation of secretory or metabolic products. Tissue viability could be demonstrated over 6 h by in vivo toluidine blue exclusion, histology and electron microscopy. Lactate dehydrogenase leakage from cells over 6 h was only 2.4% of total content. No activated trypsin was detected in the perfusion medium. A biphasic dose response was established for cholecystokinin stimulation with a maximal response at 10(-8) M. We conclude that kinetic perfusion and incubation are technically feasible with rat pancreatic lobules. This in vitro model appears particularly suited for the investigation of pharmacologic and metabolic effects on the pancreatic acinar cell when rapid changes of the cellular environment are required and when the accumulation of secretory and metabolic products must be avoided. The technique described requires neither protease inhibition in the medium nor collagenase treatment of the cells. PMID- 1724600 TI - Antiallergic effects of major metabolites of astemizole in rats and guinea pigs. AB - Antiallergic effects of astemizole (CAS 68844-77-9) and its metabolites were studied using rats and guinea pigs. All the metabolites of astemizole tested i.e., desmethylastemizole, 6-hydroxydesmethylastemizole and norastemizole, were more active than astemizole in inhibiting the contraction of the ileum as well as the bronchoconstriction induced by histamine in guinea pigs. Desmethylastemizole was about the same as the parent compound in inhibiting the 3H-mepyramine binding in guinea pig cerebellum. In heterologous passive cutaneous anaphylaxis (PCA) and homologous PCA, the metabolites caused almost equipotent inhibition to that seen in astemizole. On the other hand, in the studies of histamine release from rat peritoneal mast cells induced by compound 48/80 or from lung fragments in actively sensitized guinea pigs, desmethylastemizole, 6 hydroxydesmethylastemizole and norastemizole were much less potent than was astemizole. No H2-antagonistic activity was observed with either astemizole or desmethylastemizole. PMID- 1724601 TI - Studies on CSF tryptophan metabolism in infantile spasms. AB - Cerebrospinal fluid from 8 patients with infantile spasms (mean age: 6.1 months) was collected before treatment. The concentration of cerebrospinal fluid tryptophan metabolites was analyzed using high-performance liquid chromatography and compared to metabolite concentrations in cerebrospinal fluid from 20 age matched controls (mean age: 5.8 months). The levels of cerebrospinal fluid serotonin, 5-hydroxyindoleacetic acid, and kynurenine were significantly lower in infantile spasm patients compared to controls (P less than .05). In contrast, the levels of cerebrospinal fluid 3-hydroxykynurenine were significantly higher in infantile spasm patients than in controls (P less than .05). There were no significant differences in the levels of cerebrospinal fluid tryptophan and 5 hydroxytryptophan. Although the study population was small, these findings suggest that the presence of seizures in infantile spasms is associated with a decrease in serotonergic metabolites which, in turn, may indicate a decrease in serotonergic activity, altered clearance of these metabolites, or altered turnover in the direction of 3-hydroxykynurenine. The perturbance caused by increased 3-hydroxykynurenine and decreased kynurenine in the homeostatic balance between these 2 tryptophan metabolites could further contribute to the pathogenesis of infantile spasms. PMID- 1724602 TI - Changes in CSF neurotransmitters in infantile spasms. AB - Cerebrospinal fluid (CSF) from 7 patients with infantile spasms (mean age: 6.7 months) was collected before and after treatment with adrenocorticotropic hormone (ACTH). The concentration of neurotransmitter metabolites was analyzed using high performance liquid chromatography and compared to the metabolite concentration in the CSF from 7 age-matched controls (mean age: 6.1 months). Pretreatment levels of CSF 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid, 3-methoxy-4 hydroxyphenyl glycol (MHPG), and kynurenine were significantly lower in infantile spasm patients compared to controls. Following treatment, marked increases in 5 HIAA and decreases in kynurenine levels were observed in the CSF of the 5 infants whose seizures were eliminated or reduced by ACTH. In the 2 nonresponders 5-HIAA levels decreased. The level of MHPG was reduced slightly in 5 infants, including the 2 nonresponders, and was increased in 2 responders. CSF homovanillic acid levels increased in 4 infantile spasm infants and decreased in 3 following ACTH. These data demonstrate that the presence of seizures in infantile spasms is associated with a significant decrease in serotonergic activity and that elimination of seizures by ACTH is accompanied by increased serotonin turnover. The simultaneous increase of 5-HIAA and decrease of kynurenine, an alternate metabolite of tryptophan, suggests an underlying disturbance of tryptophan metabolism in infantile spasms. The possibility that elimination of seizures by ACTH may be related to decreased production of certain kynurenine metabolites, particularly quinolinic acid, is discussed. PMID- 1724603 TI - Chronic mumps virus encephalitis. AB - A previously well 4-year-old girl developed frequent seizures and mental deterioration after mumps parotitis. Direct IgG antibody capture enzyme-linked immunosorbent assay revealed a high titer of anti-mumps viral antibodies in the cerebrospinal fluid. Chronic mumps encephalitis was diagnosed. Her mental state and seizures improved markedly with inosine pranobex therapy. PMID- 1724604 TI - In vivo effects of acute EtOH on rat alpha and beta luteinizing hormone gene expression. AB - The suppressive effects of ethanol (EtOH) on the male rodent reproductive axis, especially on serum luteinizing hormone (LH) levels, are well known. In this study we examined, in coordinate fashion, the effects of EtOH on LH secretion and on steady-state levels of the mRNA for the two genes that direct LH synthesis, namely alpha- and beta-LH. A single intraperitoneal (IP) injection of EtOH given to castrated male rats produced a statistically significant fall in serum LH levels at 1.5 (p less than 0.05) and 3 hours (p less than 0.01) after injection compared to saline-injected controls. This effect had dissipated by 24 and 72 hours after treatment. Intrapituitary LH content was significantly increased in EtOH-compared to saline-treated animals 1.5 hours after injection (p less than 0.05) at the same time there was a significant decrease in serum LH. Steady-state levels of beta-LH mRNA were significantly diminished in EtOH-treated animals at 1.5 (p less than 0.05) and 3 hours (p less than 0.001) after injection, but returned to control levels thereafter. Since the half-life of beta-LH mRNA is greater than 8 hours, this fall is most likely due to decreased beta-LH mRNA stability and may also involve decreased beta-LH gene transcription. alpha mRNA levels were unchanged at all time points investigated. These findings were verified in four repetitions of this experiment. These data suggest that the EtOH induced fall in serum LH is due to impaired secretion of LH and to decreased LH synthesis as indicated by diminished steady-state levels of beta-LH subunit mRNA, secondary mainly to altered mRNA stability. PMID- 1724605 TI - Characterization of monoclonal antibodies directed against domains of pertussis toxin involved in receptor recognition. AB - The exotoxin pertussis toxin (PT) produced by virulent Bordetella pertussis bacteria is regarded as the main virulence factor of the organism and held responsible for most of its pathological effects. Identification of functional sites on PT would greatly facilitate site-specific detoxification and thus also the development of a new vaccine. For the investigation of structure-function aspects of PT we have prepared and characterized eleven monoclonal antibodies (mAbs) (UB-A1, UB-A2, UB-A10, UB-B7, UB-B12, UB-D4, UB-D7, UB-D10, UB-F7, UB-G1, and UB-G12) directed at the native toxin. Only UB-B12 and UB-D10 recognized PT in Western blotting indicating that most of the mAbs were directed against conformational epitopes. The mAbs were assayed for their ability to interfere with the binding of PT in model receptor systems like a solid phase binding assay using fetuin as receptor moiety, hemagglutination of chymotrypsin-sensitized goose erythrocytes, and the PT-mediated induction of the clustered growth pattern (CGP) of Chinese hamster ovary (CHO) cells. Five of the eleven mAbs (UB-A1, UB A2, UB-B7, UB-B12, and UB-D7) interfered with the binding of PT to fetuin on solid phase and with PT-mediated hemagglutination. UB-A2, UB-B7, and UB-B12 also inhibited the induction of the clustered growth pattern of CHO-cells. This indicates that the determinants recognized by these mAbs are associated with the formation of the carbohydrate recognition sites of PT. Thus, the monoclonal antibodies described in this study will be valuable tools in the further analysis of the structure-function relationship of pertussis toxin with respect to receptor recognition and binding. PMID- 1724606 TI - The establishment and evaluation of luminescent-labelled immunometric assays for prostate-specific antigen-alpha 1-antichymotrypsin complexes in serum. AB - Prostate-specific antigen is found in the prostate in two forms, one with a low (30,000) and one with a high (100,000) relative molecular mass. The latter has recently been found to be a complex of prostate-specific antigen with alpha 1 antichymotrypsin. Immunoluminometric assays were designed for the prostate specific antigen-alpha 1-antichymotrypsin complex as well as for alpha 1 antichymotrypsin, the former being compared with a commercially available radioimmunoassay for prostate-specific antigen (ProsChek RIA-Yang Laboratories). The precision of the immunoluminometric assays was acceptable (intra-assay variation less than 7%; inter-assay variation less than 8.5%) in the measuring ranges 0-90 micrograms/l for the prostate-specific antigen-alpha 1 antichymotrypsin complex and 0-12 g/l for alpha 1-antichymotrypsin. The correlation between the assays for prostate-specific antigen and prostate specific antigen-alpha 1-antichymotrypsin complex was acceptable, showing a correlation coefficient r = 0.83 after double logarithmic transformation, or r = 0.85 using the Spearman rank correlation on 131 data pairs. Extremely high alpha 1-antichymotrypsin levels (above 2 g/l) caused interference in the prostate specific antigen-alpha 1-antichymotrypsin complex assay. Such levels, although rare, are encountered in pulmonary inflammatory disease. The reference ranges for the three assays were found to be as follows: prostate-specific antigen 0.13-4.63 micrograms/l, prostate-specific antigen-alpha 1-antichymotrypsin complex 0.08 1.78 microgram/l, and for alpha 1-antichymotrypsin 0.27-0.61 g/l. These values were obtained from 82 hospitalised males for the first two assays and from 80 males and females free from infection for the latter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724607 TI - Liver uptake of trypsin-inhibitor complexes; differences between suckling and adult rats. AB - Previous studies have demonstrated increased intestinal trypsin uptake in newborn rats compared to adults. The mechanisms that protect tissues against proteolytic damage by trypsin include trypsin-inhibitor binding and subsequent liver uptake. In order to examine these mechanisms, bovine trypsin (1.25 mg/100 g body weight) plus trace 125I-trypsin were injected into the portal vein of 2-week-old and adult rats. Liver, kidney and plasma 125I activity were assessed at 1, 5 or 15 min following infusion and the different trypsin inhibitor fractions were separated and examined for 125I activity. Newborn rats had significantly increased plasma levels of 125I-trypsin and significantly decreased liver 125I levels 1 min after infusion compared to adults. In addition, the slow decline in liver 125I activity seen in the adult rats did not occur in the newborns. The pattern of trypsin-inhibitor binding was not significantly different at 1, 5 and 15 min and there were no differences at these time intervals between newborns and adults. We suggest that the newborn liver is less effective in clearing infused trypsin leading to increased plasma levels, and this increased plasma trypsin concentration subsequently leads to increased liver levels of 125I-trypsin. The increased trypsin levels in the liver may predispose newborns to protease-induced liver damage. PMID- 1724608 TI - Role of antigen-binding B lymphocytes in the formation of antigen-dependent nonspecific immunoglobulin-forming cells. AB - A role for antigen-binding B cells in the induction of antigen-dependent B cells producing antigen-nonspecific immunoglobulins (nIFC) is analyzed. An in vitro immune response system was used; sheep red blood cells were employed as the antigen. It is shown that cocultivation of antigen-binding B cells with resting naive splenocytes in the presence of homologous antigen resulted not only in an increase in the number of antibody-forming cells (AFC), but also in a sharp increase in nIFC. Cocultivation of G0 splenocytes with primed B cells depleted for antigen-binding cells did not affect AFC induction, and only slightly augmented the number of nIFC. It is suggested that some of the nIFC appearing after antigenic stimulation arise from resting B lymphocytes in response (direct or indirect) to nonspecific B-cell factor(s). PMID- 1724609 TI - Mapping of an epitope of human leukocyte alpha interferon A which is recognized by the murine monoclonal antibody NK2. AB - An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays. The results from these assays suggested that the epitope is within the sequence 112-121. Analysis of a hybrid protein IFN-A(1-92)/F(93-166) revealed that the N-terminal region of IFN-A played no significant role in NK2 binding. Three residues of IFN-F (Asn113, Val114, and Lys121) were substituted for the corresponding residues from IFN-A (Lys113, Glu114, and Arg121) by site-directed mutagenesis of the gene encoding IFN-F. NK2 was able to bind the mutated protein, IFN-F(A 113, 114, 121), as well as unmodified IFN-A. The data show that the epitope recognized by NK2 is located within the C-terminal region of IFN-A (residues 112-121). This epitope consists of the essential residues 114 and 116, and residues 112, 113, 115, 117, and 121 presumably contribute the configuration of the epitope. PMID- 1724610 TI - Targeting of liposomes to hepatocytes. AB - We began our investigations about 15 years ago with the concept that it should be possible to deliver drugs or enzymes intracellularly into lysosomes for diseases associated with lysosomal enzymes (e.g., sphingolipidosis). The results have been very gratifying. It now has been possible to extend our in vitro studies on the kinetics of lectin-liposome interaction to the targeting of glycolipid liposomes to specific liver cell types in vivo by simply varying the sugar residue on the surface of liposome. Nevertheless, much remains to be done to ascertain the stability of the external enzyme and the expression of its activity after the delivery into the lysosomes. PMID- 1724611 TI - Receptor-dependent targeting of lipoproteins to specific cell types of the liver. PMID- 1724612 TI - Genetic changes in methotrexate-resistant mosquito cells. AB - A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes. PMID- 1724613 TI - Percutaneous transhepatic placement of biliary endoprostheses: results in 100 consecutive patients. AB - One hundred patients with malignant biliary obstruction underwent palliative therapy by means of percutaneous transhepatic placement of 114 biliary endoprostheses. All patients were then followed up for at least 18 months or until death. Retrospective evaluation of the 95 patients who died showed an average survival time of 5.0 months. The five remaining patients have survived an average of 29.8 months. During the 1st week after stent insertion, a second manipulation was performed to improve stent function in nine patients. Overall, 14 (12.3%) of the stents became obstructed and six (5.2%) migrated; 86 patients required no further therapy for biliary obstruction or stent malfunction. The 30 day mortality rate was 12%; none of the deaths were directly attributable to a complication of the stent placement procedure. PMID- 1724614 TI - Percutaneous ethanol injection for the treatment of symptomatic cystic metastases from ovarian carcinoma. Work in progress. AB - Two patients with symptomatic cystic metastases from ovarian epithelial carcinoma underwent ultrasound (US)-guided percutaneous aspiration and temporary injection of 99% ethanol into the cyst. In the first case, the patient initially underwent surgical resection of the mass and received systemic chemotherapy, but the cyst recurred 2 months later. Percutaneous aspiration and ethanol sclerotherapy were performed twice in the second case; fluid reaccumulated 2 months after the initial procedure. No side effects occurred. During the follow-up period (8 months in the first case and 4 months in the second), no clinical recurrence of the initial symptoms was noted. At the end of that period, a recurrent but asymptomatic cystic lesion was revealed at US examination in the first case. In the second case, a minimal asymptomatic residual collection was depicted with computed tomography. The results indicate that this technique should be considered in patients with symptomatic cystic metastases from ovarian carcinoma and may have potential benefit in the palliative treatment of such lesions. PMID- 1724615 TI - Peptide immunization can elicit malaria protein-specific memory helper but not proliferative T cells. AB - We have studied the proliferative and helper T cell responses in mice to a malaria sporozoite vaccine candidate currently undergoing human trials. Following immunization of B10 (I-Ab) mice with the purified recombinant baculovirus expressed Plasmodium falciparum circumsporozoite protein, draining lymph node cells were challenged in vitro with a series of overlapping synthetic peptides which span the construct. Surprisingly, only a single peptide from the protein was immunodominant in that it could reproducibly elicit a significant proliferative response from the immunized lymph node cells. This epitope, (NANP)n, is also the repetitive immunodominant B cell epitope of the protein. However, immunization of mice with synthetic peptides revealed at least 3 cryptic proliferative epitopes--epitopes not revealed by protein immunization--two of which represent conserved regions of the protein. While cryptic peptide immunization did not elicit protein-specific proliferative T cells, it did reveal protein-specific helper T cells, as shown by an in vivo assay. Identification of "cryptic" epitopes not only for malaria but for other infectious diseases may aid vaccine design, especially in situations where subunit vaccines are sought. PMID- 1724616 TI - Toleration of amino acid substitutions within hepatitis B virus envelope protein epitopes established by peptide replacement set analysis. I. Region S(139-147). AB - B and T cell epitopes expressed on the surface of S-protein, a major constituent of the envelope of hepatitis B virus (HBV), are essential for eliciting protective immunity against HBV infection. A segment of the S-protein sequence encompassing residues S(139-147) is a portion of overlapping B and T cell epitopes. This sequence is conserved among distinct serological subtypes of HBV and has a 77.8% homology with an analogous sequence in S-proteins of nonhuman mammalian hepadnaviruses. Rare subtypes and variants of HBV having amino acid replacements within the S(139-147) sequence were discerned recently. The impact of amino acid replacements within this sequence on its immunological recognition at both the B and T cell levels was explored by peptide replacement set analysis. Results of the analysis permit discrimination between tolerated and forbidden amino acid replacements and provide a background for the development of reagents and immunogens specific for emerging HBV variants. PMID- 1724617 TI - The molecular specificity of linear B-epitopes in the E7 open reading frame protein of human papillomavirus 16 defined by monoclonal antibodies. AB - Human papillomavirus (HPV) 16 is highly associated with premalignant and malignant anogenital epithelial lesions. The transforming function resides within the viral E7 open reading frame protein. We have defined three immunodominant linear B-epitopes in the E7 protein. In the present study, we determine the contribution of individual amino acid residues to antibody binding of these three epitopes using replacement set analysis. In this approach, each epitope residue is substituted in turn by each of the other 19 genetically encoded amino acids to produce analogues which are tested for specific monoclonal antibody binding. We demonstrate the specificity of the monoclonal antibodies for epitopes of HPV 16 E7 in binding studies using synthetic epitope analogues of other HPV genotypes. Comparison between HPV 16 and other HPV genotypes suggests that variability in amino acid composition at the E7 epitopic sites does not appear to be host antibody driven. PMID- 1724618 TI - Interaction of cyclic peptides and depsipeptides with calmodulin. AB - A variety of small peptides bind calmodulin (CaM) and inhibit CaM-dependent enzyme activity. The cyclic peptides cyclosporin A (CSA) and gramicidin-S (GRS) are shown to bind CaM and inhibit 3',5'-cyclic nucleotide phosphodiesterase (PDE) in a calcium-dependent manner. The cyclic peptide microcystin-LR (MLR) and the depsipeptides, valinomycin (VLM) and enniatin-B (ENB), bind to CaM and inhibit PDE activity. Spectral changes exhibited by the binding of MLR, VLM and ENB to dansyl-CaM as compared to that of CSA and GRS reflected different binding sites and/or different conformational changes. The apparent binding constants (Kd) for CaM-peptide were estimated and found to be 4.8 microM for CSA, 2.85 microM GRS, 12.99 microM MLR, 4.29 microM VLM and 41.26 microM ENB. Although these peptides did not inhibit baseline PDE activity, they did inhibit CaM-dependent PDE activity in a dose-dependent manner. Half-maximal inhibition (IC50) of PDE occurred approximately at 0.11 microM MLR; 0.45 microM GRS; and greater than 5 microM for ENB, CSA and VLM. This may be the first observation that these peptides (MLR, VLM and ENB) bind to a known cytoplasmic protein and inhibit an enzyme system dependent on that protein for optimal activity. Interaction of these peptides with CaM may be responsible for creating conformational-functional changes in CaM, thus altering the signal transduction mechanism required for CaM dependent enzymes, such as cyclic nucleotidase, protein kinases and phospholipase A2. PMID- 1724619 TI - A method for the rapid identification of T lymphocyte epitopes. AB - Mounting interest in host immunity and understanding the pathogenesis of disease has focused attention on the identification of T lymphocyte epitopes within various antigens. This effort has proven troublesome because of time and financial constraints. We have developed a cost-effective technique that allows for the rapid identification of these antigenic sequences within proteins of interest. Using a panel of in vitro synthesized peptides and a CD4+ bovine T lymphocyte cell line, we have identified a single epitope on a herpesviral envelope glycoprotein. In the future, this technology should allow epitope mapping to be more practical for a large number of antigens. PMID- 1724620 TI - The use of antithyroid drugs in the medical management of feline hyperthyroidism. AB - Antithyroid drugs are widely used in human medicine for the medical management of Graves' disease. Because patients with Graves' disease may undergo spontaneous remission, antithyroid drugs are preferred for long-term therapy because they do not permanently affect thyroid function. Hyperthyroidism in cats is somewhat different, in that spontaneous remission has not been reported and therefore ablative treatment (surgery or radioiodine) is often preferred. However, antithyroid drugs are essential for preoperative stabilization of cats with hyperthyroidism and often are used for long-term management of certain cases. This chapter will review the various drugs available for the medical management of hyperthyroidism, their mechanisms of action, indications for use, and adverse side effects. PMID- 1724621 TI - In vivo effects of ecdysterone on puff formation, and RNA and protein synthesis in the salivary glands of Rhynchosciara americana. AB - 1. Fourth-instar larvae of Rhynchosciara americana were injected with the insect molting hormone, ecdysterone, giving final hemolymph concentrations from 4.46 to 223 microM. 2. Induction of the DNA puff, B2b, in the proximal (S1) region of the salivary glands of Rhynchosciara americana by 22.6 microM ecdysterone, was accompanied by the production of an mRNA and a polypeptide with the same characteristics as B2b products produced during normal development. This mRNA and polypeptide were restricted to the proximal region of the gland, as is the B2b puff. 3. Synthesis of other poly(A)+RNAs was also stimulated in S1 by ecdysterone, and other puffs that appear during normal development were induced. However, rRNA production in S1 goes through a pattern of inhibition, followed by recovery when B2b is puffed, and subsequent inhibition. 4. Low molecular weight RNA, with a peak in the region of 4S, is stimulated after ecdysterone administration. PMID- 1724622 TI - Serologic characterization of Paracoccidioides brasiliensis E2 antigen. AB - 1. The behavior of the specific E2 antigen of Paracoccidioides brasiliensis was studied by agarose gel counterimmunoelectrophoresis. When the gel was read immediately after the electrophoretic run no precipitation band was visible. Visualization of the complex was possible only after incubation of the gel at room temperature overnight. 2. At alkaline pH, the E2 antigen migrates in the direction of the cathode, as do the immunoglobulins. The higher sensitivity of counterimmunoelectrophoresis when compared to double immunodiffusion for the diagnosis of paracoccidioidomycosis is due to the presence of antibodies directed against antigens which migrate to the anode. 3. The use of specific antiserum to E2 antigen as reference permits the double immunodiffusion method to be a very sensitive test for the specific serodiagnosis of paracoccidioidomycosis. PMID- 1724623 TI - Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle. AB - Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non adrenergic non-cholinergic neurones, had no NO synthase activity. PMID- 1724624 TI - Ruthenium-red inhibits CGRP release by capsaicin and resiniferatoxin but not by ouabain, bradykinin or nicotine in guinea-pig heart: correlation with effects on cardiac contractility. AB - 1. The possible influence of ruthenium-red (RR) on contractility and outflow of calcitonin gene-related peptide (CGRP)-like and neuropeptide Y (NPY)-like immunoreactivity (LI) from the heart of the guinea-pig induced by capsaicin, resiniferatoxin, nicotine, ouabain or bradykinin was studied in vitro. 2. In the isolated right atrium, exposure to capsaicin evoked an increase in contractile rate and tension simultaneously with an enhanced outflow of CGRP-LI, indicating release from the atria. Repeated administration of capsaicin induced tachyphylaxis. Incubation with RR markedly attenuated the capsaicin-evoked release of CGRP-LI while no clear-cut effects were seen on contractile tension or rate. 3. In the isolated whole heart, perfusion with capsaicin induced an increased outflow of CGRP-LI and stimulated heart rate, while a negative inotropic effect was observed. A second administration of capsaicin to the same preparations failed to influence the CGRP-LI outflow and in these experiments the positive chronotropic effect was absent while the negative inotropic action remained unchanged. Capsaicin-perfusion in the presence of RR failed to induce any increased outflow of CGRP-LI from the hearts or changes in contractile activity. However, after 1 h of rinsing with Tyrode solution repeated capsaicin perfusion in the absence of RR caused a clear-cut (60% of control) release of CGRP-LI and contractile responses were restored. 4. Perfusion with resiniferatoxin evoked a RR-sensitive, clear-cut increased CGRP-LI output without any effects on contractile force or heart rate. Repeated administration of resiniferatoxin induced tachyphylaxis with respect to outflow. Capsaicin perfusion after resiniferatoxin did not influence cardiac rate, force or CGRP-LI outflow suggesting development of cross-tachyphylaxis. 5. Perfusion with RR did not influence the outflow of CGRP-LI or contractility changes evoked by perfusion with nicotine, ouabain or bradykinin. In addition, the release of NPY-LI by nicotine remained unchanged in the presence of RR. Furthermore, the positive chronotropic effect of human CGRP alpha remained intact in the presence of RR. 6. It is concluded that RR selectively inhibits capsaicin- and resiniferatoxin induced excitation of cardiac sensory nerves as revealed by inhibition of both CGRP-LI release and the cardiostimulatory action of capsaicin. RR also seems to protect the cardiac capsaicin-sensitive fibres from the development of tachyphylaxis to capsaicin. Finally, RR prevents the capsaicin-evoked negative inotropic effect which is not related to activation of sensory nerves. PMID- 1724625 TI - Effects of calcium modulators on vagally-mediated constriction in the guinea-pig isolated trachea. AB - 1. The effects of calcium modulators on tracheal constriction evoked by vagal stimulation were examined in the isolated, innervated trachea of the guinea-pig. Responses were assessed in the Krebs-filled trachea as changes in intraluminal pressure (ILP), increases and decreases reflecting constriction and dilatation, respectively. 2. Preparations had a positive resting ILP, indicating significant spontaneous tone. Verapamil and nifedipine reduced baseline ILP, whilst Bay K 8644 had mixed effects. 3. Verapamil and nifedipine attenuated vagal responses in a concentration-dependent manner. At lower concentrations attenuation was due entirely to postjunctional effects but at higher concentrations prejunctional effects may have contributed to attenuation. 4. Verapamil and nifedipine attenuated vagal responses in the absence or presence of flurbiprofen, indicating that their effects are largely independent of the generation of cyclo-oxygenase products. Nifedipine, however, was less effective in reducing responses to low frequency vagal stimulation (up to 5 Hz) when flurbiprofen was present. 5. Bay K 8644 augmented vagal responses, the degree varying widely between preparations. 6. It was concluded that influx of Ca2+ through voltage-operated Ca2+ channels contributes significantly to vagally-mediated tracheal constriction in normal trachea and in trachea where endogenous release of cyclo-oxygenase products is inhibited. PMID- 1724626 TI - Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non parietal cells. AB - 1. Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N-acetyl-[14C]-D glucosamine and [3H]-L-leucine, respectively, into cellular and released acid precipitable material. 2. Histamine and activators of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 microM), forskolin (10-100 microM), 3-isobutyl-1-methylxanthine (100 microM), and dibutyryl cyclic AMP (1-3 mM), respectively. In the presence of 3-isobutyl-1-methylxanthine (30 microM) histamine stimulation was enhanced. 3. As shown by gel chromatography, stimulation by histamine (100 microM), forskolin (10 microM), 3-isobutyl-1-methylxanthine (100 microM) and dibutyryl cyclic AMP (1 mM) resulted in a release of high molecular weight (approximately 2 x 10(6) daltons) glycoproteins from the cells. The histamine H2-receptor antagonist, ranitidine (100 microM), blocked the effect of histamine. 4. We conclude that cyclic AMP dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non-parietal cells and that histamine may be a physiological activator of this system. PMID- 1724627 TI - Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase. AB - 1. Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM). 2. The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3. The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM). Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM). 4. Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.5. These findings support the idea that non treated or LPS-treated cultured SMC can produce an NO-like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO-like factor may play a role in the auto-regulation of smooth muscle cell reactivity through a cyclic GMP-dependent mechanism. PMID- 1724628 TI - Contraction of vascular smooth muscle induced by phorbol 12,13 dibutyrate in human and rat pulmonary arteries. AB - 1. The effect of phorbol 12,13 dibutyrate (PDB) on vascular tone was studied in both human and rat isolated pulmonary arterial strips (HPA and RPA, respectively). 2. PDB (1 nM to 2 microM) produced slowly developing, sustained and concentration-dependent contractions in HPA (mean EC50 = 3.5 nM, n = 5) and RPA (mean EC50 = 120 nM, n = 5). The maximal response was 185.6 +/- 25% and 207 +/- 27.5% (n = 5) of that induced by K(+)-rich (80mM) solution, and 223 +/- 34.5% and 176.5 +/- 38.6% of the noradrenaline (10 microM)-induced contraction in HPA and RPA, respectively. 3. PDB-induced contractions were not altered either by the presence of atropine (10 microM), propranolol (5 microM), phentolamine (5 microM) or tetrodotoxin (10 microM) in the bathing solution, or by the removal of endothelium from pulmonary arteries. 4. In HPA, the amplitude of PDB-induced contractions was significantly reduced by removal of external calcium ions, addition of verapamil (10 microM) or trifluoperazine (TFP, 5 microM) and significantly increased by Bay K 8644 (0.5 microM). In contrast, in RPA, calcium free solution and verapamil had only a moderate effect on the maximal PDB-induced contraction (approximately 20% reduction), whereas Bay K 8644 and TFP had no significant effect. In both HPA and RPA, PDB-contractions in calcium-free solutions were not modified by ryanodine (25 microM) or by 8-(N,N diethylamino)octyl-3,4,5, trimethoxybenzoate hydrochloride (TMB-8, 50 microM). 5. PDB-induced contractions were inhibited by protein kinase C (PKC) antagonists. The maximal response was decreased by 60 +/- 10.5% and 35 +/- 11.5% (n = 5) by 145-isoquinolinesulphonyl)-2-methylpiperazine (H7, 50 microM), 70.5 +/- 12.2% and 56 +/- 18% (n = 5) by phloretin (100 microM) and 80.7 +/- 8.4% and 71 +/- 14% (n = 5) by staurosporine (25 nM) in HPA and RPA, respectively.6. Long term treatment (15-20 h) of arterial strips with phorbol esters (phorbol 12,13 didecanoate, or PDB) abolished the contractile response to subsequent addition of PDB.7. These results show that PDB is a potent vasoconstrictor agent in human and rat pulmonary arteries. Unlike the rat, part of the PDB response depends on calcium influx in human preparations. PDB action appears mainly mediated by the activation of protein kinase C. PKC could thus play a major role in the control of vascular pulmonary reactivity. PMID- 1724629 TI - Salmeterol: a potent and long-acting inhibitor of inflammatory mediator release from human lung. AB - 1. The effects of salmeterol, a novel long-acting beta 2-adrenoceptor agonist, have been investigated on antigen-induced mediator release from passively sensitized fragments of human lung in vitro. 2. Salmeterol was a potent inhibitor of the release of histamine (-log IC50 = 8.54), leukotriene C4 (LTC4)/LTD4 (-log IC50 = 9.07) and prostaglandin D2 (-log IC50 = 8.81). It was slightly less potent (1-3 fold) than isoprenaline, but significantly more potent (10-35 fold) than salbutamol. 3. Propranolol competitively antagonized the inhibitory effects of salmeterol on histamine release (pA2 = 8.41) and LTC4/LTD4 release, (pA2 = 8.40) indicating an action via beta-adrenoceptors. 4. The inhibitory effects of isoprenaline (20 nM) and salbutamol (200 nM) were removed after washing the lung tissue for 2 h and 4 h respectively. In contrast, the inhibitory effects of salmeterol (40 nM) were much longer-lasting, and were still evident after 20 h. 5. Salmeterol therefore exhibits potent and persistent inhibition of anaphylactic mediator release from human lung. This anti-inflammatory effect may be important for the therapeutic potential of salmeterol in the treatment of bronchial asthma. PMID- 1724630 TI - Action of heptaminol hydrochloride on contractile properties in frog isolated twitch muscle fibre. AB - 1. Heptaminol stopped or delayed the progressive decline in tension which characterizes the phenomenon of fatigue in frog isolated twitch muscle fibre. 2. Heptaminol had no action on the sodium, potassium and calcium voltage-dependent ionic conductances. 3. The hypothesis of an action via an internal alkalinization was tested by comparison with the action of NH4Cl. Both substances increased the tension. 4. The action of heptaminol was suppressed in sodium-free (TRIS) solution or in the presence of amiloride while the action of NH4Cl was always observed. 5. These results could be explained by a stimulation of the Na/H antiport by heptaminol. PMID- 1724631 TI - Stereoselective total synthesis of glycopeptides bearing the dimeric and trimeric sialosyl-Tn epitope. AB - The dimeric and trimeric sialosyl-Tn epitopes, [alpha-D-Neup5Ac-(2----6)-alpha-D GalpNAc-(1----3)-L-Ser]n-L-Val (n = 2 and 3), which represent part of a clustered carbohydrate region of glycophorin A, a human erythrocyte glycoprotein, have been synthesised stereoselectively. 2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-D galactopyranosyl fluoride (GalpNAc unit), Fmoc-L-serine phenacyl ester (Ser unit), and benzyl 5-acetamido-4,7,8,9-tetra-O-benzyl-5-deoxy-3-S-phenyl-3-thio-D erythro-L - gluco-2-nonulopyranosylonate bromide (Neup5Ac unit) were the key intermediates for stereoselective glycosylation. 2-Ethoxy-1-ethoxycarbonyl-1,2 dihydroquinoline-promoted elongation of the peptide chain and then hydrogenolysis afforded the title compounds. PMID- 1724632 TI - Synthesis of allyl glycosides for conversion into neoglycoproteins bearing epitopes of mycobacterial glycolipid antigens. AB - Neoglycoproteins bearing key glycosyl substituents of several glycopeptidolipid antigens of pathogenic Mycobacterium species have been synthesized. Allyl glycosides of the terminal 6-deoxyhexose-containing units of the antigens were prepared, with appropriate ether and ester substituents in place. Ozonolysis of the allyl glycosides was then followed by reductive coupling with epsilon-amino groups of lysine residues in bovine serum albumin, using sodium cyanoborohydride at pH 7.8. The resulting neoglycoproteins emulated the antigenicity of the native molecule in several serological tests. PMID- 1724633 TI - Occult metastases in the axillary lymph nodes of patients with breast cancer node negative by clinical and histologic examination and conventional histology. AB - We examined axillary lymph nodes from 80 women with node-negative breast cancer, by immunohistochemistry, utilizing polyclonal antibodies to cytokeratins and carcino-embryonic antigen and monoclonal antibodies to cytokeratins and milk fat globulin. Occult metastatic tumor, undetectable in hematoxylin and eosin stained slides, but visible by immunohistochemistry, was detected in 23 of 80 patients (29 per cent). Occult tumor was observed in patients with invasive ductal carcinoma (21/76-28 per cent) and in individuals with invasive lobular carcinoma (2/4-50 per cent). In patients with occult metastases the primary tumors were slightly larger (mean 2.39 cm, range 1.00-5.00 cm) than those of patients whose nodes were negative for tumor cells (mean, 2.03 cm, range, 0.60-4.50 cm). Information concerning clinical outcome is available for 61 patients followed for between 1 and 7 years (mean 3.2 years). Three of 17 patients (18 per cent) who had occult tumor in the nodes developed distant metastases, all less than 3 years after initial surgery. One of the 44 patients (2 per cent) whose nodes were free of occult tumor developed distant metastases 5 years following surgery. Local recurrences in the area of the mastectomy occurred in one of 17 patients with occult nodal tumor (6 per cent), less than 1 year after surgery. Local recurrences were seen in three of 44 patients without occult metastases (7 per cent), in two patients 5 years after mastectomy and in one patient 7 years after mastectomy. PMID- 1724634 TI - A new radioimmunoassay detecting early stages of colon cancer: a comparison with CEA, AFP, and Ca 19-9. AB - A previous study reported the results of a radioimmunoassay that analysed immune complexes (IC) with a specific labeled polyclonal antibody for the detection of the early stages of colon cancer. In order to investigate further the possible clinical use of this assay, a blind study that screened 505 patients referred for colonoscopy, compared their pathology reports with the results of four assays that measure levels of circulating tumor markers. These were CEA, AFP, Ca 19-9, and our previously described radioimmunoassay (RIA). Of the patients with no malignancies, the results that were in the normal range were as follows: CEA 473/495 (95.6 per cent), Ca 19-9-486/495 (98.2 per cent) and our RIA-488/495 (98.6 per cent). AFP levels were in the normal range for all patients in the study. The only assay to identify any Dukes' C and D patients was Ca 19-9, which detected 2/3 (67 per cent). Of the patients with Dukes' A and B colon cancer, CEA only identified 1/7 (14 per cent), AFP and Ca 19-9 0/7 (0 per cent), and our own RIA 5/7 (71 per cent). The positive results of our assay were significantly different from those of the other three assays with p values all less than 0.05. These preliminary results suggest that this RIA, because of its ability to detect the early stages of colon cancer, may be an effective complement to the currently available assays. The combination may provide a more comprehensive evaluation in monitoring colon cancer. PMID- 1724635 TI - Evaluation of calcofluor white [CFW] stain for detection of Pneumocystis carinii. PMID- 1724636 TI - Interference of elevated fetal hemoglobin on HbA1c measurements in adult type I diabetic patient. PMID- 1724637 TI - Lomefloxacin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. AB - The antibacterial efficacy of oral lomefloxacin has been investigated in a wide variety of infections, including respiratory and uncomplicated and complicated urinary tract infections, obstetric, gynaecological, joint, skin, oral, ear, nose, throat and eye infections. It has also been used as an otic solution in patients with otitis media and as an ophthalmic solution in the treatment of eye infections. In clinical trials its efficacy is equivalent to that of other quinolones and it is at least as effective as other antibacterial drugs ordinarily used in these infections. Lomefloxacin offers certain advantages compared with other quinolone antibacterial drugs in that it may be conveniently administered once daily and theophylline dosage adjustment does not appear to be necessary in patients receiving this bronchodilator concomitantly. Thus, orally administered lomefloxacin should prove a useful broad spectrum antibacterial drug for a wide variety of clinical infections. PMID- 1724638 TI - Flumazenil. A reappraisal of its pharmacological properties and therapeutic efficacy as a benzodiazepine antagonist. AB - Flumazenil is a specific benzodiazepine antagonist which is indicated when the central effects of a benzodiazepine need to be attenuated or terminated. Following intravenous administration of up to 1 mg, flumazenil effectively reverses sedation and improves psychomotor performance following administration of short and longer acting benzodiazepines used for sedation, or general anaesthesia supplemented with benzodiazepines. The duration of action is short at generally 30 to 60 minutes and supplemental doses of flumazenil may be needed to maintain the desired level of consciousness in some patients. After poisoning with high dosages of benzodiazepines alone or combined with other drugs, the initial single dose of flumazenil will require supplementing with repeated low intravenous doses or an infusion to maintain wakefulness. In such patients, flumazenil also facilitates differential diagnosis and reduces the necessity for interventions. Flumazenil thus enhances recovery and allows more rapid discharge of patients sedated with benzodiazepines for diagnostic procedures and facilitates management of patients during the initial recovery period following general anaesthesia supplemented with benzodiazepines, but does not preclude normal monitoring during the recovery period. Flumazenil is clearly very useful in treating drug poisoning when benzodiazepines are a major component. By virtue of its specific benzodiazepine antagonist effects, flumazenil provides an innovative and well tolerated approach in clinical situations requiring rapid reversal of benzodiazepine-induced central nervous system depressant effects. PMID- 1724639 TI - The place of intracardiac injections in the treatment of cardiac arrest. PMID- 1724641 TI - Regression of increased left ventricular mass by antihypertensives. AB - Left ventricular hypertrophy (LVH) is both a target organ response to chronic arterial hypertension and a disorder that may be responsible for cardiovascular events. Although an increase in ventricular wall thickness may initially be compensatory and decrease wall stress, numerous studies have indicated that LVH is associated with a reduction in coronary flow reserve, diastolic and systolic ventricular dysfunction, and ventricular arrhythmias, all of which predispose to morbid cardiovascular events, including acute coronary syndromes, congestive myocardial failure, and sudden death. Reduction in LVH has been accomplished with beneficial effects on diastolic function and ventricular arrhythmias, without pressure-induced deterioration in systolic or diastolic function. Although therapy with diuretics and vasodilators effectively lowers arterial pressure, these therapies are not usually associated with significant regression of LVH. Calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors, beta adrenergic blockers, central adrenergic blocking agents, and possibly alpha adrenergic blockers and diuretics with vasodilatory properties are associated with reductions in LV mass. However, studies are still needed to determine the relative effects of various antihypertensive therapies on LVH regression, coronary flow reserve, ventricular function, ventricular ectopy, and most importantly, on protection against major cardiac morbidity and mortality inherent to LVH. PMID- 1724642 TI - Drug treatment of pneumonia in the hospital. What are the choices? AB - Mortality and morbidity of nosocomial pneumonia remain high. Successful treatment of pulmonary infections depends on several factors including type of infection, offending pathogen, status of host defences, and adequate choice of antibiotic therapy. The physician's decision should aim at achieving antibiotic concentrations beyond the MIC at the site of infection. Gram-negative bacilli, notably Pseudomonos aeruginosa, Klebsiella pneumoniae and Escherichia coli, remain the most frequent agents in nosocomial pneumonia. Staphylococcus aureus and Streptococcus pneumoniae predominate among the Gram-positive cocci. Pneumocystis carinii predominates in immunocompromised patients. Protected sample bronchoscopy associated with quantitative cultures of samples, and quantification of intracellular microorganisms in cells recovered by broncho-alveolar lavage are two promising procedures which might replace previous, more aggressive methods. Penetration of antibiotics into lung tissue depends on physicochemical properties of the drug and the degree of inflammation of lung tissue. Quinolones, macrolides, tetracyclines and trimethoprim penetrate well into bronchial secretions. Penetration is moderate to low for aminoglycosides and beta-lactams. Fluoroquinolones and new beta-lactam agents, including third-generation cephalosporins imipenem, aztreonam and ticarcillin-clavulanate, showed comparative clinical efficacy in treatment of nosocomial pneumonia, with an efficacy rate close to 80%. Aminoglycosides should not be used alone. Combination therapy reduces but does not eliminate the risk of selection of Gram-negative resistant mutants. It should not be used routinely except for P. aeruginosa, Enterobacter cloacae and Serratia marcescens infections. PMID- 1724640 TI - Bisphosphonates. Pharmacology and use in the treatment of tumour-induced hypercalcaemic and metastatic bone disease. AB - The geminal bisphosphonates are a new class of drugs characterised by a P-C-P bond. Consequently, they are analogues of pyrophosphate, but are resistant to chemical and enzymatic hydrolysis. The bisphosphonates bind strongly to hydroxyapatite crystals and inhibit their formation and dissolution. This physicochemical effect leads in vivo to the prevention of soft tissue calcification and, in some instances, inhibition of normal calcification. The main effect is to inhibit bone resorption, but in contrast to the effect on mineralisation, the mechanism involved is cellular. These various effects vary greatly according to the structure of the individual bisphosphonate. The half life of circulating bisphosphonates is very brief, in the order of minutes to hours. 20% to 50% of a given dose is taken up by the skeleton, the rest being excreted in the urine. The half-life in bone is far longer and depends upon the turnover rate of the skeleton itself. Bisphosphonates are very well tolerated; the relatively few adverse events that have been associated with their use are specific for each compound. Bisphosphonates have been used to treat various clinical conditions, namely ectopic calcification, ectopic bone formation, Paget's disease, osteoporosis and increased osteolysis of malignant origin. The three compounds commercially available for use in tumour-induced bone disease are in order of increasing potency, etidronate, clodronate and pamidronate. Most data have been obtained with the latter two agents. By inhibiting bone resorption, they correct hypercalcaemia and hypercalciuria, reduce pain, the occurrence of fractures, as well as the development of new osteolytic lesions, and in consequence improve the quality of life. In view of these actions, of their excellent tolerability and of the fact that they are active for relatively long periods, these compounds are, after rehydration, the drugs of choice in tumour induced bone disease and an excellent auxiliary to the drugs used in oncology. PMID- 1724643 TI - Cardiac arrhythmias in childhood. Diagnostic considerations and treatment. AB - Determining safe and effective antiarrhythmic therapy in paediatric patients requires definition of the mechanism of the arrhythmia, determination of associated risk factors for treatment (such as the presence of congenital cardiac defects, myocarditis or cardiomyopathy), and monitoring for potential drug side effects related to the treatment. A number of modalities for non-invasive evaluation of arrhythmias is available, including ECG, 24-hour ambulatory Holter monitoring, and transtelephonic ECG transmission. Arrhythmias requiring medical treatment in children with normal cardiac anatomy and function include supraventricular tachycardia (SVT), ventricular tachycardia (VT) and primary atrial tachycardias. SVT is treated acutely with vagal manoeuvres or drugs which slow AV conduction [adenosine (adenine riboside), edrophonium, phenylephrine or verapamil]. When medical conversion is not achieved, transoesophageal overdrive pacing or direct current (DC) cardioversion may be required. Long term drug therapy for SVT includes first-line treatment with digoxin, verapamil or propranolol. Ventricular tachycardia is managed acutely with DC cardioversion and intravenous lidocaine (lignocaine). Chronic drug regimens include mexiletine, propranolol or amiodarone. In children with structural congenital heart disease or myocardial dysfunction, hazards of drug therapy for arrhythmias include depression of cardiac function, proarrhythmia (drug-induced worsening of arrhythmias), and conduction abnormalities. Care must be taken to choose medication regimens which are likely to be effective with minimum risk of potentiating abnormal haemodynamics or conduction. PMID- 1724644 TI - Treatment of lower extremity infections in diabetics. AB - Despite recent medical advances in the treatment of diabetes mellitus, foot infection remains a major cause of morbidity and mortality in patients with this disorder. Three main factors are responsible for this: neuropathy, angiopathy and immunopathy. Neuropathy is probably the most important factor: minor irritations and trauma can lead to limb-threatening infections without the patient feeling the changes. Angiopathy plays only a minor role, while immunopathy has implications for antibiotic treatment, in that bactericidal agents are needed. A classification scheme that incorporates clinical and laboratory findings can direct the selection of empirical antibiotic therapy in patients with foot infections. These infections may be defined as mild, moderate and severe. In less severe cases, there are effective oral agents that can stop the progress of the infection and obviate the need for patient hospitalisation. Moderate to severe infections require hospitalisation with the use of parenteral agents. With some of the new broad spectrum drugs, single agent therapy is now possible, eliminating the need for expensive, potentially toxic combinations. Antibiotics, however, are only part of the cure. Aggressive surgical debridement followed by conscientious local wound care plays an equal role. The ultimate goal is foot salvage, and the clinical judgement of the practitioner is paramount in determining the treatment strategies needed to achieve this objective. PMID- 1724646 TI - Staining methods for the diagnosis of cryptosporidiosis: appraisal. AB - Simple methods which can be used in any modest laboratory for the diagnosis of Cryptosporidiosis in human and animal faeces are described. These methods may be applied to duodenal and lung aspirations. The Ziehl-Nelseen and Koster modified methods were found to be suitable in the diagnosis of Cryptosporidium due to their specificity and simplicity as compared to other staining methods. Koster modified method is more specific in demonstrating the sporozoites. PMID- 1724645 TI - Enoximone. A review of its pharmacological properties and therapeutic potential. AB - Enoximone is an imidazolone derivative currently undergoing trials in patients with congestive heart failure refractory to conventional therapy. It is a phosphodiesterase inhibitor with both positive inotropic and vasodilator properties, and is active by both oral and intravenous routes of administration. While pharmacodynamic studies have documented beneficial haemodynamic effects after short term oral administration, and objective and subjective improvement relative to placebo during some short term trials, its clinical efficacy during continuous longer term therapy remains uncertain. Enoximone is of potential benefit as an adjunct in short term management of patients with end-stage cardiac failure awaiting cardiac transplantation. In the usually small studies reported to date enoximone was generally better tolerated at oral dosages of less than 2 mg/kg than at higher dosages. Thus, while its pharmacodynamic profile holds potential promise in a difficult therapeutic area, the long term clinical efficacy and tolerability of enoximone remain yet to be determined in controlled trials of adequate size and duration. PMID- 1724647 TI - Tuberculosis in children: treatment evaluation and results in a 5 year cohort of children with tuberculosis in Turiani Hospital, Tanzania. AB - Between January 1983 and January 1988, a total of 146 children started TB treatment in Turiani Hospital, Tanzania. During the treatment period 16 children died and another 16 have been transferred out. From the remaining 114, 84 could be traced and were visited at home. Out of this group, 85% were found to be in good clinical condition, and 1% was in bad shape. Death had occurred in 7% after finishing their treatment. Medical records of all children were analysed. Tuberculin sensitivity testing has been carried out in 53 children from the follow-up group. The indications for treatment and the results of the follow-up study are discussed. PMID- 1724648 TI - (+/-)-Terodiline: an M1-selective muscarinic receptor antagonist. In vivo effects at muscarinic receptors mediating urinary bladder contraction, mydriasis and salivary secretion. AB - The affinity and selectivity of racemic terodiline (N-tert-butyl-1-methyl-3,3 diphenylpropylamine HCl) for muscarinic receptor subtypes was determined from functional responses of rabbit vas deferens (M1), guinea pig atria (M2) and bladder detrusor muscle (M3). (+/-)-Terodiline was found to be about as potent as pirenzepine in the rabbit vas deferens (Kb = 15 and 31 nM, respectively) and at least as selective for M1 relative to M2 (11-fold) and M3 (19-fold) receptors. Like pirenzepine, (+/-)-terodiline does not distinguish between M2 and M3 receptors in vitro. The peripheral actions of (+/-)-terodiline were evaluated in vivo in terms of its ability to induce mydriasis, and to inhibit salivary secretion and urinary bladder contraction. (+/-)-Terodiline given s.c. was equipotent in inhibiting intravesical bladder pressure and carbachol-induced salivary secretion (ID50 = 24 and 35 mg/kg, respectively), and in increasing pupil diameter (ED50 = 59 mg/kg). These results suggest that the in vivo actions of racemic terodiline at (M3) receptors mediating bladder contraction may not be separable from its actions at receptors mediating mydriasis and salivation. Moreover, its effects on the pupil and salivary glands are apparently not mediated through M1 receptors. Together, these findings help clarify the action of (+/-)-terodiline in the treatment of neurogenic bladder. PMID- 1724649 TI - Different effects of muscarinic agonists in rat superior cervical ganglion and hippocampal slices. AB - In this study the effects of muscarinic antagonists and agonists on M1 muscarinic receptors in the isolated rat superior cervical ganglion and the rat hippocampal slice were investigated. Oxotremorine and APE but not pilocarpine, McN-A-343 or 4 Cl-McN-A-343 induced small M2 muscarinic receptor-mediated hyperpolarizations in the rat superior cervical ganglion. Nevertheless, for all the agonists investigated the pA2 values of the muscarinic antagonists pirenzepine, AF-DX 116 and p-fluoro-hexahydro-sila-difenidol indicated the presence of only M1 muscarinic receptors in the rat superior cervical ganglion and hippocampal slice. Full agonistic behaviour with respect to depolarization of the rat superior cervical ganglion was observed for pilocarpine, McN-A-343 and 4-Cl-McN-A-343. Oxotremorine and arecaidine propargyl ester were partial agonists in this preparation, with maximal effects of 35 and 46% of the maximum obtained with pilocarpine, respectively. Pilocarpine, oxotremorine and arecaidin propargyl ester displayed full agonistic behaviour on the increase in firing rate of pyramidal cells in rat hippocampal slices. Whereas 4-Cl-McN-A-343 was a partial agonist (maximal effect of 63% of the maximum obtained with pilocarpine), McN-A 343 displayed no agonistic or antagonistic activity in rat hippocampal slices. It remains to be established whether the heterogeneous behaviour of the agonists in both preparations reflects as yet unknown differences in the M1 receptor protein or results from differences in the coupling of receptor to second messenger. PMID- 1724650 TI - Phorbol esters induce oscillatory contractions of intestinal smooth muscles. AB - The actions of tumor-promoting phorbol esters in smooth muscle excitation contraction coupling were studied in isolated guinea pig ileum in the presence of various contractile agents. Muscarinic agonists, histamine and bradykinin elicited an initial transient phasic contraction and a subsequent sustained tonic contraction in guinea pig ileum. The Ca2+ channel antagonist nifedipine selectively inhibited the tonic contraction. Phorbol esters, protein kinase C activators, induced immediate muscle relaxation followed by oscillatory contractions when added during the tonic phase of contraction. Phorbol esters, when added in advance, slightly altered the ligand-induced phasic contraction but converted tonic contractions into oscillatory spikes. The amplitude, frequency and shape of the oscillation induced by phorbol esters were dependent upon the dose of phorbol ester: amplitude was increased and frequency was decreased by increasing the doses of phorbol ester. In contrast, the phorbol ester potentiated the tonic contraction induced by high potassium chloride with little effect on the phasic component. It also sensitized the muscles to Bay K 8644. Bay K 8644, which was ineffective in stimulating muscle contraction at 1 nM, became a very effective stimulator in the presence of the phorbol ester. All of these phorbol ester-induced potentiations and oscillations were sensitive to inhibition by staurosporine or nifedipine. These data suggest that in guinea pig ileum, protein kinase C plays a positive regulatory role in Ca2+ channel activation and promotes a complex regulatory effect on Ca(2+)-mobilizing ligand-stimulated Ca2+ channel activity, which results in oscillatory contractile responses to carbachol, methacholine, histamine and bradykinin. PMID- 1724651 TI - Species differences between [3H] substance P binding in rat and guinea-pig shown by the use of peptide agonists and antagonists. AB - The affinities of various substance P agonists and antagonists for NK1 receptors in rat and guinea-pig tissues were compared. Striking species differences were observed. Both septide and [D-Pro4,D-Trp7,9]SP-(4-11) possessed much higher affinity for sites in the guinea-pig (brain and ileum) than for sites in the rat brain. These results could be explained by differences in the structure of the NK1 receptor according to the species, although the existence of various subtypes of NK1 binding sites in the two species cannot be excluded. PMID- 1724652 TI - Galanin-induced inhibition of insulin secretion from rat islets: effects of rat and pig galanin and galanin fragments and analogues. AB - The 29-amino acid neuropeptide galanin occurs in intrapancreatic nerves and inhibits insulin secretion. To study the structure-activity relations of galanin, we examined the effects of pig and rat galanin, three galanin fragments (galanin (1-11), galanin-(1-16) and rat galanin-(17-29) and four galanin analogues ([Ala2]pig galanin, [Ala2]rat galanin, [D-Trp2]rat galanin and [D-Trp2]galanin-(1 16] on glucose-stimulated insulin secretion from isolated rat islets. Pig and rat galanin and galanin-(1-11) equipotently inhibited glucose-stimulated (8.3 mM) insulin secretion at and above 10(-7) M (P less than 0.05), whereas galanin-(1 16), inhibited insulin secretion at 10(-6) M (P less than 0.01). In contrast, the C-terminal rat galanin-(17-29) and the galanin analogues did not influence insulin secretion. Thus, rat and pig galanin are equipotent in inhibiting glucose stimulated insulin secretion from rat islets. The active site resides in the N terminal part of the molecule. Furthermore, the binding of galanin to its receptor depends on structural characteristics governed by the N-terminal position and in particular by the Trp2 residue. PMID- 1724653 TI - Contractile responses and calcium mobilization in renal arteries of diabetic rats. AB - The causes of diabetes-associated change of renal artery vasomotion have not been established. We investigated both contractile responses to KCl and norepinephrine (NE) in renal arteries of rats with streptozotocin-induced diabetes and age matched controls and the effects on Ca2+ mobilization. Renal arteries from diabetics had greater maximum contractile responses to KCl and NE, but the threshold concentrations and EC50 values of KCl and NE were similar in controls and diabetics. The concentration-response for Bay K 8644, a dihydropyridine Ca2+ channel agonist in the presence of 20 mM KCl was significantly greater in diabetics than in controls. The maximum contractile responses to Ca2+ in the presence of 10(-6) M NE were significantly (P less than 0.05) greater in diabetics than in controls. The increased contractile response at low concentrations of Ca2+ (0.01-0.05 mM) was inhibited in both preparations by 10( 6) M nifedipine, but at high concentrations of Ca2+ (0.1-2.5 mM) the inhibition by nifedipine was significantly less in diabetics than in the controls. 45Ca2+ uptake had significantly greater resting levels in diabetics than in controls. The uptake of 45Ca2+ induced by 10(-5) M NE was significantly greater in diabetics than in controls, and 10(-7) M prazosin diminished both responses. The results suggest hyperreactivity of contractile responses to KCl or NE, and hyperpermeability of renal artery smooth muscle membrane to Ca2+ in streptozotocin-induced diabetics. PMID- 1724654 TI - Various Ca2+ entry blockers prevent glutamate-induced neurotoxicity. AB - In the present study we investigated the effect of different Ca2+ entry blockers on the onset of neuronal damage induced by glutamate, kainate or alpha-amino-3 hydroxy-5-methyl-5-isoxazolo propionate (AMPA) in primary culture of rat cerebellar granule cells. We found that the dihydropyridine derivative, nifedipine used at 100 nM concentration, significantly counteracted the neuronal death induced by 15 min application of 50 microM glutamate. This effect was dependent on the presence of nifedipine before the exposure of granule cells to glutamate and was dose-related (IC50 = 10 nM). The nifedipine response was reproduced by isradipine and by verapamil with IC50 values of 9 and 100 nM, respectively. The activation of voltage sensitive Ca2+ channels elicited by 100 nM Bay K 8644, greatly enhanced glutamate-mediated neurotoxicity. Moreover, 100 nM isradipine was significantly active in blocking the neuronal death produced by 24 h exposure of cerebellar granule cells to 10 microM AMPA or 60 microM kainate. These results reveal a 'preventive' role of the Ca2+ entry blockers on the development of the neurodegeneration induced by overstimulation of various glutamate receptor subtypes. PMID- 1724655 TI - Ruthenium red and capsaicin induce a neurogenic inflammatory response in the rabbit eye: effects of omega-conotoxin GVIA and tetrodotoxin. AB - The effects of ruthenium red, an inorganic dye with known capsaicin antagonist properties, was investigated in the rabbit eye. At a dose of 0.24 nmol ruthenium red inhibited the inflammatory effects of capsaicin (1 or 8 nmol). Unexpectedly, when the dye was injected in doses ranging from 0.24 to 7.4 nmol, it caused an inflammatory response with constriction of the pupil (miosis) and a breakdown of the blood-aqueous barrier, leading to a rise intraocular pressure. Tetrodotoxin (30 nmol) inhibited the ruthenium red-induced rise in intraocular pressure but had less effect on the miotic response. The tachykinin antagonist spantide inhibited the miosis but had no effect on the rise in intraocular pressure. Ruthenium red induced an increase in substance P-like immunoreactivity and calcitonin gene-related peptide-like immunoreactivity in the aqueous humor. These levels were positively correlated with the rise in aqueous humor protein concentration. The ruthenium red-induced miosis and, to a less extent, the rise in intraocular pressure were inhibited by the Ca2+ channel-blocking agent omega conotoxin GVIA (CTX), indicating a partial dependence on an influx of extracellular Ca2+. CTX also attenuated the miotic effect of capsaicin but had no effect on the capsaicin-induced rise in intraocular pressure. It is concluded that, in the rabbit eye, ruthenium red induces a neurogenic inflammatory response besides its capsaicin antagonist effects. PMID- 1724656 TI - Monoclonal antibodies against merokeratin from sheep's wool. AB - Four monoclonal antibodies designated as AMK-6, AMK-10, AMK-16 and AMK-27, were raised against merokeratin prepared from sheep's wool, and their cross-reactivity to epithelial cytokeratins were examined using immunoblot analysis and immunohistochemistry. AMK-16 which was specific to merokeratin in immunoblot analysis, demonstrated specific immunohistochemical staining of wool fibers of the sheep's skin. The other 3 monoclonal antibodies (AMK-6, -10, and -27) showed cross-reactivity towards epithelial cytokeratins with several molecular weights when examined by immunoblot analysis. However, each immunohistochemical staining of the sheep's skin with these monoclonal antibodies appeared to be cell type specific. These results indicate that merokeratin and certain epithelial cytokeratins share many antigenic epitopes in common, and these monoclonal antibodies against merokeratin from sheep wool will be useful for the analysis of wool-forming cell differentiation. PMID- 1724657 TI - [The effect on the cerebral microcirculation of in vivo stains used in biomicroscopy]. AB - Vasoactive properties of semi-colloidal acid stains of the Evan blue and tripane blue, were revealed in rabbits and rats. The microphoto-technique revealed a dose depending vasodilating effect of the stains. The response of the arteries had sometimes a biphasic character: constriction--dilation. I. v. administration of the stains induced a dose-dependent significant reduction of local blood flow in the cortex, subcortical white substance and other parts of the brain, of total cerebral blood flow in the venous sinus; a drop of systemic arterial pressure in normo- and hypertensive animals; a change in cerebral bioelectrical activity with increasing theta-waves, appearance of spikes and polyspikes in the EEG. Acting upon the formation of vascular tonus, these stains can interfere with the mechanisms of regulation of cerebral blood flow. PMID- 1724658 TI - Diagnostic and prognostic utility of C-reactive protein, alpha-1-antitrypsin and alpha-2-macroglobulin in neonatal sepsis: a comparative account. AB - C-reactive protein (CRP), alpha-1-antitrypsin (alpha-1-AT) and alpha-2 macroglobulin (alpha-2-MG) levels were evaluated serially in 25 healthy and 20 septicemic neonates and then compared as early diagnostic aids and prognostic indicators in this illness. Compared to healthy controls, septicemic neonates had significantly higher mean CRP levels (p less than 0.01). Neonates with septicemia, who recovered, had higher mean CRP levels than the group which died (p less than 0.05). As an early diagnostic aid CRP had a low Youden index, whereas for prognosis its index was higher. Septicemic neonates also had significantly higher mean alpha-1-AT levels (p less than 0.05), 12-24 hours after onset of illness, as compared to healthy neonates. Alpha-1-antitrypsin could not be used as an early diagnostic aid in septicemia, but was useful for predicting outcome. Mean alpha-2-macroglobulin levels did not show significant variation in healthy and septicemic neonates. Lower mean alpha-2-MG levels were observed in neonates recovering from septicemia. As an early diagnostic aid alpha-2-MG had a low Youden index, whereas for prognosis its index was higher. CRP had a higher Youden index than alpha-2-MG for early diagnosis of neonatal septicemia and had a higher index than both alpha-1-AT and alpha-2-MG for predicting outcome in septicemia. Serial use of CRP alone is, therefore, recommended for both purposes. PMID- 1724659 TI - Current management of homozygous beta thalassemia. PMID- 1724660 TI - [Effects of a defined infusion of 10% HAES 200/0.5 in the early phase of a septic syndrome on hemodynamic parameters]. AB - In an animal model the hemodynamic effects of three infusions of HES during the early stage of a septic syndrome were examined. In nine pigs with a body weight between 29 and 36 kg a septic syndrome was produced by infusion of E. coli endotoxin. 1, 4 and 7 hours after beginning of the endotoxin infusion 250 ml HES 200/0.5 were infused. After the 1st and 3rd infusion an increase of the cardiac output resulted, which was previously lowered, without changes of the CVP. During normal CO the 2nd infusion shows an increase of CVP and MAP. PWP did not change. The reactions of the hemodynamic parameters can be well explained physiologically. A volume supply at a reduced CO increases the latter without influence of the CVP. Normalized CO before the infusion resulted in a CVP increase. The PWP is not evaluable under the conditions of an elevated pulmonary resistance, as it exists at the septic syndrome. PMID- 1724661 TI - [Tolerance of Haemofusin in hemodilution and volume substitution]. AB - In an open and uncontrolled trial the frequency and the type of adverse events were registered in patients who received Haemofusin, a hydroxyethyl starch solution, either for volume substitution or haemodilution. A total of 379 patients were investigated. No serious allergic reaction occurred. Headache, fever, rigor, light allergic reactions and nausea were documented in 17 patients. Even if one assumes that these observed concomitant events were caused by the hydroxyethyl starch, Haemofusin proved to be a well-tolerated colloid with a low rate of side effects (4.5%). PMID- 1724662 TI - Application of carboxylic-phosphinic mixed anhydrides in the fragment peptide synthesis of protected analogues of substance P. AB - Mixed carboxylic-phosphinic anhydrides derived from peptide acids and 1-oxo-1 chlorophospholane have been applied in the synthesis of the protected [Leu11]-SP by the fragment coupling strategy. The yields from fragment couplings were ca. 75%, the products were of high purity while the conditions of formation and coupling of the corresponding mixed phosphinic anhydrides, for optimum yields, have been evaluated. PMID- 1724663 TI - Synthesis and NMR conformational analysis of a beta-turn mimic incorporated into gramicidin S. A general approach to evaluate beta-turn peptidomimetics. AB - The solution structure of a gramicidin S (GS) analog containing a beta-turn mimic [BTD4-5, Lys2.2']GS has been compared to that of native GS. The linear [BTD4-5, Lys2.2']GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD4-5, Lys2.2']GS, the intramolecular hydrogen bonding pattern, inter-residue NOEs, including a transannular H alpha-H alpha NOE, and JN alpha coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' beta-turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test beta-turn mimics. PMID- 1724664 TI - Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of substance P. Structure activity relationships. AB - Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11 residue is replaced by glutamate gamma-alkylesters. These analogues were tested in three in vitro preparations representative of NK-1, NK-2, and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonists in NK-1 and NK-2 preparations but show little agonist activity in the NK 3 preparation. When the SCH3 group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH3 at NK-1 receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by gamma-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Orn6]-SP6-11. Other gamma-alkyl esters of the glutamic acid reduce its biological activity. PMID- 1724665 TI - Endoscopy of esophageal cancers. AB - In this review about endoscopy of esophageal cancers, two main chapters are considered. Diagnostic endoscopy deals with the modalities, characteristic features and caveats essential for accurate assessment of esophageal malignancies. Therapeutic endoscopy deals with the palliative endoscopic treatment of the disease. Endoscopic dilatation, intubation and laser photocoagulation are discussed. It is stressed that these techniques should be tailored to each individual situation, especially if combination therapies (multimodality protocols) are considered. PMID- 1724666 TI - Surgery for esophageal carcinoma. AB - From 1975 through 1988, 257 patients with carcinoma of the thoracic esophagus have been treated in our Department. Operability was 90% (232/257), overall resectability 77% (198/257) and for the operated group 85% (198/232). Hospital mortality was 9.6% but decreased to 3% over the period 1986-1988. There were 65% squamous cell epitheliomas and 35% adenocarcinomas. pTNM staging was as follows: Stage I: 11.6%; Stage II: 23.2%; Stage III: 37.9%; Stage IV: 27.3%. Overall survival was 62.5% at 1 year, 42.4% at 2 year and 30% at 5 year. According to the pTNM staging 5-year survival was 90% for Stage I, 56% for Stage II, 15.3% for Stage III and 0 for Stage IV. There were no statistically significant differences according to tumor localisation, pathologic type, sex, age. Introducing extensive resection and extended lymphadenectomy seems to improve significantly survival in the patients in whom an operation with curative intention was performed, the 1 year survival being 90.8% versus 72%, 2-year survival: 81% versus 46%, and 5-year survival 48.5% versus 41% for respectively radical and non radical resections. Barrett adenocarcinomas have no worse prognosis than other esophageal carcinomas with a 5-year survival of 91.5% if lymphnodes negative, and a 54% overall 5-year survival. Functional results after restoration of continuity with gastric tubulation were judged excellent to very good in 86.5% at 1 year, but infra aortic anastomoses have a much higher incidence of peptic esophagitis: 53% versus 8% for cervical anastomoses. From this study it can be concluded that in experienced hands surgery today offers the best chances for optimal staging, potential cure, and prolonged high quality palliation. PMID- 1724667 TI - Radiotherapy in squamous cell esophageal cancer. AB - As the classical surgical treatment of squamous cell esophageal carcinoma is associated with poor 5-year survival rates and high operative mortality, radiotherapy and chemotherapy are being used as adjuvant or alternative primary treatments. The authors present their view on several therapeutic approaches and describe their irradiation technique. PMID- 1724668 TI - The role of laser therapy in the palliative treatment of esophageal cancer. AB - Laser has become in the recent years an important therapeutic tool for treating esophageal cancer in a palliative setting aiming to reopen the tumor stenosis. The literature shows that the initial enthusiasm for laser, compared with other methods--especially endoprosthesis--has somewhat been tempered because the success rate in restoring a normal food intake was lower than expected (65-68%). Although the complication rate is low, repeated therapy sessions are needed to maintain the lumen open. This constitutes a major drawback in terms of patient acceptability. Variations in the therapeutical methods (contact laser, photodynamic therapy, ...) have not added up to now significant value to laser therapy. Specific indications for laser therapy versus endoprosthesis are discussed and should bring both therapeutic modalities to a more considered application. PMID- 1724669 TI - Reduction of ischaemic rest pain in advanced peripheral arterial occlusive disease. A double blind placebo controlled trial with iloprost. AB - Severe pain at rest is a major symptom of advanced peripheral arterial occlusive disease (PAOD). Preliminary studies of treatment with iloprost, a prostacyclin analogue, showed encouraging results in patients with ischaemic rest pain. Therefore a randomized, placebo-controlled multicentre study was undertaken in 113 patients admitted to hospital with rest pain of at least 2 weeks duration caused by severe PAOD. The patients were randomly assigned to receive 2-week placebo or iloprost infusions for 6 hours per day at a dose of 0.5-0.2 ng/kg/min in addition to conventional care. Demographic data and arteriographic findings were similar in the two groups. Eleven patients withdrew from the study before completion and 102 patients could be included in the final analysis. Significantly more patients in the iloprost group (62.5% of 48) than in the placebo group (42.6% of 54) had complete relief of pain without analgesic therapy during at least five consecutive days at the end of the treatment period (p less than 0.05, chi 2-test). Facial flush, headache and nausea were the most common side effects during iloprost infusion. Serious adverse reactions did not occur. Thus, a 2-week iloprost infusion was shown to be safe and effective as a treatment for ischaemic rest pain caused by PAOD. PMID- 1724670 TI - Enteric spherules diastase in enzyme preparations. AB - Enteric diastase spherules were prepared using cellulose acetate phthalate and eudragit RS-100 by emulsion solvent removal method. Factors that could affect the size shape and diastatic activity were studied. The studied factors include pH of aqueous media, temperature of dispersion media and concentration of surface active agent. Prepared enteric diastase spherules were investigated for stability and diastatic activity at different temperatures and pH in various buffer systems, in the presence and absence of pepsin, a gastric enzyme. Eudragit RS-100 based spherules were noted to be spherical and uniform in shape with an appreciable level of diastatic activity. However, diastase was observed to be released from such a system in a slow but controlled manner. Eudragit RS-100 was found to provide more stabilization to the diastase than the stabilization provided by cellulose acetate phthalate. The spherules when tested in in vitro and in situ were recorded to exhibit diastatic activity at 99 per cent potency level (based on an actually incorporated amount of diastase). The study reveals that enteric or pH sensitive diastase releasing systems hold promise and potential in the therapeutic application of digestive enzyme(s). PMID- 1724671 TI - Generation of slow-wave-type action potentials in canine colon smooth muscle involves a non-L-type Ca2+ conductance. AB - 1. The hypothesis was addressed that a non-L-type calcium conductance is involved in the generation of the initial part of the slow-wave-type action potential in the canine colon. 2. In the absence of a sodium and chloride gradient (NaCl replaced by glucamine), and in the presence of nitrendipine (in 'glucamine nitrendipine' Krebs solution), a major portion of the upstroke potential of the slow wave persists at unchanged frequency. 3. In 'glucamine-nitrendipine' Krebs solution, the rate of rise and amplitude of the upstroke potential is reduced by removal of extracellular calcium in a concentration-dependent manner. 4. The rate of rise and the amplitude of the upstroke potential is in a concentration dependent manner reduced by Ni2+ greater than Cd2+ greater than Co2+ greater than Mg2+. 5. In 'glucamine-nitrendipine' Krebs solution, Ba2+ cannot replace Ca2+ in the generation of the upstroke potential. 6. Positive evidence was obtained for the hypothesis that a non-L-type calcium conductance is involved in the initiation of the slow-wave-type action potential in colonic smooth muscle. PMID- 1724673 TI - Myelin basic protein specific T cell lines and clones derived from the CNS of rats with EAE only recognize encephalitogenic epitopes. AB - The epitope specificities of myelin basic protein (BP) specific T cell lines derived from the spinal cords (SC) and lymph nodes (LN) of rats with experimental autoimmune encephalomyelitis (EAE) were compared. To induce EAE, Lewis rats were immunized with guinea pig (GP)-BP and complete Freund's adjuvant. Mononuclear cells from the SC and LN of these animals proliferated in response to BP and the purified protein derivative (PPD) of mycobacterium. After initially being cultured in growth medium, SC mononuclear cells had an enhanced response to BP and lost their response to PPD. LN cells cultured in identical conditions lost their response to both BP and PPD. LN-derived BP specific cell lines recognized only two epitopes of GP-BP: an encephalitogenic epitope in residues 72-89 and a non-encephalitogenic epitope in residues 43-68. SC-derived BP specific cell lines and clones recognized the 72-89 epitope and a second encephalitogenic epitope contained in residues 87-99 but not the non-encephalitogenic 43-68 epitope. Unlike those from LN, BP-specific T cell lines and clones derived from the CNS appear to recognize only encephalitogenic epitopes, including epitopes not recognized by LN lines. PMID- 1724672 TI - Effect of external Cd2+ and other divalent cations on carbachol-activated non selective cation channels in guinea-pig ileum. AB - 1. Inhibitory actions of the external divalent cations, Cd2+, Ni2+, Mn2+ and Co2+ on carbachol (CCh)-induced inward current (Ins,ACh) were investigated in caesium aspartate-loaded single longitudinal smooth muscle cells of guinea-pig ileum, using a rapid solution-switching device, under voltage-clamped conditions. 2. Cd2+ (20-1000 microM), when added to the external solution, reduced Ins,ACh rapidly and reversibly. This effect occurred dose dependently, the relationship being adequately described by a Michaelis-Menten equation with a Hill coefficient (n) of 1.0 and a dissociation constant (Kd) of 98 microM. 3. The inhibitory action of Cd2+ was associated neither with agonist concentration (CCh) nor with changes in reversal potential, and was voltage independent. 4. The appearance and removal of the effect of Cd2+ were both rapid (a few hundred milliseconds), in sharp contrast with a relatively slow time course of atropine or CCh action (of the order of seconds). 5. Other divalent cations (Ni2+, Mn2+, Co2+, Mg2+) applied externally also suppressed Ins,ACh, but less potently. The sequence of apparent potency was Cd2+ (98 microM) greater than or equal to Ni2+ (131 microM) much greater than Co2+ (700 microM) greater than or equal to Mn2+ (1000 microM) much greater than Mg2+ (approximately 10 mM). 6. External Ca2+ increased Ins,ACh dose dependently and antagonized the inhibitory effect of Cd2+. However, this effect may not be a simple competition with Cd2+. 7. These results show that external divalent cations strongly modulate Ins,ACh channels, possibly through direct interaction with the channel protein. PMID- 1724674 TI - Histamine effects on the 5-HT1c receptor expressed in Xenopus oocytes. AB - To analyze the cross-reactivity between serotonin (5-HT) and histamine, the in vitro transcribed RNA for the 5-HT1c receptor was functionally expressed in Xenopus oocytes. 5-HT significantly increased 45Ca2+ efflux in RNA-injected oocytes, but not in uninjected and water-injected control oocytes. Furthermore, histamine and the H1 receptor agonists, but not the H2 and H3 agonists, significantly induced 45Ca2+ efflux in 5-HT1c receptor RNA-injected oocytes, but not in uninjected and water-injected oocytes. However, the H1, H2, and H3 antagonists failed to inhibit histamine-induced 45Ca2+ efflux at 10(-6) M. This finding suggests that the 5-HT1c receptor can be activated by both 5-HT and histamine, although the action of histamine is different from classic histamine pharmacology. PMID- 1724675 TI - Molecular modeling of the weak glycine antagonist iso-THAO. AB - When compared to strychnine, a potent glycine antagonist, iso-THAO, a bicyclic 5 isoxazolol zwitterion, has been reported to be a weak glycine antagonist. Since there are so few glycine antagonists, and there is a striking lack of similarity between the structures of these two antagonists, iso-THAO was studied using current molecular modeling techniques and quantum mechanical calculations in order to compare the structural features and charge distributions of iso-THAO with glycine. The results of this study confirm our earlier hypothesis that an antagonist to inhibitory neurotransmitters like glycine and GABA has at least three binding sites to the natural receptor that are very similar to three such binding sites in the transmitter and its agonists, and each antagonist has an additional negative binding site. We speculate that the latter negative binding site can attach to the top of the chloride channel within the receptor complex. The diminished inhibitory activity of iso-THAO is attributed to its poor structural congruence with the three atom attachment sites used by glycine at its natural recognition site. PMID- 1724676 TI - Treatment of infective endocarditis with granulocyte colony-stimulating factor. AB - Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein hormone which stimulates the proliferation and differentiation of a subset of granulocyte precursors and enhances some functional activities of mature neutrophils. We tested the effects of G-CSF on a patient with intractable infective endocarditis. The white blood cell count was increased 3-fold and the inflammatory reactions such as C-reactive protein and erythrocyte sedimentation rate were completely normalized without any side effects. This is the first report describing the use of G-CSF for infective endocarditis. Administration of G-CSF might be suitable for treating intractable infections which cannot be controlled by antibiotics alone. PMID- 1724677 TI - Characterization of temperature-sensitive mutants of measles virus. AB - Ten temperature-sensitive (ts) mutants derived from the Edmonston strain of measles virus were characterized by the complementation test and were shown to have four defective sites (A, B, C, and D). Five ts mutants which were confirmed to have a defective site D, induced neither cytopathic effect (CPE) nor an infectious virus. Among the other five ts mutants which produced viral proteins with CPE and showed positive HAD at 39.5 degrees C, the three ts mutants (P253 505, P333, and F2-104) were studied in detail. P253-505 had a defective site C and both P333 and F2-104 had a defective site A. P253-505 and F2-104 produced neither a cell-free nor cell-associated infectious virus and P333 produced only a low level of cell-associated infectious virus. P253-505 and P333 produced virus particles at 39.5 degrees C, while F2-104 did not. The pulse-chase experiment showed a normal pattern of synthesis and processing of viral proteins, but immunofluorescence tests using monoclonal antibodies indicated that P253-505 lacked two epitopes of the M protein at 39.5 degrees C, and both P333 and F2-104 lacked one epitope of the P protein. The lack of these viral epitopes was shown to correlate with the temperature-sensitivity of the three ts mutants. PMID- 1724679 TI - Examination of subconductance levels arising from a single ion channel. AB - Single-channel records often show frequent currents at a main conductance level and occasional currents at subconductance levels. In some instances, the conductances occur at regular levels that are multiples of a minimum conductance. It is well-appreciated that multiple conductance levels may arise either from the co-operative gating of more than one pore or from changes that occur in a single pore. In this paper, we used theoretical models of ion permeation to examine subconductances arising in a single-pore channel. In particular, the work focuses on the following question: how can an ion channel that provides only one aqueous pore through the membrane produce regular subconductances and a main conductance that all have the same selectivity and the same ion binding affinity? The three types of ion permeation models used in this study showed that a single-pore channel can have subconductances because of long-lived conformational states, because of alterations in rapid fluctuations between conformational states, or because of slight alterations in the electrostatic properties in the channel's entrance vestibules. Regular subconductances with the same selectivity and binding affinity can arise in a single pore even if the energy profile changes do not meet the constant peak offset condition. The results show that the appearance of regular subconductance levels in a single-channel recording is not sufficient evidence to conclude that identical pores have co-operative gating, as would arise in a channel that is a multi-pore complex. PMID- 1724678 TI - The role of oxygen-derived free radicals in two models of experimental acute pancreatitis: effects of catalase, superoxide dismutase, dimethylsulfoxide, and allopurinol. AB - The role of oxygen-derived free radicals was evaluated in two models of experimental acute pancreatitis by testing the effects of agents which either reduce oxygen-derived free radical generation or scavenge those free radicals. Those agents (catalase, superoxide dismutase, polyethylene glycol-superoxide dismutase, dimethylsulfoxide, and allopurinol) were evaluated using the choline deficient ethionine-supplemented diet-induced model of acute hemorrhagic pancreatic necrosis and the supramaximal caerulein stimulation model of acute interstitial edematous pancreatitis. In both models, the only effect associated with administration of the test agents was a reduction in the degree of pancreatic edema. These results suggest that oxygen-derived free radicals may play an important role in the development of pancreatic edema during pancreatitis but that those free radicals do not play an important role in the development of acinar cell injury. PMID- 1724680 TI - Double-labeling of saphenous nerve neuron pools: a model for determining the accuracy of axon regeneration at the single neuron level. AB - We have developed a labeling procedure which accurately and consistently labels the original sensory pools projecting to their respective nerve branches as a model to quantify the accuracy of nerve regeneration at the single neuron level. Adult and juvenile rats had the saphenous branch of the femoral nerve transected just distal to the bifurcation of the nerve into a sensory branch (saphenous nerve) and a motor branch (nerve to the quadriceps muscle) and exposed to a 3% solution of 1,1'-di-octadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) for 1 h, and then reanastomosed. Two weeks later the sensory branch was redivided proximal to the previous site and labeled with a second tracer (5% fluorogold) using similar procedures. Five days later the animals were killed and cryostat sections were prepared and analyzed with a fluorescence microscope to score single- and double-labeled primary sensory neurons. The results show that the primary sensory neurons which project into the saphenous nerve can be reliably prelabeled by exposure of the saphenous nerve to DiI, and two weeks later approximately 99% of the same population of neurons can be labeled by exposure of the nerve to a second dye, fluorogold. This model system will be very powerful for future studies concerning target reinnervation following nerve regeneration. PMID- 1724681 TI - The anterograde and retrograde transport of neurobiotin in the central nervous system of the rat: comparison with biocytin. AB - In order to test whether neurobiotin, an analogue of biotin, is transported by neurones in the central nervous system, injections of varying volumes of a 5% solution were made into different regions of the rat brain. Following perfusion fixation, the sites of injection and possible sites of transport were sectioned and incubated with an avidin-biotin-peroxidase complex and then subjected to a peroxidase reaction. All injection parameters and sites were directly compared to equivalent injections of the closely related substance, biocytin, which is effectively transported in an anterograde fashion. The sites of injection of neurobiotin were characterised by large areas of labelling that were more extensive than those produced by equivalent injections of biocytin but with less intense labelling of individual neurones. Each of the injections of neurobiotin gave rise to marked anterograde labelling, the characteristics of which were similar to those produced by biocytin, but due to the larger injection sites was heavier than that produced by biocytin. Each of the injections of biocytin or neurobiotin also gave rise to retrograde labelling; the degree of labelling was far greater with neurobiotin but varied between pathways. Retrograde labelling only occurred to a minor degree in some pathways but was particularly marked in others, e.g., the striatonigral pathway. As with biocytin, tract-tracing with neurobiotin can be applied to electron microscopy and can readily be combined with immunocytochemistry for endogenous substances. It is concluded that neurobiotin is an effective anterograde and retrograde marker that may be of use in studies in the central nervous system, particularly in large homogeneous structures such as cortical fields and the striatum. PMID- 1724682 TI - A simple and multi-purpose "concentration-clamp" method for rapid superfusion. AB - A simple method for the rapid exchange of the medium used for a variety of electrophysiological experiments including patch-clamp experiments on cultured cells is described. The advantages of this method over the conventional ones are that this solution exchange system has a very simple structure and thus is inexpensive and can be easily installed in already existing experimental setups. The method involves the use of a fine double-barrelled polyethylene needle placed near the cell or other isolated preparations under recording. Electromagnetic valves are used to switch the solutions in the polyethylene tubes which enable a rapid and localized application of test solutions as well as their rapid washout. The latency of solution exchange, as measured by a change in the holding current under voltage-clamp experiments was fairly rapid (about 120 ms). Neither was the recording interrupted nor the sealing resistance affected during repeated exposure to test solutions. This method is proving to be useful, particularly for electrophysiological and pharmacological studies on immovable cells such as those in culture. PMID- 1724683 TI - Calcitonin gene-related peptide (CGRP) causes endothelium-dependent cyclic AMP, cyclic GMP and vasorelaxant responses in rat abdominal aorta. AB - Calcitonin gene-related peptide (CGRP), a neuropeptide found in nerves surrounding most blood vessels, is a potent hypotensive agent in both humans and rats. In isolated strips of rat thoracic aorta, CGRP has been reported to cause endothelium-dependent relaxation. To study the cellular and molecular mechanisms involved in CGRP-induced vasodilation, we investigated the roles of two second messengers, cyclic AMP and cyclic GMP, as potential mediators of the signal transduction mechanism leading to vasodilation in response to CGRP in rat aorta. In the present study, the abdominal aorta, rather than thoracic aorta, was used because of its higher content of endogenous CGRP and, therefore, the greater likelihood of regulation by CGRP in vivo. Each abdominal aortic ring was precontracted with norepinephrine (NE) at its EC50 concentration (10-20 nM). CGRP (3-300 nM) caused concentration-dependent relaxations (reducing the NE-induced contractions by 34%) that were completely dependent on endothelium. The relaxations in response to CGRP were correlated in a time- and concentration dependent manner with increases in aortic levels of both cyclic AMP and cyclic GMP. CGRP (100 nM) caused significant elevations of cyclic AMP levels (1.4 to 3.2 pmol/mg protein, at 1 min) and cyclic GMP levels (1.6 to 3.6 pmol/mg protein, at 30 s). Like the vasorelaxant responses, both cyclic AMP and cyclic GMP responses to CGRP were totally dependent on the endothelium. Pre-incubation with indomethacin (3 microM, 15 min) did not alter cyclic AMP responses to CGRP (100 nM), suggesting that prostaglandins are not involved. Therefore, CGRP-induced vasodilations of abdominal aorta involve an endothelium-dependent mechanism associated with cyclic GMP elevations, similar to the mechanisms of vasodilation in response to acetylcholine and other endothelium-dependent vasodilators. However, CGRP-induced relaxations of aorta involve an additional mechanism (i.e., endothelium-dependent cyclic AMP elevations), which may also contribute to the intracellular mechanism of aortic vasodilation in response to CGRP. PMID- 1724684 TI - Substance P and neurodegenerative disorders. A speculative review. AB - The causes of the neurodegenerative disorders of Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are unknown. It is proposed that all these disorders result primarily from a loss of trophic peptidergic neurotransmitter, possibly Substance P (SP). This loss in turn produces the classical neuronal degeneration seen in each of these diseases and occurs due to a combination of natural aging and chronic autoimmune destruction following a viral infection of the CNS, early in life. The loss is therefore slow and by the time of clinical presentation the inflammatory process is disappearing as the antigenic stimulus lessens with its removal. The implications of the theory in terms of future research and therapy are briefly discussed. PMID- 1724686 TI - [Toxic effects of bupivacaine on heart conduction during peridural anesthesia]. AB - Cardiac rhythm disorder like ventricular extrasystole, has been observed into two of the twenty-eight patients with concomitant pathology, undergone to the epidural anesthesia with 0.50% Bupivacaine. The A.A., think that the strong sensitivity of the patients to the B., has been caused by concomitant symptoms from which they were suffering. PMID- 1724685 TI - Investigation of the importance of the N-terminal peptide bonds of substance P by synthetic pseudopeptide analogs. AB - Synthetic Substance P analogs where each of the first five peptide bonds are replaced by the peptide bond surrogate Gly psi [CH2CH2]Gly are investigated as ligands to the Neurokinin 1 (NK1) receptor from rat cerebral cortex. The affinities of these analogs are then compared with the corresponding Gly-Gly analogs. With this model system replacement of the peptide bond between position 4 and 5 with psi [CH2CH2] resulted in a 7-fold reduction of affinity. Substitution of other peptide bonds resulted in only minor changes in affinity. Although the N-terminal hexapeptide is important for high affinity binding to the NK1 receptor the present study provides evidence for that this effects is not caused by the side chains of the amino acids in position 1-6 or as results of intramolecular hydrogen bonding with the C-terminal part of the peptide. A possible exception is the peptide bond that in the native peptide connects Pro4 and Gln5. PMID- 1724687 TI - [Postoperative analgesia in transvesical prostatic surgery with spinal morphine]. PMID- 1724688 TI - [Propofol and histamine liberation in gynecologic surgery]. PMID- 1724690 TI - Cationic colloidal gold--a novel marker for the demonstration of glomerular polyanion status in routine renal biopsies. AB - Investigations of glomerular anionic charge status in human renal biopsies have previously been restricted, by the techniques and markers used, to staining of sites in pre-embedded tissue. The introduction of a novel marker, cationic colloidal gold, which demonstrates fixed anionic sites in hydrophilic resin (LR Gold)-embedded, ultrathin tissue sections, has now enabled glomerular charge to be evaluated in routine biopsy material. The cationic gold marker detects components which express anionic charge under different pH conditions. The patterns of staining in tissue showing minor glomerular pathology and low proteinuria, together with enzyme-digestion studies indicate that anionic sites are normally associated with heparan sulphate proteoglycans, glycocalyx sialoproteins, hyaluronic acid, and other GBM components which have not yet been characterised. Several charge aberrations involving different pathological mechanisms have been identified using cationic gold. These aberrations may be categorised according to the pathological basis of the charge pattern defect, rather than glomerular disease classification, as a prelude to the precise identification of the anionic sites and their functional importance in relation to the glomerular charge selectivity barrier. The categories which have been defined are: (1) 'Normal', (2) interrupted, (3) neutralised, (4) structurally disorganised, and (5) depleted. As sites are further characterised sub categorisation is likely. We anticipate that this approach will help to elucidate both the participation of charged components in disease pathogenesis and their role in relation to glomerular proteinuria. PMID- 1724691 TI - Hepatitis C infection among dialysis patients: a comparison between patients on maintenance haemodialysis and continuous ambulatory peritoneal dialysis. AB - Three hundred and thirty-nine dialysis patients from two centres (278 patients on continuous ambulatory peritoneal dialysis (CAPD) and 61 on maintenance haemodialysis (HD) were tested for antibody against hepatitis C virus (anti-HCV) using first-generation enzyme immunoassay kits (Ortho Diagnostics). Anti-HCV was detected in five (1.8%) CAPD patients and ten (16.4%) HD patients (P less than 0.00001). Anti-HCV was confirmed to be positive in three (1.1%) CAPD patients and eight (13.2%) HD patients using neutralisation enzyme immunoassay kits (Abbott Laboratories). The marked difference in prevalence of anti-HCV among CAPD and HD patients was related to a significantly greater transfusion requirement of the HD patients. All the anti-HCV positive patients had been transfused. The risk of HCV infection was significantly increased in those who had received more than five units of blood. Four (26.7%) anti-HCV positive patients had one or more episodes of elevated serum alanine aminotransferase (ALT) values. PMID- 1724692 TI - The calcium current of turtle cone photoreceptor axon terminals. PMID- 1724694 TI - An analysis of retinal L-type horizontal cell responses by the ionic current model. PMID- 1724689 TI - Expression of VCAM-1 in the normal and diseased kidney. AB - We have conducted an immunocytochemical analysis to investigate the presence of the recently described vascular cell adhesion molecule-1 (VCAM-1) in human kidney, using the anti-VCAM-1 monoclonal antibody 1.4C3. In normal control tissue VCAM-1 was present on some (but not all) parietal epithelial cells lining Bowman's capsule. Forty-nine of fifty clinical biopsy specimens were characterised by the additional presence of VCAM-1 on proximal tubular cells. This was most marked in biopsies of patients with interstitial nephritis or systemic vasculitis with crescentic nephritis, but was also observed in biopsies with minimal change, IgA or lupus nephropathy, or from patients with diabetic nephropathy, amyloid, or gout. Proximal tubule VCAM-1 correlated significantly with the number of transferrin-receptor-positive leukocytes (r = 0.607, p less than 0.0001) in the interstitium, but not with expression of HLA-DR by tubular cells. Surprisingly, VCAM-1 was not observed on vascular endothelial cells in these biopsies, even in the presence of a marked infiltrate; this contrasts with other tissues (e.g. skin and synovium). The presence of VCAM-1 on tubular cells in the inflamed kidney indicates the potential for these cells to interact with mononuclear cells, either as accessory cells or as cytotoxic targets. The unexpected absence of VCAM-1 in renal vascular endothelial cells suggests local differences in the endothelial cells of this organ. PMID- 1724693 TI - The transmission of signals from photoreceptors to postsynaptic cells in the vertebrate retina. PMID- 1724695 TI - [The structure and composition of the complex that provides the adhesion of the cellular layers during the morphogenesis of the human embryonic adenohypophysis]. AB - The area of contact between adenohypophysis and diencephalon rudiments of human embryos (5-7 weeks of development) was studied using immunohistochemistry and electron microscopy. Basement membranes of Rathke's pouch ectoderm and of diencephalon bottom neuroectoderm are connected by means of a complex consisting of thin fibrous material and collagen-like fibrils. After Ca+2 and Mg+2 ions removal, this complex with basement membranes was separated from epithelial layers. The material in the contact area differed in its composition from the basement membranes covering the adenohypophysis and diencephalon rudiments by the high content of tenascin and the absence of EDB-fibronectin. Other basement membrane components (collagen IV, heparan-sulphate proteoglycans, entactin and laminin) were also present in this area. Tenascin accumulation was also found in Rathke's pouch epithelium and cavity. PMID- 1724696 TI - Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1. AB - Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates. PMID- 1724697 TI - [False positive and false negative results in testing for pyrogenic impurities]. AB - Causes of the occurrence of pyrogenic impurities in injection and infusion preparations as well as false-positive and false-negative results possible in the pyrogen test are reported. Possibilities of excluding pyrogenic impurities in the preparations concerned and of avoiding false-positive and false-negative results in the pyrogen test are discussed. PMID- 1724698 TI - Subcellular localization of porphyrins using confocal laser scanning microscopy. AB - The in vitro subcellular distribution patterns of 10 porphyrins, varying in hydrophobicity and charge, were studied using confocal laser scanning microscopy on two cell lines (V79 and C6 glioma cells) for incubation times up to 24 h. All of the porphyrins were taken up rapidly by both cell lines and distinct classes of subcellular distribution patterns were observed: general cytoplasmic staining; localization in lysosomes (usually associated with general cytoplasmic staining); localization in mitochondria (and general cytoplasmic staining); localization in mitochondria with subsequent uptake into lysosomes. Structure-localization relationships which have emerged are that porphyrins with dominantly cationic side chains localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes. PMID- 1724699 TI - Influence of litter size on the RNA content of VMH and LHA in preweaning rats. AB - The nutritional status in the early postnatal period may significantly influence the development and functional activity of food intake regulating mechanisms. Therefore we investigated the weight gains and total RNA content of the ventromedial (VMH) and lateral (LHA) hypothalamic cells of rat pups in normal litters (8 pups/nest) and in reduced litters (2 pups/nest) reared permanently (from birth to weaning) or temporarily (from 1st to 5th day) in reduced nests. A significant decrease of weight gains was found in pups reared in reduced litters during the first 5 days which resulted in significant differences of body weight between the pups from normal and reduced litters on 5th day of life. On the 15th and 30th days, there were no significant differences in body weight between these animals, but the RNA content in VMH cells of pups reared in reduced litters was significantly increased on the 15th and 30th days. The RNA content in the LHA cells was unchanged. The results show that reducing a rat litter to 2 pups caused short-term undernutrition leading to permanent changes of the RNA content in the VMH neurons. The possible consequences of the permanently changed RNA content of this hypothalamic structure are discussed. PMID- 1724700 TI - [Maternal representations during pregnancy and early mother-infant interactions]. AB - In this paper we aim to explore how maternal representations during pregnancy influence the early mother-infant interactions and the style of attachment of the baby. Two different cases are presented in order to focus on how maternal representations of herself as a mother and of the future baby influence the early interactions and the style of attachment of the children. It is stressed in one case the particular fixity of the maternal phantasmatic world and in the other the modifiability which can be influenced, in the latter case, by the conscious fantasies and the relationship with her own family and her own child. PMID- 1724701 TI - [Early disturbances of development and their impact on the goals of analysis: symbolization and the establishment of the therapeutic alliance]. AB - The author examines some early disturbances in the capacity of some patients to become aware of their psychic and affective states. These exert a profound effect on the establishment of the treatment alliance and the ability to communicate with the analyst. She compares the difficulties sometimes encountered by child analysts, working with small children who do not communicate mainly verbally, with those encountered with adults whose awareness of their mental processes and affects reveal grave disturbances. Examples are given of patients who make extreme use of metaphor and/or symbolic enactments in the analytic situation for purposes of communication with the analyst. PMID- 1724702 TI - [Intervention strategies in the analysis of children]. PMID- 1724703 TI - [Childhood in East Germany. Repressive education and its consequences]. AB - The author analyzes the process by which various educational institutions (nurseries, nursing schools, primary and secondary school) are moulded by a repressive society and the process by which families are bound by the ideology of that society. Consequences on the child are tragic (developmental lags, immaturity) and hinder it's internal and external functioning. PMID- 1724704 TI - [A guide for preparing didactic medical slides]. PMID- 1724705 TI - [Leukocyte migration inhibitory factor and basophil degranulation in drug reactions]. AB - We performed a prospective study in patients with a medical history of adverse reaction to drugs with the purpose of rule out allergy. We included 31 patients who were attended in the Allergy Service. We compared the sensibility and specificity of the test of inhibition factor of leucocytes migration and degranulation basophil against the exposition. After the statistical analysis, we concluded: the laboratory test, we have already mentioned, have little sensibility and specificity so the exposition test in the quickest, useful, and more simple method to determine drugs allergy, but more dangerous. PMID- 1724706 TI - Expression of the epitope recognized by the monoclonal antibody 44-3A6 during human fetal development. AB - In this report we describe the expression of the adenocarcinoma associated antigen recognized by the monoclonal antibody 44-3A6, in various tissues during normal human fetal development. Conventional, formalin-fixed and paraffin embedded sections of normal organs were examined from fetuses ranging from 9 to 42 weeks of gestation. Immunohistochemical localization of antigen-antibody complexes was accomplished using the avidin-biotin complex (ABC) method using horseradish peroxidase. The monoclonal antibody (MAb) 44-3A6 detects a cell surface 40 kD protein which is frequently expressed by adenocarcinomas and by select normal glandular tissues. Detectable expression of this protein was seen at different time periods during fetal development depending on the tissue. This expression was confined to a relatively small range of cell types and tissues; immunostaining was noted in select epithelial cells of the aerodigestive tract, exocrine pancreas, neural tissues, renal tubules, and transitional urothelium, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances, once expression occurred within a tissue, it continued through gestation. These data show that this tumor associated gene product is differentially expressed in a broad range of normal developing human fetal tissues. PMID- 1724707 TI - Blood transcobalamin levels in malignant hepatoma. AB - Alpha-Fetoprotein (AFP) levels measured by RIA show a strong sensitivity for the biological diagnosis of malignant hepatoma (MH). However, this parameter lacks specificity. Previous observations of an alteration in vitamin B12 metabolism in the presence of hepatoma led us to study vitamin-B12-binding proteins. Vitamin B12, also called cobalamin, is transported in the blood by two proteins or transcobalamins: one is haptocorrin (HC), which is linked to most of the cobalamin, and the other is transcobalamin II, which is involved in tissue exchanges. In this work, the levels of AFP and transcobalamins were determined by RIA and radioisotope dilution assay, respectively. They were measured in patients with MH (group A) and in patients with other liver diseases (group B). Compared with group B, group A showed a significant increase in total serum HC (p less than 0.005). In conclusion, it was observed that MH is accompanied by increased levels of HC. The origin of these changes could be due to either an increase in HC synthesis or a catabolic defect. PMID- 1724708 TI - [Morphological and morphometric characteristics of the large-intestinal mucosa in Campylobacter infections]. AB - Twenty-two biopsy specimens of colon mucosa were obtained from 16 patients with gastrointestinal campylobacteriosis histologically, histochemically and morphometrically. At the height of the disease colon mucosa of these patients showed a morphological picture of acute hemorrhagic and erosive--hemorrhagic colitis. When compared with such for other intestinal infections (shigellosis, salmonellosis, rotaviral gastroenteritis), the morphological features appeared to vary permitting a differential diagnosis with acute colitis due to above infections, but objective criteria to differentiate campylobacter-induced colitis from colitis in aggravation of nonspecific ulcerative one and Crohn's disease still remains to be found. PMID- 1724709 TI - [Pathomorphosis of acute leukemia]. AB - Macro- and microscopic examinations of the viscera and hemopoietic organs of 405 dead patients who had suffered from various forms of acute leukemia were performed as well as microscopic investigations of 560 punch biopsies obtained from 372 patients at different stages of the disease. Pathomorphological findings on acute leukemias are presented, changes in the disease in the course of cytostatic antileukemia treatment are observed. Morphological signs of therapeutic pathomorphosis of acute leukemia involve: reduction or eradication of leukemic growths in the viscera and hemopoietic organs; hemopoietic hypo- and aplasia; dystrophic and necrobiotic alterations in the organs; topographic changes of leukemic proliferates (extramedullary localization of the process); augmentation of infectious and inflammatory complications; other causes of death. PMID- 1724710 TI - Islet amyloid polypeptide has no effect on amylase release from rat pancreatic acini stimulated by CCK-8, secretin, carbachol and VIP. AB - Islet amyloid polypeptide (IAPP), a novel peptide recently isolated from the amyloid substance of pancreatic islets, has been localized in normal islet B cell granules, suggesting its physiological action in exocrine pancreatic function. In the present study, the direct effect of IAPP on secretagogue-stimulated pancreatic enzyme secretion was investigated using dispersed rat pancreatic acini. Various concentrations of IAPP (10(-5) -10(-10) M) showed no significant influence on amylase release stimulated either by submaximal doses of CCK-8 (10( 11) M), secretin (0.5 x 10(-6) M) or carbachol (10(-6) M), or by a maximal dose of vasoactive intestinal peptide (10(-8) M). These results may provide negative evidence for a physiological significance of IAPP in rat pancreatic enzyme secretion. PMID- 1724711 TI - [The use of alginic acid-based preparations for treating suppurative wounds of the maxillofacial area and neck]. AB - The effects of algin-based drugs--algipor, algimaf, teralgim--on the course of maxillofacial purulent wounds healing were under study. Cytologic and morphometric data and findings of pH measurements evidence a high efficacy of teralgim and of algipor and algimaf combinations with trypsin immobilized on a cloth base during the first phase of the wound process. Application of these drugs was conducive to a sooner wound purification and to stimulation of the reparative processes. PMID- 1724712 TI - [The use of the ultrasonic spraying of interferon and larifan solutions for treating acute herpetic stomatitis in children]. AB - Ultrasonic spraying of drug solutions was used in the treatment of 100 children suffering from acute herpetic stomatitis and its efficacy compared to that of common spraying of drugs in 100 children of the reference group. The results evidence that ultrasonic spraying accelerates cure by 2-3 days and is drug saving. Interferon was found to be the most effective drug for acute herpetic stomatitis; larifan, used for the first time in the treatment of this condition, was inferior to it, though still effective enough. PMID- 1724713 TI - Carcinoid tumor of the kidney with morphologic and immunohistochemical profile of a hindgut endocrine tumor: report of a case. AB - A 54-year-old man underwent a radical nephrectomy for a presumed renal cell carcinoma. The tumor was large, showed areas of cystic degeneration and calcification, and had completely obliterated the normal renal parenchyma. The light microscopic appearance was atypical for renal cell carcinoma, and when electron microscopy revealed innumerable neurosecretory granules a diagnosis of carcinoid tumor was made. The tumor cells were argentaffin- and argyrophil negative but were chromogranin-, neuron-specific enolase-, and leu-7-positive. When tested with a battery of antibodies against specific polypeptide hormones, the tumor exhibited diffuse pancreatic polypeptide and focal somatostatin immunoreactivity. Our case represents only the 16th case of carcinoid tumor of the kidney to be reported and the first with demonstrated pancreatic polypeptide immunoreactivity. The predominantly trabecular histology, nonreactivity with silver stains, and immunohistochemical profile of this case are common characteristics of hindgut carcinoids, suggesting that, like rectal carcinoids, renal carcinoids are tumors of hindgut endocrine cells. PMID- 1724714 TI - [The effect of synchronization on estrus and pregnancy in sheep and on levels of thyroxine, triiodothyronine, 17 beta-estradiol, progesterone and cholesterol]. AB - Knowledge of pathogenesis of sexual dysfunctions at altered thyroid activity is limited by the knowledge of multiple and ubiquitous action of its hormones throughout the organism. One of the possibilities of modulatory influence of thyroid hormones on sexual functions can be realized through the participation of thyroxine and triiodothyronine in the synthesis and metabolism of primary substrate of steroid synthesis--cholesterol. The presented work is aimed at the study of simultaneous dynamic changes of concentrations of thyroxine (T4), triiodothyronine (T3), 17 beta-estradiol (E2), progesterone (P4) and cholesterol (Chol) during synchronization of the rutting period and gravidity at parallel correlative evaluation of mutual relations of the followed parameters in ten Merino sheep in the seasonal period. Synchronization was achieved by chlorsuperlutin (Agelin--vaginal swabs, Spofa; 20 mg of chlorsuperlutin/swab) and PMSG (500 I. U./animal). Blood was sampled by means of a jugular vein puncture at the time of swab insertion (-13th day) and after three (-10th day) and seven ( 7th day) following days, at the removal of swabs and application of PMSG (-3rd day), on the day of insemination (zero day), on the 7th, 14th and 17th day and in the middle of the 2nd, 3rd, 4th and 5th month of gravidity. In the phase of oestrus synchronization a significant increase of E2 concentrations on days -7 and -3 of the experiment (0.47 +/- 0.079 and 0.542 +/- 0.177 nmol.l-1 of serum, P less than 0.001; P less than 0.001) was observed compared to the E2 values on day -13 (0.084 +/- 0.036 nmol.l-1 of serum). Parallel to these observations, marked intermittent changes of T4 (Tab. I, Graph 1) were recorded with the lowest values of this parameter observed on days -10 (41.75 +/- 20.23, P less than 0.05) and -3 (50.22 +/- 18.77, P less than 0.05) and the highest on day -7 (96.77 +/- 17.51 nmol.l-1, P less than 0.01) and day zero (85.40 +/- 19.59 nmol.l-1 of serum, P less than 0.05) in comparison with the -13th day (67.22 +/- 18.29 nmol.l-1 of serum). Concentrations of P4 (Tab. I, Graph 4) declined to the lowest values on day zero observation (0.09 +/- 0.08 nmol.l-1 of serum, P less than 0.05 vs 3.40 +/- 3.61 nmol.l-1 on day -13). No significant changes of concentrations of T3 (Tab. I, Graph 2) and Chol (Tab. I, Graph 5) were observed during oestrus synchronization. During gravidity, concentrations of E2 (Tab. I Graph 3) showed an increasing trend compared to the -13th day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724715 TI - Delayed motor development in relation to nutritional status among children under two years of age in two districts of Simbu Province. AB - The median ages of attainment of independent sitting, crawling, standing and walking were investigated in relation to nutritional status in 866 children aged under 24 months in Gumine District and 270 children in the same age range in Karimui District of Simbu Province. Sitting ability in these children was found to be comparable to western children of the same age but subsequent development was delayed in relation to western standards. This is particularly the case for Karimui children, who are almost 18 months old before they walk independently. Growth retardation, particularly the manifestation of chronic undernutrition in terms of length, is associated with the delay of important motor skills and hence delays the child's opportunities for actions independent of caregivers. In both districts solid food is introduced early and before the median age at which children sit independently, that is, at an age when motor development should still be conforming to western standards. In Gumine District dietary supplements initially consist mainly of fruit and other soft foods while in Karimui there is a more rapid transition to an adult-type diet of sweet potato and green vegetables as the daily staples. PMID- 1724716 TI - Effects of d- and dl-chlorpheniramine on serotonin and 5-hydroxyindoleacetic acid levels in the regional parts of rat brain. AB - The purpose of this study was to further investigate the effects of chlorpheniramine (d- and dl-forms) on the levels of serotonin (5-HT) and 5 hydroxyindoleacetic acid (5-HIAA) in the regional parts of rat brain. After the intravenous administration of each form of chlorpheniramine, the 5-HT and 5-HIAA levels were measured by HPLC system. Each form of chlorpheniramine is effective in reducing 5-HIAA, but not 5-HT levels in most of the regional brain of rats. The degree of reducing 5-HIAA levels does not correlate with the antihistaminic potency of these drugs. Our previous results showed that chlorpheniramine reduced the 3,4-dihydroxyphenylacetic acid levels, but not the dopamine levels in the brain of rats. Taken together, these findings raise problems regarding possible physiological and behavioral effects by using chlorpheniramine as a histamine H1 blocker in animal experiments. PMID- 1724717 TI - [Molecular and cellular analysis of the development and differentiation of the brain]. PMID- 1724718 TI - Possible role of aphidicolin-sensitive DNA polymerase(s) in N-methyl-N'-nitro-N nitrosoguanidine and bleomycin induced cellular repair synthesis and virus reactivation. AB - Treatment of human embryonic cell line CLV 102 with N-methyl-N'-nitro-N nitrosoguanidine or bleomycin was used to induce DNA repair synthesis. Both the repair synthesis induced by these agents and reactivation of UV-damaged pseudorabies virus were inhibited by aphidicolin. PMID- 1724719 TI - A general approach for characterizing glycosylation sites of glycoproteins. AB - Recent advances in glycobiology have greatly stimulated carbohydrate research; however, improving techniques for identification and isolation of specific glycosylation sites in protein structure analysis remains a challenge. We report here a practical approach utilizing a membrane staining technique on Problott, a PVDF-type membrane, to screen glycoproteins and glycopeptides derived from enzymatic digests of glycoproteins. To improve the detection sensitivity, an amplified staining technique using biotinylated lectins, avidin, and biotinylated peroxidase was employed. In addition, we describe a micro-batch affinity binding procedure to isolate glycopeptides from complex glycoprotein enzymatic digests. These protocols allow us to start with a subnanomole quantity of glycoprotein and locate the glycosylation sites; isolate glycopeptides in a homogeneous form; and perform amino acid composition, amino acid sequence, and mass analyses on the isolated glycopeptides. The characterization of glycosylation site of a model glycoprotein, carboxypeptidase P, of which the structure is still largely unknown, has been investigated. PMID- 1724720 TI - Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. AB - A method for fast and simple covalent immobilization of proteins to a carboxymethyldextran-modified gold surface intended for surface plasmon resonance sensors is described. The method utilizes the formation of N-hydroxysuccinimide esters from a fraction of the carboxyl groups of the carboxymethyldextran matrix via reaction with N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride in water. In a second step the protein is passed over the surface in a solution of low ionic strength with a pH value below the isoelectric point of the protein. The protein is thereby concentrated in the matrix by electrostatic attraction forces and a simultaneous reaction with the active esters takes place. In a final step, the remaining active esters are transformed into amides via reaction with ethanolamine. This sequence is performed automatically in a system comprising an integrated microfluidic cartridge and an autosampler. Typical reaction times of less than 30 min are required for the immobilization of proteins at surface concentrations in the region of 70 fmol mm-2. Parameters such as protein concentration, protein solution ionic strength, pH, reaction times, and reagent concentration can be varied in order to control the immobilized amount of ligand. The biospecific interaction of the immobilized ligand with its biological counterpart is illustrated by the effects on the interaction of immunoglobulins with immobilized Staphylococcus aureus protein A for various amounts of protein A. PMID- 1724721 TI - A novel approach to nonradioactive hybridization assay of nucleic acids using stained latex particles. AB - The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods. PMID- 1724722 TI - Drug interference in the Bradford and 2,2'-bicinchoninic acid protein assays. AB - The interference of a range of drugs and related substances has been investigated in the Bradford Coomassie brilliant blue (CBB) protein dye-binding assay and the 2,2'-bicinchoninic acid (BCA) protein assays. Chlorpromazine was the only substance to interfere in the CBB assay but the interference was slight. In contrast, the BCA reagent interacted strongly with chlorpromazine, the penicillins, vitamin C, and paracetamol and the mode of interference varied with the test substance. The chlorpromazine produced turbidity and an atypical color. The penicillins show a slow but normal color response while vitamin C and paracetamol gave an immediate and intense response. PMID- 1724723 TI - [Scleroderma and environment. I. Scleroderma and sclerodermiform states induced by silica and chemical agents or drugs]. PMID- 1724724 TI - Combination drug therapies for immunosuppression in transplantation. AB - Current combination immunosuppression protocols used worldwide include azathioprine/prednisolone, cyclosporine protocols, cyclophosphamide/steroids and FK506/steroids. The fact that many different immunosuppression protocols are currently in use, demonstrates that none is ideal. Major determinants in the choice of any protocol include graft and patient survival, side effects and cost. While most protocols may offer one year graft survival rates of 80% to 90% in renal transplantation, it is the long term results which are becoming increasingly important, but as many protocols have only been recently introduced, it may be sometime before these answers are known. While the most effective regimens include cyclosporine, long term nephrotoxicity remains a problem. Furthermore, the cost of the drug may be prohibitive in many countries worldwide. Azathioprine and low dose steroids still provide acceptable results with lesser expense and where cost and drug availability are critical, cyclophosphamide may even be introduced in place of azathioprine in living-related renal transplant recipients. The role of the newer immunosuppressive agents such as FK506 remains unclear, as the results of prospective randomised studies are not yet available. With excellent results now obtained with many different protocols, it is apparent that the choice of the most suitable immunosuppressive regimen is no longer dictated by graft survival alone. PMID- 1724725 TI - [The sources of the innervation of the sphincter of the esophagogastric junction in rats]. AB - The investigation has been performed by means of the luminescent microscopical method. The retrograde axonal transport of the fluorescent marker primuline has demonstrated that a definite amount of labelled cells are observed in the celiac plexus, in nodes of the thoracic part of the sympathetic trunk (predominantly in Th6-Th8). Innervation of the EGP sphincter is mainly performed from the sympathetic trunk nodes (Th6-Th8) and from the celiac plexus. PMID- 1724726 TI - Chemical semisynthesis of aprotinin homologues and derivatives mutated in P' positions. AB - An extended concept for the replacement of amino acids in the P' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys15-carboxyl group) lacking the amino acids Ala16 and Arg17 leads to aprotinin homologues and derivatives mutated in the P'1 and P'2 position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala17]BPTI and [seco-17/18]BPTI is described in detail. PMID- 1724727 TI - Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization. AB - Two specific alkaline phosphatase forms were identified in the integument of wild type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KCl, respectively. Both isoenzymes have a molecular weight of approximately 180,000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and beta glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2. PMID- 1724728 TI - The use of interferon in renal cell carcinoma. AB - Metastatic renal cell carcinoma remains an incurable disease and current modalities can only offer major palliation to a small percentage of patients. Since treatment is palliative, choice and type of therapy must be carefully considered and reconciled with patient desires. When possible, patients should be offered participation in a clinical trial. For patients choosing progestin therapy, treatment with interferon (IFN) or other biological response modifiers can be instituted at the time of progestin failure. Those patients who have slow tumour progression and maintain a high quality of life can be observed without continued progestin therapy. Although pretreatment characteristics predict response to biologicals, no pretreatment characteristic should preclude an individual patient from a trial of IFN therapy. Whether high-dose interleukin-2 (IL-2), IL-2/lymphocyte-activated killer cells, or IL-2/IFN are superior to IFN alone is uncertain, but clinical trials currently underway should help resolve these issues. PMID- 1724729 TI - [A rare case of adult-onset Becker muscular dystrophy diagnosed by dystrophin staining]. AB - Different diagnosis of Becker muscular dystrophy (MD) from limb-girdle MD mainly depends on the differences of heredity form and age at onset. However, sporadic cases with either type of MD often occur, and occasionally Becker MD can occur in adult age when limb-girdle MD commonly occurs. We reported the male sporadic case of Becker MD with the onset at 30 year old who was diagnosed by dystrophin staining. At the age of 30, he noticed mild difficulty to stand up and instability when hurrying up stairs. His weakness of lower limb-girdle gradually progressed, but he is able to walk without any support at the present age of 54, and he never showed weakness in upper limbs. Neurological and laboratory examination revealed that severe atrophy of lower limb-girdle, mild calf hypertrophy and moderate elevate of serum CK level. These history and symptoms hardly distinguish between Becker and limb-girdle MD. Immunostaining of biopsy muscle from the patient using the antiserum against synthetic peptide fragment of dystrophin revealed faint and patchy pattern, and immunoblot revealed 380 kd of abnormal size dystrophin. These dystrophin testing confirmed that this case was a rare case of Becker MD with adult-onset and mild clinical course. PMID- 1724730 TI - [Applications of Q beta replicase for diagnostic purposes]. PMID- 1724731 TI - Antigen specificity and cross-species reactivity of a monoclonal antibody (mAb 72.11) against porcine pancreatic procolipase. AB - We have studied the antigen specificity and cross-reactivity of a monoclonal antibody (mAb 72.11) of subclass IgG1, raised against the precursor form of porcine colipase (procolipase), whose epitope lies near the amino terminal region of the polypeptide. mAb 72.11 cross-reacts with native porcine, equine and human procolipase, as shown by immuno-inactivation and ELISA titration studies carried out on pure proteins, pancreatic tissue homogenate or pancreatic juice. The epitope site recognized by mAb 72.11 was further characterized by studying antibody binding to denatured procolipase. Reduced carboxymethylated procolipase reacted with mAb 72.11 in ELISA. Heat inactivated or reduced carboxymethylated porcine procolipase displaced antigen from the complex formed between antibody and native procolipase. The lack of sensitivity of epitope recognized by mAb 72.11 on procolipase to heat denaturation or reduction of the disulfide bridges is indicative that antigen specificity of mAb 72.11 is not dependent on the conformation of the antigenic site. Cross-reactivity of mAb 72.11 with procolipase from the three species demonstrates that substitution of amino acid at positions 1 and 3 causes no loss of antigenicity. Finally, mAb 72.11 was coupled to sepharose to isolate human procolipase from human pancreatic juice and to separate the precursor form from activated colipase non-adsorbed on the column. PMID- 1724732 TI - Quantitative assessment of the tissue response to implanted biomaterials. AB - The tissue response to a small number of polymeric biomaterials was studied using monoclonal antibodies specific for certain inflammatory cell types, to develop a reliable and accurate method for the quantitative evaluation of biocompatibility. The sites of antibody binding were identified using an avidin-biotin staining procedure and the sections evaluated using a computer-aided image analysis system. The staining technique successfully demonstrated both polymorphonuclear leucocytes and macrophages in tissue samples containing polymeric biomaterials. The image analysis system facilitated the measurement of up to 30 cell-related parameters and allowed a large number of cells to be analysed. PMID- 1724733 TI - Alteration of the inner surface of venous catheters by antineoplastic drugs. AB - Septic complications and thrombosis are frequent causes of long-term venous catheter implantation failure and tend to occur more frequently in oncology than in patients using catheters for hyperalimentation only. The purpose of this in vitro study was to study extensively the inner surface behaviour and the possible changes in their mechanical properties of various silicone and polyurethane catheters after exposure to a flow of the most common antineoplastic drugs. Silicone catheters appeared to be the best choice for cytostatic drug infusions because of their chemical stability, but the addition of an opacifier imposes a protective inner and outer layer to improve their surface properties for biocompatibility. PMID- 1724734 TI - In vitro studies of elastin-fibrin biomaterial degradation: preservative effects of protease inhibitors and antibiotics. AB - The degradability of a new elastin-fibrin material was tested in vitro versus human pancreatic elastase (HPE) and plasmin (PL) activities. It is shown that aprotinine Iniprol, a well-known protease inhibitor and Eglin C, a new potent inhibitor of HPE, especially when used in synergy, efficiently protected the material. A small amount of specific antibiotics was incorporated into the material. The two products will allow the material to be used in digestive surgery with improved safety. PMID- 1724735 TI - Structure and specificity of the T cell antigen receptor. AB - The antigen receptor on T lymphocytes is a multi-chain complex of which two chains determine its specificity for antigen. Although these receptor chains possess genetic and structural similarities with membrane-bound immunoglobulin, the B cell antigen receptor, they endow T cells with a distinct specificity. Unlike B cells, T cells recognize foreign antigenic properties only in the form of proteolytic fragments bound to molecules encoded by the major histocompatibility complex. In addition, one stage of the development of T cells in the thymus is dependent upon an antigen receptor-mediated event. Utilizing the response to a well defined antigen as a model system, we discuss the relationship between receptor structure and the specificity of these recognition events. PMID- 1724736 TI - Structure/function analysis of the invariant subunits of the T cell antigen receptor. AB - The T cell antigen receptor (TCR) is a multimeric surface receptor on T cells responsible for recognizing MHC-restricted antigens and initiating the cellular immune response. The clonotypic nature of the TCR resides in the antigen binding TCR-alpha beta or TCR-gamma delta heterodimer. The CD3 complex of gamma, delta and epsilon and the zeta-family disulfide dimer comprise the invariant TCR chains. Assembly of the mature TCR complex requires specific subunit interactions, the detailed nature of which is becoming more evident. Surface targetting of the TCR appears dependent on a region of the zeta chain near its transmembrane domain. A functional role attributable to zeta residing in the cytoplasmic tail is the capacity to couple antigen stimulation to IL-2 secretion, likely through activation of a tyrosine kinase. Assignment of functional roles for the remaining CD3 chains is not as clear; the removal of the cytoplasmic tail of CD3-delta does not affect TCR-mediated IL-2 signalling. Mounting evidence indicates that the structural complexity of the TCR is further enhanced by the various zeta family disulfide dimer pairs that can associate with the other TCR chains. This subunit diversification may offer the possibility of multiple coupling pathways available to the TCR as it responds to foreign antigens in various contexts. PMID- 1724737 TI - Multiple signal transduction pathways activated through the T cell receptor for antigen. AB - The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL 2 response. Two proteins known to be involved in growth regulation--p21ras and c raf--have now been shown to be downstream targets of the PLC/PKC pathway. PMID- 1724738 TI - Synthesis and transport of storage proteins by testes in Heliothis virescens. AB - The synthesis of two storage protein subunits, 76,000-Mr and 82,000-Mr polypeptides, by the testes sheath has been studied in Heliothis virescens. Like fat body, which is the primary site of synthesis for the large extratesticular pool, cells of the testes sheath secrete glycosylated storage proteins assembled into hexamers. The testis sheath differed from fat body in several important respects, including the failure to synthesize an abundant (in the hemolymph) 74,000-Mr storage protein, its relatively reduced expression of the 76,000-Mr polypeptide, and the absence of resorption of storage proteins from the lumen of the testis during pupal development. Cyst cells were also shown to import actively the 82,000-Mr storage protein by pinocytosis of testicular fluid and transfer it to the developing spermatids. Unlike other cell types that sequester storage proteins in the form of cytoplasmic granules, their localization within spermatids was exclusively mitochondrial. These observations suggest that expression of the storage protein genes is regulated tissue specifically and reveal novel pathways for their transport and, perhaps, utilization and function during development. PMID- 1724739 TI - Angiogenesis after hepatic arterial occlusion in liver transplant patients. AB - The authors describe 10 liver transplant recipients who developed occlusion of the hepatic artery or aortic conduit. Since all potential collateral arterial supply to the transplant is severed at hepatectomy, hepatic artery occlusion is usually a catastrophic event that necessitates repeat transplantation. Four patients died within 8 weeks of transplantation. The remaining six developed spontaneous arterial liver revascularization. This phenomenon is believed to be an example of neovascularization through angiogenesis. Radiologic studies, particularly duplex sonography and angiography, were helpful in the evaluation of transplant vascular integrity. The tissues of the omentum and the mesentery have known angiogenic ability. The authors postulate that in transplantation techniques in which these tissues are placed close to the transplanted liver (eg, Roux-en-Y choledochojejunostomy), the omental and mesenteric tissues may be the source of neovascularity. PMID- 1724740 TI - Rostral ventrolateral medulla: a source of the glutamatergic innervation of the sympathetic intermediolateral nucleus. AB - To determine whether the sympathoexcitatory projection from the rostral ventrolateral medulla (RVL) to the sympathetic intermediolateral nucleus (IML) of the spinal cord might use glutamate as an excitatory transmitter, we performed a dual-label, transport and immunocytochemical ultrastructural study. Axon terminals within the IML were examined to determine whether anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L) following injections into the RVL, was colocalized with glutamate immunoreactivity using an antibody to hemocyanin-conjugated L-glutamate (Hepler et al., J. Histochem. Cytochem., 36 (1988) 13-22). Transported PHA-L was visualized with the peroxidase antiperoxidase technique while glutamate-like immunoreactivity was localized within the same section of the thoracic spinal cord with immunoautoradiography. By light microscopy, PHA-L immunoreactivity was found within a plexus of fine fibers and varicose processes localized to the IML. Silver grains indicative of glutamate immunoreactivity were concentrated over the IML and also over the superficial layers of the dorsal horn. Electron microscopic analysis revealed PHA L immunoreactivity in axons and axon terminals within the IML. They ranged in diameter from 0.5 to 2.0 microns, contained numerous small clear and 0-3 large, dense-core vesicles, and formed primarily asymmetric synaptic contacts on small dendrites of IML neurons. Some of the PHA-L immunoreactive terminals making asymmetric (excitatory) synaptic contacts on the small dendrites of IML neurons also contained glutamate-like immunoreactivity. We conclude that at least a portion of the input to the IML from the RVL uses glutamate as its transmitter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724741 TI - Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system. AB - The developmental maximum expression of the large myelin-associated glycoprotein isoform (L-MAG) protein prior to that of the small myelin-associated glycoprotein isoform (S-MAG) in both the central and peripheral nervous systems (CNS, PNS) in mice was shown by immunoblotting techniques using specific antibodies to the L MAG protein and the S-MAG protein. Both the L-MAG protein and the S-MAG protein were expressed earlier in the PNS than in the CNS, which reflects earlier myelination in the PNS. The peak of the L-MAG protein concentration was 8 days in the sciatic nerve and 15 days in the brainstem. The concentration of the S-MAG protein in the sciatic nerve reached a peak at 15 days, whereas in the brainstem it increased rapidly between 15 and 20 days and gradually thereafter. Thus, the preceding maximum expression of the L-MAG during active myelination in the PNS demonstrated here as well as in the CNS strongly suggests an important role for L MAG in myelin formation. PMID- 1724742 TI - DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, is present in corticothalamic neurons of the rat cingulate cortex. AB - DARPP-32 immunocytochemistry and retrograde axonal labeling were combined to determine whether DARPP-32-containing neurons of the rat anterior cingulate cortex project to thalamus. Following injections of fluorescent latex microspheres into the mediodorsal thalamic nuclei, a large proportion of the DARPP-32 immunostained neurons in layer VI were also retrogradely labeled. In area 24a, these neurons were mostly found in layer VIb, whereas in area 24b, they were visible throughout layer VI. The presence of DARPP-32 in certain corticothalamic neurons suggests that these cells may be modulated by dopamine, which increases DARPP-32 phosphorylation, and possibly by glutamate, which antagonizes DARPP-32 phosphorylation via the N-methyl-D-aspartate (NMDA) receptor. PMID- 1724743 TI - Interaction of bleomycin with deoxyribonucleic acid oligomer: proton nuclear magnetic resonance titration study using novel bleomycin complexes with Ni2+ and VO3+. AB - Two metal complexes of bleomycin (BLM), BLM-Ni2+ and BLM-VO3+ are used for studying interactions between BLM and deoxyribonucleic acid (DNA) by nuclear magnetic resonance. Although these BLMs do not mediate DNA strand scission under the usual conditions, they bind to DNA in the same manner as the active metal complexes of bleomycin (BLM-Fe2+ and BLM-Co3+). A self-complementary dodecanucleotide, d(CCCCAGCTGGGG), having a single site for cleavage was synthesized. d(CCCCAATTGGGG), which contains no -GpC- sequence, was also synthesized. The BLM-metal complexes were shown to bind specifically to the GpC site by circular dichroism and fluorescence titration studies. We assigned all the resonances for imino protons and phosphorus, and most of the nonexchangeable proton resonances of d(CCCCAGCTGGGG). No substantial change in the chemical shifts of these signals was observed upon titration with either BLM-Ni2+ or BLM VO3+. This result is not consistent with a model of the strong intercalation of the BLMs between the base-pairs. The BLMs bind to DNA in a different manner, and DNA does not change its conformation upon binding with BLMs. PMID- 1724744 TI - New hydroxyl protecting groups of a safety-catch type removable by reductive acidolysis. AB - New hydroxyl protecting groups of a safety-catch type, i.e., 4 methylsulfinylbenzyl-oxycarbonyl (Msz) group for Tyr and 4-methylsulfinylbenzyl (Msob) ether for Thr, have been developed. O-Msz and O-Msob groups are stable under both acidic and basic conditions and can be removed by a one-pot reaction involving reductive acidolysis using tetrachlorosilane-trifluoroacetic acid (TFA) scavengers. Using these new protecting groups, a 17 residue-peptide, gamma endorphin, was successfully synthesized by the efficient solid phase method. PMID- 1724745 TI - Purification, characterization, and acute phase response of plasma alpha-1 antiproteinase in the hamster, Mesacricetus auratus. AB - 1. alpha-1-Antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) with a molecular mass of 60 kDa was purified to apparent homogeneity from hamster plasma. 2. It inhibited elastase, chymotrypsin and trypsin, but did not significantly affect pancreatic kallikrein, plasma kallikrein or plasmin. 3. It has the same N-terminal heptapeptide sequence as that of rat alpha-1 antiproteinase. 4. Its plasma level decreased after injection of bacterial lipopolysaccharide. PMID- 1724746 TI - Biochemical changes in skeletal muscles of denning bears (Ursus americanus). AB - 1. Biopsies of the extensor hallucis longus (EHL) and gastrocnemius (G) muscles of four captive black bears (Ursus americanus) were obtained prior to denning (PRE), during denning (DEN) and following the Spring arousal (POST). 2. Glycogen, triglyceride and protein concentrations did not differ significantly between the three groups. Likewise, the activity of citrate synthase, a mitochondrial oxidative enzyme, was not significantly different between the three groups. 3. DNA concentrations in DEN samples increased 30% compared to other groups while RNA concentrations were significantly elevated in POST samples. The RNA/DNA ratios were significantly depressed during DEN. 4. These results suggest a degree of muscle atrophy during DEN, with the potential for an increased capacity for muscle protein synthesis following the Spring arousal. PMID- 1724747 TI - Proteolysis of interleukin-2, interferon and immunoglobulin by venoms. AB - Limited proteolysis by venoms was analysed by the cleaved peptide band(s) in SDS polyacrylamide gel electrophoresis. The venom from Crotalus atrox degraded interferon, interleukin-2, IgG, IgM, and a crude form of acetyl cholinesterase but had no effect on IgA. Although the venom from Androctonus australis did not exert appreciable proteolysis on any of the immunoglobulins it had potent proteolytic activities against interferon and interleukin-2. The venom from Vespula maculifrons had only a minor proteolytic effect on interferon. The proteolysis by venoms was not effectively inhibited by alpha 1-antitrypsin or a2 macroglobulin. Moreover, no appreciable proteolytic activity was detected in the venoms from Bufo arenarum, Apis mellifera and Heloderma suspectrum. PMID- 1724748 TI - A brief comparison of methods for preparing fish chromosomes: an overview. AB - Methods for preparing fish chromosomes are presented for comparison, and their differences and similarities are given in tabular form. Many of the seemingly new and improved methods are merely modifications of known techniques. Differences between the methods used to prepare slides complicate the direct comparison of results. The development of standard methods appears to be desirable. PMID- 1724749 TI - Ultrastructural characteristics of substance P-immunoreactive terminals in marginal division of rat striatum. AB - In our previous work using immunocytochemical method combined with tract tracing techniques a new subdivision was described in the striatum of the rat. This "marginal division" is more densely filled with substance P, enkephalin and dynorphin B terminals than the rest of the striatum. In the present study, the synaptic organization of the substance P immunoreactive (SPIR) terminals in the marginal division of the rat striatum was studied using electron microscopy and immunocytochemistry for substance P (SP). Four major types of SPIR synapses were identified in the marginal division: axodendritic, axospinous, axo-axonal, and compound synapses. Axodendritic and axospinous synapses, in which the postsynaptic targets were small or large dendrites or spines, were the most common. A few axo-axonic synapses were observed as were several subtypes of compound synapses with more than two synaptic components. SPIR axon terminals formed the presynaptic components of all these synaptic types, but in one case an unlabeled bouton was observed making a synaptic connection onto a SPIR dendrite. Both symmetric and asymmetric SPIR synapses were observed in the marginal division. The vesicles in the SPIR presynaptic boutons were mostly pleomorphic although a few of them were round. The existence of asymmetric synapses, round synaptic vesicles and small postsynaptic dendrites distinguishes the ultrastructure of the marginal division from that of the other parts of the striatum. The complex characteristics of the synaptic organization in the marginal division implies that the SPIR terminals in the marginal division originate from a different source than those in the rest of the striatum. The complexity of the synaptic organization further suggests that the function of the marginal division is different from that of the rest of the striatum. PMID- 1724750 TI - Paracelsus: founder of medical chemistry. PMID- 1724751 TI - Carbohydrates and the chemical industry: achievements and prospects. AB - Carbohydrates are man's largest organic source of renewal energy both on land and in the sea. Whether consumed by man or animal, evolution has ensured that the enzymes in living cells use carbohydrates with conservation of that energy. Industries that make monomer carbohydrates dissipate the energy stored in its polymers. Prospects for future chemical and biochemical utilisation depend on remedying this tragedy and learning the lesson taught by nature. PMID- 1724752 TI - Patterns of actin filaments during cell shaping in developing mesophyll of wheat (Triticum aestivum L.). AB - Young leaves of wheat exhibit a smooth developmental gradient with meristematic cells at the base and highly differentiated cells at the tip. During differentiation, mesophyll cells attain a lobed outline resembling tube-shaped balloons with almost regularly spaced isthmi. Microfilament patterns in developing wheat mesophyll cells were investigated using fluorescent-labeled phalloidin. Various patterns were found, including delicate arrays of transversely oriented microfilaments in the cortex of the cytoplasm. A close correlation between changes in the patterns of cortical microfilaments, microtubules, cell wall microfibrils, and cell shape was observed. The fine arrays of transversely oriented microfilaments coaligned with bands of microtubules occurring during cell elongation. These bands were found beneath sites of intense wall deposition. It has recently been proposed that the resulting hoops of wall reinforcement prevent cell expansion in the corresponding regions and thus give rise to the peculiar cell shape. When cell expansion ceased, and the typical lobed cell shape was attained, a dense network of microfilaments was retained in the cytoplasm, which was in contrast to what has been described for the microtubular arrays. PMID- 1724753 TI - Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface. AB - Hyaluronic acid binding protein (HABP) has been purified to homogeneity from normal adult rat kidney by hyaluronate Sepharose affinity chromatography, and its apparent molecular mass was found to be 68 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of HABP under reducing as well as nonreducing conditions revealed a single protein band of 34 kDa, thus indicating that kidney HABP is a homodimer and lacks interchain disulfide bond. Its glycoprotein nature was demonstrated by Con-A binding analysis. The pI value of kidney HABP was 6, indicating its acidic nature. Polyclonal antibodies were raised against it, and the monospecificity of the antibodies towards HABP was confirmed by Western blot analysis of tissue extracts. Immunoblot analysis has elucidated the occurrence of this glycoprotein in various tissues. Moreover, HABP present in these tissues are shown to be structurally and immunologically identical. However, this glycoprotein is antigenically distinct from other well characterized extracellular proteins, e.g., fibronectin, laminin and collagen type IV. With the help of enzyme-linked immunosorbent assay (ELISA) and iodinated [125I]HABP, it has been shown that kidney HABP binds specifically to hyaluronic acid (HA) amongst all the glycosaminoglycans (GAGs), however, HABP can interact with other matrix proteins, e.g., laminin, fibronectin, and collagen type IV. The apparent dissociation constants of HABP for HA, laminin, fibronectin, and collagen type IV were approximately in the range of 10(-9) M, and kinetic analysis showed that these binding interactions were complex and of positive cooperative nature. Indirect immunofluorescence staining demonstrated its localization on human fetus lung fibroblast cell surface. Detection of 34 kDa HABP in the serum-free supernatant culture medium of fibroblasts was further evident by immunoblot analysis, thus confirming the secretory nature of HABP and its occurrence in the extracellular matrix. PMID- 1724754 TI - Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L. AB - Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins. PMID- 1724755 TI - An essential 45 kDa yeast transmembrane protein reacts with anti-nuclear pore antibodies: purification of the protein, immunolocalization and cloning of the gene. AB - A yeast membrane protein was isolated by its binding to tRNA Sepharose column. The 45 kDa protein shares characteristics with rat liver nuclear pore proteins in having reactivity with a monoclonal antibody (RL1) raised against rat liver nuclear pore proteins and by the binding of wheat germ agglutinin (WGA), indicating the presence of N-acetylglucosamine (GlcNAc) moieties. Immunofluorescence microscopy and cell fractionation experiments indicate that the protein is located in the nuclear envelope and the endoplasmic reticulum of the cell. The gene for the 45 kDa protein was cloned using degenerate oligonucleotides derived from the N-terminal protein sequence and confirmed by internal peptide sequences. The gene was named WBP1. The protein coding sequence of the WBP1 gene reveals an ER entry signal peptide and a C-terminal membrane spanning domain. Topological studies indicate that the C-terminus of the protein is located in the cytoplasm. The cytoplasmic tail of the protein contains the K-K X-X signal known to be sufficient for retention of transmembrane proteins in higher eukaryotic cells. Gene disruption experiments show that the 45 kDa protein is essential for the vegetative life cycle of the yeast cell. PMID- 1724756 TI - Assembly of carboxy-terminally deleted desmin in vimentin-free cells. AB - Using a vimentin-free expression system we were able to demonstrate that the carboxy terminus of desmin is necessary for filament assembly in the living cell. Desmin subunits missing only 4 carboxy-terminal residues of their rod domain are incapable of homopolymeric filament assembly. Moreover, even single amino acid substitutions in the conserved carboxy-terminal part of the rod domain prevent desmin subunits from homopolymeric filament assembly. Desmin subunits missing 18 or more carboxy-terminal residues of their rod domain (including the complete conserved carboxy-terminal region) are unstable in cells devoid of intact type III intermediate filaments (IFs). Interaction with an intact type III IF, however, stabilizes these mutated desmin subunits. Expression of a desmin subunit missing both its non-helical end domains in vimentin-containing cells disrupts the endogenous vimentin network completely. PMID- 1724757 TI - The presence of NK3 tachykinin receptors on rat uterus. AB - The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified. PMID- 1724758 TI - Bay K 8644 enhances Ca2+ channel activities in embryonic chick heart cells without prolongation of open times. AB - The effects of the Ca2+ channel agonist, Bay K 8644, on the slow (L-type) Ca2+ channels was examined in young (3-day-old) embryonic chick heart cells, which naturally exhibit long-lasting openings. Bay K 8644 (5 microM) increased (a) the peak amplitude of the ensemble-averaged current by 3.9 +/- 0.9-fold (mean +/- S.E.) and (b) the maximal number of simultaneous opening from 2.6 +/- 0.4 to 4.4 +/- 0.9. Bay K 8644 had no effect on the unitary conductance (27 pS in control), and relatively little effect in the open-close kinetic analysis. The mean open times were 4.2 ms and 5.2 ms, in control and Bay K 8644, respectively. These results suggest that the agonistic effect of Bay K 8644 involves a mechanism other than open-time prolongation, such as activation of silent channels. PMID- 1724759 TI - Antagonists at the NMDA recognition site and blockers of the associated ion channel induce spontaneous tail-flicks in the rat. AB - The non-competitive N-methyl-D-aspartate (NMDA) antagonists (channel blockers), MK 801, phencyclidine (PCP) and ketamine induced spontaneous tail-flicks in rats. Their order of relative potency (MK 801 greater than PCP greater than ketamine) corresponds to their relative affinities for the ion channel coupled to NMDA receptors. Drugs interacting with their other potential targets (sigma receptors as well as dopamine, serotonin and noradrenaline uptake sites) failed to induce spontaneous tail-flicks. In addition, the catecholamine stimulants, methylphenidate and cocaine were inactive. CPP and CGS 19755, antagonists at the NMDA recognition site, also dose dependently elicited spontaneous tail-flicks: their maximal effect was equal to that of the channel blockers. In contrast, HA 966 and ifenprodil, putative antagonists at the glycine and polyamine recognition sites, respectively, failed to elicit spontaneous tail-flicks. These data demonstrate that both antagonists of the NMDA recognition site and non competitive blockers of the associated channel induce spontaneous tail-flicks in rats. PMID- 1724760 TI - Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4 dihydropyridine receptors. AB - The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [3H](-)-S-Bay K 8644 and the antagonist [3H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37 degrees C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with KD = 3.57 nM and Bmax = 330 fmol/mg.protein at 25 degrees C. The association and dissociation rate constants were 3.4 x 10(7) min-1 M-1 and 0.095 min-1 respectively at 25 degrees C and decreased slightly at lower temperatures. In contrast, [3H](+)-PN 200-110 bound to high (KD(H) = 0.032 nM, Bmax(H) = 316 fmol/mg.protein) and low affinity sites (KD(L) = 27.6 nM and Bmax(L) = 6432 fmol/mg.protein) at 25 degrees C in rat heart preparation. A similar two-site binding of [3H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [3H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [3H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [3H](+)-PN 200-110 were 3.94 x 10(8) min-1 M-1 and 7.86 x 10(-3) min-1 at 25 degrees C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4 degrees C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven. PMID- 1724761 TI - MC903, an analogue of 1,25-dihydroxyvitamin D3, increases the synthesis of nerve growth factor. AB - The effect of MC903, an analogue of 1,25-dihydroxyvitamin D3, on the expression of the nerve growth factor (NGF) gene has been studied in L cells. MC903 induces an increase in both NGF mRNA and protein with a time course similar to that obtained with 1,25-dihydroxyvitamin D3. This finding points to the potential importance of 1,25-dihydroxyvitamin D3 derivatives in the treatment of NGF sensitive disorders. PMID- 1724762 TI - [The use of a vibration photometric method for studying the pharmacological regulation of reversible erythrocyte aggregation]. PMID- 1724763 TI - Cell proliferation kinetics in the marginal mucosa of gastric ulcer evaluated by immunostaining of DNA polymerase alpha. AB - We studied the proliferative ability of the marginal mucosal cells surrounding the ulcer in the healing processes of gastric ulcers. We obtained a labeling index (LI) at the neck and generative zone of gastric pit using a monoclonal antibody against DNA polymerase alpha for tractable and intractable gastric ulcers located at the fundic mucosa during each endoscopic stage. The LI during the healing stage was higher than that during the active stage in both the tractable and intractable cases. However, in each stage, the LI of the tractable gastric ulcers was higher than that of their intractable counterparts. Finally, we analyzed the LI in tractable gastric ulcers after setting two groups: one treated with anti-ulcer drugs and the other untreated. There were no significant differences between these two groups. We believe that investigation of proliferative abilities in the marginal mucosa of gastric ulcers is important to understand the nature of gastric ulcers and to assess therapeutic efficacy. PMID- 1724764 TI - Determination of various molecular forms of cholecystokinin from canine mucosa by radioimmunoassay and bioassay. AB - Bioassays using amylase release from isolated pancreatic acini measure only cholecystokinin (CCK) forms with an intact carboxyl terminus ending with phenylalanine amide, but it cannot be excluded that peptides not structurally related to CCK are also responsible for CCK-like bioactivity. CCK exists in several molecular forms in intestinal mucosa which are released into the circulating blood. We studied the molecular forms of CCK in canine intestinal extracts after separation by high performance liquid chromatography by bioassay and compared them with those detected by radioimmunoassay. For the radioimmunoassay, an antibody was used which needs the carboxyl terminal phenylalanine amide for recognition. Three immunoreactive peaks were reproducibly seen in HPLC eluates which eluted in the regions of synthetic CCK-8, purified porcine CCK-33-39 (which co-elute using this gradient) and purified canine CCK 58. All these peaks were bioactive for amylase release from isolated pancreatic acini. No further bioactive peaks were detected in the HPLC eluates. When an antibody was used which recognizes the midregion of CCK-58, an additional peak was detected which eluted between CCK-33-39 and CCK-58. This form presumably represents an amino terminal fragment of CCK lacking the carboxyl terminus. It can be concluded that bioassays of CCK measure only CCK bioactivity in intestinal mucosal extracts, whereas radioimmunoassays may detect biologically inactive forms depending on the antibody recognition site. PMID- 1724765 TI - Immunopathological and immunochemical analysis of autoimmune enterocolitis in mice. AB - BALB/c (H-2d) and C57BL/6 (H-2b) mice were immunized with one or two doses of allogeneic or syngeneic mucosal antigen (MA) from small intestinal mucosa. This antigen was also characterized by immunoenzymatic assay (ELISA), by SDS-PAGE and by Western blotting. Single-dose immunized mice of either strain, sacrificed 30 days after immunization, synthesized marginal levels of anti-MA antibodies, as assessed by ELISA tests. BALB/c mice receiving two doses of the immunogen produced antibodies at a highly significant level (p less than 0.001) when compared to C57BL/6 mice belonging to the same experimental group. BALB/c mice immunized with syngeneic MA gave a humoral anti-MA response similar to that of BALB/c immunized with allogeneic MA. All experimental mice, irrespective of their genetic background or the number of doses of MA received, develop a cell-mediated immune response to MA. BALB/c mice immunized with two doses of allogeneic or syngeneic MA developed lesions mainly located in the small intestine, characterized by macroscopical and microscopical vascular congestion and hemorrhaging, epithelial cell loss, mononuclear cell infiltration of the lamina propria, cryptal degeneration and granuloma formation. Immunochemical analysis showed varying degrees of cross-reactivity between MA, liver and kidney saline extracts when heterologous anti-MA sera were tested by ELISA. Absorption of these sera with mouse liver or kidney saline extract lyophilizates reduced reactivity of anti-MA antibodies, although the degree of residual activity remained high. SDS-PAGE of mouse MA, kidney, and liver extracts revealed the presence of two polypeptidic bands of less than 17 kD molecular weight belonging to MA. Immunoblotting (Western blot) analysis revealed that heterologous or isologous anti-MA antibodies incubated with mouse MA reacted with the same epitopes as well as with others shared by other organs. Absorption of these sera with liver or kidney saline extracts revealed two organ-specific epitopes belonging to MA. Data presented here support the possibility of a genetic control of the susceptibility of BALB/c mice to the development of an acute autoimmune enterocolitis (AEC). This response appears to be a function of the amount of immunogen administered as well as the genetic background of immunized mice. The presence of a cell-mediated immune response to MA alone, however, is not sufficient for lesions to appear the histopathological alterations characteristic of AEC are present only in experimental groups where detectable levels of anti-MA antibodies are found. The antibody response seems to be directed against two unique epitopes belonging to MA.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1724766 TI - Management of disseminated midgut carcinoid tumours. AB - Forty-one patients with disseminated midgut carcinoid tumours were treated over a 6-year period according to a strict programme including primary surgical treatment. In 10 patients, a total remission of the disease was obtained. Patients with bilobar hepatic disease had ischaemic treatment of their liver metastases by hepatic arterial embolisation after primary surgical and medical treatment (low dose octreotide). Thus, by combining surgical, radiological and medical treatment modalities, we wanted to offer these patients optimal palliation. This treatment programme resulted in good symptomatic relief in all patients accompanied by a marked reduction in 5-hydroxyindoleacetic acid (5-HIAA) levels. At recurrence of symptoms in combination with rising 5-HIAA levels, embolisation was repeated. Ten of the treated patients have deceased during the observation period, but only 5 from their carcinoid disease. PMID- 1724767 TI - Role of cholecystokinin, gastrin and gastrin-releasing peptide in the regulation of pancreatic secretion in cats. AB - This study performed on 6 conscious cats with chronic pancreatic fistulas was designed to determine the role of cholecystokinin (CCK), gastrin and gastrin releasing peptide (GRP) in stimulation of pancreatic secretion in this species. Pancreatic response to GRP infused intravenously in graded doses appears to be mediated predominantly by CCK because a CCK receptor antagonist, L-364,718, abolished this response. Also, gastrin appears to mediate in part the secretory response to GRP because blockade of gastrin receptors by L-365,260, given at the dose that completely abolished the pancreatic response to exogenous gastrin, caused a significant reduction in the bombesin-induced pancreatic secretion. CCK and partly gastrin appear to mediate the postprandial pancreatic secretion in cats as the administration of L-364,718 and L-365,260 inhibited this secretion by over 90 and 30%, respectively. In contrast, GRP does not seem to contribute to food-induced pancreatic secretory stimulation, because the blockade of GRP receptors using novel bombesin/GRP antagonist (RC-3100) failed to affect this secretion. We conclude that CCK and partly gastrin, but not GRP, play an essential role in the postprandial pancreatic secretion. PMID- 1724768 TI - [Transrectal ultrasound of the prostate]. AB - We studied 100 cases of prostatic disease with transrectal ultrasound; all were confirmed by histopathology. 83 cases were diagnosed as benign hyperplasia, 76 were confirmed 7 were cancer. Sensitivity 89.4%. 17 cases were diagnosed as cancer, biopsy confirmed 8, the other 9 were hyperplasia (sensitivity 53.3%). The more reliable ultrasonographic signs of cancer are the interruption on the continuity of the prostatic capsule, the extrinsic compression of the bladder and/or the seminal glands. The echogenicity of the prostatic tissue can have variations, it can be iso-, hypo- or hyperechoic in cancer and also in benign hyperplasia. We conclude, as in other reports, that transrectal ultrasound can demonstrate abnormal prostatic tissue, yet there is not enough sensitivity. Therefore biopsy is necessary and it can be performed by means of this procedure. PMID- 1724769 TI - Immunoglobulins of the snake Psammophis sibilans. Studies using a monoclonal antibody. AB - A mouse monoclonal antibody (mAb) raised against serum immunoglobulins (Ig) of the snake. Psammophis sibilans stained in indirect immunofluorescence a proportion of snake splenic and peripheral blood lymphocytes, whereas it did not react with thymocytes, erythrocytes, brain, heart, lung, liver or kidney cells. The mAb, designated SR-2, combined in enzyme-linked immunosorbent assay (ELISA) with serum proteins of each of 20 individual P. sibilans tested. On Western blots of P. sibilans reduced whole serum proteins, purified Ig, or anti-rat erythrocyte (RRBC) antibodies eluted from glutaraldehyde-fixed RRBC, mAb SR-2 identified two bands of apparent molecular weight (m.w.) of 60,000 and 51,000 daltons. These bands were due to distinct polypeptides and not resulting from heterogeneous glycosylation of a single polypeptide, as they both were readily detected after periodate oxidation or endoglycosidase-F treatment of serum proteins and isolated Ig. MAb SR-2 bound to CNBr-activated Sepharose 4B precipitated from P. sibilans 125I-labeled serum proteins under non-reducing conditions a band that did not enter 7.5 or 9-16% gel and one of about 150,000 daltons. Under reducing conditions, two heavy bands of approximately 63,000 and 50,000 daltons and two light chains of apparent mass 23,000 and 20,000 bands were precipitated. The data presented provide, for the first time, substantial information on the molecular characteristics of snake Ig. PMID- 1724770 TI - Inhibition of HLA B8-restricted recognition by unrelated peptides: evidence for allosteric inhibition. AB - A panel of synthetic peptides representing human lymphocyte antigen (HLA) B8, other class I and class II restricted T cell epitopes and two B cell epitopes, were all able to compete with recognition of a HLA B8 restricted epitope by a cytotoxic T cell clone. Competition was obtained when the competitor peptides were added either before or after the target epitope. The target epitope also had a slow off rate, implicating allosteric inhibition. The presence of non-specific, allosteric binding sites may interfere with experiments attempting to define immunologically relevant MHC binding specificities. PMID- 1724771 TI - Chromium-cage complex as contrast agent in MR imaging--biodistribution studies of the [57Co]cobalt analogue. AB - The synthesis and T1 and T2 relaxivities of the Cr(III)-(NH2)-Sar-cage complex is reported. An outer-sphere relaxation mechanism is postulated for the relaxivity of the complex. Tissue distribution studies in mice using a [57Co]cobalt analogue as a radioactive tracer showed that the complex is excreted rapidly in the urine. Some renal uptake of the complex is seen. Appreciable uptake of labelled cage complex was observed in 3-methylcholanthrene induced murine rhabdomyosarcoma. PMID- 1724772 TI - General principles of retrovirus immunodetection tests. AB - Because infecting retroviruses contain protein and glycoprotein antigenic determinants that can be readily distinguished from host cell determinants, the development of immunologic detection systems, immunodetection tests, or immunoassays capable of identifying antigens of some retroviruses (oncoretroviruses) in blood, body fluids, or cells is possible. Conversely, detection of antibodies produced by animals against some infecting retroviruses can also be used to identify current infections of lentiretroviruses and some oncoretroviruses. Studies of various microorganisms by various immunodetection systems indicate that the most specific and sensitive assays are immunofluorescence, radioimmunoassay, and immunoblot (western blot) analysis, followed by sensitive but less specific ELISA and agglutination assays, and finally by even less sensitive but very specific isolation in culture and double immunodiffusion techniques. The first test used routinely for clinical detection of any retrovirus was the immunofluorescent antibody test, introduced in 1972, for detection of FeLV infection in pet cats. Since then, tests for human retroviruses, the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2 and the human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II) have been introduced for routine use in human medicine. Recently, retroviral tests for a second feline retrovirus, the feline immunodeficiency virus (FIV) have been introduced in veterinary medicine. General principles of sensitivity, specificity, true-positive and -negative rates, false-positive and -negative rates, and positive and negative predictive values apply to all methods used for detection of retroviral infections. PMID- 1724773 TI - Use of immunomodulators as an aid to clinical management of feline leukemia virus infected cats. PMID- 1724774 TI - Metastatic malignant insulinoma: debulking for palliation of symptoms. AB - This unusual case is that of a patient who had recent onset of sudden severe hypoglycemic symptoms. CT scan and arteriography demonstrated a large, widely metastatic tumor of the pancreas involving liver, regional lymph nodes, and the superior mesenteric vein. Tissue diagnosis was obtained by percutaneous needle biopsy of a liver metastasis. The patient had to be maintained on a continuous D10W drip until a successful debulking total pancreatectomy was done. She is currently at home on pancreatic enzyme replacement. PMID- 1724775 TI - Human leucocyte antigen and insulin dependent diabetes mellitus. AB - HLA typing was done in 25 cases of insulin dependent diabetes mellitus (IDDM) and compared with 60 healthy controls. There was a significantly increased frequency of HLA B-8, HLA B-12 and HLA DR-3 in IDDMO. The odds ratio (relative risk) of developing IDDM for HLA B-8 was 4.42 (p less than 0.10), for HLA B-12 was 3.56 (p less than 0.10) and for HLA DR3 9.75 (p less than 0.001). There was no correlation of HLA specificity with complications of diabetes. PMID- 1724776 TI - Adsorption and elution of bovine gamma-globulin using an affinity membrane containing hydrophobic amino acids as ligands. AB - A hollow-fibre affinity membrane containing hydrophobic amino acids as ligands was prepared by the radiation-induced grafting of glycidyl methacrylate onto a porous polyethylene hollow fibre and subsequent phenylalanine (Phe) or tryptophan (Trp). The densities of the Phe and Trp ligand of the resulting affinity membrane were 0.4 and 0.4 mol/kg, respectively. The Trp-containing affinity membrane exhibited a higher amount of adsorbed bovine gamma-globulin (BGG) than the Phe containing membrane. To evaluate the adsorption behaviour of the membrane, the BGG-containing buffer solution was permeated from the inside to the outside of the Trp-containing hollow-fibre affinity membrane through the ligand-immobilized pores. The breakthrough curves as a function of effluent volume coincided irrespective of the flow-rate, i.e. the residence time (55-220 s) of the solution across the membrane (thickness 0.83 mm), as a result of negligible mass transfer resistance. A series of chromatographic procedures, (adsorption-washing-elution) was repeated twice and a satisfactory quantitative elution was attained. The reproducible profile of the flux and the protein concentration assured a quantitative cycle of chromatography using the affinity membrane containing Trp as a ligand. PMID- 1724777 TI - Differentiation disorders of keratinocytes in seborrheic keratosis (acanthotic type). AB - Since the differential disorder of keratinocytes in seborrheic keratosis remains to be elucidated, the differentiation of seborrheic keratosis (acanthotic type) was examined immunohistochemically using a lectin and two anti-keratin monoclonal antibodies. A lectin, peanut agglutinin (PNA), and anti-keratin monoclonal antibody, 34 beta B4, recognize the whole epidermis except for the basal layer in the normal epidermis. In seborrheic keratosis (acanthotic type), cells unstained with either PNA or 34 beta B4 were found throughout the entire tumor. In the upper part of the tumor, some cells appeared to undergo keratinization without expressing the differential markers recognized by PNA or 34 beta B4. Another anti keratin monoclonal antibody, 34 beta E12, stained the tissues in the same way as in the normal epidermis. Thus it was indicated that, in seborrheic keratosis (acanthotic type), although differentiation was partially maintained, some cells might undergo maturation without expressing the differentiation markers recognized by PNA or 34 beta B4. PMID- 1724778 TI - [A study on incorporation of daunomycin, amylase antibody conjugate into submandibular gland]. PMID- 1724779 TI - Distinct effect of pH 2 on a common antigenic structure found in human interferons-alpha 1 and -alpha 2 in the region 30-35. AB - The antigenic similarity between molecules of recombinant human interferon-alpha 1 (IFN-alpha 1) and recombinant human IFN-alpha 2 was demonstrated with neutralizing monoclonal antibody (mAb) 1-46. The common epitope for the mAb 1-46 was localized into amino-terminal region of IFN-alpha molecule around residues 30 35. Following pH 2 treatment, the biological activity of both IFN-alpha 1 and IFN alpha 2 was retained but the antigenic relatedness between corresponding sequences 30-35 was diminished. The common structure on the IFN-alpha 1 molecule proved acid stable and the mAb 1-46 retained the ability to neutralize the pH 2 treated IFN-alpha 1. However, the neutralization of pH 2-treated IFN-alpha 2 by specific antibody was completely suppressed. These results complemented our earlier finding of the dramatic effect of acidic pH on the antigenic structure of region 132-137 of the IFN-alpha 2 molecule. We conclude that pH 2 may induce a conformational rearrangement of the IFN-alpha 2 molecule, resulting in an altered tertiary structure with deviating antigenic characteristics. PMID- 1724780 TI - Evaluation of enzyme-linked immunosorbent assay for detection of Salmonella typhi antigen and antibodies. AB - Enteric fever is considered a major health problem in developing countries. The need for a rapid, accurate and conclusive method for diagnosis is important for adequate and proper treatment. The usefulness and reliability of the ELISA test in detection of S. typhi O antigen and specific IgG and IgM antibodies were assessed using sera obtained from 63 subjects clinically suspected to have enteric fever, 22 febrilenon-enteric subjects and 20 normal subjects. ELISA detection of S. typhi somatic antigen was positive in 75% of subjects with positive clot cultures. IgG and IgM antibodies to S. typhi lipopolysaccharide (LPS) were detected in sera from 83% and 88% of enteric fever subjects, respectively. While anti-LPS IgM was negative in all sera from febrile non enteric subjects, 9% were positive for anti-LPS-IgG. The use of an ELISA for detection of anti-S. typhi LPS antibody in combination with the Widal test and/or the O antigen detection ELISA would provide a useful (95.8% sensitivity) adjunct to standard culture methods and allow for an earlier and more rapid diagnosis of enteric fever. PMID- 1724781 TI - Changes in serum thyroglobulin and thyroid autoantibodies in patients with Graves' disease treated with antithyroid drug and their relationship to relapse. AB - Serum thyroglobulin (Tg) levels are elevated in some patients with Graves' disease. Several studies have pointed out the usefulness of this fact in predicting the outcome of the disease. Among the various antibodies, the antithyroglobulin (ATA) and antimicrosomal (AMA) antibodies are valuable in screening for thyroid autoimmunity. The aim of this study was to analyze the changes in serum Tg and autoantibodies during the course of antithyroid drug therapy and to evaluate their prognostic value. Serum Tg was measured by double antibody RIA, and ATA and AMA by the semiquantitative tanned red cell hemagglutination method in 65 patients before and after antithyroid therapy. Forty-two patients (64.6%) experienced a relapse and 23 patients (35.4%) remained in remission at the end of the study. The changes in the ATA or AMA titers, measured prior to and following therapy were nonsignificant in both groups. While the titers of ATA and AMA before and after therapy were not different between the two groups, there was a tendency for the patients with a positive ATA after therapy to have a relapse. When ATA-positive cases were excluded, there was no change in the Tg levels in either group. The Tg levels of the relapse group either before or after therapy were significantly higher than those of the remission group. There was no difference in T3 or T4 before therapy between the two groups. There was no relationship between the Tg levels after therapy and the duration of remission in either group. In conclusion, during the course of antithyroid therapy, patients with a relapse of Graves' disease had higher serum Tg levels, either before or after therapy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724782 TI - [Expression of keratin 16 in normal and lesional skin of solitary and multiple Bowen's disease using monoclonal antibody Ks 8.12 staining]. AB - Bowen's disease is an intraepidermal squamous cell carcinoma usually consisting of a solitary lesion. However, multiple Bowen's lesions are one of the characteristics of arsenicalism in endemic areas where people drink deep well water containing high concentrations of arsenic along the southwest coast of Taiwan. This work seeks to clarify the differences between multiple Bowen's disease in the blackfoot disease endemic area, and solitary Bowen's disease in a non-endemic area by means of Ks 8.12 monoclonal cytokeratin antibody staining. Ks 8.12 may be regarded as a specific antibody for Keratin 16 in the skin and is used as a marker for hyperproliferation. Our results show that Ks 8.12 staining of normal skin in patients with a solitary Bowen's lesion is patchy and always restricted to the basal cell layer. By contrast, the normal skin of patients with multiple Bowen's lesions shows diffuse Ks 8.12 staining of the basal cell layer and various degrees of staining of the suprabasal layers. Similar results were observed in both solitary and multiple Bowen's lesions showing diffuse Ks 8.12 staining of the epidermis. Our results revealed clear differences in keratin expression between the clinically normal skin of patients with solitary Bowen's disease and that of patients with chronic arsenicalism. Finally the clinically normal skin of patients with multipole Bowen's disease showed characteristic changes in the expression of Keratin 16 in the suprabasal layers. PMID- 1724783 TI - Whipple's disease confined to the CNS presenting with multiple intracerebral mass lesions. AB - A patient with isolated cerebral Whipple's disease presented with signs of raised intracranial pressure and multiple ring enhancing intracerebral mass lesions evident on CT and MRI imaging. Characteristic intracellular bacilliform inclusions were identified in a brain biopsy. Clinical improvement followed treatment with parenteral antibiotics for two weeks and long term sulphamethoxazole-trimethoprim. As CNS relapse of Whipple's disease may occur after several years, long term treatment should include antibiotics that are able to cross the blood-brain barrier. PMID- 1724784 TI - Establishment and characterization of a human leptomeningeal cell line. AB - Human leptomeningeal cells grown in culture were immortalized via transfection with an SV40 large T antigen gene construct under the control of the Rous sarcoma virus promoter. This cell line, designated LTAg2B, maintained the polygonal morphology characteristic of primary cultures, and stained positively in early passage for cytokeratin, a specific marker for arachnoid cells of the leptomeninges. Additional immunofluorescent staining revealed that these cells express vimentin and desmoplakin as well; these antigens have been found together only in normal arachnoid tissue and in meningiomas, which are the neoplastic derivatives of leptomeningeal cells. Significantly, LTAg2B cells demonstrate a greatly increased life span and growth rate relative to primary cultures. The establishment of this cell line should thus facilitate studies on the cellular and molecular biology of leptomeningeal cells, as well as elucidate their roles in certain pathological situations involving the leptomeninges, such as meningitis and meningioma tumor formation. PMID- 1724785 TI - Immunohistochemical evidence for the coexistence of substance P, thyrotropin releasing hormone, GABA, methionine-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: a dual labeling in the rat. AB - By means of dual immunohistochemical labeling on the same brain section examined with a light microscope, the present study reports the presence with serotonin (5 hydroxytryptamine; 5-HT) of gamma-aminobutyric acid (GABA), substance P (SP), thyrotropin-releasing hormone (TRH), leucin-enkephalin (LEU-enk), or methionine enkephalin (MET-enk), within the same neuron in the nuclei raphe magnus, raphe obscurus, and raphe pallidus of the rat. On the one hand, peptides or GABA are detected with specific rabbit antibodies by indirect peroxidase labeling using peroxidase-conjugated Fab fragments, and on the other, 5-HT is detected with a rabbit antibody against the BSA-serotonin conjugate by radio-immunocytochemistry using [125I]-labeled protein A. The possible coexistence of TRH and SP in these neurons is also investigated by using peroxidase labeling and radio immunocytochemical detection, respectively. In the whole caudal raphe nuclei the proportion of each coexisting peptide with 5-HT appears in decreasing order as: TRH greater than SP greater than MET-enk # LEU-enk greater than GABA. In all instances the level of coexistence differs considerably in B1-B2 vs. B3 cell groups. No SP/TRH dually labeled cells have ever been found in any of the serotonergic nuclei of the caudal raphe. Given the evidence that these raphe nuclei project possibly to the spinal cord, these data constitute an anatomical substrate for the several distinct physiological functions presumably subserved by 5-HT in the cord, namely the modulation of nociception, motor, and autonomic functions. PMID- 1724786 TI - Characterization of a panel of neurofilament antibodies recognizing N-terminal epitopes. AB - Peptides corresponding to sequences from the amino-terminal "head" regions of the low, middle, and high molecular weight neurofilament proteins (NF-L, NF-M, and NF H) were synthesized by a modification of the Merrifield solid-phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS-1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters. PMID- 1724787 TI - Sugar-specific inhibitory effects of wheat germ agglutinin and phytohemagglutinin E4 on histamine release induced by basic secretagogues from rat peritoneal mast cells and their possible action sites. AB - The histamine release induced by compound 48/80, bradykinin or polyethylenimine with a molecular weight of 600 (PEI6) was inhibited by wheat germ agglutinin (WGA) and phytohemagglutinin E-subunits (PHA-E4), and the inhibition was specifically reversed by N-acetyl glucosamine and N-acetyl galactosamine, respectively. Concanavalin A (Con A) and phytohemagglutinin L-subunits (PHA-L4) did not inhibit the histamine release induced by compound 48/80, bradykinin or PEI6. The histamine release induced by substance P was also inhibited sugar specifically by WGA and PHA-E4. The binding sites for compound 48/80, bradykinin, PEI6 and substance P, therefore, seemed to especially overlap each other. These binding sites were found to be glycoproteins having affinities to WGA and PHA-E4, but not to Con A and PHA-L4. The binding of WGA and PHA-E4 to the glycoproteins resulted in inhibition of the interaction between the basic secretagogues including bradykinin and substance P and their binding sites on the mast cells. The bindings of five lectins to mast cell glycoproteins were examined by lectin blotting. Several glycoproteins, which had specific affinities to WGA and PHA-E4, but not to Con A and PHA-L4 were detected. We assumed that the binding sites for basic secretagogues which are coupled with histamine-releasing mechanisms exist among these glycoproteins. A 41-kDa protein (alpha-subunit of pertussis toxin sensitive G protein) was not detected by WGA, suggesting that the binding sites for the basic secretagogues were not G proteins. PMID- 1724788 TI - The effects of four days of continuous striatal microdialysis on indices of dopamine and serotonin neurotransmission in rats. AB - The effects of 4 days of continuous microdialysis with a small-diameter concentric-style probe on indices of striatal dopamine (DA) and serotonin neurotransmission were assessed. It was found that over 4 days of dialysis, there was a marked time-dependent decrease in the basal concentrations of 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in dialysate and in amphetamine-stimulated DA release. In contrast, there was no decrease in basal DA or in the ability of cocaine to elevate the concentration of DA in dialysate over the same period of time. There were only very modest changes in dialysate levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), relative to the marked changes in DA metabolites. It is suggested that 4 days of continuous dialysis does not result in a non-specific decrease in diffusibility of these compounds into the dialysis probe, but that the changes are more likely due to probe-induced damage to the nigrostriatal DA system. It is also suggested that a "stable" basal concentration of DA in dialysate is an especially poor indicator of the integrity of the dopaminergic input to the striatum. The implications of these findings for within-subjects design microdialysis experiments are discussed. PMID- 1724789 TI - Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae O1. AB - A toxin-coregulated pilus (TCP), that is important for intestinal colonization of Vibrio cholerae O1, may be produced by vibrios of both classical and EI Tor biotypes. By comparing TCP produced by various strains of the two biotypes in immunoblotting and enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies (mAbs) and polyclonal antisera against TCP from classical vibrios, we have found biotype-related epitope differences in TCP. Our results indicate that TCP of classical strains has an epitope in the TcpA-subunit (20.5 kDa) that is missing in EI Tor TcpA, and an additional epitope that is more strongly expressed in classical TcpA. A polyclonal antiserum reacted strongly with TcpA from strains of both biotypes in immunoblotting suggesting both the presence of major shared TcpA epitopes and that the low or absent reactivity of EI Tor TcpA with the mAbs was not due to lower production of TcpA by EI Tor strains. Whereas all the TcpA positive classical strains inhibited the binding of polyclonal antiserum and mAbs to solid phase-bound TCP-positive bacteria in an inhibition ELISA, practically no inhibition was observed with TcpA-positive EI Tor strains. This together with findings in immunoelectron microscopy studies that TCP 'bundles' were only detected on classical strains, suggest that TCP is poorly expressed on EI Tor vibrios. PMID- 1724790 TI - The surface structure seen on gonococci after treatment with CMP-NANA is due to sialylation of surface lipopolysaccharide previously described as a 'capsule'. AB - Phenotypic serum resistance of gonococci in urethral exudates is due to sialylation of lipopolysaccharide (LPS) by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). A surface structure was visible on gonococci [strain BS4 (agar)] that had been stained with ruthenium red after incubation with CMP NANA. This structure was not visible after neuraminidase treatment, which released sialic acid but not LPS. The LPS profiles of strain BS4 (agar) had another in vivo-selected strain Gc40 (variant D1), were similar. A monoclonal antibody (mAb) which recognises epitope C on the LPS of 'capsulated' gonococci was shown by immunoblotting to react with several LPS components, including one of about 4.5 kDa which contains the probable site of sialylation by CMP-NANA. The reactions with the mAb were not affected by growing the strains with CMP-NANA nor by neuraminidase treatment of the sialylated LPS. The mAb also gold-labelled the surface of both strains before and after treatment with CMP-NANA. These data indicate that sialylation by CMP-NANA and staining with ruthenium red renders more visible the surface LPS which, sometimes in the past, has been seen as a 'capsule'. PMID- 1724791 TI - Mutation screening of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerase chain reaction. AB - We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses. PMID- 1724792 TI - Immunopathologic features of retinal lesions in multiple sclerosis. AB - To further characterize the nature of retinal periphlebitis and retinitis in multiple sclerosis, immunoperoxidase studies were performed on retinal tissue from multiple sclerosis patients at autopsy. Antibodies against myelin basic protein stained the optic nerve but not the retina. Both normal and multiple sclerosis retinas showed staining of Muller cells with Leu-7 (a monoclonal antibody that cross-reacts with myelin associated glycoprotein and natural killer cells). Nerve fiber bundles of the optic nerve in cases with multiple sclerosis and controls also showed staining with Leu-7 antibody. Tissue-bound IgG was demonstrated on retinal ganglion cells in six of seven multiple sclerosis cases but not in controls. PMID- 1724793 TI - Phthalocyanine photodynamic therapy of experimental iris neovascularization. AB - Photodynamic therapy using chloroaluminum sulfonated phthalocyanine (CASPc) effectively closed experimental iris neovascularization induced in 6 eyes of cynomolgus monkeys by argon laser retinal vein occlusion. Neovascularization was followed by iris photography, fluorescein angiography, and histopathologic examination by light and electron microscopy. Intravenous injection of CASPc followed by irradiation with 675 nm light damaged endothelial cells and pericytes, leading to exposure of the basal lamina and thrombotic occlusion of the blood vessels. Surrounding tissue appeared preserved without evidence of thermal damage. Resorption of occluded vessels by macrophages began 2 to 3 days after photodynamic therapy. Neovascularization reappeared 7 days after photodynamic therapy, probably representing growth of new vessels. Photodynamic therapy with CASPc may be a useful adjunct in the treatment of iris neovascularization. The model is useful in elucidating the ultrastructural changes observed after photodynamic therapy using phthalocyanines. PMID- 1724794 TI - An evaluation of the tumour affinity of the radioactive cobalt chelate of tallysomycin S10b in lymphoma-bearing mice. AB - The tumour affinity of the radioactive cobalt chelate of tallysomycin S10b (Tlm S10b), a promising structural analogue of Bleomycin, was assessed using a mouse lymphoma model. The highest concentrations (%dose/gram tissue) were observed in the kidneys, followed by the tumour (4.1%/g 1 h p.i.) at 1 h, 4 h and 48 h. The tumour had the highest concentration among all tissues at 24 h. The biodistribution profile of 60Co-Tlm S10b was distinctly different from that obtained with 60CoCl2, demonstrating the in vivo stability of the chelate. More than half of the chelate (56.8% of the injected dose) was excreted in the urine at 1 h. The highest tumour/non-tumour ratios were obtained for blood (41.3, 4 h), bone (30.5, 4 h) and muscle (29.2, 48 h). Scintigraphy at 24 h, using 57Co-Tlm S10b, showed the tumour, liver, kidney and bladder clearly. The similarities and differences exhibited by Co-Tlm S10b with reference to the literature on cobalt chelates of Bleomycin and naturally occurring tallysomycin (A + B) are discussed. PMID- 1724795 TI - Effect of bombesin, bradykinin, substance P and CGRP in prostate, bladder body and neck. AB - Lower urinary tract tissues respond heterogeneously to adrenergic and cholinergic agents. However, the action of bioactive peptides on these tissues has not been extensively studied. The contractile and relaxant effects of nine peptides bradykinin, cholecystokinin, vasoactive intestinal polypeptide, gastrin, substance P, bombesin, neuropeptide Y, calcitonin gene-related peptide, and motilin-have been compared in the rat bladder body, bladder neck, and left ventral prostate in vitro. All three tissues contracted to bombesin and to bradykinin, although the bladder neck was less sensitive to the contractile effects of bradykinin than the other two tissues. Substance P only contracted the bladder body. Of all the peptides tested, relaxation was only observed to calcitonin gene-related peptide, which relaxed the bladder neck and prostate (phenylephrine-contracted) but not the bladder body (carbamylcholine-contracted). Thus lower urinary tract tissues are responsive to certain bioactive peptides in a nonhomogeneous fashion. These studies raise the possibility that selective modulation of peptide function may be an approach to therapy of urogenital disorders. PMID- 1724796 TI - Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y. AB - The effects of substance P, cholecystokinin and neuropeptide Y were examined on rabbit distal colonic motility. All three agents produced increased contractile activity but the mechanisms responsible differed depending on the agent tested. In the intact animal, peptide effects were measured under basal conditions and following exposure to atropine, tetrodotoxin and the alpha-adrenergic antagonist phentolamine. Administration of all three peptides resulted in a stimulation of colonic motility. Phentolamine did not significantly effect substance P-, cholecystokinin- or neuropeptide Y-induced activity. By contrast, the in vivo activity induced by cholecystokinin and neuropeptide Y, but not substance P, was nearly eliminated by tetrodotoxin. Only the neuropeptide Y response was partially atropine sensitive. In isolated colonic strips, cholecystokinin-induced activity, but not that produced by neuropeptide Y or substance P, was blocked by tetrodotoxin. Atropine did not significantly inhibit any of the hormone-induced contractions. PMID- 1724797 TI - VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis. AB - Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities. PMID- 1724798 TI - Regulation of histamine release in rat hypothalamus and hippocampus by presynaptic galanin receptors. AB - The effect of galanin, a peptide present in a subpopulation of histaminergic neurons emanating from the rat posterior hypothalamus, was investigated on K(+) evoked [3H]histamine release in slices and synaptosomes from rat cerebral cortex, striatum, hippocampus and hypothalamus. Porcine galanin (0.3 microM) significantly inhibited histamine release induced by 25 mM K+ in slices from hypothalamus and hippocampus, but not from cerebral cortex and striatum, i.e., only in regions in which a colocalization of histamine and galanin has been described. The inhibitory effect of galanin was concentration dependent, with an EC50 value of 5.8 +/- 1.9 nM. The maximal inhibition was of 30-40% in hypothalamic and hippocampal slices depolarized with 25 mM K+. The galanin induced inhibition observed in hypothalamic slices was not prevented in the presence of 0.6 microM tetrodotoxin and also occurred in hippocampal and hypothalamic synaptosomes, strongly suggesting the activation by galanin of presynaptic receptors located upon histaminergic nerve endings. The maximal inhibitory effect of galanin in slices or synaptosomes was lower than that previously reported for histamine acting at H3-autoreceptors, possibly suggesting that not all histaminergic axon terminals, even in the hypothalamus and hippocampus, are endowed with galanin receptors. It increased progressively in hypothalamic and hippocampal synaptosomes as the strength of the depolarizing stimulus was reduced. It is concluded that galanin modulates histamine release via presynaptic receptors, presumably autoreceptors located upon nerve terminals of a subpopulation of cerebral histaminergic neurons. PMID- 1724799 TI - Substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract. AB - Immunoreactive substance P and neurokinin A were measured with radioimmunoassay in extracts of different segments of porcine gastrointestinal tract using C terminally directed antisera. In all segments, the concentrations of substance P and neurokinin A were similar. The largest concentrations of both peptides were found in the mid-colon. By gel chromatography and reversed-phase high pressure liquid chromatography the immunoreactivity in extracts from ileum eluted as homogenous peptides at the positions of synthetic substance P and neurokinin A, respectively. No neurokinin B was found. By immunohistochemistry of porcine duodenum, jejunum, ileum and mid-colon, identical localization patterns were found for substance P and neurokinin A, and the two peptides demonstrated by double immunofluorescence to be colocalized in the enteric nervous system of the ileum. We conclude that the tachykinins substance P and neurokinin A are codistributed and colocalized in the procine gastrointestinal tract and suggest that the two peptides are produced from a common precursor, beta- and/or gamma preprotachykinin, in the same neurons. PMID- 1724800 TI - Effects of spantide on guinea pig coronary resistance vessels. AB - Effects of spantide ([D-Arg1,D-Trp7,9,Leu11]substance P) on coronary resistance vessels were studied in isolated guinea pig hearts perfused at constant rate with isotonic buffer containing 20 or 40 mM KCl. Spantide (1 microM) caused a 20-fold rightward shift of the substance P (SP) dose-response curve for vasodilation with no change in maximum (KB = 5.3 x 10(-8) M). Bolus injections of 0.25 to 250 pmol spantide had no effect, but higher doses caused a brief vasodilation followed by a larger, more prolonged vasoconstriction. Histamine produced similar changes in perfusion pressure. Antihistamines (H1 and H2) reduced or blocked responses to spantide and histamine. These findings indicate spantide is a competitive antagonist to SP in guinea pig coronary resistance vessels. In addition, high doses of spantide can cause prominent vascular effects which are mediated by histamine. PMID- 1724802 TI - Prenatal screening for fetal Down's syndrome. PMID- 1724801 TI - First-trimester aneuploidy screening using serum human chorionic gonadotropin (hCG), free ahCG, and progesterone. AB - Immunochemical serum assays for human chorionic gonadotropin (hCG), the free ahCG subunit, and progesterone (P) were considered separately and in combination for their ability to screen for chromosomally abnormal pregnancies in the first trimester. Maternal serum was collected from 141 women undergoing chorionic villus sampling at 9-12 menstrual weeks. Trisomy 21 pregnancies had significantly higher hCG levels, while trisomy 18 and 13 pregnancies had markedly lower hCG and progesterone levels than those of chromosomally normal pregnancies. However, the discrimination of normal from aneuploid pregnancies was poor with either hCG alone, progesterone alone, or free ahCG alone. Much improved discrimination was obtained by combining hCG, free ahCG, and P into an aneuploidy index [(P/hCG)(free ahCG/hCG)]. This index distinguished 9 out of 17 (53 per cent) of the trisomy 21 pregnancies, while only misidentifying 5 out of 112 (4.5 per cent) of the normal pregnancies. The aneuploidy index thus appears promising as a first trimester biochemical screen for aneuploid pregnancies. PMID- 1724803 TI - The chronicle of influenza epidemics. AB - Epidemics that were probably influenza have been reported throughout recorded history. There were 13 fairly severe epidemics during the 18th century and 12 during the 19th century. Probably 8 of these 25 were influenza pandemic. In the 20th century there have been 4 pandemic (1918/19, 1957/58, 1968/69 and 1977) due to the emergence of new subtypes of influenza A virus. The great pandemic of 1918/19 caused an estimated 20 million deaths. Between pandemics usually there have been epidemics of varying severity at intervals of one to three years and a trickle of sporadic cases every winter. The morbidity and mortality rates have varied greatly from epidemic to epidemic and from place to place during the same epidemic. Generally the morbidity has been lowest in people over 60 years of age, but, except for 1918/19, the mortality has been predominantly in old people. The epidemic behaviour of influenza has been so erratic and difficult to understand that there are still a few scientists who consider that extraterrestrial influences operate. These views are not taken seriously by most virologists but there are puzzling aspects of influenza that are not yet understood. PMID- 1724804 TI - [A palliative solution]. PMID- 1724805 TI - Dw14(DRB1*0404) is a Dw4-dependent risk factor for rheumatoid arthritis. Rethinking the "shared epitope" hypothesis. AB - HLA-DR4 has been shown to be associated with risk for developing rheumatoid arthritis (RA) in multiple populations and racial groups. The allelic variants of DR4 share the DR4 serologic specificity but differ by 1 to 3 amino acids in the third hypervariable region (positions 67 to 74) and at positions 57 and 86 of the DR beta 1 chain. We have examined DR4 variants in 61 DR4+ RA cases and 55 DR4+ healthy controls. Dw14 was not associated with RA risk in DR4 heterozygous (DR4,X) cases. Only 15% of DR4,X cases had the Dw14 allele compared with 28% of DR4,X controls. In homozygous (DR4,4) individuals who also expressed Dw4, however, Dw14 was associated with increased RA risk. Moreover, the relative risk for Dw4,Dw14 (16.1, p = 0.001) actually exceeded that of Dw4,Dw4 (2.2, p = ns). Thus Dw14 is not an independent risk factor for RA but is a synergistic risk factor for individuals who also have the Dw4 allele. PMID- 1724806 TI - Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin. AB - Twelve monoclonal antibodies against cobrotoxin from Naja naja atra venom were tested for cross-reactivity with eight different snake toxins, binding to linear epitopes, prevention of cobrotoxin binding to acetylcholine receptor (AchR) in vitro, and protection in mice concomitantly given a lethal dose of cobrotoxin. The antibodies were highly specific, as evidenced by little reactivity with other snake toxins. None of the monoclonal antibodies bound to reduced cobrotoxin or synthesized 8-mer regions spanning the whole molecule, thus suggesting the recognition of conformational epitopes. The in vitro binding of toxin to AchR was competitively inhibited (23-79%) with a 1.66:1 mole ratio of antibody:AchR. Preincubation of monoclonal antibody with toxin before adding AchR (3:1 mole ratio of AchR:antibody) inhibited the in vitro binding of toxin to AchR by 20 80%. Monoclonal antibodies added after the preincubation of toxin with AchR did not dissociate the toxin-AchR complex. An antibody:toxin mole ratio of 2.5:1, with 6 micrograms of cobrotoxin, delayed the time to death of mice 3.7-23.8-fold over control mice. The monoclonal antibodies that most effectively prevented in vitro binding of toxin to AchR also provided the longest delay in time to death in mice. PMID- 1724807 TI - [The morphological and functional characteristics of the human aortic endothelium. I. 2 variants of the organization of the endothelial monolayer in atherosclerosis]. AB - The en face organization of human aortic endothelium in zones of low (LP) and high (HP) probability of sudanophilia was examined in preparation impregnated with silver nitrate. It is found that the heterogeneity of endothelial cells (EC) by area is "random" or "clusterized". In the latter case the major part of small and medium-sized EC (less than 800 microns 2) is associated in distinct groups ("clusters") and form foci with high monolayer density. The luminal surface outside the clusters was formed by preferentially large and giant EC. Clusterized endothelium was found with statistically significant higher frequency in HP zones of both "normal" and "atherosclerotic" vessels. The maximum clusterization of EC was revealed on the shoulder region and on the periphery of atherosclerotic plaques which was speculated to be a growth zone of the lesion. It is suggested that the appearance of clusterized endothelium is associated with the active development of atherosclerosis. PMID- 1724808 TI - [General immunologic fundamentals and an example of virally induced immunosuppression]. AB - Cell mediated and humoral immune protection against virus infections are discussed in relation to development and use of recombinant vaccines in the murine lymphocytic choriomeningitis virus infection. In the same model, immunopathological aspects of T cell immunity to viruses are examined in the murine lymphocytic choriomeningitis virus infection. This virus causes an acquired immunosuppression that is not caused by the virus itself but by cytotoxic antiviral T CD8+ effector T cells which destroy virus infected macrophages and antigen presenting cells. This paradoxical immunopathological mechanism may apply partially to HIV induced immunosuppression in humans. PMID- 1724809 TI - [Morphological alterations of lymph nodes and thymus during the early course of SIV infection of rhesus monkeys]. AB - Rhesus monkeys (M. mulatta) were i.v. infected with SIV mac251. Three phases of lymph node changes were observed. 1: physiological follicular hyperplasia (3 and 6 weeks p.i.). 2: Alterations of germinal centers: loss of follicular mantle zone, fragmentation or sclerosis (12 and 24 weeks p.i.). 3: Partial depletion of T-lymphocytes, accumulation of plasma cells, increased numbers of syncytial giant cells, hemophgocytosis in the sinuses (about 1 year p.i.). The thymus of the juvenile animals showed first changes 12 and 24 weeks after infection with focal loss of immature (and Ki-67 positive) cortical thymocytes, leading to severe accidental involution of the thymuses one year after infection and reduced numbers of Hassalls corpuscles. These investigations show the value of this animal model for the study of morphology and pathogenesis of AIDS. PMID- 1724810 TI - [Histological and cytofluorometric investigations of immunodeficient CB17 scid/scid mice]. AB - Blood cell differentiation and development is under the control of a number of genes, and mutations in one of these genes could result in an immunodefective phenotype. Mice of the CB17 scid/scid ("severe combined imunodeficient") strain are characterized by a mutation which affects early lymphoid differentiation and lack functional T and B lymphocytes. We investigated the serum level of murine immunoglobulins of CB17 scid/scid mice in comparison with the wild type CB17. Furthermore we performed FACS-analysis of the peripheral blood cells as well as cells from the spleen and thymus of these animals. Histological methods were used to study the morphology of the lymphoid tissues. In the sera of CB17 scid/scid mice murine immunoglobuline levels below 50 ng/ml were found. Mice with a level between this and the normal level (5-10 mg/ml) were called "leakys". Cytofluorometric as well as histological investigations revealed no differences between scid and leaky mice, which both appeared quite different than immunocompetent CB17 (wildtype). On the other hand animals with lymphomas show mononuclear infiltrations in the lymphoid tissues. Inflammatory reactions in scid are characterized by leukocytic infiltrations in the draining lymphnodes. PMID- 1724811 TI - [Lymphocytes, Langerhans cells and CD68-positive monocytes/macrophages in the skin of HIV-infected patients and normal controls]. AB - Human epidermal Langerhans cells play an important role in the immunoregulation of the skin. We measured the numbers of CD(3+)-, CD(8+)-, CD1a(+)-, HLADR(+)-, IL2R(+)-, CD(4+)- and CD68 positive cells in the skin of 8 asymptomatic HIV infected Persons, 3 Patients with AIDS and 11 healthy volunteers by suction blister technique. Our results indicate increased numbers of CD1a+ cells and increased numbers of CD4+ cells in the epidermis in asymptomatic HIV-infection. At the same time CD68+ cells are decreased already in an early stage of HIV Infection. The number of CD1a/CD4+ cells is related to the degree of immunodeficiency. This fact might be caused by the activation of MPS. PMID- 1724812 TI - [Morphology and function of macrophages in AIDS in broncho-alveolar lavages (BAL)]. AB - HIV infects susceptible T-cell and mononuclear phagocyte targets. Unlike the dramatic changes that occur with T-cells during HIV disease, changes in monocyte and macrophage phenotype and function are qualitatively minimal. The aim of the study was to quantitatively analyze the morphology of macrophages in broncho alveolar lavages in patients without AIDS, with AIDS, with AIDS complicated by Pneumocystis carinii and in carcinoma patients. In patients with AIDS, the nuclei of macrophages demonstrate a higher integrated optical density (2P less than 0.10), are bigger in size (2P less than 0.05) and have less notches (2P less than 0.02). PMID- 1724813 TI - [Pathogenesis and histomorphology of the so-called Omenn syndrome]. AB - 1. The Omen-syndrome is not a disease on its own, but a complication of congenital SCID. 2. In contrast to patients with classical SCID, patients with Omenn-syndrome possess mature T-cells, which are either of maternal or of host origin. 3. These T-cells are involved in the pathogenesis of the characteristic tissue changes, in particular of skin and lymph nodes (Langerhans-histiocytosis with eosinophilia). 4. The detection of immunodeficiency in Omenn-syndrome is difficult since the lymph nodes are enlarged in contrast to patients with classical SCID. The histomorphological analysis of lymph nodes in Omenn-syndrome is considerably complicated by secondary changes closely resembling dermatopathic lymphadenopathia. PMID- 1724814 TI - [Immunohistochemistry and morphometry of bone marrow in acquired immunodeficiency syndrome with special emphasis on megakaryopoiesis and macrophages]. AB - An immunomorphometric study was performed on formalin-fixed and paraffin-embedded bone marrow biopsies in 20 patients with AIDS (18 males, 2 females-stage IV A-D). In comparison with a control group megakaryocytes (CD61-Y2/51) revealed not only a significant hyperplasia, but remarkably irregular shapes of cells and nuclei, together with a disturbance of the nuclear-cytoplasmic ratio. No predominance of micromegakaryocytes as in myelodysplastic syndromes was observable. Contrasting idiopathic (immune)-thrombocytopenia, HIV-infected patients with a pronounced depression of the platelet count did not show a significant elevation of the number of promegakaryoblasts. This feature is in keeping with findings of a severe impairment of progenitor cell proliferation and differentiation in AIDS. There was a pronounced increase in the macrophage population (PG-M1). This alteration may be related to inflammatory lesions accompanying this disorder as well as to an enforced and premature destruction of hematopoietic cell elements in the myeloid stroma. PMID- 1724815 TI - [Histopathology and morphometry of hematopoiesis in bone marrow of AIDS patients in comparison with patients with myelodysplastic syndrome]. AB - Hematological data and histopathological bone marrow findings were compared in 60 patients with acquired immune deficiency syndrome (AIDS), 40 patients with myelodysplastic syndrome (MDS) and concurrent inflammation, and 39 HIV-negative patients with severe inflammation. Results show that the hematologic, histologic, and morphometric findings in patients with AIDS have a closer resemblance to the findings in patients with severe inflammation than to those in patients with MDS. It is concluded, that the peripheral cytopenia frequently observed in AIDS patients is rather a consequence of severe inflammation, than of ineffective hematopoiesis. PMID- 1724816 TI - [Immunohistologic investigations of paraffin-embedded lymph nodes after bone marrow transplantation in autopsy samples]. AB - A histological and immunohistochemical analysis of paraffin-embedded lymph nodes from 31 autopsies after bone marrow transplantation (BMT) was performed (20 allogeneic, 10 autologous, 1 syngeneic). Monoclonal antibodies against CD 45RB, CD 20, CD 21, CD 35. CD 43, CD 45RO, CD 76 and Ki-B3, and antisera for detection of S 100-protein and immunoglobulin isotypes were used. None of the lymph nodes showed a regular reconstitution. The lymphoid cells, scattered in a diffuse pattern, were mainly CD 43-positive. Most of them also expressed the CD 45RO antigen. CD 20- and Ki-B3 positive lymphoid cells were nearly absent within the first 100 days after BMT. After that time B cell follicles were detectable in a few cases. Surprisingly, in nearly all cases with infectious complications, numerous plasma cells could be found. The origin of this plasma cells is discussed. PMID- 1724817 TI - [Immunologic defects in the lymphatic system of the intestinal mucosa in common variable immunodeficiency (CVID) syndrome]. AB - The humoral immune system of the intestinal mucosa of patients with common variable immuno deficiency (CVID) syndrome was studied immunohistologically using antibodies against immunoglobulin (Ig) A1-2, M and G1-4, against the J chain and the secretory component. In 9/13 CVID-patients IgA-positive cells were totally absent whereas a total IgM-defect was found only in 3/14 patients. Considerable numbers of J chain-positive cells were present in all CVID-patients irrespective of the extent of the Ig-defect indicating the presence of early B-cells unable to differentiate and to produce Ig. There was a strong expression of the secretory component in the cytoplasm and at the surface of enterocytes even in those CVID patients who were totally defective in IgA- and IgM-positive cells. PMID- 1724818 TI - [Distribution of immunocompetent cells small intestine transplantation in rats]. AB - Heterotopic small bowel transplantation was performed in allogeneic rats with and without cyclosporine. Syngeneic animals served as controls. Small bowel allografts, lymphoid tissues and small bowel of the host were investigated by immunohistochemistry using antibodies against T-cells and macrophages. During rejection, increasing numbers of macrophages infiltrated preferentially the deep layers of the small bowel wall, whereas only a slight T-cell infiltration was observed. No histological changes were noted in the organs of the host. Cyclosporine prevented lymphoid as well as macrophage infiltration of the transplant. Rejection monitoring of small bowel transplants is possible by investigation of macrophage infiltration in deep biopsies including the submucosa. PMID- 1724819 TI - [Morphologic HIV demonstration in formalin embedded material--techniques, problems, results]. AB - Formalin-fixed, paraffin-embedded material of archival specimens is suitable for a morphological HIV-detection: Infected cells with HIV in the proliferative phase can be demonstrated with reliable results on tissue sections by immunohistological technics using new antibodies. In situ nucleic acid hybridisation technics can also show HIV in the expression phase on paraffin embedded material, but often fail in demonstrating latently HIV-infected cells. The DNA-Polymerase chain reaction can detect latent Provirus in morphologically defined areas of paraffin sections even in autopsy material, i.e. lymphnodes and even eyes of patients with HIV-Infection, but requires precaution and control with respect to contamination. PMID- 1724820 TI - [Morphology of pulmonary complications in AIDS--a study of 208 biopsy and 231 autopsy cases]. AB - In AIDS patients infectious pulmonary complications may very frequently be demonstrated by both biopsy and autopsy. Bacterial pneumonias occur much more frequently than classic opportunistic infections, e.g. P. carinii pneumonia. Usually, several complications are present concomitantly which impairs diagnosis as well as therapy. Increased survival and modern therapeutic modalities change the spectrum of AIDS-associated pulmonary complications as well as their morphology. In the present study pulmonary complications are directly responsible for the patients' death in more than 70% of the cases. PMID- 1724821 TI - [Extrapulmonary manifestations of Pneumocystis carinii infection in AIDS]. AB - Between 1986 and 1990 we found in 7 out of 100 continuously performed AIDS autopsies at Auguste-Viktoria-Hospital (AVH) an extrapulmonary manifestation of Pneumocystis carinii (Pc). 4 of these cases showed only a singular infiltration of pulmohilar lymphnodi, while the remaining 3 cases presented various other organ involvements: spleen, liver, kidney, adrenal gland, prostate gland, pancreas, myocardium, thyroidea and eyes. All these AIDS-patients had a chronic or relapsing Pc-pneumonia with focal interstitial fibrosis, emphysema, and cavernous-cystic lesions. 6 patients developed a spontaneous pneumothorax due to ruptured subpleural bullae or cystic changes. Apparently the rare dissemination of Pc develops in the context of ruptured tissue and vessels in the pneumothorax lung of AIDS-patients during the final stage of the immunodeficiency associated with chronic lung changes. PMID- 1724822 TI - [Epidemiological and pathomorphological autopsy findings in AIDS]. AB - The autopsy findings in 41 AIDS patients (1982-1991), including a rather large group of 8 patients without one of the classical risk factors for HIV, showed a much higher rate of opportunistic infections, especially CMV and fungal infections, as well as Kaposi's sarcoma in homosexuals than in the others. There was no decline in the incidence of Kaposi's sarcoma over the last few years. The bone marrow findings, which presented a remarkable high cellularity in these patients, well correlated with the so-called HIV-myelopathy, which is highly suggestive to represent a direct HIV-related damage of the bone marrow. PMID- 1724823 TI - [Immunohistochemical characterization of HIV-and non HIV-associated lymph node tuberculosis]. AB - Cells of MPS and lymphatic system in lymph nodes from eighteen patients with culture proven tuberculous lymphadenitis were examined by histological and immunohistochemical technics. Ten patients suffered from symptomatic HIV infection and eight patients were immunocompetent individuals without HIV serology. Characteristic granulomas with or without caseation were observed in the eight immunocompetent and the four HIV-infected patients with less marked lymphopenia of CD4 positive peripheral blood lymphocytes. In lymph nodes from the other HIV-infected patients with more severe depression of CD4 positive peripheral blood lymphocyte count no epitheloid cell formation was present. Instead of these cells foamy macrophages were found. The phenotype of macrophages underwent progressive changes parallel to decreasing numbers of CD4 positive peripheral blood lymphocytes. Foamy macrophages in mycobacterium avium intracellulare infection may represent an end-stage phenotype. While many macrophages and lymphocytes expressed IL-2 receptors in cases with typical granulomas there was no such CD25 expression in cases without any epitheloid cell formation. Our results suggest that T-cell activation is necessary for epitheloid granuloma formation in human tuberculosis and preliminary in situ data support the assumption that in vivo the HIV-infection provokes an excess production of cytokines which in turn causes an exhaustion of the immune system and finally AIDS. PMID- 1724824 TI - [Large cell anaplastic lymphomas in the immunocompromised host]. AB - Large cell anaplastic (LCA) lymphomas are a newly defined tumor entity, which has recently been integrated in the updated Kiel classification. The occurrence of CD30+ LCA lymphoma in the setting of renal transplant patients has so far been reported only once. This report describes two LCA lymphomas of B-cell phenotype in renal transplant patients. The clonal evolution and possible etiologic role of Epstein-Barr Virus (EBV) in LCA lymphoma was studied. Our findings give further evidence that clonal evolution of B-cell populations is a second event in lymphomagenesis and is, at least in the cases studied, preceeded by either reactivated latent or primary EBV-infection with clonal EBV proliferation. PMID- 1724825 TI - [Expression of latent membrane proteins (LMP) of Epstein-Barr virus in malignant lymphomas]. AB - The presence of Epstein-Barr virus (EBV) DNA and antigens was assessed by polymerase chain reaction and immunohistology, respectively, in a total of 92 cases of Hodgkin's disease, angioimmunoblastic lymphadenopathy, CD30-positive anaplastic large cell (ALC) lymphomas, and AIDS-associated atypical lymphoproliferations (ALP). Proportions of the EBV DNA-positive lesions showed latent membrane protein (LMP) expression; some of the LMP-positive ALC lymphomas and ALP cases also displayed EBNA2 immunostaining. BZLF1-protein and gp250/350 immunoreactivity were absent in all instances indicating latent EBV infection. Since the LMP gene has transforming potential, our findings support the concept of a pathoetiological role for EBV in these lymphoproliferative lesions. PMID- 1724826 TI - [Brain biopsies in HIV-infected patients]. AB - Stereotactic brain biopsies of 25 HIV-seropositive patients (age range between 20 and 56 years, 23 males, 2 females) were retrospectively studied. Biopsy material was examined cytologically, histologically, immunohistochemically and electron microscopically. A definitive diagnosis could be established in 23 cases (92%). Diagnosis included non-Hodgkin's lymphoma (10 cases), toxoplasmosis (10 cases), progressive multifocal leukoencephalopathy (PML) (2 cases) and combined toxoplasmosis and lymphoma (1 case). Two biopsies were non-diagnostic. All lymphomas were B-cell lymphomas of high malignancy including one K1-lymphoma. In six cases, in which autopsy was performed, biopsy diagnosis could be confirmed. In one patient suffering from toxoplasmosis, autopsy demonstrated an additional cytomegalovirus infection. Conventional histology was not sufficiently decisive for toxoplasmosis, for some lymphomas and for PML. Stereotactic brain biopsy appears to be an effective method in the diagnosis of HIV-associated brain lesions. PMID- 1724827 TI - [The CNS in AIDS and in asymptomatic HIV positive patients]. AB - Neuropathological investigations were carried out on 166 autopsies of HIV seropositive patients, with and without AIDS. Opportunistic infections and lymphomas were present in about 50% of cases; 65 patients were bearers of HIV encephalopathy. HIV core protein p24 was detected in few mono- and multinucleated macrophages (HIV-cells), only in cases with HIV-encephalopathy. In the CNS of HIV positive, asymptomatic patients no histological or immunohistochemical abnormalities were seen. These findings let suppose that AIDS-Dementia is a result of a late infiltration of HIV-infected macrophages from the bloodstream into the brain and not due to an impairment of neuronal or glial cells infected by HIV in the early stages of the disease. PMID- 1724828 TI - [Immunohistochemical demonstration of extracerebral toxoplasmosis in AIDS]. AB - Tissue slides obtained at autopsy from 80 cases with AIDS were studied immunhistochemically for infection with Toxoplasma gondii. In 35 cases (43.75%) toxoplasmosis could be found: in 22 cases (27.5%) only cerebral, in 9 cases (11.25%) cerebral and extracerebral and in 4 cases (5%) only extracerebral. Necrotizing lesions, due to the parasite could be seen in brain, heart, lungs, pancreas, adrenal glands and testis, only intracellular trophozoites without tissue damage in GIT, liver, lymphnodes, spleen, prostate, kidney and gl. parotis. The trophozoites and pseudocysts could be clearly demonstrated by immunohistochemistry. PMID- 1724829 TI - [Progressive multifocal leukoencephalopathy (PML) in AIDS: morphological and topographical characteristics]. AB - 15 autopsy cases of PML in AIDS are presented. Frequent findings were: Very large, confluent PML-lesions (7 cases), lesions with prominent necrotic features (10 cases) and with marked perivascular mononuclear infiltrates (6 cases). In 9 cases additional PML-foci were seen in the cerebellum and/or the brain stem, and in 1 case there was even an exclusive involvement of those structures. Such characteristics regarding severity and topography of PML in AIDS, may be due to additional toxic factors. In 7 cases HIV-encephalopathy with multinucleated macrophages was present. PMID- 1724830 TI - [The central nervous system of infants born to HIV-positive mothers-- neuropathology, immunohistochemistry in in situ hybridization]. AB - The central nervous system (CNS) of 12 fetuses from human-immunodefiency-virus seropositive mothers was investigated. Postconceptional ages ranged between 15 and 21 weeks. 3 preterm infants born to HIV positive mothers whose survival ranged from 6 hours to 6 months were included in the study. In all cases, the development of the CNS was macroscopically normal. The microscopic examination showed many ectopic neurons indicating disturbances of normal neuronal migration. As a regular finding there was a significant increase in microglia in all areas of the CNS but no microglial nodules were found. In all cases haematogenous monocytes were scattered in the perivascular space of the cerebral parenchyma as well as in the chorioid pexus. The virus-antigen p24 could neither be detected immunohistochemically in the paraffin sections nor in the frozen material of the brain or by in-situ-hybridization. In contrast, in five of the cases investigated the placenta showed macrophages, socalled Hofbauer cells with p24-associated antigen. PMID- 1724831 TI - [Histomorphology of BCG infections in patients with severe combined immunodeficiency]. AB - Most inborn immunodeficiency syndromes clinically become overt by complicating infections, e.g. by BCG-infection after BCG-vaccination. The diagnostic approach often is hampered by an atypical histomorphological picture of the infection caused by the underlying immune defect. We examined biopsies of different organs in 18 infants with severe combined immunodeficiency syndrome and BCG-infection. A number of unusual histomorphological patterns of BCG-infection were found not yet published previously. There was a correlation between histomorphological features and immunological data or clinical course in most cases. PMID- 1724832 TI - [The reticuloendothelial system in immunodeficiency]. AB - The concept of the mononuclear phagocyte system (MPS) as advanced by van Furth et al. does not embrace other cell types which by virtue of their accessory cell function are inextricably linked to the immune response. Therefore, a modified functional definition of reticuloendothelial system (RES) encompassing the non lymphatic effector and accessory cells of the immune response, i.e. monocytes/ macrophages, follicular and interdigitating reticulum cells as well as endothelial cells is proposed. In addition to their morphologic and functional characteristics, their behaviour in the setting of human immunodeficiency virus (HIV) infection is discussed. As examples serve the role of RES cells as target cells and reservoirs for HIV and their dysfunctions with regard to different HIV associated conditions, e.g. tuberculosis and Kaposi's sarcoma. PMID- 1724833 TI - [Familial hemophagocytic lymphohistiocytosis]. AB - 7 cases of familial hemophagocytic lymphohistiocytosis were investigated by morphology and immunohistochemistry. Typical infiltrates composed of macrophages and lymphoid cells were found in various locations such as lymph nodes, spleen, liver, brain, and skin. The macrophages were positive for typical macrophage antibodies (Ki-M1, Ki-M1P) and showed features of activation by displaying a strong reaction with antibodies against lysosomal antigens (Ki-M1P, Ki-M6, Ki-M7, Ki-M8). In addition, they showed antigenic similarity to antigen-presenting cells (Vit-6 pos., S 100 pos.) and no conclusive evidence of in situ proliferation (Ki 67 neg). The lymphoid cells were mainly composed of more or less proliferating T cells and a few B-cells. PMID- 1724834 TI - [Virus induced acquired immunodeficiency: correlation with T cell mediated destruction of spleen and lymph node architecture]. AB - Mice infected with the non-cytopathic lymphocytic choriomeningitis virus (LCMV) develop a severe acquired immune suppression. This is caused by the elimination of virus infected antigen presenting cells by cytotoxic T cells leading to a severe destruction of the follicular organization of lymphoid organs. The impairment of immune functions may allow the virus to establish persistent infections in initially immunocompetent hosts. PMID- 1724835 TI - [Rare inflammatory reactions in infants and their correlation with pathomorphologic alterations of the immune system]. AB - Autopsy-findings in infants with rare inflammatory disorders or infectious diseases esp. opportunistic infections may suggest defects of the unspecific and specific defence. The histopathological appearances of different lesions were correlated with possible underlying alterations or generalized defects of the immune system. PMID- 1724836 TI - [Deep mycoses in leukemia and malignant lymphoma]. AB - 1053 autopsies were performed from 1976 to 1990 in patients with leukemia and malignant lymphomas. At autopsy 184 of these (17.4%) presented with deep seated mycoses. There was an increasing percentage of mycoses per year with a maximum of 30% in 1990. Today deep seated mycoses are the most frequent letal complication in hematologic neoplasias. As expected their number was especially high in patients with acute leukemia but in recent years they were nearly just as numerous in myeloproliferative disorders. Among NHL they were twice as frequent in low grade cases as in high grade cases possibly due to a different extent of bone marrow infiltration. In contrast to former years more aspergilloses than candida infections are found, probably as a result of antimycotic therapy. PMID- 1724837 TI - [Infectious causes of death in renal transplant patients. A clinical pathological autopsy study]. AB - The infectious consequences of immune deficiency after kidney transplantation are reflected in the spectrum of the causes of deaths in a renal transplant population. From 1976 to 1991, 650 kidney transplantations were performed in Rostock. An autopsy was made in 92 of the altogether 106 deaths; 45 (49%) showed infectious and 47 (51%) non-infectious causes of death. A significant decrease of bacterial causes of death was found in the transplant group from 1985 to 1991 (3/25 = 12%) in comparison to the group from 1976 to 1985 (30/67 = 45%). The important increase of patient survival is attributed to the progress of transplantation management in general as well as to the striking decrease of lethal bacterial infections due to increasing ciclosporin application in renal transplantation. PMID- 1724838 TI - [Invasive, metastatic growth of lymphoblastoid B cells in immunodeficient SCID mice]. AB - A Burkitt's lymphoma (BL, EBV +, 8/22 translocation), the EBV immortalized lymphoblastoid cell line from the same individual (LCL) and somatic cell hybrids (HYB) between both cell lines were inoculated in immunedeficient scid mice. Subcutaneous injected BL cells produced local tumor masses without distant metastases. In contrast LCL and hybrids show invasive and disseminated growth. Peripheral lymph nodes and thymic tissue were preferentially colonized. A detailed phenotypic analysis of BL, LCL and 3 different hybrids was performed using differntiation and adhesion molecules. The preferential "homing" of LCL and hybrids seem to be correlated with the expression of the lymphocyte homing receptor (CD44). The growth of LCL in scid mice shows, that additional genetic alterations are not necessary to cause a malignant phenotype. Activation of myc seem to play a minor role, because hybrids mimic the LCL phenotype and distribution pattern. PMID- 1724839 TI - [Membranoproliferative glomerulonephritis in non-Hodgkin's lymphoma nad myeloproliferative syndrome--a causal relationship?]. AB - A pathogenetic relationship is postulated for the development of membranoproliferative glomerulonephritis type I in non-Hodgkin's lymphoma (B-cell lymphoma of low-grade malignancy) and myeloproliferative syndrome, which we have observed in eight patients. This hypothesis is supported by the fact that chronic lymphatic leukaemia and immunocytoma are often associated with immunodysregulative phenomena, and by the immunohistological and ultrastructural findings in the kidney, especially the frequent electron-microscopic finding of cryoglobulins, which results in the membranoproliferative type of immune-complex glomerulonephritis, an expression of a disturbance in immune balance. The pathogenetic mechanism may involve cryoglobulins themselves as immune complexes; it is also possible that monoclonal cryoglobulins combine with an antigen to form immune complexes or lead to in situ formation of immune complexes. In addition, other immune complexes, for example with endogenous tumour-associated antigens and exogenous antigens (e.g. hepatitis antigens), may be involved in the pathogenesis. PMID- 1724840 TI - [Inborn immunodeficiencies]. AB - Primary immunodeficiency syndromes may be seen as "experiments of nature", giving insights into the organization and function of the human immune system. The principal categories of primary immunodeficiency syndromes: severe combined immunodeficiency, agammaglobulinemia and isolated T-cell defects (e.g. Di George Syndrome) are still used in view of their leading clinical presentations. However, detailed analysis of individual cases and families now shows a plethora of different diseases in each category. In this review the relationship of primary immunodeficiency diseases of the B-cell system and autoimmune phenomena are discussed. The pathology of thymus in severe combined immunodeficiency is shown: central maturation defects of the T-cell system are not due to "dysplasia" of the thymus but rather to enzyme defects of the lymphatic cells. Severe alterations of the thymus may also be caused by graft versus host disease. The clarification of genetic defects of lymphoid differentiation and maturation today may lead to improved early and prenatal diagnosis as well as specific gene therapy. The success of bone marrow transplantation in many cases of primary immunodeficiency disease syndromes may be considered as a consequence of successful gene therapy. PMID- 1724841 TI - [New cytogenetic classification of precancerous lesions and carcinomas of the endometrium: possibilities and limits of immunohistochemical differentiation]. AB - Various grades of adenomatous hyperplasia represent precancerous states leading to the most common type of adenocarcinoma with endometrial differentiation. Grades 1 and 2 are characterized by architectural abnormalities only (complex hyperplasia). They rarely progress to carcinoma. Grade 3 in addition is characterized by cytological atypicalities (atypical hyperplasia) and shows a much higher progression rate into invasive carcinoma. Endometrial carcinomas may originate from endometrial glandular epithelium and show endometrial differentiation, or from various types of metaplasias developing in the endometrium from pluripotent Mullerian epithelium. They then show endocervical or serous papillary differentiation. Because of their differences in spread, speed of growth and survival rates, it is important to subclassify these endometrial carcinomas. Immunohistochemically, adenocarcinomas with endometrial differentiation including adenoacanthomas and adenosquamous carcinomas can be recognized by their coexpression of cytokeratin 8 and vimentin, and by their negative reaction for CEA. Distinction from adenocarcinomas with mucinous differentiation, including muceopidermoid adenocarcinomas, is possible by their negative reaction for vimentin and by their positive reaction for CEA. On the other hand, carcinomas with mucinous differentiation primarily located in the endometrium can not be distinguished from those primarily located in the endocervix by immunohistochemistry; that distinction must be made topographically. The same holds true for clear cell carcinomas of both locations. Over the past decade, mucinous adenocarcinomas and clear cell carcinomas originating from the endometrium have increased, whereas adenocarcinomas with endometrial differentiation have become less frequent. This shift is closely related to the altered postmenopausal hormone substitution with the addition of the synthetic gestagens. These apparently stimulate proliferation of endocervical epithelium not only in the endocervix, but also that arising in endocervical metaplasias of the endometrium. PMID- 1724842 TI - [Demonstration and biological significance of viral and cellular oncogenes in uterine carcinomas]. AB - Premalignant and malignant lesions of the cervix uteri and endometrium were analyzed for the presence of human papillomavirus (HPV) DNA and c-myc or c erbB2/neu oncogene expression. HPV DNA was detected by PCR in 100% of the cervical carcinomas and CIN lesions, but also in endometrial lesions (3/8 adenocarcinomas, two hyperplasias and one adenomyosis uteri). Myc-overexpression was found in 25-30% of the cervical carcinomas and severe dysplasias, but not in endometrial lesions. C-erbB2 was overexpressed in 4/11 endometrial carcinomas and 3/19 CIN3 lesions. The implications of these results are discussed. PMID- 1724844 TI - The identification of stromal invasion in the distinction of atypical endometrial hyperplasia from well differentiated adenocarcinoma. PMID- 1724843 TI - [Role of steroid hormones and related biological substances in the etiology of endometrial carcinomas]. AB - Synthetical oestrogens, as well as gestagens may cause metaplastic changes of the endometrial epithelia. While squamous and tubal metaplasia are most often due to oestrogenic stimulation and develop in hyperplastic endometria, mucinous and clear cell metaplasia are stimulated by gestagens and tamoxifen. Tamoxifen seems to act gestagen-like on the endometrium. We present data of 31 patients, who were treated with tamoxifen for breast cancer disease. 3 of these 31 developed endometrial adenocarcinoma. PMID- 1724845 TI - Grading of endometrial carcinoma. PMID- 1724846 TI - [Differentiated operative therapy of endometrial carcinomas]. AB - The traditional surgical procedure to treat operable endometrial cancer is the removal of the uterus and both adnexa. In the trend of modern gynecological oncology this standardized operation should be changed in favour to a more individual procedure adapted to preoperative and intraoperative stage of the disease. A carefully fractioned curettage (of the cervix and the corpus uteri) is necessary to differ stage I (T1) and stage II (T2). Further important prognostic factors as myometrial invasion, nodal status, lymph vessel and blood vessel involvement and the intraabdominal findings (T3) are details of the post-surgical evaluation. A differentiated surgical treatment of the endometrial cancer includes for a stage I disease hysterectomy, bilateral adnectomy and pelvine lymphonodectomy. We recommend this additional procedure in all stages of the disease: The nodal status is for almost all genital cancers and breast cancer the most important prognostic factor. Postoperative adjuvent therapy (radiotherapy, hormonal therapy, chemotherapy) may be indicated by that. The surgical procedure for stage II (corpus and cervix involved) disease is a radical hysterectomy (Wertheim), bilateral adnectomy and pelvine lymphonodectomy. A paraaortal lymphonodectomy may be recommended, but most of the patients are 70 years and more and have a multimorbidity. Therefore, we indicated this additional procedure only in 7.5% of the operated patients. The surgical strategy in the (rare) stage III individual cancer is similar to the procedure in progressive ovarian cancer: Cytoreduction and debulking (e.g. omentum majus, peritoneum, involved bowel) has to be performed subsequent to hysterectomy, bilateral adnectomy and lymphonodectomy to improve the poor prognosis. PMID- 1724847 TI - [Classification of malformations of colorectal innervation]. AB - Inborn errors of colorectal innervation can be classified in four different forms: 1. Aganglionosis (including total aganglionosis of the colon, Hirschsprung's disease, ultrashort Hirschsprung's segment and neurogenic sphincter achalasia) 2. Hypoganglionosis 3. Congenital malformation of sympathetic innervation of the colon (Neuronal Intestinal Dysplasia Typ A or NID A) 4. Congenital malformation of the submucous plexus (Neuronal Intestional Dysplasia Typ B or NID B). We find in 52.2% of our biopsies with a characteristic malformation of the colorectal innervation an aganglionosis. 40.6% are malformations of the submucous plexus (NID B). Half of the cases with Hirschsprung's disease are combined with NID B. 5.0% of the classified colorectal malformations of the innervation are a hypoganglionosis and 2.2% a NID A. An additional half of our biopsies cannot be introduced into the above given classification due to moderate malformations of the colorectal innervation (mild dysganglionosis, hypogenetic nerve cells of the submucous plexus, heterotopic nerve cells of the submucous and myenteric plexus). PMID- 1724848 TI - [International documentation system for colorectal cancer-- reporting pathological findings]. AB - An international Working Party has achieved agreement on an "International Documentation System for Colorectal Cancer (IDS for CRC)". It includes the essential clinical and pathological data required for estimation of prognosis and evaluation of treatment results. These data are subdivided into 3 types of information: (i) basic patient information; (ii) variables of proven prognostic significance (anatomical extent of disease, i.e. pTNM, and residual tumor classification, some other independent variables); and (iii) information of probable prognostic significance. Recommendations for data collection, pathological techniques and reporting pathology are added. PMID- 1724849 TI - [Lymphoid tissues and AIDS: role of lymphocytes and follicular dendritic cells (FDC)]. AB - Currently discussed models of AIDS pathogenesis attribute a pivotal role to HIV variants developing in T cells during the course of the disease resulting in an increasingly rapid depletion of the infected T cells. Such models do not however explain the morphological findings observed in AIDS lymphadenopathy. To clarify the significance of the hyperplasia and subsequent destruction of the lymphoid follicles in HIV-related lymphadenopathy, we used in situ hybridization in combination with immunohistological labelling techniques to identify the phenotype of HIV-infected cells in lymph nodes. In addition to few T helper cells, mainly in germinal centres, large amounts of HIV-RNA were found in CD4 negative follicular dendritic cells (FDC) rather than in macrophages or other cells. This finding is well in accordance with the recent observations made by other authors suggesting that purified FDC can be infected with HIV in vitro. Furthermore, TNF alpha expression is localized mainly in centroblasts (activated B cells) of germinal centres. TNF alpha, released by antigen-activated B cells in vitro, has been shown to induce HIV replication in latently infected T cells. On the basis of these observations we propose that latently infected CD4+ T cells, having entered germinal centres, start HIV replication under the influence of cytokines present in this microcompartment of all lymphoreticular tissues. Here the T cells infect FDC which, in turn, pass on the virus to new healthy T helper cells. This model may explain both peculiarities of the lymphadenopathy syndrome as well as the long latency period of HIV-infection, ultimately leading to AIDS. PMID- 1724850 TI - [What role does informatics play in pathology?]. AB - Informatics can be understood as an element of management, dealing with structures of files, algorithms and hardware. The most important prerequisite to successful use of informatics is an optimal compatibility between these single elements. To realize this compatibility and to ascertain a permanent operability of informatics in pathology, the realization of the following two conditions are advisable: 1) The differentiation between a general administrative system of the whole department of pathology and systems of special units (e.g. laboratories); 2) the modularity of the laboratory systems. PMID- 1724851 TI - [Differentiation and new differentiation reflected in intermediate filament expression: investigation of normal, aged and malignant with emphasis on cytokeratin]. PMID- 1724852 TI - [HIV encephalopathy]. AB - The early manifestation of HIV infection of the brain, HIV encephalitis, is due to the invasion of HIV infected mono- and multinuclear macrophages into the brain tissue and cerebrospinal fluid. These cells are intensely PAS reactive, auto fluorescent and express the macrophage antigen CD68. They clearly prefer the white matter of cerebral hemispheres, corpus callosum and internal capsule. Lymphocytic infiltrates and microglial nodules are additional, unspecific changes. HIV encephalopathy following HIV encephalitis is patho-anatomically characterized by brain tissue damage. Its clinical correlate is so-called AIDS dementia complex. It presumably is caused by neurocytotoxic and myelinotoxic factors released by activated macrophages and/or by shedding HIV retrovirus proteins. HIV encephalopathy includes leukoencephalopathy, diffuse poliodystrophy, disseminated basal ganglia damage and brain atrophy. In some cases, granulomatous angiitis may occur. PMID- 1724853 TI - [The epidemiology and acquired immunodeficiency syndrome--status and trends]. AB - Up to June 1991 a total of 6,604 AIDS cases were reported to the central AIDS registry at the Federal Health Office. As typical for "pattern I" countries most of the AIDS-cases are homo/bisexual men (70%), followed by i.v. drug users (IDU, 13%). However, the proportion of homo/bisexual men is constantly decreasing since 1986 while the proportion of IDU's is increasing. As also observed in other industrialized countries a flattening off in the AIDS incidence curve is seen since 1989. Probable reasons for this observation are a decrease of new infections since 1984/85 (due to early saturation of the populations at highest risk and to the early onset of prevention campaigns in these populations) and improved therapeutic strategies in the prevention of AIDS indicating diseases. However, since about 60,000 people are estimated to be HIV infected in the FRG today AIDS incidence will remain on a stable level for the next years regardless the number of new infections occurring today. Since 1988 major changes in the distribution of AIDS indicating diseases are seen. While Kaposi's sarcoma is constantly decreasing non Hodgkin lymphomas, HIV encephalopathy and wasting syndrome are increasing. Due to the effective primary prophylaxis of pneumocystis carinii pneumonia (PCP) by pentamidine the proportion of PCP as AIDS-indicating opportunistic infection decreased from more than 60% in 1988 to 41% in 1991. The second most frequent opportunistic infection is now toxoplasmosis (19%). The changes in the distribution of AIDS-indicating diseases and the increasing proportion of IDU's have major implications on patient care as well as diagnostic and therapeutic procedures. PMID- 1724854 TI - [List of members]. PMID- 1724855 TI - [Histologic classification of HIV-1 induced lymphadenopathy. Current results on the pathogenesis of germinal center changes]. AB - At the IV International Conference on AIDS in Stockholm, a Symposium on the pathology of HIV-related diseases was organized. During this Symposium the "European Study Group on HIV-Pathology" presented a histological classification of lymph node alterations seen in HIV-associated lymphadenopathy. The goal of the Study group was to offer a common terminology for histologic changes previously described with different terms by different authors. In the present review the most important histological criteria used in the proposed classification are summarized. Since the classification is based on the conspicuous changes of the follicles, a brief review of the possible pathogenic mechanisms leading to disintegration of the germinal centers will also be given. PMID- 1724856 TI - [Tumors in immunodeficiency syndromes]. AB - It is the purpose of this review to analyse prevalence and pathogenesis of tumours occurring in the diverse conditions of primary and secondary immunodeficiencies. In general, most states of immunodeficiences are connected with a highly increased tumour risk. However, there is circumstantial evidence that immunodeficiency-related malignancies do not primarily result from an impaired immunosurveillance. With special regard to ataxia-telangiectasia it has been found that congenital chromosomal instability deregulates the maturation of immunocompetent lymphoid cells by interfering with their natural DNA rearrangements and eventually leads to false recombinatory events with oncogenetic consequences in a parallel way. Malignancies in primary common variable or in secondary (e.g. AIDS) immunodeficiency may result from an impaired immunity to oncogenic viruses or from chronic immune damage due to secondary autoimmune disorders. PMID- 1724857 TI - [Therapeutically induced immunodeficiency]. AB - Therapeutically induced immundefects principally can be the result of pathophysiological influences on autoimmune diseases by immunomodulation on the one hand or loss of resistance against bacteria, fungi or viruses as a consequence of immunomodulation on the other hand. Further complications such as increasing incidence of neoplasms after long-term therapy, hypersensitivity reactions, especially damage of bone marrow, can be recognised after application of many different drugs. The immunesuppressive drugs are discussed in detail. PMID- 1724858 TI - [Regulation of thrombocyte stimulating and other activities of thrombin by modulators of the recognition site]. AB - The structural and functional features of thrombin are under discussion: combination of restricted specificity and a central regulatory role in hemostasis. Thrombin specificity is mainly connected with special regions of the enzyme molecule--an additional recognition binding site for high molecular substrates. One can consider the additional site of thrombin as a kind of the allosteric centre changing thrombin-catalyzed functions at binding with modulator. Specific site of substrate (inhibitor or receptor) is used in the role of modulator. A computer search of that modulator was fulfilled by means of the program DOTHELIX. The peptides thymosin I and substance P which have regions similar to those of hirudin were shown to inhibit thrombin activity. The kinetic data point to the noncompetitive type of inhibition. The data on the high reactivity of the thrombin-activated protein C system confirm the idea of protein C to be the first defensive mechanism when thrombin is generated in blood and interacts with thrombomodulin. PMID- 1724859 TI - [The parasitism characteristics of the causative agent of melioidosis in the host cells]. AB - The electron microscopic study of the damaged areas of the lungs, liver, spleen and lymph nodes of guinea pigs infected with two P. pseudomallei virulent strains C-141 and 100 has revealed that this organism is a facultative intracellular parasite and exhibits tropism to reticuloendothelial cells of the body, parasitizing both in "professional" phagocytes (mononuclear, phagocytes, polymorphonuclear leukocytes) and "nonprofessional" phagocytes (capillary endotheliocytes, splenic and lymph-node reticulocytes). In the course of their intracellular development P. pseudomallei have been shown to form capsules protecting the pathogens from the unfavorable action of the protective mechanisms of the host cells. PMID- 1724860 TI - [The efficacy of exogenous and endogenous interferonization in preventing influenza]. AB - In this work the results of using interferon (IFN), Dibasol and the combination of these preparations for the urgent prophylaxis of influenza and acute respiratory diseases (ARD) among the employees of the Gamaleia Research Institute of Epidemiology and Microbiology (USSR Acad. Med. Sci.) are summarized. Reaferon and Dibasol decrease ARD morbidity 2 times and leukocytic IFN decreases it 1.4 times, while the combined administration of Dibasol and IFN has proved to be ineffective. Reaferon is mainly a prophylactic remedy; it has been found to bring about almost no decrease in the number of patients at the peak of morbidity, while pronouncedly decreasing it in two weeks after the administration of the preparation. Dibasol has a curative effect, sharply interrupting the beginning rise of morbidity. Reaferon normalizes the characteristics of the IFN status, decreasing the amount of circulating IFN and enhancing the capacity of leukocytes for producing alpha-IFN and gamma-IFN. For the prophylaxis of respiratory infections the use of Reaferon is advisable 3-4 weeks prior to the beginning of the epidemic and then, when the first cases of infection are registered, the course of prophylaxis with Dibasol should be carried out. PMID- 1724861 TI - [Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]. AB - A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA. PMID- 1724862 TI - [The role of the surface antigens of Pseudomonas pseudomallei in the pathogenesis of melioidosis]. AB - As established with the use of electron-immunochemical techniques, glycoprotein antigen 6 is the outer membrane component of P. pseudomallei cell wall, while glycoprotein antigen 8 is localized on the cell surface as a capsule-like formation. Antigen 6 plays no perceptible role in the realization of the pathogenic properties of the infective agent, but serves as a reliable sign in the differentiation of P. pseudomallei strains into serovars. Subcultures, defective in the synthesis of antigen 8, have sharply reduced virulence for laboratory animals. As revealed in this study, the pathogenetic action of antigen 8 is linked with its pronounced antiphagocytic function. Thus, antigen 8 is considered to be one of the key pathogenicity factors of P. pseudomallei. PMID- 1724863 TI - Expression of cytokeratin 7 in human glandular epithelium of fetal stomach. AB - Previously we reported the neo-expression of cytokeratin 7 in metaplastic and dysplastic stomach mucosa. In this study using immunohistochemistry and immunoblotting technique, we observed an cytokeratin 7 expression in the fetal and "infantile" human stomach, thus indicating an "oncofetal" behaviour of some simple epithelial type cytokeratins. PMID- 1724864 TI - [Victoria blue: staining properties in orthology and pathology]. AB - The staining properties of the cationic dye Victoria Blue are summarized in consideration of the findings in the literature and own findings. Dependent on the staining conditions (with or without preoxidation of tissue sections), different structures and substances are stained by Victoria Blue. Without preoxidation elastic fibres, acid glycosaminoglycanes and DNA predominantly stained, but with preoxidation the dye stained insulin, some peptide hormones and secretion products, HBs-antigen, and lipofuscin. The chemism of the histochemical reaction is not quite understood yet. PMID- 1724865 TI - A case for transmitter plasticity at the molecular level: axotomy-induced VIP increase in the upper spinal dorsal horn is related to blockade of retrograde axoplasmic transport of nerve growth factor in the peripheral nerve. AB - Blockade of retrograde axoplasmic transport in peripheral nerves, by means of perineurally applied microtubule inhibitors, results in an increased vasoactive intestinal polypeptide (VIP) reaction of the segmentally related, ipsilateral upper dorsal horn. Similar effect is elicited by the perineural application of an anti-Nerve Growth Factor (anti-NGF) serum. At the same time, both treatments result in depletion of Substance P from the same region of the spinal cord. It is assumed that this striking example of transmitter plasticity, obviously taking place at the molecular level, is due to a stimulating effect of NGF upon the perikaryal Substance P-synthesizing mechanism in dorsal root ganglion cells, and the inhibitory effect of NGF upon the VIP synthesizing machinery in these same nerve cells. PMID- 1724866 TI - Complex carbohydrate histochemistry of the lateral prostate and seminal vesicle of the guinea pig. AB - A systematic histochemical study of the complex carbohydrates of the lateral prostate and seminal vesicle of the guinea pig has been made. The complex carbohydrates of the guinea pig male accessory sex glands were partially characterized by various conventional carbohydrate histochemical methods including periodic acid-Schiff, selective periodate oxidation-Schiff reaction, Alcian blue staining at pH 2.5 and 1.0, and high iron diamine. The results indicated that neutral glycoconjugates with 1,2-glycol groups and sialic acids were present in the luminal border and apical cytoplasm of the glandular cells, basement membrane and connective tissue in the lamina propria of the lateral prostate. Similar patterns were demonstrated in the seminal vesicle except that there were relatively fewer or no neutral carbohydrates in the apical cytoplasm of the vesicular epithelial cells. The epithelial basement membrane and connective tissue at the epithelial-stromal interface of both glands were rich in acidic and sulphated glycosaminoglycans. Partial characterization by bovine testicular hyaluronidase indicated the presence of chondroitin sulphates in the lamina propria of the glands. PMID- 1724867 TI - Lymphocyte-induced angiogenesis: a morphometric study in the chick embryo chorioallantoic membrane. AB - A morphometric analysis was carried out on the chorioallantoic membrane (CAM) vasculature in chicken embryos treated (1) with phytohemagglutinin (PHA), (2) with supernatants of cell cultures of normal human T lymphocytes and (3) of T lymphocytes stimulated with PHA in order to evaluate their angiogenic properties. In the left chorioallantoic artery, the number of primary collaterals, and the number and length of secondary and tertiary collaterals were measured. In the series treated applying procedures 1 and 3, the mean value of the total number of all the collaterals was higher than that of the normal-developing CAM vessels, while the length of the secondary and tertiary collaterals was lower than that of the normal-developing blood vessels. In the series treated following procedure 2, no statistically significant differences were observed between experimental and control series in any parameters evaluated. The possible significances of these morphological findings are discussed with reference to the angiogenic roles played by human T lymphocytes, probably determined by lymphokines released after the activation of these cells by PHA, and by PHA alone, probably dependent on its aspecific mitotic activity. PMID- 1724868 TI - Problems related to infarct size measurements in the rat heart. AB - The feasibility of measuring the extent of hypoperfused myocardium and the infarct size was examined in rat hearts after occlusion of the left coronary artery. The extent of hypoperfused myocardium was examined by autoradiography and after perfusion with fluorescent microspheres. Both methods appeared unreliable in this model. Triphenyltetrazolium chloride (TTC) staining, however, provided a distinct demarcation line between viable myocardium, which was stained red, and the necrotic myocardium, consistent with the ultrastructural border between normal and severely damaged myocytes 5 h after coronary occlusion. TTC staining gives the best demarcation of ischemic tissues. In verapamil-treated rats, there was an apparent reduction in infarct size as compared with untreated rats; 20% reduction in infarct size 5 h after coronary occlusion and 12% reduction after 24 h. There was, however, a large postoperative mortality among the verapamil treated rats. These problems, and the nonuniform infarct size in rats, may in part explain why infarct size limitation by verapamil has been reported from rat experiments, but not from clinical trials. PMID- 1724869 TI - Chemotherapy combined with involved-field radiotherapy for 177 children with Hodgkin's disease treated in 1983-1987. PMID- 1724870 TI - Ionic channels of vascular smooth muscle in hypertension. PMID- 1724871 TI - Molecular aspects of voltage-dependent ion channels. AB - Voltage-dependent ion channels appear to form a large family whose individual structures suggest a lineage to a common ancestral channel protein. These channels share the feature of having a number (usually four or five) of homologous subunits or homologous internal repeat domains. Each contains a positively charged amphipathic helix at a conserved location within each of these subunits or repeat domains; voltage-dependent gating may be a property of this conserved S4 helix. In each channel, the ion pore itself is thought to be formed by contributions from helices of each of the subunits or domains which are themselves disposed in a pseudosymmetrical fashion around the aqueous pore. In spite of these similarities, each channel type, as well as subtypes within each type, exhibit unique kinetic and pharmacological properties, and varying patterns of tissue and cellular expression. Presumably these unique aspects of channel function are contributed by the variable regions of the protein structure and in this regard the cytoplasmic loops connecting the repeat domains or the amino- and carboxy-termini of the individual subunits may play a particular role. An appreciation of both the common aspects of channel structure and the unique characteristics of the specific channel of interest will be useful in determining its contribution to the pathobiology of hypertension and in planning therapeutic approaches in which the channel may play a role. PMID- 1724872 TI - Regulation of ionic channels by G proteins. PMID- 1724873 TI - Biochemical and molecular genetics of cystic fibrosis. PMID- 1724874 TI - Antigenic analysis of recent H1N1 influenza viruses with monoclonal antibodies. AB - Antigenic analysis of recently isolated H1 influenza viruses was performed using haemagglutination inhibition (HI) assay with monoclonal antibodies to the haemagglutinin (HA) subunit. Tests using monoclonal antibodies against the HA of the A/England/333/80 (H1N1) and A/Yamagata/120/86 (H1N1) viruses revealed that the major antigenic drift occurred in 1985 or 1986 and A/Dunedin/6/83-like virus became a major strain after 1986. PMID- 1724875 TI - Failure of azidothymidine to inhibit human immunodeficiency virus (HIV) replication in a promonocytic cell line (U937). AB - The inhibitory effect of azidothymidine (AZT) on HIV replication in the promonocytic cell line U937 was investigated. After infection with HIV-1/LAV virus, U937 cells were cultured for prolonged period in the presence of the drug at a final concentrations of 20 mumol/l or 50 mumol/l, respectively. The antiviral activity was determined according to the inhibition of viral reverse transcriptase activity, and of viral antigen production (immunofluorescence assay). We conclude that virus production was not efficiently influenced during long term passage even at high drug concentrations. PMID- 1724876 TI - Substance P-induced inhibition of Leydig cell steroidogenesis in primary culture. AB - The effect of substance P (SP) on hamster Leydig cell steroidogenesis in primary culture was investigated. Purified Leydig cells were cultured with or without SP for 24 h. The levels of testosterone and progesterone in the culture media were 174 +/- 20 pg ml-1 and 105 +/- 12 pg ml-1, respectively. In the presence of SP (10(-7) mol l-1), testosterone concentration significantly decreased to 123 +/- 19 pg ml-1, e.g. with 29.3%. By contrast, at the concentration used, SP had no effect on progesterone secretion. The possible molecular mechanism of the SP action on Leydig cell function was discussed. The reported results indicate that SP can modulate Leydig cell steroidogenesis in culture. PMID- 1724877 TI - On the expression of protamine genes in the testis of man and other mammals. AB - Protamines are low molecular weight, highly basic nuclear proteins involved in the condensation of sperm chromatin. cDNA clones for human protamine 1 and 2 (PRM1 and PRM2) were used for Northern blot experiments with RNA from different human tissues. Protamine transcripts, 0.6 kb and 0.9 kb in lengths for PRM1 and PRM2, respectively, were detected only in testicular RNA. The hybridization signals though did not produce sharp bands but enclosed a minor fraction of significant smaller transcripts. When polysomal RNA fractions were used in the hybridization, these shorter transcripts, 0.45 kb in length for PRM1 and 0.7 kb for PRM2, were specifically enriched. As demonstrated by in situ hybridization on human testis sections, the transcripts of both protamine genes are restricted to the central cell layer of the tubuli seminiferi corresponding to the spatial arrangement of postmeiotic cells. This result indicates that the protamine genes in the human are postmeiotically and haploid expressed. When cDNA clones of both protamines of the boar (BPrm-1 and BPrm-2) were used for hybridization experiments with the testicular RNA of those mammalian species which lack protamine 2 in their spermatozoa, the presence of transcripts for both protamines was detected. It can be assumed that mammals in general are endowed with at least two protamine genes which are both transcribed but are translationally regulated in a species-specific manner. PMID- 1724878 TI - Combination of alkaline phosphatase anti-alkaline phosphatase (APAAP)- and avidin biotin-alkaline phosphatase complex (ABAP)-techniques for amplification of immunocytochemical staining of human testicular tissue. AB - An amplification procedure was developed for the visualization of antigens in human testis using monoclonal antibodies against desmin and vimentin. The technique combines the high sensitive and specific APAAP- and ABAP-methods. Depending on the quality of the antibodies used and the processing of the material prior to the immunocytochemical staining the amplification technique may be applied either as a single APAAP and ABAP- or as a double APAAP and ABAP combination. Especially after the double amplification reaction a distinct increase of the staining intensity of the vimentin- (in Sertoli cells, myofibroblasts of the lamina propria, and fibroblasts of the interstitium) and desmin- (in myofibroblasts of the lamina propria and smooth muscle cells of the blood vessels) like immunoreactivity was observed. If different diazonium salts were used for the visualization of the alkaline phosphatase activity (e.g. Fast Red TR Salt, Fast Blue BB Salt) desmin- and vimentin-like immunoreactivity can be demonstrated in the same tissue section in a double sequential staining approach. For double staining, the alkaline phosphatase technique may be combined successfully with a technique or a combination that uses peroxidase as a marker. PMID- 1724879 TI - [Superficial vascular (arterial and venous) malformations: clinical aspects and complementary tests]. AB - A simple and precise classification has been established during a ten-year international cooperation. There are two major groups: hemangiomas, always regressive; and vascular malformations, which never regress; they grow, throughout life, to varying degrees. Management of vascular malformations has been clearly defined. Capillary malformations do not require examination, unless they are associated with other anomalies, such as hypertrophic underlying bone, or the leptomeningeal vascular anomaly of the Sturge-Weber syndrome. Venous type malformations are diffuse, and consist of entirely anomalous channels, with low flow. CT scan and MRI clearly demonstrate the extent of tissue involvement in venous malformations; for these slow-flow malformations the evaluation is performed without resorting to invasive diagnostic techniques: phlebography is rarely performed, and arteriography is unnecessary. Arteriovenous malformations are dangerous high-flow vascular malformations, leading to skin ischemic necrosis, and congestive cardiac failure. Arteriography shows enlarged tortuous arteries, with arteriovenous shunting, and early venous drainage. CT scan and MRI show the deep components of the lesions. Doppler ultrasound evaluation is used to follow the course of the disease. PMID- 1724880 TI - [Use of frequency doubled Nd-YAG laser and automatic scanning in the treatment of portwine hemangioma]. AB - The authors report their experience in the treatment of portwine stains by means of two recent improvements. The first improvement concerns the use of frequency doubled Nd-YAG laser which emits at 532 nanometres. This wave-length corresponds to an absorption peak of haemoglobin and is therefore more selectively absorbed by haemangiomatous tissue. This helps to explain the better clinical results obtained and the elimination of the other effects classically induced by argon laser, with complete preservation of the healthy, non-angiomatous skin as well as skin and scalp hair. Treatment is consequently facilitated, as illustrated by a series of 79 patients who received a total of 92 treatment sessions over a period of 14 months. The second improvement consists of the addition of an automat which allows the beam to be used continuously with scanning of adjacent bands. This automat not only allows a very homogeneous treatment by eliminating all of the errors associated with manual scanning, but can also be used to treat large portwine stains in a single session instead of the multiple sessions required previously. An interval of 5 to 6 months should be respected before treating the residual part of the portwine stain situated more deeply in the dermis, which should be considered to be a new portwine stain. The combination of these two apparatuses allows fluences (fluence is the product of the power selected by the exposure time per unit area) of only 8 to 15 joules/cm2 due to the dynamics of the treatment in contrast with other static modalities which require higher fluences.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724881 TI - [Surgical treatment of mature extracephalic vascular malformations (excluding portwine hemangioma). Experience of this department]. AB - The department's experience of mature extracephalic vascular malformations shows that, regardless of the site and the type of the lesion, only surgical excision is able to ensure a good result for vascular malformations of the limbs. The results were almost always excellent and only the diffuse forms benefitted from preoperative sclerosant injections. Palliative treatments such as elastic compression still have a place in these indications. Arterial embolisations are only of limited value, particularly when they are selective, as a complement to surgery (risk of necrosis in peripheral lesions). All sites may be observed, but the authors particularly emphasise perineoscrotal lymphangiomatous lesions. Recurrences do occur, but are rare when the lesion is widely resected and a good quality reconstruction is performed. PMID- 1724882 TI - [Surgical excision of giant venous angioma of the face under extracorporeal circulation: apropos of a case by a multidisciplinary team]. AB - Because of the risk of haemorrhage related to their resection, deep angiomas of the face are often considered to be inaccessible to treatment. The use of cardiopulmonary bypass with profound hypothermia allows the surgeon to operate in a bloodless field, enabling almost oncological resection of the tumour. The authors report a case of venous angioma of the submaxillary region treated in this way after failure of limited surgery and embolisation. Complete resection of the lesion was achieved and the defect was repaired with a pectoralis major flap and a latissimus dorsi flap. The authors stress the value of a multidisciplinary approach: a cardiac surgeon for CPB, an ENT surgeon for resection and a plastic surgeon for reconstruction. PMID- 1724883 TI - [Giant lingual hemangioma: apropos of a surgically treated case]. AB - This paper presents the very unusual case of a woman who, up until the age of 24 years, had been locked away by her parents in order to hide her deformity. She consequently suffered from chronic malnutrition, speech disability and was unable to apply any oral hygiene. During the preoperative period she was treated with corticosteroids leading to thrombosis of the tumour and subsequent inflammation followed by acute airways obstruction requiring emergency tracheotomy. During the operation, the large neck vessels were dissected bilaterally to avoid any haemorrhage. The lingual arteries were tied bilaterally. Haemostatic sutures were applied before section and 95% of the tumour was resected. The residual tissue was used to reconstruct the tongue. A total of 100 ml of blood was lost. Surgery changed the patient's life. She was able to eat correctly, receive dental treatment and, most importantly, speak, which she had been unable to do previously. She was subsequently integrated into society. PMID- 1724884 TI - [Orbital lymphangioma. Apropos of 2 uncommon cases]. AB - Lymphangiomas can be considered as hemodinamically isolated vascular hamartomas. They are rare tumors, and their orbito-palpebral location can be cosmetically and functionally detrimental. In severe cases, the treatment is mainly surgical and effective only if complete removal is performed. The result is often deceiving because of the diffuse extension of these tumors. We hereby report two exceptionally severe cases that both presented with major cosmetic impairment and visual loss. We discuss literature available, the management and the methods of treatment and the non-surgical alternatives. PMID- 1724885 TI - [The inframammary fold: myth or reality?]. AB - Is the inframammary fold an anatomical entity? In order to answer this question, the authors conducted an anatomical, histological and radiological study. The results of these studies were concordant and were able to anatomically define the inframammary fold. The inframammary fold corresponds to a division of the fascia superficialis. It contains numerous fibres stretched between the fascia superficialis and the fascia prepectoralis. In contrast, there are no fibres connecting the fascia superficialis to the deep dermis. PMID- 1724886 TI - [Breast reconstruction with a latissimus dorsi musculocutaneous flap after amputation for cancer. Apropos of 45 cases with a 9-year follow-up]. AB - Breast reconstruction following mastectomy for cancer using a latissimus dorsi myocutaneous flap must always be completed by insertion of a prosthesis. In 76% of our cases, it was associated with surgery on the contralateral breast to produce symmetry. By following this therapeutic plan using a flap as large as possible taken in the line of the strap of the brassiere, and raised with a large expanse of muscle to completely cover the prosthesis, the aesthetic results were uniformly good. This applies to the mastectomy patients without adjuvant therapy. All the mediocre and poor results were observed in the patients who had undergone radiotherapy. The most severe post radiotherapy complications (8.7% of our series) with progressive fibrosis and chest wall retraction, fortunately rare today, are a contraindication to this type of reconstruction. This case requires very large soft tissue transfer with a rectus abdominis myocutaneous flap. This is a much larger procedure for the patient. 91% of post radiotherapy patients are satisfied with the result with a follow-up of up to 9 years demonstrating good stability. Despite this only 74% of the results were considered to be good by the surgeon. So 17% could be improved and this suggests new efforts in two directions: 1) To avoid the most frequent cause which is the formation of a capsule. The availability of the new textured prosthesis is a good reason to hope for a decreased incidence, but the follow-up 18 month is not yet long enough to confirm this. 2) To routinely position the flap in the lower outer quadrant respecting the principle of aesthetic units. PMID- 1724887 TI - [Degeneration of scars. Apropos of 14 cases]. AB - The authors report 14 cases of scar carcinomas, arising in different types of scars, over a 5-year period. The histological type of tumour was squamous cell carcinoma. The different sites of occurrence, from those of spontaneous skin neoplasms reflect their different pathogenesis. The diagnosis must be made early, before lymph node involvement, which is the most significant prognostic factor. Treatment should be first prophylactic to ensure good quality healing. Once the tumour has appeared, only aggressive wide excision provides a hope of cure. PMID- 1724888 TI - [Covering of ischial pressure sores using a fasciocutaneous flap from the posterior surface of thigh (modified Griffith method) after mattressing with the biceps femoris. Apropos of 11 cases]. AB - The ischial region is the commonest site for pressure sores in paraplegics capable of sitting. The choice of treatment must be guided by two imperatives: the need for a thick tissue cover (mattressing) and perfect trophicity, the risk of recurrence which always remains possible. The association of a modified Griffith fasciocutaneous flap from the posterior surface of the thigh and a hamstring muscle flap (preferably biceps femoris) providing mattressing of the ischial region, appears to satisfy these imperatives. The other repair procedures are discussed in order to ensure the best management of the paraplegic patient's cutaneous capital, due to the continual risk of a new pressure sore or a recurrence of the ischial pressure sore. PMID- 1724889 TI - [Perineal complications and gracilis musculocutaneous flap in patients with paraplegia. Apropos of 19 cases]. AB - The authors reviewed the indications for gracilis musculocutaneous flap in paraplegic patients with an ischial pressure sore. After analysing the causes and types of perineal complications associated with this type of pressure sore, it would appear that this flap should be used more widely to prevent essentially urethral complications. The many advantages are emphasised: thickness of the flap, urethral protector muscle, large amount of good quality skin cover, ample rotation arc, especially as an island flap, easy closure of the donor site, preservation of the posterior musculocutaneous capital of the thigh. However, despite these advantages, the drawback, responsible for the poor reputation of this flap, must not be over-looked: the poor contiguity between the muscle volume and the volume of skin cover. The surgeon is therefore able to transform a disadvantage into an advantage by respecting rigorous technical rules. PMID- 1724890 TI - [Procedure to follow in post-traumatic reimplantation of the external ear]. AB - Cases of complete traumatic amputation of the auricle successfully treated by reimplantation are rare. The author used the Baudet technique: it consist of suppression of the skin cover of the posterior aspect of the mutilated fragment and creation through the cartilage of appropriately sized and arranged windows. This mutilated fragment is then sutured by its anterior edge to the remaining portion of the auricle and by its peripheral rim to a posteriorly based mastoid flap. PMID- 1724891 TI - [Reconstruction of the orbital floor using skull grafts obtained during the treatment of craniostenoses and irradiated by gamma rays]. AB - The authors use skull grafts obtained during the treatment of craniostenoses and sterilized by gamma radiation to reconstruct the orbital walls. They describe the practical modalities of this technique and report a series of 26 cases in which no incidents of complications were observed. PMID- 1724892 TI - Broad-spectra human monoclonal antibodies that protect mice infected with Pseudomonas aeruginosa. PMID- 1724893 TI - Pore-forming cytotoxin of Pseudomonas aeruginosa: the molecular effects and aspects of pathogenicity. PMID- 1724894 TI - [Physiopathology of cystic fibrosis]. AB - Cystic Fibrosis (CF) is the most common lethal genetic autosomic disease in Caucasians. The disease expresses itself in airway and other epithelial cells as a defective chloride ion absorption and secretion. At least, an abnormal cAMP dependent regulation of an apically located chloride channel has been proposed as the underlying molecular defect. The gene responsible for CF has been identified and predicted to encode a membrane protein termed cystic fibrosis transmembrane conductance regulator (CFTR). The functional role of the predicted protein remains unclear, although strong evidence suggest that it is directly or indirectly involved in regulation of the apical chloride permeability in epithelial cells. This review discusses the fundamental issues currently being investigated in CF. PMID- 1724895 TI - Keratohyalin granules are heterogeneous in ridged and non-ridged human skin: evidence from anti-filaggrin immunogold labelling of normal skin and skin of autosomal dominant ichthyosis vulgaris patients. AB - Recent biochemical and morphological investigations have provided evidence for a heterogeneous composition of keratohyalin in human skin. A major component is filaggrin. In interfollicular epidermis the heterogeneity of keratohyalin is not directly visible, whereas in normal ridged skin bicomponent keratohyalin is revealed by electron microscopy. Skin biopsies of ridged and non-ridged skin of normal individuals and patients with autosomal dominant ichthyosis vulgaris (ADI) -characterized by defective keratohyalin synthesis and lack of filaggrin--were investigated by routine transmission electron microscopy and immunogold postembedding techniques using a commercial monoclonal anti-filaggrin antibody. In normal interfollicular epidermis filaggrin labelling was demonstrated on keratohyalin granules and in the lowermost cornified cells, whereas in ADI patients crumbly keratohyalin granules were present that did not show specific labelling for filaggrin. In normal ridged skin only the major (more electron dense) component reacted with anti-filaggrin, whereas the attached (less electron dense) component did not react. Ridged skin of ADI patients contained globular keratohyalin that did not react with anti-filaggrin, thus corresponding to the attached keratohyalin component in normal ridged skin. Our results provide a visible counterpart to the recent biochemical investigations of keratohyalin protein heterogeneity and contribute to the understanding of terminal differentiation in human skin and of the defective keratohyalin synthesis in ADI. PMID- 1724896 TI - Quantitative analysis of contact sites between mast cells and sensory nerves in cutaneous psoriasis and lichen planus based on a histochemical double staining technique. AB - The aim of the present study was to test further our previous hypothesis that the inflammatory reaction in psoriasis is neurogenic. For this purpose, contact sites between mast cells and sensory nerves were morphometrically analysed in the basement membrane zone, papillary dermis and three dermal zones of lesional/non lesional psoriatic and lichen planus skin as well as in healthy control skin. The analyses were made on sections stained with a histochemical double stain developed for this study. With the double stain, active mast cell tryptase was stained blue enzyme histochemically, and the sensory nerves black using specific monoclonal anti-neurofilament antibodies with immunogold. In psoriatic lesions, both mast cells and mast cell--nerve contacts were markedly more frequent in the basement membrane zone and in the papillary dermis when compared with the corresponding areas in the other groups. Mast cell numbers were increased in both lesional and symptom-free skin in lichen planus, but no increase was found in the mast cell--nerve contacts. Increased contacts between mast cells and sensory nerves indicate that the elements exist for neurogenic inflammation in psoriatic lesions. These increased contacts are not due to the extensive inflammatory reaction only, because they were not observed in lichen planus lesions. PMID- 1724897 TI - Normal psoriatic epidermis expression of hyperproliferation-associated keratins. AB - Keratin expression in lesional, marginal and uninvolved psoriatic epidermis was analysed by one- and two-dimensional gel electrophoresis and immunoblotting. Keratins K1, K5, K6, K10, K14, and K16 were identified in lesional epidermis. Keratins K6 and K16 were found in all epidermis probes of uninvolved skin, but never occurred in normal epidermis of control skin samples. By means of laser densitometric evaluation of one-dimensional gels a downregulation of K1 and K10 and an upregulation of K6 and K16 was found in psoriatic epidermis. Unexpectedly, the level of K5 was considerably lower and the level of K14 considerably higher in lesional skin than in normal epidermis. These results demonstrate that not only basal keratinocytes in lesional epidermis but also suprabasal keratinocytes in uninvolved psoriatic epidermis express an altered differentiation pattern. The latter phenomenon could be very important in understanding the development of the so-called "Kobner effect" in psoriatic epidermis. PMID- 1724899 TI - Treatment effects of GnRH agonist on the binding of estrogen and progesterone, and the histological findings of uterine leiomyomas. AB - The changes in the histology and steroid hormone binding capacity of the uterine leiomyomas treated with; GnRH agonists (GnRHa buserelin acetate in, 900 micrograms/day for 16 weeks) were investigated. The occurrence of hyaline degeneration in the myometrium was significantly higher in the GnRHa-treated group than in the control group, and the grade of hyaline degeneration was more advanced in the GnRHa group. After the GnRHa treatment, the Bmax of the estrogen receptor increased significantly in the leiomyomas and myometrium. The Bmax of progesterone receptors in the myometrium decreased significantly and the reduction rates of leiomyomas (% of the initial volumes) measured by MRI correlated (r = 0.775) with the Bmax of progesterone receptors. In summary, GnRHa caused hyaline degeneration of the uterine leiomyomas which were responsible for the shrinkage. The shrunk leiomyomas have the potential for regrowth in response to estrogen as they still have high concentrations of the estrogen receptors. PMID- 1724898 TI - Keratin 17: a useful marker in anti-psoriatic therapies. PMID- 1724900 TI - Cytokine therapy in canine and primate models of radiation-induced marrow aplasia. PMID- 1724901 TI - Studies on the efficacy of mast cell growth factor (c-kit ligand) in vitro as well as in vivo. AB - We tested the ability of recombinant human (rhu) mast cell growth factor (MGF), also known as c-kit ligand, to stimulate the colony formation of human bone marrow cells in semisolid medium alone and in combination with rhu erythropoietin (EPO), rhu Interleukin 3 (IL-3), rhu granulocyte colony stimulating factor (G CSF) and rhu granulocyte-macrophage colony stimulating factor (GM-CSF). The addition of MGF to cultures containing EPO or EPO + IL-3, GM-CSF and G-CSF, resp., resulted in macroscopic erythroid burst-forming units (BFU-E). Multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]) progenitors were stimulated by MGF in the presence of EPO. Colony-forming unit granulocyte-macrophage (CFU-GM) were activated by MGF only in combination with GM-CSF. The combination of MGF with EPO was used for synergism studies in healthy cynomolgus monkeys. In the chosen concentration MGF alone had no effect on white blood cell (WBC) counts and on platelets, but a slight effect on reticulocytes. EPO by itself increased reticulocyte counts with no effects on WBC or platelets. The combination of both factors resulted in a significant increase of reticulocytes. No other effects were seen. These studies demonstrate the potent synergistic interaction of MGF and other hematopoietic growth factors. PMID- 1724902 TI - Molecular and cellular biology of mast cell growth factor: the gene product of the murine steel locus. PMID- 1724903 TI - Activation of the ubiquitin-ATP-dependent proteolytic system in skeletal muscle during fasting and denervation atrophy. AB - The rapid atrophy of skeletal muscles upon fasting or denervation is due largely to an increased rate of protein breakdown. Blocking the lysosomal or the Ca(2+) dependent pathways did not prevent increased proteolysis in muscles from fasted animals or following denervation. In contrast, upon food deprivation, the nonlysosomal ATP-dependent process increased by 150-350%. After refeeding, this process returned to control levels by 24 h. Similarly, within one day after denervation of the soleus, proteolysis increased by 50-250%. By contrast, the residual energy-independent process did not change in fasting or denervation. Because the ATP-dependent process might involve activation of the ubiquitin-ATP dependent pathway, we measured the levels of mRNA for ubiquitin [Ub] in the atrophying muscles. After food deprivation, the levels of polyUb transcripts increased 2- to 4-fold in the soleus and extensor digitorum longus (EDL) muscles, and returned to control levels within 1 day of refeeding. After denervation of the soleus, a 2- to 3-fold increase in polyUb mRNA also occurred within 1 day. The muscle content of ubiquitinated protein also changed in parallel with Ub mRNA levels under these conditions. Thus, polyUb genes appear to be selectively induced in atrophying muscle, and levels of Ub mRNA and ubiquitin-protein conjugates change coordinately with the rate of ATP-dependent proteolysis. These data suggest that in atrophying muscle the ATP-Ub-dependent system plays an important physiological role in the degradation of the bulk of cell proteins. PMID- 1724904 TI - Role of cytokines and growth factors in the induced synthesis of proteinase inhibitors belonging to acute phase proteins. AB - The effects of various cytokines on synthesis and secretion of albumin and some proteinase inhibitors belonging to the class of macroglobulins, serpins and cysteine proteinase inhibitors were studied in the primary cultures of rat and mouse hepatocytes and established human hepatoma cell line Hep G2. In all tested systems interleukin 6 depressed the synthesis of albumin and enhanced the synthesis of antichymotrypsin (or contrapsin) and alpha-1-proteinase inhibitor, while in the rat alpha-2-macroglobulin and T-kininogen (thiostatin) were the major acute phase reactants. Smaller and variable effects were observed with interleukin-1, tumour necrosis factor, interferon-gamma, transforming growth factor-beta and epidermal growth factor. Searching for the feed-back regulatory mechanism responsible for induced synthesis of proteinase inhibitors we found that cultured human lung fibroblasts exposed to human alpha-1-antichymotrypsin or antichymotrypsin-cathepsin G complexes produce significantly more interleukin 6 which stimulates Hep G2 cells to augmented synthesis of several acute phase proteins. PMID- 1724905 TI - Components of the multicatalytic proteinase complex. AB - The multicatalytic proteinase complex is a high molecular weight nonlysosomal proteinase. Kinetic studies of the proteolytic activities of the complex have shown that there are at least three distinct types of catalytic centre, each of which has a different specificity. All of the activities can be inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin. Viewed under the electron microscope, the multicatalytic proteinase purified from rat liver appears to have a hollow cylindrical structure. It is composed of many different types of subunit and on two-dimensional polyacrylamide gels gives rise to a complex pattern of about 20 spots with pI values ranging between 5 and 8.5 and molecular masses between 22 and 34 kDa. Immunoblot analysis has shown that many of the major polypeptides are antigenically distinct. However, there are some relationships between the proteinase polypeptides. For example, although N-terminal sequences of five of the polypeptides are unique, they show considerable sequence similarity suggesting that these proteins are encoded by members of the same gene family. Also, there is some cross-reactivity between certain polypeptides when blots are probed with affinity purified, subunit-specific antisera. In addition to the variety of polypeptide components of the proteinase, a small RNA species (80 nucleotides) can be found associated with the complex even after purification by chromatographic procedures. PMID- 1724906 TI - (K15R M52E) aprotinin is a weak Kunitz-type inhibitor of HIV-1 replication in H9 cells. AB - The (K15R M52E) aprotinin is a recombinant molecule with a broader inhibition spectrum against serine proteinases and a higher affinity towards certain proteinases as compared to native aprotinin. This aprotinin variant was produced in E. coli, isolated and purified to homogeneity. The inhibitor was further tested for its effectiveness to reduce human immunodeficiency virus type 1 (HIV 1) replication. Virus growth was followed by an ELISA which detects the amount of virus core protein p24. At a concentration of 50 microM, the recombinant (K15R M52E) aprotinin clearly reduced HIV-1 replication in H9 cells. PMID- 1724907 TI - Collagen I, laminin, and tenascin: ultrastructure and correlation with avian neural crest formation. AB - We have investigated the distribution of type I collagen, tenascin, and laminin in younger chick embryos than have previously been studied in detail. The initial appearance of type I collagen, but not tenascin and laminin, is exactly correlated with the beginning of neural crest migration, suggesting a role for collagen I in the migration. Light microscopy of whole mounts of 2-day-old chick embryos reveals that type I collagen is expressed in a rostral to caudal gradient; it localizes to the notochord sheath before accumulating around the neural tube and somites. Collagen I and tenascin also associate with central somite cells. Surprisingly, no extracellular matrix can be detected among the early sclerotomal cells, which suggests that little or no cell migration is involved in this epithelial-mesenchymal transformation. Electron microscopy using peroxidase antiperoxidase reveals that tenascin is present in nonstriated, 10 nm wide fibrils and in interstitial bodies, both of which have previously been reported to contain fibronectin. However, collagen I only occurs in the 10 nm fibrils and larger striated fibrils. This is the first ultrastructural study to assign tenascin to fibrils and interstitial bodies and to describe its appearance and disappearance from embryonic basement membranes. The discussion emphasizes the possible importance of type I collagen in neural crest cell migration and compares the ultrastructural associations of the ECM molecules present at this early embryonic stage. PMID- 1724908 TI - Stage II large B-cell lymphoma with sclerosis treated with MACOP-B. AB - In a series of 193 patients with advanced stage diffuse large-cell lymphoma (DLCL) treated with MACOP-B, 18 (11%) were defined as having a stage II large B cell lymphoma with sclerosis of the mediastinum. This type of lymphoma has been reported to have a highly aggressive behaviour and special histological and clinical features. In our series young women were more commonly affected and the most striking clinical feature was the presence of a bulky mediastinal mass in 81%. A comparison was made between stage II patients with DLCL with and without sclerosis. The group of patients with sclerosis had prognostic parameters significantly worse than those of the patients without sclerosis, namely, elevated LDH level and bulky disease. The complete remission rates (89% vs 76%) were similar in the two groups and, with a median follow-up of 23 months, survival and disease-free survival rates were also superimposable. MACOP-B chemotherapy has been proven effective in this subgroup of lymphoma patients with sclerosis that had thus far been reported to have a poor prognosis. PMID- 1724909 TI - The effects of milrinone in the neonatal pig heart. AB - Milrinone, a selective inhibitor of phosphodiesterase (PDE), was examined in neonatal hearts and in ventricular myocytes. Isolated, paced (180 beats/min), isovolumically beating hearts from pigs, less than 3 days of age, were perfused with an erythrocyte-enriched solution. In one group (control, n = 6), milrinone was studied at perfusate concentrations of 1, 10, and 100 micrograms/ml. In a second group (postischemia, n = 10), hearts were subjected to 30 minutes of no flow ischemic arrest, prior to the addition of milrinone. Left ventricular peak systolic pressure (PSP) and end-diastolic pressure, coronary flow (CF), heart rate (HR), and myocardial oxygen consumption (MVO2) were measured. The PSP averaged approximately 100 mmHg during the baseline periods for both groups and decreased to approximately 85 mmHg in those hearts subjected to ischemic arrest. In both groups, PSP increased approximately 14% at the 1 micrograms/ml concentration of milrinone. No additional increases in PSP were observed in the control group at the higher concentrations. However, PSP increased 28% and 41% (p less than 0.05), in the postischemia group at the 10 and 100 micrograms/ml concentrations, respectively. The CF averaged approximately 3 ml/min/g during the baseline periods of both groups and increased significantly at each milrinone concentration. The HR in both groups increased to approximately 200 and approximately 250 beats/min at the 10 and 100 micrograms/ml concentrations, respectively. Additionally, milrinone's effects in intact hearts were found to be comparable to those of isobutylmethyl xanthine (IBMX), a nonspecific PDE inhibitor. In isolated myocytes, however, milrinone produced only modest increases in cAMP levels, compared to IBMX.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724910 TI - Effect of oral glucose feeding on thyroid hormone action in human mononuclear blood cells. AB - The effect of glucose feeding on serum concentrations of thyroid hormones, nuclear T3 binding and thyroid hormone stimulated oxygen consumption and glucose uptake was evaluated. Six persons without thyroid diseases were fed exclusively glucose (2500 kcal/day) for two days. Glucose feeding resulted in the following serum hormone levels (before vs after glucose feeding) T3: 1.07 vs 1.16 nmol/l (p less than 0.05); FT4I: 76 vs 102 a.u. (p less than 0.05) and TSH: 1.48 vs 1.14 mU/l (n.s.). Nuclear T3 binding decreased after glucose feeding; before: maximal binding capacity (MBC) 1.3 +/- 0.5 fmol/100 ug DNA, MBC after glucose feeding: 0.8 +/- 0.2 fmol/100 ug DNA (p less than 0.05). The Ka's were identical in both groups before glucose, Ka: 4.2 +/- 1.2 x 10A10 l/mol, after glucose, Ka: 3.5 +/- 1.2 x 10(10) l/mol. Conversely did glucose feeding increase thyroid hormone stimulated oxygen consumption by approximately 100% and thyroid hormone stimulated glucose uptake by approximately 200%. In conclusion, our results show that glucose feeding 1) increases serum T3 levels, 2) decreases nuclear T3 binding, 3) enhances thyroid hormone stimulated oxygen consumption and glucose uptake. PMID- 1724911 TI - Plasma neuropeptides in hyperthyroidism. AB - Plasma levels of the neuropeptides, vasoactive intestinal polypeptide (VIP, neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), substance P, and galanin were determined in 15 hyperthyroid patients before and at 3 occassions during 38 weeks of treatment. Treatment was performed with either 131I alone or with carbimazole, with combination of carbimazole and thyroxine, or with subtotal thyroidectomy. Before and after 11 (+/- 4), 24 (+/- 6) and 38 (+/- 5) weeks of treatment, plasma neuropeptide levels were analysed. A group of 9 premenopausal women served as controls. During hyperthyroidism, mean plasma level of CGRP was higher than in controls (P less than 0.001). In contrast, the mean plasma levels of the other measured neuropeptides did not differ from those in the controls. Mean serum level of tree T4 was lowered from 81.9 +/- 30.1 to 23.9 +/- 8.6 pmol/l and mean serum level of free T3 was lowered from 27.3 +/- 7.9 to 6.7 +/- 2.3 pmol/l during the course of the treatment. After 11 weeks of treatment, mean plasma NPY level was significantly increased (P = 0.004) compared to pretreatment levels. However, after 38 weeks of treatment, mean plasma NPY level had returned to control values. The mean plasma CGRP level was significantly reduced at 11 and 38 weeks of treatment compared to pre-treatment value (P = 0.002 and P = 0.004, respectively). Mean plasma level of neurotensin slowly declined during the treatment (P = 0.003). In contrast, mean plasma level of VIP, of substance P, and of galanin did not differ from control value before or after treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724912 TI - Restricted clonality of antithyroglobulin antibodies in an M-component of Hashimoto's serum. AB - In serum of a 70 y. female with Hashimoto's thyroiditis we have identified an M component IgG which is predominantly composed by a single class of antihuman thyroglobulin antibody. Our assumption is supported by Scatchard analysis and by the high concentration of lambda chains in serum and IgG fraction which are absorbed by h-thyroglobulin. Attempts to dissociate the monoclonal antibody treating the immunoprecipitate at acidic pH, were unsuccessful; only a few percent (3.7) of the antibodies could be released from immunoprecipitate and those were polyclonal. In order to explain the immunoprecipitating reaction of the monoclonal antibody, it is suggested that the antibody is reacting with a repetitive structure of the h-thyroglobulin molecule. PMID- 1724913 TI - Ouham-Pende: a new endemic goiter area in the Centroafrican Republic (CAR): epidemiological survey on 7621 subjects. AB - The authors report epidemiological observations about a new endemic goiter area in the North western region of the Centro-African Republic (CAR). The research was carried out in 7 quarters of the chief-town (Bocaranga) and in 8 rural villages; 7621 people were examined by the same three of the present Authors who filled in a sample individual form (proposed by WHO) with the generalities and the goiter grading. The data were analyzed according to sex, age and place of residence by means of the common position parameters and the cumulative frequencies, taking into account the goiter grading of each group. The goiter prevalences observed in the rural areas varied from 70.9% (males) to 82.6% (females), whereas in the chief-town they varied from 29.3% (males) to 57.9% (females). Neurological and myxedematous cretins were seen to constitute 3.4% of the visited population. The M/F goitrous ratio was near unity under the age of 6; above this age, females are more widely and severely affected than males. An important fall in goiter prevalence was observed in adult males after the age of 16. The gradings observed in the rural villages, in both females and males of different ages (0-5; 6-15; 16-45; greater than 45 ys.) were significantly more severe than those observed in the chief town (P less than 0.01). The results obtained confirm those of a preliminary survey which the Authors previously carried out on a sample of school children living in the same region of CAR. PMID- 1724914 TI - Clinical, laboratory and immunologic effects of the treatment of endemic goiter with T4, T3 and KI. AB - We treated 204 patients with endemic nontoxic goiter with T4, T3 and KI, singly or in combination. Definitely nodular goiters were excluded, since the possibility of autonomy would be increased. Goiter size was evaluated before and 6 months after treatment clinically in a blind way, i.e. the observer (always the same) did not know either the pretreatment goiter size or the treatment the patient had received. At the same time various laboratory parameters were recorded. All the active treatments (but not placebo) resulted in a highly significant decrease in the gland size. The effectiveness decreased in the following order: 1) T3 50 micrograms/d (most effective), 2) (T4 50 micrograms/d + T3 12.5 micrograms) x 2, 3) T4 150 micrograms + iodide 150 micrograms/d, 4) T4 75 micrograms + T3 18.75 micrograms/d, 5) T4 200 micrograms/d, 6) T3 37.5 micrograms/d, 7) Iodide 300 micrograms/d, 8) T4 150 micrograms/d, 9) Iodide 150 micrograms/d (least effective) and 10) Placebo (not effective). The results show that T4 200 micrograms and T3 50 micrograms are roughly equipotent, and slightly more effective than 300 micrograms of Iodide. Taking into consideration the side effects (increase in pulse rate, shortening of the Achilles tendon reflex) did not change the order of effectiveness in an important way. The clinical outcome correlated in general with the suppression of the 131I uptake (r = 0.220, p = 0.03) and the TRH test (r = 0.248, p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724915 TI - Low protein supplemented diet corrects altered serum thyroid hormone and TSH concentrations in patients with chronic nephrotic syndrome. AB - Creatinine clearance, daily urinary protein excretion, serum concentrations of thyroid hormones (TT4, TT3, fT4, fT3), TSH and parathyroid hormone (PTH), have been measured before and after 2 or 6 months of a low protein diet supplemented with aminoacids and ketoanalogues in 18 patients affected by chronic nephrotic syndrome without significant impairment of renal function. Mean creatinine clearance and mean serum protein concentration (79.5 +/- 13.8 ml/min and 5.4 +/- 0.6 g/dl, mean +/- S.D., respectively) did not significantly change (79.1 +/- 17.3 ml/min and 5.5 +/- 0.6 g/dl) after the diet. Mean daily urinary protein excretion (7.1 +/- 2.2 g/day basally) significantly decreased (5.5 +/- 1.9 g/day) after the diet (p less than 0.05). Mean serum TT4 concentration (5.6 +/- 1.8 micrograms/dl basally) significantly increased (6.7 +/- 2 micrograms/dl, p less than 0.05) after the diet. Mean serum TT3 concentration (106.7 +/- 28.5 ng/dl, basally) significantly increased (126.7 +/- 22.6 ng/dl) after the diet (p less than 0.01). Mean serum fT4 and fT3 concentrations (8.0 +/- 2.9 pg/ml and 4.5 +/- 1.6 pg/ml, respectively) did not significantly change (9.4 +/- 2.7 pg/ml, and 4.9 +/- 1.9 pg/ml, respectively) after the diet. In some patients low basal serum concentration values of TT4, TT3, fT4, fT3 became normal after the diet. Mean serum TSH concentration (3.1 +/- 2.3 microU/ml basally), significantly decreased (1.5 +/- 1.3 microU/ml) after the diet (p less than 0.05). Mean serum PTH concentration (0.7 +/- 0.3 ng/ml basally) significantly decreased (0.4 +/- 0.2 ng/ml) after the diet (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724916 TI - Ectopic production of beta-subunit of human chorionic gonadotropin in malignant lymphoma. AB - Ectopic production of human chorionic gonadotropin (HCG) in tumor cells is an uncommon phenomenon that has rarely been documented. The immunoperoxidase method was used to demonstrate the presence of beta-subunit of HCG in malignant lymphoma cases, using paraffin-embedded sections of lymph nodes. 3 of 11 cases of malignant lymphoma (27%) were positively stained with beta-subunit of HCG. All positive cases were T-cell type malignant lymphomas, but all adult T-cell leukemia lymphoma cases did not react with HCG. All B-cell type malignant lymphoma cases were not stained by HCG. Hence, this evidence suggested that beta subunit of HCG may be useful as a tumor marker in certain malignant lymphoma patients. To the best of our knowledge, production of beta-subunit of HCG in the cytoplasm of malignant lymphoma cells has not been reported. PMID- 1724917 TI - Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells. AB - Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors. PMID- 1724919 TI - Immunolocalization of cytokeratins in some freshly fixed bovine epithelia. AB - Various freshly fixed bovine epithelia were studied with regard to their cytokeratin expression. The epithelia were tested by the ABC immunoperoxidase method with AE1, AE3, anti-keratin B and anti-keratin C monoclonal antibodies. While the hair follicle and nasolabial gland express both small acidic and large basic cytokeratins, the epidermal cells, sebaceous gland and mucosae appear to contain only the latter, and the mammary gland, only the former cytokeratins. Our results on fixed and paraffin embedded material are compared with those reported by others in cell cultures from human and bovine specimens. PMID- 1724918 TI - Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine. AB - Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products. PMID- 1724920 TI - Static cytometry of DNA in breast lump with reference to grading. AB - Fresh smears of 400 consecutive cases of breast lumps stained with Feulgen stain were measured with densitometric technique in static cytometry. A correlation was observed between DNA content expressed by the DNA index and histologic grading of malignant lesions; relatively high values were observed in the group of "hyperplasias", which were similar to G1. Values of DNA content in different categories: benign, hyperplastic (nodular and sclerosing adenosis, epitheliosis . . .) and all malignant types variously graded in G1, G2 and G3, show broad areas of overlapping, suggesting that the parameter is not always discriminating between benign, hyperplastic and malignant, but it can identify different amounts of DNA (high and low degrees) within a homogeneous category. Correlation between number of hypertetraploid nuclei (4c) and histologic grading was also evident. Therefore, fresh smears, in relation to DNA content and to the percentage of hypertetraploid elements allow a better definition of "high and low degree" lesions in a short time with possible prognostic involvement as regards tumoral progression. PMID- 1724921 TI - Fine needle aspiration of a pancreatic oxyphilic carcinoma with pulmonary and subcutaneous metastases. AB - The cytological appearances of an oxyphilic pancreatic carcinoma are reported and the histological and clinical features are described. Despite the presence of pulmonary and subcutaneous metastases this tumour did not behave in an aggressive fashion. PMID- 1724922 TI - Acecainide pharmacokinetics in normal subjects of known acetylator phenotype. AB - The purpose of this study was to determine the pharmacokinetics of acecainide (formerly N-acetylprocainamide) in six normal subjects of known acetylator phenotype. Three subjects were fast acetylators and three slow acetylators by sulfapyridine phenotyping criteria. Each subject received a 20-min, 3 mg kg-1 intravenous acecainide infusion. Concentrations of acecainide, procainamide, and their deethylated metabolites were measured in serum and urine samples using HPLC. Acecainide renal clearance, nonrenal clearance, steady-state volume of distribution, and other pharmacokinetic parameters were estimated using standard approaches. Acecainide renal clearance and steady-state volume of distribution were (mean +/- SD) 13.6 +/- 1.581 h-1 and 135 +/- 20.31, respectively, and were not significantly different in fast and slow acetylators. Acecainide nonrenal clearance in the six subjects was 3.0 +/- 1.01 h-1; however, nonrenal clearance in slow acetylators was 1.8 times that in fast acetylators (3.9 vs 2.21 h-1, p = 0.012) with clear separation of the subjects into two groups when the data were grouped by acetylator phenotype. The nonrenal clearance of acecainide was inversely correlated with percentage sulfapyridine acetylation. Computer simulations were conducted to explore possible explanations for the observed difference in nonrenal clearance. PMID- 1724923 TI - Experimental skin calciphylaxis induced by iron citrate sorbitol in young dogs. AB - An experimental model of skin calciphylaxis using iron citrate sorbitol is presented. There were used for the experiment 12 young dogs sensitized with D3 vitamin and then injected with 0.2 ml Jectofer on the internal face of the shank. Macroscopic lesions become evident after 2-3 days last drug was administered. Nodular calcifications occurred after 7-8 days as white, hard and irregular lesions, when sectioned, presenting dissociable crystals. From the microscopic point of view fatty cysts, dermic granuloma and an amorphous irregular material are described. In the early period Perls stain is positive and becomes negative after 3 or 4 days. Von Kossa reaction is positive after 4 or 5 days and alizarin S after 7. Degenerative lesions of elastic fascicles are noticed and discussed in relationship with localization of calcium salts. On the basis of these data the possible succesion of skin calciphylaxis steps is discussed, but many things remain unknown. PMID- 1724925 TI - FK-506 (fujimycin) reverses the multidrug resistance of tumor cells in vitro. AB - Cyclosporin A (CsA) and FK-506 have similar immunosuppressive activity profiles and cyclophilin-like intracellular targets. Since CsA can reverse the multidrug resistance of tumor cells showing P-glycoprotein-mediated drug efflux, the possible resistance-modulating activity of FK-506 was evaluated in vitro with multidrug-resistant P388 cells and their sensitive parental controls. Higher concentrations of FK-506 than CsA were needed to achieve a similar degree of chemosensitization, suggesting that FK-506 might interact less efficiently than CsA with the P-glycoprotein expressed in multidrug-resistant tumor cells. However, FK-506 was active on a broader range of concentrations than CsA, particularly because of direct cytostatic effects of CsA which appeared at concentrations only slightly higher than those required to show a significant resistance-modulating activity. PMID- 1724924 TI - Antiviral therapeutics in dentistry. AB - Rapid progress is being made in the development of antiviral therapy. This review restricts its focus to antivirals that may be used by dental practitioners, principally those agents active in herpesvirus infections. PMID- 1724926 TI - Sequence-informative fragmentation of peptides up to a molecular weight of 4.6 kDa in plasma-desorption mass spectrometry. AB - The potential of plasma-desorption mass spectrometry (PDMS) in peptide sequencing is investigated. This paper shows that PDMS spectra recorded for longer times and using larger amounts of peptide than used for obtaining molecular weight information, provides sequence information on peptides of up to 4.5 kDa molecular weight. This approach strongly enhances the utility of PDMS for validation of the sequence of recombinant peptides. PMID- 1724927 TI - BHC as a xenobiotic, showing histopathological changes in the developing ovaries and variability of the behaviour of Poekilocerus pictus. AB - The leaf-hopper P. pictus is a well-known pest of Calotropis plants. Adult females and males were treated with a sublethal BHC concentration to control fecundity and viability. Marked histological changes occurred in the follicular epithelium of both pathological and immature oocytes. Vitellogenesis was completely arrested and the PYP bodies present prior to treatment disintegrated and were resorbed by the pycnotic follicular epithelium. Fibrogenesis and thickening of the tunica propria gradually decreased. The sublethal BHC concentration injected into the haemolymph severely damaged the fat bodies and thus adversely affected the developing ovaries. When adults were treated and kept in separate pairs, mating was prolonged; it was followed by ovulation, but the number of eggs was always smaller than in the control females and they did not develop any further, although provided in the laboratory with all normal conditions. PMID- 1724928 TI - The nuclear matrix: defining structural and functional roles. PMID- 1724929 TI - Infantile cortical hyperostosis (Caffey disease): ultrastructural and immunohistochemical characterization of the peritrabecular cells. AB - The ultrastructure and the immunohistochemical pattern of the cells which are responsible for the bone resorption in the cortical infantile hyperostosis were investigated. The osteoclasts present a great positivity to MB1 antigen and a low positivity to OKM5. Mononuclear cells with primary lysosomes, looking like osteoclast ones are present in high concentration in peritrabecular spaces. These cells show a high positivity to OKM5 antigen and a low positivity to MB1 antigen. The mononuclear granulated cells are positive to tartrate-resistent acid phosphatase. The possible common origin and their co-operation in bone resorption is discussed. PMID- 1724930 TI - Studies with the Golgi method in central gangliogliomas and dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos disease). AB - The rapid Golgi method, combined with current optical and electronmicroscopical techniques, was used in three central gangliogliomas and in one dysplastic gangliocytoma of the cerebellum to study the morphology of ganglionic cells. Gangliogliomas were composed of bipolar, fusiform and radiate cells with dense core and clear vesicles in the perikaryon and cellular processes, the number of each cellular type varying from one case to another. These features, together with the fact that isodendritic neurons are considered to be phylogenetically old neurons, suggest that these tumours are composed of "primitive" neurons that are not homogeneous with regard to their morphology. In contrast, ganglionic cells in dysplastic gangliocytoma are huge cells with long, stereotyped neurites that establish unique asymmetric contacts with neighbouring perikarya and neurites by means of claw-shaped processes covered with synaptic buttons. These morphological characteristics are different from those of any other neuron of the CNS. PMID- 1724931 TI - Changes in the number and distribution of Langerhans cells in the hydantoin hyperplasic gingiva as compared with the clinically normal one. AB - A study was made of the number of Langerhan's cells (LCs) per mm2 of section which express the antigens T6 and/or HLA-DR in seriated gingival sections of diphenylhydantoine-induced hyperplasia (HG) and clinically normal gingivae (NG). NG showed histological correlation with its macroscopic appearance. In HG the classical histopathological findings were verified, as well as the epithelial maturation irregularities, conductive to the development of epithelial gaps. In the immunostained samples, LCs appear amply distributed in the epithelium in greater numbers than in NG and more branched except in the immature areas, where they mostly express HLA-DR. In HG keratinocytes, HLA-DR+ are observed in the basal layer, except in developing epithelial gap zones. The Wilcoxon test for the NG-T6/NG-DR and HG-T6/HG-DR was not significant; but the Mann Whitney test for NG T6/HG-T6 and NG-DR/HG-DR was significant to p less than 0.05. It is understood that the increase in LC numbers in HG is a manifestation of their active participation in local immune reactions. The presence of DR+/T6- LCs in the less keratinized areas seems to indicate the relationship of LCs with epithelial proliferation and/or differentiation. PMID- 1724932 TI - Heredopathia atactica polyneuritiformis (Refsum's disease). AB - A female patient started to develop deafness and vertigo at the age of 29. In the following years she became atactic and retinitis pigmentosa was discovered. The diagnosis of Refsum's disease was reached on the grounds of the high concentration of phytanic acid in plasma. The patient died 23 years after onset of the first symptoms. Liver, spleen and kidney showed lipofuscinosis and pigment laden macrophages. The retina was atrophic and its pigment discontinuous. The meninges contained lipid-laden macrophages. The nerve cells in brain and spinal cord as well as the astrocytes and perivascular macrophages stored substances weakly PAS-positive and sudanophilic. The nerve cells accumulated lysosomes and residual bodies. In the astrocytes, the residual bodies were extremely polymorphous and contained inclusions with bilamellar ribbon-like structures. In the oligodendroglia the residual bodies displayed high electron density and finger print-like pattern. Peroxisomes were found in glial cells and microperoximes in neurons. The ultrastructural findings in the present case demonstrate that in terminal stages phytanic acid can reach the brain parenchyma passing through the BBB. Further autopsy studies will be necessary to determine whether these changes are consistent findings in Refsum's disease. PMID- 1724933 TI - Electron microscopic histochemical and immunochemical analyses of heparan sulfate proteoglycan distribution in renal glomerular basement membranes. AB - Renal glomerular basement membranes (GBMs) exhibit a charge-selective barrier, comprised of anionic sites, that restrict the passage of anionic molecules into the urine. These sites are located primarily in the laminae rarae interna (LRI) and externa (LRE) of the GBM and consist of heparan sulfate proteoglycan (HSPG). Previous efforts to localize HSPG core protein within various layers of the GBM have been contradictory. In the present study when rat renal cortex blocks were treated by immersion with the cationic probe, polyethyleneimine (PEI), GBMs exhibited anionic sites concentrated primarily in the LRE and more irregularly within the LRI and lamina densa. All sites were heparitinase sensitive indicating that PEI positive sites represent negatively charged groups associated with heparan sulfate. In order to gain information on the distribution of the HSPG protein core, antibodies to HSPG from the EHS tumor matrix [anti-(EHS) HSPG] and GBMs [anti-(GBM) HSPG] were used together with immunogold to label thin sections of Lowicryl embedded kidney cortex. Depending upon the antisera used, markedly different distributions of HSPG were obtained. Immunolabelling with anti-(GBM) HSPG suggested a distribution of HSPG which was restricted to the laminae rarae, whereas labelling with anti-(EHS) HSPG indicated that the protein core penetrates through all layers of the GBM. PMID- 1724934 TI - Immunocytochemistry of epithelial markers in citral-induced prostate hyperplasia in rats. AB - Immunocytochemical characterization of several epithelial markers using the PAP technique was analyzed during different stages of induced prostatic hyperplasia in rats. Intact adolescent rats (42 days old) were treated with citral (3,7 dimethyl-2,6 octadienal) for 10, 30 and 100 days and their ventral prostate compared to untreated, matched-age animals. Among the epithelial markers studied the prostatic specific acid phosphatase was present in hyperplastic prostates of rats. The immunoreaction showed a fair correlation with the severity of lesion and duration of treatment. The prostatic specific antigen showed equally immunoreactive in both control and treated rats. The hyperplastic and normal rat prostates did not show immunoreactivity towards the other epithelial cell markers such as epithelial membrane antigen, carcinoembrionic antingen and alpha fetoprotein antisera. It is concluded that prostatic specific acid phosphatase, and to a lesser extent prostatic specific antigen, might represent valuable markers for comparative studies of prostatic hyperplasia in rodents. PMID- 1724935 TI - Immunohistochemical demonstration of neuronal and astrocytic markers and oncofoetal antigens in retinoblastomas. AB - General opinion is that retinoblastomas, though not everyone agrees with that view. Some authors suggest that retinoblastomas are derived from a primitive retinal cell able to differentiate into both neuronal and glial cell lines. The aim of the present work was to study immunohistochemically the expression of neuronal and astrocytic markers in retinoblastomas and at the same time the presence of the oncofoetal antigens carcinoembryonic antigen (CEA) and alpha Foeto Protein (AFP), since patients with retinoblastomas often show high oncofoetal antigen in serum levels. For this purpose we employed the streptavidin biotin immunoperoxidase technique in 13 cases of retinoblastoma to evaluate the presence and distribution of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP), S-100 protein, CEA and AFP. All 13 tumours studied stained for NSE. Seven of them showed GFAP- and S-100 positive perivascular glial cells as well as cells distributed randomly in the tumour that were interpreted as non tumour cells. All 13 retinoblastomas lacked detectable NF, CEA, and AFP. These results support the idea that retinoblastomas are neuronal tumours, although retinal glial cells may become incorporated in the tumour and proliferate in response to the tumour. PMID- 1724936 TI - Degeneration and ossification of the yellow ligament in unstable spine. AB - Lumbar yellow ligaments were obtained from 20 cases of lumbar spinal canal stenosis and 20 cases of degenerative olisthesis with slippage of more than 3 mm. The ligaments were stained with safranin-O, von Kossa, and immunohistochemical staining methods (S-100 protein). In the unstable group the safranin-O staining was more intense, microscopic ossification and chondroblasts were noted more frequently, and S-100 protein was more abundant. These findings show that instability of the lumbar spine accelerates degeneration and chondrometaplasia of the yellow ligament, which may lead to the enchondral ossification of the ligament. PMID- 1724937 TI - Speeding up the dynamic algorithm for planar RNA folding. AB - The simplest dynamic algorithm for planar RNA folding searches for the maximum number of base pairs. The algorithm uses O(n3) steps. The more general case, where different weights (energies) are assigned to stacked base pairs and to the various types of single-stranded region topologies, requires a considerably longer computation time because of the partial backtracking involved. Limiting the loop size reduces the running time back to O(n3). Reduction in the number of steps in the calculations of the various RNA topologies has recently been suggested, thereby improving the time behavior. Here we show how a "jumping" procedure can be used to speed up the computation, not only for the maximal number of base pairs algorithm, but for the minimal energy algorithm as well. PMID- 1724938 TI - Graphs, random sums, and sojourn time distributions, with application to ion channel modeling. AB - This paper considers the distribution of a sojourn time in a class of states of a stochastic process having finite discrete state space where sojourn times in any individual state are independent and identically distributed, and transitions between states follow a Markov chain. The state space and possible transitions of the process are represented by a graph. Class sojourn time distributions are derived by modifying this graph using 'composition' of states, defining a new Markov chain on the modified graph, and expressing the sojourn time in a composition state as a random sum. Appropriate compositions are chosen according to the possible "cores" of sojourns in the particular class, where a core describes the structure of a sojourn in terms of a single state or a chain in the original graph. Graph methods provide an algorithmic basis for the derivation, which can be simplified by using symmetry results. Models of ion-channel kinetics are used throughout for illustration; class sojourn time distributions are important in such models because individual states are often indistinguishable experimentally. Markov processes are the special case where sojourn times in individual states are exponentially distributed. In this case kinetic parameter estimation based on the observed class sojourn time distribution is briefly discussed; explicit estimating equations applicable to sequential models of nicotinic receptor kinetics are given. PMID- 1724939 TI - Stationary-state distributions in membrane channels. I. Conservative flux systems. AB - The population distribution of cations at binding sites in membrane channels is determined for stationary states characterized by a net ion flux. For conservative systems, the net flux conserves the total intra-channel cation population for an ensemble of channels. The requisite matrix formalism is developed and illustrated for some homogeneous channels with N single-ion occupancy states with chemical and electrochemical transmembrane gradients. The lowest eigen-value approximation, which is used effectively for systems with no net cation flux, is applied to these stationary-state systems for comparison with the exact solutions. The stationary states are characterized by special thermodynamic functions for entropy, internal energy, and free energy defined by using information theory. The changes in these parameters for the transition from the equilibrium to the stationary state depend on the ratio of the next flux to the transition velocity for nearest-neighbor transitions within the channel. The free energy changes with the square of this ratio while the internal energy changes linearly. PMID- 1724940 TI - Comparison of effects of long-chain and medium-chain triglyceride emulsions during hepatic regeneration in rats. AB - We examined the effect of long-chain triglyceride (LCT) and medium-chain triglyceride (MCT) emulsions on hepatic regeneration. After approximately 70% hepatectomy, Sprague-Dawley rats were maintained for 96 hours on total parenteral nutrition (TPN) (250 kcal/kg per day; nonprotein calories-nitrogen 160:1) with LCT or MCT as 30% of nonprotein calories. There were no significant differences in the body weight, cumulative nitrogen balance, urinary 3-methylhistidine excretion, or changes in the energy stores between the two groups; but the fatty acid composition of the phospholipid fraction of the regenerating liver differed significantly between the LCT and the MCT groups. The extent of hepatic regeneration by weight was 88.7 +/- 10.5% in the MCT group and 99.1 +/- 10.6% in the LCT group by 96 hours after hepatectomy. Furthermore, the incorporation of 3H orotic acid into DNA and RNA of regenerating liver cells in the LCT group was higher than in the MCT group 24 hours after hepatectomy. These observations indicate that essential fatty acids--components of the cell membrane and precursors as functional mediators--are very important to hepatic regeneration. PMID- 1724941 TI - Improvement in immune function in ICU patients by enteral nutrition supplemented with arginine, RNA, and menhaden oil is independent of nitrogen balance. AB - Hypermetabolism and multiple organ failure syndrome (MOFS) after trauma, surgery, or sepsis is associated with accelerated catabolism, the rapid onset of malnutrition, and immune system failure. Current nutritional support, enteral or parenteral, can achieve an acceptable nutritional response but appears unable to improve immune function. Nutrients such as arginine, refined menhaden oil, and RNA have been found to have immune-stimulating properties. This randomized blind prospective trial compared two nutritionally complete enteral formulas, one supplemented with arginine, menhaden oil, and RNA, on the disease-specific effects of anergy and suppression of in vitro tests of immune function in intensive-care patients and the nutritional outcome of nitrogen balance. After 7 10 days of enteral nutrition in patients with persistent sepsis syndrome, both formulas were associated with the achievement of net nitrogen retention and improved visceral protein status but with nonresolution of anergy. However, the supplemented formula was associated with marked stimulation of in vitro lymphocyte proliferative responses and a significant reduction in 3 methylhistidine excretion. Six and 12-mo follow-up data demonstrated no long-term effects. Nutrients targeted to effect the disease-induced in vitro suppression of immune function in MOFS appear to achieve that end independent of the nutritional outcome of nitrogen balance and without adverse clinical outcome. PMID- 1724942 TI - Independent effects of protein and energy deficiency on acute-phase protein response in rats. AB - The effect of restricting either protein intake alone or all dietary constituents on the acute-phase protein response was assessed during a 3-wk period after a standardized "injury" (i.e., subcutaneous injection of turpentine 0.5 ml/kg body wt) to rats. Sequential measurements were made of the circulating concentrations of alpha 2-macroglobulin (alpha 2-M), albumin, and total protein. Results were compared with those obtained from normally fed rats that were also injected with turpentine and with saline-injected rats (controls) receiving each dietary regimen. Turpentine-treated rats showed a decrease in albumin concentration by approximately 10 g/L within 2-4 days. In the protein-deficient rats, recovery to preinjection levels had not occurred by 3 wk, but in the other groups, it was complete by 7-9 days. The protein-deficient animals also showed an attenuated response in alpha 2-M and total protein and delay in their recovery toward normal. Maximal alpha 2-M and total-protein responses in this group of animals was only 44 and 39%, respectively, of those found in the control group. We conclude that protein deficiency attenuates the magnitude and alters the time profile of the positive (alpha 2-M and total protein) and negative (albumin) acute-phase protein responses to injury, independent of the severity of the stimulus. PMID- 1724943 TI - GnRH complementary peptide antibodies: outcome in GnRH receptor immunoanalysis. AB - The aim of this study was to obtain gonadoptropin-releasing hormone (GnRH) receptor antibodies of high affinity for receptor immunoanalysis. According to the complementary peptide theory, complementary nucleic acid segments encode for the hormone ligand and the receptor binding site, respectively. On this premise, we used as immunogen, GnRH complementary peptide [N-terminal]Ser-Arg-Ala-Gln-Ser Ile-Gly-Pro-Val-Leu conjugated with a carrier protein. High antibody titers were obtained in rabbits, rats and mice. Our antisera recognized the hydrophobic middle part of the GnRH complementary peptide. A band of protein with a molecular weight similar to that of the GnRH receptor (60 kDa) was specifically detected by immunoblot of solubilized rat pituitary membranes with the highest titering rabbit antiserum. In bioassays on sheep pituitary cells in vitro, some antisera inhibit basal or GnRH-induced LH secretion. In order to elicit antibodies of high affinity, we used a selective receptor assay on rat brain and pituitary sections where the ligand was the labeled agonist Des-Gly10-D-Ala6 GnRH. None of the highest titering antisera prevented the binding of such a high affinity ligand. The complementary peptide approach thus appears not to be optimal for obtaining high affinity antibodies against the GnRH receptor binding site. PMID- 1724944 TI - Identification of phosphotyrosine residues during protein sequence analysis. AB - Synthetic tyrosine-phosphorylated peptides were subjected to protein sequence analysis using a gas-phase sequencer and on-line phenylthiohydantoin (PTH) amino acid analysis. Our data show that phosphotyrosine is stable to the gas-phase sequencing chemistry and can be detected as its PTH-derivative during routine sequence analysis without the need of prior tyrosine radiolabeling. PMID- 1724945 TI - Epitopic characterization and vaccinal potential of peptides derived from a major antigen of Schistosoma mansoni (Sm28 GST). AB - The P28 antigen of Schistosoma mansoni has been shown to induce protective immunity against schistomiasis in rodents and primates. The analysis of the primary structure of the P28 molecule using various predictive algorithms led to the synthesis of seven peptides which were used to localize the major T cell epitopes of the P28. Two of these synthetic peptides (amino acids 24-43 and 115 131) were found to contain major T cell sites of the P28 antigen. Indeed, both 24 43 and 115-131 peptides stimulate T lymphocytes from Fischer rats and Balb/c mice immunized with the recombinant P28. Moreover, these localized moieties are exposed to the host's immune system during natural S. mansoni infection since they activate T cell populations of infected rats, mice and chronically infected Kenyan children. A multiple antigen peptide containing eight copies of peptide (MAP 115-131) has been prepared and used as immunogen in rats, mice and baboons. In all these models, the MAP 115-131 induces both humoral and cellular responses towards the P28. Preimmunization with the MAP 115-131 before a challenge with the whole P28 molecule increases T cell proliferation and antibody production. The active immunization of rats with the MAP 115-131 before a challenge with the whole P28 molecule increases the T cell proliferation and the antibody production. The active immunization of rats with the MAP 115-131 before challenge infection with the parasite achieved significant protection. PMID- 1724946 TI - Novel markers for the detection of platelet activation. PMID- 1724947 TI - The molecular biology of platelet membrane glycoproteins. PMID- 1724948 TI - The effects of neonatal capsaicin administration on trigeminal nerve chemoreceptors in the rat nasal cavity. AB - Trigeminal nerve fibers in the nasal cavity respond to a variety of volatile chemical stimuli. Some of these trigeminal nerve fibers have been suggested to be capsaicin-sensitive and thus belong to a class of pain receptor rather than constituting a separate class of chemoreceptor. Our current results confirm this suggestion. Trigeminal nerve responses to volatile chemical stimuli were eliminated in rats which were injected with capsaicin on the second day of life. Animals whose nerves were unresponsive to chemical stimuli also exhibited a loss of intraepithelial peptide-immunoreactive fibers in their nasal cavities. The results of this study suggest that trigeminal nerve fibers in the nasal cavity which respond to chemical stimuli may be polymodal nociceptors which contain substance P, calcitonin gene-related peptide, or perhaps other neuropeptides. PMID- 1724949 TI - Emergence of the Ly-6 superfamily of GPI-anchored molecules. PMID- 1724950 TI - Probing the signal for glycophosphatidylinositol anchor attachment using decay accelerating factor as a model system. PMID- 1724951 TI - [Two cases of cerebral embolism showing global aphasia without hemiparesis]. AB - We report two cases of typical global aphasia without hemiparesis due to cerebral embolism. Case 1 was a 65-year-old right-handed man with a history of old myocardial infarction. No spontaneous speech was noted by his family. Neurological examination upon admission revealed confusional state, global aphasia, conjugate deviation to the left and slight drift of the outstretched right limbs. The right hemiparesis rapidly recovered after admission. CT scan performed on the second hospital day showed discrete low density areas in the left posterior frontal lobe and left temporo-parietal regions. The extent and severity of his global aphasia were unchanged. The second case was an 82-year-old right-handed man with a history of atrial fibrillation. He was admitted to our hospital one hour after he was found unable to speak. Neurological examination upon admission revealed global aphasia, conjugate deviation to the left and suspected right homonymous hemianopia by confrontation. There was no sign of hemiparesis. CT scan showed extensive low density area in the left temporo parietal regions. In both cases, cerebral angiography failed to demonstrate any occlusion of intra- and extra-cranial blood vessels. IMP-SPECT showed a depression of cerebral blood flow in the left anterior and posterior watershed areas in case 1 and 2. In the literature, there have been 20 cases of global aphasia without hemiparesis including our two cases. In many cases, the initial symptom was inability or difficulty in speaking.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724952 TI - [Immunogenetics of rheumatoid arthritis]. PMID- 1724953 TI - Laboratory diagnosis of amebiasis. AB - The procedure used to diagnose amebiasis varies depending on the geographic location of the laboratory. However, in general, the microscopic diagnosis and differentiation of E. histolytica from other intestinal amebae is most satisfactorily achieved when the specimen is preserved immediately and both a concentration and stained smear are examined. The number of additional positive findings achieved by the cultivation of fecal material does not justify the time and cost involved. On the other hand, serology is a useful adjunct to diagnosis, especially in patients with extraintestinal amebiasis. The commercial development of antigen detection kits and DNA probes may provide more rapid, accurate, and less costly diagnostic procedures for the future. New guidelines need to be formulated regarding the number of specimens to be submitted, and the cost effectiveness of various diagnostic procedures should be evaluated. PMID- 1724954 TI - Inhibition of terminal complement complex formation and cell lysis by monoclonal antibodies. AB - Three monoclonal antibodies (mabs), two against C5 and one against C6, were identified and characterized. They inhibited the generation of the terminal complement complex (TCC) in serum to over 90% as assayed by a sensitive ELISA based on a neoepitope-specific mab, which recognized TCC-integrated C9. The haemolytic function of the TCC was markedly reduced by all three mabs implying that they are directed to epitopes on C5 and C6 which are essential for TCC formation in both the fluid phase and on erythrocyte membranes. Since the generation of C5a was also impaired by these mabs, they may serve as tools in investigations of the sequelae of the generation of C5a and of TCC. PMID- 1724955 TI - Evidence for keratin proteins in normal and abnormal human meibomian fluids. AB - Hyperkeratinization of meibomian glands has been postulated to cause gland dysfunction. Recent investigations on rabbits show that keratin proteins are indeed present in the meibomian fluids of these animals. In this report we present our findings on the presence of these water-insoluble proteins in human meibomian secretions. 6 anti-cytokeratin antibodies, CK8, 18, 19, CK7, CK8, CK14, CK19 and AE1/AE3 were used against the keratin proteins expressed from the human meibomian fluids. Using the immunoblotting (dot blot) technique, abnormal waxy meibomian fluids obtained from subjects diagnosed to have meibomian gland dysfunction (MGD) were compared to normal clear meibomian fluids. The results show that keratins are present in a higher concentration (10%) in the abnormal human meibomian excreta as compared to the normals. Even though the presence of protein markers for keratinization in the abnormal meibomian excreta were not shown, the increased presence of keratin proteins in the abnormal meibomian fluids suggests that, in MGD patients, hyperkeratinization of ductal epithelium may have taken place. More keratin proteins (possibly those of higher molecular weights) were produced in addition to the keratin proteins normally produced by the duct epithelium. The increased amount of keratin proteins in the abnormal meibomian fluids may be explained by the susceptibility of duct epithelium to undergo the process of hyperkeratinization as postulated by other researchers. PMID- 1724956 TI - Polarized distribution of coated pits and coated vesicles in the rat lens: an electron microscopy and WGA-HRP tracer study. AB - The presence and distribution of coated pits (CPs) and coated vesicles (CVs) in the rat lens were studied by thin-section electron microscopy (TEM) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) as a tracer. TEM revealed that CPs and CVs were approximately 150 nm in diameter, of which the characteristic clathrin coat was approximately 20 nm thick. CPs and CVs were found in both epithelium and superficial fiber cells of the entire lens, and were distributed preferentially along the basal membrane facing the lens capsule. It was estimated that more than 80% of CPs and CVs in the entire epithelium were seen along the basal membrane. The number of CPs and CVs along the basal membrane in the equatorial epithelium (4.4 per 10 microns membrane) was similar to that at the central zone (3.8 per 10 microns membrane), but there was a significant increase along the apical and lateral surfaces of the equatorial epithelium compared to that of the central epithelium, although the overall number was considerably smaller. In the lens fibers, CPs and CVs were usually found within 2-3 superficial layers of fiber cells. The number of CPs and CVs along the basal membrane of young fibers at the post-equatorial region (3.1 per 10 microns membrane) was 3-fold greater than that of the mature fibers at the posterior polar area (1 per 10 microns membrane). Thus, CPs and CVs along the entire basal membrane showed a gradual decrease in number from the anterior (and equatorial) regions to the posterior polar surface of the lens. WGA-HRP experiments showed that approximately 80% of tracer-carrying pits and vesicles were also found along the basal surface of the equatorial epithelium. This study suggests that a polarized distribution of CPs and CVs along the basal surface of epithelium and superficial fiber cells may facilitate receptor-mediated endocytosis of important macromolecules directly from the aqueous humor and vitreous body into metabolically active lens cells. PMID- 1724957 TI - T cell recognition of superantigens. PMID- 1724958 TI - T cell activation by superantigens--dependence on MHC class II molecules. PMID- 1724959 TI - Sex differences in the pharmacokinetics of recombinant human granulocyte colony stimulating factor in the rat. AB - The pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were studied in male and female rats. The serum concentration of rhG CSF after iv and sc administration to male and female Sprague-Dawley rats at a dose of 5 and 100 micrograms/kg was investigated by a sandwich enzyme-linked immunosorbent assay. After iv administration, AUC and half-lives of rhG-CSF in female rats were smaller than those for male rats. The volume of distribution of rhG-CSF in female rats was not significantly different from that in male rats. After sc administration, AUC, mean residence time, and half-lives of elimination phase in female rats were smaller than those for male rats. The in vitro biological activities of rhG-CSF were investigated using [3H]thymidine uptake assay in cultures of bone marrow cells obtained from male and female rat femur. Female rat bone marrow cells showed a similar dose-response profile to rhG-CSF to that of male rat bone marrow cells. The effect of rhG-CSF administration in rats was a specific activity on the neutrophil lineage with an increase of neutrophils in peripheral blood. The in vivo effects of rhG-CSF after iv and sc administration to male and female rats at 5 and 100 micrograms/kg doses were determined. After 100 micrograms/kg administration, the neutrophil count in female rats was similar to that in male rats in the early period; however, the neutrophil count in female rats was lower than that in male rats 24 hr after administration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724960 TI - Regulation of protein secretion by crinophagy in perfused rat liver. AB - We examined the secretion of three serum proteins, albumin (RSA), alpha 2 mu globulin (alpha 2 mu G), and transferrin (Trf), in the isolated perfused liver. Within 4 h of perfusion, only 20 to 35% of previously synthesized proteins were secreted by the liver into the recirculating medium. Low temperature inhibited the secretion of alpha 2 mu G and Trf, but not RSA. The amount of RSA secreted by the liver increased twofold in the presence of leupeptin, a proteinase inhibitor, or primaquine, a weak base capable of neutralizing acidic compartments. Neither drug affected Trf secretion, while the release of alpha 2 mu G was enhanced threefold by primaquine treatment. Only 55 to 70% of the total amount of these serum proteins present in the liver at the onset of perfusion could be accounted for after 4 h of perfusion. Our evidence suggests that these losses are due to protein degradation. The degradation of RSA and alpha 2 mu G was inhibited at 15 degrees C and by both leupeptin and primaquine. Contrary, RSA degradation was not altered when livers were perfused at 20 degrees C. Morphological techniques combined with immunological probes were utilized to identify possible intracellular sites of RSA degradation. RSA and cathepsin L were colocalized to large vacuoles found near the cell periphery. Entry of RSA into these vacuoles occurred at 20 degrees C but not at 15 degrees C. Our results using perfused rat livers suggest that as much as 40% of hepatic serum proteins are degraded via fusion of secretory vesicles with lysosomes (e.g., crinophagy). PMID- 1724961 TI - Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope. AB - We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA. PMID- 1724962 TI - Immunolocalization of an extracellular domain of connexin43 in rat heart gap junctions. AB - Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs. PMID- 1724963 TI - Immunocytochemical localization of the Golgi apparatus using protein-specific antibodies to galactosyltransferase. AB - Polyclonal antisera to human milk galactosyltransferase have been widely used as immunocytochemical reagent to visualize the Golgi apparatus. Immunochemical analysis revealed partial specificity of these antisera to glycan epitopes expressed on galactosyltransferase and other gene products (R. A. Childs et al., Biochem. J. 238, 605-611 (1986)). Since glycan-specific reagents such as lectins are known to label the Golgi apparatus by virtue of their exclusive glycan specificity, Golgi localization of galactosyltransferase by use of these polyclonal antisera remained elusive. In order to demonstrate authentic Golgi localization of the galactosyltransferase peptide, we expressed the enzyme in Escherichia coli as non-glycosylated beta-galactosidase-fusion proteins and used them as affinity matrix to protein-epitope purified polyclonal antibodies from antisera to the milk enzyme, and to induce protein-specific antisera by injecting them into rabbits, respectively. A short fusion protein comprising most of the substrate binding sites and a protein which included the stem region of galactosyltransferase were expressed. The short fusion protein was poorly immunogenic whereas the long fusion protein induced a high-titer antiserum. Protein-epitope purified antibodies derived from polyclonal antisera to the milk enzyme as well as antibodies to the long fusion protein cross-reacted with milk galactosyltransferase and with fusion protein as demonstrated by immunoblotting and enzyme-linked immunosorbent assay (ELISA) and produced typical Golgi morphologies in both HeLa and CaCo2 cells when used for immunocytochemical localization of galactosyltransferase. We conclude that previous immunolocalization studies have correctly been interpreted as Golgi localization of the galactosyltransferase protein and that antigenicity to the protein moiety is confirmed to the N-terminal third of the enzyme. PMID- 1724964 TI - Functional and morphological changes of the exocrine pancreas in ciclosporin treated rats. AB - We have examined whether or not ciclosporin A (CsA) affects structure and function of the exocrine pancreas. Male Wistar rats were treated once a day for 7 days with intraperitoneal injections of vehicle or 5, 15, 30, or 50 mg/kg of CsA. Following the CsA treatment, incorporations of [3H]thymidine into DNA and [3H]orotic acid into RNA were found to be significantly reduced in a dose dependent manner up to 30 and 15 mg/kg, respectively, at which doses the incorporations reached a plateau. The incorporation of [3H]-L-leucine into protein also decreased by 36% at the dose of 5 and by 53% at the dose of 50 mg/kg of CsA. The activities of amylase and lipase in the pancreas also decreased in the CsA-treated rats. Furthermore, a significant reduction in the specific binding of [3H]N-methylscopolamine to muscarinic receptor was observed following the administration of CsA. The Scatchard analysis for the [3H]N-methylscopolamine binding to the pancreatic membrane revealed a significant decrease in Bmax, but not in Kd values in the CsA-treated animals. In addition, it was found histologically that administration of CsA induced cellular atrophy, cytoplasmic vacuolization, and cellular necrosis in the exocrine pancreas. These results strongly suggest that prolonged treatment with CsA may induce the suppression of pancreatic exocrine functions by decreasing the contents of amylase and lipase as well as the number of muscarinic receptors, possibly by the inhibition of the syntheses of protein and DNA in the exocrine pancreas. PMID- 1724965 TI - A comparison of endothelium-derived relaxing factor activity in the coronary and renal arteries of the pig. AB - A cascade bioassay system has been used to quantify the basal and receptor mediated endothelium-derived relaxing factor (EDRF) activity of pig coronary artery and pig renal artery. When exposed to EDRF released from a common donor vessel, pig coronary artery smooth muscle showed a greater sensitivity to EDRF than pig renal artery, and these differences were paralleled by differential responses to sodium nitroprusside. When mounted as donors in the bioassay, and EDRF detected using a common recipient, pig coronary artery and renal artery endothelium showed similar basal EDRF release rates. EDRF release in response to substance P was greater from pig coronary artery donors, but EDRF release in response to bradykinin was greater from pig renal artery donors. The data indicate that differences in EDRF response and EDRF release occur in different vessels, and that certain EDRF-releasing agents may exert regional vasodilator effects. PMID- 1724966 TI - Segment-specific effects of the heat-stable enterotoxin of E. coli on electrolyte transport in the rat colon. AB - The heat-stable enterotoxin of E. coli (STa) induced an increase in short-circuit current (Isc) in the rat colon. The maximal increase in Isc was about three times larger in the proximal than the distal colon. The action of STa was mimicked by 8 Br-cyclic GMP. Unidirectional flux measurements revealed that STa decreased Na+ and Cl- absorption in the distal colon, while it decreased Na+ absorption and activated Cl- secretion in the proximal colon. In the distal, but not in the proximal colon, indomethacin inhibited the action of STa and of 8-Br-cyclic GMP. Inhibition by indomethacin could be overcome by addition of prostaglandin E2 or forskolin, but not by addition of a non-hydrolysable analogue of cyclic AMP, suggesting an action of STa on cyclic AMP hydrolysis. Amrinone and trequinsin, two inhibitors of cyclic GMP-inhibited phosphodiesterases, mimicked the action of STa on Isc and inhibited the response to a subsequent administration of the toxin indicating the modulation of a cyclic GMP-inhibited phosphodiesterase by STa in the distal colon. The results give evidence for different intracellular action sites of STa in the two parts of the rat colon. PMID- 1724967 TI - Spinal cord substance P mediates the inhibition of gastric acid secretion induced by electrical stimulation of the preoptic area. AB - A possible role of spinal substance P (SP) in the mediation of signals to inhibit gastric acid secretion by central activation of the sympatho-adrenomedullary system was examined in urethane-anesthetized rats. Intrathecal (i.t.) administration of SP (1-10 nmol) inhibited vagally induced acid output. I.t. administration of spantide, a SP receptor antagonist, reduced the inhibitory effect of 3 nmol SP. I.t. administration of spantide (0.1-1 nmol) blocked the inhibition of vagally induced gastric acid output evoked by electrical stimulation of the preoptic area. Atropine, hexamethonium, phentolamine, propranolol, DL-para-chlorophenylalanine (PCPA) and 5,7-dihydroxytryptamine (5,7 DHT) were without effect. Repeated i.t. administration of 10 nmol SP produced desensitization to the SP-induced inhibitory response on gastric acid output. In these animals, electrical stimulation of the preoptic area did not inhibit vagally induced gastric acid output. These results suggest that electrical stimulation of the preoptic area excites SP-containing neurons in the spinal cord, and a resultant sympatho-adrenomedullary system-mediated inhibition of gastric acid secretion occurs. PMID- 1724969 TI - [A physiological validation for the early diagnosis of hypogalactia in women]. AB - Colostrum cell composition observed during the first 24 hrs following delivery objectively reflects the degree of prelactation preparation of the breast. The state of neutrophilic leucocytes appeared to be the most informative in this respect. In normal lactation, maximal number of neutrophilic leucocytes was found during the first 24 hrs after delivery. The leucocyte inflow to the colostrum is highly increased in hypogalactia. Leucocytes aggregation which is the most characteristic for normal lactation did not occur in hypogalactia. The activity of alkaline phosphatase and myeloperoxidase of leucocytes is much higher in normal lactation and much lower in hypogalactia. The evaluation of alkaline phosphatase activity and sudanophilic lipids content shows no differences related to the lactation level. PMID- 1724968 TI - Concentration-related effects of extracellular application of ATP on the action potential and membrane currents of the guinea-pig vas deferens. AB - The ionic mechanisms underlying concentration-related alterations in the action potential configuration caused by ATP were studied using preparations of the guinea-pig vas deferens voltage-clamped by a double sucrose gap method. Under current-clamp conditions, ATP at concentration of 1.6 microM enhanced the rates of rise and of repolarisation of the action potential whereas at concentration of 1.6 mM it reduced both rates. Under voltage-clamp conditions, lower concentrations increased the maximum inward Ca current without altering kinetics or reversal potential. Higher concentrations reduced the maximum inward Ca current with slowing of rates of activations and inactivation, but also caused a negative shift in reversal potential without affecting conductance. These results suggest that a low ATP concentration activates the voltage-dependent Ca current channels and that the action of a high ATP concentration is related to the internal Ca ion concentration. PMID- 1724970 TI - Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers. AB - Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984). PMID- 1724971 TI - Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane. AB - Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes. PMID- 1724972 TI - [Newly available: Neupogen]. PMID- 1724973 TI - Solution synthesis of the glyco-hexapeptide sequence of the human oncofetal fibronectin defined by monoclonal antibody FDC-6. AB - The glyco-hexapeptide sequence H-Val-(GalNAc-alpha)Thr-His-Pro-Gly-Tyr-OH, was synthesized in solution by the segment condensation procedure and the stepwise procedure. A peracetylated, O-galactosaminyl threonine derivative was used for incorporating the glycosylated amino acid residue into the peptide chain. A consistent racemization occurred during the acylation of H-His-Pro-Gly-Tyr(Bzl) OBzl with Z-Val-[GalNAc(Ac)3-alpha]Thr-OH by the BOP-HOBt procedure and the D allothreonine containing glyco-hexapeptide was isolated in about 20% yield. Stepwise elongation of the C-terminal tetrapeptide with Fmoc-[GalNAc(Ac)3 alpha]Thr-OH and Z-Val-OH, in the presence of the same coupling reagents, yielded the L-threonine containing diastereoisomer without detectable racemization. A side product, the Nim-ethoxycarbonylated hexapeptide derivative, formed during the EEDQ-mediated condensation of Fmoc-[GalNAc(Ac)3-alpha]Thr-OH with the C terminal tetrapeptide, was isolated and characterized. Preliminary studies showed that the synthetic glycohexapeptide is a good competitive inhibitor of the binding of the FDC-6 monoclonal antibody to the oncofetal fibronectin, supporting the idea that it should represent the minimum essential structure required for the FDC-6 activity. PMID- 1724974 TI - Glycosylation of synthetic peptides breaks helices. Phosphorylation results in distorted structure. AB - Two proposed glycosylation sites are located within T cell epitopes of rabies virus glycoprotein, namely VVEDEGCTNLSGF (VF13; amino acids 29-41) and GKAYTIFNKTLM (GM12; amino acids 312-323). To explore the effects on peptide conformation due to post-translational modifications, we synthesized glycosylated and phosphorylated versions of the two peptides and compared their structures with the native peptide using CD and FT-IR spectroscopy. After the modifications, i.e., glycosylation on Asn with one or two N-acetyl-glucosamine or glucose residues or phosphorylation on Ser, the low to medium degree of helicity of the unmodified peptides disappears as indicated by CD measurements in water trifluoroethanol mixtures. Incorporation of one sugar moiety into either peptide resulted with a high probability in a type I (III) beta-turn formation with almost identical spectra for the different peptides. Elongation of the carbohydrate in GM12 only slightly enhanced this effect. In contrast, phosphorylation of VF13 caused distorted conformation of the peptide backbone. This novel and direct demonstration of a change in secondary structure by glycosylation (or phosphorylation) might be an important element in determining peptide antigen structure and function. PMID- 1724975 TI - Conformational prediction studies on pancreatic colipase. AB - Comparison of the primary structures of pancreatic colipases from man, pig, horse and rat shows a high degree of homology between proteins. Fifty-two out of the 95 residues of the polypeptide are identical. All colipases contain 10 half-cystines which are located at invariant positions. The secondary structure of colipases has been predicted from the sequence using the statistical method of Chou and Fasman and the method of Gibrat, Garnier and Robson based on information theory. Predictions indicate that colipases have a low content of alpha-helix and beta strand structure. The two segments at positions 7-10 and 56-59, assumed to be part of the lipid binding domain, have predicted beta-sheet conformation and should be in close spatial vicinity to each other in the proteins. Four beta turns are predicted in all colipases at positions 3-6, 46-49, 61-64, and 81-84. They might contribute, with the five disulfide bridges, to a tight packing of the protein molecule. Surface residues and major sequential antigenic determinants of mammalian colipases have been predicted using methods based either on hydrophilicity/hydropathy scales or amino acid mutability. From these studies, it appears that colipases exhibit large conformational homologies. In the absence of data on the tertiary structure of colipase, predictive methods, together with physico-chemical and immunological studies, provide valuable information on the conformation of the protein in relation to the topology of residues involved in the functional and antigenic sites. PMID- 1724976 TI - The Xmn I site (-158, C----T) 5' to the G gamma gene: correlation with the Senegalese haplotype and G gamma globin expression. AB - There are three major African haplotypes associated with the sickle mutation: Benin (#19), Senegalese (#3), and Central African Republic (#20). Previous studies have suggested that the Xmn I site (-158 bp 5' to the G gamma gene) is associated with elevated levels of G gamma and with the Senegalese haplotype, while other investigators questioned this association. In order to clarify the issue, we have determined beta haplotypes, tested for the presence of the Xmn I site, and measured Hb F and G gamma expression levels in 143 American Black patients with sickle cell anemia. Haplotypes were determined using eight polymorphic sites in the beta-like globin gene cluster: Hinc II 5' to epsilon, Hind III in IVS-II G gamma and A gamma, Hinc II within and 3' to psi beta, Ava II in IVS-II of beta, and Hpa I and Bam HI 3' to beta. The G gamma /A gamma ratio was analyzed by high performance liquid chromatography using a C18 column. The Xmn I site was present in all 31 chromosomes with the Sengalese haplotype. Of the remaining 255 chromosomes with other haplotypes, only 2 (0.8%) had the Xmn I site present. There was significant correlation between the presence of the Xmn I site and increased G gamma /A gamma ratio in a dose-dependent manner. The Hb F level was not significantly increased in the presence of the Xmn I site. The data indicate that the Xmn I site maintains a G gamma /A gamma ratio typical of fetal life but does not necessarily cause elevation of Hb F. The latter seems to depend on factors other than the Xmn I site. PMID- 1724977 TI - Alpha 1-adrenergic regulation of thyrotropin-stimulated release of 3, 5, 3' triiodothyronine and thyroxine from perifused mouse thyroid. AB - The effect of methoxamine, a specific alpha 1-adrenergic agonist, on the release of T3, T4 and cAMP from perifused mouse thyroid was studied to clarify the role of the alpha 1-adrenergic receptor in the regulation of thyroid hormone secretion. TSH-stimulated T3 and T4 release was inhibited significantly by methoxamine. With regard to cAMP release, methoxamine inhibited TSH-stimulated cAMP release in the presence of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone but did not inhibit TSH-stimulated cAMP release in the presence of 3-isobutyl-1 methylxanthine. Methoxamine did significantly suppress TSH-stimulated release of T3 and T4 in the presence of each phosphodiesterase inhibitor. Depletion of Ca2+ in the perifusion buffer abolished completely the inhibitory effect of methoxamine on TSH-stimulated T3 and T4 release. The present study suggests that activation of the alpha 1-adrenergic receptor inhibits TSH-stimulated T3 and T4 secretion through a Ca(2+)-dependent mechanism in the mouse thyroid gland. PMID- 1724978 TI - Fibre divergence in the distal optic radiation: possible basis of functional plasticity in adult primate visual cortex. AB - The precision of retinotopy in primate visual cortex is commonly thought to result from highly ordered arrangement of fibres in the visual pathways. However, rigid point-to-point representation is hardly compatible with findings of a substantial reorganization of visual cortical maps after peripheral and central lesions. Such observations could be accounted for by divergence in the optic radiation. To explore the hypothesis of fibre divergence, we made small knife cuts in the distal optic radiation of macaca fascicularis. After subsequent axonal tracing by injecting WGA-HRP into lateral geniculate nucleus, we studied the course of distal fibres in white matter. The amount of divergence was assessed by measuring, relative to the prevailing fibre course, length and orientation of labelled fibres between lesion and entry into cortex. Lesion sizes between 1 mm to 3 mm did not result in any detectable diminution of terminal labelling in layer IVC of striate cortex. Individual labelled fibres were found to diverge symmetrically from both sides into the gap distal to the lesion. Divergence starts at a distance of about 3 mm before cortex. At the white matter boundary, less than 10% of all fibres still retain the original direction, with the remaining fibres taking any other orientation without preference. We estimate that this corresponds to a divergence of visual afferents encompassing about 6-10 mm of cortical distance, if intracortical arborization of terminal fibres is taken into account. Possible consequences for functional plasticity in the adult primate visual cortex are discussed. PMID- 1724979 TI - Ontogeny of ribonucleic acid content in rat cortical neurons influenced by early stimulation. AB - Developmental changes in the total RNA content were studied in pyramidal cells of the frontal, parietal (somesthetic), temporal (auditory) and occipital (visual) cortical areas in Wistar rats aged 13, 14, 15, 21, 28, 35 and 56 days, and compared with those after complex, visual and acoustic stimulation. In all areas neuronal RNA content reached peak values at the age of 3 weeks, except the parietal cortex, where the values were highest as early as at 15 days (in 21-day pups they were only insignificantly lower). Between the age of 3 and 4 weeks, RNA content of cortical neurons decreased in all three projection areas, whereas in the frontal cortex a significant decline came later--in the 5th week. In the occipital cortex neuronal RNA at 8 weeks reached reliably lower values than in 13 day pups. Neuronal RNA increased at the time of eye-lid opening (13-15 days), highly significantly in the visual cortex. Complex visual and acoustic stimulation raised neuronal RNA content in projection areas of modality involved in stimulation and, moreover, acoustic stimulation did so in the frontal and occipital cortex. On the contrary, complex and visual stimulation decreased RNA contents in the frontal cortex. Developmental dependence, stimulation contingency, and specificity vs. nonspecificity of the observed changes are discussed, and compared with other biochemical, morphological and functional changes that have been found in the period of peaking RNA content, or immediately thereafter. PMID- 1724980 TI - Ruffed cells in the olfactory bulb of freshwater teleosts. II. A Golgi/EM study of the ruff. AB - The morphological characteristics of the synaptic contacts in the ruff of the cichlid fish Hemichromis bimaculatus were studies using the combined Golgi electron microscope technique. Ruffed cells were located in the glomerular and plexiform layers and exhibited a pyriform or round cell body and numerous thin dendritic branches that were highly ramified. Four different segments could be distinguished on the initial portion of the axon (IP) according to the number and density of protrusions. These protrusions or lateral appendages are highly interdigitated, forming a characteristic synaptic field: the ruff. The ruff displayed a very high number of synapses with terminals showing a varied morphology. Protrusions of the ruff were both presynaptic and postsynaptic, taking also part in reciprocal pairs of synapses. Synapses from the ruff to the adjacent prolongation are asymmetrical, the prolongation to protrusion synapses being symmetrical. The axonal shaft participates in fewer synaptic contacts. Boutons contacting with one protrusion can synapt with other one, and can also receive an asymmetric synapse from another terminal, forming a serial synapse. This constitutes the most complex synaptic system observed in the glomerular layer of the olfactory bulb in any vertebrate. The synaptology of the ruffed cell IP is compared with previous reports on other species, with the teleostean mitral cells and with the IP of higher vertebrates neurons, the ruffed cells showing a completely different synaptic pattern. PMID- 1724981 TI - Serum antibodies against peripheral nervous system antigens in leprosy. AB - Since antibodies against peripheral nervous system (PNS) antigens may play a pathogenetic role in the mechanism of nerve damage in leprosy, sera from leprosy patients and contacts were investigated for anti-PNS antibodies by ELISA and immunoblot. In ELISA, elevated anti-PNS antibody levels were detected in 4 of 98 (4.1%) leprosy patients (4 of 52, 7.7%, lepromatous leprosy patients), in 1 of 28 (3.6%) contacts, and in 1 of 18 (5.6%) normal controls. There was no correlation between anti-PNS antibody levels and the bacterial index or neuropathy in leprosy. Immunoblot with a sample of six leprosy and five control sera showed that the antigenic binding pattern (mainly within the 100-200-kDa region) was very similar in patients and controls. Staining intensity, however, appeared to be higher with the leprosy sera than with the control sera. IgM and IgG were found to contribute to the staining pattern: IgM in the 150-200-kDa range, IgG with multiple bands between 25 kDa and 200 kDa. Thus, the presence and levels of serum anti-PNS antibodies in leprosy appear to be unrelated to parameters of disease activity, neuropathy in particular, and do not seem to be critically involved in the pathogenesis of nerve damage. PMID- 1724982 TI - Low prevalence of hepatitis C virus and infrequent perinatal or spouse infections in pregnant women in Taiwan. AB - To assess the prevalence of hepatitis C virus (HCV) infection in the pregnant women in Taiwan, we investigated two groups of pregnant women, 944 women without serum alanine aminotransferase (ALT) screening (group A) and 197 women with abnormal ALT (greater than 45 IU/L) (group B). They were checked for anti-HCV (anti-C100-3) with HCV EIA kit (Abbott Lab., North Chicago, IL). The results showed that 21 (2.2%) in group A and 5 (2.5%) in group B were anti-HCV-positive. However, 15 out of 21 in group A had an optical density (O.D.) of anti-HCV less than 1.0, were negative by recombinant immunoblot assay (RIBA), and were regarded as false-positive. Nine infants delivered by those 11 cases were negative for anti-HCV at 6 months of age, while none of the 8 husbands were anti-HCV-positive. It is concluded that the prevalence of anti-HCV in pregnant women in Taiwan is low (6/944, 0.63%), even in the cases with abnormal ALT (5/197, 2.5%). There was no serologic evidence for perinatal transmission or spouse infection. PMID- 1724983 TI - Hepatitis C virus RNA in blood donor sera detected by the polymerase chain reaction: comparison with supplementary hepatitis C antibody assays. AB - The low specificity of screening ELISAs for antibodies to hepatitis C virus in blood donors has called for confirmatory tests. Two types of supplementary antibody assays are available, recombinant immunoblot assays (RIBA-1 and RIBA-2) and an antibody consumption test referred to as a neutralization assay. Amplification of viral nucleic acid by the polymerase chain reaction (PCR) provides an antibody independent mode of detecting viral infection. We applied reverse transcription-double PCR to detect an HCV 5'-noncoding viral RNA sequence in serum specimens and compared PCR findings with confirmatory antibody tests. This study includes sera from 37 blood donors found positive by the Ortho anti HCV (C100-3) ELISA out of 14,591 donations. Of the 37 positive sera, 8 were positive by RIBA-1 and 1 further by RIBA-2. Seven of the RIBA positive sera contained HCV RNA by PCR. Among the 8 indeterminate and the 21 negative donor sera by RIBA-1, no PCR positive serum was found. The 37 anti-HCV positive donor sera identified by Ortho ELISA were also tested by Abbott anti-HCV (C100-3) ELISA whereby 22 were positive. Of these 22 sera plus 1 further with ELISA OD just below cutoff, 8 were positive by the "neutralization assay," (Abbott Laboratories, North Chicago, IL, USA) and 6 of these, including the borderline serum, were PCR positive. One of the two neutralizable but PCR negative sera was RIBA positive and the other was indeterminate. However, one ELISA (Abbott Laboratories) positive (OD 1.99) serum was not neutralizable but nevertheless contained HCV RNA by PCR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724984 TI - Antibodies to hepatitis C virus in uremic patients on continuous ambulatory peritoneal dialysis. AB - The prevalence of antibody to hepatitis C virus (anti-HCV) among 101 uremic patients receiving continuous ambulatory peritoneal dialysis (CAPD) was evaluated using a synthetic peptide-based HCV antibodies enzyme immunoassay. Thirty (29.7%) were found anti-HCV positive. This is significantly higher than 500 unselected paid blood donors (4.2%, P less than 0.0001). Among CAPD patients, anti-HCV positivity was found more frequently in patients who had received frequent and longer duration of hemodialysis previously (40.4% vs. 20.4%, P less than 0.05). These findings suggest that hemodialysis patients have a higher risk of HCV infection. At present, CAPD may be a suitable way to reduce the incidence of HCV infection in uremic patients. PMID- 1724985 TI - Levels of pre-S antigens and HBV DNA in sera from high and low viremic HBV carriers. AB - The biological and clinical significances of pre-S antigens and HBV replication were investigated. Some 125 sera, 28 from HBeAg and 97 from anti-HBe-positive HBsAg, carriers were studied. The aim was to verify whether pre-S antigens could be expressed in serum in complete absence of viremia. Pre-S proteins, determined by an enzyme immunoassay, were found in sera regardless of the presence of HBV DNA, as detected by spot-hybridization. The sera without detectable HBV DNA were investigated further by PCR using specific primers for the S and C regions of HBV. PCR analysis of samples revealed that 4 out of 5 HBeAg and 33 out of 41 (80.5%) anti-HBe positive sera contained HBV-amplified sequences of S and C regions. Pre-S antigen values correlated well with the amounts of HBV DNA in serum detected by PCR in anti-HBe-positive subjects with high titers of pre-S antigens (10(4)-10(6)). In addition, PCR highlighted the presence of HBV DNA sequences in 8 out of 17 (47.1%) pre-S-negative HBsAg-positive sera. PMID- 1724986 TI - Surrogate markers are not useful for identification of HCV carriers in chronic hemodialysis patients. AB - Several diagnostic hepatitis C assays have been developed for the detection of antibodies to different antigens of the virus. This virus is the major cause of non-A, non-B hepatitis. Seventy-nine patients undergoing chronic hemodialysis and/or hemofiltration were tested for the presence of anti-HCV antibodies (anti-C 100-3 antibodies and anti-core antibodies), anti-hepatitis B core antibodies (anti-HBc), and aminotransferases (ALT). Seven patients were positive by one or more of the anti-HCV enzyme linked immunoassays (EIAs), while HCV-RNA was detectable in only four patients. These four patients had at least one, but not necessarily the same, positive anti-HCV EIA. HCV-RNA was not detected in patients who had no antibodies as determined by all six anti-HCV EIAs. All patients with a marker for HCV infection had persistent normal levels of transaminases. Three patients had elevated ALT values without a marker for HCV infection and suffered from hepatitis B virus infection. Anti-HBc was detected in 27/72 patients without any marker and in four patients with a marker of HCV infection. However, HCV-RNA was detectable in only one of these four anti-HBc positive patients. It is concluded that surrogate markers (anti-HBc and serum transaminases) are not useful for identification of HCV carriers in chronic hemodialysis patients. PMID- 1724987 TI - Effect of pH, dietary proteins and trypsin inhibitors on the hydrolytic rate of human granulocyte colony-stimulating factor (G-CSF) by rat digestive enzymes. AB - The effects of pH, dietary proteins and trypsin inhibitors on the hydrolytic rate of recombinant human granulocyte colony-stimulating factor (G-CSF) by digestive enzymes were studied by in vitro incubation experiments. The bile obtained by cannulation into the rat common bile duct and the eluates obtained by infusing distilled water into the gastrointestinal tract were used as sources of digestive enzymes. Some proteins including dietary proteins such as casein and ovalbumin, and two kinds of trypsin inhibitors, chicken egg white and soybean ones, were used to examine the protective action on the hydrolysis of G-CSF by digestive enzymes. With an experiment using the digestive enzyme fluids obtained after centrifugation of bile by ultra-filters with a molecular weight (Mr) cut off of 30000, 10000 and 5000, the proteolytic activity to G-CSF decreased as the cut off Mr decreased. The enzyme solution obtained with a membrane with a Mr cut off of 5000 still had an enzyme activity against G-CSF. The protease activity was mainly ascribed to the pancreatic fluid, but the hepatic bile still had an enzyme activity. The hydrolytic rate of G-CSF was dependent on the pH of the enzyme fluid, and the intestinal fluid. The hydrolytic rate of G-CSF was studied at pHs of 1.68, 4.01, 6.86 and 9.18. Especially, as the pH decreased to 1.68, the hydrolytic rate of G-CSF considerably decreased. Some proteins including dietary proteins also affected the hydrolytic rate of G-CSF. The strength of the inhibitory effect of the proteins is the following order; ovalbumin greater than casein greater than mucin greater than keratin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1724988 TI - Relationship between substance P-induced initial transient contractions and inositol phospholipid hydrolysis in guinea pig ileum. AB - The effects of neomycin on substance P (SP)-induced inositol phospholipid hydrolysis and muscle contraction were studied in longitudinal smooth muscle of guinea pig ileum. Various tachykinin peptides including SP stimulated inositol phospholipid hydrolysis in guinea pig ileum. Neomycin (2.5 mM) reduced both the contractions and the accumulation of [3H]inositol phosphates induced by SP. These results suggest that the initial transient contractions induced by SP are coupled to inositol phospholipid hydrolysis. PMID- 1724989 TI - Alcoholic hepatitis with hyperleukocytosis. AB - We report a fatal case of alcoholic hepatitis with hyperleukocytosis mainly consisting of mature granulocytes in a 43-year-old woman. White blood cell count was increased in parallel with clinical deterioration to 54,800/mm3 with no immature neutrophils on a differential count. The bone marrow aspirate revealed normal maturation and no evidence of hematological malignancy. It has been postulated that severe leukocytosis accompanied by alcoholic hepatitis may be provoked by release of high levels of colony stimulating factor from damaged hepatic cells. However, the present patient showed a normal level of serum granulocyte colony stimulating factor, and could not prove the above assumption. PMID- 1724990 TI - Axon reflex vasodilatation in human skin measured by a laser Doppler technique. AB - This study was designed to investigate whether or not an objective method to measure a blood flow value by laser Doppler technique is possible to study the mechanism of an axon reflex vasodilatation elicited by i.d. injection of histamine or substance P into human skin. The probe of laser Doppler flowmeter was placed 10 mm apart from the injection site in the ventral forearm. The blood flow increased after the i.d. injection of histamine, substance P, but not after injection of Ringer solution. Penetration by the hypodermic needle produced an increase in the blood flow, but this increase was masked by the injection of the Ringer solution. The histamine- or substance P-induced blood flow increase was dose-dependent in both duration and intensity. Pretreatment with diphenhydramine ointment or xylocaine jelly 1 h prior to i.d. injection of histamine or substance P significantly attenuated the blood flow increase elicited by either vasoactive substance. Pretreatment of the skin with capsaicin (1% alcoholic solution) for 2 or 3 days markedly reduced the histamine- or substance P-induced blood flow increase. These results show that the measurement of cutaneous blood flow by laser Doppler flowmeter is useful in studying the mechanism of vasoactive substances-induced axon reflex vasodilatation. PMID- 1724991 TI - Inhibition of ion channels by hirsutine in rat pheochromocytoma cells. AB - Effects of hirsutine, an alkaloid that produces a potent ganglion blocking effect, were investigated using rat pheochromocytoma PC12 cells. Hirsutine (1 to 10 microM) suppressed dopamine-release evoked by 100 microM nicotine. In voltage clamped cells, hirsutine (1 to 10 microM) inhibited the inward current activated by 100 microM nicotine. Hirsutine was equipotent to hexamethonium in blocking the nicotine-activated current. The voltage-dependency of the nicotine activated current was not modified by hirsutine. Effects of hirustine on other ion channels were tested to determine its selectivity. Inward currents mediated through ATP activated channels were scarcely affected by hirsutine (up to 100 microM). However, hirustine (10 microM) inhibited Ba currents passing through Ca channels and K currents activated by depolarizing voltage steps. The results suggest that hirsutine potently blocks nicotinic receptor-channels, but hirsutine also inhibits voltage-gated Ca and K channels. Roles of the inhibition of these channels in the pharmacological effects of hirsutine were discussed. PMID- 1724993 TI - Non invasive analysis of hemodynamic changes in dogs with acute pancreatitis. AB - To elucidate the hemodynamic changes in the course of acute pancreatitis in dogs, various parameters were monitored non-invasively and real-timely by the ultrasonic flow probes which had been placed in advance. The dogs were divided into two groups, i.e., control group (CG) and infusion group (IG). In the infusion group, lactated Ringer solution was infused intravenously for 12 hours in order to maintain LAP or CVP at the mean +/- 1 mmHg of its control value before inducing pancreatitis. In the control group, lactated Ringer solution was not infused. Cardiac output (CO) and LV dp/dt abruptly decreased in a few hours and remained low levels in a control group (CG), but not in an infusion group (IG). It shows that the decreased CO was caused by a decrease in preload but not by a depression of contractility. Gastroduodenal arterial flow (GDAF) and GDAF/CO decreased in a few hours and kept low level in CG, but not in IG. Therefore, a decrease in GDAF is mainly caused by an increase in vascular resistance due to an increase in Hematocrit. Superior pancreatico-duodenal venous flow (SPDVF) and SPDVF/CO decreased immediately in both groups, revealing that there is severely depressive outflow from pancreas because of hemorrhage and destruction of pancreatic parenchyma immediately after the onset of pancreatitis. In CG, Total hepatic flow (THF) and Portal vein flow (PVF) gradually decreased in a few hours and kept low levels, but Common hepatic arterial flow (CHAF) did not decrease. This suggests that decrease of THF is due to decrease of PVF. THF/CO increased in CG. PVF/CO decreased in CG, while CHAF/CO increased in both groups. The increased CHAF compensated for the decreased PVF, which may play an important role in protecting the liver. PMID- 1724992 TI - NIK-247 blocks voltage-dependent ionic currents in crayfish axon. AB - NIK-247 (9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta[b]-quinoline hydrochloride hydrate), an acetylcholinesterase inhibitor, is structurally related to 4 aminopyridine (AP). Its effects on ionic currents were examined in the artificial node of the crayfish axon under voltage-clamp. When applied externally, NIK-247 reversibly suppressed both inward and outward currents. Effects on K currents were further studied in the presence of tetrodotoxin. NIK-247 suppressed the K current dose-dependently, but with an IC50 at of 10(-3) M. THA (9-amino-1,2,3,4 tetra-hydroacridine hydrochloride hydrate), a related inhibitor, similarly suppressed the K current with an IC50 of 5 x 10(-4) M, in comparison with 3-AP and 4-AP which had IC50's of 3 x 10(-5) M and at 10(-5) M, respectively. Furthermore, NIK-247 (and THA) suppressed the K current uniformly for the whole time course, whereas AP the AP's suppressed mainly the fast activating and inactivating K current with a voltage- and frequency-dependent recovery. Therefore, NIK-247 and THA seem to be neither potent nor very specific as ionic channel blockers. With respect to the K current, however, they clearly differ from the AP's in their mode of suppression. PMID- 1724994 TI - Possible collaboration between c-fos and c-myc proto-oncogene products in in vivo lymphomagenesis. AB - Transgenic mice carrying the exogenous c-myc gene under regulation of the Ig enhancer (Ig-c-myc) were mated with mice carrying exogenous c-fos gene under control of the H-2Kb promoter (H2-c-fos) to examine their functional collaboration in in vivo lymphomagenesis. Two of the 33 (c-fos x c-myc) mice developed pre-B cell lymphomas within 22 weeks of age. None of the other F1 progeny expressing c-fos or c-myc alone showed malignant change within 14 months of age, suggesting that the exogenous c-fos and c-myc collaborate in in vivo lymphomagenesis. The exogenous c-myc RNA was overexpressed in the lymphomas, but the amount of exogenous c-fos RNA was not affected, suggesting that the large abundance of c-myc protein is a prerequisite for lymphoma onset or progression and c-fos protein plays a complementary role. C-fos protein induced immunodeficiency in the (c-fos x c-myc) mice like H2-c-fos mice. Natural killer cell activity of (c-fos x c-myc) mice was partially impaired. Therefore, these lymphomas may be a consequence of the synergism of two independent actions caused by the exogenous c-myc (lymphomagenesis) and the exogenous c-fos (low NK activity) in (c-fos x c-myc) mice. PMID- 1724995 TI - Calcium antagonists in cardiovascular care. Proceedings of the Second International Symposium. Basel, Switzerland, February 13-15, 1991. PMID- 1724996 TI - Calcium-channel drugs: structure-function relationships and selectivity of action. AB - Calcium channels are ubiquitously distributed in excitable cells. The calcium channel antagonists interact specifically at the L subclass of channels to mediate cardiovascular effects. These channels may be considered as pharmacologic receptors with specific drug binding sites and subject to a variety of regulatory influences. Each site is specific for agents of the three principal structural classes--the phenylalkylamines, the 1,4-dihydropyridines, and the benzothiazepines--and each exhibits defined structure-activity relationships. The 1,4-dihydropyridine structure exhibits both potent antagonistic and activator properties. The calcium channel antagonists exhibit considerable selectivity of action in the cardiovascular system, both between and within structural groups. This selectivity has a variety of causes including voltage dependence of the interaction, whereby the apparent affinity of the antagonist is determined by the membrane potential and stimulus pattern. Experimental evidence underlying the structure-activity relationships and the voltage-dependent behavior of 1,4 dihydropyridines is reviewed. PMID- 1724997 TI - Calcium-channel blockers as adjuvant therapy postthrombolysis. AB - Following successful thrombolytic therapy for evolving acute myocardial infarction, the inevitable process of transmural necrosis is favorably altered in a majority of patients, resulting in an aborted, or interrupted, myocardial infarction. Presumably, such myocardial salvage results in an "incomplete" infarction, which is often associated with a patent (but residually stenotic) infarct-related coronary artery. In this regard, the acute myocardial infarction successfully reperfused with thrombolytic therapy resembles non-Q-wave infarction. The subsequent pharmacologic therapy of the "incomplete" infarction remains ill-defined. This article reviews the features which "naturally occurring" non-Q-wave infarction shares with the successfully reperfused myocardial infarction. In addition, the role of adjunctive pharmacologic therapy with calcium-channel-blocker therapy is addressed; in particular, the results of the Diltiazem Reinfarction Study and the non-Q-wave infraction subset analysis of the Multicenter Diltiazem Post-Infarction Trial are discussed to provide the conceptual basis for considering such treatment appropriate adjunctive pharmacologic therapy for the postthrombolysis patient. PMID- 1724998 TI - The antiarrhythmic and antifibrillatory effects of calcium antagonists. AB - Calcium-channel antagonists are widely used in the treatment of supraventricular tachyarrhythmias. The potential benefits of these agents in the management of ventricular arrhythmias, however, are not widely appreciated. Ventricular arrhythmias result from abnormalities of impulse generation and/or impulse conduction. The calcium ion has been implicated in both mechanisms. Myocardial cytosolic calcium increases during ischemia and sympathetic nervous system activation. Elevations in cytosolic calcium provoke oscillatory depolarizations of the cardiac membrane, triggering sustained action potential generation and extrasystoles. Myocardial ischemia also results in a dispersion of refractory period, producing random re-entrant circuits and ventricular fibrillation. Alterations in action potential duration that produce this dispersion of refractory period are associated with spatial and temporal nonuniformities of intracellular calcium transients. Calcium-channel antagonists have been shown to prevent afterdepolarizations and to reduce the endocardial to epicardial dispersion of the refractory period during myocardial ischemia, and therefore could prevent malignant arrhythmias. A slow inward calcium current may, in fact, be required for the initiation and maintenance of ventricular fibrillation. Calcium antagonists reduce, whereas calcium agonists enhance, the susceptibility to ventricular fibrillation induced by the combination of exercise and acute myocardial ischemia. The cardioprotection occurs independently of actions on vascular smooth muscle. Thus, calcium antagonists that exert direct myocardial actions may prove to be particularly effective in the prevention of sudden cardiac death in patients. The challenge remains to develop calcium antagonists that selectively modulate myocardial calcium entry without compromising cardiac mechanical function. PMID- 1724999 TI - Endothelin. AB - Endothelial cells can produce contracting factors; endothelin, a 21-amino acid peptide that can control local vascular tone, is the most potent of these factors. Of the three isoforms of endothelin, endothelial cells appear to release primarily endothelin-1. The peptide is formed from its precursor big endothelin via the activity of the endothelin converting enzyme. The basal production of the peptide is stimulated by epinephrine, angiotensin II, arginine vasopressin, transforming growth factor beta, thrombin, interleukin-1, and the calcium ionophore A23187. In vascular smooth muscle cells, endothelin binds to a specific receptor that activates phospholipase C and leads to the formation of inositol trisphosphate, diacylglycerol, and increased intracellular calcium levels. In certain blood vessels, the endothelin receptor is linked to a voltage-operated calcium channel via a Gi protein. This may explain why calcium antagonists inhibit endothelin-induced contractions only in certain blood vessels. In the human forearm circulation, calcium antagonists of different classes prevent endothelin-induced contractions. In hypertension, the circulating endothelin levels appear to be normal, whereas the vascular sensitivity to the peptide is reduced in most vascular tissues, but normal and enhanced responses have also been reported. In atherosclerosis and other forms of vascular disease, circulating endothelin levels are augmented, a phenomenon that may be related to an increased formation of the peptide induced by modified forms of low-density lipoproteins. PMID- 1725000 TI - Subcellular localization of calcium release in isolated rat myocardial cells. AB - Independent determinations of Ca2+ by two indicators showed that subcellular Ca2+ activity (intracellular free calcium concentration) was heterogeneous in rat myocardial cells. Arsenazo III (Az III), a membrane-impermeant absorbance indicator for Ca2+, was loaded into cardiac muscle via liposomes and calcium quantitated at two wavelengths through a focal point diaphragm in 5-100 microns 2 regions by a photometer. Fura-2, a high-affinity fluorescent calcium indicator, was loaded into cells as the ester form, and the light intensity was measured by digital-imaging microscopy using a photon-counting camera. Calcium activity at each picture element was quantitated by division of fluorescence excited by two ultraviolet wavelengths, and corrected for concentration differences in fura-2 by a third, Ca(2+)-insensitive wavelength. Both methods revealed hot spots of Ca2+ release and uptake that could be enlarged, added to, or altered. Addition of 1-10 nM norepinephrine caused up to a 500% increase in Ca2+ in localized regions, although whole cell average Ca2+ increased by only 0-80%, suggesting the importance of localized intracellular Ca2+ release for contraction. PMID- 1725001 TI - Low-dose felodipine treatment attenuates endothelial dysfunction in rabbits fed an atherogenic diet. AB - Loss of endothelium-dependent relaxation is an early step in atherogenesis. To test the effect of low-dose felodipine on the progression of this dysfunction, male New Zealand white rabbits were rendered hypercholesterolemic with a diet containing 0.25% cholesterol and 3% coconut oil. After a 1-month induction period on this diet, during which the rabbits were identified as low or normal responders to cholesterol, 0.46 mg/kg of felodipine (FELO) or placebo (CON) were given by gavage once daily for a further 3 months. This regimen established FELO plasma levels (14.2 +/- 1.3 nM, week 9) corresponding to therapeutic concentrations in humans and an average 13-fold increase in plasma cholesterol from below 1 mM. At the end of the treatment period, relaxation of norepinephrine (1 x 10(-8) M)-precontracted proximal thoracic aorta strips to acetylcholine (ACh: 1 x 10(-8)-1 x 10(-5) M) was determined. Cholesterol exposure was calculated as the area under the curve for serum cholesterol x time [AUC (mM x day 1)]. Despite equal cholesterol load [FELO (n = 17): 1,856 +/- 182 mM x day) and CON (n = 22): 1,851 +/- 167 mM x day], maximal relaxation to 1 x 10(-7) M ACh was well preserved in strips from FELO-treated rabbits (29.5 +/- 5.7%) but suppressed in strips from untreated rabbits (11.0 +/- 2.9%). For comparison, relaxation in strips from standard diet controls was 49.8 +/- 2.9% (n = 15). Moreover, there was a significant inverse correlation (r = -0.74) between percentage ACh relaxation and cholesterol exposure in FELO-treated rabbits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725002 TI - Calcium antagonists and cholesteryl ester metabolism in macrophages. AB - Experimental data indicate that calcium antagonists modify cellular lipid metabolism in the arterial wall as part of their antiatherosclerotic action observed in animal models. In the present study, we investigated the effect of verapamil, nifedipine, and lacidipine (a new dihydropyridine derivative) on cholesteryl ester metabolism in cultured mouse peritoneal macrophages (MPMs). Cholesteryl esters are formed in the cell via acyl-CoA:cholesterol acyltransferase (ACAT), which senses free cholesterol supplied by lysosomal hydrolysis of lipoprotein cholesterol ester. Verapamil inhibited up to 99% the ability of acetyl-low-density lipoprotein (acLDL) to stimulate cholesterol esterification in macrophages, but was less effective in 25-hydroxycholesterol stimulated MPMs and in cholesterol-loaded cells after acLDL removal. Cells incubated with [3H]cholesterol ester-acLDL and verapamil showed a reduction in the cholesterol/cholesteryl ester ratio. In the same experimental conditions, nifedipine displays minor or no effects on cholesterol esterification and in the cholesterol/cholesteryl ester ratio. On the contrary, the nifedipine-like lacidipine was active in inhibiting cholesterol esterification in macrophages elicited by acLDL. Our data indicate that calcium antagonists of different structure, even within the same class, may have various effects on cholesterol esterification in macrophages in culture. PMID- 1725003 TI - Hypercholesterolemia modulates the effects of nitrendipine on blood pressure and platelet function in essential hypertension. AB - Both marked hypercholesterolemia and severe hypertension have been reported to be associated with an enhanced sensitivity of blood platelets to activating agents. To investigate a possible mutual synergistic effect of moderate hypercholesterolemia and mild hypertension on platelet reactivity, we studied in 29 patients the response to aggregating agents, ADP and collagen, and the intracellular cyclic AMP content and cytosolic Ca2+ concentration that participate, respectively, as inhibitory and stimulatory mediators in platelet responses. When compared to age- and blood pressure-matched patients with normal or slightly elevated plasma cholesterol, the patients with total platelet cholesterol higher than 6.4 mM were characterized by a decreased response to collagen and ADP (14.5 +/- 3.0 vs. 23.8 +/- 2.0 a.u. and 17.7 +/- 4.5 vs. 26.9 +/ 2.7 a.u., respectively), a tendency to a reduced cAMP content both in the basal state and after phosphodiesterase inhibition by Ro-15 2041 (2.83 +/- 0.18 vs. 3.26 +/- 0.22 mumol/10(8) cells and 4.57 +/- 0.29 vs. 5.38 +/- 0.36 mumol/10(8) cells, respectively), and no change in cytosolic Ca2+ concentration (190 +/- 11 vs. 203 +/- 13 nM). After a chronic treatment with nitrendipine (20 mg/day for 6 months), blood pressure, platelet [Ca2+]i and cAMP content decreased in the patients with normal or moderately elevated hypercholesterolemia (p less than 0.001, less than 0.001, and less than 0.05, respectively), but these effects were attenuated or absent in the patients with higher hypercholesterolemia. Plasma lipids and the platelet-aggregating response to ADP and collagen were unchanged by this long-term nitrendipine treatment in both groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725004 TI - Nifedipine vs. enalapril in treatment of hypertensive patients with glucose intolerance. AB - To compare the metabolic effects of nifedipine and enalapril, we studied 21 obese patients (BMI of 31.6 +/- 1.1) with mild-to-moderate hypertension and glucose intolerance. None of the patients were receiving insulin or hypoglycemic agents. After a washout period of 15 days, blood pressure was recorded and fasting blood glucose, insulin, and lipid concentrations were determined. At random, 11 patients were started on nifedipine and 10 patients on enalapril. At the 90th day of treatment, clinical and laboratory tests were again performed. Both of the drugs reduced blood pressure values comparably. No significant variation of metabolic parameters was found after 90 days of treatment in the enalapril group. In the nifedipine group, the fasting insulin level was decreased and the glucose/insulin ratio was significantly increased, suggesting an improvement in insulin sensitivity; moreover, total plasma cholesterol was significantly reduced. Enalapril and nifedipine seem to be effective and safe drugs in the treatment of hypertensive subjects with obesity and glucose intolerance. Nifedipine can ameliorate insulin resistance and the lipid state. PMID- 1725005 TI - Hemodynamic profile of Ro 40-5967 in conscious rats: comparison with diltiazem, verapamil, and amlodipine. AB - Ro 40-5967 is a new calcium antagonist that binds to the same site as verapamil but that has been shown to have a much lesser negative inotropic effect than verapamil. The goal of the present study was to assess the hemodynamic profile of Ro 40-5967 not only in comparison with verapamil but also with diltiazem and amlodipine. For this purpose, hemodynamic parameters were assessed in conscious normotensive rats by measuring mean arterial pressure (MAP), left ventricular (LV) dP/dtmax, and heart rate. Dose-response curves were obtained with intravenous injection of the four drugs. Despite similar decreases in arterial pressure, the effects of the four drugs on left ventricular contractility and heart rate were different. Verapamil and diltiazem were markedly negative inotropic. Amlodipine decreased left ventricular contractility only at the highest dose. Ro 40-5967 was less negative inotropic than amlodipine. Verapamil, diltiazem, and Ro 40-5967 did not alter heart rate or slightly decreased it. In contrast, amlodipine induced a reflex tachycardia. In conclusion, because of its very low negative inotropism and its lack of reflex tachycardia, Ro 40-5967 seems to have a unique hemodynamic profile among calcium antagonists. PMID- 1725006 TI - New aspects of calcium antagonists for treatment of cerebrovascular disease. AB - Cerebrovascular infarction can be subdivided into two different categories: cerebral hemorrhage and cerebral ischemia. Although the use of calcium antagonists for the treatment of subarachnoid hemorrhage has been established in many series, the benefit of these drugs for the treatment of cerebral hemorrhage is inconclusive, and we lack considerable evidence of its application in acute ischemic stroke. Inconclusive data have been reported from experimental studies using different pharmacological models and different protocols; however, various clinical studies have suggested that calcium antagonists may be useful in improving the neurological and functional outcome of the patient due to reduced infarct size and the properties of the drug to improve tissue metabolism in remote cortical territories. So far, however, these studies failed to establish the efficacy of the drug unequivocally. This may be due to the considerable amount of patients included with small or rapidly recovering neurological deficits and to the delay between onset of symptoms and start of treatment (ranging between 48 and 72 h). PMID- 1725007 TI - Calcium antagonists and the kidney: implications for renal protection. AB - During the past decade, attention has focused on the effects of calcium antagonists on renal function. When administered in vitro to the isolated perfused kidney, calcium antagonists exhibit consistent actions permitting characterization of their renal effects. Calcium antagonists do not affect the vasodilated isolated perfused kidney (IPK), but they do dramatically alter the response of this preparation to vasoconstrictor agents. Our recent studies using the isolated perfused hydronephrotic rat kidney model, which permits direct visualization of afferent and efferent arterioles, have demonstrated that the preferential augmentation of the glomerular filtration rate observed in the IPK is attributable to preferential vasodilation of preglomerular vessels. Although the clinical implications of such observations have not been fully delineated, the results of recent studies indicate that calcium antagonists exert salutary effects on renal function in patients with impaired renal hemodynamics. Such disorders include radiocontrast-induced nephrotoxicity and transplant-associated acute renal insufficiency. It is apparent, however, that the effects of calcium antagonists on renal blood flow commend their use in the management of essential hypertension. PMID- 1725008 TI - Modulation of electrical activity and of intracellular calcium oscillations of smooth muscle cells by calcium antagonists, agonists, and vasopressin. AB - The A7r5 smooth muscle cell line, which originally was derived from fetal rat aorta, shows spontaneous calcium oscillations associated with electrical activity (frequency of 0.2-0.5 Hz). Organic calcium antagonists such as isradipine (10(-8) M) stopped the calcium oscillations whereas calcium agonists (e.g., Bay K 8644, 10(-8) M) increased the frequency and amplitude of calcium oscillations without changing the shape of the electrical spikes. The enantiomers of the dihydropyridine SDZ 202-791 known to have opposite activity with respect to L type Ca2+ channels antagonized each other when tested for their effects on the calcium oscillations. The modulation of the activity of these cells by inorganic ions that affect Ca2+ and K+ channels was also investigated. The addition of barium chloride (10(-4) M) to the bathing solution increased the spiking rate whereas cadmium chloride (10(-6) M) abolished the spikes. The vasoconstrictor peptide vasopressin first induced a hyperpolarization associated with the cessation of spiking activity followed by a slow depolarization. The intracellular Ca2+ concentration ([Ca2+]i), measured with the calcium indicator fura-2, was increased transiently to a level about 10-fold above basal and then gained a new steady state at about twice the basal level. Vasopressin stimulated Ca2+ release from intracellular stores (via InsP3), resulting in membrane hyperpolarization through activation of Ca(2+)-activated K+ channels. The late and long-lasting [Ca2+]i elevation was due to Ca2+ influx through dihydropyridine insensitive channels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725009 TI - Calcium antagonists and atherosclerosis. AB - Numerous trials have used calcium antagonistic substances to inhibit or prevent genesis and progression of atherosclerosis, as well as disintegration of intimal proliferates and calcium deposition in atheromas or in matrix tissue of artery walls. In experimental models, various components involved in the development of atherosclerosis can be influenced by calcium antagonists. Transendothelial transport through interendothelial spaces and through the endothelial cells are sensitive to calcium antagonists of various types. Some drugs with calcium antagonistic properties are capable of inhibiting the development of stenosing proliferates caused by ballooning or other types of local artery wall stimulation. With cultures of cells obtained from normal and atherosclerotic arteries, insights into the mechanisms are possible by which various calcium antagonists influence the development and the progression of atherosclerosis. PMID- 1725010 TI - Calcium antagonists in antihypertensive combination therapy. AB - Calcium antagonists can be used with a number of other antihypertensive compounds. The combined use of a dihydropyridine calcium antagonist and beta blocker is well established and is probably the most efficacious combination for routine use. Angiotensin-converting enzyme (ACE) inhibitors may also constitute a suitable addition to calcium antagonist therapy, whereas little benefit appears to be derived from the combined use of a calcium antagonist and a diuretic. Other compounds, e.g., alpha-adrenoceptor blockers, can also be given together with calcium antagonists, although this should be done with great caution because dramatic reductions in blood pressure may result. It is worth noting that in addition to the improved antihypertensive efficacy produced by some of the calcium antagonist combinations, potentially positive metabolic changes may also occur, e.g., a reduction in serum cholesterol, when a calcium antagonist and a beta-blocker are combined. PMID- 1725011 TI - Calcium antagonists, ventricular arrhythmias, and sudden cardiac death: a major challenge for the future. AB - Intracellular overloading by calcium ions may play an important role in the development of ischemic ventricular fibrillation, as shown by experimental data. Mechanisms are those that develop secondary to the rise in cytosolic calcium thought to occur in ischemia. After-depolarizations are stressed as a possible cause of automaticity and triggered arrhythmias in ventricular tissue. The ideal calcium antagonist should be able to inhibit such afterdepolarizations in ischemic tissue without having a negative inotropic effect on nonischemic tissue. Hence, it might be possible to extend the beneficial results of verapamil found in a recent postinfarct trial to show even better postinfarct protection by a calcium antagonist with these specific properties. On the other hand, a highly vascular-selective calcium antagonist, by avoiding any effect on the myocardium, could also be desirable in postinfarct patients with heart failure. Future trials should compare these two types of calcium antagonists. If the adverse effect of calcium antagonists on heart failure could be avoided, then the calcium antagonists would stand along-side the beta-blockers as agents able to prevent postinfarct sudden death. Furthermore, the combination of calcium antagonists and beta-blockade could then become attractive as potentially powerful postinfarct protection. PMID- 1725012 TI - Protective effects of calcium antagonists on energy and substrate metabolism during ischemia and reperfusion in hypertensive myocardial hypertrophy. AB - The aim of the present study was to define the protective effects of verapamil and nifedipine on mechanical performance and energy and substrate metabolism of the postischemically reperfused myocardium in a chronic pressure overload cardiac hypertrophy model. The isolated beating rat heart preparation was used and left ventricular pressures and high-energy phosphates were continuously monitored during 30 min of global ischemia and reperfusion, respectively. Recovery of mechanical performance and high-energy phosphate and sugar monophosphate metabolism was significantly impaired in untreated hypertrophied hearts compared with normal control hearts and hypertrophied hearts treated with calcium antagonists. In hypertrophied hearts with chronic verapamil treatment, recovery was significantly improved compared to acute verapamil treatment, nifedipine treatment, and normal control hearts. Thus, verapamil and nifedipine exerted a protective effect on the postischemic recovery of the hypertrophied myocardium that was most prominent following long-term pretreatment with verapamil. Prolonged maintenance of adequate plasma levels and tissue distribution of calcium antagonists before an ischemic event may improve the postischemic prognosis in the presence of pressure-induced myocardial hypertrophy. PMID- 1725013 TI - Calcium antagonists and the stunned myocardium. AB - "Stunned myocardium" is defined as the prolonged but transient contractile dysfunction of viable myocardium salvaged by reperfusion. For example, a brief 15 min episode of coronary artery occlusion does not result in myocyte necrosis, yet contractile function of the previously ischemic tissue remains profoundly depressed at 0-30% of baseline values for hours to days following reflow. This phenomenon, first characterized in the experimental canine model, has more recently been documented in clinical instances of angina, following cardiac surgery, after angioplasty, and following successful reperfusion for the treatment of acute myocardial infarction. Considerable evidence indicates that calcium antagonists administered prior to coronary occlusion attenuate postischemic stunning in the canine model: verapamil, diltiazem, and amlodipine have been shown to restore contractile function to 50-100% of baseline values during the initial hours following relief of ischemia. Furthermore, both verapamil and nifedipine improved systolic contraction of stunned myocardium even when treatment was "delayed"--i.e., when the agents were administered 30 min after reflow had been established. This improved recovery of contractile function associated with calcium antagonist treatment may be due in part to the well documented afterload reducing and coronary vasodilatory properties of these agents. However, as low doses of intracoronary nifedipine infused after reperfusion restored systolic contraction to 75-90% of baseline values in the absence of afterload reduction or increases in coronary blood flow, these data suggest that calcium antagonists may act in part by favorably modulating calcium flux within the stunned, previously ischemic myocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725014 TI - ACE inhibition - present and future. The role of ramipril. Proceedings of International Symposium. London, October 12-13, 1990. PMID- 1725015 TI - Molecular biology of the renin-angiotensin system: implications for hypertension and beyond. AB - In the last decade, nucleic acid sequences coding for all three components of the renin-angiotensin cascade have been cloned. This has led to increased understanding of the transcriptional and translational regulation of renin, angiotensinogen, and angiotensin-converting enzyme (ACE). This review discusses the impact of the availability of these clones in three clinically relevant areas -the role of the renin-angiotensin system in hypertension, the role of tissue renin-angiotensin systems, and the development of renin inhibitors. PMID- 1725016 TI - The Acute Infarction Ramipril Efficacy (AIRE) Study: rationale, design, organization, and outcome definitions. AB - The rationale, design, organization, and outcome definitions of the Acute Infarction Ramipril Efficacy (AIRE) Study are described prospectively. A total of 2,000 patients (1,000 per treatment group) will be recruited to this multicenter, multinational, double-blind, randomized, placebo-controlled study investigating the effect of oral treatment with ramipril (2.5 or 5 mg twice daily) on the total mortality of survivors of an acute myocardial infarction (AMI) with early clinical evidence of heart failure. Secondary outcomes of the study include progression to severe/resistant heart failure (at which time the patient will be withdrawn from the study treatment), reinfarction, and stroke. Treatment will be initiated in hospital between day 3 and day 10 following AMI, and follow-up continued for an average of 15 months and a minimum of 6 months. The study data will be analyzed on an intention-to-treat basis: a single formal interim analysis will be conducted after 175 deaths. An Independent Adjudicating Panel will act as the overall ethical supervisory body for the study and will retain the randomization code. An International Steering Committee will be responsible for the clinical definitions of the secondary study outcomes, and will regularly review progress of the study. We believe that early treatment with ramipril may reduce the total mortality of patients surviving an AMI with clinical evidence of heart failure. PMID- 1725017 TI - Preservation of endothelial function by ramipril in rabbits on a long-term atherogenic diet. AB - Hypertension and hypercholesterolemia predispose to atherosclerosis. Ramipril, known to lower blood pressure, was used to study the effect of converting-enzyme inhibition on impairment of endothelium-derived relaxation and changes in basal cGMP content in rabbits fed an atherogenic diet (0.25% cholesterol). The generation of cGMP in the presence of bradykinin and ramiprilat was studied in vitro in aortic segments from normal untreated rabbits as well as in bovine endothelial cells. The ability to relax in response to acetylcholine was almost abolished in aortic segments from the vehicle-treated rabbits fed the atherogenic diet for 4 months. The basal cGMP content was substantially reduced. Aortic segments from rabbits concomitantly treated with ramipril (0.3 and 3.0 mg/kg/day) for 3 months showed well-preserved relaxation and matching basal cGMP content compared to normal controls. The relaxation was not significantly greater in aortic segments from ramipril-treated rabbits fed the standard diet, but the cGMP content was more than doubled. In vitro studies in aortic segments and in endothelial cells showed that both the ramiprilat and bradykinin concentrations dependently stimulated cGMP formation, which serves as a biochemical marker of nitric oxide or EDRF release. Thus, the observed endothelial protection against hypercholesterolemia by ramipril may be the result of continuously increased cGMP formation due to preserved EDRF release. This is presumably produced by enhanced bradykinin activity through inhibition of degradation by converting-enzyme inhibition with ramipril. PMID- 1725018 TI - Preliminary results of biliary excretion of ramipril after T-drainage in cholecystectomy patients. AB - Four cholecystectomy patients, aged between 52 and 56 years, weighing between 64 and 90 kg, received 5 mg of ramipril as a single dose in order to investigate the pharmacokinetics and excretion pattern of ramipril. All patients had a T-drainage that allowed bile collection. Serum was collected at regular intervals, bile was collected hourly for 6 h followed by a 6- and 12-h fraction, and urine was collected every 2 h for 8 h followed by a 4- and 12-h fraction. The concentrations of ramipril and ramiprilat in serum and ramipril, ramiprilat, ramipril glucuronide, ramiprilat glucuronide, diketopiperazine, and diketopiperazine acid in bile and urine were determined and the amounts excreted in urine and bile over 24 h were calculated. There were great interindividual differences in maximum concentrations as well as in the time to reach maximum concentrations in plasma and bile as well as in the excretion pattern between urine and bile. The highest concentrations in bile were found for diketopiperazine acid (3,080 ng/ml) and ramipril glucuronide (2,414 ng/ml). In general, only minimal amounts of unchanged ramipril (prodrug) were detected in the bile. In the urine, the major metabolites excreted were diketopiperazine acid, ramiprilat, and diketopiperazine in amounts of 537, 188, and 124 micrograms, respectively. In bile, the main substances excreted were diketopiperazine acid and ramiprilat glucuronide, which amounted to 501 and 314 micrograms, respectively. Biliary excretion may be the explanation for the noncomplete urinary recovery of ramipril and its metabolites. PMID- 1725019 TI - Effect of angiotensin-converting enzyme inhibition on human tissue renin. AB - This investigation was performed to assess the effect of treatment with an angiotensin-converting enzyme (ACE) inhibitor, ramipril, on human tissue renin levels. A total of 41 patients were studied (32 males and 9 females, aged 48-71 years). Twenty-two of these patients received up to 5 days treatment with the ACE inhibitor ramipril (5 mg p.o.) prior to surgery, the remaining 19 patients served as controls (no ramipril treatment). Under surgery vascular tissues were removed and renin-like activities measured in carotid and renal arteries. Plasma renin and ACE activity were determined before and after surgical intervention. Vascular renin values were two- to threefold higher in ACE-treated hypertensive patients compared to normotensive and untreated hypertensive controls (irrespective of concomitant atherosclerotic lesions). There were no differences in vascular renin observed between primary (essential) and secondary hypertension. In the hypertensive patients treated with ramipril, blood pressures were markedly (but not significantly) lowered in 12 patients. A similar pattern was observed for treatment-induced changes in plasma renin (increased) and ACE activity (decreased). The findings of this study demonstrate that treatment with an ACE inhibitor, such as ramipril, evokes changes in extrarenal tissues similar to those demonstrable in plasma. Conceivably, the blood pressure-lowering effect of ACE inhibition on human tissue is partially induced at the vascular level. PMID- 1725020 TI - The pharmacokinetics of ramipril in a group of ten elderly patients with essential hypertension. AB - This open, general practice study looked at the pharmacokinetics of ramipril over a period of 1 year in elderly (greater than 65 years) hypertensive patients. Ten patients with a diastolic blood pressure between 95 and 125 mm Hg were treated with 5 mg of ramipril daily for 4 weeks, followed by an additional 11 months of treatment in which the dose was titrated against the blood pressure. Pharmacokinetic data were collected at baseline, 4 weeks, and 1 year. Mean peak concentrations of ramiprilat (the active diacid) were 11.5 ng/ml acutely, 18.5 ng/ml at 1 month, and 31.3 ng/ml at 1 year, and these were all statistically significantly different. Mean half-lives of ramiprilat were 5.5 h acutely, 5.2 h at 1 month, and 4.6 h at 1 year, and these were not statistically significantly different. Mean areas under the curve excluding the component for saturable binding were 106.8 ng/h/ml acutely, 115.7 ng/h/ml at 1 month, and 248 ng/h/ml at 1 year; the latter was statistically significantly different from the earlier readings. There is no evidence from this study that the pharmacokinetics change in any clinically relevant way. PMID- 1725021 TI - Dose-response relationship of ramipril in patients with mild-to-moderate hypertension. AB - The dose-response relationship of ramipril was examined in 216 subjects with mild to-moderate essential hypertension in a double-blind, placebo-controlled, multicenter study. Ramipril capsules (1.25, 2.5, 5, or 10 mg) or placebo capsules were administered once daily for 12 weeks. Significant reductions in supine and standing diastolic and systolic blood pressures were seen at end point in the ramipril 2.5, 5, and 10 mg treatment groups compared with placebo. The antihypertensive effect was greater at higher doses. The minimum effective dose of ramipril was 2.5 mg once daily. PMID- 1725022 TI - The effect of ramipril on ambulatory blood pressure: a multicenter trial. AB - The antihypertensive efficacy of ramipril was evaluated using 24-h noninvasive ambulatory sphygmomanometry in this double-blind, placebo-controlled study. One hundred subjects with mild-to-moderate essential hypertension were randomized to ramipril, 10 mg or placebo once daily for a 4-week treatment period. Ramipril decreased systolic and diastolic blood pressures throughout the 24-h period after dosing. Blood pressures measured manually 24 h postdose also showed that ramipril significantly reduced supine and standing blood pressures when compared with placebo. Incidences of adverse events were similar in the two study groups. Ramipril proved to be a well-tolerated agent with a sustained 24-h antihypertensive effect in this study. PMID- 1725023 TI - Antihypertensive efficacy, tolerance, and safety of ramipril in young vs. old patients: a retrospective study. AB - The efficacy, tolerance, and safety of ramipril, an angiotensin-converting enzyme inhibitor, were assessed in 502 patients from five multicenter, double-blind studies who had mild-to-moderate essential hypertension. Each study was designed with a 4-week placebo run-in phase followed by 6 weeks of treatment with ramipril or one of five other antihypertensive treatments. A total of 412 young patients (17-65 years of age) and 90 old patients (66-87 years) in these studies received single daily doses of 5 or 10 mg of ramipril. At the end point of treatment, mean reductions in supine systolic blood pressure (19.4 mm Hg in young patients, 17.8 mm Hg in old) were significantly different, whereas mean reductions in supine diastolic blood pressure (13.3 mm Hg in young patients, 12.5 mm Hg in old) showed no significant difference. The number of responders was similar in both age groups: 68.6% and 71.1% of young and old patients respectively. No clinically relevant trends were observed in biochemical and hematological variables. Ramipril was well tolerated by both young and old patients, and there was little evidence that it was less safe in the elderly. PMID- 1725024 TI - Antihypertensive efficacy, tolerance, and safety of long-term treatment with ramipril in patients with mild-to-moderate essential hypertension. AB - A total of 555 hypertensive patients took part in a 2-year multicenter, open label study to determine the efficacy, tolerance, and safety of long-term therapy with ramipril. In the beginning, all patients were to receive 5 mg of ramipril/day. The dosage was then adjusted in accordance with response to treatment and ranged from 1.25-20 mg of ramipril daily. Of these patients, 129 also received 25 mg of hydrochlorothiazide daily at some point during the trial. To evaluate whether tolerance to ramipril developed during long-term treatment, a subgroup of 202 patients was analyzed for efficacy maintenance. Prior to enrolling in the 2-year study, these patients had received ramipril monotherapy in a short-term, double-blind study and had been classified as responders, i.e., their diastolic blood pressure had been maintained at less than or equal to 90 mm Hg. At the end of 104 weeks of treatment, 45.9% of patients were on 2.5 mg of ramipril alone and 43.6% were on 5 mg of ramipril alone. Only four patients required the addition of 25 mg of hydrochlorothiazide. No clinically important changes occurred, and kidney function was well maintained. The most frequently reported adverse events excluding intercurrent illnesses were dizziness/vertigo (6%), asthenia (4%), nausea (3%), headache (2%), and abdominal pain, gastrointestinal disorder, rash, and increased cough (1% each). Ramipril was safe, effective, and well tolerated in the long-term treatment of patients with mild-to-moderate essential hypertension. PMID- 1725025 TI - Angiotensin II: vasoconstrictor or growth factor? AB - The consequence of a sustained rise in blood pressure is an adaptive change in the structure of the heart and the vasculature. In humans, the left ventricle, aorta, and medium-sized arteries undergo hypertrophy whereas the changes in resistance vessels are unclear at present. Recent studies suggest that left ventricular hypertrophy is a better predictor of stroke or cardiac risk than age or blood pressure. Therefore, the cellular mechanisms that bring about these structural alterations to the circulation in hypertension are the subject of intense research at this time. The main factor was assumed to be the pressure load imposed on the wall of the left ventricle and the arteries. However, recent work has suggested that pressor hormones can act as growth factors in vascular tissues by pressure-independent actions. This article examines the cellular mechanisms activated by angiotensin II that could be involved in producing growth and the evidence available that this nonpressor role is important in vitro and in vivo. PMID- 1725026 TI - Tolerability of ramipril in a multicenter study of mild-to-moderate hypertension in general practice. AB - In this study, the tolerability and safety of ramipril, as monotherapy and in combination with a low dose of furosemide, were assessed in patients with mild-to moderate hypertension in general practice. After a placebo run-in phase, patients received ramipril as monotherapy in a dose of 2.5 to 5 mg daily for 6 weeks. Nonresponders (diastolic blood pressure greater than 90 mm Hg) entered a double blind treatment period, and received either 10 mg of ramipril daily, or 5 mg of ramipril in combination with 20 mg of furosemide daily. The tolerability of the study medication was assessed by reported adverse events, and by monitoring blood cell count, electrolytes, serum creatinine, fasting blood glucose, and apolipoproteins AI and B. Of a total of 770 patients who entered the placebo run in phase, 661 patients were enrolled in the first active treatment period. The most commonly reported adverse events were headache, cough, dizziness, asthenia, cramps, diarrhea, and nausea, but not all of these events were related to ramipril treatment. A total of 38 patients discontinued active treatment due to nonserious adverse events, mainly cough, dizziness, or diarrhea. There appeared to be a relationship between the prevalence of cough and ramipril dosage; however, an increased incidence of cough was also observed during outbreaks of influenza in France. There were no significant changes in laboratory variables during the study. PMID- 1725027 TI - Comparison of response rates to the angiotensin-converting enzyme inhibitor ramipril in mild-to-moderate hypertension in a double-blind, parallel-group study and an open single-blind study. AB - Appropriate clinical trial methodologies in general practice and suitable end points for dose-finding studies are discussed with reference to antihypertensive drugs in general and angiotensin-converting enzyme (ACE) inhibitors in particular. Two clinical studies were conducted with ramipril, a new nonsulfhydryl ACE inhibitor, to identify the minimum effective dose for the management of mild-to-moderate hypertension. Study 1 was a double-blind, parallel group, randomized design with three treatment groups (placebo and 2.5 and 5 mg of ramipril), and study 2 was an open, single-blind design with individual dose titration from 2.5 to 5 mg of ramipril if the diastolic blood pressure (DBP) was greater than 90 mm Hg after 3 weeks. Response rates and DBP reductions with 2.5 mg of ramipril were similar in both studies, although overall response rates, DBP reductions, and side effect incidence appeared to be greater in the open, single blind, dose-titration study. It is concluded that study methodology apparently influences efficacy and tolerability. PMID- 1725028 TI - Efficacy and safety of a new angiotensin-converting enzyme inhibitor, ramipril, vs. enalapril in essential hypertension: a multicenter trial. AB - The antihypertensive effect of ramipril, a new angiotensin-converting enzyme (ACE) inhibitor, was evaluated and compared to enalapril in a double-blind, randomized, controlled trial. Subjects received either 2.5, 5, or 10 mg of ramipril given once daily or 5, 10, or 20 mg of enalapril once daily for 4 weeks. Significant decreases from baseline in supine and standing diastolic blood pressures were seen in all dosage groups at end point. There were significant decreases at end point for supine and standing systolic blood pressures at the higher doses. Side effects were minimal and were similar for all treatment groups. PMID- 1725029 TI - Comparison of ramipril against atenolol in controlling mild-to-moderate hypertension. AB - This trial was a multicenter, double-blind, randomized, parallel-group, variable dose study comparing ramipril with atenolol, and incorporating a 4-week run-in period on placebo. A total of 92 patients suffering from mild to moderate hypertension were randomized to the trial. Following a 4-week run-in period on placebo, newly diagnosed patients were only continued if their blood pressure remained within the defined range throughout the placebo run-in period. Known hypertensive patients who were safely switched to the trial therapy were only continued if their blood pressure was within the defined range at the end of this placebo washout period. An analysis of the data at baseline showed no statistical differences between treatment groups. Both systolic and diastolic blood pressures were significantly reduced from baseline in each group, but there were no differences between treatments. The study showed that ramipril and atenolol were both successful at reducing and controlling mild-to-moderate hypertension, but it showed no difference between treatments. PMID- 1725030 TI - Effects of ramipril on arterial hemodynamics. AB - The acute and chronic arterial effects of the angiotensin-converting enzyme (ACE) inhibitor ramipril were studied in hypertensive patients. Hemodynamic and biological parameters were measured 3 h after the first dose of 5 mg of ramipril, and then again after 4 weeks of treatment, 3 and 24 h after drug administration. Brachial and carotid artery hemodynamics were evaluated using a two-dimensional pulsed Doppler system. Arterial distensibility was studied noninvasively in three arterial segments (carotidofemoral, brachioradial, and femorotibial) by evaluating the pulse wave velocity. Ramipril lowered the blood pressure significantly after acute and chronic administration. Chronic treatment with ramipril was followed by a long-lasting increase in brachial artery diameter, a decrease in forearm vascular resistance, and an improvement in aortic distensibility. The other arterial segments studied did not show any significant changes. Our results suggest that the long-lasting arterial effects of the ACE inhibitor ramipril are partly pressure independent and are related to effects on arterial tone that may reduce the cardiovascular abnormalities associated with hypertension. PMID- 1725031 TI - The effects of ramipril on glucose tolerance, insulin secretion, and insulin sensitivity in patients with hypertension. AB - To evaluate a possible influence of the angiotensin-converting enzyme inhibitor ramipril on glucose tolerance and insulin sensitivity, an oral glucose tolerance test (oGTT) and an euglycemic clamp were performed in 10 nonobese, nondiabetic patients with mild hypertension before and after treatment with ramipril for 14 days. Following ramipril treatment, systolic and diastolic blood pressures were significantly lower (152.5 +/- 10.6/97.5 +/- 4.3 vs. 136.5 +/- 18.9/79.5 +/- 16.4 mm Hg). Therapy with ramipril showed no influence on glucose tolerance (serum glucose of 106.8 +/- 32.8 vs. 109.1 +/- 33.9 mg/dl at 120 min during the oGTT), insulin secretion (53.0 +/- 45.7 vs. 41.1 +/- 10.6 microU/ml at 120 min), and insulin sensitivity (glucose infusion rate of 180.8 +/- 60.7 vs. 199.8 +/- 77.5 mg/m2/min after 3 h of clamp). In conclusion, short-term treatment of hypertension with ramipril has no influence on glucose metabolism in nondiabetic patients. PMID- 1725032 TI - Assessment of the efficacy, tolerance, and safety of ramipril in diabetic patients with mild-to-moderate hypertension: a retrospective analysis. AB - Six double-blind studies were designed to assess the efficacy, tolerance, and safety of the angiotensin-converting enzyme inhibitor ramipril in patients with mild-to-moderate essential hypertension. Of 1,189 hypertensive patients in these studies, 105 patients were diabetic. They were randomly assigned either to a ramipril monotherapy group (1.25-10 mg/day) or to one of the following treatment groups: ramipril (5 mg/day) plus piretanide (3 mg/day), captopril (100 mg/day), enalapril (10-20 mg/day), hydrochlorothiazide (50 mg/day), or atenolol (100 mg/day). In all studies, a 4-week single-blind placebo run-in phase was followed by a 6-week double-blind active treatment phase. Significant reductions in blood pressure were achieved with all antihypertensive agents. No statistically significant deleterious effects were observed on concentrations of blood glucose, although diabetics who received hydrochlorothiazide showed slight increases in blood glucose levels. Ramipril was well tolerated by diabetic patients, and no serious adverse events occurred. Adverse events reported were typical of ACE inhibitors. PMID- 1725033 TI - Small doses of ramipril to reduce microalbuminuria in diabetic patients with incipient nephropathy independently of blood pressure changes. AB - The mechanism of action of angiotensin-converting enzyme (ACE) inhibitors on urinary albumin excretion (UAE) in diabetic patients remains controversial. Sixteen type 1, insulin-dependent diabetics with incipient nephropathy received ramipril, a long-acting ACE inhibitor, at hypotensive doses (treatment A: 5 mg/day, n = 8) or at nonhypotensive doses (treatment B: 1.25 mg/day, n = 8) during a 6-week, double-blind, parallel study to establish whether its antihypertensive effects could be dissociated from its local renal effects. Blood pressure, UAE, glomerular filtration rate (GFR), effective renal plasma flow (ERPF, constant [125I]iodothalamate + [131I]hippurate infusion), and ACE activity were measured before and after treatment. Blood pressure was lowered with treatment A but not with treatment B. UAE and ACE activity were reduced with both treatments. Baseline GFR and ERPF were not altered by either treatment. In the patient population as a whole, ACE inhibition correlated with a rise in ERPF and with a reduction in filtration fraction (GFR/ERPF), but not with the changes in blood pressure. Changes in UAE correlated with the changes in filtration fraction. It is concluded that renal hemodynamics may be modified by ramipril independently of blood pressure changes. PMID- 1725034 TI - Efficacy and safety of ramipril in combination with hydrochlorothiazide: results of a long-term study. AB - Hypertensive patients from a double-blind study comparing 5 mg of ramipril, 10 mg of ramipril, and 5 mg of ramipril + 25 mg of hydrochlorothiazide (HCTZ) were enrolled in an open 1-year extension study with ramipril and concomitant HCTZ. The starting dose of ramipril was 5 mg/day. Patients were given 25 mg of HCTZ in addition only if their diastolic blood pressure (DBP) was higher than 90 mm Hg. During treatment, the investigator was permitted to adjust the dosage of ramipril and HCTZ according to BP response and tolerance. A total of 159 patients were included in the 1-year study (86 men, 73 women, mean age of 54 years). One hundred twenty-one of the 159 patients received the combination of ramipril + HCTZ at some point in the study, 83 of them for more than 50 weeks. Thirty-eight patients were treated with ramipril alone over the entire study. In patients treated with ramipril monotherapy throughout the study, the largest drop in blood pressure occurred before visit 1. In patients treated with ramipril + HCTZ for greater than or equal to 50 weeks, the mean blood pressure continued to fall up to around week 10, while the therapy was being adjusted. Subsequently, the mean blood pressures remained low and fairly stable in both treatment groups. Similar results were seen in patients treated with the combination for less than 50 weeks. Adverse events were reported in 11 of the 38 patients in the ramipril group, in 22 of 83 patients treated with the combination for more than 50 weeks, and in 9 of 38 patients treated with the combination for 50 weeks or less. The analysis of the laboratory values revealed no general deterioration. It can be concluded that ramipril alone and in combination with HCTZ is an effective and safe drug for the long-term treatment of essential hypertension. PMID- 1725035 TI - Influence of ramipril on renal function in patients with chronic congestive heart failure. AB - Ramipril was administered to 13 patients, aged 49-80 years, weighing 53.5-90.3 kg, with congestive heart failure (CHF) for 2 weeks in an open trial. One patient dropped out after day 1 and another accidentally received another angiotensin converting enzyme (ACE) inhibitor, so that 11 patients remained for analysis. Each patient received 5 mg of ramipril daily for 2 weeks, followed by a 1-week washout period. Urine was collected in 4-h fractions for 12 h followed by a 12-h fraction on days 1, 8, and 15. Creatinine clearance, as well as the excretion of albumin and the urinary brush border enzymes gamma-glutamyl transferase (GGT), alanine aminopeptidase (AAP), N-acetyl-beta-D-glucosaminidase (NAG), and lactate dehydrogenase (LDH) were determined. The values for albumin excretion, creatinine clearance, GGT, AAP, NAG, and LDH obtained for the various days during the time course before and during multiple dosing were subjected to an analysis of variance followed by Scheffe's test for means. Daily albumin excretion decreased during treatment with ramipril from 23.8 +/- 27.4 mg/24 h (baseline) to 12.9 +/- 15.1 (day 1), 10.8 +/- 15.6 (day 8), and 12.4 +/- 12.7 mg/24 h (day 15). The differences were significant (alpha = 0.05) when compared to pretreatment albumin excretion. Creatinine clearance increased from 78.8 +/- 38.3 ml/min (baseline) to 82.4 +/- 34.6 (day 1), 85.1 +/- 28.5 (day 8), and 91.7 +/- 36.4 ml/min (day 15). The change on day 15 was significant (alpha = 0.05) when compared to pretreatment values.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725036 TI - A comparison of the efficacy and safety of ramipril and digoxin added to maintenance diuretic treatment in patients with chronic heart failure. AB - The efficacy and safety of ramipril were compared with that of digoxin in a prospective, randomized, double-blind, crossover study of 35 patients with congestive heart failure (CHF), New York Heart Association (NYHA) grades II to IV, stabilized on diuretic maintenance therapy. Major assessments were conducted at baseline and at the end of each 10-week treatment period: primary efficacy variables were total exercise duration (modified Bruce, treadmill), NYHA grade, and clinical signs and symptoms (by visual analogue score) of heart failure. Twenty-seven patients completed the study. There were two deaths (one on each study drug) and six patient withdrawals (one on ramipril and five on digoxin). Although the NYHA grade was significantly better on ramipril than on digoxin, there were no other important differences in the relief of either signs or symptoms of heart failure. A significant order effect was observed with the exercise testing data and therefore only data in the first active treatment period were analyzed; no significant differences were noted. There were fewer reports of adverse effects, and no clinically significant episodes of hyperkalemia or renal impairment on ramipril. We conclude that ramipril seems to be better tolerated and marginally more effective than digoxin in the management of patients with moderate to severe chronic CHF, stabilized on maintenance diuretic therapy. PMID- 1725037 TI - Tissue renin-angiotensin systems: fact or fiction? AB - The discovery of the components of the renin-angiotensin system (RAS) in various tissues gave rise to the idea that functional "tissue" RASs exist that are more or less independent of the hormonal RAS. Further support for this notion came from the recent demonstration of the mRNA for the protein components of the RAS in a number of organs. Last but not least, the introduction of the converting enzyme (CE) inhibitors, generating an enormous scientific interest in the role of the RAS, has contributed to the concept of "tissue" RASs, since some of the effects of these drugs were thought to be reconciled better with "tissue" than with plasma RAS inhibition. However, this model of plasma vs. "tissue" RAS still suffers from a number of conceptual problems. For instance, with respect to the "tissue" RAS in the vascular wall, it is not clear at present whether angiotensin converting enzyme (ACE) is at all active inside endothelial cells. In addition, all evidence available speaks against its localization in the vascular media, while there may be some activity of the enzyme in the adventitial layer. Concerning the heart, there is at present no unequivocal evidence for a local angiotensin II (Ang II) production through ACE in cardiac tissue outside the coronary vessels. The localization of Ang II generation within tissue RASs in the adrenal gland or in the kidney, where Ang II may be generated through CE at the tubular brush border, are far from being elucidated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725038 TI - Experimental cardiovascular benefits of angiotensin-converting enzyme inhibitors: beyond blood pressure reduction. AB - In an attempt to separate the cardiac effects of converting-enzyme (CE) inhibition from those on blood pressure, experiments were performed in rats and dogs with nonantihypertensive (subhypotensive) doses of the CE inhibitor ramipril. (a) Left ventricular hypertrophy: Rats with aortic constriction treated with a nonantihypertensive dose of ramipril (10 micrograms/kg/day) for 6 weeks showed the same prevention and regression of cardiac hypertrophy as groups receiving the antihypertensive dose of 1 mg/kg/day. Comparable results were obtained in animals treated for 1 year. (b) Ischemia-reperfusion injuries: In rats, subchronic oral administration of ramipril in a subhypotensive dose (10 micrograms/kg/day) prevented ex vivo postischemic reperfusion arrhythmias and improved cardiodynamic and metabolic parameters. Almost complete inhibition of cardiac CE was achieved with this low dose. (c) Acute myocardial infarction: Ramiprilat (40 ng/kg/min) was infused for 6 h into the left coronary artery of anesthetized dogs with a ligation of the descending branch of this artery. This route and the low dose were chosen to achieve local cardiac effects without affecting systemic hemodynamics. Ramiprilat significantly reduced the infarct area expressed as a percentage of the area at risk. This cardioprotective effect of ramiprilat was mimicked by bradykinin and abolished by coadministration of a bradykinin antagonist. Thus, factors beyond blood pressure reduction and load changes may add to the cardiovascular benefits of CE inhibitors. This may indicate local (cardiac) paracrine and/or autocrine effects of the renin angiotensin system and/or participation of kinins. PMID- 1725039 TI - Hypertension and coronary artery disease: an unsolved problem. AB - Drug treatment of hypertension reduced the incidence of stroke by 40% and prevented cardiac and renal failure, but had less effect on coronary artery disease in the major trials. Coronary events were reduced by about 14% (confidence limits of 4-22%), which compares to the reduction of 20-25% anticipated from epidemiological studies. Any shortfall in coronary prevention that could be attributable to adverse effects of antihypertensive drugs would therefore amount to only 6-11% of all coronary events, and could easily arise by chance. At present, there are no grounds for preferring one class of drug over another from the point of view of coronary prevention, or for altering the "target" diastolic pressure during treatment. Strategies other than blood pressure reduction aimed at preventing coronary disease in hypertensive subjects need to be explored. They should, however, be examined in large controlled outcome trials, and should not be introduced to ordinary practice on the basis of the inadequate evidence currently available. PMID- 1725040 TI - Treatment of hypertension: a clinical epidemiologist's view. AB - The majority of deaths attributable to hypertension are coronary artery disease (CAD) deaths and, consequently, the prevention of CAD should be the primary aim of hypertension management. Recent meta-analyses confirm the results of the individual hypertension intervention trials, which demonstrated a disappointing shortfall in the observed prevention of CAD events and mortality from lowering blood pressure compared with the expected benefits. These trial results, rather than challenging the validity of the causal nature of the association between hypertension and CAD may be interpreted to suggest that the management of hypertension in the trials was suboptimal. The drugs used in the trials were almost exclusively thiazide diuretics and to a lesser extent beta-blockers. Both of these drug groups have been shown to have adverse effects on lipid profiles--a pivotal risk factor for CAD. In addition to the effects on lipids, diuretics also adversely affect potassium, uric acid, glucose metabolism, and insulin resistance, all of which directly or indirectly affect the incidence of CAD. In the face of a major shortfall in the overall benefit from managing hypertension with diuretics and to a lesser extent beta-blockers, it therefore seems more logical to recommend for the management of hypertension the use of agents with a more metabolic-friendly profile. PMID- 1725041 TI - Is there a cellular abnormality in hypertension? AB - There are probably weak associations between altered blood cell ion handling and hypertension. This has been most consistently demonstrated for sodium-lithium countertransport and erythrocyte binding of calcium. These associations can be demonstrated when allowance is made for such confounding factors as age, weight, sex, and ethnic background. It is proposed that there is a disturbance in the physicochemical properties of the cell membrane in essential hypertension in humans and in the spontaneously hypertensive rat. Although alterations in smooth muscle growth or contractility can be demonstrated in some situations, there is no evidence that this is of primary pathogenetic importance. It is accordingly suggested that blood cell ion transport disturbances are serving as markers of a primary disorder in autonomic rather than vascular smooth muscle function. PMID- 1725042 TI - Hypertension, insulin, and atherogenesis. AB - Essential hypertension and non-insulin-dependent diabetes mellitus are both associated with hyperinsulinemia and it has been proposed that this might contribute to increased atherogenesis in these conditions. In hypertension, hyperinsulinemia probably reflects reduced insulin-stimulated glucose uptake, but the reason for this, and the contribution of hyperinsulinemia (or of resistance to insulin) to the development of hypertension and atheroma, remains unclear. As well as glucose uptake, insulin has important effects on other aspects of cell function; for example, the hormone is an important regulator of the expression and function of the major inhibitory guanine nucleotide binding protein Gi. In insulin deficiency, Gi levels and function are greatly reduced and are restored by insulin treatment. We have examined whether in human hypertension or in animal models of hypertension there is evidence of abnormal regulation of this protein. Platelet membranes from humans and rat membranes from a range of tissues, including myocardium and vasculature, were studied. No alteration in Gi levels or function was found in these studies, and there is no evidence that this aspect of insulin action on cell function is abnormal. Insulin is also involved in the regulation of cell growth, and in vascular smooth muscle cells there is evidence that this effect involves action of other growth factors, such as PDGF. If the growth regulatory actions of insulin are also unimpaired despite limitation of insulin-stimulated glucose uptake, chronic hyperinsulinemia could lead to increased vascular smooth muscle cell growth and contribute to development of atheroma. PMID- 1725043 TI - Microvascular hemodynamics in hypertension and diabetes. AB - Microvascular damage occurs in both diabetes and hypertension and hypertension is a risk factor for diabetic microangiopathy. In both conditions, indirect evidence suggests that capillary pressure might be raised. A servonulling pressure measuring technique has been used in conjunction with direct micropuncture of finger nailfold capillaries to determine capillary pressure dynamically. In patients with essential hypertension, capillary pressure is raised compared to matched normotensive controls. In insulin-dependent diabetic patients, capillary pressure is also raised, to a degree that correlates with recent diabetic control. In a pilot study of hypertensive diabetic patients, elevated capillary pressure has been normalized using an angiotensin-converting enzyme inhibitor. Manipulation of microvascular hemodynamics in diabetes and hypertension may provide a means of protecting against the microvascular complications of these two conditions. PMID- 1725044 TI - Preservation of renal function in diabetic patients. AB - Hypertension in type I diabetes appears to be causally related to nephropathy, whereas the relationship between hypertension and type II diabetes is more complex, hypertension being present in the absence of clinically overt nephropathy and even preceding the onset of diabetes. In the diabetic, hypertension is exquisitely sodium-sensitive. It is in diabetic nephropathy that the best evidence has been obtained that treatment of hypertension retards the progression of renal failure. Consensus is still lacking with respect to the point when antihypertensive treatment should be started and the target blood pressure that should be aimed at. Currently, there is no evidence in humans that converting enzyme inhibitors are superior to alternative antihypertensive agents in retarding progression, but tantalizing preliminary evidence on this has been reported in nondiabetic patients with renal failure. PMID- 1725045 TI - Effects of antihypertensive treatment on coronary artery disease: directions for future research. AB - Previous trials have shown that a 5-6 mm Hg reduction in diastolic blood pressure produced by antihypertensive treatment (mainly diuretics) reduces the risk of coronary artery disease (CAD) by 14% SD5 (2p less than 0.01). However, the 95% confidence limits for this estimate of treatment effect are wide and consistent, with true reductions as small as 4% or as large as 22%. For this reason, it is not possible to determine whether the treatment benefit is of a worthwhile magnitude. Because CAD remains the leading cause of death in hypertensive patients (and normotensive patients) in most Western populations, further studies are required to determine more precisely the effect of blood pressure reduction on the incidence of CAD. This could be achieved by further large-scale studies comparing antihypertensive treatment with no treatment. It could also be achieved by comparing the effects of more and less intensive antihypertensive treatment regimens. Additional relevant information might also be generated by studies comparing the effects on CAD of new classes of antihypertensive drugs (such as angiotensin-converting enzyme inhibitors and calcium antagonists) with those of older classes (in particular diuretics). In all such studies, the detection of plausible treatment effects or of plausible treatment differences requires the recruitment of large study populations with follow-up continued for several years. This is only feasible if study methods are kept simple to insure the widest possible collaboration. PMID- 1725046 TI - The treatment of hypertension: a therapeutic philosophy for the 1990s. AB - To date, a range of drugs are available that are generally well tolerated and effective in lowering blood pressure. Although they are successful in reducing stroke, renal failure, and cardiac failure, they have a disappointing and less than expected influence on coronary artery disease and its manifestations. The genetic and environmental factors determining susceptibility to atherosclerosis and coronary artery disease are now more clearly defined and interactions between risk factors and protective mechanisms recognized. Drug treatment of hypertension must become a part of the overall approach to prevention of cardiovascular disease and possible health promotion. Dietary and hygienic measures (cessation of smoking and control of alcohol intake) should be combined where necessary with specific treatment of hypertension and hyperlipidemia. Future drug treatment must not only be effective and well tolerated but should complement other preventive approaches. In view of the increasing recognition that blood pressure treatment with a single drug is unlikely to be successful in all patients, there is likely to be a role in the future for pharmacologically coherent low-dose combinations of antihypertensive drugs. PMID- 1725047 TI - The cardiomyopathy of overload: a hypothesis. AB - Progressive deterioration of the failing heart is now recognized to be a major cause of disability and death in patients with congestive heart failure. It appears that although the normal human heart functions for at least 90-100 years, overload-induced hypertrophy shortens the heart's life span to about 5 years. This accelerated deterioration of the failing heart can be viewed as a cardiomyopathy of overload in which chronic overloading causes changes in the myocardial cells that, while increasing cell mass, reduce their long-term viability. Although the pathogenesis of this putative cardiomyopathy remains poorly understood, chronic energy starvation and altered myocardial cell growth and composition appear to be contributing factors. Preferential expression of fetal isoforms of key muscle proteins, which accompanies accelerated growth of the overloaded heart, may contribute to this cardiomyopathy. The hypothesis that the cardiomyopathy of overload is due in part to a growth abnormality is supported by evidence that deterioration of the failing heart is slowed by the converting enzyme inhibitors, which may attenuate important effects of angiotensin II to stimulate cellular growth. PMID- 1725048 TI - Molecular and cell biology of angiotensin receptors. AB - The sites that initiate angiotensin effects on vascular and extravascular tissues have historically been identified as "the angiotensin II (Ang II) receptor." However, this unitary perspective of a single receptor responding to a single hormone has been revised with the molecular cloning of angiotensin-responsive receptors. The mammalian proto-oncogene MAS has been identified as a novel neuronal angiotensin receptor, which responds preferentially to Ang III, and has other unusual pharmacological properties. Although its tissue distribution suggests it may function normally in the brain in sites not normally associated with angiotensins, it shares many structural and functional features with the cloned vascular Ang II receptor. The understanding of the MAS/angiotensin receptor may compel a basic rethinking of many aspects of the cardiovascular biology of the renin-angiotensin system. PMID- 1725049 TI - Myocardial mechanics and arrhythmia. AB - The initiating cause of the first ectopic beat and its precipitation of sustained lethal arrhythmia in acute myocardial ischemia is not clear. Comparable uncertainties surround sudden death in myocardial failure. Progress in control of ventricular fibrillation has been slow, perhaps because diagnosis and treatment have been based on the premise that ischemic biochemical changes solely cause the alterations in electrophysiological behavior. Alternative approaches need exploration. Evidence that mechanical changes can initiate electrophysiological changes by a process sometimes referred to as "mechanoelectric feedback" is accumulating. It operates when any primary mechanical change in ventricular muscle, e.g., contraction produces a change in its electrical properties. Mechanical changes are prevalent in ischemia and cardiac failure. If mechanoelectric feedback operates here, it is not surprising that arrhythmias in some of these pathologies parallel the degree of mechanical myocardial dysfunction rather than electrophysiological changes, independent of etiology. This mechanoelectric feedback system may exist as an intrinsic property of normal myocardium, providing a feedback control of activation processes at both the cellular and gross levels. Its disruption during pathological events produces instability in the system and thus ventricular arrhythmia. This concept provides a new potential avenue for arrhythmia therapy. PMID- 1725050 TI - Why do patients die after myocardial infarction? AB - Death during and following myocardial infarction can arise from a number of different causes. Some of them, such as early ventricular fibrillation and cardiac rupture, seem unrelated to infarct size. However, deaths occurring later during the course of infarction do seem to be related to the extent of myocardial damage and to such phenomena as infarct extension and expansion, and the mechanisms involved include cardiac failure and shock, and late arrhythmias. Preventive measures must be directed at the various mechanisms involved. Thrombolytic drugs, by limiting infarct size, prevent death from several causes, whereas beta-blockers seem mainly to operate by preventing early rupture, reinfarction, and late ventricular fibrillation. Aspirin prevents reinfarction. Angiotensin-converting enzyme (ACE) inhibitors may prove to have a beneficial role in the early phase by unloading the heart and later by preventing infarct expansion and subsequent cardiac failure. PMID- 1725051 TI - Pathophysiologic factors influencing prognosis after myocardial infarction. AB - The major determinants of outcome after surviving the acute phase of myocardial infarction include the extent of cumulative myocardial damage, infarct type and location, ventricular electrical instability, the extent of residual jeopardized ischemic myocardium, rheologic conditions, and neurohumoral factors. These several factors are intimately related to each other, yet each contributes prognostic information. Patients with multiple myocardial infarctions are at considerably greater mortality risk than those with first infarctions. Patients with first infarctions are approximately 2 years younger, have less advanced coronary disease, have less left ventricular dysfunction, and have a better outcome than those with one or more prior infarctions. Recently, the importance of nonfatal reinfarction as a time-dependent predictor of subsequent cardiac death has been appreciated. The relative contribution of nonfatal reinfarction to the risk of cardiac death is highest in patients with a first myocardial infarction. These findings indicate that prevention of further myocardial damage (reinfarction), optimization of left ventricular function, and reduction in myocardial ischemia should improve prognosis after myocardial infarction. PMID- 1725052 TI - Thrombolysis, left ventricular function, and mortality. AB - Coronary thrombolysis is now accepted as effective treatment for coronary thrombosis and myocardial infarction. The a priori hypotheses that thrombolysis works by restoring coronary patency and that coronary reperfusion preserves myocardial function have been disputed but not disproved. This report discusses the effects of reperfusion on ventricular function, the limitations of present techniques for assessing myocardial salvage, and the implications for future clinical studies of these limitations. PMID- 1725053 TI - Antiarrhythmic therapy and survival following myocardial infarction. AB - Arrhythmias remain a major cause of late mortality following myocardial infarction. They arise due to fibrosis within the infarct, which creates the conditions of slow conduction necessary for re-entry. In individual patients who have already manifested a malignant arrhythmia, antiarrhythmic drug therapy, guided by invasive electrophysiological testing, is of proven benefit in prolonging survival. By contrast, when used on a population basis, antiarrhythmic drug therapy has proved singularly ineffective. This is illustrated by the recent Cardiac Arrhythmia Suppression Trial (CAST) study--far from improving survival, antiarrhythmic therapy increased mortality. The use of antiarrhythmic drugs on a population basis is therefore fundamentally flawed. Hemodynamic intervention provides an alternative strategy in arrhythmia prevention. Hemodynamic changes may influence electrophysiological parameters and arrhythmogenesis in a number of ways. First, myocardial stretch may contribute to arrhythmogenesis through contraction-excitation feedback. Second, hemodynamic changes can influence ventricular remodeling following infarction, which may be an important determinant of subsequent arrhythmogenesis. Hemodynamic intervention, therefore, represents a promising new approach to arrhythmia prevention following myocardial infarction. PMID- 1725054 TI - Angiotensin-converting enzyme inhibitors in heart failure: a role after myocardial infarction. AB - The prognosis for clinical congestive heart failure remains poor even with modern treatment as severe ventricular dysfunction is often present at the time of clinical presentation. A substantial improvement in prognosis might be achieved through earlier intervention and a preventive approach to treatment following myocardial infarction to delay progressive ventricular dilation and the occurrence of clinical heart failure. The rationale for treatment of left ventricular dysfunction following myocardial infarction is further supported by the prognostic importance of ventricular dilation and by experimental animal studies that demonstrate that converting enzyme inhibition can improve ventricular function and survival following myocardial infarction. Similarly, clinical studies have demonstrated that converting enzyme inhibition can improve ventricular function during the year following transmural myocardial infarction. Possible mechanisms of action that require further understanding include ventricular afterload reduction and direct coronary and tissue effects of angiotensin II blockade. The further benefit to be obtained from very early intervention following myocardial infarction is currently being addressed in several studies. Large-scale studies also in progress should determine the mortality benefit of such treatment. Further questions that remain include the optimal timing of intervention, comparison with other treatments such as nitrates and beta-blockade, and also the use of appropriate combination treatments. PMID- 1725055 TI - Prevention of chemotherapy-induced leukemia and of leukemia relapses. AB - Fifty percent of patients with the myelodysplastic syndrome, frequently following treatment by radiation or chemotherapy, have prognostically unfavorable deletions of the long arms of chromosomes 5 and 7, or trisomy 8, as have the 25% of patients with acute myeloblastic leukemia where remissions last 6-12 months, and where relapse cannot be prevented. In contrast, patients with prognostically favorable cytogenetics (translocation 15; 17 or 8; 21 or inversion 16) maintenance chemotherapy may prevent relapses. Of chronic myelocytic leukemia patients, 85% can achieve hematological remission with interferon alpha, and 40% a partial cytogenetic remission, which probably delays relapse. PMID- 1725056 TI - Methods for field detection of resistance to temephos in simuliids. Larval esterase level and topical application of the insecticide to adults. AB - Two practical field methods for indirect detection of simuliid populations resistant to temephos are proposed. The first is based on high esterase activity in resistant larvae and involves adaptations of a filter paper test in which faintly stained spots indicate susceptible populations and strongly stained ones reveal populations resistant to temephos. The second is based on the resistance to the larvicide when adults are topically exposed, and involves the use of diagnostic doses obtained by the comparison between the LD50 for susceptible and resistant populations. The relevance of such methods is discussed in order to help resistance detection in Simulium pertinax Kollar control programmes. PMID- 1725057 TI - [Aphasia without alexia after surgical treatment of aneurysm of the right middle cerebral artery--incomplete lateralization of verbal functions?]. AB - The authors present a case of aphatic disorders after surgical treatment of the right middle cerebral artery aneurysm. A prominent dissociation of verbal function was noticed. PMID- 1725058 TI - Molecules specific to pigment epithelial cells: expression during in situ development and in vitro lens transdifferentiation of chick embryo pigment epithelium. AB - The retinal pigment epithelium (PE) is a monolayer of cells and plays a vital role in the regulation of the neural retina. We prepared monoclonal antibodies directed against retinal PE cells to analyze the specificity and differentiation of these cells. Spleen cells from BALB/c mice immunized with chick embryo retinal PE cells were fused with myeloma cells. Seven independent monoclonal antibodies were obtained which specifically recognized PE cells but did not react with any other tissues examined. None of the monoclonal antibodies reacted with the choroid and skin of pigmented chicks, suggesting that these antigens were unrelated to melanogenesis. Two of the 7 antibodies reacted with the PE cells in the retina, ciliary body and iris; the remaining 5 antibodies were specific to the PE cells in the retina. In the process of in vitro lens transdifferentiation from PE cells, the distribution of an antigen detected by one monoclonal antibody changed from the cytoplasmic granules to the actin fibers and then its immunoreactivity declined. The other monoclonal antibodies did not react with the differentiated PE cells and transdifferentiated lens cells, suggesting that the antibodies might be specific to the PE cells in the differentiated state, both in vivo and in vitro. During the in situ developmental process, each monoclonal antibody began to be immunoreactive to future PE cells in the optic eye cup at various stages from 72 to 120 h. The molecules common to all types of PE cells were expressed earlier than those specific to PE cells of the retina. Future ciliary and iridial PE cells appeared to transiently express the molecules specific to the retinal PE cells before the tip of eye cup contacted the lens vesicle. These data suggest that the monoclonal antibodies established in this study are powerful probes for exploring the functions and differentiation of PE cells. PMID- 1725059 TI - Insulin-like growth factor production by childhood solid tumors. PMID- 1725060 TI - [Surfactant-inducted suppressive action on fibroblasts of pulmonary macrophages from rats with fibrosis after intratracheal administration of bleomycin]. AB - Influence of lung macrophages and their contact with a surfactant on the fibroblast proliferative capacity was studied. It was found that fibroblasts added to the medium after culturing macrophages isolated from the rat lungs 3 weeks after intratracheal bleomycin administration led to a lower incorporation of 3H-thymidine into the fibroblast fraction precipitated with trichloroacetic acid. This phenomenon partially depended on prostaglandin synthesis in both types of cells. Preliminary macrophage incubation with the lung surfactant during 8 hours potentiated the inhibitory effect greatly. The use of indomethacin eliminated the surfactant-induced effects of macrophages on the fibroblasts. The surfactant isolated from the lung at the peak of bleomycin-induced fibroblast formation had a more pronounced suppressive action than that obtained from the control animals. Discussion of the results leads to the concept of the protective role of the lung surfactant in the formation of interstitial pneumofibrosis, which is aimed at lowering the proliferative capacity of fibroblasts and the hyperproduction of collagen. PMID- 1725061 TI - Endogenous opioid dependence--a basic pathophysiological phenomenon of stress induced and genetically fixed disturbances in adaptation--influence of substance P. AB - Disturbances in adaptive processes can be induced by chronic exposition to stress or can result from a genetical predisposition. Experimental data of chronically stressed Wistar rats and of spontaneously hypertensive rats (SHR) demonstrate a relation between a decreased level of substance P (SP) in adrenals, the existence of a dependence on endogenous opioid peptides and an increased regulatory level of blood pressure. The endogenous level of SP was determined by using a RIA. The dependence (physical) on endogenous opioid peptides was detected by using the method of "gut dependence". SP injection i.p. once a day for 4 d antagonized the dependence on endogenous opioid peptides and normalized the increased level of blood pressure in both animal models. Investigations on SHR had shown that the adaptive effect of SP on blood pressure and endogenous opioid dependence is bound to the premise of an acute stimulated endogenous opioid system at the moment of SP-application. Experimental findings suggest that different systems of opioid peptides take part in the etiopathogenesis of genetically predisposed hypertension of SHR and in stress-induced increase of blood pressure level of Wistar rats. The effect of SP on blood pressure and endogenous opioid dependence will be discussed as a result of the modulatory influence on the cholinergic opioid-peptidergic interaction. PMID- 1725062 TI - Mapping of linear B-cell epitopes of hepatitis B surface antigen. AB - Eight monoclonal antibodies directed against the surface protein of hepatitis B virus (HBV) were tested using an epitope-mapping system (Pepscan) for characterizing antigenic domains. Four different amino acid sequences corresponding to linear epitopes were identified: one in pre-S1 corresponding to the sequence 29-36, two in pre-S2 corresponding to overlapping sequences 134-141 and 137-144, and one in the S region of the protein corresponding to the amino acid sequence 117-126. PMID- 1725063 TI - [Determining the levels of selected pesticides, nitrates, nitrites, ammonium ions, sulfates, chlorides and urea in surface and underground waters. VII]. AB - The subjects of the study was determination of the residues of pesticides: lindane, metoxychlor and chlorphenvinphos, nitrates, nitrites, ammonium ions, sulphates, chlorides and urea in surface waters and underground waters in the southeastern part of the Province of Szczecin in 1983-1988. The certain pesticides and urea in none of determined samples of waters were present. The concentrations of nitrates, ammonium and chlorides below the permitted value have been. Nitrites in surface waters and in underground waters in 48 per cent and 82 percent of the samples have been respectively. PMID- 1725064 TI - Prevention of cytomegalovirus disease in renal allograft recipients. AB - Cytomegalovirus (CMV) disease in renal allograft recipients continues to cause morbidity, mortality and high medical expenses. In addition to damaging organs directly, CMV has been associated with allograft rejection, and bacterial, fungal and parasitic superinfections. Dramatic strides have been made in our efforts to prevent CMV disease. The prophylactic strategies have included passive immunization, interferon, vaccine, and antiviral agents. This article summarizes the experience to date in efforts to prevent CMV disease, emphasizing our current approach using high-dose oral acyclovir. It concludes with a review of potential future directions in prophylaxis with new antiviral agents alone or in combination. PMID- 1725065 TI - Effect of pentosan polysulfate on fibrinolysis: basic tests and clinical application. PMID- 1725066 TI - Effect of pentosan polysulfate on activated partial thromboplastin time, thrombin time, euglobulin clot lysis, and on tissue-type plasminogen activator and plasminogen activator inhibitor activities in patients with thromboembolic disease. PMID- 1725067 TI - Effect of heparin on the activation of factor XII and the contact system in plasma. AB - We examined in purified systems and in human plasma whether heparin serves as a contact system activating compound. Purified human factor XII zymogen was not activated by heparin through an autoactivation mechanism, but was activated in the presence of purified prekallikrein. Zn2+ (12 microM) did not support autoactivation by heparin. The activation of factor XII and the contact system by heparin in plasma anticoagulated with citrate or with hirudin (not chelating ions) was examined by the cleavage of 125I-labeled factor XII and high molecular weight kininogen (HK). Heparin at 1.6 and 16 USP U/ml was not able to produce activation, in contrast to dextran sulfate (20 micrograms/ml) which supported activation of both factor XII and HK. This study indicates that heparinized plasma does not support activation of the contact system mediated through activation of factor XII. It is not expected that heparin anticoagulant therapy will contribute to activation of the contact system. PMID- 1725068 TI - Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator bm 06.022 in healthy volunteers. AB - In a randomized, single-blind, placebo-controlled, cross-over Phase-I study pharmacokinetic and hemostatic properties of BM 06.022 were investigated in seven healthy, male human volunteers. The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 domain and the protease domain of human t-PA and is unglycosylated due to its expression in Escherichia coli cells. Vehicle or 6 MU (= 10.4 mg) BM 06.022 was administered as a single i.v. bolus injection of 10 ml over 2 min. BM 06.022 was well tolerated. Fibrinogen levels and clotting times remained unchanged at baseline levels after 6 MU BM 06.022; plasminogen and alpha 2-antiplasmin (collected on chloromethylketone) decreased maximally to 83 +/- 1% and 64 +/- 3%, respectively, of baseline. D-dimers and fibrinogen degradation products increased to 1,006 +/- 234 ng/ml and 555 +/- 155 ng/ml, respectively, after BM 06.022. Half-life of BM 06.022-activity was 11.2 +/- 0.4 min and of antigen was 13.9 +/- 0.7 min, followed by a terminal half-life only for antigen of 173 +/- 33 min. Plasma clearance of BM 06.022 was 371 +/- 13 ml/min for activity and 183 +/- 15 ml/min for antigen. Thus, BM 06.022 is not fibrinogenolytic at 6 MU and is a fibrinolytic agent with a longer half-life than t-PA. PMID- 1725069 TI - Alpha 2-antiplasmin, plasminogen activator inhibitor (PAI) and dilute blood clot lysis time in selected disease states. AB - This paper is an attempt to assess the relevance of the inhibitors of fibrinolysis for clot lysis in selected disease states and to discuss the mechanisms leading to acquired abnormal levels of such inhibitors. When compared to 20 control subjects the 30 hypertriglyceridemic patients (14 with type IIb and 16 with type IV) displayed significantly (p less than 0.001) increased plasma plasminogen activator inhibitor (PAI) activity (221 +/- 88% and 290 +/- 104% respectively; mean +/- SD), moderately (p less than 0.01) increased alpha 2 antiplasmin (alpha 2AP) level (112 +/- 11% and 115 +/- 16%) and accordingly an obviously prolonged dilute blood clot lysis time (DBCLT). Neither PAI activity and alpha 2AP level nor DBCLT were significantly different from controls in the 10 patients with hyperlipoproteinemia type IIa. The 18 patients with severe hepatic cirrhosis had low alpha 2AP level (59 +/- 19.7%) and accelerated clot lysis, while mean PAI activity (160 +/- 87%) was slightly (p less than 0.05) increased. In the 17 nephrotic patients alpha 2AP was increased (115 +/- 12%) while PAI activity was similar to controls and DBCLT rather shorter. Two liver secretion enzymes, namely serum cholinesterase and plasma protein C, were found to be decreased in cirrhotic patients, similar to control values in hyperlipoproteinemia type IIa and obviously increased in nephrotic patients as well as in hypertriglyceridemic subjects. The relevance of PAI and alpha 2AP for clot lysis was considered in relation to data in the literature concerning the behaviour of t-PA and factor XIII.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725070 TI - [The interferon-inducing and immunomodulating action of bonafton in chronic diseases of the oral mucosa]. AB - The immunocorrective activity of bonaphthon, an antiviral agent, was studied in 27 patients with chronic diseases of the buccal mucosa (recurrent aphthous stomatitis, lichen ruber planus, Melkersson-Rosenthal's syndrome). Bonaphthon therapy resulted in quite a number of patients in elevation of serum interferon titer and of the titer of specific antiherpetic antibodies. These results evidence that, besides the antiviral effect, bonaphthon is characterized by an immunomodulating effect and is conducive to fortification of the defense potential of the body in patients with stubborn chronic diseases of the buccal mucosa. PMID- 1725071 TI - Molecular immunological analysis of meningococcal class 1 outer membrane protein and lipopolysaccharide. PMID- 1725072 TI - Meningococcal molecular mimicry and the search for an ideal vaccine. AB - The carbohydrates expressed on the surface of meningococcal strains of groups B and C mimic those commonly found on human cells and thus are not functionally antigenic in infancy. In order to develop an effective vaccine, it will be necessary to find ways of circumventing this molecular mimicry. Three possible ways of achieving this are discussed. (i) The surface polysaccharides can theoretically present conformationally different epitopes, some of which might be recognized as antigenic by the host. Experimental evidence is presented that such differences do indeed exist; what is needed is to determine which of these conformations are unique to the organism and hence potentially antigenic. (ii) Precursors of the surface lipooligosaccharides may be unable to mimic human antigens, and so may be potential candidates for vaccine development. (iii) Natural immunity to some strains of meningococci develops in young children who are colonized with strains of Neisseria lactamica, and it is possible that its development could be enhanced by widespread intentional colonization by N. lactamica strains that are particularly efficient inducers of broad immunity. PMID- 1725073 TI - [Cytoskeletal changes in A-431 cells under the action of epidermal growth factor]. AB - By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A 431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges. PMID- 1725074 TI - [The circadian rhythms of myocardial ischemia and ectopic activity in patients with unstable stenocardia]. AB - Holter monitoring was used in 81 patients with non-stable stenocardia with the purpose of establishing the relationship between the development of myocardial ischemia and cardiac rhythm disorders in the course of 24 hours. It was found that cardiac arrhythmia and painless ischemia develops mainly from 0 to 6 hours. Anti-ischemic therapy proved rather effective in relation to to controlling ventricular extrasystole. The dependence of ventricular extrasystole on myocardial ischemia requires further investigation. PMID- 1725075 TI - [The incidence of detecting antibodies to the hepatitis C virus in different population groups in the USSR]. AB - Markedly unequal distribution of hepatitis C virus (HCV) among blood donors in different regions of the USSR was established. The necessity of introduction into the blood service of regular donor blood screening for anti-C100-3 is substantiated. The portion of chronic hepatitis associated with HCV in the structure of chronic virus hepatitis was established. The results of the study indicate the expedience of specific virus hepatitis C prophylaxis in contingents of high risk of HCV infection. PMID- 1725076 TI - [New sites in the hemagglutinin composition of epidemic variants of the influenza virus A (H3N2) from 1989-1990]. AB - Immunological analysis of the antigenic structure of hemagglutinin of newly isolated variants of influenza (H3N2) virus carried out using monoclonal and monospecific antibodies to individual antigenic sites of hemagglutinin showed the 1989-1990 isolates to be markedly different in their antigenic properties from the variants isolated in previous years. Sites with new antigenic properties were determined in hemagglutinin of the isolates. Wide variability was found in the region of three immunodominant sites. The fact of circulation in the human population of influenza viruses of one subtype with different antigenic structures within the limits of one epidemic season was established. PMID- 1725077 TI - [The interferon status and circulating immunoglobulins during the interferon therapy of respiratory papillomatosis in children]. AB - Studies on the functional activity of interferon (IF) system (interferon status and serum immunoglobulins levels) and its changes in the course of interferon therapy of juvenile respiratory papillomatosis are presented. The initial disorders in alpha-IF production and levels of circulating IF came to normal after interferon therapy. Correlative changes of the following parameters occurred: a decrease in the level of circulating IF and IgM and simultaneous increase in the level of alpha-IF and IgG which, however, did not always correlate strictly with the course of the disease and the kind of preparation used. PMID- 1725078 TI - [The detection of the Marburg virus antigen by solid-phase immunoenzyme analysis]. AB - Comparative studies of two variants of the enzyme-linked immunosorbent assay (ELISA) were carried out to determine the sensitivity of the detection of Marburg virus antigens in Vero cells. Both competitive and two-antibody ELISA variants detected as little as 5 ng of Marburg virus antigen. The Vero cell monolayer was found to produce 5-50 ng/0.05 ml of the virus-specific proteins at 6 to 8 days postinfection. PMID- 1725079 TI - Hypertensive crisis from chronic intoxication with nasal decongestant and cough medications. AB - Cardiac hypertensive structural changes, catecholamine-related cardiomyopathy, and congestive heart failure (CHF) have been encountered in pheochromocytoma, as a result of prolonged exposure to high concentrations of endogenous catecholamines. A 34-year-old man presented with severe hypertension, cardiomegaly, and CHF, presumably as a result of continuous alpha-adrenergic intoxication with oxymetazoline hydrochloride, phenylephrine hydrochloride, and ephedrine hydrochloride, consumed in massive doses by an overuse of nasal decongestants and cough syrup (daily doses of 20, 100, and 300 mg, respectively). Coadministered chlorpromazine hydrochloride and trimeprazine tartrate may have also contributed to the clinical presentation through their anticholinergic and antihistaminic properties. The possibility of an overuse of these over-the counter drugs should be considered in the differential diagnosis of hypertensive emergencies, especially with the simultaneous use of anticholinergic and antihistamine medications, beta-blocking agents, or monoamine oxidase inhibitors. PMID- 1725081 TI - Coexistence and plasticity of neurotransmitters and neuropeptides in the rat iris. AB - The presence, distribution and origin of substance P (SP), neuropeptide Y (NPY), and CGRP-immunoreactive axons in rat iris were investigated in whole mount preparations, with special respect to the localization of the "classical" adrenergic and cholinergic ground plexuses. SP-IR fibres are distributed parallel to the pupillary margin in the sphincter muscle, and in an irregular plexus in the dilator muscle. The distribution of CGRP-IR fibres was similar to this. Both SP- and CGRP-IR elements originated from the Gasserian ganglion. Following electrocoagulation of the ophthalmic nerve, both SP- and CGRP-IR nerves completely disappeared, while in the caudal nucleus of the trigeminal nerve a substantial decrease of the immunoreactivity was found. NPY-IR fibres have also been demonstrated in the anterior uvea, displaying a pattern similar to that of the adrenergic nerves. In the sympathectomized iris, there was a marked decreased in the density of NPY-IR fibres indicating that NPY most likely coexists with the classical sympathetic neurotransmitter, noradrenalin in the sympathetic nerve supply deriving from the superior cervical ganglion. 1 month after sympathectomy, there was an increase in the density (and possibly also in the number) of both SP and CGRP-IR fibres in the denervated iris. Subsequent immuno-electron microscopic analysis has demonstrated that both SP- and CGRP-IR fibres are unmyelinated axons, embedded in a common Schwann cell cytoplasm together with a number of axons devoid of immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725080 TI - Topical aspirin for postherpetic neuralgia. PMID- 1725082 TI - Substance P- and neuron-specific enolase-like immunoreactivity of rodent Leydig cells in tissue section and cell culture. AB - Comparative immunocytochemical studies concerning the presence of a neurotransmitter (substance P), and a marker of neuroendocrine cells (neuron specific enolase), in the Leydig cells of 3 mammalian species (golden hamster, guinea pig, and rat) were carried out on tissue sections and cell cultures. Substance P(SP)-like immunoreactivity (-LI) was found to be present in both fetal and adult generation of Leydig cells in hamster and guinea pig, while neuron specific enolase (NSE)-LI was detected in Leydig cells of the 3 species at all stages studied: fetal, neonatal and adult. In primary cultures of Leydig cells isolated from adult hamster testes, SP- and NSE-LI was also established. This result was considered as an indirect evidence for the synthesis of the substances under study by the steroidogenic cells of the testis. A comparison of these results with data obtained in vivo suggests that Leydig cells may be related to the APUD- or the diffuse neuroendocrine system. PMID- 1725083 TI - Light and electron microscopic studies of lectin binding on the glycocalyx of rat pancreatic cells. I. Normal tissue and isolated cells. AB - Lectin binding of the glycocalyx of pancreatic tissue sections as well as of isolated pancreatic acini and acinar cells was studied of healthy wistar rats by light and electron microscopy. For light microscopy, we used FITC (WGA, RCAI, LCA) or peroxidase marked (WGA, RCAI, PNA, PHA, LCA, UEAI, LPA) as well as unmarked lectins (Con A, VAA I). Gold marked lectins were used for electron microscopy (WGA, RCAI, LCA, HPA, PNA, VAAI-B). Intact acinar cells in pancreatic tissue sections and isolated acini showed a strong binding of WGA, RACI, and HPA on the apical cell surface, whereas VAAI, UEAI, LCA, and Con A reacted strongly with the basolateral glycocalyx, but not with the apical surface. The 2 main domains of the glycocalyx of pancreatic cells showed their specific lectin binding so long as the junctional complexes between the cells are intact. The polarity of the cell surface of pancreatic acinar cells is discussed in regard to the possible function of the 2 domains. PMID- 1725084 TI - Acidic and basic fibroblast growth factor levels in spinal cord cultures are not regulated by alterations in heparan sulfate proteoglycan expression. AB - The present study was undertaken to assess both the levels of acidic and basic fibroblast growth factors in spinal cord cultures and to determine how they were presented to responsive cells. Western blots detected a single acidic fibroblast growth factor-like protein (17 kDa) and two (18 kDa, 24 kDa) basic fibroblast growth factor-immunoreactive proteins, the levels of which varied with the antibody used. Levels of all three proteins were unaltered in cultures grown in the presence of a mitotic inhibitor, which greatly reduced the number of astrocytes. Cell blots showed increased survival of spinal cord neurons at Mr that corresponded with the three proteins detected immunologically. Solubilized cultures separated on a P100 column showed mitogenic activity for NIH3T3 cells from 17-18 and 24 kDa fractions. Treatment of the cultures with heparitinase did not decrease the levels of acidic and basic fibroblast growth factors detected by Western blots, suggesting that these proteins were not associated with extracellular membrane heparan sulfate proteoglycans. The major fraction of both proteins appeared to be intracellular with a minor amount complexed with extracellular matrix proteins. An inhibitor of xylose-linked proteoglycan synthesis significantly altered heparan sulfate proteoglycan deposition into extracellular matrix, but did not alter the levels of acidic or basic fibroblast growth factors detected by Western blots, or the levels of choline acetyltransferase, glutamic acid decarboxylase, or aspartate aminotransferase activities. These results indicate that both acidic and basic fibroblast growth factors are stored predominantly intracellularly, with only a minor fraction complexed with extracellular proteins. We suggest that these intracellular proteins may be released following injury in the CNS and mediate a cascade of neuroprotective events. PMID- 1725085 TI - Expression and quantitative changes of carbonic anhydrase in developing neurones of rat central nervous system. AB - Postnatal changes in carbonic anhydrase activity were investigated in the islands of Calleja, which have been previously reported to contain the enzyme. Results obtained with a new modified method of Hansson provided further evidence for the distinction between the medial and lateral islands of Calleja. The enzyme was localized mainly in the nucleus and cytoplasm of granule cells without showing binding to any cytoplasmic organelle. No large neurons of the islands displayed carbonic anhydrase reactivity. The time course and rate of increase of carbonic anhydrase expression were different in the giant island of Calleja and lateral islands and this finding may strengthen the hypothesis regarding the medio lateral diversity of Calleja's islands. On the other hand, at the end of the maturation process the granule cell complexes showed no significant difference in the proportion of carbonic anhydrase positive neurones. The almost equal rate of appearance of carbonic anhydrase reactive granule cells raises the possibility of a basic common role of both medial and lateral islets. PMID- 1725086 TI - Short- and long-term effects of combined pre- and postnatal ethanol exposure (three trimester equivalency) on the development of myelin and axons in rat optic nerve. AB - This study evaluated the effects of a combined gestational and 10 day postnatal alcohol exposure (human three trimester equivalency) on the development of myelin and axons in rat optic nerve. Rats were exposed during gestation via liquid diet, then their artificially reared pups were further exposed for 10 postnatal days via an ethanol-containing diet fed by gastrostomy. Control animals from pair-fed dams were artificially reared for 10 days on pair-fed isocaloric diets. Anesthetized animals were perfused with fixative on gestational days (G) 15 and 20 and postnatal days (P) 5, 10, 15, 20, and 90, then optic nerve tissues prepared for electron microscopy. Optic nerve cross-sectional areas were generally less from G20 through P90 in ethanol exposed animals. Counts of the number of myelinated nerve fibers per unit area and of the numbers of fibers in different stages of myelin development revealed that alcohol exposure caused a delay in myelin acquisition at 10 and 15 days that was compensated for at 20 and 90 days. Myelin thickness as a function of axon diameter was decreased in the alcohol exposed animals from 10 through 90 days, indicating a permanent reduction in the relative thickness of myelin. These results show that alcohol exposure for all of gestation and 10 postnatal days in the rat (human three trimester equivalency) causes a permanent reduction in myelin thickness along with a delay in myelin acquisition in the optic nerve. Such alterations in developing and adult myelin could help to explain some of the neurological and visual dysfunctions associated with developmental alcohol exposures. PMID- 1725087 TI - What is the role of cytokines in human colostrum? AB - No one has ever doubted that maternal milk, in comparison to formula milk, has a far superior nutritional value. Colostrum has a well acknowledge crucial value for the survival of the animal species that cannot receive immunoglobulins through the placenta. Until recently the presence of cytokines in colostrum was unsuspected but it has been now clarified that normally there are at least four cytokines, namely interleukin 1 and 6, tumor necrosis factor and interferon gamma, that may exert an important immunostimulatory role particularly on the oropharyngeal-associated lymphoid tissue. As a corollary, physiological concentration of cytokines administered per os may exert a useful adjuvant activity in aged or immunodeficient people. PMID- 1725088 TI - The identification of the bacteriophage HP1c1 and S2 integration sites in Haemophilus influenzae Rd by field-inversion gel electrophoresis of large DNA fragments. AB - The resolution of high molecular weight DNA fragments by field-inversion gel electrophoresis (FIGE) demonstrate the presence of two phage (S2 and HP1c1) integration sites (attB) in the Haemophilus influenzae Rd chromosome. In a population of wild-type cells either prophage site appears to be occupied in a single cell by one to at least three, tandemly repeated, amplified phage DNA molecules. The attL of the second bacterial attachment site present in the host SmaI fragment 7 and the leftmost part of phage S2 type B DNA of its genome organization (Piekarowicz et. al., 1986) have been sequenced. A comparison of the two bacterial att sites demonstrated that their homology is limited to the core region. A comparison of the DNA sequences of phage S2 type B and HP1c1 type C revealed a 530-bp insertion in the HP1c1 type C (not present in S2 type B) in addition to DNA variants due mostly to single-base mismatches. We postulate that phage S2 and HP1c1 genome variants (A, B, and C) evolved from a single phage origin and might stem from passage history arisen through accumulation of mutations. PMID- 1725089 TI - Proteins of Caulobacter crescentus strongly binding to DNA. Spectroscopic analysis of major histone-like proteins. AB - The protein composition of deoxyribonucleoprotein (DNP) from Caulobacter crescentus, protected from exogenous nucleases, was analysed. This fraction was obtained by purification of cell lysate on Sephacryl S-400. It contained the following proteins: 13.4 kDa (HCc), 15.2 kDa, 17.5 kDa, 28 kDa and 40 kDa. The strength of protein 15.2 kDa binding to ds-DNA was the same as that of the eukaryotic histones H2A and H2B. Proteins 13.4 kDa (HCc) and 17.5 kDa purified to homogeneity have a UV spectrum identical to protein HU of Escherichia coli which lacks tryptophane and tyrosine. This confirms the classification of protein HCc to the class of HU-like proteins. PMID- 1725090 TI - Decreased chemical mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae. AB - In the DNA replication mutant of yeast cdc8 the frequency of chemically induced reversion of lys2-1 and hom2-1 was found to be reduced. Mutation induced by ethyl methanesulfonate (EMS) were greatly diminished in the strain homozygous for the cdc8-1 gene. PMID- 1725091 TI - Influence of cadmium ions on Streptomyces strains. AB - The sensitivity of 19 strains of Streptomyces to cadmium ions was studied by the disk method using cadmium chloride in quantities: 2, 5, 10 ug Cd2+. Sensitivity was estimated by measuring the size of zones of the growth inhibition. Also four selected strains were spread on substrates containing 1, 5, 10 ppm Cd2+ and the number of colonies was countwed. In all tests a comparison was made between the 2 kinds of inoculum: spores suspended in physiological salt solution and spores prior preincubation on a medium. Differences of the reaction to cadmium ions among tested strains were observed. Spores after germination were more resistant to cadmium ions than non-germinating spores of the same strain. Five ppm Cd2+ added to the medium decreased the number of colonies, especially in the case of sensitive strains. Ten ppm of cadmium ions decreased the number of colonies of the resistant strains and inhibited the growth of sensitive strains. PMID- 1725092 TI - The effect of some physical and serological treatments on haemofusing activity of bovine parainfluenza virus type 3. AB - Bovine parainfluenza virus type 3 irrespective of the time of its harvesting from Madin-Darby bovine kidney cells, expressed little or no haemofusing activity. Treatment of the virus by freezing and thawing, sonication or antibody and complement enhanced this activity. Moreover haemofusing activity did not correlate with the viral capacity for fusion of susceptible cells in monolayer cultures. PMID- 1725093 TI - Impaired bactericidal activity of serum of a child suffering from focal proliferative glomerulonephritis. AB - The serum of a child with focal proliferative glomerulonephritis was found to exhibit a weaker bactericidal activity against Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis and Escherichia coli strains as compared with sera of the child's parents. The child's serum showed a low haemolytical activity of complement as well as a low C3 concentration. The authors believe that the abnormal complement concentration could cause the impaired bactericidal activity of the patient serum. PMID- 1725094 TI - Enzymatic characterization of Vibrionaceae strains isolated from environment and cold-blooded animals. AB - Enzymatic profiles were determined by the API ZYM system for 15 strains of non 01 Vibrio cholerae, 4 strains of V. metschnikovii, 9 strains of V. anguillarum, 6 strains of Plesiomonas shigelloides and 115 strains motile Aeromonas sp. All of the tested strains produced alkaline phosphatase, leucine aminopeptidase and did not possess alpha-fucosidase and alpha-mannosidase. Some differences in enzymatic activities among the tested Vibrionaceae strains were noted. The strains of non 01 V. cholerae, V. metschnikovii, V. anguillarum and P. shigelloides did not produce trypsin, whereas all of the tested Vibrio sp. strains appeared to be positive for this enzyme. Only the strains of P. shigelloides produced BI-Phospho hydrolase. The lack of acid phosphatase activity was observed among the strains of V. anguillarum. PMID- 1725095 TI - Serological activity of antigens isolated from 11 Bacteroides vulgatus strains. AB - Capsular (CPS) and phenol water (PW) extracts were prepared from 11 serogroup specific strains of B. vulgatus isolated from normal gut flora. The extracts were active in immunodiffusion (ID) and passive hemagglutination (HA) tests. There was no correlation between sugar and protein contents and serological activity of these extracts. PMID- 1725096 TI - Comparative studies of serotype-specific Clostridium difficile strains. AB - The following properties of serotype-specific Clostridium difficile strains were studied: toxigenicity, encapsulation, susceptibility to certain antibiotics, biochemical properties, enzymatic activity. No correlation between toxin titer and frequency of capsule production as well as serogroup affiliation and sensitivity to antibiotics was observed. The strain representative of serogroup C attracts attention because of its distinct properties. PMID- 1725097 TI - Altered excitation-contraction coupling in hypertension: role of plasma membrane phospholipids and ion channels. PMID- 1725098 TI - Cloning phospholamban cDNA from rat aortic smooth muscle. PMID- 1725099 TI - Control of HCO3-dependent exchangers by cyclic nucleotides in vascular smooth muscle cells. PMID- 1725100 TI - Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome. AB - Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6 deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not. PMID- 1725101 TI - Russel bodies-containing cells in the Harderian gland of the chicken in relation to different immune conditions. AB - The modulation of the number of Russel bodies-containing plasma cells in the Harderian gland under different experimentally induced immune conditions is reported. The findings support the conception of an immunologic function of the Harderian gland which is similar to the function of the thymus on the one hand, and to the function of the bursa of Fabricius on the other. PMID- 1725102 TI - 5-Hydroxytryptamine releasing activity of the supernatants from cultured mouse spleen cells. AB - Mouse spleen cells have been shown to produce a histamine releasing factor (HRF) after stimulation with mitogen or specific antigen in vitro. The supernatants from the cultures of mouse spleen cells released not only histamine but also 5 hydroxytryptamine (5-HT) from homologous mast cells in a dose-dependent manner. The time-course of this release was similar to that observed in antigen or anti IgE reaction. Heavy water (D2O) enhanced supernatant-induced mediator release. PMID- 1725103 TI - Combined action of interferons and transforming growth factor beta on the proliferation of human fibroblasts. AB - The effects of interferons (IFNs) and transforming growth factor-beta (TGF-beta) used alone and in combination on the multiplication of human embryonic diploid fibroblasts were studied. The experimental conditions were standardized and medium containing 2% of a single batch of fetal calf serum, which was almost minimal dose required for good the cell attachment and growth, was used. The observed effects were dose related. IFNs (types alpha, beta, or gamma) at concentration of 1 to 100 U/ml or TGF-beta at concentration of 0.1 to 1.0 ng/ml stimulated the cell multiplication by 20 to 25% when compared to the control cultures incubated without the factors. At higher doses both IFNs (10(2) to 10(5) U/ml) or TGF-beta (1 to 10 ng/ml) inhibited the proliferation of the cells. The antimitogenic action of IFNs and TGF-beta was synergistic. We suggest that IFNs as well as TGF-beta are bifunctional growth regulators although their antimitotic action dominates over their possible growth stimulatory activity. The latter action may be a secondary phenomenon due to interaction of IFNs or TGF-beta with other factors present in cell culture. We have shown that epidermal growth factor (EGF) may increase the growth stimulatory action of low doses of IFN-gamma or TGF beta. PMID- 1725104 TI - Acute phase reactants and circulating immune complexes in patients with ovarian carcinoma. AB - Serum levels of haptoglobin (HP), sialic acid total (NAN) and lipid-bound (NAL), seromucoid (SER), its content in total protein (%SER), as well as circulating immune complexes (CIC), were measured in sera of women with ovarian carcinoma, prior to their treatment and through the course of chemotherapy, remission and recurrence of malignancy, respectively. Control groups consisted of healthy women and patients with benign tumors (ovarian cysts and uterine myomas). Pretreatment measurements of acute phase reactants discriminated cancers (FIGO stages I+II, III, IV) from healthy group, however differences between benign tumors and stages of ovarian cancer were not so distinct. Changes in the examined parameters (acute phase reactants) indicated satisfactorily a response to the administered chemotherapy and early signs of the progression of the disease. Because of great variations in serum CIC concentrations, they were found to be of no value either in diagnosis or in the surveillance of the disease status. PMID- 1725105 TI - The main serum protease inhibitors; alpha-1-proteinase inhibitor and alpha-2 macroglobulin inhibit H2O2 release from human polymorphonuclear leukocytes stimulated with phorbol myristate acetate. AB - Since various functions of phagocytes can be affected by protease inhibitors, the ability of alpha-1-proteinase inhibitor (alpha 1PI) and alpha-2-macroglobulin (alpha 2M) to modulate the H2O2 production by human polymorphonuclear leukocytes (PMNL) was studied. The preincubation of PMNL for 30 min with these protease inhibitors at concentrations which may occur in human blood diminished in a dose dependent manner their H2O2 generation induced with phorbol myristate acetate. At alpha 1PI 600 mg/dl and alpha 2M 800 mg/dl the H2O2 response decreased to 61 +/- 2 and 58 + 3% (p less than 0.001, n = 4) of the control value obtained with cells preincubated in phosphate buffered saline with glucose, respectively. Autologous serum alone and with addition of pure alpha 1 PI or alpha 2M also suppressed H2O2 release from PMNL. It is suggested that inhibition of H2O2 generation from PMNL may be another additional way by which these serum protease inhibitors may protect tissues (especially lungs) from acute injury related to inflammation. PMID- 1725106 TI - [Cyclic analogs of substance P. III. Cyclo (6(sup)gamma----oligomethylenediamine- --11) substance P(6-11)-hexapeptides]. AB - Cyclic analogues of substance P of the formula cyclo-[Glu-Phe-Phe-Gly-Leu-Met NH(CH2)nNH-], where n = 3-10, 12, and open-chain analogues (XVIIIa, b) H Glu.(NHR)-Phe-Phe-Gly-Leu-Met-NHR, where R = -CH3, -CH2CH2CH3, were synthesized. By NMR spectroscopy it was found that cyclo-compounds with n = 3-8 have regularly arranged structures, stabilized by intramolecular hydrogen bonds. Substances of this type showed less than or equal to 0.1% of the substance P activity on the guinea pig ileum, but some of them antagonize the natural peptide (for compound with n = 5 IC50 = 3.2.10(-6) M). The open-chain compounds proved to have rather high myotropic activity, viz., 22% (R = -CH3) and 8% (R = -CH2CH2CH3) of the substance P activity. PMID- 1725107 TI - Alpha-fetoprotein alterations in alcoholics with liver disease. V.A. Cooperative Study Groups. AB - Sera on 409 male alcoholics with liver injury were assayed for alpha-fetoprotein (AFP) as part of a VA co-operative study on the natural history and therapy of alcoholic liver disease. In 78% of the patients values below normal were observed and 42% had undetectable levels. Clinically the lowest AFP concentrations were observed in the more severely ill patients with the poorest 1 year survival. Furthermore, improvement in AFP was associated with improved survival. Correlation analysis showed a relationship of AFP to (1) visceral protein concentrations (i.e. albumin, transferrin, retinal binding protein); (2) variables related to hepatic fibrogenesis (i.e. Ito cell activity, quantitative estimates of fibrosis and Kupffer cell abnormalities); and (3) changes in immunoglobulin levels particularly IgG. These findings suggest that AFP is a good index of disease prognosis. PMID- 1725108 TI - Improved expression vectors for eukaryotic promoter/enhancer studies. AB - We describe two transfectable vectors designed to facilitate the functional analysis of eukaryotic promoter/enhancer sequences. The first, pJFCAT1, is an improved chloramphenicol acetyltransferase (CAT) reporter gene expression vector with two features that distinguish it from the majority of other CAT vectors currently in use: 1) it carries a trimer cassette of the simian virus 40 major late polyadenylation site to block plasmid-initiated read-through expression of CAT, and 2) it includes the phage f1 origin of replication, permitting generation of single-stranded copies to serve as templates for oligonucleotide-directed mutagenesis or single-strand DNA sequencing. The promoterless pJFCAT1 directs little if any CAT activity in transfected mouse L cells and, therefore, may be particularly useful for the analysis of weak promoters whose activity is otherwise masked by background CAT expression. The second vector, pTAG-1, uses human beta-globin as a reporter gene and was designed to facilitate the analysis of reporter gene expression at the RNA level. Like pJFCAT1, pTAG-1 also includes the simian virus 40 polyadenylation site trimer cassette located just upstream of the promoter insertion site. We have used each of these vectors to study functional elements in the human and mouse thymidine kinase promoters. PMID- 1725109 TI - Use of riboprobes for northern blotting analysis. AB - We compared cDNA and riboprobes for their use in Northern blotting analysis and found that higher hybridization signal can be obtained using riboprobes. Comparison of five nylon membranes for Northern blotting using riboprobe revealed that each nylon membrane differed in their performance. Background noise was minimal for all five membranes for two riboprobes. However, because of the higher thermal stability of RNA.RNA hybrids, the riboprobes could not be stripped out from the membrane even after five hours of treatment at 80 degrees C. PMID- 1725110 TI - A rapid and inexpensive method for the addition of poly A coding tracts to transcription vectors. PMID- 1725111 TI - A general method for quantitative PCR analysis of mRNA levels for members of gene families: application to GABAA receptor subunits. AB - We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits. PMID- 1725112 TI - A rapid method for purification of synthetic oligoribonucleotides. AB - A procedure for large-scale purification of synthetic oligoribonucleotides has been developed that has significant advantages over gel purification techniques currently in use. Synthesis was performed using commercially available 2'-O silylated ribonucleoside 3'-O-phosphoramidites, and coupling efficiencies were consistently greater than 97% for oligoribonucleotides up to 31 residues in length. Using C4 reverse-phase chromatography to remove material not deprotected by treatment with tetrabutylammonium fluoride, we have eliminated reactants in which the 2'-O-silyl group is only partly removed, thus ensuring a homogeneous population of oligoribonucleotide. PMID- 1725113 TI - Older ("traditional") methods for thumb reconstruction with special regard to bad results. PMID- 1725114 TI - Complications and bad results in pollicization of the index finger (in congenital cases). PMID- 1725115 TI - Bad results after large toe to thumb transplantation. PMID- 1725116 TI - A critical study of thumb reconstruction by second toe transfer. PMID- 1725117 TI - Complications and bad results of thumb reconstruction by the microvascular "wrap around" technique. PMID- 1725118 TI - Complications and bad results of toe partial transfers in thumb reconstruction. PMID- 1725119 TI - Late lesions of the brachial plexus after fracture of the clavicle. AB - Fractures of the clavicle, particularly those which are markedly displaced, may, in rare instances cause injury to the subclavian vessels and the brachial plexus which manifest progressively days or weeks after the initial trauma. More often than not, however, a costo-clavicular compression syndrome appears months or years after the clavicular fracture as a result of constriction by scar which invests the neuro-vascular bundle, by a secondary aneurysm or by hypertrophic callus. The authors report 16 such cases, one of which was treated conservatively, thirteen treated by surgical intervention while two cases are awaiting operation. These patients represent just over 1% of brachial plexus lesions seen over a period of twenty years in two surgical centres. Operative treatment consists of reduction of the clavicular deformity, possibly first rib resection, liberation of the plexus and correction of a vascular lesion as required. The outcome is usually good. PMID- 1725120 TI - Palmar subluxation of the carpus in rheumatoid disease: a radiological evaluation. AB - The authors present a radiological assessment of palmar subluxation of the carpus in rheumatoid disease. The radiological measurements have been taken with reference to the long axis of the radius, the radiological centres of the lunate and the capitate and expressing their reciprocal distances as a ratio which we have termed the radio-carpal and inter-carpal indices. Using a control group of a 100 serial radiographs of normal wrists it was possible to determine the normal indices and their normal range. A subsequent analysis of 100 rheumatoid wrists enabled them to accurately determine the degree of anterior carpal subluxation, and allowed this form of radio-carpal malalignment to be differentiated from other forms of carpal instability. PMID- 1725121 TI - The Herbert screw for the treatment of scaphoid fractures. AB - Forty-nine patients with fifty fractures of the scaphoid were reviewed more than six months after surgical treatment using the Herbert bone screw. Twenty-nine patients (mainly those with delayed union and non-union) also had bone grafts. The mean period of follow-up was 18.3 months. In 47 patients (94%) the fracture had definitely or probably united. The three patients whose fractures did not unite had proximal pole fractures with pre-existing avascular necrosis and osteoarthritis. Immediate post-operative mobilisation was possible in thirty patients. Wrist function as measured by grip strength and range of motion was excellent or good in most patients. Twenty patients had proximal pole fractures and their results are analysed separately. PMID- 1725122 TI - Treatment of fracture-dislocation of the proximal interphalangeal joint using extension-block Kirschner wire. AB - Fourteen cases of fracture-dislocation of the proximal interphalangeal joint (PIPJ) were treated using extension-block Kirschner (K)-wire after closed or open reduction of the dislocation. The postoperative ROM of the PIPJ in the 14 patients averaged 94.4 degrees. A full ROM of the PIPJ was regained in 10 cases. This wire provides an alternative method to the use of extension block splintage in the management of such injuries. PMID- 1725123 TI - Anatomical study of distal insertion of the abductor pollicis longus. Concept of a new musculo-tendinous unit: the abductor carpi muscle. AB - Having performed 100 anatomical dissections we found that in the first dorsal compartment of the wrist besides other tendons there are one or two tendons belonging to a musculo-tendinous unit, not yet described, inserting in the trapezium and acting almost together with the other units going to the first metacarpal. This is present in 71% of cases. Its function is to abduct the carpus. Therefore we suggest to call this unit the "abductor carpi" muscle. If it is missing, which is "abnormal", instability and arthritis of the trapezio metacarpal joint occur in a higher percentage because the other abducent forces pull the base of the first metacarpal with a shearing effect on the TM joint. Besides other less frequent variants, other "anomalies" such as the presence of a tendon going to the abductor pollicis brevis (59%) or to the opponents muscle (15%) have been found. PMID- 1725124 TI - Surface topography and molecular stoichiometry of the mitochondrial channel, VDAC, in crystalline arrays. AB - The mitochondrial outer membrane contains a protein, called VDAC, that forms large aqueous pores. In Neurospora crassa outer membranes, VDAC forms two dimensional crystalline arrays whose size and frequency can be greatly augmented by lipase treatment of these membranes (C. Mannella, Science 224, 165, 1984). Fourier filtration and surface reconstruction of freeze-dried/shadowed (45 degrees) arrays produced detailed images of two populations of crystals, whose lattices are mirror images of each other. Most likely, this technique has revealed both surfaces of the same two-dimensional crystal with lattice parameters: a = 12.3 +/- 0.1 nm, b = 11.2 +/- 0.1 nm, and theta = 109 +/- 1 degree. Three-dimensional reconstructions of the surface reliefs on both sides of the crystal show them to be very similar. The majority of the protein forming the channel appears to be at or below the level of the membrane. To address the issue of the number of 30-kDa polypeptides that form a VDAC channel, measurements of mass per unit area were carried out by analyzing scanning transmission electron micrographs of unstained, freeze-dried arrays. The crystal form used for mass analysis contained the same motif of six stain-accumulating centers per unit cell, with p2 symmetry as in the oblique configuration, but it had a different orientation relative to the lattice lines. These data yielded a surface density of 1.9 +/- 0.2 kDa/nm2, indicating that there is a one-to-one ratio between VDAC polypeptides and the channels visualized in filtered electron micrographs, and that VDAC membrane crystals contain 68% protein and 32% lipid by mass. PMID- 1725125 TI - Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy. AB - Two views of native alpha 2-macroglobulin are revealed by electron microscopy of negatively stained samples; in one view the molecule resembles a padlock and in the other, a pair of lips. Interconversion of the two views upon tilting establishes that these are two different projected views of the same structure. Furthermore, the two views are related by a 45 degrees rotation about their major axis because they interconvert when the specimens are titled +/- 22.5 degrees. Negatively stained molecules on Butvar films present a nearly equal distribution of the two views, whereas in frozen-hydrated samples the molecules almost exclusively are oriented in the lip view. Measurements from both views indicate that the alpha 2-macroglobulin molecule is approximately 200 A long and approximately 140 A wide. Our results suggest that alpha 2-macroglobulin is composed of two protomeric units, each in the shape of a twisted letter S. These units are joined together at their ends to form a complex with point group symmetry 222. The 45 degrees interconversion angle between the lip and padlock views support this arrangement. Average images of unstained and stained lips are quite similar, indicating that the native structure is consistently preserved by the two electron microscopy procedures used in this investigation. This is substantiated by the interconversion between the lip and padlock views that occurs when the molecule is rotated 45 degrees [corrected] about its major twofold axis. PMID- 1725126 TI - The maturation-dependent conformational change of phage T4 capsid involves the translocation of specific epitopes between the inner and the outer capsid surfaces. AB - After polymerization of the phage T4 prohead is complete, its capsid expands by approximately 16%, is greatly stabilized, and acquires the capacity to bind accessory proteins. These effects are manifestations of a large-scale, irreversible, conformational change undergone by the major capsid protein, gp23 (521 residues) which is cleaved to gp23* (residues 66-521) during this maturation process. In order to explore its structural basis, we have performed immunoelectron microscopy with antibodies raised against synthetic peptides that correspond to precisely defined segments of the amino acid sequence of gp23. These antibodies were used to label purified polyheads (tubular polymorphic variants of the normal icosahedral capsid), in experiments designed to impose constraints on the possible foldings of the gp23/gp23* polypeptide chains in their successive conformational states. Peptide 1 (residues 48-57), part of the gp23-delta domain that is excised when gp23 is converted to gp23*, resides on the inner surface of the precursor surface lattice, but--if not proteolyzed--is found on the outer surface of the mature surface lattice. Peptide 2 (residues 65-73), immediately distal to the cleavage site, is located on the inside of the precursor surface lattice, and remains there subsequent to expansion. Peptide 3 (residues 139-146) is translocated in the opposite direction from peptide 1, i.e., from the outer to the inner surface upon expansion; moreover, expansion greatly increases the polyheads' affinity for these antibodies. Peptide 5 (residues 301-308) is located on the inside in both the precursor and the mature states. Taking into account data from other sources, these observations imply that the conformational change that underlies capsid expansion involves a radical reorganization of the proteins' structure, in which at least three distinct epitopes, situated in widely differing parts of the polypeptide chain, are translocated from one side to the other. Moreover, the amino-terminal portion of gp23/gp23*, around the cleavage site, is particularly affected. PMID- 1725127 TI - Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin. AB - Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725128 TI - Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase. AB - We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5 isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA 1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein. PMID- 1725129 TI - Alternative patterns of mitogenesis and cell scattering induced by acidic FGF as a function of cell density in a rat bladder carcinoma cell line. AB - The dual function exerted by acidic fibroblast growth factor (aFGF) in a rat bladder carcinoma cell line has been explored under two different conditions of culture density. At low cell density, aFGF promotes the epithelium-to-mesenchyme transition of NBT-II cells characterized by cell dissociation, morphological changes toward a fibroblastic-like phenotype, and acquisition of cell motility. Under these conditions, NBT-II cells are unresponsive to the growth-promoting effect of aFGF. At high cell density, aFGF is a potent mitogenic factor, but its scattering activity is essentially abrogated. Slight modifications in the binding of aFGF to its specific receptors were observed at high cell density; these changes correlated with a downregulation of receptors with no apparent change in their molecular form. NBT-II cells located at the edge of artificial wounds mimicked the behavior of subconfluent cells, because they did not proliferate upon aFGF treatment. Furthermore, in large-sized NBT-II colonies, peripheral cells were the first to dissociate in response to aFGF. Altogether, our results suggest that the cellular response to multifunctional growth factors might depend on the localization within the responding cell population. PMID- 1725130 TI - Decrease in cytokine production by HIV-infected macrophages in response to LPS mediated activation. AB - The capacity of human monocytes/macrophages (M/M) infected with a human immunodeficiency virus-1 (HIV-1) isolate to produce several immunomodulating cytokines including interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-8, and macrophage chemoattractant and activating factor (MCAF) was examined. Although HIV infection itself induced significant increases in the level of mRNAs for IL-1 beta, TNF-alpha, IL-6, and IL-8, the levels of lipopolysaccharide (LPS)-induced mRNAs for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, IL-8, and MCAF were decreased over those of uninfected LPS stimulated cells. In addition, HIV-infected M/M produced lower amounts of IL-8 protein, as measured by radioimmunoassay over an 18-day culture period. These results suggest that HIV infection generally suppresses the LPS-inducible cytokine production in human M/M. The impact of the role of these cytokines in the immunity and pathogenesis of HIV-1 infection is discussed. PMID- 1725131 TI - Interleukin 4 down-regulates the expression of CD14 and the production of interleukin 6 in acute myeloid leukemia cells. AB - On normal monocytes, interleukin 4 (IL-4) down-regulates the production of several proteins that are produced in response to bacterial cell wall lipopolysaccharides (LPS), among others IL-6. In addition, IL-4 was shown to down regulate the expression of the monocytic differentiation marker CD14, recently identified as a receptor for LPS. Since (autocrine) IL-6 is possibly involved in the proliferation and differentiation of acute myeloid leukemia cells with myelomonocytic differentiation (AML-M4/M5), we studied the effects of IL-4 on IL 6 production and CD14 expression by these cells. We found that IL-4 down regulates both the expression of CD14 (n = 6) and the production of IL-6 (n = 9) in AML cells. Using Northern blot analysis, we found that IL-4 down-regulates the level of steady-state mRNA for both CD14 (two of three cases tested) and IL-6 (three cases tested). PMID- 1725132 TI - [An interferon inhibitor in regulating the activity of human natural killer cells]. AB - The action of mouse serum interferon--alpha/beta (IFN) at the dose of 100 U/ml, of its inhibitor (I) at the dose of 8 U/ml as well as of their combination with the above doses on sensitivities of mouse target cells (TC) of sensitive to IFN line L 929 and resistant to the one line MCB in natural cytotoxic reaction was studied. Cytotoxic activity of human natural killer cells was detected in 14 hrs cytotoxic test using 3H-uridine for labelling of TC. IFN, I, and IFN+I were added to cell cultures for 24 hrs at 37 degrees C with following removing of preparations. It has been shown that I abolished protective effect of IFN on TC L 929 whereas the one possessed the protective action on TC MCB in natural cytotoxic reaction. These data confirmed a suggestion about immunoregulatory properties of I which displayed in abolition or realization of protective effect on TC in natural cytotoxic reaction in dependence on initial sensitivity of TC to antiviral IFN action. PMID- 1725133 TI - [Histochemical and clinical-diagnostic study of placental alpha 1-microglobulin using monoclonal antibodies]. AB - Five stable hybridomas against placental protein PAMG-1 were obtained. Monoclonal antibodies were not cross reactive with other placental proteins and human serum albumin. The content of PAMG-1 during a physiological pregnancy increased to 40 ng/ml. 23 women with repeated abortions had an increased content of PAMG-1. PMID- 1725134 TI - Erythrocyte vesiculation in paroxysmal nocturnal hemoglobinuria. AB - Red blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) were found to be liable to vesiculate, as demonstrated by both DMPC liposome- and CaCl2-induced vesiculation and measured in terms of acetylcholinesterase activity and 3H-inositol radioactivity in the supernatant. Membrane proteins released from the cells during vesiculation included several constituents with molecular weights identical to those of some complement regulating factors (e.g. DAF) which play an essential role in complement-mediated hemolysis. Red blood cells from both normal and PNH patients showed decreased deformability after vesiculation. Liability to vesiculate and the consequential loss of certain essential membrane proteins and decreased deformability might be a factor contributing to the mechanism of hemolysis in PNH. PMID- 1725135 TI - Antitumor effect of 22-oxa-1 alpha,25-dihydroxyvitamin D3, a potent angiogenesis inhibitor, on rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene. AB - The effect of 22-oxa-1 alpha,25-dihydroxyvitamin D3 (22-oxa-1,25(OH)2D3) on the growth of autochthonous rat mammary tumors induced by 7,12 dimethylbenz[a]anthracene (DMBA) was examined on the basis of our previous finding that this synthetic vitamin D3 analog has a potent angiogenesis inhibitory effect. Two doses of 22-oxa-1,25(OH)2D3, 0.1 and 1 microgram/kg of body weight, due to the limited amount of the compound available, were used. The daily administration of 22-oxa-1,25(OH)2D3 at the dose of 1 microgram/kg/day resulted in significant inhibition of the growth of these mammary tumors at 1, 2 and 3 weeks after the administration of this agent, although the agent caused little or no regression of the tumors. After daily administration for 3 weeks, a significant antitumor effect was also observed in the group treated with 0.1 microgram/kg/day. Treatment with 22-oxa-1,25(OH)2D3 did not affect the serum calcium levels in the treated rats. The lower dose of 22-oxa-1,25(OH)2D3 neither affected weight gain nor caused a decrease in body weight, while the higher dose, although having some effect on weight gain, did not induce a decrease in body weight. There were no significant differences in the weights of adrenals, uteri and ovaries between the treated groups and controls. These results suggest that 22-oxa-1,25(OH)2D3 has a significant growth inhibitory effect on DMBA-induced autochthonous mammary tumors in rats, without producing severe side effects, including hypercalcemic activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725136 TI - The effect of education on physicians' knowledge of a laboratory test: the case of maternal serum alpha-fetoprotein screening. PMID- 1725137 TI - Ethical issues described by NICU nurses. PMID- 1725138 TI - The distribution of peptide-containing nerves in the synovia of the cat knee joint. AB - Synovial capsule in cats is extensively innervated by a network with axonal diameter ranging from 0.6-3 microns according to its position and neuropeptide content. Nerve markers such as Neuron Specific Enolase (NSE) and Neurofilament triplet protein (NF) could be observed only when the axonal fibre attained a critical diameter of over the 3 microns limit. The relatively thick fibres (1-3 microns) show positive immunoreactivity for Substance P (SP), 5-hydroxytryptamine (5-HT), and Vasoactive Intestinal Peptide (VIP), and seldom coreact with NSE and NF, whereas, the thinnest fibres (0.6-0.8 microns) characterized to contain either Methionine or Leucine Enkephalin (M-Enk, L-Enk) did not coreact positively with axonal markers. We found that different anesthetics may effect variably the immunoreactivity of some neuropeptides (SP, L-Enk, 5-HT) while others (VIP, M Enk) remained unaffected. Based on our data and the few reported ones in the pertinent literature, it is judged that urethane is the anesthetic of choice in experimental studies of neuropeptides. Our findings of isolated positive immunoreactive cell bodies to enkephalin in synovia might suggest the presence of intrinsic relay system, where the enkephalin acts as suppressor of SP and VIP release from the sunovium nerve terminals. Such a local inter-relationship between different neuropeptide systems might have a practical role on the understanding of the pathogenesis of different arthritic processes as well as therapeutic strategy in the future. PMID- 1725139 TI - Benign multicystic mesothelial proliferation of the peritoneum: immunohistochemical and electron microscopical study of a case and review of the literature. AB - We report a case of benign multicystic mesothelial proliferation (the so-called multicystic peritoneal mesothelioma) arising multifocally in the abdomen of a 46 year-old white man. His anamnesis showed an 8-year history of intermittent pain in the right lower abdominal quadrant. Mucin stains, immunohistochemistry, and electron microscopy confirmed the mesothelial origin of the lesion. Review of the available literature allowed us to find another 85 reported cases of benign multicystic mesothelial proliferations of the peritoneum. Out of these cases, eighteen only occurred in men, the majority being reported in middle-aged women mostly with complaints of abdominal pain. Electron microscopy or immunohistochemistry are needed to make a differential diagnosis towards other multicystic lesions, such as peritoneal cystic lymphangioma. Although multicystic mesothelial proliferations of the peritoneum have often been regarded as benign neoplasms, the true nature--neoplastic or hyperplastic--of these lesions still remains greatly elusive. Therefore, we believe that the unbinding term benign multicystic mesothelial proliferation (first used with regard to the unique hitherto reported case arisen in the pleural cavity) should be considered at present more appropriate to indicate even these peritoneal lesions. PMID- 1725140 TI - Lung concentrations of 5-hydroxytryptamine, tryptophan, 5-hydroxytryptophan and 5 hydroxyindole acetic acid in rats of different ages. PMID- 1725141 TI - Spatial relationship between cardiac mast cells and coronary capillaries in neonatal rats with cardiomegaly. AB - Density of cardiac mast cells and their localization with respect to coronary capillaries was studied in two experimental situations. First, cardiac hypertrophy produced by aortic constriction in 5-day-old rats was studied. Left ventricular weight increased more than twofold in this experimental situation, while the increases in total capillary length and total number of cardiac mast cells were much smaller, resulting in decreased densities of both tissue components. In the second series of experiments, localization of cardiac mast cells at two distinct portions of coronary capillaries was studied in normal hearts of adult rats. Differential histochemical staining enabled us to distinguish between the portions of capillaries close to arterioles and portions on the venular side. The number of mast cells close to arteriolar portions of coronary capillaries was significantly higher than one would expect in the case of their even distribution along the capillary wall. The relationship between the mast cells and formation of new capillaries is discussed. PMID- 1725142 TI - The structure of Proteus penneri strain 14-O-specific polysaccharide containing D and L-alanine. PMID- 1725143 TI - Importance of the L-arginine-nitric oxide pathway in NANC nerve function of the opossum esophageal body. AB - Circular muscle strips from the opossum esophageal body obtained 3-5 cm above the esophagogastric junction were suspended in organ baths for measurement of isometric tension. Stimulation of nonadrenergic, noncholinergic (NANC) inhibitory nerves was performed using transmural field stimulation (TMS). During TMS, no mechanical response was elicited. After cessation of the stimulus a short period, also without mechanical response, intervened, and this period is called latency. The latency was followed by the 'off'-contraction. In control preparations, the latency and the amplitude of the 'off'-contraction were 1.47 +/- 0.17 s, and 3.8 +/- 0.9 mN, respectively. The inhibitor of the L-arginine-nitric oxide (NO) pathway, NG-nitro-L-arginine (L-NNA) concentration-dependently reduced the latency at concentrations greater than 10(-6) M (n = 6-7). At the highest concentration of L-NNA (10(-4) M), 'off' contractions were no longer seen. In 5 out of 7 preparations exposed to L-NNA (10(-4) M), a small contraction was seen during stimulation, and this contraction was abolished by atropine (10(-6) M) in all strips. L-NNA concentration-dependently reduced the amplitude of contractions at concentrations greater than 10(-6) M (n = 6-7). At 10(-4) M, the amplitude was reduced to 3 +/- 2% of that of the initial contraction. Preincubation with L arginine (10(-5) M) had no influence on the latency. The effects of L-NNA on both latency and the amplitude of contraction were antagonized by preincubation with L arginine (10(-5) M). Atropine (10(-6) M had no effect on the amplitude of the 'off'-contraction in control preparations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725144 TI - Effect of substance P on right colonic pressure: a dose response study in pigs. AB - The purpose of the present investigation was to elucidate the influence and mechanism of the action of substance P (SP) upon intraluminal pressure in proximal porcine colon. In 9 pigs, SP was infused intra-arterially in varying doses from 0.5 to 160 ng/kg/min. Mean baseline colonic pressure was 8.9 mm Hg increasing in a dose-dependent manner to 34.6 mm Hg at an SP dose of 40 ng/kg/min. Hereafter, the colonic pressure remained constant. In other series, SP infusion (5-80 ng/kg) was preceded by infusion of hexamethonium (0.5 mg/kg/min, continuous i.v.; n = 7), atropine (0.01 mg/kg/30 min, i.v.; n = 6), by naloxone (0.01 mg/kg/15 min, i.a.; n = 5) or tetrodotoxin (1 microgram/kg/h, i.a.; n = 7). Atropine significantly inhibited the effect of SP on colonic pressure, whereas the other blockers were without influence on the SP-induced colonic pressure. It is concluded that SP has a potent dose-dependent stimulatory effect upon right colonic intraluminal activity of the pig, mediated partly by atropine-sensitive receptors. PMID- 1725145 TI - Postural sway analysis of a teenager with childhood lead intoxication--a case study. AB - We had an opportunity to study a 15-year-old boy, evaluating the long-term effect of early childhood lead (Pb) poisoning on the maturation of postural balance. Postural balance (or sway) was quantified using a microprocessor-based Force Platform System along with four postural tasks specifically designed to indirectly challenge and/or minimize the effect of vision, proprioception and vestibular systems relevant for postural stability. The Pb-intoxicated patient showed increased postural sway compared to those of non-poisoned young adults and a 14-year-old boy for postural tasks requiring input from higher centers. A review of historical information and results of the physical and neurological examinations did not reveal an alternative explanation for this patient's postural sway abnormalities. These responses are also comparable to those noted in younger children with chronic Pb-exposure histories. In summary, this case study gives suggestive evidence that early excessive exposure to Pb can have long term, detrimental neurological effects as reflected by postural stability. Measurement of postural balance appears to be a sensitive research methodology to assess subclinical neurological effects of remote lead intoxication. PMID- 1725146 TI - [Tyrosine hydroxylase activity in Drosophila virilis in normal conditions and under heat stress]. PMID- 1725147 TI - The use of oral antibiotics in daily clinical practice. AB - In the treatment of infectious diseases in daily clinical practice, the physician is faced with a wide choice of antibiotics. Rational antibiotic use requires knowledge of the pathogens causing disease at that site, and the prevalence of resistance. In outpatient respiratory tract infection, for example, 3 pathogens, Branhamella (Moraxella) catarrhalis, Haemophilus influenzae and Streptococcus pneumoniae, predominate, beta-Lactamase production by the first 2 is a significant factor in antibiotic selection for respiratory tract infection. Empirical antibiotics are selected for efficacy, cost-effectiveness, safety and patient compliance. PMID- 1725148 TI - Single dose oral administration of cefixime 400mg in the treatment of acute uncomplicated cystitis and gonorrhoea. AB - A placebo-controlled prospective randomised double-blind study was performed in 80 consecutive female outpatients with acute cystitis. Single dose oral antibiotic treatment was successful in 89.4% of patients treated with cefixime 400mg or ofloxacin 200mg and in 84.2% of those receiving cotrimoxazole (160/800mg). Bacteriuria was eradicated in 26.3% of patients in the placebo group. Two noncomparative clinical trials involving a total of 43 male patients with acute gonococcal urethritis reported a 100% cure rate after administration of a single 400mg dose of cefixime. Such single dose regimens offer the advantages of reduced expense, good tolerability, minimal alteration of normal bacterial flora, and the potential for improved patient compliance, compared with multiple dose antibacterial therapy. PMID- 1725149 TI - Direct and indirect pathogenicity of beta-lactamase-producing bacteria in respiratory tract infection in children. Role of cephalosporins resistant to enzymatic hydrolysis. AB - A recent increase in the incidence of beta-lactamase-producing aerobic and anaerobic bacteria in upper respiratory tract infection has been associated with an increase in the failure rate of penicillin treatment of these infections. Experimental evidence for this correlation has been reported by many investigators, who have described the sheltering of susceptible pathogens by beta lactamase-producing organisms as 'indirect pathogenicity'. The organisms implicated in mixed infections that cooperate are Staphylococcus aureus, Haemophilus influenzae, Branhamella (Moraxella) catarrhalis and Bacteroides spp., which are pathogenic and also normal oropharyngeal inhabitants. Since these bacteria exhibit both direct and indirect pathogenicity, effective treatment requires administration of antimicrobial therapy active against all microorganisms present in mixed infection. Oral third generation cephalosporins, which are resistant to enzymatic hydrolysis, seem to be suitable initial therapy. PMID- 1725150 TI - Clinical efficacy and tolerability of cefixime in the treatment of acute sinusitis. AB - In a noncomparative trial, 73 adults with acute sinusitis confirmed by x-ray received cefixime 400mg once daily for approximately 10 days. At the end of treatment, 60 patients (82%) were cured, 2 (2.7%) had improved and 7 (9.6%) had failed therapy; 4 patients were not evaluable. No relapses were observed at follow-up. Haemophilus influenzae, Streptococcus pneumoniae and Branhamella (Moraxella) catarrhalis were the main pretreatment pathogens, accounting for 65% of all bacterial isolates. Overall, 84% of pathogens were eradicated after treatment. Cefixime was well tolerated, moderate gastrointestinal disturbances being the most frequent adverse effects noted (3 of 4 patients with adverse effects). These results are comparable to those obtained with cefixime 400mg administered orally in 2 divided doses. PMID- 1725151 TI - Cefixime vs amoxicillin in the treatment of acute otitis media in infants and children. AB - Cefixime is a new oral cephamycin antibiotic with a broad spectrum of antibacterial activity in vitro. It is resistant to hydrolysis by most beta lactamases and has pharmacokinetic characteristics which allow administration in a single daily dose for the treatment of some bacterial infections. The aim of this study was to compare the clinical efficacy of cefixime with that of amoxicillin in the treatment of acute otitis media in 40 children. Cefixime 8 mg/kg was given once daily at bedtime, whereas amoxicillin 50 mg/kg/day was administered in 3 divided doses; both drugs were given for 10 days. 15 days after starting the trial, a favourable clinical response was demonstrated in 18 of 20 children in both treatment groups. Cure rates, recurrences and persistent middle ear effusions were not significantly different in the 2 study groups during a 3 month follow-up. It was concluded that cefixime is clinically effective and well tolerated in the treatment of children with acute otitis media. PMID- 1725152 TI - Efficacy and tolerability of cefixime in otitis media. A multicentre study in over 25,000 children. AB - A large scale multicentre clinical study was undertaken to assess the efficacy and tolerability of cefixime in the treatment of acute otitis media in children. A total of 25,863 evaluable children with acute otitis media received cefixime 8 mg/kg once daily for at least 10 days. At the end of treatment, 86% of patients were considered to be either cured or improved. These results are consistent with those achieved with cefixime in controlled clinical trials. Adverse effects were reported in 11.5% of patients, but were judged to be related to cefixime therapy in only 9.4% of patients. The results of this study demonstrate that cefixime is an effective and well tolerated treatment for acute otitis media in children. PMID- 1725153 TI - Comparative bioavailability study of cefixime administered as tablets or aqueous solution. AB - The relative bioavailability of cefixime was studied in 24 healthy male volunteers, with each subject receiving a single 400mg dose of cefixime administered as an aqueous solution, a 400mg tablet and two 200mg tablets, in a randomised crossover sequence. Serum and urine samples were analysed using high performance liquid chromatography. Peak cefixime levels were achieved 3 hours after administration of the solution vs 4 hours for the 2 tablet formulations; however, the extent of absorption was only slightly improved with the solution (by 14 and 7% compared with the 1 x 400 and 2 x 200mg tablets, respectively). The 400mg and 2 x 200mg tablets were found to be bioequivalent. The pharmacokinetic profile of the 400mg cefixime tablet (mean maximum plasma concentrations of 4.4 mg/L at 4 hours, area under the concentration-time curve of 34.4 mg/L.h, and apparent terminal elimination half-life of 3.7 hours) supports the clinical evaluation of a 400mg once-daily dosage regimen for cefixime. PMID- 1725154 TI - Ion channel agonists: expectations for therapy. AB - Some animal or plant toxins and man-made drugs exert agonist activity on Na+, Ca2+ and K+ channels. The increase in current through these channels is essentially due to an increase of 'open probability' and not of single channel conductance. The enhanced open probability is caused by a prolongation of the open time. In the case of voltage-operated channels this change in open time can be accompanied by increased reopenings and thus slowing of inactivation, or a shift in the activation process to more negative potentials. In the case of the ligand-operated K+ channel, a decrease in the affinity for the normal physiological ligand, ATP, is the mechanism underlying the enhancement of open probability. Agonists show potential clinical applications for Na+ and Ca2+ channels more specifically as positive inotropic agents in cardiac tissue. For K+ channels, the potential therapeutic field is even broader and spans from relaxation of smooth muscle (hypertension, asthma, bladder, uterus), reduction in excitability (arrhythmias, certain skeletal muscle myopathies) to inhibition of neurotransmitter release (depression, epilepsy). PMID- 1725155 TI - Triggered activity in the heart: cellular mechanisms of early after depolarizations. AB - The arrhythmogenic effects of ischaemia and reperfusion result from the complex interplay of normal ion channels reacting to the ischaemic environment, channels made abnormal by ischaemic modification, the appearance of new currents normally not present, and possible ischaemic alteration of metabolic electrogenic processes. In this report the cellular mechanisms thought to underlie the different types of triggered activity will be discussed. The role of Ca2+ channels and Ca2+ 'window' current in the generation of early after depolarizations (EADs) will be elucidated. PMID- 1725156 TI - Evidence for protein kinase C modulation of the ciliary muscle response to carbachol and desensitization. AB - The role of protein kinase C (PKC) in the desensitization of muscarinic receptor mediated responses in bovine ciliary muscle was examined. Exposure of the bovine ciliary muscle to phorbol esters, used to activate PKC, resulted in antagonism of muscarinic receptor-mediated contraction. On the other hand, staurosporine, a known PKC inhibitor, caused a significant potentiation of the contractile effect induced by carbachol. Staurosporine reduced the desensitization induced by repeated additions of carbachol and completely suppressed that induced by phorbol esters. The results also indicate that desensitization mediated by phorbol esters as well as that mediated by muscarinic receptor agonists is heterologous. PMID- 1725157 TI - Centrally coinjected galanin and a 5-HT1A agonist act synergistically to produce vasodepressor responses in the rat. AB - The present study was undertaken to evaluate the possible functional implications of the previously demonstrated in vitro interactions between galanin and 5-HT1A receptors. To this end we analysed the interactions between galanin and the 5 HT1A receptor agonist 8-OH-2-(di-n-propylamino)-tetralin (8-OH-DPAT) in central cardiovascular regulation. 8-OH-DPAT given intracisternally (i.c.) produced a dose-dependent reduction of blood pressure, the peak action being 32% at 10 nmol of 8-OH-DPAT. Heart rate and respiration rate were not affected. The vasodepressor action of 8-OH-DPAT was counteracted by the 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190). A threshold dose (1 nmol) of galanin given i.c. was shown to enhance the vasodepressor effect of both an ED50 dose and a threshold dose of 8-OH-DPAT. Quantitative receptor autoradiography showed that the IC50 values for [125I]galanin binding sites were reduced in the presence of 8-OH-DPAT (10 nM) by approximately 40% in the dorsal region of the nucleus of the solitary tract, the area postrema, and the raphe pallidus and obscurus nuclei. Galanin (10 nM) also significantly increased the IC50 value for [3H]8-OH-DPAT binding sites within the nucleus of the solitary tract. The results provide evidence for a synergistic interaction between 8-OH-DPAT and galanin in cardiovascular regulation after their central administration, an interaction possibly related to the ability of 8 OH-DPAT to enhance the affinity of the galanin receptor within regions of the medulla oblongata involved in cardiovascular control. PMID- 1725158 TI - Metastatic prostatic adenocarcinoma-diagnosed by bronchoalveolar lavage and tumour marker determination. AB - We report a patient who presented with a diffuse interstitial lung disease in whom clinical and radiologic investigation led to the suspicion of lymphangitis carcinomatosa. Bronchoalveolar lavage (BAL) was performed and revealed the presence of prostatic specific antigen (PSA) positive cells. A prostatic needle aspiration confirmed the diagnosis of adenocarcinoma. This case demonstrates the value of tumour marker determination in BAL. PMID- 1725159 TI - Cytoprotective and dose-dependent inhibitory effects of prostaglandin E1 on rat pancreas treated with ciclosporin A. AB - The prostaglandin E1 analogue rioprostil protects the pancreas from the noxious effect of ciclosporin A (CsA). To determine whether this cytoprotective action of rioprostil is dependent on or independent of inhibitory effects on pancreatic exocrine and endocrine secretion we studied the effect of different doses of rioprostil on pancreatic exocrine and endocrine secretions in the presence or absence of CsA. Rats received either CsA at a dose of 10 mg/kg body weight by tube feeding once in the morning, rioprostil at linearly increasing doses from 1.8 to 120 micrograms/kg body weight subcutaneously twice daily, a combination of both substances or NaCl. After 8 treatment days, the animals were operated on, and the pancreas isolated and arterially perfused. Insulin secretion was determined after stimulation with glucose, and amylase secretion after stimulation with CCK-8. Insulin and amylase secretion were significantly impaired by CsA. Rioprostil at doses of 1.8, 3.6 and 7.5 micrograms/kg body weight had no significant effect on insulin secretion in the absence of CsA but significantly improved insulin secretion in the presence of CsA. Higher doses of rioprostil significantly inhibited insulin secretion both in the presence or absence of CsA. Amylase secretion was not influenced by rioprostil at doses up to 15 micrograms/kg body weight but improved significantly amylase secretion in the presence of CsA. CsA blood and pancreatic tissue levels were not influenced by rioprostil at doses up to 120 micrograms/kb body weight. We conclude that the cytoprotective effect of the prostaglandin E1 analogue rioprostil against the noxious effect of CsA is dose dependent and is not related to its inhibitory action on endocrine and exocrine pancreatic secretion. PMID- 1725160 TI - Galanin and vasoactive intestinal polypeptide: coexistence and corelease from the vascularly perfused pig ileum during distension and chemical stimulation of the mucosa. AB - By immunohistochemistry and double staining technique, almost complete coexistence of galanin-like immunoreactivity (GAL-LI) and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) was demonstrated in submucosal ganglionic cells and mucosal nerve fibers of the porcine ileum. The release of the two neuropeptides was studied in isolated, vascularly perfused pig ileum. Distension (increasing intraluminal pressure by 10 mm Hg) and intraluminal instillation of homologous gallbladder bile, amino acids, 0.1 M HCl, hypertonic NaCl (3,400 mosm x kg-1) and hypertonic glucose (1,100 mosm x kg-1) all increased the release of GAL-LI and VIP-LI into the venous effluent in parallel. Being the most potent stimulus, bile increased the output of GAL-LI from 0.74 +/- 0.12 to 2.44 +/- 0.81 pmol/min (mean +/- SE, p = 0.018) and the output of VIP-LI from 3.29 +/- 0.19 to 12.68 +/- 4.01 pmol/min (p less than 0.001), respectively. In conclusion, the coexistence and parallel release of GAL and VIP suggest that GAL/VIP neurons may be involved in intramural secretory and motor reflexes. PMID- 1725161 TI - Evidence for the extralenticular expression of members of the beta-crystallin gene family in the chick and a comparison with delta-crystallin during differentiation and transdifferentiation. AB - The beta-crystallins are major water soluble proteins of vertebrate lens fibre cells and have previously been regarded as lens-specific proteins: however beta B2-and beta A3/A1-crystallin RNAs are transcribed and beta-crystallin polypeptides are detectable in the developing chick retina. The beta-crystallin RNA is transcribed in a subpopulation of retina cells and the number of transcribing cells and the level of beta-crystallin polypeptides increase during the differentiation of the retina. Several tissues express beta-crystallin polypeptides, but individual tissues are characterised by qualitative and quantitative differences in the beta- and delta-crystallin polypeptides expressed. The expression of beta-crystallins appears to be non-random as defined by tissue distribution, cellular localisation and ontogeny, implying a function for extralenticular beta-crystallins and a complex mechanism for the regulation of their expression. PMID- 1725162 TI - The expression of the genes for entactin, laminin A, laminin B1 and laminin B2 in murine lens morphogenesis and eye development. AB - The temporal expression of the genes for the excellular matrix proteins entactin and the A, B1, and B2 chains of laminin was examined in the eye of the developing mouse embryo by in situ hybridization of their messenger RNAs. Entactin messenger RNA was found in abundance in specific cells. In the 25 somite embryo entactin message was synthesized by mesenchymal cells and, at later stages, by hyalocytes and lens cells in addition. The message was not detectable in corneal epithelium at embryonic stages E15 and E18.5 and at birth but was present in adjacent stromal cells. At the 28 and 38 somite stages, before pigment granules interfered with the detection of silver grains, no entactin message was detected in pigmented epithelial cells, in contrast to the messages for laminin B1 and B2. Entactin was not found in the neural epithelium at any time during development. The distribution of the laminin B1, B2 and A chain messenger RNAs was distinctly different from that of entactin. In particular, during the early stages of development B1 and B2 messages were synthesized by ectodermal, lens, corneal, pigment epithelial and hyaloid cells. In the older embryos cells in the ganglion layer of the retina synthesized B1 and B2 messages but undetectable amounts of entactin or the A chain messages. In general the A chain message was in lower abundance throughout development. The distribution of laminin and entactin messages suggested that the extracellular matrices, which contained both proteins, can be derived either from a single cell type or from the contributions of multiple cell types. The data demonstrate the complexity of extracellular matrix synthesis and assembly in the diverse structures of the developing eye where the temporal expression of specific molecules are tailored to the specific developmental requirements of particular structures. PMID- 1725163 TI - Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. AB - A defined cultivation system was developed for the differentiation of pluripotent embryonic stem cells of the mouse into spontaneously beating cardiomyocytes, allowing investigations of chronotropic responses, as well as electrophysiological studies of different cardioactive drugs in vitro. The beta adrenoceptor agonists (-)isoprenaline and clenbuterol, the mediators of cAMP metabolism, forskolin and isobutylmethylxanthine (IBMX), the alpha 1-adrenoceptor agonist (-)phenylephrine, and the heart glycoside digitoxin induced a positive, the muscarinic cholinoceptor agonist carbachol and L-type Ca2+ channel blockers nisoldipine, gallopamil and diltiazem induced a negative chronotropic response. In early differentiated cardiomyocytes beta 1-, alpha 1-, but not beta 2 adrenoceptors, cholinoceptors, as well as L-type Ca2+ channels participated in the chronotropic response. In terminally differentiated cardiomyocytes beta 2 adrenoceptors and digitoxin responses were also functionally expressed. The contractions of spontaneously beating cardiomyocytes were concomitant with rhythmic action potentials very similar to those described for embryonic cardiomyocytes and sinus-node cells. We conclude that cardiomyocytes differentiating from pluripotent embryonic stem cells are able to develop adrenoceptors and cholinoceptors and signal transduction pathways as well as L type Ca2+ channels as a consequence of cell-cell interactions during embryoid body formation in vitro, independent of the development in living organisms. The cellular system described may be useful as in vitro assay for toxicological investigations of chronotropic drugs and a model system for studying commitment and cellular differentiation in vitro. PMID- 1725164 TI - Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation. AB - The patterns of cytoskeletal differentiation were studied in 20 testicular non seminomatous germ cell tumors by immunohistochemistry, using diverse monoclonal antibodies specific for different intermediate filament (IF) proteins and for desmoplakin. Immunofluorescence and immunoperoxidase methods on both formalin fixed and frozen tissues were applied, in some cases together with a gel electrophoretic analysis of IF proteins. The tumors examined included embryonal carcinoma (EC), endodermal sinus tumor (EST), choriocarcinoma and teratoma. Nine of the tumors were composed of only one histological type, the others showed mixed components. Cytokeratins 8 and 18 were identified in all these neoplasms, but their immunostaining was weak in ECs. Cytokeratin 19 was absent or very scarce in ECs, but strongly expressed in ESTs, choriocarcinomas and teratomas, thus allowing the identification of small EST and choriocarcinoma elements in ECs even when they were morphologically not obvious. Occasionally, some cells in ECs and ESTs also stained for cytokeratins 4 and/or 17, indicating potential for epithelial stratification. The majority of the germ cell tumors showed varied amounts of vimentin, often in co-existence with cytokeratins. Neurofilaments were demonstrated in scattered tumor cells in a single case of EST. In the teratomas studied, each type of tissue component present showed the expected IF protein. However, in many germ cell tumors some stromal cells and blood vessels contained, in addition to vimentin and desmin, also cytokeratins 8 and 18. This heterogeneity of the cytoskeletal profile of germ cell tumors is indicative of the varied differentiation potential inherent in these neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725165 TI - Variable expression of retinoic acid receptor (RAR beta) mRNA in human oral and epidermal keratinocytes; relation to keratin 19 expression and keratinization potential. AB - Previous studies have revealed that the cells that form the different regions of the oral and epidermal stratified squamous epithelia represent a number of intrinsically distinct keratinocyte subtypes, each of which is developmentally programmed to preferentially express a particular pattern of keratins and type of suprabasal histology. Retinoic acid (RA) is known to modulate stratified squamous epithelial differentiation, including expression of the basal cell keratin K19 and the suprabasal keratins K1/K10 and K4/K13. We have found that all keratinocyte subtypes are similar in their steady state levels of RAR alpha and RAR gamma mRNAs in culture and that these levels are only minimally affected by RA. In contrast, RAR beta mRNA expression varies greatly among keratinocyte subtypes and, in eight of ten cell strains examined, directly correlated with their levels of K19 mRNA. Exposure to 10(-6) M RA increases the levels of RAR beta and K19 mRNA; conversely, complete removal of RA from the medium results in reduced levels of these messages. RA does not coordinately induce RAR beta and K19 messages in nonkeratinocyte cell types: fibroblasts cultured in the presence of 10(-6) M RA express very high levels of RAR beta mRNA but do not express detectable K19, and mesothelial cells decrease their levels of RAR beta and K19 mRNA in response to 10(-6) M RA. The correlation between RAR beta and K19 mRNA levels in most keratinocyte subtypes suggests a role for RAR beta in specifying patterns of keratin expression and suprabasal differentiation in stratified squamous epithelia. PMID- 1725166 TI - Protective effects of human recombinant MnSOD in adjuvant arthritis and bleomycin induced lung fibrosis. AB - We have previously shown that human recombinant methionyl manganese superoxide dismutase (MnSOD) is more efficient than CuZnSOD as an anti-inflammatory agent in a model of acute inflammation (Carrageenan-induced paw edema). This effect was attributed to the prolonged half-life of MnSOD in blood (t1/2 = 6 h vs. 10 min, respectively). In the present study, the two enzymes were compared in terms of their effectiveness in two systems: (1) Adjuvant-induced arthritis in rats, which is considered to be a model for the chronic situation of rheumatoid arthritis and (2) Bleomycin-induced lung fibrosis, which is a chronic situation believed to be mediated by oxygen free radicals. Rats inflicted with adjuvant arthritis were treated during the period of maximal joint swelling (Days 15-21 after adjuvant injection) with MnSOD or CuZnSOD (12 to 50 mg/kg, i.p. daily). MnSOD administration resulted in a 50-75% reduction of paw swelling, as well as inhibition of the elevation of serum globulins. A similar treatment with CuZnSOD gave merely marginal effects. In the second system, lung fibrosis was induced in rats by intratracheal administration of bleomycin. MnSOD (50 mg/kg, s.c.), administered 2 h before and then 2 and 4 days after bleomycin, markedly inhibited lung fibrosis, as evident from lung weight and collagen content measured by the 3rd week. By contrast, CuZnSOD administration did not give a significant effect. The results indicate that MnSOD is superior to CuZnSOD in the treatment of chronic inflammatory processes. In addition, they lend further support to the involvement of oxygen free radicals in bleomycin toxicity. PMID- 1725167 TI - Identification of molecules detected by three different anti-H-2Kk monoclonal antibodies. AB - Three different monoclonal antibodies specific for murine Class I H-2Kk-encoded determinants have been found to bind to molecules other than the classical 45 Kd Class I glycoprotein. These results were obtained after electrophoresis of a detergent lysate of spleen cells, and extraction of molecules present in multiple gel fractions. The antigenic activity in each gel fraction was assessed after removal of detergent by capacity to inhibit the binding of each of these antibodies to Ig-capped spleen cells in a rosetting assay. Only the H100-27R9 antibody was inhibited by an extract representing 45-50 Kd proteins resembling Class I molecules. This antibody also bound material extracted from a higher 65 70 Kd fraction of the gel. The binding of the 11-4.1 and H100-30R3 antibodies was inhibited by unique molecules in the molecular weight range of less than 20 Kd. The inhibitory material was not sensitive to pronase but was sensitive to glycosidases. This material could be absorbed by each of the H100-30R3 and 11-4.1 antibodies but not by H100-27R9. All data are consistent with unique, low molecular weight carriers of carbohydrate-defined epitopes which can bind monoclonal antibodies having specificity for H-2K-encoded gene products. It is predicted that these are glycolipids, but it is not yet known whether they are cell-surface or cytoplasmic molecules. PMID- 1725168 TI - Immunological and biochemical characterization of the nonspecific cross-reacting antigen epitopes using twenty-three monoclonal antibodies. AB - Twenty-three monoclonal antibodies (MAbs) reactive with nonspecific cross reacting antigen (NCA) were prepared and used for constructing a serological map of the NCA molecule. The MAbs were generated using purified NCA or carcinoembryonic antigen (CEA) as immunogen. The MAbs could be divided into two groups: Group X, 10 clones reactive with NCA and CEA; and Group Y, 13 clones specific for NCA. Cross-competition enzyme immunoassays between MAbs of the individual groups revealed that at least 8 different subgroups can be defined i.e., 5 and 3 subgroups in Groups X and Y, respectively. The chemical nature of the epitopes recognized by those MAbs was tested using chemically or enzymatically treated antigens; all MAbs reacted with periodate-treated NCA and deglycosylated NCA, indicating that all the epitopes identified appeared to be protein in nature. Reduction and alkylation, pepsin digestion or pronase treatment of NCA, however, gave some differential results with respect to MAb binding. The serologic mapping and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA. PMID- 1725169 TI - Antitumor monoclonal antibodies generated by immunization with mucins. AB - This report describes the development and characterization of monoclonal antibody EG2.3. Although produced from a fusion that used splenocytes from donor mice immune to bovine salivary mucin (BSM), EG2.3 bound selectively to a number of human tumor cell lines including colon adenocarcinoma LS174T. Therefore, EG2.3 was compared to B72.3, another mucin (TAG-72) binding monoclonal antibody that also binds to LS174T. Like B72.3, EG2.3 reacted with an epitope on TAG-72. However, these two MAbs differed in a number of ways. Treatment of mucin or TAG 72 with periodate did not reduce the binding of EG2.3 to either antigen. In contrast, B72.3 did not react with either periodate treated antigens. Removal of sialic acid from either BSM or TAG-72 compromised the reactivity of both EG2.3 and B72.3. It was concluded that the EG2.3 binding site was distinct from the carbohydrate structure detected by B72.3. PMID- 1725170 TI - Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin. AB - Seven high affinity antibodies to human serum transferrin which recognize at least four different epitopes are described. Apparent dissociation constants (Kd's) have been determined for the binding of the antibodies to human transferrin in the presence and absence of iron. Small differences in reactivity were found. Five of the antibodies bind to the isolated amino-terminal half molecule of human transferrin. Two of the antibodies appear to be to the C terminal lobe since they bind to holo-transferrin but do not recognize the N terminal half-molecule. Immunoblotting shows that six of the antibodies recognize both reduced and nonreduced transferrin. In addition, all of the antibodies bind with sufficiently high avidity to transferrin to make them useful as probes in studies in which binding of transferrin to the specific transferrin receptor is examined. PMID- 1725171 TI - 16-kilodalton rice protein is one of the major allergens in rice grain extract and responsible for cross-allergenicity between cereal grains in the Poaceae family. AB - Cross-allergenicity between five cereal grains including rice, wheat, corn, Japanese millet (Panicum crus-galli L. var. frumentaceum Trin.) and Italian millet (Setaria italica Beauv. var. germanica schrad.) was examined by radioallergosorbent test (RAST) and RAST inhibition assay. There were significant close correlations between every combinations of RAST values for the five cereal grain extracts. RAST inhibition assay of each extract against RAST discs coupled with other cereal grain extracts indicated marked cross-reactivity of IgE binding between these cereal grain extracts. Rice protein 16KD (RP16KD) was shown to be one of major allergens in rice grain extracts by immunoblotting analysis, histamine release assay from human leukocytes and RAST inhibition. Next, the involvement of RP16KD in the cross-allergenicity between these cereals was investigated. RAST values for RP16KD significantly correlated with that for Italian millet as well as rice but not with those for corn and wheat. There was a trend of positive correlation between RAST values for RP16KD and Japanese millet. In the RAST inhibition assay using sera with positive RAST for these five cereal grain extracts and RP16KD, RP16KD inhibited IgE binding to these all cereal discs in a dose-dependent manner. Similarly, all of the five cereal grain extracts showed an effective decrease in IgE binding to the RP16KD disc. These results indicated possible participation of IgE binding structure on RP16KD in cross allergenicity between these cereal grain extracts in the Poaceae family. PMID- 1725172 TI - Phase II study of fazarabine in advanced head and neck cancer. A Southwest Oncology Group study. PMID- 1725173 TI - Human T-lymphoblastoid cells selected for growth in serum-free medium provide new tools for study of HIV replication and cytopathogenicity. AB - Human T-lymphoblastoid cells H9, CEM and CEM-clone 5 were selected for growth in RPMI 1640 supplemented with transferrin 5 micrograms/ml, insulin 5 micrograms/ml and sodium selenite 5 ng/ml. After 40 days of adaptation to serum-free medium, these cells displayed growth, morphology, and expression of CD4 similar to serum supplemented cultures. Infection of these cells with two strains of HIV-1 (LAV and NDK) and a strain of HIV-2 (ROD) was as efficient in serum-free as in serum supplemented medium as demonstrated by reverse transcriptase activity in the culture supernatants of infected cells. Furthermore, HIV-induced cytopathogenicity was observed in serum-free cultures, demonstrating that both HIV infection and cytopathic effect did not require the presence of serum components. Electron microscopy showed that mature viral particles were produced from infected cells cultured in serum-free medium. Finally, the ability of monoclonal antibody OKT4 A to inhibit infection by HIV-1 LAV but not by HIV-1 NDK was the same with and without serum in the culture medium, demonstrating that both CD4-dependent and CD4-independent infections can occur in the total absence of serum. Human T-lymphoblastoid cells adapted for growth in serum-free medium provide therefore a complementary tool for the study of HIV infection and cytopathogenicity under defined conditions. PMID- 1725174 TI - [Changes with time in incomplete root and periodontal tissues of young dog teeth with torque loading applied]. AB - We investigated, using labeling techniques (tetracycline, calcein) and routine histopathological methods (H. E. and Azan stainings), the histological changes with passage of time in incomplete root and the periodontium of young dog teeth with torque loading applied. The maxillary left first, second and third incisor teeth were used as anchorage teeth, and the maxillary right second incisor was loaded with either 3 degrees or 9 degrees torque. The orthodontic forces of these are 20-30 g and 80-90 g, respectively. Some temporal disturbances (such as mild, moderate, or severe bendings of labial and palatal apexes) were observed in certain regions of the apex of the incomplete root. However, with subsequent development of the root, the disturbances were repaired and pulp of the teeth was normally formed. Furthermore, although periodontium was also temporarily disturbed, it developed normally with the passage of time. PMID- 1725175 TI - Parameters of immunity acute phase reaction in men in relation to exposure duration to mercury vapours. AB - The study was carried out in 89 men aged 21 to 57 years with a history of exposure to mercury vapour from 2 to 26 years during occupational work involving chlorine production by the method of mercury electrolysis. The workers were divided into three groups depending on the duration of occupational exposure: 1) 32 workers with a short history of exposure 2-10 years, 2) 37 workers with medium long exposure - 11-20 years, and 3) 20 workers with a history of long exposure - 21-26 years. The urinary concentrations of mercury in these individuals was 73 +/ 60 microliters x 1(-1), and in blood this concentration was not exceeding 50 microliters x 1(-1). The control group comprised 40 men aged 17 to 52 years. They had not had any occupational exposure to chemicals, or harmful physical factors. On the basis of clinical, haematological and biochemical studies 89 workers with occupational exposure to mercury vapour were regarded as clinically healthy. None of them had any symptoms and signs of the complete neurasthenic syndrome or organic brain injury. Increased nervous excitability was the complaint of 24 workers, 9 had headaches, sleep disturbances were reported by 5, and a feeling of tiredness and apathy was mentioned by 5 men. EEG recording demonstrated 81 normal tracings, and moderately pathological records in 8 men. The parameters of immunity and proteins acute phase reaction were determined, measuring the concentration of immunoglobulins, lysozyme, C3c, C4, alpha 1-acid glycoprotein, haptoglobin and ceruloplasmin in serum. A lower level of IgA, IgG and lysozyme was only noted in individuals with occupational exposure exceeding 20 years.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725176 TI - Recent advances in the development of techniques for diagnosis and epidemiology of leprosy. PMID- 1725177 TI - [New classification of anti-arrhythmia agents according to the effects on ion channels]. PMID- 1725178 TI - Antibody to hepatitis C virus in cryptogenic chronic liver disease. AB - The prevalence of antibody to hepatitis C virus (HCV) was studied in 207 patients with chronic liver disease of unknown etiology, in relation to clinical, epidemiological and histological features. Serum antibody to C-100 epitope of HCV was detected by ELISA in 82.6% of patients, with a significant difference compared with a group of patients with primary biliary cirrhosis (10%). The presence of anti-HCV antibody in serum did not correlate with age, sex, histological diagnosis, and activity and duration of the disease, nor with serum anti-HBc, used as a marker of exposure to hepatitis B virus infection. These results strongly support the view that most cases that were previously defined as cryptogenic forms of chronic liver disease are in fact related to HCV infection. There was a correlation between serum anti-HCV antibody and history of risk for parenteral exposure or of acute hepatitis. This correlation was particularly evident for transmission by parenteral route, suggesting that HCV infection may be transmitted often by this route (36.8% among anti-HCV antibody-positive patients and 11.1% among anti-HCV-negative patients). Liver disease in patients without risk factors for parenteral transmission and with lower prevalence of anti-HCV antibody may be caused by other as yet unidentified non-A, non-B (non-C) agents or may be of nonviral origin. PMID- 1725179 TI - Prevalence and significance of antibodies to hepatitis C virus among Saudi haemodialysis patients. AB - Seventy-four patients who were maintained on chronic haemodialysis in King Khalid University Hospital, Riyadh, Saudi Arabia were tested using the recently available ELISA to determine the prevalence of antibodies to hepatitis C virus (anti-HCV) in a haemodialysis unit. The prevalence rate of anti-HCV antibodies of 41.9% in the haemodialysis patients was significantly higher than the rate of 3.9% detected in 488 asymptomatic blood donors who were similarly tested. In the haemodialysis patients, anti-HCV positivity was related to previous blood transfusion (greater than 5 units of blood) and to the duration of haemodialysis (greater than 4 years); but was unrelated to sex, age, positive HBV markers or to past or current elevation of serum ALT. The results indicate a relatively higher prevalence of anti-HCV antibodies in our patients compared to rates of 1-20% reported from Europe and the U.S.A. An effective control strategy for HCV infection in this high risk group is urgently indicated. PMID- 1725180 TI - Role of disulphide bonds in burst-like activity of nicotinic acetylcholine receptor channels in rat sympathetic neurones. AB - 1. The effects of reduction of disulphide bonds in nicotinic acetylcholine receptors (nicotinic AChRs) with dithiothreitol (DTT) were studied in rat superior cervical ganglion neurones using the patch-clamp method in whole-cell and cell-attached recording modes. 2. Dithiothreitol (1 mM) markedly reduced the ACh-induced membrane current, while the action of ACh remained reversible. Conversely, bromoacetylcholine (BrACh), if applied after the treatment with DTT, caused irreversible activation of nicotinic AChRs manifested in the appearance of a non-declined steady-state component in BrACh-induced currents accompanied by increased membrane current fluctuations. The successive reoxidation of sulphydryl groups by potassium ferricyanide (1 mM-ferricyanide) restored the response to ACh. Ferricyanide itself had a weaker inhibitory effect on the ACh-induced current, compared to the effect of DTT. 3. As a result of the action of DTT (1 mM), the spectrum of BrACh-induced current noise shifted to a higher frequency range. 4. The distributions of durations of the gaps (closed states) and the bursts (the states identified as open states after the shortest gaps were ignored) in single-channel activity of native (non-treated with DTT) nicotinic AChRs caused by ACh (30 microM) and BrACh (30 microM) were similar and both revealed four to five and two to three components for gap intervals and burst durations respectively. 5. Single-channel behaviour of reduced nicotinic AChRs was similar for both ACh and BrACh as agonists, but significantly differed from that in the native one. The first difference was the marked increase in the frequency of the appearance of long closed states of the channel that was presumably due to enhanced receptor desensitization. The second difference was an almost complete disappearance of long bursts associated with disappearance of the fastest component in gap interval distribution. 6. Mean conductance of single nicotinic AChR channels decreased by approximately 20% in the reduced receptor compared with that in the native one, for both agonists. 7. The results suggest a critical role of disulphide bonds for the functioning of native neuronal nicotinic AChRs: the disruption of disulphide bonds leads to the loss of burst like kinetics of the nicotinic AChR ionic channel. PMID- 1725181 TI - ATP activates cationic currents and modulates the calcium current through GTP binding protein in rabbit portal vein. AB - 1. Effects of adenosine 5'-triphosphate (ATP) on ionic currents of dispersed smooth muscle cells of the rabbit portal vein were investigated using the voltage clamp procedure. 2. ATP (greater than or equal to 300 microM) produced transient and maintained inward currents. The former was inactivated within a few seconds, but the latter lasted more than several minutes. The transient but not the maintained current was blocked by pre-treatment with alpha,beta-methylene adenosine 5'-triphosphate (AMP-CPP). The amplitude of the latter was increased by ATP in a concentration-dependent manner. The following investigations were made on the ionic mechanism of the ATP-induced maintained inward current. 3. In 2.5 mM Ca(2+)-containing tetraethylammonium chloride (TEA-Cl) solution (2.5 mM-Ca(2+) TEA+ solution), the reversal potential for the ATP-induced inward current was close to the Cl- equilibrium potential, and in 140 mM-Na+ (nominally Ca(2+)-free or 0.3 mM-EGTA-containing) solution, the reversal potential was coincident with the Na+ equilibrium potential. 4. In 2.5 mM-Ca(2+)-TEA+ solution but not in 140 mM-Na+ solution and in physiological salt solution (PSS), niflumic acid (10 microM), a Cl- channel blocker, and Cl(-)-deficient perfusate in the pipette markedly inhibited the ATP-induced inward current. These results imply that in 2.5 mM-Ca(2+)-TEA+ solution the ATP-activated ion channel may admit Ca2+ which then accelerates the Ca(2+)-dependent Cl- current, but in 140 mM-Na+ solution and in PSS this channel may admit only Na+. 5. Intracellular perfusion of guanosine 5'-O-(3-thio triphosphate (GTP gamma S) did not provoke the current, but significantly increased the amplitude of the ATP-induced inward current in 2.5 mM Ca(2+)-TEA+, 140 mM-Na+ and 2.5 mM-Ba(2+)-containing TEA+ (2.5 mM-Ba(2+)-TEA+) solutions. On the other hand, intracellular perfusion of guanosine 5'-O-(2 thiodiphosphate) (GDP beta S) reduced the amplitude of the ATP-induced inward current in the above solutions. 6. A low concentration of ATP (30 microM) transiently augmented the amplitude of the voltage-dependent Ca2+ current recorded in both 2.5 mM-Ca(2+)-TEA+ solution and PSS, but a high concentration of ATP (3 mM) consistently inhibited the voltage-dependent Ca2+ current in both solutions (4 mM-EGTA in the pipette). Such inhibition was partly prevented by application of 20 mM-EGTA in the pipette.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725182 TI - Blockage and permeation of divalent cations through the cyclic GMP-activated channel from tiger salamander retinal rods. AB - 1. Blockage and permeation of divalent cations through channels activated by guanosine 3',5'-cyclic monophosphate (cyclic GMP) were studied in membrane patches excised from retinal rods of the tiger salamander Ambystoma tigrinum by rapidly changing the ionic medium bathing the intracellular side of the excised membrane. 2. The Na+ current, observed when 110 mM-NaCl was present on both sides of the membrane patch, was reduced by the addition of 1 mM of the chloride salts of Ca2+, Mg2+, Sr2+, Ba2+ or Mn2+ to the bathing medium. The sequence of blocking potency at +60 mV was Mg2+ greater than Mn2+ approximately Ba2+ greater than Ca2+ greater than Sr2+, while at -60 mV it was Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ approximately Mg2+. For all divalent cations the blocking effect depended, in a complex way, on the membrane potential. 3. The blocking effect of Ca2+ and Mg2+ increased when the concentration of cyclic GMP was reduced from 100 to 5 microM. At -60 mV 1 mM-Ca2+ blocked about 34% of the Na+ current in the presence of 100 microM-cyclic GMP, while in the presence of 5 microM-cyclic GMP, 1 mM-Ca2+ blocked about 56% of the Na+ current. 4. When, in the presence of 100 microM-cyclic GMP, 110 mM-NaCl at the intracellular side was replaced by equiosmolar amounts of chloride salts of divalent cations (73.3 mM) a small outward current carried by divalent cations could be observed at large positive membrane potentials. At +60 mV the ratio between the current carried by Na+, Sr2+, Ca2+, Ba2+, Mg2+ and Mn2+ was 83.3:1.4:1:0.58:0.33:0.25. 5. In agreement with previous observations the dependence of the Na+ current on the concentration of cyclic GMP shows a clear co-operativity among cyclic GMP molecules.4+ cyclic GMP-gated channel in excised patches is similar to but not identical to the selectivity sequence of divalent cations through the channel in intact rods. PMID- 1725183 TI - ATP-induced Ca2+ release and Cl- current in cultured smooth muscle cells from pig aorta. AB - 1. The effect of exogenous ATP on transmembrane currents and on the cytoplasmic Ca2+ has been investigated in single cultured smooth muscle cells of pig aorta. 2. ATP applied to cells held at a potential of -50 mV evoked a transient inward current and a transient rise in [Ca2+]i. At a potential of +20 mV the ATP-induced increase in [Ca2+]i was accompanied by an outward current. 3. At a potential of 50 mV, ATP evoked in Ca(2+)-free solution an inward current which was similar to that in the presence of external Ca2+. A second application of ATP in Ca(2+)-free solution induced a much smaller current. 4. ATP induced in Ca(2+)-free solution a pronounced transient stimulation of the 45Ca2+ efflux from confluent smooth muscle monolayers. 5. The I-V curve of the ATP-activated current has a reversal potential close to 0 mV. A reduction of external Cl- shifts this reversal potential in accordance with the change of the Cl- equilibrium potential. 6. It is concluded that ATP causes a release of calcium from intracellular stores. The ensuing increase of [Ca2+]i activates a Cl- current, which can depolarize the cell membrane and thereby promote a voltage-gated Ca2+ entry. PMID- 1725185 TI - Vascular expansion in chronic periodontitis. AB - Animal studies have demonstrated expansion and remodelling of gingival blood vessels in inflamed gingival tissues. However, there is a paucity of information, regarding the vascular response in human gingivitis and periodontitis. In this study, gingival biopsies were obtained from 51 separate patients. Fifteen minimally inflamed, 16 gingivitis and 20 periodontitis specimens were studied. Gingival biopsies were divided into five fields, and a quantitative survey of vascular changes was performed. In fields adjacent to the bacterial plaque irritant, vessel profiles were increased in number with the development of the advanced periodontal lesion. The diameter of blood vessels throughout the entire thickness of gingival biopsies was found to increase with advancing periodontal disease. It is concluded that considerable remodelling of the gingival vasculature occurs in chronic periodontitis, and that this may contribute to the tissue destruction seen in this disease. PMID- 1725184 TI - The role of the beta 4-subunit in determining the kinetic properties of rat neuronal nicotinic acetylcholine alpha 3-receptors. AB - 1. Single-channel currents were recorded from Xenopus oocytes which had been injected with complementary RNAs (cRNAs) for the neuronal nicotinic acetylcholine receptor subunits alpha 3 and beta 4. The co-expression of alpha 3 and beta 4 gave rise to three different channel types with conductances of 22, 18 and 13 pS. 2. The activity arising from the expression of these two subunits was compared with that observed when the alpha 3 subunit was co-expressed with an alternative beta-subunit, beta 2. The alpha 3 beta 4-receptors differed from alpha 3 beta 2 receptors in conductance, open times and most notably, in burst kinetics. The association of the beta 4-subunit with the alpha 3-subunit results in receptors which have a high probability of re-opening after closing, yielding protracted bursts of activity not observed when the alpha 3-subunit is associated with the beta 2-subunit. 3. All three alpha 3 beta 4-channel types had similar burst kinetics. However, the 13 pS conductance channels showed an additional long-lived open state to varying degrees when clusters of activity within an individual record were examined. PMID- 1725186 TI - Sequence of changes in rat buccal mucosa induced by zinc deficiency. AB - In weanling rats, after receiving a zinc-deficient diet (less than 1 ppm) for 4 wk, the buccal mucosa appears hyperplastic. This study determines changes at earlier stages of the lesion. After 9 days of deficiency, the keratin layer had partially converted to parakeratosis and thickened, and the size of the capillary bed was increased. After 18 days, the keratin layer was fully parakeratotic and thickened further. The cellular layer was thickened. The mitotic rate was doubled and rete ridges were convoluted. After 27 days, the keratin and cellular layers were further thickened, and the mitotic rate remained elevated. The rete ridges were further convoluted. The number of mast cells was doubled and the size of the vascular bed had increased further. These findings suggest early and late interactions between the epithelium and lamina propria. After four days on a control diet following 27 days of zinc deficiency, the mucosa returned normal. PMID- 1725187 TI - Ameloblastic carcinoma: case report and review. AB - The histologic classification for odontogenic carcinomas is still under revision; thus, the differentiation between the terms "malignant ameloblastoma" and "ameloblastic carcinoma" has not been definitely stated. Nevertheless, it is recommended to reserve the former for those lesions that, in spite of an apparently innocuous histology, have given origin to metastatic growths, and to apply the latter for those ameloblastomas in which there is histologic evidence of malignancy in the primary, recurrent or metastatic lesions. A case of an ameloblastic carcinoma in the mandible is presented. Histologically, it was characterized by areas with features of a typical ameloblastoma and areas with anaplastic appearances. PMID- 1725188 TI - [Analysis of antigenic structure on lipopolysaccharides (LPSs) from Eikenella corrodens and endotoxin-neutralizing antibody production]. PMID- 1725189 TI - Renal cell carcinoma extending into the vena cava: a multi-institute study. AB - The present paper reports the clinical results from cases of renal cell carcinoma with vena caval tumor thrombus. Fifty patients were entered into the study between 1980 and 1989. The primary tumor and vena caval tumor thrombus were completely removed in 39 (78%) patients, and 41 (82%) received postoperative treatment. The overall 12-, 36-, 60- and 90-month actuarial survival rates were 72, 41, 19 and 10%, respectively. Only tumor stage affected survival, however, other factors such as tumor grade, nodal status, metastatic status and level of vena caval involvement not affecting it. Postoperative interferon (IFN) treatment affected survival, whereas degree of curative surgery did not. The results of our study were, however, examined retrospectively in several institutes. Further investigation of the postoperative treatment for renal cell carcinoma with vena caval tumor thrombus will be necessary. PMID- 1725190 TI - Primary endodermal sinus (yolk sac) tumor of the anterior mediastinum: a report of a successfully treated eleven-year-old boy. AB - An asymptomatic 11-year-old Japanese boy with an endodermal sinus (yolk sac) tumor of the anterior mediastinum was successfully treated with cisplatinum-based combination chemotherapy and subsequent surgical excision. The patient is alive and in good health 60 months from diagnosis. PMID- 1725191 TI - [Pharmacodynamics of allapinine during long-term infusion in patients with chronic ischemic heart disease and frequent ventricular extrasystole]. AB - During a prolonged (24-hour) intravenous administration in a dose of 126 mg, allapinine produced a stable pronounced antiarrhythmic effect in 90% of the patients with frequent premature ventricular contraction and adverse reactions in the central nervous system in 65% of the cases. However, the latter effect of allapinine is short-term and ceased independently without altering the infusion rate. More serious adverse effects (hypotensive reactions, proarhythmic effects) occurred in 4% of the cases. PMID- 1725192 TI - [Extrasystolic activity of the myocardium in pipe-rolling workers during their work]. AB - A total of 30 apparently healthy male pipe-rolling workers having various working conditions and no resting ESG abnormalities in cardiac arrhythmias. A continuous ECG recording was performed in all the examinees during a working day, followed by interpretation of the records separately at rest and during work and automatic calculation of heart rate per min and of the number of supraventricular and ventricular premature contractions per hour. There was a heating microclimate- and hard labour-induced increase in heart rate and the number of premature contractions, ventricular premature contractions in particular. PMID- 1725193 TI - [Effect of lymph-stimulating drugs on the clinical manifestations and parameters of lipid metabolism in patients with post-infarction cardiosclerosis and angina pectoris]. AB - As many as 64 patients with coronary heart disease (CHD) were examined. They were divided into 3 groups: (1) control subjects; (2) patients on supplementary rheogluman and (3) those on supplementary haemodesum (neocompensan). Their status was assessed from the frequency and intensity of anginal attacks, the magnitude of changes in lipid metabolic parameters prior to and following the therapy. Rheogluman and haemodesum were ascertained to have a favourable effect on the course of CHD, rheogluman producing a more beneficial effect. Supplementation of rheogluman to the treatment of CHD resulted in better correction of lipid metabolic disturbances in postinfarction cardiosclerosis to a greater extent than in angina. The hypolipidemic effect of haemodesum was slightly weaker than that of rheogluman. Rheogluman contributes to better correction of lipid parameters mainly in Type IIb hyperlipidemia, whereas haemodesum is more effective in type IIa. PMID- 1725194 TI - Angiotensin-converting enzyme inhibitors in the treatment of hypertension. AB - This article examines the evidence justifying the use of angiotensin-converting enzyme (ACE) inhibitors as first-line antihypertensive drugs. ACE inhibitors are as effective as traditional antihypertensive agents and, in addition, exert their blood-pressure-lowering effect with near optimal hemodynamic alterations. These drugs have a good tolerance and safety profile although they induce cough in an appreciable number of patients. They can be safely associated with other antihypertensive agents to provide therapeutic benefit in a large proportion of the hypertensive population. ACE inhibitors are contraindicated only in bilateral renal artery stenosis or stenosis of a single kidney. Although to date no prospective study has examined the ability of ACE inhibitors to reduce cardiovascular morbidity and mortality in hypertension, several features of these drugs suggest that this may be the case as several effects of ACE inhibitors in hypertension are potentially nephroprotective and cardioprotective. PMID- 1725195 TI - ACE inhibition--prospects for the future. Satellite symposium to the XIIth Congress of the European Society of Cardiology. Stockholm, September 19, 1990. PMID- 1725196 TI - Angiotensin-converting enzyme inhibition and vascular structure in hypertension. AB - The pathogenesis of hypertension is associated with a remodeling of vascular structure. Folkow has postulated that the decreased luminal area and thickened medial layer in hypertensive vessels enhance the vasoconstrictive response to vasoactive agents. It is hypothesized that this increase in vascular reactivity may serve to perpetuate hypertension. These same structural changes also predispose to the end-organ damage associated with hypertension. A growing body of evidence suggests that autocrine-paracrine vasoactive substances and growth factors modulate vascular structure in hypertension. It is speculated that therapeutic interventions that normalize blood pressure as well as reverse the vascular remodeling process may have special clinical value. The roles of the paracrine renin-angiotensin system and angiotensin-converting enzyme inhibitors in hypertension are discussed in this context. PMID- 1725197 TI - Vascular effects of perindopril: from experimental to clinical investigation. AB - Vascular remodeling is central to the pathophysiology of hypertension and atherosclerosis. The effects of antihypertensive drugs on this process are important to consider from a mechanistic and a pathogenetic point of view in relation to vascular complications of hypertension, e.g., decrease in vascular reserves, shift in cerebral blood flow autoregulation and atherosclerosis development. There is now evidence that, in addition to several other growth factors, vasoactive peptides such as angiotensin II may act as vascular smooth muscle growth promoting substances. Based on these data, the effects of perindopril, a potent and long-lasting angiotensin-converting enzyme (ACE) inhibitor, on structural and mechanical properties of the arterial wall, have been studied in animal models of hypertension as well as in humans. Perindopril completely reversed aortic medial hypertrophy and arterial stiffening observed in renovascular hypertensive rats. Similar benefits were reported in mesenteric resistance vessels of spontaneously hypertensive rats. The effect of perindopril was totally in keeping with potent inhibition of vascular ACE and emphasized the potential role of angiotensin II as a vascular growth modulator. Clinical studies confirmed animal experiments; both suggest that increases in arterial compliance and distensibility following perindopril is likely to be related to drug-induced modification of the arterial wall, at least partially independently of blood pressure reduction. The increase in arterial compliance was associated with a selective decrease in pulse pressure, a finding that is important, not only for the arterial wall, but also for the structure and function of the hypertensive heart. PMID- 1725198 TI - Hemodynamic and humoral interactions between perindopril and indomethacin in essential hypertensive subjects. AB - To evaluate whether perindopril, a carboxyl-containing new angiotensin-converting enzyme (ACE) inhibitor, exerts its antihypertensive action through the stimulation of prostaglandin synthesis, 10 uncomplicated essential hypertensive patients randomly received indomethacin (50 mg b.i.d.) or the corresponding placebo for 1 week and the reverse treatment after 2 weeks. Perindopril alone tended to reduce serum and urinary thromboxane B2 (TxB2) and to raise urinary 6 ketoPGF1 alpha and PGE2 and inhibited serum ACE activity 24 h post dosing by about 85%. Indomethacin, which significantly inhibited serum TxB2 and urinary TxB2, 6-ketoPGF1 alpha, and PGE2 without interfering with the inhibitory effect of perindopril on ACE, significantly reduced the antihypertensive action of perindopril alone by about 30%, but decreased though not significantly that of perindopril plus placebo. Although the size of the study limits the interpretation, these findings suggest that the stimulation of prostaglandin synthesis plays only a minor role in the antihypertensive action of perindopril. PMID- 1725199 TI - Cardiac hypertrophy and arterial compliance following drug treatment in hypertension. AB - The load of the heart in hypertension is related both to increased peripheral vascular resistance and decreased aortic compliance. From noninvasive studies involving determinations of pulse-wave velocity and systolic-diastolic variations of aortic arch diameter, it can be shown that increased aortic elastic modulus is strongly related to increased cardiac mass. The relationship is observed even after adjustment for the level of mean arterial pressure. It is suggested that decreased aortic compliance in hypertension causes a disproportionate increase in systolic pressure and end-systolic stress, thus contributing to promote cardiac hypertrophy. Such a possibility may have consequences for long-term antihypertensive therapy. Following converting enzyme inhibition and calcium blockade, important dissociations may be observed between the antihypertensive effect and the cardiac and arterial changes. PMID- 1725200 TI - Angiotensin-converting enzyme inhibition: prospects for the future. AB - Angiotensin-converting enzyme (ACE) is a widely distributed dipeptidyl carboxydipeptidase. Using computer analysis of the binding of radiolabeled ACE inhibitors, we have mapped the distribution of ACE in normal animals and in models of disease, and have studied the tissue effects of ACE inhibitors. In the myocardium, ACE is located in vascular and valvular structures as well as in the atria and ventricles. Its concentration is increased in myocardial hypertrophy in the infarct model of heart failure. Chronic ACE-inhibitor therapy prevents myocardial hypertrophy. In the brain, ACE is localized in multiple specific sites where its role is unknown. Following oral ACE-inhibitor treatment, effects are restricted predominantly to areas devoid of blood-brain barrier. Early studies suggest ACE inhibitors enhance cognition. In the testis, ACE is located in the seminiferous tubules where its function is unknown. It is not inhibited following oral ACE-inhibitor treatment. In the kidney and gastrointestinal tract, ACE is in high concentration in the epithelial brush border where its function is unknown. In clinical use ACE inhibitors primarily affect the cardiovascular system. With pharmacological developments, ACE inhibitors targeted for specific organs may have effects predominantly in the brain, heart, reproductive system, or gastrointestinal tract. PMID- 1725201 TI - Long-term tolerance of perindopril in hypertensive patients with impaired renal function. AB - Thirty-six hypertensive patients with impaired renal function entered a long-term study to assess the safety of perindopril. There were 28 men and 8 women of mean age 57.1 +/- 2.0 years (mean +/- SEM). The duration of documented hypertension was 7.3 +/- 1.2 years. Perindopril was given orally in single daily doses. The initial dosage was chosen according to the degree of renal function impairment: 29 patients received 4 mg o.d. [creatinine clearance (Clcr), 42.2 +/- 3.2 ml.min 1] and 7 patients received 2 mg o.d. (Clcr, 22.3 +/- 3.1 ml.min-1). Patients in whom blood pressure was not controlled had their dose doubled and then, if necessary, an additional diuretic therapy was added at subsequent visits. Six patients were withdrawn for adverse events (myocardial infarction, pneumonia, leucopenia in a patient who had lupus, diabetes mellitus, skin rash, epigastric pain), two patients were withdrawn for poor compliance, and three for personal convenience. The mean duration of treatment was 10.2 months with a range of 3-12 months (excluding one patient who died from myocardial infarction in the first days of the study and was not included in the analysis). Systolic and diastolic blood pressure decreased significantly (from 170.5/100.6 +/- 3.4/1.8 mm Hg to 151.8/88.8 +/- 3.0/1.7 mm Hg, n = 35, p less than 0.001). Baseline and final values of plasma creatinine (from 223.7 +/- 22.7 to 234.7 +/- 28.5 mumols/l), Clcr (42.5 +/- 3.2 to 45.7 +/- 4.6 ml.min-1), and kalemia (from 4.4 +/- 0.1 to 4.7 +/- 0.1 mmol/L) were not statistically different.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725202 TI - Assessment of antihypertensive efficacy of perindopril: results of double-blind multicenter studies versus reference drugs. AB - Perindopril (4 mg) was compared to atenolol (50 mg), captopril (25 mg b.i.d.), or a diuretic (hydrochlorothiazide 50 mg and amiloride 5 mg) in three studies involving a total of 503 hypertensive patients with diastolic blood pressure (DBP) of 95-125 mm Hg. A 4-week single-blind placebo period preceded 12 weeks of active treatment. Dose titration was at weeks 4 and 8 if the supine DBP was greater than 90 mm Hg. The dose was doubled and, if necessary, a diuretic was added in atenolol or captopril comparison and atenolol added in the diuretic study. The fall in supine blood pressure (BP) was 27/17 mm Hg with perindopril and 21/16 mm Hg for atenolol. Fifty-five percent on perindopril and 48% on atenolol were controlled on monotherapy, increasing to 78 and 58% with addition of hydrochlorothiazide. Captopril caused a BP fall of 19/12 mm Hg compared to 27/18 mm Hg for perindopril with 49% of both groups being controlled on monotherapy. Diuretic addition produced a greater antihypertensive effect with perindopril 75% compared to 57% of the captopril patients achieving control. Perindopril caused a comparable fall in supine BP to the diuretic combination 27/19 mm Hg and 31/18 mm Hg but the fall in erect systolic BP was significantly greater for the diuretic. At 3 months, 84% of the diuretic and 78% of the perindopril group achieved the target BP. There were no adverse effects on the blood count or blood chemistry with a low incidence of side effects and withdrawals from treatment. These studies show that perindopril compares favorably with the standard antihypertensive drugs. PMID- 1725203 TI - Pharmacokinetics of perindopril in high-risk populations. AB - The aim of this article is to review the pharmacokinetics of perindopril and its active metabolite perindoprilat in high-risk populations in comparison with their basic features in healthy volunteers. As it has been shown that the kinetics of perindoprilat are mainly affected by renal insufficiency, a dosage reduction is therefore recommended on initiation of treatment in elderly patients and in those with renal failure according to the degree of renal failure. In patients with chronic heart failure, the kinetics of both perindopril and perindoprilat were shown to have been altered, also indicating the need for an initial dosage reduction in such patients. Hepatic impairment had no significant effect on the kinetics of either the prodrug or the active metabolite. PMID- 1725204 TI - Twelve tips on using double slide projection. AB - Double slide projection is not a technique that everyone will choose to use. It is a technique, however, which does offer the lecturer a number of advantages and it is not difficult to implement in the average lecture theatre. Careful consideration, however, must be given to the use to which the two projection system is to be put. In this paper 12 tips are given in the use of double slide projection and 10 possible uses for double slide projection are described. Three ways in which the lecturer can control the slide changes are presented. PMID- 1725205 TI - Relating media with staff development in medical education. AB - Medical education nowadays experiences an increasing gap between the potential didactic value of educational technology and the practical implementation thereof. One of the important obstacles is the tendency to separate media and media agencies from staff development. A new conceptual model was designed for staff development in medical education, which incorporates media as one of the fundamental components. In application of this model the Bureau for Medical and Dental Education at the University of Stellenbosch medical school directs its media activities and services to the improvement of instruction and learning. For example instead of only offering technical audio-visual production services, medical teachers are also taught how to use different media effectively in teaching and learning. In this way media are directly related to staff development. PMID- 1725206 TI - Teaching acute respiratory infection using low-cost aids. An experience in Pondicherry, South India. AB - The topic of acute respiratory infections (ARI) was taught to two successive batches of medical students in their first clinical year. The low-cost aids in the form of slide set, tape recorder and video cassette were used. The knowledge and attitude of students was assessed using a pre-test and a post-test, which showed a statistically significant improvement. PMID- 1725207 TI - [SEM-studies on the osseous healing and changes of the microvascular architecture in supporting tissues of endosseous implants]. AB - Relations between successive changes of the microvascular architecture and osseous healing in the restorative process of supporting structures of endosseous implants were experimentally investigated on plastic microcasts of blood vessels under SEM. Sixty days after upper incisor extractions of Japanese monkeys (Macaca fuscata), titanium, notch and screw-typed, and bioceram-anchorpin were implanted. The animals were sacrificed at postoperative 1, 2, 4, 9 weeks, respectively. Acryl plastic was injected via the common carotid arteries. Microvascular casts together with bone element of the maxillae were prepared for SEM as well histological slides for light microscope. One week after implantation, sinusoidal vessels sprouted out of pre-existing vessels including leakages of the injected plastic. Two weeks, these sinusoids began to be sorted out. Newly formed, stalked bone trabeculae reached the implant surface, where each of them has enlarged as an island like piece. Four weeks, these pieces have been connected with each other by adhesion. Outside the new bone, blood capillaries sorted out have formed a cylindrical plexus. These successive osseous integration surrounding the implant has continued until about 9 weeks. In conclusion, it can be said that the period of 4-6 weeks after implantation was most important and significant to be established the osseous integration. PMID- 1725208 TI - Sequential administration of interleukin-6 and granulocyte-colony stimulating factor in newborn rats: modulation of newborn granulopoiesis and thrombopoiesis. AB - During states of increased demand, neonatal host defense is characterized by dysregulation of granulopoiesis, resulting in a high incidence of neutropenia. This study investigated the modulation of neonatal rat hematopoiesis by 14-d administration of recombinant human (rh) IL-6, rh-granulocyte-colony stimulating factor (G-CSF), or sequential combination of rhIL-6 and rhG-CSF. Specifically, newborn Sprague-Dawley rats were treated with either rhIL-6 (5 micrograms/kg/d for 14 d), rhG-CSF (5 micrograms/kg/d for 14 d), rhIL-6 for 7 d followed by rhG CSF for 7 d, PBS/BSA for 7 d followed by rhG-CSF for 7 d, or PBS/BSA for 14 d. RhIL-6 alone significantly increased the peripheral platelet count during the latter part of the 2nd wk of administration (d 13: 980 +/- 42 versus 716 +/- 23 x 10(3)/mm3) (p = less than 0.001) (mean +/- SEM). Treatment with rhIL-6 for 7 d followed by rhG-CSF significantly increased the peripheral neutrophil count compared with 7 d of PBS/BSA and 7 d of G-CSF (d 14 absolute neutrophil count 4888 +/- 12 versus 2720 +/- 317/mm3) (p = less than 0.05). Similarly, sequential rhIL-6/rhG-CSF significantly increased the d-14 bone marrow neutrophil storage pool (9873 +/- 882 versus 3564 +/- 159/mm3) (p = less than 0.005). Lastly, sequential rhIL-6/rhG-CSF induced the highest increase in bone marrow (p less than 0.01) and liver/spleen CFU-GM pool (p less than 0.001) compared with any other treatment group. These studies suggest that rhIL-6 alone is associated with a significant increase in the neonatal platelet count.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725209 TI - Long-term cultures of mast cells: a new model for studying the allergic response. AB - Mast cells are the key cells of allergic reactions and probably play also a role in chronic inflammatory reactions resulting in fibrosis. Although their biochemical and functional properties have been extensively investigated, several studies have been hampered by the absence of a tissue culture system to keep these cells alive and functionally active for long periods of time. Recently we have developed an in vitro system in which rat peritoneal mast cells are cocultured with 3T3 mouse skin derived fibroblasts (MC/3T3). Under these tissue culture conditions, mast cells do not proliferate and maintain their viability and functional activity for more than a month. This system allowed us to carry out long-term studies on the functional properties of mast cells. We have found that mast cells activated both by IgE-dependent and IgE-independent stimuli survive, and slowly replenish their histamine content. After a non-immunological challenge mast cells retain their full potential to release histamine upon a repeated similar challenge. In contrast, immunologically challenged mast cells become partially unresponsive to a similar activation event for up to 3 weeks. By exploiting this long-term culture system we described a novel type of mast cell activation induced by cytokines. The onset of this activation is slow and its course appears to be chronic-continuous, very different from the classical anaphylactic type activation that is completed within few minutes. Since MC/3T3 release higher amounts of histamine, this system is a sensitive tool to investigate the antiallergic properties of various drugs. By employing MC/3T3 cultures we were able to show that the gold salt auranofin inhibits histamine release from mast cells stimulated by different secretagogues. In addition, salbutamol inhibited histamine release from repeatedly challenged mast cells; and nedocromil sodium was effective in preventing mast cell activation when incubated for a week with MC/3T3. PMID- 1725210 TI - Women's opinions on the offer and use of prenatal diagnosis. AB - We have studied the opinions and attitudes of women towards prenatal diagnosis (amniocentesis/chorionic villus sampling/ultrasound/serum AFP testing). A questionnaire was sent to 185 women who had had their first baby a few months before. The respondents have a strong positive attitude towards the diagnostic procedures, especially if treatable abnormalities can be detected. Younger women and women with a high level of education were less inclined to make use of prenatal diagnosis. If tests were made more widely available, this might lead to a significant increase in the use of prenatal diagnosis. PMID- 1725211 TI - Comparative audit of booking and mid-trimester ultrasound scans in the prenatal diagnosis of congenital anomalies. AB - During 1988 and 1989, 3565 women booked under consultants who performed an ultrasound scan at booking, whilst 4984 booked under consultants who performed a formal mid-trimester scan between 16 and 18 weeks. All significant anomalies diagnosed prenatally and in the neonatal period were recorded, the incidence in each group being 12.9/1000 and 9.83/1000, respectively (NS). The sensitivity of diagnosis before 20 weeks was 45 percent in the 'mid-trimester' group (kappa 0.62) compared with 30 percent in the 'booking' group (kappa 0.46), overall sensitivity of prenatal diagnosis, however, being similar in both groups (63 vs. 65 percent, kappa 0.77 vs. 0.79). Cardiac anomalies were the single largest group which were not detected equally prenatally in both groups. This study shows that formal mid-trimester scanning leads to anomalies being detected significantly earlier in the antenatal period. Although not statistically significant, three lethal anomalies were missed prenatally in the 'booking' group which we would have expected to diagnose on a mid-trimester scan. These figures are discussed in the light of previous reports. PMID- 1725212 TI - High serum levels of beta subunit human chorionic gonadotropin in trisomy X. PMID- 1725213 TI - Increased maternal serum human chorionic gonadotropin level associated with Klinefelter's syndrome. PMID- 1725214 TI - Minimal fetomaternal transfusion in ultrasound-guided fetal skin sampling. PMID- 1725215 TI - Trace expression of the germ-cell alkaline phosphatase gene in human placenta. AB - We have developed hybridization probes that clearly distinguish the RNA transcripts of the two closely related human heat-stable alkaline phosphatase genes. RNA from the PLAP-1 gene, encoding the term placental alkaline phosphatase, is the predominant transcript in placenta from 8 weeks to term. Transcripts from the PLAP-2 gene, encoding the germ-cell or PLAP-like enzyme, are also detectable in the placenta, but at no more than 2 per cent the level of PLAP 1 transcripts. PMID- 1725216 TI - The effect of tryptophan supplementation on autotomy induced by nerve lesions in rats. AB - Rats were fed an artificial diet containing either their normal, or five times their normal, daily requirement of tryptophan for up to five weeks and were tested in an animal model of deafferentation pain, nerve lesion-induced autotomy. In this model one of the hind limbs of the animal was denervated, and the extent to which the animal attacked its denervated paw was assessed. Rats receiving the high-tryptophan diet showed significantly lower levels of autotomy, compared to rats fed the control diet. 5-Hydroxytryptamine and 5-hydroxyindoleacetic acid in the brain and spinal cord were significantly elevated in rats receiving the high tryptophan diet, indicating that the supplemented diet produced a chronic increase in CNS indoleamine metabolism. Currently there is no accepted pharmacological treatment of deafferentiation pain. Our results suggest that tryptophan should be tested in phantom limb pain and other deafferentation pain syndromes. PMID- 1725217 TI - The functional significance of biochemical alterations in streptozotocin-induced diabetes. AB - These experiments examined the effects of restraint stress on dopamine (DA) and 5 hydroxytryptamine (5-HT) and their principal metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA), respectively, in 4 brain regions, as well as on plasma corticosterone concentration (CORT) and behavior in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats had widespread reductions in DA and 5-HT turnover (DOPAC/DA and 5-HIAA/5-HT ratios). Restraint led to equivalent increases in DA turnover in diabetics and nondiabetics, but attenuated increases in 5-HT turnover in diabetic rats. CORT concentration of diabetics and nondiabetics measured in complete quiet did not differ. Relative to these measures, only diabetics had elevated CORT when either restrained or kept in the same room with restrained rats with food and water removed. Open-field exploration was suppressed by restraint in diabetics only. All diabetic rats showed decreased locomotion in a novel environment which was normalized during a second exposure to the apparatus. Together, these results suggest that diabetes-induced disruptions in open-field activity are related to anxiety rather than to motor or energy deficits, and may be related to impaired 5 HT and CORT systems. PMID- 1725218 TI - Naloxone administration in vivo stereoselectively alters antigen-dependent and antigen-independent immune responses. AB - An investigation was conducted to determine the role naloxone has on immunocompetence in vivo. Mice (n = 7) injected with sheep red blood cells and treated with naloxone (0.1-10.0 mg/kg) show an enhanced production of total and antigen-specific IgM antibody by splenic lymphocytes compared to control (mock treated) mice. The response was dose-dependent, with the greatest effect occurring at 0.1 mg/kg naloxone. A naloxone dose of 0.001 mg/kg was not active. In addition, natural killer activity was enhanced in the naloxone-treated mice compared to the controls. The effects on antigen-specific antibody production and natural killer activity were stereoselective, since (-)-naloxone is active whereas (+)-naloxone was not. These results illustrate the ability of an opioid receptor antagonist administered in vivo to regulate immunocompetence to antigen specific and antigen-nonspecific immune responses, which may be useful during selective inflammatory processes. PMID- 1725219 TI - Pituitary-adrenal-immune system in normal subjects and in patients with anorexia nervosa: the number of circulating helper T lymphocytes (CD4) expressing the homing receptor Leu8 is regulated in part by pituitary-adrenal products. AB - The association between plasma pituitary-adrenal (PA) hormones and the number of certain populations of peripheral blood lymphocytes (PBL) was examined in subjects with normal PA function and in patients with anorexia nervosa (AN). AN patients display several neuroendocrine dysfunctions, including hypercortisolemia. In the normal subjects there were positive correlations between adrenocorticotropic hormone (ACTH) and the number of PBL and helper T lymphocytes expressing the homing receptor Leu8 (CD4+Leu8+); there was a negative relationship between cortisol and these lymphocyte populations. These latter, inverse correlations did not occur in the AN patients, either while underweight or after weight recovery, with some persistence of hypercortisolemia. Administration of dexamethasone (DEX) suppressed cortisol levels and reduced, perhaps via a receptor-mediated mechanism, the number of circulating PBL and CD4+Leu8+ in the normal subjects but not in the AN patients. These results support the physiological relevance of PA-CMI interaction in subjects with normal PA function and indicate that the PA-CMI interrelationship is disrupted in AN patients with hypercortisolemia. PMID- 1725220 TI - Immunocytochemical localisation of pancreastatin and chromogranin A in porcine neuroendocrine tissues. AB - Pancreastatin is a 49 amino acid peptide with a C-terminal glycine amide originally isolated from porcine pancreas. In the present study the cellular localisation of pancreastatin in porcine neuroendocrine tissue was examined immunocytochemically using an antiserum raised against porcine pancreastatin (33 49) that does not cross-react with porcine chromogranin A. In order to study the possible precursor-product relationship between chromogranin A and pancreastatin the cellular localisation of both peptides was examined in peripheral tissues using simultaneous double immunostaining. The pancreastatin antiserum immunostained cells and nerve fibers throughout the neuroendocrine system. In most of the examined tissues we found colocalisation of pancreastatin and chromogranin A immunostaining. These results support the precursor-product concept for chromogranin A and pancreastatin. However, in the gastrointestinal tract and the adenohypophysis a minor population of the endocrine cells exhibited immunostaining with only one of the two antibodies. This discrepancy between immunostaining with pancreastatin antiserum and monoclonal chromogranin A antibody could be due to absence of, or extensive, processing of chromogranin A in certain cell populations. PMID- 1725221 TI - Fate of the major outer membrane protein P.IA in early and late events of gonococcal infection of epithelial cells. AB - We investigated the fate of the major outer membrane protein of Neisseria gonorrhoeae, P.IA, during gonococcal infection of Chang conjunctiva epithelial cells by using immunoelectron microscopy. Probing of P.IA epitopes with mono- and polyclonal antibodies revealed variable, fixation-dependent P.IA epitope exposure in the gonococci used as an inoculum in the infection experiments. Detection of invariable exposed P.IA epitopes in cryosections of infected epithelial cells with a polyclonal antiserum revealed unaltered P.IA exposure on the bacterial membranes during early attachment of the bacteria to the eukaryotic cells. Upon entry of the bacteria into the host cells, however, labelling was extended to membraneous structures that intercalated between the bacteria and the host cell surface, and, occasionally, to the host cell plasma membrane. The latter observation is consistent with the suggested active role of P.I. in the uptake process (as shown in 1985 by E.C. Gotschlich). Once inside the epithelial cells, both morphologically intact and disintegrating bacteria could be distinguished. The disintegration of the bacteria was accompanied by a loss of P.IA immunoreactivity. PMID- 1725222 TI - [Anti-nucleic acid antibodies in children with systemic lupus erythematosus and systemic scleroderma and in their relatives living with the probands]. AB - The nature of antibodies to nucleic acids was studied in children of a Caucasian region suffering from systemic lupus erythematosus (SLE) and systemic sclerodermia (SSD). Two types of antibodies (to two- and one-filament DNA) were revealed in SLE, and in SSD mainly--to one-filament DNA. Antibodies to RNA in patients with SLE were specific for two-filament conformation. Immunogenesis disorders of different degree with respect to DNA and RNA were found; this expressed itself in the synthesis of various classes of immunoglobulins to these antibodies (IgG to DNA and IgM to RNA). High incidence of the RNA-binding activity of relatives living together with probands is noted. These data point to a high occurrence of antibodies to denatured DNA and low incidence of the cases of DNA-binding activity in the blood sera of the patients' fathers and brothers. Thus a combination of three factors: viral, genetic and hormonal in the pathogenesis of this disease is possible. PMID- 1725223 TI - [Prevention of postoperative venous thrombosis and pulmonary embolism. Consensus conference 8 March 1991 by Assistance Publique-Hopitaux de Paris]. PMID- 1725224 TI - [The use of rheopolyglucin in diabetic patients with a microcirculatory disorder]. PMID- 1725225 TI - [The effect of combined therapy with the use of thymalin and piracetam on the level of middle-molecule peptides in the blood and on the lipid peroxidation activity in patients with diffuse toxic goiter]. AB - The content of medium-weight molecular peptides (MMP), lipid peroxidation (LPO) activity, the profile of blood thyroid hormones, and radioactive iodine absorption by the thyroid were determined in 104 patients with light, moderate and grave diffuse toxic goiter (DTG). The content of MMP in the blood correlated with the gravity of DTG and thus may serve an additional test for its determination. Depending on the treatment 66 patients with DTG of medium gravity were distributed into 5 groups: group I included patients given mercazolyl, group II patients on lithium carbonate, group III patients who received mercazolyl and thymalin, group IV patients on mercazolyl and piracetam, and group V included patients given mercazolyl, thymalin and piracetam. A study was made of the blood content of MMP and LPO activity before therapy and after euthyroidism attainment. The treatment with the use of mercazolyl, thymalin and piracetam was found to produce a beneficial effect on the parameters under study and the clinical course of DTG. PMID- 1725226 TI - Comparison of the effects of sodium pentosanpolysulfate and unfractionated heparin on venous thrombosis; an experimental study in rats. AB - Sodium pentosanpolysulfate (Fibrezym) and unfractionated heparin (Liquemin) had significant antithrombotic efficacy in rats. 1 mg/kg body weight (BW) FibrezymR had its maximal effect 2-4 hours after subcutaneous administration; 200 U/kg BW LiqueminR s.c. were most effective 4-6 hours after application. Both drugs caused frequent embolic break-offs of thrombi during the time periods of maximal antithrombotic efficiency. PMID- 1725227 TI - Functionally active protein C inhibitor/plasminogen activator inhibitor-3 (PCI/PAI-3) is secreted in seminal vesicles, occurs at high concentrations in human seminal plasma and complexes with prostate-specific antigen. AB - Protein C inhibitor (PCI) is a heparin-dependent serpin present in a native form in plasma at concentrations of 5 micrograms/mL. In vitro, PCI inhibits activated protein C (APC), thrombin, plasma kallikrein (KK) and urokinase-(uPA) and tissue type plasminogen activator (tPA), and we have shown in vivo inhibition of APC, uPA and KK by PCI. In order to further characterize the physiological role of PCI, we have measured the level of PCI in several biological fluids. PCI antigen was assayed by ELISA and PCI activity was measured by its capability to form complexes with APC in the presence of heparin. Seminal plasma from voluntary donors had PCI levels (160 +/- 20 micrograms/mL, mean +/- SD) about 30 or 40 times higher than those found in blood plasma. Patients under a fertilization program had significantly reduced PCI seminal levels (110 +/- 35 micrograms/mL). Seminal plasma PCI retained about 45% of its activity immediately after ejaculation, and the activity rapidly decreased following incubation of seminal plasma at 37 degrees C, in parallel with the appearance of complexes of PCI with prostate-specific antigen (PSA). PCI was present in seminal vesicle secretion, obtained by autopsy, at concentration similar to that observed in semen, was mostly active and was not inactivated by incubation of secretion at 37 degrees C. The mean functional and antigen levels of PCI in urine from normal donors were 0.58 and 0.25 micrograms/mL, respectively, whereas in saliva these levels were 20 and 0.8 ng/mL, respectively. Amniotic fluid contained PCI antigen levels of 2.1 +/- 0.2 microgram/mL. These results show that PCI is secreted in the seminal vesicles in a functional form, and suggest that PSA, a major secretory component of the prostate, is responsible for its inactivation. They also suggest a physiological role of PCI in reproduction, and show that PCI is present in various biological fluids. PMID- 1725228 TI - Influence of histochemical preparation on acoustic parameters of liver tissue: a 5-MHz study. AB - In this study the influence of various histological techniques on the acoustic parameters of liver tissue was investigated. Radiofrequency (RF) echographic data were obtained in vitro from 21 liver samples taken from 8 white New Zealander rabbits. The samples were measured in four different subsequent histological tissue processing conditions (freshly excised, 4% buffered formalin fixed, after it went through a paraffin cycle and after staining with hematoxylin and eosin). The acoustic parameters that were obtained from the rf data were velocity of sound, slope of the attenuation coefficient versus frequency between 1.9 and 6.9 MHz, attenuation coefficient at 4.4 MHz, slope of the backscattering spectrum between 1.9 and 6.9 MHz, and intercept of the backscattering spectrum. It was found that fixation by formalin preserves the acoustic properties of the tissue to a reasonable extent. Embedding in paraffin and deparaffinizing induces large changes in the acoustic properties of the tissue. As an alternative, freezing prior to cutting, rather than the paraffin cycle, was investigated also in 10 liver samples obtained from 4 New Zealander rabbits. This method produced no significant changes of the acoustic parameters and should therefore be preferred in acoustic microscopy. PMID- 1725229 TI - Molecular basis of beta-thalassemia intermedia in a southern Italian region (Puglia). AB - We investigated the molecular bases for a mild phenotype by alpha-, beta- and gamma-globin gene analyses in 22 patients with transfusion-independent thalassemia intermedia (15) or a late-presenting form of thalassemia major (7) originating from Puglia, a region of southern Italy. Twenty-two patients with thalassemia major served as controls. The beta+ IVS-I nt 6 of the beta-globin gene and the C----T substitution at position -158 5' of the G gamma-globin gene were detected more frequently in patients with thalassemia intermedia or late presenting thalassemia major considered together as compared to those affected by typical transfusion-dependent thalassemia major. Three of 15 patients with thalassemia intermedia had the triple alpha-globin gene arrangement in the heterozygous (2) or homozygous state (1) in association with heterozygous beta zero-thalassemia. From these results, we may conclude that the inheritance of a mild beta-thalassemia allele such as the beta+ IVS-I nt 6 mutation, in the homozygous or heterozygous state, the coinheritance with homozygous beta zero thalassemia of the -158 (C----T) G gamma gene promoter mutation and the presence of heterozygous beta-thalassemia/triple alpha-globin gene arrangement are the most common reasons accounting for the development of attenuated forms of beta thalassemia in Puglia. PMID- 1725230 TI - [Combined pharmacokinetic-pharmacodynamic model of procainamide in rabbits with induced ventricular fibrillation threshold (VFT) changes]. AB - The change of electrically induced VFT was chosen as index of effect in anesthetized rabbits for study of pharmacodynamics of PA and NAPA. We analyzed the pharmacokinetic properties of PA and NAPA and elucidated their effect kinetics with a pharmacokinetic-pharmacodynamic (PK/PD) model in view of different transfer qualities. A linear-addition effect model was used to describe the relationship between the effect and the amount of drug and its metabolite in the effect compartment. PA was found to be eliminated faster than NAPA and distributed more extensively in rabbits. The effect per unit concentration of PA was shown to be larger than that of NAPA. PMID- 1725231 TI - Multiple forms of synaptic coordinative interactions in the autonomic neurotransmission. Tensiometric evaluations in peripheral tissues. AB - The existence of co-transmitters interacting in the complex regulation of autonomic neurotransmission, has led to investigate the interactive effects between the conventional neurotransmitters and other drugs, such as histamine, serotonin, substance P, strictly related to inflammatory mediators, in both vas deferens and urinary bladder of guinea pig. The results suggest that synergistic interactions, involving post-synaptic sites, exist between co-transmitters; furthermore, inflammatory mediators are able to modify the responses to endogenous (via intramural nerves stimulation) and exogenous conventional neurotransmitters. It is conceivable that inflammatory mediators, released by activation of mast cells either within or delivered to the tissues by the vascular supply, may alter the physiological responses of several organ systems. PMID- 1725232 TI - Oral delivery of antigens in live bacterial vectors. PMID- 1725233 TI - Modified-live infectious bovine rhinotracheitis virus (IBRV) vaccine expressing foot-and-mouth disease virus (FMDV) capsid protein epitopes on surface of hybrid virus particles. PMID- 1725234 TI - HIV-1 neutralizing antibody and approaches to the envelope diversity problem. PMID- 1725235 TI - Epitope mapping studies of snake venom phospholipase A2 using monoclonal antibodies. AB - Fifteen different monoclonal antibodies developed against pseudexin, a snake venom phospholipase A2 with presynaptic neurotoxicity, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin were synthesized by using alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies bound to toxin peptides and were detected with an enzyme-linked immunosorbent assay. Three of the bound antibodies recognized a site important in calcium binding and the interlocking of dimeric forms of snake venom phospholipase A2. Analogous regions from other phospholipases were synthesized and probed with the four reactive antibodies. A good correlation was found between the reactivity of whole molecule phospholipases and peptide regions with the antibodies. Monoclonal antibodies neutralizing the lethal or enzymatic effects of pseudexin did not recognize any linear epitopes. PMID- 1725236 TI - Identification of epitopes of the receptor binding subunit of cholera toxin by synthetic peptide and CBIB approaches. PMID- 1725237 TI - Maternal antibody epitope mapping in mother-to-child transmission of HIV. PMID- 1725238 TI - Immunogenicity of synthetic peptides corresponding to various epitopes of the human immunodeficiency virus envelope protein. PMID- 1725240 TI - Complexes and conjugates of cis-Pt for immunotargeted chemotherapy. PMID- 1725239 TI - Isolation and characterization of the neutralizable epitope of simian retrovirus 1 (SRV-1) and of the cell receptor for the virus. AB - An area encompassing residues 142-167 of the envelope protein of type D simian retrovirus (SRV-1) has been shown to contain the epitope to which neutralizing antibodies are directed. This area has been synthesized and shown to bind to monkey and mouse antiviral antibodies and to a virus neutralizing mouse monoclonal antibody. Protein conjugates of this peptide as well as the cross linked or the free peptide induce antibodies capable of neutralizing, in vitro, viral infectivity. The cell receptor to the virus was isolated following extraction of Raji cells with non-ionic detergents. The receptor was isolated and characterized following radioimmuno-precipitation of 125I labeled cell extract bound to viral envelope protein. This immunoprecipitation could be inhibited by antiserum to peptide 142-167. Analysis in gels indicate that the receptor is of molecular weight of approximately 60 KDa. These results indicate that the neutralizing antibodies and the receptor recognize the same area on the viral envelope protein and that neutralization is the result of blocking the virus receptor interaction by antibodies. PMID- 1725241 TI - A new monoclonal antibody to an age sensitive band 3 transmembrane segment. AB - The appearance of band 3 structural modifications related to aging could be evidenced by means of monoclonal antibodies against senescence antigen. Hence in the attempt to provide an immunological marker of erythrocyte aging, we raised a monoclonal antibody against native band 3 (B6 MoAb), which seems to detect differences in the band 3 molecule from erythrocytes of different ages separated by density gradient. Densitometric evaluation of immunoblotting patterns indicates that the in vivo aging is associated with band 3 monomer degradation. The Percoll separated fractions show a significant increase of those proteolytic fragments that bind the B6 antibody. Finally, protease digestions of unsealed membrane ghosts have been performed to test the binding site of the B6 antibody on the band 3 molecule. The data show that the B6 antibody binds a 19 KDa chymotryptic-tryptic fragment which corresponds to a segment of the looped membrane domain whose steric structure appears to be sensitive to age. PMID- 1725242 TI - Lung cancer in filling station attendants. AB - At the Danish census on 9 November 1970, 4,055 men and 1,195 women aged 20-64 years indicated an employment that was coded as retail sale of oil and gasoline; almost all individuals probably worked as filling station attendants. Record linkage at Danmarks Statistik showed that 529 of the men had died during the following 17 years. Respiratory cancer (75 deaths) was the only cause of death that showed a significant excess (standardized mortality ration, 1.58; 95% confidence interval, 1.25-2.00) when compared to all men gainfully employed at the time of the census. An increased mortality due to cardiovascular disease could not be related to any particular diagnostic subgroup; the mortality in women did not differ from expected rates. These results are in accordance with data from other countries on occupational groups exposed to high levels of exhaust fumes. PMID- 1725243 TI - A nonprescription cerumenolytic. AB - Cerumen impaction and removal is a very common otologic problem. The perfect cerumenolytic agent has yet to be developed. After years of trying multiple different cerumenolytics without satisfaction, a stool softener, docusate sodium, has become our agent of choice. A commonly used stool softener, it is widely available without prescription. It is a highly effective cerumenolytic, but remains relatively unknown in this capacity. PMID- 1725244 TI - Demonstration of changes in cytokeratin expression in condylomata accuminata in relation to the presence of human papilloma virus as shown by a combination of immunohistochemistry and in situ hybridization. AB - Human papillomavirus (HPV) can be detected in, and is probably involved in the etiology of, the majority of anogenital neoplasias. Infection with the virus induces a number of events in the infected epithelial cells that may lead to the development of benign or malignant tumors. One change that can be detected in the infected cells is in squamous differentiation, which is reflected by the pattern of cytokeratin polypeptide expression. By studying this pattern in relation to the presence of the virus, an indication may be obtained of the influence of the virus on the cellular differentiation in individual cells. By using a combination of DNA in situ hybridization and immunohistochemistry, for HPV and cytokeratin polypeptides, respectively, we studied the presence of HPV6 or HPV11 in condylomata accuminata derived from anogenital skin in relation to the cytokeratin polypeptides K1, 4, 8, 10, 14, and 18. We found that in many samples the presence of the skin-type cytokeratins K1 and K10 was decreased, whereas K13, and to a lesser degree K4, appeared. The cellular localization of these aberrations in cytokeratin expression could be related to the presence of HPV6 or 11 DNA in the tissue. PMID- 1725245 TI - HMB-45 staining in benign and malignant melanocytic lesions. A reflection of cellular activation. AB - The antibody HMB-45 used as an immunohistochemical reagent has often been labeled as a marker for melanoma, even though some benign lesions have been noted to show positive staining reactions with this reagent. Biopsy specimens from 225 benign and malignant melanocytic lesions were examined after immunoperoxidase staining for S-100 protein and HMB-45. The lesions studied included common acquired nevi, spindle cell and epithelioid cell nevi (Spitz nevi), cellular blue nevi, deep penetrating nevi, congenital nevi, nevi from hormonally reactive areas (genital), malignant melanoma, and desmoplastic malignant melanoma. A positive reaction for HMB-45 was seen in the dermal component in a high percentage of each of these types of lesions except for the common acquired nevi and the desmoplastic malignant melanomas that were uniformly negative for HMB-45 in the dermal component. HMB-45 correlates with melanosome production and thus a melanocytic origin of HMB-45-positive cells. HMB-45 may correlate best with factors that stimulate melanocytic proliferation and production of melanosomes. PMID- 1725246 TI - Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937). AB - Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus. This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc receptor, one mAb against the sialosyl-Tn epitope enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody mediated enhancement may depend on location of the epitope on gp120 rather than whether the antibody binds Fc-receptors. PMID- 1725247 TI - A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymer templates. AB - R82913, (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (a TIBO derivative), inhibited the replication of thirteen different strains of HIV-1 in CEM cells with a median IC50 of 0.15 microM. The concentration of compound that killed 50% of the cells was much higher (46 microM), indicating that R82913 has a high selectivity index. R82913 was 20-fold more potent than AZT-TP in the inhibition of HIV-1 reverse transcriptase in an assay using a naturally occurring template (ribosomal RNA) that more accurately resembles native viral RNA than a synthetic homopolymer. With this template, R82913 inhibited HIV-1 reverse transcriptase with an ID50 (0.01 microM) that is equal to, or lower than, the IC50 for this compound in all of our cell culture assays (0.01-0.65 microM). R82913 has no effect on the replication of HIV-2 in CEM cells and does not inhibit the reverse transcriptase from this virus. PMID- 1725248 TI - [Problems of resistance of the causative agent of melioidosis to antibiotics]. AB - It was shown that the pathogen causing melioidosis was highly resistant to antibiotics including beta-lactams. Antibiotic sensitive mutants of P. pseudomallei were isolated after mutagenesis induced by nitrosoguanidine. Permeability for 3H-tetracycline and tetracycline sensitivity of the mutant cells was respectively 3 and 20 times as high as those of the initial parent strain. Gas liquid chromatography revealed quantitative and qualitative changes in separate sugars of the lipopolysaccharide structure in antibiotic sensitive mutants as compared to the initial strain. PMID- 1725249 TI - [Increasing the effectiveness of immunotherapy of rabies by using rifampicin in experimental studies]. AB - The experiments showed that in a dose of 35-70 mg/kg rifampicin inhibited reproduction of the fixed rabies virus in the brain of infected animals. The drug had no inhibitory effect on synthesis of the virus-neutralizing antibodies after vaccination. Combination of rifampicin with antirabies gamma-globulin had a marked synergistic effect. The animal survival after the combination use amounted to 75-100 per cent and depended on the infective dose of the virus and the scheme of the drug administration. It was concluded that rifampicin might be used in complex therapy of rabies during the incubation period (along with gamma-globulin and the vaccine) for inhibiting virus reproduction at early infection stages. PMID- 1725250 TI - Alpha-fetoprotein in tumours derived from cystic and simple embryoid bodies. AB - Cellular aggregates called embryoid bodies (EB) have been obtained from the experimental teratocarcinoma (TC) 0TT6050. Two morphological types of EB can be differentiated, which are injected subcutaneously into isogenic 129/Sv mice. The tumors are collected 20 and 30 days after EB injection and processed histologically, and immunohistochemically with anti-alpha-fetoprotein (alpha-FP) antibodies. Our results indicate that the histological pattern of the tumors is related to the degree of morphological organization of the EB used. PMID- 1725251 TI - Histology and histochemistry of the intermoult integument in the ghost crab Ocypoda platytarsis (Milne-Edwards) (Crustacea: Brachyura). AB - The histological and histochemical aspects of the integument have been described and discussed during the intermoult period of Ocypoda platytarsis. Histological observations revealed that the cuticle comprises of four layers namely epicuticle, exocuticle, endocuticle and membranous layers. Various types of cells in the subepidermal tissue have also been elucidated. PMID- 1725252 TI - Influence of bile acids on the development of hepatic transport of organic anions. PMID- 1725253 TI - Myocardial infarction (Part 2) PMID- 1725254 TI - [The surgical treatment of malignant neoplasms of the pancreas: resection or palliative intervention?]. AB - The authors retrospectively evaluate their 10-year experience in the surgical management of pancreatic cancer, and analyze their results in terms of morbidity and long-term survival. The comparison between curative and palliative surgery shows, in this series, a better long-term survival and a better performance status for the patients in the curative group, although postoperative morbidity and mortality are higher. The difficulty of an early diagnosis as well as a correct preoperative staging is confirmed. Finally, the authors propose a personal, totally mechanic technique of digestive tract restoration after gastric resection during pancreatic surgery underlining this procedure is easy, safe, fast and functional. PMID- 1725255 TI - A novel heterobifunctional linker for formyl to thiol coupling. AB - A maleimide hydrazide has been synthesized as a heterobifunctional cross-linking agent for thiol to formyl coupling. This linker has been applied to the coupling of the monoclonal antibody 17-1A, or an Fab' derived therefrom, to polyaldehyde dextran onto which the antineoplastic agent ellipticine has been attached. High binding avidities for the unshed antigen on the SW1116 colorectal tumor cell are retained in these drug-dextran-linker-antibody conjugates. PMID- 1725256 TI - The interaction between streptomycin and ribosomal RNA. AB - The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results. PMID- 1725257 TI - Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli. AB - The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous ribosome population, which consequently hinders direct probing of mutant rRNAs. Here, we describe how nonconserved helical regions of plasmid-coded rRNA have been altered in a manner that preserves their secondary structures while creating new sites for primer extension of mutant rRNAs. This facilitates specific biochemical probing of mutagenised rRNA regions despite the background of wild-type molecules. Four priming sites have been made to investigate the structural effects of mutations in the GTPase centre, helix 1200-1250, the peptidyl transferase region and the alpha-sarcin loop of 23S rRNA. PMID- 1725258 TI - Translation and ribosome assembly in extremely thermophilic archaebacteria. AB - Several features of translation and ribosome structure in extremely thermophilic, sulfur-dependent archaebacteria are described, including: i) a peculiar mechanism of transfer RNA-mediated 70S ribosome formation from free subunits; ii) poly(U)translation by hybrid ribosomes composed by one archaebacterial and one eucaryotic subunit; iii) ribosome assembly and homologous and heterologous RNA/protein recognition. PMID- 1725259 TI - Regulation of the heat shock response in E coli: involvement of positive and negative cis-acting elements in translation control of sigma 32 synthesis. AB - When cells of E coli are transferred from 30 to 42 degrees C, the cellular level of sigma 32 (rpoH gene product) increases transiently, resulting in increased transcription of a set of heat shock genes. Both increased synthesis and increased stability of sigma 32 contribute to transient accumulation of sigma 32. Evidence suggests that synthesis of sigma 32 is enhanced primarily at the level of rpoH translation. We have constructed and examined the expression of deletion derivatives of rpoH-lacZ gene fusion at 30 degrees C and after shift to 42 degrees C. It was revealed that two cis-acting sequences within the rpoH coding region are involved in thermal regulation of fusion protein synthesis. One region immediately downstream of the initiation codon is required for high level expression, whereas the other internal region is involved in repression at low temperature. Thus, these regions act as positive and negative cis-elements in thermal regulation of rpoH translation. The rpoH mRNA secondary structure model suggesting an interplay between the two regions has been proposed to account for the temperature-induced sigma 32 synthesis as a primary cellular response to the heat shock stress. PMID- 1725260 TI - Properties of an abundant RNA-binding protein in yeast mitochondria. AB - We have previously identified a protein with Mr approximately 40,000 (p40) that binds with high specificity and affinity to the 5'-untranslated leaders of mitochondrial mRNAs in yeast. Here we show that this protein is abundant, comprising about 0.4% of total mitochondrial protein. p40 is present in a cytoplasmic (rho degree) petite mutant that lacks mitochondrial protein synthesis and is therefore nuclear encoded. p40 can be detected by immunological techniques in cell lysates of several different pet mutants, specifically disturbed in the translation of individual mitochondrial mRNAs. It is thus not one of the translation factors defined by any of these mutations. In the case of a pet111 mutant, which is specifically blocked in the translation of COX2 mRNA, extracts still display COX2 mRNA binding activity, indicating that p40 complex formation in vitro is not dependent on the presence of PET111. PMID- 1725261 TI - Interaction between 16S ribosomal RNA and ribosomal protein S12: differential effects of paromomycin and streptomycin. AB - Strains containing a series of restrictive and non-restrictive mutations in ribosomal protein S12 have been transformed with plasmids carrying the rrnB operon with mutations at positions 1409 and 1491 in 16S rRNA. The effects of the double-mutant constructs have been measured by growth rate, paromomycin and streptomycin sensitivity, resistance and dependence. The results demonstrate a functional interaction between the 1409-1491 region of rRNA and ribosomal protein S12. PMID- 1725262 TI - Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA(Gln) interaction. AB - This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA(Gln). Temperature sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA(Gln).GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA(Gln) species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3' terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA(Gln) are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed. PMID- 1725263 TI - Identification of nuclear genes which participate to mitochondrial translation in Saccharomyces cerevisiae. AB - The mitochondrial protein synthesis presents specific features and uses specific components different from their cytoplasmic counterparts. Since most genes which code for these components are localized in the chromosomes and only a small number are encoded by the mitochondrial DNA, it is important to identify and characterize the nuclear genes involved in this process. In order to do this, we have used a genetic screening which implies the selection and study of nuclear suppressors of mitochondrial mutations (or the reverse situation) which affect the mitochondrial protein synthesis. Three mutations have been used for this purpose. Two of them (ts 1398, cs 909) impair the mitochondrial ribosome; they were used to characterize new interacting components as well as two genes, MBR1 and MBR2, which control the assembly or the regulation of other genes involved in mitochondrial protein synthesis. The third mutation (ts 932), blocks the 3'-end maturation of the mitochondrial aspartyl tRNA. A nuclear suppressor has been obtained which presents all the characteristics of a mutation in the gene encoding the enzyme responsible for this process. PMID- 1725264 TI - The relation between translation and mRNA degradation in the lacZ gene. AB - The technique of gene fusion, in which the gene of interest, severed from its 3' end, is in-phase fused to a reporter gene--usually lacZ--is widely used to study translational regulation in Escherichia coli. Implicit in these approaches is the assumption that the activity of the ribosome binding site (RBS) fused in-phase with lacZ, does not per se modify the steady-state level of the lacZ mRNA. Herein, we have tested this hypothesis, using a model system in which the RBS of the lamB gene is fused to lacZ. Several point mutations affecting translation initiation have been formerly characterized in this RBS, and we used Northern blots to study their effect upon the lacZ mRNA pattern. Two series of constructs were assayed: in the first one, a 51-bp fragment centered around the lamB initiator codon, was inserted in front of lacZ within the natural lactose operon, whereas in the second the lacZ gene was fused to the genuine malK-lamB operon just downstream from the lamB RBS. We observed that in the first series, the concentration and average molecular weight of the lacZ mRNA dropped sharply as the efficiency of the RBS decreased. This apparently arose from a decreased stability of the message, since the mRNA patterns are equalized when the endonuclease RNase E is inactivated. We suggest that in this case the rate limiting step in the decay process is an RNase E cleavage that is outcompeted by translation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725265 TI - Ribosome-mRNA contact sites at different stages of translation initiation as revealed by cross-linking of model mRNAs. AB - Two model mRNAs, one with and one without the Shine-Dalgarno (SD) sequence, were bound to Escherichia coli 30S ribosomal subunits in the presence and absence of initiation factors and initiator tRNA and then cross-linked by diepoxybutane. The distribution of the cross-linked mRNA among rRNA and ribosomal proteins (r proteins) and the extent to which individual r-proteins react was found to be affected by the presence or absence of the SD sequence and by the initiation factors and initiator tRNA. The results are consistent with the hypothesis that the position of the 30S-bound mRNA is shifted under the influence of the initiation factors and fMet-tRNA from a stand-by position towards a second site where the decoding of the initiation triplet by the initiator tRNA occurs. PMID- 1725266 TI - Ribosomal RNA and peptidyl-tRNA hydrolase: a peptide chain termination model for lambda bar RNA inhibition. AB - We propose here a model to explain the inhibition of bacteriophage lambda (lambda) vegetative growth and the killing of E coli cells defective in peptidyl tRNA hydrolase (Pth) by lambda bar RNA. The model suggests that bar RNA, which contains a characteristic UGA triplet, base-pairs in an anti-parallel fashion with the 1199-1205 region of E coli 16S rRNA. In doing so, it prevents the required functioning of that region of 16S rRNA in UGA-specific peptide chain termination. Pth is implicated in peptide chain termination because a defect in Pth is required for the achievement of the bar RNA inhibitory effects. We make certain predictions that flow from the model, predictions involving suppression of nonsense mutations, and present preliminary experimental results that demonstrate the fulfillment of those predictions. PMID- 1725267 TI - [Character and reasons for change in mitochondrial ATP content during auto oscillation of ion currents]. AB - The changes in the adenosine triphosphate content in the course of ionic flux oscillations in mitochondria were estimated by using the chemiluminescence method. The ATP concentration changes were shown to be of cyclic character; the oscillations in the ATP content were shifted by 180 degrees C against those of K+ fluxes. The oligomycin-induced oxidative phosphorylation blocking changed (but did not eliminate) the oscillational character of the ATP content in mitochondrial suspensions. It was concluded that substrate phosphorylation is the source of ATP under conditions of oxidative phosphorylation inhibition. Incubation of mitochondria in the presence of exogenous ATP led to suppression of ionic flux oscillations. PMID- 1725268 TI - [Properties of mitochondrial porin from cattle heart]. AB - Porin, a protein able to form ionic channels in model phospholipid membranes, has been isolated for the first time from bovine heart mitochondria. One-dimensional electrophoresis in the presence of sodium dodecyl sulfate revealed a major band with Mr of 32-34 kDa. On two-dimensional electrophoregrams this protein is represented by four components with pI ranging from 6.5 to 7.1. Porin spots were identified on two-dimensional electrophoregrams in a complete mixture of mitochondrial proteins. The presence of porin in bovine heart submitochondrial particles was demonstrated by two-dimensional electrophoresis. PMID- 1725269 TI - [Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]. AB - The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested. PMID- 1725270 TI - Expression of messenger RNAs encoding insulin-like growth factor-I, -II, and insulin-like growth factor binding protein-2 in bovine endometrium during the estrous cycle and early pregnancy. AB - The present study characterized the changes in concentrations of insulin-like growth factors-I and -II (IGF-I and IGF-II) in uterine luminal flushings, and the endometrial mRNA levels of IGF-I, IGF-II, and IGF binding protein-2 (IGFBP-2) obtained from Days 0 through 18 of the bovine estrous cycle and early pregnancy. Concentrations of IGF-I and IGF-II in uterine flushings were greater on Days 0 and 5 than on other days of the estrous cycle or pregnancy. Northern blot analysis of endometrial poly(A)+ RNA revealed two major transcripts of 7.5 and 1.0 Kb for IGF-I, one major transcript of 4.0 Kb for IGF-II, and one major transcript of 1.3 Kb for IGFBP-2. RNA dot-blot analyses indicated that endometrial expression of IGF-I mRNA was unaffected by day of the estrous cycle or status (cyclic vs. pregnancy). However, endometrial expression of IGF-II mRNA was greater in pregnant than in cyclic endometrium on Days 15 and 18. Levels of endometrial IGFBP-2 mRNA increased (p less than 0.05) between Days 10 and 18 of the estrous cycle and early pregnancy. Results suggest that the presence of the bovine conceptus has a stimulatory effect on endometrial expression levels of IGF II, whereas progesterone appears to be involved with enhancement of IGFBP-2. PMID- 1725271 TI - Pharmacokinetic behavior of [57Co]bleomycin liposomes in mice: comparison with the unencapsulated substance. AB - The distribution and excretion of [57Co]Bleomycin, dissolved in saline or encapsulated in liposomes, was studied in normal or tumor-bearing [P388 leukemia, reticulum cell sarcoma (RS)] mice. The free substance is cleared relatively quickly from the organism both after intravenous and intraperitoneal administration (t1/2 0.17 and 2.41 h, respectively), and is excreted predominantly via the urinary tract. In contrast, following entrapment in small unilamellar vesicles (SUV) or multilamellar vesicles (MLV), the 57Co radioactivity remains 7- to 30-fold longer in the blood stream and is detectable in considerable amounts in liver, spleen, lung and tumor of the RS model even after 48 h. Concomitantly, the renal excretion is diminished to about 50% of the free drug and the feces excretion is slightly increased, possibly due to the higher concentrations in the liver. Whereas the renal levels of radioactivity were similar with all application forms of [57Co]Bleomycin, there were marked differences in all the other tissues studied. After administration of SUV there was a higher activity in liver, brain and tumor, whereas MLV were more concentrated in spleen and lung. Therapeutic experiments confirmed the favorable results obtained with liposomes. While the free Bleomycin in the P388 leukemia had only a moderate influence on the lifetime of the animals with a treated/control value of 111%, encapsulation of the drug in SUV or MLV improved the results to 194 and 167%, respectively. In the intramuscular transplanted RS model, the SUV in a day 1 schedule had the same effect on tumor growth as the free drug in a day 1-4 schedule.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725272 TI - Modeling RNA secondary structures. I. Mathematical structural model for predicting RNA secondary structures. AB - A mathematical model for analyzing the secondary structures of RNA is developed that is based on the connection matrix associated with the planar p-h graph. The classification of the elementary structures allows the introduction of the basis of structural space from which to build the global secondary structure. All admissible solutions belong to the configuration space and can be obtained directly from its basis. PMID- 1725273 TI - Modeling RNA secondary structures. II. The geometric structural solution for tRNA. AB - The set of admissible solutions of the configuration space of tRNA molecules is obtained after incorporating further restrictions based on the principles of local stability and homology as well as on geometric and energetic considerations. PMID- 1725274 TI - Complementary replication. AB - Differential equations for the kinetics of complementary replicating macromolecules in a flow reactor are derived. It is shown that such a model has many features in common with the differential equation for direct replication, the replicator equation. Two special cases of replication, and the influence of mutation on them, have been studied in detail. In the case of first-order mass action kinetics--the quasi-species model--complementary replication, like direct replication, exhibits an error threshold for the replication accuracy, below which the genetic information is lost. In turns out that the long-time behavior of many special cases of the second-order kinetics model can be described in terms of second-order replicator equations, although this is not possible in general. PMID- 1725275 TI - Effects of milrinone, sulmazole and theophylline on adenosine enhancement of antigen-induced bronchoconstriction and mediator release in rat isolated lungs. AB - The effects of adenosine and some of its analogues on bronchoconstriction and mediator release were studied in isolated lungs of actively sensitized rats. The influence of two novel cardiotonic drugs, milrinone and sulmazole on these adenosine-induced effects was compared with that of theophylline, a well known adenosine antagonist. Adenosine (ADO) and its analogues N-ethyl-carboxamide adenosine (NECA) and R-phenyl-isopropyladenosine (R-PIA), dose-dependently enhanced antigen-induced bronchoconstriction. The enhancement of anaphylactic bronchoconstriction by adenosine and its analogues was accompanied by a rise in histamine release. The rank order of potency for adenosine and analogues with respect to enhancement of anaphylactic bronchoconstriction, was NECA greater than or equal to R-PIA greater than ADO. An unequivocal classification of the adenosine receptor involved, was therefore not possible. Dipyridamole and S-(p nitrobenzyl-6-thioinosine) (NBTI), both inhibitors of adenosine uptake, had no inhibitory influence on the adenosine-induced enhancement of anaphylactic bronchoconstriction, indicating that this enhancement is mediated by an extra cellular receptor. Theophylline, milrinone and sulmazole inhibited the enhancement of anaphylactic bronchoconstriction, without affecting preformed mediator release. Theophylline and sulmazole were both more effective as inhibitors of adenosine-enhanced bronchoconstriction than as inhibitors of antigen-induced bronchoconstriction, suggesting adenosine antagonism. Milrinone was equi-effective as inhibitor of both types of bronchoconstriction. Since adenosine antagonism has been associated with the side effects of theophylline it will be interesting to further investigate the therapeutic merits of novel cyclic nucleotide phosphodiesterase inhibitors in the treatment of asthma. PMID- 1725276 TI - [Diversity and relative inaccuracy of internal images of antigen group A revealed by IEF analysis]. AB - The diversity of a polyclonal anti-idiotypic response (Ab2) to a murine monoclonal anti-A (Ab1) was investigated after purification of two Ab2 populations. One was eluted from human polyclonal anti-A column and the other from Ab1. Analysis of the Ab specificity, as well as screening of the clonotypic distribution, were achieved after splitting Ab by IEF; this was followed by immunoblotting and probing with various anti-ABH mAb. The first population reacted with almost all the murine anti-ABH mAb, as well as with four human anti A mAb, and consequently consisted of Ab2 beta. The second was composed of "true" Ab2 directed against Ab1. In the first population internal images mimicked either A Ag, or H Ag, or some epitopes common to both. This study demonstrates the plurality of internal images-bearing Ig molecules, some mimicking completely, and some only partially or even unfaithfully the nominal A determinant. The analysis of this idiotypic cascade proves the existence of a degeneracy of the initial restricted antigenic specificity. The consequences of such a process are discussed. PMID- 1725277 TI - Central depressant effects of N3-substituted 6-azauridines in mice. AB - Central depressant effects in mice of N3-substituted 6-azauridines (6-AzUd) (1) were examined by intracerebroventricular (i.c.v.) injection. Eleven derivatives including alkyl-, benzyl-, xylyl- and phenylethyl-substitution onto the N3 position of 1 were synthesized and their pharmacological effects were evaluated using hypnotic activity, locomotor activity, motor incoordination and pentobarbital-induced sleep prolongation as indices. Six of 12 compounds showed the hypnotic activity. At a dose of 2 mumol/mouse, the mean sleeping time induced by 1, N3-benzyl-6-AzUd (7), N3-o-xylyl-6-AzUd (8), N3-m-xylyl-6-AzUd (9), N3-p xylyl-6-AzUd (10) and N3-alpha-phenylethyl-6-AzUd (11) was 14, 11, 45, 12, 9 and 16 min, respectively. These derivatives and N3-beta-phenylethyl-6-AzUd (12) (1.5 mumol/mouse) significantly prolonged pentobarbital-induced (40 mg/kg, i.p.) sleeping time, whereas none of the N3-alkylated derivatives (methyl-, ethyl-, n propyl-, n-butyl- and allyl-substitution) exerted the hypnotic activity or pentobarbital-induced sleep prolongation. Nucleoside 1 and its xylyl-derivatives (1.5 mumol/mouse) significantly decreased locomotor activity of mice, their effects paralleled the hypnotic activity. These compounds (1.5 mumol/mouse) also produced motor incoordination and potentiated the effect of diazepam-induced motor incoordination. These results indicate that 1 and its benzyl-related derivatives, but not alkyl-derivatives have a depressant effect on the central nervous system. PMID- 1725278 TI - Purification and characterization of a nuclease from Lentinus edodes. AB - An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum. PMID- 1725279 TI - [Effects of different administration of Salvia miltiorrhiza and heparin on antithrombin IIIAg, antithrombin III: A and alpha 2-macroglobulin in patients with cor pulmonale]. AB - The effects of different administration of Salvia miltiorrhiza (SM) and heparin on ATIII and alpha 2-M in 102 patients with cor pulmonale were studied. The results showed that (1) the level of ATIII in patients was significantly lower than that in the controls (P less than 0.01). Heparin intravenous drip induced ATIII decreased; (2) the level of ATIII:A in patients with heparin vapour inhalation treatment was significantly higher than that with regular treatment and with heparin intravenous drip (P less than 0.05) and (P less than 0.005); (3) SM presents ATIII-like activity, after treatment with SM, the level of ATIII:A was significantly higher than that with heparin vapour inhalation, heparin intravenous drip and regular treatment (P less than 0.05-0.005), heparin may enhance the ATIII-like activity of SM; (4) the treatment combining SM intravenous drip with heparin vapour inhalation is an efficient therapy of anticoagulation on the patients with cor pulmonale. The level of alpha 2-M in each group did not reveal significant change. PMID- 1725280 TI - [Randomized trial of combined chemotherapy including high dose cisplatin and radiotherapy for esophageal cancer]. AB - A prospective randomized trial in 64 esophageal cancer patients was conducted from 1986 to 1989. The patients were randomized into two groups-PPF (DDP 100 mg/m2 + pingyangmycin + 5-FU) regimen plus radiotherapy (combined group) or radiotherapy alone (control group). The overall response rate and CR rate in the combined group were 87.5% (28/32) and 40.6% (13/32). Those of the control group were 81.3% (26/32) and 21.9% (7/32), respectively. The overall response rates were similar in these two groups, but the CR rate in the combined groups was higher than that of the control group. The 1-year survival rates in the combined and control groups were 77.4% (24/31) and 45.2% (14/31) (p less than 0.01). The 2 year survival rates were 56.3% (9/16) and 34.6% (9/26), respectively (p greater than 0.05). Our preliminary results show that combined chemotherapy including high dose cisplatin and radiotherapy may improve the therapeutic result of esophageal cancer. PMID- 1725281 TI - [Radiotherapy of cancer of the gastric cardia--report on 40 patients]. AB - Fourty patients with cancer of the gastric cardia as proven by pathology or cytology were treated by radiation therapy from January 1985 to October 1986. All patients except four who refused operation had lesions too advanced for surgical intervention. Telecobalt was used to deliver 60-70 Gy/6-7 wk through 3 or 4 portals. In some patients, 45-60 Gy was also given to the supraclavicular area. 90% of patients had symptomatic improvement and 80% showed objective improvement on X ray barium meal. The 1-, 2- and 3-year survival rates were 83%, 43% and 23%. Ten percent of patients survived the third year without evidence of disease. The influence of sex, age, siege of lesion and X ray findings at the conclusion of treatment were analyzed. Female patients and those whose lesions completely resolved at the completion of radiotherapy had more favorable outcome than the others (P less than 0.01, P less than 0.05). It is suggested that radiation therapy may be an effective means in the combination treatment or palliative treatment of cancer of the gastric cardia. PMID- 1725282 TI - A pharmacokinetic analysis of 3,4-methylenedioxymethamphetamine effects on monoamine concentrations in brain dialysates. AB - Interpretation of the in vivo actions of 3,4-methylenedioxymethamphetamine (MDMA) is complicated by the formation of the active metabolite, 3,4 methylenedioxyamphetamine (MDA). This study evaluates the role of MDA in the dopamine releasing actions of (+)-MDMA. In the study, rats were given subcutaneous doses of (+)-MDMA and concentrations of monoamines and their metabolites in striatal dialysate were measured at 15 min intervals. In parallel experiments, plasma concentrations of (+)- and (-)-MDMA and MDA were determined by GC/MS procedures. The time course of MDMA levels was comparable for the two isomers as were their bioavailabilities. In contrast, the plasma levels of MDA were about three times higher after (+)-MDMA. (+)-MDMA caused a rapid increase in striatal dialysate levels of dopamine and decreased extracellular levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). There was a significant correlation between dopamine concentration in striatal dialysate and plasma MDMA concentration, but not with plasma MDA. These results indicate that MDMA itself has stereoselective actions on dopamine neurons. However, the higher plasma MDA levels after (+)-MDMA may account for part of the enantiomeric differences in the behavioral and neurotoxicological effects of MDMA. PMID- 1725283 TI - Non inherited hemoglobin anomalies. AB - A number of genetic or acquired conditions in which hemoglobin anomalies occur without detectable modifications of globin genes are reviewed. They include increased fetal hemoglobin (alpha 2 gamma 2), variations in hemoglobin A2 concentration, the presence of Hb H (beta 4), Bart's Hb (gamma 4), Hb Koelliker, glycosylated, carbamylated and acetylated hemoglobins. PMID- 1725284 TI - Location of the quinine-dependent epitope on platelet glycoprotein IIIa. AB - Quinine-dependent (Q) IgG antibodies (Q.Ab) in drug-induced immune thrombocytopenia are heterogeneous and bind to different platelet surface glycoproteins (GP), namely GPIb, IX, IIb, IIIa and an unidentified 57-kDa membrane proteins. Although both the Q-dependent epitope on GPIIIa and the P1A1 antigen require intact disulphide bonds for their expression, they are distinct because Q.Ab bind to GPIIIa lacking P1A1. Epitopes for both antigens were examined on Western blots of either intact washed human platelets or purified GPIIIa. When intact platelets were digested with trypsin and washed and solubilised prior to electrophoresis, membrane-associated fragments of GPIIIa of 78 kDa were found to be reactive with both antibodies. In addition, 60- and 68 kDa fragments bound anti-P1A1 but not Q.Ab. Similar digestion with chymotrypsin produced only 60-kDa fragments containing both epitopes. Digestion of purified GPIIIa with chymotrypsin produced 60-kDa peptides reactive with Q.Ab and anti P1A1 in immunoblotting studies. Similar digestion with elastase produced 58-kDa fragments also containing the epitopes for both antibodies. Longer digestion times or sequential digestion with different enzymes did not reveal extra fragments. However, immunoprecipitation of trypsin-digested 125I-labelled GPIIIa with affinity-purified Q.Ab produced a 17-kDa fragment containing the Q-dependent epitope. PMID- 1725285 TI - Effect of L-asparaginase on insulin secretion from isolated rat islets of Langerhans. AB - The effects of L-asparaginase were evaluated on glucose-induced insulin release from isolated rat islets of Langerhans. Islets were obtained by enzymatic digestion of pancreas from Sprague-Dawley rats. The study of L-asparaginase effects on insulin secretion was performed in a static incubation of islets. Insulin secretion was measured at 60 min of incubation with different secretagogues with and without L-asparaginase. L-Asparaginase at concentrations from 310 to 5,000 U/ml could inhibit the glucose-induced insulin secretion in a dose-dependent manner. This effect was not recovered after incubation in the absence of the drug for another 2 h. The half-maximal inhibitory effect of the enzyme on insulin secretion was observed at L-asparaginase concentrations of 1,000 U/ml. Tolbutamide (200 microM) and ketoisocaproic acid (20 mM) did not induce insulin secretion in the presence of moderately high L-asparaginase concentrations. L-Asparaginase did not inhibit glucose-induced insulin secretion in the presence of isobutyl-methyl-xanthine (IBMX) (20 microM) or forskolin (20 microM). L-Asparaginase promoted a decrease in total c-AMP in isolated rat islets at concentrations from 500 to 1,500 U/ml when they were stimulated by glucose. If islets were treated with IBMX or forskolin, L-asparaginase did not inhibit the glucose-induced total c-AMP levels in islets. PMID- 1725286 TI - Lyophilized type-I collagen and chronic leg ulcers. AB - Lyophilized type I collagen (L.C.) can stimulate wound healing by recruiting a number of different cell types (i.e. platelets and macrophages) and proteins (i.e. fibronectin). Platelets and macrophages produce locally-acting growth factors that in turn induce fibroblast and epidermal migration, angiogenesis and increase matrix synthesis. Chronic leg ulcers (C.L.U.) are the end result of microvascular failure owing to ischemia and stasis. When L.C. has been used in the treatment of C.L.U. we have observed that: a) it is significantly more effective in stimulating the healing of chronic venous ulcers when compared to hydrocolloids (p less than .05), the two products being applied upon half of the same ulcer; b) in the treatment of C.L.U. due to arterial obstruction L.C. is more effective than hydrocolloids without achieving statistical significance; c) it is very effective in the treatment of C.L.U. in thalassaemic patients; d) telethermographic studies have demonstrated an increase of blood perfusion and histological studies have shown the stimulation of angiogenesis, fibropoiesis and epidermal growth; e) the application of L.C. determines the maximum obtainable increase also under conditions of proven cicatrization difficulty; and f) enzymatic degradation of L.C. has not promoted any bacterial infection and no local or generalized sensibilization phenomena have been observed. We can conclude that L.C. is a pharmacological approach to wound healing, directly interfering with cellular and non-cellular components, and significantly improves the reparative process when delayed. PMID- 1725287 TI - Combined supravital staining and hypoosmotic swelling test. AB - The parameter of sperm viability in hypoosmotic solution (VHOS) provides information concerning the membrane integrity of the sperm head. The objective of this study was to determine the association between the VHOS parameter and the sperm penetration assay. The VHOS parameter correctly predicted 70.0-71.4% of the failed sperm penetration assay samples in the short duration preincubation groups. A combination of both the VHOS parameter and the hypoosomotic sperm swelling (HOS) test significantly reduced the number of false negative results. In general, a sperm sample with an abnormal VHOS result and an abnormal HOS test result would be associated with a negative sperm penetration assay. Washing by centrifugation appeared to weaken the sperm head membranes while the swim-up method selected for sperm with strong head and tail membranes. After the various processing methods unique changes in the integrity of the sperm head and tail membranes for each sperm sample may help to identify the optimal method of preparation for individual patients undergoing the newer assisted reproductive technologies such as sperm microinjection. PMID- 1725288 TI - Human sperm tail fibrous sheath undergoes phosphorylation during its development. AB - The phosphorylation of the human sperm tail fibrous sheath as a maturational step during its development is reported for the first time. This was demonstrated using GDA-J/F3 and RT97 monoclonal antibodies (MoAb) which recognize the fibrous sheath. In indirect immunofluorescence microscopy of frozen sections of human adult testes, the two antibodies reacted with the assembled fibrous sheath only, but the numbers of sperm tails stained with RT97 were consistently lower than those treated with GDA-J/F3. Furthermore, by using double indirect immunofluorescence, although the majority of spermatozoa were doubly stained with the two MoAbs, some GDA-J/F3-positive sperm tails were negative with RT97. In epididymal and ejaculated spermatozoa, the two antibodies stained all the tails. This indicated that the ontogenic appearance of the GDA-J/F3 epitope precedes that of RT97. In Western blotting and/or indirect immunofluorescence of spermatozoa, treatment of samples with alkaline phosphatase abolished the reactivity of RT97 while that of GDA-J/F3 MoAb was not affected. This finding indicated that the RT97 but not the GDA-J/F3 epitope was phosphorylated. Together, these results therefore reveal that during tail morphogenesis, the fibrous sheath undergoes phosphorylation as part of its structural maturation. Screening of sperm cell precursors recovered from oligozoospermic donors showed reaction of some abnormal germ cells with GDA-J/F3 MoAb but not with RT97, suggesting the possible failure of phosphorylation of the fibrous sheath protein in these cells. The significance of these findings is discussed together with the biological importance of phosphorylation to the fibrous sheath. PMID- 1725289 TI - A rapid latex agglutination test for detection of antibodies in tuberculosis and Hansen's disease. AB - Antigens of Mycobacterium w, a saprophytic fast growing organism having antigenic epitopes cross-reactive with Mycobacterium leprae and Mycobacterium tuberculosis, were coated on to latex beads (0.33 micron Zn size), and the reactivity tested with sera of tuberculosis and Hansen's disease (HD) patients. Seventy nine percent of lepromatous leprosy (LL) and eighty five percent of pulmonary tuberculosis (TB) patients sera showed an agglutination reaction easily read by naked eye. Specificity of the test was further checked by testing sera of non mycobacterial infection cases and all of them were found negative. Among apparently healthy controls, 4.3% were found positive from non-endemic and 8.8% from endemic area. The sensitivity of the assay is further enhanced from 78.7% to 90.4% and 85.7% to 91.6% in both LL HD and pulmonary tuberculosis respectively, by using immune complexes extracted from the patients sera. Potential of these antigen coated beads to detect the two major human mycobacterial disease, LL HD and pulmonary TB was also put evidence in a double blind study on coded sera samples obtained from various hospitals in India. The antigen coated beads are stable for upto 6 months at 4 degrees C. The latex slide agglutination test reported here, is simple, rapid, easy to perform and can be used even in rural areas of developing countries. PMID- 1725290 TI - Microleakage around direct composite inlays. AB - The purpose of this study was to compare the microleakage around direct composite inlays bonded with a dual cure luting composite into Class V type inlay cavities in extracted molar teeth. Bonding methods which included two cavity cleansing regimes and three bonding treatments were used. Either pumice and rinse or rinse only cavity cleansing was used to remove the separator agent (agar/alcohol) from the cavity surface prior to inlay bonding. Restorations were thermocycled between 5 degrees C and 55 degrees C (with intermediate baths at 36 degrees C) before (240 cycles) and during (12 cycles) silver staining. Microleakage around the sectioned restorations was quantified using digital imaging microscopy at x 40 magnification. Data analysis indicated that failure to include pumice slurry application as part of the cavity cleansing regime prior to bonding lead to a marked increase in microleakage at the enamel/restoration interface following one of the three bonding treatments. PMID- 1725291 TI - Keratin expression in Merkel cells of fetal rat skin. AB - The cytokeratin expression of Merkel cells in fetal rat skin was studied by light and electron microscopy. Employing a pre-embedding staining method, 2 monoclonal anti-keratin antibodies (RCK-102, MA-902) were shown to stain Merkel cells specifically. Neighbouring keratinocytes were unstained. The staining reaction seems to be based on the expression of 52.5 kD cytokeratin. PMID- 1725292 TI - Evidence that human and porcine insulin differently affect the human insulin receptor: studies with monoclonal anti-insulin receptor antibodies. AB - Binding studies have been carried out with radioiodinated monoclonal antibodies directed to various epitopes of the insulin receptor in order to detect differences between human and porcine insulin in the interaction with the human insulin receptor. Human insulin was more effective that porcine insulin at inhibiting the binding of 125I-MA-5 to IM-9 cells, Hep-2 human larynx cells and human placenta membranes. On the contrary, human and porcine insulin showed similar inhibitory effect on the binding of two other labeled anti-insulin receptor monoclonal antibodies, thus ruling out the possibility that results were due to experimental artifacts. Although several interpretations are possible, data reported suggest that human insulin and porcine insulin might differently affect the insulin receptor, even if, the biological significance of these findings remains unknown. PMID- 1725293 TI - Effects of local prostatic hyperthermia on human NK and T cell function. AB - Studies on lymphocyte subsets, mitogen transformation and NK cytotoxicity of blood mononuclear cells (BMNC) were performed in 30 patients who received transrectal microwave hyperthermia (TRHT) of the prostate. Of the 30 patients, 15 had advanced adenocarcinoma of the prostate (CAP) and 15 had severely symptomatic benign prostatic hyperplasia (BPH). Local TRHT was given twice a week for a total of six sessions. The treatments were administered at 2450 MHz or 434 MHz using a water-cooled rectal applicator. Each TRHT session lasted for 30 min at steady state temperature controlled on the rectal mucosa at 45 degrees C. Studies of immune reactions were performed before TRHT, at the completion of six TRHT sessions, and at 1, 2, 4, and 6 months from therapy. Identical studies at the same time-interval were performed in 30 healthy male volunteers. In the 15 CAP patients the results of the immune studies obtained before TRHT, including CD4+/CD8+ ratio, PHA and Con-A transformation indices were significantly lower (p less than 0.01) than in the 15 BPH patients and in the 30 normal volunteers. The 15 BPH patients and the 30 normal volunteers all had immune parameters within the normal limits. Following the administration of TRHT in the 15 CAP patients, a transient significant (p less than 0.01) stimulation of the tested cell-mediated immune parameters was observed when compared with the pretreatment values. The peak effect of this stimulation was noted at 2 months with a subsequent decrease. In the 15 BPH patients a lesser degree of immune stimulation was noted. As expected there was no substantial change in the measured cell-mediated immune parameters in the 30 normal volunteers. A significant increase of NK cytotoxic activity was noted following TRHT in CAP patients when compared with the pretreatment results. This activity reached 120-130% of the individual initial values, being significant at p less than 0.01. The finding of transient stimulation of cell-mediated immune reaction, following local hyperthermia in patients with CAP, may be of some clinical relevance and of clinical importance. Additional studies are being formulated to confirm these interesting findings. PMID- 1725294 TI - [Mechanism of the modulation of pain transmission at the subnucleus caudalis of the trigeminal sensory nuclear complex in rabbits]. AB - The modulation of dental pain transmission at the subnucleus caudalis of the trigeminal sensory nuclear complex (SpVc) was investigated in rabbits in vivo. The superficial layers of SpVc were perfused with artificial cerebrospinal fluid using a push-pull cannula system. Immunoreactive substance P (SP) released into the perfusates following electrical stimulation of the lower incisor pulp was measured. The obtained results were as follows. 1. An increase in the release of SP and [Met5]-enkephalin was observed by the electrical stimulation with 40 V. 2. The increase of SP release following electrical stimulation was inhibited by systemic administration of morphine (10 mg/kg i.v.) or local application of morphine (10(-6) M) to SpVc. The stimulus-evoked SP release was also inhibited by local application of [D-Ala2, Met5]-enkephalinamide (an analog of [Met5] enkephalin; 10(-4) M). 3. Spontaneous release of serotonin (5-HT) into the perfusates was observed, while that of norepinephrine was not. Tooth pulp stimulation tended to increase the level of 5-HT. Systemic administration of morphine (10 mg/kg i.v.) and electrical stimulation of the nucleus raphe magnus (NRM) significantly enhanced the release of 5-HT. 4. The release of SP evoked by tooth pulp stimulation was inhibited by local application of 5-HT (10(-6)M) and electrical stimulation of NRM. These results suggest that there are two modulatory systems controlling the delivery of the ascending sensory message at the superficial layers of SpVc. One is an intrinsic mechanism associated with the segmental enkephalinergic system, the other is a descending monoaminergic system originating in NRM. It is also suggested that these two systems play an important role in producing the analgesic effect of morphine. PMID- 1725295 TI - [Status of the protective mucous barrier of the stomach after organ-sparing operations for duodenal ulcer]. AB - A protective mucous barrier of the stomach was studied in 153 patients with duodenal ulcer disease. Before the operation in 70 patients, decrease in concentration of protective factors of gastric mucus, especially in complicated form of ulcer disease was noted. More complete restoration of a protective barrier was noted after organ-preserving operations with excision of ulcerous substrate (in 54 patients) than after those without its excision (in 29). PMID- 1725296 TI - Tissue specificity of the endothelin-induced responses. AB - Endothelin (ET-1) has many activities in various tissues and organs. Even in the vascular system, ET-1 elicits vasodilation as well as vasoconstriction. These diverse responses induced by ET-1 are ascribed to the balance between ET-1 and ET 3 effects that are mediated by (a) at least two types of calcium channel activated by ET, (b) three distinct prototypes of ET-receptor subtypes, (c) the diversity of the intracellular signal transduction system, especially cyclooxygenase products, and (d) modulation of hormonal and neuronal factors. PMID- 1725297 TI - Evidence against a role for aspartyl proteases in intracellular processing of big endothelin. AB - CPAE endothelial cells were cultured in the presence of pepstatin, NH4Cl, or chloroquine in order to assess their effects on the secretion of endothelin-1 (ET 1). The first of these is an inhibitor of aspartyl proteases and the last two are known to neutralize acidic intracellular compartments. The pepstatin was encapsulated into liposomes to aid in its uptake, and uptake was confirmed by measuring the residual aspartyl protease activity in washed, lysed cells. Pepstatin had no effect (less than 5%) on the secretion of ET-1, 25 mM NH4Cl decreased secretion by 30-47%, and 25 microM chloroquine increased secretion by 37-79%. In contrast, each of these reagents is known to inhibit lysosomal degradation of intracellular proteins by 75-90%. Additionally, big ET was shown to be a very poor substrate, in terms of kcat/Km values, for aspartyl proteases. The rate constants were less than 10(4) M-1 s-1, which is approximately 1% of the value for the best substrates. The data, therefore, do not support a role for aspartyl proteases in the formation of ET-1. Similar to chloroquine, 0.5 microM monensin increased the secretion of ET-1 by 40-60%. Both of these reagents have previously been shown to increase the rate of constitutive secretion of peptides by affecting their partitioning between packaging into storage granules and constitutive secretion. The results would therefore provide supportive evidence for the existence of a storage form of ET-1 in endothelial cells. PMID- 1725298 TI - Endothelin in human brain and pituitary gland: comparison with rat. AB - To investigate the physiological roles of endothelin (ET) in the brain and pituitary gland, the presence of immunoreactive (ir) ET and ET receptors was studied by radioimmunoassay and receptor assay in humans and rats. ir-ET concentrations in human brain (6-10 fmol/g of wet weight) were comparable with the levels in the rat brain (5-9 fmol/g of wet weight). Higher concentrations of ir-ET were found in human pituitary glands (147 +/- 30 fmol/g of wet weight, mean +/- SEM) and rat posterior pituitary lobes (88 +/- 26 fmol/g of wet weight). Fast protein liquid chromatography (FPLC) showed that the ir-ET in human hypothalamus and brainstem was mainly ET-1, while the ir-ET in human pituitary was mainly ET 3. FPLC of the whole rat brain extract showed a larger peak in the position of ET 3 and a smaller peak in the position of ET-1. Receptor assay showed that [125I]ET 1 binding sites were present in very large numbers in all five human brain regions and four rat brain regions examined but were much less abundant in the human pituitary. ET mRNA was detected by Northern blot hybridization in human pituitary but not in human hypothalamus. These findings are in accord with the possibility that ET acts as a neurotransmitter, neuromodulator, or neurohormone. PMID- 1725299 TI - Binding characteristics of endothelin isoforms (ET-1, ET-2, and ET-3) in vascular smooth muscle cells. AB - The existence of distinct endothelin (ET) receptor subtypes has been reported in several tissues. In the present study, we investigated the binding characteristics of the three endothelin isoforms to cultured rat aortic smooth muscle cells. [125I]ET-1, [125I]ET-2, and [125I]ET-3 bound to an apparent single class of binding sites with apparent dissociation constants (Kd) of 111, 123, and 1410 pM and binding capacities (Bmax) of 54.1, 46.0, and 7.9 fmol/10(6) cells, respectively. The binding of the three radiolabeled endothelin isoforms was equally inhibited by ET-1 and ET-2. ET-3 was more effective in competing with [125I]ET-3 than with [125I]ET-1 or [125I]ET-2. In contrast to ET-1 and ET-2, the binding of ET-3 was reversible. Furthermore, 18 h of pre-exposure of the cells to 1 nM ET-1 or ET-2 decreased the ET-1 binding capacity, whereas ET-3 (10 nM) was ineffective. ET-3 binding characteristics were not affected by pretreatment of the cells with any of the endothelin isoforms. These results suggest the presence of two distinct endothelin receptor subtypes in rat aortic smooth muscle cells. The ET-1 and ET-2 preferring receptor (80-85%), sensitive to downregulation or internalization, elicits an irreversible binding. The second subtype (15-20%) binds the three endothelin isoforms with the same affinity in a reversible manner, and is insensitive to downregulation or internalization. PMID- 1725300 TI - Heterogeneity of endothelin receptor. AB - The complex formed between endothelin (ET) and its binding protein was adequately stable to be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) at low temperature. Cross-linking was not necessary. This simple method was applied for both qualitative (determination of molecular weight of ET binding protein) and quantitative (determination of content of ET binding protein) analyses of ET-binding protein for various organs from different mammals. Molecular weights of the major ET binding proteins were around 35 and 55 kDa in several canine organs examined. The distribution of these protein species among organs was quite different. In the case of human placenta, the predominant one showed a molecular weight of 35 kDa. We obtained several monoclonal antibodies that could immunoprecipitate ET binding activity from the solubilized human placenta membrane fraction. One of the monoclonal antibodies recognized approximately 58-kDa protein, as determined by Western blot analysis, when the gel is run in the absence of beta-mercaptoethanol. PMID- 1725301 TI - Production of endothelin by vascular smooth muscle cells. AB - Endothelin (ET) production by cultured vascular smooth muscle cells isolated from rat and rabbit aortae was measured by a specific radioimmunoassay. Vascular smooth muscle cells released ET at a rate of 0.6 (range of 0.1-1) fmol/10(5) cells/24 h compared to 45 (range of 10-80) fmol/10(5) cells/h for endothelial cells. ET immunoreactivity was confirmed as ET-1 by fast protein liquid chromatography. Calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) suppressed basal release of ET by 24-66% from both rat and rabbit vascular smooth muscle. Forskolin and dibutryl-cAMP similarly suppressed ET release by 33-86%, suggesting that increased intracellular cAMP may account for the mechanism of action of CGRP and VIP. In contrast, CGRP and VIP did not have any effect on ET release from endothelial cells. The characteristics and regulation of ET binding sites on vascular smooth muscle cells were measured using [125I]ET-1 as the radioligand. Pretreatment with CGRP, VIP, forskolin, and dibutryl-cAMP increased the ET receptor density on both smooth muscle cell types. The low level production of ET-1 by vascular smooth muscle cells may have an important autocrine function and may be under regulation of neuropeptides localized to perivascular nerves. PMID- 1725302 TI - Separation and purification of 34- and 52-kDa species of bovine lung endothelin receptors and identification of the 34-kDa species as a degradation product. AB - We solubilized bovine lung endothelin receptors in a nonaggregated state and identified 34- and 52-kDa species of endothelin receptor by affinity labeling. The ratio of 34-kDa species to 52-kDa species varied widely from sample to sample, suggesting that the 34-kDa species is a proteolytic product; we therefore examined the effect of various proteinase inhibitors and found that EDTA (greater than 50 mM) was very effective in preventing the conversion. These results indicate that the lower Mr species arose from the 52-kDa receptor as a result of limited proteolysis by metal proteinase(s). The resulting 34-kDa species had endothelin-binding activity, was very stable, and was not processed further even in the absence of proteinase inhibitors. We therefore first tried to purify the 34-kDa species by affinity chromatography. Bovine lung extracts were mixed with biotinylated endothelin and the ligand-receptor complexes formed were isolated with avidin-agarose. Starting from 3.5 kg of bovine lung, approximately 200 micrograms of the pure receptor was obtained. Microsequencing of two well separated tryptic fragments yielded the following partial amino acid sequences: Ala-Asn-Asp-His-Gly-Tyr-Asp-Asn-Phe and Ser-Gly-Met-Gln-Ile-Ala-Leu-Asn-Asp-His Leu-Lys. PMID- 1725303 TI - Two endothelin receptor subtypes in porcine arteries. AB - Endothelin-1 (ET-1) and ET-3 caused constrictions of endothelium-denuded porcine coronary artery strips with different concentration-response curves: a typical sigmoidal curve to ET-1 and a two-phase sigmoidal curve to ET-3. Binding assays using a membrane preparation demonstrated different Bmax values for [125I]ET-1 and [125I]ET-3 binding. In addition, [125I]ET-1 binding was inhibited by ET-1 and ET-3 with different potencies (ET-1 greater than ET-3), while [125I]ET-3 binding was inhibited by both ETs equally. From these results, two distinct ET receptor subtypes were proposed in the artery; site 1 (selective to ET-1) and site 2 (equally sensitive to both ETs). However, only site 1 was identified on cultured arterial smooth muscle cells (VSMCs) by the binding assay, and this was confirmed since only ET-1 (not ET-3) caused a significant increase in the intracellular free Ca2+ concentration. Therefore, it seems likely that vasoconstriction is mediated via the binding of ET-1 to site 1 (VSMCs) and site 2 (non-VSMCs), or the binding of ET-3 to site 2 (non-VSMCs). Furthermore, site 2 was predominant in nonvascular tissues such as lung, kidney, and cerebellum, thereby suggesting that site 1 may exist in limited tissues such as VSMCs. PMID- 1725304 TI - Characterization of endothelin receptor subtypes. AB - Scatchard-plot analysis of [125I]ET-1 and [125I]ET-3 binding to rat lung membranes exhibited almost the same Kd values whereas the concentration of the binding sites of ET-1 is approximately four times higher than that of ET-3. This result suggests the presence of at least two distinct subtypes of ET receptors: an ET-1-specific type and an ET-3-specific or ET-1 and ET-3 nonselective type. On the other hand, in rat brain membranes, a curvilinear Scatchard plot was obtained for [125I]ET-3 in contrast to a linear plot for [125I]ET-1. This finding also demonstrates the existence of the two different receptor subtypes having the same affinity for ET-1, but one of which has a high affinity and the other has a low affinity for ET-3. Moreover, these data indicate that an ET receptor subtype exists in rat brain different from the subtype in rat lung. To obtain the bases for a further detailed characterization of the receptor, we have purified the rat lung ET receptor to homogeneity by ET-1-affinity chromatography. The purified receptor exhibits a molecular mass of 45 kDa, in good agreement with that estimated from the affinity labeling. PMID- 1725305 TI - Interaction between endothelin-induced Na+ and Ca2+ kinetics in cultured rat vascular smooth muscle cells. AB - The present study was undertaken to examine the interaction between endothelin-1 (ET-1)-induced Ca2+ and Na+ kinetics in cultured rat vascular smooth muscle cells (VSMCs) by measuring cytosolic free Ca2+ ([Ca2+]i) and intracellular Na+ ([Na+]i) using intracellular fluorescent dyes (fura-2 and SBFI, respectively). ET-1 increased both [Ca2+]i (basal, 58.9 nM; 10(-9) M, 119.8 nM; 10(-8) M, 265.0 nM; 10(-7) M, 419.0 nM) and [Na+]i (basal, 13.4 mM; 10(-9) M, 17.1 mM; 10(-8) M, 21.6 mM; 10(-7) M, 25.2 mM) in a dose-dependent manner in cultured rat VSMCs. The Na(+)-free solution enhanced the 10(-7) M ET-1-mobilized [Ca2+]i (419 vs. 1,192 nM) but reduced the 10(-7) M ET-1-induced increase in [Na+]i (25.2 vs. 6.1 mM). The Ca(2+)-free solution decreased the peak levels of 10(-7) M ET-1-induced mobilization of [Ca2+]i (411 vs. 350 nM) and the sustained increase in [Ca2+]i and totally diminished the 10(-7) M ET-1-induced increase in [Na+]i (25.2 vs. 12.8 mM). These results therefore indicate that ET-1 increases [Na+]i in association with an increase in cellular Na+ and Ca2+ uptake in VSMCs. PMID- 1725306 TI - Multiple subtypes of endothelin receptors in human and porcine tissues: characterization by ligand binding, affinity labeling, and regional distribution. AB - To characterize the properties and the distribution of endothelin (ET) receptor subtypes, we have examined the ligand selectivity and the molecular weight (Mr) of [125I]ET-1 and [125I]ET-3 binding sites in various tissues of human and pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound [125I]ET 1 in all of the tissues examined. ET-3, sarafotoxin S6b (SRTX-b), and sarafotoxin S6C (SRTX-c) displaced the [125I]ET-1 with nearly the same sensitivity as ET-1 (IC50 = 0.07-3.0 nM) in brain, kidney, liver, and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against [125I]ET-1 binding in atria, aorta, lung, stomach, and uterus. The Bmax value for [125I]ET-3 was 83% of that for [125I]ET-1 in human liver membranes, whereas the Bmax for [125I]ET-3 was only 12% of that for [125I]ET-1 in human atrial membranes. [125I]ET-3 bound to liver and atrial membranes was displaced by ET/SRTX isopeptides almost equipotently. Two proteins with Mr of 110 and 50 kDa were specifically affinity-labeled with [125I]ET-1 in porcine lung membranes. The above findings indicated that two distinct subclasses of ET receptors, namely ET 1/ET-2-specific and ET/SRT family common receptors, were distributed in various proportions in mammalian tissues. PMID- 1725307 TI - Isolation and characterization of porcine endothelin-like peptide. AB - Two endogenous molecules possessing an endothelin-like sequence with an apparent molecular weight of 7 kDa were isolated from serum-free culture medium of porcine aortic endothelial cells, using a specific radioimmunoassay. N-terminal sequences of two molecules were identical to each other and were Ser-Leu-Lys-Asp-Leu-Phe Pro-Ala-Lys-Ala-Ala-Asp-Arg-Arg-Asp-Arg-X-Gln-X- Ala-X- Gln-Lys-Asp, which corresponds to the sequence [94-117] of preproendothelin-1. This finding indicates that these two molecules may be closely related peptides such as the oxidized form or disulfide analogues and also suggests that the endogenous peptide possessing an endothelin-like sequence is generated by proteolytic cleavage at paired basic amino acids Arg92-Arg93. Further studies on the structure and function of new endogenous peptides possessing an endothelin-like sequence are ongoing in our laboratory. PMID- 1725308 TI - Functional properties of high- and low-affinity receptor subtypes for endothelin 3. AB - Endothelial cells from brain microvessels express two types of endothelin (ET) receptor. The first receptor subtype (defined as E alpha) shows a high affinity for ET-1, a low affinity for ET-3, and it is coupled to phospholipase C. The second subtype (E beta) shows a high affinity for both ET-1 and ET-3. It is not coupled to phospholipase C, but its activation leads to an increased activity of the Na+/H+ exchanger via a protein kinase C-independent mechanism. Brain astrocytes also express a high-affinity ET-3 receptor. However, unlike that of brain capillary endothelial cells, this receptor is coupled to phospholipase C and it may be a third type of endothelin receptor (E gamma). Thus, it seems that by using both binding and functional criteria, at least three subtypes of endothelin receptor can be distinguished: a low-affinity ET-3 receptor and two high-affinity ET-3 receptors that are coupled to different intracellular signaling pathways. PMID- 1725309 TI - Evidence of glycosylated sites on the endothelin-1 receptor in Swiss 3T3 cells. AB - The effects of incubation of intact cells with six different lectins on the specific binding of [125I]endothelin-1 (ET-1) were determined in Swiss 3T3 fibroblasts. ET-1 binding was unaffected by pretreatment of cells for 1 h at 37 degrees C with concanavalin A, soybean agglutinin, Ulex europaeus agglutinin I, peanut agglutinin, or Galanthus nivalis agglutinin. However, preincubation of cells with 300 micrograms/ml of wheat germ agglutinin resulted in a 70% decrease in specific binding of ET-1 to cell-surface receptors. The inhibitory effects of wheat germ agglutinin were diminished by brief incubation of lectin-treated cells with 100 mM N-acetylglucosamine, a monosaccharide specifically recognized by wheat germ agglutinin. Neither glucose nor mannose had any effect on wheat germ agglutinin-mediated inhibition of the specific binding of ET-1. These results suggest that the ET-1 receptor on 3T3 cells is a glycoprotein that contains one or more N-acetylglucosamine residues at or near the ligand binding site. PMID- 1725310 TI - Receptors for endothelin in the central nervous system. AB - The quantitative receptor autoradiographic method we used revealed that specific [125I]endothelin-1 ([125I]ET-1) binding sites are highly concentrated in the choroid plexus (ChP), subfornical organ (SFO), lacunosum molecular layer of the hippocampus (LMol), and granular layer of the cerebellum (GC) of the rat brain. [125I]ET-1 bound to the rat ChP and LMol with a high affinity and to the SFO and GC with a low affinity. The possibility that ET-1 acts as a neuropeptide within the central nervous system by interacting with specific receptors would have to be considered. PMID- 1725311 TI - Endothelin-3 stimulates prostacyclin production in cultured bovine endothelial cells. AB - Among three endothelin (ET) isopeptides, ET-3 shows the most potent initial depressor response through endothelium-dependent mechanism. We have recently reported that ET-3 induces receptor-mediated phosphoinositide breakdown, which increases the intracellular Ca2+ concentration [( Ca2+]i) and stimulates synthesis of endothelium-derived relaxing factor (EDRF), or nitric oxide (NO), in endothelial cells (ECs). We examined whether ET-3 has any effect on synthesis of prostacyclin, a potent vasodilator prostanoid in bovine ECs. ET-3 dose dependently (10(-10) - 10(-8) M) stimulated the formation of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, in culture media of ECs, whose stimulatory effect was inhibited by indomethacin, a cyclooxygenase inhibitor. These data suggest that ET-3 stimulates, in addition to EDRF (NO), the synthesis and release of prostacyclin. PMID- 1725312 TI - Scintillation proximity assay: competitive binding studies with [125I]endothelin 1 in human placenta and porcine lung. AB - Scintillation proximity assay (SPA) technology has been used to investigate the competitive binding of [125I]endothelin-1 (ET-1) by ET-1, endothelin-2 (ET-2), endothelin-3 (ET-3), sarafotoxin 6b (SFTX-b), and big endothelin (big ET) to endothelin receptors in human placenta and porcine lung. Specific binding of [125I]ET-1 to high-affinity receptors was detected in membranes coupled to wheat germ agglutinin (WGA)-coated beads impregnated with scintillant (fluomicrospheres). The binding characteristics of ET-1 were similar in both of the tissues studied. In contrast, the receptor-binding properties of ET-2, ET-3, and SFTX-b were different in human placental and porcine lung membranes. This suggests that different endothelin receptor populations exist in these tissues. In addition, the binding characteristics of ET-3 and SFTX-b differed markedly to those of ET-1 and ET-2 in both tissues. This may be due to the four amino acid residue substitution in the N-terminal loop of both ET-3 and SFTX-b. We have also demonstrated that the ET-1 precursor, big ET, competitively inhibits [125I]ET-1 binding in these tissues, but is 100-fold less potent than ET-1. PMID- 1725313 TI - Intracellular signaling pathway of endothelin-1. AB - The intracellular signaling pathway of endothelin-1 (ET-1) was studied in individual mesangial cells (MCs) and vascular smooth muscle cells (VSMCs) using microspectrofluorimetry of fura-2 ([Ca2+]i), SPQ ([Cl-]i), and bisoxonol (membrane potential). ET-1 elicited a five-fold increase in [Ca2+]i that showed immediate and sustained phases. Both the Ca(2+)-free medium and nifedipine pretreatment curtailed the sustained phase of the response to ET-1. ET-1 resulted in sustained membrane depolarization of MCs and VSMCs. This depolarization was not attributed to Na influx, as Na-free medium did not abolish it. A Cl(-) channel inhibitor, IAA-94, blunted the depolarization and sustained elevation of [Ca2+]i in response to ET-1. In aortic rings, both nifedipine and IAA-94 attenuated ET-1-induced contraction. No additivity in the effect of nifedipine and IAA-94 was detected. Studies of SPQ fluorescence changes induced by ET-1 revealed an immediate and sustained increase in fluorescence intensity consistent with the decrease in [Cl-]i. The sustained but not immediate increase in SPQ fluorescence was virtually abolished in Ca(2+)-free medium with or without pretreatment with the intracellular Ca2+ chelator BAPTA. In conclusion, we hypothesize that ET-1 results in Ca2+ mobilization and Ca(2+)-dependent and independent activation of Cl- channels. Ensuing Cl- efflux causes membrane depolarization and, in turn, activation of voltage-gated Ca2+ channels in MCs and VSMCs. The latter results in sustained elevation of [Ca2+]i that is indispensable for the full-scale contractile response to ET-1. PMID- 1725314 TI - Endothelin-1 receptor binding assay for high throughput chemical screening. AB - An endothelin-1 (ET-1) receptor-binding assay, in microtiter format, has been developed for use in high throughput chemical or natural product screening. A rat smooth muscle, clonal cell line derived from embryonic thoracic aorta and designated A10 has been shown to consistently express high-affinity ET-1 receptors. A 96-well microtiter filtration plate, with individual PVDF membranes attached to the bottom of each well, was used for separation. In saturation binding assays, using [125I]Tyr13-ET-1, Scatchard analysis was monophasic, indicating a single high-affinity population of receptors with Kd = 0.12 nM with approximately 40,000 receptors per cell. The Ki values for peptides, known to bind at the ET receptor, were as follows: ET-1, 0.14 nM; ET-2, 0.16 nM; sarafotoxin S6b, 0.6 nM; VIC, 0.2 nM; ET-3, 16 nM; and big human ET, greater than 1 microM. ET receptors on A10 cells were stable for at least 2 months when stored at -20 degrees C. The assay is suitable for automation, because it is stable and reproducible. This method gave a 90% reduction in radioactive waste compared to tissue homogenate assays that use glass fiber filtration and cell harvesters. PMID- 1725315 TI - Localization of endothelin-1 (ET-1), ET-2, and ET-3, mouse VIC, and sarafotoxin S6b binding sites in mammalian heart and kidney. AB - Quantitative in vitro receptor autoradiography was used to identify and compare binding sites for endothelin-1 (ET-1), ET-2, and ET-3, mouse vasoactive intestinal contractor (VIC), and sarafotoxin S6b in human, porcine, and rat cardiac and renal tissues. In the rat kidney, the highest densities of binding sites for all five labeled peptides were present in the glomeruli, with lower levels in the papilla, cortex, and inner band of the medulla. A similar pattern was found in porcine and human kidney except that no discrete glomerular binding could be detected. In the heart, higher densities were detected in the atria compared to the ventricle. Although the pattern of binding in cardiac and renal tissue was similar for all four labeled ET isoforms, nonspecific binding was consistently higher with ET-3, resulting in lower levels of specific binding. These results suggest that these peptides could be acting via the same population of binding sites, but multiple receptor subtypes may exist that differ in their rank order of affinity for the ET isoforms and sarafotoxin S6b, as proposed in other tissues. PMID- 1725316 TI - Dynamics of the secretory response evoked by endothelin-1 in adrenal chromaffin cells. AB - Recently, we have observed that endothelin-1 (ET-1) evokes the release of catecholamines (CA) from adrenal chromaffin cells. We have investigated this secretagogue action of ET-1 on cultured adrenal chromaffin cells using a real time monitoring system. Pulsatile exposure to ET-1 evoked transient secretory responses in a dose-dependent manner, whereas repetitive stimulation produced a rapid tachyphylaxis. This secretagogue activity was dependent on extracellular Ca2+ and was blocked by 85% by 10(-5) M nifedipine. The onset of the secretory response to ET-1 was very slow, and the duration of the response was much longer than that seen with acetylcholine (ACh). These novel dynamics may well be related to the mechanism of action of ET-1 in these cells. In addition, ET-1 produced a synergistic increase in ACh-evoked release, suggesting that ET-1 may physiologically modulate ACh-evoked CA release in adrenal chromaffin cells by a mechanism involving dihydropyridine-sensitive Ca2+ channels. PMID- 1725317 TI - Electrophysiological effects of endothelin-1 on canine myocardial cells. AB - Endothelin-1 (ET-1) has been shown to induce severe ventricular arrhythmias associated with myocardial ischemia. However, ET-1 may have a direct arrhythmogenic action that is not related to myocardial ischemia. To examine this possibility, we studied the electrophysiological effects of ET-1 on cardiac tissues. The right bundle branch, false tendon, ventricular muscle, and atrial muscle were isolated from the dog, and transmembrane potentials were recorded by the conventional microelectrode technique. ET-1 prolonged the action potential duration (APD) in all of the tissues tested except in the atrial muscle, where the APD was shortened. Bay K 8644, a calcium channel agonist, prolonged the APD in all cardiac tissues. Spontaneous firing of the right bundle branch was suppressed by ET-1 but not Bay K 8644. The prolongation of the APD by ET-1 was far more marked in the right bundle branch than in other tissues, and it was followed by the development of early after depolarizations (EADs) only in the right bundle branch. The EADs induced by ET-1 or Bay K 8644 were abolished by nicardipine. These data suggest that L-type calcium current is involved in the genesis of EADs by ET-1, although other ionic mechanisms can not be ruled out. Since EADs underlie some types of arrhythmias, arrhythmias caused by ET-1 are at least partly attributable to the direct actions of the agent on myocardial cells. PMID- 1725318 TI - Effect of endogenous digitalis-like factor on endothelin secretion from bovine endothelial cells. AB - We have recently isolated two endogenous digitalis-like factors (EDLFs) from human urine. The polar EDLF (ouabain-displacing compound 1; ODC-1) fulfills the criteria for the putative natriuretic and vasoactive digitalis-like factor. In this study, we examined the effects of ODC-1 and ouabain on endothelin-1 (ET-1) secretion from cultured endothelial cells. ODC-1 was partially purified from human urine with use of reverse-phase HPLC based on the inhibitory effects on [3H]ouabain binding to intact human erythrocytes. A serial dilution curve of the extract of the serum-free culture medium from CPAE (bovine pulmonary artery endothelium) cells was parallel to that of standard ET-1. Immunoreactive (ir-) ET 1 was secreted in a linear fashion over 5 h from CPAE cells. ODC-1 stimulated ET 1 secretion in a dose-dependent manner. In contrast, ouabain did not affect ET-1 secretion at the concentration of 10(-9)-10(-5) M. These results indicate that human urine-derived ODC-1 is distinct from plant-derived ouabain and acts as a stimulator of ET-1 secretion, and also suggest that ODC-1 may play an important role as a direct and indirect modulator of vascular tone. PMID- 1725319 TI - Differential inhibitory action of phorbol-12,13-dibutyrate on the positive inotropic effect of endothelin-1 and Bay K 8644 in the isolated rabbit papillary muscle. AB - Endothelin-1 (ET-1) elicited a concentration-dependent positive inotropic effect on the rabbit isolated papillary muscle (electrically driven at 1 Hz at 37 degrees C). The duration of isometric contractions was prolonged by ET-1 in a concentration-dependent manner mainly by prolongation of relaxation time. A tumor promoting phorbol ester, i.e., phorbol-12,13-dibutyrate (PDBu), inhibited selectively the positive inotropic effect of ET-1 at the concentration that it did not (10 nmol/L) or only slightly (10-20% at 100 nmol/L) reduced the basal force of contraction and the positive inotropic effect of Bay K 8644. The positive inotropic effect of 10 mumol/L of phenylephrine mediated via myocardial alpha 1-adrenoceptors (in the presence of 0.3 mumol/L of bupranolol) was likewise inhibited by PDBu in the same concentration range as it suppressed the ET-1 induced positive inotropic effect. PDBu at concentrations higher than 100 nmol/L inhibited the positive inotropic effects of Bay K 8644 and isoproterenol, and decreased the basal force of contraction in a concentration-dependent manner to a similar extent. Thus, PDBu exhibited selective and potent inhibitory action on the ET-1-induced and alpha 1-adrenoceptor-mediated positive inotropic effect (compared with that on the effect of the Ca2+ channel agonist Bay K 8644 and a beta-adrenoceptor agonist). The present findings indicate that ET-1 elicits a positive inotropic effect on the rabbit ventricular myocardium, the characteristics of which are similar to those of myocardial alpha-adrenoceptor activation, which may involve the phosphoinositide hydrolysis. PMID- 1725320 TI - Role of endothelium-derived relaxing factor in endothelin-induced renal vasoconstriction. AB - It has been suggested that endothelin (ET) induces the release of endothelium derived relaxing factor (EDRF). To explore the possible modification of ET induced renal vasoconstriction by EDRF, we examined the effects of ET on renal vascular resistance (RVR) and urinary Na excretion (UNaV) in the rat isolated perfused kidney before and after the administration of EDRF antagonists. ET at 2 x 10(-11) to 2 x 10(-9) M elevated the RVR in a dose-dependent fashion, whereas it lowered the RVR at 10(-12) M. ET decreased UNaV significantly only at the highest dose. Acetylcholine at 10(-7) M decreased the RVR (-19%, p less than 0.05) and increased UNaV (+177%, p less than 0.05). In contrast, a soluble guanylate cyclase inhibitor, methylene blue (MB; 10(-5) M), increased the RVF by 30% (p less than 0.05) and decreased UNaV by 48% (p less than 0.05). Pretreatment with MB significantly augmented the ET-induced renal vasoconstriction by about 80%. However, UNaV was not influenced significantly. ET increased the urinary excretion of prostaglandin (PG) E2 and 6-keto-PGF1 alpha. Pretreatment with indomethacin (10(-5) M) also significantly enhanced the response of RVR to ET by 60% without changing UNaV. These results suggest that the vasoconstrictor, but not the antinatriuretic, activity of ET may be modified by the release of EDRF and prostacyclin. PMID- 1725321 TI - Conversion of big endothelin isopeptides to mature endothelin isopeptides by cultured bovine endothelial cells. AB - Both the soluble and membrane fractions prepared from cultured bovine endothelial cells (ECs) possessed the converting activities to metabolize big endothelin-1 (big ET-1) to endothelin-1 (ET-1) at neutral pH. Metal chelators inhibited the activities of both fractions, whereas phosphoramidon, a metalloprotease inhibitor, strongly inhibited only the activity of the membrane fraction. Phosphoramidon reduced the secretion of ET-1 and concomitantly enhanced the release of big ET-1 from cultured ECs. The incubations of big ET-1, big ET-2, and big ET-3 with cultured ECs resulted in their conversions to mature ETs. Phosphoramidon also abolished these conversions. These results indicate that vascular endothelium can convert not only endogenous big ET-1 but also exogenous big ET isopeptides to their mature ETs through a phosphoramidon-sensitive neutral metalloprotease. PMID- 1725322 TI - Comparative effects of cyclosporine A and FK-506 on endothelin secretion by a cultured renal cell line, LLC-PK1. AB - Cyclosporine A (CSA) stimulated endothelin secretion by a cultured renal epithelial cell line, LLC-PK1. A less nephrotoxic immunosuppressant (FK-506) did not affect endothelin secretion. Putative endothelin converting enzyme inhibitors or specific receptor antagonists may therefore prevent CSA nephrotoxicity. PMID- 1725323 TI - Effects of ouabain and verapamil on endothelin-1-induced contraction of mesenteric artery in young spontaneously hypertensive rats. AB - We studied the effects of ouabain and verapamil on endothelin-1(ET-1)-induced contraction of the mesenteric artery in prehypertensive spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. Rings of mesenteric artery of SHRs and WKY rats aged 4 weeks were superfused with physiological saline solution and isometric tension was measured. ET-1 (10(-9) or 10(-8) M)-induced contractions were significantly larger in WKY rats than in SHRs. ET-1 (10(-7) M)-induced contractions were not significantly different between SHRs and WKY rats. Verapamil markedly inhibited ET-1 (10(-7) M)-induced contractions both in SHRs and in WKY rats. Although the percent inhibition tended to be larger in SHRs than in WKY rats, the difference was not significant. Ouabain significantly increased ET-1 (10(-7) M)-induced contraction in the presence of verapamil both in SHRs and in WKY rats. The increase was significantly greater in SHRs than in WKY rats. As a result, ET-1 (10(-7) M) induced contractions were significantly greater in SHRs than in WKY rats in the presence of verapamil and ouabain. These results suggest that in ET-1-induced arterial contraction, [Na+]i-mediated [Ca2+]i regulation such as the Na+/Ca2+ exchange system might be greater in SHRs than in WKY rats, and that this might be, at least in part, involved in the pathogenesis or maintenance of hypertension in SHRs. PMID- 1725324 TI - Inhibition of endothelin-1-induced DNA synthesis by prostacyclin and its stable analogues in vascular smooth muscle cells. AB - The effects of prostacyclin and its stable analogues on endothelin-induced DNA synthesis were investigated in cultured vascular smooth muscle cells. Aortic smooth muscle cells were obtained from male Wistar rats. In order to eliminate endothelial cells, the cells were cloned. DNA synthesis in intact cells was estimated by [3H]thymidine incorporation. Prostacyclin and its stable analogues (TEI-7165, TEI-9090, TEI-1324, TEI-3356, and TEI-9063) inhibited the endothelin induced DNA synthesis with an IC50 of 1-30 nM. These results indicate that prostacyclin and its stable analogues are possibly effective in preventing the proliferation of vascular smooth muscle cells under some pathological situations, including atherosclerosis. PMID- 1725325 TI - Synergistic coronary vasoconstriction produced by endothelin-1 in combination with 5-hydroxytryptamine. AB - The present study was undertaken to examine the action of endothelin-1 (ET-1) per se, or the combined effects of ET-1 with vasoconstrictor agonists such as acetylcholine (ACh), histamine (His), and 5-hydroxytryptamine (5-HT), all of which have been implicated in the genesis of coronary spasm. Isometric tension development and the cytosolic Ca2+ concentration [( Ca2+]i) in a ring segment of porcine coronary artery loaded with fura-2 were measured simultaneously with a mechanoelectric transducer and a fluorometer, respectively. ET-1 (30-100 pM) specifically potentiated the 5-HT-induced contraction without causing a significant increase in [Ca2+]i. This effect of the peptide seems to be qualitatively similar to that produced by the tumor-promoting phorbol ester, DPB. These results suggest that the combined stimulation of ET-1 and 5-HT augments the Ca2+ sensitivity of the contractile elements through the possible activation of protein kinase C. PMID- 1725326 TI - Endothelin-1 stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serum-free medium. II. AB - The effect of endothelin-1 (ET-1) on rat cardiac myocytes cultured in a serum free medium was determined. Cardiac myocytes cultured in our medium showed an increased rate of protein synthesis, morphological size, contraction rate, and Ca2+ uptake when ET-1 was added. These actions of ET-1 were inhibited by a protein kinase C inhibitor, H-7. Among these four phenomena, contraction and Ca2+ uptake were inhibited by a Ca2+ channel blocker, nicardipine. Therefore, it is likely that the signal for ET-1 to induce these phenomena is transduced by kinase C, and Ca2+ uptake is related to maintaining a high contraction rate. PMID- 1725327 TI - Cell growth-dependent expression of endothelin-1 provocable Ca2+ channels in cloned vascular smooth muscle cells. AB - To analyze the action of endothelin-1 (ET-1) on intracellular Ca ion ([Ca2+]i) handling, we have measured the [Ca2+]i transients in a cultured vascular smooth muscle cell (VSMC) line, A7r5, using two-dimensional microfluorophotometry. In the presence of 1 mM extracellular Ca2+ ([Ca2+]e), ET-1 (100 nM) induced a transient increase in [Ca2+]i due to Ca2+ release from sarcoplasmic reticulum and a subsequent sustained [Ca2+]i increase, suggestive of the entry via the voltage dependent Ca2+ channel. The transient phase was heterogenous in the VSMC population; only two-thirds of VSMCs showed the phase, whereas all cells showed the sustained phase. To elucidate the cause of heterogeneity, we measured the [Ca2+]i using VSMCs cultured under two extreme conditions. When the cell cycle was arrested at the quiescent phase in the defined serum-free medium, the transient phase was observed in most cells. In contrast, when cell growth was promoted by the above medium plus PDGF (50 ng/ml), the [Ca2+]i response was completely abolished, whereas both the voltage-dependent Ca2+ channel and vasopressin-operated Ca2+ mobilization mechanism were universally preserved, irrespective of the culture conditions. These results may imply that the ET-1 mediated Ca2+ release mechanism is closely influenced by cell growth and preferentially effective at the quiescent phase. PMID- 1725328 TI - The importance of tachyphylaxis and release of cyclooxygenase products in the modulation of endothelin-1-induced responses in rat and guinea pig tracheal chains. AB - Responses to endothelin-1 (ET-1) were studied in both rat and guinea pig tracheal chains in vitro. Indomethacin potentiated contractile responses to ET-1 in the guinea pig but had no effect in the rat. Furthermore, relaxation responses to ET 1 observed in some guinea pig preparations only were abolished by indomethacin. Studies using repeated full cumulative dose-response curves and repeated single dosing techniques demonstrated tachyphylaxis to ET-1 in both rat and guinea pig. However, the degree of tachyphylaxis in the guinea pig was dependent on the initial response to the peptide, and in single-dose studies in this species, tachyphylaxis to ET-1 was more prominent in the presence of indomethacin. It is suggested that both tachyphylaxis and inconsistent release of cyclooxygenase products may be responsible for the wide differences in potency estimates for ET 1 that have been observed in airways smooth muscle. PMID- 1725329 TI - Pharmacological analysis of the positive inotropic effect of endothelin-1 in guinea pig left atria. AB - Endothelin-1 (ET-1) increased the force of contraction and stimulated phosphoinositide hydrolysis in guinea pig left atria. ET-1 at 20 nM produced a dual-component positive inotropic effect composed of an initial increasing phase (early component) and a second and late developing, greater positive inotropic phase (late component). The late component was preferentially and markedly inhibited by nifedipine and the protein kinase C inhibitors H-7 and staurosporine. In guinea pig left atria, the activation of protein kinase C might contribute to the establishment of the positive inotropic effect of ET-1 mediated by modulation of voltage-dependent Ca2+ channels. PMID- 1725330 TI - Endothelin-3 inhibits ganglionic transmission at preganglionic sites through activation of endogenous thromboxane A2 production in dog cardiac sympathetic ganglia. AB - The effects of endothelin-3 (ET-3) on ganglionic transmission of dog cardiac sympathetic ganglia and possible mechanisms involved were investigated in vivo and in vitro. Positive chronotropic responses to preganglionic stellate stimulation and those to dimethylphenylpiperazinium as well as McN-A-343 administered to the ganglia were inhibited by ET-3. The amount of acetylcholine released by preganglionic stimulation was reduced dose dependently after exposure to ET-3. The reduction elicited by ET-3 was antagonized by pretreatment with phospholipase A2 inhibitors (dexamethasone and methylprednisolone) and cyclooxygenase inhibitors (aspirin and indomethacin). In addition, the reduction of acetylcholine release was similarly induced by exposure to exogenously applied STA2, a stable thromboxane A2 analogue; U-46619, a TXA2/PGH2 receptor agonist; and prostaglandin E2. Furthermore, the reduction produced by ET-3 was antagonized by pretreatment with a thromboxane A2 synthetase inhibitor (OKY-046) and a specific thromboxane A2 receptor antagonist (S-145), but not by a specific prostaglandin E2 receptor antagonist (SC-19220). These results indicate that ET-3 inhibits the sympathetic ganglionic transmission via reducing acetylcholine release from the presynaptic nerve terminals of ganglia and that this inhibition involves the activation of endogenous thromboxane A2 production. PMID- 1725331 TI - Characterization and partial purification of endothelin-converting enzyme in rat lung. AB - Endothelin-1 (ET-1) was originally identified in the culture supernatant of porcine aortic endothelial cells. From the deduced amino acid sequence, a biosynthetic pathway has been proposed that a prepro- form of porcine ET-1 is initially processed by dibasic pair proteolysis to a 39-amino acid intermediate form (big ET), which is then converted to ET-1 by specific proteolytic cleavage between Trp21 and Val22. We have identified an enzyme activity that converts human big endothelin[1-38] to endothelin[1-21] and a C-terminal fragment (CTF, 22 38) in a homogenate fraction from rat lung. The conversion activity was enriched threefold in a plasma membrane fraction. Metal ions activated the activity by about 1.5- to 2.5-fold, in the order of Mn2+ greater than Zn2+ = Ca2+ greater than Mg2+ greater than Ba2+. The conversion activity was optimal at pH 4.0, was inhibited by pepstatin-A (IC50 = 20 nmol), but not affected by TLCK, aprotinin, PMSF, E-64, bestatin, phosphoramidon, or thiorphan at 40 microM. The converting enzyme was partially purified from rat lung plasma membranes by sequential HPLC on Mono Q, Superose 12, and Mono P. The enzyme has an apparent molecular mass of 90 kDa as estimated by SDS-PAGE or gel filtration and appears to be a single peptide protein. The enzyme may exist as isozymes with isoelectric point (pI) values at 6.2 and 6.3. PMID- 1725332 TI - Endothelin-3 directly affects neurons in the anteroventral third ventricle region and supraoptic nucleus of the rat hypothalamus in vitro. AB - Extracellular recordings were made from anteroventral third ventricle (AV3V) and supraoptic nucleus (SON) neurons of rat hypothalamus in slice preparations. ET-3 was applied at concentrations of 10(-10) to 3 x 10(-7) M. Of 119 AV3V neurons tested, 21 (18%) were excited, 8 (6%) were slightly inhibited, and 90 (76%) were unaffected. The threshold concentration to evoke responses was approximately 10( 8) M. Excitatory responses of the AV3V neurons to ET-3 remained in a Ca(2+)-free medium. Of 120 SON neurons tested, 46 were phasic (putative vasopressin neuron) and 74 were nonphasic (putative oxytocin neuron). Of 46 phasic neurons tested, 26 (56.5%) were inhibited by ET-3, 20 (43.5%) were nonresponsive, and none was excited. Of 74 nonphasic neurons tested, 14 (19%) neurons were inhibited by ET-3, 60 (81%) were nonresponsive, and none was excited. These results suggest that ET 3 has dual effects on AV3V and SON neurons and the mechanisms of the responses may be different in the AV3V and SON neurons. PMID- 1725333 TI - Endothelin modulates L-NG-nitroarginine-induced enhancement of vasoconstriction evoked by norepinephrine. AB - The interaction of endothelin-1 (ET-1), a novel vasoconstrictor peptide synthesized according to the encoded amino acid sequence, with L-NG-nitroarginine (NOARG), an L-arginine-reversible inhibitor of endothelium-derived relaxing factor (EDRF) on adrenergic receptors, was investigated. Male Sprague-Dawley rats were used in this study. Mesenteric arteries were isolated and perfused with Krebs-Ringer solution at a constant flow rate. The vasoconstrictor responses to exogenous norepinephrine were determined. Low concentrations of ET-1 (10(-11) and 10(-10) M) potentiated the pressor responses to norepinephrine without affecting the baseline perfusion pressure. Simultaneous NOARG (10(-5) M) infusion with ET-1 (10(-11) M) into the mesenteric arteries caused further enhancement of the pressor responses to exogenous norepinephrine (200 ng) without affecting the baseline perfusion pressure: 126 +/- 15% to 262 +/- 45% (p less than 0.05, vehicle control: 100%). This facilitatory effect of NOARG on the pressor response to norepinephrine was attenuated with L-arginine (10(-4) M) infusion, but not D arginine. These results suggest that ET, one of the endothelium-derived vasoconstricting factors, interacts with EDRF to modulate the tone of resistance vessels. PMID- 1725334 TI - Functional and autoradiographic studies of endothelin-1 and endothelin-2 in human bronchi, pulmonary arteries, and airway parasympathetic ganglia. AB - We compared the contractile potency and efficacy of two of the forms of endothelin (endothelin-1 and endothelin-2) on human bronchi and pulmonary arteries in vitro and examined the effects of sarafotoxin on the bronchial preparations. In addition, we used autoradiographic methods to investigate the location of binding sites for both forms of endothelin within human lung. Endothelin-1 (ET-1) was 2.5 times more potent than endothelin-2 (ET-2) in both the airway and vascular tissues, and both forms of the peptide were 5 times more potent in the isolated pulmonary artery than in the bronchial tissue. There were no differences in the magnitude of the contractions generated by ET-1 and ET-2 in either tissue type. Sarafotoxin also produced a contractile response in the bronchi that was equipotent to ET-1 but of a greater magnitude. Specific binding on the smooth muscle of bronchus, pulmonary artery, and alveolar walls to both ET 1 and ET-2 was detected in autoradiographic studies. In addition, binding sites for both forms of the peptide were localized to parasympathetic ganglia within the airways, where the presence of muscarinic receptors was also confirmed with [3H]QNB. There appeared to be no difference between the endothelins in the location or the density of binding sites. These results indicate that endothelin may have a neuromodulatory role in the airways as well as a direct contractile action on the bronchial and arterial smooth muscle. PMID- 1725335 TI - Endothelin-1-induced relaxation of guinea pig trachealis muscles. AB - Our accompanying paper demonstrated that endothelin-1 (ET-1 constricts guinea pig airways directly and indirectly through mediators such as histamine and arachidonate metabolites. In order to exclude the role of mast cells in bronchoconstriction, we sensitized guinea pigs and challenged them in vitro with an antigen (ovalbumin). In the postanaphylactic trachea, ET-1 caused a transient relaxation followed by constriction. Such relaxation by ET was also observed in the tracheas constricted with carbamylcholine. The relaxation was completely blocked by nordihydroguaretic acid and AA861, lipooxygenase inhibitors, but not by indomethacin, a cyclooxygenase inhibitor, and FPL 55712, a leukotriene antagonist. Because the relaxation was not affected even in the presence of methylarginine, an inhibitor of NO synthesis, and superoxide dismutase, an enzyme for destroying NO radical, we concluded that ET-1 induces the relaxation of the tracheal muscles by producing lipooxygenase products, probably hydroperoxides of arachidonic acid. PMID- 1725336 TI - Multiple mechanisms of bronchoconstrictive responses to endothelin-1. AB - We investigated the mechanism of the endothelin-1 (ET-1)-induced bronchoconstriction of guinea pig tracheal smooth muscles. ET-1 contracted the tracheas in a dose-dependent manner. A combination of FPL55712 (leukotriene antagonist), diphenhydramine (histamine antagonist), and indomethacin (cyclooxygenase inhibitor) shifted the dose-response curve of ET-1 to the right and suppressed the maximal constriction. Azelastine, an antiallergic agent, exerted essentially similar results. The present data suggest that ET-1 constricts the airway smooth muscles not only by direct action on the tracheal smooth muscles but also by indirect action mediated through production of various chemical mediators in cells other than muscles. PMID- 1725337 TI - Antiaggregatory and hypotensive effects of endothelin-1 in beagle dogs: role for prostacyclin. AB - The effects of endothelin-1 (ET-1) on blood pressure and platelet aggregation were studied in anesthetized beagle dogs. Platelet aggregation was monitored in vivo by a filter-loop technique and in vitro by using platelet-rich plasma and whole blood. ET-1 (0.03-0.3 nmol/kg) induced a transient dose-dependent hypotension and inhibited platelet aggregation in vivo. These changes were accompanied by dose-dependent elevation of plasma 6-keto-PGF1 alpha levels. Pretreatment of animals with indomethacin (2 mg/kg plus 3 mg/kg/h) completely abolished the antiaggregatory action of ET-1 and significantly attenuated the hypotensive response to ET-1. Intra-arterial infusion of prostacyclin at concentrations similar to those observed following ET-1 injections mimicked the antiaggregatory and hypotensive actions of ET-1. ET-1 (0.1-100 nM) did not modify platelet aggregation induced by ADP, collagen, and platelet-activating factor in vitro, suggesting that dog platelets do not possess ET-1 receptors. These findings indicate that the antiaggregatory and hypotensive actions of ET-1 in beagle dogs are mediated through release of prostacyclin. PMID- 1725338 TI - Characterization of the effect of endothelins in canine cerebral arteries. AB - In a few populations of endothelial cells of dog basilar arteries, endothelin (ET)-like immunoreactivity was detected to be present. ET-1, ET-2, and ET-3 caused vasoconstrictor responses but not vasodilator responses in isolated ring preparations of dog cerebral arteries in vitro. The ED50 values for the contractions were 411 pM, 478 pM, and 26.5 nM for ET-1, ET-2, and ET-3, respectively. NiCl2 (10(-3) M) attenuated the contractions induced by ET-3 (10( 8)-3 x 10(-7) M) and those to relatively low doses (10(-9) M) but not higher doses (10(-8)-10(-7) M) of ET-1 and ET-2. The contractions in response to ET-1, ET-2, and ET-3 were greatly attenuated in Ca(2+)-free solutions, although high concentrations of ET-1 and ET-2 still evoked contractions. These results suggest that the vasoconstriction induced by ET-3 and lower doses of ET-1 and ET-2 largely depends on the influx of Ca2+ ions. Furthermore, additional distinct mechanisms may contribute to the vasoconstrictor effects of high concentrations of ET-1 and ET-2. The presence of endothelin-like immunoreactivity in endothelial cells suggests that endothelin is a potential endogenous spasmogen. PMID- 1725339 TI - Mechanisms of endothelin-1 mRNA and peptides induction by TGF-beta and TPA in MDCK cells. AB - Mechanisms of prepro-ET-1 mRNA expression and mature endothelin-1 (ET-1) peptide secretion in MDCK cells (dog collecting duct origin) were investigated. MDCK cells constitutively expressed prepro-ET-1 mRNA (approximately 2.3 kb). TGF-beta time-dependently increased prepro-ET-1 mRNA levels between 30 min and 6 h. Induction of the mRNA 6 h following TGF-beta addition was dose-dependent with a half-maximal concentration of 10 pM. The half-life of prepro-ET-1 mRNA was 15 min in controls when the cells were treated with 10 micrograms/ml of actinomycin D, whereas it was extended to 30-45 min by TGF-beta treatment. Prepro-ET-1 mRNA was rapidly (15-30 min) induced by 0.5 microM TPA, one of the phorbol esters, but downregulated below baseline after 1 h. Our data show that MDCK cells constitutively secrete ET-1 and increase its production in response to TGF-beta. An axis of TGF-beta-ET-1-collecting duct may play an important role in regulation of electrolyte transport and cell growth of the renal tubules. PMID- 1725340 TI - Polarized secretion of endothelin-1 and big ET-1 in MDCK cells is inhibited by cell Na+ flux and disrupted by NH4Cl. AB - We recently found that TGF-beta increases ET-1 secretion in MDCK, a renal tubular cell line. The secretion of ET-1, by a confluent monolayer of MDCK cells grown on a filter, to the basolateral side of the epithelium was two times greater than that to the apical side. However, TGF-beta-increased ET-1 and big ET-1 secretion were exclusively released to the basolateral side. We investigated the mechanism of polarized secretion of ET-1 and big ET-1 in MDCK cells. After 24 h of incubation with 80 pM TGF-beta, basolateral secretion of both ET-1 and big ET-1 increased two to threefold without a significant increase on the apical side. TGF beta-stimulated ET-1 and big ET-1 secretion in the basolateral bath were inhibited when 10 microM amiloride (Na+ channel blocker) or 1 microM ouabain (Na+, K(+)-ATPase inhibitor) was added for 3 h. Polarized secretion of ET-1 and big ET-1 was not affected. In contrast, 10 mM NH4Cl or 0.2 mM chloroquine (both lysosomal function inhibitors) reduced TGF-beta-stimulated ET-1 secretion in the basolateral bath whereas big ET-1 secretion in the apical bath increased two times. Thus, the polarity of big ET-1 secretion was reversed. In addition, the conversion rate from big ET-1 to ET-1 was significantly decreased from 80 to 55% by inhibiting lysosomal function. Prepro-ET-1 mRNAs 3 h after these perturbations were virtually unaltered. Our data show that ET-1 and big ET-1 secretion may be regulated by cell Na+ flux and that lysosomal functions may play some roles in the conversion of big ET-1 to mature ET-1 and in polarized secretion of big ET-1. Translational or posttranslational regulation may play an additional role in ET-1 secretion as well as the mRNA level. PMID- 1725341 TI - Effects of endothelin-1 and endothelin-3 on the release of prostanoids from isolated perfused rat kidney. AB - Endothelin-1 (ET-1) or endothelin-3 (ET-3) injected (0.1-100 pmol) into the renal artery of the isolated perfused rat kidney resulted in a dose-dependent increase in perfusion pressure (delta PP). In this respect, ET-1 was 30 times more potent than ET-3. Infusion of ET-1 (3 nM) for 3 min induced a pronounced and long lasting rise in delta PP, in contrast to a transient increase in delta PP after a 3-min infusion of ET-3 (10 nM). The pressure effect of endothelins was associated with the appearance of 6-keto-PGF1 alpha and PGE2 but not TXB2 in the perfusate. ET-1 produced a transient release of comparable amounts of prostacyclin and PGE2. In contrast to ET-1, ET-3 evoked a sustained release of prostacyclin and PGE2 at a ratio 3:1. It is assumed that PGE2 contributed to the vasoconstrictor responses of the endothelins in the rat kidney because indomethacin (5.6 microM) attenuated the pressor responses induced by ET-1 to a larger extent than that induced by ET 3. These results are in line with earlier observations that PGE2 is a vasoconstrictor in rat kidney. The differential release of prostanoids by ET-1 and ET-3 suggests the existence of separate receptors for ET-1 and ET-3 in the rat kidney. ET-3 appears to be a good candidate for an endogenous regulator of the release of prostacyclin. PMID- 1725342 TI - Activation of guinea pig alveolar macrophages by endothelin-1. AB - Endothelin-1 (ET-1) induced a concentration- and time-dependent increase in arachidonic acid release from guinea pig alveolar macrophages, a maximum being reached at 1 nM ET-1. Regarding the time-course study, maximal release of arachidonic acid was observed after 30 s of incubation with ET-1 and then followed by a marked decrease, suggesting a reuptake of free arachidonic acid. ET 1 (0.1-10 nM) induced a concentration- and time-dependent release of TXB2, the stable metabolite of TXA2, evaluated by an ELISA technique. Maximum release was also obtained with 1 nM ET-1 but only after a 1-min incubation period. By contrast, ET-1 (1 pM-1 microM) did not stimulate the release of superoxide anion at any time point of the kinetic study. These results suggests that ET-1 may activate guinea pig alveolar macrophages, probably through a mechanism involving the arachidonic acid pathway. PMID- 1725343 TI - Effect of endothelin-1 on responses of isolated blood vessels to vasoconstrictor agonists. AB - The effect of endothelin-1 (ET-1) on vasoconstrictor responses of the endothelium denuded perfused rabbit ear artery to 5-hydroxytryptamine (5-HT), histamine, and vasopressin (VP) was studied. In low concentrations with no vasoconstrictor action (0.1 and 0.3 nM), and in a concentration that increased the perfusion pressure by 70 mm Hg (1 nM), ET-1 significantly enhanced responses to the agonists studied; this enhancement was apparently mediated by an increased influx of extracellular calcium through voltage-operated channels, as it was abolished by the calcium channel antagonist nicardipine (10 nM). In contrast, higher concentrations of ET-1 (3 and 10 nM) inhibited responses to VP and 5-HT and this inhibitory effect was accentuated in the presence of nicardipine. The possible mechanism by which ET-1 exerts this inhibitory effect on vasoconstrictor responses is discussed. Because ET-1 is released from endothelial cells that are immediately adjacent to vascular smooth muscle cells, these modulatory effects of ET-1 on responses to endogenously present vasoconstrictors may play a role in vascular function. PMID- 1725344 TI - Effect of endothelin-1 on the vascular smooth muscle cell cycle. AB - Rabbit vascular smooth muscle cells were cultured in various concentrations of endothelin-1 (ET-1) at 37 degrees C for 24 h. The vascular smooth muscle cell cycle distribution was determined by flow cytometry according to DNA content. In the control group, the mitotically active phase (S + G2 + M phase) of vascular smooth muscle cells was 11.94%. In ET-1-cultured smooth muscle cells, the mitotically active phase was 11.69% at a concentration of 1 pM ET-1, whereas the mitotically active phases were 18.98 and 26.91% at concentrations of 0.1 and 10 nM ET-1, respectively. The findings show that ET-1 significantly increased mitotic activity of cultured vascular smooth muscle cells. PMID- 1725345 TI - Functional study and immunocytochemical identification of endothelin in cultured epididymal cells and intact epididymis of the rat. AB - Endothelin, a novel potent vasoconstrictor peptide produced by vascular endothelial cells, stimulated anion secretion by a cultured secretory epithelium derived from the rat epididymis as measured by changes in short-circuit current (SCC). Stimulation of the SCC was observed when endothelin was added to the basolateral or the apical side of the epithelium. The response to basolateral application was greater than that to apical application. The EC50 values were found to be 1.3 and 3.0 nM for basolateral and apical application, respectively. These values were about one-half to one order of magnitude higher than that required for its vasoconstrictor action. The stimulation of SCC by endothelin was likely to be due to an increase to anion secretion as removal of Cl from the incubation medium markedly reduced the SCC response to endothelin. Diphenylamine 2-carboxylate (DPC, 0.1 mM), a Cl-channel blocker, added to the apical side also inhibited the endothelin-induced rise in SCC. The stimulation of SCC by endothelin was accompanied by a rise in the intracellular cyclic AMP content in epididymal monolayers. Immunofluorescence staining has shown the presence of immunoreactive endothelin-like compound in the interstitium and epithelial cells of the rat epididymis. It is speculated that endogenous endothelin plays an important role in the control of water and electrolyte transport in the epididymis. PMID- 1725346 TI - Endothelin-1 stimulates cyclic GMP formation in porcine kidney epithelial cells via activation of the L-arginine-dependent soluble guanylate cyclase pathway. AB - Endothelin-1 (ET-1) elevated cyclic GMP levels in cultured porcine kidney epithelial cells (LLC-PK1) in a concentration-dependent manner with an EC50 value of about 5 x 10(-10) M. NG-methyl-L-arginine and NG-nitro-L-arginine inhibited cyclic GMP responses to 10(-8) M ET-1 with IC50 values of 1.2 x 10(-6) and 7.6 x 10(-8) M, respectively, and the inhibition was prevented with L-arginine. ET-1 induced cyclic GMP accumulation was enhanced with superoxide dismutase and diminished with oxyhemoglobin and methylene blue. Furthermore, the effect of ET-1 on the cyclic GMP levels was totally dependent on extracellular Ca2+. ET-3, but not big ET-1 and ET C-terminal hexapeptide16-21, elicited similar cyclic GMP responses as observed with ET-1 at the same concentration range. These data strongly suggest that, in LLC-PK1 cells, ET-1 stimulates formation of an endothelium-derived relaxing factor-like substance from L-arginine in a Ca(2+) dependent fashion, which in turn activates soluble guanylate cyclase to elevate cellular cyclic GMP levels. The effects of ET on cyclic GMP accumulation in the kidney epithelial cells may be related to the natriuretic effects of ET in vivo. PMID- 1725347 TI - Human big endothelin releases prostacyclin in vivo and in vitro through a phosphoramidon-sensitive conversion to endothelin-1. AB - Human big endothelin (big ET) and endothelin-1 (ET-1) induce similar increases in left ventricular systolic pressure in the anesthetized rabbit. Unlike ET-1, human big ET does not induce an initial transient hypotension. Human big ET (3 nmol/kg) inhibits ADP-induced platelet aggregation ex vivo by 60% whereas ET-1 at 1 nmol/kg inhibits platelet aggregation by more than 80%. The C-terminal fragment, big ET[22-38] (3 nmol/kg), has no antiaggregatory properties. Inhibition of ex vivo platelet aggregation by human big ET and ET-1 was not seen in rabbits pretreated with indomethacin (5 mg/kg). Human big ET (10(-7) M) or ET-1 (2.5 x 10(-9)-10(-8) M) induced the release of prostacyclin (PGI2) from rabbit, guinea pig, and rat lungs. Phosphoramidon (50 microM, infused 45 min prior to and during administration of peptides) inhibited the prostanoid-releasing properties of human big ET without affecting the release induced by ET-1. Intravascular administration of human big ET (1 nmol/kg) significantly increased the circulating levels of immunoreactive ET-1 (ir-ET-1) for 30 min whereas administration of ET-1 at the same concentration increased the plasma level of ir ET-1 for 5 min only. Our results suggest that human big ET is converted to ET-1 in the rabbit in vivo. We further suggest that to induce the release of prostanoids in perfused lungs, human big ET needs to be converted to ET-1 by a phosphoramidon-sensitive endothelin-converting enzyme (ECE). PMID- 1725348 TI - Endothelin-1 and endothelin-3 stimulate ovarian steroidogenesis. AB - The possible direct involvement of endothelin (ET) in preovulatory follicular steroidogenesis was investigated in vitro. Follicular development in immature rats was induced by pregnant mare's serum gonadotropin (PMS). Twenty-four and 48 h after PMS injection, ovarian follicles were incubated in vitro with or without 4 x 10(-11) to 4 x 10(-5) M endothelin-1 (ET-1) or endothelin-3 (ET-3) for 2 h, or perifused in vitro with 4 x 10(-7) M of ET-1 or ET-3 for 3 h. In the incubation study, ET-1 and ET-3 significantly stimulated progesterone, testosterone, and 17 beta-estradiol production by follicles 24 and 48 h after PMS injection. Progesterone, testosterone, and 17 beta-estradiol were increased in a dose-dependent manner by 4 x 10(-11) to 4 x 10(-6) M ET-1 or ET-3; in the case of ET-3, this increase occurred only at 24 h after PMS injection. In addition, ET-1 stimulated ovarian steroid production more effectively than ET-3. In addition to their effects on ovarian steroidogenesis, both ET-1 and ET-3 increased tissue cyclic adenosine-3',5'-monophosphate (cyclic AMP) production. The steroidogenic effect of ET-1 or ET-3 was most pronounced by follicles 48 h after PMS injection. In the perifusion experiment, ET-1 and ET-3 also stimulated progesterone, testosterone, and 17 beta-estradiol secretion. These indicate the involvement of ET-1 or ET-3 in the regulation of ovarian differentiation by stimulating steroidogenesis, at least partially through the mediation of cyclic AMP. PMID- 1725349 TI - Endothelin-converting enzyme and its in vitro and in vivo inhibition. AB - We investigated endothelin (ET)-converting enzyme and its localization in the vasculature. The membrane and cytosol fractions of cultured endothelial cells of bovine carotid artery contain phosphoramidon-sensitive ET-converting enzymes, and their molecular weights are about 100 and 540 kDa, respectively. The specific conversion of big ET-1 by these enzymes proceeds at pH 7.0 +/- 0.5, and it is inhibited by EDTA, o-phenanthroline, and phosphoramidon. Big ET-3 is converted by the membrane enzyme at a rate about one-tenth that of big ET-1, but it is not converted by the cytosol enzyme. Big ET-1 (but not ET-1)-induced hypertension in rats was remarkably suppressed by pretreatment with phosphoramidon, and big ET-1 (but not ET-1)-induced contraction of isolated coronary arteries, either with or without the endothelium, was substantially suppressed by phosphoramidon. These results suggest an essential role of phosphoramidon-sensitive enzyme(s) in the vascular conversion of big ET-1, and the existence of such enzymes also in nonendothelial cells. We found three converting enzymes operating at different optimal pH values in noncultured vascular smooth muscle cells; two pepstatin sensitive, cytosolic acid proteinases and a phosphoramidon-sensitive neutral enzyme(s) in the membrane and cytosol. All of these findings strongly suggest the importance of phosphoramidon-sensitive neutral enzymes in the vascular conversion of big ET-1. PMID- 1725350 TI - The role of cerebral microvessel endothelium in regulation of cerebral blood flow through production of endothelin-1. AB - We have previously reported the production of endothelin-1 (ET-1) by the cerebral microvessel endothelia and suggested an important role of ET-1 and microvessel endothelia in the regulation of local blood flow within the brain. In the present study, radioimmunoassay of ET-1 revealed that ET-1, produced by cultured cerebral microvessel endothelia grown on a filter, is released mainly to the basal side (corresponding to the basement membrane side), not to the apical side (corresponding to the vascular lumen side). This might indicate that ET-1 constricts arterioles locally at the same place where it is produced by endothelia. The present results also show that cultured cerebral microvessel endothelia produce less ET-1 under low oxygen pressure and that they produce more ET-1 under low carbon dioxide pressure. Taken together, our results may suggest that the negative feedback regulation of cerebral blood flow through oxygen and carbon dioxide pressure is mediated by ET-1 produced locally by cerebral microvessel endothelia. PMID- 1725351 TI - Differential effect of cyclic GMP on the release of endothelin-1 from cultured endothelial cells and intact porcine aorta. AB - The effect of the inhibition of the release of endothelium-derived nitric oxide (NO) by L-NG-monomethylarginine (L-NMMA) on the production of endothelin-1 (ET-1) was studied both in intact porcine aorta and in cultured porcine aortic endothelial cells. The basal production of endothelin was not affected by L-NMMA or by SIN-1, but was significantly increased in the presence of thrombin under both experimental conditions. In the intact porcine aorta, the thrombin stimulated release of the peptide was potentiated in the presence of L-NMMA, demonstrating that the thrombin-stimulated release of NO downregulates the concomitant production of endothelin. In contrast, in cultured endothelial cells obtained from the porcine aorta, L-NMMA did not affect the thrombin-stimulated release of the peptide, suggesting that the regulatory mechanism in cultured endothelial cells is not linked to the production of ET-1. PMID- 1725352 TI - The basal and stimulated release of EDRF inhibits the contractions evoked by endothelin-1 and endothelin-3 in aortae of normotensive and spontaneously hypertensive rats. AB - Endothelin-1 (ET-1) evoked concentration-dependent contractions that were slow in onset and sustained in aortae from both normotensive (Wistar and Wistar-Kyoto rats) and spontaneously hypertensive rats. The presence of a functional endothelium reduced the contractions evoked by low concentrations of ET-1 in the aortae from normotensive rats and shifted the concentration-contraction curves to the right in the hypertensive rat. NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide (NO) synthase, inhibited the influence of the endothelium. Endothelin-3 (ET-3) evoked contractions in aortae from both normotensive and hypertensive rats at concentrations greater than 3 x 10(-8) M, which were reduced by the presence of a functional endothelium. ET-1 and ET-3 evoked concentration- and endothelium-dependent relaxations in aortae contracted submaximally with phenylephrine, from both types of rats. The relaxations were reversed by methylene blue, an inhibitor of soluble guanylate cyclase, and nitro L-arginine, a competitive inhibitor of NO synthase. These observations demonstrate that the endothelium modulates the contractile response evoked by ET 1 and ET-3 in the aorta of the rat. This inhibition is more pronounced in aortas from hypertensive compared to normotensive rats and is mediated, at least in part, by an enhanced production of endothelium-derived NO. PMID- 1725353 TI - Modulation of norepinephrine-induced vasoconstriction by endothelin-1 and nitric oxide in rat tail artery. AB - The effects of endothelin-1 (ET-1) and an inhibitor of the synthesis of nitric oxide (NO) have been examined on vasoconstrictor responses to nerve stimulation and norepinephrine in the rat isolated perfused tail artery. In endothelium denuded preparations, a 60-min exposure to 0.3 nM ET-1 had no effect on the basal perfusion pressure, but significantly enhanced responses to stimulation (1 Hz, 10 s) and norepinephrine (10 ng) to 124 +/- 9% (n = 6) and 139 +/- 14% (n = 8), respectively, of control responses. In endothelium-intact preparations, inhibition of NO synthesis by NG-nitro-L-arginine (NOLA, 10 microM) enhanced responses to stimulation (5 Hz, 10 s) and norepinephrine (10 ng) to 171 +/- 12% (n = 6) and 222 +/- 9% (n = 4), respectively, of control responses. The NOLA induced enhancements were prevented by 100 microM L-arginine but not by 100 microM D-arginine, and did not occur in endothelium-denuded arteries. In other experiments, segments of rat tail artery were perfused in a low-volume (3.5 ml) recirculating system. The basal perfusion pressure was consistent for at least 60 min. In endothelium-denuded segments, vasoconstrictor responses to nerve stimulation (0.5 Hz, 10 s) or norepinephrine (10 ng) remained constant. However, in endothelium-intact segments, responses to stimulation and norepinephrine gradually increased to 158 +/- 13% (n = 6) and 152 +/- 8% (n = 5), respectively, of initial responses, after 45 min of recirculation. The increases were not related to NO, as they remained unchanged in the presence of 10 microM NOLA. Thus, it appears that a vasoconstriction-enhancing factor, possibly ET-1, is released from endothelial cells and accumulates in the perfusion fluid. PMID- 1725354 TI - Effects of endothelin-1 on coronary microcirculation in isolated beating hearts of rats. AB - An intravital fluorescence videomicroscope system was used to investigate the pharmacological effects of endothelin-1 (ET-1) on the coronary microcirculation in isolated beating hearts of rats. The heart was perfused by retrograde aortic steady flow with an oxygenated Krebs-Ringer solution containing FITC-dextran. Cumulative injections of ET-1 (1-30 pmol) elicited a dose-dependent increase in perfusion pressure from 52 +/- 15 mm Hg (mean +/- SEM; n = 6) before the ET-1 injection to 104 +/- 23 mm Hg at the ET-1 dose of 30 pmol. A dose-dependent narrowing of microvessels was also observed on a monitor screen. This diffuse vasoconstriction was especially prominent in small arterioles; the maximum vasoconstriction of the smaller arterioles was significantly higher than that of the larger arterioles. These findings suggest that ET-1 may have an important role in governing the coronary resistance and regulating the capillary flow in myocardium. PMID- 1725355 TI - Involvement of platelet-activating factor in endothelin-induced vascular smooth muscle cell contraction. AB - This study was designed to elucidate the participation of platelet-activating factor (PAF) in endothelin-induced vascular constriction in vivo and in vitro. The microvascular hemodynamic changes in rat mesentery induced by the superfusion of endothelin-1 (ET-1) were visualized through an intravital microscopic system. It was revealed in vivo that ET-1 in a range of 100 fM-10 pM caused a sustained arteriolar constriction in a dose-dependent manner. Pretreatment with CV-6209, a selective PAF antagonist, significantly attenuated the constrictive change in arterioles. Changes of intracellular Ca2+ mobilization after treatment with ET-1 were investigated in vitro using a cell line (A-10 cell) derived from rat arterial smooth muscle cells. ET-1 caused a prompt rise in the fura-2-associated fluorescence intensity in the individual A-10 cell and it fell to a lower plateau level that was still higher than the baseline value. CV-6209-pretreated cells did not show the rapid-phase mobilization of Ca2+, but showed the slow late phase of Ca2+ activation. The present study demonstrates that PAF may be involved in endothelin-induced microvascular constriction by mediating the mobilization of Ca2+ in vascular smooth muscle cells. PMID- 1725356 TI - Pharmacology of endothelin-1 in vivo in humans. AB - Endothelin-1 (ET-1) is a potent vasoconstrictor and pressor agent with a prolonged action in animal preparations. We have investigated the vascular pharmacology of ET-1 in healthy human volunteers. In arterial studies, blood flow was measured in both forearms and the left brachial artery cannulated for infusion of drugs and vehicle. Local blood flow was reduced in a dose-dependent manner by ET-1. At 5 pmol/min, the response was maximal by 60 min, and flow returned to baseline only after a further 120 min. ET-1 and angiotensin II, both at 5 pmol/min, reduced flow by 40% at 60 min. These responses were reversed to a similar degree by the dihydropyridine calcium antagonist nicardipine (0.3-10 micrograms/min). At 1 pmol/min, ET-1 did not affect sympathetic vasoconstriction to arterial norepinephrine or lower body negative pressure. In venous studies, the internal diameter of a dorsal hand vein was measured during local infusion of drugs and vehicle. ET-1 (5 pmol/min) produced local venoconstriction, with a maximal reduction in vein size of 83 +/- 12% at 60 min. Reversal of responses was slow, and unaffected by nicardipine (1.5 micrograms/min). We have shown that ET-1 is a potent vasconstrictor producing prolonged effects in resistance and capacitance vessels in humans. Low-dose ET-1 does not appear to affect sympathetically mediated vasoconstriction. Studies with nicardipine do not provide support for the hypothesis that ET-1 is an endogenous ligand of the dihydropyridine-sensitive calcium channel. PMID- 1725357 TI - Effects of intracerebroventricular and intravenous injections of endothelin-1 on blood pressure and sympathetic activity in urethane-anesthetized rats. AB - Because endothelin-1 (ET-1) is believed to be an endogenous calcium-channel agonist, it was thought that it may affect not only vascular smooth muscle cells but also nerve activity. Intravenous injections of ET-1 (0.01 nmol) did not affect cardiovascular parameters or renal sympathetic activity. ET-1 (1 nmol) initially decreased blood pressure for a few minutes, and then caused an increase for 5-10 min. Blood pressure then returned to baseline but later there was a rise in pressure for more than 60 min. Sympathetic activity was markedly suppressed during the initial hypertensive phase but increased during the later phase. Intracerebroventricular injections of ET-1 increased blood pressure even with the small dose (0.01 nmol). The increase was maintained for about 10 min, and returned to baseline. It then decreased abruptly with marked bradycardia, and finally returned to the baseline 60-90 min later. These results indicate that ET 1 affects not only the vascular smooth muscle but also the autonomic nervous system to influence cardiovascular functions. PMID- 1725358 TI - Phosphoramidon blocks the pressor activity of big endothelin[1-39] and lowers blood pressure in spontaneously hypertensive rats. AB - In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET 1) is formed from a 39-amino acid intermediate big endothelin (big ET) by a putative endothelin-converting enzyme (ECE) that cleaves the 39-mer at the Trp21 Val22 bond. Because big ET has less than 1% of the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of big ET. Big ET injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET to ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were ineffective at blocking the pressor response to big ET. However, the metalloprotease inhibitors phosphoramidon and thiorphan both dose-dependently inhibited the pressor response to big ET, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on blood pressure when administered alone as an i.v. bolus to the ganglion blocked anesthetized rat. However, phosphoramidon infused intravenously at 20 mg/kg/h for 4 h lowered the mean arterial pressure (MAP) in conscious spontaneously hypertensive rats (SHRs) whereas kelatorphan at the same dose did not. Our results suggest that ECE is a novel metalloprotease and that ECE inhibitors could have therapeutic potential for the treatment of hypertension. PMID- 1725359 TI - Role of endogenous endothelin in renal function during altered sodium balance. AB - This study was conducted to determine the involvement of endogenous endothelin-1 (ET-1), a potent vasoconstricting peptide, in systemic and renal hemodynamics and in the renin-angiotensin system, by inhibiting ET-1 action with an infusion of specific ET-1 antiserum during altered sodium balance. Infusion of 1:50 diluted ET-1 antiserum, which completely inhibited renal vasoconstriction caused by exogenously administered ET-3 (0.25 to 1.0 nmol/kg), increased urinary sodium excretion (UNaV) and fractional excretion of sodium (FENa), and decreased the plasma renin concentration (PRC) without significant changes in blood pressure, heart rate, GFR, RPF, or urine volume (UV) in conscious rats fed a low-salt diet, but not those on a high-salt diet. A time-control study showed no significant changes in any of these parameters. These results suggest that during low salt intake, in comparison to high salt intake, endogenous ET is more potent. Endogenous ET may thus contribute to the adaptive modulation of sodium excretion by a renal tubular action, and of renin release in association with a change in sodium balance. PMID- 1725360 TI - Hemodynamic responses to sarafotoxin 6 peptides. AB - Hemodynamic responses to sarafotoxin (SFTX6) peptides and endothelin-1 (ET-1) were compared in the anesthetized cat. SFTX6a and ET-1 at a dose of 0.3 nmol/kg i.v. produced a biphasic change in arterial pressure characterized by an initial decrease followed by a secondary increase in pressure. In contrast, similar doses of SFTX6b and SFTX6c produced mainly decreases in arterial pressure. Each peptide produced increases in central venous pressure, pulmonary arterial pressure, left atrial pressure, and cardiac output. Secondary decreases in cardiac output could be observed in response to SFTX6a and ET-1. SFTX6a and ET-1 produced biphasic changes, whereas SFTX6b and SFTX6c produced only decreases in systemic vascular resistance. In contrast, each peptide produced biphasic changes in pulmonary vascular resistance. The initial fall in pulmonary vascular resistance may be passively related to an increase in pulmonary blood flow because it occurs at a time when cardiac output is increased and no initial fall in pulmonary arterial pressure is observed. The present data show that the SFTX6 peptides can produce different patterns of responses in the systemic vascular bed of the cat, whereas responses in the pulmonary vascular bed are similar. PMID- 1725361 TI - Blood pressure responses to intravenous or intracerebroventricular endothelin-1 in spontaneously hypertensive rats. AB - Responses of blood pressure to intravenous (i.v.) or intracerebroventricular (i.c.v.) endothelin-1 (ET-1) in spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were investigated. Conscious male SHRs and WKY rats were used for the experiments. ET-1 (300-1,000 ng/kg) was injected intravenously in the i.v. experiments, or 30 and 100 ng/kg of ET-1 were injected into the lateral ventricle in the i.c.v. experiments. The initial depressor response to i.v. ET-1 was greater in SHRs (n = 10) than in WKY rats (n = 10) at 1,000 ng/kg (-54 +/- 6 vs. -41 +/- 3 mm Hg, mean +/- SEM, p less than 0.05). The subsequent pressor response to i.v. ET-1 was smaller in SHRs than in WKY rats (3.5 +/- 0.8 vs. 7.0 +/- 0.9 mm Hg at 300 ng/kg, p less than 0.05, and 11 +/- 2 vs. 17 +/- 2 mm Hg at 1,000 ng/kg, p less than 0.05). Pressor responses to i.c.v. ET-1 were not different in SHRs (n = 8) and WKY rats (n = 8) at both doses (10 +/- 4 vs. 10 +/- 3 mm Hg at 30 ng/kg, and 46 +/- 10 vs. 49 +/- 10 mm Hg at 100 ng/kg). A greater initial depressor response and a smaller subsequent pressor response to i.v. ET-1 were observed in SHRs than in WKY rats. The attenuated pressor response to intravenously administered ET-1 may be unique since vasoconstrictor responses to other known vasoactive substances such as angiotensin, catecholamines, or vasopressin are reportedly augmented in SHRs. We did not find any difference in responsiveness to i.c.v. ET-1 between SHRs and WKY rats. PMID- 1725362 TI - Subepicardial microischemia formation induced by epicardial application of endothelin-1. AB - The effects of endothelin-1 (ET-1), applied topically on the epicardium, on the coronary microcirculation of the beating canine heart were investigated in situ. ET-1 (1, 10, 100, and 1,000 pmol) induced a dose-dependent elevation of the ST segment in the epicardial ECG (n = 5). After application of ET-1 (100 pmol), the beating hearts were rapidly cross-sectioned and freeze-clamped in 120 ms using a specially developed device (n = 6). By NADH fluorescence photography of the cross sectioned frozen sample, an increased fluorescent area in the subepicardium was clearly observable. The fluorescent dye injected from the left atrium was negative in the NADH fluorescent area. It can be concluded that ET-1, when administered extravascularly, induces severe vasoconstriction and myocardial ischemia in the microcirculation of the beating canine heart. PMID- 1725363 TI - Differential effects of endothelin-1 and big endothelin on canine kidneys. AB - Renal effects of endothelin-1 (ET-1, human, 1-21) and the equivalent molar concentration of big ET (human, 1-38) and the contributions of prostaglandins and endothelium-derived relaxing factor to these actions were examined. Intrarenal infusion of ET-1, at a dose of 0.4 pmol/kg/min, decreased renal blood flow (RBF), glomerular filtration rate (GFR), and urine output (V) to 62, 70, and 45% of control values, respectively. Additional aspirin-DL-lysine (ASP) treatment (25 mg/kg) caused marked decreases in RBF, GFR, and V to 20, 14, and 9% of control values, respectively. During ET-1 infusion, intrarenal infusion of NG-monomethyl L-arginine (L-NMMA, 1 mumol/min) caused decreases in RBF, GFR, and V. In another group, intrarenal infusion of big ET at a dose equivalent to that of ET-1 did not significantly affect RBF, GFR, or urine output, which were 95, 107, and 84% of control values, respectively. Additional ASP treatment decreased these values to 78, 69, and 52% of control values, respectively. L-NMMA infusion during big ET infusion showed little effect on renal function. Our study demonstrated that intrarenal infusion of ET-1, at a dose of 0.4 pmol/kg/min, caused significant hemodynamic and functional changes in the canine kidney compared with an equivalent molar concentration of big ET. PMID- 1725364 TI - Nicardipine inhibits the endothelin-1-induced constriction of systemic capacitance and resistance vessels. AB - We estimated the effects of intravenous injection of endothelin-1 (ET-1; 400 pmol/kg) on the systemic capacitance and resistance vessels in anesthetized mongrel dogs by measuring changes in the mean circulatory pressure (MCP) and total peripheral resistance (TPR), respectively. In addition, the present study investigated the inhibiting action of nicardipine (NIC; 10 micrograms/kg bolus injection followed by 1.0-2.0 micrograms/kg/min continuous infusion) against the vasoconstrictor action of ET-1. We examined the effects of ET-1 (30 to 1,000 pmol/0.1 ml) on the femoral arterial system by measuring the percent changes in the perfusion pressure (% delta PP) of the femoral artery perfused with a constant flow, and investigated the effect of NIC on the vasoconstrictor action of ET-1 in this preparation. We obtained the following results: (a) Intravenous injection of ET-1 raised both TPR and MCP. However, in the presence of NIC, ET-1 raised TPR without influencing MCP. (b) Intra-arterial injection of ET-1 caused a dose-dependent increase in % delta PP. NIC shifted the dose-response curve of ET 1 for % delta PP to the right in a parallel fashion. PMID- 1725365 TI - Comparison of endothelin-1 concentrations in normal and complicated pregnancies. AB - Endothelin-1 (ET-1) may be important in regulating vascular tone in humans. To understand the role of ET-1 during pregnancy, we measured the concentrations of plasma immunoreactive ET-1 in normal, toxemic (pre-eclamptic or eclamptic), and other complicated pregnancies. There were no significant differences between ET-1 concentrations in normal pregnancy and those in toxemic pregnancy, except in one case with IgA nephropathy and severe toxemia. There also was no significant difference between the ET-1 concentrations in pure toxemia of pregnancy compared with superimposed toxemia of pregnancy. Furthermore, there was no correlation between ET-1 concentrations, blood pressure, or renal function during toxemic pregnancy. Similarly, there were no significant differences between ET-1 plasma levels in normal pregnancies and other complicated pregnancies except for two cases in which much blood was lost due to uterine atony and abruptio placenta. Thus, plasma ET-1 concentrations showed no significant relationship to blood pressure or renal function during normal or toxemic pregnancy, and were not related to the type (pure or superimposed type), grade, or severity of toxemic pregnancy. PMID- 1725366 TI - Relative preservation of the responsiveness to endothelin-1 during reperfusion following renal ischemia in the rat. AB - Reperfusion after renal ischemia is characterized by a preglomerular vasoconstriction. As endothelin-1 (ET-1) is a potent preglomerular vasoconstrictor, we designed a study to investigate the role of ET-1 in postischemic renal vasoconstriction. Renal ischemia was induced by 45 min of left renal artery clamping in anesthetized rats. After ischemia, renal blood flow was restored to only 61 +/- 4% of its preischemic value. The sensitivity to exogenous ET-1 after renal ischemia was decreased, but much less than the sensitivity to angiotensin II, which almost lost its vasoconstrictor effect, and to norepinephrine, which became a vasodilator. In addition, the binding affinity of [125I]ET-1 in the kidney increased significantly after renal ischemia, despite no change in the density of binding sites. These findings may be in favor of a role of endogenous ET-1 in postischemic renal vasoconstriction. PMID- 1725367 TI - Central effects of endothelin-1 on vasopressin and atrial natriuretic peptide release and cardiovascular and renal function in conscious rats. AB - In order to investigate the central effect of endothelin-1 (ET-1) on arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) release and cardiovascular and renal function, either a high dose of ET-1 (3.5 ng/kg/min, HET-1) or a low dose (0.35 ng/kg/min, LET-1) was administered into the cerebral third ventricle in conscious rats. ET-1 increased the mean arterial blood pressure, but not heart rate, in a dose-related manner. LET-1 slightly stimulated AVP release, but not ANP release. HET-1 increased plasma AVP and ANP. Pretreatment with a V1 antagonist (TMeAVP) attenuated the blood pressure response to ET-1 and completely abolished increases in plasma ANP. Moreover, prazosin given i.v. completely prevented the residual pressure response. ET-1 did not significantly affect renal electrolytes and water handling. These results suggest that centrally administered ET-1 may increase the activity of the sympathetic nervous system and vasopressinergic neurons, resulting in an increase in blood pressure. PMID- 1725368 TI - Cicletanine modulates endothelin-induced renal vasoconstriction in the rat. AB - Endothelin (ET-1), a recently discovered endothelium-derived peptide, has been reported to produce potent vasoconstriction in various isolated vessels of experimental animals. Cicletanine (CIC) is a novel antihypertensive agent. This study concerns the effect of CIC on the vascular actions of ET-1 (0.2 nM/kg) and epinephrine (1 microgram/kg) in normotensive Wistar rats. The hemodynamic effects of ET-1 and epinephrine were also tested in the presence of molsidomine (MOL), a vasodilator that releases nitric oxide. Rats were treated for 15 days with CIC (10 mg/kg/day) or gum arabic p.o. Subsequently, the animals were anesthetized and renal and aortic blood flow (BF) determined by pulsed Doppler flowmetry. ET-1 or epinephrine was injected. After return to the basal level, MOL (5 mg/kg) was injected; 10 min later, the mean arterial pressure (MAP) was decreased and then ET-1 or epinephrine was administered. The vascular resistance was calculated by the MAP/BF ratio and expressed as a percentage. In CIC-treated rats, ET-1 induced a renal vasoconstriction smaller than in control rats (+27.2 +/- 5.95 and +60.4 +/- 11.95%, respectively, p less than 0.01). In the presence of MOL, ET-1 produced a smaller increase in MAP (+9.7 +/- 1.34 and +16.9 +/- 2.49 mm Hg, p less than 0.05). Epinephrine injected after MOL in CIC-treated rats induced a smaller renal vasoconstriction than in control rats (+98.8 +/- 29.83 and 185 +/- 30.33%, p less than 0.05). Thus, CIC partially reduced the hypertensive and renal vasoconstrictor effects of ET-1. A combination of CIC and MOL diminished the renal effects of epinephrine. In conclusion, CIC could be used to attenuate the hypertensive status or renal ischemia disorders where ET-1 seems to be implicated. PMID- 1725369 TI - Hemodynamic effects of endothelin-1 in the newborn piglet: influence on pulmonary and systemic vascular resistance. AB - The aim of this study was to investigate the influence of endothelin-1 (ET-1) on the pulmonary and systemic circulations in newborn piglets. Twelve piglets (mean age of 6.8 days, range of 1-12 days) were anesthetized with chloralose-urethane and ventilated at FiO2 = 1.0. Animals were instrumented to measure cardiac output (CO), pulmonary artery pressure (PAP), aortic pressure (AoP), left atrial pressure (LAP), and right atrial pressure (RAP). Six piglets received a control saline injection into the pulmonary artery (PA), and six piglets received one injection of ET-1 (500 ng/kg) into the PA while PAP, AoP, LAP, RAP, CO, and heart rate (HR) were measured simultaneously at 20-s intervals with pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) calculated by computer. Administration of ET-1 into the PA led to (a) a transient decrease in mean PAP (delta max of -4 +/- 2 mm Hg, p less than 0.001); (b) a biphasic response (an initial decline followed by a substantial increase) in PVR (delta max of -760 +/- 460 dyn cm/s5, p less than 0.01, and delta max of +1,490 +/- 1,360 dyn cm/s5, p less than 0.05) and in AoP (delta max of -12 +/- 7 mm Hg, p less than 0.005, and delta max of +11 +/- 6 mm Hg, p less than 0.005); (c) an increase in SVR (delta max of +5,930 +/- 2,330 dyn cm/s5, p less than 0.05); and (d) an initial increase in CO (delta max of +0.06 +/- 0.03 L/min, p less than 0.05) followed by a decrease (delta max of -0.09 +/- 0.05 L/min, p less than 0.05). These responses have not been observed in adult species and may indicate a developmental difference in the response of the pulmonary circulation to ET-1 administration. PMID- 1725370 TI - Bronchopulmonary and pressor activities of endothelin-1 (ET-1), ET-2, ET-3, and big ET-1 in the guinea pig. AB - In anesthetized and ventilated guinea pigs, intravenous injection of ET-1, ET-2, or ET-3 induced similar rapid and dose-related increases in pulmonary inflation pressure (PIP) and mean arterial blood pressure (MABP). Indomethacin inhibited the increase in PIP evoked by ET-1, ET-2, or ET-3, whereas the changes in MABP following injection of the various ET isotypes were not significantly affected. Injection of ET-1, ET-2, or ET-3 via the pulmonary artery of isolated guinea pig lungs induced similar dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TxB2) release, and formation of lung edema. Indomethacin (5 microM), added to the perfusion medium, significantly inhibited the alterations of PIP, PPP, TxB2 release, and lung edema formation evoked by the three ET isoforms. Intravenous injection of 1 nmol/kg of big ET-1 to guinea pigs did not induce significant changes in PIP and MABP. When administered at a dose of 10 nmol/kg, big ET-1 provoked marked slow-developing and sustained increases in PIP and MABP. When big ET-1 was incubated in vitro with either alpha chymotrypsin or pepsin and injected into guinea pigs at a dose of 1 nmol/kg, marked rapid bronchoconstrictor and pressor responses were observed. The present results demonstrate that ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig. On the contrary, big ET-1 exhibits moderate direct bronchoconstrictor and pressor effects and its hydrolysis by proteases appears to be essential for expression of its full activity. PMID- 1725371 TI - Endothelin-1 and bronchial hyperresponsiveness in the guinea pig. AB - Exposure of phosphoramidon-treated (0.1 mM solution by aerosol for 15 min) guinea pigs to an aerosol of endothelin-1 (ET-1) (10 micrograms/ml) for 30 min induced after 18-24 h an enhanced bronchopulmonary response (BR) to rechallenge with ET-1 (3 micrograms/ml) administered by aerosol. Compared with saline-exposed animals, aerosol exposure of guinea pigs to ET-1 (10 micrograms/ml) for 30 min did not alter the dose-related BR to acetylcholine (ACh) administered by aerosol 18-24 h following challenge with the peptide. In phosphoramidon-treated guinea pigs, ET-1 (5 micrograms/ml) exposure for 30 min evoked a slight but non-significant enhancement of the ACh-induced BR. Administered by aerosol in phosphoramidon treated guinea pigs, ET-1 did not induce eosinophil accumulation in the lung, as demonstrated by examination of histological preparations and the assessment of the cell composition of bronchoalveolar lavages. The present data indicate that in spite of treatment of guinea pigs with phosphoramidon, ET-1 administration does not lead to the development of bronchial hyperresponsiveness. PMID- 1725372 TI - Effects of NG-nitro-L-arginine on renal hemodynamic responses to endothelin-3 in anesthetized dogs. AB - The effects of NG-nitro-L-arginine (L-NNA), a nitric oxide (NO) synthase inhibitor, on renal vascular response to endothelin-3 (ET-3) were studied in anesthetized dogs. Intrarenal arterial (i.r.a.) infusion of ET-3 (5 and 25 ng/kg/min for 2 min) elicited a dose-dependent increase in renal blood flow (RBF) with no change in the systemic blood pressure (BP). Marked attenuation of the renal vasodilatory response to ET-3 was observed following the administration of L-NNA (75 micrograms/kg/min i.r.a. for 20 min), and there was a partial reversion when L-arginine (100 mg/kg i.v.) was given. Continuous administration of ET-3 at a rate of 5 ng/kg/min i.r.a. for 25 min increased RBF with no change in BP and heart rate (HR). The glomerular filtration rate (GFR) remained unchanged throughout the experiment, the result being a significant decrease in the filtration fraction (FF). The significant increase in urine flow rate (UF) was associated with a marked reduction in urine osmolarity (Uosm) during ET-3 infusion, but urinary excretion of sodium (UNaV) remained unchanged. Pretreatment with L-NNA abolished the ET-3-induced renal vasodilation. RBF decreased immediately after the start of ET-3 infusion into animals treated with L-NNA. Thus, ET-3 is a diuretic and renal vasodilatory peptide, the vascular effects of which may be mediated by production of endothelial NO in the kidney. PMID- 1725373 TI - Endothelin modulation of neuroeffector transmission in smooth muscle. AB - In a series of muscle preparations, the peptides endothelin-1 (ET-1) and endothelin-3 (ET-3) were investigated for effects on basal muscle tone, responses to transmural nerve stimulation, and release of [3H]norepinephrine or [3H]acetylcholine. ET-1 and ET-3 contracted rat vas deferens and guinea pig ileum, ET-1 being the most potent. In the guinea pig taenia coli, ET-1 induced a relaxation whereas ET-3 was almost without relaxing effect. In the rat vas deferens, ET-1 and ET-3 enhanced contractile responses to nerve stimulation, whereas the nerve-induced release of [3H]norepinephrine was inhibited by ET-1 but not by ET-3. Contractions to exogenous ATP were increased by ET-1 whereas contractions to norepinephrine were not. In the guinea pig ileum, nerve-induced contractions were inhibited by ET-1 and ET-3 as was acetylcholine release, whereas contractions to exogenous acetylcholine were enhanced by ET-1. The inhibition of nerve-induced contractions by the endothelins was not affected by treatment with 8-(p-sulfophenyl)theophylline, BW755C, or indomethacin. The relaxation by ET-1 in the guinea pig taenia coli was not affected by treatment with NG-monomethyl-L-arginine, BW755C, or indomethacin. In conclusion, ET-1 exerted a stimulatory postjunctional effect and concomitantly an inhibitory prejunctional effect on adrenergic and cholinergic neurotransmission. Also, ET-3 exerted a stimulatory postjunctional effect, whereas a prejunctional inhibitory effect of ET-3 only was evident on cholinergic neurotransmission. Blockade of the production of nitric oxide or arachidonic acid metabolites or application of an adenosine antagonist did not alter the effects of ET-1 or ET-3. PMID- 1725374 TI - Human polymorphonuclear leukocytes generate and degrade endothelin-1 by two distinct neutral proteases. AB - Human polymorphonuclear leukocytes (PMNs, 4 x 10(6)/ml) converted human big endothelin (bET) to an endothelin-1 (ET-1)-like contractile factor, as assessed by bioassay. The formation of this ET-1-like activity from bET was partially inhibited by phosphoramidon (54 micrograms/ml), but not by pepstatin-A (1 microgram/ml), epoxysuccinyl-L-leucylamido(guanidino)butane (E-64, 10 micrograms/ml) or phenylmethylsulfonyl fluoride (PMSF, 25 micrograms/ml). In addition, nonactivated PMNs converted [125I]bET to [125I]ET-1, thus confirming the bioassay results. Incubation of ET-1 with fMLP-activated PMNs or cell-free supernatants from activated PMNs resulted in the loss of its contractile activity, and this loss of activity was paralleled by the metabolism of [125I]ET 1. The metabolism of [125I]ET-1 by PMNs or leukocyte cathepsin G (5 micrograms/ml) was prevented by PMSF (25 micrograms/ml), but not by phosphoramidon (54 micrograms/ml) or pepstatin-A (1 microgram/ml). Thus, PMNs can form ET-1 from bET via a neutral protease and degrade ET-1 via a serine protease, an observation that may have important pathophysiologic implications in disease states associated with PMN infiltration. PMID- 1725375 TI - Responses of blood flow, extracellular lactate, and dopamine in the striatum to intrastriatal injection of endothelin-1 in anesthetized rats. AB - Recently a histological study has demonstrated that intrastriatally injected endothelin-1 (ET-1) produced ischemia-like lesions in the neostriatum. The present study was undertaken to investigate whether intrastriatally injected ET-1 produces ischemic responses such as a decrease in the striatal blood flow and increases in extracellular lactate and dopamine levels in the neostriatum as seen in other models of ischemia. A small needle (for injection of ET-1), a microdialysis probe (for collecting extracellular substances), and a probe of a laser Doppler flowmeter (for measuring local cerebral blood flow) were implanted with their tips close to each other in the neostriatum of halothane-anesthetized rats. Focal administration of ET-1 (430 pmol) into the neostriatum resulted in a marked decrease in striatal blood flow without any change in systemic blood pressure. It also markedly increased extracellular lactate and dopamine levels, whereas it decreased pyruvate and dopamine metabolite levels. These changes agreed well with those known to be produced by ischemia. Intracerebral injection of ET-1 will therefore provide a new model for production of local ischemia in experimental animals. PMID- 1725376 TI - Cardiorespiratory effects of topical application of endothelin-1 to the ventral surface of the rat medulla. AB - In urethane-anesthetized and vagotomized rats, we examined the cardiorespiratory effects of a topical application of endothelin-1 (ET-1) to the ventral surface of the medulla (VSM) and surveyed subregions of the VSM influenced by these effects. ET-1 (0.1 fmol) delivered to the S area of the VSM via a needle (i.d. of approximately 100 microns) gently pressed on the VSM had no effect on mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve activity (RSNA), phrenic nerve activity (PNA), or the burst rate of PNA. However, a dose of 1 fmol of ET-1 induced transient but significant increases in MAP, HR, RSNA, and burst rate while at a dose of 10 fmol or more, PNA also increased and simultaneously longer-lasting decreases in MAP, RSNA, and PNA followed the initial increase. The subregion of the VSM in which ET-1 most prominently elicited these effects was the S and caudal part of the M area, where topical application of 50 nmol of L-glutamate caused cardiorespiratory changes. Additionally, there was a restricted region within the caudal VSM in which ET-1 caused a decrease in PNA with an increase in burst rate. These results support our hypothesis that the VSM is crucially involved in the cardiorespiratory changes induced by centrally administered ET-1. PMID- 1725377 TI - Hypothalamic endothelin: presence and effects related to fluid and electrolyte homeostasis. AB - Endothelin-3-like immunoreactivity (ET-3-ir) was detected in extracts of rat hypothalamic median eminence, and in the anterior and neurointermediate lobes of the pituitary at levels (ng ET-3/mg protein) exceeding those present in extracts of abdominal aorta. This ET-3-ir appeared authentic because radioimmunoassay (RIA) dose-response curves parallel to those of synthetic ET-3 could be constructed and this ET-3-ir comigrated on C-8 high-pressure liquid chromatography (HPLC) with synthetic ET-3. Endothelin-1-like immunoreactivity, on the other hand, was abundant in extracts of abdominal aorta and cerebral cortex and only minimally present in hypothalamus and anterior pituitary gland. Central administration of 11 and 23 pmol ET-3 resulted in significant (4.2- and 5.7-fold, respectively) elevations of plasma levels of vasopressin. Oxytocin levels were transiently, yet significantly, elevated (1.8-fold) by the higher dose of ET-3. These results, and our findings that central administration of ET-3 inhibits stimulated water drinking, suggest a physiologically important role for endogenously produced endothelin in the central mechanisms regulating fluid and electrolyte homeostasis. PMID- 1725378 TI - Evidence for release of endothelin-1 in pigs and humans. AB - The release and pharmacokinetics of endothelin-1 (ET-1) in plasma were studied in pigs and humans in vivo. Between 50-90% of plasma ET-1-like immunoreactivity (LI) was cleared by the pig and human kidney, splanchnic circulation, and skeletal muscle. The precursor big ET-1 was only cleared to a moderate extent (34%) by the kidney with progressive formation of ET-1-LI in the pig. The half-lives of circulating ET-1-LI and big ET-1-LI were about 1 and 10 min, respectively. The threshold vasoconstrictor effects for plasma ET-1-LI in the splanchnic and renal circulation in humans were around 30 pM. ET-1-LI in fetal umbilical arterial plasma was very high (15 pM before and 94 pM after establishment of breathing) compared with about 2 pM in maternal plasma. Bacterial endotoxin or sepsis increased ET-1-LI in plasma more than fivefold in both pigs and humans reaching levels close to threshold vasoconstriction. However, hemorrhagic shock or hypotension did not alter plasma ET-1-LI. It is concluded that ET-1 has a short half-life with very high regional plasma clearance, which limits detection of overflow into the systemic circulation. However, release of ET-1 reaching vasoconstrictor levels seems to occur both upon special physiological circulatory changes in the newborn and in septic shock. PMID- 1725379 TI - Reduction in local cerebral blood flow induced by endothelin-1 applied topically to the middle cerebral artery in the rat. AB - The role of endothelin-1 (ET-1) in cerebral ischemic injury remains the subject of much debate. Vasoconstriction in large conduit vessels may not be associated with reductions in flow at the tissue level. We present two studies examining the effects on local cerebral blood flow of topical application of ET-1 to the surgically exposed middle cerebral artery (MCA) in adult male Sprague-Dawley rats. In the first series using 14C-iodoantipyrine autoradiography, 10 min following application of ET-1 (1 nmol) to the MCA, up to 80% reduction in blood flow in the territory of distribution of the MCA is seen (e.g., dorsolateral caudate nucleus--flow reduced from 131 +/- 3 ml/100 g/min to 29 +/- 25 ml/100 g/min). These levels of flow are comparable with those seen with permanent bipolar diathermy occlusion and division of the proximal MCA--a standard rat model of focal cerebral ischemia. In a second series using hydrogen clearance technique for measurement of local cerebral blood flow in the caudate nucleus, we have shown that flow ipsilateral to application of ET-1 (0.25 nmol) is significantly reduced compared with saline controls for 80 min. Such reduction of flow, at the tissue level, sustained over this duration is consistent with the induction of ischemic cell damage by ET-1. PMID- 1725380 TI - Pulmonary vascular reactivity to endothelin-1 in normal and chronically pulmonary hypertensive rats. AB - The pulmonary vascular reactivity to endothelin-1 (ET-1) was assessed in rats previously exposed to 11% O2 (hypoxic) or room air (controls) for 3 weeks. In isolated control lung preparations studied during conditions of increased tone by U46619 (50 pmol/min) and treated with meclofenamate (3 microM), low doses of ET-1 (30 and 100 pM) reduced the pressor response to U46619 by 58 +/- 5% (p less than 0.01). Vasodilation induced by ET-1 was not abolished by the antagonist of endothelium-dependent relaxing factor (EDRF) NG-monomethyl-L-arginine (5 x 10(-4) M), which suppressed vasodilator response to ionophore A23187 (10(-8)-10(-7) M). Higher doses of ET-1 (300 and 1,000 pM) induced vasoconstriction during conditions of basal tone, and the pressor response to 300 pM ET-1 was enhanced by EDRF antagonists. Administration of ET-1 to lungs from hypoxic rats failed to cause pulmonary vasodilation and instead induced a greater pulmonary pressor response (300 pM) than in control rat lungs (7 +/- 1.5 vs. 1.6 +/- 0.5 mm Hg, p less than 0.01), which was not further potentiated by EDRF antagonists. Infusion of 300 pM ET to conscious catheterized animals induced a sustained increase in pulmonary resistance only in the hypoxic group (from 305 +/- 37 to 389 +/- 55 mm Hg/L/min, p less than 0.01) (n = 7). The results suggest that depending on the dose, ET-1 can cause pulmonary vasodilation (independent of EDRF release) or vasoconstriction (opposed by EDRF). During chronic hypoxic pulmonary hypertension, ET-1 behaves only as a pulmonary vasoconstrictor. PMID- 1725381 TI - Differential pharmacological profile of endothelin-1 and its precursor, big endothelin. AB - In pentobarbital-anesthetized rats endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3), and mouse ET-2 (mET-2), in contrast to human big ET-1 (h-big ET), administered as i.v. bolus injections (0.25 nmol/kg i.v.) produced rapidly appearing and short-lasting blood pressure decreases. This effect was markedly inhibited (80-100%) after an 8-min i.v. infusion (0.1 nmol/kg/min over 10 min) of any of the ET studied, but not by h-big ET, the precursor of ET-1. Similarly, in pithed rats given a 10 min i.v. infusion of an equipressor dose (0.1 nmol/kg/min) of ET-1 or h-big ET, the hypotensive effects of ET-1 were entirely blocked only in the group of animals pretreated with ET-1. In pithed rats, ET-1 (0.25 nmol/kg i.v.) and h-big ET (0.5 nmol/kg i.v.) produced equivalent maximal pressor responses and the same pattern of increase in systemic, hindquarter, and renal vascular resistance. However, ET-1 was three times more potent than h-big ET as a vasoconstrictor of the mesenteric bed. Also the pressor response to h-big ET, but not ET-1 (0.25 nmol/kg i.v.), was markedly inhibited by the metalloprotease inhibitor phosphoramidon (5 mg/kg i.v.). These results indicate that the hypotensive effects of ET isopeptides have a common mechanism because they elicit cross tachyphylaxis, although h-big ET did not inhibit the decrease in blood pressure produced by ET-1. A possible explanation for this finding is that h-big ET has intrinsic pressor activity but does not have affinity for receptors mediating the vasodepressor effects of ET isoforms. Alternatively, h-big ET is converted into ET-1 too slowly to yield biophase concentrations of ET-1 necessary for lowering blood pressure and developing tachyphylaxis to ET-isoform-induced hypotension. Finally, if the pressor effects of h-big ET are mediated by ET-1 formation, phosphoramidon can be considered as an inhibitor of the endothelin converting enzyme. PMID- 1725382 TI - Cross-desensitization between angiotensin II and endothelin-1 in the pithed rat. AB - It has been shown that in vitro angiotensin II (Ang II) potently downregulates endothelin-1 (ET-1) binding sites. In this study, we investigated in vivo the interactions between the renin-angiotensin system and ET-1. Sprague-Dawley rats were pithed, ventilated, and diastolic blood pressure (DBP) was recorded. ET-1 (1 nmol/kg) induced a biphasic response: a transient hypotension followed by a sustained increase of DBP. Captopril (5 mg/kg, i.v.) or Sar1-Ile8-Ang II (10 ng/kg/min) did not affect ET-1 responses. In other experiments, DBP was increased by infusion of methoxamine (MTX: 10, 20, 25, 32.5 micrograms/kg/min) or Ang II (50, 100, 150, 200 ng/kg/min). ET-1-induced hypotension correlated with the level of DBP (r = 0.94) for both agonists. The elevation of DBP in response to ET-1 was also related to the vascular tone but was dose-dependently attenuated by Ang II infusion when compared with MTX. Conversely, infusion of ET-1 (0.1 nmol/kg/min) blunted the pressor response to Ang II (0.1 micrograms/kg) but not to MTX (50 micrograms/kg). These doses induced the same increase of DBP in pithed control rats. Similarly, increased plasma renin activity induced by chronic salt depletion (0% NaCl) in pithed rats provoked a shift to the right of the dose response curves to Ang II and ET-1 but not to MTX. These results suggest an in vivo cross-desensitization between ET-1 and Ang II. PMID- 1725383 TI - Evidence for distinct endothelin receptors in the pulmonary vascular bed in vivo. AB - The present study was undertaken to investigate the effects of endothelin (ET) isopeptides on the pulmonary vascular bed of the intact, spontaneously breathing cat under conditions of constant pulmonary blood flow and left atrial pressure. When pulmonary vasomotor tone was actively increased by intralobar infusion of U46619, intralobar bolus injections of ET-1 (1 micrograms), ET-2 (1 micrograms), and ET-3 (3 micrograms) produced marked reductions in pulmonary and systemic vascular resistances. The pulmonary vasodilator response to each ET isopeptide was not altered by atropine (1 mg/kg i.v.), indomethacin (2.5 mg/kg i.v.), or ICI 118551 (1 mg/kg i.v.), but was significantly inhibited by an intra-arterial (i.a.) infusion of glybenclamide at 5 mg/kg. This dose of glybenclamide significantly inhibited the decrease in lobar arterial and systemic arterial pressures in response to intralobar injection of pinacidil (30 and 100 micrograms), whereas the pulmonary vasodilator responses to acetylcholine (0.03 and 0.1 micrograms) and prostaglandin I2 (0.1 and 0.3 micrograms) were not altered. The systemic vasodilator response to each ET isopeptide was not changed by glybenclamide or by the other blocking agents studied. The present data demonstrate for the first time that ET-1, ET-2, and ET-3 dilate the pulmonary vascular bed in vivo. The present data suggest that the pulmonary vasodilator response to ET isopeptides depends, in part, on activation of potassium channels and is mediated differently from the systemic vasodilator response to these substances. Contrary to earlier work, the present data indicate the pulmonary vascular response to ET isopeptides depends on the pre-existing level of pulmonary vasomotor tone. Furthermore, the present data suggest that in the lung ET-1, ET-2, and ET-3 may serve as endogenous agonists for potassium channels, a newly described vasodilator mechanism in the pulmonary vascular bed of intact adult animals. PMID- 1725384 TI - Pulmonary vascular and airway responses to endothelin-1 are mediated by different mechanisms in the cat. AB - The role of cyclooxygenase product formation and thromboxane A2 receptor activation in the response to endothelin-1 (ET-1) was investigated and compared in the airways and in the pulmonary vascular bed of the intact-chest cat. Intravenous injections of ET-1, 0.3 nmol/kg, increased lung resistance and decreased dynamic compliance. Bronchoconstrictor responses to ET-1 were decreased significantly by a cyclooxygenase inhibitor and by a thromboxane receptor blocking agent. In the pulmonary vascular bed of the cat under constant flow conditions, ET-1 increased lobar arterial pressure in a dose-related manner, and pulmonary vasconstrictor responses to the peptide were not altered by a cyclooxygenase inhibitor or thromboxane receptor blocking agent. The cyclooxygenase inhibitor blocked responses to the prostaglandin precursor, arachidonic acid; and the thromboxane receptor blocking agent reduced responses to the thromboxane mimic, U-46619. The present data suggest that bronchoconstrictor responses to ET-1 are dependent on the release of arachidonic acid, the formation of prostaglandins, and activation of thromboxane A2 receptors whereas pulmonary vasoconstrictor responses to the peptide are mediated by a different mechanism. PMID- 1725385 TI - Localization of endothelin binding sites and endothelin-like immunoreactivity in human fetal heart. AB - The localization of [125I]endothelin-1 ([125I]ET-1) and [125I]endothelin-3 ([125I]ET-3) binding sites, as well as ET-like immunoreactivity, was investigated in sections of human fetal heart, using in vitro autoradiographic and immunohistochemical techniques. High-affinity [125I]ET-1 binding sites showed a tissue-specific distribution pattern, with high-density binding to the atria, ventricles (77-100 amol/mm2), and cardiac valve cusps (120.6 +/- 2.6 amol/mm2). Specific high-density binding of [125I]ET-3 was also exhibited on valve cusps (143.2 +/- 2 amol/mm2), whereas a much lower density of binding was displayed on atria and ventricles (10-15 amol/mm2). Microautoradiographic examination demonstrated binding sites on the wall of the aorta, pulmonary and coronary arteries, myocardium, ventricular conduction system, endocardium, and endothelial lining of valve cusps. Regional differences in the density and affinity of ET binding sites suggest that subpopulations of receptors are present in the human fetal heart. ET-like immunoreactivity was localized to a heterogeneous population of endothelial, endocardial, and epicardial mesothelial cells. The concordant localization of specific binding sites and ET-like immunoreactive cells indicates that locally released peptide might have a paracrine or autocrine role, possibly influencing cardiovascular development and function. PMID- 1725386 TI - Localization of [125I]endothelin binding sites in the region of the carotid bifurcation and brainstem of the cat: possible baro- and chemoreceptor involvement. AB - We have demonstrated by autoradiography, displaceable binding for [125I]endothelin-1 ([125I]ET-1), [125I]endothelin-2 ([125I]ET-2), and [125I]endothelin-3 ([125I]ET-3) in the cat carotid bifurcation as well as in the nucleus of the tractus solitarius, where baroreceptor and chemoreceptor afferents from the carotid body and sinus terminate. There was also significant binding in the nodose and superior cervical ganglia. Barosensory and chemosensory discharge was recorded from filaments of the carotid sinus nerve in cats anesthetized with pentobarbitone. Intra-carotid injection of ET-1 or ET-3 (4-402 pmoles) caused transient dose-related depression of baroreceptor discharge without any immediate effects on systemic blood pressure (BP) or heart rate; there was a delayed biphasic effect on BP. ET-1 had little effect on chemosensory discharge during the first 15 s post-injection, but there was a delayed (45-90 s) dose-related increase in discharge. The effects of all three ETs were qualitatively similar, and ET enhanced chemoexcitation evoked by either acetylcholine or sodium cyanide. Our results show that (a) ET binding sites are located in the baroreceptor and chemoreceptor afferent pathways and (b) ETs can influence afferent activity of baroreceptors and chemoreceptors. Further studies are needed to determine the significance of these findings, particularly with regard to reflex control of the cardiovascular system. PMID- 1725387 TI - Expression of endothelin-2 (ET-2) gene in a human renal adenocarcinoma cell line: purification and cDNA cloning of ET-2. AB - It has been found that human renal adenocarcinoma ACHN cells synthesize and secrete immunoreactive endothelin (ir-ET) in the culture medium. Partial characterization of this material with reverse-phase high-performance liquid chromatography (RP-HPLC) suggested that ACHN cells synthesized only human endothelin-2 (ET-2). Isolation and characterization of this ir-ET-2 has revealed that this peptide has almost the same amino acid sequence and molecular weight as that of human ET-2 deduced from the nucleotide sequence of cloned human ET-2 gene. To delineate the precise structure of human ET-2 precursor, ET-2 cDNAs were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET-2 cDNA that has the longest open reading frame encodes prepro-ET-2 protein, consisting of 178 amino acid residues. The ET-like sequence found in the prepro-ET-1 and -ET 3 was also conserved in this prepro-ET-2. The Northern blot analysis of mRNA revealed that the transcript of the human ET-2 gene was 1.4 kb. PMID- 1725388 TI - Immunoreactive endothelin in human plasma, urine, milk, and saliva. AB - Using a highly sensitive radioimmunoassay, elevated plasma immunoreactive endothelin (ir-ET) levels were found in patients with diabetes mellitus (1.88 +/- 0.12 pmol/L, mean +/- SEM, n = 100), patients undergoing maintenance hemodialysis (4.28 +/- 0.76 pmol/L, n = 14), patients with acute myocardial infarction (3.43 +/- 1.03 pmol/L, n = 6), and patients with subarachnoid hemorrhage (4.92 +/- 0.64 pmol/L, n = 14) (normal controls: 0.54 +/- 0.05 pmol/L, n = 19). ir-ET was also present in urine (2.1 +/- 0.3 pmol/L, n = 12), breast milk (6.8 +/- 1.6 pmol/L, n = 16), and saliva (2.0 +/- 0.2 pmol/L, n = 15) obtained from healthy subjects. Chromatography studies verify the identity of endothelin. Fast protein liquid chromatography (FPLC) showed one peak in the normal plasma extract, three peaks in the plasma extracts from diabetic patients and patients undergoing maintenance hemodialysis, three peaks in the urine extract, four peaks in the milk extract, and five peaks in the saliva extract. When the materials eluting in the void volume on FPLC of urine and saliva extracts were loaded onto a Sephadex G-25 column, the ir-ET was eluted in a higher molecular weight region. Incubation of endothelin-1, endothelin-2, and endothelin-3 in urine for 5 h showed that the total amount of ir-ET decreased to less than 30% of the initial levels, suggesting that endothelins are very unstable in urine. PMID- 1725389 TI - Plasma concentrations of endothelin-1 and endothelin-3 are altered differently in various pathophysiological conditions in humans. AB - Several studies have indicated that endothelin-1 (ET-1) and endothelin-3 (ET-3) are produced by different cells. Although ET-1 is produced by vascular endothelial cells, these cells do not produce ET-3. In the present study, we measured plasma concentrations of both ET-1 and ET-3 by sandwich-enzyme immunoassays which we developed recently in patients on chronic hemodialysis, age matched normal subjects, patients with acute myocardial infarction, patients undergoing surgery, and healthy subjects before and after strenuous endurance exercise. Plasma levels of ET-1 and ET-3 were demonstrated to be altered differently in the above conditions in humans. Although the exact origin of circulating endothelins has yet to be elucidated, the different alterations of plasma levels suggest that both ET-1 and ET-3 may play different roles in physiological and/or pathophysiological responses to various conditions in humans. PMID- 1725390 TI - Endothelin-secreting tumor. AB - Recently, we have reported two cases with endothelin (ET)-secreting tumor presenting with hypertension and elevated plasma endothelin-1 (ET-1) levels. The present study examines the histopathology of the ET-secreting tumor in one of these cases. The neoplasm was located in the skin and microscopically characterized as a malignant hemangioendothelioma by remarkable intravascular proliferations of atypical endothelium with the expression of Factor VIII-related antigen in the tumor cells. The tumor enlarged rapidly with rising ET-1 and blood pressure levels. The ET-1 content and its messenger RNA expression in the tumor extract were higher than those from normal parts of skin. ET-1 may play a pathophysiological role in patients with ET-secreting malignant hemangioendothelioma. PMID- 1725391 TI - Increased plasma concentrations of endothelin-1 during and after pulmonary surgery. AB - We studied the involvement of endothelin-1 (ET-1) in the physiological response to surgical stress. Plasma concentrations of ET-1 were measured by a sandwich type enzyme immunoassay. The blood samples were collected from the pulmonary artery (PA), the left atrium (LA), and the median cubital vein (MCV) in four patients who had undergone pulmonary lobectomy. The samples were collected before, during, and after surgery. Plasma ET-1 concentrations increased in the blood from all sampling sites. The highest concentration of plasma ET-1 was observed at 6 h after surgery in MCV (3.97 +/- 1.47 pg/ml, mean +/- SD) and at the end of surgery in PA (1.79 +/- 0.48 pg/ml) and in LA (1.93 +/- 0.43 pg/ml). These values returned to the baseline value within 72 h after surgery [1.21 +/- 0.19 (MCV), 1.00 +/- 0.18 (PA)]. Although the lung has a large capacity to remove ET-1 from the circulating blood, ET-1 concentrations measured in plasma samples obtained from the LA were not different from those obtained from the PA. This may suggest that the lungs have the ability to absorb and release ET-1 simultaneously during pulmonary surgery. The increase of ET-1 in MCV was greater than that in PA or in LA. This suggests the possibility of ET-1 release from the forearm. PMID- 1725392 TI - Plasma endothelin levels in healthy children: high values in early infancy. AB - We measured plasma endothelin-1-like immunoreactivity (ET-1-LI) levels in the peripheral vein of 70 healthy children (37 males and 33 females) aged 5 days to 15 years to establish the normal range in children, and compared them with those in 11 normal adults (age, mean +/- SD; 29.5 +/- 5.7 years, 6 males and 5 females). Plasma ET-1-LI levels (pg/ml, mean +/- SD) in children of each age group and adults were as follows: children younger than 1 month = 23.4 +/- 3.9 (n = 10), 1-3 months = 25.0 +/- 6.1 (n = 8), 3-6 months = 17.0 +/- 1.4 (n = 8), 6 months to 1 year = 14.0 +/- 1.7 (n = 13), 1-5 years = 13.8 +/- 2.6 (n = 11), 5-10 years = 13.6 +/- 1.6 (n = 10), 10-15 years = 12.9 +/- 2.2 (n = 10), and adults = 13.2 +/- 2.4 (n = 11). There was no significant difference in plasma ET-1-LI levels between males and females in each age group. Plasma ET-1-LI levels in children younger than 3 months were significantly (p less than 0.01) higher than those in older children or adults. After 3 months of age, plasma ET-1-LI levels were nearly constant at all ages. This is the first report to establish the normal range of plasma ET-1-LI levels in children. PMID- 1725393 TI - Endothelin-1 secretion from cultured rabbit gastric epithelial cells. AB - It has been shown that endothelin-1 (ET-1) is synthesized in various extraendothelial tissues. Although ET-1 has been reported to have potent ulcerogenic action in the stomach, the synthesis and physiological roles of ET-1 in the gastric mucosa are poorly understood. The aim of the present study was to investigate whether or not cultured gastric epithelial cells secrete ET-1 and possess autocrine functions. Gastric epithelial cells from rabbits were cultured in medium supplemented with 10% FBS after isolation. ET-1 was extracted by C18 columns from serum-free culture media and measured by radioimmunoassay (RIA). Effects of ET-1 on the intracellular concentration of calcium of the cultured cells were examined with Indo-1. Prostaglandin E2 (PGE2) was measured by RIA. Primary cultures of gastric epithelial cells were mainly composed of mucous cells. ET-1 was detected in the culture medium by RIA, and 70 pg/10(6) cells/24 h of ET-1 was secreted by cultured cells. Tumor growth factor-beta (4 ng/ml) and thrombin (8 U/ml) significantly increased ET-1 secretion. Exogenously administered ET-1 up to 10(-6) M neither modulated the intracellular calcium concentration nor affected PGE2 release by these cells. These results suggest that gastric mucous cells in culture secrete ET-1. Further studies are needed to explore the possible involvement of such paracrine function in the reported ulcerogenic action of ET-1. PMID- 1725394 TI - Endothelin-1 release from mesenteric arteries of spontaneously hypertensive rats. AB - Release of endothelin-1 (ET-1) from the mesenteric arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were measured by radioimmunoassay after purification using immunoaffinity column. The identity of ET-1 in perfusate was established using reversed-phase high-performance liquid chromatography. The mesenteric arteries from 5- to 6- and 9- to 10-week-old SHR released a significantly higher level (mean +/- SEM) of ET-1 (32.8 +/- 2.8 and 47.5 +/- 4.1 pg/h, respectively) than WKY age-matched rats (23.4 +/- 2.1 and 34.8 +/- 3.7, respectively). There was an age-related increase in ET-1 release in both groups of rats. Though the SHR studied were in the hypertensive stage, the present results suggest that locally produced ET-1 may contribute to the development of hypertension independent of the plasma levels. PMID- 1725395 TI - Stimulatory effect of thrombin on endothelin-1 production in isolated glomeruli and cultured mesangial cells of rats. AB - To determine whether endothelin-1 (ET-1) is released in close proximity to its binding sites on glomerular mesangial cells, and also to elucidate the regulatory factors responsible for its release, we measured immunoreactive ET-1 (ir-ET-1) in the incubation media of isolated glomeruli and cultured mesangial cells of rats using enzyme immunoassay under both basal and thrombin-stimulated conditions. ir ET-1 was released time-dependently, from isolated glomeruli and cultured mesangial cells. Thrombin (2 U/ml) stimulated the release from both preparations. The release of immunoreactive big endothelin-1 (ir-big ET-1), which was assayed by using enzyme immunoassay, was also time-dependent, and the release was increased by thrombin (2 U/ml; at 4 h, 8 h, and 24 h) in cultured mesangial cells. The releases of big ET-1 and its converted product ET-1 from mesangial cells, an established target for ET-1, suggests that ET-1 acts as an autocrine factor for the cells. PMID- 1725396 TI - Immunoreactive endothelin in plasma of nonmammalian vertebrates. AB - Immunoreactive endothelin (ir-ET) was estimated in plasma of nonmammalian vertebrates, using radioimmunoassay (RIA) for endothelin-1 (ET-1). Blood samples were collected from unanesthetized animals. Plasma ET was determined by RIA after extraction. Plasma levels of ir-ET were 0.7 +/- 0.2 pg/ml (n = 6) in the hagfish Eptatretus burgeri, 4.3 +/- 0.9 pg/ml (n = 5) in the banded dogfish Triakis scyllia, 3.6 +/- 0.8 pg/ml (n = 3) in the common Japanese conger Conger myriaster, 9.6 +/- 1.4 pg/ml (n = 7) in the carp Cyprinus carpio, 6.4 +/- 0.8 pg/ml (n = 5) in the bullfrog Rana catesbeiana, 6.7 +/- 0.6 pg/ml (n = 3) in the soft-shelled turtle Trionyx sinensis japonica, and 3.3 +/- 0.6 pg/ml (n = 8) in the Japanese quail Coturnix coturnix japonica. The dilution curves of the plasma extracts from each species almost paralleled the standard curve for ET-1. Analysis of the plasma extracts of the carp by reverse-phase high-performance liquid chromatography revealed that ir-ET consisted of three components, a predominant peak being located at the elution position of synthetic ET-1. The present results demonstrate clearly that an ET-1-like substance circulates in blood of nonmammalian vertebrates, suggesting an endocrine function of the peptide in these species. PMID- 1725397 TI - Immunoreactive endothelin-1 contents in brain regions from spontaneously hypertensive rats. AB - To investigate the possible role of brain endothelin-1 (ET-1) in hypertension of spontaneously hypertensive rats (SHRs), we measured immunoreactive (ir) ET-1 contents in brain regions as well as plasma ir-ET-1 levels in SHRs aged 4-5 and 12-14 weeks and age-matched Wistar-Kyoto rats (WKY) with a radioimmunoassay for ET-1. Systolic blood pressures of SHR aged 4-5 and 12-14 weeks were significantly higher than those of corresponding WKY. Significant amounts of ir-ET-1 were detectable throughout the discrete brain regions analyzed in both strains; higher ir-ET-1 contents in structures such as thalamus, hypothalamus, midbrain, pons, medulla, and cerebellum, with the lowest in cerebral cortex, were observed. A reverse-phase high-performance liquid chromatography of the brain extracts revealed the presence of both a major component identical to the elution position of synthetic ET-1 and a minor component possibly corresponding to its oxidized form. When compared, ir-ET-1 contents in all brain regions analyzed were lower in SHRs than in WKY rats. This strain-related change of ir-ET-1 contents was significant in the medulla at 4-5 weeks of age, and in all brain regions except hypothalamus at 12-14 weeks of age. Plasma ir-ET-1 levels, in contrast, were comparable between SHRs and WKY rats. These results suggest that brain ET-1 may be involved in the development and the maintenance of hypertension in SHRs. PMID- 1725398 TI - Sandwich-enzyme immunoassays for endothelin family peptides. AB - Sandwich-enzyme immunoassays (sandwich-EIAs) for endothelin-1 (ET-1), endothelin 3 (ET-3), big ET-1, big endothelin-2 (big ET-2), and big endothelin-3 (big ET-3) have been established using two antibodies directed against the N-terminal and C terminal portions of each ET and big ET. These sandwich-EIAs are sensitive enough to detect 0.1-0.4 pg/well of each ET or big ET with cross-reactivity of less than 1% with other ETs and big ETs (except in the case of the EIA for ET-1, which detects immunoreactivity of ET-2 as well as ET-1). Plasma levels of immunoreactive (ir-) ET-1, big ET-1, and ET-3 in normal humans were approximately 2.0, 5.1, and 0.9 pg/ml, respectively, when corrected for recovery efficiency. In reverse-phase high-performance liquid chromatography (HPLC) analysis, immunoreactivities detected by these sandwich-EIAs were eluted at the positions of authentic ET-1, big ET-1, and ET-3, indicating that plasma levels of ET-1, big ET-1, and ET-3 were precisely measured by our methods. Levels of ir-big ET-2 and ir-big ET-3 were also detected in normal human plasma; however, further studies are needed to determine their precise values. The establishment of reliable sandwich-EIAs and the measurement of specific ETs and big ETs in normal human plasma will contribute to clarifying the biological and clinical significance of ET family peptides. PMID- 1725399 TI - Immunoreactive endothelin in urine of patients with and without diabetes mellitus. AB - Immunoreactive (ir)-endothelin (ET) in urine was studied with a radioimmunoassay in patients with diabetes mellitus (DM) and non-DM diseases including endocrine disorders, primary glomerular diseases, autoimmune diseases, and hematological malignancies. Twenty-four-hour excretions (mean +/- SEM) of ir-ET were 8.0 +/- 0.9 pmol/day in the DM group (n = 13) and 9.5 +/- 1.2 pmol/day in the non-DM group (n = 51). No significant differences among DM and other disease groups were noted with respect to 24-h ir-ET excretion. Reverse-phase high-performance liquid chromatography of a normal urine extract revealed a major peak eluting later than ET-1. Gel chromatography revealed a single major peak in a smaller molecular weight (MW) region in normal urine and an additional peak in larger MW region in a urine extract from a DM patient. Urinary ir-ET consists of at least two components which may be metabolites of ET or ET precursors in plasma or peptides derived from the kidney. PMID- 1725400 TI - Development and application of three radioimmunoassays of endothelin of varying specificities. AB - We have developed three radioimmunoassays (RIAs) of varying specificities toward the endothelin (ET) isoforms. The assays are called the endothelin-1,2[125I] assay system (RPA535), the endothelin 1-21 Specific [125I] assay system (RPA555), and the endothelin-1,2 high-sensitivity [125I] assay system (RPA545). We have fully characterized their cross-reactivities: RPA535-ED50 approximately 12 fmol/tube, ET-1 100%, ET-2 204%, ET-3 0.0024%, hBig ET 37.9%, pBig ET 32.9%; RPA545-ED50 approximately 4.8 fmol/tube, ET-1 100%, ET-2 1,300%, ET-3 less than 0.001%, hBig ET 189%, pBig ET 63%; RPA555-ED50 approximately 3.6 fmol/tube, ET-1 100%, ET-2 144%, ET-3 52%, hBig ET 0.4%, pBig ET 0.26%. These assays have also been applied to the measurement of ET immunoreactivity (ir) in normal human plasma following Amprep extraction. Further validation of the use of these assays has included the measurement of endothelin-1 (ET-1) derived from big ET by in vitro enzymatic conversion by cathepsin E, which has demonstrated that cathepsin E possesses the appropriate specificity of cleavage to be considered the endothelin-converting enzyme (ECE). PMID- 1725401 TI - Immunoreactive endothelin in pheochromocytomas. AB - The presence of endothelin (ET) in tumor tissue and plasma of patients with pheochromocytoma was studied by radioimmunoassay. Immunoreactive (ir-) ET concentrations in 12 pheochromocytomas ranged from 66 to 253 fmol per gram wet tissue (gwt) (146 +/- 20 fmol/gwt, mean +/- SEM). These values were not significantly higher than tissue ir-ET concentrations of two primary aldosteronism (66 and 132 fmol/gwt) and three normal adrenal glands (71-120 fmol/gwt) (0.05 less than p less than 0.1). However, tumor tissue ir-ET concentrations in six of the 12 pheochromocytomas were higher than 132 fmol/gwt (the upper value of the control tissues). Sephadex G-50 column chromatography and reverse-phase high-performance liquid chromatography of pheochromocytoma tumor extracts showed a major peak eluting at an identical position to synthetic ET-1. Plasma ir-ET concentrations of pheochromocytomas (1.4 +/- 0.9 fmol/ml, n = 17) were not significantly different from those of patients with essential hypertension (1.0 +/- 0.7 fmol/ml, n = 20) and normal subjects (1.0 +/- 0.4 fmol/ml, n = 18) (0.05 less than p less than 0.1). This study has shown that high concentrations of ET-1 are present in tumor tissues of 50% of pheochromocytomas. PMID- 1725402 TI - Big endothelin in plasma and amniotic fluid. AB - We previously demonstrated that endothelin-1 (ET-1) exists both in the peripheral circulation and the amniotic fluid (AF). The objects of the present study were (a) to measure the concentrations of ET-1 and big endothelin (big ET) in plasma and AF by radioimmunoassay (RIA) and (b) to characterize the molecular forms of endothelin in human AF by high-performance liquid chromatography (HPLC) coupled with RIAs. Plasma samples from healthy male and female volunteers, and AF samples from full-term and mid-trimester pregnant women were extracted by C18 cartridges. Big ET, like ET-1, exists both in plasma and in AF. Significant increases (p less than 0.01) of big ET and ET-1 in AF from mid-trimester to full-term pregnancy were observed. The reverse-phase HPLC elution profile of immunoreactive (ir-) ET 1 presented a single major peak at a position corresponding to that of the standard ET-1 in the extracted AF. Two major components of ir-big ET were revealed: one corresponded to the elution position of standard big ET; the other one to that of the standard endothelin fragment [22-38] (EF). IN CONCLUSION: (a) Three molecular forms of ET exist in AF: ET-1 as a major form, big ET, and EF. (b) ET-1 and big ET are both present in higher concentrations in AF at full-term pregnancy than during mid-trimester. PMID- 1725403 TI - Endothelin-1- and endothelin-3-like immunoreactivity in human cerebrospinal fluid. AB - Using highly specific and sensitive radioimmunoassays (RIAs) for endothelin-1 (ET 1) and endothelin-3 (ET-3), we have determined the concentrations of ET-1- and ET 3-like immunoreactivities (LI) in human cerebrospinal fluid (CSF). The concentration of ET-3-LI (82.5 +/- 4.8 pg/ml) was about 1.5-fold greater than that of ET-1-LI (34.3 +/- 1.3 pg/ml). There was no significant correlation between ET-1-LI and ET-3-LI concentrations in human CSF (r = 0.44). Reverse-phase high-performance liquid chromatography (HPLC) revealed a single major component of ET-1-LI and ET-3-LI coeluting with authentic ET-1 and ET-3, respectively. It is suggested that ET-1 and ET-3 play a neuropeptide-like role in the central nervous system. PMID- 1725404 TI - Endothelin in patients with chronic renal failure. AB - To investigate the pathophysiological significance of endothelin-1 (ET-1) in renal failure, we measured plasma and urine concentrations of ET-1-like immunoreactivity (LI) by radioimmunoassay (RIA). The plasma ET-1-LI level was significantly increased in hemodialyzed and nonhemodialyzed patients with chronic renal failure (CRF). The increase in the plasma ET-1-LI level in CRF was much larger than that in other diseases in which the plasma ET-1-LI level was reported to be increased. The urine ET-1-LI level was lower, and daily excreted amounts of ET-1-LI were smaller in CRF patients than in normal subjects. These findings indicate that the increased plasma level of ET-1-LI in CRF results from the decreased clearance rate of ET-1-LI in the kidney. Gel permeation chromatographic analysis revealed that ET-1-LI in plasma from CRF consisted of small and large molecular forms of ET-1-LI. The small molecular form is presumably ET-1, and large molecular forms are big ET-1 and another component with the molecular weight of 6,000 as is the case of that in normal plasma (1). However, the ratio of the smaller to large molecular forms of ET-1-LI in CRF was 1:13 and was quite different from that in normal plasma (1:4). These observations clearly indicate that the elevation of plasma ET-1-LI level in CRF was mainly due to the increase in the large molecular form of ET-1-LI. PMID- 1725405 TI - Micelle formation of endothelin-1. AB - Circular dichroism of endothelin-1 synthesized through a continuous flow process reveals, when dissolved in water, a strong concentration dependence of the spectrum. Furthermore, the general feature of the spectrum rules out the possibility of the existence of any alpha and beta structures. In addition, surface tension and conductivity measurements suggest that the peptide aggregates through formation of micelles. PMID- 1725406 TI - Concentrations of endothelin-1 in human amniotic fluid at various stages of pregnancy. AB - To examine the possible role of endothelin in the initiation of parturition, endothelin-1-like immunoreactivity (ET-1-LI) in the amniotic fluid (AF) was determined by a sensitive radioimmunoassay (RIA) system. The concentrations of ET 1-LI in AF were 24.7 +/- 6.1 pg/ml (mean +/- SD, n = 6) at the second trimester and were significantly increased to 40.4 +/- 15.1 pg/ml (n = 6) at term. These ET 1-LI concentrations are about three- to fourfold higher than those observed in the maternal plasma. The gel permeation chromatography (GPC) profile of ET-1-LI extracted from second-trimester AF indicated that the ET-1-LI consisted of only one component of 6kET, the molecular weight of which was 6,000, larger than big ET-1. In contrast, ET-1-LI in the AF at term consisted of two components, ET-1 and 6kET. These findings suggest that the level of biologically active ET-1 in the AF increases markedly from second trimester toward term. A significant amount of ET-1-LI was also detected in the supernatant of amnion cell culture. The level of ET-1-LI in the culture medium after 12 h of incubation was almost comparable with that in AF at term. Thus, it is suggested that amnion tissue may be the major source of ET-1-LI in the AF. Together with the fact that ET-1 increases cytoplasmic calcium ion in the target cell, the results of present study raise the possibility that endothelin plays an important role in the initiation of parturition by triggering the production of prostaglandins in fetal membranes. PMID- 1725407 TI - Concentrations of endothelin-1 in maternal and umbilical cord blood at various stages of pregnancy. AB - To examine the role of endothelin (ET) in the maternal and fetal circulation, the levels of endothelin-1-like immunoreactivity (ET-1-LI) in the plasma of maternal vein (MV), umbilical vein (UV), and umbilical artery (UA) were determined by a sensitive radioimmunoassay (RIA). Levels of ET-1-LI in MV did not show any significant change (9.9 +/- 1.5 pg/ml, n = 26) throughout normal pregnancy and were similar to those of normal nonpregnant women (10.7 +/- 2.5 pg/ml, n = 5). Levels of ET-1-LI in UV and UA obtained at normal deliveries at term were about three times higher than those in MV. In the patients with mild and severe pre eclampsia, the levels of plasma ET-1-LI were significantly higher than those of normal pregnancy (14.3 +/- 2.2 pg/ml, n = 5 and 27.2 +/- 8.6 pg/ml, n = 5, respectively). However, in pregnant women with chronic hypertension, the levels of ET-1-LI did not increase when the hypertension did not worsen during pregnancy (11.4 +/- 1.6 pg/ml, n = 7). Moreover, in two pregnant women with abnormally stimulated coagulation, such as acute or subacute DIC, the levels of ET-1-LI were extremely high and returned gradually to those of normal nonpregnant women after the coagulation was normalized by treatment. These results suggest the possibility that ET-1 plays an important role in the pathophysiology of preeclampsia. PMID- 1725408 TI - Plasma immunoreactive endothelin-1 in pregnant women with and without pre eclampsia. AB - In normotensive nonpregnant women (n = 23), the plasma concentrations of immunoreactive endothelin-1 (ir-ET-1) were not different from nonpregnant women with essential hypertension (n = 15): 3.6 (2.0-5.4) ng/L (mean, range) versus 3.8 (2.4-5.8) ng/L. In normotensive pregnant women (n = 25), the plasma level of ir ET-1 was 2.1 (1.3-3.4) ng/L (p less than 0.01) lower than in normotensive nonpregnant women. Pre-eclamptic patients (n = 25) had elevated ir-ET-1 plasma levels of 5.0 (2.1-12.4) ng/L (p less than 0.001 for normotensive pregnant women, p less than 0.01 for nonpregnant women). The low level of ir-ET-1 in normotensive pregnant women may be explained by the increase in the distribution volume of ir ET-1 during the course of pregnancy. Damage of vascular endothelium is a consistent morphological abnormality in pre-eclampsia. Elevated ir-ET-1 in plasma might be a biochemical marker of this abnormality and may contribute to the pathogenesis of pre-eclampsia. PMID- 1725409 TI - Expression of endothelin-like peptide in the nervous system of the marine mollusk Aplysia. AB - The distribution of endothelin (ET)-like peptide immunoreactivity and mRNA in the nervous system of Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Using specific antisera to human endothelin-1 (ET-1) and to the c-terminal peptide of human big endothelin-1 (22 38) (big ET-1), ET-like immunoreactivity was localized to various types of neurons. Immunoreactive nerve fibers for both antisera were prominent throughout the nervous system. Hybridization of radiolabeled complementary RNA probes for human ET-1 or rat endothelin (endothelin-3, ET-3) to paraformaldehyde-fixed, paraffin-embedded sections of various ganglia revealed the presence of a large number of labeled neurons. The number of labeled neurons was higher than those that were immunoreactive for the ET antisera. Preabsorption of the ET antisera with their respective synthetic peptide or hybridization of sections with the sense probes confirmed the specificity of the data. The present study shows that members of the endothelin family may be found in very distant phylogenetic organisms. This conservation suggests a possible role for this peptide as a neurotransmitter and/or neuromodulator in the central nervous systems of diverse species. PMID- 1725410 TI - Localization of [125I]endothelin-1 in rat tissues observed by electron microscopic radioautography. AB - [125I]Endothelin-1 (ET-1) was administered intravenously into rats, and binding sites in various tissues were examined by electron-microscopic radioautography. Labeling was observed predominantly on the glomerular fenestrated endothelial cells of kidney, alveolar capillary endothelial cells of lung, fat-storing cells in the sinusoids of liver, and subepithelial myofibroblasts of the small intestine. At first, silver grains were observed mostly on the plasmalemma of these cells and with time translocated into the cytoplasm of these cells. Myofibroblasts of the small intestine were cultured to study the cellular effects of ET. By fura-2 fluorescence method, transient increase and oscillation of intracellular Ca2+ level were observed in the cultured myofibroblasts following the application of ET-1 or endothelin-3 (ET-3). These results suggest that ET induces diverse physiological reactions in specific cells of various tissues. PMID- 1725411 TI - Localization of endothelin-1 and its binding sites to the nervous system of the human colon. AB - Endothelin-1 (ET-1) has been reported to possess a wide variety of biological activities, including neurotransmission. Our aim was to demonstrate ET-like immunoreactivity (ET-LI) and its binding sites in human enteric nervous system using immunohistochemistry and in vitro autoradiography. ET-LI was displayed in nerve bundles and most of the ganglion cells in both myenteric and submucous plexuses, many of which costored VIP. [125I]ET-1 binding sites were identified, especially to plexuses, mucosa, and blood vessels. High-affinity (Kd = 0.35 +/- 0.014 nM; mean +/- SEM) binding sites, with a maximum binding capacity (Bmax) of 92 +/- 6.3 amol/mm2, were demonstrated in the myenteric plexus. This study provides evidence that ET-1 is a neuropeptide in the human colon with binding sites on neural plexuses and mucosa, indicating a possible role in the modulation of motility and secretion in the human intestine. PMID- 1725412 TI - Autoradiographic localization of [125I]endothelin binding sites in human blood vessels and coronary tissue: functional correlates. AB - The distribution of endothelin-1 (ET-1) receptors on human vascular tissue has been studied. High- and low-resolution autoradiography was used to determine the distribution of [125]ET binding sites in human blood vessels and ventricular myocardium. Dense, displaceable [125I]ET binding was associated with cardiac myocytes and the smooth muscle layer of all vessels were examined. There was also dense binding to vasa vasora. There was increased [125I]ET binding to atheromatous coronary arteries and vein graft, which was associated with the tunica media and vasa vasora or regions of neovascularization. Vasoconstrictor and positive inotropic activity of ET-1 has been established in vitro. The vasoconstrictor effect of ET-1 is likely to be mediated via the binding sites identified on vascular smooth muscle. The striking perivascular [125I]ET-1 binding suggests that ET-1 may also have constrictor activity on vasa vasora. There is experimental evidence that ET-1 has mitogenic activity on vascular smooth muscle cells in culture. The increased binding to both smooth muscle and regions of neovascularization in atheromatous vessels suggests that ET-1 may play a role in atherosclerosis. PMID- 1725413 TI - Endothelin-like immunoreactivity in the nervous system of invertebrates and fish. AB - There have been no reports on the distribution of immunoreactive endothelin (ir ET) in lower vertebrates and invertebrates, except for our previous studies on the nereid Neanthes diversicolor and the earthworm Eisenia foetida. In the present study, we found ET-like immunoreactivity in five species of invertebrates and two species of fish with antiserum against synthetic endothelin-1 (ET-1). Immunoreactive perikarya and nerve fibers were observed in the central nervous system of the slug Limax marginatus, the freshwater snail Indoplanorbis exustus, and the mussel Mytilus edulis in mollusks, the field cricket Gryllus bimaculatus in insects, and the tube tunicate Ciona intestinalis in protochordates. In the medaka, Oryzias latipes, ir-ET was found in the hypothalamoneurohypophysial system, the caudal neurosecretory system, the gill, and the kidney. Immunoreactive cells were also found in the mucous gland of the slug and in the adenohypophysis of the lamprey, Lampetra japonica. The wide distribution of ET like substances in invertebrates and fish provides evidence for the case that ET found in mammals has a long evolutionary history. PMID- 1725414 TI - Endothelin-1 in human skin: immunohistochemical, receptor binding, and functional studies. AB - The morphological distribution of endothelin-1 (ET-1) was investigated in human digital skin biopsies by immunohistochemistry using endothelin antisera, and by in vitro autoradiographic binding studies with 125I-labeled ET-1. In vitro studies on the responsiveness of cutaneous microvascular endothelium were carried out using human dermal microvascular endothelial cells (HDMEC) cultured from neonatal foreskin. Endothelin immunoreactivity was present in the endothelial cells of cutaneous blood vessels, in large and small arteries, veins in the dermis and capillaries, including those of the dermal papillae. Autoradiographic studies showed the presence of 125I-labeled ET-1 binding sites on the blood vessels and sweat glands. In confluent monolayers of HDMEC, ET-1 (100 pM-100 nM) significantly inhibited basal release of prostaglandin E2 (PGE2) (p less than 0.05), while causing a dose-dependent increase of intracellular cyclic adenosine monophosphate (cAMP) levels. cAMP accumulation, induced by 100 nM ET-1, was blocked by extracellular Ca2+ chelator EGTA, the intracellular Ca2+ chelator TMB 8, and dihydropyridine Ca(2+)-channel antagonist nifedipine (p less than 0.05), whereas ET-1 inhibition of PGE2 release was unaffected. These findings indicate that ET-1 may be important in the control of blood flow and vascular tone in the cutaneous microvasculature. PMID- 1725415 TI - Big endothelin-converting enzyme activities in subcellular fractions of bovine aortic endothelial cells. AB - A combination of HPLC elution patterns and peptide sequencing has been used to characterize two distinct activities present in subcellular fractions of bovine aortic endothelial cells (BAECs) capable of converting human big endothelin-1 (big ET-1) to mature (ET-1). A pepstatin-inhibitable activity with an acidic pH optimum present in a lysosome-enriched fraction cleaved big ET-1 at positions 18 and 21 at similar rates. A neutral pH activity present in a postlysosomal organelles subfraction was also able to convert big ET-1, and was inhibited by EDTA, but not by 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), an inhibitor of chymotrypsin-like serine proteases. PMID- 1725416 TI - Plasma immunoreactive endothelin, but not thrombomodulin, is increased in patients with essential hypertension and ischemic heart disease. AB - To ascertain an involvement of vascular endothelial cells in cardiovascular disease, we have determined plasma levels of two endothelium-derived substances, endothelin (ET) and thrombomodulin (TM), in essential hypertension (EH) and ischemic heart disease. Plasma ET was determined by radioimmunoassay (RIA) after extraction. Plasma TM levels were determined by enzymunoimmunoassay. Plasma ET levels were significantly elevated in patients with EH involving target organ damage, vasospastic angina pectoris (VSA), and acute myocardial infarction (AMI), especially in those associated with cardiogenic shock. There was a weak but significant correlation between plasma ET levels and serum creatinine concentration in patients with EH. Plasma ET levels were elevated even before the coronary spasm in patients with VSA, whereas they did not show any further increase during the spasm. In contrast, plasma TM levels in patients with EH and VSA did not show a significant difference from that in normal subjects. These results suggest that ET plays an important role in the pathophysiology of EH and ischemic heart disease, and also that increases in plasma ET cannot be simply attributed to a leakage of the peptide from the injured endothelial cells. PMID- 1725417 TI - Plasma endothelin-1 levels in patients with diabetes mellitus with or without vascular complication. AB - Plasma endothelin-1 (ET-1) concentrations were measured in 25 patients with non insulin-dependent diabetes mellitus (11 with angiopathy and 14 without angiopathy) and 21 normal subjects using radioimmunoassay specific to ET-1. Basal plasma immunoreactive (ir) ET-1 levels in diabetic patients with and without angiopathy were 1.73 +/- 0.29 and 1.68 +/- 0.20 pg/ml, respectively. Although high glucose levels may stimulate ET-1 release from vascular endothelial cells in vitro, our data suggest that circulating ET-1 may not be elevated in most diabetic patients with or without angiopathy. PMID- 1725418 TI - Pathophysiological role of endothelin in renal transplantation. AB - To assess the pathophysiological role of endothelin (ET) in the early post transplantation period, we followed plasma ET concentrations for 2 months from 1 week prior to surgery in four renal transplant patients. Plasma ET concentrations were elevated before transplantation in all patients. In two patients who developed marked pretibial edema, plasma ET concentrations increased before the onset of edema and before the increase in plasma atrial natriuretic peptide (ANP) concentrations, and remained high while the edema persisted. In the other two patients without edema, plasma ET and ANP concentrations fell toward normal immediately after transplantation. Plasma ET concentration increased transiently again in one of these patients when acute rejection occurred. After recovery from this rejection episode, the plasma ET concentration again fell to the normal range. There were significant correlations between plasma ET concentrations and changes in body weight and urine volume. These results suggest that the plasma ET concentration may reflect the function of transplanted kidneys and that ET may modulate body fluid volume regulation after transplantation. In addition, ET may participate in the pathogenesis of acute rejection. PMID- 1725419 TI - Possible role of endothelin in the pathogenesis of cerebral vasospasm. AB - Since the discovery of endothelin-1 (ET-1), its involvement in cerebral vasospasm after subarachnoid hemorrhage (SAH) has been suspected. We performed various experiments, first to demonstrate the presence of ET in both patients and dogs with SAH, and second to examine the effects of ET synthesis inhibition in experimental vasospasm. Here we report that ET was present in both plasma and cerebrospinal fluid (CSF) in SAH, but did not correlate with vasospasm. However, ET was locally expressed in the vascular endothelium in vasospasm. Several therapeutic approaches causing the inhibition of ET synthesis were effective in preventing the development of vasospasm. Such approaches utilized drugs that inhibited RNA and DNA synthesis. Among them, actinomycin D treatment was most effective. We also utilized phosphoramidon, a recently found conversion inhibitor of big ET to ET. However, this product failed to ameliorate the development of vasospasm. Therefore, although we cannot yet conclude that ET is the main cause of cerebral vasospasm, it may, at least, act as one of the modifying factors in cerebral vasospasm. PMID- 1725420 TI - Role of endothelin in the development of Dahl hypertension. AB - To evaluate the possible role of endothelin in the development and/or maintenance of hypertension in Dahl rats, we examined the responsiveness of isolated vascular smooth muscle and glomerular mesangial cells, as well as deendothelialized vascular ring preparations to endothelin-1 (ET-1). Production of immunoreactive endothelin (ir-ET) was studied in freshly isolated glomeruli and renal medullary slices. Both glomerular mesangial cells and vascular smooth muscle cells obtained from prehypertensive Dahl-S rats exhibited an exaggerated [Ca2+]i response to ET 1, as compared with cells obtained from Dahl-R rats. This was paralleled by the enhanced isometric contraction of vascular rings obtained from prehypertensive Dahl-S rats. ir-ET production was doubled in response to 0.1 mM ouabain in tissue samples obtained from prehypertensive Dahl-S, but not Dahl-R rats. This effect was not observed in tissues obtained from animals fed a 4% NaCl diet (hypertensive). Immunocytochemistry of ET distribution in the outer medullary stripe showed approximately a 40% higher fluorescence intensity in sections obtained from Dahl-S rats fed 4% NaCl diet as compared with Dahl-R rats fed the same diet. Northern blot analysis of poly(A)+ RNA extracted from medullae of prehypertensive Dahl-S and -R rats using a full-length cDNA probe for rat ET-1 revealed a marginal induction of pre-pro-ET-1 message in Dahl-S samples after 30 and 60 min of incubation with 0.1 mM ouabain. In conclusion, increased responsiveness of target cells to ET-1 and inducibility of ir-ET production in prehypertensive Dahl-S rats are in favor of a possible role of this peptide in the pathogenesis of Dahl hypertension. We hypothesize that ouabain-like factor(s) may trigger production of ET, thus serving as a link between high-salt intake and the development of hypertension in Dahl-S rats. PMID- 1725421 TI - Endothelin-1 and big endothelin cause subarachnoid hemorrhage in the anesthetized rabbit. AB - Intra-arterial injection of endothelin-1 (ET-1) (1 nmol/kg; n = 8) or human big endothelin-1 (b-ET-1; 3 nmol/kg; n = 8) into anesthetized rabbits produced a significant rise in left ventricular systolic pressure (LVSP) and caused subarachnoid hemorrhage (SAH) in 75 +/- 17% and 88 +/- 12% of the experiments, respectively. In all animals, the SAH occurred in the subarachnoid space around the distal part of the basilar artery complex. The cyclooxygenase inhibitor indomethacin (5 mg/kg i.v.) significantly potentiated the pressor effect of both peptides, and all animals pretreated with indomethacin prior to ET-1 (n = 3) or b ET (n = 3) developed SAH. In contrast, rabbits treated with vehicle (saline; n = 7), indomethacin alone (n = 3), or the carboxy-terminal fragment of b-ET (CT 22 38; 3 nmol/kg i.a.; n = 3) developed neither a rise in LVSP nor SAH. A rise in blood pressure alone is unlikely to account for the SAH brought about by the peptides for angiotensin II (1 nmol/kg/min for 30 min; n = 7) produced a significantly greater increment in LVSP than ET-1 or b-ET, but did not cause SAH. In addition, there was no significant correlation between the rise in LVSP produced by ET-1 or b-ET and the severity of the SAH. PMID- 1725422 TI - Endothelin-1-induced hypertension: a consequence of medullary ischemia? AB - The central hemodynamic effects of the peptide endothelin-1 (ET-1) have been investigated in the conscious, normotensive rat. Intracisternal administration of ET-1 (0.01-0.03 nmol) gave rise to an increase in mean arterial pressure with minimal effects on heart rate and was accompanied in some cases by barrel rolling activity. Intracisternal administration of 0.03 nmol ET-1 gave rise to a significant elevation in plasma noradrenaline and adrenaline levels. This elevation in plasma catecholamines was present only in those animals that also exhibited marked behavioral changes. Autoradiographic measurement of cerebral blood flow carried out during the maximum response to 0.03 nmol of intracisternal ET-1 revealed a widespread and profound ischemia throughout the caudal brainstem. Cerebral ischemia is known to activate compensatory circulatory reflexes in the medulla oblongata that result in increased sympathetic and vagal outflow. This is the most likely cause of intracisternal ET-1-induced hypertension. ET-1 is unique in its ability to override the brain's autoregulatory mechanisms and induce ischemia of pathological magnitude. PMID- 1725423 TI - The biological activity of endothelin-1 analogues in three different assay systems. AB - Endothelin (ET) is a vasoconstrictor peptide with 21-amino acid residues. In this study, we determined the relative potencies of ET-1 analogues to investigate the essential moiety of ET-1 for expression of its biological effect. We synthesized ET-1 analogues with the substitution of one amino acid at positions 2-18 and 21. All ET-1 analogues had the proper intramolecular disulfide bonds. We have conducted a systematic survey to compare the biological activity of ET-1 analogues in three different assay systems: the vasoconstrictor activity in the rat pulmonary artery, the nonvascular smooth muscle contracting activity in the rat trachea, and the pressor activity in the conscious rat. In the pulmonary arterial preparation, the carboxyl groups of Asp8, Glu10, and Asp18, the aromatic groups of Phe14, His16, and Trp21, and the hydrophobic group of Leu17 contributed to the expression of the vasoconstrictor activities. However, the contracting activities in the tracheal preparations were retained even upon replacement of these amino acid residues by alanine or other amino acids. Moreover, substitution of the amino acid residue of ET-1 at positions Ser5, Lys9, and Tyr13 for alanine was found to increase the potency of ET-1 by a factor of about 2. With respect to vasopressor effects, the activities were retained, and all ET-1 analogues showed relative potencies ranging from 5 to 90% of ET-1. These findings suggest that different types of receptors may be present in different organs. PMID- 1725424 TI - Down-regulation of endothelin-1 receptors by protein kinase C in streptozotocin diabetic rats. AB - The vasoconstrictor response is defective in diabetes mellitus (DM). Activation of protein kinase C (PKC) is also known to prevail in diabetes mellitus, and it is thought to be secondary to abnormal diacylglycerol metabolism. To ascertain whether this PKC activation in diabetes underlies the vasomotor defect by regulating biological receptors, we studied the characteristics of the receptor for endothelin (ET), "the vasoconstrictor of injury." For this purpose, diabetes was induced in rats by intravenous streptozotocin. One to 2 weeks after streptozotocin treatment (average glucose at time of experiments: 518 mg/dl), glomeruli were isolated and assessed for ET receptor and PKC activity. ET receptor characteristics were also assessed following infusion of a specific PKC inhibitor, 1-(5-isoquinolinesulfonyl)piperazine (CI). For comparison, nondiabetic controls with and without PKC inhibitor were studied. No differences in high affinity ET-1 receptor (ER-1) characteristics were found among the diabetic and normal rats. In contrast, receptor density for the lower-affinity receptor (ER-2) was significantly depressed in DM without changes in the equilibrium dissociation constant. Infusion of CI 20 min before glomerular harvesting did not affect the glomerular PKC activity in controls (particulate: 28.0 +/- 4.0% of total activity to 22.0 +/- 3.9%, n = 3). In contrast, in diabetes mellitus rats infused with CI, PKC activity decreased (particulate: from 44.7 +/- 2.9% of total activity to 18.5 +/- 3.2%, n = 3, p less than 0.05). This CI-induced suppression of PKC in DM was accompanied by complete reversal in down-regulation of ER-2 receptors. Thus, DM is characterized by down-regulation in low-affinity ET-1 receptors. Furthermore, this receptor down-regulation can be reversed by abolishing abnormally enhanced PKC activity. These results indicate that abnormal activation of PKC may underlie the profoundly vasodilative status and defective vasoconstrictor response characterizing DM. PMID- 1725425 TI - Is big endothelin converted to endothelin-1 in circulating blood? AB - Although evidence has been accumulating to support an intracellular processing of big endothelin-1 (big ET-1) to ET-1, molecular conversion in the circulating blood remains to be elucidated. The present study was undertaken to investigate whether big ET-1 was converted to ET-1 in human blood. In the first experiment, normal serum with synthetic big ET-1 exogenously added or serum from patients with chronic renal failure was incubated in vitro at 37 degrees C for 1 h. In the second experiment, synthetic big ET-1 was incubated in the whole blood at 37 degrees C for 1 h. In the third experiment, synthetic big ET-1 was administered intravenously in anesthetized rat and a plasma sample was obtained before and after 15 min and 1 h. After respective incubation, molecular forms of ET were determined by a combination of reverse-phase high-performance liquid chromatography and radioimmunoassay. There was no significant conversion of big ET-1 to ET-1 in the serum obtained from normal and CRF patients. However, there was a slight but significant increase of ET-1 after incubation of big ET-1 in the whole blood or after administration of big ET-1 in anesthetized rat. The conversion in the whole blood was inhibited by 5 mM EDTA. These results suggest that circulating blood may not be a major site of molecular conversion from big ET-1 to ET-1, although conversion does occur in the circulation by blood cell mediated process. PMID- 1725426 TI - Molecular form of immunoreactive endothelin in plasma and urine of normal subjects and patients with various disease states. AB - To elucidate the pathophysiologic significance of the family of endothelin (ET) peptides, we have investigated plasma and urinary immunoreactive (ir-) ET levels and its molecular forms in normal and pathological conditions. Plasma and urine ET were extracted with an Amprep C2 column. The molecular form of ET was determined by a combination of radioimmunoassay and reverse-phase high performance liquid chromatography. Although plasma ir-ET was composed mainly of big ET and endothelin-1 (ET-1) in normal subjects, that in acute myocardial infarction, chronic renal failure (CRF), essential hypertension, and vasospastic angina pectoris was characterized by an increase of high molecular ir-ET in addition to increases in big ET and ET-1. Urinary ir-ET in both normal subjects and patients with CRF was composed mainly of a high molecular form in addition to big ET and ET-1. These results suggest that the biosynthetic and/or degradation process of ET under pathological conditions appears to be different from that under normal conditions. PMID- 1725427 TI - Relationship between fetal hypoxia and endothelin-1 in fetal circulation. AB - The role of endothelin-1 (ET-1), a potent vasoconstrictor peptide secreted by endothelial cells, in fetal circulation was investigated in relation to fetal hypoxia. Umbilical venous blood was obtained from 23 subjects who delivered between 36 and 41 weeks of gestation. In all cases, pH of umbilical venous blood was measured immediately after the delivery of placenta. ET-1 was extracted from the umbilical venous plasma by an Amprep C2 column and determined by a specific radioimmunoassay. Immunoreactive ET levels in the umbilical cord plasma (pg/ml, mean +/- SEM) from subjects with umbilical venous blood pH levels below 7.30 (n = 9) were 12.53 +/- 2.03, significantly higher than those with pH levels above 7.30 (6.44 +/- 1.11, n = 14). These results indicate that ET-1 in the fetal circulation may be involved in the regulation of the circulation in response to changes in acid-base balance related to fetal hypoxia. PMID- 1725428 TI - Acute and chronic effects of anti-endothelin-1 antibody on blood pressure in spontaneously hypertensive rats. AB - To evaluate the significance of endogenous endothelin (ET) in the maintenance of blood pressure in hypertensive and normotensive conditions, we examined the acute and chronic effects of anti-ET-1 antibody in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Immunoglobulin (Ig) solution purified from anti-ET-1 rabbit serum was injected intravenously in conscious rats aged 12-14 weeks (SHR: n = 7, WKY: n = 7). In the control group (SHR: n = 5, WKY: n = 4), Ig prepared from nonimmune rabbit serum was administered. One week after the first injections of Ig, second intravenous injections were given. After the intravenous injections of ET-1 antibody, there were no significant changes in directly measured blood pressure (BP) for up to 60 min in both strains. On the first, third, and seventh days after the first administration of ET-1 antibody, as well as on the first, third, and seventh days after the second injection, there were no appreciable differences in BP between SHR treated with anti-ET-1 antibody and SHR with control Ig. No significant changes in BP were observed throughout the observation period in WKY groups. Plasma levels of immunoreactive ET-1 were similar in SHR and WKY. This study suggests that circulating ET may not play a significant role in the maintenance of hypertension in SHRs or in the regulation of blood pressure in WKY rats. PMID- 1725429 TI - Chronic synergistic effect of endothelin-1 and angiotensin II on blood pressure in conscious rats. AB - We assessed whether there is an interaction between angiotensin II (Ang II) and endothelin-1 (ET-1) in the regulation of blood pressure and sodium and water metabolism in rats. Male Sprague-Dawley rats were divided into four groups. Group I rats received Ang II at a subpressor dose (400 micrograms/kg/day i.p.) for up to 6 days. Group II rats received ET-1 at a subpressor dose (3 micrograms/kg/day i.v.) for up to 6 days. Group III rats received both the subpressor dose of Ang II and the subpressor dose of ET-1. Group IV rats received vehicle only. There was no significant difference in systolic blood pressure (SBP) among groups I, II, and IV during the study. On day 6, SBP in groups I, II, and IV was 148.0 +/- 3.0, 142.7 +/- 2.9, and 143.5 +/- 3.0 mm Hg, respectively. On the other hand, SBP in group III was higher than those of the other groups on day 2 and remained elevated thereafter. On day 6, SBP in group III rats was 189.0 +/- 12.5 mm Hg. There were no significant differences in body weight, fluid intake, urine volume, urinary sodium excretion, or urinary potassium excretion among the four groups. The present results suggest that Ang II and ET-1 exert their pressor effects synergistically and might play a role in controlling blood pressure. They also suggest the possibility of the existence of a common pathway in the hypertension producing mechanism of these two peptides. PMID- 1725430 TI - The evolutionary history of the sarafotoxin/endothelin/endothelin-like superfamily. AB - The evolutionary relationships among 17 protein and nucleic acid sequences from the sarafotoxin/endothelin/endothelin-like superfamily of peptides were studied. The endothelin/endothelin-like gene family has diverged from an ancestral gene that has experienced an exon duplication event followed by two complete gene duplications. The sarafotoxin lineage diverged from the ancestral gene prior to the first gene duplication event. In several lineages, the peptides have independently accumulated identical amino acid replacements in position 2. This finding supports the hypothesis that residue 2 is crucial to biological activity. PMID- 1725431 TI - Establishment of a human cell line highly expressing endothelin in serum-free medium. AB - Human preproendothelin-1 (prepro-ET-1) was transfected into an immortal human endothelial cell line that had been cultivated in a serum-free medium. Several transformants selected with Ecogpt selection were probed to have prepro-ET insert, express prepro-ET-1 mRNA, and secrete immunoreactive ET (ir-ET). One of the transformants, t-HUE2-1, secreted 30 times the amount of ET-1 and the ratio of mature ET-1 to big ET-1 was 11-fold higher compared with normal human endothelial cells. Thus, this transformant might be of use to study regulation of the posttranscriptional process of prepro-ET-1 in human cells. PMID- 1725432 TI - Structure of the precursor for vasoactive intestinal contractor (VIC): its comparison with those of endothelin-1 and endothelin-3. AB - Vasoactive intestinal contractor (VIC) is a member of the endothelin (ET) peptide family, which evokes a strong contractile response in the ileum, its gene being expressed only in the intestine. Using dot blot analysis, we carried out an interspecies comparison of the nucleotide and deduced amino acid sequences of the precursor for VIC with those of ET-1 and ET-3 to investigate the physiological significance of processing of the precursor for VIC. The highly conserved amino acid sequence was observed between the big form region (big VIC, big ET-1, and big ET-3) of about 40 amino acids and the like peptide region (VIC-like peptide, ET-1-like peptide, and ET-3-like peptide) of 15 amino acids downstream from the big form region. Sequence identity of amino acids of the precursors of ET-1 and ET-3 with that of VIC was 29 and 28%, respectively. Thus, the precursors for the three peptides might have arisen from a common progenitor gene. However, apparent cleavage sites of the like peptide regions are rather unique in the VIC-like peptide, i.e., it had dibasic amino acids at the amino and carboxy termini. Therefore, we suggest that the VIC-like peptide might be liberated from its precursor protein and play some role in the intestine in vivo. PMID- 1725433 TI - Structure-activity studies of the C-terminal region of the endothelins and the sarafotoxins. AB - Peptides corresponding to the C-terminal 16-21 hexapeptide of the endothelins ( His-Leu-Asp-Ile-Ile-Trp) and sarafotoxins (a-c) (-His-Gln-Asp-Val-Ile-Trp) were prepared to study the role of the individual amino acids in receptor recognition and activation. Receptor binding in rabbit aorta, rabbit pulmonary artery, and rat heart ventricle is reported for all analogues. In addition, selected C terminal hexapeptides have been evaluated functionally in two tissues (rabbit pulmonary artery and rat left atria). The C-terminal carboxylate, indole nitrogen, and nature of the aromatic residue are all important for receptor binding, but N-terminal acetylation has no effect. L-Amino acids are required in positions 19 and 21, whereas D-amino acids are tolerated in 17 and 18. D-Amino acids in positions 16 and 20 enhance the binding affinity of the hexapeptide in all three tissues. The nature of the basic residue at position 16 is important. Glu and Asn are acceptable substitutions for Asp18, although Ala leads to a substantial loss in binding. The binding of the C-terminal hexapeptide of SRTX-a, -b, and -c is less than ET[16-21] and this appears to be primarily due to the substitution of Gln for Leu17. None of the 16-21 hexapeptides showed any functional activity in the tissues studied. PMID- 1725434 TI - Monocyclic endothelins: examination of the importance of the individual disulfide rings. AB - Monocyclic fragment analogues of endothelin-1 (ET-1) were prepared by standard solid-phase peptide synthetic methods. The analogues were designed to determine the importance of the unique bicyclic structure of the endothelins, vasoactive intestinal contractor, and the sarafotoxins to their binding affinity and functional activity. The binding affinity of the monocyclic analogues to the endothelin receptor was examined in three tissue preparations: (a) rabbit pulmonary artery, (b) rabbit aorta, and (c) rat heart ventricle. Functional activity was assessed in two tissues: (a) rabbit pulmonary artery and (b) rat left atria. Binding was not observed for the disulfide bridged cyclic 3-11 loop of ET-1 at concentrations of up to 100 microM. Attachment of the weakly binding 16-21 C-terminal hexapeptide of ET-1 to the cyclic 3-11 loop did not significantly enhance the observed receptor binding over that of the hexapeptide. Monocyclic Cys-Ser-Aoc-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp (Aoc = 8 aminooctanoic acid) exhibited the greatest binding affinity (1.0 to 1.6 microM) in all three tissues, but was still approximately 1,000-fold less effective than ET-1. At concentrations of up to 30 microM, none of the analogues exhibited any functional activity. PMID- 1725435 TI - Conversion of big endothelin-1 to endothelin-1 by phosphoramidon-sensitive metalloproteinase derived from aortic endothelial cells. AB - When big endothelin-1 (big ET-1, 1-39) was incubated with the membrane fraction obtained from cultured endothelial cells (ECs) at pH 7.0 for 6 h, the immunoreactive (ir) ET in the reaction mixture was markedly increased. Phosphoramidon, a metalloproteinase inhibitor, as well as metal chelators specifically suppressed the above increase. Using reverse-phase high-performance liquid chromatography, ir-ET was confirmed to be ET-1[1-21]. In addition, we noted that the alterations in ET-1 correlated with those in the C-terminal fragment (CTF, 22-39) of big ET-1. When cultured ECs were incubated with phosphoramidon, time-dependent secretion of ET-1 and CTF from the cells was markedly suppressed. In contrast, the secretion of big ET-1 was increased by phosphoramidon. Thiorphan, a specific inhibitor of neutral endopeptidase 24.11, was without effect on the secretion of ET-related peptides. Moreover, phosphoramidon potently inhibited the hypertensive effect of big ET-1 without affecting the ET-1-induced hypertension in anesthetized rats. From these findings, it seems reasonable to consider that phosphoramidon-sensitive and membrane-bound metalloproteinase, which is not a neutral endopeptidase 24.11, is the most plausible candidate for big ET-1-converting enzyme in vivo. PMID- 1725436 TI - Mode of cleavage of porcine big endothelin-1 by aspartic proteinases. AB - Cleavage sites of porcine big endothelin-1 (big ET-1, 1-39) by cathepsin D were examined and compared with those by other aspartic proteinases including pepsin. Cathepsin D cleaved not only the Trp21-Val22 bond, but also the Asp18-Ile19 bond of big ET-1[1-39]. The mature ET-1[1-21], generated by the cleavage between Trp21 and Val22, was subsequently degraded by removal of the C-terminal tripeptide (Ile19-Ile20-Trp21). On the other hand, pepsin cleaved the Trp21-Val22 bond of big ET-1[1-39] to produce ET-1[1-21], but did not degrade the generated ET-1[1 21]. These results indicate that aspartic proteinases such as cathepsin D and pepsin are capable of converting big ET-1[1-39] to ET-1[1-21], whereas the former proteinase is by no means specific for the Trp21-Val22 bond of big ET-1[1-39]. PMID- 1725437 TI - Smooth muscle cells contain a factor responsible for decreasing big endothelin and endothelin-1 produced by cultured endothelial cells. AB - The production of big endothelin-1 (big ET-1) and its processing to endothelin (ET-1) were examined in cocultures of bovine pulmonary artery endothelial cells (BPECs) and human bronchiolar smooth muscle cells (HBSMCs). The BPECs produced immunologically reactive ET-1 (ir-ET-1) and big ET-1 (ir-big ET-1) that increased linearly in the cell culture media over a 72 h time course when confluent monolayers were incubated at 37 degrees C in serum-free media. HBSMCs maintained in serum-free media over a 72-h period did not produce any detectable ir-big ET-1 or ir-ET-1. After coculture of these two cell types for 24 h (the monolayers were physically separated), the culture medium contained no detectable ir-big ET-1 or ir-ET-1. When medium conditioned for 24 h with the HBSMCs was transferred to the BPEC monolayers, the accumulation rates of ET-1 and big ET-1 were attenuated 70 and 10%, respectively. When medium conditioned for 24 h with the BPECs was transferred to the HBSM cell monolayers, the amount of ir-ET-1 and ir-big ET-1 in the medium decreased greater than 90% over the next 24 h. Cellular binding and uptake could not account for this decrease as ascertained by quantitation of cellular levels of ET-1 and big ET-1. When conditioned medium from the HBSMCs was added to conditioned medium from the BPECs in a 1:1, 2:1, 3:1, 1:2, or 1:3 ratio (v/v) in the absence of cells, the amount of ET-1 or big ET-1 present in the EC medium did not decrease significantly over the following 24 h. These data imply that HBSMCs contain a factor that is responsible for decreasing the accumulation of ir-big ET-1 and ir-ET-1 in the cell culture media of BPECs. The data also suggest that the most likely mechanism for this effect is a factor that degrades BET-1 and ET-1 at different rates. PMID- 1725438 TI - Endothelin-1 as an autocrine/paracrine factor for human tumor cell lines. AB - We determined whether endothelin-1 (ET-1) is produced and secreted by human tumor cell lines and whether ET-1 can affect cytosolic free Ca2+ concentrations ([Ca2+]i) in these cells. Dilution curves of extracts of cultured conditioned media from several human cancer cell lines (HeLa, HEp-2, DLD-1, and Ca9-22) were parallel to those of standard ET-1 in radioimmunoassay (RIA). Amounts of ET-1 like immunoreactivity released into conditioned media were reduced by omitting fetal bovine serum from culture media. Synthetic ET-1 dose-dependently induced gradual and sustained increases in [Ca2+]i in HeLa, HEp-2, GOTO, and Lovo cells, an effect completely abolished by a Ca2+ channel blocker or by chelating extracellular Ca2+. These data suggest an autocrine/paracrine role of ET-1 in human tumor cell lines. PMID- 1725439 TI - Endothelin receptors and coupled GTP-binding proteins in glomerular mesangial cells. AB - Endothelins (ETs) are a family of vasoactive peptides with profound biological actions in diverse cell systems. Among its varied actions, ET stimulates phospholipase C (PLC) in cultured mesangial cells. We investigated the presence of specific ET receptors in rat mesangial cells in culture, and studied the role of GTP-binding proteins (G proteins) in coupling PLC to the endothelin receptor. [125I]ET binding was time- and temperature-dependent, and Scatchard analysis of saturation data showed a single class of high-affinity binding sites. Heterologous displacement with two related peptides, ET-3 and sarafotoxin (SFTX), revealed the presence of two binding sites for these isopeptides. Preincubation of cells with ET-1 reduced the receptor number without affecting Kd, and this effect was not prevented by protein kinase C inhibition or downregulation. We confirmed the presence of a 41- to 43-kDa pertussis toxin substrate in rat mesangial cell membranes in an ADP ribosylation assay. ET-1 inhibits and GDP beta S enhances toxin-catalyzed transfer of ADP-ribose to this substrate. ET-1 potentiated GTP gamma S-induced phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. In addition, pertussis toxin partially inhibited ET-stimulated PI hydrolysis in intact mesangial cells. Pertussis toxin also reduced the magnitude of ET-stimulated intracellular free calcium [( Ca2+ )i]. Thus, ET-1 binds to specific receptors on rat mesangial cells and activates PLC, in part, through a pertussis toxin-sensitive G-protein. PMID- 1725440 TI - Endothelin-induced activation of phosphoinositide turnover, calcium mobilization, and transmitter release in cultured neurons and neurally related cell types. AB - Endothelin (ET)-related peptides robustly stimulated [3H]-inositol phosphate (IP) formation in cultured cerebellar granule cells, astrocytes, and C6 glioma cells. Their agonist selectivities were ET-1 = ET-2 greater than or equal to sarafotoxin S6b greater than ET-3 greater than big ET-1 for granule cells and ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for cerebellar astrocytes and C6 glioma cells. These effects were Ca(2+)-dependent but insensitive to antagonists of L-type Ca2+ channels and the Na+/Ca2+ antiporter. Pretreatment of cells with ET-1 or S6b induced homologous desensitization of phosphoinositide (PI) response mediated by ET receptors. Long term pertussis toxin (PTX) treatment attenuated the phosphoinositide (PI) response in astrocytes and glioma but not in granule cells. ET-1 and its related peptides increased [Ca2+]i in C6 glioma by two distinct pathways: IP3-induced Ca2+ mobilization or receptor-operated Ca2+ influx. La3+, Mn2+, and Cd2+ inhibited the Ca2+ influx and sustained PI turnover, while Ca2+ mobilization was attenuated by phorbol ester and TMB-8. ET-induced Ca2+ influx was essential for the sustained [Ca2+]i increase and PI turnover. Homologous desensitization of [Ca2+]i increase was also noted. In cerebellar granule cells, ET evoked the release of [3H]D-aspartate from these neurons. This action appears to be dependent on PI hydrolysis and [Ca2+]i increase and modulated by protein kinase C. PMID- 1725441 TI - Regulation of intracellular Ca2+ and gene expression by endothelin-1. AB - In addition to its powerful vasoconstrictive activity, endothelin-1 (ET-1) is a potent agonist for stimulating a multitude of second messenger pathways. In the Rat-1 fibroblastic cell line, ET-1 induces a robust elevation of the intracellular levels of Ca2+, diacylglycerols (DGs), and inositol trisphosphate (IP3). Although low concentrations of ET-1 stimulate a significant increase in the rate of Ca2+ influx, this Ca2+ influx is not required for the observed increases in either IP3 or DG levels following ET-1 treatment, as both of these effects are observed even in the absence of extracellular Ca2+. The ability of ET 1 to stimulate Ca2+ influx shows a biphasic pattern, such that Ca2+ influx is stimulated at low ET-1 concentrations and inhibited at high concentrations. Investigations of the molecular mechanisms underlying this biphasic response indicate that elevated intracellular Ca2+ levels exert a negative feedback inhibition on Ca2+ influx, which can be relieved by the chelation of intracellular Ca2+. The ability of ET-1 to activate a number of distinct signal transduction pathways appears to have direct functional significance in determining the targeting of ET-1 activation. Short-term effects of ET-1 stimulation such as the induction of gene expression appear to be independent of ET-1's ability to activate protein kinase C (PKC) by elevating DG levels, as depletion of PKC activity has little or no effect on gene expression. In contrast, the ability of ET-1 to induce the rapid expression of the VL30 gene is totally dependent upon the ability of ET-1 to elevate intracellular Ca2+ levels above a specific threshold. Activation of PKC by ET-1, however, is essential for the long-term effects of ET-1 on cell proliferation and anchorage-independent growth, as the ability of ET-1 to promote DNA synthesis and to synergize with epidermal growth factor in augmenting anchorage-independent growth is significantly inhibited by prior PKC depletion. Thus, in fibroblasts, ET-1 appears to activate at least two bifurcating pathways: a Ca(2+)-sensitive pathway involved in the regulation of gene expression, and a PKC-dependent pathway required for the mitogenic effects of ET-1. PMID- 1725442 TI - Induction of endothelin secretion by angiotensin II: effects on growth and synthetic activity of vascular smooth muscle cells. AB - Angiotensin II induces the synthesis and secretion of endothelin by cultured rat vascular smooth muscle cells. Previous studies demonstrate that angiotensin II also activates the synthesis of extracellular matrix-associated glycoconjugates (glycopeptides and proteoglycans) by cultured smooth muscle cells. Furthermore, in culture medium containing mitogen-depleted, plasma-derived serum (1.0%), angiotensin II stimulates the growth of rat smooth muscle cells. Therefore, the influence of endothelin-1 (ET-1) on the growth and matrix elaboration by cultured rat smooth muscle cells was studied to assess its contribution to the stimulatory activity of angiotensin II. In quiescent cultures of smooth muscle cells, no stimulation of the incorporation of [3H]thymidine into DNA by ET-1 was apparent, even at maximal doses (10(-7) M). Under the same conditions, the synthesis of extracellular matrix glycoconjugate material was not enhanced by ET-1, unlike angiotensin II. In cultures maintained in medium containing 1.0% plasma-derived serum, ET-1 was incapable of stimulating either proliferation or incorporation of [3H]thymidine into DNA whereas angiotensin II stimulated both activities. ET-1, however, did induce the expression of growth factor and thrombospondin genes in quiescent smooth muscle cultures. The data suggest that growth stimulation of smooth muscle cells by angiotensin II does not occur as a consequence of stimulated ET-1 production. PMID- 1725443 TI - Functional interaction of lacidipine with calcium channels in vascular smooth muscle. AB - We investigated the effect of lacidipine on 100-mM K(+)-evoked contractions and on 100-mM K(+)-evoked 45Ca2+ influx in rat aorta. Moreover, we compared the effect of prolonged depolarization on lacidipine inhibition and (+)isradipine inhibition of 100-mM K(+)-evoked contractions and the reversal of this inhibition by washing with a Ca(2+)-free physiological solution or with a Ca(2+)-free 40-mM K+ depolarizing solution. Lacidipine dose-dependently depressed the response to depolarization, and the pattern of inhibition was typical of voltage-dependent dihydropyridines. The concentration-inhibition curves on K(+)-evoked contraction and on K(+)-evoked 45Ca2+ influx were superimposed; the IC50 was 0.09 nM for the former and 0.11 nM for the latter. The inhibitory effect of lacidipine 60 pM or (+)isradipine 60 pM on rat aorta contraction was significantly enhanced when the preparations had been preincubated in the presence of the drugs in a K(+)-rich depolarizing solution. The inhibitory effect of lacidipine 60 pM or (+)isradipine 100 pM on rat aorta contraction was fully reversed after washing with a physiologic solution. When the washout was performed with a 40-mM K+ depolarizing solution, the inhibition was maintained in lacidipine-treated preparations, whereas it was reversed in the (+)isradipine treated ones. We conclude that lacidipine inhibits rat aorta contraction by interacting specifically with voltage-operated Ca2+ channels. Moreover, unlike with (+)isradipine, the complex formed with lacidipine and inactivated Ca2+ channels seems to not dissociate, because the inhibitory effect persisted in spite of removal of the drug from the depolarizing solution in which the arteries were immersed. PMID- 1725444 TI - The pharmacokinetic and pharmacodynamic interaction between lacidipine and propranolol in healthy volunteers. AB - The pharmacokinetic and pharmacodynamic profiles of lacidipine, a 1,4 dihydropyridine calcium antagonist, and the beta-adrenoceptor blocker propranolol were determined alone and in combination in 24 healthy male volunteers. One group (I) of 12 subjects received a single oral dose of 4 mg lacidipine on two separate occasions, which was taken together with a single oral dose of either 160 mg propranolol or placebo; a second group (II) of 12 subjects received propranolol on two occasions, taken with either lacidipine or placebo. Propranolol significantly decreased the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) of lacidipine (by 38% and 42%, respectively) whereas lacidipine significantly increased the Cmax and AUC of propranolol (by 35% and 26%, respectively); neither the time to maximum plasma concentration (tmax) nor the terminal half-life (t 1/2) were affected. With regard to the pharmacodynamics, in Group I, there was a greater reduction in supine systolic blood pressure (6 mm Hg) and diastolic blood pressure (4 mm Hg) compared to the reduction produced by lacidipine alone, and pulse rate was approximately 5 beats/min less. In Group II, a significantly greater reduction (6 mm Hg) in supine systolic blood pressure compared to the reduction produced by propranolol alone occurred, but there was no marked difference in supine diastolic blood pressure and pulse rate. In conclusion, a modest pharmacokinetic and pharmacodynamic interaction is evident and should be evaluated further in patients with hypertension. PMID- 1725445 TI - Clinical position of lacidipine, a new dihydropyridine calcium antagonist, in the treatment of hypertension. AB - Lacidipine is a calcium antagonist of the dihydropyridine group that is highly selective for vascular tissues. It has been tested during the last few years for the treatment of arterial hypertension. We evaluated the clinical position of this calcium antagonist on the basis of clinical results. From the dose-ranging studies carried out to determine the efficacy of lacidipine, it has been shown that the dose range of 2-6 mg of lacidipine given once daily is effective in reducing blood pressure levels. Lacidipine efficacy (4-6 mg/o.d.) also has been compared with that of the diuretic hydrochlorothiazide, the calcium-antagonist nifedipine, and the beta-blocker atenolol in three different clinical trials according to the same protocol. The results indicated that lacidipine, hydrochlorothiazide, nifedipine, and atenolol caused similar reductions both in systolic and diastolic blood pressure after 1 and 2 months of treatment. Similar blood pressure levels were also reached when another antihypertensive agent (atenolol or hydrochlorothiazide) had been added in nonresponders to monotherapy. Heart rate showed a significant decrease only under atenolol treatment, either monotherapy or associated to the previous treatment. In these comparison studies, tolerability was also evaluated. The incidence of side effects during lacidipine treatment was lower than during nifedipine or atenolol treatment, but higher than during hydrochlorothiazide therapy. Lacidipine did not cause any changes in the laboratory parameters evaluated during the 2-month monotherapy period. From a trial conducted in elderly patients, lacidipine 2 and 4 mg/o.d. has been shown to cause similar diastolic blood pressure reduction after 1 month of treatment, the effect being more gradual with the lower dose.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725446 TI - Long-term antihypertensive treatment with lacidipine, a new long-acting calcium antagonist. AB - Eleven centers in Tuscany, Italy, recruited 96 patients (aged 21-75 years) with mild-to-moderate essential hypertension [diastolic blood pressure (DBP) 95-115 mm Hg; systolic blood pressure (SBP) less than or equal to 200 mm Hg]. After a 4 week, single-blind, placebo run-in period, patients received lacidipine 4 mg once daily. If blood pressure was not controlled after 1 month (control = DBP less than or equal to 90 mm Hg, or less than or equal to 95 mm Hg if reduced by greater than or equal to 15 mm Hg from baseline), the dose was increased to lacidipine 8 mg once daily. Atenolol 50 mg once daily was added after 2 months' monotherapy, if required for blood pressure control. The study continued for 13 months. About 40% of patients were titrated to 8 mg lacidipine; only 7% required addition of atenolol. Lacidipine significantly reduced blood pressure, with 84% of patients showing control of pressure values 24 h after the previous dose on completion of 5 months' monotherapy (63% controlled with lacidipine 4 mg/day; 21% with lacidipine 8 mg/day). There was no clinically relevant difference in the first-dose effect between the two doses of lacidipine (4 mg and 8 mg); both smoothly reduced blood pressure, with maximum effect after 2 h. Lacidipine was well tolerated. Adverse events were those expected with dihydropyridines, were mainly mild and occurred early, disappearing without discontinuation of treatment. Results indicate that 4-8 mg of lacidipine, administered once daily, is effective and well tolerated in mildly to moderately hypertensive patients. PMID- 1725447 TI - Effect of lacidipine on pituitary function in essential hypertension. AB - Calcium plays an important role in endocrine reactions such as hormone biosynthesis, release, secretion, and action on target organs. The aim of this study was to evaluate the effects of a new long-lasting calcium-channel blocker, lacidipine, on basal and stimulated anterior pituitary hormone secretion. In a single-blind crossover study comparing lacidipine 4 mg p.o. once daily with placebo, variations in plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), thyroid-stimulating hormone (TSH), and adrenocorticotropic hormone (ACTH) were evaluated in 10 hypertensive patients. Basal or stimulated anterior pituitary hormone secretion was similar after lacidipine and placebo. Lacidipine treatment significantly reduced blood pressure. It can thus be concluded that lacidipine is an effective calcium antagonist that has no effect on pituitary function. PMID- 1725448 TI - Lacidipine: prevention of vascular damage induced by hypertension. AB - Lacidipine is a long-lasting 1,4-dihydropyridine calcium antagonist that has been reported to protect salt-loaded Dahl-S rats from vascular damage and accelerated mortality when it is administered prophylactically at 0.1 and 0.3 mg/kg p.o. once a day (equivalent to the recommended dose in humans). The aim of this study was to investigate the vasoprotective properties of lacidipine in Dahl-S rats that had already developed sustained hypertension after 4 weeks of a salt-rich diet. Although none of the dosages of lacidipine (0.3, 1, and 3 mg/kg) reduced the elevated values of blood pressure, an almost complete protection from mortality was obtained. Moreover, lacidipine dose-dependently inhibited the development of macroscopic and microscopic alterations in the distal branches of mesenteric arteries and in the brain. A clear regression of vascular damage and cardiac hypertrophy was observed at the highest dose tested (3 mg/kg). These findings further support the assumption that the protective properties of lacidipine are not restricted to a reduction in blood pressure. PMID- 1725449 TI - Interleukin 6 response factor binds co-operatively at two adjacent sites in the promoter upstream region of the rat alpha 2-macroglobulin gene. AB - Transcription of the alpha 2-macroglobulin gene (alpha 2M) in rat hepatocytes is strongly induced during acute inflammations by interleukin 6 (IL6). An IL6 response region has previously been mapped in the promoter upstream sequence of this gene. The region consists of two adjacent elements (IL6-REs), the IL6-RE core (CTGGGAA, -164 to -158 bp) and the core homology (CTGGAAA, -184 to -178 bp), elements, that are located 20 bp apart. Both elements bind nuclear factors with very similar protein-DNA contact patterns when they are contained in their original sequence context. A protein-DNA complex III was obtained in gel mobility shift experiments using a probe individually representing the core site. With probes containing both the core and core homology sites, a hormone inducible complex II of slower mobility was obtained. Complex II consisted of multiple copies of the same protein or proteins with very similar molecular masses bound at both sites. The core homology site was the weaker binding site. With a probe containing two tandem copies of the core site, binding at the second site occurred with 81 times greater affinity when the first site was occupied, than when it was free. Thus, the factor binding at the IL6-REs, the IL6-RE binding protein (IL6 RE-BP), was capable of co-operatively interacting with itself. Another factor, IL6-DBP/LAP, has recently been shown to be involved in the regulation of a major subgroup of acute phase genes by IL6. Using recombinant IL6 DBP/LAP and corresponding antisera, we demonstrated here that the IL6 RE-BP of the alpha 2M gene was distinct from IL6-DBP/LAP and from the related factor DBP. Thus, two major IL6-response elements can be distinguished: type 1 elements occurring in the human C-reactive protein, hemopexin and haptoglobin genes and utilizing IL6-DBP/LAP; and type 2 elements occurring in the rat alpha 2M, and alpha 1-acid glycoprotein genes, and utilizing a different IL6 RE-BP. The IL6 RE BP of the alpha 2M gene was also shown to be distinct from the transcription factor NF kappa B. The IL6RE-BP had relative molecular mass of Mr = 46,000, distinct from IL6-DBP/LAP (Mr = 32,000) and NF kappa B (Mr = 50,000) and its overall DNA binding capacity was induced under acute phase conditions. PMID- 1725450 TI - Sequence of rat alpha 1-macroglobulin, a broad-range proteinase inhibitor from the alpha-macroglobulin-complement family. AB - The alpha-macroglobulin-complement family of plasma proteins includes at least three different alpha-macroglobulins in rats, alpha 2-macroglobulin (alpha 2M), alpha 1 inhibitor III (alpha 1I3), and alpha 1-macroglobulin (alpha 1M). These high molecular weight polypeptides (Mr 160,000 to 200,000) are broad-range proteinase inhibitors. They utilize an internal thiolester function to trap proteinases that cleave their bait regions. The amino acid sequences of alpha 2M and two variants of alpha 1I3 are known. The isolation of alpha 1M cDNA clones and the determination of their cDNA and derived amino acid sequences are reported here. Alpha 1M shares 57.2% and 53.0% overall amino acid sequence identity with alpha 2M and alpha 1I3, respectively, but differs significantly from both in its bait region, suggesting that each inhibitor addresses a different spectrum of proteinases. The disulfide bridge structure of alpha 1M was deduced from its sequence and showed extensive similarities with the experimentally determined structures of other alpha-macroglobulins, suggesting similar overall tertiary structures. Alpha 1M mRNA was detected in 13 different rat tissues tested, whereas alpha 2M and alpha 1I3 mRNAs showed a far more restricted tissue distribution. While alpha 2M and alpha 1I3 mRNA and protein concentrations are significantly altered during acute and chronic inflammations, alpha 1M plasma concentrations are changed only twofold. Thus, in contrast with alpha 2M and alpha 1I3, the functions provided by alpha 1M may be ubiquitously and constitutively required in a broad range of tissues. PMID- 1725451 TI - The zona-free hamster oocyte penetration test: a simple procedure using a DNA fluorescent stain to detect the male pronucleus formation. AB - In order to increase the value of the zona-free hamster oocyte penetration test, a comparatively simple and fast method using the fluorochrome Hoechst 33342 was developed. Human spermatozoa were washed and incubated 1 hr medium BWW for capacitation. Hamster oocytes were stripped of cumulus oophorus and zona pellucida with hyaluronidase and trypsin, washed and used immediately. Thirty oocytes were placed in a drop of BWW containing 3,5.10(6)/ml of human spermatozoa under mineral oil. The sperm-oocyte preparation was incubated for 3 hr at 37 degrees C, during the last 15 min of incubation, the fluorochrome Hoechst 33342 (H) was added and incubation was allowed to proceed until the incubation time was over. Observations showed that the female pronucleus, eccentrically placed, gives a bright green-bluish fluorescence whereas chromatin of sperm heads shows different stages of decondensation and also a bright fluorescence. This inexpensive method has given consistent results in a large number of cases and provides an additional new approach to the "penetration test" as a proof of the capacity to form a "male pronucleus". PMID- 1725452 TI - [Psychomotor development in children born with anoxia--perinatal risk factors]. AB - In 1989 a control of psychomotor development was conducted in 318 children born with the Apgar score of 7 and less. The children controlled were of the age of 3 months to 6.5 years old. Clinical examination and the testing of children of up to the age of one year according to Brunet Lezin have shown a decrease in the number of points of nine children (4.05%). The finding points to a deviation in psychomotor development of a mild degree. Out of 80 preschool children, 8% of them had clinically evident signs of a possible cortical lesion, which were discreet and regarded the visuomotor, in other words visuoconstructive functions. PMID- 1725453 TI - [A pathological and histochemical study of capsules of impacted teeth with special reference to keratin immunohistochemistry in the lining epithelium]. AB - The purpose of this study was to examine and understand the nature of capsules consisted of fifty cases of unerupted wisdom teeth using radiological, pathological and immunohistochemical methods. Radiologically, the pericoronal space was measured with slide caliper and divided into three groups (-0.9 mm, 1.0 1.9 mm, 2.0mm). Pathologically, all specimens were examined by the routine paraffin method and were stained with hematoxylin.eosin and PAS-alcian blue pH 2.5 stainings. Immunohistochemically, PAP (peroxidase-antiperoxidase) method using various keratin antibodies, such as, anti-keratin K 528 (40-68 KD), anti cytokeratin PKK 1 (40, 45, 52.5 KD), anti-cytokeratin PKK 2 (40, 46, 48, 54 KD), anti-keratin A 575 (56, 64 KD) and anti-cytokeratin high molecular weight (68 KD), and other various antibodies of anti-EMA, anti-IgA and anti-SC was used in order to study the nature of the epithelium of capsule of impacted teeth. The following results were obtained; 1. Radiologically, the width of the capsules was 1.6 mm in the average. Histopathologically, the inner surface of the capsule was covered with two types of epithelium which were composed of non-typical epithelium with cuboidal (basal layer) and columnar (superficial layer) cells, and typical stratified squamous epithelium. The epithelium lining was observed in 74% of all cases. Among the cases, cyst like structure was more often seen in the cases with typical squamous epithelial lining and with pericoronal space more than 1.0 mm width. 2. In the connective tissue's findings of capsules of impacted teeth, fibrosis was found in almost all the cases. Inflammatory and myxomatous changes were recognized in ca. half cases. Epithelial rest and calcification with dystrophy and epithelial product were observed in about one third of the cases. The epithelial proliferation in the capsule was also rarely seen in the specimens. 3. Immunohistochemically, keratins of 56 KD and 64 KD were identified in the superficial layer of non-typical epithelium and basal, intermediate and superficial layers of the typical squamous epithelium of the capsules of imparted teeth. Keratin of 68 KD was only observed in superficial layer of squamous epithelium. 4. Epithelial membrane antigen was positive in the intermediate and superficial layers of typical squamous cell epithelium. However, it was not seen in the non-typical epithelium of capsule of impacted teeth. 5. Immunoglobulin A and secretory component were negative in the epithelia of capsules. PMID- 1725454 TI - [Hydroxyethyl starch as diluent of choice during intentional normovolemic hemodilution]. PMID- 1725455 TI - [Mixed venous oxygen saturation during infusion of PGI2 in the critically ill patient]. PMID- 1725456 TI - [A method of mathematical prognosis of Sudeck's syndrome after radius fracture at a typical site]. PMID- 1725458 TI - Perceptions of Hamlet. PMID- 1725457 TI - [Effect of heparin, obsidan and rheogluman on lymph coagulating activity and flow rate in acute myocardial infarction]. AB - Experiments were conducted on dogs to study the effect of heparin, obsidan, and rheogluman on the rate of lymph flow from the thoracic duct and the lymph coagulation potential in acute myocardial infarction. Rheoglumin produced the most marked lymph stimulating effect. The effect of heparin and partly that of rheogluman on lymph coagulability is mediated. PMID- 1725459 TI - New treatments for lung cancer. PMID- 1725460 TI - THC does not affect striatal dopamine release: microdialysis in freely moving rats. AB - The hypothesis that cannabinoids potentiate the motor effects of neuroleptics and produce their abuse potential by stimulating dopaminergic activity was tested by measuring the ability of THC to increase extracellular dopamine concentrations. Male Long-Evans rats were implanted with guide cannulae for the striatum or nucleus accumbens. Fifteen hours prior to testing, removable microdialysis probes were inserted through the guide cannulae. Dialysis samples were collected during resting baseline, after 1.0 mg/kg, 10 mg/kg THC, or vehicle of olive oil with 5% ETOH (by gavage) followed by amphetamine (1.5 mg/kg) or fluphenazine (0.3 mg/kg). THC produced no change in the extracellular concentrations of DA, DOPAC, and HVA, nor in 5-HIAA. THC also had no effect on the enhancement of extracellular DA produced by amphetamine nor on the transient increase in DA, DOPAC, and HVA produced by fluphenazine. There were also no behavioral differences between groups during any of these treatments. PMID- 1725462 TI - [Vertebral prostheses in surgical treatment of metastatic disease]. AB - Metastasis disease is often located at the spine, especially with solid tumors where frequency can reach 80%. Indications for metastasis operational approach are threatening or existing neurologic deficit, static dislocations or even unbearable pain. Operation treatment may be extensive, with radical metastasis removal or even partial one with decompressive laminectomy and tumor reduction and spine stabilization. Using various kinds of alenthesis, the authors have used their own vertebra artificial limb in 10 cases, which enables spine stabilization after radical vertebra removal. They report their experience in the essay. PMID- 1725461 TI - Evidence for a direct anterior pituitary site of delta-9-tetrahydrocannabinol action. AB - The effects of delta-9-tetrahydrocannabinol (THC), alone and in the presence of estradiol (E), on several estrogen-sensitive parameters in the immature female rat were examined, and it was demonstrated that THC administration antagonized the stimulatory effects of E on anterior pituitary weight and on both the secretion and pituitary content of prolactin. In the current study, the anterior pituitary gland was examined as a potential site of THC action in the ability of this cannabinoid to antagonize E-induced stimulation of pituitary function. A stimulatory dose of E (1 nM) significantly elevated prolactin levels in pituitary cells derived from either immature or retired breeder animals. Whereas THC (1 microM) alone had no effect on prolactin levels when compared to controls, THC completely prevented the E-induced increase in media prolactin levels. Moreover, THC blocked the ability of E to desensitize pituitary cells to the inhibitory influence of dopamine. Together with the findings that THC inhibited E-induced stimulation of total RNA synthesis in pituitary cell cultures, these data strongly suggest that THC antagonizes the stimulatory effect of E on the pituitary by a direct action at the adenohypophyseal level. PMID- 1725463 TI - Is the fluorescein dilaurate test suitable for monitoring the effectiveness of pancreatic enzyme replacement therapy? AB - We have employed an oral test of pancreatic digestive function using fluorescein dilaurate (a substrate for pancreatic cholesterol ester hydrolase), administered together with pancreatic enzyme supplements, in order to test the hypothesis that urinary excretion of fluorescein provides a simple method for determining the optimal dose of oral pancreatic enzymes. Commercially available pancreatic enzyme supplements had negligible cholesterol hydrolase activity in vitro, and did not increase the urinary excretion of fluorescein when administered together with fluorescein dilaurate in 16 patients with pancreatic exocrine insufficiency. Inhibition of gastric secretion by ranitidine resulted in a statistically significant increase in urinary fluorescein excretion during the fluorescein dilaurate test. The fluorescein dilaurate test is not, therefore, suitable for determining the effectiveness of oral pancreatic enzyme replacement therapy. Moreover, the manufacturer's advice to stop oral pancreatic enzyme therapy for 5 days before performing the fluorescein dilaurate test appears unnecessary, since oral pancreatic enzymes do not alter test results. PMID- 1725464 TI - [Experimental studies of direct pulp capping in permanent teeth with incompletely formed apices]. AB - In permanent teeth described as having incompleted roots, dentin is still in the process of deposition on internal dentin walls and the root is in the process of completion. The purpose of this study of dog's immature permanent teeth was to investigate pulp healing and continuous root and internal dentin-wall developments after direct pulp capping with two types of calcium hydroxide paste on mechanically exposed pulp. Material and methods In this study, 148 vital permanent premolars with incompletely formed apices were obtained from 26 dogs (6 months old). The animals were generally anesthetized with 5% sodium pentobarbital, and a cavity was prepared on the occlusal surface of each experimental tooth using an air turban handpiece with diamond bur. After the cavity preparation, the central pulp horn of each tooth was mechanically exposed with a sterile round bur (ISO No. 007, 2/0). In experimental teeth on the right side, a calcium hydroxide-iodoform paste "Calvital" was applied on the exposed pulp, in experimental teeth on the left side, the exposed pulp was capped with a hard-setting calcium hydroxide paste "Dycal". The cavities were lined with first a zinc-oxide eugenol cement and then a zinc-phosphate cement. Then the cavities were filled with silver amalgam. The dogs were sacrificed by means of sodium pentobarbital at intervals of 3, 7, 14, 28, and 56 days after direct pulp capping. Their jaws and teeth were removed, fixed with formalin, and 100 cases out of 148 teeth with bones from 20 dogs were decalcified, and embedded in celloidin. Each specimen was serially sectioned, stained with hematoxylin-eosin, thionin-picric acid and van Gieson's stain, and evaluated microscopically. In order to identify postoperative dentin formation, intravenous injections of tetracycline were administered to the remaining 6 dogs (48 teeth) at various intervals after the experimental procedure. Two dogs were sacrificed each of the experimental periods. Specimens were embedded in resin and sectioned to a thickness of 50 microns. Undecalcified ground sections were observed by contact microradiography, and tetracycline labeling was evaluated by ultraviolet light. Results and Conclusion 1. Pathological evaluations of the 2 groups "Calvital" group: 49 (98.0%) of 50 cases were evaluated as good, and 1 case (2.0%) as moderately good. "Dycal" group: 31 (62.0%) of 50 cases were evaluated as good, 15 (30.0%) as moderately good, and 4 (8.0%) as bad.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725465 TI - [A study on changes of liver and salivary glands in rats with experimentally induced liver injuries. Pathological and biochemical observations]. AB - Changes in the salivary glands, liver and pancreas in rats with experimentally induced liver injuries were examined. The injuries (experimental group) were induced by subcutaneous injections of carbon tetrachloride (0.01ml/kg body weight) in a 50% olive-oil solution. The injections were administered twice weekly for 10,20 and 40 weeks. Control animals received the same doses of olive oil during the same periods. 1. In the experimental group, serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) increased remarkably, whereas serum albumin decreased. 2. Swelling of the liver and multiple nodular formations occurred in the experimental group. Liver fibrosis with the formation of pseudolobules indicated a form of liver cirrhosis. 3. No significant histological changes were observed in the pancreases of animals in the 10- and 20-week experimental groups. Vacuolation in the acinar cells was observable in 3 of 8 cases in the 40-week experimental group. 4. In connection with histological findings of parotid glands, vacuolation of the acinar cells occurred in 7 of 12 cases in the 10-week experimental group, in 7 of 8 cases in the 20-week experimental group, and in all 8 cases in the 40-week experimental group. Vacuole numbers and sizes increased as the experimental period was prolonged. 5. Immunohistochemical investigation showed strong positive reactions to the anti-amylase antibody around vacuoles in acinar cells of parotid glands. In unvacuolated acinar cells, on the other hand, only slight positive reaction was observed. 6. Electronmicroscopic observation of the acini revealed greatly enlarged lumina and dilated intercellular canaliculi connected to the lumina. Small vacuoles were observed on the basement of the acini. 7. No such significant changes as fibrosis, vacuolation, and atrophy of acinar cells were observed in the submandibular and sublingual glands of the experimental animals. 8. Serum amylase activity increased more in the experimental than in the control rats. Electrophoretic patterns suggested that in the control group 95 percent of serum amylase was parotid type, and also in the experimental group 95 percent of serum amylase was parotid type. 9. Amylase activity in the parotid glands also increased more in the experimental than in the control animals. PMID- 1725466 TI - Significance of human T-lymphotropic virus type I indeterminant serological findings among healthy individuals. AB - Follow-up studies on 67 blood donors with indeterminant serological findings for human T-lymphotropic virus (HTLV) type I by standard immunoassays showed no evidence of infection by polymerase chain reaction analysis for HTLV-I or HTLV-II nucleic acids or by antibody reactivity to a unique HTLV-I recombinant envelope protein, MTA-4. Among HTLV-I- or -II-infected individuals, a history of blood transfusion, past residence in established HTLV-I endemic areas or some association with intravenous drug use were common. In contrast, 85% of indeterminant cases had none of these risk factors. These observations suggest that healthy individuals with indeterminant serology for HTLV-I should not require additional studies. PMID- 1725467 TI - Effects of adenosine and its derivatives on cultured myocardial cells and detection of adenosine receptors in rat. AB - Adenosine and its derivatives 1 nmol.L-1 -0.1 mmol.L-1 elicited negative chronotropic effect on cultured rat myocardial cells. The respective IC50 were: L PIA 85 +/- 10 greater than NECA 160 +/- 64 greater than adenosine 361 +/- 41 nmol.L-1. The negative chronotropic effect was antagonized by IBMX. [3H]Adenosine bound to myocardial cells of rat in a saturable way with a Kd of 238 nmol.L-1 and a Bmax of 7.6 pmol/10(8) cells. The binding of [3H]adenosine was competitively antagonized by adenosine, L-PIA, NECA, and IBMX. These results indicated the existence of adenosine receptor in rat myocardial cells. PMID- 1725468 TI - Effects of substance P on electric activity of mouse pancreatic islet cells in vitro. AB - The effects of substance P (SP), [D-Pro2, D-Trp7,9]SP and verapamil on the electric activity of B cells of Langerhans islets of mice were investigated. Addition of SP 100 nmol.L-1 to the perfusion medium stimulated spontaneous electric activity induced by glucose 5.5 mmol.L-1 with an increase in frequency and amplitude of the spikes. [D-Pro2, D-Trp7,9]SP 50 nmol.L-1 reversed the stimulative effect of SP. There was no evidence for an enhancing interaction between SP and acetylcholine 1 mumol.L-1. Verapamil 60 mumol.L-1 blocked the stimulative effect of SP. These results suggest that the stimulative effect of SP on the electric activity of B cells may be due to the increase in Ca2+ influx through the voltage-dependent Ca2+ channels. PMID- 1725469 TI - [Antagonistic effect of phencyclidine on cerebral ischemic damage of rats]. AB - Dynorphin and catecholamine were measured in ischemic rat produced by four-vessel (2 vertebral arteries and 2 common carotid arteries) occlusion for 10 min. The results showed that: (1) The contents of dynorphine (pg/mg tissue) in cerebral cortex were 5.5 +/- 0.6 (n = 7) in normal rats and decreased to 4.9 +/- 0.5 (n = 9, P less than 0.05) in cerebral ischemic rats; with immediate ip phencyclidine (1-(1-phenylcyclophexyl)piperidine, PCP, 1 mg.kg-1), the contents of dynorphin were increased to 5.3 +/- 0.4 (n = 5, P less than 0.05 vs the ischemic rats). (2) The contents of DOPAC (pg/mg tissue) in cerebral cortex were 38 +/- 6 (n = 7) and increased to 120 +/- 60 (n = 5, P less than 0.05) in 10 min cerebral ischemic rats; with immediately ip PCP (1 mg.kg-1), the contents of DOPAC were decreased to 26 +/- 13 (n = 7, P less than 0.05 vs the ischemic rats). (3) The release of DA (pg/mg tissue) in cortical slices in vitro, in high K+ solution were 24 +/- 3 (n = 5) and significantly increased to 57 +/- 15 (n = 5, P less than 0.05) in ischemic rat brain slices; with immediate ip PCP (1 mg.kg-1), the contents of DA were decreased to 38 +/- 10 (n = 5, P less than 0.05 vs the ischemic rats). These results suggest PCP play an antagonistic role in cerebral ischemic damage of rats. PMID- 1725470 TI - [Effects of morphine on cutaneous blood flow and substance P release evoked by electric stimulation of rat sciatic nerve]. AB - Electric stimulation of the rat sciatic nerve containing sensory afferent fibers produced an increase in cutaneous blood flow. Morphine (10, 30 mumol.kg-1 ia infusion) inhibited the electric stimulation-induced increase of the cutaneous blood flow velocity, and its effect was antagonized by naloxone (2 mg.kg-1 ip). In order to investigate the cause of this effect, we determined immunoreactive substance P (iSP) levels in the sc perfusate of hind paw. We found that electric stimulation of the sciatic nerve led to a significant increase of iSP release into the sc perfusate. Morphine (30 mumol.kg-1 ia infusion) inhibited the electrical stimulation-induced release of iSP, and this effect was completely antagonized by naloxone (2 mg.kg-1 ip). These result suggest that morphine induced inhibition of the electrical stimulation-evoked increase in cutaneous blood flow could result from inhibition of the release of SP from peripheral sensory nerve endings. PMID- 1725471 TI - [Testicular metastasis of prostatic origin]. PMID- 1725472 TI - [Treatment of hormone-refractory prostate cancer with estramustine phosphate]. AB - Twenty-one patients with hormone refractory prostate cancer were treated with oral estramustine phosphate, 600 mg/day, and the response was monitored through serial dosages of prostatic specific antigen (PSA). Patients showed biological response (67%) and clinical response (43%). The duration of the biological response was 6 months in 50% cases and over 12 months in 18%. Survival was higher for responders than non responders. It is concluded that estramustine phosphate is effective in this type of therapy and that PSA allows to measure the type of response and its duration. PMID- 1725473 TI - [Paratesticular rhabdomyosarcoma with intracaval neoplastic involvement: presentation of a case and review of the literature]. AB - One case of paratesticular embryonal rhabdomyosarcoma (RMS), affected during its evolution by a tumoral thrombus in the inferior vena cava. This unusual association forced the use of a cardiopulmonary by-pass, profound hypothermia and circulatory arrest, in order to carry out complete exeresis of the damage. Also, revision of the literature emphasizing that today's therapeutical approach for RMS should essentially be multidisciplinary. PMID- 1725474 TI - Quantitation of DNA and RNA in crude tissue extracts by flow injection analysis. AB - An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 micrograms DNA and 0.10 micrograms RNA), automation, and a high sample throughput. PMID- 1725475 TI - Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid-phase sequence analysis. AB - A method for the determination of the sites of tyrosine phosphorylation in proteins and peptides at the low picomole level for "cold" phosphopeptides and at the subpicomole level for 32P-labeled phosphopeptides is presented. The procedure is based on solid-phase sequence analysis of phosphopeptides immobilized on carrier discs and the "on-line" detection by reverse-phase high-performance liquid chromatography of the phenylthiohydantoin derivative of phosphotyrosine. The procedure is sensitive and automated and allows the identification of phosphotyrosine derivatives in the same operation as the detection of the derivatives of the other common amino acids. Essentially quantitative extraction of the phosphotyrosine derivatives from the sequencer makes this method ideally suited for the quantitative assessment of protein-tyrosine kinase and protein phosphatase activities and for the determination of their respective recognition sequences. PMID- 1725476 TI - Ro (SS-A) and La (SS-B) ribonucleoprotein complexes: structure, function and antigenicity. PMID- 1725477 TI - Quantifying heterogeneity: flow cytometry of bacterial cultures. AB - Flow cytometry is a technique which permits the characterisation of individual cells in populations, in terms of distributions in their properties such as DNA content, protein content, viability, enzyme activities and so on. We review the technique, and some of its recent applications to microbiological problems. It is concluded that cellular heterogeneity, in both batch and continuous axenic cultures, is far greater than is normally assumed. This has important implications for the quantitative analysis of microbial processes. PMID- 1725478 TI - [Treatment of obstruction caused by prostatic hypertrophy with prazosin. 1-year follow-up]. AB - Since Caine demonstrated the presence of alpha-adrenergic receptors in prostate tissue in 1975, an alpha-blocker capable of producing maximum relaxation of prostatic tissue with minimum side effects has been sought. Prazosin, a selective alpha-1 blocking agent, must meet these requirements. We report the results of a study undertaken in 28 patients with benign hyperplasia of the prostate demonstrated by flowmetry, clinically and by ultrasound. A significant improvement was observed in all patients except for the ultrasound measured prostate size, which remained unchanged. Undesired effects were observed in 36% of the patients. Despite the initial improvement, 89.3% had abandoned treatment at 1 year principally owing to deterioration of the clinical picture and because they were submitted to surgery (the majority) or treatment with other drugs. We can conclude from our results that prazosin is useful for short-term therapy; i.e., until the patient can be referred for surgery. PMID- 1725479 TI - [Epitope specificity of hemagglutinating monoclonal anti-B antibodies]. AB - Fine epitope specificity of ten monoclonal antibodies (MA) agglutinating red blood cells B was studied. Three methods were used: 1) inhibition of MA binding to natural antigen by synthetic oligosaccharides (OS) and their polyacrylamide conjugates, 2) direct MA binding to a series of synthetic OS-polyacrylamide conjugates differing in carbohydrate epitope density, 3) direct MA binding to the affinity sorbents. It is shown that all antibodies studied prefer trisaccharide B determinant Gal alpha 1-3(Fuc alpha 1-2) Gal independently of their ability to discriminate serological subgroups of B erythrocytes (B, Bweak, B3). The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells B subgroups is discussed. Of an interest is that MAs which are able to agglutinate any B subgroups also bing the synthetic tetrasaccharide Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc, a B type 3 determinant. PMID- 1725480 TI - [Direct breaks in a single-stranded DNA fragment by a bleomycin-derived oligonucleotide]. AB - A method for coupling bleomycin to oligonucleotides is suggested. The reaction was carried out between the amino group of the spermidine residue of the bleomycin A5 Cu(II)-complex (Cu(II)Blm-RH) and the 5'-phosphate group of the oligonucleotide pd(CCAAACA) (I) activated with a mixture of triphenylphosphine and 2,2'-dipyridyldisulphide in the presence of 4-N,N-dimethylaminopyridine-1 oxide. The resultant compound (Ia) (yield 70%) forms more stable complementary complexes than the parent oligonucleotide (delta Tm = 11 degrees C). When Cu(II) ion was removed from (Ia), compound (Ib) formed which effectively (80%) cleaved pd(TGTTTGGCGAAGGA). Neither pd(TCCTTCG) nor the oligonucleotide tail of the reagent (Ib) were destroyed under the cleavage conditions. Free Blm-RH and bleomycin bound in the reagent (Ib) damage different regions of the target. PMID- 1725481 TI - Characterization of two monoclonal antibodies against teguments of adult and schistosomula of Schistosoma japonicum. AB - Two monoclonal antibodies (McAbs) were produced from BALB/c mice hyperimmunized with tegumental extract of Schistosoma japonicum (Chinese strain). The two McAbs were characterized with regard to antibody isotype, antigen binding specificity and parasite stage specificity. One McAb, 8G9-5, was identified as IgM, whereas the other McAb, 9E7, was determined to be IgG2a. Immunoblotting assay indicated that McAb 8G9-5 binds strongly to the band of tegumental antigens of Mr 64 kDa but also binds weakly to other bands at 116, 105, 97, 54, 50, 47 and 45 kDa, whereas 9E7 McAb reacts specifically at Mr 54 kDa. Anatomical localization of the antigens in the adult worm by indirect immunofluorescence assay indicated that the target epitopes of McAb 8G9-5 are in the intra-tegumental structure, whereas the McAb 9E7 epitope is on the surface membrane. The two McAbs also react at similar sites within the teguments of schistosomula and lung worms. PMID- 1725482 TI - Morphometric study on the CH4 of the nucleus basalis of Meynert in Alzheimer's disease. AB - Ch4 neurons were studied morphometrically in three normal controls and three patients with Alzheimer's disease (AD). Two series of preparations stained with cresyl violet and with the indirect immunoperoxidase method using a monoclonal antibody to acetylcholinesterase (AChE) counterstained with cresyl violet were morphometrically analyzed. The cholinergic neurons were identified as positive reaction products of AChE in the perikarya. The cross-sectional area of all the Ch4 neurons with clearly visible nucleoli in one preparation was measured using a computer imaging system. Evaluation of these data provided a mean value of the cross-sectional neuronal area (CNA), number of neurons, standard deviation of the CNA and coefficient of variation of the cholinergic and noncholinergic neurons. The cholinergic neurons were decreased in number but showed hypertrophy in the early stage of AD, and they gradually became atrophic in the advanced stage. The noncholinergic neurons showed only atrophy without definite neuronal cell depletion. These findings indicate that cholinergic neurons have a plasticity and remodeling property against the AD process and suggest that hypertrophy of the cholinergic neurons in the Ch4 is the result of the effect of neurotrophic factors. PMID- 1725483 TI - [Early intervention in infants with intrauterine growth retardation: its effects on neuropsychological development]. AB - Forty-eight term infants in a tertiary center in Puerto Rico during 1985-86 were monitored prospectively since birth completing a neuropsychological evaluation using the Mental Developmental Index of Bayley Scales. All infants were free from perinatal complications and chronic diseases by clinical evaluation. 25 infants with intrauterine growth retardation (IUGR) and 23 adequate for gestational age (AGA) infants were evaluated. Fourteen IUGR infants were controls and eleven IUGR infants were intervened. However, all AGA infants were used as controls. Mother infant relationship at one month was scored and none of intervened infants had poor neuropsychological behavior with normal or near normal MDI values. Other relationships and possible explanations are discussed in the article. PMID- 1725484 TI - Quantitative assessment of chromatin stability alteration in human spermatozoa induced by freezing and thawing. A flow cytometric study. AB - The authors examined the effect of cryopreservation on chromatin stability in human spermatozoa from 21 ejaculates. Each ejaculate was divided into four aliquots: (1) fresh aliquot, (2) frozen and thawed aliquot, (3) fresh aliquot subjected to an in vitro decondensation method, and (4) frozen and thawed aliquot subjected to the same in vitro decondensation method; all were then fixed with an ethanol fixative agent. Chromatin stainability was quantified by flow cytometric measurement of fluorochrome uptake by DNA. Study of 21 fresh aliquots showed that 37.9% of the DNA was accessible to propidium iodide. The comparative stainability between the 21 fresh and 21 frozen-thawed, undecondensed aliquots demonstrated a low but significant increase in accessibility of DNA by propidium iodide for the thawed samples: 38.7 +/- 1.7% (mean +/- SD) versus 37.9 +/- 1.3%. The biochemical action of the nuclear decondensation solution increased the accessibility of propidium iodide, but in different ways: 57.2 +/- 12.9% versus 54.7 +/- 13.7%, respectively, for fresh and frozen-thawed aliquots. Analysis of the flow cytometric histograms revealed an intermediate population of spermatozoa adjacent to the main germinal peak. This population increased significantly: 9.6 +/- 1.9% for the fresh versus 12.3 +/- 4.9% for the frozen-thawed undecondensed aliquots and 8.6 +/- 3.5% versus 12.3 +/- 4.9%, respectively, for fresh and frozen-thawed, decondensed aliquots. Because the chromatin stability of thawed spermatozoa may be a critical factor in assisted procreation, the authors discuss the effect of thermal denaturation on the nucleoprotein structures and the origin of the intermediate population of spermatozoa. PMID- 1725485 TI - Automated selection of the most epithelium-rich areas in gynecologic tumor sections. AB - The paper describes an image analysis technique for automated selection of the epithelium-rich areas in standard paraffin tissue sections of ovarian and endometrial premalignancies and malignancies. Two staining procedures were evaluated, Feulgen (pararosanilin) and CAM 5.2, demonstrating the presence of cytokeratin 8 and 18; both were counterstained with naphthol yellow. The technique is based on the corresponding image processing method of automated estimation of the percentage of epithelium in interactively selected microscope fields. With the technique, one image is recorded with a filter to demonstrate where epithelium and stroma lie. This filter is chosen according to the type of staining: it is yellow (lambda = 552 nm) for Feulgen and blue (lambda = 470 nm) for anticytokeratin CAM 5.2. When stroma cannot be distinguished from lumina with the green filter or from epithelium with the blue filter, a second image is recorded from the same microscope field, with a blue filter (lambda = 420 nm) for Feulgen and a yellow filter (lambda = 576 nm) for anticytokeratin CAM 5.2. Discrimination between epithelium and stroma is based on the image contrast range and the packing of nuclei in the yellow image and on the automated classification of the gray value histogram peaks in the blue image. For Feulgen stain the method was evaluated on 30 ovarian tumors of the common epithelial types (8 borderline tumors and 22 carcinomas with various degrees of differentiation) and 30 endometrial carcinomas of different grades.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725486 TI - Experience of palliative medicine among trainees. PMID- 1725487 TI - Headaches caused by exhaust fumes. PMID- 1725488 TI - A common channel-forming motif in evolutionarily distant porins. AB - Four new crystal packings of Escherichia coli porins are presented (phosphoporin, maltoporin, and two crystal forms of matrix porin). These were determined by molecular replacement methods using a polyalanine trial model acquired from the refined coordinates of porin from Rhodobacter capsulatus. The successful molecular replacement shows that the dominant motif found in R. capsulatus porin (a 16-stranded antiparallel beta-barrel) also applies to the E. coli porins, despite the lack of significant amino acid sequence homology. A 30 degrees-40 degrees tilt of the beta-strands with respect to the membrane normal was derived from the intensity distributions in the X-ray diffraction patterns for each porin studied, stressing their similarity. In view of the evolutionary distance between enteric and photosynthetic bacteria, the antiparallel beta-barrel may have significance as a basic structural motif for the formation of bacterial membrane channel structures. PMID- 1725489 TI - Organization of coiled-coil molecules in native mouse keratin 1/keratin 10 intermediate filaments: evidence for alternating rows of antiparallel in-register and antiparallel staggered molecules. AB - There is considerable diversity of opinion in the literature concerning the organization of two-chain coiled-coil molecules in intermediate filaments. I have reexplored this issue using the limited proteolysis paradigm with native mouse epidermal keratin intermediate filaments (KIF), consisting of keratins 1 and 10. KIF were harvested as cytoskeletal pellets, dissociated into subfilamentous forms at pH 9.8, 9.0, or 2.6, and were subjected to limited proteolytic digestion to recover alpha-helix-enriched particles that derived from the rod domains of the constituent chains, using conditions that do not promote reorganization of the constituent protein chains or coiled-coil molecules. The multichain particles were subjected to physicochemical analyses, amino acid sequencing, and electron microscopy in order to determine their composition, structure, and organization within the intact KIF. The results predict two principal modes of alignment: neighboring molecules may be aligned in register and antiparallel or staggered and antiparallel. From known structural constraints, this permits construction of a two-dimensional surface lattice for KIF which consists of alternating antiparallel rows of in-register and staggered molecules. These data establish the level of hierarchy at which the well-known antiparallelity and staggered features of KIF are introduced. This model supports the proposals of KIF structure based on theoretical considerations of ionic interactions scores (Crewther et al., 1983). When the KIF are dissociated at extremes of pH, this structural model allows for disruption along alternate axes; the in-register antiparallel alignment is seen only when KIF are dissociated at high pH values; below pH 9, only the staggered antiparallel alignment is seen. The process of molecule realignment especially in concentrated urea solutions indicates that the staggered antiparallel alignment is the more thermodynamically stable form in solution. PMID- 1725490 TI - Analysis of the mechanism of assembly of mouse keratin 1/keratin 10 intermediate filaments in vitro suggests that intermediate filaments are built from multiple oligomeric units rather than a unique tetrameric building block. AB - The question as to whether keratin intermediate filaments (KIF) are built from a unique "building block" consisting of a pair of coiled-coil molecules has been studied by examining the earliest stages of reassembly of mouse K1/K10 KIF in vitro. Particles formed in protein solutions of about 45 micrograms/ml (near or below the critical concentration for assembly) or 0.5-1.65 mg/ml were monitored by turbidity, visualized by electron microscopy, and their structures resolved biochemically using crosslinking, limited proteolysis, and amino acid sequencing. The rate of KIF reassembly in vitro is limited by an initial slow step involving the formation of a three- or four-molecule oligomer. At 2 min, the particles in solution are about 65 nm long and consist of two molecules aligned antiparallel and staggered. A few minutes later, a three- and/or four-molecule species appears that may be the rate-limiting particle(s). It is also 65 nm long, but contains one or two additional molecules aligned in register but antiparallel with respect to one of the molecules on the two-molecule particle. The present data cannot establish whether the rate-limiting particle contains three or four molecules, or in fact consists of a mixture of both. Below the critical concentration for KIF assembly, it exists in solution in rapid exchange with particles containing one and two molecules. In solutions above the critical concentration for assembly, once this oligomer has formed in sufficient quantity, further assembly into KIF occurs rapidly; 90, 110, and 130-nm particles soon appear by apparent addition of a single molecule or oligomers containing two, three, four, or even several molecules. Within about 20 min short KIF about 200-500 nm long appear which later elongate to long (greater than 1 micron) KIF. These data suggest that KIF assembly requires the initial correct alignment of three or four molecules which, once formed, provides a template for further rapid addition of molecules leading to KIF assembly. Furthermore, the data establish that KIF are built from alternating rows of in-register and staggered antiparallel molecules. The present data confirm independently the observations of the previous paper and do not support earlier notions that IF are built from a tetrameric building block consisting of a pair of in-register molecules. Finally, the data suggest that the mechanism of assembly in vitro and the dynamic in vivo assembly-disassembly characteristics of KIF in particular and IF in general are mediated through a variety of small oligomeric species ranging in size from one to several molecules. PMID- 1725491 TI - The Trypanosoma brucei cytoskeleton: ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy. AB - We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quick-freezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction wit the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules. PMID- 1725492 TI - A preliminary scanning tunnelling microscopy study of the surface-organised structures of gramicidin S hydrochloride on highly oriented pyrolytic graphite. AB - For an initial study of potentially surface-structural self-organising systems of biological significance by scanning tunnelling microscopy (STM), gramicidin S, a pseudocentrosymmetric cyclodecapeptide with antibiotic properties, was chosen as prototype, recognising its structure as having both intramolecular and intermolecular hydrogen-bond forming propensity. The surface-organised structures, based on gramicidin S hydrochloride deposited on a highly oriented pyrolytic graphite (HOPG) substrate, have been observed by STM in air under ambient conditions. These are characterised in the main by rectangular or rectangle-like structural elements identified with the individual gramicidin S hydrochloride molecules. Two kinds of arrangements of gramicidin S hydrochloride in a two-dimensional array are found, i.e., as a centred rectangular lattice and a primitive rectangular lattice. The STM topographical arrays and the molecular dimensions obtained are in good quantitative agreement with the corresponding X ray crystallographic data. The differences between the STM results, the theoretical models, and the X-ray crystallographic data are attributed to the intermolecular interactions present in the three-dimensional gramicidin S crystal but absent in the lower dimensional arrays and to the environments in which a gramicidin S hydrochloride molecule finds itself during deposition and drying on the HOPG substrate. PMID- 1725493 TI - Toward a more complete 3-D structure of the nuclear pore complex. AB - The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent "knobs" that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a "fishtrap." These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed. PMID- 1725494 TI - [Ribonucleotide reductase--transition enzymes from RNA metabolism to DNA metabolism]. AB - Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides, this reaction being vitally important for all organisms. The enzyme provides a link between RNA and DNA metabolisms. Several forms of the enzyme occur in nature and those differ in their structure and catalytic mechanisms. While the direct reduction of ribonucleotides via a radical mechanism is a general mode of dNTP synthesis in all organisms, the way in which this is achieved varies. The ability of the enzyme to control DNA synthesis is of considerable clinical interest. Selective inhibition of DNA synthesis is desired, for example, in clinical applications, such as restriction of tumour growth or virus replication. PMID- 1725495 TI - Molecular recognition between ligands and nucleic acids: DNA binding characteristics of analogues of Hoechst 33258 designed to exhibit altered base and sequence recognition. AB - The DNA binding characteristics of new analogues (2-8) of Hoechst 33258 (1), containing pyridine and benzoxazole units and designed for altered base specificity, were evaluated using UV, fluorescence, and circular dichroism studies. Like Hoechst 33258 the new analogues also bind through the minor groove of B-DNA in a nonintercalative fashion. The interaction of the compounds with poly(dA-dT) is salt independent. The studies with poly(dA-dT), ct DNA, and poly(dG-dC) indicated a decrease in the relative binding strength of the new analogues to DNAs compared with the parent molecule, Hoechst 33258. Compounds 5 and 7 showed acceptance of GC bases adjacent to AT base pairs. None of the compounds studied exhibited affinity for A-DNA, double-stranded RNA, or Z-DNA. Structure-DNA binding relationships of the new analogues compared with their parent molecule, Hoechst 33258, are discussed. PMID- 1725496 TI - Vaginal cyclicity, sexual receptivity, and eating behavior of the female rat following treatment with chlordecone. AB - The effects of 25, 50, or 75 mg/kg chlordecone on vaginal and behavioral estrus were examined following treatment of intact rats during estrus, diestrus 1, or diestrus 2. Chlordecone accelerated vaginal estrus, but sexual behavior was eliminated, delayed, or reduced. Chlordecone treatment led to the presence of vaginal estrus within 2 days, but reduced or eliminated sexual behavior on the evening of predicted proestrus. Of the females that received chlordecone, 20% to 50% showed some behavior on the day after the evening of predicted proestrus and 20% to 35% never showed behavior during the 8-day observation period. Although the lordosis to mount ratio was still reduced, the occurrence of behavior a day late suggested that the pesticide had delayed behavioral estrus. Chlordecone also rapidly suppressed food intake and led to a significant decline in body weight; these nutritional factors could have contributed to the disrupted estrous cycle. Some support for this possibility was derived from a reduced sexual receptivity on the evening of proestrus when the caloric intake of untreated female rats was matched to that of the chlordecone treated animals. However, the effects of caloric reduction on proestrous lordosis behavior were less robust than seen following chlordecone. Chlordecone treatment on diestrus 2 reduced the number of progesterone receptors in uterine tissue of females on the predicted day of proestrus. This suggested that the tissue sensitivity to circulating levels of progesterone would be reduced within 2 days after chlordecone treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725497 TI - An alternative to menus in multimedia presentations. PMID- 1725498 TI - [Application of a monoclonal antibody SZ-51 specific for activated human platelets in canine thrombosis imaging]. AB - To detect specifically the localization of thrombus in vivo, we prepared a monoclonal antibody (McAb) SZ-51 specific for an alpha-granule membrane protein (GMP-140) exposed on the surface of activated human platelets. 131I labeled SZ-51 antibody combined preferentially with thrombus induced by thrombin in vitro. Owing to McAb SZ-51 crossreaction with the activated platelets of dogs, thrombi in both femoral artery and vein were prepared and imaged with SPECT in dogs. The ratio of radioactivity between thrombi and blood pool was significantly increased in both arterial and venous thrombus after injection of 131I-SZ-51, especially at the 4th hour. Moreover, the imaging of arterial thrombus is better than that of venous thrombus. However, both arterial and venous thrombi could not be imaged by the injection of 131I-nonimmune IgG. These findings were in agreement with the radioactivity ratios between the removed thrombi and blood 24 hours after injection. Therefore, these results indicate that McAb SZ-51 has the power of detecting thrombi in vivo. PMID- 1725499 TI - Keratin expression in cultures of adult human epidermal cells. AB - Keratins are complex fibrous proteins characteristic of epithelial cells. We have developed a procedure that allows us to culture and passage adult human dermal keratinocytes in the absence of mesenchymal substrates. Electron microscopic examination of stratifying cultures showed the presence of numerous filament bundles, desmosomes and electron dense granules. The expression of different classes of keratin was examined by immunofluorescence, SDS-PAGE and immunoblots using monoclonal antibodies. The analysis of water-insoluble proteins revealed the presence of keratins of molecular weights 40 Kda, 50-52 Kda, 56 Kda and 65-67 Kda. Our results indicate that the terminal differentiation of keratinocytes may not require dermal factors. PMID- 1725500 TI - Contact patterns in concanavalin A agglutinated erythrocytes. AB - Agglutination of human erythrocytes by the lectin concanavalin A is enhanced when the erythrocytes are pretreated with neuraminidase, which removes sialic acids, or with pronase, which degrades both the glycophorins and band 3 protein. In the present work transmission electron microscopy of the enzymatically pretreated erythrocytes shows a regular pattern of interruption of contact between interacting plasma membranes. The lengths characteristic of the pattern were 0.66 and 0.50 microns for pronase- and neuraminidase-pretreated cells, respectively. Agglutination of normal erythrocytes and of neuraminidase-pretreated erythrocytes can be fully reversed by exposure to the competitive inhibitor methyl alpha-D mannopyranoside. Complete reversal of contact does not occur with pronase pretreated cells. The comparatively greater tenacity of contact between cells that were treated with pronase before exposure to lectin argues for an involvement of nonspecific interactions in the agglutination process. The results are compared with previously published studies of spatially periodic contact patterns induced by a range of other polymers. PMID- 1725501 TI - Light-induced photon emission by rat hepatocytes and hepatoma cells. AB - We have investigated spontaneous and light-induced photon emission of suspensions of rat hepatocytes and of HTC hepatoma cells. Rat hepatocytes exhibit spontaneous biophoton emission, but from hepatoma cells this was not detectable. In contrast, after irradiation with white light, the reemission intensity was found to be lower for hepatocytes than for the tumor cell line. Induced photon emission was neither influenced by anaerobiosis nor by the intactness of the cells. Cell fractionation studies demonstrate that the induced photon emission was caused by the nuclear fraction and by isolated chromatin. Phenol-extracted DNA, however, has lost this capacity. Our data suggest that differences in the chromatin structure may explain the cell-specific induced photon emission. PMID- 1725502 TI - Dimensional changes in cells and tissues during specimen preparation for the electron microscope. AB - Studies on dimensional changes incurred during preparation of tissue specimens for the transmission and scanning electron microscopes are reviewed, with emphasis on quantitative measurements pertinent to morphometry and three dimensional reconstruction. The scope of the review includes fixation, dehydration, plastic embedment, critical-point drying, and freeze-drying. Recommendations are presented for monitoring dimensional changes; a strategy for the choice of method of specimen preparation is outlined. PMID- 1725503 TI - Effects of doxorubicin on the sensitivity of L1210 leukemia cells to deformation associated trauma. AB - Most of the cancer cells arrested in the microcirculation during hematogenous metastasis are rapidly killed; one major mechanism is surface-membrane rupture, associated with the mechanical deformation of cancer cells in capillaries. The feasibility of increasing the susceptibility of cancer cells to lethal, deformation-associated trauma by doxorubicin, was tested in an in vitro mechanical model system, by filtering suspensions of L1210 leukemia cells through 8-microns pore-size Nuclepore membranes, with or without prior incubation with 10(-7)M doxorubicin. The results showed that mechanically-induced loss of cancer cells immediately after filtration was increased from 18 to 55% in cells previously exposed to doxorubicin for 48 h. The results indicate the feasibility of chemotherapeutic enhancement of the mechanical killing-action of the microvasculature as a potential rate-regulator of hematogenous metastasis. PMID- 1725504 TI - Improvement in survival from choriocarcinoma in Harare. AB - Thirty-nine patients with choriocarcinoma and one with post molar trophoblastic tumour are presented. It was often found that there had been a long interval between the preceding pregnancy and the time of diagnosis or presentation. As a result, there was a high incidence of non-gynaecological presenting symptoms and 95 pc of our patients belonged to the high risk or poor prognosis group. The highest rate of cerebral metastases from choriocarcinoma so far reported in the world literature is presented here. At the beginning of 1987, a modified EMA regimen as introduced and has produced a great improvement in survival. The mortality from choriocarcinoma in Harare from 1985 to 1986 was 81 pc. In 1987 to 1988, this has fallen to 31 pc. The follow-up for the patients on the modified EMA regimen varies from four to twenty-four months. PMID- 1725506 TI - A case of homozygous sickle cell disease in Sri Lanka. AB - An 11 year old Muslim boy with a 2 month history of fever, loss of appetite, pallor and abdominal distension, had hepato-splenomegaly. Haemoglobin electrophoresis showed the presence of haemoglobins S and F, with complete absence of haemoglobin A. The sickling test was positive. Marital consanguinity was present. In both parents, the sickling test was positive and haemoglobin electrophoresis showed the presence of haemoglobins A and S. This is the first report of homozygous sickle cell disease in Sri Lanka. PMID- 1725505 TI - Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs. AB - To investigate whether RNA editing in plant mitochondria modifies structural RNAs as well as protein-coding RNAs we compared the genomic-encoded information with the respective transcripts of several genes in Oenothera. The genes analysed are the 5S, 18S and 26 S rRNAs, the alpha-subunit of ATPase (atpA), cytochrome b (cytb), orfB, which is located upstream of cytochrome oxidase subunit III, and the respective leader, trailer and spacer sequences. All open reading frames were found to be edited to some degree. The atpA coding region has the least edited mRNA in Oenothera mitochondria, with only four nucleotides altered in the 1533 nucleotide open reading frame. From this analysis we conclude that frequent RNA editing is indicative of functional protein coding regions in plant mitochondria. The extensive editing in orfB, for example, suggests that this orf codes for a mitochondrial protein. No RNA editing event was found in the 5S rRNA or in the 1824 nucleotides analysed of the 18S rRNA, but two nucleotides were found to be altered in the 1970 nucleotides compared for the 26S rRNA. One nucleotide alteration has changed C to U, the other in reverse U to C. However, only one of five cDNA clones covering this region shows the modifications, similar to many silent editing events in open reading frames. RNA editing in the structural RNAs thus does not seem to be essential for their function in the mitochondrial ribosome. PMID- 1725507 TI - The effect of ligands on the uptake of iron by cells in culture. AB - Uptake of iron by a mammalian epithelial cell line (CNCM I-221) was shown to be dependent on the nature of the iron complex. Iron uptake was demonstrated by cytochemical staining and determination of redox-reactive iron in cell lysates. Three classes of ligands were investigated: (i) low molecular weight hydrophilic compounds, represented by ethylenediamine-tetraacetic acid (EDTA) and other charged ligands such as adenosine phosphates (ATP, ADP, AMP) and diethylenetriaminepentaacetic acid (DTPA), (2) low-molecular weight lipophilic ligands such as 8-hydroxyquinoline (8-HQ) and (3) a high molecular mass ligand, dextran. Iron complexed to 8-HQ accumulated intracellularly, the uptake rate of iron being 4.16 fmoles cell-1 h-1 of exposure at 37 degrees C or 3.86 fmoles cell 1 h-1 at 4 degrees C. Iron-dextran was endocytosed and retained in phagosomes. The uptake rate of iron following exposure to iron dextrans was found to be 5.6 fmoles cell-1 h-1 of exposure at 37 degrees C. In contrast to iron/8-HQ, uptake of iron dextran by cells was inhibited at 4 degrees C. Iron complexed to low molecular weight hydrophilic ligands was not taken up by cells. Cytotoxicity was measured by reduction of plating efficiency or tritiated thymidine incorporation. These tests showed that toxic effects of added iron were demonstrable only in cells exposed to the complex with 8-HQ. PMID- 1725508 TI - Cytophotometric assessment of Feulgen-DNA and coomassie-total cell protein in adrenocortical fasciculata cells of noise-exposed Wistar rats. AB - The present investigation was undertaken to examine possible noise-induced alterations in adrenal fasciculata cell (AFC) metabolism, and also to determine if the magnitude of these changes differs in male versus female rats. Wistar rats approximately 3 months old were exposed to intense noise for 60 min (100 dB, re 2 x 10(-5) N(m2)-1, 350-20,000 Hz); control rats were housed under identical conditions, at an ambient noise level of 40-60 dB. Adrenal fasciculata cells (AFC) from each animal were examined for noise-induced alterations in Feulgen-DNA reactivity (as an indicator of chromatin template activity) and Coomassie-total cell protein levels using scanning-integrating cytophotometry. The results provide evidence that intense noise elicited a marked AFC metabolic enhancement in both male and female rats; the degree of this enhancement was more pronounced in males. This disparity may be due to pre-existing differences in male versus female AFC enzymatic capability and subsequent responsiveness to noise-induced activation. PMID- 1725509 TI - Comparison of normal subjects and asthma patients with respect to age-related elastase-binding activity of alpha 2-macroglobulin in plasma. PMID- 1725510 TI - Alpha 2-macroglobulin and generation of oxygen radicals by granulocytes: potential role in prevention and treatment of reperfusion injury. PMID- 1725511 TI - Two unknown renal complications in primary distal renal tubular acidosis. PMID- 1725512 TI - Single channel and monolayer studies of acylated gramicidin A: influence of the length of the alkyl group. AB - Three different gramicidin A analogues bearing acyl chains of various length on the ethanolamine moiety have been studied by investigating their single channel behaviour and their monolayer properties. It is shown that the single channel conductance does not depend on the substitution of the ethanolamine OH group and that the channel lifetime is roughly proportional to the length of the alkyl chain. The monolayer study indicates that acylation of gramicidin A produces compounds which have medium-dependent conformations. These acylated compounds are miscible with lipids, while GA is not, and the surface potential is not modified by the esterification of the alcohol group. PMID- 1725514 TI - [Role of substance P on neurogenic inflammation in the rat dental pulp and inferior lip]. AB - A physiological role of substance P (SP) in inflammatory reaction was examined in the rat incisor pulp and inferior lip. SP content in pulps and lips significantly increased after antidromic stimulation of the inferior alveolar nerve. Following the same stimulation, vascular permeability also increased significantly in pulps and lips, and this permeability response was significantly inhibited by an SP antagonist. Morphine reduced the permeability response to antidromic stimulation in pulps but had no effect in lips. N-methyl levallorphan (a peripherally selective narcotic antagonist) prevented the morphine-induced reduction, and was more potent than naloxone. Morphine caused a marked increase of SP content in pulps following antidromic stimulation of the inferior alveolar nerve but failed in lips. These suggest a possibility that a peripheral SP release-suppressive mechanism by opiates may exist in pulps but not in lips. The permeability response to antidromic stimulation was also reduced by aspirin and a bradykinin antagonist in both of the tissues, indicating that prostaglandin and bradykinin may be related to this response. Since mepyramine and methysergide inhibited the permeability response in lips but were inactive in pulps, there is a difference in participation of histamine and serotonin between the two tissues. SP injection into the dental pulp and lip induced dye leakage. This response was inhibited by compound 48/80 pretreatment in lips whereas it was resistant in pulps. Histamine content in lips decreased significantly after antidromic stimulation and compound 48/80 pretreatment, but it was not changed in pulps. The present results suggest that in lips after being released from the peripheral sensory nerve endings SP may act on vascular system through histamine release from mast cells, while in pulps SP may directly cause vascular response because mast cells may be few or not exist. PMID- 1725513 TI - Ion channels formed by amphipathic helical peptides. A molecular modelling study. AB - Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an alpha-helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K+ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) delta-toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K(+)-channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. delta-Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K(+)-channel interactions. Rotation of W15 about its C beta-C gamma bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating. PMID- 1725515 TI - Triiodothyronine (T3) inhibition of growth hormone secretion by chicken pituitary cells in vitro. AB - These studies examined the cellular basis for the inhibitory effects of triiodothyronine (T3) on growth hormone-releasing factor (GRF)-evoked growth hormone (GH) release from chicken anterior pituitary cells in vitro. A primary monolayer culture of anterior pituitaries from 4- to 8-week-old White Leghorn cockerels was performed as previously described by this laboratory. Following a 72-hr preincubation period, cells were washed and incubated (2 hr) with either secretagogues or media alone (control). T3 (20 ng/ml) or vehicle was added to cells during both the preincubation (48-72 hr) and incubation (2 hr period. Triiodothyronine reduced (P less than 0.05) GH release (ng/ml) in response to (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) the cAMP analog and protein kinase A activator, 8-bromo cAMP; and (4) the phorbol ester and protein kinase C activator, phorbol 12-myristate 13-acetate. Triiodothyronine reduced (P less than 0.05) the intracellular content of GH and total GH (released GH and intracellular GH) irrespectively of whether secretagogues were also present. When GH release was expressed as a percentage of total GH [released GH/(intracellular GH + released GH)], percentage GH released in response to GRF, or the protein kinase A, protein kinase C, or calcium pathway activators was not as great in T3 treated versus non-T3-treated cells. These data indicate that T3 inhibits GRF evoked GH release by reducing the availability of intracellular stores of GH and by also inhibiting second messenger-stimulated GH release pathways. PMID- 1725516 TI - Immune response of the long-tailed field mouse (Apodemus sylvaticus) to tick borne encephalitis virus infection. AB - The immune response following infection with a virulent strain of Central European encephalitis (CEE) virus in a natural host, long-tailed field mouse (Apodemus sylvaticus L.) and white laboratory-bread ICR mouse, was compared. Viraemia was demonstrated in ICR mice after intraperitoneal infection with a dose of 10(5) LD50/0.5 ml. The virus titres were high in the spleen and, particularly, in the brain. In A. sylvaticus the virus was detected in the blood and spleen, but not in the brain. CEE virus multiplied in peritoneal macrophages from ICR mice, but not from A. sylvaticus. The infection induced a strong interferon response in both hosts. The natural killer (NK) cell activity increase was twice as high in A. sylvaticus compared to ICR mice. The neutralization antibodies appeared sooner in A. sylvaticus and reached higher titres in the early phases of infection. PMID- 1725517 TI - Vertex evoked potentials to tonal, verbal and white noise stimuli in children with developmental dysphasia and dysarthria. PMID- 1725518 TI - Simplified developmental assessment. PMID- 1725519 TI - Trivandrum Developmental Screening Chart. AB - The Trivandrum Developmental Screening Chart (TDSC) was designed by selecting 17 test items from BSID (Baroda Norms). It was validated both at the hospital and community level against the standard DDST. TDSC had a sensitivity of 66.7% and specificity of 78.8%, which makes it an acceptable simple screening tool even for the community level worker. PMID- 1725520 TI - Relaxometry of dextran and albumin labeled with Gd-chelates. PMID- 1725521 TI - Paramagnetic dextrans as magnetic resonance contrast agents. PMID- 1725522 TI - Blood-pool x-ray contrast agents. Evaluation of a new iodinated polymer. PMID- 1725523 TI - Iodinated polymer as blood-pool contrast agent. Computed tomography evaluation in rabbits. PMID- 1725524 TI - Effect of the beta-adrenergic agonist L644,969 on muscle growth, endogenous proteinase activities, and postmortem proteolysis in wether lambs. AB - To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins. PMID- 1725525 TI - Dictyostelium discoideum contains two profilin isoforms that differ in structure and function. AB - Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a lambda gt11 cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13,064 Da vs 12,729 Da), has a more acidic isoelectric point (calc. pI 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most preparations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd 1.8 x 10(-6) M) as compared to that of profilin I (approx. Kd 5.1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725526 TI - Differential pathways of recruitment for centrosomal antigens to the mitotic poles during bipolar spindle formation. AB - As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine-HCl at pH 1.0 for 1 h to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluorescein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtK1 cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways. PMID- 1725527 TI - Long-term persistence of hepatitis C virus antibodies in a single source outbreak. AB - The occurrence of antibodies to hepatitis C virus (HCV) was investigated in 81 patients who developed hepatitis non-A, non-B (HNANB) after parenteral administration of contaminated immunoglobulin to prevent Rh sensitization. Sera from 74 of the 81 patients (89.9%) were anti-HCV positive at either 6-12 months or 9-10 years after administration of immunoglobulin. Sera were not available from any patients at either of the times: however, 52 of 56 sera (92.9%) were anti-HCV positive 6-12 months after use of immunoglobulin, and anti-HCV was present in 45 of 65 sera (69.2%) 9-10 years after immunoglobulin treatment. Of the latter, only two of 13 (15.4%) sera from patients who recovered from hepatitis were anti-HCV positive, whereas 43 of 52 patients (82.7%) with chronic disease were anti-HCV positive. The ELISA using a recombinant antigen was found a good detector as marker for a HCV infection because 90% of patients infected by a common source became anti-HCV positive. However, 10 years after infection most patients who did not develop chronic disease no longer had detectable antibodies. PMID- 1725528 TI - The serum concentrations of the aminoterminal propeptide of procollagen type III and the hepatic content of mRNA for the alpha 1 chain of procollagen type III in carbon tetrachloride-induced rat liver fibrogenesis. AB - Serum concentrations of the aminoterminal propeptide of procollagen type III (PIIIP) are elevated in fibrogenic diseases of the liver, but the mechanism of elevation is not fully understood. To investigate the mechanism, we compared serum concentrations of PIIIP with total liver content of mRNA for the pro alpha 1 (III) chain, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Adult male rats received CCl4 in mineral oil twice weekly for 8 weeks and were compared with age-matched controls. Serum concentrations of PIIIP were measured by a specific radioimmunoassay; molecular sizes of PIIIP in serum were also determined. Pro alpha 1 (III) mRNA content in the liver was quantitated by RNA slot-blot hybridization and chemical measurement of total hepatic RNA content. Total collagen content of the liver was estimated by hydroxyproline measurement. All CCl4-treated animals had septal fibrosis after 4 weeks, and evidence of cirrhosis (regenerative nodules, ascites) was seen after 7 weeks of treatment. Serum concentrations of PIIIP and pro alpha 1 (III) mRNA content in the liver were correlated well until cirrhosis has established. They increased simultaneously after 3 weeks of treatment, 1 week before any elevation of hepatic hydroxyproline could be detected. After cirrhosis has established, pro alpha 1 (III) mRNA content in the liver decreased markedly, but serum PIIIP levels continued to be elevated. Hepatic hydroxyproline plateaued after 5 weeks. The molecular sizes of serum PIIIP indicate the release of intact native procollagen peptide during the development of cirrhosis. In conclusion, at least in CCl4 induced liver fibrosis in the rats, serum PIIIP levels can be used as a fibrogenic marker for the period progressing to cirrhosis. But the use of the serum PIIIP levels in cirrhosis seems to be limited by factors other than liver fibrogenesis. PMID- 1725529 TI - Hepatitis C virus replication in 'autoimmune' chronic hepatitis. AB - Both high and low anti-hepatitis C virus antibody (anti-HCV) prevalence has been reported in autoimmune chronic active hepatitis. Therefore, we studied 15 consecutive HBsAg-negative, ELISA anti-HCV-positive, autoantibody-positive patients with biopsy proven chronic active hepatitis in order to confirm ELISA specificity by immunoblot test (RIBA-HCV), and to evaluate HCV replication by serum HCV-RNA. Nine patients were anti-nuclear, three type 1 anti-liver-kidney microsomal and three anti-smooth muscle antibody positive. None had associated autoimmune disease. All cases showed mild clinical disease and only moderate necroinflammatory activity. Response to prednisone was poor. RIBA-HCV confirmed ELISA results in all patients. HCV-RNA was found in the serum from 10 patients. Institution of alpha-interferon treatment in three steroid non-responsive patients was followed by prompt normalization of transaminases. Thus, a subgroup of autoantibody-positive chronic active hepatitis can be recognized as HCV related and should be clinically and etiologically distinguished from autoimmune chronic active hepatitis. Trials of alpha-interferon treatment are worthwhile in this condition. PMID- 1725530 TI - Selection for a pre-C stop codon mutation in a hepatitis B virus variant with a pre-C initiation codon mutation during interferon treatment. AB - Hepatitis B virus (HBV) variants with a stop codon, a mutated translation initiation codon or other mutations in the pre-C region which prevent e-antigen expression are highly prevalent in anti-HBe chronic carriers and can be positively selected from a mixed virus infection. Our laboratories recently described pre-C variants with two pre-C mutations which prevent HBeAg expression. Here we have investigated whether there is a selective pressure for acquisition of the second pre-C mutation. By direct sequencing of amplified HBV DNA from sera of a chronic carrier taken during a 6-year follow-up, we found that genomes of a virus population virtually all had a pre-C translation initiation codon mutation and about 50% had an additional stop codon mutation. With the onset of interferon treatment, the genomes with the stop codon mutation increased to more than 95% while the frequency of the translation initiation codon mutation in all genomes remained constant. These data indicate positive selection (possibly immune mediated and HBeAg-targeted) for a second pre-C mutation. This putative enhancement of negative translational control may be present because a pre-C translation initiation codon mutation cannot totally prevent HBeAg expression and is therefore less frequent. PMID- 1725531 TI - Hepatitis C virus and hepatocellular carcinoma in France: detection of antibodies using two second generation assays. PMID- 1725532 TI - Tissue distribution of 111In-labeled uricase conjugated with charged dextrans and polyethylene glycol. AB - Uricase (UC) was conjugated with dextran, cationic diethylaminoethyl-dextran (DEAED), and anionic carboxymethyl-dextran (CMD) giving a molecular weight of 10000 by the periodate oxidation method. Their disposition characteristics were studied after intravenous injection (i.v.) in mice. Disposition of the conjugate with activated polyethylene glycol (PEG2) with a similar molecular weight was also studied for comparison. Tissue distribution of the 111In-labeled UC in these conjugates was evaluated by a tissue uptake clearance index calculated in terms of clearance. After i.v. injection, 111In-UC was slowly eliminated from the circulation and gradually accumulated in the liver, spleen, and kidney. Conjugation with neutral dextran slightly enhanced the uptake of 111In-UC by the liver and spleen, while PEG2 conjugation decreased the tissue uptake and resulted in extremely long plasma retention. On the other hand, DEAED and CMD conjugation resulted in significant enhancement and reduction of hepatic uptake, respectively. These results demonstrated that the pharmacokinetic behaviour of UC can be widely controlled by chemical modification with macromolecules having adequate physiochemical properties. PMID- 1725533 TI - [Growth hormone for therapy of development disorders in pediatric patients with hemodialysis for chronic kidney failure]. PMID- 1725534 TI - [Studies on antinephritic effect of Lipo PGE1 (2). The suppressive action of Lipo PGE1 on the development of accelerated passive Heymann nephritis in rats]. AB - The present study was designed to examine the suppressive action of Lipo PGE1 on the development of accelerated passive Heymann nephritis in rats. Lipo PGE1, given i.v. twice a day at 20, 40 and 80 micrograms/kg from the day after immunization with rabbit gamma-globulin (gamma-G) (the 1st day), remarkably inhibited the urinary protein excretion as well as glomerular histopathological changes such as thickening of basement membrane and spike formation. Lipo PGE1 at doses which the development of nephritis was suppressed, significantly inhibited the elevation of plasma antibody titer against rabbit gamma-G from the day before the appearance of the heavy proteinuria and apparently reduced the deposition of rat IgG in glomeruli. In addition, a single i. v. administration of Lipo PGE1 remarkably recovered the diminished renal blood flow induced by nephritis. These results suggest that intravenous Lipo PGE1 is effective in suppressing the development of the experimental membranous nephropathy. This agent may mainly prevent the development of nephritis by reducing the deposition of rat IgG in glomeruli via the suppression of host antibody formation. Furthermore, the increasing action of Lipo PGE1 on renal blood flow may be also in part related to a beneficial effect of this agent. PMID- 1725535 TI - New concepts for the treatment of unstable angina: role for intravenous diltiazem. AB - The management of unstable angina continues to undergo rapid evolution as new therapies and techniques become increasingly available to clinicians. It appears that the pathogenesis of unstable angina involves endothelial factors (plaque rupture of fissure resulting in a complex coronary stenotic lesion) together with dynamic factors (including platelet aggregation, thrombosis, and altered coronary vasomotor tone), and therefore it is clear why the approach to the patient with acute coronary syndromes has become multidimensional. This article summarizes the current views of the pathogenesis of unstable angina, and the role that the above factors may play in the clinical syndrome of acute coronary insufficiency. In addition, the newer therapeutic approaches to the pharmacologic management of unstable angina are discussed, including the use of nitrates, heparin, aspirin, and beta-blockers. Increasingly, calcium-channel blockers are being utilized in the early pharmacologic management of unstable angina, particularly because these agents have a salutary effect on reducing increased coronary vasomotor tone, reducing myocardial oxygen demand while at the same time augmenting coronary blood flow, and decreasing platelet aggregation. Numerous small clinical trials have examined the role of intravenous calcium-channel-blocker therapy for the acute management of unstable angina. In particular, intravenous diltiazem appears to be both safe and efficacious in this setting, and may offer some advantages to intravenous nitroglycerin, when used in the Coronary Care Unit setting. Because diltiazem is a heart rate-lowering vasoactive drug, it may attenuate myocardial ischemia without causing reflex tachycardia associated with other vasoactive pharmacologic therapies. Several of these studies utilizing intravenous diltiazem in unstable angina are reviewed and discussed. PMID- 1725536 TI - Target-organ protection by diltiazem. Satellite symposium to the Second International Symposium on Calcium Antagonists in Cardiovascular Care. Basel, February 15, 1991. PMID- 1725537 TI - Role of diltiazem in the treatment of silent myocardial ischemia. AB - Silent myocardial ischemia is a frequent finding when Holter monitoring is done in patients with advanced coronary disease. Silent ischemia is associated with a worse prognosis in patients with stable or unstable angina, survivors of myocardial infarction, and populations at risk for coronary disease. Whether medical therapy for silent ischemia improves prognosis is not known. In a randomized, placebo-controlled, multicenter trial of 60 patients with documented coronary disease, positive exercise tests, and ischemic episodes on Holter monitoring, long-acting diltiazem reduced ischemic episodes by 50% compared to placebo, from a mean of 5.6 to 2.8 (p less than 0.0001). Efficacy was maintained over 24 h and diltiazem also significantly improved exercise test parameters. Three smaller studies also demonstrated that diltiazem effectively reduces ambulatory ischemia; however, results with nifedipine are conflicting, with several studies showing no benefit. In contrast, beta-blockers reliably reduce ischemic episodes. The role of medical therapy for silent ischemia will be clarified only when its effect upon morbidity and mortality are determined. PMID- 1725538 TI - Diltiazem and renal hemodynamics: implications for renal protection. AB - During the past decade, attention has focused on the effects of calcium antagonists on renal function. When administered in vitro to the isolated perfused kidney, calcium antagonists (including diltiazem) do not affect the vasodilated isolated perfused kidney, but they do dramatically alter the response of this preparation to vasoconstrictor agents. Our recent studies using the isolated perfused hydronephrotic rat kidney model, which permits direct visualization of afferent and efferent arterioles, have demonstrated that the preferential augmentation of glomerular filtration rate induced by calcium antagonists in the isolated perfused kidney is attributable to preferential vasodilation of preglomerular vessels. Although the clinical implications of such observations have not been fully delineated, the results of recent studies indicate that calcium antagonists exert salutary effects on renal function in patients with impaired renal hemodynamics, such as radiocontrast-induced nephrotoxicity and transplant-associated acute renal insufficiency. PMID- 1725539 TI - Diltiazem: its place in the antihypertensive armamentarium. AB - In 15 randomized, double-blind studies with blood pressure measured at the end of the dosing interval, diltiazem sustained-release or conventional tablets were found to be equal in efficacy to hydrochlorothiazide, beta-blockers, angiotensin converting enzyme inhibitors, and other calcium-channel antagonists. The total number of patients with adverse effects and those who drop out due to adverse effects are similar for diltiazem and the other drugs. Combination therapy with diltiazem and captopril showed additive effects, and combination of diltiazem with hydrochlorothiazide or atenolol showed additional, but perhaps less than additive, effects. Six studies in older and younger patients have shown no overall effect of age on the antihypertensive effect of diltiazem. Two studies showed no difference in mean antihypertensive response between black and non black patients. In contrast to diuretics and beta-blockers, diltiazem does not have adverse metabolic effects on electrolytes, carbohydrate metabolism, and lipid metabolism. Diltiazem is an excellent antianginal agent. It has been shown in one study to decrease proteinuria as effectively as lisinopril, and it may have renal protective effects. The antihypertensive efficacy of diltiazem as monotherapy is equal to that of all other antihypertensive classes, and it is tolerated as well or better than most other antihypertensive drugs. Diltiazem is particularly indicated in patients with hypertension and concurrent angina pectoris, diabetes, hyperlipidemias, and, perhaps, chronic renal disease. PMID- 1725540 TI - The effects of nisoldipine, diltiazem, nifedipine, and verapamil on hemodynamic parameters and red blood cells-platelet interactions in patients with arterial hypertension. AB - In 80 patients with moderate hypertension the effects of nisoldipine 10 mg b.i.d., nifedipine 10 mg and 20 mg t.i.d., diltiazem 60 mg and 120 mg t.i.d., and verapamil 40 mg q.i.d. (all after single dose and 14 days' treatment) on blood pressure; hemodynamic parameters (cardiac output, stroke volume, left ventricular ejection fraction, and total peripheral resistance); and red blood cell and platelet functional state parameters (platelet aggregation, erythrocytal mechanical resistance, and free hemoglobin and ADP levels in plasma) were studied. All of the drugs studied in doses mentioned produced statistically significant hypotensive effects. Nifedipine 20 mg and nisoldipine 10 mg produced the most peripheral vasodilatator activity after a single dose and chronic treatment. Diltiazem had the same effect only in doses of 120 mg and after chronic treatment. However, all the drugs significantly normalized the red blood cell and platelet functional state disturbances in 70-80% of patients with moderate hypertension, even 2 h after a single dose. Disaggregation effect was parallel to clinical improvement. These data make it possible to predict the individual response of most patients to antihypertensive drugs on the basis of acute pharmacological tests, with determination of disaggregation effect. PMID- 1725541 TI - Chronic effects of diltiazem on the hemodynamic and sympathoadrenal tone in responsive and nonresponsive hypertensive patients at rest and during isometric exercise. AB - The objective of this study was to identify hemodynamic and sympathetic parameters that could be predictive of the hypotensive response to diltiazem (DTZ). Parameters of cardiovascular functions were measured from M-mode echocardiography and the index of sympathoadrenal tone was given by circulating catecholamine levels in 25 normotensive subjects and in 19 mild-to-moderate hypertensive patients before and after 2 months as well as 12 months (responders only) of treatment with DTZ (SR 120 or 180 mg b.i.d.). The responder (R) subgroup (63% of total population) consisted of patients who showed a decrease in mean arterial pressure (MAP) greater than or equal to 5 mm Hg (day average) by ambulatory blood pressure (BP) monitoring. Before treatment, R patients were characterized by higher circulating norepinephrine (NE) levels and by hyperkinetic cardiac functions [increased heart rate (HR), cardiac index (CI), and mean velocity of circumferential fiber shortening, p less than 0.05] while peripheral resistance was normal. In contrast, nonresponders (NR) were characterized by higher peripheral resistance (p less than 0.05) and normal cardiac functions. Following treatment, hyperkinetic cardiac functions were normalized but the peripheral resistance was unchanged in the R subgroup whereas in the NR subgroup, cardiac parameters were slightly increased and the peripheral resistance was normalized. During isometric exercise, cardiac performance was found to be impaired (p less than 0.05) and the increase in peripheral resistance was greater (p less than 0.05) in the R subgroup before treatment, whereas those responses were normal in the BR group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725542 TI - Platelet function and fibrinolytic activity in hypertension: differential effects of calcium antagonists and beta-adrenergic receptor blockers. AB - Platelet function was investigated in healthy volunteers and patients with essential hypertension by measurement of thresholds for ADP and adrenaline induced aggregation and plasma concentrations of platelet factor 4 (PF-4) and beta-thromboglobulin (beta-TG) after administration of antihypertensive drugs. Fibrinolytic activity was investigated by the euglobulin clot lysis time (ECLT) and tissue plasminogen activator (t-PA) activity. Compared to normotensive controls, patients with essential hypertension showed increased aggregation as evidenced by a decrease in ADP thresholds for ex vivo platelet aggregation. ECLT was significantly prolonged and t-PA significantly lowered, indicating impaired fibrinolytic activity in mild hypertension. In different studies, we have shown that various antihypertensive drug regimens differ in their effects on platelet function and fibrinolytic activity when given to healthy volunteers or patients with mild-to-moderate essential hypertension. In normal volunteers, treatment with the calcium antagonists verapamil, nifedipine, and felodipine lowered plasma concentrations of PF-4 and beta-TG, indicating a reduced platelet activity in vivo. Fibrinolytic activity was not influenced by calcium antagonist treatment in the normal volunteers. Interestingly, however, t-PA increased significantly in the hypertensive group. When compared to placebo or beta 1-selective blockers, propranolol, a non-selective beta-adrenergic blocker without partial agonist activity, reduced ADP and adrenaline threshold values for ex vivo platelet aggregation in hypertensive subjects and impaired fibrinolytic activity in the normal volunteers as well as in the hypertensive groups by increasing ECLT and reducing t-PA. Hypothetically, the effects of antihypertensive drugs on platelet function and fibrinolytic activity could be of importance for their proposed actions on cardiovascular morbidity and mortality. PMID- 1725543 TI - Treatment of mild-to-moderate hypertension: comparison between a calcium-channel blocker and a potassium-sparing diuretic. AB - In a multicenter study, 61 patients, 18-70 years of age, with mild-to-moderate hypertension [diastolic blood pressure (DBP) 95/114] completed a 28-week treatment. After initial placebo washout, patients were randomly allocated either to diltiazem or hydrochlorothiazide/triamterene. At the end of 12 weeks, the patients continued on the same medication if their goal blood pressure achieved (DBP less than 90; or 10 mm Hg below baseline). If not, the alternate agent was added (either diltiazem + hydrochlorothiazide/triamterene or hydrochlorothiazide/triamterene + diltiazem). At the end of 28 weeks, the intent to-treat analysis showed that 90% on diltiazem alone, 73.7% on hydrochlorothiazide/triamterene alone, 71.4% on (diltiazem + hydrochlorothiazide/triamterene), and 57.1% on (hydrochlorothiazide/triamterene + diltiazem) achieved goal BP. End point mean values of BP and heart rate after adjusting for sex, baseline values, age, and weight showed no significant difference between groups. Forty-six percent on hydrochlorothiazide/triamterene alone and 24% on diltiazem alone reported one or more adverse events, possibly related to study medication. Patients with diltiazem as the first choice had better BP control than those on hydrochlorothiazide/triamterene alone (81.5% vs. 69.7%). Furthermore, among non-goal achievers at week 12, there was a greater response in the group when hydrochlorothiazide/triamterene was added to diltiazem than when diltiazem was added to hydrochlorothiazide/triamterene. This study suggests that in mild-to-moderate hypertension, diltiazem is better than hydrochlorothiazide/triamterene as first line therapy. PMID- 1725544 TI - Studies of captopril alone and in combination with the benzothiazepine diltiazem or the dihydropyridine nifedipine in treating essential hypertension. AB - Calcium antagonists and converting enzyme inhibitors are now widely used as first line therapy for high blood pressure. This gradual move from the diuretics and beta-blockers is, in part, due to fewer or different side effects and the perceived lack of deleterious metabolic effects. Calcium antagonists may be more effective in elderly and low-renin patients. However, this is likely to be due in part to the finding that calcium antagonists are more effective with a higher initial pressure. The efficacy of converting enzyme inhibitors is related to the initial level of plasma angiotensin II (Ang II) or plasma renin activity. However, patients with low plasma renin activity also have a fall in blood pressure with converting enzyme inhibitors, illustrating the importance of differences in Ang II receptor sensitivity to the prevailing level of Ang II. Many patients require the combination of more than one drug to control their blood pressure. Combining a converting enzyme inhibitor with either a dihydropyridine or a benzothiazepine calcium antagonist is a particularly effective approach to the treatment of patients with more severe essential hypertension. PMID- 1725545 TI - Long-term efficacy of diltiazem controlled release versus metoprolol in patients with stable angina pectoris. AB - In a randomized, double-blind, multicenter study, the efficacy of diltiazem controlled-release (CR) 120 mg b.i.d. was compared with metoprolol 100 mg b.i.d. in 56 patients with stable exertional angina pectoris. Fifty-one patients (28 receiving diltiazem CR, 23 receiving metoprolol), completed a follow-up period of 8 weeks. Thirty-nine patients (20 receiving diltiazem CR, 19 receiving metoprolol), completed a follow-up period of 32 weeks. Maximal exercise testing was performed at baseline and after 8, 20, and 32 weeks of treatment. Most exercise parameters were not significantly different between the patients on diltiazem CR and those on metoprolol. However, exercise duration was longer and maximal work load was higher in patients on diltiazem CR than in patients on metoprolol, and significant differences were observed at 20 weeks of treatment (p = 0.006 and p = 0.008, respectively). At all times during treatment, heart rate at maximal exercise and rate-pressure product at maximal exercise were significantly lower in the patients treated with metoprolol. In conclusion, monotherapy with diltiazem CR is at least as effective as monotherapy with metoprolol in patients with stable angina pectoris. As compared to metoprolol, diltiazem CR has a minor depressing effect on rate-pressure product, resulting in a favorable effect on exercise duration. PMID- 1725546 TI - Effect of therapy with streptokinase, diltiazem, and heparin on ventricular arrhythmias in unstable angina. AB - We evaluated the effect of three different anti-ischemic therapeutic regimens on ventricular arrhythmias in 25 patients hospitalized for unstable angina. All patients were randomized to receive (in addition to infusion of heparin, diltiazem, and nitrates), either placebo, or streptokinase (SK) 1,500,000 U in 1 h, or SK 250,000 U in 30 min followed by 100,000 U/h for 48 h. Patients underwent ECG monitoring during the first 72 h after admission and for 24 h after 15 days of oral therapy with diltiazem, aspirin, and transdermal nitrates. Premature ventricular complexes (PVC) and ventricular tachycardia episodes (VT) were significantly reduced during the early phase of hospitalization and after 15 days, in patients treated with prolonged SK infusion. Ventricular arrhythmias are a frequent finding in unstable angina; they are correlated neither to the severity of coronary disease nor to ventricular function; prolonged infusion of SK added to heparin, diltiazem, and nitrates seems to reduce the number and severity of ventricular arrhythmias. PMID- 1725547 TI - Changes in human coronary flow reserve after administration of intracoronary diltiazem. AB - Epicardial coronary artery diameter (ECAD), coronary blood flow velocity (CBFV), and coronary flow velocity reserve (CFVR) were analyzed at baseline and after a 500 micrograms i.c. bolus of diltiazem in nonstenotic coronary arteries of awake humans. Furthermore, patients (n = 25) were first randomized to pretreatment either with placebo (n = 12) or isosorbide dinitrate (0.5 micrograms/kg/min infusion) (n = 13). Diltiazem resulted in a significant increase in epicardial diameter (+10%; p = 0.001) and in coronary blood flow (CBF) (+30%; p = 0.0001). Whereas basal CBFV only slightly increased (+7%; NS), there was a significant fall in CFVR (-11%; p = 0.001). The increase in coronary diameter and CBF after administration of i.c. diltiazem was comparable in placebo- and nitrate pretreated patients. The decrease in CFVR, however, was restricted to the placebo pretreated patients (-21%; p = 0.0004). Apparently, diltiazem attenuated the CFVR but only in the absence of nitrates. Thus, diltiazem i.c. appears to enhance myocardial oxygen supply without deleterious effects on the distal coronary perfusion pressure. PMID- 1725548 TI - Improvement of left ventricular function in silent ischemia following myocardial infarction, after administration of diltiazem. AB - In a group of 72 patients with acute myocardial infarction who underwent a maximal symptom-limited predischarge exercise test in conjunction with radionuclide angiography, 25 (35%) showed greater than 1 mm asymptomatic ST-T segment depression during exercise. All 25 patients underwent repeated exercise radionuclide angiography 2 days later, 2 h after oral intake of 120 mg diltiazem. Double product was not significantly different before and after diltiazem both at rest and during exercise. Maximal ST-T-segment depression after diltiazem was reduced from 2.4 +/- 0.9 mm to 0.8 +/- 0.6 mm (p less than 0.01). Left ventricular ejection fraction (LVEF) at rest was (before diltiazem) 52.1 +/- 8.9% and (after diltiazem) 55.1 +/- 12.3% (NS). During exercise, LVEF improved after diltiazem from 42.8 +/- 12.1% to 49.1 +/- 10.8% (p less than 0.05). Regional wall motion score (1 = normal, 2 = hypokinetic, 3 = akinetic, 4 = dyskinetic) at rest before diltiazem was 9.9 +/- 2.3 and, after diltiazem, was 9.0 +/- 1.9 (NS). During exercise, regional wall motion score improved after diltiazem from 5.9 +/- 1.3 to 4.2 +/- 1.2 (p less than 0.02). We conclude that diltiazem has acute beneficial effects on asymptomatic ST-T-segment depression and on global and regional left ventricular function in post-infarction patients with silent ischemia. PMID- 1725549 TI - New concepts in ischemia prevention. AB - Transient myocardial ischemia may result from obstruction to flow in the large epicardial coronary arteries or diminished flow reserve due to small vessel disease or left ventricular hypertrophy. In patients with coronary heart disease, calcium blockers have proven to reduce stress induced ischemia in patients with normal left ventricular function and in those with ischemic cardiomyopathy. However, recent studies indicate a need for caution when giving calcium antagonists to patients with postinfarction left ventricular systolic dysfunction. Moreover, calcium antagonists that reduce heart rate (diltiazem) are able as a monotherapy to reduce total ischemic burden. Calcium antagonists that may increase rate (dihydropiridines) have to be combined with beta-blockers to achieve this goal. For 24-h control of ischemia the ischemic threshold should be determined for a differentiated therapy in the individual patient. Is the ischemic threshold of the majority of episodes lower than the exercise threshold, a calcium blocker should work. Angiotensin-converting enzyme (ACE) inhibitors are not effective in stress-induced ischemia, but may reduce total ischemic burden, although this effect is not significant. In patients with left ventricular hypertrophy and/or small vessel disease, calcium blockers and ACE inhibitors are probably effective in regression of left ventricular hypertrophy and vascular hypertrophy. However, it remains to be shown that ischemia is reduced by these drugs. PMID- 1725550 TI - Comparison of diltiazem, nitrendipine, and their combination for systemic hypertension and stable angina pectoris. AB - The relative efficacies of 240 mg/day diltiazem and 20 mg/day nitrendipine were compared in a placebo-controlled, randomized, crossover study of 48 patients who had both moderate systemic hypertension and stable angina pectoris. Hemodynamic indices were measured by impedance cardiography and echocardiography and bicycle ergometry was performed. Compared with placebo, both diltiazem and nitrendipine decreased systolic (p less than 0.05 for both) and diastolic (p less than 0.05 for both) blood pressure, the total peripheral vascular resistance (p less than 0.01 for both), the frequency of anginal attacks (p less than 0.001 for both) and nitroglycerin usage (p less than 0.01 for both), and increased exercise duration (p less than 0.01 and p less than 0.05), exercise time to angina (p less than 0.01 and p less than 0.05) and time to the onset of ST-segment deviation (p less than 0.01 and p less than 0.05). There were no significant differences in the antihypertensive and antianginal effects of the two drugs. Resting heart rate was increased from placebo baseline with nitrendipine (p less than 0.01) during the first week of the treatment, and the reduction of the rate-pressure product and the decrease of the left ventricular mass were greater using diltiazem (p less than 0.05). Sixteen patients had insufficient responses to monotherapy and continued in the study for additional treatment with the combination 120 mg/day diltiazem and 20 mg/day nitrendipine. The normalization of diastolic blood pressure was achieved and more pronounced rate-pressure product reduction was found. No additional side effects were mentioned.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725551 TI - Inhibitor of interferon-induced double-stranded RNA-dependent protein kinase and its relevance to alteration of cellular protein kinase activity level in response to external stimuli. AB - In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA dependent activation. The inhibitor was readily dissociated from PK-I by DEAE cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein. PMID- 1725552 TI - Comparison of immunochemical specificities of Vibrio parahaemolyticus O10 and O12 antigens using monoclonal antibodies. AB - The monoclonal antibodies against Vibrio parahaemolyticus O10 and O12 antigens (lipopolysaccharide, LPS) were prepared and specificities of the antibodies were examined. Five of six anti-O10 antibodies reacted with O10 antigen, but none of them reacted with O12 antigen. On the contrary all of the five anti-O12 antibodies reacted with O10 antigen as well as homologous O12 antigen. O10 and O12 antigens were subjected to alkali treatment or periodate oxidation, and reactivities of these chemically modified preparations with the monoclonal antibodies were examined. Reactivities of O10 with anti-O10 and anti-O12 antibodies were reduced by the above two chemical treatments, but that of O12 with anti-O12 was not. O-Deacetylation of O10 LPS by the alkaline treatment was confirmed by NMR spectroscopy. These results suggest contributions to O10 specificity of O-acetyl group and periodate sensitive sugar residue. Inhibition experiments of O10 and O12 homologous precipitations were also carried out with various sugars. From the results we concluded that O10 and O12 antigenic determinants were distinct entities, although O10 and O12 antigens have been reported to be similar and cross-reactive. PMID- 1725553 TI - Steroid hormone receptors in prostatic hyperplasia and prostatic carcinoma. AB - One hundred and six prostatic tissue samples obtained from transurethral resection were analysed for androgen and estrogen receptors. In 62 of these, progesterone and glucocorticoid receptors were also assayed. Steroid receptors were assayed using single saturation dose 3H-labelled ligand assays. Ninety percent of the 97 prostatic hyperplasia tissues and six of the nine prostatic carcinoma tissues were positive for androgen receptors. Estrogen receptors were only present in 19% and 33% respectively. Progesterone receptors were present in 70% of the tissues, but glucocorticoid receptors were present in only 16% of prostatic hyperplasia and none in prostatic carcinoma. PMID- 1725554 TI - [Comparison of the results of the treatment of patients with SSPE using various immunomodulating preparations]. AB - The authors analysed the results of controlled treatment by three methods of patients in the 1st or 2nd phase of SSPE in a randomized group. The groups received: I. Propionibacterium granulosum KP-45 (interferon inducer) + isoprinosine, II--TFX (thymus extract) + isoprinosine, III--only isoprinosine. In all groups the treatment was continued during 6 months. The analysis of the clinical results of these methods failed to demonstrate a statistically significant superiority of any of these methods (among others, due to small number of cases) but an evident statistical tendency was revealed suggesting a better effectiveness of combined treatment (immunostimulator + isoprinosine) in relation to treatment with isoprinosine only. The final evaluation of the results requires further observations of these patients. PMID- 1725555 TI - Continuous subcutaneous infusions. New use for an old route. PMID- 1725557 TI - Cumulative 14-year index. Volumes 1-14: 1978-1991. PMID- 1725556 TI - [Cytological picture of the damage and regeneration of the nasal epithelium]. AB - The cytological pictures of the degeneration and regeneration of the columnar and squamous nasal epithelium were presented. Squamous metaplastic changes of the nasal epithelium were also discussed. PMID- 1725558 TI - [Characteristics of cell membrane of B-lymphocytes in chronic lymphocytic leukemia. I. Expression of class II antigens (HLA-D)]. AB - Surface expression of class II antigens (HLA-D) on B lymphocytes was studied in 47 patients with chronic lymphatic leukemia (CLL) with an aid of monoclonal antibodies against monomorphic determinants of DR, DP and DQ. An association was found between progression of disease and gradual loss of those markers, especially DP and DQ. PMID- 1725559 TI - [Cell membrane characteristics of B-lymphocytes in chronic lymphocytic leukemia. II. Expression of B-cell markers]. AB - Surface immunoglobulin, CD20 antigen and spontaneous and inducible interleukin 2 receptor (CD25) expression was assessed in chronic lymphatic leukemia. Most patients had but a weak expression of SmIg and CD20. Expression of CD25 was heterogenous and unrelated to disease activity. PMID- 1725560 TI - Functional neurosurgery--a future for the gamma knife? AB - The Gamma Knife is currently the only radiosurgical device which has been used in functional neurosurgery. This mode of utilization is possible because the instrument can make lesions in normal brains with a volume as small as 50 mm3. The experience of functional radiosurgery accumulated at the Karolinska Institute over 21 years is reviewed, and the possible implications of the new developments in imaging techniques for the future of functional radiosurgery are considered. The review covers gamma thalamotomy for pain and tremor, radiosurgery for trigeminal neuralgia, gamma capsulotomy for severe anxiety and obsessive compulsive neurosis, and Gamma Knife surgery for focal epilepsy. The important role of stereotactic MRI localization in functional radiosurgery is pointed out, and a preliminary report of the recent experience with stereotactic magnetoencephalography combined with stereotactic MRI for physiological and anatomic target localization is given. It is concluded that functional radiosurgery should only be performed with radiation of very small volumes of brain, as the very high doses required would be devastating if delivered to even small volumes. PMID- 1725561 TI - Scanning electron microscopy of corrosion casting in medicine. AB - The aims of this review are: 1. to provide a bibliography of the publications that have used the corrosion casting technique; 2. to describe the advantages and limitations of the methodology; 3. to illustrate possible applications in the field of medicine, and 4. to highlight the significance of this method in the teaching of medical students. Thus, this paper is primarily focused on the scanning electron microscopical examination of vascular corrosion casts. The unsurpassed three-dimensionality of the corrosion casting technique compared to any other means stands out in particular. This can be especially useful when complex vascular-anatomical relationships are present. This applies not only to the portrayal of the modes of branching and varying vascular densities but also to regulatory arrangements, such as sphincters and arteriovenous anastomoses. Between 1966 and 1990, a total of 549 publications were found in the Medline literature data bank, containing the key words "corrosion casting", "microvascular cast", or "vascular cast" (as of August, 1990). Of those publications, most dealt with applications to experimental animals. By contrast, only 142 reports were mainly or partially concerned with human investigational material. The normal vascular system of nearly all organs, insofar as this is of direct medical relevance, has been largely resolved. In our opinion, one of the most important potential applications of the corrosion casting technique lies in the investigation of gastrointestinal, renal or hepatic ailments, which coincide with the reconstruction or rarefication of the vascular bed, e.g., in ulcers, ileitis terminalis, colitis ulcerosa, cirrhosis or glomerulonephritis. PMID- 1725562 TI - [Home care--hospital in one's own home]. PMID- 1725563 TI - Studies on the importance of the proposed release of nitric oxide from platelets. AB - The importance of the presence of the L-arginine (L-Arg)-nitric oxide (NO) pathway in platelet function was investigated. This was achieved by studying the interactions between anti-platelet agents such as glyceryl trinitrate (GTN) or iloprost (Ilo) and the NO precursor L-Arg. Thrombin or collagen-induced aggregation of human washed platelets was inhibited in a concentration-dependent fashion by a 3 min incubation with GTN (40-200 microM). This effect was reversed by oxyhaemoglobin (oxyHb, 10 microM). GTN at the lowest concentration tested (40 microM) potentiated the anti-aggregatory activity of subthreshold concentrations of Ilo (0.2 nM). GTN did not potentiate the action of L-arginine (100 microM) and L-Arg by itself failed to influence thrombin or collagen-induced platelet aggregation. The platelet responses to thrombin or collagen were not affected by a 3 min or 1 h treatment with the NO synthesis inhibitor NG-monomethyl-L-arginine (MeArg, 300 microM). This treatment did not alter the platelet inhibitory effects of GTN and in the presence of MeArg, exogenously applied L-Arg did not potentiate the anti-platelet activity of GTN. These results indicate that under our experimental conditions human washed platelets do not generate sufficient amounts of NO to modify (1) platelet aggregation or (2) the anti-platelet activity of GTN or iloprost. PMID- 1725564 TI - Oxygen free radicals and corneal endothelium: effect on fluxes and permeability. PMID- 1725565 TI - [Tumor-associated and epithelial markers in gastrointestinal tumors]. PMID- 1725566 TI - [Regulation of the endothelium by TNF]. PMID- 1725567 TI - [Lasers in gastroenterology]. PMID- 1725568 TI - [The multivariant realization of elementary functional tasks and the simplification of the system of molecular interactions as a pattern in functional evolution (a problem-oriented paper)]. PMID- 1725569 TI - Protective effect of liposome-entrapped superoxide dismutase and catalase on bleomycin-induced lung injury in rats. II. Phospholipids of the lung surfactant. AB - The influence of liposome-entrapped catalase and/or superoxide dismutase on phospholipids of the lung surfactant in bleomycin-treated rats was investigated. Changes in phospholipid composition of lung surfactant were much pronounced in animals supplemented with antioxidant enzymes-loaded liposomes. It is suggested that liposomes are good carriers for drugs in bleomycin-induced lung injury treatment. PMID- 1725570 TI - Growth factor signal transduction in human intestinal cells. PMID- 1725571 TI - Transfer of enterally administered proteins from lactating mouse to neonate: the potential role of environmental contamination. PMID- 1725572 TI - Effect of hemodialysis on the concentration of the seven tumor markers carcinoembryonic antigen, alpha-fetoprotein, squamous cell carcinoma-related antigen, neuron-specific enolase, CA 125, CA 19-9 and CA 15-3 in uremic patients. AB - The incidence of the 7 tumor markers carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), squamous cell carcinoma-related antigen (SCC), neuron-specific enolase (NSE), CA 125, CA 19-9 and CA 15-3 was studied before and after hemodialysis (HD) in 144 uremic patients who had no malignancies. Before HD, of all tumor markers, the mean concentration of SCC only exceeded the normal value. The positive rate was highest in SCC (95.1%), and that of CEA and NSE was 25.7 and 10.6%, respectively. However, AFP was within the normal range in all cases. Among CA antigens, the positive rate of CA 125 was 7.6%, of CA 19-9 was 6.3% and of CA 15-3 was 3.5%. After HD, the incidence as well as the mean concentration of all tumor markers increased. A parallel increment of total protein was observed after HD. The membrane filter used in HD appears to be insufficient to remove tumor marker proteins during HD. It is necessary to consider the clinical interpretation of elevated tumor markers in patients with uremia. PMID- 1725573 TI - Urinary kallikrein excretion in chronic pancreatic diseases. AB - Variations in urinary kallikrein in pancreatic diseases were ascertained, and possible influencing factors were investigated. Serum amylase and urinary excretion of glandular kallikrein, pancreatic ribonuclease (RNase), gamma glutamyltransferase (GGT) and amylase were measured in 24 control subjects, 39 patients with pancreatic cancer, 49 with pancreatitis and 63 with extra pancreatic diseases. Urinary kallikrein was found to be elevated in a substantial number of patients with pancreatitis. Higher levels were detected in patients with a relapse, which was diagnosed using clinical and biochemical examinations. RNase was also increased in a high number of patients with pancreatic diseases, but was not correlated with pancreatic damage. In patients with pancreatitis, a correlation was found between urinary kallikrein and RNase excretions. No correlations were found between kallikrein and serum or urinary amylase and GGT. We can conclude that urinary kallikrein excretion increases in pancreatitis, especially when a phlogistic involvement of the pancreas is present; this condition may lead to a release of this ultrafiltrable enzyme in the circulation. Renal tubular damage, which determines a reduced reabsorption of this enzyme, seems to play a concomitant but minor role in this process. PMID- 1725574 TI - [Properties of experimental high abrasion resistance plastic teeth]. AB - High abrasion resistance plastic teeth made of microfilled urethane dimethacrylate resin were developed around ten years ago. In recent years, several of this same type of resin teeth were developed and introduced commercially in Japan. Many studies have reported the properties of restorative resin materials, but few comparative studies have been conducted among the various resin teeth on the market. The purpose of this study was to compare the basic physical properties of conventional acrylic resin teeth as dental materials with an experimental high abrasion resistance plastic for posterior teeth made of microfilled urethane dimethacrylate. In addition, we compared the properties of the experimental teeth with the other high abrasion resistance plastic teeth including a comparative study of anterior and posterior teeth already on the market. The results indicated that basic physical properties of the experimental posterior resin teeth were better than those of the conventional acrylic resin teeth, but the staining resistance of the former was a little lower than that of the latter. Further, the experimental posterior teeth appeared to have physical properties equal to those of other resin teeth of the same type, but the staining resistance of the experimental resin teeth was higher. PMID- 1725575 TI - [A carbohydrate histochemical study of the growth of rat parotid glandular cells]. AB - OBJECTIVE: The submandibular gland is used in most studies of the development and differentiation of the salivary glands. There are only a few reports on the genesis and growth of the parotid gland: reports by Emi (1939), Uehashi (1960), Harold (1961), Redman and Sreebny (1971), Eguchi (1975), Takeuchi (1978) and Redman et al. (1980) who used rats and those by Akiyoshi (1929) and Iwata (1958) who used humans. Since there are no reports on the carbohydrates in parotid glandular cells, we carried out histochemical studies of changes in the carbohydrates of the secretory granules in parotid glandular cells in young rats. MATERIALS AND METHODS: Wistar rats were mated, and 5 male offspring were killed with chloroform daily between the time of birth (day 0) and day 13 and weekly between the 2nd and 8th weeks after birth. Their parotid glands were immediately resected, fixed in buffered formalin, embedded in paraffin and cut into 6 mu sections for the following histochemical reactions (Tables 1 and 2): PAS reaction (Lillie's technique), PAS-dimedone reaction reaction and salivary digestion test for the determination of glycogen; acetylation-PAS reaction, acetylation saponification-PAS reaction and sulfation-toluidine blue reaction (TB; pH 2.5) for the determination of neutral mucopolysaccharides; Sugiyama's neutral red technique and Alcian blue staining (AB; pH 2.5) for the determination of weekly acidic mucopolysaccharides; Sugiyama's neutral red technique and Alcian blue staining (pH 1.0 and 0.5) for the determination of strongly acidic mucopolysaccharides; high iron diamine (HID) test, periodic acid-treated HID test, HID-AB (pH 2.5) test, low iron diamine (LTD) test and periodic acid-treated LID test for the determination of compound carbohydrates; and PA-Con A-HRP-AB test (pH 2.5) and PA-red-Con A-HRP-AB test (pH 2.5) as paradox lectin tests. RESULTS: Histological findings: The terminal of the parotid gland of the young rats showed two types of granule-containing cells. One of the two types was mucoid cells with large irregular massive granules strongly positive for PAS present above the nucleus (L cells), and the other, serous cells with fine granules moderately to weakly positive for PAS (S cells). L cells were present between days 1 and 11 after birth, being most abundant between days 4 and 7. In S cells, the morphology of granules began to be obvious on day 1 and to be similar to that of cells in adult rats at week 4 after birth. 1. Glycogen: i) PAS reaction: L cells showed moderately positive reaction between days 1 and 3 after birth and strongly positive reaction between days 4 and 11. S cells showed weakly positive reaction between days 1 and 3 after birth and moderately positive reaction between day 4 and week 8. ii) PAS-dimedone reaction: No glycogen was detected in the L or S cells of any animal. iii) Salivary digestion test: No glycogen was detected in the L or S cells of any animal. 2. Neutral mucopolysaccharides: i) Acetylation-PAS reaction: L cells showed slightly positive reaction only on days 3, 6 and 7. ii) Acetylation-saponification-PAS reaction: L cells showed slightly positive reaction, and S cells, weakly positive reaction starting on day 10 after birth. iii) Sulfation-TB reaction (pH 2.5): L cells showed mild metachromasia between days 2 and 9 after birth, and S cells, starting at week 3. 3. Weakly acidic mucopolysaccharides: i) Sugiyama's neutral red technique: L cells showed weak metachromatic reaction on days 3, 4 and 5 after birth and slight metachromatic reaction between days 8 and 11. S cell were negative. ii) AB staining (pH 2.5): L cells showed slightly positive reaction on days 1 and 2 after birth and moderately positive reaction between days 3 and 11. S cells showed slightly positive reaction on days 3, 4, 7 and 8 after birth. 4. Strongly acidic mucopolysaccharides: i) Sugiyama's neutral red technique: Both L and S cells were negative. ii) AB staining (pH 1.0): L cells were slightly positive betwee PMID- 1725576 TI - Standardization of immunoassays. AB - The aim of standardization is to ensure that assays of the same analyte in the same samples, done at different places or at different times or both, can be readily compared. Standardization is especially desirable for immunoassays because all external quality assessment surveys have shown that this type of method involves a much greater variability than traditional assays. A major problem in immunoassays is that the recognition of the analyte is determined by the reagent (i.e. an antibody or conversely, if antibodies are to be determined, an antigen) using a limited portion of the molecule, the epitope or the paratope, respectively. Therefore, the specificity of whole process is questionable, even if the specificity of the epitope/paratope reaction is fair. Biological variability is such that many molecules possessing the same recognition site coexist in biological fluids. A first example is the different secretion forms of hormones, their fragments, and other related hormones which occur simultaneously in the blood. A second example is the high variety of antibodies possessing the same paratope. Many other examples can be cited. Thus, a precise definition of the analyte should be given, dependent on the aim of the assay. This is a major conceptual difficulty because several different molecules having the same recognition site may cooperate in a physiological function. In this context, it may be useful to assay them simultaneously, but it is impossible to express their concentrations in common mass units. Another major difficulty in immunoassay standardization is due to the fact that such methods operate at very low concentrations and are sensitive to the environmental conditions created by ambient molecules existing in much higher concentrations. These "non-specific" effects create problems of non-comparability due to the prevalent conditions in different samples. From the different causes mentioned above, it follows that the major difficulty is to ensure perfect identity of the conditions in the calibrator and in the sample (analyte and matrix). The current procedures of assay standardization are reviewed and their deficiencies are stressed. Recent proposals for rationalization of standardization of immunoassays are described. PMID- 1725577 TI - Regulation of keratin gene expression in hair follicle differentiation. AB - In hair growth, as the follicle bulb cells rapidly differentiate into either cortical or cuticle hair keratinocytes, about 50-100 keratin genes are transcriptionally activated. However, this complexity can be reduced to several, highly conserved gene families. In studying the regulation of keratin gene expression in the hair follicle we have isolated genes from most of these families and have examined their expression patterns by in situ hybridization. In the cortical keratinocytes striking patterns of keratin gene expression exist, suggesting that different transcriptional hierarchies operate in the various cell types. Comparisons of the keratin gene promoter regions indicates conserved sequence motifs that could be involved in determining these cell specificities. Similarly, we have isolated related sheep and human cuticle keratin genes and find conserved DNA motifs and expression patterns in cuticle cell differentiation. Additionally, the expression of sheep wool follicle IF and high sulfur keratin genes in transgenic mice suggests that the regulatory DNA elements and proteins of hair keratin genes are functionally conserved between mammals. PMID- 1725578 TI - Experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle outer root sheath and matrix cells. AB - Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner root sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725579 TI - An in vitro model for the study of human hair growth. AB - Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermosubcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watch makers' forceps. Isolated hair follicles maintained free floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinized hair shaft, and was not associated with the loss of hair follicle morphology. [Methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labeling of keratins showed that their patterns of synthesis did not change with maintenance. Serum was found to inhibit hair follicle growth in vitro; and when follicles were maintained in serum-free medium, they grew for up to 10 days, suggesting that in vitro the hair follicles are able to regulate their own growth, possibly by the production of relevant growth factors. This may prove useful in identifying the autocrine/paracrine mechanisms that operate in the hair follicle. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth regulatory effect on hair follicles in vitro and that EGF and its other receptor ligand TGF-alpha mimic the in vivo depilatory effects of EGF that have been reported for sheep and mice. PMID- 1725580 TI - Stem cells in hair follicles. Cytoskeletal studies. PMID- 1725581 TI - Chromosomal localization of mouse hair keratin genes. AB - Many genetic defects are known to cause abnormal development of the coat in mice. Hair keratin genes would seem to be particularly promising candidates among the potential targets of these mutations in mice and of inherited hair-related abnormalities in humans as well. We used specific probes from cloned and sequenced mouse hair keratin cDNAs (MHKA-2, MHKB-1, and MHKB-2) to assess linkage of hair keratin genes and mouse mutations. We analyzed DNA from the progeny of interspecies backcrossed mice for segregation of hair mutations, hair ("hard") keratin alleles, and epidermal ("soft") keratin alleles (Krt-1 and Krt-2 loci). The results suggest that most, if not all, hair keratin genes (types Ia and IIa) are part of the Krt-1 locus on chromosome 11 and Krt-2 locus on chromosome 15, respectively. Linkage of the hair keratin genes and the mutations Re, Den, and Bsk on chromosome 11, and Ca, Sha, and Ve on chromosome 15 suggests that these mutations may possibly involve altered hair keratin expression or structure. In addition, the nondispersion of homologous keratin genes in the mammalian genome suggests that a domain organization of the genes has influenced evolution of the keratin gene family and that the organization may play a significant role in tissue-specific and developmental regulation of keratin gene expression as well. PMID- 1725582 TI - myc protooncogenes of wool and hair growth. AB - The growth of hard keratin fibers such as wool and hair is dependent on the proliferation of cells in the follicle bulb. If the cells leaving the bulb could be induced to undergo an extra division, then fiber growth should increase. The cellular division within the follicle is complex and probably involves one or more growth factors, which act by altering the expression of transcription factors and other nuclear proteins. We propose that the expression of the myc protooncogenes is a central part of this mechanism. In support of this hypothesis we have detected the mRNAs for TGF-beta 1, basic FGF, TGF-alpha, and c-myc in plucked wool follicles using PCR amplification. We have also shown that the TGF beta 1, TGF-beta 2, TGF-beta 3, EGF, TGF-alpha, basic FGF, N-myc, and c-myc genes are expressed in mouse skin, and we looked for changes during the hair cycle. The PCR data suggest that in whole skin the levels of mRNA for TGF-beta 1, TGF-beta 2, TGF-alpha, and c-myc do not change. In Quackenbush mice the levels for N-myc, TGF-beta 3, and basic FGF mRNA appear to be lower at the end of the hair cycle. We have confirmed in CBA/C57 black mice that lower levels of N-myc mRNA are detected when hair growth ceases in catagen and telogen. To test our hypothesis further and to assess its practical application, we are making transgenic mice in which the N-myc gene is overexpressed in the hair follicle by way of a wool keratin promoter. The transgene consists of 3.3 kb of 5' sequence from an ovine type 1 IF gene, the murine N-myc genomic coding sequence, and an SV40 polyadenylation signal. The native keratin type 1 IF gene is expressed exclusively in the wool follicle, as shown by in situ hybridization. However, in mice the injection of the transgene has resulted in high embryonic mortality and some embryos with large body size and head malformations. Since these mice were not transgenic, this is likely to be an effect of transient expression of the transgene during embryogenesis. The two transgenic mice produced so far have a normal phenotype. PMID- 1725583 TI - Differential expression of the Hox 3.1 gene in adult mouse skin. PMID- 1725584 TI - A new method of quantitating damage to the hair shaft: its application to ultraviolet- and radio frequency-treated hair. PMID- 1725585 TI - Interaction between dermal papilla and bulge: the rhino mouse mutation as a model system. PMID- 1725586 TI - The role of trichohyalin in hair follicle differentiation and its expression in nonfollicular epithelia. PMID- 1725588 TI - Benign prostatic hypertrophy. PMID- 1725587 TI - Interferon-induction in mouse spleen cells by the Newcastle disease virus (NDV) HN protein. AB - Newcastle disease virus (NDV) envelope glycoproteins that are expressed at the surface of fixed NDV (Ploufragan strain)-infected chick fibroblasts induce interferon (IFN) in mouse spleen cells. HN protein appears to be involved, since an anti-HN monoclonal antibody (Mab 3115) reduces the IFN production to 6% at most. However, the precise site of the molecule responsible for IFN induction is probably not exactly superimposed on the Mab 3115-epitope, since the NDV (83309 strain)-HN protein, which exhibits a modified Mab 3115-epitope, is also able to induce IFN. These preliminary results require further investigation in order to characterize the IFN herein demonstrated, to establish whether this induction mechanism exists in chicken lymphoid cells and to more accurately define the part of the HN molecule involved. PMID- 1725589 TI - Interactions between the human viruses and unicellular algae in marine environment. PMID- 1725590 TI - [The immune status versus poliomyelitis in a sample of the population of the provinces of Cremona and Mantova]. PMID- 1725591 TI - [Natural disaster emergencies: coordinated interventions and health management]. PMID- 1725592 TI - [Risk factors for tumors of the female genitalia]. PMID- 1725593 TI - [The epidemiology and prevention of endometriosis]. PMID- 1725594 TI - [The epidemiology and prevention of endometrial tumors]. PMID- 1725595 TI - [A phase-I clinical trial of a new antitetanus/antidiphtheria vaccine for adults]. PMID- 1725596 TI - [Traffic noise and the response of the population in the Trastevere quarter of Rome]. PMID- 1725597 TI - [The evaluation of the environmental hygiene aspects in a wet area of international interest in Sardinia]. PMID- 1725598 TI - RNA template-specific PCR: an improved method that dramatically reduces false positives in RT-PCR. AB - We report a novel modification of the reverse transcription PCR method, designated RNA template-specific PCR. With this approach, the 5' end of the first strand is tagged with a unique nucleotide sequence during reverse transcription that may then be exploited to amplify preferentially RNA-derived sequences. In our hands, RNA template-specific PCR retains the sensitivity of the traditional method, but greatly reduces the frequency of false positives, and virtually eliminates carryover contamination from PCR products amplified in previous experiments. PMID- 1725599 TI - Signal transduction in the visual system of Drosophila. PMID- 1725600 TI - Structure and biological role of vitronectin. PMID- 1725601 TI - Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues. AB - In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices. PMID- 1725602 TI - Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. AB - We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment. PMID- 1725603 TI - Healing of experimental colon anastomosis. AB - The healing of colonic anastomoses is slower and accompanied by more complications than healing elsewhere in the gastrointestinal tract. The purpose of this study was to investigate local energy metabolism and the synthesis and accumulation of extracellular matrix components central in wound healing (fibronectin, laminin and collagen types I, III, IV and V) in the healing colon anastomosis. Also, the local synthesis of EGF and EGF-receptors during normal healing were of interest because EGF is a potent enhancer of wound healing and it is found in the gastrointestinal tract. 47 female rats had an anastomosis of the sigmoid colon and eight rats served as unoperated controls. The animals were killed 1-21 days after anastomosis and the anastomotic area was removed for study. The following histologic investigations of the anastomotic area were made: conventional histology, immunohistology (with antibodies for type I, III, IV and V collagen, fibronectin and laminin), enzyme-histochemistry (for SDH, LDH, Glu-6 PhDH, NADH, NADPH, acid phosphatase, leucylaminopeptidase and GLDH) and in-situ hybridizations with cDNA clones (for pro alpha 1 (I) and pro alpha 1 (III) collagens, fibronectin, EGF and the EGF-receptor). Further studies were made by Northern hybridizations to estimate the activity of the genes expressed in the anastomotic area. The normal colon has a strong aerobic and anaerobic glycolytic metabolism. Anastomosis causes a deep and long lasting reduction in the energy metabolism in the anastomotic area. These changes are especially extensive in the mucosa and the muscle layers. On the other hand, the energy metabolism in the submucosal layer remains intact after surgery. Aerobic metabolism is not normalized in the mucosa and muscle layers until three weeks after surgery. The repair tissue in the anastomotic gap shows the first LDH activity on day three which is also the time when revascularization and accumulation of fibrillar collagens begin. A fibronectin reaction was seen from day one with maximum on day five. Some of the fibronectin is locally synthesized, according to our gene expression studies. Preexisting collagens and laminin decrease during the first two postoperative days after which they increase to preoperative values or more within a week. New laminin and type IV collagen are also synthesized as basal laminae are formed during revascularization and reepithelization. Activation of type I and III collagen genes are seen in the submucosal layer and in the serosal surface from day one and in the anastomotic repair tissue from day two. The highest expression of the collagen genes is not seen until one week after surgery.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725605 TI - [Transfusion medicine. Symposium. Kassel, September 1990]. PMID- 1725604 TI - Antigen-dependent leukotriene synthesis and histamine release from IgG1 passively sensitized guinea pig lungs ex vivo: relationship between serum levels of antigen specific IgG1 and mediator synthesis/release. AB - Naive guinea-pigs were passively sensitized with varying amounts of affinity column purified, homologous, anti-ovalbumin IgG1 (anti-OA IgG1) and then examined for a) the capacity of lung tissue to release mediators (histamine and LTB4/LTD4) in response to antigen-challenge ex vivo and b) the attendant circulating levels of anti-OA IgG1. Intraperitoneal administration of anti-OA IgG1 (0.125-0.75 mg/kg) to guinea-pigs facilitated the synthesis of LTB4 (8-25 ng/g lung) and LTD4 (18-80 ng/g) and the release of histamine (1-7 ug/g) from lung tissue after exposure to 10 micrograms/ml of ovalbumin for 20 min ex vivo. Peak levels of mediators were found using 0.5 mg/kg anti-OA IgG1 with an ED50 = 0.35 mg/kg. LTD4/LTB4 synthesis and histamine release were both antigen concentration- and time-dependent, and LT synthesis was observable in non-perfused lungs and in lungs perfused free of blood. Maximum sensitization occurred at 1-2 days post i.p. administration of anti-OA IgG1 and was maintained up to 7 days. Measurement of anti-OA IgG1 using an enzyme-linked immunosorbent assay demonstrated that circulating antibody levels were 2-6 micrograms/ml at the doses which caused sensitization. The level of anti-OA IgG1 found in passively sensitized animals was at least 100-fold less than that found in actively-sensitized guinea-pigs despite the similar magnitude in LTD4/LTB4 synthesized and the amount of histamine released. Using purified antibody, the results demonstrate that in guinea-pigs, IgG1 can play a prominent role in regulating lung LT synthesis and histamine release, and that microgram per ml circulating levels of this antibody are sufficient to sensitize naive lungs. PMID- 1725606 TI - [Current aspects of HIV infection]. AB - The epidemiology of HIV-1 and HIV-2 infection in 1989/90 shows a further spread of both viruses, especially in southern Europe, eastern Asia and central Africa. The sequence analysis of an immunodeficiency virus of a chimpanzee indicates the presence of SIV-1, in contrast to all other monkey isolates analyzed hitherto as members of SIV-2. The occurrence of eight newly HIV-infected hemophiliacs by one factor IX product gives rise to the question whether a 100% safety of blood products can be achieved. Analysis of HIV nucleic acids by PCR leads not to an improvement compared to the serological tests for the screening of blood donors. Despite all the efforts in the recent years, a safe and effective HIV vaccine will not be available in the near future. PMID- 1725607 TI - [Important laboratory parameters of deep frozen coagulation active fresh plasma and virus inactivated lyophilized pool plasma]. AB - Fresh Frozen Plasma is the most important therapeutic agent in acquired coagulation disorders. We investigated the quality of a new virus-inactivated lyophilisated human pool-plasma (VFL) and conventional fresh frozen plasma (FFP). In VFL we found a significantly reduced coagulation activity, a significantly higher pH-value, a highly significant rise of heparin values and a significantly lower number of thrombocytes. Electrolytes, protein levels and complement activity showed no significant difference. In conclusion, this virus-inactivated lyophilisated human pool plasma does not seem to be susceptible to therapy coagulation disorders like the conventional fresh frozen plasma. PMID- 1725608 TI - [Clinical importance of human plasma proteins and their future development]. AB - For more than 50 years now, proteins based on human blood plasma are used as drugs. More than 20 different protein preparations: Albumin, immunoglobulins, coagulation factors and inhibitors are currently available for substitution therapy. In the near future, antihemophilic globulins and perhaps Factor IX, too, will be produced by genetic engineering, independent from human blood plasma. Albumin and immunoglobulins as well as a number of plasma proteins for which only a small field of indication exists will continue to be produced from blood plasma. On a long-term basis, part of the immunoglobulins with special antibody titers will be replaced by human monoclonal antibodies. PMID- 1725609 TI - Quality control of immunoglobulins for intravenous administration. AB - Since there is as yet no official European pharmacopoeia or monograph for intravenously administrable immunoglobulins (IVIGs) and the manufacturers of IVIGs use different ACA methods to test their products until today, the study group 'Blood Plasma Constituents' of the DGTI carried out a multicenter study with six IVIGs from five manufacturers. The results of this first multicenter study have not been analysed statistically, because the participants have decided to carry out a second multicenter study using a uniform ACA method. PMID- 1725610 TI - [Hepatitis C virus]. AB - The hepatitis C virus is a single-stranded RNA virus and is composed of at least three structural proteins. Viral RNA can be detected in the serum shortly after infection (earliest after three days). Specific antibodies, however, are detected not earlier than 3-4 months after infection. The second generation ELISA detects antibodies to structural (core/C22) and nonstructural (C100, 3c) proteins. Patients with complete recovery may lose antibodies within 2-3 years. However, patients with chronic outcome show a long time persistence of antibodies (anti C100). Risk groups, such as hemophiliacs (70-95%), i.v. drug addicts (30-80%) and hemodialysis patients (0-45%), show a high prevalence of chronic HCV infection. The current tests seem to be good diagnostic tools for chronic HCV infection, but not for acute and past HCV infection. PMID- 1725611 TI - [Immunologic aspects of thrombocyte transfusion]. AB - This review surveys immunological aspects of platelet transfusions with regard to definition of refractoriness, clinical factors influencing platelet increments, possibilities to delay or prevent immunization, antibody specificities involved in refractory states, immunological complications of platelet transfusions and associations between production of platelet-specific antibodies (i.e. anti-Zwa and anti-Bra) and HLA class II antigens. PMID- 1725612 TI - Platelet transfusion in aplastic patients: fatal thrombocytopenic hemorrhage in myeloblastic leukemia. PMID- 1725613 TI - Bio- (hemo- plus eco-) compatibility of blood collection- and apheresis harness. PMID- 1725614 TI - [Particle formation in disposable systems for the V.50.1 (Haemonetics) cell separator]. AB - Recently, we described small, free particles in the disposable cell separator platelet apheresis systems for the cell separator V.50.1 (Haemonetics). In the meantime, the production procedure for this system has been modified and a new material (Ceramic-polypropylene) is in use for the rotating seal. We could not find the formerly described particles in 32 of these newly developed systems. PMID- 1725615 TI - [Comparison of plateletpheresis systems]. AB - Important criteria for assessing a cell separator are thrombocyte yield, separation efficiency and purity of the thrombocyte concentrates. Based on a Multicentric Counting Study in which twelve centers participated, we conclude that it is very difficult to compare the results of the various centers in regard to the separation efficiency. This is especially true for the comparison of different separation procedures. In Marburg we compared three different cell separators of the newest generation. COBE Spectra (n = 71), Fresenius AS-104 (n greater than 1100) and Fenwal CS-3000 TNX (n = 79). The COBE Spectra exhibited the best separation efficiency with the lowest leukocyte contamination (thrombocytes 4.3 x 10(11) (72.2%), leukocytes 0.5 x 10(7)) on the condition that the ACD-blood ratio did not differ more than -15% from the required algorithm. In order to reduce the risk to the donor, the system correspondingly reduces the donor's blood flow, resulting in a longer donation time (on the average 89-100 min). When the ACD ratio was reduced further, a considerable number of spontaneous and sometimes irreversible platelet aggregation occurred, increasing the risk of reduced platelet function and shortened survival. The AS-104 and the modified CS-3000 (TNX) had similar separation efficiencies (approx. 60%). While the platelet concentrates (PC) of the AS-104 almost reached the purity of that from the COBE Spectra, the leukocyte contamination of the CS-3000 PC's was still about four times higher. Other results published show that morphology, in vitro function and in vivo survival of thrombocytes collected with the AS-104 are significantly better than those with the CS-3000. Based on further criteria, the three systems were compared with each other. However, we could not come to a definite recommendation for any of the systems. PMID- 1725616 TI - [Evaluating a new, fully automated blood separator with continuous blood flow]. AB - The AS 104 separator provides for platelet yields in agreement with given standards. The number of undesired cells (leukocytes, erythrocytes) compares favorably with other technical separation systems. Concentrate volumes, total amount of ACD-A used, extracorporal blood volume and the reduction of donor blood cells are no challenge for the donor. The number of technical problems is not unusual for a new separation system entering into the first clinical evaluation. These data, as well as the deviation of yields, recommend further optimation. The technical optimation achieved so far and an expanded evaluation using statistical procedures permit an optimal, donor-independent (using variance analysis) or individual (using regression analysis) equilibration of the machine and permit the recognition of potential differences between different separators, centers or software versions, thus allowing the calculation of the number of runs needed to recognize differences from optimal standards. PMID- 1725617 TI - [Optimizing cell separation with the Cell Separator AS-106]. AB - Seventeen different separation protocols based on more than 1100 thrombocytaphereses were tested. Twelve of these are reported on in this study. It became apparent that the most important variable is the centrifugation speed (1900 rpm at a blood flow of 50 ml/min). This made it possible to collect 3.5 to 3.8 x 10(11) thrombocytes (approx. 60% extraction efficiency) from 3.15 l blood. Further optimization of this procedure is desirable, since 10% of the thrombocytes still remain in the separation chamber. However, our optimization work was successful as far as the reduction of leukocyte and erythrocyte contamination is concerned, which ultimately resulted in leukocyte and erythrocyte contamination of about 1 x 10(7). PMID- 1725618 TI - [Plateletpheresis with the new cell separator A 201 (Baxter)]. AB - This report deals with our first experiences in testing the new system A 201 (Baxter Fenwal Division) for plateletpheresis in routine application. The apparatus developed from the well-known plasmapheresis equipment Autopheresis C only requires a single venipuncture and uses two separation devices. The platelet cell device produces a primary platelet concentrate. After the donor's disconnection, the platelet-containing plasma is separated into 400 ml of red cell and leucocyte-free plasma and the platelet concentrate, which is done by a rotating membrane filter of the same instrument. Plateletpheresis was performed on 34 healthy donors (26 male and 8 female), formerly experienced by other apheresis procedures. Different samples were drawn from the donors before, during and after apheresis. In comparison with other apheresis procedures there were no significant differences in the harvested platelets (3 +/- 0.7 x 10(11), but the preparations showed a higher contamination of leucocytes (3.7 +/- 3.7 x 10(8). This means a considerable disadvantage and requires improvement. Yet the advantages of this separator are obvious: Reduction of needed anticoagulation to only 6% of processed blood volume, single needle technique, safety of operation and, due to less size, more mobility. PMID- 1725619 TI - Comparison of plateletpheresis with two different cell separators using 100 identical donors. AB - Plateletpheresis with the cell separator COBE Spectra and Fenwal CS 3000 was compared in 100 identical donors. The Spectra was significantly superior with regard to collection efficiency (53% vs. 42%, p less than .001) and leukocyte contamination of the platelet products (.1 x 10(8) vs. 1.4 x 10(8), p less than .001). Additionally, it offers a selectable volume of the concentrate and a better control of the ACD infusion. The Spectra standard procedure needs more ACD, resulting in more citrate dependent donor reactions (5% vs. 1%). In conclusion, the Spectra system shows a more efficient and pure platelet separation compared with the CS 3000. PMID- 1725620 TI - [Transfusion effect with platelet concentrates from various cell separators]. AB - 115 patients with bone marrow aplasia/hypoplasia received a total of 567 transfusions of fresh HLA-selected platelet concentrates at random from the AS 104 and CS-3000 and, whenever possible, from both separators using the same donor. By daily platelet counting pre and up to seven days post transfusion, the posttransfusional increments per 10(11) platelets transfused were calculated. Fresh platelets collected from the AS-104 showed comparable in vivo recovery at the first day post transfusion but significantly better survival compared to those from the CS-3000. This is in line with in vitro studies published before, where better in vitro function and morphology were observed. Increased platelet yields and improval of the platelet survival of the PC's from the AS-104 should result in prolonged transfusion intervals. When additionally evaluating a limited number of PC's from the AS-104 stored in teflon bags up to five days before transfusion (n = 12), our results were not favorable compared to PC's from the CS 3000 stored in polyolefine bags. As this seemed to be due to the geometry of the bags, this was consequently changed. PMID- 1725621 TI - [Prevention of HLA-alloimmunization of recipients of platelet concentrates]. AB - Over a period of twelve months 7163 HLA-matched, single-donor platelet concentrates were prepared for 557 thrombocytopenic patients with hematologic or neoplastic disorders. 80% of the platelet recipients remained HLA-antibody negative. Patients who developed HLA-antibodies received random platelets and/or leukocyte-contaminated blood-components simultaneously. Thus, HLA-matching, single-donor-preparations and removal of contaminating leukocytes may prevent HLA alloimmunisation, and therefore improve the efficacy of platelet-transfusion. PMID- 1725622 TI - [Neonatal alloimmune thrombocytopenia--can cryopreserved thrombocytes help? (Case report)]. AB - We present a case report of the transfusion of cryopreserved HPA 1-negative platelets to a newborn suffering from neonatal alloimmune thrombocytopenia (NAT). A boy weighing 3450 g, born of a HPA1-negative mother with a platelet count of 9 Gpt/l after delivery, received two transfusions of cryopreserved HPA1-negative platelet concentrates. The first transfusion resulted in a platelet increment to 32 Gpt/l (CCI 11). A second transfusion was needed after a further drop of the platelet count on day 4 to 18 Gpt/l, resulting in an increment to 64 Gpt/l (CCI 36). Normal development during the first six months was obtained. In our opinion, cryopreserved HPA1-negative platelets could be helpful in the therapeutical strategy of NAT. PMID- 1725623 TI - [Comparative evaluation of 3 leukocyte filters for thrombocyte concentrates]. AB - Several polyester adsorption filters are available for the removal of leukocytes during bedside filtration of platelet concentrates (PC). We tried to evaluate the efficacy of three leukocyte filters: PL-100 (Pall, USA), Sepacell PL-5A and PL 10A (both Asahi, Japan) using single donor PCs. All filters were used according to the manufacturer's instructions. The main difference in handling is the recommendation of priming and rinsing the Asahi filters with saline. Platelet counts were performed by an automatic cell counter, leukocyte (WBC) counts using a modified chamber method. Biocompatibility was examined by assessing anaphylatoxin, PMN-elastase and LDH levels, platelet function by measuring induced aggregation, hypotonic shock response, beta-thromboglobulin levels and the mean platelet volume. According to the different initial WBC contamination, three groups of PCs were tested. Group I and II were processed one hour after apheresis. Group III (n = 30) was stored at 12 degrees C up to five days before filtration. Function and integrity of post-filtration platelets were not affected as compared to the prefiltration status and bioincompatibility could not be detected. Our results indicate that the WBC reduction capacity of the filters is related to the initial WBC contamination of the PC. Beyond an initial contamination of 10(9) WBC, none of the filters is capable of reducing the leukocyte count below 10(6) WBC/PC. All but PL-5A reduce the WBC reliably below 10(6)/PC as determined from PCs routinely prepared. The Asahi filters cause significantly less platelet loss apparently due to the suggested postfiltration rinsing. Storage of PC does not influence the WBC reduction capacity, however increases the platelet loss for the PL-100. PMID- 1725624 TI - [HCV antibodies in selected patient groups and blood donors]. AB - In a multicenter study in three eastern German blood banks, we searched for HCV antibodies in polytransfused patients, unpaid donors, hemodialysis patients and hemophiliacs. The high seropositivity of HCV antibodies in polytransfused patients shows the danger of the transmission of HCV through the transfusion of blood. Therefore, blood banks should consider making HCV antibody screening of the blood of donors a part of the policy to reduce NANB PTH. PMID- 1725625 TI - [A new procedure for lymphocyte depletion of thrombocyte concentrates with immunomagnetic beads]. AB - In a series of pilot experiments we depleted platelet concentrates from contaminating leukocytes by use of immunmagnetic beads. The beads were coated with monoclonal antibodies directed against the leukocyte/lymphocyte antigens CD 2, CD 19 or CD 45. The amount of isolated cells depended on the dose of the used beads and was analogous to the observed decrease of leukocytes in platelet concentrates. The total amount of necessary beads for a complete leukocyte depletion depends on the grade of leukocyte contamination, which shows a high variation among different platelet preparations. PMID- 1725626 TI - Flow cytometric analysis of lymphocyte contamination of plateletpheresis products. PMID- 1725627 TI - [Therapeutic cytapheresis]. AB - In myeloproliferative disorders many complications are caused by circulatory problems due to high leukocyte or platelet numbers and by hyperviscosity. With cytaphereses and mild cytostatics like Azathioprine, these problems are solved quickly and without major side effects. We report about plateletaphereses for polycythemia vera and megacaryocytic myelosis and leukocytaphereses for chronic myelogenous leukemia. In addition, erythrocytaphereses were carried out successfully in a patient with a combination of heterozygous sickle cell anemia and thalassemia minor. PMID- 1725628 TI - [The role of stem cell mobilization in the scope of autologous blood stem cell transplantation]. AB - It is widely believed that hemopoietic stem cells have to be mobilized from extravascular sites into the circulation to guarantee a sufficient and safe blood stem cell autograft. The question of using mobilized or non-mobilized stem cells for transplantation purposes addresses the quality of hemopoietic engraftment rather than its feasibility. Two aspects are of clinical relevance: 1. The increment of peripheral cell concentration per time, and 2. shortening the duration of total aplasia following myeloablation and stem cell transplantation. When comparing the various stem cell mobilization techniques the CFU-GM yield per apheresis was highest during rh GM-CSF application (250 micrograms/m2/day continuous i.v. infusion), whereas the MNC yield was not greatly affected. More severe side effects were seen during rh GM-CSF infusion: One patient experienced an axillary phlebothrombosis. In a series of 15 advanced stage Hodgkin's lymphoma patients the reconstitutive ability of the various stem cell autografts, whether chemotherapy-, cytokine-, or non-mobilized, did not vary. Particularly in acute leukemias, mobilization of hemopoietic precursor cells does not necessarily exclude a concomitant mobilization of clonogenic tumor cells, and, therefore, the probability of disease-free survival after ABSCT might be lower when using mobilized stem cells for transplant. PMID- 1725629 TI - [Collection and transfusion of peripheral blood stem cells]. AB - Harvesting of peripheral blood stem cells (PBSC) by cytapheresis was performed in 57 patients, who underwent chemotherapy. The best yields were obtained when the leukocyte count was above 1 x 10(9)/l and the platelet count was raised above 80 x 10(9)/l. Using a Haemonetics V-50 or a Baxter CS-3000, 374 PBSC-aphereses were performed with a median of six aphereses per patient. The median number of PBSC (CFU-GM) retransfused in 22 patients who received PBSC for hematological reconstitution only was 3.26 x 10(4)/kg. For 22 patients who received autologous bone marrow plus BPSC, the median number of retransfused PBSC was 2.14 x 10(4)/kg. Myeloid engraftment was achieved in all patients, but megakaryopoiesis was delayed when the number of PBSC was less than 5.0 x 10(4)/kg. The results demonstrate that harvesting of a sufficient number of PBSC after chemotherapy is feasible but further measures like the use of rh GM-CSF will be necessary to reduce apheresis procedures and to obtain high yields to ensure rapid and complete engraftment. PMID- 1725630 TI - [Stem cell separation with different cell separators]. AB - For some years, there has been an increasing success in transplanting peripheral blood stem cells (PBSC) instead of autologous bone marrow in patients suffering from different malignancies. While collecting PBSC for autologous transplantation, we compared four different separation techniques and three different cell separators (COBE Spectra, Fresenius AS 104, Haemonetics V50) routinely used for platelet production. Our results suggest that continuous flow separators seem to have some advantage over discontinuous flow machines in harvesting PBSC. PMID- 1725631 TI - [Hepatitis C virus antibodies (anti-HCV) prevalence rate in anti-HBc-negative blood donors]. AB - Since 1986 only anti-HBc negative blood donors had been admitted for blood donations at the Heidelberg University Blood Bank. The maximum ALT-level for blood donors was set at less than or equal to 30 U/l. Four of 1514 blood donors (0.26%) were found to be anti-HCV positive. The routine screening of blood donors for anti-HBc in combination with an ALT-level less than or equal to 30 U/l leads to the observed lower prevalence compared to the rate found in German blood donors (0.26%/0.42% [1]). PMID- 1725632 TI - [Clinical use of peripheral stem cells for autologous transplantation]. AB - Peripheral blood derived hematopoietic stem cells (PBSC) have been transplanted successfully to eight patients suffering from different malignancies. The separations were started during chemotherapy-induced cytopenia and large volumes of peripheral blood were processed. Using this separation concept, it was possible in all cases to obtain the stem cell amount necessary for hematopoietic reconstitution. PMID- 1725633 TI - [Future perspectives of bone marrow and stem cell activation for autologous transplantation]. AB - Autologous bone marrow transplantation has gained an increasing role in modern oncology. The use of circulating progenitors from the peripheral blood as autografts might have some important advantages, since leukaphereses are better tolerable than a BM harvest, could be done immediately following chemotherapy, are possible in patients with previous irradiation to the pelvis and might be less contaminated with malignant progenitor cells. We have shown that monitoring transplants with the relatively simple in vitro assay for myeloid progenitor cells is predictive for hematopoietic recovery in animal models as well as in the clinical setting. Moreover, stem cells harvested during repeated leukapheresis were able to repopulate permanently the irradiated BM. Using recombinant human granulocyte/macrophage colony stimulating factor, large quantities of progenitor cells can be mobilized into the peripheral blood and collected for autotransplantation, in particular if used directly following chemotherapy. Cell separation studies showed that a bone marrow/blood barrier retains some subpopulations of progenitor cells which might lead to the preferential circulation of normal stem cells following chemotherapy. Methods are described to distinguish normal and leukemic progenitor cells which will help in the future to assay autografts both for their repopulating ability and their contamination with tumor cells. PMID- 1725634 TI - [Long-term cultivation of human bone marrow and its importance for clinical practice]. AB - In vitro analysis of early steps in hemopoiesis has become feasible by culture systems allowing long-term maintenance and proliferation of self-renewing pluripotent stem cells. In this long-term bone marrow culture system, hemopoietic stem cells can be maintained for weeks due to the inductive capacity of a marrow derived stromal microenvironment. In order to be able to analyze multiple samples, we have miniaturized the culture system. Studies were performed on the proliferative potential of pluripotent hemopoietic stem cells after intensive polychemotherapy of patients with either lymphoma or leukemia. Moreover, in long term bone marrow cultures, the effects of cytokines on the pluripotent stem cell compartment were studied. PMID- 1725635 TI - [Detection of high interleukin 6 concentrations in miniaturized human bone marrow long-term cultures]. AB - The long-term bone marrow culture system (LTBMC) consists of an inductive environment provided by a marrow-derived adherent layer which induces pluripotent stem cell replication. The activity of IL-6 was analyzed in micro LTBMC as a potential stem cell regulator. Immediately after initiation there was a tremendous increase in IL-6 activity. With each refeeding, IL-6 levels rose until confluency of the stromal layer was reached at week two to three post initiation. As a working hypothesis for further investigations, we suggest the immediate shift of stem cells after refeeding from a non-cycling into a cycling state might be mediated by the steep increase of IL-6 alone or in cooperation with other factors. PMID- 1725636 TI - [The role of blood banks in bone marrow transplantation]. AB - The transfusion service (TS) plays an important role in bone marrow transplantation (BMT). Many of the techniques and methods employed are also used in the daily work of a TS like tissue typing, apheresis techniques, handling of blood and its components under sterile conditions. In the pretransplantation phase the TS is responsible for the typing of recipient and presumptive donors, harvesting of autologous blood and selection of appropriate blood components. During BMT the TS can perform bone marrow harvesting, depletion of red cells in case of ABO-incompatibility and bone marrow manipulation when T-cell depletion or purging procedures are considered. Peripheral stem cell harvest by apheresis is also best performed by the TS experienced in such techniques. Storage of hematopoietic cells in liquid nitrogen and thawing are also techniques already used in most of the transfusion services. Post BMT, the support with blood components, irradiated and almost free of white cells to avoid TA-GVH and CMV infection, is a major job of the TS. These facts demonstrate that a well organized transfusion service is a 'conditio sine qua non' for successful BMT. PMID- 1725637 TI - [Hepatitis C virus antibodies in patients following blood transfusion and kidney transplantation]. AB - The prevalence of anti-HCV in patients after kidney transplantation was tested by HCV-Antibody-ELISA (Ortho Diagnostics). In addition, reactive samples were tested by HCV-EIA (Abbott Laboratories), neutralization, anti-HBc (Corzyme, Abbott) and by HBs-Ag (Auszyme, Abbott). 27 of 271 patients (10%) were anti-HCV positive. Receiving more than one kidney graft (TPL) or the transfusion of more than four blood units (BU) increases the risk of HCV infection four times (OR: 4.1; p less than 0.01) or 2.5 times (OR: 2.5; p less than 0.05), respectively, compared with one TPL or less than 4 BU. Receiving more than one kidney graft and transfusion of more than four BU raises the risk of HCV infection 6.8 times. 52% of anti-HCV positive patients were anti-HBc positive, 48% were anti-HBc negative as well as HBs-Ag negative. PMID- 1725638 TI - [Experiences with autologous donation and transfusion equipment]. AB - For autologous plasma predeposit there are commonly two types of cell-separators in use: 1. Discontinuous centrifugation (Plasma Collection System PCS, Haemonetics Company). 2. Discontinuous membrane filtration (Plasmapur-Monitor, Organon Teknika Company). Normally donation volume is 900 ml. The rate of undesired secondary effects does not exceed those numbers known of regular homologous donors. Shed wound blood processing by a wash-centrifuge cell separator (Type Cell-Saver, Haemonetics Company) stands for optimal quality of the refusable red blood cells. Depending on the type of operation and the accuracy of wound blood suction, up to 75% of the red blood cells lost may be harvested. In order to achieve more widespread mechanical autotransfusion, one issue is to lower the rather high costs of the disposables by simplifying the too highly sophisticated systems. From clinical experience with orthopedic patients, at least practical advice is given on how to use the single autologous transfusion methods in a comprehensive strategy. PMID- 1725639 TI - [The "concept of autologous transfusion"]. AB - This study analyzes the need for homologous blood in a prospectively studied group of 4,357 orthopedic-surgical patients after having established the 'Concept of Autologous Transfusion' (CAT) in 1989, in comparison to a retrospectively studied group of 7,485 orthopedic-surgical patients that had been treated exclusively with homologous blood (in 1986 and 1987). Despite an increase by 18% of the number of operations performed (in 1986: 3,698 operations vs. 4,357 operations in 1989), the need for homologous blood has been reduced by more than 80% (in 1986: 12,600 units of homologous packed red blood cells vs. 1989: 2,145 units of homologous blood). This effect has been achieved by the combination of various blood-saving techniques, namely by normovolemic hemodilution in 3,591 patients (82.4%), intra- and post-operative blood salvage in 1,936 patients (44.4%) as well as by 2,261 preoperative autologous blood donations, and by 5,279 preoperative plasmaphereses. Preoperative autologous donations have been accompanied by side effects in 1.3% (mild and moderate); no serious or even fetal complications occurred and all ambulatory patients coming to the hospital just for an autologous donation left for home on the same day. PMID- 1725640 TI - [Risks and side effects of autologous transfusion]. AB - Several versions of autologous transfusion are becoming increasingly popular. This review deals with their risks and side effects, which are still insufficiently documented. Basically, a number of preexisting conditions amounting to a subnormal performance and reserve capacity of several organ systems contraindicates preoperative donations, but 'critical' values of relevant parameters are still under debate. Since a preoperative deposit implies that the same individual donates several units within a few weeks, reaction rates must be related to the number of donors rather than that of donations. Viewed this way, the risk of--usually cardiocirculatory and sometimes serious--incidents is two times higher in autologous than in homologous donors. The reduction of oxygen transport capacity is a matter of concern. In order to avoid a mixed-venous p02 less than 35 mm Hg, which indicates an impending oxygen deficit, all 'non hemoglobin' factors impacting on tissue oxygen supply must be normal even with 10 g/dl of hemoglobin; lower Hb values are undesirable. The use of erythropoietin to boost the preoperative harvest of red cells may not, by itself, be risky, but the accelerated pace of donations obviously is. The quality of cellular blood products prepared by intraoperative machine autotransfusion still leaves much to be desired. The suspicion that adenine and, especially, fibrinogen degradation products may be immunosuppressive, raises questions about the liberal use of autologous fresh frozen plasma. Bacterial contamination of reservoirs and the presence of tumor cells as well as fat droplets from bone marrow cannot be dismissed as being unimportant. Suction devices activate coagulation and fibrinolysis. Altogether, the risks of autologous transfusions, though different from those of homologous blood, require further efforts aimed at their reduction. PMID- 1725641 TI - [HTLV-I antibody screening of blood donors]. AB - Routine screening of 344,266 blood donations of approximately 200,000 donors for anti-HTLV-I was done by enzyme immuno assay (EIA). Repeatedly reactive samples were confirmed by Western Blot (WB) and radio immuno precipitation assay (RIPA). The prevalence of anti-HTLV-I in Bavarian blood donors is 0.02-0.026%. The specificity of HTLV-I-EIA is 99.95%. PMID- 1725642 TI - [Risks of autologous blood transfusion]. PMID- 1725643 TI - [Preoperative autologous blood donation--analysis of side effects and complications]. AB - From August 1989 to April 1990, 511 patients donated preoperative blood for elective cardiothoracic, orthopedic or orthodontic operations. No side effects related to donations showed 64% of the patients, anemia 22%, vaso-vagal reactions 11%. From the autologous units 39% were transfused to the cardiothoracic, 63% to the orthopedic and 68% to the orthodontic patients. PMID- 1725644 TI - [Risks of autologous blood transfusion]. AB - Underutilization of autologous blood for transfusion is widespread. Our results suggest that this problem could only gradually be corrected, as some patients show insufficient erythropoietic recovery after a predeposit autologous donation. PMID- 1725645 TI - [The need for thorough infection screening in donors of autologous blood]. AB - To avoid the risk of diseases transmitted by homologous blood, predeposited autologous blood is useful for elective surgical patients. World-wide, there is disagreement over whether complete donor testing should or should not be done on autologous collections. There are a variety of testing concerns which have evolved into reasons for testing all autologous units for the presence of infections disease markers. One of the reasons is the risk of giving the wrong (untested) blood to the wrong patient and transmitting HIV (and thus AIDS) or viral hepatitis. Another stated concern is the risk of creating additional problems in the laboratory by treating some units differently than other units. The argument continues that without this testing, blood bank and hospital personnel may be unnecessarily exposed to blood that has the potential to cause illness. Another justification for testing is given when the blood bank participates in crossover (transfusing blood to recipients other than the autologous donor/patient). PMID- 1725646 TI - [Risks and side effects of intraoperative autotransfusion]. AB - At the Institute of Anesthesiology of the University of Wurzburg, blood has routinely been replaced by autotransfusion in orthopedic and surgical patients since the mid-seventies. At present the Haemonetics-Cell-Saver 3 is used to prepare autologous erythrocyte concentrates. When using this or similar, older, autotransfusion machines, the most dangerous hazard is venous air embolism during manual use despite blood centrifugation and preparation. To avoid this danger, the connecting tube to the patient must be clamped during filling of the autotransfusion bag. During autotransfusion the connecting tube between blood centrifuge and retransfusion bag must be clamped. The time loss due to this management has to be accepted. Regarding coagulation disorders, autotransfusion of large amounts of blood resembles massive transfusion with homologous blood. To maintain coagulation, hemostaseological parameters (Quick, thrombin time, ATIII) should be analyzed at the latest after replacement of half the estimated blood volume. On principle, blood components should be substituted only according to measured values. The substitution of ATIII is most frequently necessary to decrease the hazard of vein thrombosis and pulmonary embolism in these patients. The hazard of blood contamination by suctioning of operating room air should be considered. The number of operating room personnel should be as low as possible. Additionally, the suction device could be constructed to function only when necessary and not continuously. When these safety measures are followed, risks of this effective blood-saving procedure are minimized. PMID- 1725647 TI - [Retrospective study of a borreliosis infected blood donor]. AB - Lyme disease is caused by a spirochete, Borrelia burgdorferi, and represents a potential transfusion hazard. Some Borrelia burgdorferi-infected blood donors may not be disqualified by standard donor selection procedures, thus possibly transmitting the disease. In a follow-up of 14 recipients of blood products donated by such a donor, no clinical signs or serologic evidence of a transfusion transmitted borreliosis could be demonstrated. PMID- 1725648 TI - [The safety of preoperative mechanical autologous plasma donation]. AB - Preoperatively performed plasmaphereses in elective surgery enables the patients not only to have an autologous product retransfused but also to receive a product that contains proteins, coagulation factors and immunoglobulins, both in physiological concentration and composition. Moreover, it is a very effective volume substitute, especially in cases in which a great intraoperative blood loss and/or an intense hemodilution may have caused disorders of the coagulatory system due to loss of coagulation factors as well as due to their dilution. Despite the fact that in our study (7540 preoperative autologous plasmaphereses in 4157 patients in 1989) approximately one-third of the patients was older than 70 years and more than 50% had to be put into group IV of the extended/modified ASA-Score, the rate of side effects accompanying autologous plasmapheresis is only 1.3%. No serious or even fatal complications occurred, and all the day-case patients coming to the hospital just for autologous donations left for home on the same day. Our results demonstrate that a preoperatively performed autologous donation is not only a very effective, but also a very safe method to reduce the need for homologous blood products. If, in elective surgery, a preoperative autologous donation is considered necessary due to the expected blood loss, the general rule for autologous donation is: A patient who is not fit for autologous donation is not fit for elective surgery, either. PMID- 1725649 TI - [Disorders of primary hemostasis caused by albumin infusion in hemodilution]. AB - Previous studies using the In-vitro Bleeding Test have shown that albumin impairs primary hemostasis more pronouncedly than other infusion solutions. For this reason we compared ADP (4 microM)-induced platelet aggregation in samples of platelet rich plasma containing 50% albumin. We added the same amount of platelet poor plasma to the controls. Using this method we could show the inhibitory effect of albumin once more. This means that not only changes in rheology are responsible for the impairment of hemostasis by albumin. Platelet aggregation itself is influenced. PMID- 1725650 TI - [Autologous hemotherapy in heart transplantation: a case report]. AB - The use of autologous blood in patients with organ transplantations is feasible only when the blood is stored in the frozen state, since the timing of the operation is not predictable. A 53 year-old patient with dilatative cardiomyopathy was selected for transplantation. By preoperative donation, four erythrocyte concentrates were prepared and frozen by high-glycerol low temperature technique. The frozen blood was thawed, manually washed and made available for transfusion as required. The increment of hemoglobin after transfusion was 0.9 g/dl per unit. Quality control of the prepared erythrocyte concentrates was performed: Resistance to osmotic fragility of the erythrocytes was in the normal range, beginning hemolysis was found at 0.40 +/- 0.02% NaCl, complete hemolysis was found at 0.30 +/- 0.02% NaCl. The osmolarity of the supernatant fluid of the final washing was 0.314 +/- 0.116 mosmol/l. This case report stresses the feasibility to prepare and store autologous blood even if the time when the blood is needed cannot be determined. PMID- 1725651 TI - [Kinetics of bacterial wash-out in the Haemonetics Cell Saver III]. AB - Autologous, outdated blood units were inoculated with four types of bacteria- Pseudomonas aeruginosa, Escherichia coli, Streptococcus faecalis, Staphylococcus aureus--in two concentrations (10e4, 10e6) to test the effect of the washing procedure (Cell Saver III Haemonetics) on the elimination of bacteria. The elimination rate ranged from 17% (Streptococcus faec. 10e4) to 96% (Pseudomonas aer. 10e6). These results confirm the routine use of wash centrifuge systems (type Cell Saver) to process shed wound blood as well as drainage blood for autologous transfusions. PMID- 1725652 TI - [Essential standards of mechanical autologous donation and transfusion]. AB - Apparative autologous blood donation and transfusion can be performed by simple devices (Bentley ATS, Sorenson, Solcotrans) or by using cell separation and RBC washing (Dideco, Haemonetics). Due to many problems in retransfusion of recovered whole blood, simple devices should no longer be used. By mechanical autotransfusion including cell separation and RBC-washing, an autologous RBC concentrate of high quality is reached. Considering essential standards, mechanical autotransfusion is a safe method to reduce the risks of homologous blood transfusion. PMID- 1725653 TI - [Quality control in autologous blood donation and transfusion]. AB - In Germany the quality control for homologous blood preparations is well established. On the other hand, the quality assurance for autologous blood products is much more complicated, due to more problems concerning blood collection and preparation. Therefore, quality control is even more strongly indicated in autologous hemotherapy. On the basis of existing standards for the preparation of homologous blood components, we defined guidelines for the quality control of the autologous preoperative blood deposit, plasmapheresis, acute normovolemic hemodilutions and the intraoperative autotransfusions. We took into account that particularly the perioperative techniques only allow the collection of blood components with a reduced quality. The guidelines include the recommendation of adequate schooling for the staff. PMID- 1725654 TI - [Therapeutic use of fresh plasma and "virus-safe" plasma]. AB - The use of plasma and factor concentrates for the treatment of coagulation disorders is well established, but many questions remain. The indications for fresh frozen plasma are still not clearly established and excessive use is rampant. The safety of all blood products has not yet been firmly established. Peer review of transfusion practice in a hospital can be achieved by a Hospital Transfusion Committee. The review and analysis of the statistical reports of the transfusion service and strategies for enhancing quality of patient care will be presented. PMID- 1725655 TI - [Effect of various blood components and additive solutions on phytohemagglutinin (PHA)-induced lymphocyte proliferation]. AB - The effect of red blood cells (RBC), fresh plasma (FP), fresh frozen plasma (FFP) and different storage solutions (e.g. CPDA-1, CPD, PAGGS-M, SAG-M, Adsol) on the immune response was investigated in human lymphocyte cultures. The inhibitory activity was measured by determining the effect of different components on the PHA-induced T-cell proliferation. Our results indicate that the inhibitory effect is caused by an as yet unidentified plasma factor (e.g. anti-idiotypic antibodies, soluble HLA-class I or II antigens). In contrast, pure RBC or blood storage solutions revealed only minimal effects on the T-cell response. PMID- 1725656 TI - [Report of the Second International Workshop and Symposium on Monoclonal Antibodies against human erythrocytes and related antigens in Lund]. AB - In about 200 papers the results of the workshop on approx. 210 clones, including 67 laboratories, are reported. The present status of techniques in production and use of monoclonal antibodies against blood group antigens, human gammaglobulins, and some complement factors is demonstrated. PMID- 1725657 TI - [Preliminary results of the DGTI Workshop for evaluating the reactivity of monoclonal anti-D]. AB - Within a working group of the German transfusion society, a workshop was conducted to evaluate the reactivity of monoclonal anti-D supernatants. During this evaluation 10,000 samples were tested, including 325 Du-samples, with 3-5 IgM type antibodies and 3-4 IgG type antibodies, which were selected to also find 'D-variants'. In general, the reactivity with D+ red blood cells was good, and specific in respect to D negative samples. Some samples have been found to have a different pattern of reactivity than the monoclonal antibodies used. Dependent on the method and on the antibody, in some studies up to 100% of Du samples could be detected by anti-D of the IgM type. Some data indicate that the reaction with Du samples is not dependent on titer. In addition, in separate studies the data show a correlation of reactivity in respect to the Rh-phenotype (CcDuee greater than ccDuEe). PMID- 1725658 TI - [Electronic data transfer from computer to computer in blood banks using HL7]. AB - To date on national, european and worldwide level, different groups and committees are working on making a compatible transfer of data in all areas possible. The Working Party on Automation and Data Processing of the International Society of Blood Transfusion (ISBT) has implemented a task force, which, together with HL7 (Health Level 7), is trying to realize a practical concept for a flexible and standardized transfer of all relevant data on blood banking. PMID- 1725659 TI - [Experiences with a Token Ring Network in blood bank administration of the Hannover medical university]. AB - The Token Ring Network is a Local Area Network of IBM. It is a very helpful tool for modulating working processes by personal computers. The Token Ring is a network of the third generation and constructed like a star net. The blood bank of the Medical University of Hannover (MHH) is using the Token Ring for the administration of blood storage and donors. Medical data of foreign laboratories are transmitted into the blood bank computer. During the last year, we had no difficulties working with this software tool in response time and data security and it seemed to us a very cheap opportunity for data integration and data collection on decentralized systems. PMID- 1725660 TI - [Quality standards and quality control of plasma]. PMID- 1725661 TI - [Efficiency of mechanical plasmapheresis]. AB - In a clinical study the automated donor plasmapheresis devices Autopheresis-C A 100, Plasmapur-Monitor, and Ultralite-PCS were tested. Including also manual plasmapheresis, we analyzed the efficiency of donor plasmapheresis, the reliability and breakdown susceptibility of the methods, the quality of the collected fresh plasma, and the suitability of the different machine plasmapheresis devices for routine application, using a total of 2,372 donor plasmaphereses. We also studied the behavior of some individual laboratory parameters due to plasmapheresis with Plasmapur-Monitor. The data allow us to recommend machine plasmapheresis for routine operation (Ultralite-PCS greater than Autopheresis-C A 100 greater than Plasmapur-Monitor). PMID- 1725662 TI - [Comparison of 4 plasmapheresis procedures]. AB - In recent years the collection of plasma has been increasingly carried out by apparative plasmapheresis. In the University Hospitals of Gottingen and Wurzburg the plasmapheresis machines PCS from Haemonetics, Autopheresis-C from Baxter, as well as Plasmapur-Monitor from Organon-Teknika, were compared with each other and with the conventional centrifugation of blood bags; experiences in routine use were complemented by specific studies. Altogether, the apparative methods distinguished themselves through a high quality of retransfused blood and collected plasma. Furthermore, practicability, donor compatibility and donor safety resulted in good acceptance by both donors and staff. PMID- 1725663 TI - [HIV study of German Red Cross blood banks in Germany and Berlin]. AB - In the HIV study of the German Red Cross blood banks the overall rate has remained constantly low, with less than two Western blot positive donors in 100,000 donors since 1987. However, the rate of HIV positivity tends to increase in male first time and in male repeat blood donors. Despite this fact, the overall rate stays constant since the number of young first time donors has decreased with increasing donations by women. Therefore the blood banks once again have to take newly intensified but also sophisticated measures to guarantee further as well a sufficient number of blood products as their HIV safety. PMID- 1725664 TI - [Side effects and risk factors of various plasma donation methods]. AB - Weekly plasma donations of 600 ml were carried out for ten weeks on 20 probands to compare the Plasma Collecting System, Autopheresis C, Plamapur Monitor and the conventional double bag centrifugation. In terms of compatibility for the donor the different systems appear comparable. The PCS tubing set has to be emptied by isotonic saline at the end of the procedure. In the Autopheresis C and Plasmapur Monitor special care should be taken not to mix up isotonic saline and anticoagulant solutions. The protein concentration is higher in the centrifugation systems, but platelet contamination is higher, too. PMID- 1725665 TI - [Virus inactivation of plasma]. AB - A procedure for the virus inactivation of single donor plasma is described. Virus inactivation is achieved by the combination of 1 mumol of phenothiazin dye, e.g. methylene blue and toluidine blue, and visible light. Using this method VSV, HSV and HIV, as well as other viruses, were inactivated. It could be shown that virus inactivation caused only a minimal reduction in the biological activities of coagulation factors and inhibitors. Furthermore, there was no indication for either neo-antigens or IgE-antibodies. In tolerance studies on dogs it was demonstrated that virus-inactivated autologous plasma caused no toxic effects. PMID- 1725666 TI - [Comparative study of 8 combined HIV-1,2-antibody tests]. AB - The sensitivity and specificity of eight different' combined' HIV-1,-2 antibody EIA's were compared by investigating serial dilutions of 22 HIV-1 and 11 HIV-2 antibody-positive sera, as well as 520 HIV-negative donor sera. All eight EIA's detected our neat seropositive samples, however with marked differences in diluted samples. For routine purposes six out of seven HIV-EIA's (and possibly also a rapid test pack) proved to be suitable. PMID- 1725667 TI - Results of a quality-control study of lyophilized pooled plasmas which have been 'virally inactivated' using a solvent detergent method (modified Horowitz procedure). AB - The 'virus-free' lyophilized pooled plasmas supplied to our institute by the German Red Cross in Hagen did not meet the quality norms found in standard products. Indeed, with respect to all the major parameters, they deviated greatly from standard coagulative fresh frozen plasmas. In order to achieve a suitable substitution effect and approximate the properties of fresh plasma, it would be necessary to administer two to three times the amount of 'virus-free' plasma. At the same time it should be noted that by contrast to coagulative fresh plasma, the new product neither compensates for factors which activate or inhibit coagulation, nor for fibrinolytic factors. On the contrary, the ratio between these factors deviates dangerously from their physiological equilibrium. Grave therapeutic consequences can be expected therefrom. In addition, most of the tested batches already contain heparin in quantities within the therapeutic range in spite of the fact that the manufacturer neglects to mention this detail. Finally, the method of preparation pushes the pH values far into the alkaline range. This fact alone could have fatal consequences if this product was administered to severely ill patients. In general, we can only express our surprise that the Bundesgesundheitsamt (German Health Board), as the official body responsible for approving this product, has agreed to its distribution on the basis of notification that changes would be made to an existing approved product. The following facts, in our opinion, should have determined the renewal of the licence for sale: As opposed to the approved product--a plasma derived from individual donors--the 'virus-free' plasma is a pooled plasma. As opposed to the approved product--a deep-frozen plasma derived from individual donors--the 'virus-free' plasma is a pooled lyophilized plasma. As opposed to the approved product which is not subjected to any chemical processing, the 'virus-free' product is treated with tri(n)butylphosphate and an unspecified detergent. These virucidal properties are assumed on the basis of consequential logic rather than proven by hard experimentally determined fact. Data confirming the efficiency of the process based on legitimate animal experiments has not be presented. In spite of all the efforts to improve safety in transfusion medicine we believe that if certain nonprofit-making blood banks are to retain their credibility, they should be subjected to the same stringent laws and regulations that are applied to the rest of the German pharmaceutical industry.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725668 TI - [Evaluation of intratumoral bleomycin application in a tumor model and in a clinical pilot study]. AB - In a total of 21 patients with squamous cell carcinomas of the oral mucosa 26.9% of the total volume of 57Co-Bleomycin injected into the tumor was found in the blood as early as 10 min following injection. Examinations using a gamma camera demonstrated that 94.6% of the substance have cleared the tumor tissue after just 1 h. This sharp and rapid drop in activity, which was particularly pronounced in carcinomas of the tongue and the floor of the mouth, might be caused by rapid resorption due to the high degree of vascularization of the maxillofacial region. Autoradiography showed deposits of the substance in tumor-free areas of the operation specimens. It seems that the postulated advantages of intratumoral application--increased concentration and depot effect in the tumor tissue--are no longer tenable, thus large-scale clinical trials with intratumoral bleomycin treatment cannot be justified. PMID- 1725669 TI - Identification of contact allergens in C.I. Solvent Red 23 (commercial Sudan III) by chemical analysis and animal testing. AB - C.I. Solvent Red 23, commercial Sudan III, is widely used in cosmetic products. Chemical analyses and guinea pig sensitization tests were carried out to identify its contact allergens. In the Magnusson & Kligman guinea pig maximization test, C.I. Solvent Red 23 showed 20% positive reactions. By conducting chemical analyses with HPLC and GLC, 2-naphthol (82 ppm), azobenzene (48 ppm), Sudan I (570 ppm) and many unknown impurities, as well as the main constituent pigment Sudan III (87%), were found. The chemical structure of one unknown impurity was identified as an isomer of Sudan III. We found that purified Sudan III showed no positive reaction, while the isomer elicited 30% positive reactions, in the same guinea pig test. Furthermore, cross-sensitization with p-phenylenediamine was investigated using the guinea pig test. Animals sensitized with p phenylenediamine also showed positive elicitation reactions with purified Sudan III. From these results, the contact allergenicity of C.I. Solvent Red 23 is considered to be due to impurities, including the isomer of Sudan III, 1-(o phenylazophenylazo)-2-naphthol. Positive reactions to Sudan III previously demonstrated in hairdressers are due to cross-sensitivity with p phenylenediamine. PMID- 1725670 TI - Phosphorylation of myelin-associated glycoprotein in cultured oligodendrocytes. AB - The myelin-associated glycoprotein (MAG) in primary cultures of oligodendrocytes is subject to phosphorylation in the absence of neurons. The positive response of this phosphorylation to vanadate suggests that one of the modified sites in MAG is phosphotyrosine. We found that phosphorylation was enhanced by brief treatment of the cells with insulin-like growth factor I and active phorbol ester, agents that stimulate oligodendrocyte differentiation. Preliminary observations suggest that phosphorylation enhances the association of MAG with the cytoskeleton. PMID- 1725671 TI - Granulocyte kinetics in neutrophilia induced by recombinant human granulocyte colony-stimulating factor in mice. AB - To investigate the mechanisms of neutrophilic expansion induced by granulocyte colony-stimulating factor (G-CSF), granulocyte kinetics was studied by means of an autoradiographic method in G-CSF treated mice. Daily intraperitoneal injections of recombinant human G-CSF (rhG-CSF; 2.5 micrograms/day for 5 days) markedly increased the white blood cell count, especially granulocytes in circulating blood. Whole body bone marrow cellularity, quantitated using the radiodilution principle described by Donohue and Finch, increased from 30.0 x 10(7) cells/body (15.2 x 10(9) cells/kg) to 83.5 x 10(7) (41.8 x 10(9)) after the administration of rhG-CSF for 3 days. Generation time of myeloblasts, mitotic pool transit time, and post-mitotic pool transit time, assessed by autoradiography with 3H-thymidine, were significantly shortened in rhG-CSF treated mice compared with the control mice. Daily neutrophil production rates, calculated from these parameters, were 15. 61 x 10(7) cells/day in rhG-CSF mice and 1.93 x 10(7) in the controls. rhG-CSF had no significant effect on the survival time of granulocytes in the circulation, assessed by T1/2 of 3H thymidine labeled granulocytes. Thus, neutrophilia induced by rhG-CSF is partly due to shortening of the generation time, mitotic pool transit time and post mitotic pool transit time of myeloid cells. PMID- 1725672 TI - Final Exit: a wake-up call to hospice. AB - The publication of Final Exit resulted in a public response that was exuberant, largely sympathetic and, to many within hospice, disquieting. The book and the public response it engendered can not be understood without exploring the Hemlock Society and the political agenda which both the Society and book advance. Hospice must begin a response to this book, and any discussion with Hemlock supporters, from a basis of consensus. Hospice must acknowledge that those within the euthanasia/assisted suicide movement believe as deeply as we in hospice in the need to address the suffering of people enduring the effects of terminal illness. We must further acknowledge that there remain unmet needs in the care of the dying which for primarily socio-political reasons hospice has been unable to resolve. There are several compelling reasons for hospice as an organized movement to oppose the political initiatives of the Hemlock Society--at least in their present form and within the current social context. These reasons involve core ethical issues and issues of direct social consequence, each of which seems sufficient to reject the current proposals. Hospice programs and personnel must enter this debate in earnest. Before serious consideration is accorded to legalization of euthanasia/assisted suicide, we must insist that genuine access to comprehensive hospice/palliative care becomes a reality for all dying patients and their families. PMID- 1725673 TI - Hospice and the do-not-resuscitate order. AB - Recent passage of the Patient Self-Determination Act will require health care providers to develop policies concerning patients' wishes for life prolonging therapy. Since American hospice programs have generally had do-not-resuscitate (DNR) policies since their inception we thought it timely to review the experience of hospice programs with the DNR order. Many programs assume that a signed DNR order is a prerequisite to being accepted as a hospice patient. Other programs are more flexible. This lack of uniformity exposes the unresolved issue within the hospice community as to what is considered appropriate hospice or palliative care. Problems with paramedics responding to 911 calls and not respecting DNR orders or living wills are also discussed. PMID- 1725674 TI - Extracellular phospholipase A2 and histamine release from rat peritoneal mast cells. AB - Phospholipase A2 (PLA2) from cobra (Naja naja) venom and PLA2 from porcine pancreas accelerated IgE antibody-mediated histamine release from rat peritoneal mast cells. These enhancements were clearly abrogated by heating the enzymes and pretreatment with parabromophenacyl bromide, mepacrine and antiflammin. Indomethacin (cyclooxygenase inhibitor) and AA-861 (lipoxygenase inhibitor) did not affect the enhancement by PLA2. These results indicate that extracellular PLA2 enhances the IgE antibody-mediated histamine release from rat peritoneal mast cells without the participation of arachidonate. PMID- 1725675 TI - Effects of N-acetylcysteine on histamine release by sodium fluoride and compound 48/80 from isolated rat mast cells. AB - N-acetylcysteine (NAC) enhances the release of histamine induced by the fluoride calcium system but not by compound 48/80. After preincubation of the cells for 2 h at room temperature (RT) as well as at 37 degrees C, NAC was found to enhance histamine release also when induced by compound 48/80. Both fluoride treatment and prolonged incubation at 37 degrees C (but not at RT) for 2 h decreased the ATP content of the cells. NAC was found to counteract the fall in ATP caused by prolonged incubation of the cells at 37 degrees C but not when induced by exposure to sodium fluoride. The results do not favor the concept that free radicals generated by fluoride treatment are responsible for the subsequent sensitivity of the cells to the secretory action of calcium. On the other hand, it cannot be excluded that free radicals generated during prolonged incubation of the cells at 37 degrees C might be involved in the decrease of the sensitivity of the cells to the secretory action of the fluoride-calcium system and of compound 48/80. This is supported by the finding that the presence of NAC not only activated the secretory response but also counteracted the decrease of the cellular ATP content noted following preincubation of the cells for 2 h at 37 degrees C. PMID- 1725676 TI - Neuroendocrine peptides in bone. AB - A method for demineralization of bone, preserving the antigenicity of neuroactive peptides, was developed. In all parts of rat long bones, nerves immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were detected after immunohistochemical staining. The majority of nerves were vascular, although several non-vascular endings were observed at the growth plate and amidst marrow cells. An abundance of nerves were demonstrated near the epiphyseal plate and in the periosteum, regions of high osteogenic activity. The occurrence of different nerve types was analyzed at different stages of heterotopic osteogenesis, induced by allogeneic bone matrix. Nerve fibres immunoreactive to SP, CGRP, NPY and TH occurred amidst differentiating chondroblastic cells in the second week. They gradually increased in number during the ensuing eight weeks. In an in vitro study of osteoblastic cells (UMR 106-01, ROS 17/2.8, Saos-2, MC3T3-E1) receptors to CGRP, VIP, noradrenaline (NA) and NPY were demonstrated as assessed by analysis of cyclic AMP formation. In UMR cells, NPY inhibited the effects of NA and parathyroid hormone (PTH), which is the first demonstration of a receptor interaction between a local neuropeptide and a systemic calcium regulating hormone. The combined findings indicate a neuroendocrine influence on bone physiology. PMID- 1725677 TI - Bone metastases as the first manifestation of a tumour. AB - We have reviewed 29 patients whose first sign of a tumour was a bone metastasis. Two primaries were identified, lung adenocarcinoma and uterine adenocarcinoma and in 2 cases a presumptive diagnosis of tumours of the breast and prostate was made. The mean survival time was 3 months. When bone metastases are found in the absence of a primary tumour, investigation must include a clinical history, physical examination, routine laboratory tests and chest radiographs. Mammography should be done in women, particularly when there are palpable axillary nodes. Abdominal CT scanning and bronchoscopy should only be undertaken when there is a clinical indication. PMID- 1725678 TI - Grosse-Kempf nail combined with scan hip prosthesis. PMID- 1725679 TI - Studies on tumor induced angiogenesis. AB - Methods were developed to test the angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Human malignant melanoma tissue and crude platelet derived growth factor (PDGF) preparations had significant angiogenic effects. Purified, recombinant PDGF preparations were also effective initiators. Hemorheologic agents which also inhibit platelet aggregation [e.g. pentoxifylline (Px) (Trental) (also found to release PgI2 and tissue plasminogen activator (t-PA)] inhibited human tumor implant-induced angiogenesis. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as immune stimulating activity, was shown to be anti-angiogenic and to increase the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranexamic acid (t-AMCHA) were anti-angiogenic. PMID- 1725680 TI - Chronic adult periodontitis and burst progression may reflect local neutrophil defects due to perivascular hyaline deposits. AB - Chronic adult periodontitis (CAP) is a common disease of the supporting tissues of teeth, and is a major cause of tooth loss. This disease is distinguished from more rare rapidly progressing forms of periodontitis, in which a variety of neutrophilic polymorphonuclear leukocyte (PMN) defects have been identified. PMN dysfunctions have, however, not been observed in CAP. In CAP, destructive episodes of the disease occur sporadically and independently in different parts of the mouth. In this paper, it is proposed that CAP is due to highly localized defects in PMN function. Impaired PMN function is suggested as resulting in the formation of a virulent bacterial plaque, which is capable of initiating periodontal pocket formation. A previously reported perivascular hyaline material may account for localized PMN defects, by reducing the number of PMNs entering affected sites. The proposed model may explain both the presence of CAP in otherwise normal patients, and the sporadic pattern of tissue destruction seen in this disease. PMID- 1725681 TI - [The role of granulocytes on the murine liver metastasis: preliminary report]. PMID- 1725682 TI - [Neurospecific proteins and the function of the hemato-encephalic barrier in patients with meningococcal and pneumococcal infections]. PMID- 1725683 TI - The biological and clinical significance of nicks in human chorionic gonadotropin and its free beta-subunit. AB - Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits, alpha and beta. Nicks or missing peptide linkages have been found in the beta 44-52 region of the beta-subunit of hCG, whether from pregnancy or trophoblast disease. This article reviews recent reports about the location of nicks in hCG, their origin and occurrence, their effects on the steroidogenic and receptor-binding activities of hCG, and on the immunological activities of hCG and its free beta-subunit. Taken together, the reports show: (1) nicks occur primarily between beta 47 and beta 48, and to a lesser extent between beta 44 and beta 45; (2) the extent of nicking in hCG samples varies widely, from undetectable to 100 percent of molecules; (3) nicks greatly reduce the steroidogenic activity of hCG in vitro (nicked molecules have less than 20 percent of the activity of the intact hormone); (4) nicks may occur at the trophoblast-myometrial interface or in the circulation by the action of human leucocyte elastase or similar leucocytic protease; (5) hCG testing kits using dimer-specific antibodies may not detect nicked molecules and may give different results from those using other antibodies; (6) hCG international reference preparations and the CR series of hCG standards are variably nicked (10 percent to 20 percent), complicating the problem of discordant hCG results in nick sensitive assays; (7) results from commonly used immunoassays for measurement of the hCG free beta-subunit vary by as much as tenfold because some of the antibodies employed do not detect nick free beta-subunit. PMID- 1725684 TI - Interferon enhances fibronectin expression in various cell types. AB - Fibronectin (FN), a normal plasma and extracellular matrix glycoprotein, plays a significant role in various phases of wound healing. At wound site FN is synthesized locally by various cell types involved in the healing process (viz. epithelial, endothelial, fibroblast and macrophage cells) or deposited from the plasma. The present study was undertaken to investigate the in vitro effect of IFN on FN synthesis as well as release in the culture medium by various cell types. Indirect immunofluorescence and immunoelectron microscopy studies, using specific antibodies, revealed that IFN treatment resulted in significantly more staining for FN as compared to untreated control cells. Metabolic labeling with 35S-methionine, immunoprecipitation and SDS-page studies showed an increase in FN synthesis and release by IFN treated cells. In addition, to determine whether this increased synthesis was reflected at mRNA levels, poly (A)+ RNA was isolated from human lung epithelial cells (A549) and probed with FN specific cDNA. We found that IFN treatment increased the level of FN mRNA. PMID- 1725685 TI - Thyroid hormone control of brain and motor development: molecular, neuroanatomical, and behavioral studies. PMID- 1725686 TI - Reversible rat mesenteric mast cell swelling caused by vagal stimulation or sham feeding. AB - Fragments of rat mesentery were examined using acetylthiocholine to detect cholinergic nerve fibers and toluidine blue to identify mast cells. 59.2 +/- 2.6 percent of mast cells were at less than one-half mean cell diameter (4-5 microns), from the nerve fibers. Under the electron microscope, the membrane of mast cells was within less than 50 nm from axon membranes, suggesting a synaptic type of connection. Average mast cell area in fasted rats increased following feeding, stimulation of the left abdominal vagus nerve or exposure of the animal to the smell of food. It returned to control values within 60-80 min. Granule exocytosis was not observed. Mast cell swelling was prevented by atropine and induced by intravenously administered carbamylcholine. It appears that in rat mesentery, impulses travelling via cholinergic, parasympathetic fibers innervating mast cells, cause mast cell swelling. Compound 48/80 administered to rats at doses causing little degranulation and minimum release of histamine, caused extensive, reversible swelling of mesentery mast cells. PMID- 1725688 TI - Angiogenesis in pannus formation. PMID- 1725687 TI - Wheal and flare responses to muscle relaxants in humans. AB - Chemically and pharmacologically unrelated molecules release histamine in humans to produce both cutaneous and systemic responses. It has been suggested that molecular changes in the new benzylisoquinoline-derived muscle relaxant, atracurium, make it less likely to cause histamine release. We therefore injected volunteers intradermally with equimolar concentrations of various muscle relaxants, morphine, papaverine (a benzylisoquinoline), and histamine, to evaluate the relative ability of these drugs to cause wheal and flare responses, and mast-cell degranulation. There were no significant differences in wheal and flare responses among the three benzylisoquinoline-derived muscle relaxants, D tubocurarine, metocurine, and atracurium. The cutaneous effects of morphine were significantly greater than those of the benzylisoquinoline muscle relaxants, suggesting both direct vascular changes and histamine release. Papaverine injection was followed by a significant wheal but no flare. Skin biopsies from vecuronium- and papaverine-induced wheals revealed normal intact mast-cell granules, suggesting a direct cutaneous vascular response rather than histamine release. Skin biopsies after morphine and atracurium injections revealed mast cell degranulation. All evaluated benzylisoquinoline muscle relaxants are equipotent histamine releasers at equimolar concentrations. A hydrogenated, benzylisoquinoline-nitrogen-containing ring, present in atracurium but not in papaverine, appears to be the molecular conformation responsible for mast-cell degranulation by atracurium. PMID- 1725689 TI - Angiogenesis and chronic inflammation. Meeting of the British Inflammation Research Association (BIRAs) 5th June, 1990. Roche Products, Welwyn Garden City. PMID- 1725690 TI - Thiol-containing compounds inhibit the production of monocyte/macrophage-derived angiogenic activity. AB - Macrophage (M phi)-mediated angiogenesis is believed to play an important role in the pathogenesis of rheumatoid arthritis. Gold sodium thiomalate, which is used in the treatment of rheumatoid arthritis, is a potent inhibitor of the production of m phi-derived angiogenic activity. To determine the mechanism of this inhibition, we studied the effects of thiol containing compounds (TCCs) on elicited mouse peritoneal m phi and lipopolysaccharide stimulated normal human monocytes. Monocyte/m phi conditioned media were potently angiogenic when assayed in rat corneas, while conditioned media from viable monocyte/m phi s treated with TCCs (at concentrations of 8.3-16.6 x 10(-5) M) were not. TCCs inhibited production of angiogenic activity by the m phi s rather than affecting other components of the angiogenic response such as the angiogenic factors or the target microvasculature of the rat cornea. Levels of the angiogenic mediator tumor necrosis factor-alpha (TNF-alpha) were not decreased in conditioned media of monocyte/m phi s treated with TCCs. We conclude that TCCs are potent inhibitors of the production of m phi-mediated angiogenic activity. This action of TCCs on m phi s may be in part responsible for the mechanism of action of therapeutic gold compounds in rheumatoid arthritis. PMID- 1725691 TI - The rat female protein, a pentraxin with lectinic properties. AB - The C-reactive protein is the major acute phase protein (APP) in humans which binds lectin-like to different membraneous structures and exerts an important function in non-specific defense. Because of a pentameric molecular symmetry CRP as well as serum amyloid P component (SAP) and hamster female protein (FP) was merged into a special protein family named pentraxins. In rats a protein was found referred to as rat FP which was close related to hamster FP with respect to hormonal regulation and APP nature as well. Based on this conformity the molecular structure of rat FP was analyzed and as the results a pentameric structure could be demonstrated for rat FP, too. Furthermore, the response of rat CRP and FP on injection of adrenal hormones, agents being involved in acute phase reaction, was investigated. Epinephrine administration led to an increase in CRP and a decrease in FP serum concentration. Dexamethasone has the same effect in case of FP and changed the CRP concentration in a biphasic way with a maximum at about 0.01 mg/kg, a minimum at 0.6 mg/kg and a return to control values at 1.8 mg/kg. Thus, the results indicate a neuroendocrine control of CRP and FP but probably in a different way. Using FITC-labelled lectin the exposition of galactose-containing membraneous structures could be demonstrated in carbon tetrachloride-injured liver tissue in contrast to controls. These binding sites are in accordance with increased FP-binding shown by immunofluorescence histochemistry. Thus, lectin-like properties may be ascribed to rat FP comparable to CRP and SAP activity. The results are discussed with respect to findings from literature that also the acetylcholine receptor seems to have a pentameric structure. PMID- 1725692 TI - Differential inhibitory effects of sulfated polysaccharides and polymers on the replication of various myxoviruses and retroviruses, depending on the composition of the target amino acid sequences of the viral envelope glycoproteins. AB - Sulfated polysaccharides (i.e., dextran sulfate) and sulfated polymers (i.e., sulfated polyvinylalcohol and sulfated copolymers of acrylic acid with vinylalcohol) were found to be potent and selective inhibitors of the replication of respiratory syncytial virus (RSV) and influenza virus type A (influenza A virus) but not of other myxoviruses (parainfluenza 3, measles, and influenza B viruses). The compounds were also inhibitory to human immunodeficiency virus type 1 (HIV-1) and HIV-2 and simian immunodeficiency virus but not simian AIDS-related virus. The mode of antiviral action of the sulfated polysaccharides and polymers can be attributed to an inhibition of virus binding to the cells (HIV-1), inhibition of virus-cell fusion (influenza A virus), or inhibition of both virus cell binding and fusion (RSV). The fact that the sulfated polysaccharides and polymers are inhibitory to some myxoviruses and retroviruses but not to others seems to depend on the composition of the amino acid sequences of the viral envelope glycoproteins that are involved in virus-cell binding and fusion. All myxoviruses and retroviruses that are sensitive to the sulfated polysaccharides and polymers share a tripeptide segment (Phe-Leu-Gly). This tripeptide segment may be involved either directly (as a target sequence) or indirectly in the inhibitory effects of the compounds on virus-cell binding and fusion. PMID- 1725693 TI - Non-phosphorylative transglucosylation and other biochemical indices in serum and uterine myoma in women. AB - Non-phosphorylative transglucosylation, GGT and LAP activity, the level of some "acute phase" proteins i.e. sialic acids, ceruloplasmin, papain and trypsin inhibitors were examined in the serum, normal myometrium around tumours and in the uterine myoma of women. It was observed that the contents of proteins in the "acute phase" as well as GGT and LAP activity increased in the serum, whereas a decreased activity of these enzymes accompanied by increased transferase activity was observed in uterine myoma. PMID- 1725694 TI - Chymotrypsinogen: an activating enzyme in the mitochondrial membrane of rat submaxillary gland. AB - A peripheral membrane protease was purified from mitochondria of rat submaxillary gland. On non-denaturing PAGE the purified enzyme showed a single protein band with the enzyme activity. It yielded two protein bands with molecular weights of 39 KDa and 20 KDa on SDS-PAGE, indicating that the enzyme is composed of two protein components. The enzyme activity was strongly inhibited by SBTI, aprotinin and benzamidine. PMSF, TLCK and EDTA did not produce inhibition. The enzyme could hydrolyze different synthetic substrates having arginine at the P1 position with highest affinity for the substrate Bz-Phe-Val-Arg-p-nitroanilide was noted. The enzyme showed significant activation of chymotrypsinogen A. PMID- 1725695 TI - Identification of pyruvate carboxylase in 3T3-L1 cells by transblot and its correlation with differentiation into adipocytes. AB - The 3T3-L1 preadipocytes treated with insulin, dexamethasone and 3-methyl-1 isobutylxanthine (IBMX) two days before reaching monolayer undergo differentiation into adipocytes. Cell lysates were prepared from these cells under various conditions and analyzed by SDS-PAGE and transblot. Peroxidase conjugated avidin used to detect endogenous proteins interacted strongly with a protein with an estimated molecular weight of 120 kDa, corresponding to pyruvate carboxylase, in the differentiated 3T3-L1 cells. On the other hand, this protein was not detected in undifferentiated 3T3-L1 cells. PMID- 1725696 TI - Presynaptic facilitation by the new nootropic drug nebracetam, of ganglionic muscarinic transmission in the dog cardiac sympathetic ganglion. AB - Effects of nebracetam (4-aminomethyl-1-benzylpyrrolidine-2-one hemifumarate, WEB 1881 FU, CAS 118607-07-1), a new nootropic drug, on impulse transmission in the cardiac sympathetic ganglia were studied in spinal dogs by monitoring heart rate as an indicator of the ganglionic function. The ganglionic stimulants were given directly into the cardiac sympathetic ganglia through the right subclavian artery (i.a.). Nebracetam, 5 mg/kg, i.v. caused a slight and temporal increase in heart rate. After nebracetam, the frequency-response curves of heart rate for preganglionic stellate stimulation (0.25-4 Hz) were not altered in the untreated and atropine-pretreated animals, but the curves (2.5-40 Hz) were shifted to the left in the hexamethonium-pretreated animals. The enhancement of ganglionic muscarinic transmission was dose-dependent on nebracetam i.v. at doses ranging from 0.5 to 15 mg/kg, with a maximal effect at 5 mg/kg. This enhanced muscarinic transmission by nebracetam was almost abolished after subsequent administration of pirenzepine 0.5 mg/kg i.v. The enhancement in the muscarinic transmission by nebracetam was also eliminated after depletion of acetylcholine at preganglionic sites caused by treatment with hemicholinium-3 in combination with preganglionic stimulation. Furthermore, nebracetam failed to affect dose-dependent post ganglionic stimulation by McN-A-343 (1-32 micrograms), 1,1-dimethyl-4 phenylpiperazinium (1-32 micrograms) and angiotensin II (0.1 and 0.2 micrograms) administered i.a. directly to the ganglia. These results suggest that nebracetam facilitates the ganglionic muscarinic transmission through acting on presynaptic sites. PMID- 1725697 TI - Effect of the new antiallergic drug epinastine on chemical mediator induced bronchoconstrictions in guinea pigs. AB - Recently, mast cell stabilizers, so called antiallergic drugs, that also have blocking effects on receptors for chemical mediators (CM) have been developed. The present study investigated the effects of a new antiallergic drug, epinastine (3-amino-9,13b-dihydro-1H-dibenz[c,f]imidazol [1,5-a]azepine hydrochloride, WAL 810 CL; CAS 80012-43-7) on the in vivo bronchoconstriction (BC) induced by several CM as compared with those of other antiallergic drugs such as ketotifen, azelastine and oxatomide. Male Hartley guinea pigs were used. The BC was measured with a modified Konzett-Rossler method and expressed as a change in ventilation overflow (VO) under anesthesia. Antiallergic drugs were given orally 1 h before i.v. administration of CM. I.v. administrations of histamine (His), U-46619 (thromboxane A2 mimetic), leukotriene D4 (LTD4), prostaglandin D2 (PGD2), substance P (SP), neurokinin A (NKA), bradykinin (BK) and endothelin-1 (ET-1) increased VO in a dose dependent manner. The potency was as follows; ET-1 greater than LTD4 greater than NKA much greater than U-46619 greater than SP greater than His greater than BK much greater than PGD2. All the antiallergic drugs used markedly inhibited the His-induced BC. Epinastine, ketotifen and azelastine also significantly inhibited the BK-induced BC; epinastine had the strongest anti-BK effect among them. On the other hand, the four antiallergic drugs did not inhibit the BC induced by the other CM except for His and BK. Based on the above results, it is suggested that epinastine possesses anti-His and BK effects, and therefore could be promising as a new antiallergic drug without sedative effect. PMID- 1725698 TI - In vitro microleakage of dentin adhesives. AB - This in vitro study measured the microleakage of current dentin bonding agents and glass-ionomer bases. Freshly extracted human molars were prepared to a flat surface, and dentin adhesives and composite resins were applied in a plastic matrix. Samples were stored in water at 37 degrees C, thermocycled, stained with AgNO3, embedded in epoxy resin, and sectioned for evaluation of stain penetration at the composite resin/tooth interface. The reliability index of the dentin adhesives varied significantly between materials. The enamel control had essentially no microleakage, and the aluminum oxalate dentin adhesive on dentin had significantly less microleakage than other dentin adhesives tested. Present dentin adhesives were unable to prevent, but may reduce, microleakage. PMID- 1725699 TI - Human T cells recognize multiple epitopes of an immediate early/tegument protein (IE62) and glycoprotein I of varicella zoster virus. AB - Infection with varicella zoster virus (VZV) elicits persistent cell-mediated immunity directed against the immediate early (IE62) protein and the glycoprotein I (gp I) in most healthy subjects. In these experiments, synthetic peptides corresponding to residues of the IE62 protein and gp I were used to identify linear amino acid sequences of these immunogenic VZV proteins that were recognized by peripheral blood T lymphocytes from VZV-immune individuals of known major histocompatibility complex (MHC) type. All of 12 VZV-immune donors had T cell proliferative responses, defined as a stimulation index (SI) greater than or equal to 2.0, to at least two of ten synthetic IE62 peptides; the mean number of IE62 peptides recognized by T cells from VZV-immune donors was seven. Five of the ten IE62 peptides stimulated T cells from 75% to 83% of the VZV-immune donors; the other five IE62 peptides were recognized by T cells from 42% to 67% of the subjects. All VZV-immune donors also had T proliferation responses to at least two of ten synthetic gp I peptides; the mean number of peptides recognized was six. Six of the ten gp I peptides were recognized by T cells from 67% to 92% of the VZV-immune donors; the frequency of donors responding to the other gp I peptides ranged from 42% to 58%. None of five nonimmune donors demonstrated T cell proliferation to any of the IE62 or gp I peptides. A combination of two IE62 peptides provided epitopes that could be recognized by T cells from all twelve VZV-immune donors, regardless of DR type. Similarly, one gp I peptide in combination with either of two other gp I peptides induced proliferation of T cells from all immune subjects. Memory T cells with specificity for multiple short amino acid sequences of the IE62 protein and gp I were detected in subjects who had had primary VZV infection more than 20 years earlier. These observations indicate that natural VZV infection elicits a diverse cell-mediated immune response to viral proteins that is not restricted to only one or two immunodominant regions. Although the usefulness of peptide vaccines remains to be established, multiple epitopes of the IE62 protein and gp I were identified that could be presented by antigen-presenting cells (APC) and recognized by T cells from most subjects in an "outbred" human population. PMID- 1725700 TI - Clonal analysis of the cytotoxic T-lymphocyte response to reovirus. AB - Reovirus has previously been classified into three serotypes based on hemagglutination inhibition assays. In the present study, the specificity of cytotoxic T lymphocytes (CTL) generated in reovirus type 3-infected C3H/HeN mice was investigated at the population and clonal levels. Short-term CTL lines generated in response to reovirus type 3 preferentially lysed cells infected with reovirus type 1 or 3 and, in some instances, type 2-infected cells as well. Eleven CTL clones established from the lines demonstrated two unique patterns of recognition. A single clone was exquisitely specific for reovirus type 3-infected cells and did not cross-react on reovirus type 1- or 2-infected cells. Ten of the clones recognized reovirus type 1- and type 3-infected cells. These clones had low levels of cross-reactivity on reovirus type 2-infected cells that was revealed only at high effector:target cell ratios. Precursor frequency analysis further revealed that the majority of the CTL generated against reovirus type 3 could cross-react on both reovirus type 1- and type 2-infected cells. Some CTL could be detected that had a more restricted pattern of recognition and recognized reovirus type 3-infected cells exclusively or recognized reovirus type 3-infected cells and either reovirus type 1- or type 2-infected cells. These results indicate that a minimum of four epitopes are recognized by reovirus specific CTL and that the response is dominated by CTL that recognize an epitope common to all three serotypes of reovirus. PMID- 1725704 TI - Further observations on pineal brain sand formation and evolution in man. AB - Acervuli obtained from fragments of the human pineal glands of subjects of both sexes and age ranging from 23 to 87 years were analyzed by light microscopy after histochemical stains and by EDS-microanalysis. It was found that the sub-units and acervuli are positive to P.A.S., Alcian Blue pH 2.5, Gomori-Bargmann procedures for the presence of proteins, glycoproteins, proteoglycans and of the neurosecretory material in different layers of the sub-units and acervuli. We suppose that the increase of Mg++ in the sub-unit from the core to the periphery is responsible for the inhibition of following hydroxyapatite deposition and for growth. We suggest that the presence of glycoproteins and proteoglycans can represent the aggregation factor for bindings between disulphide bonds and calcium. PMID- 1725701 TI - Ligand-gated ion channels. Homology and diversity. PMID- 1725705 TI - [Local and regional anesthesia of the upper limb in emergency hand surgery]. AB - The very conditions of the emergency led the authors to define the indications for the various modalities of local and regional anaesthesia: intravenous regional anaesthesia, nerve trunk blocks, plexus blocks, interdigital block and local infiltration. The parallel development of anaesthetic drugs with variable systemic toxicity and a duration of action inversely proportional to the toxicity now allows precise adaptation of the anaesthesia to the type of lesion, the patient's general condition, the practical conditions of the emergency and the surgical technique selected, provided the anaesthetist is fully aware of the traps to be avoided, which can only be based on a long practice of local and regional anaesthesia in elective surgery. PMID- 1725703 TI - The molecular biology of addictive drugs. AB - The additive drugs alcohol, morphine, cocaine, and amphetamine are each associated with the development of tolerance and physical dependence. Changes in gene expression occur in cell culture and in vivo with the administration of these centrally-acting drugs. This article reviews those experiments that have studied drug-induced alterations in gene transcription. Ethanol has diverse effects on the amounts of messenger RNA molecules within the central nervous system. Ion channels, neuropeptides, membrane receptors, and immediate early genes represent several regulated mRNAs. The effects are selective, however, as many other specific products are not altered. Evidence for a genetic predisposition to ethanol use reinforces the importance of the genotype. Opioids, cocaine, and amphetamine also affect gene transcription. Messenger RNAs studied have included many of those demonstrated to be altered by alcohol use. Interestingly, use of any of these drugs alters the expression of immediate early genes. These genes may represent an initial step in the pathway that leads to drug addiction. The composite of drug-induced changes in gene expression results in the cellular responses of tolerance and dependence. The characterization of these changes should provide a better understanding of the molecular mechanisms of drug addiction. PMID- 1725702 TI - The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies. AB - Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation. PMID- 1725706 TI - [Digital reimplantations and revascularizations. Factors of preservation. 183 fingers]. AB - The causes of vascular failures of a series of 183 injured fingers treated by microsurgery (80 reimplantations, 103 revascularisations) were investigated. This consecutive series was characterised by the fact that the operation was performed even in the presence of classical factors of poor prognosis: patients over the age of 40 years (27%), smoking (51%), crush injury or avulsion (32%), ischaemia time longer than 7 hours (46%). The overall intraoperative and postoperative failure rate was 49% (69% in reimplantations and 31% in revascularisations). Preoperative factors (age, smoking, mechanism, ischaemia time) were not sufficiently important to constitute contraindications to vascular microsurgery. Only storage of the segment in contact with iced water made failure almost certain. Suture of 2 arteries and one or several veins (in the absence of a skin bridge) improved the prognosis. The postoperative use of subcutaneous heparin in reimplantations significantly decreased the failure rate from 77% to 55%. Postoperative surveillance is essential to rapidly detect the arterial or venous mechanism responsible for vascular disorders and to decide appropriate emergency treatment. Arterial ischaemia warrants revision of the sutures with a success rate of about one in two. Disturbances of the venous drainage should initially be treated medically (2/3 of preservation after drainage by leeches), but, when this is not effective, surgical revision salvages one third of failures. Emergency microsurgery units must therefore have access to a specialised postoperative surveillance unit. PMID- 1725707 TI - Four finger amputation injuries. Concept of treatment. AB - Fourteen patients with an amputation injury of four fingers are reported. Fifty one of these 56 fingers were replanted with a survival rate of 88%. To achieve optimal results replantation of all fingers should be attempted. Dividing the operative procedure into a macrosurgical and a microsurgical part helps to economize both tourniquet time and total operative time. In case of arterial spasm or other difficulties to reestablish blood flow, a check list can be of great value. To achieve good results in such severe and difficult procedures a special organisation of replantation centers is needed. PMID- 1725708 TI - [Digital flap autografts for pulp coverage in distal amputations of the fingers. 68 flaps]. AB - Sixty-one patients underwent 68 digital pulp amputations involving the distal phalanx (Zone 2 and 3). 46 unipedicular Vankataswami-Subramanian island flaps (VS flap) and 22 bipedicular Moberg-O'Brien flaps (MOB flap) were performed. All distal thumb amputations were treated by MOB flap, whereas all lateral finger distal amputations of the long digits were treated by VS flap. Both flaps were used for the other types of amputation. Four digits developed complications and needed a delayed regularisation. The mean follow-up for the 46 flaps was two years. Pulp reconstruction was satisfactory in all flaps. Sensation was normal or slightly decreased in 66%. Nail dystrophy was considered to be poor in 56% and was attributed to initial trauma and the distal phalanx shortening. 20 degrees of extension deficit was found in 8 patients. We obtained 61 excellent and good results justifying our indications: MOB flap for all types of distal amputation of thumb in zone 2 and 3, VS flap for lateral distal amputations of the long fingers. In transverse amputation of the long fingers, MOB flaps seem to give better results than VS flaps. PMID- 1725709 TI - [Digital island autograft flaps in pulp defects. 18 cases]. AB - Eighteen homodigital island flaps were performed for pulp defects over a period of two years in the Hand Emergency unit. Apart from one failure due to an incorrect indication, the results confirmed the excellent trophicity of the flap and its aesthetic appearance was appreciated by the 17 patients. Discrimination on Weber's test, the possibilities of advancement, and the fact that the size of the flap can be tailored to individual needs make it one of the most attractive alternatives in this type of indication. However, postoperative stiffness of the PIP joint is an almost constant disadvantage requiring an extension splint as soon as possible. The homodigital island flap is an elegant, minimally disfiguring flap indicated in pulp defects or distal palmar or lateral pulp amputations. PMID- 1725710 TI - [Emergency pulp reconstructions]. AB - Pulp reconstructions use numerous techniques ranging from simple debridement to local and microsurgical flaps. The authors analyse the various treatments, in which directed healing still retains numerous indications. Local flaps have a definite place, but they are not devoid of risks (iatrogenic stiffness). The indications are presented schematically in table form together with several examples. A wide range of techniques is necessary in order to treat the various lesions appropriately. Large defects are best treated by microsurgical transfer of great toe pulp with satisfactory results, which justifies their management in specialised centers. PMID- 1725711 TI - [The inguinal pedicle flap in emergency traumatic hand surgery: value of the long pedicle permitting an early rehabilitation]. AB - The main drawbacks of the use of a groin pedicle flap in emergency surgery for hand injuries are the declivitous position of the upper limb with a tendency towards oedema and the difficulty of instituting early rehabilitation because of the fact that the injured hand is fixed to the abdomen. Based on an extensive experience of 76 emergency flaps out of more than 100 flaps performed over the last ten years, the authors describe their flap raising technique which allows the formation of a long, closed tube compatible with early rehabilitation and also allowing the possible use of splints. PMID- 1725712 TI - [Ballistic hand trauma]. AB - 46 victims of projectile accidents or explosions were treated over a 5 years period between 1984 and 1989. 3 categories were distinguished: injuries due to a single projectile (12 cases), only inducing serious bone damage and, apart from 3 immediate amputations, the final result was satisfactory. Injuries due to multiple scattered projectiles (11 cases), less severe in terms of the initial lesions, not requiring any amputations, with good results in 8 cases. Explosion injuries (23 cases) in which the effect of the explosion induced considerable initial lesions leading to one hand amputation and 33 finger amputations; the association of skeletal and soft tissue lesions raises the problem of excision and primary cover, requiring large flaps. The course is long and 8 out of 26 hands had serious sequelae, while the reconstruction of an elementary pinch can be considered to be an acceptable result in the other cases. PMID- 1725713 TI - Firework injuries to the hand. AB - Hand injuries by fireworks occur every year especially around New Year's Eve. These complex injuries show a combination of avulsion, laceration, blast, crush and burns. Three typical cases are presented and their treatment is outlined. PMID- 1725714 TI - [Microsurgical emergencies with the exception of reimplantations]. AB - Although reimplantations are very well defined operations, the indications for emergency free flaps and the management of nerve injuries are much less so. Using several examples, the authors define the conditions in which a free flap is justified and the most useful procedures for nerve repair. An emergency free flap should constitute the best solution, with a reasonable chance of success, without compromising other possibilities of reconstruction in the event of failure. The authors present the three situations observed in the case of nerve lesions: clean nerve sections, nerve defects, cases with an unclear diagnosis and cases in which the conditions are unsuitable for immediate permanent repair. PMID- 1725715 TI - [Irreducible palmar dislocation of the metacarpophalangeal thumb joint by a skiing accident]. AB - Amongst the large number of severe sprains of the lateral ulnar ligament of the metacarpophalangeal joint of the thumb by skiing accidents which we treat each year as a result of our geographical situation, we have encountered an original lesion on two occasions. The patients were referred with unstable palmar dislocation of the metacarpophalangeal joint of the thumb despite an attempt of reduction under local anaesthesia performed at the ski resort by practitioners particularly well trained in this form of traumatology. On examination, the thumb was globally painful and presented a defect of active extension of the MP joint suggestive of a complex lesion of the extensor apparatus. X-rays showed palmar dislocation of the MP joint of the thumb. Surgical exploration in both cases revealed identical lesions: complete rupture of the two bundles of the lateral ulnar ligament of the MP joint of the thumb, whose distal extremities could be seen as soon as the skin was opened with incarceration of the extensor apparatus between the palmar surface of the neck of the first metacarpal and the dorsal surface of the first phalanx. Perfectly classical treatment consisted of a dorso lateral incision to reduce the extensor apparatus and to suture the two bundles of the lateral ulnar ligament; immediately restoring the stability which is usually observed in "classical" severe sprains. The current result is good, entirely comparable to that of severe sprains of the MP joint of the thumb.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725716 TI - [Bundled central medullary bone wiring. Method of choice in the treatment of fractures of the neck of the fifth metacarpal necessitating a reduction. 30 cases]. AB - The authors present a short series of 30 displaced fractures of the neck of the fifth metacarpal. All fractures were treated by K-wire nailing according to the method described by Fourcher in 1976. The good results confirm the advantages of this technique, allowing early mobilization and rehabilitation. The poor results are due to faults in the use of the method, and the authors emphasize these technical errors. PMID- 1725717 TI - [Bundled wiring in fractures of the neck of the fifth metacarpal. 5 cases]. AB - By reviewing a series of 50 patients operated on using the Foucher technique for a closed isolated fracture of the neck of the fifth metacarpal bone, the authors determine efficiency of this surgical technique which gives satisfactory results when the operation performed is early enough (before 10 days). They explain the modification of the technique, insisting on the importance of a post-operative follow-up which guarantees a good result and they propose other surgical indications whether they concern fractures of the neck of the fifth metacarpal bone with a relatively small displacement, mainly in young subjects and labourers or indications concerning special cases. PMID- 1725718 TI - [Urgent nature and severity of lesions due to high pressure injections in the hand]. AB - High pressure injections in the hand and fingers are relatively rare accidents, but are responsible for serious lesions. Only extremely urgent treatment can minimise the aesthetic and functional sequelae. This treatment must include debridement of the portal of entry, exposure of all the contused and devitalised tissues and broad spectrum antibiotics. The postoperative course must include long and patient rehabilitation for these lesions which actually correspond to severe contusion of the hand and fingers associated with a high risk of septic dissemination. The authors describe these lesions based on a series of 20 consecutive cases. PMID- 1725719 TI - [Traumatic lesions of the hand: narcissistic wound, functional and psychic spin, psychological management]. AB - In our unit, the clinical psychological approach is a constant concern of the therapeutic team. Individual conversations allow verbalisation of personal ideas about the accident, the narcissistic wound, the idealisation of surgeons and surgery, guilt feelings, rehabilitation, the necessity for informations, ... for the discussion of these issues has therapeutic effects for the injured person and improves his or her capacities for reinvestment. PMID- 1725720 TI - [Value of a polyurethane dressing in hand surgery. Preliminary study]. PMID- 1725721 TI - [When continuity is told to us. Mental health and symbolization of filiation in aged persons]. AB - The author examines the links between the feeling of continuity and the mental health of 25 elderly people who recount their perception of how material and cultural values are transmitted in their line of descendants and in their social community. In considering the aging process as a cultural system, the author builds a model depicting the symbolization of the rule of filiality, for which there are three categories developed in function of manifestations of a more or less strong feeling of continuity or of a feeling of rupture. Beyond their psychological vulnerability and the "deadline" to which they are confronted, these people find themselves in one of the three categories of this model through their relationships with their ascendants and descendants. PMID- 1725722 TI - Angiogenesis and its inhibition: potential new therapies in oncology and non neoplastic diseases. AB - To summarise the key points: The ability to mount an angiogenic response is probably present in all tissues, and stimulation of endothelial cells by any one of a wide variety of factors initiates a cascade of events leading to angiogenesis. In most tissues the overall lack of angiogenesis in normal situations probably results from the interaction of a complex series of multifactorial systems, each of which maintained in a state of balance between stimulation and inhibition. An imbalance in any one of these systems, for example by an increase in the concentration of a growth factor, may lead to angiogenesis. Inhibition of angiogenic stimuli is unlikely to be effective as an approach to new angiostatic drugs, given the multiple stimulatory pathways available. Tumour cells for example may induce angiogenesis via release of numerous growth factors, prostaglandins etc, and by their attraction of inflammatory cells which in turn release multiple angiogenic stimuli. Inhibitory modulation of many of the individual steps of capillary growth which occur following an angiogenic stimulus can block the angiogenic response. This leads to the expectation that an effective inhibitor of a single key step in this cascade would be able to completely suppress angiogenesis. Inappropriate angiogenesis is an important factor in many diseases including cancer and arthritis. In particular angiogenesis is an absolute requirement for neoplastic growth of solid tumours, and the establishment of secondary growths. There is also a strong link between induction of angiogenesis by a tumour and its ability to metastasise. Several drugs with proven clinical effects in diseases involving angiogenesis have recently been found to be angiogenesis inhibitors, and this may be their primary mechanism of action. In particular the activities of methotrexate and gold compounds in arthritis, and alpha-interferon and medroxyprogesterone in cancer therapy may be due to inhibition of angiogenesis. In animal models, treatment with angiogenesis inhibitors has proven anti-tumour effects in vivo, and can both reduce metastases and lead to regression of the primary growth by necrosis following capillary retraction. In man the success of alpha-interferon and TNF alpha in AIDS related Kaposi's sarcoma may be due to inhibition of angiogenesis. Interferon has also been successfully used to treat pulmonary hemangiomatosis, in which angiogenesis in the lung may be the pathogenic basis of the disease. PMID- 1725723 TI - Novel naphthalenedisulfonic acid anti-HIV-1 agents. Synthesis and activity against reverse transcriptase, virus replication and syncytia formation. AB - Several naphthalenedisulfonic acid derivatives have been synthesized and evaluated in four anti-HIV-1 screens. Antiviral activity was evaluated in the assays that measure inhibition of HIV-1 reverse transcriptase (RT), HIV-1 induced giant cell (syncytia) formation, and HIV-1 induced cytopathogenicity in both a laboratory and clinical strain of HIV-1. 4-acetylamino-5-hydroxy-2,7- naphthalenedisulfonic and a bis naphthalenedisulfonic acid derivative emerged as the most active compounds exhibiting activity in all assays at concentrations non toxic to the host cells. The bis 3-amino-1,5-naphthalenedisulfonic acid derivative was the most active compound, but less active than the bis 4-amino-5 hydroxynaphthalenedisulfonic acid analog in the assays that measure RT and syncytia inhibition. However, in the assay that measured inhibition of cytopathogenicity in a clinical HIV-1 isolate, 4-acetylamino-5-hydroxy-2,7 naphthalenedisulfonic acid was the most potent analog, demonstrating an in vitro therapeutic index greater than 54. This study shows that for certain naphthalenedisulfonic acid moieties a bis derivative is not a requirement for significant anti-HIV-1 activity. These agents represent a new class of lead non nucleoside anti-HIV-1 compounds worthy of further development for potential anti AIDS therapy. PMID- 1725724 TI - In vitro interferon and virus production at in vivo physiologic oxygen tensions. AB - Rhabdomyosarcoma (TE) and cervical carcinoma (MS) cells 24 h previously brought from medium with a PO2 of 18 kilo Pascal (kPa) (ambient air) to PO2 of 6 or 3 Kpa (in vivo physiologic values) were infected with Sendai virus, and the interferon (IFN) and virus production was followed in the ensuing 24 h period. With TE cells the IFN production decreased when moving from 18 to 6 Kpa and ceased completely at 3 Kpa, while the virus production responded inversely. MS cells produced most IFN at the lowest oxygen tension, and virus production was only moderately affected. Growth for three months at the three different oxygen tensions prior to infection reduced the difference in IFN production at the different oxygen tensions. On the same target cells different human virus responded in different ways to the tested oxygen tensions. It is concluded that mimicking the in vivo situations might require primary cultures of virus and cells to be started and passaged at in vivo physiological oxygen tensions. PMID- 1725725 TI - Occurrence and distribution of non-NMDA glutamate receptor mRNAs in the cochlea. AB - The expression of mRNAs encoding five putative non-NMDA glutamate receptors was investigated using in situ hybridization with radiolabeled riboprobes. Hybridization was observed in spiral ganglion neurons with probes complementary to mRNA products of the glutamate receptor genes GluR2 and GluR3. No specific hybridization was observed with probes for GluR1, GluR4 or GluR5. The results support the hypothesis that glutamate is the transmitter between cochlear inner hair cells and spiral ganglion neurons, and that it acts via non-NMDA glutamate receptors. PMID- 1725726 TI - Desmethylimipramine potentiates NMDA responses in a mouse cortical slice preparation. AB - Desmethylimipramine (DMI) has been shown to interact with the N-methyl-D aspartate (NMDA) receptor complex. Its probable action is through blockade of the cationic channel at the phencyclidine site and as a result it has potential anticonvulsant action. In this present study we have investigated the effects of DMI and ketamine on both NMDA-induced and spontaneous depolarizing shifts in cortical wedges prepared from genetically epilepsy-prone mice (DBA/2). Contrary to published reports, DMI potentiated the effects of NMDA and increased the frequency of spontaneous depolarizations. The actions of ketamine were inhibitory and these were reversed by DMI. Presynaptic mechanisms may be involved in the DMI induced potentiation and this may explain the lowering of convulsive thresholds seen clinically with tricyclic antidepressants. PMID- 1725727 TI - The expression of the c-src+ proto-oncogene mRNA in foetal striatal tissue grafts. AB - We have studied by in situ hybridization the mRNA expression of the pp60c-src proto-oncogene in grafts of foetal striatal neurons implanted into the ibotenic acid lesioned adult rat neostriatum. Animals were studied at 5, 10, 15, 30 days and 4 months after transplantation. Using 35S-labelled specific oligonucleotide probes, high levels of mRNA for pp60c-src+ but not pp60c-src- were found in the normal embryonic striatum and in striatal grafts up to 1 month after implantation; no hybridization signal was obtained in the normal adult striatum or in 4-month old grafts. The data suggest that pp60c-src+ is highly expressed in developing, but not mature, neurons and may play a fundamental role in the maturation and differentiation of grafted embryonic striatal neurons. PMID- 1725728 TI - Acetylcholine receptor channel properties are modified by benzyl alcohol. AB - The effect of the general anaesthetic, benzyl alcohol, on the nicotinic cholinergic receptor (AChR) was evaluated at the single channel level using the patch-clamp technique. Benzyl alcohol decreases both the conductance (about 2 fold with 40 mM benzyl alcohol) and the mean open time (about 2.5-fold) of the AChR channels. When modified channels are activated by high ACh concentrations, groups of brief channel openings are observed. Each group is in turn composed of a higher number of openings than in non-treated receptors. Similar modifications are observed when benzyl alcohol is applied from the cytoplasmic side of the membrane, suggesting that the general anaesthetic interacts with a nonspecific site, possibly the lipid-protein interface. PMID- 1725729 TI - Properties of the leakage current pathway: effects of the gadolinium ion on myelinated axons. AB - Voltage-clamp experiments on myelinated axons revealed that the trivalent gadoliniumion ion blocked the leakage current in a dose-dependent manner. The block showed 1:2 stoichiometry, and 50% reduction at 700 microM. Na+ and K+ currents were blocked at ten times lower concentration and showed 1:1 stoichiometry. Comparisons with biochemical studies suggest that the leakage current directly depends on a phospholipid pathway in contrast to the protein channel dependent Na+ and K+ currents. The results may be explained by a leakage current pathway located at the interface between channel proteins and the lipid phase. PMID- 1725730 TI - Carbohydrate antigens on cancer-associated mucin-like molecules. AB - Mucins are large molecular weight glycoproteins characterized by carbohydrate sugars attached via 'O-glycosidic' linkages to serine or threonine. Mucins are synthesized by a variety of epithelial tissues as membrane-bound or secreted proteins, encoded by several distinct mucin genes. Numerous alterations of mucin associated carbohydrates can be detected in neoplastic epithelial tissues and on circulating mucins in patients with adenocarcinomas. The expression of various sialyosylated-carbohydrate epitopes may correlate with poor prognosis and enhanced metastatic disease in colorectal adenocarcinomas. Mucin-associated carbohydrate and peptide antigens are currently being investigated for their role in cancer diagnosis, monitoring for progression or metastases, immunotherapy and immunosuppression. PMID- 1725731 TI - Humoral immune responses to tumor-associated carbohydrate antigens. AB - Antibodies to carbohydrate structures are quite commonly found in human sera. Normal individuals, as well as individuals with autoimmune diseases and cancer, produce antibodies reacting with a variety of carbohydrate determinants. These determinants can be expressed on glycoproteins, mucins or glycosphingolipids. For understanding a possible immune response to cancer, the challenge is to determine which, if any, of the serum antibodies are related to the tumor state. This problem has been approached by analyzing the specificity of serum antibodies and, more recently, by producing monoclonal antibodies from the lymphocytes of cancer patients. Malignant melanoma and lung cancer have been the main focus of the monoclonal antibody approach. Melanoma patients' lymphocytes have, in general, yielded anti-ganglioside antibodies whereas anti-neutral glycolipid antibodies were isolated from lung cancer patients. Although these studies provide reagents for potential use in the in vivo diagnosis and therapy of cancer, the significance of these antibodies for an immune response to cancer needs to be further explored. PMID- 1725732 TI - Belgian Society of Biochemistry. 147th reunion. Mons, 25 May 1991. Abstracts. PMID- 1725733 TI - Excitatory responses of the dorsal root discharge in L7 segment elicited by ascending and descending volleys in the cat. AB - Excitatory responses of the dorsal root discharge (DRD) which consist in short lasting increase in its frequency have been recorded from the central cut ends of single dorsal root fibres of L7 dorsal root in spinal cats. They were evoked by stimulation of afferent nerves entering the cord below and above the level of recording of antidromic discharge and then ascending and descending to this level. Ascending volleys were provided by stimulation of caudal femoral posterior and pudendal nerves and descending volleys by stimulation of saphenous and quadriceps nerves. The excitatory responses of the DRD were elicited by stimulation of low threshold cutaneous afferents and group II and III muscle afferents while high threshold cutaneous and group I muscle afferents were ineffective. The excitatory responses of the DRD evoked by ascending volleys were larger than the effects elicited by descending volleys. The incidence of the excitatory effects to ascending volleys was significantly higher than to descending volleys. The size and incidence of the excitatory responses to ascending and descending volleys were, however, smaller than those of the effects elicited by stimulation entering the cord at the level of recording of antidromic discharge (i.e. in the posterior tibial nerve). It is suggested that the size and incidence of the excitatory responses of the DRD are related to the direction of spread and the level of entry of afferent volleys. PMID- 1725734 TI - Impairment of maximal bilirubin secretion by cyclosporin A in the rat. AB - The effect of cyclosporin A on the maximal secretion of bilirubin was investigated in male Wistar rats. Following a saturating load of the pigment, intravenous administration of cyclosporin A as a single bolus (20 mg/kg) significantly reduced bile flow and bile acid secretion. The bilirubin secretory transport maximum was alos significantly reduced. The decreased excretion of the bile pigment corresponded both to bilirubin mono- and di-conjugates, the excretion of unconjugated bilirubin being slightly and non-significantly reduced. Plasma and liver bilirubin concentrations were significantly increased, while liver UDP-glucuronosyltransferase activity did not significantly differ between control and cyclosporin A-treated rats. Our data indicate that, although in cyclosporin A-treated rats there are no changes in the activities of others markers of hepatocellular dysfunction, the overall plasma to bile transfer of bilirubin is altered. The reduced hepatobiliary transport of the pigment is mainly caused by an impairment of its intracellular/canalicular transport. PMID- 1725735 TI - Thyroid state and electrical properties of rat papillary muscle fibres. AB - We have studied the effects of in vivo alterations of thyroid state on the electrophysiological properties, measured in vitro, of rat papillary muscle fibres. The duration of the action potential, recorded from single cells of papillary muscle obtained from 60 day old rats, thyroidectomized at weaning, was larger than in preparations of euthyroid animals of same age. The treatment of thyroidectomized rats with T3 physiological doses restored the values of action potential duration present in euthyroid animals of same age. The data dealing with the time course of these variations indicate a late effect of thyroidectomy or T3 treatment. The hyperthyroid state, obtained by treatment of thyroidectomized rats with larger doses of T3, was associated with a significant reduction in the action potential. Action potential duration is frequency dependent. As the stimulation rate was increased from 1 to 5 Hz, this duration increased in all groups. However the difference found among the rat groups remained significant. Propranolol had virtually no effect on action potentials from fibres of euthyroid animals and those in altered thyroid state. PMID- 1725736 TI - Testosterone effects on staining density and autoradiographic investigations of the alpha 2-adrenergic receptor in the medial preoptic nucleus of the Japanese quail: relationship to the activation of reproductive behavior. AB - The effects of gonadectomy combined or not with testosterone (T) therapy on the sexual behavior, cloacal gland area, staining density and alpha 2-adrenergic receptors in the medial preoptic nucleus (POM) were studied in male and female Japanese quail. In castrated males, T increased the cloacal gland size and activated copulatory behavior. In agreement with previous reports, these effects were sexually differentiated: under the influence of T, cloacal gland growth was smaller and this treatment did not activate masculine sexual behavior in females. The optical density of the medial preoptic nucleus stained with thionein blue (Nissl stain) was decreased by castration and increased by T in both males and females. This suggests that T has a profound effect on the synthesis of proteins in the POM and, since POM is a critical site in the activation by T of masculine sexual behavior, these induced proteins are, in all probability, a part of the causal chain of biochemical events giving rise to copulatory behavior. The treatment with T had, by contrast, no clear effect on the number of binding sites and on the affinity of the alpha 2-adrenergic receptors in the POM. Therefore, if the noradrenergic transmission is involved in the induction by T of copulatory behavior, it is probably acting either through the alteration of another adrenergic receptor subtype or through the modulation of the baseline levels or turnover of the transmitter itself. PMID- 1725737 TI - Suppression of the diurnal rhythm of oxygen uptake by eyestalk ablation in the crab Oziotelphusa senex senex Fabricius. AB - Oxygen uptake of the whole animal, of the isolated hepatopancreas and of some muscles of Oziotelphusa senex senex exhibits a diurnal rhythm with a maximum uptake at 00 h, and a minimum at 12 h. The rhythm is related to the animal's motor function which is active during the night. Bilateral eyestalk ablation increases motor activity and oxygen uptake during the day. The diurnal rhythm of oxygen consumption is suppressed. PMID- 1725738 TI - Possible thromboxane A2 mediated effect of angiotensin II in the rabbit isolated perfused kidney. AB - Angiotensin II (A II) when given through the renal artery of the rabbit isolated perfused kidney elicited a concentration-dependent increase in perfusion pressure (PP) and urine flow (UF). Noradrenaline (NA) produced similar effect in PP but slightly not significantly increased UF. Addition of UK 38,485, a thromboxane A2 (TXA2)-synthetase inhibitor, to the bathing medium at the concentration of 10(-7) M caused a significant decrease in the pressor effect of A II but significantly enhanced UF. BM 13,177, a TXA2-receptor blocker, in the bathing medium (10(-6) M) produced identical effect in both PP and UF. The responses to NA were not significantly altered by both compounds. A II-induced enhancement of UF in presence of BM 13,177 was significantly lower than that obtained in presence of UK 38,485. These results were taken as an evidence that A II may increase the production of TXA2 in kidney probably originated from vascular bed or tubuli and part of the pressor effect of the peptide is due to this metabolite of arachidonic acid. Enhancement of UF following UK 38,485 is probably due to the shift of the synthesis toward E series of PGs. In addition this effect seems to be specific for A II since no such effect was seen with NA. PMID- 1725739 TI - Renin-angiotensin system in thyroidectomized rats at different periods of development. AB - The relationship between the renal function and some components of the renin angiotensin system has been studied in hypothyroid rats thyroidectomized surgically at different periods of their life. Changes in plasma renin concentration (PRC) depending on the period hypothyroidism were induced. Results showed that the renin release control could result from an equilibrium between the reduced beta-adrenergic activity and the marked natriuresis observed in hypothyroidism. A reduction in plasma angiotensinogen concentration (PAC), due to a decrease in its hepatic production, was observed in thyroidectomized animals. PAC reduction was independent of the hypothyroidism induction period. Alterations in plasma renin activity (PRA) were a consequence of PRC and PAC changes in thyroidectomized animals, as an increase in fractional sodium excretion (FENa) time course dependent, was found in these rats. PMID- 1725740 TI - Crustacean hyperglycemic hormone induced alterations in millepede (Spirostreptus asthenes) carbohydrate metabolism. AB - Following intersegmental injection of the hyperglycemic hormone (HGH) obtained from fresh water crab (Oziotelphusa senex senex) and marine tiger prawn (Penaeus monodon), hemolymph sugar concentration was significantly increased in millepede (Spirostreptus asthenes). Glycogen and total carbohydrates of fat body decreased in injected animals indicating that the source of hyperglycemia was carbohydrates from the fat body. Correlating with the decreased glycogen content, phosphorylase activity of the fat body was elevated. The results strongly suggest that crustacean hyperglycemic hormone exerts influence on non-crustacean species also. PMID- 1725741 TI - Cyclic AMP increases electrical capacitance of apical membrane of toad urinary bladder. AB - We have measured the effects of oxytocin and three other compounds (chlorophenyl thio-cyclic AMP, forskolin and theophylline) that increase cytoplasmic cyclic AMP on the impedance of the toad urinary bladder. Membrane capacitance was calculated from transepithelial impedance measured by a computerized sine wave method. All four agents increased tissue capacitance. Since in these tissues this parameter is proportional to apical membrane area our results suggest that cAMP can be a second messenger involved in the action of agents that promote fusion of exocytotic vesicles with the apical membrane. PMID- 1725742 TI - Physico-chemical properties of hepatocyte plasma-membrane-bound arginase. AB - Rat liver plasma-membrane-bound arginase was investigated in order to obtain data regarding its physico-chemical properties. Arginase bound to plasma membrane presented a specific activity of 0.74 +/- 0.09 IU/mg for the fully-activated enzyme, the pH of maximum activity being 9.8. Maximum stability was recorded at two pH values, 7 and 10.5 respectively. Mn2+ activated the enzyme, while Cu2+ and Zn2+, and to a lesser extend Co2+, showed a strong inhibitory effect. Ca2+ and Mg2+ had no effect at the concentrations assayed. The influence of temperature was studied in the presence and in the absence of Mn2+. The enzyme was stable up to 65 degrees C in both cases. Membrane- bound arginase showed an activation energy of 11.5 +/- 1.4 Kcal/mol between 20 and 40 degrees C, and 13.3 +/- 2.5 Kcal/mol between 40 and 60 degrees C. The Q10 for the same temperature ranges were 1.78 and 1.9 respectively. The membrane-bound enzyme presented two different Michaelis constants, one with high affinity (2.05 +/- 0.73 mM) and the other with low affinity for arginine (130 +/- 27.2 mM). Solubilized arginase showed very similar values. Among all the structural analogous assayed, only L-canavanine proved to be substrate for arginase, with and L-arginine/L-canavanine hydrolysis ratio of 5.8 +/- 0.28. No reactivity was found between plasma-membrane-bound arginase and anti-rat liver arginase antibodies raised in rabbits. PMID- 1725743 TI - [Mobilization of leukocytes during sub-maximal dynamic exercise]. AB - Increase of blood leucocytes induced by a dynamic exercise has been studied in 10 adult male subjects (age: 22 +/- 2 years; body weight 73 +/- 10 kg; VO2max: 54 +/ 8 ml O2/kg.min. The exercise consisted of a 20 minutes test on a cycle ergometer at a work rate eliciting a total energy expenditure roughly equal to 80% of the maximal aerobic power (80% VO2max). During exercise the absolute number of circulating leucocytes (n Leu) increased significantly toward a maximal value reached in most cases at the end of exercise. The maximal values of n Leu- expressed as percentage of n Leu measured at rest--in the different blood leucocyte types (monocytes:n Mono; granulocytes:n GR; natural killer cells:n NK) and lymphocyte subsets (lymphocytes:n LY; lymphocytes T:n T; lymphocytes B:n B; lymphocytes T helper:n T4; lymphocytes T suppressor:n T8)--were as follows:n Mono:240%; n GR:170%; n NK:490%; n LY:220%; n T:200%; n B:190%; n T4:140%; n T8:190%. With the exception of n GR, n Leu decreased during recovery at a rate depending on the leucocyte type. The n T4/n T8 ratio, equal to 1 at rest, was reduced by about 40% during exercise. This ratio rose after exercise and reached a value equal to that measured at rest after 20 minutes recovery. In agreement with FERRY (1989), we concluded that exercise is accompanied by a selective mobilization of the different leucocyte types. The magnitude of this phenomenon and the kinetics of n Leu recuperation depend on cell types. PMID- 1725744 TI - Regional development of alpha-methyl-D-glucoside transport in the small intestine of chick embryos and newly-hatched chicks. AB - A regional study of the intestinal hexose transport shows the role played by duodenum, jejunum and ileum during the chick perinatal development. From at least two days before hatching the three regions of small intestine accumulate alpha Methyl-D-Glucose (alpha-MG) by mediated transport mechanisms, and phloridzin inhibit about 90% of the uptakes. This ability reaches the maximal level at 1 day after hatch in the three regions. Before hatching the jejunum shows higher transport levels than the observed values in the duodenum and ileum, but the three regions show similar values at 1 day after hatch. In the following days, the alpha-MG transport ability is strongly reduced in the duodenum, slightly reduced in the jejunum and maintained in the ileum until at least 7 day-old chicks. PMID- 1725745 TI - [Motility of antrum, pylorus, and duodenal bulb: importance of radiologic series analysis in man]. AB - The quantification of gastroduodenal motility uses invasive methods. The aim of our study was to further characterize the contractility of antrum, pylorus and duodenal bulb in two states, using a new, non invasive fluorographic method. Healthy volunteers (mean age = 28) were involved in this study under fasting conditions (n = 20) and after a meal (n = 20). In both conditions, the subjects have swallowed 250 ml of a barium sulfate solution for fluoroscopic gastrographies taken every 2 seconds, during 30 s. Spot films were analysed using a graphic table and locally developed microcomputer program to generate space time diagrams of antral and duodenal areas and of pylorus diameters. For each subject, the antral CA, duodenal CB contractilities (maximal variation of antral, duodenal areas in p 100) and pylorus contractility CP (closure during the observation time in p 100) were calculated. CA values were high in the two situations, but did not differ (81.1 p 100 vs 82.5 p 100 post-prandial). In contrast, CP was higher under fasting (54.0 p 100) than in post-prandial (15.0 p 100; p less than .001). Two pyloric motor patterns were observed in post prandial: A--pyloric contractions in relation with the end of antral contraction B--augmented pyloric resistances observed during the end of antral contraction and also associated with duodenal contractions. CB was low in post-prandial (27.0 p 100) compared to fasting CB (50.8 p 100; P less than .001). This method is reproducible and simple to use. It allows quantification of contractility and coordination in the antrum, pylorus and duodenal bulb. It may be useful to assess gastroduodenal motility under pathological conditions. PMID- 1725746 TI - [Can the efferent auditory system be explored via early evoked potentials? Preliminary results in normal hearing subjects and people with tinnitus]. AB - The use of masking in brainstem auditory evoked potential technic remains one of the important issue regarding the evaluation of different auditory control systems. Brainstem auditory evoked response wave form latencies and amplitudes have been studied on 14 normal hearing subjects. On one hand the contralateral masking which theoretically stimulates the medial contralateral efferent bundle did not modify the responses. On the other hand, the ipsilateral masking stimulating the lateral bundle reduced the amplitude of wave I and lengthened the latency of interwave I to V. The same protocol has been applied to 21 tinnitus patients. In these subjects before masking, the waves amplitude was lower and inter wave I-V latency was lengthened. The ipsilateral did not modify the responses as in the normal population. Comparing brainstem responses obtained in masking and no masking condition offers the possibility of evaluating the lateral efferent system function but we can conclude that the contralateral masking is of no use studying the medial system and that dysfunctions of the lateral efferent system could be one mechanism responsible for tinnitus. PMID- 1725747 TI - The effects of serotonin (5-HT) on twitch contractions induced by electrical stimulation of guinea-pig common bile duct. AB - In guinea-pig common bile duct serotonin (5-HT) (10(-7)-10(-5) M) administered cumulatively caused dose-dependent inhibition of the twitches evoked by electrical field stimulation (0.3 ms, 0.1 Hz, supramaximal voltage). Maximum inhibition was 86 +/- 2% of the control twitch amplitude. The effect was mainly due to a presynaptic mechanism since acetylcholine induced contractions were not influenced by 5-HT. This inhibitory action was unaffected by ketanserin (10(-6) M), ICS 205-930 (10(-6) M) and yohimbine (10(-6) M) indicating the lack of involvement of 5-HT2, 5-HT3 and alpha-2 adrenergic receptors respectively. Metoclopramide (3 x 10(-6)-3 x 10(-5) M) per se increased the twitch height and antagonized the 5-HT effect in a non-competitive manner. Methysergide (10(-7)-10( 5) M) also prevented the twitch inhibition and the mode of blockade was not consistent with simple competitive antagonism. The results suggest that presynaptically located 5-HT receptor differs in its pharmacological properties from other neuronal 5-HT receptors. PMID- 1725748 TI - Body water distribution after intracerebroventricular injection of NaCl in rats. AB - The aim of the investigation was to evaluate the effects of the intracerebroventricular (ICV) injection of 0.1 ml of 1 M NaCl, on the body water distribution in nephrectomized male rats. In comparison with the control rats injected with an equal volume of isotonic saline, the ICV injection of 1 M NaCl elicited 90 min later a significant increase of plasma volume whereas the total water, the inulin space, the calculated interstitial space and the intracellular water were not altered. The increase of plasma volume could be explained by a decrease of capillary efflux to the interstitial space. These results suggest that the ICV injection of 1 M NaCl releases a substance which could regulate the plasma volume. PMID- 1725749 TI - [Gastrin, cholinergic and alimentary stimulation of gastric acid secretion in awake rabbits with a Heidenham pouch]. AB - Gastric acid secretion was studied in awake rabbits equipped with a Heidenhain pouch. The realisation of a pouch in the rabbit is reported for the first time. Post surgical complications were observed such as shocks, anaesthesia toxic effects, electrolytic impairments, skin burns and gastric ulcers. The continuous gastric secretion and the thinness of the muscularis are mainly responsible for the technical problems not observed in other species. Gastric acid secretion was high during basal periods in rabbits after 24 h of fasting with a collar to prevent any caecotrophia, which gives an empty stomach. It is equivalent to 20% of the acid output produced by pentagastrin (32 micrograms.kg-1.h-1). The highest acid output was obtained during carbaminoylcholine intravenous infusion (20 micrograms.kg-1.h-1) i.e. about twice that induced by pentagastrin. Regular dry diet did not produce any stimulation nor 2 DDG used as a control of complete vagal denervation of the pouch. Unlike in other species, gastric acid secretion of awake rabbit displays a preferential sensitivity to cholinergic stimulants and an insensitivity to regular diet which releases endogenous hormones humorally transmitted to the pouch. The use of rabbit isolated parietal cells or gastric glands to demonstrate the mechanisms of gastric acid secretion, makes it necessary to perform a complete physiological study of gastric secretion in the rabbit with the technique of the isolated pouch we describe here. PMID- 1725750 TI - Dietary regulation of fructose metabolism in the intestine and in the liver of the rat. Duration of the effects of a high fructose diet after the return to the standard diet. AB - In this research on metabolic effects of a high fructose diet, we studied the duration of these effects by measuring the specific activity of 8 enzymes stimulated by such a diet, on days, 0, 3, 6, 9, 15, after the return to a normal diet. In the intestinal mucosa, ketohexokinase, aldolase, triokinase, fructose diphosphatase, and glucose-6-phosphatase specific activities were still entirely or partially stimulated on the 15th day after return to the standard diet. The stimulation of glucose-6-phosphatase and pyruvate kinase specific activities stopped quickly. In the liver, with the exception of fructose-diphosphatase, the return to basic values was much quicker than in intestine. In 7 enzymes out of 8 it was realized in 9 days or less. When a high fructose diet gives way to a normal one, return to basic values comes so much the quicker as activation has needed longer to appear. PMID- 1725751 TI - Hyperventilation does not increase alveolar surfactant phospholipids in the anesthetized rat. AB - Pulmonary distension elicits an increase of the surfactant secretion. Effects of hyperventilation on this same secretion are less precise since they were observed under particular experimental conditions. We report a study of the effects of hyperventilation on the phospholipid content of alveolar lining fluid in the rat. One hour's hyperventilation induced by addition of a dead space to the tracheal cannula of anesthetized rats did not affect the phospholipid content of broncho alveolar lavage fluid collected in situ immediately after killing. Phospholipid content (4.82 +/- 1.39 mg.g-1 dry lung weight) did not differ significantly from that in anesthetized spontaneously breathing rats (4.00 +/- 1.09 mg.g-1 dry lung weight). Furthermore, phospholipid content was not found to increase in animals maintained at 37 degrees C for 20 min (4.43 +/- 1.30 mg.g-1 dry lung weight) or 60 min (3.55 +/- 0.88 mg.g-1 dry lung weight) after killing. In conclusion the constancy of phospholipid content can be due either to a normal secretion or to a hypersecretion with a concomitant removal. PMID- 1725752 TI - Effects of breathing dense gas mixtures on cardiovascular function and EEG rhythms. AB - Gas mixtures containing oxygen and nitrogen or dense inert gases (sulfur hexafluoride, SF6, or argon, Ar) were inhaled by anaesthetized, artificially ventilated rabbits and cats. SF6 and Ar were compared to nitrous oxide (N2O), a dense inert gas well known for its anaesthetic properties. Concentrations in these gases were 50% (SF6) and 60% (Ar or N2O). In cats, there was a significant increase in total lung resistance with SF6 or Ar associated with a slight but significant drop in arterial blood pressure and heart rate (-5% and -10% with SF6 and Ar, respectively), with no change in electrocardiogram activity. N2O induced in some cats a slower electroencephalographic (EEG) pattern constituted by delta waves of higher amplitude, whereas neither SF6 nor Ar changed the EEG rhythms in this species. In the rabbits, the unsteadiness of the EEG rhythms did not make it possible to assess the cortical effects observed with SF6 or N2O. PMID- 1725753 TI - Human neonatal red cells. Regulatory volume response under anisotonic conditions. AB - Studies have been carried out in human neonatal red cells (cord blood) to examine the outcome of cellular volume under anisotonic media. When exposed to hypertonicity these cells showed, after an initial shrinkage phase, a volume expansion that reached short of the original volume at 60 minutes. The chloride replacement by nitrate as well as amiloride (in chloride media) inhibited this regulatory volume response. The cellular sodium increase that paralleled the volume expansion was also amiloride-sensitive. We suggest that a Na+/H+ antiport mechanism could mediate the response. Cell volume increase in hypotonic media was not followed by a shrinkage phase. PMID- 1725755 TI - [XVth Congress of the Society of Biomechanics. Cluny, 18-19 September 1990. Abstracts]. PMID- 1725754 TI - Are adrenal catecholamines involved in the enhancement of aldosterone production in cold acclimated rats? AB - In order to determine the possible role of adrenal catecholamines in the enhancement of aldosterone production in cold acclimated rats, dopamine (DA), noradrenaline (NA) and adrenaline (A) contents of adrenals were investigated in cold acclimated (or not), hypophysectomized (or not) rats. The DA content, DA being an inhibitor of aldosterone synthesis, increased in hypophysectomized rats and remained unchanged in sham-operated ones following cold exposure. The NA and A contents, these being, in vitro, stimulators of aldosterone production did not increase, in either group of cold acclimated rats. It is concluded that adrenal catecholamines do not seem to be implicated in the enhancement of aldosterone production. PMID- 1725756 TI - [Dopamine antibodies in the pathogenesis of parkinsonism]. AB - L-DOPA and dopamine (DA) binding antibodies were found in the blood serum of Parkinsonian patients and middle-aged and elderly normal persons. DA-binding serum gamma-globulins of parkinsonian patients injected into rat caudate nuclei induced the pathogenetic mechanism of Parkinson's syndrome (generator of pathologically enhanced excitation) in these brain part and evoked main parkinsonian symptoms (oligokinesia, rigidity, tremor). The serum gamma-globulins of Parkinsonian patients without Da-antibodies caused less pronounced EEG disturbances. Parkinsonian symptoms developed rarely and were shorter and less pronounced compared with the DA-antibody effect. The DA binding antibodies role in Parkinson's syndrome pathogenesis and is L-DOPA therapeutic tolerance formation was discussed. PMID- 1725757 TI - [The structural changes in the lungs and the phospholipids of the pulmonary surfactant in experimental bleomycin-induced pneumosclerosis in rats]. AB - Light microscopy was used to study rat lungs. Some morphological and biochemical characteristics after single bleomycin intratracheal administration in a dose of 10 mg/kg were studied. Some metabolic changes in the alveolar epithelium and vascular endothelium were estimated quantitatively. The peculiar picture of acute toxic alveolitis in the early stage of the injury and the features of fibrotic alveolitis by the 30-60th day were observed. The LDH and succinate dehydrogenase activities in the alveolar epithelium increased by the 3-7th day and returned to normal by the 30th day. At the same time the PL content increased 2-fold by the 30-60th day. The lysolecithin, sphingomyelin and phosphatidylserine concentration increased on the 3-7th day and returned to normal level on the 14th day. The phosphatidylglicerol content decreased significantly by the 60th day. PMID- 1725758 TI - Insecticide-induced inhibition of thyroid activity in the fish Oreochromis mossambicus and recovery in insecticide-free water. AB - The histological and histochemical state of the thyroid gland of Oreochromis mossambicus was analyzed after 20 days' exposure of the fish to BHC (p less than 0.001 ppm). Histological changes after exposure included acolloidal and atrophied follicles and goitre formation. Follicular and nuclear diameter and the E/T ratio were significantly (p less than 0.001) higher than in the corresponding controls. The above insecticide led to thyroid dysfunction. The histochemical characteristics of the gland also changed after BHC treatment. When exposed fish were transferred to normal, clear, dechlorinated water, the altered follicles displayed remarkable recovery of activity and thyroid function returned to almost the same state as in normal controls. Histological and histochemical evaluation at 10-day intervals revealed healthy functional restitution of the gland, indicating that BHC-induced changes are reversible. PMID- 1725759 TI - Endocrine profile in gastric carcinomas. An immunohistochemical study. AB - 22 gastric carcinomas (13 intestinal type and nine diffuse type) were immunostained for neuron specific enolase, chromogranin, Leu-7 and a panel of fifteen different peptide hormones. Five out of the 13 tumours of intestinal type and four out of the nine diffuse carcinomas expressed immunoreactivity for one or more of the pan endocrine markers. Seven out of the 13 tumours of intestinal type and five out of the nine diffuse carcinomas also expressed immunoreactivity for gastrin (3), ACTH (3), serotonin (7) and calcitonin (7). Immunoreactivity for somatostatin (1) and substance P (1) were also seen in two tumours of intestinal type. Seven out of 18 cases with benign mucosa adjacent to the tumours expressed a focal immunoreactivity for chromogranin (6), serotonin (6), gastrin (5) and calcitonin (1). All hormone-producing tumours also expressed immunoreactivity for carcino-embryonic antigen. Our results confirm that a high proportion of gastric carcinomas are hormone producing. PMID- 1725760 TI - Pathological changes in dendrites of substantia nigra neurons in Parkinson's disease: a Golgi study. AB - Neurons of the substantia nigra show severe morphological changes in Parkinson's disease. Pathological alterations of cell bodies have been described, whereas those of neuronal processes have hardly been investigated. Golgi impregnation has been the chosen method for demonstrating neuronal processes and dendritic and somatic spines. We therefore used the Golgi-Braitenberg method to qualitatively and semi-quantitatively study the substantia nigra of eight patients with Parkinson's disease compared with eight control cases. Golgi impregnation of substantia nigra neurons was good in all control cases. In full agreement with the analysis of Braak and Braak (1986) three neuronal types within the substantia nigra were found. In cases of Parkinson's disease, severe pathological changes such as decrease of dendritic length, loss of dendritic spines and several types of dendritic varicosities were found only in the melanin-containing pars compacta neurons. Pars reticulata nerve cells were intact. These findings support the predominant role played by the dopaminergic efferent pathway in the degenerative process. The afferent pathway was not affected. This suggests that the substantia nigra lesion is primary in Parkinson's disease. Loss of neurons found in H & E sections corresponded to a lesser amount of impregnated pars compacta neurons in cases with Parkinson's disease when compared to controls. Evidences exist that the duration of the disease may be related to the extent of pathologically altered Golgi-impregnated pars compacta cells. The amount of Lewy bodies in H & E sections corresponded to the quantity of round varicosities in impregnated pars compacta neurons. These round dendritic varicosities were considered to be Lewy body inclusions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725761 TI - cAMP-dependent regulation of P450-catalyzed dealkylation in rat conceptal tissues. AB - Exposures of cultured whole rat conceptuses to varying concentrations of dibutyryl cyclic AMP or isobutylmethylxanthine, alone or in combination, resulted in significant increases in rates of cytochrome P450-dependent depentylation of pentoxyphenoxazone in cell-free preparations. Lesser increases in rates of debenzylation of benzyloxyphenoxazone were also observed. In cultured whole conceptuses, basal depentylase and debenzylase activities in the visceral yolk sac were approximately sixfold higher than in the embryo. The ectoplacental cone and decidual tissues exhibited no detectable depentylase activity. Only the visceral yolk sac exhibited increased depentylase activity in response to dibutyryl cyclic AMP and isobutylmethylxanthine. Inhibitory antibodies raised against adult hepatic P450s IIB1, IIC11, and IA1 failed to significantly inhibit the yolk sac depentylase activities of noncultured conceptuses. The results suggested that the conceptal depentylation reaction may be catalyzed by a unique P450 isoform(s) that is not expressed during adult life. PMID- 1725762 TI - Effects of vasoactive intestinal peptide, helodermin and galanin on responses of guinea-pig lung parenchyma to histamine, acetylcholine and leukotriene D4. AB - 1. The effect of vasoactive intestinal peptide (VIP) was studied on the contractile response of guinea-pig lung parenchymal strips (GPP) induced by bronchoconstrictor agonists, such as leukotriene D4 (LTD4), histamine and acetylcholine (ACh). This effect of VIP was compared with helodermin, a peptide that is structurally related to VIP, and galanin, another neuropeptide that is thought to co-exist with VIP. 2. VIP (10 nM) induced a potent and reversible inhibition of the contractions of GPP induced by LTD4 (1-30 pmol) but did not affect those due to ACh (1-100 nmol) or histamine (1-30 nmol). A ten fold higher concentration of VIP (100 nM) did not further inhibit LTD4-induced responses or reduce those induced by histamine or ACh. 3. Helodermin (10 nM) had a similar inhibitory effect on contractions of GPP induced by LTD4 (3-30 pmol) but did not affect contractions induced by histamine (1-10 nmol). 4. Indomethacin (2.8 microM) and salbutamol (10 nM) significantly reduced responses elicited by LTD4 and histamine but not those due to ACh. A ten fold higher concentration of salbutamol (100 nM) further inhibited the contractions due to LTD4 and histamine and at this concentration responses induced by ACh were inhibited. 5. VIP (10 nM) and helodermin (10 nM) significantly reduced the LTD4-induced release of thromboxane A2 (TXA2), measured as TxB2 by radioimmunoassay, from GPP. The smaller release of TxA2 induced by histamine was not significantly reduced in the presence of VIP. 6. In comparative studies, galanin (10-100 nM) did not affect contractions of GPP induced by either LTD4, histamine or ACh. In contrast to VIP and helodermin, both at 0.1-3 nmol, which induced doserelated relaxations of guinea-pig trachea, galanin was inactive on this preparation in doses of up to 3 nmol.7. In conclusion, our results show that contractions of GPP induced by LTD4 are more sensitive to inhibition by VIP and helodermin than are contractions due to histamine or ACh. This inhibition appears to be associated with the different contribution of released TxA2 to contractions evoked by the agonists. VIP and helodermin inhibit the cyclo-oxygenase-dependent component of the LTD4-induced response, as in the case of indomethacin. PMID- 1725763 TI - Neuropeptide Y, peptide YY and C-terminal fragments release histamine from rat peritoneal mast cells. AB - 1. Neuropeptide Y (NPY) and peptide YY (PYY) seem to act on at least two receptor subtypes, Y1 and Y2. The Y1-receptor requires the whole C-terminally amidated NPY/PYY molecule whereas the Y2-receptor in addition recognizes C-terminal fragments of the two peptides. The present study was designed to elucidate whether NPY and related peptides were able to release histamine from isolated peritoneal mast cells of the rat. 2. NPY, NPY 15-36, NPY 22-36, NPY 26-36 and desamido-NPY evoked a concentration-dependent release of mast-cell histamine. The pEC15 values for NPY 15-36 and NPY 22-36 were higher, while the pEC15 value for NPY 26-36 was lower than that for NPY. At the highest concentration tested (0.1 mM), NPY and its C-terminal fragments released between 30 and 40% of the total histamine content. At the same concentration desamido-NPY released about 20%. 3. PYY and PYY 15-36 also evoked a concentration-dependent release of mast-cell histamine. PYY was more effective than PYY 15-36 since, at 0.1 mM, PYY released about 33%, while PYY 15-36 released about 15% of the total histamine content. Pancreatic polypeptide (PP) and the Y1-receptor-selective agonist [Pro34]NPY were virtually inactive. 4. The effect profile of the NPY/PYY-related peptides suggests that they act on the mast cells by a mechanism that does not involve either of the receptor subtypes hitherto described. The kinetics of the NPY evoked histamine release may suggest that positively charged amino acid residues of NPY/PYY release mast-cell histamine by a non-receptor mechanism, as has been suggested for substance P and other basic peptides. PMID- 1725764 TI - Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection. AB - 1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2. Carbenoxolone (100-300 microM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)- 3 morpholino-syndnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (500 microM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300 microM) antagonized the reversal of the RPN- or SIN-1-induced antiaggregatory effect by oxyhaemoglobin (10 microM). 3. Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.3 microM) were relaxed by carbenoxolone (100-300 microM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10 microM) or NG-nitro-L-arginine (L-NNA, 100 microM). Sodium nitroprusside (10 nM) induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 microM). . The carbenoxolone (200 mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-40 mg kg-1) in a dose-dependent manner. Pretreatment of rats with indomethacin (10 mg kg-1, s.c.) increased the effect of L-NNA. 5. The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone. PMID- 1725765 TI - Histamine-induced increases in cyclic AMP levels in bovine adrenal medullary cells. AB - 1. The effect of histamine on cellular cyclic AMP levels in cultured bovine adrenal medullary cells has been studied. 2. Histamine (0.3-30 microM) increased cyclic AMP levels transiently, with a maximal response after 5 min, a smaller response after 20 min, and no increase seen after 80 or 180 min. The EC50 at 5 min was approximately 2 microM. Histamine had no effect on cyclic AMP release from the cells over 5 min, but increased it after 90 min. 3. The cyclic AMP response to 5 microM histamine was reduced by 45% by 1 microM mepyramine and by almost 30% by 1 microM cimetidine, and was abolished by the combination of both antagonists. Cimetidine at 100 microM did not inhibit the response to histamine more than 1 microM cimetidine. The H3-receptor antagonist, thioperamide (1 microM), had no effect on the response to histamine. 4. The H1-receptor agonist, 2-thiazolyethylamine (5-100 microM) and the H2-receptor agonist, dimaprit (5-100 microM), each induced a cyclic AMP response, and gave more-than-additive responses when combined. The H3 agonist (R) alpha-methylhistamine (100 microM) had no effect either on its own or in combination with either the H1 or the H2 agonist. The response to 100 microM 2-thiazolylethylamine was unaffected by cimetidine (100 microM). 5. The cyclic AMP responses to 5 microM histamine, 100 microM thiazolylethylamine and 100 microM dimaprit were each weakly enhanced in the presence of 1 mM 3-isobutyl-1-methylxanthine. The response to dimaprit was enhanced more than 10 fold in the presence of 0.3 microM forskolin, while the responses to histamine and thiazolylethylamine were weakly enhanced.6. The cyclic AMP response to 5 microM histamine was partially reduced in the absence of extracellular Ca2 and the residual response was fully antagonized by 1 microM cimetidine and was unaffected by 1 microM mepyramine.In the absence of Ca2 , the cyclic AMP response to 100 microM thiazolylethylamine was abolished, while that to 100 microM dimaprit was unaffected.7. Reincubation of 5 microM histamine solutions with a second set of chromaffin cells, following prior incubation with another set of cells, induced a cyclic AMP response in the fresh cells. This response was reduced by a combination of mepyramine and cimetidine to the same degree as the response to fresh 5 microm histamine solutions.8. The results indicate that histamine increases cellular cyclic AMP levels in bovine chromaffin cells by three mechanisms: by acting on H1 receptors, by acting on H2 receptors, and by an interaction between H, and H2 receptors. The H1 response does not require concomitant activation of H2 receptors, is fully dependent on extracellular Ca2 +, does not depend on secreted chromaffin cell products, and is not due to reduced cyclic AMP degradation or export. The H2 cyclic AMP response is the first functional response reported for H2 receptors on chromaffin cells, is independent of Ca2 , is not due to reduced cyclic AMP export or degradation, and is likely to be mediated via a direct action through Gs. The role of these different mechanisms in the regulation of cyclic AMP-dependent processes in chromaffin cells by histamine is under investigation. PMID- 1725767 TI - Interferon inhibits synaptic potentiation in rat hippocampus. AB - The effects of rat interferon (IFN) on the electrically-induced potentiation of the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. The treatment with rat IFN (120 U/ml) reduced the size of short-term potentiation (STP) and suppressed long-term potentiation (LTP). These IFN-induced effects were dose-dependent in the range of 50-500 U/ml. In addition, IFN slightly attenuated the potentiation when applied during the maintenance of LTP. Basal synaptic transmission was affected by IFN at concentrations greater than or equal to 250 U/ml. Following an acute exposure to IFN (500-200 U/ml), cultured embryonic neurones from rat hippocampus often exhibited an attenuation of N-methyl-D-aspartate-induced currents and a variation (increase or decrease) of voltage-activated Ca2+ current amplitude. A possible role of IFN as neuromodulator in mammalian brain during immune responses is discussed. PMID- 1725766 TI - Differential effects of phosphoramidon on neurokinin A- and substance P-induced airflow obstruction and airway microvascular leakage in guinea-pig. AB - 1. The effects of the inhaled neuropeptides, neurokinin A (NKA) and substance P (SP) on lung resistance (RL) and airway microvascular permeability were studied in anaesthetized guinea-pigs. 2. Single doses of inhaled NKA (3 x 10(-5), 1 x 10( 4), 3 x 10(-4) M; 45 breaths) and SP (1 x 10(-4), 3 x 10(-4), 1 x 10(-3); 45 breaths) caused a dose-dependent increase in both RL and airway microvascular leakage, assessed as extravasation of the albumin marker, Evans blue dye. 3. NKA at 1 x 10(-4) and 3 x 10(-4) M resulted in a significantly higher increase in RL than SP at the same doses. 4. Inhaled SP (3 x 10(-4) M; 45 breaths) caused significantly higher Evans blue dye extravasation in main bronchi and proximal intrapulmonary airways compared to the same dose of NKA. 5. Pretreatment with the specific inhibitor of neural endopeptidase (NEP24.11), phosphoramidon, caused an approximately 100 fold leftward shift of the RL responses to inhaled NKA and SP. 6. Phosphoramidon significantly potentiated both NKA- and SP-induced airway microvascular leakage at proximal intrapulmonary airways, but not at any other airway level. 7. Inhibition of NEP24.11 potentiate both the SP- or NKA-induced airflow obstruction to a larger extent than the induced airway microvascular leakage, suggesting that NEP24.11 is more important in the modulation of the airflow obstruction observed after these mediators. PMID- 1725768 TI - The kappa opioid-related anticonvulsants U-50488H and U-54494A attenuate N-methyl D-aspartate induced brain injury in the neonatal rat. AB - The neuroprotective effects of the kappa opioid-related anticonvulsants U-50488H and U-54494A were tested in a model of N-methyl-D-aspartate (NMDA)-induced brain injury in the neonatal rat. Seven-day-old rat pups were injected intracerebrally with 7.5 nmol NMDA. Five days later, the ensuing unilateral hemisphere weight reduction was measured and used to assess the severity of insult. Control animals (n = 85) exhibited a 21.7 +/- 0.5% hemisphere weight reduction. Animals treated with U-54494A in split doses before and after NMDA administration showed significant neuroprotection at 10, 15, and 20 mg/kg, with the maximum effect observed at 15 mg/kg (33.8% protection). Animals treated with U-50488H on a similar dosing schedule showed significant neuroprotection at all doses tested, with peak protection observed at 30 mg/kg (51.8% protection). Both compounds exhibited a neuroprotective effect when hemisphere cross-sectional area and hippocampal histology were assessed. Treatment with U-54494A after NMDA administration also afforded neuroprotection at various doses, as measured by hemisphere weight disparity, with peak protection occurring at a dose of 20 mg/kg (32.4% protection). These data show that both U-50488H and U-54494A afford neuroprotection against NMDA-induced neuronal injury in the neonatal rat brain. PMID- 1725769 TI - Determination of chlorhexidine in saliva and in aqueous solutions. AB - A new method is presented for the determination of chlorhexidine in centrifuged saliva and in aqueous solutions by means of fluorescence spectroscopy. The method relies on complex formation between chlorhexidine and eosin. The fluorescence value of the chlorhexidine-eosin system decreases with increasing chlorhexidine concentrations. In centrifuged saliva a linear relation was found between the fluorescence at 541 nm and the chlorhexidine content up to about 15 ppm; the reproducibility of the method was found to be better than 1 ppm chlorhexidine. The biological spreading for centrifuged saliva from different participants as well as the spreading due to the period of saliva collection are about 3%. In whole uncentrifuged saliva the fluorescence method has been a detection limit of about 8 ppm chlorhexidine, due to the binding of the compound to salivary constituents. Above 8 ppm chlorhexidine, the biological variation in saliva is such that chlorhexidine determinations by means of fluorescence can be done only with a reproducibility of +/- 10 ppm. Advantages and disadvantages of the ultraviolet spectroscopy method, the radioactive labelling technique, and the fluorescence method for chlorhexidine determinations are discussed. PMID- 1725770 TI - Evidence for biological and structural diversity among scrapie strains. PMID- 1725771 TI - Ultrastructural studies of prions. PMID- 1725772 TI - Esculine, ranitidine and carbenoxolone: different modes of action on gastric mucosa. AB - 1. This study was designed to determine the antiulcerogenicity of esculine in various types of experimentally induced gastric ulcers in which the appearance of lesions is due to an ischemic process: cold-restraint stress and pylorus-ligated induced ulcers. 2. In the first experimental model, esculine (50 mg/kg) produced a significant diminution not only in the number of haemorrhagic stomachs (21.5% by 37.5% of the controls) but also in the ulcer index, U.I. (1.00 +/- 0.63, P less than 0.05). 3. When the mucosal damage was induced as a consequence of the pylorus-ligated gastric secretion, pretreatment of esculine (25 and 50 mg/kg) prevented the formation of gastric lesions (12.4 +/- 2.8, P less than 0.05 and 12.2 +/- 1.20, P less than 0.005), although it was less effective than ranitidine (2.8 +/- 1.8, P less than 0.025). However a significant reduction on the acidity with a dose of 25 mg/kg was observed (31.69 +/- 6.42, P less than 0.025). For the rest of the studied parameters: pepsin, histamine and Na and K electrolytes no differences with regard to the control groups were produced. 4. The effects of esculine on mucosal lesions produced by intragastric instillation of 1 ml of absolute ethanol, were also studied. In this model esculine did not show any protective effect and high U.I. values were obtained. PMID- 1725773 TI - Angiogenesis: modulation with opioids. AB - 1. The effect of beta-endorphin (beta-EP) and morphine sulfate (MS), in presence and absence of naloxone (NX), on chicken chorioallantoic membrane was studied as a function of blood vessel proliferation. 2. A 50% reduction in blood vessel proliferation occurred by 10 micrograms of beta-EP or by 5 micrograms of MS per egg compared to controls. 3. An individual dose, i.e. 5 micrograms of beta-EP, did not significantly inhibit blood vessel counts after initial 24 hr period of the drug application when given alone compared to inhibition occurring with combined use of NX. 4. NX (1 microgram) did not significantly reverse the angiostatic effects of MS (10 micrograms) or of beta-EP (5 micrograms). 5. The observed modulation of angiogenesis by opioids suggests involvement of beta-EP and MS in the proliferation of vascular endothelial cells. 6. This may be due to an effect of beta-EP and MS on cell-mediated immunity factors such as interferons, interleukins and prostaglandin E2. PMID- 1725774 TI - Effects of steroidal and non-steroidal antiandrogens on the left atrium of the rat in vitro. AB - 1. The effect of steroidal-cyproterone acetate (CPA, 10(-7)-10(-5) M), clormadinone acetate (CMA, 10(-7)-10(-5) M), medroxyprogesterone acetate (MPA, 10(-7)-10(-5) M) and spironolactone (SPI, 10(-7)10(-5)M) and non-steroidal flutamide (F, 10(-7)-6 x 10(-5) M) and cimetidine (C, 10(-7)-10(-4)M) antiandrogens on contractile force of electrically stimulated left atria of the rat have been assayed. 2. CPA, CMA, MPA and F inhibit, in a dose-dependent way, the contractile force of left atria. SPI enhanced (P less than 0.01 at 10(-6) M), and cimetidine did not modify the contractile force. 3. F, but not CPA, CMA, MPA or SPI, inhibit the contractile force induced by CaCl2 (0.9-7.2 mM) on electrically stimulated left atria. 4. The inhibitory effect of CPA (10(-7)-10( 5) M) was significantly increased by atropine (10(-6) M) and diltiazen (10(-5) M), and significantly reduced by yohimbine (10(-7) M) and in reserpinized (2.5 mg/kg, 48 and 24 hr before experiments) rats. 5. Our results suggest that the negative inotropism of steroidal antiandrogens could be related to inhibition of noradrenaline release, presumably through an alpha 2 adrenergic effect. The negative inotropism produced by F is related to inhibition of calcium dependent process on left atria contraction. PMID- 1725775 TI - Alterations in the potassium-evoked release of substance P from the spinal cord of streptozotocin-induced diabetic rats in vitro. AB - 1. The release of SPLI evoked by high levels of K+ (50 mM) from the spinal cord of diabetic rats was greater than in the case of spinal cord from control rats. 2. Morphine (10(-5) M) significantly inhibited the K(+)-evoked release of SPLI release in both the groups. However, in spinal cord from diabetic rats, morphine reduced the K(+)-evoked release of SPLI only to the levels that were observed in material from control rats prior to treatment with morphine. 3. Glucose (20 mM) and dibutyryl cyclic-AMP (10(-4) M) significantly potentiated the K(+)-evoked release of SPLI in spinal cord from control rats. 4. These findings suggest that excessive release of SPLI from the spinal cord may be associated with the reported abnormalities in nociceptive transmission in diabetic rats, and that excessive release of SPLI may be modulated by levels of glucose and/or cyclic-AMP in the spinal cord. PMID- 1725776 TI - Peptides do not induce contractions in gastrointestinal smooth muscle in calcium free solution. AB - 1. Smooth muscle from six sites in the cat gastrointestinal tract was evaluated with respect to its ability to generate contractions in calcium-free solutions. 2. Membrane depolarization and carbachol, but not cholecystokinin or neurotensin, increased tension in smooth muscle segments of esophagus, corpus, duodenum, ileum, proximal colon and distal colon in calcium-free solution. 3. Substance-P produced a contractile response in the absence of calcium but only in the corpus and distal colon. 4. These findings indicate that peptide mediated release of intracellular calcium plays a minimal role in activation of cat gastrointestinal smooth muscle. PMID- 1725777 TI - Neurotransmitter metabolites in medicated epileptic patients. AB - The serotonin, dopamine, and noradrenaline metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and methoxyhydroxyphenylglycol (MHPG), were measured in urine of 38 male patients with idiopathic epilepsy on a single drug regime, and in 36 male healthy controls. Twenty patients were on carbamazepine and 18 on diphenylhydantoin. The concentrations of HVA and MHPG did not differ from normal, but 5-HIAA was significantly reduced in both patient groups. The reduced serotonin catabolism may be the result of a better utilisation of serotonin in the synapses, caused by the antiepileptic medication. PMID- 1725778 TI - Separation and characterization of sialic acid-containing salivary-type amylase from patients' sera with immunoglobulin A-type myeloma. AB - Isoamylases, with an abnormal anodic migration, were detected by an electrophoretic technique in the sera from two patients with immunoglobulin A type myeloma. The abnormal isoamylase bands migrate towards the anode faster than the salivary isoamylase (S2) band and were stained more strongly than the S2 sub band. The abnormal isoamylase could be separated from patients' sera by using size-exclusion high-performance liquid chromatography. The serum abnormal isoamylases were showed to be sialic acid residues containing amylase, after the study of treatment with neuraminidase (EC 3.2.1.18), and to be salivary-type amylase, after the study of reaction with human salivary monoclonal antibody. The abnormal bands were not detected in the saliva from one patient. The two patients had no detectable malignancies except myeloma. These findings strongly suggest that the sialic acid-containing salivary-type amylases were produced ectopically from myeloma cells. In this regard the ectopic amylase production by myeloma cells is discussed. PMID- 1725779 TI - An outbreak of lindane-resistant scabies treated successfully with permethrin 5% cream. AB - An outbreak of scabies in 12 persons, after contact with a hospitalized patient with lindane-resistant Norwegian scabies, is described. Resistance to treatment was documented in 7 of 10 patients treated with lindane. Of these, one responded to crotamiton 10% cream, and the remaining six were cured by a single treatment with permethrin 5% cream. PMID- 1725780 TI - Immunohistochemical detection of proliferation and differentiation in discoid lupus erythematosus. AB - Discoid lupus erythematosus lesions show hyperkeratosis and atrophy, which may reflect abnormal epidermal proliferation, differentiation, or both. In this investigation, markers for epidermal proliferation, differentiation and inflammation were studied in cutaneous lesions of discoid lupus erythematosus. Frozen sections of biopsy specimens from 20 patients were examined immunohistochemically regarding Ki-67 staining and keratin 16 expression (parameters for proliferation), and the expression of keratin 10, involucrin, and filaggrin (parameters for differentiation). The inflammatory infiltrate was characterized with the use of antibodies against T lymphocytes, monocytes/macrophages, and Langerhans cells. With these markers, epidermal proliferation was found to be increased in discoid lupus erythematosus. Keratin 10 expression, a marker for early differentiation, showed the pattern of normal skin. Involucrin and filaggrin, markers for terminal differentiation, were expressed already in the lower part of the stratum spinosum, whereas in normal skin these markers were restricted to the stratum granulosum and the upper layers of the stratum spinosum, and the stratum granulosum and stratum corneum, respectively. Infiltrate analysis revealed the well-established picture. We conclude that in cutaneous lesions of discoid lupus erythematosus, hyperproliferation is combined with normal early differentiation and premature terminal differentiation of keratinocytes. PMID- 1725781 TI - Organization of cortical and subcortical projections to the feline insular visual area, IVA. AB - Extracellular recordings with carbon fiber-filled microelectrodes were used to identify the visually responsive area within the insular cortex (referred to hereafter as the insular visual area, IVA) of anaesthetized cats. Broadly speaking, IVA comprises the cortex surrounding the anterior ectosylvian sulcus (AEs) along its ventral bank and the major portion of the anterior sylvian gyrus. Visually sensitive cells were recorded along the whole length of the AEs. In the same animals, the afferent connections of IVA were studied through the use of the retrograde tracers wheat germ agglutinin-conjugated horseradish peroxidase (WGA HRP) and fluorescent Diamidino yellow (DY), in combination with standard electrophysiological stimulation and recording techniques. The results indicate that: (1) the IVA receives a wide variety of telencephalic inputs, not only from visual, sensorimotor, auditory, limbic and association cortical areas, and from the claustrum, amygdala and basal nucleus of Meynert, as well, but also from the diencephalic projections arising mainly from the lateralis medialis-suprage niculate nuclear complex (LM-Sg) and the ventral medial nucleus (VM). (2) The gyral part of IVA (gIVA) receives afferents mainly from the lateral part of the lateral suprasylvian visual area (LS) throughout almost its entire length, as well as from area 20, the posterior suprasylvian sulcal area (PS), the frontal eye fields, areas 6 and 36, and almost the whole length of the cortical area lying along the anterior ectosylvian sulcus (AEs). (3) By contrast with (2), the sulcal part of IVA (sIVA) which corresponds to the anterior part of the anterior ectosylvian visual area (AEV) of Norita et al. ('86), receives cortical projections mainly from the lateral and medial parts of the anterior half of LS, area 20, PS, the frontal eye fields, area 36, and most parts of the cortical area extending along the AEs. (4) Subcortically, IVA receives thalamic afferents mainly from VM and LM-Sg. The connections between IVA and LM-Sg are organized topographically, with the more anterior part of IVA being related to the more ventral portion of LM-Sg, and with sIVA being related chiefly to the mid-portions of LM-Sg. These results thus suggest that IVA may function as an integrative centre among structures belonging to the extrageniculostriate system, the sensorimotor system, as well as to the limbic system. Furthermore, our electrophysiological and anatomical findings, together with previous reports concerning AEV, suggest that the posterior part of AEV (AEV proper) is distinctive from gIVA, and that the sIVA apparently serves as a transitional region between AEV and gIVA. PMID- 1725782 TI - Cytokeratin expression in human spinal meninges and ependymal cells. AB - The expression of intermediate filament protein in human spinal cord arachnoid cells and ependyma was studied by immunohistochemistry and immunoblotting. Monoclonal antibodies specific for individual cytokeratin polypeptides indicated a developmental change in the presence of cytokeratin 8 and 18 in spinal leptomeninx and tanycytes of the spinal cord ependyma. While in fetal material cytokeratin 8 and 18 were abundant in arachnoid cells, in adults immunoreactivity was restricted to a few cells. Immunoblots prepared from adult as well as fetal arachnoid membranes showed significant amounts of cytokeratin 8. These findings indicate that although cytokeratin is represented in both fetal and adult arachnoid cells there is development regulation of its specific localization. PMID- 1725783 TI - Normalization of protein synthesis in dystrophic neurons of cerebral cortex in rats after hypoxia, opening of the blood-brain barrier and treatment with organospecific RNA. AB - The possibility of normalization of protein synthesis intensity was explored in dystrophic neurons and in the total brain cortex of rats after acute hypoxic hypoxia. We avoided transplantation of embryonic nervous tissue (ENT) into the rat brain, as we did before, as well as operations and brain damages in opening of the blood-brain barrier (BBB) caused by hypoxia and intramuscular injections of organo (brain)-specific RNA. As shown by the autoradiographic and biochemical methods using radioactive isotopes (3H-leucine), hypoxia causes a statistically significant reduction in the intensity of protein synthesis which increases and becomes normalized after injection of brain-specific RNA into femoral muscles of animals. Thus, it is possible to normalize hypoxia-inhibited compensation restoration processes in the brain cortex of animals and, probably, the function of the higher nervous activity using the new simple and harmless biological method. The data presented are of priority significance and important both for development of a number of fundamental biological problems and for medicine since the described method permits the treatment of some serious nervous and mental diseases in humans. PMID- 1725784 TI - Geniculo- and subthalamohypothalamic connections in the lizard: HRP study. AB - Reciprocal connections of the hypothalamus with the ventral nucleus of the lateral geniculate body and ventrolateral (subthalamic) nucleus were demonstrated in the lizard Ophisaurus apodus using the retrograde and anterograde axonal tracing method following local HRP injection into the mamillary complex. PMID- 1725785 TI - Immunohistochemical localization of substance P, enkephalin and serotonin in the developing human retina. AB - The localization of substance P (SP), enkephalin (ENK) and serotonin (5-HT) in the retinae of 12 human embryos/fetuses ranging in age from 6-30 weeks was determined immunohistochemically using the PAP method. At the 6 week stage [crown rump length (CRL) unknown due to incomplete specimen], the developing retina consisted of a single undifferentiated cell mass from which immunoreactive cells were absent. By 90 mm CRL (10th week of gestation), the retina was composed of an outer neuroblastic layer, an inner plexiform layer and an innermost layer of ganglion cells. At this stage, SP, ENK and 5-HT positive cells were detected solely in the outer neuroblastic layer. By 140 mm CRL (17 weeks), the retina consisted of cell layers similar in number and type to those of the adult retina. In specimens 140-216 mm CRL (gestation ages 17-24 weeks), SP, ENK and 5-HT neurons were present in the outer nuclear, inner nuclear and inner plexiform layers. In addition, 5-HT positive neurons and fibers were evident in the outer plexiform layer. By 285-295 mm CRL (26-30 weeks), neurons in the ganglion cell layer and the fovea were also SP and ENK positive. In earlier specimens, the cell bodies alone were immunopositive and not until 142 mm CRL (17 weeks) were positive processes observed. Finally, the presence of SP, ENK and 5-HT immunopositive structures occurred in a sequence from outer to inner layers of the developing human retina. PMID- 1725786 TI - A neuroanatomical analysis of the rostral striatopallidal pathway in the rat. AB - Anterograde and retrograde experiments were made in the rat using either a mixture of horseradish peroxidase free and wheat germ-agglutinated (HRP-WGA) or HRP-WGA unmixed, in an attempt to investigate how the striatopallidal pathway is organized from rostral territories of the rat's striatum. The main findings emerging from this study are: a) a remarkable projection is directed from the rostral pole of the caudate-putamen complex to rostral sectors of the globus pallidus (GP) and entopeduncular nucleus (EP) and its is organized in a topographical fashion; b) this projection presents a remarkable heterogeneity in the distribution of terminal labeling into the GP and EP; c) a heterogeneous distribution of retrogradely labeled cells was seen in the rostral striatum after retrograde tracer injections in the GP that might explain, at least partially, the heterogeneity observed in the anterograde experiments. PMID- 1725787 TI - [Studies on the pathophysiology of paraneoplastic syndromes: both cancer cells and host immune cells are responsible for the pathophysiology of leukocytosis associated with oral cancer]. AB - Leukocytosis associated with malignant disease has been known as a paraneoplastic syndrome and occurs occasionally in patients with oral malignancies. In this study, mechanisms underlying leukocytosis associated with malignancy was investigated, using a squamous cell carcinoma of the maxilla from a patient who manifested marked leukocytosis. When the patient's tumor was inoculated into nude mice, it formed squamous cell carcinoma (MH85) and induced leukocytosis and splenomegaly. Leukocytosis and splenomegaly paralleled tumor growth. Surgical excision of MH85 tumor resulted in a dramatic reduction of leukocyte count and spleen weight, indicating an involvement of humoral mediators released by MH85. MH85 cells conditioned medium (MH85CM) were shown to contain granulocyte-colony stimulating factor (G-CSF) activity, which is a potent growth factor specific for granulocytes. These results suggest G-CSF or G-CSF like substance secreted by MH85 cells is responsible for leukocytosis in MH85 bearing nude mice (MH85 mice) and in the patient. MH85 cell growth was stimulated by G-CSF and inhibited by anti-G-CSF antibody, thus suggesting that G-CSF like substance is a autocrine growth factor for MH85 cells. Splenectomized MH85 mice developed less severe leukocytosis than did non-splenectomized mice. This finding indicated that not only G-CSF like substance secreted by MH85 cells but other humoral factors released by the hyperplastic spleen contribute to the development of leukocytosis. Splenic monocytes derived from MH85 mice and MH85CM-stimulated splenic monocytes showed increased secretion of tumor necrosis factor (TNF) and interleukin-1 (IL-1), both of which have been reported to induce neutrophilia in animals. Moreover, injection of anti-TNF-antibody into neutrophilic MH85 mice significantly, although not completely, decreased leukocyte count. Thus, it seemed likely that increased secretion of TNF and IL-1 by spleen cells that are stimulated by humoral factors released from MH85 also contributes to the progression of leukocytosis. In splenectomized mice, enlargement of MH85 tumor was retarded and metastases were impaired compared these in nonsplenectomized mice. Coculture of splenocytes from MH85 mice with normal spleen cells, inhibited blastogenesis in response to mitogen. The result suggests that splenocytes from MH85 mice played as immune suppressive cells. MH85CM conferred immune suppressive activity on normal spleen cells. This suppressor cell-inducing factor (SCIF) in MH85CM was found to have an apparent molecular weight of approximately 25kd, and its biological activity was neutralized by anti-G-CSF antibody. Therefore, SCIF secreted by MH85 cells was likely to be G-CSF like substance.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725788 TI - [A pharmacological study of the participation of the peripheral endings of primary afferent neurons in the inflammatory response evoked by heat and mechanical noxious stimulation]. AB - To investigate the participation of neuropeptides present in the peripheral endings of primary afferent neurons in the inflammatory response, immunoreactive substance P (iSP), calcitonin gene-related peptide (iCGRP) and neurokinin A (iNKA) levels in the s.c. perfusate, and inflammatory response (edema and plasma protein extravasation) evoked in rat paw by noxious stimulation were determined. The effects of these peptides on plasma protein extravasation in the skin of the hind paw of mice were also examined with the pontamine sky blue protein labelling method. The following results were obtained. 1) Immersion of the rat hind paw for 30 min into hot water adjusted to 47 degrees C led to a marked increase in the release of iSP and iCGRP in the subcutaneous perfusate with the formation of thermal edema. 2) Mechanical stimulation (600 g, 10 min) to the hind paw or electrical stimulation of the saphenous and sciatic nerves (10 V, 2 Hz, 1msec duration, 10 min) evoked the increase of iSP release in the perfusate with plasma protein extravasation. 3) iNKA release was not affected by neither heat nor mechanical stimulation. 4) Intraplantar injection of SP, CGRP and NKA induced plasma protein extravasation, the order of potencies being SP greater than CGRP greater than NKA. The action of SP was antagonized by spantide, an SP antagonist. The injection of CGRP with SP produced a synergistic action on plasma protein extravasation. 5) Neonatal pretreatment with capsaicin, which is known to degenerate small-diameter primary afferent neurons, caused the decrease in amount of iSP and iCGRP released during noxious heat stimulation. 6) Pretreatment with Compound 48/80, or stem bromelain and emorphazone, or des-Arg9-[Leu8]-BK, inhibited the iSP release evoked by noxious heat stimulation. 7) Opioids such as morphine (mu-agonist) and ethylketocyclazocine (kappa agonist) inhibited the heat stimulus-evoked iSP release and thermal edema, and the inhibitory effects were antagonized by pretreatment with their antagonists. 8) Morphine or ethylketocyclazocine or [D-Ala2,D-Leu5]-enkephalin (delta-agonist) inhibited the release of iSP evoked by electrical stimulation of the saphenous and sciatic nerves. These results indicate that SP and CGRP present in peripheral endings of small-diameter primary afferent neurons play an important role in the inflammatory response, and that opioids are involved in the regulation of inflammatory response through the inhibition of SP release. PMID- 1725789 TI - [Immunohistochemical studies on the peptidergic nerve distribution in hard palate mucosa and gingiva of the rat]. AB - The present study demonstrates the distribution of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in the hard palate mucosa and gingiva of the rat by the use of an indirect immunofluorescence method. Nerve fibers containing CGRP, SP and NKA showed very similar distribution patterns. Numerous nerve fibers containing CGRP, SP and NKA were observed in the lamina propria of hard palate mucosa. They formed dense subepithelial plexuses in incisive papilla and transverse palatine ridges. Some of them penetrated deeply into the epithelium and terminated as free nerve endings. In gingiva, nerve fibers containing these peptides were found especially near gingival sulcus. They were abundantly distributed beneath the junctional epithelium and formed nerve plexus. In all sampling regions, these nerve fibers were seen around the large- and medium-sized blood vessels in lamina propria and submucosal layer. NPY- and VIP-containing nerve fibers were observed in close association with blood vessels of hard palate mucosa. These nerve fibers were located around the large-sized blood vessels in the submucosal layer of hard palate and a few fibers were seen running along the small blood vessels in the lamina propria of the anterior half of hard palate mucosa. Nerve fibers containing these peptides were never found in gingiva. PMID- 1725790 TI - Duplex pulsed Doppler sonography of hepatocellular carcinoma treated with transcatheter arterial embolization. AB - Thirty-one patients with hepatocellular carcinoma underwent duplex scanning of their lesions with comparative angiography and serum alpha-fetoprotein determinations before and after transcatheter arterial embolization. Duplex ultrasound correctly identified 26 patients with patent tumor vessels after embolization. The Doppler signals became undetectable after embolization in the other five patients. Comparative angiography demonstrated good devascularization of their lesions. However, tiny tumor blush or small satellite nodules around the periphery of the devascularized masses were found. Therefore, the presence of signals after therapy indubitably needs further embolization. The timing of the second embolization for the patient with undetectable Doppler signals after the first embolization needs to be justified by further evaluations. PMID- 1725791 TI - Spontaneous fetal-maternal hemorrhage resulting in hydrops and elevated maternal serum alpha-fetoprotein levels. PMID- 1725792 TI - [Study on the histochemical staining of boric acid]. AB - The detection of boric acid in the tissue is of significance in investigating its toxicity. Because of this, we have devised a histochemical staining method to detect the presence of boric acid. The outline of this method follows. Frozen 12 14 microns sections, cut by a cryostat, are fixed in anhydrous ethanol and stained for 20 minutes in a protonated curcumin solution. Washing in acetic acid follows, and a red stain results if boric acid is present. This method causes a reaction, in which rosocyanin is formed by the reaction of boric acid and the protonated curcumin, and this principle is now used when an analysis of boric acid is needed. As to procedure, a 1 N concentration of sodium hydroxide is dropped onto a part of the stain to be tested, and the presence of rosocyanin is confirmed if the stain turns blue. Consequently, this staining confirms the presence of boric acid. PMID- 1725793 TI - New aspects of calcium antagonism in the treatment of hypertension. Proceedings of the Nitrendipine Symposium. Cairo, Egypt, September 27-30, 1990. PMID- 1725794 TI - Long-term protective effects of nitrendipine in experimental hypertension. AB - The antihypertensive and tissue-protective effects of nitrendipine were studied after long-term treatment of rats with experimental hypertension. Nitrendipine had opposite effects in comparison with vasodilators like hydralazine or minoxidil. Nitrendipine lowered heart weights, plasma atrial natriuretic peptide levels, and plasma renin activity, and was diuretic. Also, in spontaneously hypertensive rats that had been hypertensive for more than half of their life span prior to treatment, nitrendipine still had these effects. Nitrendipine prevented hypertension-induced mortality, even at subantihypertensive doses. Nitrendipine inhibited endothelin-1-induced DNA synthesis in isolated vascular smooth muscle cells as well as atherosclerotic plaque formation in cholesterol fed rats. PMID- 1725795 TI - Renal effects of nitrendipine. AB - Calcium-entry blockers exert several characteristic effects on the kidney that potentiate their antihypertensive effect. Long-term therapy with nitrendipine, a dihydropyridine derivative, lowers blood pressure while maintaining renal hemodynamics within limits similar to pretreatment values in essential hypertension with normal renal function. This is accompanied by a persistent natriuretic effect that probably facilitates its antihypertensive action and is not followed by changes of the renin-angiotensin-aldosterone system. Preliminary data also seem to indicate that nitrendipine could be safely used in patients with arterial hypertension and chronic renal failure. PMID- 1725796 TI - Arteriosclerosis and antihypertensive response to calcium antagonists in end stage renal failure. AB - The relationship between the presence of arterial calcinosis and the antihypertensive response to calcium blockers was studied in 40 hypertensive patients with end-stage renal failure (ESRF) on chronic hemodialysis, before and during 16 weeks after administration of nitrendipine in monotherapy. In a double blind, placebo-randomized study, nitrendipine reduced systolic blood pressure regardless of the presence or absence of arterial calcifications. The antihypertensive effects were significantly more pronounced in subjects with aortic calcium deposits in comparison with patients without clinical signs of arteriosclerosis (p less than 0.01). Diastolic blood pressure was significantly reduced only in patients with aortic calcifications, and remained unchanged in subjects with noncalcified aorta. Aortic pulse wave velocity decreased significantly in patients with aortic calcifications (p less than 0.001), but remained unaffected in patients with noncalcified vessels. Multivariate regression analysis showed that antihypertensive action of nitrendipine was correlated with the presence of aortic calcium deposits independently of age or baseline blood pressure levels. The results of the present study indicate that an overt arteriosclerosis as demonstrated by the presence of aortic calcifications on abdominal radiographs is a good indication for use of dihydropiridines in patients with ESRF. PMID- 1725797 TI - Nitrendipine and metabolic balance. AB - Abnormalities of glucose and lipoprotein metabolism have frequently been found in hypertensive patients in both epidemiological and clinical studies. Reduction of blood pressure favorably affects the rate of cardiovascular diseases mainly when concomitant with a decrease in glucose and lipid serum levels. For these reasons antihypertensive drugs without untoward metabolic side effects should be preferred, particularly in hypertensive patients with metabolic impairment. Nitrendipine, a dihydropiridine derivative, like other calcium-entry blockers, has been proved not to deteriorate fasting glucose (75 +/- 12 vs. 75 +/- 10 mg/dl) and serum insulin (8.5 +/- 3 vs. 10.8 +/- 5 microU/ml) levels. Glucose and insulin response to an oral carbohydrate challenge (glucose % removal rate 1.4 +/ 0.2 vs. 1.5 +/- 0.3%/min; incremental area for serum insulin 955 +/- 279 vs. 905 +/- 458 microU/ml/min), serum cholesterol (192 +/- 33 vs. 200 +/- 43 mg/dl), triglyceride (83 +/- 57 vs. 84 +/- 37 mg/dl) and high-density lipoprotein cholesterol (46 +/- 9 vs. 50 +/- 14 mg/dl) were not affected as well. It is therefore possible to conclude that nitrendipine may be safely prescribed when the antihypertensive activity of a calcium-entry blocker is required. PMID- 1725798 TI - Regression of cardiovascular structural changes after long-term antihypertensive treatment with the calcium antagonist nitrendipine. AB - Regression of cardiovascular structural changes is a main goal of antihypertensive treatment. Nitrendipine is a calcium antagonist of the dihydropiridine group that may be given once daily. In different animal models of experimental hypertension, nitrendipine was shown to reduce blood pressure (BP) and left ventricular (LV) mass, to prevent early mortality, and to limit the development of vascular lesions. It has also been proposed that nitrendipine is able to preserve tissue integrity and increase life span in malignant hypertension, because it prevents a deleterious calcium overload in the heart and arterial vessels. In humans, the effect of nitrendipine on LV mass has been evaluated in a limited number of studies with conflicting results. It has been shown that nitrendipine is able to increase the compliance of large arteries; its effect on vascular structural changes has never been reported. In this study, nitrendipine (20 mg o.d.) was given to 10 hypertensive patients. BP, (ambulatory BP monitoring) heart rate (HR), LV mass and function (TM echo, 2D guided), forearm minimal vascular resistance (min VR = BP/max blood flow--venous occlusion plethysmography--taken as an index of vascular STC), plasma renin activity (PRA), plasma catecholamines (NE and E), and aldosterone (ALD) were measured during placebo, after 2 and 6 months of treatment. BP was significantly reduced and HR was slightly increased. After 6 months of treatment a significant reduction of LV mass index (p less than 0.001) and of min VR (p less than 0.002) was observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725799 TI - Biochemical fractionation of oocytes. PMID- 1725800 TI - Fluorescent dextran clonal markers. PMID- 1725801 TI - Histological preparation of Xenopus laevis oocytes and embryos. PMID- 1725802 TI - Whole-mount staining of Xenopus and other vertebrates. PMID- 1725803 TI - Expression of ion channels by injection of mRNA into Xenopus oocytes. PMID- 1725804 TI - The fabrication and collagenous substructure of the eggshell membrane in the isthmus of the hen oviduct. AB - The eggshell of the chicken consists of a bi-layered shell membrane overlaid with a thick, calcified shell matrix. The shell membranes and matrix are deposited onto the egg as it passes through the oviduct. To assess the temporal and spatial aspects of the fabrication of type X collagen within the eggshell extracellular matrix, the immunohistochemical localization of type X collagen was studied in three regions of the hen oviduct (magnum, isthmus and uterus), in the membranes of uncalcified eggshells obtained from the oviduct prior to mineral deposition and in eggshell membrane and calcified eggshell matrix. Additionally, immunohistochemical localization of type I and III collagens was done in order to determine possible co-localization of collagen types or to define tissue compartments. None of the collagen epitopes assayed was found in the shell matrix. Type X collagen epitope was immunohistochemically localized only to the epithelial cell layer lining the isthmus region of the oviduct and in the shell membranes of both uncalcified and calcified eggshells. Antitype III collagen monoclonal antibody delineated the inter-tubular gland connective tissue of the oviduct and was negative in the shell layers under conditions which gave strong connective tissue reactivity. Type I collagen epitope was exposed after pepsin treatment of the tissue and co-localizes with the distribution of type III collagen. Type I collagen co-localized with type X collagen in the shell membranes of uncalcified shells. The type I collagen epitope was reactive in the shell membrane of the uncalcified shells, but could only be detected in calcified shells following pepsin digestion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725805 TI - Distribution of cartilage proteoglycan (aggrecan) core protein and link protein gene expression during human skeletal development. AB - The distribution of cartilage proteoglycan core protein (aggrecan) and cartilage proteoglycan link protein was investigated by in situ hybridization during different stages of human skeletal development. Aggrecan and link protein expression were confined to chondrocytes of the developing skeleton and other cartilaginous structures. Distribution and intensity of the signal was identical with aggrecan as compared to link protein probes. Parallel to the calcification of cartilaginous matrix, chondrocytes of this area lost the expression of aggrecan and link protein specific mRNA and stayed negative throughout the following stages of skeletal development. Highest expression was found in the lower proliferative and upper hypertrophic zone whereas the resting zone showed less expression. Aggrecan gene expression was additionally investigated in iliac crest biopsies of 3 patients with pseudoachondroplasia and compared to age matched controls. Distribution and intensity of staining revealed no abnormalities. Thus, the phenotypic changes during chondrocyte maturation are accompanied by distinct changes in aggrecan and link protein gene expression. This pattern was maintained in the growth plate of patients with pseudoachondroplasia. PMID- 1725806 TI - The effects of capsaicin upon electrogenic ion transport in rat descending colon. AB - Preparations of rat descending colon mucosa have been used to record changes in short circuit current (SCC) under voltage clamp conditions. When added to the basolateral compartment capsaicin (8-methyl-N-vanillyl-6-nonenamide, 0.1-3 microM) caused an initial transient increase in SCC, followed by a more prolonged reduction in SCC, that lasted for 20-30 min. Repeated applications of 3 microM capsaicin caused desensitisation of the initial secretory response. The antisecretory effects (i.e. reduction in SCC from the original baseline) remained, although they were significantly reduced. In some preparations described as "non-responders", 3 microM capsaicin did not elicit a secretory response. No desensitization of the remaining antisecretory responses was observed in these tissues; in fact these reductions in SCC were consistently larger than those from tissues which responded with a secretory response. Tetrodotoxin (100 nM), hexamethonium (10 microM), and yohimbine (50 microM) had no significant effect upon either secretory or antisecretory responses. Ruthenium red (10 microM) abolished the secretory response to 3 microM capsaicin, but had no effect upon the antisecretory responses. Pretreatment of the tissues with 1 microM substance P (SP) resulted in significant desensitisation to the peptide and abolished the secretory response to 3 microM capsaicin. The antisecretory responses remained, and were significantly larger compared with responses from control tissues. PMID- 1725807 TI - 5-Hydroxytryptamine is emetogenic in the house musk shrew, Suncus murinus. AB - The emetic effects of 5-hydroxytryptamine (5-HT) and 5-HT3 receptor agonists were investigated in the house musk shrew, Suncus murinus. 5-Hydroxytryptamine (5-HT; i.p., i.v., s.c.) and 2-methyl-5-HT (2-Me-5-HT; i.p.) but not 5 hydroxyindoleacetic acid (i.p.) or 5-methoxytryptamine (i.p.) induced emesis with very short latency. Tropisetron (ICS 205-930, a 5-HT3 receptor antagonist, s.c.) blocked the emesis induced by 5-HT (10 mg/kg, i.p.) and 2-Me-5-HT (5 mg/kg, i.p.) with respective ID50 values of 7.8 and 70.9 micrograms/kg. Pindolol (5-HT1 receptor antagonist) and ketanserin (5-HT2 receptor antagonist) were about 100 times less potent than tropisetron. The emesis induced by 5-HT was prevented by surgical vagotomy but not by pretreatment with a combination of atropine (0.1 mg/kg, s.c.) and hexamethonium (10 mg/kg, s.c.). These results clearly indicate that 5-HT is emetogenic probably through a stimulation of peripheral 5-HT3 receptors. PMID- 1725808 TI - Hemodynamic study of posterior circulation using hydraulic vascular model- critical stenosis of the vertebrobasilar artery and tolerance to occlusion therapy. AB - A hydraulic vascular model with glass and silicone tubes of the intracranial portion of the vertebro-basilar artery was used to determine the critical stenosis causing vertebrobasilar insufficiency, and the minimum diameter of the posterior communicating arteries (PComAs) necessary to tolerate therapeutic vertebrobasilar occlusion for unclippable aneurysms. The critical stenosis of one vertebral artery (VA) or basilar artery (BA) differed greatly depending upon the anatomical variations of the PComA and the posterior cerebral artery (PCA): 1.14 mm diameter when the artery supplies 80 ml/min to the cerebellum and brainstem only, 1.33 mm when 140 ml/min to these structures and one PCA, and 1.56 mm when 200 ml/min to these structures and both PCAs. The minimum PComA diameter to tolerate therapeutic occlusion depended largely upon the occlusion site: one PComA with 1.54 mm diameter for bilateral VA occlusion and 1.25 mm for BA occlusion distal to the branching of the superior cerebellar arteries. The total volume of collateral flow through both PComAs can be estimated by summing the squares of the diameters. These values cannot be applied rigidly to clinical cases, but are useful standards to evaluate the stenotic lesion or tolerance to occlusion. PMID- 1725809 TI - Expression of MHC class II antigens on human glioma cells modulated by transfection with genes encoding these antigens. AB - Four glioma cell lines not expressing human leukocyte antigen (HLA)-DR or DQ did so after transfection with genes encoding HLA-DR and DQ. The glioma cells had enhanced ability to stimulate allogeneic and autologous responding lymphocytes in the mixed lymphocyte response (MLR). Glioma cells expressing only HLA-DR had weaker MLR enhancement than those expressing both HLA-DR and DQ or only HLA-DQ. Stimulation by transformed glioma cells increased the killing activity of responding lymphocytes against autologous glioma cells 2.0 times. Both HLA-DR and DQ are important in MLR, and the immunogenicity of glioma cells might be increased by transfection with genes encoding these antigens. PMID- 1725810 TI - The role of the paraventricular nucleus and pituitary gland in morphine analgesia. AB - The authors investigated the analgesic effects of small morphine doses injected into the paraventricular nucleus (PVN) of normal rats and hypophysectomized (Hx) rats. An injection cannula was stereotactically inserted into the PVN or third ventricle. On the 5-7th postoperative day, morphine (7 micrograms) was injected and the pain threshold (paw lick latency: PLL) was measured using a hot plate analgesia meter (52.0 +/- 0.1 degrees C). PVN morphine injection caused significantly longer PLL than the control (physiological saline) in both normal and Hx rats. Ventricular morphine injection did not increase PLL over the control. PVN is a site of morphine action. The analgesia induced by PVN morphine injection was not affected by hypophysectomy, or induced by leaking of morphine into the third ventricle. PMID- 1725811 TI - Effect of intracisternal injection of endothelin-1 on regional cerebral blood flow in cats. AB - The effect of endothelin-1 on regional cerebral blood flow (rCBF) was investigated in adult mongrel cats. Endothelin-1 was injected into the cisterna magna and rCBF was measured by the hydrogen clearance method before and every 30 minutes for 180 minutes after the injection. Endothelin-1 (10(-11) mol and 10(-9) mol) induced a significant decrease in rCBF and an increase in arterial pressure. The effects of endothelin-1 on rCBF and arterial pressure were mediated, at least in part, by Ca2+, because pretreatment with intracisternally injected nicardipine prevented the changes. Cerebral angiograms obtained before and after endothelin-1 injection demonstrated severe constriction of the basilar artery, but constriction of the internal carotid and middle cerebral arteries was mild. Since the rCBF measured was in the territory of the middle cerebral artery, the decrease in blood flow was probably not solely due to the artery constriction. Other mechanisms such as arteriole constriction and/or regulation of rCBF by the central nervous system may also occur. PMID- 1725812 TI - Dynamic computed tomography for the evaluation of cerebrovascular disease. AB - Dynamic computed tomography (DCT) was evaluated as a diagnostic indicator for chronic supratentorial ischemia in 50 cases with or without minor neurological deficits. Peak height (PH, the maximum value of the gamma fitted curve), peak time (PT, the time to PH from the start of DCT), transit time (TT, the time between the first and second inflection points of the gamma fitted curve), and their functional maps were analyzed. Cerebral angiography was then performed in all cases to identify stenotic or occlusive vascular lesions in major cerebral arteries. DCT clearly detected 12 of 13 occlusions of the internal carotid artery (ICA) or middle cerebral artery (MCA), although one ICA occlusion was masked by the contralateral MCA occlusion. However, DCT detected only severe ICA or MCA stenosis (more than 90%). Probably, stenotic lesions of less than 90% did not cause detectable hemodynamic compromise. DCT using PH, PT, and TT functional maps is a useful diagnostic method for hemodynamic changes in ischemic cerebrovascular disease, although bilateral lesions and less stenotic lesions (less than 90%) are difficult to detect. PMID- 1725813 TI - Local complications in transbrachial cerebral angiography using the 4-F catheter. AB - Local complications of cerebral angiography using the transbrachial technique were assessed. In 333 patients with cerebrovascular disease 342 catheterizations using a sheathless 4-F catheter were attempted, with 337 successes (98.5%). There were two major and five minor complications (2.1%). A massive hematoma and a pulse deficit were overcome without sequelae. The overall complication incidence was significantly higher in females (5.7%) than in males (0.4%). This procedure requires extra care in female patients. PMID- 1725814 TI - Early MR abnormality indicating functional recovery from spontaneous intracerebral hemorrhage. AB - Magnetic resonance (MR) imaging as an indicator of recovery from hemiparesis was evaluated in 60 patients with spontaneous intracerebral hemorrhage. T2-weighted MR images revealed early MR abnormality (EMA) of the corticospinal tract within 1 week of ictus. Most patients without EMA recovered beyond Brunnstrom's Recovery Stage 3 while only a few patients with EMA did so. Patients with EMA cannot regain motor function because EMA is almost always followed by complete tract degeneration. EMA in the brainstem and poor motor function recovery are closely correlated. PMID- 1725815 TI - Treatment of malignant glioma with high dose intra-arterial ACNU and autologous bone marrow transplantation--case report. AB - A 44-year-old female with malignant astrocytoma received subtotal removal and high dose (200 mg/m2) intra-arterial 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3 (2- chloroethyl)-3-nitrosourea hydrochloride (ACNU) with autologous bone marrow transplantation. Tumor remission with minimal bone marrow suppression was achieved. However, she developed severe encephalopathy and computed tomographic scans revealed a low-density area at the ACNU delivery site. She received glycerol solution to treat the brain edema and recovered completely from the encephalopathy. Intra-arterial ACNU exceeding 200 mg/m2 possibly causes neurotoxicity. PMID- 1725816 TI - Revascularization of the calcarine artery in moyamoya disease: OA-cortical PCA anastomosis--case report. AB - A 38-year-old male presented with moyamoya disease. Occlusion of the bilateral proximal posterior cerebral arteries (PCAs) resulted in homonymous 3/4 (right half and left lower quadrant) anopsia and various mental symptoms. To prevent impending cortical blindness, revascularization to the right PCA was performed by occipital artery (OA) to calcarine artery anastomosis. His neurological state was stabilized. OA-cortical PCA (calcarine artery) anastomosis is an alternative to omentum transplantation or occipital burr hole procedures for impending cortical blindness. PMID- 1725817 TI - Intraventricular hemangiopericytoma--case report. AB - A very rare large intraventricular hemangiopericytoma occurred in a 41-year-old male with a 2-month history of headache and paresthesia of the right shoulder and arm. The tumor was partially removed, followed by 50 Gy local Linac irradiation given over 6 weeks. Four months later the residual tumor demonstrated a marked decrease in size and vascularity. The residual tumor was totally removed with less operative bleeding than at the initial operation. This is the first reported case of hemangiopericytoma located in the trigone of the lateral ventricle. PMID- 1725818 TI - [Function and diseases of peroxisomes]. AB - The properties and metabolic functions of peroxisomes are discussed. Classification and clinical symptoms of various diseases resulting from deficiencies of those organellae are presented. Most of the diseases involve the nervous system. The detection and determination of long-chain fatty acids (containing over 26 carbon atoms) is the principal diagnostic method in peroxisomal diseases. PMID- 1725819 TI - Vaccination with 'concealed' antigens: myth or reality? AB - Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well characterized antigen from the tick gut. Several factors explain how cattle could produce antibody to such antigens, which one would expect to be 'concealed' from the host's immune system, during natural infestation. It has been shown that the carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and that the reaction of bovine antibodies with intrinsic membrane glycoprotein is at least partially blocked by low molecular weight carbohydrate. Further, antisera from cattle exposed to ticks react with a glycosylated, native Bm86 but not with a non-glycosylated, recombinant Bm86. Thus the reaction of concealed antigens with antibodies produced as a result of tick infestation appears to be due to a relatively non-specific reaction with carbohydrate determinants on tick glycoprotein. Evidence is also presented that antibodies directed against carbohydrate determinants of Bm86 are not protective. Care must therefore be exercised in interpreting the results of antibody reaction with glycoproteins in such complex organisms. PMID- 1725820 TI - Antigenic variation in Giardia lamblia: infection of congenitally athymic nude and scid mice. AB - Athymic nude mice of the outbred Zur:ICR-nu and inbred BALB/c strain and scid mice were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7). Changes in the expression of the major surface epitope of the intestinal trophozoites (characterized by the binding capacity of monoclonal antibody MoAbG10/4) as well as cellular and humoral immune parameters of the hosts were followed during the course of infection. Self-cure was observed in heterozygous (nu/+) BALB/c mice by day 22 post-infection (p.i.) and in heterozygous (nu/+) Zur:ICR-nu strain by day 65 p.i. Homozygous (nu/nu) mice of both strains remained chronically infected until end of the experiments (day 45 p.i. for BALB/c mice and day 122 p.i. for Zur:ICR-nu mice, respectively). Only heterozygous (nu/+) mice were able to mount a gut-associated (Peyer's patch) lymphoproliferative response to G. lamblia antigen. Therefore, T-cell dependent mechanisms were necessary for a self-cure. Antigenic variation occurred in all nu/+ and nu/nu animals of both strains. Trophozoites expressing the major surface epitope (assessed by direct immunofluorescence with FITC-labelled MoAb G10/4) decreased to zero by day 22 p.i. In contrast, the proportion of trophozoites expressing the major surface epitope in infected scid mice remained at the initial level (greater than 99%) until termination of the experiment (day 25 p.i.); therefore, antigenic variation did not occur. All nu/nu and nu/+ mice but not scid mice demonstrated a humoral immune response to G. lamblia antigen. These experiments suggest functional B-cell dependent mechanisms are most likely responsible for the surface antigen switch. Transfer of infection occurred naturally from experimentally infected scid-mice to their mother, proving the initial antigenic surface variant remains unchanged after encystment and subsequent excystment followed by infection in a new host. PMID- 1725821 TI - Epitopes of the Plasmodium falciparum clustered-asparagine-rich protein (CARP) recognized by human T-cells and antibodies. AB - Linear B- and T-cell epitopes have been identified in the Plasmodium falciparum clustered-asparagine-rich-protein (CARP). Twenty-six synthetic peptides, 15-25 amino acids in length, were assayed for their ability to stimulate purified, human T-cells primed to P.falciparum by natural infection to proliferate and/or secrete gamma-interferon (IFN gamma). The plasma of malaria exposed individuals were tested for antibody reactivity with peptides coupled to bovine serum albumin in a semiquantitative ELISA. Two of the peptides (NNFMNRNMKNKNMN/NAKNVNDMYRDGEMS) induced T-cells from many malaria exposed donors to proliferate and/or secrete IFN gamma. Six peptides bound antibodies from a large number of the plasma samples, the amounts ranging from ten to more than 200 micrograms specific antibody/ml. T-cell activation was most pronounced when the T-cells were from highly immune donors. In contrast, high anti-peptide specific antibody levels were usually detected in the plasma of less immune donors, recently exposed to infection. One short sequence (NAKNVNDMYRDGEMS) was found to contain both T- and B-cell epitopes. Thus, CARP includes both T- and B-cell reactive elements recognized by the human immune system following exposure to the parasite by natural infection. PMID- 1725822 TI - Schistosoma japonicum (Chinese strain): characterization of two monoclonal antibodies which recognized common epitopes in the tegument of adult worms and schistosomula. AB - Two monoclonal antibodies have been produced that bind to the common epitopes on adult and schistosomula of S. japonicum (Chinese strain). The isotypes of these monoclonal antibodies were determined to be IgM (designated 8G9-5) and IgG2a (designed 9E7) respectively. The target epitopes of 8G9-5 and 9E7, as analysed by immunoblotting assay, are at Mr of 64 kDa and 54 kDa respectively. Indirect immunofluorescence assay using frozen sections of adult worms, intact mechanically transformed schistosomula and 5-day-old lung schistosomula indicated that both epitopes are located in the tegument of both stages of parasite. These epitopes appear to be two of several major immunogenic proteins on the tegument of both young and adult worms. PMID- 1725823 TI - [Rheological behavior of blood substitutes with a perfluorocarbon base]. AB - Oxygen uptake and desoxygenation in blood take place in the capillary system of the organism so that flow characteristics of blood substitutes should be adapted to that of microcirculation. Therefore, appropriate viscosity of blood substitutes is required. In addition to this, studies on the viscosity of blood substitutes made on the basis of perfluorocarbons are also of interest for considering the deemulsificability during circulation. Viscosity measurements carried out on highly polymerized dextran solutions determining the colloid osmotic pressure in blood substitutes, perfluorocarbon emulsions as oxygen carriers, blood as well as mixtures of these components showed that the rheological behaviour of blood substitutes examined did not lead to organic disorders. PMID- 1725824 TI - Effects of insulin on brain monoamine metabolism in the Zucker rat: influence of genotype and age. AB - Disturbances of insulin or brain monoamine metabolism may play a role in the impaired regulation of food intake and body weight in the obese Zucker rat. We investigated a possible insulin-monoamine interaction by measuring monoamine levels in the hypothalamus and striatum of obese (fa-fa) and lean (Fa-Fa and Fa fa) Zucker rats after peripheral insulin administration. The classically reported effects of insulin, i.e., increases in tryptophan, 5-hydroxy-indolacetic acid (5 HIAA) and dihydroxyphenylacetic acid (DOPAC) levels, were observed in the hypothalamus of Fa-Fa and Fa-fa rats, but not in obese fa-fa rats. Given the mechanism of action of insulin, this lack of effect in the obese rats may be related to the peripheral insulin resistance they exhibit. Furthermore, given the role of these monoaminergic systems, this reduced effect may be related to the impaired regulation of food intake and body weight. At 8 wk of age, however, insulin restored the decreased basal 5-HIAA levels observed in the obese rats. Increase in 5-HIAA levels following insulin administration appeared in the striatum of Fa-Fa rats only, suggesting that, as for brain insulin content, other central insulin-related disturbances may be related to the presence of the "fa" gene. In addition, certain effects of insulin on striatal dopamine release were observed in only the Fa-Fa and fa-fa rats, suggesting a particular disturbance related to the heterozygous character. This latter point calls for further investigations on the central dopaminergic effects of insulin. PMID- 1725826 TI - Patterns of cytokeratins and lamins in rat liver and in rat liver cell lines as shown by immunoblotting using the monoclonal antibodies A 45-B/B3 and A 51-B/H4. AB - The cytokeratins and lamins of rat liver nuclei, nuclear lamina, and cytoskeleton preparations as well as of rat liver cell lines were studied by immunoblotting using the monoclonal broad-range cytokeratin antibodies A 45-B/B3 and A 51-B/H4. A 45-B/B3 is bound to 64-, 58-, 55-, 52.5-, 49-, 45-, 43-, 39-, and 35-kDa proteins, which are already known as rat liver cytokeratins, excepted the 64- and 35-kDa bands. This antibody can therefore be considered to be a pan-cytokeratin marker for the rat. A 51-B/H4 detected an epitope occurring in both lamins (70, 67, and 60-63 kDa) and cytokeratins (49, 45, 43, and 39 kDa). In a few experiments, this monoclonal antibody also indicated a 55-kDa protein. Furthermore, in some cell lines a 80-kDa protein was detected which possibly may correspond to a higher molecular member of the lamin family. In subcellular preparations, the full set of cytokeratins was found to be associated with the nuclei and the nuclear lamina fraction, suggesting a tight connection of the intermediate filaments with the nuclear lamina. Cell lines derived from fetal or neonatal rat liver exhibited cytokeratin patterns which resembled the phenotype of immature or atypically differentiated parenchymal liver cells. PMID- 1725825 TI - Modification of gonadectomy-induced increases in brain monoamine metabolism by steroid hormones in male and female rats. AB - Concentrations of monoamines (dopamine, DA; serotonin, 5-HT) and their major metabolites (homovanillic acid--HVA; dihydroxyphenylacetic acid--DOPAC; 5 hydroxyindolacetic acid--5-HIAA) were measured in selected brain areas of chronically gonadectomized, steroid- or oil-treated male and female rats. Concentrations of DOPAC and HVA were markedly increased in the hypothalamus (male, female), striatum (male, female) and brainstem (male) following gonadectomy, whereas the levels of DA remained unaltered in most of the brain areas examined. Most of the changes were reversed or attenuated by chronic estradiol (EB) substitution. In contrast, chronic treatment with physiological concentrations of testosterone (TP) reduced indexes of DA turnover only in the striatum of ovariectomized (OVX) and brainstem of orchidectomized (ORDX) rats. ORDX-related increases in striatal levels of DOPAC and HVA were not reversed by either EB or TP. ORDX increased the levels of 5-HIAA (hypothalamus, striatum) and decreased those of 5-HT (hypothalamus, hippocampus). These changes were reversed by chronic treatment with either TP or EB. Brain metabolism of 5-HT remained unaltered following OVX. Gonadectomy and chronic steroid replacement therapy appear to alter brain monoamine metabolism in a brain region and sex-dependent manner. Our data demonstrate that gonadectomy-related increases in the activity of brain monoaminergic neurons in both male and female rats was attenuated more effectively with physiological concentrations of estradiol than with testosterone. Insensitivity of monoaminergic neurons in a number of brain areas (e.g., hypothalamus, striatum) to the action of testosterone was evident in both sexes. PMID- 1725827 TI - Molecular diversity of cytokeratins: significance for cell and tumor differentiation. AB - Normal and transformed epithelial cells are characterized by the expression of a distinct class of intermediate filaments (IFs), the cytokeratin (CK) filaments. Their constituents, the CKs, comprise a complex multigene family of related proteins which can be subdivided into two sequence types (I and II). In the various epithelial cell types (excluding trichocytes), 19 different CK polypeptides (CKs 1-19) have been distinguished until recently. An additional cytoskeletal polypeptide of Mr 46,000 has been detected, by gel electrophoresis and by immunocytochemistry, in certain types of epithelia including gastric foveolar epithelium, small and large intestinal epithelium, urothelium, and epidermal Merkel cells. On the basis of its biochemical properties and considerable sequence homologies with several type I CKs, this new cytoskeletal protein is suggested to be included in the catalogue of human CKs as CK 20. The various CK polypeptides, as detected by gel electrophoretic and/or immunocytochemical analysis, are expressed in different epithelia and carcinomas in various combinations in a differentiation-dependent manner. Moreover, co expressions of CKs with other IF classes (vimentin, neurofilaments, glial filaments) are also characteristic of certain epithelial differentiation lineages. Both non-neoplastic epithelial alterations as well as malignant transformation may result in similar modifications of the IF expression profiles although there is a considerable tendency of conservativity. In several instances, a reduced degree of differentiation is paralleled by an increased complexity of the IF protein pattern. CK (and IF) typing can be successfully used in the tracing of developmental lineages as well as in the histological differential diagnosis of primary and metastatic carcinomas. Certain CK polypeptides (CKs 5, 6, 14, 16, 17) identify squamous cell carcinomas including poorly differentiated ones, while other CKs, including CK 20, are typical of primary and metastatic urothelium-derived carcinomas. Among simple-epithelial tumors, CK analysis is of potential diagnostic value in the distinction of mesotheliomas from adenocarcinomas, the discrimination of different carcinomas within the gastrointestinal tract, and the distinction of certain gastrointestinal carcinomas from other adenocarcinoma types including breast and lung carcinomas. Among the individual CKs, CKs 5, 7, 13, 14, 19, and 20 appear to be particularly useful for diagnostic purposes. An important task in the future will be the development of additional monospecific CK-antibodies, particularly such which work on routine paraffin sections. PMID- 1725828 TI - Role of Ca2+ and cAMP in rat spermatogenesis--ultrastructural evidences. AB - On the ultrastructural level, the role of Ca2+ and cAMP in the rat spermatogenesis was studied. Using a K-pyroantimonate method for intracellular localization of Ca2+, precipitates were found within (i) the vesicular component of the chromatoid body, (ii) vesicular elements of the trans-Golgi area including coated vesicles, (iii) smooth endoplasmic reticulum vesicles and cisterns accompanying the perinuclear (manchette) microtubules, and (iv) mid-piece mitochondria of germ and Sertoli cells. The results suggest a close relationship between the Ca(2+)-containing smooth endoplasmic reticulum and the control of microtubule assembly within microdomains. Theophylline (a cAMP-phosphodiesterase inhibitor) treatment of rats (96 mg/kg daily i.p. over 5 days) caused a significant increase of (a) the GERL-related coated vesicles and (b) the number of manchette microtubules. PMID- 1725829 TI - Analysis of the gliding, fishtailing and circling motions of native microtubules. AB - In this report we describe the different forms of motile behavior of individual native microtubules from squid giant axons. The three major types of motile behavior of native microtubules are gliding, fishtailing and circling. Gliding, the type of movement observed most often, is the straight translocation of an unbent microtubule segment. Gliding velocities observed in the population ranged from 0.2 to 0.7 microns/s with an average velocity of 0.45 microns/s. The direction of gliding was random with respect to the surface suggesting that physical features of the surface did not influence the direction of gliding. Microtubules are able to glide over objects on the surface and over each other without changing velocity or direction. These observations prove that gliding can continue under conditions where direct contact of the microtubule with the glass surface is not possible along its entire length. When a frontal segment of a microtubule becomes slowed down or attached to the surface, the microtubule begins to fishtail, a process whereby bends form in the frontal part and propagate rearward. The shapes of a fishtailing microtubule resemble that of a beating flagellum. Microtubules with focal attachment near the tip do not propagate bending waves but assume a spiral or circular shape and rotate horizontally (circling). The frontal end of these microtubules stays or rotates in place as pushing forces from the rear turn the microtubule in a circular pattern. An analysis of these data shows that all forms of motion can be explained by pushing forces due to kinesin acting along the length of the microtubule. In an attempt to transport the kinesin-covered cover glass as if it were a big organelle, microtubules translocate themselves in the opposite direction. We estimated the minimum density of force generating enzymes on the surfaces of our preparations as well as that required to maintain active gliding of microtubules. We concluded that the heads of the surface-bound kinesin molecules must display extreme rotatory freedom in order to explain the observed smoothness and straightness of microtubule motion. Few, but usually at least two molecules of kinesin have to work simultaneously to generate the forms of motility observed. PMID- 1725830 TI - Tubulin orientation in microtubules probed with domain-specific antibodies. AB - A panel of monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in microtubules. Limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of tubulin subunits. Antibodies against N-terminal domains were tested for their ability to interfere with the formation of microtubules in vitro. Although the antibodies exhibited similar association constants when tested on immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. The sedimentation assay using microtubules prepared from purified tubulin showed an almost undetectable binding of the antibodies with the strongest inhibitory power to the microtubules. Immunofluorescence staining of unfixed detergent-extracted cells revealed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies against N-terminal domains. The same results were obtained after a microinjection of antibodies into living cells. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of microtubules, considerable regions of the N-terminal domains are not. The surface regions of N-terminal domains appear to be involved in the formation of microtubules. PMID- 1725831 TI - [Novel method to observe the interface between adhesive resin and dentin by staining Fe3+ with tannic acid]. AB - The reaction of Fe3+ and tannic acid was used in a new visual method to confirm the Fe3+ incorporated with dentin during the pretreatment with a 10% citric acid 3% ferric chloride solution or EDTA ferric ammonium salt solution. The combination of Fe3+ and tannic acid was also effective to investigate the state of incorporated resin (resin tags and resin reinforced dentin) with the dentin. The adhered dentin treated by staining with tannic acid was examined. The incorporation of Fe3+ with dentin enhanced the monomer infiltration and resulted in a high bond strength. Addition of 4-META into the MMA-TBB resin increased the resin content in dentin by promoting the rate of monomer diffusion. PMID- 1725832 TI - Role of interferon in clinical practice. AB - Interferons are currently the most widely used biological response modifiers. They are of high clinical value in haematological malignancies (chronic myelogenous leukaemia, multiple myeloma, non-Hodgkin lymphoma), in solid tumours (malignant melanoma, hypernephroma, pancreas neoplasms, carcinoid tumours, Kaposi's sarcoma, glioma, in ovarium, cervix and bladder carcinoma, and in basalioma) and in infectious diseases (chronic hepatitis B, chronic non-A/non-B hepatitis, chronic delta hepatitis, AIDS, Papova virus and Rhinovirus infections, leishmaniasis, leprosy) and some other conditions. Although the mechanism of action of interferons has not been explained in every detail these agents are promising therapeutic means in a number of diseases. PMID- 1725833 TI - Atypical clinical features of hypo- and hyperthyroidism in elderly age. AB - The clinical features of senile hypothyroidism and hyperthyroidism differ from the general characteristics of these conditions. This may be explained primarily by the changed response of peripheral tissues to thyroid hormones. Because of the atypical clinical symptoms the recognition of these conditions meets difficulties and, in general, occurs late. Therapy raises further special questions considering primarily cardiac complications and other accompanying diseases (for instance arteriosclerosis, osteoporosis, diabetes mellitus.). The authors analyse data of 171 hypothyroid and 219 hyperthyroid patients as a function of age, primary disease, and the most often occurring impulse generation, impulse conduction disorders. PMID- 1725834 TI - Diurnal patterns of serotonin, 5-hydroxyindoleacetic acid, tryptophan and fibrinolytic activity in blood of depressive patients and healthy volunteers. AB - Diurnal changes of serotonin-related factors in whole blood and fibrinolytic activity were determined in depressed patients and healthy controls. Whole blood serotonin concentration of depressed patients showed marked changes throughout daytime, with maximum values in the evening and lowest values in the morning, whereas its metabolite 5-HIAA followed a contrary pattern. The circadian rhythm of 5-HT and 5-HIAA in the control group was quite different from depressed patients. Plasma levels of tPA decreased from 12:30 to 16:30. Concentrations of free plasminogen activator inhibitor (PAI-1) and complex of tPA-PAI-1 decreased from 8:30 to 16:30. Plasma levels of total PAI-1 decreased from 8:30 to 16:30. Plasma levels of the fibrinolytic parameters may be lower in depressive patients than in normal controls. These results support the changes in the circadian rhythm of serotonin and its related substances in the blood of depressive patients. PMID- 1725835 TI - Mobilising the appropriate T-cell subset: the immune response as taxonomist? PMID- 1725836 TI - Severe skin reactions to thiacetazone in east Nepal. PMID- 1725837 TI - Oral immunization with Salmonella typhi Ty21a-based clones expressing Vibrio cholerae O-antigen: serum bactericidal antibody responses in man in relation to pre-immunization antibody levels. AB - Previous studies have shown that oral immunization with Salmonella typhi Ty21a based clones expressing Vibrio cholerae O-antigen elicits serum antibody responses against the foreign polysaccharide in human volunteers. These responses are conveniently assayed by complement-dependent bacteriolysis of V. cholerae. In this report the bactericidal responses generated by two such clones are analysed in relation to the pre-immunization titres of various serum antibodies. A significant association was found, in that recipients with higher prevaccine titres of anti-V. cholerae bactericidal antibodies were less likely to register significant bactericidal responses following immunization. These results are discussed in relation to the concept of vaccine exclusion. PMID- 1725838 TI - [The extracorporeal immunostimulating effect of terrilytin in staphylococcal infection]. AB - The intravenous injection of terrilytin-treated lymphocytes into rats infected with staphylococci enhances the formation of staphylococcal alpha antitoxin in the animals and the development of immune response to T-dependent antigen, such as sheep red blood cells (SRBC), but produces no effect on the development of immune response induced by T-independent antigen (lipopolysaccharide). Terrilytin treated lymphocytes induce the release of the factor promoting the development of immune response to staphylococcal antigens and SRBC by spleen cells, incapable of adherence to plastic, but have no influence on the development of immune response to lipopolysaccharide in rats infected with staphylococci. At the same time in such rats spleen cells adhering to plastic take part in the transfer of signals from terrilytin-treated lymphocytes to nonadhering spleen cells of recipients. PMID- 1725839 TI - [The enhancement of the immunogenicity of streptococcal group-A M protein by conjugation with a synthetic polyelectrolyte]. AB - Immunization with the polypeptide fragment of group A streptococcal protein M conjugated with the copolymer of acrylic acid and N-vinylpyrrolidone in complete Freund's adjuvant has been found to lead to a sharp increase in the level of antibodies to the type-specific determinants of protein M, detected in the enzyme immunoassay (EIA). The possibility of the application of such sera to preliminary typing of streptococci in EIA with the use of whole microbial cells as antigens has been shown. The data on high activity of the sera thus obtained in the bactericidal test with streptococci of the homologous type are presented. Recommendations on the use of sera obtained by the above method for highly precise typing of the virulent cultures of group A streptococci in the bactericidal test are given. PMID- 1725840 TI - Biological effects of interferons and their role in onco-hematological conditions. PMID- 1725841 TI - Amylolytic activity of Humicola sp. AB - The thermophilic and cellulolytic fungus Humicola sp. secretes amylase in the liquid culture medium. This activity is induced by starch, maltose and cellobiose. Glucose impairs accumulation of amylolytic activity in the culture medium. The enzyme hydrolyzes starch, maltose and pullulan to glucose as the end product. PMID- 1725842 TI - [Characteristics of the family structure of school children with antecedents of severe and early malnutrition which nowadays present different intellectual levels]. AB - Children who present early and severe malnutrition show a deficit in their psychomotor development and specific behaviour disturbances which are recovered to a large extent when receiving integral rehabilitation. During their growth and development process, these children have differences in their intellectual development that cannot be explained as a consequence of the nutritional deficit alone. Therefore, the purpose of this study was to describe and compare from a systemic perspective, some characteristics of the family structure of these children with a history of early and severe malnutrition and who showed a differing intellectual performance at school age. Seven families with infants who had been admitted and treated in a Closed Nutritional Recovery Center of the Corporation for Infantile Nutrition (CONIN) were studied. All the children showed a moderate delay in their psychomotor development on admission, but all had significantly recovered at their discharge. At school age, in four families, the index subject presented an intellectual capacity of greater than or equal to 70 less than 80 and in the other three, children showed an intelligence quotient greater than or equal to 85. Each family was studied through a semi-structured interview held at the family's home with participation of all its members. Each interview lasted approximately one hour and was filmed with an audio-video equipment. The interviews were later coded using a checklist of behaviour indicators for family interaction. The results revealed differences between the two family groups, both with respect to their parental sub-systems and also in the relations established between the parents and those children with a history of early and severe malnutrition, especially with respect to the paternal sub system and their index children. The observed differences between the two groups centered mainly on the amount of help that parents give to their children with a history of malnutrition, and the orders given to them. The results obtained are discussed in relation to sexual roles in low-income families and their relation to socioeconomic conditions. PMID- 1725843 TI - [Amylase 2 phenotypes in Central Hessia and value in paternity assessment]. AB - In 677 unrelated persons from the Central Hessian region, the following distribution of amylase 2 phenotypes was found: type 1 in 89.22%, type 2-1 in 8.57%, type 2 in 0.30%. The special bands 3 and 4 could be detected in 0.59% and the new bands F1-6 (on the anodal side of the 1 band) we have described could be demonstrated in 1.33%. In 244 cases, paternity could be excluded on the basis of amylase 2 in seven cases (i.e. 2.9%); this was a classic exclusion in six cases. In the biostatistical calculation of the probability of paternity, this enzyme system provides probabilities which justify routine determination of amylase 2. PMID- 1725844 TI - [Code of codon roots of amino acids and idiotypic networks]. AB - Novel models of idiotype nets of antibodies have been developed to study the code responsible for the amino acid interaction and complex formation of proteins. It is shown that the interaction of protein active centres in idiotype nets can be interpreted and predicted basing on the structure of code of codon roots of amino acids and polarity principle. "Internal images" of the sequence antigen determinants of proteins in immunoglobulin molecules are built mainly from the amino acid groups having common codon roots, which is in agreement with the conception of the structure of the root code. PMID- 1725845 TI - [Synthesis of peptides of the preS1-region of the viral hepatitis B envelope protein and localization of antigenic determinants]. AB - We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides. PMID- 1725846 TI - [Trityl-cyanoethylidene condensation of azidosaccharide derivatives as a route to hexosaminoglycans. Synthesis of the O-antigenic polysaccharide of Pseudomonas aeruginosa X (Meitert)]. AB - Derivatives of azidosugars were shown to be stable under conditions of trityl cyanoethylidene condensation. Tritylated 1,2-O-(1-cyano)ethylidene derivative of 2-azido-2-deoxy-beta-D-mannopyranosyl-(1----4)-L-rhamnopyranose was used as a starting material for the synthesis of [----3)-beta-D-ManNAc-(1----4)-alpha-L-Rha (1----]n, the O-specific polysaccharide of Pseudomonas aeruginosa X (Meitert). PMID- 1725847 TI - Immunopathology of urticaria. AB - Actual concepts of urticaria immunopathology are briefly discussed, emphasizing the cytokine and inflammatory cells network. PMID- 1725848 TI - [Sensitivity and specificity of alpha-1-antitrypsin and acid alpha-1-glycoprotein in colorectal carcinoma]. AB - In a group of 80 patients with colorectal cancer and a control group of 91 persons and in 15 patients with acute diverticulitis of the sigmoid colon acid alpha-1-glycoprotein (AGP) and alpha-1-antitrypsin (AAT) were measured. The median of AAT in the carcinoma group was 3.34 g/l, in the control group 2.39 g/l. AGP was in the carcinoma group 1.19 g/l, in the control group 0.79 g/l. Both differences are significant. The sensitivities of AGP and AAT were compared with the sensitivities of CEA and CA 19/9 using ROC-curves. AAT and AGP have a distinctly lower sensitivity than CEA, but only on the basis of a high specificity of 95%. In the region of lower specificities AAT has the highest sensitivity. Therefore, the sensitivity of CEA for colorectal carcinoma is not reached by AAT and AGP, when a high specificity of the test is required. The relevance of AAT and AGP determinations is further reduced because in the diverticulitis group the levels were as high as in the carcinoma group. PMID- 1725849 TI - Influence of aprotinin and promazine on survival of isolated pancreatic acinar cells. AB - Aprotinin, a protease inhibitor, and promazine, an inhibitor of phospholipase A2, were tested for possible inhibition of pancreatic acinar cell (PAC) decline induced by uncoupling of oxidative phosphorylation with 2,4-dinitrophenol (DNP) or by temporary anoxia/reoxygenation. In incubates of acinar cells isolated from rat pancreas the presence of aprotinin did not influence the survival of cells treated with these noxae. This finding excludes that extracellulary acting trypsin, possibly released from damaged cells, contributes to further cell death. While promazine at concentrations of 15 to 20 nmol.(10(6) cells)-1 was well tolerated by untreated PAC, higher concentrations caused a clear reduction of cell viability. At optimum concentration promazine was without influence on DNP treated cells, but it had a beneficial effect on survival and morphology of anoxia-treated PAC (p less than or equal to 0.05). Therefore, it can be assumed that after anoxia/reoxygenation the membrane phospholipase A2 becomes stimulated and causes phospholipid depletion with final death of the cells. It is suggested that such a mechanism may contribute to the initial cell damage in the pathogenesis of acute pancreatitis, too. PMID- 1725850 TI - [Effect of gentamicin on the concentration of calcium, magnesium and zinc in perilymph of the guinea pig]. AB - The calcium, magnesium and zinc ions in perilymph of the guinea pig were measured after hearing loss induced by gentamicin. The concentration of the calcium ions and magnesium ions increased, the zinc ions decreased in perilymph. The ototoxicity of gentamicin was related to the changes of these electrolytes in inner ear circumstance. The results showed that the changes of ions were the step of gentamicin ototoxicity. PMID- 1725851 TI - Hemopoiesis stimulating and radioprotective effects of carboxymethylglucan. AB - Carboxymethylglucan, a novel soluble derivative of beta-1,3-glucan, was found to enhance hemopoietic recovery in sublethally gamma-irradiated mice and to increase survival in lethally irradiated animals when given 24 hours prior to irradiation. Postirradiation treatment with carboxymethylglucan also induced favourable effects in terms of survival when used in combination with preirradiation cystamine administration. PMID- 1725852 TI - A quick-mixed aluminum hematoxylin stain. AB - Two stock solutions are composed as follows: A) aluminum sulfate, sodium iodate and acetic acid in aqueous propylene glycol and B) hematoxylin in pure propylene glycol. When combined in specified proportions the stock solutions yield aluminum hematein dissolved in nontoxic propylene glycol. The ready-to-use stain, prepared in small volumes as needed, performs well in paraffin sections of plant tissues. PMID- 1725853 TI - The standard Romanowsky-Giemsa stain in histology. AB - A new and technically simple Romanowsky-Giemsa (RG) stain is proposed as a standardized technique for use in histology. An RG stock solution (pure azure B 7.5 g/l, eosin Y as eosinic acid 1.2 g/l in dimethylsulfoxide) is diluted to form the working solution with HEPES-buffer, pH 6. Staining time is 30-90 min after formol-calcium solution (or 2-4 hr after formaldehyde-organic acid mixtures). The resulting overstained sections are to be differentiated. A tannic acid-acetic acid combination in an isopropanol-water mixture was found to give optimum results within 100 sec. Subsequent dehydration is in isopropanol only. The staining pattern obtained is polychrome. The distribution of colors in detail is influenced by the modes of pre- and posttreatment. Of practical interest is the development of green and greenish blue colors on collagen fibrils which contrast strongly against the pink of sarcoplasm. For this and other reasons, this RG stain version seems suitable to replace the trichrome Gomori-type trichrome stains under appropriate processing conditions. PMID- 1725854 TI - DAPI as a useful stain for nuclear quantitation. AB - A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples. PMID- 1725855 TI - Differentiation of cancellous bone and medullary bone in laying hens: a novel technique for image analysis. AB - A selective staining technique for the identification and differentiation of cancellous bone from medullary bone of the laying hen by image analysis is described. Undecalcified Polymaster resin sections were oxidized in acidified potassium permanganate and oxalic acid before being immersed in an ammoniacal silver solution. The sections were reduced in formalin, fixed in sodium thiosulfate and counterstained in naphthalene black 10B which was dissolved in picric and acetic acids. Intensely stained cancellous bone was prominent with this technique compared with a paler medullary bone component which permitted the former to be easily recognized and measured by image analysis. PMID- 1725856 TI - Decontamination of aqueous solutions of biological stains. AB - Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins. Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions. Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16. Reaction times varied from 10 min to 18 hr. Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16. Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium. None of the completely decontaminated solutions were found to be mutagenic. PMID- 1725857 TI - A new stain for pollen fertility studies. AB - The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P.H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive. PMID- 1725858 TI - Biochemical and immunological identification of human neutrophil elastase on nitrocellulose membranes. AB - Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils. PMID- 1725859 TI - Characterization of the gene-product of the Steel locus. AB - Steel factor (SLF) (aka: mast cell growth factor, stem cell factor, kit ligand) is the product of the murine Steel locus on chromosome 10 and is a ligand for the c-kit protooncogene. Isolation of cDNAs for SLF revealed that it was a membrane bound growth factor. Proteolytic processing releases a soluble version of the growth factor which has been shown to promote a wide variety of biological functions. In this review we focus on the cellular and molecular biology of SLF. PMID- 1725860 TI - Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6). AB - Six different insulin-like growth factor binding proteins (IGFBPs) have been identified by molecular cloning of their cDNAs from rat and human tissues and designated as IGFBP-1, -2, -3, -4, -5 and -6. The total number of amino acid residues for the mature rat BPs ranges from 201 for IGFBP-6 to 270 for IGFBP-2, while the human homologs range from 216 for IGFBP-6 to 289 for IGFBP-2. Except for IGFBP-6, all rat and human IGFBPs contain 18 homologous cysteines; twelve are located at the N-terminal and span approximately one-third of the total amino acid sequence, while the remaining six are distributed at the C-terminal and span the last one-third of the protein sequence. Both rat and human IGFBP-4 possess two extra cysteines at the mid-region of the molecule. By contrast, rat and human IGFBP-6 contain only 14 and 16 cysteines, respectively. Absence of the two and four cysteines in the N-terminal region in the human and rat IGFBP-6 resulted in the deletion of the invariant Gly-Cys-Gly-Cys-Cys sequence which is present in all the other five IGFBPs. Both rat and human IGFBP-3 possess multiple N-linked glycosylation sites at the mid-region of the molecule, which accounts for their apparent molecular size being larger than the calculated molecular weight, based on the amino acid sequence. One potential N-linked glycosylation site is located at the mid-region of rat and human IGFBP-4, whereas only human but not rat IGFBP 6 possesses one N-linked glycosylation site at the extreme C-terminal of the molecule. An RGD sequence is found in the C-terminal of IGFBP-1 and -2. In this short review, updated information on the structural identification and molecular cloning of the six IGFBPs will be presented. In addition, the potential regulation of the BPs at the transcriptional and translational levels will be discussed. PMID- 1725861 TI - Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum). AB - Adult rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with capsaicin, substance P, serotonin, or a control of saline vehicle or bovine serum albumin (0.5 microgram/g body weight). Fish were sacrificed 30 min and 1, 2, and 4 h post-injection, the gut was dissected out, and a small section of the upper intestine was processed for electron microscopy. A significant proportion of eosinophilic granule cells (EGCs) of the intestine were in close association with non-myelinated neuronal bundles in all fish (4 fish per treatment and time period), but there was no significant difference between treatment or time, suggesting that the association was unaffected by these factors. Close examination of EGC ultrastructure showed that fish treated with capsaicin and substance P exhibited limited degranulation of the EGCs in the stratum compactum and extensive crinophagic-like degranulation in the lamina propria. Cells of the lamina propria contained characteristic multivesicular-like bodies. The degranulation was reminiscent of both mast cell degranulation and endocrine cell crinophagy. EGCs of fish treated with serotonin or a control were unaffected, suggesting that the serotoninergic neurons, believed to be involved in gut motility, were not responsible for degranulation. It is apparent that EGCs of the trout intestine may be under nervous control, as has been demonstrated previously for mammalian mast cells. PMID- 1725862 TI - Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins. AB - The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick beta 1 subunit of integrin, suggesting that beta 1-integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (greater than 1 microgram ml-1; Low-LM), but not at high coating concentration of laminin (10 micrograms ml-1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required greater than 0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the beta 1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of beta 1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin. PMID- 1725863 TI - [Treatment of keratocysts in deciduous and mixed dentitions]. AB - A survey is given of patients with odontogenic keratocysts in childhood and adolescence which were treated at the Clinic for Maxillofacial Surgery Tubingen in the last 10 years. Odontogenic keratocysts occurring in the growth period are treated in most cases by enucleation or partial marsupialization. To avoid damaging the nerve or dental germs, fixation with Carnoy's solution is not performed. In a second part an experimental study dealing with the effects of Carnoy's solution on peripheral nerves is presented. After incubating the sciatic nerve of rats with Carnoy's solution lumbar SEP, M-response and Hoffmann's reflex were recorded. It was possible to demonstrate the detrimental effect of Carnoy's solution on peripheral nerves. After incubation periods of more than 2 minutes almost complete functional loss was observed. There was a strong correlation between electrophysiological and histological findings. PMID- 1725864 TI - Studies on the mechanism by which galanin inhibits insulin secretion in islets. AB - The mechanism by which the neuropeptide galanin inhibits insulin secretion in normal islets is not yet fully elucidated. Isolated rat or mouse islets were perifused in a medium containing glucose (8.3 mM) and galanin (10(-6) M) or the sulphonamide diazoxide (400 microM). In rat islets prelabelled with 86Rb+ or 45Ca2+, galanin inhibited glucose-induced insulin secretion at the same time as increasing 86Rb+ efflux and reducing 45Ca2+ efflux. The diazoxide-induced 86Rb+ efflux was not affected by galanin, indicating that galanin activates ATP regulated K+ channels in rat islets. In mouse islets prelabelled with 86Rb+, galanin (10(-6) M) decreased 86Rb+ efflux. These results suggest that galanin inhibits insulin release in isolated islets by increasing K+ and decreasing Ca2+ permeability. The increased K+ permeability, which is probably regulated differently in rat and mouse islets, is followed by a reduced Ca2+ influx, possibly through voltage-dependent Ca2+ channels. In addition, during a 60-min incubation with isolated islets, galanin inhibited insulin secretion induced by forskolin (1 microM), dibutyryl cyclic AMP (1 mM), or TPA (12-O tetradecanoylphorbol-13-acetate; 0.1 microM). Galanin also reduced the content of cyclic AMP in islets stimulated by 16.7 mM glucose. We therefore conclude that the inhibitory action of galanin on insulin secretion in normal islets includes increasing K+ permeability as well as interference with the activation of adenylate cyclase and the activity of protein kinase C and cyclic AMP. PMID- 1725865 TI - Muscarinic inhibition of pancreatic B-cells. AB - Muscarinic agonists are known to potentiate insulin secretion and increase glucose-induced firing of pancreatic B-cells. Here we report two experimental situations in which inhibitory effects of muscarinic agonists may be observed. (1) Muscarinic agonists delay the onset of the 11.1 mM glucose-induced cell depolarization. (2) At concentrations between 10(-9) and 10(-7) M, acetyl-beta methylcholine (and also bethanechol and MCN-a-343) decreases cell input resistance and decreases insulin release of islet cells exposed to 5.6 mM glucose. PMID- 1725866 TI - Alteration of cholecystokinin receptor binding after caerulein-induced pancreatitis in rats. AB - The alteration of CCK receptor binding on the pancreatic acini in vitro following the induction of pancreatitis was investigated in male rats. Pancreatitis was induced by administering 5 intraperitoneal injections of caerulein, 40 micrograms/kg each at hourly intervals. The uptake of [3H]-thymidine in the pancreatic acini increased on day 7 following caerulein administration. The release of amylase stimulated by CCK-8, and CCK receptor analysis using bioactive [125I]-BH-CCK-8, were performed at regeneration stage, on days 14 and 28 following the injections. The maximal release of amylase stimulated by CCK-8 was reduced on day 14 by about 40% and recovered on day 28. On day 14 there was a decrease of 60% in the number of high-affinity receptors and an increase of 161% in the number of low-affinity receptors. On day 28 there was a 128% increase in the number of low-affinity receptors. Accordingly, we suggest that the CCK receptors of the regenerating cells following caerulein-induced pancreatitis differ from those of the intact cells. PMID- 1725867 TI - Conjugation of proteins and enzymes with hydrophilic polymers and their applications. AB - The efficacy of a number of therapeutically active proteins and peptides is severely limited due to their instability in circulation. Of the various approaches used to stabilise these proteins, the one more successful is covalent modification of the protein or enzyme with some hydrophilic polymers such as dextran or PEG. These conjugates are more stable than the native protein both in vitro as well as in vivo. They exhibit enhanced resistant to proteolytic degradation, have a long-life in circulation and exhibit reduced immunogenicity. The therapeutic efficacy of these conjugates is also greatly enhanced compared to the native protein or enzyme. PMID- 1725868 TI - Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally. AB - The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori. PMID- 1725869 TI - Histologic evaluation of dental endosseous implants placed in surgically created extraction defects. PMID- 1725870 TI - Analysis of neural crest cell lineage and migration. AB - In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo. PMID- 1725871 TI - Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors. AB - Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis. PMID- 1725872 TI - Molecular determinants during dental morphogenesis and cytodifferentiation: a review. AB - Craniofacial development provides a number of opportunities to investigate the cellular and molecular biology of morphogenesis, cytodifferentiation, tissue specific extracellular matrix (ECM) formations, and biomineralization. Regulatory processes associated with mandibular morphogenesis and specifically tooth formation are being investigated by the identification of when and where molecular determinants such as cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), and tissue-specific structural gene products are expressed during sequential developmental stages. Based upon in vitro organotypic culture studies in serumless, chemically defined medium, instructive and permissive signaling has been found to be required for both mandibular and dental morphogenesis and cytodifferentiation. For example, intrinsic developmental instructions (autocrine and paracrine factors), independent of long-range hormonal or exogenous growth factors, mediate morphogenesis from the initiation of the dental lamina through crown and initial root stages of tooth development. This review summarizes recent results using experimental embryology, organ culture, recombinant DNA technology, and immunocytology to elucidate mechanisms responsive to instructive epithelial-mesenchymal interactions associated with mandibular morphogenesis, tooth positional information, and subsequent tooth crown and initial root development. PMID- 1725873 TI - Larval susceptibility status of Culex (culex) gelidus Theobald, against four organophosphorus compounds in Mysore City. PMID- 1725874 TI - EM Golgi study on neurons of ectostriatum centrale in telencephalon of one-day old chicken's brains. AB - In Golgi impregnated ectostriatum centrale of telencephalon of one-day-old chicken two types of neuron: projection neuron and interneuron with large axonal field and varicose axonal preterminals and terminals, were identified and examined in EM preparates. In the serial EM sections the morphological features of the two neurons types were observed. The projection neuron is characterized by the few axo-somatic synapses on the cell membrane, which are of symmetrical type. The INs, however, have numerous axo-somatic synapses on cell membrane both of symmetrical and asymmetrical types. Also the axonterminals were analysed. PMID- 1725875 TI - Microvascular density, microvascular surface area and endocrine cell volume in pituitary transplants to the fourth ventricle of adult rats. AB - Pituitary gland endocrine tissue was transplanted to the fourth ventricle of the brain of adult rats. The grafts were left in place for eight to ten weeks. One micron thick Epon sections were cut for microscopical and morphometrical analysis. The microvascular length density, the microvascular surface area density and the volume weighted mean pituitary cell volume were measured. The transplants were connected to the recipient brains through a few rather large vessels vessels either from the arachnoidea or the choroid plexus. Neuronal connections were never established. The microvascular length and surface area densities were significantly increased in the grafts, whereas the weighted mean cell volume was reduced. The results indicate that pituitary tissue can be transplanted to the fourth ventricle of the brain and the blood supply reestablished. The increased vascular densities are due to lower cell volume and probably also to cell loss and not to proliferation or angiogenesis. The reduced pituitary cell volume may be the result of missing trophic factors which normally are secreted to the pituitary in the sella turcica. PMID- 1725876 TI - Projections from neurons innervating the masseter muscle to the subnucleus oralis of the spinal trigeminal nucleus and adjacent lateral reticular formation in the rat. AB - The projection from the masseter to the subnucleus oralis of the spinal trigeminal nucleus (Vo) and adjacent parvicellular reticular nucleus (PCRt) was studied with retrograde and transganglionic transport of horseradish peroxidase (HRP) combined with fluorescent double labeling method. The labeled axons and terminals within the medulla showed a specific distribution. The field of labeling formed a continuous band from rostro-laterally to caudo-medially. At the level of caudal Vo the labeling region became the most expanded in the medulla, which occupied dorso-medial Vo (Vodm) and adjacent PCRt about half by half. The ratio of masseter primary afferent neurons in the mesencephalic trigeminal nucleus (Vme) to that in the trigeminal ganglion (TG) was found to be 7.7-8.5:1 following injection of HRP into the masseter nerve. However, the proportion between the Vme and the TG neurons which build up the pathway from the masseter to the Vodm and the adjacent PCRt was 8.7-10:1, revealed by means of fluorescent double labeling when different tracers were applied at the peripheral and central targets. PMID- 1725877 TI - Normalization of protein synthesis and the structure of brain dystrophic neurons after the action of hypoxia, 10% NaCl and organ-specific RNA. AB - It was shown previously (Polezhaev and Alexandrova, 1986) that hypoxic hypoxia causes mass (up to 30%) diffuse dystrophy of brain cortex and hippocamp neurons in rats, disturbances in the higher nervous activity, reduction of protein, RNA synthesis in neurons and of DNA synthesis in the whole brain cortex. Transplantation of embryonic nervous tissue (ENT) in one of the hemispheres normalizes all the above abnormalities observed in some neurologic and mental diseases in humans. However, transplantation may entail injuries of parenchyma and brain blood vessels. This forces researchers to search for another biological method similar by its action but safer and simpler. ENT transplantation has a dual action: 1) formation of biologically active substances (BAS) releasing from the ENT transplant and from the host brain nervous tissue upon operation; 2) establishment of synaptic connections between the transplant and host neurons. Previously we (Vitvitsky, 1987) described the isolation of BAS from rat forebrain in the form of organ-specific RNA. The latter was injected intraperitoneally several times to post-hypoxic rats in which 30 min prior to that the blood-brain barrier (BBB) was opened by injecting intravenously and intraperitoneally 10% NaCl solution without damaging the host brain. At the beginning 10% NaCl increased the destruction of brain cortical neurons and then stimulated protein synthesis in them. RNA injections stimulated the synthesis in cortical neurons and normalized their structure. Thus, we propose a safe and simple method for normalization of dystrophic neurons which can be used after certain improvement for curing neurodegenerative and neuropsychic diseases in humans. PMID- 1725879 TI - Simulation of the bursting activity of neuron R15 in Aplysia: role of ionic currents, calcium balance, and modulatory transmitters. AB - 1. An equivalent circuit model of the R15 bursting neuron in Aplysia has been combined with a fluid compartment model, resulting in a model that incorporates descriptions of most of the membrane ion channels that are known to exist in the somata of R15, as well as providing a Ca2+ balance on the cell. 2. A voltage activated, calcium-inactivated Ca2+ current (denoted the slow inward current ISI) was sufficient to produce bursting activity without invoking any other calcium dependent currents (such as a nonspecific cation current, INS, or a calcium activated K+ current, IK,Ca). Furthermore, many characteristics of a typical R15 burst could be simulated, such as a parabolic variation in interspike interval, the depolarizing afterpotential (DAP), and the progressive decrease in the undershoots of spikes during a burst. 3. The dynamic activity of R15 was analyzed by separately characterizing two different temporal domains; the fast dynamics associated with action potentials and the slow dynamics associated with low amplitude oscillations lasting tens of seconds ("slow waves"). The slow dynamics were isolated by setting the Na+ conductance (gNa) to zero and then studied by the use of a system of equations reduced to two variables: intracellular concentration of Ca2+ and membrane potential. The fixed point of the system was located at the intersection of the nullclines for these two variables. A stability analysis of the fixed point was then used to determine whether a given set of parameters would produce slow-wave activity. 4. If the reduced model predicted slow-wave oscillations for a given set of parameters with gNa set to zero, then bursting activity was observed for the same set of parameters in the full model with gNa reset to its control value. However, for certain sets of parameters with gNa at its usual value, the full model exhibited bursting activity because of a slow oscillation produced by the activation of INS by action potentials. This oscillation resulted from an interaction between the fast and slow dynamics that the reduced model alone could not predict and was not observed when gNa was subsequently set to zero. If gNS was also set to zero, this discrepancy disappeared.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725878 TI - Biological activity of novel macrocyclic alkaloids (budmunchiamines) from Albizia amara detected on the basis of interaction with DNA. AB - Extracts derived from Albizia amara were found to demonstrate activity in a recently developed hplc system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. All four isolates interacted with calf thymus DNA and were generally cytotoxic with a battery of cultured mammalian cells. As determined with Salmonella typhimurium strain TM677, isolates X1 and X3 were bactericidal, but not mutagenic. Isolate X1 was found to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. With DNA polymerase, the reaction was shown to be inhibited in a manner that was competitive with respect to DNA. In addition, isolate X1 inhibited each of the following: platelet aggregation, human lymphocyte transformation, phorbol-ester-induced chemiluminescence with human granulocytes, and cyclooxygenase activity. Detection of these alkaloids on the basis of their interaction with DNA exemplifies the validity of this approach. PMID- 1725880 TI - Discharge characteristics of medial rectus and abducens motoneurons in the goldfish. AB - 1. The discharge of antidromically identified medial rectus and abducens motoneurons was recorded in restrained unanesthesized goldfish during spontaneous eye movements and in response to vestibular and optokinetic stimulation. 2. All medial rectus and abducens motoneurons exhibited a similar discharge pattern. A burst of spikes accompanied spontaneous saccades and fast phases during vestibular and optokinetic nystagmus in the ON-direction. Firing rate decreased for the same eye movements in the OFF-direction. All units showed a steady firing rate proportional to eye position beyond their recruitment threshold. 3. Motoneuronal position (ks) and velocity (rs) sensitivity for spontaneous eye movements were calculated from the slope of the rate-position and rate-velocity linear regression lines, respectively. The averaged ks and rs values of medial rectus motoneurons were higher than those of abducens motoneurons. The differences in motoneuronal sensitivity coupled with structural variations in the lateral versus the medial rectus muscle suggest that symmetric nasal and temporal eye movements are preserved by different motor unit composition. Although the abducens nucleus consists of distinct rostral and caudal subgroups, mean ks and rs values were not significantly different between the two populations. 4. Every abducens and medial rectus motoneuron fired an intense burst of spikes during its corresponding temporal or nasal activation phase of the "eye blink." This eye movement consisted of a sequential, rather than a synergic, contraction of both vertical and horizontal extraocular muscles. The eye blink could act neither as a protective reflex nor as a goal-directed eye movement because it could not be evoked in response to sensory stimuli. We propose a role for the blink in recentering eye position. 5. Motoneuronal firing rate after ON-directed saccades decreased exponentially before reaching the sustained discharge proportional to the new eye position. Time constants of the exponential decay ranged from 50 to 300 ms. Longer time constants after the saccade were associated with backward drifts of eye position and shorter time constants with onward drifts. These postsaccadic slide signals are suggested to encode the transition of eye position to the new steady level. 6. Motoneurons modulated sinusoidally in response to sinusoidal head rotation in the dark, but for a part of the cycle they went into cutoff, dependent on their eye position recruitment threshold. Eye position (kv) and velocity (rv) sensitivity during vestibular stimulation were measured at frequencies between 1/16 and 2 Hz. Motoneuronal time constants (tau v = rv/kv) decreased on the average by 25% with the frequency of vestibular stimulation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1725881 TI - Successful tracheal autotransplantation with a vascularized omental flap. AB - A major problem in tracheal transplantation is the restoration of an adequate vascular supply to the transplanted trachea. In 12 piglets, a segment (6 rings) of thoracic trachea was removed and the excised segment was then sutured back in place. In 9 animals (group A), a vascularized omental flap was wrapped around the autotransplanted trachea. In the other 3 pigs (group B), the omentum was not used. Eight of 9 group A pigs were killed, 1 or 2 months later, having had no signs of airway obstruction; the 9th pig was killed after 14 days because of airway obstruction. The 3 pigs in group B were killed after 11 to 13 days because of progressive respiratory obstruction. In the 8 asymptomatic pigs in group A, the omental flap was viable and tracheal growth was normal with no differences in diameter between normal and autotransplanted trachea. Histologically intact cartilage was lined with respiratory epithelium. In the one group A pig who was killed early, the omental flap was necrotic. In this pig and in the 3 group B animals, extensive tracheal necrosis and nonviable cartilage were observed. These findings indicate that in the pig, a 6-ring segment of trachea can be transplanted with vascularization provided by an omental flap. PMID- 1725882 TI - The metabolic effects of guanyl nucleotides on rat pancreatic acini permeabilized with streptolysin O suggest a widespread use of G proteins. AB - In streptolysin O permeabilized acini that were normally responsive to carbamylcholine and cholecystokinin octapeptide, amylase secretion was stimulated: a) by the stable guanyl nucleotides with a potency decreasing as follows: guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanylyl imidodiphosphate (GMP-PNP) = guanylyl-(beta, gamma-methylene)-diphosphonate (GMP PCP), in the presence of 0.5 mM calcium and b) by calcium alone (EC50 3 microM). The maximal secretory effect of calcium alone (a 2-fold increase) was less effective than that of GTP gamma S and calcium offered in combination (an 8-fold increase). In the virtual absence of Ca2+, GTP gamma S still stimulated amylase release (a 3-fold increase) while 12-O-tetradecanoylphorbol 13-acetate (TPA) did not. The relative potencies of guanyl nucleotides were GTP gamma S greater than GMP-PNP = GMP-PCP = GTP on phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, GTP gamma S greater than GMP-PNP greater than GMP-PCP = GTP on 45Ca2+ efflux, and GTP GMP-PNP = GMP-PCP = GTP gamma S on [1-14C]arachidonate efflux. Based on these data, the contribution of G proteins to stimulus-secretion coupling beyond the transduction of receptor signal is considered. PMID- 1725883 TI - Effects of recombinant human granulocyte colony-stimulating factor on neutrophil function in vitro and in vivo following chemotherapy and autologous bone marrow transplantation. AB - Recombinant granulocyte colony-stimulating factor (rG-CSF) primed the ability of human neutrophils to generate increased levels of reactive oxidants in response to fMet-Leu-Phe, and also resulted in an increased rate of protein biosynthesis which was similar to that induced by granulocyte-macrophage colony-stimulating factor. However, rG-CSF reduced the chemotactic activity of neutrophils in response to endotoxin and did not result in an enhanced rate of killing of Staphylococcus aureus. rG-CSF was administered to patients after high dose chemotherapy and autologous bone marrow transplantation for the treatment of either Hodgkin's disease or multiple myeloma. This cytokine decreased the period of neutropenia following such treatment. Neutrophil function in two patients, measured seven days after the final administration of rG-CSF, was severely impaired as indicated by a greatly decreased ability to generate reactive oxidants. However, seven days later (i.e. 14 days post-therapy), the functional activity of the neutrophils from these patients had returned to normal. These data indicate that assays of neutrophil function together with morphological assessment of neutrophil numbers and maturity should be performed in order to evaluate the immune status of patients undergoing such therapy. PMID- 1725884 TI - Fatty acid profile and acid phosphatase activity of fresh isolates of Pseudomonas pseudomallei. AB - Eighty-one fresh isolates of Pseudomonas pseudomallei from melioidosis patients were subjected to the analysis for the fatty acid composition by gas-liquid chromatography (GLC) and pH-dependent pattern of nonspecific phosphatase activity. All the test strains were identical in the GLC profile showing the three peaks of characteristic hydroxy acids (3-OH 14:0, 2-OH 16:0, 3-OH 16:0) and the two prominent peaks of cyclopropane acids (17:0 delta, 19:0 delta). They had also basically the same pH-dependent curves of the enzymatic activity with paranitrophenyl phosphate as substrate, showing two to three peaks or shoulders only in the acidic side of the curve. These two biochemical characteristics could differentiate P. pseudomallei distinctly from P. aeruginosa, but not from P. cepacia. PMID- 1725885 TI - Demonstration of acid phosphatase activity in antigenic glycoprotein fractions obtained from the culture filtrate of Pseudomonas pseudomallei. AB - Pseudomonas pseudomallei, the causative microorganism of melioidosis, was grown in Mueller-Hinton liquid medium, and glycoprotein fractions were separated from the culture filtrate by ammonium sulfate precipitation, gel-filtration with Sephadex G-75, and column chromatography with DEAE-cellulose. The fractions revealed acid phosphatase activity, and reacted to the sera from melioidosis patient in gel-diffusion precipitation assay. PMID- 1725886 TI - Substrate response in acid phosphatase activity of Pseudomonas pseudomallei and Pseudomonas cepacia, with special reference to tyrosine phosphatase. AB - The substrate response in acid phosphatase activity of Pseudomonas pseudomallei and Pseudomonas cepacia was examined with different phosphate esters including hexose phosphates and phosphoaminoacids in a whole cell assay system. The enzymatic activity against each substrate was evaluated in terms of percent activity to that against para-nitrophenyl phosphate set as 100. A remarkable finding was that the phosphatase reaction was the highest with phosphotyrosine or phosphoserine as substrate showing 180% activity. This tyrosine phosphatase activity was resistant to heating at 60 C for 20 min and inhibited greatly by 0.1% ZnCl2. Pseudomonas cepacia showed the same pattern of substrate response and the same characteristics of tyrosine phosphatase activity. PMID- 1725887 TI - Modificational changes in function and morphology of cultured macrophages by geraniin. AB - Treatment of peritoneal macrophages with geraniin, isolated from Geranium funbergii, markedly induced the phagocytosis of living yeasts. Marked increases in phagocytosis and acid phosphatase activity in macrophage lysates were observed 24 hr after the beginning of geraniin treatment. As observed by electron microscopy, macrophages that had been treated for 24 hr with geraniin had a markedly thickened surface layer which was positive to ruthenium red, compared to the control cells. In addition, geraniin treatment of macrophages appeared to induce remarkably large mitochondria, more coated pits and prominent lysosomal granules. In conclusion, the stimulation of phagocytosis and acid phosphatase activity of macrophages by geraniin treatment may involve alterations of the plasma membrane and cytoplasmic reorganization. PMID- 1725888 TI - Antithrombotic activity of the phosphodiesterase III inhibitor pelrinone in a canine model of coronary artery thrombosis: enhancement of efficacy with concurrent alpha 2-adrenergic antagonism. AB - The purpose of this study was to determine if idazoxan, an alpha 2-adrenergic antagonist, could enhance the antithrombotic activity of pelrinone, a PDE III inhibitor, in a canine model of coronary thrombosis that uses electrical current to injure the coronary endothelium. Thrombus mass in vehicle-treated animals was 37.9 +/- 8 mg. Pelrinone, 0.625 and 2.5 mg/kg decreased thrombus size by 46 and 21%, respectively, while idazoxan, 0.75 mg/kg decreased thrombus mass by 43%. When this dose of idazoxan was combined with pelrinone, 0.625 and 2.5 mg/kg, thrombus mass was decreased by 71 and 91%, respectively. Antithrombotic efficacy correlated with the ability of these treatments to inhibit epinephrine sensitized, collagen-induced platelet aggregation. Sixty minutes following drug administration, idazoxan, 0.50 mg/kg inhibited aggregation by 50%, while pelrinone, 0.625 and 2.5 mg/kg inhibited aggregation by 55 and 68%, respectively. Combined administration of idazoxan with pelrinone, 0.625 and 2.5 mg/kg resulted in 80 and 95% inhibition of aggregation, respectively. Similar trends in inhibiting platelet aggregation to epinephrine-sensitized ADP and arachidonic acid were also observed. Experimental treatments did not affect hematocrit or circulating platelet count, although pelrinone was observed to prolong prothrombin time slightly. To examine the effect of drug-induced increases in coronary blood flow on thrombus formation, the potassium channel activator drug cromakalim was studied at a dose (0.1 mg/kg) that increased coronary blood flow by 25-35 ml/min above baseline in sham control animals. Animals treated with cromakalim showed a shorter time to coronary occlusion (103 +/- 11 min) vs. vehicle (173 +/- 24 min) and developed larger thrombi (53.7 +/- 19 mg). These results demonstrate that coronary vasodilation does not contribute to antithrombotic activity in this model. Results from the study also show that alpha-adrenergic inhibition of platelet function can potentiate phosphodiesterase inhibitor antiaggregatory and antithrombotic activity. PMID- 1725889 TI - Vascular contractions to serotonin are augmented by cooling. AB - Human saphenous veins (HSV; n = 17) were obtained at surgery and assayed immediately. The veins were cut into rings, suspended in organ chambers and connected to force transducers for the recording of isometric tension. Tail arteries (RTA) from male Sprague-Dawley rats were similarly prepared. In quiescent rings, cooling from 37 to 24 degrees C had no significant effect. In the presence of alpha-adrenoceptor blockade, potassium chloride (KCl, 10-80 mM) caused concentration-dependent contractions that were inhibited slightly by cooling. Serotonin (5-HT, 10(-8)-10(-5) M) elicited concentration-dependent contractions in both the HSV and RTA with EC50's (-log concentration required to induce contractions 50% of maximal) of 6.31 +/- 0.03 and 6.55 +/- 0.06, respectively. These contractions were subject to blockade with the S2-selective 5 HT antagonist, ketanserin. When either HSVs or RTAs were contracted with 5-HT and cooled, a potent augmentation of the contractions ensued. The data indicate that, while acute moderate cooling does not significantly augment either resting tone or KCl-induced contractions of HSV or RTA, S2-receptor-mediated events are enhanced, yielding potent augmentations of the vascular response to the platelet derived compound, serotonin. The relationship between temperature and serotonergic receptor responsiveness may be of clinical importance as a mechanism contributing to cold-induced vasospasm. PMID- 1725890 TI - Effects of the potassium channel activator, cromakalim, on arterial and cardiac responses to norepinephrine, angiotensin II, and isoproterenol in normotensive men. AB - Cromakalim relaxes vascular smooth muscle by increasing potassium channel conductance, thus hyperpolarizing cell membranes. Its interactions in humans with the cardiovascular effects of norepinephrine, angiotensin II, and isoproterenol were investigated. Eight young normotensive male volunteers received on three different study days, 7-14 days apart, placebo, cromakalim at 1 mg, or cromakalim at 2 mg in a single oral dose, followed 2 h later by incremental intravenous doses of norepinephrine, angiotensin II, and isoproterenol. The diastolic blood pressure and total peripheral resistance (TPR) did not decrease significantly, but significant positive inotropic and chronotropic responses were noted after cromakalim. Cromakalim, both at 1 and 2 mg, blunted (p less than 0.05) the increase in TPR induced by norepinephrine, but did not interfere with its positive inotropic effect (P/V ratio). During angiotensin II infusion, TPR was also lower on both doses of cromakalim (p less than 0.05). Cardiac effects and aldosterone release were not affected. Cromakalim blunted the peripheral vasodilation induced by beta-receptor stimulation by isoproterenol, but did not affect the cardiac stimulation. The potassium channel activator, cromakalim, therefore antagonized the changes in TPR induced by norepinephrine, angiotensin II, and isoproterenol, but did not interfere with the cardiac responses, suggesting selectivity for arterial smooth muscle. PMID- 1725891 TI - Comparative effects on blood pressure and regional hemodynamics of nicardipine and captopril. AB - This randomized, double-blind, crossover study was aimed at detecting efficacy differences in blood pressure control and peripheral circulation in cerebral aortic and femoral districts of slow-release nicardipine, 40 mg, and captopril, 50 mg, given twice daily to 20 primary hypertensive patients (9 male, 11 female; age range 33-60 years) for a period of 4 weeks for each treatment. Blood flow velocities in the aorta, common carotid artery, internal carotid artery, middle cerebral artery, and common femoral artery were noninvasively measured using a 2.0- or 5-MHz Doppler probe. Systolic blood pressure fall was similar with nicardipine (from 156 +/- 12 to 139 +/- 8 mm Hg, p less than 0.001) and captopril (from 158 +/- 13 to 146 +/- 13 mm Hg, p less than 0.001) while significant efficacy difference (p less than 0.005) in diastolic pressure control was detected (from 104 +/- 6 to 91 +/- 8 mm Hg, p less than 0.001 with nicardipine and from 102 +/- 6 to 98 +/- 7 mm Hg with captopril). Regarding regional hemodynamics, nicardipine increased (from 119 +/- 24 to 133 +/- 27 cm/s), and captopril did not affect systolic velocity in the aorta (difference between final data, p less than 0.03). In the common carotid artery systolic velocity was increased solely by nicardipine. No significant changes were detected in the other vessels explored.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725892 TI - Double-blind placebo-controlled crossover study of oral nicardipine in the treatment of Raynaud's phenomenon. AB - The purpose of this study was to measure the efficacy and side effects of oral nicardipine in the treatment of Raynaud's phenomenon (RP). The study consisted of a 3-week baseline period followed by a double-blind, randomized, two-period placebo-controlled, balanced crossover design. Both treatment periods of 3 weeks with either oral nicardipine (3 x 30 mg) or matching placebo were interrupted by a 2-week washout period. Twenty-five patients with either primary (n = 16) or secondary (n = 9) RP participated. Twelve of them had taken part in a previous study on the acute effects of i.v. nicardipine to find out whether long-term efficacy could be predicted by the acute circulatory effects. No statistically significant differences were found between nicardipine and placebo for number, duration, or severity of vasospastic attacks or for any of the microcirculatory parameters (finger skin temperature and laser Doppler flux) measured during a finger cooling test. In patients with primary RP, heart rate significantly increased during nicardipine treatment (mean +/- SD: 9 +/- 6 beats/min; p less than 0.05). The long-term effects could not be predicted by the outcome of the i.v. study. Plasma nicardipine concentrations varied considerably, but in general were on the low side (17.4 +/- 3.7 ng/ml; range, 0-55.4 ng/ml). The adverse effects reported with nicardipine were similar to those with placebo, and required withdrawal of two patients on nicardipine and one on placebo. In conclusion, the results show that oral nicardipine does not significantly alter the course of RP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725893 TI - Importance of central noradrenergic and serotonergic pathways in the cardiovascular actions of rilmenidine and clonidine. AB - We examined the role of central monoamine neurotransmitters noradrenaline and serotonin in the cardiovascular actions of the alpha 2-adrenoceptor agonist rilmenidine in the conscious rabbit and compared it to clonidine. Rilmenidine and clonidine were equieffective in producing a maximum 24% reduction in blood pressure and heart rate when given intracisternally (i.c.), but rilmenidine was approximately 20-30 times less potent than clonidine. The effective i.c. doses of both drugs were 25-30 times lower than those required peripherally (i.v.). Comparison of the time course of an equieffective dose of rilmenidine and clonidine showed that the time to reach maximum effect was 10 min for both drugs but time to recovery was greater for rilmenidine (2.5-3 h vs. 1.5 h for clonidine). Destruction of central noradrenergic neuron pathways with i.c. 6 hydroxydopamine attenuated the hypotensive and bradycardic actions of both rilmenidine and clonidine at 2, 4, and 8 weeks after treatment. The maximum observed attenuation of the hypotension was at 8 weeks (40% of control for rilmenidine and 29% for clonidine), while for the bradycardia this was at 2 weeks (9 and 2% of control, respectively) with some recovery by 8 weeks (29 and 50% of control). Destruction of the central serotonergic neurons with i.c. 5,6 dihydroxytryptamine also attenuated the hypotension and bradycardia to rilmenidine and clonidine but was slower in onset, needing the full 8 weeks to reach its maximum effect. At this time the hypotension and bradycardia to rilmenidine and clonidine were reduced to between 35 and 48% of control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725894 TI - Effect of antiischemic therapy on coronary flow reserve and the pressure-maximal coronary flow relationship in anesthetized swine. AB - The effect of nifedipine (0.5, 1.0, and 2.0 micrograms/kg/min), metoprolol (0.1, 0.5, and 1.0 mg/kg), the beta 1-selective adrenoceptor partial agonist epanolol (10, 50, and 200 micrograms/kg), or equivalent volumes of isotonic saline (n = 6, in each group), on coronary blood flow capacity were studied in anesthetized swine. Intracoronary bolus injections of adenosine (20 micrograms/kg/0.2 ml) were administered without and during three levels of coronary stenosis, prior to and following each dose of drug, to obtain maximal coronary blood flows at different perfusion pressures in the autoregulatory range. Coronary perfusion pressures were varied by partial inflation of a balloon around the left anterior descending coronary artery. Special care was taken that the stenoses not lead to myocardial ischemia. Three indices of coronary blood flow capacity were used: absolute coronary flow reserve (ACFR, the ratio of maximal to resting coronary blood flow), the slope and the extrapolated pressure at zero flow (Pzf) of the pressure maximal coronary flow (PMCF) relationship, and relative coronary flow reserve (RCFR, the ratio of maximal coronary blood flow with a stenosis to maximal coronary blood flow without a stenosis) at two of the three levels of stenosis. Nifedipine decreased ACFR from 4.5 +/- 1.9 to 1.9 +/- 0.3 (mean +/- SD; p less than 0.05), reflecting in part the increase in resting coronary blood flow. The nifedipine-induced changes in maximal coronary blood flow were not only due to a drop in perfusion pressure, as the slope of the PMCF relationship decreased from 2.27 +/- 0.49 ml/(min.mm Hg) to 1.54 +/- 0.51 ml/(min.mm Hg) (p less than 0.05), and Pzf decreased from 30 +/- 4 mm Hg to 20 +/- 7 mm Hg (p less than 0.05). Consequently, calculated maximal coronary blood flow was attenuated from 114 +/- 31 ml/min to 93 +/- 37 ml/min at 80 mm Hg, but was enhanced from 23 +/- 13 to 37 +/- 24 ml/min at 40 mm Hg coronary perfusion pressure. In concert with the change in the PMCF relationship, RCFR at equivalent severe stenosis increased from 0.33 +/- 0.06 to 0.47 +/- 0.10 (p less than 0.05). No changes were observed with metoprolol, epanolol, or saline. The effect of nifedipine on the PMCF relationship not only provides a mechanism for the drug's antiischemic action, but should also be considered in the interpretation of coronary flow reserve measurements in patients on nifedipine treatment. PMID- 1725895 TI - Characterization of the beta-adrenoceptor blocking property of diprafenone in rats: stereoselective interaction, subtype specificity, and sensitization. AB - Because of their structural relationship to propranolol, propafenone and diprafenone display beta-adrenoceptor blocking activity in addition to their class Ic antiarrhythmic property. As demonstrated in membranes derived from rat ventricle (predominantly beta 1-adrenoceptors) and rat lung tissue (predominantly beta 2-adrenoceptors), the (-)-enantiomer of diprafenone was about four times more potent (Ki 6.6 nmol/L) than the (+)-enantiomer in displacing [125I]iodocyanopindolol (ICYP) binding. The Ki values for the (+)- and (-) stereoisomer, racemic (+/-)-diprafenone, and 5-hydroxydiprafenone, the main metabolite of diprafenone in humans, were approximately 2.5 times lower in lung than in ventricular membranes, suggesting very low beta 2-selectivity for diprafenone. The regulatory effect of diprafenone on ventricular beta adrenoceptors was studied in rats in vivo by prolonged i.p. administration of the drug. Density of beta-adrenoceptors was estimated by ICYP saturation binding after 2-day (4 or 20 mg/kg, b.i.d.) and after 7-day treatment (4 mg/kg, b.i.d.), respectively. For control purposes, different groups of animals were treated with propranolol (1.7 mg/kg, b.i.d., i.p.), isoprenaline (0.1 mg/kg/h via subcutaneously implanted osmotic minipumps), and vehicle (0.9% NaCl) only. Whereas propranolol and isoprenaline produced an increase (7-day treatment) and a decrease (2- and 7-day treatment) in beta-adrenoceptors, respectively, diprafenone did not produce any change in beta-adrenoceptor number, irrespective of the dose and duration of treatment used. Furthermore, the combined administration of diprafenone and isoprenaline did not antagonize isoprenaline induced down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725896 TI - Nifedipine improves left ventricular function in patients with hypertension. AB - The effects of the nifedipine gastrointestinal therapeutic system (GITS) on blood pressure (BP), systemic vascular resistance (SVR), and left ventricular (LV) performance were determined in eight patients with essential hypertension. LV systolic and diastolic performance were assessed by first-pass radionuclide cineangiography at rest and during upright bicycle exercise after initial and long-term BP reduction. After initial treatment, end-diastolic volume (LVEDV) increased in association with an increase in stroke volume (SV), cardiac output (CO), and peak ejection rate. After long-term treatment, LVEDV decreased, SV and CO returned to pretreatment values, early diastolic filling fraction increased, and time to peak filling rate decreased. These hemodynamic changes are consistent with an initial predominant effect of vasodilation on LV function. With long-term treatment, the effects on LV diastolic performance are consistent with a positive lusitropic effect of nifedipine GITS. Nifedipine GITS is an effective agent for control of hypertension; its hemodynamic effects are consistent with both an effect on SVR due to decreased vascular smooth muscle contraction and a direct lusitropic effect on myocardial function. PMID- 1725897 TI - Effect of pentisomide (CM 7857) on myocardial excitation, conduction, repolarization, and refractoriness. An electrophysiological study in humans. AB - The electrophysiological effects of pentisomide upon the intact human heart were evaluated using programmed stimulation and recording of intracardiac monophasic action potentials (MAP) in 17 patients with various ventricular arrhythmias. After i.v. administration of pentisomide, 85-135 mg, the atrial-His interval increased by 8 +/- 12 ms (p less than 0.05) during sinus rhythm and by 13 +/- 21 ms (p less than 0.05) at atrial pacing of 600 ms cycle length (600 ms pacing). The His-ventricular interval also increased by 6 +/- 10 ms during sinus rhythm (p less than 0.05) and by 5 +/- 9 ms at 600 ms pacing (NS). The QRS duration prolonged by 9 +/- 10 ms (p less than 0.01) and 6 +/- 8 ms (p less than 0.01) during 600 and 500 ms ventricular pacing, respectively. The right ventricular MAP duration to 90% repolarization was significantly shortened, by 20 +/- 21 ms (p less than 0.01) during sinus rhythm, by 16 +/- 17 ms (p less than 0.01) at 600 ms ventricular pacing, and by 11 +/- 16 ms (p less than 0.01) at 500 ms ventricular pacing. The corrected QT interval was shortened by 21 +/- 28 ms (p less than 0.01). The present study supports that pentisomide is a class-I antiarrhythmic agent with a marked effect on depolarization (action of class Ia and Ic) and on repolarization (action of class Ib). This unique combination of cellular electrophysiological properties indicates that the clinical antiarrhythmic efficacy of pentisomide may differ from that of hitherto available antiarrhythmic drugs. PMID- 1725898 TI - Antihypertensive effect of a novel calcium antagonist, SD-3211, in experimental hypertensive rats. AB - The antihypertensive effect of SD-3211, a structurally novel type of nondihydropyridine calcium antagonist, was assessed using several types of experimental hypertensive rats. Oral administration of SD-3211 (10, 20, and 30 mg/kg) to conscious spontaneously hypertensive rats (SHR), deoxycorticosterone acetate-salt hypertensive rats (DHR) and 2-kidney, 1-clip renal hypertensive rats (RHR) resulted in a dose-dependent decrease in systolic blood pressure (SBP). The hypotensive effect of SD-3211 in these hypertensive rats was more pronounced than in normotensive rats (NR). The potencies of SD-3211 for the hypotensive effect in the hypertensive rats and NR were 5-7 times greater than that of diltiazem but 2 3 times less than that of nicardipine. Furthermore, SD-3211 showed longer-lasting hypotensive action than diltiazem and nicardipine, at the respective equihypotensive dose. During the course of hypotension, SD-3211 did not exert any influence on heart rate (HR) in any type of hypertensive rats or NR, in contrast to the appearance of tachycardia with nicardipine in SHR, DHR, and NR and of bradycardia with diltiazem in DHR. At doses of 10 and 30 mg/kg, the hypotensive doses, SD-3211 elicited a dose-dependent natriuresis but no kaliuresis in SHR. In the chronic study using SHR, SD-3211 at 10 mg/kg/day showed an antihypertensive effect during an administration period of 12 consecutive weeks. These results allow us to conclude that SD-3211 has a potent and long-lasting hypotensive action with little cardiac effect. PMID- 1725899 TI - Beneficial effects of volatile anesthetics on decrease in coronary flow and myocardial contractility induced by oxygen-derived free radicals in isolated rabbit hearts. AB - Oxygen-derived free radicals have been implicated in reperfusion injury whereas volatile anesthetics have been shown to enhance myocardial recovery during reperfusion. To explore the mechanism by which these agents improve myocardial recovery, we measured the effect of volatile anesthetics on the free radical induced reduction in left ventricular pressure (LVP), coronary flow, and endothelium-dependent dilation induced by acetylcholine (Ach). Isolated rabbit hearts were perfused in a Langendorff apparatus. Isovolumetric LVP and coronary flow were measured throughout the study. Oxygen-derived free radicals were produced by the electrolysis (direct current of 0.6 mA) of the perfusate. The following volatile anesthetics were used: halothane 0.5 or 1.0%, isoflurane 0.7 or 1.4%, and enflurane 1.0 or 2.0%. Oxygen free radicals induced a significant decrease in systolic LVP and coronary flow. Pretreatment of the heart with enflurane 1.0 or 2.0%, halothane 1.0%, or isoflurane 0.7% attenuated the effect of the free radicals on both systolic LVP and coronary flow. Free radicals reduced the dilating response induced by 0.1 microM Ach with or without addition of volatile anesthetics. These data suggest that the volatile agents have beneficial effects on the free radical cell damage pathway and that this protection is not related to the preservation of endothelium-dependent dilation. PMID- 1725900 TI - Modification of nitrovasodilator effects on vascular smooth muscle by exogenous GTP and guanosine. AB - The effects of exogenous guanosine 5'-triphosphate (GTP) and guanosine on nitroglycerin-, sodium nitrite- and SIN-1-induced guanosine 3',5'-cyclic monophosphate (cyclic GMP) accumulation and smooth muscle relaxation were studied using endothelium-denuded rat mesenteric artery rings precontracted with noradrenaline. Preincubation of contracted artery rings with GTP (100 microM) or guanosine (100 microM) before eliciting relaxations with nitrovasodilators significantly shifted the dose-response curves of nitrocompounds to the left and augmented the increases in cyclic GMP. GTP and guanosine alone also induced cyclic GMP accumulation in pre-contracted artery rings. These effects of GTP and guanosine on nitrovasodilator responses were not related to the preincubation period (0-30 min). The present results raise the possibility of a cell membrane site of action for GTP and guanosine, which mediates the activation of soluble guanylate cyclase and leads to increased nitrovasodilator-induced cyclic GMP accumulation and arterial smooth muscle relaxation. PMID- 1725901 TI - Atrial natriuretic peptide in diabetic patients before and after control of blood sugar level. AB - Atrial natriuretic peptide (ANP) was examined in 20 diabetic patients: 10 patients referred from the emergency room with severe hyperglycemia, (Group A), and 10 patients with uncontrolled diabetes referred by the outpatient clinic (Group B). Seven patients from Group A reached the nadir of less than 10 pg/ml, and three reached 12-16 pg/ml; following equilibration of sugar level, mean ANP level rose to 51.4 pg/ml (SD +/- 5.6). In Group B mean ANP level before treatment was 19.2 pg/ml +/- 11.4, and after diabetes control reached 40.4 pg/ml (SD +/- 10.04). Findings demonstrate a significant decrease in ANP in the acute hypoglycemic state, and a return to normal levels when sugar is controlled and hypovolemia corrected. Patients with chronic hyperglycemia exhibit compensation of intravascular volume. It seems that the equilibration system functioning via ANP is highly sensitive as a result of acute changes in total body fluid, and becomes desensitized during chronic disequilibrium. PMID- 1725902 TI - Prolonged action potential duration and positive inotropy induced by the novel class III antiarrhythmic agent H 234/09 (Almokalant) in isolated human ventricular muscle. AB - The electromechanical properties of H 234/09 (Almokalant), a novel class III antiarrhythmic agent, was examined in isolated human ventricular muscle strips excised from patients undergoing mitral valve replacement. Using transmembrane microelectrode recording techniques, we demonstrated that H 234/09 markedly prolonged the action potential duration (APD) without affecting the maximal rate of depolarization or action potential amplitude. At 75 and 90% repolarization APD was prolonged to a similar extent, whereas the lengthening at 50% repolarization was somewhat less marked. In isometrically contracting muscle strips, H 234/09 increased peak developed force and its maximal rate of rise (dF/dt) and fall ( dF/dt) in a concentration-dependent manner, whereas time to peak developed force was unaltered. We conclude from these studies that H 234/09 is a class III agent in human ventricular muscle and that the class III effect is linked with a positive inotropic response. PMID- 1725903 TI - Hemodynamic response to molsidomine in patients with ischemic cardiomyopathy tolerant to isosorbide dinitrate. AB - Unlike nitrates, molsidomine is able to relax vascular smooth muscle without depending on the availability of sulfhydryl groups. To assess the clinical relevance of this property, the hemodynamic effects of a 24-h i.v. infusion of molsidomine were studied in 14 patients with ischemic cardiomyopathy rendered tolerant to i.v. isosorbide dinitrate. In order to determine the role of neurohormonal activation, six of these patients were studied in the presence of an angiotensin-converting enzyme (ACE) inhibitor (enalapril, 5 mg, b.i.d.) (group 2). Six patients out of eight in group 1 (without ACE inhibition) and all patients in group 2 responded to molsidomine with a marked reduction of pulmonary artery wedge pressure (PAWP) (49% +/- 5 and 50% +/- 4 versus baseline value, respectively). However, the reduction of PAWP in group 1 was no longer significant at 12 h, and at 24 h the loss of the peak effect reached 67% +/- 7. On the contrary, PAWP remained persistently reduced in group 2 (loss of peak effect, 20% +/- 3 at 24 h, p less than 0.005). In addition, a significant decrease in hematocrit and increase in epinephrine occurred in group 1 but not in group 2. These results suggest that both the absence of dependence on sulfhydryl groups and the blockade of neurohormonal reactions are needed to avoid nitrate tolerance. PMID- 1725904 TI - Modulation of circulating endothelin levels in hypertension and endotoxemia in rats. AB - We have developed separate radioimmunoassays to measure circulating ET-1 and ET-3 levels in normotensive and different hypertensive rat models so that the role of endothelin in the regulation of vasomotor function can be studied. We also assessed the stimulatory effects of endotoxin on plasma and liver lymph ET-1 and ET-3 levels. The circulating ET-1 levels in normotensive rats, SHRs, and DOCA salt hypertensive rats were 2.3 +/- 0.5, 2.1 +/- 0.4, and 2.1 +/- 0.9 pg/ml, respectively. Similarly, the plasma ET-3 levels in normotensive and different hypertensive rats were similar, ranging from 19.7 +/- 1.5 to 24.7 +/- 2.2 pg/ml. The data indicate that steady-state circulating levels of endothelins are a poor correlate of the hypertensive state. Endotoxin (30 mg/kg i.v. over 15 min) reduced blood pressure significantly and augmented plasma ET-1 levels by sevenfold (29.1 +/- 3.7 vs. 4.1 +/- 0.6 pg/ml in the vehicle group; p less than 0.05) and ET-3 levels by twofold (47.7 +/- 7.0 vs. 22.7 +/- 4.0 pg/ml in the vehicle group; p less than 0.05). Human TNF-alpha (30 ng/kg/min x 30 min), a putative mediator of endotoxin shock, enhanced plasma ET-1 (18.3 +/- 1.0 vs. 2.7 +/- 0.4 pg/ml in the vehicle group; p less than 0.05) by sevenfold and ET-3 levels by twofold (45.7 +/- 2.0 vs. 27.1 +/- 4.0 pg/ml in the vehicle group; p less than 0.05) without affecting blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725905 TI - Coronary effects of a combined beta adrenoceptor blocking and calcium antagonist therapy in running dogs. AB - The effects of YM-16151 (1 mg/kg, i.v.), a combined beta 1-adrenoceptor blocking and calcium antagonist drug, on large (circumflex artery) and small coronary arteries and on systemic hemodynamics were investigated in chronically instrumented conscious dogs at rest and during treadmill exercise. These effects were compared to those in the same animals of nicardipine (0.1 mg/kg, i.v.), atenolol (1 mg/kg, i.v.) and their combination (at the same doses). Circumflex artery diameter (CxAD) and coronary blood flow increased and coronary vascular resistance (CVR) decreased during control exercise under saline. YM-16151 and the nicardipine-atenolol combination similarly dilated large and small coronary arteries at rest, but dilation of large conductance vessels was abolished during exercise, while CVR decreased further. Both YM-16151 and the nicardipine-atenolol combination only slightly increased the rate-pressure product at rest, but strongly opposed its exercise-induced rise. Nicardipine maximally increased CxAD, decreased CVR, and enhanced the rate-pressure product at rest and during exercise. Conversely, atenolol decreased CxAD and the rate-pressure product and increased CVR at rest, but large coronary arteries remained constricted during exercise despite the concomitant dilation of small resistance vessels. Thus, in the coronary vascular bed of conscious dogs, YM-16151 really behaves as a hybrid drug, combining beta 1-adrenoceptor blocking and calcium antagonist properties, both at rest and during exercise. As a result, YM-16151 increases oxygen supply at rest and decreases oxygen demand during exercise. Finally, this study emphasizes the major role of beta 1-adrenoceptors in the mediation of exercise induced dilation of large coronary arteries. PMID- 1725906 TI - Comparative frequency-dependent effects of three class Ic agents, Org 7797, flecainide, and propafenone, on ventricular action potential duration. AB - The frequency-dependent electrophysiological effects of three class Ic agents, Org 7797 (0.5-5 microM), flecainide (1-20 microM), and propafenone (1-10 microM), were investigated using isolated guinea pig papillary muscle. Transmembrane cellular action potentials were recorded using conventional microelectrode techniques at driving frequencies of 0.3, 1.0, and 2.0 Hz. In concentrations inducing similar frequency-dependent decreases in Vmax, both flecainide and propafenone shortened action potential duration (APD) at the two lower stimulation frequencies, whereas increasing the driving frequency to 2 Hz attenuated drug-induced APD shortening. In contrast, Org 7797 did not shorten APD at any stimulation frequency, and indeed APD lengthening was observed at 2.0 Hz. Increases in the effective refractory period (ERP) were seen at 1.0 and 2.0 Hz in the presence of Org 7797 and at 2.0 Hz in the presence of flecainide, but ERP was either unchanged (2.0 Hz) or shortened by propafenone. The rate of onset of sodium channel block was faster in response to propafenone compared to Org 7797 and flecainide. It was concluded that all three drugs may inhibit outward repolarising potassium currents, especially at higher stimulation frequencies. However, the concentrations of Org 7797 necessary to influence repolarisation may be closer to those required to induce sodium channel block compared to flecainide and especially propafenone. These differences may influence drug-induced effects on wavelength resulting in differences in antifibrillatory efficacy. PMID- 1725907 TI - Characterization of Ca(2+)-antagonistic effects of three metabolites of the new antihypertensive agent naftopidil, (naphthyl)hydroxy-naftopidil, (phenyl)hydroxy naftopidil, and O-desmethyl-naftopidil. AB - The newly developed antihypertensive agent naftopidil blocks alpha 1 adrenoceptors and inhibits Ca2+ entry via potential-dependent channels in vascular and cardiac muscle. It is extensively metabolized in vivo. Since it is of interest whether its metabolites are still pharmacologically active, we have characterized the effects of (naphthyl)hydroxy-naftopidil (NHN), (phenyl)hydroxy naftopidil (PHN), and O-desmethyl-naftopidil (DMN) in various isolated preparations of the guinea pig heart. In constant-flow Langendorff hearts, the compounds decreased force of contraction by 66-81% and slowed spontaneous heart rate by 28-48%. DMN reduced perfusion pressure by 33%. The fibrillation threshold, which was measured as the strength of alternating current required to induce ventricular fibrillation, increased more than 10-fold. In papillary muscles, 3 x 10(-5) M of all compounds reduced force of contraction (pD2 values approximately 5.5) and shortened the action potential duration in the plateau phase. The maximum depolarization velocity (dV/dtmax) was slightly reduced (10 21%) by NHN, PHN, and DMN. In voltage-clamped ventricular cardiomyocytes, the calcium current ICa was depressed by the three compounds (10(-6)-10(-4) M) in a concentration-dependent manner. In conclusion, the three naftopidil metabolites investigated have pharmacological activities similar to those of their parent compound and hence could contribute to the in vivo effects of naftopidil. PMID- 1725908 TI - Kinins and endothelium-dependent relaxations to converting enzyme inhibitors in perfused canine arteries. PMID- 1725909 TI - Differentiation between calcium- and cholesterol-dominated types of arteriosclerotic lesions: antiarteriosclerotic aspects of calcium antagonists. AB - In a series of animal studies we have clearly demonstrated since 1970 that calcium overload of the arterial wall is crucially involved in the pathogenesis of arteriosclerotic lesions. Following excessive calcium uptake, vascular stretch compliance and distensibility were lost together with structural integrity. Conversely, calcium antagonists that counteract abundant calcium uptake were found to prevent pathogenic mural calcium accumulation and damage. These effects were realized with light and electron microscopy as well as with measurements of calcium accumulation using atomic absorption spectrometry and radiocalcium. The experiments were carried out on rats exposed to various well-known risk factors such as nicotine, alloxan diabetes, high doses of vitamin D3, or dihydrotachysterol. Another particularly vasotoxic factor appeared to be hypertension, which develops in spontaneously hypertensive Okamoto rats (SHRs), in hypertensive NaCl-loaded salt-sensitive Dahl-S rats, and in rats with nephrogenic Goldblatt hypertension. Interestingly, aging arteries in animals and humans also exhibit, as a characteristic phenomenon, a steady increase in arterial calcium content. This natural age-dependent calcium accumulation is further enhanced in severe diabetics, heavy smokers, and hypertensive patients. However, the most excessive degree of toxic calcium overload, correlated with dramatic structural damage, occurred in human coronary artery plaques. Here, in "fatty streaks" (type I plaques according to World Health Organization classification), the mural calcium content exhibited a rise by 13 times above normal; in type II lesions (fibrous plaques), the increase in calcium was 24 fold; and in type III plaques, i.e., in "complicated lesions of stenosing character," calcium overload amounted to a value greater than 80 times above that found in healthy coronary segments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725910 TI - Cardioprotective aspects of calcium antagonists. AB - The use of calcium antagonists as cardioprotective agents is based on the assumption that uncontrolled Ca2+ gain is a key factor in causing cell death and tissue necrosis. This uncontrolled gain in Ca2+ is the ultimate expression of a series of metabolic events triggered by inadequate perfusion. One of the early events is a rise in cytosolic Ca2+ (Cai2+). Using 1,2-bis(e-amino-5 fluorophenoxy)ethan-N1N1N11N11tetraacetic acid and nuclear magnetic resonance spectroscopy to monitor this early rise in Cai2+, it is possible to show that, in isolated perfused rat hearts, Cai2+ increases (p less than 0.01) within the initial 10 min of ischemia and that the increase progresses with time. Possible causes of this early rise in Cai2+ include activation of the endothelin-1 receptors with the subsequent inositol triphosphate-induced activation of sarcoplasmic (SR) Ca2+ release, enhanced Ca(2+)-induced Ca2+ release from the SR reticulum, displacement of bound Ca2+ by the accumulating protons and entry of Ca2+ in exchange for Na+, or through the voltage-sensitive Ca2+ channels. Using the d and l isomers of verapamil it is possible to show that verapamil slows the early rise in Cai2+, the l isomer being more effective (p less than 0.01) than the d isomer. This, in addition to its established energy-sparing effect, may contribute to the effectiveness of verapamil as a cardioprotective agent when used prophylactically. PMID- 1725911 TI - Prevention of restenosis after PTCA: role of calcium antagonists. AB - The recurrence of coronary obstruction after initially successful percutaneous transluminal coronary angioplasty (PTCA) represents a major problem of this interventional procedure at present. In the pathogenesis of restenosis the growth factor-dependent proliferation and migration of medial smooth muscle cells into the intima of the vessel wall may play a very important role. In experimental studies this process could be inhibited by calcium antagonists. However, the first two placebo-controlled clinical trials showed disappointing results: the restenosis rate was not decreased by the treatment with 10 mg of nifedipine four times daily or by the treatment with 90 mg of diltiazem three times daily. The influence of high-dose verapamil treatment [Isoptin RR (240 mg) twice daily] on the recurrence of coronary stenosis has been investigated by a recently completed double-blind, placebo-controlled trial, the verapamil angioplasty study (VAS). The VAS included 196 consecutive patients with at least one risk factor for restenosis after successful PTCA for stable angina pectoris (n = 75) or unstable angina pectoris/non-Q-wave infarction (n = 97). Eighty-eight percent of the patients underwent follow-up angiography at 4.3 +/- 2.3 months. The publication of detailed data of the VAS including restenosis rates in both clinical subgroups is being prepared. PMID- 1725912 TI - Treatment with verapamil during and after an acute myocardial infarction: a review based on the Danish Verapamil Infarction Trials I and II. The Danish Study Group on Verapamil in Myocardial Infarction. AB - The effect of verapamil on death and reinfarction after an acute myocardial infarction was studied in two double-blind, randomized, placebo-controlled multicenter trials, the Danish Verapamil Infarction Trials I and II (DAVIT I and II). The studies demonstrated that verapamil 360 mg/day from the 2nd week after an acute myocardial infarction, prevented death and reinfarction. Meta-analyses of the results of DAVITs I and II resulted in a reduction of pooled ratios of 22% (95% confidence limits 1-37, p = 0.04) for death, 21% (5-35, p = 0.02) for first major events (first reinfarction or death), and 27% (6-43, p = 0.02) for first reinfarctions. The effect of verapamil was to prevent myocardial ischemia and reduce sudden death and reinfarction. It is concluded that long-term treatment with verapamil after an acute myocardial infarction may be recommended with the object of reducing overall mortality, major events and reinfarction. PMID- 1725913 TI - Effect of verapamil on ischemia and ventricular arrhythmias after an acute myocardial infarction: prognostic implications. The Danish Verapamil Infarction Trial II Study Group. AB - This article is a review of presented subsets of the Danish Verapamil Infarction Trial II (DAVIT II) regarding the effect of verapamil on postinfarction ischemia, ventricular arrhythmias, and heart rate (HR), and the prognostic implications of these findings. Patients underwent Holter monitoring for 24-48 h at 1 week, i.e., before randomization to long-term treatment with placebo or verapamil, and after 1 month and about 1 year of study treatment. Ischemia: 18% of the patients had transient ST-segment deviation before randomization; 24% of the placebo- and 8% of the verapamil-treated patients (p = 0.04) showed ischemia after 1 month; and after 1 year, the figures were 26 and 4%, respectively (p = 0.02). The 18-month major event rate, i.e., first reinfarction or death, in patients with ischemia before randomization were 40 and 23.8% in patients without ischemia (p = 0.06). Arrhythmias: In the placebo group the prevalence and incidence of many ventricular ectopic beats (VEBs), i.e., more than 10 VEBs/h, increased significantly during the first years after infarction; this was not the case in the verapamil patients group. The mean HR was significantly reduced by verapamil treatment after 1 month and after 16 months of treatment. Multivariate analysis demonstrated the presence of more than 10 VEBs/h only early (i.e., 1 week) but not late (i.e., 1 month) after infarction, to be an independent predictor of major events during 18 months' follow-up observation. A HR above 80 beats/min independently predicted major events when appearing both early and late after infarction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725914 TI - The effect of verapamil on major events in patients with exercise-induced ischemia recovering from acute myocardial infarction: a review. The Danish Study Group on Verapamil in Myocardial Infarction. AB - The effect of verapamil on death and reinfarction in patients with and without exercise-induced myocardial ischemia after acute myocardial infarction was studied in a double-blind, placebo-controlled multicenter study. Two hundred ninety-eight patients were included. A symptom-limited exercise test was carried out 9 days after the myocardial infarction but before randomization. Forty-four patients with and 111 without exercise-induced ischemia were randomly assigned to verapamil and 39 and 104, respectively, to placebo treatment. The patients were observed for 18 months. The overall 18-month event rate was 12.1%. The lowest event rate was seen in patients with myocardial ischemia and verapamil treatment (9.1%), and the highest in patients with myocardial ischemia receiving a placebo (15.4%), while rates of patients without ischemia were sandwiched in between (12.5 and 12.6%). The differences were not statistically significant. PMID- 1725915 TI - Hydrochlorothiazide and verapamil in the treatment of hypertension. The Verapamil Versus Diuretic (VERDI) Trial Research Group. AB - The efficacy and tolerability of hydrochlorothiazide, sustained-release verapamil, and their combination was compared in patients with mild to moderate hypertension. The design was a multicenter, randomized, double-blind clinical trial with a single-blind placebo baseline period of 2 weeks. Three hundred sixty nine patients with a diastolic blood pressure of 95-120 mm Hg were included. Treatment consisted of 12.5 mg of hydrochlorothiazide once daily, then 25 mg once daily, and finally 25 mg twice daily; the other group received verapamil at a dose of 120 mg once daily, then 240 mg once daily, and finally 240 mg twice daily. Patients not achieving target blood pressure (less than 90 mm Hg diastolic) were given the combination of hydrochlorothiazide and verapamil, that is, 25 and 240 mg once daily and twice daily. During all time points during the study patients receiving verapamil achieved target blood pressure more often than patients receiving hydrochlorothiazide (after 8 weeks, 57.7 and 42.7%; after 24 weeks, 46.8 and 26.4%; and after 48 weeks, 44.8 and 24.7%, respectively). Adding verapamil to hydrochlorothiazide was more effective in lowering blood pressure than adding hydrochlorothiazide to verapamil. The number of treatment withdrawals was essentially the same with the two drugs. Therefore, in the doses currently used in antihypertensive treatment, verapamil was more effective than hydrochlorothiazide as a single agent and in combination in mild to moderate hypertension. PMID- 1725916 TI - Role of calcium-channel blockers in preventing acute and chronic renal injury. AB - Calcium-channel blockers (CCBs) have been shown to afford protection against acute (ARF) and chronic renal failure (CRF). The effects of CCBs against acute renal injury occur at both the vascular and tubular epithelial level. At the vascular level, experimental ARF-associated loss of renal autoregulation and hypersensitivity to renal nerve stimulation has been shown to be reversed by CCBs. These beneficial vascular effects of CCBs occur on the background of the finding that renal ischemic injury is associated with an increase in cellular Ca2+ concentration. A rise in tubular epithelial Ca2+ concentration also occurs very early after a renal ischemic insult. This effect of ischemia is associated with evidence of membrane depolarization, opening of slow calcium channels, increased cellular Ca2+ uptake, and reversal by CCBs. There is evidence that the increased cellular Ca2+ uptake activates phospholipases, which prolong and increase membrane damage. Experimental CRF is also associated with increased renal cellular Ca2+ concentration, an effect that can be attenuated by CCBs. The CCBs probably slow progression of CRF by both cytoprotective and antihypertensive effects. These findings of vascular and tubular effects of CCBs in experimental ARF and CRF have led to their clinical use to prevent initial dysfunction of cadaveric kidney transplants, cyclosporine nephrotoxicity, radiocontrast-induced ARF, and progression of CRF. Randomized clinical studies are necessary to further examine the efficacy of CCBs in ARF and CRF. PMID- 1725917 TI - Rationale and applications for macromolecular Gd-based contrast agents. PMID- 1725918 TI - 31P spectroscopy of the human prostate gland in vivo using a transrectal probe. AB - Using a transrectal probe, good quality 31P magnetic resonance spectroscopy of the human prostate was performed safely, consistently, and in a reasonable amount of time (average of 60 min). Initial results indicate that transrectal 31P MRS has the ability to characterize the phosphorylated metabolites of normal, hyperplastic, and malignant prostates. This study demonstrated that malignant prostates are characterized by significantly decreased levels of phosphocreatine (PCr) and increased levels of phosphomonoesters (PME) as compared to healthy prostates. PMID- 1725919 TI - Blood clearance of dextran magnetite particles determined by a noninvasive in vivo ESR method. AB - Dextran magnetite (DM) is a potential MR contrast agent with superparamagnetic properties. Its fast clearance from the blood and selective uptake by tissue macrophages provide advantages for imaging tumors in the liver and spleen. DM consists of a suspension of solid particles with a wide distribution of sizes. In this study we have used ESR spectroscopy to determine the blood clearance of DM injected iv in mice. The spectra are obtained on living animals by inserting the tail of a mice into the waveguide cavity of the ESR spectrometer and recording the ESR spectrum continuously. This procedure allows the direct measurement of the plasma clearance of DM from individual animals, without blood sampling. We applied this method to study the clearance of suspensions of DM particles with different average sizes. PMID- 1725920 TI - [Value of biochemical and immunological studies in the diagnosis of silicosis]. AB - A series of biochemical and immunological investigations was performed in a group of 33 silicosis patients and in 34 controls. The results obtained suggest future prospects of utilizing the following tests as silicotic-fibrosis markers: alpha 2 globulin and ceruloplasmin levels, activity of certain lysosomal enzymes (beta galactosidase and beta-acetylglucosaminidase), IgM and IgG immunoglobulins, elastases, and the C4 component of the complement. PMID- 1725921 TI - Modulation by adrenergic transmitters of the efferent function of capsaicin sensitive nerves in cardiac tissue. AB - In atrial preparations obtained from reserpine-pre-treated guinea-pigs, incubated in the presence of 1 microM atropine plus 1 microM CGP 20712A (a beta 1 blocking drug), a positive inotropic effect due to CGRP release from capsaicin-sensitive sensory neurons was induced by electrical field stimulation (EFS). This response was concentration-dependently reduced by noradrenaline (0.01-3 microM), neuropeptide Y (NPY, 3-300 nM) and adenosine triphosphate (ATP, 1-30 microM). On the other hand, the overflow of [3H]-noradrenaline from sympathetic nerve terminals induced by EFS in isolated atria obtained from normal untreated animals was not modified in 10 nM calcitonin gene-related peptide (CGRP). Substance P (SP) and neurokinin A (NKA), at concentrations ranging from 0.01 to 1 microM did not affect the cardiac response to field stimulation of adrenergic terminals of atrial tissue. These findings demonstrate that all the co-transmitters stored in adrenergic nerve terminals have a modulatory role on the efferent function of cardiac capsaicin-sensitive sensory neurons, while cardiac adrenergic neurotransmission is not influenced by the peptidergic transmitters released from sensory neurons. PMID- 1725922 TI - Second generation meningococcal OMP-LPS vaccines. PMID- 1725923 TI - [Molecular basis of cystic fibrosis]. PMID- 1725924 TI - [Synthetic immunomodulators designed on the basis of natural products of microorganisms]. PMID- 1725925 TI - Efficacy and tolerability of a new antipsychotic compound (risperidone): results of a pilot study. AB - Risperidone is a new benzisoxazole derivative displaying a very potent serotonin antagonism and a potent dopamine antagonism in pharmacological studies. These properties suggest the hypothesis that risperidone may exert antipsychotic effects and be superior to classic neuroleptics in its beneficial effects on negative and affective symptoms and its low extrapyramidal side-effect propensity. In an open pilot study 13 patients suffering from acute schizophrenic psychosis were treated with risperidone within an individually adapted dose range from 1 to 10 mg per day. A good antipsychotic efficacy could be demonstrated in 6 of the 8 patients who completed the trial. Risperidone was very well tolerated. The substance possesses a low EPS-inducing profile. Future research has to test the suggested advantage of risperidone over other neuroleptic drugs and its performance in the treatment of chronic schizophrenic patients. PMID- 1725926 TI - [Prevention of recurrence of Sudeck's disease with calcitonin]. AB - In patients suffering from, or with a history of, reflex sympathetic dystrophy (RSD), there is always a risk of exacerbation or reactivation of this disease after surgery or trauma. The use of calcitonin in the treatment of this type of disorder is well established. Starting in 1984, 18 patients with clinical symptoms of RSD or a history of this disease were given daily prophylactic treatment with salmon calcitonin (100 IU subcutaneously or as a spray). Ten of the patients underwent orthopaedic surgery and 8 received conservative treatment following trauma. The mean duration of prophylactic treatment was 4 days before and 23 days after surgery or trauma. Only one recurrence was observed (3 per cent). For comparison, a retrospective analysis of a group with the same inclusion criteria who underwent surgery or received conservative treatment after trauma, without prophylactic administration of calcitonin showed a recurrence of the underlying disease 28 per cent of the cases. It is therefore possible to recommend prophylactic administration of calcitonin, to patients with a history of RSD who are about to undergo orthopaedic surgery or conservative treatment following trauma. PMID- 1725927 TI - [Identification of varieties of Cryptococcus neoformans by the use of culture media]. AB - Ten Cryptococcus neoformans strains of human origin were studied in order to know their variety by using 2 different culture mediums: creatinine-bromothymol-blue dextrose and glycine-cycloheximide-phenol red. The results obtained in the two mediums were not totally coincidental. The possible causes and the specificity of these mediums for identifying C. neoformans varieties are discussed. PMID- 1725928 TI - [Diagnostic certainty in intestinal amebiasis in Managua]. AB - Three surveys were carried out in Managua. Nicaragua. In the first one it was found that 71% of the intestinal amebiasis cases of obligatory notification had no comparable diagnostic foundations; in the second one, 11.7% of the laboratory diagnoses as Entamoeba histolytica referred by 4 health centers were correct; and in the third one, it was obvious that only 9.4% of the doctors knew well what intestinal amebiasis is. The results suggest that intestinal amebiasis is being diagnosed excessively in Managua. PMID- 1725929 TI - Evidence of a direct TRH effect on the rat exocrine pancreas. AB - In the present study, the effect of TRH on amylase secretion was determined both in vivo, by cannulating the pancreatic duct of rats, as well as in vitro, by using isolated lobules and dissociated acini. The results show that TRH inhibited both basal and stimulated in vivo amylase secretion. Nevertheless, the in vitro experiments failed to show a TRH-related inhibitory effect when TRH was used alone, although the hormone did blunt the secretion elicited by CCK8 and bethanechol from isolated lobules and dissociated acini. Results suggest that TRH can inhibit stimulated amylase secretion in rats through a direct effect on acinar cells. PMID- 1725930 TI - On multiple forms of NO synthase and their occurrence in human cells. PMID- 1725931 TI - NO synthases: mechanism of activation, identity of NOX and expression in human cells. PMID- 1725932 TI - Antiproliferative effects of NO synthase products. PMID- 1725933 TI - Two unique aspects of inducible .N = O synthase in liver cells and accessory cells: hepatic damage is minimized by hepatocyte .N = O production and immunoregulation is mediated by macrophage .N = O production. PMID- 1725934 TI - Regulation of nitric oxide biosynthesis: cofactor requirement and enzymatic characteristics. PMID- 1725935 TI - A commentary: inducible nitric oxide synthase. PMID- 1725936 TI - [The use of human fibrin glue in retropubic adenectomy by the Millin's technique]. AB - The authors take into consideration the use of human fibrin glue as a completion of the retropubic adenomectomy according the classic Millin technique. Human fibrin glue is applied on the suture line of the prostatic capsule and in the retropubic space. The Authors compare two series of ten operated with and without the use of this biological glue. PMID- 1725937 TI - [Pharmacologic treatment of benign prostatic hypertrophy (BPH): a combination of mepartricin and simvastatin. Analysis and results]. AB - The Authors propose a medical treatment for Benign Prostatic Hyperplasia (BPH) using mepartricina-simvastatina, two drugs with a hypocholesterolemizing action. We have treated fifty patients with BPH (twelve with permanent catheters) obtaining a complete clinical results in 40% of the total and a partial clinical result in 38%. This therapy, in association with a correct diet and adequate physical activity, significantly improved not only the cervicurethral obstruction but also general hematochemical and, above all, lipidemic parameters. PMID- 1725938 TI - [The direct demonstration of the tyrosine phosphorylation of epidermal growth factor receptors internalized in the endosomes of A431 cells]. AB - Phosphorylation of the epidermal growth factor (EGF) receptors in endosomes isolated from A431 cells was studied using antiphosphotyrosin antibody (anti-P Tyr). A431 cells were preincubated with EGF and then washed with acid buffer to remove surface-bound EGF. Endosomes were isolated from such cells by the method of subcellular fractionation on Percoll density gradient. Addition to isolated endosomes of anti-P-Tyr complexes with immunogold resulted in a significant shift of endosome peak to the high density region. This fact indicates that anti-P-Tyr interacts with phosphotyrosine residues of EGF receptors localized in endosomes. PMID- 1725939 TI - [Effect of prenatal neutron irradiation on gene expression in the developing rat brain]. AB - .5 Gy prenatal neutron irradiation at the 17 day of gestation resulted in activation of gene expression in brain. Elevated level of neuronal (GAP-43, NCAM, protein kinase C, calmodulin) and protooncogene (c-foc, c-jun, c-mas) gene transcripts in brain of 2-4 weeks old rats well correlated both with brain maturation in normal animals and development of brain weight deficiency in the irradiated rats. Activation of gene expression appears to be a compensatory response of surviving brain cells to irradiation. Suppression of the gene activity in brain of 18 months old rats correlated with impairment of brain functions at later periods after prenatal irradiation. PMID- 1725940 TI - [Use of monoclonal antibodies to intermediate filament cytoskeletal proteins in diagnosis human tumors]. PMID- 1725941 TI - Prostate-specific antigen. AB - The Scientific Board of the California Medical Association presents the following inventory of items of progress in urology. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome, and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, researchers, or scholars to stay abreast of these items of progress in urology that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.The items of progress listed below were selected by the Advisory Panel to the Section on Urology of the California Medical Association, and the summaries were prepared under its direction. PMID- 1725942 TI - Medical treatment of benign prostatic hyperplasia. PMID- 1725943 TI - Studies on the prenatal chromosomal analysis and the changes of maternal serum alpha-fetoprotein following chorionic villus sampling. AB - Transcervical chorionic villus sampling (CVS) was performed in 174 patients between 7 & 12 menstrual weeks of pregnancy opting for prenatal diagnosis. Advanced maternal age was the most common indication for CVS (39.7%). The sampling success rate was 95.4% (166/174), representing 88.9% at 7 to 8 weeks, 98.9% at 9 to 10 weeks & 92.7% at 11 to 12 weeks gestation. In 139 of 174 patients (80%), successful sampling was accomplished in one or two catheter passages only. Four spontaneous fetal losses (2.3%) occurred. The cytogenetic analysis routinely used was the direct overnight & long-term culture methods which revealed 4 abnormalities (2.4%). To date, 90 of the women have been delivered & all infants are doing well and the remaining 65 pregnancies are continuing uneventually. Maternal serum alphafetoprotein (MSAFP) concentration was determined in 72 patients immediately before & after CVS. A significant increase of 20% or more, comparable to pre CVS levels, was noted immediately after sampling in 56 of 72 patients (77.8%). The increase in MSAFP concentration correlated with the amount of villi sampled (r = 0.498, p less than 0.001) & with the number of sampling attempts (p less than 0.05). Estimated CVS related fetomaternal hemorrhage (FMH) ranged from 0.005 to 0.1552 ml and in 5 of 72 patients (6.90%) 0.06 ml or more of FMH was noted. Two of the 5 patients had FMH of 0.1 ml or more. PMID- 1725944 TI - Enzymatic investigation for delayed-type hypersensitivity reaction. Assay for thrombin and plasmin activities in tuberculin reaction sites. AB - Induration is a prominent feature of delayed hypersensitivity reaction (DHR) and is associated with fibrin deposition. To determine whether thrombin and plasmin mediate the development of induration, we examined guinea pig skin extracts of tuberculin reaction sites for the protease activities. To measure their low activities without inactivation by the inhibitors in the extracts, fluorogenic peptide substrates specific to each of the proteases were used. Because thiol proteases in the extracts hydrolyzed the substrates, the two serine protease activities were selected using a serine protease inhibitor. The extracts contained alpha 2-macroglobulin (alpha 2-MG)-trapped thrombin that hydrolyzes the substrate, but no alpha 2-MG-trapped plasmin. To exclude alpha 2-MG-trapped thrombin activity, the extract treated with 0.2 M methylamine was incubated for two hours and the residual activity was excluded from the initial thrombin activity. Thrombin activity paralleled increasing intensity of induration, and plasmin activity was associated with the reduction of induration. Neither induration nor an increase of protease activity was observed at control sites. The results show that thrombin and plasmin mediate the development of induration probably by regulating fibrin deposition in DHR sites and that the present method can measure the protease activities in biological fluids and tissue extracts. PMID- 1725945 TI - Clinicopathologic and immunohistochemical characteristics of goblet cell type adenocarcinoma of the lung. AB - Twenty-two resected goblet cell type adenocarcinomas of the lung were examined clinicopathologically and immunohistochemically. The stage and survival curve of goblet cell type adenocarcinomas were compared with those of 44 cases of pure or mixed Clara cell and bronchial surface epithelial cell (Clara and BSE) type adenocarcinomas. Each case of goblet cell type was matched with two cases of Clara and BSE type as to sex, age and date of surgery. In goblet cell type adenocarcinomas, lymph node metastasis was less frequently and intrapulmonary metastasis was more frequently detected than in other types of adenocarcinomas (p less than 0.001 and p less than 0.05, respectively). Goblet cell type adenocarcinomas showed better prognoses than Clara and BSE type adenocarcinomas. However, the estimated survival curves of those two groups become similar after adjustment of the TNM condition using Cox's proportional-hazard general linear model. This result indicated that the longer survival of goblet cell type adenocarcinoma was due to the characteristic distribution of TNM conditions, that is, unique local growth and low incidence of lymph node metastasis. When goblet cell type adenocarcinoma was macroscopically classified into two types, i.e. solitary peripheral nodule type (nodular type) and multifocal nodular type or consolidation of all or part of a lobe (diffuse type), the nodular type had better prognosis than the diffuse type (p less than 0.05). Immunohistochemically, 83%, 11%, and 0% of goblet cell type adenocarcinomas were positive for NCC-CO 450, carcinoembryonic antigen (CEA), and surfactant apoprotein, respectively. Most Clara and BSE type adenocarcinomas were negative for NCC-CO-450, but positive for CEA and surfactant apoprotein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1725946 TI - Production of prochymosin in lactococci. PMID- 1725947 TI - Origins of the multiple cathepsin E transcripts observed in human gastric mucosa and gastric adenocarcinoma. PMID- 1725948 TI - N-myristoyl transferase assay using phosphocellulose paper binding. AB - N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract. PMID- 1725949 TI - Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels. AB - In a previous work (J.-R. Daban, M. Samso, and S. Bartolome, Anal. Biochem. 199, 162-168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[alpha] phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 M Tris and 0.192 M glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680-685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethyl-rhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass. PMID- 1725950 TI - Chemiluminescent assay of various enzymes using indoxyl derivatives as substrate and its applications to enzyme immunoassay and DNA probe assay. AB - Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H2O2). Hydrogen peroxide is detected chemiluminescently using isoluminol microperoxidase. Alkaline phosphatase (ALP), beta-D-galactosidase (beta-gal), and beta-glucosidase were assayed by this method using 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and 5-bromo-4-chloro-3-indolyl-beta-D-glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10(-19) mol of ALP and beta-gal, respectively. This assay system can be applied to enzyme immunoassay and DNA probe assay. PMID- 1725951 TI - Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5 pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins. AB - A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods. PMID- 1725952 TI - A general radiochemical-color method for quantitation of immunoblots. AB - Quantitative interpretation of protein immunoblotting procedures is hampered by a variety of technical liabilities inherent in the use of photographic and densitometric methods. In this paper, we present a novel, simple, and generally applicable alternative procedure to acquire quantitative data from immunoblots. Our strategy employs both the standard alkaline phosphatase color reaction and radiolabelled Protein A. The color reaction is used to localize the polypeptide of interest after transfer to a solid support. The colored bands are then excised and the radioactivity in the colocalized Protein A is quantitated in a gamma counter. In addition to avoiding the problems associated with photographic and densitometric procedures, our assay also overcomes common problems associated with variable gel lane width and individual band distortion. The resulting data is linear over a range of at least 50-fold (10-500 ng of specific protein, for the example used in this study) and is highly reproducible. PMID- 1725953 TI - Different types of messenger RNA editing. PMID- 1725954 TI - Shades of gray: a personal view of palliation by radiotherapy. AB - Palliation is a significant part of the work of a radiation oncologist and yet is discussed infrequently. All too often it is considered simple when in many cases the contrary is the case. The philosophy of palliation is discussed and is defined as non-curative treatment. Further sub-division into symptom control, growth restraint/local control is helpful. The aim of palliation must be clearly defined if it is to be achieved, taking into account not only the lesion causing the problem but also many other factors including the patient's general condition, the clinical evolution of the tumour, previous treatment and social factors. The aim should be communicated to the patient and relatives. Doses for effective palliation vary from low to high and require different fractionation regimens. Examples are given from clinical practice with emphasis on difficult and controversial areas. There are as many diseases as patients and the need to individualise treatment is stressed. PMID- 1725955 TI - Detection of lipopolysaccharide-binding proteins on membranes of murine lymphocyte and macrophage-like cell lines. AB - Lipopolysaccharide-(LPS) binding proteins present on murine-lymphocyte and macrophage-like cell lines were identified by a ligand-blotting method and subsequent immunological detection of bound LPS. Membrane proteins of the murine pre-B-cell line 70Z/3 were separated by SDS-PAGE, transferred electrophoretically onto nitrocellulose, and the blot was incubated with LPS of the Salmonella minnesota Re-mutant R595 (mRe-LPS). LPS bound to proteins on nitrocellulose was immunologically detected by anti-mRe-LPS antibodies; LPS was associated with one of the membrane proteins of 70Z/3 cells. This protein was 40 kDa under reducing and 45 kDa under non-reducing conditions, respectively. Treatment of 70Z/3 cells with pronase led to the disappearance of the LPS-binding protein indicating its surface location. Excess free lipid A, which represents the biologically active region of LPS, inhibited the binding of mRe-LPS to the protein. This LPS-binding protein was also identified on the pre-B-cell line CYG8, the B-cell line CYG101 and the murine-T-cell line BW5147. It was, however, not detectable on the B-cell line CYG34 and the myeloma-cell line P3-X63-Ag8.653. No other LPS-binding protein could be detected on these cell lines. In the murine-macrophage-like cell line J774.1, two LPS-binding proteins, one of 40 kDa and one of 80 kDa, were detected. These results indicate that mRe-LPS is specifically bound to a 40-kDa protein of lymphocytes, whereas in the case of macrophages it is associated with two LPS binding proteins of 40 and 80 kDa. PMID- 1725956 TI - Immunological characterization of a low molecular mass polypeptidic antigen of Borrelia burgdorferi. AB - The presence of a low molecular mass polypeptidic antigen in Borrelia burgdorferi was described. The protein was exposed at the bacterial surface since it was clearly identified by mAb 3H4 using the immunofluorescence test performed with living bacteria. This antigen was cleaved by proteinase K treatment, whereas it was resistant to the action of chymotrypsin, trypsin and thermolysin. Western blotting analysis of the immunological reactivity of this antigenic structure performed using monoclonal antibody, mouse-immune ascitic fluids raised against B. burgdorferi and other spirochetes, sera from patients with Lyme disease and other infirmities in which false positive results in serological tests for B. burgdorferi have been described, demonstrated that this protein expresses only species-specific epitopes which may be recognized during human B. burgdorferi infections. PMID- 1725957 TI - The immunohistochemical detection of involucrin in denture induced fibrous inflammatory hyperplasia of oral mucous membrane. AB - Involucrin is a major structural protein specific to the cross-linked cell envelope found in the stratum corneum of stratified squamous epithelium. This protein is considered to be an excellent immunohistochemical marker of normal squamous differentiation. Detection of variations to the patterns of immunostaining for involucrin may also be of value in the differential diagnosis between benign and malignant lesions. Previous studies of involucrin expression in oral mucosa have failed to clarify the effect of chronic inflammatory change upon the patterns of immunoreactivity. This study investigated involucrin staining patterns in fibrous inflammatory hyperplasia of oral mucous membrane (FIH). The results suggest that in FIH an altered pattern of involucrin immunostain occurs in areas of severe inflammatory change. This may reflect changes to the pattern of squamous differentiation in this tissue. PMID- 1725958 TI - Ribosomal inhibitory proteins from plants inhibit HIV-1 replication in acutely infected peripheral blood mononuclear cells. AB - Peripheral blood mononuclear cells from seronegative donors were stimulated with phytohemagglutinin and then infected with human immunodeficiency virus (HIV-1). Using this experimental system, the antiviral activity of two translation inhibitory proteins (pokeweed antiviral protein, PAP-S, and Luffa ribosomal inhibitory protein, LRIP-I) isolated from plants and a recombinant form of ricin A chain were studied. Previously, it had been shown that toxin polypeptides linked to monoclonal antibodies could inhibit HIV-infected cells. In the present study, the free, unconjugated, proteins were found to inhibit HIV replication at doses in which they were nontoxic to uninfected peripheral blood mononuclear cells. Among the inhibitory proteins, PAP-S and recombinant ricin A chain markedly reduced the reverse transcriptase activity and the expression of p24 core protein in infected cultures. Dose response studies indicate that the anti HIV activity of PAP-S was comparable to AZT. The other ribosome inhibitory proteins (RIPs) showed moderate but significant antiviral activity. PMID- 1725959 TI - Production and characterization of mouse monoclonal antibodies against the transmembrane protein of a human immunodeficiency virus type 2. AB - We established seven hybridoma clones producing monoclonal antibodies (MAbs) against the envelope transmembrane protein (TMP) of a Ghanian isolate of human immunodeficiency virus type 2 (HIV-2[GH-1]) from mice immunized with the detergent-disrupted purified whole virus. The MAbs were found to react with 35 kilodalton (kD) TMP of the HIV-2[GH-1] virus in a Western blot assay (WB). Two of these MAbs recognized 135 kD proteins in addition to TMP in the lysate of HIV 2[GH-1]-infected cells. Two other MAbs cross-reacted with viral components corresponding to TMPs of HIV-2ROD and SIVMAC isolates in a Western blot. Results of competitive binding assay suggest that there are at least three epitopes on a TMP molecule of the HIV-2[GH-1] isolate. The MAbs did not inhibit syncytium formation between HIV-2[GH-1]-infected MOLT-4 cells and MOLT-4 clone 8 cells, nor virus infection to MOLT-4 clone 8 cells. PMID- 1725960 TI - The HIV-1 Rev protein: a model system for coupled RNA transport and translation. AB - The impact of the Rev protein of the human immunodeficiency virus type 1 (HIV-1) on RNA transport, intranuclear RNA distribution, and gene expression was examined for two Rev-dependent expression systems by means of fluorescence in situ hybridization, immunofluorescence, S1 nuclease protection, and functional assays. In the pgTat expression system, which utilizes authentic HIV-1 splice signals, unspliced mRNA remained entrapped in the nucleus in the absence of Rev and was exported to the cytoplasm in its presence, consistent with published findings. In the pSVAR expression system, significant levels of mRNA were found in the nucleus and cytoplasm in both the presence and absence of Rev, but only in the presence of Rev was mRNA translated into protein. The presence of cytoplasmic untranslated mRNA in the absence of Rev was demonstrated by in situ hybridization analysis of individual cells as well as by S1 nuclease analysis of cell populations. The results indicate that Rev has the potential to affect translation as well as transport, suggesting the possibility that cellular mechanisms exist whereby the translational efficiency of an mRNA may be affected by the manner in which it is transported from the nucleus. Fluorescence hybridization also provided high resolution visualization of the intranuclear distribution of RNAs containing the Rev response element. This demonstrated for both expression systems that mRNA was not highly localized in tracks or around the nucleolus in the presence or absence of Rev, a nucleolar protein, but was more widely distributed throughout the nucleus. In pgTat transfectants, HIV-1 RNA often became localized in 5 to 20 discrete large intranuclear clusters in the presence of Rev, the potential significance of which is discussed. PMID- 1725961 TI - Alternative forms of the human G-CSF receptor function in growth signal transduction. AB - Two forms of the human granulocyte colony-stimulating factor (G-CSF) receptor (HuG-CSFR), differing only at the carboxyl terminus, were recently identified by cDNA cloning. In this report we show that transfection and subsequent expression of either cDNA clone in the interleukin-3 (IL-3)-dependent murine cell line BAF/BO3 converts the cells to G-CSF-responsiveness. The transfected cells bound HuG-CSF in a manner indistinguishable from the native receptors. Expression of a mutant form of the HuG-CSFR, with a deletion in the cytoplasmic domain, in BAF/BO3 cells failed to convert the cells to HuG-CSF-responsiveness. In a similar manner, expression of these two HuG-CSFRs in the interleukin-6 (IL-6)-dependent murine hybridoma B9 resulted in the ability of these cells to grow in HuG-CSF [corrected]. These results strongly suggest that sequences in the first 96 amino acids of the cytoplasmic domain of the HuG-CSFR are required for signal transduction in response to ligand binding. PMID- 1725962 TI - The beta-adrenergic receptor-regulated 1,4-dihydropyridine calcium channel receptor sites in coronary artery smooth muscle. AB - The cross-regulatory communication from beta-adrenergic receptors to 1,4 dihydropyridine (DHP) Ca2+ channel agonist and antagonist binding sites and cooperativity between DHP binding sites were studied in microsomal membranes of canine coronary artery (purified to a factor 2.9 for DHPs). The maximal number of binding sites (Bmax) identified in coronary artery microsomal membranes (CAM) with Ca2+ channel agonist (-)-S-(3H)BAY K 8644 was two times higher than Bmax of sites labelled with Ca2+ channel antagonist (+)-(3H)PN 200-110. The exposure of CAM to isoprenaline was accompanied with down-regulation of beta-adrenergic receptors and with increase in binding capacity for DHPs. The increase in Bmax was proportional in both groups of experiments and was related to increased affinity of DHPs. The 1,4-DHP binding sites identified in vascular smooth muscle showed characteristics typical for classification of specific 1,4-DHP receptor on Ca2+ channels. The binding was of high affinity, saturable and reversible, it showed stereoselectivity and it was positively modulated by beta-adrenergic stimulation and its showed cAMP and GTP sensitivity. The results support the hypothesis that beta-receptors also regulate the mode of Ca2+ channels in coronary artery smooth muscle. PMID- 1725963 TI - Thermospray tandem mass spectrometric analysis of oxygen incorporation into citrulline by nitric oxide synthase. AB - Citrulline is formed as a co-product in the biosynthesis of nitric oxide from L arginine by the action of either constitutive or inducible nitric oxide synthase which is present in a variety of cells. We have previously shown that the oxygen atom incorporated into both nitric oxide and citrulline derives from molecular oxygen and not water. This paper describes the tandem mass spectrometric analysis of citrulline synthesized by the macrophage cell line J774 in the presence of native or guanidino-labelled arginine and air or isotopically enriched oxygen. The results confirm that oxygen is incorporated into the ureido position of citrulline. PMID- 1725964 TI - In vitro production of anti-neutrophilocyte-cytoplasm-antibodies (ANCA) by Epstein-Barr virus-transformed B-cell lines in Wegener's granulomatosis. AB - The frequent detection of anti-neutrophilocyte-cytoplasm-antibodies (ANCA) in patients with Wegener's granulomatosis (WG) led to the supposition that this disease might be of autoimmune nature. For some authors assume that Epstein-Barr virus (EBV) infection of human B-lymphocytes besides polyclonal activation could reveal the cryptic immune status against different autoantigens in patients with autoimmune diseases we investigated EBV-transformed B-lymphocytes from patients with Sjogren's syndrome, mixed connective tissue disease, WG and healthy blood donors. Two stable B-cell lines (Ho3, We1) could be established. Inhibition experiments showed that antibodies produced by transformed B-lymphocytes and serum ANCA (C-ANCA type) of 10 WG patients recognized the identical antigen. Stimulation of one clone (Ho3) with interleukin 6 (IL-6) led to a switch from IgM to IgG production. Antibodies produced by this clone also stained glomeruli of human frozen kidney sections. Western blot analysis using immunoaffinity purified antigen prepared from human granulocytes revealed a reaction with a protein of approx. 29 kD MW. Our data underscore some new aspects concerning the direct pathogenicity of C-ANCA confirming the hypothesis that the autoimmune B-cell repertoire in WG not only reflects a polyclonal B-cell activation but is shaped by antigen driven responses. PMID- 1725965 TI - Antibodies to myelin basic protein and measles virus in multiple sclerosis: precursor frequency analysis of the antibody producing B cells. AB - Antibody-producing B lymphocytes were polyclonally activated and transformed, by Epstein-Barr virus (EBV), into multiple B lymphoblastoid cell lines in a microculture system. The frequencies of B precursor cells producing antibodies to myelin basic protein (MBP) and measles virus were analyzed in peripheral blood of patients with multiple sclerosis (MS) and control subjects. Measles virus specific B cells were detected at a significantly higher frequency in MS patients (n = 10, P less than 0.005) than patients with other neurological diseases (n = 10) and normal subjects (n = 10). In contrast, the frequencies of B cells producing anti-MBP antibodies and natural antibodies did not differ statistically among the three groups tested (P greater than 0.05). In addition, the anti-MBP antibodies produced by a panel of stable B cell lines obtained were found to react selectively with an epitope(s) within the C-terminal half fragment 90-171 of the human MBP molecule. In our experiments, no antibody cross-reactivity between MBP and measles virus could be detected in a total of 2760 B cell cultures. PMID- 1725966 TI - The acute phase response: from Hippocrates to cytokine biology. PMID- 1725967 TI - Cytokine control of acute phase protein expression. PMID- 1725968 TI - Studies on monoclonal antibody against recombinant human granulocyte colony stimulating factor. AB - Three hybridoma cell lines producing monoclonal antibodies (McAb) against recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been established by fusing mouse myeloma cell line SP2/0 cells with spleen cells from a BALB/c mouse immunized with rhG-CSF. Ascites was obtained from BALB/c mice by injecting the hybridoma cells intraperitoneally. Three McAbs were purified by the caprylic acid-ammonium sulfate method. For each, the IgG subclass, valence and activity, molecular weight, specificity and affinity were determined. The applications of McAbs against rhG-CSF in the clinic and laboratory are discussed. PMID- 1725969 TI - Capillary supply and cardiac performance in the rabbit after chronic dobutamine treatment. AB - STUDY OBJECTIVE: Growth of capillaries in the adult heart can occur after long term vasodilator treatment. This study investigated the effects of increased stroke volume and/or force of contraction in addition to coronary blood flow on myocardial capillary growth. DESIGN: Coronary blood flow, cardiac function, and capillary supply were evaluated after chronic treatment with the inotrope dobutamine, at a dose shown to increase contraction force (dP/dt) acutely by 20% (p less than 0.05) and coronary blood flow by 60% (NS). EXPERIMENTAL MATERIAL: New Zealand Red rabbits received dobutamine (20 micrograms.kg-1.min-1 by intravenous infusion) for 2 weeks prior to measurements, under pentobarbitone anaesthesia, of coronary blood flow by radiolabelled microspheres, left ventricular dP/dtmax and maximum noradrenaline induced cardiac work, computed from cardiac output (thermodilution) and blood pressure. Capillary density.mm-2 was estimated in frozen 12 microns sections of left ventricular myocardium stained for alkaline phosphatase. MEASUREMENTS AND MAIN RESULTS: Capillary density was 29% greater after 2 weeks dobutamine infusion than in saline infused controls, which exceeds increases observed after longer infusions of vasodilators alone. Cardiac function was improved; maximum stroke work was significantly higher, stroke volume capacity larger, and dP/dt increased more during noradrenaline challenge. Coronary blood flow was unchanged in dobutamine treated hearts, hence efficiency of utilisation of coronary flow in terms of work was enhanced. Heart to body weight ratio was also greater after dobutamine, at 0.238(SEM 0.008) v 0.206(0.120), p less than 0.05. CONCLUSIONS: Greater capillary growth can be elicited in the heart by increased stretch and/or force of contraction of myocytes in addition to moderate changes in blood flow than by increased blood flow alone. Chronic dobutamine treatment induced adaptive enhancement of maximal cardiac work capacity. PMID- 1725970 TI - Changes in beta-tubulin isoforms and their RNA level in synchronized tobacco cells. AB - Tobacco BY-2 cells were synchronized by an aphidicolin treatment, and their beta tubulin isoforms and their mRNA were analyzed by Western, Northern and dot blottings. The relative ratio of the beta-tubulin isoforms changed with the progress of cell cycle stage. By Northern blot hybridization of poly(A)+RNAs with a cloned carrot beta-tubulin cDNA probe, a single band of about 1.6 kb was detected throughout the cell cycle. Dot blot hybridization showed that beta tubulin mRNA existed in all stages in the cell cycle at a relatively constant level, though it accumulated slightly more than average at M phase and decreased during G1 phase. PMID- 1725971 TI - [Extraskeletal myxoid chondrosarcoma: a clinicopathological study of six cases]. AB - The clinicopathologic, ultrastructural, and immunohistochemical features of six cases of extraskeletal myxoid chondrosarcoma are described. Light microscopic examination disclosed a lobulated neoplasm consisting of cells that tended to be round or polygonal with plenty of eosinophilic cytoplasm. The tumor cells were arranged in strands and small nests surrounded by abundant myxoid extracellular matrix. Ultrastructurally, these tumor cells displayed the signs of chondroblastic differentiation. The cytoplasm contained numerous rough endoplasmic reticulum, mitochondria and scattered glycogen granules. The intercellular matrix showed amorphous material and electronic dense and fine granules. The tumor cells in all these six cases showed diffuse immunostaining for S-100 protein but were negative for keratin. The data obtained tend to support the chondroblastic origin of this tumor. PMID- 1725972 TI - [Experimental study on the treatment of hypertension with combined traditional Chinese and Western medicine]. AB - In order to study the connection of diastolic activity of smooth muscles of blood vessels with development of hypertension, plasma cAMP, cGMP, TXB2, 6-K-PGF1 alpha, ANP, SP were determined with radioimmunoassay, of 173 hypertension patients with Liver Yang exuberance (LYE) 91 cases, and Yin deficiency and Yang exuberance (YDYE) 82 cases. In addition, 228 health subjects served as control. The results showed that the levels of cAMP, cGMP and TXB2 in both LYE and YDYE groups were higher than those in the control group, but the levels of ANP, SP and cAMP/cGMP ratio in LYE and YDYE groups were lower than those in the control. As to the level of 6-K-PGF1 alpha, no significant variance was found between these groups. After TCM-WM treatment, the levels of cAMP, cGMP and TXB2 in LYE and YDYE groups got down, as compared with those in the control, adversely the levels of ANP, SP and 6-K-PGF1 alpha in LYE and YDYE groups turned up significantly. However the cAMP/cGMP ratio had no remarkable change between these groups. The linear regression analyses between the diastolic pressure and ANP or SP both proved negative correlation (r = -0.36, P less than 0.05; r = -0.35, P less than 0.05). The findings indicated that the TCM-WM treatment was the most effective among the therapies employed in the study, and that this therapy affected the diastolic activity of smooth muscles by modulating the above factors existing in the nervous and endocrine systems of the patients with hypertension. PMID- 1725973 TI - Radioimmunoscintigraphy of ovarian cancer. PMID- 1725974 TI - The mucin antigens: what are we measuring? AB - Serum of breast cancer patients contains high molecular weight, mucin-like glycoproteins which are held to be differentiation markers for certain types of normal epithelia, in particular mammary epithelium. These components have primarily been identified using monoclonal antibodies raised against human milk fat globule membranes, tumour extracts or purified mucins. Even so, many of the antibodies produced react with a discrete region of the mucin protein core involving the hydrophilic turn domain APDTRPAP. The present investigation using the anti-urinary mucin antibody, C595, illustrates both the clinical potential of the mucin antigens in breast cancer studies as well as the exquisite specificity of immune recognition of a complex polymorphic glycoprotein at the level of the individual amino acids. PMID- 1725975 TI - Absence of O6-alkylguanine-DNA alkyltransferase induction in chick embryo liver and brain following X-irradiation or treatment with bleomycin. AB - 1. The presence of O6-alkylguanine-DNA alkyltransferase (AT) in liver and brain of chick embryos, chicks and hens was demonstrated. An induction of AT activity has only been found in the liver of chicks and hens 48 hr after X-irradiation (5, 10 or 12 Gy). 2. The administration of methylmethanesulphonate to the chick embryo resulted 3-24 hr later in strong inhibition of AT activity accompanied by DNA alkylation. Under the same conditions, X-irradiation, dimethylnitrosamine and bleomycin exhibited no effect. 3. The results are compared with those obtained in mouse, rat and human foetal tissues. PMID- 1725976 TI - The effect of iron and ethanol on rat hepatocyte collagen synthesis. AB - 1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes. 2. Acute exposure of cultured hepatocytes from control and iron loaded rats to ethanol (25-100 mM) resulted in a significant inhibition of protein synthesis. 3. Inhibition of protein synthesis in hepatocytes from iron loaded rats was primarily due to impaired amino acid uptake by these cells. 4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats. 5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA. 6. Acute administration of ethanol did not inhibit procollagen synthesis. 7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes. 8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol. PMID- 1725977 TI - [Practical value of prostatography]. AB - From September 1988 to September 1989 perineal puncture prostatography with 60% urografin was performed in 21 patients with the prostate enlargement. Prostatographic findings suggested that 15 patients had benign prostatic hyperplasia and 6 had prostatic cancer. The diagnosis had been histologically proved after prostatectomy in 19 cases. The procedure was easily and safely conducted at outpatient clinic without complications. We believe that prostatography is useful in differential diagnosis of prostatic diseases and in selecting operative methods. PMID- 1725978 TI - Changes in RNA content of the hypothalamus of mice following insulin administration. AB - Cytoplasmic total RNA in neurons of the supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and arcuate nucleus was determined cytophotometrically following a single intramuscular dose of 4 IU/kg insulin semilente. A significant decrease was observed after 3, 6, and 12 hours. A return towards normal values was evident after 24 hours and was full after 48 hours only in suprachiasmatic nucleus. Other hypothalamic nuclei did not reach the full normal value in the time interval examined, i.e. 48 hours post-injection. PMID- 1725979 TI - Preparation and characterization of a chimeric CD19 monoclonal antibody. AB - FMC63 is an IgG2a mouse monoclonal antibody belonging to the CD19 cluster. CD19 antibodies react with a 95kDa protein expressed by cells of the B lymphocyte lineage, from pre-B cells to mature B lymphocytes. CD19 antibodies have been suggested as candidates for immunological attack on leukaemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind, we have produced a mouse-human chimera in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed. PMID- 1725981 TI - Addition of cetyl pyridinium chloride to histological demineralizing agents to improve the demonstrability of gram-positive bacteria. AB - It has previously been shown experimentally using S. faecalis that the relative number of Gram-positive bacteria is reduced by demineralizing agents. To achieve better bacterial preservation after demineralization, cetyl pyridinium chloride (CPC) and neutral formaldehyde solution (NF) were added, separately and in combination, to formic acid and EDTA. The number of S. faecalis organisms in suspension was determined using a haemocytometer, and the percentage of Gram positive bacteria was ascertained from smears. Both CPC and NF accomplished better bacterial preservation; when both substances were added together preservation was improved still further. The best results were achieved when CPC and NF were added to EDTA. If these findings could be extrapolated to tissue sections, and if it were intended to demonstrate Gram-positive bacteria, 10 per cent EDTA to which 0.5 per cent CPC and 4 per cent NF had been added would be recommended for demineralization. PMID- 1725982 TI - The cumulative effect of disinfection, storage, histological fixation and demineralization on number and staining ability of gram-positive bacteria. AB - It has previously been shown experimentally using S. faecalis that the relative number of blue-staining Gram-positive bacteria was reduced by demineralizing agents as well as by disinfection (Wijnbergen & van Mullem 1987, van Mullem & Wijnbergen 1989). In this study the cumulative effect of disinfection, experimental period, fixation and demineralization was investigated. The use of additives to the fixative and the demineralizing agents to limit the loss of blue staining bacteria was also studied. The numbers of bacteria were determined using a haemocytometer, and the percentage of blue-staining organisms were ascertained from smears. When compared with the start of the experiment, the sequence of disinfection with chlorhexidine, storage in PBS, fixation with neutral formaldehyde and demineralization with formic acid or EDTA appeared to reduce the relative numbers of Gram-positive staining bacteria. For storage periods of 0 and 4 days the reduction factors were 100 and 600, respectively, using formic acid. These factors were 50 and 95, respectively, using EDTA. Addition of cetyl pyridinium chloride to the fixative and the demineralizing agents, and addition of neutral formaldehyde to the demineralizing agents lowered these reduction factors to 80 and 200, respectively, for formic acid, and 40 and 85, respectively, for EDTA. If the results are extrapolated to animal experiments where disinfection, experimental period, fixation and demineralization form part of the experimental framework, even the lowest reduction in the number of blue staining bacteria could lead to false interpretation of tissue sections. PMID- 1725980 TI - Surgical management of congenital heart defects: current trends. AB - Surgical treatment for congenital heart disease has become available over the last five decades. Palliative procedures have been designed to improve physiologic abnormalities, for example systemic artery (or venous) to pulmonary artery shunts of various types to increase the pulmonary blood flow, pulmonary artery constriction (banding) to decrease the pulmonary blood flow, and surgical or transcatheter atrial septostomy to augment intracardiac mixing. These can be performed with a low mortality. The majority of congenital heart defects can be corrected by open heart surgical techniques; some require prior palliation and others can be operated without prior palliative surgery. Recent surgical advances include early total surgical correction for tetralogy of Fallot, arterial switch procedure for transposition of the great arteries, Fontan operation and its modifications for tricuspid atresia and single ventricle, new operations for hypoplastic left heart syndrome, newer prosthetic valves, myocardial preservation and cardiac transplantation. PMID- 1725983 TI - Growth retardation during the suckling period in expanded litters of rats: observations of growth patterns and protein turnover. AB - Postnatal patterns of growth and protein turnover were studied in suckling rats derived from expanded (EL) as compared to contracted (control) suckling rats (Widdowson and McCance, 1960). Maximum deviation of body weight growth velocity in EL vs. control litters of animals occurred early in the postnatal period (days 3-9). Fractional protein synthesis (Ks) in heart, diaphragm and gastrocnemius at 15 postnatal days were not significantly different in animals from expanded vs. control litters (e.g., Ks in heart 35.5 +/- 3.8% per day in control and 36.1 +/- 9.2% in expanded litters). Fractional growth rates in these 3 tissues were comparable in EL and controls, while fractional rates of protein breakdown (Kd) were somewhat reduced in diaphragm and gastrocnemius from EL animals (e.g., Kd in diaphragm 10.4% per day in control and 6.1% per day in EL). Absolute rates of protein synthesis and degradation were lower in EL animals, with rates of degradation reduced more than rates of synthesis. RNA activity of striated muscle tissues at 15 postnatal days in EL animals was not reduced vs. controls in contrast to results obtained in malnourished weanling and adult rats. PMID- 1725984 TI - Clarification on use of chlorpyrifos. PMID- 1725985 TI - Epidemiology and demographic update in oral cancer: California 1973-1990. PMID- 1725986 TI - Do's and don'ts of oral soft tissue biopsy in dental practice. PMID- 1725987 TI - The pulsed Nd:YAG dental laser: review of clinical applications. AB - Since the first demonstration of a laser in 1960, numerous applications of this unique form of energy have been developed for the manufacturing, electronic, consumer and medical industries. Recent technological innovations have permitted development of lasers appropriate for use in the dental operatory. The carbon dioxide laser has been used for soft tissue surgery; the Nd:YAG laser has both soft and hard tissue applications. Advantages of laser treatment over conventional methods include minimal cellular destruction and tissue swelling, hemostasis, increased visualization of surgical sites and reduced post-operative pain. Additionally, it is possible to perform many procedures without needing anesthesia. Soft tissue clinical applications of the Nd:YAG laser include gingivectomies, gingivoplasties, operculectomies, biopsies, incising and draining procedures, frenectomies and treatment of aphthous ulcers; hard tissue clinical applications include vaporizing decay, etching enamel and dentin, desensitizing exposed root structure and creating temporary analgesia. As both clinical experiences and scientific investigations expand, possible future applications of the dental laser may well include development of new dental adhesives and composite systems, new methods for managing caries and new endodontic treatments. With its numerous benefits, the laser is having a positive impact on patients and the dental team. PMID- 1725988 TI - Dental lasers and science. PMID- 1725989 TI - Patient response to dental laser treatment; a preliminary report. PMID- 1725990 TI - A preliminary report: YAG laser treatment in pediatric dentistry. AB - Pediatric dentistry offers many opportunities to employ lasers. Observations of laser uses with the primary dentition and evaluation of laser anesthesia for permanent premolars are presented in this preliminary report. Soft tissue surgery with children and adolescents is described. Dentin became desensitized with normal pulp test readings and histology when premolars were given laser exposure. Patient acceptance was high. Enamel conditioning for bonding, decay removal, root canal cleansing and exposing unerupted teeth are the most common procedures. PMID- 1725991 TI - Guided tissue regeneration by intermittent Nd:YAG de-epithelialization. PMID- 1725992 TI - Nd:YAG pulsed infrared laser for treatment of root surface. PMID- 1725993 TI - Degranulation of acinar cells in von Ebner's gland of the rat. AB - We investigated the effect of sympathetic agonists, parasympathetic muscarinic agonists and substance P on depletion of secretory granules in acinar cells of rat von Ebner's gland. Drugs were injected intraperitoneally at several different concentrations. Antagonists were given 15 minutes before injection of the agonist, and the extent of depletion of secretory granules in glandular acini was calculated using a computerized color image analyzer. The specific alpha 2 sympathetic agonist clonidine and the beta 1-sympathetic agonist dobutamine produced a depletion of secretory granules. When combined with injections of the alpha 2-sympathetic antagonist yohimbine and the beta 1-sympathetic antagonist acebutolol, depletion of secretory granules was blocked. The parasympathetic muscarinic agonist carbachol also produced a depletion of secretory granules. QNB blocked the depletion caused by carbachol, while atropine partially inhibited depletion. The specific M1-muscarinic agonist McN-A-343 caused some depletion, although there was no significant differences between it and the control. Complete depletion of the secretory granules was achieved by carbachol stimulation superimposed on substance P stimulation. We concluded that the activation of the sympathetic alpha 2- and beta 1-receptors, as well as the M2 (M2 beta)-muscarinic and substance P receptors, results in degranulation of acinar cells in von Ebner's gland of the rat. PMID- 1725994 TI - The role of substance P in parasympathetic nerve-induced secretion in the rat submandibular gland. AB - Fluid secretion from rat submandibular saliva evoked by electrical stimulation of the chorda tympani (parasympathetic secretory stimulation) was greatly reduced in the presence of atropine. However, some secretion was always evident at frequencies of 5 Hz and higher. The substance P-antagonist ([D-Arg1, D-Pro2, D Trp7,9, Leu11]-substance P) also reduced the chorda-evoked saliva. Salivary secretion induced by the electrical stimulation of the superior cervical ganglion (sympathetic secretory nerve) was not affected by either atropine or substance P antagonist. Continuous chordal stimulation reduced the glandular content of substance P to approximately 50% after 60 min, but similar stimulation of the superior cervical ganglion failed to produce such a reduction. Electron histochemical observation showed that the chordal stimulation caused depletion of substance P from the appropriate nerve fibers within the gland. The reduced secretory capability after continuous stimulation of the chorda at high frequency (20 Hz) was reversed by infusion of a subthreshold dose of exogenous substance P. Histological examination of granular duct cells revealed no degranulation in either chorda-stimulated or substance P-infused rats. The summation of findings suggests that endogenous substance P plays a complementary role in the regulation of parasympathetic nerve-induced fluid secretion in the acinus but is minimally involved in degranulation from granular duct cells. PMID- 1725995 TI - The blood binding of cefotiam and cyclohexanol, metabolites of the prodrug cefotiam hexetil, in-vitro. AB - The binding of cefotiam and cyclohexanol to human serum, isolated proteins and erythrocytes has been studied in-vitro by equilibrium dialysis. The two molecules are 50% bound to serum proteins and the free fraction for both compounds remained constant within the therapeutic concentration range. Human serum albumin (HSA) was exclusively responsible for the cefotiam binding (48%) with a saturable process characterized by one binding site (n = 1.00 +/- 0.14) with a very weak affinity (Ka = 1457 +/- 352 M-1). Like other cephalosporins, cefotiam showed no binding to alpha 1-acid glycoprotein, lipoproteins or gamma-globulins. Cyclohexanol is mainly bound to HSA with a weak affinity (Ka approximately 1,800 M-1) but lipoproteins and alpha 1-acid glycoprotein bind about 30% of bound cyclohexanol in serum. Interactions with free fatty acids (FFA) or bilirubin were studied at physiopathological concentrations. HSA-bound cefotiam was displaced by FFA (1260 microM) and bilirubin (330 microM), whereas the cyclohexanol binding was inhibited only by FFA. The cefotiam binding site seems to be close to the warfarin site (site I) whereas cyclohexanol probably shares the diazepam site (site II) on HSA. There is no mutual inhibition of binding between cefotiam and cyclohexanol at therapeutic levels. The binding of both compounds to erythrocytes is low and restricted when measured in the presence of plasma. PMID- 1725996 TI - Effects of clomipramine on the concentrations of catecholamines, indoleamines and their metabolites in 11 rat brain regions. AB - The effects of clomipramine (CMP) treatment on the brain concentrations of catecholamines, indoleamines and their metabolites were evaluated in 11 rat brain regions. An acute CMP treatment (15 mg/kg) reduced only the 5-hydroxyindole-3 acetic acid (5HIAA) concentrations in all regions. A long-term CMP treatment (14 days, 10 mg/kg/day) decreased the concentrations of dopamine (DA), 5 hydroxytryptamine and 5HIAA in the mesencephalon, and increased the DA concentrations in the hippocampus. In comparison with the values for the long term treated rats, an acute injection of CMP (15 mg/kg) after a long-term treatment increased the norepinephrine concentrations in the hypothalamus and amygdala, and the concentrations of DA, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the basal ganglia. On the other hand, it failed to decrease the 5HIAA concentrations in the basal ganglia, frontal cortex, hypothalamus, cerebellum, pons + medulla oblongata, and mesencephalon. PMID- 1725997 TI - Introduction. Why a new calcium antagonist? PMID- 1725998 TI - Pharmacology of lacidipine, a vascular-selective calcium antagonist. AB - Lacidipine is a new 1,4-dihydropyridine (DHP) that induces a potent calcium antagonistic activity on vascular preparations. It has a markedly slow rate of onset, but its effect is still apparent after 9 h of drug washout. Calcium antagonistic activity has been evaluated on nonvascular smooth muscle, and lacidipine has proved to be more vascular-selective than standard DHP derivatives. In cardiac preparations, the concentrations required to induce negative inotropic effects are approximately 100 times higher than concentrations needed to antagonize calcium-induced contractions in vascular smooth muscle. In addition, lacidipine exhibits antioxidant properties in tests based on the autoperoxidation of rat cerebral cortical membranes. In spontaneously hypertensive rats (SHRs), lacidipine induces a potent and long-acting blood pressure reduction (ED25 = 0.19 mg/kg orally and 0.006 mg/kg intravenously, with durations greater than 12 and 3 h, respectively). In saline-loaded SHRs and at antihypertensive dosages, lacidipine increases urine volume as well as urinary excretion of sodium. In one-clip, two-kidney renal hypertensive dogs, the antihypertensive properties of lacidipine after both oral and intravenous administration were confirmed. The marked vascular selectivity of lacidipine was clearly evident both in pithed rats infused with angiotensin II and in anesthetized dogs, in which preferential and long-lasting coronary and vertebral blood flow increases were also observed. PMID- 1725999 TI - Expanding the role of calcium antagonists in hypertension. First International Lacidipine Symposium, Marbella, Spain, September 7-8, 1990. PMID- 1726000 TI - Selection of initial and maintenance dosages for lacidipine, a new once-daily calcium antagonist for the treatment of hypertension. AB - Selection of the appropriate dose of a new drug for initiation of treatment and then for maintenance is a complicated task. It requires the careful assessment and weighing of benefit vs. risk. A series of studies was carried out with lacidipine to determine the optimal initial dose in a general hypertensive population as well as in special populations in whom the pharmacokinetics may be different. The conclusions of these studies are that (a) in a general adult population, the initial dose should be 4 mg once daily; (b) in the elderly and in those with impaired liver function, the starting dose should be 2 mg once daily; and (c) renal dysfunction does not alter the usual starting dose of 4 mg once daily. PMID- 1726001 TI - Lacidipine and circadian variation in blood pressure: considerations for therapy. AB - The circadian rhythm of blood pressure has been shown to be an accurate and repeatable method of characterizing blood pressure over each 24-h period, and probably provides the best index of cardiovascular risk. Many studies have confirmed that these curves can be used to define accurately the duration and degree of antihypertensive effect of drugs, and are not influenced by the administration of placebo. Furthermore, these curves represent an ideal method for testing the efficacy of once-daily drugs. Lacidipine has been tested against these curves in 12 hypertensive patients, and has been shown to be an effective antihypertensive agent throughout the day, with a 24-h duration of action after a single oral dose and without reflex tachycardia. PMID- 1726002 TI - A double-blind comparison of the efficacy and safety of lacidipine with atenolol in the treatment of essential hypertension. The United Kingdom Lacidipine Study Group. AB - Thirty-one centers in the U.K. recruited 637 patients (aged 21 to 75 years) with mild-to-moderate essential hypertension [diastolic blood pressure (DBP) of 95 to 115 mm Hg, and systolic blood pressure (SBP) less than or equal to 200 mm Hg on three occasions]. After a 4-week placebo run-in period, 533 patients were randomized to receive double-blind 4 mg of lacidipine once daily (n = 268) or 50 mg of atenolol once daily (n = 265). If blood pressure was not controlled after 1 month (control = DBP less than or equal to 90 mm Hg, or less than or equal to 95 mm Hg if reduced by greater than or equal to 15 mm Hg from baseline), dosages were increased to 6 mg of lacidipine once daily or 100 mg of atenolol once daily. Hydrochlorothiazide (HCTZ, 25 mg once daily) was added after 2 months of active treatment if required for blood pressure control. Both lacidipine and atenolol reduced blood pressure to a similar degree over the 5 months of double-blind active treatment. The reduction achieved was maintained for the duration of the open phase of the study (to month 14). The incidence of adverse events was also similar for both drugs, and serious adverse events were rare and thought to be unrelated to the study drug therapy. The results indicate that lacidipine once daily for mild-to-moderate hypertension has an efficacy and safety similar to that of atenolol. PMID- 1726003 TI - Comparative study of lacidipine and nifedipine SR in the treatment of hypertension: an Italian multicenter study. The Northern Italian Study Group of Lacidipine in Hypertension. AB - The aim of this study was to compare the antihypertensive efficacy and tolerability of lacidipine, a new dihydropyridine calcium antagonist, and slow release nifedipine (SR) in patients with mild-to-moderate essential hypertension. After a 1-month placebo run-in period, 435 patients were randomized into a double blind, parallel-group study to receive either 4 mg of lacidipine once daily (n = 220) or 20 mg of nifedipine twice daily (n = 215). After 4 weeks, the doses for "nonresponders" were increased to 6 mg of lacidipine once daily (n = 82) and to 40 mg of nifedipine SR twice daily (n = 62). After 4 weeks, 50 mg of atenolol once daily was added to the regimens of patients still uncontrolled by lacidipine (n = 32) and nifedipine SR (n = 23) as monotherapy. Sitting blood pressure and heart rate were measured at 22-24 h after lacidipine and 10-12 h after nifedipine SR. Both calcium antagonists similarly and significantly reduced the blood pressure at rest (sitting) and on exercise, while the heart rate did not change significantly. The nature and incidence of side effects and withdrawals also did not differ between the two. In conclusion, lacidipine once daily is as effective and well tolerated as nifedipine SR twice daily in patients with mild-to-moderate essential hypertension. PMID- 1726004 TI - A double-blind comparison of the efficacy and safety of lacidipine and hydrochlorothiazide in essential hypertension. The Southern Italy Lacidipine Study Group. AB - This multicenter study was designed to assess the clinical efficacy and safety of the new once-daily calcium antagonist lacidipine in the treatment of mild-to moderate essential hypertension. Patients were randomly assigned to receive, double-blind, either lacidipine (n = 180) or hydrochlorothiazide (HCTZ, n = 182) following a 1-month placebo run-in period. Both drugs were titrated after 1 month if blood pressure was not controlled: lacidipine, from 4 to 6 mg once daily; HCTZ, from 25 to 50 mg once daily. Atenolol was added later if necessary to achieve blood pressure control. Lacidipine and HCTZ were equally effective in controlling the blood pressure levels in the majority of patients (approximately 90% after 4 months). Adverse events were those to be expected with these classes of drug and were reported in 48 (26.7%) of the patients receiving lacidipine treatment and in 34 (18.7%) of those receiving HCTZ. The diuretic produced a significantly higher incidence of hypokalemia. PMID- 1726005 TI - Efficacy and safety of lacidipine, a new long-lasting calcium antagonist, in elderly hypertensive patients. AB - Elderly patients are becoming a greater proportion of the hypertensive population. In addition, they may respond differently to drugs, both pharmacodynamically and pharmacokinetically. Therefore, approximately 25% of the patients treated with lacidipine in early studies were over 65 years old. In three double-blind, parallel-group comparative trials, 118 elderly hypertensive patients were treated with lacidipine or comparators, either atenolol, nifedipine SR, or hydrochlorothiazide (HCTZ). Lacidipine was at least as effective in lowering blood pressure as the comparators, as well tolerated as nifedipine SR (but with a significantly lower incidence of edema) and HCTZ, and better tolerated than atenolol. In a double-blind, placebo-controlled, parallel-group, dose-response study of 131 elderly hypertensive patients randomized to receive either lacidipine or placebo, lacidipine significantly reduced the diastolic blood pressure compared with placebo. In the long-term evaluation, more patients required titration with 2 mg of lacidipine than with 4 mg. These results suggest that 4 mg once daily is an effective and well-tolerated antihypertensive treatment in the elderly and, as with the general adult population, represents the optimal long-term maintenance dose. PMID- 1726006 TI - Safety profile of lacidipine: a review of clinical data. AB - The clinical safety of the new dihydropyridine calcium antagonist lacidipine was assessed in 14 clinical trials, including 1,372 patients treated with lacidipine and 687 treated with an alternative antihypertensive agent. The type and incidence of adverse events seen with lacidipine were characteristic of this class of drug, being mainly those associated with the pharmacologic effect of vasodilation, but with a lower incidence of edema than seen with nifedipine. There were no unexpected adverse effects. Hence, lacidipine is likely to be well accepted in general clinical use. Furthermore, as it has the advantages of a long duration of action and once-daily dosage, the benefit/risk ratio indicates that lacidipine is a suitable agent for the first-line treatment of hypertension across a wide range of patients. PMID- 1726007 TI - Antihypertensive treatment: is blood pressure-lowering the only goal? PMID- 1726008 TI - Effects of lacidipine on the carotid and cerebral circulations in essential hypertension. AB - Arterial diameter, blood flow, and vascular resistance of the common carotid artery were studied, using a pulsed Doppler system, in patients with uncomplicated sustained essential hypertension compared with age-matched normotensive subjects. In the hypertensive subjects, arterial diameter and blood flow remained close to the normal range while vascular resistance was increased. Following antihypertensive drug treatment with converting enzyme inhibitors, nitrates, or calcium antagonists, dilation of small or large arteries, or both, may be obtained, depending on the administered compound. Calcium blockade by lacidipine produced a predominantly carotid arteriolar dilation with no change in arterial diameter, suggesting independent consequences on cerebral circulation and baroreflex mechanisms. PMID- 1726009 TI - Effects of oral lacidipine on cardiopulmonary function at rest and during exercise in normal subjects. AB - A randomized, double-blind, crossover, placebo-controlled study was carried out to evaluate the effects of a single oral 4-mg dose of lacidipine vs. placebo on cardiopulmonary circulation at rest and during exercise. Twelve healthy volunteers were randomized to receive either placebo or 4 mg of lacidipine once daily for 2 days, followed by a 3-day washout period, after which they received alternate treatment. Patients were assessed before and at 60, 90, and 180 min after dosing. At 120 min, a maximum exercise test with a treadmill was performed according to the Bruce protocol. No relevant changes with placebo or lacidipine were observed in the respiratory function tests whereas 4 mg of lacidipine increased pulmonary effective blood flow (Qp. eff.) and stroke volume index (SVI) at 60 min, reaching a peak at 90 min; at 180 min, these effects, although diminished, were still present. The arteriovenous oxygen difference [C(a-v)O2] decreased, but reverted to normal values by 180 min. No differences in maximum attained Qp. eff. and oxygen consumption (VO2) during exercise were observed. Only the heart rate was higher both before and after treatment with lacidipine. Lacidipine increased Qp. eff. in these normal subjects without relevant effects on respiratory function. Performance on exercise testing after dosing was normal, although drug-induced vasodilation was present. PMID- 1726010 TI - Left ventricular hypertrophy: an independent risk factor. AB - Left ventricular hypertrophy (LVH), an increase in the muscle mass of the left ventricle, has been identified as a powerful risk factor for future cardiovascular morbidity and mortality. The risk of acute myocardial infarction, congestive heart failure, sudden death, and other cardiovascular events increases six- to eightfold with the presence of LVH. The increase in myocardial mass lowers coronary reserve and enhances cardiac oxygen requirements, gives rise to ventricular ectopy, and impairs left ventricular filling and contractility. Hypertension, obesity, advanced age, valvular heart disease, and other disorders that cause an increase in the hemodynamic burden can lead to LVH. Left ventricular hypertrophy and its sequelae can be reduced by specific antihypertensive therapy but, despite these promising findings, future epidemiological studies are necessary to document the clinical benefits of a reduction of LVH. PMID- 1726011 TI - Remodeling of left ventricular geometry and function induced by lacidipine and nifedipine SR in mild-to-moderate hypertension. AB - The effects of nifedipine SR and lacidipine on blood pressure, left ventricular (LV) hypertrophy, and diastolic function were evaluated in 15 male patients (mean age of 45 +/- 8 years) with mild-to-moderate hypertension. Clinical evaluation and complete echocardiography were performed at baseline (after 15 days of washout). Seven patients received nifedipine SR (20-40 mg twice daily) and eight lacidipine (4-6 mg once daily). Clinical evaluation and complete echocardiography studies were repeated after 1 and 6 months of active treatment. In addition, full echocardiographic evaluations were carried out in 10 normotensive subjects (for comparison). Results were similar with either drug. After 1 month of therapy, systolic and diastolic blood pressures were significantly decreased (p less than 0.05); after 6 months of therapy, further changes were observed, confirming their significance. Analysis of our data suggests that (a) mild essential hypertension induces early modifications of LV geometry with consequent LV diastolic function characterized by a prolonged and incomplete diastolic filling; thus, LV wall thickness may be increased with a simultaneous reduction in LV end-diastolic short-axis diameter and volume; and (b) antihypertensive treatment with calcium antagonists such as nifedipine SR and lacidipine leads to early normalization of LV geometry and diastolic function without a significant change in total LV mass. PMID- 1726012 TI - Vascular protection of lacidipine in salt-loaded Dahl-S rats at nonsustained antihypertensive doses. AB - The aim of this study was to characterize the antihypertensive and vasoprotective properties of lacidipine in salt-loaded Dahl-S rats, a suitable animal model of malignant hypertension. After 9 weeks of a high (8%) sodium chloride (NaCl) diet, 80% of the untreated Dahl-S rats died (20% survival rate) whereas a 100% survival rate was observed with chronic treatment with lacidipine at doses of 0.1 (equivalent to the recommended dose in humans), 0.3, 1, and 10 mg/kg once daily by gastric gavage. The most interesting results included the following: (a) Only the highest dose tested (10 mg/kg once daily) was able to control the increase in blood pressure, which was measured 24 h after the preceding administration of drug, yet a 100% survival rate was maintained. (b) There appeared to be prevention of brain lesions, which is very likely the cause of the survival of all of the lacidipine-treated rats in this study. (c) A clear dose-related vascular protection was observed in other tissues. In conclusion, lacidipine protects against the vascular damage and concomitant increase in mortality of salt-loaded Dahl-S rats even at doses that do not adequately control the development of hypertension. PMID- 1726013 TI - Antiatherogenic properties of calcium antagonists. AB - Atherosclerosis results from multiple factors, and involves several mechanisms including endothelial monocyte and smooth muscle cell changes, cholesterol accumulation, lumen stenosis, necrosis, mineralization, plaque hemorrhage, rupture, and thromboembolism. Calcium antagonists have been shown in hypercholesterolemic animal models to reduce atherosclerosis. This effect cannot be explained on the basis of changes in blood pressure, therefore suggesting that calcium channel antagonists have a direct effect on arterial wall processes associated with plaque evolution. The antiatherosclerosis properties of calcium antagonists have been tested in human subjects and suggest that these compounds inhibit new lesion development. Recent developments in B-mode ultrasonography allow investigators to detect and monitor atherosclerosis noninvasively. This method is being used in several trials within the U.S. and Europe to evaluate treatment effects on carotid atherosclerosis. Carotid artery disease is associated with transient ischemic attacks, ischemic cerebral infarction, and with risk for coronary artery disease. B-mode ultrasonography is a powerful method for monitoring atherosclerosis progression. The combination of this technology with calcium antagonist treatment will allow evaluation of the efficacy of intervention on the arterial wall during the asymptomatic stages of atherosclerosis evolution. PMID- 1726014 TI - Clinical pharmacology of lacidipine. AB - The safety and tolerability of lacidipine was assessed in a volunteer population, and its pharmacodynamic and pharmacokinetic profiles evaluated. In normotensive subjects, single oral doses of 3-5 mg of lacidipine produced a dose-related fall in peripheral vascular resistance. This was accompanied by reflex-mediated increases in heart rate and cardiac output to maintain blood pressure. Adverse events were those typically related to the vasodilatory action of lacidipine, such as flushing and headache. A 4-mg dose of lacidipine elicited a cardiovascular response equivalent to that with 10 mg of nifedipine, given as a single oral dose. Lacidipine did not affect sinoatrial or atrioventricular conduction in the healthy subjects studied. Two specialized electrophysiologic studies in patients confirmed that lacidipine does not affect pacemaker tissue and that it exhibits relative selectivity for the vascular smooth muscle. Lacidipine is eliminated primarily by hepatic metabolism, and extensive first pass loss occurs after oral dosing. Absolute bioavailability is less than 10%. The systemic availability of lacidipine was increased in healthy elderly subjects and in patients with impaired hepatic function, but not in patients with impaired renal function. PMID- 1726015 TI - The CD4 receptor: post binding events, conformational change and the second site. PMID- 1726016 TI - A proposed molecular model for the carboxy terminus of HIV-1 gp120 showing structural features consistent with the presence of a T-cell alloepitope. AB - A computer graphics molecular model of the C terminus of gp120 of HIV has been constructed using predicted secondary structure based on homologies with proteins for which X-ray crystallographic data have been published. The model shows sequences known to be important in CD4 binding in close proximity to regions with a high probability of forming alpha helical and beta strand motifs. The orientation adopted by these domains approximates to the known 3D structure of HLA-A2 alpha 2 chain without constraints based on HLA-A2 as a template being introduced. The model may therefore represent an energetically favourable conformation for a part of gp120 which mimics the binding domain for the T-cell receptor on MHC molecules. Recognition of gp120 as an alloepitope in high affinity association with CD4 would explain many of the sequelae of acquired immune deficiency on HIV infection. PMID- 1726017 TI - Vascular endothelium: a potential role in HIV infection and the pathogenesis of Kaposi's sarcoma: observations and speculations. AB - Kaposi's sarcoma (KS) has become a source of interest in recent years primarily for its strong association with the acquired immune deficiency syndrome (AIDS). Endothelial cells (EC) are central to inflammation and can regulate coagulation and leucocyte emigration and may be central to the development of the disease. As they are also capable of being infected by HIV in vivo, this infection may contribute to the immunosuppressive effects of HIV seen in AIDS. Recent work has shed new light on the mechanisms involved in EC proliferation. The aim of this article is to review such evidence implicating EC in the development of KS. Additionally, hypotheses will be put forward to explain the mechanism of the vascular proliferation in KS and the possible role of EC in HIV infection. There is therefore enormous potential for the therapeutic targeting of endothelium to control these diseases. PMID- 1726018 TI - Chemotherapy against human immunodeficiency virus infection. PMID- 1726019 TI - [The existence of the ribonucleoprotein branching enzyme in rabbit skeletal muscle and ribozyme corresponding to it]. AB - Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously. PMID- 1726020 TI - [Hybridase cleavage of RNA. V. Complementary precision of cleavage]. AB - The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch. PMID- 1726021 TI - [Properties of the replicator region of the natural plasmid pLG13 containing genes of the EcoRV restriction-modification system]. AB - The previously constructed plasmid pILRV8 that induces endonuclease EcoRV gene overexpression kills cells of some E. coli strains under the induction of this enzyme synthesis. Cell transformation by natural plasmid pLG13 carrying genes of the EcoRV restriction--modification system was found to appreciably enhance cell viability ("survival") under endonuclease overproduction. A plasmid pLG13 region located in immediate proximity to the methylase gene was shown to be responsible for the above effect. This region was also capable for autonomous replication. The analysis of the DNA primary structure in the found replicator region allowed to refer the pLG13 to ColE1 family plasmids. Perturbations in the region lead to loss of the "survival" effect and change of the plasmid replicative properties. A relationship between the replicon elements, the EcoRV genes region and "survival" effect is discussed. Based on the replicon found multicopy vector molecules have been constructed. PMID- 1726023 TI - Do astroglial cells participate in the process of human spinal cord myelination? AB - The material comprised spinal cord segments C8 or Th1 of 6 human fetuses aged from 16-17 up to 34 weeks and 6 infants aged from one day to 3 years. On formalin fixed, paraffin-embedded sections, peroxidase-antiperoxidase Sternberger's et al. (1970) method for visualization of astrocytic proteins (S-100 and GFAP), myelin basic protein (MBP) and neuron specific enolase (NSE), was used. Parallelly to the increasing immunoreactivity to MBP, more S-100 and GFAP-positive cells were observed. Immunoreactivity to S-100 was more distinct in astrocytic perikarya, whereas, in GFAP reaction immunostaining of astrocytic processes was more pronounced. It is very difficult to explain why reactivity of astrocytes appears during myelination. PMID- 1726022 TI - [Formation of phosphonoester bonds, catalyzed by DNA polymerases]. AB - 3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma diphosphate (II) were synthesised. Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E. coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective. Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues. Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I. The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E. coli. PMID- 1726024 TI - [Hematopoietic growth factors]. AB - In the paper the role of interleukin-3, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF), in the proliferation and differentiation of haemopoietic cells and pathogenesis of leukaemia are reviewed. Role of erythropoietin, thrombopoietin and other thrombopoiesis-stimulating factors in the development of hematopoietic is presented. Potential applications of recombinant haemopoietic growth factors in the treatment of myelodysplastic syndromes. AIDS and other haematologic, infections and neoplastic disorders are also discussed. PMID- 1726025 TI - Synaptic potentials in locus coeruleus neurons in brain slices. AB - Neurons of the locus coeruleus (LC) fire action potentials spontaneously in vitro in the absence of any stimulation. This spontaneous activity is thought to arise from intrinsic membrane properties that include a balance between at least two ion conductances. One is a persistent inward sodium current that is active near the threshold for action potential generation. The second is a calcium-dependent potassium current that is activated following the entry of calcium during the action potential, is responsible for the after-hyperpolarization following the action potential, and decays over a period of 1-2 sec following the action potential. The spontaneous activity of LC neurons can be altered by both excitatory and inhibitory synaptic inputs. One excitatory input has been described that is mediated by glutamate receptors of both the non-NMDA and NMDA subtypes. Inhibitory synaptic potentials include those mediated by GABA (acting on GABAA-receptors), glycine (acting on a strychnine-sensitive receptor) and noradrenaline (acting on alpha 2-adrenoceptors). The presence of synaptic potentials mediated by these transmitters, studied in vitro, correlate with studies made in vivo and with histochemical identification of synaptic inputs to the locus coeruleus. PMID- 1726026 TI - Angiotensin II and the locus coeruleus. AB - The locus coeruleus (LC) is a putative site of action for angiotensin II in the brain. Immunocytochemical studies have identified angiotensin II-like immunoreactive material in nerve terminals innervating the LC, and the LC contains one of the highest densities of angiotensin II receptor binding sites in the rat brain. Recent studies using selective neurotoxins suggest that the binding sites for angiotensin II in the LC are present on noradrenergic perikarya. Angiotensin II receptors are now known to exist as two subtypes that are distinguishable both pharmacologically and biochemically. Radioligand binding studies using agonists and antagonists selective for these angiotensin II receptor subtypes indicate that the rat LC contains a mixture of the two known angiotensin II receptor subtypes, but that the PD123177-sensitive AII beta receptor subtype is predominant. Comparisons of spontaneously hypertensive rats with normotensive rats indicates that angiotensin II and its receptors in the LC are elevated in the hypertensive rat strain. Studies of the biochemical and physiological actions of angiotensin II in the LC have not yet established an agreed-upon function for angiotensin II in this nucleus. PMID- 1726027 TI - Selective effects of DSP-4 on locus coeruleus axons: are there pharmacologically different types of noradrenergic axons in the central nervous system? AB - There is considerable evidence from biochemical studies that the transmitter depleting action of drugs and neurotoxins which act upon central noradrenergic (NA) axon terminals is not uniform in different brain regions. Among NA axons, those originating in the locus coeruleus (LC) have been proposed to be most susceptible to the action of NA neurotoxins such as N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine (DSP-4). The studies described here were conducted to determine whether this differential susceptibility to DSP-4 reflects a pharmacological heterogeneity between different populations of NA axons. To determine whether DSP 4 acts selectively upon LC axons, we have characterized the effects of this drug on NA axons in different brain regions, by using noradrenaline and dopamine-beta hydroxylase (D beta H) immunohistochemistry. Following systemic administration of DSP-4, there was an almost complete loss of noradrenaline and D beta H staining in brain regions innervated by LC axons. No effects of the drug treatment were detected in brain regions innervated primarily by non-coerulean NA axons. These results demonstrate that both the transmitter-depleting and the neurodegenerative action of DSP-4 are restricted to NA axons originating in the LC. To explore the basis for this selectivity, noradrenaline uptake studies were conducted using synaptosomes from brain regions in which NA axons differ in their response to DSP 4. The results reveal a significant difference in the affinity of DSP-4 for the noradrenaline uptake carrier in cortical and hypothalamic synaptosomes. This finding is compatible with the hypothesis that the noradrenaline uptake carrier is pharmacologically distinct in LC and non-coerulean NA axons. This heterogeneity in noradrenaline uptake raises the question whether other drugs may also have differential actions on LC and non-coerulean NA neurons. PMID- 1726028 TI - Actions of norepinephrine in the cerebral cortex and thalamus: implications for function of the central noradrenergic system. AB - Norepinephrine (NE) has potent and long-lasting ionic effects on cortical and thalamic neurons. In cortical pyramidal cells, activation of beta-adrenergic receptors results in an enhanced excitability and responsiveness to depolarizing inputs. This enhanced excitability is expressed as a reduction in spike frequency adaptation and is mediated by a marked suppression of a slow Ca(++)-activated potassium current known as IAHP. In the thalamus, application of NE results in the suppression of ongoing rhythmic burst activity and a switch to the single spike firing mode of action potential generation. This effect is mediated through an alpha 1-adrenergic suppression of a resting leak potassium current, IKL, and through a beta-adrenoceptor-mediated enhancement of the hyperpolarization activated cation current Ih. Together with the actions of other neuromodulatory neurotransmitters (i.e., acetylcholine, histamine, serotonin) these effects facilitate the switch of these neurons from a state of rhythmic oscillation and low excitability during drowsiness and slow-wave sleep to a state of increased excitability and responsiveness during periods of waking, attentiveness and cognition. PMID- 1726029 TI - Neurochemicals in the dorsal pontine tegmentum. AB - Detailed maps of neurochemicals in the locus coeruleus and adjacent dorsal tegmental areas are discussed in this chapter. The locus coeruleus appears to be one of the most complex brain regions with six neurochemicals (acetylcholinesterase, tyrosine hydroxylase, galanin, neuropeptide Y, neurotensin, and vasoactive intestinal protein) contained within the cell bodies. PMID- 1726031 TI - Chloride channel inhibition by the venom of the scorpion Leiurus quinquestriatus. AB - The venom of the scorpion Leiurus quinquestriatus produced a significant, reversible inhibition of reconstituted Cl- channels of the small conductance type found in rat colonic epithelial cells. The kinetics of single-channel block by this venom were consistent with a first-order binding reaction in which the binding of one ligand molecule is sufficient to induce channel block. Single channel mean block times were c.6 sec at -20 mV, and a KI in the submicromolar range is predicted. The active component has a mol. wt of roughly 5000 as judged by molecular sieve chromatography. PMID- 1726030 TI - Alterations in the locus coeruleus in dementias of Alzheimer's and Parkinson's disease. AB - For diagnostic purposes, a differentiation can be made between the locus coeruleus (LC) in normal brain and the LC, in senile dementia of the Alzheimer's type (SDAT) and Parkinson's disease (PD). The differentiation is based on findings concerning the morphological alterations of the TH-immunoreactive; neurons, on the topographical distribution of neuron loss within the length of the LC, and on the total reduction in cell number. PMID- 1726032 TI - Interferon therapy in hepatitis C virus (HCV) induced chronic hepatitis: clinical significance of pretreatment with glycyrhizine. PMID- 1726033 TI - [Total protein and RNA biosynthesis in the liver cells of the long-tailed suslik Citellus undulatus in different functional states]. AB - The synthesis of proteins and RNA have been studied by means of labeled precursors of 14C-amino acids and 14C-orotic acid in liver cells of hibernating, arousing and active ground squirrels (Citellus undulatus). The investigations conducted testify to the possibility of CHI-inhibited synthesis of proteins in liver cells of hibernating ground squirrels, which occurs, apparently, on preexisting polyribosomes. The low level of protein biosynthesis in this state is stipulated not only by the Arrhenius dependence, but by the dissociation of polyribosomes into monosomes as well. In the course of arousal, an intensive assembling of polyribosomes was detected even under the inhibition of RNA synthesis by actinomycin D, testifying to the presence of a pool of mRNA [correction of iRNA] in liver cells, which are necessary for the observed activation of translation. The intensity of protein synthesis on arousal is also stimulated by the activation of transcription. PMID- 1726034 TI - [Aphasia--intensive training for rehabilitation]. PMID- 1726035 TI - Regulation of acute phase reaction by transforming growth factor beta in cultured murine hepatocytes. AB - Transforming growth factor-beta (TGF beta 1), a multipotent immunoregulatory peptide produced by human platelets, has been shown to stimulate the synthesis of fibrinogen, contrapsin, complement component C3, and alpha-1-proteinase inhibitor by murine hepatocytes cultured for 2 days in DMEM containing 1 microM insulin and dexamethasone and 0.2% BSA. In the range of 10 pg to 10 ng/ml TGF-beta 1 did not elicit any change in albumin secretion. Two main inflammatory cytokines: interleukin-6 (IL-6) and interleukin-1 (IL-1), known to stimulate two different subsets of murine acute phase plasma proteins, failed to increase contrapsin and alpha-1-proteinase inhibitor production. Epidermal growth factor (EGF) in the concentration 1 ng to 10 ng/ml effectively counteracted the stimulatory effect of TGF-beta 1 on acute phase protein production. TGF-beta 1-induced fibrinogen protein levels were associated with increased beta-fibrinogen mRNA content. TGF beta 1 appears to be an additional physiological factor responsible for the direct stimulation of normal mouse hepatocytes to acute phase response. PMID- 1726036 TI - Prophylaxis of thromboembolism in orthopaedics and traumatology. AB - In a retrospective study we collected patient data relating to clinically detected postoperative thromboembolic complications before (n = 1710) and after (n = 2212) adopting a protocol for prophylaxis of deep vein thrombosis (DVT). A relatively low incidence of clinically diagnosed DVT, four per thousand, before systematic instructions for prophylaxis in 1981 could be explained by the frequent use of dextran as a volume expander in critically ill patients. However, following systematic prophylaxis the frequency of DVT decreased further (one per thousand). PMID- 1726037 TI - SV40 T antigen transgenic mice: cytotoxic T lymphocytes as a selective force in tumor progression. PMID- 1726038 TI - Alpha-Fetoprotein and renal dysfunction of the fetus. AB - Alpha-fetoprotein (AFP) levels were measured in 25 pregnancies with renal dysfunction of the fetus. Oligohydramnios was found in 14 of 16 patients diagnosed by 20 weeks of gestation and in all of those diagnosed later. AFP levels in maternal serum were normal in 17 (68%), elevated in five (20%), and low in three patients (12%). AFP serum levels up to 24 weeks of gestation were significantly higher than after 24 weeks. AFP levels in amniotic fluid obtained from amniocentesis in six patients were normal or slightly elevated. We suggest that the concentration of AFP in amniotic fluid and maternal serum does not only depend on the physiologic proteinuria of the immature fetal kidneys but also on the permeability of the not yet keratinized fetal skin and the diluting volume of the amniotic fluid. PMID- 1726039 TI - Inhibition of HIV-1 infection by alkylureas. AB - The risk of infection by HIV-1 through transfusion of contaminated blood products has been markedly decreased but not eliminated by serological screening of donors. Methods are required to further minimize or eliminate the risk of infection of blood product recipients. We therefore examined the capacity of alkylureas to inhibit infectivity of HIV-1. Incubation of free HIV-1 virions with alkylureas suppressed their infectivity, and the minimal inhibitory concentration of the alkylureas was related to the length of the alkyl chain. Butylurea, the most potent inhibitor of HIV-1, inhibited the infectivity of 10(5) median tissue culture infective dose (TCID)50 of HIV-1, chronically HIV-1-infected H9 cells and mononuclear cells from two HIV-1-infected patients. Size fractionation of HIV-1 following incubation with butylurea indicated that the structure of the virus was disrupted by butylurea. This study demonstrates that butylurea, at a concentration that has been shown not to affect red blood cell function, can inhibit infectivity of extracellular and intracellular HIV-1. Since the HIV-1 inhibitory capacity of the alkylureas increases with the length of the alkyl side chain, it is likely that hydrophobic interactions between the alkylureas and HIV 1 are responsible for the observed effect. PMID- 1726040 TI - MS-8209, a new Amphotericin B derivative that inhibits HIV-1 replication in vitro and restores T-cell activation via the CD3/TcR in HIV-infected CD4+ cells. AB - A new Amphotericin B derivative, MS-8209, which retains high antifungal activity with greatly reduced toxicity and improved solubility, has been developed. We investigated the antiviral properties of MS-8209 in Jurkat and CEM T-cell lines and in peripheral blood mononuclear cells infected in vitro with HIV-1BRU. Our results demonstrate, by determination of reverse transcriptase activity and p24 antigen level titration in cell culture supernatants, that MS-8209 inhibits HIV-1 replication in all cell types at concentrations without cytotoxicity. MS-8209 also prevents membrane expression of the HIV-1 large envelope glycoprotein gp120 and the decrease in CD4 level at the surface of infected cells. HIV-1-infected Jurkat cells exhibit a severe signalling defect at CD3 stimulation. Treatment with MS-8209 restores normal responsiveness at CD3 as assessed by measurement of inositol triphosphate accumulation and calcium flux. Finally, our results indicate that MS-8209 inhibits HIV-1BRU replication without preventing virus binding and penetration into target cells. PMID- 1726041 TI - Pharmacokinetics of intravenous pentosan polysulphate (HOE/BAY 946) in HIV positive patients. PMID- 1726042 TI - RNA editing. AB - Since its discovery, RNA editing in kinetoplastid mitochondria has proven a fascinating topic of study, and the last one and a half years have witnessed enormous advances in our understanding of this unprecedented form of RNA processing. The information flow in this RNA editing, once considered a candidate for defying the central dogma, is now known to conform to the DNA-to-RNA-to protein paradigm, with the novel feature that the sequence of an edited region is not actually present in any DNA segment, but instead derives by a novel micro interdigitating of information encoded in multiple DNA regions. PMID- 1726043 TI - The kit ligand encoded at the murine Steel locus: a pleiotropic growth and differentiation factor. AB - The c-kit receptor and its recently identified ligand are allelic with the murine White Spotting and Steel loci, respectively. These observations brought to light the functions of the c-kit receptor system in melanogenesis, gametogenesis and hematopoeisis during embryogenesis and in postnatal life. The recent molecular analysis of several White Spotting and Steel alleles has provided insights into the mechanism of c-kit ligand-mediated processes, including cell proliferation, cell migration and cell survival. Furthermore, the availability of the kit ligand has allowed in vitro investigations of the pleiotropic functions of c-kit in development and cell differentiation to be carried out. PMID- 1726044 TI - Differential and coordinate regulation of TH and PNMT mRNAs in chromaffin cell cultures by second messenger system activation and steroid treatment. AB - Primary cultures of chromaffin cells were prepared from bovine adrenal medullae and the levels of mRNA for tyrosine hydroxylase (TH) and phenylethanolamine N methyltransferase (PNMT) determined. The cells expressed moderate levels of TH mRNA and low levels of PNMT mRNA. The latter appeared to be more sensitive than TH mRNA to variations in the culture medium. The treatment of cultures with agents that activate signal transduction pathways, forskolin or phorbol esters, dramatically enhanced the expression of both mRNAs. The forskolin-induced increases in the steady-state levels of TH and PNMT mRNAs occurred rapidly and were apparent within 5 hours. These data suggest that the TH and PNMT genes can be regulated by second messengers. In contrast, dexamethasone treatment dramatically increased PNMT mRNA with no change in TH mRNA. The increase in PNMT mRNA was apparent within 6 hours of addition of the drug to the culture medium. PMID- 1726045 TI - Phosphoprotein B-50: localization of proteolytic sites for S. aureus V8 protease using truncated cRNAs for cell-free translation. AB - B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15 kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme. PMID- 1726046 TI - Protein parameters of differential gene activation during development and tumorigenesis. AB - Various models of normal and abnormal developmental systems were addressed to get an insight into molecular parameters of cell differentiation at the level of protein gene products. Electrophoretic analysis of heterogeneous protein mixtures permitted qualitative analysis of developing systems, particularly during organogenesis in mammals, as well as of neoplastic growth in the animal and plant kingdoms. From our earlier findings indicating that the definite protein patterns characteristic of adult organs are acquired long after the adult morphological and histological characteristics of these tissues have developed, it has been repeatedly proven that quantitative changes in whole proteins is not a dependable indicator of cell differentiation. PMID- 1726047 TI - Prolonged osmification as an indicator of the differentiation process in the endomembrane system. AB - The technique of prolonged osmification was used in the analysis of reducing capacity of perinuclear space, endoplasmic reticulum and cis-Golgi cisternae in different epithelial cells during embryonic differentiation and immediately after the birth. Cells of the mouse gastric and intestinal epithelium and of the exocrine pancreas and mammary gland were analyzed. It was shown that endomembrane compartments exhibit high variability in their capacity to reduce OsO4 into lower valency oxides. Typical staining of cis-Golgi cisternae by osmium black does not occur before the cells achieve the developmental state in which production of specific products starts. The changes in stainability occurring from the perinuclear space and endoplasmic reticulum towards the cis-Golgi cisternae indicate a maturation pathway with no direct correlation to the chemical characteristic of the substances produced in different cell types. In the mammary gland the reduction capacity of endoplasmic reticulum disappeared with the intensive synthesis of lipids. Considering our previous results and those of other authors, the possible reasons for the observed dynamics in reducibility in particular segments of endomembraneous space are discussed. PMID- 1726048 TI - Effect of insecticides (Dimiline WP 25, Torak EC 24 and Gamacide 20) on hydra (Hydra vulgaris Pallas). AB - Investigations showed that the three insecticides used had the most damaging effect upon hydra immediately after treatment. The tentacles and the hypostome are the parts most often damaged. Inse the affected cells, lesions appear in the intracellular membranes, the nucleus shell and the membranes of the mitochondria, Golgi complex and the endoplasmic reticulum, while the cell membrane is preserved. The damaged parts of the body regenerate within three days. Zymogen cells play a significant role in the course of regeneration. They dedifferentiate into gastrodermal interstitial cells and later into other types of cells of the ectoderm and the gastroderm. Apart from their intense participation in regeneration, these totipotent cells also invariably participate in the formation of new hydra buds. It was observed that Dimiline WP 25 and Torak EC 24 in the concentrations used stimulate asexual reproduction of this animal. PMID- 1726049 TI - Development of the notochord in normal and malformed human embryos and fetuses. AB - In view of its possible involvement in early embryogenesis and teratogenesis, the developmental characteristics of the human notochord were studied by light and electron microscopy and immunohistochemistry on 20 human conceptuses (5th-22nd week). At the earliest embryonic stages examined, the notochord is closely related to both the pharyngeal endoderm and the neuroectoderm of the posterior (tail) end of the neural tube. In both regions the interspace is bridged by slender cytoplasmic processes, lined with basal lamina and filled with amorphous extracellular material containing collagen types III and IV and laminin. The notochordal cells express cytokeratin brightly and vimentin weakly. As embryonic age progresses, the notochord gradually separates from the epithelia, becomes the axis of developing spinal column and undergoes progressive cell degeneration and rearrangement within the vertebral bodies. This is associated with extensive production of extracellular material and the first appearance of fibronectin. Intracellularly, the expression of vimentin gradually increases, while that of cytokeratin slightly weakens. Changes in the notochord parallel other developmental events in axial organs. In anencephalic fetuses the course of the notochord is irregular and partly interrupted with segments outside the basichondrocranium in specimens with craniorachischisis. PMID- 1726050 TI - An aphasia group intensive efficacy study. AB - The response of five adults with chronic aphasia to a 4-week intensive treatment course is presented. Using four language tests, pre-intervention stability was demonstrated over the month preceding the treatment programme. Following the intervention period, two subjects showed improvement in one test, and more widespread changes occurred in three subjects. On further assessment 1 month after treatment had finished, one of the two subjects who had made least progress performed better than at the end of the course, and one had returned to pre treatment level. Although there were indications of regression in two of the three other subjects, this was not to pre-treatment level. Only one subject maintained the gains made fully. Given the short duration of the course, the changes in performance noted suggest that, providing transport is available, intensive treatment of aphasia should be available to patients with sufficient motivation and stamina. PMID- 1726051 TI - A profile of aphasia services in three health districts. AB - The study examines language rehabilitation provisions for aphasic people and their families in three health districts, as perceived by speech and language therapists. The study is exploratory. Semi-structured interviews were carried out with each District Speech Therapist and with speech and language therapists whose caseloads regularly included aphasic people, and documentary evidence was used where available. Comparison is made of: speech and language therapy expenditure and staffing in the three districts (West Suffolk, West Essex and Newcastle-upon Tyne); aphasia therapy staffing; caseloads of aphasic people; patterns of treatment (inpatient/outpatient/community; individual/group) and reasons for these; provision for relatives; relationships between speech and language therapy and other services; volunteer involvement; and speech and language therapists' work situations (support from colleagues, post-qualification training, secretarial support and accommodation). Differences are found in levels of provision, with Newcastle having considerably more resources devoted to aphasia services than the other two districts, and modes of service delivery. Some shared concerns are identified (e.g. relationships with other professions, accommodation and transport). Implications of the findings are discussed and areas for further research identified. PMID- 1726052 TI - [Molecular mechanisms of the antiviral effect of interferon]. AB - The report presents the main aspects of the problem of the interferon action on viruses: definition and types of interferon, antiviral effect of the many species of alpha interferon, receptors for interferon, molecular effectors inducing the antiviral state, molecular cloning of proteins mediating gene transcription activation induced by the interferon. PMID- 1726053 TI - Serum, phorbol ester, and polypeptide mitogens increase class 1 and 2 heparin binding (acidic and basic fibroblast) growth factor gene expression in human vascular smooth muscle cells. AB - Vascular smooth muscle cell proliferation is regarded as a key early event in the pathogenesis of atherosclerosis. Heparin-binding growth factor (HBGF)-1 and HBGF 2, also referred to as acidic and basic fibroblast growth factor, are potent mitogens for human vascular smooth muscle cells. These cells coexpress HBGF-1 and HBGF-2 and thus represent a vessel wall source for both polypeptides. In this report, we demonstrate that HBGF-1 and HBGF-2 expression is increased when quiescent human smooth muscle cells are treated with fetal bovine serum. The kinetics of HBGF-1 and HBGF-2 mRNA accumulation following serum treatment are distinct. In addition, HBGF-1 transcripts remain elevated for a longer time period; this may reflect the different decay rates of the HBGF-1 and HBGF-2 mRNAs. Serum-inducible HBGF-1 and HBGF-2 mRNA expression does not occur when RNA synthesis is repressed by actinomycin D but can occur in the presence of cycloheximide, an inhibitor of protein synthesis. Immunoprecipitation experiments indicate that serum treatment also increases HBGF-1 and HBGF-2 production. Smooth muscle cells treated with phorbol 12-myristate 13-acetate or certain combinations of polypeptide growth factors also express increased levels of HBGF-1 and HBGF-2 transcripts. Potential sources for these growth factors in vivo include platelets, macrophages, and T lymphocytes; thus, smooth muscle cells located at sites of vascular injury or inflammation may express elevated levels of HBGF-1 and HBGF-2. PMID- 1726054 TI - Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells. AB - Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells. PMID- 1726055 TI - The extraction and characterization of human nail keratin. AB - We investigated nail keratins obtained by sequential extraction with increasing reducing agent concentrations (50 mM and 200 mM 2-mercaptoethanol) and examined each of the extracted keratins by gel electrophoresis followed by immunoblotting. It was found that nail contained epidermal and hair keratins. With 50 mM 2 mercaptoethanol, only epidermal type of keratins including skin differentiation specific-keratin could be extracted, whereas hair type of keratins required 200 mM 2-mercaptoethanol for extraction from nail. These results may indicate that nail has unique properties with respect to it's keratin compositions. The difference in durabilities of epidermal and hair keratins in nail against 2 mercaptoethanol suggests that two different types of the keratin filaments which are composed of epidermal or hair keratin polypeptides may have differing structural features, despite of co-existence in nail. PMID- 1726056 TI - In vitro IgE eluation and histamine releasability from peripheral leukocytes of atopics and normals. AB - The relation between the amount of cell-bound IgE and the histamine 'releasability' of peripheral leukocytes was studied in 28 patients with atopic diseases and 26 non-atopic controls after in vitro stimulation with anti-IgE. Cell-bound IgE was eluted in acid buffer (pH 3.7) and the amount of histamine released (HR) into supernatant at this pH was measured. Incubation with acetate buffer (pH 3.7) induced significantly higher spontaneous HR (32 net percent) in atopics compared to 18% in controls. The amount of IgE eluted was significantly higher in atopics: The calculated number of IgE molecules/basophil was 332,000 in atopics compared to 177,000 in controls. There was a significant positive correlation between plasma IgE and in vitro elutable IgE in atopics (r = 0.73) compared to controls (r = 0.24). After a careful washing procedure attempts were made to 'resensitize' the basophils through incubation with autologous plasma or standard IgE solutions. When resensitization was possible, there was no correlation between histamine releasability after resensitization and original IgE content of basophils. It is concluded that the increased histamine releasability from leukocytes of atopic individuals after stimulation with anti IgE is only in part due to an increased number of IgE molecules per basophil surface. A non-specific increased releasability was demonstrated by increased spontaneous HR rates in acid buffer (pH 3.7). A resensitization with autologous plasma-IgE was possible only in half of the subjects investigated, most of them being atopic. The data support the concept of an altered releasability both towards IgE-dependent and independent stimuli being one possible factor in the pathogenesis of atopic eczema. PMID- 1726057 TI - [Standardization of the keratinization-index by filter imprints]. AB - Exfoliative-cytological procedures can demonstrate pathologic changes of the oral gingival epithelium. Filter-imprints of the gingiva of 31 male test persons were taken 6 times at intervals of 8 days under standardized conditions. The statistical evaluation of the "keratinization index" showed, that the extent of cornification of the healthy gingiva represents an individually different value for every test person. Variations in the course of 5 weeks were without statistical significance. PMID- 1726058 TI - Fine needle aspiration of the prostate: a histo-cytological correlation. AB - There were studied 84 patients with benign and malignant hypertrophia of the prostate from a cytomorphological point of view. Fine needle aspiration and transurethral resection was practised and a comparison between cytologic and histologic results was made. There were 14 adenomas, 3 adenomas with associated atypic canalicular hyperplasia, 6 adenomas with associated carcinoma and 61 carcinomas. Cytologic observations revealed malignant cells in 59 from 61 cases of carcinoma and in 4 from 6 adenomas with carcinoma. General accuracy of the method was appreciated at 96.8%. The incidence, causes and significance of false negative and one false positive results are discussed. Using Gaeta system as standard for histologic grading we may conclude that information (as well as histological form of the tumor and invasion) is difficult or even impossible to obtain only by the study of the smears. The importance of the method for detection of "early cancer", its inocuity, repeatability and the reduced number of complications recommend it as the most useful in the diagnosis of prostate carcinoma. PMID- 1726059 TI - Lysosomal activity at nodes of Ranvier in dorsal column and dorsal root axons of the cat after injection of horseradish peroxidase in the dorsal column nuclei. AB - The occurrence of acid phosphatase (AcPase)-positive bodies, i.e. lysosomes, in dorsal column and dorsal root axons of the spinal cord segments C8 and L7 in adult cats was analyzed by light and electron cytochemical methods after injection of horseradish peroxidase (HRP) in the dorsal column nuclei. Axonal lysosomes were, with few exceptions, concentrated at the nodes of Ranvier. We found no changes in nodal occurrence and distribution of lysosomes in axons of the HRP-injected sides, as compared to axons of the uninjected sides or of animals not exposed to HRP. Axonal lysosomes were very rare in the dorsal columns, where the frequency of nodes containing light microscopically detectable AcPase-positive bodies was 0-5% at the HRP-injected sides, 0-6% at the contralateral sides, and 0-3% in control animals. The corresponding values in the cervical and lumbar dorsal roots were 6-23%, 9-20%, 10-12% and 19-37%, 21-40%, 26 43%, respectively. In view of our recent observations in alpha-motor neurons, the results point at a noteworthy difference in local degradative ability between dorsal column axons and alpha-motor axons, the latter being able to accumulate intramuscularly injected and retrogradely transported HRP at their PNS nodes of Ranvier for 48-60 h, during which period the axoplasmic AcPase activity/concentration increases at some nodes. Such a degradative activity, which could protect the motor neurons by restricting axoplasmic transport of exogenous materials imbibed by their axon terminals outside the CNS, may not be of the same significance for neurons, e.g. dorsal root ganglion neurons, the axon terminals of which are located within the CNS. PMID- 1726060 TI - Selective expression of Jun proteins following axotomy and axonal transport block in peripheral nerves in the rat: evidence for a role in the regeneration process. AB - Expression of the protein products of the immediate-early genes (IEGs), members of the fos, jun and krox families (Jun, Fos, and Krox, resp.) was investigated in the spinal cord and sensory ganglia (DRG) of normal rats; and following transection of, block of axonal transport in, or electrical stimulation of their peripheral axons. The nuclei of many moto- and DRG neurons showed a faint basal immunoreactivity (IR) for Jun proteins, but not for Fos or Krox proteins. There was a strong and selective induction of Jun-IR in moto- and DRG neurons after peripheral nerve transection or crush, or colchicine- or vinblastine-induced block of axonal transport. The Jun-IR induced by nerve transection disappeared after nerve regeneration. In contrast, Jun, Fos and Krox proteins were all induced transynaptically in spinal dorsal horn neurons following electrical stimulation of the C-fibers in the afferent nerves. Thus in differentiated neurons in vivo these IEG proteins can be expressed either independently or concomitantly depending on the type of stimulus. PMID- 1726061 TI - Distribution of excitatory and inhibitory amino acid, sigma, monoamine, catecholamine, acetylcholine, opioid, neurotensin, substance P, adenosine and neuropeptide Y receptors in human motor and somatosensory cortex. AB - Autoradiography was used to visualise N-methyl-D-aspartate, phencyclidine, strychnine-insensitive glycine, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainic acid, benzodiazepine, gamma-aminobutyric acid type A, sigma, serotonergic, dopaminergic, alpha 2-adrenergic, beta-adrenergic, muscarinic cholinergic, nicotinic, opioid, neurotensin, substance P, adenosine A1 and neuropeptide Y receptors in the human primary motor (Brodmann's area 4) and somatosensory cortex (Brodmann's areas 3, 2 and 1). With the exception of serotonin type 2 receptors, all receptor types examined had a similar distribution in area 4 which showed little dependence on the underlying distribution of cell somata, often continuing unaltered through the somatosensory cortex despite marked cytoarchitectural changes. The highest densities occurred in the outer (most superficial) 30-40% of the cortical grey matter, followed by a band of relatively low binding and then moderate levels in the inner (deeper) region. In many instances, an additional band of dense binding could be discerned in the region of laminae IV/Va running unbroken through both gyri. The distribution of most receptor types in the somatosensory cortex also followed this pattern, except for opioid and kainic acid receptors which showed higher levels in the inner rather than the outer third of this region. At the edge of area 4, a change occurred such that a high density outer band appeared, giving these receptor types the same pattern in area 4 as the majority. Serotonin type 2 receptor levels were quite low in the outermost region of area 4, although the pattern was otherwise similar to that of the other receptors. Thus, with the exception of serotonin receptors, the similarity in many binding site distributions recently noted in area 4 of the rhesus monkey also tends to occur in the human area 4, to the extent that 2 ligands will reverse their usual cortical binding pattern to conform with the common area 4 pattern. PMID- 1726062 TI - Efferent projections of the infralimbic (area 25) region of the medial prefrontal cortex in the rat: an anterograde tracer PHA-L study. AB - The efferent projections of the infralimbic region (IL) of the medial prefrontal cortex of the rat were examined by using the anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L). Major targets of the IL were found to include the agranular insular cortex, olfactory tubercle, perirhinal cortex, the whole amygdaloid complex, caudate putamen, accumbens nucleus, bed nucleus of the stria terminalis, midline thalamic nuclei, the lateral preoptic nucleus, paraventricular nucleus, supramammillary nucleus, medial mammillary nucleus, dorsal and posterior areas of the hypothalamus, ventral tegmental area, central gray, interpeduncular nucleus, dorsal raphe, lateral parabrachial nucleus and locus coeruleus. Previously unreported projections of the IL to the anterior olfactory nucleus, piriform cortex, anterior hypothalamic area and lateroanterior hypothalamic nucleus were observed. The density of labeled terminals was especially high in the agranular insular cortex, olfactory tubercle, medial division of the mediodorsal nucleus of the thalamus, dorsal hypothalamic area and the lateral division of the central amygdaloid nucleus. Several physiological and pharmacological studies have suggested that the IL functions as the 'visceral motor' cortex, involved in autonomic integration with behavioral and emotional events. The present investigation is the first comprehensive study of the IL efferent projections to support this concept. PMID- 1726063 TI - A glycinergic projection from the ventromedial lower brainstem to spinal motoneurons. An ultrastructural double labeling study in rat. AB - In the present study it was determined whether glycine was present in the descending brainstem projections to spinal motoneurons in the rat. For this purpose injections of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) were made in the ventromedial part of the lower brainstem at the levels of the rostral inferior olive and the caudal facial nucleus. After perfusion, WGA-HRP histochemistry was performed, followed by the postembedding immunogold technique with an antibody against glycine. Electron microscopical examination of the lumbar motoneuronal cell groups showed that 15% of the WGA-HRP labeled terminals, derived from the ventromedial reticular formation, were also labeled for glycine. The majority (91%) of these double labeled terminals were of the F-type (containing many flattened vesicles), while the remaining 9% were of the S-type (containing mostly spherical vesicles). Many of the double labeled terminals established a synapse, mostly with proximal and distal dendrites. The present data, combined with our previous findings that 40% of the projections from the same ventromedial brainstem area to lumbar motoneurons contained gamma aminobutyric acid (GABA), indicate that over 50% of these brainstem projections contain GABA and/or glycine, exerting a direct inhibitory effect on spinal motoneurons. The possibility that the glycinergic fibers within these projections play an important role in producing muscle atonia during rapid eye movement (REM) sleep is discussed. PMID- 1726064 TI - Different pathways by which extracellular Ca2+ promotes calcitonin gene-related peptide release from central terminals of capsaicin-sensitive afferents of guinea pigs: effect of capsaicin, high K+ and low pH media. AB - Different modes by which Ca2+, entering the nerve terminal, promotes transmitter secretion as well as the ability of protons to release neuropeptides, have been shown in peripheral endings of capsaicin-sensitive afferents. We have studied these two aspects in the central endings of these neurons by measuring the release of calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) from slices of the dorsal half of the guinea pig spinal cord. Although capsaicin (1 microM) released both CGRP-LI and substance P-like immunoreactivity (SP-LI), CGRP LI was chosen as the sole suitable marker of peptides released from central terminals of capsaicin-sensitive afferents, since after in vitro desensitization to capsaicin (1 microM capsaicin for 20 min), high K+ (80 mM) failed to evoke CGRP-LI release, whereas SP-LI release was still observed. The capsaicin (1 microM)-evoked CGRP-LI release was entirely dependent on extracellular Ca2+. It was unaffected by 0.3 microM tetrodotoxin (TTX), slightly reduced by 0.1 microM omega-conotoxin (CTX) and blocked by 10 microM Ruthenium red (RR). The Ca(2+) dependent K+ (80 mM)-evoked CGRP-LI release was unaffected by TTX, markedly reduced by CTX and only moderately inhibited by RR. Low pH (pH 5) produced a remarkable increase in CGRP-LI outflow that was abolished after exposure to capsaicin, reduced by about 50% in Ca(2+)-free medium and unaffected by TTX (0.3 microM). The Ca(2+)-dependent component of the proton-evoked CGRP-LI release was abolished in the presence of RR (10 microM) and slightly inhibited by CTX (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726065 TI - Amyotrophic lateral sclerosis: changes of noradrenergic and serotonergic transmitter systems in the spinal cord. AB - Noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured in discrete subdivisions of cervical, thoracic and lumbar spinal cord segments obtained at autopsy of 4 subjects with amyotrophic lateral sclerosis (ALS) and 7 control patients. NA concentrations in thoracic and lumbar spinal cord of ALS patients were 2- to 4-fold higher compared with values obtained in control patients. 5-HT levels were unchanged at the cervical and thoracic level and slightly above normal in lumbar spinal cord, while the concentration of 5-HIAA was lowered in cervical and thoracic, but within the control range, in lumbar spinal cord. As a result, the molar ratios of 5-HT/5 HIAA were increased at all spinal levels in ALS. No difference in spinal DA concentration was found between ALS and control patients. The changes in the noradrenergic and serotonergic transmitter systems reported here most probably reflect a decreased release of these transmitter substances in ALS spinal cord. Since lack of the facilitatory monoaminergic influence would necessitate an increase in the excitatory, potentially neurotoxic glutamatergic input onto the motoneurones, we hypothesize that this could contribute to the progressive loss of spinal motoneurones in amyotrophic lateral sclerosis. PMID- 1726066 TI - Release of substance P into the superficial dorsal horn following nociceptive activation of the hindpaw of the rat. AB - Substance P (SP) has been widely proposed as being involved in the transmission of nociceptive information in the dorsal horn of the spinal cord. Formalin injected into the hindpaw as a nociceptive stimulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. Much is known concerning the release of SP from tissue slices or from the entire spinal cord in vivo. However, less is known about the release patterns of SP in the superficial dorsal horn during the activation of peripheral nociceptors. In this study, noxious pinch applied to and formalin injection into the hindpaw were used as nociceptive stimuli while a stereotaxic push-pull cannula was used to perfuse the L5 dorsal horn. Experiments were conducted in unanesthetized decerebrate/spinal rats, and radioimmunoassay was used to determine the SP-like immunoreactivity (SPLI) content of collected perfusates. Results demonstrate that graded intensities of noxious mechanical pinch produced progressively increased release of SPLI into the dorsal horn; SPLI release returned to baseline rates following termination of the stimulus. The injection of 100 microliters of 5% formalin into the hindpaw produced a biphasic inhibition of SPLI release 0-40 min and greater than 60 min after formalin injection. The application of a noxious pinch following formalin injection produced an increase in SPLI release which did not return to baseline rates; this may be indicative of production of a hyperalgesic state caused by formalin injection. The results of this study support the concept that formalin injected into the hindpaw activates segmental antinociceptive systems which block SP release and limit nociceptive transmission.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726067 TI - Substance P-immunoreactive innervation from the retina to the suprachiasmatic nucleus in the rat. AB - The present study examined substance P (SP) innervation in the suprachiasmatic nucleus (SCN) of the rat. In the colchicine-untreated rat, SP-immunoreactive fibers formed a dense oval plexus in the ventral part of the SCN. After bilateral eye enucleation, there was a marked reduction in SP-immunoreactive fibers in the ventral part of the SCNs. The SP-immunoreactive neurons in the retinal ganglion cell layer were retrogradely labeled after injection of Fluoro-gold into the SCN. These findings indicate the presence of the SP innervation from the retina to the SCN in the rat. The role of SP in the retino-hypothalamic tract was discussed from the light-dark cycle. PMID- 1726068 TI - Galanin inhibits the potassium-evoked release of acetylcholine and the muscarinic receptor-mediated stimulation of phosphoinositide turnover in slices of monkey hippocampus. AB - In the ventral hippocampus of Cynomologus monkey, galanin, a 29 amino acid long neuropeptide, reduced the potassium-evoked release of [3H]acetylcholine from slices preloaded with [3H]choline and diminished the carbachol-stimulated accumulation of [3H]inositol polyphosphates in hippocampal microprisms preincubated with myo-[2-3H]inositol. Using receptor autoradiography a strong, specific binding of iodinated galanin was observed in the molecular layer of the dentate gyrus. These may thus be the sites where galanin exerts its inhibitory effects on acetylcholine (ACh) release and phosphoinositide breakdown. These data provide evidence that galanin is a modulator of cholinergic function in septo hippocampal neurons of primates. PMID- 1726069 TI - Preservation of [125I]galanin binding sites despite loss of cholinergic neurones to the hippocampus in Alzheimer's disease. AB - [125I]Galanin binding sites were examined in the hippocampal region of patients with Alzheimer's disease (AD) and age-matched control subjects using quantitative autoradiography. In control subjects, [125I]galanin binding sites were highly concentrated in the presubicular parvopyramidal layer (5.60 +/- 0.74 pmol/g), while other hippocampal regions had considerably lower levels of binding (0.56 1.73 pmol/g). In the majority of hippocampal regions, [125I]galanin binding was similar in AD patients to that in controls with a significant reduction being observed only in the deep layers of the parahippocampal gyrus. The activity of choline acetyltransferase, determined in the same tissue samples used for autoradiographic studies, was markedly reduced in AD (controls 6.8 +/- 0.6; AD 2.7 +/- 0.6 nmol/h/mg protein, mean +/- S.E.M.). These data are not consistent with a presynaptic location on cholinergic terminals of galanin binding sites in the human hippocampus unless upregulation has occurred. PMID- 1726070 TI - Differential thalamic connectivity of rostral and caudal parts of cortical area Fr2 in rats. AB - Thalamocortical connections were studied after injections of retrograde tracers were made into rostral or caudal parts of cortical area Fr2 in adult rats. The data reveal that both rostral Fr2 (rFr2) and caudal Fr2 (cFr2) receive input from the centrolateral, central medial, interanteromedial, mediodorsal, paracentral, parafascicular, posterior, reuniens, rhomboid, ventrolateral, ventromedial and zona incerta nuclei. In addition, cFr2, but not rFr2, receives input from the anteromedial, anteroventral, laterodorsal and lateral posterior nuclei. These findings provide further evidence that Fr2 is connectionally and functionally heterogeneous, with rFr2 connected with somatomotor-related nuclei and with cFr2 connected with somatomotor- and visuomotor-related nuclei. PMID- 1726071 TI - Presynaptic dopaminergic neurotransmission mediates amphetamine-induced unconditioned but not amphetamine-conditioned locomotion and defecation in the rat. AB - A series of experiments were conducted to investigate the role of presynaptic dopamine (DA) and noradrenaline (NA) neurotransmission in stimulant-unconditioned and conditioned locomotion and defecation. (+)-Amphetamine (AMP, 1.5 mg/kg, s.c.) increased both locomotion and defecation in rats, and both of these effects were conditioned to environmental stimuli. Some groups of rats were treated with DSP4 (50 mg/kg, i.p.), a selective, long-lasting NA neurotoxin, given 7 days prior to conditioning with AMP. This treatment depleted forebrain NA to between 1% and 54% of control levels, depending on the brain region, but did not attenuate either AMP-unconditioned or conditioned locomotion. These results indicate that NA does not mediate either AMP unconditioned or conditioned locomotion. alpha-Methyl-para tyrosine methyl ester (alpha MPT, 25-50 mg/kg, s.c.), a selective inhibitor of catecholamine synthesis given during conditioning with AMP, attenuated unconditioned AMP-induced locomotion and defecation but did not influence AMP conditioned locomotion and defecation. Thus, alpha MPT blocked AMP-induced unconditioned locomotion, supporting the hypothesis that the locomotor and defecation stimulant effects of AMP are mediated by DA release. In spite of the attenuation of the direct effects of AMP, alpha MPT did not attenuate AMP conditioned locomotion or defecation. It is concluded that AMP-induced release of dopamine is responsible for the unconditioned behavioral effects of amphetamine but not for the conditioning of amphetamine-induced locomotion and defecation. PMID- 1726072 TI - Ethanol-induced Cl- flux in rat cerebellar granule cells as measured by a fluorescent probe. AB - Cl- fluxes through the GABAA receptor gated ion channels in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl- ions. The cells were bathed in a low Cl- medium so that the Cl- gradient was directed outward. Ethanol increased the SPQ fluorescence indicating a decrease in intracellular Cl- due to Cl- efflux. Picrotoxin inhibited the effect at low concentrations of ethanol (less than 50 mM) in a concentration dependent manner. The effects of ethanol were potentiated at low concentrations (less than 10 microM) of gamma-aminobutyric acid (GABA), but inhibited at higher concentrations (0.3-2.0 mM). The results support the hypothesis that ethanol may act via the GABAA receptor gated ion channel. The results also suggest that SPQ is a suitable probe for measuring GABAA receptor coupled Cl- fluxes through the GABAA receptor-gated channels in living cells. PMID- 1726073 TI - Serotonin, but not dopamine, metabolites are increased in selected brain regions of subordinate male rats in a colony environment. AB - Subordinate male laboratory rats maintained in mixed-sex groups in a Visible Burrow System habitat show a complex pattern of stress-related changes including enhanced defensive behavior, early mortality and increased voluntary ethanol consumption. Analysis of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels indicated that 5-HT levels do not differ between colony subordinates, colony dominants, and singly-housed control animals. However, 5-HIAA levels were higher in subordinates than either dominants or control animals in the preoptic area, amygdala, hippocampus, and spinal cord, and, were higher than dominants only, in entorhinal cortex. Subordinates' regional 5-HIAA/5-HT ratios were reliably higher than those of dominant or control animals in midbrain and spinal cord and reliably higher than dominants only, in hypothalamus. Dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) levels and DA/DOPAC ratios were affected neither in hypothalamus nor midbrain. These findings suggest that a consistent increase of 5-HIAA levels in selected brain regions of subordinate rats may represent a biological substrate for a well-characterized pattern of alterations in defensive behaviors for these animals. PMID- 1726074 TI - Biologically active constituents of Magnolia salicifolia: inhibitors of induced histamine release from rat mast cells. AB - The extracts of the flower buds of Magnolia salicifolia showed remarkable anti allergy effects in passive cutaneous anaphylaxis (PCA) test. The bioactive constituents of this medicinal drug were isolated by monitoring their activities with an in vitro bioassay system measuring inhibitory effects on induced histamine release from rat mast cells. Of the ten isolated compounds magnosalicin is a new compound of neolignan structure. In addition to the isolated compounds samples of coumarins and lignans were evaluated their biological activities with the in vitro bioassay. PMID- 1726075 TI - Biologically active constituents of Centipeda minima: sesquiterpenes of potential anti-allergy activity. AB - Ether, methanol and aqueous extracts of Centipeda minima (Compositae) herbs were found to have significant anti-allergy activities in passive cutaneous anaphylaxis (PCA) test. Three flavonoids, two sesquiterpene lactones and an amide were isolated from this plant material as inhibitors to induced histamine release from mast cells. The sesquiterpenes were identified as isobutyroylplenolin and senecioylplenolin by spectral investigations. The flavonoids and sesquiterpenes exhibited significant anti-allergy activity in PCA test with p.o. administration. PMID- 1726076 TI - Biologically active constituents of Melaleuca leucadendron: inhibitors of induced histamine release from rat mast cells. AB - Chloroform and methanol extracts of the fruits of Melaleuca leucadendron strongly inhibited histamine release from rat mast cells induced by compound 48/80 or concanavalin A. Ursolic acid, a triterpene, was the most active compound contained in the chloroform extract and two stilbenes, piceatannol and oxyresveratrol, were isolated as active compounds from the methanol extract. Several other stilbenes and related compounds were examined to obtain information on the structure activity relationships of stilbenes. PMID- 1726077 TI - Inhibitory effects of 3,3',4,5'-tetrahydroxystilbene and 3,3',4,5' tetrahydroxybibenzyl, the constituents of Cassia garrettiana on antigen-induced histamine release in vitro. AB - 3,3',4,5'-Tetrahydroxystilbene (I) and 3,3',4,5'-tetrahydroxybibenzyl (II), isolated from the heartwood of Cassia garrettiana Craib (Leguminosae), showed inhibitory effects on antigen-induced histamine release from rat peritoneal mast cells in vitro. The inhibitory effect of I (IC50 = 30.2 microM) was much stronger than that of II (greater than 100 microM). Compound II, as well as I (IC50 = 7.3 microM) reported previously, also inhibited the histamine release from human peripheral basophils induced by anti-immunoglobulin E (IgE) in vitro, and its IC50-value was 68.0 microM. These results suggest that the trans-olefin structure in the molecule may be necessary for I to have an inhibitory effect on histamine release. Considering that disodium cromoglycate did not show any significant inhibitory effect on anti-IgE-induced histamine release from human basophils, the strong inhibitory effects of I in both tests are of considerable interest. PMID- 1726078 TI - Sandwich enzyme immunoassay for serum integrins using monoclonal antibodies. AB - We produced monoclonal antibodies (mABs) against human integrins. Competitive enzyme-linked immunosorbent assay (ELISA) revealed that each mAB bound to different antigenic determinants. We then developed sandwich-type enzyme immunoassays (EIAs) to measure the concentration of fibronectin receptor (FNR) and vitronectin receptor (VNR). Serum immunoreactive integrin levels were measured using these EIAs in various liver and malignant diseases. In almost all cases of liver cirrhosis (LC) and hepatocellular carcinoma (HCC), serum integrin levels were significantly elevated, but were in the normal range in gastric, colon, lung cancer, and acute hepatitis (AH). The correlation between serum FNR and VNR levels was statistically significant in all cases of liver disease, and no correlation was observed between these integrin levels and conventional biochemical markers such as AST, ALT, and GGT. The serum integrin levels were demonstrated to be a potential diagnostic marker for hepatic fibrogenesis and carcinogenesis, and these sandwich EIAs could be useful for determination of these integrins in clinical laboratory tests. PMID- 1726079 TI - Schistosoma mansoni and Biomphalaria glabrata have some common epitopes. I. Common epitopes are present on the surface of early stages of S. mansoni. AB - 1. The presence of snail and glycocalyx antigens in the Schistosoma mansoni cercarial surface and their permanence throughout development in vitro and in vivo was investigated. 2. Rabbit antisera raised against two fractions of glycocalyx released from cercariae and Biomphalaria glabrata soft tissues or haemolymph were obtained. 3. All four antisera bound to cercariae and schistosomula kept in vitro or in vivo for up to 24 hr. 4. No binding to schistosomula kept in vivo for 5 days or longer was observed. 5. Schistosomula cultured in vitro for up to 12 days bound the antisera throughout the period of culture. PMID- 1726080 TI - Six years of protein structure determination by NMR spectroscopy: what have we learned? AB - Nuclear magnetic resonance (NMR) spectroscopy in solution is a second technique, in addition to X-ray diffraction in single crystals, for the determination of three-dimensional protein structures at atomic resolution. Structures of proteins derived by NMR have now been with us for six years, and here I entertain the following question: what information have we gained that would not be available if X-ray crystallography were still the only method for protein structure determination? Answers include that NMR structures are available of proteins that have not been crystallized, that the two techniques afford different insights into internal mobility of proteins, and that one gets different views of protein hydration and hence the molecular surface when using NMR spectroscopy or X-ray diffraction. PMID- 1726081 TI - Aminopyrine-N-demethylase. I. Directed modification of substrates' structure as a way of production of inducer of the monooxygenase isoform P-450b. AB - The effect of the phenobarbital-type monooxygenase inducers is accomplished via the native molecule. This made it possible to transform the typical substrate of cytochrome P-450b aminopyrine into an inducer of this isozyme by blocking the substrate molecule position undergoing monooxygenation. Substitution of two methyl groups in the aminopyrine -N(CH3)2 position by an isopropyl group gave rise to a clear-cut inducive effect. This was registered by spectral, kinetic, radiological and immunochemical methods. PMID- 1726082 TI - Aminopyrine-N-demethylase. II. Characterization of a unique monooxygenase isoform P-450ap. AB - A previously unidentified cytochrome P-450ap possessing the highest aminopyrine-N demethylase activity has been isolated from liver microsomes of 4 isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on a hydroxyapatite column. The isolated cytochrome P-450ap has the following characteristics: Mr = 49 kD, CO-peak maximum at 450.5 nm, rate of demethylation in a reconstituted system for aminopyrine of 25.5 nmoles of HCHO/min per nmole of P-450, and for benzphetamine a rate of 17.0 nmoles of HCHO/min per nmole of P 450. The hemoprotein synthesis is paralleled by the synthesis of a protein with Mr of 51 kD. Immunochemical analysis permitted the identification of the latter protein as cytochrome P-450b. It was, demonstrated that cytochrome P-450ap does not interact with the antibodies to the major phenobarbital induced form, i.e. with cytochrome P-450b. PMID- 1726083 TI - Anatomy of the prostate: review of the different models. AB - Current understanding of the anatomic subdivision of the adult prostate gland is secondary to two major studies successively published by Gil Vernet in 1953 and by McNeal in 1968. In Gil Vernet's report, glandular tissue consists of a caudal gland, a cranial gland and a intermediate gland. These 3 glandular areas were defined according to the location of their glandular openings into the urethra. In McNeal's report, the urethra is taken as the primary anatomic reference point. The four glandular areas are best visualized by examination of the selected coronal plane of sections along the proximal and distal prostatic urethra. The peripheral zone is located posterolaterally, the central zone is located at the base, and the 2 lobes of the transition zone are located along the proximal urethra. Fibromuscular regions are concentrated anteromedially. Most of the anatomic regions are similar between the two models, however, in McNeal's model, accuracies concerning the boundaries of the central zone and transition zone ultimately make this model superior for the study of both the physiology and pathology of the prostate. PMID- 1726084 TI - Subjective and clinical results after transurethral resection and suprapubic prostatectomy in benign prostatic hypertrophy. AB - This study was performed after a mean postoperative period of approximately 2 years. Results after transurethral resection (TUR) appeared to be inferior to results after suprapubic prostatectomy in patients' subjective symptomatology concerning frequency, nocturia, urinary stream complaints and incontinence. Late postoperative complications were found in a greater extent after TUR. TUR showed more often pathologic uroflow and pathologic residual urine data. Generally speaking, the results after suprapubic prostatectomy were substantially more satisfactory than the results after TUR. PMID- 1726085 TI - Incidental adenocarcinoma of the prostate: a retrospective analysis. AB - Between January 1980 and June 1990, 5875 patients underwent either TURP (5,529) or open prostatectomy (346) for the management of benign prostatic hyperplasia. Incidental carcinoma was identified in 53 patients (0.9%) - 49 in the TURP group and 4 in the open prostatectomy group. The overall incidence of stage A prostate carcinoma during the same period was 13.1% (53/406). Of those 50 regularly followed patients, only 6 (12.0%) underwent subsequent radical prostatectomy with pelvic lymphadenectomy, and residual tumors were identified in 4 patients (66.7%). The progression rate was 33.3% (3/9) in high-grade tumors (Gleason's score greater than 6) and 7.7% (2/26) in low-grade tumors (Gleason's score less than 5). In those 50 patients, 41 (82.0%) presented no evidence of disease and 9 (18.0%) stayed alive with progression. The mean length of follow-up was 34 months (range: 2-108 months). Our study demonstrates that the outcome of incidental carcinoma of the prostate is good. However, the residual tumor rate still remains high at 66.7%. Radical prostatectomy is recommended in young patients or in patients with high grade tumor. PMID- 1726086 TI - Activation of the hypothalamic serotoninergic system by central interleukin-1. PMID- 1726087 TI - Separate sites for the dantrolene-induced inhibition of contracture of the rat diaphragm preparation due to depolarization or to caffeine. AB - Addition of dantrolene 8.5 x 10(-5) M caused a mono-exponential decay of the depolarization contractures caused by inhibition of the sarcolemmal Na,K-ATPase with propranolol 1 mM or by depolarization of the sarcolemma and T tubular membranes with KCl 100 mM. The half-times of the inhibitory effects were 6 s for the propranolol contracture and 11 s for the KCl contracture. The inhibition of both contractures was complete. Inhibition of the caffeine (10 mM) contracture was bi-exponential with half-times of 45 s and 9.5 min. Inhibition was incomplete; 29.6 +/- 5.0% of the contracture tension could not be inhibited. The inhibition of twitch contractions was similar to that of the caffeine contracture, with half-times of 48 s and 9.1 min, and 20.6 +/- 1.2% of the initial twitch tension could not be inhibited. The contracture tensions induced by release of Ca from the mitochondria with dicumarol, and by actin-myosin binding with the sulfhydryl inhibitor, N-ethyl-maleimide, could not be inhibited by dantrolene. The present results indicate that dantrolene inhibits depolarization signals from the sarcolemma and the T tubular membranes, in addition to inhibition of the coupling between the T tubules and the sarcoplasmic reticulum, and of the release of Ca from the sarcoplasmic reticulum. All these effects of dantrolene may contribute to its therapeutic effect in malignant hyperthermia. PMID- 1726088 TI - Effects of the cyclic nucleotide phosphodiesterase inhibitors, rolipram, 3 isobutyl-1-methylxanthine, amrinone and zaprinast, on pancreatic exocrine secretion in dogs. AB - The effects of the cyclic phosphodiesterase (PDE) inhibitors, rolipram, 3 isobutyl-1-methylxanthine (IBMX), amrinone and zaprinast on pancreatic exocrine secretion were investigated in anesthetized dogs. Rolipram (1-30 nmol), IBMX (44 440 nmol) or zaprinast (1-10 mumol) injected i.a. elicited a dose-dependent increase in the secretion of pancreatic juice, but amrinone (up to 53 mumol) did not. The bicarbonate concentration in pancreatic juice was increased and the protein concentration was decreased by rolipram and IBMX, but neither was affected by zaprinast. Rolipram elicited more than the respective additive secretory response when added together with secretin, although the stimulatory effects of CCK-8 with rolipram were additive. Rolipram and IBMX, but not zaprinast, increased cyclic AMP concentration but did not affect cyclic GMP concentration. These results suggest that rolipram, IBMX and zaprinast have direct secretory properties on pancreatic exocrine glands of the dog, which may be mediated through the increase of intracellular cyclic AMP concentration, by inhibiting PDE activity. Furthermore, the pancreatic PDE enzyme in the dog pancreas may be mainly a type IV. PMID- 1726089 TI - Involvement of NK1 receptors and importance of the N-terminal sequence of substance P in the stimulation of protein secretion in rat parotid glands. AB - Recent in vitro studies have shown that the dose-response curve of substance P on [3H]protein secretion from rat parotid glands is biphasic. Such a response could result either from the activation of tachykinin receptors or from the amphiphilic character of substance P, since it has previously been shown that the N-terminal part of substance P may play an important role in the activation of phosphoinositides in rat parotid glands. To investigate these possibilities, we studied the effects of selective NK1, NK2, NK3 receptor agonists and C-terminal fragments of substance P and neurokinin A on protein secretion from rat parotid lobules. The poor activity of NK2 (neurokinin A-(4-10) and [beta-Ala8]neurokinin A-(4-10)) as well as of NK3 ([MePhe7]neurokinin B) selective agonists allowed us to rule out a possible involvement of NK2 and NK3 receptors in the parotid gland secretory process. Conversely, the selective NK1 receptor agonist, [Sar9,Met(O2)11]substance P, reproduced the biphasic dose-response curve for [3H]protein secretion typical of native substance P. However, a biphasic response was not observed with peptides deprived of the N-terminal moiety of substance P, such as substance P-(4-11) or [AcArg6,Sar9,Met(O2)11] substance P-(6-11). Our data therefore indicate that the [3H]protein secretion obtained with substance P results from the activation of NK1 receptors. Moreover, our data suggest that the N-terminal tripeptide of substance P is also active, and could stimulate different phospholipases either by acting through a second functional site on the NK1 receptor or by directly activating G-proteins. PMID- 1726090 TI - Use of an in vitro model to assess the effects of APF gel treatment on the staining potential of dental porcelain. AB - Porcelains and resin composites exposed to acidulated phosphate fluorides (APF) have been reported to result in increased roughness, loss of weight, and loss of specular reflectance (gloss). Six samples of five commercial porcelains were subjected to five four-minute treatments with APF gels. Samples were then subjected to a nine-day cyclic staining procedure that utilized a tea, coffee, and mucin mixture. Changes in reflectance were then measured by means of a Minolta Chromameter (CR121) and converted to CIE L* a* b* values at illuminant D65 against a white background. delta L*, delta a*, delta b*, and delta E values were calculated. There was a substantial decrease in the L* value (lightness) for all porcelains. The average L* value for APF-treated and then stained porcelains was 43.6, for the stained-untreated samples, 48.2, and for untreated-unstained porcelain, 53.5. For three of the five porcelains, the differences in L* between treated and untreated stained porcelains were statistically significant. Changes in a* and b* values were also found to be consistent with but not as large as the changes in L*. PMID- 1726091 TI - [Radioimmunoassay of serum myelin basic protein]. AB - Using purified human brain myelin basic protein (MBP) to raise antiserum in rabbits and to prepare 125I-labelled MBP (chloramine-T method), We have established a high specific, precise and sensitive double-antibody radioimmunoassay for the measurement of human serum MBP. The sensitivity was 0.5 ng/ml. In the present study, the serum samples of thirty patients with various neurological diseases were detected. An important clinical implication is that serum MBP level should serve as an index for the damage degree of central neurological diseases. PMID- 1726092 TI - Modulation of humoral immune responses by Tinospora malabarica in experimental animals. AB - Effect of alkaloidal fraction of aqueous extract of T. malabarica (Tm) was studied on humoral antibody responses in rats and guineapigs. The anti-SRBC haemagglutination titre was found to be enhanced in rats pretreated with Tm (2.5 mg/kg). Passive cutaneous anaphylaxis (PCA) in rats was also increased in Tm treated group. In vitro experiments with sensitized rat peritoneal mast cells showed a significant decrease in antigen-induced various spasmogens on isolated guineapig ileum. PMID- 1726093 TI - Unexpected up-regulation of gene expression by cyclosporin A and FK-506 in a T cell lymphoma: both immunosuppressants augment Ly-6E antigen induction by interferon-gamma in the presence of ionomycin. AB - Cyclosporin A (CsA) and FK-506 inhibit lymphokine gene activation in T-cells. In the present study, we investigated the effects of these immunosuppressants on the regulation of a non-lymphokine molecule, the Ly-6E surface antigen, in the YAC-1 T-cell lymphoma. These cells do not normally express Ly-6E mRNA or Ly-6E surface molecules but are induced to do so upon treatment with IFN-gamma. At submicromolar concentrations, CsA or FK-506 did not alter this induction. However, at higher concentrations (1-12 microM), they both increased the induction of Ly-6E mRNA expression. Cyclosporin A or FK-506 also markedly affected Ly-6E induction when the cultures were co-treated with the calcium ionophore, ionomycin. In the absence of CsA or FK-506, ionomycin suppressed Ly-6E induction by IFN-gamma. Both immunosuppressants reversed this inhibitory effect and increased Ly-6E mRNA and Ly-6E surface expression to levels that were 2- to 3 fold higher than in cells induced with IFN-gamma alone. In this system, the two immunosuppressants were active at pharmacologically relevant concentrations, similar to those inhibiting normal T-cell activation, with FK-506 being 30- to 50 fold more potent than CsA. The ability of CsA analogs to enhance Ly-6E induction in the presence of ionomycin also correlated with their immunosuppressive activity. Therefore, through mechanisms apparently related to those involved in their immunosuppressive action, both CsA and FK-506 convert the negative effect of ionomycin on IFN-gamma-mediated Ly-6E induction into an overall positive effect. The YAC-1 cell model, described here, provides a unique example of upregulation of gene expression by these two immunosuppressants. PMID- 1726094 TI - Hb Mizuho or alpha 2 beta (2)68(E12)Leu----Pro in a Caucasian boy with high levels of Hb F; identification by sequencing of amplified DNA. AB - Hb Mizuho, an unstable beta chain variant with a Leu----Pro substitution at position beta 68, was observed in a young Caucasian boy from Kentucky. Identification was made by sequence analyses of amplified DNA and by hybridization of amplified DNA with specific probes. The patient had high Hb F levels up to the present age of nearly 5 years; this high Hb F might in part be responsible for his condition which was considerably milder than that seen in the two patients described in earlier reports. The increased gamma chain synthesis may be due to special characteristics of the beta-globin gene cistron which are comparable to those observed in sickle cell anemia patients with a relatively mild disease. PMID- 1726095 TI - Hb F-Cosenza or G gamma 25(B7)Gly----Glu: a new fast-moving fetal hemoglobin variant. AB - A fast-moving gamma chain variant was discovered in the cord blood of a newborn during a screening program carried out in the northern region of Calabria, Southern Italy. The structural analysis showed a Gly----Glu substitution at position 25 of the G gamma chain indicating a new fetal hemoglobin variant that was named Hb F-Cosenza. PMID- 1726096 TI - Detection of the Hb Quong Sze mutation in a Chinese family by selective amplification of the alpha 2-globin gene and restriction map analysis with Msp I. AB - The polymerase chain reaction technique combined with restriction map analysis with Msp I and hybridization with synthetic oligonucleotide probes has been used to identify Hb Quong Sze [alpha 125(H8)Leu----Pro] in a Chinese family in Guangxi (Quong Sze), P. R. China. Our data and those described in an earlier publication (1) indicate that the Hb Quong Sze carriers originate from the same province of the People's Republic of China, namely Guangxi. PMID- 1726097 TI - Hb F-Xinjiang or alpha 2A gamma T(2)25(B7)Gly----Arg identified by reversed phase HPLC; second observation. PMID- 1726098 TI - A second observation of Hb F-Lodz or alpha 2G gamma (2)44(CD3)Ser----Arg. PMID- 1726099 TI - Electrocardiographic changes during upper gastrointestinal endoscopy in ambient hypoxia. AB - Electrocardiographic (ECG) changes were studied in 120 consecutive subjects during and after upper GI endoscopy done in ambient hypoxia (PO2-120 mmHg) at Shimla (2200 m). No premedication was given to any of the subjects. There were 75 men and 44 women. Fifty three subjects were aged 40 years or below (Group I) and 67 subjects were above 40 (Group II). There were 29 subjects with and 91 subjects without cardiac diseases. Increase in heart rate was seen in 96.6% of subjects. Maximum rise in heart rate was found in cardiac patients. ST depression was seen in 14.2%, T wave inversion in 13.3%, supraventricular tachycardia in 5.8% and ventricular ectopics in 1.6%. ST depression was more frequent in cardiac than in non cardiac patients (P less than 0.001) and T wave inversion was more frequent in women than in men (P less than 0.001). All the changes reverted to normal within 10 minutes. ECG changes notwithstanding, upper GI endoscopy without premedication in the presence of ambient hypoxia is a safe procedure. PMID- 1726100 TI - A 33 kDa protein with sequence homology to the 'laminin binding protein' is associated with the cytoskeleton in hydra and in mammalian cells. AB - In hydra and in mammalian cells the monoclonal antibody V recognises an epitope which colocalises with cytoskeletal structures. Using this antibody for expression screening, a cDNA clone (955 bp) was isolated from hydra, which covers an open reading frame for a protein of 294 amino acids with a calculated molecular mass of 32.8 kDa. Northern blot analysis of hydra RNA resulted in a single mRNA species of 1.2 kb, and primer extension experiments proved this to be the full length message. 218 residues at the amino terminus of the hydra protein show extensive homology (73.5%) to a human protein designated 'laminin binding protein'. The carboxyl-terminal 76 amino acids possess no significant similarity (20%). The monoclonal antibody V, which recognises an epitope in this carboxyl terminal part, reacts in Western blots, both in hydra and in mammalian cells, with a protein of 33 kDa and not with the 45 kDa 'laminin binding protein'. The 33 kDa protein is not extracellular or transmembrane, but has a strictly intracellular location as indicated by its amino acid sequence and by immunocytochemical and cell fractionation studies. In non-dividing mammalian cells the 33 kDa protein colocalises with filamentous structures; in dividing cells it dissociates from it and concentrates centrally. Presence of the SPLR sequence, which is the consensus phosphorylation motif for the p34cdc2 kinase, links this 33 kDa protein to events occurring during the cell cycle. PMID- 1726101 TI - Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture. AB - In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures. PMID- 1726102 TI - Nature and origin of gap filaments in striated muscle. AB - Immunoelectron microscopy was used to study the nature and origin of 'gap' filaments in frog semitendinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-11, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 nm beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 microns. Above 3.6 microns, the span between the epitope and the end of the A-band abruptly increased, and above 4 microns, the antibodies could be seen to decorate the gap filaments. Between 5 and 6 microns, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 microns implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain. PMID- 1726103 TI - Protein myristoylation in human mononclear phagocytes: modulation by interferon gamma and tumor necrosis factor-alpha. AB - Labelling of cells with [3H]myristic acid and analysis of labelled proteins by SDS-PAGE and fluorography, enabled the identification of a limited number of myristoylated proteins in human monocytes and monocyte-derived macrophages. In human monocytes, cultivated for one to three days, major myristoylated proteins observed were of 18 kDa, 44 kDa, 60-62 kDa, 90 kDa, and a doublet of 38-40 kDa. Differentiation of monocytes to macrophages by in vitro cultivation was accompanied by a selective decrease in the 60-62 kDa protein. Cultivation of the cells in the presence of the macrophage-activating cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), prevented the decrease in the expression of the 60-62 kDa myristoylated protein. The effect of cytokines was observed when monocytes were treated with IFN-gamma or TNF-alpha for 24 or 48 h and protein myristoylation analyzed at day four of culture. Maintenance of monocytes in culture for up to nine days in the presence of cytokines prevented the decrease in the expression of the 60-62 kDa myristoylated protein. IFN-gamma had additional effects on myristoylation of macrophage proteins. Treatment of monocytes with IFN-gamma for a few hours caused the induction of a 66 kDa protein. Induction of this myristoylated protein by IFN-gamma was time-dependent and peaked at six hours. Analysis of the subcellular distribution of the 66 kDa protein induced by IFN-gamma showed that, analogously to other myristoylated proteins, most of it was associated with cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726104 TI - Deoxyguanosine enhances the cytotoxicity of the topoisomerase I inhibitor camptothecin by reducing the repair of double-strand breaks induced in replicating DNA. AB - Deoxyguanosine (dG) enhances the S phase cytotoxicity of camptothecin (CPT), a topoisomerase I (topo I) inhibitor, but by contrast does not affect the toxicity of VM26, a topoisomerase II inhibitor. The 80% survival of S phase human fibroblasts after a 60 min exposure to 0.2 microM CPT is reduced by half in the presence of 25 microM dG. G1 cells are resistant to CPT toxicity, though the levels of the single-strand DNA breaks induced by the drug are similar in G1 and S phase cells. Higher concentrations of dG retard the recovery of RNA and DNA synthesis and inhibit recovery from the S-G2 cycle block after CPT removal. At 100 microM dG the number of CPT-induced protein-linked single-strand DNA breaks is almost doubled, suggestive of a direct effect of dG on the cellular activity of topo I. In the presence or absence of dG, single-strand breaks disappear within minutes of the removal of CPT. We found that the inhibition of topo I by CPT induces the formation of double as well as single-strand breaks in the chromosomal DNA. Previously we have shown, using a pulse-field gel electrophoresis technique, that the double-strand breaks (DSBs) are generated predominantly at sites of replication and not in the bulk DNA. A number of these DSBs are long-lived. The present study shows that dG affects the repair of these DSBs in a dose-dependent manner, and that a higher proportion of the initial lesions induced in nascent DNA remain 24 h after removal of CPT. We suggest that the long-lived double-strand breaks, formed in replicating DNA at the time of CPT exposure, are the lethal drug-induced lesions, which explains both the selective cytotoxicity of CPT towards S phase cells and the enhancement of CPT cytotoxicity by dG. PMID- 1726105 TI - Afferent and sympathetic neurons projecting into lumbar visceral nerves of the male rat. AB - The cell bodies of thoracolumbar sensory and sympathetic pre- and postganglionic neurons that project to the colon and pelvic organs of the male rat were labeled retrogradely with horseradish peroxidase (HRP) in order to study numbers, segmental distribution, and location of the somata of these neurons quantitatively. HRP was applied to one hypogastric nerve (HGN), to the lumbar colonic nerves (LCN) and to the intermesenteric nerve (IMN). In order to estimate the significance of the branching of one axon into both hypogastric nerves a double-labeling technique with fluorogold and HRP was used. About 2640 neurons project into the two HGN added together (800 afferent, 1320 pre-, and 520 postganglionic), 4650 neurons into the LCN (360 afferent, 0 pre- and 4290 postganglionic), and 5990 into the IMN (1500 afferent, 1250 pre-, and 3240 postganglionic). About 4190 sympathetic postganglionic prevertebral neurons innervate the colon and pelvic organs, 1900 are located in the inferior mesenteric ganglion and 2290 in ganglia of the IMN. Considering the efferent component, the HGN mainly are preganglionic and the LCN exclusively postganglionic nerves. Branching of one axon into both HGN is a rare event and quantitatively negligible (less than 3%). Afferent neurons of all three nerves were found in the dorsal root ganglia (DRG) T12-L2 with the maximum in L1 and L2. The distribution of afferent neurons projecting into the LCN is shifted slightly more rostrally compared to neurons projecting into the HGN. The IMN distribution is located in a position in between. Preganglionic neurons projecting into the IMN are located in the spinal cord segments T12-L3 with the maximum in L1 and L2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726106 TI - Peripheral and central aspects of the acoustic and lateral line system of a bottom dwelling catfish, Ancistrus sp. AB - The topographical relationship between the swim bladder, the inner ear, and the otic lateral line was studied in the bottom dwelling catfish, Ancistrus sp. In addition, afferent and efferent subcomponents of the eighth and lateral line nerves were labelled with horseradish peroxidase (HRP) or with differently fluorescing dextran amines. The swim bladder of Ancistrus consists of two separate, transversely oriented parts of each of which is connected to the sinus impar of the inner ears via two Weberian ossicles and the perilymphatic sac. The osseous capsula of the ear has two foramina other than the nerve foramina. One is for the sinus impar. The other foramen, which also separates two fluid-filled spaces, exits where the horizontal canal of the ear contacts the otic lateral line. Both the otic and the postotic lateral line canal run deep below the epidermis. Each canal contains a neuromast that is innervated by the middle lateral line nerve. Further caudally, the otic lateral line canal gives rise to the postotic and finally to the truck canal whose nonossified anterior part travels through an ossified chamber that surrounds the swim bladder. Thus the anterior part of each trunk lateral line canal is in contact with a bipartite sound pressure receiver, the swim bladder. Anterior and posterior lateral line afferents terminate ipsilaterally throughout the neuropil of the electroreceptive lateral line nucleus and the mechanoreceptive nuclei medialis and caudalis of the medulla. Middle lateral line afferents terminate between the projection sites of anterior and posterior lateral line afferents. Some primary mechanosensory anterior lateral line nerve fibers continue into the ipsilateral eminentia granularis and the valvula cerebelli. In the electroreceptive lateral line projection, anterior lateral line fibers terminate more medially and posterior fibers more laterally. This somatotopy is not as clear-cut in the mechanosensory lateral line. Afferents of the sacculus and the lagena terminate predominantly in the saccular nucleus. Afferents of the utriculus, the horizontal canal, and the anterior vertical canal terminate in the magnocellular vestibular nucleus and in the medial octavolateral nucleus. The projection sites of the anterior part and the posterior part of the eighth nerve show little overlap. Eighth nerve projections to the valvula cerebelli are less prominent than the projections from the lateral line. Eighth nerve and lateral line nerve efferents arise from a common nucleus, the octavolateralis efferent nucleus. Axons of efferent cells may divide to supply two or more branches of the eighth nerve and some axons supply both lateral line and eighth nerve endorgans.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1726107 TI - The distribution of neuropeptides in the dorsomedial telencephalon of the pigeon (Columba livia): a basis for regional subdivisions. AB - The distribution of six neuropeptides [substance P (SP), leucine (leu5-) enkephalin (LENK), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), neuropeptide Y (NPY), and somatostatin (SS)] in the dorsomedial telencephalon (hippocampal region) of the pigeon was studied by immunohistochemistry. All six peptides were found in fibers passing through the septo-hippocampal junction and along the medial wall of the hippocampal region. NPY-, SS-, and VIP-like staining of fibers was seen in the hippocampal commissure. NPY and SS had similar distributions within the hippocampal region, both being most conspicuous in cell bodies, terminals, and fibers of the medial hippocampal region. VIP-positive cells were found in an area dorsal to the SS/NPY cell region. CCK-like immunoreactivity was found in terminal baskets surrounding large cells of a v-shaped structure in the ventromedial hippocampal region. SP- and LENK-like immunoreactivity was found in neuropils in a lateral-dorsal region, the two substances showing similar distributions. This region is thought to lie lateral to the limit of the hippocampal region. Parallels with the distribution of immunoreactivity in the mammalian hippocampus are used to suggest possible equivalent subdivisions of the avian and mammalian hippocampal regions. PMID- 1726108 TI - Thalamic projections to the lateral suprasylvian visual area in cats with neonatal or adult visual cortex damage. AB - Previous transneuronal anterograde tracing studies have shown that the retino thalamic pathway to the posteromedial lateral suprasylvian (PMLS) visual area of cortex is heavier than normal in adult cats that received neonatal damage to visual cortical areas 17, 18, and 19. In contrast, the strength of this projection does not appear to differ from that in normal animals in cats that experienced visual cortex damage as adults. In the present study, we used retrograde tracing methods to identify the thalamic cells that project to the PMLS cortex in adult cats that had received a lesion of visual cortex during infancy or adulthood. In five kittens, a unilateral visual cortex lesion was made on the day of birth, and horseradish peroxidase (HRP) was injected into the PMLS cortex of both hemispheres when the animals were 10.5 to 13 months old. For comparison, HRP was injected bilaterally into the PMLS cortex of three cats 6.5 to 13.5 months after they received a similar unilateral visual cortex lesion as adults. In cats with a neonatal lesion, retrograde labeling was found in the large neurons that survive in the otherwise degenerated layers A and A1 of the lateral geniculate nucleus (LGN) ipsilateral to the lesion. Retrograde labeling of A-layer neurons was not seen in the undamaged hemisphere of these animals or in either hemisphere of animals that had received a lesion as adults. As in normal adult cats, retrograde labeling also was present in the C layers of the LGN, the medial interlaminar nucleus, the posterior nucleus of Rioch, the lateral posterior nucleus, and the pulvinar nucleus ipsilateral to a neonatal or adult lesion. Quantitative estimates indicate that the number of labeled cells is much larger than normal in the C layers of the LGN ipsilateral to a neonatal visual cortex lesion. Thus the results indicate that the heavier than normal projection from the thalamus to PMLS cortex that exists in adult cats after neonatal visual cortex damage arises, at least in part, from surviving LGN neurons in the A and C layers of the LGN. Although several thalamic nuclei, as well as the C layers of the LGN, continue to project to PMLS cortex after an adult visual cortex lesion, these projections appear not to be affected significantly by the lesion. PMID- 1726109 TI - Development of the projections from the dorsal lateral geniculate nucleus to the lateral suprasylvian visual area of cortex in the cat. AB - In the study reported in the preceding paper, we used retrograde labeling methods to show that the enhanced projection from the thalamus to the posteromedial lateral suprasylvian (PMLS) visual area of cortex that is present in adult cats following neonatal visual cortex damage arises at least partly from surviving neurons in the dorsal lateral geniculate nucleus (LGN). In the C layers of the LGN, many more cells than normal are retrogradely labeled by horseradish peroxidase (HRP) injected into PMLS cortex ipsilateral to a visual cortex lesion. In addition, retrogradely labeled cells are found in the A layers, which normally have no projection to PMLS cortex in adult cats. The purpose of the present study was to investigate the mechanisms of this enhanced projection by examining the normal development of projections from the thalamus, especially the LGN, to PMLS cortex. Injections of HRP were made into PMLS cortex on the day of birth or at 1, 2, 4, or 8 weeks of age. Retrogradely labeled neurons were present in the lateral posterior nucleus, posterior nucleus of Rioch, pulvinar, and medial interlaminar nucleus, as well as in the LGN, at all ages studied. Within the LGN of the youngest kittens, a small number of retrogradely labeled cells was present in the interlaminar zones and among the cells in the A layers that border these zones. Such labeled cells were virtually absent by 8 weeks of age, and they are not found in normal adult cats. Sparse retrograde labeling of C-layer neurons also was present in newborn kittens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726110 TI - Transabdominal placental biopsy in the second and third trimester of pregnancy. AB - Transabdominal placental biopsy under ultrasound guidance was carried out in 260 cases in the second trimester and 50 cases in the third trimester of pregnancy. Placental tissue was aspirated using an 18 or 20 gauge needle. In a total of 310 placental biopsies in the second and third trimester, 100 were performed because of suspicious ultrasonographic findings. Placental biopsy is simple in the presence of severe oligohydramnios where fetal blood sampling is usually more difficult. Oligohydramnios and polyhydramnios were the ultrasonographic findings in 50% of cases and were found to be associated with 30% of abnormal chromosomal findings. There was one (0.3%) abortion within two weeks following placental biopsy. Placental biopsy did not affect the outcome of the pregnancy. PMID- 1726111 TI - Oxygen transport in newborns at different gestational ages. AB - Oxygen (O2) transport was assessed through the affinity between O2 and hemoglobin (Hb) in 123 newborns of 28 to 40 week gestational ge, with a minimum of 9 newborns for each gestational age group (see table). In order to assess the O2-Hb affinity, we studied the correlation between the pO2 and the Hb saturation for each gestational age, obtaining estimates of the oxy-hemoglobin dissociation curves corresponding to each gestational age (see fig. 3). The pO2 levels corresponding to the 50% saturation (P50) for each gestational age were estimated from there. All newborns were from single vaginal deliveries with no fetal distress before birth and with an adequate weight for gestational age. The latter was calculated according to the date of the last menstrual period (78% of the cases), echography (10.6% of the cases) or neonatal physical exam (11.4% of the cases). A P50 vs. gestational age linear regression showed a high determination rate (r2 = 0.957, p less than 0.00001) (see fig. 2) which supports the hypothesis of the P50 linear growth; decrease in the Hb-O2 affinity with increasive gestational age (Hb-O2 affinity is different in newborns of different gestational ages). With these results one may conclude that the Hb-O2 uptake varies according to gestational age (P50 changes linearly as gestational age increases) and that a single measurement of pO2 in a newborns, blood does not accurately evaluate the amount of O2 that is transported to the tissues, because the transport capacity depends, among other factors, upon gestational age. The Hb saturation better represents the amount of O2 that can get to the cell level.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726112 TI - Intracellular delivery of drugs by liposomes containing P0 glycoprotein from peripheral nerve myelin into human M21 melanoma cells. AB - The effect of P0 protein (a cell adhesion molecule from avian peripheral nerve myelin) on the rate of interaction of liposomes with human M21 melanoma cells was investigated. Liposome uptake by the cells was quantitated using radioactive lipids and liposome-entrapped drugs under various conditions. Liposomes containing P0 protein and [14C]dipalmitoylphosphatidylcholine:cholesterol (10:1 molar ratio) had an interaction rate with M21 cells three times higher than control vesicles of the same lipid composition but without the protein after incubation at 37 or 4 degrees C. The presence of P0 protein could be detected on the surface of melanoma cells by immunofluorescence after incubation. Binding to the cell surface and endocytosis of P0 liposomes was suggested from the sensitivity of cell-associated proteoliposomes to trypsin, metabolic inhibitors, and low temperature. Liposomal encapsulation highly increased the association of model compounds [( 3H]methotrexate and [3H]inulin) with cells. The proteoliposomes appeared to be leaky in the incubation medium, which led to the delivery of a lower amount of drug into cells than could be expected from their initial drug content. The results suggest that the attachment of liposomes to the cell surface can increase their drug delivery potential, because the binding triggers endocytic processes or a juxtapositional temporary permeability increase of liposome and cellular membrane that can lead to the uptake of drug from liposomes. PMID- 1726113 TI - Differential effects of inhibitors of cellular function on inflammatory mediator stimulated increases in vascular permeability. AB - The suffused noneverted cheek pouch of pentobarbital anesthetized hamsters was used to study the effects of various inhibitors of receptor/cellular function on inflammatory mediator stimulated increases in vascular permeability. Fluorescein isothiocynate dextran (FITC-D, 70,000 Da) was utilized as a tracer, and intra vital light microscopy was employed to monitor the formation of vascular leakage sites while direct measurement of plasma and suffusate tracer concentrations were used to monitor tracer clearance. Vascular permeability increases were triggered by suffusing the cheek pouch with histamine, bradykinin, or Compound 48/80 which stimulated the formation of focal FITC-D leakage sites in the postcapillary venules resulting in marked increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. Saline, calmidazolium, and papaverine lacked intrinsic permeability increasing activity, and failed to alter histamine, bradykinin, and compound 48/80 stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. In contrast, treatment with cytochalasin B, DDAVP, diphenhydramine, tubulazole C, or verapamil inhibited histamine and Compound 48/80 stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. Bradykinin stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance were not affected by treatment with calmidazolium, cytochalasin B, DDAVP, diphenhydramine, tubulazole C, or verapamil. These findings demonstrate that inflammatory mediator stimulated increases in vascular permeability may be differentially affected by inhibitors of receptor/cellular function. PMID- 1726114 TI - Effects of local mast cell degranulation on vascular permeability to macromolecules. AB - The suffused noneverted cheek pouch of pentobarbital anesthetized hamsters was used to study the effects of localized, selective mast cell degranulation on vascular permeability. Fluorescein isothiocynate dextran (FITC-D, 70,000 Da) was utilized as a tracer, and intra-vital light microscopy was employed to monitor the formation of vascular leakage sites while direct measurement of plasma and suffusate tracer concentrations were used to monitor tracer clearance. Varying the time at which the FITC-D tracer was injected i.v. relative to the start of the Compound 48/80 suffusion permitted direct determination of the duration of any observed increase in vascular permeability. Selective, local mast cell degranulation was triggered by suffusing the cheek pouch with Compound 48/80 for 10 minutes which stimulated the formation of focal FITC-D leakage sites in the postcapillary venules resulting in increases in [FITC-D]S, [FITC-D]S/[FITC-D]P. 10(-6), and FITC-D clearance. In contrast, suffusion of the cheek pouches with saline failed to trigger the formation of venular FITC-D leakage sites or to promote increases in [FITC-D]S, [FITC-D]S/[FITC-D]P. 10(-6), and FITC-D clearance. The increase in permeability produced by Compound 48/80 was marked but transient (duration less than 20 minutes), and subject to inhibition by treatment with either the H1 receptor antagonist diphenhydramine or the endothelial cell stabilizer isoproterenol. There was no evidence for for a non-histamine mediated or delayed-onset increase in vascular permeability to macromolecules during the course of these experiments. PMID- 1726115 TI - Acidic fibroblast growth factor (aFGF)-like immunoreactivity in the optic nerve. AB - Acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined in the optic nerves of 1- to 25-month-old Wistar rats, 0.5- to 7-year-old bovine animals and normal human adults (24 and 35 years old), using cryostat sections incubated with a rabbit polyclonal antibody specific for aFGF. The immunoreactivity was associated with glial cells, and was localized predominantly in the nucleus. The presence of endogenous aFGF in the optic nerve of adult subjects and 'old' rats suggests that aFGF could play a role in the survival of retinal ganglion cells and their axons during aging. PMID- 1726116 TI - Serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons projecting from the periaqueductal gray to the ventromedial hypothalamic nucleus in the rat. AB - Direct projections from serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) to the ventromedial hypothalamic nucleus (VMH) in the rat were investigated by the retrograde horseradish peroxidase tracing method combined with the immunocytochemical technique. Serotonin-, substance P- and tyrosine hydroxylase like immunoreactive PAG neurons sending their axons to the VMH were distributed in the ventrolateral subnucleus and ventral portion of the medial subnucleus of PAG at the middle and caudal levels. PMID- 1726117 TI - Capsazepine, a novel capsaicin antagonist, selectively antagonises the effects of capsaicin in the mouse spinal cord in vitro. AB - The mouse hemisected spinal cord with attached dorsal roots and spinal ganglia in vitro preparation was used to investigate the effects of the capsaicin antagonist, capsazepine (2-[2-(4-chlorophenyl)ethylamino-thiocarbonyl]-7,8 dihydroxy-2,3,4 ,5- tetrahydro-1H-2-benzazepine). The spinal cord and the ganglia were separated by a perspex gap, allowing application of drugs separately to each compartment. Intracellular recordings were made from 37 cells in laminae II-VI of 12 to 20-day-old mice. Brief applications (30 s) of capsaicin (0.8 microM) excited dorsal horn neurones by activating small diameter primary afferent fibres. The response to capsaicin administered to the spinal cord or to the spinal ganglia was antagonised by the capsaicin antagonist, capsazepine (1.5 microM), administered to the same site. Excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the dorsal root were not affected by capsazepine. Capsazepine itself (5 microM) did not affect the membrane potential of the dorsal horn cells. Capsazepine did not depress the depolarization evoked by substance P. When capsazepine was applied to the spinal cord and capsaicin to the dorsal root ganglion the capsaicin effect was not antagonised. These data suggest that capsaicin-induced depolarization of spinal dorsal horn neurones was mediated via activation of a specific receptor on primary afferent neurones. PMID- 1726118 TI - Periodontists warn against inappropriate dental laser use. PMID- 1726119 TI - Rapid degradation of neurotensin by stimulated rat mast cells. AB - A RIA towards neurotensin (NT) using C-terminal- and N-terminal-specific antisera was used to study degradation of this tridecapeptide by isolated rat mast cells. Incubation of NT (10 microM) with peritoneal or pleural mast cells resulted in a rapid loss of NT immunoreactivity (iNT), as measured by C-terminal-directed antiserum, with little effect on N-terminal iNT. The rate of the reaction was faster with pleural cells (T1/2, 30 s) than with peritoneal cells (T1/2, 180 s) and was greater than 10-fold slower in the presence of metabolic poisons. The enzyme(s) involved is most likely released from the cells during secretion, as NT was degraded by media conditioned by compound 48/80-stimulated mast cells 40-60 times faster than by media from unstimulated cells. This degradation by conditioned media was concentration dependent, pH dependent, and temperature sensitive. HPLC analyses indicated a near stoichiometric conversion of NT to NT(1 12) (66%) and NT(1-11) (34%) after incubation for 10-30 s with conditioned media. By 30 min only NT(1-11) and NT(1-10) were present. Phenanthroline (1 mM), an inhibitor of carboxypeptidase, prevented the loss of C-terminal iNT and the generation of NT(1-12) and NT(1-11). While NT(1-12) was effective in releasing histamine from mast cells in vitro and increasing vascular permeability in vivo, NT(1-11) was not. These results suggest that carboxypeptidase-like enzyme(s) could modulate the level and form of NT-related peptides in various states involving activation of mast cells. PMID- 1726120 TI - Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord. AB - Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities. PMID- 1726121 TI - Vasopressin elevates cytosolic calcium in small cell lung cancer cells. AB - The ability of vasopressin to elevate cytosolic Ca2+ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca2+ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca2+ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca2+ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca2+. The response to vasopressin was strongly blocked by [(beta-mercapto-beta,beta cyclopentamethylene propionic acid)1,O-MeTyr2,Arg8]vasopressin and weakly blocked by [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,O MeTyr2,Orn8]vasotocin. These data suggest that V1 vasopressin receptors are present on SCLC cells. PMID- 1726122 TI - Acute effect of substance P in immunologic vasculitis in the rat colon. AB - Substance P has been implicated as a neuronal mediator of inflammation in various inflammatory conditions. However, the exact role played by substance P in inflammatory bowel diseases or in experimental colonic vasculitis has not been clearly understood. In this study, we examined the effect of close superior mesenteric artery injection of substance P under prevailing inflammatory conditions induced by intravenous human albumin antialbumin immune complex followed by intracolonic perfusion of 2.5% formaldehyde in rats or intracolonic perfusion of 5% alcohol alone. The immune complex- and formaldehyde-treated rats showed severe microvascular changes such as microvascular plugging by red blood cells, endothelial breakage and extravasation of plasma proteins and red blood cells. The bolus injection of 10(-8) M substance P reduced extravasation of Evans blue dye by 50% and the tissue wet to dry ratio by 20% in immune complex- and formaldehyde-perfused rats. Myeloperoxidase activity was not changed. Substance P also significantly inhibited (44%) the extravasation in alcohol-perfused rats. Pretreatment of immune complex- and formaldehyde-treated rats with substance P antagonist reversed the effect of substance P. These findings suggest that the most immediate effect of substance P may be vasodilation and clearing of vascular plugs induced by immune complex and formaldehyde. This effect of substance P differs from its chronic effect, which causes vasodilation and extravasation. PMID- 1726123 TI - Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme. AB - In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (less than 1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15-5.91 nmol/min/ml), which sequentially converted SP to SP(3 11) and SP(5-11). In turn, the SP(5-11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2-25.5 nmol/min/ml). The Km values of SP for DAP IV and of SP(5-11) for AmM ranged from 32.7 to 123 microM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76-10.8 nmol/min/ml; Km = 90.7 microM). These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma. PMID- 1726124 TI - Inhibitory role on gastric secretion of a central NK-3 tachykinin receptor agonist, senktide. AB - When administered intracerebroventricularly, the highly selective NK-3 tachykinin receptor agonist senktide possesses a potent and dose-related inhibitory effect on gastric acid secretion. The central mechanism governing the antisecretory effect of senktide was examined in perfused-stomach rats by studying its influence on gastric acid secretion elicited by the secretagogues histamine, pentagastrin and bethanechol. Given intracerebroventricularly, senktide reduced the acid response to histamine, but not that to pentagastrin or bethanechol. Stimulation of NK-3 receptors in rat brain thus appears to inhibit gastric acid secretion through histaminergic pathways. PMID- 1726125 TI - Interrelationships between prostanoids and skin flap survival: a review. PMID- 1726126 TI - Interaction(s) between essential fatty acids, eicosanoids, cytokines, growth factors and free radicals: relevance to new therapeutic strategies in rheumatoid arthritis and other collagen vascular diseases. AB - Eicosanoids, lymphokines, and free radicals are known to participate in the pathogenesis of inflammation. Tumour necrosis factor (TNF), interleukin-1 and 6 (IL-1 and IL-6) and colony stimulating factor -1 (CSF-1) are secreted mainly by activated macrophages, whereas T-cells secrete IL-2, IL-3, IL-4 and interferon gamma (IFN-gamma). In addition, activated macrophages and lymphocytes can also produce eicosanoids and free radicals which have potent pro-inflammatory actions. Eicosanoids, lymphokines, and free radicals can modulate the immune response, cell proliferation, stimulate collagenase and proteases secretion and induce bone resorption; events which are known to be associated with various collagen vascular diseases. On the other hand transforming growth factor-beta (TGF-beta) produced by synovial tissue, platelets and lymphocytes can inhibit collagenase production, suppress T-cell and NK-cell proliferation and activation and block free radical generation and seems to be of benefit in rheumatoid arthritis. Drugs such as cyclosporine, 1,25,dihydroxycholecalciferol and pentoxyfylline can block lymphokine and TNF production and thus, may inhibit the inflammatory process. Essential fatty acids, the precursors of eicosanoids, are suppressors of T-cell proliferation, IL-1, IL-2 and TNF production and have been shown to be of benefit in rheumatoid arthritis, systemic lupus erythematosus and glomerulonephritis. Thus, the interactions between essential fatty acids, eicosanoids, lymphokines, TGF-beta and free radicals suggest that new therapeutic strategies can be devised to modify the course of collagen vascular diseases. PMID- 1726127 TI - [Evaluation of culture media for detecting the starch hydrolysis reaction in pathovars of Xanthomonas campestris]. AB - Sixty strains of different pathovars of Xanthomonas campestris have been tested for the evaluation of various starch agars and compounds of starch degradation on six media: soluble starch, potato insoluble starch, corn insoluble starch, potato amylopectin, corn amylopectin and potato amylose. The purpose of the present investigation was the selection of the most suitable medium for the visualization of the starch hydrolysis test, presenting this reaction as a distinct character between pathovars of the Xanthomonas campestris group. From 60 strains tested, 74% gave positive reactions. Pathovars holcicola, pelargonii, pruni and vitians were negative. Regarding X. campestris pv. vesicatoria cultures, results were variable. Potato and corn insoluble starch agars were the most suitable media for the visualization of the starch hydrolysis reaction and at the same time the most appropriate for direct isolation. Differentiation at species level could be practicable, but within the Xanthomonas campestris group, variation amongst pathovars suggest the unsuitability of the test in spite of the high percentage of positive reactions. PMID- 1726128 TI - Immunocytochemical identification of Leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies. AB - The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 microns thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-Leishmania sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by anti-Leishmania sera only at low dilutions (between 1:60-1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes. PMID- 1726129 TI - Kinetics of bioaccumulation and clearance of isomeric hexachlorocyclohexanes. AB - Bioaccumulation experiments were performed on the hexachlorocyclohexane isomers alpha-HCH, beta-HCH, gamma-HCH and delta-HCH, testing them simultaneously. Bioaccumulation factors (BCFs) were calculated from the uptake rate constant (k1) and the clearance rate constant (k2) as: BCF = k1/k2. The BCFs of alpha-HCH (1100 +/- 175) and gamma-HCH (920 +/- 131) were similar. The BCFs of beta-HCH (1520 +/- 276) and delta-HCH (1640 +/- 269), also similar, were significantly higher than those of the alpha- and gamma-isomers. PMID- 1726130 TI - Chromatographic measurement of mucin production in cultures of gastric mucous cells. AB - The use of a miniature column chromatographic assay (using Sepharose CL-4B columns) for measuring mucin production in guinea pig gastric mucous cell cultures is described. The assay was based upon the ability of radiolabelled precursors ([14C]serine and [3H]galactose) to incorporate with high specificity into mucins which thereby appeared in the excluded material. Rates of excluded material radiolabelling by both precursors were constant for incubations up to 24 hours, and substantially reduced by cycloheximide co-incubation (25 microM). Labelled excluded material was completely degraded by mild alkaline borohydride treatment, only partially degraded by HNO2 (pH 1.5), and not degraded by chondroitinase ABC. Thus the major radiolabelled product measured in this system was mucin, although we found that it was less glycosylated than gastric mucins obtained from other sources. In addition, the technique employed to separate and measure mucin production proved rapid and consistent. PMID- 1726131 TI - Allosteric inhibition of the water-soluble C-terminal fragment of Sendai virus neuraminidase. AB - Trypsin solubilized hemagglutinin-neuraminidase of Sendai virus (cHN) displays michaelian kinetics, with native fetuin as substrate, at 37 degrees C. Vmax and Km values are only marginally altered, as compared to intact viral neuraminidase. At lower temperatures, cHN follows non-michaelian kinetics, with marked substrate inhibition at 4 degrees C. With denaturated fetuin, michaelian kinetics are observed in all conditions, while asialo fetuin was an uncompetitive inhibitor of cHN, with native fetuin or sialyl lactose as substrates. These results can be explained assuming that the protein moiety of fetuin acts as an allosteric inhibitor of cHN. PMID- 1726132 TI - Changes in stainability observed by light microscopy in the brains of ataxial mice subjected to three generations of manganese administration. AB - Two neonates of mice which manifested abnormal motions in their gait in the third generation litter, following the start of manganese (Mn) administration, were selected. One was severely affected by Mn and the other was only moderately affected. Various regions in the brains of the neonates were subjected to histochemical examination under a light microscopy. The losses of stainability in granular cells in the external layer of the cerebral cortex, and Purkinje cells in the cerebellar cortex, and the increase in stainability of the nerve fibers in the cerebellar medulla were in parallel to the degree of abnormal movement in the gait; the greater loss or gain in stainability, varying according to the regions, was associated with the more severe damages to motion. Meanwhile, the changes in the stainabilities of nerve cell nuclei in the lamellar structure of cerebral motor areas and the Nissl bodies in the cerebral medulla were already maximal in the moderately affected neonate. These results indicate that the Mn effect covers a broad area of the extrapyramidal tract even though there are some differences in the sensitivity to Mn in different regions. PMID- 1726133 TI - Removal of 3' nontranslated cDNA sequence improves interferon gamma synthesis in Escherichia coli. AB - The cDNA coding for human interferon gamma contains approximately 600 basepairs not translated sequence with 4 adenine/thymidine (AT)-rich clusters at its 3'end. The influence of this sequence on the expression of recombinant human Interferon gamma in Escherichia coli was investigated. Removal of the non-translated sequence and especially of the AT-rich clusters yielded an increase in expression of interferon gamma from 10 to 40% of total protein. These results may also be important for the production of other recombinant human proteins with similar 3' sequences in their cDNA. PMID- 1726134 TI - Synthesis of tolerogenic monomethoxypolyethylene glycol and polyvinyl alcohol conjugates of peptides. AB - Recent studies from this laboratory showed that tolerogenic peptide conjugates are very effective reagents for obtaining epitope-specific immunosuppression of antibody responses to immunopathogenic sites on multideterminant complex protein antigens. This paper describes the procedure for synthesis of well-defined conjugates of peptides to monomethoxypolyethylene glycol (mPEG) or to polyvinyl alcohol (PVA). The first step involves succinylation of the hydroxyl groups on the polymers by reaction with succinic anhydride. The polymer is then coupled via the carboxyl of the succinyl group to the alpha-NH2 of the completed peptide on the synthetic resin, while maintaining intact all the side-chain protecting groups on the peptide. The mPEG or PVA-peptide conjugates are cleaved from the resin and purified by standard procedures. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is coupled to the N terminal of an otherwise unaltered peptide molecule. PMID- 1726136 TI - Molecular characterization of the murine mutation myelin deficient. PMID- 1726135 TI - Genetic analysis of ion channels in Drosophila. PMID- 1726137 TI - The time dependent UV resonance Raman spectra, conformation, and biological activity of acetylcholine analogues upon binding to acetylcholine binding proteins. AB - In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1726138 TI - Immunohistochemical study of sensory nerve formations in human glabrous skin. AB - The sensory nerve formations (or corpuscles) of normal human glabrous skin from hand and fingers, obtained by punch biopsies, were studied by the streptavidin biotin method using monoclonal antibodies directed against neurofilament protein (NFP), S-100 protein, glial fibrillary acidic protein (GFAP), cytokeratins, and vimentin. NFP immunoreactivity (IR) was observed in the central axons of most sensory formations, while S-100 protein IR was restricted to non-neuronal cells forming the so-called inner cells core or lamellar cells. Furthermore, vimentin IR was found in the same cells of Meissner's and glomerular corpuscles. None of the sensory nerve formations were stained for GFAP or keratin. The present results suggest that the main nature of the intermediate filaments of the non neuronal cells of sensory nerve formations from human glabrous skin is represented by vimentin and not by GFAP. Thus, our findings suggest that lamellar and inner core cells of SNF are modified and specialized Schwann cells and not epithelial or perineurial derived cells. PMID- 1726139 TI - Neonatal whisker removal in rats stabilizes a transient projection from the auditory thalamus to the primary somatosensory cortex. AB - A normally transient cross-modal thalamocortical projection from the magnocellular subdivision of the medial geniculate nucleus (MGm) to the primary somatosensory (SI) cortex of rats was found to remain unchanged throughout adulthood following unilateral removal of whiskers in newborn animals. The normal MGm projection to the auditory cortex is not lost in these neonatally whisker deprived adults rats but some of the MGm neurons send collaterals to both primary auditory and SI cortices. Parallel electrophysiological experiments demonstrated the multimodal character of some MGm neurons, since they responded to both auditory and cutaneous stimulation. These results suggest that the areal distribution in the cortex of thalamocortical projections arising from a multimodal thalamic nucleus, such as the MGm, may be determined during early postnatal development by the normal flow of sensory information from the periphery to the thalamus and that an early postnatal somatosensory deprivation may prevent the normal withdrawal of a cross-modal projection from the MGm to the SI. PMID- 1726140 TI - Dopaminergic activity is reduced in the prefrontal cortex and increased in the nucleus accumbens of rats predisposed to develop amphetamine self-administration. AB - Individual vulnerability to the reinforcing effects of drugs appear to be a crucial factor in the development of addiction in humans. In the rat, individuals at risk for psychostimulant self-administration (SA) may be identified from their locomotor reactivity to a stress situation such as exposure to a novel environment. Animals with higher locomotor responses to novelty (High Responders, HR) tend to acquire amphetamine SA, while animals with the lower responses (Low Responders, LR) do not. In this study, we examined whether activity of dopaminergic (DA) and serotoninergic (5-HT) systems differed between HR and LR animals. These transmitter systems are thought to be involved in the reinforcing effects of psychostimulants. Animals from both groups were sacrificed under basal conditions and after exposure for 30 or 120 min to a novel environment, and the DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindolacetic acid (5-HIAA) contents were determined in the prefrontal cortex, nucleus accumbens and striatum. The HR rats displayed a specific neurochemical pattern: a higher DOPAC/DA ratio in the nucleus accumbens and striatum and a lower one in the prefrontal cortex. Furthermore, HR animals had lower overall 5-HT and 5-HIAA levels, corresponding to the mean of these compounds for the three structures studied over the three environmental conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726141 TI - Presynaptic M2-muscarinic receptors on noradrenergic nerve endings and endothelium-derived M3 receptors in cat cerebral arteries. AB - The muscarinic (M) receptors involved in the vasodilation elicited by acetylcholine (ACh) and in the carbachol inhibition in electrically induced [3H]noradrenaline (NA) release in cat cerebral arteries was investigated. For this, atropine, pirenzepine, AF-DX 116, 4-DAMP, non-specific, M1, M2 and M3 receptor antagonists, respectively, were used. ACh elicited concentration dependent relaxations up to 10(-6) M which were attenuated by these antagonists; the order of potency (pA2 values) to inhibit the ACh-induced relaxation was: atropine (10.1) 4-DAMP (8.9) greater than pirenzepine (7.6) greater than AF-DX 116 (5.9). The electrical stimulation (200 mA, 0.3 ms, 2 Hz, during 1 min) of these arteries preincubated with [3H]NA caused tritium release which was inhibited by carbachol (10(-6) M). The 4 antagonists attenuated the action of the M agonist; the order of potency (pIC50 values) was: atropine (8.7) greater than 4 DAMP (8.1) greater than AF-DX 116 (7.9) greater than pirenzepine (5.8). The action of McN-A-343, a putative M1 agonist, was also investigated. This agent produced small vasodilator responses and elevated concentrations (5 x 10(-5) M) inhibited the stimulated NA release, which was partially antagonized by atropine (10(-7) M) and pirenzepine (10(-8) and 10(-7) M). These results suggest the existence of M3 and M2 receptors mediating the relaxation induced by ACh and the NA release inhibition evoked by carbachol, respectively. PMID- 1726142 TI - [Parameters of non-specific immunity in umbilical cord blood in response to the stress of labor and surgery in healthy mothers and patients with late gestoses]. AB - The authors investigated six proteins of the acute phase in the serum of mothers and in the umbilical blood of the foetus during spontaneous delivery and after Caesarean section in 40 women and their neonates. Half the women were in good health, half suffered from late gestoses. The patients' age, weeks of delivery and operation, the mean body weight of the neonates and evaluation of their condition after delivery by the Agpar score were comparable. The results were processed statistically. Mean levels of six proteins of the acute phase were assessed in the serum of the mother and the umbilical blood in all four groups of women and their neonates and percentage ratios of proteins in mother and foetus were assessed. On evaluation by the two-sample t-test in the umbilical blood of spontaneous deliveries in late gestoses significantly lower prealbumin values were found, and the same applied to Caesarean sections. Conversely a significant increase of A2MG levels and orosomucoid was found in the umbilical blood of children from mothers with late gestoses after Caesarean section. The values of the orosomucoid/prealbumin index according to Hollinshead were markedly elevated in late gestoses in the maternal blood as well as in the umbilical blood of the foetus. PMID- 1726143 TI - [Sensitivity of human squamous and adenoid cystic carcinoma cell lines with cisplatin and it's combined chemotherapy in vitro]. AB - This was a report on the sensitivity of human squamous (Tca-8113) and adenoid cystic carcinoma (Acc-2) cell lines with Cisplatin (DDP) and it's combined chemotherapy (DDP+VCR+PYM, or PVP). DDP could kill both these two cell lines in vitro depending on the concentration of agent in given time. The rate of 3H-TdR incorporation, plating efficiency and DNA synthesis of carcinoma cells were prevented in drug-exposing groups. Tca-8113 cell line was more sensitive to DDP than Acc-2 cell line (P less than 0.01). PVP combined chemotherapy could enhance DDP's anticancer efficiency. PMID- 1726144 TI - [Biphenotypic acute leukemia. Clinical, morphological, cytochemical and immunophenotypic studies]. AB - The blast cells from 2 cases of acute leukemic patients classified as M1 type by FAB criterion simultaneously expressed lymphoid markers such as SmIgG, CD19, CD20, DR, PAS in case 1 and CD9, CD10, DR, PAS in case 2. The blast cells of these two cases also expressed CD38 antigen. The data on phenotype and cytochemistry in these two cases fulfil the criteria of biphenotypic acute leukemia proposed by Dr. Gale. The problems in diagnosis, treatment and prognosis of this kind of mixed acute leukemia were discussed. PMID- 1726145 TI - [Advances in the research of clinical use of recombinant human granulocyte colony stimulating factors]. PMID- 1726146 TI - Language activation studies with positron emission tomography. AB - Behavioural tasks produce changes in regional cerebral blood flow (rCBF), the result of increased local neural activity. These changes can be measured with positron emission tomography (PET). Language activation studies by means of PET are being used to relate regional patterns of cerebral activation to information processing models of speech and reading. Significant activation confined to both superior temporal gyri has been observed when normal subjects hear words played backwards, listen to non-words, and perform category judgements on pairs of heard real words. Prestriate cortex is activated by seeing strings of letter-like symbols, consonant strings, pronounceable non-words and real words, with additional activation in left medial prestriate cortex in response to the non words and real words. Left posterior superior temporal gyrus (PSTG), left dorsolateral prefrontal cortex (DLPFC) and supplementary motor area (SMA) are engaged when subjects retrieve verbs from memory to match nouns. Finally, primary sensorimotor cortex is activated during articulation. There is particular interest at present in the precise roles of left PSTG and DLPFC in single-word comprehension and generation, and interpretation of the results depends critically on the design of the single-word tasks used for behavioural activation. PMID- 1726147 TI - [Adverse drug reaction of dextran-40: analysis of 57 cases]. AB - Dextran-40 has long been used as a potential anticoagulant in hand surgery. From 1982 to 1989, it was given to 148 cases undergoing hand surgery. 57 of them developed adverse drug reaction due neither to overdose nor inappropriate use. Clinical manifestations and varieties of these reactions were verified. According to the standards issued by WHO, the reactions in these 57 cases were classified as having Grade I 9 cases, Grade II 26 cases; and other reactions 22 cases. The factors inducing reaction and the characteristics of the clinical manifestations were discussed. In spite of adverse reactions, Dextran-40 is still an effective anticoagulant widely used in the field of hand surgery. But it should be used with care. PMID- 1726148 TI - [The use of anti-keratin and anti-CEA monoclonal antibodies in the study of lacrimal epithelial tumors]. AB - 34 cases of lacrimal epithelial tumor were studied with the anti-keratin and anti CEA monoclonal antibody peroxidase-anti-peroxidase staining and the AB/PAS mucohistochemical staining. It was found that HK2 was positive in the gland luminal cells and cancer cells in the squamous metaplastic portion of the tumor; K12 was positive in the mucoid, fibroid, and cartilaginoid metaplastic tumor cells, while K27 was positive in less number of the squamous cells; CEA was positive in tumor cells of the nest and glandular lumina. Mecohistochemically, the tumor cells were 100% positive. The authors were of the opinion that keratin HK2 and K12 and CEA were ideal markers for lacrimal epithelial tumors. PMID- 1726149 TI - [Periodontal side effects of the immunosuppressant cyclosporin A. Clinical study]. AB - Gingival hyperplasia caused by the immunosuppressant cyclosporin A seems to be another variant of pharmacologically induced gingival enlargements. Clinical studies were carried out to sheed light on the differences and common features of cyclosporin-vs. diphenyl hydantoinate-induced gingival hyperplasia. A marked difference was the pronounced vascularization observed both clinically and histologically in cyclosporin A-induced hyperplasia. PMID- 1726150 TI - A new staining method for the detection of activities of H2O2-producing oxidases on gels and blots using cerium and 3,3'-diaminobenzidine. AB - Cerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown-black precipitate by the subsequent reaction with 3,3'-diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecificity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms. PMID- 1726151 TI - Pharmacological evidence of alpha 1- and alpha 2-adrenergic supersensitivity in orthostatic hypotension due to spinal cord injury: a case report. AB - Sympathetic efferent pathways and alpha-adrenergic receptivity were investigated in one patient with spinal cord transection (D1 level) and orthostatic hypotension. The lack of increase in catecholamine plasma levels during orthostasis and the paradoxical pressor effect of clonidine (2 micrograms/kg orally) suggested complete interruption of efferent sympathetic pathways. The pressor response to exogenous noradrenaline was significantly higher in the patient than in 6 normal controls (0.09 vs 0.72 micrograms.kg-1), indicating supersensitivity of vascular alpha-adrenoceptors. The platelet alpha 2-adrenergic receptor number, measured with [3H]yohimbine, was 507 in the patient vs 178 fmol.mg-1 protein in controls. The increase in systolic blood pressure induced by 10 mg midodrine, a specific alpha 1-agonist, was significantly higher in the patient (delta = 56 mm Hg) than in controls (delta = 15 mm Hg). The results indicate that in the patient there was alpha-adrenergic supersensitivity both of alpha 1- and alpha 2-adrenoceptors. This led to successfully oral treatment of the orthostatic hypotension with clonidine 150 micrograms bd and midodrine 10 mg bd. PMID- 1726152 TI - Platelet-derived growth factor inhibits cytokine induction of nitric oxide synthase in rat renal mesangial cells. PMID- 1726153 TI - Cytochalasin B stimulates insulin-like growth factor-binding protein-1 production by Hep G2 cells. AB - Hep G2 cells were used to study the early sequence of events regulating production of insulin-like growth factor-binding protein-1 (IGFBP-1). Cytochalasin B (100 microM) specifically inhibited 2-deoxyglucose uptake by Hep G2 cells and stimulated IGFBP-1 production 2-fold. Insulin (300 nM) did not stimulate hexose uptake but inhibited IGFBP-1 production more than 50%. A change in IGFBP-1 secretion was observed as early as 2 h after a 15-min or 2-h pulse exposure to either effector. In contrast to IGFBP-1, albumin production was diminished in the presence of cytochalasin B and increased by insulin. From these results we conclude that IGFBP-1 synthesis is (i) stimulated by transient inhibition of cellular glucose uptake and further stimulated by long-term glucose deprivation, and (ii) inhibited by transient exposure to insulin with further inhibition on long-term exposure. These effects are consistent with the dynamic regulation of IGFBP-1 by nutritional status. PMID- 1726154 TI - Medium hyperosmolarity depresses thyrotropin-releasing hormone-induced Ca2+ influx and prolactin secretion in GH4C1 cells. AB - We studied the influence of graded degrees of hyperosmolarity on the dynamics of the thyrotropin-releasing hormone (TRH)-induced rise in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion in GH4C1 cells. TRH caused two phases of increase in [Ca2+]i that were differentially altered by hyperosmolarity: 100% hyperosmolarity (600 mOsm) depressed only 20% of an initial high-amplitude [Ca2+]i burst (first phase) dependent on Ca2+ mobilized from intracellular pools, but it abolished a sustained low-amplitude second phase dependent on extracellular Ca2+ influx. Low degrees of hyperosmolarity suppressed PRL secretion due to Ca2+ influx while high degrees suppressed secretion due to mobilized Ca2+. These data suggest that in GH4C1 cells hypertonic inhibition of secretion may result from both blocking Ca2+ influx and mechanisms unrelated to [Ca2+]i. PMID- 1726155 TI - Expression of transforming growth factor-alpha and epidermal growth factor receptor in human fetal kidneys. AB - Beginning at the fifth week of fetal life, successive generations of individual nephrons are induced by contact between metanephric mesenchyme and ureteric bud. Following phenotypic transformation, cells of each primitive renal vesicle undergo a phase of rapid cell division. In order to identify genes which might regulate nephron development in man, we screened adult and fetal kidney RNA for expression of a panel of growth-related genes. Among the genes which were expressed at higher levels in fetal kidney was the epidermal growth factor (EGF) receptor. There is controversy as to the most likely physiologic EGF receptor ligand in fetal kidney; we were able to identify a transcript for transforming growth factor-alpha (TGF-alpha) but not EGF on Northern blots of fetal kidney RNA. Since the abundance of TGF-alpha mRNA is low, we confirmed its presence by polymerase chain reaction amplification. Using specific radioimmunoassays, we also provide direct evidence for TGF-alpha but not EGF peptide in extracts of fetal kidney and mid-gestational amniotic fluid. We suggest that TGF-alpha/EGF receptor interactions may serve an important function in development of human fetal kidney. PMID- 1726156 TI - [Morpho-functional changes in the myocardium after exposure to infrasound]. AB - Influence of infrasound with frequency of 8 and 16 Hz, pressure from 120 to 140 dB and duration 3 h a day from 1 to 40 day was studied. Increasing of the myocardium functional activity was noted. No destructive changes were discovered. PMID- 1726157 TI - Galanin stimulates release of LH in young chickens. PMID- 1726158 TI - Increased angiogeneic activity of spleen cells in spontaneous hypertensive rats (SHR). PMID- 1726159 TI - Inhibition of plasminogen activation by monoclonal antibodies to the kringle 5-B chain segment of human plasminogen. AB - Three murine monoclonal antibodies (designated alpha Pg-28, alpha Pg-96, and alpha Pg-247) against human plasminogen were prepared. All three antibodies bound plasminogen and the elastase-digestion product of plasminogen consisting of residues Val442-Asn790 (miniplasminogen). The epitopes recognized by each antibody were distinct. Antibodies alpha Pg-96 and alpha Pg-247 blocked tissue plasminogen activator-dependent lysis in a fibrin plate assay while antibody alpha Pg-28 had no effect. Antibody alpha Pg-96 blocked urokinase- and tissue plasminogen activator-catalyzed, and streptokinase-mediated, plasminogen activation. Antibodies alpha Pg-28 and alpha Pg-247 partially inhibited tissue plasminogen activator-catalyzed plasminogen activation. Antibodies alpha Pg-28 and alpha Pg-247 also inhibited streptokinase-mediated plasminogen activation, but not urokinase-catalyzed activation. Antibody alpha Pg-247 inhibited plasmin catalysis of substrate S-2251 by decreasing the VMAX and increasing the KM of plasmin for the synthetic substrate S-2251 four-fold. The other antibodies had no significant effect on plasmin activity. This differential inhibition of plasminogen activation suggests that activation by urokinase, tissue plasminogen activator, and streptokinase possibly involve distinct regions of miniplasminogen structure. PMID- 1726160 TI - Preparation and characterization of monoclonal antibodies to substance P. AB - A series of mouse hybridomas that produced monoclonal antibodies reactive with Substance P was prepared in an attempt to find a surrogate NK-1 receptor. When the binding profile of one particular antibody, SubP14.36.1, was compared in a rank order plot with NK-1 receptors against various agonists and antagonists of Substance P, a high correlation was found between these two binding structures. In addition, this monoclonal antibody inhibited a functional assay for Substance P. This result indicated that the pharmacological effects of this peptide can be blocked by an antibody (Ab). PMID- 1726161 TI - A novel monoclonal antibody directed against the heat-inducible 68 kDa heat shock protein. AB - A novel monoclonal rat IgM antibody (8F7) is described which detects the inducible 68 kDa heat shock protein (hsp68) in man, mouse, rat, pig and cattle. Hsp68 expression is analysed in various cell types and cell lines by immunoblot with antibody 8F7 and in parallel by autoradiography after metabolic labeling with [35S]methionine. The new antibody cross-reacts with proteins of 180 and 48 kDa, which are not heat shock-inducible and show a pattern of expression different than hsp68. PMID- 1726162 TI - Use of Chronogest implants and Folltropin for estrus synchronization and superovulation in goats. AB - Six Barbari goats each were assigned randomly to treatments 1,2 or 3, comprising im injections of FSH (folltropin) at 12, 14 or 16 mg dose level respectively. Estrus was synchronized with intravaginal sponge impregnated with flugestone acetate (30 mg; chronogest) inserted for 12 days and cloprostenol (125 micrograms) im at the insertion as well as at removal of sponge. FSH treatment started 48 hr before the sponge removal as 4-day declining dose scheme. Estrus could be effectively synchronized in all goats under the study, with significant difference (P less than 0.05) in the onset of estrus between the treatment groups. All goats were administered with 750 IU hCG i.v. at estrus. Recording of ovarian response and embryo recovery was done 45 hr after the onset of estrus. The prime aim of superovulation was effectively achieved in Barbari goats with the use of chronogest implants and folltropin. There was no difference (P greater than 0.05) between the treatment groups in recovery of transferable embryos, however, 14 mg folltropin appeared to be near optimal dose. There was no adverse effect on the quality of recovered embryos with high doses of folltropin. PMID- 1726163 TI - Current prevalence of hepatitis B, A and C in a healthy Spanish population. A seroepidemiological study. AB - A seroepidemiological study was carried out in order to determine the prevalence of markers of viral hepatitis infection in employees of five health-care companies and their cohabiting family members. Each participating family unit was required to fill out a questionnaire, in which, among other data, the employee was requested to indicate his or her job category. Markers of hepatitis B infection (anti-HBs, anti-HBc or HBsAg) were observed in 11.7% (58/497) of all subjects. When employees and family members were analysed according to the employee's job category, significant differences were found between "staff" (3%) and "administrative personnel" (13.3%; p less than 0.01) or "factory workers" (16.9%; p less than 0.01). Of 489 individuals tested for the presence of anti-HAV and anti-HCV, 59.1% and 0.6% respectively, were positive. There was a correlation between the prevalence of anti-HAV and age; a large proportion of the subjects under the age of 30 years had no evidence of prior HAV infection. PMID- 1726164 TI - Antibodies against hepatitis C virus in mixed cryoglobulinemia patients. AB - The prevalence of antibodies against hepatitis C virus (anti-HCV) in an unselected series of 45 mixed cryoglobulinemia patients was assessed by an enzyme linked immunosorbent assay (Chiron ELISA HCV, Second Generation). The anti-HCV specificity was evaluated by a recombinant based immunoblot assay (Chiron RIBA HCV, Second Generation Assay). HBV-related markers and HIVAb were detected in the same samples. The prevalence of anti-HCV observed in mixed cryoglobulinemia was compared with 80 patients with other immunological systemic diseases. Anti-HCV were found in 91% of mixed cryoglobulinemia patients, and confirmed by RIBA in all cases; on the other hand, anti-HCV were practically absent in other control diseases. HBV markers were recorded in 49% of mixed cryoglobulinemia subjects; while HIVAb were constantly absent. These data give us new insights into the etiopathogenesis of mixed cryoglobulinemia. PMID- 1726165 TI - Antibody prevalence of parenterally transmitted viruses (HIV-1, HTLV-I, HBV, HCV) in Austrian intravenous drug users. AB - From 1985 to 1990 the sera of 372 newly imprisoned intravenous drug users (IDU) were tested for HIV-1 antibodies. The seroprevalence was 18%, males 16% and females 31%. HIV-1 seroprevalence in Austrian IDU has not increased since 1986. All sera tested for HTLV-I-antibodies were negative. The majority of the HIV-1 seropositive drug users had been infected before 1985. The reported frequency of needle sharing has decreased since 1986. Of 151 IDU tested for HCV antibodies 75% were positive for anti-HCV, 68% were positive for hepatitis B markers, 59% were positive for both HBV and HCV markers, and 13% were positive for HIV-1 antibodies. IN CONCLUSION: in Austrian IDU HCV seems to be the most frequent parenterally transmitted virus; HIV-1 seroprevalence has not increased since 1986; HLTV-I infection has not yet begun. PMID- 1726166 TI - Intrafamilial spread of hepatitis C virus. AB - We studied the prevalence of antibodies against hepatitis C virus (anti-HCV) among 530 household contacts of 225 anti-HCV-positive subjects (index cases). Twenty-six (4.9%) relatives had anti-HCV, a proportion higher than that found among blood donors (175 of 22,435; 0.78%) (p less than 0.001). We did not find any differences regarding the type of relation with the index case (sexual or nonsexual). The prevalence of anti-HCV increased with the age of the relatives, with the contact time with the index case, and with the time of exposure to HCV. On the other hand, the anti-HCV was associated mainly with the existence of cirrhosis or hepatocellular carcinoma in the patient. We concluded that intrafamilial transmission may be an important mechanism in the spread of HCV. PMID- 1726167 TI - Sensitivity of second generation anti-HCV test for diagnosis of acute hepatitis C. PMID- 1726168 TI - Mechanisms of angiogenesis in normal and diseased skin. PMID- 1726170 TI - Acquired haemoglobin H (HbH) disease. PMID- 1726169 TI - The in-vitro antimicrobial effects of azelaic acid upon Propionibacterium acnes strain P37. AB - The in-vitro antimicrobial activity of azelaic acid a new topical acne treatment, upon Propionibacterium acnes strain P37 was studied. In phosphate buffer at pH 6.0 500 mM azelaic acid had bactericidal activity whilst the addition of nutrients reduced susceptibility. Bactericidal activity was greatly enhanced by reducing the pH to 5.6. In a simple defined medium growth was inhibited by 100 microM azelaic acid. The accumulation of 14C azelaic acid was pH and temperature dependent with maximum uptake occurring at pH 4.6, 30 degrees C. Valinomycin, nigericin and CCCP (membrane-active inhibitors of energy transduction) inhibited uptake and azelaic acid was not accumulated by non-viable cells. The degradation of azelaic acid was repressed by glucose, and acetic acid was the major end product of azelaic acid degradation in glucose depleted media. The incorporation of radiolabelled precursors into protein, DNA and RNA were inhibited in a dose dependent manner, and 50% inhibition occurred at 313, 3639 and 9226 microM respectively. The synthesis of proteins was shown to be significantly more sensitive to the action of azelaic acid than both RNA and DNA synthesis. PMID- 1726171 TI - Epitope mapping of HBsAg using a panel of human anti-HBs antibodies. AB - Mapping of B cell epitopes on HBsAg was performed using a panel of human anti-HBs antibodies. Synthetic peptides representing different regions of HBsAg failed to inhibit the binding of two antibodies which recognized non-conformational HBsAg determinants in dot-blot ELISA and HBsAg polypeptide bands in immunoblot analysis. Cross-inhibition studies using five of the antibodies conjugated to horseradish peroxidase suggested that at least three different epitopes are recognised by the panel of antibodies, two of which are within the 'a' group determinant. PMID- 1726172 TI - NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. AB - Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1). PMID- 1726173 TI - Co-amplification of specific sequences of HCV and HIV-1 genomes by using the polymerase chain reaction assay: a potential tool for the simultaneous detection of HCV and HIV-1. AB - A rapid and simple method using the polymerase chain reaction (PCR) was devised for the co-amplification and simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) specific sequences in the same serum sample. Genomic RNA was extracted from 13 blood donor sera that were reactive in ELISA for both anti-HCV and anti-HIV-1. The extracted RNA was reverse transcribed into cDNA and amplified using nested primer pairs (SN01 and SN04; SN02 and SN03) based on the HCV prototype sequence of clones 37b and 81, and SK 38/39 for HIV-1 simultaneously. PCR products were analyzed by liquid hybridization or Southern blot hybridization with 32P end-labeled oligonucleotide probes from the regions between the primer pairs, excluding the primer sequences. HCV-RNA was detected in all 13 (100%) samples tested; HIV-RNA was detected in 11 (85%) samples. The ability to co-amplify specific sequences from two different viral genomes in the same reaction mixture offers the possibility of simultaneous detection and diagnosis of more than one viral agent in serum samples of infected individuals. PMID- 1726174 TI - Organization of the superior olivary complex in the guinea pig. I. Cytoarchitecture, cytochrome oxidase histochemistry, and dendritic morphology. AB - The superior olivary complex is a prominent component of the auditory system. It consists of the lateral and medial superior olivary nuclei and a large number of smaller cell groups known as the periolivary nuclei, which are sources of both ascending and descending projections. The goal of this study was to establish criteria for identifying the periolivary nuclei in the guinea pig. Use of Nissl stains, the Golgi impregnation technique, and cytochrome oxidase histochemistry allowed us to distinguish eleven periolivary nuclei on the basis of differences in the types of cells they contain, in the distribution of cell types, and in the cytochrome oxidase staining characteristics of both the cells and the neuropil. The nuclei, named according to their position with respect to the lateral and medial superior olivary nuclei, can be divided into four groups: (1) a lateral group comprising the lateral nucleus of the trapezoid body and the anterolateral and posteroventral periolivary nuclei, (2) a dorsal group comprising the dorsal and dorsolateral periolivary nuclei, (3) a ventral group comprising the ventral nucleus of the trapezoid body and the anteroventral, ventromedial and rostral periolivary nuclei, and (4) a medial group comprising the medial nucleus of the trapezoid body and the superior paraolivary nucleus. Cytological distinctions among the periolivary nuclei are consistent with other evidence that they serve different functions and highlight the need for detailed study of their connections, immunocytochemistry and physiological response properties. PMID- 1726175 TI - Properties of single fast chloride channels from rat cerebral cortex neurons. AB - 1. Properties of Cl- channels from surface membranes of acutely dissociated rat cerebral cortical neurons were studied with the patch clamp technique. These channels were present in the majority of excised inside-out membrane patches. 2. Cl- channels were rarely observed in cell-attached membrane patches, and usually several minutes elapsed following excision of the patch before Cl- channels became active. 3. Under asymmetric ionic conditions (1000 mM-KCli, 140 mM-KClo), neuronal Cl- channels are fairly selective for Cl- over K+ and Na+, with permeability ratios, determined by reversal potential shifts of 4.8 for both PCl/PK and PCl/PNa. 4. Neuronal Cl- channel kinetic activity remained stable over periods of time long enough to collect up to 500,000 open and closed intervals. Occasionally, the channels entered altered modes of activity. In the 'buzz mode' the open and closed interval durations became much shorter than normal for several hundreds of intervals. In the 'subconductance mode' the channel opened to a current level about two-thirds of the normal level. 5. Using the method of maximum likelihood, sums of exponentials were fitted to the distributions of open and closed interval durations. Open interval distributions required at least two exponential components with time constants of less than 1 ms. At least six or seven exponential components were required to fit the closed interval distributions with time constants ranging from 30 microseconds to several hundreds of milliseconds. This suggests that neuronal Cl- channels enter at least two open and six or seven closed kinetic states during normal activity. 6. Cl- channels often entered long-duration closed states of several minutes which could not be accounted for by the sums of exponentials fitted to the distribution of closed interval durations. 7. Neuronal Cl- channels exhibit a marked voltage dependence with the percentage of time the channels are open increasing with depolarization. Most of the observed voltage dependence can be accounted for by a decrease in the mean closed interval duration with depolarization. The mean open interval was relatively independent of voltage. 8. These results suggest a high degree of similarity in kinetic behaviour and conductance properties between the fast Cl- channels of tissue-cultured rat skeletal muscle and fast Cl- channels in acutely dissociated rat cerebral cortical neurons. PMID- 1726176 TI - Parathyroid hormone selectively inhibits L-type calcium channels in single vascular smooth muscle cells of the rat. AB - 1. The active synthetic N-terminal fragment of bovine parathyroid hormone, bPTH (1-34) at a concentration of 1 microM, decreased the peak amplitude of the long lasting (L-type) calcium channel current by 37% (n = 14, P less than 0.01) in rat tail artery smooth muscle cells. By contrast, this fragment of parathyroid hormone (PTH) (1 microM) had no effect on the transient (T-type) calcium channel current in the same cell preparation. 2. The inhibitory effect of bPTH-(1-34) on L-channel currents was reversible and could be antagonized by the L-channel agonist, Bay K 8644. In contrast, bPTH-(1-34) inhibited Bay K 8644-induced amplification of L-channel currents. 3. The inhibitory effect of bPTH-(1-34) on L Channel currents was dose dependent with a threshold concentration of less than 10(-7), and voltage dependent with increased inhibition at more positive holding potentials. However, this effect of bPTH-(1-34) was not dependent on different pulse lengths or interpulse intervals. 4. The kinetics of deactivation of L channel currents were not changed although the instantaneous amplitude of the L channel tail current was reduced by bPTH-(1-34). 5. Application of bPTH-(1-34) antagonists (10(-6) M-bPTH-(3-34) and 10(-5) M-bPTH-(7-34] did not result in any significant change in the magnitude of L-channel currents (n = 15 and n = 7, respectively). 6. Pre-incubation of cells with bPTH-(3-34) for more than 15 min abolished the inhibitory effect of bPTH-(1-34) on L-channel currents. 7. The present study provides direct evidence to demonstrate the PTH, an endogenous circulating hormone, is a selective inhibitor of L-channel currents in vascular smooth muscle cells. PMID- 1726177 TI - Multiple antepartum amnioinfusions in selected cases of oligohydramnios. AB - In general the perinatal outcome of oligohydramnios is poor. The diverse etiology of the entity is difficult to pinpoint. Multiple antepartum amnioinfusions can be beneficial in selected cases not associated with malformations, abnormal karyotypes or premature rupture of the membranes. We treated 38 patients with 105 such instillations. PMID- 1726178 TI - [Activity of the kallikrein-kinin system of blood during combined treatment of patients with high and stable arterial hypertension]. AB - The activity of the blood kallikrein-kinin system was evaluated in 18 patients with refractory arterial hypertension who received a combined therapy by using prostaglandin E2 (PGE2) infusions. PGE2 infusions produced a direct effect in modulating the vasoactive peptide system. There was activity mobilization at low baseline values of blood kallikrein and its inhibition at high values. The activating effect of PGE2 remained for further 2 weeks following the infusion. The response of the patients to previously low effective antihypertensive agents could be enhanced. PMID- 1726179 TI - [Blood vessels of the myocardial microcirculatory bed after radical correction of tetralogy of Fallot]. AB - The intraoperative right ventricular myocardial biopsy specimens taken from 54 patients during radical correction of Fallot's tetrad were used to examine the microcirculatory vessels during combined pharmacological ice-chip cardioplegia (PICC) and reperfusion. The combined PICC was shown to fail to exclude myocardial microcirculatory vessel lesions, namely: ultrastructural evidence for edema and destructive changes in the membrane structures of endothelial cells, expanded interendothelial contacts, basal membrane stratification. The lesions were more marked in patients with cyanotic heart disease in cardioplegia lasting more than 65 min. Coronary blood flow recovery was accompanied by evolving endotheliocytic cytoplasmic edema, phenomena of microclasmatosis, microvascular obstruction with cell fragments and membrane structures. This impaired microcirculation, promoted interstitial edema and dystrophic cardiomyocytic changes and might be followed by acute heart failure in the postoperative period. PMID- 1726180 TI - [Use of a Soviet-made device for recording late ventricular potentials]. AB - Late ventricular potentials (LVP) were detected with an ECG signal-averaging installation developed at the Institute of Radio Engineering and Electronics, USSR Academy of Sciences. A total of 103 patients were examined: 33 with sustained ventricular tachycardia (VT), 30 with unsustained VT and high-grade ventricular premature contraction, 20 with coronary heart disease, postinfarction cardiosclerosis without arrhythmias and 20 apparently healthy subjects (controls). LVPs are the most susceptible to sustained VT in the presence of postinfarction cardiosclerosis (77%), their specificity is lower (70%). LVPs are a low sensitive marker (35%) for sustained idiopathic VT. In unsustained VT and high-grade premature ventricular contraction, LVPs were recorded infrequently in 1 (3%) of the 30 patients. PMID- 1726181 TI - [Filtration angles in pseudophakic eyes]. AB - Examination of the filtration angle comprised 86 eyes with implanted artificial intraocular lenses in the 8th postoperative month. The evaluation detected in both groups the appearance of pigmentary deposits in the trabecular area, of circumferential anterior synechiae and of vascular proliferation of the circumference of the iris. In some patients with anterior chamber lenses were observed abnormalities in the position of the implant. PMID- 1726182 TI - [Etiopathogenetic problems in primary open-angle glaucoma]. AB - The authoress discusses the contemporary opinions on the genetically conditioned predisposition to primary open angle glaucoma, histopathological changes in the structures responsible for the increase of the resistance of the outflow and on the mechanism of their formation. The pathogenesis of glaucomatous neuropathy is considered in the view of the damage caused by a direct compression of the nervous fibres and their chronic ischaemia. Both these mechanisms have an influence on the transport of axoplasma, the obstruction of which leads to atrophic changes of the nervous fibres and of the ganglion cells of the retina. The place of the primary lesion of these elements is considered. PMID- 1726183 TI - Molecular cloning and expression of the 01 rfb region from a pyelonephritic Escherichia coli 01:H1:K7. AB - The genes responsible for the biosynthesis of the O1 polysaccharide from a human pyelonephritic Escherichia coli were cloned and expressed in a rfb-deleted E. coli K-12 strain. Deletion analysis of the clone demonstrated that a DNA fragment size larger than 6.7 kb and smaller than 10 kb is responsible for O1-antigen biosynthesis. PMID- 1726184 TI - Stretch sensitivity of the lateral wall of the auditory outer hair cell from the guinea pig. AB - The inner and outer hair cells of the mammalian hearing organ are mechano transducer cells. Here we report evidence that the lateral wall of outer hair cells (OHCs) is a mechano-receptor. This mechano-sensitivity appears to complement that of the stereocilia. Patch clamping studies showed that stretching of the membrane patches by suction at the pipette activated potassium channels with 130 pS unit conductance specifically localized in the lateral wall. Application of an osmotic tension to the entire cell membrane under whole-cell recording produced a 10 mV hyperpolarization. The reversal potential and the magnitude of the macroscopic current under voltage clamp were consistent with the single-channel properties of stretch-activated potassium channels. The elongated cylindrical cell body of the OHC is optimally positioned in the cochlea to sense axial force due to the vibrations of the basilar membrane during sound stimulation. This sensitivity can explain the production of a predominantly hyperpolarizing response to sound stimuli, unique to the OHC. Coupled with voltage-dependent OHC motility, the stretch-activated channels may play an important role in producing a mechanical feedback, an indispensable element in cochlear tuning. PMID- 1726185 TI - Neurotensin and growth hormone-releasing factor-containing neurons projecting to the median eminence of the rat: a combined retrograde tracing and immunohistochemical study. AB - The origins of the neurotensin (NT)-containing nerve terminals in the median eminence were determined in rats by a combination of retrograde labeling with True blue and immunofluorescence staining. Moreover, the distribution of NT and growth hormone-releasing factor (GRF) was analyzed in the arcuate nucleus (ARC) with an elution-restaining procedure. The retrogradely labeled NT neurons were found mainly in the ARC. Only a few labeled NT neurons were seen in the periventricular and the paraventricular nuclei. The elution restaining procedure demonstrated that an average of 43% of the labeled NT cells in the ventral part of the ARC restained for GRF. These findings support the hypothesis of co expression of NT with GRF from the ARC. PMID- 1726186 TI - A chloride channel in rat and human axons. AB - Current recordings from single chloride channels were obtained from excised and cell-attached patches of rat and human axons. In rat axons the channels showed an outwardly rectifying current-voltage relationship with a slope conductance of 33 pS at negative membrane potentials and 65 pS at positive potentials (symmetrical 150 mM CsCl). They were measurably permeable for cations (PNa/PCs/PCl = 0.1/0.2/1). Channel currents were independent of cytoplasmatic calcium concentration. Inactivation was not observed and gating was weakly voltage dependent. Cl- channels in human axons showed similar gating behavior but had a lower conductance. PMID- 1726187 TI - Neurochordins of higher vertebrates: similarities in P-epitope-bearing polypeptide composition and a study of P-epitope expression in cultured neural tissue cells. AB - Neurochordins are a family of high Mr P-epitope-bearing neural tissue glycoproteins. Comparison of SDS-agarose electrophoretic patterns of extracts from human, rat, mouse and chicken brain after immunostaining of blots with an anti-P-epitope MAb At5 demonstrated that in each case neurochordins from the A, B and C group were present. A similar result was obtained when polyclonal serum against total human neurochordin was used. The data favours the idea of high evolutional conservatism of neurochordins of higher vertebrate species. Expression of the P-epitope on glial cells in culture was studied. Strong immunostaining of oligodendrocytes with MAb At5 and the absence of this staining in astrocytes and Schwann cells make it possible to use At5 for identification of cell types in tissue culture experiments as well as for differential diagnostics of brain tumours. PMID- 1726188 TI - Computer-generated slides: tips and traps. PMID- 1726189 TI - Serotonin neurotoxicity in rats after combined treatment with a dopaminergic agent followed by a nonneurotoxic 3,4-methylenedioxymethamphetamine (MDMA) analogue. AB - There is increasing evidence linking dopamine (DA) to the long-term serotonergic (5-HT) neurotoxic effects of certain substituted amphetamines such as 3,4 methylenedioxymethamphetamine (MDMA). The present study was undertaken to examine the importance of DA metabolism, uptake inhibition and release in the long-term effects of these drugs by combining various dopaminergic agents with an analogue of MDMA that had low neurotoxic liability, namely 5,6-methylenedioxy-2-aminoindan (MDAI). Monoamine and metabolite levels and the number of 5-HT uptake sites (using [3H]paroxetine binding) were determined 3 hours or 1 week after treatments. Combining the monoamine oxidase inhibitors, clorgyline (MAOA selective) or deprenyl (MAOB selective) with MDAI did not result in any long-term reductions of serotonergic markers. Similarly, combining the DA uptake inhibitor GBR-12909 with MDAI did not result in any long-term changes in monoamine levels at 1 week. In contrast, a single pretreatment of posttreatment with the nonvesicular DA releaser S-amphetamine and MDAI resulted in small but significant long-term changes in monoamine levels. More importantly, if a subacute dosing regimen (every 12 hours for 4 days) was utilized, the combination of S amphetamine with MDAI resulted in a marked long-term decrease in the levels of cortical, hippocampal and striatal 5-HT, 5-HIAA and the number of 5-HT uptake sites. The results are discussed in terms of the significance of DA and especially nonvesicular DA release in the long-term effects of MDMA-like drugs. PMID- 1726190 TI - [Fetal hemoglobin in children with different neoplasms]. AB - The rate of HbF was studied in 123 children with different malignancies in order to assess its changes and relationship with other values of peripheral blood. The cases were distributed into 34 with acute lymphoblastic leukaemia (ALL), 19 with acute myelogenous leukaemia (AML), 23 cases of Hodgkin's disease (HD), 16 of non Hodgkin lymphoma (NHL) and 31 of different solid tumors (ST). ALL and AML groups had the highest HbF rates (2.88 +/- 1.93% and 2.63 +/- 2.7%, respectively), followed by HD and NHL (1.89 +/- 1.09% and 1.81 +/- 1.68%, respectively), whereas the ST group showed the lowest values (1.32 +/- 1.74%). When comparing these figures with the findings in a group of adult leukaemia and lymphoma (1.6 +/- 0,7% and 1.2 +/- 0.5%, respectively), it was found that HbF was increased in the children to a higher extent. The analysis of the gamma G and gamma A chains of HbF, performed on 14 of the patients, showed in 7 cases a correlation similar to the newborn pattern, while this was similar to the adults in the remainders. These findings suggest that some factors intrinsic to the neoplasm might favour, in a different degree, the re-expression of HbF, which, in turn, would not necessarily mean a reversion to the foctal stages since the correlation of the gamma chains was similar to the adult pattern in 50% of the cases studied. PMID- 1726191 TI - [Retinal projections to the pretectum in the fowl (Gallus gallus domesticus)]. AB - The retinal projection to the pretectum was examined in the adult domestic fowl by means of anterograde transport of horseradish peroxidase (HRP). At the same time, the cytoarchitecture of the pretectum was studied in Nissl preparation. Fifteen fowls (Gallus gallus domesticus) were used for the HRP study, and four were used for the cytoarchitectural study. One hundred microliters of a 30% HRP solution in physiological saline were injected into the vitreous body of the unilateral eye under sodium pentobarbital anesthesia (30 mg/kg body weight). After a postoperative period of 48 hours, the animals were deeply anesthetized and perfused intracardially with 1,000 ml of Ringer solution, followed by 2,000 ml of 1% paraformaldehyde and 1.25% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) which was then followed by 1,000 ml of 10% sucrose in the same buffer. The brain was cut into serial transverse sections at 60 microns on a freezing microtome. Every section was treated with tetramethyl benzidine (TMB). Based on cytoarchitectural and HRP results, the pretectum of the adult hen was divided into the following eight nuclei: nucleus principalis pretectalis (P), nucleus subpretectalis (SP), nucleus principalis precommissuralis (PPC), nucleus medialis pretectalis (PTM), nucleus pretectalis dorsalis (APd), nucleus area pretectalis (AP), nucleus spiriformis lateralis (SPL), and nucleus spiriformis medialis (SPM). The SPL and SPM lay closely adjacent to each other, although the boundary between them can be easily defined in any section. Furthermore, the SPM could be divided into two subnuclei: the dorsal part of the SPM (SPMd) located just under the SMT and dorsomedial to the P, and the ventral part of the SPM (SPMv), which was located above the PPC and ventromedial to the P. Retinal projections were found in the APd, the PTM, the AP and the SPM, contralaterally. APd was the smallest nucleus among the pretectal nuclei lying just above the tractus septomesencephalicus (SMT) and received the heaviest retinal inputs. In the PTM, clear terminals were distributed over the whole of the nucleus, whereas strong labeled terminals were also found around the principal pretectal nucleus. Slight retinal inputs were found in the ventral part of the SPM. Furthermore, heavily labeled terminals were found in the medial part of the AP designated as the interstitial nucleus of the posterior commissure by Niimi. Terminals in the ventral part of the SPM and the medial part of the AP have not been reported in the previous studies in the chick and pigeon. There were no terminals in the contralateral P, SPL, SP, PPC, and the ipsilateral brain stem. PMID- 1726192 TI - Ion permeability through bilayer lipid membranes for biosensor development: control by chemical modification of interfacial regions between phase domains. AB - Based on the results of studies on cystic fibrosis, which implicated hydroxystearic acid (HSA) as a contributing factor in altered biomembrane function, solvent-free bilayer lipid membranes (BLMs) and monolayer films were prepared from a lipid mixture containing (by mass) 34% phosphatidylcholine, 19% dipalmitoylphosphatidyl serine, 47% cholesterol and variable amounts of 10- and 12-HSA (0-50%). Ion currents, resulting from K+ permeation through BLMs that were supported in 0.1 mol dm-3 KCl solutions buffered to pH 7.4, were monitored with use of a d.c. circuit. The structures of monolayer films at the air-water interface of a Langmuir-Blodgett trough were studied by pressure-area correlations and by further correlation with microscopic phase separation as revealed by fluorescence microscopy. In order to elucidate the role of the hydroxyl moieties in ion permeability, the transmembrane ion current was corrected for the effect of the negative surface charge of the carboxylic acid by replacement of the HSA component with stearic acid. The ion current was found to increase with the molar proportion of the HSAs. Two models for ion conduction through BLMs were considered: 'hopping' via hydrophilic sites within the hydrophobic zone of the BLMs, introduced by the hydroxyl moiety of 10- or 12-HSA; and transport through interfacial regions between phase domains that represent areas of low steric density and low structural order within monolayers. Although the two mechanisms are not distinct, the ion permeability results indicate a change in the response of ion current to HSA concentration at 35 mol-%, suggesting a change in the relative proportion of the mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726194 TI - The symbolic experience of hysterectomy. PMID- 1726193 TI - Pharmacological study of bacterial lipopolysaccharide-induced airway hyperresponsiveness in guinea-pigs. AB - Bacterial lipopolysaccharide-induced airway hyperreactivity in guinea-pigs was investigated pharmacologically. Inhalation of bacterial lipopolysaccharide resulted in an increase in airway muscarinic reactivity, as measured by intravenous acetylcholine administration. Metopirone, an inhibitor of 11 beta hydroxylase, increased the excretion of urinary 17-ketosteroid, displayed a tendency to lower plasma cortisone levels and increased lipopolysaccharide induced bronchial hyperreactivity. After bacterial lipopolysaccharide was inhaled by metopirone-treated guinea-pigs, increased vascular permeability and an increased number of leukocytes in the pulmonary capillaries were observed. When prednisolone, alone or in combination with cyclophosphamide, was injected into the guinea-pigs, lipopolysaccharide-induced airway hyperreactivity was inhibited, along with the simultaneous disappearance of inflammatory signs in the airway. Tranilast and ketotifen, reported in clinical studies to inhibit airway hyperreactivity, inhibited hyperreactivity induced by lipopolysaccharide and increased vascular permeability in the pulmonary capillaries, without affecting the increase in leukocytes. These results indicate that increased permeability in pulmonary capillaries is an important factor in the induction of lipopolysaccharide-induced bronchial hyperreactivity in guinea-pigs, and that this model is useful for the pharmacological study of airway hyperreactivity. PMID- 1726195 TI - Characterisation of the N-linked oligosaccharides of the light chain of human glycoprotein IIb by f.a.b.-m.s. AB - The glycosylation of the light chain (GPIIbL) of glycoprotein IIb, one of the glycoproteins constituting the receptor for fibrinogen, fibronectin, and the von Willebrand factor on platelet cell surfaces, was investigated using fast-atom bombardment mass spectrometry (f.a.b.-m.s.). Complex-type N-glycans were observed, attached to Asn-60. The most abundant oligosaccharide is a disialylated biantennary structure substituted with fucose on the chitobiose core. Mono sialylated biantennary, and di- and tri-sialylated triantennary structures were found as minor constituents of the N-glycan population. The amino acid sequence of GPIIbL was fully mapped by f.a.b.-m.s., thereby providing the first direct evidence for the absence of O-glycosylation. PMID- 1726196 TI - Characterisation of minor tetra- to hepta-saccharides O-linked to human meconium glycoproteins by t.l.c.-m.s. microsequencing of neoglycolipid derivatives in conjunction with conventional m.s. and 1H-n.m.r. spectroscopy. AB - As part of the establishment of a data base for core and backbone sequences of O linked oligosaccharides of human meconium glycoproteins, the minor tetra- to hepta-saccharides released from mild-acid treated blood group [corrected] H active glycoproteins have been studied. These oligosaccharides are heterogeneous and difficult to isolate, and a t.l.c.-m.s. microsequencing procedure has been applied to the neoglycolipid derivatives, in conjunction with 1H-n.m.r. spectroscopy, methylation analysis, and mass spectrometry (m.s.) of native and methylated oligosaccharides. Among an array of oligosaccharides characterised are those having the branched beta-GlcNAc-(1----6)[beta-Gal- (1----3)]-GalNAcol core, and others with the following linear sequences not characterised previously from this source: beta-Gal-(1----3)-beta-GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, beta-Gal-(1----4)-beta-GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, alpha-GalNAc- (1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-GalNAcol, beta-GlcNAc- (1----3) beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)- GalNAcol, beta-Gal-(1----3/4)-beta GlcNAc-(1----3)-beta-Gal-(1----3/4)-beta- GlcNAc-(1----3)-beta-Gal-(1----3) GalNAcol, and beta-GlcNAc-(1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-beta Gal- (1----3/4)-beta-GlcNAc-(1----3)-GalNAcol. PMID- 1726197 TI - Measurement of the JC-1,H-1 coupling constants for the Escherichia coli O1A O polysaccharide, a comparison of some n.m.r. experiments. PMID- 1726198 TI - [Pain treatment in advanced head and neck tumors. Pilot study with continuous subcutaneous administration of morphine]. AB - In the pain treatment of patients with incurable head and neck tumors it may be difficult to provide adequate oral drug therapy in the advanced stages. In these cases continuous subcutaneous application of morphine by means of external infusors is an alternative. This mode of application was investigated in the long term treatment of 20 patients with pain that had been therapyresistent until then. Producing sufficient analgesia and having, all told, comparable side effects, this method was experienced by all patients as being more agreeable than the previously used oral mode of application. Technical simplicity of handling makes this safe and effective treatment concept also a valuable means for outpatient care in tumor clinics. PMID- 1726199 TI - [Study on screening for primary liver cancer in high risk population of an endemic area]. AB - Positive HBsAg males aged 30-59 years in Qidong were considered a high risk population for primary liver cancer (PLC). A screening study was conducted among 3,958 high risk population which were assigned randomly to a screening group or to a non-screening group. 2,580 persons were screened by alpha-fetoprotein (AFP) once every six months, the incidence rate of PLC was 1,599.38 per 100,000 person years (PYs), and that in non-screening group with 1,378 persons was 729.39 per 100,000 PYs. Screening survey was also done among 2,332 males aged 30-59 years with negative HBsAg at one-year interval, and the incidence was 128.76 per 100,000 PYs. 26 (86.67%) of 30 PLC patients found in the screening survey were stage I PLCs, but none in 11 out-patients was found in this stage. This study suggests that periodic AFP screening for high risk population is suitable for the high-incidence area, and is one of the effective ways of secondary prevention for PLC. PMID- 1726200 TI - [Effects of high-pass filters with different frequencies on signal-averaged electrocardiograms]. AB - The results of the root mean square voltage of the terminal 40 ms (V40), the duration of the low amplitude signal of less than 20 muV (D40) and the duration of the signal-averaged QRS complex (QRSr) after high-pass filters with 5, 10, 15, 20, 25, 40, 80, 100, 150 and 200Hz were reported. The results showed that the signal-averaged electrocardiogram (SAECG) was highly influenced by the frequencies of the filter. According to the specificity and sensitivity after different high-pass filters, 25Hz was considered as the optimal high-pass filtering frequency. PMID- 1726201 TI - [Assessment of late potential as a screening test for programmed stimulation detecting high risk patients with ventricular tachycardia]. PMID- 1726202 TI - Summary of clinical experience with cefpodoxime proxetil in adults in Japan. AB - Cefpodoxime proxetil is an oral cephem antibiotic of a new ester type, developed by Sankyo Co., Ltd in Japan. It has a broad antibacterial spectrum, which includes Staphylococcus, and a long half-life, allowing twice-daily administration. In Japan, clinical studies on this drug were performed in various fields, including internal medicine, surgery, urology, otorhinolaryngology, and obstetrics and gynaecology. Good or excellent clinical responses were observed in 2275 of 2902 patients analysed, giving a 78.4% efficacy rate overall. Side effects occurred in 98 patients (2.7%); these were mainly gastrointestinal and included diarrhoea, nausea, and vomiting. Abnormal laboratory test results observed included increased AST in 2.8% (55 of 1973), increased ALT in 3.2% (63 of 1965), and eosinophilia in 2.4% (36 of 1521). PMID- 1726203 TI - A review of the pharmacokinetics of cefpodoxime proxetil. AB - Cefpodoxime proxetil is an orally absorbed broad spectrum third generation cephalosporin antibacterial. It is a prodrug that is de-esterified in vivo to its active metabolite, cefpodoxime. After single- and multiple-dose (12-hourly) administration of cefpodoxime proxetil in the therapeutic dose range of 100 to 400mg of cefpodoxime equivalents, average peak plasma concentrations of cefpodoxime range from 1.0 to 4.5 mg/L and occur between 1.9 and 3.1 hours after administration. The half-life of cefpodoxime ranges from 1.9 to 2.8 hours. The absolute bio-availability of cefpodoxime proxetil tablets is 50%, and absorption is enhanced by concomitant administration of food. Raising gastric pH by pretreatment with antacids or H2-receptor antagonists results in reduced absorption. Binding of cefpodoxime to human plasma or serum protein is low (18 to 23%), suggesting that cefpodoxime should readily transfer across the capillary lining into tissues. Cefpodoxime undergoes minimal metabolism in humans. Drug not absorbed is degraded in the gastrointestinal tract and excreted in the faeces. As expected for a drug eliminated primarily by renal excretion, the disposition of cefpodoxime is altered in patients with impaired renal function; the half-life increases, while apparent plasma clearance and renal clearance decrease. The pharmacokinetics of cefpodoxime after oral administration of cefpodoxime proxetil are not affected by age. PMID- 1726204 TI - Microbiological conclusions. PMID- 1726205 TI - Cefpodoxime proxetil in upper respiratory tract infections. AB - Cefpodoxime proxetil is a new third generation oral cephalosporin, which shows potent antibacterial activity against both Gram-positive and Gram-negative bacteria, and high stability in the presence of beta-lactamases. Low concentrations of cefpodoxime inhibit most respiratory pathogens, including Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella (Branhamella) catarrhalis. Cefpodoxime reaches concentrations of 0.24 +/- 0.06 mg/kg in tonsils, 0.89 +/- 0.80 mg/kg in lung parenchyma, and 0.91 +/- 0.01 mg/kg in bronchial mucosa; these values exceed by far the minimum inhibitory concentrations (MICs) of cefpodoxime for respiratory pathogens. Preliminary clinical studies were carried out in 181 patients with upper respiratory tract infections: the results indicated an overall clinical response in 88.4% of patients; in 30% the clinical efficacy was excellent and in 58.5% it was good. Further studies showed clinical cure in 90.3% of patients with pharyngotonsillitis, and clinical efficacy (cure plus improvement) in 95% of those with acute sinusitis. Moreover, bacterial eradication was obtained in 78 to 96.7% of cases, most of which involved H. influenzae, streptococci, or M. catarrhalis. Cefpodoxime appears to be an effective new antibacterial that can be recommended as a drug of first choice in the treatment of most upper respiratory tract infections. PMID- 1726206 TI - Cefpodoxime proxetil in the treatment of lower respiratory tract infections. AB - Cefpodoxime proxetil is the orally absorbed ester of cefpodoxime, a new third generation cephalosporin. In the gastrointestinal tract, cefpodoxime proxetil is hydrolysed to cefpodoxime, which has potent antibacterial activity against the major bacterial pathogens involved in lower respiratory tract infections: Haemophilus influenzae, Moraxella (Branhamella) catarrhalis (including beta lactamase-producing strains), and Streptococcus pneumoniae (including amoxicillin resistant strains). Six randomised comparative studies in patients with lower respiratory tract infections, 5 of which were large (enrollment of more than 200 patients) and double-blind, examined the efficacy and safety of cefpodoxime proxetil. Cefpodoxime proxetil (at a dosage equivalent to 200mg of cefpodoxime) administered twice daily for 5 to 10 days was similar in clinical and bacteriological efficacy to the following: amoxicillin 500mg 3 times daily in the treatment of community-acquired pneumonia; intramuscular ceftriaxone Ig once daily in the treatment of pulmonary infections in hospitalised patients; and to amoxicillin/clavulanic acid 500/125mg 3 times daily in the treatment of acute exacerbations of chronic bronchitis (AECB). Additionally, a dosage equivalent to 100mg or 200mg of cefpodoxime twice daily was similar in clinical and bacteriological efficacy to amoxicillin 250mg 3 times daily in the treatment of bronchitis (acute or AECB). The adverse events noted with cefpodoxime proxetil administration were similar to those associated with other beta-lactam antibacterials and most commonly involved the gastrointestinal tract and skin or mucous membranes. Thus, cefpodoxime proxetil is a useful addition to the antibacterials available for the treatment of infections of the lower respiratory tract. PMID- 1726207 TI - Review of clinical experience in the United States with cefpodoxime proxetil in adults with uncomplicated urinary tract infections. AB - Two controlled United States trials compared the safety and efficacy of cefpodoxime proxetil (100mg twice daily) with either cefaclor (250mg 3 times daily) or amoxicillin (250mg 3 times daily) in patients with uncomplicated urinary tract infections. Treatment duration was 7 days. 307 of 762 patients treated with cefpodoxime proxetil, 99 of 190 treated with cefaclor, and 57 of 185 treated with amoxicillin were evaluable for efficacy. 311, 99 and 59 pathogens were isolated from cefpodoxime proxetil, cefaclor and amoxicillin patients, respectively, the most common pathogens being Escherichia coli, Klebsiella spp., Proteus mirabilis, and Staphylococcus saprophyticus. Bacteriological cure rates were 80% (247/307), 82% (81/99) and 70% (40/57) for cefpodoxime proxetil, cefaclor and amoxicillin, respectively. Respective clinical cure rates were 79% (242/307), 79% (78/99) and 72% (41/57). Cefpodoxime proxetil was well tolerated, and there was no significant difference between the groups in the overall incidence of adverse experiences. Thus, cefpodoxime proxetil is efficacious and safe in the treatment of patients with uncomplicated urinary tract infections and compares favourably with cefaclor and amoxicillin. PMID- 1726208 TI - Cefpodoxime proxetil in the treatment of skin and soft tissue infections. AB - Patients with skin and soft tissue infections were enrolled in a study comparing 2 dosage regimens of orally administered cefpodoxime proxetil; 204 patients with mild to moderate infections received cefpodoxime proxetil 200mg twice daily and 47 patients with severe infections received 400mg twice daily. Both dosage regimens were given for 7 to 14 days. 132 of 142 (93.0%) evaluable patients in the 200mg group and 22 of 29 (75.9%) in the 400mg group were clinically cured post-therapy, the remainder in both groups being classified as improved. The pathogen eradication rate at the end of therapy in the 200mg group was 161 of 165 (97.6%), and 38 of 38 (100%) in the 400mg group. Adverse reactions (drug-related) were reported by 20 (8.0%) patients overall, and there was no apparent relationship between the dosage group and the incidence of adverse reactions. The most commonly reported reactions involved the gastrointestinal tract (diarrhoea) or female genital tract (vaginitis). Cefpodoxime proxetil appears to be a useful and safe agent in the therapy of skin and soft tissue infections. PMID- 1726209 TI - Clinical trials of cefpodoxime proxetil suspension in paediatrics. AB - The pharmacokinetics, bacteriological and clinical efficacy, and safety of the suspension formulation of cefpodoxime proxetil, an oral cephalosporin antibacterial, were evaluated in paediatric patients with various infections. With single doses of 3 and 6 mg/kg (cefpodoxime equivalent) a dose response was evident in the serum concentration values. Absorption, as evidenced by serum concentrations and areas under the concentration-time curve, was enhanced when the suspension was administered before a meal. The overall clinical efficacy (defined as an excellent or good response) in evaluable patients (those from whom a pathogen was isolated) was 94.7% (451 of 476). Bacteriological eradication rates were as follows: Gram-positive bacteria 91.3%; Gram-negative bacteria 88.6%, and 90.0% overall. Side effects occurred in 17 (2.29%) patients, and transient and reversible abnormal laboratory values were found in a few patients. PMID- 1726210 TI - Microbiological evaluation of cefpodoxime proxetil. AB - Cefpodoxime, the active de-esterified molecule of the orally absorbable cephalosporin cefpodoxime proxetil, inhibits streptococci, Neisseria spp., and most Enterobacteriaceae, with MIC50 and/or MIC90 values of less than or equal to 2 mg/L; with regard to the latter family of bacteria, the MIC50 and/or MIC90 values of cefpodoxime are consistently greater than or equal to 4 mg/L for only Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, and Morganella morganii. The MIC50 of cedpodoxime for coagulase-negative staphylococci is greater than 2 mg/L, while the MIC for Staphylococcus aureus strains is 4 mg/L. In comparison with other orally absorbable cephalosporins, cefpodoxime is slightly less active than cefixime, cefetamet, and cefotiam against Gram-negative bacteria, but more active than cefuroxime, cefaclor, and cefalexin. Against staphylococci, the activity of cefpodoxime is comparable to that of cefotiam and cefuroxime, and superior to that of cefaclor, while cefixime and cefetamet have insufficient activity against these species. In common with other cephalosporins, cefpodoxime has no activity against enterococci. In vitro models simulating human serum cefpodoxime concentrations demonstrate that a dosage regimen of 200mg is probably sufficient to treat most infections. However, further study is needed to clarify whether infections due to bacteria such as S. aureus, with higher cefpodoxime MICs, can be treated with this dose regimen. PMID- 1726212 TI - Outcome of transient ischaemic attacks and stroke. AB - Much of the published data concerning the outcome in patients with transient ischaemic attacks and stroke was produced before the development of computerised tomography, and when the risk factors for cerebrovascular disease were different in quality and quantity. Outcome data also depend on the definition of the original disorders, which remain controversial and are often poorly described. Recent advances in laboratory diagnosis, such as imaging of the brain and extra- and intra-cranial blood vessels, have also made definition more complex. Strokes due to carotid artery stenosis differ in outcome from those due to cardiac embolism; the most benign group is that of lacunar strokes. Recent trials indicate a more serious prognosis for patients with carotid stenosis than previously believed, although these data are affected by referral bias. Outcome data will need to be updated in the future as more becomes known about the aetiology and pattern of cerebrovascular disease. PMID- 1726211 TI - Vessel wall-related risk factors in acute vascular events. AB - Angiography in patients with unstable angina or myocardial infarction with subtotal coronary occlusion often reveals eccentric stenoses with irregular borders, suggesting ruptured atherosclerotic plaques and thrombosis, as documented by angioscopy and at autopsy. We have studied these processes in an ex vivo perfusion chamber, an in vivo swine model, and in human subjects. Our results, and those of other investigators, suggest that specific local risk factors at the time of plaque disruption influence the degree of thrombogenicity and, therefore, the various clinical syndromes. These risk factors can be divided into 2 groups: local vessel wall-related factors, and local (focal action) systemic factors. These risk factors include the following: 1) Rheological factors. It has been demonstrated that the more severe the stenotic lesion after plaque rupture, the higher the local shear rate with enhanced platelet deposition and thrombus formation; platelet deposition and thrombosis are particularly likely if the rupture includes the apex of the stenotic plaque, because of the high shear rate induced. 2) Degree of plaque damage. Plaque rupture produces a rough surface and stimulates an occlusive thrombus, which is enhanced depending on the degree of damage or amount of collagen type I and macrophage-dependent tissue factor exposed. 3) Residual thrombus. After spontaneous or pharmacological reperfusion, the surface of the residual thrombus is very thrombogenic and may contribute to reocclusion; this is partially due to thrombin bound to fibrin in the original thrombus. 4) Systemic factors. There is clinical and experimental evidence to suggest that 3 systemic factors at the time of plaque rupture may enhance thrombogenicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726213 TI - Coronary disease and stroke in patients with large-vessel peripheral arterial disease. AB - Previous reports have indicated an excess of mortality from coronary heart disease (CHD), cardiovascular disease (CVD), and all causes in subjects with large-vessel peripheral arterial disease (LV-PAD). However, there is little information available concerning the risk of nonfatal events (morbidity) in this patient group. In a population-based study of 67 patients with LV-PAD and 408 control subjects without this condition, nonfatal CHD and stroke, and total CVD events, morbidity and mortality were evaluated in both men and women. Those with LV-PAD had a 3-fold excess of CVD morbidity at baseline compared with control subjects of the same sex. However, the absolute CVD rates were greater in men than women. During the 10 years of follow-up, women with LV-PAD had more nonfatal CVD events than men, resulting in comparable overall morbidity rates. In logistic regression models adjusted for other CVD risk factors, total CVD morbidity and mortality combined with 2.5 times as great in men and 5 times as great in women with LV-PAD as in those without peripheral arterial disease. These results suggest that the total morbidity and mortality burden are dramatically increased in both men and women with LV-PAD. PMID- 1726216 TI - Prophylactic antiplatelet therapy in peripheral arterial disease. AB - Prophylactic therapy with antiplatelet drugs aims to improve both the general prognosis of patients and the local progression of peripheral arterial disease. A recent meta-analysis of 28 trials revealed that the proportional risk reduction in serious vascular events is similar to that in cardio- and cerebrovascular disease. A decreased incidence of vascular complications was also found in a meta analysis of 4 trials with ticlopidine, and a recent Swedish study [Swedish Ticlopidine Multicentre Study (STIMS)] confirmed that long term ticlopidine reduces both mortality and cardio- and cerebrovascular morbidity. There is also evidence that aspirin (acetylsalicylic acid) and ticlopidine retard progression of atherosclerosis and the occurrence of its thrombotic complications, in patients with arterial disease of the legs. Meta-analysis has recently indicated that antiplatelet agents reduce the incidence of graft occlusion in arterial surgery. These drugs are also traditionally prescribed after percutaneous revascularisation procedures. The efficacy of antiplatelet therapy in patients with peripheral arterial disease emphasises the role of platelets in this condition. PMID- 1726215 TI - Antiplatelet therapy in the prevention of stroke. AB - Aspirin (acetylsalicylic acid) is effective in reducing vascular outcome events in patients with atherosclerosis: a relative risk reduction of about 30% for stroke, 22% for stroke and death, and 15% for vascular mortality. It is probable that low and high dose aspirin are similar in efficacy. Complications are more frequent with high dose aspirin than with low doses. Four randomised trials evaluating sulfinpyrazone vs placebo, and 3 trials evaluating sulfinpyrazone vs aspirin, showed more cerebrovascular events in the sulfinpyrazone group than in the aspirin and placebo groups. One small trial comparing dipyridamole with placebo in patients with cerebrovascular disease found no difference between the 2 groups in outcome. No other studies have compared dipyridamole alone with placebo or aspirin. The European Stroke Prevention Study II is currently in progress and is comparing dipyridamole + aspirin, dipyridamole, aspirin, and placebo. In the first year, the Ticlopidine Aspirin Stroke Study (TASS) showed a 42% risk reduction for stroke and death using the efficacy analysis and a 47% risk reduction for stroke and stroke death. Ticlopidine was more effective than aspirin in reducing stroke in both males and females. Apart from a reversible severe neutropenia in 0.86% of patients, ticlopidine-related adverse effects were relatively benign and reversible. The Canadian-American Ticlopidine Study (CATS) compared ticlopidine with placebo in patients with completed major strokes. The cumulative event rates for the primary outcome events of stroke, myocardial infarction and vascular death, using the efficacy approach, show clear evidence of separation almost immediately after randomisation, consistent with a constant risk reduction of about 30% in the ticlopidine group. These data provide strong evidence that ticlopidine conveys a clinically important reduction in the risk of thromboembolic events in patients with a history of completed thromboembolic stroke. In conclusion, aspirin is effective in preventing atherothrombotic morbidity and mortality. It reduces the overall vascular event rate by about 25%. Sulfinpyrazone and dipyridamole appear to add nothing important over aspirin alone. Ticlopidine is more effective than aspirin in preventing stroke. The modest, reversible risk of neutropenia, affecting less than 1% of patients, makes the benefit: risk ratio a reasonable one. PMID- 1726214 TI - Risk factors, interventions and therapeutic agents in the prevention of atherosclerosis-related ischaemic diseases. AB - Of the major risk factors for atherosclerosis, high factor VII and fibrinogen levels, genetic predisposition, gender and age cannot be influenced. Reduction of high blood pressure reduces the cerebral but not the coronary vascular risk and correction of dyslipidaemia correlates with cardiovascular risk. Other major risk factors (tobacco consumption, obesity, sedentary lifestyle and diabetes) can also be modified. Aspirin in doses of approximately 300 mg/day may be recommended for the primary prevention of myocardial infarction (MI), but only in those patients with a moderate to high risk of cardiovascular disease. Aspirin reduces the risk of fatal and nonfatal MI by about 50% and also decreases the overall mortality rate among patients with unstable angina. A lower dose of aspirin (150 mg/day) also reduces mortality by 23% in the acute phase of MI. In doses of 300 mg/day, aspirin is useful in the secondary prevention of MI and reduces the overall mortality rate by 15%. Various antiplatelet agents, including aspirin (alone or combined with dipyridamole) and ticlopidine, have proved useful in the prevention of thrombosis in aorto-coronary grafts, provided treatment begins at the latest 6 hours after surgery. The usefulness of antiplatelet drugs has been well established in the prevention of immediate reocclusion following coronary angioplasty, but not in the prevention of late reocclusion. Aspirin and ticlopidine are also beneficial in extracorporeal circulation techniques. In patients with a synthetic cardiac valve prosthesis, antivitamin K-anticoagulants are still indispensable lifelong, but their antithrombotic effect can be reinforced by dipyridamole or aspirin. Diuretics probably provide the best primary protection against cerebrovascular accidents, although medium doses of aspirin may be considered in elderly people at high risk of such accidents. Aspirin (alone or combined with dipyridamole) and ticlopidine may be recommended for the secondary prevention of cerebral ischaemic accidents. Aspirin (with or without dipyridamole) and ticlopidine reinforce the treatment of obliterative arterial disease in the lower limbs. PMID- 1726217 TI - The first Alzheimer disease case: a metachromatic leukodystrophy? AB - A reconsideration of the original report of Alzheimer and of the description of case 1 by Perusini in 1908, published in 1910, suggests that they were describing the same case. Both the temporal evidence and the clinical description make this conclusion inescapable. The histopathology of this first case shows some features that are not characteristic of the histological pattern of the modern Alzheimer, namely demyelination of the central white matter and metachromatic deposits in the spinal cord. PMID- 1726218 TI - Uranaffin reaction of Merkel corpuscles in the lingual mucosa of the finch, Lonchula striata var. domestica. AB - Subepithelial Merkel cells in the tongue of the finch were investigated by use of the uranaffin reaction. The results indicate that the secretory granules of subepithelial Merkel cells in this animal are positive for the uranaffin reaction, and in this respect are cytochemically similar to those reported in mammalian intraepithelial Merkel cells. PMID- 1726219 TI - Molecular aspects of the neuronal noradrenaline transporter. AB - The neuronal noradrenaline transporter was partially purified by means of low and high pressure liquid chromatography using anion exchange, gel filtration and lectin affinity columns. A protein characterized by a molecular weight of 50-53 kilodalton was enriched; it may represent the transporter or a component of it. In addition, a RNA fraction characterized by a mean size of 2 kilobases was isolated from PC12 rat phaeochromocytoma cells and from bovine adrenal medulla; this RNA fraction caused expression of the noradrenaline transporter after microinjection into Xenopus laevis oocytes. PMID- 1726220 TI - Comparisons of peripheral blood and hepatic lymphocyte subpopulations and interferon production in chronic viral hepatitis. AB - Studies were undertaken to clarify the immunological mechanism in patients with chronic hepatitis C compared with chronic hepatitis B. Phenotypic study by flow cytometry showed that CD8+ T cells and HLA-DR+ cells isolated from liver biopsy specimen were significantly increased in their proportions as compared with those in peripheral blood, while intrahepatic CD4+ T cells were significantly lower than peripheral blood CD4+ T cells in both types of patients with chronic active hepatitis (CAH). Furthermore two-color analysis revealed that CD8+ CD11- and CD3+ HLA-DR+ cells were significantly elevated in liver tissue than in peripheral blood in both patients groups. In in vitro tests, mononuclear cells obtained from the liver of type B and type C CAH had a reduced capacity to produce IFN-gamma in response to pokeweed mitogen, while they produced equal amounts of IFN-alpha under stimulation with Newcastle disease virus as compared with control peripheral blood mononuclear cells. In contrast, spontaneous productions of both IFNs were greater in liver infiltrates of the two patients groups. These results indicate that functionally activated T cells are distributed in a similar manner in the liver of both chronic hepatitis B and C, suggesting that cytotoxic T cell plays a major role in the hepatocellular injury of both groups of patients. PMID- 1726221 TI - Regulation of isotype immunoglobulin and interleukin production by adjuvants in vitro. AB - An in vitro system has been developed in which antibodies to fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulfate (DXS), are produced in the presence or absence of different adjuvants. The antibody response of in vitro cultures was measured by assaying the total Ig secreting cells and FITC-specific plaque-forming cells (PFC). The presence of low levels of antigen and various cytokines were necessary for the production of isotypes other than IgM. Our results indicate that the regulation of isotype switching in vitro is dependent upon the non-specific stimuli-adjuvants, which probably activate different types of cells for cytokine production. The pre activation of spleen cells, in our system, by antigen, seems to play a decisive role in the subclass of IgG produced. The adjuvant may still be able to influence the precommitted cell isotype to switch to another subclass but only in a "down stream" direction i.e., IgG3----IgG1----IgG2b----IgG2a. PMID- 1726222 TI - [Diagnostic value of serum level of alpha 2-macroglobulin-trypsin complex in pancreatic diseases using a colorimetric assay with a synthetic chromogenic substrate]. AB - We measured serum level of alpha 2-macroglobulin-trypsin complex (alpha 2M-T complex) using a colorimetric assay with a synthetic chromogenic substrate, D gamma-tert-butyloxy-Gly-Arg-3-carboxy-4-hydroxyanilide dihydrochloride. Serum level of alpha 2M-T complex was greater in acute pancreatitis patients than in chronic pancreatitis or pancreatic cancer patients. In severe acute pancreatitis patients, both mean level and frequency of abnormal value of serum alpha 2M-T complex were significantly greater than in mild acute pancreatitis patients (13.1 +/- 12.9 vs 2.9 +/- 3.5 U/L, p less than 0.01; 100 vs 41%, p less than 0.01). In conclusion, the determination of serum level of alpha 2M-T complex can be useful for the diagnosis of severe acute pancreatitis. PMID- 1726223 TI - Murine model of leptomeningeal dissemination using human medulloblastoma cells. AB - An experimental model of meningeal dissemination was developed by intracisternal inoculation of human medulloblastoma (ONS-76) cells into nude mice. All mice died within 65 days after inoculation of 1 x 10(7) tumor cells. The median survival time was 56 days. Clinical signs and histological findings were similar to those in medulloblastoma patients with meningeal dissemination. Immunohistochemical studies showed that ONS-76 cells in the subarachnoid space expressed major histocompatibility complex (MHC) class I antigens until 20 days after inoculation. After 30 days, expression of MHC class I antigens decreased and cells began to proliferate rapidly. Expression of MHC class I antigens on tumor cells may result in effective recognition by the host immune system. PMID- 1726224 TI - Portable digital subtraction angiography in the operating room and intensive care unit. AB - A simple, inexpensive method of portable digital subtraction angiography (DSA) using an image processor (Sigma X), still-videorecorder and control panel combined with a surgical x-ray television unit can provide real time subtraction images on the monitor. This portable DSA unit was used in 161 cases (130 in the operating room and 31 in the intensive care unit). In the operating room it is useful: 1) to confirm patency of the parent artery and its branches after aneurysm clipping, 2) to identify feeding arteries of arteriovenous malformation and to confirm total extirpation, 3) to confirm the patency of extracranial intracranial bypass, 4) to confirm patency of the internal carotid artery and absence of flap formation after carotid endarterectomy. In the intensive care unit, it is particularly useful for visualizing cerebral vasospasm after subarachnoid hemorrhage and recanalization of an occluded major intracranial artery. Absence of intracranial circulation can be demonstrated in patients with suspected brain death. PMID- 1726225 TI - Primary intracranial amelanotic melanoma--report of an autopsy case. AB - Primary intracranial amelanotic melanoma was verified at autopsy in a 38-year-old male. Correct diagnosis of amelanotic melanoma needs electron microscopy or immunohistochemistry, since Masson staining is negative due to the absence of melanin pigment. We adopted the following criteria for clinical use: macroscopically not dark and microscopically negative for Masson staining, but ultrastructurally various melanoma types present. Although the clinical profile of this case is consistent with melanotic melanoma, the more detailed features of primary intracranial amelanotic melanoma require future study. PMID- 1726226 TI - Cerebellar ganglioglioma--case report. AB - A 14-year-old boy presented with cerebellar ganglioglioma manifesting as severe headache and confusion. Computed tomographic scans showed a huge, partly enhanced cystic cerebellar tumor. The tumor was totally removed. Histological examination disclosed glial cells and mature ganglion cells. The latter were identified by Nissl's staining and immunostaining for neurofilaments. Ganglion cells were present in the cerebellum and the surrounding subarachnoid space. This heterotopic growth of ganglion cells enabled a firm diagnosis of cerebellar ganglioglioma. PMID- 1726227 TI - Central nervous system metastasis from gallbladder carcinoma--case report. AB - Systemic metastases from gallbladder carcinoma occur frequently, but involvement of the central nervous system is rare. We describe such a case in a 68-year-old female. Solitary brain metastasis from gallbladder carcinoma was completely removed 4 months after operation for the primary tumor. Planned chemotherapy was then given to prevent recurrence. She was leading a normal life 4 years later. Patients with solitary brain metastasis from gallbladder carcinoma can achieve a better outcome and longer survival after removal of the brain metastasis if there is no other metastasis. PMID- 1726228 TI - Multiple calcified metastatic brain tumor--case report. AB - The authors describe an unusual case of pulmonary small cell carcinoma with numerous calcified metastatic nodules of the brain in a 62-year-old female. Computed tomographic scanning and magnetic resonance imaging demonstrated these lesions with ring enhancement. Autopsy revealed about 30 calcified metastatic nodules in the brain, but no calcification in the pulmonary lesion. Histological examination of the brain lesions disclosed necrosis and calcification in the center and small cell carcinoma cells in the periphery. PMID- 1726229 TI - Delayed traumatic intracerebellar hematoma: correlation between the location of the hematoma and the pre-existing cerebellar contusion--case report. AB - A rare case of delayed traumatic intracerebellar hematoma (DTIC1H) in a 54-year old male achieved an excellent outcome without surgery. Analysis of this case and other reported cases suggests that DTIC1H occurs in two types: Group I with hematoma developing in previously contused areas, and Group II with hematoma developing in areas appearing normal on the initial computed tomographic scan. Group I hematomas occurred in the cerebellar cortex, but Group II hematomas occurred in the subcortical region or vermis where direct impact is less likely to have an effect. This suggests different mechanisms of development for DTIC1H. PMID- 1726230 TI - Ruptured cerebral aneurysm successfully clipped in idiopathic thrombocytopenic purpura after high-dose gamma-globulin therapy--case report. AB - A ruptured cerebral aneurysm in a 50-year-old female with idiopathic thrombocytopenic purpura was successfully clipped after preoperative high-dose gamma-globulin therapy to control the hemorrhagic diathesis. High-dose gamma globulin therapy with or without steroid and/or platelet transfusion is recommended for such cases if the blood pressure can be controlled and the neurological condition permits delayed surgery. PMID- 1726231 TI - Intracranial carotid artery occlusion with telangiectasia (moyamoya disease) associated with persistent primitive trigeminal artery--case report. AB - A 35-year-old female presented with moyamoya disease coincidentally associated with a persistent primitive trigeminal artery (PPTA). Bilateral encephalo-duro arterio-synangiosis was performed and the postoperative course was uneventful. Moyamoya disease and PPTA might have a congenital origin, but moyamoya disease may be promoted by a PPTA. The hemodynamics of PPTA during progressive occlusive change in the internal carotid artery in moyamoya disease should be re-evaluated to determine its function in the development of moyamoya vessels. PMID- 1726232 TI - Large arteriovenous malformation associated with persistent primitive hypoglossal artery--case report. AB - A rare case of large arteriovenous malformation (AVM) with persistent primitive hypoglossal artery in a 43-year-old male was treated by staged embolization, followed by total removal. AVM associated with carotid-basilar anastomosis is quite rare, but the incidence of AVM in patients with carotid-basilar anastomosis is high. AVM associated with persistent carotid-basilar anastomosis has no distinguishing features compared with ordinary AVM, but participation of the posterior circulation as a feeder is characteristic. PMID- 1726233 TI - [Disproportionately large, communicating fourth ventricle. Case report]. AB - A 39-year-old male was admitted because of slowly progressive disturbance of consciousness, diplopia, and ataxia after laparotomy. Ventriculoperitoneal shunting and removal of an arteriovenous malformation had been performed previously. Neurological examination on admission revealed drowsiness, rotatory nystagmus, Parinaud's sign, and truncal ataxia. Computed tomography scan revealed extraordinary dilatation of the fourth ventricle compared with other dilated ventricles, and old low-density areas in the cerebellar hemispheres. After an external ventricular drainage (EVD) was inserted, all the ventricles decreased in size and the symptoms disappeared. The authors confirmed the patency of the aqueductal canal. One week later, the EVD was replaced by a ventriculoperitoneal shunt. A disproportionately large, communicating fourth ventricle (DLCFV) should be differentiated from an isolated fourth ventricle, which consists of marked enlargement of the fourth ventricle with obstruction of both the inlet and outlet of the fourth ventricle. The authors propose the importance of the fragility to pressure of the brain parenchyma and cerebellar hemispheres around the fourth ventricle as the mechanism of producing DLCFV. PMID- 1726234 TI - [Binswanger's disease with normal pressure hydrocephalus. Case report]. AB - A 72-year-old hypertensive male was hospitalized with progressive gait disturbance (small step gait), urinary incontinence, and dementia. Computed tomography (CT) showed ventriculomegaly with periventricular lucency. T2-weighted magnetic resonance imaging revealed wide periventricular high-signal intensity and small infarctions in the basal ganglia. CT cisternography demonstrated ventricular stasis and convexity stasis of the contrast medium. Continuous intracranial pressure monitoring showed increased B wave percentage, low pressure volume index, and high outflow resistance. These findings indicated the coexistence of normal pressure hydrocephalus. After ventriculoperitoneal shunting, gait disturbance was greatly improved and urinary incontinence disappeared. The development of Binswanger's disease may be partially due to disturbed cerebrospinal fluid (CSF) dynamics. The possible pathophysiology of CSF dynamics in relation to Binswanger's disease is discussed. Detailed investigations of CSF dynamics are important in patients with Binswanger's disease, especially in the early stage. PMID- 1726235 TI - [Abdominal metastasis of a pineal region tumor through ventriculoperitoneal shunt. Case report]. AB - An 18-year-old male was admitted with headache, nausea, and vomiting. Computed tomography (CT) revealed an enhanced tumor of the pineal region and hydrocephalus. The tumor was partially resected via a parieto-occipital craniectomy. The histological diagnosis was germinoma. No serum tumor markers such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) were detectable. A ventriculo-peritoneal (V-P) shunt was emplaced and radiation therapy (whole brain 59 Gy) given. The tumor and the hydrocephalus regressed completely and he returned to work. Six years later, he experienced constipation and general fatigue. CT and echotomography of the abdomen showed a large peritoneal tumor and ascites. Laboratory investigation demonstrated serum levels of AFP 7640 ng/ml and HCG 150 IU/l, and high ascitic levels of AFP 12,890 ng/ml and HCG 1030 IU/l. AFP and HCG levels regressed after combined chemotherapy. However, he died due to leukopenia and pneumonia. Autopsy found no metastasis of tumor cells to the central nervous system. The peritoneal cavity contained hemorrhagic fluid and a large tumor 4100 g in weight. The tip of the V-P shunt tube was in front of the tumor. No neoplasm was found in the testis, retroperitoneal cavity, thymus, and other organs. The microscopic appearance of the peritoneal tumor was different to the first pineal tumor. The neoplasm was confirmed as a mixed germ cell tumor with teratoma components and suspected to be a metastasis of the pineal tumor through the V-P shunt system. PMID- 1726236 TI - [Primitive neuroectodermal tumor with Wilms' tumor. Case report]. AB - Occurrence of embryonal kidney tumors in patients with primitive neuroectodermal tumors, so-called central nervous system-renal neoplasia has been reported. An infant who presented with masses in the right lateral ventricle and the cerebellar vermis is reported. Histological examination showed primitive neuroectodermal tumors. Further investigation revealed tumors in the bilateral kidneys, which were removed subtotally and pathologically shown to be Wilms' tumors. The patient was then treated with anticancer drugs and irradiation. However, he developed lung metastases from the renal tumors and expired. At autopsy, a small tumor was found in the inferior horn of the left lateral ventricle. Histological finding showed a primitive neuroectodermal tumor. Also, bilateral large masses of recurrent Wilms' tumors, multiple metastases to the lungs and peritoneal dissemination were found. There is no evidence that this association is based on the selective neoplastic transformation of embryonal cells of similar histogenetic or cytogenetic origin. Several reports demonstrate the presence of embryonal cells in the nervous tissue which could imply a neuroepithelial origin for Wilms' tumors. PMID- 1726237 TI - [Diffuse cerebrospinal gliomatosis. Case report]. AB - A 65-year-old male was admitted with memory and gait disturbance. A computed tomography (CT) scan showed bilateral, diffuse, low-density areas with two round, slightly enhanced masses. T1-weighted magnetic resonance image revealed a low intensity area in the left paraventricular region, which converted to increased signal intensity, extending to the right paraventricular region through the splenium, on T2-weighted images. The tumor was diagnosed as glioblastoma multiforme after needle biopsy and treated by irradiation and chemotherapy. Seven months after admission, a CT scan revealed subependymal infiltration of the tumor with spotty calcification. He died of respiratory complications 11 months after the onset of symptoms. The autopsy showed brain swelling with flattened gyri. Horizontal sections of the brain showed diffuse enlargement of the white matter and basal ganglia with scattered hemorrhage and necrosis. Microscopically, the lesion was far more extensive and diffuse than was suspected from gross examination. Wide glial tumor cell infiltration was observed in the cerebral hemispheres, basal ganglia, brainstem, cerebellum, and even the cervical spinal cord with minimum destruction of the pre-existing architecture. Calcification was found around the thrombosed vessels and necrotic lesions. The clinical diagnosis and histological features of gliomatosis cerebri are discussed with reference to reported cases. PMID- 1726238 TI - [Intraosseous meningioma. Case report]. AB - An 82-year-old female was admitted with slowly progressive aphasia and right hemiparesis, accompanied by a hard, 5 x 5 cm subcutaneous swelling in the left frontotemporal region. Plain X-ray film showed a well-circumscribed round radiolucency in the left pterional region. Computed tomography (CT) scans showed an intraosseous mass lesion, homogeneously enhanced postcontrast, extending to the intracranial cavity. Bone CT demonstrated a concave appearance and partial destruction of the inner table, strongly suggesting an intradiploic origin of the tumor. Left carotid angiography revealed the mass supplied by the middle meningeal artery. Left frontotemporal craniectomy demonstrated that the inner surface of the skull was destroyed, and the dura was compressed but not invaded. Histological examination found meningotheliomatous meningioma with many psammoma bodies. PMID- 1726239 TI - [Ossifying fibroma in the cranial vault. Case report]. AB - Ossifying fibroma is relatively common in the maxilla, but rare in the cranial vault. A 10-year-old girl was referred for painful swelling of the left temporal region. On admission, she presented no abnormal physical and neurological findings except for the painful swelling. Plain skull X-ray films showed a radiolucent lesion of the left temporal bone about 4 cm in diameter, with a hyperostotic area of the parietal side. Computed tomography scan using bone window level also showed an abnormal density lesion in the same site. Curettage of this tumor was performed from a cosmetic point of view and at the family's petition. Histological examination showed vascular fibrous tissue in which lamellar bone was surrounded by osteoblasts. PMID- 1726240 TI - [Amplification of L-myc oncogene in malignant meningioma. Case report]. AB - Amplification of L-myc oncogene was noticed in a malignant meningioma originating from the right sphenoidal wing of a 54-year-old female. The patient underwent three surgical resections plus radiotherapy over a period of 11 years and then the growth rate of the tumor became much greater with a severely invasive appearance. Using Southern blot hybridization, L-myc amplification was examined on the specimen resected at the fourth operation. As a result, approximately five fold amplification was confirmed, which has not been previously reported except for that in a small cell carcinoma of the lung. This result may suggest that L myc amplification is responsible to some extent for the malignant transformation in this meningioma. PMID- 1726241 TI - [A basic study on omental transplantation. Vascular endothelial cell growth factor in human omentum]. AB - Omentum tissue has potent angiogenic activity, so transplantation is often effective in treatment of moyamoya disease. Omentum was homogenized and fractionated to investigate the presence of an endothelial cell growth factor. The effectiveness of extracts was measured by the growth activity of bovine aortic endothelial cell incubated for 6 days with various omentum extracts. The lipid fraction had no growth promoting activity. However, the water soluble extracts were active. The activity could be increased by ammonium sulfate precipitation (60-80% saturation). Gel permeation chromatography of the ammonium sulfate fraction showed that the endothelial growth factor had an apparent molecular weight of 96,000. Heparin affinity chromatography revealed poor heparin affinity. The activity was stable at pH 3.5 and pH 9.5, but inactivated by heating at 70 degrees C for 10 minutes or 100 degrees C for 2 minutes. These properties clearly distinguish the omentum-derived growth factor (OmDGF) from the heparin binding growth factor. OmDGF is probably distinct from other vascular endothelial cell growth factors. PMID- 1726242 TI - [Establishment of ACNU-resistant rat gliosarcoma cell lines and their characteristics]. AB - One of the most serious problems in cancer chemotherapy is the acquired drug resistance of tumor cells. In order to investigate the mechanism of acquired resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3 nitrosourea hydrochloride (ACNU), nine resistant cells were isolated from rat gliosarcoma 9L cells and characterized. ACNU-resistant cells were easily isolated after a single treatment even with low-dose ACNU (5 micrograms/ml). However, in spite of repeatedly high-pressure dose of ACNU, more than 30 fold increase to parental 9L cell resistance was not achieved. At this time, further increase in selection pressure resulted in cell death. The resistant ratios of these resistant cells were stable in the absence of ACNU for more than 1 year. Luria and Delbruck's fluctuation test indicated that the appearance of ACNU-resistant cells occurred spontaneously at a rate of (4.40-8.02) x 10(-7)/cell/generation. In karyotypic analysis, the mode was higher in the resistant cells than in 9L cells, but a homogeneously staining region or double minute chromosome, which was considered to be a result of gene amplification, was not observed. In growth kinetics, all the resistant cells except R1 and R12 cells had higher saturation density and plating efficacy than parental 9L cells. These findings seemed to be due to cellular modification associated with ACNU resistance. PMID- 1726243 TI - [Thermosensitivity of glioma cells with special reference to changes in cytoskeletons]. AB - The relationship between the thermosensitivity of cultured brain tumor cells and cytoskeleton was studied. C6 rat glioma cell line (C6 cells) and U-373-MG human glioblastoma cell line (MG cells) were used in monolayer culture. Survival rates at various temperatures were calculated by colony forming assay 10 days after heat treatment. Actin filaments, the main components of microfilaments, were observed by the 7-chloro-4-nitrobenzo-2-oxadiazole phallacidin staining and indirect immunofluorescence staining methods. Alpha-tubulins, the main components of microtubules, were also stained with an indirect immunofluorescence staining method. The morphological changes were investigated by scanning electron microscopy (SEM). Both the C6 cells and the MG cells showed moderate thermosensitivity on the survival curves. Actin filaments were revealed at stress fibers and the ruffles of the leading edge on both cell lines. Stress fibers were well developed in MG cells but were only minor in C6 cells. After heat treatment ruffles and stress fibers were disrupted. However, alpha-tubulins were not affected by heat treatment. SEM showed Swiss-cheese like change of cell surfaces due to many pores with disruption of ruffles and stress fibers after heat treatment. These results suggest that the cytoskeleton, especially microfilaments, may be damaged by hyperthermia. PMID- 1726244 TI - [Immunohistochemical study for choroid plexus papillomas and ependymomas]. AB - An immunohistochemical study of glial fibrillary acidic protein (GFAP), S-100 protein, cytokeratin (CKER), epithelial membrane antigen (EMA), and transthyretin (TTR) was carried out on 11 cases of choroid plexus papilloma (CPP) and 19 of ependymoma, using the peroxidase antiperoxidase technique. Among the 11 cases of CPP, all 11 were positive for CKER, EMA, and TTR, 10 for S-100 protein, and five for GFAP. Most of the GFAP-positive papilloma cells were simultaneously positive for CKER. However, these GFAP-positive cells were negative for TTR. Among the 19 cases of ependymoma, 16 were positive for GFAP, 17 for S-100 protein, three for CKER, eight for EMA, but none for TTR. Some GFAP-positive cells were also stained for CKER. However, TTR was not found in any of the ependymal cells. These findings suggested that CPP cells which show ependymal or glial differentiation lost the ability to synthesize TTR which is known to be synthesized in the epithelial cells of the choroid plexus. The more GFAP-positive cells present in a CPP, fewer TTR-positive cells are present. Though CPPs are usually easily distinguishable from ependymomas, occasional doubt arises concerning the differential diagnosis between CPP and papillary ependymoma. TTR can be a very useful diagnostic marker of CPP. PMID- 1726245 TI - [Effects of perfluorochemicals on the depressed rCBF and brain metabolism shortly after the hypertensive ICH]. AB - The effect of perfluorodecalin and perfluorotripropylamine, Fluosol DA-20% (FDA 20%), on the ischemic brain after hypertensive intracerebral hemorrhage (ICH) was evaluated and the mechanism investigated. Regional cerebral blood flow (rCBF), and cerebral metabolic ratios of oxygen (CMRO2) and glucose (CMRGlu) were measured before and after infusion of FDA-20%. The rCBF was estimated by single photon emission computed tomography using the 133Xe inhalation method, and CMRO2 and CMRGlu were determined by calculating arteriovenous difference between the femoral artery and the jugular bulb. The results showed: 1) The mean cerebral blood flow (mCBF) in both affected and non-affected hemispheres significantly increased by 10-20% over the control during 3 hours and 6-12 hours after infusion of 500 ml and 1000 ml FDA-20%, respectively. 2) Administration of 500 ml FDA-20% caused a maximal increase of 15% in CMRO2 and 24% in CMRGlu, and 1000 ml FDA-20% administration caused a maximal increase of 30% in CMRO2 and 75% in CMRGlu. 3) There was no significant change in mean blood pressure, arterial partial pressure of carbon dioxide and oxygen, or hematocrit before and after FDA-20% infusion. The results indicate that FDA-20% improved rCBF and brain metabolism in the ischemic brain of hypertensive ICH. FDA-20% may operate as an excellent oxygen carrier, and stimulate the brain metabolism directly. PMID- 1726246 TI - [A study of ionized calcium in the whole blood of patients with subarachnoid hemorrhage]. AB - In 14 normal volunteers and 22 consecutive patients with subarachnoid hemorrhage (SAH) due to a ruptured cerebral aneurysm, ionized calcium (Ca2+) concentrations in whole blood were measured using an automatic calcium analyzer. The values were corrected against pH 7.4. The relationships between the Ca2+ concentration and the following aspects were studied: neurological grading by Hunt and Kosnik, grade of SAH on computed tomography scan by Fisher's definition, and the presence of vasospasm. Clinical vasospasm was recognized in eight cases. The averaged Ca2+ value of the control group was 1.23 +/- 0.02 mmol/l. In patients with a poor neurological grade or severe SAH, the Ca2+ level was apt to be lower than that of the control group. In patients with vasospasm, the values of Ca2+ were significantly decreased, especially between 8 and 14 days after SAH, compared with those patients without vasospasm and the control group (p less than 0.05). These results indicate that measurement of Ca2+ concentration in whole blood may give a useful clue to treatment of vasospasm by calcium antagonist and that it may also provide a possible indicator as to the time of vasospasm in patients with severe SAH. However, it is very difficult to conclude whether decreased level of Ca2+ in patients with vasospasm is caused by the vasospasm itself. PMID- 1726247 TI - [Hypothalamic and pituitary function in brain death]. AB - Hypothalamic and pituitary hormone levels were measured in 56 patients meeting the criteria of brain death proposed by the Japanese Ministry of Welfare. Pituitary hormone releasing tests were carried out in 39 patients. In addition, cerebral angiography and transcranial Doppler (TCD) were performed in 13 and six patients, respectively, just after hormone measurements. Serum hypothalamic and pituitary hormone levels were inconsistently high based on the half life time in the presumed absence of cerebral blood flow shown by angiography. The responses to releasing hormones were normal in 16 patients. TCD detected cerebral blood flow in the middle cerebral artery or ophthalmic artery in three patients who showed non-filling on angiography. Postmortem microscopic examination of the hypothalamus and anterior pituitary lobe revealed normal structure and cells intermingled with lytic changes and necrosis. This series suggests that some part of the hypothalamus and hypophysis may still be alive after brain death, although the function of these regions may be clinically insignificant. PMID- 1726248 TI - [Study on recurrence of hypertensive intracerebral hemorrhage]. AB - Recurrence of hypertensive intracerebral hemorrhage confirmed by computed tomography scan has rarely been reported. The authors have experienced nine cases of recurrent hemorrhage among 494 cases of hypertensive intracerebral hemorrhage. Recurrence rate was 1.8%. There were eight males and one female with an average age of 60.5 years. Six cases had their first and second attacks in ganglionic regions. Among them, three cases had the second attack in the ipsilateral side, and the other three cases had the second attack in the contralateral side. Two cases had the first attack in the thalamic and the second in the cerebellar regions. One case had the first attack in the pontine and the second in the putaminal regions. Intervals between the first and second attacks were within 6 months for ipsilateral ganglionic attacks and over 4 years for contralateral ganglionic attacks. In all cases systemic blood pressure was normalized or well controlled by antihypertensive agents after the first attack. The mechanism of rebleeding has not been clarified. PMID- 1726249 TI - [Problems of surgical treatment for multiple intracranial aneurysms]. AB - A series of 105 patients presenting with multiple aneurysms and subarachnoid hemorrhage (SAH) were operated on for ruptured and unruptured aneurysms between 1976 and 1984. Clinical factors other than the severity of SAH affecting the outcomes included: 1) Misdiagnosis of the location of a ruptured aneurysm among multiple aneurysms resulted in poor outcomes because of multiple surgical approaches or rebleeding during the acute period. 2) Combinations of aneurysmal locations requiring multiple surgical approaches, such as interhemispheric and transsylvian, during the acute stage caused worse outcomes than with multi-stage surgeries. If an unruptured aneurysm could not be reached during the initial exposure, multi-stage surgery was safe if the ruptured aneurysm had been clipped during the acute period. 3) Complications occurring during unruptured aneurysm surgery. The patient's age, the location and size of the unruptured aneurysms were significant factors in the clinical prognosis. Surgery for unruptured aneurysm caused 1.8% morbidity in patients between 28 and 55 years, but 18.0% morbidity in patients over 56 years of age. Surgery for internal carotid artery aneurysms resulted in 14.8% overall morbidity. Surgery for middle cerebral and anterior cerebral artery aneurysms caused below 5% morbidity. Postoperative morbidity in patients with aneurysms less than 5 mm in diameter was 1.3%, and with aneurysms measuring 10 mm or more, 20%. The optimum treatment for multiple aneurysms with SAH should be based on all factors of the patient's condition, including the unruptured aneurysms. PMID- 1726250 TI - [Neurosurgical management of dialyzed patients]. AB - Cerebrovascular disease is a lethal complication for patients with renal failure because of the hemostatic disturbance and equivocality about management of the central nervous system. Appropriate management of the renal failure in neurosurgical patients was considered on the basis of experience. Hemodialysis (HD), which is the most common dialysis method, has a serious disadvantage: the elevation of intracranial pressure during dialysis due to the "disequilibrium syndrome." It is important to stabilize the serum osmolarity during dialysis in order to prevent the disequilibrium syndrome. From this point of view, continuous ambulatory peritoneal dialysis (CAPD) has great advantages; serum osmolarity is not rapidly changed and no anticoagulants are required during dialysis. CAPD is recommended as the first method of choice in neurosurgical management of renal failure patients. However, if a patient has to be maintained with HD because of a history of laparotomy or peritonitis, it is essential to keep the serum osmolarity as stable as possible using the extracorporeal ultrafiltration method, hypernatremic HD, bicarbonate HD, and intravenous administration of glycerol. PMID- 1726251 TI - [Clinico-histological study of low-density non-enhancing glioma]. AB - Some gliomas are not noticeably enhanced on initial computer tomography (CT) scans. Such low-density non-enhancing gliomas have a relatively long period between onset and surgery ranging from 5 to 25 months. The mechanism causing the low density over a certain period of time in the sequential CT findings was retrospectively investigated. Characteristic histological findings associated with such low-density areas were microcystic formation, necrosis, intratumoral edema, infiltrative growth, and absence of capillary proliferation. Proliferation of capillary vessels is characteristic of CT enhancement. PMID- 1726252 TI - [Efficacy of preoperative irradiation in midline oligodendrogliomas. Report of two cases]. AB - Two midline oligodendrogliomas in young males were successfully totally removed after preoperative irradiation. A 33-year-old male with right lower extremity weakness had a large hypervascular mass occupying the left lateral ventricle. Even after 31 Gy whole-brain irradiation, massive bleeding occurred at surgery and resulted in only partial removal. The residual tumor markedly regressed with disappearance of abnormal vascularity after subsequent local boost irradiation. At the second operation, the tumor was totally removed. A 32-year-old male with progressive headache had a hypervascular mass with gross calcification in the right lateral ventricle. The tumor was partially resected due to its abundant vascularity and blood loss. After 60 Gy local irradiation, the tumor was moderately shrunk with a significant reduction in vascularity. At the second operation, the tumor was totally removed. Preoperative irradiation as an adjunct to surgery may increase the resectability of highly vascular tumors such as midline oligodendrogliomas. PMID- 1726253 TI - [A comparative study of multimodality evoked potentials and computed tomography in head injuries]. AB - Multimodality evoked potentials (EPs) or three types of EPs--auditory brainstem response (ABR), somatosensory evoked potential (SEP), and visual evoked potential (VEP)--were recorded in 51 cases of traumatic intracranial hemorrhage within 3 days after injury. In order to assess these EPs, five EP grades were constructed, from normal, Grade I, to highly abnormal, Grade V. Furthermore, an EP pattern classification was devised to integrate the respective EP grades. Namely, PA, consisting of all three EPs within Grades I-III; PB, composed of one type of EP or both ABR and VEP at Grades IV and V; PC, consisting of both SEP and VEP at Grades IV and V; PD, comprising both ABR and SEP at Grades IV and V; and PE, covering all three EPs at Grades IV and V. PA signifies "no severe damage," PB, "localized damage," PC, "severe cerebral damage," PD, "severe brainstem damage," and PE, "severe diffuse damage." The results when compared with computed tomography (CT) findings were as follows: 1) The size of hematoma correlated with the SEP grade in 16 cases of acute epidural hematoma; a hematoma diameter of 17.5 mm was the threshold value at which SEP abnormalities developed. Eleven patients who underwent surgical removal of the hematoma showed "no severe damage," and the outcome was good. 2) In 13 cases of acute subdural hematoma, six cases revealed "severe cerebral damage" or "severe diffuse damage." In such cases, the degree of damage was not related to the hematoma size, and the outcome was very poor. 3) In 13 cases of a solitary contusional hemorrhage, only one case revealed "severe diffuse damage" and subsequently died.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726254 TI - [Delayed evolution of post-traumatic contralateral extracerebral hematoma after evacuation of initial hematoma]. AB - 105 patients over a 5-year period underwent emergency evacuation of traumatic intracranial hematomas. Seven (6.7%) developed delayed contralateral extracerebral hematomas (5 epidural and 2 subdural hematomas). These hematomas were insignificant or not present on initial computed tomography (CT) scan, but repeat CT scan after craniotomy showed sizable hemorrhage. In one patient, neurological deterioration heralded the delayed onset. In one case, intraoperative ultrasound imaging disclosed an epidural hematoma. Ultrasound examination is recommended in cases with a skull fracture contralateral to the initial hemorrhage. PMID- 1726255 TI - [Post-traumatic syringomyelia. Report of three cases]. AB - Three cases of post-traumatic syringomyelia are presented and the mechanism of syrinx formation is discussed. Two cases were examined radiologically. Computed tomography and magnetic resonance images (MRI) showed an expansive syrinx with adhesive arachnoiditis in the thoracic levels below the injury site and a localized syrinx on the posterolateral gray matter in the cervical levels above the injury site. These syrinxes existed below the C2 level and had no communication with the fourth ventricle. The other was an autopsy case. Postmortem examination revealed that a syrinx existed from C2 to Th6 and had no communication with the fourth ventricle or the central canal. It is concluded that small traumatic cavities in the gray matter evolve to an extensive syrinx by cerebrospinal fluid (CSF) entering via the posterior root entry zone, and adhesive arachnoiditis is an important factor in increasing the CSF which is entering. MRI was useful for the diagnosis. PMID- 1726256 TI - [Clinical analysis of ossified thoracic ligaments and thoracic disc hernia]. AB - Thoracic lesions present several clinical problems, particularly in their diagnosis and treatment, compared with cervical or lumbar lesions. Since 1983, 18 cases of thoracic space lesions, excluding spinal tumors or trauma have been experienced: nine cases of ossification of yellow ligament (OYL), five of ossification of posterior longitudinal ligament (OPLL), and four of disc hernia (DH). In these 18 patients, problems of clinical manifestations, neuroradiological examination, and surgical approaches are analyzed and discussed. As clinical manifestations, there was a preponderant occurrence in males in the OYL group, while in the OPLL group all the patients were females. OYL and DH occurred at lower thoracic levels. Thirteen of the 18 patients showed combined lesions either in the cervical or in the lumbar regions, such as cervical OPLL, cervical spondylosis, lumbar DH, and lumbar canal stenosis. In the neuroradiological examinations diagnosis of the upper thoracic lesions was difficult. Computed tomography (CT) scan with intrathecal metrizamide injection seemed essential for examination of ossified thoracic lesions. However, because CT imaging of the entire spine is impractical, efficient use of this examination requires previous localization of the offending vertebral level from either the neurological findings or other neuroradiological examinations such as myelography. Magnetic resonance imaging seemed most useful for ruling out the thoracic compressing lesions. As for surgical approaches, posterior decompression was effective for OYL and the anterior approach was useful for OPLL and DH. In patients with "tandem lesions," neurological and neuroradiological findings played an important role in deciding the responsible site. PMID- 1726257 TI - [Ventricular dilation during the treatment of subdural hygromas]. AB - Fifty-one cases with subdural hygroma experienced in the past 20 years were retrospectively reviewed. Eight patients showed definite ventricular dilation differing from simple restoration of the ventricles following disappearance of the cavity. The mean age was 72.4 years. Six patients presented with mental change as the initial symptom. On the initial computed tomography (CT), 75% of the cases had bilateral lesions, all were low density, and 88% were crescent shaped. Trepanation performed on six patients yielded watery clear or xanthochromic fluid. Nine to 61 days (mean 4 weeks) after admission, definite ventricular dilation was observed. Cisternography performed in four patients was all abnormal, although cerebrospinal fluid (CSF) pressure was within normal range. Cerebral blood flow images using 123I-iodoamphetamine and single photon emission CT in four patients revealed periventricular low uptake which was disproportionately large compared with the ventricular span on CT. A ventriculoperitoneal shunt was placed in four patients. The final outcome, however, was poor irrespective of treatment. These findings indicate that an impairment of the CSF circulation was not the sole cause of the ventricular dilation. Low CSF pressure and the disproportionately large periventricular low perfusion, compared with the ventricular span on CT scan, suggest a pre-existing periventricular parenchymal damage, which had been subsequently compromised by the presence of subdural mass lesion. Therefore, attention should be paid in aged patients with bilateral low dense, crescent-shaped subdural hygroma, presenting with mental change, for the risk of subsequent ventricular dilation which may affect the functional outcome. PMID- 1726258 TI - [Improvement of ischemic symptoms and cerebral blood flow after percutaneous transluminal angioplasty for renovascular hypertension. Report of a case with multiple cerebrovascular occlusive disease]. AB - A 53-year-old male complained of frequent left motor-sensory transient ischemic attack for 4 months. On admission, he demonstrated mild right hemiparesis, dysarthria, and right hemisensory disturbance of all modalities. Cerebral angiography demonstrated complete occlusion of the left internal carotid artery just above the origin of the ophthalmic artery and a stenotic lesion at the horizontal segment of the right middle cerebral artery. Renal angiography showed severe stenosis of the right renal artery. Systolic blood pressure was over 200 mmHg and marked circadian variation of blood pressure was noted. Serum renin was 4.0 ng/ml/hr. Four months after superficial temporal artery-middle cerebral artery anastomosis, left carotid angiography showed good patency of the bypass and the ischemic symptoms completely disappeared. Single photon emission computed tomography (SPECT) showed increased cerebral blood flow (CBF), especially in the left hemisphere after surgery. Six months after the bypass surgery, he complained of mild right hemiparesis again. Shortly after percutaneous transluminal angioplasty (PTA) for renal arterial stenosis, his hemiparesis was improved and the systolic blood pressure stabilized to 150-170 mmHg. SPECT showed the CBF had also recovered in both hemispheres. The improvement in ischemic symptoms and increased CBF after PTA were probably related to stabilization of the systemic blood pressure or inhibition of serum renin-angiotensin. PMID- 1726259 TI - [Giant aneurysm of the middle cerebral artery presenting with complex partial seizure. Case report]. AB - A 24-year-old female who had had several attacks of complex partial seizures was admitted after minor head trauma. There was no neurological deficit on admission, but a large oval calcification was incidentally found in the left temporal region on the plain skull film. Computed tomography scan and carotid angiography revealed a giant thrombosed aneurysm which arose from the M2 portion of the left middle cerebral artery. Focal spike discharges were found on the left temporal region on conventional electroencephalography. Left frontotemporal craniotomy and opening of the left sylvian fissure disclosed a giant aneurysm at the M2 portion of the left middle cerebral artery. The neck of the aneurysm was buried in the dome of the aneurysm and the parent artery was curved at an acute angle at the site of the neck. The aneurysm was excised and end-to-end anastomosis of the main stem of the M2 portion was successfully performed. Postoperative course was uneventful and the patient became completely free from seizures. The surgical technique and the possible mechanism of complex partial seizure in this patient are described. PMID- 1726260 TI - [Unilateral development of moyamoya vessels after contralateral cervical internal carotid artery occlusion. Case report]. AB - A 41-year-old female suffered transient ischemic attack. Cerebral angiography revealed occlusion of the left internal carotid artery at the cervical portion and collateral pathways consisting of transpial anastomosis and parenchymal anastomosis from the posterior circulation. Five years later, the second angiography was carried out. Left carotid angiography revealed appearance of transdural anastomosis from the middle meningeal artery and the anterior ethmoidal artery. Right carotid angiography revealed severe stenosis at the carotid fork with moyamoya vessels. Bilateral encephalomyo-arterio-synangiosis was performed for revascularization. Postoperative bilateral carotid angiograms revealed good neovascularization on both sides. In typical moyamoya disease, occlusive change of the carotid fork with moyamoya vessels appeared symmetrically and simultaneously on both sides. Although this case is not a typical moyamoya disease, its pathogenesis is quite similar to that of moyamoya disease. PMID- 1726261 TI - [Neurological deterioration due to cerebral hyperperfusion following carotid endarterectomy. Case report]. AB - Recently attention has been drawn to postoperative cerebral hyperperfusion after carotid endarterectomy (CEA) associated with a preoperative state of impaired cerebral hemodynamics. Rarely postoperative neurological deficits are caused by cerebral edema due to hyperperfusion. The patient was a 65-year-old male with dysarthria and right hemiparesis. Because of the presence of severe stenosis of the left carotid artery, CEA was performed. On the 6th postoperative day, he developed severe right hemiparesis and aphasia due to cerebral edema in the subcortical region of the left cerebral hemisphere. Left carotid angiography showed normal circulation without evidence of the carotid stenosis. Later the cerebral edema and the neurological deficits gradually disappeared. PMID- 1726262 TI - [Spontaneous disappearance of aneurysm after total removal of accompanying intracranial arteriovenous malformation. Case report]. AB - A 55-year-old male was hospitalized with severe headache. On admission, neurological examination revealed no abnormal findings. Plain computed tomography (CT) showed a slightly high-density area in the medial surface of the right parietal lobe. A marked enhancement in the same region was noted in enhanced CT. Cerebral angiography showed an arteriovenous malformation (AVM) in the medial surface of the right parietal lobe and two aneurysms on the right pericallosal artery which fed the AVM. In addition, a saccular aneurysm was noted at the anterior communicating artery. It was not possible to treat the AVM, two aneurysms nearby the AVM, and the unruptured anterior communicating artery aneurysm simultaneously with a single craniotomy. It was therefore decided to perform surgery for the AVM and two aneurysms nearby the AVM prior to clipping of the anterior communicating artery aneurysm. Total excision of the AVM and two aneurysms nearby the AVM was performed. Cerebral angiography performed 18 days after surgery revealed no AVM and also reduction in size was noted of the anterior communicating artery aneurysm. Three months later, repeated cerebral angiography showed disappearance of the aneurysm. This was further confirmed 15 months after surgery by angiography. From the literature, 117 cases of coexistence of AVM and aneurysms of the brain were collected and classified into three types according to their anatomical and hemodynamic correlation. It is suggested that hemodynamic stress, due to increased blood flow caused by the AVM, played a major role in the development of the aneurysm.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726263 TI - [Bilateral aneurysms of extracranial internal carotid arteries. Case report]. AB - A 52-year-old male was admitted because of right hemiparesis. Computed tomography scan showed a low-density area in the basal ganglia on the left side. Left carotid angiography showed an aneurysm of the extracranial internal carotid artery at the level of the C1-C2 vertebral body. Right carotid angiography also showed an aneurysm of the extracranial internal carotid artery. Because there were neither steno-occlusive changes in the intracranial vessels nor abnormality in the heart, the right hemiparesis seemed to be due to embolism from the extracranial aneurysm. Aneurysmectomy and end-to-end anastomosis of the left internal carotid artery were performed. Extracranial carotid aneurysms are rare conditions. In surgery on these aneurysms, ischemic changes of the brain during arterial clump must be detected and treated. Hypothermia, induced hypertension, and/or internal shunting have been used during arterial clump. The pathogenesis, symptoms, prognosis, and surgical treatments of these aneurysms are discussed. PMID- 1726264 TI - [Superior sagittal sinus thrombosis complicated with multiple aneurysms presenting as subarachnoid hemorrhage. Case report]. AB - A 48-year-old female suffered from severe headache, vomiting, and disturbance of consciousness. On admission, she was somnolent with mild paresis of the left leg. Precontrast computed tomography (CT) scans showed a high-density area in the left sylvian fissure and the posterior horn of the left lateral ventricle. Angiographically, a right middle cerebral artery aneurysm and a basilar artery aneurysm were recognized. Furthermore, on the venous phase of bilateral carotid angiograms, superior sagittal sinus (SSS) thrombosis was recognized. Subarachnoid hemorrhage (SAH) was probably induced by rupture of a dilated vein associated with SSS thrombosis, because high-density area on CT scan and location of the aneurysms were different. The patient was initially treated conservatively. Two months later, craniotomy was performed which did not disclose any trace of hemorrhage around the aneurysms and aneurysms themselves. Postoperatively, acute brain swelling and generalized convulsion were induced. The patient became ambulatory 5 months after surgery. In SAH cases, the venous phase should be examined at least in one side of the carotid arteries. In such a SAH case induced by venous thrombosis complicated by aneurysms it is very difficult to decide the timing of surgery for aneurysms. PMID- 1726265 TI - [Traumatic sinus pericranii. Case report]. AB - The patient was involved in a traffic accident at the age of 1 and the left parieto-occipital scalp was contused without skull fracture. At the age of 5, an extracranial scalp mass was first noticed just beneath the multiple scalp scars. Angiography through the mass revealed that the extracranial mass cavity was connected to the superior sagittal sinus through the emissary veins. The mass located in the subgaleal, epiperiosteal space was totally resected and the connection with the intracranial sinus was closed with bone wax. Histologically, there were many capillaries and some large blood cavities with only one layered endothelium and connective tissue. Therefore, the mass was diagnosed as sinus pericranii and considered to be secondary to previous head trauma because: 1) The patient had a history of head trauma with considerably severe scalp injuries. 2) The extracranial blood sinus was located exactly beneath the traumatic scar. 3) There was no neoplastic tissue histologically. 4) No scalp mass was noticed before the traffic accident. 5) There was an elapsed time between the trauma and the growth of the mass. 6) No scalp nevus such as port-wine stain existed. PMID- 1726266 TI - [Multiple aspergillus brain abscess complicated with systemic lupus erythematosus -case report]. AB - A 15-year-old female was hospitalized for the treatment of systemic lupus erythematosus complicated with nephritis. She improved with administration of steroid hormones and an immunosuppressant, plasma exchange, and dialysis. However, a lung abscess developed 6 months after admission, and multiple brain abscesses appeared 2 months after the onset of the lung abscess. The lung abscess faded with oral administration of fluocytosine and intravenous administration of miconazole, but the brain abscesses enlarged. Intrathecal administration of miconazole was not effective. Therefore, the abscess in the right frontal lobe was surgically removed and an Ommaya's reservoir was placed in the anterior horn of the right lateral ventricle. Aspergillus was identified in the removed abscess. Subsequently, miconazole was administered intraventricularly through the Ommaya's reservoir 10 mg daily for 1 month. The abscesses in the left parietal lobe gradually diminished. One year later, she complained of right hypesthesia again. Computed tomography scan revealed enlargement of the abscess. Miconazole was administered intravenously and intraventricularly for 1 month. Second craniotomy was performed 16 months after the first surgery and the abscess was completely removed. She was discharged with mild hypesthesia of the right leg. It is concluded that intraventricular administration of miconazole through an Ommaya's reservoir is an effective therapy for central nervous system aspergillosis. PMID- 1726267 TI - [Intracranial tuberculoma with difficult preoperative diagnosis. Case report]. AB - Intracranial tuberculoma is a relatively rare tumor in developed countries. A 41 year-old Japanese male with a personal history of pulmonary tuberculosis at the age of 20 was referred because of continuous headache. Computed tomography scan revealed multilocular ring-like enhancement in the right deep temporal region with massive brain edema. Angiography showed an avascular mass with narrowing of the carotid bifurcation. The preoperative diagnosis was glioblastoma. This mass was successfully removed and the histological diagnosis was tuberculoma. This case suggested that tuberculoma is still one of the differential diagnoses of an enhanced mass lesion even without any active extracranial tuberculous lesion. PMID- 1726268 TI - [Bilateral fenestrations of the vertebrobasilar artery with trigeminal neuralgia. Case report]. AB - Fenestration of cerebral vessels is congenital and usually of no clinical significance. A 58-year-old female presented with left trigeminal neuralgia associated with double fenestrations of the vertebrobasilar artery. Vertebral angiography showed bilateral fenestrations in the intracranial segment. The left fenestrated artery originated at the distal portion of the vertebral artery and terminated at the middle portion of the basilar artery, compressing the left Vth cranial nerve root. The neuralgia improved after microvascular decompression. Fenestration of cerebral vessels is usually single. Five of eight reported cases with double fenestrations had bilateral extracranial fenestrations at the atlantoaxial portion of the vertebral artery. Bilateral fenestrations of the vertebrobasilar artery with trigeminal neuralgia have not been previously reported. PMID- 1726269 TI - [Ossification of the thoracic ligamentum flavum presenting with intercostal neuralgia. Case report]. AB - A 46-year-old female was admitted complaining of progressive, severe girdle pain consistent with the left Th3 dermatome. Neurological examination on admission revealed dysesthesia and radiating pain in the left Th3 territory. Plain X-rays and tomograms of the thoracic spine revealed a beak-like bony excrescence arising from the lamina and projecting moderately to the Th3/4 intervertebral foramen, suggesting ossification of the thoracic ligamentum flavum (OYL). Myelography showed the dural sac compressed from laterally just below the left Th3 pedicle, which suggested that the Th3 nerve root was compressed by the OYL. After Th3 nerve root decompression through hemilaminectomy and foraminotomy, the girdle pain disappeared. OYL is known to cause thoracic radiculomyelopathy, but presentation with intercostal neuralgia only is very rare. The authors review the literature and stress the importance of myelography for diagnosis. PMID- 1726270 TI - Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors. AB - The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2 ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed. PMID- 1726271 TI - Defining topological equivalences in macromolecules. AB - We describe a completely automated and objective method for defining topological equivalents in macromolecules. The method is based on well established techniques for identifying topologically and topographically equivalent atoms in small molecules and has been used in structural alignment of proteins and RNA molecules, and to extract fragments of molecules (protein secondary structures and RNA and DNA double helices) from structural databases consistent with some specified template structure. PMID- 1726272 TI - Cross-reacting epitopes shared between Plasmodium falciparum and its host: the origin of autoreactive antibodies? PMID- 1726273 TI - Heat shock proteins as "super"-carriers for sporozoite peptide vaccines? PMID- 1726274 TI - [Levels of tumor necrosis factor and interleukin 1 in uremia and in hemodialysis]. AB - The present study aimed to assess the serum levels of tumor necrosis factor (TNFalpha) and Interleukin-1 (IL-1) in uremic patients and those patients undergoing extracorporeal replacement therapy in relation to the duration of dialysis and the type of membrane. Serum cytokine values were assayed using an immunoradiometric technique (monoclonal antibodies) in 28 uremic patients and 7 healthy controls. TNFalpha levels were normal in control subjects but increased in uremic patients. Patients undergoing hemodialytic treatment showed increases which were statistically significant in comparison to basal levels, with a trend which was directly correlated to the duration of dialysis. No significant variations were induced by the different types of dialysis membranes used. In virtually all patients IL-1 values could not be assayed using this method. PMID- 1726275 TI - Diagnosis and therapy monitoring of liver metastases from neuroendocrine tumours. PMID- 1726276 TI - [Palliative treatment in neoplastic jaundice. Personal experience]. AB - The palliative treatment of biliary duct neoplastic obstruction represents a problem of great importance and frequently can't leave out of consideration patients clinical conditions and phase of neoplastic disease. Authors, in this article refers their experience on palliative treatment of neoplastic jaundice and indications for surgical or endoscopic treatment. Their experience shows that surgical palliation must be performed in patients with preoperative instrumental investigations without "surgical risk", this vouches for a better quality of life than endoscopic procedure performed with diffuse neoplastic disease and in patients with surgical risk. PMID- 1726278 TI - [Vacation and health. Seminar on tourist medicine]. PMID- 1726277 TI - [Postoperative sclerosis of the bladder neck: pathogenic mechanisms]. AB - A study was conducted in 69 patients with postoperative sclerosis of the vesical neck to analyze the different pathogenic mechanisms that have been put forward. This complication, however, could not be ascribed to any underlying cause. It presents in patients who undergo surgery of the prostate early, when their adenoma is generally small and frequently with inflammatory lesions as those of chronic prostatitis. Surgery appears to be the triggering mechanism in these patients who are likely to be predisposed to developing this condition as some patients are predisposed to develop keloid from cicatricial hypertrophy. PMID- 1726279 TI - [Tourism medicine: a new branch of medicine]. PMID- 1726280 TI - [Vaccinal and chemoprophylaxis of tourists]. PMID- 1726281 TI - [The information and prevention intervention directed at tourism operators through the Fiavet health service for tourism]. PMID- 1726282 TI - [The tourist trip as a state of well-being: the sociological and psychological motivations]. PMID- 1726283 TI - [Hot springs, tourism and climatology]. PMID- 1726284 TI - [Community rules in tourist matters: the duties of the travel organizer and agent]. PMID- 1726285 TI - [Environmental rules in the tourism sector: the role of local regulation]. PMID- 1726286 TI - [Tourism and restaurants]. PMID- 1726287 TI - [Viral infections on trips: viral hepatitis B in the world]. PMID- 1726288 TI - [The epidemiology and prevention of AIDS in travelers]. PMID- 1726289 TI - [The epidemiology and prevention of traveler's diarrhea]. PMID- 1726290 TI - [Sexually transmitted diseases]. PMID- 1726291 TI - [International health regulation]. PMID- 1726292 TI - [The international convention concerning the travel contract: the outline of the responsibility of the tourism operator]. PMID- 1726293 TI - [An "objective" project: epidemiological research and the organizational proposal for Service No. 1 of U.S.L. 28/Locri]. PMID- 1726294 TI - [Airport malaria: the diffusion of the phenomenon and preventive measures]. PMID- 1726295 TI - [New frontiers in tourism medicine]. PMID- 1726296 TI - [Anesthetics in operating rooms: environmental and biological monitoring in relation to functional restructuring]. PMID- 1726297 TI - [The health surveillance of those exposed to low concentrations of anesthetic gases]. PMID- 1726298 TI - [The epidemiology of domestic accidents: the trend of the phenomenon in Italy and prevention rules]. PMID- 1726299 TI - [The costs of cesarean section compared to vaginal delivery: an economic analysis as related to an average-size hospital]. PMID- 1726300 TI - Use and relevance of action-research for tackling health care delivery problems at district level in developing countries. PMID- 1726301 TI - [The possibilities of controlling severe forms of periodontopathy via antibiotics]. PMID- 1726302 TI - [Legionellosis: some epidemiological and preventive aspects]. PMID- 1726303 TI - [Industrial treatments for the preservation of food: the potential genotoxic effects]. PMID- 1726304 TI - [The problems of choosing standards for selecting girls at risk of low stature. 1]. PMID- 1726305 TI - [Low statute at first observation. What stature at the end of growth? 2]. PMID- 1726306 TI - [Low stature at the end of the observation in girls with normal stature (beyond the 10th percentile) at the first observation. 3]. PMID- 1726307 TI - Change of cerebral glucose metabolism by antineoplastic drug. AB - To estimate change of cerebral glucose metabolism by anticancer drugs, positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) was performed in 11 patients with malignancies who did not show neurological symptoms. In a patient treated with intravenous high-dose methotrexate (HD-MTX) and intrathecal MTX, apparent decrease of glucose metabolism was observed. HD-MTX and intrathecal MTX may cause cerebral glucose metabolism disorder primarily. Cerebral glucose metabolism was also mildly reduced by chemotherapy with drugs other than MTX and by radiotherapy outside the brain, with a corresponding change of blood data. It may reflect a psychosomatic change in whole body. PMID- 1726308 TI - Biochemical modifications in the bursa of fabricius, thymus and adrenals of chickens injected with an anti-bursal serum. AB - Cornish-Rock chickens were given 0.3 ml anti-bursal serum in the pectoral muscle on the first day of life. Three days after serum administration, the modifications were evident both in the bursa of Fabricius and thymus (ponderal involution and decrease of RNA and glycogen contents). Pronounced thymo-bursal differences appeared at 13 days: in the bursa the values were normalized; in the thymus inverse modifications appeared as compared to the 3 days values. The modifications in the adrenals are in agreement with those in the lymph organs. PMID- 1726309 TI - Role of RNA as a dietary source of pyrimidines and purines in immune function. PMID- 1726310 TI - Effect of diet on transfusion induced immune suppression. PMID- 1726311 TI - The effect of dietary glutamine and dietary RNA on ileal flora, ileal histology, and bacterial translocation in mice. PMID- 1726312 TI - The effect of a uniquely formulated diet (supplemented with arginine, RNA, and menhaden oil) on ileal flora, ileal histology, and bacterial translocation in mice. PMID- 1726313 TI - Biochemical characterization and anatomical distribution of a major form of unamidated precursor of substance P in rat brain. AB - Previous work from this laboratory has provided biochemical characterization of several posttranslational processing intermediates of the neuropeptide substance P (SP) in central nervous system (CNS) tissues, including the COOH-terminal glycine-extended dodecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (SP-G). SP-G is a major species of unprocessed SP found in rodent CNS tissues, and is the likely immediate precursor form of SP in the biosynthetic scheme. Here we present extensive characterization of the normal regional distribution of SP G, as compared to SP, throughout the rat CNS via coordinated biochemical and morphological analyses. By radioimmunoassay (RIA), an approximate 10-fold variation in regional levels of SP-G-like immunoreactivity (SP-G-LI) was observed, ranging from 0.30 pmol/g in the amygdala, to 6.49 pmol/g in the medulla. On a normalized basis, the regional variation of unamidated precursor relative to mature peptide (SP-G-LI/SP-LI molar ratio) ranged from 0.30% in the amygdala to 5.15% in the dorsal root ganglia (DRG). Overall, the highest SP-G LI/SP-LI ratios were found in DRG, medulla, and spinal cord, i.e. CNS areas associated with primary sensory afferent innervation via capsaicin-sensitive unmyelinated small diameter fibers. In addition, chromatographic and RIA analyses of extracted brain tissues indicated that the quantified immunoreactivities corresponding to SP, SP-G, as well as an additional COOH-terminal Gly-Lys extended precursor, i.e., SP-G-K, displayed very similar chromatographic behavior as demonstrated for chemically authentic standards. These biochemical data were complemented by immunohistochemical analyses demonstrating a pattern of immunohistochemical staining for the presence of SP-G-LI as a defined subset of SP-LI-containing neural elements. Here, reaction product was localized to dendritic, axonal, and terminal neuronal elements in representative CNS regions of the rat, with relatively high levels of SP-G-LI found within anatomical areas containing a high density of sensory terminal structures. In an attempt to provide correlative functional anatomy, a group of rats was treated with colchicine, in order to differentially localize SP-LI- and SP-G-LI-containing somata after inhibition of axoplasmic transport. Most prominently, colchicine administration engendered immunohistochemical visualization of both SP-LI- and SP G-LI-positive cells in mesencephalic and brainstem regions associated with stress, pain responses, and central control of autonomic function. Within this context, the coordinate expression of both SP-LI- and of SP-G-LI-positive somata in discrete brain areas is probably indicative of high ongoing rates of tachykinin synthesis coupled to utilization. PMID- 1726314 TI - Dissociation of neurite-promoting activity and protease-inhibiting function of alpha 2-macroglobulin in culture. AB - The relationship between the neurite-promoting effect and the protease-inhibiting function of alpha 2-macroglobulin (alpha 2M) was examined in a culture of dissociated neurons from embryonic rat neocortical tissue. The neurite-promoting effect of various protease inhibitors (soybean trypsin inhibitor, alpha 1 antitrypsin, aprotinin, phenylmethanesulfonyl fluoride, leupeptin, antipain, human alpha 2M and purified rat alpha 2M) was investigated. At lower concentrations, only alpha 2M significantly promoted neurite outgrowth. Furthermore, alpha 2M-trypsin complexes, which had lost their protease-inhibiting function and exposed their receptor-recognition site, promoted neurite outgrowth more efficiently than native alpha 2M. These results indicate the possibility that the neurite-promoting effect of alpha 2M is mediated by its receptor-binding activity but is not directly related to its protease-inhibiting function. PMID- 1726315 TI - [Resistance of isolated cells from squamous cell carcinomas of the oral cavity to cytotoxic drugs]. AB - Human tumor cells from 22 patients with squamous cell carcinomas of the oral cavity were cultivated in vitro and then subjected to sensitivity tests to cytotoxic drugs such as methotrexate, 5-fluorouracil, cisplatin and bleomycin. Tumor cell proliferation showed drug-specific reaction patterns and time-dose dependent variations. While the effect of methotrexate on cell proliferation was insignificant, 5-fluorouracil caused a growth suppression of 50%. Cisplatin and bleomycin suppressed cell growth by more than 90%. PMID- 1726316 TI - [Long-term study on the effects of the width of keratinized gingiva on the localization of the gingival margin]. AB - In a 5-year longitudinal study we assessed the amount and frequency of gingival recessions in relation to the width of the keratinized gingiva and the already existing recessions in two groups (each n = 15). Group I (dental students) had nearly no recessions and group II (patients) had exposed roots on 24% of their teeth. In both groups there existed no correlation between the width of the keratinized gingiva or, respectively the existing recessions and the recessions occurring. Only 36 sites had a recession greater than or equal to 1 mm, these were accumulated in just a few probands. PMID- 1726317 TI - Sequence complexity of polyadenylated RNA from seizure-susceptible El mouse brain polysomes. AB - The hybridization kinetics in the complementary DNAs (cDNA) to polyadenylated messenger RNA (mRNA) in the brain of seizure-susceptible El mice were examined to elucidate the correlation between seizure susceptibility and genetic expression. Homologous and heterologous saturation hybridizations indicated that cDNA of seizure-nonsusceptible ddY mice hybridized with less mRNA in seizure-experienced El(+) or seizure-nonexperienced El(o) mice than that in ddY mice, cDNA of El(+) mice hybridized to a lesser extent with mRNA of El(o) mice than that of ddY or El(+) mice, and no difference was observed with cDNA hybridization results of El(o) mice to individual mRNAs of ddY, El(+) or El(o) mice. The results suggest that some sequences found in mRNA of ddY are lacking in both El strains and are responsible for the inbred predisposition, while those present in El(+) mRNA but missing in El(o) may associate with seizure susceptibility, since common sequences are present in the mRNA of those groups of animal. The sequence diversity among the 3 groups of mice was mainly observed with the rare class of mRNA. Molecular cloning of the cDNA corresponding to this class of mRNA found in the El(+) mice would clarify the gene expression in the brains of epileptics. PMID- 1726318 TI - Nerve-induced tachykinin-mediated vasodilation in skeletal muscle is dependent on nitric oxide formation. AB - Nerve-induced vasodilatation was studied by intravital microscopy of the rabbit tenuissimus muscle, pretreated with pancuronium, phentolamine, and guanethidine. Nerve stimulation of the tenuissimus nerve induced a vasodilatation which was frequency and pulse duration-dependent and insensitive to atropine and propanolol but abolished by tetrodotoxin. The nitric oxide synthase inhibitor, N omega-nitro L-arginine methyl ester (L-NAME, 100 microM), but not its enantiomer, D-NAME, markedly inhibited the vasodilation induced by nerve stimulation or by exogenous substance P or neurokinin A. Vasodilatation due to calcitonin gene-related peptide, prostaglandin E2 or nitroprusside was unaffected. The substance P antagonist, spantide (30 microM), significantly attenuated nerve-induced vasodilatation, in parallel with L-NAME. Our results indicate that nerve-induced vasodilatation in skeletal muscle can be attributed to the release of substance P and/or other tachykinins and that nitric oxide subsequently mediates the response to endogenous tachykinins released from nerves. PMID- 1726319 TI - Selective activation of quisqualate metabotropic receptor potentiates NMDA but not AMPA responses. PMID- 1726320 TI - [Diagnostic and therapeutic guidelines in arrhythmias with onset in childhood]. PMID- 1726321 TI - Angiocentric lymphoma--a case report. PMID- 1726322 TI - Mast cells as a source of superoxide anions and nitric oxide-like factor: relevance to histamine release. AB - Rat serosal mast cells (MCs, 85-90% pure), obtained from peritoneal washing of Wistar albino rats, produced a significant amount of superoxide anions (O2.-) as measured by the increase in absorbance due to the reduction of ferricytochrome c; they were also able to generate a nitric oxide (NO)-like factor, as measured by two bioassay systems: i) inhibition of platelet aggregation and ii) stimulation of MCs guanylate cyclase. Incubation of MCs with human washed platelets resulted in an inhibition of thrombin-induced platelet aggregation which was proportional to cell number. The inhibitory activity of MCs was potentiated by substances which preserve NO (superoxide dismutase, SOD), and reversed by compounds which inactivate NO (oxyhaemoglobin, oxyHb) or which inhibit its synthesis (NG monomethyl-L-arginine, MeArg). Mechanical stimulation of MCs produced a time dependent increase in the levels of their cGMP but not cAMP; this increase was enhanced by E. coli lipopolysaccharide (LPS). NO generators such as sodium nitroprusside (NaNp) also augmented the levels of cGMP in MCs. NaNp inhibited in a dose-dependent manner the release of histamine evoked by compound 48/80 (0.5 microgram/ml), but not by the O2.--generating system (xanthine-xanthine oxidase), suggesting a bidirectional regulation of histamine release afforded by O2.- and NO. PMID- 1726323 TI - Cyclic nucleotides in human macrophages: effects of atrial natriuretic factor and nitroprusside on cGMP and cAMP production. AB - Atrial natriuretic factor (ANF, 10(-7) M) and sodium nitroprusside (SNP, 10(-5) 10(-3) M) stimulated cGMP production in human peritoneal macrophages (HPM). This suggests the existence of two separate forms of guanylate cyclase in HPM, e.g. the receptor-related form by ANF and the soluble form by SNP. In parallel with the rise in cGMP levels, both agents provoked a decrease in cAMP levels. Increasing the concentration of the phosphodiesterase inhibitor IBMX (0.2 mM to 1.0 mM) in the incubation media resulted in a significantly greater rise in cGMP levels which was accompanied by a profound decrease in cAMP levels. ANF did not exert any direct or GTP-related effect on cAMP production, which is in contrast to its action in other tissues. These results suggest that cAMP levels can be modulated through a cGMP signal, most likely at the production level. Results also give substantial evidence for the presence of a ANF receptor site on human peritoneal macrophages. PMID- 1726324 TI - A common antigenic epitope expressed on human Pneumocystis carinii. AB - Recently antigenic heterogeneity in human Pneumocystis carinii (Pc) isolates was observed in several laboratories. Monoclonal antibodies (MAb) were produced to human Pc (PcH) from a lung autopsy sample from a non-AIDS patient (MAb Group I, n = 10), or from bronchoalveolar lavage (BAL) fluid from AIDS patients (MAb Group II, n = 8). To detect Pc antigen from specimens, indirect immunofluorescence and immunoblotting techniques were used. The reactivity was evaluated by using one autopsy sample from the non-AIDS patient and 14 BAL samples from AIDS patients. The MAb in group I (C5-9, E9) stained a part of PcH from all isolates. On the other hand, several MAb in group II (L20-5, M34-2, M78-3, M79-5, N23-4) stained all PcH from all isolates. Some MAb (C5-9, E9, M34-2, M78-3) stained cysts as well as trophozoites. Immunoblot studies detected a 92 kDa molecule as a common antigen by all of these MAb. Therefore, we have found a common antigenic epitope on PcH and MAb that recognize this epitope may become useful for diagnosis of infection and for biological characterization studies on the organism. PMID- 1726325 TI - Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii. AB - The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development. PMID- 1726326 TI - New rapid staining methods of Cryptosporidium oocysts in stools. AB - Two new extemporaneous negative-staining methods are proposed to detect Cryptosporidium oocysts in stools, using light-green and merbromine in 1% and 2% aqueous solution, respectively. A Ziehl-Neelsen stain as modified by Henriksen and Polhenz was used as a reference technique. A comparison between these two new stains and the reference, a modified Ziehl-Neelsen mod. method gave almost identical sensitivity and specificity. We propose their use in routine diagnosis for enteric cryptosporidiosis. PMID- 1726327 TI - Epitope study and cDNA screening of major surface glycoprotein of Pneumocystis carinii. AB - Use of monoclonal antibodies against the major glycoprotein of Pneumocystis carinii (P115) implicated the sugar moiety as being strongly antigenic. Furthermore, monoclonal antibodies directed against the peptide portion of P115 were generated by using synthetic oligopeptides after amino acid sequencing was done on P115 proteolytic fragments. PMID- 1726328 TI - Sporozoites and merozoites of Cryptosporidium parvum share a common epitope recognized by a monoclonal antibody and two-dimensional electrophoresis. AB - Sporozoites and merozoites of Cryptosporidium parvum were analyzed for the presence of a 15 kDa surface antigen using a monoclonal antibody probe. Both were found to possess the antigen by immunofluorescence, and further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed these observations. When separated by two-dimensional electrophoresis the isoelectric point was found to be similar, with major spots at 4.25 and minor spots at 4.15. PMID- 1726329 TI - Assays for testing Pneumocystis carinii viability. AB - A series of classical vital stains and fluorescent indicator compounds were evaluated as viability assays of P. carinii. The combination of the acetoxymethyl ester of calcein with either ethidium homodimer or propidium iodide distinguished between live, dead and moribund organisms and provided high fluorescence intensity and low bleaching enabling photodocumentation at high magnifications. PMID- 1726330 TI - The effects of in vitro hyperthermia on natural killer activity from lung, blood and spleen. AB - The in vitro effects of hyperthermia on natural killer (NK) activity from rat lung, peripheral blood and spleen were assessed. NK activity of all three compartments was very sensitive to heat shock. Exposure at 42.5 degrees C for 30 min resulted in more than 95% inhibition of NK activity. Conjugate-formation assays revealed that the mechanisms of hyperthermic NK inactivation are associated with post-binding lytic events. However, hyperthermia treated NK cells could partially recover their cytotoxic activity. Unheated lung lymphocytes (LL) were more sensitive to hyperthermic inhibition than spleen (SL) and blood (PBL) lymphocytes but they were also able to recover to a greater extent from such inactivation. Moreover, the responsiveness of NK cells from lung, blood and spleen to interleukin-2 (IL-2) and interferon (IFN-alpha/beta) was altered differently by heat shock. Hyperthermia treatment increased the ability of NK cells from blood to respond to IL-2 or IFN-alpha/beta. Similarly hyperthermia treated NK cells from spleen were more responsive to IFN-alpha/beta. By contrast such treatment did not change significantly the responsiveness of lung NK cells to these agents. Taken together, our findings indicate that NK cells from various compartments behave differently in response to hyperthermia treatment. Moreover, it suggests that hyperthermia treatment does not irreversibly comprise the host natural killer response and may even in some cases increase NK cell responsiveness to biological response modifiers (BRM). PMID- 1726331 TI - Bovine coronary artery endothelium: culture, characterization, angiogenesis and sensitivity to laser photodynamic treatment modalities. AB - Culture, characterization and sensitivity to laser and photosensitizers of bovine coronary artery endothelium is presented. Endothelial cells from bovine coronary artery specimens obtained after sacrifice were successfully cultured. Endothelial cells were obtained from the anterior descending coronary artery. Cells were grown in RPMI-1640 with 20% fetal bovine serum. Preconditioned medium was required to enhance the efficiency of the initial inoculum. The resulting cultures could be passed for up to 15 times and maintained a stable, normal karyotype throughout this period. The culture reached a stable confluency packing density by two to three weeks (5 x 10(5) to 10(6) cells/cm2). When cultures were maintained at the confluency packing density for three to four weeks, the cells had the unique tendency to assume capillary-like networks of cell cords around which neighboring cells showed polar orientation and migration towards the apparent tube-like structures without the need for added extracellular matrix. All cultured cells were stained positive with mouse anti-human factor VIII monoclonal antibody tagged with either Texas red-streptavidin or fluorescein isothiocyanate (FITC). All cultures were negative for staining with mouse monoclonal antibody to alpha actin tagged with FITC, suggesting absence of smooth muscle cells or fibroblasts. Cells were negligibly sensitive to argon laser irradiation at wave lengths 620 nm at 37 J/cm2. Cultured cells showed dose dependent sensitivity to both unactivated HPD and phycocyanin with minimal cytotoxicity (less than 20%) at concentrations below 0.5 and 50 micrograms/ml, respectively. Laser activation of the photosensitizers at these concentrations resulted in similar but significant cell death, 40% and 41% respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726332 TI - [The enzyme release by Trichophyton rubrum is dependent on nutritional substance supply]. AB - Enzymes liberated by growing dermatophytes are of pathogenetic importance in tinea. To investigate the influence of nutrients on this enzyme release, Trichophyton rubrum was grown in media containing peptone, keratin and lipids, to which glucose was added in separate assays. The culture supernatants were compared for extracellular enzyme activities by use of the api-zym-test. Our results clearly show that the extracellular enzyme activity is dependent on the nutrients supplied. Seven different enzymes were released when keratin was supplied, as compared to only 5 and 2, respectively, when lipids or peptone were available. Among these enzymes alkaline phosphatase and N-acetyl-beta glucosaminidase were detected in all cultures lacking glucose. Enzyme release was inhibited completely when glucose was added to the media, except for N-acetyl beta-glucosaminidase in peptone cultures. This dependency of enzyme release on fungal nutrition can be expected to occur in vivo, too. In addition, it has to be considered for in vitro cultural conditions. Alkaline phosphatase and acetyl glucosaminidase may be more important in tinea than has been assumed so far. PMID- 1726333 TI - Granulocytic colony-stimulating factors (G-CSF and GM-CSF) in the treatment of adult acute myeloid leukemia. AB - Large randomized trials have shown that granulocytic colony-stimulating factors (G-CSF and GM-CSF) may be interesting in vivo in promoting neutrophil recovery after high-dose myelosuppressive therapy in some clinical settings. However, any beneficial effect on survival was not yet demonstrated. Granulocytic CSFs act as growth and viability factors on myeloid leukemic cells and their use in acute myeloid leukemias (AML) is theorically not without risk. Paradoxically, these CSFs will maybe be usefull in the future as a part of AML treatment. First, the high early mortality rate in elderly AML patients after intensive chemotherapy should allow a demonstration of a CSF-induced improvement of survival. Secondly, GM-CSF should increase the efficacy of cell cycle dependent cytotoxic drugs based on recruitment of quiescent leukemic cells. Third, granulocytic CSFs should be used to induce programmed cell death in myeloid leukemic cells. PMID- 1726334 TI - Production of a monoclonal antibody (2B44) reactive on a shared epitope on dendritic reticulum cells, smooth muscle cells of vessels and Reed-Sternberg cells. AB - Lymph node cells from a patient with Hodgkin's disease (HD) were cultured without Epstein-Barr virus (EBV) or leukine adjuvant. A cell line (719-AB) emerged from the culture after four weeks. The cell line express CD20 (79%), CD 21 (30%), CD30 (63%), CD 35 (61%) antigens and weakly CD25 (19%). using Southern Blot technique, the existence of specific EBV DNA and polyclonal immunoglobulin genes rearrangement were observed in the cell line. In order to obtain a monoclonal antibodies (MoAb), mice Balb/C were immunized with this cell line. The splenic cells suspension of immunized animals were fused with the mouse myeloma NS1. Antibody IgM kappa from secreting clones 2B44 was studied using both indirect immunofluorescence with labeled anti-mouse immunoglobulin and immunohistochemistry based on alkaline phosphatase/antiphosphatase complex (APAAP) and ModAMeX technique on a panel of normal or pathological cells. Normal peripheral lymphocytes, monocytes, polymorphonuclear cells, and erythrocytes, did not react. The MoAb 2B44 recognized the dendritic reticulum cells and the smooth muscle cells of vessels on frozen section and paraffin section from HD or reactive lymph nodes. On specially processed paraffin sections (ModAMeX) Reed Sternberg cells (RSC) were reactive with 2B44 MoAb (in 2 cases out of 5 tested). The molecular weight of the antigen recognized by 2B44 MoAb is of 37 kd. The description of a new epitope shared by different histological components might be of interest for defining a new cluster and better understanding the nature of RSC. PMID- 1726335 TI - Structure of the human interferon alpha receptor. AB - The structure of the IFN alpha receptor has been studied by methods such as affinity crosslinking and gel chromatography over the last 8 years. The recent development of monoclonal antibodies against the receptor, and the cloning of an IFN alpha receptor cDNA has provided new important tools to understand the IFN alpha receptor structure. Thus, it has become obvious that the IFN alpha receptor has a more complex structure than first anticipated, probably involving more than one subunit. This review analyzes the present knowledge about the structure of the IFN alpha receptor, as well as many unresolved issues concerning this topic. PMID- 1726336 TI - [Agreement among observers in the classification of acute leukemias]. AB - This study was carried out to establish the level of concordance between two observers from two different health institutions in Mexico City, in the diagnosis of acute leukemias and their different varieties. We studied 73 consecutive cases of adults with these diseases. Each one of the two observers established their diagnosis on two occasions at least 15 days apart. They first made their diagnosis taking as a base the neoplastic cells morphology in bone marrow smears, and after that, with morphology plus specific cytochemistry. The outcomes of the two observers were also compared with the official diagnosis. Kappa test was performed to know interobserver and intraobserver concordance. The kappa values for the diagnosis myeloid/lymphoid were found among the highest (51 to 91). Weighted kappa was also applied to know the level of concordance in the diagnosis of the different varieties of acute leukemia, myeloid and lymphoid. In these cases the weighted kappa values were lower compared with the previous values (lymphoid, from 47 to 82; myeloid, from 30 to 66). Cytochemistry paradoxically was a confusing factor when it was used: in these cases the kappa values were lower (32 to 84) than morphology alone (39 to 91). The outcomes showed the subjective level in the diagnosis of the myeloid subtypes was more important in them than in the lymphoid subtypes. PMID- 1726337 TI - Studies on the 52 kDa antigen of Salmonella typhi: physicochemical stability, purification by affinity chromatography and immunochemical specificity. AB - The 52 kDa specific protein antigen of Salmonella typhi, as identified by monoclonal antibodies (Ekpo et al. 1990) has been studied with respect to its physicochemical stability, purification by affinity chromatography and immunochemical specificity. It was found that the 52 kDa protein was degraded into smaller antigenic fragments of MW 30-51 kDa when treated with acetone, ethanol, sodium thiocyanate, 0.3M sodium chloride and Veronal and Tris buffers. The exact chemical nature of the degradation of the protein under these conditions is not known but digestion by conventional proteases and dissociation of the non-covalent subunit type have been ruled out. It is proposed that the degradation may be the result of yet unidentified enzyme(s) which become activated by various physical or chemical treatments. Affinity chromatography using a specific monoclonal antibody has been carried out in an attempt to purify the 52 kDa protein. The binding of S. typhi protein to the column was saturable at 65.6 microgram protein/ml gel. The amount of S. typhi protein adsorbed on the column was 0.51% of the total sonicated cell protein. SDS-PAGE of the immunoadsorbent purified protein revealed bands at Mr 15-58 kDa, indicating that the protein obtained had been severely degraded. However, Western blot of the purified protein stained with a specific monoclonal antibody and with rabbit polyclonal antibody against S. typhi showed striking similarity, indicating that the protein obtained was close to immunochemical purity. The 52 kDa protein purified by affinity adsorbent was used as an antigen for the detection of specific IgM in sera of patients. It was shown that sera of patients infected with S. typhi as well as those infected with other bacteria, contained specific IgM against the 52 kDa protein. Thus, it appears that the 52 kDa protein contains species specific as well as cross-reacting epitopes. The possible development of specific diagnosis of S. typhi based on the present experimental results in discussed. PMID- 1726338 TI - High-dose intravenous immunoglobulin in the management of immune hemolysis in patients with thalassemic disease: factors which determine refractoriness. AB - We report our experience with high dose intravenous immunoglobulin (IVIg) in 3 thalassemic patients who had evidence of possible immune hemolysis. In 2 patients who had serious sepsis, their responses to IVIg were only partial and transient. The other patient who had marked splenomegaly had no evidence of response to IVIg. Both serious infections and large spleen may hamper the effect of IVIg and should be considered before IVIg is to be used in thalassemia. PMID- 1726339 TI - Molecules, maps and gradients in the retinotectal projection. PMID- 1726340 TI - Transgenic modelling of neurodegenerative events gathers momentum. PMID- 1726341 TI - GABA: an excitatory transmitter in early postnatal life. AB - In the adult mammalian CNS, GABA is the main inhibitory transmitter. It inhibits neuronal firing by increasing a Cl- conductance. Bicuculline blocks this effect and induces interictal discharges. A different picture is present in neonatal hippocampal neurones, where synaptically released or exogenously applied GABA depolarizes and excites neuronal membranes--an effect that is due to a different Cl- gradient. In fact, during the early neonatal period, GABA acting on GABAA receptors provides most of the excitatory drive, whereas excitatory glutamatergic synapses are quiescent. It is suggested that during development GABA exerts mainly a trophic action through membrane depolarization and a rise in intracellular Ca2+. PMID- 1726342 TI - Olfaction in Drosophila: genetic and molecular analysis. AB - Drosophila melanogaster has a sophisticated yet relatively simple olfactory system, the function of which can be studied in vivo by either physiological or behavioral methods. Several genetic and molecular approaches have been applied to isolating and characterizing genes required for the function or development of the olfactory system. Recent analysis of some of these genes is beginning to provide insight into their functions. PMID- 1726343 TI - Two distinct receptor subtypes for mammalian bombesin-like peptides. AB - The mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), are structurally related neuropeptides that elicit a wide spectrum of biological activities including regulation of smooth muscle contraction, stimulation of secretion, modulation of neural activity, and growth regulation. Earlier studies have shown that GRP and NMB are expressed in different regions of both the CNS and peripheral organs. Recent ligand-binding and molecular-cloning studies have revealed two pharmacologically distinct G protein-coupled receptor subtypes for mammalian bombesin-like peptides that have different relative affinities for GRP, NMB and bombesin receptor antagonists. Similar to the peptide ligands, the two receptor subtypes are expressed in a distinct but overlapping set of CNS regions, some of which have been identified in functional studies as sites where bombesin peptides elicit defined biological responses. Delineation of these peptide ligands and receptor subtypes will be important in future studies that explore the molecular basis for the heterogeneous nature of the responses to bombesin observed in mammalian systems. PMID- 1726344 TI - Polysialic acid on the surface of axons regulates patterns of normal and activity dependent innervation. AB - Studies of the cell-cell adhesion molecules NCAM and L1 have indicated that their combined action is an important determinant in establishing normal patterns of muscle innervation. Moreover, they participate in activity-dependent changes in axonal sprouting. Recent findings in vivo, however, suggest that the central variable in both events is not altered NCAM or L1 expression, but rather changes in the amount of polysialic acid (PSA) at the cell surface. This finding is consistent with the proposed role of PSA as a regulator of cell-cell interactions. Because these molecular entities are present in most of the nervous system, it is likely that this mechanism can influence many aspects of axonal behavior during development and regeneration. PMID- 1726345 TI - Human cytotoxic effector cells: definition and analysis of activity. AB - Studies have indicated that the complexity of NK and LAK activities goes beyond the question of which effector cell mediates most of this activity. Examination of Nk activity reveals that numerous effector phenotypes can mediate spontaneous, non-MHC-restricted cytotoxicity. These effector cells are predominantly the CD3-, Leu19 (NKH1)+ and the CD3+, Leu19 (NKH1)+ lymphocytes. However, the existence of other effector cells capable of mediating NK activity and the relative contribution of each type of effector cell in vivo situations requires further study. In addition, LAK activity has been attributed to numerous cell types, and an indepth examination of the regulation and recognition events involved in LAK activity indicates a greater degree of heterogeneity than was initially anticipated. The preliminary data presented above support the contention that although NK and LAK activities are quite similar with regard to their lytic properties (e.g. effector phenotype, target selection), their mechanisms of targets cell recognition and lytic attack appear to be quite different. Our investigation of recognition events indicated that IL-2-activated effector cells do not recognize the same structure that is recognized by freshly isolated NK cells. These data suggest that a recognition event occurs on activated cells that can be dissociated from the events involved in NK binding by fresh CD3- LGL. With regard to CD3+ effectors, the role of the TcR and the nature of the receptor mediating LAK activity require further study to determine whether multiple receptors or a single, unique receptor class are expressed after IL-2 stimulation. In addition, the role of IFN gamma and other regulatory cytokines must be further examined to determine their importance in human LAK activity. PMID- 1726346 TI - [Experimental concept of periprosthetic membrane neo-flap with axial vascular pedicle]. AB - This study was designed to demonstrate that progressive inflation of an expansion prosthesis placed under the epigastric pedicle of the rat induced the formation of vascular connections between this vessel and the periprosthetic membrane. 17 Wistar rats were operated under general anaesthesia. The 7 cc prosthesis was implanted into the inguinal fold and the valve was placed in a dorsal position via limited incisions. 11 rats were studied, divided into 3 series: 3 control rats (non-inflated prosthesis), a series in which the prosthesis was inflated up to 20 cc (at a rate of 5 cc per week) and a series in which the fate of the capsular flap isolated as a vascular island flap in a prefabricated silicone block was studied. The assessment was threefold: clinical, microradiographic after injection of lipiodol into the epigastric artery and histological. The clinical and microradiographic studies demonstrated the reality of the blood supply of part of the periprosthetic membrane and its connections with the epigastric axial pedicle. This membrane surface, constituting a real biological flap prefabricated by expansion, corresponds to the peripedicular zone (2 x 1 cm). The viability of the island flaps was confirmed after 8, 14 and 21 days. Histological studies confirmed the hypervascularisation and richness of the collagen fibres as a result of expansion. PMID- 1726347 TI - [Median cleft of the lower lip. Apropos of a case]. AB - The authors report a case of median cleft of the lower lip and mandible. This is rare malformation (60 cases published in the literature) with anomalies of the tongue and sometimes midline cervical cord. Cleft of the lower lip is repaired early between one and six months, but the correction of mandibular defect by bone graft will be done at the age of ten years. In this way the authors hope to preserve mandibular and teeth growth. PMID- 1726348 TI - [Treatment of buccal epidermoid carcinoma. Up-to-date review]. AB - This review systematically analyses all of the current data concerning the treatment of oral cancer. Each therapeutic modality involving surgery, radiotherapy, chemotherapy and immunotherapy is detailed and an exhaustive review of the literature about each therapeutic strategy is presented. PMID- 1726349 TI - [Use of injectable micro-implants in esthetic surgery of the face. Initial report]. AB - The authors present a series of 74 patients who underwent injections of a biphasic copolymer (Bioplastique) to improve the facial contours or to fill deep creases and folds. The follow-up period is ten months. This preliminary report is designed to define the possibilities of the new product. PMID- 1726350 TI - [Treatment of axillary burn scars by a scapular island flap with a vascular pedicle]. AB - Type 3 axillary burn retractions, involving the neck, the axilla, the upper arm and the lateral aspect of the chest, can cause difficult static and dynamic deficits altering the aesthetic appearance and the function of the shoulder. The posterior scapular area can provide an autonomized skin flap, based on the circumflex scapular vessels. This scapular flap, used as an island vascular flap, easily covers the anterior and posterior aspects of the axilla, obtaining a smooth skin surface, with a good underlying subcutaneous fatty tissue. Its elastic properties are well adapted to the dynamic requirements of the separate movements of the shoulder joint. Performed on 25 axillary burn retractions in 20 patients, this flap provided a convincing improvement in the shoulder range of movement and a return to a normal activity. PMID- 1726351 TI - [Value of a scapular flap in the treatment of axillary hidradenitis. Apropos of a clinical case]. AB - A severe case of axillary hidradenitis suppurativa, treated bilaterally by large excision followed by reconstruction with a pedicled scapular flap is presented. This reliable and thin fasciocutaneous flap provides good cover of large axillary defects with minimal donor site consequences. Its advantage is discussed versus the other surgical procedures used in this pathology for axillary reconstruction. PMID- 1726352 TI - [Necrotizing fasciitis of the upper limb. Apropos of a case]. AB - Necrotizing fasciitis is a rare infection, serious because of its risk of functional sequelae and mortality. The first clinical and histological descriptions of acute Streptococcal infection of the fascia were published in 1924 by Meleney, who referred to this condition as "Streptococcal haemolytic gangrene". However, since Meleney, numerous authors have reported cases of necrotizing fasciitis due to a wide range of bacteria. The authors report a case of necrotizing fasciitis of the upper limb in a 45 year old man following a superficial lesion of the dorsum of the hand. Despite the use of antibiotics, the morbidity and mortality of this disease are still high. Treatment is surgical and must be performed urgently. A review of the literature groups together the published cases and analyses the various aetiologies and risk factors. PMID- 1726353 TI - [War injuries of the femoral nerve. Apropos of a series of 27 cases]. AB - The authors report a series of 27 war injuries to the femoral nerve which represent approximately 1% of all of the war injuries to peripheral nerves operated in our department since the Islamic revolution in Iran in 1978 and during the 8 years of the war between Iran and Iraq. All of the victims presented with associated lesions: abdominal (24 laparotomies, including 10 colostomies) and/or vascular (3 iliac artery by-pass grafts). We divided the cases into 3 groups. In Group 1 (18 cases), the femoral nerve had a mean defect of 8 centimetres which was grafted. In Group 2 (4 cases), the nerve was simply compressed by fibrosis and/or metallic bodies and was released. In Group 3 (5 cases), the nerve trunk had a partial loss of substance which was grafted. The motor results were very satisfactory (M3 to M5) in the 3 groups with muscular recovery in about 2 years. PMID- 1726354 TI - [Treatment of loss of substance of the ankle or heel in adults. Apropos of a series of 88 cases]. AB - Cover of ankle and heel defects is always a difficult problem for the plastic surgeon. This complexity is due to the poor blood supply and the limited amount of surrounding soft tissue, as well as the high incidence of adjacent lesions. Apart from pressure sores in bedridden patients, skin defects in active patients, which require reconstruction to a state as close as possible to the premorbid state, are relatively rare. A plastic surgery unit is faced with this problem four or five times a year, on average. This low incidence means that each patient constitutes a particular case. There are no series in the international literature sufficiently large to establish any statistically valid data. The authors analyse a series of 88 cases of ankle or heel defects treated between 1982 and 1989 in the plastic surgery unit of CHU de Tours. They report their experience of the techniques used and define the indications in relation to the nature of the structures exposed and the weight-bearing or non-weight-bearing nature of the defect to be covered. PMID- 1726355 TI - [Internal plantar flap on external plantar pedicle: a technique for the coverage of the whole surface of the foot. Initial report]. AB - The authors present a new type of the classical medial plantar flap which derives its main blood supply from the medial plantar artery, a terminal branch of the posterior tibial artery. Our approach concerning flap dissection has not been modified, but the vascular supply of the flap has been shifted and a reverse blood flow coming from the lateral plantar artery is created after dividing the posterior tibial artery just proximal to its distal bifurcation. The reverse flow medial plantar artery island flap survival is ensured by rich anastomoses that usually occur between the dorsalis pedis and lateral plantar arteries. However, the venous return is also ensured by a reverse flow as in the case of lateral plantar artery. In addition, this flap presents a wide arc of rotation that is suitable for a variety of local defects, allowing cover of the foot. This new technique mainly provides a perfect indication for cover of distal weight bearing area defects of the foot when the distally based plantar flap is not possible for anatomical aberrations and/or in the presence of an unreliable blood supply of the flap itself. Moreover, it deserves a general description of a new type of flap pedicle design that we denote by the term "reverse flow Y-V extended flap pedicle". We intend to provide the readers with more details concerning clinical applications of this new surgical procedure. PMID- 1726356 TI - [May a general surgeon still operate breast cancer without the help of a plastic surgeon?]. PMID- 1726357 TI - Non-seminomatous germ cell tumours of the testis: game and set but not match. PMID- 1726358 TI - Single institutional experience with non-seminomatous germ cell tumours of the testis: local perspectives on a curable cancer. AB - We have reviewed 77 patients with Non-seminomatous germ cell tumour of the testis (NSGCTT) treated at a single institution. A residual mass following definitive treatment occurred in 16 patients (35%), 13 of whom had a resection of the mass, yielding active tumour in only one patient. Nine patients (12%) relapsed including four of the 14 with Stage I disease who were treated by orchidectomy alone. Four relapses occurred at more than two and a half years after primary treatment. Relapse prior to the development of clinical symptoms or signs was evident in three of nine patients; in two patients by routine imaging and one with elevated routine serum markers. Three of the nine patients who relapsed had elevated serum markers. Two patients died from disease but there were four treatment-related deaths (7%). Overall, 64 patients (83%) remain disease free at the time of follow-up. A further seven (9%) have been lost to follow-up but were disease-free at a minimum of 26 months after diagnosis. This study confirms features of this disease including the excellent prognosis when adequately treated. However, it also reveals the problems of late or marker negative relapses, the implementation of an observation policy in Stage I disease and treatment related mortality in young men. PMID- 1726359 TI - Recombinant alpha-2b interferon in patients with malignant carcinoid tumour. AB - Seventeen patients with malignant carcinoid tumour, ten of whom had the malignant carcinoid syndrome, were treated with recombinant alpha-2b interferon by subcutaneous injection (3 MU per dose) three times per week for a median of 12 weeks (range 4-48). No objective tumour responses were observed; however, there was a greater than 50% reduction in 24-hour urinary 5-hydroxyindolacetic acid (5 HIAA) excretion in four of ten patients (40%) with elevated pretreatment levels. Five of ten patients (50%) with flushing, five of seven patients (71%) with diarrhoea and both patients with wheezing experienced relief of symptoms. Three of four patients (75%) with weight loss as their only problem experienced weight gain. Responses occurred within the first eight weeks of treatment, but were generally of short duration. Toxicity occurred in all patients, and consisted mainly of fever, chills, anorexia, fatigue and weight loss. Four patients ceased therapy due to toxic reactions. Although interferon has activity against carcinoid tumours, its benefits are short-lived and toxicity limits its use with increasing dose. Patients with carcinoid syndrome appear to achieve the best therapeutic response, and it is likely that low doses (9-20 million IU weekly) are as effective as higher doses (36-72 million IU weekly). PMID- 1726360 TI - Interventional radiology--a review. PMID- 1726361 TI - [Basis of the functions of keratinocytes]. PMID- 1726362 TI - Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella virginia O:8. AB - The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure: [formula; see text] The disaccharide structure alpha-D-Abep-(1----3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of alpha-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum. PMID- 1726363 TI - Health in the Americas XX/XXI centuries. Perspectives in international health. AB - A case is made that a new frontier of international health is developing technical cooperation among countries of the Americas for narrowing and eventually filling the gap between them. Several initiatives of PAHO are cited. PMID- 1726364 TI - Prenatal care, screening, and complications. AB - Articles are reviewed that give the clinician new guidelines to diagnose neural tube defects without using amniocentesis. Cervical measurement using ultrasound as a tool to objectively evaluate and follow patients at risk for premature labor and incompetent cervix are reviewed. The utility of transabdominal versus transvaginal ultrasound is discussed. Two papers are presented that look at the use of aspirin in preeclampsia. One study looks at metabolic degradation of the prostaglandins associated with pregnancy-induced hypertension and shows that there is a heterogeneity in response to aspirin therapy. However, once the patient has pregnancy-induced hypertension, aspirin does not seem to be effective. A paper is presented that looks at the safety of autologous blood donation for both the mother and the baby and confirms its usefulness in obstetrics as in the nonpregnant patient. PMID- 1726365 TI - Nucleotide sequence of murine PCNA: interspecies comparison of the cDNA and the 5' flanking region of the gene. AB - Proliferating cell nuclear antigen (PCNA) RNA levels are regulated by transcription as well as changes in stability, in growing cells. We have cloned the murine PCNA cDNA and a fragment of the murine PCNA gene flanking the transcription initiation site. Comparison of the murine deduced amino acid sequence with the PCNA sequence from rat, human, Drosophila, Saccharomyces cerevisiae, and higher plants, reveals extensive homology between species. The homology is likely to be related to the fundamental role of PCNA as an auxiliary protein for DNA replication. Consensus sequences for transcriptional regulatory factors identified within 520 bp 5' of the cap site of the murine PCNA gene include: an inverted CCAAT site, an enhancer core element (EBP-1), three cAMP response elements (CRE-BP), one AP-2 site, three Sp1 sites, and two octamer sequences. The first 20 bp of the transcriptional unit are homologous to an initiator element, which may direct transcription from RNA polymerase II in the absence of a TATAA box. The consensus elements in the murine PCNA gene are similar in sequence and/or location to elements identified in the genes for human, Drosophilia, and yeast PCNA. PMID- 1726366 TI - [Determination of elastase and alpha 2-macroglobulin in bronchoalveolar lavage fluid in patients with interstitial diseases and its clinical significance]. AB - Determination of elastase and its inhibitor alpha 2-Macroglobulin was done in BALF in 40 patients with ILD (study group) and 10 healthy volunteers (control group). The results showed that BALF levels of elastase and alpha 2-macroglobulin in study group were significantly higher than those in control group (P less than 0.01; P less than 0.05). Correlation was found between BALF elastase levels and cell count (r = 0.3214, P less than 0.05). It was suggested that elastase may play a role in the development of pulmonary fibrosis in interstitial lung diseases. PMID- 1726367 TI - [A study on the inhibition effect of tetrandrine on the pingyangmycinum induced pulmonary fibrosis]. AB - A model of pulmonary fibrosis caused by a single intratracheal injection 0.1 mg pingyangmycinum in mice was reported. The effect of tetrandrine, a herbal powder, 50 mg/kg time, on the pathological development of pulmonary fibrosis, and the activity of superoxide dismutase (SOD), angiotensin converting enzyme (ACE), quantity of hydroxyproline (HYP) in this mice model were investigated. We also contrast tetrandrine with cortisol. The result showed that tetrandrine may inhibit the formation and development of pulmonary fibrosis. On the other hand, tetrandrine and cortisol may increase the activity of SOD of lung tissue, reduce the quantity of HYP of lung tissue and decrease the activity of serum ACE. PMID- 1726368 TI - Molecular epidemiology of Klebsiella pneumoniae. AB - Plasmid profile, restriction fragment length polymorphism (RFLP) patterned by an rRNA probe, and capsule serotyping of 143 Taiwan local bacteriemic isolates of Klebsiella pneumoniae were analyzed. Our results indicate that K1 and K2 are probably the most prevalent serotypes in Taiwan, accounting for 39% of total isolates tested. A remarkable genotypic heterogeneity was observed among these isolates by the analysis of rRNA gene RFLP patterns and plasmid profile, which highlighted differences that were not discerned by the serological tests used to differentiated these strains. On the basis of these analyses, 27 rRNA gene restriction patterns and 57 plasmid profiles were identified. PMID- 1726369 TI - Acute effects of iloprost on blood pressure, heart rate, renal hemodynamics, diuresis, natriuresis, plasma renin activity and plasma norepinephrine in uninephrectomized hypertensive mongrel dogs. PMID- 1726370 TI - [Bicolor peroxidase anti-peroxidase staining]. PMID- 1726371 TI - Effect of loading conditions on peak aortic flow velocity and its maximal acceleration. AB - Aortic flow measurement with Doppler echocardiography has become a non-invasive technique in clinical practice. In the present animal study, we evaluated the flow-derived parameters such as peak velocity (PV) and its maximal acceleration (MA) as indices of ventricular contractility independent of the loading status. Eight pentobarbital-anesthetized cats were maintained with artificial ventilation. The chest was opened to place an electromagnetic flow probe around the ascending aorta for recording pulsatile aortic flow. PV and MA were measured from the flow tracing and on-line electronic differentiation. Intravenous infusions of dobutamine (DT), angiotensin II (AII) and dextran (DN) were used to alter the cardiac inotropism, afterload and preload, respectively. At a steady state (approximately 5 min after infusion), DT increased the PV from 56 +/- 9 to 78 +/- 14 cm/sec (p less than 0.05) and MA from 1302 +/- 108 to 1699 +/- 117 cm/sec2 (p less than 0.05). In response to AII infusion, PV was slightly reduced (60 +/- 7 to 55 +/- 6 cm/sec, p less than 0.05) while MA was also reduced mildly but significantly (1219 +/- 109 to 1099 +/- 109 cm/sec2, p less than 0.05). Dextran infusion produced a marked increase in PV (48 +/- 7 to 82 +/- 13 cm/sec, p less than 0.05) while the increase was slightly less for MA (1089 +/- 95 to 1604 +/- 109 cm/sec2). The results indicated that inotropic stimulation markedly increased both PV and MA. PV and MA responded slightly but significantly to afterload alterations. (8.3% vs 9.8%, respectively). Both PV and MA increased markedly to the preload increment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726372 TI - Innervation of the knee joint in the rabbit and the Formosan rock-monkey (Macaca cyclopis): a retrograde HRP study. AB - The number and segmental distribution of cell bodies of sensory afferents and sympathetic efferent innervating to the knee joint of the rabbit and the Formosan rock-monkey were investigated using retrograde transport with horseradish peroxidase (HRP). After injecting HRP into the articular knee joint capsule of the rabbits, labeled neurons were found in the ipsilateral L4-S2 dorsal root ganglia (DRG). However, following injection of HRP into the articular cavity of the knee joint in the rabbit and the monkey, labeled neurons were found in both the ipsilateral DRG (L5-S2 and L4-S1 of the rabbit and monkey, respectively) and in the ipsilateral sympathetic ganglia (SG) (L4-S3 (rabbit) and L3-S1 (monkey)). The majority of labeled neurons within the DRG and the SG were composed of medium and large neurons in the monkey and the rabbit, respectively. The present findings suggest that the sensory projections from and sympathetic projection to the knee joint in rabbits and monkeys are similar, but that both projections of monkeys were "shifted" one segment cranially compared to the rabbit on both projections. PMID- 1726373 TI - [Common variable immunodeficiency (hypogammaglobulinemia of late onset or acquired hypogammaglobulinemia): initial follow-up of 11 cases]. AB - The present paper describes the clinical and laboratory follow-up of 11 patients with the diagnosis of common variable immunodeficiency. Their age varied from 8 to 45 years. The mean disease time was 12.6 years and mean diagnosis time 4.3 years. Infectious manifestations, mainly of the respiratory and digestive tracts, occurred in all patients. Polyadenomegaly was noted in seven, hepatomegaly in six, splenomegaly in five and arthralgia in four patients. All of them presented serum IgG less than 250 mg/dl. IgA less than 33 mg/dl and IgM less than 31 mg/dl, except one with IgM = 176 mg/dl. The isohaemagglutinin titers were less than 1/20 in all but one patient. The determination of the number of B lymphocytes in the peripheral blood revealed normal counts in three, elevated in one and decreased in five patients. The CD-4/CD-8 ratio was less than 1 in 8 and greater than 1 in three of them. Five patients had positive cutaneous late reactions to at least one of the following antigens: PPD, SK-SD (Varidase), Trichophytin and Levedurin (Candidin). A decrease of the proliferative activity of peripheral blood mononuclear cells stimulated by lectins (PHA, Con-A, PWM) was also noted. Natural killer function was decreased. The association a possible role of regulatory lymphocytes in the immunopathogenesis of this disease. The data presented here emphasize the diversity of clinical and immunological manifestations of this disease, which could be noted between diverse patients and in the follow-up of a single one. In our cases the disease had an evolutive character, with a primarily humoral dysfunction followed by cellular immunity disturbances that determined poorer prognosis and progressive difficulties in the therapeutics. We suggest a conceptual reevaluation of this condition and a new denomination, for instance "Late-Onset Combined Immunodeficiency". The long delay between the initial clinical manifestations of the disease and its diagnosis was a handicap for an adequate treatment. Early intervention could certainly decrease the morbidity and mortality of the disease. PMID- 1726374 TI - [Accidental hypothermia: glycemic, hematologic and blood amylase changes]. AB - In twenty patients with accidental hypothermia the plasma levels of amylase and glucose, hematologic aspects, and the response to the treatment were studied. The treatment was made at the Intensive Care Unit of the Medical Division of the Hospital of the University of Sao Paulo. The data showed that high levels of serum amylase without clinical manifestations may be observed in accidental hypothermia. Hyperglycemia was more frequently observed than hypoglycemia. Patients with body temperature lower than 30 degrees C may show leukopenia which returns to normal values when the temperature is corrected. The pneumonia and sepsis were the causes of death in accidental hypothermia. PMID- 1726375 TI - [Ionic, electrical and water balance in the animal cell. The system with active cation transport, Goldman's channels and the Na + K +2Cl-type symport]. AB - The relations between intracellular potassium, sodium water content and resting potential on the one hand and the ion transport parameters and intracellular electrical charge on the other hand were computed for a model of animal cell with a several ion transporters and variable intracellular charge. The case of the balanced ion distribution is considered. The results are presented in a graphical form. PMID- 1726376 TI - [Ion transport and cell proliferation]. AB - Recent studies of potassium fluxes and intracellular potassium content is mitogen activated cells have shown that the stimulation of G0----G1----S transition in arrested cell cultures in associated with both immediate (early) and prolonged (delayed) increase in potassium influx due to elevation of ouabain-inhibitable transport by Na,K-ATPase. The early and the delayed changes in ion transporters of plasma membrane can be disrupted, mechanisms of these changes being presumably different. The dissociation between the early and delayed ionic events were demonstrated in cell cultures activated to proliferate by growth factors, hormones, cAMP-elevating agents, as well as in the presence of cycloheximide. The early ionic events are related to the primary transduction of membrane signal, whereas the delayed modulation of ion transport via Na,K-ATPase has another function and is associated with cell growth. The increase in cell potassium content per gram of protein is typical of the successful G1----S transition in mitogen-activated cell cultures. PMID- 1726377 TI - [The transport and distribution of monovalent cations during the blast transformation of human peripheral blood lymphocytes activated by phytohemagglutinin]. AB - Potassium (rubidium) influx, sodium and potassium contents, as well as size distribution, DNA and protein contents and synthesis have been examined in PHA activated human lymphocytes within 0.5-72 h. A complex set of ionic events was found to include at least two stages of the increase in potassium and sodium contents per g cell protein and in ouabain-sensitive potassium influx which are preceded by a decrease in potassium content by almost 17% within the first 2-5 h. The kinetics of potassium and sodium changes has own pattern for each of cations, thus indicating definite changes in the ouabain-resistant transport of potassium and sodium during the G0----G1----S progression. The late increase in potassium content per g cell protein was found to correlate with the growth in cell size. This finding confirms the rule which was stated earlier for other animal cells, i. e. cells that prepare to proliferate are to raise their potassium per g cell protein up to the level of 0.8-1.0 mmole (Vereninov. Marakhova, 1986). PMID- 1726378 TI - Use of tumor receptors for diagnostic imaging. A review. AB - The article reviews briefly the binders which carry 'warheads' to tumors and bind with receptors. If the combination between binders and receptors is specific and tight, the warheads may accumulate only on/in the cells of the tumor but not in host tissues. The higher the specificity of the binder-receptor reactions, the higher is the therapeutic effect and the lower the side effects. The antibody is an effective binder, but only a fraction of the total activity administered stays in target cells (homing), usually less than 10 per cent. Another 90 per cent of the warheads are distributed in normal tissues or secreted outside the body. Very often injuries from irradiation or chemotherapy shorten the life span of the patients. Radioimmunoimaging does not yet yield better results than conventional imaging techniques, such as computed tomography, ultrasonography and angiography. PMID- 1726379 TI - The iconography in use for battle against drunk driving. AB - The origin of driving accidents is polyfactorial. If the mediocrity of the road network, the technical failures of the vehicles, the violation of the highway code represent an important percentage in the cause of accidents, the great purveyor remains "drunk driving" in the global number of accidents as well as in their importance. The public authorities have not only established laws in this matter so as to penalize alcohol consumption, but furthermore, have initiated a wide programme of verbal information (radio - television), written information (brochures) and iconographic information (posters) to sensitise public opinion on the risks of driving under the influence of alcohol. PMID- 1726380 TI - Correlation between IEF and SDS-PAGE phenotypes of keratins. PMID- 1726381 TI - The effects of cosmetic treatment on the IEF pattern of human hair keratins. PMID- 1726382 TI - [Value and limitations of urinary cytology in the diagnosis of bladder tumors]. AB - There were studied 222 patients with bladder tumors, 64 with operated transitional cell carcinoma and 166 with non-malignant diseases from cytologic and histologic point of view. Each patient was investigated by urinary cytology, endoscopy, and during the intervention biopsies were taken, which helped us to establish the histological form, the degree of differentiation and "T" element, using TNM system. All smears were stained by blue polychrome-tannin Dragan method and correlated with histological results which allowed us to appreciate cytodiagnostic as real positives, false negatives, real negatives and false positives. We signalled out some morphological aspects of malignant cells revealed by blue polychrome-tannin Dragan stain, which identified malignant elements in 87% tumors of the bladder G1, in 91.5% in G2 and 93.5% in G3; results were enhanced using washings of the bladder. There was some causes for false negatives results: a calcified tumor in its surface, tumors developed in vesical diverticulum, a limited exfoliation and a wrong interpretation of exfoliated cells. In patients transurethrally operated for tumors of the bladder the urinary cytology identified malignant elements in 22 of them; four presented tumors in the moment of endoscopic examination and 18 were considered as "apparently false positive"; eleven of them developed tumors in the next 6-24 months from the intervention. In patients with non-malignant diseases false positives results were related specially to urinary lithiasis and chronic renal failure. Results of initial and survey cytodiagnosis allowed us to increase the period between traditional cystoscopies. PMID- 1726383 TI - [Reconstruction of the medial canthus in oncology. Apropos of 105 cases over 9 years]. AB - Without forgetting that resection of a malignant tumour must always take priority over the problems of reconstruction, based on a series of 105 cases of medial canthus lesions, the authors believe that: for tumours with a reserved prognosis, particularly sclerodermiform basal cell carcinomas of the orbit, full thickness skin graft is the most appropriate technique; for cases with a good prognosis, the cosmetic result takes priority. An intersuperciliary island flap is currently the method of choice; for advanced cancers of the medial canthus, the combination of frontal flaps and nasolabial flaps is the procedure which gives the best aesthetic and functional results. This series includes three cases of radical orbital exenteration, including two because of sclerodermiform basal cell carcinomas, and the authors stress the very serious prognosis of these tumours in the orbit, especially in the presence of a recurrence in an irradiated territory. PMID- 1726384 TI - [Indications for bone autografts and allografts in cranioplasties. Apropos of a series of 51 cases, 30 of them with follow-up]. AB - Based on a series of 51 cranioplasties performed between 1978 and 1988 with follow-up of 30 cases, the authors define the respective indications for bone autografts and allografts. The various donor sites of autografts are reviewed, particularly in relation to the history of their use. Similarly, the history of the use of allografts is also reviewed and the various methods of sterilisation are considered. The authors review all of the literature on this subject and compare their results with those of other authors. PMID- 1726385 TI - [Failure and problems: apropos of a cranioplasty]. AB - The authors report a case of post-traumatic cranioplasty associated with various complications. They stress the difficulties in managing this type of reconstruction when a vast cerebral defect is associated with ventriculo peritoneal by-pass. PMID- 1726386 TI - [Value of fibrin glue in surgery of patients with severe burns]. AB - From January 1985 to May 1986, fibrin glue was used for graft sealing in 158 cases of our 200 skin grafts performed for the treatment of burns. When the graft area was less than 200 cm2, primary and complete healing was routinely observed. In the remainder, we noticed a higher quality of healing when fibrin glue was used compared to the other grafts. In 2 patients, infection of the wound was responsible for a total graft lysis which occurred immediately in the non-sealed grafts and was delayed in the sealed ones. Fibrin glue shortens skin graft healing time while it procures a better quality of life in patients with burns during in hospital stay. However the use of this healing-facilitating compound has to be limited to well-defined indications. PMID- 1726387 TI - [The muscular vascular index. Analysis of the vascularization of the muscles of the forearm]. AB - The results of an anatomic study based on 50 fresh adult cadaver upper extremities are analysed. All the arterial pedicles of each forearm muscle were counted, and each muscle was weighed. Each forearm contained an average number of 264 muscular pedicles. The relative mass fraction of each muscle was calculated, as was its "muscular vascular index" or MVI (number of pedicles divided by the weight of the muscle in grams). Half of the forearm muscles had a significant statistical relationship between their weight and their number of vascular pedicles. The other half had no statistical relationship. These two statistical muscular groups (with and without a statistical relationship) did not clearly correspond to anatomic or functional groups. No muscular group based on the average of MVI was found. Each of the 20 forearm muscle had finally its own characteristics of weight, number of pedicles, and MVI. The average MVI was 0.9 (from 0.4 to 1.8). The global muscular vascular index (GMVI) is the division of the total number of muscular pedicles of a forearm by the total muscular weight of this forearm. The average GMVI was 0.8 (from 0.4 to 1.6). In spite of its theoretical and practical limits, the MVI concept approximately reflects the high vascular density of the forearm muscles. PMID- 1726388 TI - [Arterial anatomy of peroneal flaps assessed by Doppler study and arteriography]. AB - Clinical applications of fasciocutaneous peroneal island flaps are important. 25 patients with cutaneous or composite defects of the lower extremity have been investigated by preoperative Doppler flowmeter and angiography. This before and after reviewed study allow the authors to establish a clinically useful peroneal arterial map which particularly concern the number and distribution of the postero-lateral septocutaneous arteries. The false positive and negative results of these two examinations are presented and explain differences with anatomic findings. Two anatomic variations are revealed by arteriography and are important for the surgical procedure. The first is the inconstant "circumflex peroneal artery" (12 cases on 22) which is the uppermost posterolateral cutaneous vessel which arises from the posterior tibial artery. The second is the angiographic view of a proximal bifurcation of the peroneal artery (9 cases). Regarding these findings, the authors classify the indications of preoperative angiography: absolute, useful, useless according to each case. PMID- 1726389 TI - [The gracilis muscle as musculocutaneous flap. Evaluation of 20 cases]. AB - The use of gracilis as muscular or myocutaneous flap is very well-known. The authors report 20 cases of gracilis flap including 13 reconstructions of the vaginal cavity following extended abdomino-perineal resection. Some technical points concerning the localization of the cutaneous part of the flap and the pedicle dissection are discussed. The use of gracilis flap is still limited in surgical teams following extended abdomino-perineal resection, nevertheless it is a very useful flap because of its low morbidity, the shortening of patient hospitalization and the very satisfying aesthetic result of the neo-vaginal cavity. PMID- 1726390 TI - [One-stage columellar reconstruction. Use of a vascularized double labial flap]. AB - The author describes a technique for one-stage repair of the columella which he has used since 1984. The procedure is based on the anatomical concept of the venous drainage of the columellar region and philtrum. Two vascularised para-alar flaps are raised from the philtrum then raised 90 degrees with an equivalent horizontal rotation. The donor site is closed by primary suture with no cicatricial sequelae. A columellar support can be inserted between the two flaps. The long-term results were considered to be good in every case. PMID- 1726391 TI - [Septal deposition in difficult rhinoseptoplasties. Surgical techniques. Experience. Results]. AB - The authors describe their septoplasty technique of performing an extramucosal dissection with excision, revision and replacement of the cartilaginous septum during difficult rhinoplasties. They analyse the main septal deformations which may benefit from this technique and the functional and esthetic results. PMID- 1726392 TI - ["Shark tooth" in the excision of so-called giant cell circumdigital tumors of the tendon sheaths. Results apropos of a series of 74 patients]. AB - The authors propose an easily extended incision around the finger for giant cell tumors of the tendon sheath: two distally based triangles, one palmar, one dorsal, joining at the midlateral line. A V-Y plasty allows further enlargement and decreases the excess of skin bulkiness. Used in circumferentially extensive tumors, a decreased recurrence rate (4%) was noted compared to the classical approach (15%). PMID- 1726393 TI - [Crossed leg expanded flaps]. AB - The authors report their experience of three cases of crossed leg expanded flaps. They describe the operative technique and discuss the advantages and disadvantages. Prior expansion of the crossed leg flap reduces the donor site scar. PMID- 1726394 TI - [Dermoid cyst of the scalp. Apropos of a case]. AB - A subgaleal dermoid cyst of a 32 years old white adult is reported. The lesion appeared at the age of several years and ten regularly. Clinical examination, complementary exams as well as the result of the operation are reported. Embryology, histological classification and the main clinical, laboratory and histological characteristics of such lesions are defined by the review of the literature. Subgaleal dermoid cyst is rare in childhood and exceptionally occurs in adults. Only six cases of subgaleal dermoid cyst of adult are mentioned in the literature. Most of the time, the lesion is situated on the anterior fontanelle. The treatment is surgical and the prognosis is excellent. PMID- 1726395 TI - [Supernumerary mammary gland. An unusual case]. AB - The authors report a rare case of supernumerary mammary gland resulting in a double breast appearance. An embryological explanation for this anomaly is proposed. PMID- 1726396 TI - [Changes of lysosomal and digestive enzymes in rat caerulein pancreatitis]. AB - We evaluated the changes of lysosomal and digestive enzymes in the exocrine pancreas after caerulein induced acute pancreatitis in rats. The serum amylase levels and water content as well as pancreatic amylase and cathepsin B contents increased significantly in the early stage (0-12 h) after caerulein was administered, however, returned to the normal levels at 36 h. In the early stage, colocalization of lysosomal enzyme and digestive enzyme was found. Histologically, in the early stage, there were remarkable changes such as acinar cell vacuolization and interstitial edema, but these changes disappeared at 36 h. Furthermore, amylase and cathepsin B outputs decreased significantly in the early stage (12 h) but at 24 h, these increased significantly. LDH discharge from dispersed acini and cathepsin B leakage from lysosomes also increased in the early stage (0-12 h), but these values returned to the normal levels at 36 h. These results indicate that exocrine pancreas needs about 36 h to recover from the caerulein induced acute pancreatitis, and in this recovering process, secretion of colocalized digestive enzyme and lysosomal enzyme seem to play an important role. PMID- 1726397 TI - Design, development, and interpretation of HIV neutralization assays. AB - In developing therapeutic reagents for the control of HIV infection, it is necessary to screen candidate products in vitro for their ability to reduce or neutralize viral infection. Although the current literature describes numerous neutralization assays, no universally accepted standards have been adopted. In this article, we briefly review the available neutralization assays and describe in detail the methods we have selected in our laboratory for the screening and characterization of reagents with potential anti-HIV properties. After evaluating many different technical protocols and experimental procedures, we have found the syncytium inhibition and syncytial focus assays to be particularly useful and have found p24 gag antigen production to be an excellent objective measure of HIV infection under a variety of conditions. These assays proved reproducible and sensitive and are suitable for use in the majority of laboratories. PMID- 1726398 TI - Broadly reactive antibody-dependent cellular cytotoxic response to HIV-1 envelope glycoproteins precedes broad neutralizing response in human infection. AB - To determine if and when the antibody-dependent cell-mediated cytotoxic (ADCC) response of human serum exhibits broad reactivity across HIV-1 strains, multiple sera were tested for their ability to mediate ADCC against target cells infected with recombinant vaccinia vectors expressing envelope genes of HTLV-IIIB or HTLV IIIRF. These vectors were found to express the envelope glycoproteins of the two HIV-1 strains and so were appropriate targets for ADCC assays. All the HIV-1 positive sera were able to mediate ADCC against both HTLV-IIIB and HTLV-IIIRF envelope-expressing targets at similar titer. In sera from early seroconverters, the ADCC response was again broadly reactive, even in those sera that exhibited strain-specific neutralizing antibody responses. The ADCC response to natural infection with HIV-1 is therefore broadly reactive and precedes the development of a broad neutralizing antibody response. The broad reactivity of HIV-1-specific ADCC responses may be important for protection against cell-associated virus in vaccine development. PMID- 1726399 TI - Antibody response to reverse transcriptase in cats infected with feline immunodeficiency virus. AB - The antibody response in cats to feline immunodeficiency virus (FIV) reverse transcriptase (RT) was followed for 3 years. Eight of the nine cats used in this study produced reverse transcriptase-inhibiting (RTI) antibodies. Relative inhibitory means of 2.9%, 18.4%, 33%, and 47% were found 6, 12, 24, and 36 months, respectively, after infection with FIV. The enzyme activity was suppressed by greater than or equal to 78% with the use of 100 micrograms of FIV associated IgG. The RTI antibodies were FIV-specific, as they did not inhibit other mammalian retroviral polymerases, including feline leukemia virus RT. An RT inhibition assay with sera in the presence of protein A and immunoblot analysis showed that antibody binding to FIV RT protein p62 is independent of antibody ability to block enzyme activity. Viral RT released by detergent-treated virus was stable for more than 6 weeks at 4 degrees C, whereas its activity was reduced by 50% after 2 weeks at 37 degrees C. Because significant concentrations of RTI antibodies are detected only at 1 to 2 years after infection, they can be used to determine the approximate time of virus infection and as a marker for disease progression. PMID- 1726400 TI - Demonstration of the in vitro antiviral properties of bovine lymphokine-activated killer (LAK) cells. AB - In cattle, cells with functional characteristics similar to those of natural killer (NK) cells are difficult to detect. However, lymphokine-activated killer (LAK) cells can be detected readily after in vitro activation of peripheral blood mononuclear leukocytes (PBML) with interleukin-2 (IL-2). In the present study, we demonstrated that IL-2-activated PBML preferentially lyse bovine herpesvirus type 1 (BHV-1)-infected cells and that the cell responsible for the lysis copurified with the cell responsible for lysis of K562. The IL-2-activated effector cells were capable of significant reducing virus production. The reduction in virus yield seemed to be by an interferon (IFN)-independent mechanism, as the amount of IFN induced in effector cells by BHV-1 was not altered by the addition of IL-2. Furthermore, enrichment of cytotoxic cells by passage through nylon wool columns removed the capability of PBML to produce IFN in response to the virus. These results suggest that activation of LAK mechanisms in cattle plays a role in controlling virus spread. PMID- 1726401 TI - Zinc and copper of fetal organs during the second trimester of pregnancy. AB - In fetus with a mean gestational age of 18 weeks (range 15-25, n = 14), zinc and copper concentrations in liver, femur, rib, and skeletal muscle were measured. Zinc and copper concentrations are highest in liver. A trend of decreasing liver zinc concentrations during gestational age is suggested. Zinc concentrations are significantly correlated with copper concentrations in liver and in femur, suggesting steady growth in both organs. Femur zinc values rank ca. 30% of those in liver, femur copper, ca. 2%. Zinc or copper concentrations in rib are of the same levels as in skeletal muscle. Their concentration for zinc ranks ca. 20%, for copper, ca. 5% of the values in liver. All zinc and copper values are lower than reported in third trimester fetal organs. Calculated zinc/copper molar ratios are distinctive for the various organs: in liver, 6 +/- 1, in femur, 73 +/ 8, and in soft tissues, 26 +/- 3. Calculated ratios from published values obtained from the third trimester of pregnancy show that the ratios in liver and skeletal muscle maintain these levels. The zinc/copper molar ratio can serve as an internal reference in zinc and/or copper measurements. PMID- 1726402 TI - Effect of sex and time of sampling on selenium and glutathione peroxidase activity in tissues of mature rats. AB - Sprague-Dawley rats were used to investigate variations in measures of glutathione peroxidase (GSH-Px) and selenium (Se) concentration resulting from diurnal cycles and sex. Mature rats (equal numbers of males and females) were killed at 4 h intervals over a 48 h period (0200, 0600, 1000, 1800 and 2200 h each day). Selenium and GSH-Px were measured in plasma, erythrocytes, and liver and kidney cytosols. Selenium concentrations did not vary diurnally, but plasma GSH-Px activities were higher during the light than dark periods. Males had greater plasma GSH-Px activities and Se concentrations (42 EU and .45 mg/kg, respectively) than females (35 EU and .41 mg/kg respectively). GSH-Px activities were also higher in male kidney cytosols than females (117 and 76 EU, respectively). Selenium and GSH-Px activities, however, were lower in male liver cytosols (.48 mg/kg and 272 EU) than females (1.19 mg/kg and 795 EU, respectively). These data suggest that Se is distributed differently in male and female rats and the difference in Se distribution is accomplished by differences in GSH-Px activities. PMID- 1726403 TI - Inhibition of copper absorption by zinc. Effect of histidine. AB - Copper and zinc interact at the intestinal mucosal level, affecting copper absorption. Amino acids, such as histidine, may affect the absorption of these two elements by chelating these cations. The two mechanisms could have additive potential. This possibility was investigated using a duodenal-jejunal single-pass perfusion procedure in anesthetized rats. Copper absorption and tissue retention from solutions containing 0.1 mM copper were determined in the presence of either no zinc or equimolar zinc, or at a zinc/copper ratio of 10/1, either without histidine or with histidine at a 10/1 or 20/1 ratio to copper. Copper removal from the intestinal lumen was decreased by zinc, and further reduced by increasing concentrations of histidine. There was a greater accumulation of copper in the small intestine, reaching a maximum with a 10-fold excess of histidine. With zinc at a 10/1 ratio to copper, the addition of a 10- or 20-fold molar excess of histidine further decreased the net uptake of copper from the perfusate while greater copper accumulation in the tissue occurred. Histidine thus enhances the inhibitory effects of zinc on copper absorption, suggesting the application of convergent mechanisms for diminishing copper uptake. This could be relevant for the treatment of Wilson's disease. PMID- 1726404 TI - Teratogenicity of Ni2+ in Xenopus laevis, assayed by the FETAX procedure. AB - The teratogenicity of Ni2+ was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In seven assays, beginning at 5 h postfertilization, groups of Xenopus embryos were incubated for 96 h in media that contained Ni2+ (added as NiCl2) at concentrations ranging from 1 x 10(-7) to 3 x 10(-3) mol/L; control groups were incubated in the same medium without added NiCl2. At 101 h postfertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to determine their developmental stages, malformations, and head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 7%. Malformations were found in greater than 95% of embryos exposed to Ni2+ concentrations greater than or equal to 5.6 mumol/L. The most frequent malformations in Ni(2+)-exposed embryos were ocular, skeletal, and intestinal deformities; less common malformations included facial, cardiac, and integumentary deformities. Other abnormalities, not categorized as malformations, included stunted growth, dermal hypopigmentation, and coelomic effusions or hemorrhages. The median embryolethal concentration (LC50) of Ni2+ was 365 (SE +/- 9) mumol/L; the median teratogenic concentration (EC50) was 2.5 (SE +/- 0.1) mumol/L; the Teratogenic Index (TI = LC50/EC50) was 147 (SE +/- 5), indicating that Ni2+ is a potent teratogen for Xenopus laevis. Experiments in which Ni(2+) exposures were limited to specific 24 h periods showed that Xenopus embryos were most susceptible to Ni(2+)-induced malformations on the second and third days of life, during the most active period of organogenesis. PMID- 1726405 TI - Interaction of dietary calcium, manganese, and manganese source (Mn oxide or Mn methionine complex) on chick performance and manganese utilization. AB - Two trials were conducted to determine the utilization of manganese (Mn) as influenced by the level and source of Mn and the level of dietary calcium (Ca) in broiler chickens. Trial One was a 2 x 2 x 3 factorial arrangement of two Mn sources (Mn methionine or manganous oxide), two levels of dietary Ca (1.8 or 1.0), and three levels of supplemental Mn (30, 60, or 200 mg/kg) fed until 4 wk of age. Total phosphorus (available phosphorus) levels were 0.70% (0.48%) during all ages. High levels of dietary Ca caused a slower early rate of growth (0.53 vs. 0.64 kg) for chicks fed 1.8 vs 1.0% Ca, respectively. Chick weight was equivalent for all diets within the Ca-treatment group, except the dietary combination of high Ca and 200 mg/kg Mn as Mn methionine. Bone and liver Mn were significantly increased as the Mn level increased, but were not affected by the Mn source. Chicks fed 1.8% Ca had higher levels of bone Mn (9.28 ppm) than chicks fed 1.0% Ca (7.23 ppm). High levels of dietary Ca and 200 ppm Mn methionine dramatically depressed early growth, feed intake, and bone ash in this trial, raising the question of a diet x environment (heat-stress) effect. Trial Two was a 2 x 2 factorial arrangement of two levels of dietary Ca (1.8 or 1.0%) and two Mn sources (200 mg/kg Mn as Mn methionine or MnO) up to 3 wk of age in a controlled heat-stress environment. No growth depression in the chicks fed high levels of Ca and Mn methionine was observed. In the presence of high levels of dietary Ca, bone Mn was significantly higher when chicks were fed the MnO source. In summary, dietary Ca did not decrease Mn utilization in these trials, and availability of Mn in Mn methionine as a source compared to MnO depended on dietary Ca levels. PMID- 1726406 TI - Zinc in teeth from children with and without cystic fibrosis. AB - This study analyzes Zn concentration levels in teeth from children with and without cystic fibrosis with respect to different variables, namely: gender, age, type of teeth, area, fluoridation of water supply, term of pregnancy, maternal smoking habit, and maternal drinking habit. The method of analysis is proton induced X-ray emission on crown samples. Atomic absorption spectrometry (AAS) studies on some of these samples show close correlation to the PIXE data. In general, the concentration level of zinc in teeth of children greater than 10 years old is less than the concentration level in teeth of children aged 10 years or younger for children who took nontetracycline antibiotics. Breast feeding mixed feeding-bottle feeding show no significant difference in the zinc concentration in teeth. PMID- 1726407 TI - The effect of various dietary zinc concentrations on the biological interactions of zinc, copper, and iron in rats. AB - Three groups (14 rats each) were fed one of the following diets for 8 wks: a control purified basal diet containing 12 ppm zinc, 5 ppm copper, and 35 ppm iron; the basal diet with less than 2 ppm zinc; or the basal diet supplemented with 1000 ppm zinc. Rats fed the zinc-deficient diet had decreased weight gain, moderate polydipsia, and intermittent mild diarrhea. The zinc-supplemented rats had a cyclical pattern of food intake and weight loss from weeks 5 to 8. Tissue concentrations suggest that zinc and copper were not mutually antagonistic with chronic dietary imbalances. If tissue element concentrations reflected intestinal uptake, then competition and/or inhibition of intestinal uptake occurred between zinc and iron. The fluctuations in tissue element concentrations that occurred with increased duration of the study were at variance with previous studies of shorter time periods. The dietary proportions of zinc, copper, and iron appear to influence zinc, copper, and iron metabolism at the intestinal and cellular transport levels over a given period of time. PMID- 1726408 TI - Effect of dietary iron deficiency on mineral levels in tissues of rats. AB - To clarify the influence of iron deficiency on mineral status, the following two synthetic diets were fed to male Wistar rats: a control diet containing 128 micrograms iron/g, and an iron-deficient diet containing 5.9 micrograms iron/g. The rats fed the iron-deficient diet showed pale red conjunctiva and less reactiveness than the rats fed the control diet. The hemoglobin concentration and hematocrit of the rats fed the iron-deficient diet were markedly less than the rats fed the control diet. The changes of mineral concentrations observed in tissues of the rats fed the iron-deficient diet, as compared with the rats fed the control diet, are summarized as follows: . Iron concentrations in blood, brain, lung, heart, liver, spleen, kidney, testis, femoral muscle, and tibia decreased; . Calcium concentrations in blood and liver increased; calcium concentration in lung decreased; . Magnesium concentration in blood increased; . Copper concentrations in blood, liver, spleen and tibia increased; copper concentration in femoral muscle decreased; . Zinc concentration in blood decreased; . Manganese concentrations in brain, heart, kidney, testis, femoral muscle and tibia increased. These results suggest that iron deficiency affects mineral status (iron, calcium, magnesium, copper, zinc, and manganese) in rats. PMID- 1726409 TI - Nephrotoxicity and neurotoxicity in humans from organogermanium compounds and germanium dioxide. AB - There is no known biological requirement for germanium (Ge), germanates, or any organogermanium compound. Ge deficiency has not been demonstrated in any animal. The estimated average dietary intake of Ge in humans is 1.5 mg/d. Ge is widely distributed in edible foods, all of which, with few exceptions, contain less than 5 ppm Ge, since higher levels are toxic to most plants. Ingestion of Ge compounds has been shown to produce toxic effects in experimental animals. In recent years inorganic germanium salts and novel organogermanium compounds, such as carboxyethyl germanium sesquioxide (Ge-132) and lactate-citrate-germanate (Ge lactate citrate) have been sold as "nutritional supplements" in some countries for their purported immunomodulatory effects or as health-producing elixirs, resulting in intakes of Ge significantly exceeding the estimated average dietary intake. Since 1982, there have been 18 reported cases of acute renal dysfunction or failure, including two deaths, linked to oral intake of Ge elixirs containing germanium dioxide (GeO2) or Ge-132. In these cases, biopsies show vacuolar degeneration in renal tubular epithelial cells, without proteinuria or hematuria, in the absence of glomerular changes. Serum creatinine levels have been well above 400 mumol/L in such patients. In 17 of 18 cases, accumulated elemental Ge intakes reportedly ranged between 16 to 328 g over a 4-36 mo period, or between 100 to 2000 times the average estimated dietary intake for human. In surviving patients, renal function improved after discontinuation of Ge supplementation. However, in no case was recovery complete. One organogermanium compound, an azaspiran organogermanium compound, 2-aza-8-germanspiro[4,5] decane-2-propamine 8,8-diethyl-N,N-dimethyl dichloride (spirogermanium), has been found to cause both neurotoxicity and pulmonary toxicity in phase I and II studies examining its chemotherapeutic potential as an antitumor drug in the treatment of various malignancies. In cancer patients given the drug spirogermanium, 40% experienced marked, yet transient neurotoxicity. Two patients suffered from pulmonary toxicity. Results of phases I and II human cancer trials for spirogermanium have not been favorable, with the exception of moderate benefits for three types of malignancies. It is recommended that patients exposed to long-term (greater than 3 mo) Ge supplementation at levels well above the estimated daily intake be medically supervised and monitored for potential renal-, pulmonary- or neurotoxicity. Further study regarding the mechanism of Ge-induced nephrotoxicity in human is warranted. PMID- 1726410 TI - Changes in pulmonary Cu-Zn contents in superior mesenteric artery occlusion shock of rabbit. AB - Contents of Cu and Zn of into-pulmonary blood (IPB), out-pulmonary blood (OPB), Lung tissue, and supernatant and macrophages of Lung Lavage were determined in superior mesenteric artery occlusion (SMAO) shock of rabbits. Zn of pulmonary tissue was 11.42 +/- 0.60 and 14.52 +/- 1.78 (micrograms/g wet wt) in SMAO shock and control groups, respectively. Content of Zn was found to be lower, Cu was not changed, and Cu/Zn ratio increased in lung tissue in SMAO shock. Contents of Cu and Zn in other samples were not changed. The results suggest that lower Zn in lung tissue related to acute lung injury. PMID- 1726411 TI - A preliminary report on the intervention trials of primary liver cancer in high risk populations with nutritional supplementation of selenium in China. AB - The purpose of this study was to evaluate the effect of selenium (Se) in the prevention of human primary liver cancer. Three intervention trials were conducted among the residents at high risk to primary liver cancer (PLC) in Qidong county, Jiang-su province, the People's Republic of China. This area has the second highest rate of PLC in China. One trial was undertaken among the general population in a township with supplement of table salt fortified with 15 ppm anhydrous sodium selenite (Se-salt) for 5 y and the other four townships with similar PLC incidence rate served as the controls using normal table salt. The second trial was undertaken among hepatitis B virus surface antigen carriers (HBVsAg+) receiving supplement of 200 micrograms Se in form of selenized yeast (Se-yeast) daily vs placebo for 4 y. The third trial was carried out in members of families with high PLC incidence using Se-yeast (200 micrograms of Se daily) vs placebo for 2 y. The results showed that nutritional supplement of Se could reduce the PLC incidence significantly. PMID- 1726412 TI - Population survey of thyroid autoimmunity in Italy. Three year follow up. AB - Thyroid autoimmunity and primary hypothyroidism have been evaluated in a 3 year interval in 4 samples of general population from Bologna, Napoli, Palermo, Rome and Cagliari. The average prevalence of Mic-Abs was in males 4.1% and in females 10%. A significantly higher prevalence of Mic-Abs was found in the Cagliari sample where it reaches in males 6.1% and in females 20.4% (p less than 0.0005). Likewise Tg-Abs have been found increased in Cagliari (males: 4.6% vs 10.7%; females: 10.0% vs 16.6%; p less than 0.01). In the 3 year follow up Mic-Abs titre increases significantly whereas the Tg-Abs titre remains substantially unchanged. In the initial screening 1.65% of subjects were found to be latent (1.2%) or overt (0.45%) hypothyroids. Hypothyroidism was particularly frequent in the Cagliari sample and was constantly associated with Mic-Abs. In the 3 year follow up only 1 of 8 latent hypothyroids became overt. PMID- 1726413 TI - Intervention policy in endemic goitre areas. AB - The multifactorial nature and complex interactions of regionspecific environmental conditions with host factors in the pathogenesis of endemic goitre constitute a major challenge to the understanding and control of the problem in endemic areas. However, to control and prevent this important public health problem, the most obvious but difficult initial step requires substantial socioeconomic improvements in the affected areas of less developed countries, including, first, provision of efficient iodine prophylaxis programs, second, diversification of dietary constituents with adequate daily protein-calorie intake, and third, institution of proper sanitary conditions with effective water treatment to eliminate organic and bacterial pollutants. This last intervention is also a requirement to control and prevent goitre in the iodine-sufficient more developed countries. In this regard, more research is needed to provide effective ways of water treatment that can be applied in individual households or at the community level. PMID- 1726414 TI - Three years of experience of the congenital hypothyroidism National Register. AB - Mental retardation caused by congenital deficiency of thyroid hormones can be prevented by early diagnosis and therapy which are assured by neonatal thyroid screening. Congenital hypothyroidism screening is performed in Italy by regional centres which in 1989 have screened more than 82% of neonatal population. Since 1987 a National Register of children affected by CH has been instituted. The results of the analysis of data collected in the first three years are reported. PMID- 1726415 TI - Physiopathology of thyroid cysts in euthyroid nodular goiter. AB - Following the introduction of echography it has became evident that a large proportion of nodules (15-37%) in euthyroid nodular goiter is entirely or partially cystic (20% in the A. experience). It is thought that cystic areas are subsequent to hemorrhagic necrosis occurring during goitrogenesis and therefore the term "pseudocysts" seems to be preferred. The article summarizes some hypothetical mechanisms of pseudocyst formation. In pseudocysts of recent onset a direct or indirect viral or bacterial etiology is suggested; in longstanding pseudocysts a deficient angiogenesis or immunotoxic mechanisms are proposed. PMID- 1726416 TI - The clonal origin of thyroid nodules in euthyroid nodular goiter. AB - Prevalence of euthyroid nodular goiter varies widely according to the endemia of the region studied. The goiter hyperplasia under goitrogenic stimuli progresses in the formation of nodules. Presently the pathophysiology of the thyroid nodules' formation in euthyroid nodular goiter is unknown. The A. reviews the two current hypothesis on the clonal origin of thyroid nodules. According to one hypothesis the nodule issues as the result of a coordinated replication of a cluster of follicles with an increased growth potential. According to the second hypothesis the formation of new follicles during goitrogenesis is due to a sprouting of one or a few cells from the follicles shell; nodules formation is the result of post-necrotic fibrosis and uneven growth. PMID- 1726417 TI - Advances in pathogenesis of goitre. AB - Growth regulation of the thyroid has been reinvestigated using an ex vivo system of isolated porcine thyroid follicles. Not only the direct effect of TSH, EGF, IGF I as well as iodine on growth of these follicles has been investigated but also the paracrine communication of these follicles with endothelial cells and fibroblasts. The results of recently published investigations with this culture system are summarized in this article. We could demonstrate that IGF I and EGF have a dose related effect on thyroid cell proliferation, whereas TSH has no effect on thyroid cell growth, if the iodine content of follicles is kept normal. Saturation of thyroid follicles with increasing amounts of iodine (1-40 microM K1) inhibit dose dependent EGF, IGF I or fetal calf serum induced thyroid cell proliferation. Inhibition of iodide organification with PTU or MMI abolish the growth inhibitory effect of iodide indicating that an organified iodinated product is responsible for the growth inhibitory effect of iodide on thyroid cell proliferation. Thyroid follicles secrete a FGF like substance which stimulates the growth of fibroblasts as well as endothelial cells. The secretion of FGF into the culture medium is decreased during TSH stimulation and enhanced during stimulation with EGF. The pretreatment of follicles with iodide abolishes the growth promoting effect of conditioned medium from thyroid follicles on fibroblasts and endothelial cells. We conclude that local growth factors like IGF I and EGF are responsible for thyroid cell proliferation whereas TSH stimulates specific function and hypertrophy of thyroid cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726418 TI - Thyroid growth stimulating immunoglobulins in sporadic and endemic colloid goitre. AB - Several methods have been described to measure immunoglobulins stimulating the growth of thyroid cells in vitro, the so called Thyroid Growth stimulating Immunoglobulins (TGI). The methods make use of either Ig-stimulated guinea pig thyroid (organ) cultures (the cytochemical bioassays; growth is measured via Feulgen-densitometry or pentose shunt analysis), the culture of Ig-stimulated rat or porcine thyroid follicles (growth is measured via 3H-thymidine incorporation), or of Ig-stimulated rat FRTL-5 cells (growth is measured via 3H-thymidine incorporation, or counting the number of mitoses in arrest). The various methods have now been validated: (a) the data obtained with TGI preparations in the Feulgen Cytochemical Bioassay (CBA's) correlate well with those obtained in the FRTL-5 mitosis arrest assay with the same TGI preparations, (b) the growth stimulation can not be ascribed to hormonal contaminations of the used Ig preparations, since protein A-sepharose purified IgG is active in the assays and since anti human IgG's neutralize the growth stimulating effects of the preparations. Using the assays TGI has been found positive in goitrous Graves patients, in sporadic goitre patients and in endemic goitre patients. Particularly, patients complaning of recurrent goitre after thyroidectomy or with recent goitre growth are TGI positive. TGI occurs in sporadic goitre in the absence of TSH-receptor antibodies. The autoimmune-prone animal model of the BB rat also proved to be TGI-positive; the animal shows an increased thyroid glandular weight. Both the histomorphology of human goitres as well as the goitre of the BB rat indicate that the dendritic cell plays a prominent role in initiating the thyroid autoimmune reaction. PMID- 1726419 TI - Purification and biological properties of vasculotropin, a new angiogenic cytokine. AB - Angiogenesis is a key step in organ development and remodeling during embryogenesis or tissue regeneration. Some pathological events such as tumor growth or diabetic retinopathy also lead to angiogenesis formation. Several molecules have already been identified as promoting angiogenesis in vivo. Whether their bioactivity is mediated by other angiogenic growth factors or not is still unclear. We identified and purified recently a new angiogenic growth factor. Its unique specificity for vascular endothelial cells led us to provisionally name it vasculotropin (VAS). We describe the biochemical properties of VAS and its biological functions. Structural data showed that VAS is related to the SIS family. In vivo VAS was recognized as an inducer of angiogenesis and vascular permeability. In vitro, despite a moderate action on proliferation, VAS strongly stimulates the cell migration. The screening of the presence of cellular receptors and VAS production showed that the cells which bind VAS do not synthesize it, whereas the cells which synthesize VAS do not bind it. Thus, VAS seems to act through a paracrine pathway. We also present data suggesting that VAS has a lymphokine activity. PMID- 1726420 TI - Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number. AB - Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726421 TI - Comparative analysis of four parameters involved in puffing activity along chromosome arm 2L of D melanogaster. AB - Autoradiographic and immunofluorescent techniques have been used to analyse the relationship between puffing, transcription and occurrence of DNA/RNA hybrids in D melanogaster salivary gland chromosome 2L. Experiments of 3H-uridine incorporation have indicated that similar rates of RNA synthesis are observable in well developed puffs as well as in some diffuse bands and interbands. On the other hand, puffs of similar size incorporate 3H-uridine at quite different rates. The presence of RNA polymerase II seems to follow a coincident pattern with that of 3H-uridine incorporation. Our results indicate that the rate of transcription does not determine either the formation of a puff or its potential size. Instead, we have found a positive correlation between the amount of DNA/RNA hybrids and puff size, independently of the transcription rates. Transient accumulation of transcribed RNAs in their chromosomal compartment could therefore play a relevant role in the determination of puff size. PMID- 1726422 TI - [Assessment of induced noninvasively postextrasystolic potentiation on the right ventricular function in patients with cor pulmonale]. AB - The indices of postextrasystolic potentiation degree (Rpesp: Rcdz/dt, RHI, delta Q-Zc, delta PEP/RVET) were observed in the patients with cor-pulmonale, chronic obstructive pulmonary disease (COPD) and normal subjects using extrasystole induced by transesophageal atrial pacing combined with differential impedance rheopneumography. The results shows that Rpesp of the patients with cor-pulmonale was larger than that of the patients with COPD and the normal subjects. The positive rates of RCdz/dt, RHI, delta Q-Zc, delta PEP/RVET was detected respectively in 95%, 100%, 85%, 85% of the cor-pulmonale and in 60%, 60%, 70%, 60% in COPD. It suggested that this method be a safe, simple and reliable one to assess right ventricular contraction function and to diagnose early cor pulmonale. PMID- 1726423 TI - [Nonradioactive molecular hybridization]. AB - Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The new technology of non-radioactive probes (cold probes) allows the routine use of this method outside of the specialized molecular biology laboratory. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in biological fluid or in a biopsy tissue. Hybridization leads to the formation of a double-stranded molecule called hybrid or duplex, which can be detected with a great sensitivity by using high energy radioisotope. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, radiolysis of the probe and the radioactive hazards. In order to overcome autoradiography delay and the drawbacks of techniques employing radioisotopes, various non-radioactive labels have been proposed. Labeling and detection systems are currently designed in two ways: The label molecule can be attached directly to the DNA probe (direct labelling) or it can be attached to a molecule which binds either to a modified probe or specifically to the duplex (indirect labeling). Various substances has been used to label directly a nucleic acid probe. The assay detection limit depends largely on the detection limit of the label, therefore, probe assay based on label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). In the indirect labeling, the probe cannot be detected alone; rather, it requires the addition of a detection system.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726424 TI - 3-Hydroxy-3-methylglutaric aciduria: a possible pitfall in diagnosis. PMID- 1726425 TI - Novel mammalian cyclins (CYL genes) expressed during G1. PMID- 1726426 TI - A CD strategy for the study of polypeptide folding/unfolding. A synthetic foot and-mouth disease virus immunogenic peptide. AB - The circular dichroism spectrum of the 20-residue immunogenic peptide from the foot-and-mouth disease virus (VP1; 141-160 of serotype A, subtype 12) was solvent and temperature-dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left-handed extended helix, the alpha-helix, and the 3(10) helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D-NMR studies. A new mechanism for the stabilization of left-handed extended helices and destabilization of alpha helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X-ray crystallography. PMID- 1726427 TI - Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi. AB - In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the D forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13 psi (CH2NH)Leu14-bombesin(6-14) and Leu13 psi(CH2NH)Phe14-bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I-Tyr4-bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3095) was slightly more potent in these assays than D-Trp6,Leu13 psi (CH2NH)Leu14 bombesin (6-14)(RC-3125). Nevertheless, D-Trp6,Leu13 psi (CH2NH)Phe14-bombesin (6 14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726429 TI - Periodontal basics for the general practitioner. PMID- 1726428 TI - Perio made basic. PMID- 1726430 TI - Periodontal examination, diagnosis and treatment. PMID- 1726431 TI - The general dentist/periodontist team--an unrecognized synergy? PMID- 1726432 TI - Using tooth movement to reshape periodontal structures. PMID- 1726433 TI - The periodontal approach to implant dentistry. PMID- 1726434 TI - Implant maintenance. PMID- 1726435 TI - The ailing implant. PMID- 1726436 TI - Current trends in soft tissue. PMID- 1726437 TI - Periodontal flap surgery. PMID- 1726438 TI - Guided tissue regeneration in periodontal therapy. PMID- 1726439 TI - Looking for a quick fix. PMID- 1726440 TI - Monoclonal antibodies to chick renal calcium regulating hemeproteins. Biochemical and immunohistochemical interactions with mitochondrial 25-hydroxyvitamin D3 hydroxylases. AB - The kidney mitochondrial monooxygenases known as 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases are two analogous enzymes which utilize the vitamin as a common substrate for the catalytic production of 1 alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3. These two enzymes are complexes of NADPH ferredoxin reductase, and an (Fe-S)-cluster containing ferredoxin with a redox potential that allows the ultimate transfer of reducing equivalents to the terminal oxidases distinctly known as cytochromes P-450(1) alpha and P-450(24). We have used in vitro immunizations of splenocytes obtained from mice sensitized with the purified cytochrome P-450(1) alpha to generate three hybridoma clones from fusion with p3 x 63.Ag8.653 myeloma ATCC cells which selectively secrete monoclonal antibodies (MAbs) of the IgM class. The MAbs have been partially purified by ammonium sulfate fractionation followed by size separation chromatography on Sephacryl S-200-HR. We have compared the structural similarities and differences between the two kidney enzymes in Western Blot analyses using horseradish peroxidase conjugated goat anti-mouse Igs specific for the heavy and light chains of mouse IgA, IgG and IgM. The MAbs from all three clones recognized and interacted with apparent common epitopes of the two hydroxylases but selectively discriminated against liver microsomal P-450LM2 type and adrenal mitochondrial P-450SCC cytochromes. The cytochromes P-450(1) alpha and P-450(24) were detected as two separate bands with approximate molecular weights of 57 and 55 KDa, respectively. In reconstitution of hydroxylase activities in vitro, the MAbs were equally effective in inhibiting the 1 alpha hydroxylation and 24R-hydroxylation reactions. The ratio of micrograms of Igs to pmol cytochrome P-450 for a 50% inhibition of either activity was approximately 25. These results, collectively, seem to suggest the existence of a precursor product relationship between the kidney mitochondrial 1 alpha- and the 24R hydroxylases, or perhaps, a common ancestral origin. Immunochemical peroxidase anti-peroxidase staining of kidney tissue first exposed to the MAbs revealed that only the proximal tubular segment of the nephron was specifically enriched with the cytochromes. PMID- 1726441 TI - Epitope binding of human thyroglobulin autoantibodies of different IgG subclasses. AB - Autoantibodies against thyroglobulin in patients with Hashimoto's thyroiditis are believed to recognize only three main epitopes. The possibility that antibodies of different IgG subclass recognize these separate epitopes has been suggested by preliminary studies with monoclonal antibodies against thyroglobulin. To assess whether this is a more general phenomenon, sera from 9 Hashimoto patients were fractionated into subclasses IgG1, IgG2 and IgG4 by negative selection on affinity columns. Binding of each subclass to thyroglobulin both in a competition ELISA and an assay of radiolabelled thyroglobulin immunoprecipitation was significantly inhibited by the other subclasses. These results indicate that there is no restriction of thyroglobulin epitope recognition by different IgG subclasses in unselected patients with autoimmune thyroiditis. PMID- 1726442 TI - The effect of inhibitors of platelet aggregation on the metabolism of platelet activating factor (PAF) in washed rabbit platelets. AB - The rabbit platelet metabolizes platelet-activating factor (PAF) intracellularly. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I2 (PGI2), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI2) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor. PMID- 1726443 TI - Platelet-activating factor (PAF) in allergic diseases: inhibitory effects of anti allergic drugs, ketotifen and three kampo medicines on PAF production. AB - Platelet-activating factor (PAF) is considered an important mediator in allergic and inflammatory reactions. To further investigate the role of PAF in allergic diseases, we examined the in vitro and ex vivo effect of anti-allergic drugs on PAF production by human neutrophils. Ketotifen and three Kampo medicines, which are widely used in the treatment of allergic diseases in Japan, inhibited PAF production by normal human neutrophils dose-dependently, whereas disodium chromoglycate (DSCG) and tranilast did not have any inhibitory effect. Ketotifen also suppressed ex vivo PAF production by neutrophils from healthy subjects. PAF production decreased from 40.5 +/- 5.0 units to 21.6 +/- 4.7 units after oral administration of 2 mg of ketotifen per day for one week. We also measured the concentration of lysoPAF in serum from patients with Japanese cedar pollinosis during the pollen season and followed the effect of ketotifen on lysoPAF levels in the serum from these patients. We found that the concentration of lysoPAF, a precursor and degradation product of PAF, in patients with cedar pollinosis (87.8 +/- 8.7 units) was significantly higher than in healthy subjects (54.7 +/- 7.7 units). After two weeks, lysoPAF levels decreased significantly (82.6 units to 41.3 units) only in the group treated with nasal aerosol and ketotifen. These results suggest that PAF may play a role in allergic diseases and that the clinical effects of anti-allergic drugs may at least partially be due to their anti-PAF action. PMID- 1726444 TI - [The level of spontaneous phage production and sensitivity to melioidosis phages of museum cultures of Pseudomonas pseudomallei]. AB - The phage-producing activity and sensitivity of museum cultures of a melioidosis agent to melioidosis phages have been studied. Most cultures show spontaneous phage-production, though its level differs in strains. Some of the melioidosis cultures demonstrate lack of phages. The fact that these strains have different sensitivity to chloroform lysate of cultures producing phages indicates their possible lysogenic state. Electron microscopy has shown the presence of at least tow morphological types of phages in P. pseudomallei C-141. These data confirm existence of polysogeny in the melioidosis agent. PMID- 1726445 TI - [The use of a CHO cell culture in studying Vibrio cholerae grown under different conditions]. AB - Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells. PMID- 1726446 TI - [Human fibroblast interferon therapy alone and human fibroblast interferon combined with thymostimulin in genital papillomavirus infection associated with cervical intraepithelial neoplasia]. AB - Eighteen patients suffering for HPV infections associated with CIN were treated using human fibroblast interferon, via an intramuscular route, with positive results. Five cases of recurrent HPV within nine months of treatment received thymostimulin therapy, via an intramuscular route, with positive results. PMID- 1726447 TI - [AIDS in the world]. PMID- 1726448 TI - Hyperploid cells in the limb buds of rats and 5-azacytidine-induced digital deformities. AB - To clarify the mechanism underlying the digital teratogenesis of 5-azacytidine (5 AC), the DNA contents of mesenchymal cells were examined in the limb buds of the rat by microspectrophotometry. 5-AC induced hyperploid cells: 2.4% at 18 h after injection and 3.2% at 24 h. No hyperploid cells were noted 30 h after the treatment. Because posttreatment caffeine suppressed 5-AC-induced digital defects, the actions of caffeine on 5-AC-induced hyperploid cells were further studied; no hyperploid cells were detected at any sampling point. Caffeine suppressed hyperploid cells as well as digital defects. These results suggest that the causative factors of digital defects by 5-AC may involve hyperploid cells. However, in that there were too few hyperploid cells to cause the defects, further studies are required to clarify the nature of the action of 5-AC. PMID- 1726449 TI - Effect of different intravenous nutrients on upper gastrointestinal secretion in rats. AB - Total parenteral nutrition has been advocated for nutritional support of patients with proximal small bowel fistula. Data on the direct effects of different nutrients on upper gastrointestinal secretion are controversial. Therefore, we examined the effects of glucose, amino acid, fat and mixed parenteral solutions on gastric juice secretion, hydrochloric acid and pepsin content, duodenal juice secretion, and pancreatic protein, amylase, bilirubin and bicarbonate content in thirty rats with chronic gastric and duodenal fistulas. Gastric secretion was increased by glucose solution (D10W) and by amino acid solution (5% AA) (13% and 12% increase respectively) but not to significant degree by fat emulsion (2% F). Both D10W and 5% AA caused comparable increase in hydrochloric acid secretion (D10W: +34%; 5% AA: +28%) and the pepsin secretion (D10W: +37%). The single infusion of hypertonic glucose, amino acids and fat were associated with decreases in duodenal fistula volume (p less than 0.05) or content output. Mixed nutrients decreased both gastric secretion (volume: -28%; HCl: -19%; pepsin: 65%) and duodenal juice volume (p less than 0.05), protein and amylase content (p less than 0.05). The elevated bilirubin in reduced volume of duodenal juice caused by mixed nutrients may be responsible for bile sludge formation. This study indicates that the use of parenteral nutrition can provide full nutritional support while decreases the fistula output. PMID- 1726450 TI - Hodgkin's disease. A historical perspective. AB - Two hundred and sixty-four patients with Hodgkin's disease (HD) forming the basis of our 15 year experience are retrospectively analyzed. Three therapeutic periods are recognizable. 1. The 1974-76 period was characterized by the increasing knowledge of staging procedures and therapeutic approaches. The 81 patients treated in this period experience 67 and 60% survival at 5 and 10 years, respectively. 2. The 1977-80 period was characterized by a large combination of MOPP and radiotherapy. The 87 patients who entered this period experienced 75 and 72% survival at 5 and 10 years, respectively. 3. The last therapeutic period 1981 84 is characterized by the increasing relevance of prognostic factors and alternating the use of MOPP and ABVD as non-cross resistant regimen. The 96 patients who entered this period showed 96% survival at 4 years. Both survival and disease-free survival were positively influenced by the change of therapeutic strategies during the three periods (p less than .005). Although better results have been recorded moving from one to the next therapeutic period, the present policy has been also based on the recognition of a high number of late complications due to therapy. Preliminary results about the present therapeutic experience seem to indicate both a good remission rate and low incidence of complications. PMID- 1726451 TI - 3T3 cells in adipocytic conversion. AB - 3T3 are murine cells of an established heteroploid cellular line. Some clones of this cellular line, when cultured under adequate conditions differentiate into adipocytes. During the process of differentiation, the cells undergo a change from the elongated fibroblastic shape to a round or oval form and accumulate small drops of lipids within their cytoplasma. These lipid drops fuse into one large drop which displaces the nucleus towards the periphery, giving the cell the aspect of a mature adipocyte of white adipose tissue. The cells not only change their morphology, but they also present important biochemical changes. They show a simultaneous increase in triglyceride synthesis and activity of lipogenic enzymes. There is also an increase in the response of the activity of various hormones and the de novo synthesis of the receptors to such hormones, as insulin and ACTH. During the process of differentiation important changes occur in the synthesis of various proteins, such as actin, tubulin, and other proteins which also make up the cellular cytoskeleton, forming part of the lipid transportation within the adipose cell. The adipocytic differentiation of 3T3 cells depends on adipogenic serum factors used in the supplementary culture medium. These adipogenic factors seem to play an important role in the development of adipose tissue. There are hormones, chemical agents and serum factors which modulate adipocytic differentiation. The clone must be susceptible to adipocytic differentiation, it must reach a quiescent state and find itself in adipogenic conditions for the 3T3 cells to differentiate into adipocytes. It must also carry out an DNA synthesis which is an expression of the new phenotype. The differentiation of 3T3 cells in terminal. The fact that these cells present an adipocytic conversion under physiologic conditions and with adipogenic hormones which exist in the whole animal has been demonstrated. All of these characteristics show that the 3T3 cells may be used as an adequate experimental system to analyze the events which occur during the differentiation and development of adipose tissue. PMID- 1726452 TI - [An experimental study on the pathogenesis of pulmonary edema in acute pancreatitis, with special interest to the effects of colloid infusion and the role of endotoxin]. AB - This experimental study was undertaken to clarify the role of pancreatic enzymes and endotoxin in the pathogenesis of pulmonary edema in acute pancreatitis, paying special attention to the effects of two different intravenous infusions: lactated Ringer's solution (LR) and Dextran 40 (D40). After acute pancreatitis was induced in dogs by injecting autologous gallbladder bile into the main pancreatic duct, plasma endotoxin levels increased markedly in both the LR and D40 groups, and PaO2 decreased more significantly in the D40 group. Extravascular lung water (EVLW) increased more significantly in the D40 group than in the LR group, in spite of the fact that colloid-hydrostatic pressure gradient (CHPG) had been maintained more efficiently in the D40 group. Significant correlation between EVLW and plasma endotoxin level was delineated in both groups, but the slope of the regression line in the D40 group was much greater than that of the LR group. Infusion of trypsin and elastase into the pulmonary artery in normal dogs caused moderate elevation of EVLW in the D40 group, but there was no significant alteration in the LR group. The changes of PaO2, EVLW, and CHPG after infusion of endotoxin were similar to those in the animals with experimental acute pancreatitis. In conclusion, endotoxin appears to play an important role in the pathogenesis of pancreatitis-induced pulmonary edema by causing an increase in pulmonary vascular permeability, and under these circumstances the infusion of large amount of colloid solution promotes the development of pulmonary edema. PMID- 1726453 TI - [Changes of acinar cells in the pancreato-biliary duct ligation with exocrine pancreatic stimulation model in rats; protective effects of a new potent protease inhibitor, ONO3307]. AB - To evaluate the changes of pancreatic acinar cells in the pancreatic duct obstructed animals as well as the protective effects of a new potent protease inhibitor, ONO3307, we measured the serum amylase levels, pancreatic water content, histological changes, lysosomal fragility in in-vitro incubation, cathepsin B distribution in acinar cells, and cathepsin B and amylase output into pancreatic juice after short-termed (3 hrs) pancreatic duct obstruction with caerulein (0.2 micrograms/kg.hr) infusion in rats. Serum amylase levels, pancreatic water content, and lysosomal fragility in duct obstructed with caerulein infused animals were significantly increased compared with the control groups, and remarkable shift of cathepsin B from lysosomal fraction to zymogen fraction was observed in this group. These changes tended to continue 24 hours after removal of duct obstruction. But with infusion of ONO3307, these changes observed in duct-obstructed with caerulein infusion groups were significantly, almost completely attenuated. These results indicate the intimate relationship between the pathogenesis of acute pancreatitis and lysosomes and some known proteases which are inhibited by ONO 3307 and suggest the usefulness of such a kind of protease inhibitor in the treatment of acute pancreatitis. PMID- 1726454 TI - Pancreatic lysosomal enzyme secretion via gut-hormone-regulated pathway in rats. AB - To explore the secretory profiles of lysosomal enzyme in pancreatic juice, we stimulated the secretion of lysosomal enzyme by intravenous pancreatic secretagogues and intraduodenal instillation of liquid meals in rats. Lysosomal hydrolases, such as cathepsin B, are secreted from the apices of pancreatic acinar cells via a hormone-regulated pathway, as in the secretion of pancreatic digestive enzymes. The intravenous infusion of the cholecystokinin analogue caerulein, or the intraduodenal administration of nutrients results in a closely related secretion of both amylase and cathepsin B from the apices of acinar cells, suggesting that they are discharged from the same presecretory compartment (zymogen granules). Lysosomal enzymes appear to enter into the secretory compartment as a result of malsorting, but the cause of this anomaly is not known. We found small amounts of lysosomal enzymes colocalized with digestive enzymes within zymogen granules in normal acinar cells and in normal pancreatic juice, suggesting some physiological roles of lysosomal enzymes in pancreatic ducts. Furthermore, lysosomal enzymes appear to play important roles in the pathogenesis of pancreatic disease, such as pancreatitis, from both inside and outside the pancreas, since cathepsin B can probably activate trypsinogen. PMID- 1726455 TI - Enzymatic fragment substitution as a tool in protein design. AB - An easy and rapid enzymatic method is described which allows replacement of P' residues in bovine pancreatic trypsin inhibitor. Insertion of Xaa-Arg or Xaa-Lys into a BPTI fragment lacking P1' = Ala16 and P2' = Arg17 was carried out in a "one pot" reaction catalysed by trypsin in the presence of 80% 1,4 butanediol. PMID- 1726456 TI - The kit receptor and its ligand, steel factor, as regulators of hemopoiesis. AB - Mouse strains carrying mutations at the Dominant White Spotting (W) locus or the Steel (Sl) locus are anemic and display defects in pigmentation and gametogenesis. In W mutants the anemia is due to a deficiency of hemopoietic stem cells and, in Sl mutants, to a deficiency of supporting stromal cells in the bone marrow. The W locus encodes the c-kit proto-oncogene product, a cell surface receptor with protein-tyrosine kinase activity, and the Sl locus encodes its ligand, a hemopoietic cytokine known variously as Steel factor (SLF), mast cell growth factor, stem cell factor, and Kit ligand. SLF can synergize with a number of other cytokines to stimulate growth of hemopoietic progenitors in vitro and stimulates blood cell production in vivo in animals. Here we review the biological activities of SLF, with particular emphasis on its effects on hemopoietic stem and progenitor cells. We also discuss present knowledge of the molecules involved in SLF-triggered signal transduction, and speculate on potential therapeutic applications for SLF in human disease. PMID- 1726457 TI - Early prognosis of survival or death after a recent stroke by blood levels of acute-phase proteins. AB - From 129 patients with a recent stroke 105 survived and 24 died within 3 weeks from stroke-onset. At around 40 hours after the latter, the blood-levels of the acute-phase proteins ceruloplasmin and albumin did not forecast the death of the respective patients, but, in contradistinction, the level of fibrinogen was significantly higher in those who eventually died, than in those who survived. Therefore, a higher level of fibrinogen could be a risk-factor for death after stroke. PMID- 1726458 TI - Liver transplantation at the University of Pittsburgh, 1984 to 1990. AB - Patient and primary graft survival for 2,090 patients who received primary liver transplants at the University of Pittsburgh from 1984 through 1990 are presented. Observed (actual) 3- and 12-month patient and primary graft survival rates were compared for 3 periods: 1) January 1984 to September 1987 (cyclosporine, OKT3, and Euro-Collins preservation period); 2) October 1987 to December 1988 (University of Wisconsin solution preservation period); and 3) January 1989 to December 1990 (FK506 period). Data for results according to age group, medical urgency, and primary diagnosis are provided. In addition, estimated survivor and cumulative hazard functions (life-table method) for patient and primary graft survival out to 60 months after transplantation are presented. Overall results have improved significantly in recent experience. Most notable are the improved results seen in liver transplantation for patients with biliary atresia (especially in infants), primary sclerosing cholangitis, fulminant hepatic failure, and chronic active hepatitis B. For all but a few conditions, most of the mortality after liver transplantation occurred in the first 3 months after surgery. Less than 2% of patients were lost in each 6-month interval beyond the first 6 months after transplantation. Outcome was related to patient condition at the time of surgery. Observed survival rates at 3 and 12 months for patients called in the hospital to receive a transplant were 88.6% and 86.5%, respectively, compared with 81.9% and 73.7% for patients in critical condition. The continuing shortage of organs for transplantation, which often forces patients to wait longer for an organ than they can afford to, continues to impose a significant penalty. PMID- 1726460 TI - HLA class I epitopes accounted for by single residues. AB - We examined the serological reaction of 50,000 HLA antisera for correlations with amino acid substitutions at 16 variable residues on the A locus and 8 variable residues on the B locus. We identified antisera that produced mutually exclusive allelic reactions corresponding to amino acid variants at the various residues. We assumed that by this procedure, epitopes that were determined by a single amino acid substitution could be identified. Among 55 variable residues on the A locus, antibodies that reacted in an allelic fashion were found for 16, or 32%. These serologically determined epitopes were on the alpha helix for 13 of the 16 residues, and 3 were in the beta sheet. Among 54 variable residues for the B locus, antibodies were found to 8 which reacted in a mutually exclusive pattern. Thus, 13% of the residues were serologically defined. Sixteen "monospecific" HLA specificities were determined by a single residue. Interestingly, many "multispecific" sera could be accounted for by reactions to a single amino-acid defined epitope. We assume that most of the other monospecific and multispecific specificities are determined by conformational epitopes, as described in an accompanying article (Ch. 33). PMID- 1726459 TI - Orthotopic liver transplantation at Addenbrooke's Hospital Cambridge 1968 to 1991. AB - Liver grafting is now established as the optimal treatment for patients with end stage parenchymal liver disease. Twenty-three years of experience at a single center is presented. The 1-year actuarial patient survival rate for all cases transplanted in Cambridge has now risen from 10% in 1968 to 1970 to 80% in 1990 to 1991. Increasing numbers of patients are being referred for transplantation with an ever-increasing range of indications being developed. Many inborn errors of metabolism can now be cured by liver grafting. There is still, however, considerable scope for improvement in many areas of patient treatment from operative and postoperative care to long-term immunosuppressive management. Much remains to be done to minimize early sepsis- and rejection-related deaths and late immunosuppression-related morbidity. PMID- 1726461 TI - Association of high sensitization to the structure of HLA class I alleles. AB - Some HLA Class I alleles or groups of alleles can be explained by exclusive residues which may explain their structural uniqueness. However, many sera are directed to alleles whose structural uniqueness is explained only by a combination of nonexclusive residues. A program implementing a combinatorial search algorithm was developed to analyze serological data by associating the primary structure of specific alleles to reaction patterns of broad multispecific sera. The method determines the residue or residues whose associated alleles best describe the serum specificity. The raw data reaction patterns direct the search, which utilizes known primary sequence information of HLA Class I alleles. Three hundred highly positive sera from parous females were analyzed; 74 were highly correlated to the A locus and 125 to the B locus. Some of the remaining sera were associated with multiple loci. Reorganization methods were applied to the data. Clusters of residues associated with certain alleles focus attention on specific locations on the HLA Class I molecule. These regions may be included in, or in close proximity to, the serological determinant. Serological descriptions of certain broad specific sera have been termed "public epitopes." Specificities associated with distinct conserved regions of the Class I molecule offer a molecular basis for many public epitopes. Similar reaction patterns to 3 serologically allelic regions were found in pregnancy allosera, monoclonal antibodies, and transplant recipient sera. The correlated areas were: positions 166 and 167 of the alpha 2 domain alpha helix on the A locus, positions 79-83 of the alpha 1 domain alpha helix on the A locus, and positions 80-83 on the A and B loci. PMID- 1726462 TI - HLA epitopes and graft survival. AB - 1. The fraction of patients and graft survival was provided for amino acid residues, heptapeptides, and conformational epitopes with HLA specificities of up to 7 antigens mismatched. 2. Mismatching large numbers of serologically defined residues was detrimental to graft survival, whereas nonserologically defined residues did not have an effect. 3. The difference in graft survival for those with a few and those with many conventional A,B,DR mismatches was doubled by counting serologically defined peptide epitopes. 4. The attempt to create conformationally defined epitopes by taking adjacent variable residues did not provide a good correlation with graft outcome. PMID- 1726463 TI - International Cell Exchange, 1991. AB - 1. During 1991, lymphocytes from 40 individuals of various races and 20 cultured B-cell lines were sent to 290 laboratories (listed in appendix) for typing. The results of the blind tests were analyzed and reported monthly. This is a summary of the year's 10 monthly exchanges. 2. Ten A-locus antigens showed 95% or greater agreement among laboratories, and 100% agreement was achieved for A2 and A3. Agreement of 95% or more was reached for 7 B-locus specificities. Both Bw54 and Bw73 showed marked improvement in detection during the last 3 years. 3. Six laboratories did not miss the assignment of any A- or B-locus antigens. The false positive and false-negative assignment rates are given for the A- and B-loci. 4. The following variants were studied: B5-53 variants from various races, BN21, B7x40 (DT), and B7x27. 5. Aw66 assignment was found to be discrepant between the United States and European laboratories. 6. The A28 splits, Aw68 and Aw69, were studied, as were cells with different Aw68 splits. 7. Cell lines were typed by 150 laboratories, including 19 DNA laboratories. Six Class II antigens had 95% or higher detection levels, and 5 others had higher than 90% detection levels. 8. Both DNA and serology laboratories had difficulty in assigning the DRw6 splits, DRw13 and DRw14. The definition of the splits of DR3, DQw1, and DQw3 was clarified with the additional DNA typing results. 9. Amino acid sequencing confirmed a new variant of Bw53, as indicated by the assignments based on serology. PMID- 1726465 TI - The tumor markers, sialyl Le(a) and sialyl Le(x) bind ELAM-1. PMID- 1726464 TI - HLA class II gene frequencies in Italy. AB - The frequency of HLA alleles at HLA-DR and DQ loci, and that of the related HLA-D specificities, were estimated in the Italian population. 109 healthy unrelated subjects, born in several Italian regions and living in the district of Torino, were studied. DNA typing was achieved by the restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta, DQ alpha and beta genes, hybridizing specific probes with TaqI digested DNAs. The present study allowed to define in more detail the HLA class II polymorphisms in the Italian population. PMID- 1726466 TI - N-linked sugar chains of mouse B16 melanoma cells and their low-metastasizing variant selected by wheat germ agglutinin. AB - The N-linked sugar chains of metastasizing mouse B16 melanoma cells (F1) and their wheat germ agglutinin-resistant variant (Wa4-b1) showing a dramatic decrease in metastasizing and tumorigenic potentials were liberated from their membrane glycoproteins by hydrazinolysis, and their structures were comparatively analysed. The results indicated that both cell lines contain high-mannose-type and bi-, tri- and tetraantennary complex-type oligosaccharides, and their ratios are similar. However, outer chain moieties of tri- and tetraantennary oligosaccharides of the variant are extensively fucosylated, resulting in the formation of X-antigenic determinants, Gal beta 1----4(Fuc alpha 1----3)GlcNAc. Oligosaccharides containing X-antigenic determinants amounted to 71% of the total complex-type oligosaccharides. Fucosylation occurs on every N-acetyllactosamine unit and the number of the determinants ranges from one to three in triantennary oligosaccharides and one to four in tetraantennary oligosaccharides. The determinants occur predominantly in non-sialylated forms, although some are in sialylated forms. Oligosaccharides containing non-sialylated X-antigenic determinants and those containing sialylated X-antigenic determinants are approximately in the ratio 6:1. Since no significant difference was found in the extent of branching of complex-type oligosaccharides of the two cell lines, it is suggested that non-fucosylated outer chains are important for the expression of metastasizing potential by the tumour cells. PMID- 1726467 TI - Polyadenylation precedes splicing in vitro. AB - Vertebrate premessenger RNAs are usually spliced and polyadenylated. In vivo analysis of the relative kinetics of the two reactions is difficult. We have used in vitro processing systems to investigate the order of splicing and polyadenylation of chimeric precursor RNAs containing a single intron and a poly(A) site. Polyadenylated, but not spliced, intermediate RNA appeared first and reached a low steady-state level early during incubation, properties consistent with its being a reaction intermediate in the production of doubly processed spliced and polyadenylated product RNA. The kinetics of polyadenylation suggested that polyadenylated RNA was the only intermediate in the production of doubly-processed RNA. Spliced, but not polyadenylated, RNA also appeared. This species, however, continued to accumulate during reaction, and could not be chased into product spliced and polyadenylated RNA. These data support a preferred order of reaction for 3' terminal introns and exons in which polyadenylation precedes splicing. PMID- 1726468 TI - Developmental changes in the subcellular distribution of the 43K (v1) polypeptides in Torpedo marmorata electrocyte: support for a role in acetylcholine receptor stabilization. AB - Analysis of the relative amounts of the acetylcholine receptros (AChR) and of the 43K protein present in the membrane of developing electrocyte shows that massive accumulation of 43K protein is not required for induction of early AChR clustering. Furthermore, we demonstrate the existence o of cytosol- and membrane associated 43K polypeptide pools in Torpedo electrocyte. Epitope analysis shows that both pools of 43K protein are related to the long mRNA transcript and share similar antigenic determinants distributed throughout the protein sequence. Their partition between the cytosol and membrane fractions abruptly increases in favor of the membrane during the postsynaptic maturation phase of development, supporting a role for 43K protein in the stabilization and maintenance of the postsynaptic domain. PMID- 1726469 TI - Death anxiety and job stress in hospice and medical-surgical nurses. AB - This study compared death anxiety and frequency and severity of job stress in 30 hospice and 40 medical-surgical nurses. Death anxiety was assessed through the Templer/McMordie Death Anxiety Scale, job stress through the Gray-Toft/Anderson Nursing Stress Scale. There were no significant group differences in the death anxiety scores nor in the total scores for the frequency segment of the nursing stress scale. The medical-surgical nurses scored a significantly higher total for the severity segment of the nursing stress scale. Death anxiety correlated significantly with frequency and severity of job stress for medical-surgical nurses but not for hospice nurses. Death anxiety correlated highly significantly with death and dying as a source of stress for the medical-surgical nurses. PMID- 1726470 TI - Electrophysiologic mechanisms of ventricular arrhythmias. AB - In this work the electrophysiologic mechanisms of ventricular arrhythmias have been briefly summarized. Ventricular arrhythmias can be caused either by pacemaker activity or by reentrant excitation. Enhancement of normal automaticity can generate a parasystolic rhythm in normal fibers. Abnormal automaticity may arise from fibers in which maximum diastolic potential has been reduced by a variety of interventions. Triggered activity is caused by either an early (EAD) or delayed (DAD) afterdepolarization and requires a prior normal action potential for initiation. While there is growing evidence that EAD-induced triggered activity plays a significant role in the Long QTU syndrome and Torsade de Pointes, no clinical arrhythmias has definitely been ascribed to DADs, although DADs have been recorded in man after acute digoxin intoxication. Ventricular arrhythmias can be also caused by reentrant excitation, which can be subdivided into reflection or circus movement reentry (CMR). In the reflection model impulses in both directions are transmitted over the same pathway. In the CMR three models can be differentiated: the ring model, which requires a fixed anatomical obstacle; the figure-eight model and the leading circle model, where functional rather than fixed anatomical obstacles are involved. PMID- 1726471 TI - Clinical magnetocardiography: experience with a biomagnetic multichannel system. AB - The magnetic fields caused by the human heart's electrical activity were coherently recorded with a biomagnetic multichannel system (KRENIKON) during 1 to 10 minutes in 49 patients. 31 to 37 magnetic channels were recorded simultaneously with the ECG and respiration. Comparison of a magnetic index and the Sokolow-Lyon index to echocardiographic findings in the quantification of left ventricular hypertrophy demonstrated the superiority of the magnetocardiogram (MCG) as compared to the ECG. The magnetocardiographic investigation of patients with WPW-Syndrome, ventricular extrasystoles, ventricular tachycardia, and paced ventricular beats demonstrated that multichannel magnetocardiography permits the non-invasive three dimensional localization of arrhythmogenic tissue with high spatial accuracy. PMID- 1726472 TI - Natural modifiers of the inflammatory process in the human dental pulp. AB - Concentrations of the protease inhibitors alpha 1-antitrypsin and alpha 2 macroglobulin were determined in normal and inflamed human dental pulps. Carious pulpal exposure which is associated with polymorphonuclear leukocyte infiltration and release of lysosomal enzymes was chosen as the point of verifiable inflammatory activity in the pulp. Normal samples were collected from nondiseased third molar teeth treatment planned for extraction and inflamed human pulps were collected from teeth with deep carious lesions. One half of each sample was assayed for concentration of protease inhibitors by enzyme-linked immunosorbent assay and the remaining half was examined histologically to verify the clinical diagnosis and categorize the extent of the inflammatory process. alpha 1 Antitrypsin and alpha 2-macroglobulin were detected in normal and inflamed human dental pulps in the nanogram per milliliter range. Statistically significant differences were found in the concentrations of alpha 2-macroglobulin (p less than 0.01) in moderate to severe inflammation versus normal pulp categories and between mildly inflamed pulps and moderate to severely inflamed pulps (p less than 0.05). Although differences in concentrations of alpha 1-antitrypsin were seen between inflamed and normal pulps, the differences were not statistically significant. The presence of these two protease inhibitors in the human dental pulp tissue and the increase in their concentration in acute inflammation indicates that these proteins play a role in the pathogenesis of pulpal inflammatory disease. PMID- 1726473 TI - Dihydropyridine effects on skeletal muscle fatigue. AB - 1. The purpose of this investigation was to determine if alterations in extracellular calcium (Ca2+) influx by the dihydropyridine derivatives Bay K 8644 and nifedipine affected skeletal muscle fatigue. 2. Tetanic contractions (80 Hz, 100 msec) of frog sartorius muscles were evoked every sec for 3 min. Muscles were fatigued in normal Ringer's solution (NR), in NR containing 1 microM nifedipine of 10 microM Bay K 8644 or in low Ca2+ Ringer's. 3. In each case, the experimental conditions increased the rate and magnitude of fatigue. Rate constants of fatigue obtained during Bay K 8644, nifedipine and low Ca2+ conditions (-.0122 +/- .0016, -.0397 +/- 0022 and 0.0169 +/- .0064 sec-1, respectively) were significantly greater than NR (-.0104 +/- .0006 sec-1, p less than .05). In addition, tetanic forces developed at the end of the stimulation period under the experimental conditions (3.90 +/- 0.81, 1.21 +/- 1.40 and 2.04 +/- 1.10% of initial) were significantly less than NR (7.18 +/- 1.27%, p less than .05). 4. Caffeine contracture forces (10 mM) evoked immediately after stimulation were not significantly different between conditions. 5. These results suggest that alterations in sarcolemmal Ca2+ exchange has some influence on the fatigue process. PMID- 1726474 TI - Growth factors promote high-dose chemotherapy. PMID- 1726475 TI - Enzyme release by Trichophyton rubrum depends on nutritional conditions. AB - Enzymes liberated by growing dermatophytes are of pathogenetic importance in tinea. To investigate the influence of nutrients on this enzyme release, Trichophyton rubrum was grown in media containing peptone, keratin and lipids, to which glucose was added in separate assays. The culture supernatants were compared for extracellular enzyme activities by use of the api-zym test. Our results clearly show that the extracellular enzyme activity is dependent on the nutrients supplied. Seven different enzymes were released when keratin was supplied, as compared with only five and two respectively when lipids or peptone were available. Among these enzymes alkaline phosphatase and N-acetyl-beta glucosaminidase were detected in all cultures lacking glucose. Enzyme release was inhibited completely when glucose was added to the media, except for N-acetyl beta-glucosaminidase in peptone cultures. This dependency of enzyme release on fungal nutrition can be expected to occur in vivo too. In addition, it has to be considered for in vitro cultural conditions. Alkaline phosphatase and acetylglucosaminidase may be more important in tinea than has been assumed so far. PMID- 1726476 TI - Effects of interferons and other cytokines on tumors in animals: a review. AB - The term cytokine describes a group of protein cell regulators involved in the control of cell growth and differentiation in embryogenesis, immunity and inflammation. They are of low molecular weight, are produced locally, and act in an autocrine or paracrine manner. In the past decade their use as cancer therapy has become a reality. Thirty years ago mice were treated with the antiviral protein interferon (IFN) which not only produced a reduction in the incidence of virus-induced tumors but also slowed the development of transplantable tumors. This was one of the first indications that cytokines can be negative regulators of cell growth. Here we outline current knowledge of the actions of IFNs and other cytokines in animal models, and draw parallels with clinical trials to illustrate the invaluable nature of this preclinical and mechanistic work. PMID- 1726477 TI - Angiogenesis in wound healing. AB - Angiogenesis is an essential component of wound healing. Vessel growth is controlled by the local actions of chemical mediators, the extracellular matrix, metabolic gradients, and physical forces. Manipulation of some of these factors can improve healing in experimental wounds. The clinical potential and specific application of 'angiomodulatory' strategies are discussed. PMID- 1726478 TI - [53-year-old patient with fever, polyarthritis and adenopathies]. PMID- 1726479 TI - [Electrolyte secretion in saliva]. AB - Because they are easily accessible and because their secretion can be obtained in an almost pure form, the salivary glands are an adequate model for the study of hydroelectrolytic secretion of secreting epithelia which cannot generate an action potential. It is generally accepted that the salivary secretion occurs in two stages: in the acini, where primary saliva is formed similar in ionic constituents to plasmatic water, and in the ducts of the adenomer, where such original saliva is modified by absorption and secretion of electrolytes. Departing from Thaysen's hypothesis, the mechanisms responsible for the preparation of final saliva are reviewed. The authors briefly discuss the evolution of the ideas about salivary secretion emphasizing recent works which modified early theories, particularly the role of the Na-K ATPase and the cotransportation of the Cl- ion. Ideas about what occurs along the ducts are also reviewed, starting with the hypothesis of Brusilow and Cook, which was later on gradually modified. Recent works are compared to those of the authors. Certain topics, such as the impermeability of duct walls to water, existence of a threshold for the transport of Na+, possibility of experimentally obtaining saliva with higher osmotic pressure than plasma are stressed. Finally, the role of loop diuretics which interfere in potassium transport channels is discussed. PMID- 1726480 TI - Susceptibility of Aedes aegypti larvae to temephos and Bacillus thuringiensis var israelensis in integrated control. AB - The susceptibility of field collected Aedes aegypti larvae was evaluated in terms of median lethal time (LT50) and final mortality, when treated with temephos, Bacillus thuringiensis var israelensis as well as mixtures of these two agents. Third instar larvae were shown to be more susceptible than early and late fourth instar ones to the entomopathogen. Survival of some individuals when exposed to temephos suggest possible resistance. Temporal synergism in early fourth instar larvae was detected when they were exposed to mixtures of Bti-temephos. The possibility of this integrated treatment is commented on. PMID- 1726481 TI - [Susceptibility of populations of Simulium (Chirostilbia) pertinax Kollar, 1832 (Culicomorpha, Simuliidae) to eemephos and to Bacillus thuringiensis var. israelensis-based formulation]. AB - The use of wooden troughs on stream beds, artificially colonized by blackfly larvae, is proposed for larvicide evaluations. Mortality was recorded 3 or 4 hours after treatment. Larval susceptibility was also evaluated utilizing the LT50 criterion. In there field assays Simulium (C.) pertinax populations from the litoral of S. Paulo and Rio de Janeiro States were shown to be resistant to temephos, even when subjected to high concentrations. Vectobac 12 AS, a Bacillus thuringiensis var. israelensis product, was shown to be more potent against late instar larvae and efficient in concentrations higher than 7,200 ITU/l (10 min). The LT50 to 3,744 ITU/l (10 min) was calculated as 70.9 min. PMID- 1726482 TI - Calcium induced differentiation of SV40 immortalized human epidermal keratinocytes cultured in a defined medium. AB - Human epidermal keratinocytes, immortalized by the introduction of SV40 adenovirus recombinants (9), were maintained in a low calcium (0.15 mM) defined medium. When differentiation was induced by a higher extracellular calcium concentration (1.5 mM), these cells altered in shape and developed stratification with formation of desmosomes, while they demonstrated limited terminal keratinization. The expression of involucrin, one of the precursor proteins of the cornified envelope, became elevated as the cell density increased, but was not affected by calcium. These results indicate that the SV40 immortalized human keratinocytes, maintained in a low calcium defined medium, partially respond to changes in extracellular calcium concentration. PMID- 1726483 TI - Possible mechanisms for the maintenance of polymorphisms in Plasmodium populations. AB - There are two views on the origin and maintenance of the high levels of polymorphism found in antigenic Plasmodium proteins. Immune selectionists consider that mutations which avoid stimulating a host response are frequent and advantageous. Proponents of the random genetic drift of selectively equivalent mutations hold that Plasmodium antigens are relatively unconstrained and can tolerate considerable structural diversity. Both sides agree that antigenic diversity is advantageous although selectionists see benefits in individual mutations whereas the proponents of random genetic drift see the advantage in the parasite's capacity to tolerate diversity per se. PMID- 1726484 TI - Study on staining methods for mast cells in the nasal mucosa. AB - Mast cells are widely distributed in various organs. Two classes of mast cells, the mucosal mast cell (MMC) and the connective tissue mast cell (CTMC), have been shown to exist in the intestine of experimental animals. In the present study, we investigated the method of staining suitable for observing the mast cells distributing in the nasal mucosa, and also examined by the use of two fixatives whether the mast cells have properties of MMC or those of CTMC. A neutral buffered formalin solution and Carnoy's solution were used as fixative. For staining, five solutions, i.e., toluidine blue (TB) solution (pH 0.5, 2.5, and 4.0), 0.4 M MgCl2-alcian blue (AB) solution, and naphthol AS-D chloracetate (NAS DC) solution, were tested. In the specimen fixed with Carnoy's fixative, staining with pH 0.5 TB showed the largest number of mast cells in all mucosal layers, particularly in the epithelial layer. The number of these mast cells agreed with that of the cells positive to pH 0.5 AB and also with that of the tryptase positive cells stained immunohistochemically with a mouse monoclonal antitryptase antibody. Compared with formalin-fixed specimens, those fixed with Carnoy's fixative and stained with pH 0.5 TB showed significantly (p less than 0.01) many mast cells in the epithelial layer and in the subepithelial layer of lamina propria. To identify mast cells in the nasal mucosa with nasal allergy, fixation with Carnoy's fixative and staining with pH 0.5 TB were found to be most effective and simplest.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726485 TI - Expression of the Arabidopsis floral homeotic gene AGAMOUS is restricted to specific cell types late in flower development. AB - Mutations in the AGAMOUS (AG) gene cause transformations in two adjacent whorls of the Arabidopsis flower. Petals develop in the third floral whorl rather than the normal stamens, and the cells that would normally develop into the fourth whorl gynoecium behave as if they constituted an ag flower primordium. Early in flower development, AG RNA is evenly distributed throughout third and fourth whorl organ primordia but is not present in the organ primordia of whorls one and two. In contrast to the early expression pattern, later in flower development, AG RNA is restricted to specific cell types within the stamens and carpels as cellular differentiation occurs in those organs. Ectopic AG expression patterns in flowers mutant for the floral homeotic gene APETELA2 (AP2), which regulates early AG expression, suggest that the late AG expression is not directly dependent on AP2 activity. PMID- 1726486 TI - [Primary culture of human gingival tissue cells in vitro]. AB - In order to establish and understand the in vitro human gingival cell culture system, this study presents newly developed and characterized primary culture cell types derived from human gingival tissues. Cell cultures were established from human gingival tissues by means of the explant technique and monolayer culture. Cells were studied under stable growth conditions and were characterized in terms of their morphology, Giemsa staining, anti-epithelial cytoskeletal staining, and proliferative parameters. At confluence, disoriented fibroblast cells formed the multilayered culture. The epithelial nature of the epithelioid cells was confirmed by staining for cytoplasmic keratin which is an exclusive epithelial cell protein. The growth curve and cell doubling time of the fibroblasts were evaluated. The results indicate that both epithelial cells and fibroblasts can be cultured from human gingival tissue. This technique provides us with a stable source of normal cells for further in-depth in vitro studies. PMID- 1726487 TI - Piroxicam-natural polymers interactions. AB - The interaction of the anti-inflammatory drug, piroxicam, with some naturally occurring polymers-viz gamma-globulin, bovine serum-albumin, bovine synovial fluid and casein was characterized in this study. This was done using equilibrium dialysis method where the dialysis membrane was permeable only to the drug molecules while polymers and formed complex(es) cannot pass through. The amount of permeable piroxicam molecules was determined spectrophotometrically. It was found out that increasing the concentration of piroxicam was accompanied by an increase in the amount of piroxicam bound to the investigated natural polymers. The maximum binding capacity of the investigated polymers for the drug as revealed by Langmuir plots, could be arranged in ascending manner as follows: synovial fluid, albumin, casein and gamma-globulin. The binding parameters K (association constant) and n (the number of binding sites available on the polymer) were determined using Sandberg plots. It was found that two classed of binding sites were involved in the interaction of piroxicam with gamma-globulin, albumin and casein. Increasing salt concentration was accompanied by a decrease in piroxicam binding with the investigated polymers till a constant value of ionic strength (0.6 mole/liter). This result may suggest the ionic nature in binding of piroxicam with the investigated polymers. It was concluded that piroxicam is strongly bound to the investigated natural polymers with different degree of binding, such binding may affect the pharmacological effect of piroxicam. An in-vivo absorption study was performed on eight male healthy volunteers to compare the bioavailability of piroxicam in presence and absence of casein using plasma data after 100 mg single dose treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726488 TI - [Regulation of hormone biosynthesis in guinea pig adrenal glands by potassium ions as affected by dihydropyridines. 1. Analysis of changes in steroidogenesis evoked by dihydropyridines]. AB - Influence of Ca2+ channel modulators BAY K 8644, nitrendipine and newly synthesized derivative of 1,4-dihydropyridine: 4-(3', 4'-dimethoxyphenyl)-2,6 dimethyl-3,5- diethoxy-carbonyl-1,4-dihydropyridine-(DHP-51) on aldosterone production by adrenocortical slices depends on K+ content in the incubation medium. The modulators only slightly influence the hormone output at low K+ level in the medium. Intensive synthesis of aldosterone at high level of potassium in the medium was prevented in the presence of DHP-51 and low concentration of BAY K 8644. DHP-51 inhibited [3H]-cholesterol incorporation into all main corticosteroids in the high-potassium medium. PMID- 1726489 TI - Intermediate filaments in normal thyrocytes: modulation of vimentin expression in primary cultures. AB - In dog thyrocyte primary cultures, the antagonistic effects of thyrotropin (TSH) and epidermal growth factor (EGF) on differentiation expression were accompagnied by distinct long-term morphological changes: TSH-treated cells showed an epitheloid morphology; EGF reversibly induced a fusiform shape. Using indirect immunofluorescence microscopy and two-dimensional gel electrophoresis, we studied the modifications in the distribution and synthesis of the intermediate filament proteins of the cytoskeleton in response to TSH and EGF. These factors had little effect on the expression of cytokeratins 8 and 18, which were expressed in 98% of cells. However, TSH induced a profound redistribution of cytokeratins (and actin) with the appearance of a marked staining of cell junctions. Vimentin was coexpressed with cytokeratins in about 40% of cells from normal thyroid follicles freshly isolated by collagenase. During culture, immunostained vimentin network progressively developed in 90% of control and EGF-treated cells simultaneously with vimentin synthesis. In contrast, only 20% of TSH-treated cells reacted with vimentin antibody and we observed a marked decrease in vimentin synthesis in response to TSH. Therefore, vimentin synthesis, which should occur in at least some normal thyroid follicles in vivo, was inhibited in vitro by TSH which promotes differentiation expression. However, EGF-treated cells thereafter cultured with TSH regained an epitheloid morphology and differentiation in spite of the persistency of a complete network of vimentin. PMID- 1726490 TI - Identification and androgen-regulated expression of two major human glandular kallikrein-1 (hGK-1) mRNA species. AB - The screening of an oligo(dT)-primed prostate cDNA library with a human glandular kallikrein-1 (hGK-1) genomic DNA fragment resulted in the isolation of two different hGK-1 cDNAs. A 1.2 kb cDNA (pGK-1) contains an open reading frame of 510 bp, encoding the major part of the previously predicted hGK-1 protein (Schedlich et al. (1987) DNA 6, 429-437). This cDNA contains a 3'-untranslated region of 677 nucleotides and terminates in a poly(A) stretch, preceded by the canonical AATAAA polyadenylation signal. A second cDNA (pGK-10A), with a size of 1.5 kb, contains an open reading frame of 669 nucleotides preceded by 16 nucleotides of the 5'-untranslated region. pGK-10A differs from pGK-1 by the presence of an additional 37 bp fragment, interrupting the protein coding region of hGK-1, which results from the use of an alternative splice donor site of intron IV of the hGK-1 gene. The mature protein (excluding presumed pre- and propeptides) as deduced from the pGK-10A cDNA sequence, has a size of 199 amino acids and differs at the COOH-terminus from the 237 amino acid hGK-1 protein. The alternatively spliced mRNA comprises approximately 20% of the hGK-1 transcripts, as deduced from analysis of mRNA from prostate cells by PCR amplification of specific fragments. The regulation of hGK-1 mRNA expression was studied in different human prostate tumors and cell lines by Northern blotting, using a hGK 1-specific oligonucleotide probe. A high level of hGK-1 expression was found in the androgen-dependent tumors PC 82 and PC EW. hGK-1 mRNA was also present in the androgen-sensitive LNCaP cell line, but undetectable in the androgen-insensitive prostate tumors PC 133, PC 135 and the PC 3 cell line. In LNCaP cells, the expression of hGK-1 mRNA was strongly induced by androgens. Regulation of expression of the closely related prostate-specific antigen (PA) gene showed a similar pattern. PMID- 1726491 TI - Effects of androgens on the transcription of secretory protein genes in rat seminal vesicle. AB - Run-on transcription in isolated nuclei has been used to study the effects of testosterone on gene expression in rat seminal vesicles. General transcriptional rates were increased by about 6-fold with an additional 2- to 3-fold differential stimulation of the genes for secretory proteins IV and V. These transcriptional changes are insufficient to explain overall changes in cellular mRNA levels, indicating that androgens must also have major effects on post-transcriptional processing of RNA transcripts or on mRNA stability. Analysis of nuclear RNA by Northern blotting with intron probes suggests substantial androgen effects on primary transcript processing. PMID- 1726492 TI - Intensive treatment of stage III-IV aggressive malignant lymphomas (protocol TPL 84). AB - BACKGROUND: Much progress has been made in the last ten years in the treatment of non Hodgkin's lymphomas by increasing drug schedules and by using non cross resistant regimens. METHODS: So we decided in 1984 to test a new multiple drug protocol (Tours-Poitiers-Limoges = TPL protocol) which used a sequence of three courses of classical high-dose induction therapy, three courses of consolidation therapy using Teniposide, Cytosine Arabinoside, L Asparaginase and high-dose Methotrexate, and three courses of late intensification using the same drugs as induction therapy. Results. Thirty-eight patients younger than 60 years were included. Complete remission was obtained in 27 patients (71%). The median follow up was 3 years and 9 months with one third of CR patients having been followed beyond 5 years. Seven patients relapsed (26% of CR patients) and one died of toxicity in complete remission. At present 22 patients (58%) are in complete remission, 19 in first CR, 1 in first CR after allogenic bone marrow transplantation, and 2 in second prolonged CR after autologous bone marrow transplantation. The median survival time is 48 months and the actuarial disease free survival curve seems to have a plateau at 48.5%, with no relapse after 24 months. CONCLUSIONS: These results confirm the efficacy of alternating high-dose conventional chemotherapy in the treatment of intermediate and high-grade NHL, with about half of the patients being cured. However, more intensive chemotherapy regimens are needed to improve cure rates. PMID- 1726493 TI - [Proliferative vitreoretinopathy as a cause of early recurrence of retinal detachment]. AB - Analysed were the results of surgical treatment, causes of the failure and early recurrence in 108 patients with retinal detachment in whom was performed an indentation of the sclera by means of a balloon (1st group--50) or by an episcleral implant (2d group--58). In the first group the apposition was achieved in 92 p.c., in the 2d group in 90 p.c. An early recurrence (2 weeks to 2 months) was observed in 9 eyes which were operated again achieving an apposition of the retina in 6 eyes. There were following causes of the failure: the existence of a second hole, rupture of the balloon, PVR before surgery and traction of the retina in the region of the hole. An early recurrence occurred in the consequence of intensification of the PVR together with inaccurate localization of the implant, existence of the PVR before the operation and traction of the retina in the vicinity of the hole, formation of a new hole. PMID- 1726494 TI - [Risk factors of proliferative vitreoretinopathy]. AB - Presented is the personal material comprising cases treated surgically for primary retinal detachment; analysed is the causal connection between postoperatively occurring proliferative vitreoretinopathy and the risk factors. The comparison of personal results with results of the other authors shows that in case of diagnosis of risk factors the reduction of the surgical trauma reduces the threat of proliferative vitreoretinopathy after operation of retinal detachment. PMID- 1726495 TI - [Results of laser photocoagulation in diabetic retinopathy with maculopathy]. AB - Examined were 91 eyes with non-proliferative and proliferative retinopathy with coexisting maculopathy in 51 patients in whom segmental or retinal pan photocoagulation were performed. The period of observation--3 to 4.5 years. Stabilization of the pathological process was observed in 37.3 p.c. of cases, 54.9 p.c. of patients showed further deterioration and 7.6 p.c. loss of vision. The most frequent cause of deterioration was the progression of the macular lesions (72 p.c.), a smaller number of cases (28 p.c.) was due to other changes. PMID- 1726496 TI - [Laser coagulation of the iris surface with neovascularization in glaucoma]. AB - Photocoagulation of the iris surface in the course of rubeosis iridis was performed in 11 patients (12 eyes) with secondary absolute glaucoma. The intraocular pressure was lowered down to 30-35 mm Hg in 8 eyes, in the remaining 4 eyes it oscillated between 45 and 50 mm Hg. The pain receded in all the cases. One considers the possibility that in the disappearance of pain--besides the lowering of the IOP--a substantial factor was the lesion of the innervation of the iris. PMID- 1726497 TI - [Indicators of nonspecific immunity in patients with non-Hodgkin's lymphoma of high grade malignancy in the remission phase of the disease]. AB - Parameters of unspecific immunity (absolute numbers of peripheral blood lymphocytes and their subpopulations, monocytes, blood concentrations of G, A, M immunoglobulins and skin tests with recall-antigens) were evaluated in 25 patients with high grade malignancy non-Hodgkin lymphomas at diagnosis. The frequency, site and gravidity of infections were recorded during the cytostatic treatment with the use of CHOP or CBVPP/ABVD regimens. The same immunological parameters were reevaluated in 9 patients in remission, within 6-52 months following treatment's cessation. Disturbances in at least 2 out of 10 studied parameters were found in all 25 patients at diagnosis. While on treatment, 19 out of 25 patients suffered from various bacterial, viral or fungal infections. The diminished frequency of infections was observed after treatment cessation, in contrast to persisting immunological disturbances. The usefulness of the immunological status monitoring and of immunomodulatory treatment during the remission phase of NHL is postulated. PMID- 1726498 TI - Pharmacological interaction between neuropeptides and morphine or pentazocine in rat spinal cord. AB - Male Wistar rats were treated with morphine or pentazocine subcutaneously (sc) and then intrathecally (it) by methionine- or leucine-enkephalin, neurotensin, substance P or cholecystokinin octapeptide 26-33. Then antinociceptive effect was measured during 1 h using tail-immersion test. Leucine-enkephalin potentiated and methionine-enkephalin antagonized morphine or pentazocine analgesia. Neurotensin, substance P and cholecystokinin acted biphasically on morphine induced antinociception. After short elevation, the diminution of antinociceptive effect was seen. Neurotensin diminished but substance P and cholecystokinin elevated analgesic effect elicited by pentazocine. Experimental model employed by us may be useful for preliminary screening of pharmacological interactions between neuropeptides and opioid analgesics on the spinal level. Our data confirm the results of other authors that individual enkephalins have different pharmacological features. PMID- 1726499 TI - Histamine of mouse lungs after single exposure to nitrogen dioxide. AB - Pulmonary histamine and the weight of lungs were studied in mice, exposed to a single one-hour effect of high concentrations of edemagenic gas nitrogen dioxide in a metabolic chamber. Nitrogen dioxide concentrations were chosen according to the results of nitrogen dioxide analysis of the mining atmosphere immediately after the mining blasts. The results were estimated by the method of paired comparison with the findings registered in mice, exposed to the effect of air atmosphere under identical experimental conditions. The intervals following immediately the exposure and 5 hours after were chosen for the evaluation, with regard to the dynamics of the early and late stages of hypersensitivity of the first type. i) Immediately after the exposure to the concentrations of 43, 250, 387 and 540 mg.m-3, no significant differences were observed in the amount of pulmonary histamine. In concentrations higher than 43 mg.m-3, the weight of lungs increased (the proportion of pulmonary water and the dry tissue). ii) Five hours after the exposure (nitrogen dioxide concentrations 66, 130, 137 and 270 mg.m-3), pulmonary histamine decreased, at the concentration of 137 mg.m-3 in a significant way, on the other hand, it increased significantly at the concentration of 270 mg.m-3. Both concentrations higher than 130 mg.m-3 manifested an increased weight of lungs (increased proportion of dry tissue and pulmonary water). The obtained data do not allow to establish unambiguously the part of histamine on the pulmonary changes following the effect of nitrogen dioxide. The edemagenic effect of nitrogen dioxide estimated after one-hour influence can be considered as reversible up to five hours after the exposure in concentrations lower than 130 mg.m-3. The metodical part of the study gives detailed description of exposure technique and it brings a survey of methods of histamine determination in blood and tissues. PMID- 1726500 TI - Demonstration of the nucleolar organizer region by silver staining (AgNOR method) in research and in histopathological practice. AB - The silver-staining method for the detection of nucleolar organizer regions (AgNOR) was tested on a large material and the AgNORs evaluated in normal and pathological tissues, especially in various benign and malignant neoplasms. Pathological lesions were represented by dysplasias and in situ carcinoma of the uterine cervix, dysplasias and various histological forms of breast carcinoma, carcinomas of the lung, of urinary bladder, of prostate, various neoplasms of the gastrointestinal tract, trophoblastic disease, naevocellular naevi and melanomas of the skin, myeloproliferative diseases and haematological malignancies, and also by several cases of xenotransplanted human neoplasms in athymic nude mice. The AgNORs were evaluated using several procedures. Marked AgNOR heterogeneity in individual malignant tumours was found to be a common feature suggesting the presence of cellular subpopulations or clones with different proliferative activity and metabolic properties. Close correlation between the proliferative behaviour and the quantitative and qualitative change of AgNORs was found in most obviously malignant neoplasms. The method, however, could not recognize neoplastic cells as such and is therefore not a specific oncological marker. This limits the diagnostic contribution of the silver staining method in diagnostic pathology, where it does not exceed the value of routinely stained slides of good quality. In spite of these limitations, the AgNOR staining method seems to represent a valuable contribution in the study of some aspects of the proliferative lesions, such as their proliferative and metabolic activity, hormonal dependence etc. PMID- 1726501 TI - Dying with dignity. AB - People facing death because of incurable illness are likely to suffer intense physical and mental distress. The provision of help and support for them can be seen as a medical discipline in its own right, demanding special training and attitudes. Each country needs to work out the best ways of looking after dying people in accordance with its culture and resources. PMID- 1726502 TI - Palliative care--an urgent but neglected public health priority. PMID- 1726503 TI - A study of lectin histochemistry in ameloblastoma. AB - The lectin histochemistry of ameloblastoma was investigated in order to understand the nature of the carbohydrate residues in this tumor, in accordance with the WHO subclassification. Mannose and glucose were generally common in almost all cells in both the follicular and plexiform types, especially stellate cells and cystic lesions of the follicular type. N- acetylglucosamine (GlcNac), N acetylneuraminic acid (NANA) and galactose were much more abundant in stellate cells of the plexiform type than in those of the follicular type. Thus, there is a slight difference in lectin histochemistry of the stellate cells between the follicular and plexiform types of ameloblastoma. PMID- 1726504 TI - Anti-phosphotyrosine antibodies--obtaining and purification. AB - Anti-phosphotyrosine antibodies were obtained by rabbits immunization using bovine serum albumin-phosphotyrosine complex. Hyperimmune rabbit serum was purified on affinity chromatography column obtained by phosphotyramine binding on Sepharose 4 B, CNBr-activated. The antibodies obtained after purification were tested by ELISA and immunoblotting. PMID- 1726505 TI - Anti-phosphotyrosine and anti-phosphoserine antibodies in SLE sera. AB - By using an ELISA method, we identified antiPTyr and antiPSer antibodies in the sera of some SLE patients. Afterwards, antiPTyr and antiPSer antibodies were purified by affinity chromatography on phosphotyramine-CNBr-Sepharose column and on phosphoethanolamine-CNBr-Sepharose, respectively, and the specificity of the purified antibodies was demonstrated by inhibition assays. The study pointed out a higher incidence of antiPTyr than anti PSer antibodies in the sera of these patients, which suggests that some "autoantigens" from membrane might be involved. PMID- 1726506 TI - Enhancement of carcinoembryonic antigen (CEA) expression in colorectal tumors by neuraminidase. AB - Twelve colorectal carcinomas with transitional mucosa and 10 colorectal adenomas which previously displayed weak or no carcinoembryonic antigen (CEA) expression were selected to verify whether neuraminidase unmasks CEA carbohydrate epitopes and, consequently, enhances the CEA expression. Peroxidase-antiperoxidase (PAP) method was performed on routinely processed tissues, without and with neuraminidase pretreatment of the sections. Lysine, without and with neuraminidase pretreatment of the sections. Lysine, as a modifier of electrostatic charge at cell surface, instead of neuraminidase was used to clarify whether the enzyme yields a specific or non-specific influence on CEA expression. All colorectal tumors exhibited more CEA after neuraminidase pretreatment, while previous negative specimens developed CEA expression. The same effect was observed in some transitional mucosa sections. This has not occurred in normal mucosa, probably owing to a resistant sialylation. The enhancement effect of lysine, although more weakly and not entirely superimposed to that or neuraminidase, suggests non-specific mechanisms of enzyme action. The removal of the negative charge at cell surface, especially due to sialic acid, allows more anti-CEA antibodies to react. The neuraminidase pretreatment of the sections is a useful method to demonstrate the real incidence of CEA in the colorectal tumors. PMID- 1726507 TI - Time course of changes in rat pancreatic synthesis rates and retention thresholds of four hydrolases during consumption of a low-protein followed by a balanced diet. AB - Synthesis rates of pancreatic amylase, trypsinogen 2 (Tg2), chymotrypsinogen 1 (Chtg1), and lipase, were measured in control rats fed a 20% protein diet for 46 days and in malnourished rats fed a 5% protein diet for 23 days (protein malnutrition) followed by refeeding a balanced diet for 23 days. In the control group, a progressive pancreatic maturation (namely an increase in hydrolase synthesis per gram of tissue) appeared with age. In the malnourished group, pancreas maturation of the four hydrolases was inhibited. Synthesis rates of the four hydrolases were reduced to a lesser extent from day 2 of protein malnutrition. With continued protein malnutrition, Tg2 synthesis rates remained steady, whereas the lipase synthesis rate continued to decrease and Chtg1 and amylase rates started to increase. As soon as refeeding was initiated, an important enhancement in synthesis was observed. The synthesis rates of Tg2, Chtg1, and amylase actually showed a rebound effect, then decreased with the refeeding time to reach control values, except for Chtg1, which remained higher than control values throughout the refeeding phase. Lipase synthesis rate rose more slowly and reached the control values only after 9 days of refeeding. The retention threshold (pancreas tissue versus pancreatic secretion) values showed that the storage levels were different from one hydrolase to the other and were variable with age in the control group, and with protein malnutrition and refeeding times in the malnourished group. In the control group, a preferential secretion of newly synthesized enzymes was observed in young rats, whereas with age, the proportion of newly synthesized hydrolases excreted decreased slowly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726508 TI - Human colostrum as a source of organohalogen xenobiotics for a breast-fed neonate. AB - The concentrations of p,p'-isomers of DDT, DDE, and DDD, and alpha, beta, and gamma isomers of hexachlorohexane (HCH), hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs) were determined by means of gas-liquid chromatography in 3/4 postpartum day colostrum of 54 normal women. The milk levels of t-DDT, t-HCH, HCB, and PCBs correlated significantly with one another. The contents in milk of all the studied organohalides significantly increased with maternal age. The average daily intakes of t-DDT and PCBs were estimated for the studied neonates. Values exceeding the Acceptable Daily Intake values (ADIs) recommended by the WHO for t-DDT and PCBs were found for 70.4% and 24.1% of subjects, respectively. The present study confirms the trends in organohalogen residues of human milk observed by us in the studied region's inhabitants during the 17 years of monitoring (1970-1987), i.e., a consistent decline in t-DDT levels and an increase in PCB content in the present decade as compared to the 1970s. In conclusion, despite legal restrictions in their usage, the contamination with organohalides persist in human milk at a level that may result in neonatal alimentary exposure exceeding the recommended daily intakes. PMID- 1726509 TI - The lack of mutagenicity of aryl 4-guanidinobenzoates in the transplacental micronucleus assay in mice. AB - Two acrosin inhibitors, 4'-methylumbelliferyl 4-guanidinobenzoate and 2' carbomethoxyphenyl 4-guanidinobenzoate, were tested for mutagenicity in the transplacental micronucleus assay and the mouse bone marrow micronucleus assay. The compounds were administered intraperitoneally at doses of 125 mg/kg and 250 mg/kg to pregnant mice. Fetal peripheral blood and maternal bone marrow cells were examined at 36 h for the frequency of micronucleated polychromatic erythrocytes. Neither compound induced micronuclei in maternal or fetal tissues. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was not affected by the drug treatments indicating that the compounds had no effect on the cell cycle or mitosis in these tissues and that they were not cytotoxic. Both compounds, which show promise as vaginal contraceptives, were not mutagenic in this study. PMID- 1726510 TI - Characteristics of the limb malformations induced by maternal exposure to cadmium in the mouse. AB - Single doses of 2,3,4,6,8,10, and 15 mg/kg of cadmium chloride were administered (SC) to groups of MF1 mice on one of days 7 to 12 of gestation. Fetuses collected on day 18 were observed for limb malformations, and alizarin red-S stained skeletons were examined for their skeletal bases. Ectrodactyly, postaxial polydactyly, syndactyly, brachydactyly, adactyly, phocomelia, meromelia, and malrotation of the limbs were detected in a significant number of fetuses. Days 7 to 10 were greatly susceptible for induction of these malformations. Postaxial ectrodactyly was more frequent in the forepaws, and sidedness was not significant. Preaxial ectrodactyly preferentially affected the left hindpaws in the 9th-day treatment group. Postaxial polydactyly was predominently right sided and mostly involved the forepaws. Day 8 was particularly susceptible for induction of adactyly. Malrotation of the limbs together with edema and caudal narrowing resulted in a 'penguin-like' appearance. Ossification of the long bones, the carpals, tarsals, and phalanges were affected. Even those limbs that were not externally malformed had skeletal dysgenesis. Limb buds examined histologically at midgestation showed scanty and poorly organized mesenchyme, extensive elaboration of marginal sinsus, reduced thickness of the apical ectodermal ridge (AER), and discontinuous basement membrane. It is speculated that such histologic alterations at early stages of development could have contributed to the defective morphogenesis of limbs in this animal model. PMID- 1726511 TI - Protective role of vitamin A in the male reproductive toxicity of hexachlorocyclohexane (HCH) in the rat. AB - The reproductive toxicity of the organochlorine insecticide, hexachlorocyclohexane (HCH), was investigated in male albino rats fed a diet free of vitamin A or containing vitamin A at 2000 or 100,000 IU/kg diet. Diets containing 1000 ppm HCH for 7 weeks did not cause testicular toxicity in the vitamin-A-deficient and supplemented rats. However, reproductive toxicity was clearly manifested 2 weeks after withdrawing HCH from the diets and was more pronounced in the vitamin A deficient rats compared to their vitamin A supplemented counterparts. Reduction in the testicular weights was accompanied by atrophy of epididymides and seminal vesicles in the vitamin A deficient rats alone. Inhibition of spermatogenesis was further confirmed by decreased sperm count in the epididymis. Biochemically, the activities of the steroidogenic enzymes were drastically reduced. Supplementation of vitamin A after withdrawal of HCH accelerated the recovery and restored spermatogenesis and enzyme activities in the deficient rats. These results demonstrate the greater susceptibility of the male reproductive system to HCH toxicity during vitamin A deficiency and also the protective effect of vitamin A supplementation. PMID- 1726512 TI - Relationship between cumulative occupational exposure to asbestos fibres and respiratory symptoms. AB - A survey of chronic respiratory symptoms was undertaken in 1127 asbestos workers engaged in asbestos mining, asbestos cement production, production of friction materials or in the manufacture of asbestos textile. A control group of 593 persons was also surveyed. The exposure of asbestos workers was analysed by evaluating separately the cumulative exposure to total airborne particles and to airborne asbestos fibres. The prevalences of all the respiratory symptoms (chronic cough, chronic phlegm, chronic bronchitis, dyspnoea grade 3+) were significantly higher in asbestos workers compared to controls in both nonsmokers and smokers (p less than 0.01). The prevalences of all the respiratory parameters in asbestos workers increased with both the length of employment and cumulative exposure to total airborne particles. Prevalences of chronic cough, chronic phlegm and chronic bronchitis did not show an increase with cumulative exposure to airborne asbestos fibres expressed as fibres/cc years. It is concluded that the development of chronic cough, chronic phlegm and chronic bronchitis in asbestos workers is likely to be an unspecific effect of the exposure to the difficulty soluble airborne particles rather than a specific effect of the exposure to airborne asbestos fibres. PMID- 1726513 TI - Persistence of immunity against measles in persons immunized with Edmonston Zagreb vaccine. AB - Cohorts of 30 children--470 in all--vaccinated against measles 2 to 17 years earlier were tested for measles heminhibiting antibodies. The vaccinees were randomly selected from a semi-urban, semi-rural population where the circulation of the wild measles virus has never been interrupted. The vaccinal status of the vaccinees varied widely, some were vaccinated only once, some twice or even three times, in some the primary vaccination was performed with a measles monovalent and in some with a combined MMR vaccine, only the revaccination having always been performed with a monovalent vaccine. The results of this serologic study reveal that the vaccine-induced immunity against measles does not fade with time, on the contrary, as judged by the mean geometric antibody titers, it rises. This is speculatively attributed to the booster effect of natural reinfection(s). Thereafter it is demonstrated that revaccination(s) do not yield a better immunity than the primary vaccination alone. This "revaccination", if withheld, helps only to attain a better coverage of children that have escaped primary vaccination. PMID- 1726514 TI - [Risk factors and satisfactory living circumstances in 103 patients with acute coronary disease under 50 years of age]. AB - For 30 months, from 1988, some risk factors and the satisfaction of basic needs, such as food, habitation, clothes, transport, health, security, family relation, possibility of affirmation, possibility of creation and cultural needs, were studied in 103 under 55-year-old patients with acute myocardial infarction and unstable angina pectoris. The control group matched the coronary group in age, sex, marital status, education, and working place. A cardiovascular clinical examination and ECG recording were performed in all subjects. In the coronary group also CPK and glucose in serum were recorded several times. Each person estimated the fulfillment of his basic needs from 1 to 5. The family history of coronary heart disease was positive in 43 (41.7%) coronary patients and in 12 (11.6%) control subjects (P less than 0.05). The prevalence of arterial hypertension in the coronary group was 46.6% and of diabetes mellitus 12.6%. In the coronary group 74.7% patients smoked and in the control group 47.5% (P less than 0.01). The mean body index in the coronary group was 27.5 +/- 0.36 and in the control group 27.9 +/- 0.36 (P less than 0.05). The basic needs (food, habitation, clothes, transport, and cultural needs) were given a significantly higher mark in the coronary than in the control group (P less than 0.05). The study has shown significant differences in the presence of classical risk factors between the group of 103 acute coronary patients and the control group. The socio economic was higher in the coronary than in the control group. PMID- 1726515 TI - The relationship between skinfold thicknesses, body circumferences, plasma lipids and glucose in the elderly. AB - A total of 699 inhabitants of Ljubljana (269 men and 430 women) aged over 60 years were examined. Weight and height were measured, the body mass index (g/cm2) was summed, skinfold thickness on 14 sites (biceps, abdomen /on the right side/, subscapular, suprailiacal, thigh, calf, lower arm /on the right and left side/) and 8 body circumferences (chest, abdomen, upper arm, thigh and calf /on the right and left side/) were determined. Fasting venous blood was taken for determining plasma glucose, total plasma cholesterol, triglycerides and HDL cholesterol. Plasma cholesterol, both in men and women, is not associated with predictors of fatness - skinfold thicknesses and body circumferences. There are statistically significant correlations between plasma triglycerides and predictors of fatness, particularly in the body, both in men and women. The correlation of plasma HDL-cholesterol is inversely proportional to the predictors of fatness (circumferences and skinfold thicknesses) at the central and peripheral sites of the subcutaneous fat, in both sexes. Relations between plasma glucose and parameters of fatness (body circumferences) are weak and less frequent. They are noticed only in the female population. Body circumferences, predictors of fatness and the distribution of body fat can be as good factors as skinfold thicknesses in the population aged over 60 years. PMID- 1726516 TI - [Histologic criteria for the diagnosis of superficially spreading melanoma in patients with proven metastases]. AB - The majority of authors agree that the prognosis of malignant melanoma depends on the depth of extension of malignant melanocytes into the dermis. A number of studies were performed to determine histologic characteristics of primary malignant melanoma that already metastasized. Their goal was to predict the biological behaviour of malignant melanoma by the histological picture. The study was designed to establish histological characteristics of superficial spreading melanoma. To prove the malignant biological behaviour, only superficial spreading melanomas with metastases in lymph nodes were analysed. The aim of the study was to see whether the melanoma's biological behaviour could be predicted on the basis of its histological picture. A total of 39 cases of superficially spreading malignant melanoma from the Department of Pathology, University Hospital Center Rebro, Zagreb, were analysed. The sections were taken from 5 different places, fixed in 10% purified formalin, paraffin embedded and stained with hematoxylin eosin. In some cases the sections were additionally stained with Fontana to prove melanin. The sections were analysed by light microscopy. The following histological characteristics were analysed: 1. poor circumscription of the intraepidermal melanocytes; 2. individual melanocytes extending laterally; 3. number of atypical melanocytes above the basal membrane; 4. marked variation in the size and shape of melanocytic nests; 5. tendency of melanocytic nests towards confluence; 6. presence of melanocytes with nuclear atypia; 7. absence of maturation of melanocytes with descent into the dermis; 8. intradermal necrosis and degeneration of melanocytes; 9. lymphocytic infiltrate; 10. desmoplasia and ulceration. There were 22 male (56.4%) and 17 female (43.6%) patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726517 TI - [Cytoplasmic proteins in the uterine cervix]. AB - The uterine cervical tissue was homogenized in STM buffer (0.25 M sucrose, 10 mM MgCl2, 10 mM Tris, pH 7.5). A soluble fraction with cytoplasmic proteins was obtained in the supernatant by centrifugation at 100,000 g for 40 minutes. According to the Davis method, there was almost no difference between the most abundant native cytoplasmic proteins (0.8/0.4/0.1/0.3) and the control serum proteins (0.8/0.1/0.4/0.3). Otherwise, according to the Laemmli method, the third and the fourth number of the "four-part numerical code" were strongly different for cytoplasmic proteins (67, 41, 186, 294) toward serum proteins (67, 41, 112, 235). Those data confirm Irwin's suggestion about the specificity of the "tissue codes". PMID- 1726518 TI - Lactate and glucose in cerebrospinal fluid heavily contaminated with blood. AB - To simulate a traumatic lumbar puncture, blood was added to 33 normal cerebrospinal fluid (CSF) specimens. A hypothesis was tested if the CSF glucose and CSF lactate were unchanged after contamination with blood. Lactate and glucose here measured in both normal and blood-stained CSF. The estimated contamination of the normal CSF with red cells ranged from 84000 to 676500 cells per cubic millimeter. CSF lactate was unchanged by the addition of blood (P = 0.8), whereas CSF glucose was significantly higher in the blood-stained CSF (P = 0.0005). Therefore, the determination of lactate levels in the CSF heavily contaminated with blood could be useful in differentiating viral from bacterial meningitis. PMID- 1726519 TI - Histochemical aspects and fine structural characteristics of thyreocytes in pinealectomized and melatonin treated rats prior to irradiation. AB - The authors were investigating the histochemical and electron microscopic characteristics of thyreocytes in pinealectomized rats treated with melatonin, prior to irradiation. They observed that the animals that did not receive melatonin show a higher degree of the destruction appearing at the level of all ultrastructural organelles and a very low expression of the DNA and RNA histochemical reaction. These results suggest the role of melatonin in the determination of the thyreocyte behaviour under given irradiation conditions. PMID- 1726520 TI - [Prenatal, perinatal and neonatal factors in infantile autism]. AB - Analysing prenatal, perinatal, and neonatal complications in autistic children by a specially prepared questionnaire, the authors aimed at establishing whether there is a connection between prenatal, perinatal, and neonatal complications and autism in comparison with normal and mentally retarded children. The results have shown that there is a statistically significant connection of some of these factors with autism in relation to the control group of healthy children, while these differences proved less significant in relation to the group of mentally retarded children. The results obtained partly correspond to the results of similar investigations in the world, indicating a complex etiology of autism. PMID- 1726521 TI - [Evaluation of the effect of local corticosteroids in the phototherapy of psoriasis]. AB - An open clinical trial was carried out in order to establish to what extent the application of topical corticosteroids with the UVB therapy influences the complete dosage of the UVB radiation required for the clearing of psoriasis and the duration of remission. Out of 30 patients who were administered the suberythemogenic UVB dosage, 15 received a topical corticosteroid and 15 an indifferent ointment (u. emolliens). Although the therapeutic response was initially somewhat more immediate in the corticosteroid--UVB group, there is no statistically significant difference between these two groups in the total UVB dosage required for the remission of the disease. The patients in the topical corticosteroid--UVB group remained in remission longer than the patients in the control group receiving no local corticosteroid. PMID- 1726522 TI - Postnucleation orbital implants. AB - A follow-up study of a group of 40 patients after the enucleation completed by the intra-orbital acrylic spherical magnetic implant (Roper-Hall style) is presented. The mean postoperative observation time is 3.3 years. Late complications were present in two patients: the extrusion of the implant in one of them was probably caused by his poor state due to chronic dialysis, while the traumatic luxation of the implant in the other was surgically repaired. Postoperative results in all other patients were satisfactory with the adequate orbital volume substitution and the ability of the prosthesis to twist and turn in any axis without fitting problems. PMID- 1726523 TI - [Comparative analysis of the results after implantation of a "one piece" and other types of intraocular lenses]. AB - The authors analysed the results obtained in 100 patients with the "one piece" posterior chamber lens and compared them with those in 85 patients with soft haptics. All procedures followed the extracapsular cataract extraction. The incidence of intraoperative and postoperative complications was compared. Special attention was paid to the positioning of the implant after a period of at least 6 months. The advantages of "one piece" implant lenses were established and better postoperative results obtained in this group of patients. PMID- 1726524 TI - Surgical management of pain. AB - This article presents an overview of the neuroanatomical, neurochemical, and neurophysiological substrates of nociception relevant to the neurosurgical treatment of chronic pain. Consideration is given to the various procedures currently employed in the treatment of patients suffering from medically intractable chronic pain of both benign and malignant diseases, including their indications, techniques, and results. Particular attention is given to the modern neuroaugmentative methods, such as electrical stimulation and CNS drug infusion, that are progressively overshadowing the previously developed ablative procedures. PMID- 1726525 TI - Transglutaminase stabilizes melanoma adhesion under laminar flow. AB - To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN. PMID- 1726526 TI - Simulation of cell rolling and adhesion on surfaces in shear flow. Microvilli coated hard spheres with adhesive springs. AB - The adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This article describes a calculational method that allows simulation of the interaction of a single cell with a ligand-coated surface. The cell is idealized as a microvilli coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal, and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the springs, the response of springs to extension, and the magnitude of hydrodynamic stresses. By varying these parameters, the model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the model can provide meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the extension of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same extension. Changes in the fractional spring slippage can radically change the adhesive behavior of a cell. We show that stiffer springs will only serve to increase adhesion if the fractional slippage remains small. In addition, our simulations emphasize the importance of reaction rates between receptor and ligand, rather than affinity, as being the key determinant of adhesion under flow. These results suggest reaction rates and response to stress of adhesion molecules must be independently measured to understand how adhesion is controlled at the molecular level. PMID- 1726527 TI - Deformation-driven, lethal damage to cancer cells. Its contribution to metastatic inefficiency. AB - Direct and indirect, in vivo and in vitro observations are in accord with the hypothesis that as a consequence of their deformation within capillaries, cancer cells undergo sphere-to-cylinder shape-transformations that create a demand for increased surface area. When this demand cannot be met by apparent increases in surface area accomplished by nonlethal, surface "unfolding," the cell surface membrane is stretched; if expansion results in more than a 4% increase in true surface area, the membrane ruptures, resulting in cancer cell death. It is suggested that this deformation-driven process is an important factor in accounting for the rapid death of circulating cancer cells that have been trapped in the microvasculature. Therefore, this mechanism is thought to make a significant contribution to metastatic inefficiency by acting as a potent rate regulator for hematogenous metastasis. PMID- 1726528 TI - In vitro studies of deformation and adhesion properties of transformed cells. AB - The micropipet aspiration technique and the parallel-plate flow chamber were used to investigate the deformation and detachment properties, respectively, of normal and transformed rat fibroblasts. The normal Cloned Rat Embryo Fibroblasts (CREF) cell line was transfected with the T24 ras oncogene to produce the transformed cell line CREF T24. The CREF T24 cell line was transfected with a Kirsten ras revertant gene (K-rev 1a suppressor) to produce the CT24HKB1 cells, which have the same morphological characteristics as the cells in the CREF line. The cells utilized in this investigation were derived from the parent cell line CREF, the only differences being the presence or absence of the T24 ras oncogene and the Kirsten ras revertant gene. The detachment and deformation properties, therefore, could be related to the metastatic phenotype of the cell rather than inherent differences between disparate cell lines. Results indicated that transfecting the CREF cell line with the ras oncogene greatly modified the detachment and deformation properties. The CREF T24 cells were more easily detached from normal cells and were 50% more deformable. Both CREF and CT24HKB1 showed similar detachment properties. Based on these results, it is speculated that K-rev 1a reversed ras-induced membrane alterations in these cells. Preliminary investigations have demonstrated that both CREF and CREF T24 cells in different phases of the cell cycle differed in morphological characteristics. However, the majority of the cells within a given cell line showed similar deformation characteristics. Current investigations are focusing on characterization of both detachment and deformation properties of these cells as a function of the cell cycle using synchronization techniques. PMID- 1726529 TI - Possible role of cell cycle-dependent morphology, geometry, and mechanical properties in tumor cell metastasis. AB - Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37 degrees C, an increase in cell volume from 1400 microns 3 to 5700 microns 3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the "older" cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system. PMID- 1726530 TI - Boundaries in gravitational and magnetic activation of cells for sorting. AB - Standard deviations in the distribution of radii of cells and particles are considered to arrive at realistic limits in the use of gravitational and magnetic activation of cells for sorting. Using a specific fractionation design, it is shown that the radius of particles (or cells) may be fractionated down to a precision of +/- 0.76%. Although higher precisions could be obtained with other designs, the number of particles available per fraction is inversely proportional to the precision desired. Thus, one would prefer to keep the precision as moderate as permissible by the experiments. PMID- 1726531 TI - Studies of the T1 and T2 of intracellular water as a function of frequency in normal and transformed fetal cells. AB - Frequency-dependent values of the spin-lattice relaxation time (T1) and the spin spin relaxation time (T2) have been obtained for intracellular water in normal and transformed Syrian hamster fetal fibroblasts. Values of T1 and T2 were obtained for normal and transformed cells at 24.3 (0.57 T), 100 (2.4 T), 300 (7.0 T), and 400 MHz (9.4 T). At each frequency, values of T1 were the same for both normal and transformed cells, whereas values of T2 were lower for one passage of transformed cells. As expected, T1 increased with frequency. However, T2 decreased with frequency for both normal and transformed cells. The frequency dependence of T2, was similar for all cells; thus, the ability of T2 to make a distinction between normal and transformed cells did not change with field. PMID- 1726532 TI - Attenuation of spontaneous pseudopod formation in human neutrophils by pentoxifylline. AB - The effects of pentoxifylline (PTX) on spontaneous pseudopod formation in neutrophils in response to the tripeptide formyl-Met-Leu-Phe (fMLP), endotoxin, human complement C5a, and leukotriene B4 (LTB4) were examined in autologous plasma. Unseparated supernatant leukocyte suspensions from fresh heparinized venous human blood were incubated with PTX (0-5 mM) for 25 min and then stimulated for 5-25 min within a range of concentrations of fMLP, endotoxin, complement C5a, and LTB4. The cell suspensions were fixed with glutaraldehyde and stained with crystal violet in acetic acid; the percentage of neutrophils with pseudopods was determined under high-resolution light microscope. The results show that PTX significantly decreases formation of pseudopods in the presence of all four stimulators. The mechanism of pseudopod suppression appears to be independent of the adenosine receptor. PTX and its analogues, HWA 138 and HWA 448, decreased pseudopod formation by similar amounts when stimulated with 10( 8)M fMLP. These results suggest that PTX may improve microvascular perfusion and attenuate neutrophil-mediated injury by reducing the degree of neutrophil pseudopod formation in free suspension and microvascular entrapment. PMID- 1726533 TI - Chronobiologic approach to beat-to-beat variations of cultured murine myocardial cells. AB - An earlier demonstration of a circadian rhythm in rat atria by others is complemented herein by observations in culture: A single murine myocardial cell and two sets of grouped cells beating in culture for several days reveal several features of an anticipated, presumably built-in spectrum of multifrequency rhythms and trends, the chronome. Circadian and about 12-h (circasemidian) components are modulated by an approximately 84-h (circasemiseptan) component, which cannot be separated from trends in view of the brevity of the series. The circumstance under which the culture is aging and in which fibroblasts proliferate is a further complication that limits the findings to a single cycle reproduced in three separate cultures. Whether it is a rhythm that repeats itself of a response to placement into culture, an approximately 3.5-d component in the beating of myocardial cells in culture is to be aligned with a very prominent similar component found in the incidence of 85,819 human myocardial infarctions. PMID- 1726534 TI - Red blood cell mechanics and capillary blood rheology. AB - Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessels walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic "tank-treading" motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow. PMID- 1726535 TI - Interaction between advancing ice fronts and erythrocytes. Mechanism of erythrocyte destruction upon freezing and influence of cryoprotective agents. AB - It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) are excluded by advancing freezing fronts. This puts the cells under considerable osmotic stress and may be considered to be the major source of cell destruction upon freezing. It is also shown theoretically and experimentally that in aqueous suspensions, admixed with appropriate concentrations of a cryoprotectant (e.g., glycerol), cells are engulfed by advancing freezing fronts: Under such conditions, cells do not undergo any osmotic stress and remain undamaged when frozen. The influence of various common cryoprotectants is discussed, as is the reason why penetrating as well as nonpenetrating agents can be equally effective cryoprotective agents. The reason why leukocytes require lower cryoprotectant concentrations than erythrocytes is also elucidated. PMID- 1726536 TI - A study of deoxyribonucleotide metabolism and its relation to DNA synthesis. Supercomputer simulation and model-system analysis. AB - A model system (1) was established to analyze purine and pyrimidine metabolism. This system has been expanded to include macrosimulation of DNA synthesis and the study of its regulation by terminal deoxynucleoside triphosphates (dNTPs) via a complex set of interactions. Computer experiments reveal that our model exhibits adequate and reasonable sensitivity in terms of dNTP pool levels and rates of DNA synthesis when inputs to the system are varied. These simulation experiments reveal that in order to achieve maximum DNA synthesis (in terms of purine metabolism), a proper balance is required in guanine and adenine input into this metabolic system. Excessive inputs will become inhibitory to DNA synthesis. In addition, studies are carried out on rates of DNA synthesis when various parameters are changed quantitatively. The current system is formulated by 110 differential equations. PMID- 1726537 TI - Acoustic microscopy of cultured cells. Distribution of forces and cytoskeletal elements. AB - The scanning acoustic microscope (SAM) allows one to measure mechanical parameters of living cells with high lateral resolution. By analyzing single acoustic images' sound attenuation and sound velocity, the latter corresponding to stiffness (elasticity) of the cortical cytoplasm can be determined. In this study, measurements of stiffness distribution in XTH-2 cells were compared with the organization of F-actin and microtubules. Single XTH-2 cells exhibit relatively high stiffness at the free margins; toward the cell center this value decreases and reaches a sudden minimum where the slope of the surface topography enlargens at the margin of the dome-shaped cell center. The steepness of the increase in slope is linearly related to the decrease in sound velocity at this site. Thus, a significant determinant of cell shape is paralleled by an alteration of stiffness. In the most central parts, no interferences could be distinguished, therefore, this region had to be excluded from the calculations. Stiffness distribution roughly coincided with the distribution of F-actin, but no correlation to microtubule arrangement was found. Following the treatment of XTH 2 cells with ionomycin in the presence of calcium (in the culture medium), the cell cortex first contracted as indicated by shape changes and by a marked increase in stiffness (deduced from sound velocity). This contraction phase was followed by a phase of microtubule and F-actin disassembly. Concomittantly, sound velocity decreased considerably, indicating the loss of elasticity in the cell cortex. No structural equivalent to sound attenuation has been identified. PMID- 1726538 TI - A comparative study of the cellular, exudative and histological responses to carrageenan, dextran and zymosan in the mouse. AB - A murine 6-day air-pouch model of inflammation has been developed and used to compare the patterns of acute and chronic inflammatory response to three irritants, carrageenan, dextran and zymosan, each injected into the cavity of the pre-formed pouch. The inflammation was assessed by measurement of exudate volume and numbers of infiltrating leucocytes over a 30-day time course. Histological changes in the inflamed air-pouch lining tissue were also investigated. The inflammatory response to carrageenan was acute with moderate exudate formation and cell numbers. Dextran produced a mild inflammatory reaction with low cell infiltration into exudate. In contrast, the inflammatory response to zymosan was greater in terms of cell migration, but smaller in terms of exudate volume and occurred later in the time course. Histological changes in the inflamed air-pouch tissue were also markedly different in response to the three irritants. Carrageenan induced a rapid, mainly polymorphonuclear leucocyte (PMN) infiltrate into the tissue and deposition of fibrin on the luminal surface. The response to dextran was characterized by a rapid resolution of the inflammatory response, with fewer leucocytes present in the lining and no fibrin deposition. In contrast, zymosan caused a marked but slower leucocyte influx, with greater numbers of monocytes, and clearance of the zymosan particles from the air-pouch lining by macrophages. This study indicates that by using different irritants to produce inflammation, it may be possible to dissect the roles played by various cells and inflammatory mediators during acute and chronic inflammation. PMID- 1726539 TI - Ultrastructure of phagocytizing cells of the rat hypothalamic neurosurgery nuclei in a remote period after the sustained clinical death. AB - Ultrastructural studies were carried out on neurosecretory nuclei taken from brains of rats which have survived for 16 weeks following clinical death of 5 or 10 min duration. The reported observations indicate that brain lesions were caused by changes due to ischemia and superimposed secondary changes resulting from the maturation of pathological processes. In animals subjected to clinical death for 5 min the observed cells in the perivascular area were integrated in the capillary wall and wrapped by the basement membrane. Sometimes they were accompanied by cells engaged in the process of phagocytosis. Phagocytizing cells were more frequently noted in the animals subjected to clinical death for 10 min and then their cytoplasmic processes were connected with the adjacent capillaries. We assume that these cells, which may represent cerebral macrophages occur by transformation of pericytes or blood derived monocytes. PMID- 1726540 TI - Significant non-serotonergic raphe projection to the visual cortex of the rat. An immunohistochemical study combined with retrograde tracing. AB - The present study investigated the distribution of serotonergic and non serotonergic raphe neurons with direct projections to the visual cortex. The study employed the WGA-apoHRP-Au retrograde transport technique combined with 5 HT immunohistochemical staining. Retrogradely labeled cells were observed in the dorsal raphe nucleus, the median raphe nucleus, and in the B9 and B6 cell groups. One notable finding was the great number of retrogradely labeled, non-5-HT immunoreactive cells. The average percentages of such cells in the various raphe regions were as follows: DR: 52% (n = 401); MR: 35% (n = 311); B9: 24% (n = 129); B6: 95% (n = 200). The present study demonstrated the presence of a significant proportion of non-serotonergic raphe region neurons projecting to the primary visual cortex in the rat. It is suggested that these neurons may complement the aminergic neurons as part of the ascending system which controls the functions of the visual cortex. PMID- 1726541 TI - Age-related changes in serotonin-immunoreactivity in the telencephalon and diencephalon of rats. AB - Comparison of the appearance and density of the serotoninergic fibers and terminals in some tel- and diencephalon areas of young adult (3 months) and aged (28 months) rats was made immunohistochemically using an antibody to serotonin. In young adult rats a characteristic arrangement of the serotonin-immunoreactive (SER-IR) fibers and terminals in the tel- and diencephalon was observed. In aged rats the distribution pattern was the same but changes in the appearance and density of the SER-IR fibers were demonstrated. Many swollen and folded fibers forming cluster-like structures as well as reduced fiber networks were seen in the neocortex, hippocampal formation and striatum of aged rats. Aberrant fibers and decreased fiber density were also observed in the bed nucleus of the stria terminalis, lateral septum, nucleus accumbens and thalamic nuclei. Reduced fine granular immunostaining and scattered swollen varicosities were found in some amygdaloid and hypothalamic nuclei of aged rats. These results provide further morphological evidence for changes in the serotoninergic innervation during aging. PMID- 1726542 TI - [The transition zone of the central nervous system-peripheral nervous system of the adult rat; ultrastructural and immunocytochemical studies: a new function of the astroglia?]. AB - At the transition between central nervous system (CNS) and peripheral nervous system (PNS), the CNS compartment forms cone-shaped incursions into the peripheral part of the dorsal root. The ultrastructural study of the CNS-PNS transitional zone shows that this region is particularly rich in astrocyte processes. In an attempt to investigate the possible role of the CNS-PNS interface astrocytes in myelin formation, a photonic microscopy immunocytochemical study has been done with anti-GFAP and anti-MBP sera. The CNS glial expansion shows an important GFAP immunoreactivity with intimate association between astrocyte processes and myelinated axons. This may indicate that the transitional myelin originates from astrocytes. The same region is also MBP-positive. Two explanations are considered: some astrocytes form transitional myelin sheathes and express MBP epitopes, or oligodendrocytes, with cell bodies distant from the CNS-PNS interface, send myelinating cytoplasmic expansions which are not shown by the techniques we used. PMID- 1726543 TI - Distribution and immunochemical specificities of fimbriae of Porphyromonas gingivalis and related bacterial species. AB - Rabbit polyclonal antibody (Poly-1) and mouse monoclonal antibodies (mAbs) TO-11, TO-14 and TO-M1 specific for Porphyromonas gingivalis 381 fimbriae were prepared. Poly-1 and the 3 mAbs were screened for their reactivity with whole cells oral and nonoral black-pigmented bacterial species by enzyme-linked immunosorbent assay (ELISA) and the binding experiment using [125I]Poly-1 and [125I]mAbs. ELISA revealed that Poly-1 definitely reacted with whole cells of all the 11 strains of P. gingivalis tested. However, 8 of 11 P. gingivalis strains reacted with mAbs TO 11, TO-14 and TO-M1. These results were confirmed by the specific binding of radiolabelled Poly-1 and mAb TO-11 to the 8 strains. The M(r) of the fimbrial subunit protein (fimbrilin) isolated and purified from P. gingivalis strains 381, BH18/10, HW24D-1, 6/26 and OMZ 314 was 41 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis. It was found by immunoblotting that mAbs TO 11 and TO-14/TO-M1 recognized different epitopes of fimbrial protein from P. gingivalis strains. Immunoelectron micrographs of whole cells and the purified fimbriae of P. gingivalis strains visualized similar serotype-specific antibody bindings to the fimbriae. These results indicate that 11 strains of P. gingivalis could be divided into at least 2 separate groups based on the immunochemical specificities of the fimbriae. PMID- 1726544 TI - Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica. AB - Human isolates of Porphyromonas gingivalis (n = 16), Prevotella intermedia n = 82) and Prevotella melaninogenica (n = 18) from diseased periodontal pockets were examined for interaction with human lactoferrin (HLf) in a standardized 125I labeled protein binding assay. The highest HLf binding was found in P. intermedia strains, followed by P. gingivalis and P. melaninogenica. Further characterization of the interaction was performed with 1 representative strain from each species. HLf binding to P. gingivalis reached a saturation instantly and was optimal at pH 5.0-6.5. The corresponding values for P. melaninogenica were 90 min and pH 3.0-5.5. The HLf binding to the 2 strains seem to be nonspecific. In contrast, P. intermedia demonstrated specific binding, and a time saturability within 60 min with an optimal uptake at pH 6.0-7.5. Scatchard analysis implied 45,000 receptors per cell with an affinity constant of 5.5 x 10( 7) M on P. intermedia strain 4H. The binding capacity in all 3 strains was affected by the culture medium. HLf binding components in these strains were susceptible to heat or proteases. Binding was eliminated in P. gingivalis and was enhanced in P. intermedia and P. melaninogenica by periodate treatment. Unlabeled HLf or bovine lactoferrin effectively displaced labeled HLf binding. Various proteins and carbohydrates did not inhibit HLf binding. Our data suggest that HLf binds to these periodontitis-associated species and that this mechanism is distinct from the previously known ligand interactions in oral bacteria. PMID- 1726545 TI - Immunohistochemical study of cholecystokinin peptide in rat spinal motoneurons. AB - With the aid of indirect immunofluorescence histochemistry and sequence specific antibodies a possible localization of cholecystokinin (CCK) peptide in spinal motoneurons has been analyzed. To increase peptide levels, the sciatic nerve was ligated, and the area around the ligation was studied 24 hours later. For comparison, antisera raised against calcitonin gene-related peptide (CGRP) and substance P were employed. With CCK specific antisera (directed to the N-terminal portion of CCK-8 or the midportion of CCK-33) accumulation of peptide-like immunoreactivity (LI) was observed in large, dilated axonal swellings proximal to, but at some distance from, the ligature. Such accumulations were also observed with C-terminally directed CCK antiserum, but in addition numerous axons of smaller diameter extending up to the ligation contained this type of immunoreactivity. The latter antiserum is thought to cross-react with CGRP. In fact, this staining pattern was indistinguishable from the one seen after incubation with CGRP antiserum. In contrast substance P-LI could not be seen in the larger dilated axons but only in large numbers of thinner fibers close to the ligation. Double staining experiments revealed that the large dilations contained both CGRP- and CCK-specific LI. Distal to the ligation CGRP- and substance P- but no specific CCK-LI could be observed. The present findings support the view that CCK mRNA in spinal motoneurons is translated into CCK peptide, at least after axotomy, and that the peptide is transported into the motoneuron axon. However, compared to CGRP the CCK levels are presumably low, and the functional role of CCK peptide in motoneurons remains to be established. PMID- 1726546 TI - [The isolation and characteristics of monoclonal antibodies to the HNK-1 antigen of natural killers]. AB - Monoclonal antibodies to the HNK-1 differentiated antigen of natural killer cells have been obtained. A glycoprotein of the white human brain connected with myelin was used as an antigen for immunization of mice. The monoclonal antibodies obtained are shown to reduce the cytotoxic activity of a human blood mononuclear fraction as much as by 65% in relation to the human lymphoblastoma cell culture K 562. Their interaction with the surface antigen of mononuclears was shown by immunofluorescent method. Monoclonal antibodies belong to the class of immunoglobulins M. PMID- 1726547 TI - Substance P in chronic pain. PMID- 1726548 TI - [Maternal serum alpha fetoprotein: the implications for embryofetal status]. PMID- 1726549 TI - Mechanisms regulating glycosylation of human acute phase proteins. PMID- 1726550 TI - The regulation of complement on cell surfaces. AB - The regulation of complement at the surface of cells is mediated by both plasma and membrane proteins. These molecules act in concert to prevent the accelerated catabolism of complement proteins and the concomitant lysis of homologous blood cells. The deficiency of either a plasma or membrane protein predisposes to the development of disease. PMID- 1726551 TI - Glial ion channels. AB - It now appears that most of the ion channels discovered in glia are similar or identical to their neuronal equivalents. Recent studies show that glial cells can sense and respond to neuronal signals and that neurons may influence both the development and maintenance of ion channel expression of certain glial cells. Although they lack excitability, glia are probably active participants in brain function. PMID- 1726552 TI - Axonal transport: beyond kinesin and cytoplasmic dynein. AB - In vitro and in vivo studies of specific neuronal fast and slow transport components are presently reshaping our understanding of how the processes of vesicular and cytoskeletal transport are regulated in axons and dendrites. Evidence suggests that vesicles possess an inherent directionality, possibly the result of their motor receptor proteins responding to intracellular cues, which then allows movement with either kinesin or cytoplasmic dynein. PMID- 1726553 TI - Structure of germline immunoglobulin heavy-chain gamma 1 transcripts in interleukin 4 treated mouse spleen cells. AB - Antibody class switching is mediated by a DNA recombination event that replaces the C mu gene with one of the other heavy (H) chain constant region (CH) genes located 3' to the C mu gene. The regulation of this process is essential to the immune response because different CH regions provide different biological functions. Correlative evidence indicates that the isotype (class) specificity of the switch is determined by the accessibility of specific CH genes as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germline CH genes is unknown. In this report, we describe the structure of RNA transcribed from unrearranged gamma 1 genes in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4. The germline gamma 1 RNA is initiated at multiple start sites 5' to the tandem repeats of the gamma 1 switch (S gamma 1) region. As is true for analogous RNAs transcribed from unrearranged gamma 2b and alpha genes, the germline gamma 1 RNA has an I exon transcribed from the region 5' to S gamma 1 sequences, which is spliced at a unique site to the C gamma gene. The germline gamma 1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acid in length. PMID- 1726554 TI - Ontogeny of rat thymic epithelium defined by monoclonal anticytokeratin antibodies. AB - Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunofluorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall's corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life. PMID- 1726555 TI - CD3- leukocytes present in the human uterus during early placentation: phenotypic and morphologic characterization of the CD56++ population. AB - In this study, the CD3- LGL/NK cells present in the pregnant human uterus have been characterized. Phenotypic and morphologic analyses of decidual LGL revealed many similarities to the minor CD56bright+, CD16- subset in peripheral blood, but there were some important differences. The relative surface density of CD56+ is greatly increased on decidual LGL to 22x that found on the majority of CD56+ peripheral blood NK cells. The CD56bright+ cells in decidua show LGL morphology, whereas in peripheral blood, they are mainly agranular. Proliferation of CD56+ cells occurs predominantly during the nonpregnant secretory (luteal) phase, indicating these CD56+ uterine LGL do not migrate as terminally differentiated cells. The appearance of CD56+ cells was examined at the ultrastructural level using immunoelectron microscopy. Cells with phenotypic characteristics of decidual LGL occur in a higher percentage (1.11%) in the peripheral blood of women of reproductive age than in men (0.66%). On the basis of these results, it is proposed that the CD56bright+ uterine leukocytes represent a distinctive, hormonally regulated subset possibly adapted to control human placentation. PMID- 1726556 TI - Functional relationship between T15 and J558 idiotypes in BALB/c mice. AB - In inbred strains of mice, antiphosphorylcholine (PC) and anti-alpha 1,3 dextran (DEX) antibodies are structurally distinct from each other and have been shown to exhibit noncross-reactive antigen binding and idiotypic specificities. However, the prototype anti-PC and anti-DEX antibodies, TEPC15 and J558, respectively, were shown to be connected via a common autoantiidiotypic monoclonal antibody isolated from newborn BALB/c mice. The capacity of various monoclonal anti-PC and anti-DEX antibodies as well as the antigens PC and DEX to modulate T15 and J558 idiotypes in BALB/c mice was tested by their administration to newborn mice. Anti PC antibodies of the T15 idiotype injected into 2-4-day-old mice, at a time when T15+ anti-PC precursors develop in BALB/c mice, suppressed the anti-PC response of these mice at 6 weeks of age. Similarly, J558 antibodies injected into 8-12 day-old mice, at a time when J558 precursors normally develop, suppressed the response to DEX. As a further demonstration of this connectivity, the injection of J558 into 4-day-old mice led to a down modulation of T15 idiotype, whereas both T15 and a minor idiotype-expressing antibody M167 when injected into 8-12 day-old mice caused a reduction in expression of the J558 idiotype. As predicted from in vitro analysis, injection of anti-PC antibodies of the M167 idiotype 2 to 4 days after birth enhanced the subsequent response to PC. However, anti-PC antibodies expressing another minor M603 idiotype did not affect the PC response. The results parallel the in vitro enhancement of M167 antibodies but not M603 on T15 binding to antiidiotype in vitro. Similarly, anti-DEX antibodies expressing the M104E idiotype had no detectable effects on the capacity to respond to PC or DEX or on the expression of T15 and J558 idiotypes as adults. Exposure of newborn mice to PC led to a dramatic reduction in the response to DEX as adults, whereas exposure to DEX at this stage of development had no effect on response to PC as adults. Collectively, these observations provide evidence for a complex functional connectivity between T15 and J558 idiotype-bearing B cells during ontogeny and extend our previous observations that development of these idiotypes is regulated by idiotype-directed interactions between B cells or their immunoglobulin products. PMID- 1726557 TI - Practitioner's guide to industrial back problems. AB - If return to work is part of the expected outcome, more and more data indicate that medical care alone does not hold the key to providing success. Our modern physical treatments may seem a humane alternative to no treatment, but they have not been proven to significantly alter the natural course of back problems. Even the results of strongly indicated surgical treatment differ little from doing nothing at all after a 4-year period. From the physical standpoint, activation has the best track record for recovery and can be simply provided in a manner that can be used lifelong. The real key to return to work goes beyond the physical treatment by addressing the predicament of what the patient will do until age 65. This humane approach to care has evolved from common frustrations of dealing with patients with back problems, observations in the third world, and information gained from scientific studies. Medical pain, and physical models alone are unsuccessful. To be humane and successful, we can no longer ignore the nonphysical factors that can, and do, influence patients' responses to physical treatment, especially when return to work is part of the expected outcome. PMID- 1726558 TI - Expression of the wheat mitochondrial nad3-rps12 transcription unit: correlation between editing and mRNA maturation. AB - In plant mitochondria, RNA editing involves the conversion of cytidines in the genomic DNA into uridines in the corresponding RNA. Analysis of cDNAs prepared by reverse transcription of mitochondrial RNAs has shown that partially edited RNAs are present in wheat mitochondria. The extent of this partial editing as well as its potential influence on the corresponding protein sequence were studied along with the expression of a wheat mitochondrial locus. The sequence, expression, and RNA editing of the wheat mitochondrial transcription unit containing four open reading frames (nad3, rps12, orf299, orf156), all cotranscribed into a same predominant precursor RNA, have been studied. The product of orf156 is an 18-kD mitochondrial membrane protein of unknown function, whereas the product of orf299 could not be detected and this sequence seems to be a pseudogene. Sequences of cDNA clones derived by the polymerase chain reaction technique show that nad3, rps12, and orf156 transcripts are edited, whereas orf299 is not edited, except for a sequence identical to part of the coxII gene. Analysis of cDNA clones obtained from the precursor RNA shows the presence of a large number of partially edited nad3-rps12 transcripts with no evident polarity for the editing process. This shows that RNA editing is a post-transcriptional event. In addition, study of partial editing at the level of precursor, mature, and polysomal transcripts shows that mainly mature, completely edited sequences are used for translation. Deletions of a nucleotide at editing sites were observed in a number of cDNA clones, suggesting that C----U RNA editing in plant mitochondria would be achieved by nucleotide replacement. PMID- 1726559 TI - Identification of cultured cells expressing ligand-gated cationic channels. AB - We have identified cultured cells that express ligand-gated cation channels using a simple method which may also be applied to the screening of chemical agents for their use as agonists or antagonists. This assay is based upon the observation that many ligand-gated cation channels are permeable to lithium and agonists induce the flux of lithium into the cells which contain them. Since the accumulation of intracellular lithium can alter the cell cycle, the measurement of [3H]thymidine ([3H]thy) incorporation should reflect this occurrence. This expectation was realized using the PC12 cell line which expresses neuronal-like nicotinic acetylcholine receptor (nAChR). When cholinergic agonists are applied to PC12 cells in the presence of lithium-containing buffer and cells are subsequently pulsed with [3H]thy, the radiolabel incorporation into these cells relative to controls is reduced. If cholinergic antagonists are included or if the concentration of agonist either rapidly desensitizes receptors or is insufficient to induce channel opening, the reduction in [3H]thy incorporation is not observed. This method also provides a rapid way to screen cultured cell lines for those that express ligand-gated cation channels. This assay offers the potential to be automated for the low cost screening of drugs which act upon ligand-gated ion channels. PMID- 1726560 TI - Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage. AB - A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography. PMID- 1726561 TI - Ataxia telangiectasia presenting as an extrapyramidal movement disorder and ocular motor apraxia without overt telangiectasia. AB - Ataxia telangiectasia may present with few, if any, of its typical extraneurological manifestations, but the combination of an extrapyramidal movement disorder, ocular motor apraxia with head thrusting and cerebellar incoordination is characteristic. In this sporadic case there was no overt immune dysfunction, oculocutaneous telangiectasia were inconspicuous and the neurological presentation was atypical with dystonia predominating over cerebellar incoordination. The uncontrollable and disabling involuntary movements, which have not to our knowledge been described in ataxia telangiectasia before, showed a partial response to moderately large doses of benzhexol, but were refractory to all other medications. Treatment in the future is to be with increasing doses of benzhexol until the dystonia is controlled or larger doses cannot be tolerated. PMID- 1726562 TI - Drosophila PS integrins recognize vertebrate vitronectin and function as cell substratum adhesion receptors in vitro. AB - Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 x 10(3) Mr and other components. To characterize the 120 x 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 x 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion. PMID- 1726563 TI - Biologically active kit ligand growth factor is produced by mouse Sertoli cells and is defective in SId mutant mice. AB - In order to define the role of Kit ligand (KL) growth factor encoded at the mouse steel (SI) locus in spermatogenesis, we have examined its production in Sertoli cells. As a measure KL growth factor bioactivity, the ability to support proliferation and maintenance of mast cells was used in co-culture with primary mouse Sertoli cells. On the sertoli cells derived from +/+ and Wv/Wv mice, +/+ mast cells proliferated and were supported for more than 2 weeks, but not W/Wv mast cells. In contrast, Sertoli cells from SId/SId mice could not support +/+ mast cell proliferation under similar conditions. The supportive effect required close-range interaction of Sertoli cells with cultured mast cells. These results indicate that Sertoli cells derived from +/+ and Wv/Wv but not SId/SId mutant mice produce biologically active KL growth factor as a membrane-bound form. The biologically active KL of Sertoli cells may also play an important role in germ cell growth and differentiation. PMID- 1726564 TI - The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave. AB - Events in the morphogenetic furrow set the stage for all subsequent compound eye development in Drosophila. The periodic pattern of the adult eye begins in the furrow with the spaced initiation of ommatidial rudiments, the preclusters. A wave of mitosis closely follows the furrow. A cell-by-cell analysis reveals details of these events. Early stages of ommatidial assembly can be resolved using a lead sulfide stain. Overt ommatidial organization begins in the morphogenetic furrow as cells gather into periodically spaced concentric aggregates. A stereotyped sequence of cell rearrangements converts these aggregates into preclusters. In the furrow, new rows of ommatidia are initiated at the equator and grow as new clusters are added to the peripheral ends. Mitotic labeling using BrdU feeds shows that all cells not incorporated into a precluster divide. BrdU injections show that cells divide roughly simultaneously between two adjacent rows of ommatidia. PMID- 1726565 TI - Associations between transforming growth factor beta 1 RNA expression and epithelial-mesenchymal interactions during tooth morphogenesis. AB - We have studied the expression of transforming growth factor beta-1 (TGF-beta 1) RNA during mouse tooth development, using in situ hybridization and experimental tissue recombinations. Analysis of the serial sections revealed the appearance of local expression of TGF-beta 1 RNA in the dental epithelium at bud-staged teeth (13-day embryos). Just before transition to the cap stage, TGF-beta 1 RNA expression rapidly increased in the epithelial bud, and it also extended to the condensed dental mesenchyme. At cap stage (14- and 15-day embryos), there was an intense expression of TGF-beta 1 RNA in the morphologically active cervical loops of the dental epithelium. During early bell stage (16- and 17-day embryos), TGF beta 1 RNA expression was detected in the inner enamel epithelium where it subsequently almost disappeared (18-day embryos). After birth TGF-beta 1 transcripts transiently appeared in these cells when they were differentiating into ameloblasts (1-day mice). The transcripts were lost from the ameloblasts when they became secretory (4-day mice), but the expression continued in ameloblasts in enamel-free areas. Transient expression of TGF-beta 1 RNA was also detected in epithelial stratum intermedium cells at the time of ameloblast differentiation. In the mesenchyme, TGF-beta 1 RNA was not detected during bell stage until it appeared in differentiated odontoblasts (18-day embryos). The secretory odontoblasts continued to express TGF-beta 1 RNA at all stages studied including the odontoblasts of incisor roots. Analysis of the distribution of bromodeoxyuridine (BrdU) incorporation indicated apparent correlations between TGF-beta 1 RNA expression and cell proliferation at the bud and cap stages but not at later stages of tooth development. Tissue recombination experiments of bud staged (13-day embryos) dental and non-dental tissues showed that tooth epithelium, when cultured together with tooth mesenchyme, expressed TGF-beta 1 RNA. When the tooth epithelium was combined with non-dental jaw mesenchyme, TGF beta 1 transcripts were not expressed. However, TGF-beta 1 RNA expression was seen in oral epithelium cultured with dental mesenchyme, while no expression of TGF-beta 1 transcripts was seen in the oral epithelium during normal development. Thus, TGF-beta 1 RNA expression seems to be regulated by epithelial-mesenchymal interactions. PMID- 1726566 TI - Survival of cardiac allograft in highly sensitized and nonsensitized rats treated with FK506. AB - The effect of FK506 was studied in sensitized and nonsensitized recipients using a rat cardiac allograft model. Heart grafts from Male ACI rats were heterotopically transplanted to Male LEW rats. The sensitized rat models were established by using donor type blood, admixed with immunoadjuvant (Adjuvant Complete Freund), seven days prior to transplantation. FK506 was administrated from day 0 to day 14 posttransplantation. The results showed that FK506 could prolong allograft survival in both sensitized and nonsensitized rat recipients in a dose-dependent manner, although, the dose required in sensitized recipients was higher than in nonsensitized recipients. The minimal effective dose of FK506 was 0.32 mg/kg/day in sensitized recipients and 0.1 mg/kg/day in nonsensitized recipients. PMID- 1726567 TI - Effect of human alpha-fetoprotein on native and in vitro-stimulated NK activity. AB - We have examined the effect of human alpha-fetoprotein (h-alpha FP), at concentrations ranging from the physiological to pathological circulating levels, on the native and alpha IFN- and IL2-boosted NK activity of normal peripheral blood lymphocytes (PBL). The cytotoxic function of unstimulated PBL was unchanged after a 16 hr incubation with 200 to 8000 ng/ml h-alpha FP. By contrast, this treatment significantly reduced the responsiveness of PBL to the NK-enhancer factors IFN and Interleukin 2. Optimal inhibition was observed when cells were pre-incubated with h-alpha FP before being stimulated with IFN. The sensitivity of PBL to the h-alpha FP mediated inhibition was restricted to the very early times (30 min) of incubation with the stimulating agents. PMID- 1726568 TI - Human brucellosis: immunoblotting analysis of three Brucella abortus antigenic fractions allows the detection of components of diagnostic importance. AB - Results indicating that analysis of the immune humoral response of brucellosis patients by immunoblotting provides useful information for the characterization of antigenic fractions of possible diagnostic importance in human brucellosis are presented. Sera of 90 patients were obtained: 23 suffering from chronic brucellosis, 20 from acute brucellosis and 47 belonging to the group of serologically positive individuals (SPI) without clinical evidence of active infection at the time of examination, and 35 healthy volunteers. They were tested against three antigenic fractions: cytoplasmic (CYT), outer membrane (OM) and inner membrane (IM). These fractions, which include virtually all the bacterial protein components, were prepared from Brucella abortus 1119/3 strain by detergent solubilization, enzymatic digestion and ultracentrifugation. Results obtained with these fractions showed the existence of antigens that permit the detection of brucellosis patients and their differentiation from SPI patients, with very high sensitivity. PMID- 1726569 TI - [The importance of methylation reactions in the biochemistry of catecholaminergic and serotoninergic neurons]. PMID- 1726570 TI - Adsorption staining of freeze-substituted and low temperature embedded frog skeletal muscle with cesium: a new method for the investigation of protein-ion interactions. AB - A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs(+)-ions which are known to accumulate in living cells like K(+)-ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be stained with electron-dense Cs(+)-ions: protein sites of preferential ion adsorption are visualized. These sites are similar to those accumulating monovalent ions in living cells as had been shown previously with frozen-hydrated preparations. An observed pH-dependency of the adsorption staining is consistent with the view that the ion adsorption sites are beta- and gamma-carboxyl groups of cellular proteins. The results obtained so far indicate that the new method can be used to investigate weak interactions between cellular proteins and different ions by electron microscopic methods. PMID- 1726571 TI - The matrix of urinary tract stones: protein composition, antigenicity, and ultrastructure. AB - We have extracted proteins from urinary tract stones by electrodialysis and have developed antisera to the core and the shell of a renal stone. The protein composition varies between stones but is identical in the core and the shell of the same stone. One stone antigen is present in the urine of normal individuals and stone formers, as well as in cholesterol gallstone extracts. Electron microscopy of the core of a urate-calcium oxalate stone before and after demineralization reveals a fibrillar structure associated with mineral deposits, as well as aggregates of crystals. PMID- 1726572 TI - The development of the vascular system in quail embryos: a combination of microvascular corrosion casts and immunohistochemical identification. AB - Although vascular casts, obtained by injection with methacrylates, are frequently used to investigate the adult vascular system, little data are available for embryonic stages. In this paper we use Mercox in quail embryos in the period of 2 to 7 days after incubation. The microvascular corrosion casts were evaluated in the scanning electron microscope (SEM) with special attention to the development and remodelling of the large arteries and veins. Our results show that the remodelling of the large arteries and veins together with their developing tributary vessels can be visualized from very early embryonic stages onwards. However, complete replication of a developing vascular system depends on diameter and regularity of the lumen. In the stages investigated, the vascular lumen, even of the largest vessels, is still very irregular. Detailed cellular characteristics like nuclear impressions of endothelial cells, as often seen in adult material, were seldom found in the embryos. To examine whether blind-ending sprouts are completely or incompletely replicated in a developing vascular system, additional series of quail embryos were stained immunohistochemically with a monoclonal antibody (MB1) specific for endothelial and hemopoietic cells. It seems that a plexus consisting of endothelial precursors (endothelial cells lacking a lumen) is present in the developing organ before the formation of a lumen and assembly into vessels, which are connected to an adjacent artery or vein. Expansion of the vascular system may in part be due to incorporation of these endothelial precursors in the wall of existing vessels. PMID- 1726573 TI - [Use of acetocarmines for staining of larvae of digenetic Trematoda in situ]. AB - Qualities of microscopical pictures of digeneic larvae total slides stained with different acetocarmines were compared. Semichon's acidifying carmine for topographical staining, Schneider's and Belling's carmines for chromosomes as well as iron acetocarmine (modification of the last two) were used. The last stain permits to obtain the slides, that in respect of their colour and quality of microscopical picture may be compared with slides stained with Semichon's carmine. Contrast, colour and reaction of staining depend on concentration of iron acetate in the staining solution. Total slides of investigated larvae stained respectively with Schneider's and Belling's carmines are not enough readable. PMID- 1726574 TI - [Detection of pneumococcal capsular polysaccharide antigen in urine by counterimmunoelectrophoresis. Technical features and correlation with serotype]. AB - The effectiveness of CIE in detecting capsular polysaccharide antigen of pneumococci in urine is revised. Using CIE, we studied urine samples from 57 patients with systemic pneumococcal infections, proved bacteriologically by means of isolation of the microorganisms from sterile sites. We compare the usefulness of CIE with the microorganism isolation from organic products. We also determine the sensitivity gain after passive diffusion at 4 degrees C and bright blue Coomasie staining (CBB R-250). We correlate the CIE results with the serotype of isolated pneumococci. CIE was positive in 17 of all 35 pneumonia cases (48.6%), in 2 of all 5 bacteremias (40%), 4 of all 15 meningitis (26.7%) and in none of all two peritonitis. Direct urine examination was positive in 12 out of 23 patients with positive CIE (52.2%) and concentrated urine in 22 patients (95.6%). Passive diffusion at 4 degrees C and CBB R-250 stain increases the positive rate of the test. The correlation of serotype with our results is difficult due to the wide variety of serotypes identified. However, particular serotypes such (3, 4 or 8) had been identified with increased sensitivity. Global sensitivity of CIE in detecting capsular polysaccharide antigen in urine samples is not high enough (40.3%), even under the best circumstances. Antigen detection in urine is more sensitive than blood cultures, and therefore we believe that could be used in clinical microbiology laboratories until a more effective method is available. PMID- 1726575 TI - [Utility of the L-alanine-aminopeptidase test for differentiating the cell wall structure of bacteria]. AB - Evaluation of a detection test for L-alanine-aminopeptidase enzyme (Bactident Aminopeptidase) for determining the structure of bacterial cell wall. In a total of 246 clinical isolates of aerobic, microaerophilic and anaerobic bacteria, we detect the presence or absence of L-alanine-aminopeptidase using commercial kits (Bactident Aminopeptidase, Merck Diagnostica). We also identify and further classified the 246 strains. In nearly all gram-negative bacteria L-alanine aminopeptidase was found, with the exception of Campylobacter spp and gram negative anaerobic bacilli. All gram-positive and gram-variable bacteria were negative for the L-alanine-aminopeptidase presence. The results suggest a good correlation between the presence or absence of L-alanine-aminopeptidase and Gram stain method. PMID- 1726576 TI - Characterization of polyamino acids by use of GPC-viscometry technology. AB - The properties of polybenzyl glutamate, a well known polyamino acid vary considerably with molecular weight in both solid state and in solution. Therefore, accurate determinations of the molecular weights of these polyamino acids is essential. The dual viscometer/refractometer when used as detector system for size exclusion chromatography provides a way of determining accurate molecular weights. An indirect method of determining the molecular weight distribution (MWD) and the radius of gyration distribution (RgD) of polybenzyl glutamate is described. The MWD is calculated from the measured value of intrinsic viscosity (IV) and the known IV-to-MW relationship, at every SEC retention volume slice. Such a technique of determining MWD requires no calibration and is more precisely measurable than conventional SEC methods. PMID- 1726577 TI - Design of liposomes for circumventing the reticuloendothelial cells. AB - Two different aspects of liposomal drug delivery to non-RES cells have been described. In one of the systems, by incorporating neutral glycolipids, with terminal beta-galactoside residue into liposomes, it is possible to target liposomes to the liver parenchymal cells, partially bypassing the RES. Asialoganglioside seems to be the most suited for this purpose. In another approach, various factors that prolong the lifespan of circulating liposomes have been discussed. It is possible to design such liposomes by imparting hydrophilicity to the liposomal surface. The effectiveness of a number of possible candidates, such as dextran, GM1 ganglioside and PEG, has been discussed in this context. PMID- 1726578 TI - Biosynthesis in vitro of SA-Lex and SA-diLex by alpha 1-3 fucosyltransferases from colon carcinoma cells and embryonic brain tissues. AB - The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAc alpha 2 3Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta 1-3Gal beta 1-4Glc-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an alpha 1-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-substituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4 Glc-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726579 TI - LINEs. AB - LINEs are ubiquitous transposable elements of eukaryotes. Significant information has accumulated in the past year concerning the mechanism of transposition and the intermediates involved. In addition, progress has been made in the understanding of LINE structure and evolution, and several laboratories have exploited the interspersed, repetitive nature of LINEs for use in genome mapping. PMID- 1726580 TI - Ionic conductance mechanisms contributing to the electrophysiological properties of neurons. AB - Neurons have a multiplicity of ionic conductance mechanisms, the interactions of which determine in part the response of a neuron to chemical and electrical synaptic interactions, firing patterns, excitability, membrane potential and action-potential waveform. Several papers published in the past year have provided important new information on the role that ionic conductance mechanisms play in determining the electrophysiological properties of neurons, and how subtle differences can contribute to considerable variability of response. PMID- 1726581 TI - Signalling mechanisms. PMID- 1726582 TI - Receptors coupled to ionic channels: the glutamate receptor family. AB - Glutamate-gated ion channels belong to a complex family of receptors containing several pharmacological subtypes. They are thought to be essential for the acquisition of associative memory and for activity-dependent synaptogenesis, and have been implicated in several central nervous system diseases. Within the past year, molecular cloning of the first glutamate receptor channel and several related subunits has opened new approaches for understanding the basis of these important phenomena. PMID- 1726583 TI - Ionic channels: modulation by G proteins and by phosphorylation. AB - The gating of ion channels may be modulated by G proteins or by phosphorylation. Direct coupling between G proteins and ion channels has been shown in excised patches of membrane. Steps must now be taken to study the protein domains of G proteins and ion channels involved in the mutual interaction. The concept of channel modulation by protein kinases has recently been extended to include additional types of ion channel. PMID- 1726584 TI - Capsaicin and substance P. PMID- 1726585 TI - Malignant melanoma: biotherapeutic strategies for management with the interferons. PMID- 1726586 TI - Cytokines in anticancer therapy. PMID- 1726587 TI - Phyllogenetically conserved epitopes of the keratin 8 polypeptide recognized by a novel set of monoclonal antibodies. AB - In an attempt to raise a set of monoclonal antibody (MAb) probes for the study of biology and diagnostic value of human keratin No. 8, a series of hybridomas was prepared and their characteristics investigated by immunohistochemistry and immunoblotting. The polypeptide specificities of individual MAbs ranged from monospecificity for keratin No. 8 (MAbs C-15, C-23, C-36, C-43, C-47, C-51, and C 61) up to the "pan-keratin" MAbs C-11 and C-66 recognizing a broad range of keratins. The target epitopes of the remaining 4 MAbs (C-10, C-22, C-50, C-69) appear to be shared by various pairs of keratin polypeptides. The immunohistochemical investigation of tissues and/or cultured cells from 10 animal species revealed considerable variability of the interspecies crossreactivity of individual MAbs. The target epitopes of 5 MAbs appear to be phyllogenetically conserved from man to Xenopus, 6 of 12 MAbs tested show reactivity with several mammalian species (but not chicken or Xenopus) and the MAb C-15 reacts with human and sheep keratin only. The data obtained demonstrate that at least 9, and possibly as many as 12, nonidentical epitopes of the human keratin 8 polypeptide can be distinguished by this set of antibodies. The results of the present study suggest that our MAbs represent a significant contribution to the list of reagents applicable in both research of intermediate filament (IF) biology and diagnostic histopathology. PMID- 1726588 TI - Independent emergence of a vaccine-induced escape mutant of hepatitis B virus. AB - We investigated the case of a child who was vaccinated at birth against hepatitis B and was also given hepatitis B immune globulin, but who nevertheless later became infected with the virus. Hepatitis B virus-specific deoxyribonucleic acid covering the region of the genome encoding the predominant a epitope of hepatitis B surface antigen was amplified using the polymerase chain reaction and the nucleotide sequence determined. We present evidence for the independent emergence of an escape mutant, similar to that previously identified in Italy, where a substitution of arginine for glycine has occurred in the second loop of the a epitope. PMID- 1726589 TI - Sequence heterogeneity of the hepatitis C virus envelope region. Implications for the location of predicted antigenic determinants. PMID- 1726590 TI - Reverse transcription and packaging of hepatitis B virus (HBV)-RNA generates in vivo defective serum HBV particles. PMID- 1726591 TI - Mutations in the S-gene affecting the immunologic determinants of the envelope protein of hepatitis B virus. PMID- 1726592 TI - Characterization of a Ca(2+)-dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers. AB - 1. Anion-selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation-regulated chloride transport in this tissue, whilst a calcium-dependent pathway appears to function normally. 2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion-selective channel from sheep tracheal epithelium. 3. In symmetrical 200 mM-NaCl solutions the channel showed rectification, with a chord conductance at negative voltages of 107 pS and at positive voltages of 67 pS. The channel characteristically demonstrated subconductance states at 1/3 and 3/4 of the fully open level. Selectivity for chloride over sodium was approximately 6:1. 4. The channel required a minimum of approximately 100 microM-calcium on the presumed cytoplasmic surface (cis) for opening events to be observed. Open probability (Po) of the fully open state was markedly voltage dependent, but little effect of voltage was seen on the 1/3 subconductance state. 5. The relative permeabilities of monovalent anions monitored under bi-ionic conditions gave the following sequence: NO3- greater than I- greater than Cl- = Br- much much greater than F-. The order of conductances in symmetrical solutions was Cl- = NO3- greater than Br greater than I- much much greater than F-. 6. The chloride channel blocker 5 nitro-2-(3-phenylpropylamino)-benzoate (NPPB) produced a dose-related reduction in Po with a flickering block at 10-50 microM and complete block at higher concentrations. 7. ATP produced a dose-related reduction in Po with effects at 1 microM and complete closing at 1 mM. These effects were only seen with addition to the cis chamber. 8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect. 9. Channels with very similar properties were seen from transfected human tracheo-bronchial cells. 10. Recent whole-cell patch-clamp studies have suggested a distinct calcium-activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single-channel basis. PMID- 1726593 TI - Properties of the late transient outward current in isolated intestinal smooth muscle cells of the guinea-pig. AB - 1. Whole-cell membrane currents in voltage-clamped single isolated cells of longitudinal smooth muscle of guinea-pig ileum were studied at room temperature using patch pipettes filled with either high-K+ solution or high-Cs+ solution, to suppress K+ outward current, and containing 0.3 mM-EGTA. 2. In the presence of high-K+ solution in the pipette, membrane depolarization from the holding potential of -50 mV evoked an initial inward calcium current (ICa) followed by a large initial transient outward current and a sustained outward current with spontaneous oscillations superimposed. Prolonged depolarization above -20 mV produced a late transient outward current which reached a maximum (up to several nanoamps at +10 mV) within approximately 1 s and lasted several seconds. 3. The late outward current (ILTO) was voltage dependent and reversed at the EK (potassium equilibrium potential) in cells exposed to high-K+ external solution. It was blocked by TEA+ (tetraethylammonium) or Ba2+ applied externally (calculated Kd (dissociation constant) values were 0.67 and 4.43 mM, respectively) or by high-Cs+ solution perfusing the cell. The removal of extracellular Ca2+, application of Ca2+ channel blockers (3 mM-Co2+, 0.2 mM-Cd2+ or 1 microM-nifedipine) or perfusion of 5 mM-EGTA inside the cell also abolished the current. Thus, the current seems to be a Ca(2+)-activated K+ current. 4. There is a great discrepancy between the time course of the ICa and that of the late ILTO, which suggests that Ca2+ release from intracellular storage sites may contribute to the generation of the ILTO. 5. Bath application of caffeine (10 mM) during the development of ILTO enhanced the current. However, in the presence of caffeine ILTO was inhibited. Moderate inhibition of ICa by caffeine was also observed. 6. Ryanodine (5 microM) applied to the bathing solution completely inhibited ILTO within 3.5 min; however, it had no or little effect on the ICa. 7. Ruthenium Red (10 microM) completely blocked the ILTO and slightly and more slowly inhibited the ICa. 8. Increasing Mg2+ concentration in the pipette solution from 1 to 6 mM abolished the ILTO. 9. It was concluded that the ILTO was activated mainly by Ca2+ released from the intracellular storage sites following Ca2+ entry, presumably by a Ca(2+)-induced Ca2+ release mechanism. PMID- 1726594 TI - Voltage-dependent block by magnesium of neuronal nicotinic acetylcholine receptor channels in rat phaeochromocytoma cells. AB - 1. The effects of Mg2+ on the single-channel conductance of neuronal nicotinic acetylcholine receptors were examined using receptors expressed by the rat phaeochromocytoma cell line, PC12. PC12 cells express at least three conductance classes of channels that are activated by acetylcholine, the largest conductance class being the most prevalent. This receptor channel is blocked by intracellular and extracellular Mg2+. 2. The effects of Mg2+ are asymmetrical; at a given concentration, internal Mg2+ is more effective at blocking outward currents than external Mg2+ is at blocking inward currents. Receptor channels are blocked at concentrations of Mg2+ that are low compared to the concentration of the main permeant cation, Na+, and the block is voltage dependent. 3. The block by Mg2+ is not complete as Mg2+ can permeate the channel. With 80 mM-extracellular Mg2+ (no extracellular Na+), the channel has an inward slope conductance of 2.9 pS. 4. The block by extracellular Mg2+ can be described by a one site, two barrier model for the channel which includes a negative surface charge on the external surface of the membrane. The parameters of the model place the binding site for Mg2+ at 52% of the membrane field from the outside with an apparent dissociation constant of 14 mM. However, the same parameters cannot describe the block by intracellular Mg2+. The deviations from the model suggest that the receptor channel may have more than one binding site for Mg2+. PMID- 1726595 TI - Mechanical and morphological properties of chronically inactive cat tibialis anterior motor units. AB - 1. The lumbar spinal cord was functionally isolated in ten cats by cord transection at the junctions of segments T12-T13 and L7-S1 and cutting bilaterally all dorsal roots between the two transections. Two 24 h EMG recording sessions were used to verify that muscles in the lower limb were virtually electrically silent. The cats were maintained in excellent health for 6 months. 2. Six months after spinal cord isolation, an acute experiment was performed to isolate a single motor unit from the tibialis anterior of each hindlimb using ventral root splitting techniques. Each motor unit was characterized physiologically as either fast fatigable (FF, n = 11), fast fatigue resistant (FR, n = 4), fast intermediate (FI, n = 2), or slow (S, n = 1), and repetitively stimulated to deplete the motor unit of its glycogen. 3. Maximum tensions of the fast motor units were lower than mean maximum tensions of control, whereas the S motor unit remained within the range observed in controls. In general, the isometric contractile properties, as well as fatigability, were within the ranges for each of the motor unit types in control cats. The mean fibre cross-sectional areas of the fibres within the FR and FF motor units were approximately 40 and 50% smaller than control, while the mean fibre size of the fibers within the S motor unit was similar to control. 4. Innervation ratios and specific tensions for all experimental motor units were within the ranges of those reported for tibialis anterior motor units in control cats. Thus, it appears that the decrease in maximum tension of the fast motor units was primarily related to a reduction in fibre size. 5. The spatial distribution of the fibres within fast motor units of a spinally isolated cat, as measured by interfibre distances of the motor unit fibres, was similar to that reported for control tibialis anterior motor units. 6. These data suggest that factors independent of activity play a prominent, if not dominant, role in maintaining the complement of motor unit types typical of adult cat muscles. In addition, normal innervation patterns appear to be maintained in the absence of activity. PMID- 1726596 TI - Three types of bovine chromaffin cell Ca2+ channels: facilitation increases the opening probability of a 27 pS channel. AB - 1. Cell-attached patch recordings from bovine chromaffin cells were performed with 90 mM-Ba2+ in the patch pipette and with isotonic potassium aspartate in the bathing solution to zero the membrane potential. Three different types of unitary Ca2+ channel activity could be distinguished in these recordings. 2. A 27 pS Ca2+ channel was distinguished by constructing amplitude histograms and measuring slope conductance. This channel activated over a broad range of potentials (depolarizations greater than -10 mV). 3. A second Ca2+ channel with a slope conductance of 14 pS could also be detected with amplitude histograms. This channel activated with depolarizations greater than -20 mV. 4. An 18 pS Ca2+ channel was observed infrequently indicating that this channel may carry only a small amount of the whole-cell current. This 18 pS channel was sensitive to changes in holding potential. Depolarizing the patch to +10 mV from a holding potential of -80 mV elicited robust unitary activity. Changing the patch holding potential to -40 mV while maintaining test depolarizations to +10 mV completely inactivated the 18 pS channel. Neither the 25 pS nor the 14 pS Ca2+ channels were affected by changes in holding potential in the range from -80 mV to -40 mV, indicating the 18 pS channel was a different type of channel. As the 18 pS channel was observed so infrequently, no detailed studies of it were possible. 5. Chromaffin cell Ca2+ currents exhibited facilitation. Large pre-depolarizations greatly augmented whole-cell currents observed in these cells. Whole-cell currents could double or triple after recruiting facilitation. The application of large pre-depolarizations altered the gating behaviour of the 27 pS Ca2+ channel manifested as dramatically increased channel opening probabilities measured during subsequent test pulses. Large pre-depolarizations induced unitary activity in the 27 pS Ca2+ channel similar to the long-lived openings exhibited by L-type Ca2+ channels in the presence of Bay K 8644. Large pre-depolarizations did not change the gating behaviour of the 14 pS Ca2+ channel. 6. Repetitive depolarizations in the physiological range could also induce facilitation. At the single-channel level facilitation was manifested as a striking increase in opening probability of the 27 pS Ca2+ channel. No effect of repetitive activity was observed on 14 pS channel gating. At the whole-cell level, repetitive depolarizations dramatically increased the current observed. 7. Facilitation of 27 pS Ca2+ channel activity could be induced by changing the holding potential to a depolarized level (greater than or equal to -10 mV).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1726598 TI - Excitation-contraction coupling in skeletal muscle fibres of rat and toad in the presence of GTP gamma S. AB - 1. Rapid force responses were elicited in single mechanically skinned fibres from extensor digitorum longus (EDL) muscle of the rat when the fibres were depolarized by substituting K+ in the bathing solution with Na+. The properties of these depolarization-induced responses, the responses to lowered [Mg2+], and the characteristics of the slow prolonged response ('second component') produced in 'loaded' fibres by choline chloride (ChCl) substitution, were virtually identical to those observed previously in skinned fibres from toad muscle. 2. At physiological levels of [Mg2+] (1 mM) and Ca2+ loading, application of 50 microM- to 1 mM-GTP gamma S (guanosine-5'-O-(3-thiotriphosphate), a non-hydrolysable analogue of GTP) did not produce a response in any mammalian or amphibian fibre, even though the depolarization-induced coupling was totally functional. Furthermore, the presence of GTP gamma S had no apparent effect on the size, the threshold or the maximum number of responses which could be elicited by depolarization. 3. GTP gamma S did not elicit any response when excitation contraction coupling was abolished by prolonged depolarization or by chemically skinning the fibre with saponin or by 24 h exposure to low [Ca2+] (5 mM-EGTA). 4. GDP beta S (guanosine-5'-O-(2-thiodiphosphate), 250 microM or 1 mM) neither evoked a response nor affected the responses to depolarization or caffeine. 5. When the [Mg2+] was lowered to 0.2 mM and the fibres were heavily loaded with Ca2+, addition of GTP gamma S (250 microM or 1 mM) induced a small response in about 50% of fibres, but depolarization-induced responses were not affected in any fibres. 6. Asymmetric charge movement recorded in EDL fibres with the vaseline-gap voltage clamp was not affected by the application of 1 mM-GTP gamma S to the cut ends of the fibres for up to 1 h. 7. These data imply that GTP binding proteins (G-proteins) are not involved in coupling the voltage sensors to Ca2+ release in skeletal muscle. Furthermore, there was no evidence that G proteins play any role in modulating the voltage sensors, though this possibility could not be totally excluded. PMID- 1726597 TI - Influence of perivascular peptides on endoneurial blood flow and microvascular resistance in the sciatic nerve of the rat. AB - 1. A variety of vasoactive peptides has been identified in the axon terminals innervating vasa nervorum but their function is unknown. In mesenteric arterioles, substance P (SP) and calcitonin gene-related peptide (CGRP) have been postulated to have a role in tonic vasodilatation. 2. We explored the effect of epineurial capsaicin, SP, CGRP, spantide (SP antagonist), and hCGRP (8-37) (CGRP antagonist) on blood flow (EBF) and microvascular resistance (EMR) in the endoneurial compartment of the rat sciatic nerve, as measured by hydrogen clearance. 3. Epineurial capsaicin induced a prompt, intense and prolonged increase in EBF and lowering of EMR as compared to epineurial application of the carrier alone in a separate animal group. The hyperaemic response was also confirmed by studying serial clearance curves in individual animals. 4. Multifibre sciatic-tibial motor conduction was not changed by epineurial capsaicin. 5. When co-administered with capsaicin, hCGRP (8-37) completely blocked the hyperaemic response and increased EMR above the pooled control range. Spantide also blocked the capsaicin response. 6. When administered alone, both epineurial hCGRP (8-37) and spantide lowered EBF below and increased EMR above the control measurements in the same animals. 7. At 10(-5) M epineurial CGRP, but not SP lowered EMR. Vasodilatation from intra-arterial administration of CGRP was much greater and was more prolonged compared with that induced by SP. hCGRP (8 37), but not spantide reduced the intra-arterial response to CGRP. 8. The findings suggest that epineurial peptidergic terminals mediate a vasodilatory response (particularly through CGRP) that increases blood flow in the 'downstream' endoneurial compartment. Physiological peptide release (blocked by SP and CGRP receptor antagonism) may be important in maintaining tonic vasodilatation. PMID- 1726599 TI - Calcium oscillations in guinea-pig pancreatic acinar cells exposed to carbachol, cholecystokinin and substance P. AB - 1. Cytoplasmic Ca2+ ([Ca2+]i) responses were studied in guinea-pig pancreatic acinar cells during stimulation with cholecystokinin octapeptide (CCK-8), substance P (SP) and carbachol. 2. Individual cells exhibited [Ca2+]i responses to all three agonists. 3. In the absence of external Ca2+, all the agonists initiated [Ca2+]i peaks which, particularly at high agonist concentrations, rapidly declined. 4. SP induced repetitive monophasic [Ca2+]i transients which started from basal [Ca2+]i even after elevation of the external Ca2+ concentration. 5. CCK-8 triggered similar oscillations, which particularly at high agonist concentration or after elevating external Ca2+ became superimposed upon a sustained elevation of [Ca2+]i. 6. Carbachol-induced oscillations were more complex with [Ca2+]i transients superimposed on slower waves. 7. At high carbachol concentrations or elevation of external Ca2+ the slow waves fused into a sustained increase of [Ca2+]i. 8. The protein kinase C (PKC) activator 12-O tetradecanoylphorbol-13-acetate attenuated the agonist-induced [Ca2+]i responses, and this effect was reversed by the PKC activator staurosporine. 9. The results indicate that oscillations of [Ca2+]i induced by SP, CCK-8 and carbachol involve intracellular mobilization of Ca2+. 10. CCK-8 and carbachol also cause a rise of [Ca2+]i by a mechanism more directly dependent on the presence of extracellular Ca2+. 11. In the case of carbachol the latter component is subject to oscillatory control. 12. The transition from oscillatory [Ca2+]i to sustained increase may be associated with inhibition of amylase release. PMID- 1726600 TI - Effect of a prostacyclin analogue on the vasopressin and oxytocin secretion in vitro. AB - Iloprost (ZK 36374; a stable prostacyclin analogue) increases basal as well as potassium-evoked vasopressin and oxytocin secretion from rat neurointermediate lobes in vitro. This finding suggests a possible regulatory role of endogenous prostacyclin in the release of neurohypophysial hormones. PMID- 1726601 TI - The lack of influence of some neuropeptides present in the posterior pituitary lobe on the frequency of spontaneous contraction of the isolated heart auricle. AB - Investigations have shown the presence of a cardiodepressant factor in the fluid incubating the posterior pituitary lobe "in situ", which decreased contraction frequency of the isolated heart auricle (Acta Physiol. Pol., 1984, 35: 460-468). The influence on the spontaneous contraction frequency of the isolated heart auricle of the following synthetic neuropeptides was determined: substance P, leu enkephalin, met-enkephalin, angiotensin II, arg-vasopressin, oxytocin, delta sleep-inducing peptide and atrial natriuretic factor. It was found that the investigated neuropeptides had no effect on the contraction frequency of the isolated auricle of the heart right atrium of two-day-old rat in a concentration from 2.1 x 10(-7) to 1 x 10(-3) mol/l in the bathing medium and it was concluded that their biological properties differ from the cardiodepressant factor. PMID- 1726602 TI - [Detection of cytomegalovirus in urine by the shell-vial technique: usefulness of diluting the sample]. AB - To assess the toxicity of urine samples on human fibroblast cell monolayer using Shell-vial technique for cytomegalovirus antigen detection. A total of 227 urine samples were processed, inoculated on Shell-vial simultaneously, being either diluted (1:1) and non diluted in transport media. After 24-48 hours incubation, the fibroblast cells monolayer was stained using fluorescein-marked monoclonal anti-cytomegalovirus antibodies. Cytomegalovirus was detected in 9.6% of samples inoculated with diluted urine, and in 5.2% of samples inoculated with non-diluted urine. Destruction of monolayer cells was seen in 13.2 and 22.4, respectively. With Shell-vial technique for detecting cytomegalovirus in urine samples, a high number of positive results was obtained using diluted urine. Also a significant minor destruction of cells monolayer was observed with the same inoculum. PMID- 1726603 TI - Newborn basal forebrain lesions disrupt cortical cytodifferentiation as visualized by rapid Golgi staining. AB - We have previously shown that neonatal lesions of the basal forebrain cholinergic afferents result in transient cholinergic depletion concomitant with abnormal morphogenesis of cerebral cortex in Balb/CByJ mice (Hohmann et al., 1988). Here, we have utilized the rapid Golgi method to further characterize these previously observed abnormalities. We compared layer V pyramidal neurons in somatomotor cortex ipsi- and contralateral to the lesion at postnatal days (PND) 7 and 14. Quantitative evaluations showed a significant reduction in all aspects of the dendritic tree as well as in cell body size in ipsilateral cortex at PND 7. Differences between ipsi- and contralateral pyramidal cells had attenuated by PND 14, but significant somatic size differences persisted, as did changes in the apical branching pattern. Qualitative differences between ipsilateral and contralateral hemispheres included the relatively more immature appearance of ipsilateral neurons at both ages, in addition to unusual dendritic morphology, particularly at PND 14. A close correlation was apparent between the magnitude of cholinergic depletion in cortex (larger at PND 7 than at PND 14) and the severity of abnormalities in pyramidal cell morphogenesis. We conclude that a normal cholinergic innervation to neocortex is instrumental in the timely differentiation of cortical neurons, because neonatal nBM lesions disrupted the time schedule of differentiation, but did not preclude the pyramidal neurons from further differentiation at a later time. PMID- 1726604 TI - Efferent and afferent connections of mouse sensory-motor cortex following cholinergic deafferentation at birth. AB - The present study investigates the effect of cholinergic basal forebrain lesions at birth on cortical connectivity in adulthood. We have previously shown that such neonatal lesions result in extensive cortical cholinergic deafferentation during early postnatal development, which is accompanied by abnormal morphogenesis of cortical cytoarchitecture (Hohmann at al., 1988). Here, we have used WGA-HRP to label anterogradely and retrogradely afferent and efferent projections of dorsal neocortex. Our results show an altered projection pattern from dorsal thalamus to layer IV of sensory-motor cortex following lesions among the cholinergic basal forebrain neurons (nBM), while corticothalamic projections from layer VI appear normal. In addition, corticofugal projections from layer V, labeled by striatal injection, appear to be expanded following the lesion. This indicates that cortical layers undergoing differentiation after the newborn nBM lesion present with long-term abnormalities in connectivity. The present results are compatible with the hypothesis that cholinergic afferents are instrumental in the regulation of cortical morphogenesis. Furthermore, our data show that ontogenetic disturbances can lead to structural abnormalities that persist long after the initial deficiency has abated. We discuss the significance of these results in relationship to human neurological disorders. PMID- 1726605 TI - Interhemispheric integration: I. Symmetry and convergence of the corticocortical connections of the left and the right principal sulcus (PS) and the left and the right supplementary motor area (SMA) in the rhesus monkey. AB - The relationship between the termination zones of projections from paired homotopic areas in the frontal lobe was examined in the cerebral cortex of the macaque monkey. Injections of WGA-HRP and tritiated amino acids were made in topographically matched regions of the principal sulcus (PS) or the supplementary motor area (SMA) in each hemisphere, such that the projections from the same area on each side were differentially labeled in the same animal. Adjacent sections through the cortical regions that received bilateral inputs from these areas were processed for the respective tracers, permitting the relationship between the converging projections to be defined. Comparison of the cortical connections of the left and right PS or of the left and right SMA yielded two major findings. First, only minor differences in the topographic distribution and strength of connections of homotopic areas were observed, providing little evidence of asymmetry in the connections of either the PS or the SMA in the macaque. Second, with the exception of interdigitation observed in a portion of the dorsal bank of the PS, the cortical projections from both the left and the right PS and SMA converged (overlapped) in common columnar territories. These termination patterns allow for a remarkable degree of interhemispheric integration. PMID- 1726606 TI - Interhemispheric integration: II. Symmetry and convergence of the corticostriatal projections of the left and the right principal sulcus (PS) and the left and the right supplementary motor area (SMA) of the rhesus monkey. AB - The relationship between the termination zones of projections from paired homotopic areas of the frontal lobes was examined in the caudate nucleus and the putamen of the macaque monkey. Injections of WGA-HRP and tritiated amino acids were made in topographically matched regions of the principal sulcus (PS) or of the supplementary motor area (SMA) in each hemisphere, such that the projections from the same area on each side were differentially labeled in the same animal. Adjacent sections through the neostriatum were processed for the respective tracers, permitting the relationship between the converging projections to be defined. The topographic distribution and strength of ipsilateral corticostriatal projections observed for separately labeled left and right hemispheres were strikingly similar. Projections from the left and the right PS terminated preferentially in central parts of the left and right neostriata, respectively, while projections of the left and right SMAs terminated preferentially in dorsolateral parts of respective left and right neostriata. Therefore, little evidence for asymmetry of corticostriatal projections was found. The projections of the left and the right PS to the same neostriatum were also compared. Remarkably, whether in the left or right hemisphere, projections from the left and the right PS were in precise register in topographically specific territories of the caudate and putamen. Likewise, projections of the left and right SMAs converged in both the left and right neostriata. Such convergence allows for a remarkable degree of interhemispheric integration in the descending corticostriatal networks. PMID- 1726607 TI - Layer IVA of rhesus monkey primary visual cortex. AB - Layer IVA of rhesus monkey striate cortex contains pyramidal cells arranged in distinct groups. Their cell bodies are in a configuration of flat cones, each with an average diameter of 60 microns, and their apical dendrites aggregate into bundles that ascend toward the pial surface. Nissl-stained sections suggest that these pyramidal cell cones have their bases in layer IVB, with their tops extending into layer IVA. The neurons in the cones are readily apparent in MAP2 antibody-stained material, and in cytochrome oxidase-reacted tissue it is evident that the pyramidal cell cones occupy the pale spaces that are surrounded by the darkly reactive honeycomb lattice. This lattice of neuropil around the cones contains some axons and boutons that are immunoreactive for parvalbumin, and it is within the lattice that other investigators have shown afferents from the parvocellular (P)-layers of the dLGN to terminate. Because of this input, it is likely that the pyramidal cell cones of layer IVA are involved with color and form perception. The relationship between the layer IVA cones of neurons and the underlying system of previously described pyramidal cell modules (Peters and Sethares, 1991) is discussed, as well as the possibility that the pyramidal cell cones might represent aggregations of neurons, which receive input from basic sets of P-like afferents originating from color-responsive ganglion cells of the retina, as described by Schein and de Monasterio (1987). PMID- 1726608 TI - Occurrence of keratinophilic fungi on Indian birds. AB - Keratinophilic fungi were isolated from feathers of most common Indian birds, viz. domestic chicken (Gallus domesticus), domestic pigeon (Columba livia), house sparrow (Passer domesticus), house crow (Corvus splendens), duck (Anas sp.), rose ringed parakeet (Psittacula krameri). Out of 87 birds, 58 yielded 4 keratinophilic fungal genera representing 13 fungal species and one sterile mycelium. The isolated fungi were cultured on Sabouraud's dextrose agar at 28 +/- 2 degrees C. Chrysosporium species were isolated on most of the birds. Chrysosporium lucknowense and Chrysosporium tropicum were the most common fungal species associated with these Indian birds. Maximum occurrence of fungi (47%) was recorded on domestic chickens and the least number of keratinophilic fungi was isolated from the domestic pigeon and duck. The average number of fungi per bird was found to be the 0.44. PMID- 1726609 TI - Amplification of invasiveness in organotypic cultures after NBT-II rat bladder carcinoma stimulation with in vitro scattering factors. AB - Acidic fibroblast growth factor (aFGF) or transforming growth factor-alpha (TGF alpha), in addition to being mitogenic, induce individual scattering of NBT-II rat bladder carcinoma cell clusters on tissue culture dishes, suggesting that they may contribute to tumor cell dissemination. To assay their scattering potential and their effect on cell invasiveness in a more complex and physiologically relevant model, we analyzed the behavior of NBT-II spheroids confronted with urinary bladder in organotypic cultures. NBT-II spheroids progressively replaced the urothelium at the site of contact with the bladder explant. In the absence of aFGF or TGF-alpha, inserted cells grew in a pattern suggestive of local hyperplasia, with occasional invasive cell protrusions. Exogenous scattering growth factors elicited a more rapid appearance of these protrusions, which were also more numerous. NBT-II cells transfected with cDNA constructs bearing the gene of aFGF, TGF-alpha or the oncogene hst/KFGF were also used. After exogenous or autocrine stimulation of NBT-II cells with the growth factors, a deeper penetration of the bladder wall in the form of nodular outgrowths and clusters of infiltrating cells was always observed. Altogether these observations suggest that the stimulation of NBT-II clusters by scattering/growth factors can promote cell shedding and amplify invasiveness in the complex extracellular environment of bladder tissues. PMID- 1726610 TI - Reduced tenascin expression in colonic carcinoma with lymphogenous metastasis. AB - We have studied expression of tenascin (TN) in colonic carcinoma cells from 81 patients with colonic carcinoma without (20) and with (61) lymphogenous metastases, in order to assess whether TN plays a role in local invasiveness and metastasis of tumors. In metastatic colonic carcinoma tissues from 52 cases, moderate, partial expression of TN was observed in the primary foci but no TN expression was observed in tissues from 9 others. However, in every case of nonmetastatic colonic carcinoma, very strong TN expression was observed in the tissues. Furthermore, the presence of dense TN accumulation correlated well with the prognoses (1-year survival) of colonic cancer patients (p less than 0.01). Weak TN expression was observed in 24/61 of the metastatic lymph nodes examined. In 2 patients with metastatic colonic carcinoma, the colonic cancer cells produced TN. TN may, therefore, play a role in limiting or preventing local tumor invasion rather than preventing metastasis and is a useful marker for predicting the prognoses of patients with colonic cancer. PMID- 1726611 TI - Prevalence of antibody to hepatitis C virus in blood donors, high-risk groups and patients with liver diseases in Hungary. A multicentre study using ABBOTT EIA test and a comparison with an ORTHO ELISA test system. AB - Serum samples from 1185 individuals (blood donors, health-care workers, patients on haemodialysis, those from other high-risk groups and those suffering from non A, non-B hepatitis or other liver diseases) were examined for antibody to a recombinant HCV antigen. An ABBOTT HCV EIA system was used throughout and in addition a parallel study with ORTHO HCV ELISA was done in 380 of the samples to compare the two anti-HCV tests. A confirmatory neutralizing ABBOTT ELISA probe was also performed in 45 cases. The anti-HCV test was positive in 1.60% of the healthy blood donors and in 9% of subjects excluded from donation for elevated aminotransferase. In patients on haemodialysis 47%, in other high-risk-group subjects 33% anti-HCV prevalence was found. Patients with acute and chronic post transfusion NANB hepatitis showed 40% and 70% prevalence, respectively. The two ELISA tests revealed 95% agreement in the parallel determinations. Serial end point-dilution studies of anti-HCV-positive sera suggest that the ABBOTT test was of superior sensitivity. The results of the confirmatory test suggest that reactive (positive) samples of low optical density near to the cut-off value, required a confirmation with the naturalization test. HCV infection seems to be a common aetiological factor in PT-NANB hepatitis in Hungary, therefore, screening of blood donors for anti-HCV may be justified. PMID- 1726612 TI - Diagnosis of clonorchiasis by ELISA-inhibition test using a Clonorchis sinensis specific monoclonal antibody. AB - The ELISA-inhibition test using Clonorchis sinensis specific monoclonal antibody (CsHyb 0605-23) for diagnosis of clonorchiasis was carried out. It demonstrated sensitivity and high specificity in comparison with the conventional ELISA. PMID- 1726613 TI - Bacterial determinants of intracellular growth of Listeria monocytogenes. PMID- 1726614 TI - Staphylococcal L-asparaginase: antilymphoma and immunosuppressive action. AB - Staphylococcal L-asparaginase inhibits blastic transformation of human lymphocytes and growth of mice leukemia lymphoblasts L5178Y-R. The enzyme is removed from blood stream of DBA/2 mice very rapidly. PMID- 1726615 TI - Staphylococcal L-asparaginase: enzyme kinetics. AB - The ph optimum of purified staphylococcal L-asparaginase (EC 3.5.1.1) was found to be between 8.6 and 8.8. The temperature optimum was 30 degrees-32 degrees C and the highest reaction rate occurred at 30 degrees C. The KM of the enzyme calculated from Lineweaver-Burk plot was 3.71 x 10(-2) M. Besides L-asparaginase, the substrate specificity of enzyme was restricted to N-alpha-acetyl-L asparagine. D-asparagine, L-aspartic acid and D-glutamic acid were competitive inhibitors. Hg2+ and Cu2+ cations strongly inhibited the enzyme while Na+ and K+ cations strongly stimulated activity. Two SH-groups could be detected after enzyme denaturation with guanidine. PMID- 1726616 TI - Preparation of antibacterial and antitoxic Clostridium difficile sera. AB - Preparation of Clostridium difficile antibacterial and antitoxic sera is presented. Fifty one strains (72%) were typeable within Delmee scheme. Twenty strains (28%) belonged to new Polish serogroups designated 18, 27, 70, 71, 72, 88, 89 and NICH. Supernatants of all toxigenic Clostridium difficile strains were neutralized by gamma-globulin fraction of goat Clostridium difficile antitoxin in neutralization assay when it was performed on McCoy cell line. Only 8 toxigenic strains (21%) were positive in counterimmunoelectrophoresis. PMID- 1726617 TI - Methylammonium as a substrate for autotrophic growth by wild type Thiobacillus versutus, and mutant strains with lesions in chemolithotrophic metabolism. AB - Methylammonium consumption and ribulose 1-5-bisphosphate carboxylase (RuBisCO) activity were monitored in cultures of wild type Thiobacillus versutus and mutants deficient in autotrophic metabolism grown under various growth conditions. Only mutants 22, 72, 73 (deficient in ability to oxidize thiosulphate) could grow and develop RuBisCO activity on methylammonium, and assimilate 14CO2 generated as a result of methylammonium metabolism. Mutants 40 and 76, deficient in autotrophic CO2 fixation, showed no 14C methylammonium assimilation and did not oxidize it as a sole substrate within normal incubation periods. Relations of substrate metabolism and RuBisCO regulation in cultures grown on mixtures of thiosulphate or sucrose and methylammonium are described. Further genetic analysis of mutants with defects in autotrophic metabolism may allow localisation of genes responsible for the metabolic effects described. PMID- 1726618 TI - Ethanol production by thermotolerant yeast and its UV resistant mutants. AB - Six thermotolerant yeasts were isolated at 37 degrees C from over-ripe grapes by serial dilution technique using glucose yeast extract medium. Purified yeast cultures were screened for ethanol production at 37 degrees C by batch fermentation, using cane molasses containing 20% sugars. Sugar conversion efficiency of these isolates varied from 66.0 to 88.5% and ethanol productivity from 1.11 to 1.73 ml/l/h. The highest ethanol producing isolate was exposed to UV radiations and 13 mutants were picked up from the UV treatment exhibiting 0.1 to 1.0%, survival. The UV mutants varied in cell size from parent as well as among themselves. Determination of ethanol produced by all the mutants revealed that only five mutants resulted in 4.5 to 6.2% increase in sugar conversion and 8.25 to 18.56% increase in ethanol concentration coupled with maximum ethanol productivity of 2.4 ml/l/h in 48 h of batch fermentation of cane molasses (20% sugars) at 37 degrees C temperature. PMID- 1726619 TI - The effect of aromatic compounds on the work of activated sludge. AB - The resistance of bacteria occurring in activated sludge purifying refining petrochemical wastewaters to phenol, cresol and pirocatechin as well as the possibility of the purification of synthetic wastewaters carrying high concentrations of these compounds by sludge composed of resistant strains was studied. The most toxic of the studied compounds was phenol. Six out of 29 strains were resistant to high concentration of phenol (1000 mg/l). Activated sludge synthesized from strains resistant to phenol, cresol and pyrocatechin was tolerant to high concentrations of these compounds. Worsening of the work of the sludge, expressed by decrease of GOD, was observed at the concentrations of phenol, cresol and pyrocatechin of 2000, 400 and 300 mg/l, respectively. PMID- 1726620 TI - Improvement of the biodegradation of some cellulosic wastes by acid pretreatment. AB - Acid pretreatment of cellulosic wastes improved their susceptibility to Fusarium acuminatum enzymes. The effectiveness of acid pretreatment was demonstrated with an increase in both fungal growth and enzyme activities. A growth yield of 0.15 g/100 ml was achieved on medium containing 5% acid pretreated pods of bean for 60 minutes. Avicelase (C1), carboxymethylcellulase (Cx) and B-glucosidase (C2) reached their maximal biosynthesis on acid pretreated wheat bran, sugar-cane bagasse and sawdust-containing media, respectively. Xylanase and pectinase attained their highest accumulation on pretreated pods of bean media. A mixture of free sugars has been released by acid pretreatment. O.199 g dry mycelium was obtained when the fungus was grown on 100 ml of medium containing hydrolysate of 10% H2SO4 pretreated pods of bean for 30 min. No cellulase enzymes could be detected on hydrolysate medium at the time that low contents of both xylanase and pectinase were accumulated. PMID- 1726621 TI - Glucose oxidase synthesis by Aspergillus niger GIV-10 on starch. AB - The effect of various kinds of starch, as the sole source of organic carbon, on the biosynthesis of glucose oxidase by A. niger GIV-10 was examined. A. niger grown on 6% wheat starch medium provided extracellular and intracellular glucose oxidase with the highest enzymatic activities. A new method of intracellular glucose oxidase extraction (without disruption of mycelium), developed and discussed in this paper, increased 2 to 3.8-times glucose oxidase yield, as compared to that described earlier. PMID- 1726622 TI - The metabolism of anthracene and 9,10-dimethylanthracene by bacteria isolated from waters. AB - The metabolism of two polycyclic aromatic hydrocarbons i.e. anthracene and 9,10 dimethylanthracene by Micrococcus sp., Pseudomonas sp. and Bacillus macerans was examined. The above compounds were used as a sole carbon source for their growth. Using the reversed-phase thin layer chromatography techniques a number of anthracene and 9,10-dimethylanthracene metabolites were isolated and their structures identified spectroscopically. These included anthracene and 9,10 dimethylanthracene cis-dihydrodiols, hydroxy-methyl-derivatives and various phenolic compounds. Bacteria metabolise hydrocarbons using the dioxygenase enzyme system, which differs from the mammalian cytochrome P-450 monoxygenase. Hence in addition rat liver microsomal metabolism of the above hydrocarbons was investigated using the same separation techniques. PMID- 1726623 TI - Changes in development and ultrastructure of Aspergillus niger mycelium in the presence of toxic substances from molasses in citric acid fermentation medium. AB - Effects of a surface-active agent (Spumol BJ) and toxic molasses compounds on development and ultrastructure of Aspergillus niger strain R-16 mycelium producing citric acid in surface fermentation on molasses medium with 80% yield are presented. Microscopic observations showed that Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Giant conidia unable to germinate, appeared along with typical ones. Delicate mycelium outgrowing from germinating conidia however, sank after 20-24 h culture. Observations of hyphae in electron microscope showed changes in the ultrastructure and dimensions of mitochondria followed by successive degeneration of protoplast. The data obtained indicate that one of the reasons disqualifying raw molasses for biotechnological processes might be the excessive amount of surface-active antifoaming additives. PMID- 1726624 TI - Changes in development and ultrastructure of Aspergillus niger mycelium, strain with increased tolerance to toxic substances from beet molasses. AB - Effects of a defoamer and toxic molasses compounds on development and ultrastructure of A. niger mycelium, strain Z, characterized by high tolerance to these substances and producing citric acid in surface fermentation on proper molasses media with 70% yield were presented. Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Moreover, giant conidia, unable to germinate, appeared. Developing mycelium with dispersed hyphae became mucilaginous after 17-20 h culture, which indicated the process of sinking but after 24 h some part of the mycelium developed normally. Electron microscopic observations of mycelium developing in the presence of the toxic substances showed along with electron-transparent cytoplasm in a consequence of decrease in ribosome number, changes in ultrastructure of mitochondria. It may be assumed that one of the reasons of the above described abnormalities in development and ultrastructure of mycelium was a disturbance of respiration processes. The appearance of deposits of electron-dense material in mitochondria suggested the existence of a defence mechanism, eliminating toxic substances. PMID- 1726625 TI - Biological activity of rhizobial siderophore. AB - Non-nodulating mutant of Rhizobium leguminosarum biovar trifolli produces the phenolate type of siderophore consisting of 2,3-dihydroxybenzoic acid and threonine. The activity of this compound against the various bacteria was tested. Only, the growth of R. leguminosarum strains was stimulated by siderophore. The other species of Rhizobium, especially R. meliloti, were sensitive to this agent. The growth of R. meliloti was also inhibited by agrobactin and pseudobactin. This effect was reversed by ferric iron. PMID- 1726626 TI - Case-control study on risk factors for caesarean section: methodological issues. PMID- 1726627 TI - Possible sources of hexachlorobenzene (HCB) in the Italian environment. PMID- 1726628 TI - [Acute narcotic addiction deaths in Milan in the years 1985 and 1988: a comparison of diverse trends of the phenomenon]. PMID- 1726629 TI - [A preliminary study on hygienic conditions of lagoon areas destined for aquaculture]. PMID- 1726630 TI - [Quality of waters of the Marche Region rivers: the state of the Foglia River]. PMID- 1726631 TI - [Epidemiology and prevention of hospital infections in the Local Health Unit of Sassari: profile of bacterial resistance and antimicrobial agents of large usage. 1. Antibiotics]. PMID- 1726632 TI - [Epidemiology and prevention of hospital infections in the Local Health Unit of Sassari:profile of bacterial resistance and antimicrobial agents of large usage. 2. Antiseptics/disinfectants]. PMID- 1726633 TI - Sialyltransferases of developing rat brain. AB - The activity of four different sialyltransferases acting on N- or O-linked chains of glycoproteins was studied in brains of 19 days-old embryos, 1-day-old newborns and adult rats. By using asialofetuin, fetuin and N-acetyllactosamine as acceptors, it has been possible to measure independently the following enzyme activities: CMP-NeuAc:Gal beta 1-3GalNAc alpha(2-3)-sialyltransferase (EC 2.4.99.4), CMP-NeuAc:Gal beta 1-4GlcNAc alpha(2-3)-sialyltransferase (EC 2.4.99.6), CMP-NeuAc:Gal beta 1-4GlcNAc alpha(2-6)-sialyltransferase (EC 2.4.99.1) and CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-3GalNAc alpha(2-6) sialyltransferase (EC 2.4.99.7). The specific activity of the first three enzymes which act on asialylated acceptors showed a 2.6-fold decrease in a parallel manner after ontogenic development, while the activity of NeuAc alpha 2-3Gal beta 1-3GalNAc alpha(2-6)-sialyltransferase was four times lower in adult than in embryonic brain, showing a stronger dependence on ontogenic development. Despite the higher level of sialyltransferases able to act on glycoproteins, in fetal brain these glycoproteins do not contain a higher amount of sialic acid. PMID- 1726634 TI - Involvement of protein kinase C in angiotensin II-mediated release of 12 hydroxyeicosatetraenoic acid in bovine adrenal glomerulosa cells. AB - The present study was conducted to determine whether protein kinase C was involved in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid (12-HETE) from bovine adrenal glomerulosa cells. Activators of protein kinase C, 12-O-tetradecanoylphorbol 4-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG), significantly increased release of 12-HETE. The effect of OAG was potentiated by BAYK8644, a stimulator of calcium entry. Sphingosine, H-7 and staurosporine, which inhibited the activity of protein kinase C in vitro, almost completely blocked 12-HETE release induced by TPA. These agents also significantly reduced angiotensin II-mediated 12-HETE release. When time course of the liberation of 12 HETE was measured, angiotensin II elicited sustained release of 12-HETE, which was inhibited by staurosporine. These results indicate that angiotensin II induces sustained release of 12-HETE, a feed forward regulator of aldosterone secretion, and that protein kinase C may be involved in this process. PMID- 1726635 TI - Serum free triiodothyronine to free thyroxine ratio enables early prediction of the outcome of antithyroid drug therapy in patients with Graves' hyperthyroidism. AB - This study scrutinizes the correlation between serum free triiodothyronine (FT3) to free thyroxine (FT4) ratios and the eventual outcome of antithyroid drug (ATD) therapy in patients with Graves' disease. Forty-four patients with Graves' thyrotoxicosis were treated with methylmercaptoimidazole (methimazole). During the follow-up, 16 patients relapsed in the short period of one to five months after cessation of the drug (relapse group), and 28 patients remained in remission when checked at 12 to 20 months after treatment (remission group). Serum FT3 to FT4 ratios [(pg/ml/ng/dl) x 10] were less than 55 throughout ATD therapy in 27 of the 28 remission patients whereas the ratios of the relapse group exceeded 55 from the early phase of methimazole treatment in 10 of 16 patients. In eight of these 10 patients the increased ratios were detected within three months of therapy (1 month, 3 patients; 2 months, 4 patients; 3 months, 1 patient). The ratios for the remaining two patients rose above 55 at the fifth and sixth months. There was no statistical difference between the remission and relapse groups in the FT3 to FT4 ratios either before nor at the completion of the treatment. However, a clear difference could be measured at a point during the therapy. Those in whom this difference was pronounced later underwent relapse.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726636 TI - Transurethral versus open prostatectomy: do these procedures apply to the same patients and the same disease? PMID- 1726637 TI - Too stringent patient selection criteria in a clinical trial of alpha-blockers reduce the value of the results. AB - The patient selection criteria in a clinical trial of an alpha-blocker for the treatment of benign prostatic hypertrophy were such that 85% of the patients on the waiting lists of 4 hospitals had to be excluded. The value of such a trial is discussed. Trial protocols should be designed to allow the enrollment of patients truly representative of the patients for which the treatment is intended. PMID- 1726638 TI - Focussed extracorporeal pyrotherapy: experimental results. AB - A new device made of piezoelectric ceramic placed in a semispherical dish and focussed at 320 mm was developed in order to generate heat and cavitation responsible for coagulative necrosis of deep tissues. The target to be treated is located with a central ultrasound probe of 3.5 MHz. In vitro studies with polyurethane phantoms showed that the ultrasound melted a surface of 2 x 12 mm within 1 s. The temperature recorded at the focus was 270 degrees C. In tissue samples (prostate cancer and benign prostate hyperplasia), the temperature rose to 85 degrees C in vitro and a hyperechoic zone appeared at the focus during shots. In vivo 8-mm plastic spheres, introduced surgically into the bladder of pigs, were melted by repeat shots without burning of crossed tissues. These studies were performed in the kidney and the liver. Autopsy performed on day 0 showed congestion, autopsy performed between day 6 and day 11 showed necrosis, whereas at 3 months the focussed area was fibrosed. This technique, which we called 'focussed extracorporeal pyrotherapy', combines phenomena of cavitation and high heat at the focus. Prostate tumors, bladder tumors, kidney tumors and liver metastases are potential indications for pyrotherapy. PMID- 1726639 TI - Randomized, double-blind trial of Lithostat (acetohydroxamic acid) in the palliative treatment of infection-induced urinary calculi. AB - In a prospective, double-blind, placebo-controlled study, the efficacy and safety of acetohydroxamic acid (AHA) in preventing urinary calculogenesis was evaluated in 94 patients with chronic urinary infection. Stone growth occurred in 17% of the AHA group and in 46% of the placebo group (p less than 0.005). Completely reversible side effects consisting predominantly of psychoneurologic and musculo integumentary symptoms were more prevalent in the AHA group (p less than 0.01). Side effects which were judged 'intolerable' were experienced by 10 (22.2%) of patients in the AHA group and 2 (4.1%) in the placebo group. It is concluded that AHA treatment is effective, relatively safe, and clinically useful in preventing infection-induced urinary calculogenesis. PMID- 1726640 TI - Possibilities for application of radioimmunological analysis of CEA, AFP, SP1 and ferritin in patients with basal cell carcinoma of the eyelids. PMID- 1726641 TI - A mathematical model for the diffusion of tumour angiogenesis factor into the surrounding host tissue. AB - Unless they are furnished with an adequate blood supply and a means of disposing of their waste products by a mechanism other than diffusion, solid tumours cannot grow beyond a few millimetres in diameter. It is now a well-established fact that, in order to accomplish this neovascularization, solid tumours secrete a diffusable chemical compound known as tumour angiogenesis factor (TAF) into the surrounding tissue. This stimulates nearby blood vessels to migrate towards and finally penetrate the tumour. Once provided with the new supply of nutrient, rapid growth takes place. In this paper, a mathematical model is presented for the diffusion of TAF into the surrounding tissue. The complete process of angiogenesis is made up of a sequence of several distinct events and the model is an attempt to take into account as many of these as possible. In the diffusion equation for the TAF, a decay term is included which models the loss of the chemical into the surrounding tissue itself. A threshold distance for the TAF is incorporated in an attempt to reflect the results from experiments on corneal implants in test animals. By formulating the problems in terms of a free boundary problem, the extent of the diffusion of TAF into the surrounding tissue can be monitored. Finally, by introducing a sink term representing the action of proliferating endothelial cells, the boundary of the TAF is seen to recede, and hence the position and movement of the capillaries can be indirectly followed. The changing concentration gradient observed as the boundary recedes may offer a possible explanation for the initiation of anastomosis. Several functions are considered as possible sink terms and numerical results are presented. The situation where the tumour (i.e. the source of TAF) is removed is also considered. PMID- 1726642 TI - Alpha-fetoprotein in hepatocellular carcinoma: a serological and histochemical study in Malaysian patients. AB - Serum alpha-fetoprotein (AFP) levels and its expression in liver tissue was studied in 50 cases of histologically confirmed hepatocellular carcinoma (HCC). Serum AFP levels were elevated (greater than 20iu/ml) in 35/50 (70%) of the cases, 28 of whom had levels greater than 500 iu/ml, which is highly suggestive of HCC. These results indicate that serum AFP, by itself, is a relatively insensitive diagnostic test for HCC. Although elevated levels in high risk patients provide a specific clue, a negative result does not exclude the diagnosis of HCC. Expression of AFP by tumour cells paralleled that of serum in the majority of cases. However, tissue AFP was negative in 7 patients who had markedly elevated serum AFP. This observation may be a reflection of preferential excretion of the tumour antigen or differential expression of the antigen by the tumour cells. None of the patients with normal serum AFP demonstrated a reaction for tissue AFP. There was no correlation between AFP production and tumour differentiation. PMID- 1726643 TI - Analysis of the proteolytic degradation products of hyaline cartilage proteoglycans. AB - The proteolytic degradation products of nasal hyaline cartilage proteoglycans produced by polymorphonuclear leukocyte lysosomal enzymes were investigated. The protein content of the degradation products is 7.0-8.6% corresponding to a peptide chain of 24-28 amino acids and the relative molecular mass of the total fragment is M(r) = 37,600-39,200. On an average, each proteoglycan fragment contains two chondroitin-sulphate chains (M(r) = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (M(r) = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin-4-sulphate, 12.9-19.4% chondroitin-6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the degradation products resemble those from native proteoglycans, it is suggested that the degradation of the proteoglycans occurs by proteinases that attack preferably the chondroitin sulphate region of the core protein. PMID- 1726644 TI - Prenatal development of cutaneous afferent connections in the spinal cord of fetal sheep. A physiological and neurochemical study. AB - In this study we have examined the physiological and neurochemical development of the cutaneous afferent pathways from the hindlimb to the spinal cord in fetal sheep. We have shown that somatosensory input from the hindlimb evokes activity in DRG neurons at 87d gestation and in cells in the dorsal horn at 92d (term, 146d). There is evidence of immunoreactivity for substance P, calcitonin gene related peptide and glutamine several days prior to this at 77-80 days. The implication of these findings are discussed. PMID- 1726646 TI - Expression of dolichol-linked saccharide intermediate synthesis during the development of B lymphocytes. AB - There are large developmental increases in the rates of dolichol-linked oligosaccharide synthesis and protein N-glycosylation when resting murine splenic B lymphocytes are activated by bacterial lipopolysaccharide (LPS). These in vivo and in vitro studies were carried out to investigate the underlying biochemical mechanisms involved in the dramatic increase in the rate of oligosaccharide-lipid biosynthesis in LPS-stimulated B cells. Metabolic labelling experiments showed that the rate of synthesis of N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc P-P-Dol), mannosylphosphoryldolichol (Man-P-Dol) and glucosylphosphoryldolichol (Glc-P-Dol) increased 4- to 15-fold between 20 and 40 h after exposure to LPS. When the glycosyltransferase activities catalysing the formation of the three dolichol-bound monosaccharides were assayed in vitro with endoplasmic reticulum (ER)-enriched fractions, the initial rates were found to be elevated 4-fold prior to the major increases in oligosaccharide-lipid intermediate biosynthesis observed in vivo. Based on kinetic analyses, the higher enzyme activities were due to an increase in the amount of the three glycosyltransferases in activated cells. The time courses for elevated cellular content and rate of synthesis of guanosine-diphosphomannose (GDP)-Man corresponded to the developmental increase in oligosaccharide-lipid synthesis. The kinetics and magnitude of the induction of oligosaccharide-lipid synthesis were similar whether the initial rates were calculated on the basis of [2-3H]mannose-labelling or the specific activity of the GDP-[2-3H]mannose pool.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726645 TI - Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease. AB - The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation. PMID- 1726647 TI - Experimental hyaline cartilage lesions: two-dimensional spin-echo versus three dimensional gradient-echo MR imaging. AB - The value of magnetic resonance (MR) imaging, with two-dimensional (2D) spin-echo and FISP (fast imaging with steady-state precession) and FLASH (fast low-angle shot) three-dimensional (3D) gradient-echo sequences, for the detection of hyaline cartilage defects of the femoral condyle and the tibial plateau, was investigated in an animal model. In eight dogs, the anterior cruciate ligament was transected in one knee joint, resulting in rapid development of osteoarthritis with degeneration of the hyaline cartilage. At autopsy, 24 cartilage lesions were found, which were classified into four grades. The overall detection of cartilage lesions with MR imaging was poor. Only five of the 24 lesions were visible on 2D spin-echo images, while 11 of 24 were visible on 3D FISP images and 15 of 24 were seen on 3D FLASH images. The best results were obtained in advanced stages of cartilage degeneration, involving ulceration and complete abrasion of the cartilage layer. Signal loss or signal intensity increase in the cartilage layer was seen inconsistently in grades 3 and 4 degeneration. In this animal model, 2D spin-echo imaging was inadequate for the diagnosis of hyaline cartilage lesions, while 3D gradient-echo imaging permitted satisfactory diagnosis in only grade 4 cartilage disease. PMID- 1726648 TI - Assessment of human cytotoxic T cell activity using synthetic peptides: potential for field application. AB - The use of synthetic peptides to delineate epitopes recognized by major histocompatability complex-restricted cytotoxic T lymphocytes has been well documented. In the present study, we report a method for the generation and assay of peptide-specific cytotoxic T lymphocytes adapted for the field situation where quantities of blood are often limited. From a single bleed of the donor and using minimal quantities of peptide (less than 5 micrograms/ml), up to 100 potential epitopes could be screened within a short time frame. Subsequent cloning of positive cultures for further analysis of defined epitopes is permissible. The system is readily adaptable to a wide range of viral, parasitic or bacterial infections. PMID- 1726649 TI - Systematic screening for bioactive peptides. AB - Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature. PMID- 1726650 TI - Partial characterization of human amniotic membrane interferon. AB - 1. The molecular weight and the electrofocusing profile of human amniotic membrane interferon (IFN-AM) were determined. 2. When submitted to gel filtration, IFN-AM showed a single 26-28 kDa component; in polyacrylamide gel electrophoresis one component of 19,500. 3. In electrofocusing, IFN-AM displayed a heterogeneous profile with 5 to 7 peaks, but different from human alpha or beta IFNs. This heterogeneity was reduced by previous treatment of IFN-AM with neuraminidase. 4. IFN-AM is a sialoglycoprotein similar to human beta IFN in terms of antigenicity but different from it in electrofocusing profile. PMID- 1726651 TI - Effects of acute 2,4-dichlorophenoxyacetic acid intoxication on some rat serum components and enzyme activities. AB - The present study was undertaken to investigate the effects of acute 2,4 dichlorophenoxyacetic acid (2,4-D) intoxication (0.6 g/kg, po) on lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, amylase, creatinine, glucose, total protein and albumin levels in rats. Serum levels of lactate dehydrogenase, alkaline phosphatase and creatinine increased from 1- to 4-fold at 5, 8 and 24 h after 2,4-D administration, whereas serum levels of aspartate and alanine aminotransferase were higher only at 8 and 24 h. Amylase levels were only increased 8 h after administration of 2,4-D and then returned to normal levels. In contrast, 2,4-D reduced the serum levels of glucose and total protein 5, 8 and 24 h and serum albumin levels 5 h after herbicide intoxication. Thus, acute intoxication with 2,4-D disrupts serum levels of several enzymes and components which are considered to be indicators of tissue injury. Most likely these alterations mainly reflect hepatic and muscle tissue damage induced by the herbicide, but significant pancreatic and kidney toxicity may also have occurred. PMID- 1726652 TI - The neurons of the retinal ganglion cell layer of the guinea pig: quantitative analysis of their distribution and size. AB - 1. The topographical distribution of ganglion cells and displaced amacrine cells in the guinea pig retina is described. 2. Neurons were counted in the ganglion cell layer of retinal whole mounts stained by the method of Nissl or retrogradely labeled with horseradish peroxidase. Neuronal soma size was estimated from samples taken from different retinal regions. 3. We estimate that a total of 295,000 neurons comprise the guinea pig ganglion cell layer and they consist of 159,000 ganglion cells and 136,000 displaced amacrine cells. 4. The visual streak is poorly differentiated. Ganglion cell density reaches a peak of 2,272 cells/mm2 in a temporal expansion of the visual streak, 4-5 mm toward the optic disk. The visual streak temporal expansion may represent the analogue of the area centralis for this species. The ventral hemi-retina has a higher ganglion cell density than the dorsal hemi-retina. The displaced amacrine cells are more uniformly distributed than the ganglion cells. 5. The present paper provides relevant data concerning the number and distribution of the neurons of the retinal ganglion cell which were not available or were very contradictory in the literature. PMID- 1726653 TI - Immunological memory for HIV-1 induced in rabbits by immune poly(A)+ RNA. AB - New Zealand rabbits were used to demonstrate the in vitro and in vivo transfer of reactivity, including immunological memory, to a synthetic peptide corresponding to residues 586-606 of the gp-160 protein of human immunodeficiency virus (HIV 1). The transfer were mediated by immune poly (A)+ RNA from lymphoid organs (spleen and mesenteric nodules) harvested after immunization of a sheep with the peptide (8 subcutaneous injections plus glucan and complete Freund's adjuvant using a total of 1750 micrograms peptide). Immunological reactivity was detected by the leukocyte adherence inhibition (LAI) test for cellular immunity. A dose of 150 micrograms poly(A)- RNA ml-1 10(7) leukocytes-1 or 2.0 micrograms poly(A)+ RNA ml-1 10(7) leukocytes-1 was used for in vitro transfer. For in vivo transfer the recipient rabbits received 3,000 micrograms poly(A)- RNA or 20 micrograms poly(A)+ RNA. The mean non-adherence index (NAI) obtained in vitro was 10 +/- 7 for leukocytes treated with poly(A)-RNA and 60 +/- 10 leukocytes treated with poly(A)+ RNA. The poly(A)+ RNA fraction induced a primary-like response and memory cells in vivo. The poly(A)-RNA fraction had no effect. Since sheep are refractory to, and rabbits are sensitive to HIV-1, we suggest the use of this animal model for testing the immunomodulating effect of anti-HIV-1 immune poly(A)+ RNA. PMID- 1726654 TI - ATP-sensitive potassium channels do not mediate vasorelaxation by acetylcholine or iloprost. AB - The influence of glibenclamide (GBC), a blocker of ATP-sensitive K+ channels, on relaxation caused by cromakalim (CKL), acetylcholine (ACh) and iloprost (ILO) was assessed in aortic rings (AR) with (E+) or without endothelium (E-) and in the perfused arterial mesentery (MES) of the rat. In AR preconstricted with noradrenaline, CKL (0.03-10 microM) and ILO (5.5 nM-1.6 microM) caused graded vasodilations which were not modified by endothelium removal. ACh (0.01-3 microM) only relaxed E+AR preparations. GBC (3 microM) markedly reduced responses to CKL in E+AR and E-AR, but did not affect vasodilation induced by ILO in E+AR or E-AR and by ACh in E+AR. In MES preconstricted with methoxamine, bolus injections of CKL (10 or 30 nmol) or ACh (0.03-1 nmol) caused graded reductions of perfusion pressure. Only the responses to CKL were significantly inhibited by GBC (10 microM). We conclude that AR and MES contain functional ATP-sensitive K+ channels, which, however, do not play a significant role in the endothelium dependent vasodilation triggered by ACh or in the endothelium-independent relaxation induced by ILO. PMID- 1726655 TI - Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes. PMID- 1726656 TI - Twice-daily tapering dexamethasone treatment during cranial radiation for newly diagnosed brain metastases. AB - Twenty evaluable patients with newly diagnosed brain metastases underwent treatment with a novel dose/schedule of dexamethasone aimed at reducing steroid toxicity during palliative radiation therapy. All patients received twice daily dexamethasone starting at 8 mg bid for four days then 4 mg bid for four days then 2 mg bid until the last day of radiation therapy. The radiation prescriptions were not standardized varying from 2000 cGy/5 fractions to 5800 cGy/29 fractions. Fourteen patients received dexamethasone for a minimum of 24 hours before their first radiation treatment and 7 (50%) experienced improvement in neurologic symptoms/signs prior to starting radiation treatments. Fourteen patients completed the planned course of radiation and dexamethasone. Only 1 patient needed to restart dexamethasone within 30 days of finishing radiation because of steroid reversible neurologic deficits. Steroid toxicity was mild including hyperglycemia (1), candida esophagitis (1), steroid pseudorheumatism (2), peripheral edema (1) and steroid withdrawal syndrome (1). Only two toxic events were recorded in patients receiving steroids less than 21 days. Twice daily dexamethasone appears to provide good clinical results with minimal morbidity. PMID- 1726657 TI - Visual scotomata resulting from lupus anticoagulant in a patient with lymphoma in remission. AB - Episodic cerebro or retinovascular ischemic events without apparent cause occur in patients with cancer. We report a patient in remission from lymphoma whose multiple episodes of presumed ocular ischemia occurred in the setting of a circulating lupus anticoagulant. Symptoms resolved following therapy with Warfarin. PMID- 1726658 TI - Homer Wright rosettes in ependymoma. AB - A brain tumor in the fourth ventricle of a 26-year-old woman displayed numerous Homer Wright rosettes by conventional histology, but immunostaining and electron microscopy revealed the tumor an ependymoma. Since the presence of Homer Wright rosettes in ependymoma has not been well appreciated in the past, the diagnostic importance of this finding is discussed. PMID- 1726659 TI - [The treatment of acute paraproctitis in an emergency proctology department]. AB - An experience with treatment of 3160 patients with acute paraproctitis under conditions of a specialized department is presented. Rational curative methods allow to achieve healing of 2280 patients (72.2%) with small amount of complications (22 cases). The duration of treatment was shortened to 11.2 days. No recurrences were noted. PMID- 1726660 TI - [Role of leukocytes and amebic proteases in experimental testicular necrosis produced in the rat by Entamoeba histolytica]. AB - We examined the participation of polymorphonuclear leucocytes and amebic proteinases upon tissue damage by means of an experimental model of acute amebic lesions developed in rat's testicle. In leukopenic rats (less than 1,000 leucocytes/ml) intratesticular injection of axenic E. histolytica's trophozoites (HM-1) produced lesions undistinguishable from the normal controls. On the other hand, inhibition of 80% (average) of the proteinase activity by means of previous incubation of the trophozoites with human a2M gave way to minimal inflammatory lesions almost undistinguishable from the controls which were injected with PBS A. Our data suggest that in this experimental model of acute amebiasis polymorphonuclear leukocytes do not participate in the tissue damage and that amebic proteinases are responsible for Entamoeba histolytica's virulence. PMID- 1726661 TI - [Obtaining monoclonal antibodies against outer membrane glycoproteins of Entamoeba histolytica]. AB - The goal of this paper was the production of monoclonal antibodies capable of detecting relevant antigens from the surface of Entamoeba histolytica trophozoites, with the purpose of using them as a diagnostic test. The cellular fusion for obtaining the monoclonal antibodies (mAb) was done with spleen cells from BALB/c mice, previously immunized with glycoproteins from the membrane, as well as Sp2/0 cells. The hybridoma supernatants were tested with ELISA, using glycoproteins and lipopeptide phosphoglycans (LPPG) as antigens. Seven hybridomas producing mAb against the glycoproteins were found. Among these, three recognize LPPG. The ability of reacting with the mAb against two molecules disappeared for all the LPPG positive ones when were treated with meta-periodate, and only three reacted against the glycoproteins. All of the mAb were of the Ig M isotypes. They were characterized by Dot blot and Western blot assays. From the results, one may deduce that some mAb recognize as epitopes the polysaccharide portion, and thus infer that they are directed of against the surface and therefore, in the future, could be used with a diagnostic purpose. PMID- 1726662 TI - [Antigens specific to pre-cysts and in vivo chitin synthetase activity in Entamoeba invadens]. AB - In this paper we studied the transformation of a trophozoite into a cyst in Entamoeba, using E. invadens as a model. We had the following objectives: a) identification of precyst-specific proteins (P), by a monoclonal antibody against E. invadens and heterologous polyclonal antibodies against cellular (165) fractions of Mucar rouxii, which are chitin synthetase activity rich; and b) in vivo determination of the time required for the expression of activity of the chitin synthetase during encystment. We found P markers which are not found in either trophozoites nor cysts. Monoclonal F507 antibody recognized a 33 kDa protein in P and the polyclonal anti-16S antibodies reacted with a 90 kDa protein to P. Even though the 33 and 90 kDa proteins have a different molecular weight from the chitin synthetase described in fungi (57 kDa and 65 kDa) and yeasts (63 kDa), we conclude that these proteins are specific to P and that the 90 kDa one shares epitopes with chitosomal fractions of M. rouxii. Also, the mayor accumulation of alkali-resistant material, sensitive to chitinase, occurred during the formation of P, between 40 and 50 hours post incubation, during encystment. One may conclude that chitin polymers are synthesized during the P phase. PMID- 1726663 TI - [The eosinophil and Entamoeba histolytica. II. Testicular amebic lesions produced in eosinophilic rats]. AB - Even though eosinophiles are not characteristic of amebiasis, polymorphonuclear eosinophilic cells are regularly found in the inflammatory lesion which occurs in the early phases of amebic invasion. To acquire better knowledge of the intervention of these cells when confronted with E. histolytica, testicular lesions were produced by means of direct injection of amebas in rats with eosinophilia. Eosinophilia (greater than or equal to 4%) was induced in male Sprague Dawley rats by an intravenous injection of Sephadex G200 suspension. Later, both the eosinophilic and normal (control) rats were challenged with an intratesticular injection (on the left side) of 2 x 10(6) amebas of the virulent strain HM1-IMSS. The other testicle was injected with BFS-A to serve as control. The testicles were removed 5 hours later and evaluated histologically. The size and celullarity of the testicular lesions produced by the ameba on both eosinophilic and controls were similar, whereas lesions were not found in testicles injected with BSF-A. Within the limits of our experimental model, the results suggest that eosinophils do not contribute substantially to the genesis of tissue lesions produced by E. histolytica. PMID- 1726664 TI - Use of non-cellular models to study the interaction of E. histolytica with mammalian cells. AB - We have utilized liposomes constructed with individual mammalian cell membrane glycosphingolipids and latex beads conjugated with glycoproteins as models to investigate the molecular specificity and mechanism of interaction of E. histolytica with target cells. Synthetic liposomes constructed with a variety of glycosphingolipids bearing neutral, straight chain glycans with galactose or N acetylgalactosamine termini stimulated rapid (90 sec), contact dependent polymerization of E. histolytica actin. Glycans with terminal N-acetylglucosamine residues were not, or only weakly, stimulatory. Attachment of N-acetylneuraminate to the terminal residue of stimulatory glycosphingolipids eliminated activity. Attachment of fucose to the penultimate sugar reduced glycan recognition. Latex beads conjugated with asialofetuin (galactose beta 1-4 glycan termini) adhered to amoebae more effectively than fetuin conjugated beads (N-acetylneuraminate termini) or agalactosyl asialofetuin conjugated beads (N-acetylglucosamine termini). However, none of the glycoprotein conjugated beads stimulated contact mediated polymerization of E. histolytica actin. The carbohydrate specificity of E. histolytica interaction with non-cellular particles resembles that observed with whole target cells our results demonstrate that carbohydrate recognition specificity extends to lipid as well as protein bound glycoconjugates. Further, these studies suggest that the biochemical consequences of binding to glycosphingolipids differ from those resulting from interaction with glycoprotein. This may be relevant to the mechanism of mammalian cell attack by this pathogen. PMID- 1726665 TI - Formalin is deleterious to cytoskeleton proteins: do we need to replace it by formalin-free Kryofix? AB - Formalin is hazardous for the environment and for the laboratory personnel and deleterious to cytoskeleton proteins. The pathology and anatomy laboratory can be formalin-free when Kryofix is used as a substitute fixative. In four years experience with Kryofix, we learned that immunostaining on paraffin sections is reproducible and of excellent quality. The current study concerns the comparison of interfilament staining on paraffin sections made from formalin or from Kryofix treated tumors. For both keratin and vimentin there were false-negative findings in the formalin-fixed tumors. Of these two, vimentin was het most susceptible to formalin fixation. Three of the ten adenocarcinomas were positive for both keratin and vimentin, indicating that this double staining pattern is not uncommon. Postfixation of Kryofix-treated tissue with formalin results in false negative immunostaining. PMID- 1726666 TI - Microwave applications in classical staining methods in formalin-fixed human brain tissue: a comparison between heating with microwave and conventional ovens. AB - The quality of microwave adaptations of three classical neuroanatomical staining methods (the Nissl, Kluver-Barrera and Haggqvist stains) was tested on frozen serial sections from human brain specimens which has been stored for up to 10 years in 10% formalin. The conclusion was that the use of microwave irradiation reduces processing time and/or concentrations of the chemicals used, whereas the light microscopical quality of the stains considered is equal or improved as compared to their original counterparts. Next, a comparison was made between microwave adapted stains and classical procedures, which, except for the use of a conventional oven as heat source together with pre-heated solutions, were entirely identical. It appeared, that at light microscopical level no difference can be appreciated between the effect of internally (using microwave irradiation) and externally (using a conventional oven) supplied heat on the staining result. PMID- 1726667 TI - Microwave fixation provides rapid, primary fixation for light and electron microscopy and for immunohistochemistry and immunocytochemistry. AB - A relatively new approach to specimen preservation for morphologic studies uses microwave energy and chemicals. Microwave fixation can produce fixation results equal in quality to chemical fixation methods and equal in speed to freeze fixation methods. The importance of this microwave fixation technology lies in its potential to provide a standardized fixation approach in histopathology, immunohistochemistry, and immunocytochemistry. PMID- 1726668 TI - Immunohistochemical visualization of the nervous system in the porcine small intestine using antisera raised against the cytoskeletal proteins MAP1 and MAP2, in combination with neuropeptide immunocytochemistry. AB - This investigation was performed to determine whether antisera raised against microtubule-associated proteins, i.e. MAP1 and MAP2, may constitute an alternative to the silver-impregnation studies for the identification of the distinct morphological enteric neuronal cell types in the porcine small intestine. MAP1-immunostaining seems less suited since it preferentially stains the neuronal somata and axons and hardly permits to observe the dendritic processes. MAP2-immunostaining chiefly visualizes the perikaryal-dendritic domain and the proximal part of the axonal processes in the enteric neurons of the porcine gut. Hence, MAP2-immunostaining enables for the first time the unambiguous immunocytochemical identification of enteric multi(short)dendritic uniaxonal type I neurons. Double labelling techniques using antisera against MAP2 and substance P indicate that part of the type I neurons in the myenteric plexus of the porcine small intestine, which are taking part in an ascending pathway, are substance P-immunoreactive, whereas the substance P/neuromedin U-minineurons in the Meissner's plexus do not stain for MAP2. We may conclude that, although MAP2-immunostaining falls short of the quality achieved with silver-impregnation, the possibility to combine MAP2-immunostaining with neuropeptide immunocytochemistry to study the intestinal neurons has the advantage that part of the enteric neuron types stained with a distinct neurotransmitter or neuromodulator can be classified morphologically. PMID- 1726669 TI - Generation of DM-20 splice site in myelin proteolipid protein gene: a hypothesis based on analysis of the amphibian protein. AB - The DM-20 isoform of proteolipid protein (PLP) of central nervous system myelin is undetectable in amphibia, but readily detectable in reptiles, birds and mammals. To explain the phylogenetic origin of DM-20, it has been proposed that a donor-acceptor RNA splice site was generated by the introduction of a single base change in the codon encoding amino acid 116 of PLP. We tested this hypothesis by isolating and sequencing peptides from bullfrog PLP. One of the peptides corresponded to mammalian residues 110-122, which contain the N-terminal boundary region for the domain excluded from DM-20 in higher vertebrates. We found that the Thr115-Val116 sequence is conserved between frog and mammal. Therefore, we propose that a single base change in the third position of the codon for Thr115 would account for the appearance of the new donor-acceptor splice site. Three amino acids elsewhere in this thirteen-residue peptide were found to differ between bullfrog and mammal, which could account for the previously reported weak recognition of amphibian PLP by an antiserum specific for peptide 109-128. A second peptide from bullfrog PLP had a sequence identical to that of residues 45 52 in mammalian PLP. Our findings explain how a specific change in the myelin PLP gene could generate a new form of the PLP protein. PMID- 1726671 TI - Synthesis of the trisaccharide, 2-(p-trifluoroacetamidophenyl)ethyl O-alpha-D galactopyranosyl-(1-4)-O-beta-D-galactopyranosyl-(1-4)-2-acet amido-2-deoxy-beta D-glucopyranoside, corresponding to the blood group P1 determinant. AB - The 2-(p-trifluoroacetamidophenyl)ethyl beta-glycoside of the P1 antigen trisaccharide, O-alpha-D-galactopyranosyl-(1-4)-O-beta-D-galactopyranosyl-(1-4)-2 acetamido-2-deoxy-D-glucopyranose, was synthesized. Thioglycoside intermediates were used as building blocks. PMID- 1726670 TI - A monoclonal antibody for terminal beta-galactose. Use in analysis of glycosphingolipids. AB - A monoclonal antibody (mAb 8281) specific for the terminal beta-galactose (beta Gal) of glycosphingolipids (GSL) and glycoproteins was produced from mice immunized with lipid extract from fresh acute lymphocytic leukemia (ALL) cells. Immuno-thin layer chromatography (ITLC) and competition assays with purified neutral GSL standards, free sugars, and synthetic neoglycoproteins showed mAb 8281 to be strongly reactive with LacCer, GalCer and Gal-beta-O-(CH3)2S(CH3)2 CONH-(Gal-beta-O-CETE) linked to bovine serum albumin (BSA). The penultimate sugar also played a role in binding. The antibody was not reactive with carbohydrates with terminal alpha Gal structures and unrelated terminal moieties. Indirect immunoperoxidase staining and flow cytometry with mAb 8281 demonstrated positive staining on numerous tissues, including smooth muscle, gastrointestinal mucosa, lymph node B cells and monocytes. ITLC analysis of the GSL composition of fresh B cell neoplasms using mAb 8281 confirmed the presence of lactosylceramide and galactosylceramide in neoplasms of varying stages of differentiation. Because of its specificity for terminal beta Gal carbohydrate residues, mAb 8281 may be useful in structural and functional analyses of GSL. PMID- 1726672 TI - Access to fluorescent probes via allyl glycosides: the synthesis of a Brucella trisaccharide epitope linked to a coumarin. AB - Oligosaccharide allyl glycosides are demonstrated to provide a route to fluorescent probes and simple inhibitors. Ethyl 2-O-acetyl-4-azido-3-O-benzoyl 4,6-dideoxy-1-thio-alpha-D-mannopyranosid e (6) was used as glycosyl donor in the preparation of the trisaccharide [alpha-D-Rha p4NFo-(1----2)-]2-alpha-D-Rha p4NFo O-allyl (16). Thioglycoside 6 was activated with N-iodosuccinimide and triflic acid or by bromine in the glycosylations and the inhibitor 16 was obtained after deprotection by transesterification, reduction of the azido groups with hydrogen sulfide, and N-formylation with ethyl formate. Ozonolysis of the allyl glycoside in 16 and reductive amination with 7-amino-4-methylcoumarin then gave the target fluorescent trisaccharide conjugate. PMID- 1726673 TI - A fluorometric rapid microassay to identify anti-proliferative compounds for human melanoma cells in vitro. AB - A simple, rapid and reproducible assay for the determination of melanoma cell proliferation in vitro is described, based on the hydrolysis of a fluorogenic substrate by cell esterases in the cytoplasm of living cells. Human melanoma cells were cultured at several densities in 96-well culture plates for 24 h and were then incubated with 4-methylumbelliferyl heptanoate. The generated fluorescence showed a strong correlation with the cell numbers, similar to those assessed by determining the [3H]thymidine incorporation into cellular DNA and by quantifying the fluorescence obtained after DNA labelling with Hoechst 33258. The latter, however, was less sensitive and exhibited higher standard deviations. In addition, the method reliably detected the anti-proliferative effects of the anti cancer compounds cisplatin and vindesine. It is, therefore, suggested that the fluorometric assay with 4-methylumbelliferyl heptanoate as substrate could prove useful for the screening of potential anti-cancer agents with anti-proliferative activity. PMID- 1726674 TI - [Simultaneous predictions of disposition kinetics of procainamide and its metabolite N-acetylprocainamide in rat by a physiological pharmacokinetic model]. AB - Disposition kinetics of procainamide (PA) and its metabolite N-acetylprocainamide (NAPA) in rats was simulataneously predicted by a physiological pharmacokinetic model. The parameters, such as clearances in kidney and liver and tissue/blood concentration ratios, which were needed for simulations, were determined. The estimated clearances of PA in rat blood, kidney and liver were 47. 28, 13. 56 and and 33. 71 ml.kg-1.min-1, respectively. Tissue/blood drug concentration ratios were obtained after iv administration according to Gallo's method and demonstrated that heart, liver, kidney, muscle and small intestine have greater affinity for PA than do blood components. The concentrations of PA and NAPA in rat tissues following iv administration of PA.HCl 75 mg/kg were predicted and compared with observed values. The results showed that a good agreement between predictions and observed data was found in most of rat tissues. Concentrations of PA and NAPA in plasma of man, based on scaling-up of kinetics of PA and NAPA from rat to man, was also simulated. PMID- 1726675 TI - Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants. AB - Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L pre-S2/15 induced anti-HBsAg a-determinant antibody as well. PMID- 1726676 TI - [Spontaneous variability of ventricular extrasystole in chronic Chagas cardiopathy]. AB - PURPOSE: To study the spontaneous variability of single (VPCs) and coupled (CVPCs) in patients with chronic Chagas' disease (CCD). PATIENTS AND METHODS: Twenty patients with CCD, 14 male, in class I and II NYHA, with frequent VPCs and VCPCs, free of drug therapy were studied. 21 hour Holter monitoring was done for 4 subsequent days. The data analysis assessed the variation in the frequency of VPCs and CVPCs between patients, seven hour periods one hour periods in a hierarchical model by a Poisson process. RESULTS: a) the frequency of VPCs follows a circadian rhythm, closely related to the hourly variations of the mean heart rate; b) disregarding the heart rate influence on the variability of the ventricular arrhythmia, its behavior was at random and unpredictable; c) the minimal percentual reduction in VPCs/h that discriminated between antiarrhythmic effect and spontaneous between antiarrhythmic effect and spontaneous between antiarrhythmic effect and spontaneous variability was 121.86% for seven-hour, 58.42% for 21-hour and 38.45% for 42-hour electrocardiographic monitoring periods; d) the same approach for the VCPCs revealed values of 133.6%, 63.21% and 41.3% respectively. CONCLUSION: The large variability of ventricular arrhythmia in CCD during a 24 hour period makes necessary observations always longer. The minimal percentual reduction in VPCs/h that discriminated between antiarrhythmic effect and spontaneous variability might be 58.42% for two 21-hour electrocardiography monitoring periods. PMID- 1726677 TI - Experience with palliative [131I]metaiodobenzylguanidine therapy in advanced neuroblastoma. AB - Our experience with palliative [131I]metabenzylguanidine (131I-MIBG) therapy in 7 patients (6 children and 1 adult) affected by advanced neuroblastoma is reported. All patients (classified as IV stage) showed a progression following initial intensive therapy, including chemotherapy and, in some cases, hemi-body irradiation and surgery for their primary tumor. 131I-MIBG activity ranged for a single course between 2.77 GBq to 5.55 GBq on the basis of age, intensity of uptake, and the hematological assessment. Four patients received only one course of therapy due to progressive disease (2), early death (1) or persistent thrombocytopenia unrelated to 131I-MIBG therapy (1). Two patients received two courses and showed a partial response lasting 4 months and stable disease lasting 3 months respectively. Therapy was thereafter discontinued due to progression. One patient received 4 courses of therapy (cumulative activity = 19.61 GBq) in 5 months. A partial response for 9 months in the bone metastases was documented, but the therapy was discontinued due to persistent thrombocytopenia (58,000 plts/microL) lasting 4 months. Thrombocytopenia was the major side-effect, occurring in 5/7 patients over 8 courses of therapy for a mean period of 37 days (7-120 d). Thus, in our experience thrombocytopenia is the major factor limiting the therapeutic effect of 131I-MIBG therapy in palliative treatment. PMID- 1726678 TI - Role of [131I]metaiodobenzylguanidine therapy in carcinoids. AB - From cumulative reported data the sensitivity of [131I]metaiodobenzylguanidine (131I-MIBG) scintigraphy of carcinoids appears to be greater than 60%; at our Institute 131I-MIBG scintigrams were positive in 51 of 70 patients with metastatic carcinoid. Twenty patients with symptomatic, metastatic disease have received 7.4 GBq doses of 131I-MIBG for palliation. Most of these patients had multiple large metastases showing no response to other therapies. No objective response (greater than 50% tumor volume reduction) was ever observed; however, 13 patients were relieved of symptoms, such as flushes, diarrhea, anorexia and pain. Palliation in some of these patients was meaningful and long lasting. Possible explanations for a palliative effect in the absence of objective remission are discussed. Treatment with escalating doses of stable MIBG (up to 80 mg) in 9 patients does not support the hypothesis that the palliation is due to a purely pharmacological effect. Palliation might be explained by the observation that carcinoid liver metastases may present both as hot and cold lesions; 131I-MIBG therapy will thus target exclusively at metabolically active metastases, which are responsible for the patient's symptoms. PMID- 1726679 TI - [Effect of immunomodulating drugs on the release and activities of lysosomal proteinases of the liver of rats with ethanol poisoning]. AB - Increased activity of cathepsin A and D in the cytosol fraction and homogenate of the liver of rats intoxicated for 4 weeks with ethanol (0.6 g/100 g of the body weight) was found. The cytosol cathepsin A and D activities were unaffected under the influence of Levamisole and isoprinosine++. Encorton reduced the activity of both cathepsins in the cytosol fraction while it did not diminish their activities in the liver homogenates. Encorton, and to a markedly lesser degree, Levamisole and isoprinosine++ caused a regression of vacuolar degeneration and of necrotic lesions and an increase in the number of glycogen granules in the livers of ethanol-intoxicated rats. PMID- 1726680 TI - [Regressive changes in the placenta in early pregnancy in women with primary arterial hypertension]. AB - Morphological methods, in particular histochemical technique of secondary fluorescence according to Bertalanffy were used for the assessment of regressive changes of placenta. In women with primary arterial hypertension during early pregnancy fibrinoid masses in decidua, chorion, anchoring willi, villous plate, basal plate, villous trunks and terminal villi appear. These changes are followed from one side, by an atrophy of the blood vessels and connective tissue cells, and from the other, there increases the number of decidua giant cells, as well as of syncytium and cytotrophoblast. The areas of impaired blood supply contain giant cells and intervening foam cells. These cells according to the authors can be the forms of regressive changes exhibiting relative biological activity influencing trophic status of the placenta. Possible importance of the fibrinoid masses for the immunology of pregnancy which is developing under maternal morbid conditions has been discussed. PMID- 1726681 TI - Development of a radioimmunoassay for human group-II phospholipase A2 and demonstration of postoperative elevation. AB - A radioimmunoassay (RIA) for the determination of human group-II phospholipase A2 (M-PLA2) has been developed. M-PLA2 was purified from human spleen. Monoclonal antibody (IgG) was prepared by fusion of splenic cells from immunized mice with M PLA2 and the mouse myeloma cell line NS-1. The RIA was carried out by a single antibody method. The assay is sensitive (0.78 micrograms/l), reproducible and specific. In healthy individuals, the serum M-PLA2 concentration ranges from 1.4 to 4.2 micrograms/l, the average being 2.2 +/- 0.1 micrograms/l (mean +/- SE). Using the RIA, we found increased serum M-PLA2 in patients with various infections and malignant tumors. We also showed the postoperative transient elevation of serum M-PLA2 in cases without any infectious complications. The elevation was independent of the surgical procedure or site. The maximum serum M PLA2 level was seen on the 2nd to 4th postoperative day. In these patients, the serum M-PLA2 and C-reactive protein levels were significantly correlated. The present study indicated that serum M-PLA2 is an acute phase reactant. PMID- 1726682 TI - Determination of leukocyte elastase concentration in plasma and serum by a simple method using a specific synthetic substrate. AB - The concentration of leukocyte elastase (ELP) in plasma and serum was determined by an amidolysis method using a specific synthetic substrate for ELP, Suc-Ala-Tyr Leu-Val-pNA. Results were compared with those using ELISA. ELP levels in plasma from healthy donors were similar to those determined by ELISA; however, the levels in serum were lower than those determined by ELISA. Correlation coefficients of ELP levels in plasma and serum as measured by the two methods were 0.75 (amidolysis) and 0.90 (ELISA). On the other hand, the correlation coefficient between serum ELP by the two methods was 0.83. Half of the ELP levels in plasma from 150 patients and serum from 400 patients were significantly elevated when compared with those from healthy donors, and the ELP elevation determined by amidolytic assay correlated with some variables in blood, namely fibrin(ogen)-degraded products, fibrinogen, GOT, GPT, gamma-GTP, LDH and leucine aminopeptidase. Despite the fact that the amidolysis method detects the alpha 2 macroglobulin-ELP complex while ELISA detects the alpha 1-antitrypsin-ELP complex, a comparative study showed amidolysis to provide sufficiently sensitive measurement of both plasma and serum ELP. PMID- 1726683 TI - Inosine pranobex in the treatment of HIV infection: a review. AB - Inosine pranobex (InPx) could prove a valuable and innovative approach to the treatment of HIV-infected patients, since InPx administration has been shown in two multicenter trials to effectively delay the progression of HIV infection to overt AIDS. However, further studies are strongly required to optimize both the dosage of inosine pranobex and the administration schedules. Furthermore, clinical trials evaluating combination therapy of HIV infection with both InPx and zidovudine should ultimately provide an important advance in the management of HIV-infected patients. Our finding that concomitantly administered InPx to zidovudine-receiving patients increased the plasma levels of zidovudine as well as prolonged zidovudine mean half-life during InPx treatment suggests several potential advantages of the combination treatment with both InPx and zidovudine, such as a need for lower zidovudine dosage and a longer interval period between administering zidovudine to obtain sustained plasma levels as well as a potential to enhance residue immune function resulting from inosine pranobex treatment. PMID- 1726684 TI - Rationale for a combined use of antiretroviral and immunomodulatory therapies in HIV infection. AB - To investigate a possible potentiation of Azidothymidine activity by immunomodulators, two in vitro models of HIV infection were analyzed. Peripheral blood lymphocytes on one hand, and cells from the CEM line on the other hand, were infected in vitro with HIV1 and reverse transcriptase activity was monitored daily in the cultures. When Azidothymidine was added, reverse transcriptase activity was significantly reduced. When Isoprinosine or Diethyldithiocarbamate was added in vitro, the reverse transcriptase was only slightly decreased (to a much less significant degree). When Azidothymidine was added together with either Isoprinosine or Diethyldithiocarbamate, a synergistic effect was observed with a very potent inhibition of reverse transcriptase activity. Before a possible application of such a combined therapy to patients, the pharmacokinetics of Azidothymidine was analyzed after the compound had been given alone or in combination with Isoprinosine. No alteration of pharmacokinetics was observed, suggesting that the immunomodulator will not alter the metabolism of the antiretroviral therapy in vivo, and may even enhance its biological activity, as it does in vitro. PMID- 1726685 TI - Cytokine regulation of the human immunodeficiency virus (HIV). AB - The remarkable ability of HIV to insinuate itself into the working of the immune system is the key of its success as an infectious agent. Given that the cytokine network regulates the immune responses, it is not surprising that cytokines can modulate HIV infection. GM-CSF, IL6 and TNF-alpha enhance HIV, but TGF-beta and HIF inhibits the virus. However, the anti-HIV activity of TGF-beta is restricted to myeloid cells, while HIF inhibits HIV in myeloid cells and in T-lymphocytes. HIF is produced by CEM cells after induction by an extract from pine cones. It is not an interferon and is likely a novel cytokine. It is pepsin-sensitive but trypsin-resistant and has an apparent molecular weight of 7-12 KDa. Apart from having anti-HIV activity, crude preparations of HIF also inhibit HTLV-1 virus but not HSV virus replication. PMID- 1726686 TI - Chemotherapy of the acquired immune deficiency syndrome (AIDS): non-nucleoside inhibitors of the human immunodeficiency virus type 1 reverse transcriptase. AB - Several classes of non-nucleotide analogues (i.e. TIBO and HEPT derivatives) have been identified that specifically interact with the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These derivatives inhibit the replication of HIV-1 in various cell lines, including peripheral blood lymphocytes and monocytes/macrophages, at concentrations that are 10,000- to 100,000-fold lower than the cytotoxic concentrations. At the HIV-1 RT level, they appear to interact with a specific allosteric "TIBO" site, which may be functionally and also structurally associated with the substrate binding site. The TIBO and TIBO-like compounds are orally bioavailable. In vivo they sustain plasma drug levels that are well above the concentrations required to inhibit virus replication in vitro. PMID- 1726687 TI - Effects of 8-bromo-cyclic GMP on slow channel mediated action potentials of 3 days-old embryonic chick ventricle. AB - The role of cyclic GMP in modulation of cardiac slow channel activity was investigated by observing the effects of 8-bromo-cyclic GMP (8-Br-cGMP) on action potentials of isolated ventricle of 3-days-old chick embryo, which exhibit upstroke primarily due to slow channels. 8-Br-cGMP (0.5 & 1 mM) reduced the maximum diastolic potential, maximal upstroke velocity (+Vmax) and overshoot in 30-60 min. 8-Bromo-cyclic AMP (8 Br-cAMP, 0.5 & 1 mM), isoproterenol (Iso, 0.5-5 microM) and forskolin (0.5-2 microM) caused an increase in +Vmax and overshoot. 8 Br-cGMP antagonised this enhancement of +Vmax. Increase in +Vmax and overshoot by Bay-K-8644 (1 microM) was also blocked by 8-Br-cGMP. These findings show that 8 Br-cGMP inhibited the early embryonic cardiac slow channel activity, which contributes significantly to the upstroke of action potential, under basal conditions as well as after its accentuation by elevation of cyclic AMP levels (by 8-Br-cAMP, Iso & Forskolin) or by direct stimulation of the channel activity (by Bay-K-8644). It is suggested on the basis of these findings that cyclic GMP plays a key role in down modulation of the cardiac slow channel activity in early embryonic chick heart. PMID- 1726688 TI - Children at home on mechanical assistive devices and their families: a retrospective study. AB - Few assessments have been done of the influence of caregiver and family interactions on the developmental status of children discharged on mechanical assistive devices. The ages of the 25 children in this study ranged from 2-30 months. Seven were on apnea monitors at the time of the home interviews, and 18 had been off the monitors for 18 months or more. Mothers were administered six questionnaires: Family Adaptability and Cohesion Evaluation Scales (FACES III), Duke-UNC Functional Social Support Questionnaire, Families Coping Strategies (FCOPES), Self-Esteem Scale, Home Observation for Measurement of the Environment, and the Receptive-Expressive Emergent Language Scale (REEL). Results on the FACES III revealed that families were not rigid in adaptability; 61% were chaotic, and 33% were balanced. On the cohesion scale, 25% were disengaged, 30% were enmeshed, and 45% were balanced. On the FCOPES, and it's subscales, 36-40% of the respondents scored below the established norms. Social support was strongly correlated with Cohesion and Adaptability (r = .47, .67), HOME scores correlated with Self-Esteem (r = .47) and FCOPES Reframe scale (r = .45), and REEL scores correlated with Social Support (r = .47, .52). Path-analysis revealed two paths: help-seeking behaviors of the mother and the child-mother interactions. PMID- 1726689 TI - [Quinidine-induced thrombocytopenia]. AB - We present the case of drug-dependent thrombocytopenic purpura due to the treatment with quinidine. IgG quinidine-dependent antibodies in patient serum were detected in the platelet immunofluorescence test. They were bound to GP Ib on platelet membrane. PMID- 1726690 TI - Hyperfunctioning thyroid nodules. AB - The authors describe the principal clinical and pathological aspects of the solitary hyperfunctioning adenoma or the multifocal hyperfunction of a multinodular goitre. Successively they report the incidence of these conditions in countries with different iodine intake as well as the age distribution of the examined patients. In the area with low iodine intake the incidence of hyperthyroidism caused by multinodular goitre is 10 times higher than in the high iodine intake area. Finally, the role of the laboratory in the diagnosis of hyperthyroidism and in identifying the type of hyperthyroidism is discussed; an up-todate flow-sheme is also reported. PMID- 1726691 TI - Isoelectric focusing and immunoblotting analysis of thyroglobulin from different thyroid diseases. AB - Thyroglobulins (Tgs) have been obtained from macro-microfollicular goiter, toxic adenomas, papillary carcinomas and metastatic lympho-node and chromatographed by gel filtration. The 19S Tgs so obtained have been characterized by isoelectric focusing (IEF) and immunoblotting. IEF showed microheterogeneity pattern of Tgs from normal tissue, macro-microfollicular goiter and toxic adenomas to be within pH range from 4.2 to 4.5, while that from papillary carcinomas and metastatic lympho-node showed a wider microheterogeneity. Passive blotting of focused Tgs and immunoreaction with rabbit anti-Tg peroxidase conjugated antibody have been carried out and a positive reaction for all examined samples have been evidenced. Also two bands, focused at more cathodic pH value (pH 4.7), observed in IEF patterns of Tgs from carcinomas and metastatic lympho-node, gave a positive reaction with anti-Tg conjugated antibody. These differences observed in IEF and immunoblotting patterns of Tgs from papillary carcinomas and metastatic lympho node are discussed in this report. PMID- 1726692 TI - The thyroid nodule: diagnostic strategy. AB - This paper comments on the principal techniques (nuclear imaging echography and needle aspiration) and the most widely used protocols available for preoperative selection of thyroid nodule patients. Successively, the authors report the principles to define a new appropriate protocol based on needle aspiration and hormone assays and evaluate their experience with this new simple protocol. Long term clinical practices shows that surgical excision can be avoided for more than 90% of the nodule patients and that thyroid scintigraphy can be avoided for most of these patients, without appreciable disadvantage. PMID- 1726693 TI - Analysis of the DNA content in the management of thyroid tumours. AB - Thyroid carcinoma is rare and constitutes about 1% of all malignant tumours. For some time it has been known that age, sex, histology, and tumour grade are factors of importance for prognosis. In Stockholm we have analyzed the amount of nuclear DNA in thyroid tumours for the last 10 years. In our studies and those of other nuclear DNA-analysis has given important prognostic information for differentiated thyroid tumours in addition to that provided by histopathology, and may be of value preoperatively for treatment planning. DNA-analysis is well established in our clinical work and is one of the factors considered in a prospective randomized study concerning patients with papillary carcinoma in the Stockholm-Uppsala area. Tumours with diploid nuclear DNA pattern are randomized between lobectomy and total thyroidectomy in this study. PMID- 1726694 TI - The measurement of plasma D-dimer in the follow-up after thyroidectomy for cancer: preliminary data. AB - Plasma concentration of fibrinopeptide A (FpA) and D-dimer (DD), sensitive indicators of coagulation and fibrinolysis activation, were measured in 21 patients thyroidectomized for differentiated cancer (4 had distant metastases) and in 27 control subjects. Only two patients (one with distant metastases) presented elevated FpA values. All the four patients with distant metastases showed elevated DD levels. In three patients who developed distant metastases the time course of the circulating levels of plasma DD and serum thyroglobulin (Tg) was monitored. Monitoring of plasma DD concentration correctly indicated the time course of all the 3 metastatic patients while serum Tg levels failed to adequately describe the outcome of one metastatic patient. These data suggest that fibrinolysis is enhanced in thyroid cancer patients with distant metastases and that plasma DD levels are useful in the monitoring of this condition. PMID- 1726695 TI - Monoclonal antibodies define structural alterations of the thyroglobulin secreted by well-differentiated thyroid carcinomas. AB - We have studied the sorting and alteration of the thyroglobulin (tg) secreted by well-differentiated carcinomas using a group of anti-tg monoclonal antibodies. A previously described monoclonal antibody, specific for the hormogenic iodinated epitopes, shows that in carcinomas the iodinated tg is not secreted in the colloid as in normal glands but is accumulated in the cytoplasm of the cells. Separation of tg in a concanavalin A column, to isolate the correctly glycosilated protein, reveals that hormogenesis is present only in glycosilated tg with a correct final configuration. An other monoclonal antibody, specific for an epitope connected with the carbohydrate moiety of the tg, reacts with tgs from normal glands and from any thyroid lesion except tg from carcinomas. This tg of carcinomas presents an alteration in the oligosaccharide chains that can be detected by this antibody. The alteration is specific for malignant tumors and could be connected with the missing exocytosis of the iodinated tg in carcinomas. PMID- 1726696 TI - [The problem of excitability in the works of L. L. Vasil'ev and its further development]. AB - Using microelectrode recording of the mollusks Planorbis corneus and Lymnaea stagnalis neurones, protracted depolarisation current was shown to induce first increased and then decreased excitability. Protracted hyperpolarization current entails a transmembrane potential difference and eliminates the inactivation of ion channels. Rhythmic depolarisation stimulation causes inactivation of inward ion current. PMID- 1726697 TI - Is aging preprogrammed? Observations from the brain/gut axis. AB - Age related differential gene expression occurs in the neuro-enteral axis. Brain and gut organ weight, total RNA, total protein and three peptides were quantified in 4-, 10- and 37-week-old Sprague-Dawley rats. As animals aged, total RNA decreased in the brain (0.65 +/- 0.3-0.28 +/- 0.03 mg/g), but remained stable in the gut (2.6 +/- 0.3-2.9 +/- 0.4 mg/g). Total protein concentration rose in the duodenum (612 +/- 28-734 +/- 34 mg/g), while levels remained stable in the brain (641 +/- 54-666 +/- 34 mg/g). Three peptides were studied, cholecystokinin (CCK), VIP and secretin. With increasing age, significant changes were found only in CCK a true neural-enteral peptide. The concentration of smaller molecular forms of CCK decreased in the brain (248 +/- 18-188 +/- 21 pmol/g), while they remained stable in the duodenum (33 +/- 2-36 +/- 3 pmol/g). By contrast, the concentration of the larger forms of CCK were stable in the brain (36 +/- 3-40 +/- 4 pmol/g), but rose in the gut (89 +/- 14-134 +/- 17 pmol/g). These data indicate that as rats age there is preprogrammed differential control of gene expression between brain and intestine. PMID- 1726698 TI - Myosin light chain 1 isoform expression remains constant during ageing in Wistar F455 rats. AB - In order to study muscle gene expression during ageing, we examined both protein and total cellular RNA from Wistar F455 rat soleus and extensor digitorum longus (EDL) muscles at a variety of chronological ages. We found no evidence of the reappearance of the fast protein isoform of myosin light chain 1 [MLC1] in the slow soleus muscle during ageing previously reported by Syrovy and Gutmann, Pflugers Arch., 369 (1977) 85-89. We used both SDS-PAGE analysis of MLC1 proteins and slot blot RNA analysis with a probe specific for rat fast MLC1 mRNA (pC91), and found no changes in fast MLC1 expression during ageing in soleus or EDL muscles from these rats. These results indicate that re-expression of the fast MLC1 isoform is not a universal property of ageing soleus muscle. PMID- 1726699 TI - Human B cell proliferative responses during aging. Reduced RNA synthesis and DNA replication after signal transduction by surface immunoglobulins compared to B cell antigenic determinants CD20 and CD40. AB - Age-related reductions in the DNA replication of human peripheral blood B cells have been reported after stimulation by cross-linking surface immunoglobulins (sIg) with the polyclonal activator Staphylococcus aureus Cowan I (SAC). However, little is known about the mechanisms of these age-related impairments. To examine whether these impairments represented defects unique to sIg mediated signalling, B cells from elderly humans were stimulated with SAC, immobilized anti-IgM and with monoclonal antibodies (mAbs) specific for B cell CD20 and CD40 determinants. Regardless of the stimuli or combinations of stimuli, the proliferative responses of B cells from elderly subjects remained 50% or less of the values observed for B cells from young subjects. Also, the failure to fully restore the age-related impairments of B cells could not be attributed to an absolute lack of potentially reactive cells. Supplementation of anti-IgM stimulated B cells from elderly subjects with IL-2, IL-4 or B cell growth factor (BCGF) revealed that BCGF was able to improve the reduced responses to levels approximating B cells of young subjects. The age-related defects were not restricted to B cell DNA replication because reductions in G1 progression of stimulated B cells from elderly subjects were directly demonstrated by decreased [3H]uridine incorporation into de novo RNA synthesis. However, the age-related impairments in RNA synthesis were less severe than those in DNA replication consistent with progressively greater reductions in the abilities of B cells to traverse the entire cell cycle. Other results showed that the reduced DNA replication of B cells from elderly subjects to immobilized anti-IgM with and without IL-2 did not represent a premature exit of B cells from DNA replication and accelerated maturation into antibody producing cells. Thus, these studies demonstrate that age-related impairments exist in activation signals mediated by several types of human B cell determinants and that abnormalities can be detected during pre-S phase events. PMID- 1726700 TI - [Treatment of Hodgkin's disease. National Protocol of Antineoplastic Drugs: preliminary report]. AB - We report preliminary results of treatment of Hodgkin disease according to the National Protocol on Antineoplastic Drugs. 37 males and 22 females with a median age of 34 years (range 16 to 76) were treated. Patients with stages I or II received radiotherapy alone or chemotherapy (C-MOPP) followed by radiotherapy. Patients with stages III or IV received radiotherapy and chemotherapy or alternating courses of C-MOPP and ABVD. Complete remission was observed in 74% of 39 patients completing full therapy. Complete remission was more common in patients with nodular sclerosis without B symptoms and under 45 years of age. Actuarial survival at 22 months was 79%, significantly higher for patients with nodular sclerosis compared to patients with lymphocytic depletion. Different treatments in patients at stages I and II or III and IV gave comparable results. Complications included infection with the Chickenpox-Zoster virus, pneumonia and fever of unknown origin. Mortality was associated to older age, complications of treatment or failure to comply with therapy. PMID- 1726701 TI - Hyaline droplet accumulation in kidney proximal tubules of mice with histiocytic sarcoma. PMID- 1726702 TI - [Mechanisms involved in normal cardiac automatism]. PMID- 1726703 TI - Human burst-forming units-erythroid need direct interaction with stem cell factor for further development. AB - To understand the factors that regulate the early growth and development of immature erythroid progenitor cells, the burst-forming units-erythroid (BFU-E), it is necessary to have both highly purified target cells and a medium free of serum. When highly purified human blood BFU-E were cultured in a serum-free medium adequate for the growth of later erythroid progenitors, BFU-E would not grow even with the addition of recombinant human interleukin-3 (rIL-3), known to be essential for these cells. However, the addition of recombinant human stem cell factor (rSCF), which supports germ cell and pluripotential stem cell growth, stimulated BFU-E to grow equally well in serum-free as in serum-containing medium. Limiting dilution studies showed that rSCF acts directly on the BFU-E that do not require accessory cells for growth. Furthermore, rSCF was necessary for BFU-E development during the initial 7 days of culture, until these cells reached the stage of the late progenitors, the colony-forming units-erythroid (CFU-E). These studies indicate that early erythropoiesis is dependent on the direct action of SCF that not only affects early stem cells but is continually necessary for the further development of committed erythroid progenitor cells until the CFU-E stage of maturation. PMID- 1726704 TI - Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils. AB - We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5 mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis. PMID- 1726705 TI - Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs. AB - Amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (adenosine aminohydrolase, EC 3.5.4.4; ADA) were used to transduce canine marrow cells. In one approach, dogs were treated for 7 days with recombinant human granulocyte colony-stimulating factor to stimulate hematopoietic cell division. Bone marrow cells were collected and transduced by 24 hours of cocultivation on vector producing cells followed by incubation in a vector-containing long-term marrow culture system for 4 days. Transduced autologous marrow (0.4 to 1.0 x 10(8) cells/kg) was infused into dogs administered otherwise lethal total body irradiation (TBI) of 920 cGy. Two of four dogs engrafted, and their marrows showed intermittently between 1% and 11% G418-resistant colony-forming unit granulocyte-macrophage (CFU-GM) colonies for up to 2 years after transplantation. In a different experimental approach, autologous marrow, obtained at the time of the PB neutrophil nadir 7 days after a single cyclophosphamide injection (40 mg/kg intravenously), was cocultivated for 24 hours on vector-producing cells and infused at doses of 0.06 to 0.18 x 10(8) cells/kg into dogs administered 920 cGy TBI. One of three dogs engrafted, and the marrow showed intermittently 1% to 10% G418-resistant CFU-GM colonies for at least 2 years. Culture results were confirmed by polymerase chain reaction (PCR) showing the presence of the neo gene in marrow cells, peripheral blood (PB) granulocytes, and PB and lymph node lymphocytes. Dilution experiments indicated that up to 10% of marrow, lymph node, and PB cells contained the neo gene, consistent with the culture results. Samples harboring the neo gene also contained the gene for human ADA. However, repeated analyses of PB and marrow cells for human ADA gene expression by starch gel electrophoresis were negative. PB samples of all dogs were free of helper virus, and no long-term side effects from the transduction were observed. PMID- 1726706 TI - Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking. AB - To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi-Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor-mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets. PMID- 1726707 TI - Elevated serum levels of soluble interleukin-2 receptor: a marker of disease activity in the hypereosinophilic syndrome. AB - We report here the presence of very high serum levels of the soluble interleukin 2 receptor (sIL-2R) in patients with blood hypereosinophilia with or without detectable markers of malignancy or signs of visceral involvement. The highest sIL-2R levels were observed in 16 eosinophilic patients with T-cell lymphoma (3,440 to 79,500 U/mL). Elevated levels of sIL-2R were also present (1,330 to 22,500 U/mL) in sera from 38 patients with the hypereosinophilic syndrome (HES) without detectable T-cell lymphoma. In this group of patients, the highest levels were noted in the patients with the malignant form of HES. Significantly lower levels were measured in sera of patients with hypereosinophilia associated with parasitic diseases, allergic disorders, or other miscellaneous diseases. Elevated serum sIL-2R levels were not closely paralleled by changes in the number of CD25 positive peripheral blood mononuclear cells as assessed by flow cytometric analysis. However, expression of IL-2R messenger RNA was detected in blood mononuclear cells collected from HES patients. In eight eosinophilic patients with T-cell lymphoma, the serum sIL-2R levels were significantly correlated with the eosinophil counts, and with the total number of blood hypodense eosinophils. alpha-Interferon (alpha-IFN) therapy resulted in both a dramatic clinical improvement and a rapid decrease in sIL-2R levels and blood hypereosinophilia. Similar beneficial effects of alpha-IFN were noted in patients with malignant HES who lacked a detectable T-cell lymphoma. Our data indicate that HES is associated with elevated serum IL-2R levels. The highest levels were observed in the most severe forms of HES with hematologic markers of malignancy or evident visceral involvement. Serum levels of sIL-2R might represent a useful indicator for the management of HES patients. In addition, the respective changes of sIL-2R and blood eosinophilia might reflect distinct processes of mononuclear cell activation affecting the eosinophil lineage. PMID- 1726708 TI - Eosinophils from patients with blood eosinophilia express transforming growth factor beta 1. AB - The infiltration of eosinophils into tissues during pathologic responses is often associated with extracellular matrix alterations such as fibrosis. Transforming growth factor-beta 1 (TGF-beta 1) is a well-characterized multifunctional cytokine known to exert potent effects on the extracellular matrix. In this report, we showed the production of TGF-beta 1 by human eosinophils from patients with blood eosinophilia. Northern blot analysis using RNA isolated from eosinophils purified from a patient with the idiopathic hypereosinophilic syndrome (HES) detected the 2.5-kb TGF-beta 1 transcript. In situ hybridization and immunohistochemistry of leukocytes from two patients with HES and two patients with blood eosinophilia localized TGF-beta 1 messenger RNA (mRNA) and protein to eosinophils. No other cell type contained TGF-beta 1 mRNA by in situ hybridization, whereas other leukocytes contained detectable TGF-beta 1 protein by immunohistochemistry. Eosinophils from four normal donors contained little or no detectable TGF-beta 1 protein by immunohistochemistry, whereas eosinophils from two of these four normal donors labeled weakly for TGF-beta 1 mRNA by in situ hybridization. These results show that eosinophils in the peripheral blood of patients with blood eosinophilia can express TGF-beta 1, but that eosinophils in the blood of normal donors contained little or no TGF-beta 1. PMID- 1726709 TI - Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors. AB - Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time-courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM-CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G CSF, or FMLP at the inflammatory sites. PMID- 1726711 TI - Role of sulfation in the processing of gastric mucins. AB - The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity. PMID- 1726710 TI - Neutrophil and monocyte adherence to and migration across monolayers of cytokine activated endothelial cells: the contribution of CD18, ELAM-1, and VLA-4. AB - Pretreatment of endothelial cells with cytokines enhances the adherence of leukocytes, a process that is mediated by surface proteins expressed on both cell types. A three-dimensional model system for the simultaneous determination of leukocyte adherence and migration was used to study the contribution of CD11/CD18, endothelial leukocyte-adhesion molecule-1 (ELAM-1) and VLA-4 in neutrophil and monocyte adherence to and migration through cytokine-activated endothelial cells. Pretreatment of endothelial cells for 4 hours with recombinant interleukin-1 beta (rIL-1 beta) was found to enhance neutrophil adherence and migration to a much greater extent than monocyte adherence and migration. Neutrophil adherence was almost completely prevented by the combined use of monoclonal antibodies (MoAbs) against ELAM-1 and CD18. Although ELAM-1 has been designated an endothelial cell-specific cytokine-inducible receptor for neutrophils, we observed that ENA2, an anti-ELAM-1 MoAb, significantly reduced monocyte adherence about 30%. MoAbs against VLA-4, the ligand of the cytokine inducible receptor VCAM-1, did not affect monocyte adherence. However, the combined use of the MoAbs against CD18, ELAM-1, and VLA-4 had a very strong and additive inhibitory effect on rIL-1 beta-induced monocyte adherence. The anti CD18 MoAb reduced both rIL-1 beta-induced neutrophil and monocyte migration far below the level of the unstimulated controls, whereas neither the anti-ELAM-1 nor the anti-VLA-4 MoAb significantly affected the process of migration. Our results indicate that neutrophils and monocytes initially adhere to cytokine-activated endothelial cells by CD18-independent and (to a lesser extent) by CD18-dependent mechanisms and subsequently change gears to a completely CD18-dependent migratory mechanism. PMID- 1726712 TI - Averaging time modeling of exposure simulation with application to the El Camino Real vehicle data. AB - When profiles of activity patterns are used to generate time series of simulated exposure, one typically samples from exposure distributions which are microenvironment-specific to each activity. If the simulation time step is short, then independent sampling at each time step, ignoring autocorrelation, will result in aggregates with too little variability from one simulation to another. Autocorrelation can often be modeled with one or two extra parameters and then used in the simulation. Furthermore, one may substantially reduce computation by generating a single averaged exposure for each activity segment whose distribution depends in a simple way on the activity duration and the modeled autocorrelation. The process is illustrated using the El Camino Real commuting exposure study data of Ott, Switzer, and Willits. PMID- 1726713 TI - The pathobiology of lost human potential: schizophrenia as a neurodevelopmental disorder. PMID- 1726714 TI - Experimental autoimmune uveoretinitis and pinealitis induced by interphotoreceptor retinoid-binding protein and S-antigen: induction of intraretinal and subretinal neovascularization. AB - Experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in Lewis rats following hind foot pad injection of interphotoreceptor retinoid-binding protein (IRBP) or S-antigen. A comparison is made in this study of the in vivo and histological changes in uveoretinitis and pinealitis induced by administering similar doses of highly-purified IRBP and S-antigen emulsified in complete Freund's adjuvant (CFA). The time of onset of ocular inflammation after inoculation was slightly later in S-antigen (14-18 days) as compared with IRBP inoculated animals (10-14 days), while the severity of the inflammation was lower in the latter group. The distribution of inflammation in the anterior segment was similar in both the S-antigen and IRBP sensitized animals but there was major variation in the location of the posterior segment disease. Vasculitis was a predominant feature of IRBP induced disease while chorioretinitis and photoreceptor destruction was more prominent in the S-antigen sensitized animals. A striking feature of this study is that both antigens induced intraretinal and subretinal neovascularization, an observation which has not been reported previously. Inflammation was induced in all pineal glands and as with EAU the severity was closely related to the type of antigen inoculated. PMID- 1726715 TI - [Expression regulation of insulin-like growth factor binding proteins (IGFBP). Tissue-specific expression of IGFBP-1]. AB - We have investigated the liver-specific expression of IGFBP-1 gene, an Insulin like Growth Factor binding protein. Northern blot as well as transient transfection experiments, both carried out in differentiated (H4II, C2Rev7) and dedifferentiated (H5, C2) hepatoma cell lines, yielded clear cut data allowing the involvement of HNF1, a liver-specific trans acting factor, in basal IGFBP-1 liver-specific expression. PMID- 1726716 TI - The 5'-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23. AB - The growth-related protein p23 of the Ehrlich ascites tumor (EAT) is preferentially expressed in the exponentially growing tumor; its synthesis is translationally controlled. p23 mRNA is efficiently translated in the wheat germ cell-free lysate. In contrast, p23 mRNA present in poly(A)+RNA isolated from EAT is not translated in cell-free systems of EAT and reticulocytes. Moreover, translation of a p23 transcript is inhibited in the presence of total poly(A)+RNA. This inhibition is abolished by the removal of the 5'-UTR of the p23 transcript. Solution hybridization/RNase protection experiments point to the presence of a nucleotide sequence complementary to the 5'-UTR of p23 mRNA which might be involved in p23 mRNA inhibition. PMID- 1726717 TI - Transient inactivation of an angiogenesis suppressor gene during palatogenesis. PMID- 1726718 TI - Effect of injection of RNA isolated from normal and epileptic cortexes of rabbits into functioning mollusc neurons. AB - Epileptogenic foci were formed in rabbit visual cortex by freezing with liquid nitrogen. RNA isolated from the epileptogenic cortex (RNAepl), or from the frontal lobes (RNAcont) was injected into spontaneously active neurons of the mollusc Planorbarius corneus. The amplitude and duration of the spontaneous action potentials generated following the injection of RNAepl were reproducibly higher than those produced following the introduction of RNAcont. But the time interval between injection and cessation of spontaneous activity was considerably shorter after RNAepl-injection than after RNAcont-injection. Perfusion of neurons with a solution containing puromycin substantially prolonged the period of spontaneous discharge generation in both cases. The addition of Co2+ to the perfusion solution restored the spontaneous rhythmic activity to cells in which the generation of spikes had ceased following the injection of RNAepl. The mechanism underlying these effects is discussed. PMID- 1726719 TI - Viral chimeric protein including a determinant of myelin basic protein is capable of inducing allergic encephalomyelitis in guinea pigs. AB - A hybrid vaccinia virus expressing a chimeric protein consisting of thymidine kinase and the encephalitogenic determinant, S1, from guinea pig myelin basic protein was constructed. Infection of guinea pigs with the virus resulted in the development of allergic encephalomyelitis. PMID- 1726720 TI - [Quantitative analyses of human prostatic alpha-adrenoceptors and effects of terazosin on the alpha-adrenoceptor activity]. AB - The adrenergic alpha-1 and -2 adrenoceptors in human hypertrophied and non hypertrophied prostatic adenomas were measured in the saturation experiment using 3H-prazosin and 3H-yohimbine. Not only alpha-1 adrenoceptor but also alpha-2 adrenoceptor were found to exist with great amount in prostatic adenomas. In the inhibition experiment selective alpha-1 antagonists, prazosin and terazosin inhibited the 3H-prazosin or 3H-yohimbine bindings to prostatic adenomas. The ability as alpha-1 antagonist is ten times greater in prazosin than in terazosin, but that as alpha-2 antagonist is greater in terazosin than in prazosin. These data suggest that terazosin may have other effects than the relaxation of prostatic smooth muscles in hypertrophied prostatic adenoma. PMID- 1726721 TI - Probing the basic defect in cystic fibrosis. AB - The concurrent developments in electrophysiology studies and the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has provided a unique opportunity to probe the basic cellular defect underlying cystic fibrosis. Various properties of the CFTR protein have been deduced from its primary sequence, the variety of mutations in patients and genotype-phenotype correlations, as well as the results of more recent DNA transfection studies. The most exciting observation is the fact that CFTR acts like a cAMP-regulated Cl- channel. PMID- 1726722 TI - A trans-splicing model for the expression of the tripartite nad5 gene in wheat and maize mitochondria. AB - The mitochondrial single-copy gene nad5 of wheat and maize consists of 5 exons located on three widely separated regions of the genome that are independently transcribed. The first region contains exons I and II separated by an atypical group II intron; in the second region is exon III (only 22 bp long), which is flanked upstream by a maturase-related open reading frame (ORF) and exon e of the nad1 gene, and downstream by a previously unidentified ORF (ORF143); in the third region are exons IV and V separated by a group II intron. In maize, this last domain is flanked upstream by the genes rps12, nad3, and tRNA(Ser) and downstream by a chloroplast tRNA(Cys). RNA editing occurs in wheat exons IV and V as C-to-U changes. A detailed analysis of the transcription of the nad5 gene in wheat and maize reveals that the exons are assembled into a 2.4-kb mRNA after two cis splicing (between exons I and II and exons IV and V) and two trans-splicing events. The trans-splicing process involves the sequences flanking exons II, III, and IV that feature group II introns. A model is proposed for the assembly and maturation of the nad5 transcripts. PMID- 1726723 TI - [Cervicoprostatic section, an endoscopic alternative in treating periurethral adenoma]. AB - Uni- and bilateral endoscopic sectioning of the vesical collum and of the prostatic tissue has become an efficient therapeutic alternative for small and middle adenomas and cervicoprostatic scleroses. The authors recommend the use of bilateral cervicoprostatic endoscopic sectioning, with resection of the intermediary tissue (that adds important advantages), harvesting of prostatic tissue for anatomopathologic examination and removal of the remaining tissular isle that might later obstruct the urethra. This original therapeutic method was used in 38 cases (added to 25 patients treated only by uni- or bilateral cervico prostatic section). The good results obtained, without important complications, lasted in time on the dysectatic syndrome, in the patients with cervicoprostatic sclerosis and small and middle adenomas. PMID- 1726724 TI - [Biliary decompression by tumor drilling and endoprosthesis with a hepatocholedochal tube in a case of cholangiocarcinoma]. AB - The paper reports on the case of an 82-year-old patient, operated for a cholangiocarcinoma with high location, involving the bifurcation of the hepatic ducts. The surgery consisted of an endo-lumen tumoral drilling, followed by assembling two tubes introduced in the hepato-choledochal canal for ensuring the internal biliary drainage. A survival of more than 30 months was obtained. The authors show the possibility of using this palliative method, followed by a good result when a radical surgery cannot be used. PMID- 1726725 TI - Effect of the combination of low-dose mexiletine and metoprolol on ventricular arrhythmia. AB - Antiarrhythmic drug therapy is often ineffective or poorly tolerated. Combining antiarrhythmic agents with different electrophysiologic properties may have a synergistic antiarrhythmic effect when compared with each drug alone. If a lower dose of each drug can be used, combination therapy may also result in lower incidence of side effects. The goal of our study was to assess the complementary effect of low-dose mexiletine and metoprolol, when compared with either drug alone. Ten patients with frequent ventricular arrhythmias including 7 patients with nonsustained ventricular tachycardia were evaluated in an open-label sequential study. The response to drug therapy was evaluated by 24-h continuous EKG monitoring, exercise stress testing, and echocardiogram after each treatment. Combination therapy effectively reduced ventricular arrhythmias in 8 patients (80%) in contrast to only 1 patient (10%) on metoprolol alone and 4 patients (40%) on mexiletine alone. In 5 patients (71%) ventricular tachycardia was abolished. The number of couplets was reduced from 51 +/- 39 to 1.9 +/- 2.4 (p less than 0.01) and total premature ventricular beats from 7790 +/- 9047 to 597 +/- 515 (p = 0.06). Combination therapy was well tolerated without proarrhythmia or precipitation of congestive heart failure. It is concluded that low-dose mexiletine combined with metoprolol is effective in suppressing ventricular arrhythmias in selected patients, and enhances the antiarrhythmic effect of either drug alone without significant side effects. PMID- 1726726 TI - [The effect of high-molecular compounds on the rheological properties of the blood and on the reactivity of the skeletal muscle vessels]. AB - The administration of polymers into the vascular bed of the m. gastrocnemius and in vitro did not affect the postocclusion hyperemia but in changed the venous outflow from the muscle, the change having a phasic character. The effects of concentrations and molecular mass of the polymers on the blood rheological properties were more obvious in vitro. Dextran-20 thousand most favourably affects the blood viscosity whereas the least favourable one belongs to polyoxyethylene-20 thousand. The polymers with molecular mass about 500 thousand in small concentrations reduce the viscosity of the blood, in larger concentrations they enhance it increasing the erythrocytes aggregation. PMID- 1726727 TI - Antibodies to T. cruzi cytosol acidic antigens (FIV) in Chagas' disease recognize parasite cell surface and human heart epitopes. AB - The expression of T. cruzi electronegative antigens (FIV) on the parasite surface and their cross-reactivity with heart tissue antigens was studied. For the former purpose epimastigotes (EPI) treated with glutaraldehyde were used to absorb antibodies against surface antigens. Glutaraldehyde fixed heart tissue was used for absorption of antibodies in sera from two groups of chagasic patients with normal and altered electrocardiogram. The absorption of sera from normal electrocardiogram group with EPI significantly reduced the anti FIV activity by ELISA (p less than 0.001). The decreased reactivity was observed with the antigenic bands focused at pI about 4.5. Thus, the results indicate that chagasic patients without electrocardiographic alterations have a high percentage of antibodies reactive with T. cruzi cell surface antigens. Serum absorption with glutaraldehyde fixed heart tissue reduced the anti FIV activity from both groups of patients by ELISA and diminished the intensity of several bands focused at pI 4-6. PMID- 1726728 TI - Calcium channels in the brain as targets for the calcium-channel modulators used in the treatment of neurological disorders. AB - Recent investigations of calcium channels in brain cells by voltage-clamp techniques have revealed that, in spite of electrophysiological similarities, the pharmacological properties of these channels differ considerably from channels in peripheral tissues, e.g., heart and smooth muscle. Therefore, instead of extrapolation from results obtained on cardiac or smooth muscle cells, a reclassification of calcium-channel modulators applied in disorders of the brain appears necessary. The present article reviews some pertinent observations from groups involved in neuronal calcium-channel characterization and draws the attention to major pharmacological differences on neurons in comparison to those in the heart and in smooth muscle. For certain therapeutic applications in neurology particularly, the low-voltage activated types of calcium channels deserve considerable attention. PMID- 1726729 TI - Update on calcium antagonists in cerebrovascular diseases. AB - Clinical management of patients affected by subarachnoid hemorrhage has been modified by the use of nimodipine. Although no differences in overall neurologic outcome and rates of symptomatic spasm have been observed between nimodipine and control patients, severity of permanent neurologic deficits consequent to cerebral vasospasm is reduced in the former. On the other hand, clinical trials with nimodipine in ischemic stroke did not substantiate the expected neurologic benefits. A meta-analysis of the two phase IV studies published thus far shows that of 350 patients examined, mortality rate was 11.5% and 19% in subjects given nimodipine and placebo, respectively (n.s.). Cerebral death accounted for 30% of cases in both groups, whereas a lower percentage of cardiac and pulmonary fatal events were observed among nimodipine-treated subjects. Moreover, neurologic outcome of survivors was not significantly different. These results may be associated with the notion that the voltage-operated channel blockade exerted by calcium antagonists is only a part of the complex events leading to the enhancement of calcium ion intracellular concentration as a "common final pathway." However, difficulties encountered in planning clinical trials in acute ischemic stroke also might explain the lack of conclusive results. The feasibility of randomization of an adequate sample of patients and of very early therapeutic intervention after stroke onset are discussed. PMID- 1726730 TI - Flunarizine in migraine attack. AB - The usual drugs for migraine attacks carry risks of increased frequency, resistance to other treatment, drug dependency, and abuse. Ergotamines may also be vascular risk factors. Alternative drugs without these risks would be useful. Flunarizine could be an alternative. Migraine cannot be reduced to molecular pathophysiology; it is a disorder of higher brain functions. Flunarizine exhibits the profile of a psychotropic drug that fits in with this situation. In double blind placebo-controlled studies, it was shown that 20 mg flunarizine i.v. was superior to placebo in suppressing migraine attacks and was well tolerated. These results should be further investigated, especially concerning reduction of rebound attacks. PMID- 1726731 TI - The place of calcium antagonists in the prophylactic treatment of migraine. AB - Migraine patients suffering from frequent and severe attacks may need prophylactic treatment. Propranolol, a beta-receptor blocker, and flunarizine, a calcium antagonist, are considered to be the most effective compounds for the prophylaxis of migraine. In a number of controlled studies, flunarizine has been shown to reduce the number of migraine attacks significantly. In migraine studies, sedation and weight gain are the most frequent side effects of flunarizine. PMID- 1726732 TI - Flunarizine in migraine prophylaxis: the clinical experience. AB - Apart from the treatment of migraine attacks, prophylaxis may be required when certain criteria of frequency, duration, or severity are met. In a series of placebo-controlled, double-blind studies, the effectiveness of the cerebral calcium antagonist flunarizine (Sibelium) in migraine prophylaxis was shown. In further investigations, the effectiveness of flunarizine was similar to that of propranolol, metoprolol, pizotifene, and methysergide. The side effects described for treatment with flunarizine were somnolence, weight gain, and, in rare cases, depressive mood and extrapyramidal motor disorders. Considering the benefit/risk relation, flunarizine and the beta-adrenergic agents propranolol and metoprolol are now regarded as the drugs of choice. The mechanism of action of flunarizine in migraine prophylaxis is largely unexplained, but the antihypoxic effect of flunarizine is discussed in this context. The search for predicting factors for a successful treatment with flunarizine and the investigation of an injectable solution for the treatment of acute migraine attacks must be left to future research. PMID- 1726733 TI - Flunarizine in the treatment of vestibular vertigo: experimental and clinical data. AB - Because maintenance antivertiginous treatment with commonly used drugs is only moderately effective, there is still need for new therapeutic concepts in the therapy of vestibular vertigo. The cerebral calcium antagonist flunarizine (Sibelium) revealed positive vestibular effects in experimental animal studies and in healthy volunteers. Clinical trials versus placebo and reference drugs proved flunarizine to be effective in the treatment of vestibular disorders. Somnolence, weight gain, and, in rare cases, extrapyramidal symptoms and depression were discussed as side effects. Further studies on flunarizine's mechanism of action are needed to elucidate whether its clinical effects are indeed due to calcium-entry blockade. PMID- 1726734 TI - Medical treatment of acute ischemic stroke. AB - Progress in general symptomatic therapy such as avoidance of complications through nursing care, rehabilitation, and secondary prevention of stroke yield a better quality of life to stroke patients. Unfortunately, no specific drug therapy for acute stroke has been proven to be of benefit in controlled trials. However, new drugs and drug therapies such as thrombolytic therapy, 5 hydroxytryptamine agonists, NMDA receptor antagonists, and free radical scavengers are being studied. PMID- 1726735 TI - New insights on the comma-less theory. AB - The comma-less hypothesis represents a theoretical effort to describe one of the steps in the early evolution of the translation apparatus. This hypothesis emphasizes the advantages that a RNY coding pattern would have provided in a primitive RNA adaptor-catalyst system. This theory has been debated for years, both in conceptual and statistical terms, and no consensus about its validity has been ascertained. In this work, a statistical model refuting this theory was reconsidered. This new approach eliminates the bias due to the absence of stop codons in the open reading frame, and to the amino acid composition of bacterial genes. The results obtained support the biological significance of the RNY coding pattern. PMID- 1726736 TI - Retrotransposons and evolution in phlebotomines. AB - The polymerase chain reaction was used to amplify a segment of the reverse transcriptase (RT) gene of putative retrotransposons from Phlebotomus (Larroussius) perniciosus, P. (L.) perfiliewi, P. (Phlebotomus) papatasi and Lutzomyia (Lutzomyia) longipalpis. Based on amino acid sequence comparisons with known RT genes, the amplified products of these species were shown to be derived from non-LTR retrotransposons related to the F element of Drosophila melanogaster. The usefulness of this technique is discussed in relation to taxonomy and genetic manipulation. PMID- 1726737 TI - Geographical distribution of the subgenus Helcocyrtomyia, genus Lutzomyia (Diptera: Psychodidae--Phlebotominae). AB - The known geographical distribution of species included in the subgenus Helcocyrtomyia Barretto is reviewed. It is suggested that the vexator series of the subgenus originated in the Nearctic Region and extended southwards along the Andes (peruensis series) whereas the oswaldoi series is a South American assemblage originating in Gondowana. It is concluded that Helcocyrtomyia cannot be accepted as a monophyletic group. PMID- 1726738 TI - Inhibition of reverse transcription by unmodified and modified antisense oligodeoxynucleotides. AB - We used oligodeoxynucleotides to prevent reverse transcription of beta-globin mRNA by reverse transcriptase of avian myeloblastosis virus. Unmodified oligomers hybridized to the template arrested synthesis of cDNA in a dose dependent manner. The longer the oligomer the more efficient the inhibition, 50% inhibition being achieved at 0.3 and 30 microM of a 17- or a 12-mer, respectively. The use of complementary oligonucleotides with a 3' end blocked either by a dideoxy residue or by a dodecanol group also induced inhibition of cDNA synthesis. PMID- 1726739 TI - Investigation of DNA biosynthesis catalyzed by DNA polymerases: approach of synthesis of compounds with anti-HIV activity. PMID- 1726740 TI - Hexopyranosylnucleoside 6'-triphosphates are not substrates for DNA polymerases. PMID- 1726741 TI - Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches. AB - The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described. PMID- 1726742 TI - Oligonucleotides and their derivatives as tools for investigations of protein nucleic acid interactions in template biocatalysis. AB - On the basis of quantitative characteristics (Kd, Km, Gibbs energy values) for the interaction of oligonucleotides with template and primer site of eucaryotic and procaryotic DNA polymerases, a general model of template-primer interaction with these enzymes was suggested. The interactions of AMV- and HIV- reverse transcriptases with various 5'-derived oligonucleotides and with human DNA polymerase alpha and Klenow fragment are compared. The results obtained suggest a method to improve selectively the affinity of an oligonucleotide primer to RNA template with AMV- and HIV-reverse transcriptases. PMID- 1726743 TI - Synthetic oligonucleotides for biomedical applications. AB - New developments of oligonucleotides for biomedical applications are surveyed. Diagnostic probes were conveniently labelled by enzymatic immunogenic tailing with 5-bromo-deoxyuridine triphosphate. "Antisense" oligonucleotides of potential therapeutic value were stabilized against nucleolytic decay by inversion of terminal internucleotidic linkages. Introduction of 2'-deoxy-2'-fluoro-nucleotide units enhances duplex stability and conveys resistance to ribonucleases. PMID- 1726744 TI - Synthesis and modification of genes through artificial splicing by directed ligation (ASDL). AB - An approach to the directed genetic recombination in vitro mediated by synthetic oligodeoxynucleotides and polymerase chain reaction (PCR) is devised, which allows the joining, in a predetermined chemical-enzymatic way, of a series of DNA segments to give a precisely spliced polynucleotide sequence (Artificial Splicing by Directed Ligation, ASDL). The approach can thus lead to the totally processed eukaryotic genes using genomic DNA, with no mRNA needed. This approach has been used for the synthesis of artificial genes of interleukin-1 alpha and, in combination with PCR on the mRNA-cDNA duplex as template, of interleukin-1 receptor antagonist and their analogues, as well as for the modified genes. PMID- 1726745 TI - Nucleoside 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphates as substrates for DNA polymerases. PMID- 1726746 TI - The synthetic oligonucleotides as a model for studying of DNA folding. PMID- 1726747 TI - 'Quasi-enzyme' cleavage of a DNA target by a bleomycin A5 oligonucleotide derivative. PMID- 1726748 TI - Studies in the synthesis of oligo- and poly-ribonucleotides. PMID- 1726749 TI - Biological and physico-chemical study of ara-AMP conjugates. PMID- 1726750 TI - Cocrystallization of Escherichia coli RNase H with synthetic DNA/RNA hybrid oligomers. PMID- 1726751 TI - Oligonucleotide derivatives resistant to the cell nuclease degradation and effective for the RNA hydrolysis by RNase H. PMID- 1726752 TI - The hybridase RNA hydrolysis can be altered by oligonucleotide probes. PMID- 1726753 TI - The application of the method of ligation-mediated, single-sided polymerase chain reaction for the analysis of heterogeneity of 5'-untranslated regions of specific mRNA. PMID- 1726754 TI - Direct non-radioactive detection of virus RNA by a novel RNA-PCR test. PMID- 1726755 TI - 2'-5'-linked oligonucleotides form stable complexes with complementary RNA and DNA. PMID- 1726756 TI - Synthesis of branched nona and deca-RNA modelling the lariat formed in pre-mRNA processing reaction (splicing). PMID- 1726757 TI - Synthesis and biological studies with dithioate DNA. PMID- 1726758 TI - [Effect of isoprinosine and acyclovir on the clinical course of chickenpox and herpes zoster]. AB - The therapeutic effect of isoprinosine and acyclovir have been studied in 352 and 284 patients with chicken-pox and herpes zoster respectively. The patients were divided into 4 groups: the first one was given palliative treatment only, the second--both palliative and isoprinosine ones, the third--palliative and acyclovir treatment, and the fourth group was given all these. The best therapeutic effect was achieved when acyclovir and isoprinosine was applied jointly, the one of acyclovir alone was less pronounced and that of isoprinosine only was the smallest. According to the authors acyclovir should be the treatment of choice in the very severe and severe cases of chicken-pox and herpes zoster; in the early stage of disease it should be supplemented with isoprinosine and passive immunotherapy. PMID- 1726759 TI - The influence of humidity on the residual action of benzene hexachloride (BHC). AB - In controlled humidity chambers in the laboratory differences in the absorption velocity of BHC were observed depending on the substrate sprayed. While BHC is no longer used in Chagas' disease control this data could have relevance to spraying houses in a control programme with other insecticides. PMID- 1726760 TI - The revenge of the kainate receptor. PMID- 1726761 TI - Paradoxical movement in Parkinson's disease. AB - Patients with Parkinson's disease, although impaired, can sometimes move effectively under visual guidance. The stimuli that often elicit such paradoxical movement are similar to those that relay visual information to the cerebellum. We suggest that many instances of paradoxical movement may be explained by the fact that the pathways relaying those visual stimuli can bypass the damaged basal ganglia and allow an intact cerebellar circuit to be used for visuomotor control. PMID- 1726762 TI - Cholinergic controversies. PMID- 1726763 TI - Cholinergic controversies. PMID- 1726764 TI - Cholinergic controversies. PMID- 1726765 TI - The molecular genetics of invertebrate phototransduction. AB - Phototransduction, the primary event in the processing of visual stimuli, is the conversion of light energy into a change in the ionic permeabilities of the photoreceptor cell membrane. In both vertebrates and invertebrates, this process is carried out through a specialized form of a G-protein-coupled receptor cascade. The mechanisms that mediate visual excitation in the vertebrate photoreceptor have been physiologically and biochemically well characterized, and many aspects of this system have served as prototypes for other transduction cascades. However, there are still many unresolved issues in vertebrate phototransduction. The study of phototransduction in Drosophila offers a unique opportunity to make use of powerful molecular genetic techniques to identify novel transduction molecules, and then to examine the function of these molecules in vivo, in their normal cellular environment. The results of a combination of molecular, genetic, physiological and biochemical studies are beginning to produce a clearer model for the complex mechanisms involved in invertebrate visual transduction. PMID- 1726766 TI - The basal forebrain-cortical cholinergic system: interpreting the functional consequences of excitotoxic lesions. AB - Ibotenic acid and kainic acid lesions of the basal forebrain induce profound deficits in performance on a wide variety of tasks involving discrimination learning and memory. These observations have been widely taken to reflect damage of cholinergic projections from the nucleus basalis magnocellularis (NBM) to the neocortex, and to provide an animal model of dementia. However, injections of the toxins quisqualic acid and, more recently, alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA) into the same site, which produce at least as extensive cholinergic cell loss, induce only marginal impairments on the same range of cognitive tasks. Further analysis suggests that the cholinergic regulation of the neocortex may influence specific aspects of discrimination learning and visual attention. Conversely, we propose that many of the functional consequences of ibotenic acid lesions on mnemonic tasks cannot be attributed to disruption of basal forebrain cholinergic systems, but may instead result from damage in the globus pallidus to corticostriatal output pathways. PMID- 1726767 TI - Neurofilament phosphorylation: a new look at regulation and function. AB - Dynamic remodeling of cytoskeleton architecture is necessary for axonal growth and guidance, signal transduction and other fundamental aspects of neuron function. Protein phosphorylation plays a key part in these remodeling processes. Since neurofilaments are major cytoskeletal constituents and are among the most highly phosphorylated neuronal proteins, the control of their behavior serves as a possible model for understanding how phosphorylation regulates the many other phosphoproteins in the cytoskeleton. Recent studies show that neurofilament protein subunits are phosphorylated on both their amino-terminal head domains and carboxy-terminal tails by different protein kinases. This review considers the implications of this complex regulation for neurofilament function in normal neurons and in disease states characterized by neurofibrillary pathology. PMID- 1726768 TI - Hematopoietic growth factors for the treatment of myelodysplastic syndromes. PMID- 1726769 TI - [Acute phase factors in anemia]. AB - In various anaemias the values of 8 acute phase factors were determined simultaneously before and at the end of treatment: seromucoid, sialic acid, acid alpha 1-glycoprotein, alpha 1-antitrypsin, haptoglobin, ceruloplasmin, transferrin and fibrinogen. In iron-deficiency anaemia without coexistent inflammatory changes in organs the levels of 4 proteins--seromucoid, alpha 1 antitrypsin, ceruloplasmin and transferrin, were consistently raised. In iron deficiency anemia with concomitant infection 4 proteins also were increased, but in place of alpha 1-antitrypsin the haptoglobin level was raised. In megaloblastic anaemia the ceruloplasmin level was increased, and in haemolytic anaemia one factor--sialic acid--was decreased. At the end of treatment the concentrations of certain proteins were changed depending on their specific role in various forms of anaemia and on various additional factors. In iron-deficiency anaemia without coexistent infection the concentration of seromucoid was decreased, and in this anaemia with coexistent infection alpha 1-antitrypsin, haptoglobin, and fibrinogen levels were raised, in haemolytic anaemia only fibrinogen was increased, and megaloblastic anaemia was associated with raised seromucoid level. The therapeutic result was good in all these anaemias with the exception of iron-deficiency anaemia associated with infection in which it was less propitious. PMID- 1726770 TI - [Interactions of leukocytes with endothelial cells]. PMID- 1726771 TI - Goat antibodies to amino acid sequences of human chorionic gonadotropin (hCG) AB - Human chorionic gonadotropin, its two subunits and its conjugate with thyroglobulin were used for immunization. The strongest immunogens were demonstrated to be the intact hormone and beta subunit, the weakest was alpha subunit. Using three peptides corresponding to hormone subunits coupled to Sepharose 4B various antibodies were isolated from goat antiserum by immunoaffinity chromatography. Specificity of the purified antibody preparations was studied. It was demonstrated that enzyme linked immunosorbent assay with two goat antibodies--one for coating of microtiter plates and the other one conjugated with peroxidase--is suitable for sensitive assays of both human chorionic gonadotropin and luteinizing hormone. Specific and sensitive assays were performed using combination of goat antibody anti-122-145-beta hCG and monoclonal antibody anti-alpha hCG. PMID- 1726772 TI - Peripheral blood lymphocyte subsets in urinary bladder carcinoma patients. AB - The percentages of pan T (CD3+), T helper (CD4+), T cytotoxic/suppressor (CD8+), B (CD22+) and natural killer (CD57+) cells in peripheral blood lymphocytes of 15 urinary bladder carcinoma patients and in parallel, 10 healthy donors were estimated, using monoclonal antibodies in indirect membrane immunofluorescence. A significant decrease in the percentage of CD3+ lymphocytes and a highly significant decrease in the proportion of CD8+ cells was revealed in urinary bladder cancer patients. This change was accompanied by a significant increase in the CD4/CD8 ratio and in the frequency of CD57+ (HNK-1+) cells. Our data document, for the first time, the complete lymphocyte profile of patients with advanced (T3) urinary bladder carcinoma. The reason and significance of the decline in CD8+ lymphocyte percentage and the increase of CD57+ cells are discussed. PMID- 1726773 TI - Subretinal neovascular membrane in an infant with a retinochoroidal coloboma. PMID- 1726774 TI - Retinal and vitreal neovascularization in retinopathy of prematurity. A scanning electron microscopic study in the kitten. AB - The angioarchitecture of vitreal and retinal neovascularizations produced experimentally in the eyes of kittens aged 2 to 9 weeks was studied with scanning electron microscopy. Various forms of new retinal and vitreal vessels were observed depending on topographic locations. Intraretinal neovascularization was observed at the retinal periphery as it grew toward the avascular zone in forms of short vascular buds, aneurysmal outgrowths, and neovascular loops. Posterior or to this frond of neovascularization, intertwining intraretinal telangiectasia was observed. At the posterior pole, capillaries with microaneurysms extended posteriorly toward the deeper layers of the retina from the vascular trunks at the nerve fiber layer. Vitreal neovascularization broke through the internal limiting membrane and exhibited aneurysmal outgrowths, clusters of glomerular swellings, and sinusoidal vascular channels. At the optic disc, vitreous neovascularization took the form of aneurysmal outgrowths and long vascular buds. Vitreal neovascularization showed different characteristics from the intraretinal neovascularization. We hypothesize that the topographic variation of the angioarchitecture of retinal and vitreal neovascularizations depends on the maturity of the vessels and might be related to the hemodynamics at each site. PMID- 1726775 TI - Investigation of antigenic structure of attenuated and virulent Venezuelan equine encephalomyelitis virus by means of monoclonal antibodies. AB - A comparative study of the antigenic structure of virulent strains and attenuated vaccine strains of Venezuelan equine encephalomyelitis virus (VEEV) by means of monoclonal antibodies has made it possible to investigate the antigenic structure of the envelope glycoproteins E1 and E2, and to specify their role in the development of antiviral immunity. On the E1 glycoprotein there are five nonoverlapping antigenic sites consisting of eight epitopes that are recognized by monoclonal antibodies; six sites consisting of twenty epitopes were found on the E2 glycoprotein. The monoclonal antibodies against four sites protect the animals from lethal infection with the virulent strain, Trinidad donkey. Out of the thirteen epitopes identified as being responsible for antiviral immunity, three are changes in the TC-83 strain, and six belong to two sites in the strain 230. The results obtained indicate the necessity for further improvement of the available vaccine preparations against this dangerous infectious disease. PMID- 1726776 TI - Matrix-assisted laser desorption ionization with a magnetic mass spectrometer. AB - Matrix-assisted laser desorption ionization has been carried out with a high mass double-focusing magnetic mass spectrometer. The pulsed ion signal, generated by irradiation of the sample (substance P, ubiquitin, and cytochrome c) embedded in 2,5-dihydroxybenzoic acid with a XeF excimer laser (353 nm, 12 ns pulse, 10-160 Hz), was recorded with an integrating array detector. Good resolution of 2600 (full width at half maximum) and high sensitivity (a few pmol) were obtained. The loss of small neutral fragments was observed, supporting the notion that the peak broadening observed in the time-of-flight mass spectrometers commonly used for this ionization mode is due to such metastable decomposition. PMID- 1726777 TI - Ribozyme-mediated cleavage of an HIV-1 gag RNA: the effects of nontargeted sequences and secondary structure on ribozyme cleavage activity. AB - Catalytic antisense RNAs, or ribozymes, have great potential as inhibitors of gene expression and as antiviral therapeutic agents. The major advantage of ribozymes versus standard antisense RNAs is their catalytic capability, enabling these RNAs to cleave multiple substrates. We have been investigating the antiviral activity of ribozymes targeted to the HIV-1 genome. The successful use of these antisense agents in an intracellular milieu requires stabilization of the ribozymes by flanking, non-base-pairing sequences, or some modification of the sugar-phosphate backbone. We describe a systematic investigation of the effects of flanking, non-base-pairing sequences on the catalytic activity of an anti-HIV-1 gag ribozyme embedded in radically different transcripts. Amazingly, these complex ribozyme-containing transcripts maintain substantial catalytic activity. Finally, we describe a bacterial gene fusion system that has potential for the large scale production of catalytically active ribozymes. PMID- 1726778 TI - Antiangiogenic effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides in the chick embryo chorioallantoic membrane. AB - The inhibiting effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides on the normal outgrowth of capillaries was tested in the chick embryo chorioallantoic membrane (CAM) with and without the presence of hydrocortisone. An antiangiogenic response to 50 micrograms of heparin and heparan sulphate (without hydrocortisone present) was observed in 38.8% and 23.1% of the CAMs, respectively, while the antiangiogenic response rate for dermatan sulphate, chondroitin sulphate A or C, hyaluronic acid and keratan sulphate was 15.9-0%. All sulphated homopolysaccharides tested were more effective than the naturally occurring glycosaminoglycans. Nonsulphated dextran and (methyl) cellulose had no antiangiogenic effect, while largely desulphated heparin retained such an effect. Hydrocortisone generally improved the antiangiogenic effect, a 100% response was obtained when it was combined with cellulose sulphate or fucoidan (polyfucose sulphate derived from marine algae), but the antiangiogenic effect of the largely desulphated heparin was unaffected by the presence of hydrocortisone. The results show that different polysulphated polysaccharides also have an antiangiogenic effect, without the addition of corticosteroids. The effect was apparently independent of their degree of sulphation, but the glycosidic structure may be of critical importance. PMID- 1726779 TI - Concanavalin A-stimulated expression of gangliosides with GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta structure in murine thymocytes. AB - We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml-1 Con A, 10 U ml-1 rIL 2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL 2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II3Neu-Gg4Cer, Gg4Cer and IV3GalNAc alpha-Gb4Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II3Neu-Gg3Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV3Neu-Gg5Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc beta 1-4(NeuAc or NeuGc alpha 2-3)Gal beta-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation of N acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726780 TI - Efficient analysis of protein 2D NMR spectra using the software package EASY. AB - The program EASY supports the spectral analysis of biomacromolecular two dimensional (2D) nuclear magnetic resonance (NMR) data. It provides a user friendly, window-based environment in which to view spectra for interactive interpretation. In addition, it includes a number of automated routines for peak picking, spin-system identification, sequential resonance assignment in polypeptide chains, and cross peak integration. In this uniform environment, all resulting parameter lists can be recorded on disk, so that the paper plots and handwritten notes which normally accompany manual assignment of spectra can be largely eliminated. For example, in a protein structure determination by 2D 1H NMR, EASY accepts the frequency domain datasets as input, and after combined use of the automated and interactive routines it can yield a listing of conformational constraints in the format required as input for the calculation of the 3D structure. The program was extensively tested with current protein structure determinations in our laboratory. In this paper, its main features are illustrated with data on the protein basic pancreatic trypsin inhibitor. PMID- 1726781 TI - Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy. AB - Solid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the 15N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2 delta, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides. PMID- 1726782 TI - Protein hydration studied with homonuclear 3D 1H NMR experiments. AB - Homonuclear 3D 1H NOESY-TOCSY and 3D 1H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H2O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4 degrees C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface. PMID- 1726783 TI - RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing. AB - RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated. PMID- 1726784 TI - Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein. AB - A gene encoding a temperature-sensitive mutant (MPP154A) of the 30-kilodalton movement protein (MP) of tobacco mosaic virus (TMV) was transformed into Nicotiana tabacum cv Xanthi. Transgenic plants expressing the MPP154A gene complemented local and systemic movement of an MP-defective mutant of TMV (U3/12MPfs) at the permissive temperature of 24 degrees C but not at 32 degrees C, the nonpermissive temperature. A microinjection procedure was used to investigate the effects of the modified TMV MP on plasmodesmatal size-exclusion limits. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran), with an average molecular mass of 9.4 kilodaltons, was detected between leaf mesophyll cells of the transgenic plants at 24 degrees C; however, no movement of either 3.9-kilodalton or 9.4-kilodalton F-dextrans was detected when the transgenic plants were held for 6 hours (or longer) at 32 degrees C. When these plants were shifted back to 24 degrees C for 6 hours, cell-to-cell movement of the F-dextrans was again observed. Accumulation of MPP154A was not affected by the temperature regime, nor was the subcellular distribution of the MP altered. These results are consistent with a change in the protein conformation of MPP154A at the nonpermissive temperature, which gives rise to a protein that fails to modify the molecular size-exclusion limits of plasmodesmata to the same extent as wild-type MP. Surprisingly, at 32 degrees C, movement of the F-dextrans was inhibited in transgenic plants expressing the wild-type MP gene; however, the inhibition was transient and was no longer detected after 48 hours at this elevated temperature. This transient inhibition of plasmodesmatal function was alleviated with Sirofluor, an inhibitor of callose ([1----3]-beta-D-glucan) synthesis. This result provides experimental evidence that callose deposition is involved in regulating the molecular size-exclusion limit of plasmodesmata in plants. Sirofluor had no effect on the inhibition of F-dextran movement at 32 degrees C in plants expressing the MPP154A gene, indicating that callose formation was not responsible for the failure of the temperature-sensitive mutant protein to alter the size-exclusion limit of plasmodesmata. PMID- 1726785 TI - The extracellular domain of the TSH receptor has an immunogenic epitope reactive with Graves' IgG but unrelated to receptor function as well as determinants having different roles for high affinity TSH binding and the activity of thyroid stimulating autoantibodies. AB - The possibility that thyroid-stimulating antibodies (TSAbs) might interact with receptor determinants different from those important for high affinity TSH binding has been evaluated. Deletion mutants of the extracellular domain of the rat TSH receptor as well as point mutations of potential N-linked glycosylation sites were created. TSH binding and the ability of TSH or a TSAb to increase cAMP levels after transfection in Cos-7 cells were then measured. Mutation of two glycosylation sites (residues 77 and 198) was shown to significantly decrease high affinity TSH binding but not the activity of a TSAb. A third glycosylation site mutant (residue 302) was identified that enhanced TSAb activity but had no effect on high affinity TSH binding, and a deletion mutant (residues 308-410) lost TSAb activity but preserved TSH binding. The last two mutations are within a region having low homology with gonadotropin receptors. This same region has, in addition, a determinant that is not important for receptor activity, yet is reactive with Graves' IgG. Thus, a deletion of residues 339-367 has no effect on TSH binding or TSH/TSAb activity, yet contains a peptide (residues 352-367) reactive in ELISA assays with IgG from greater than 80% of Graves' patients but not with IgG from normal individuals, patients with nonautoimmune thyroid disease, or patients with autoimmune disease not related to the thyroid. We, therefore, identify different receptor determinants for TSAb and high affinity TSH binding, consistent with predictions from TSH receptor monoclonal antibody studies. In addition, we identify a receptor peptide that is reactive with TSH receptor antibodies in Graves' patients, despite its having no determinants important for TSH or autoantibody activity in functional assays. PMID- 1726786 TI - Normal thyroglobulin and thyroperoxidase gene expression in thyroid congenital defective thyroglobulin synthesis. AB - We have studied a member (JBM) of a family MO previously described, with congenital goiter, hypothyroidism, and presence of hyposialylated Tg in the follicular lumen. Other congenital goiters (MA and JNA) with virtual absence of Tg were studied similarly. The presence of apparently normal-sized Tg in JBM tissue was confirmed in the present study by radioimmunoassay, Sephacryl S300 column chromatography, immunoelectrophoresis, and SDS agarose gel electrophoresis. Dot blot hybridization analysis with Tg and TPO probes indicated that mRNA hybridization levels of JBM tissue were similar to control thyroid tissues. Congenital goiter tissues showed relatively lower TSH receptor mRNA content in comparison with normal thyroid tissues. DNA was digested with five restriction endonucleases (Taq I, Eco Rv, Pvu II, Pst I, and Eco RI), and the results revealed polymorphisms previously described with the Tg gene. No significant differences in the TPO Pst I pattern were observed in comparison with control samples. We conclude that no major alterations of the Tg and TPO gene expression are detectable and that no significant deletions of these genes are present. The biochemical abnormality in the JBM Tg molecule may be a posttranslational error during the assembly of the protein. PMID- 1726787 TI - Polypeptide modulators of prostatic growth and development. AB - Normal and abnormal developmental events in the prostate are strongly influenced by androgens. There is abundant evidence, however, that androgens are not the only substances present that have the capacity to influence prostatic growth. A number of polypeptides that either stimulate or inhibit growth have now been identified in the prostate. These include members of the HBGF family, TGF-beta family, EGF and TGF-alpha, PDGF, NGF, and the less well characterized osteoblast growth factors. In some cases, the prostatic cell population, stromal or epithelial, that synthesizes the growth factor and its receptor is known. This information and the properties of the growth factors suggest ways in which these polypeptides may be involved in regulating growth of the prostate, including benign prostatic hyperplasia and prostate cancer. PMID- 1726788 TI - Molecular methods for predicting the metastatic potential of prostate cancer. AB - As yet, few molecular markers are available that are likely to be useful in predicting the metastatic potential of prostate cancer cancer cells. The need for such "progression markers" is indicated by the expectation that more localized cancers (ie stage A-2, B-1,2) will be clinically diagnosed in the near future, owing to improvements in diagnostic techniques (eg transrectal ultrasound) and the screening of population groups at risk (males over 50 years old). Few model systems are available for such studies. Animal models are unsuitable for the isolation of monoclonal antibodies that detect epitopes associated with the progression of prostate cancer. Since few cell lines are available, an approach using primary cancer tissue should be undertaken. For the differential hybridization approach described here, the choice of species presents no difficulty, since many DNA sequences are conserved between species. However, no model system fully represents the human situation. Hence, differential hybridization studies using primary prostate cancer tissue need to be considered. Moreover, the group of genes/proteins with potential relevance for cancer progression (eg oncogenes, tumour suppressor genes, genes encoding cell adhesion molecules or growth factors) has not been studied extensively in prostate cancer. Because of the intrinsic heterogeneity of prostate tumours, the use of pathologically defined tissue sections is imperative for a reliable study. This could be achieved either by in situ techniques (whereby tissue morphology is conserved) or by step sectioning. The difficulties associated with the small amount of material obtained from such sections can be overcome by the use of techniques based on the polymerase chain reaction. Taking these considerations into account, a systematic screening of prostate cancer with probes representing the above mentioned genes should be undertaken. PMID- 1726789 TI - Experimental oncogene induced prostate cancer. AB - The mouse prostate reconstitution model exploits the ability of the fetal urogenital sinus to differentiate into a mature prostate when grafted under the renal capsule of an adult isogenic male host. By use of a recombinant retroviral vector, the ras and myc oncogenes are introduced singly or in combination into the fetal urogenital sinus--resulting in distinct phenotypes of prostatic pathology: dysplasia (caused by ras), hyperplasia (caused by myc) and frank carcinomas (caused by a combination of ras+myc). This unique experimental model creates in vivo conditions that mimic the natural initiation and progression of cancer. An expanded MPR protocol allows restricted retrovirus infection of the mesenchyme or epithelial compartments to evaluate paracrine activities. It enables almost unparalleled flexibility in addressing fundamental questions in prostate cancer. We have identified genetic variance in the susceptibility to tumour induction between two different strains of mice (mimicking the observation of racial variability in the predisposition to clinical prostate cancer). The MPR model supports data from other tumour models and implicates TGF-beta 1 and TGF beta 3 as being strongly associated with tumour progression. Finally, with this model, we have established clonal prostate adenocarcinomas to study directly the affects of castration on gene expression. Not only are TGF-beta 1 and TGF-beta 3 mRNA levels increased in association with malignancy but they are also further enhanced by castration treatment. Based on these experimental studies, we believe that TGF-beta 1 and TGF-beta 3 expression strongly influences the progression of prostate cancer. This information will hopefully impact on the development of more effective therapy for this important malignancy. PMID- 1726790 TI - Reciprocal mesenchymal-epithelial interaction affecting prostate tumour growth and hormonal responsiveness. AB - A novel cell-cell recombination model was established to test the reciprocal mesenchymal (fibroblast)-epithelial interaction in the prostate gland. Both growth factors and ECM pathways were found to be actively engaged during cellular communications. The application of this cell-cell recombination concept to prostate cancer established a new human prostate cancer animal model in which the tumours actively secrete prostate specific antigen, a known human prostate cancer marker. This review explores the significance of mesenchymal-epithelial interaction in determining prostate hormonal responsiveness and prostate cell transformation and speculates on the potential roles of mesenchymal-epithelial interaction in prostate cancer growth and progression. PMID- 1726791 TI - [The characteristics of the morphofunctional organization of the raphe nucleus neurons in tissue culture]. AB - Morpho-functional development of the raphe nuclei neurons was studied in long term (up to 20 days) organotypic cultures taken from newborn rats. Differentiated neurons typical for raphe nuclei were identified in explants after 10 days in vitro. The synthesis of serotonin (5-HT) and uptake of 3H-5-HT were more intense in the raphe neurons culture than in hippocampal and neocortical cultures. The cultured raphe nucleus neurons generate low-frequency (1.2 Hz on the average) and regular (rhythmic) spike activity which corresponds to the data obtained in vivo. The data obtained confirm the idea of autonomic activity of the raphe neurons. PMID- 1726792 TI - Molecular genetics--new horizons in haematology. AB - The unprecedentedly swift developments in molecular genetics has opened up a new era in biology and medicine. The powerful methods of recombinant DNA (rDNA) technology are fast moving into the fields of diagnostics and therapy since the newly found ability to define physiological and pathological cell functions at a molecular level. The centerpiece of molecular genetics is the possibility to map and determine the fine structure of human genes and to define in molecular terms how each gene controls all the enzymes of energy metabolism, structural proteins of cells, the membrane proteins, including transport proteins and receptors, the plasma proteins and those proteins which participate in the synthesis of complex lipids, carbohydrates, lipoproteins and glycoproteins. The fundamental change of emphasis in cellular and clinical research in medicine started in haematology. No wonder, since the circulating blood cells, the bone marrow, and lymphoid tissue cells are easily available for investigation and most of the haematological diseases are well defined entities. This review will try to present the increasing depth and broadening spectrum of molecular haematology by arbitrary chosen examples: 1) Molecular regulation of cell specific gene expression and of age specific switch of the globin genes, 2) Revisiting the haemolysis of paroxysmal nocturnal haemoglobinuria (PNH) cells--the phosphatidylinositol-glycan (PIG) anchored membrane proteins, 3) Molecular genetics of the ABO and Rh blood group specificity, 4) The regulation of stem cells and the multistep process of their malignant transformation. 5) The impact of gene technology on diagnostics, prevention and therapy in haematology, 6) The present state of art and future possibilities of the treatment of genetic diseases. PMID- 1726793 TI - Colicin E1 export in Salmonella typhimurium wild-type and lipopolysaccharide mutants. AB - Colicin export was studied in different Salmonella typhimurium strains lacking the O-antigen repeating units (O-) and different strains with different chemotypes for the lipopolysaccharide core, as well as the wild-type strain (O+), to determine the role of lipopolysaccharide length on colicin E1 export. While the lipopolysaccharide length influences the levels of external hemolytic activity in S. typhimurium, no effect was detected on colicin E1 export. PMID- 1726794 TI - Effects of 2,3-butanedione monoxime on whole-cell Ca2+ channel currents in single cells of the guinea-pig taenia caeci. AB - 1. The inhibitory actions of cadmium (Cd2+), nifedipine and 2,3-butanedione monoxime (BDM) on whole-cell Ca2+ channel currents in single cells of the guinea pig taenia caeci were investigated using a single-electrode whole-cell voltage clamp technique. 2. Calcium channel currents were isolated using pipette solutions containing Cs+, tetraethylammonium and ATP (3 mM). Ca2+ or Ba2+ (7.5 mM) in the bathing solution acted as the charge carrier during inward current flow. Ca2+ channel currents in 7.5 mM-Ba2+ (IBa) were recorded at potentials positive to -40 mV, were maximal near 0 mV and reversed near +60 mV. Ca2+ channel activation showed a sigmoidal relationship with potential, which was half-maximal at -13 mV. 3. Both the inward and outward flow of current was depressed and eventually blocked by 0.3-100 microM-Cd2+, 0.1-10 microM-nifedipine and 2-20 mM BDM. Half-maximal blockade of IBa at 0 mV was achieved with approximately 3 microM-Cd2+, 1 microM-nifedipine and 10 microM-BDM. Steady-state activation curves were not affected by Cd2+ or BDM, but were shifted in the hyperpolarizing direction by nifedipine at concentrations > 1 microM. 4. Calcium channel currents in single cells and K+ contractures in intact strips were both blocked in a voltage-dependent manner. Steady-state inactivation curves (f infinity (V)) for IBa were shifted 20 mV in the hyperpolarizing direction by 0.3 microM-nifedipine and 4 mV by 10 mM-BDM. From these shifts a dissociation binding constant to inactivated Ca2+ channels for nifedipine was estimated as 78 nM, and for BDM, 5 mM. 5. At 10 microM Cd2+ produced a 43 +/- 6% (n = 3) block of the inward current at 0 mV when Ca2+ (7.5 mM) was the charge carrier (ICa), compared with the 36 +/- 3% block of IBa induced by 1 microM-Cd2+, consistent with the suggestion that Ca2+, Ba2+ and Cd2+ compete for the same binding site. In contrast, nifedipine (1 microM) and BDM (10 mM) blocked ICa more effectively than IBa. 6. Bay K 8644 (1.0 microM) increased Ca2+ channel currents two- to fourfold at all potentials due to a shift, of approximately 10 mV in the negative direction, of their activation curve and an equal shift in the positive direction of their inactivation curve. BDM (5-10 mM) could antagonize the action of Bay K 8644, shifting both curves back towards their control.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1726795 TI - Protons activate a cation conductance in a sub-population of rat dorsal root ganglion neurones. AB - 1. The responses of adult and neonatal rat dorsal root ganglion (DRG) neurones to buffered acidic solutions were studied with both voltage clamp and radioactive ion flux techniques. Electrophysiological experiments were made on acutely isolated neurones and ion flux experiments were made on cells that had been in culture for 3-6 days. 2. Acid solutions of pH < 6.2 evoked a sustained, slowly inactivating inward current in neurones voltage clamped at negative holding potentials. The size of the current increased with increasing proton concentrations. This response was restricted to a sub-population (approximately 45%) of adult and neonatal rat DRG neurones and was distinct from a rapidly activating and inactivating proton-induced inward sodium current that was also found in DRG neurones. 3. The proton-activated sustained current was due to an increase in cation conductance that allowed K+, Cs+ and Na+ to pass with PK/PNa = 1.32 and PCs/PNa = 1.12. 4. Radioactive ion efflux experiments made on neonatal rat cultured DRG neurones showed that protons also increased the permeability to both [14C]guanidinium and 86Rb+ ions. The half-maximal increase in efflux rate for 86Rb+ occurred at pH 5.8. Acid solution also stimulated the efflux of 86Rb+ in cultures of adult rat neurones. 5. Cells that showed a late, sustained proton activated current also responded to capsaicin. In addition, no proton-activated fluxes of either [14C]guanidinium or 86Rb+ ions were observed in cultures of DRG neurones that had been treated with high concentrations of capsaicin (10 microM) to kill the capsaicin-sensitive neurones. Thus this proton-activated current is restricted largely, if not exclusively, to capsaicin-sensitive peripheral sensory neurones. PMID- 1726796 TI - Modulation of voltage-activated channels by calcitonin gene-related peptide in cultured rat neurones. AB - 1. Whole-cell currents were recorded from cultures of dissociated neocortical neurones of the rat. Rat alpha-calcitonin gene-related peptide (CGRP; 1 nM-1 microM) caused significant dose-dependent decreases in the voltage-activated transient (A-current) and delayed rectifier K+ currents. Forskolin (10 nM-20 microM) mimicked this effect. Peak K+ currents were gradually decreased after loading neurones with cyclic AMP (100 microM) through patch pipettes. CGRP was ineffective in neurones loaded with cyclic AMP. 2. CGRP (0.5-2 microM) increased cytosolic cyclic AMP concentration and this effect was mimicked by forskolin (5 40 microM). 3. CGRP (0.1-1 microM) reduced high-threshold Ca2+ currents; as did forskolin (5-20 microM) and cyclic AMP loaded into the neurones. In contrast, low threshold Ca2+ currents were not affected by any of these agents. 4. Voltage activated Na+ currents were significantly reduced by both CGRP (0.1-1 microM) and forskolin (5-20 microM). A similar effect was observed when cells were loaded with cyclic AMP. 5. We conclude that, in neocortical neurones, CGRP attenuates voltage-activated currents by stimulating the intracellular cyclic AMP signalling system. PMID- 1726797 TI - Pharmacological properties and H+ sensitivity of excitatory amino acid receptor channels in rat cerebellar granule neurones. AB - 1. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor channels have been examined in rat cerebellar granule neurones with whole-cell and single-channel patch-clamp methods. The whole-cell peak and steady-state aspartate and NMDA currents were reversibly inhibited by extracellular protons; the IC50 (concentration producing half-maximal inhibition) for the full H+ inhibition curve for NMDA receptors corresponded to pH 7.3, near to physiological pH. (S)-AMPA and kainate whole-cell currents were inhibited by protons with IC50 values that corresponded to pH 6.3 and 5.7, respectively; these receptors were, however, insensitive to H+ concentrations that inhibited NMDA receptor responses. 2. Proton inhibition of the NMDA, AMPA and kainate receptor-mediated responses was voltage insensitive, and did not involve a shift in reversal potential. 3. The EC50 (concentration producing half-maximal effect) for aspartate calculated from the whole-cell dose response curve was similar at pH 6.8 and 7.6 (mean 11.2 microM). Although the EC50 for glycine potentiation of the aspartate response was marginally increased from 273 nM at pH 7.6 to 373 nM at pH 6.8, H+ inhibition was not overcome by up to 1 mM-external glycine. Inhibiting concentrations of H+ appropriate for AMPA and kainate receptors did not markedly alter the EC50 values determined for (S) AMPA (3.4 microM) and kainate (114 microM) at pH 7.2. 4. Treatment of neurones with N-ethylmaleimide, iodoacetic acid, dithiothretiol or diethyl pyrocarbonate did not influence proton inhibition of NMDA receptor responses. However, treatment with diethyl pyrocarbonate, which potentiated aspartate responses, appeared to reduce the effectiveness of Zn2+ inhibition of NMDA receptors. 5. Desensitization of whole-cell NMDA and (S)-AMPA currents was studied with ionophoretic application of agonist to the cell soma. Whole-cell aspartate currents desensitized rapidly, irrespective of the glycine concentration. Increased H+ concentrations did not detectably alter the ratio of peak/steady state current, or the time constants describing the onset of, or recovery from, desensitization. The time constant describing desensitization of (S)-AMPA-induced whole-cell currents also appeared unchanged by inhibiting pH (6.2). 6. The amplitudes of aspartate- or NMDA-activated single-channel multiple conductance levels were unchanged by decreasing the pH to 6.8.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1726798 TI - Activation of ion channels in the frog endplate by several analogues of acetylcholine. AB - 1. Single-ion-channel recording has been used to estimate the equilibrium concentration-response relationship for several acetylcholine analogues. The response, corrected for desensitization, was taken as the probability of a channel being open during clusters of openings that were separated by desensitized periods. 2. All agonists were able to block the channels which they themselves opened. Carbachol, suberyldicholine and the sulphonium analogue of acetylcholine were all found to be efficacious agonists in the sense that the results indicate that all of them, in sufficiently high concentration, would be able to open 90% or more of channels if it were not for channel block. 3. In the case of suberyldicholine the results are much as predicted by the interpretation of the fine structure of channel openings at low agonist concentrations. 4. The maximum probability of opening that could be obtained with decamethonium and with phenyltrimethylammonium was low (below 4%), and it was not possible to distinguish whether this was wholly a result of the powerful (relative to activation potency) channel-blocking action of these agonists, or whether it was to some extent attributable to their being genuine partial agonists. 5. The results suggest that, for a range of agonists, differences in equilibrium potency are usually more strongly influenced by affinity for binding to the resting state of the receptor than by ability to activate the receptor once bound, though in the case of suxamethonium (relative to acetylcholine) the contributions of each factor are similar. PMID- 1726799 TI - Purified Trypanosoma cruzi specific glycoprotein for discriminative serological diagnosis of South American trypanosomiasis (Chagas' disease). PMID- 1726800 TI - Functional and antigenic properties of the major cysteine proteinase (GP57/51) of Trypanosoma cruzi. PMID- 1726801 TI - Autoantibodies in Chagas' heart disease: possible markers of severe Chagas' heart complaint. PMID- 1726802 TI - Estimation of whole-body RNA catabolism based on the urinary modified nucleoside levels of cancer and AIDS patients. AB - From the relationship between the molar ratio of nucleosides calculated stoichiometrically from modified nucleoside occurrences in major RNA species and the proportion of rRNA to all of RNA contents in average tissues, the increase of rRNA contents in cancer tissues growing rapidly was found. Thus, we found that selected urinary modified nucleoside levels were very useful as a biological marker of cancer and AIDS, as well as a good indicator of whole-body metabolic conditions of RNAs. PMID- 1726803 TI - The molecular recognition mechanism of DNA by bleomycin. AB - The interaction between the antibiotic bleomycin (BLM) and an oligonucleotide d(CCACCTAGGTGG) was studied by 1H NMR. The DNA oligomer was titrated with BLM VO(III), which is an 'inactive analogue' of BLM-Fe(II). A marked upfield shift was observed for the T10N3H signal, but no other imino proton signals either shifted or broadened. When BLM-VO(III) was titrated with d(CCACCTAGGTGG), a large upfield shift (0.27 ppm) was observed for the delta NH signal of butylguanidinium residue (G). Analysis of NOESY spectra of a 1:1 complex of DNA:BLM-VO(III) strongly suggests that the connectivities of the sequential NOEs of the DNA duplex are the same as those of free DNA. These results indicate that BLM does not intercalate between the base-pairs but binds to DNA in a different manner. A model of 'minor-groove binding' is more likely to explain our NMR data. PMID- 1726804 TI - Cell-free translation system using phosphorothioate-containing mRNA. AB - Phosphorothioate-containing RNAs were generated by transcription of template DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) (NTP alpha S) and T7 RNA polymerase. The substitution of mRNA by phosphorothioate increased the efficiency of protein synthesis by stabilizing the mRNAs in prokaryotic cell-free translation systems. The substituted mRNAs were also shown to be applicable to the continuous cell-free translation system developed by Spirin and coworkers. PMID- 1726805 TI - Identity determinants of E. coli tRNAs. AB - Identity determinants of E. coli tRNA(Val) and three class II tRNAs, tRNA(Ser), tRNA(Tyr) and tRNA(Leu), are studied by using various variants of tRNA transcripts. Anticodon, discriminator base and acceptor stem are involved in the identity elements for tRNA(Val). Discrimination among class II tRNAs are considered to be dependent on the bases at positions 2, 71 and 73 as well as their different tertiary structures including the long variable arm. PMID- 1726806 TI - Mechanisms of molecular recognition of tRNAs by aminoacyl-tRNA synthetases. AB - Interactions of Escherichia coli isoleucyl- and glutamyl-tRNA synthetases and their cognate tRNAs were analyzed by phosphate-alkylation mapping with N-nitroso N-ethylurea and/or by 1H-NMR analysis. When E. coli tRNA(Ile) was bound with isoleucyl-tRNA synthetase, many of the phosphate groups in the anticodon loop and stem and in the D-stem were protected from alkylation. This result is consistent with that of analysis of imino proton resonances due to the secondary and tertiary base pairs. These analyses also suggested that the L-shaped tertiary structure of tRNA(Ile) is distorted upon complex formation with IleRS because of disruption of some tertiary base pairs. In the case of E. coli tRNA(Glu), several phosphate groups in the D-stem and the variable loop were significantly protected by the cognate synthetase. These results indicate that the two tRNAs, unlike other tRNAs studied so far, have some of the "identity determinants" in the D stem and/or in the anticodon stem. PMID- 1726807 TI - Structure of mitochondrial tRNA. AB - Some mitochondrial (mt) tRNAs are presumed to have unusual secondary structures on the basis of DNA sequence analyses. However there are few tRNAs whose structures have been investigated at RNA level. Thus we isolated some mt tRNAs to investigated their structures and also synthesized mt tRNA by run-off transcription. In these experiments, following results have been obtained. 1) Bovine mt tRNA(Ser)(AGY) needs the interaction between the T-loop and D-arm for its aminoacylation activity. 2) The secondary structure of bovine mt tRNA(Ser)(UCN) is difficult from that predicted previously from its gene. 3) It has been confirmed by the hybridization using oligonucleotide probes that some Ascaris suum mt tRNAs possessing the unusual T-arms actually exist in its mitochondria. PMID- 1726808 TI - Molecular structure of extraordinarily stable RNA: model building and X-ray analysis. AB - A tridecaribonucleotide, rUGAGCUUCGGCUC which adopts the extraordinarily thermally stable hairpin structure, was chemically synthesized. The crystallographic study of this RNA molecule was done. Crystals grew as plates belonging to space group C2 with a = 38.49A b = 32.30A c = 38.76A and beta = 117.56. and one molecule per asymmetric unit. Intensity data were collected at a resolution 2.1A. Structure determination using molecular replacement method has been underway. The favorable three-dimensional model of hairpin was built by molecular dynamics and molecular mechanics calculations. PMID- 1726809 TI - Facile cleavage of RNAs by oligoamines. Correlation between amine structure and catalytic activity. AB - Oligoamines such as ethylenediamine and diethylenetriamine exhibit remarkable catalysis for the hydrolysis of RNAs. Methyl substitution of ethylenediamine at the nitrogen atoms causes virtually no effect on the catalytic activity, indicating that attachment of the oligoamines to sequence-recognizing moiety provides superb artificial ribonuclease. PMID- 1726811 TI - Relation between functions and conformational characteristics of modified nucleosides found in tRNAs. AB - Conformational characteristics of N4-acetyl-2'-O-methylcytidine (ac4Cm), 5-methyl 2'-O-methylcytidine (m5Cm) and N2-dimethyl-2'-O-methylguanosine (m2(2)Gm) found in tRNAs from extremely thermophilic archaebacteria were analyzed by proton NMR spectroscopy. The 2'-O-methylation of ac4C, m5C and m2(2)G was found to stabilize the C3'-endo form and therefore cause "conformational rigidity". In particular, the ac4Cm was found to be extremely rigid due to additive effects of the N4 acetylation and 2'-O-methylation. Therefore, tRNAs from the extremely thermophilic archaebacteria use the base modifications in combination with the 2' O-methylation, resulting in stabilization of the A-type conformation at specific positions in the tRNAs even at very high temperatures. In contrast, mesophile tRNAs use for a given site only one of these ribose and base modifications each of which is effective enough by itself at ordinary temperatures. These findings are consistent with our previous findings that roles of a variety of post transcriptional modifications are to regulate the conformational rigidity/flexibility which is essential for the tRNA functions. PMID- 1726810 TI - Inhibitory effect of N3-methyl derivative of 3'-azido-3'-deoxythymidine 5' triphosphate on the activity of HIV-1 reverse transcriptase. AB - N3-Methyl derivative of 3'-azido-3'-deoxythymidine 5'-triphosphate (Me-AZTTP) showed a potent inhibitory effect on HIV-1 reverse transcriptase using MS2 phage RNA as the template. The inhibition mechanism of MeAZTTP was noncompetitive with respect to any of the template MS2 RNA, dATP and dCTP. On the other hand, MeAZTTP showed a mixed-type inhibition with respect to dGTP and dTTP. These results indicate that MeAZTTP competes not only with dTTP but also with dGTP. PMID- 1726812 TI - Chemical synthesis of branched RNAs: a new method for construction of synthetic units for branched RNAs by use of 2'-phosphorylation on the basis of steric control. AB - A 3',5'-unprotected 2'-O-bis(tert-butoxy)-phosphinyl-6-N-benzoyladenosine derivative was prepared as a key intermediate for the synthesis of branched RNAs, and used for construction of building units from which chain elongation in any of 2'-, 3'-, and 5'-directions would be possible. From this synthetic intermediate, 2'-phosphorylated ApC dimer was also synthesized. PMID- 1726813 TI - Selective and effective cleavage of RNAs by vinyladenine-vinylamine copolymer. AB - Poly(uridylic acid) (poly[U]) and poly(adenylic acid) (poly[A]) are efficiently cleaved, via phosphodiester linkage hydrolysis, at pH 8, 50 degrees C by use of poly(vinyladenine-co-vinylamine) as catalyst. The catalytic activity of the copolymer surpasses the value of poly(vinylamine), indicating a significant role of the adenine residue in the copolymer for the effective catalysis. PMID- 1726814 TI - [Physiopathology of migraine]. AB - The basic theories on the origin of migraine are reviewed. A vascular paroxysm was the initial hypothesis, followed by the "spreading depression" phenomenon based of regional flow studies of Danish authors. A third hypothesis relates the origin of migraine to liberation of substance "p" and others (K, neurotransmitters and polypeptides) from trigeminal nerve endings. Finally, a behavioral disturbance originating an adrenergic response and a loss of central nervous system defense mechanisms, has been also postulated. PMID- 1726815 TI - [Atmospheric pollution in the city of Santiago. A statement from the Chilean Society of Respiratory Diseases]. AB - Air pollution was the subject of the 1990 Fall Meeting of the Chilean Society of Respiratory Diseases. The potential danger of air pollution was considered a serious challenge. Groups at special risk are children under 5 years of age, patients with chronic respiratory diseases (COPD) or cardiovascular disease, the aged and pregnant women. Santiago is located in a valley surrounded by mountains, with a large population and many industries. The anticyclonic conditions determine a dominant semiarid climate and air circulation is prevented by a thermal inversion layer. Unacceptably high levels of total suspension particles (TSP) and carbon monoxide have been measured by the network of air pollution monitoring stations during winter and fall. 70% of TSP comes from diesel engines of the public transportation system. Most of the CO comes from automobiles. High levels of O3 have been detected in the east and north areas of the city. Nitrous oxide interference may account for an underestimation of the O3 level in the downtown area. Errors in the methodology for measurement of SO2 interfere with adequate knowledge about this pollutant. Actions to control the level of pollutants should include modification of industrial processes, changes in the transportation systems and others that should be enforced by law. Cooperation of the population and a well defined political will are urgently needed to implement solutions. PMID- 1726816 TI - [Diagnostic use of neutrophil segments negative for peroxidase, chloroacetate esterase and sudanophilia in peripheral blood in leukemia]. AB - The paper contains the results of three cytochemical reactions used for detection of myeloid differentiation (peroxidase, sudanophilia, chloroacetate esterase) in neutrophil segments of the peripheral blood stream in 107 patients with acute myeloid or lymphatic leukaemia. Enzymatically deficient segments were detected in 23 (34.8%) patients with acute myeloid leukaemia. They were not found in any patients lymphatic leukaemia not in healthy subjects. The concurrent deficit of all three reactions was found in 69% of the cases with defective neutrophil segments. However, also isolated affection of any of these reactions was found. In acute leukaemias, not differentiated from the cytochemical and immunophenotypical aspect, we may consider simple evidence of the presence of the mentioned abnormal neutrophil segments in peripheral blood as a highly probable sign of myeloid differentiation of acute leukaemia. PMID- 1726817 TI - Effect of nitrous oxide on the concentrations of opioid peptides, substance P, and LHRH in the brain and beta-endorphin in the pituitary. AB - Many studies have indicated that nitrous oxide (N2O) exposure results in specific effects on the reproductive system, some of which are antigonadotropic. The neurochemical events regulating the pituitary-gonadal axis are probably influenced by N2O, but precise documentation is lacking. The effects of exposure to 30% N2O in air on the brain tissue concentrations of luteinizing hormone releasing hormone (LHRH), substance P (SP), met-enkephalin, and beta-endorphin and on beta-endorphin concentrations of the pituitary gland are described in this study. Female rats were exposed to either N2O or air for 8 hr a day over one estrous cycle, and the brain and pituitary tissues were collected and processed. Neuropeptide concentrations were measured by specific radioimmunoassays. Exposure to N2O resulted in significant elevation of LHRH in the preoptic area, with a concomitant decrease in SP. The SP concentration of the medial basal hypothalamus was significantly elevated in N2O-exposed animals. Exposure to N2O resulted in significant increases in met-enkephalin in the brainstem area and beta-endorphin in the pituitary. These results suggest that exposure to N2O alters the interactive neural system activity regulating gonadotropin secretion from the pituitary. The significance of increased met-enkephalin in the brainstem of N2O exposed animals is not known. PMID- 1726818 TI - Detection of submicroscopic lymph node metastases in patients with melanoma. AB - A cell culture technique was developed to investigate submicroscopic lymph node metastases in patients with stage 1 or 2 malignant melanoma. Lymph nodes were isolated from standard dissections and bivalved. Half of the node was evaluated by routine histopathologic examination, while the other half was processed and placed into tissue culture. Three hundred twenty-three lymph nodes were collected from 41 patients. The cell culture technique identified 155 of 323 lymph nodes containing micrometastases, while only 20 of 323 lymph nodes tested positive with routine histochemical processing. Nine patients were upgraded from stage 1 or 2 to stage 3 disease after micrometastases were identified in lymph node cultures. Identification of melanoma was confirmed by cytologic examination, immunohistologic staining, and the presence of GD3 ganglioside and 250-kd glycoprotein melanoma-associated antigens. This study provides evidence that the culture of lymph nodes is a sensitive method for the detection of micrometastases. In addition, this procedure may change prognosis and identify candidates for adjuvant therapies. PMID- 1726819 TI - Human lymphokine-activated killer cell activity. Role of IL-2, IL-4, and IL-7. AB - The T-cell growth factors interleukin 2 (IL-2) and interleukin 7 (IL-7) induce lymphokine-activated killer (LAK) cell activity in short-term cultures of human peripheral blood mononuclear cells. Interleukin 4 (IL-4), another T-cell growth factor, induces LAK cell activity in IL-2-prestimulated lymphocytes only and inhibits LAK cell generation in normal peripheral blood mononuclear cells. Our studies of the processes involved using 21-mer phosphorothioate antisense oligonucleotides to the sequence adjacent to the start codon of IL-2 mRNA or IL-4 mRNA (effective concentration, 5 to 10 mumol/L) and cyclosporine (0.01 to 1.0 microgram/mL) or FK506 (0.01 to 1.0 ng/mL) demonstrate that IL-7-induced LAK cell activity is independent of IL-2 production and is regulated by endogenously generated IL-4. Like IL-2, IL-7 stimulated production of tumor necrosis factor alpha, but we failed to detect interferon gamma in IL-7-stimulated cultures. The implication of this regulatory feedback in IL-7-induced LAK cell generation for clinical applications is discussed. PMID- 1726820 TI - Biochemical correlate of depression in children. AB - The degree of depression in 88 abandoned children was analysed through a depression rating scale adapted to prepubertal children. The items were grouped into three dimensions: sociological-relational, psychological and biological. In 46 children from this sample it was dosed plasmatic cortisol and the urinary excretion of catecholamine, VMA, HVA and 5-HIAA. When analysing the principal components, the sociological and psychological dimensions were the most important ones in the sample, followed by age and catecholamine variables. The group of male depressed children presented a higher level of catecholamine urinary excretion and a lower peak of plasmatic cortisol than the non-depressed group. The variable age, in both sexes, was correlated with the biochemical variable catecholamine. Biochemical alterations are present in depressed children but it is difficult to show a correlation of dependence between them and the phenomenological aspects of depression. PMID- 1726821 TI - Microscopic anatomy of the human vertebro-basilar system. AB - Concerning the structure of connective-muscular components the authors studied the walls of the terminal segments of the vertebral arteries as well as the basilar artery, utilizing the following staining methods: Azan modified by Heidenheim, Weigert's resorcin-fuchsin, and Weigert modified by van Gieson. It was established that wall of the vertebro-basilar system exhibits a mixed structure, muscular and elastic, by means of which the vessels are adjusted to the specific blood circulation conditions. Thus, vertebral arteries show in the most external layer of tunica media an evident external elastic lamina. In contrast, in the basilar artery the elastic tissue is localized mainly in the tunica media, and is distributed heterogeneously. In its caudal segment the elastic fibers are situated in the most internal layer of tunica media, and in the cranial segment the elastic component is homogeneously distributed in the whole of tunica media. PMID- 1726822 TI - Cytokeratin expression in human stomach. Immunofluorescence study. AB - Twenty one samples of normal human stomach were examined for the cytokeratin pattern by immunofluorescence microscopy of cryostat sections. In all cases epithelial cells of stomach showed reactivity with monoclonal antibodies directed against individual cytokeratins No. 8,18 and 19. In 5 cases epithelial cells of stomach were also positive for keratin 7. The results show that there are "subtype" of gastric epithelial cells, positive for keratin No. 7. PMID- 1726823 TI - Early detection of disturbances of epithelial keratinization. AB - Unsatisfactory results treatment of the hypopharyngeal and upper respiratory tract carcinoma lead us to investigations on the efficient diagnostic methods of keratinization disturbances. We used a method worked out earlier in our Clinic and based on cytologic examinations of Papanicolaou. In present series we performed cytologic examinations in patients from high-risk groups of the upper respiratory tract and hypopharyngeal cancer. In any doubt cytology was accompanied by histological examination. Our material judges the statement that cytological examination may be used as a screening method of examination in prophylaxis of hypopharyngeal cancer. PMID- 1726824 TI - Vitamin E protects human leucocytes against toxic effects of lindan in vitro. AB - Vitamin E (15 micrograms/ml and 60 micrograms/ml) was used for protection of human leucocytes against toxic effects of lindan (150 microM and 200 microM) in vitro. The protective effect of vitamin E manifested itself as maintenance of the phagocytic activity of granulocytes, the value of nucleologram in lymphocytes, and the proportion of euchrysin-negative lymphocytes near the values in controls. PMID- 1726825 TI - A case of organic aciduria--suspected 3-hydroxy-3-methylglutaric aciduria. AB - In a girl from a family with muscular hypotonia, hypoglycaemia, lactic acidosis and delayed development the analysis of organic acids in urine suggested a defect in leucine metabolism--3-hydroxy-3-methylglutaric aciduria. A good therapeutic effect was obtained with low-protein diet. PMID- 1726826 TI - [Factorial analysis of the "proper" angles involved in ethnic prognathism]. AB - This work rest on 10 populations, chiefly arising from "musee de l'Homme" (Paris). 9 angles was considered in squeletal profile, and "reciprocal averaging" analysis was made. It shows the main function of peri-buccal segments situation, relatively to palatal and mandibular planes, as well as participation of facial whole situation relatively to anterior neurocranium. This analysis confirm the peculiar position of leucoderm men with reference to other groups. PMID- 1726827 TI - [Do magnetic forces potentiate the histophysiology of orthodontic movement?]. AB - A comparative clinical research has been made to prove if magnet forces can be superior to mechanical forces for tooth movement. Six dogs went through a distalazing movement of their lower premolars on one side with a magnet system and on the other side with a mechanical system producing similar forces. A histological analysis of the moved premolars has showed a more important rate of osteoclasts and a reduction of the hyalinisation area with the magnet system. PMID- 1726828 TI - [Orthodontic movement through compact bone and spongious bone. The difference in tissue reaction with 2 different forces]. AB - The objective of this study, was to study the contribution of the osseous density factor at the orthodontic movement. For this purpose we compared the histophysiological responses frontally to the roots of the mandibular first molar (at the experimental Wistar rat) according to the direction of displacement (mesio-lingual region). In fact, frontally to the mesial and lingual roots (at the mesio-lingual region) there is compact bony tissue in contrary at the vestibular and distal roots the density is less and there is spongiose osseous tissue. We found the following: the region of pressure is subjected to compression and hyalinisation periodontally from the first day, even with the "ideal" force; the extent of compression and hyalinisation periodontally, reaches the maximum approximately after two days; the extent of compression and hyalinisation periodontally is greater frontally to spongiose osseous tissue and even more greater with a heavy force; the immediate displacement of a tooth is greater frontally to spongiose bone and even more greater with a heavy force; there is bony resorption of the internal alveolarwall adjacent to the hyaline zone, resorption (direct laterally or peripherically) frontally to the compact and to the spongiose osseous tissue; the direct lateral resorption: is slower for the compact bone; frontally to the spongiose bone the lateral direct resorption is associated to an indirect resorption at the endoostic side; the real displacement of a tooth is less rapid in compact bone because the lateral direct resorption is slower and also because of the absence of indirect endoostic resorption; at the time of the real dental displacement, the periodontal space has reestablished; on regard of the spongiose bone, the periodontal space not only reestablishes, but also increases during the real dental displacement; at the level of the periosteum, we can observe a compensatory osseous apposition to the direct lateral bony resorption, only with light forces, on the contrary with heavy forces there is periostic resorption and the tooth movement occurs within the bone (especially frontal to a compact bone); the threshold for stimulating the osteoclasts is lower than the ideal force frontal to the compact bone. On the contrary, frontal to the spongiose bone, the threshold for stimulating the osteoclasts, is situated higher to the ideal force. PMID- 1726829 TI - [Use of the Gallyas impregnation method in the study of angioarchitecture in cerebrospinal gliomas in humans]. AB - The Gallyas' method based on impregnation of endothelial basal lamina and reticulin fibres, allowing to reveal the microcirculatory bed, was used to demonstrate the angioarchitecture of brain and spinal cord tumours. This technique, for the first time, enables a more complex view on tumours tissue microvascular architecture than previously. Using of thick paraffin sections enables to incorporate this method into the set of routine diagnostic histology. PMID- 1726830 TI - Microinjection of fluorescent tracers to study neural cell lineages. AB - The examination of cell lineages is an important step towards understanding the developmental events that specify the various cell types in the organism. The mechanisms that control which cell types are formed, their locations, and their numbers remain unknown. Analyses of cell lineage in the frog neural retina have revealed that individual precursors are multipotent and are capable of producing almost any combination of cell types. In addition to giving rise to a wide range of phenotypes, the precursors can give rise to a wide range of clone sizes. Cell lineage studies in other systems indicate that some precursors are multipotent, like those in the retina, while others appear to produce a more restricted range of descendants, perhaps even a single phenotype. These differences in the developmental potential of precursor cells suggest that the nervous system uses several strategies for producing its many cell types. Investigation of these strategies, at the cellular and molecular level, requires more than a description of the normal cell lineages. We are now exploiting the frog neural retina to perform the experimental manipulations needed to elucidate these strategies. PMID- 1726831 TI - Cell lineage analysis of the avian neural crest. AB - Neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. A major unanswered question concerning the neural crest is when and how the neural crest cells become determined to adopt a particular fate. We have explored the developmental potential of trunk neural crest cells in avian embryos by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells within the dorsal neural tube. We find that premigratory and emigrating neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. These results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after emigration from the neural tube either during their migration or at their sites of localization. To determine whether neural crest cells become restricted during their migration, we have microinjected individual trunk neural crest cells with dye shortly after they leave the neural tube or as they migrate through the somite. We find that a majority of the clones derived from migrating neural crest cells appear to be multipotent; individual migrating neural crest cells gave rise to both sensory and sympathetic neurons, as well as cells with the morphological characteristics of Schwann cells, and other non neuronal cells. Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. These data demonstrate that migrating trunk neural crest cells, like their premigratory progenitors, can be multipotent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726832 TI - Visual rehabilitation in low vision patients with age-related macular degeneration. AB - Using optical visual aids, visual rehabilitation was performed in 14 low vision patients (25 eyes) with age-related macular degeneration. With distance aids, visual acuity improvement appeared in 24 eyes (95%) out of the 25 eyes. Twelve eyes (48%) obtained a visual acuity equal to or better than 0.4. With near visual aids, near acuity of all eyes (100%) was improved. Thirteen eyes (52%) got the near vision equal to or better than 0.5. Ten patients could read No. 5 Chinese Reading Card. The reading success rate was 71.4%. The results suggest that application of visual aids is an effective method to improve the distant and near visual acuity of low vision patients with AMD. PMID- 1726833 TI - [Interferon treatment of chronic viral hepatitis]. PMID- 1726834 TI - Immunocytochemical demonstration of calcitonin in the Didelphis albiventris thyroid. PMID- 1726835 TI - Evidence that the inhibition of TE85 human bone cell proliferation by agents which stimulate cAMP production may in part be mediated by changes in the IGF-II regulatory system. AB - Our previous studies have shown that insulin-like growth factor-II (IGF-II) is an important autocrine/paracrine regulator of human bone cell proliferation. In this study, we sought to look at the regulation of IGF-II production by human bone cells since IGF-II synthesis is a key variable that regulates the actions of IGF II at a local site of bone. For studies of IGF-II regulation, we used TE85 human osteosarcoma cells as a model system since these cells exhibited several characteristics which are similar to that of untransformed normal human bone cells. In this study, we investigated the effects of agents which increase intracellular cAMP (forskolin, isobutylmethyl xanthine and prostaglandin E2) and N6,O2' dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on the IGF-II regulatory system. It was found that these agents caused an increase in the production of an IGFBP in TE85 cells. Subsequent studies on the purification and characterization of IGFBP from DBcAMP treated TE85 cell conditioned medium revealed that the IGFBP produced by TE85 cells in response to DBcAMP treatment was identical to that of 25 kDa inhibitory IGFBP (now designated as IGFBP-4) purified from TE89 human bone cells based on: 1) N-terminal amino acid sequence, 2) amino acid composition, 3) molecular weight and 4) inhibitory actions on basal and IGF-II induced bone cell proliferation. In addition, forskolin and DBcAMP also caused a dose dependent decrease in the production of IGF-II. Consistent with these results, DBcAMP and agents which increase intracellular cAMP inhibited TE85 cell proliferation in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726836 TI - Insulin-like growth factor binding proteins (IGFBPs) in acromegaly. AB - Hypersecretion of growth hormone (GH) is the principal feature of acromegaly and is accompanied by an excess of IGF-I production which mediates some of the actions of GH. The activity of IGF-I is modulated by a number of specific binding proteins (IGFBPs) which form complexes with IGF-I in the circulation. In this study, the technique of Western Ligand Blotting followed by 2-dimensional radioactive scanning was employed to investigate the correlations between relative levels of the IGFBPs and the main factors implicated in their regulation: IGF-I, GH and insulin, in a group of acromegalics with varying disease activity. The two glycosylated forms of IGFBP-3 correlated with increased levels of IGF-I (40.5 kD, r = 0.468, p < or = 0.01 and 36.5 kD, r = 0.809, p < or = 0.001), but did not relate to mean GH levels. Quantification of IGFBP-2 on the ligand blot showed an association with RIA levels of IGFBP-1 (r = 0.35, p < or = 0.05). IGFBP-1 RIA levels did not relate to the radioactivity in the assumed IGFBP-1 region of the ligand blot. This may be explained by fragments of IGFBP-3 running in this region and could account for the correlation seen between radioactivity in the 29 kD band with both forms of IGFBP-3 as well as with IGFBP 4. IGFBP-3 levels were normal in biochemically cured acromegalics with normal GH levels, although fasting insulin levels remained higher than normal (mean 16 +/- 4 vs normal 7.4 +/- 0.4 mU/L).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726837 TI - Purification from human cerebrospinal fluid of insulin-like growth factor binding proteins (IGFBPs). Isolation of IGFBP-2, an altered form of IGFBP-3 and a new IGFBP species. AB - Cerebrospinal fluid insulin-like growth factor binding proteins (CSF IGFBPs) characteristically have a preferential affinity for IGF-II, which is largely accounted for by a 32-30 kDa IGFBP(Ka = 10(11) M-1) previously purified from human CSF, with an N-terminal sequence unlike any other IGFBP identified at the time. We have now used procedure (including ammonium sulphate precipitation, acidic gel filtration, affinity chromatography and reverse phase chromatography) and purified three further IGFBPs to homogeneity from child CSF. Apart from the 32-30-kDa form, the predominant IGFBP in CSF is a 34-kDa form (non-reduced in SDS polyacrylamide gel electrophoresis). Like the 32-30-kDa IGFBP, it has a preferential affinity for IGF-II (Ka = 2 x 10(10) M-1). Its N-terminus, Phe-Arg (/)-Pro-Pro-(/)-Thr-Pro-Glu-Arg-Arg-(/)-Gly-Pro-Pro-Pro-Val, corresponds to that deduced for IGFBP-2, except for the first three residues which were not found in the CSF IGFBP. Another form, of 30 kDa, has an N-terminus identical to IGFBP-3's over the first 18 residues determined and seems to be an altered form of IGFBP-3. A third minor species, of 22 kDa, with a preferential affinity for IGF-II similar to that of the 34-kDa IGFBP, has an N-terminal sequence, (/)-(/)-Asp-Ser-Phe-Val Pro-(/)-Glu-Pro-Ser-Asp-Glu-Lys-Ala-Leu-Ser-(/)- (/)-Pro, which bears some analogy with those of other known human IGFBPs, particularly IGFBP-3 (7 homologous residues), and appears to be related to, but distinct from, other IGFBP species. PMID- 1726838 TI - [The influence of intratracheal bleomycin instillation on peroxidative processes in rat lung tissue]. AB - The influence of intratracheally instilled bleomycin (10 mg x kg-1) on antioxidant enzyme activity as well as on lipid peroxidation product levels after 7 and 14 days from drug administration in rat lungs was investigated. The 200 400% increase in superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase and glucose-6-phosphate dehydrogenase activities were observed, as compared to control group. The levels of malondialdehyde, conjugated dienes and lipid hydroperoxides in lung tissue of bleomycin-treated rats were also higher than those in control group. These phenomena are the signs of adaptative mechanisms induction in lungs, protecting the tissue from dangerous effect of bleomycin-generated free radicals. PMID- 1726839 TI - The effect of adrenal cortex hormones on heart and skeletal muscles amylases activity under conditions of alpha- and beta-adrenergic stimulation. AB - Influence of hydrocortisone (H), deoxycorticosterone (D), phenylephrine (F) and isoprenaline (I) on activity of alpha amylase (AA) = 4-glucohydrolase alpha-1, 4 glucan [3, 2, 1, 1] and glucoamylase (GA) = glucohydrolase alpha-1, 4-glucan [3, 2, 1, 3] was investigated in heart and skeletal muscle in rats, before and after adrenalectomy (AT). It was found that AT inhibits a rise in the activities of AA and GA caused by F in both investigated muscles, whereas the I given after AT loses its ability to inhibit GA activity in heart and skeletal muscle. Both H and D administered after AT recover the ability of F to an increase in the investigated enzyme activity whereas recovers I ability to inhibit amylase activity in the presence of H. It can be concluded that inhibition of amylase activity is muscles results from interaction between beta-adrenergic stimulation with glucocorticoids, while the increase in activities of these enzymes is a result of interaction between alpha-adrenergic stimulation with mineral tropic action of both D and H. PMID- 1726840 TI - [The effect of a synthetic fragment of human interleukin-2 on the growth and vascularization of sarcoma 180 in mice]. AB - The effect of a modified peptide of the middle part of the human interleukin-2 molecule (C-1-6) on the growth of transplantable sarcoma 180 of mice was studied. C-1-6 injected intraperitoneally in the dose of 0.5, 5 and 50 micrograms 4 times every other day was shown to stimulate tumor growth and enhance angiogenesis induced by the tumor transplanted intracutaneously. Stimulation of tumor growth was most apparent when the agent was given late (on day 4) after transplantation. Supernatants of murine peritoneal macrophages activated by 1 microgram/ml solution of C-1-6 proved capable of stimulating tumor growth, too. It is suggested that the stimulating effect of C-1-6 on tissue regeneration be investigated. PMID- 1726841 TI - [The treatment of patients with stage-IV stomach cancer]. PMID- 1726842 TI - [The clinical information value of an immunoenzyme study of the tumor markers CA 19-9, CEA and AFP in cancer of the stomach, pancreas, colon and rectum]. AB - Blood levels of CEA, CA 19-9 and AFP were assayed by immunoenzyme technique in 60 cases of gastric cancer, 15 patients with pancreatic cancer and 30 patients with colorectal cancer. CEA and CA 19-9 levels were found to depend upon stage and degree of tumor differentiation. Changes in the antigen levels in the course of treatment reflected the degree of its radicality. In application of the immunoenzyme assay, CA 19-9 level appeared most clinically relevant in gastric, pancreatic and colorectal cancers. CEA concentration can serve as an indicator of liver metastases. CA 19-9 and CEA levels can be used for monitoring and objective evaluation of treatment for gastric, pancreatic and colorectal cancer as well as for predicting response. PMID- 1726843 TI - Morphological heterogeneity within the cingulate cortex in rat: a horseradish peroxidase transport study. AB - We compared the connections of two areas within rat cingulate cortex, the Cg1/Cg2 area vs the Cg3 area, by iontophoresing small quantities of wheatgerm agglutinin horseradish peroxidase (WGA-HRP) into either of these two divisions and identifying afferent and efferent connections. Cortical projections were more widespread for the cingulate cortex (Cg3) area than for the Cg1/Cg2 area and included the dysgranular and agranular insular cortex, and perirhinal cortex. The Cg3 area received input from the CA1 layer of the hippocampus while the Cg1/Cg2 area was interconnected primarily with retrosplenial cortex. In the brainstem, both received input from Barrington's nucleus however, many of the subcortical connections of the two areas differed and supported the hypothesis that the Cg3 area is part of the limbic and visceral motor system while the Cg1/Cg2 area is more closely allied with somatic motor control. The Cg3 area received input from the basolateral nucleus of the amygdala, the supramammillary hypothalamic nucleus, the laterodorsal tegmental nucleus, and the lateral parabrachial nucleus. The Cg1/Cg2 area received input from the substantia nigra and targeted deep layers of the superior colliculus. Thus, rat cingulate cortex is a heterogeneous area that can be further subdivided into separate limbic/autonomic (Cg3) and somatic motor areas (Cg1/Cg2). PMID- 1726844 TI - Isolation and amino terminal sequence of beta-trace, a novel protein from human cerebrospinal fluid. AB - beta-Trace, a 23.5 kDa glycoprotein of unknown biological functions, is present in all body fluids tested. It is found in higher concentration in human seminal fluid and cerebrospinal fluid (CSF) than in serum. A one-step procedure for the isolation of beta-trace from pooled CSF is described, by affinity chromatography using a specific antibody made against beta-trace. Amino terminal sequence analysis yields the sequence A P E A Q V S V Q P N F Q Q D K F L G with no homology to known proteins, indicating that beta-trace is a novel CSF protein. PMID- 1726845 TI - A new translaparoscopic approach in endometriosis treatment: a. CO2 laser endometriosis excision and/or vaporization. b. CO2 laser uterine nerve ablation. c. Uterine suspension with Falope Rings. d. Intraperitoneally 32% Dextran-70 installation. AB - During a period of 18 months with a history of chronic pelvic pain symptomatology (severe dysmenorrhea, severe dyspareunia, extramenstrual pain) retroverted or retroflexed uterus, and infertility were subjected to laparoscopy for diagnostic and therapeutic purposes as well. These women were able to follow up this protocol. After informed consent had been presented patient decided, in a case of endometriosis being verified by the tissue pathology intraoperatively, which one mode of therapy (Group I or Group II) would be administered in her case. All women failed to respond to non-steroidal, antiinflammatory medication, as well as to oral contraceptive treatment. Proposed intraoperative staging of pelvic endometriosis that has not yet been published, was utilized by the author. Group I twenty women were subjected to a translaparoscopic CO2 laser excision and (or vaporization of endometriosis implants, CO2 laser uterine nerve ablation, uterine suspension with Falope Rings and intraperitoneally 32% Dextran was installed. Group II twenty women were subjected only to a translaparoscopic CO2 laser endometriosis excision and/or vaporization and intraperitoneally 32% Dextran-70 was installed. In Group I extramenstrually pain was 90%, severe dysmenorrhea 85%, and infertility 90% were cured. Ten per cent of extramenstrual pain, 5% of severe dysmenorrhea, and 15% of severe dyspareunia were improved. Infertility in this group was unchanged in 10%. Patients' symptoms were not worsened during the 18 months of observation. In Group II only 60% infertility was curred. In 60% extramenstrual pain, in 35% severe dysmenorrhea, in 5% severe dyspareunia were improved. Symptoms were noted to worsen in 5% extramenstrual pain, in 5% severe dysmenorrhea, in 10% severe dyspareunia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726846 TI - [High biliary obstructions--their diagnosis, treatment and complications]. PMID- 1726847 TI - [A method for placing and using an indwelling epidural catheter for analgesia in incurable cancer patients]. AB - Operative technique for implantation of indwelling epidural catheter produce of the firm "Braun" (Germany) is described. During the implantation the analgesics zone is checked with Lidocaine. The catheter pouch is fixed over a bone pad in a place well visible by the patient. For permanent pain relief Dipidolor is applied twice daily one quarter of the ampoule. This catheter is implanted for pains originating from the lower extremities, the pelvis and the abdomen. The method was applied on 2 patients with very good results obtained with minimal amount of opiates. PMID- 1726848 TI - Electron microscopic observation of communication between inner ear stereocilia under normal and noise stimulated conditions. AB - The ultrastructure of communication between inner ear stereocilia under normal and noise stimulated conditions was studied in the guinea pig. With ruthenium red added to the pre- and post fixation solutions, the connections between the stereocilia could be observed in more detail than with ordinary fixation. After exposure to 1,000 Hz 110 dBSPL sound for 3 h, parts of the connections between the stereocilia had disappeared, and adherence between the adjoining stereocilia was observed. The results of our study lend support to the fact that the congregation of stereocilia after noise stimulation is due to the sticky adjoining stereocilia membrane. PMID- 1726849 TI - Imprints of the tectorial membrane following acoustic overstimulation and kanamycin treatment. AB - Normal imprints in guinea pigs were examined mainly using a scanning electron microscope (SEM). Morphological changes in imprints after loading of the conditions mentioned below were also investigated. Imprints observed on the normal tectorial membrane were composed of small concavities lined on a W-shaped line and the W-type was of a V-type in proportion to ascend to the upper turn. The imprints occurred at the rate of one for every outer hair cell and in 3-4 lines corresponding to the outer hair cell hairs. With exposure to a high intensity of sound, small concavities of the imprints were deformed and remnant sensory hairs were observed, sporadically. Another line of new imprints was sometimes evident in the lower turn of the cochlea, in addition to the original imprints. In case of inner ear disturbances due to kanamycin (KM) loading, the imprints were little deformed, but the remnant sensory hairs were numerous and their time-course revealed a gradual decrease and the disposal required a longer time than seen in the degenerated cell fractions on the reticular membrane. With exposure to high intensity sound after KM loading, KM-type changes or high intensity sound-type changes were observed and depended on severity of the KM induced disturbance. The imprints existed for a considerably long time even when the lower region sensory cells degenerated or disappeared in the presence of various conditions. PMID- 1726850 TI - Whole blood histamine release rate during immunotherapy for nasal allergy. AB - In treating nasal allergies, desensitization is widely used on a daily basis, and its efficacy has been highly valued. With the purpose of predicting therapeutic efficacy before carrying out immunotherapy, we compared whole blood histamine release rate and various subjective symptoms before and after immunotherapy. We also compared the degrees of efficacy. Immunotherapy brought about a tendency for a reduction in the whole blood histamine release rate. This tendency appeared most marked in those patients in whom an improvement of subjective symptoms was considerable. Among subjective symptoms, the reduction in whole blood histamine release rate was strongest against nasal discharge and sneezing. In patients with a low histamine release rate prior to the initiation of immunotherapy, the improvement in subjective symptoms tended to be low. PMID- 1726851 TI - [Future of immunologic therapy of bronchogenic carcinoma]. AB - The AA. report their experience as to the treatment applied to patients suffering from unremovable bronchial cancers. They can therefore stress the results obtained by chemotherapeutic treatment associated to thymopentin as well as interferon-thymopentin treatment. Elderly patients were subjected to interferon thymopentin therapy, which helped in improving their life standard and survival. PMID- 1726852 TI - The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria. AB - Variable regions of the 16s ribosomal RNA have been frequently used as the target for DNA probes to identify microorganisms. In some situations, however, there is very little sequence variation observed between the 16s rRNA genes of closely related microorganisms. This study presents a general method to obtain species specific probes using the spacer (intergenic) region between the 16s and 23s rRNA genes. The overall strategy is analogous to that which has previously been developed for the variable regions of the 16s rRNA genes. Sequence analysis of the 16s rRNA and 23s rRNA coding sequences flanking the spacer regions resulted in the design of PCR primers that can be used to amplify the spacer regions of a wide range of eubacteria. Sequencing the amplified spacer region then gives rise to the information that can be used to select specific DNA sequences for use as a DNA probe or for the generation of specific PCR primers to a microorganism of interest. In this study the approach to develop specific DNA markers for members of the genus Clostridium is described in detail. A specific DNA oligonucleotide probe and PCR primers have been designed for Clostridium perfringens that distinguish it from other organisms in the genus. PMID- 1726853 TI - RT-PCR artifacts from processed pseudogenes. PMID- 1726854 TI - Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. AB - By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase. We have tested this method of ligation-independent cloning by using UDG to create complementary single-stranded sticky ends between vector and Alu-PCR products generated from cosmid clones containing DNA from human chromosome 21. Using a single primer, Alu-PCR amplifies the sequence between appropriately oriented, repetitive (Alu) sequences in human DNA that are no more than 2 to 3 kb apart. Nineteen Alu-PCR products were observed in four human chromosome 21 cosmids. Thirteen of these products were detected among 48 subclones picked at random after cloning of the Alu-PCR products using UDG. The size or abundance of an Alu-PCR product did not appear to affect significantly the efficiency of cloning. Eight of the subclones were tested and all hybridized to human chromosome 21 DNA. UDG cloning should prove to be a general PCR cloning method that allows one to rapidly subclone small fragments from human genomic DNA. PMID- 1726855 TI - [Palliative management in advanced bronchopulmonary cancers]. PMID- 1726856 TI - [Palliative treatment of terminal stages of bronchopulmonary cancers]. PMID- 1726857 TI - Molecular biology of hemoglobin: its application to sickle cell anemia and thalassemia. AB - During the past 15 years there have been remarkable advances in our understanding of the molecular biology of hemoglobin synthesis and the abnormalities in hemoglobinopathies and thalassemia. The globin genes were among the first mammalian structural genes that were cloned and the DNA sequence of the human globin gene clusters has since been completely delineated. During the last ten years, we have also learned of the many deletions and point mutations that give rise to hemoglobin-opathies and thalassemia. In addition, the sequences that control erythroid specific expression of the globin gene has also been revealed. These findings have contributed to our understanding of the pathophysiology of the diseases and have allowed the institution of accurate DNA diagnostic methods to be applied to prenatal diagnosis. As increased fetal hemoglobin synthesis is known to ameliorate the severity of the disease in disorders such as sickle cell anemia and thalassemia, agents which increase the level of fetal hemoglobin synthesis are being tested. Also, the discovery of DNA sequences and transacting factors which are responsible for high erythroid globin gene expression [4] may provide more effective means of gene therapy. PMID- 1726858 TI - Allogeneic bone marrow transplantation for patients with hematologic malignancies. AB - Allogeneic marrow transplantation is an effective modality for the treatment of patients with hematologic malignancies. Limitations of success are donor availability, posttransplant relapses, and transplant-related morbidity and mortality. Progress is being made in improving the results of partially HLA matched family member donor transplants and in the use of HLA matched unrelated donors. Increases in dose intensity of pretransplant regimens are limited by toxicity and in order to be effective methods need to be developed to ameliorate side effects. The use of anti-inflammatory agents and recombinant growth factors show promise in decreasing transplant-related deaths. PMID- 1726859 TI - [Euthanasia?]. PMID- 1726860 TI - HLA antigens and Vogt-Koyanagi-Harada's disease. AB - Thirty patients with Vogt-Koyanagi-Harada's disease were typed for HLA-A and HLA B antigenic determinants by a microlymphocytotoxicity technique. HLA-B22 antigen showed an increased frequency of 43.3% in the patient group (relative risk = 8.69; exact P < 0.0001; corrected P < 0.0025) compared with normal control group (frequency = 7.69%). This association suggests that immunogenetic factor may play an important role in the pathogenesis of Vogt-Koyanagi-Harada's disease. PMID- 1726861 TI - [The detection of the c-src protein gene product in human lung tumors]. AB - Immunoblotting and immunochemistry were used to study the expression of a c-src gene-encoded protein in human lung tumors. The authors were the first to identify this otherwise rarely expressed protooncogene in as many as 60% of lung malignancies of various histogenesis. The expression of c-src protein was increased not only in neuroendocrine tumors (small-cell cancer and atypical carcinoid) but also in non-small-cell tumors such as adenocarcinoma, bronchoalveolar and squamous-cell lung cancer. A weak correlation between protein expression and degree of cell differentiation was established for squamous-cell tumors. The study failed to identify the oncoprotein in the normal tissue and benign lesions. Immunoblotting using Mab 327 monoclonal antibodies and the immunohistochemical method employing TBR polyclonal serum yielded similar data. To summarize, an increased expression of pp60c-src was observed in lung cancer of various histology. PMID- 1726862 TI - [Enteroparasitoses in the V Region, Chile. A study of rural school children from Santo Domingo, 1987]. AB - The results of a new enteroparasitological survey carried out by the authors are analyzed with the aim of contributing to the knowledge of the situation of these infections in the V Region, Chile. In 1987 the children of five rural schools of Santo Domingo were studied by means of the modified Telemann method, the Ziehl Neelsen stain and seried Graham test. The parasites more frequently found were: E. vermicularis (50.4%) and G. lamblia (10.8%). No E. histolytica was found in these children. The most frequent commensal was E. nana (21.9%). Cryptosporidium sp. presented a low frequency (0.9%), a figure that in these asymptomatic subjects is in accordance with that found in outpatients with chronic diarrhea in Valparaiso, V Region. PMID- 1726863 TI - [Prefabricated free grafts and neovascularized free grafts. 150 cases in rats]. AB - The use of vascularized, composite and prepared free transplants is a recent technique. Two types of transplants were grafted in rats (150 operated cases). For the prefabricated free transplants, each component remains vascularized by rami of the main pedicle: vessels, nerves, bone, periosteum, cartilage, muscle, skin (50 cases). The graft is transplanted immediately. Neovascularized free transplants are quite different, the various components being laid around the vascular pedicle (100 cases). Neovascularization revascularizes the components. The free graft is transplanted 5 weeks after being prepared. The period of observation ranges from 3 to 12 months. Observation includes macroscopy, arteriography, histology and intravascular dye injections. The results with prefabricated free transplants show normal vascularization of soft tissues. There is bone in all cases, it is normal in 80% of cases. In neovascularized transplants, the bone is normal in 33% of cases only, and totally resorbed in 46%. Prefabricated free transplants produce better results than neovascularized free transplants and must be preferred. The merit of these composite free transplants is that they produce free transplants selectively, using various tissues and chosen vascular pedicles. These transplants are a useful and futuristic alternative for highly sophisticated reconstructive surgery. PMID- 1726864 TI - Histo-bacteriological investigation on borderline tuberculoid leprosy. AB - The multi-sections, which were stained by Fite method, of skin biopsies taken from twelve active BT cases were examined under the guidance of special stains for demonstration of nerve components. All cases were AFB positive. Bacilli were found in infiltrated nerves in 11 cases, of which, in 7 cases, bacilli were detected in nerve fragments within epithelioid cell granuloma. And bacilli were seen in arrector pili muscles in 2 cases. No bacilli were detected in other sites. Since the survival of M. leprae in nerves is one of the reasons causing relapse, this paper suggests that it would be better to treat active BT cases with multibacillary regimen recommended by WHO even though smear-negative. PMID- 1726865 TI - In-vivo treatment with 5-azacytidine causes degeneration of central lymphatic organs and induces autoimmune disease in the chicken. AB - In-vitro evidence suggests that DNA methylation may be involved in the development of forbidden immune responses that can result in autoimmune disease. In the present study we examined in-vivo effects of 5-azacytidine (5-azaC), a substance that inhibits DNA methylation, on the immune system and the occurrence of a spontaneous autoimmune disease in the chicken model. We found that (1) treatment of young normal chickens with 1.0 mg/kg 5-azaC on 7 consecutive days caused a rapid degeneration of the central lymphoid organs thymus and bursa; (2) this regimen with 5-azaC apparently inhibited B cell maturation, as the frequency of cytoplasmic Ig+ plasma cells in the bone marrow was found to be significantly reduced, whereas the total number of bone marrow cells was unchanged; and (3) a chronic low-dose (0.5 and 1.0 mg/kg) application of 5-azaC through 6 weeks was found to significantly enhance the spontaneous autoimmune thyroiditis in newly hatched chickens of the Cornell C strain, as determined by anti-thyroglobulin autoantibody titres and histological analysis of thyroid gland infiltration. The possible implications of these data for the generation of pathogenic autoimmune responses are discussed. PMID- 1726866 TI - Stimulatory effect of epidermal growth factor on the development of mouse early embryos in vitro. AB - Effects of epidermal growth factor (EGF) on the development of mouse 2-cell embryos cultured in vitro were investigated. The addition of EGF at a concentration of 0.5 ng/ml enhanced the development of 2-cell embryos during 24 h of incubation. As expected, EGF stimulated the synthesis of DNA in the 2-cell embryos about 4-fold over the control. The growth-promoting effect of EGF seemed to be specific in that other growth factors, such as transforming growth factor alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF) had no effect on the embryonal development. The addition of EGF also increased the rate of RNA synthesis in a dose-related manner between 0.1 and 50 ng/ml. However, protein synthesis was unaffected by EGF. These results raise the possibility that EGF may participate in the process of early embryogenesis in vivo. PMID- 1726867 TI - Re-investigation of ontogenesis of epidermal growth factor receptor mRNA in the liver of rats: quantitative evaluation of northern blot analysis. AB - Changes in the expression rate of epidermal growth factor receptor (EGFR) mRNA with age in the liver of rats were examined to assess the contribution of EGF to the proliferation of hepatocytes in younger animals. Northern blot analyses with a 32P-labeled probe derived from the extracellular domain of EGFR cDNA were evaluated quantitatively by normalizing the densitometric scanning data with the relative amounts of poly(A)+ RNA in the samples. The expression of EGFR mRNA was low during the early period and increased towards the adult level after 21 days of birth. In contrast, the rate of beta-actin mRNA expression was significantly higher immediately after birth and showed a tendency to decrease with time. PMID- 1726868 TI - Effect of aging on the sensitivity of pancreatic beta-cells to insulin secretagogues in rats. AB - Glucose-stimulated insulin release from rat pancreas is known to be blunted by aging. In the present study, we examined the effect of aging on insulin release induced by various secretagogues using the isolated perfused pancreas of female rats. Insulin release from the perfused pancreas in response to 16.7 mM glucose in 8-month-old rats (older rats) was much less than that in 2-month-old rats (young rats). The first phase of insulin release after glucose stimulation was attenuated in older rats. The addition of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) potentiated glucose-induced insulin secretion in both groups of rats. However, the second phase of insulin secretion in older rats was lower than that in younger rats. The phorbol ester 12-O-tetradecanoyl phorbol ester (TPA, 200 nM) enhanced both the first and the second phases of insulin release induced by glucose in both groups of rats. The amount of first phase insulin release induced by TPA with glucose in young rats was greater than that in older rats, whereas the second phase of insulin release was similar in both groups of rats. On the other hand, tolbutamide (200 uM) similarly stimulated the first phase of insulin release in both age groups of rat. In addition, the amount of cumulative insulin secretion induced by tolbutamide during the second phase was slightly but significantly greater in older rats than in young controls. Insulin content in the pancreas was significantly greater in older rats than in young rats and increased after the stimulation with TPA and tolbutamide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726869 TI - Molecular biology of trophoblast interferons and studies of their effects in vivo. AB - Southern blotting of bovine genomic DNA indicated the presence of at least 3 bovine tIFN genes. The full DNA sequence of one of these genes, thought to be expressed in trophoblast, has been determined, including 193 bp of 5' non-coding region. The inferred amino acid sequence of bovine tIFN is more similar to ovine tIFN (80%) than to bovine IFN-alpha II (70%). The 5' flanking sequence has some similarity with bovine IFN-alpha II, and may contain a viral response element. A recombinant bovine alpha I interferon (Ciba Geigy; brIFN), resembling tIFN, extended oestrous cycle length in sheep when administered by intrauterine infusion over the period, Days 12-15 after oestrus, when maternal recognition of pregnancy occurs. Intramuscular injection was only effective at the doses used if given over a longer period (i.e. Days 9-15). Our experiments indicate that both tIFN and brIFN inhibit luteolysis by preventing a rise in endometrial oxytocin receptor concentrations, and suggest that tIFN achieves this by extending the time for which progesterone suppresses oxytocin receptor development. Further studies are required to confirm this hypothesis and to elucidate the interaction of the effects of progesterone and tIFN in endometrial cells. PMID- 1726870 TI - The ovarian insulin-like growth factor system. PMID- 1726871 TI - [Cellular and fibrillar organization of the locomotor regions of the brain stem in cats]. AB - Cellular and fibrillar organization of the hypothalamic (HLR) and mesencephalic (MLR) locomotor regions of the brain stem has been studied in cats by means of staining the nervous tissue after Nissl and Sokolyansky. Morphometric investigations of neurons in these regions has been performed in frontal and sagittal slices. Within the limits of the HLR ad MLR there are about 45,000 of neurons, which are organized as various nuclei, the MLR cells arranging in them more compactly in comparison to diffusely ++ scattering HLR neurons. Within the limits of the locomotor regions, together with the cells and powerful compact fasciculi of fibers in the tract passing, that is especially specific for MLR, there are also numerous diffusely ++ scattered fibers. A possible role of the neurons and the fibers passing in starting locomotion at activation of the locomotor areas of the brain stem is discussed. PMID- 1726872 TI - [Morphological criteria of early embryonal histogenesis exemplified by the development of the auxiliary ocular apparatus in humans]. AB - In 50 human embryos at the age of 21 days--12 weeks of development, including the stages X-XXIII and beginning of the fetal period according to Carnegie's classification, spatial-temporal regularities on rearrangement of cellular material in the mesenchymal derivatives of the ocular auxiliary apparatus have been investigated. The main attention has been paid to ascertain the moments, when the first signs of differences appear in the previously homogenous cellular material of the differentiating mesenchyme around the ocular cup and in the eyelids, taking into account appearance of derivates in the ocular auxiliary apparatus: sclera, oculomotor muscles, stroma of the lacrimal gland, cartilagenous laminae in the eyelids and in the orbicular muscle. Regularities of cytochemical differentiation of the mesenchyme and its derivatives have been studied, taking into account the organ's changing topography. Morphometrical investigations of cells in the anlages mentioned are expanded and correlated with the time of their cytochemical differentiation. Correlation of the morphometrical and cytochemical data with the histological investigations has been performed. PMID- 1726873 TI - [Comparative anatomical characteristics of the histological structure of the arteries at the base of the brain in various mammalian orders and species]. AB - In 30 mammalian species specific peculiarities of the arterial structure has been revealed and their dependence on the type of the brain blood supply has been demonstrated. The architectonics of the blood supply sources is correlated with the structure of the wall in the arteries, immediately participating in dumping the blood stream during its transport to the brain. PMID- 1726874 TI - [Embryogenesis of the human tongue]. AB - Developmental peculiarities of the tongue as a whole and its main structural elements (muscles, membranes, glands, lingual tonsil) have been studied in 120 human embryos and fetuses 5-week-old--9-month-old. Transmissive electron microscopy, electron histochemistry (for estimation of 5'-nucleotidase activity), staining of semithin slices with toluidine blue and aniline++ pink have been applied. Problems on migration of myogenic elements in the developing tongue have been discussed. Ultrastructure of gustatory bulbs of the human fetus tongues has been investigated. A complete formation of the tongue takes place by the 8th-9th months of the prenatal development. PMID- 1726875 TI - [Endocrine cells of the gastric mucosa in patients with duodenal ulcer after vagotomy]. AB - Bioptates of the stomach mucous membrane (SMM) have been investigated in 169 patients suffering from duodenal ulcer (DU). According to the nocturnal gastric secretion test among them there are "hypersecretors" and persons with moderate elevation of acid formation. In conformity with the efficiency of the operative treatment among the patients examined, groups are defined: those with recurrent disease and those recovered after vagotomy. The DU endocrine apparatus undergoes both qualitative and quantitative alterations after vagotomy. When recovery after vagotomy takes place, the number of endocrine cells only slightly exceeds these parameters in the patients with a moderately manifested acid production. These alterations are adaptive. The recurrence of DU in patients with moderately manifested acid production before the operation can be explained by hyperplasia of G-cells. A high degree of hyperplasia of all elements of the endocrine apparatus in the "hypersecretors" can be one of the causes of the DU recurrence. The data about the state of G-, Ec- and EcL-cells before and after vagotomy can be used at prognostication the results of surgical treatment of patients with DU. PMID- 1726876 TI - [A method of intracellular administration of lucifer yellow for the study of human brain using autopsy specimens]. PMID- 1726877 TI - [Transmitral flow velocity patterns as influenced by preload, afterload and heart rate alterations: pulsed Doppler echocardiographic study]. AB - The influences of preload, afterload and heart rate alterations on the pattern of left ventricular filling were investigated using pulsed Doppler echocardiography (PDE) in humans. Transmitral flow at the level of the valvular tip was recorded during dextran infusion, lower body negative pressure, angiotensin II infusion, and atrial and atrioventricular sequential pacings. Peak velocity of rapid filling (R), peak velocity of atrial contraction (A), the ratio of peak velocities (A/R), flow velocity integrals of the rapid filling phase (IR) and atrial contraction (IA) were obtained. PDE and the measurement of hemodynamics during lower body negative pressure (0, -10 mmHg, -20 mmHg) and dextran infusion (100 ml, 200 ml) were studied in 22 patients with ischemic heart disease. R decreased significantly after lower body negative pressure, and increased significantly during dextran infusion. Before and during angiotensin II infusion, PDE and the measurement of hemodynamics were studied in 14 patients with ischemic heart disease. The patients were categorized into 2 subgroups according to left ventricular function. During afterload stress, the A/R and IA increased in patients with normal left ventricular function; whereas, the A/R decreased in patients with poor left ventricular function. PDE was recorded during right atrial and atrioventricular sequential pacings at the heart rates of 60 to 100 beats/min in 29 patients with ischemic heart disease. When the heart rates increased, R decreased during atrial and atrioventricular sequential pacings. The A increased after the occurrence of the summation between the rapid and atrial filling waves. The A/R gradually increased with incremental heart rate. These results indicate that changes in the preload alter the peak velocity of left ventricular filling pattern of transmitral flow. The effects of the increasing afterload depend on the basal left ventricular function, with an increase in the peak velocity of atrial contraction being observed in the presence of normal left ventricular function. Both the peak velocity of rapid filling and atrial contraction were related to the heart rate and atrioventricular conduction delay. In assessing left ventricular filling dynamics using PDE, the influence of the preload, afterload and heart rate must be considered. PMID- 1726878 TI - Two cases of bi-ventricular dysplasia associated with ventricular tachycardia and familial occurrence of sudden death. AB - Two strikingly similar patients with arrhythmogenic right ventricular dysplasia which severely impaired not only the right ventricle but also the left ventricle are described in association with familial occurrence of sudden death. A 49-year old man experienced syncope which was due to ventricular tachycardia. Electrocardiography revealed a first degree atrioventricular block, incomplete right bundle-branch block, T wave inversions in leads II, III, a VF and V1 to V5, and multiformal ventricular extrasystoles. Echocardiography and ventricular cineangiography showed not only the right ventricular dilatation with an aneurysm in the right ventricular apex, inflow and outflow tracts, but also mild dilatation of the left ventricle with left ventricular apical and posterior aneurysms. Radionuclide angiography also disclosed dysfunction of both ventricles, especially during exercise. His family history revealed that 3 members of his family died of sudden deaths. A 56-year-old woman experienced syncope secondary to ventricular tachycardia, with left bundle-branch block. Electrocardiography showed complete right bundle-branch block, left axis deviation, and T wave inversions in leads V1 to V4. Echocardiography and ventricular cineangiography revealed not only marked right ventricular dilatation with the "triangle of dysplasia", but also a left ventricular aneurysm in the apex and posterior portion. Her elder brother died of a sudden death, and electrocardiograms of 2 members of her family showed ventricular extrasystoles and T wave inversions. These 2 cases may well be termed "familial bi-ventricular dysplasia". PMID- 1726879 TI - Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes. AB - Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif+-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10(4) cells. PMID- 1726880 TI - Control of protein topology at the endoplasmic reticulum. AB - I have described recent work that supports several conclusions that might not have been previously expected: first, that stop transfer, like the initiation of translocation, is receptor-mediated; second, that at least some of the topology determining events at the ER membrane can be regulated (an example is provided where regulation may occur developmentally [PrP] and a possible example where receptor interactions for stop transfer seem to have been dissociated from those of integration in the membrane, in the course of evolution [apo B]); third, that these variations on the universal mechanism of eukaryotic secretory and transmembrane protein biogenesis can occur either through the variations in sequences presented to the common machinery of translocation or through variations in the machinery with which these sequences interact. Thus, on the one hand, at least some of these variations are directed by signal and stop transfer sequence subtypes and, on the other hand, in at least one case, a special cytoplasmic factor distinct from the core machinery for chain translocation also seems to be involved (RRL cytosolic factor effect on PrP topology) in the special handling of the STE stop transfer sequence subtype. In another case, the conserved universal machinery is engaged by a protein (apo B) to carry out an unusual, if not unique, mechanism presumably related to the lipid carrying role of this soluble secretory protein. Whether stop transfer sequence subtypes are involved here remains to be demonstrated, but it is a tempting hypothesis. Taken together, these findings suggest that the ER is more than a barrier to be overcome in protein export. In some cases, it plays a regulatory role in gene expression (e.g., alternate fates of PrP), and in other cases, it plays a role as a specialized assembly line for biogenesis of proteins with unusual properties. It seems likely that many other examples of proteins using these two mechanisms will be found, as well as entirely different variations on the mechanisms of protein biogenesis. A common conceptual theme is likely to be that they are all directed by discrete sequences within the particular newly synthesized proteins engaging both/either the common and/or distinctive component of the cellular machinery for protein biogenesis. PMID- 1726881 TI - Regulation of prolactin storage. PMID- 1726882 TI - Secretory granule formation. The morphologist's view. PMID- 1726883 TI - Mannose-6-phosphate receptors and their role in protein sorting along the pathway to lysosomes. PMID- 1726884 TI - Ribosome binding to endoplasmic reticulum. PMID- 1726885 TI - An analysis of BET1, BET2, and BOS1. Three factors mediating ER to Golgi transport in yeast. PMID- 1726886 TI - Biochemical analysis of constitutive secretion in a semiintact cell system. PMID- 1726887 TI - Processing of peptide precursors. Identification of a new family of mammalian proteases. PMID- 1726888 TI - The enzymology of proinsulin conversion. PMID- 1726889 TI - Processing and sorting of human prorenin. PMID- 1726890 TI - Intracellular trafficking and processing of pro-opiomelanocortin. PMID- 1726891 TI - What the granins tell us about the formation of secretory granules in neuroendocrine cells. PMID- 1726893 TI - [Alternative chemotherapy of malignant bone neoplasms in children]. AB - The authors propose alternative chemotherapy of osteosarcoma and Ewing's sarcoma in children. The aim of this proposal was elaboration of effective and, at the same time, less expensive and less toxic therapeutic regimens. The authors recommend open surgical biopsy with doxorubicin for 3 consecutive days as a protection against the released circulating neoplastic cells. After completion of histopathologic examination, one of two types of chemotherapy is chosen randomly. In osteosarcoma, there was induction chemotherapy for 4 or 9 weeks (according to the type of operation--conservative amputation or limb salvage surgery). In the I type of induction chemotherapy, high doses of methotrexate with vincristine and citrovorum factor rescue are administrated weekly, in the II type--the combination of BCD (bleomycin, cytoxan, actinomycin D) and CDDP (cisplatin). On the regimen of intensification chemotherapy decides the degree of tumour response to induction chemotherapy assessed as tumour necrosis in histopathologic examination. Maintenance chemotherapy is the same in two types of regimen and is continued for the period up to 2 years. The authors elaborated concomitantly the regimen of high methotrexate doses administration with rescue procedure in the case of elevated serum methotrexate levels, and regimen of cisplatin administration aiming at maximal patients protecting against the toxic effects of both drugs. In Ewing's sarcoma the randomisation differentiates between T-9 Rosen's regimen of chemotherapy and own modification of Memphis group regimen. The primary tumour is treated by radiotherapy with lower doses adjusted to the tumor response to induction chemotherapy (30-50 Gy or 50 Gy) and the irradiation port limited to the residual bone lesion plus a 2-3 centimeter margin. Surgical excision of bone with tumor depends on special tumor localisation as the clavicula, rib or fibula. The results of discussed treatment regimens will be subsequently published. PMID- 1726892 TI - Cellular and viral components that mediate glucocorticoid-regulated processing of retroviral envelope proteins. PMID- 1726894 TI - [Cellular analysis of bronchoalveolar lavage fluid. I. Detection of macrophage populations]. AB - The aim of the study was to compare the results of routine counts of percentage of BAL macrophages, using morphological differential staining method--Pappenheim stain (M) and staining for nonspecific esterase activity (E) with those obtained by APAAP technique, using specific for monocytes (macrophages monoclonal antibodies (Ki-M1) and the complex APAAP (alkaline phosphatase--mouse anti alkaline phosphatase complex). The mean counts were comparable, although different criteria of estimation were used. Significant differences were seen in smokers when the criteria were based on morphology and expression of surface antigens. The results indicate that correct estimation of macrophage counts and their characteristics (enzymatic activity, expression of surface antigens) can only be made using many methods based on various criteria of estimation. PMID- 1726895 TI - FK506 lacks the ability to inhibit expression of interleukin-2 receptor beta chain on human lymphocytes. AB - Proliferation of T-lymphocytes is induced by interleukin-2 binding to the interleukin-2 receptor. This receptor consists of an alpha and a beta chain. FK506 is a new immunosuppressant known to inhibit synthesis of interleukin 2 and expression of the membrane bound alpha chain. In this study we show that FK506 does not suppress the expression of the beta chain on human lymphocytes, in the concentration range 1 pM to 1 microM, as measured by flow cytometry. PMID- 1726896 TI - Characteristics of single-channels activated by quisqualate and kainate in teleost retinal horizontal cells. AB - There is increasing evidence in the teleost retina that the excitatory amino acid glutamate is the neurotransmitter used by some photoreceptors. Single kainate and quisqualate channels were recorded on isolated white bass horizontal cells using patch-clamp techniques. Two categories of channels were observed. The first, labelled a slow-channel, exhibited conductance and open time averages for channels activated by quisqualate of 8.5 pS and 8.8 msec, and for kainate 8.5 pS and 4.5 msec. The closed times of these channels could be described by two time constants. The second channel category was termed a fast-channel. Quisqualate and kainate activated channels in this category with two prominent conductances in the range of about 10 pS and 20-30 pS and open times of 1-2 msec. These channels demonstrated closed times with only a single time constant. Openings of slow channels elicited by the agonists tended to occur in bursts. Activity of the fast channels was noisy and no bursting behavior could be seen. Both channels exhibited multiple conductance states. PMID- 1726897 TI - Voltage clamp study of electrophysiologically-identified horizontal cells in carp retina. AB - Passive membrane properties and electromotive force of light modulated currents of L-, R/G-type and rod-driven horizontal cells were studied by voltage-clamp using double-barrelled micro-electrodes whilst perfusing with 5 microM dopamine to uncouple the gap junctions. Input impedances of horizontal cells in darkness were 31 +/- 1.4 M omega (mean +/- SE, n = 63); the resting potentials were -37 +/ 1.3 mV. Current-voltage relationships had regions of both inward and outward rectification and a region of negative resistance was commonly observed. Reversal potentials of light modulated currents were estimated on average to be -7 +/- 4 mV (n = 14), which is consistent with the involvement of K+ and Na+ and/or Ca2+ gradients. Importantly in R/G cells both depolarizing and hyperpolarizing components of the response had essentially the same reversal potential. PMID- 1726898 TI - Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6 phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase. AB - The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high affinity site with (mean +/- SD, n = 7) Ka1 3.0 +/- 0.3 * 10(9) M-1 and maximal binding capacity (MBC1 112.1 +/- 20.7 fmol/mg DNA, and to a low affinity site with (median, (range), n = 7) Ka2 1.4 (0.6 - 2.6) * 10(7) M-1 and MBC2 766 (461-2687) fmol/mg DNA. Incubation of cells with T3 6 nmol/l for 20 hours reduced the area under the T3 binding curves (AUC) to 80.9% +/- 10.0% of AUC in cells incubated without T3 (p < 0.01, n = 7). The downregulation, being reversible and associated with receptor saturation, was caused by a reduction in MBC1 of the high affinity site to 66.6 fmol/mg DNA, whereas Ka1 was unchanged. T3 stimulated cell growth (p < 0.05, n = 8), but had no effect on the activities of ME, G6PD, and 6PGD. Insulin (1 mumol/l) enhanced the activities of ME (p < 0.01, n = 6) and 6PGD (p < 0.05, n = 6). IN CONCLUSION: The cellular effects of T3 in the human hepatocyte cell-line was: 1) a reversible modulation of NBT3 associated to receptor saturation; 2) stimulation of cell growth; 3) contrary to the findings in rat hepatocytes no stimulation of ME, G6PD or 6PGD. Insulin enhanced ME and 6PGD. PMID- 1726899 TI - Thyroxine and reverse T3 5'deiodination in the cells of human adipose tissue. AB - The intracellular 5'deiodination (5'D) of T4 and rT3 has been investigated in human adipose tissue. We studied 5'D in intact adipose tissue, its morphological components and in 3T3-L1-cells. 5'D as assessed by T3-production out of T4 and by rT3-decomposition was not inhibited by propylthiouracil (PTU), but by iopodate (IOP). The apparent Michaelis constants were kM = 3 nM for rT3 and kM = 1 microM for T4. The rT3-degradation was linear over 25 h (115 pg/h.mg (prot.)) both at 37 degrees C and at 4 degrees C. The same type of 5'D was observed in adipocytes, stromal-vascular cells and in 3T3-L1-cells regarding T4 to T3 degradation (244 +/ 30, 181 +/- 27, 227 +/- 37 pg T3/mg.min), resp.; PTU did not exert any influence upon 5'D in the cells investigated. We conclude, that i. the intracellular generation of T3 in adipose tissue does not derive from type I deiodination; ii. 5'D in adipocyte precursors and differentiated adipocytes is identical and iii. there is no difference between human cells and 3T3-L1-cells regarding 5'D. PMID- 1726900 TI - Thyrotropin-releasing hormone degradation in patients with insulin dependent diabetes mellitus. Effects of metabolic control. AB - We investigated thyrotropin releasing hormone (TRH) degradation in terms of half life (t1/2) and metabolic clearance rate (MCR) in eight subjects with insulin dependent diabetes mellitus (IDDM) before and after strict metabolic control. The results were compared with those of six healthy control subjects. The basal plasma TRH-IR levels (31 +/- 9 fmoles/ml) were on the lowest normal limit in the IDDM patients and were not considerably changed (24 +/- 10) after strict metabolic control. The basal and delta max rise of TSH to TRH (200 micrograms i.v.) were not significantly different before or after improved metabolic control in IDDM and as compared to controls. The TRH-degradation curves showed similar exponential decay before and after improvement of metabolic control (t1/2: 7.6 +/ 0.4 min and 7.3 +/- 0.3 respectively; 6.5 +/- 0.4 min for the controls). The MCR of exogenously administered TRH in IDDM before (65.5 +/- 8.6 l/m2/day) and after (65.0 +/- 8.9) control was not different compared to the normals (76.5 +/- 9.6). The area under the plasma concentration-time curve (AUC) in IDDM before (52.193 +/- 6.773 fmoles.ml-1.min) and after improvement of metabolic control (53.186 +/- 7.856) was slightly higher than in the healthy subjects (40.151 +/- 3.741, n.s.). These findings demonstrate that a) the degradation of exogenous TRH is not dependent on the glucose metabolic state, b) insulin deficient diabetes mellitus does not affect the enzymatic system responsible for TRH degradation and, c) the hypothalamic-pituitary axis appears to be intact in IDDM. PMID- 1726901 TI - Thyroid stimulating antibodies in patients with Graves' ophthalmopathy. AB - The possible pathogenetic role of thyroid stimulating antibodies (TSAb) was studied in 24 patients. The TSAb were measured by their ability to stimulate concentration of cAMP in human thyroid slices incubated in vitro with the immunoglobulins of these patients. The patients were divided in two groups: Group I, 16 patients with eye disease and hyperthyroidism and Group II, eight patients with eye disease but without hyperthyroidism. TSAb, were found to be high in 15 patients of the Group I (3.22 +/- 1.74 pmol cAMP/mg of wet tissue, mean +/- S.E.M.) whilst were normal in six patients of the Group II (0.98 +/- 0.53 pmol cAMP/mg of wet tissue, mean S.E.M.) (p < 0.001). These findings indicate that TSAb is not a marker of the Graves' ophthalmopathy and support the concept that hyperthyroidism and exophthalmos are two separate disorders. PMID- 1726902 TI - Hormonal and biochemical patterns in subjects from a new endemic goiter area in the Central African Republic. AB - Hormonal serum patterns of 268 subjects, living in rural villages and in the chief town of the Ouham province (Bocaranga), were studied to accomplish a survey in this endemic goiter area of the Central African Republic (CAR). Circulating TSH, TT3, TT4, FT4, rT3 and TG were determined in an accurately randomized population sample. Urinary excretion of iodine and thiocyanate (SCN) was also measured. The comparison of the mean values of such parameters with control values showed statistically significant differences (P < or = 0.05 divided by 0.001) in all the parameters studied, excepting TT3. The urinary iodine excretion was lower than 35 micrograms/l in the chief town and of 25 micrograms/l in the rural villages. The thiocyanate excretion was constantly greater than 8 mg/l; the I/SCN ratio = 3.7 was clearly lower than in controls. In a sample of 41 subjects, whose hormonal patterns were in the physiological range, significant differences were found between their TT3, TT4, FT4, TSH and TG serum concentrations and the respective control values. The severe iodine deficiency and the high consumption of manioc as a staple food are the prominent etiopathogenic factors of the hyper endemic goiter in this region. The authors believe that extensive iodoprophylactic action is urgently needed. PMID- 1726903 TI - Basal and stimulated TSH serum concentrations in euthyroid thyroid nodule patients. AB - Serum TSH concentrations were determined by a sensitive second generation immunoradiometric (IRMA) assay, basally and 20 min after i.v. injection of 200 micrograms of TRH, in 630 consecutive ambulatory clinically and biochemically euthyroid patients with palpable thyroid nodules. The TSH response was defined as normal when the stimulated TSH values was higher than the basal one by at least 2 microU/ml, as suppressed when the difference between the two TSH values was less than 1 microU/ml and as blunted when this difference was between 1 and 2 microU/ml. The TSH response was normal in 511 patients (81.1%), suppressed in 78 (12.4%) and blunted in 41 cases (6.5%). Nodule patients with suppressed responses showed significantly higher mean age (52.7 vs 45.8 years; p < 0.05) and mean serum concentrations of TT4 (9.32 vs 8.71 micrograms/dl; p < 0.05), TT3 (161 vs 137 ng/dl; p < 0.01) and fT3 (4.94 vs 3.86 pg/ml; p < 0.01) than those with normal TSH secretion. Analysis of the distribution of the different TSH responses in the patients grouped according to basal TSH concentration values showed that 50% of the patient group with basal TSH concentration between > 0.2 and 0.3 microU/ml had normal TSH response to TRH. A normal response occurred in 86.5% of patients with basal TSH between > 0.4 and 0.5 microU/ml and in 95.3% of those with basal TSH between > 0.7 and 0.8 microU/ml. The proportion of normal responses in the patients with basal TSH up to 0.1 microU/ml was 15.7% and that of abnormal responses in those with basal TSH > 1.5 microU/ml was 2.7%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726904 TI - The outcome of benign thyroid nodules correlates with the findings of fine needle biopsy. AB - 204 patients with a single or dominant 'cold' thyroid nodule were treated with l thyroxine, 150-250 micrograms/d, for 4 to 12 months. The two clinicians who evaluated the evolution of the nodules knew that fine-needle biopsy (FNB) had excluded malignancy, but ignored any other morphologic data of the FNB smears. Good outcome, i.e. disappearance of the nodule or definite decrease in its size, was observed in 66 cases (32%). This good clinical outcome was correlated with various morphologic characteristics of the FNB aspirate. The nodule was more likely to regress if FNB showed much colloid, many degenerative changes and many phagocytes, whereas the presence of extensive hyperplasia, lymphocytes or fibrosis were associated with an unfavourable prognosis. With logistic regression, controlling for the confounding effects of sex, age, clinical and histological picture and thyroxine dose, significant correlations were shown with the presence of colloid, hyperplasia and fibrosis. It is concluded that the morphologic characteristics of the FNB aspirate may, to some extent, predict the outcome of cold thyroid nodules. PMID- 1726905 TI - Graves' orbitopathy and restrictive myopathy. AB - Restrictive myopathy in Graves' orbitopathy is a disabling disorder, difficult to treat, involving clinically the inferior and medial recti. Most patients do not return to the normal, premorbid state of muscular alignment and the goal of treatment is to reach binocular vision in the important directions of gaze- straight ahead (primary position) and downgaze (reading position). The treatment in the acute, congestive stage is optical, prisms or patching, or medical. The medical treatment principally consists of a course of oral or intravenous corticosteroid treatment, and in selected patients orbital irradiation can be of benefit. Surgery is performed on the euthyroid patient after the muscular imbalance has stabilized, usually six months from the onset of the disorder. The mainstay of therapy is a large recession of the involved muscles, if possible on an adjustable suture. Sometimes marginal myotomies are added to the muscle recession. A fadenoperation on the contralateral, minimally involved inferior rectus has been suggested as a means of increasing muscular alignment in both primary position and downgaze. Thorough evaluation of the course of the disease, and the benefit of different treatment modalities is possible only with long term follow-up. PMID- 1726906 TI - Treatment of supraventricular tachyarrhythmias associated with hyperthyroidism by radioiodine, amiodarone and propylthiouracil. AB - Beta-blockers and calcium antagonists have been advocated for thyrotoxicosis induced tachyarrhythmias. Amiodarone is generally considered as contraindicated because of its high iodine content. Since amiodarone combined with propylthiouracil induced a greater fall in serum thyroid hormone concentrations than propylthiouracil alone, we treated 2 hyperthyroid patients with supraventricular arrhythmias by radioiodine (day 0) followed after 24 h by amiodarone and propylthiouracil. Serum T3 was normalized on day 2 (patient 1) and 3 (patient 2). Effective t1/2 of intrathyroidal 131I were 6.6 and 4.3 days (versus 5.9 days for 131I given alone). In patient 1, atrial fibrillation, reverted to sinus rhythm after verapamil and digoxin, and did not recur. In patient 2, conversion of atrial fibrillation to sinus rhythm occurred on day 11; from day 0 to day 11, ventricular rate decreased and was significantly correlated to T3 (r = 0.82; p < 0.05). In conclusion, amiodarone may be beneficial in thyrotoxicosis associated tachyarrhythmias, given with propylthiouracil 24 h after radioiodine, it did not decrease thyroid irradiation and rapidly decreased serum T3. PMID- 1726907 TI - Vitiligo in autoimmune thyroid disease. AB - The authors studied the association between vitiligo and autoimmune thyroid disease. Vitiligo was found in 20 of 293 patients with autoimmune thyroid disease (6.83%), 2 out of 227 patients with nonautoimmune thyroid disease (0.88%), and 3 out of 386 control group (0.78%). These results showed that vitiligo is closely associated with autoimmune thyroid disease (chi 2 = 24.33, p < 0.0001), but not with nonautoimmune thyroid disease. Prevalence of vitiligo in nonautoimmune thyroid disease was not different from that in control. Vitiligo in autoimmune thyroid disease was most frequently found on dorsum hands and forearms, and usually preceded the onset of thyroid disease. Four out of twenty patients with vitiligo associated autoimmune thyroid disease had another presumed autoimmune disease, that is, alopecia areata, alopecia totalis, and rheumatoid arthritis. These findings suggested that autoimmunity plays an important role in the pathogenesis of vitiligo. PMID- 1726908 TI - Autonomously functioning thyroid adenoma in a seven year old boy. AB - Hyperfunctioning thyroid adenoma is an extremely rare disorder in childhood. A case of a seven year old boy is reported. Clinical and laboratory findings were similar to those seen in adults. Recovery of thyroid function was prompt after ablative surgery and no substitutive therapy was required. PMID- 1726910 TI - Specific opiate effects on dextran-induced inflammation in rats. AB - The effects of opiates were investigated in two models of acute inflammation in rats. Morphine (0.1-10 mg/kg, i.p.) inhibited by 50% the paw edema induced by interdigital injection of 1% dextran solution. Low but not high doses of naltrexone produced a similar degree of inhibition. Naltrexone (10 mg/kg, i.p.) completely prevented morphine antiedema effect. Local anesthesia of the hindleg with lidocaine neither modified dextran-induced paw edema nor morphine inhibitory effects. Morphine (10 mg/kg, i.p.) enhanced by 50% skin vascular permeability induced by intradermically injected 1% dextran solution. Again, naltrexone prevented morphine effects. The obtained results suggest a specific modulatory role of opiates in the acute inflammatory responses of the rat. PMID- 1726909 TI - Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-C. AB - Insulin and insulin-like growth factor I promote the growth of rat neuronal cells in primary culture. In order to investigate the mechanism of hormone signalling in this biological system, we studied the effect of cyclic AMP agonists and a protein kinase C stimulator on basal and hormone-induced RNA synthesis. Agents elevating endogenous cyclic AMP levels (forskolin, dibutyryl cyclic AMP, cholera toxin) blocked the stimulatory effects of both insulin and the growth factor; dibutyryl cyclic AMP, however, altered the binding of neither hormone. Although, unlike the aforementioned agents, phorbol ester significantly elevated basal RNA synthesis; it nevertheless inhibited the stimulation by insulin; this latter effect probably being mediated by an increase in intracellular cyclic AMP levels, as has been found in other cell types. Staurosporine, an inhibitor of protein kinase C, also blocked the effects of insulin on RNA synthesis. PMID- 1726911 TI - [Pulmonary ventilation after administration of lymphocytic supernatant to patients with chronic spastic bronchitis]. AB - In 10 patients of chronic bronchitis culture of mononuclear cells were performed. Supernatant was applied in inhalation. Bronchospasm was not observed in spastic bronchitis patients but it was present in asthmatic patients. In 50% of chronic bronchitis patients the inhalation test to histamine was positive. PMID- 1726912 TI - [Histologic study of Helicobacter pylori in 265 consecutive gastric biopsies]. AB - We evaluated 265 consecutive endoscopic gastric biopsies of subjects attending the gastric cancer mass survey that is taken place in the State of Tachira, Venezuela. They were 152 males and 113 females between 18 and 70 years of age. We established the prevalence of Helicobacter pylori in relation with histologic diagnosis and location of the lesions in the stomach. We performed hematoxylin eosin, Giemsa and Gimenez stains. The bacteria was identified in chronic non atrophic gastritis in 81% of the cases, in 76% of erosive gastritis, in 72% of gastric ulcers, in 36% of chronic atrophic gastritis and in 16% of neoplastic tissues. Helicobacter pylori was found in 82% of antral biopsies, and in 9% of the body, 7% of the fundus and 2% of the cardiac biopsies. From the total of 265 biopsies HP was positive in 172. The prevalence of this bacteria was 69.9% in this investigation. PMID- 1726913 TI - [Antibodies against hepatitis C virus. Experience with a second generation test]. AB - A second generation recombinant immunoblot assay to detect antibodies against Hepatitis C virus (RIBA II, Ortho Diagnostic Systems) was applied to 30 serum samples repeatedly reactive to ELISA anti-HCV c100-3, 11 from hemodialysis patients, 11 from patients with chronic hepatitis and 8 from patients with cirrhosis. The assay detects individual antibodies directed to 4 antigenic components of the C virus, 2 antigens, c100 and 5-1-1 contained in the original ELISA assay and 2 new antigens, c33c and c22-3 the latter structural in nature. From the total of 30 samples, 23 (77%) were reactive, 4 (13%) indeterminate and 3 (10%) non reactive to the RIBA II assay. Much variation was observed regarding the response to each individual antigen. c22-3 was the most frequently reactive with the highest intensity. RIBA II assay confirmed a high percentage of the reactive results with the ELISA c-100-3, serum samples that were non reactive to RIBA showed a low optical density with ELISA. c22-3 was the most sensitive antigenic component. PMID- 1726914 TI - [Helicobacter pylori]. PMID- 1726915 TI - [Ultrastructural study of the antral mucosa to determine the presence of Helicobacter pylori and its association with chronic active gastritis]. AB - We present the results of ultrastructural studies of gastric mucosa obtained through upper digestive endoscopy of eight patients with suspicious symptoms of non ulcer dyspepsia or peptic ulcer. Two were male and six female with a median age of 54 years. The urease test to determine the presence of HP and Hematoxylin Eosin and Warthin-Starry staining techniques were practiced with the purpose of a better detection of bacteria and gastritis. We did not find any correlation between the endoscopic results and the presence of gastritis or HP. Of the 8 patients only one had negative results for HP and for Electron microscopy studies. Chronic active gastritis was seen with light microscopy in all of the cases. Three in this group presented mild focal dysplasia and one case intestinal metaplasia type IIa. The main ultrastructural findings were: a) diminished or absent microvilli underneath the bacteria; b) HP inside phagolysosomes in the cytoplasm of the epithelial cells; c) in some cases, the bacteria was attached to the cell membrane; d) the cellular wall of HP contains mucopolysaccharides and up to four polar flagella; e) there are polymorphonuclear leucocytes in the epithelium. We conclude that Electron Microscopy is not a routine method for studying HP, but it constitutes a good method to study pathogenicity of the bacteria. PMID- 1726916 TI - [Detection of salmonella in food products]. AB - The Rappaport-Vassiliadis (RV) medium prepared under laboratory conditions from its different components was found to be equally suitable as the commercial RV Oxoid medium for routine detection of Salmonella in food products. The best detection (30 of 113 examined samples) was obtained using 0.1 ml of culture and 10 ml of medium. In case of 1 ml of culture and 10 ml of medium Salmonella was isolated only from 3 samples. Only 7 positive samples were obtained using MK medium. Necessity of preliminary toxicity verification for malachite green oxalate colour used in RV medium, to standard Salmonella strains, prior to routine food products tests, was found. PMID- 1726917 TI - Revascularized jejunal grafts in oral reconstruction. AB - Since 1983 we have used the vascularized small bowel graft for oral reconstruction after resection of extensive carcinomas at different locations within the oral cavity and oropharynx. Our experience with this technique in 27 cases over a period of six years is discussed with particular reference to the specific problems encountered. The future place of the free jejunum as an ideal tissue in oral reconstructions restoring speech and deglutition will become apparent. PMID- 1726918 TI - The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria. PMID- 1726919 TI - [Experimental antitriatomic program in Santiago]. AB - The rural migration to urban centers occurred since the 40s in many Latin American countries, including Santiago the capital city of Chile, originated a growing belt of premises built with light poor material (the rests of previous rural habitations, mud, pieces of timber, plastic and cardboard for walls, and cane stalks and artificial clinkstones for roofs) giving raise to many types of slums. This situation facilitated the passive transport of the different instars, including eggs, of triatomine bugs. Due to the fact that in the 1959-1960 warm seasons, the Santiago province health institutions had received an increasing reported number of triatomine bugs (Triatoma infestans) in dwellings from different periurban, even urban and rural sections of the province, the central local health authorities with the advise of the University of Chile, Department of Parasitology decided to carry out an experimental program against these vectors of Chagas' disease. The program consisted basically in an spray and thorough application of liquid forms (emulsion, suspension, solution) of 1% lindane (average > or = 500 mg per 1 m2), depending on the material of the constructions, to all the surfaces of walls, ceilings, attics and peridomiciliary structures of all the infested dwellings in a sector and those located less than 100 m around. In order to reach triatomine bugs not affected, for different reasons, in the first spraying, a second application, identical to the first was performed to the total number of premises between 30 and 120 days later. Periodical evaluations were made, and positive dwellings found and neighboring ones were sprayed again. During insecticide applications adequate protection measures for spraying workers, inhabitants, domestic animals, household goods and food were adopted. All the steps of the program were accompanied by health education activities directed to individuals, families, school teachers and community institutions, tending to motivate the people to an active participation, as in the report of the presence or reappearing of triatomines in premises as in cooperating in the sprayings and improving the material conditions of their properties. Thus, in the period 1960-1972 the following goals were achieved: 1) Spraying twice with 1% lindane 32,708 dwellings located in 199 quarters from 26 periurban and rural sections. 2) Protection to 191,090 people against T. infestans bites and the eventual acquired chagasic infection. 3) Percentages of triatomines infested dwellings decreased from to 18.7 to 3.0 according to residents and from 3.0 to 0.3 according insects collection. At present, a significant proportion of the sprayed dwellings has been demolished and replaced by new constructions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1726920 TI - Histological preparation of the Echinococcus sp. tapeworms: staining affinity of the adult form. AB - Thirty specimens of Echinococcus sp. obtained by anti-helminthic treatment using arecoline hydrobromide from a dog from an Echinococcosis endemic region were fixed in 10% formalin. The material was processed histologically by a rapid manual method and stained using eleven different techniques. Green Trichrome staining as modified in the present study permitted the observation of large structures of Echinococcus sp. tapeworms. PMID- 1726921 TI - Activity of a platelet protein kinase that phosphorylates fibrinogen and histone in Argentine hemorrhagic fever. AB - The activity of a platelet protein kinase that phosphorylates the alpha-chain of fibrinogen and the exogenous substrate histone was evaluated in 28 patients with Argentine hemorrhagic fever, grouped into: 13 mild, 6 moderate and 9 severe clinical forms. Blood samples were obtained before treatment with immune plasma, 4 days later and at recovery. Exogenous histone and fibrinogen phosphorylation were assayed with 25 Ci/mmol (gamma-32P)-ATP. Platelet counts and interferon (IFN) activity were performed simultaneously. Histone phosphorylation was found below normal in all patients during the acute phase of illness. This reduction was coincident with the lowest platelet count and the highest IFN titers. Fibrinogen phosphorylation was similarly reduced. Histone and fibrinogen phosphorylation were still low after 4 days of treatment, when IFN levels were almost undetectable. The low level of phosphorylation was not simply due to the reduced number of platelets and may be another evidence of a platelet abnormality in patients with Argentine hemorrhagic fever. PMID- 1726922 TI - The effect of aprotinin (trasylol) on postoperative bleeding in cyanotic congenital heart disease. AB - Despite the widespread clinical success in open-heart surgery, bleeding after cardiopulmonary by-pass (CPB) has been a common problem especially in cyanotic congenital heart disease. Recently, there have been reports demonstrating that treatment with high doses of aprotinin reduces postoperative bleeding. We studied the effect of aprotinin on postoperative bleeding in patients with tetralogy of Fallot who had undergone total correction in the Department of Thoracic and Cardiovascular Surgery of the Hacettepe University Faculty of Medicine, and compared our results with those in the literature. Ten patients out of 20 in the study were given high doses of aprotinin and were compared with the remaining 10 patients who had not received the drug. Standard anesthesia, perfusion and surgical techniques were used in all operations. The total amount of bleeding in the aprotinin-treated group was found to be 1530 ml, while in the other group it was 4185 ml (p < 0.05). The total quantity of blood transfused in the aprotinin treated patients was 3250 ml while it was 5865 ml in the control group (p < 0.05). No significant effect of aprotinin was found on Hb, Hct, PT, aPTT and thrombocyte counts (p > 0.05). However, the effect of the drug on bleeding and coagulation time was found to be statistically significant (p < 0.05). PMID- 1726923 TI - TSH, IGF-1 and activated ras protein induce DNA synthesis in cultured thyroid cells. AB - TSH, IGF-1 and cellular ras genes have been proposed to function in thyroid cell transformation. Using cultured follicular cells, we demonstrate that TSH, IGF-1 and microinjected activated ras protein individually stimulate DNA synthesis. TSH stimulated pathways include Gs at the plasma membrane and cyclic AMP response elements in the nucleus. The pathways and nuclear targets of IGF-1 and ras action appear at least partially distinct from those used by TSH. PMID- 1726924 TI - Various facets of the intercellular heterogeneity in thyroid primary culture. AB - The mechanisms that generate the intercellular heterogeneity of functional and proliferation responses in a tissue are generally unknown. In thyroid gland, this heterogeneity is peculiarly marked and it was suggested that it could result from the coexistence of (epi)genetically different subpopulations of thyrocytes. As summarized in this short review, qualitative or quantitative intercellular heterogeneities have been found at each levels of our "in situ" investigations of morphological, differentiation and proliferative responses of unselected dog thyrocytes in primary culture. These different heterogeneities are unrelated at the individual cell level, which does not indicate the coexistence in thyroid of cell subpopulations stably expressing special properties. Nevertheless, using a double labeling methodology that allows to trace the proliferative behavior of some cells, we show how cell division may stably affect further proliferation responses and how a local synchrony of the dividing cells can result in a stable heterogeneity with a regional, patchy pattern, which resembles the one characterizing multinodular goiter. PMID- 1726925 TI - Thyroid cell lines in research on goitrogenesis. AB - Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID- 1726926 TI - Thyroid autocrine factors: regulation of secretion of insulin-like growth factor binding proteins from sheep thyroid cells by autocrine basic fibroblast growth factor. AB - The mRNA for basic fibroblast growth factor has been detected in primary cultures of sheep thyroid cells. RNA species of 2.0 kb, 3.3 kb and 7.2 kb have been identified. The effects of this autocrine factor on the production of insulin like growth factor binding proteins (IGFBPs) have been examined. bFGF enhanced the secretion of IGFBP-2 and, like epidermal growth factor (EGF) and the tumour promoting phorbol ester (TPA), induced the appearance of IGFBP-3. We conclude that bFGF is synthesized by sheep thyroid cells and has autocrine potential since it can regulate the secretion of other thyroid autocrine factors which, in turn, may regulate thyroid growth and function. PMID- 1726927 TI - Reconstruction of the thyroid follicle with long-term cultured cells. AB - The reconstruction of the thyroid follicle was studied in vitro using the porcine thyroid cell line ATHOS. When performed monolayers of ATHOS cells grown after more than 50 passages are cultured in a sandwich of collagen in the presence of thyrotropin (10 mU/ml), figures resembling follicles are observed under light microscopy. By electron microscopy cells appear correctly polarized, microvilli facing follicle lumina. These histiotypical figures that mimic the thyroid architecture are maintained 4-5 weeks. PMID- 1726928 TI - The molecular biology of the human anaplastic thyroid carcinoma cell. AB - In the present investigation we show data from our studies of anaplastic human thyroid carcinoma cell lines. The cell lines employed in the study were HTh 7, HTh 74, C 643 and SW 1736, all derived from tumours diagnosed as anaplastic thyroid carcinomas. Northern blot analysis with four different thyroid specific cDNA probes showed a varying pattern of expression. Thyroglobulin mRNA was found in three of the carcinoma cell lines, although the signal was very weak compared to the expression in tissue from a toxic goitre, used as positive control. Interestingly, two of the cell lines expressed the receptor for thyrotropin, but none of them contained thyroperoxidase mRNA. Three of the cell lines expressed mRNA for receptors platelet-derived growth factor, PDGFR-alpha and/or PDGFR-beta type. Messenger RNA of a thyroid specific transcription factor, TTF-1, known to regulate the normal function of thyrocytes, was found in the toxic goitre but not in the anaplastic thyroid carcinoma cell lines. Lack of expression of TTF-1 might the immediate cause of the anaplastic phenotype, considering the possibility that TTF-1 functions as a master regulatory gene in thyroid cell differentiation. PMID- 1726929 TI - Epidermal growth factor stimulates thyroid follicle neogenesis in collagen gel culture. AB - The mechanism involved in the neoformation of thyroid follicles is poorly understood. In the present study, whole porcine thyroid follicles were cultured as "miniorgans", embedded within collagen gels. Incubation was performed up to 4 days with or without EGF (10 ng/ml) and TSH (2 mU/ml). A single dose of 3H thymidine was added at the start of the experiments in some cases. Light microscopy and autoradiography was performed. EGF induced a dramatic migration of follicles cells; these were seen to back out from the mother follicle and formed microfollicles with normal polarity including microvilli at the apical border. As evident from the analysis of 3H-thymidine incorporation, most microfollicles were comprised of newly divided cells. The results infer a new mechanism for the neoformation of follicles in the thyroid. PMID- 1726931 TI - Proceedings of the International Symposium: Goitrogenesis. Munich, December 5-6, 1991. PMID- 1726930 TI - Increased cellular growth of FRTL-5 thyroid cells mediated by discontinuous stimulation with bTSH. AB - Previous reports showed pulsatile secretion of TSH in man. Therefore, we investigated the effect of discontinuous versus continuous TSH stimulation on the cellular level of FRTL-5 thyroid cells, namely the expression of a nuclear proliferation antigen (NPAg). The expression of this antigen correlates linearly with the 3H-thymidine incorporation and is a marker for cellular growth. The FRTL 5 cells were stimulated for 4 hours with bTSH (0.01-1.0 U/l). Compared to continuously stimulated cell cultures the discontinuous stimulation of FRTL-5 cells with bTSH showed a significant higher rate of NPAg expression, i.e. cellular growth. PMID- 1726932 TI - The TSH cyclic AMP cascade in the control of thyroid cell proliferation: the story of a concept. AB - Thyrotropin stimulates the growth and proliferation of thyroid cells in vivo. Starting in the 1970, we have progressively shown that these effects could be reproduced in vitro in dog thyroid cells in primary culture. They are accompanied by the expression of differentiation. All the effects of thyrotropin are mediated by the cyclic AMP cascade. The best argument that this concept applies in vivo is the generation of hyperfunctioning adenoma involving the whole gland in transgenic mice expressing the constitutively active adenosine A2 receptor in the thyroid. PMID- 1726933 TI - Switch to the angiogenic phenotype during tumorigenesis. AB - Tumor growth and metastasis are angiogenesis-dependent. Virtually all solid tumors are neovascularized by the time they are detected. However, there is a prevascular phase during early tumor development where few or no tumor cells are angiogenic and expansion of the tumor is restricted to a few mm3. When enough tumor cells become angiogenic, the tumor can expand progressively and shed metastatic cells. This angiogenic switch has recently been quantitated for human breast cancer, as well as for prostate cancer. We have studied the problem of how tumors switch to the angiogenic phenotype by using transgenic mice in which tumors develop at a predictable time and in discrete prevascular and vascular stages. When the transgene is the bovine papilloma virus (BPV) genome, angiogenic fibrosarcomas develop from non-angiogenic precursors called fibromatoses. The fibrosarcomas secrete growth factors for capillary endothelial cells. In contrast, the fibromatoses do not secrete endothelial cell growth factors. When the transgene consists of the large "T" antigen of SV40 under the control of the rat insulin promoter, 70% of pancreatic islets become hyperplastic and 4-10% of these become angiogenic at 6-7 weeks. Tumors arise from these neovascularized hyperplastic islets and reach > 1000 x the volume of the preangiogenic islets. The onset of angiogenic activity coincides with the secretion of acidic fibroblast growth factor (aFGF) and other growth factors not fully identified at this writing. These studies help to explain the switch to the angiogenic phenotype during tumorigenesis and provide models to discover antiangiogenic therapies directed at the source of angiogenic activity. Such therapy, when developed, may be co-administered with currently available angiogenesis inhibitors which are directed at the target of angiogenic activity, vascular endothelial cells. PMID- 1726934 TI - Pathology and molecular mechanisms of multistage human hepatocarcinogenesis. AB - Recent fundamental research has disclosed the presence of multiple genetic alterations including activation of oncogenes and inactivation of tumor suppressor genes in various human cancers. These multiple genetic alterations are thought to be correlated with multiple stages of carcinogenesis and further progression. Hepatocellular carcinoma (HCC) is a typical example. The majority of HCCs are associated with infection by hepatitis virus B or C. In the damaged liver, small nodular lesions develop due to clonal expansion of hepatocytes. Some of these nodules are diagnosed as early HCC of the well differentiated type and correspond to in situ or microinvasive carcinoma. Within these nodules, moderately or poorly differentiated HCCs often emerge as nodule-in-nodule lesions when the diameter of the nodules exceeds 1.5 cm. Ordinary HCCs formed by progression show highly increased cell proliferation, neovascularization, production of high-molecular-mass forms of basic fibroblast growth factor and aneuploidy in some tumors. Corresponding to this stage of malignant progression, HCCs show loss of heterozygosity for multiple chromosomes including chromosomes 4, 16q and 17p. Tumor suppressor gene p53, located on 17p, is frequently mutated in high-grade, but not in early, HCCs. Thus, it is strongly suggested that inactivation of multiple tumor suppressor genes plays an important role in progression, and probably directly or indirectly causes chromosome instability, enhanced cell proliferation and neovascularization. PMID- 1726935 TI - Exposure to nitrogen oxides and other air pollutants from automobiles. AB - We have conducted an epidemiological study to investigate the association between exposure to automobile exhaust and respiratory health. Three zones were selected in Tokyo according to expected exposure levels. Zone A is within 20 meters from the roadside of major roads with heavy traffic. Zone B is 20-150 meters away from the roadside of the same road. Zone C is the residential district in a suburb of Tokyo. Zone A, which was nearest to the roads, had the highest mean levels of personal exposure to nitrogen dioxide for housewives, followed by Zone B and Zone C. Ambient levels of nitrogen oxides, and mass concentrations, polycyclic aromatic hydrocarbons and mutagenicity of suspended particulate matter were higher at the roadside within Zone A. A cross-sectional study of respiratory symptoms and repeated examinations of pulmonary function were also performed in each zone. These results suggest that exposure to automobile exhaust may be associated with respiratory symptoms. Nevertheless, pulmonary function didn't show consistent differences over all examinations. We should have further analyses about a decline of pulmonary function with age. PMID- 1726936 TI - Antigen presentation and the association of class-I molecules. AB - We have identified two mutant cell lines which are not able to present epitopes of influenza virus synthesized in the cytoplasm but can present the same epitope when exposed to it as a peptide in the extracellular medium. The cell lines also have a defect in class-I assembly, with reduced expression of assembled alpha chain: beta 2M heterodimers at their cell surface. This led to the suggestion that the two traits were the result of the same mutation and that stable assembly of class-I molecules is dependent on peptide binding. Consistent with this idea was the finding that exposure to specific peptides in the extracellular fluid promotes stable association of class-I heavy chains with beta 2M and restores expression of class-I at the cell surface. We have gone on to show that stable assembly of class-I molecules can be supported in detergent extracts of the mutant cells when specific peptides are added. Peptides stabilized a conformational change in the class-I heavy chain and association with beta 2M by binding to the complexes. This effect is apparent at peptide concentrations around 100-fold lower than required in "peptide feeding" experiments with whole cells. We have also demonstrated that the conformational change induced in heavy chain is influenced by the concentration of beta 2M, and consequently have been able to demonstrate the formation of empty class-I molecules. PMID- 1726937 TI - Neuropeptides in atrial subepicardial ganglia of rats. AB - The distribution of several neuropeptides in subepicardial atrial ganglia was examined in the rat heart by using immunohistochemical techniques. Some of the ganglion cells in the right atrium exhibited neuropeptide Y-immunoreactivity while no immunostaining was found with antisera against atrial natriuretic polypeptide (ANF), substance P, vasoactive intestinal polypeptide (VIP), and calcitonin gene-related peptide (CGRP). Nerve fibers in and around the ganglia showed neuropeptide Y, calcitonin gene-related peptide and vasoactive intestinal polypeptide immunoreactivities. Some of these immunoreactive fibers (substance P, calcitonin gene-related peptide, neuropeptide Y) formed relatively dense networks around blood vessels and capillaries. Atrial natriuretic polypeptide-like immunoreactivity which was present in atrial myocytes failed to show up in any neuronal elements in the rat heart. PMID- 1726938 TI - Thymic dendritic cells and B cells: isolation and function. AB - The thymus is the primary organ in which T cells undergo rearrangement of T cell receptor alpha and beta genes, positive selection for affinity to self MHC products, and elimination (negative selection) of reactivity to self antigens. These events require an interaction of the developing T cell with other cell types in the thymus. The latter include epithelial cells, macrophages, dendritic cells, and the recently described thymic B cells the majority of which are CD5+. Here we review the identification and isolation of thymic dendritic cells and CD5+ B cells. We consider phenotype, ontogeny, and function, including possible contributions to the induction of self tolerance. Thymic dendritic cells are similar to spleen dendritic cells, but are larger and exhibit a few differences in phenotype. Dendritic cells from both organs are equally potent accessory cells for the MLR and lectin-induced, T cell proliferation. Thymic dendritic cells have higher levels of Fc receptors and support anti-CD3 dependent mitogenesis. Thymic CD5+ B cells share phenotypic features with peritoneal CD5+ B cells. However thymic B cells neither proliferate nor form antibody producing cells in response to the stimulation with LPS or anti-IgM plus IL-4, but do respond to stimulation with MHC class II-restricted helper T cells. Thymic dendritic cells and CD5+ B cells both appear at a similar time in ontogeny, about 14 d of gestation, which is the time T cell differentiation begins to take place. Dendritic cells from spleen, which are potent activators for peripheral T cells, are also potent inactivators for thymic-derived cytotoxic T cells. A correlation between reactivity to MIs products and the expression of TCR-V beta genes is well documented, and B cells are the primary APC for this antigen. Therefore, thymic CD5+ B cells may be a good tool for the investigation of tolerance to M1s products. PMID- 1726939 TI - [The development of transurethral resection in prostatic hypertrophy]. AB - Some hitherto unknown facts on the development of transurethral prostate resection in a historical aspect are reported. Creation of the modern resectoscope from the first punch-instrument till nowadays is followed up in detail, quoting original sources from the literature. Each stage of development of transurethral resection is briefly discussed. PMID- 1726940 TI - Human bcr-abl gene has a lethal effect on embryogenesis. AB - The chimaeric bcr-abl oncogene is thought to have a crucial role in the development or maintenance of chronic myelogenous leukaemia. To study this oncogene in a more direct way, the bcr-abl gene encoding the P210 protein under control of the bcr gene promoter was introduced into fertilized one-cell embryos, which were then re-implanted into foster mothers. Our data, obtained after several experiments, demonstrate that no live transgenic progeny could be obtained using this bcr-abl construct. The bcr gene is expressed in the course of embryogenesis and the bcr-abl gene product appears to have a pleiotropic lethal effect during this period of development. In concordance, several gross abnormalities were observed while no evidence of neoplastic formation was found. These results suggest that the bcr-abl encoded protein severely affects the process of normal embryogenesis. PMID- 1726941 TI - Ossification of the N-methyl-N'-nitro-N-nitrosoguanidine-induced small intestine adenocarcinomas in rats. AB - Eighty rats out of 233 developed malignant tumors in the stomach and small intestine by administration of 100 micrograms/ml N-methyl-N'-nitro-N nitrosoguanidine (MNNG) in drinking water for 28 weeks. Fifteen lesions (30%) among the 50 small intestinal carcinomas showed ossification in the tumor, while none in the sarcomas (12 lesions) or gastric adenocarcinomas (59 lesions) showed ossification. Multifocal heterotopic bone formation was found within stroma in close approximation to the neoplastic glands. The islands of bone trabeculae were covered by osteoblast-like cells, and abundant fibroblasts in loose stroma gathered around the bony islands which enclosed osteocytes in lacunae. Neither osteoclast nor cartilage was identified. In 5 cases, ossification was extensive, which comprised the major portion of the stroma. In contrast, intraluminal calcification without ossified foci were occasionally seen in the gastric carcinoma. Ossification of the intestinal tumors correlated to the degree of mucin content (p < 0.05, chi square with Yates' correction), degree of neutrophilic infiltration (p < 0.05), and size of the tumor (p < 0.1). (The average size of the ossified tumor was 21.5 +/- 4.0 mm, while that of nonossified tumors was 12.5 +/- 1.9 mm). The degree of tumoral necrosis, desmoplasia or depth of invasion did not seem to be related to the ossification of the tumor. The ossification rate of this experimental model was much higher than in human cases. Various histologic alterations, such as mucin leakage, inflammatory cell infiltration, necrosis and/or fibrosis, which might be caused by continuous stimulation of the strong carcinogen, may play some role in the ossification of experimental tumors. PMID- 1726942 TI - [Prostatic specific antigen (PSA). Interpretation of results as a function of the assay method]. AB - The authors compare the two PSA assay methods most widely used in France. The first method (RIA Baxter) uses an isotope marker (Iodine 125), the other (EIA Biotrol) uses an enzymatic marker (alkaline phosphatase). PSA was assayed by means of these two techniques in 2 groups of patients: one group of 49 men considered to be free of any prostatic disease, recruited from blood donors; another group of 87 male patients in whom a PSA assay was performed prospectively at the first urology outpatients visit. The two PSA assay techniques gave different results, but the values obtained by these two methods were not discordant. It is therefore possible to define a coefficient of proportionality of 1.47 regardless of the PSA concentration or the urological disease considered (EIA Biotrol x 1.47 = RIA Baxter). PMID- 1726943 TI - [Circulating prostatic-specific antigen in benign hypertrophy and localized prostate cancer: can PSA be considered a screening examination for localized cancer?]. AB - Prostate-specific antigen (PSA) is increasingly used in the diagnosis of prostatic pathology. Its usefulness in the early diagnosis of prostatic cancer is controversial. The aim of the study is to evaluate the sensitivity and specificity of PSA in a population with prostate diseases. Moreover, we wanted to know if the measure of the prostate volume may increase the sensitivity of the test. In benign prostatic hypertrophy (88 patients), a good correlation exists between circulating PSA and the prostatic volume or the volume of the adenoma. This correlation disappears in the presence of an adenocarcinoma at the profit of the tumor volume (46 patients). Used as a means of screening for cancer, the serum level of PSA with a threshold value of 2.5 ng/ml has a sensitivity of 91% and a specificity of 32%. The sensitivity is 50% and the specificity is 85% at a level of 15 ng/ml. Taken alone, the level of PSA is inadequate for diagnosis: If the lower level is chosen (2.5 ng/ml), the majority of benign prostatic hypertrophies will be the object of a biopsy. If the higher level is chosen (15 23 ng/ml), 50% of localized cancers of the prostate will escape detection. Nevertheless, a level of PSA < 15 ng/ml is an argument for a strong suspicion in favor of an adenocarcinoma of the prostate. The capacity of BPH to "secrete" serum PSA is five times greater than that of the normal peripheral prostate, and the capacity of cancer is 20 times greater than that of an adenoma. The individual variability of serum PSA per cubic centimeter of prostatic tissue is too great to allow a precise interpretation as a function of volume. PMID- 1726944 TI - [Prostatic cancer before the age of 50--report of 7 cases]. AB - Based on a retrospective series of 7 cases, the authors study the particular features of prostatic cancer before the age of 50 years. These 7 patients all had an advanced tumour with, in 4 cases, documented metastases. A single patient had a well differentiated tumour, while the others had a moderately or poorly differentiated or undifferentiated tumour. All patients died rapidly, except one who is currently receiving treatment with an LHRH agonist, with a follow-up of 25 months. On the basis of this study, the authors distinguish between prostatic cancers clinically detectable before the age of 50 years, and those which remain occult, leading to a histological discussion between a benign disease and true early cancer. In the light of these findings, systematic screening for prostatic cancer before the age of 50 years does not appear to be justified. PMID- 1726945 TI - [Major urological cancer excisions after the age of 75]. AB - 32 patients more than 75 years old had major surgical procedures for urologic neoplasms: radical nephrectomy for renal cell cancer (9 cases), nephroureterectomy for upper urinary tract tumor (3 cases), radical cystectomy for invasive bladder cancer (20 cases). Postoperative mortality was 12.5%. In the nephrectomy group, 3 palliative procedures gave a mean survival time of 7 months. On 9 curative procedures, 7 patients are alive and free of disease with a mean follow-up of 45.6 months. In the cystectomy group, 5 palliative procedures gave a mean survival time of 7 months. On 15 curative procedures, 6 patients are alive and free of disease with a mean follow-up of 18.6 months. Our data confirm that curative procedures can be performed in the elderly. Mean survival time and quality of life after palliative procedures suggest that only true comfort procedures have to be performed. PMID- 1726946 TI - World Health Organization Consensus Committee recommendations concerning the diagnosis of BPH. AB - The expert committees, which met in Paris in June 1991 under the patronage of WHO to establish a consensus concerning BPH, adopted the recommendations summarised in this report. Despite certain criticisms which can be made, these recommendations offer the advantage of simplicity and uniformity, thereby constituting a "universal language". This will facilitate comparison of patients and therapeutic results, both in everyday practice and in the course of clinical trials. These recommendations will be periodically re-evaluated in the light of clinical experience and technological progress. PMID- 1726947 TI - [Etiologic diagnosis of pneumonia in adults: usefulness of bronchoalveolar lavage]. AB - Quantitative evaluation of bacteria in bronchoalveolar lavage fluid from 15 patients with pneumonia was compared to that in 29 healthy controls. Aerobic and anaerobic cultures were used as well as cultures and staining for fungi and mycobacteria. A total of 1000 colony forming units per ml was considered a cutoff mark between colonization and infection. A positive result was obtained in 13 of 15 patients with pneumonia, allowing the identification of the causing agent. Counts below the indicated level were observed in 23 of 29 controls. There was no morbidity associated to the procedure. Thus, an 85% sensitivity and 87% specificity for bronchoalveolar lavage in the etiologic diagnosis of pneumonia may be estimated from this study. PMID- 1726948 TI - Various signal molecules modulate voltage-activated ion currents on snail neurons. AB - The modulatory effects of interleukin-1 (IL-1), an immunotransmitter, and FMRFamide, a molluscan neuropeptide, were studied on identified neurons of Helix pomatia L. (Mollusca, Gastropoda) by using the method of two microelectrode voltage clamp. IL-1 and FMRFamide uniformly decreased the voltage-activated inward current (ICa), while the voltage-dependent outward potassium current (Inet K) increased. IL-1 and FMRFamide were shown to use the same cellular targets as used by the low molecular weight neurotransmitters. PMID- 1726949 TI - Assessment of naming failures in neurological communication disorders. PMID- 1726950 TI - Human CYP1A1 (cytochrome P(1)450) gene: lack of association between the Msp I restriction fragment length polymorphism and incidence of lung cancer in a Norwegian population. AB - In this study of 221 lung cancer patients and 212 controls, no association between a Msp I polymorphism in the CYP1A1 gene and an increased risk of lung cancer was found. Histological type, smoking habits and family history were also examined. No associations between the Msp I restriction fragment length polymorphism in the CYP1A1 gene and any of these parameters were found. These results are in contrast to a previous report by a Japanese group (Kawajiri et al., 1990) who found an association between the less common allele and an increased susceptibility to lung cancer in their population. The frequency of the less common Msp I 1.9 kb fragment allele (C2) appears to be three times greater in the Japanese population than in the Norwegian population and a Caucasian population of North America. It is possible that in the Asian population this Msp I polymorphism is in linkage disequilibrium with another mutation important for CYP1A1 gene expression, whereas in the Caucasian population these mutations are in equilibrium. PMID- 1726951 TI - Understanding biological variability in susceptibility to respiratory disease. PMID- 1726952 TI - Lethality of triatomines (Hemiptera: Reduviidae), vectors of Chagas' disease, feeding on blood baits containing synthetic insecticides, under laboratory conditions. AB - A laboratory study was conducted to test the toxicity of synthetic insecticides added to defibrinated sheep blood kept at room temperature and offered as food to the following triatomine species: Triatoma infestans, Panstrongylus megistus, Triatoma vitticeps, Triatoma pseudomaculata, Triatoma brasiliensis and Rhodnius prolixus. The insecticides used, at a concentration of 1 g/l, were: HCH, DDT, Malathion and Trichlorfon, and the lethalithy observed at the end of a 7-day period varied according to the active principle of each. HCH was the most effective by the oral route, killing 100% of the insects, except P. megistus (95.7%) and T. pseudomaculata (94.1%). Trichlorfon killed the insects at rates ranging from 71.8% (T. vitticeps) to 98% (R. prolixus). Malathion was slightly less efficient, killing the insects at rates from 56.8% (T. vitticeps) to 97% (T. brasiliensis). DDT was the least effective, with a killing rate of 10% (T. vitticeps) to 75% (T. brasiliensis). Since the tests were performed at room temperature, we suggest that baits of this type should be tried for the control of triatomines in the field. PMID- 1726953 TI - HIV-1 neutralization directed to epitopes other than linear V3 determinants. PMID- 1726954 TI - HIV epitopes recognized by cytotoxic T-lymphocytes. PMID- 1726955 TI - Antiretroviral treatment: state of the art and future directions. PMID- 1726956 TI - Antiretroviral drug resistance. PMID- 1726957 TI - [Sea-blue histiocyte syndrome]. AB - The sea-blue histiocyte syndrome, similar to Niemann-Pick disease, is a congenital, hereditary histiolipidosis due to an inborn enzymatic error. Accumulation of non saturated, oxidated, polymerized lipids is observed; ceroids of lipofuscin, glycophospholipids and sphingomyelin, like bulky granules 1 to 3 u in diameter, turn blue with May Grunwald staining, orange reddish with PAS and black with Sudan III and osmic acid. The sea-blue histiocytes are preferably located at the bone marrow, liver and spleen and less frequently in lymph nodes, lungs and some other organs. The prognosis is variable: fatal in the central nervous system location, relatively mild in cases of spleen and bone marrow location. The possibility of complicating hepatic cirrhosis and/or pulmonary fibrosis is always present. Seven cases are described in this paper, 4 of them family related. Acute myelomonocytic leukemia in one case and histioimmunoblastic lymphoma in another were complications not yet reported in the literature. PMID- 1726958 TI - [Malignant lymphomas. Preliminary report of the National Protocol of Antineoplastic Drugs]. AB - The first results of the management of adult non-Hodgkin lymphoma according to the National Protocol are reported. 164 pts (71 female and 93 male), aged 50 (range 15 to 79 years) were included. 89 pts had lymphomas with intermediate, 34 with low and 24 with high grade malignancy. 65% had advanced stages (III and IV). In the low grade malignancy group 38% were in complete remission and actuarial survival at 20 months was 80%. In the intermediate grade group, stages I and II the corresponding values were 79 and 85%; in the intermediate grade stages III and IV, 58 and 69%, and in the high grade group 61 and 77%. Bone marrow toxicity and infections were the main complications. 32 pts have died, 10 of them with drug toxicity complications. PMID- 1726959 TI - Palliative care in advanced HIV disease: presentation, problems and palliation. PMID- 1726960 TI - HIV vaccine development: lessons learned to date. PMID- 1726961 TI - Progress toward an artificial vaccine for HIV: identification of helper and cytotoxic T-cell epitopes and methods of immunization. AB - Design of a synthetic vaccine for HIV requires basic knowledge of the structure of helper and cytotoxic T-cell epitopes and neutralizing antibody epitopes, of ways to couple these to produce an effective immunogen, and of the role of viral sequence variation on MHC presentation of antigen. T-cell recognition, and cross reactivity. We have been addressing all these issues for the HIV envelope and more recently also for the reverse transcriptase. We have now identified antigenic sites or epitopes from HIV envelope and reverse transcriptase recognized by cytotoxic T cells from both mice and humans in association with murine class I H-2 and human class I HLA antigens, as well as epitopes recognized by helper T cells in association with class II MHC molecules from both mice and humans. We have identified residues affecting interaction of peptides with MHC molecules and T-cell receptors and have examined the role of viral variability on presentation of these peptides by MHC molecules and recognition by T cells. One CTL epitope peptide was found to be presented by class II MHC molecules as well as class I MHC molecules and to be able to elicit CD4+ helper cells to aid in the induction of CD8+ CTL against the same peptide. One of the helper epitope peptides has been shown to be a powerful carrier for inducing neutralizing antibodies, and we have shown in rhesus monkeys that some of these helper peptides are immunogenic in primates and can elicit helper T cells that greatly augment the antibody response to a challenge in vivo with a suboptimal dose of HIV envelope protein compared to monkeys not given peptides, as one would want a vaccine to do. We have also identified multideterminant regions of the HIV-1 envelope and have made peptides corresponding to these that elicit helper T-cell responses in a large fraction of mouse strains and of outbred humans, as an approach to overcoming the problem of genetic restriction of T-cell responses. We have also developed a way of using purified recombinant proteins to elicit cytotoxic T cells in vivo by immunizing with the proteins incorporated into ISCOMs, and this method could be applied to an artificial vaccine as well. Some of these peptides should be candidates for immunotherapy trials in HIV-infected humans, as well as for vaccine development and diagnostic use. PMID- 1726962 TI - Identification of HIV-1 cytotoxic T-lymphocyte epitopes and implications for vaccine development. PMID- 1726964 TI - Protection of mammalian cells from complement-mediated lysis by transfection of human membrane cofactor protein and decay-accelerating factor. PMID- 1726963 TI - Importance of conformation on the neutralizing antibody response to HIV-1 gp120. AB - We have investigated the role of conformation of HIV-1 gp120 on its potential efficacy as a subunit vaccine. The questions that we set out to answer were: 1) Are there neutralizing antibodies directed to conformational epitopes in gp120? 2) If so, what is the spectrum of virus isolates neutralized by these antibodies? 3) Is a conformationally correct gp120 subunit more effective in the induction of neutralizing antibodies than a denatured subunit? 4) Does native gp120 subunit vaccination induce a broader neutralizing response than a gp120 antigen that cannot display conformational epitopes? To address these questions, we characterized the gp120-specific antibody response of HIV-1-infected humans and of experimental animals immunized with recombinant native and nonnative gp120 subunits. Two versions of recombinant gp120 produced from the HIV-SF2 isolate of HIV-1 were employed in these studies: 1) a nonglycosylated, denatured version produced in genetically engineered yeast, which we presume is capable of presenting only linear determinants, and 2) a fully glycosylated, native version, produced in genetically engineered mammalian cells, that is capable of displaying linear as well as conformational epitopes. Antibodies directed exclusively to conformational epitopes in gp120 were purified from pooled HIV antibody-positive human sera using these two versions of HIV-SF2 gp120. These antibodies exhibited neutralizing activity, and this activity was effective in the neutralization of a different, broader spectrum of HIV-1 isolates than that of antibodies to linear determinants in gp120 purified from the same serum pool. When these two versions of HIV-SF2 gp120 were used as subunit immunogens in baboons, clear differences in their abilities to elicit neutralizing antibodies were observed. The native version was more effective in the induction of neutralizing antibodies effective against HIV-SF2, the homologous virus isolate. The isolate specificity of the neutralizing response to these two versions of HIV-SF2 gp120 also differed. The nonglycosylated version induced neutralizing antibodies that were effective against only the isolate, or closely related isolates, from which the antigen was derived. In contrast, the native version induced a neutralizing response that was effective against a broad panel of HIV-1 isolates, including at least one isolate that one would not expect to be neutralized by antibodies to the PND of HIV-SF2 gp120. PMID- 1726965 TI - Characterization of an early thymocyte cell line which is chronically infected with the human immunodeficiency virus. PMID- 1726966 TI - The daily intravenous administration of acidic fibroblast growth factor induces goiter in rats. PMID- 1726967 TI - Mutagen sensitivity: a biological marker of cancer susceptibility. AB - We intend to continue our exploration of the bleomycin assay as a biological marker for the development of environmentally induced cancers. The impetus for such efforts would be enhanced through effective integration of cancer screening and intervention to achieve diminished cancer mortality. Currently, we are integrating combining bleomycin sensitivity screening to chemopreventive therapy against second primary malignancies in head and neck cancer patients. Previous studies have demonstrated that cis-retinoic acid, the agent used in our chemopreventive trial, is effective in such circumstances (35). Our purpose is to identify a high-risk subpopulation through application of the bleomycin sensitivity assay and then demonstrate that we can modulate the carcinogenic process with cis-retinoic acid. The use of genetic markers clearly enhances the power and precision of epidemiological research. The preventive implications of precise and valid markers for carcinogen sensitivity are obvious. We are aware of the need for extensive validation of the assay and for rigorously designed and conducted epidemiological studies. The strength of the association between cancer risk and mutagen sensitivity, despite the inherent problems in the size and design of the studies, is noteworthy. The thesis that chromosome instability and defective DNA repair may underlie susceptibility to environmental carcinogenesis is plausible and presents a promising avenue for further multidisciplinary research. PMID- 1726969 TI - The use of the Picrosirius-polarization method for the study of the biopathology of collagen. PMID- 1726970 TI - Autoantibodies from ITP patients recognize hydropathically generated epitopes. PMID- 1726971 TI - Histochemical and ultrastructural study on the presence of elastic microfibrils in the myotendinal junction of mouse gastrocnemius. PMID- 1726972 TI - Cutaneous reactions to azacitidine. PMID- 1726973 TI - HIV, HBV and HCV seropositivity in hemophiliacs. AB - Eleven cases of severe type hemophiliacs who had received long-term factor VIII injections were tested for the serological markers of human immunodeficiency virus (HIV), hepatitis B virus and hepatitis C virus (HCV). The period of factor VIII concentrate injections ranged from 2 to 32 years. The seropositive rates of HIV and HCV were 9/11(82%) and 11/11(100%), respectively. The seropositive rate of hepatitis B surface antigen was only 1/11(9%), while the seropositive rates of antibody to hepatitis B core antigen and antibody to hepatitis B surface antigen were 9/11(82%) and 7/11(64%), respectively, Although the patients had no symptoms related to acquired immunodeficiency syndrome, they were noted to have inverted helper/suppressor T-lymphocyte ratio, suggesting that hemophiliacs with long-term factor VIII injections have a high incidence of HIV and HCV infection, with immunological aberration. PMID- 1726974 TI - Measurement of the inflammatory activity by the help of serum acute-phase proteins in juvenile chronic arthritis. AB - We measured the level of serum haptoglobin, transferrin, alfa-I antitrypsin, orosomucoid, beta-2-microglobulin, ferritin in the case of 30 children (aged 11 16 years) with juvenile chronic arthritis. We divided the patients into two groups. In the first group there were 15 patients with active disease under continuous treatment and in clinical remission (We 20 mm/hour). In the second group there were 15 patients without active disease and they were not given continuous treatment for two Years. These groups were studied, by a control one. If we measure more phase-proteins together, they are suitable for the demonstration of the inflammatory activity in juvenile chronic arthritis. We made a points system for the evaluation of activity. PMID- 1726975 TI - [Mutagens and carcinogens in city air]. AB - Many urban air pollutants are recognized to be mutagenic and carcinogenic agents. Combustion processes, which generate gaseous and particulate complex mixtures, are responsible for the major part of urban air pollution. Emissions from automotive sources are estimated to be nowadays the main cause of mutagenic/carcinogenic risk for people living in urban areas of industrialized countries. Increasing vehicle traffic also contributes to the presence in urban air of valuable concentrations of asbestos fibers and benzene, both well-known potent carcinogens. PMID- 1726977 TI - [Microwaves in morphologic examinations: technologic progress or whim?]. AB - Microwave-ovens shorten the time and eliminate or reduce the formaldehyde concentration in the process of tissue-fixation for light- and electron microscopy. These devices can be also applied in dehydration, and in impregnation of tissues with paraffin as well as in staining of paraffin or cryostat cut slices. The use of microwaves improves results of immunohistochemical and ultracytochemical stainings. The best results are given by precise laboratory microwave-devices, but domestic microwave-ovens can be also used in this procedure. PMID- 1726976 TI - Corticotropin-releasing factor (CRF) gene family in the brain of the teleost fish Catostomus commersoni (white sucker): molecular analysis predicts distinct precursors for two CRFs and one urotensin I peptide. AB - Molecular cloning experiments indicate the presence of two distinct CRF genes in the sucker genome encoding independent 162-amino-acid precursors, which both consist of a signal sequence, succeeded by a cryptic peptide and subsequently by the hormone moiety. The two 41-amino-acid CRF peptides differ by an Ala-->Val substitution at amino acid position 28. CRF transcripts are primarily found in the sucker pre-optic nucleus (PON), to a much lesser extent in the lateral tuberal nucleus (LTN). In contrast, urotensin I (U I) encoding mRNA is equally present in both tissues. In urophysectomized fish, U I mRNA is elevated especially in LTN tissue, while CRF mRNA levels remain more or less constant in the PON and LTN regions. PMID- 1726978 TI - [Placental changes in toxicoses in the second half of pregnancy]. AB - An investigation of 88 placentas in toxicosis of the second half of pregnancy has detected symptoms of the immune alteration detailed by means of an electron microscopic analysis and immunomorphological investigations with the help of luminescent sera against immunoglobulins A, M. G and C3 fraction of complement. The alterations found are indicative of the participation of immune mechanisms in the formation of placental insufficiency and their important role in pathogenesis of gestational toxicosis. PMID- 1726979 TI - [The intraorganic lymphatic bed of the diaphragm, liver and kidneys of the dog under conditions of hypokinesia]. AB - In the experiment performed on 49 dogs architectonic peculiarities of the intraorganic lymphatic bed have been investigated under conditions of round-the clock mobility restriction from 1 up to 90 days. Morphological and morphometrical results have demonstrated that rearrangement of the lymphatic bed elements occurs in parallel with certain changes in vessels and tissues of the organs studied (liver, diaphragm, kidneys, lung, pancreas). The course of the changes can be divided into 3 periods: 1) the first 10 days, 2) 11-30 days, 3) from 31 up to 90 days. As demonstrates the analysis during the whole experiment, in the third period (stabilization of the adaptive changes) the reaction of the deep and superficial vessels and capillaries is different in its direction. PMID- 1726980 TI - [The formation of the insulo-acinar portal system in the pancreas of human fetuses]. AB - In human fetuses 12-20-week-old peculiarities in creation and development of the pancreatic hemo-microcirculatory bed have been studied in connection of its incretory part formation. The vascular glomerulus begins to form on the 12th-14th week as transformation of capillaries of the exocrinic parenchyma into the glomerular capillaries, which, on their getting out of the glomeruli, turn into vessels. The latter participate in formation of the insulo-acinar portal system. Certain structures have been revealed for adaptation of an elevated volume of the blood stream in the glomeruli place, where the glomerular microvessels pass into the acinar anastomoses. The latter perform shunting of the glomerular capillaries with the arteriole, which brings blood, enriched in insular hormones, to distantly situating acinar cells. PMID- 1726981 TI - Determinants of alcohol preference in the AA and ANA rat lines selected for differential ethanol intake. AB - A selective breeding program conducted in this laboratory has resulted in the establishment of the alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Nonalcohol) rat lines. These lines have been used as a tool for attempting to identify the behavioral, neurochemical, and biochemical correlates of differential voluntary ethanol consumption. Some of the differences that have been found between the lines involve differential reinforcement: AA rats, but not ANA rats, rapidly acquire an ethanol-reinforced operant response. The AA's greater development of tolerance to the depressant effects of ethanol and their faster ethanol metabolism would also allow them to drink more. Neurochemical studies have suggested differential functioning of brain monoaminergic mechanisms. The activity of tyrosine hydroxylase and dopa decarboxylase, and the brain dopamine concentrations are higher in the AA rats than in the ANA rats, and the maximal number of dopamine D2 receptors is lower in the AA rats. The concentration of noradrenaline is higher in the brain of ANA rats than in that of AA rats, while the 5-hydroxytryptamine levels do not seem to differ greatly. The importance of these differences to the line difference in ethanol intake is not, however, clear, since there appears to be no difference in the sensitivity of monoamine systems of the two lines to ethanol. PMID- 1726982 TI - Alcohol inhibition of NMDA channel function. AB - In mammalian central neurons, intoxicating concentrations of ethanol inhibit the ion current activated by the glutamate agonist N-methyl-D-aspartate (NMDA). Electrophysiologic analysis of the molecular mechanism involved in this inhibition indicates that ethanol does not inhibit NMDA-activated ion current by voltage-dependent block of the channel, altering the ion selectivity of the channel, or altering the affinity of binding sites for NMDA, glycine or substances known to regulate the function of this channel (Mg2+, Zn2+ and ketamine). The potency for inhibiting the NMDA-activated current by different alcohols is linearly related to their hydrophobicity, suggesting that alcohols may inhibit the NMDA-activated current by a novel type of interaction with a hydrophobic region of the channel. PMID- 1726983 TI - Demonstration that ethanol potentiates the GABAA-activated Cl- current in central mammalian neurons. AB - Previous studies have suggested that ethanol potentiates GABA-mediated responses in the mammalian brain. In the present study, using patch-clamp techniques, we show that the GABAA-activated Cl- current was potentiated by ethanol in a subpopulation of mouse hippocampal cells. PMID- 1726984 TI - Electrophysiological approaches to studying ethanol targets. AB - In this paper, we describe two approaches to the study of alcohol action in the nervous system. The first approach involves the use of model systems to elucidate the nature of the interactions between ethanol and membrane elements. In this approach, it is common to use model targets which may not actually prove to be physiologically relevant. We describe data obtained from Aplysia studies and oocyte expression studies which use this approach. A second approach examines the effects of ethanol on elements known to underlie some aspect of behavior altered by the drug, in the hope that the altered function accounts for the behavioral effect. We describe data obtained from isolated peptide-releasing nerve terminals of the rat neurohypophysis, which uses this approach. PMID- 1726985 TI - Are chronic alcohol-induced central vasopressin changes determinant in strain dependency of tolerance in mice? AB - As vasopressin (VP) has been related to tolerance, we were interested in following central VP levels after chronic alcohol exposure of two selected mouse lines (C57Bl and Balb/c). Strongly elevated VP and VP mRNA levels have been noted, in particular in the hypothalamus. The phenomenon is much more marked in Balb/c mice than in C57Bl; in extrahypothalamic areas in the changes in VP noted in septum and amygdala are only apparent in Balb/c mice. Hypothalamic norepinephrine and serotonin, known to partly control VP release, also reacts in a strain dependent manner to alcohol. This study provides neurochemical evidence that long term ethanol intoxication selectively activates central vasopressinergic and aminergic neurons in mice. Such activation appears to be strain dependent; therefore it may be related to the unequal capacities of these strains to adapt to chronic alcohol intoxication. Such phenomena may partly account for differences between individuals in tolerance to chronic alcohol in men. PMID- 1726986 TI - Serotonin and dopamine systems regulating alcohol intake. AB - Neurochemical and neuropharmacological studies were undertaken to assess the involvement of CNS serotonin (5-HT) and dopamine (DA) pathways in regulating the alcohol intake of rats selectively bred for their high alcohol seeking behavior (P and HAD lines). Neurochemical data indicate that high alcohol seeking behavior (when compared with data from rats with low alcohol seeking characteristics) is associated with (a) lower contents of 5-HT in certain limbic regions, e.g., n. accumbens (Acb), frontal cortex, (b) a lower content of DA in the Acb, and (c) higher densities of 5-HT1A receptors in certain limbic regions, e.g., cerebral cortex. Neurochemical data also suggest that ethanol can activate the DA and 5-HT systems projecting to the Acb. Neuropharmacological studies demonstrated that local microinfusion of a 5-HT agonist into the Acb of the P line of rats enhanced ethanol drinking. Intracranial self-administration studies established that P rats will self-administer ethanol directly into the ventral tegmental area (VTA). Overall the data suggest the involvement of certain VTA DA and dorsal raphe nucleus 5-HT pathways in regulating high alcohol drinking behavior. PMID- 1726987 TI - The renin-angiotensin system: a multidimensional source of control over alcohol consumption. AB - While the renin-angiotensin system has traditionally been considered part of a larger homeostatic process that regulates blood pressure and fluid/electrolyte balance, the data summarized in this review suggest a new and different kind of function- that of regulating the behavior of alcohol consumption. Using a wide variety of drug, genetic, dietary, surgical and neurosurgical manipulations all of which share the common property of altering activity in the renin-angiotensin system, the picture emerges of an inverse relationship between activity in the renin-angiotensin system and alcohol consumption. One possible mechanism of this effect may be a process of satiety where levels of the bioactive peptide angiotensin II exceeding a critical level, set behavioral processes into action that lead to a cessation in alcohol intake. PMID- 1726988 TI - [Effect of thromboxane A2 synthetase inhibitor and prostacyclin analogue on arterial blood pressure, fibrinolysis and platelet function in patients with hypertension]. AB - An effect of the specific thromboxane A2 synthetase inhibitor and stable prostacyclin analogue on arterial blood hypertension was investigated in 12 patients with spontaneous hypertension of II degree and in 12 healthy subjects. The patients were given a 3-hour intravenous infusion of Iloprost (Schering) in the dose of 2 ng/kg b.w. per minute and OKY-046 (ONO, Japan) in a single oral dose of 400 mg. Iloprost shortened euglobin fibrinolysis time without an effect on tissue plasminogen activator levels or blood pressure. OKY-046 decreased TBX2 to undetectable values, increased 6-keto-PGF1 alpha by 8-fold, and significantly reduced both systolic and diastolic blood pressures in hypertensive patients. Such effects may dependent upon an increase in the endogenous prostacyclin or an inhibition in thromboxane production in the affected arterial walls. If the present observations would be confirmed by double blind trial, they would constitute the base for new pharmacotherapy of hypertension. PMID- 1726989 TI - [Determination of susceptibility to pharmacologic treatment in Graves'-Basedow disease]. AB - The authors present the results of research on the usefulness of clinical data, thyroid hormones, TSH, antithyroid antibody, HLA estimations and a type of behavior, for prognosing the susceptibility to pharmacological therapy in thyrotoxicosis, specially in Graves disease. The highest chance of predictability was found in patients with small goiter, HLA-DR5 positive with onset of the disease at the age before 40. In general it is difficult to predict the reaction to therapy in individual case with high probability. PMID- 1726990 TI - [Salivary gland adenolymphomas as seen in current histochemical and immunomorphological research]. AB - Samples of 32 adenolymphomas and 10 intact salivary glands were studied using histological, histochemical (Grimelius and congo red staining), microscopic and immunohistochemical methods employing polyclonal antibodies to myosin, human carboanhydrase III and monoclonal antibodies to cytokeratin polypeptides No. 8 and 17, epithelial membrane antigen and T- and B-lymphocytes as well as peroxidase--antiperoxidase reaction. Binding of monoclonal antibodies to cytokeratin No. 8 and epithelial membrane antigen was observed in glandular cells of cysts whereas proliferating cells forming "Sandersen's cushions" showed binding of polyclonal antibodies to myosin, carboanhydrase III and monoclonal antibodies to cytokeratin No. 17. Tumor stroma revealed T- and B-lymphocytes incretory granulocytes and amyloid spaced along the fiber structures. It is inferred that adenolymphoma is a tumor arising exactly in salivary glands. The epithelial component of this tumor forms following tumor transformation of epithelial and myoepithelial cells of the distal part of salivary gland ducts. T- and B-lymphocytes contribute to formation of tumor stroma. Epithelial membrane antigen is an effective marker of the epithelial component of tumor. PMID- 1726991 TI - [Monoamine concentration in the cerebrospinal fluid of patients with subarachnoid aneurysm]. AB - Using a spectrofluorometric method, the concentration of the major monoamines, norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-), and 5 hydroxyindoleacetic (5-HIAA) was determined in cerebrospinal fluid (CSF) of patients with subarachnoid aneurysm before its rupture. DA, 5-HT, and 5-HIAA levels in CSF were found significantly increased when compared with the corresponding values found in persons with no clinical alterations of central nervous system (CNS). No significant differences were seen in NE values in CSF when data from patients with subarachnoid aneurysm were compared to their control subjects. Data obtained suggest that of NE diffusion to CSF could be at a different rate than that of the other monoamines measured. Alternatively, the reuptake of NE could vary in relation with that to the other monoamines in the affected brain tissue during the onset and development of the aneurysm. Results may also indicate a possible accumulation of NE in brain tissue adjacent to the damaged areas as a result of an increased monoaminergic activity, which, could be involved in the pathophysiology of aneurysm rupture. PMID- 1726992 TI - Effects of diet composition on the reactivity pattern to the zinc-iodide/osmium tetroxide mixture of the rat pancreatic acinar tissue. AB - When adult rat pancreatic exocrine tissue is incubated in zinc-iodide/osmium tetroxide (ZIO) mixture, 3 cell types are recognized depending their degree of impregnation: intensely impregnated (I-PAC), moderately impregnated (M-PAC), and non-impregnated (N-PAC) cells. Their distribution in pancreatic head was 8%, 46% and 46% respectively; whereas in the tail, it was 7%, 35% and 58% respectively. With the purpose to know whether those variations in ZIO-impregnation had some relation with the diet components, 7 groups of 30-day old male rats (n = 5 each) were maintained for 40 days with the following diets: control (C), high-protein (HP), low-protein (hP), high-lipid (HL), low-lipid (hL), high-dextrin (HD) and low-dextrin (hD) diets. Fragments of head and tail were incubated in ZIO mixture, embedded in epoxy resins and cut. Sections were examined unstained under a light microscope. The most striking features in the head were seen with hP, hL and particularly with HD diets. In tail tissue, the most relevant changes were seen with hP, hD and especially with HL diets. It is concluded that there could be a regional acinar cell populations related to preferential production of digestive enzymes in pancreatic head, body and tail, which would need to be further investigated. PMID- 1726994 TI - [Pertinence of animal and human models in the evaluation of ventricular anti arrhythmia agents]. AB - The development of antiarrhythmic agents for the treatment of ventricular arrhythmias depends to a large extent on their effects in different animal and human models. The clinical relevance of the data so obtained is debatable. Firstly, in vitro animal models of arrhythmias are not very predictive of the multiple clinical forms of ventricular arrhythmias. Secondly, the intermediary criteria of evaluation of the effects of antiarrhythmic drugs in humans are generally not valid in terms of criteria of substitution for the evaluation of therapeutic effects. Nevertheless, cellular and hemodynamic studies of the electrophysiological properties of drugs are essential for correct clinical usage of antiarrhythmics. They help predict the principal clinical electrocardiographic changes and their modulation with respect to parameters such as ischemia or heart rate, their hemodynamic tolerance and certain undesirable, especially proarrhythmic, effects. However, the clinical pertinence of these studies remains limited for a number of reasons. In particular, most antiarrhythmic agents have multiple electrophysiological effects, the resultant of which is difficult to predict in the clinical situation. In addition, many of these drugs have active metabolites, the formation of which varies from person to person, which also reduces the clinical relevance of studies of the parent molecule alone. Clinical trials in appropriate patient populations should therefore be preferred to the multiplication of studies on experimental models of uncertain relevance. PMID- 1726993 TI - [Characterization of SH groups in the membrane proteins of human spermatozoa]. AB - Membrane proteins rich in SH- groups were identified in human ejaculated spermatozoon, using the alkylation reaction with radioactive iodoacetic acid with a subsequent determination of the electrophoretic characterization of the proteins, as well as their comparison with the proteins in seminal plasma and blood serum of the same individuals. The determination of the incorporated radioactivity shows that out of 20 proteic bands identified by staining with Coomassie Blue, only 9 were alkylated with the iodoacetate. Among these 9 proteic bands, 4 are glycoproteins since they give a PAS positive reaction. All the proteins which incorporated the marked iodoacetate had a molecular weight below 67,000 daltons with a predominance of molecular weight below 25,000 daltons. A comparison of the electrophoresis obtained from the spermatozoon's plasmatic membrane, seminal plasma and blood serum, one finds that among the 9 proteins which were alkylated with the iodoacetate in the plasmatic membranes, two, bands I and IV are also found in the seminal plasma and blood serum, two more, bands VII and IX are found in the membranes and seminal plasma, but not in blood serum, while the rest are only found in the spermatozoon. PMID- 1726995 TI - [The role of Holter monitoring and electrophysiological studies in the evaluation of ventricular anti-arrhythmia agents]. AB - The roles of Holter monitoring and electrophysiological studies (EPS) in the evaluation of antiarrhythmic drugs for ventricular arrhythmias are to record their effects on ventricular extrasystoles (VES) and ventricular tachycardia (VT), and to search for undesirable rhythmological effects. The usual protocol is to perform baseline studies and then repeat them after a certain period of treatment using a number of modalities, comparison with placebo, control group, in acute or oral administration. Holter monitoring is an economical non-invasive method which carries no risk. Spontaneous arrhythmias of sufficient frequency to be recorded during the monitoring period can be studied. When applied to VES, it provides quantitative rather than qualitative information despite classifications such as Lown's. The results should be analysed taking spontaneous variations of the arrhythmia into consideration. Holter monitoring may also reveal proarrhythmic drug effects (bradycardia, torsades de pointe). However, there are no absolute criteria of efficacy except total suppression of VES, unexplainable by spontaneous variability. Holter antiarrhythmic studies require stable VES which creates a bias in the evaluation of results due to the special selection of patients. Electrophysiological studies are costly, invasive and uncomfortable but they are the only way of assessing paroxysmal VT apart from clinical follow-up. This method is only applicable to inducible VT, which is the commonest form. The investigating protocols are specific and reproducible: a tachycardia which is non inducible does not recur in 90% of cases, which enables prediction of the antiarrhythmic effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726996 TI - [Unwanted cardiovascular effects of anti-arrhythmia agents]. AB - Antiarrhythmic drugs can cause many undesirable side effects affecting a number of organs and functions. Of those which affect the cardiovascular system, the proarrhythmic and negative inotropic effects are the most serious. Proarrhythmic effects, suggested by an aggravation of an arrhythmia or the induction of a previously undocumented arrhythmia, may be favorised by the presence of an arrhythmogenic substrate (unidirectional lock, delayed conduction, dual conduction pathways, low thresholds of depolarisation or fibrillation, presence of zones of hyperautomaticity...), "triggering" mechanisms (extrasystoles, variations of heart rate, after-depolarisation) and by changes in the cardiac environment (variations of autonomic nervous tone and hormonal changes, electrolytic or metabolic disorders...). An antiarrhythmic may have a beneficial action on one of these factors (for example, suppression of extrasystoles) but an aggravating effect on others (for example, an increase in the heart rate, creation of zones of reentry). This probably explains the fact that, for the moment, only molecules which have multifactorial modes of action have been shown to be beneficial in arrhythmias after myocardial infarction. Negative inotropic effects may be directly responsible for a deterioration in the hemodynamic status of patients on antiarrhythmics and indirectly responsible for aggravating arrhythmia by altering the anatomical substrate, so favorising proarrhythmic effects. The negative inotropic action may be related to ionic mechanisms (lowering intracellular calcium concentration due to blockade of the sodium channel by Class I antiarrhythmics) or to indirect mechanisms (reduced sympathetic tone, non-specific beta inhibition, calcium channel blockade, decreased left ventricular compliance, vasoconstrictor effects...).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1726997 TI - [How to develop an anti-arrhythmia agent in 1990. The cardiologist's viewpoint]. AB - The development of an antiarrhythmic drug in 1990 follows well established pathways. The first step is animal experimentation to establish the antiarrhythmic effect and to estimate the risk of toxicity, but clinical trial is the fundamental stage which confers on a product the title of antiarrhythmic. Several models may be used, one of which is ventricular extrasystoles. However, the data so obtained must not be extrapolated to a wider context. All methods for evaluating an antitachycardia effect have imperfections. Obviously, arrhythmias treated in hospital are privileged although they may only represent a special subgroup of more severe arrhythmias. Potential antiarrhythmics are more often assessed by their effects on tachycardias reproduced by programmed pacing, leaving other arrhythmias, including atrial fibrillation, to one side. In addition, long-term efficacy is more often suspected than demonstrated. The action on malignant ventricular tachyarrhythmias escapes all valid assessment mainly because of the difficulty of using a control group of patients with life threatening arrhythmias. The use of automatic defibrillator devices could, in the near future, bring a more rigorous approach to this problem. One of the most important objectives of antiarrhythmic drugs is the prevention of sudden death. Results have been relatively disappointing until now. This may be due to deleterious, above all proarrhythmic, side-effects counterbalancing the benefits of the drug. The unwanted side-effects must be evaluated systematically in the future. The methodology needed is not yet established and requires further research. PMID- 1726998 TI - [Amiodarone and secondary prevention. The EMIAT study]. AB - The EMIAT is a randomized, double-blind trial versus placebo, designed to evaluate the efficacy of amiodarone in the prevention of total mortality in patients with left ventricular ejection fraction of under 40% after myocardial infarction. This multicenter European trial initiated by SANOFI Research in association with the Arrhythmia Working Group of the European Society of Cardiology, is part of a larger program of evaluation (3,400 patients) of the benefit-risk ratio of the use of amiodarone for secondary prevention in high risk patients. PMID- 1726999 TI - [A retrospective study of the serum CEA, AFP, TPA and ferritin values in 500 patients with chronic liver diseases]. AB - The authors evaluate the positivity of the tumor markers CEA, AFP, TPA and ferritin among an homogeneous group of 500 patients suffering from a chronic hepatopathy and positive for HBsAg. The obtained results show a significant increase of TPA, AFP and Ferritin (70.4%, 20% and 24% respectively of the examined patients), while CEA is increased only in the 3.2%. The correlation between the positivity of these markers and possible evolution of the chronic hepatopathy is at present under investigation. PMID- 1727000 TI - Cytoarchitecture, neuronal composition, and entorhinal afferents of the flying fox hippocampus. AB - In a comparative approach, the anatomical organization of the hippocampus was investigated in two species of megachiropteran bats, the grey-headed flying fox, Pteropus poliocephalus, and the little red flying fox, Pteropus scapulatus. In general, the cytoarchitectonic appearance of the flying fox hippocampus corresponded well with that of other mammals, revealing all major subdivisions. While the dentate fascia was trilaminated with a molecular layer, a granule cell layer, and a distinct polymorphic layer, the ammonic subfields were subdivided into stratum lacunosum molecular, stratum radiatum, stratum lucidum or mossy fiber layer (restricted to the CA3 region), pyramidal cell layer, and stratum oriens. In Ammon's horn, only subfields CA1, CA3, and CA3c were clearly discernible, whereas the CA2 region remained indistinct. In some cytoarchitectonic features, such as the dispersion of the pyramidal layer in CA1, the megachiropteran hippocampus resembled the corresponding region in primates. Five characteristic neuronal cell types of the megachiropteran hippocampus were studied in fixed slice preparations after intracellular injection with Lucifer Yellow. While the morphological appearance of CA3 pyramidal cells, horizontal stratum oriens cells, aspiny stellate cells, and mossy cells strongly resembled their counterparts in rodents, primates, and carnivores, granule cells showed an interesting variation from the nonprimate pattern. Like a subset of granule cells in the primate dentate gyrus, 75% of flying fox granule cells revealed 1-2 basal dendrites that ramified in the polymorphic layer. These processes are presumed to form the morphological substrate for recurrent excitation. Entorhinal afferents to Ammon's horn and the dentate fascia were revealed by employing the method of tract tracing in fixed tissue with the carbocyanine dye DiI. Similar to the rat and cat, but unlike the monkey, the entorhino-dentate projection in the flying fox is bilaminate, with medial entorhinal afferents occupying the middle third of the molecular layer and lateral entorhinal axons ramifying closer to the hippocampal fissure. The remaining inner third of the molecular layer was free from entorhinal input. In contrast to the radial organization of the projection to dentate gyrus and subfield CA3, entorhinal afferents to region CA1 followed a proximo-distal gradient, with medial entorhinal afferents terminating closer to the CA3/CA1 border. Photoconverted preparations were used to determine the trajectory of individual axons. The majority of entorhino-dentate axons traversed the hippocampal fissure, usually close to the crest region, and gave rise to several terminal branches with numerous en passant varicosities. Individual fibers coursed for considerable distances parallel to the granule cell layer, thus presumably activating a large number of postsynaptic granule cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727001 TI - Organization of CA1 projections to the subiculum: a PHA-L analysis in the rat. AB - The organization of CA1 projections to the rat subiculum was investigated with the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Discrete iontophoretic injections of PHA-L were placed into various transverse positions of the CA1 field at different septotemporal levels of the hippocampus. The distribution of CA1 projections was observed in dissected and extended hippocampal preparations. CA1 cells located proximally in the field, i.e., close to the CA2 field, gave rise to projections that terminated in the distal third of the subiculum, i.e., close to the presubiculum. CA1 cells located distally in the field, i.e., close to the subiculum, gave rise to projections that terminated proximally in the subiculum, i.e., just across the CA1/subiculum border. CA1 cells in the middle of the field projected to a midtransverse portion of the subiculum. The same general pattern of projections was observed at all septotemporal levels of the hippocampus. Varicose fibers from the CA1 neurons terminated among the basal dendrites of the subicular pyramidal cells, within the pyramidal cell layer, and in the deep portion of the molecular layer. In addition to the CA1 to subiculum projections, the discrete PHA-L injections provided the opportunity of examining the extent of local and associational connections within CA1. In general, associational connections in CA1 are far less extensive than in CA3. CA1 is not entirely without local connections, however. CA1 cells located close to the subicular border, for example, originated axons that first innervated the proximal subiculum and then reentered the CA1 field at the interface between stratum radiatum and stratum lacunosum-moleculare. In most of the experimental cases, there were collaterals located in stratum oriens of CA1 that branched from the fibers directed toward the subiculum. Thus, the basal dendrites of CA1 cells may receive associational inputs. The organization of the CA1 projections to the subiculum is discussed in relation to the organization of CA3 projections to CA1 and the differential output of transverse regions of the subiculum. The possibility is raised that information may be "channeled" through the hippocampal formation via the transverse organization of these connections and ultimately distributed to different recipients of hippocampal efferent projections. PMID- 1727002 TI - Cystic fibrosis--from gene to therapy. PMID- 1727003 TI - [Hydrolysis of raw keratin wastes with use of certain sulfonic acids. II. Analysis of optimal conditions for hydrolysis]. AB - We have fixed optimal conditions to hydrolysis of the waste keratin material cerebellum, hoof and bristle. With apply 1 mol/dm3 solutions of acids: amide-, benzene-, 4-methylbenzene- and naphtalene-1-sulfonate, the best results obtained by degradation protein for 24 hours in closed glass tube in the argon in the temperature 110 degrees C by means of benzenesulfonate acid; materials after hydrolysis contain all exogenous amino acids. PMID- 1727004 TI - Laparoscopic pelvic lymphadenectomy. AB - Eleven male patients with pelvic malignancy underwent laparoscopic lymphadenectomy for staging of their tumors. The technique allowed removal of pelvic lymph nodes in all 11 patients and metastatic disease was found in five cases, resulting in a change of recommended therapy. The technique was via a three-port exposure, although a fourth suprapubic port was occasionally used for additional retraction. A bladder laceration, which was repaired laparoscopically, was the only intraoperative complication. A pelvic hematoma was the only significant postoperative complication. Laparoscopic pelvic lymphadenectomy appears to offer a less morbid staging for those patients with a high likelihood of nodal metastasis. Laparoscopic detection of positive pelvic lymph nodes may alter the management of genitourinary malignancy and improve overall patient care. PMID- 1727005 TI - Studies on sex related differences in elimination of theophylline in the rat after pretreatment with phenobarbital, chrysene, Aroclor 1254 or lindane. AB - Cytochrome P-450 in liver microsomes plays a central role as a drug metabolizing enzyme. The sex-dependent differences in the properties of cytochrome P-450 have been extensively studied using liver microsomes, and marked differences have been found in microsomal metabolism of xenobiotic. Effect of pretreatment with phenobarbital, chrysene, lindane and polychlorinated biphenyls (Aroclor 1254) on the theophylline elimination in male and female rats was studied. Levels of theophylline were measured spectrophotometrically. PMID- 1727006 TI - Kallikrein-thrombolytic and hypotensive action in cats--preliminary results. AB - An intravenous injection of kallikrein produced hypotensive and thrombolytic effects in anesthetized cats, using the blood superfused tendon technique. The thrombolytic action of kallikrein was mediated by an unstable substance. The generation of this substance was abolished by either acetylsalicylic acid (ASA) or aprotonin and enhanced by captopril. The hypotensive action of kallikrein was only partially inhibited by ASA. It is proposed that both these pharmacological effects of kallikrein are mediated by bradykinin which in turn releases prostacyclin from the endothelium. However, in contrast to the thrombolytic effect of kallikrein which is totally mediated by prostacyclin the hypotensive action of kallikrein depends not only on prostacyclin but also on another endothelium-derived vasorelaxant, e.g. EDRF. PMID- 1727007 TI - Number of beta-receptors in patients allergic to Dermatophagoides pteronyssinus. AB - This study was carried out on 96 atopic patients with monosensitization against Dermatophagoides pteronyssinus and 30 control individuals. The patients were divided into 3 subgroups: 27 asymptomatic patients, 27 patients who only presented nasal symptomatology, and 27 patients with marked bronchial symptomatology. Total serum IgE, antigen-specific histamine release, and the number of beta 2-adrenergic receptors in peripheral blood lymphocytes in all patients were determined. The control group presented the highest number of beta receptors in comparison with the bronchial symptomatic and nasal symptomatic patients. However, asymptomatic patients presented numbers of beta-receptors similar to controls, with no significant differences between both groups. No significant correlation was found between beta-adrenergic receptors and levels of total serum IgE. On the other hand, correlation between beta-receptors and specific histamine release was detected only in symptomatic nasal patients. It can be postulated that the decrease in beta-receptors is a consequence, and not the cause, of atopy. PMID- 1727008 TI - Influence of seasonal variations on histamine release and other immunological parameters in pollinosis. AB - One hundred and fifty-six pollen-sensitive patients with sensitization to grass pollen only were chosen for this study; 65 patients were allergic to Dermatophagoides, while 45 were non-allergic subjects with no atopic history. The following tests were carried out on all patients: histamine release of total blood, basal histamine, total histamine, determination of total IgE and specific IgE (RAST) serum levels, serum hemagglutinating antibodies and skin tests. Seasonal increase of IgE was observed, with post-seasonal fall; 50% of the cases reached normal values. However, RAST as a semiquantitative method did not prove to be useful in the detection of variations in specific IgE levels. During pollination, an increase in titers in pollen-sensitive subjects was seen. This was attributed to a humoral immune response epiphenomenon which was independent of IgE. The histamine release test was significantly greater during pollination in pollen-sensitive subjects. The total of stored histamine increased during the months of allergenic exposure. PMID- 1727009 TI - Number of beta-receptors in rhinitic pollinic patients. AB - The study was carried out on venous blood from 67 patients with seasonal rhinoconjunctivitis caused by sensitization to grass pollen and 30 control individuals. Total IgE determination, antigen-specific histamine release test against two concentrations of Phleum pratense, and quantification of beta 2 adrenergic receptor numbers in lymphocyte membrane of peripheral blood were done on all patients. Those pollinic patients who were asymptomatic at the time of the study had 500.07 +/- 237.27 receptors/cell; no significant differences were established with the control group, with 541.53 +/- 123.63 receptors/cell. However, both the control group and asymptomatic patients had receptor numbers which were significantly higher than those of symptomatic pollinic patients, with 376.81 +/- 158.65 receptors/cell (p < 0.01 and p < 0.05, respectively). The average decrease in number of receptors in symptomatic pollinic patients was 30.42% in relation to controls and 24.65% in relation to asymptomatic patients. Within the subgroup of pollinic patients, studied both in and out of season, the number of beta 2-adrenergic receptors had an average decrease of 13.22% during pollination, with 363.7 receptors/cell. Once the pollination season was over, this figure increased to 419.1 receptors/cell, establishing significant differences with p < 0.025. The number of beta 2-adrenergic receptors did not correlate with total seric IgE figures or with antigen-specific histamine release. These data indicate that the decrease of these receptors does not constitute the causal factor of atopic diseases; it seems more likely to be a consequence of the same. PMID- 1727010 TI - Determination of phagocytosis of Leishmania spp. by human polymorphonuclear leukocytes using anti-Leishmania monoclonal antibodies. AB - Phagocytosis of Leishmania is an early event in the capacity of human polymorphonuclear cells to limit the spread of this infectious agent. We compared two methods to assess the phagocytosis of Leishmania by PMN cells; the first using histochemical staining with Wright or Giemsa, and the second using the immunoperoxidase technique with anti-Leishmania monoclonal antibodies. The quantitative results obtained with either of the cytochemical methods were comparable with the immunoperoxidase technique, but the latter offered the advantage of an easier identification of the intracellular parasites. This improvement greatly reduced the time required to quantify phagocytosis compared to the conventional staining techniques. PMID- 1727011 TI - Study of IgE-dependent basophil releasability in allergic patients. AB - For this study, venous blood from 160 patients with a diagnosis of asthma and/or rhinitis was used. Histamine release test (H.R.T.) with anti-IgE at 1/5 and 1/25 dilutions and with causal antigen in the case of atopic patients (144) was carried out on all patients. Basophils from atopic patients released more histamine than those from nonatopic patients (p < 0.001). Basophils of atopic patients released more histamine with 1/5 anti-IgE dilution (p < 0.01), while non atopic patients did so with the 1/25 dilution (p < 0.05). On grouping atopic patients according to positive or negative results in Ag-specific histamine release, 14% of patients presented negative Ag-specific H.R.T. and 85.7% of the cases did not respond to anti-IgE stimulus; this was 12% of the total number of atopics. On the other hand, 17% of the patients studied did not show positive histamine release against anti-IgE and 70.6% of them had negative Ag-specific release. As for the effect of age on IgE-dependent histamine release, the group of patients who presented greater releasability corresponded to those older than 6 years of age. The discrepancies observed between the clinical history, skin tests, serum IgE and antigen-dependent histamine release in some patients could be related to the individual basophil ability to release histamine. Therefore, this basophil releasability evaluation has an important practical application in discerning false negatives in antigen-specific H.R.T. PMID- 1727012 TI - [Is the multiple of the median a common statistical expression in our medical practice?]. PMID- 1727013 TI - [Trachoma and the conjunctival impression test]. AB - The Transferred Conjunctival Impression test (test d'Impression Conjonctivale Transferee) is a simple, sensitive and non-invasive method enabling populations at risk from Xerophthalmia to be identified. Nevertheless, the results of the test do seem to be influenced by the conjunctival pathology, trachomatous in particular. It appears that conjunctival deficit is noticeably more frequent in trachomatous than non-trachomatous subjects. The trial, involving a random sample of 450, four- to six-year-old children living in the Sahelian zone, enabled us to confirm and quantify this relation. In terms of relative risk, with age, place of residence and nutritional state all being equal, the rate of abnormal TCI tests was 1.7 times higher in trachomatous than non-trachomatous children. Using the Transferred Conjunctival Impression test is thus especially indicated in zones with a low prevalence of trachoma. When high, an eye examination must be carried out during the trial in order to present the results in two categories: trachomatous and non-trachomatous. Failing this, estimates may be obtained using the formulae put forward by the authors. PMID- 1727014 TI - Interaction of T lymphocytes with cerebral endothelial cells in vitro. AB - As a prerequisite of inflammatory lesion formation in (auto-)immune disease of the central nervous system, lymphocytes have to interact with brain endothelia. In recent years much progress has been made towards a better understanding of mechanisms and factors involved in organ specific homing of lymphocytes. Many lines of evidence indicate that T lymphocytes recognizing antigens which are exclusively beyond the blood-brain barrier cross this barrier only when they are in an activated state, irrespective of their antigen specificity. Antigen presentation by blood-brain barrier endothelia, however, may play a role in later stages of florid inflammation. PMID- 1727015 TI - Alzheimer's disease: a description of the structural lesions. PMID- 1727016 TI - Anti-cytoskeletal autoantibodies: diagnostic significance for liver diseases, infections and systemic autoimmune diseases. PMID- 1727017 TI - Comparison of aromatase activity in human prostatic, testicular and placental tissues. AB - The aromatase enzyme was quantified by the release of tritiated water from [1 beta-3H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0 microM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC50 values of 6.4, 0.17 and 0.0017 microM, respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075 microM, respectively). PMID- 1727018 TI - The relationship between autoantibodies to triiodothyronine (T3) and thyroglobulin (Tg) in the dog. AB - The relationship between T3 autoantibodies (T3AA) and thyroglobulin autoantibodies (TgAA) in dogs was investigated by determining the inhibitory effect of triiodothyronine (T3), thyroxine (T4) and thyroglobulin (Tg) on T3AA and TgAA binding activity and by determining the pattern of occurrence of the two activities in canine serum samples. Strong similarity in binding characteristics between the two activities, as one might expect if T3AA activity were merely a cross-reactivity of TgAA, was not observed. Canine T3AA activity exhibited a cross-reactivity to purified canine Tg that was intermediate between that of T3 and T4, indicating an antigenic relationship to an epitope of Tg. Average affinity constants of canine T3AA (N = 11) for T3, Tg and T4 were 1.76 x 10(10) M 1, 2.29 x 10(9) M-1, and 1.02 x 10(8) M-1, respectively. Canine TgAA activity, however, did not cross-react significantly with T3 or T4. Canine TgAA (N = 21) binding to canine Tg was not inhibited by T4 or T3 at concentrations up to 2 x 10(-4) M. Each of 23 canine serum samples containing T3AA also exhibited TgAA activity, although there was poor correlation between the magnitudes of the two activities. Neither T3AA nor TgAA activity was observed in serum samples from 16 euthyroid dogs; however, 46.7% of the samples from 15 hypothyroid dogs had detectable TgAA activity. T3AA is so rare that is was not observed in this small population of samples from hypothyroid dogs. The [125I] T3 binding in serum from hypothyroid dogs was elevated compared to that in euthyroid dogs, but was considerably lower than in samples generally designated as containing T3AA. These results suggest that T3AA found in occasional canine serum samples are due to the presence of autoantibodies recognizing a T3 containing epitope of Tg that is different from the epitopes involved in eliciting the predominant population of canine Tg autoantibodies. PMID- 1727019 TI - The 65kD heat shock protein: a key molecule mediating the development of autoimmune arthritis? PMID- 1727020 TI - Autoantibodies to neutrophil cytoplasm (ANCA) in Wegener's granulomatosis. Clinical significance and antigen specificity. PMID- 1727021 TI - Chlamydia trachomatis infection in "sine causa" recurrent abortion. AB - One hundred and one women suffering from "sine causa" recurrent abortion were screened for Chlamydia trachomatis (C.T.) infection by using direct examination, cultural and serological procedures. In this series, C.T. infection did not appear to be related to increased risk of recurrent abortion. The culture positive and serology-positive rates (14.85% and 34.65%, respectively) did not differ from other unselected populations. Neither time from last abortion nor type of abortion were significantly related to C.T. infection. Nonetheless, the women who underwent examination within one year from last abortion and had a culture-positive partner as well, were more likely to present with a C.T. positive culture. PMID- 1727022 TI - Epidemiological evaluation by rRNA-DNA hybridization of strains of Salmonella enterica subsp. enterica serovar Abortusovis isolated in southern Italy in the years 1981-1989. AB - Salmonella enterica subsp. enterica serovar Abortusovis is a major agent of abortion of the sheep and is firmly established, although at low prevalence, in Sicily. This paper describes the application rDNA gene restriction pattern fingerprinting to investigate relatedness among 7 serovar Abortusovis strains isolated at the "Istituto Zooprofilattico Sperimentale" of Sicily and 29 isolates identified at the Southern Italy Centre of Enterobacteriaceae between 1981 and 1989. Although Abortusovis serovar has exhibited a remarkable degree of homogeneity, genomic DNA polymorphisms, that have emerged, suggest possible importation of bacterial clones from different geographic areas. PMID- 1727023 TI - [Care of the terminally ill]. PMID- 1727024 TI - [Clinical use of hematopoietic growth factors]. PMID- 1727025 TI - [Interferon in the treatment of renal cancer]. PMID- 1727026 TI - Small bowel obstruction in patients with a prior history of cancer. AB - To assess the efficacy of operative and nonoperative therapy of small bowel obstruction (SBO) in patients with a previous diagnosis of cancer, a review of 54 cases was carried out. The 32 men and 22 women had a mean age of 58 years. At presentation with SBO, 26 patients (48%) had known recurrent cancer. Forty patients were initially treated nonoperatively; 11 (28%) had resolution of their SBO after a mean of 7 days of nasogastric suction. Five of 11 patients developed recurrent SBO prior to death. Thirty-seven patients underwent laparotomy, 14 on the day of admission and 23 after failure of nasogastric suction. Twenty-five of 37 (68%) had obstruction due to recurrent carcinoma. Small bowel obstruction due to recurrent cancer occurred earlier (21 +/- 5 months) than SBO from benign causes (61 +/- 18 months; p < 0.01). Mean survival for patients with malignant obstruction (5 +/- 1 month) was significantly shorter than for those with benign obstruction (50 +/- 10 months; p < 0.001). The 30-day and in-hospital mortality rates for the 25 surgically treated patients with malignant SBO were 24% and 28%, respectively; in 9 of 25 (36%), the obstruction failed to fully resolve. The only factor predictive of in-hospital mortality was obstruction secondary to cancer (p < 0.05). The median posthospital survival for surgically treated patients with malignant SBO was only 2.5 months. We conclude that: (1) patients should be given an initial trial of nonoperative therapy; (2) patients with no known recurrence or a long interval to the development of SBO should be aggressively treated with early surgery if nonoperative treatment fails; and (3) for patients with known abdominal recurrence in whom nonoperative therapy fails, the results of surgical palliation are grim. Innovative approaches are needed to maximize palliation while also limiting morbidity and mortality. PMID- 1727027 TI - Modulation for antigen presentation in tuberculosis by using synthetic peptides. AB - Competition assay technology has been a very useful tool in the study of parasite antigens and has been inferred but never proven that this approach can be applied to select T-cell epitopes by using another microorganisms. In this study, HLA restricted T-cell clones specific to synthetic peptides derived from the 65 kDa mycobacterial protein were used to investigate whether these peptides are able to compete with each other at the level of MHC-binding sites in tuberculosis. Fixed APCs were pulsed with suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptide. The results showed that two peptides from this protein were able to compete with each other inducing a significant inhibition in the proliferation assays while there was no competition by using a control peptide. The amount of cross-reactivity was influenced by the peptide concentrations. More important was the observation that these peptides were able to bind to the same HLA-class II molecules therefore blocking the binding of each other. The fact that these peptides have not an identical amino acid sequence support the idea that the MHC-peptide interaction must have a broad specificity to be able to bind a large number of peptides. These results demonstrate that it is possible to modulate the antigen presentation by blocking the peptide MHC-class II interaction in tuberculosis and support the idea that this approach facilitates the selection of appropriate T-cell epitopes to be incorporated in a vaccine. PMID- 1727030 TI - [The treatment of advanced breast cancer is changing]. PMID- 1727028 TI - [Functional and evolutionary aspects of the aminoacyl-tRNA synthetases]. AB - A main event in protein bioshynthesis is the esterification of the correct aminoacid to cognate tRNA catalized by the aminoacyl-tRNA synthetases. The central role of this family of enzymes in metabolism is an evidence of their ancient origin. As it is the case in many others molecules involved in protein synthesis, the emergence of the aminoacyl-tRNA synthetases appears to be a problem that is not yet solved in order to understand the origin of the genetic translation. To obtain a comprehensive view of the evolution of the relationship between each one of the twenty aminoacyl-tRNA synthetases from one organism as well as from different sources (eubacteria, archaebacteria and eukaryotes) we review the information collected from the structural and catalytic properties of these enzyme. The results allow us to establish the following relationship between aminoacyl-tRNA synthetases. On one side there is a monofiletic origin for glutamyl, glutamynil and argynil-tRNA synthetases from Escherichia coli and for valyl, leucyl, metionyl, isoleucyl and phenylalanil-tRNA synthetases from eubacterias, archaebacterias and eukaryotes. On the other side there is an evolutionary relationship between aminoacyl-tRNA synthetases of eubacteria and organelles (plastids and mitochondria) and among eukaryotes and archaebacteria. PMID- 1727031 TI - [Affective levels]. AB - An naturalistic and ethological approach of early affective development in childhood is described. This approach leads to define different levels of affectivity and is illustrated by a case report. PMID- 1727032 TI - Picture naming deficits: a single case study. AB - A single case study of a 68 year old patient, with picture naming deficits, has been reported. The aim of this study was to determine the level of breakdown in the picture naming process and to examine the nature of the impairment utilizing an information processing framework. The results of this investigation demonstrate, that DA's difficulties are not attributable to breakdown either at the level of access to semantics or to speech output lexicon as suggested in the literature, but to breakdown beyond that level. PMID- 1727033 TI - [Radiation therapy in the management of epidemic Kaposi's sarcoma]. PMID- 1727034 TI - [Is the lack of interferon causing overproduction of IgE?]. PMID- 1727035 TI - [Risk of hepatitis after blood transfusion in Finland]. PMID- 1727036 TI - Countercurrent chromatography for the purification of peptides. AB - In Table 3 are listed the types of peptides separated by CCC, including the instruments used and the solvent conditions. In the last 20 years, intriguing discoveries about the hydrodynamic behavior of liquids in open-coiled tubing have introduced many possibilities for chromatographic separations. A fruitful area of endeavor certainly lies in further elucidation of the mechanisms. However, it is evident that CCC has the capability to separate all types of substances: organic compounds, proteins, membrane proteins, subcellular particles, as well as peptides, the intermediate-sized molecules. The potential for applications also lies at various levels of the scale such as analytical, laboratory, and, very importantly, industrial processes. PMID- 1727037 TI - Chromatographic methods for determining carcinogenic benz(c)acridine. PMID- 1727038 TI - Endorectal sonography. AB - Endorectal sonography is a technique that has been developed recently to visualize the rectal wall and perirectal tissues with a high degree of clarity. Studies utilizing endorectal sonography in the preoperative staging of rectal carcinoma have reported an accuracy of between 67 and 92% in the visualization of the depth of tumor invasion in the rectal wall. This surpasses the accuracy reported for digital exam and other preoperative imaging methods such as CT and MRI. Perirectal lymphadenopathy is also well visualized by this method and guided biopsy of perirectal nodes has been reported. Precise preoperative staging of rectal carcinoma by endorectal sonography is an important technique that can: (1) improve surgical planning, (2) provide prognosis in nonsurgical candidates, and (3) select patients suitable for local excision therapy. PMID- 1727039 TI - Current imaging concepts of thoracic intervertebral disks. AB - This article reviews the current concepts of thoracic herniated disks using the radiologic literature as well as our own experience with more than 100 thoracic HNPs. The relative frequency of asymptomatic thoracic HNPs is documented. Points of interest include the optimal technique, criteria for interpretation, strengths and weaknesses of various imaging modalities, including water-soluble myelography, CT myelography, and magnetic resonance imaging. Additionally, the protean clinical manifestations of thoracic HNPs and current operative management are briefly addressed. PMID- 1727040 TI - Diagnostic imaging of liver abscess. AB - This article on imaging of liver abscess discusses the appearance of pyogenic, amebic, and fungal abscesses using a multimodality approach, as well as imaging pathologic correlation. It summarizes the experience obtained reviewing 100 cases of hepatic abscesses from two geographical areas that have different incidences of etiologic factors. Sixty abscesses were collected at the Hospital General de Mexico, where there is a high incidence of amebic abscesses, and 40 cases were collected at the University of Florida College of Medicine, Gainesville, Florida, where pyogenic and fungal abscesses predominate. This article includes sections on pathology, etiology, epidemiology, clinical findings, prognosis, and therapy of hepatic abscesses, as well as imaging findings. Imaging is divided by techniques, including plain films, nuclear medicine, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), and angiography. It also includes a differential diagnosis section where different imaging findings of cystic lesions of the liver other than abscesses are displayed. Thirty-three references from both U.S. and Mexican authors are included, as well as 20 illustrations. In summary, in this article, we demonstrate how imaging is useful in diagnosing hepatic abscesses and how a focal mass in the liver may be distinguished between an abscess and a necrotic tumor. A practical approach is used so radiologists can benefit from the authors' experience and apply the findings described in their daily practice. PMID- 1727041 TI - Easily missed trauma of bones and ligaments. AB - While most fractures and dislocations are easily detected by radiographic and physical examination, some traumatic events are overlooked because the normal radiographic anatomy is not appreciated. Furthermore, certain routine radiographic views are inadequate to detect specific injuries. "Chip" or "sprain" fractures are often considered minor injuries when they actually connote significant injury to ligaments and capsules. These injuries are usually dismissed by the primary care physician, who does not believe them serious enough to warrant consulting an orthopedic surgeon. However, some are also overlooked by radiologists and orthopedic surgeons. If these injuries are not treated adequately, permanent loss of function or death may result. With these thoughts in mind, we will describe some of these injuries and discuss the radiographic anatomy, the knowledge of which should preclude some of the serious mistakes of diagnosis. PMID- 1727042 TI - Reduced cell-cell communication between mitotic and nonmitotic coupled cells. AB - The effects of mitosis on gap junctional intercellular communication (GJIC) were quantified in a clonal cell line of spontaneously immortalized rat granulosa cells (SIGC) using a fluorescence recovery after photobleaching assay. Reduction of GJIC was associated with the process of mitosis and was first apparent at the onset of prophase. Resumption of GJIC between newly divided cells and surrounding cells occurred slowly, requiring several hours following cytokinesis before reestablishment of maximal rates. Mitotic rates of GJIC in SIGC were comparable to values obtained in interphase cells partially uncoupled by 0.5 mM octanol. Limited studies of other cell lines generalized the mitotic-associated reduction of GJIC observed in SIGC. The data suggest that mitosis is one process which alters GJIC. This could be of significance when there is a change in the rate of proliferation, such as in the acquisition of immortalization, an early stage of transformation. PMID- 1727043 TI - Double-stranded RNA regulation of DNA synthesis in fibroblasts. AB - The double-stranded RNA molecule polyinosinic-polycytidylic acid (poly IC) has been found in some studies to have a mitogenic effect on fibroblast proliferation while other studies found poly IC to have an inhibitory effect on proliferation. In this study, we investigated whether a stabilized form of poly IC complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC) had a bidirectional effect on DNA synthesis in fibroblasts from four different cell lines and determined factors that potentially influence this bidirectional effect. In medium containing fetal bovine serum, poly ICLC slightly increased the levels of [3H]thymidine incorporation in growing fibroblasts in three of the four fibroblast cell lines tested, while poly ICLC increased [3H]thymidine incorporation in confluent, quiescent fibroblasts in two of four cell lines. Poly ICLC did not induce DNA synthesis in subconfluent, quiescent or in confluent, quiescent fibroblasts under serum-free conditions. Poly ICLC significantly suppressed serum-induced [3H]thymidine incorporation by quiescent fibroblasts in all cell lines. We conclude that the stimulatory and inhibitory effects of poly ICLC on DNA synthesis are influenced by both the cell line and the presence of serum components in the culture medium but not by population density. PMID- 1727044 TI - Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells. AB - We tried to make a well-characterized bacterial protein function in mammalian cell nuclei. For this purpose we chose Escherichia coli RecA protein and fused its carboxy terminus to the nuclear location signal of SV40 large T-antigen by oligonucleotide-dependent modification of the gene. When injected into the cytoplasm, the modified RecA protein (T-RecA for the T-antigen signal) accumulated efficiently in the nuclei, whereas the wild-type RecA protein remained in the cytoplasm. The T-RecA protein retained its original in vivo activity, judging from the finding that uv-sensitive bacteria (recA- E. coli) became uv-resistant on transformation with the T-recA plasmid as well as the recA plasmid. For expression of the T-recA gene in mammalian cells, the 5' region was replaced by the chicken beta-actin promoter and Kozak's initiation signal. A high level of expression was observed when Chinese hamster ovary (CHO-K1) cells were transfected with this plasmid. Indirect immunofluorescence examination revealed that the T-RecA protein in nuclei of mammalian cells bound to chromatin. PMID- 1727045 TI - Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells. AB - The laminin-like protein merosin was purified from human placenta in intact form and as pepsin fragments and compared to laminin in heparin affinity chromatography and cell binding assays. Intact merosin and a small fragment of merosin comprising the last two repeats of the heavy chain g domain bind to heparin. Intact merosin and large pepsin fragments of merosin, but not the small C-terminal fragment, mediate the attachment and spreading of several types of cells and promote neurite outgrowth from neuronal cells similar to laminin and its corresponding fragments. Cells with various integrin-type receptors for laminin attached equally well to merosin and laminin, suggesting that several of the known laminin binding receptors also bind to merosin. Antibodies to the beta 1 subunit of integrins inhibited neurite outgrowth on merosin as well as on laminin, confirming the involvement of integrin-mediated interaction of cells with both merosin and laminin. Schwannoma cells, which have previously been shown to produce a laminin-like, neurite-promoting factor, synthesize merosin in vivo and in vitro as shown by protein and mRNA analysis. The results suggest that merosin, which is the more abundant basement membrane protein in the laminin family, has properties very similar to laminin despite differences in the structure of the heavy chain. Furthermore, merosin may be identical to or a component of the neurite-promoting factors previously reported from heart, muscle, and Schwann cells. PMID- 1727046 TI - Micropatterned substratum adhesiveness: a model for morphogenetic cues controlling cell behavior. AB - It is generally considered that tracks of cell adhesiveness are important in controlling cell migration during the development and regeneration of many tissues. In order to investigate this experimentally, a number of techniques have in the past been employed to make patterns of differential adhesiveness for in vitro studies. However, practical limitations on patterning resolution and the introduction of residual topography to the experimental substrata have restricted their usefulness. Here we describe a simplified photolithographic technique for patterning cell adhesiveness which allows a high degree of flexibility and precision. We have quantified, using adhesion and spreading characteristics of BHK cells, the differential adhesiveness that can be created on patterned surfaces, how this alters with the duration of exposure to serum proteins, and how this, in turn, relates to the persistence of cell patterning despite increases in cell density. We believe that this technique will prove extremely useful for the detailed in vitro examination of the mechanisms controlling cell behavior as it offers a degree of precision and ease of fabrication that has previously been unavailable. PMID- 1727047 TI - Human ribosomal RNA gene repeats are localized in the dense fibrillar component of nucleoli: light and electron microscopic in situ hybridization in human Sertoli cells. AB - The distribution of the human ribosomal gene repeat within human Sertoli cell nucleoli was investigated with the help of DNA-DNA in situ hybridization at the light and electron microscopic level. Probes from both the transcribed part of the gene repeat and the "non-transcribed" spacer were found to hybridize predominantly to the dense fibrillar component of nucleoli. It therefore can be concluded that the dense fibrillar component of nucleoli is the major site of the intranucleolar location of the ribosomal DNA. This holds true not only for the dense fibrillar component adjacent to fibrillar centers, but also for the dense fibrillar component remote from the fibrillar centers. PMID- 1727048 TI - Calcium ions are required for the intracellular routing of insulin and its receptor. AB - We have studied the role of the cytosolic-free calcium concentration ([Ca2+]i) on the early and later internalization steps of insulin and its receptor. As before, we find that the rate of 125I-insulin internalization in HL60 cells remains normal when [Ca2+]i is lowered 10 times below normal resting level by the use of an intracellular Ca2+ chelator. By contrast, the subsequent intracellular steps, i.e. insulin receptor recycling and insulin degradation, are inhibited in calcium depleted cells. Under low [Ca2+]i conditions, the association of 125I-insulin with late endosomes and lysosomes is also reduced. This suggests that calcium ions are required for fusion processes occurring at the endosomal or postendosomal stage of internalization. Thus, by regulating insulin receptor recycling and by controlling insulin degradation, Ca2+ ions play a key role in the regulation of insulin action. PMID- 1727049 TI - Somatic and germ cell interactions during histogenetic aggregation of mouse fetal testes. AB - In the present study we examined the capacity of somatic and germ cells dissociated from fetal mouse testes at various stages to reform seminiferous cords in culture. We found that after 12 h in culture, seminiferous cords became segregated from stromal cells. Although Sertoli cells were incorporated into seminiferous cords at all stages studied, the germ cells dramatically changed their histogenetic behavior with age. Most germ cells which had been dissociated at 12.5 days postcoitum (dpc) were incorporated into the seminiferous cords, whereas at 14.5 dpc or later the majority remained among the stromal cells or as clusters on the surface of the aggregates. We considered three possible causes for this change in behavior of germ cells: (i) Failure to deposit some extracellular matrix components in the aggregates. (ii) Decrease in adhesiveness of prospermatogonia to either extracellular matrix components or Sertoli cells. (iii) A change in adhesiveness of Sertoli cells to germ cells with age. We found that laminin and fibronectin were similarly deposited in aggregates at 12.5 and 15.5 dpc. When prospermatogonia at 15.5 dpc labeled with colloidal gold were reaggregated with somatic cells at 12.5 dpc, 50% were incorporated into seminiferous cords. Moreover, [3H]thymidine-labeled Sertoli cells at 15.5 dpc formed heterochronic seminiferous cords with Sertoli cells at 12.5 dpc. These results suggest that mouse Sertoli cells change their surface property which is essential for binding to germ cells when they enter the mitotic resting stage (T prospermatogonia). PMID- 1727050 TI - Increased mitochondrial uptake of rhodamine 123 by CDDP treatment. AB - Rhodamine 123 (R 123) is a positively charged dye at physiological pH that accumulates specifically in the mitochondria of living cells without cytotoxic effect. In the present study, the uptake of R 123 by EL-4 lymphoma cells in culture with anticancer agents was measured by flow cytometry. Changes in R 123 uptake during the cultivation period were compared with cell distribution at different phases of the cell cycle. According to the increase in the proportion of S phase cells, mitochondrial synthesis increased, giving rise to a maximal fluorescence intensity of about 1.3-fold. Synchronous cultures showed the same relationship between increased mitochondrial uptake of R 123 and the S phase fraction as was observed in normal cultures. After treatment with 10(-3) M 5 fluorouracil (5-FU) for 1 h, EL-4 cells showed an increased binding of R 123 per cell followed by an accumulation of early S phase cells transiently. However, uptake of R 123 decreased 24 h later. On the contrary, after treatment with 10 micrograms/ml of cis-diamminedichloroplatinum (CDDP), a G2 + M block was observed from 12 h of reseeding and accumulation of the G2 + M cells continued. In this case, high uptake of R 123 continued during the observation period. From these results, mitochondrial synthesis seemed to increase according to the increment in proportion of S phase when the acceleration of the cell cycle turnover was augmented or the cycle was blocked in S phase by 5-FU. CDDP inhibited the cell division at G2 + M phase and caused increased R 123 fluorescence per cell. The stainability of R 123 may indicate the activity of cell division and may be a good way of evaluating the efficacy of antitumor drugs on the cells. PMID- 1727051 TI - The effect of carboxyl-terminal deletions on the nuclear transport rate of rat hsc70. AB - Rat brain hsc70 is a constitutively expressed member of the 70-kDa family of heat shock proteins that is capable of bidirectional transport across the nuclear envelope when microinjected into Xenopus oocytes [1]. The objective of this study was to identify domains involved in its bidirectional transport. Limited proteolytic digestion with chymotrypsin generated three major truncated proteins of approximately 67.5, 59.5, and 56.5 kDa. Reactivity with NH2-terminal-specific antibodies showed that carboxyl-terminal fragments were removed. Nuclear uptake studies were performed by microinjecting 125I-labeled proteins into the cytoplasm and determining their subsequent nucleocytoplasmic distribution. The accumulation rates, while faster than bovine serum albumin controls, were inversely related to the size of the truncated proteins and greatly reduced compared to undigested hsc70. Nuclear efflux was assayed by microinjecting labeled proteins directly into oocyte nuclei. The relative efflux rates of the truncated polypeptides were less than the undigested protein, and, as observed for uptake, were inversely related to size. These results indicate that the carboxyl-terminal domain of hsc70 is involved in its bidirectional exchange. PMID- 1727052 TI - Lack of correlation between the activation of the Ca(2+)-phosphatidylinositol cascade and the regulation of DNA synthesis in the dog thymocyte. AB - Changes in the [Ca2+]i and/or activation of phospholipase C are thought to participate in the control by several growth factors of the mammalian cell proliferation. It has even been claimed that activation of the Ca(2+) phosphatidylinositol cascade is sufficient to elicit cell proliferation [Jackson et al. (1988) Nature 335, 437-440; Julius et al. (1989) Science 244, 1057-1062]. In this work, we have evaluated the control of DNA synthesis by this cascade in a differentiated epithelial cell model: the dog thyrocyte in primary culture. We first observed that potent activators of the dog thyrocyte (2+) phosphatidylinositol cascade such as carbachol or bradykinin failed to promote the onset of DNA synthesis in these cells. Moreover, carbachol inhibited the mitogenic effect of thyroid stimulating hormone (TSH) and of epidermal growth factor (EGF). The mitogenic effect of EGF was also reduced by bradykinin. Nevertheless, carbachol enhanced the expression of the protooncogenes c-fos and c myc mRNAs. The time course of this enhancement was identical to the time course for the induction of c-fos and c-myc mRNAs by phorbol esters or EGF. On the other hand, in most experiments, TSH and EGF were able to trigger the onset of dog thyrocyte DNA synthesis without affecting their intracellular free Ca2+ concentration [Ca2+]i, 45Ca2+ efflux, or inositol phosphate generation. In several experiments, TSH increased the dog thyrocyte 45Ca2+ release and promoted a rise in the [Ca2+]i or the inositol phosphate accumulation but these effects were weak. In contrast to the effect of carbachol, the TSH effects on the [Ca2+]i and the 45Ca2+ efflux appeared slowly, were sustained, and were extremely sensitive to extracellular Ca2+ depletion. They were observed at hormone concentrations higher than the concentration achieving maximal stimulation of DNA synthesis. Similarly, in a few experiments, a slight increase in the [Ca2+]i or in the inositol trisphosphate generation were provoked by EGF. However, these modifications were not associated with an increased mitogenic potency of EGF. Finally, in all experiments, fetal calf serum slightly accelerated the dog thyrocyte 45Ca2+ efflux and increased their inositol phosphate generation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727053 TI - Novel nucleolar and nuclear morphology in a vincristine-dependent human leukemia cell line (L100). AB - Acquired resistance to chemotherapeutic drugs by tumor cells is an important obstacle to effective therapy of human malignancy. We now describe a vincristine (VCR)-induced multidrug-resistant (MDR) human acute lymphatic leukemia cell line, the sustained in vitro growth of which is dependent on vincristine. The doubling time for parental drug-sensitive cells (L0) is 40.2 +/- 13.2 h and for the MDR subline (L100) 62.5 +/- 11.3 h. L100 cells have similar G2 and mitotic phase to parental cells, express the MDR phenotype, and are characterized by novel morphologic features with multilobulated nuclei and multiple small nucleoli. Compared with L0 cells which have 2-3 nucleoli per cell, L100 cells have 7-8 nucleoli per cell. Average nucleolar area is 11.3 +/- 7.3 microns 2 for L0 and 2.5 +/- 2.4 microns 2 for L100 cells determined by the laser scanning method. The striking morphologic abnormalities of L100 cells suggest a drug-induced cytoskeletal abnormality. The relationship of these abnormalities to the VCR growth dependence of L100 cells is discussed. PMID- 1727054 TI - Estimating percentage constitutive heterochromatin by flow cytometry. AB - Flow cytometry is a powerful method for the assessment of both plant and animal genomes. One of the most interesting aspects is the analysis of chromatin structure. By using intercalating and base pair-specific fluorochromes, the chromatin structure in various cell cultures and microorganisms has been determined. In this study, several maize lines of known heterochromatic composition were analyzed. The nuclei of each line were isolated and stained with DAPI (base pair specific) and PI (intercalator) separately. For each maize line, the PI/DAPI ratio was determined. A significant negative correlation was observed between C-band number and PI/DAPI ratio (r = 0.920) and between percentage heterochromatin and PI/DAPI ratio (r = 0.997). Flow cytometry with use of the fluorochromes DAPI and PI was found to be a rapid and efficient method of determining heterochromatin amount in maize. PMID- 1727055 TI - Transforming growth factor-beta 1 acts cooperatively with sodium n-butyrate to induce differentiation of normal human keratinocytes. AB - Growth factors with established biological activity toward cultured normal human epidermal keratinocytes (NHEKs) (e.g., transforming growth factor-beta, TGF-beta; retinoic acid, RA) initiate programmed changes in cellular maturation which differ with regard to the specific differentiation pathway (normal or abnormal) analyzed. Sodium butyrate (NaB) initiates one form of epidermal differentiation leading to enhanced cornified envelope (CE) formation which involves abrogation of the normally inhibitory effect of RA on NHEK maturation. NaB also induces TGF beta mRNA in the maturing suprabasal compartment, suggesting that TGF-beta may play a role in NaB-initiated NHEK differentiation. Treatment with TGF-beta 1 alone, however, only marginally increased (by twofold) the number of detergent resistant CEs compared to control NHEKs and did not alter the prevalence of fully mature enucleated CEs. TGF-beta 1 was quite effective in inducing significant levels of CE expression when used simultaneously with suboptimal concentrations of NaB. The cooperative action of suboptimal NaB and TGF-beta 1 generated numbers of CEs which, in fact, exceeded the incidence of mature CEs formed in response to optimal levels of NaB alone. Neutralizing antibodies to TGF-beta, moreover, effectively reduced the incidence of CE formation in cultures treated with optimal NaB concentrations, further implicating endogenous TGF-beta activity in the NaB-initiated NHEK differentiation model. It is suggested, therefore, that within the NaB-induced pathway of NHEK differentiation, TGF-beta can positively modulate expression of the differentiated phenotype but alone is insufficient for generation of mature CEs. PMID- 1727056 TI - Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme. AB - The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells. PMID- 1727057 TI - Interaction of gelonin with macrophages: effect of lysosomotropic amines. AB - The cytotoxic effects of gelonin on various phagocytic and nonphagocytic cells were studied. Peritoneal exudate cells (PEC) are found to be more sensitive to gelonin compared to P388D1 and J774A.1 cells, nonphagocytic cells being the least sensitive. While chloroquine markedly enhances the cytotoxicity of gelonin in macrophages (greater than 100-fold) ammonium chloride confers protection. A higher rate of uptake of 125I-gelonin in PEC (7 times the rate observed in other cells) is probably mediated by an interaction of terminal mannose residues of gelonin with mannose receptors on PEC plasma membrane as inferred from a pronounced inhibitory effect of mannan. In contrast to a pronounced inhibitory effect of mannan on the uptake of gelonin in PEC (7-fold), the cytotoxicity is reduced only by 2-fold. On the other hand, mannan has little or no effect on enhancement of the toxicity of gelonin by chloroquine. The studies have suggested that the internalization of gelonin in PEC may involve two pathways: (a) mannose receptor-mediated endocytosis, which plays a minor role in the intoxication process, and (b) nonspecific fluid phase pinocytosis, which is susceptible to enhancement by chloroquine and has a major role to play in the manifestation of the toxic effect of gelonin. In macrophage-like cells only the latter pathway operates. PMID- 1727058 TI - Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration. AB - Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte specific functions. PMID- 1727059 TI - Basal cells are the progenitors of primary tracheal epithelial cell cultures. AB - The goal of this study was to identify the cells from the rat tracheal epithelium which attach and proliferate in primary culture. When cells isolated from tracheas by enzymatic digestion were held in suspension at 37 degrees C for several hours most of the differentiated cells died. The kinetics of this selective cell death were not dependent on the constituents of the holding medium. With time in suspension, the colony forming efficiency of the surviving cells increased two- to threefold. Comparison of the growth curves of cells held or plated directly showed no difference in the number of cells in the proliferating populations. Using two lectins, it was possible to monitor the loss of specific populations in suspension. BS1-B4 is a marker for basal cells and UEA 1 is a secretory cell marker. Only those cells that were BS1-B4 positive survived in suspension. Further, the colonies that formed in primary culture were positive for this marker. Single cell suspensions of cells were sorted by flow cytometry and a fivefold increase in the colony forming efficiency of BS1-B4 positive cells compared to that of the negative cells was observed. These findings suggest that the cells that survived in suspension and proliferated in culture originated from the basal cells of the trachea. PMID- 1727060 TI - Evolutionary conservation of a germ cell-specific lamin persisting through mammalian spermiogenesis. AB - We had identified earlier a germ cell-specific lamin of 60 kDa in rat which is related to somatic lamin B. This polypeptide was shown to be the only major component organizing the lamina structure of round spermatids. In the present study, we find that this 60-kDa polypeptide persists in the testicular and epididymal sperms of rat. We also show, by indirect immunofluorescence studies, that the 60-kDa protein is antigenically conserved in the germ cells of grasshopper, rooster, and frog and in plant meiocytes. The distribution of fluorescence among the various germ cell populations shows that the antigen is located around the nuclear cortex of pre- and postmeiotic germ cells, while it is distributed all over the pachytene nuclei. The anti-60-kDa polyclonal antibodies also reacted with a 60-kDa polypeptide in the Western blot analysis of nuclear matrix proteins of grasshopper germ cells. The similar fluorescent localization pattern of the antigen observed in various eukaryotic species strongly suggests that this germ cell-specific lamin may play a very crucial role during meiotic prophase, particularly during homologous chromosome pairing and recombination. PMID- 1727061 TI - Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators. AB - The human U373 glioblastoma/astrocytoma cell line was found to constitutively produce and secrete a plasminogen activator and a plasminogen activator inhibitor. The plasminogen activator was identified as urokinase based on apparent molecular weight, immunoblotting with anti-urokinase antibodies, and Northern blotting with a human urokinase cDNA probe. The inhibitor secreted by U373 cells was found to be related to the PAI-1 molecule based on reactivity with anti-human PAI-1 antibodies, apparent molecular weight, and Northern blot analysis with a human PAI-1 cDNA probe. The expression of both urokinase and the PAI-1-like molecule by U373 cells could be modulated by phorbol myristate acetate or by inflammatory mediators such as interferon-gamma and interleukin-1. In the case of interleukin-1, the alpha form exhibited no detectable effect while the beta form not only elevated inhibitor levels, it also appeared to induce the production of tissue plasminogen activator. Thus, in these cells interleukin-1 beta induces alterations in PA and PAI expression and interleukin-1 alpha does not, even though the two forms are reported to utilize the same cellular receptor. PMID- 1727062 TI - Role of reduced suppression of glucose production and diminished early insulin release in impaired glucose tolerance. AB - BACKGROUND: Insulin resistance and impaired insulin secretion both occur in non insulin-dependent diabetes (NIDDM), but their relative importance is unclear. Hyperglycemia itself has adverse effects on tissue insulin sensitivity and insulin secretion that make it difficult to distinguish between primary and secondary abnormalities. To avoid this problem we studied subjects with postprandial glucose intolerance but not sustained hyperglycemia. METHODS: We compared the rate of systemic appearance and disappearance of glucose, the output of endogenous hepatic glucose, splanchnic and muscle uptake of glucose, and plasma insulin and glucagon responses after the ingestion of 1 g of glucose per kilogram of body weight in 15 subjects with impaired glucose tolerance (8 of them nonobese and 7 obese) and in 16 normal subjects (9 nonobese and 7 obese) who were matched for age and weight. RESULTS: After glucose ingestion the mean (+/- SE) rate of total systemic appearance of glucose was significantly higher in both the nonobese subjects (455 +/- 12 mmol per five hours) and the obese subjects (486 +/ 17 mmol per five hours) with impaired glucose tolerance than in the respective normal subjects (411 +/- 11 and 436 +/- 7 mmol per five hours). This difference was fully accounted for by the reduced suppression of endogenous hepatic glucose in the subjects with impaired glucose tolerance (a reduction of about 28 percent, vs. 48 percent in the normal subjects; P less than 0.01). Despite late hyperinsulinemia, at 30 minutes the subjects with impaired glucose tolerance had smaller increases in plasma insulin and smaller reductions in plasma glucagon (both P less than 0.01). Molar ratios of plasma insulin to plasma glucagon levels correlated inversely (r = -0.62, P less than 0.001) with the rates of systemic glucose appearance; the latter correlated positively (r = 0.72, P less than 0.0001) with peak plasma glucose concentrations. CONCLUSIONS: Impaired glucose tolerance, the precursor of NIDDM, results primarily from reduced suppression of hepatic glucose output due to abnormal pancreatic islet-cell function. The late hyperinsulinemia may be the consequence of an inadequate early beta-cell response rather than of insulin resistance. PMID- 1727063 TI - Medical problems associated with underwater diving. PMID- 1727064 TI - Pulmonary hemorrhage in a patient with fibrillary glomerulonephritis. PMID- 1727065 TI - Do the right thing. Pain relief in infants and children. PMID- 1727066 TI - Angioplasty as a treatment for coronary artery disease. PMID- 1727067 TI - Clinical problem-solving--a new feature in the Journal. PMID- 1727068 TI - Physicians and healers--unwitting partners in health care. PMID- 1727069 TI - Sensory reinnervation of the heart after cardiac transplantation. PMID- 1727070 TI - Oral anticoagulant drugs. PMID- 1727071 TI - Presence of the French Canadian deletion in a French patient with familial hypercholesterolemia. PMID- 1727072 TI - Intrauterine growth retardation, perinatal death, and maternal homocysteine levels. PMID- 1727073 TI - The Food and Drug Administration and its problems. PMID- 1727074 TI - The Food and Drug Administration and its problems. PMID- 1727075 TI - Radium exposure in U.S. military personnel. PMID- 1727076 TI - Are tick-borne diseases also horse-borne? PMID- 1727077 TI - Radiation-induced edema after radiosurgery for pontine arteriovenous malformation. A case report and detection by magnetic resonance imaging. AB - A 29-year-old woman, who had undergone stereotactic radiosurgery for a pontine arteriovenous malformation, experienced sudden onset of facial nerve palsy with trigeminal nerve disturbance 19 months after irradiation. Magnetic resonance imaging revealed significant radiation-induced edema surrounding the nidus. Angiography demonstrated total obliteration of the arteriovenous malformation 24 months after irradiation. Further magnetic resonance imaging studies, performed 28 months after treatment, showed that, despite the persistence of symptoms, the radiation-induced edema had subsided. PMID- 1727078 TI - Ganglioglioma of the optic pathway. A case report. AB - A case of ganglioglioma of the optic pathway associated with congenital exophthalmos and strabismus is presented. Since the tumor extended from the right optic nerve to the right geniculate body, it was diagnosed as an optic glioma before operation. However, optic nerve biopsy showed that the lesion was a ganglioglioma. Although a literature review yielded two previous cases of ganglioglioma of the optic pathway, this is the first case in which the tumor involved the whole optic pathway. PMID- 1727079 TI - Single cervical exostosis. Report of a case and review of the literature. AB - The authors present a rare case of solitary cervical osteochondroma. Because of its rarity and its predilection for the atlantoaxial area, the diagnosis may be overlooked in evaluating patients having cervical myelopathy. Surgical decompression usually improves the patient's neurologic status. PMID- 1727080 TI - Shifting of dural arteriovenous malformation from the cavernous sinus to the sigmoid sinus to the transverse sinus after transvenous embolization. A case of left spontaneous carotid-cavernous sinus fistula. AB - The angiographic features of left spontaneous carotid-cavernous sinus fistula and multiple dural arteriovenous malformations that developed after transvenous embolization are described. A dural arteriovenous malformation involving the left sigmoid sinus was demonstrated, along with a marked decrease in size of the left carotid-cavernous sinus fistula and the disappearance of venous drainage from the left cavernous to the right cavernous sinus after embolization with spring coils via the left superior ophthalmic vein. The dural arteriovenous malformation of the left sigmoid sinus subsequently extended to the transverse sinus after partial embolization of the sigmoid sinus. Finally, a dural arteriovenous malformation involving the left transverse sinus developed, with the disappearance of the arteriovenous malformation affecting the sigmoid sinus and left carotid-cavernous sinus fistula following complete embolization of the sigmoid sinus via the left transverse sinus. PMID- 1727081 TI - Recurrent brain abscess due to an unexpected foreign body. AB - We report an exceptional case of a patient with chronic frontal sinusitis complicated by chronic osteomyelitis and a cutaneous fistula. A recurrent brain abscess developed and was only cured after a very unusual wooden retained foreign body was removed at surgery. The hazards of wood as a foreign body are discussed and it is stressed that the possibility of a retained foreign body, even unsuspected, must always be borne in mind. PMID- 1727082 TI - Intramedullary meningioma: case report and review of the literature. AB - Intramedullary meningioma is a rarely reported clinical entity. As far as we know, only three cases have been reported to date. We describe a further case at the cervical level and review the few published cases. PMID- 1727083 TI - Atypical Moyamoya disease associated with brain tumor. AB - A 4-year-old boy with right retinal hemorrhage, mental retardation, and multiple minor anomalies was referred to our hospital. Computed tomography scanning revealed a cystic brain tumor at the vermis. Angiography showed stenosis of both internal carotid arteries at the supraclinoid portion and the Moyamoya vessels. The right ophthalmic artery was dilated as wide as the internal carotid artery. Stenosis of the basilar artery was also observed. Collateral circulation via the posterior inferior cerebellar artery and Moyamoya vessels in the area of the posterior cerebral artery was observed. PMID- 1727084 TI - Adult "congenital" bilateral occlusion of the foramina of Monro. AB - Bilateral occlusion of the foramina of Monro was detected and treated in a hydrocephalic adult who developed rapid striking recent memory loss. She was treated by midline windowing of the third ventricle into the dilated lateral ventricles at a location 2 cm posterior to the occluded foramina of Monro. No inflammation was present. A biopsy specimen showed no evidence of malignancy. A reservoir was placed for long-term measurement of intraventricular pressure. Ten year follow-up with pressure measurements, serial computed tomography scans, and magnetic resonance imaging showed no evidence of tumor. PMID- 1727085 TI - Primary Ewing's sarcoma of the temporal bone. AB - Primary cranial Ewing's sarcoma is exceptionally rare. Only ten cases of such a tumor had been reported heretofore in the literature. We describe a case of primary Ewing's sarcoma occurring in the temporal bone. The tumor was surgically excised, and the patient underwent radiation and chemotherapy. Neither recurrence nor distant metastasis was noted at 12 months after surgery. Although the prognosis of Ewing's sarcoma in general is often poor because of early metastasis to the lungs and/or to other bones, a review of the literature suggested that the same tumor occurring in the cranium can often be successfully managed by intensive therapy with radical excision and radiochemotherapy. This inference was supported by the case reported here. PMID- 1727086 TI - Traumatic aneurysm of the middle meningeal artery presenting as delayed onset of acute subdural hematoma. AB - A case is presented in which recurrence of acute subdural hematoma developed 29 days after head trauma. An emergency craniotomy was complicated by intraoperative profuse bleeding, which was caused by the rupture of a large false aneurysm of the middle meningeal artery. A pitfall in the surgical treatment of this rare lesion is discussed. PMID- 1727087 TI - Is the hydrocephalic state progressive to become irreversible during fetal life? PMID- 1727088 TI - The uncitedness index. PMID- 1727089 TI - The course of lymphocytic hypophysitis. PMID- 1727090 TI - Effects of theophylline on the selective increases in intratumoral blood flow induced by intracarotid infusion of adenosine and adenosine triphosphate in C6 glioma-transplanted rat brains. AB - We have previously reported that the intracarotid administration of adenosine or adenosine triphosphate (ATP) selectively increased blood flow in intracerebrally transplanted C6 glioma cells in rats, using the hydrogen clearance method. In the present paper, we studied the difference between the effects of adenosine and ATP, using theophylline, a P1 purinoceptor blocker. The selective enhancement of the tumor blood flow by intracarotid administration of adenosine was almost totally inhibited by theophylline. In contrast, the selective enhancement by ATP was shown definitely not to be inhibited by theophylline. Therefore, it is supposed that the selective increase of intratumoral blood flow by the intracarotid infusion of adenosine is closely related to the P1 purinoceptor, and the effect of the intracarotid infusion of ATP is composed not only of the effect as degraded into adenosine but also of the effect of ATP itself. PMID- 1727091 TI - Predictors of thromboembolism in atrial fibrillation: I. Clinical features of patients at risk. The Stroke Prevention in Atrial Fibrillation Investigators. AB - OBJECTIVE: To identify those patients with nonrheumatic atrial fibrillation who are at high risk and those at low risk for arterial thromboembolism. DESIGN: Cohort study of patients assigned to placebo in a randomized clinical trial. SETTING: Five hundred sixty-eight inpatients and outpatients with nonrheumatic atrial fibrillation assigned to placebo therapy at 15 U.S. medical centers from 1987 to 1989 in the Stroke Prevention in Atrial Fibrillation study. Patients were followed for a mean of 1.3 years. MEASUREMENTS: Clinical variables were assessed at study entry and correlated with subsequent ischemic stroke and systemic embolism by multivariate analysis. MAIN RESULTS: Recent (within 3 months) congestive heart failure, a history of hypertension, and previous arterial thromboembolism were each significantly and independently associated with a substantial risk for thromboembolism (greater than 7% per year; P less than or equal to 0.05). The presence of these three independent clinical predictors (recent congestive heart failure, history of hypertension, previous thromboembolism of 2.5% per year (no risk factors), 7.2% per year (one risk factor), and 17.6% per year (two or three risk factors). Nondiabetic patients without these risk factors, comprising 38% of the cohort, had a low risk for thromboembolism (1.4% per year; 95% Cl, 0.05% to 3.7%). Patients without clinical risk factors who were under 60 years of age had no thromboembolic events. CONCLUSION: Patients with atrial fibrillation at high risk (greater than 7% per year) and low risk (less than 3% per year) for thromboembolism can be identified by readily available clinical variables. PMID- 1727092 TI - Obesity at diagnosis of breast carcinoma influences duration of disease-free survival. AB - OBJECTIVE: To study disease-free survival at 10 years in relation to obesity at the time of diagnosis. DESIGN: A prospective study of consecutively treated patients with primary breast cancer. SETTING: Memorial Sloan-Kettering Cancer Center, New York. PATIENTS: Nine hundred twenty-three women treated by mastectomy and axillary dissection. MAIN RESULTS: Women who were obese (25% or more over optimal weight for height) at the time of primary breast cancer treatment were at significantly greater risk for recurrence (42%) compared with nonobese patients (32%) 10 years after diagnosis (P less than 0.01). In multivariate analyses, obesity remained a statistically significant prognostic factor after controlling for measured tumor size, number of positive axillary lymph nodes, age at diagnosis, and adjuvant chemotherapy with a hazard ratio of 1.29 (95% CI, 1.0 to 1.67). When analyses were restricted to the 557 patients free of lymph node metastases, the hazard ratio of recurrence associated with obesity was 1.59 (CI, 1.06 to 2.39); 32% of obese patients developed recurrent disease compared with 19% of nonobese women. CONCLUSIONS: Obesity at the time of diagnosis is a significant prognostic factor that may limit the reduction in breast cancer mortality attainable through detection at an early stage of disease. Because obesity and the risk for breast cancer increase with age, interventions that encourage weight control may influence breast cancer survival rates. PMID- 1727093 TI - Treatment of toxoplasmic encephalitis in patients with AIDS. A randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulfadiazine. The California Collaborative Treatment Group. AB - OBJECTIVE: To compare pyrimethamine plus clindamycin (PC) to pyrimethamine plus sulfadiazine (PS) as a treatment for toxoplasmic encephalitis (TE) in patients with the acquired immunodeficiency syndrome (AIDS). DESIGN: Randomized, unblinded phase II, multicenter trial with provision for crossover for failure or intolerance of the assigned regimen. SETTING: University hospitals. PATIENTS: Eighty-four patients with presumptive TE were entered. Thirteen were excluded when they were found to have another diagnosis, and 12 were excluded because they did not meet entry criteria. The baseline characteristics in the remaining 26 patients randomized to PC and 33 randomized to PS were comparable. INTERVENTIONS: Patients were treated for 6 weeks with pyrimethamine and folinic acid plus either sulfadiazine or clindamycin. Clindamycin was given intravenously during the first 3 weeks. MEASUREMENTS AND MAIN RESULTS: There was a trend toward greater survival in patients randomized to PS (hazard ratio, 3.25; 95% CI, 0.63 to 16.8; P = 0.13), but most study deaths were not directly related to TE. In contrast, patients randomized to PC appeared more likely to achieve complete clinical (odds ratio, 0.67; CI, 0.2 to 1.97; P greater than 0.2) and radiologic responses (odds ratio, 0.28; CI, 0.08 to 0.96; P = 0.02). Multivariate analysis revealed drug effects to be largely independent of other variables. Similar efficacy of the treatments was also suggested by a hazard analysis of resolution of abnormal mental status, fever, and headache. Skin rash was the most common adverse event in both treatment arms. Because of toxicity, six patients randomized to PC and 11 patients randomized to PS had to switch to the alternate treatment, but only three were unable to complete therapy after crossover. CONCLUSIONS: The results of several end points of efficacy, taken together, suggest that the relative efficacy of PC approximately equals that of PS. PC appears to be an acceptable alternative in patients unable to tolerate PS. PMID- 1727094 TI - Usefulness of complete blood counts as a case-finding tool in medical outpatients. AB - OBJECTIVE: To determine the usefulness of routine complete blood counts as a case finding tool in medical outpatients. DESIGN: Prospective evaluation in a consecutive, unselected cohort. SETTING: A university-based outpatient clinic providing primary and referral care in general internal medicine. PATIENTS: A total of 595 patients (mean age [+/- SD], 40 +/- 15 years) making their initial clinic visit. INTERVENTION: A complete blood count was done in all patients. Components of the complete blood count that were not indicated for diagnostic or management purposes at the initial visit were classified as routine tests. MAIN OUTCOME MEASURES: The number and nature of new diagnoses leading to new patient management; the number of additional visits and diagnostic tests precipitated by positive results. MAIN RESULTS: Of the 2378 tests done, 1540 (65%) were routine tests. The percentages of abnormal test results for the four hematologic test components were as follows: leukocyte count, 7.6%; hemoglobin, 5.8%; mean corpuscular volume (MCV) of red blood cells, 7.9%; and platelet count, 4.5%. In only three patients (0.5%) was a new diagnosis made that led to a new management strategy (treatment of iron deficiency). Further work-up was unprofitable in 14 patients (2.4%). Two additional visits were necessary because of abnormal results on routine tests. No abnormality-associated clinical disease developed during a 15-month follow-up period in patients with unexplained abnormal results on routine hematologic tests. CONCLUSIONS: The routine complete blood count has limited usefulness as a case-finding tool and has a minimal effect on care in middle-aged medical outpatients. PMID- 1727095 TI - Autoimmune chronic hepatitis exacerbated by alpha-interferon. PMID- 1727096 TI - Diabetes mellitus induced by megestrol acetate in a patient with AIDS and cachexia. PMID- 1727097 TI - Predictors of thromboembolism in atrial fibrillation: II. Echocardiographic features of patients at risk. The Stroke Prevention in Atrial Fibrillation Investigators. AB - OBJECTIVE: To identify echocardiographic predictors of arterial thromboembolism in patients with nonrheumatic atrial fibrillation and to determine whether these add to clinical variables for risk stratification. DESIGN: Cohort study of patients assigned to placebo in a randomized clinical trial. SETTING: Five hundred sixty-eight inpatients and outpatients with nonrheumatic atrial fibrillation assigned to placebo therapy at 15 U.S. medical centers from 1987 to 1989 in the Stroke Prevention in Atrial Fibrillation study. Patients were followed for a mean of 1.3 years. MEASUREMENTS: M-mode and two-dimensional (2-D) echocardiograms performed at study entry and interpreted by local cardiologists. The predictive value of 14 echocardiographic variables for later ischemic stroke or systemic embolism was assessed by multivariate analysis. MAIN RESULTS: Left ventricular dysfunction from 2-D echocardiograms (P = 0.003) and the size of the left atrium from M-mode echocardiograms (P = 0.02) were the strongest independent predictors of later thromboembolism. Multivariate analysis of these two independent echocardiographic predictors with the three independent clinical predictors of thromboembolism (history of hypertension, recent congestive heart failure, previous thromboembolism) identified 26% of the cohort with a low risk for thromboembolism (1.0% per year; 95% Cl, 0.2% to 4.0%). Compared with risk stratification using clinical variables alone, echocardiographic results altered thromboembolic risk stratification in 18% of the entire cohort and in 38% of those without clinical risk factors. CONCLUSIONS: Both left ventricular and left atrial variables are significant predictors of thromboembolism in patients with nonvalvular atrial fibrillation. Our results challenge traditional views of the pathogenesis of ischemic stroke in patients with atrial fibrillation and suggest that standard echocardiography contributes to risk stratification, differentiating the one third of patients without clinical risk factors who are at increased risk for stroke from the remainder who may not need antithrombotic prophylaxis. PMID- 1727098 TI - Alpha-interferon treatment of chronic hepatitis C: need for accurate diagnosis in selecting patients. PMID- 1727099 TI - Evidence for sexual transmission of human T lymphotropic virus type II. PMID- 1727100 TI - Cocaine and chest pain. PMID- 1727101 TI - Treating hypertension in women. PMID- 1727102 TI - Cyclophosphamide-induced myopia. PMID- 1727103 TI - The Tinu syndrome or the Sjogren syndrome? PMID- 1727104 TI - Eggs and Salmonella enteritidis transmission. PMID- 1727105 TI - Setting probabilities: lost in the translation. PMID- 1727106 TI - Equivalence of continuous and pulse simulated low dose rate irradiation in 9L gliosarcoma cells at 37 degrees and 41 degrees C. AB - The development of a brachytherapy technique that will use a scanning source to simulate continuous low dose rate irradiation holds the possibility of improving dose distributions and other clinically relevant factors as well as enhancing radiation safety. Rat 9L gliosarcoma cells growing in vitro have been used as a model to determine the role of fraction size when individual pulses of irradiation are given at appropriate intervals to result in an overall dose rate that is identical to currently applied continuous low dose rate irradiation. With an overall dose rate of 0.5 Gy/hr, cell killing was identical for fractionation schemes of 0.25 Gy every 0.5 hr, 1.00 Gy every 2.0 hr, and 3.00 Gy every 6.0 hr. The cell sensitivity of these schemes was also identical to continuous irradiation at the same overall dose rate. Increasing the fraction size to 6.0 Gy with intervals of 12 hr increased the cytotoxicity. This breaking point was above the Dq (3.9 Gy) of acutely irradiated 9L cells. These data support the hypothesis that continuous low dose rate irradiation can be simulated by fractionated high dose rate irradiation as long as the fraction size remains less than the Dq of the acute radiation response of the cells and the overall dose rate remains constant. The role of simultaneous heating at 41 degrees C during pulsed and continuous low dose rate irradiation was also investigated. Substantial sensitization was observed for both continuous low dose rate irradiation and pulse simulated low dose rate irradiation. The Do thermal enhancement ratios were 1.98 and 1.92, respectively. The above results demonstrate that modalities utilizing intermittent high dose rate irradiation can be designed such that they will be equivalent to continuous low dose rate irradiation, and that in either case simultaneous extended low temperature heating can greatly enhance the cytotoxic effects of these irradiations. PMID- 1727107 TI - Interaction of misonidazole, hyperthermia, and irradiation in a C3H mammary carcinoma and its surrounding skin in vivo. AB - The interaction of irradiation, Misonidazole (MISO), and hyperthermia was studied in a C3H mouse mammary carcinoma and its surrounding skin in vivo. MISO (0.5-1.0 mg/g) was injected 30 min before irradiation. Hyperthermia (41.5 degrees-43.5 degrees C for 60 min) was given either simultaneously, 0.5 hr, or 4 hr after X rays. The results were evaluated as the radiation dose to achieve tumor control (TCD50) or moist desquamation of the skin (DD50) in half of the treated animals. A therapeutic gain was found when the enhancement in tumors were greater than that found in skin. The combination of simultaneous heat and irradiation caused great enhancement in radiation response, but with no therapeutic gain. A slightly lower enhancement of the damage in both tissues was found with a 30 min interval between irradiation and hyperthermia, whereas heat 4 hr after X rays gave a small, but significant therapeutic gain. MISO significantly enhanced the response in tumors but not in skin. Combined trimodality treatment with MISO, irradiation, and hyperthermia resulted in enhancement ratios up to 15, dependent on temperature, radiation-heat interval, and to a lesser extent the MISO dose. The enhancement was for all schedules most pronounced in the tumors, resulting in an improved therapeutic effect. The combination of MISO and hyperthermia may be a valuable addition to radiotherapy, especially if heat and irradiation can be applied with close interval and with one of the modalities given selectively to the tumor. PMID- 1727108 TI - Relationship between vascular thermotolerance and intratumor pH. AB - The changes in blood flow, intratumor pH, and clonogenicity of tumor cells after one and two heatings were studied in SCK tumors of A/J mice. When SCK tumors were heated at 42.5 degrees C for 1 hr, vascular thermotolerance promptly developed and peaked at 18 hr post-heating. The intratumor pH was 7.05 +/- 0.14 (mean +/- S.D.) in control SCK tumors of A/J mice, with a significant decrease (p less than or equal to 0.001) to 6.86 +/- 0.08 and 6.70 +/- 0.08 when heated for 1 hr at 43.5 degrees C and 44.5 degrees C, respectively. However, when the vascular thermotolerance was at its peak, heating at the same doses caused little change in the intratumor pH. When SCK tumors were heated for the first time at 44.5 degrees C for 1 hr and left in situ, the number of clonogenic cells significantly declined. Such a secondary cell death could be attributed to the deterioration of the intratumor environment ensuing from the vascular damage. When the tumor vessels were thermotolerant, however, virtually no secondary cell death occurred after heating. PMID- 1727109 TI - Prophylactic cranial irradiation dose effects on late cognitive function in children treated for acute lymphoblastic leukemia. AB - Prophylactic central nervous system treatment has dramatically improved the disease-free survival of children with acute lymphoblastic leukemia (ALL). Long term neuropsychological sequelae are documented in children who received 2400 cGy prophylactic cranial irradiation. The dose was reduced to 1800 cGy. Available reports on developmental consequences, with short follow-up, have yielded inconsistent results. This study assesses radiation dose effects on cognitive function in children with leukemia who received central nervous system prophylaxis with 2400 cGy versus 1800 cGy whole brain radiotherapy. All leukemic children also received intrathecal methotrexate. A control group of children (treated for Wilms' tumor) received no central nervous system therapy. Nineteen children were treated with 2400 cGy, 16 children with 1800 cGy. The 12 control children received no irradiation. All patients were off therapy for at least 70 months. The 1800 cGy and 2400 cGy patient groups were off therapy for equivalent periods of time (range 70-123 mo) at follow-up testing. Mean age at diagnosis was 49 months, at testing: 142 months. The male to female ratio was 1/1. Standardized psychological tests were administered. Full-Scale, Verbal, and Performance IQ were measured with the Wechsler Intelligence Scale for Children-Revised. Wide Range Achievement Testing evaluated reading, spelling, and arithmetic abilities. Children treated with 1800 cGy performed significantly better than those who received 2400 cGy, and at the same level as controls. There were statistically significant differences between the 1800 cGy and 2400 cGy subjects in all measures. 2400 cGy patients had deficiencies in IQ and academic performance. 1800 cGy patients scored approximately 12 points higher than 2400 cGy children. Eleven children, two in the control group, two in the 1800 cGy, and seven in the 2400 cGy group had IQ scores of less than 90. Eight of the nine irradiated children with deficits had radiotherapy before age 5. These results indicate a mild, but diffuse information processing deficit in children who received 2400 cGy, but not in children who received 1800 cGy. These findings with a minimum of 6 years of follow-up provide new information on late effects of CNS prophylaxis in ALL. Reducing the cranial RT dose from 2400 cGy to 1800 cGy reduced neurotoxicity to acceptable levels. PMID- 1727110 TI - Three-dimensional theoretical temperature distributions produced by 915 MHz dipole antenna arrays with varying insertion depths in muscle tissue. AB - Interstitial microwave antenna array hyperthermia (IMAAH) systems are currently being used in the treatment of cancer. The insertion depth of an interstitial microwave antenna, defined as the length of the antenna from the tip to the point of insertion in tissue, affects its ability to produce uniform power deposition patterns in tumor volumes. The effect of varying insertion depths on the ability of an IMAAH system to heat two theoretical tumor models was examined. Four dipole microwave antennas were implanted in a 2 x 2 cm array and driven at 915 MHz in muscle tissue. The explicit power deposition patterns were calculated for each insertion depth using known theory. The bioheat transfer equation was solved for the 3-dimensional steady-state temperature distributions in cylindrical and ellipsoidal tumor models using a finite element method. Homogeneous and nonhomogeneous blood flow models were considered. As a basis of comparison of the various temperature distributions, the volume of tumor heated to greater than or equal to 43 degrees C was calculated. Under the conditions of this study, the insertion depth was shown to have a significant effect on the ability of an IMAAH system to heat the tumor volumes. A sharp decrease in the percentage of tumor volume heated to greater than or equal to 43 degrees C was seen for insertion depths between 7.8 and 14.6 cm. At an insertion depth of 11.7 cm (3/4 lambda) there was virtually no heating of the tumor. Regions of elevated power occurred outside of the desired treatment volume, stressing the importance of adequate thermometry techniques and demonstrating the need for hyperthermia treatment planning prior to implantation of an antenna array. Plots of the power deposition patterns and the corresponding temperatures produced in the diagonal plane of the antenna arrays are present. PMID- 1727111 TI - Phase I trial of postoperative 5-FU, radiation therapy, and high dose leucovorin for resectable rectal cancer. AB - Following surgery for Stages T3-4N0-2M0 primary and recurrent resectable rectal cancer limited to the pelvis, 25 patients have been entered on a Phase I trial of pelvic radiation therapy (RT) [5040 cGy] and 12 cycles of postoperative 5-FU and high dose Leucovorin (LV) chemotherapy. 5-FU was escalated 50 mg/m2 while the LV remained constant at 200 mg/m2. The initial doses of 5-FU were: combined RT/chemotherapy = 200 mg/m2 and post-RT chemotherapy = 325 mg/m2. The median F/U was 25 months (range: 13-36). Two maximum tolerated doses (MTD's) have been determined, one for combined-RT/chemotherapy and one for post-RT chemotherapy. The MTD for combined-RT/chemotherapy was 250 mg/m2; therefore, the recommended dose of 5-FU is 200 mg/m2. The MTD for post-RT chemotherapy was 375 mg/m2; therefore, the recommended dose of 5-FU is 325 mg/m2. The dose limiting toxicities were diarrhea, tenesmus, frequent bowel movements, dysuria, and myelosuppression. For the nine patients who received 5-FU at the recommended dose level the median low counts were WBC 3.5 (2.2-4.0), HGB 10.3 (9.0-12.3), and PLT (x 1000) 167 (133-280), and the incidence of any grade greater than or equal to 3 toxicity was 22% diarrhea, 17% tenesmus, and 22% frequent bowel movements. The recommended dose of combined-RT/chemotherapy as used in this protocol was relatively well tolerated. However, optimal doses of 5-FU cannot be delivered until the fourth postoperative month. Therefore, despite the encouraging results reported with high dose LV in patients with advanced disease, we do not recommend that high dose LV be used with combined RT and 5-FU in the treatment regimen as presently designed. PMID- 1727112 TI - Radiation oncology residents' computer workstation. AB - We are investigating the feasibility of using the Macintosh computer as a workstation platform for radiation oncology residents because of its ease of use, graphics capability, and low cost. Hypercard was chosen as the programming environment because it easily mixes graphics, text, and control functions in an integrated screen display. Furthermore, it results in a system that can be relatively easily extended and customized by individual users with varying degrees of computer skills. We have developed several software modules in order to test the ability of this environment to support the demands of such a workstation. Modules created thus far include various clinical physics aids and tutorials, treatment planning guides, oncology databases, and others. The software runs on all Macintosh configurations, but calculation speeds are improved when a 68020 or greater processor is used. In general, we have been pleased with the implementation thus far. Graphics display capability is good, but design and entry of graphics have proved labor-intensive. Searching is fast and text is easily entered and manipulated. Finished modules can be customized with minimal computer training, but implementing complex new functions requires familiarity with Hypercard's programming language. New modules, once developed, are easily integrated into the workstation universe, suggesting that cooperative development of the workstation by multiple contributors is realistically achievable. PMID- 1727113 TI - Daily monitoring and correction of radiation field placement using a video-based portal imaging system: a pilot study. AB - We have developed a video-based portal imaging system for radiotherapy localization. The system can acquire high quality portal images automatically using short (1-3 monitor unit) irradiations and immediately display the images. The major advantage of the imaging system is that it can be used routinely to check and correct patient positioning before much of the daily irradiation has been delivered. The portal imaging system has been used in a pilot study to monitor five patients during each of their daily treatments. The study has shown that: (i) image quality is sufficiently high to detect discrepancies in field placement from that prescribed on the simulator film; (ii) discrepancies in field placement occur frequently; and, (iii) routine correction of patient and block positioning can reduce the size of these discrepancies. This is the first time that field placement in radiation therapy has been checked and corrected routinely, before the treatment irradiation. However, limitations in the size of the field of view and in the methods of extracting and presenting the geometric information to the users limits the clinical utility of the imaging system. Solutions to these limitations are currently under development. PMID- 1727114 TI - Custom beam profiles in computer-controlled radiation therapy. AB - A computer-controlled radiation therapy technique is demonstrated which uses multiple concurrent boost fields to modify the beam profile of a conventional treatment beam. A principal field, identical to that of a corresponding conventional treatment plan, delivers the major component of the prescribed dose. Dose increments given from boost fields placed within this principal field compensate for variations in patient anatomy, for variations in target volume shape, and/or for imperfect beam characteristics, such as excessive off-axis dose or inadequate beam wedge angle. This concurrent boost field technique is demonstrated for several treatment sites. It produces significant improvement in uniformity of dose delivered to the target compared to conventional treatment. Implementation of these treatments requires a computer-controlled linear accelerator with independently-movable collimator jaws, an automatic beam set-up procedure, and a patient prescription database. Since all fields are delivered under computer control, concurrent boost technique treatment times are not much longer than those of conventional treatments. PMID- 1727115 TI - Computer controlled stereotaxic radiotherapy system. AB - A computer-controlled stereotaxic radiotherapy system based on a low-frequency magnetic field technology integrated with a single fixation point stereotaxic guide has been designed and instituted. The magnetic field, generated in space by a special field source located in the accelerator gantry, is digitized in real time by a field sensor that is six degree-of-freedom measurement device. As this sensor is an integral part of the patient stereotaxic halo, the patient position (x, y, z) and orientation (azimuth, elevation, roll) within the accelerator frame of reference are always known. Six parameters--three coordinates and three Euler space angles--are continuously transmitted to a computer where they are analyzed and compared with the stereotaxic parameters of the target point. Hence, the system facilitates rapid and accurate patient set-up for stereotaxic treatment as well as monitoring of patient during the subsequent irradiation session. The stereotaxic system has been developed to promote the integration of diagnostic and therapeutic procedures, with the specific aim of integrating CT and/or MR aided tumor localization and long term (4- to 7-week) fractionated radiotherapy of small intracranial and ocular lesions. PMID- 1727116 TI - Computed tomography treatment planning in IR-192 brachytherapy in the head and neck. AB - Brachytherapy dose prescription and treatment planning lag behind the state-of the-art for external beam therapy. As altered fractionation of external beam therapy improves patient outcome in head and neck cancer, there will be an increased need to compare the two radiotherapy techniques. Currently, implant techniques and dose prescription documentation are not uniform, dose prescription to a target volume is subjective, and implant quality is poorly understood and not routinely assessed. All contribute to a lack of scientifically rigorous brachytherapy clinical trials. Studies designed to combine tumor imaging and dosimetry data are important in the evolution of brachytherapy treatment planning. Head and neck implants, which often require nonparallel, arching, or looping source carriers for all but small tumors in order to encompass the target volume adequately, were used to evaluate the clinical utility and feasibility of computed tomography as a treatment planning tool in brachytherapy. Following placement of plastic afterloading tubes under general anesthesia, orthogonal radiographs with dummy sources in the afterloading tubes are obtained as customary for source localization. With the patient in the same position, axial CT scans are obtained with the dummy seeds still in place for treatment planning. The implant physician, using data from the pre-treatment diagnostic CT scan, outlines target areas on sequential images creating a 3-dimensional target volume. By superimposing anatomic data with isodose curves one can objectively define implant parameters important in clinical trials analysis. These include minimum target absorbed dose, implant uniformity, and treatment to target volume ratio. The results of the first 10 patients are presented and implications of these data regarding the analysis of implant technique, implant quality, and implant optimization are discussed. The technique as performed is laborious but practicable in the clinical research setting of head and neck implant. Further research efforts should improve, simplify, and objectify brachytherapy and hasten the time when rigorous multi-institutional brachytherapy trials will be reality. PMID- 1727117 TI - Study of elasto-gel pads used as surface bolus material in high energy photon and electron therapy. AB - Bolus materials are occasionally used during the high energy photon and electron radiation treatments of head, neck, breast, and chest wall areas in order to deliver the full prescribed dose to the skin surface and underlining tissue. Where skin surface curvatures make uniform contact between the bolus material and skin surface difficult, we have studied a new bolus material called Elasto-gel. Elasto-gel, packed in a sterilized envelope, is routinely used in the treatment of superficial burns and wounds. The adhesive nature of Elasto-gel bolus material provides excellent skin contact without air gaps. The Elasto-gel is made of non toxic material and is transparent. High dose irradiation has no effect on the appearance and property of the Elasto-gel pad. We are presenting in this note the radiation attenuation and build up effects of the Elasto-gel pads of photon energies of 6 and 18 MV and of electron energies of 6 MeV and 20 MeV. There is little difference between Elasto-gel and Polystyrene in radiation dosimetry. PMID- 1727118 TI - A simple CT aperture emulator for use with a radiotherapy simulator. AB - Tumor localization in radiation treatment planning often involves the generation of quantitative anatomical data from multiple imaging modalities. It is desirable to take all of the images in the selected treatment position, which is usually decided upon during the initial simulator session. The different scanning modalities are often operated by different staff, at different times and in different locations; thus, it is difficult to ensure consistency in the position of the patient's body, and its documentation, at various times and places. Also, devices such as CT and MR scanners frequently pose restrictions due to their limited apertures. Failure to consider the physical limitations of such scanning equipment at the time of simulation or localization may result in placing the patient in a treatment position which will not fit through the aperture of the CT (or MRI) scanner, or which will result in a clinically important portion of the anatomy being "cut off" in the resulting scans. This can lead to re-simulation of the patient or result in a lack of accurate coordination of simulator and CT scan data. To minimize problems such as these, we have developed a CT Aperture Emulator which can be used at the time of the initial simulation. This is a lightweight "halo" easily attached to the simulator, which mimics the size and shape of the CT aperture. It permits reproducible adjustment of the patient's position, while allowing technologists and physicians to set up the patient with respect to potential CT constraints, in particular with regard to the use of immobilization and support devices. The emulator device also facilitates reproducing a patient's treatment position on the CT scanner. The concept has been found to have additional clinical uses and can be extended to a variety of imaging equipment. PMID- 1727119 TI - Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole. AB - Fluoromisonidazole (FMISO) has been shown to bind selectively to hypoxic cells in vitro and in vivo at radiobiologically significant oxygen levels. When labeled with the positron emitter fluorine-18 (F-18), its uptake in tissue can be detected quantitatively with high precision by positron emission transaxial tomography (PETT). This paper presents the first experiences with PETT imaging of [F-18]FMISO uptake in human malignancies, and describes the development of this technique as a tool for the non-invasive assessment of tumor hypoxia. Eight patients with selected cancers were imaged prior to primary radiotherapy, and 3 returned for follow-up scans, for a total of 11 imaging studies. Six of eight pre radiotherapy studies revealed retention of [F-18]FMISO in tumors that significantly exceeded plasma concentrations by 2 hr after drug injection; all five patients with head and neck primaries had such "positive" scans. An analytic method for the interpretation of [F-18] FMISO PETT images is presented, defining hypoxic elements within a tumor volume as regions with a threshold regional tumor:plasma [F-18]FMISO ratio of greater than or equal to 1.4 by 2 or more hours after injection. Toward the end of a course of fractionated radiotherapy, three repeat studies in patients with initially positive scans showed no tumor accumulation of drug above the threshold ratio of 1.4, suggesting reoxygenation had occurred. Pharmacokinetic and dosimetry data support continued use of [F 18]FMISO as a safe hypoxia probe. Two imaging protocols have been developed for human studies; a long protocol allows for more complete biodistribution and dosimetry information, and a shorter protocol facilitates increased patient accrual by applying a simple, clinically expedient imaging procedure. When correlated with tumor outcome, [F-18]FMISO PETT imaging may be developed as a predictor of tumor response to conventional radiotherapy. The implications of this technique in addressing persistent questions of tumor hypoxia in human oncology is discussed. PMID- 1727120 TI - Chemotherapy of testis cancer: a review. PMID- 1727121 TI - HDR brachytherapy for carcinoma of the cervix: fractionation considerations. PMID- 1727122 TI - Fractionation is important for HDR cervix cancer brachytherapy. PMID- 1727123 TI - Critique of 'a management approach to incompletely excised basal cell carcinoma of the skin'. PMID- 1727124 TI - Increased acute mucosal and cutaneous radiation toxicity in two patients taking amiodarone. PMID- 1727125 TI - The use of high dose rate endobronchial brachytherapy to palliate symptomatic endobronchial recurrence of previously irradiated bronchogenic carcinoma. AB - Thirty-eight patients were treated with high dose rate endobronchial brachytherapy to palliate symptoms (cough, hemoptysis, fever, and/or shortness of breath) caused by endobronchial of previously irradiated (greater than or equal to 5000 cGy) bronchogenic carcinoma. The dose per fraction was 600 cGy at a radius of 1 cm from the center of the linear path of the source, and each patient received three fraction, each fraction separated by a 1-week interval. Twenty nine patients (76%) had symptomatic improvement, 16 with complete and 13 with partial relief of symptoms. The likelihood of symptom relief was greater in those patients who had extra-bronchial tumor measuring less than 5 cm (15/15) compared to those with extra-bronchial tumor measuring greater than or equal to 5 cm (2/8). The median duration of symptom relief was 7.5 months. Repeat bronchoscopy done 3 months after brachytherapy revealed that 41% (11/27) had complete tumor regression and another 41% (11/27) had partial regression. Nine of 14 patients with post-obstructive atelectasis/pneumonitis had radiographic improvement. Twelve patients (32%) died from massive hemoptysis occurring 2-56 weeks (median 10 weeks) after brachytherapy. Location of the recurrence was the most important predictor of pulmonary hemorrhage. It occurred only in patients with recurrence in the right upper lobe, right mainstem, or left upper lobe bronchus. Whether this high rate of fatal pulmonary hemorrhage was a real phenomenon or a statistical fluke of small numbers remains an unanswered question. PMID- 1727126 TI - Dose, time, and fraction size issues for late effects in head and neck cancers. AB - This paper contains a statistical analysis of the dose-time factors influencing late complications in 784 patients with squamous cell carcinomas of the pharynx or larynx treated with external beam irradiation only at the University of Florida. The patients include 560 who received continuous course once-a-day therapy, 116 who received twice-a-day treatment, and 108 who received a once-a day split course regimen. Both 2+ and 3+ complications were considered. Fifty-six patients developed either of these complications. The factors included in the analysis were site and size of the primary, total dose, fraction size, and treatment time. The linear-quadratic model was used to incorporate fraction size into the analysis. Proportional hazards analysis, which models the time to occurrence of the late complication, was used to quantify the joint influence of the above patient and fractionation variables on the incidence of late effects. The occurrence of the late effects was heterogeneous, with only a weak relationship to the patient and fractionation variables. The influence of the size of the primary was significant, with larger primaries associated with higher complication rates independent of fractionation variables. For oropharynx primary sites there was no significant effect of the fractionation variables. For larynx and hypopharynx, excluding T1-T2 true vocal cord, there was a significant effect of total dose and fraction size. The alpha/beta ratio was estimated to be 7.8 Gy (95% confidence interval, 3.0, infinity). There was no significant effect of overall treatment time. The estimated 2+ complication rate at 1 year from 68 Gy given in 2 Gy fractions in 50 days is 0.1% for T 1-2 vocal cord, 4.1% for T1-T2 supraglottic larynx, 3.8% for T3 supraglottic larynx and vocal cord, 14.9% for T4 supraglottic larynx, 6.7% for T1-T2 tonsil and soft palate, 7.6% for T3-T4 tonsil and soft palate, 7.0% for T1-T2 pyriform sinus and pharyngeal wall, and 13.0% for T3-T4 pyriform sinus and pharyngeal wall. PMID- 1727127 TI - Patterns of failure and survival in locally advanced carcinoma of the uterine cervix treated with high dose-rate intracavitary irradiation system. AB - We retrospectively analyzed 71 patients with locally advanced carcinoma of the uterine cervix treated by irradiation using high dose-rate intracavitary brachytherapy between 1978 and 1985. Seven patients were Stage IIIa, 46 Stage IIIb, and 18 Stage IVa. Five-year survivals for Stage IIIa, IIIb, and IVa were 71.4, 60.9, and 16.7%, respectively. An analysis of patterns of failure demonstrated that loco-regional recurrences were observed in 1 (14.3%) for Stage IIIa, 6 (13.0%) for Stage IIIb, and 9 (50.0%) for Stage IVa. The incidence of recurrence outside the pelvis observed in Stage IIIb patients (7 para-aortic nodes, 5 distant metastases) was much higher than that of local recurrence. Five patients (7.0% of the total: 1 with Stage IIIa, 3 with Stage IIIb, 1 with Stage IVa) required surgery to manage the complications. These data suggest that a high dose-rate intracavitary irradiation system is an effective tool for the treatment of cervical cancer. Further efforts to control metastatic lesions outside the pelvis are required for patients with Stage IIIb. To increase a loco-regional control rate for patients with Stage IVa disease, it is important to give additional treatment such as chemotherapy in conjunction with radiation therapy. PMID- 1727128 TI - Selection of reagents for human radioimmunotherapy. AB - Promising response rates are noted in patients with refractory Hodgkin's disease after radioimmunoglobulin therapy (RIT) with Yttrium-90 labeled polyclonal antiferritin. To explore the most efficacious selection of RIT reagents for use in humans, experimental animal data are reviewed for radiolabeled antiferritin and B72.3. Nude mice with subcutaneously implanted human malignancies provide an excellent primary screen for radiolabeled antibodies under consideration for use in humans. They provide information on the potential of a new reagent to target a human malignancy in vivo. The other determinant of the therapeutic ratio of RIT reagents--normal tissue toxicity--is best analyzed in large animals, such as dogs. Hematologic toxicity is dose limiting in all species and best predicted by a prescription of radiolabeled antibodies in mCi per kilogram body weight and the presence or absence of bone marrow targeting. Per cGy, RIT is more effective in causing BM damage in dogs than in rats. In dogs, bone marrow transplantation with autologous cryopreserved bone marrow cells or G-CSF treatment can accelerate hemopoietic recovery and granulopoiesis, respectively, after RIT. When dose escalation beyond bone marrow toxicity is performed, the liver (dog) or the intestinal tract (rat) become the next dose limiting tissue in dose escalation studies. Significant improvement in RIT results will be achieved when the normal liver uptake of chelated monoclonal antibody in dogs and in human patients can be prevented. The described animal models and continued investigations of RIT in patients with endstage Hodgkin's disease will allow for further improvement in the therapeutic ratio of RIT and the applicability of RIT in humans. PMID- 1727129 TI - High-dose single-fraction brain irradiation: MRI, cerebral blood flow, electrophysiological, and histological studies. AB - Radiation-induced alterations in cerebrovascular and metabolic function form the basis for the radiosurgical treatment of selected intracranial vascular malformations and tumors in human patients. However, the underlying mechanisms, temporal progression, and modifying factors involved in the radiosurgical obliteration of these intracranial lesions as well as the risks of delayed radiation injury to surrounding normal brain remain poorly understood. In this report, the rabbit brain was used as an animal model to examine the effects of high-dose single-fraction X-irradiation on magnetic resonance imaging (MRI) appearance, neurophysiologic function, and histological integrity. At approximately 10 weeks following left-hemisphere irradiation with 60 Gy (225 kVp) X rays, MRI studies showed radiation-induced changes including blood-brain barrier (BBB) perturbations in the white matter regions and the hippocampus. Significant reductions in regional cerebral blood flow (rCBF) ratios were found in the hippocampus and certain regions of the cortex in irradiated animals. However, no changes in somatosensory evoked potentials (SEP) were observed. Histological studies demonstrated telangiectatic vessels, spreading edema in the white matter, and focal regions of necrosis and hemorrhage in the irradiated cortices and hippocampi. These results demonstrate that the irradiated rabbit brain may be used as an experimental model to correlate the spatiotemporal pattern of functional changes with radiologic and histological changes in delayed radiation injury. PMID- 1727130 TI - The effect of single doses of radiation on mouse spinal cord. AB - We have used a mouse model to study spinal cord injury following single doses (12 to 75 Gy) of radiation. The spinal cord (T9,10-L4,5) of C3Hf/Sed//Kam mice was irradiated and response graded using four levels of neurological change. Findings were: (a) the doses required to paralyze 50% of animals (ED50) were 19.79, 20.77, and 21.85 Gy for mild, partial, and complete paralysis, respectively, as measured 200-360 days after radiation. (b) Most damage was progressive but it was not necessarily so; after doses up to 28 Gy recovery was occasionally seen. (c) Latency depended on the dose and the level of damage. Following doses around the ED50, paralysis occurred between 180 to 300 days. (d) There were significant fluctuations in the dose-latency relationship at doses less than 35 Gy. Latency may be not a reliable endpoint to compare biological effects of radiation in this dose range. (e) The radiosensitivity of mouse spinal cord was similar to that reported for rats. (f) Histologically, demyelination was the dominant lesion in the paralyzed animals. We conclude that the mouse is a good animal model to study radiation damage to the spinal cord, at least when a 2.2 cm length is irradiated. PMID- 1727131 TI - Effects of hyperbaric oxygen and a perfluorooctylbromide emulsion on the radiation responses of tumors and normal tissues in rodents. AB - Perfluorochemical emulsions are being examined in many laboratory and clinical studies as possible adjuncts to radiotherapy and chemotherapy. The studies reported here examine the clinical potential of hyperbaric oxygen (HBO) in combination with a highly concentrated perfluorochemical emulsion (Oxygent) containing 100% w/v perfluorooctylbromide (PFOB). HBO alone produced only a small improvement in the radiation response of BA1112 tumors in WAG/rij rats, while regimens combining HBO with Oxygent produced much greater radiation sensitization. A sham emulsion, formulated without the O2-carrying PFOB, did not alter the radiation response of the tumors in comparison with that seen with HBO alone. Neither HBO nor Oxygent plus HBO altered the radiosensitivity of bone marrow progenitor cells in BALB/c mice. HBO alone augmented skin reactions in BALB/c mice, but addition of Oxygent did not alter the skin reactions in comparison to those seen with HBO alone. Regimens combining Oxygent with HBO selectively increased the radiation sensitivity of tumors relative to normal tissues, thereby enhancing the therapeutic ratio. These results support the potential usefulness of perfluorochemical emulsions and HBO in clinical radiation therapy. PMID- 1727132 TI - Energy metabolism, pH changes, and lactate production in RIF-1 tumors following intratumoral injection of glucose. AB - The metabolic consequences of increased glucose availability were examined in subcutaneous RIF-1 tumors in vivo, using 13C and 31P NMR spectroscopy. Significant increases in the levels of nucleotide triphosphates and phosphocreatine relative to low energy phosphates and in tumor pH were observed within 30 min following injection of 1 g/kg of glucose directly into the tumor. These changes did not occur following an equivalent intratumoral dose of the non metabolizable sugar alcohol, mannitol. When [1-13C]-glucose was administered, [3 13C]-lactate and [3-13C]-alanine were the only labeled metabolites detected in the in vivo 13C NMR spectra during the period of bioenergetic improvement. Biochemical analysis revealed a substantial increase in tumor and plasma glucose concentration, but no increase in either tumor or plasma lactate, consistent with the absence of acidosis. Evaluation of the distribution of glucose in the tumor by quantitative autoradiography of [1-14C]-2-deoxyglucose administered with the glucose indicated that, on average, 7 mM of the added glucose distributed over the entire tumor within 10 min. The significant improvement in overall metabolic status of the tumors following glucose administration is attributed to the existence of substrate limited regions within the tumor. PMID- 1727133 TI - Effects of L-NMMA and L-NNA on the selective ATP-induced enhancement of intratumoral blood flow. AB - We studied the effects of NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L arginine (L-NNA) on the selective ATP and adenosine-induced enhancement of intratumoral blood flow in rats measured by the hydrogen clearance method. Both adenosine and ATP produced a selective enhancement of the intratumoral blood flow. Neither L-NMMA nor L-NNA had a significant effect on either the CBF or the intratumoral blood flow. Adenosine-induced enhancement was not inhibited by L NMMA or L-NNA. On the other hand, the ATP-induced enhancement was totally inhibited by both L-NMMA and L-NNA. The inhibitory action of L-NMMA against ATP was blocked by L-arginine, but not by D-arginine. It is suggested that the ATP induced increase of intratumoral blood flow is evoked by nitric oxide synthesized from the endothelium of the intratumoral blood vessels. PMID- 1727134 TI - Blood flow in portal systems with special reference to the rat pituitary gland. AB - Regional pituitary blood flow has been studied in adult female Fischer 344 rats by [14C]iodoantipyrine autoradiography. A general mathematical solution has been derived to allow the calculation of blood flow in the second compartment of a portal system and the proportion of blood "shunted" through the first compartment without exposure to tissue uptake from a knowledge of (a) the volume ratios of the two compartments, (b) the tissue tracer uptakes of the two compartments, and (c) the arterial tracer concentration with respect to time of a freely diffusible tracer. Significant diffusion limitation and/or arteriovenous shunting has been demonstrated in the neurohypophysis, suggesting that the majority of incoming blood is "shunted" unchanged to the adenohypophysis. The mean value of the shunt is 89% (range of 84-93%) for the median eminence and lies between 72% (range of 52-82%) and 73% (range of 59-81%) for the posterior pituitary. Neurohypophysial flow rates of 1.20 (range of 0.99-1.55) ml g-1 min-1 for the median eminence and 1.68 (range of 0.83-3.53) ml g-1 min-1 for the posterior pituitary were measured. These values represent "tissue-available" (nonshunted) flow; estimated mean total (shunted plus nonshunted) neurohypophysial flow rates were 11.7 (range of 9.5 17.5) ml g-1 min-1 for the median eminence and 6.1 (range of 3.1-8.9) ml g-1 min 1 (minimum) for the posterior pituitary. Adenohypophysial blood flow is heterogeneous. In the long portal territory, the flow rate was 1.18 (range of 0.95-1.75) ml g-1 min-1 but short portal territory flow calculation is complicated by an unquantifiable nonportal venous drainage; using the natural limits of zero and 100% gives a minimum adenohypophysial flow rate of 1.42 (range of 0.76-2.07) ml g-1 min-1 and a maximum value of 1.97 (range of 1.03-2.82) ml g 1 min-1. PMID- 1727135 TI - In vivo muscarinic cholinergic receptor imaging in human brain with [11C]scopolamine and positron emission tomography. AB - Cerebral muscarinic cholinergic receptors were imaged and regionally quantified in vivo in humans with the use of [11C]scopolamine and positron emission tomography. Previous studies in experimental animals have suggested the utility of radiolabeled scopolamine for in vivo measurements, on the bases of its maintained pharmacologic specificity following systemic administration and the exclusion of labeled metabolites from the brain. The present studies describe the cerebral distribution kinetics of [11C]scopolamine in normal subjects following intravenous injection. Scopolamine is initially delivered to brain in a perfusion directed pattern. After 30 to 60 min, activity is lost preferentially from cerebral structures with low muscarinic receptor density including the cerebellum and thalamus. Activity continues to accumulate throughout a 2 h postinjection period in receptor-rich areas including cerebral cortex and the basal ganglia. The late regional concentration of [11C]scopolamine does not, however, accurately parallel known differences in muscarinic receptor numbers in these receptor-rich areas. Tracer kinetic analysis of the data, performed on the basis of a three compartment model, provides receptor binding estimates in good agreement with prior in vitro measurements. Kinetic analysis confirms significant contributions of ligand delivery and extraction to the late distribution of [11C]scopolamine, reconciling the discrepancy between receptor levels and tracer concentration. Finally, a novel dual-isotope method for rapid chromatographic processing of arterial blood samples in radiotracer studies is presented. The combination of rapid chromatography and compartmental analysis of tracer distribution should have broad utility in future in vivo studies with short-lived radioligands. PMID- 1727136 TI - Validation of 133Xe clearance as a cerebral blood flow measurement technique during cardiopulmonary bypass. AB - 133Xe clearance to measure cerebral blood flow (CBF) was examined in 10 dogs during cardiopulmonary bypass. As a reference method, a continuous Kety-Schmidt technique (CBFKS) with 133Xe as indicator was used. Extracranial tissue was removed to directly place the 133Xe detectors on the skull, and the head was covered with a 3 mm lead shield to minimize contamination of the 133Xe clearance curve with extracranial radiation. 133Xe detectors for the Kety-Schmidt technique were embedded in a shielded brass block to minimize interference with radiation from the animal's body. 133Xe clearance data were analyzed using stochastic (CBF10, CBF15, and CBFINF) and initial slope methods (CBFIS), and the results were compared with CBFKS using linear regression. CBF15 and CBFINF yielded similar CBF values as CBFKS (CBFKS = 0.97.CBF15-2.08, r = 0.92, p less than 0.01; CBFKS = 1.13.CBFINF-1.21, r = 0.92, p less than 0.01). CBF10 slightly overestimated CBFKS but still showed a close correlation to CBFKS (CBFKS = 0.89.CBF10-2.58, r = 0.92, p less than 0.01) and CBFIS considerably overestimated CBFKS (CBFKS = 0.60.CBFIS-1.27, r = 0.87, p less than 0.01). With extracranial contamination of the 133Xe clearance curve minimized, all 133Xe clearance techniques used to measure CBF were consistently related to CBFKS in a constant, significant manner. 133Xe clearance therefore is a valid method to assess CBF during cardiopulmonary bypass. PMID- 1727137 TI - Evaluation of cerebral vasomotor reactivity by various vasodilating stimuli: comparison of CO2 to acetazolamide. AB - To evaluate the role of different vasomotor stimuli for the measurement of cerebrovascular vasomotor reactivity (VMR), 47 patients (i.e., 93 hemispheres) with various degrees of internal carotid artery (ICA) occlusive disease were studied. Patients were divided into clinical [asymptomatic, transient ischemic attack (TIA) or completed stroke] as well as angiological subgroups. Low-grade or high-grade unilateral ICA lesions were compared to bilateral ICA occlusive disease. Relative flow velocity changes within the middle cerebral artery were measured by means of transcranial Doppler during hyper- and hypocapnia (VMRTOT), during hypercapnia alone (VMRCO2), and after injection of 1 g acetazolamide (VMRACE). VMR was expressed as the percentage change in flow velocity after stimulus application as compared with flow velocity at rest. There was a close and statistically highly significant correlation of CO2-induced with acetazolamide-induced VMR (r = 0.69 in VMRTOT versus VMRACE and 0.79 in VMRCO2 versus VMRACE; P less than 0.0001; linear regression), indicating a strong similarity of the vasodilatative effects of CO2 and acetazolamide on cerebral arteries. Both stimulation techniques highly significantly differentiated between asymptomatic patients and those with TIA or completed stroke. Angiological subgroups were separated best by the acetazolamide test. Reclassification of patients into angiological subgroups by linear discriminant analysis was equally good with all three methods. We conclude that both acetazolamide- and CO2-induced stimulation of the cerebral vasomotors are valid techniques to measure reduction in perfusion reserve due to extracranial cerebrovascular occlusive disease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727138 TI - Hyperglycemia suppresses c-fos mRNA expression following transient cerebral ischemia in gerbils. AB - The c-fos proto-oncogene is activated by transient cerebral ischemia. This activation may signify a specific genetic response to ischemia affecting tolerance to ischemia and ultimate cell survival. Hyperglycemia, which enhances brain injury from transient ischemia, was studied for its effects on this gene system in gerbils by measuring c-fos mRNA 2 h after 20 min of bilateral carotid artery occlusion. Brain c-fos mRNA was increased by ischemia (11.7 +/- 5.0, p less than or equal to 0.05, fold increase) compared to nonischemic controls (1.0 +/- 1.3). Pretreatment with 1 g/kg of glucose partially reduced postischemic c fos mRNA (6.3 +/- 1.6, p less than or equal to 0.05) while 4 g/kg of glucose completely suppressed postischemic c-fos expression (0.7 +/- 0.3, p less than or equal to 0.05). These data indicate that hyperglycemia suppresses normal postischemic gene expression and suggest the possibility that such suppression is a predictor or even a contributor to hyperglycemia-enhanced ischemic brain damage. PMID- 1727139 TI - In vivo nonspecific binding parameters of (R,R)-[125I]4IQNB estimated from the pharmacokinetics of the (S,S)-[125I]4IQNB stereoisomer. PMID- 1727140 TI - Focal and perifocal changes in tissue energy state during middle cerebral artery occlusion in normo- and hyperglycemic rats. AB - The objective of the present study was to assess changes in cellular energy metabolism in focal and perifocal areas of a stroke lesion and to explore how these changes are modulated by preischemic hyperglycemia. A model for reversible occlusion of the middle cerebral artery (MCA) in rats was used to study changes in energy metabolism. Following MCA occlusion for 5, 15, or 30 min in normoglycemic rats, the tissue was frozen in situ, and samples from the lateral caudoputamen and from two neocortical areas were collected for metabolite analyses, together with a control sample from the contralateral, nonischemic hemisphere. Two other groups, subjected to 30 min of MCA occlusion, were made hyperglycemic by acute glucose infusion or by prior injection of streptozotocin. Enzymatic techniques were used for measurements of phosphocreatine, creatine, ATP, ADP, AMP, glycogen, glucose, pyruvate, and lactate. The neocortex of the contralateral, nonischemic hemisphere had labile metabolites that were similar to those measured in control animals. Ipsilateral neocortex bordering the focus, and thus constituting the "penumbra," showed mild to moderate ischemic changes. In the "focus" (lateral caudoputamen plus the overlying neocortex), deterioration of energy state was rapid and relatively extensive (ATP content 20-40% of control). After 5 min of occlusion, no further deterioration of metabolic parameters was observed. Substrate levels were markedly reduced, and lactate content rose to approximately 10 mM kg-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727141 TI - Glycolysis, oxidative metabolism, and brain potassium ion clearance. AB - Studies were directed toward defining relationships between brain ion transport, glycolysis, and oxidative phosphorylation. This was done by examining the relative sensitivity to hypoxemia and to iodoacetate (IAA)-induced inhibition of glycolysis in rats anesthetized with pentobarbital. Both insults had minimal effects on K+o baseline. In response to neuronal activation, IAA increased the time required for K+o clearance from maximal values to half-recovery of baseline. Hypoxemia slowed the later phase of K+o clearance, when K+o was approaching "resting" levels. Hypoxemia produced greater declines in high-energy intermediates than did IAA, which indicated that the IAA effect was not due to a greater overall insult to metabolism and suggested a direct link between ATP produced by glycolysis and ion transport activity. These data demonstrate that K+o clearance requires energy from glycolysis and oxidative phosphorylation for different phases of the recovery process and that inhibition specific to glycolysis or oxidative phosphorylation may be temporally resolved within a single stimulus. PMID- 1727143 TI - Focal cerebral ischemia in rats: effects of induced hypertension, during reperfusion, on CBF. AB - The effect of phenylephrine-induced hypertension on CBF was investigated after 120 min of middle cerebral artery occlusion in rats. Blood pressure was manipulated by one of the following schedules during a 90-min period of reperfusion: 90/NORM, 90 min of normotensive reperfusion; 90/HTN, 90 min of hypertensive reperfusion (MABP increased by 30 mm Hg); or 15/HTN, the 90-min period of reperfusion was divided into 30 min of normotension, followed by 15 min of hypertension and 45 min of normotension. At the end of reperfusion, 100 microCi kg-1 of [14C]iodoantipyrine was given and an autoradiographic analysis of CBF performed. In the coronal brain section at the center of middle cerebral artery distribution, the area (percentage of hemisphere, mean +/- SD) with a CBF of 0-20 or 21-40 ml 100 g-1 min-1 was less (p less than 0.05) in the 15/HTN group (1 +/- 2 and 5 +/- 3%, respectively) versus the 90/HTN group (12 +/- 4 and 10 +/- 4%), which was in turn less than in the 90/NORM group (18 +/- 5 and 22 +/- 6%). These data are consistent with the hypothesis that during reperfusion a short interval of hypertension effectively augments CBF via an abrupt opening of collapsed vessels and that a more sustained interval of hypertension conveys no added benefit. PMID- 1727142 TI - Effect of superoxide dismutase on intracellular calcium in stroke. AB - To clarify the relationship between calcium metabolism and free radical damage during the reperfusion period following ischemia, we investigated the effect of superoxide dismutase (SOD) on changes in cytosolic free calcium, cortical blood flow, and histologic changes following focal cerebral ischemia and reperfusion in 12 cats. Using indo-1, a fluorescent intracellular Ca2+ indicator, we simultaneously measured changes in the Ca2+ signal ratio (400:500 nm), NADH signal (464 nm), and reflectance (340 nm) during ultraviolet excitation (340 nm) directly from the cortex in vivo. The middle cerebral artery (MCA) was occluded for 1 h; only cats in which the EEG amplitude was depressed to less than 10% of control during the occlusion were entered into the study. Starting 2 min prior to release of the occlusion and continuing for 4 min, SOD (10,000 U/kg) was slowly infused in six cats, while in six cats, the vehicle only was infused. During MCA occlusion, the Ca2+ signal ratio increased significantly in both groups with no significant difference between the groups. During reperfusion, the Ca2+ signal ratio remained at a high level in the vehicle-treated group, while in the SOD treated group, the Ca2+ signal ratio decreased. There was a statistically significant difference between the two groups at 10, 20, and 30 min after reperfusion (p less than 0.01). The histologically damaged area in the SOD treated group was significantly smaller than that in the vehicle-treated group (p less than 0.01). These data suggest that the histoprotective action of SOD may be due to its ability to attenuate increases in intracellular calcium during the recirculation period following focal cerebral ischemia. PMID- 1727144 TI - Effect of ischemia and reperfusion on lambda of the lumped constant of the [14C]deoxyglucose technique. AB - We measured the parameter lambda, which is the ratio of the distribution spaces of 2-deoxy-D-glucose (DG) and glucose in the brain, in a model of focal cerebral ischemia in the cat. lambda is the parameter in the lumped constant of the [14C]DG technique most susceptible to changes in ischemia. Cats were subjected to occlusion of the middle cerebral artery for a period of 2 h. During the last 60 min of occlusion, [14C]DG was infused in a programmed fashion so as to obtain a stable arterial blood [14C]DG concentration. The brain was funnel-frozen to preserve tissue metabolites and the frozen brain was sampled regionally (4 to 7 mg samples) for local concentrations of glucose, ATP, phosphocreatine (PCr), and lactate. In a separate series of cats, the infusion of [14C]DG was started after 2 h of occlusion and 3 h of recirculation. In both series, lambda declined slightly for increased levels of tissue glucose and increased appreciably as tissue glucose decreased. A similar relationship was observed between lambda and ATP and PCr, although the correlation was not as clear. Since lambda, and hence the lumped constant, increases in ischemia as well as in postischemic tissue, it is important to measure tissue glucose concentration if quantitative values of local cerebral glucose metabolism are desired in this condition. PMID- 1727145 TI - Eicosanoid production in the caudate nucleus and dorsal hippocampus after forebrain ischemia: a microdialysis study. AB - Thromboxane (Tx)B2 and 6-keto-prostaglandin (6-keto-PG) F1 alpha formation in the hippocampus and caudate nucleus were evaluated by microdialysis during and following forebrain ischemia. Spontaneously hypertensive rats were subjected to bilateral carotid artery occlusion with simultaneous hypotension for 8, 14, or 20 min. Dialysate was collected during the ischemic interval and during the reperfusion period. TxB2 and 6-keto-PGF1 alpha levels were measured by radioimmunoassay. In both structures, TxB2 production increased significantly during the reperfusion period in all three ischemic groups. By contrast, increased 6-keto-PGF1 alpha elaboration was observed after only the longest ischemic duration. While TxB2 levels gradually decreased during the 3-h reperfusion period in all groups, the levels in the group subjected to 8 min of ischemia returned to control values most rapidly. A relationship between the duration of ischemia and TxB2 production was therefore evident. 6-Keto-PGF1 alpha levels increased in only the group subjected to 20 min of ischemia and, by contrast to the pattern of TxB2 change, 6-keto-PGF1 alpha levels remained elevated throughout the reperfusion period. During reperfusion, the ratio of TxB2 to 6-keto-PGF1 alpha increased substantially versus the preischemic period in both structures. The data demonstrate that eicosanoid elaboration following cerebral ischemia can be evaluated by the microdialysis technique. In addition, they indicate that the thresholds (duration of ischemia) for the postischemic production and the temporal profiles of TxB2 and 6-keto-PGF1 alpha in the caudate and hippocampus differ. They also demonstrate that there is regional heterogeneity in the patterns of eicosanoid elaboration after forebrain ischemia. PMID- 1727146 TI - Extracellular antioxidants and amino acids in the cortex of the rat: monitoring by microdialysis of early ischemic changes. AB - Extracellular concentrations of ascorbic acid, glutathione, cysteine, uric acid, tyrosine, and tryptophan were monitored using intracerebral microdialysis in the left frontoparietal cortex of spontaneous hypertensive rats before, and for 3 h after, either focal ischemia [left middle cerebral artery occlusion (MCAO)] or sham operation. The size of the ischemic area and the position of the microdialysis probe were checked using the enzyme histotopochemical acid phosphatase reaction. The probe was always located in the cortex inside the stained area. Ascorbic acid levels rose immediately after MCAO and remained at about 12-fold for 3 h. There was a transient release of glutathione during 1-1.5 h. Uric acid concentrations were also increased but the differences did not reach significance. The levels of the amino acids tyrosine and tryptophan increased steadily after MCAO. The increases in cysteine were variable but significant. In some experiments, the pH of the dialysate was measured online. The parameters ascorbic acid, glutathione, cysteine, and pH are suitable for the early detection of cortical ischemic events by microdialysis. PMID- 1727147 TI - Immunotoxins and central nervous system neoplasia. AB - The poor prognosis associated with central nervous system (CNS) malignancy has led investigators to seek new, innovative treatment modalities. Immunotoxins, carrier molecules linked to toxic agents, combine high specificity for tumor associated antigens with extreme potency. The rationale for both the development of these compounds and for their application to CNS neoplasia is explained. This report discusses the design and construction of immunoconjugates, using toxins that differ in their mechanism of action bound to ligands directed against various antigens. A comparison is made between the in vitro efficacy of standard chemotherapy and immunotoxins in glioblastoma- and medulloblastoma-derived cell lines. A review is included of the results of experiments in animals with leptomeningeal neoplasia, where prolongation of survival following intrathecal administration of immunotoxins has been reported. The obstacles encountered in clinical trials with other types of cancer are addressed and approaches to optimize the use of these novel agents in the context of treating malignant disease of the CNS are suggested. PMID- 1727148 TI - Efficacy of LY233053, a competitive glutamate antagonist, in experimental central nervous system ischemia. AB - Antagonists of excitatory amino acids appear to serve a neuroprotective role during ischemic conditions in a variety of in vivo and in vitro models. The usefulness of such agents in the clinical setting, however, may be limited by poor central nervous system (CNS) entry and intolerable side effects. The authors report high efficacy in reducing neurological damage and relatively limited side effects of LY233053, a novel competitive glutamate antagonist, in two models of experimental CNS ischemia in the rabbit. PMID- 1727149 TI - Central and peripheral biogenic amine effects of brain missile wounding and increased intracranial pressure. AB - This study was performed to ascertain the acute effects of brain missile wounding on brain-stem and hypothalamic biogenic amines in a group of cats anesthetized with pentobarbital (40 mg/kg). Brain wounding is associated with a concomitant increase in intracranial pressure (ICP); to separate the effects of elevated ICP alone from the effects of wounding, a second group of cats had ICP artificially increased from a normal level of approximately 5 mm Hg to approximately 140 mm Hg by infusion of mock cerebrospinal fluid into the cisterna magna. In both groups, significant epinephrine depletions (47% to 74%) occurred in the nucleus tractus solitarius, area A1C1, locus ceruleus, raphe nuclei, and posterior hypothalamus. Epinephrine levels were also significantly decreased in the anterior hypothalamus in the wounded cats. In addition, both brain wounding and artificially induced ICP increases caused significant decreases of norepinephrine in the posterior hypothalamus, and of serotonin, 5-hydroxyindoleacetic acid, dopamine, and homovanillic acid in the raphe nuclei. Only brain wounding, however, caused significant reductions of norepinephrine, dopamine, and homovanillic acid in the nucleus tractus solitarius and area A1C1. The plasma catecholamine levels resulting from brain wounding or artificially induced ICP increases were dissimilar only in the amount of time required to attain maximum plasma levels, with the wounded animals responding faster. It is concluded that the hypothalamic and brain-stem biogenic amine changes resulting from either brain wounding or increased ICP alone are reflective of a stress response. Brain-stem distortion caused by brain wounding did not appear to be a factor and monoaminergic systems appeared to remain intact despite a severe and eventually lethal brain injury.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727150 TI - The neuroprotective pharmacology of methylprednisolone. AB - A 24-hour intensive intravenous dosing regimen with the glucocorticoid steroid methylprednisolone has recently been shown to be effective in enhancing neurological recovery in spinal cord-injured patients when initiated within 8 hours after injury. The state of knowledge concerning the neuroprotective pharmacology of methylprednisolone, including mechanism(s) of action, dosing requirements, and time-action considerations is reviewed, as are the results of studies with high doses in experimental and clinical head injury, subarachnoid hemorrhage, and cerebral ischemia. A primary neuroprotective mechanism of action in each of these cases is hypothesized to involve the ability of high doses of methylprednisolone to inhibit oxygen free radical-induced lipid peroxidation, although additional mechanisms may contribute. Unresolved issues are also addressed, including the therapeutic window, optimum duration of treatment, and rational combination with other neuroprotective agents. A newer methylprednisolone pro-drug with improved solution stability is discussed, together with a brief consideration of novel nonglucocorticoid steroids that surpass methylprednisolone's lipid antioxidant effects without unwanted glucocorticoid properties. PMID- 1727151 TI - Iatrogenic arteriovenous fistula presenting as a recurrent subdural hematoma. Case report. AB - An unusual case of an iatrogenic dural arteriovenous fistula is reported. The patient presented with a history of progressive generalized headache over a period of 3 to 4 weeks. Computerized tomography demonstrated a chronic subdural hematoma that was successfully evacuated by burr-hole drainage. The patient's postoperative course was complicated by recurrent acute subdural hematomas at the drainage site. Coagulation studies were unremarkable. Selective external carotid angiography demonstrated a small dural arteriovenous fistula adjacent to the burr hole used for the initial operative procedure. Extension of the bone flap and coagulation of the fistula resulted in a good outcome. In the patient with recurrent acute subdural hematoma, the possibility of a vascular malformation must be considered. Selective internal and external carotid angiography is key to the correct diagnosis. PMID- 1727152 TI - Remote congenital cerebral arteriovenous fistulae associated with aortic coarctation. Case report. AB - A neonate presented with anatomically discrete cerebral arteriovenous fistulae located in the right sylvian fissure and the cerebellar vermis that were initially detected by prenatal ultrasonography. Following delivery of the baby by caesarean section, both malformations were treated by surgical obliteration. These intracranial vascular lesions were associated with cardiac anomalies and a periductal coarctation of the aorta, which was treated with a left subclavian rotational arterial pedicle repair. Follow-up examination of the infant at age 13 months demonstrated an excellent clinical result with normalization of the circulation. The pathophysiology of this syndrome is discussed and the literature reviewed. PMID- 1727153 TI - Fibroma of the meninges in a child: immunohistological and ultrastructural study. Case report. AB - A case of meningeal fibroma in a 5-year-old girl is described. The lesion presented as a benign intracranial tumor, eroding the frontal bone and protruding under the skin. It was composed of fibroblasts and collagen, embedded in a loose background with focal myxoid changes. The authors describe the patient's clinical presentation and the tumor's histological, immunohistochemical, and ultrastructural features, and discuss its differential diagnosis. It is concluded that fibromas of the meninges should be distinguished from fibroblastic meningiomas. PMID- 1727154 TI - Metastatic choriocarcinoma with neoplastic aneurysms cured by aneurysm resection and chemotherapy. Case report. AB - A case of choriocarcinoma with brain and lung metastasis is reported. The patient was admitted for treatment of a cerebral hemorrhage from neoplastic aneurysms and, following removal of the hematoma and resection of the aneurysms, her carcinoma was successfully managed with chemotherapy. She has survived for 6 years after onset without neuroimaging evidence of recurrence. Surgical treatment of metastatic lesions followed by prolonged intensive chemotherapy are indicated for the improved prognosis of choriocarcinoma. PMID- 1727155 TI - Relief of akinetic mutism from obstructive hydrocephalus using bromocriptine and ephedrine. Case report. AB - The case of a 20-year-old man with obstructive hydrocephalus who suffered multiple shunt failures and shunt revisions is presented. The patient gradually developed a clinical syndrome of akinetic mutism. This behavioral syndrome failed to respond to shunt revisions, but did improve after the administration of a combination of bromocriptine and ephedrine. PMID- 1727156 TI - Instrumentation for microsurgical osseous dissection. Technical note. AB - The use of the operating microscope has revolutionized the surgical approach to many neurosurgical diseases. The microscope has provided magnification, binocular vision, and excellent lighting in the depths of neurosurgical wounds, allowing the performance of exceedingly delicate procedures that were previously impossible. Occasionally, an operative approach demands microscopic bone dissection. Instrumentation has been developed for working with soft tissue, but special instruments for osseous dissection have not been available. A set of newly developed punches and curettes with a bayonetted offset is described. These keep the surgeon's hand out of the operating field and allow unimpeded visualization through the operating microscope. These prototype instruments have been used successfully in over 100 microscopic neurosurgical procedures. PMID- 1727157 TI - Anterior cervical vertebrectomy and interbody fusion. Technical note. AB - This report discusses the authors' technique in performing anterior cervical vertebrectomy and interbody fusion for multilevel cervical disease. The technique is performed with a high-speed drill and bone-bank fibular strut graft. After decompression of the cervical canal, ledges are made in the intact vertebral bodies to create a rectangular bed for safe seating of the bone graft. The bone bank fibular strut graft is a feasible alternative to autograft. The simplified and safe nature of this procedure reduces postoperative morbidity as well as the length of hospital stay. PMID- 1727158 TI - Thrombolytic therapy for vascular occlusions. PMID- 1727159 TI - Aneurysmal dilatation of ICA after tumor resection. PMID- 1727160 TI - Nutrition in head-injured patients. PMID- 1727161 TI - Increased MR signal intensity and myelopathy. PMID- 1727162 TI - Radiation-induced optic neuropathy. PMID- 1727163 TI - Thecoperitoneal shunting. PMID- 1727164 TI - Approach to skull-base tumors. PMID- 1727165 TI - Methylprednisolone or naloxone treatment after acute spinal cord injury: 1-year follow-up data. Results of the second National Acute Spinal Cord Injury Study. AB - The 1-year follow-up data of a multicenter randomized controlled trial of methylprednisolone (30 mg/kg bolus and 5.4 mg/kg/hr for 23 hours) or naloxone (5.4 mg/kg bolus and 4.0 mg/kg/hr for 23 hours) treatment for acute spinal cord injury are reported and compared with placebo results. In patients treated with methylprednisolone within 8 hours of injury, increased recovery of neurological function was seen at 6 weeks and at 6 months and continued to be observed 1 year after injury. For motor function, this difference was statistically significant (p = 0.030), and was found in patients with total sensory and motor loss in the emergency room (p = 0.019) and in those with some preservation of motor and sensory function (p = 0.024). Naloxone-treated patients did not show significantly greater recovery. Patients treated after 8 hours of injury recovered less motor function if receiving methylprednisolone (p = 0.08) or naloxone (p = 0.10) as compared with those given placebo. Complication and mortality rates were similar in either group of treated patients as compared with the placebo group. The authors conclude that treatment with the study dose of methylprednisolone is indicated for acute spinal cord trauma, but only if it can be started within 8 hours of injury. PMID- 1727166 TI - Central neurocytoma: histopathological variants and therapeutic approaches. AB - The central neurocytoma has recently been added to the differential diagnosis of intraventricular tumors. Histopathologically, this tumor is characterized by a uniform neoplastic cell population with features of neuronal differentiation. Central neurocytomas occur in young adults, develop in the area of the foramen of Monro, and are usually associated with the septum pellucidum. Initial reports appeared to indicate that these tumors are benign lesions with a favorable postoperative prognosis. The authors present clinical and neuropathological findings in a series of eight patients with central neurocytoma. An anterior transcallosal microneurosurgical approach yielded good outcomes. Postoperative radiation therapy was restricted to two patients with a malignant variant of central neurocytoma and one patient with a recurrent tumor. Observations of anaplastic variants of this neoplasm in two cases and local tumor recurrences in three indicate that the biological behavior and postoperative prognosis of central neurocytoma may not always be as favorable as previously assumed. PMID- 1727167 TI - Cavernous angiomas of the central nervous system in children. AB - A surgical series of 19 patients under the age of 18 years with pathologically verified cavernous angioma is presented. Most lesions were located in the cerebral hemispheres, but four were in the pons or midbrain, two in the diencephalon, and one in the spinal cord. Fourteen patients presented with an acute or progressing neurological deficit, three with seizures, one infant with irritability, and one with headache alone. Five patients had family histories of vascular malformations of the central nervous system, and five had multiple lesions. Surgery for small or deep lesions was aided considerably by intraoperative ultrasonographic or stereotactic localization techniques. Pathological examination of the resected malformations revealed a complex histology containing not only typical closely approximated cavernous vessels, but also areas of marked proliferation of granulation tissue and partially re endothelialized hemorrhage, suggesting a mechanism for the apparent growth of certain cavernous angiomas. The postoperative results were good, with only one patient suffering a permanent worsening of neurological status after surgery. Incomplete resection was initially carried out in five patients, two of whom rebled within 1 year after operation. Long-term follow-up findings in these patients have emphasized the unusual history of certain of these malformations. PMID- 1727168 TI - Aggressive surgical management of craniopharyngiomas in children. AB - The cases of 50 patients with craniopharyngioma operated on at The Hospital for Sick Children in Toronto between January, 1975, and December, 1989, are reviewed. All patients were under 18 years of age (mean 9.39 years). Headaches, endocrine deficiencies, and visual deficits were the most common symptoms on admission. Forty-five patients underwent what was considered by the surgeon to be total excision of their tumor, and five had subtotal excision. Tumors recurred in 17 patients (mean time of recurrence 32.6 months after surgery). One patient died in the postoperative period and three have been lost to follow-up study. Of the remaining 46 patients, 28 are leading a normal or nearly normal life, although all are receiving endocrine replacement and some have required help to overcome mild deficits in memory or visual acuity. Twelve patients are able to function reasonably well and attend school despite being hampered by intellectual or visual deficits or problems with weight control; four have a significant handicap, and two have died. PMID- 1727169 TI - Leksell's posteroventral pallidotomy in the treatment of Parkinson's disease. AB - Between 1985 and 1990, the authors performed stereotactic posteroventral pallidotomies on 38 patients with Parkinson's disease whose main complaint was hypokinesia. Upon re-examination 2 to 71 months after surgery (mean 28 months), complete or almost complete relief of rigidity and hypokinesia was observed in 92% of the patients. Of the 32 patients who before surgery also suffered from tremor, 26 (81%) had complete or almost complete relief of tremor. The L-dopa induced dyskinesias and muscle pain had greatly improved or disappeared in most patients, and gait and speech volume also showed remarkable improvement. Complications were observed in seven patients: six had a permanent partial homonymous hemianopsia (one also had transient dysphasia and facial weakness) and one developed transitory hemiparesis 1 week after pallidotomy. The results presented here confirm the 1960 findings of Svennilson, et al., that parkinsonian tremor, rigidity, and hypokinesia can be effectively abolished by posteroventral pallidotomy, an approach developed in 1956 and 1957 by Lars Leksell. The positive effect of posteroventral pallidotomy is believed to be based on the interruption of some striopallidal or subthalamopallidal pathways, which results in disinhibition of medial pallidal activity necessary for movement control. PMID- 1727170 TI - Angiographic frequency of saccular intracranial aneurysms in patients with spontaneous cervical artery dissection. AB - The pathogenesis of intracranial aneurysms and spontaneous cervical artery dissection is incompletely understood but a primary arteriopathy, possibly similar in both disorders, may be of importance. To investigate the frequency of intracranial aneurysms in patients with spontaneous cervical artery dissection, the angiograms of 164 patients who were diagnosed at the Mayo Clinic as having spontaneous extracranial carotid or vertebral artery dissection were reviewed. Thirteen intracranial aneurysms were detected in nine (5.5%) of the 164 patients: eight (8.8%) of the 91 female patients and one (1.4%) of the 73 male patients. The frequency of intracranial aneurysms in these patients was significantly higher (p less than 0.01) than that observed in a recent angiographic study from the same institution, estimating the frequency of intracranial aneurysms in the general population (1.1%). The significance of these findings is discussed. PMID- 1727171 TI - Significance of positive Queckenstedt test in patients with syringomyelia associated with Arnold-Chiari malformations. AB - Ten patients with syringomyelia associated with Arnold-Chiari Type I malformations were evaluated. In each patient, a manometric Queckenstedt test was performed with the neck in various positions. No patient showed evidence of a block to the flow of cerebrospinal fluid (CSF) with the neck in the extended position; however, all showed a complete CSF block with the neck in a flexed position. Posterior fossa decompression with a C1-2 laminectomy was performed in nine cases, after which Queckenstedt test demonstrated free CSF communication in all nine with the neck in extension, in a neutral position, and in flexion. Postoperative magnetic resonance imaging showed shrinkage of the syrinx in the patients who underwent surgery. It is suggested that obstruction of the CSF pathway at the foramen magnum produced by neck movement is of importance in the formation and progression of a syrinx. PMID- 1727172 TI - Chemical monitoring of neurosurgical intensive care patients using intracerebral microdialysis. AB - The authors have used intracerebral microdialysis to develop a method for routine monitoring of disturbances in brain energy metabolism in patients in the neurosurgical intensive care unit. Microdialysis was conducted for periods ranging from 2.3 to 8.3 days in four patients (three with severe head injuries and one with severe subarachnoid hemorrhage). Altogether, 4447 chemical analyses from 587 dialysis samples were carried out. Concentrations of the energy-related metabolites lactate, pyruvate, and hypoxanthine were measured, and the lactate:pyruvate ratio was calculated. In addition, the acids glutamate, aspartate, taurine, glutamine, asparagine, and glycine were measured in one patient. The microdialysis data were matched with various clinical events, including intracranial hypertension and therapeutic interventions such as initiation or withdrawal of barbiturates and cerebrospinal fluid drainage. The present study shows that microdialysis can be used for long-term measurement of extracellular fluid (ECF) energy-related metabolites and amino acids in the frontal cortex of neurosurgical patients in a clinical setting. Fluctuations of the measured ECF energy-related substances corresponded to various clinical events presumably involving hypoxia/ischemia. The authors found a 25-fold increase in ECF glutamate, aspartate, and taurine under conditions of energy perturbation, as indicated by high levels of the lactate:pyruvate ratio, lactate, and hypoxanthine. The use of long-term intracerebral microdialysis in patients opens a new field of clinical research, with many possibilities for improving insight into intracranial dynamics in acute cerebral conditions. PMID- 1727173 TI - Cytoskeletal and extracellular matrix proteins in cerebral arteries following subarachnoid hemorrhage in monkeys. AB - It is unclear if vasospasm after subarachnoid hemorrhage (SAH) is predominantly due to smooth-muscle contraction, proliferative vasculopathy, or other changes within the arterial wall such as fibrosis or change in smooth-muscle phenotype. In this study, immunohistochemistry was used to examine changes in extracellular and cytoskeletal proteins in cerebral arteries after SAH that might support one of these mechanisms. Following baseline cerebral angiography, bilateral SAH was created in nine monkeys. Three animals each were killed 7, 14, or 28 days after SAH. Cerebral angiography was repeated on Day 7 in all animals and immediately prior to sacrifice in animals killed on Days 14 and 28. Both middle cerebral arteries and four control basilar arteries were examined using fluorescent antibody techniques with antisera to alpha-actin, myosin, fibronectin, fibrinogen, vimentin, desmin, laminin, and collagens (types I, III, IV, and V). Angiography showed that vasospasm was most severe on Day 7, present but resolving on Day 14, and completely resolved by Day 28. Microscopic study of arterial sections and blinded review of microphotographs of arterial sections by five independent observers did not reveal changes in intensity of density of staining for collagens, desmin, myosin, laminin, or alpha-actin in the tunica media of tunica adventitia. Fibronectin immunoreactivity increased 14 days after SAH. Seven days after SAH, occasional areas of tunica media showed immunoreactivity to fibrinogen. On Day 28, intimal thickening was observed in four of six middle cerebral arteries and this tissue demonstrated immunoreactivity to alpha-actin, myosin, vimentin, desmin, fibronectin, laminin, and each type of collagen. No significant increases in the number of intimal cells showing immunoreactivity to alpha-actin were seen and no significant changes in the hydroxyproline content of cerebral arteries developed at any time after SAH. These results suggest that rigidity and lumen narrowing of vasospasm are not due to increased arterial collagen, although other proteins in the arterial wall or an alteration in cross linking of existing proteins could produce these changes. There is no indication that smooth-muscle contractile proteins change during vasospasm or that increases in the number of alpha-actin-containing myointimal cells contribute to vasospasm. The occurrence of intimal thickening and increased tunica media fibronectin after vasospasm suggests that vasospasm damages smooth muscle, possibly as a result of intense prolonged smooth-muscle contraction. PMID- 1727174 TI - Intravenous fluid tonicity: effect on intracranial pressure, cerebral blood flow, and cerebral oxygen delivery in focal brain injury. AB - An investigation into the role of intravenous fluid tonicity in determining intracranial pressure (ICP) after brain injury is described. The authors compare the results of infusion of a hypotonic fluid (Ringer's lactate, 270 mOsm/liter) to those of a hypertonic fluid (hypertonic sodium lactate, 500 mOsm/liter) in a porcine model of focal cryogenic brain injury. Hemodynamic parameters (ICP, regional cerebral blood flow (CBF), and oxygen delivery) and serum osmolarity were measured every 3 hours for 24 hours after injury. At sacrifice, the water content of the lesioned and nonlesioned cortex was determined by specific gravity. The cryogenic injury produced a significant increase in ICP and a significant decrease in CBF in all experimental groups. Maintenance infusion of hypertonic sodium lactate for 24 hours resulted in significantly lower ICP, higher CBF and oxygen delivery, and higher serum osmolarity than Ringer's lactate infusion. Cortical water content in the area of the lesion was similar in both groups, but in the uninjured hemisphere it was significantly lower in the hypertonic group. These data suggest that hypertonic maintenance fluid improves intracranial compliance by dehydrating uninjured cortex. Improved CBF in the hypertonic group may be due to dehydration of cerebrovascular endothelium and erythrocytes. By reducing ICP and improving CBF, hypertonic fluid administration may thus reduce secondary brain injury after head trauma. PMID- 1727175 TI - Endothelin-1 of canine basilar artery in vasospasm. AB - Cerebral vasospasm was induced in adult mongrel dogs by a two-hemorrhage method. The basilar arteries were quickly frozen after careful removal of surrounding blood clot and their level of immunoreactive endothelin-1, a strong vasoconstrictor produced by the endothelial and vascular smooth-muscle cells, was measured by sandwich-enzyme immunoassay. The levels of immunoreactive endothelin 1 (mean +/- standard deviation) were 112.9 +/- 7.0 pg/mg protein prior to vasospasm, 180.4 +/- 24.7 pg/mg protein on Day 2 after vasospasm, and 115.0 +/- 24.0 pg/mg protein on Day 7, showing a significant increase (p less than 0.01) in immunoreactive endothelin-1 only on Day 2. In addition, vasospasm was moderately reversed by the topical application of monoclonal antibody against endothelin-1 on Day 2 but rather resistant to topical monoclonal antibody on Day 7. It is suggested that endothelin-1 could act as a trigger in the early stages of cerebral vasospasm, but that the maintenance of cerebral vasospasm at later stages might be independent of endothelin-1. PMID- 1727176 TI - Childhood cholesterol screening: contraindicated. PMID- 1727177 TI - Journal reviews in JAMA. PMID- 1727178 TI - 1992 could be pivotal year in efforts to improve health of people everywhere. PMID- 1727179 TI - Computer systems for medical diagnoses. PMID- 1727180 TI - Informatics: development and evaluation of information technology in medicine. PMID- 1727181 TI - Computer-based patient records: the next generation of medicine? PMID- 1727182 TI - Palmtop computers on the medical wards. PMID- 1727183 TI - What AIDS patients won't tell their doctors. PMID- 1727184 TI - From the Assistant Secretary for Health, US Public Health Service. PMID- 1727185 TI - From the Centers for Disease Control. HIV/AIDS knowledge and awareness of testing and treatment--behavioral risk factor surveillance. PMID- 1727186 TI - Limiting specific interventions in advance directives. PMID- 1727187 TI - Preemployment drug screening. PMID- 1727188 TI - The health hazards of asbestos removal. PMID- 1727189 TI - Group A streptococcus septicemia in children. PMID- 1727190 TI - Licensing of international medical graduates. PMID- 1727191 TI - Needlestick injury associated with venipuncture. PMID- 1727192 TI - Haloperidol: did it cause the respiratory arrest? PMID- 1727193 TI - Of mugs and marketing: the Robin Hood solution. PMID- 1727194 TI - Continuous quality improvement. PMID- 1727195 TI - Beeper costochondritis. PMID- 1727196 TI - Endocarditis after acupuncture and injection--treatment by a natural healer. PMID- 1727197 TI - House calls. PMID- 1727198 TI - Prevalence of migraine headache in the United States. Relation to age, income, race, and other sociodemographic factors. AB - OBJECTIVE: To describe the magnitude and distribution of the public health problem posed by migraine in the United States by examining migraine prevalence, attack frequency, and attack-related disability by gender, age, race, household income, geographic region, and urban vs rural residence. DESIGN: In 1989, a self administered questionnaire was sent to a sample of 15,000 households. A designated member of each household initially responded to the questionnaire. Each household member with severe headache was asked to respond to detailed questions about symptoms, frequency, and severity of headaches. SETTING: A sample of households selected from a panel to be representative of the US population in terms of age, gender, household size, and geographic area. PARTICIPANTS: After a single mailing, 20,468 subjects (63.4% response rate) between 12 and 80 years of age responded to the survey. Respondents and non-respondents did not differ by gender, household income, region of the country, or urban vs rural status. Whites and the elderly were more likely to respond. Migraine headache cases were identified on the basis of reported symptoms using established diagnostic criteria. RESULTS: 17.6% of females and 5.7% of males were found to have one or more migraine headaches per year. The prevalence of migraine varied considerably by age and was highest in both men and women between the ages of 35 to 45 years. Migraine prevalence was strongly associated with household income; prevalence in the lowest income group (less than $10,000) was more than 60% higher than in the two highest income groups (greater than or equal to $30,000). The proportion of migraine sufferers who experienced moderate to severe disability was not related to gender, age, income, urban vs rural residence, or region of the country. In contrast, the frequency of headaches was lower in higher-income groups. Attack frequency was inversely related to disability. CONCLUSIONS: A projection to the US population suggests that 8.7 million females and 2.6 million males suffer from migraine headache with moderate to severe disability. Of these, 3.4 million females and 1.1 million males experience one or more attacks per month. Females between ages 30 to 49 years from lower-income households are at especially high risk of having migraines and are more likely than other groups to use emergency care services for their acute condition. PMID- 1727199 TI - Plasma cholesterol concentration and mortality. The Whitehall Study. AB - OBJECTIVE--To examine the relationship between plasma cholesterol concentration and mortality from major causes of death. DESIGN: --Cohort study. SETTING--Civil service offices in London, England. PARTICIPANTS--There were 17,718 male civil servants aged 40 through 64 years at the time of study entry between 1967 and 1969. MAIN OUTCOME MEASURE--Mortality from major cause groups. RESULTS--There were 4022 deaths in the cohort over the 18 years of follow-up. Total mortality increased with cholesterol level, although mortality in the small group with very low cholesterol levels (5% of study population) was nonsignificantly higher (P greater than .5) than that of the remainder of the lowest quintile cholesterol group. Coronary heart disease mortality increased with increasing cholesterol concentration from the lowest levels (P less than .001 for trend). The cancer mortality rate in the group below the fifth centile of the cholesterol distribution was higher than in the remainder of the cohort for lung (P less than .001), pancreas (P = .05), liver (P = .09), and all smoking-related cancers (P = .02). Only for lung cancer was there a consistent inverse trend with cholesterol level (P less than .01). Rates of mortality due to non-neoplastic respiratory disease were inversely related to cholesterol level (P less than .001). Health state at the time of examination and socioeconomic position were related to cholesterol concentration--subjects in lower employment grades, with disease at baseline, with a history of recent unexplained weight loss, or who had been widowed had lower initial cholesterol levels. These associations largely accounted for the relationships between cholesterol level and noncardiovascular mortality. CONCLUSIONS--The inverse associations between plasma cholesterol concentration and mortality from certain causes of death seen in cohort studies could be because the participants with low cholesterol levels possess other characteristics that place them at an elevated risk of death. PMID- 1727200 TI - Eosinophilia-myalgia syndrome in L-tryptophan-exposed patients. AB - OBJECTIVES: To study the incidence of eosinophilia-myalgia syndrome, the risk factors associated with the syndrome, and the clinical spectrum of illness associated with L-tryptophan use in an exposed population. DESIGN: Retrospective cohort and nested case-control studies of risk factors for eosinophilia-myalgia syndrome using inpatient and outpatient chart reviews, telephone interviews, and in-person patient interviews. Descriptive study of clinical course of L tryptophan users. SETTING: Office practice of one psychiatrist based in a small city (population 43,467) in South Carolina. PATIENTS: Eligible subjects were all patients from the practice who used L-tryptophan during the 1989 study interval. Of these, 418 (87%) were interviewed. MAIN OUTCOME MEASURES: Clinical spectrum of illness associated with L-tryptophan use, including definite and possible cases of eosinophilia-myalgia syndrome. RESULTS: Among the 418 interviewed L-tryptophan users, we identified 47 definite cases (11%) and 68 possible cases (16%) of eosinophilia-myalgia syndrome, most of which involved patients who were using one retail brand of L-tryptophan (brand A). Among the 157 brand A users, we identified 45 definite cases (29%) and 36 possible cases (23%) of eosinophilia myalgia syndrome, and the risk for the syndrome increased as the brand A dose increased. Fifty percent (19 of 38) of those using more than 4000 mg/day developed definite eosinophilia-myalgia syndrome, and 84% (32 of 38) developed either definite or possible eosinophilia-myalgia syndrome. On multivariate analysis, risk for definite eosinophilia-myalgia syndrome was associated with brand A dose and age of the patient; however, gender, race, and use of other medications were not associated with the syndrome. CONCLUSIONS: These results suggest that many people exposed to the agent causing eosinophilia-myalgia syndrome may develop illness, and dose of presumably contaminated L-tryptophan is the single most important predictor of eosinophilia-myalgia syndrome. The broad range of signs and symptoms reported by patients using L-tryptophan illustrates that a strict case definition may identify only about half of those affected. PMID- 1727201 TI - Effects of ranitidine on blood alcohol levels after ethanol ingestion. Comparison with other H2-receptor antagonists. AB - OBJECTIVE: To determine whether the H2-receptor antagonist, ranitidine, which is a potent inhibitor of gastric alcohol dehydrogenase activity in vitro, increases the bioavailability of orally administered ethanol (0.3 g/kg of body weight) and to compare the resulting blood alcohol concentrations with those of two other H2 antagonists, cimetidine and famotidine, the latter of which does not inhibit gastric alcohol dehydrogenase. DESIGN: For each of the H2-receptor antagonists, a different group of subjects was used. In each group, a paired design was adopted with each subject serving as his own control. SETTING: Hospital laboratory. SUBJECTS: Normal, healthy men aged 24 to 46 years. INTERVENTION: Eight men were treated for 1 week with ranitidine (300 mg/d), six with cimetidine (1000 mg/d), and six with famotidine (40 mg/d). MEASURES: Peak blood alcohol concentrations, areas under the blood alcohol curve, first-pass metabolism, and bioavailability of orally consumed ethanol. RESULTS: Relative to baseline, ranitidine increased the mean peak concentration and the area under the curve of blood alcohol concentrations by 34% (P less than .05) and 41% (P less than .01), respectively. First-pass metabolism of ethanol was decreased from 70 +/- 10 to 31 +/- 9 mg/kg of body weight, with a corresponding increase in ethanol bioavailability of 79.6% to 92.6%. By comparison, cimetidine had even a greater effect on blood alcohol levels, while famotidine had no significant effects. CONCLUSION: Patients treated with ranitidine or cimetidine should be warned of possible functional impairments after consumption of amounts of ethanol considered safe in the absence of such therapy. PMID- 1727202 TI - The Department of Veterans Affairs smoke-free policy. PMID- 1727203 TI - Costs and benefits of preemployment drug screening. AB - OBJECTIVE: This study provides a cost-benefit analysis of preemployment drug screening and evaluates the sensitivity of this analysis to variation in its underlying assumptions. DESIGN: Cost-benefit analysis, based on a cohort analytic study previously reported. SETTING: Employees of the US Postal Service in Boston, Mass. PARTICIPANTS: Estimates of costs and benefit are based on a cohort of 2533 postal workers in Boston and on average costs for the Postal Service in Boston and nationwide. RESULTS: Drug screening would have saved the Postal Service $162 per applicant hired. However, these results were sensitive to the assumptions in the model. If the prevalence of drug use in the population screened were 1% rather than 12%, the program would lose money. Similarly, if the cost per urine sample screened were $95 rather than the $49 assumed, then the program would lose money, even if the prevalence of drug positives was as high as 9%. CONCLUSIONS: Because of the sensitivity of this analysis to changes in its underlying assumptions, any company considering preemployment drug screening should carefully weigh the costs and benefits in its own industry. PMID- 1727204 TI - Passive smoking and the risk of heart disease. AB - OBJECTIVE: This paper reviews the evidence that exposure to environmental tobacco smoke (ETS) increases the risk of heart disease death among persons who have never smoked (never-smokers). The annual number of heart disease deaths in the United States attributable to ETS is estimated, as is the individual risk of heart disease death for exposed never-smokers. DATA SOURCES: Nine epidemiologic studies and numerous experimental studies are available to evaluate the association of ETS and heart disease. DATA SYNTHESIS: The relative risk for never smokers living with current or former smokers, compared with never-smokers living with nonsmokers, has ranged from 0.9 to 3.0 in nine studies. Seven studies were positive, one was positive for women but not men, and one was negative. Several studies have shown a dose-response relationship and have controlled for other risk factors. Evidence from experimental studies suggests that ETS can damage the cardiovascular system, via both short-term and long-term mechanisms. Assuming that the observed heart disease risk for those exposed to ETS is not an artifact of misclassification or confounding, approximately 35,000 to 40,000 deaths from ischemic heart disease among never-smokers and long-term former smokers are estimated to have occurred annually in the United States as a result of ETS exposure in the early 1980s. An individual male never-smoker living with a current or former smoker is estimated to have an approximately 9.6% chance of dying of ischemic heart disease by the age of 74 years, compared with a 7.4% chance for a male never-smoker living with a nonsmoker. The corresponding lifetime risks for women are 6.1% and 4.9%. CONCLUSIONS: The public health burden due to ETS exposure is likely to be much greater for heart disease than for lung cancer, which has been the focus of most debate to date. Individual lifetime excess risks of heart disease death due to ETS of one to three per 100 can be compared with much lower excess risks of one death per 100,000, which are often used in determining environmental limits for other toxins. Exposure to ETS is not currently regulated at the federal level, except for domestic air traffic. PMID- 1727205 TI - Points and end points in pediatric oncology. PMID- 1727206 TI - Behavioral effects of corticosteroids in children with acute lymphoblastic leukemia. AB - We evaluated the behavior of 38 children with standard-risk and high-risk acute lymphoblastic leukemia who received corticosteroids as part of their antileukemia chemotherapy. Each patient was assessed on two occasions: 16 weeks following remission and 1 year thereafter. Parent reports on emotional lability, attention span/hyperactivity, sleep disturbance, listlessness, peer relations, and depressed mood were obtained for 4 consecutive weeks: week before, week during, and 2 weeks after treatment administration. At the 16 week evaluation, standard risk (N = 17) and high-risk (N = 21) treatment differed by steroid dose and systemic chemotherapy, while at the 1 year testing, treatment differed only by steroid dose (prednisone 40 mg/m2 vs. 120 mg/m2). Statistically significant changes in all measures were observed during treatment as compared with before and after treatment for both risk groups and assessment times. High-risk patients exhibited greater behavioral effects than standard-risk patients only for emotional lability, listlessness, and depressed mood at the 16 week testing, when both steroid dose and chemotherapy differed. Girls had slightly greater behavioral effects than boys, while no influence of age was observed. At the doses tested, steroid dose per se does not appear to be the primary variable affecting behavioral changes. PMID- 1727207 TI - Complications associated with indwelling catheters. AB - Between 1983 and 1985, 170 consecutive patients received doxorubicin-containing adjuvant chemotherapy through central venous catheters, and four via a long indwelling catheter in the antecubital fossa. The objective of this retrospective study is to determine the acute and chronic complications associated with indwelling catheters. Ninety-four (56%) patients did not experience any complications. The incidence of acute complications was 2%, which included three pneumothorax. Twenty-nine (17%) patients experienced chronic complications. Of those, 20 (12%) developed infectious complications, and 9 (19%) developed thrombus. In addition, 12 (7%) developed multiple complications, and 32 (19%) patients had other local complications at the catheter site, which included allergic reactions and catheter breakage. Each of these complications was resolved with appropriate treatment. None of the patients died from complications of the indwelling catheter. PMID- 1727208 TI - Serum-soluble interleukin-2 receptors in B-cell lymphoproliferative malignancies. AB - The levels of soluble interleukin-2 receptors (sIL-2R) were determined in the serum of 53 patients with B-cell lymphoproliferative malignancies, including 31 patients with non-Hodgkin lymphomas (NHL), 16 with chronic lymphocytic leukemia (CLL), and 6 with multiple myeloma. In addition, serum samples from 40 patients with various solid tumors as well as from 53 healthy individuals were used as controls. It was found that the mean serum levels of sIL-2R were significantly increased (P less than 0.001) in NHL (mean +/- standard error of the mean 2,327 +/- 320 units/ml) and CLL patients (2517 +/- 451 units/ml) as compared to normal controls (207 +/- 17 units/ml). No such difference was observed when the serum sIL-2R levels of patients with multiple myeloma or solid tumors were analyzed. Serum sIL-2R levels were closely related to the clinical stage, the presence of B symptoms, and the disease activity of patients with NHL and CLL. In fact, response to chemotherapy was followed by marked decrease or normalization of sIL 2R levels, while in a number of patients sIL-2R values were even able to predict disease relapse. Finally, no association with histologic grade in NHL patients, could be demonstrated. We conclude that serum sIL-2R (1) are increased only in B NHL and B-CLL but not in myeloma patients, (2) are related to the tumor burden, and (3) can serve as a valuable tumor marker for the monitoring of patients treatment. PMID- 1727209 TI - Surgical complications in acute leukemia in childhood: a 20 year experience. AB - A retrospective study of all patients presenting with acute leukemia to a single institution during the period 1968-1988 was undertaken to determine the type and incidence of surgical complications. One hundred twenty-eight of 296 patients were identified as requiring a surgical consultation for complications occurring during the course of the disease and many surgical disciplines were involved. Operative intervention was often required for these complications. Pediatric surgical specialists need to be aware of the range of surgical complications that can occur in children with acute leukemia and they must work in close co operation with pediatric medical oncologists to ensure optimal treatment for all patients. PMID- 1727210 TI - Hyperaminoaciduria identifies patients at risk of developing renal tubular toxicity associated with ifosfamide and platinate containing regimens. AB - We monitored renal tubular function in 18 neuroblastoma patients treated with chemotherapeutic regimens containing Ifosfamide and platinates. The total IFO dose ranged from 30 to 48 g/m2. After each IFO course and at regular intervals during follow-up 24 hour urinary exception of aminoacids, qualitative excretion of protein and glucose, tubular reabsorption of phosphate, serum pH, and liver enzyme (SGOT and SGPT) were measured. The ratio of alpha-amino-Nitrogen/total Nitrogen (normal less than 2.5%) and the urinary excretion pattern were used to quantify the aminoaciduria in 12 out of 15 evaluable patients some degree of tubular toxicity occurred during treatment, slowly progressing to a Debre-de Toni Fanconi syndrome (DTFS) in 7 patients. The DTFS was fully developed in most cases 3-9 months after the end of treatment and was reversible in 3 cases. Hyperaminoaciduria (HAA) occurred in all patients during treatment, preceding other signs of tubular toxicity. The maximum ratio measured before development of a DTFS was significantly higher in the patients with severe toxicity (P less than .01). HAA characterized by a ratio greater than 10% predicts the development of a DTFS with a sensitivity of 71.4% and a specificity of 87.4%. PMID- 1727211 TI - Acute nonlymphoblastic leukemia in children treated for acute lymphoblastic leukemia with an intensive regimen including teniposide. AB - Some cases of conversion from acute lymphoblastic leukemia (ALL) to acute nonlymphoblastic leukemia (ANLL) at relapse have been reported recently. We report three cases initially diagnosed as having ALL and showing morphological, cytochemical, and immunophenotypic features of ANLL at relapse (lineage switch). Conversion was observed among 14 patients who developed bone marrow relapse while undergoing intensive treatment with our ALL protocol, which includes teniposide, and that had been administered to 62 patients. The three cases converted at first relapse, with a mean time of 20 months (13-29 months). Clinical and immunologic characteristics of T-cell leukemia were present in one patient. Changes documented in cytogenetic studies are discussed. The underlying mechanisms for the lineage switch remain unclear as does its relation with mixed lineage leukemias, but we believe that drugs employed in our therapy protocol could have had an influence on this conversion. PMID- 1727212 TI - The pediatric patient with suspected adrenal neoplasm: which radiological test to use? AB - To establish which radiological test to use in a pediatric patient with suspected neoplasm, we retrospectively studied 19 children with proven adrenal neoplasm who had a combination of ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI). The results show that US should remain the initial imaging modality for evaluating abdominal masses in children but that MRI is more accurate than CT and US in detecting the organ of origin and the extent of an adrenal lesion and should, therefore, be the modality of choice for the definitive evaluation of adrenal neoplasm in children. PMID- 1727213 TI - Bone marrow necrosis and thrombotic complications in childhood acute lymphoblastic leukemia. AB - Two children with bone marrow necrosis at diagnosis or at relapse of acute lymphoblastic leukemia (ALL) had thrombotic complications 15 and 17 days after starting remission induction therapy including prednisone, vincristine, and L asparaginase. The close temporal relationship of these two relatively rare events suggests that bone marrow necrosis is a predisposing factor to the development of thrombosis. PMID- 1727214 TI - Late effects of therapy in adult survivors of osteosarcoma and Ewing's sarcoma. AB - To study the late consequences of primary bone cancer, we interviewed 82 osteosarcoma and 29 Ewing's sarcoma survivors regarding their health, fertility and offspring, employment, annual income, and activities of daily living. All subjects had been diagnosed before age 20 (mean age, 14.6 years), had survived at least 5 years from diagnosis, and were at least 21 years of age. On average, they were 32.5 years of age at interview. As controls, 151 siblings were interviewed. During the follow-up period, eight survivors had died, and eight survivors had been diagnosed with a second malignancy (7.2%; P = .002). No other health condition distinguished survivors from controls. Although the survivors were more likely than controls to have some difficulty climbing stairs and to have had employment disability, employment status and annual income at follow-up were similar. Deficits in marriage and fertility were not significant. Adult survivors of primary bone tumors diagnosed during childhood or adolescence are at high risk for second malignancies and premature death, making continued medical follow-up of utmost importance. Despite the physical impairment following limb amputation for many, the majority of outcomes we measured did not differ from controls, suggesting few adverse psychosocial outcomes in this group of cancer survivors. PMID- 1727215 TI - Secondary acute non-lymphoblastic leukemia in two children following treatment with a cis-diamminedichloroplatinum-II-based regimen for osteosarcoma. AB - Acute nonlymphocytic leukemia (ANLL) developed in 2 of 142 pediatric patients with osteosarcoma treated with a cis-diamminedichloroplatinum-II (CDP)-based regimen: acute monomyelogenous leukemia (M4) with a normal female karyotype in one and acute myelogenous leukemia (M2) with t (8,21) in the other. The ANLLs occurred, respectively, in each patient 3 and 4 years after the initial diagnosis of osteosarcoma. In contrast to most of the adult experience and consistent with the majority of reported ANLL in children, the disease was characterized by an absence of the smoldering phase and cytogenetic findings similar to those seen in de novo ANLL. PMID- 1727216 TI - Malignant melanoma: primary presentation in bone marrow and lymph node. AB - We describe an unusual presentation of malignant melanoma with simultaneous lymph node and bone marrow metastasis. In addition the disease was associated with immune-mediated thrombocytopenia. A brief remission was obtained with combination chemotherapy. PMID- 1727217 TI - Bone metastases in malignant mesothelioma are not uncommon. PMID- 1727218 TI - Manipulating the immune system with immune globulin. PMID- 1727219 TI - Causes of iron overload. PMID- 1727220 TI - AIDs, activism, and the politics of health. PMID- 1727221 TI - Drug promotion and scientific exchange. PMID- 1727222 TI - Drug promotion and scientific exchange. PMID- 1727224 TI - Drug promotion and scientific exchange. PMID- 1727223 TI - Drug promotion and scientific exchange. PMID- 1727226 TI - Screening for colorectal cancer. PMID- 1727225 TI - Screening for colorectal cancer. PMID- 1727227 TI - Screening for colorectal cancer. PMID- 1727228 TI - Absence of birth defects in offspring of women treated with dactinomycin. PMID- 1727229 TI - Hazard of lead in infant formula. PMID- 1727230 TI - Sensitivity of erythrocyte protoporphyrin as a screening test for lead poisoning. PMID- 1727231 TI - Balloon aortic valvuloplasty. PMID- 1727232 TI - Prophylactic immune globulin in chronic lymphocytic leukemia. PMID- 1727233 TI - Malpractice and negligence. PMID- 1727234 TI - Malpractice and negligence. PMID- 1727235 TI - The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. AB - BACKGROUND: Dilated cardiomyopathy is characterized by an increase in ventricular size and impairment of ventricular function. Most cases are believed to be sporadic, and familial dilated cardiomyopathy is usually considered to be a rare and distinct disorder. We studied the proportion of cases of idiopathic dilated cardiomyopathy that were familial in a large sequential series of patients whose first-degree relatives were investigated regardless of whether these relatives had cardiac symptoms. METHODS: We studied relatives of 59 index patients with idiopathic dilated cardiomyopathy of obtaining a family history and performing a physical examination, electrocardiography, and two-dimensional, M-mode, and Doppler echocardiography. A total of 315 relatives were examined. RESULTS: Eighteen relatives from 12 families were shown to have dilated cardiomyopathy. Thus, 12 of the 59 index patients (20.3 percent) had familial disease. There was no difference in age, sex, severity of disease, exposure to selected environmental factors, or electrocardiographic or echocardiographic features between the index patients with familial disease and those with nonfamilial disease. A noteworthy finding was that 22 of 240 healthy relatives (9.2 percent) with normal ejection fractions had increased left ventricular diameters during systole or diastole (or both), as compared with 2 of 112 healthy control subjects (1.8 percent) who were studied separately. CONCLUSIONS: Dilated cardiomyopathy was found to be familial in at least one in five of the patients in this study, a considerably higher percentage than in previous reports. This finding has important implications for family screening and provides direction for further investigation into the causes and natural history of dilated cardiomyopathy. PMID- 1727236 TI - Comparison of amphotericin B with fluconazole in the treatment of acute AIDS associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group. AB - BACKGROUND: Intravenous amphotericin B, with or without flucytosine, is usually standard therapy for cryptococcal meningitis in patients with the acquired immunodeficiency syndrome (AIDS). Fluconazole, an oral triazole agent, represents a promising new approach to the treatment of cryptococcal disease. METHODS: In a randomized multicenter trial, we compared intravenous amphotericin B with oral fluconazole as primary therapy for AIDS-associated acute cryptococcal meningitis. Eligible patients, in all of whom the diagnosis had been confirmed by culture, were randomly assigned in a 2:1 ratio to receive either fluconazole (200 mg per day) or amphotericin B. Treatment was considered successful if the patient had had two consecutive negative cerebrospinal fluid cultures by the end of the 10 week treatment period. RESULTS: Of the 194 eligible patients, 131 received fluconazole and 63 received amphotericin B (mean daily dose, 0.4 mg per kilogram of body weight in patients with successful treatment and 0.5 mg per kilogram in patients with treatment failure; P = 0.34). Treatment was successful in 25 of the 63 amphotericin B recipients (40 percent; 95 percent confidence interval, 26 percent to 53 percent) and in 44 of the 131 fluconazole recipients (34 percent; 95 percent confidence interval, 25 percent to 42 percent) (P = 0.40). There was no significant difference between the groups in overall mortality due to cryptococcosis (amphotericin vs. fluconazole, 9 of 63 [14 percent] vs. 24 of 131 [18 percent]; P = 0.48); however, mortality during the first two weeks of therapy was higher in the fluconazole group (15 percent vs. 8 percent; P = 0.25). The median length of time to the first negative cerebrospinal fluid culture was 42 days (95 percent confidence interval, 28 to 71) in the amphotericin B group and 64 days (95 percent confidence interval, 53 to 67) in the fluconazole group (P = 0.25). Multivariate analyses identified abnormal mental status (lethargy, somnolence, or obtundation) as the most important predictive factor of a high risk of death during therapy (P less than 0.0001). CONCLUSIONS: Fluconazole is an effective alternative to amphotericin B as primary treatment of cryptococcal meningitis in patients with AIDS. Single-drug therapy with either drug is most effective in patients who are at low risk for treatment failure. The optimal therapy for patients at high risk remains to be determined. PMID- 1727237 TI - Iron overload in Africa. Interaction between a gene and dietary iron content. AB - BACKGROUND AND METHODS: In contrast to hemochromatosis, which in white populations is inherited through a gene linked to the HLA locus, iron overload in sub-Saharan Africa is believed to result solely from increased dietary iron derived from traditional home-brewed beer. To examine the hypothesis that African iron overload also involves a genetic factor, we used likelihood analysis to test for an interaction between a gene (the hypothesized iron-loading locus) and an environmental factor (increased dietary iron) that determines transferrin saturation and unsaturated iron-binding capacity. We studied 236 members of 36 African families chosen because they contained index subjects with iron overload. Linkage to the HLA region was tested with use of lod scores. RESULTS: In the index subjects, increased iron was present in both hepatocytes and cells of the mononuclear-phagocyte system. Among family members with increased dietary iron due to the consumption of traditional beer, transferrin saturation in serum was distributed bimodally, with 56 normal values (less than 60 percent saturation) and 44 elevated values; the mean serum ferritin concentration was five times higher in the subjects with elevated transferrin saturation (P less than 0.005). The pedigree analysis provided evidence of both a genetic effect (P less than 0.005) and an effect of increased dietary iron (P less than 0.005) on transferrin saturation and unsaturated iron-binding capacity. In the most likely model, increased dietary iron raised the mean transferrin saturation from 30 to 81 percent and lowered the mean unsaturated iron-binding capacity from 38 to 13 mumol per liter in subjects heterozygous for the iron-loading locus. The hypothesis of tight linkage to HLA was rejected. CONCLUSIONS: Iron overload in Africa may be caused by an interaction between the amount of dietary iron and a gene distinct from any HLA-linked gene. PMID- 1727238 TI - Lengthening the human mandible by gradual distraction. AB - Lengthening of the mandible by gradual distraction was performed on four young patients (average age 78 months). The amount of mandibular bone lengthening ranged from 18 to 24 mm; one patient with Nager's syndrome underwent bilateral mandibular expansion. Following the period of expansion, the patients were maintained in external fixation for an average of 9 weeks to allow ossification. The patients were followed for a minimum of 11 months to a maximum of 20 months with clinical and dental examinations as well as photographic and radiographic documentation. The technique holds promise for early reconstruction of craniofacial skeletal defects without the need for bone grafts, blood transfusion, or intermaxillary fixation. PMID- 1727239 TI - Adaptive hypertrophy of the digit following little finger to thumb transposition. AB - This is a long-term retrospective study of eight patients who had undergone little finger to thumb transposition after traumatic thumb loss in order to evaluate the presence of long-term changes in the transposed digit. The transposed little finger, contralateral (nontransposed) little finger, and contralateral thumb were compared using standardized measurements of size, comparison photographs, x-rays, and volume determination using silicone mold impressions of these digits. Significant and marked hypertrophy of the transposed digit was demonstrated in all these patients. Comparison radiographs demonstrated that this enlargement was due to hypertrophy of both soft-tissue and osseous components. This study demonstrates that the little finger transposed to the thumb position undergoes an adaptive hypertrophy to become more thumblike in appearance as well as function. PMID- 1727240 TI - A comparative analysis of healing of surgical cleft lip corrected in utero and neonates. AB - We studied the healing process in surgically created cleft lips in fetal mice and compared it with that in newborn mice with cleft lips. Our purpose was to determine the time for optimal healing, defined as minimal scarring, for a repaired cleft lip. Full-thickness paramedian lip incisions were made in NMRI mice in utero, in 2- and 4-day-old neonates, and in adults (n = 10 in each experimental and control group). The healing process was studied by biochemical analysis of hyaluronic acid and hydroxyproline content in the repaired cleft tissue. We found that the production of hyaluronic acid remained stable during the healing period and was similar in all experimental groups. However, there was an unexplained but consistent depression in the hyaluronic acid content of fetal tissue 2 days after repair. Hydroxyproline was present in the fetal healing tissue, but in a low concentration, starting 4 days after surgical incision of the lip. The production of hydroxyproline in 2-day-old neonates was similar to that in the fetuses throughout the healing period (p less than 0.0005). However, the production of hydroxyproline increased in 4-day-old neonatal and adult tissues. In conclusion, we found an optimal healing period for mice with minimal collagen production in the late fetal stage, and this lasted 2 days after birth. PMID- 1727242 TI - Photographs: are they misleading? PMID- 1727241 TI - The microcirculation of myocutaneous island flaps in pigs studied with radioactive blood volume tracers and microspheres of different sizes. AB - In order to further improve the understanding of hemodynamic changes in the immediate postoperative phase after elevation of myocutaneous flaps, regional blood flow and arteriovenous (A-V) shunting were measured in rectus abdominis island flaps in 8 pigs. Radioactive microspheres of two sizes (15 and 50 micron) were used. Approximately half (53.4 +/- 6 percent) of the 15-micron microspheres and one-fourth (24.1 +/- 6 percent) of the 50-micron microspheres entering the flap appeared in the venous outflow. Compared with the control area, A-V shunting was significantly increased in muscle and substantially more pronounced in skin. Nutritional blood flow, total blood flow, and vascular volume were increased in muscle and unchanged in skin and subcutis. The lowest tissue hematocrit of 7 +/- 1 percent was found in skin as compared with a central hematocrit of 35 +/- 2 percent. Tissue hematocrit in flap muscle was decreased to 17 +/- 2 percent when compared with control muscle (22 +/- 3 percent), and the mean transit time for blood was correspondingly decreased. Thus vasodilation provided increased perfusion through muscular capillaries and through A-V shunts. Shunting of 15 micron microspheres appeared to take place not only in skin, but also in subcutis and muscle, which challenges the widespread belief that A-V shunting does not occur in muscle. PMID- 1727243 TI - The insurance game. PMID- 1727244 TI - Pedunculated club-shaped swelling in the nostril. AB - This paper presents a skin mass found in the nostril of a boy who had no other anomalies. The skin mass was similar to the pedunculated masses with median cleft that have been reported previously. The literature is reviewed, and the relationship between skin masses associated with true median cleft and the skin mass in our patient is discussed. PMID- 1727246 TI - Pencil-core granuloma. AB - Noncaseating granulomas present as a delayed foreign-body reaction to retained pencil-core fragments. The clinical appearance of the lesion can closely resemble melanoma. Surgeons who use pencils as surgical marking implements should be aware that there is a potential risk of developing late pencil-core granulomas. This risk can be reduced by carefully removing any pieces of pencil lead from the wound. PMID- 1727245 TI - Nasal reconstruction with autologous rib cartilage: a 43-year follow-up. AB - Autogenous costal cartilage has long been a popular material for nasal augmentation. The history of autogenous cartilage transplantation is reviewed. Two patients are presented who underwent nasal augmentation with autologous costal cartilage with a 43-year follow-up on each patient. PMID- 1727247 TI - Surgical model for in vivo evaluation of cultured epidermal sheets in mice. AB - The objective of this study was to establish a novel surgical model for the grafting of cultured epidermal sheets in mice in order to optimize studies on the behavior of these grafts. Graft-related skin immunology and wound-healing studies also would benefit from this adapted surgical approach. Adapted tie-over surgical procedures were established for mice and promptly applied. Early-stage observation of grafts was made possible by replacing the cotton dressing with a saddle-like plastic tube dressing with a screw cap. We grafted normal Balb/c mice and athymic nude (nu/nu) mice with cultured human epidermis. Evaluation of graft rejection was carried out with the first group, whereas the second provided information on epidermal stratification and terminal differentiation. This innovation permitted direct evaluation of the grafted tissue at any time. Advances in applied transplantation research will certainly provide additional tools for human applications. PMID- 1727248 TI - A new and simple way to clear the hair from operating fields in the scalp. AB - The McGivney instrument has been designed for rubber band ligation of hemorrhoids. A simple way to clear the hair from the operating field with the aid of this device during surgery on the scalp is described. The method has been found to be quick and easy, and the tuft of hair is also held in a firmer position than with other methods used for this purpose. PMID- 1727249 TI - Harelip surgery in ancient China: further investigations. AB - The various statements of medical historians concerning harelip surgery in Ancient China have been studied with the help of eminent historians and based on reliable documents. The text of the Chin Shu adds a very important contribution to our knowledge of ancient cleft lip repair: the first use of the word harelip, the first short but rather accurate description of the operation and the postoperative care, and the social and psychological impact of facial disfigurement. The assertion regarding Fang Kan as the doctor of lip repair during the T'ang Dynasty turned out to be completely erroneous. Fang Kan was a poet whose life and lack of acceptance were dominated by his external appearance: a harelip deformity that was operated on late in life. PMID- 1727250 TI - Adequate preclinical testing of bioplastique injectable material? PMID- 1727251 TI - Prosthesis radiolucency. PMID- 1727252 TI - A better way of performing photography in the operating room. PMID- 1727253 TI - The ocular danger of Hibiclens (chlorhexidine) PMID- 1727254 TI - Skoog remembered in the repair of unilateral cleft lip. PMID- 1727255 TI - Nasotracheal intubation in patients with facial fractures. PMID- 1727256 TI - Clomipramine for obsessive pain-type syndromes. PMID- 1727257 TI - Treatment of exophthalmos. PMID- 1727258 TI - Recollections of Michael L. Lewin, M.D. PMID- 1727259 TI - In memoriam, Michael L. Lewin, M.D. PMID- 1727260 TI - Frontal plagiocephaly: synostotic, compensational, or deformational. AB - Plagiocephaly is a term commonly used to describe congenital forehead asymmetry. Sixty patients with frontal plagiocephaly were evaluated retrospectively and separated into three types: synostotic (N = 24), compensational (N = 3), and deformational (N = 33). Categorization of frontal plagiocephaly as synostotic or deformational was reliably made by physical examination, focusing on the supraorbital rims, nasal root, ears, and malar eminences. Other anatomic parameters useful in the differential diagnosis included chin point, palpebral fissures, and facial height. This study documented that birth histories were similar for synostotic and deformational frontal plagiocephalic infants. However, other deformational anomalies were more common in deformational frontal plagiocephalic infants, whereas malformations had an equal incidence in deformational and synostotic frontal plagiocephalic infants. Torticollis was an associated finding in 64 percent of infants with deformational frontal plagiocephaly; almost all were ipsilateral. In contrast, head tilt, usually to the contralateral side, was noted in 14 percent of patients with synostotic frontal plagiocephaly. Female preponderance was noted in both synostotic (79 percent) and deformational (76 percent) frontal plagiocephaly. Left-sided involvement was seen in 73 percent of patients with deformational frontal plagiocephaly and in 46 percent of patients with synostotic frontal plagiocephaly. Premature pelvic descent, in the left occipital anterior position, may account for the high incidence of left-sided deformational plagiocephaly and ipsilateral torticollis. PMID- 1727261 TI - Indirect intracranial volume measurements using CT scans: clinical applications for craniosynostosis. AB - This paper describes a method for obtaining indirect intracranial volume measurements using CT scans with CTpak, a software package for quantitative analysis of CT scan data. The validity of this technique was confirmed by comparing direct measurement of the intracranial volume of five dry skulls with axial scans at 1.5- and 4-mm slice intervals to determine indirect volume. The indirect intracranial volume measurement technique was then used to compare preoperative and postoperative intracranial volume in 30 patients with craniosynostosis who underwent cranial vault and orbital osteotomies with reshaping and advancement. Our findings show that the suture release and simultaneous reshaping procedures usually carried out are, in fact, associated with increased intracranial volume. The observed intracranial volume gain is attributable to a combination of factors, including the surgical procedure carried out and ongoing growth. These factors are further modified by the diagnosis, age of the patient, and time interval between CT scans. PMID- 1727262 TI - Distinguishing soft-tissue hemangiomas from vascular malformations using technetium-labeled red blood cell scintigraphy. AB - Soft-tissue vascular lesions in children can be classified as either hemangiomas or vascular malformations. The distinction between the two has important prognostic and therapeutic implications. Over the past 8 years, we have evaluated 64 vascular lesions with the technetium-labeled red blood cell (Tc-RBC) scan. Twenty-eight lesions imaged as hemangiomas with intense focal uniform uptake. This diagnosis was confirmed in 27 lesions, or 96 percent. Thirty-six lesions imaged as vascular malformations with abnormal vessels or diffusely increased activity. This diagnosis was confirmed in 35 lesions, or 97 percent. Overall, the Tc-RBC scan was 97 percent accurate in distinguishing hemangiomas from vascular malformations. It is particularly useful when the clinical diagnosis of the lesion may not be evident. Not only can biopsy be avoided, but parents can be reassured at an earlier age and given accurate information regarding prognosis. PMID- 1727263 TI - Early, aggressive management of postoperative oropharyngocutaneous fistulas. AB - Oropharyngocutaneous fistulas remain a serious and potentially lethal complication. Advantages from surgical repair and the use of musculocutaneous flaps have been demonstrated. Timing of the procedure, however, has not been adequately addressed or emphasized. This report presents our experience with early, aggressive management of postoperative orocutaneous fistulas. Patients were reoperated at an average of 12 days after the initial surgery and underwent exploration, debridement of all devitalized tissues, and closure by reelevation of previously used flaps or with additional flaps. All wounds healed without further problem. We conclude that as long as the patient's general condition permits, early, aggressive management of fistulas should be the procedure of choice to reduce hospital stay and costly wound care and to avoid maceration and partial or complete necrosis of flaps and the potential rupture of the carotid artery. Timely radiotherapy can then be delivered, and quality of life can be significantly improved. PMID- 1727264 TI - The vascular anatomy of the galeal flap in the interparietal and midline regions. AB - The potential extension of the galeal flap in the interparietal area was studied on 17 fresh human cadaver heads by intravascular dye injection technique. It was demonstrated that an ipsilateral superficial temporal artery that supplies the galeal flap does not cross the midline or anastomose with the contralateral superficial temporal artery but ensures the survival of a flap extended up to 1 cm proximal to the sagittal suture line. The width of the temporoparietal flap can be extended up to 15 cm, depending on the vascular pattern of the superficial temporal artery. When required, the lateral extension may provide the required soft-tissue bulk despite the reduced flap length. PMID- 1727265 TI - Long-term effect of retrobulbar hematomas on the vision of cynomolgus monkeys. AB - Using the monkey model previously developed, we investigated the long-term effects of retrobulbar hematoma-induced retinal ischemia on functional vision and retinal histology. In this experimental model, ischemic periods of up to 120 minutes did not cause permanent visual deficits, as measured by flashed evoked visual potentials. Similarly, retinal histology showed no evidence of ischemic injury. From this we conclude that blindness after blepharoplasty is not due to retrobulbar hematomas alone and that additional predisposing factors are involved. The most likely additional factor is preexisting occult vascular ocular pathology. PMID- 1727266 TI - A comparison of conventional and low-bleed implants in augmentation mammaplasty. AB - We conducted a double-blind, retrospective comparison between low-bleed and non low-bleed (conventional) mammary implants because no controlled study has shown a difference in the degree of capsular contracture between the two types of implants. Twenty-five patients had conventional implants and form group A; twenty eight patients had low-bleed implants and form group B. All patients had submuscular augmentation. The mean Baker score was 1.51 for group A and 1.04 for group B for the entire patient population and 1.65 for group A and 1.07 for group B for patients with more than 1 year of follow-up. For the entire population, 34 percent of group A and 3.6 percent of group B had a Baker score of 2 or greater. For the population with more than 1 year of follow-up, 42 percent of group A and 7 percent of group B had a Baker score of 2 or greater. There was significantly (p less than 0.007) less contracture with the low-bleed implants for the entire population as well as for those patients with greater than 1 year of follow-up (p less than 0.015). PMID- 1727267 TI - Results of reaugmentation with MISTI prostheses after failure of smooth silicone prostheses. AB - In 1988 and 1989, we replaced smooth silicone double-lumen implants with Molecular Impact Surface Textured Implants (MISTIs) in 28 of our patients. Of these, 20 had experienced recurrent capsular contracture and sought an alternative prosthesis that would provide long-term relief from this problem, and 8 simply wanted larger prostheses. Among the reaugmentation patients who had experienced recurrent contractures, 4 have had problems with the texturized implants; 1 developed an infection, and 3 developed unilateral fibrosis within weeks of surgery. The infection was resolved with antibiotics, and the fibrosis was resolved with capsulectomy, biweekly methylprednisolone irrigation in the surgical pocket, and lenticular suction drainage over a 3-week period. After a 2 year follow-up, these 4 problematic patients have remained soft and asymptomatic following their treatment, and the remaining 16 patients have remained soft and asymptomatic since their surgery. PMID- 1727268 TI - The inferiorly based rectus abdominis myocutaneous flap for reconstruction of recurrent pressure sores. AB - Eight neurologically impaired patients underwent reconstruction of chronic perineal and ischial pressure sores utilizing an inferiorly based rectus abdominis myocutaneous flap. Other local and regional flap options had been previously used or were not feasible. In six patients, healing was uncomplicated. One patient required local debridement and flap readvancement. The second involved minor separation of a suture line and healed by secondary intention. All donor sites were closed directly and healed by primary intention. There was no evidence of hernia formation, and no functional deficit was detected from removal of the rectus muscle in any of the patients. In conclusion, it was felt that the inferiorly based rectus abdominis myocutaneous flap should be considered a reconstructive option in dealing with perineal and ischial pressure sores. Furthermore, for reasons discussed, we found distinct advantages to using this flap in spinal cord injury patients. PMID- 1727269 TI - MR imaging for pulmonary metastases? PMID- 1727270 TI - Recurrent breast cancer: stereotaxic localization for fine-needle aspiration biopsy. Work in progress. AB - The efficacy of stereotaxic localization for fine-needle aspiration biopsy in the detection of recurrent cancer manifested as calcifications on mammograms was evaluated in 43 patients that had been treated with local resection and radiation therapy. Six patients had malignant aspirates and one had an atypical aspirate; examination of the surgical specimens revealed all seven of these to be malignant. Thirteen patients underwent surgical biopsies, the results of which were malignant in seven and benign in six. The remaining 30 patients were followed up with mammography. The follow-up mammograms were obtained at 6-month intervals and demonstrated no change in appearance. On the basis of this initial experience, stereotaxic localization for aspiration biopsy offers the potential to accurately distinguish benign from malignant lesions. PMID- 1727271 TI - Distribution of a breast-directed I-131-radiolabeled monoclonal antibody in blood and bone marrow: implications for radiation immunotherapy. AB - Bone marrow is most often the dose-limiting organ in radiation immunotherapy. Controversy exists over optimal methods of estimating radiation dose to bone marrow. The authors compared findings in serial blood samples to findings in bone marrow biopsy samples as measures of bone marrow activity from which to calculate bone marrow dose. Peripheral blood samples and bone marrow biopsy samples were obtained from 11 female patients at 48 and 168 hours after infusion of iodine-131 labeled Mc5 antibody. Bone marrow biopsy data demonstrated markedly decreased specific activity compared with that measured in the peripheral blood. Activities at 48 hours after infusion ranged from 3% to 22% of the peripheral blood activity. These results indicate that activity concentration in the bone marrow is not equivalent to activity concentration in the blood for the Mc5 antibody. These results imply that a dose three to four times that indicated from serial blood samples could be tolerated by patients, provided the antibody-radioisotope does not bind to the marrow. PMID- 1727272 TI - Missed bronchogenic carcinoma: radiographic findings in 27 patients with a potentially resectable lesion evident in retrospect. AB - Eighteen radiologists failed to detect 27 potentially resectable bronchogenic carcinomas revealed retrospectively on serial chest radiographs. Most of the cancers were in an upper lobe (n = 22 [81%]), especially the right upper lobe (n = 15 [56%]). More of the cancers were in women (n = 18 [67%]) than in men (n = 9 [33%]). The mean diameter of the missed lesions was 1.6 cm +/- 0.8 (range, 0.6 3.4 cm). Only two lesions (7%) were well defined around their entire extent. A lateral radiograph (available for 23 patients) revealed the missed lesion better than the posteroanterior radiograph in four patients (17%). Six consultant radiologists, who were biased by knowledge that the cases were of missed bronchogenic carcinoma, were individually shown the radiographs in 22 of the cases. Each consultant missed a mean of 26% (5.8 +/- 1.7) of the lesions. At least one of the six consultants missed the lesion in 16 (73%) of the cases. The predominant characteristics of radiographically missed and potentially resectable bronchogenic carcinomas were difficulty in radiographic detection, female gender, and location in an upper lobe, especially on the right side. PMID- 1727273 TI - Contrast-enhanced MR imaging of the liver. PMID- 1727274 TI - Pulmonary metastases: MR imaging with surgical correlation--a prospective study. AB - The sensitivity of magnetic resonance (MR) imaging for detection of pulmonary metastases in 11 patients scheduled for thoracotomy and curative resection of metastases was evaluated with a prospective, controlled study. MR imaging performed at 0.5 T was compared with chest radiography, computed tomography (CT), and thoracotomy in 12 cases. (One patient had two separate occurrences of pulmonary metastases.) All images were interpreted in blinded fashion. When all MR sequences were interpreted together, MR imaging enabled correct identification of all patients with pulmonary nodules (100%). CT enabled detection of at least one nodule in all 12 cases (100%) by design; the sensitivity of chest radiography was only 64%. For individual nodules, MR imaging was at least as sensitive as CT (P2 less than .25 [two-sided value]) and significantly more sensitive than chest radiography (P2 less than .01). Among all MR sequences, short inversion time inversion-recovery sequences had the highest sensitivity for detection of individual nodules (82%). PMID- 1727275 TI - Pneumopericardium secondary to esophageal carcinoma. AB - The authors describe a case of squamous cell carcinoma of the esophagus that was initially diagnosed as hydropneumopericardium at radiography. The patient subsequently underwent computed tomography (CT) and esophagography. CT revealed pneumopericardium, pericardial thickening, a mass in the esophagus, and bilateral effusion. Esophagography demonstrated perforation of the mid-thoracic esophagus and marked irregularity of the esophageal mucosa. To the authors' knowledge, this is the only reported instance of esophageal carcinoma initially diagnosed as hydropneumopericardium. CT can be used as a primary method to investigate spontaneous pneumopericardium. PMID- 1727276 TI - Peritoneal carcinomatosis: preoperative CT with intraperitoneal contrast material. AB - Thirty-five abdominal computed tomographic (CT) scans of 27 patients with peritoneal metastases from a mucin-producing tumor of the appendix, colon, small bowel, or ovary were retrospectively reviewed. Fifteen scans were obtained of 15 patients after CT with intraperitoneal infusion of contrast material (IP), and 20 scans were obtained of 16 patients with CT without IP. Subsequent exploratory laparotomy revealed that all 27 patients had multi-focal spread of peritoneal metastases. The sensitivity of CTIP and CT without IP for detection of peritoneal metastases at all sites of involvement was 61% and 59%, respectively. For CTIP, the highest sensitivity was in the right subphrenic space (88%), splenic hilum (86%), and left subphrenic space (83%). For CT without IP, the highest sensitivity was noted in the splenic hilum (100%), left subphrenic space (75%), and left paracolic gutter (75%). CTIP and CT without IP had low sensitivity for detection of disease in the greater omentum (50% each) and small-bowel mesentery (38% and 59%, respectively), two areas that had the highest frequency of metastases. PMID- 1727277 TI - Patterns of intrahepatic bile duct dilatation at CT: correlation with obstructive disease processes. AB - The authors performed a blinded, retrospective analysis of 100 computed tomographic (CT) scans of patients with proved extrahepatic bile duct obstruction, including primary sclerosing cholangitis (PSC), to determine whether certain patterns of intrahepatic bile duct dilatation are suggestive of specific disease processes. Among 30 patients with benign obstructive disease, CT showed pruning of the intrahepatic ducts in four patients (13%), beading in four (13%), and skip dilatations in one (3%). Among 54 patients with malignant obstructive disease, CT illustrated pruning in eight (15%) patients, beading in 11 (20%), and skip dilatations in two (4%). Among 16 patients with PSC, CT demonstrated pruning in four (25%), beading in two (13%), and skip dilatations in five (31%). The majority of patients with malignant or benign obstructive disease or PSC had intrahepatic duct dilatation in both lobes of the liver. It extended into the periphery in 46 of 54 patients (85%) with malignant obstructive disease, in 20 of 30 (67%) with benign obstructive disease, and in 10 of 16 (63%) with PSC. The CT finding of skip dilatations is strongly suggestive of PSC. The CT findings of pruning and beading are nonspecific and may be observed at CT in patients with bile duct obstruction due to a wide variety of causes. The distribution and extent of intrahepatic duct dilatation at CT do not differ among biliary disease processes. PMID- 1727278 TI - Techniques for high-resolution echo-planar MR imaging of the pancreas. AB - Recent technical advances in echo-planar magnetic resonance (MR) imaging prompted an investigation of these new techniques in pancreatic MR imaging and evaluation of bowel lumen enhancement with an aqueous bowel contrast agent. In 42 subjects (36 healthy, six with pancreatic disease), various T1-weighted inversion-recovery and T2-weighted spin-echo fat-suppressed pulse sequences were assessed with an echo-planar technique implemented with a modified clinical MR imager. Single excitation imaging (echo time, 26 msec) provided a higher (P less than .05) signal-to-noise ratio than did conventional spin-echo and all other echo-planar techniques. In 13 (72%) of 18 healthy subjects who did not undergo administration of the contrast agent, the entire pancreas was distinguished from adjoining bowel. In all 18 subjects who underwent contrast-enhanced imaging, a significantly greater (P less than .05) intraluminal signal intensity was apparent with all echo-planar pulse sequences and the entire pancreas was identified. In six patients with pancreatic disease, lesions could be identified by their difference in signal intensity. PMID- 1727279 TI - Extraintestinal amebiasis. AB - Extraintestinal involvement is a dreaded complication of amebiasis, with a reported mortality rate of 7%-14%. The authors retrospectively studied 188 patients with extraintestinal amebiasis confirmed by means of clinical, surgical, and radiologic criteria over a 45-month period. Ultrasonography (US) was the mainstay of radiologic investigation. Liver abscess was present in 183 patients (97%); five patients (3%) had other organ involvement but a normal liver. The majority of liver abscesses were in the right lobe. US is recommended for the diagnosis and follow-up of patients with extraintestinal amebiasis. It is noninvasive, simple, easily reproducible, and less expensive than computed tomography. Portable models can be taken into remote areas of the less-developed world. PMID- 1727280 TI - PET evaluation of soft-tissue masses with fluorine-18 fluoro-2-deoxy-D-glucose. AB - Positron emission tomography (PET) with fluorine-18 fluoro-2-deoxy-D-glucose (FDG) was performed in 19 patients referred for clinical evaluation of soft tissue masses. These patients had 20 different lesions and had been evaluated previously with computed tomography (CT) and/or magnetic resonance (MR) imaging. The diagnoses were subsequently confirmed with open biopsy or excision (19 lesions) or by clinical and radiographic follow-up (one lesion). Semiquantitative assessment of FDG accumulation (differential uptake ratio) within the suspected tumor helped correctly separate the 10 malignant tumors from the 10 benign lesions. In contrast, a simple ratio of FDG uptake within the suspected tumor to that within comparable normal soft tissue was less successful in helping make this distinction, with overlap in 12 of the 20 cases. Careful comparison with findings from other available imaging studies is essential for accurate interpretation of PET studies of soft-tissue masses, but in many cases, PET may be a useful adjunct in the preoperative evaluation of suspected soft-tissue tumors, yielding valuable information that is not provided with CT or MR imaging. PMID- 1727281 TI - Diagnosis of bone, joint, and joint prosthesis infections with In-111-labeled nonspecific human immunoglobulin G scintigraphy. AB - The diagnostic accuracy of scintigraphy performed after injection of indium-111 labeled nonspecific human immunoglobulin G (IgG) was studied in 113 patients with 120 foci of suspected infection in bone (52 chronic and eight acute infections), joints (15 localizations), joint prostheses (37 prostheses), and soft tissue of the locomotor system (eight localizations). All patients also underwent standard three-phase scintigraphy after injection of technetium-99m-labeled methylene diphosphonate. A scan obtained with In-111-labeled IgG was considered positive if focal increasing accumulation was noted over time. In 51 patients (45.1%), the results of scintigraphy were verified with intraoperative cultures, and in 21 patients (18.6%), with needle aspiration. The prevalence of infection was 59%; overall sensitivity, 97%; and specificity, 85%. Use of In-111-labeled IgG enabled correct identification of the presence, site, and extent of infection in 69 of 71 proved foci of infection; 41 of 48 negative studies were correct. Only two infections proved with culture were missed; in both patients, the cultures revealed growth of Staphylococcus aureus in low counts. PMID- 1727282 TI - Nondisplaced fractures of the greater tuberosity of the humerus: sonographic detection. AB - In this retrospective study, the sonographic appearance of fracture of the greater tuberosity of the humerus was evaluated in 17 men and 14 women aged 20-69 years with acute, semiacute, or remote shoulder trauma in whom results of rotator cuff sonography had suggested the diagnosis of such a fracture. Clinical data, radiologic reports, sonograms, and initial plain radiographs of the shoulder were analyzed; clinical follow-up information was assessed in 22 patients. Sonography showed discontinuity and irregularity of the humeral cortex in all patients. In 25 patients (81%), displaced fracture fragments could be seen. Sonographic findings were suggestive of, but not specific for, fracture. Cortical abnormalities of the humerus were identified without modification of standard scanning protocols. A humeral fracture was confirmed with radiography in 24 patients; in 10 of them, the fracture had been missed initially on plain radiographs. It is concluded that, in evaluation of soft tissues in shoulder trauma, sonography may define rotator cuff abnormalities and occasionally help in detection of occult humeral fractures. PMID- 1727283 TI - Chronic wrist pain: spin-echo and short tau inversion recovery MR imaging and conventional and MR arthrography. AB - The accuracy of T1-, proton-density-, and T2-weighted magnetic resonance (MR) imaging sequences and gadolinium-enhanced MR arthrography in evaluation of the triangular fibro-cartilage complex (TFCC) and the scapholunate (SL) and lunotriquetral (LT) ligaments was studied in 15 patients with chronic wrist pain. Arthrography and arthroscopy were used as standards of reference. Twelve patients also underwent imaging with short tau inversion recovery (STIR) sequences. MR imaging was more reliable in evaluation of the morphology of the TFCC and SL ligament than in that of the LT ligament. With arthrography as the standard, sensitivity was 0.721, specificity was 0.947, and accuracy was 0.887 for the TFCC; these values were 0.500, 0.864, and 0.765 for the SL ligament and 0.519, 0.455, and 0.490 for the LT ligament. No visualization of the SL ligament indicated a tear, but this sign was not helpful in evaluation of the LT ligament. Fluid in the distal radioulnar joint had a high association with TFCC tears. Accuracy with MR arthrography was higher than with the other sequences. STIR images were effective in evaluation of the TFCC. The combination of proton density-and T2-weighted images appears to be useful because morphologic characteristics and the presence of fluid can be evaluated. PMID- 1727284 TI - Rapid destructive osteoarthritis: clinical, radiographic, and pathologic features. AB - Twenty-seven cases of an unusual, poorly recognized destructive hip arthropathy with radiographic findings of rapid severe joint destruction are presented. Radiographic findings mimicked those of other disorders such as septic arthritis, rheumatoid and seronegative arthritis, primary osteonecrosis with secondary osteoarthritis, or neuropathic osteoarthropathy, but none of the patients had clinical, pathologic, or laboratory evidence of these entities. All patients underwent hip arthroplasty, and osteoarthritis was confirmed at pathologic examination. Rapid progression of hip pain and disability was a consistent clinical feature. The average duration of symptoms was 1.4 years. Radiographs obtained at various intervals before surgery (average, 18 months) in nine patients documented rapid hip destruction. Involvement was unilateral in 89% (24 of 27 cases). Twenty patients (83%) were elderly women. The authors postulate that these cases represent an uncommon, rapidly destructive subset of osteoarthritis. PMID- 1727285 TI - Fat-suppressed MR imaging of myositis. AB - A hybrid fat-suppression sequence in magnetic resonance (MR) imaging was used to evaluate inflammatory muscle disorders in seven children: five patients with dermatomyositis, one patient with vasculitis, and one patient with viral myositis. Fat-suppressed multisection axial images obtained with the same repetition and echo times as those used to obtain standard spin-echo (SE) images enabled direct comparison of images, with little variation of T1 and T2 weighting. In six patients, the contrast on images obtained with T2 fat suppression was 15%-20% greater than contrast on conventional T2-weighted SE images. In all seven patients, the subjective judgment was that T2-weighted fat suppression sequences improved visualization of muscle abnormalities. It is concluded that T2 fat suppression is useful in evaluation of inflammatory muscle disorders in children because it increases contrast and eliminates fat as a cause of muscle abnormality. PMID- 1727286 TI - Bone signal abnormalities in the posterolateral tibia and lateral femoral condyle in complete tears of the anterior cruciate ligament: a specific sign? AB - Thirty-two patients with acute, complete tears of the anterior cruciate ligament (ACL) proved at surgery underwent examination with magnetic resonance (MR) imaging. Bone impaction sites were present in the posterolateral tibial plateau in 30 patients (94%) and in the lateral femoral condyle (LFC) in 29 patients (91%). The bone abnormalities had low signal intensity on T1-weighted images and high signal intensity on T2-weighted images when compared with the signal intensity of normal marrow. It is assumed that the bone changes occur during injury when the LFC impacts into the posterior tibia, either during the initial rotary subluxation or as the LFC recoils to return to anatomic alignment. Only one of six partial ACL tears had a bone signal change. In patients with acute knee injury, bone impaction sites in the posterolateral tibia and the LFC suggest that a complete ACL tear is present. PMID- 1727287 TI - Left ventricular thrombi: evaluation with spin-echo and gradient-echo MR imaging. AB - Gradient-echo (GRE) and spin-echo (SE) magnetic resonance (MR) imaging was performed in 31 patients with chronic left ventricular (LV) thrombi. Thrombi were confirmed or excluded at surgery or by means of other corroborative diagnostic techniques. MR images were evaluated by three reviewers without knowledge of results of corroborative studies. Diagnoses were graded unequivocal if agreed on by three observers and probable if agreed on by two observers. With SE imaging, 12 of 18 confirmed thrombi were detected unequivocally, five were considered probable, and one was not detected. With GRE imaging, 16 of the 18 thrombi were visualized unequivocally; two were considered probable. With SE technique, thrombus was unequivocally excluded in nine of 13 cases and exclusion was considered probable in four. One finding was false-negative. Exclusion of thrombus with GRE imaging was unequivocal in 10 of 13 cases and probable in two, and one finding of thrombus was false-positive. GRE imaging resulted in improved differentiation of thrombi from the surrounding blood pool and myocardium and thus was diagnostically superior to SE imaging in detection of LV thrombi. PMID- 1727288 TI - Fibromuscular elements of the right atrium: pseudomass at MR imaging. AB - Results of 20 random cardiac spin-echo and cine gradient-echo magnetic resonance (MR) imaging examinations were reviewed for nodular and/or linear soft-tissue structures projecting from the right atrial wall. A nodular soft-tissue structure (mean diameter, 6 mm) isointense with myocardium was observed along the posterior atrial wall in 18 of 20 patients (90%). The structure extended between the orifices of the superior and inferior venae cavae. Linear strand-like projections originating from the structure coursed across the atrial chamber in 5 of 20 patients (25%). Similar findings were present in one patient who underwent cardiac surgery for a suspected atrial mass. The region of nodular thickening was shown histologically to represent myocyte hypertrophy and fibrosis. The nodular and linear structures correlated anatomically with the crista terminalis muscle bundle and Chiari network, respectively. These structures are frequently visualized on cardiac MR images, are of variable prominence, and may closely simulate the appearance of tumor or other cardiac masses in some patients. Awareness of the location and MR imaging features of these structures will help prevent misdiagnosis. PMID- 1727289 TI - Zollinger-Ellison syndrome: technique, results, and complications of portal venous sampling. AB - All 95 portal venous sampling (PVS) procedures performed in patients with Zollinger-Ellison syndrome in the past 10 years at the authors' institution were reviewed. It was possible to catheterize at least one branch of the pancreaticoduodenal venous arcade in all but two procedures (98%). The highest concentration of gastrin was found in a selective sample from the pancreaticoduodenal venous arcade or the transverse pancreatic vein in 56 of 91 procedures (62%). Selective sampling of pancreatic head veins yielded a gastrin gradient sufficient for localization in 60 patients (63%). Among 55 solitary sporadic gastrinomas identified at surgery, PVS allowed correct localization of the tumor in 32 (58%); if selective samples had not been obtained, only eight (15%) would have been localized (P less than .0005). Sensitivity was the same for tumors in the gastrinoma triangle (64%) and the body or tail of the pancreas (60%). There were no false-positive results. The overall complication rate was 20%, but most complications were abdominal pain lasting 3 days or less. Six patients (6%) had serious complications. PMID- 1727290 TI - Arteriographic complications in the DSA era. AB - Prospective data were collected on complications associated with intraarterial digital subtraction angiography in 2,475 consecutive patients at a 650-bed Melbourne teaching hospital. Carotid or cerebral studies were performed in 939 patients, and the prevalence of stroke (ie, permanent neurologic deficit) was 0.3%. The overall prevalence of systemic complications was 1.8%, with no patients requiring hemodialysis because of renal failure. Comparison was made with previously reported complication rates for conventional film angiography. PMID- 1727291 TI - Acute traumatic aortic rupture: intravascular US findings. AB - Traumatic aortic rupture is a lethal injury that requires immediate diagnosis and surgical repair. The authors report a case of acute aortic rupture in which aortography demonstrated a subtle intimal discontinuity at the cephalic margin of the aortic spindle. Intravascular ultrasound (US) imaging of the aorta demonstrated a mural flap and a small, contained hematoma. Intravascular US may have a role in enabling confirmation or clarification of subtle aortic abnormalities. PMID- 1727292 TI - Radiologic placement of peritoneal dialysis catheters: preliminary experience. AB - The authors percutaneously placed 45 catheters for peritoneal dialysis in 32 patients, aged 31-83 years, in a radiology department. In all patients, the procedure was modified by use of the Hawkins needle, and in response to the high frequency of extrusion of the proximal cuff, the deep cuff of the 16th and each subsequent catheter was sutured to the rectus muscle or fascia. After 17 catheters were placed, the catheter was modified with a permanent bend, or "U" neck, between the two cuffs, which were then thickened. All procedures were performed with use of local anesthesia, and all catheters were successfully placed. Acute complications included bowel perforation associated with peritonitis in one patient (2%). Delayed complications included cuff extrusion in nine patients (20%), obstruction in nine patients (20%), and peritonitis requiring removal of the catheter in three patients (7%). This study shows the feasibility of percutaneous placement of peritoneal dialysis catheters by radiologists despite the need for improved technique and equipment. PMID- 1727293 TI - Rheolytic catheter for percutaneous removal of thrombus. AB - The authors present a percutaneous thrombectomy system (rheolytic thrombectomy catheter [RTC]) in which high-velocity jets of saline solution are used to lyse and remove thrombus. The catheters (4-6 F) direct a 10,000-15,000-psi (0.7-1.05 x 10(5)-kPa) jet of saline solution onto an exhaust port from orifices at the end of the catheter. The jet entrains clot and resulting fragments and brings them into the high-velocity region for lysis and removal. Whole blood clots (10-15 cm) placed in 6-9-mm-diameter tubing were completely dissolved and removed with the RTC in less than 1 minute. In vivo use in a canine model resulted in lysis and removal of clots from a femoral artery, without vessel damage. The small caliber, flexibility, and effective lysis of this system suggest its potential usefulness in large central vessels that are difficult to access surgically and in small diameter vessels that require more rapid removal of thrombus than can be achieved with thrombolytic therapy. PMID- 1727294 TI - Renal carcinoma: detection of venous extension with gradient-echo MR imaging. AB - Magnetic resonance (MR) imaging has been proposed as a noninvasive alternative to vena cavography and computed tomography for the detection of venous extension of renal adenocarcinoma. However, spin-echo MR images may be compromised by the presence of flow-related artifacts, extrinsic compression, and respiratory or cardiac motion artifacts. Use of gradient-recalled echo (GRE) sequences is advantageous for imaging of vascular structures. To investigate the detection of vascular extension of tumor with the GRE technique, findings in the preoperative GRE MR images of 26 patients with renal adenocarcinoma were compared with findings at surgery and pathologic examination. Vena cava thrombus was correctly identified in 13 of 13 patients (100%). Renal vein thrombus was correctly identified in 23 of 26 patients (88%), and right atrial thrombus was correctly identified in four of five patients (80%). Use of GRE sequences allows accurate assessment of vascular structures that is sufficient for surgical planning. PMID- 1727295 TI - Early-stage glottic cancer: importance of dose fractionation in radiation therapy. AB - The treatment results in 85 patients with T1N0M0 squamous cell carcinoma of the glottic larynx who were treated with primary radiation therapy were reviewed to analyze for local control. After a minimum follow-up period of 2 years, 13 patients had local recurrence of disease, which yielded a local control rate of 84.7%. Local control was then reassessed as a function of substages (T1a and T1b) and dose fractionation. No difference in local control was seen in T1a and T1b neoplasms. However, after undergoing standard once-a-day fractionation, patients treated with fractions of 200 cGy had a local control rate of 96%, while those receiving 180 cGy had a local control rate of 79% (P = .05). Mean total dose for each patient group was comparable, and the median number of days of treatment interruption was the same for both groups. These data corroborate the recent findings of other authors regarding the importance of fraction size in facilitating local control of early-stage glottic cancer. PMID- 1727296 TI - CT of scoliotic patients after myelography: value of lateral decubitus positioning. AB - In routine computed tomography (CT) of scoliotic patients, multiple scans through each intervertebral disk level must be made, which can make interpretation of disks and neural foramina difficult. Use of lateral decubitus positioning for postmyelography CT allows use of gantry angulation to achieve true axial scans through disks, which permits easier evaluation of individual disks and foramina. PMID- 1727297 TI - Contrast medium gel for marking vaginal position during defecography. AB - A tampon soaked with contrast medium, which had been inserted into the vagina as part of standard defecography procedure, obscured signs of anterior rectocele and rectal intussusception in a 34-year-old woman. A contrast medium gel for marking vaginal position was formulated, and postsurgical examination with use of the gel revealed improved rectal function and no intussusception. The gel provided excellent contrast without obscuring important diagnostic information. PMID- 1727298 TI - Temporomandibular joint: improved MR image quality with decreased section thickness. AB - To determine if the quality of spin-echo magnetic resonance (MR) images of the temporomandibular joint (TMJ) could be improved by reducing section thickness, coronal and sagittal 3.0- and 1.5-mm MR images of the same joints were evaluated. Depiction of the disk, trabecular pattern, and cortex of the condyle was better on coronal 1.5-mm images than on 3.0-mm images (P less than .01), and 1.5-mm sagittal images were better for depiction of the trabecular pattern of the condyle than were 3.0-mm images (P less than .05). The ability of MR imaging with thinner sections to reveal more anatomic details should result in improved diagnostic accuracy. PMID- 1727299 TI - Intravascular foreign bodies: loop-snare retrieval system with a three-lumen catheter. AB - A foreign body retrieval system composed of a three-lumen catheter, a guide wire, and a wire loop nearly perpendicular to the axis of the catheter was used to retrieve catheter fragments located in the inferior vena cava in two patients and in the superior vena cava in one patient. Fragments were looped within 1 minute of catheterization and were retrieved without complication. Catheter fragments placed in the pulmonary artery of one dog were also successfully retrieved. PMID- 1727300 TI - Plantar fasciitis: US imaging. PMID- 1727301 TI - Renal lesion characterization. PMID- 1727302 TI - Patient and physician radiation exposure during fluoroscopy. PMID- 1727303 TI - Potential hazard of metal-filled sandbags in MR imaging. PMID- 1727304 TI - Attitudinal expressions as a measure of reviewer fairness. PMID- 1727305 TI - Decreased phosphorus metabolite concentrations and alkalosis in chronic cerebral infarction. AB - A study was performed to determine quantitatively the alterations in phosphorus metabolite concentrations and pH in regions of the human brain damaged by chronic stroke. Image-guided phosphorus-31 magnetic resonance spectroscopy was performed on the brains of eight healthy subjects and six patients with cerebral infarction of more than 3 months duration. Phosphorus metabolite concentrations in infarcted regions were reduced 8%-67%. Significant decreases occurred in phosphomonoester (PME), phosphodiester (PDE), and adenosine triphosphate (ATP) concentrations, while inorganic phosphate (Pi) and phosphocreatine (PCr) concentrations showed smaller, nonsignificant decreases. The PCr/ATP ratio was significantly increased, while the ATP/Pi ratio was somewhat lower. The phospholipid ratio PDE/PME was also significantly increased, while the ratios of phospholipid (PME, PDE) to phosphate (PCR, Pi) metabolites were significantly decreased. The pH of the infarcted region indicated significantly more alkalinity than in the normal brain. The results suggest that chronic stroke is associated with significant changes in brain metabolite concentrations and pH that are different from those reported for other brain diseases. PMID- 1727306 TI - Detection of internal carotid artery stenosis: comparison of MR angiography, color Doppler sonography, and arteriography. AB - Findings of two-dimensional time-of-flight magnetic resonance (MR) angiography projection angiograms were prospectively compared with those of color Doppler sonography by using angiography as a standard in 23 consecutive patients (42 carotid bifurcations) to evaluate their utility in determining the presence of carotid artery stenosis. MR angiography helped detect 50% or greater lumen diameter stenosis (sensitivity, 0.96; specificity, 0.64). Color Doppler sonography with 1.25 m/sec peak systolic velocity as a threshold had a sensitivity of 0.96 and a specificity of 0.71. Statistical analysis showed a correlation between percentage of lumen diameter narrowing and the length of the zone of signal intensity loss with MR angiography (r = .69; P less than .0001). A stronger relationship was obtained between angiographic narrowing and peak systolic velocity derived from color Doppler sonography (r = .80; P less than .0001). Two-dimensional time-of-flight MR angiography displayed as projection angiograms and combined with carotid artery and combined with carotid artery sonography is a useful approach for helping detect and potentially grade the severity of stenoses of the carotid artery. PMID- 1727307 TI - Acute cerebral ischemia: evaluation with dynamic contrast-enhanced MR imaging and MR angiography. AB - Dynamic contrast-enhanced T2-weighted magnetic resonance (MR) imaging and MR angiography (MRA) were used to evaluate cerebral blood volume and the intracranial arterial system in 34 patients within 48 hours after the onset of cerebral ischemia. In 24 of the patients, an abnormality identified on T2 weighted images corresponded to the acute clinical deficit. Intracranial MRA demonstrated occlusions or severe stenoses of major vessels supplying the area of infarction in 16 of these patients, and decreased blood volume correlated well with MRA abnormalities. Infarcts less than 2 cm in diameter were not reliably shown with MRA or blood volume studies. Correlation between lesions seen with MRA and decreased blood volume in acute infarcts was good, and both techniques demonstrated lesions early in the clinical course. By providing information about hemodynamics not available with conventional T1- or T2-weighted images, MRA and dynamic MR imaging could prove helpful in describing the pathophysiologic characteristics of stroke and in guiding early therapeutic intervention. PMID- 1727308 TI - Traumatic lesions of the suprasellar region: MR imaging. AB - The authors studied nine patients with injuries to the suprasellar region with 1.5-T magnetic resonance (MR) imaging. Five patients had chiasmal injuries diagnosed by means of clinical examination. MR imaging demonstrated complete transection in two of these five patients, contusion of the chiasm by inferior herniation of the gyrus rectus in one, and a normal chiasm in two. Two patients had large tears of the floor of the third ventricle resulting in wide communication between the third ventricle and the prepontine cistern. One of these patients also had an avulsed third nerve. Transection of the pituitary stalk was seen in two patients. MR imaging can demonstrate injuries to the suprasellar structures. The MR imaging appearance of optic chiasm correlates with different types of injury to the chiasm described in the clinical literature and may alleviate the need for additional diagnostic studies to help explain the patient's symptoms. PMID- 1727309 TI - Brain hemorrhage: evaluation with fast spin-echo and conventional dual spin-echo images. AB - Signal intensity of blood products on proton-density- and T2-weighted images obtained with spin-echo (SE) and fast SE (FSE) sequences was evaluated in 15 patients with central nervous system hemorrhage to determine the extent of differences between the two techniques when signal loss from magnetic susceptibility effects in hemorrhagic lesions is considered. Within operator defined regions of interest, signal intensity of hemorrhage, iron-containing nuclei, white matter, scalp fat, and noise was measured along the phase-encoding direction. Hemosiderin, deoxyhemoglobin, and iron-containing nuclei had slightly higher signal intensity on FSE images than on SE images, but the differences were not statistically significant. Signal intensity of methemoglobin was similar with both sequences, whereas that of scalp fat was higher on FSE images. Signal intensity measurements for most tissues studied were comparable, but the signal to-noise ratios with FSE imaging were less than those with SE imaging. Although paramagnetic blood products may show slightly higher signal intensity with FSE imaging, contrast with the two sequences was comparable and lesion conspicuity was nearly identical. PMID- 1727310 TI - Contrast-enhanced MR imaging performed after successful lumbar disk surgery: prospective study. AB - A prospective study was undertaken to establish the normal spectrum and timing of gadolinium-enhanced magnetic resonance (MR) imaging findings in 15 patients who had resolution of symptoms after successful lumbar disk surgery. Enhancement of the facet joints (in 88% of disk levels) and paraspinal muscles (100%) decreased gradually after surgery. Enhancement of the decompressed nerve root tracked proximally toward the conus medullaris in 62% at 3 weeks and was absent in all by 6 months postoperatively. Areas of intermediate signal intensity with peripheral enhancement and mass effect were seen on T1-weighted images at the site of the original disk herniation in 38% at 3 weeks and 12% at 3 months, despite complete relief of leg pain. These results reveal that even in successfully treated (asymptomatic) patients, residual mass effect on the neural elements may frequently simulate a recurrent or residual disk fragment. There is an orderly progression of imaging changes during the first 6 months after lumbar surgery that limits the interpretation of MR examinations during that period. PMID- 1727311 TI - Cerebral vasculitis: MR imaging and angiographic correlation. AB - Cerebral vasculitis is an unusual disorder with numerous causes. One such entity, noninfectious granulomatous angiitis of the nervous system (GANS), is an extremely rare disease with a predilection for leptomeningeal and parenchymal arteries and veins. Isolated involvement of the central nervous system is characteristic of GANS, which has also been referred to as primary angiitis of the central nervous system (PACNS). The results of magnetic resonance (MR) imaging and angiography in seven patients with presumed PACNS were retrospectively analyzed and correlated. MR images were positive in every case. Characteristically, lesions were multiple, bilateral, and supratentorial. Both gray- and white-matter infarcts were identified in four of seven patients; infarcts were most common in the deep white matter. PACNS can also appear as primary parenchymal hemorrhage or simulate low-grade glioma. All lesions identified on MR images were associated with positive angiographic findings of cerebral vasculitis in the corresponding vascular distribution. However, for 12 of 33 vascular distributions with angiographic evidence of cerebral vasculitis, no lesions were identified on MR images. These correlative observations suggest that some patients with proved PACNS may have normal MR imaging results. PMID- 1727312 TI - Echogenic material in the fetal gallbladder: sonographic and clinical observations. AB - Obstetric sonograms of 26 fetuses with echogenic material in the gallbladder were reviewed to describe the sonographic findings and clinical significance. Gestational age at the time of diagnosis ranged from 28 to 42 weeks (mean, 36.2 weeks). The echogenic foci were associated with distal shadowing in eight fetuses (30%), comet-tail artifact in nine (35%), and no distal artifact in nine (35%). No hemolytic anemias, other predisposing risk factors, or clinical sequelae associated with biliary tract disease were identified in any of the infants. Postnatal sonographic or pathologic follow-up studies were available in 17 cases. In nine of these 17 infants, the echogenic foci had resolved. In three, the foci have persisted, but none of the children have become symptomatic; the longest period of follow-up with stones still present is 4 1/2 years. Whether all echogenic foci in the fetal gallbladder represent true gallstones remains unknown. Echogenic foci may be seen in the fetal gallbladder during the third trimester. No predisposing fetal risk factors or clinical sequelae were evident in our series. Many echogenic foci, but not all, will resolve. PMID- 1727313 TI - Childhood intussusception: US-guided hydrostatic reduction. AB - Over a 30-month period, real-time ultrasound (US) was performed in 116 children with suspected intussusception. US findings were positive in all 75 cases of intussusception. Except in one case of transient small-bowel intussusception, the authors immediately attempted US-guided hydrostatic reduction in all cases. Reduction was successful in 63 cases (85%), as demonstrated with US and resolution of signs and symptoms of intussusception. Negative sonograms were confirmed with clinical follow-up. Among 11 failed cases, reduction with barium enema was attempted in six, but all attempts failed. No complications have occurred to date. The authors conclude that US is a reliable diagnostic screening modality in cases of suspected intussusception and that US-guided hydrostatic reduction is a promising technique in nonoperative treatment. PMID- 1727314 TI - Missed lung nodules: lost opportunities for cancer cure. PMID- 1727315 TI - Nonobstructive posterior urethral widening (spinning top urethra) in boys with bladder instability. AB - Seven boys between the ages of 5 and 10 years with symptoms of urinary frequency and urgency and daytime wetting were studied with urodynamics and were shown to have bladder instability and a dilated posterior urethra. In two the dilatation occurred predominantly during bladder filling. Unstable contractions caused filling of the posterior urethra, and leakage was prevented by voluntary contraction of the distal urethral sphincter; with voiding, the urethra showed a more normal appearance. In the remaining five, there were similar changes during filling, but dilatation persisted during voiding. In six the measured urine flow rate was normal, and none showed any evidence of anatomic obstruction. The mechanism of urethral distention appears to be similar to that previously shown in girls with spinning top urethra: Unstable contractions resisted by voluntary sphincter contraction cause posterior urethral dilatation. Boys with dilated posterior urethras who have urinary frequency and urgency and daytime wetting and normal urine flow rates should be assumed to have bladder instability. PMID- 1727316 TI - Primary tuberculosis in childhood: radiographic manifestations. AB - The aim of the study was to review the radiologic features of primary tuberculosis in childhood and to determine whether differences in patterns of disease occur among age and ethnic groups. Chest radiographs of 191 children with pediatric primary tuberculosis were reviewed by two observers. Lymphadenopathy, present in 92% of cases, was the most common abnormality identified on the initial chest radiograph and typically involved the hilar and paratracheal regions. Parenchymal abnormalities, identified in 70% of cases, occurred more commonly in the right lung (P less than .001). Children 0-3 years of age had a higher prevalence of lymphadenopathy (P less than .01) and a lower prevalence of parenchymal abnormalities (P less than .001) than older children. A lower prevalence of lymphadenopathy was found in whites than in nonwhites (P less than .02). The radiologic abnormalities often progressed in the initial follow-up. Lymphadenopathy, with or without concomitant parenchymal abnormality, is the radiologic hallmark of primary tuberculosis in childhood. However, distinct age related and racial differences in presenting patterns of disease exist and should be recognized. PMID- 1727317 TI - Juvenile rheumatoid arthritis of the knee: MR evaluation with Gd-DOTA. AB - Synovial hypertrophy, effusion, and articular cartilage status were evaluated with gadolinium tetraazacyclododecanetetraacetic acid (DOTA)-enhanced magnetic resonance (MR) imaging in 24 knees in 24 pediatric patients (17 female, seven male; mean age, 10 years; range, 3-18 years) with juvenile rheumatoid arthritis (JRA). T1-weighted spin-echo sequences were performed with a 0.5-T unit before and immediately after injection of Gd-DOTA (0.1 mmol/kg). Substantial enhancement of synovial proliferation was seen in 23 of 24 knees, allowing precise assessment of pannus extension (n = 23), joint effusion (n = 21), cartilage loss (n = 21), and meniscal hypotrophy (n = 23). On T1-weighted images without contrast enhancement, cartilage thickness, loculation of joint effusion, and pannus extension were underestimated. Thus, Gd-DOTA-enhanced MR imaging is mandatory in the assessment of knee involvement in children with JRA and may prove to be useful in the evaluation of response to therapy. PMID- 1727318 TI - Emotional support for patients with cancer who are undergoing CT: semistructured interviews of patients at a cancer institute. AB - To understand and improve the experience of cancer patients undergoing computed tomography (CT), 79 patients who underwent CT at a cancer institute participated in semistructured interviews about their experiences with CT. All patients had previously undergone CT; 75% (n = 59), three times or more. Anxiety about results was the most common concern during first and subsequent CT examinations. Technical aspects were a common concern during initial scanning, but not subsequently. Methods of relaxation most used by patients during CT were following instructions (56% [n = 44]), meditating and visualizing (44% [n = 35]), and praying (42% [n = 33]). Patients suggested several ways in which the radiology staff can support them during the evaluation of their malignancy. Fifty five (70%) of the patients said they would like the radiologist to tell them the results of their scanning. Optimal care of patients with cancer who undergo CT goes beyond technical to emotional and spiritual support. PMID- 1727319 TI - Overview of surgery: 1992. PMID- 1727320 TI - Fractures of the distal radius. PMID- 1727321 TI - Integrated ambulatory foregut monitoring in patients with functional foregut disorders. PMID- 1727322 TI - Surgical applications of the function of the pylorus. PMID- 1727323 TI - Liver transplantation in children: an update. AB - Liver transplantation in children has progressed to the point where much of the initial skepticism surrounding the value of this extraordinary endeavor has been overcome, and the results clearly justify the widespread use of this procedure in children with limited life expectancy secondary to severe liver disease. Advances in the areas of organ preservation and reduced-size liver transplantation have increased organ availability for children and significantly decreased mortality on transplant waiting lists. Changes in the ways pediatricians and pediatric surgeons think about children with EHBA have led to an increasing level of sophistication in combining the traditional surgical and medical treatments of this disease with the developing field of transplantation to maximize the chances for a normal life for all children with this problem. Newer immunosuppressive agents and more rational use of available medications have led to fewer graft losses to rejection while minimizing the undesirable side effects of individual drugs. As our understanding of the delicate interaction between the immune system and the graft increases, newer methods of immunomodulation may yet lead to the eventual goal of donor-specific tolerance, in which all immunologic reactivity remains normal except with respect to donor antigens on the graft. More specific immunosuppressive agents and more effective antiviral strategies have led to a decreased mortality from viral infections and may lead to a decrease in the mortality from secondary malignant disease. As mere survival after liver transplantation becomes more commonplace, more effort can be directed into meeting the long-term psychological and social needs of children with liver transplants to ensure that children develop and grow in as normal a manner as possible. PMID- 1727324 TI - Current concepts in neuroblastoma. AB - With the fund of knowledge presented here, clinical practice can begin to apply what basic science has discovered. New approaches to immunotherapy and genetic engineering will supplant the current surgical tools for our fight against this tumor. PMID- 1727325 TI - Peritoneal adhesions. Incidence, cause, and prevention. PMID- 1727326 TI - Management of severe intra-abdominal infection. PMID- 1727327 TI - Porous graphitic carbon in biomedical applications. PMID- 1727328 TI - The use of chromatography in forensic science. PMID- 1727329 TI - Tryptic mapping by reversed phase liquid chromatography. AB - This chapter reviews the technique of tryptic mapping by reversed phase liquid chromatography by describing its fundamental principles, present status, and recent advances as well as its uses and limitations. Two commonly used S carboxymethylation/tryptic digestion procedures are documented. Column and instrumental requirements are described and illustrated with examples. Mobile phase selection and operating variables (tG, F) are also discussed in terms of their effects on both peptide resolution and detection sensitivity. PMID- 1727330 TI - Determination of dissolved gases in water by gas chromatography. PMID- 1727331 TI - Separation of polar lipid classes into their molecular species components by planar and column liquid chromatography. PMID- 1727332 TI - Caldwell Lecture. Respiratory problems of early life now allowing survival into adulthood: concepts for radiologists. AB - Many patients with illnesses that once were fatal at birth or during childhood now survive into adult life. This article considers four respiratory illnesses of early life in which long-term survival now occurs frequently: cystic fibrosis, diaphragmatic hernia, esophageal atresia-tracheoesophageal fistula, and bronchopulmonary dysplasia. In cystic fibrosis, although the median age at death is now 25 years, chronic pulmonary infection due ultimately to the abnormal composition and clearance of airway mucus is still the usual cause of death. Earlier survivors of congenital diaphragmatic hernia had only minor diminution of perfusion and ventilation of the lung on the side of the hernia as adolescents or young adults; however, as infants with greater degrees of pulmonary hypoplasia have successful repair of their hernias, more long-term respiratory impairment will probably be found. The esophageal atresia tracheoesophageal fistula complex leaves all esophagi and many tracheas permanently abnormal; recurrent aspiration, repeated pneumonia, and an unduly collapsible trachea are the result, although symptoms may be few. Survivors of bronchopulmonary dysplasia have decreased exercise capacity, wheezing, and recurrent pneumonia, although their chest radiographs may become normal or almost normal. PMID- 1727333 TI - Chondromalacia patellae: diagnosis with MR imaging. AB - Most previous studies of MR imaging for detection of chondromalacia have used T1 weighted images. We correlated findings on axial MR images of the knee with arthroscopic findings to determine MR findings of chondromalacia patellae on T2 weighted and proton density-weighted images. The study population included 52 patients who had MR examination of the knee with a 1.5-T unit and subsequent arthroscopy, which documented chondromalacia patellae in 29 patients and normal cartilage in 23. The patellar cartilage was assessed retrospectively for MR signal and contour characteristics. MR diagnosis based on the criteria of focal signal or focal contour abnormality on either the T2-weighted or proton density weighted images yielded the highest correlation with the arthroscopic diagnosis of chondromalacia. When these criteria were used, patients with chondromalacia were detected with 86% sensitivity, 74% specificity, and 81% accuracy. MR diagnosis based on T2-weighted images alone was more sensitive and accurate than was diagnosis based on proton density-weighted images alone. In conclusion, most patients with chondromalacia patellae have focal signal or focal contour defects in the patellar cartilage on T2-weighted MR images. These findings are absent in most patients with arthroscopically normal cartilage. PMID- 1727334 TI - Chondromalacia patellae. PMID- 1727335 TI - Diagnosis of pelvic fractures in patients with acute pelvic trauma: efficacy of plain radiographs. AB - Although CT is widely recognized as an important adjunct to plain films in the evaluation of patients with acute pelvic trauma, accurate diagnosis of orthopedic injuries with plain films alone is often important to determine if immediate external fixation is necessary. The purpose of this study was to determine the efficacy of plain radiographs in the detection of pelvic fractures and dislocations in patients with acute pelvic trauma by using CT as the gold standard. CT scans and plain films collected prospectively in 50 patients with acute pelvic injuries were evaluated independently, and fractures and dislocations were identified and tabulated. Of a total of 162 fractures and dislocations seen on CT, only 14 (9%) were misdiagnosed on plain films. None of these misdiagnoses altered patients' management. Sixteen (80%) of 20 cases of intraarticular fragments in the hip joint associated with acetabular fractures were not identified on plain films. We conclude that plain film examination of the patient with pelvic trauma is sufficient to identify virtually all clinically important fractures and dislocations. Plain radiographs alone are not accurate in detecting fracture fragments within the hip joint. PMID- 1727336 TI - Hemophilia: evaluation of musculoskeletal involvement with CT, sonography, and MR imaging. AB - The purpose of this essay is to illustrate features of the musculoskeletal complications of hemophilia as shown by CT, sonography, and MR imaging. MR can be used to detect early synovial and cartilaginous changes that may not be evident on conventional radiography and to differentiate between acute and chronic bleeding in soft tissues. CT is useful in evaluating subtle bony erosion and intra- and extraosseous pseudotumors. Sonography is valuable in following progression and regression of soft-tissue hematomas. PMID- 1727337 TI - Benign gaseous distension of the bowel in premature infants treated with nasal continuous airway pressure: a study of contributing factors. AB - Continuous positive airway pressure (CPAP) administered as a mixture of oxygen and compressed air via nasal prongs has dramatically improved survival rates and lessened the frequency of barotrauma and bronchopulmonary dysplasia in the premature infant with respiratory distress syndrome. Associated with the increased use of nasal CPAP has been the development of marked bowel distension (CPAP belly syndrome), which occurs as the infant's respiratory status improves and the baby becomes more vigorous. To identify contributing factors, we prospectively compared 25 premature infants treated with nasal CPAP with 29 premature infants not treated with nasal CPAP. Infants were followed up for development of distension, defined clinically as bulging flanks, increased abdominal girth, and visibly dilated intestinal loops. We evaluated birth weight, weight at time of distension, method of feeding (oral, orogastric tube), and treatment with nasal CPAP and correlated these factors with radiologic findings. Of the infants who received nasal CPAP therapy, gaseous bowel distension developed in 83% (10/12) of infants weighing less than 1000 g, but in only 14% (2/14) of those weighing at least 1000 g. Only 10% (3/29) of infants not treated with nasal CPAP had distension, and all three weighed less than 1000 g. Presence of sepsis and method of feeding did not correlate with occurrence of distension. Neither necrotizing enterocolitis nor bowel obstruction developed in any of the patients with a diagnosis of CPAP belly syndrome. Our study shows that nasal CPAP, aerophagia, and immaturity of bowel motility in very small infants were the major contributors to the development of benign gaseous bowel distension. PMID- 1727338 TI - Sonography of hypertrophic pyloric stenosis: frequency and cause of nonuniform echogenicity of the thickened pyloric muscle. AB - In hypertrophic pyloric stenosis, the muscle is typically described as hypoechoic on sonography. However, we have frequently noted a nonuniform pattern; the pyloric muscle seen in the transverse plane is more echogenic in the near and far fields and less echogenic on the sides. The muscle also appears almost as echogenic as the liver on midline longitudinal sonograms. To establish the frequency of these findings, we reviewed the sonograms of 71 infants with hypertrophic pyloric stenosis. The muscle was imaged directly during surgery in three patients. In an in vitro experiment, muscle arranged to stimulate the pyloric ring was scanned in a water bath. Then, using two sections of muscle, we compared the echogenicity when scanning in a plane perpendicular to the long axis of the muscle fibers with that seen with the beam parallel to the long axis of the muscle fibers. In the transverse plane, nonuniform echogenicity of the pyloric muscle was seen in 59 (98%) of 60 patients. In the midline longitudinal plane, the muscle was equal to or slightly less echogenic than the liver in all patients. Both the in vivo and in vitro studies show that the echogenicity varies with the relationship of the ultrasound beam to the orientation of the circular muscle fibers; this phenomenon is known as the anisotropic effect. Our results show that nonuniform echogenicity of the hypertrophied pyloric muscle is a characteristic sonographic finding caused by the anisotropic effect, which is related to the orientation of the ultrasound beam with respect to the circular fibers of the pyloric muscle. PMID- 1727339 TI - Correlation between omphalocele contents and karyotypic abnormalities: sonographic study in 37 cases. AB - To evaluate the observation that fetuses with omphaloceles containing only bowel have an especially high prevalence of karyotypic abnormalities, we retrospectively reviewed the sonograms and case records of 37 fetuses with omphaloceles detected sonographically between 1984 and 1990. Nine fetuses had concomitant morphologic abnormalities characteristic of the amniotic band syndrome. Of the remaining 28 fetuses, karyotypic correlation was available in 22, and the karyotype was abnormal in five of these (23%). The omphaloceles contained liver in 22 fetuses and only bowel in six fetuses. Among fetuses with exteriorized liver, karyotypes were abnormal in one (6%) of 16 tested. In contrast, four (67%) of the six fetuses whose omphaloceles contained only bowel had abnormal karyotypes; for each of these four, sonograms showed morphologic abnormalities in addition to the omphalocele. In the two fetuses with bowel-only omphaloceles and normal karyotypes, the omphalocele was the only abnormality seen on sonograms, and these children are well after surgical repair. When fetuses with the amniotic band syndrome were excluded, sonograms showed concomitant anomalies in 15 fetuses with liver-containing omphaloceles, and the karyotype was abnormal in only one of these 15. The results of this study support previous observations that karyotypic abnormalities are more common in association with omphaloceles that contain only bowel compared with those that contain only liver. If we combine our data with data from three other studies that address this issue, 87% of fetuses with omphaloceles containing only bowel had an abnormal karyotype, a significantly higher rate than in those fetuses whose omphaloceles contained liver also (9%). PMID- 1727341 TI - Hepatic masses in infants and children: CT evaluation. AB - Hepatic masses are uncommon in children. Although some lesions may have overlapping appearances on CT, a careful analysis of the CT characteristics of the lesion, in combination with the age of the patient and the clinical and laboratory data, can lead to a specific diagnosis in many cases [1-5]. In this pictorial essay, the spectrum of hepatic masses in children is reviewed, with emphasis on the key CT findings that help in differentiating the various lesions. PMID- 1727340 TI - Voiding cystourethrography in children: value of digital fluoroscopy in reducing radiation dose. AB - Voiding cystourethrography is a commonly used fluoroscopic procedure in children that can directly irradiate the gonads. As a consequence, much attention has been given to reducing the dose of radiation received during the procedure. A digital fluoroscope, especially adapted for use in children, was evaluated for potential reduction of the dose of radiation during the procedure. Entrance and midplane doses were calculated on child-sized phantoms by using the digital fluoroscope, digital spot films, and 105-mm spot films. Subsequently, data were collected on 47 children, grouped by ages (neonate to 1 year, 1-5 years, and 5-7 years), in whom voiding cystourethrography was performed by using the same exposure factors as those for the phantoms. On the basis of the exposure doses for the phantoms and recorded clinical peak kilovoltages, milliamperes, milliseconds, and fluoroscopic time, average skin and ovarian doses were calculated for each group of children. These doses were compared with previously reported doses for fluoroscopic and radionuclide voiding cystourethrography. Results of line-pair resolution studies for the digital spot films and 105-mm spot films were similar. Images from the digital device and 105-mm images obtained on a conventional fluoroscope were considered equally adequate for clinical decision making. The average midplane and skin doses with digital spot films for children less than 5 years old were equal to or less than 0.66 and 2.37 mGy, respectively, as opposed to 1.37 and 5.32 mGy with the 105-mm spot films. Previously reported ovarian doses range from 2.52 to 10.0 mGy for fluoroscopic voiding cystourethrography and from 0.04 to 0.05 mGy for radionuclide voiding cystourethrography. The use of digital spot films reduced dose approximately 50% compared with 105-mm spot films; the ovarian dose was 0.62 mGy greater than that for radionuclide voiding cystourethrography. PMID- 1727342 TI - Real-time sonography in ocular trauma. AB - Real-time sonography was evaluated retrospectively in 71 consecutive patients with ocular trauma. A total of 51 vitreous hemorrhages, 20 hemorrhages in the anterior chamber, 22 retinal detachments, seven choroidal detachments, five foreign bodies, and 12 dislocated lenses were identified sonographically. In 10 instances (three choroidal detachments, six retinal detachments, and one lens dislocation), these sonographic findings were not apparent on clinical examination. One hemorrhage of the anterior chamber was missed on sonography. Both sonography and clinical examination failed to visualize one retinal detachment. The results of this study show that real-time sonography is valuable in the assessment of ocular trauma and supplements clinical examination with valuable information. PMID- 1727343 TI - MR lymphography with iron oxide particles: dose-response studies and pulse sequence optimization in rabbits. AB - Superparamagnetic iron oxide (SPIO) particles are a promising contrast agent for MR lymphography. The effect of SPIO on MR imaging of normal lymph nodes and the impact of the size of the dose have not yet been investigated in detail. Therefore, we performed dose-response and pulse sequence optimization studies. MR images of the iliac lymph nodes of 15 normal rabbits were obtained at 1.5 T with 12 different spin-echo (SE) and gradient-echo (GRE) sequences before and after SPIO administration. The contrast agent was injected into a femoral lymph vessel at five different doses (0.02-2.0 mumol Fe/animal). The dose that reduced signal intensity by half (ED50) was determined for each sequence, and images were evaluated qualitatively. Doses of 0.2 and 1.0 mumol Fe caused a complete signal loss throughout the lymph node. In this dose range, proton density-weighted SE sequences showed a profound signal loss (ED50, 0.132 mumol Fe), and lymph nodes were sharply demarcated. The GRE sequences (ED50, 0.027-0.070 mumol Fe) and the T2-weighted SE sequence (ED50, 0.014 mumol Fe) showed an even more pronounced signal loss but insufficient anatomic resolution. Underdosing (less than or equal to 0.1 mumol Fe) caused only a focal signal loss in the lymph nodes. Oversaturation (2.0 mumol Fe for SE sequences, greater than or equal to 1.0 mumol Fe for GRE sequences) led to image distortion and did not allow assessment of lymph node morphology. Our results show that optimal contrast enhancement of normal lymph nodes with SPIO can be achieved in the dose range of 0.2-1.0 mumol Fe on proton density-weighted SE sequences. Our results may serve as a basis for further development of noninvasive MR lymphography. PMID- 1727344 TI - Computed radiography in musculoskeletal imaging: state of the art. AB - Computed radiography is a 2K x 2K x 10 bit digital radiographic system that replaces the film-screen combination with a photo-stimulable phosphor plate. The advantages of this relatively new technology include linear detector response, improved detector efficiency, and digital processing capabilities. Musculoskeletal applications benefit significantly from these attributes, which result clinically in the ability to reduce both radiation dose and number of exposures. Studies of observers' performance have shown no statistically significant difference in diagnostic accuracy between film-screen and computed radiographic musculoskeletal images. Computed radiography is particularly useful in the evaluation of the musculoskeletal system in traumatized patients with portable radiographs, spine radiographs, scoliosis studies, and depiction of soft tissue abnormalities. Limitations include change in image format and size, high cost, decreased spatial resolution, restricted throughput, increased perception of noise, and new artifacts that must be recognized. Spatial resolution limitations of computed radiography in identification of fine detail information can be improved by using magnification techniques. Radiation dose reduction with an exposure decrease of 25-50% can be achieved without loss of diagnostic accuracy, although this depends on the examination and the abnormality. An interactive workstation is important in the use of a computed radiographic system with capabilities to adjust display parameters to best depict images and disease. We conclude that computed radiography is an alternative to film-screen radiography without significant differences in diagnostic quality in the evaluation of musculoskeletal images. PMID- 1727345 TI - A new electronically enhanced biopsy system: value in improving needle-tip visibility during sonographically guided interventional procedures. AB - Sonographically guided fine-needle biopsy procedures are hampered by poor visibility of the needle tip. This study was performed to evaluate a new system for placing needles under sonographic guidance. The Biosponder needle (Advanced Technology Laboratories, Bothell, WA) incorporates a specialized stylet with a passive sensor at its tip. When an ultrasound pulse is detected by the sensor, an electrical signal is transmitted to the sonographic unit by a battery-powered electronic module connected to the stylet and to the scanner. This signal is converted into a bright, flashing marker on the screen at the precise location of the needle tip. The Biosponder system, which uses 20- or 22-gauge needles, was compared with a 20-gauge Turner needle (Cook, Bloomington, IN) in 18 patients with masses or fluid collections and two patients requiring nephrostomy tube placement. The tip of the Turner needle could not be localized precisely in any patient. The shaft of the Turner needle was seen clearly in 13 patients, poorly in three, and was not visualized at all in four patients. Excluding four instances of mechanical failure, the Biosponder system allowed precise localization of the needle tip in every patient and was consistently rated as easier to use than the Turner needle. We conclude that the Biosponder needle, with its precise tip localization and ease of use, is a valuable tool for sonographically guided needle placement. PMID- 1727346 TI - Preparation of manuscripts for radiology journals: advice to first-time authors. PMID- 1727347 TI - Controlled circulation journals. PMID- 1727348 TI - Lung cancer in a drug addict seropositive for human immunodeficiency virus. PMID- 1727349 TI - Endobronchial lipoma associated with lipomatosis. PMID- 1727350 TI - Hydatid disease: CT demonstration and follow-up of a cystogastric fistula. PMID- 1727351 TI - Hypoplasia of the left lobe of the liver. PMID- 1727352 TI - Uterine adenofibroma mimicking endometrial carcinoma: MR findings. PMID- 1727353 TI - Prostatic and renal stones and unilateral obstruction of the urinary tract caused by ochronosis. PMID- 1727354 TI - Congenital constricting ring and amputations of the fingers due to amniotic bands. PMID- 1727355 TI - Duplex sonography of the cerebral arteries: efficacy, limitations, and indications. AB - This review considers the capabilities and limitations of duplex sonography in the diagnosis of abnormalities of the cerebral vasculature. Duplex sonography is an elegant union of B-mode and Doppler sonography that provides valuable information about atherosclerotic obstruction of the carotid arteries. Duplex sonography also can be used to evaluate, in a general way, the composition of carotid atherosclerotic plaque, and in this respect, it is unique among imaging procedures. Duplex sonography is not very effective in providing a "global" perspective of the cerebral vasculature, because only the cervical portion of the carotid arteries can be examined in detail. The best documented and most clearly effective use of duplex sonography is for detecting severe obstructive lesions in the carotid artery that might warrant endarterectomy in patients with cerebral hemispheric symptoms. The role of duplex sonography in the choice between medical and surgical therapy in asymptomatic patients with carotid artery stenosis is controversial, because the indications for endarterectomy are unclear in these patients. The capacity of duplex sonography to assess plaque composition may ultimately prove to be quite valuable for selecting therapeutic options and for evaluating the effectiveness of medical therapy. Meanwhile, information concerning the clinical value of this use of duplex sonography remains limited. PMID- 1727356 TI - The future of carotid sonography. PMID- 1727357 TI - Design and conduct of a low-cost mammography screening project: experience of the American Cancer Society, Texas Division. AB - To improve compliance with recommendations for screening mammography, the American Cancer Society (ACS) Texas Division designed and conducted a media promoted screening project in 1987. The project was planned during a 2-year period by a task force made up of physicians and lay members of ACS division committees. Radiology centers desiring to participate in the project were asked to submit information about the number of patients they could screen and their equipment, along with physics data, to a review committee. Of 306 facilities that responded, 266 (87%) passed the initial review. Thirteen facilities (4%) submitted images from two examinations using a dedicated mammography phantom, and 27 sites (9%) entered the project by agreeing to adhere to the project standards and guidelines without undergoing formal review. All facilities agreed to provide mammograms for $50 to women scheduling appointments during a 2-week media campaign in February 1987. The project generated 64,459 mammographic screening examinations. Our experience indicates that a media campaign can encourage women to have screening mammograms and that screening facilities will agree to screen a large number of women at reduced cost. This strategy, if widely applied, can improve compliance with mammographic screening recommendations and reduce breast cancer mortality. PMID- 1727358 TI - Low-cost mass screening for breast cancer with mammography. PMID- 1727359 TI - Colonic contour changes in chronic ulcerative colitis: reappraisal of some old concepts. PMID- 1727360 TI - Menetrier disease. PMID- 1727361 TI - Intestinal stricture due to lap-belt injury. PMID- 1727362 TI - Splenic and hepatic peliosis: MR findings. PMID- 1727363 TI - Percutaneous drainage of hydatid cysts: use of a new cutting device to avoid leakage. PMID- 1727364 TI - Pleomorphic pancreatic sarcoma mimicking pancreatic pseudocyst: CT appearance. PMID- 1727365 TI - The scintigraphic diagnosis of osteomyelitis. AB - Osteomyelitis is a serious health problem that results in multiple limb amputations annually. This article reviews the current scintigraphic procedures used in the diagnosis of osteomyelitis and discusses some of the newer radiopharmaceuticals now being developed. The goal is to understand the strengths and weaknesses of each method so that the procedure most effective for specific clinical settings can be selected. In general, the three-phase bone scan is the procedure of choice if the suspected osteomyelitis is not superimposed on another disease that causes increased bone remodeling (i.e., findings on the radiograph are normal). If the suspected osteomyelitis is superimposed on a disease that causes increased bone remodeling, the combined 111In-labeled leukocyte-99mTc bone scan is the procedure of choice in the non-marrow-containing skeleton and the 111In-labeled leukocyte and 99mTc bone marrow scans are the procedures of choice in the marrow-containing skeleton. PMID- 1727366 TI - Symptomatic renal obstruction or urosepsis during pregnancy: treatment by sonographically guided percutaneous nephrostomy. AB - Seven pregnant women with symptomatic hydronephrosis had sonographically guided percutaneous nephrostomy for pyosepsis (five patients) or for pain with azotemia (two patients with renal transplants). Antibiotics had been ineffective in controlling pyosepsis in each patient; retrograde ureteral catheterization via cystoscopy was unsuccessful in one patient. After percutaneous nephrostomy, prompt clinical improvement was observed in all patients (i.e., sepsis was relieved and pain abated). Labor was not induced in any of the patients, and no adverse effects occurred to any fetus or mother. Eleven (eight percutaneous nephrostomy, three catheter exchanges) of the 12 procedures were done without conventional radiography and with sonographic guidance alone. After percutaneous nephrostomy, maneuvers to obtain a diagnosis and to treat the obstruction (if necessary) were delayed until after delivery. The causes of ureteral obstruction were calculi (four patients) and a gravid uterus (three patients). After delivery, stones were removed either percutaneously (one patient) or cystoscopically (two patients) or passed spontaneously (one patient); resolution of obstruction by the gravid uterus was proved by Whitaker test after delivery. Sonographically guided percutaneous nephrostomy is an effective and safe method to treat pregnant women who have symptomatic obstructive hydronephrosis associated with either pyosepsis or azotemia. The procedure is rapid, requires minimal anesthesia, has no radiation, and is safe for the fetus. The technique is a useful and perhaps preferable alternative to more invasive surgical therapy or retrograde stenting. PMID- 1727367 TI - MR appearance of the normal and abnormal vagina after hysterectomy. AB - To define the MR appearance of the vagina after hysterectomy, we reviewed the MR examinations of 43 women who had undergone hysterectomy for a variety of benign and malignant indications. In eight of the patients, MR examinations showed a mass lesion involving the vagina. The masses included four recurrent primary gynecologic malignant neoplasms (one endometrial, one ovarian, and two cervical carcinomas) and four primary vaginal carcinomas. The remaining 35 patients had no evidence of vaginal disease. Of these 35 patients, the repaired vaginal apex, or vaginal cuff, was linear and smooth in appearance in 23 and partially obscured by surgical clip artifacts in seven. In the other five patients, the vaginal cuff had a nodular appearance that was indistinguishable from a vaginal mass on T1 weighted images. However, the vagina had a normal appearance in these patients on T2-weighted images, on which the low-signal-intensity layer of vaginal smooth muscle could be distinguished from the bright outer layer of connective tissue. In patients with recurrent vaginal tumors, the tumor was relatively high in signal intensity on T2-weighted images and obliterated the low-signal-intensity vaginal muscularis. Our experience shows that the normal posthysterectomy vagina may have a nodular appearance on T1-weighted images mimicking a vaginal mass. This appearance can be distinguished from that of a true vaginal mass on the basis of different signal intensity characteristics on T2-weighted images. PMID- 1727368 TI - Preclinical evaluation of bryostatin as an anticancer agent against several murine tumor cell lines: in vitro versus in vivo activity. AB - We have examined the ability of bryostatin 1 to inhibit the in vitro growth and in vivo development of a panel of four murine tumors of diverse tissue origins. A wide range of antiproliferative responses was observed for the four tumors. At 100 ng/ml the in vitro growth of the Renca renal adenocarcinoma, the B16 melanoma, the M5076 reticulum cell sarcoma, and the L10A B-cell lymphoma were inhibited by 0, 40, 40, and 94% respectively. All three cell lines sensitive to bryostatin in vitro responded to multiple dose, 1 microgram/injection/day in vivo i.p., bryostatin therapy. Only the in vitro resistant Renca tumor failed to respond to bryostatin in vivo. The correlation between in vitro and in vivo antitumor efficacy suggests a direct mechanism of antitumor activity for bryostatin. Both local regional therapy (M5076 i.p.) and systemic therapy (B16 lung metastases and L10A s.c. tumors) with bryostatin were successful at prolonging survival time. Multiple i.p. doses of bryostatin at a minimum level of 0.5-1.0 microgram/injection were required to observe significant in vivo antitumor effects. The success of in vivo administration of bryostatin in mice bearing 8-10-mm s.c. masses of L10A lymphoma (5-10 x 10(9)) and our further observation that five of a panel of six human B-cell lymphoma cell lines were sensitive to the growth inhibitory effects of bryostatin in vitro suggest that bryostatin may be effective in treating lymphoid malignancies in humans. PMID- 1727369 TI - Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction. AB - A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100 fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations. PMID- 1727370 TI - Abundance of the primary transcript and its processed product of growth-related genes in normal and leukemic cells during proliferation and differentiation. AB - The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was determined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the S14 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells. PMID- 1727371 TI - Interaction of photodynamic treatment and either hyperthermia or ionizing radiation and of ionizing radiation and hyperthermia with respect to cell killing of L929 fibroblasts, Chinese hamster ovary cells, and T24 human bladder carcinoma cells. AB - Both hyperthermia and photodynamic therapy of cancer are frequently used in combination with other treatment modalities in order to improve tumor control with minimal damage to normal tissues. The present results indicate that the most effective combination of treatment modalities is different in different cell types. For instance, ionizing irradiation and hyperthermia exhibited additivity when applied to L929 fibroblasts, in contrast to the synergistic interaction described before in many other cell lines. This aberrant behavior of L929 cells could be explained by the relative insensitivity of DNA repair in these cells to hyperthermia. Conversely, a synergistic interaction between photodynamic treatment and ionizing irradiation was observed with L929 fibroblasts, whereas these treatments were additive with Chinese hamster ovary and T24 cells. The synergistic interaction with L929 cells could be explained by the high sensitivity of DNA repair in these cells to photodynamic treatment. Photodynamic treatment and hyperthermia exhibited a synergistic interaction in L929, Chinese hamster ovary, and T24 cells. The critical target for cell killing by the combined treatment protocol in these cell lines has not yet been elucidated. In all three cell lines, however, analysis of the results according to the Arrhenius equation revealed a photodynamically induced change of both the frequency factor and the activation energy of subsequent thermal cell killing. It is considered that this may indicate a basic mechanism, in which a particular protein is a common, critical target of the two modalities of treatment. PMID- 1727372 TI - Immunotherapy of human glioma xenografts with unlabeled, 131I-, or 125I-labeled monoclonal antibody 425 to epidermal growth factor receptor. AB - Monoclonal antibody (mAb) 425 (IgG2a) binds to the external domain of the epidermal growth factor receptor. This determinant is highly expressed by human glioma tissues but rarely by normal brain tissues, and is absent on peripheral blood lymphocytes and bone marrow cells. The mAb exerts variable cytotoxic effects against cultured human glioma cells in conjunction with human and murine effector cells. Inhibition of growth of s.c. glioma xenografts in nude mice by the mAb may be mediated by murine macrophages or may be related to the capacity of the mAb to antagonize growth stimulation of glioma cells by epidermal growth factor. In approaches to radioimmunotherapy of human glioma with mAb 425, the 125I-labeled mAb 425 exhibited more significant antitumor effects than the 131I labeled mAb both in vitro and in vivo in xenotransplanted nude mice. These differences may be due to enhanced nuclear damage caused by 125I-labeled versus 131I-labeled fragments following their internalization into the glioma cells. Our studies provide the rationale for immunotherapy of glioma patients with either unlabeled or 125I-labeled anti-epidermal growth factor receptor mAb 425. PMID- 1727373 TI - Immunoconjugates containing novel maytansinoids: promising anticancer drugs. AB - The potential of immunoconjugates of cytotoxic drugs for the treatment of cancer has not yet been realized owing to the difficulty of delivering therapeutic concentrations of these drugs to the target cells. In an effort to overcome this problem we have synthesized maytansinoids that have 100- to 1000-fold higher cytotoxic potency than clinically used anticancer drugs. These maytansinoids are linked to antibodies via disulfide bonds, which ensures the release of fully active drug inside the cells. The conjugates show high antigen-specific cytotoxicity for cultured human cancer cells (50% inhibiting concentration, 10 to 40 pM), low systemic toxicity in mice, and good pharmacokinetic behavior. PMID- 1727374 TI - Melphalan penetration of the blood-brain barrier via the neutral amino acid transporter in tumor-bearing brain. AB - Melphalan, a nitrogen mustard derivative of the neutral amino acid L phenylalanine, was transported across the rat blood-brain barrier by the large (L system) neutral amino acid transporter in tumor-bearing brain, but no evidence for blood-brain barrier transport by the alanine-serine-cysteine system carrier was obtained in the present study. The ability of melphalan to inhibit phenylalanine uptake was compared in rats implanted with two experimental CNS tumors: the C-6 glioma (a model of primary brain tumors) and Walker carcinoma (a model of metastatic brain tumors). The melphalan concentration which caused 50% inhibition of blood-brain barrier (BBB) phenylalanine uptake (Ki) was 0.49 +/- 0.18 mM in the Walker tumor, compared with 0.46 +/- 0.19 mM in the contralateral control brain. In the ipsilateral hemisphere (Ki = 0.59 +/- 0.25 mM) and contralateral hemisphere (Ki = 0.45 +/- 0.19 mM), drug entry was also via the neutral amino acid transporter. In C-6 gliomas (Ki = 0.77 +/- 0.20 mM) and contralateral control brain (Ki = 0.84 +/- 0.29 mM), melphalan also inhibited BBB phenylalanine transport. A major finding was that, at melphalan concentrations greater than 1.0 mM, BBB permeability of radiolabeled indium (chelated to EDTA) increased in proportion to melphalan concentration. In the contralateral hemisphere of rats implanted with C-6 gliomas, brain extractions of indium-EDTA measured 3 to 4% in the absence of drug, 5 to 6% at 2.5 mM melphalan, and 9 to 10% at 5 mM melphalan. A similar phenomenon was observed in the nontumoral brain regions of rats implanted with Walker carcinoma cells. In normal (nonimplanted) rats, melphalan's inhibition (Ki = 0.29 mM) of phenylalanine and tryptophan (Ki = 0.20 mM) uptake was confirmed, and brain extraction of sucrose (a nonspecific marker which does not penetrate the intact BBB) was observed to increase in proportion to melphalan concentration. We conclude that melphalan not only enters the brain via the neutral amino acid transporter, but at higher concentrations (greater than 1 mM) may open the blood-brain barrier in a nonspecific manner. PMID- 1727375 TI - Intracellular acidification is associated with enhanced morphological transformation in Syrian hamster embryo cells. AB - A series of studies has indicated that the frequency of morphological transformation induced by chemical carcinogens in early passage Syrian hamster embryo (SHE) cells is significantly higher when these cells are cultured in medium of reduced bicarbonate concentration and pH (6.70) compared with cells cultured in medium of higher pH. It has also been shown that intercellular gap junctional communication is decreased in these cells when they are cultured at pH 6.70 compared with medium of higher pH. The purpose of the studies reported here was to characterize the effect of changing extracellular pH on intracellular pH in SHE cells. The frequency of morphological transformation induced by benzo(a)pyrene was established at various extracellular pHs and compared with intracellular pH values. Cells cultured in medium of pH ranging from 6.70 to 7.35 were loaded with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6 carboxyfluorescein, and either the steady-state intracellular pH values or the kinetics of change in intracellular pH following refeeding of the cultures with medium of pH ranging from pH 6.70 to pH 7.35 was monitored via image analysis techniques. Results from these studies indicate that, at culture medium pH above 6.95, SHE cells were relatively insensitive to changes in extracellular pH, maintaining an intracellular pH of 7.30 to 7.35 in medium containing 0% serum or pH 7.05 to 7.10 in medium containing 20% fetal bovine serum. At extracellular pHs below 6.95, intracellular pH decreased and, in the presence of serum, equilibrated with extracellular pH. The decrease in intracellular pH was closely associated with an increase in benzo(a)pyrene-induced morphological transformation frequency observed in parallel studies. These results indicate that SHE cells have active intracellular pH regulatory activities and suggest that intracellular acidification plays a role in the increased frequency of transformation observed in SHE cells cultured under acidic conditions. PMID- 1727376 TI - Persistence of platinum-ammine-DNA adducts in gonads and kidneys of rats and multiple tissues from cancer patients. AB - The persistence of platinum-DNA adducts was investigated using normal rats as well as tissues from cancer patients receiving either cisdiamminedichloroplatinum(II) (cisplatin) or diamminecyclobutanedicarboxylatoplatinum(II) (carboplatin) for cancer chemotherapy. These studies used an enzyme-linked immunosorbent assay, established with a rabbit anti-cisplatin-DNA that is specific for intrastrand platinum-DNA adducts. The gonads and kidneys of male and female rats, sites for antitumor activity and toxicity, respectively, were monitored for cisplatin-DNA adduct formation after a single dose of drug and during multiple-dose exposures (once a wk for 3 wk). DNA adducts were measured by enzyme-linked immunosorbent assay 4 h and 2, 4, 7, and 14 days after administering a single i.v. injection of 8 mg/kg of cisplatin. Adduct profiles in renal tissues were similar in both males and females with adduct levels increasing between 4 h and 2 days, decreasing between Days 2 and 7, and stable between Days 7 and 14. In both sexes, levels of kidney DNA adduct measured 7 to 14 days after cisplatin injection comprised about 30% of the highest (Day 2) value. In testes and ovaries, adduct removal was complete by 4 days, and 40 to 50% of adducts present at Day 2 persisted until Days 7 and 14. A study of multiple dosing showed that adducts in renal and testicular DNA from rats given three weekly doses of 5 mg/kg of cisplatin had different accumulation profiles. In the testis there was a 2-fold accumulation of adduct after the third dose, while in the kidney adducts dropped with repeated dosing. In humans, the persistence of platinum-DNA adducts was studied in tissues from eight cancer patients who received their last dose of cisplatin or carboplatin chemotherapy between 1 day and 15 mo before autopsy. The patients had either ovarian cancer, breast cancer, or lymphoma, and the tissues studied included ovarian tumor, bone marrow, kidney, liver, spleen, lymph node, peripheral nerve, and brain. When samples were available from tumor tissues and from bone marrow within the same patient, adduct levels were similar in the two tissues. In addition, adducts were persistent for many months, since half of the individuals received their most recent platinum-drug therapy 7 to 15 mo before death. Overall, these studies demonstrate a widespread distribution and high degree of platinum-DNA adduct persistence in both animal and human tissues subsequent to cisplatin or carboplatin treatment. PMID- 1727377 TI - Morphological reversion of sis-transformed NIH3T3 cells by trichostatin A. AB - Trichostatin A (TSA) induced the normal and flat phenotype of sis-transformed NIH3T3 cells at quite a low concentration of 1 ng/ml. Although morphological changes were found in other oncogene-transformed cells, they were not the same as those seen for the sis-transformed cells. Almost complete reversion into the flat phenotype was seen at 6 h after administration of the compound, suggesting that the morphological change was caused not merely by selection of TSA-resistant cells of the flat phenotype. The effect of TSA was reversible when the cell culture was incubated after its removal. Synthesis of sis-mRNA did not decrease with the treatment of TSA at a concentration sufficient to reverse the transformed morphology. Cycloheximide abolished the activity of TSA, showing that TSA required new protein synthesis to express its activity. PMID- 1727378 TI - Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. AB - K1 is a monoclonal antibody that reacts with a cell surface antigen (CAK1) found in human mesothelia and nonmucinous ovarian tumors. In this article, the characteristics of the CAK1 antigen have been examined in detail. Using immunofluorescence microscopy, we have found that the CAK1 signal is removed from the cell surface by treatment with proteases or by phosphatidylinositol phospholipase C, but not by neuraminidase and beta-galactosidase. The phosphatidylinositol-phospholipase C-released material was found to contain the CAK1 antigen which was detected by a competition radioimmunoassay. The phosphatidylinositol-phospholipase C-released CAK1 antigen was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and found to be approximately 40 kDa protein. The CAK1-K1 antibody complex remains on the cell surface and is poorly internalized, as shown by an acid wash immunofluorescence internalization assay. An immunotoxin composed of K1 and Lys PE40, a mutant form of Pseudomonas exotoxin lacking the cell binding domain, was not cytotoxic, supporting the conclusion that the CAK1-K1 antibody complex is not internalized. However, an immunotoxin composed of K1 and native Pseudomonas exotoxin was selectively cytotoxic to cells expressing the CAK1 antigen. This cytotoxicity is due to the fact that domain I of Pseudomonas exotoxin promotes internalization of antigens which are not internalized or bound to antibody alone. Our results suggest that CAK1 is a polypeptide that is expressed on mesothelial cells and many ovarian cancers, and that K1 may be useful as a targeting agent for the immunotherapy of human ovarian cancer. PMID- 1727379 TI - Comparative study of doxorubicin, mitoxantrone, and epirubicin in combination with ICRF-187 (ADR-529) in a chronic cardiotoxicity animal model. AB - In this study doxorubicin, epirubicin, and mitoxantrone were compared for their cardiotoxic potential in a chronic mouse model in an effort to identify and compare their mechanism(s) of toxicity. In addition, the cardioprotective ability of ICRF-187 [(+/-)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane] with each anticancer drug was evaluated in this model. The antioxidant capacity (superoxide dismutase, reduced glutathione, catalase, and glutathione peroxidase) was assessed following drug treatment. Five-week-old BALB/c mice received weekly i.p. injections of each drug or the drug and ICRF-187 over a 3-month period. ICRF-187 was administered 30 min prior to the anticancer drug. The hearts were examined by electron and light microscopy to assess subcellular changes, and the cardiac and hepatic antioxidant levels were measured concurrently. Chronic treatment with these drugs or each combined with ICRF-187 did not change the antioxidant levels relative to the control values. However, all three drugs caused cardiac damage during chronic exposure. Both epirubicin and mitoxantrone caused less severe damage than doxorubicin, and epirubicin was the least cardiotoxic of the three. ICRF-187 was cardioprotective for epirubicin and doxorubicin but not for mitoxantrone. These results suggest epirubicin acts by a mechanism similar to that of doxorubicin that is probably mediated by oxygen-free radicals, while mitoxantrone acts by a different mechanism to cause cardiotoxicity. PMID- 1727380 TI - Specific binding to protein kinase C by ingenol and its induction of biological responses. AB - We have examined the ability of ingenol to bind to and activate protein kinase C and to induce similar responses to the phorbol esters in biological systems. The rationale was that ingenol possesses the critical functionalities of the phorbol ester pharmacophore with the exception of the hydrophobic domain; it might therefore possess weak potency, although previous reports had indicated that ingenol was biologically inactive. Our data demonstrate that ingenol indeed binds to protein kinase C with a Ki of 30 microM and activates the enzyme. In addition, ingenol was biologically active in 3 separate cell systems, showing effects similar to the phorbol esters on morphological change, cell-cell communication, epidermal growth factor binding, arachidonic acid metabolite release, and ornithine decarboxylase activity. The 50% effective concentration values for the biological activity of ingenol were between 30 microM and 1 mM, varying somewhat with the cell system and type of response. The biological activity of ingenol in general supports the proposed models of the phorbol ester pharmacophore and imposes additional experimental constraints that the modeling must satisfy. PMID- 1727381 TI - Altered p53 gene structure and expression in human epithelial cells after exposure to nickel. AB - The carcinogenicity of certain nickel compounds is well known. We have previously shown that human kidney epithelial cells were immortalized by treatment with Ni(II) and in cooperation with the v-Ha-ras oncogene transformed the cells to acquire tumorigenicity in athymic nude mice. Immunocytochemistry and sequence analysis of DNA from the nickel-immortalized cells revealed abnormal p53 expression and a T----C transition mutation in codon 238. These data are consistent with the hypothesis that Ni(II)-induced mutation in the p53 gene can be involved in the escape from senescence of kidney epithelial cells. PMID- 1727382 TI - Suppression of acute lymphoblastic leukemia by the human wild-type p53 gene. AB - Independent mutations in both alleles of the p53 tumor suppressor gene are a frequent finding in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines and in the cells of some T-ALL patients in relapse. One major goal of studying the status of p53 (and other tumor suppressor genes) in human cancer is to facilitate the suppression of the tumorigenic phenotype through the restoration of the expression of the wild-type allele. While the efficient insertion of a suppressor into all cells of solid/metastatic human tumors may at present be impossible, insertion into leukemia cells may be feasible due to the accessibility of the leukemia cells in the body. To examine the feasibility of suppressing the tumorigenicity of human T-leukemia cells, the human T-ALL cell line Be-13, which lacks endogenous p53 protein, was infected with a recombinant retrovirus encoding the wild-type allele of human p53 (hwtp53). Expression of p53 reduced the growth rate of infected Be-13 cells in vitro, suppressed colony formation in methylcellulose cultures, and abrogated their tumorigenic phenotype in nude mice in vivo. These results suggest that suppression of the leukemic phenotype of relapse T-ALL-derived Be-13 cells is feasible. Acute leukemia cell suppression via high-efficiency infection with retroviruses encoding wtp53 may be feasible and beneficial in T-ALL cases as part of a bone marrow transplantation regimen in an effort to reduce the frequency of posttransplantation relapse. PMID- 1727383 TI - Role of tamoxifen in the induction of hormone-independent rat mammary tumors. AB - Using the dimethylbenzanthracene-induced rat mammary tumor model, we examined the appearance and growth of tumors in animals given tamoxifen (TAM) either coincident with dimethylbenzanthracene or after initial tumor formation. While fewer tumors arose after coadministration of antiestrogen and carcinogen and TAM treatment caused regression of most existing tumors, new tumors developed in the presence of TAM in both studies. Since none of these new tumors regressed following ovariectomy, all were classified as hormone independent. Furthermore, these independent tumors grew more rapidly than both control-dependent and independent tumors, resulting in a greater average volume. These data suggest that a more aggressive form of hormone-independent tumor appears during TAM treatment. PMID- 1727384 TI - Expression of human O6-methylguanine-DNA methyltransferase in a DNA excision repair-deficient Chinese hamster ovary cell line and its response to certain alkylating agents. AB - A plasmid has been constructed in which the expression of human O6-methylguanine DNA methyltransferase (MGMT) complementary DNA is driven by the Rous sarcoma virus promoter sequence. We had previously shown that transfection of this plasmid into Chinese hamster ovary (CHO) cells results in the expression of MGMT and in increased cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine and 1-(2-chloroethyl)-1-nitrosourea (CNU) but not N-nitroso-N-ethylurea (ENU). In the present study, the Rous sarcoma virus promoter-MGMT was transfected into DNA excision repair-deficient CHO UV41 cells to investigate the phenotype associated with MGMT expression in the absence of DNA excision repair. Both the UV41/MGMT and CHO/MGMT cells expressed similar levels of MGMT and exhibited a similar increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine. The UV41 cells were 20-fold more sensitive to CNU than the wild-type CHO cells. Expression of MGMT increased the resistance to CNU about 6-fold in both cell lines, but the difference between the two cell lines attributable to the excision repair defect still persisted. The UV41 cells were 2- to 3-fold more sensitive than the wild type CHO cells to the monofunctional alkylating agents 1-(2-hydroxyethyl)-1 nitrosourea and ENU, but the MGMT phenotype did not alter sensitivity. This suggests that alkylation at the O6 position of guanine has no role in cytotoxicity of ethylating agents and that monofunctional DNA damage has little role in the cytotoxicity of CNU. Since MGMT can prevent the formation of G-C interstrand cross-links formed by CNU, other excision repair-sensitive DNA adducts must play a major role in the sensitivity of UV41 cells to this bifunctional alkylating agent. These results suggest that DNA intrastrand cross links may be major contributors to the cytotoxicity of CNU. PMID- 1727385 TI - Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi Autonomous Region, People's Republic of China. AB - Hepatocellular carcinoma is one of the five leading human cancers causing at least 250,000 deaths each year. One of the major risk factors for this disease is exposure to dietary aflatoxins, and the development of appropriate molecular dosimetry biomarkers would facilitate the identification of individuals at risk. This study was undertaken to explore the relationship between dietary intake of aflatoxins and the excretion of the major aflatoxin-DNA adduct and other metabolites into the urine of chronically exposed people. The following protocol was developed for this investigation in Guangxi Autonomous Region, People's Republic of China, where the diets of 30 males and 12 females (ages, 25-64 years) were monitored for 1 week and aflatoxin intake levels determined each day. Starting on the fourth day, total urine volumes were obtained in consecutive 12-h fractions for 3 or 4 days. High performance liquid chromatography and competitive radioimmunoassay analyses were done on each of the urine samples, and the relationships between excretion of total aflatoxin metabolites, aflatoxin-N7 guanine, aflatoxin M1, aflatoxin P1, and aflatoxin B1, and aflatoxin B1 intake values were determined. The average intake of aflatoxin B1 by men was 48.4 micrograms/day, giving a total mean exposure during the study period of 276.8 micrograms. The average daily intake by women was 77.4 micrograms/day, resulting in a total average exposure during the 7-day period of 542.6 micrograms aflatoxin B1. Initial efforts to characterize aflatoxin metabolites in urine samples were with an analysis by competitive radioimmunoassay. The analysis by linear regression of the association between aflatoxin B1 intake/day and total aflatoxin metabolite excretion/day showed a correlation coefficient of only 0.26. These findings stimulated the immunoaffinity/analytical high performance liquid chromatography analysis for individual metabolites. When the data were analyzed by linear regression analysis, the aflatoxin N7-guanine excretion and aflatoxin B1 intake from the previous day showed a correlation coefficient of 0.65 and P less than 0.000001. Similar analysis for aflatoxin M1 resulted in a correlation coefficient of 0.55 and P less than 0.00001, whereas there was no positive statistical association between exposure in the diet and aflatoxin P1 excretion, despite aflatoxin P1 being quantitatively a major metabolite. Analysis of the total aflatoxin-N7-guanine excretion in the urine during the complete collection period plotted against the total aflatoxin B1 exposure in the diet for each of the individuals, smoothing the day to day variations, revealed a correlation coefficient of 0.80 and P less than 0.0000001.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727386 TI - Activation of the gadd153 promoter by genotoxic agents: a rapid and specific response to DNA damage. AB - Accumulation of gadd153 mRNA is strongly stimulated in mammalian cells by treatments which arrest growth or damage DNA (A. J. Fornace, Jr. et al., Mol. Cell. Biol., 9: 4196-4203, 1989). In previous studies, we demonstrated that the increased expression of gadd153 following treatment with several DNA-damaging agents was mediated transcriptionally (J. D. Luethy et al., J. Biol. Chem., 265: 16521-16526, 1990). To better define the specificity of this response, we have established a sensitive reporter system in which we have stably integrated a chimeric gene containing the gadd153 promoter linked to the coding region of the chloramphenicol acetyltransferase (CAT) gene into the genome of HeLa cells. Transcriptional activation from the gadd153 promoter was monitored by determining levels of CAT activity in cellular lysates prepared from gadd153CAT/HeLa cells treated with a variety of agents. The gadd153 promoter was strongly activated by a broad spectrum of genotoxic agents including UV-mimetic agents, DNA-cross linking and alkylating agents, DNA intercalators, and topoisomerase inhibitors. Of the DNA-damaging agents tested, only X-irradiation and bleomycin treatments failed to induce gadd153 promoter activity. Agents which inhibit replication and cell division and agents which otherwise result in cytotoxicity or growth arrest also had little influence on gadd153 promoter activity. Expression of the gadd153CAT chimeric gene in xeroderma pigmentosum Group A cells, which are deficient in nucleotide excision DNA repair of pyrimidine dimers, was maximally induced at UV doses at least 6-fold lower than those required for similar induction in repair-proficient HeLa cells. However, the methyl methanesulfonate induced gadd153 promoter activities were similar in both cell lines. Novobiocin pretreatment inhibited both UV- and methyl methanesulfonate-induced gadd153CAT expression. Collectively, these data indicate that: (a) the gadd153 promoter is activated rapidly and specifically by DNA damage; (b) the altered DNA structure is the inducing signal for the activation of the signal transduction pathway responsible for enhanced gadd153 expression; and (c) regulation of gadd153 by growth arrest is distinct from that of DNA damage. Thus, the gadd153CAT/HeLa cells are a useful model for examining the molecular mechanisms associated with the response to DNA damage and provide a reporter system for the screening of potential genotoxic agents. PMID- 1727388 TI - Selective depletion of tumor ATP by 2-deoxyglucose and insulin, detected by 31P magnetic resonance spectroscopy. AB - The purpose of this study was to investigate whether substrate deprivation acutely and selectively decreases ATP concentration in an experimental sarcoma. Two methods of substrate deprivation were examined: glycolysis was inhibited using 2-deoxyglucose (2DG), and plasma substrate levels were reduced using insulin. The effects of treatment on tumor ATP, inorganic phosphate, and pH were studied by 31P nuclear magnetic resonance spectroscopy. 2DG (2 g/kg) was administered i.p. to rats bearing s.c. methylcholanthrene-induced sarcomas. Inhibition of glycolysis by 2DG caused a 52 +/- 13% (SE) decrease in the tumor ATP to inorganic phosphate ratio, associated with a decrease in pH of 0.38 +/- 0.10 unit. The same dose of 2DG caused no significant change in the ratio of phosphocreatine to ATP in brain. Insulin (125 units/kg, i.p.) caused a 68% decline in plasma glucose and a 71% decline in betahydroxybutyrate compared to saline-treated animals. Concomitantly, 31P nuclear magnetic resonance spectroscopy detected a 48 +/- 13% decrease in sarcoma ATP, with a reciprocal elevation of inorganic phosphate in insulin-treated animals. In contrast, the brain phosphocratine/ATP ratio was unaffected by insulin. These results suggest that large tumors are acutely sensitive to inhibition of glycolysis and reductions in plasma levels of substrates for oxidative phosphorylation and glycolysis, while the brain is unaffected. In addition, this work provides support for the use of 31P nuclear magnetic resonance spectroscopy to monitor tumor response to therapy. PMID- 1727387 TI - Use of adaptive control with feedback to individualize suramin dosing. AB - Suramin is the first putative growth factor inhibitor in clinical trial that has demonstrated antitumor activity. Administration of suramin is complicated by a narrow therapeutic index and significant interpatient variability of measured pharmacokinetic parameters. Because both antitumor response and dose-limiting toxicities are related to plasma suramin concentration profiles, individualized dose schedules are required for optimal administration of the compound. In this report, the use of optimal sampling theory to derive sparse data monitoring and control strategies for use with suramin is described. A fixed rate continuous infusion schedule was used in seven patients, and the time to peak concentration (280-300 micrograms/ml) ranged from 7.7-21 days (mean, 13.2 days) with a decline to 150 micrograms/ml in 3-22 days (mean, 11 days). An initial population pharmacokinetic model was fit using a maximum likelihood algorithm. The mean volume of the central compartment was 4.5 +/- 6.7 liters/m2, volume of the peripheral compartment 10.6 +/- 1.4 liters/m2, distributional half-life 25 +/- 5.4 h, and elimination half-life 29.7 +/- 6.9 h. The terminal half-life was shorter than previously reported. These parameters were used as the initial population model for an iterative 2-stage analysis. The resulting distributional half-life of 22.3 +/- 2.7 h and elimination half-life of 28.2 +/- 5.0 h were similar, reflecting the intensive sampling. The iterative 2-stage analysis model was then used to determine the optimal sampling times and to simulate 20 data sets for a protocol designed to maintain plasma concentrations in a defined concentration range. This strategy is currently under investigation in phase I clinical trials. PMID- 1727389 TI - Human colon adenocarcinoma cell lines display infrared spectroscopic features of malignant colon tissues. AB - Seven human colon cell lines were studied by infrared spectroscopy including study of several spectral parameters under high pressure (pressure tuning spectroscopy). The results were compared to those obtained from the study of normal and malignant colon tissues (B. Rigas et al., Proc. Natl. Acad. Sci. USA, 87: 8140-8144, 1990; P. T. T. Wong and B. Rigas., Appl. Spectrosc., 44: 1715, 1990). The seven adenocarcinoma cell lines displayed almost all of the important spectroscopic features of colon cancer tissues: (a) increased hydrogen-bonding of the phosphodiester groups of nucleic acids; (b) decreased hydrogen-bonding of the C--OH groups of carbohydrates and proteins; (c) a prominent band at 972 cm-1; and (d) a shift of the band normally appearing at 1082 cm-1 to 1086 cm-1. These cell lines differed spectroscopically from the colon cancer tissues in that: (a) they displayed a band at 991 cm-1, which is weak in colon tissues; and (b) the packing and degree of disorder of membrane lipids were close to those observed in normal colonic tissues. These findings (i) establish IR spectroscopy, used in combination with pressure tuning, as a useful method to address problems of tumor biology in cell culture systems, (ii) indicate that these cell lines offer a useful experimental model to explore the origin of the spectroscopic changes that we observed in colon cancer tissues, and (iii) support the idea that the malignant colonocyte is the likely source of all or most spectroscopic abnormalities of human colon cancer. PMID- 1727390 TI - Identification of the major protein components in breast secretions from women with benign and malignant breast diseases. AB - The protein composition of breast secretions from 99 premenopausal women with benign or malignant breast diseases and from 70 control women without breast pathologies has been studied by using polyacrylamide gel electrophoresis. These fluids have been classified into two types according to their major polypeptide components. Type I fluids are defined by three major distinctive bands at Mr 44,000, 24,000, and 17,000, while those designated Type II present distinctive bands at Mr 80,000, 15,000, and 14,000. Amino acid sequencing and immunoblotting analysis demonstrated that proteins in Type I secretions correspond to Zn-alpha 2 glycoprotein, apolipoprotein D, and gross cystic disease fluid protein-15, while those from Type II fluids have been identified as lactoferrin, lysozyme, and alpha-lactalbumin. Most women (93%) without breast pathology and most patients (88%) with benign diseases had secretions with a Type I polypeptide pattern. By contrast, a large percentage (57%) of secretions from women with breast carcinoma presented a Type II protein pattern. Further studies with a large number of women will be useful for corroborating the potential clinical interest of breast fluid protein analysis. PMID- 1727391 TI - Alzheimer's disease. AB - Alzheimer's disease is one of the most severe and most common chronic diseases of older persons. Because occurrence of the disease is strongly related to age, its public health impact is likely to continue to increase as the population ages. As with many other diseases, a diagnosis of Alzheimer's disease is made through a combination of clinical history, physical, and neurologic examination, and laboratory evaluation. Because the dominant feature of this disease is its effect on cognition, its diagnosis requires careful evaluation of cognitive function usually with formal neuropsychological performance testing. Clinical evaluation of persons for Alzheimer's disease has four objectives: (1) to determine as accurately as possible if the person has dementia; (2) if dementia is present, to determine whether its presentation and course are consistent with a diagnosis of Alzheimer's disease; (3) to assess evidence for any alternate diagnoses, especially if the presentation and course are atypical for Alzheimer's disease; and (4) to evaluate evidence of other, coexisting, diseases that may contribute to the dementia, with strong emphasis on conditions that might respond to treatment. There is no reliable antemortem diagnostic test for Alzheimer's disease; the main purpose of laboratory testing is to identify other conditions that might cause or exacerbate dementia. Pathologically, Alzheimer's disease is characterized by the presence of two lesions on microscopic examination of the brain: neuritic plaques and neurofibrillary tangles. Both lesions can be seen in the brains of older persons without dementia. However, they are found in greater numbers in the neocortex and hippocampus with Alzheimer's disease. Caring for patients with Alzheimer's disease is demanding and requires compassion and skills that go beyond the choices among sophisticated and effective therapies that characterize much of modern medical practice. The current lack of effective pharmacotherapy for cognitive dysfunction in Alzheimer's disease should not obscure that there are many areas in which intervention can improve quality of life for both the patient and the caregiver. Achieving success in these areas typically requires that the physician work effectively with providers of many other medical and nonmedical services. Community resources, advocacy, behavior management, and experimental therapies and procedures, should be discussed with the family of each patient. In addition, persons with mild disease should be promptly informed of their diagnosis in order to obtain their wishes regarding life prolonging measures and extended care options. PMID- 1727392 TI - American Society for Biochemistry and Molecular Biology/Biophysical Society, joint meeting. Houston, Texas, February 9-13, 1992. Abstracts. PMID- 1727393 TI - Data watch. AHA: third-quarter HAS/Monitrend II data. PMID- 1727394 TI - Administrative ethics in the 1990s: CEOs confront payment, access dilemmas. AB - The 1990s are presenting new challenges to hospital CEOs. One theme currently emerging is the ethics of resource allocation. As they search for ways to fulfill their community responsibilities under pressures related to reimbursement, technology and community desires, hospitals are finding that they must make some very tough choices in how they allocate resources. Given the opportunity, how would you respond? Compare your response to an ethical scenario with those of three nationally recognized hospital CEOs. PMID- 1727395 TI - Outcomes research: hospitals face confidentiality concerns. PMID- 1727396 TI - Financial performance data: HCIA, HFMA battle over methodology, market share. AB - The leading health care financial data services are battling for market share with new products and refined FY 1990 performance indicators. But each service uses a different methodology, resulting in very different indicators. PMID- 1727397 TI - Critics examine economic merits, drawbacks of reform plans. PMID- 1727398 TI - Finding jobs for 1,200 laid-off employees: Health One's goal. PMID- 1727399 TI - Orthopedics offers hospitals niche opportunities in clinical areas. PMID- 1727400 TI - Home health, PPOs top diversification options. PMID- 1727401 TI - Three hospitals link clinical, financial data. PMID- 1727402 TI - ED overcrowding, preventive care topics of studies. PMID- 1727403 TI - Community college link: a hospital training bargain. PMID- 1727404 TI - CEOs: civic leadership boosts hospitals' image. PMID- 1727405 TI - A 5-y follow-up of the radiation exposure to in-room personnel during cardiac catheterization. AB - This study documents the radiation doses received by all in-room personnel of three cardiac catheterization laboratories where more than 15,000 cardiac procedures have been performed over a 5-y period. It is shown that all in-room personnel was exposed to a body dose equivalent well below any regulatory limits. However, some workers may have exceeded the occupational 150 mSv y-1 recommended limit for the lens of the eye. The physicians-in-training and the staff physicians are the two groups more likely to reach this limit. It is also demonstrated that a low correlation exists between the annual number of procedures and the annual head dose equivalent of a physician, but more variation is likely to originate from his/her working attitude and techniques. The mean dose equivalent at the collar level of the physicians is estimated to be 0.04 +/- 0.02 mSv per procedure. PMID- 1727406 TI - Dosimetry associated with exposure to 210Po by Mound workers. PMID- 1727407 TI - QUEST expands 30%, with abstracts. PMID- 1727408 TI - Fallout risk following a major nuclear attack on the United States. AB - Fallout distributions are calculated for nuclear attacks on the contiguous United States. Four attack scenarios are treated, including counterforce and counterforce-countervalue attacks, for meteorological conditions associated with a typical day in summer and one in winter. The countervalue attacks contain mostly airbursts. To determine fallout effects, the population surviving the prompt effects is first calculated. For the prompt effects, a "conflagration type" model is used. The counterforce attack produces about 8 million prompt deaths, and the counterforce-countervalue case projects 98 million prompt deaths. Partial relocation before attack to low-risk fallout areas at least 15 km from potential strategic targets would result in a decrease in projections of deaths by tens of millions. For fallout risk calculations, only the dose received in the first 48 h (the early or local fallout) is considered. Populations are assumed to be sheltered, with a shelter protection factor profile that varies for a large urban area, a small urban area, or a rural area. With these profiles, without relocation, the fallout fatalities for all four attack scenarios are calculated to be less than one million people. This can be compared to fallout fatalities of about 10 million for a hypothetical unsheltered "phantom" population. PMID- 1727409 TI - Determining the lower limit of detection for personnel dosimetry systems. AB - A simple method for determining the lower limit of detection (LLD) for personnel dosimetry systems is described. The method relies on the definition of a critical level and a detection level. The critical level is the signal level above which a result has a small probability of being due to a fluctuation of the background. All results below the critical level should not be reported as an indication of a positive result. The detection level is the net signal level (i.e., dose received) above which there is a high confidence that a true reading will be detected and reported as a qualitatively positive result. The detection level may be identified as the LLD. A simple formula is derived to allow the calculation of the LLD under various conditions. This type of formula is being used by the Department of Energy Laboratory Accreditation Program (DOELAP) for personnel dosimetry. Participants in either the National Voluntary Laboratory Accreditation Program (NVLAP) for personnel dosimetry or DOELAP can use performance test results along with a measurement of background levels to estimate the LLDs for their dosimetry system. As long as they maintain their dosimetry system such that the LLDs are less than half the lower limit of the NVLAP or DOELAP test exposure ranges, dosimetry laboratories can avoid testing failures due to poor performance at very low exposures. PMID- 1727410 TI - Internal dose following a major nuclear war. AB - The PATHWAY model results were used, in conjunction with a hypothetical major nuclear attack on the U.S., to arrive at the ratio of internal to external dose for humans from early (48 h) fallout. Considered were the four nuclides (137Cs, 89Sr, 90Sr, 131I) that account for most of the reconstructed whole-body committed equivalent dose from internal radiation in people who lived downwind of the Nevada Test Site during atmospheric tests. Effects of climate perturbations (the "nuclear winter" effect) on food crops were considered. These could increase internal dose estimates, depending on the severity of the climate perturbations. Internal and external doses to humans for 10 locations within the U.S. have been calculated, with varying local conditions and varying assumption about their shelters. The estimated 50-y internal dose commitment ranged from 0.0-0.17 Sv, the 48-h external dose from 0.15-4.6 Sv. The resultant ratios of internal to external committed dose received in the first months (until food transport was restored) varied from less than 0.01 to about 0.2. In all cases examined, the total dose from early fallout was found to be dominated by the external dose. PMID- 1727411 TI - Indoor 222Rn concentrations in a probability sample of 43,000 houses across 30 states. AB - The U.S. Environmental Protection Agency has assisted 30 of the 48 conterminous states in completing statistically designed surveys of indoor 222Rn over the past 4 y. In all states, the lowest livable level of 43,054 randomly selected houses was tested using charcoal canisters exposed for 48 h. The sampled population included owner-occupied ground-level houses having listed telephone numbers. Summary statistics along with the percentage of houses exceeding various concentration levels are given by state and over all states for houses with basements, for houses without basements, and for all houses. As expected, 222Rn concentration varies widely from one state to another and, in every state, basement houses exhibit higher concentrations than nonbasement houses. The lognormal distribution is shown to be a good approximation to the distribution of screening measurements over the 30-state area. There is, however, some evidence that the lognormal distribution underestimates, by a narrow margin, the upper tail of the observed distribution of basement measurements. PMID- 1727412 TI - Environmental dose assessment for low-level radioactive effluents discharged from Tokai reprocessing plant. AB - In the normal operation of the Tokai reprocessing plant, low-level radioactive effluents are discharged to the atmosphere and the ocean under rigid control. Radiation exposures to the population around the plant have been estimated for potential pathways with site-specific parameters such as food consumption, concentration factors of marine organisms, and meteorological conditions. External exposures to a radioactive cloud and internal exposures from inhalation and oral intake of radionuclides are evaluated for airborne effluents. External exposures to a contaminated fishing net and fishing boat are considered pathways for fishermen. External exposure to a contaminated beach and internal exposure via oral intake of marine products are evaluated for liquid effluents. Since the plant began operation in 1977, estimated effective dose equivalents for the public have been less than the annual effective dose equivalent limit of 0.1% recommended by the International Commission on Radiological Protection (1977). PMID- 1727413 TI - Absorption and retention of uranium from drinking water by rats and rabbits. AB - Uranium in the form of uranyl nitrate hexahydrate was administered in drinking water to Sprague-Dawley rats for periods of 28 and 91 d and New Zealand White rabbits for 91 d. The animals consumed food and water ad libitum. Subgroups of rabbits were followed for recovery periods of up to 91 d; 24-h collections of urine and feces were performed for some of the rabbits at various times during the exposure and recovery periods. At the end of the experiment, all animals were sacrificed and femur and kidney samples were analyzed for uranium residues. The results show that both rats and rabbits absorb about 0.06% of ingested uranium in the gastrointestinal (GI) tract. The distribution and retention of uranium in the skeleton and kidneys of rats are comparable to parameters reported for humans. The retention half-time in rabbit bone is substantially longer than for humans. The implications of extrapolating from animal data to effects on humans are discussed. PMID- 1727414 TI - Accidental overexposure to 192Ir source in industrial radiography: a follow-up study. AB - The consequences of an accidental overexposure from an 192Ir radiography source are being followed up to assess the long-term effects to the victim. The estimated dose equivalents resulting from the accident were approximately 24 Sv to the fingertips and 2-3 Sv to the body. Progressive tissue deterioration has set in and has led to multiple surgeries and successive amputations of the fingertips. PMID- 1727415 TI - Terrestrial gamma radiation dose in Hong Kong. AB - The first reactor of the Daya Bay nuclear power plant, 30 km from Hong Kong, is expected to begin operation in 1992. As a result, residents in Hong Kong are concerned with potential exposure to man-made and natural radiation. Therefore, the Radioisotope Unit of the University of Hong Kong performed studies to estimate Hong Kong's natural background radiation and the potential exposure to local residents, the results of which are presented here. PMID- 1727416 TI - Elevation correction factors for E-PERM radon monitors. AB - E-PERM radon monitors are based on the principle of electret ion chambers and are usually calibrated in a standard radon chamber located at sea level. Corrections are needed if the monitors are used at elevations other than sea level. These were experimentally determined for three models of commercially available electret ion chambers (E-PERM) as functions of elevation above sea level. These corrections are minor and should be applied for obtaining more accurate results. PMID- 1727417 TI - A multiyear quality control study of alpha-track radon monitors. AB - Quality control exposures of commercial alpha-track radon monitors have been conducted approximately weekly at the U.S. Department of Energy (DOE) Grand Junction Projects Office since early 1987 in support of DOE remedial action programs. The results of these exposures provide a historical record of the comparative performances of these radon monitors. PMID- 1727418 TI - Permeability of caulking compounds to 222Rn. AB - Caulking compounds have been tested as 222Rn barriers. Of these, the most effective is GE acrylic Latex Silicone Caulk, 0.2 cm of which attenuates 222Rn by 98% to 99.9%. The same thickness of two commercial 222Rn barriers attenuates radon by 75% to 90%. Permeabilities range from 0.3 to 140 x 10(-8) cm2 s-1. PMID- 1727419 TI - 222Rn in groundwater in Nigeria: a survey. AB - 222Rn in groundwater was measured in wells of different rock types in Nigeria by direct gamma counting. Concentrations ranged between 1.7 +/- 0.1 and 161.6 +/- 2.4 Bq L-1, with only slight variations (maximum 2.1 sigma from the mean) in a year in a typical well. PMID- 1727420 TI - Using a computer to manage the radiation safety and quality control activities in diagnostic radiology. AB - Software has been developed to aid in the management of a radiation safety quality control program in diagnostic radiology. The core of the system is a data base of radiation safety activities. Software design goals were the prioritization, scheduling, and reporting of these activities. Computerization has helped organize the timely performance of surveys necessary for compliance with regulatory and accreditation agencies. Clear, concise program documentation and reporting are also provided. PMID- 1727421 TI - The cerebral microvessels in culture, an update. AB - Recent advances in our knowledge of the blood-brain barrier (BBB) have in part been made by studying the properties and function of cerebral endothelial cells in vitro. After an era of working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro BBB model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results obtained from this rapidly growing field. It can be stated with certainty that, in addition to providing a better insight into the chemical composition of cerebral endothelial cells, much has been learned from these studies about the characteristics of transport processes and cell-to-cell interactions during the last 12 years. With the application of new technologies, the approach offers a new means of investigation, applicable not only to biochemistry and physiology but also to the drug research, and may improve the transport of substances through the BBB. The in vitro approach has been and should remain an excellent model of the BBB to help unravel the complex molecular interactions underlying and regulating the permeability of the cerebral endothelium. PMID- 1727422 TI - Effect of hydroperoxy fatty acids on acylation and deacylation of arachidonoyl groups in synaptic phospholipids. AB - The effect of hydroperoxy fatty acids on reactions involved in the acylation deacylation cycle of synaptic phospholipids was studied in vitro, using nerve ending fraction isolated from rat forebrain. 15-Hydroperoxyeicosatetraenoic acid (15-HPETE), 13-hydroperoxylinoleic acid (13-HP 18: 2), and hydroperoxydocosahexaenoic acid (22:6 Hpx), at 25 microM final concentration, all inhibited the incorporation of [1-14C]arachidonate into synaptosomal phosphatidylinositol (PI), phosphatidylcholine (PC), and triacylglycerides by 50 80%. The lowest effective concentration of 15-HPETE and 13-HP 18:2 resulting in significant inhibition of the reacylation of PI was 5 microM, whereas the inhibition of [1-14C]arachidonate incorporation into PC required 10 and 5 microM hydroperoxy fatty acids, respectively. Cumene hydroperoxide and tert-butyl hydroperoxide at concentrations of 100 microM did not inhibit reacylation of PI and PC. Synthesis of labeled arachidonoyl-CoA from [1-14C]arachidonate was decreased by about 50% by 25 microM hydroperoxy fatty acids both in synaptosomes and in the microsomal fraction. Use of [1-14C]arachidonoyl-CoA as a substrate, to bypass the fatty acid activation reaction, revealed that activity of acyltransferase was not affected significantly by 25 microM 15-HPETE and 13-HP 18:2. At the same time, however, the hydrolysis of labeled arachidonoyl-CoA was substantially enhanced. Exposure of synaptosomes to 25 microM fatty acid hydroperoxides did not affect significantly the endogenous concentrations of five major free fatty acids. It is concluded that (1) among synaptic phospholipids, reacylation of PI and PC is the most susceptible to the inhibitory action of fatty acid hydroperoxides, and (2) the enzymes affected by these compounds in nerve endings are arachidonoyl-CoA synthetase and hydrolase. PMID- 1727423 TI - Determination of acidic metabolites of biogenic amines in human aqueous humour by gas chromatography--negative ion chemical ionisation mass spectrometry. AB - The concentrations of acidic metabolites derived from the biogenic amines o-, m-, and p-tyramines (o-, m-, and p-hydroxyphenylacetic acids), p-octopamine/p synephrine (p-hydroxymandelic acid), and dopamine (homovanillic acid and 3,4 dihydroxyphenylacetic acid) were measured in human aqueous humour obtained from patients undergoing elective surgery for cataract removal or for trabeculectomy as a treatment for chronic open-angle glaucoma. There were no clear differences in the pattern of metabolism of neurotransmitters between the two groups. An unexpected finding was that the o-tyramine metabolite, o-hydroxyphenylacetic acid, was present in aqueous humour. PMID- 1727424 TI - Decarboxylation of exogenous L-3,4-dihydroxyphenylalanine in rat striatum as studied by in vivo voltammetry. AB - An in vivo voltammetric technique was used to determine whether striatal nondopaminergic neurons take up and decarboxylate exogenous L-3,4 dihydroxyphenylalanine (L-DOPA) and release it as dopamine. After the striatal serotonergic neurons of the rat had been destroyed by intraventricular injection of 5,7-dihydroxytryptamine, L-DOPA was administered intraperitoneally. It was found that changes in the dopamine concentration in the striatal extracellular fluid of the rat were the same as those in the nonlesioned rat. L-DOPA was also administered to the rat after the striatal perikarya had been destroyed by the intrastriatal injection of kainate. The striatal dopamine concentrations of the lesioned rat changed in parallel with 5,7-dihydroxytryptamine-lesioned rats, as well as the nonlesioned rats. Moreover, when normal rats were administered L DOPA, the dopamine concentration was not increased in the cerebellum, where dopamine neurons do not exist. From these observations, it is concluded that exogenous L-DOPA is taken up, decarboxylated to dopamine, and released only in the striatal dopamine neurons. PMID- 1727425 TI - Developmental and age-dependent changes of 28-kDa calbindin-D in the central nervous tissue determined with a sensitive immunoassay method. AB - For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4-5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727426 TI - Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate. AB - Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE. PMID- 1727427 TI - Purification of a synaptic membrane Na+/Ca2+ antiporter and immunoextraction with antibodies to a 36-kDa protein. AB - The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter. PMID- 1727428 TI - Increased intracellular gamma-aminobutyric acid selectively lowers the level of the larger of two glutamate decarboxylase proteins in cultured GABAergic neurons from rat cerebral cortex. AB - The regulation of glutamate decarboxylase (GAD; EC 4.1.1.15) was studied by using cultures of cerebral cortical neurons from rat brain grown in serum-free medium. About 50% of the neurons in the cultures were gamma-aminobutyric acid (GABA)ergic as determined by two double-staining procedures. Immunoblotting experiments with four anti-GAD sera that recognize the two forms to varying degrees, demonstrated that the cultures contained the two forms of GAD that are present in rat brain (apparent molecular masses = 63 and 66 kDa). GAD activity was reduced by 60-70% when intracellular GABA levels were increased by incubating the cultures with the GABA-transaminase inhibitor gamma-vinyl-GABA for greater than 5-10 h or with 1 mM GABA itself. Neither baclofen nor muscimol (100 microM) affected GAD activity. Immunoblotting experiments showed that only the larger of the two forms of GAD (66 kDa) was decreased by elevated GABA levels. These results, together with previous results indicating that the smaller form of GAD is more strongly regulated by pyridoxal 5'-phosphate (the cofactor for GAD), suggest that the two forms of GAD are regulated by different mechanisms. PMID- 1727429 TI - In vivo distribution of CGS-19755 within brain in a model of focal cerebral ischemia. AB - The blood-brain barrier permeability of the competitive N-methyl-D-aspartate receptor antagonist CGS-19755 [cis-4-(phosphonomethyl)-2-piperidine carboxylic acid] was assessed in normal and ischemic rat brain. The brain uptake index of CGS-19755 relative to iodoantipyrine was assessed using the Oldendorf technique in normal brain. The average brain uptake index in brain regions supplied by the middle cerebral artery was 0.15 +/- 0.35% (mean +/- SEM). The unidirectional clearance of CGS-19755 from plasma across the blood-brain barrier was determined from measurements of the volume of distribution of CGS-19755 in brain. These studies were performed in normal rats and in rats with focal cerebral ischemia produced by combined occlusion of the proximal middle cerebral artery and ipsilateral common carotid artery. In normal rats the regional plasma clearance across the blood-brain barrier was low, averaging 0.015 ml 100 g-1 min-1. In ischemic rats this clearance value averaged 0.019 ml 100 g-1 min-1 in the ischemic hemisphere and 0.009 ml 100 g-1 min-1 in the nonischemic hemisphere. No significant regional differences in plasma clearance of CGS-19755 were observed in either normal or ischemic rats except in cortex injured by electrocautery where a 14-fold increase in clearance across the blood-brain barrier was measured. We conclude that CGS-19755 crosses the blood-brain barrier very slowly, even in acutely ischemic tissue. PMID- 1727430 TI - Evidence for a hyperexcitability state of staggerer mutant mice macrophages. AB - We recently reported an abnormal production of interleukin-1 (IL-1) in peripheral macrophages of several neurological mutant mice that exhibit patterns of neuronal degeneration, especially in the cerebellum. After in vitro activation by lipopolysaccharide acid (LPS), these macrophages hyperexpress IL-1 beta mRNA and hyperproduce IL-1 protein in comparison with +/+ controls. In the present study, focused on the staggerer mutant mice, we investigate if this genetic dysregulation is specific for IL-1 beta or if it reflects a generalized hyperexcitability of these macrophages. The hyperexpression of IL-1 beta mRNA in sg/sg macrophages is present whatever the duration of LPS stimulation, even for periods as short as 15 min, although it reaches a maximum after 4 h of stimulation. The hyperinducibility of sg/sg macrophages is observed even when very low doses of LPS are used (0.01 microgram/ml) and reaches its maximum for 5 micrograms/ml LPS. Synthetic molecules (muramyl dipeptides), such as N acetylmuramyl-L-alanyl-D-isoglutamine or murabutide, known as macrophage activators, are also efficient in revealing the cytokine hyperexpression in sg/sg macrophages. In addition, hyperexpression of two other cytokines, i.e., tumor necrosis factor-alpha and IL-1 alpha mRNAs, is also detected in LPS-stimulated macrophages of mutant mice. Finally, the effect of an inhibitor of protein synthesis, cycloheximide, is similar in +/+ and sg/sg macrophages. As a whole, these data lead us to conclude that the sg/sg macrophages are in a state of general hyperexcitability when compared with +/+ ones. PMID- 1727431 TI - Extracellular concentration and in vivo recovery of dopamine in the nucleus accumbens using microdialysis. AB - The present study compared two different in vivo microdialysis methods which estimate the extracellular concentration of analytes at a steady state where there is no effect of probe sampling efficiency. Each method was used to estimate the basal extracellular concentration of dopamine (DA) in the nucleus accumbens of the rat. In the first method, DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 2.5, 5, and 10 nM) and DA is measured in the dialysate from the brain to generate a series of points which are interpolated to determine the concentration of no net flux. Using this method, basal DA was estimated to be 4.2 +/- 0.2 nM (mean +/- SEM, n = 5). The slope of the regression gives the in vivo recovery of DA, which was 65 +/- 5%. This method was also used to estimate a basal extracellular 3,4 dihydroxyphenylacetic acid (DOPAC) concentration in the nucleus accumbens of 5.7 +/- 0.6 microM, with an in vivo recovery of 52 +/- 11% (n = 5). A further experiment which extended the perfusate concentration range showed that the in vivo recovery of DA is significantly higher than the in vivo recovery of DOPAC (p less than 0.001), whereas the in vitro recoveries of DA and DOPAA are not significantly different from each other. The in vivo difference is thought to be caused by active processes associated with the DA nerve terminal, principally release and uptake of DA, which may alter the concentration gradient in the tissue surrounding the probe.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727432 TI - Comparative metabolism of fluorinated 3,4-dihydroxyphenylalanine isomers in aggregating brain cell cultures. AB - Fluorinated analogues of 3,4-dihydroxyphenylalanine (DOPA) were tested for intracellular metabolic conversion in aggregating cell cultures prepared from fetal rat brain. 5-Fluoro-D/L-DOPA was methylated almost exclusively to 3-O methyl-5-fluoro-D/L-DOPA. Metabolism of 6-fluoro-D/L-DOPA resulted in 6 fluorodopamine, 6-fluoro-3,4-dihydroxyphenylacetic acid, and 3-O-methyl-6-fluoro D/L-DOPA, but with a qualitatively and quantitatively different metabolite pattern compared with that of L-DOPA and D/L-DOPA, respectively. Homovanillic acid and fluorohomovanillic acid have not been found intracellularly in the cultures. On the basis of these data, the model development of the cerebral metabolism of tracers used in positron emission tomography can be improved. PMID- 1727433 TI - Carbon dioxide fixation in neuronal and astroglial cells in culture. AB - The concentration of potassium in the extracellular fluid has been found to stimulate the rate of CO2 fixation by astroglial cells grown in primary culture. Raising the concentration of extracellular potassium increased both the initial rate of formation of the 14C-labeled products of 14CO2 fixation and the final steady-state level of these products within the cells. In contrast, neither veratridine nor L-glutamate affected the rate of CO2 fixation in astroglial cells. The very low rate of CO2 fixation found in primarily neuronal cultures was unaffected by increased extracellular potassium as was CO2 fixation in fibroblasts. When cultured alone, astroglial cells release a large fraction of the 14C-labeled products of CO2 fixation into the surrounding medium. Mixed cultures of astroglia and neurons also fix CO2 but, in contrast to astroglia cultured alone, release only a small fraction of the 14C-labeled products into the culture medium. PMID- 1727434 TI - Relationship of intracellular calcium to dependence on nerve growth factor in dorsal root ganglion neurons in cell culture. AB - During development, neural crest-derived sensory neurons require nerve growth factor (NGF) for survival, but lose this dependency postnatally. Similarly, dissociated embryonic sensory neurons lose their NGF dependence during the first 3 weeks in cell culture. It has been hypothesized that, in sympathetic neurons, intracellular levels of calcium are related to trophic factor dependence. In vitro during the period in which embryonic-day-15 sensory neurons become independent of NGF, intracellular calcium concentrations progressively increased in parallel to the decline in NGF dependence. This elevation of intracellular calcium was directly related to the absolute age of the neurons, not to the length of time in culture. Without NGF, immature sensory, i.e., dependent, neurons survived in the presence of high extracellular potassium, a condition that produces elevated intracellular calcium. In another paradigm, measurements of intracellular calcium were determined in NGF-dependent neurons "committed to die" after NGF withdrawal. These measurements were determined prior to the time that extensive morphological changes, consistent with cell death, were noted by phase-contrast microscopy. No elevation in intracellular calcium was found in these dying neurons, but rather, a small decrease was observed prior to the disintegration of the neurons. These findings support the hypothesis that trophic factor dependence of neurons may be inversely related to levels of intracellular calcium. PMID- 1727435 TI - Stathmin phosphorylation is regulated in striatal neurons by vasoactive intestinal peptide and monoamines via multiple intracellular pathways. AB - Stathmin is a ubiquitous soluble protein whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell proliferation, differentiation, and functions by extracellular effectors. It is present in the various tissues and cell types as at least two distinct isoforms in their unphosphorylated (Mr approximately 19,000; pI approximately 6.2-6.0) and increasingly phosphorylated forms. Stathmin is particularly abundant in brain, mostly because of its high concentration in neurons, where the protein is a major phosphorylation substrate. In intact striatal neurons grown in primary culture, the cyclic AMP-increasing drug forskolin and the protein kinase C-activating agent 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a potent phosphorylation of stathmin. Their actions were at least partially additive, appearing actually most likely "sequential" on various phosphorylated states of stathmin. Vasoactive intestinal peptide (VIP) reproduced the forskolin-like stimulation but stimulated also other, TPA, and/or Ca2(+)-like protein phosphorylations. These actions of VIP were already maximal after 5 min and were long lasting, still important after 2 h. In addition, concentrations as low as 1 nM were enough to obtain a significant effect, on both cyclic AMP-dependent and independent phosphorylations. Dopamine and the beta-adrenergic agonist isoproterenol were also able to stimulate stathmin phosphorylation, but only with a forskolin-like pattern. Their actions were not additive to those of VIP, confirming previous results on the colocalization of both dopamine D1 and noradrenaline beta 1 receptors with VIP receptors on striatal neurons.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727436 TI - Inhibition of ischemia-induced dopamine release by omega-conotoxin, a calcium channel blocker, in the striatum of spontaneously hypertensive rats: in vivo brain dialysis study. AB - The effect of omega-conotoxin GVIA (CgTX), an N-and L-type voltage-sensitive calcium channel (VSCC) blocker, on the release of dopamine and 3,4 dihydroxyphenylacetic acid (DOPAC) in the striatum before and during transient cerebral ischemia in spontaneously hypertensive rats was studied using an in vivo brain dialysis technique. Continuous perfusion of CgTX in the striatum was started 20 min before ischemia and concentrations of dopamine and DOPAC in the dialysate were measured using HPLC with an electro-chemical detector. Before ischemia, both 10 and 100 microM CgTX significantly lowered the concentration of dopamine, to 49% of the basal values. DOPAC concentrations also decreased significantly, by 28 and 17%, respectively. Forebrain ischemia, produced by bilateral carotid artery occlusion, reduced striatal blood flow to less than 6% of the resting value in each group. During 20 min of ischemia, the vehicle group showed a marked increase in dopamine (175 times the basal concentration). In the 10 or 100 microM CgTX perfusion group, in contrast, dopamine release was significantly attenuated, to 38 or 29% of the vehicle group, respectively. DOPAC concentrations decreased during ischemia to 58% of the basal value in the vehicle group and 49% in both CgTX groups. These results indicate that the massive release of striatal dopamine during ischemia depends largely on the influx of extracellular calcium via CgTX-sensitive VSCCs. PMID- 1727437 TI - Transcription of the rat cholecystokinin gene is initiated at multiple sites: verification by an in vitro transcription system. AB - The cDNA that accommodates the most distal 5'-end region of cholecystokinin mRNA was isolated from an internally primed cDNA library. Using primer extension and S1 nuclease protection analyses, we demonstrated multiple RNA molecules generated from the rat cholecystokinin gene, a single-copy sequence. The longest RNA is transcribed at position--225 upstream relative to the translation start site. The major transcription, more than 95% of the total cholecystokinin mRNA in rat brain, occurred at--59 and its promoter activity was determined by in vitro RNA synthesis in a HeLa cell extract. Deletion to--105 demonstrated an approximately 60% decrease in transcriptional level compared with the full promoter activity. At least the upstream region between--254 and--105 is necessary for transcription initiated at--59 of the cholecystokinin gene by the cell-free system. PMID- 1727438 TI - Differential effects of presynaptic phospholipase A2 neurotoxins on Torpedo synaptosomes. AB - The effects of several phospholipase A2 neurotoxins from snake venoms were examined on purely cholinergic synaptosomes from Torpedo electric organ. The noncatalytic component A of crotoxin had no effect, whereas its phospholipase component B, used alone or complexed to component A, elicited a rapid and dose dependent acetylcholine (ACh) release and a depolarization of the preparation. Subsequent ACh release evoked by high K+ levels or calcium ionophore was identical to the control after the action of component A but reduced after the action of crotoxin or of component B. These effects were not observed when the phospholipase A2 activity of the toxin was blocked either by replacing Ca2+ by Ba2+ (respectively, activator and inhibitor of phospholipase A2) or by alkylation of component B with p-bromophenacyl bromide. beta-Bungarotoxin, another very potent phospholipase A2 neurotoxin, induced release of little ACh, did not affect ionophore-evoked ACh release, but significantly reduced depolarization-induced ACh release. The single-chain phospholipase A2 neurotoxin agkistrodotoxin behaved like crotoxin component B. A nonneurotoxic phospholipase A2 from mammalian pancrease induced release of an amount of ACh similar to that released by crotoxin but did not affect the evoked responses. The obvious differences in effect of the various neurotoxins suggest that they exert their specific actions on the excitation-secretion coupling process at different sites or by different mechanisms. PMID- 1727439 TI - Phosphorylation of glial fibrillary acidic protein and vimentin by cytoskeletal associated intermediate filament protein kinase activity in astrocytes. AB - These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727441 TI - Design of an affinity matrix for purification of the histamine H1 receptor from guinea pig cerebellum. AB - H1 receptors from guinea pig cerebellum were solubilized using digitonin, and [125I]iodobolpyramine was used as a probe. [125I]Iodobolpyramine binding to this solubilized preparation occurred with a KD of 0.1 nM and a Bmax of 220 fmol/mg of protein and was inhibited by various H1 ligands with the expected potencies. Using a gel filtration procedure, a very sensitive radioassay was set up for detecting H1 activity in the solubilized preparation: 0.1 nM [125I]iodobolpyramine specific binding represented greater than 90% of total binding. Moreover, the synthesis is described of potent H1 antagonists that are mepyramine derivatives with an amino alkyl acylamido alkyl spacer arm. One of them, UCL 1057 (Ki = 0.5 nM), has been coupled to a Sepharose epoxy-activated resin. The resulting affinity matrix adsorbed selectively [125I]iodobolpyramine binding sites from the guinea pig cerebellum soluble preparation. In contrast, a Sepharose-glycine matrix was not able to adsorb these sites. PMID- 1727440 TI - 21-aminosteroids interact with the dopamine transporter to protect against 1 methyl-4-phenylpyridinium-induced neurotoxicity. AB - U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4 phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U 78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma [3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter. PMID- 1727442 TI - Different natures of supersensitivity of adenylate cyclase stimulated by calcitonin gene-related peptide and isoproterenol in rat diaphragm after denervation and reserpine treatment. AB - In skeletal muscles, calcitonin gene-related peptide (CGRP) released from motor nerve terminals and humoral catecholamines stimulate adenylate cyclase (AC) and enhance muscle contraction. The effects of denervation and treatment with reserpine on twitch contraction and the AC system in rat diaphragm were investigated. The basal levels of twitch contraction and AC activity of the diaphragm of rats were both increased 2 weeks after phrenic nerve denervation but were not altered by treatment with reserpine. Reserpine treatment provoked supersensitivity of AC to isoproterenol, without affecting the response to CGRP. On the other hand, denervation decreased the activation of AC and enhancement of twitch contraction by CGRP, without affecting the responses to isoproterenol. These data suggest that denervation causes up-regulation of AC as a result of loss of CGRP release from nerve terminal and that depletion of catecholamines by reserpine treatment supersensitizes the responses at the beta-adrenoceptor level. Thus, nervous and humoral factors regulate the AC system in striated muscle by different mechanisms. PMID- 1727443 TI - Inhibition of brain glutamate decarboxylase activity is related to febrile seizures in rat pups. AB - Because previous work showed that in the newborn brain, but not in the adult brain, glutamate decarboxylase (GAD) is notably susceptible to heat, we have studied the possible involvement of GAD inhibition in febrile convulsions and the related changes in gamma-aminobutyric acid (GABA) content. Rats of different ages were subjected to hyperthermia, and GAD activity was determined in brain homogenates by measuring the release of 14CO2 from labeled glutamate and by measuring the formation of GABA. The latter method gave considerably lower values than the former in the youngest rats, and was considered more reliable. With this method, we found a 37-48% inhibition of GAD activity in rat pups 2-5 days old, which showed febrile seizures at progressively higher body temperatures, whereas in 10- and 15-day-old animals, which did not show convulsions, GAD activity was not affected by hyperthermia. Whole-brain GABA levels, however, did not change at any age. In contrast to GAD, choline acetyltransferase and lactic dehydrogenase activities were not altered by hyperthermia at any of the ages studied. These results suggest that a decreased efficiency of the inhibitory neurotransmission mediated by GABA, consequent to the inhibition of GAD activity, may be a factor related to febrile convulsions. PMID- 1727444 TI - Differential metabolism of hydroxyeicosatetraenoic acid isomers by mouse cerebromicrovascular endothelium. AB - Hydroxyeicosatetraenoic acid (HETE) derivatives of arachidonic acid are produced in the brain and have been implicated as pathologic mediators in various types of brain injury. To understand better their fate in the brain, particularly in cerebral microvessels, several HETEs were incubated with cultured mouse cerebromicrovascular endothelium for 1, 2, and 4 h, followed by HPLC analysis of medium and cellular lipids. 5(S)-, 8(RS)-, and 9(RS)-HETE were not metabolized by the cells, but were extensively incorporated, unmodified, into cell lipids. On the other hand, 11(RS)-, 12(S)-, and 15(S)-HETE were extensively metabolized and only minimally incorporated into cell lipids. Previously, the major 12-HETE metabolite was identified as 8-hydroxyhexadecatrienoic acid. In the present study, we identified the major 11-HETE metabolite as 7-hydroxyhexadecatrienoic acid and the major 15-HETE metabolite as 11-hydroxyhexadecatrienoic acid. omega-3 compounds, 15(S)- and 12(S)-hydroxyeicosapentaenoic acids (HEPE), were also metabolized to more polar compounds, but to a lesser extent than their tetraenoic acid, omega-6 counterparts. Comparison of 5-, 12-, and 15-HETE enantiomers revealed no differences in metabolism or incorporation between the R and S stereoisomers. These data suggest that many isomers of HETE and HEPE can be incorporated into cell lipids or metabolized by pathways that do not distinguish between enantiomers. These pathways, however, are sensitive to the position or number of double bonds and are selective based on the position of the hydroxyl group. PMID- 1727445 TI - Induction of interleukin-1 beta mRNA in rat brain after transient forebrain ischemia. AB - The expression of interleukin-1 beta (IL-1 beta) mRNA in the cerebral cortex, hippocampus, striatum, and thalamus of rats was studied after transient forebrain ischemia. IL-1 beta mRNA was not detected in all these regions of sham-operated control rats. IL-1 beta mRNA was induced after transient forebrain ischemia and reached a detectable level in all regions examined 15 min after the start of recirculation. The induction of IL-1 beta mRNA had a few peaks, that is, peaks were observed at 30 and 240 min in the four regions examined, and another peak was observed at 90 min in the striatum. One day after the start of recirculation, IL-1 beta mRNA levels were markedly decreased, but even 7 days after that, IL-1 beta mRNA was found at very low levels in all regions examined. The amounts of c fos and beta-actin mRNAs on the same blots were also examined. The induction of c fos mRNA was transient and had only one peak in all regions examined, whereas the levels of beta-actin mRNA in these regions were fairly constant throughout the recirculation period. Thus, we provide the first evidence for a characteristic expression of IL-1 beta mRNA in several brain regions after transient forebrain ischemia. PMID- 1727446 TI - Neuraminidase activities in oligodendroglial cells of the rat brain. AB - Neuraminidase activities in oligodendroglial cells were characterized using rats of different ages. Rat oligodendroglial cells had intrinsic neuraminidase activities directed toward GM3 and N-acetylneuramin(2-3)lactitol (NL). Developmental profiles of the neuraminidase activities toward the two substrates in oligodendroglial cells were different from each other. The neuraminidase activity toward GM3 increased rapidly with the onset of active myelination and, after 26 days of development, reached the adult level which was about 18 times higher than that in myelin. At the adult age, oligodendroglial cells had the highest neuraminidase activity toward GM3 among the individual brain cell types examined. The activity of NL-neuraminidase showed a less remarkable developmental profile, with a peak value at 26 days. The UDP-galactose:ceramide galactosyltransferase activity in oligodendroglial cells increased during the period of active myelination and, afterward, returned to the basal level. The enrichment and unique developmental profile in oligodendroglial cells of the neuraminidase activity toward GM3 suggest that this enzyme may play an important role in the formation and maintenance of the myelin sheath. PMID- 1727447 TI - Role of myelin-associated neuraminidase in the ganglioside metabolism of rat brain myelin. AB - The role of myelin-associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di- and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di- and polysialo-species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin-associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin. PMID- 1727448 TI - Characterization of the serpin, alpha 1-antichymotrypsin, in normal human cerebrospinal fluid. AB - Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1 antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727449 TI - Who's really at risk for AIDS? PMID- 1727450 TI - Informed consent for the dual degree. PMID- 1727451 TI - Problems facing our specialty. PMID- 1727452 TI - Walter Guralnick to receive 1992 Donald B. Osbon Award for outstanding educator. PMID- 1727453 TI - Leonard Kaban to receive Foundation's 1992 Research Recognition Award. PMID- 1727454 TI - Ampicillin concentrations in human radicular granuloma following a single oral dose of bacampicillin. AB - Ampicillin concentrations in human serum and radicular granulomas of 42 patients were determined after a single oral dose of bacampicillin (equivalent to 500 mg of ampicillin). Although wide variations were found among both serum and radicular granuloma ampicillin concentrations, measurable concentrations were found in all cases. The mean peak ampicillin concentrations in serum and radicular granulomas occurred at identical times, 1.5 hours, and were 11.19 micrograms/mL (range, 1.30 to 21.00 micrograms/mL) and 5.12 micrograms/g (range, 0.50 to 10.50 micrograms/g), respectively. The mean radicular granuloma/serum ampicillin concentration ratio at the peak time was 0.42. Ampicillin concentrations in radicular granulomas exceeded most of the minimum inhibitory concentrations for bacteria commonly isolated from odontogenic infections. PMID- 1727455 TI - Evaluation of mandibular reconstruction techniques following resection of malignant tumors in the oral region. AB - Over the past 15 years, reconstruction following excision of malignant oral tumors was performed on 27 patients with segmental resection and five patients with hemiresection of the mandible. Following segmental resection, the mandible was reconstructed using an autogenous bone graft in eight patients in whom the surrounding soft tissues were fairly well preserved. Bony union was achieved in six of them. In the remaining two, the graft was removed because of postoperative infection, and one patient underwent secondary bone grafting. A pedicled myocutaneous flap and bone graft was used in seven patients who underwent extensive resection of the surrounding soft tissue. Bony union was achieved in three patients, and one developed pseudoarthrosis. The graft was removed in the remaining three because of postoperative infection. Reconstruction with only a metallic plate for stabilization of the mandible was carried out in six aged or sarcoma-affected patients. In two of them, the postoperative course was uneventful for 4 to 7 years. In the remaining four patients, plate removal was required because of exposure or tumor recurrence. In 5 of 11 patients in whom reconstruction was carried out with a combination of a pedicled myocutaneous flap and metallic plate, the postoperative course was uneventful for 2 to 8 years. Two of these five patients underwent secondary bone grafting. In four of the remaining six patients, the plate was removed because of exposure or improper adaptation to the stump. Two others died of disseminated intravascular coagulation syndrome within 1 month. A prosthesis was used more frequently by patients when reconstruction was performed using a pedicled osteomyocutaneous flap. The metallic reconstruction plate was helpful for restoring mandibular contour.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727456 TI - Three-dimensional visualization of the temporomandibular joint: a computerized multisectional autopsy study of disc position and configuration. AB - To demonstrate disc position and disc configuration of the temporomandibular joint, three-dimensional multisectional computer reconstructions were made of 20 autopsy specimens (11 female, 9 male; mean age, 40.4 years). The presence of a distinct occlusion was the only criterion for selection. Normal disc position was found in 13 joints, partial anterior disc position was found in 5 joints, and complete anterior disc position was seen in 2 joints. Fifteen joints had biconcave disc configuration and 5 joints had deformed discs. Considering the high incidence of disc position deviating from the normal superior position, it is suggested that in some cases a so-called abnormal disc position can be regarded as within the limits of anatomic and physiological variability. PMID- 1727458 TI - The third molar as a cause of deep space infections. AB - An 81-month review of patients with deep space infections attributed to third molars requiring hospital admission is presented. Thirty-one patients were identified, with males predominating 2:1 and mandibular third molars as an etiology predominating 15:1. All patients were aged 23 years or older. Most patients identified (24) had one or more medical problems or other risk factors, the most frequent of which was smoking (18). All patients developing postoperative infections (9) had complete or partial bone impactions and a preoperative diagnosis of pericoronitis. PMID- 1727457 TI - Information seeking and interactive videodisc preparation for third molar extraction. AB - Patient response to interactive videodisc preparation for third molar extraction surgery was examined as a function of self-reported information-seeking style. Amount learned was compared among patients informed via an interactive videodisc, noninteractive videotape of the same material, or surgeon only. Anxiety levels and satisfaction with preparation were compared between the videodisc and videotape groups. At consultation, patients (n = 35) were randomly assigned to either the disc- or the tape-viewing group. First, subjects completed a demographic survey, state anxiety scale, quiz on knowledge about third molars and surgery risks and complications, and information-seeking scales. Immediately after viewing the video, subjects completed another anxiety scale and a multiple choice quiz covering the material presented. Subsequently, another 25 patients undergoing the routine (surgeon-only) consultation procedure were given the same multiple-choice quiz following consultation. Quiz scores differed significantly among the groups; mean percent correct for the tape-viewing subjects was 85; for disc-viewing subjects 72.6; for surgeon-only subjects, 40. Self-rated information seeking was unrelated to amount of video viewed by disc subjects (on average, 64% of the videodisc was viewed), and disc subjects who rated themselves higher in information-seeking achieved the lowest postpreparation quiz scores. Subjects in the disc group were significantly more satisfied with the amount of preparation than the tape group. Although disc group subjects were significantly less knowledgeable following consultation than were tape group subjects, interactive videodisc preparation for third molar extraction appears to have some advantages over more traditional approaches. Further research is needed to determine whether this approach to preparing patients is suitable for widespread clinical use. PMID- 1727459 TI - Utility of capnography in predicting venous carbon dioxide partial pressure in sedated patients during outpatient oral surgery. AB - Twelve ASA class I patients scheduled for removal of third molars under intravenous sedation were included in the study. Samples for venous blood gas analysis were drawn every 5 minutes and the venous partial pressure of carbon dioxide was compared to the end-tidal CO2 recorded from a modified nasal cannula at the same time the samples were drawn. Correlation analysis was performed using the Pearson correlation coefficient. The overall correlation between end-tidal CO2 and PVCO2 was .54 (P = .0001). The results of the investigation indicate that through simple modifications of the end-tidal CO2 monitoring device, the correlation between end-tidal CO2 and serum PCO2 in a nonintubated patient can be improved. PMID- 1727460 TI - Effect of chitosan on lingual hemostasis in rabbits with platelet dysfunction induced by epoprostenol. AB - Chitosan, a complex carbohydrate derivative of shellfish exoskeleton, is shown to enhance lingual hemostasis in rabbits treated with a known antagonist of platelet function, epoprostenol (prostacyclin or PGI2). Bleeding times were measured for bilateral (15 mm x 2 mm) tongue incisions in 10 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan and the other was treated with control vehicle without chitosan. Extraoral bleeding and coagulation times were measured for each animal before, during, and after infusion of epoprostenol. Continuous infusion of epoprostenol increased mean systemic bleeding time 95%. In this platelet dysfunction animal model, lingual incisions receiving the experimental substance showed a 56% improvement in bleeding time in comparison with lingual incisions receiving control solution (P = .003). PMID- 1727461 TI - Comparison of torque measurements between cortical screws and emergency replacement screws in the cadaver mandible. AB - The so-called emergency screws are designed for use in a drill hole where the conventional screw has failed following a stripped bone thread. It is not known, however, if these screws are as well anchored in bone as cortical screws, nor is it known how well they secure contact to the previously damaged screw bed. On the basis of torque measurements in mandibles of fresh cadavers, emergency screws were compared with cortical screws. Further, the contact between emergency screw and bone was investigated on interfacial sections after embedding in methyl methacrylate resin. The maximum torque reached during insertion of the AO emergency screws was about 70% of the value measured with 2.7-mm conventional cortical screws and 67% of this value when the 3.2-mm emergency screws were used. While the emergency screws had good contact to the bone in the thread crest area, no bone contact was evident at the bottom of thread groove. This experimental situation can not be entirely extrapolated to the clinical situation. However, the study seems to suggest that emergency screws do not fully meet the demands of rigid internal fixation. PMID- 1727462 TI - A survey of oral and maxillofacial surgeons concerning their knowledge, beliefs, attitudes, and behavior relative to parameters of care. AB - In December 1990, a survey was sent to 1,296 randomly selected members of the American Association of Oral and Maxillofacial Surgeons (AAOMS) to determine their previous experience with standards and criteria of care, their type of practice, the educational methods that influence their professional decisions, and their attitudes about the development and use of parameters of care. A 55.7% response was obtained. This article reports the results of this survey. The average age of responding surgeons was 45 years and they had been in practice an average of 15 years. The majority were in private practice, had hospital staff privileges, worked between 31 and 55 hours per week, spent 90% of their working week in direct patient care, and devoted an average of 9.16 hours per month to professional affairs outside of their practice. Forty-two percent (42%) of the practitioners were in solo practice, whereas 50% practiced in groups. Surgeons concentrated 65% of their patient care time on dentoalveolar surgery and a significant number planned increases in practice activity in implant, orthognathic, and temporomandibular joint surgery. They learned new clinical skills in various ways, there being a difference between the most convenient and effective methods of learning. A majority of surgeons had been involved with quality assurance activities in the past 5 years. They were predominantly favorable to parameters now and when they first learned about them, but few thought they had a clear understanding about how parameters of care would be used.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727463 TI - Painful mass of the palate. PMID- 1727464 TI - Calcification of the cricoid cartilage mistaken for a foreign body: report of a case. PMID- 1727465 TI - Grooved palate associated with prolonged use of orogastric feeding tubes in premature infants. PMID- 1727466 TI - Squamous cell carcinoma of the oral cavity after irradiation for nonmalignant lesions: report of four cases. PMID- 1727467 TI - Resorption of the mandibular angle in progressive systemic sclerosis: case report. PMID- 1727468 TI - Transient facial nerve palsy following orthognathic surgery: a case report. PMID- 1727469 TI - Osteoporotic bone marrow defect of the mandible: report of a case diagnosed by computed tomography scanning. PMID- 1727470 TI - Simultaneous reconstruction of maxillary and nasal deformity in a patient with Binder's syndrome (maxillonasal dysplasia). PMID- 1727471 TI - Facial width problems associated with rigid fixation of mandibular fractures: case reports. PMID- 1727472 TI - A semirigid stent for use after auricular cartilage graft harvest. PMID- 1727473 TI - UV activation of human immunodeficiency virus gene expression in transgenic mice. AB - Human immunodeficiency virus (HIV) infection is associated with a clinical latency of as long as 10 years before the development of disease. One explanation for this delay is the requirement of cofactors such as other DNA or RNA viruses, cytokines critical for immune modulation, or environmental UV light. At least in tissue culture studies, these agents are capable of inducing HIV gene expression in cell lines which either harbor the entire viral genome or contain a reporter gene under the control of the viral long terminal repeat regulatory region. The role of these cofactors in terminating clinical latency and inducing disease has been difficult to ascertain because of the lack of an appropriate animal model. We now report that UV light can markedly induce HIV gene expression in transgenic mice carrying both the cis-acting (long terminal repeat) and trans-acting (the tat gene) elements which are essential for viral transactivation and replication in infected cells. Our finding may explain the clinical observations that cutaneous lesions in HIV-infected individuals are often seen in the sunlight exposed areas of the skin, including the face and neck. PMID- 1727474 TI - In vitro selection of variants of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine. AB - Drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) have been isolated by in vitro selection. MT-4 cells were infected with either a laboratory strain (HIV-IIIB) or a clinical isolate (no. 187) of HIV-1 and maintained in medium containing subeffective concentrations of the drugs 3'-azido-3' deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). By gradually increasing the drug concentration in the culture medium during propagation of the virus on fresh MT-4 cells, we were able to isolate variants of HIV-IIIB and clinical isolate 187 which showed up to 100-fold increases in resistance to the drugs. The drug resistance phenotypes remained stable after propagation of the variants in the absence of drug pressure for over 2 months. However, variants resistant to one drug showed little or no cross-resistance to the other, suggesting that the genetic bases for resistance to the compounds differed. Genotypic analysis of these nucleoside-resistant variants by polymerase chain reaction (PCR) with primer pairs previously shown to correspond to mutations responsible for resistance to AZT was also carried out. A heterogeneity of genotypes was observed, with known mutations at pol codons 70 and 215 occurring in most of the AZT-resistant variants generated from either HIV-IIIB or clinical strain 187. However, mutations in codons 67 and 219 were less frequently detected, and none of these changes were observed in each of four variants resistant to ddI. Cloning and sequencing studies of the reverse transcriptase coding region of two of the isolates were also performed and confirmed the PCR data that had been obtained. In addition to previously described mutation sites responsible for resistance to AZT, an HIV-IIIB-resistant variant was shown to be mutated at positions 108 (Val- --Ala) and 135 (Ile----Thr), while a resistant variant of strain 187 was mutated at positions 50 (Ile----Val) and 135 (Ile----Val). PMID- 1727475 TI - Kinetics of soluble CD4 binding to cells expressing human immunodeficiency virus type 1 envelope glycoprotein. AB - The high-affinity interaction between the envelope glycoprotein (gp120-gp41) of the human immunodeficiency virus type 1 and its receptor, CD4, is important for viral entry into cells and therapeutical approaches based on the soluble form of CD4 (sCD4). Using flow cytometry, we studied the kinetics of binding of sCD4 to gp120-gp41 expressed on the cell surface. sCD4 binding was dependent on sCD4 concentration and temperature and exhibited bimolecular reaction kinetics. Binding was very slow at low sCD4 concentrations (below 0.2 micrograms/ml) and low temperatures (below 13 degrees C) but increased sharply with increasing temperature. The rate constant for association at 37 degrees C (1.5 x 10(5) M-1 s 1) was 14-fold higher than at 4 degrees C, but the affinity of sCD4 to membrane bound gp120-gp41 was not significantly affected. The activation energy at higher temperatures (28 to 37 degrees C) was less than at lower temperatures (4 to 13 degrees C). After long periods of incubation, we observed a decrease of surface bound sCD4 and gp120, even at low temperatures, which was attributed to sCD4 induced shedding of gp120. The rate of gp120 shedding was much lower than the rate of sCD4 binding and was dependent on sCD4 concentration and temperature. The finding that sCD4 binding is slow, especially at low sCD4 concentrations, can be of critical importance for efficient blocking of viral infection by sCD4 and should be considered when designing new protocols in the therapy of AIDS patients. PMID- 1727476 TI - Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat. AB - We have analyzed the contributory role of the human immunodeficiency virus type 1 (HIV-1) promoter and enhancers in basal and Tat-induced transcription. We found that a minimal promoter competent for basal expression is contained within sequences spanning nucleotides -43 to +80. Basal expression from this HIV-1 promoter was boosted more by the additional presence of the NF-kappa B elements than by the Sp1 elements. The minimal long terminal repeat promoter (-43 to +80), while having an intact TAR sequence, was not Tat inducible. However, the simple addition of short synthetic enhancer motifs (AP1, Oct, Sp1, and NF-kappa B) conferred Tat responsiveness. This ability to respond to Tat was in part dependent on the presence of the HIV-1 promoter. Changing the HIV-1 TATA to other eucaryotic TATA or non-TATA initiators minimally affected basal expression but altered Tat inducibility. Our findings suggest a specific context of functional promoter and enhancer elements that is optimal for Tat trans activation of the HIV-1 long terminal repeat. Our results do not allow conclusions about whether Tat acts at the level of initiation or at the level of elongation to be drawn. PMID- 1727477 TI - Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. AB - The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host. PMID- 1727478 TI - Definition of functional domains in P135gag-myb-ets and p48v-myb proteins required to maintain the response of neuroretina cells to basic fibroblast growth factor. AB - The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus. PMID- 1727479 TI - Generation of deletion mutants of simian immunodeficiency virus incapable of proviral integration. AB - Deletion mutants of simian immunodeficiency virus (SIVmac) which were unable to integrate into host cells were generated by removing a portion of the integrase (IN) domain of the pol gene. The resulting plasmid was transfected into HUT-78 and human rhabdomyosarcoma cells. In comparison with the parental plasmid DNA transfected in parallel, the deletion mutant was found to direct efficient production of virus in both cell systems. Viruses derived from wild-type and mutant proviral DNAs were also tested for their relative replicative abilities in HUT-78 and U937 cells, and the kinetics of virus production was found to vary between these two cell systems. Analysis of DNA from infected cell nuclei showed that the deletion mutant lacked the ability to integrate despite being able to produce infectious virus. Using the sensitive polymerase chain reaction technique, we have clearly demonstrated the absence of the IN domain in the deletion mutant after infection and replication in HUT-78 cells. Such mutants might form the basis for the development of an experimental live attenuated vaccine. PMID- 1727480 TI - Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons. AB - The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2 3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates. PMID- 1727481 TI - Insertional inactivation of the vaccinia virus 32-kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells. AB - The mechanism of poxvirus attachment to cells is poorly understood. We have identified a 32-kDa envelope protein of vaccinia virus which binds to the surface of cultured cells. This binding is specific and selective (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:22174-22180, 1990; C. Lai, S. Gong, and M. Esteban, J. Virol. 65:499-504, 1991). In this investigation, we studied the effect of inactivating the 32-kDa gene (32K gene) on the biology of vaccinia virus. We show that inactivation of the 32K gene decreases by 80% the mortality of mice infected with 32K- vaccinia virus. This reduction in mortality correlates with diminished viral gene expression in target tissues. In highly polarized epithelial cells, viral gene expression of 32K- virus was reduced (50 to 60%) at both the apical and basolateral surfaces in comparison with a 32K+ virus. Restriction of virus gene expression in polarized cell surfaces occurs for both intracellular and extracellular forms of infectious 32K- vaccinia virus. The two infectious forms of vaccinia virus 32K+ infect polarized cells preferentially by the basolateral surface. Our findings provide evidence of the importance of the 32-kDa protein in viral pathogenesis. PMID- 1727482 TI - Construction of a transducing virus from double-stranded RNA bacteriophage phi6: establishment of carrier states in host cells. AB - Bacteriophage phi 6 contains three double-stranded RNA (dsRNA) genomic segments. We have constructed a plasmid that contains a cDNA copy of the middle (M) segment, with a gene for kanamycin resistance (kan) inserted into the PstI site. A transcript of this cDNA was incorporated in vitro into procapsids along with natural transcripts of the S and L segments. The procapsids were coated with nucleocapsid surface protein P8 and transfected into Pseudomonas syringae pv. phaseolicola. The resulting infectious virus, phi 6 K1, was found to contain an M segment that was 1.2 kbp larger than the normal 4.1 kbp. K1 formed small, turbid plaques, and its genome was unstable. Preparations of K1 contained from about 0.1 to 10% large, clear-plaque forms of the virus which were usually missing the kan gene, and in some cases, the resulting segment M was smaller than its normal size. Cells picked from lawns of host cells infected with K1 yielded colonies that were resistant to kanamycin (Kan). These colonies could be passaged on kanamycin-containing medium. The cells were found to contain large amounts of dsRNA corresponding to the viral genomic segments. Some strains continued to produce viable phage, while others lost this ability. One strain completely lost the small genomic segment S. Approximately 1 in 10,000 infected cells acquired the carrier state with the original phage isolate K1. However, we isolated a viral mutant that was able to induce the carrier state in 10 to 20% of the infected cells. The ability to use drug resistance as a test for the carrier state makes this system very useful for the study of the mechanisms of induction of persistent infections. PMID- 1727483 TI - Evidence for intracellular down-regulation of the epidermal growth factor (EGF) receptor during adenovirus infection by an EGF-independent mechanism. AB - We have reported previously that human group C adenoviruses down-regulate the epidermal growth factor (EGF) receptor (EGF-R) (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). Expression of a 13.7-kDa protein encoded by a gene in the E3 transcription unit is necessary and sufficient for this effect (Carlin et al., Cell, 1989; B. L. Hoffman, A. Ullrich, W. S. M. Wold, and C. R. Carlin, Mol. Cell. Biol. 10:5521-5524, 1990). We show here that EGF-R down-regulation is accelerated in cells which overexpress the receptor when these cells are infected with virus mutants that overproduce the 13.7-kDa protein compared with wild-type virus. This is in contrast to EGF stimulation, for which others have shown that high concentrations of ligand are associated with low rates of receptor internalization in EGF-R-overexpressing cells (D. Kuppuswamy and L. J. Pike, J. Biol. Chem. 264:3357-3363, 1989; H. S. Wiley, J. Cell Biol. 107:801-810, 1988). We also show that the E3 protein is not present in media conditioned by infected cells and that it does not induce secretion of an EGF-like autocrine factor. Moreover, while mature membrane-bound EGF-R is down-regulated, the precursor of the membrane-bound form is not. Adenovirus infection also does not affect receptor-related molecules expressed in the secretory pathway. Interestingly, adenovirus-induced down-regulation is not regulated by concentrations of EGF associated with a slow rate of internalization in A431 cells. This suggests that 13.7-kDa protein expression triggers receptor entry by a novel ligand-independent pathway or, alternatively, that it compensates for a cellular factor that may be rate limiting during EGF-mediated endocytosis. PMID- 1727484 TI - Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b. AB - The nonconditional RNA packaging mutant SE21Q1b contains cis- and trans-acting defects which cause cellular mRNA, rather than viral genomic RNA, to be nonspecifically packaged into SE21Q1b viral particles. Using genomic libraries of the c-SE21Q1b quail cell line, we have been able to construct a molecular clone of the SE21Q1b provirus. Upon transfection into primary quail embryo fibroblasts, the SE21Q1b molecular clone is able to recapitulate the nonspecific RNA packaging phenotype of the c-SE21Q1b cell line. The RNA packaging phenotypes displayed by several SE21Q1b/avian sarcoma-leukemia virus hybrid provirus constructs have further indicated that sequences responsible for the altered RNA packaging phenotype of SE21Q1b are localized in the left third of the SE21Q1b proviral genome. DNA sequence analysis of this region has revealed that the 5' SE21Q1b deletion has removed 179 bp from the SE21Q1b left long terminal repeat and leader regions. Several differences were detected at the carboxyl terminus of the deduced SE21Q1b nucleocapsid protein sequence in comparison with that of Rous sarcoma virus PR-C. Results of site-directed oligonucleotide mutagenesis experiments indicate, however, that the presence of these residues in the nucleocapsid protein alone is not responsible for the decreased RNA packaging specificity of SE21Q1b. PMID- 1727485 TI - Pathogenesis of early and late disease in mice infected with Theiler's virus, using intratypic recombinant GDVII/DA viruses. AB - Intratypic recombinant Theiler's viruses prepared between GDVII and DA strains were used to identify genomic sequences important in neurovirulence, virus persistence, and demyelination and to clarify the mechanisms involved in disease induction. The coding region between 1B and 2C of the highly virulent GDVII strain contains a determinant partly responsible for neurovirulence (early paralysis and death) which correlates with elevated levels of infectious virus and the presence of virus antigen within neurons of the brain stem and gray matter of the spinal cord. Both the GDVII and the DA strains of virus contain genetic determinants for late demyelination in spinal cord. However, quantitative analysis of demyelination produced by recombinant GDVII/DA viruses suggest that multiple gene segments influence the number and extent of demyelinating lesions. PMID- 1727486 TI - Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes. AB - Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes. PMID- 1727487 TI - Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates. AB - Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4 induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins. PMID- 1727488 TI - Regulation of human immunodeficiency virus enhancer function by PRDII-BF1 and c rel gene products. AB - The human immunodeficiency virus (HIV) enhancer element is important in the regulation of HIV gene expression. A number of cellular proteins have been demonstrated to bind to the NF-kappa B motifs in this element. The genes encoding several of these proteins, including members of the rel family and PRDII-BF1, have been cloned. We characterized the binding of proteins encoded by the human c rel and PRDII-BF1 genes to HIV NF-kappa B motifs and related enhancer elements. Both the human c-rel protein and two proteins derived from the PRDII-BF1 gene by alternative splicing bound specifically to the HIV NF-kappa B motif and related enhancer elements found in the immunoglobulin kappa, class I major histocompatibility complex, and interleukin-2 receptor genes. To determine the role of these factors in regulating HIV gene expression, we fused the cDNAs encoding either of the two proteins derived by alternative splicing of the PRDII BF1 gene or the c-rel gene to the DNA binding region of the yeast transcription factor GAL4. GAL4 binding sites were inserted in place of the native HIV enhancer sequences in an HIV long terminal repeat chloramphenicol acetyltransferase construct. Cotransfection of these constructs revealed that c-rel was a strong activator of basal HIV gene expression but did not result in synergistic effects in the presence of tat. PRDII-BF1-derived cDNAs did not result in stimulation of either basal or tat-induced activated gene expression. These results indicate that multiple enhancer binding proteins may potentially regulate HIV in both a positive and negative manner. PMID- 1727489 TI - The cellular proto-oncogene product Myb acts as transcriptional activator of the long terminal repeat of human T-lymphotropic virus type I. AB - The proto-oncogene c-myb encodes a nuclear transcription factor that binds to DNA in a sequence-specific manner and activates transcription of several viral and cellular genes. Expression of the c-myb gene is induced in mitogen- and/or antigen-stimulated T lymphocytes, which are also the preferential target cells of human T-lymphotropic virus type I (HTLV-I) in vivo and in vitro. We report here that Myb binds to the HTLV-I long terminal repeat (LTR) in four different regions in a sequence-specific manner. Electrophoretic mobility shift assay using labeled LTR fragments as well as labeled double-stranded oligonucleotides show that there are two high-affinity and two low-affinity Myb-binding sites present in the HTLV I LTR. DNase I footprinting analysis and oligonucleotide competition experiments indicate that this binding is sequence specific. Cotransfection experiments in HeLa cells, using a Myb expression vector and chloramphenicol acetyltransferase reporter gene linked to the HTLV-I LTR, show that Myb activates HTLV-I LTR mediated transcription by a factor of four-to sixfold. Thus, in HTLV-I-infected T cells, Myb protein binding to the HTLV-I LTR may constitute one of the signal that regulate HTLV-I transcription in vivo. PMID- 1727490 TI - The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism. AB - Different isolates of human immunodeficiency virus type 1 (HIV-1) vary in the cell tropisms they display, i.e., the range of cell types in which they are able to establish a productive infection. Here, we report on the phenotypes of recombinants between two molecularly cloned strains of HIV-1. Our results prove that the envelope glycoprotein gp120 is solely responsible for the difference in cell tropism between the two parental isolates and that no other genes or sequences are involved in determining the cell tropism of these strains. The region of the envelope involved in the determination of cell tropism includes sequences which encode the V3 loop of gp120. Control of cell tropism by this region of the virus env gene is a general phenomenon which applies to many different HIV-1 isolates. PMID- 1727491 TI - Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease. AB - Studies of cultivatable human immunodeficiency virus type 1 (HIV-1) from plasma samples from infected patients have shown a correspondence between increasing viral burden and disease progression, but these measurements are selective and thus nonrepresentative of the in vivo viral load. Quantitation of proviral DNA sequences by the polymerase chain reaction in purified CD4+ T cells has shown a similar relationship but does not provide a measure of viral gene expression. We have studied viral DNA, genomic RNA, and spliced mRNA expression of HIV-1 in infected patients with a quantitative polymerase chain reaction assay. Viral RNA expression is detected in all stages of infection. These data show that the natural history of HIV infection is associated with a shift in the balance of viral expression favoring the production of genomic RNA without a preceding period of true viral latency. PMID- 1727492 TI - Similar replication capacities of primary human immunodeficiency virus type 1 isolates derived from a wide range of clinical sources. AB - Numerous studies have suggested that there are significant differences in replication capacities and cytopathicities among human immunodeficiency virus type 1 (HIV-1) isolates and that these differences correlate with the clinical status and geographical origin of infected individuals. However, it has been difficult to assess whether reported distinctions could be attributed to the methods used or whether they imply a true disparity between viral isolates. We thus attempted to characterize the replication properties of HIV-1 isolates directly recovered from infected patients (primary isolates) by using a standardized infection assay. Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by a single coculture with normal donor PBMC stimulated with phytohemagglutinin. Replication curves and cytopathic effect of a standard inoculum (1 ng of p24) of 66 primary HIV-1 isolates were similar regardless of clinical stage of the patient (asymptomatic, AIDS-related complex, or AIDS) and evolutive feature (rate of progression to AIDS). There was no difference between viruses derived from patients sensitive to zidovudine and those derived from patients resistant to zidovudine. Moreover, no difference was found among viral isolates of different geographical origins (Central Africa, Zaire, Brazil, or France). Similarly, the replication patterns and cytopathicities of isolates from bronchoalveolar lymphocytes did not differ from those of isolates derived from PBMC. In contrast, the same amount of viral inoculum of five laboratory HIV-1 strains (HIV-1, EL1, SF, MN, and RF) produced different replication curves and were much less cytopathic. In contrast to laboratory viral strains, it appears that the primary HIV-1 isolates tested, whatever their clinical status and source, exhibited similar replication capacities and cytopathicities in allogeneic donor PBMC. PMID- 1727493 TI - Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide. AB - We have analyzed the translocation of hepatitis B virus (HBV) precore (PC) proteins by using Xenopus oocytes injected with a synthetic PC mRNA. The PC region is a 29-amino-acid sequence that precedes the 21.5-kDa HBV capsid or core (C) protein (p21.5) and directs the secretion of core-related proteins. The first 19 PC amino acids provide a signal peptide that is cleaved with the resultant translocation of a 22.5-kDa species (p22.5), in which the last 10 PC residues precede the complete p21.5 C polypeptide. Most p22.5 is matured to 16-20 kDa species by carboxyl-terminal proteolytic cleavage prior to secretion. Here we show that some four unexpected PC proteins of 24 to 25 kDa are produced in addition to the secretion products described above. Protease protection and membrane cosedimentation experiments reveal that all PC proteins behave as expected for proteins that are translocated into the lumen of the endoplasmic reticulum except for the single largest PC protein (p25), which is not translocated. Like p21.5, p25 is a phosphoprotein that localizes to the oocyte cytosol and nucleus, and protease digestion studies suggest that the two molecules have similar two-domain structures. Radiosequencing of immobilized p25 demonstrates that it contains the intact PC signal peptide and represents the unprocessed translation product of the entire PC/C locus. Thus, while many HBV PC protein molecules are correctly targeted to intracellular membranes and translocated, a significant fraction of these molecules can evade translocation and processing. PMID- 1727494 TI - Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans. AB - Vaccinia virus (VV) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gt11 expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of VV, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins. PMID- 1727495 TI - Genetic differences accounting for evolution and pathogenicity of simian immunodeficiency virus from a sooty mangabey monkey after cross-species transmission to a pig-tailed macaque. AB - We determined the nucleotide sequences of two related isolates of simian immunodeficiency virus from the sooty mangabey monkey (SIVsmm) that exhibit dramatic differences in virulence. These isolates are separated by one experimental cross-species transmission, from sooty mangabey to pig-tailed macaque. The parental virus (SIVsmm9), nonpathogenic in the original host (sooty mangabeys), causes a chronic AIDS-like disease in macaques. In contrast, the variant virus (SIVsmmPBj14) induces an acute lethal disease in various macaque species and is also pathogenic for sooty mangabeys. The combination of necessary and sufficient mutations that determined the acutely lethal phenotype on the SIVsmm9 genetic background is included within a maximal set of 57 point mutations, plus two insertions located in the long terminal repeat (22 bp spanning an NF-kappa B-like enhancer element) and in the surface envelope glycoprotein (5 amino acids). Comparisons of synonymous and nonsynonymous nucleotide substitutions in the genome of SIVsmm indicated that selective pressures, probably due to the host immune response, favored amino acid changes in the envelope. This immunoevolutionary mechanism could explain the increase in diversity and the apparition of new virulent phenotypes after cross-species transmission. PMID- 1727496 TI - The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine. AB - The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability. PMID- 1727497 TI - Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity. AB - The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry. PMID- 1727499 TI - Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation. AB - The active form of the retroviral proteinase (PR) is a homodimer of monomeric subunits expressed as integral parts of the viral gag-pol precursor polyproteins, and dimerization of polyproteins is presumed to be important for regulation of PR activity. Expression of a single-chain dimer of the human immunodeficiency virus (HIV) type 1 PR as a component of the viral polyprotein has been shown to prevent particle assembly and viral infectivity (H.-G. Krausslich, Proc. Natl. Acad. Sci. USA 88:3213-3217, 1991). Ro31-8959, a specific inhibitor of HIV PR, blocked proteolysis of polyproteins containing either wild-type or single-chain dimer PR at the same inhibitor concentration. Different inhibitor concentrations gave three phenotypic effects for the linked PR: at a concentration of 10 nM, cytotoxicity was prevented yet viral polyproteins were almost completely processed and no particles were released. The majority of HIV capsid proteins was found in the soluble cytoplasmic fraction, whereas at a concentration of 1 microM inhibitor most HIV gag proteins were associated with an insoluble fraction. Release of particles consisting of partially processed polyproteins was observed at 100 nM Ro31-8959, and polyprotein processing was blocked at 10 microM. Particles derived from the dimer-containing provirus were noninfectious independently of the inhibitor concentration. Production of infectious HIV after transfection of wild-type provirus was abolished at 100 nM and markedly reduced at 10 nM Ro31-8959. PMID- 1727498 TI - Transient expression of the vaccinia virus DNA polymerase is an intrinsic feature of the early phase of infection and is unlinked to DNA replication and late gene expression. AB - We have studied the expression pattern of the vaccinia virus DNA polymerase during the viral replicative cycle. To monitor polymerase synthesis, a polyclonal antiserum was raised against a TrpE-DNA polymerase fusion protein. Immunoprecipitation and S1 analyses revealed that polymerase synthesis and mRNA levels peak by 2 to 3.5 h postinfection during wild-type infections and then decline, becoming barely detectable by 5 to 6.5 h postinfection. Blocking viral DNA replication by performing infections with temperature-sensitive DNA- mutants at the nonpermissive temperature or by performing wild-type infections in the presence of cytosine beta-D-arabinofuranoside had no effect on polymerase expression. These results indicate that the transient expression of the DNA polymerase is regulated independently of intermediate and late viral gene expression. Cycloheximide, which inhibits protein synthesis and prevents secondary uncoating, caused prolonged and elevated levels of polymerase transcription. Early viral proteins and uncoating, rather than exhaustion of the encapsidated transcription machinery, are presumed to mediate the cessation of polymerase transcription. In the presence of aphidicolin, the polymerase transcripts were maintained at maximal levels rather than exhibiting their normal decline. This inhibition of RNA decay was seen even in infections performed with isolates encoding aphidicolin-resistant DNA polymerases, suggesting that aphidicolin may interfere directly with the process of RNA degradation. Under these conditions, polymerase synthesis remained transient and was not prolonged, despite the continuing presence of available mRNA. These observations suggest that early mRNAs may experience a loss in translation efficiency as infection progresses. PMID- 1727500 TI - Infection of cord blood monocyte-derived macrophages with human immunodeficiency virus type 1. AB - We have investigated the susceptibility of cord blood monocyte-derived macrophages to human immunodeficiency virus type 1 (HIV-1) infection in vitro. Cord blood monocytes were maintained in vitro for 10 to 15 days and then infected with HIV-1. Syncytia were observed 14 days after infection by light microscopy. Viral proteins were detected by immunofluorescence assay. Electron microscopic examination demonstrated typical lentivirus particles within cytoplasmic vacuoles. The supernatants from the HIV-1-infected cultures also contained significant reverse transcriptase activity and p24 antigen. Like adult monocyte/macrophages, cord-derived monocyte/macrophages expressed the CD4 receptor molecule. Pretreatment with blocking antibody prior to infection with HIV-1 Bal significantly reduced or blocked infection of cord monocyte/macrophages. When cord and adult monocyte/macrophages were infected with HIV-1 Bal or Ada-M and directly compared, higher reverse transcriptase activities and p24 antigen expression were obtained with cord monocyte/macrophages. However, no significant difference was found between adult and cord monocyte/macrophages infected with HIV-1 IIIB. These observations suggest that cord monocyte-derived macrophages may be important in the pathogenesis of pediatric AIDS and that the increased susceptibility of cord monocyte/macrophages to HIV-1 infection in vitro may be relevant to the enhanced susceptibility of neonates to HIV-1 diseases in vivo. PMID- 1727501 TI - Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion. AB - Selected strains of the human immunodeficiency virus (HIV) types 1 and 2 are able to infect human colon epithelial cells in vitro, suggesting a mechanism for the anal route of HIV transmission. In some cases, HIV is not produced by infected colon cells but can be rescued after coculture with T-lymphoid cells. One of the HIV strains (HIV1-NDK) replicated well in colonic cells. A transmission electron microscope study demonstrated two major structural perturbations in producer colon cells: an unusual number of secretion bodies and the appearance of intracellular lumina with disorganized microvilli, indicating a defect in brush border assembly and differentiation. Either abnormality could account for HIV induced enteropathy consisting of chronic diarrhea and malabsorption in the absence of enteric pathogens. Moreover, HT29 cells infected with HIV provide a unique model for selection of enterotropic HIV strains. PMID- 1727502 TI - Glucocorticoid receptor-binding site in the human immunodeficiency virus long terminal repeat. AB - Previous reports (P. D. Katsanakis, C. E. Sekaris, and D. A. Spandidos, Anticancer Res. 11:381-383, 1991; J. Laurence, M. B. Sellers, and S. K. Sikder, Blood 74:291-297, 1989; R. Miksicek, A. Heber, W. Schmid, U. Danesch, G. Posseckert, M. Beato, and G. Schutz, Cell 46:283-290, 1986) have suggested the existence of a glucocorticoid response element in the long terminal repeat of human immunodeficiency virus (HIV) type 1. This study demonstrated a sequence specific interaction of the glucocorticoid receptor DNA-binding domain with the previously predicted HIV glucocorticoid response element. This interaction may be relevant to the steroid responsiveness of HIV (P. A. Furth, H. Westphal, and L. Hennighausen, AIDS Res. Hum. Retroviruses 6:553-560, 1990; J. Laurence, M. B. Sellers, and S. K. Sikder, Blood 74:291-297, 1989; J. Laurence, H. Cooke, and S. K. Sikder, Blood 75:696-703, 1990; D. A. Spandidos, V. Zoounpovilis, A. Kotsinas, C. Tsiripotis, and C. E. Sekeris, Anticancer Res. 10:1241-1246, 1990). PMID- 1727503 TI - Molecular characterization of a unique retrovirus associated with a fish tumor. AB - The walleye dermal sarcoma is a mesenchymal tumor which seasonally affects up to 27% of adult walleye fish (Stizostedion vitreum). It arises multicentrically in the dermis, in which its development remains restricted. We report the molecular cloning of a type C retrovirus from this tumor. The genome of this virus (13.2 kb) is larger than that of all retroviruses and in that respect is approximated only by the recently characterized spumaviruses. In tumors, the predominantly unintegrated linear viral DNA has a single-stranded gap region which is similar to the structure found in some lentiviruses and all spumaviruses. The presence of at least four viral transcripts suggests that this virus has the capacity to encode accessory functions and is reminiscent of the transcriptional complexity of lentiviruses and spumaviruses. PMID- 1727505 TI - Largest-ever antismoking effort aims to form grass-roots coalitions. PMID- 1727506 TI - Manufacturer of 'death cigarettes' says he's working to bring about the death of smoking. PMID- 1727504 TI - Inhibition of human immunodeficiency virus type 1 Rev-Rev-response element complex formation by complementary oligonucleotides. AB - Complementary 18-mer oligodeoxynucleotides (oligonucleotides) specifically inhibited the formation of human immunodeficiency virus Rev-Rev-response element (RRE) complexes. Inhibition of Rev-RRE binding required blockage of G-7819 to G 7820 in band shift assays. Structural studies revealed both local and distal effects. RRE structure was also disrupted by oligonucleotides targeted to other minor stems, by altering RNA renaturation conditions, or by reducing Rev concentrations--indicating a dynamic RRE structure and involvement of a minor RRE stem in the maturation of initial Rev-RRE complexes. Thus, complementary oligonucleotides alter RRE structure and may prove useful for the design of therapeutic anti-RRE oligonucleotides. PMID- 1727507 TI - Acellular pertussis vaccines available soon for fourth, fifth doses of DTP immunization. PMID- 1727508 TI - From the Centers for Disease Control. Annual and New Year's Day alcohol-related traffic fatalities--United States, 1982-1990. PMID- 1727509 TI - From the Centers for Disease Control. Mycobacterium haemophilum infections--New York City metropolitan area, 1990-1991. PMID- 1727510 TI - From the Centers for Disease Control. Influenza activity--United States, 1991 1992. PMID- 1727511 TI - Patients leaving emergency departments without being seen by a physician. PMID- 1727512 TI - Patients leaving emergency departments without being seen by a physician. PMID- 1727513 TI - Patients leaving emergency departments without being seen by a physician. PMID- 1727514 TI - Patients leaving emergency departments without being seen by a physician. PMID- 1727515 TI - Queuing in Canada. PMID- 1727516 TI - Serum-plasma differences in total cholesterol: what correction factor should be used? PMID- 1727517 TI - The office of decedent affairs. PMID- 1727519 TI - Levothyroxine. PMID- 1727518 TI - Excretion of HTLV-I in saliva. PMID- 1727520 TI - The potential supply of organ donors. An assessment of the efficacy of organ procurement efforts in the United States. AB - OBJECTIVES: To estimate the potential supply of organ donors and to measure the efficiency of organ procurement efforts in the United States. METHODS: A geographic database has been developed consisting of multiple cause of death and sociodemographic data compiled by the National Center for Health Statistics. All deaths are evaluated as to their potential for organ donation. Two classes of potential donors are identified: class 1 estimates are restricted to causes of death involving significant head trauma only, and class 2 estimates include class 1 estimates as well as deaths in which brain death was less probable. RESULTS: Over 23,000 people are currently awaiting a kidney, heart, liver, heart-lung, pancreas, or lung transplantation. Donor supply is inadequate, and the number of donors remained unchanged at approximately 4000 annually for 1986 through 1989, with a modest 9.1% increase in 1990. Between 6900 and 10,700 potential donors are available annually (eg, 28.5 to 43.7 per million population). Depending on the class of donor considered, organ procurement efforts are between 37% and 59% efficient. Efficiency greatly varies by state and organ procurement organization. CONCLUSIONS: Many more organ donors are available than are being accessed through existing organ procurement efforts. Realistically, it may be possible to increase by 80% the number of donors available in the United States (up to 7300 annually). It is conceivable, although unlikely, that the supply of donor organs could achieve a level to meet demand. PMID- 1727521 TI - Factors affecting the waiting time of cadaveric kidney transplant candidates in the United States. AB - OBJECTIVE--To evaluate the relative impact of various factors that could account for differences in waiting time of cadaveric kidney transplant candidates (eg, black and sensitized patients). DESIGN: --A cohort study using multivariate analyses to identify associations between 36 patient, donor, and center factors with waiting time for all US cadaveric kidney transplant candidates listed between October 1, 1987, and June 30, 1990. SETTING--All US kidney transplant centers. PATIENTS--The study included 23,468 cadaveric renal transplant candidates on active waiting status. RESULTS--The patient characteristics most significantly associated with increased waiting time (adjusted for all other variables) were immunologic and included presensitization to HLA antigens, O or B blood type, candidacy for a repeat transplantation, and expression of rare HLA-A or HLA-B antigen phenotypes. Nonimmunologic factors also affected waiting times, which were significantly shorter for patients younger than 15 years vs those aged 15 through 44 years (8.4 vs 12.9 months, respectively; P less than .0001), for those listed at multiple centers vs one center (7.0 vs 13.3 months, respectively; P less than .0001), or for white vs black patients (11.9 vs 15.4 months, respectively; P less than .0001). Local transplant center characteristics associated with a significantly shorter waiting time included a small number of transplantation candidates, a high (greater than 35 per million population) local kidney organ recovery rate, and an approved variance from the Organ Procurement and Transplantation Network allocation algorithm. CONCLUSIONS--The time renal transplant candidates must wait for kidney transplantation is influenced by several factors in addition to those expected due to immunologic reasons of donor incompatibility, the algorithms used for organ distribution, or the effectiveness of local kidney recovery. The impact of these factors should be considered as the current US system for allocating scarce donor organs for kidney transplantation is modified. PMID- 1727522 TI - Variations in methadone treatment practices. Results from a national study. AB - OBJECTIVE: To examine the extent to which outpatient methadone maintenance treatment units are engaging in treatment practices that previous research indicates are ineffective (eg, inadequate dose levels); to examine factors that may be related to variation in methadone treatment practices. DESIGN: Survey of unit directors and clinical supervisors. SETTING: The study includes units that vary in terms of ownership (public, private for-profit, or private not-for profit) and setting (eg, hospital-based, mental health center-based, or free standing facility). PARTICIPANTS: A national random sample of 172 units participated, for an 82% response rate; the data were weighted to ensure that they were nationally representative. MAIN OUTCOME MEASURES: Clients' awareness of and influence on doses; units' use of take-home dosages; upper limits on doses; average dose levels; unit emphasis on decreasing dosages; time when clients are encouraged to detoxify; average length of treatment. RESULTS: The data indicate that many units have treatment practices such as low average dose levels that are not effective according to the majority of previous studies. Units with higher average dose levels have longer average lengths of time in treatment. CONCLUSIONS: Steps should be taken to monitor and, if necessary, change the treatment practices of methadone units that are providing inadequate dose levels with little client input. PMID- 1727523 TI - Inconsistencies in coding of race and ethnicity between birth and death in US infants. A new look at infant mortality, 1983 through 1985. AB - OBJECTIVE: To ascertain the consistency of the racial and ethnic classification of US infants between birth and death and its impact on infant mortality rates. SUBJECTS: All US infants born from 1983 through 1985 who died within a year. DESIGN: We used the national linked birth/infant-death computer tape, augmented with information on infants' race and ethnicity at death, to compare the coding of race and Hispanic ethnicity at birth and at death. We also assessed infant mortality rates by race and ethnicity as defined (1) by the standard algorithm and (2) by the rule that, beginning in published tabulations for 1989, assigns newborns the race of their mothers. Finally, we estimated infant mortality rates based on consistent coding of race and ethnicity at birth and death. RESULTS: Inconsistency in the coding of race is low for whites (1.2%), greater for blacks (4.3%), and greatest for races other than white or black (43.2%). Most infants reclassified at death (87.3%) are classified as white at death. Inconsistency in coding is lower for non-Hispanic whites (3.5%) and non-Hispanic blacks (3.3%) than for Hispanic populations (30.3%). Compared with the standard algorithm for calculation of infant mortality, consistent definition at birth and death produces rates 2.1% lower for whites, and higher for all other groups--3.2% for blacks, 46.9% for American Indians, 33.3% for Chinese, 48.8% for Japanese, 78.7% for Filipinos, and 8.9% for Hispanics. CONCLUSIONS: The coding of race and ethnicity of infants at birth and death is remarkably inconsistent, with substantial impact on the estimation of infant mortality rates. A need exists to reconsider the nature and definition of race and ethnicity in public health. PMID- 1727524 TI - Treatment of streptococcal endocarditis with a single daily dose of ceftriaxone sodium for 4 weeks. Efficacy and outpatient treatment feasibility. AB - OBJECTIVE--To evaluate the efficacy and safety of ceftriaxone sodium in the treatment of streptococcal endocarditis. DESIGN--An open, multicenter, noncomparative study with a follow-up of patients for 4 months to 5 years. SETTING--Internal medicine wards and outpatient clinics of hospitals of various sizes in three European countries. PATIENTS--Fifty-nine patients with defined criteria for streptococcal endocarditis. INTERVENTION--Ceftriaxone sodium administered at a once-daily dose of 2 g for 4 weeks. MAIN OUTCOME MEASURES- Clinical outcome and microbiological cure rate. RESULTS--Among the 59 patients, 55 completed the treatment and were followed up for 4 months to 5 years. No patients showed evidence of relapse. Treatment was completely uneventful in 42 patients (71%). A cardiac valve was replaced in four patients (7%) receiving antimicrobial therapy and in six patients (10%) who had completed antimicrobial therapy. One of the 10 valves taken for culture at surgery was positive, but only for microorganisms that were different from the microorganism isolated before the treatment. The treatment had to be interrupted in four patients because of drug allergy. Other side effects were mild except for two cases of reversible neutropenia. The treatment was easy to administer: 27 patients (46%) had no permanent intravenous catheter at any time, seven patients (12%) had such a catheter for less than 4 days. Twenty-three patients (39%) were discharged from the hospital less than 2 weeks after admission. CONCLUSIONS: --Ceftriaxone sodium administered at a once-daily dose of 2 g appears to be an effective and safe treatment of streptococcal endocarditis. In hospitals, this agent may be more convenient to administer than penicillin G with or without aminoglycosides. Some patients may even be treated as outpatients. PMID- 1727525 TI - The state of federal health statistics on racial and ethnic groups. AB - OBJECTIVE: To examine assumptions underlying federal health statistics on racial and ethnic groups in the United States. DATA SOURCES: Studies conducted by federal agencies and other investigators, and technical appendices of published vital statistics and census reports. DATA SYNTHESIS: Several assumptions underlying federal health statistics on racial and ethnic groups are not well supported. Conceptual (as opposed to operational) definitions of race and ethnicity are not available, and scientific grounds for definition are not considered. Procedures for the ascertainment of race and ethnicity vary within and among data-collection agencies. Miscounting and misclassification may vary by an order of magnitude between whites and other races. The responses of individuals to questions of racial and ethnic identity differ for different indicators, in different surveys, and at different times. As a result, counts, rates, and rate ratios may not be meaningful or accurate. Particularly for Hispanics and for races other than whites or blacks, there are inconsistencies in statistical information that may hinder health research and program development. CONCLUSIONS: Improvement of federal health statistics for racial and ethnic groups requires (1) clarification of goals for classification, (2) adoption of scientific principles for the validation and definition of the categories "race" and "ethnicity," (3) assessment of perceived social identity in the population, and (4) periodic evaluation. PMID- 1727526 TI - Case and survival definitions in out-of-hospital cardiac arrest. Effect on survival rate calculation. AB - OBJECTIVE: To determine the effect of different case and survival definitions of out-of-hospital cardiac arrest on survival rate calculations. DESIGN: A 22-month case series of nontraumatic, out-of-hospital cardiac arrests. SETTING: Southwestern city (population, 400,000; area, 390 km2) with a two-tiered emergency response system consisting of emergency medical technicians and paramedics. PATIENTS: A consecutive sample of 372 patients found without palpable pulse of spontaneous respiration. MAIN OUTCOME MEASURES: Survival rate after cardiac arrest was calculated using three case definitions of arrest and two definitions of survival. RESULTS: Twenty percent of all patients survived to hospital admission and 6% survived to hospital discharge. Twenty-six percent of adults whose collapse was witnessed survived to hospital admission, and 10% survived to hospital discharge. Patients whose collapse was witnessed and who experienced initial ventricular fibrillation survived to hospital admission in 38% and to hospital discharge in 15% of cases. CONCLUSIONS: The survival rate after out-of-hospital cardiac arrest varies widely depending on the case and survival definitions selected. To facilitate intersystem comparison and assessment of interventions designed to improve outcome, the Utstein Consensus Conference recommended that case and survival definitions should be adopted by all prehospital emergency systems. PMID- 1727527 TI - The use of race in medical research. PMID- 1727528 TI - Ceftriaxone sodium therapy of penicillin G-susceptible streptococcal endocarditis. PMID- 1727529 TI - Ineffective use of psychoactive drugs. Methadone treatment is no exception. PMID- 1727530 TI - Diagnostic and therapeutic technology assessment. Measurement of bone density with dual-energy X-ray absorptiometry (DEXA) PMID- 1727531 TI - A piece of my mind. A doctor in her house. PMID- 1727533 TI - Medical management of AIDS patients. PMID- 1727532 TI - HIV-1 infection. Diagnosis and management. AB - This article discusses the epidemiology, diagnostic methods, prevention of complications, and medical management of HIV-1 infection. Many different therapeutic strategies for HIV-1 infection are being evaluated, but until the immune suppression is eradicated, presumably through more effective antiviral therapy, complications of AIDS and HIV-1 infection will continue to be encountered. PMID- 1727534 TI - Pneumocystosis. AB - In much of the world, pneumocystosis remains the most common life-threatening opportunistic infection among patients with HIV disease. The infection is caused by Pneumocystis carinii--an organism whose identity as a fungus or parasite is still debated. What is no longer debated, after a decade of AIDS, is that pneumocystosis is almost entirely preventable and eminently treatable. Understanding has improved concerning when prophylaxis should be initiated. It is also recognized that, at least with the agents available today, antiretroviral therapy alone will not prevent pneumocystosis. Sputum induction and the use of monoclonal antibodies have modestly improved our ability to diagnose the infection; however, invasive procedures are still required for most patients, and unusual presentations of the disease, such as cavitary lesions, apical infiltrates, pneumothoraces, and extrapulmonary infection, are not infrequently seen. For treatment, trimethoprim-sulfamethoxazole and intravenous pentamidine remain the mainstays; oral therapy with dapsone and trimethoprim can be as effective as conventional therapy in mild disease, permitting treatment on an outpatient basis. Adjunctive steroids are useful for treatment of moderate to severe pneumocystosis, but clinicians should be alert to the possibility of activation of other latent infections during and after courses of steroids. Both aerosol pentamidine and trimethoprim-sulfamethoxazole are effective prophylaxis. The latter appears to be more effective and costs much less, but the results of comparative trials are not yet available. More data are also needed on the safety, efficacy, and relative advantages of dapsone for prophylaxis. The first decade of the AIDS epidemic has been a decade of progress against pneumocystosis. In the next decade, the emergence of new technologies for diagnosis and of new agents for prophylaxis and treatment will bring us closer to the goal of controlling this serious infection. PMID- 1727535 TI - Medical management of AIDS patients. Tuberculosis and nontuberculous mycobacterial disease. AB - AIDS has been responsible for a significant increase in mycobacterial disease, which in this setting is often extrapulmonary. In contrast to HIV-associated Mycobacterium avium complex disease, HIV-associated tuberculosis is normally transmissible between humans by the aerosol route, occurs earlier than most AIDS related infections, and is readily treatable and preventable with conventional drugs. PMID- 1727536 TI - Medical management of AIDS patients. Bacterial and fungal infections. AB - The rapid and thus far generally inexorable rise in HIV infections has led to a series of opportunistic infection that includes those caused by bacteria, yeasts, and members of the Eumycetes. The infections range in prevalence from occasional to highly prevalent, in severity from trivial to fatal, and in anatomic areas involved from local to disseminated. They occur as isolated, concurrent, or sequential infections with regard to other opportunistic diseases. Some vary in their geographic distribution. They may be newly acquired or reactivated and occur early or late in the course of HIV infection. Bacterial infections are usually easily treated, although they frequently disseminate and often recur after seemingly appropriate treatment. In contrast, all but the mildest fungal infections are difficult to treat and even more difficult or impossible to eradicate. The diagnosis of bacterial and fungal infections begins with clinical suspicion and involves relatively standard methodology. Treatment of the systemic mycoses and some bacterial infections in HIV infected patients is punctuated by exaggerated side effects of therapy, frequent relapses, and the need for maintenance suppressive therapy. PMID- 1727537 TI - Medical management of AIDS patients. Pulmonary disease. AB - The authors provide an overview of the spectrum of pulmonary disorders commonly encountered in the HIV-infected patient. Then an approach to assessment of patients and the limitations and indications for specific diagnostic techniques are discussed. Finally, recommendations for prophylactic measures are given. PMID- 1727538 TI - Coccidian infections in AIDS. Toxoplasmosis, cryptosporidiosis, and isosporiasis. AB - Cryptosporidium sp. and Isospora belli are coccidian protozoan parasites that were long recognized as pathogens for many animal species. The medical community became acquainted with these organisms with the advent of AIDS. Both parasites are associated with persistent, debilitating enteritis and, in the case of Cryptosporidium, biliary tract involvement in patients with AIDS. For the immunocompetent host, infection with these two pathogens usually results in self limited diarrhea. Cryptosporidiosis appears to occur more often than isosporiasis, but the true prevalence of both infections for various populations of humans is unknown. Clinically, cryptosporidiosis is indistinguishable from isosporiasis. Diagnosis is based on finding the acid-fast (red staining oocyst in stained fecal specimens). There is no known effective therapy for cryptosporidiosis, whereas patients with isosporiasis respond promptly to treatment with trimethoprim-sulfamethoxazole. Patients with AIDS and isosporiasis have a high relapse rate after achieving complete remission and therefore need to be maintained on suppressive therapy. Much more needs to be learned about these two fascinating, "newly recognized" parasites. PMID- 1727539 TI - AIDS-associated malignant lymphoma. AB - Lymphoma has been well described in various states of congenital, acquired, and iatrogenic immune dysfunction, and the clinical and pathologic characteristics in these settings are similar to those seen in patients with HIV-induced immunodeficiency. High grade B cell lymphomas are expected, with widespread extranodal disease at the time of initial presentation. Unusual sites of disease may be seen, such as the CNS. Factors predictive of short survival include low performance status, history of AIDS prior to the diagnosis of lymphoma, stage IV or bone marrow involvement, and low CD4 cells. Use of intensive multiagent chemotherapy may be associated with demise from opportunistic infections. Less intensive regimens may be indicated in patients with poor prognostic indicators, whereas patients lacking these factors may be able to tolerate greater dose intensity. PMID- 1727540 TI - HIV infection and the healthcare worker. AB - The perception of degree of risk can vary markedly from actual risk. About 5% of the cases of AIDS and HIV infection in the United States have occurred in healthcare workers, a percentage that has remained stable over time. Nearly all of these infections are related to lifestyle factors, not occupational risk. The risk to patients appears to be very much smaller, but has received even more publicity. Apprehension exists concerning the future framework of our medical care delivery system and who will care for whom. The sensitive handling of legitimate fears and the minimization and balancing of conflicting risks will be a challenging task in the decades ahead. PMID- 1727541 TI - Medical management of AIDS patients. Gastrointestinal manifestations. AB - Gastrointestinal manifestations of AIDS are common. Opportunistic infections and tumors may affect any portion of the GI tract from oral cavity to anus. Esophageal involvement may result from Candida, CMV, HSV, HIV, and tumors. Biliary tract and pancreatic disease may cause abdominal pain. Diarrhea occurs in over 50% of AIDS patients and is multifactorial. PMID- 1727542 TI - Medical management of AIDS patients. Central and peripheral nervous system abnormalities. AB - This article discusses the wide range of neurologic complications of HIV infection according to degree of advancement of systemic HIV disease. The focus is principally on those disorders that appear at least in part to be directly related to HIV: AIDS dementia complex, peripheral neuropathy, and myopathy. Unusual disturbances such as seizures and transient neurologic disorders are also discussed. PMID- 1727543 TI - Medical management of AIDS patients. Psychiatric disturbances. AB - Despite the complexity and magnitude of the psychiatric disturbances associated with HIV illness, a good deal is understood about their clinical presentation. Techniques for psychopharmacologic and psychotherapeutic management have been well established and are readily available. The expertise developed for management of psychiatric disturbances in other medical illnesses applies quite well to HIV-related conditions. Although the HIV epidemic challenges us with new difficulties, our experience with other illnesses provides us with a basis to respond. PMID- 1727544 TI - Evidence that dyslexia may represent the lower tail of a normal distribution of reading ability. AB - BACKGROUND: Dyslexia is now widely believed to be a biologically based disorder that is distinct from other, less specific reading problems. According to this view, reading ability is considered to follow a bimodal distribution, with dyslexia as the lower mode. We hypothesized that, instead, reading ability follows a normal distribution, with dyslexia at the lower end of the continuum. METHODS AND RESULTS: We used data from the Connecticut Longitudinal Study, a sample survey of 414 Connecticut children who entered kindergarten in 1983 and were followed as a longitudinal cohort. Dyslexia was defined in terms of a discrepancy score, which represents the difference between actual reading achievement and achievement predicted on the basis of measures of intelligence. Data were available from intelligence tests administered in grades 1, 3, and 5 and achievement tests administered yearly in grades 1 through 6. For each child there were 108 possible discrepancy scores ([3 x 3 years] x [2 x 6 years]) based on combinations of the ability scores (full-scale, verbal, and performance IQ) in each of three years and two achievement scores (reading and mathematics) in each of six years. We demonstrated that each of the discrepancy scores followed a univariate normal distribution and that the interrelation of two different discrepancy scores followed a bivariate normal distribution. At most, only 9 of 108 discrepancy scores (8.3 percent) and 171 of 3402 pairs of discrepancy scores (5.0 percent) were significantly different (at the 5 percent level) from the expected scores--well within the expected values for data with univariate and bivariate normal distributions, respectively. We also examined the stability of dyslexia over time. The normal-distribution model predicted (and the data indicated) that only 7 of the 25 children (28 percent) classified as having dyslexia in grade 1 would also be classified as having dyslexia in grade 3. CONCLUSIONS: Reading difficulties, including dyslexia, occur as part of a continuum that also includes normal reading ability. Dyslexia is not an all-or none phenomenon, but like hypertension, occurs in degrees. The variability inherent in the diagnosis of dyslexia can be both quantified and predicted with use of the normal-distribution model. PMID- 1727545 TI - Potable water as a cause of sporadic cases of community-acquired legionnaires' disease. AB - BACKGROUND: The environmental sources of sporadic, community-acquired legionnaires' disease are largely unknown, and culturing of water sources after identification of a case is currently not recommended. We conducted a prospective study of sporadic cases of community-acquired legionnaires' disease to determine whether the environmental reservoirs could be identified. METHODS: We cultured samples of potable water obtained from sources to which each of 20 patients with culture-confirmed, community-acquired legionnaires' disease had been exposed during the two weeks before the onset of symptoms. Monoclonal-antibody subtyping and restriction-endonuclease analysis were performed on the legionella isolates recovered from both the patients and the associated environmental cultures. RESULTS: For 8 of the 20 patients, isolates of Legionella pneumophila with identical subtypes were identified in cultures from both the patient and the potable water to which the patient had been exposed. The environmental reservoirs linked to the infections were the water supplies of two private residences, two nursing homes, two hospital outpatient clinics, and an industrial plant. CONCLUSIONS: Potable-water supplies that harbor L. pneumophila are an important source of community-acquired legionnaires' disease. Future studies should include attempts to identify the environmental sources of this infection. PMID- 1727546 TI - Reasons that patients with acute myelogenous leukemia do not undergo allogeneic bone marrow transplantation. AB - BACKGROUND: Numerous reports suggest that allogeneic bone marrow transplantation prolongs the survival of adult patients with acute myelogenous leukemia (AML) in first remission. However, it is unclear how many such patients actually undergo this procedure. METHODS: We reviewed the case records of 350 consecutive adult patients with AML treated with chemotherapy at a single institution from 1979 (when the policy of offering allogeneic transplantation to all such patients in first remission was introduced) through 1990. The criteria for exclusion before transplantation included age greater than 40 and, beginning in 1984, a diagnosis of acute promyelocytic leukemia. RESULTS: One hundred forty-two patients (41 percent of the study population) were 40 years of age or under. HLA testing was performed for 120 of these patients (85 percent). Sixty-seven patients (47 percent) had an HLA-identical sibling as a potential donor. One hundred three patients (73 percent) entered remission during treatment according to one of five chemotherapy protocols. Of the 52 patients who both entered remission and had an HLA match, 30 underwent transplantation while they were in first remission. These 30 patients constituted 21 percent of all study patients 40 years old or under, 29 percent of all patients 40 or under who entered remission, 45 percent of all patients with an HLA match, 58 percent of all patients who had both a remission and a match, and 9 percent of all patients treated according to a protocol. Among patients with a match who did not undergo transplantation, those with primary refractory disease were the largest subgroup. CONCLUSIONS: These findings suggest that allogeneic bone marrow transplantation is performed in less than 60 percent of adult patients with AML who are potentially eligible for the procedure. PMID- 1727547 TI - Hypogonadism caused by a single amino acid substitution in the beta subunit of luteinizing hormone. PMID- 1727548 TI - Dyslexia--is it a disease? PMID- 1727549 TI - Expanding the differential diagnosis of male hypogonadism. PMID- 1727550 TI - Journals in bits and bytes. Electronic medical journals. PMID- 1727551 TI - Euthanasia--the need for procedural safeguards. PMID- 1727552 TI - Relation of meat, fat, and fiber intake to the risk of colon cancer in women. PMID- 1727553 TI - Relation of meat, fat, and fiber intake to the risk of colon cancer in women. PMID- 1727554 TI - Relation of meat, fat, and fiber intake to the risk of colon cancer in women. PMID- 1727555 TI - Relation of meat, fat, and fiber intake to the risk of colon cancer in women. PMID- 1727556 TI - Relation of meat, fat, and fiber intake to the risk of colon cancer in women. PMID- 1727557 TI - Relation of meat, fat, and fiber intake to the risk of colon cancer in women. PMID- 1727558 TI - Effect of very low birth weight on cognitive abilities at school age. PMID- 1727559 TI - The intrauterine device and primary tubal infertility. PMID- 1727560 TI - Treatment of recurrent respiratory papillomatosis. PMID- 1727561 TI - The health care industry--where is it taking us? PMID- 1727562 TI - The health care industry--where is it taking us? PMID- 1727563 TI - The health care industry--where is it taking us? PMID- 1727564 TI - Tattooing should be regulated. PMID- 1727565 TI - The physician and the social contract. AB - Expenditures for health care are growing at a staggering rate, yet over 30 million Americans lack access to care. A growing population with needs, increasing administrative costs, expanding biomedical technology, and the ever increasing expectations of patients are among the explanations for this cost. To resolve the problem, we must establish priorities, evaluate technology more carefully before application, reassess our fees as they relate to effort, and evaluate what we do for our patients as it relates to society generally. Many of the problems in health care are societal problems, but physicians as a particularly responsible and knowledgeable part of society must take the lead in finding and implementing the solutions. PMID- 1727566 TI - Endocervical glandular atypia in Papanicolaou smears. AB - The importance of endocervical glandular atypia in a cervicovaginal Papanicolaou smear has not been fully investigated. Between July 1988 and June 1989, 21,930 cervicovaginal smears were reviewed by the Massachusetts General Hospital Cytopathology Laboratory. One hundred smears with endocervical atypia were identified, an incidence of 0.46%. Follow-up was available on 63 cases: Seven had negative follow-up smears for at least 2 years, 15 had negative biopsies, seven had endocervical polyps, two had endometrial hyperplasia, eight had mild dysplasia, five had moderate dysplasia, six had severe dysplasia, six had squamous carcinoma in situ, five had adenocarcinoma in situ, and two had invasive adenocarcinoma. Twelve women's smears showed endocervical atypia with features suggestive of reactive atypia; three of these had dysplasia. Twenty-six (41%) of the Papanicolaou smears with endocervical atypia had coexisting squamous atypia or dysplasia. We conclude that endocervical atypia may be associated with substantial cervical disease in as many as half of cases. PMID- 1727567 TI - Results of conservative management of cervical intraepithelial neoplasia. AB - Cryotherapy and laser surgery have been the most frequently used conservative methods to treat cervical intraepithelial neoplasia (CIN) in the past decade. This report documents our experience using these modalities to treat 2773 patients between the years 1984-1989. One thousand eight hundred eleven women received laser surgery and the remaining 962 were treated with cryotherapy. In the first 2 years of the study period, only 78 patients were treated with laser surgery. Conversely, only 69 of the 979 patients treated in 1988 and 1989 had cryotherapy. As greater experience was gained with laser surgery, the success rates rose from 58.3% in 1984 to 95.5% in 1988. The success rate was similar for all grades of CIN. Overall, 11.2% of all patients were lost to follow-up. Among patients treated with laser surgery, 4.8% had postoperative bleeding that required either packing or, in two instances, sutures for hemostasis. Success with these methods appeared to be related to the size of lesion and not to the degree of histologic abnormality. The shift toward increasing use of laser surgery in our clinic was due to its precision in destroying identified lesions in the transformation zone. Our results indicate that both cryotherapy and laser surgery are simple, effective methods for the treatment of CIN. PMID- 1727569 TI - Prazosin: a neglected cause of genuine stress incontinence. AB - Prazosin, a common antihypertensive drug, lowers peripheral vascular resistance by selectively blocking alpha-1 adrenergic receptors on arteriolar smooth muscle. Alpha-1 adrenoceptor inhibition also has a relaxant effect on smooth muscle present in the urethra. Between 1985-1990, 58 of 1335 women (4.3%) seen in our urodynamic clinic with urinary incontinence and other urinary symptoms were taking prazosin. The incidence of genuine stress incontinence was significantly higher in women taking prazosin (86.2%) than in the non-prazosin group (65.7%) (P less than .01). Twenty-five of the 45 women contacted had their urinary incontinence improved or cured by prazosin withdrawal. All of these 25 women with prazosin-related urinary incontinence had stress incontinence. The incidence of previous bladder neck surgery in this group was over 50%, with 11 previous vaginal repairs, one Burch colposuspension, and one Aldridge sling procedure. Seven women who were continent after prazosin withdrawal had their urodynamic studies repeated. There was a significant increase in functional urethral length, maximum urethral closure pressure, and abdominal pressure transmission to the urethra following prazosin withdrawal, although no significant change was found in other cystometric measurements including peak flow rate and residual urine volume. In this study, prazosin was a frequently unrecognized cause of stress incontinence in women, many of whom had unsuccessful and possibly unnecessary surgery. PMID- 1727568 TI - Postmenopausal bleeding from unusual endometrial polyps in women on chronic tamoxifen therapy. AB - There are recent reports of postmenopausal bleeding from endometrial polyps in women receiving tamoxifen therapy for breast cancer. We describe four additional patients who presented with vaginal bleeding, and emphasize the pathology. These polyps demonstrated cystically dilated glands in all cases and stromal decidualization in two; in one instance, metastatic breast carcinoma was present in the polyp. The mechanisms by which tamoxifen may affect the development of these polyps are discussed. PMID- 1727570 TI - Retrograde ejaculation: successful treatment with artificial insemination. AB - Retrograde ejaculation is characterized by aspermia or oligospermia and results from an incompetent bladder neck, often due to a dysfunction of the internal sphincter. In almost 3 years, eight couples who suffered from infertility due to retrograde ejaculation were treated with inseminations with spermatozoa gained from the urine. Ovulation was predicted on the basis of blood LH levels. The urine-semen sample was collected in 100 mL of Hepes medium and 5 mL 1% human albumin (pH 7.4). After centrifuging, the remaining sperm pellet was dispersed on a Percoll gradient. After centrifuging and resuspending, followed by two washing procedures with Ham's F-10 and human albumin 1%, the remaining sample was used for intrauterine insemination. Twelve pregnancies were thus achieved; two women became pregnant twice and one three times. The pregnancy rate per cycle was 44.4%. In seven couples, pregnancy was achieved within three cycles. Four pregnancies ended in spontaneous abortion and five ended in the birth of a healthy child; three pregnancies were continuing at the time of writing. Retrograde ejaculation can be treated successfully with inseminations using spermatozoa obtained from urine. It seems important to collect the urine-semen sample in a buffered medium and to time the insemination on the basis of the LH surge. PMID- 1727571 TI - Cushing syndrome in pregnancy. AB - The occurrence of pregnancy in the face of untreated Cushing syndrome is rare because of the high incidence of ovulatory disturbances experienced by patients with the disorder. A total of 58 patients with 65 pregnancies has been reported in the literature to date. Although pituitary-dependent adrenal hyperplasia is the most common etiology of Cushing syndrome in the general population, adrenal adenoma is more common in the pregnant population. Significant maternal morbidity is attributable to hypertension, congestive heart failure, and poor tissue healing. Prematurity and intrauterine growth retardation account for most of the perinatal morbidity; perinatal mortality is substantial. Treatment directed at relieving hypercortisolism has been instituted during pregnancy: Pituitary or adrenal surgery, chemotherapy, and pituitary irradiation have all been reported, with variable results. Information is lacking on any alteration of maternal morbidity after treatment. The impact of therapy on perinatal outcome appears limited to a reduction in the prematurity rate, but overall numbers are small and such a conclusion should be viewed with caution. No significant maternal or perinatal complications secondary to treatment itself were reported. PMID- 1727572 TI - Randomized controlled trials of home uterine activity monitoring: a review and critique. AB - Home uterine activity monitoring has been proposed as an effective technique for reducing the incidence of preterm birth by early recognition of incipient labor. Five randomized controlled trials evaluating this technique have been published in peer review journals. As judged by accepted criteria for such trials, all have serious methodologic deficiencies. Four of the five trials demonstrated no significant benefit from this monitoring. Two other trials not published in peer review journals support the hypothesis that home uterine activity monitoring is no more effective than daily nursing contact. Until the efficacy of this technology has been established, home uterine activity monitoring should not be used clinically. PMID- 1727573 TI - Coagulation profile in severe preeclampsia. AB - One hundred women with severe preeclampsia or chronic hypertension with superimposed preeclampsia were seen during a 2-year period. We sought to determine whether a normal platelet count assures that no other clinically significant clotting abnormalities are present, and what level of thrombocytopenia predicts a risk of abnormalities in other coagulation indices. Fifty women had platelet counts below 150,000/microL, of whom 13 had a fibrinogen level below 300 mg/dL and two had a prolonged prothrombin time (PT) or partial thromboplastin time (PTT). The admission platelet count was an excellent predictor of subsequent thrombocytopenia (r = 0.829, P less than .001). No subject had an abnormal fibrinogen level or prolonged PT or PTT in the absence of thrombocytopenia. When monitoring intrapartum coagulation indices in preeclampsia, one can safely follow only the platelet count at admission and subsequently, reserving PT and PTT and fibrinogen levels for those cases complicated by counts less than 100,000/microL. PMID- 1727574 TI - Comparison of specimens removed by CO2 laser conization and the loop electrosurgical excision procedure. AB - A comparison is made of the histologic changes in the cervical epithelium and stroma following CO2 laser conization and office excisional biopsy of cervical tissue using the loop electrosurgical excision procedure. In both types of specimens, two zones of thermal injury were detected. The zone at the margin of resection measured approximately 50 microns in thickness and was characterized by extensive carbonization and charring. The other zone was much more variable in thickness and characterized by tissue coagulation, but lacked charring. No significant difference in the biocharacteristics or extent of thermal damage was detected between the methods. For 11 specimens obtained with the CO2 laser, the coagulated zone ranged from 130-750 microns in greatest thickness; the mean thickness was 411 microns. For 40 specimens obtained using the loop procedure, the range of thickness of the coagulated zone was 150-830 microns, and the mean thickness was 396 microns. The difference in the mean value of thermal injury (measured in microns) between the laser and loop procedures was not significant (Student t test; P = .79). Extensive areas of carbonization and epithelial distortion at the margins of excision were only occasionally present in specimens obtained by the electrosurgical excision procedure but almost invariably present in CO2 laser specimens. However, in all cases it was possible to evaluate the epithelium and the stroma both histologically and cytologically. PMID- 1727575 TI - Oxytocin augmentation of labor and perinatal outcome in nulliparas. PMID- 1727577 TI - Pregnancy outcome in an active-duty population. PMID- 1727576 TI - Effect of epidural analgesia on the primary cesarean rate. PMID- 1727578 TI - Relationship of small-for-dates sac size to crown-rump length and spontaneous abortion in patients with a known date of ovulation. PMID- 1727579 TI - Complement, neutrophil, and macrophage activation in women with severe preeclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelet count. AB - Activation of complement, neutrophils, and macrophages was studied in 14 women with severe preeclampsia, 11 of whom had the syndrome of hemolysis, elevated liver enzymes, and low platelet count; in 14 women with normal pregnancies; in seven normal pregnant women undergoing cesarean deliveries; and in 15 healthy nonpregnant women. Activation of complement, neutrophils, and macrophages was measured by plasma determinations of complement split products, polymorphonuclear (PMN) elastase, and neopterin, respectively. Women with severe preeclampsia had increased levels of C5a, terminal complement complex, PMN elastase, and neopterin at delivery and 1 day postpartum as compared with the normal pregnant group. One week postpartum, neopterin remained higher in preeclamptic women, whereas the complement components and PMN elastase had returned to normal. Cesarean delivery after normal pregnancy did not increase the levels of complement split products, PMN elastase (except for one value), or neopterin. The nonpregnant women had normal PMN elastase and neopterin levels. Accordingly, complement, neutrophils, and macrophages are activated in women with severe preeclampsia at delivery. The plasma levels of PMN elastase correlated positively to the formed terminal complement complexes in vivo. An in vitro study was performed to elucidate further the connection between complement and leukocyte activation. Recombinant C5a incubated in whole blood and in a neutrophil cell suspension gave a dose dependent release of PMN elastase. Both the clinical and the in vitro results indicate that activation of the complement system may affect the function of neutrophils. This study supports the theory that the pathologic manifestations of severe preeclampsia may be explained by complement-induced release of biologically active substances from activated leukocytes. PMID- 1727580 TI - Maternal cerebral hemodynamics in the supine hypotensive syndrome. AB - Cerebral hemodynamics were studied in eight nonpregnant women and 24 women in late pregnancy by internal carotid artery velocimetry with a 3.5-MHz continuous wave Doppler system. Criteria for supine hypotensive syndrome were a mean blood pressure decrease of 15 mmHg and a 2-minute sustained increase in pulse of 20 beats per minute under postural change from the left lateral to supine position. Nonpregnant and normal pregnant controls not meeting these two criteria displayed decreases of 22.9 and 21.7%, respectively, in time-averaged mean peak velocity (mean velocity) in the supine position compared with the left lateral position. Five subjects with subclinical supine hypotensive syndrome who met one of the above criteria showed a 37.0% decrease in internal carotid artery mean velocity in the supine position. Two patients with supine hypotensive syndrome could not tolerate the supine position for more than 6 minutes, at which time internal carotid artery mean velocity fell below 10 cm/second, reverse flow was observed, and they complained of dizziness, nausea, and syncope. Internal carotid artery mean velocity in all women showed no change in the sitting position compared with the left lateral position. These results indicate that the supine position should be avoided in late pregnancy, especially by women with cerebrovascular complications. PMID- 1727581 TI - Intrauterine PGF2 alpha infusion for termination of pregnancies with second trimester rupture of membranes. AB - Intrauterine prostaglandin (PG) F2 alpha infusion and intravenous (IV) oxytocin infusion were compared to evaluate the effectiveness of the two methods for termination of pregnancies with second-trimester rupture of membranes. Twenty-two women with this complication were randomly allocated to receive either 20 mg PGF2 alpha, diluted in 500 mL of NaCl 0.9% and administered through a Foley catheter inserted through the cervix, or IV oxytocin infusion in increasing doses. All subjects in the PGF2 alpha group aborted after the first administration. Repeat infusion was necessary in three oxytocin-treated subjects. The mean (+/- SD) induction-abortion interval was significantly shorter in those receiving PGF2 alpha (6.7 +/- 1.2 hours) than in those receiving oxytocin (8.8 +/- 2.7 hours). Minor side effects, such as nausea and vomiting, were observed in three women during PGF2 alpha infusion and were treated symptomatically and by temporary interruption of the infusion. Uterine hypertonus, observed in one subject in each group, was treated by temporary cessation of the infusion. We conclude that intrauterine PGF2 alpha infusion seems more effective than IV oxytocin for termination of pregnancies with second-trimester rupture of membranes. PMID- 1727582 TI - The changing pattern of fetal death, 1961-1988. AB - The aim of this study was to assess any changes in cause-specific fetal death rates in the nonreferred population of a tertiary care unit. The fetal death rate (per 1000 births) among 88,651 births diminished from 11.5 in the 1960s to 5.1 in the 1980s. Fetal death due to intrapartum asphyxia and Rh isoimmunization has almost disappeared. Toxemia and diabetes continue to make similar and small contributions to fetal death rates. There has been a significant decline in unexplained antepartum fetal deaths and in those caused by fetal growth retardation, but no significant change in the death rate due to intrauterine infection or abruptio placentae. During the 1960s, the risk of fetal death was increased in women with hypertension, diabetes, or a history of stillbirth; during the 1980s, only women with a history of insulin-dependent diabetes were at risk. Improved application of current knowledge may help decrease the fetal death rate caused by fetal growth retardation. Reduction in deaths due to abruptio placentae, intrauterine infections, or lethal malformations, as well as unexplained antepartum deaths, appears to depend on better understanding of the etiology of these disorders. PMID- 1727583 TI - The impact of prenatal care on fetal and neonatal death rates for uninsured patients: a "natural experiment" in West Virginia. AB - A three-county program in southern West Virginia was developed by an obstetric practice to deliver prenatal care to a population of uninsured patients. Between January 1984 and December 1986, 1331 (29.4%) of 4534 patients were delivered at a level 2 hospital after prenatal care within the clinic program. The hospital-wide fetal death ratio declined from 11.8 to 7.2 per 1000 live births during the years of clinic operation, a statistically significant reduction (P = .02). Uninsured patients experienced a statistically significant reduction in fetal death ratio during the program, from 35.4 to 7.0 per 1000 live births (P = .02), whereas those covered by medical assistance did not experience a reduction. Privately insured patients also had a significant decrease, from 10.0 to 3.1 per 1000 live births (P less than .001). The increasing operating expense, mainly due to rising malpractice insurance premiums, required suspension of the program in December 1986. The fetal death ratio returned to 10.3 deaths per 1000 live births in 1987. Factors that varied significantly during the "clinic" phase included: higher rates of cesarean, diagnosed maternal hypertension, and diabetes mellitus; and lower rates of premature rupture of membranes and non-white population. Other factors, including age over 35 years, postdatism, incidence of twins, incidence of lethal congenital anomalies, and single marital status, did not vary significantly before, during, or after the clinic program. This study identified a high-risk population of patients who did not qualify for medical assistance coverage and were de facto "uninsured." The results suggest that prenatal care for this high-risk population of uninsured patients can reduce the fetal death rate. PMID- 1727584 TI - Evaluation of fetal growth by estimation of neonatal body composition. AB - To characterize the variation in normal fetal growth by body composition analysis, 188 neonates from uncomplicated singleton term pregnancies were evaluated within 24 hours of birth. Anthropometric measures used to estimate lean body mass and body fat included the following: birth weight 3553 +/- 462 g, lean body mass 3060 +/- 377 g (86.3%), fat mass 495 +/- 196 g (13.7%), and ponderal index 2.65 +/- 0.25. There was a significant linear correlation between birth weight and lean body mass (r2 = 0.83, P = .0001), fat mass (r2 = 0.46, P = .0001), and ponderal index (r2 = 0.22, P = .001). Although the ponderal index has been used as an index of corpulence, the correlation between ponderal index and percent body fat was poor (r2 = 0.15). These results suggest that although neonatal fat mass constitutes only 14% of total birth weight, it explains 46% of its variance. In contrast, the ponderal index explains only 22% of the variance in birth weight and correlates poorly with percent body fat. Body composition analysis explains a significant amount of the variance in normal birth weight. PMID- 1727585 TI - Pregnancy outcome following first-trimester varicella infection. AB - Varicella infection in the first trimester has been associated with a constellation of congenital abnormalities. The incidence of the congenital varicella syndrome is unknown, although it has been reported to be as high as 9%. In a prospective study performed between 1986-1990, 40 patients were identified who had first-trimester varicella infection. Pregnant patients were referred from physicians in the perinatal regional network after developing the classical picture of varicella infection. Targeted fetal ultrasound examinations were performed between 16-20 weeks' gestation in all cases and neonatal outcome was determined. Of the 40 patients, three had first-trimester losses and another underwent an elective termination of pregnancy after counseling. Of the remaining 36 women, one had fetal omphalocele. Thirty-five pregnancies continued until term, and no infant had features of the congenital varicella syndrome at birth. Other than the case of omphalocele, no major congenital anomalies were identified. This study, the largest series of patients with first-trimester varicella infection, showed an incidence of congenital varicella syndrome of 0% and an incidence of congenital anomalies of 3% (range 0-8% at 95% confidence level). PMID- 1727586 TI - Interventricular septal thickness in fetuses of diabetic mothers. AB - Sixty-four diabetic women had their fetuses studied by M-mode echocardiogram between 20-41 weeks of gestation. The mean septal size during both diastole and systole increased in a linear fashion with advancing gestational age in both the normal and diabetic groups. Ventricular septal hypertrophy (ie, more than 2 standard deviations above the normal mean) was present in 48 of 64 (75%) of the fetuses of diabetic women. The ratio of septal size to the anteroposterior cardiac dimension was significantly greater in the diabetic than in the normal group (12 versus 8%; P less than .05). The anteroposterior cardiac dimension indexed to the estimated fetal weight was also greater in the diabetic group. Because both septum and cardiac dimension are larger with maternal diabetes, there may be a specific diabetic cardiomyopathy that originates in utero. PMID- 1727587 TI - Sonographic diagnosis of the large for gestational age fetus at term: does it make a difference? AB - We evaluated 406 women with late third-trimester ultrasound examinations to determine whether the sonographic diagnosis of a large for gestational age (LGA) fetus, defined as an estimated fetal weight at or above the 90th percentile, altered the management of labor and delivery. The sonographic prediction of LGA fetuses had a sensitivity, specificity, and positive predictive value of 50, 90, and 52%, respectively. Women without the sonographic diagnosis of an LGA fetus (N = 338) differed from those with the diagnosis (N = 68) in the frequency of diagnosed labor abnormalities (19 versus 30%, P = .03), use of epidural anesthesia (57 versus 74%, P = .01), and the incidence of cesarean deliveries (32 versus 53%, P = .004). To determine whether it was the sonographic prediction of an LGA fetus or the actual fetal weight that altered clinical management and perinatal outcomes, we stratified the study population into four groups and compared the true negatives with the false positives and the false negatives with the true positives. The incorrect sonographic diagnosis of an LGA fetus had a statistically significant effect on both the diagnosis of labor abnormalities (P = .04) and the incidence of elective cesareans (P = .04) in pregnancies with appropriate for gestational age birth weights. PMID- 1727588 TI - Discordant umbilical arteries: ultrasonographic and Doppler analysis. AB - Discordancy in size between the umbilical arteries was detected during real-time ultrasonographic study of six patients. Cross-sectional and longitudinal views from multiple locations of the three-vessel umbilical cords documented the size discrepancies in a total of 23 serial examinations. The size difference was defined by cursor measurement of the artery diameters. Pulsed- and continuous wave Doppler evaluation of the dissimilar arteries in each cord demonstrated discordant flow velocity waveforms. The mean difference between small and large artery systolic-diastolic ratios (S/Ds) was significant (P less than .0001). Two of the six patients studied had poor perinatal outcomes. Pathologic confirmation of the artery discordance was provided by gross and microscopic examination of an undrained cord segment from a seventh patient who had dissimilar artery sizes and S/Ds on ultrasonographic inspection. The finding of umbilical artery discordance has not been previously described in the obstetric ultrasound literature, and further investigation is warranted. PMID- 1727589 TI - Necrotizing funisitis. AB - Necrotizing funisitis is an umbilical cord lesion characterized by perivascular bands of necrotic Wharton jelly containing inflammatory cells in various stages of degeneration. Sixty cases were reviewed histologically. Clinical information was available in 45. Forty-five age-matched infants with acute (nonspecific) funisitis only were used as controls. Infants with necrotizing funisitis had more stillbirths, birth weights below the tenth percentile (small for gestational age [SGA]), infectious complications, and necrotizing enterocolitis. No consistent infectious agents or predisposing maternal factors were found. Cord neovascularization correlated with SGA infants. Necrotizing funisitis occurred in 0.1% of deliveries greater than 20 weeks' gestation. The perivascular bands, likened to the pattern of an Ouchterlony diffusion plate, suggest the presence of a diffusible toxin in the amniotic fluid. The stillbirths and SGA infants may represent the toxin's effect on the fetus. The lack of perivascular necrotic bands around vessels on the placental surface suggests neutralization or more effective clearing of the agent in this region, for reasons as yet undetermined. The factors underlying the cord lesion may contribute to superimposed acute nonspecific vasculitis and chorioamnionitis. PMID- 1727590 TI - Acyclovir in pregnancy registry: six years' experience. The Acyclovir in Pregnancy Registry Advisory Committee. AB - The Acyclovir in Pregnancy Registry was established to gather data on prenatal exposure to acyclovir. Exposed pregnancies are tracked prospectively to ascertain exposure, risk factors, and pregnancy outcome. Through June 30, 1990, 312 acyclovir-exposed pregnancies had been reported and followed. Of these, 239 were exposed during the first trimester; outcomes included 24 spontaneous fetal losses, 47 induced abortions, 159 live births of infants without congenital abnormalities, and nine outcomes with congenital abnormalities. Among the 73 second- and third-trimester exposures, one infant was born with an abnormality. Exposures are also reported to the registry retrospectively, ie, after the outcome of pregnancy is known. Registry findings to date do not show an increase in the number of birth defects among the prospective reports when compared with that expected in the general population, and there is no consistent pattern of abnormalities among retrospective or prospective reports. These findings should provide some reassurance in counseling women following inadvertent prenatal exposure. The cases accumulated to date represent a sample of insufficient size for reaching reliable and definitive conclusions about the safety of acyclovir for pregnant women and their developing fetuses. Therefore, until further information is available, the Acyclovir in Pregnancy Registry Advisory Committee recommends following the 1989 Centers for Disease Control Sexually Transmitted Diseases Treatment Guidelines for the use of acyclovir in pregnancy, and encourages reporting of all prenatal exposures to the registry (1-800-722-9292, ext. 8465). PMID- 1727591 TI - Preterm birth is associated with increased risk of maternal and neonatal infection. AB - Much information suggests that maternal reproductive tract infections, both recognized and unrecognized, account for an important and possibly preventable portion of preterm births. If such infections do mediate instances of preterm labor and premature rupture of the membranes (PROM), then associated risks of subsequent maternal and neonatal infections would be increased, even after controlling for confounding variables. To evaluate possible associations between preterm birth and maternal and neonatal infections, we conducted a retrospective study of 9642 births at the University of Colorado Health Sciences Center between July 1980 and June 1985. Clinical chorioamnionitis occurred more frequently among women delivering before term with intact membranes at the onset of labor (5.8% preterm versus 1.7% term) and among women with PROM (26.5% preterm versus 6.7% term). Among the women delivered by cesarean, the incidence of postpartum endometritis was higher in those with preterm PROM than in those with term rupture of membranes. The incidence of neonatal infection increased significantly as the gestational age of the neonates decreased (P less than .01). The rate of culture-proven neonatal infection was significantly higher following PROM (P less than .01) than after birth without PROM. Both neonatal infection and perinatal mortality were increased in association with chorioamnionitis in both preterm and term pregnancies. These consistent observations complement and support suggestions that reproductive tract infection plays a possibly preventable role in the pathogenesis of preterm birth. PMID- 1727592 TI - Lack of effect of maternal cocaine administration on myometrial electromyogram and maternal plasma oxytocin concentrations in pregnant sheep at 124-146 days' gestational age. AB - Although cocaine abuse during human pregnancy is associated with an increased incidence of preterm labor, there are few reports on the effects of cocaine on myometrial activity during pregnancy in experimental animals. Cocaine (0.5, 1, or 2 mg/kg) or vehicle was randomly administered intravenously to 15 pregnant ewes between 124-146 days' gestation (term is 147 days). Neither cocaine nor vehicle administration altered total myometrial electromyographic activity from pre-dose levels 1 or 6 hours after administration. Maternal arterial plasma oxytocin did not change during the study. Using a positive control, we confirmed observations of other investigators that administration of 2 mg/kg cocaine significantly increases maternal arterial blood pressure. The results indicate that cocaine does not stimulate myometrial contractility significantly in late pregnancy in sheep. PMID- 1727593 TI - The effect of cigarette smoking, Chlamydia trachomatis infection, and vaginal douching on ectopic pregnancy. AB - We conducted a case-control study of the relation between ectopic pregnancy and three exposures of interest: cigarette smoking, previous chlamydial infection, and vaginal douching. Cases were women with surgically confirmed tubal ectopic pregnancy; controls were women with intrauterine pregnancy at 14 weeks' gestation or less. All women were between the ages of 18-40 and were cared for at the same hospital. Sixty-nine case women and 101 controls were interviewed and underwent serologic tests for Chlamydia trachomatis exposure. Cases were more likely than controls to be nulliparous, non-white, and unmarried and to report a high school education or less (P less than .05). The proportions of cases and controls who reported smoking during the month of conception were 51 and 20%, respectively. The adjusted odds ratio for smoking was 2.4 (95% confidence interval 1.2-5.1) when current smokers were compared with former smokers and women who had never smoked. The proportions of women who had previous chlamydial infection (immunoglobulin G [IgG] greater than 1:64) among cases and controls were 35 and 20% (adjusted odds ratio 1.3, 95% confidence interval 0.6-3.0). Overall, 28% of cases and 19% of controls douched once or more per month (adjusted odds ratio 0.8, 95% confidence interval 0.3-2.2). We conclude that current cigarette smoking may be associated independently with ectopic pregnancy and that smoking cessation before the month of conception may reduce this risk. For these women, previous chlamydial infection and vaginal douching did not appear to increase significantly the risk of ectopic pregnancy. PMID- 1727594 TI - Turnip crinkle virus genes required for RNA replication and virus movement. AB - We have used infectious in vitro transcripts from mutagenized turnip crinkle virus (TCV) cDNA clones to identify the gene products required for viral RNA replication, virion assembly, and intercellular movement. Previous sequence analysis of the TCV genome revealed the presence of five open reading frames which had the potential to encode gene products of 88, 38, 28, 9, and 8 kDa. Inoculation of protoplasts with infectious RNA revealed that only the p28 and p88 gene products are required for viral RNA synthesis. Although the p8 and p9 gene products were dispensable for RNA replication and virion assembly in protoplasts, mutations in the p8 and p9 genes prevented the production of systemic infections in plants. No viral RNA or protein was observed in the inoculated or systemic leaves of plants inoculated with transcripts synthesized from p8 or p9 mutant cDNAs. In contrast to these results, viral RNA was recovered from the inoculated, but not the systemic leaves, of plants inoculated with an RNA lacking the coat protein (CP) gene. With the CP mutant, no symptoms were observed on normally systemic hosts, but small local lesions were induced on Chenopodium amaranticolor. These results indicate that p8, p9, and CP are required for viral movement. PMID- 1727595 TI - A human binding site for transcription factor USF/MLTF mimics the negative regulatory element of human immunodeficiency virus type 1. AB - Transcriptional regulation of the proviral form of the human immunodeficiency virus type 1 (HIV-1) is exerted by its 5' long terminal repeat (LTR), which contains recognition sites for several cell factors. By gel retardation and DNase I footprinting experiments we have identified a binding site for a human nuclear protein between nucleotides -152 to -174 upstream of transcription start site, in a region previously recognized as a negative regulator of transcription (negative regulatory element, NRE). The recognized sequence contains the dyad symmetry element CACGTG, which represents a binding motif, very conserved through evolution, present in a putative human DNA replication origin (pB48), in the upstream element of the major late promoter (MLP-UE) of adenovirus, and, as transcriptional element, upstream of many eukaryotic genes. Common binding activities exist in human nuclear extracts for pB48, MLP-UE and the HIV-1 LTR; at least three protein species recognize the LTR sequence, of 44 (corresponding to transcription factor USF/MLTF), 70, and 110 kDa, respectively. Chloramphenicol acetyltransferase assays suggest that the USF/MLTF binding site located in the HIV-1 LTR acts as a negative regulator of transcription, and that it contributes to the overall negative function exerted by the NRE. An oligonucleotide corresponding to another characterized human USF/MLTF binding site can functionally replace part of the activity of the NRE. This negative function is exerted both in presence or absence of tat transactivation, in different cell lines, and after PMA stimulation. PMID- 1727596 TI - The establishment of rodent cell lines persistently producing HIV-1. AB - Animal cells differ in susceptibility to HIV-1 infection. To identify rodent cells which are permissive to HIV-1 replication, we transfected murine and rat cells with an infectious clone of HIV-1 and a vector containing the chloramphenicol acetyl transferase gene under the control of HIV-1 LTR. Three groups of transfectants were distinguished: (i) Cells which permit neither HIV-1 LTR activation nor viral protein expression; (ii) Cells which permit activation of the HIV-1 LTR but not HIV-1 protein expression; and (iii) Cells which are fully permissive to both HIV-1 LTR activation and virus production. The latter included rat embryonal fibroblastoid (Rat2) cells, which, in short-term transfection assays, produced titers of HIV-1 proteins similar to transfected T lymphoid cells. To establish persistently infected cells, Rat2 cells were stably transfected with a plasmid containing an infectious clone of HIV-1/N1T-A and a neo gene, yielding several G-418-resistant, HIV-1-producing cell cultures. Of these, Rat2/A1 and Rat2/A2 cell cultures expressed up to 60 ng HIV-1 p24 core antigen per 1 x 10(6) cells 3 days after cell subculture over a period of 3 months. Southern blot hybridization revealed that Rat2/A1 and Rat2/A2 carried one to two HIV-1 DNA copies per cell; no rearrangements or deletions in viral DNA were present. Restriction endonuclease analysis of HIV-1 DNA in Rat2/A2 cells suggested clonal expansion of cells containing integrated HIV-1 genome. Virus produced by the Rat2/A1 cells was infectious in human T cells. These data demonstrate that some rodent cells have no inherent restriction to persistent and efficient production of infectious HIV-1. PMID- 1727597 TI - Synthesis of full-length transcripts of beet western yellows virus RNA: messenger properties and biological activity in protoplasts. AB - Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-). The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems. The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation. Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA. Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts. Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission. PMID- 1727598 TI - In vitro synthesis of an infectious viroid: analysis of the infectivity of monomeric linear CEV. AB - Infectious monomers of citrus exocortis viroid (CEV) were synthesized in vitro precisely to predetermined sequences in microgram quantities without resorting to cloning procedures. Amplification of CEV double-stranded cDNAs fused with a T7 RNA polymerase promoter was followed by transcription of the DNA resulting in the production of an infectious linear CEV monomer. This is the first demonstration of an infectious unit length viroid synthesized in vitro. Transcripts containing 3'-OH terminal groups were infectious, demonstrating that a 2',3'-cyclic phosphate terminus is not a prerequisite for viroid infectivity as previously suggested. Conversion of the 5'-triphosphate terminus to either 5'-monophosphate or 5'-OH had little effect on infectivity. The linear RNA could be circularized using T4 RNA ligase to produce an authentic CEV molecule. This procedure, which results in the production of biologically active RNA, would allow routine application of oligonucleotide-directed mutagenesis to the study of viroids and other circular RNAs. It would also enable the in vitro synthesis and mutagenesis of infectious viral RNAs containing a 5'-G residue. PMID- 1727599 TI - Site-directed mutagenesis of the C-terminal portion of reovirus protein sigma 1: evidence for a conformation-dependent receptor binding domain. AB - Oligonucleotide site-directed mutagenesis was used to modify the type 3 (T3) reovirus cell attachment protein sigma 1 at residues located in the three regions (designated C, D, and E in the C-terminal one-third of sigma 1) that are highly conserved between the three reovirus serotypes. Of the eight residues targeted for mutagenesis, five (one in region C, and two each in regions D, and E) are conserved among all three proteins. Wild-type (wt) and mutant sigma 1 forms were synthesized in an vitro transcription/translation system and subjected to structural and functional analysis. None of the mutations affected the ability of sigma 1 to form trimers. However, mutation (all representing drastic changes) in any of the five triply conserved residues (Tyr326, Asn369, Phe370, Tyr450, and Pro451) caused a complete or partial abrogation of sigma 1 cell binding function, whereas mutation in any of the other three residues (Ser325, Ser327, and Asp365) had no adverse effect. The structural integrity of the mutant proteins was then probed using trypsin, chymotrypsin, and a neutralizing monoclonal anti-sigma 1 antibody. In all cases, the loss of cell binding function was associated with a drastic conformational change in the C-terminal globular head of sigma 1. These results suggest that conserved residues in the three highly conserved regions in the C-terminal portion of sigma 1 play important structural and functional roles and are involved in proper head folding and generation of a conformation dependent receptor binding domain. PMID- 1727600 TI - A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo. AB - The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus. PMID- 1727601 TI - Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells. AB - Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag pol open reading frame did not yield particles. PMID- 1727602 TI - Identification of immunodominant epitopes in envelope glycoprotein of human T lymphotropic virus type II. AB - A series of synthetic peptides derived from the envelope glycoprotein of human T lymphotropic virus type II (HTLV-II) was used in an enzyme immunoassay to determine the immunodominant epitopes of envelope glycoprotein. Of the 11 synthetic peptides spanning the external glycoprotein of HTLV-II (gp52) and the 3 from the transmembrane protein (gp21), 3 peptides from gp52 (termed Env-20(85 102), Env-202(173-209), and Env-203(219-256] reacted with most of the polymerase chain reaction-confirmed HTLV-II specimens (83, 95, and 76%, respectively); all other peptides reacted minimally with these specimens. Env-202(173-209) reacted with a greater percentage (91 to 100%) of specimens from different risk groups, including intravenous drug users (n = 30), North American Indians (n = 13), Guaymi Indians from Panama (n = 22), and routine U.S. blood donors (n = 34), when compared with Env-20(85-102) (73 to 100%) or Env-203(219-256) (68 to 83%). Furthermore, Env-20(85-102) and Env-202(173-209) had some reactivity (8-25%) with sera from HTLV-I-infected individuals, whereas Env-203(219-256) reacted with 58% of HTLV-I specimens. We conclude that peptides Env-20(85-102) and Env-202(173 209) represent the type-specific immunodominant epitopes of HTLV-II external glycoprotein. PMID- 1727603 TI - The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2. AB - We report the sequence of 7558 nucleotides of the adenovirus type 5 genome. With this sequence and previously published data, the complete sequence of this genome is now available and can be compared with the already known sequence of the adenovirus type 2 genome. These two serotypes belong to the same subgroup and sequence comparison shows 94.7% homology between the two genomes. The differences are not at all randomly distributed. Transitions between C and T and between A and G account in total for 58.3% of the differences and even for 68.6% for the genome devoid of the fiber and the hexon genes (instead of 33% expected for an equal probability of changes). In the fiber gene the transitions account for 47% of the differences. The detailed analysis of the nucleotide substitution between the two genomes suggests that the Ad2 genome could derive from that of Ad5 one, with the exception of the fiber gene which is likely to be present in Ad2 genome as a result of genetic recombination. The homology between the amino acids sequences of the structural proteins varies from 100% (proteins pVII and IX) to only 69.2% for the fiber. PMID- 1727604 TI - Determinants of tomato golden mosaic virus symptom development located on DNA B. AB - Infectious clones have been constructed from two strains of the bipartite geminivirus tomato golden mosaic virus. The common strain and the yellow vein strain show marked phenotypic differences in Nicotiana benthamiana which are reproduced following infection with the cloned viral genomes. Pseudorecombinants between the two strains, produced by exchange of genome components (DNAs A and B), established that the difference in symptoms in several species of the Solanaceae is determined by DNA B. Recombinants produced in vitro between the DNA B components showed that determinants of symptom development map to the common region and gene BL1. DNA B is known to carry functions necessary for spread of viral DNA through the host plant. Our results emphasize the link between symptom type and virus spread. PMID- 1727605 TI - Composition of the helical internal components of influenza virus as revealed by immunogold labeling/electron microscopy. AB - The composition of the large helical internal components of influenza virus was investigated by immunogold labeling/electron microscopy with antibodies to the nucleoprotein (NP), matrix protein (M), and polymerase complex (PB1, PB2, and PA) of the virus. The morphologically intact helices, obtained by air-drying of the virions on the electron microscope grid, showed little or no labeling with any of the above antibodies. However, partial to full degradation of the helix by proteinase K (2 ng/ml) prior to immunogold labeling made the helices accessible to all three antibodies. The results are consistent with a model that the helix represents a polymer of M protein enclosing or containing the influenza ribonucleoprotein(s). PMID- 1727606 TI - Localization of a surface domain of the capsid protein of barley yellow dwarf virus. AB - We describe a method for localizing protein domains situated at the surface of virus particles. A cDNA clone of the New York PAV isolate (NY-PAV) of barley yellow dwarf virus (BYDV) containing the capsid protein gene was generated and sequenced. A defined set of overlapping cDNA fragments specific to the capsid protein ORF of NY-PAV was subcloned into the pGEX expression vectors. Cells of Escherichia coli carrying these plasmids synthesize recombinant glutathione S transferase/capsid proteins. These proteins were used in immunoblot experiments to localize the epitopes of three PAV-BYDV-directed monoclonal antibodies to a 20 amino acid-long segment of the NY-PAV capsid protein. All three monoclonal antibodies reacted with NY-PAV virions in a triple antibody sandwich enzyme linked immunosorbent assay, indicating that their epitopes are located at the virion surface. PMID- 1727607 TI - Functional roles of the V3 hypervariable region of HIV-1 gp160 in the processing of gp160 and in the formation of syncytia in CD4+ cells. AB - To study the roles of the V3 hypervariable region (amino acid residues 301-336) of the HIV-1 envelope glycoprotein (gp160) during infection, we constructed recombinant vaccinia viruses that expressed either wild-type gp160 (v-env10) or mutant gp160 in which the V3 region was deleted (v-dl29.1 and v-dl29.2). In v dl29.1 the V3 loop, formed by disulfide bonding between cysteine residues 301 and 336, was deleted from cys301 to cys336 (inclusive) and replaced by one serine residue. In v-dl29.2 the V3 loop was deleted from arg303 to ala334 and replaced by three residues: gly-ala-gly. Cells infected with all three recombinant vaccinia viruses expressed gp160 on the cell surface, but v-dl29.1-derived gp160 was not cleaved into gp120 and gp41 and did not bind the CD4 glycoprotein. In contrast, gp160 produced by recombinant v-dl29.2 was cleaved normally, and the mutant gp120 produced was secreted and retained binding activity to CD4+ cells. However, both mutants failed to induce syncytia in HeLa CD4+ cells. Thus a disulfide loop at the V3 portion of gp160 is required for cleavage into gp120 and gp41, presumably because the loop is required for proper tertiary structure. The sequence within the V3 loop, however, is not required for cleavage and secretion of gp160, or for binding to CD4+, but this region is essential for gp120-mediated syncytia formation. PMID- 1727608 TI - The hemagglutinin/esterase gene of human coronavirus strain OC43: phylogenetic relationships to bovine and murine coronaviruses and influenza C virus. AB - The complete nucleotide sequences of the hemagglutinin/esterase (HE) genes of human coronavirus (HCV) strain OC43 and bovine respiratory coronavirus (BRCV) strain G95 were determined from single-stranded cDNA fragments generated by reverse transcription of virus-specific mRNAs and amplified by polymerase chain reaction. An open reading frame of 1272 nucleotides was identified as the putative HE gene by homology to the bovine coronavirus HE gene. This open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kDa. Ten potential N-linked glycosylation sites were predicted in the HE protein of HCV-OC43 while nine of them were present in BRCV-G95. Fourteen cysteine residues were conserved in the HE proteins of both viruses. Two hydrophobic sequences at the N-terminus and the C-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. The predicted HE protein of HCV-OC43 was 95% identical to the HEs of BRCV-G95 and other bovine coronaviruses, and 60% identical to the HEs of mouse hepatitis viruses. Phylogenetic analysis suggests that the HE genes of coronaviruses and influenza C virus have a common ancestral origin, and that bovine coronaviruses and HCV-OC43 are closely related. PMID- 1727609 TI - Isolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. AB - Influenza viruses were disrupted layer by layer with the nonionic detergent NP-40 at fixed pH. Treatment of the virions with NP-40 at neutral or mildly alkaline pH (6.8-8.0) yielded viral core structures containing M1 protein. The matrix M1 protein was selectively extracted from cores at acidic pH 3.0-4.5 with citrate, acetate, and phosphate buffers or with morpholinoethanesulfonic acid. The resulting M1 protein sedimented in a glycerol gradient with a coefficient of 2.8 S and most likely existed as a monomeric form of the 27,000-Da polypeptide. An antigenic map of the monomeric protein M1 tested with a panel of monoclonal anti M1 antibodies was found to be similar to those of the assembled M1 protein in whole virions. The isolated M1 protein retained biological properties and inhibited the RNA polymerase activity of viral RNP. This transcription-inhibition function of M1 monomers was specifically restricted by one of the monoclonal antibodies studied. PMID- 1727610 TI - Oligomeric organization and strain-specific proteolytic modification of the virion M2 protein of influenza A H1N1 viruses. AB - The M2 protein of influenza A H1N1 strains PR8, WS, and WSN is present in homooligomeric forms in virions grown in the allantoic cavity of embryonated eggs. The bulk of the virion M2 is detected as tetramers and dimers. The oligomeric forms of PR8 virions differ from those of WS and WSN not only in apparent molecular weight (MW) but also in that they seem to be composed of two types of monomers differing in MW by approximately 1.5 kDa. Evidence from monoclonal antibody binding (or lack of it) and from in vitro trypsin digestion suggests that, in ovo, the external NH2 region of some PR8 M2 monomers is proteolytically trimmed, resulting in heterogeneous oligomers composed of cleaved and uncleaved monomers in different proportions. PMID- 1727611 TI - Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex. AB - To study the intracellular transport and targeting to the Golgi complex of the membrane glycoproteins G1 and G2 of Hantaan virus, we have expressed them together and separately using recombinant vaccinia viruses. When expressed from the same recombinant vaccinia virus, G1 and G2 were localized to the Golgi complex as analyzed by both immunofluorescence and subcellular fractionation. However, when the glycoproteins were expressed from separate recombinant viruses, both proteins remained in the endoplasmic reticulum. Using several monoclonal antibodies, it was found that G1 expressed alone did not acquire its correct conformation. Finally, if cells were coinfected with G1- and G2-expressing recombinant viruses, the proteins were again targeted to the Golgi complex. The N linked glycans remained in all cases largely endoglycosidase-H sensitive. With none of the recombinant viruses were expression of the glycoproteins observed on the cell surface. Neither did chasing in the presence of cycloheximide result in the surface expression of G1 or G2. Our results indicate that for transport out of the endoplasmic reticulum and proper targeting to the Golgi complex, the two glycoproteins have to be coexpressed. The most likely interpretation is that G1 and G2 have to interact with each other in the endoplasmic reticulum in order to become transport competent. PMID- 1727612 TI - A stable flank of unstable lymphotropic papovavirus integration sites is associated with a cellular S1 nuclease-sensitive sequence. AB - Integration of viral DNA sequences is associated with stable transformation by viruses of the polyomavirus genus. In LPV-HE cells, a hamster embryonal cell line transformed by the African green monkey lymphotropic papovavirus (LPV), viral integration is unstable during tissue culture passage, leading to subclones with rearrangements of the original integration locus. Three of four viral integrations that were molecularly cloned together with flanking cellular sequences are identical in one junction but are different from each other at the other flank. The "stable" flank which is already present in early passage cells contains an S1 nuclease-sensitive sequence located approximately 60 bp outside of the viral-cellular junction. The presence of this site may contribute to the stability of the flanking region. PMID- 1727613 TI - Channel catfish virus: a new type of herpesvirus. AB - Herpesviruses are large double-stranded DNA viruses which infect vertebrates from fish to man. Convincing genetic relationships between herpesviruses that infect higher vertebrates (birds and mammals) have been demonstrated previously by comparing proteins predicted from DNA sequences and have been interpreted as a result of evolution from a common ancestor. In order to evaluate how herpesviruses of lower vertebrates fit into this scheme, the 134,226-bp genome of channel catfish virus was sequenced. Genetic comparisons indicate that a separate evolutionary origin for this virus must be considered. The findings impact upon current perceptions of herpesvirus evolution and gene function. PMID- 1727614 TI - Progression and remission of renal disease in the Lupus Nephritis Collaborative Study. Results of treatment with prednisone and short-term oral cyclophosphamide. AB - OBJECTIVE: To describe the clinical course of severe lupus nephritis and to identify the risk factors for progression to renal failure among patients treated with prednisone and short-term courses of low-dose oral cyclophosphamide. DESIGN: Ancillary analyses of data from the Lupus Nephritis Collaborative Study (LNCS). SETTING: University hospital medical centers (14). PATIENTS: The 86 patients who participated in the LNCS (mean follow-up, 136 weeks [2.6 years]) and a subgroup of 63 patients with follow-up of more than 48 weeks (mean follow-up, 160 weeks [3.1 years]). MEASUREMENTS: Initial clinical and pathologic features, response to therapy within 48 weeks, and subsequent clinical events, including development of renal failure. MAIN RESULTS: Renal failure developed in 18 patients (21%). An observed elevation in serum creatinine concentration was the only initial feature predictive of subsequent renal failure. Mean (+/- SD) initial serum creatinine levels were higher in patients who subsequently developed renal failure (244 +/- 134 mumol/L [2.76 +/- 1.52 mg/dL] compared with 163 +/- 103 mumol/L [1.85 +/- 1.17 mg/dL]; P = 0.007). The risk for renal failure was higher among patients with initial serum creatinine levels greater than 106 mumol/L (1.2 mg/dL) (29% compared with 6.5%; P = 0.014). Response to therapy (defined as resolution of initial serum creatinine elevations within 48 weeks) refined the prognosis based on initial serum creatinine determinations. The risk for subsequent renal failure was higher among patients who failed to respond to therapy within 48 weeks (30% compared with 0%; P = 0.015). By comparison, 9% of patients with normal initial serum creatinine levels progressed to renal failure after 48 weeks. CONCLUSIONS: Initial serum creatinine levels and responses to initial therapy with prednisone and short-term cyclophosphamide, as used in the LNCS, can guide further therapy. Patients with normal initial serum creatinine levels or resolution of initial serum creatinine elevations within 48 weeks have a low risk for renal failure and may not require long-term treatment with cyclophosphamide. PMID- 1727615 TI - Emphysema-like pulmonary disease associated with human immunodeficiency virus infection. AB - OBJECTIVE: To describe a possible association between prolonged infection with human immunodeficiency virus (HIV) and a pathophysiologic process suggestive of pulmonary emphysema. DESIGN: Case series. SETTING: The Ohio State University Hospital, Columbus, Ohio. MEASUREMENTS AND MAIN RESULTS: We describe four HIV seropositive individuals ranging in age from 32 to 55 years who presented with dyspnea. Radiographic examination of the chest showed no infiltrates. All patients were presumed to have had prolonged HIV infection (mean CD4 count, 99.8 +/- 43 cells/mm3), but none had a previous history of pneumonia or opportunistic infections. Comprehensive examination of bronchoalveolar lavage fluid showed no pathogens or other complications of HIV infection. All patients had markedly abnormal pulmonary function tests that were suggestive of emphysema with air trapping, hyperinflation, and a markedly decreased diffusing capacity. However, only minimal evidence of airflow obstruction was noted. Three patients subsequently had high-resolution computed tomographic scans of the chest that revealed emphysema-like bullous changes. Known causes of emphysema were not present in these patients. CONCLUSIONS: Our findings support an association between prolonged HIV infection and an emphysema-like process. This syndrome may occur in the absence of previous pulmonary infections or apparent pulmonary complications and is characterized by unusual pulmonary function test abnormalities. PMID- 1727616 TI - Quality of life in persons with human immunodeficiency virus infection: measurement by the Medical Outcomes Study instrument. AB - OBJECTIVE: To assess the reliability and validity of the Medical Outcomes Study (MOS) Short Form Health Survey as an indicator for quality of life in patients infected with the human immunodeficiency virus (HIV). DESIGN: Patient interview survey. SETTING: The AIDS Health Services Program in seven sites: Newark and Jersey City, New Jersey; Nassau County, New York; Atlanta, Georgia; Dallas, Texas; Fort Lauderdale and Miami, Florida; New Orleans, Louisiana; and Seattle, Washington. PATIENTS: Patients (520) with HIV infection receiving health services at one of the above sites. MEASUREMENTS: All components of the MOS Short Form Health Survey were included in the interview. Minor modifications were made to adapt the survey to the particular circumstances of the study. Measured sociodemographic characteristics included age, sex, race, intravenous drug use, and education. Symptoms were assessed by closed-ended questions concerning memory, seizure, weakness or numbness, fever, chills, diaphoreses, dyspnea, diarrhea, and weight loss. Information on the frequency of symptoms was also collected. History of Pneumocystis carinii pneumonia and Kaposi sarcoma was noted. MAIN RESULTS: The sociodemographic characteristics resemble those of patients with the acquired immunodeficiency syndrome (AIDS) reported to the Centers for Disease Control (CDC): mean age, 36; men, 89%; nonwhite, 31%; intravenous drug use, 34%. Neurologic symptoms (memory trouble, seizures, weakness or numbness) occurred in 71% of patients; constitutional symptoms (fever, chills, night sweats, weight loss) in 69%; dyspnea in 50%; and diarrhea in 47%. Although older age, female sex, nonwhite race, and intravenous drug use were associated with lower MOS scores in several areas, the strongest single or adjusted indicator of lower MOS scores was the presence of symptoms. Finally, patients with HIV infection had significantly lower scores than did previously reported patients with other chronic medical conditions (P less than 0.001). CONCLUSIONS: The MOS survey is a reliable measure of quality of life for patients with HIV infection. These patients tend to have low scores, suggesting validity of the survey. The MOS survey is extremely sensitive to the effect of symptoms, which suggests that it might be useful as a quality-of-life indicator for AIDS clinical drug trials. PMID- 1727617 TI - Reversible orthodeoxia and platypnea due to right-to-left intracardiac shunting related to pericardial effusion. PMID- 1727618 TI - The cause and pathogenesis of the eosinophilia-myalgia syndrome. AB - OBJECTIVE: To review recent advances in the understanding of the cause and pathogenesis of the eosinophilia-myalgia syndrome associated with ingestion of L tryptophan. DATA SOURCES: Studies published from 1989 to 1991 were identified using a MEDLINE literature search. Additional references were selected from the bibliographies of identified articles. DATA SYNTHESIS: The eosinophilia-myalgia syndrome was epidemiologically associated with ingestion of L-tryptophan containing preparations. Analysis of case-associated lots of L-tryptophan has revealed several chemical impurities. One of these, labeled "peak E," is an unusual dimeric form of L-tryptophan (1,1'-ethylidenebis[tryptophan]), and its presence is associated with the eosinophilia-myalgia syndrome (P = 0.022). Evidence of abnormal metabolism of tryptophan has been found in some patients with the syndrome. Eosinophil activation and the release of major basic protein and other eosinophil-derived toxic proteins into the extracellular space is a striking feature in the eosinophilia-myalgia syndrome and implicates eosinophils or their products in the pathogenesis. Mononuclear cell activation and infiltration of various affected tissues as well as fibrosis of the integument and of the connective tissue components of blood vessels, nerves, and muscles are additional frequent findings. CONCLUSIONS: Current evidence suggests that the epidemic of the eosinophilia-myalgia syndrome was caused by contaminated L tryptophan preparations originating from a single manufacturer. Peak E or other, as yet unidentified, contaminants may trigger activation of eosinophils and inflammatory cells and increase biosynthesis of connective tissue components, resulting in the clinical and pathologic manifestations of the eosinophilia myalgia syndrome. Further studies of the interaction of eosinophils, inflammatory cells, and fibroblasts may increase the understanding of the pathogenesis of the eosinophilia-myalgia syndrome. The insights gained from the epidemic may be applicable to more common idiopathic diseases associated with eosinophilia and fibrosis. PMID- 1727619 TI - The multichain interleukin-2 receptor: a target for immunotherapy. AB - Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection. PMID- 1727620 TI - Playing God. PMID- 1727621 TI - Reflections on "Playing God". PMID- 1727622 TI - The internist as sports medicine physician. PMID- 1727623 TI - Outcomes assessment and quality of life in patients with human immunodeficiency virus infection. PMID- 1727624 TI - Transesophageal echocardiography for aortic aneurysm. PMID- 1727625 TI - Physicians and preventive services. PMID- 1727626 TI - Nonconvulsive generalized status epilepticus and AIDS. PMID- 1727627 TI - Scutwork and the real world. PMID- 1727628 TI - Surviving sudden death: whatever happened to Webster? PMID- 1727629 TI - Beta-oxidation of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid by MOLT-4 lymphocytes. AB - MOLT-4 lymphocytes metabolize 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S) HETE via beta-oxidation with retention of the hydroxyl group at the omega 9 carbon atom. The isolation of 6-hydroxy-4,8-tetradecadienoic acid documents that these cells have the capacity to catabolize the conjugated diene system. 12(S) HETE was also metabolized to 3,12-dihydroxy-8,10,14-eicosatrienoic acid and 1,9 dihydroxy-5,7,11-heptadecatriene as well as to 17- and 19-carbon aldehydes. When MOLT-4 cells were incubated with the beta-oxidation product, 10-hydroxy-6,8,12 octadecatrienoic acid, it was in part further catabolized but in addition it served as an anabolic precursor as defined by the accumulation 3,12-dihydroxy 8,10,14-eicosatrienoic acid as well as 1,11-dihydroxy-7,9,13-nonadecatriene. Neither 10-hydroxy-6,8,12-octadecatrienoic acid nor 13-hydroxy-5,8,11 octadecatrienic acid was as potent in inhibiting phytohemagglutin-induced lymphocyte mitogenesis as were their parent compounds--i.e., 12(S)- and 15(S) HETE. These findings argue against the hypothesis that beta-oxidation products of 12(S)- and 15(S)-HETE are the potential modulators of lymphocyte function. However, neither the pathway for synthesis, nor the role of odd chain aldehydes and diols as potential lipid mediators was determined in this study. PMID- 1727630 TI - Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization. AB - Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation. PMID- 1727631 TI - Stable expression of cytochrome P450IIIA7 cDNA in human breast cancer cell line MCF-7 and its application to cytotoxicity testing. AB - A mammalian cell expression plasmid containing cytochrome P450IIIA7 complementary DNA was constructed. Breast cancer cells (MCF-7) were transfected with the plasmid and neomycin-resistant selection marker plasmid. We established three cell lines, termed M13, M21, and M27, which expressed the cytochrome P450IIIA7 as examined by RNA blot and immunoblot analyses. These cell lines showed 8- to 10 fold higher sensitivity against aflatoxin B1 compared to parental MCF-7 cells, suggesting that cytochromes P450IIIA7 expressed in the cells were responsible for the production of the cytotoxic metabolite of aflatoxin B1. PMID- 1727632 TI - The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantz). AB - A microsomal system catalyzing the in vitro synthesis of the aglycones of the two cyanogenic glucosides linamarin and lotaustralin has been isolated from young etiolated seedlings of cassava (Manihot esculenta Crantz). A prerequisite to obtain active preparations is the complete removal of the endosperm pellicle covering the cotyledons before seedling homogenization. The rates of conversion of the parent amino acids valine and isoleucine to their cyanohydrins are 19 and 6 nmol/h/mg protein, respectively. The conversion rates for the corresponding oximes (2-methylpropanal oxime and 2-methylbutanal oxime) are 475 and 440 nmol/h/mg protein and for the nitriles (2-methylpropionitrile and 2 methylbutyronitrile) 45 and 75 nmol/h/mg protein. With the exception of 2 cyclopentenylglycine, none of the additionally tested amino acids are metabolized, whereas a broad substrate specificity is observed using oximes and nitriles as substrates. The in vitro biosynthesis is photoreversibly inhibited by carbon monoxide, demonstrating the involvement of cytochrome P450 in the hydroxylation processes. All tissues of the cassava seedling contain cyanogenic glucosides. The microsomal enzyme system responsible for their synthesis is restricted to the cotyledons and their petioles. This demonstrates that the cyanogenic glucosides are actively transported to other parts of the seedling. The enzyme activity decreases with the height of the etiolated seedling and is barely detectable in seedlings above 75 mm. PMID- 1727633 TI - Binding of bile acids, organic anions, and fatty acids by bovine intestinal Z protein. AB - Z protein from bovine small intestinal mucosa was purified and its binding affinities for bile acids, organic anions, and fatty acids were compared with those of bovine hepatic Z protein. Purification of Z protein from intestinal and hepatic cytosol was performed by gel filtration, chromatofocusing, and hydroxyapatite chromatography. Both purified proteins had the same molecular weight (Mr 14,000) and eluted from a chromatofocused gel at about pH 10. Binding studies were performed by the competitive displacement of 8-anilinonaphthalene-1 sulfonic acid and by equilibrium dialysis. Binding affinities for bile acids, organic anions, and fatty acids were very similar between intestinal and hepatic Z proteins. Although the real physiologic role of Z protein remains to be further elucidated, these data indicate that intestinal Z protein participates in the mechanism of intracellular bile acid transfer in enterocytes. PMID- 1727634 TI - Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction. AB - A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea. In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa). A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not. In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP thioredoxin reductase (NTR) were evidenced. Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs. Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs. Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak. The algal thioredoxin h was also slightly active with spinach and E. coli NTRs. These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments. PMID- 1727635 TI - Role of vesicular traffic in the transport of surface transferrin receptor to the Golgi complex in cultured human cells. AB - We have previously shown that transferrin receptor (TfR) recycles from the cell surface through the Golgi complex in K562 human leukemia cells. However, little is known about the transport pathway that carries these receptors to the Golgi complex. To learn more about this transport, we studied the effects of treatments that block specific types of vesicular traffic. K562 cells were cultured in test media and the transport of surface TfR to the Golgi complex was assessed by measuring the entry of asialo-TfR into the sialyltransferase compartment of the Golgi complex. Depletion of cellular potassium, which blocks formation of coated vesicles at the cell surface, stimulated asialo-TfR resialylation by 60% over controls, suggesting that coated vesicle formation is not the rate-limiting step in cell surface-to-Golgi transport. Similarly, culture in sodium-free medium, which blocks transport from endosomes to lysosomes, increased asialo-TfR resialylation by 40%, arguing that lysosomes do not lie on the transport pathway. In contrast, incubation of cells in hypertonic medium, which blocks many vesicular transport steps, inhibited TfR resialylation by 40%, confirming the importance of vesicular traffic in transport of asialo-TfR from the cell surface to the Golgi complex. These results are consistent with two possible pathways for cell surface-to-Golgi transport. Receptor could be transported via an endosomal intermediate, with the rate-limiting step occurring at a post-endosomal site. Alternatively, receptor could be transported directly to the Golgi via a pathway that does not involve endosomes. PMID- 1727636 TI - Amyloid-like properties of a synthetic peptide corresponding to the carboxy terminus of beta-amyloid protein precursor. AB - A synthetic peptide whose sequence corresponds to the 20 carboxy-terminal amino acids of beta-amyloid protein precursor (APP) was found to form fibrils in vitro. These fibrils showed birefringence in polarized light when stained with Congo red, fluoresced when bound with thioflavin S, were resistant to proteases, and had a cross-beta conformation. By contrast, peptides with other sequences from the intracellular domain of APP and a peptide corresponding to this entire domain did not exhibit the full range of beta-amyloid properties. These results suggest that a fragment from the C-terminus of the beta-amyloid protein precursor could bind to intraneuronal paired helical filaments and account for some of its amyloid-like properties. PMID- 1727637 TI - Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions. AB - The soluble P450 isolated from Bacillus megaterium (the product of the CYP 102 gene) (P450BM-3) is a catalytically self-sufficient fatty acid hydroxylase which converts lauric, myristic, and palmitic acids to omega-1, omega-2, and omega-3 hydroxy analogs. The percentage distribution of the regioisomers depends on the substrate chain length. Lauric and myristic acids were preferentially metabolized to their omega-1 hydroxy counterparts while no hydroxylation occurred when capric acid was used as the substrate. Palmitic acid, when present at concentrations greater than the concentration of oxygen in the reaction medium (greater than 250 microM), was hydroxylated to its omega-1, omega-2, and omega-3 hydroxy analogs, with the percentage distribution of the regioisomers being 21:44:35, respectively. No omega hydroxylation of any of the fatty acids was detected. When the concentration of palmitic acid was less than the concentration of oxygen in the reaction mixture, it was noted that a number of additional products were formed. Under these conditions, unlike lauric and myristic acids, it was observed that palmitic acid was first converted to its monohydroxy isomers which were subsequently metabolized to a mixture of 14-ketohexadecanoic, 15 ketohexadecanoic, 13-hydroxy-14-ketohexadecanoic, 14-hydroxy-15-ketohexadecanoic, and 13,14-dihydroxyhexadecanoic acids with a relative distribution of 8:2:40:30:20, respectively. Thus, P450BM-3 is able not only to monohydroxylate a variety of fatty acids but also to further metabolize some of these primary metabolites to secondary and tertiary products. The present paper characterizes the products formed during the sequential hydroxylation of palmitic acid and proposes reaction pathways to explain these results. PMID- 1727638 TI - Reduction of prostaglandin H synthase compound II by phenol and hydroquinone, and the effect of indomethacin. AB - The reduction of prostaglandin H synthase compound II to native enzyme by phenol and by hydroquinone, in the presence of diethyldithiocarbamate as a stabilizing agent, was studied by rapid scan spectrometry and transient state kinetics at 4.0 +/- 0.5 degrees C in 0.1 M phosphate buffer, pH 8.0. The plot of pseudo-first order rate constants for the conversion of prostaglandin H synthase compound II to native enzyme versus phenol concentration was linear with a non-zero intercept. The second-order rate constant was determined from the slope to be (5.3 +/- 0.3) x 10(5) M-1 s-1. For the reduction by hydroquinone, the second order rate constant was determined from pointwise measurements of the pseudo first-order rate constant to be (2.1 +/- 0.4) x 10(6) M-1 s-1. Rapid scan spectrum results also showed the reduction of compound I to compound II by both phenol and hydroquinone. Thus reduction of both compound I and compound II is one electron process. Our results suggest that the tyrosyl radical, detected in the presence of oxidizing agents, is formed by intramolecular electron transfer from the tyrosyl residue to the porphyrin pi-cation radical, and this reaction tends to disappear in the presence of sufficient reducing substrate. These in vitro results support speculation that there is a role of the peroxidase component of prostaglandin H synthase in benzene-induced toxicity. In the present work, the effect of indomethacin on the reduction of prostaglandin H synthase compound II by diethyldithiocarbamate, phenol, and hydroquinone was also investigated. Results revealed, for the first time, that indomethacin is an inhibitor of the peroxidase activity of prostaglandin H synthase, although not as effectively as in its well-known inhibition of cyclooxygenase activity. PMID- 1727639 TI - Mechanisms of cellular resistance to hydrogen peroxide, hyperoxia, and 4-hydroxy 2-nonenal toxicity: the significance of increased catalase activity in H2O2 resistant fibroblasts. AB - An H2O2-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line which demonstrates a 20-fold increase in catalase activity was utilized in the study of mechanisms responsible for cellular resistance to hydrogen peroxide, oxygen, and 4-hydroxy-2-nonenal toxicity. HA1 and OC14 cells were treated with 9 mM aminotriazole which resulted in a 60 to 80% reduction in catalase activity. Pretreatment with aminotriazole resulted in significant sensitization to the toxicity of 1-h exposures to exogenously applied H2O2, which was proportional to the reduction in catalase activity. Treatment with aminotriazole produced significant sensitization to the toxicity of 95% O2 after 45 h of O2 exposure but no sensitization to the toxicity of a 1-h exposure to 50 microM 4-hydroxy-2 nonenal. Inhibition of catalase activity by aminotriazole had no effect on the metabolism of 4-hydroxy-2-nonenal by either cell line tested. These results support the conclusion that in H2O2-resistant cells, catalase activity is a major determinant of cellular resistance to H2O2 toxicity, whereas catalase activity has a limited role in cellular resistance to an acute exposure to 95% O2 and is unrelated to cellular resistance to 4-hydroxy-2-nonenal. PMID- 1727640 TI - Expression of P4501A1 in a primary culture of rainbow trout hepatocytes exposed to beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. AB - Primary cultures of rainbow trout hepatocytes were used to study the expression of CYP1A1 mRNA, its protein product (P4501A1), and catalytic activities (7-ethoxy resorufin-O-deethylase, EROD) during a 96-h period after exposure of cells to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10 degrees C. Cells were exposed to the inducers for 48 h after 24 h of preculturing. The EROD activity of BNF-treated hepatocytes was higher than that of the control hepatocytes 12 h after addition of the inducer. Activities peaked after 48 h and had declined to control levels after 72 h. EROD activity in TCDD-treated cells was significantly elevated after 12 h, but in contrast to BNF-exposure, activities continued to increase during the experimental period. The content of P4501A1 protein, measured with an indirect ELISA technique (using anti-codP4501A1 IgG), increased linearly during the first 12 h and remained constant thereafter. In TCDD-exposed cells the immunochemically determined P4501A1 levels changed in parallel with EROD activity. CYP1A1 mRNA levels, determined by Northern blot and slot-blot analyses (using the trout P4501A1 cDNA pSg15 probe), were hardly detectable in control cells. In BNF- and TCDD-treated cells a 2.8-kb mRNA band was detected by the probe 6 h before the protein and catalytic activities became detectable. The elevated levels of CYP1A1 mRNA were sustained more effectively by TCDD than by BNF. In addition, a second mRNA band at 1.9 kb was seen. The results suggest that transcriptional activation is probably the prime factor even though post-transcriptional events may be involved in the regulation of P4501A1 induction by PAHs in rainbow trout hepatocytes. PMID- 1727641 TI - Hexose metabolism in pancreatic islets: unequal oxidation of the two carbons of glucose-derived acetyl residues. AB - The fate of the C1 and C2 of glucose-derived acetyl residues was examined in rat pancreatic islets. The production of 14CO2 from D-[2-14C]glucose exceeded that from D-[6-14C]glucose, in the same manner as the oxidation of [1-14C]acetate exceeded that of [2-14C]acetate. The difference in 14CO2 output from D-[2 14C]glucose and D-[6-14C]glucose was matched by complementary differences in the generation of 14C-labeled acidic metabolites and amino acids. Even the production of 14C-labeled L-lactate was somewhat higher in the case of D-[6-14C]glucose than D-[2-14C]glucose. The ratio between D-[2-14C]glucose and D-[6-14C]glucose oxidation progressively decreased at increasing concentrations of the hexose (2.8, 7.0, and 16.7 mM), was higher after 30 than 120 min incubation, and was decreased in the presence of a nonmetabolized analogue of L-leucine. These findings are consistent with the view that the difference between D-[6 14C]glucose and D-[2-14C]glucose oxidation is mainly attributable to the inflow into the Krebs cycle of unlabeled metabolites generated from endogenous nutrients, this being compensated by the exit of partially labeled metabolites from the same cycle. The present results also indicate that the oxidation of glucose-derived acetyl residues relative to their generation in the reaction catalyzed by pyruvate dehydrogenase is higher than that estimated from the ratio between D-[6-14C]glucose and D-[3,4-14C]glucose conversion to 14CO2. PMID- 1727642 TI - Kinetics of inhibition of peptide bond formation on bacterial ribosomes. AB - A cell-free system derived from Escherichia coli has been used in order to study the kinetics of inhibition of peptide bond formation with the aid of the puromycin reaction in solution. A similar study has been carried out earlier on a solid support matrix with the same inhibitors. We find that the overall pattern of the kinetics of inhibition is the same in the two systems. At low concentrations of inhibitor there is a competitive phase of inhibition, whereas at higher concentrations of inhibitor the type of inhibition becomes mixed noncompetitive. The values of Ki of the competitive phase in the system in solution are: 5.8 microM (amicetin), 0.2 microM (blasticidin S), 0.5 microM (chloramphenicol), and 0.5 microM (tevenel). The inhibitors amicetin, blasticidin S, and tevenel interact with the ribosome in a reaction which is slower than that of the substrate puromycin, showing clear-cut characteristics of slow-onset inhibition in both systems. Chloramphenicol, on the other hand does not easily show such a delay in solution. It interacts with the ribosome relatively faster than the other three antibiotics. Despite this, chloramphenicol too shows characteristics of time-dependent inhibition. PMID- 1727643 TI - Purification and characterization of monodehydroascorbate reductase from soybean root nodules. AB - Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences. PMID- 1727644 TI - Chemical modification of lysine residues in cytochrome P450LM2 (P450IIB4): influence on heme liganding of arylamines. AB - Treatment of cytochrome P450LM2 with fluorescein isothiocyanate to introduce up to two equivalents of fluorophore per polypeptide chain resulted in the selective derivatization of lysine residues. CD-spectral measurements revealed the overall conformation as well as the immediate heme environment of the hemoprotein to remain unaffected by attachment of the label. Modification caused decreased affinity of p-phenylenediamine and other 4-substituted anilines for the heme site, whereas there was a rise in the extent of substrate interaction. Experiments with pigment containing acetylated lysines gave analogous results, suggesting that the observed phenomenon was due to charge neutralization. There was linear correlation between the Hammett sigma P values and both the optical dissociation constants for arylamine binding to intact enzyme and the dipole moments of the anilines, indicating that basicity along with electronic factors controlled heme liganding; lipophilicity appeared to be of minor importance. Introduction of fluorescein isothiocyanate into the oxygenase was found to influence the bond-making process through modulating basicity of the nitrogenous compounds, but perturbation of optimal spacial orientation of the amine nitrogen toward the heme iron also might have been operative. The lysines studied seem to represent metabolically inactive elements of the substrate channel located on the cytosolic surface of the aggregates, as evidenced by steady-state fluorescence measurements. A hydrophilic segment in the cytochrome P450LM2 molecule that would accommodate the critical residues is discussed. PMID- 1727645 TI - The amino acid sequence and oxygen-binding properties of the single hemoglobin of the cold-adapted Antarctic teleost Gymnodraco acuticeps. AB - The complete amino acid sequence of the single hemoglobin of the Antarctic teleost Gymnodraco acuticeps has been determined. The alpha chain contains 142 amino acid residues; an acetylated seryl residue is at the amino terminal. The beta chain contains 146 residues. A very high degree of sequence identity has been found with hemoglobins of other Antarctic fishes. Oxygen binding is not modulated by pH and allosteric effectors. The Bohr and Root effects are absent, although specific amino acid residues, considered responsible of most of these functions, are conserved in the sequence, thus posing new questions about the molecular basis of these mechanisms. The low heat of oxygenation may be interpreted as one of the mechanisms involved in the process of cold adaptation. PMID- 1727646 TI - ZK98299--a new antiprogesterone: biochemical characterization of steroid binding parameters in the calf uterine cytosol. AB - We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11 beta-(4-dimethylaminophenyl)-17 alpha hydroxy-17 beta-(3-hydroxypropyl)- 13 alpha-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna 4,9-diene-3,20-dione]. In the freshly prepared cytosol, [3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity (Kd = 2.5 nM) interaction between [3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2-3 h at 0 degrees C, and was facilitated by 20 mM Na2MoO4. Under nontransforming conditions, [3H]ZK98299 receptor complexes sedimented as 8 S species in 8-30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5-7%) binding to DNA-cellulose. In contrast, transformed [3H]R5020-receptor complexes exhibited a greater extent of binding (25-55%) to DNA-cellulose. [3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 degrees C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as beta-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [3H]R5020 binding, there was a significant (nearly twofold) increase in the [3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid. PMID- 1727647 TI - Artificial induction of exocytosis in bull sperm. AB - We have investigated an exocytotic event, the acrosome reaction (AR), induced by treatment of bovine sperm with vesicles composed of dilauroyl phosphatidylcholine (PC12). Cell membrane permeability barriers (dye exclusion), acrosomal status (pisum sativum (PSA) lectin binding), and intracellular Ca2+ (Fluo3 fluorescence) were evaluated utilizing flow cytometry and fluorescence microscopy. By these methods the AR is resolved into four kinetically distinct steps: (a) PC12 transfer to the sperm plasma membrane (PM); (b) increased permeability of the PM to extracellular Ca2+; (c) localized leakage of acrosomal contents at the anterior tip of the sperm; and (d) vesiculation of sperm membranes and complete exposure of acrosomal contents. Evidence for PC12 transfer to sperm includes transfer of a fluorescent PC12 analogue from vesicles to cells and the absence of detectable vesicle--cell fusion. The fusion inducing properties of PC12 appear to reside in the lipid head group as neither dilauroylphosphatidylethanolamine nor dilauroylphosphatidylglycerol stimulated the AR. The effect of PC chain length on AR induction closely parallels the aqueous phase solubility of the lipid tested. The rate and extent of the AR depend on the extracellular calcium concentration. Cells treated in the absence of calcium do not undergo the AR, but do so rapidly (less than 1 min) upon subsequent addition of calcium. This role of Ca2+ is partially filled by Sr2+, but not by Ba2+ or Mg2+. The rate of the AR decreases with decreasing temperature and the AR occurs very slowly below 27 degrees C. Simultaneous evaluation of intracellular calcium and acrosomal status reveals the kinetic relationship between Ca2+ influx and the exposure of acrosomal contents. N-Ethylmaleimide preincubation arrests PC12-treated sperm at an intermediate stage in the AR, characterized by punctate PSA binding over the tip of the sperm head. The AR, a developmentally regulated, receptor-mediated fusion event, synchronously induced here in vitro, provides a useful model for mechanistic studies of exocytosis. PMID- 1727648 TI - Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. AB - Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis. PMID- 1727649 TI - Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes. AB - The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined. PMID- 1727650 TI - Succinate-ubiquinone reductase linked recycling of alpha-tocopherol in reconstituted systems and mitochondria: requirement for reduced ubiquinone. AB - Studies have demonstrated that accumulation of mitochondrial tocopheroxyl radical, the primary oxidation product of alpha-tocopherol, accompanies rapid consumption of tocopherol. Enzyme-linked electron flow lowers both the steady state concentration of the radical and the consumption of tocopherol. Reduction of tocopheroxyl radical by a mitochondrial electron carrier(s) seems a likely mechanism of tocopherol recycling. Succinate-ubiquinone reductase (complex II) was incorporated into liposomes in the presence of tocopherol and ubiquinone-10. After inducing formation of tocopheroxyl radical, it was possible to show that reduced ubiquinone prevents radical accumulation and tocopherol consumption. There was no evidence of direct reduction of tocopheroxyl radical by succinate reduced complex II. These reactions were also measured using ubiquinone-1 and alpha-C-6-chromanol (2,5,7,8-tetramethyl-2-(4'-methylpentyl)-6-chromanol) which are less hydrophobic analogues of ubiquinone-10 and alpha-tocopherol. Mitochondrial membranes were made deficient in ubiquinone but sufficient in alpha tocopherol and were reconstituted with added quinone. With these membranes it was shown that mitochondrial enzyme-linked reduction of ubiquinone protects alpha tocopherol from consumption, and there is a requirement for ubiquinone. This complements the observations made in liposomes and we propose that reduced mitochondrial ubiquinones have a role in alpha-tocopherol protection, presumably through efficient reduction of the tocopheroxyl radical. PMID- 1727651 TI - Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts. AB - The precursor protein to the chick corneal keratan sulfate proteoglycan was identified by immunoprecipitation with antiserum to its core protein from lysates of [35S]methionine-pulsed corneas and corneal fibroblasts in cell culture. Antiserum to the keratan sulfate proteoglycan immunoprecipitated a doublet of Mr 52,000 and 50,000 and minor amounts of a Mr 40,000 protein from pulsed corneas. Pulse-chase experiments, which permitted the conversion of the precursor proteins to proteoglycans and digestion of the glycosaminoglycans on immunoprecipitated proteoglycans with keratanase or chondroitinase ABC, showed that the Mr 52,000 50,000 doublet was converted to a keratan sulfate proteoglycan and the Mr 40,000 protein was converted to a chondroitin sulfate proteoglycan. Chick corneal fibroblasts in cell culture primarily produced the smaller (Mr50,000) precursor protein, and in the presence of tunicamycin the precursor protein size was reduced to Mr35,000, which indicates that the core protein contains approximately five N-linked oligosaccharides. Pulse-chase experiments with corneal fibroblasts in culture showed that the precursor protein was processed and secreted into the medium. However, its sensitivity to endo-beta-galactosidase and resistance to keratanase indicate that the precursor protein was converted to a glycoprotein with large oligosaccharides and not to a proteoglycan. This suggests that, although the precursor protein for the proteoglycan is produced in cultured corneal fibroblasts, the sulfation enzymes for keratan sulfate may be absent. PMID- 1727652 TI - Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation. AB - NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2. PMID- 1727653 TI - Role of endogenous fatty acids in the control of hepatic gluconeogenesis. AB - Inhibition of endogenous long chain fatty acids oxidation by tetradecylglycidate (TDGA) impeded gluconeogenesis from lactate or from low concentrations of pyruvate (less than 0.5 mM). The inhibitory effect of TDGA was overcome by medium and short chain fatty acid or by concentrations of pyruvate about 0.5 mM, but not by 10-fold higher concentrations of lactate. Despite decreased energy demand when gluconeogenesis was inhibited by TDGA, the pyruvate-induced increase in hepatic oxygen consumption was similar to the control, indicating that pyruvate transport across the mitochondrial membrane and/or its decarboxylation was not altered, and therefore can not be responsible for the inhibition of gluconeogenesis. Neither does a deficiency of acetyl-CoA explain the decrease in the gluconeogenic flux since high pyruvate loads (greater than 0.5 mM), beta-hydroxybutyrate or even ethanol was capable of overcoming the inhibitory effect of TDGA in the absence of significant changes in the hepatic content of acetyl-CoA. At low (less than 0.3 mM), presumably physiological, pyruvate concentrations, its rate of mitochondrial utilization is limited by the activity of the monocarboxylate transporter. Agents that reduced the mitochondrial NAD system, and therefore reduced flux through pyruvate dehydrogenase, like beta-hydroxybutyrate or ethanol, stimulated gluconeogenesis when fatty acid oxidation was inhibited. The latter observations indicate that the primary role of endogenous fatty acid, when substrate availability is limiting, is to spare mitochondrial pyruvate by decreasing its oxidation, and therefore shifting the partitioning between the carboxylation and decarboxylation reactions toward the former. PMID- 1727654 TI - An isolated nonosseous metastasis to the epidural space from an osteogenic sarcoma. AB - Metastatic disease from osteosarcoma most commonly occurs in the lung and bony sites. Both primary spinal osteosarcomas and spinal metastatic lesions are rare. A case is reported of a nonosseous epidural metastatic lesion from osteosarcoma. It was visualized best by metrizamide-enhanced computed tomographic scanning. The patient symptomatically improved with excision of the lesion although there was massive recurrence despite combined therapy. PMID- 1727655 TI - Human melanoma cell lines established from metastases of a patient with a completely regressed primary site. AB - Two unique human melanoma cell lines were established from each of two metastases, with collections separated by a 1-year interval, in a patient with a spontaneously completely regressed primary cutaneous malignant melanoma. These cell lines were distinct, and under culture, they had characteristic features that correlated with those shown by the original tumors from which they were derived. Cells derived from the second metastasis were more aggressive and had a higher proliferative growth rate, serious chromosomal abnormalities, a greater capacity to form colonies on agar, and a lesser dependence on serum-derived growth factors. This study of malignant melanoma cell lines covered the range from the stage of complete spontaneous regression of the primary lesion through the development of the first metastasis (from which the cell line designated L1M1 was established) to the second metastasis (discovered 1 year later, from which the cell line G1M2 was established). These cell lines grow continuously in the laboratory and can be carried for an unlimited number of passages. They afford an opportunity to investigate and compare the malignant pattern and behavior of human malignant melanoma originating after a completely spontaneously regressed primary lesion. PMID- 1727656 TI - Nucleolar organizer regions in aggressive and nonaggressive basal cell carcinoma of the skin. AB - Nucleolar organizer regions (NOR) were investigated on routine paraffin-embedded histologic sections of 11 aggressive basal cell carcinomas that recurred and/or metastasized (BCC2) and 11 nonaggressive basal cell carcinomas (BCC1). The absolute number of NOR per nucleus was higher in BCC2 than in BCC1, and their distribution pattern was also different. In fact, the means of argyrophilic staining of NOR (AgNOR) counts in the two groups of tumors by two observers were 6.56 with a SD of 1.98 for the nonaggressive and 9.48 with a SD of 2.12 for the aggressive basal cell carcinomas. A statistical analysis of these data using the Student's t test confirmed these observations (t = 64.49). Problems in the evaluation of NOR and possible comparison with other experiences are also discussed. The authors conclude that a quantitative assay of AgNOR and perhaps their distribution pattern may provide information useful to recognize BCC2 and hence may be of help in their prognostic prediction. PMID- 1727657 TI - Fine-needle aspiration and cytologic findings of surgical scar lesions in women with breast cancer. AB - Benign and/or malignant lesions may occur in surgical scars after mastectomy or lumpectomy (SML) in patients with breast cancer (BC). Early diagnosis of these lesions is essential for both therapeutic and prognostic evaluation. The diagnostic value of fine-needle aspiration (FNA) was determined for these scar lesions. The findings of cytologic and histologic specimens obtained from the same lesion of SML in 83 women with BC were correlated. Twenty-five FNA yielded only acellular specimens. Of the FNA done by the cytopathologist, only 6.2% were not representative. However, 45% of those done by less experienced clinicians were not representative. Representative FNA were obtained from 58 of the women who took part in the study. Based on the histologic diagnosis, 38 patients had malignant scar lesions (MSL), and 20 had benign scar lesions (BSL). In one patient of the 38 with MSL, cytologic examination did not show that the malignant lesion; in four women, the tumor was suspected cytologically; and in the remaining 33, the cytologic findings were consistent with malignancy. In 18 of the 20 patients with BSL, cytologic findings were reported as benign and in the other two, as inconclusive. The sensitivity, specificity, and positive and negative predictive values for the cytologic findings were 97.4%, 100%, 100%, and 94.7%, respectively. The diagnostic accuracy of FNA cytology was 98.2%. No complications followed the procedure. It was concluded that FNA cytologic examination of lesions in SML is a simple, safe, highly accurate, and cost effective method to distinguish malignant from benign lesions in women with BC. Lesions in SML should be explored routinely by FNA, rather than by the traditional biopsy, provided the FNA is done by an experienced operator. PMID- 1727658 TI - Relative effect of steroid hormone receptors on the prognosis of patients with operable breast cancer. A univariate and multivariate analysis of 3089 Japanese patients with breast cancer from the Study Group for the Japanese Breast Cancer Society on Hormone Receptors and Prognosis in Breast Cancer. AB - In a retrospective multicenter study to investigate the correlation between estrogen (ER) and/or progesterone receptors (PgR) in primary breast cancer with patient prognosis, 3118 patients with operable breast cancer (International Union Against Cancer Stages I, II, and III) were investigated from ten hospitals in Japan who underwent surgery from October 1972 to December 1982; 3089 were evaluable. The ER-positive and PgR-positive cancers were found in 56% and 34% of patients, respectively. The positivities decreased as the tumor size increased but were independent on lymph node metastasis. There were no significant differences in relapse-free survival (RFS) in relation to receptor status (median follow-up, 89 months [ER], 84 months [PgR]). However, in patients with four or more positive nodes, those with PgR-positive cancer had a longer RFS. The patients with ER-positive cancer survived significantly longer than ER-negative ones, with the greatest difference seen in those with four or more positive nodes. There was a significantly longer postrelapse survival (PRS) for patients with ER-positive cancer because of the different distribution of the major metastasis and better responses to first-line and subsequent treatments. Cox's multivariate analysis showed that overall survival but not PRS was affected by ER (and more weakly by PgR) because of the longer PRS in patients with ER-positive cancer. PMID- 1727659 TI - Breast cancer screening behaviors and attitudes in three racial/ethnic groups. AB - Data from a multiethnic sample of women participating in the American Cancer Society 1987 Texas Breast Screening Project was used to compare attitudes and behaviors related to breast cancer screening for whites, blacks, and Hispanics. In general, similar patterns of association were observed across racial/ethnic groups between a number of demographic and risk factors and prior mammography and recent clinical breast examination (CBE), although the magnitude of the associations varied somewhat across groups. Reasons for not having had prior mammography also were similar across groups, with lack of physician referral and cost cited as the two most important reasons. However, Hispanics were less likely than blacks or whites to report prior breast cancer screening, including mammography, CBE, and breast self-examination (BSE). This study demonstrated that women of different racial/ethnic backgrounds can be successfully recruited to participate in a patient-initiated, community-based program. However, this programmatic approach requires augmentation with other intervention strategies designed to reach low-income women because women with more years of education and higher family income were overrepresented in all three groups. PMID- 1727660 TI - Neutropenic enterocolitis. Clinical diagnosis and treatment. AB - Review of the consultation records of the Gastrointestinal Surgical Oncology service at Roswell Park Memorial Institute from 1982 to 1987 revealed 22 patients with a clinical diagnosis of neutropenic enterocolitis. Ninety-one percent of the patients had hematologic malignancies, and 95% were receiving cytotoxic chemotherapy. Sixteen patients were treated nonsurgically; 11 died. Of those 11 cases, autopsies were performed in 9. At autopsy, the clinical diagnosis was confirmed in four cases; four cases were found to have normal intestinal tracts, and one case had a small bowel volvulus. In none of the four cases for which autopsy proved neutropenic enterocolitis was transmural bowel necrosis or perforation found. Laparotomy was performed in six patients; three survived. The clinical diagnosis was verified in four of the six patients. Neutropenic enterocolitis must be considered a diagnosis of exclusion. Care of these patients should be individualized. Nonoperative management with bowel rest, decompression, nutritional support, and broad spectrum antibiotics is recommended initially. Operative intervention is recommended for those with perforation or those whose condition deteriorates clinically during close, frequent observation. PMID- 1727661 TI - High-dose-rate remote afterloading intracavitary radiation therapy for cancer of the uterine cervix. A 20-year experience. AB - Retrospective analysis was performed on 1022 patients with squamous cell carcinoma of the uterine cervix who were treated with high-dose-rate remote afterloading intracavitary irradiation at the National Institute of Radiological Sciences, Angawa, Chiba-shi, Japan, from 1968 to 1982 in comparison with low-dose rate intracavitary radiation therapy. The patient population consisted of 147 patients with Stage I disease, 256 patients with Stage II disease, 515 patients with Stage III disease, and 104 patients with Stage IV disease. Absolute 5-year survival rates for Stages Ib, IIa, IIb, IIIb, IVa, and IVb disease were 88.1%, 76.9%, 67.0%, 52.2%, 24.1%, and 13.3%, respectively. The rates of severe complication of Grades 3 and 4 were 4.1% for the rectosigmoid colon, 1.2% for the bladder, and 1.1% for the small intestine. In the case of Stage I to II disease, the optimal dose from intracavitary sources was suggested to be 2900 cGy +/- 200 cGy at point A, with 4 to 5 fractions of 600 to 700 cGy delivered over 4 to 5 weeks. These results suggested that high-dose-rate intracavitary radiation therapy provided clinical results comparable to those of a low-dose-rate technique. PMID- 1727662 TI - Multivariate analysis of the histopathologic prognostic factors of cervical cancer in patients undergoing radical hysterectomy. AB - Three hundred forty-five patients with invasive carcinoma of the uterine cervix, Stages Ib (211 patients) and II (134 patients), underwent radical hysterectomy and pelvic lymphadenectomy. The influence of histologic factors including histologic subtype, maximum depth of cervical stromal invasion, degree of stromal invasion, longitudinal tumor diameter, lymph-vascular space invasion, corpus invasion, parametrial invasion, vaginal invasion, and pelvic lymph node (PLN) metastases on survival were examined by multivariate analysis. Univariate analysis revealed that all the variables except corpus invasion and vaginal invasion were significant in survival (P less than 0.05). Among these variables, however, PLN metastases, histologic subtype, and longitudinal tumor diameter were identified as independent and significant prognostic factors by multivariate analysis using Cox regression models. The prognostic index (PI), defined by the model (an indicator of the patient's place in the prognostic spectrum), was able to divide the patients into three prognostic groups. The key factors in the definition of these groups were (1) squamous cell carcinoma, small tumor diameter, and no PLN metastases for the good prognostic group and (2) PLN metastasis in two or more node groups, adenocarcinoma with one positive PLN group, or squamous cell carcinoma with one PLN group and large diameter for the poor prognostic group. These prognostic findings could predict the prognosis more precisely than that of clinical staging. PMID- 1727663 TI - DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact. AB - Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume weighted mean nuclear volume (nuclear vv), were obtained in pretreatment biopsies from 51 patients treated for cervical cancer in clinical Stages I through III (mean age of 56 years, follow-up period greater than 5 years). In addition, conventional, two-dimensional morphometric estimates of nuclear and mitotic features were obtained. DNA indices (DI) were estimated by flow cytometry. Finally, the semiquantitative malignancy grade score value (MGS) was determined according to previously published methods. Estimates of nuclear vv were on average increased in euploid lesions (2P = 0.01), but the overall relationship between nuclear vv and DI was poor. Different clinical stages of disease did not differ with regard to nuclear vv (2P = 0.99) and DI (2P = 0.56). No relationship was disclosed between MGS and nuclear vv (2P = 0.85). Single-factor analysis showed prognostic impact of clinical stage of disease (2P = 0.0001) and DI (2P = 0.04), whereas estimates of nuclear vv were only of marginal prognostic significance (2P = 0.07). However, Cox multivariate regression analysis showed independent prognostic value of patient age and nuclear vv along with clinical stage and DI. All other investigated variables were rejected from the model. A prognostic index with highly distinguishing capacity between prognostically poor and favorable cases was constructed (2P = 1.9 x 10(-7)). It is concluded that realistic estimates of nuclear volume are independent of nuclear DNA content and are of prognostic value for objective malignancy grading in patients with squamous cell carcinoma of the uterine cervix. PMID- 1727664 TI - The new International Federation of Gynecology and Obstetrics surgical staging and survival rates in early endometrial carcinoma. AB - The medical records of patients with clinical Stage I endometrial adenocarcinoma who were treated at the Maimonides Medical Center between October 1979 and October 1987 were reviewed. There was sufficient surgical-pathologic information to allow a reclassification based on the new International Federation of Gynecology and Obstetrics (FIGO) surgical staging in 93 patients. These are the subjects of analysis. Twenty-one patients (23%) were found surgically to have more than Stage I disease. The 5-year survival rate for the whole group (N = 93) was 90%. However, it was significantly better for patients with surgical Stage I disease (98%) than for patients with surgical Stage III disease (60%) (P less than 0.001). There was no significant statistical difference in survival among patients with different substages within surgical Stage I (i.e., IA, 100%; IB, 100%; and IC, 88%), whereas the distribution of adjuvant therapy among these substages was not statistically different (P = 0.17). Thus, survival was not significantly affected by depth of myometrial invasion in patients who had negative peritoneal washing and no involvement of lymph nodes or the peritoneal cavity. PMID- 1727665 TI - A pilot trial of chemohormonal therapy for metastatic prostate carcinoma. AB - Fifteen patients with previously untreated metastatic prostate cancer were treated on a pilot trial with a combination of maximal androgen blockade plus intermittent cytotoxic therapy after androgen priming to stimulate cell division. Androgen blockage was carried out using a gonadotropin-releasing hormone analog (leuprolide) plus a nonsteroidal antiandrogen (flutamide). Carboplatin (CBDCA) (800 mg/m2) was given intravenously every 28 days, preceded for 3 days and followed for 3 days by androgen treatment with fluoxymesterone (5 mg orally twice a day), during which time flutamide was discontinued. Three patients (20%) achieved a complete response (CR), and eight patients (53.3%) achieved a partial response (PR). Four patients (26.7%) had stable disease (SD). The median progression-free survival (PFS) time was 31 months. Nine of 15 patients (60%) remain alive with a median follow-up time of 42+ months (range, 22 to 54 months). Grade 4 thrombocytopenia and Grades 3 or 4 leukopenia were experienced in 87% and 80% of patients, respectively, requiring dose reductions of CBDCA in 85% of the cycles. Six of 15 patients experienced a flare in bone pain with androgen priming. There were no associated spinal cord compressions; however, exclusion of impending spinal cord compression was required before entrance on study. PMID- 1727666 TI - Nuclear volume and expression of T-antigen, sialosyl-Tn-antigen, and Tn-antigen in carcinoma of the human bladder. Relation to tumor recurrence and progression. AB - The T-antigen system and the mean nuclear volume have been proposed as risk variables in bladder tumors. This study includes 34 patients with initially noninvasive (Ta) transitional cell carcinomas who experienced different courses of disease. Tissue specimens of primary tumors were analyzed for the expression of T-antigen, Tn-antigen, and sialosyl-Tn-antigen using monoclonal antibodies (MoAb) and the lectin peanut agglutinin (PNA) in an indirect immunoperoxidase method. In addition, the mean nuclear volume was estimated by morphometry. Tissue from 7 of 13 patients (54%) who had invasive disease during a follow-up period of 5 years expressed T-antigen, as defined by MoAb HH8 in the primary tumor, whereas tissue of only 3 of 21 patients who did not have invasive disease expressed the antigen (P less than 0.02). No association was found between tumor progression to invasion and the expression of Tn-antigen or sialosyl-Tn-antigen. Tn-antigen expression was partially lost in invasive tumors (P less than 0.03) when compared with the expression in primary noninvasive tumors. A high mean nuclear volume in tissue specimens of primary tumors correlated with a progression to invasive disease (P less than 0.01). A significantly (P less than 0.003) higher mean nuclear volume was found in tumor areas that were positive for PNA compared with areas that were negative for PNA in primary tumors. A significantly lower mean nuclear volume was found in Tn-antigen-positive invasive Grade 3 tumor areas than in Tn-antigen-negative areas (P less than 0.005). The combined use of T-antigen expression and mean nuclear volume is of potential clinical interest for determining patients who are at high risk of disease progression. PMID- 1727667 TI - Histologic grade does not predict prognosis in optimally treated, advanced-stage nodular sclerosing Hodgkin's disease. AB - Forty-two patients with advanced-stage nodular sclerosing Hodgkin's disease (NSHD) were treated uniformly with combination chemotherapy and radiation therapy at the University of Nebraska Medical Center between 1982 and 1987. The cases were subclassified into low-grade (13 cases) and high-grade (29 cases) categories using the British National Lymphoma Investigation (BNLI) histologic criteria. After a median follow-up interval of 48 months, no significant differences with regard to the complete remission rate (100% versus 90%), remission durability (85% versus 96%), or predicted 4-year actuarial survival (92% versus 86%) were observed between the two groups, respectively. It was concluded that the BNLI grading scheme for NSHD does not predict the clinical outcome of patients with advanced-stage NSHD who receive optimal therapy. PMID- 1727668 TI - Effects of chemotherapy and remission on carbohydrate metabolism in dogs with lymphoma. AB - After a 12-hour fast, blood samples were obtained from 27 dogs with previously untreated lymphoma before and 5, 15, 30, 45, and 60 minutes after an intravenous (IV) challenge with 500 mg/kg dextrose. This procedure was done for each dog before up to five treatments with the IV doxorubicin (30 mg/m2 every 3 weeks). All dogs achieved a complete remission. Samples were assayed for glucose, lactate, and insulin concentrations, and results were compared statistically with those from 16 normal control dogs of similar weight and age undergoing an identical dextrose challenge before and 3 weeks after receiving one dose of IV doxorubicin (30 mg/m2). Glucose, lactate, and insulin concentrations did not change significantly in response to glucose challenge in control dogs after doxorubicin chemotherapy. Lactate and insulin concentrations in untreated dogs with lymphoma were significantly higher than controls. This hyperlactatemia and hyperinsulinemia did not improve when dogs with lymphoma were put into remission with doxorubicin chemotherapy. The results indicate that carbohydrate metabolism is altered in dogs with lymphoma, and that these abnormalities do not improve when a complete remission is obtained with doxorubicin chemotherapy. PMID- 1727669 TI - Human T-cell leukemia virus type I associated lymphadenitis. AB - Histopathologic changes in lymph nodes were examined from ten patients with mild lymphadenopathy, a few atypical lymphocytes in their peripheral blood, skin lesions, and proviral DNA of human T-cell leukemia virus type I (HTLV-I) in their nodes. The proviral DNA of HTLV-I was detected by southern blot analysis, in situ hybridization, and/or polymerase chain reaction techniques. The lymph nodes showed preserved nodal architecture with diffuse infiltration of small to intermediate-sized lymphocytes in association with scattered transformed lymphocytes and a few immunoblast-like cells in the enlarged paracortex. The infiltrating lymphocytes were positive for CD4, but neither rearrangement nor deletion of T-cell receptors and immunoglobulin heavy chain genes was detected. Eight of ten patients received no therapy, and all patients were alive and healthy more than 5 months after the biopsies. The histologic findings resembled those of a viral infection and could be distinguished from HTLV-I associated lymphomas. PMID- 1727670 TI - Protein kinase C activity in human colonic adenoma and colorectal carcinoma. AB - To additionally understand the molecular mechanisms and biologic indicators of colonic tumorigenesis through the adenoma-carcinoma sequence, protein kinase C (PKC) activity was examined in the cytosol and particulate fraction of specimen homogenates from 18 human colonic carcinomas and seven coexisting colonic adenomas and was compared with the adjacent normal mucosal tissues. This study showed that PKC activity could be detected precisely using mini DEAE-Sephacel column purification and histone III-S as a substrate. The PKC activity in both colonic adenoma and carcinoma progressively was reduced in the particulate fraction compared with that of the adjacent normal mucosa from each patient (74.9 +/- 11.3 and 42.4 +/- 9.37 versus 112 +/- 16.8 pmol/min/mg, P less than 0.001), although PKC activity in the cytosolic fraction was not significantly different (62.6 +/- 17.7 and 63.1 +/- 8.08 versus 56.4 +/- 7.32 pmol/min/mg) with respect to protein concentration. Both colonic adenomas and carcinomas showed a significant progressive decrease in total particulate PKC activity compared with the adjacent normal mucosa of each patient (13.5 +/- 2.18 and 7.64 +/- 1.35 versus 19.8 +/- 2.74 pmol/min/g tissue, P less than 0.001) and no difference in total cytosolic PKC activity (15.2 +/- 3.80 and 16.5 +/- 2.02 versus 14.6 +/- 1.81 pmol/min/g tissue). Among PKC activities in carcinomas, there was no difference related to histologic type, Dukes' staging, or carcinoembryonic antigen values. Among PKC activities in colonic adenomas, a significant decrease in particulate PKC correlated with size. The specific PKC activity in the particulate fraction decreased with advancing adenoma size (P less than 0.05). This study showed that colonic carcinogenesis might be associated with alterations in cellular levels of PKC activity and that the decrease in particulate PKC activity in the adenoma had a possible correlation with adenoma size. PMID- 1727671 TI - Identification of previous erythrocyte alloimmunization and the type and screen at a large cancer center. A 4-year retrospective review. AB - A review of 4 years of hemolytic transfusion reactions and evidence for erythrocyte alloimmunization (RBC-A) was conducted from September 1, 1985 to August 31, 1989 to assess retrospectively the safety of pretransfusion testing using the type and screen (T & S) with immediate-spin crossmatch (IS-XM). All transfusion reaction reports were reviewed for reported "possible hemolytic transfusion reaction" and RBC-A, as identified by the reviewing pathologist. Three hemolytic transfusion reactions (HTR) and eight RBC-A were found for the 4 years. During the review period, 92,759 units of packed RBC were transfused to 22,317 patients. Of these, 76,257 (82.2%) units of packed RBC had IS-XM, 12,414 (13.4%) had an antiglobulin crossmatch, and 4088 (4.4%) were issued without crossmatch. Of the immediate reactions, two were caused by clerical errors, and one was of undetermined cause. The eight RBC-A were discovered during subsequent pretransfusion testing in the Transfusion Service and classified as anamnestic responses to prior transfusions or pregnancies. The eight RBC-A were attributed to the following antibodies: three anti-Jka, one anti-E, one anti-FYa plus unidentified antibody, one anti-Fya, one anti-Jkb, and one anti-Fy3. The low rate of HTR and prior RBC-A detected posttransfusionally in a large cancer center such as this may be used to support the conclusion that the use of T & S with IS-XM is a reasonably safe procedure. PMID- 1727672 TI - Persistent or recurrent acromegaly. Long-term endocrinologic efficacy and neurologic safety of postsurgical radiation therapy. AB - As in other clinics, pituitary surgery was definitive treatment in less than 50% of cases of acromegaly treated at one institution over several decades. From 1965 to 1989, 24 acromegalic patients who had noncurative pituitary surgery received radiation therapy at the National Institutes of Health, with a basal human growth hormone level of greater than 5 ng/ml as the criterion for active disease. Using megavoltage irradiation, more than 60% of these patients stabilized at a normal hormonal range, and the overwhelming majority had decreasing growth hormone levels with time. No major side effects of irradiation were encountered except panhypopituitarism of varying degrees. The authors evolved a policy of surgery as the first option, followed by irradiation for patients with postoperative growth hormone levels more than 5 ng/ml. PMID- 1727673 TI - National Cancer Data Base. A clinical assessment of patients with cancer. PMID- 1727674 TI - Embryonal sarcoma of the liver in an adult treated with preoperative chemotherapy, radiation therapy, and hepatic lobectomy. AB - A rare case of embryonal sarcoma of the liver in a 28-year-old man is reported. The patient was treated preoperatively with a combination of chemotherapy and radiation therapy. Complete surgical resection, 4.5 months after diagnosis, consisted of a left hepatic lobectomy. No viable tumor was found in the operative specimen. The patient was disease-free 20 months postoperatively. PMID- 1727675 TI - Undifferentiated (embryonal) sarcoma of the liver. Pathologic findings and long term survival after complete surgical resection. AB - Undifferentiated (embryonal) sarcoma of liver is a rare tumor with a reputed poor prognosis. Four patients with this tumor are reported, of whom three were alive without recurrence 1.5, 2.5, and 12 years after initial complete surgical resection, and two of whom received no adjuvant therapy. The fourth patient, in whom complete surgical resection of tumor was not achieved, died with recurrent tumor at 13 months. The latter tumor differed histologically and consisted mainly of closely packed smaller undifferentiated cells with a higher mitotic and apoptotic rate. Eosinophilic globules, characteristic of embryonal sarcoma, were found in some cases to contain condensed nuclear chromatin, evidence of origin from tumor cells dying by apoptosis. One tumor mainly contained large cysts lined by biliary-type epithelium; this suggested an origin from a multipotent precursor cell able to differentiate along both stromal and epithelial lines. PMID- 1727676 TI - Gallbladder cancer in Chile. A report on 54 potentially resectable tumors. AB - Fifty-four patients with potentially resectable gallbladder tumors, chosen from 205 cases diagnosed at the Pathologic Unit of the authors' institution, were included in a prospective protocol of management. Of the potentially resectable tumors, only four were indicated before cholecystectomy (7.4%). Inconspicuous tumors were frequently observed, explaining in part the poor results of ultrasonogram for diagnosis. Poorly differentiated tumors were related to a greater rate of metastasis and shorter survival. Likewise, younger patients were associated with a worse prognosis. Patients with tumor confined to the mucosal layer were followed-up only during their postoperative courses. Patients with tumor involvement of the subserosa or muscular layer were offered treatment of a second operation, which included a lymphadenectomy and a liver wedge resection. For patients with serosal involvement, a more aggressive approach was proposed. Metastatic lymph node involvement was found in 9 of the 25 (36%) patients in whom dissection was performed. However, tumor invasion of the liver was seen in 10 of the 24 (41.6%) patients who underwent a liver resection. Patients who had a curative resection had a significantly longer survival in comparison with those who had a palliative resection or simple cholecystectomy. PMID- 1727677 TI - A comparative cytopathologic and histologic study of atypia, dysplasia, and adenocarcinoma in Barrett's esophagus. AB - As a potentially premalignant condition, Barrett's esophagus has stimulated controversy over the need for surveillance of glandular dysplasia and early carcinoma. This prompted the authors to review their experience with endoscopic cytologic brushings and biopsies from patients with Barrett's esophagus. The authors reviewed 65 consecutive specimens from 42 patients with Barrett's esophagus in which both the concurrently obtained esophageal cytologic brushings and companion biopsy specimens were available. In addition, esophagogastrectomy specimens from 9 nine these patients were reviewed. Cytologic and histologic specimens were assigned to one of four diagnostic categories, based on specifically defined criteria: simple Barrett's esophagus with or without inflammatory atypia; dysplasia; adenocarcinoma; or suspicious for dysplasia or carcinoma. Simple Barrett's esophagus was diagnosed in 38 cytologic brushings and 44 biopsy specimens, dysplasia in 4 brushings and 7 biopsy specimens, and carcinoma in 14 brushings and 10 biopsy specimens. Nine brushings and three biopsy specimens were suspicious. In 13 cases, brushings revealed a higher grade lesion than did histology; in 5 cases, biopsy specimens showed a higher grade lesion. Agreement between the two occurred in 72% (47/65) of all specimens. Accuracy was confirmed in the histologic examinations of the resection specimens. The authors conclude that specific criteria, when consistently applied, allow accurate cytologic diagnoses of epithelial changes in Barrett's esophagus. The use of esophageal brush cytology and biopsy specimens provides two complementary techniques, which detect a greater number of serious lesions than either technique alone. PMID- 1727678 TI - Lung cancer histologic type and family history of cancer. AB - The authors studied 300 patients with pathologically confirmed cancer of the trachea, bronchus, or lung in a 16-parish (county) area of southern Louisiana. Squamous-cell carcinoma was observed most frequently among these patients (39.3%), with nearly equal numbers of adenocarcinoma (25.0%) and small cell varieties (25.5%). Patients with large cell cancer, the least frequent type (10.3%), were 4.6 years younger on average than those with small cell (P less than 0.05) or squamous cell (P less than 0.05) neoplasias. Squamous cell neoplasia was more frequent among men (45.5%) than women (22.0%) (P less than 0.05). To assess whether family history differed according to the histologic cell type of the index family member, 248 patients were interviewed with regard to a family history of neoplasia. Those with small cell cancer had the highest family size adjusted mean number of lung cancers per family (0.28). This was 2.2 times greater than the mean number of affected persons among relatives of patients with adenocarcinoma and 1.5 times greater than the mean for the families of patients with large or squamous cell types. However, none of these differences was statistically significant. Similar results were obtained when the total number of cancers at all sites was tabulated. Probands with small cell neoplasia were again most likely to have a positive family history, but the differences between histologic types were small. Although these data suggest an association, a larger study sample is required to determine conclusively whether or not a family history of lung cancer differs according to histologic type. PMID- 1727679 TI - Cystic thymomas. A clinicopathologic study of ten cases. AB - Cystic degeneration in thymoma is a relatively frequent but focal event. In rare cases, the process proceeds to the extent that most or all of the lesion becomes cystic. The authors studied ten cases of thymoma undergoing cystic degeneration of such degree that the lesions initially were mistaken grossly and microscopically for nonneoplastic thymic cysts. The patients' ages ranged from 23 to 81 years, and the sex distribution was equal. The lesions were characterized by the formation of multiple large cystic cavities filled with clear, hemorrhagic or grumose material. Histologically, residual solid islands showing the characteristic features of thymoma, i.e., biphasic cell population (epithelial cells/lymphocytes), perivascular spaces, and areas of medullary differentiation, were present within the cyst walls. In contrast with nonneoplastic thymic cysts, the walls of the cavities generally were devoid of an epithelial lining; most of the cysts appeared to predominantly result from extreme dilatation and confluence of perivascular spaces. In some instances, the cystic degeneration of the tumor was accompanied by cystic changes of an inflammatory nature in the surrounding, nonneoplastic thymic tissue leading to firm adhesions and apparent infiltration of adjacent mediastinal structures. None of the lesions in the studied patients recurred during follow-up periods of from 2 to 10 years (average follow-up, 5 years). Cystic thymomas should be distinguished from nonneoplastic congenital and acquired thymic cysts and other primary thymic neoplasms undergoing extensive cystic degeneration. It is important not to misinterpret the apparent infiltration of surrounding mediastinal structures that results from the inflammatory changes that often accompany these tumors as evidence of aggressive or malignant behavior. PMID- 1727680 TI - Functional outcome of pathologic fracture secondary to malignant disease in a rehabilitation hospital. AB - Fifty-eight patients with 62 pathologic fractures secondary to metastatic disease were admitted to a rehabilitation hospital during a 5-year period. Thirty-four patients were discharged home, 7 were transferred to other facilities, and 17 died. The average hospital stay for the patients who went home (37 days) was only 3 days longer than for patients with nonpathologic fractures. No patient could transfer independently or ambulate at the time of admission, but 26 and 23, respectively, could do so by the time of discharge; 27 patients showed significant improvement in their ability to perform activities of daily living as measured by Kenny scores. All 11 patients who had hypercalcemia died. Eleven of 13 patients requiring parenteral narcotics died. Patients with pathologic fractures secondary to metastatic disease are excellent candidates for intensive rehabilitation programs, but hypercalcemia and administration of parenteral narcotics suggest a poor rehabilitation outcome. PMID- 1727681 TI - Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats. AB - Endothelium-dependent relaxations are depressed in hypertension. In this study we investigated the possible involvement of endothelium-dependent smooth muscle hyperpolarization in this phenomenon. In isolated aortic segments from control rats, acetylcholine (10(-8)-10(-5) M) elicits relaxations after precontraction with norepinephrine (10(-7) M), and acetylcholine or carbachol (10(-5) M) induce smooth muscle hyperpolarization (10.6 +/- 0.9 mV). Both effects disappear after removal of the endothelium and are depressed by tetraethylammonium (3 x 10(-3) M), a rather nonspecific blocker of K+ channels, but not by glibenclamide (10(-5) M), a potent blocker of the ATP-regulated K+ channels, which has a marked effect on the relaxation induced by BRL 38227. The relaxation effect of acetylcholine is impaired in norepinephrine-contracted preparations from hypertensive rats but is not further depressed by tetraethylammonium. In aorta from hypertensive rats, hyperpolarization induced by carbachol was significantly reduced to a mean of only 21.8% of the values obtained in preparations from normotensive rats. From the relaxation-hyperpolarization relation obtained with BRL 38227 (opening K+ channels), it is derived that the endothelium-dependent hyperpolarization (approximately 10 mV) contributes for at least 20-30% of the maximal relaxation effect of acetylcholine on rat aorta. It is concluded that the diminished endothelium-dependent hyperpolarization may contribute to the depression of the endothelium-dependent relaxation in hypertension. PMID- 1727682 TI - Smooth muscle tone and rapid resetting of rat aortic baroreceptors. AB - Changes in conditioning mean arterial pressure (cMAP) selectively alter the set point of arterial baroreceptors and baroreflexes without affecting gain. Changes in smooth muscle tone at constant cMAPs shift the pressure-discharge curves of aortic baroreceptors in a similar manner. Using an in vitro preparation of the rat aortic arch, we tested whether near maximal changes in smooth muscle tone affect rapid resetting in single regularly discharging aortic baroreceptors. Discharge, pressure, and aortic diameter were simultaneously measured. By using vasoactive drugs (phenylephrine, angiotensin II, Bay K 8644, and nitroprusside), rapid resetting to cMAP changes was tested during different smooth muscle tone conditions (control, constricted, and dilated). Baroreceptor discharge-response curves were periodically assessed with slow ramps of increasing pressure at each of three different cMAP levels (10-15 minutes each). Rapid resetting relations were constructed for pressure threshold (Pth) and diameter threshold (Dth) plotted against cMAP and conditioning diameter (cD), respectively. Vasoconstriction decreased Pth in all baroreceptors (n = 13, p less than 0.05). Baroreceptor resetting ability as indicated by the slopes of the resetting relations (pressure- or diameter-resetting ratios, delta Pth/delta cMAP or delta Dth/delta cD, respectively) was unaffected by large increases in smooth muscle tone (p greater than 0.12). Vasoconstriction, however, offset the pressure resetting relation, shifting the linear relation in a parallel manner to higher Pth values. In contrast, Dth values during vasoconstriction were not offset but instead fell along a single diameter-resetting relation coincident with the control relation for each baroreceptor. This last result suggests that acute alteration of vessel mechanics by vasoconstriction does not alter the basic rapid resetting process.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727683 TI - Cellular and ventricular contractile dysfunction in experimental canine mitral regurgitation. AB - This study was designed to answer two questions. First, does the left ventricular contractile dysfunction resulting from mitral regurgitation (MR) reflect a primary defect in the cardiac muscle cell? Second, what is the basis for any change in cellular contractile function that might be observed? Left ventricular volume overload was produced in 10 dogs by catheter transection of mitral chordae tendineae. Three months later in these and in seven control dogs, left ventricular contractile function was characterized by the end-ejection stress volume relation (EESVR). Investigators who were blinded to these results then characterized the contractile performance of cardiac muscle cells, or cardiocytes, from these same left ventricles in terms of the viscosity (graded external load)-velocity relation. Finally, the tissue and cellular components of these same left ventricles were analyzed morphometrically. Both the left ventricles from the MR group and their constituent cardiocytes showed marked contractile abnormalities. By matching ventricles with cells from the same MR dogs, ventricular EESVR was correlated with cardiocyte peak sarcomere shortening velocity (SSV). The correlation coefficient between EESVR and SSV was 0.63, but between a size-independent measure of active ventricular stiffness and SSV, it was 0.88. No change in left ventricular interstitial volume fraction was found in MR dogs, but both ventricular and cellular contractile dysfunction strongly correlated with a decreased volume fraction of cardiocyte myofibrils. Last, in an attempt to relate the degree of contractile dysfunction to the hypertrophic response, left ventricular mass in the MR dogs was correlated with both cellular and ventricular contractile indexes; no significant correlation was found. Three conclusions are warranted by these studies. First, chronic left ventricular volume overload from mitral regurgitation leads to contractile defects at both the ventricular and cellular levels, the extent of which correlates well in individual animals. Second, no quantitative interstitial change resulted from MR. Taken together, these two findings strongly suggest that the contractile defect is intrinsic to the cardiocyte. Third, while the contractile abnormality in MR remains undefined, the most basic defects appear to be a combination of myofibrillar loss with the failure of compensatory hypertrophy to occur in response to progressive decrements in cellular and ventricular function. PMID- 1727684 TI - Reversibility of the effects of normothermic global ischemia on the ryanodine sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum. AB - The effect of normothermic ischemia and ischemia/reperfusion on the function of cardiac sarcoplasmic reticulum (CSR) was investigated using a modified Langendorff perfusion of isolated rat hearts. The function of the CSR was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates. The contribution of the ryanodine-sensitive portion of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The Ca2+ uptake rate of the CSR decreased progressively with increasing duration of ischemia, but this depression was much less when uptake was assayed in the presence of ryanodine. The depression in CSR Ca2+ uptake preceded ischemic contracture. Ryanodine and ruthenium red stimulated uptake almost equally in control hearts, but ruthenium red was much less effective than ryanodine after ischemia. This difference could not be overcome by increasing the ruthenium red concentration. These results confirm the suggestion that the Ca2+ release channel is inappropriately opened after ischemia. The CSR uptake rates were almost completely restored at 15 minutes of reperfusion after 5 and 10 minutes of ischemia but were only partially restored after 15 minutes of ischemia. At reperfusion, mechanical function (end-diastolic pressure and peak systolic developed pressure) was markedly depressed after only 15 minutes of ischemia. The degree of "stunning" correlated well with the depression of CSR function in individual hearts. The decreased Ca2+ uptake of the CSR was not due to a buildup of ADP in the homogenates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727685 TI - Naloxone potentiates cardiopulmonary baroreflex sympathetic control in normal humans. AB - Naloxone, an opioid antagonist, augments baroreflex mechanisms in animals; this occurrence suggests that endogenous opioids blunt baroreflex responses. Limited human studies suggest an inhibitory action of endogenous opioids on baroreflex mediated vagal responses during arterial baroreceptor deactivation. To evaluate the potential effect of endogenous opioids on cardiopulmonary baroreflex mechanisms in humans, we measured arterial and central venous pressures, heart rate, and efferent muscle sympathetic nerve activity (MSNA, by peroneal microneurography) during unloading of cardiopulmonary baroreceptors with incremental lower body negative pressure (LBNP, from 0 to -15 mm Hg) and during the cold pressor test in 21 normal subjects (aged 24 +/- 1 [mean +/- SEM] years). In 14 subjects, we performed LBNP before and after naloxone (0.15 mg/kg i.v.) and placebo (n = 11) on separate days. In six of these 14 subjects and an additional seven subjects (n = 13), studies were also performed before and after administration of a lower dose of naloxone (0.075 mg/kg i.v.) on separate days. Neither dose of naloxone significantly altered control arterial or central venous pressures or heart rate. Control MSNA was reduced after the higher but not after the lower dose of naloxone. Comparable reductions in central venous pressure were produced by LBNP in all groups before and after naloxone or placebo, whereas LBNP did not alter arterial pressure. Cardiopulmonary baroreflex sympathetic sensitivity, which was derived as the slope of the linear regression relation between percent change in total MSNA (units) per absolute change in central venous pressure (mm Hg) during incremental LBNP, was significantly augmented after both the high dose (from 18.6 +/- 4.7%/mm Hg to 39.3 +/- 8.1%/mm Hg, p = 0.001) and low dose of naloxone, whereas placebo had no effect. MSNA responses to the cold pressor test were not altered by either dose of naloxone. Thus, naloxone selectively potentiates cardiopulmonary baroreflex regulation of sympathetic neural activity in normal humans. These findings suggest that endogenous opioids exert a tonic inhibitory effect on sympathetic responses to orthostatic stress in normal humans. PMID- 1727686 TI - Gentamicin effects on renal ischemia/reperfusion injury. AB - This study assessed gentamicin's effects on ischemia/reperfusion renal injury to better understand when and how it worsens postischemic acute renal failure. Rats were subjected to 25 minutes of renal pedicle occlusion with and without preischemic (15-minute) or postischemic (15-minute or 8-hour) gentamicin treatment (100 mg/kg, by itself a subtoxic dose). Gentamicin's impact on hypoxia/reoxygenation injury to isolated rat proximal tubular segments was also assessed. Preischemic and postischemic gentamicin worsened the severity of acute renal failure to the same degree, suggesting that pretreatment induces its effect in the reperfusion period. Gentamicin paradoxically lessened hypoxic damage to proximal tubular segments (assessed by lactate dehydrogenase release), again implying no adverse impact on oxygen deprivation-induced tubular injury. From 0-4 hours of reperfusion, gentamicin approximately halved ATP/ADP ratios (due to increased ADP), indicating a drug-induced defect in cellular energetics. This abnormality temporally correlated with evolving morphological damage. Although antioxidants (deferoxamine and sodium benzoate) have been reported to protect against pure aminoglycoside nephrotoxicity, they did not mitigate gentamicin's adverse impact on postischemic acute renal failure. Gentamicin did not influence ischemia/immediate reperfusion deacylation/reacylation (assessed by renal free fatty acid content) despite its known antiphospholipase activity. Although in the normal kidney gentamicin preferentially accumulated in cortex, in the postischemic kidney, both cortex and outer medullary stripe developed striking (approximately threefold to fivefold) and comparable gentamicin increments. In conclusion, gentamicin appears to exacerbate postischemic acute renal failure by adversely influencing the reperfusion, not the ischemic injury, process. This may occur because increased gentamicin accumulation negatively impacts on reperfusion cellular energetics. PMID- 1727687 TI - ADP plays an important role in mediating platelet aggregation and cyclic flow variations in vivo in stenosed and endothelium-injured canine coronary arteries. AB - The goal of this study was to test the hypotheses that endogenous ADP plays an important role in vivo in mediating platelet aggregation and cyclic coronary artery blood flow variations (CFVs) in stenosed and endothelium-injured coronary arteries in an experimental canine model. Anesthetized animals were studied and coronary blood flow velocities monitored by a pulsed Doppler flow probe positioned around the left anterior descending coronary artery. CFVs were established by an external constrictor positioned at sites with injured endothelium. Apyrase, an ADP-removing enzyme, was infused into the left anterior descending coronary artery (0.3-1.8 units/min) 30 minutes or 2 hours after the establishment of CFVs. Complete abolition of CFVs was achieved in 81% (13/16) of dogs with 30-minute CFVs and in 83% (five of six) of dogs with 2-hour CFVs. In other dogs, a potent inhibitor of ADP-induced platelet aggregation, clopidogrel, was administered as a 10 mg/kg i.v. bolus and a 2.5 mg/kg/hr infusion 30 minutes and 3 hours after the establishment of CFVs. This treatment resulted in complete abolition of CFVs in 14 dogs (100%) with either 30-minute or 3-hour CFVs. Epinephrine was infused into some dogs after CFVs had ceased as a result of either apyrase or clopidogrel administration and into some dogs in whom SQ29548, a thromboxane A2 receptor antagonist, had been given when apyrase failed to abolish CFVs. Epinephrine restored CFVs in all dogs treated with apyrase alone, 67% (four of six) of dogs treated with the combination of apyrase and SQ29548, and 29% (two of seven) of dogs treated with clopidogrel. The plasma epinephrine levels required for CFV restoration were 20 times higher than baseline values in dogs receiving apyrase alone, 100 times higher when a combination of apyrase and SQ29548 had been given, and more than 5,000 times higher in dogs receiving clopidogrel. In vitro studies showed that apyrase only inhibited ADP-induced platelet aggregation, whereas clopidogrel not only inhibited ADP-induced platelet aggregation, but also reduced platelet aggregation induced by the thromboxane mimetic U46619 and serotonin. These data suggest that 1) ADP is an important mediator of platelet aggregation and CFVs in vivo and 2) combined inhibition of thromboxane A2 and ADP's effects provides marked protection against CFVs in experimentally stenosed and endothelium-injured canine coronary arteries. These data and our previous observations are consistent with the possibility that specific antagonists of thromboxane A2, serotonin, and ADP, alone and together, may provide substantial protection against platelet aggregation leading to CFVs at sites of endothelial injury and coronary artery stenosis. PMID- 1727688 TI - Expression and pharmacological characterization of a stimulatory subtype of adenosine receptor in fetal chick ventricular myocytes. AB - Ventricular and atrial myocytes cultured from chick embryos 14 days in ovo were used as model systems to study cardiac adenosine receptors. In membranes of ventricular cultures, blocking of the A1-adenosine receptor pathway by the A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or by pertussis toxin treatment of the myocyte resulted in a significant adenosine agonist mediated stimulation of the adenylate cyclase activity. The maximal increases in adenylate cyclase activity caused by the equipotent or the A2-adenosine receptor selective agonists (from 52.1 +/- 3% to 63 +/- 10% [mean +/- SEM]) were significantly greater than those caused by the A1-selective agonists (from 11 +/- 5% to 34.6 +/- 7%) (p less than 0.01, by t test, n = 4-8). However, in membranes of atrial myocytes, when A1-subtype had been blocked, the various adenosine agonists had no effect on the adenylate cyclase activity. Whether the stimulatory adenylate cyclase-coupled adenosine receptor is also capable of stimulating contractility in the intact ventricular myocyte was next investigated. In ventricular but not in atrial cells, the various adenosine agonists caused an increase in the contractile amplitude in the presence of DPCPX or in myocytes preexposed to pertussis toxin. The increase in contraction amplitude caused by each agonist was expressed as percent of maximum (maximum is the increase in contractility caused by 2.4 mM calcium). In the pertussis toxin-treated myocyte, the maximal increases caused by the equipotent or A2-agonists (NECA, MECA, CV 1808, and CGS21680, from 49.6 +/- 3% to 52.5 +/- 6%, n = 8-12) were significantly greater than those elicited by the A1-agonists (2-CADO, S-PIA, R-PIA, and DCCA, from 12 +/- 4% to 37 +/- 3%, n = 8) (p less than 0.05, by t test). These data demonstrated that a stimulatory adenosine receptor, likely the A2-adenosine receptor, was present on the ventricular but not the atrial myocytes and was linked directly to a stimulation of the cardiac contractility. The functional effects mediated by the A1-subtype became manifested in the presence of isoproterenol, as evidence by an inhibition of the isoproterenol-stimulated increases in adenylate cyclase activity and in cardiac contractility by adenosine agonists. Thus, both subtypes of adenosine receptors, each mediating opposing responses, were present on the ventricular myocytes, whereas only the A1-subtype was found in the atria. The presence of a stimulatory functional A2-adenosine receptor may help explain the absence of a direct negative inotropic response to adenosine in the ventricle. PMID- 1727689 TI - Effects of adenine nucleotides on the proliferation of aortic endothelial cells. AB - The effects of adenine nucleotides and adenosine on DNA synthesis and cell growth have been studied in bovine aortic endothelial cells (BAECs). ATP produced a small but significant (+44%) increase of the fraction of BAECs whose nuclei are labeled by [3H]thymidine. This mitogenic effect was mimicked by ADP, the phosphorothioate analogues ATP gamma S and ADP beta S, and the nonhydrolyzable analogue adenosine 5'-(beta, gamma-imido)triphosphate (APPNP), whereas adenosine 5'-(alpha, beta-methylene)triphosphate (APCPP), a selective agonist of P2x purinoceptors, had no effect at 10 microM and a small one at 100 microM; this profile is consistent with the involvement of P2y-receptors. Adenosine induced a mitogenic response of a magnitude similar to that of ATP. This effect was not reproduced by R-phenylisopropyl adenosine, by 5'-N-ethylcarboxamide adenosine, or by 2',5'-dideoxyadenosine, selective ligands of the A1- and A2-receptors and the P site, respectively, nor was it inhibited by 8-phenyltheophylline, an antagonist of both A1- and A2-receptors. The mechanism of this adenosine action thus remains unclear. ATP and ATP gamma S did not enhance the proliferation of BAECs cultured in the presence of fetal calf serum concentrations ranging from 0.5% to 10%. They inhibited the growth-promoting effect of basic fibroblast growth factor; among the various nucleotides tested, APCPP was the least effective to reproduce the action of ATP, suggesting the possible involvement of P2y-receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727690 TI - Potassium rectifier currents differ in myocytes of endocardial and epicardial origin. AB - Whole-cell voltage-clamp experiments and single-channel current recordings in cell-attached patch mode were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to compare the characteristics of inward rectifier K+ current (IK1) and delayed rectifier K+ current (IK) between endocardial and epicardial cells and to test the hypothesis that the differential characteristics of IK1 and/or IK are responsible for the differences in action potential configuration between the two cell types. IK1 in endocardial cells displayed a distinct N-shaped current-voltage (I-V) relation, with a prominent outward current at potentials between -80 and -30 mV. In epicardial cells, an outward current region was much smaller, and the I-V relation demonstrated a blunted N shaped I-V relation. In single-channel current recordings in cell-attached patch mode, neither unitary current amplitude of IK1 nor probability of channel opening was different between endocardial and epicardial cells, suggesting that the difference in the number of functional channels might be responsible for the differential IK1 I-V relations. The characteristics of IK also differed between endocardial and epicardial cells. The time course of growth of tail current of IK (IK,tail) (activation of IK) was significantly enhanced and that of IK,tail deactivation was delayed in epicardial cells compared with endocardial cells. The time constant of the slow component of IK activation at +20 mV was 3,950 +/- 787 msec in endocardial cells and 2,746 +/- 689 msec in epicardial cells (p less than 0.05); the corresponding values for IK deactivation at -50 mV were 1,041 +/- 387 msec and 1,959 +/- 551 msec, respectively (p less than 0.01). The voltage dependence of steady-state activation of IK,tail was similar between endocardial and epicardial cells, suggesting that the probability of channel opening at any potential was not different in the two cell types. The amplitude and density of fully activated IK (IK,full) were significantly greater in epicardial cells than in endocardial cells. At repolarization to -20 mV, IK,full amplitude was 452 +/- 113 pA in endocardial cells and 578 +/- 135 pA in epicardial cells (p less than 0.05), and the corresponding values for IK,full density were 2.86 +/- 0.73 and 4.21 +/- 0.83 microA/cm2, respectively (p less than 0.05). A nonstationary fluctuation analysis revealed that the amplitude of IK unitary current was similar between endocardial and epicardial cells (0.23 +/- 0.07 versus 0.22 +/- 0.03 pA, p = NS).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727691 TI - Membrane biochemistry and chemical hepatocarcinogenesis. AB - Biochemical membrane alterations appearing during the process of chemical carcinogenesis are described. Emphasis is put on membrane composition, structure, and biogenesis. In this presentation the knowledge gained from experimental studies of liver and skin in the process of cancer development is acknowledged. Important biochemical changes have been reported in lipid composition, fatty acid saturation, constitutional enzyme expression, receptor turnover and oligomerization. Functional consequences of the altered membrane structure is discussed within the concepts of regulation of cell proliferation, regulation of membrane receptor expression, redox control, signal transduction, drug metabolism, and multidrug resistance. Data from malignant tumours and normal tissue are addressed to evaluate the importance of the alterations for the process and for the eventual malignant transformation. PMID- 1727692 TI - Eukaryotic DNA replication. AB - The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication. PMID- 1727693 TI - Mucin-type glycoproteins. AB - Considerable advances have been made in recent years in our understanding of the biochemistry of mucin-type glycoproteins. This class of compounds is characterized mainly by a high level of O-linked oligosaccharides. Initially, the glycoproteins were solely known as the major constituents of mucus. Recent studies have shown that mucins from the gastrointestinal tract, lungs, salivary glands, sweat glands, breast, and tumor cells are structurally related to high molecular-weight glycoproteins, which are produced by epithelial cells as membrane proteins. During mucin synthesis, an orchestrated sequence of events results in giant molecules of Mr 4 to 6 x 10(6), which are stored in mucous granules until secretion. Once secreted, mucin forms a barrier, not only to protect the delicate epithelial cells against the extracellular environment, but also to select substances for binding and uptake by these epithelia. This review is designed to critically examine relations between structure and function of the different compounds categorized as mucin glycoproteins. PMID- 1727694 TI - Retinoic acid induces matrix Gla protein gene expression in human cells. AB - The objective of this study was to investigate the possible regulation of the vitamin K-dependent matrix Gla (gamma-carboxyglutamic acid) protein (MGP) by retinoic acid, a regulation suggested by the recent observation that the human MGP promoter has a perfect direct repeat which is nearly identical to the retinoic acid-responsive element in the retinoic acid receptor-beta gene. We report that retinoic acid strongly increases MGP mRNA levels in all human cells tested, including osteoblasts, articular cartilage chondrocytes, and fibroblasts. In osteoblastic cells, MGP mRNA levels are increased by 25-fold at 1 microM retinoic acid and achieve half-maximal levels at 0.1 microM hormone. MGP is a small secreted protein of unknown function that is synthesized in a wide variety of vertebrate tissues. The present results suggest that part of the known actions of retinoic acid on skin, bone, cartilage, and other tissues in the human may be mediated by the stimulation of MGP synthesis and the consequent effect of increased MGP secretion on nearby target cells. PMID- 1727695 TI - Activity of oxytocinergic neurons in the paraventricular nucleus mirrors the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion. AB - PRL secretion is regulated by an endogenous stimulatory rhythm of PRL-releasing factors within the hypothalamus. The endogenous rhythm has a bimodal periodicity with a nocturnal component which peaks at approximately 0300 h and a diurnal component that peaks at approximately 1700 h. Several PRL-releasing factors are known to be involved in this rhythm. Among these are oxytocin (OT), vasoactive intestinal peptide, and serotonin. We have proposed that OT is the neurohormone that stimulates PRL release from the lactotroph. In this study, we examined the activity of OTergic neurons in the paraventricular nucleus using the expression of the protooncogene c-fos (Fos) as a marker of neuronal activity. Ovariectomized rats were killed at either 0300, 1200, or 1700 h and brains quickly fixed by perfusion with 2.5% acrolein in 4% paraformaldehyde. Brains were blocked and processed for OT/Fos immunohistochemistry. Rats killed at 0300 and 1700 h had significantly greater proportion of Fos expressing OTergic neurons than control rats (1200 h). Percent of Fos-positive OTergic neurons were 2- and 1.5-fold greater at 0300 and 1700 h than 1200 h, respectively. The majority of these neurons were located in the medial parvocellular paraventricular nucleus and periventricular area. In another experiment, groups of OVX rats were killed every 2 h over a 24-h period and OT extracted from their anterior and posterior pituitaries. OT was present in the anterior pituitary in a bimodal rhythm. OT concentrations were greatest at approximately 0400 h and slowly declined to baseline by 1000 h. Another peak of OT was present in the anterior pituitary at approximately 2000 h and quickly declined to baseline by 2400 h. This rhythm of OT was not reflected in either the posterior pituitary or trunk blood. These data suggest that activity of a specific population of OTergic neurons of the paraventricular nucleus is rhythmic. The periodicity of these neurons mirrors that of the endogenous stimulatory rhythm. Furthermore, the anatomical location of these neurons suggests that they may project to the median eminence. Indeed, this heightened activity is reflected in a bimodal rhythm of OT in the anterior pituitary. Taken together, the data presented here provide compelling support for the role of OT as the neurohormone in the mechanism of the endogenous stimulatory rhythm. PMID- 1727696 TI - In vivo action of activin-A on pituitary-gonadal system. AB - Activin, a dimer of the beta-subunits of inhibin, has been found to stimulate FSH secretion from the cultured pituitary cells. However, in vivo action of activin is poorly elucidated. Daily sc injections of 40 micrograms activin-A over a period of 1-3 days to intact immature female rats caused a significant increase in serum FSH, inhibin, estradiol, uterine weight, and ovarian FSH receptors. Daily sc injections of 5 micrograms or 20 micrograms activin-A for 6 days caused a marked increase in ovarian weight and the development of large ovarian follicles. However, daily sc injections of 20 micrograms activin-A to hypophysectomized immature female rats for 3 days induced no significant changes in ovarian and uterine weight, serum inhibin, estradiol, and progesterone levels. Simultaneous injections of both activin-A and 5 IU PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels, compared to simultaneous injections of both vehicle and PMSG in the hypophysectomized immature female rats. These results demonstrate that activin-A induces not only an increase of FSH secretion from the pituitary but also a direct autocrine or paracrine ovarian stimulation resulting in an increase of the number of ovarian FSH receptors and ovarian and uterine weight, as well as an increase in the level of inhibin and estradiol secretion from the ovary. PMID- 1727697 TI - Pituitary adenylate cyclase-activating polypeptide specifically increases cytosolic calcium ion concentration in rat gonadotropes and somatotropes. AB - The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is a potent stimulator of cAMP accumulation in the anterior pituitary gland, though its physiological function has yet to be defined. To establish the target cells of PACAP action we have measured PACAP-induced changes in cytosolic free calcium ion concentration ([Ca2+]i) in single identified anterior pituitary cells. This was achieved by combining fura-2 videomicroscopy, to measure [Ca2+]i, and reverse hemolytic plaque assays, to identify the secreted hormone. PACAP (100 nM) increased [Ca2+]i in 32% of all pituitary cells. These responses were predominantly seen in identified gonadotropes and somatotropes, but rarely in corticotropes or lactotropes. PACAP induced two forms of Ca2+ response in gonadotropes; a "Ca2+ spike" (independent of extracellular Ca2+) in 72% of responding gonadotropes, and an extracellular Ca(2+)-dependent "Ca2+ plateau" (28% of cells). In somatotropes, PACAP stimulated either Ca2+ plateau responses (58% of responding somatotropes) or repetitive "Ca2+ transients" (42% of cells), both of which were dependent upon extracellular Ca2+. PACAP, therefore, produces distinct changes in [Ca2+]i in gonadotropes and somatotropes, which may be related to distinct intracellular messenger pathways. The identification of these cell types as targets of PACAP action suggests a role in the regulation of reproduction and growth. PMID- 1727698 TI - Mastoparan-induced hormone release from rat pancreatic islets. AB - Mastoparan, a tetradecapeptide purified from wasp venom, stimulates insulin and glucagon release by rat pancreatic islets in a dose-related manner. In perifusion experiments, mastoparan produces monophasic hormone release, which ceases within 10 min of removal of the peptide. After exposure of the isles to mastoparan, glucose-induced insulin release is clearly retained. In incubation experiments, mastoparan-induced insulin release is greatly blocked by pretreatment of the islets with pertussis toxin or neomycin (inhibitor of phosphoinositide turnover) or by lowering the ambient temperature to 17 C. Pretreatment of the islets with nifedipine (calcium channel blocker), H-7 (inhibitor of A- and C-kinase), somatostatin, or divalent cation-free medium does not affect the response to mastoparan. Pretreatment with parabromophenacylbromide (phospholipase-A2 inhibitor) does not block the response induced by a high concentration of (58 microM) mastoparan. The peptide does not stimulate insulin synthesis during 30 min of incubation. Mastoparan raises the cytosolic free Ca2+ concentration, measured by fura-2, in isolated islet cells at normal (1.9 mM) and very low (6.5 microM) extracellular Ca2+ concentrations. Intravenous administration of mastoparan in rats causes a significant elevation of both insulin and glucagon. Together with the previous data, we conclude that mastoparan stimulates islet hormone release through a temperature-dependent process mediated by pertussis toxin-sensitive GTP-binding protein(s). Activation of phospholipase-C and liberation of intracellular Ca2+ are likely to be coupled to exocytosis. Ca2+ influx through the Ca2+ channel and protein kinase-A and -C appear not to be involved in mastoparan's hormone-releasing action. Phospholipase-A2 may be involved in the hormone release induced by low, but not high, concentrations of the peptide. PMID- 1727699 TI - Late pregnancy and rat choriocarcinoma cells inhibit nocturnal prolactin surges and serotonin-induced prolactin release. AB - The purpose of the present study was to determine the effect of hormonal secretion by trophoblast cells on serotonin-induced release of PRL in both pregnant and nonpregnant rats. In the first experiment, three compounds that effectively lead to stimulation of serotonin receptors were injected ip or intraarterially between 0900-1200 h on day 8 or 16 of pregnancy. These included the serotonin precursor 5-hydroxytryptophan (20 mg/kg BW); a releasor of serotonin, fenfluramine (10 mg/kg BW); and a serotonin S2 receptor agonist, DOI (2,5-dimethoxy-4-iodophenyl-2-aminopropane-HCl; 500 micrograms/kg BW). When injected on day 8, these treatments significantly (P less than 0.01) increased the level of plasma PRL within 30 min after the injection. However, on day 16 the same treatments could not induce any change in the plasma PRL level. In the second experiment, rat choriocarcinoma (Rcho) cells, which secrete placental lactogen I in vivo, were injected beneath the kidney capsule on day 1 of pregnancy. Control pregnant rats injected with the cell culture medium RPMI-1640 containing 20% FBS continued to have a nocturnal PRL surges on days 7, 8, and 9, with the peak value of plasma PRL occurring at 0400 h. Rats injected with the cells had Rcho tumors at the site of injection when analyzed on day 9. These rats also had significantly (P less than 0.05) reduced nocturnal PRL surges on days 7 and 8 of pregnancy compared to the control animals, and on day 9, the PRL surge was completely blocked. In another group of day 9 pregnant rats containing Rcho tumors, DOI-induced PRL release was blocked by Rcho cells, whereas in controls, plasma PRL increased from 5 to 47 ng/ml. The final experiment tested whether the presence of Rcho cells affected serotonergic- or TRH (1 microgram/rat)-induced PRL release in cyclic rats that were ovariectomized 1 day before drug injection. Injection of Rcho cells 8 days earlier completely inhibited 5-hydroxytryptophan- or DOI-induced PRL release, but did not affect TRH-induced PRL release. These results indicate that the absence of PRL surges after midpregnancy may be due in part to the inability of serotonin to stimulate PRL at this time compared to early pregnancy. Secretion of placental lactogens or other PRL-like peptides from the placenta in the pregnant rat may be antagonistic to the normal stimuli that cause the PRL surges of early pregnancy, resulting in a loss of surges. PMID- 1727700 TI - Steroid hormones differentially modulate glycoconjugate synthesis and vectorial secretion by polarized uterine epithelial cells in vitro. AB - Characterization of glycoconjugates synthesized by polarized immature rabbit uterine epithelial (UE) cells in vitro, their vectorial patterns of secretion, and regulation by ovarian steroid hormones are reported. Large (mol wt, greater than 230 kDa) sialomucoglycoproteins and hyaluronate were primarily (86-96%) secreted from the apical cell surface domain, while heparan sulfate proteoglycans were predominantly secreted from the basal cell surface of the polarized UE cells. The polarized UE cells responded to estrogen and progesterone in vitro and exhibited distinct profiles in their synthesis and secretion of different glycoconjugates. Progesterone and/or estrogen reduced the secretion of the mucosialoglycoproteins; however, progesterone caused a 4- to 5-fold accumulation of mucosialoglycoproteins in the cell-associated fraction, suggesting regulation by the hormone at the level of secretion, rather than synthesis. Estrogen and progesterone both stimulated the synthesis and secretion of hyaluronate by the polarized UE cells. Neither hormone substantially altered the synthesis or secretory pattern of heparan sulfate proteoglycans. Collectively, these studies provide the first comprehensive characterization of the major glycoconjugates synthesized and secreted by rabbit UE cells. Furthermore, these observations demonstrate marked differential direct influences of steroid hormones on the production of distinct classes of UE cell glycoconjugates. PMID- 1727701 TI - Effects of glucocorticoids on expression of the fos protooncogene in AtT-20 cells. AB - Glucocorticoid regulation of expression of the protooncogene fos has been examined in AtT-20 cells at both the RNA and protein levels. When cells were incubated continuously in the presence of dexamethasone, an early (30 min) rise in the expression of fos mRNA was observed, which declined by 1 h, but rose again after 2 h of hormone treatment. Six hours after hormone treatment, fos mRNA levels had returned to control levels in spite of the continued presence of dexamethasone. Serum treatment resulted in a sustained increase in fos mRNA levels; however, the glucocorticoid and serum effects were additive. Dexamethasone and/or serum both increased the steady state levels of fos protein. Glucocorticoid treatment of AtT-20 cells results in complex changes in fos expression, but does not affect their viability or growth rate; these results suggest that fos may play a role in mediation or modulation of glucocorticoid effects other than growth. PMID- 1727702 TI - Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat. AB - A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane. PMID- 1727703 TI - Intraventricular corticosterone increases the rate of body weight gain in underweight adrenalectomized rats. AB - Circulating glucocorticoids are necessary for hyperphagia and excessive weight gain in obese rodents. It has been reported that intraventricular administration of selected glucocorticoids restores hyperphagia and weight gain in anorexic adrenalectomized gold thioglucose-treated mice. We wanted to determine whether administration of glucocorticoids directly into the central nervous system of normal (nonobese) rats will enhance the rate of weight gain. In the initial experiment rats were allowed ad libitum access to chow or were rendered underweight by a 7- to 10-day period of food restriction. Animals were then adrenalectomized or sham operated and allowed ad libitum access to chow. Both groups of adrenalectomized animals consumed less food and gained weight less rapidly than their respective sham controls. Previously food-restricted and therefore underweight adrenalectomized rats consumed significantly more food and gained weight more rapidly than previously ad libitum-fed adrenalectomized rats. These data support the conclusion that adrenalectomized rats, like intact rats, regulate their weight, albeit at a lower level than intact rats. To determine a possible role of central glucocorticoids in this phenomenon, food-restricted or ad libitum-fed rats received a single intraventricular injection of corticosterone or its vehicle on the day after adrenalectomy or sham surgery. Whereas intraventricular corticosterone had no effect on weight gain in ad libitum-fed rats, it significantly increased the rate of weight gain in food restricted adrenalectomized rats relative to that in vehicle-treated controls. Peripheral administration of corticosterone had no effect in this paradigm. It is concluded that glucocorticoids act directly on the central nervous system of underweight lean rats to augment weight gain. PMID- 1727704 TI - Plasma sex steroids and tissue aromatization in hatchling zebra finches: implications for the sexual differentiation of singing behavior. AB - One of the best examples for sex hormone regulation of brain development is found in songbirds. In zebra finches, only males sing because of striking sex differences in the neural circuitry that controls songs. Because developing females treated with estradiol (E2) develop a masculine song system, E2 is considered the normal masculinizing hormone. However, questions about the role of E2 in male development persist, because E2 treatments that masculinize song can demasculinize other sexual behaviors, and there exists contradictory evidence for high levels of circulating E2 in developing males. We remeasured plasma steriods in zebra finches during the first 13 days after hatching. E2 circulated at low levels, and there were no sex differences in circulating E2, estrone, testosterone, androstenedione, or dihydrotestosterone. We also measured aromatase activity [( 3H]androstenedione conversion to [3H]estrone and [3H]E2) in gonad, adrenal, brain, and other tissues of hatchlings. Aromatase was abundant in ovary, but was not definitively detected in testes, adrenals, or other nonneural tissues of males. Aromatase was also found in diencephalon and in high amounts in telencephalon, but sex differences were not detected in whole brain or cellular subfractions of telencephalon. Because ovarian steroidogenesis is high, it may be involved in differentiation of the female zebra finch, as in nonpasserine birds. By contrast, the functional estrogen necessary for masculinization of song is most likely derived from brain, supplied with substrate from the adrenals. The puzzle remains why the song system is not masculinized in females, who possess high levels of aromatizable androgens and telencephalic aromatase. PMID- 1727705 TI - Structure-function relationship of parathyroid hormone: activation of phospholipase-C, protein kinase-A and -C in osteosarcoma cells. AB - Recent evidence indicates that after PTh interaction with its receptor, both protein kinase-A (PKA) and protein kinase-C (PKC) are activated. To investigate the relationship between PTH structure and protein kinase stimulation, we have analyzed the effects of synthetic PTH fragments on PKA and PKC in the rat osteogenic sarcoma cells, UMR 106-01. Activation of PKA by 10(-7) M bovine (b) PTH-(1-34) was maximal (2.7-fold of control) at 5 min and remained elevated 15 min after hormone exposure. bPTH-(2-34), at equimolar doses, also stimulated PKA, but with a lower potency (1.4-fold of control), whereas propionyl bPTH-(2-34) [pbPTH-(2-34)], bPTH-(3-34), [Tyr34]bPTH-(7-34) amide [bPTH-(7-34)], and bPTH-(30 34) were ineffective. On the other hand, translocation of PKC activity from the cytosol to the membrane after exposure to bPTH-(1-34) was transient, with a peak at 1 min (1.9-fold of control), and returned to basal levels after 5 min. Other fragments, bPTH-(2-34), pbPTH-(2-34), bPTH-(3-34), and bPTH-(7-34), were also active on PKC, with relative potencies of 81%, 67%, 62%, and 51% of bPTH-(1-34), respectively, whereas bPTH-(30-34) was inactive. bPTH-(1-34), bPTH-(2-34), pbPTH (2-34), and bPTH-(3-34) also induced inositol 1,4,5-trisphosphate production, with a potency order of 1.6-, 1.6-, 1.5-, and 1.6-fold over the control value, respectively, thus indicating activation of phospholipase-C. Neither bPTH-(7-34) nor bPTH-(30-34) caused a statistically significant increase in inositol 1,4,5 trisphosphate production. These results demonstrate that PTH signal transduction through the two different pathways can be dissociated; while activation of the cAMP/PKA system requires amino acids 1 and 2, the phospholipase-C/PKC system is coupled to a longer domain of the hormone's N-terminus. PMID- 1727706 TI - Remembrances of our founders: will growth factors, oncogenes, cytokines, and gastrointestinal hormones return us to our beginnings? PMID- 1727707 TI - Recruitment and maturation of small subsets of luteinizing hormone gonadotropes during the estrous cycle. AB - Small and medium-sized gonadotropes may enlarge and produce more LH in order to contribute to the proestrous surge. To test this hypothesis, dispersed pituitary cells from cycling female rats were separated by centrifugal elutriation into small, medium, and large fractions and labeled for LH beta antigens or mRNA (by in situ hybridization with a biotinylated oligonucleotide probe complementary to sequences encoding amino acids 28-40). The percentage of cells bearing LH beta mRNA in the pituitary cell population increased from 6 +/- 0.4% in the evening of diestrous day 2 to 16 +/- 0.7% in the morning of estrus (average +/- SEM). Over 80% of these labeled cells were large or small subtypes. The proportion of small gonadotropes labeled with LH beta mRNA declined from 43 +/- 3% at metestrus to 29 +/- 1% on the evening of proestrus as the proportion of medium-sized gonadotropes labeled for LH beta antigens (15 +/- 1%) or mRNA (17 +/- 1%) increased to 25 +/- 2% or 38 +/- 2%, respectively. Because the overall percentage of immunoreactive LH cells did not change after diestrus, small LH cells may have enlarged or increased their density to join the medium-sized pool. During proestrus, the proportion of large immunoreactive LH gonadotropes increased from 41 +/- 2% to 65 +/- 2% (by the morning of estrus) as the proportion of small or medium-sized LH cells declined to 17-18 +/- 1%, suggesting further increases in size or density. These data suggest that small or medium-sized gonadotropes are activated during early diestrus to enlarge and produce LH beta. They contribute to the increased number of cells in medium-sized and large fractions in proestrous or estrous rats. The predominance of the smaller subtypes during metestrus and diestrus suggests that LH gonadotropes may revert to a smaller or lighter subset to await activation during the next cycle. PMID- 1727708 TI - Heightened secretion by small and medium-sized luteinizing hormone (LH) gonadotropes late in the cycle suggests contributions to the LH surge or possible paracrine interactions. AB - LH secretion from pituitary cell fractions separated by centrifugal elutriation was compared to learn whether any contributed to the LH surge. After plating (1 h) and stimulation with 0-10 nM [D-Lys6]GnRH (4 h), some fractions secreted levels that were out of proportion to their enrichment. Large cells from proestrous morning rats (3-fold enriched) secreted 4.3 times more LH, and medium sized fractions (1.5-fold enriched) secreted 2-3.4 times more LH than unseparated cells during estrus or metestrus. Normalized data (nanograms per 20,000 cells) showed that basal levels reflected the enrichment in the fractions. Data were also normalized to nanograms of LH per 1,000 LH gonadotropes to focus on LH cell activity. Basal secretion in unseparated cultures (4-6 ng/ml/1,000 LH cells) was lower than that in small or large LH cells during all stages except proestrus, when small gonadotropes became as active as those in unseparated cultures, and large gonadotropes secreted 2-3 times more LH. Basal secretion from medium-sized gonadotropes was comparable to that in unseparated cultures. [D-Lys6]GnRH mediated secretion from unseparated, small, and large LH cells was comparable during most stages. However, during proestrus, large gonadotropes secreted 2.2 times more LH than unseparated counterparts. Stimulated medium-sized LH cells were 1.3-2.3 times more active in most stages than those in unseparated cultures and 1.75-2.8 times more active than those in large cell fractions (from proestrous PM to metestrus). Whereas this enhanced secretion late in proestrus suggests that medium-sized LH cells may support the LH surge, it also may reflect the removal of regulatory factors from cells in other fractions. Studies of autocrine or paracrine interactions with gonadotropes are needed to test this hypothesis. PMID- 1727709 TI - Distribution and regulation of epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin in the rat epididymis. AB - The epithelium of the epididymis possesses an elaborate network of tight junctions between principal cells which is altered as a function of postnatal age. Cadherins are implicated in the formation of tight junctions. The objective of the present study was to determine whether RNA transcripts for cadherins were present in the epididymis, and if so, how they were hormonally regulated. Using specific cDNA probes for epithelial cadherin (E-Cad) and neural cadherin (N-Cad), Northern blot analysis was used to study steady state levels of cadherin mRNAs. A major E-Cad mRNA species of 4.7 kilobases and a weaker 4.3-kilobase species were observed in the epididymis. No signal for N-Cad was detected. Steady state mRNA levels for E-Cad were highest in the caput and corpus epididymidis and were almost 4 times higher than those in the initial segments and cauda epididymidis; no signal was detected in the vas deferens. Light microscopic immunocytochemical localization of E-Cad revealed a reaction over the principal cells of the entire epididymis. The relative intensities of the immunoreactivity suggested that the E Cad protein concentration was highest in the corpus, followed by the caput, cauda, and initial segments of the epididymis. There was no reaction over the epithelial basal and clear cells or intraepithelial halo cells. Three days after bilateral orchidectomy, E-cad mRNA was decreased by 75% in the caput epididymidis. A dose-dependent maintenance of mRNA concentration for E-Cad was observed throughout the epididymis of orchidectomized rats after replacement with testosterone. Fourteen days after unilateral orchidectomy, no differences were observed in the concentrations of epididymal E-Cad mRNA between control and unilaterally orchidectomized rats. Together, these data demonstrate that mRNA for E-Cad is present and translated in the rat epididymis, is differentially distributed along this tissue, and can be regulated by circulating androgens. PMID- 1727710 TI - Estrogen receptor-immunoreactive glia, endothelia, and ependyma in guinea pig preoptic area and median eminence: electron microscopy. AB - The presence of estrogen receptors (ERs) in nonneural cells in brain, including glia, ependyma, and endothelia, has not previously been documented with electron microscopy. This study employed immunocytochemistry to investigate whether ER immunoreactivity (ER-ir) is present in glial, ependymal, or endothelial cells in the medial preoptic area (POA) and median eminence (ME) in the brain of gonadally intact female guinea pigs. Tissue sections through these regions were immunostained with monoclonal antibody H222 for ER localization using 3,3',5,5' tetramethylbenzidine (TMB) as the chromogen. ER-ir cells were identified ultrastructurally by the presence of distinct spicule-like TMB crystals in nuclei. While neurons constituted the clear majority of ER-immunopositive cells, labeled astrocytes, ependyma, and endothelia were also present. Distinct intranuclear TMB crystals were present in astrocytes at the anterior pole of the POA within the preventricular periventricular nucleus, anterior compact subnucleus of the medial preoptic nucleus (MPNa), and organum vasculosum of the lamina terminalis, indicating ER-ir. In the MPNa, cell counts performed at the ultrastructural level revealed that 9.6% (15 of 156) of the astrocytes were ER ir. To further explore the relationship of ERs with astrocytes, ER/glial fibrillary acidic protein (GFAP) double labeling experiments were performed using TMB and diaminobenzidine tetrahydrochloride for ER and GFAP localization, respectively. These studies verified the presence of ERs in astrocytes at the anterior pole of the POA and demonstrated the presence of ERs in GFAP-ir cells in the ME. Cell counts at the ME showed that 23 of 50 (46%) GFAP-ir cells were ER ir. ER-ir was also present in scattered ependymal cells lining the third ventricle at the POA and overlying the ME. Typically, approximately four to eight ER-ir ependymal cells were present around the perimeter of the third ventricle, although occasionally small aggregations of greater numbers of labeled cells were observed. Both common ependyma and cells morphologically identified as tanycytes were ER-ir. Some endothelial cells and vascular smooth muscle cells also contained ERs. While approximately 11% of the vessels were lined by ER-ir cells in sections through the MPNa and preventricular periventricular nucleus, approximately 15% of the vessels were labeled in the organum vasculosum of the lamina terminalis. In the ME a greater percentage (59%) of the vessels contained ER-ir endothelial cells. Collectively, these results indicate that in addition to regulating the activity of neurons, estrogen may affect brain function through effects exerted on astrocytes, ependymal cells, and endothelial cells. PMID- 1727711 TI - Pancreatic islet ganglioside expression in nonobese diabetic mice: comparison with C57BL/10 mice and changes after autoimmune beta-cell destruction. AB - Recent observations have shown that the presumed target antigen of cytoplasmic islet cell antibodies (ICA) has properties of a monosialo-ganglioside migrating between GM2 and GM1 standards (GM2-1) and that ICA binding is higher in nonobese diabetic (NOD) than in C57BL/10SnJ mouse pancreatic frozen sections. This study aimed to characterize the ganglioside expression in NOD mouse islets in comparison with the control C57BL/10SnJ strain, taking into account possible sex differences, variations with age, and changes after autoimmune beta-cell destruction. Thus, acidic glycolipid composition was analyzed 1) in isolated islets from 11-week-old female and male NOD mice and age-matched female and male C57BL/10SnJ mice, and 2) in whole pancreas of both NOD and control mouse strains at different ages (4, 8, and 18 weeks) and of female NOD mice before and after diabetes onset. The acidic glycolipid GM2-1 is expressed in isolated female NOD islets, male NOD islets, and C57BL/10SnJ mouse islets, but quantitative analysis showed an increased amount of GM2-1 in NOD vs. C57BL/10 islets. GM3 is a ganglioside fraction expressed in female and male NOD mice and not in the C57BL/10 strain, whereas GD3 characterizes the C57BL/10 strain islets. GM2-1 is the sole ganglioside fraction in the whole pancreas to clearly decrease with age in the NOD mouse, and diabetes onset in this strain is associated with a significant decrease in the expression of this component as well as of GM3, whereas other pancreatic ganglioside (GD3, GD1a, and GT1b) levels did not significantly decrease; no age-related ganglioside change was observed in the C57BL/10SnJ mouse. Interestingly, the observed increased ICA binding in NOD islets is paralleled by the increased expression of GM2-1 islet ganglioside, and beta-cell destruction in NOD mice is associated with a significant decrease in the amount of this ganglioside in the pancreas. PMID- 1727712 TI - Tyrosine phosphorylation selectively regulates renal cellular phosphate transport. Evidence that it mediates the stimulatory effect of insulin-like growth factor-1. AB - The signal transduction mechanism responsible for the stimulation of the transport of inorganic phosphate (Pi) in response to mitogens is not known. In the present study, the changes in both Pi transport and tyrosine phosphorylation activity were determined in response to orthovanadate (VO4), an insulin-like agent and genistein, a specific inhibitor of tyrosine kinase activity. The results indicate that in opossum kidney epithelia, VO4 stimulated and genistein inhibited Pi transport dose dependently. The characteristics of the VO4 effect were quite similar to those described for insulin-like growth factor-1 (IGF-1) in terms of time course, selectivity (no effect on the Na-alanine transport), and dependency of the de novo synthesis of proteins. The effects of VO4 and IGF-1 on Pi transport, when tested at submaximal and maximal concentrations, respectively, were not additive suggesting that these two agents act through a common regulatory mechanism. As previously shown with IGF-1, the VO4 effect on Pi transport was additive to that of the maximal effect of Pi deprivation. Changes in tyrosine phosphorylation activity were tested in purified plasma membrane isolated from confluent OK cells using the polymer Glu:Tyr (4:1) as exogenous substrate. VO4 markedly enhanced whereas genistein inhibited the tyrosine kinase activity. The VO4 effect was dose dependent (0.1-1.0 mM), a concentration range similar to that eliciting the Pi transport response. The change in Pi transport was highly correlated with the variation in the tyrosine kinase activity induced by either vanadate (R = 0.969 P less than 0.01) or genistein (R = 0.992, P less than 0.01). In conclusion, VO4, an insulin-like agent and genistein, a specific inhibitor of tyrosine kinase activity selectively altered cellular Pi transport. These changes in Pi transport were associated with a dose-related alteration in tyrosine kinase activity measured in purified plasma membrane. The characteristics of the vanadate effects on Pi transport are similar to those reported for IGF-1 suggesting an important role of this signal transduction mechanism in mediating changes in Pi transport in response to mitogenic factors such as IGF-1. PMID- 1727713 TI - Ontogeny of 11 beta-hydroxysteroid dehydrogenase in rat brain and kidney. AB - Close regulation of circulating corticosteroid levels during the early postnatal period is crucial for normal development and maturation of the central nervous system. In the first weeks of life cerebral glucocorticoid receptor concentrations are low and the hypothalamic-pituitary-adrenal axis is relatively unresponsive to stress, which might, in part, protect the developing brain from elevated corticosteroid levels. However, central mineralocorticoid receptors are at near adult levels and free glucocorticoid concentrations may approximate adult values as corticosteroid binding globulin is absent. Thus other mechanisms controlling cerebral exposure to corticosteroids may be of importance. 11 beta Hydroxysteroid dehydrogenase (11 beta-OHSD) determines the access of corticosterone to peripheral mineralocorticoid and glucocorticoid receptors in adults in vivo by metabolizing corticosterone to inactive 11 dehydrocorticosterone. The enzyme has recently been demonstrated in brain subregions and may modulate local corticosteroid-receptor interactions. We therefore examined 11 beta-OHSD bioactivity and messenger RNA (mRNA) expression in the brain, compared with kidney, during the neonatal period. 11 beta-OHSD bioactivity (expressed as the percentage conversion of corticosterone to 11 dehydrocorticosterone) was moderately high in hippocampus and parietal cortex at birth (46 +/- 4% and 48 +/- 5%, respectively), fell significantly to a nadir (32 +/- 1% and 30 +/- 1%, respectively) at postnatal day 10 and then gradually rose to adult values (52 +/- 3% and 58 +/- 3%). By contrast, 11 beta-OHSD activity in cerebellum was high at birth (60 +/- 3%), rose significantly to a peak at postnatal day 10 (74 +/- 3%), and then fell to adult values by postnatal day 15 (64 +/- 3%). Renal 11 beta-OHSD activity was moderately high (69 +/- 3%) at birth and reached adult values (80 +/- 2%) by postnatal day 5. Northern blots showed high and similar expression of a single species of 11 beta-OHSD mRNA from birth to adulthood in the hippocampus. Only low expression of 11 beta-OHSD (two or three separate species) was found in the kidney during the first 2 weeks of life, whereas, in adults high expression of 11 beta-OHSD mRNA was detected in kidney (four species). Using in situ hybridization high 11 beta-OHSD mRNA expression was localized to the neuronal layers of the postnatal hippocampus, neocortex, and cerebellum, and low but detectable expression was found in the neonatal renal cortex. Thus, 11 beta-OHSD is highly expressed in rat brain subregions in the early postnatal period with specific developmental patterns of activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727714 TI - An evaluation of the functions of the 22-kilodalton (kDa), the 20-kDa, and the N terminal polypeptide forms of human growth hormone using transgenic mice. AB - Multiple peptide hormones can be derived from the single human GH gene. In addition to the full-length 191-amino acid 22-kilodalton (kDa) form, a 20-kDa variant can be produced by alternative splicing, and a 5-kDa variant can be produced by posttranslational cleavage. To more fully appreciate the physiological roles of these proteins, we have made a comparison of transgenic mice that constitutively overexpress one or another of these variants. We have found that both the 22-kDa and the 20-kDa forms of human GH stimulate linear growth and liver hypertrophy. The increase in linear growth in 22-kDa transgenic mice does not, however, correlate with an increase in circulating IGF-I; rather, the increase in IGF-I that does finally occur correlates with marked liver pathology. Both groups of mice also develop glomerulosclerosis and suffer from hyperinsulinemia. Although there are histologically obvious lesions in the livers of both the 22-kDa and the 20-kDa transgenic mice, only the former exhibit hyperalbuminemia and hypercholesterolemia. Both forms of GH lead to anemia, which is normocytic in the 20-kDa transgenic mice and macrocytic in the 22-kDa transgenic mice. Despite the presence of high levels of the 5-kDa N-terminal form of human GH, the transgenic mice that express this protein are indistinguishable from their nontransgenic littermates. PMID- 1727715 TI - Immunodetection of estrogen receptors in fetal and neonatal male mouse reproductive tracts. AB - Immunodetection methods were used to detect estrogen receptors (ER) in male reproductive tracts on fetal days 13, 15, and 17 and on the day of birth. Immunocytochemistry revealed that most of the cells of the gonad and associated Wolffian duct stained for ER on fetal day 13. During the next 6 days, ER distribution changed, and by the day of birth, ERs were observed only in epithelial cells of the epididymis (derived from the Wolffian duct) and in a portion of cells from the testis. Immunoblots confirmed that a band the size of the ER stained in reproductive tracts for all ages studied. Similar to the fetal female, ERs are present throughout the early development of the fetal male reproductive tract. However, in contrast to the female, ERs appear to decrease in the male fetal reproductive tract at the time of birth. PMID- 1727716 TI - Tumor necrosis factor impairs insulin action on peripheral glucose disposal and hepatic glucose output. AB - The present study examined whether a prolonged infusion of tumor necrosis factor (TNF) into rats could sustain the increased rate of whole body glucose metabolism observed with short term exposure, and whether TNF produced hepatic or peripheral insulin resistance. Basal glucose metabolism was determined with the use of [3 3H]glucose 18 h after initiating a constant infusion of recombinant human TNF (1 microgram/kg.h). Thereafter, a two-step euglycemic hyperinsulinemic clamp was performed to determine whether TNF impaired insulin action. The overnight infusion of TNF minimally elevated plasma glucose concentrations (17%), but produced large increases in the whole body rate of glucose production and utilization (133%). Under hyperinsulinemic conditions, the glucose infusion rate necessary to maintain euglycemia was 30% lower in TNF-treated rats, indicating an insulin-resistant condition. This resulted from an impaired ability of insulin to both suppress hepatic glucose production and stimulate peripheral glucose utilization in TNF-infused animals. A second series of experiments was performed, using the in vivo tracer [U-14C]2-deoxyglucose technique, to elucidate which tissues were responsible for the TNF-induced increase in basal (no exogenous insulin) glucose disposal and peripheral insulin resistance. Under basal conditions, TNF increased glucose uptake by various muscles (gastrocnemius, heart, and diaphragm) as well as nonmuscle tissues (liver, lung, spleen, gut, skin and fat). Because of their relatively large mass and/or high rate of glucose uptake, the increased uptake by skin (25%), intestine (24%), muscle (23%), and liver (15%) accounted for the majority of the TNF-induced increment in whole body glucose disposal. Under euglycemic hyperinsulinemic conditions, the increment in glucose uptake by muscle and skin (85%) accounted for the majority of the glucose disposal in control rats. However, in TNF-infused animals, hyperinsulinemia failed to increase glucose uptake by skin and blunted the insulin-mediated increase in muscle by 73%. These results suggest that sustained elevations of TNF during chronic therapy and prolonged production of TNF by patients and experimental animals with malignancies or infectious diseases may be an important mechanism for the enhanced glucose flux as well as the insulin resistance seen in these conditions. PMID- 1727717 TI - Effect of macrophage colony-stimulating factor on in vitro osteoclast generation and bone resorption. AB - To investigate the role of recombinant human macrophage colony-stimulating factor (rhM-CSF) on in vitro bone resorption, two bone explants, each at a different developmental stage, were adopted, namely 1) radii and 2) metatarsals of 17-day old embryonic mice. At this stage of gestation, bone resorption in the radii is exclusively dependent upon fusion of osteoclast precursors and activation of mature osteoclasts, whereas in metatarsals it is dependent upon the generation of new osteoclasts. rhM-CSF showed no effect in radii, but stimulated 45Ca release in metatarsals, when they were either intact or periosteum stripped in coculture with embryonal liver as a source of hemopoietic progenitors. The rhM-CSF-induced increase in 45Ca release was paralleled by a higher number of osteoclasts. The stimulating effect was found to be in a concentration range between 250-500 U/ml M-CSF. The action of rhM-CSF was blocked by irradiation, indicating that it is dependent upon cell proliferation. These results, thus, show that M-CSF stimulates bone resorption only when it is dependent on generation of new osteoclasts. M-CSF does not appear to have any effect on the activity of mature osteoclasts. The mechanism of action might be direct on osteoclast precursors or indirect on accessory cells influencing osteoclast generation. PMID- 1727718 TI - Transforming growth factor-beta stimulates ascorbate transport activity in osteoblastic cells. AB - Transforming growth factor-beta (TGF beta) modulates the proliferation and differentiation of a number of cell types, including osteoblasts. TGF beta has been shown to stimulate matrix synthesis by connective tissue cells, but its mechanism of action is poorly understood. Because ascorbate (reduced vitamin C) also influences osteoblastic differentiation and is required as a cofactor for collagen synthesis, the present study examined the effect of TGF beta on osteoblastic ascorbate uptake. Saturable Na(+)-dependent uptake of ascorbate by cultures of UMR-106 rat osteosarcoma cells proceeded linearly with time for at least 10 min at 37 C. Exposure of cultures to TGF beta 1 stimulated initial rates of saturable Na(+)-dependent ascorbate transport, but did not affect nonspecific uptake or binding of the vitamin. Cells pretreated for 24 h with either vehicle or TGF beta 1 (3 ng/ml) and then assayed for transport of L-[14C] ascorbate (10 microM) showed significantly different transport activities (vehicle, 30 +/- 2; TGF beta 1, 44 +/- 3 nmol ascorbate/g protein/min; n = 14; P less than 0.005). Kinetic studies revealed that TGF beta 1 increased the maximum velocity of ascorbate transport without changing the affinity of the transporter for the vitamin, since the apparent maximum velocity increased from 83 to 106 nmol ascorbate/g protein/min; while the apparent Km remained unchanged at 20 microM L ascorbate. The effect of this growth factor on ascorbate transport appeared to require protein synthesis, because it was completely blocked by cycloheximide. These results are consistent with TGF beta 1 increasing the rate of synthesis of either new Na+ ascorbate cotransporters or a regulatory protein that interacts with existing transporters to increase their turnover number. Enhanced uptake of ascorbate may contribute to the increase in collagen synthesis induced by TGF beta. PMID- 1727719 TI - Dynamics of gonadotropin-releasing hormone release during a pulse. AB - This study examined the nature of the GnRH signal that travels down the pituitary portal vessels and causes an LH pulse. Individual GnRH pulses were described in terms of abruptness of increase and decrease, amplitude, duration, and amount of GnRH released. Pituitary portal blood was obtained at 30-sec intervals for 2.5 or 5 h from five short-term ovariectomized ewes. Jugular blood was sampled every 10 min for LH. We examined 13 GnRH pulss; each produced an LH pulse. The contour of most GnRH pulses approximated a square wave. The rising edge of the GnRH pulse was very abrupt; GnRH secretion increased as much as 50-fold within 1 min. The mean peak amount of GnRH collected during pulses (24 pg/min, range 2-66) was 70 fold greater than the interpulse baseline (0.2-0.5 pg/min). The release period was sustained an average of 5.5 min; thereafter, GnRH fell to prepulse levels within 3 min. Overall, the larger and more prolonged pulses of GnRH were associated with higher amplitude LH pulses. To assess the distortion of the GnRH signal by the collection procedure, samples were obtained in vitro using the same technique during application of 4- and 7-min square wave GnRH pulses by means of a syringe pump. Signals were carried as square-waves through the sampling operation with minimal distoration, with the exception that amplitude decreased during the collection procedure. Our findings indicate the square-wave pulses observed in vivo are an accurate description of the dynamics of GnRH release during a pulse in short-term overiectomized ewes. PMID- 1727720 TI - The protein kinase-C activation domain of the parathyroid hormone. AB - The PTH activates both adenylate cyclase and a mechanism that increases membrane associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1 2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28 34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH (1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments. PMID- 1727721 TI - Localization of an 11 beta hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney. AB - An 11 beta hydroxysteroid dehydrogenase (11 beta HSD) activity has been localized in the rat kidney by a histochemical technique which links steroid metabolism with the production of a color reaction. Oxidation of 11 beta hydroxyandrostenedione was observed in cortical distal convoluted tubules and in medullary collecting ducts. Carbenoxolone abolished staining, no reaction was obtained with androstenedione hydroxylated at the 17 or 19 position, and oxidation of 11 beta-hydroxyandrostenedione was nicotinamide-adenine dinucleotide (NAD) dependent. These results demonstrate the presence of a dehydrogenase activity separate from the nicotinamide-adenine dinucleotide phosphate (NADP) dependent 11 beta hydroxysteroid dehydrogenase recently purified and cloned from rat liver. We have named this activity 11 beta HSD2 to distinguish it from the NADP-dependent 11 beta HSD. Histological studies showed that 11 beta HSD2 activity does not correlate with the immunocytochemical localization of the previously defined 11 beta HSD enzyme, but rather the 11 beta HSD2 activity is localized in the distal tubules of the rat kidney. In this respect 11 beta HSD2 colocalizes with the mineralocorticoid receptor. No reaction product was obtained using cortisol or corticosterone as substrate with either NAD or NADP as cofactor. Furthermore incubation of tissue sections with 11 beta androstenedione in the presence of deoxycorticosterone completely inhibited cytochemical staining. We interpret these results as evidence of 20 reductase activity which uses the reduced cofactor at the expense of the color reaction. These results support the crucial role played by an 11 beta hydroxysteroid dehydrogenase in the local protection of type I receptors in mineralocorticoid selective tissues. PMID- 1727722 TI - Detection and identification of endothelin-1 immunoreactivity in rat and porcine thyroid follicular cells. AB - Endothelin-1 immunoreactivity (irET-1) was observed in rat and porcine thyroid glands. Using a radioimmunoassay for endothelin-1, the mean concentration in extracts of rat and porcine thyroid glands were 0.75 pg/mg +/- 0.03 (n = 4) and 1.5 pg/mg +/- 0.2 (n = 8) (mean +/- SE) respectively. Gel-filtration and reverse phase HPLC showed that ir ET-1 eluted in a position identical to synthetic endothelin-1. In addition, immunohistochemical study showed that irET-1 is located within epithelial follicular cells. No immunostaining was seen in parafollicular C-cells nor in parathyroid. PMID- 1727723 TI - Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin. AB - This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2) treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727724 TI - Alfred E. Mirsky and the foundations of molecular biology and neuroendocrinology. PMID- 1727725 TI - Polarized rat uterine epithelium in vitro: responses to estrogen in defined medium. AB - Previously described procedures for the culture of immature rat uterine epithelium (UE) allowed the cells to proliferate to confluence and develop morphological and functional polarity. The present study describes the transition from culture of UE cells in serum to a serum-free defined medium. This was accomplished with no significant alteration in the ability of UE cells to attain morphological and functional polarity. In defined medium, which contained estrogen (2.5 x 10(-9) M), UE cells proliferated to confluence, demonstrated separation of apical and basal plasma membrane domains, and displayed preferential secretion of proteins and proteoglycans from the apical surface. Apical secretions of polarized cultures contained complement component C3, the secreted portion of the immunoglobulin A receptor and the secretory glycoprotein, USP-1. The cell surface adhesion molecule, CAM 105, could be demonstrated at the apical cell surface. Expression of this profile of secretory and cell surface markers, representative of the in vivo estrogen response of immature rat UE cells, correlated with an in vitro state of non-receptivity of polarized UE cells toward blastocysts which remained viable and competent to attach. We conclude that the polarized UE cell that develops in the described defined medium expresses a phenotype similar to that which characterizes the in vivo uterine estrogen response. PMID- 1727726 TI - Polarized rat uterine epithelium in vitro: constitutive expression of estrogen induced proteins. AB - The hormonal responsiveness of immature rat primary uterine epithelial (UE) cells, cultured in a serum-free, phenol red-free defined medium, was examined under conditions which allowed the UE cells to reestablish their polarized phenotype. In the absence of estradiol and phenol red UE cells proliferated to confluence, achieving cell densities equal to those reached by UE cells cultured in the presence of estradiol. The expression of marker proteins, characteristic of the in vivo response of the uterus to estrogen, i.e. the adhesion molecule cell CAM 105, complement component C3, the secretory component of the immunoglobulin A receptor, and keratan sulfate proteoglycan, by polarized cultures of UE cells proved to be independent of estrogen in vitro. Polarized UE cells required the presence of estrogen to maintain integrity of their monolayer and did exhibit a dose-dependent response to estradiol in vitro in terms of cell growth (hypertrophy) and the secretion of two proteins not previously described as estrogen response markers. UE cell secretion, in particular apical secretion, was stimulated by estradiol but not by progesterone, dexamethasone, or testosterone. Progesterone failed to down-regulate the polarized UE cell responses to estradiol. Collectively, these observations suggest that many of the responses which nominally characterize the action of estrogen on the UE cell in vivo are likely to be initiated by agents other than estrogen, e.g. growth factors. PMID- 1727727 TI - Activation of phospholipase D by glyceraldehyde in isolated islet cells follows protein kinase C activation. AB - Our recent studies have demonstrated the presence in neonatal islet cells and intact adult islets of a phosphatidylcholine-directed phospholipase D (PLD) which is activated after phorbol ester stimulation. The present study describes PLD activation in the presence of a carbohydrate insulin secretagogue. At the highest concentration tested (20 mM) the triose, glyceraldehyde, induced formation of phosphatidic acid in cells prelabeled with [14C]arachidonic acid or [3H]myristic acid (164 +/- 7 and 210 +/- 9% of basal phosphatidic acid values, respectively). Experimental confirmation of a concentration-dependent specific activation of PLD was provided by the formation of a transphosphatidylation product, phosphatidylethanol, after stimulation with glyceraldehyde in the presence of added ethanol (1.5%). Additionally, there was an early (within 5 min) rise in [14C]arachidonate-labeled diacylglycerol (139 +/- 7% of basal) accompanied by an increase in intracellular diacylglycerol mass (51 +/- 2 pmol/mg protein) and an increase in membrane-associated protein kinase C activity (183 +/- 5% of basal) which preceded the activation of PLD, as indicated by the time course of glyceraldehyde-stimulated phosphatidylethanol formation in the presence of ethanol. Pretreatment of islet cells with 2 microM 12-O-tetradecanoylphorbol-13 acetate for 18 h, to down-regulate protein kinase C, was without effect on diacylglycerol and phosphatidic acid production after 5 min but inhibited completely the production of phosphatidylethanol at 30 min. The phosphohydrolase inhibitor propranolol (100 microM) potentiated the accumulation of phosphatidic acid and phosphatidylethanol incubation following incubation with glyceraldehyde. These findings demonstrate for the first time that a physiological nutrient activates a phospholipase directed against endogenous phosphatidylcholine in intact islet cells; furthermore, they indicate a role for PLD in a delayed formation of phosphatidic acid in the islet cell. The finding of an early rise in glyceraldehyde-stimulated diacylglycerol (which may be formed de novo or by the action of phospholipase C), suggests that PLD is recruited by the activation of protein kinase C by this nutrient. PMID- 1727728 TI - History of Diabetes, the Journal (1952-1991) editor's swan song. AB - Diabetes has provided scientific information in diabetes-related areas of basic and clinical research since 1952. This brief historical overview of the journal's activities and published articles is offered as an illustration of the remarkable strides in diabetes-related research that have taken place over the past 40 years. PMID- 1727729 TI - Psychomotor slowing is associated with distal symmetrical polyneuropathy in adults with diabetes mellitus. AB - To test the hypothesis that diabetes mellitus is associated with cognitive dysfunction, a battery of neuropsychological tests was administered to 75 diabetic adults and an equal number of demographically similar nondiabetic control subjects. Compared with control subjects, diabetic subjects performed significantly more poorly on measures of psychomotor efficiency and spatial information processing. In contrast, no between-group differences appeared on measures of verbal intelligence, learning, memory, problem solving, or simple motor speed. Results from multiple regression analyses showed that clinically significant distal symmetrical polyneuropathy was strongly associated with psychomotor slowing, whereas, glycosylated hemoglobin values were weakly associated with both psychomotor slowing and spatial processing. No other biomedical variables predicted cognitive test performance. These neurobehavioral data are consistent with the hypothesis that a "central neuropathy" may be associated, at least in part, with chronic hyperglycemia. PMID- 1727730 TI - Prevention of recurrence of IDDM in islet-transplanted diabetic NOD mice by adjuvant immunotherapy. AB - Insulin-dependent diabetes mellitus (IDDM) involves the destruction of the insulin-producing cells in the islets of Langerhans. One possible cure is by transplanting the islet cells; however, transplanted islets, even between identical twins, are subject to autoimmune destruction by the disease process, resulting in diabetes recurrence. We recently reported that complete Freund's adjuvant (CFA), an immunomodulating agent, prevented development of autoimmune diabetes in the NOD mouse. In this study, we evaluated adjuvant therapy in prevention of autoimmune destruction and rejection of transplanted islets in diabetic NOD mice. After transplantation, untreated syngeneic islet recipients (n = 16) initially became normoglycemic and then hyperglycemic, with a median survival time (MST) of the graft of 17 days. When CFA was administered at the time of transplantation, 11 of 13 CFA-treated syngeneic islet recipients remained normoglycemic long term (greater than 100 days) with an MST greater than 107 days. Ten of 11 mice maintained indefinite normoglycemia until the conclusion of follow-up (101 to 172 days). When adjuvant therapy was used in conjunction with allogeneic islet transplantation, graft survival was not extended, with MST being similar to the untreated allogeneic islet recipients (12 [n = 5] and 13 [n = 5] days, respectively). The extended acceptance of second syngeneic islet grafts by CFA-treated mice indicates that the persistent autoimmunity against the transplanted islets can be reversed in the diabetic NOD mice after CFA treatment. PMID- 1727731 TI - T-lymphocyte lines specific for glutamic acid decarboxylase (GAD) the 64K beta cell antigen of IDDM. AB - Insulin-dependent diabetes mellitus (IDDM) is viewed as a thymus-dependent autoimmune disease, although the specific beta-cell autoantigen or autoantigens remain unknown. The recent identification of the beta-cell 64K antigen as the enzyme glutamic acid decarboxylase (GAD) permits investigation of GAD as a candidate for the autoantigen associated with beta-cell destruction, mediated by T-lymphocytes, in susceptible individuals. In this study, we describe the isolation of GAD-specific T-lymphocyte lines from BB rats, an animal model of IDDM. GAD (Escherichia coli) was inoculated into the footpads of diabetes resistant BB rats, and after 10 days, a popliteal lymph node cell culture suspension was prepared. GAD-specific T lymphocytes were obtained by culture with interleukin 2 and repeated stimulation with GAD in the presence of BB rat thymic antigen-presenting cells. Four stable, CD4+, MHC (RT1u)-restricted T-lymphocyte lines were isolated. They proliferate selectively in the presence of GAD and secrete interleukin 2 and interferon-gamma. T-lymphocyte lines such as these could be important in the definition of pathogenetic epitopes associated with GAD. PMID- 1727732 TI - Glucagon immunoneutralization in diabetic rats normalizes urea synthesis and decreases nitrogen wasting. AB - To study the effect of glucagon neutralization on urea synthesis in diabetic rats, animals with newly induced (75 mg/kg streptozocin) experimental diabetes mellitus were divided into two groups. One group was given one weekly injection of nonimmune rabbit serum (n = 6), and the other group was given one weekly injection of a specific high-titer antibody against pancreatic glucagon (n = 6). Four weeks later, serum-treated diabetic rats had fasting glucagon concentrations 2-3 times higher than nondiabetic controls given one weekly injection of saline (control). Plasma glucagon binding capacity of diabetic rats given glucagon antibodies was 10-15 times higher than the glucagon concentration. A second group of nondiabetic controls were given nonimmune serum. Blood glucose concentration and urinary glucose output were identical in both groups of diabetic animals. Food intake doubled in both groups of diabetic rats. In control rats, the accumulated nitrogen balance, determined weekly for 4 wk, was positive at 81 +/- 3.1 mmol/96 h; in serum-treated diabetic rats, the accumulated nitrogen balance was negative, -8.3 +/- 2.4 mmol/96 h throughout the 4 wk, whereas it was higher at 4.7 +/- 2.3 mmol/96 h in the glucagon antibody-treated diabetic rats (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727733 TI - Pituitary response to growth hormone-releasing hormone in IDDM. Abnormal responses to insulin and hyperglycemia. AB - In poorly controlled insulin-dependent diabetes mellitus (IDDM), hyperglycemia fails to inhibit the pituitary response to growth hormone-releasing factor (GRF). To evaluate whether this derangement is reversed by a simultaneous elevation of circulating insulin, 0.3 micrograms/kg i.v. GRF 1-40 was administered to nine poorly controlled IDDM subjects (HbA1 greater than 11.1%) with and without concomitant infusion of insulin. In the absence of insulin, the poorly controlled IDDM subjects demonstrated a growth hormone response to GRF similar to that of nondiabetic subjects, despite marked hyperglycemia (approximately 16.8 mM). When insulin was infused into these same patients (insulin clamp) to produce combined hyperinsulinemia (528 +/- 90 pM) and hyperglycemia (16.5 +/- 1.98 mM), the GRF induced growth hormone rise was markedly exaggerated (65 +/- 11 vs. 20 +/- 4 micrograms/L without insulin infusion, P less than 0.001). This enhancement of GRF-stimulated growth hormone release by insulin was strikingly attenuated (22 +/ 7 micrograms/L) in five well-controlled diabetic subjects studied under conditions of similar hyperinsulinemia (486 +/- 84 pM) and hyperglycemia (16.41 +/- 0.95 mM). In contrast, in nondiabetic subjects, acute hyperinsulinemia reduced the growth hormone response to GRF. We conclude that the failure of hyperglycemia to block the pituitary response to GRF in poorly controlled diabetes is not attributable to the lack of a coincident increase in circulating insulin. The paradoxical stimulatory effect of insulin on GRF-induced growth hormone release may contribute to the high spontaneous growth hormone levels characteristically seen in poorly controlled insulin-treated patients, and its attenuation after intensive insulin therapy may contribute to the reversal of growth hormone hypersecretion in well-controlled diabetic patients. PMID- 1727734 TI - Upregulation of GLUT2 mRNA by glucose, mannose, and fructose in isolated rat hepatocytes. AB - Previously, demonstrated that GLUT2 mRNA and protein are increased in liver of streptozocin-induced diabetic rats. To examine the mechanisms whereby GLUT2 mRNA is regulated, we cultured isolated hepatocytes in the absence and presence of various concentrations of glucose. Culture of hepatocytes in high glucose concentration (27.8 mM) for 20 h induced a 3.2-fold increase in GLUT2 mRNA levels compared with hepatocytes cultured without D-glucose. Interestingly, D-mannose and D-fructose could substitute for D-glucose to elevate the GLUT2 mRNA level, whereas 3-O-methyl-D-glucose, 2-deoxy-D-glucose, and sucrose, which were not metabolized or taken up by the cells, were without effect. Insulin had no significant effect on GLUT2 mRNA levels in hepatocytes in the presence or absence of D-glucose. Therefore, the regulation of the GLUT2 gene by D-glucose in hepatocytes is contrary to that reported for GLUT1 and GLUT4 genes, which are downregulated by D-glucose. These results also suggest that the elevated GLUT2 mRNA level observed in diabetic rat liver is due to the high blood glucose concentration rather than to insulin deficiency. PMID- 1727735 TI - Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine. AB - Aminoguanidine-HCl inhibits the formation of advanced glycosylation end products (AGEs) in vitro and in vivo, but the mechanism by which this occurs has not been determined. Aminoguanidine inhibited glucose-derived AGE formation on RNase A by 67-85% at aminoguanidine-glucose molar ratios of 1:5 to 1:50 without affecting the concentration of Amadori products. Fast-atom-bombardment mass spectrometry of RNase peptides incubated with glucose alone or with glucose plus aminoguanidine showed that aminoguanidine inhibited the formation of AGEs without forming an adduct with glycosylated peptide. These data suggest that the primary mechanism of aminoguanidine action is reaction with Amadori-derived fragmentation products in solution. These findings are relevant to the potential clinical use of aminoguanidine in the prevention of diabetic complications. PMID- 1727736 TI - Effect of diabetes on fast response to norepinephrine in rat aorta. AB - The fast and slow components of the mechanical response to 1 microM norepinephrine (NE) were measured in aortic rings isolated from eight spontaneously diabetic rats, six streptozocin-induced diabetic (STZ-D) rats, six STZ-D rats treated with 2.5 U insulin/day during the 4 days before being killed, and six age- and sex-matched control rats. The total contraction to NE (i.e., the sum of fast and slow components) was similar in the four groups: spontaneously diabetic, 16.53 +/- 1.72 mN; STZ-D, 15.68 +/- 1.41 mN; insulin-treated, 16.17 +/- 2.05 mN; and control, 15.27 +/- 0.96 mN (NS). The fast component, measured graphically in a total contraction in 1.35 mM Ca, was greater in spontaneously diabetic (12.61 +/- 1.07 mN, P less than 0.05) and STZ-D (12.25 +/- 0.89 mN, P less than 0.05) rats compared with control (9.14 +/- 0.74 mN) or insulin-treated (8.58 +/- 1.23 mN) rats. The same increase of the fast component was detectable after 3 min of incubation in Ca-free medium + 2 mM EGTA (control 6.54 +/- 0.47 mN, spontaneously diabetic 9.07 +/- 0.76 mN, P less than 0.05; STZ-D 8.82 +/- 0.72 mN, P less than 0.05), and it was also abolished by insulin treatment (insulin-treated 6.29 +/- 0.36 mN). We conclude that the diabetic state increases the fast component of NE-induced contraction either in the absence or presence of Ca in the medium. This suggests that such an increase depends on a larger release of Ca from intracellular stores. PMID- 1727737 TI - Effect of Mg2+ on Na(+)-dependent inositol transport. Role for Mg2+ in etiology of diabetic complications. AB - Diabetes mellitus is associated with a significant reduction in the serum concentration of Mg2+. Several studies have suggested that hypomagnesemia may be implicated in the etiology of diabetic complications; however, no mechanism has been proposed. This study demonstrates that Mg2+ is a positive effector of inositol transport and is capable of promoting a 2.5-fold increase in the affinity of the transporter for inositol. Analysis of the kinetics of inositol transport shows that, at physiological concentrations of inositol, the reductions in Mg2+ concentrations that occur in diabetic patients would result in a significant decline in the rate of inositol transport (1.5- to 2-fold). We suggest that hypomagnesemia may be linked to the development of diabetic complications via reduction in the rate of inositol transport and subsequent intracellular inositol depletion. This assertion allows hypomagnesemia and the polyol theory to be unified into one mechanistic model for the development of diabetic complications. PMID- 1727738 TI - High incidence of thyroiditis and anti-thyroid autoantibodies in NOD mice. AB - NOD mice develop spontaneous insulin-dependent diabetes mellitus (IDDM) associated with infiltration of pancreatic islets with mononuclear cells. Islet infiltration results in autoimmune destruction of insulin-secreting beta-cells. Because in humans and BB rats diabetes is often associated with autoimmune thyroid disease (ATD), the NOD mouse model was examined for evidence of thyroiditis and serum antibodies reactive with mouse thyroid membrane antigens (MTMAs). The incidence of thyroiditis was 77% in mice greater than 180 days old, 67% in mice 61-180 days old, 72% in mice 31-60 days old, 74% in mice 21-30 days old, 78% in mice 11-20 days old, and 90% in mice less than or equal to 10 days old. NOD mice less than or equal to 30 days old had less-severe thyroiditis than animals greater than 180 days old. There was no significant different in severity of thyroiditis between any of the other age-groups tested. The incidence of thyroiditis was not increased in diabetic compared with nondiabetic animals, nor was an association found between thyroiditis and sex. The high incidence of thyroiditis in the less than or equal to 30-day-old age-group indicates that infiltration of lymphocytes into the thyroid can precede initiation of insulitis in this model. Although both thyroiditis and insulitis in NOD mice began early (by the 1st and 2nd mo of life, respectively), no significant association between infiltration of these two organs was noted in individual mice.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727739 TI - Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. AB - Nonenzymatic glycosylation of structural proteins and the formation of advanced glycosylation end products (AGEs) have been involved in the pathogenesis of diabetic complications. We examined the effect of aminoguanidine, a potent inhibitor of AGE formation, on functional and structural abnormalities in peripheral nerve of streptozocin-induced diabetic rats. Diabetic rats were treated with daily injections of 25 mg/kg body wt s.c. aminoguanidine (AG) sulfate for 16 wk and compared to untreated diabetic rats. AG treatment improved motor nerve conduction velocity in 12- and 16-wk diabetic rats despite no changes in body weight, blood glucose, and HbAIc levels. AG treatment inhibited an accumulation of fluorescent AGE in diabetic nerves, and morphometric analysis of the sural nerve showed a partial effect on myelinated fiber size and axonal atrophy. These findings suggest that AG may have a beneficial effect on diabetic neuropathy and nonenzymatic glycosylation of peripheral nerve proteins. PMID- 1727740 TI - Evidence that two naturally occurring human insulin receptor alpha-subunit variants are immunologically distinct. AB - The IgG from a patient (Italy 2 [I2]) with hypoglycemia, due to autoantibodies to the insulin receptor, was purified on protein A Sepharose into two fractions that were tested in various human tissues and cells. The IgG fraction that bound protein A (absorbed IgG [IgGa]) nearly completely inhibited the binding of 125I labeled insulin to various cells or tissues (placenta, IM-9, adipocytes, HEp-2 larynx cells, Epstein-Barr virus lymphocytes) but not greater than 50% of 125I labeled insulin binding to human liver membranes. Conversely, both the IgG fraction from this patient, which did not bind protein A (flow-through IgG [IgGb]), and the IgGa fraction from a second similar patient (Italy 1 [I-1]) almost completely inhibited the binding of 125I-labeled insulin to liver membranes. The IgGa fraction from patient I-2 did not change receptor affinity because 50% inhibition of 125I-labeled insulin binding was not affected by either the presence or absence of these IgG fractions. Furthermore, liver binding data were not due to cross-reaction of 125I-labeled insulin to the insulinlike growth factor I receptor, and treatment of liver membranes with neuraminidase did not alter the inhibitory effect of the IgGa fraction from patient I-2 on 125I-labeled insulin binding to liver. Binding inhibition experiments performed with cells transfected with and overexpressing the -12 (human insulin receptor [HIR]-A) or the +12 (HIR-B) variant of HIR revealed that the IgGa fraction from patient I-2 inhibited 125I-labeled insulin binding to the HIR-A receptor but not to the HIR-B receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727741 TI - Effect of angiotensin-converting enzyme inhibition on renal function and albuminuria in normotensive type I diabetic patients. AB - Normotensive patients with insulin-dependent (type I) diabetes mellitus (n = 18) were given 25 mg captopril (b.i.d.) and placebo for 3 mo in a randomized double blind crossover study. Patients had normal renal function, and none had retinopathy. Albuminuria was less than 20 micrograms/min in 12 patients and between 20 and 200 micrograms/min in the other 6. Patients were examined at the end of the placebo and captopril phases. Captopril caused little reduction in blood pressure obtained by 24-h ambulatory monitoring (systolic 126.0 +/- 2.7 to 123.9 +/- 2.4 mmHg, P less than 0.08; diastolic 74.2 +/- 1.9 to 72.1 +/- 1.9 mmHg, P less than 0.09). Captopril lowered glomerular filtration rate from 99.5 +/- 7.7 to 71.0 +/- 5.5 ml.min-1. 1.73 m-2 (P less than 0.01), whereas renal plasma flow (443.9 +/- 15.2 ml.min-1. 1.73 m-2) remained unchanged. Filtration fraction was reduced from 22.4 +/- 1.4 to 17.4 +/- 1.4% (P less than 0.01). Urinary albumin excretion was reduced from 59.1 +/- 0.15 to 27.7 +/- 13.9 micrograms/min (P less than 0.1). Reduction was related to the extent of initial albuminuria (r = 0.997, P less than 0.001), a relationship that remained significant after logarithmic transformation (r = 0.540, P less than 0.02). Dextran clearance was used to determine glomerular capillary function. Angiotensin inhibition caused reduction in effective glomerular pore size and also reduced flow via the nondiscriminatory shunt. Angiotensin inhibition in normotensive patients with type I diabetes was well tolerated. Reduction in albuminuria is mediated by a combination of hemodynamic changes and alterations in glomerular capillary function. PMID- 1727742 TI - NOR/Lt mice: MHC-matched diabetes-resistant control strain for NOD mice. AB - NOR/Lt is an insulitis-resistant and diabetes-free strain produced from an isolated genetic contamination within an NOD/Lt pedigree line. The albino coat color phenotype, strain-specific endogenous retroviral profile, and skin graft tests indicated an NOD/Lt x C57BL/KsJ outcross-backcross segregant as the source of the contaminating genome. Analysis of 53 polymorphic DNA, biochemical, and immunologic markers distinguishing NOD/Lt from C57BL/KsJ revealed that 4 chromosomes (chromosomes 2, 4, 11, and 12) in NOR/Lt contained C57BL/KsJ-derived genes. The remaining markers on 14 chromosomes, including the diabetogenic H-2g7 complex on chromosome 17, were of NOD origin. Although completely resistant to cyclophosphamide-induced diabetes, NOR/Lt mice exhibited the same peripheral T lymphocyte accumulation characteristic of NOD/Lt. Similarly, NOR/Lt peritoneal macrophages exhibited depressed interleukin-1 secretion characteristic of NOD/Lt. In addition to their diabetes resistance, NOR/Lt mice were distinguished from NOD/Lt by exhibiting more robust suppressor T-lymphocyte function. Outcross of NOR/Lt with NOD/Lt to generate heterozygosity at those chromosomal segments, defined by C57BL/KsJ markers in NOR/Lt parentals, did not produce insulitis or diabetes in F1 females. However, these F1 females were sensitive to cyclophosphamide-induced diabetes. In summary, the NOR/Lt strain is an MHC matched diabetes-resistant control strain for NOD/Lt. Moreover, NOR/Lt will help identify the location and function of a non-MHC gene or genes capable of conferring resistance against insulitis and diabetes. PMID- 1727743 TI - Transit through the proximal colon influences stool weight in the irritable bowel syndrome. AB - The inherent variability of symptoms and motor abnormalities in patients with the irritable bowel syndrome has hampered the demonstration of motor abnormalities that could underlie symptoms. The aim in the current study was to evaluate whether altered regional capacitance or transit of solid residue through the unprepared human gut were factors in the diarrhea of patients with the irritable bowel syndrome. In 10 such patients and in 5 healthy controls, gastric and small bowel transits were evaluated scintigraphically by means of a mixed meal containing 99mTc-labeled resin pellets. Regional colonic transit was quantitated by 111In-labeled pellets delivered to the ileocecal region by a pH-sensitive, methacrylate-coated capsule. Symptomatic patients did not have significantly altered gastric or small bowel transits, but colonic transit was accelerated in 7 of 10 persons with the irritable bowel syndrome (P less than 0.02), in the proximal colon of five patients and in the left colon of two patients. The 24 hour stool weight was positively correlated with the rate at which solid residue emptied from the ascending and transverse colons (r = 0.78; P less than 0.01). There was also an inverse relationship between emptying rates and maximal volumes accommodated by the proximal colon (r = -0.58; P less than 0.05), although the maximum volume of the proximal colon was not significantly different in patients and healthy subjects. Thus, accelerated transit through the proximal colon is a factor in the pathophysiology of the irritable bowel syndrome and influences the stool weight of such patients. The capacitance of the proximal colon presumably influences its storage capacity and, hence, the rate at which it empties. PMID- 1727745 TI - Collagens facilitate epithelial migration in restitution of native guinea pig intestinal epithelium. AB - An in vitro intestinal epithelial wound/repair model in which epithelium is stripped from villus tips and the wound is resealed during the following 60 minutes has previously been described. The process, termed epithelial restitution, results in part from the rapid migration of epithelial cells shouldering the wound over the denuded basement membrane. The present report examines the requirements for epithelial cell-basement membrane interactions during restitution in this model. Addition of heparin, soluble matrix components, or a variety of antibodies to matrix components (laminin; fibronectin; collagen I, III, IV) does not impair restitution. Although inhibition of protein synthesis alone also does not retard restitution, in the simultaneous presence of antibody to type III and IV collagen restitution is impeded as judged functionally and structurally. Preincubation of tissues with 20 mmol/L cis-OH-proline (a condition known to inhibit cellular secretion of newly synthesized collagen) similarly inhibited structurally and functionally defined restitution only if antibodies to type III and IV collagen were simultaneously present. These results suggest that collagen-epithelial cell interactions are important in restitution after injury, and if necessary, collagen can be produced locally and rapidly at the site of injury to allow restitution to normally proceed. PMID- 1727744 TI - Two doses of omeprazole versus placebo in symptomatic erosive esophagitis: the U.S. Multicenter Study. AB - Two hundred thirty patients with reflux symptoms and endoscopically proven erosive esophagitis were enrolled from 15 U.S. centers into a randomized, double blind, dose-ranging study comparing placebo with omeprazole, 20 or 40 mg given once daily in the morning. Esophagitis grade 2 was present in 44% of patients, grade 3 in 37% of patients, and grade 4 in 19% of patients. Endpoints, defined as complete relief of heartburn and complete esophageal mucosal healing, were assessed after 4 and 8 weeks of treatment. Both omeprazole doses were significantly superior to placebo in complete endoscopic healing. After 8 weeks of treatment, 73.5% of patients in the 20-mg omeprazole group and 74.7% in the 40 mg omeprazole group, compared with 14.0% in the placebo group, had complete healing of the esophageal mucosa. At the end of the study, complete relief of daytime heartburn was obtained in 79.5% of patients in the 20-mg omeprazole group, 81.6% in the 40-mg omeprazole group, and 37.2% in the placebo group (P less than or equal to 0.05). Complete relief of nighttime heartburn was noted by 79.5% of patients in the 20-mg omeprazole group, 85.1% in the 40-mg omeprazole group, and 34.9% in the placebo group (P less than or equal to 0.05). The median time to complete relief of daytime and nighttime heartburn occurred earlier in the 40-mg group than in the 20-mg group (9 vs. 17 days and 9 vs. 20 days, respectively); however, these differences were not statistically significant. Relief of acid regurgitation and dysphagia also occurred earlier in the 40-mg group. Omeprazole was well tolerated in this group of patients. No unexpected adverse experiences occurred. The results of this study confirm those of six multicenter, international trials in which omeprazole in doses of 20-60 mg provided a degree of esophageal mucosal healing and complete relief of reflux symptoms superior to any other medical treatment. PMID- 1727746 TI - Cholecystokinin receptor antagonist MK-329 blocks intestinal fat-induced inhibition of meal-stimulated gastric acid secretion. AB - MK-329, a selective type A cholecystokinin (CCK) receptor antagonist, was given to dogs to test the hypothesis that CCK is one of the principal physiological enterogastrones mediating fat-induced decreases in gastric acid secretion. Gastric acid secretion in response to 300 mL 8% peptone meals was measured by intragastric titration to pH 5.5 in six awake dogs with chronic gastric, duodenal, and jejunal fistulas. Gastric emptying was measured by a dye-dilution technique. During the last hour of peptone stimulation, the intestine was perfused with either control solution or 20% lipid (Intralipid; Kabi Vitrum, Alamedo, CA) intraduodenally or intrajejunally. Compared with control perfusions, mean gastric acid outputs were decreased significantly after lipid perfusion of the duodenum (47% of control) and jejunum (24% of control). Similarly, mean gastric emptying rates were significantly less after lipid perfusion of the duodenum (56%) and jejunum (26%). Oral pretreatment with MK-329 (1 mg/kg) significantly reversed the inhibition of gastric acid output caused by lipid perfusion of the duodenum and jejunum, but fat-induced inhibition of gastric emptying was not significantly affected. These studies provide evidence for an important inhibitory role for CCK as an enterogastrone but do not implicate CCK as being important in fat-induced delayed gastric emptying of a liquid meal in dogs. PMID- 1727747 TI - Leukotriene D4 participates in colonic transit disturbances induced by intracolonic administration of trinitrobenzene sulfonic acid in rats. AB - The effects of colonic inflammation induced by trinitrobenzene sulfonic acid and influence of previous treatment with specific antagonists of inflammatory mediators (platelet-activating factor, leukotrienes, prostaglandins, and thromboxanes) on colonic transit were examined in conscious rats which were permanently fitted with an intracolonic catheter inserted into the proximal colon. Colonic inflammation was induced by intracolonic administration of trinitrobenzene acid (80 mg/kg) in 50% ethanol. Colonic transit time was evaluated by intracolonic administration of a radiolabeled marker [( 51Cr]sodium chromate) and collection of the feces per hour on a conveyor belt. Excretion of the marker was then plotted vs. time, permitting calculations of the times elapsed to recover 25%, 50%, and 75% of the marker injected (T25, T50, and T75, respectively). In control (saline) animals, excretion of the marker described a regular sigmoid curve with 50% of the marker recovered at 6.92 +/- 0.40 hours after intracolonic administration (T25 = 6.4 +/- 0.43 hours; T75 = 7.49 +/- 0.39 hours). Ethanol (vehicle), 50%, did not modify the profile of marker recovery. On the contrary, single intracolonic administration of trinitrobenzene sulfonic acid/ethanol induced a biphasic response consisting of an early pool of radiolabeled feces (T25 = 4.03 +/- 0.55 hours) with a delayed total one (T50 = 11.74 +/- 0.83 hours; T75 = 13.70 +/- 0.49 hours). Antagonists of the leukotriene pathway, i.e., MK = 886, a lipoxygenase inhibitor, and SKF 104,353 and SR 2640, two different leukotriene D4 receptor antagonists, blocked the effects of trinitrobenzene sulfonic acid on colonic transit time and restored a control profile of radiolabeled marker excretion. In contrast, indomethacin, a cyclooxygenase inhibitor, and SC 19220, a specific prostaglandin E2 receptor antagonist, were inefficient in blocking the effects of trinitrobenzene sulfonic acid on colonic transit time. Specific thromboxane A2 receptor antagonists, KT1 32 and GR 32191B, did not show any improvement in colonic transit after trinitrobenzene sulfonic acid administration. Previous injection of the specific platelet-activating factor receptor antagonists, BN 52021 or BN 50730, was also unable to restore a normal marker excretion profile after administration of trinitrobenzene sulfonic acid. It is concluded that the alterations of colonic transit immediately observed after intracolonic trinitrobenzene sulfonic acid administration are mediated through the release of leukotriene D4. In contrast, platelet-activating factor, prostaglandins, and thromboxanes are not involved in the mediation of these transit disturbances. PMID- 1727748 TI - Relationship between mast cell degranulation and jejunal myoelectric alterations in intestinal anaphylaxis in rats. AB - The effects of two degranulators of mast cells and intestinal anaphylaxis on jejunal myoelectric activity were compared in rats fasted for 15 hours. Attempts to antagonize the motility changes were performed using antagonists of histamine and serotonin and a cyclooxygenase and lipoxygenase inhibitor. Hooded Lister rats were chronically fitted with electrodes implanted in the jejunal wall. A group of rats was sensitized to egg albumin and challenged 14 days later by intraduodenal infusion of antigen. Sensitized animals had serum titers greater than or equal to 1:64. The other group was administered with mast cells degranulators. Both 48/80 (1 mg/kg), a degranulator of connective mast cells, and bromolasalocid (2 mg/kg), acting on connective and mucosal mast cells, induced a phase of total spiking inhibition followed by a progressive irregular spiking activity until the recovery of migrating myoelectric complex pattern (about 3 hours after injection). In contrast, antigen challenge disrupted the migrating myoelectric complex pattern, which was replaced by a peculiar pattern characterized by propagated spike burst, lasting 98 +/- 11.3 minutes. Chlorpheniramine (1 mg/kg) antagonized only the inhibitory phase induced by degranulators and was ineffective on the intestinal anaphylaxis-induced motor changes. Methysergide (1 mg/kg) and indomethacin (5 mg/kg) significantly reduced the degranulator effects as well as the anaphylaxis-induced alterations of intestinal motility. It is concluded that anaphylaxis-induced motor disturbances are relevant to mucosal mast cell degranulation involving 5-hydroxytryptamine and arachidonic acid derivative products, whereas histamine release appears to be a minor component. PMID- 1727749 TI - Transmucosal penetration of bismuth particles in the human stomach. AB - Electron microscopic examination of upper gastrointestinal biopsies with x-ray microanalysis was used to detect electron-dense particles of bismuth in the mucosa of the upper gastrointestinal tract, 30-60 minutes after oral dosing with either tripotassium dicitrato bismuthate [De-Noltab; Brocades (Great Britain) Ltd., Weybridge, UK; five patients] or bismuth salicylate (Pepto-Bismol; Richardson Vicks Ltd., Egham, UK; five patients), or without dosing (two patients). Transmucosal penetration of bismuth particles was observed in the gastric antral mucosa of all patients who had been dosed with tripotassium dicitrato bismuthate, but there was no penetration after oral dosing with bismuth salicylate. Persorption of bismuth particles through the gastric mucosa to the vascular endothelium provides an explanation for the rapid rise of plasma bismuth concentration observed only after oral dosing with tripotassium dicitrato bismuthate. PMID- 1727750 TI - Endogenous nitric oxide as a mediator of gastric mucosal vasodilatation during acid secretion. AB - The role of the endothelium-derived vasodilator, nitric oxide, as a mediator of the increase in gastric mucosal blood flow and as a modulator of the acid secretory response induced by pentagastrin was investigated in the anesthetised rat. Intravenous administration of the selective inhibitor of endogenous nitric oxide synthesis, NG-monomethyl-L-arginine (12.5 and 50 mg/kg), which dose dependently increased systemic arterial blood pressure, did not affect resting acid output. However, NG-monomethyl-L-arginine significantly reduced resting gastric mucosal blood flow at the higher dose, as determined by hydrogen gas clearance. Infusion of pentagastrin (80 micrograms kg-1.h-1) stimulated gastric acid secretion and elevated gastric mucosal blood flow. Pretreatment with NG monomethyl-L-arginine (12.5 mg/kg IV) did not affect this stimulation of acid output but substantially attenuated (by 65% +/- 10%; P less than 0.01) the associated increase in gastric mucosal blood flow. Pretreatment with NG monomethyl-L-arginine (50 mg/kg IV) induced a minor inhibition of pentagastrin stimulated acid secretion but abolished the increase in gastric mucosal blood flow. When administered during pentagastrin infusion, NG-monomethyl-L-arginine (50 mg/kg IV) did not affect the acid secretory response but induced a 76% +/- 8% inhibition (P less than 0.05) of the elevated gastric mucosal blood flow. The effects of NG-monomethyl-L-arginine on blood pressure, acid secretion, and gastric mucosal blood flow were abolished by pretreatment with the precursor for nitric oxide synthesis, L-arginine (300 mg/kg IV). These findings in the rat suggest that endogenous nitric oxide, synthesized from L-arginine, does not directly modulate the acid secretory response induced by pentagastrin but makes a substantial contribution to the mucosal vasodilatation associated with the stimulation of gastric acid secretion. PMID- 1727751 TI - Role of affect and personality in gastric acid secretion and serum gastrin concentration. Comparative studies in normal men and in male duodenal ulcer patients. AB - The role of mood state (affect) and personality on basal acid secretion and basal serum gastrin concentrations were examined in seven healthy men and eight patients with duodenal ulcer. In each subject, gastric secretion and affect were assessed simultaneously on 5 separate days. None of 10 self-reported affect variables correlated with daily fluctuations in basal acid secretion in either group. Three variables (tension, conflict, and anxiety) correlated significantly with serum gastrin fluctuations in normal subjects, but these relationships were not present in patients with ulcer, who were hypergastrinemic regardless of their affective state. The degree to which serum gastrin fluctuated was unrelated to personality, as assessed by Minnesota Multiphasic Personality Inventory. On the other hand, several Minnesota Multiphasic Personality Inventory scales correlated with the degree of variability in basal acid secretion, including scales that measured impulsivity and social isolation/alienation. These studies indicate that serum gastrin concentrations are related to affective state in normal men, that this relationship is altered in men with duodenal ulcer, and that certain personality traits, such as impulsivity and social isolation, are associated with more labile basal acid secretion rates. PMID- 1727752 TI - Role of platelet-activating factor in hemodynamic derangements in an acute rodent pancreatic model. AB - Systemic hemodynamics were assessed in a model of experimental pancreatitis induced in rats by the retrograde injection of sodium deoxycholate, 40%, 1 mL/kg, in the pancreatic duct, using the radioactive microsphere technique before and 25 minutes after pancreatitis induction while blood pressure was stable (n = 10). A 55% decrease in cardiac out-put, a 14% decrease in heart rate, and a 3.3-fold increase in total peripheral resistances, without significant changes in blood pressure, were observed. Renal blood flow decreased by 68%. When rats were given BN-52021, a blocker of platelet-activating factor receptors (5 mg/h, IV; n = 13) coinciding with pancreatitis induction, no significant hemodynamic changes were observed. Animals treated with BN-52021 survived 89 +/- 10 minutes, whereas death occurred 67 +/- 5 minutes after pancreatitis induction in untreated rats (P less than 0.001). A different group of rats with pancreatitis showed higher blood levels of platelet-activating factor (0.28 +/- 0.06 ng/mL; n = 11) than control rats (0.16 +/- 0.03; n = 15; P less than 0.05). Very high levels of platelet activating factor were found in peritoneal exudate from rats with pancreatitis. These data show an effective protective effect of BN-52021 on the hemodynamic impairment that follows pancreatitis induction, as well as a role of platelet activating factor in these alterations. PMID- 1727753 TI - Staging of pancreatic and ampullary carcinoma by endoscopic ultrasonography. Comparison with conventional sonography, computed tomography, and angiography. AB - In a prospective study, endoscopic ultrasonography was compared with transabdominal ultrasonography, computed tomography, and angiography in 60 consecutive patients with pancreatic (n = 46) and ampullary (n = 14) cancer considered to be candidates for surgery. The diagnostic value of these imaging procedures in determining local resectability was assessed. The diagnosis of ampullopancreatic malignancy was made by operation (n = 40) or puncture/biopsy (n = 20). In the 40 patients who underwent surgery, endoscopic ultrasonography was significantly superior to abdominal ultrasonography and computed tomography in determining tumor size and extent and lymph node metastases of pancreatic and ampullary cancer. Furthermore, involvement of the portal venous system as judged by histopathology or surgical exploration was correctly assessed by endoscopic ultrasonography in 95%, whereas angiography (85%), computed tomography (75%) and abdominal ultrasonography (55%) were less sensitive. Of 11 cases of portal venous infiltration found at surgery, endoscopic ultrasonography correctly predicted 10, abdominal ultrasonography only 1, computed tomography 4, and angiography 5 (P less than 0.05 for all three comparisons). Twenty patients did not undergo surgery for different reasons: of those, 9 patients were excluded from operation because of portal venous involvement as shown by angiography. Endoscopic ultrasonography detected portal venous invasion in all these cases. In contrast to the venous system, arterial encasement was less reliably detected by endoscopic ultrasonography. In conclusion, endoscopic ultrasonography is the most effective single imaging procedure for local tumor staging in pancreatic and ampullary cancer. Thus, endoscopic ultrasonography will improve the assessment of tumor resectability and further decrease the need for explorative laparotomy. PMID- 1727754 TI - Accelerated improvement of alcoholic liver disease with enteral nutrition. AB - This prospective study compared the effects of tube-fed nutrition with those of a regular diet in alcoholic liver disease. The high prevalence of malnutrition in patients with alcoholic liver disease requires clarification of the benefits of aggressive nutritional support. Patients were randomly assigned a regular diet without or with tube-fed supplementation, delivering 1.5 g/kg protein and 167 kJ/kg daily. Comparisons of encephalopathy, antipyrine clearance, metabolic rate, and biochemical parameters were performed weekly for 4 weeks. Sixteen patients receiving enteral supplementation had antipyrine half-life (50% vs. 3% reduction), serum bilirubin (25% vs. 0% reduction), and median encephalopathy scores that improved more rapidly than those of controls. Initially, 15 controls did not consume adequate calories to meet measured resting energy expenditure. Aggressive nutritional intervention accelerated improvement in alcoholic liver disease. Adverse effects did not offset the demonstrated benefits of a 2-cal/mL, casein-based tube-fed supplement. These findings support the use of standard, casein-based solutions in the treatment of alcoholic liver disease and as the control condition for future studies. PMID- 1727755 TI - Endoscopic variceal sclerosis does not increase the risk of portal venous thrombosis. AB - In this study the risk of thrombosis in the portal venous system was assessed in patients with chronic variceal bleeding undergoing sclerotherapy. Twenty-two patients with cirrhosis were prospectively studied with angiography before initiation of sclerotherapy and at mean (+/- SD) 26 +/- 17-month (range, 8-63 months) follow-up. Sclerotherapy consisted of flexible endoscopy, intravariceal and paravariceal, using sodium morrhuate (1.5%-2%) and sodium tetradecyl sulfate (0.5%-1.5%), to obliteration. The mean number of sessions was 6.5 +/- 2.2 (range, 3-11), with a mean total amount of sclerosant of 62 +/- 25 mL (range, 25-112 mL). No patient developed splenic or portal vein thrombosis as shown by arteriography. The flow patterns of portal perfusion, vessel size, and coronary vein visualization showed no significant change. Only one patient had spontaneous reversal of portal flow. Splenic vein histology, examined in five patients in whom sclerotherapy failed and who required shunt surgery, was not significantly different from that in eight patients who had no prior sclerotherapy. It is concluded that under the conditions of the current study, chronic sclerotherapy did not increase the risk of thrombosis in the portal venous system and did not significantly alter the histology of the portal hypertensive splenic vein. PMID- 1727756 TI - Pancreatic proteases and intestinal mucosal injury after ischemia and reperfusion in the pig. AB - Intraluminal pancreatic proteases have been proposed to play a pathogenic role in the injury seen after ischemia and reperfusion of the small intestinal mucosa. Intestinal ischemia can be detected by indirect intramucosal pH measurements using tonometry. In this study, pigs were subjected to laparotomy and ligation of the pancreatic duct (n = 10) or a sham procedure (n = 10). Three weeks later, a standardized hemorrhagic shock was induced followed by retransfusion. Central hemodynamics, portal venous flow, and duodenal and small intestinal mucosal intramucosal pH were monitored. Samples were obtained from the small intestine for microscopic examination. A typical superficial mucosal injury developed in both groups of animals after reperfusion. However, the injury developed significantly later in the duct-ligated animals. No major differences in survival, splanchnic hemodynamics, or intramucosal pH between the groups were seen during hemorrhagic hypotension or after reperfusion. These data favor the concept that intraluminal pancreatic proteases are important for the rapid development of the mucosal reperfusion injury. PMID- 1727757 TI - Mechanism of insulin resistance in CCl4-induced cirrhosis of rats. AB - Insulin action was studied in rats with CCl4/phenobarbital-induced cirrhosis of the liver using the euglycemic hyperinsulinemic clamp technique coupled with isotopic measurement of individual tissue glucose uptake, glycogen formation, and lipogenesis. In cirrhotic rats, dose response curves showed a reduction of insulin-stimulated total body glucose disposal of about 30%. Insulin action on tissue glucose uptake and initial phosphorylation (assessed with [3H]2 deoxyglucose) were unchanged; however, incorporation of [14C]glucose into lipids and particularly into glycogen was reduced substantially (being most pronounced in skeletal muscle and diaphragm) at maximally as well as half-maximally effective serum insulin concentrations during euglycemic clamping. At identical IV insulin infusion rates, steady-state serum insulin concentrations were elevated up to fourfold in cirrhotic animals. Antilipolytic action of insulin was unaltered. These data suggest that the principal metabolic pathway affected in insulin resistance of rats with experimental cirrhosis appeared to be insulin stimulated glycogen formation in muscle tissues. PMID- 1727758 TI - Oral administration of clonidine in patients with alcoholic cirrhosis. Hemodynamic and liver function effects. AB - The effects of long-term oral clonidine treatment on hepatic and systemic hemodynamics and on quantitative liver function tests were investigated in 15 patients with alcoholic cirrhosis. Clonidine was administered at a mean dose of 0.33 +/- 0.1 mg/day (mean +/- SD) for a mean period of 64 +/- 10 days. Oral clonidine induced a significant reduction in the hepatic venous pressure gradient from 18.8 +/- 3.0 mm Hg to 15.9 +/- 3.4 mm Hg (P less than 0.001), which was the result of an increase in the free hepatic venous pressure from 5.1 +/- 4.2 mm Hg to 8.7 +/- 3.8 mm Hg (P less than 0.05). In 10 of the 15 patients (67%), the reduction in the hepatic venous pressure gradient was greater than 10% of baseline values. Hepatic blood flow did not change significantly after clonidine treatment. Additionally, treatment with clonidine decreased mean arterial pressure by 15.5% +/- 6% (P less than 0.001), heart rate by 17.7% +/- 7% (P less than 0.001), and cardiac output by 14.6% +/- 7% (P less than 0.001). However, systemic vascular resistance did not change significantly. There were no adverse effects on liver function, as shown by the nonsignificant changes in galactose elimination capacity (149 +/- 59 vs. 170 +/- 58 mg/min), hepatic clearance of indocyanine green (0.19 +/- 0.10 vs. 0.17 +/- 0.07 L/min), and hepatic intrinsic clearance of indocyanine green (0.23 +/- 0.14 vs. 0.21 +/- 0.1 L/min) before and after clonidine treatment, respectively. In none of the patients was the drug withdrawn because of side effects, although 12 subjects complained of dry mouths. This study suggests that in patients with alcoholic cirrhosis, long-term oral clonidine administration achieves a reduction in the hepatic venous pressure gradient without adverse effects on hepatic blood flow and liver function. PMID- 1727759 TI - The role of endogenous prostaglandins in hormone-stimulated pancreatic exocrine secretion. AB - The authors have previously shown that neurotensin and secretin inhibit gastric acid secretion in the dog and that these actions are inhibited by the prostaglandin synthesis inhibitor indomethacin. Conversely, neurotensin and secretin share similar stimulatory effects on pancreatic exocrine secretion. In the present study, the effects of blockade of prostaglandin synthesis by indomethacin on neurotensin-, cholecystokinin-, and secretin-stimulated exocrine secretion are examined along with the effects of these same agents on the release of pancreatic polypeptide. The studies were performed on conscious dogs with chronic gastric and pancreatic cannulas. Dose-dependent increases in pancreatic exocrine secretion of water and bicarbonate were observed with IV infusion of neurotensin or secretin; however, inhibition of prostaglandin synthesis by indomethacin abolished this response. Protein secretion stimulated by either neurotensin or cholecystokinin was not affected by prostaglandin inhibition. Cholecystokinin and neurotensin infusion stimulated release of pancreatic polypeptide; only neurotensin-stimulated release of pancreatic polypeptide was inhibited by indomethacin treatment. It is concluded that intact prostaglandin synthesis is necessary for the actions of neurotensin and secretin (but not that of cholecystokinin) on pancreatic exocrine secretion of water and bicarbonate and for neurotensin- (but not cholecystokinin-) stimulated release of pancreatic polypeptide. PMID- 1727760 TI - Cholecystokinin release from isolated canine epithelial cells in short-term culture. AB - Canine jejunal epithelial cells were isolated and maintained in short-term culture to study cholecystokinin (CCK) release. Sequential digestion of jejunal mucosa with collagenase and ethylenediaminetetraacetic acid was followed by counterflow elutriation to enrich CCK-containing cells. After 40 hours in culture on collagen-coated plates, 8.4% of the initially seeded cells were attached; 8.7% of them stained positive with a C-terminal CCK/gastrin antibody and 2.5% stained positive with a gastrin-specific antibody. Basal release of CCK into the culture medium amounted to 1.3% of total cell content over 105 minutes. Receptor independent stimulation of protein kinase C by the phorbol ester beta-phorbol-12 myristate-13-acetate caused significant CCK release. The inactive form, 4 alpha phorbol-12-myristate-13-acetate, had no effect. Activation of adenylate cyclase by 10(-5) mol/L forskolin evoked a 2.5-fold increase in CCK concentrations, which was completely abolished by 10(-8) mol/L somatostatin. L-phenylalanine stimulated CCK release at 20 and 50 mmol/L, whereas D-phenylalanine caused significant hormone output only at 50 mmol/L. L-tryptophan had no effect. Cholecystokinin release stimulated by L-phenylalanine was not influenced by the addition of either somatostatin or somatostatin antibody. In conclusion, a system of isolated canine jejunal epithelial cells was developed in short-term culture. This preparation proved suitable for the study of CCK release on a cellular basis. PMID- 1727761 TI - Antral control of gallbladder cyclic motor activity in the fasting state. AB - The hypothesis that gastric antrum controls the phasic contractions of gallbladder cyclic motor activity in the fasting state was tested. Gallbladder, gastric, and small bowel motor and myoelectric activity was recorded by strain gauge transducers and bipolar electrodes. Gallbladder pressure was measured manometrically by a surgically implanted intraluminal catheter. After control recordings for 4 to 6 weeks, antrectomy and gastroduodenostomy were performed. Six weeks later, bilateral truncal vagotomy was performed in each dog. Recordings were made after each surgical procedure. In the control state, the gallbladder exhibited cyclic motor activity consisting of phasic contractions at a frequency of 0.75 +/- 0.02/min superimposed on an increase in baseline pressure. Antrectomy and gastroduodenostomy completely abolished the phasic contractions of gallbladder cyclic motor activity and significantly decreased the incidence of the cyclic increase in baseline pressure. Subsequent vagotomy had no additional effect on gallbladder cyclic motor activity. In intact dogs, the gallbladder filled from 0% to 80% and emptied from 80% to 100% of the duodenal migrating motor complex cycle, which was considered to begin at the start of phase I activity. Antrectomy significantly altered this pattern; after antrectomy, the gallbladder filled from 0% to 10% and from 90% to 100% and emptied during the remainder of the duodenal migrating motor complex cycle. Subsequent vagotomy had no additional effect on periodic gallbladder filling and emptying. It is concluded that major changes occur in gallbladder cyclic motor activity and its periodic filling and emptying pattern in the fasting state after antrectomy and vagotomy. It is hypothesized that in the absence of cyclic phasic contractions after antrectomy, periodic stirring and agitation of gallbladder bile and its mixing with fresh hepatic bile may not occur in the fasting state. The absence of this phenomenon may lead to supersaturation of bile near the mucosal surface and increase the propensity for precipitation of salts and formation of gallstones. PMID- 1727762 TI - The public policy plan of the American Gastroenterological Association. PMID- 1727764 TI - Prediction of survival of patients with primary biliary cirrhosis. Examination of the Mayo Clinic model on a group of patients with known endpoint. AB - Increasing use of liver transplantation and new treatment regimens necessitate an accurate estimate of prognosis in primary biliary cirrhosis. To test the usefulness of the Mayo model for this purpose, the R value of the model was calculated for a group of 28 patients after each patient encounter and plotted against time. The data were best described by two linear regressions. For the period 10-2 years before death, the average increase in R value was 0.23 annually [R = 7.1-0.23 x time (in years)]. In the last 2 years of life, the average increase in R value was 1.4. This period could be fit by the expression R = 8.2 1.4 x time (in years). The increase in R value represents the natural progression of primary biliary cirrhosis and can be used for evaluating treatment of patients. PMID- 1727763 TI - Systemic prostacyclin in cirrhotic patients. Relationship with portal hypertension and changes after intestinal decontamination. AB - The total body production of prostacyclin was shown to be increased in cirrhotic patients, suggesting that its synthesis by blood vessels of the systemic circulation is enhanced. However, the mechanism by which the synthesis of systemic prostacyclin is stimulated is not known. The present study investigated the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha, an index of total body prostacyclin synthesis, first, in cirrhotics with portal hypertension (n = 19) as compared with cirrhotics with reduced portal pressure after portacaval shunt surgery (n = 18) and with control noncirrhotic subjects (n = 11), and; second, in cirrhotics before and after intestinal decontamination by oral nonabsorbable antibiotics (n = 9 antibiotic treated patients, n = 10 control nontreated cirrhotics). Control noncirrhotic subjects showed lower urinary excretion of 2,3 dinor-6-keto-PGF1 alpha than both groups of cirrhotics (P less than 0.001). Interestingly, urinary excretion of 2,3-dinor-6-keto-PGF1 alpha was significantly higher in cirrhotics with portacaval shunt than in those with portal hypertension (P less than 0.01). The urinary excretion of 2,3-dinor-6-keto-PGF1 alpha decreased significantly after intestinal decontamination in the antibiotic treated group (580.1 +/- 232.4 vs. 431.2 +/- 219.2 pg/mg creatinine; P less than 0.05) but not in nontreated patients (543.9 +/- 214.4 vs. 581.2 +/- 281.4 pg/mg creatinine; P = NS). These data suggest that the increased urinary excretion of 2,3-dinor-6-keto-PGF1 alpha observed in cirrhotics is not directly related to portal hypertension itself but to portal blood factors that bypass the liver. Some such factors may be of intestinal bacterial origin. PMID- 1727765 TI - Prospective evaluation of immediate versus delayed refeeding and prognostic value of endoscopy in patients with upper gastrointestinal hemorrhage. AB - The effects of immediate vs. delayed refeeding and the prognostic value of endoscopic findings in patients with major upper gastrointestinal hemorrhage were assessed in a prospective randomized study. Entry criteria were clinical evidence of major hemorrhage and endoscopic evidence of a Mallory-Weiss tear or an ulcer with a clean base, flat spot, or clot. Two hundred fifty-eight patients were randomly assigned to groups receiving a regular diet immediately or nothing by mouth for 36 hours, then clear liquids for 12 hours, and a regular diet thereafter. Outcomes in the immediate and delayed refeeding groups were comparable: rebleeding occurred in 4% vs. 5%; urgent intervention, 2% vs. 2%; and deaths, 1% vs. 1%, respectively. Rebleeding occurred in 2 (2%) of 96 patients with cleanbased ulcers, 5 (8%) of 65 with ulcers with spots, 3 (14%) of 21 with ulcers with clots (P = 0.05, 3 x 2 chi2 test), and 1 (2%) of 66 with Mallory Weiss tears. It is concluded that the time of refeeding does not influence the hospital course of patients with a low risk of recurrent bleeding. Patients with clean-based ulcers or nonbleeding Mallory-Weiss tears may be refed and discharged home immediately after stabilization. PMID- 1727766 TI - Distal colonic hyperplastic polyps do not predict proximal adenomas in asymptomatic average-risk subjects. AB - The significance of distal colonic hyperplastic polyps was investigated in 482 asymptomatic average-risk subjects, aged 50-75 years, in whom fecal occult blood test results were negative and who underwent screening colonoscopy. The incidence of adenomas in the colon proximal to the sigmoid-descending colon junction in subjects with hyperplastic polyps distal to that point was 18% and was similar to the incidence of proximal colonic adenomas in subjects with no distal colonic polyps (15%). The incidence of proximal colonic adenomas in subjects with no distal colonic adenomas was 38% and was significantly greater than the incidence found in individuals with no distal colonic polyps or only hyperplastic polyps. Our data do not support distal colonic hyperplastic polyps as markers for proximal colonic adenomas in asymptomatic average-risk subjects. PMID- 1727767 TI - Endoscopy-negative upper gastrointestinal bleeding in a patient with chronic pancreatitis. PMID- 1727768 TI - Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity ('celiac sprue'). AB - This article examines associations between gluten, polymorphisms of the major histocompatibility complex, and mucosal pathology representative of the spectrum of gluten sensitivity. Sequences of wheat, rye, and barley prolamins contain recurring tetrapeptide motifs that are predicted to have beta-reverse-turn secondary structure and that, with in vitro assays, appear active. Structural polymorphisms of major histocompatibility complex subloci identify codon switches within the second exon that control the third hypervariable region in the outer domain of the beta chain. Observations of the intestinal response to gluten reveal five interrelated lesions (preinfiltrative, infiltrative, hyperplastic, destructive, and hypoplastic) that are interpretable as cell-mediated immunologic responses. These responses originate in the lamina propria, where a series of antigen-specific inflammatory processes has now been identified. There is no evidence that celiac sprue is a disease of jejunal enterocytes. Furthermore, the role of intraepithelial space lymphocytes in pathogenesis, if relevant, needs further experimental dissection. Also awaiting further definition are polymorphisms of the celiac lymphocyte antigen receptor and their relationship to gliadin oligopeptide(s) and predisposing genes. The nature and basis of nonresponsive celiac sprue require more thoughtful initiatives to elucidate the immunologic mechanism(s) of unresponsiveness and evaluate possible means of reversal. Finally, a more sensible definition of gluten sensitivity (unhampered by qualitative morphological imagery) is ultimately called for in order to accommodate the biomolecular advances addressed in this review. PMID- 1727769 TI - Grading and classification of chronic gastritis: one American response to the Sydney system. PMID- 1727770 TI - Histological classification of chronic gastritis: an iconoclastic view. PMID- 1727771 TI - Short-chain fatty acids and the colon. PMID- 1727772 TI - Prophylactic sclerotherapy for esophageal varices. PMID- 1727773 TI - Fasting and postprandial residual gallbladder volumes. PMID- 1727774 TI - Momentum omentum! PMID- 1727775 TI - Mucosal sensitivity to acid in nonulcer dyspepsia. PMID- 1727776 TI - MHC class II antigens on the epithelial cells of the human gastrointestinal tract. PMID- 1727777 TI - Careful observations and flawed conclusions. PMID- 1727778 TI - Alcohol and renal phosphate excretion. PMID- 1727779 TI - Symptoms and risk factors of Helicobacter pylori infection in a cohort of epidemiologists. AB - To identify symptoms and risk factors associated with Helicobacter pylori infection, a cohort of 341 epidemiologists was studied. All subjects had one banked serum (collected between 1969 and 1987) and one recent serum sample (collected in 1988) evaluated for H. pylori immunoglobulin G by enzyme-linked immunosorbent assay; subjects provided information on gastrointestinal symptoms and risk factors for gastritis and peptic ulcer disease. Prevalence of infection decreased from the early 1970s to the present. Eleven subjects (3% of the total cohort) seroconverted during the interval between serum samples, giving a crude conversion rate of 0.49% per person-year (95% confidence interval, 0.3-0.9). Nonreactors on the 1988 serum sample described similar symptoms to reactors. However, subjects who seroconverted in the interval between the two serum samples were more likely than either persistent nonreactors [relative risk (RR), 4.1] or persistent reactors (RR, 3.7) to have experienced upper gastrointestinal symptoms in the interval years. Consumption of caffeinated beverages (RR, 4.6) and residence in the northeastern United States (RR, 5.3) seemed to increase risk for infection. Because pain was similarly common in H. pylori-positive and -negative patients, H. pylori cannot be summarily accepted as the cause of dyspeptic symptoms even when infection is confirmed. PMID- 1727780 TI - Ultrastructure of interstitial cells of Cajal associated with deep muscular plexus of human small intestine. AB - Evidence showing that interstitial cells of Cajal have important regulatory functions in the gut musculature is accumulating. In the current study, the ultrastructure of the deep muscular plexus and associated interstial cells of Cajal in human small intestine were studied to provide a reference for identification and further physiological or pathological studies. The deep muscular plexus was sandwiched between a thin inner layer of smooth muscle (one to five cells thick) and the bulk of the circular muscle. Interstitial cells of Cajal in this region very much resembled smooth muscle cells (with a continuous basal lamina, caveolae, intermediate filaments, dense bodies, dense bands, and a well-developed subsurface smooth endoplasmic reticulum), but the arrangement of organelles was clearly different, and cisternae of granular endoplasmic reticulum were abundant. Interstitial cells of Cajal were distinguished from fibroblasts or macrophages in the region. They ramified in the inner zone of the outer division of circular muscle, penetrated the inner-most circular layer, and were also found at the submucosal border. They were in close, synapselike contact with nerve terminals of the deep muscular plexus, and only few gap junctions with other interstitial cells of Cajal or with the musculature were observed. Compared with interstitial cells of Cajal from other mammals, those associated with the deep muscular plexus in the human small intestine more closely resemble smooth muscle cells, and their organization appears more diffuse; however, the ultrastructure and organization of interstitial cells of Cajal is compatible with modulatory actions on the circular muscle also in humans. PMID- 1727781 TI - Spontaneous cytotoxicity of human intraepithelial lymphocytes against epithelial cell tumors. AB - Human intraepithelial lymphocytes are T cells primarily of the CD8+ phenotype located between intestinal epithelial cells. The cytotoxic and suppressor activities of these lymphocytes are largely unexplored. The spontaneous cytotoxic activity of these cells is evaluated in this study. Jejunal intraepithelial lymphocytes spontaneously lysed a variety of epithelial cell tumor lines (colonic and pancreatic adenocarcinomas and bladder epidermoid carcinoma) but not the highly natural killer-sensitive K-562 cells. Cold target inhibition studies showed that these lymphocytes preferentially bind the DLD-1 colonic adenocarcinoma cells rather than the K-562 cells. Pretreatment of the effector cells with interferon-gamma did not change their cytotoxic activity. The cytotoxic cells are T lymphocytes (expressing CD2, CD3, and CD8). In contrast, the spontaneous cytotoxic activity of peripheral blood lymphocytes is directed against both epithelial cell targets and K-562 cells, is enhanced by interferon gamma, and is effected by natural killer cells (expressing CD2, CD16, and NKH1). Thus, the spontaneous cytotoxicity of intraepithelial lymphocytes differs from that of peripheral blood lymphocytes in their target cell restriction, lack of response to interferon gamma, and effector cell phenotype. Lymphocytes in the intestinal epithelium may have a novel and important role in recognizing and destroying transformed epithelial cells and colon cancers. PMID- 1727782 TI - The evolution of public policy in the American Gastroenterological Association. PMID- 1727783 TI - Effect of CO2 on rat colonic Na absorption: studies with nystatin. AB - An increase in ambient CO2 tension from 3% to 11% augments colonic Na absorption in the rat. The membrane site of action of CO2 was examined by measuring colonic Na absorption in the Ussing chamber when nystatin was used to permeabilize the luminal (apical) membrane. The equal rates of ouabain-sensitive Na absorption at 3% and 11% CO2 in the presence of nystatin and at 11% CO2 in its absence suggested that CO2 acted at the luminal membrane. This finding was also observed at a submaximal rate of Na absorption (produced by lowering bathing solution Na from 140 to 27 mmol/L) and in a Cl-free solution (to prevent cell swelling). The basolateral membrane was indeed rate limiting for Na absorption in the presence of nystatin, because methylprednisolone (3 mg/kg SC for 3 days to increase sodium potassium--stimulated adenosine triphosphatase activity) increased Na absorption measured in the presence of nystatin and because CO2 increased absorption in steroid-treated rats in the absence of nystatin. These results validate the protocol and confirm the luminal site of action of CO2 and nystatin on colonic Na absorption. PMID- 1727784 TI - Effect of motilin on gastric emptying in patients with diabetic gastroparesis. AB - Erythromycin markedly accelerates gastric emptying, possibly because it acts as a motilin agonist. In the present study, the effect of an equipotent dose of motilin was tested. In six patients with severe diabetic gastroparesis, gastric emptying of liquids and solids was examined scintigraphically after motilin or placebo in a double-blind crossover study. Motilin (10 pmol.kg-1.min-1) or saline was infused over a 90-minute period starting 5 minutes before breakfast. Motilin markedly accelerated emptying. For liquids, the half-emptying time was reduced from 51 +/- 6 to 22 +/- 11 minutes (P less than 0.01) and for solids from 111 +/- 4 to 51 +/- 12 minutes (P less than 0.01). The mean increase in plasma motilin levels was 1315 +/- 342 pg/mL, corresponding to an effective infusion rate of about 4 pmol.kg-1.min-1. In the control experiments, basal motilin levels (173 +/ 17 pg/mL) were within the normal range but increased steadily postprandially, reaching 321 +/- 25 pg/mL at the end of the study period, probably reflecting gastric distension. The postprandial increase in pancreatic polypeptide level was blunted compared with accepted normal values but was more pronounced during motilin infusion, i.e., 650 +/- 217 vs. 279 +/- 66 pg/mL (P less than 0.01), probably because of the improved emptying. Our data show that motilin accelerates gastric emptying in diabetic gastroparesis and support the hypothesis that erythromycin's effect is mediated through motilin receptors. PMID- 1727785 TI - Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver. AB - The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 mumol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca(2+)-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25 mumol/L) on cytosolic Ca2+ in isolated hepatocytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single-pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca(2+) sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% +/- 10% (mean +/- S.D.) decrease in bile flow and a sustained 271% +/- 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 mumol/L) and the protein kinase C inhibitor H-7 (50 mumol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 mumol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% +/- 4% decrease in bile flow and a sustained 7% +/- 4% increase in perfusion pressure. Pretreatment with H-7 alone corrected the vasopressin-induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two-thirds without affecting the increase in perfusion pressure, 2,5'-di(tert-butyl)-1,4-benzohydroquinone produced a transient 65% +/- 20% decrease in bile flow and a transient 56% +/- 15% increase in perfusion pressure. In isolated hepatocytes, bromo-A23187, the nonfluorescent form of the ionophore, produced a sustained 56% +/- 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% +/- 75% increase and 2,5-di(tert-butyl)-1,4-benzohydroquinone resulted in a sustained 149% +/- 66% increase. The ionophore-induced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin-induced increase was reduced by 38%.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727786 TI - Circulatory changes induced by portal venous diversion and mesenteric hypertension in rats. AB - We studied the hemodynamics in four groups of rats with combinations of mesenteric hypertension and portal diversion. Operations created three groups with mesenteric hypertension and different degrees of portal venous diversion: mesenteric vein stenosis, portal vein stenosis and end-to-side portacaval anastomosis with mesenteric vein stenosis, the fourth group had only portacaval anastomosis. A control group had sham operations. Cardiac output, splanchnic blood flows and portosystemic shunt indices were measured with radioactive microspheres. Mesenteric venous pressures in the mesenteric-stenosed, portal stenosed, portacaval-shunted and end-to-side portacaval anastomosis with mesenteric vein stenosis rats were, respectively, 13.5 +/- 0.6, 15.3 +/- 0.7, 4.3 +/- 0.5 and 13.0 +/- 0.9 mm Hg, which were all significantly different from controls: 8.3 +/- 0.3 mm Hg. Portosystemic shunt indices were also significantly different from each other: controls, 0.4% +/- 0.02%; mesenteric-stenosed, 5.9% +/ 2.3%; and portal-stenosed, 52.1% +/- 4.9%. Cardiac output and splanchnic visceral blood flows were significantly increased in the portal-stenosed rats and the two groups with portacaval anastomoses, with the latter two groups having the highest values. The addition of mesenteric stenosis did not change the blood flows because mesenteric-stenosed rats did not differ from controls and end-to side portacaval anastomosis with mesenteric vein stenosis rats did not differ from rats with portacaval anastomosis alone. These results suggest that mesenteric venous hypertension per se does not affect hemodynamics but that diversion of portal venous blood from the liver is a critical factor in the development of hyperkinetic circulation in portal hypertension. PMID- 1727787 TI - Intrasplenic hepatocellular transplantation corrects hepatic encephalopathy in portacaval-shunted rats. AB - The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery. Spontaneous activity and nose-poke exploration by individual rats were studied in automated open field boxes equipped with infrared cells. Each cell beam interruption was automatically recorded on a microcomputer and transformed into a score index (counts/hour). Plasma levels of amino acids, ammonia and total biliary acids were measured. Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. Intrasplenic hepatocellular transplantation significantly increased spontaneous activity after 2 mo and improved nose-poke exploration after 3 wk. At 3 mo, spontaneous activity and nose poke exploration in portacaval shunt/intrasplenic hepatocellular transplantation rats were not significantly different from those of sham rats. Increases in plasma ammonia levels after portacaval shunt were not corrected. Amino acid imbalance and bile acid concentration in plasma were partially corrected by intrasplenic hepatocellular transplantation. These data show that intrasplenic hepatocellular transplantation can correct the neurological symptoms of hepatic encephalopathy in an experimental model of chronic liver failure and suggest that intrasplenic hepatocellular transplantation might be of therapeutic interest in chronic liver failure. PMID- 1727788 TI - The acute-phase response protects mice from D-galactosamine sensitization to endotoxin and tumor necrosis factor-alpha. AB - D-Galactosamine is an hepatocyte-specific inhibitor of RNA synthesis. It has been used to sensitize animals both to the lethal effects of bacterial endotoxin (lipopolysaccharide) and to a principal lipopolysaccharide-induced mediator of shock, tumor necrosis factor-alpha. The mechanism by which this sensitization occurs is unknown. Because lipopolysaccharide, acting through a network of cytokines, provokes the transcription of a number of hepatic acute-phase proteins, we postulated that the lipopolysaccharide-sensitizing effect of D galactosamine could be caused by its inhibition of acute-phase product transcription. We confirmed that the acute-phase response to lipopolysaccharide was attenuated by simultaneous administration of D-galactosamine. However, when the acute-phase response was induced by subcutaneous turpentine 24 hr before D galactosamine administration, the effect of D-galactosamine on circulating acute phase reactants was negligible. Furthermore, induction of an a priori acute-phase response protected mice from both D-galactosamine/lipopolysaccharide and D galactosamine/tumor necrosis factor-alpha-induced death. The turpentine-induced acute-phase response did not decrease endogenous tumor necrosis factor-alpha production after lipopolysaccharide, nor did it affect the clearance of larger doses of injected tumor necrosis factor-alpha. Thus we suggest that the acute phase response protects against death in D-galactosamine-sensitized mice through an interaction with mediators of shock subsequent to tumor necrosis factor-alpha release. PMID- 1727789 TI - The effect of liver denervation on hepatic hemodynamics during hypovolemic shock in swine. AB - This study tested the hypothesis that the denervated liver is more susceptible to hypovolemic shock than the normal liver. Fourteen swine, seven nondenervated and seven after liver denervation, were studied during hypovolemic shock to 50% of baseline blood pressure. Hepatic artery and portal vein flows were measured using transonic flow probes, and cardiac output and central venous pressure were measured using Swan-Ganz catheters. Hepatic artery flow fell equivalently in the two groups, from 132 +/- 71 to 94 +/- 17 ml/min in the nondenervated group compared with 149 +/- 56 to 91 +/- 55 ml/min in the denervated group. In contrast, portal flow in the denervated group (276 +/- 71 to 119 +/- 53 ml/min) fell significantly (p less than 0.001) more than in the nondenervated group (289 +/- 135 to 194 +/- 70 ml/min). The 58% reduction from baseline in portal flow in the denervated group compared with the 30% reduction in the nondenervated group suggests that the normal compensatory mechanism to maintain portal flow during hypovolemic shock is neurally mediated. It can be hypothesized that sensory afferent fibers might initiate a feedback to splanchnic vasodilatation in response to reduced portal flow. This study supports the hypothesis that the denervated liver is more susceptible to hypovolemic shock. PMID- 1727790 TI - The clinical significance of molecular variation within the hepatitis B virus genome. PMID- 1727791 TI - Hepatocyte transplantation: back to the future. PMID- 1727792 TI - Further insights into sinusoidal organic anion uptake. PMID- 1727793 TI - Yet another role for the "good" matrix protein: laminin in regenerating liver. PMID- 1727794 TI - Does the precore mutant of HBV cause fulminant hepatitis? PMID- 1727795 TI - Anti-pre-S responses and viral clearance in chronic hepatitis B virus infection. AB - Serial sera were collected prospectively during the clinical course of 13 HBsAg carriers with chronic liver disease and analyzed for ALT levels, pre-S1 and pre S2 antigens and corresponding antibodies and other serological hepatitis B virus markers. In five patients, anti-pre-S1 and anti-pre-S2 antibodies became detectable in multiple serum samples, whereas in eight patients anti-pre-S was never detected or only appeared transiently during the follow-up. The first pattern was associated with normalization of ALT levels and undetectable pre-S antigens and viral DNA by the polymerase chain reaction assay at final follow-up. HBsAg clearance occurred in two of the five patients. The second pattern was one of persistence of HBsAg and pre-S antigens, associated with the presence of serum HBV DNA detectable by spot hybridization or polymerase chain reaction regardless of clinical outcome. These findings demonstrate the occurrence of anti-pre-S antibodies in chronic hepatitis B virus-induced liver disease and associate anti pre-S appearance with the clearance of hepatitis B virus from serum. PMID- 1727796 TI - Demonstration of hepatitis B virus DNA by polymerase chain reaction in the serum and the liver after spontaneous or therapeutically induced HBeAg to anti-HBe or HBsAg to anti-HBs seroconversion in patients with chronic hepatitis B. AB - The objective was to determine the proportion of patients with chronic hepatitis B in whom hepatitis B virus DNA is demonstrated by polymerase chain reaction after HBeAg to anti-HBe or HBsAg to anti-HBs spontaneous or therapeutically induced seroconversion. Polymerase chain reaction was performed on serum 6 and 12 mo after HBeAg to anti-HBe seroconversion in 12 patients and 2, 6 and 12 mo after HBsAg to anti-HBs seroconversion in 13 patients. Polymerase chain reaction was performed on liver tissue after HBeAg to anti-HBe seroconversion in five patients and after HBsAg to anti-HBs seroconversion in one patient. Serum HBV DNA was demonstrated by polymerase chain reaction in 83% of patients 6 or 12 mo after HBeAg to anti-HBe seroconversion and in 58%, 31% and 15% of patients at 2, 6 and 12 mo, respectively, after HBsAg to anti-HBs seroconversion. Liver HBV DNA was demonstrated by polymerase chain reaction in all patients tested. Our results show that (a) a reduced level of hepatitis B virus replication persists in most of the patients after HBeAg to anti-HBe seroconversion and might be predictive of reactivation, and (b) in contrast, hepatitis B virus replication progressively disappears in most of the patients after HBsAg to anti-HBs seroconversion. PMID- 1727797 TI - Diagnosis of chronic hepatitis C after liver transplantation by the detection of viral sequences with polymerase chain reaction. AB - Chronic hepatitis frequently occurs after liver transplantation. The role of hepatitis C virus infection in patients after liver transplantation is unknown, although antibodies to HCV are detected in some of these cases. The use of polymerase chain reaction techniques for the detection of hepatitis C virus RNA should improve sensitivity and specificity, particularly in these immunosuppressed patients. Our goal was to further clarify the role of hepatitis C virus infection in chronic hepatitis occurring after liver transplantation. Patients with chronic hepatitis of uncertain origin after transplantation were identified. Serum samples taken at the time of the most recent liver biopsy that showed chronic hepatitis were tested for anti-hepatitis C virus using enzyme linked immunoassay and supplemented by recombinant immunoblot assay (recombinant immunoblot assay I and recombinant immunoblot assay II). The samples were also tested for the presence of hepatitis C virus RNA using polymerase chain reaction. Of the 25 patients with chronic hepatitis, 15 (60%) had hepatitis C virus RNA present. Only seven (47%) of these 15 patients had anti-hepatitis C virus detected. Hepatitis C virus is a major cause of chronic hepatitis occurring after liver transplantation. The magnitude of hepatitis C virus infection will be underestimated if only currently available assays for anti-hepatitis C virus are used. PMID- 1727798 TI - Ultrastructural identification of light microscopic giant mitochondria in alcoholic liver disease. AB - Ultrastructural identification of light microscopic giant mitochondria was performed on the same specimens for light and electron microscopic observations. The liver tissue specimens were fixed in OsO4, embedded in epoxy resin, cut 4 microns thick and stained with polychrome. At the beginning of the study a light microscopic observation was made, and a microphotograph was taken. The identification of light microscopic giant mitochondria by conventional microscopy was identified by the occupation rate in liver cells, the negative findings of stainability and the morphological consistency (round, cigar-shaped and granular). The specimens were subsequently embedded again in epoxy resin and cut into ultrathin sections of 400 A. A transillumination electron microscope was used for the observation, and ultrastructural images of light microscopic giant mitochondria revealed that they were crystalloid bodies with a crystalline latticelike structure. The occupation rates within liver cells and the morphological shapes of the crystalloid bodies corresponded with those of light microscopic giant mitochondria. The light microscopic giant mitochondria obviously had different features from those of electron microscopic giant mitochondria and Mallory bodies (Yokoo's type II), although Mallory bodies showed the same staining properties as light microscopic giant mitochondria. PMID- 1727799 TI - Graft regeneration and host liver atrophy after auxiliary heterotopic liver transplantation for chronic liver failure. AB - We studied the size of the liver graft and the host liver in six consecutive patients undergoing auxiliary heterotopic liver transplantation for chronic end stage liver disease. In all cases, a liver reduced in size by left lateral hepatectomy was inserted. The sizes of the graft and host liver were estimated by planimetry of two-dimensional di-isopropyl iminodiacetic acid scintigrams taken 3, 7, 21, 90 and 180 days after surgery. Graft size increased from a mean of 12.2 cm2 (95% confidence interval = 10.2 to 14.1) on day 3 to a maximum of 14.8 cm2 (95% confidence interval = 13.4 to 16.1) on day 21 and remained stable thereafter; in contrast, the host liver decreased in size from 9.6 cm2 (95% confidence interval = 6.8 to 12.3) on day 3 to 3.9 cm2 (95% confidence interval = 3.0 to 4.8) at mo 6. We conclude that in patients with chronic liver failure, an auxiliary allograft reduced in size and placed adjacent to the host liver shows regenerative growth within 3 wk, whereas the host liver atrophies in 3 to 6 mo. PMID- 1727800 TI - Plasma catecholamine concentrations are a reliable index of sympathetic vascular tone in patients with cirrhosis. AB - In patients with cirrhosis, the significance of elevated plasma catecholamine concentrations is unclear. Thus we investigated the relationship between plasma catecholamine concentrations and the hemodynamic effect of pindolol (an index of sympathetic vascular tone) in 10 patients with cirrhosis. Systemic and splanchnic hemodynamics and plasma catecholamine concentrations in the pulmonary artery and the splanchnic veins (hepatic and azygos veins) were studied before and after the oral administration of pindolol (20 mg). In basal conditions patients exhibited a hyperkinetic circulatory syndrome and elevated plasma catecholamine concentrations. Alterations in basal hemodynamics were correlated with plasma epinephrine concentrations but not with norepinephrine. Pindolol administration significantly decreased heart rate and increased right atrial pressure. After pindolol administration, individual hemodynamic changes (cardiac index, systemic vascular resistance, wedged hepatic venous pressure) were significantly correlated with plasma catecholamine concentrations. In conclusion, this study shows that in cirrhotic patients epinephrine may play a role in hemodynamic alterations, and plasma catecholamine concentrations are an index of sympathetic vascular tone. PMID- 1727801 TI - Sclerotherapy vs. esophageal transection vs. distal splenorenal shunt for the clinical management of esophageal varices in patients with child class A and B liver function: a prospective randomized trial. AB - Ninety-six patients with good liver function (Child class A or B) and esophageal varices were randomly assigned to one of three groups given different treatments: endoscopic injection sclerotherapy (n = 32), esophageal transection (n = 32) or distal splenorenal shunt (n = 32). Five patients (5.2%) had to be excluded from this study because severe chronic pancreatitis made separation of the distal splenic vein from the pancreatic bed difficult. Esophageal transection was performed for these patients. No deaths occurred during the 30 days of treatment. The 5-yr cumulative bleeding rates were 0%, 5.9% and 12.9% in the endoscopic injection sclerotherapy, esophageal transection and distal splenorenal shunt groups, respectively (no statistical significance). In no case in the three groups did death occur because of variceal bleeding. Sixteen patients died, mainly because of underlying liver disease; four were in the endoscopic injection sclerotherapy group, five were in the esophageal transection group and seven were in the distal splenorenal shunt group. No statistically significant difference in survival rate among the three groups was found. These results show that endoscopic injection sclerotherapy is a satisfactory alternative to esophageal transection or distal splenorenal shunt for the clinical management of patients with esophageal varices. PMID- 1727802 TI - Endoscopic injection sclerotherapy for 1,000 patients with esophageal varices: a nine-year prospective study. AB - We report here the results of endoscopic injection sclerotherapy performed in 1,000 consecutively treated Japanese patients with esophageal varices. This prospective study covered the period from 1982 to 1990. Variceal bleeding was controlled in 215 (97.7%) of 220 patients. Esophageal varices were completely eradicated in 778 patients (77.8%); the mean number of sessions was 4.2. In only 3 of the 778 patients did esophageal varices of the same size recur. Small, dilated, venous vessels that required additional sclerotherapy in follow-up endoscopy at 3-mo intervals appeared in 171 (22.2%) of 778 patients. The cumulative nonbleeding rate at 5 yr was 94.5% in patients in whom the varices had been eradicated. Deaths caused by upper gastrointestinal bleeding accounted for 2.6% of cases, whereas the rates of liver failure and hepatoma were 4.6% and 47.3%, respectively. The 5-yr cumulative survival rate was 54.1% in patients without concomitant hepatoma; it was 12.0% in patients with hepatomas. Multivariate analysis showed that hepatoma, Child classification, indication (acute, elective or prophylactic) and eradication were independent factors that significantly influenced survival time. This study clearly shows that close follow-up with endoscopy and complete eradication lead to significant reduction in bleeding from esophageal varices and reduction of mortality related to this bleeding. PMID- 1727803 TI - Treatment of severe alcoholic hepatitis by infusion of insulin and glucagon: a multicenter sequential trial. AB - Severe alcoholic hepatitis is still a therapeutic challenge. It has been recently advocated that a 3-wk infusion with insulin and glucagon reduces its short-term mortality rate. A multicenter, randomized, single-blind, sequential trial was designed to compare this treatment with placebo. The triangular boundary was defined with alpha = 0.05, beta = 0.10 and estimated survival at 4 wk of 50% with placebo, 75% with treatment. Patients with biopsy-proven severe alcoholic hepatitis (presence of one or more of three criteria: encephalopathy, prothrombin activity less than or equal to 50%, bilirubinemia greater than or equal to 100 mumol/L) were randomized into two groups; one treatment group received an infusion (12 hr/day) of an association of insulin (30 IU) and glucagon (3 mg), and a control group received an infusion of glucose. Treatments were administered during a 3-wk period, and the mortality rate was noted at 4 wk. The decision to discontinue the trial was reached on the basis of results from the first 44 patients. Overall results were assessed in the 72 patients included at the time of this decision (treatment group: n = 37; control group: n = 35). Fifty-three patients had cirrhosis. No significant differences were noted between the two groups at inclusion on the basis of clinical, laboratory and histological criteria. The mortality rate was not significantly different in the two groups; 10 patients (27%) in the treatment group and 5 patients (14%) in the control group died. Causes of death were similar in the two groups and consisted primarily of gastrointestinal hemorrhage, hepatic failure and infectious events.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727804 TI - The role of transcription and messenger RNA stability in the regulation of epidermal growth factor receptor gene expression in regenerating mouse liver. AB - The influence of partial hepatectomy on epidermal growth factor receptor gene expression was studied in mouse liver. Epidermal growth factor receptor binding and epidermal growth factor receptor messenger RNA levels in the liver showed a rapid peak 8 hr after partial hepatectomy, whereas the sham operation had no effects on these levels. The peak epidermal growth factor receptor messenger RNA level was approximately threefold higher than preoperative values. The increase in epidermal growth factor receptor messenger RNA levels occurred primarily as a consequence of an increase in the rate of transcription. Partial hepatectomy slightly increased the half-life of epidermal growth factor receptor messenger RNA in the liver from 2.8 to 3.6 hr. Treatment of partially hepatectomized mice with cycloheximide increased hepatic epidermal growth factor receptor messenger RNA levels about fivefold by prolonging the half-life of the messenger RNA to 11.2 hr, although this treatment inhibited the increase in transcription induced by partial hepatectomy. Cycloheximide also increased epidermal growth factor receptor messenger RNA levels in the liver or kidney of sham-operated mice about threefold, primarily through stabilizing epidermal growth factor receptor messenger RNA. In contrast, cycloheximide had no effects on beta-actin messenger RNA levels in the liver and kidney. These results suggest that transcription induced by partial hepatectomy requires protein synthesis and that labile proteins are involved in the regulation of the stability of epidermal growth factor receptor messenger RNA. PMID- 1727805 TI - Viability and primary culture of rat hepatocytes after hypothermic preservation: the superiority of the Leibovitz medium over the University of Wisconsin solution for cold storage. AB - Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency. PMID- 1727806 TI - Renin gene expression in the adrenal and kidney of patients with primary aldosteronism. AB - mRNA levels for renin in the adrenal gland and kidney were measured by ribonuclease protection assay (RPA). Renin mRNA was not detected by RPA in aldosteronoma and kidney tissues obtained from two patients with primary aldosteronism (PA). In these patients, the PRA values, plasma concentrations of active renin (ARC), and total renin (TRC = ARC + prorenin) were below the assay limit (less than 0.03 ng/L.s, 2.5 ng/L, and 10 ng/L, respectively). On the other hand, renin mRNA was recognized by RPA in aldosteronoma and kidney tissues obtained from two other patients with PA treated with 50 mg/day spironolactone for more than 2 months. Their TRC values were 49.8 and 16.6 ng/L, but their PRA and ARC were undetectable. Renin mRNA content was greater in normal adrenocortical tissue and in the normal kidneys obtained from three hypertensive patients with renal cell carcinoma. In these patients, the mean values of PRA, ARC, and TRC were 0.28 +/- 0.03 (mean +/- SD) ng/L.s, 18.4 +/- 7.8 ng/L, and 110 +/- 15 ng/L, respectively. This is the first report of the lack of renin gene expression in aldosteronoma and kidney tissues obtained from untreated patients with PA. Furthermore, treatment with spironolactone resulted in an increase in the levels of renin mRNA in the aldosteronoma and kidney tissues of patients with PA. PMID- 1727807 TI - Melatonin and melatonin-progestin combinations alter pituitary-ovarian function in women and can inhibit ovulation. AB - Although melatonin (MEL) controls seasonal reproductive cyclicity in some mammalian species, its role in women is controversial. In this study data are presented related to the influence of MEL or MEL-progestin combinations on the pituitary-ovarian axis and ovulation in 32 women. MEL was administered in a dosage of 300 mg to 12 women for 4 months [to 8 women daily (days 1-30) and to 4 women on days 5-17 of the cycle]. MEL was also combined with the synthetic progestin norethisterone (NET) in an attempt to evaluate MEL's effect on a partially suppressed pituitary-ovarian axis. In 16 women, 4 combinations were tested on 4 women each on days 1-21: dosages of 300 mg MEL/0.75 mg NET, 75 mg MEL/0.75 mg NET, 7.5 mg MEL/0.75 mg NET, and 75 mg MEL/0.30 mg NET. In addition, 2 women were medicated with 300 mg MEL alone, and 2 were medicated with 300 mg MEL/0.15 mg NET on days 1-21 for 2 months. During the study, LH, FSH, estradiol (E2), and progesterone (P4) blood levels were determined at regular intervals. After a period of 4 months, daily administration of 300 mg MEL (days 1-30) caused significantly decreased mean LH levels compared to those in 8 nonmedicated controls (P less than 0.001). Also compared to nonmedicated control data, a significant inhibition of P4 in the first and fourth medication months (P less than 0.001) was observed. LH and E2 inhibition reached significance in the fourth medication month (P less than 0.005). Also, the treatments of 300 mg MEL (days 5 17) and 75 mg MEL combined with 0.3 mg NET caused a significant decrease in LH, E2, and P4 levels compared to those in the nonmedicated control group in the first and fourth medication months (P less than 0.05). The data further suggest an additive or synergistic effect between MEL and NET. The medications did not alter sleep-wake rhythms and were not complicated by any side-effects. The presented data suggest that MEL and MEL/NET combinations inhibit ovarian function in women, and that MEL/NET combinations can emerge as effective oral contraceptives. PMID- 1727808 TI - Bone mineral status in growth hormone-deficient males with isolated and multiple pituitary deficiencies of childhood onset. AB - In order to determine whether growth hormone (GH) deficiency of childhood onset affects the adult bone mineral status, we assessed bone mineral content (BMC) by photon absorptiometry in 30 full-grown GH-deficient men (8 with isolated GH deficiency and 22 with multiple pituitary deficiencies; 28 previously treated with GH) and in 30 male controls matched for age (within 4 yr) and height (within 10 cm). Forearm BMC was measured by single photon absorptiometry just proximally of the distal one third of the nondominant forearm (PBMC-2 in arbitrary units and PBMC/bone width (BW) after normalization for bone width) and at a more distal site, close to the carpal joint (DBMC-2 and DBMC/BW). Lumbar BMC was measured by dual photon absorptiometry and reported as total BMC for L2-L4 (LBMC in g) and after normalization for projected area (LBMD in g/cm2). The patients had a significantly lower BMC, both at the forearm (P less than 0.0001) and at the lumbar spine (P less than 0.005): 35.7 +/- 1.0 vs. 50.0 +/- 1.6 and 36.9 +/- 1.2 vs. 52.8 +/- 1.9 (mean +/- SEM) for PBMC-2 and DBMC-2 in patients and controls, respectively; 1.36 +/- 0.03 vs. 1.70 +/- 0.04 and 1.07 +/- 0.03 vs. 1.35 +/- 0.04 for PBMC/BW and DBMC/BW; 34.00 +/- 1.08 vs. 42.02 +/- 1.27 g for LBMC and 0.886 +/- 0.016 vs. 0.976 +/- 0.018 g/cm2 for LBMD. Both the patients with isolated GH deficiency and the patients with multiple pituitary deficiencies were osteopenic when compared to their respective controls (P less than 0.01 to P less than 0.0001 for the patients with multiple deficiencies; statistical significance reached for PBMC-2, DBMC-2, and DBMC/BW only, P less than 0.05, in the small group of patients with isolated GH deficiency). For the patients (n = 19) who had at least three serial measurements over a period of 6 to 28 months, no decrease in BMC was detected. Our findings indicate that men with GH deficiency of childhood onset present with a low adult bone mass, despite prior GH substitution in most of these subjects. The observations of a more pronounced bone mineral deficit at the forearm (20-30% lower mean values, depending on the type of measurements) than at the lumbar spine (9-19%) and the findings of osteopenia in both the patients with isolated GH deficiency and multiple pituitary deficiencies, support the view that GH deficiency per se is responsible for part of the observed deficit.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727809 TI - Endothelin and reproduction? PMID- 1727810 TI - Energetic metabolism in hypothyroid skeletal muscle, as studied by phosphorus magnetic resonance spectroscopy. AB - Phosphorus nuclear magnetic resonance spectroscopy was used to investigate the muscle bioenergetics in different hypothyroid states. Using the thenar muscle group as reference, 2 patients with chronic and severe hormonal deficiency, 3 patients with subacute hypothyroidism, and 8 patients with moderate thyroid insufficiency with isolated high blood TSH levels were studied at rest, during exercise, and during subsequent recovery. The patients were compared with 15 control subjects. Only 1 patient presented a clinical myopathy. The intracellular pH and the relative measurements of inorganic phosphate, phosphocreatine, phosphodiesters, and ATP were directly calculated from phosphorus spectra. Resting muscle showed a significant rise in the inorganic phosphate to ATP ratio. In working hypothyroid muscle, a more important decrease in phosphocreatine levels was noted in patients with chronic and subacute thyroid deficiency, while the intracellular pH fall was greater in all hypothyroid patients than in control subjects. The phosphocreatine recovery rate was lower in all deficient patients, reflecting a probable mitochondrial metabolism impairment. These results are consistent with a defect of the high energy phosphate metabolism in hypothyroidism, even in moderate or recent hormonal deficiency. PMID- 1727811 TI - Tumor necrosis factor-alpha inhibits human chorionic gonadotropin secretion. AB - The placenta is a major source of tumor necrosis factor-alpha (TNF alpha) and is rich in TNF alpha receptors. TNF alpha inhibited hCG secretion by normal chorionic villi at 6 weeks gestation, but chorionic villi contain the heterogenous cell population. To investigate the direct effects of TNF alpha on trophoblasts, the NUC1 choriocarcinoma cell line was used as an in vitro placental cell model. TNF alpha also inhibited hCG secretion by NUC1 cells at concentrations from 1-100 U/mL. TNF alpha at concentrations of 1, 10, and 100 U/mL significantly decreased hCG secretion for 24 h to 88%, 81%, and 71% of the control level, respectively. The expression of hCG beta mRNA was also decreased to 23% of the control level after 24 h of TNF alpha treatment (100 U/mL). Inhibition occurred after 12 h of TNF alpha treatment (100 U/mL). Two measurements, trypan blue dye exclusion and 3-(4,5-dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide reduction, revealed that these inhibitory effects were not due to the cytotoxic activity of TNF alpha on NUC1. In conclusion, TNF alpha reduces hCG secretion in vitro. PMID- 1727812 TI - Immunohistochemical detection of proluteinizing hormone-releasing hormone peptides in neurons in the human hypothalamus. AB - To determine the presence of LHRH prohormone products in the human hypothalamus, antisera raised against LHRH and GnRH-associated peptide (GAP) were used to search for the presence of the corresponding antigens in the human adult and fetal hypothalamus by an immunohistochemical approach. The comparison of immunostaining on adjacent sections shows that all of the cells labeled with LHRH antiserum are also labeled with GAP antiserum and vice versa. Labeled cells are detectable during the 9th week of fetal life, this being the earliest time evaluated. At this time, the LHRH/GAP-positive cells frequently have a neuroblastic appearance. The first detectable fibers appear during the 11th week, and these were observed in the lamina terminalis cinerea and median eminence. In the adult brain, fibers and endings labeled with LHRH or GAP antiserum in the median eminence demonstrate the same topography and morphological characteristics, which are distinct from fibers labeled with other neuropeptide antisera. These results show that the LHRH precursor molecule is produced throughout life in the human hypothalamus, including the earliest stages of development of the peptidergic neurons. Moreover, the detection of LHRH- and GAP positive fibers in the median eminence by the 11th week of fetal life suggests the possibility of an early role of LHRH and, possibly, other LHRH prohormone derived peptides in the development of anterior pituitary function during the fetal period. PMID- 1727813 TI - Endothelin-1 gene expression and protein biosynthesis in human endometrium: potential modulator of endometrial blood flow. AB - We present evidence that endothelin-1 (ET-1) is produced by two distinct cell types (other than vascular endothelial cells) in human endometrial tissue. The supportive findings of this investigation are summarized as follows: 1) prepro-ET 1 mRNA is present in endometrial tissue and in separated endometrial stromal and glandular epithelial cells in culture; 2) immunoreactive ET is secreted into the medium of isolated endometrial stromal cells and glandular epithelium maintained in culture; and 3) the level of prepro-ET-1 mRNA in endometrial tissues obtained at the premenstrual-menstrual phase of the endometrial cycle is greater than that in tissues from the proliferative or early and midsecretory phases. We also found that transforming growth factor-beta and interleukin-1 alpha act to increase the levels of prepro-ET-1 mRNA in endometrial stroma cells in monolayer culture. We speculate that ET-1 derived from endometrial stromal cells may act on the adventitial surface of contiguous spiral arterioles of the endometrium to modulate endometrial blood flow. PMID- 1727814 TI - Decreased insulin clearance as a feature of essential hypertension. AB - Several studies report that essential hypertension is associated with hyperinsulinemia. This condition may depend on enhanced pancreatic insulin secretion and/or a decreased MCR of the circulating hormone. Twenty-five nonobese glucose-normotolerant patients with primary hypertension were divided into 5 groups, each consisting of 5 subjects. Each group was submitted to continuous 120 min double infusion of different doses of insulin (group I, 0.025; II, 0.05; III, 0.1; IV, 0.2; V, 0.4 U/kg.h) and glucose (I, 2; II, 3.5; III, 6; IV, 8; V, 10 mg/kg.min). The same procedures were applied to 25 healthy normotensive volunteers. Basal and steady state plasma levels of glucose, insulin, and C peptide were significantly (P less than 0.05 or less) higher in hypertensive patients than in control subjects of all groups. The MCR of insulin (milliliters per kg/min) at all insulin-glucose infusion rates was significantly (P less than 0.05 or less) lower in hypertensive than normotensive subjects. Despite the significantly higher steady state plasma insulin levels in hypertensives, the MCR of glucose (milliliters per kg/min) was significantly (P less than 0.05 or less) lower in hypertensive than normotensive subjects. These results suggest that an altered insulin removal may contribute to the hyperinsulinemia found in the essential hypertensive subjects. In addition, a defect in insulin-stimulated glucose uptake which persists at supraphysiological insulin concentrations is confirmed in this population. PMID- 1727815 TI - Markers of sodium and volume homeostasis in pregnancy-induced hypertension. AB - Normal pregnancy is associated with increased levels of digitalis-like factor (DLF) and erythrocyte sodium-lithium countertransport (RBC CTT), which return to normal levels postpartum. Patients with pregnancy-induced hypertension (PIH) have greater increases in both factors than women with normotensive pregnancies. This study was designed to determine if both abnormalities are observed concomitantly in PIH, if they correlate with blood pressure, if they correlate negatively with a hormonal index of volume status (PRA), and if they differ in women with and without proteinuria. Twenty-six normotensive women and 26 women with PIH were studied in the third trimester. Thirteen of these patients were also studied 6 months postpartum. Women with PIH, compared to those who were normotensive, had higher RBC CTT (0.49 +/- 0.04 vs. 0.36 +/- 0.03 mmol Li/L cells.h; P = 0.004) and DLF (0.30 +/- 0.3 vs. 0.20 +/- 0.03 microgram digoxin equiv./L; P = 0.01) and lower PRA [4.58 +/- 0.76 vs. 7.34 +/- 0.86 ng/mL.h (1.27 +/- 0.21 vs. 2.04 +/- 0.24 ng/L.s); P = 0.001]. All three parameters correlated significantly with diastolic blood pressures (RBC CTT and DLF positively (P less than or equal to 0.02) and PRA negatively (P = 0.03). Comparisons of DLF, RBC CTT, and PRA demonstrated a significant correlation of RBC CTT and DLF for normotensive pregnant women only (r = 0.38; P = 0.05). Patients with PIH were further analyzed according to whether proteinuria (24-h urinary protein, greater than 0.30 g; urine dipstick, greater than or equal to 2+) was present or absent. There was no significant difference in diastolic blood pressure or PRA between the hypertensive subpopulations, although there was a tendency for those without proteinuria to have lower PRAs [3.85 +/- 0.80 ng/mL.h (1.07 +/- 0.02 ng/L.s)] than those with proteinuria [5.31 +/- 1.30 ng/mL.h (1.48 +/- 0.36 ng/L.s)]. RBC CTT was significantly higher (P less than 0.05) in women with PIH without proteinuria, whereas serum DLF was significantly higher in women with PIH with proteinuria (P less than 0.05). In 13 women studied 6 months postpartum, there was a significant reduction in serum DLF, RBC CTT, and PRA for all women and in blood pressure for women who had had PIH (P less than 0.01). Thus, women with PIH, compared to normotensive pregnant women, had abnormalities in a variety of factors known to be volume sensitive or indicative of salt- and volume-sensitive forms of hypertension.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1727816 TI - Immunoscintigraphy using 111In-labeled F(ab')2 fragments of anticarcinoembryonic antigen monoclonal antibody for detecting recurrences of medullary thyroid carcinoma. AB - The only current possibility for curing medullary thyroid carcinoma (MTC), especially recurrences, is total surgical removal. Early positive diagnosis of recurrences is now possible by monitoring tumor markers such as thyrocalcitonin and carcinoembryonic antigen (CEA). However, preoperative topographic diagnosis of such recurrences remains an unresolved problem. Immunoscintigraphy (IS) using an anti-CEA monoclonal antibody is a new approach that complements morphological imaging, i.e. ultrasonography, computerized tomography, and magnetic resonance imaging. In this study, IS by means of an 111In-labeled anti-CEA monoclonal antibody F(ab')2 was performed nine times in eight patients. True positives were obtained five times (one case of cervical involvement confirmed by surgery, three cases of mediastinal involvement confirmed by computerized tomography, magnetic resonance imaging, and surgery, and one case of bone metastasis, one of them was revealed neither by x-ray nor by conventional bone scan). The remaining four tests gave a false positive, a true negative, a probably false negative, and one unconclusive result. We conclude that IS is helpful in diagnosing sites of MTC recurrence and should accompany other examinations in the evaluation of lesions. PMID- 1727817 TI - An immunologically anomalous luteinizing hormone variant in a healthy woman. AB - An investigation was undertaken to characterize an immunological LH variant in a 31-yr-old healthy woman whose serum LH was either poorly or not at all recognized by two monoclonal antibodies. The two antibodies recognize epitopes present on the intact LH dimer, but not on the free subunits. It was found that the immunologically aberrant LH of the subject was bioactive, as evidenced by an in vitro bioassay for LH. Nothing in the personal history of the subject or in the results from a number of hormone analyses revealed any endocrine abnormalities. In a GnRH stimulation test, the increase in immunoreactive LH using two reference immunometric assays for LH was less than 10% of the mean response of five control subjects. In relative terms, the maximal increase in LH in the subject was only 60-100%, in contrast to 340-560% for the control subjects. The bio/immuno ratio of the LH in the subject was high and was further increased in the GnRH stimulation test. A low proportion of acid LH isoforms in basal and stimulated samples from the subject was in agreement with the high bio/immuno ratio. Gel filtration studies showed the presence of molecular species of apparently lower molecular size than the intact LH, but different from the free beta-subunit. The results suggest the presence of fragments of the alpha-beta-dimer where at least part of the beta-subunit has been lost. A pedigree analysis involving the parents, siblings, and children of the subject strongly suggests a genetic origin of the LH variant described with an autosomal mode of inheritance. This report on an immunological variant of LH illustrates the potential dangers of using monoclonal based immunoassays where a protein hormone with fully maintained biopotency may be partially or totally missed due to the monospecificity of the immunoreagent. This possibility should be kept in mind when inappropriately low levels of gonadotropins are detected in diagnostic routine. PMID- 1727818 TI - Increased steroid production by the ovarian stromal tissue of postmenopausal women with endometrial cancer. AB - An increase in ovarian steroid secretion could play a role in the pathogenesis of endometrial cancer in postmenopausal women. The present study was undertaken to investigate steroid production by isolated ovarian stromal tissues of postmenopausal women with endometrial cancer and to study the effect of LH and insulin on ovarian steroidogenesis in postmenopausal women. Ovarian stromal tissue was obtained from 10 postmenopausal women with endometrial cancer and 8 women without cancer. The stroma was incubated in either the medium alone or the medium to which was added LH (50 ng/mL) or insulin (500 ng/mL). The ovarian stroma of postmenopausal women with cancer released significantly more androstenedione (A), testosterone, and dehydroepiandrosterone than that of women without cancer. Addition of LH resulted in a significant increase in A, testosterone, dehydroepiandrosterone, and progesterone release compared to that with vehicle alone. Addition of insulin stimulated the release of A from the ovarian stroma of women with cancer, but had no effect on the normal postmenopausal ovarian stroma. These results indicate that the ovarian stroma of postmenopausal women with endometrial cancer secrete significantly greater amounts of androgens than those of women without cancer and that both LH and insulin may be important factors contributing to this increase in ovarian steroidogenesis. PMID- 1727819 TI - Trophoblast-derived tumor necrosis factor-alpha induces release of human chorionic gonadotropin using interleukin-6 (IL-6) and IL-6-receptor-dependent system in the normal human trophoblasts. AB - The titer of tumor necrosis factor-alpha (TNF alpha) secreted by placental blocks was determined by enzyme immunoassay. The source of placental TNF alpha was immunohistochemically demonstrated with monoclonal anti-TNF alpha antibody to be only trophoblasts. Purified trophoblasts produced 174.4 ng/L TNF alpha by 24 h of culture in vitro. To investigate the role of TNF alpha in placental hormonogenesis, purified trophoblasts were stimulated with recombinant TNF alpha (rTNF alpha) to determine the hCG titer by enzyme immunoassay. Trophoblasts stimulated with rTNF alpha released hCG in a dose-dependent fashion with kinetics similar to those of recombinant interleukin-1 (rIL-1)-stimulated trophoblasts. The stimulated trophoblasts released IL-6 before hCG, but failed to show hCG release when pretreated with anti-IL-6 receptor (anti-IL-6-R) monoclonal antibody PM-1. However, the pretreatment of trophoblasts with PM-1 did not interfere with rTNF alpha-induced IL-6 release, ruling out the possibility of a nonspecific toxic effect of PM-1 on trophoblasts. These results suggest that trophoblast derived TNF alpha induced IL-6 release and then activated the IL-6-R system in trophoblasts to release hCG. Since IL-1 has also been demonstrated to induce similar release of IL-6 and hCG from trophoblasts, the effects of TNF alpha and IL-1 on these trophoblast functions were also examined. Simultaneous stimulation of trophoblasts with rTNF alpha and gamma IL-1 alpha resulted in synergistic enhancement of IL-6 release, subsequently leading to enhanced hCG release. Collectively, trophoblast-derived TNF alpha and IL-1 synergistically regulated the level of IL-6 secreted by trophoblasts, the magnitude of which determined the level of hCG released by activating the IL-6-R system in trophoblasts. PMID- 1727820 TI - Effect of insulin resistance and hyperglycemia on proinsulin release in a primate model of diabetes mellitus. AB - An elevated plasma proinsulin (PI) to immunoreactive insulin (IRI) ratio occurs in relatives of patients with insulin-dependent diabetes mellitus and in subjects with non-insulin-dependent diabetes mellitus. To determine whether this alteration is the result of B-cell dysfunction and/or insulin resistance, we infused nicotinic acid for 3 weeks to produce insulin resistance in five adolescent male baboons before and after the administration of streptozocin (200 mg/kg). We measured basal PI and IRI levels and the acute incremental PI (APIR) and IRI (AIRIR) responses to iv arginine. The quantity of IRI comprised of PI was calculated in the basal state (PI/IRI) and following arginine injection (APIR/AIRIR). Streptozocin administration did not change the fasting plasma glucose (FPG) compared to that in the normal animals (4.7 +/- 0.3 vs. 4.3 +/- 0.2 mM) but raised the PI/IRI (16.4 +/- 3.4 vs. 5.9 +/- 1.7%) and APIR/AIRIR (7.1 +/- 1.0 vs. 2.8 +/- 1.0%) due to a concurrent reduction in IRI and increase in PI concentrations. The induction of experimental insulin resistance with nicotinic acid in the normal animals had no effect on the FPG (4.4 +/- 0.2 mM) but in the streptozocin treated animals, fasting hyperglycemia (8.3 +/- 1.7 mM) developed. Neither the basal PI/IRI (10.2 +/- 2.2%) or the APIR/AIRIR (2.3 +/- 0.6%) increased in the insulin-resistant streptozocin animals thus being no different to that of normal control animals before or during experimental insulin resistance. We conclude that disproportionate proinsulinemia is a manifestation of B-cell damage from streptozocin which is not exacerbated by insulin resistance or hyperglycemia. PMID- 1727821 TI - Relative contributions of years since menopause, age, and weight to vertebral density in postmenopausal women. AB - Vertebral mineral density (VMD) was measured by quantitative computerized tomography (QCT) in 16 premenopausal and 243 untreated postmenopausal women without vertebral compression. The mean VMD in the premenopausal group was 157 +/ 10.1 mg/mL, which is close to previously reported values. In the postmenopausal women, VMD fell significantly with age and years since menopause (YSM) separately and together, but the relation to YSM was more significant than that to age. After logarithmic transformation of YSM, the fall in bone density with logYSM was highly significant (P less than 0.001), and that with age was not quite significant. In 36 pairs of women matched for YSM, there was no significant difference in VMD between the subjects up to and over 55 yr of age. In 32 pairs matched for age, VMD was significantly lower in those over 55 yr than in those up to 55 yr (P = 0.005). There was also a significant correlation between VMD and body weight. After this was allowed for, the correlation between VMD and logYSM remained highly significant, but the correlation with age was not significant. We conclude that the fall in vertebral body trabecular bone in postmenopausal women is self-limiting, amounts to about 35% bone loss in 25 yr (most of it in the first 5 yr), and corresponds to but is proportionately greater than the trabecular component in postmenopausal forearm bone loss. PMID- 1727822 TI - C-peptide suppression test: effects of gender, age, and body mass index; implications for the diagnosis of insulinoma. AB - To assess the effects of gender, age, and body mass index (BMI) on suppression of plasma C-peptide during insulin-induced hypoglycemia, 101 lean and obese, healthy men and women ages 20 to 80 yr underwent infusion of human regular insulin, 0.125 U/kg over 60 min after an overnight fast. Plasma glucose, insulin, and C-peptide were measured every 30 min for 120 min. C-peptide concentrations were influenced by gender at 30 min, by BMI at baseline and both BMI and age at all subsequent time points. Because of variations in baseline plasma C-peptide concentrations, percent decrease in C-peptide was evaluated. Significantly less percent decrease of C-peptide with increased age at 30, 60, and 90 min and with increased BMI at 30 and 60 min were noted with no effect of gender. From stepwise regression analysis using multiple, additional variables only the plasma glucose concentration at 30 min made a significant, albeit small (8%), contribution to the variability in percent decrease in C-peptide at 60 min. When C-peptide responses from eight histologically confirmed insulinoma patients were contrasted to values adjusted for age, gender, and BMI of normal subjects, all insulinoma patients had abnormal responses when percent decrease in C-peptide was used, whereas only four insulinoma patients had abnormal response when actual C-peptide concentrations were used. PMID- 1727823 TI - Trophoblast-derived transforming growth factor-beta 1 suppresses cytokine induced, but not gonadotropin-releasing hormone-induced, release of human chorionic gonadotropin by normal human trophoblasts. AB - Using a transforming growth factor-beta (TGF beta)-sensitive cell line, Mv1Lu (or CCL 64), we demonstrated that trophoblasts predominantly produced a latent form of TGF beta. After converting latent TGF beta to active TGF beta in vitro by acid (pH 2.5), alkali (pH 10.0), or heat (90 C; 10 min) treatment, addition of rabbit anti-TGF beta 1 antiserum resulted in the elimination of TGF beta activity, thus suggesting that trophoblasts produced at least a certain amount of latent TGF beta 1. To investigate the role of TGF beta 1 in placental hormonogenesis, we first studied the effect of recombinant (r) TGF beta 1 on the production of interleukin-6 (IL-6) and hCG by trophoblasts. rTGF beta 1 exerted no inhibitory activity on IL-6 and hCG production. The effect of rTGF beta 1 on cytokine induced IL-6 and hCG release was then examined. While rTGF beta 1 failed to inhibit basal hCG secretion, it did inhibit recombinant tumor necrosis factor alpha (rTNF alpha)-induced IL-6 release as well as rTNF alpha- and rIL-6-induced hCG release in a dose-dependent manner. However, rIL-1 alpha-induced IL-6 and hCG release was remarkably sensitive to rTGF beta 1-mediated suppression. In contrast, GnRH-induced hCG release, the response of which is independent of the IL-6 and IL-6 receptor system in trophoblasts, was completely resistant to rTGF beta 1. Thus, trophoblast-derived TGF beta 1 is an important regulatory molecule of cytokine-dependent, but not cytokine-independent, hCG release, possibly by converting latent TGF beta to active TGF beta at the local site of trophoblasts. PMID- 1727824 TI - Massive amounts of immunoreactive endothelin in human seminal fluid. AB - We found that immunoreactive endothelin (ET) is present in seminal fluid in very large amounts (500-5000 ng/L; quantification based on ET-1 standard). This immunoreactive ET was detected by use of a radioimmunoassay system in which the N terminal portion of ET-1 and ET-2 (and big ET-1 and big ET-2) are recognized. Thus, the immunoreactive ET in seminal fluid may include the precursors of ET-1 or ET-2 (i.e., big ET) as well as metabolites of ET-1 or ET-2 in which the N terminal region is intact. The levels of immunoreactive ET in seminal fluid from men with normal semen analyses and that in seminal fluid of vasectomized men were within the same range. Using a different radioimmunoassay system in which the C terminal portion of ET-1, ET-2, and ET-3 is recognized, we found that the levels of immunoreactive ET were much lower (or undetectable). We speculate that bioactive ET may be produced and act to promote sperm transport in the male reproductive tract; thereafter, bioactive ET may be metabolized by membrane metalloendopeptidase (which is present in male reproductive tissues and semen) to immunoreactive, inactive products. Alternatively, big ET in seminal fluid may be processed in tissues of the female internal genitalia to bioactive ET, which could act to promote sperm transport through the uterine cavity by stimulating myometrial contractions. PMID- 1727825 TI - Effect of acute variations in dietary fat and carbohydrate intake on retinyl ester content of intestinally derived lipoproteins. AB - Vitamin A was administered to eight patients with noninsulin-dependent diabetes mellitus in conjunction with the two different test meals containing (as percentage of total calories) either 15% protein, 60% carbohydrate (CHO), and 25% fat or 15% protein, 40% CHO, and 45% fat. The vitamin A and test meals were given at noon (4 h after a standard breakfast), and blood was obtained hourly from noon to midnight for measurement of plasma glucose, insulin, triglyceride (TG), and cholesterol concentrations; concentrations of TG and cholesterol in Sverdberg floatation (Sf) unit above 400 and Sf 20-400 lipoproteins; retinyl ester concentration in plasma; and both Sf more than 400 and Sf 20-400 lipoproteins. The postprandial TG response in plasma, Sf more than 400 lipoproteins, and Sf 20 400 lipoproteins from noon to midnight was only slightly higher than values seen after consumption of the 60% CHO diet, which contained much less fat (25% vs. 45%) and the retinyl ester concentration was actually higher in both lipoprotein fractions after the diet containing the smallest amount of fat (60% CHO). Furthermore, the cholesterol concentration in the plasma and two lipoprotein fractions was identical after the two diets, despite the great difference in fat content. These data indicate that the acute ingestion of high CHO (60%), low fat (25%) diets by patients with noninsulin-dependent diabetes mellitus led to little or no decrease in postprandial plasma or lipoprotein TG or cholesterol concentrations and an actual increase in concentration of potentially atherogenic small chylomicron and/or chylomicron remnants. PMID- 1727826 TI - Clinical review 29: Postpartum thyroiditis. PMID- 1727827 TI - Phorbol ester increases plasminogen activator inhibitor accumulation in cultures of human granulosa cells. AB - Proteolytic enzymes such as plasminogen activators (PAs) and collagenases are implicated in the process of ovarian follicle rupture. Data obtained in rats support the concept that PAs are both hormonally regulated and temporally related to the ovulatory process; however, such data are lacking in the human ovary. The recent identification of a family of PA inhibitors (PAIs) adds a new dimension to the control of PA activity, and in contrast to animal studies, the human preovulatory follicle is characterized by PA inhibitory activity. To initially examine the PA and PAI system in the human ovary, granulosa cells obtained from women undergoing gonadal hyperstimulation for in vitro fertilization were cultured in the presence or absence of the protein kinase-C activator, phorbol 12 myristate 13-acetate. Reverse fibrin autography of conditioned medium from control cells revealed the presence of a putative PAI with an apparent mol wt of 50,000. Phorbol ester stimulated the accumulation of this PAI in a specific and dose-dependent manner. Cumulus cells and noncumulus granulosa cells were similar in terms of presence and regulation of PAI. Immunoprecipitation with specific antisera revealed that this human granulosa cell PAI was immunochemically related to PAI-1. This identification was supported by quantitative analysis revealing a 3.7-fold increase in PAI-1 antigen, as assessed by a specific enzyme-linked immunoabsorbent assay. These findings are the first demonstration of the in vitro regulation of PAI activity in the human ovary. PMID- 1727828 TI - Hyposialylated thyroglobulin in a patient with congenital goiter and hypothyroidism. AB - A large family (14 children) with congenital goiter whose parents are first cousins was studied. Thyroid tissue was obtained, after 125I in vivo labeling, from one of the siblings (JBM). Gel filtration of thyroid proteins indicated that thyroglobulin (Tg) eluted as a single symmetrical peak in the same position as authentic 19S Tg. Gel electrophoresis in a 7.5% sodium dodecyl sulfate polyacrylamide gel revealed a major band with the same mobility and immunoreactivity as normal 19S Tg. Hydrolysis of the patient's Tg indicated that most of the radioactivity was mono- and diiodotyrosines. The yield of T4 from JBM Tg (26 pmol/mg protein) was 5-fold less than normal thyroid tissue (140 pmol/mg protein) and approximately half of that in thyroid tissue from endemic goiter (51 pmol/mg). Total T3 released from JBM Tg was similar to the other two tissues. When the carbohydrate content of normal and patient Tg was analyzed, there was no differences in glucosamine, galactose or mannose content. However, unlike normal and endemic-goiter Tg, that had a mean sialic acid content of 7.3 and 5.6 micrograms/mg protein, respectively, the sialic acid concentration of the patients Tg was only 0.3 microgram/mg. Sialyltransferase activity was readily demonstrated in homogenate from normal thyroid or endemic goiter, but no sialyltransferase activity was detectable in a homogenate of JBM-thyroid tissue. We conclude that the finding of severely hyposialylated Tg is linked to a defect in iodotyrosine coupling seen in this patient with a possibly abnormal migration of Tg into the follicular lumen. PMID- 1727829 TI - Recessive inheritance of thyroid hormone resistance caused by complete deletion of the protein-coding region of the thyroid hormone receptor-beta gene. AB - Generalized resistance to thyroid hormone is a syndrome of reduced responsiveness of target tissues to thyroid hormone. The determination of amino acid sequences of the human thyroid receptor-beta (hTR beta), deduced from cDNA sequencing, has enabled evaluation of the genetic basis for this syndrome. Distinct point mutations in the ligand-binding domain of hTR beta have been identified in affected members of unrelated families, producing single amino acid substitutions that result in products with decreased or no hormone-binding activity. Inheritance in these families was autosomal dominant. We now report the molecular basis of generalized resistance to thyroid hormone in a consanguineous family unique for its autosomal recessive mode of inheritance. Deletion of the entire coding region of both hTR beta alleles in homozygous affected members of the family was demonstrated by the failure to amplify the coding exons 3-8 by the polymerase chain reaction using primers specific for flanking intronic sequences and by the demonstration of the presence of only two noncoding exons in Southern blots hybridized with exon-specific probes. As expected, obligate heterozygotes were phenotypically normal, since, in contrast to alleles with point mutations, the deleted allele could not act in a dominant negative fashion. Survival and maintenance of a euthyroid state are presumably mediated through expression of the hTR alpha gene, present in affected subjects, and the maintenance of high thyroid hormone levels. Furthermore, the clinical manifestations were relatively more mild that those observed in a homozygous patient with a single amino acid deletion in the hTR beta gene. PMID- 1727830 TI - Genetic basis of endocrine disease 2: congenital adrenal hyperplasia due to 21 hydroxylase deficiency. PMID- 1727831 TI - Development of thyrotropin circadian rhythm in infancy. AB - Normal children and adults show a similar pattern of diurnal variation of TSH secretion with lower values at 1100 h and higher around 2300 h. The purpose of this study was to investigate the age of appearance of TSH circadian rhythm. In 57 fullterm infants 0-6 months old and in 37 premature infants 1-4 weeks old TSH was measured at 1030, 1100, 1130 h and 2230, 2300, and 2330 h. No diurnal rhythm was detected in both premature and fullterm infants less than 4 weeks of life. After the first month of life a significant difference between AM and PM values was observed in fullterm infants. In infants 1-2 months old mean +/- SEM AM and PM values were 2.8 +/- 0.2 and 3.5 +/- 0.4 mU/L, respectively (P less than 0.025), in infants 3-4 months old 3.0 +/- 0.6 and 4.1 +/- 0.8 (P less than 0.01) and in infants 5-6 months old 1.8 +/- 0.2 and 2.6 +/- 0.3 (P less than 0.0005). These data clearly indicate that the development of TSH circadian rhythm starts after the first month of life. PMID- 1727832 TI - A biodegradable testosterone microcapsule formulation provides uniform eugonadal levels of testosterone for 10-11 weeks in hypogonadal men. AB - Limitations of presently available testosterone esters (enanthate and cypionate) include the fluctuating serum testosterone levels and the need for relatively frequent injections (every 10-21 days). These limitations of testosterone esters have prompted the development of more physiological and longer acting systems for androgen delivery. This paper reports pharmacokinetic and pharmacodynamic data with a second generation long-acting testosterone microcapsule formulation in hypogonadal men. This was a single dose, open label, nonrandomized study. Ten hypogonadal men with primary (n = 6) or secondary (n = 4) hypogonadism, otherwise in good health, received 630 mg microencapsulated testosterone in dextran solution (IM) on day 1. Serum total and free testosterone; LH; FSH; dihydrotesterone; estradiol; sex hormone-binding globulin; total cholesterol; high, low, and very low density lipoprotein cholesterol; triglycerides; and apoprotein-AII and -B were measured on multiple occasions during the 2-week control period and the 16-week treatment period. In addition, on days 0, 1, 28, 56, and 84, subjects were hospitalized for detailed hormone analyses over the 24 h period. Serum total and free testosterone levels rose quickly into the midnormal range and stayed uniformly in the eugonadal range for about 70-77 days, after which serum testosterone levels declined gradually into the hypogonadal range. Testosterone release from the microcapsule formulation over the first 10 weeks approximated zero order kinetics. Serum dihydrotestosterone levels rose into the normal range, and testosterone to dihydrotestosterone ratios remained in the physiological range. Serum estradiol levels rose and stayed in the midnormal male range. Serum sex hormone-binding globulin levels decreased significantly during treatment. Serum LH and FSH levels also significantly decreased in the six hypergonadotropic men. Total cholesterol low and very low density lipoprotein cholesterol and triglyceride levels did not change, but plasma high density lipoprotein cholesterol levels decreased significantly during treatment. These data indicate that testosterone microcapsule formulation provides uniform eugonadal levels of testosterone for about 10 weeks. The long duration and zero order kinetics make it an attractive alternative to existing methods of androgen replacement. PMID- 1727833 TI - Depot gonadotropin-releasing hormone agonist blunts the androgen-induced suppression of spermatogenesis in a clinical trial of male contraception. AB - Thus far, when tested as male contraceptives, GnRH agonists in combination with androgens were not very effective in producing azoospermia. Since in previous studies androgens were always given simultaneously with the GnRH agonist or later, we tested whether GnRH agonist administration after an initial androgen suppression phase might yield better results. After a control period, 3 groups of young healthy men (n = 8/group) received an initial loading dose of 400 mg 19 nortestosterone hexyloxyphenylpropionate (19NT-HPP), followed by 200 mg of the ester every 3 weeks for 24 weeks. One week after the first 19NT-HPP injection, 2 groups were given a single sc implant injection of 3.3 or 6.6 mg of the GnRH agonist buserelin, respectively, whereas a placebo implant was given to the third group. In the group receiving only 19NT-HPP, serum LH and FSH were markedly suppressed and remained low during the treatment phase. In the 16 volunteers receiving the buserelin implant LH and FSH were also suppressed on day 7, followed by a marked increase in the gonadotropins up to 2 weeks after buserelin implant injection. While LH was consistently suppressed for the remaining treatment phase, FSH returned to almost normal values in weeks 9-15. In contrast to the group treated with 19NT-HPP alone, in which sperm concentrations were reduced to oligozoospermia after only 3 weeks of treatment, the first suppressive effect in the 19NT-HPP/buserelin-treated groups was not seen before week 9. After 30 weeks, when the maximal suppression of spermatogenesis was seen, 4 of 8 volunteers in the group treated with 19NT-HPP alone were azoospermic, and the remaining 4 volunteers were oligozoospermic. In the groups treated with 19NT HPP/buserelin, no more than 4 of 16 volunteers were azoospermic, and no more than 8 of 16 volunteers were oligozoospermic at any time point. It is concluded that GnRH agonist depot preparations have a blunting effect on the suppression of pituitary and testicular function caused by androgens in men participating in contraceptive trials. PMID- 1727834 TI - Divergent effects of short term glucocorticoid excess on the gonadotropic and somatotropic axes in normal men. AB - We investigated the effects of short term glucocorticoid excess on the gonadotropic and somatotropic axes in healthy men. Subjects (n = 5) underwent blood sampling at 10-min intervals for 6 h before and on days 2, 5, and 8 of glucocorticoid treatment, and for 24 h (n = 6) to examine pulsatile LH and GH release before and during dexamethasone administration (1.5 mg orally twice daily for 1 week). In the time-course study, we found significant decreases on day 8 in serum concentrations of estradiol (from 144 +/- 18 to 99 +/- 18 pmol/L), free testosterone (from 105 +/- 10 to 87 +/- 10 pmol/L), and dehydroepiandrosterone sulfate (from 6.0 +/- 1.6 to 1.7 +/- 0.3 mumol/L; P less than 0.05). Mean serum LH concentrations did not change (baseline, 5.3 +/- 1.2 IU/L; glucocorticoid, 4.2 +/- 0.61 IU/L). The mean plasma somatomedin-C concentration rose from 0.74 +/- 0.08 to 2.0 +/- 0.35 U/mL (P less than 0.05), and the mean serum GH concentration increased from 1.2 +/- 0.90 micrograms/L (basal) to 4.2 +/- 1.5 micrograms/L (day 8 of dexamethasone; P less than 0.01). Deconvolution analysis of 24-h serum GH and LH concentration profiles revealed that the half-life of endogenous GH and the duration and amplitude (maximal rate of secretion) of computer-resolved GH secretory bursts were not influenced significantly by dexamethasone. The mass of GH secreted per burst rose 1.6-fold. Glucocorticoid treatment also increased detectable GH secretory burst frequency from 12 +/- 1.6 to 18 +/- 1.6 episodes/24 h, decreased the GH interburst interval from 127 +/- 23 to 79 +/- 5 min, and increased the daily GH secretion rate from 41 +/- 11 to 101 +/- 11 micrograms/L.day. These effects on the somatotropic axis were specific, since the half-life of LH; LH secretory burst frequency, amplitude, mass, and duration; and the total daily LH production rate (and LH secretion in response to exogenous GnRH) were not altered by dexamethasone administration. We conclude that short term moderate glucocorticoid excess augments pulsatile GH secretion without influencing the episodic release of LH in normal men. PMID- 1727835 TI - Low to moderate intensity endurance training in healthy older adults: physiological responses after four months. AB - OBJECTIVE: To determine the physiological adaptations in previously sedentary healthy older men and women (mean age = 68) to a 16-week low-to-moderate intensity exercise program. DESIGN: Randomized, controlled trial. SETTING: An exercise facility and testing laboratory in a gerontological research institute. PARTICIPANTS: Two-hundred forty-seven community-dwelling older persons free of significant cardiovascular, pulmonary, or uncontrolled metabolic disease, anemia, electrolyte abnormality, resting BP of 165/90 or greater, or chronic disease affecting the ability to exercise on a bicycle. INTERVENTION: Subjects were randomly assigned to either an exercise (n = 166) or attention control group (n = 81). Exercisers trained thrice weekly for 40 minutes on a cycle ergometer (5 minute warm up, 30 minutes at training heart rate (THR), 5-minute cool down). THR was set at 70% of peak heart rate attained on a maximal exercise test (mean = 115 +/- 15). Control subjects attended weekly group talks. Testing took place before and after the program. RESULTS: Peak attained oxygen uptake (VO2max) increased 8.5% in exercisers and decreased slightly in controls (p less than .001) and oxygen uptake at ventilatory threshold (VeT VO2) increased by 3.5% in exercisers and decreased by 3% in controls (p less than .001). This pattern of a greater increase in VO2max than VeT VO2 is different from that seen in young and middle aged subjects. CONCLUSION: This study demonstrated that a large scale training program is feasible for healthy older people, that physiologic improvements can be measured after 16 weeks of low-to-moderate-intensity training, and that mechanisms of adaptation to exercise may be different in elderly subjects from those in younger ones. PMID- 1727836 TI - Long-term care for dementia: if appropriate, why "special"? PMID- 1727837 TI - Form of presentation of data on muscle strength. PMID- 1727838 TI - Triazolam dose in older patients. PMID- 1727839 TI - Change in functional capacity for elderly patients. PMID- 1727840 TI - Elevated C-reactive protein in older people. PMID- 1727841 TI - Treatment of hypercholesterolemia: comparison of younger versus older patients using wax-matrix sustained-release niacin. AB - OBJECTIVE: Compare lipid response, side effect profile and toxicity of younger (less than 50 years) versus older (50 to 70 years) hypercholesterolemic subjects taking wax-matrix sustained-release niacin (Endur-acin). STUDY DESIGN: An 8-week randomized double-blind placebo controlled trial. SETTING: General community. PARTICIPANTS: Volunteers from community cholesterol screening programs and chart review of patients at family practice clinics. Male and female subjects, age 20 to 70, with baseline low density lipoprotein cholesterol level within the 75th to 95th percentile, excluded if on medications that affect lipids or if a history of diabetes, gout, peptic disease, or liver disease is present. INTERVENTION: Nicotinic acid dosage schedules were 1,000 mg/day, 1,250 mg/day, 1,500 mg/day, or 2,000 mg/day for 8 weeks. MAIN OUTCOME MEASURES: Change in blood lipids and blood chemistries, side effects, and pill compliance. RESULTS: 158 subjects (79%) completed the study. Higher dose groups (1,500 mg and 2,000 mg) demonstrated improvements in total cholesterol, LDL-cholesterol, HDL cholesterol, and total-to HDL-cholesterol ratio (P less than 0.05) compared to baseline and controls. Higher-dose older subjects demonstrated significantly greater improvements than younger subjects on comparable doses for total cholesterol, HDL cholesterol, total-to-HDL-cholesterol ratio, and triglycerides, P less than 0.02). Adherence to medication schedules was better and incidence of side effects and toxicity no greater in older subjects compared to younger. CONCLUSION: Wax-matrix niacin (Endur-acin) was shown to be effective and well tolerated for the pharmacological treatment of hypercholesterolemia. Older persons, ages 50 to 70, appear to experience greater benefits with no greater side effects when compared to younger subjects on similar doses. PMID- 1727842 TI - PTCA in the elderly: the "young-old" versus the "old-old". AB - OBJECTIVE: To evaluate the use of percutaneous transluminal coronary angioplasty (PTCA) in elderly coronary artery disease (CAD) patients. DESIGN: A prospective study of patients 60 years and older undergoing de novo PTCA. We analyze patient risk factors, underlying disease, and clinical outcomes, with at least 3-year follow-up. Comparisons between different age strata among these patients are made to clarify differences between the young old (60 to 69 years), the middle old (70 to 79 years), and the very old (80 years and older). SETTING: Beth Israel Hospital, Boston, both a primary care and tertiary care teaching hospital. PATIENTS: 907 consecutive elderly cardiac patients referred for PTCA are studied. INTERVENTIONS: PTCA's were completed using the newest catheter technologies as they became available. All patients were premedicated with aspirin and dipyridamole, and all were anticoagulated with heparin. RESULTS: Subdivision by age demonstrates that the majority (67%) of patients aged 60 to 69 were males, but females were preponderant (61%) in those aged 80 and older. Octogenarians also had lower incidence of hypercholesterolemia, tobacco use, and family history of CAD, and a higher frequency of CHF, angina, and previous MI. Although total procedure-complications increased with age, critical complications (MI, reocclusion, CABG, death) did not. Primary procedural success was similar in all age strata, but older patients had a higher prevalence of multi-vessel disease and longer hospital stay. Follow-up shows that most patients did well after PTCA; there was no increase in repeat PTCA, CABG, and MI with age. CONCLUSIONS: While advanced age is associated with changes in risk and clinical parameters for CAD patients, age alone is not a reasonable criterion to limit the use of PTCA. PMID- 1727843 TI - Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy. AB - OBJECTIVE: To evaluate the effects of low-power light therapy on pain and disability in elderly patients with degenerative osteoarthritis of the knee. DESIGN: Partially double-blinded, fully randomized trial comparing red, infrared, and placebo light emitters. PATIENTS: Fifty patients with degenerative osteoarthritis of both knees were randomly assigned to three treatment groups: red (15 patients), infrared (18 patients), and placebo (17 patients). Infrared and placebo emitters were double-blinded. INTERVENTIONS: Self-applied treatment to both sides of the knee for 15 minutes twice a day for 10 days. MAIN OUTCOME MEASURES: Short-Form McGill Pain Questionnaire, Present Pain Intensity, and Visual Analogue Scale for pain and Disability Index Questionnaire for disability were used. We evaluated pain and disability before and on the tenth day of therapy. The period from the end of the treatment until the patient's request to be retreated was summed up 1 year after the trial. RESULTS: Pain and disability before treatment did not show statistically significant differences between the three groups. Pain reduction in the red and infrared groups after the treatment was more than 50% in all scoring methods (P less than 0.05). There was no significant pain improvement in the placebo group. We observed significant functional improvement in red- and infrared-treated groups (p less than 0.05), but not in the placebo group. The period from the end of treatment until the patients required treatment was longer for red and infrared groups than for the placebo group (4.2 +/- 3.0, 6.1 +/- 3.2, and 0.53 +/- 0.62 months, for red, infrared, and placebo, respectively). CONCLUSIONS: Low-power light therapy is effective in relieving pain and disability in degenerative osteoarthritis of the knee. PMID- 1727845 TI - A simple method of recognizing geriatric patients at risk for death and disability. AB - OBJECTIVE: To identify prognostic indicators for geriatric patients discharged from an acute care hospital. DESIGN: Prospective observational study. SETTING: Base line assessment at discharge from an acute care hospital; reassessment after 1 year at home. PATIENTS: One hundred-seventy-eight consecutive patients over 70 years of age (mean age +/- SD = 75.6 +/- 13.1 years, range 70-95 years, 52% males); 56% were dependent in one or more Activities of Daily Living, 21% had abnormal Mini Mental State Scores. MAIN OUTCOME MEASURES: mortality, increasing physical dependence, health care utilization. RESULTS: Mortality was directly related to a low ADL score at hospital discharges (Odds Ratio = 3.31, Confidence Limits = 1.91-5.75), neoplastic disease (OR = 3.59, CL = 2.01-6.43), cardiovascular disease (OR = 2.47, CL = 1.40-4.36), and drug use, expressed as the total number of individual preparations prescribed at discharge (OR = 1.72, CL = 1.05-2.83). Low ADL score, cardiovascular and neoplastic disease were also predictive of increasing physical dependency. The use of health care services, quantified by an appropriately designed score, did not correlate with any of the baseline variables, with the implication that the use of the health care services was not proportional to the need for care. CONCLUSIONS: Elderly subjects at major risk of death and disability can be easily identified at discharge by a simple assessment of their medical and functional state. PMID- 1727844 TI - Dementia and the nursing home: association with care needs. AB - OBJECTIVE: To determine whether RUG reimbursement categories accurately predict requirements for care in nursing homes. DESIGN: Prospective descriptive study of residents in lower reimbursement categories according to RUG. SETTING: Three nursing homes in New York City. PARTICIPANTS: Convenience sample of 173 residents who agreed to participate, not significantly different from 201 who did not agree to participate. MAIN MEASURES: Chart review; assessment of residents' cognitive and functional abilities; nursing assistants' ratings of residents' functional abilities, behavioral problems, the amount of effort required in care; and time motion studies of staff-resident interactions. RESULTS: Both the residents' RUG classification (P less than 0.01) and the level of ADL independence (P less than 0.001) had significant impacts on the staff effort required in their care, with more dependent residents requiring greater effort. The residents' level of cognitive impairment also had a significant impact on the staff effort, with the severely impaired requiring greater effort (P less than 0.05). The time-motion analysis indicated that residents within the same RUG category differed in the number of staff-resident interactions based on their level of cognitive impairment. CONCLUSIONS: Cognitive impairment is a significant morbidity (or co morbidity) in determining the quantity of staff effort required by the resident, and behavioral interventions are an important care component. There is marked heterogeneity within lower (RUG) reimbursement categories which translates into strikingly different care requirements. PMID- 1727846 TI - Ability of surrogates to represent satisfaction of nursing home residents with quality of care. AB - OBJECTIVE: To measure the ability of surrogates to accurately represent nursing home residents' satisfaction with the nursing home care. DESIGN: Comparison by correlation analysis of questionnaire answers by nursing-home residents and their designated surrogates. SETTING: Four non-profit community nursing homes. PARTICIPANTS: One-hundred fifty-two resident-surrogate pairs were included, based on the following criteria: (1) the resident was able to respond to questions verbally and in English, had cognitive abilities sufficient to understand the questions, and had a responsible party who had a telephone number in the medical record; (2) both the resident and the surrogate agreed to be interviewed. OUTCOME MEASURES: A 26-item instrument (21 specific and 5 global items) was developed to measure surrogates' perceptions of residents' satisfaction with the quality of the physician services, nursing care, and the nursing home environment. The instrument was scored on a 4-point Likert scale in which higher scores indicated greater satisfaction and paralleled a similar instrument designed for nursing home residents. Correlation of residents' with surrogates' scores on the satisfaction instruments was examined. RESULTS: The mean score for most items was greater than 3.0, indicating overall satisfaction with the care. Correlations between surrogates and residents on specific items ranged from 0.1 to 0.55. Correlations were highest for global items and items addressing satisfaction with the environment. CONCLUSION: We conclude that nursing home residents' surrogates cannot accurately express the residents' satisfaction with all areas of nursing home care and that their evaluations should not be taken in lieu of the residents' opinions. PMID- 1727847 TI - The treatment of urinary incontinence with electrical stimulation in nursing home patients: a pilot study. AB - OBJECTIVES: To test the effectiveness of electrical stimulation in the treatment of urinary incontinence in female nursing home patients. SETTING: A community long term care facility. PARTICIPANTS: Nine unselected female nursing home patients with urinary incontinence. All patients were moderately to severely cognitively impaired. By bedside cystometry, six patients had involuntary detrusor contractions while two had inconclusive results. INTERVENTION: Participants were treated with electrical stimulation for 8 weeks using the Microgyn II device. A current with a frequency of 20 hertz and a pulse width of 1 millisecond was delivered repeatedly for 2 seconds on, 4 seconds off for 15 seconds twice a week. MEASUREMENTS: The number of every-2-hour wet episodes during a 48-hour period (Wet) was recorded by a blinded observer at baseline and after 4 and 8 weeks of treatment. We evaluated the overall effect of electrical stimulation by averaging the Wet at 4 and 8 weeks for each patient and comparing it to Wet at baseline. MAIN RESULTS: The mean +/- standard deviation of intensity of electrical stimulation was 12 +/- 5 milliamps. Mean Wet at baseline was 11.8 +/- 4.2. For all patients mean Wet increased by 2.3 +/- 3.2, P = 0.07. Analysis of patients with documented involuntary detrusor contractions showed a mean increase in Wet of 2.6 +/- 3.6, P = 0.16. The volume of fluid at which an involuntary contraction occurred during cystometry showed a mean increase of 48.3 +/- 52.6 mL, P = 0.07 after 8 weeks of treatment. CONCLUSIONS: Electrical stimulation is well tolerated in elderly nursing home patients. However, it was ineffective in improving urinary incontinence. In fact, there was a tendency for the treatment to worsen the incontinence. PMID- 1727848 TI - Longitudinal prescribing patterns in a nursing home population. AB - OBJECTIVE: This study documents the patients characteristics associated with prescribed medications on entry to a nursing home and the change in prescribing patterns after 3 months. DESIGN: One-year admission cohort. SETTING: Three university-affiliated community nursing homes in Albuquerque, NM. PATIENTS: All new admissions (n = 81) to a University geriatrics team, covering intermediate and skilled levels of care during 1 year (July 1, 1988-July 1, 1989). METHODS: Outcome measures were scheduled and as-needed (PRN) medications prescribed at entry and 3 months. Data collected at entry included patient demographics, activities of daily living index, mental status score, and medical diagnoses. RESULTS: Older persons were prescribed fewer scheduled medications than younger ones, and women fewer than men. There was a positive association between the number of diagnoses and the number of scheduled medications (r = 0.25, P = 0.02). No associations were found between medications prescribed and mental status or functional level. There were no associations between as-needed (PRN) medications and any of the variables studied. Overall, there was a significant increase in the average total number of medications prescribed between admission (4.7) and 3 months (6.2). This was due to an increase in the number of PRN medications from 1.3 at admission to 3.0 at 3 months (P less than 0.001). CONCLUSIONS: Measuring medications at consistent points in a person's nursing home stay may be more informative than using cross-sectional sampling. Future studies on medications in nursing home populations should distinguish between PRN and scheduled medications because medication prescribing patterns may be different in these categories. PMID- 1727849 TI - Endoscopic biliary endoprosthesis in elderly patients with large bile duct stones: long-term follow-up. AB - OBJECTIVE: To assess the long-term evolution of elderly patients with large or impacted bile duct stones, treated by an endoscopic biliary endoprosthesis. DESIGN: Case series. SETTING: Tertiary care center. PATIENTS: Twenty-three patients with a mean (+/- SD) age of 86 +/- 5 years (range, 77-97 years). On admission, 96% were highly symptomatic. These patients represent 8.4% of a group of 273 elderly patients (greater than or equal to 70 years old) with choledocholithiasis treated by endoscopic sphincterotomy between November 1984 and May 1989. INTERVENTION: Endoscopic insertion of a biliary endoprosthesis. RESULTS: Eight-seven percent (20/23) remained completely free of biliary symptoms and died of unrelated illness (48%) after a mean follow-up of 23 months or are still alive (39%) with a mean follow-up of 52 months. In four cases, this asymptomatic evolution now extends for more than 5 years. CONCLUSION: Insertion of a biliary endoprosthesis offers an effective method for long-term treatment of non-extractable biliary stones in elderly patients. PMID- 1727850 TI - Use of a vacuum tumescence device in the management of impotence in men with a history of penile implant or severe pelvic disease. AB - PURPOSE: To evaluate the use of a vacuum tumescence device in the treatment of impotence in couples wishing to restore coital function, whose male partners had unsatisfactory results from penile implants or in whom the man was impotent following treatment for prostate or colon carcinoma. SUBJECTS: Convenience sample of seventeen couples seeking treatment of male factor sexual dysfunction. METHODS: After completion of a comprehensive diagnostic evaluation of the male partner, couples who expressed a wish to restore coital function were instructed in the use of the vacuum tumescence device. Partners each filled out and initialled a daily diary of sexual activity and returned to clinic for followup at 3 and 6 months. RESULTS: Sixteen patients were able to obtain firm to hard erections lasting an average of 14.9 minutes and had satisfactory coitus with vaginal ejaculation an average of 3.9 times per month. There were no significant complications. CONCLUSION: The vacuum tumescence device can be effective in the treatment of impotence after penile prosthesis explantation, in enhancement of inadequate erections with a prosthesis in place, and after surgical or radiation therapy for prostate or colon carcinoma. PMID- 1727851 TI - Cecal bascule: an overlooked diagnosis in the elderly. AB - This elderly male with a long history of alcohol abuse presented with an acute pleural trauma and hemopneumothorax, which may have served as the precipitating medical illness for cecal volvulus. He subsequently developed bacterial peritonitis as a complication of his bowel obstruction. It is probable that his pleural cavity was seeded hematogenously via a bacteremia from his peritonitis, thus accounting for the empyema with species typical of bowel flora. Cecal bascule is a type of cecal volvulus that causes intestinal obstruction. Diagnosis is difficult, but a delay in recognition may result in intestinal ischemia, perforation, sepsis, and even death. Cecal ischemia or gangrene cannot always be determined based on physical examination or laboratory findings. Plain films of the abdomen may be helpful, and barium enema has been advocated by some authors. However, laparotomy is often necessary for definitive diagnosis and therapy. While cecal volvulus has not been reported to occur frequently in the elderly, the relatively common occurrence of anatomic predisposition in addition to the widespread use of respirators and the increasing age and number of medical illnesses of our population make it possible that cecal volvulus will be seen with increasing frequency in the future. PMID- 1727852 TI - Undetectable serum levels of thyrotropin (TSH) in an older woman with secondary hypothyroidism: a clinical observation. PMID- 1727853 TI - Major surgery in nursing home patients: procedures, morbidity, and mortality in the frailest of the frail elderly. AB - OBJECTIVE: To determine the surgical procedures being done on long-term care (level 2) nursing home residents and the resultant in-hospital morbidity and mortality. DESIGN: A retrospective chart review of inpatient medical records from two hospitals, identified by computerized search of medical records and/or referral by directors of nursing of area nursing homes. SETTING: Patients originated in skilled-care nursing homes in New Castle County, Delaware, USA. Surgery was performed in the area's two major hospitals, one a 1000-bed regional referral and teaching hospital, and the other a 300-bed community hospital. PATIENTS: Residents of skilled-care nursing homes (level 2) who underwent major surgery between January 1979 and December 1989. MEASUREMENTS AND MAIN RESULTS: Eighty procedures were performed in 74 patients. Many different types of procedures were done. After primary repair of hip fracture the most common procedures were non-orthopedic extremity and abdominal surgeries. Three deaths occurred (mortality 3.8%), and all were in patients undergoing emergency surgery who were classified above American Society of Anesthesiology Class 3. Serious complications occurred in 43% of the procedures and were most commonly cardiopulmonary and psychiatric, including profound depression in four. Antibiotic-associated colitis occurred in three patients and required a second surgical procedure in one. Fewer adverse outcomes were seen in patients undergoing elective surgical procedures with spinal or local anesthesia than in patients receiving general anesthesia. CONCLUSIONS: Although retrospective and limited to inpatient data, in-hospital surgical mortality in this very frail population was low, comparable to series in unselected geriatric populations. However, major complications were very common. Primary hip surgery repair may have been too frequently done. A multi-institution, prospective trial would be useful to assess functional outcome of surgery in this population. PMID- 1727854 TI - Immunological principles and emerging strategies of vaccination for the elderly. PMID- 1727856 TI - Routine laboratory assessment of nursing home patients. AB - Little is known about protocols for routine laboratory testing in nursing homes. To determine what laboratory protocols are employed by community nursing homes, directors of nursing service in the 73 long-term-care facilities in Portland, Oregon were surveyed. One-hundred percent responded, and 56% reported having laboratory testing protocols. Ninety percent of protocols employed screening tests, and 88% employed monitoring tests, but content varied widely. Although physicians are ultimately responsible for ordering laboratory tests, protocols were derived from various sources including nursing staff, pharmacy and laboratory consultants, and Department of Health and Human Service guidelines. It is recommended that physicians take a leadership role in helping nursing homes develop clinically useful, cost-effective testing strategies. PMID- 1727855 TI - Ethics rounds at the nursing home: an alternative to an ethics committee. AB - The increased attention in US medicine to medical ethics reflects in large part the "new" demography of a growing elderly population and the conflict of whether decisions regarding medical care should be based on cost-effectiveness or "human effectiveness." Currently, about 40 percent of the nation's elderly end up in nursing homes where they confront ethical and legal dilemmas that would not arise in their own homes. In the nursing home, difficult medical-ethical decisions generally rely on two approaches: the often used but frequently invalid concept of informed consent and little used ethics committees. At The Jewish Home and Hospital for Aged in New York City we have developed a program of "ethics rounds" as an alternative to ethics committees. We conduct the rounds in the open style of a forum or clinical conference rather than with the aura of a decision-making group. We encourage the participation of patients and family and seek to educate the staff, any one of whom may choose to attend. The rounds consist of a multidisciplinary case presentation, an interview of patient and/or family, a discussion by the staff, and an overview by an ethicist. Staff response to the educational and interdisciplinary aspects of the rounds has been remarkably positive. PMID- 1727857 TI - A comparative study of the neutrophil stimulatory activity in vitro and pro inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8. AB - IL-8, a potent neutrophil-activating protein, can be produced by many cell types including monocytes, lymphocytes, fibroblasts, neutrophils, and endothelial cells. Depending on the cell source, the N-terminal amino acid sequence of IL-8 displays heterogeneity that has been shown to confer differences in its neutrophil stimulatory activity in vitro. Despite these observations the relative potency of different IL-8 molecules in vivo is unknown. To address this question we have investigated the biologic activity of the two predominant forms of IL-8, the 72 and the 77 amino acid proteins, in vitro and in vivo. In vitro, human rIL 8(72) and human rIL-8(77) dose dependently induced adherence of rabbit peritoneal neutrophils and human neutrophils to laminin-coated plates and elevated cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2 loaded neutrophils. In these in vitro assays human rIL-8(72) was more potent than human rIL-8(77) while inducing comparable responses to human rC5a. With respect to enhancing [Ca2+]i, neutrophils desensitized to human rIL-8(72) failed to respond to human rIL-8(77). However, neutrophils fully desensitized to human rIL-8(77) could exhibit a partial response to human rIL-8(72). Further, human rIL-8(72) was approximately 10-fold more effective than human rIL-8(77) in displacing human [125I]rIL-8(72) from rabbit peritoneal neutrophils in a receptor-binding assay. In vivo, intradermally administered human rIL-8(72) and human rIL-8(77) induced 111In neutrophil accumulation and edema formation in rabbit skin. In contrast to the in vitro studies, the two forms of IL-8 gave identical responses in vivo although they were less potent than human rC5a. Our results demonstrate that, in vitro, human rIL-8(72) is more potent than human rIL-8(77) in stimulating neutrophils. It may be that IL-8)72) has a greater affinity and/or efficacy for the neutrophil IL-8 cell-surface receptors. One possibility for the observation that both forms of IL-8 are equipotent in inducing inflammatory responses in vivo is that the extended form is proteolytically cleaved to the more biologically active IL 8(72). PMID- 1727858 TI - Lipopolysaccharide-induced inhibition of scavenger receptor expression in human monocyte-macrophages is mediated through tumor necrosis factor-alpha. AB - The exposure of mononuclear cells to LPS results in a variety of cellular alterations including changes in the expression of various membrane receptors. In human monocyte-macrophages the development of the scavenger receptor, which mediates the uptake and internalization of chemically modified proteins, was suppressed by 100 ng/ml of LPS, concomitant with a reduction in receptor mRNA. Removal of LPS from the media resulted in a rapid increase in scavenger-receptor activity and mRNA that was further enhanced by macrophage-CSF and granulocyte/macrophage-CSF. However, neither macrophage-CSF nor granulocyte/macrophage-CSF could overcome the suppression of scavenger-receptor activity in the presence of LPS. The LPS-induced suppression of the scavenger receptor could be overcome by the co-addition of neutralizing antibody to TNF alpha. TNF-alpha added to human monocyte-macrophages in the absence of LPS suppressed scavenger receptor activity to the same extent as did LPS. In contrast, the co-addition of LPS and neutralizing antibodies to either IFN-gamma or to IL-1 beta did not overcome the inhibitory effects of LPS on scavenger receptor activity. We conclude that the LPS-induced suppression of the scavenger receptor is mediated primarily through TNF-alpha. PMID- 1727859 TI - Isolation and characterization of a protein from hagfish serum that is homologous to the third component of the mammalian complement system. AB - The 192-kDa protein HX, a major component of serum that specifically binds to zymosan particles, was prepared from the plasma of the hagfish (Eptatretus burgeri) by ion-exchange chromatography and gel filtration. HX, present at a concentration of 0.8 mg/ml in the original plasma, was composed of two distinct subunits of 115 kDa and 77 kDa, respectively, which were linked by disulfide bonds. The protein had the same electrophoretic mobility as beta-globulin. Digestion by trypsin resulted in a specific cleavage of the 115-kDa subunit and a change in its immunoelectrophoretic mobility in the anodal direction, leaving the 77-kDa subunit intact. Treatment with SDS and urea resulted in the splitting of the 115-kDa subunits into 68-kDa and 45-kDa components, but this splitting was inhibited by pretreatment with methylamine, suggesting the presence of a thiol ester bond in the 115-kDa subunit. The amino acid composition of HX revealed a striking resemblance to that of human C3. We conclude, therefore, that the 192 kDa protein isolated in this study is analogous to C3, which plays a key role in the mammalian C system. PMID- 1727860 TI - Paramyosin inhibits complement C1. AB - We report here the results of studies showing that inhibition of C is a property of several invertebrate paramyosins. Paramyosins from Taenia solium, Schistosoma mansoni, and the mussel Mytilus edulis bind polymeric collagen and can be isolated from crude extracts of tissues by collagen affinity. These paramyosins inhibit C1 function whether the C1 is isolated or present in C2-deficient serum. Because T. solium paramyosin was the best inhibitor, we concentrated further studies on this molecule. T. solium paramyosin binds purified C1q in solution with a dose/response similar to C1r2S2. Further studies of the C1-paramyosin interaction indicate that: 1) C4 is not activated, 2) C4b2a decay is not affected, and 3) there is no effect on the efficiency of C3-9, as provided in EDTA-chelated guinea pig serum, in lysing SRBC. Thus, paramyosin inhibition is directed at the initiation of the classical pathway. The results suggest that paramyosins of helminthic parasites may have a role as modulators of the host immune response through C inhibition at C1. PMID- 1727861 TI - A new isoform of human membrane-bound IgE. AB - The epsilon-chain of membrane-bound IgE on the surface of B lymphocytes is known to contain a membrane-anchoring peptide segment that is encoded by two membrane exons, me.1 and me.2. In analyzing pertinent segments in mRNA from human IgE expressing B cells by using PCR methods and Northern blotting analyses, we have identified three species of mRNA of epsilon-chain with variations in the splicing of the membrane exons. The conventional species (m/s) contains the predicted me.1 and me.2; species m/1 harbors 156 extra nucleotides 5' of me.1 with unaltered reading frame; species s/t lacks me.1 and hence the segment encoding the hydrophobic transmembrane stretch and contains a shifted me.2 reading frame. Rabbit antibodies, which were prepared by immunization using a peptide of 36 amino acid residues representing an encoded segment unique to mRNA species m/l, could specifically bind to human IgE-expressing B cell lines and react with an epsilon-chain on Western immunoblots. These results indicate that there exists a previously unidentified isoform of human membrane-bound IgE. PMID- 1727862 TI - MHC class I-restricted presentation of exogenous antigen by thymic antigen presenting cells in vitro and in vivo. AB - We have used a T-T hybridoma, RF33.70, to detect the MHC class I-restricted presentation of exogenous native OVA by thymic APC in vitro. Presentation of OVA with class I molecules by thymic APC requires intracellular processing. Phenotypic analyses indicated that low bouyant density, MHC class II+, FcR+ cells are capable of using this presentation pathway. In order to determine whether thymic APC have this function in vivo, thymic APC were isolated from mice after i.v. injection of native OVA. We find that OVA is presented in association with MHC class I, but not class II, molecules in the thymus. This is in contrast to splenic APC, which present exogenous OVA with both class I and class II molecules under these conditions. Our findings have implications for the repertoire of self peptides that might be presented by thymic APC to developing T lymphocytes. PMID- 1727863 TI - Autoantibody to the alternative pathway C3/C5 convertase and its anti-idiotypic response. A study in affinity. AB - In an effort to understand the development and control of autoantibody production, we studied the affinity of autoantibody to the alternative pathway C3/C5 convertase (C3 nephritic factor (C3NeF)) and its autoanti-idiotypic antibodies, Ab2 alpha and Ab2 beta. These were isolated and purified from newborns, normal adults, and patients with membranoproliferative glomerulonephritis. In all cases, both IgG and IgM C3NeF were available for study. The affinity of IgG and IgM C3NeF for their natural Ag (10(8) liters/mol) as well as for the internal image of that Ag displayed on Ab2 beta was high (10(10) liters/mol). Furthermore, the affinity of IgG C3NeF was nearly 100-fold higher in patients than in newborns, whereas there were no significant changes with IgM C3NeF. By contrast, there were not differences in the affinity of IgG Ab2 alpha (which does not display any likeness to the native Ag) from normal adults and patients to any C3NeF isolate. There was, however, a progressive increase in affinity between both Ab2 alpha preparations and IgG C3NeF from newborns, adult normal subjects, and patients, implying an alteration in C3NeF to account for the changes in affinity. These data suggest that Ag-driven affinity maturation occurs with autoantibody but may not occur within the idiotypic network. These data also indicate that as autoantibody affinity matures, it appears to modify its idiotype, perhaps in an effort towards autoregulation. PMID- 1727864 TI - Ig V kappa family repertoire of plasma cells derived from autoimmune MRL mice. AB - V kappa gene family usage was determined in the resident in vivo-activated plasma cells of individual diseased MRL mice by using in situ hybridization. In this way, the entire autoimmune repertoire could be analyzed. Autoantibody levels and extent of glomerulonephritis were also measured, so that the severity of disease could be assessed. It was found that V kappa expression was highly variable from mouse to mouse. Some animals displayed a V kappa family repertoire similar to mitogen-stimulated cells and consistent with the size of the families. These animals tended to have lower disease indices. Other animals, which had higher disease indices, displayed considerable over- or underutilization of individual V kappa families. However, no particular V kappa families were repeatedly biased in their expression, as was found at the VH level with J558. Importantly, in the 10% of animals that expressed VH J558 exclusively, four or more V kappa families were expressed and multiple antiself specificities were produced. The data are most consistent with a number of J558 genes being expanded in a variety of self specificities. However, because only VH J558 is expressed in these sicker animals, nonspecific polyclonal activation is highly unlikely. These results underscore the continuing evolution of the autoimmune repertoire, with considerable diversity at early stages followed by a highly selected repertoire in which a potential role for nonspecific polyclonal activation is virtually excluded. PMID- 1727865 TI - T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. Evidence for different kinetics and recognition of a wide spectrum of protein antigens. AB - Recent evidence suggests the existence of at least two pathways of acquired specific resistance to Mycobacterium tuberculosis infection; the first consisting of cytokine-mediated activation of parasitized host cells by protective T cells, and the second involving the lysis of these cells by cytolytic T cells. Evidence presented in this report shows that both of the above mechanisms are operative in experimentally infected mice, but that they differ markedly in terms of their kinetics of emergence and loss. It was found that protective T cell activity was acquired very early during the course of the infection, and was temporally associated with the onset of bacterial elimination; however, cytolytic activity did not peak until 10 to 20 days later. This report shows further that the target Ag of these effector T cell populations were apparently numerous with no evidence for preferential recognition of a few immunodominant Ag. In view of the preponderance of target proteins in the bacterial filtrate, we present the hypothesis that such proteins secreted or otherwise leaked from the dividing mycobacterium are pinocytosed from the phagosome and used by the infected macrophage as the key protective Ag leading to T cell sensitization. This hypothesis thus explains the preferential requirement for the viable bacterium in the generation of specific resistance, and further explains why protective immunity is generated even while the organism is still multiplying in an apparently unrestrained manner. PMID- 1727866 TI - IL-6 induced by IL-1 inhibits malaria pre-erythrocytic stages but its secretion is down-regulated by the parasite. AB - The capacity of IL-6 to mediate the antiparasitic activity of IL-1 on intrahepatic development of malaria parasite was demonstrated. The comparisons of IL-6 levels in infected and noninfected hepatocyte cultures, either purified or enriched with nonparenchymal cells and stimulated by IL-1 or IL-6, indicate that subtle interactions exist between intrahepatocytic development of Plasmodium yoelii and liver synthesis of IL-6. During its intrahepatic multiplication, the parasite causes a decline in IL-6 production. IL-6 mRNA was not detected in the livers of infected mice during development of either hepatic or blood stage parasites although IL-6 activity was found in the sera during both stages. PMID- 1727867 TI - Protective immunization with invariant peptides of the Plasmodium falciparum antigen MSA2. AB - Three octapeptides from the N and C terminal C regions of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum elicit anti-MSA2 antibody when given as diphtheria toxoid conjugates. These antibodies also bind to the MSA2 homolog from the rodent malaria Plasmodium berghei. All mice vaccinated with these conjugates and challenged with an otherwise lethal inoculum of P. berghei showed substantial protection with most surviving. There was a inverse correlation between the development of the parasitemia and the antibody titer, with alum, algammulin, and CFA giving comparable results. These observations show that the conserved region of MSA2 could form the basis of a malaria vaccine when presented in a suitably immunogenic form, thus avoiding the problems of antigenic diversity [corrected]. PMID- 1727868 TI - A nontoxic, idiotope vaccine against gram-negative bacterial infections. AB - Experiments were performed to test the ability of mouse antiidiotopic mAb, specific for an antilipid A mAb, to act as a vaccine against gram-negative bacterial infections. Lipid A is a conserved region of bacterial LPS. Immunization with the antiidiotopic antibodies, coupled to an immunogenic carrier protein (hemocyanin), specifically induced anti-LPS antibody responses in animals from different species. In a mouse model, this immunization resulted in protection against both lethal gram-negative bacteremia and endotoxemia. The antiidiotopic antibodies, however, did not stimulate endotoxin-associated bioactivities, such as induction of TNF and IL-1. These results support the hypothesis that an idiotope vaccine can stimulate beneficial protective immunity against gram-negative infections without the toxicity inherent in LPS. PMID- 1727869 TI - Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production. AB - Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL 2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production. PMID- 1727870 TI - Polymorphism, recombination, and linkage disequilibrium within the HLA class II region. AB - Thirty-nine CEPH (Centre d'Etude du Polymorphisme Humain) families, comprised of 502 individuals, have been typed for the HLA class II genes DRB1, DQA1, DQB1, and DPB1 using nonradioactive sequence-specific oligonucleotide probes to analyze polymerase chain reaction amplified DNA. This population, which consists of 266 independent chromosomes, contains 27 DRB1, 7 DQA1, 12 DQB1, and 17 DPB1 alleles. Analysis of the distribution of allele frequencies using the homozygosity statistic, which gives an indication of past selection pressures, suggests that balancing selection has acted on the DRB1, DQA1, and DQB1 loci. The distribution of DPB1 alleles, however, suggests a different evolutionary past. Family data permits the estimation of recombination rates and the unambiguous assignment of haplotypes. No recombinants were found between DRB1, DQA1, and DQB1; however, recombinants were detected between DQB1 and DPB1, resulting in an estimated recombination fraction of greater than or equal to 0.008 +/- 0.004. Only 33 distinct DRB1-DQA1-DQB1 haplotypes were found in this population which illustrates the extreme nonrandom haplotypic association of alleles at these loci. A few of these haplotypes are unusual (previously unreported) for a Caucasian population and most likely result from past recombination events between the DR and DQ subregions. Examination of disequilibrium across the HLA region using these data and the available serologic HLA-A and HLA-B types of these samples shows that global disequilibrium between these loci declines with the recombination fraction, approaching statistic nonsignificance at the most distant interval, HLA-A to HLA-DP.DR-DQ haplotypes in linkage disequilibrium with DPB1 and B are noted and, finally, the evolutionary origin of certain class II haplotypes is addressed. PMID- 1727871 TI - Expression of a functional chimeric Ig-MHC class II protein. AB - We have generated a chimeric protein molecule composed of the alpha- and beta chains of the MHC class II I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E alpha- and beta-chains, respectively. Cells transfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated with anti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to the transfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class II protein with regard to correct chain assembly and transport to the cell surface; as well as 2) the Ag binding capacity of the antibody genes used. The generation of hybrid MHC class II molecules with highly specific, non-MHC-restricted binding capacities will be useful for studying MHC class II-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice. PMID- 1727872 TI - Protease inhibitors interfere with the transforming growth factor-beta-dependent but not the transforming growth factor-beta-independent pathway of tumor cell mediated immunosuppression. AB - Tumor cells have been reported to exert inhibitory effects on the activation of T lymphocytes in vitro. We show that the IL-2-stimulated proliferation of a Th cell line is suppressed when the T cells are cocultured with human glioblastoma and melanoma cell lines. The use of two Th cell clones that differ in their responsiveness to growth-inhibition by transforming growth factor-beta (TGF-beta) and the analysis of tumor cell-derived supernatants as well as of TGF-beta 1/TGF beta 2 gene expression allowed to distinguish two pathways of tumor-induced immunosuppression. Glioblastoma cells exert their immunosuppressive effects by producing biologically active TGF-beta 2, whereas the immunosuppressive state induced by melanoma cells is TGF-beta-independent and requires direct contact between tumor cell and T cell. The TGF-beta-dependent immunosuppression is down regulated by various protease inhibitors and up-regulated by estradiol via modulation of the production of biologically active TGF-beta 2 by glioblastoma cells leaving total activatable TGF-beta 2 unaffected. No such modulation is functional for the TGF-beta-independent pathway of immunosuppression. We conclude that the production of active TGF-beta by tumor cells is regulated at a posttranslational level by the coordinated action of several proteolytic enzymes. PMID- 1727873 TI - Anti-IgM but not anti-IgD antibodies inhibit cell division of normal human mature B cells. AB - Insolubilized anti-IgD antibody markedly increased DNA synthesis in and cell division of normal peripheral blood B cells (PBL-B) when used in combination with IL-4. Anti-IgM antibodies also induced DNA synthesis of PBL-B, but their ability to induce cell division was less than that of anti-IgD antibodies even when used in combination with IL-4. Moreover, anti-IgM antibodies inhibited cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4 without affecting DNA synthesis. Anti-IgM antibodies also inhibited Staphylococcus aureus Cowan I-induced cell division of PBL-B without affecting DNA synthesis. These results indicate that cross-linkage of surface IgM (sIgM) in mature B cells generates negative signals to inhibit cell division of mature B cells. Because anti-IgD antibodies did not inhibit cell division at all, the role of sIgD in the regulation of cell division of mature B cells may be quite different from that of sIgM. IFN-alpha/beta promoted cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4. They also counteracted the inhibitory effect of anti-IgM antibody on cell division of PBL-B. PMID- 1727874 TI - Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1). AB - We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used. PMID- 1727875 TI - Michael Heidelberger April 29, 1888-June 25, 1991. PMID- 1727876 TI - Endogenous loading of HLA-A2 molecules with an analog of the influenza virus matrix protein-derived peptide and its inhibition by an exogenous peptide antagonist. AB - Episomal plasmids (p8901) with minigenes coding for the influenza virus matrix peptide amino acids 57-68 (KGILGFVFTLTV; referred to as M57-68) or coding for a modified peptide were introduced into HLA-A2-positive target cells. The association of these peptides, synthesized in the cytoplasm, with HLA-A2 and the expression of this complex at the cell surface was evaluated with HLA-A2 restricted CTL specific for the influenza virus matrix peptide M57-68. Cells expressing M57-68 were lysed effectively, as were cells expressing a peptide that retained residues 60-64 with seven flanking alanine residues (AAALGFVFAAAA). An exogenously added synthetic analog of peptide M57-68 that inhibited sensitization of targets with synthetic peptide M57-68 also inhibited lysis of cells expressing the minigene coding for the peptide with seven alanine substitutions. These results demonstrate the utility of minigene DNA constructs in creating experimental systems to develop agents to diminish the severity of CTL-mediated tissue damage in autoimmune diseases and graft rejection. PMID- 1727877 TI - Secretion of IL-1: role of protein kinase C. AB - In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC. PMID- 1727878 TI - IL-7 is requisite for IL-1-induced thymocyte proliferation. Involvement of IL-7 in the synergistic effects of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor with IL-1. AB - In the absence of artificial comitogens murine thymocytes proliferate significantly in response to IL-1 at high but not at low cell densities. This observation has led us to examine a possible indirect mechanism requiring other thymocyte-growth factors, such as IL-2, IL-4, IL-6, and IL-7, in this phenomenon. Our data provide evidence that IL-7 is requisite for the IL-1-induced proliferative response because on the one hand the growth-promoting activity of IL-1 is completely inhibited by an anti-IL-7 mAb, and on the other hand IL-7 synergizes with IL-1 on thymocyte growth. This synergy is observed even at concentrations at which IL-7 is not detected in the pre-B cell proliferation assay, and results, at optimal doses, in TdR incorporation levels similar to those attained in response to IL-1 + IL-2. The anti-IL-7 mAb acts in a dose dependent manner and does not affect other activities of IL-1, such as its capacity to sustain the growth of the U373 astrocytoma cell line. It is also noteworthy that this mAb does not significantly impair thymocyte growth in response to IL-2 and that the growth-promoting activity of IL-1 is not affected by neutralizing mAb against IL-2, IL-4, and IL-6. In addition, we show that the potentiating effect of granulocyte-macrophage (GM)-CSF and TNF-alpha on IL-1 induced thymocyte growth is dependent on IL-7 because i) the anti-IL-7 mAb abrogates the respective synergistic interactions and ii) both factors potentiate the proliferative response to IL-7. Finally, depletion of thymocyte suspensions for Ia+ Mac-1+ accessory cells results in a considerable decrease in IL-1- and IL 1 + GM-CSF-induced TdR uptake, whereas IL-7-induced growth remains unchanged. Taken together, these results support the notion that, in the absence of artificial comitogens, thymocyte proliferation in response to IL-1 alone or in combination with GM-CSF is dependent on accessory cell-derived IL-7. PMID- 1727879 TI - Guidelines for the use of systemic glucocorticosteroids in the management of selected infections. Working Group on Steroid Use, Antimicrobial Agents Committee, Infectious Diseases Society of America. PMID- 1727880 TI - Quantitative analysis of human immunodeficiency virus type 1 antibody reactivity by western immunoblots: evaluation of relative antibody levels in seropositive individuals and mothers. AB - A quantitative analysis of antibody responses to human immunodeficiency virus type 1 (HIV-1) proteins using Western immunoblots and 125I-labeled protein A is reproducible and can be validated. The antibody levels obtained by Western immunoblots were compared with stoichiometric p24 radioimmunoassay over a wide range of antibody (correlation coefficient, .94; P less than .001). Antibody levels to gp160 and gp120 were validated using purified antigens. Analysis of antibody levels from 31 seropositive individuals revealed a statistically significant correlation between antibody levels to p24 and the other viral proteins except gp120. Anti-gag p24 antibody was strongly correlated with antibodies to other env products, specifically gp41 and gp160. Using the validated assay, HIV-1-infected mothers of infants were found to have highly variable levels of antibody to all viral proteins. Mothers of infected infants did not differ significantly from mothers of uninfected infants in antibody pattern or levels to any viral protein including gp120. PMID- 1727881 TI - Precore sequence variation in Chinese isolates of hepatitis B virus. AB - Direct sequencing of polymerase chain reaction-amplified serum hepatitis B virus (HBV) DNA was used to characterize the precore region of HBV from Chinese patients with chronic hepatitis. Two types of mutually exclusive variants were found in hepatitis B e antigen (HBeAg)-negative patients. The first (M1) contains a substitution from proline to serine at codon 15. A second group were infected with a previously described mutant (M2) containing a translational stop codon. HBeAg-positive patients were infected with the wild-type virus or the M1 containing strain. M2 emerged in patients with wild-type infection after seroconversion to anti-HBe, whereas M1 was present during the HBeAg-positive phase. In those with fluctuating HBe status, there was no correlation between prevailing HBe serology and sequence. There was an association between infection with variants and severe chronic hepatitis. Patients infected with strains containing M1 while HBeAg positive had a worse prognosis after seroconversion to anti-HBe. PMID- 1727882 TI - Randomized trial of meatal care with silver sulfadiazine cream for the prevention of catheter-associated bacteriuria. AB - A randomized, controlled, prospective clinical trial involving 696 hospitalized patients was undertaken to determine the effectiveness of 1% silver sulfadiazine cream applied twice daily to the urethral meatus in preventing transurethral catheter-associated bacteriuria. The overall incidence of bacteriuria was 11.4% (38/332) in the treated group and 13.2% (48/364) in the untreated group (P = .56; odds ratio, 0.85; 95% confidence interval, 0.53-1.37). Cox proportional hazards analysis identified female sex, lack of antibiotic use, and a positive initial meatal culture (but not treatment randomization or lack of urinemeter use) as independent variables associated with an increased risk of bacteriuria. Survival curve analysis of subgroups stratified by sex and antibiotic use failed to detect an effect of silver sulfadiazine on the rate of bacteriuria. Meatal care with silver sulfadiazine cream did not prevent the development of catheter-associated bacteriuria in short-term catheterized patients. PMID- 1727883 TI - Tissue culture-adherent Escherichia coli in infantile diarrhea. AB - To determine the association of tissue culture-adherent Escherichia coli with diarrhea, serotyped E. coli strains isolated in a yearlong case-control study of infantile diarrhea in Bangkok, Thailand, were examined for adherence to HeLa cells and for hybridization with the enteropathogenic E. coli adherence factor, the F1845, and the enteroaggregative E. coli (EAggEC) DNA probes. E. coli that adhered to HeLa cells in a localized adherence (LA) pattern (LA E. coli) was isolated from 26 of 509 infants with diarrhea (cases) and 9 of 509 age-matched controls (P = .006); E. coli with diffuse or aggregative adherence (DA or AA) to HeLa cells or that hybridized with the F1845 or EAggEC probes was not associated with infantile diarrhea. LA E. coli of classical enteropathogenic E. coli (EPEC) serotypes was isolated from 11 cases and 1 control (P = .003). EPEC O44:H18 that adhered to HeLa cells in a DA pattern and hybridized with the F1845 DNA probe was the predominant E. coli (five of five colonies tested) isolated from a 5-month old girl with diarrhea in whom no other enteric infections were identified. Although LA E. coli was highly associated with infantile diarrhea, the role of DA and AA E. coli was uncertain in this setting. PMID- 1727884 TI - Shigella and enteroinvasive Escherichia coli infections in households of children with dysentery in Bangkok. AB - Shigellae and enteroinvasive Escherichia coli (EIEC) were identified in children with dysentery and their household contacts in Bangkok. Shigellae were isolated from 49% and EIEC from 6% of 306 children with dysentery seen at the outpatient department of Children's Hospital on weekdays during January through June 1989 and October 1989 through October 1990. The same serotype infecting the index child was isolated from 21 (4%) of 522 household contacts of 151 index children with Shigella infections and from none of 60 household contacts of 19 index children with EIEC infections. Amplification of DNA sequences coding for the invasion-associated locus (ial) by polymerase chain reaction increased the identification of Shigella and EIEC infections from 57% (111/193) to 68% (132/193). ial sequences were identified in 3 of 20 drinking water specimens from which shigellae or EIEC were not isolated. Amplification of ial sequences identified more shigellae and EIEC than did bacteriologic and colony hybridization methods in children with dysentery and in drinking water in Bangkok. PMID- 1727885 TI - Nonspecific proctitis: association with human immunodeficiency virus infection in homosexual men. AB - In a cross-sectional study of 140 homosexual men attending a sexually transmissible diseases clinic, the association between the presence of antibody to the human immunodeficiency virus (HIV) and the presence of proctitis, as determined by histologic examination, as well as part or present exposure to other pathogens and details of sexual practices was analyzed. Significant associations with HIV seropositivity were found with the number of lifetime partners, positive treponemal serology, and evidence of previous infection with herpes simplex virus. However the major and unique finding was the strong and independent association between proctitis diagnosed by histologic criteria and seropositivity for HIV. Whether this is cause or effect awaits further elucidation. PMID- 1727886 TI - Respiratory syncytial virus infections in pediatric liver transplant recipients. AB - Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in infants and young children. The charts of 17 children found to have RSV among 493 children who underwent liver transplantation between February 1985 and February 1991 were reviewed. The median age at diagnosis was 20 months. Median time of diagnosis was 24 days after transplantation. Thirteen patients developed nosocomial infections while convalescing from their transplant. Common symptoms included tachypnea, cough, fever, and congestion. Acute radiographic changes were seen in 12 patients. Two deaths were associated with progressive pulmonary disease and occurred in children with infection early in the postoperative period who were intubated before the onset of symptoms. RSV in children after liver transplantation has a clinical spectrum similar to that in normal children. Early onset of infection (less than 20 days) after transplantation and preexisting lung disease may predict more severe disease. PMID- 1727887 TI - Adenovirus infection in pediatric liver transplant recipients. AB - A retrospective review of adenoviral infection in pediatric liver transplant recipients was done at Children's Hospital of Pittsburgh to define its epidemiology and clinical importance. Medical records of patients with adenovirus were reviewed and data collected regarding clinical course, microbiologic studies, biopsy results, immunosuppression, concurrent infections, and outcome. Of 484 liver transplant recipients, 49 had 53 episodes of adenoviral infection. The most common sites of adenoviral infection were the liver, lung, and gastrointestinal tract. Serotypes 1, 2, and 5 were recovered most often; type 5 was commonly associated with hepatitis. Invasive adenoviral infection occurred in 20 children, leading to death in 9. Median time from transplantation until isolation of adenovirus was 25.5 days. This timing suggests either reactivation or donor-associated transmission. Prospective studies using molecular epidemiologic techniques will be helpful in evaluating transmission patterns of adenovirus in this population. PMID- 1727888 TI - Disseminated Nocardia transvalensis infection: an unusual opportunistic pathogen in severely immunocompromised patients. AB - Nocardia infections are infrequently recognized in humans. Nocardia species may cause severe life-threatening infections among immunocompromised patients and have been reported to cause actinomycotic mycetomas, primarily in tropical areas. Two severely immunocompromised patients had disseminated N. transvalensis infections. One had underlying X-linked variant chronic granulomatous disease and died of disseminated N. transvalensis infection, which was diagnosed only at postmortem examination. The second patient developed N. transvalensis pneumonia within 3 months of undergoing renal transplantation and died of disseminated mixed Pseudallescheria boydii and N. transvalensis infections. Thus, N. transvalensis may cause invasive and potentially fatal pulmonary and disseminated infections. Accordingly, clinical microbiology laboratories should become proficient in identifying this uncommon aerobic actinomycete. PMID- 1727889 TI - Are strains of Mycobacterium avium more drug resistant when isolated from human immunodeficiency virus-infected patients? PMID- 1727890 TI - A second-generation hepatitis C virus confirmatory test for chronic hepatitis B virus infection. PMID- 1727891 TI - The antibody response to an oral Ty21a-based typhoid-cholera hybrid is unaffected by prior oral vaccination with Ty21a. PMID- 1727892 TI - Specific serotype of Campylobacter jejuni associated with Guillain-Barre syndrome. PMID- 1727893 TI - No association of chronic Chlamydia pneumoniae infection with chronic fatigue syndrome. PMID- 1727894 TI - The relationship between the mumps vaccine strain and parotitis after vaccination. PMID- 1727895 TI - Human monoclonal antibodies to glycolipid A that exhibit complement species specific effector functions. AB - Two human IgM monoclonal anti-glycolipid A antibodies (MAbs) were evaluated for their abilities to bind to various endotoxins and pathogenic gram-negative bacteria and to activate complement pathways, thereby accomplishing bactericidal and opsonic effector functions. Both MAbs cross-reacted with glycolipid A mutant lipopolysaccharides from rough colony-forming gram-negative bacteria and with selected endotoxins from smooth colony-forming bacteria. However, MAb 10235 bound to all clinical isolates of Escherichia coli and Klebsiella pneumoniae tested but only very weakly to Pseudomonas aeruginosa, whereas MAb 10058 bound to all three genera. Several strains of serum-insensitive organisms were selected for evaluation of antigen-specific, complement-mediated effector functions for the two MAbs. Assessment of bactericidal and opsonic activities showed that neither MAb was able to activate complement from nonprimate species (mouse, rat, rabbit, guinea pig, or sheep). However, both MAbs were highly effective in using primate sources of serum complement to mediate these effector functions. PMID- 1727896 TI - Antibacterial and protective properties of monoclonal antibodies reactive with Escherichia coli O111:B4 lipopolysaccharide: relation to antibody isotype and complement-fixing activity. AB - In vitro and in vivo antibacterial and protective properties of murine monoclonal antibodies (MAbs) to Escherichia coli O111:B4 lipopolysaccharide (LPS) were evaluated in relation to antibody isotype and complement-fixing activity. Six O side chain-specific MAbs, including two IgMs and one of each IgG subclass, were analyzed for quantitative binding and C3 deposition on intact bacteria, complement-mediated bactericidal and opsonophagocytic activity, and protection against intraperitoneal infections in mice. Although C3 was deposited on bacteria in the presence of normal human serum (NHS) alone, LPS-specific MAbs increased C3 attachment in a dose-dependent manner. Bacterial killing occurred only in the presence of both antibody and complement NHS and required an intact alternative pathway. The efficiency of bacterial killing varied by antibody isotype (IgM greater than IgG2a greater than other IgG subclasses) and correlated with C3 fixing capacity. Opsonophagocytic activity of MAbs exhibited a similar isotype related rank order. Likewise, IgM was more active than IgG, and IgG2a was superior to other IgG subclasses, in MAb-mediated protection against intraperitoneal infection. These data document the interdependent antibacterial and complement-fixing properties of LPS-reactive MAbs and the degree to which both activities are determined by antibody class and isotype. PMID- 1727897 TI - Resident colonic Escherichia coli strains frequently display uropathogenic characteristics. AB - Bacterial factors associated with long-term persistence in the colon have not been defined. Individual Escherichia coli strains in the colonic flora of 13 schoolgirls with asymptomatic bacteriuria were identified by electromorphic typing of chromosomally encoded enzymes and defined as resident or transient. The strains were characterized as to serotype, receptor specificity, and adherence to the human colonic epithelial cell line HT-29. Colonic resident strains expressed P fimbriae, adhered to colonic epithelial cells via a mannose-resistant mechanism, and expressed the uropathogenic serotypes O1, O2, O6, O7, O18, O25, or O75 more often than did the transient strains, which were often nontypeable. The serotype and hemagglutination pattern were generally retained during intestinal carriage, in contrast to the loss of such properties upon prolonged colonization of the urinary tract. P fimbriae with Gal alpha 1----4Gal beta-specific adherence may, in fact, have evolved to increase persistence in the colon. PMID- 1727898 TI - Macrophage-mediated killing of opsonized Treponema pallidum. AB - The ability of proteose peptone-induced normal rabbit peritoneal macrophages to kill Treponema pallidum subspecies pallidum in vitro is demonstrated. Treponemes and 10% heated immune or normal sera were incubated with macrophages at a ratio of 1:200. After 2-10 h of incubation, these mixtures were injected intradermally at duplicate sites on normal rabbits. Maximal killing (failure to develop lesions) was seen at 10 h of incubation with immune serum: Only 7% (1/14) of lesions developed compared with 90% (9/10) after incubation in the presence of normal serum (P less than .001). Maximal phagocytosis (detected by immunofluorescence) occurred by 8 h in the presence of immune serum, when 90% of macrophages had ingested treponemes. At this point, however, 70% of lesion sites from macrophages incubated with treponemes and immune serum still developed, suggesting that effective killing may require at least 2 h after phagocytosis. PMID- 1727899 TI - An animal model of Mycobacterium avium complex disseminated infection after colonization of the intestinal tract. AB - Mycobacterium avium complex infections occur in 30%-80% of patients with AIDS. Recent evidence supports the gastrointestinal tract as the source of M. avium. Although a reproducible animal model exists, a model more closely resembling the infection in AIDS patients is needed to answer pertinent questions regarding response to therapy and prophylaxis. Beige mice were infected orally (1 x 10(8) or 1 x 10(4) cfu, five doses), and consistent, reproducible disseminated infections after 4 and 8 weeks, respectively, were obtained. Bacteremia was observed in none to 70% of the animals depending on the strain used, and mortality ranged from none to 33%, also depending on the strain used. Concomitant ingestion of ethanol (4% of daily dietary calories) was associated with a significant increase in the number of viable bacteria recovered from liver, spleen, and appendix compared with animals not receiving ethanol. The orally infected animal model closely resembles M. avium infection in humans and may be important in investigating prophylaxis and therapy of this infection. PMID- 1727900 TI - Concurrent human immunodeficiency virus and mycobacterial infection of macrophages in vitro does not reveal any reciprocal effect. AB - To test whether in vitro infection of macrophages with either human immunodeficiency virus (HIV) or mycobacteria would influence the replication of the other pathogen, macrophages were infected sequentially with the macrophage tropic isolate HTLV-IIIBa-L/85 and Mycobacterium tuberculosis H37Rv or Mycobacterium avium. The intracellular growth of mycobacteria was measured by colony counting and radiometric assay of macrophage lysates and the replication of HIV by the release of p24 antigen into the culture supernatants. Phagocytosis and intracellular growth of mycobacteria was similar in HIV-infected macrophages and controls. Conversely, mycobacteria did not affect the replication of HIV in macrophages. These experiments failed to demonstrate any direct intracellular interaction between HIV and mycobacteria in cultured macrophages that would explain the increased rate of mycobacterial diseases in patients infected with HIV or that would support the hypothesis that mycobacterial infection of macrophages per se can enhance HIV replication in these cells. PMID- 1727901 TI - Prevalence of human immunodeficiency virus infection among patients attending tuberculosis clinics in the United States. AB - In 1988-1989, surveillance for human immunodeficiency virus (HIV) infection was conducted in 20 clinics providing medical care for patients with suspected and confirmed tuberculosis (TB) in 14 cities. A total of 3077 specimens from consecutive patients were tested for HIV after patient identifiers were removed. The median clinic seroprevalence rate was 3.4%, (range, 0-46.3%). The highest rates were found in the Northeast and Atlantic coastal areas. Rates by clinic were highest for persons born in the United States (median, 11.2%) and in the Caribbean region (Haitians, 36%-40%, and Cubans, 16%). Most HIV-infected patients had pulmonary TB, but HIV infections were more frequent in patients with extrapulmonary TB than in pulmonary TB patients (19.8% vs. 10.2%, P less than .0002). For US-born patients, rates did not differ by race or sex. These serosurveillance data indicate widespread HIV infection among TB patients and have important implications for clinical management of TB patients and for TB and AIDS prevention programs. Testing all HIV-infected persons and all TB patients for dual infection is essential to control the interrelated epidemics of AIDS and TB. PMID- 1727902 TI - Clinical pharmacology of 2',3'-dideoxyinosine in human immunodeficiency virus infected children. AB - The pharmacokinetics of intravenous and oral 2',3'-dideoxyinosine (ddI) and the relationships between pharmacokinetic parameters and measures of response were studied in 48 human immunodeficiency virus-infected children. Disappearance of ddI from plasma after the intravenous dose was rapid and biexponential, with half lives of 12 min and 1.0 h and a total clearance of 510 +/- 180 ml/min/m2. After oral administration, ddI absorption was limited and variable (mean bioavailability, 19% +/- 17%). A plasma ddI concentration-response relationship was observed for both decline in viral p24 antigen levels and improvement in intelligence quotient score. A limited sampling model was developed that accurately predicts the area under the ddI plasma concentration-time curve from one to three plasma samples. Although this pharmacokinetic study was done in children, the results also have relevance to adults and suggest that individualization of dose and schedule through therapeutic drug monitoring may be necessary to achieve optimal response. PMID- 1727903 TI - A prospective, randomized, double-blind study of trimethoprim-sulfamethoxazole for prophylaxis of infection in renal transplantation. Side effects of trimethoprim-sulfamethoxazole, interaction with cyclosporine. AB - Questions have been raised regarding the safety of trimethoprim-sulfamethoxazole (TMP-SMZ) in organ transplantation, particularly adverse interactions with azathioprine and cyclosporine. In a prospective randomized, double-blind, trial in 132 patients that encompassed 33,876 patient-days, long-term prophylaxis with TMP-SMZ was found to significantly reduce the incidence of bacterial infection after renal transplantation. Prophylaxis was very well tolerated; none of the 66 recipients of TMP-SMZ, who took the drug for an average of 8.9 months, was withdrawn from the study because of hypersensitivity or toxic side effects. Serial measurements of hematologic parameters and liver function tests after transplantation in the two groups showed no significant differences. Recipients of cadaveric transplants, who were all given cyclosporine, randomized to receive TMP-SMZ had serum creatinine levels approximately 15% higher than those in control patients receiving cyclosporine (p less than 0.01); comparison of renal function by 24-hour endogenous creatinine clearances and technetium 99m-labeled diethylenetriamine-penta-acetic acid glomerular filtration rates in 17 patients crossed over to the alternate treatment group for 7 weeks, however, shows that the observed differences are reversible and represent inhibition of tubular excretion of creatinine by TMP in the presence of cyclosporine. Prophylaxis with TMP-SMZ had no discernable effect on cyclosporine pharmacokinetics: recipients of TMP-SMZ had blood levels of cyclosporine similar to those in patients in the placebo group. Episodes of graft rejection occurred at a similar frequency in the two groups (placebo, 50; TMP-SMZ, 44). We conclude that long-term prophylaxis with TMP-SMZ does not produce discernable hematologic, renal, or hepatic toxicity in renal transplant recipients nor does it augment nephrotoxicity with cyclosporine or increase the risk of rejection. TMP-SMZ may be used safely and is highly cost-beneficial for prophylaxis of infection in renal transplantation. PMID- 1727904 TI - Acute effects of indomethacin on the disposition of a potassium load. AB - We examined the effects of acute indomethacin administration on the disposition of a potassium load in anesthetized rats. In response to the potassium load, indomethacin-treated animals had greater plasma potassium concentrations and smaller increases in fractional excretion of potassium than did vehicle-treated rats, but there was no change in urine flow rate. Findings were consistent with indomethacin-induced impairment of renal potassium excretion. The effects of indomethacin in adrenalectomized animals were comparable to those that were observed in intact rats, which indicates that inhibition of aldosterone release was not responsible for the acute effects of indomethacin. No differences in plasma potassium were noted after indomethacin or vehicle infusion in the animals that underwent bilateral ureteral ligation, which suggests that indomethacin did not impair extrarenal potassium disposition. These results indicate that acute administration of indomethacin impairs the response to a potassium load, not as a result of inhibition of aldosterone secretion or extrarenal potassium distribution but by means of inhibition of renal potassium excretion. PMID- 1727906 TI - Aging alters ornithine decarboxylase and decreases polyamines in regenerating rat liver but putrescine replacement has no effect. AB - Aging decreases rat liver regeneration. We (1) compared the expression of ornithine decarboxylase (ODC), a critical enzyme for liver regeneration, and polyamine levels in regenerating liver of 6-week-old and 1-year-old rats and (2) evaluated the effect of exogenous putrescine supplementation on liver regeneration in 1-year-old rats. ODC messenger ribonucleic acid (mRNA) transcript sizes were the same in rats of both ages. ODC mRNA content and enzyme activity were higher in the younger rats; however, magnitudes of increase after partial hepatectomy were greater in the older rats. From peak levels, the rate of decline of the mRNA was slower in the older rats, but enzyme activity declined at the same rate in both ages. ODC apoenzyme content was significantly less in normal liver tissue from 1-year-old rats, but there was little change after partial hepatectomy in rats of either age. No change in ODC transcriptional activity was found. Hepatic putrescine levels were lower in 48 hours regenerating liver tissue from 1-year-old rats. To determine whether supplemental putrescine would increase liver regeneration in 1-year-old rats, putrescine (600 mumol/kg IP every 4 hours) was administered beginning 4 days before or at the time of partial hepatectomy. This raised polyamine levels and decreased ODC activity significantly, but there was no change in regenerating liver weight, total DNA and RNA content, and tritiated thymidine incorporation at 48 hours. These results indicate that ODC expression is different and polyamine levels are lower in 1-year-old rats than in 6-week-old rats. However, putrescine supplementation that is sufficient to decrease ODC activity has no apparent effect on regeneration. PMID- 1727905 TI - Ethanol inhibits production of messenger ribonucleic acid for kappa-chain in stimulated B lymphocytes. AB - Alcohol consumption can adversely affect an individual's response to infection. We have shown that alcohol has a direct suppressive effect on numbers of antibody secreting cells in antigen-activated B lymphocytes but does not suppress the early membrane and intracellular events that are associated with binding of antigens to specific receptors. The studies reported here were designed to determine whether alcohol inhibits immunoglobulin synthesis. When 150 mg/dl ethanol was added to anti-mu-stimulated purified B cells, proliferation was inhibited. Similar exposure to ethanol inhibited production of messenger RNA for kappa chain in anti-mu-stimulated B cells but did not affect total RNA production or messenger RNA for beta-actin. Thus alcohol inhibits both the proliferation of antigen-activated B lymphocytes and their synthesis of immunoglobulin. PMID- 1727907 TI - Production of anti-P1A monoclonal antibodies. AB - Two peptides corresponding to the sequence of platelet glycoprotein IIIa between serine 27 and arginine 37 were synthesized and used to produce monoclonal antibodies. These two synthetic peptides were identical except for a single substitution at position 33, where a Pro/Leu polymorphism was shown to occur in human platelets and was predicted to be responsible for the P1A1-P1A2 alloantigen system (Newman et al., J. Clin Invest 1989:83:1778-81). Two monoclonal antibodies named 3C1 for the anti-"P1A1 peptide" and AD3 for the anti-"P1A2 peptide" were characterized. These monoclonal antibodies interacted with the two allelic forms of the reduced glycoprotein IIIa. They were used to type P1A1 and P1A2 homozygote as well as heterozygote platelets. Thus these two immunoprobes confirm that the Pro-Leu substitution is associated with the P1A1-P1A2 alloantigenic system. Although they interact only with reduced glycoprotein IIIa, these antibodies can be used to design simple tests for the typing of the P1A status of patients. PMID- 1727908 TI - Sexual differences in lipoprotein composition in a family with dyslipidemic hypertension with premature atheroschlerosis: deficiency of high-density lipoprotein-L and high-density lipoprotein-M "apolipoprotein-I alone" particle. AB - This article describes a family with a high incidence of premature atherosclerosis and primary hypertriglyceridemia in the women. The lipoprotein composition of this family was investigated with a new methodology that combines gradient ultracentrifugation to isolate lipoprotein subfractions with high performance liquid chromatography to quantitate apolipoproteins. The major lipoprotein abnormalities that were identified in the hyperlipidemic women in this family were (1) an increased mass of very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) with triglyceriderich VLDL but normal IDL composition; (2) triglyceride-rich low-density lipoprotein (LDL) with normal cholesterol and apolipoprotein B concentrations; (3) a relatively normal total mass of high-density lipoprotein (HDL)-L and HDL-M but with a reduction in the apolipoprotein A-I/A-II ratio and a decrease in the cholesterol to triglyceride ratio; (4) an elevation of HDL-D apolipoprotein A-I. The reduction in the apolipoprotein A-I/A-II ratio was also seen in the hyperlipidemic men and in most of nonhyperlipidemic family members and was the most common lipoprotein abnormality that was identified in this family (9 of 11 family members who were not on lipid-lowering medications were affected). The hypertriglyceridemic women appeared to have an increase in the "A-I + A-II" HDL particles in all subfractions and an increase in the "A-I alone" particles in HDL-D. These increases provided the apparently normal total mass of HDL that was observed in these women. These increases in HDL were not seen in the hypertriglyceridemic men. We conclude that a deficiency of the "A-I alone" particle in HDL-L and HDL-M may contribute to the premature atherosclerosis that was seen in this family and that it appears to precede the appearance of hypertriglyceridemia. The increase in the "A-I + A-II" HDL particles did not appear to provide the same protection as would be expected from "A-I alone" HDL. PMID- 1727909 TI - Plasmapheresis for hyperviscosity syndrome in macroglobulinemia Waldenstrom and multiple myeloma: influence on blood rheology and the microcirculation. AB - The efficacy of plasmapheresis in improving blood flow properties in patients with hyperviscosity syndrome was studied during 22 plasmapheresis treatments in four patients with hyperviscosity syndrome (three with macroglobulinemia Waldenstrom, one with multiple myeloma). Immediately before and after plasmapheresis (exchange volume 3 liters) the following parameters were determined: standard hematologic parameters, serum proteins, plasma viscosity, whole blood viscosity, and blood flow velocity in finger nailfold capillaries by video microscopy. The hematocrit remained unchanged. Paraprotein concentrations were markedly reduced by plasmapheresis (average 35%). Plasma viscosity fell from 5.0 +/- 3.3 cp to 2.1 +/- 1.0 cp (p less than 0.0001, normal range 1.1 to 1.4 cp). Whole blood viscosity changed accordingly. The plasma viscosity before plasmapheresis (x) determined the drop in viscosity after plasmapheresis, according to the following regression: y = 0.97 - 0.77 x; r = 0.962, p less than 0.001. The spontaneous capillary blood cell flow velocity increased from 0.33 +/- 0.14 mm/sec to 0.55 +/- 0.21 mm/sec (p less than 0.01) and the change in spontaneous flow velocity (y) was correlated with the change in plasma viscosity (x): y = 0.02 - 0.05 x; r = 0.833, n = 7, p less than 0.05. We conclude that plasma viscosity is a major determinant of capillary blood flow and that plasmapheresis is an efficient treatment of abnormal microcirculation caused by increased plasma viscosity. Our data make it possible to predict the benefit of plasmapheresis in a given situation and contribute to a better use of this valuable method. PMID- 1727910 TI - Sensitive antigenic determinations of high molecular weight kininogen performed by covalent coupling of capture antibody. AB - Knowledge of the role of high-molecular-weight kininogen (HK) in disease states has been limited by the lack of easy, reliable, and rapid assays for HK antigen. We have developed two immunochemical determinations, which use covalent coupling of the capture antibody, that will greatly facilitate the measurement of HK in plasma and cells. The first is an enzyme-linked immunosorbent assay (ELISA) that is performed in 96-well microplates and can be prepared up to 5 months in advance; it can, therefore, be available when needed. The second is a very rapid method that uses the technique known as particle concentration fluorescence immunoassay (PCFIA) and can be performed on 96 samples in less than 30 minutes. The HK-ELISA correlated well with HK coagulant activity in 22 normal donors (r = 0.88). The interassay coefficient of variation (CV) of 22 samples assayed on 5 separate days was 12%, whereas the interassay CV was 4%. The HK-PCFIA demonstrated an excellent correlation (r = 0.97) with HK coagulant activity in 22 normal donors, with an interassay CV of 7% and an intraassay CV of 1.5%. The HK ELISA was linear between 6 and 80 ng/ml, whereas the HK-PCFIA was linear between 5 and 800 ng/ml. Using the HK-PCFIA, we found a difference in HK antigen levels between two groups of patients with sepsis. The mean of those who survived (n = 10) was 70 micrograms/ml, whereas the mean of those who died (n = 6) was 48 micrograms/ml. Both HK-ELISA and HK-PCFIA were able to detect HK antigen in baboon plasma, enabling the monitoring of HK during experimental protocols. The HK-PCFIA was sensitive enough to detect HK antigen in the supernatant of hepatoma G2 at levels of 1 to 4 ng/ml. These assays should enable convenient and frequent measurement of HK antigen in various physiologic and pathophysiologic states, as well as in cell fractions. PMID- 1727911 TI - Osteosarcoma: good news despite crude tools. PMID- 1727912 TI - A phase II study of high-dose cyclophosphamide, thiotepa, and carboplatin with autologous marrow support in women with measurable advanced breast cancer responding to standard-dose therapy. AB - PURPOSE: The study was designed to determine the duration of complete response (CR) for patients with unresectable or metastatic breast cancer treated with high dose cyclophosphamide, thiotepa, and carboplatin (CTCb) while responding to conventional-dose therapy. METHODS: Eligibility criteria included histologically documented metastatic or unresectable breast cancer, at least a partial response (PR) to conventional-dose therapy, no prior pelvic radiotherapy, cumulative doxorubicin of less than 500 mg/m3, and physiologic age between 18 and 55 years. Patients with inadequate renal, hepatic, pulmonary, and/or cardiac function or tumor involvement of marrow or CNS were excluded. Cyclophosphamide 6,000 mg/m2, thiotepa 500 mg/m2, and carboplatin 800 mg/m2 were given by continuous infusion over 4 days. After recovery, sites of prior bulk disease were to be radiated or resected if feasible. RESULTS: Of 29 registered patients, one died of toxicity (3%; hemorrhage). CRs and PRs continued a median of 16 and 5 months after transplant, respectively (26 and 9 months from initiation of chemotherapy for metastatic disease). Of 10 patients transplanted in CR, four have not progressed at 17 to 31 months after transplantation (25 to 43 months after beginning standard-dose therapy). One of four patients with uptake on bone scan as their only sites of residual disease before transplant and one of three who converted from PR to CR with transplant have not progressed at 27 and 29 months, respectively, after transplant. CONCLUSIONS: CTCb is an intensification regimen with a low mortality that delivers a significantly increased dose of agents with known activity at conventional doses in breast cancer. Although the duration of PR is short as expected, CRs appear to be durable. PMID- 1727913 TI - ICRF-187 permits longer treatment with doxorubicin in women with breast cancer. AB - PURPOSE: To test potential protection by ICRF-187 against cumulative doxorubicin dose-related cardiac toxicity, we conducted a randomized clinical trial in 150 women with advanced breast cancer. PATIENTS AND METHODS: Patients received fluorouracil (5FU) 500 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide 500 mg/m2 every 21 days intravenously (IV) (control regimen, 74 patients), or the same regimen preceded by ICRF-187 1,000 mg/m2 IV (experimental regimen, 76 patients). RESULTS: We previously reported that ICRF-187 in this dose and schedule provides cardiac protection and does not substantially alter the noncardiac toxicity or antitumor efficacy of the control regimen. In this updated analysis of the entire patient cohort, we provide additional support for these findings and demonstrate that patients in the ICRF-187 group received more cycles (median, 11) and higher cumulative doses (median, 500 mg/m2) of doxorubicin than patients in the control group (median, nine cycles, P less than .01; and 441 mg/m2, P less than .05). Twenty-six patients in the ICRF-187 group received doxorubicin doses of at least 700 mg/m2, and among them, 11 patients received 1,000 mg/m2 or more. Only three patients in the control group received doxorubicin doses of 700 mg/m2; the maximum dose administered to one patient in this group was 950 mg/m2. ICRF-187 cardiac protection was demonstrated by difference in incidence of clinical congestive heart failure (CHF; two patients in the ICRF-187 group v 20 in the control group; P less than .0001) and by differences in resting left ventricular ejection fraction (LVEF) determined by multigated radionuclide (MUGA) scan from baselines and that required patient removal from study (five patients in the ICRF-187 group had a decrease in LVEF to less than 0.45 or a decrease from the baseline LVEF of 0.20 or more v 32 in the control group; P less than .000001). Among the 30 patients who had an assessable endomyocardial biopsy at cumulative doxorubicin 450 mg/m2, none of 16 in the ICRF 187 group and six of 14 in the control group had a score of 2 (P less than .05). ICRF-187 cardiac protection was observed in patients with and without prior chest wall radiation or other risk factors for developing doxorubicin cardiac toxicity. CONCLUSION: By protecting against cumulative doxorubicin-induced cardiac toxicity, ICRF-187 permits significantly greater doses of doxorubicin to be administered to patients with greater safety. PMID- 1727914 TI - A method of predicting adult height and obesity in long-term survivors of childhood acute lymphoblastic leukemia. AB - PURPOSE: Short stature and obesity have been reported among long-term survivors of childhood acute lymphocytic leukemia (ALL). We examined factors that contribute to these adverse sequelae. PATIENTS AND METHODS: Serial height and weight measurements were analyzed for 91 long-term survivors who were treated for ALL between 1967 and 1975 at a single institution. These patients were all younger than 12 years at diagnosis, were in continuous complete remission, had reached final height, and had height and weight measurements within 1 year of age 18 years. They had received craniospinal (n = 33) or cranial irradiation (n = 58) to total doses of 24 Gy as CNS prophylaxis. Standard deviation scores (SDS) were used to reflect the deviation of height and weight measurements from population means, and the body mass index (BMI; weight divided by height squared) was used in assessing obesity at age 18 years. RESULTS: Short stature (less than fifth percentile) was seen in 41 patients (45%), and obesity (BMI greater than or equal to 24 kg/m2) in 35 (38%). Regression formulae were developed that explain 65% and 62% of the variability in patient height and BMI, respectively. CONCLUSIONS: Risk factors were identified for abnormally short stature, which was defined to be a decrease of 1.5 SDS in height from diagnosis to age 18 years. These factors include younger age and above-average height for age at diagnosis (height SDS greater than 0), craniospinal irradiation, and greater decrease in height SDS during antileukemic therapy. Risk factors for obesity at age 18 years include weight SDS greater than 0 and greater than height SDS at 1 year after the end of chemotherapy. PMID- 1727915 TI - Comparative study of pamidronate disodium and etidronate disodium in the treatment of cancer-related hypercalcemia. AB - PURPOSE: This multicenter, double-blind, randomized trial was performed to determine the efficacy and safety of pamidronate disodium (APD) in comparison to etidronate disodium (EHDP) in the treatment of cancer-related hypercalcemia. PATIENTS AND METHODS: Sixty-five male and female adult patients with cancer and corrected calcium levels of greater than or equal to 12.0 mg/dL after 24 hours of hydration were randomized to receive either 60 mg APD given as a single 24-hour infusion or 7.5 mg/kg EHDP given as a 2-hour infusion daily for 3 days. RESULTS: APD normalized corrected calcium levels in 70% (21 of 30) of patients, whereas EHDP did so in 41% (14 of 34) of patients (P = .026). The mean corrected serum calcium level decreased from 14.6 to 10.5 mg/dL in the APD-treated group and from 13.8 to 11.6 mg/dL in the EHDP-treated group within the first week of treatment. There was no difference in response to APD in patients without versus those with bone metastases (78% v 67%). Both drugs were well tolerated. CONCLUSION: This study demonstrated that a single 60-mg infusion of APD is safe and more effective than EHDP given at the dose of 7.5 mg/kg for 3 days in the treatment of cancer related hypercalcemia. PMID- 1727916 TI - Phase I/II trial and pharmacokinetics of intrathecal diaziquone in refractory meningeal malignancies. AB - PURPOSE: Because there is a compelling need to develop new agents for intrathecal use, we investigated the safety, efficacy, and CSF pharmacokinetics of diaziquone (AZQ) following intrathecal administration in patients with refractory meningeal malignancies. PATIENTS AND METHODS: Thirty-nine patients received 45 courses of intrathecal AZQ. Two schedules were studied; twice-weekly administration of a 1- or 2-mg dose and "concentration times time" (C x T) administration of 0.5 mg every 6 hours for three doses, administered once weekly. RESULTS: Dose-limiting toxicity consisting of headache, nausea, or vomiting occurred in only three patients and only at the 2-mg, twice weekly dose. The schedules of 1 mg twice weekly and 0.5 mg every 6 hours for three doses were well tolerated. Thirty-seven courses were assessable for response. The overall response rate was 62%. Complete responses (CRs) occurred in 14 of 37 courses (38%) and partial responses (PRs) occurred in nine of 37 courses (24%). Among patients with meningeal leukemia, CRs were observed in 11 of 26 courses (42%) and PRs in nine of 26 courses (35%). There was no difference in response rate related to dose or schedule. The pharmacokinetic behavior of intrathecally administered AZQ was characterized by biexponential disappearance from ventricular CSF, with mean half-lives of 18.2 and 78.6 minutes. The mean clearance rate was 0.37 mL/min. CONCLUSION: Intrathecal AZQ is safe, well tolerated, and highly active against refractory meningeal malignancies. PMID- 1727917 TI - Current attitudes and practice of American Society of Clinical Oncology-member clinical oncologists regarding cancer prevention and control. AB - PURPOSE AND METHODS: A nationwide needs assessment survey including a validated Cancer Prevention and Early Detection Attitude Inventory of 1,500 randomly selected American Society of Clinical Oncology (ASCO)-member clinical oncologists was conducted via a 67-item, mailed questionnaire to assess practice and attitudes regarding cancer prevention and control. RESULTS: Responses of 729 physicians from 48 states representing medical (57%), radiation (17%), surgical (16%), and pediatric oncology (6%), and hematology/other (4%) fields were obtained. Except for ambivalence regarding an important role for diet in cancer causation, cancer prevention and control recommendations were widely endorsed despite skepticism about their impact on reducing deaths from cancer. Surprisingly, a significantly (P less than .001) more favorable attitude for cancer prevention and control issues was found in physicians with greater than 20 years practice compared with younger oncology colleagues, as measured by a 22 item Cancer Prevention and Early Detection Attitude Inventory. Among all physicians, participation in cancer therapy trials exceeded that in cancer prevention and control trials (91% v 27%, P less than .01). Formal instruction during postgraduate training in cancer screening (34%) or prevention (23%) was received by few oncologists; nonetheless, 69% considered themselves a resource for cancer prevention and control issues in their practice communities. Of potential barriers to cancer prevention and control activity, only lack of patients without cancer (53%) and difficulty in including such activity economically into clinical practice (65%) were majority selections. Importantly, 64% agreed they could "motivate their patients to change lifestyle to reduce cancer risk." CONCLUSION: Clinical oncologists may represent a potential resource for implementation of cancer prevention and control objectives if economically feasible models for their use in practice settings can be identified. PMID- 1727918 TI - Amsacrine retains a special role supporting Food and Drug Administration approval. PMID- 1727919 TI - Cytarabine and CNS toxicity. PMID- 1727920 TI - Lymphomatoid papulosis: response to treatment with recombinant interferon alfa 2b. PMID- 1727922 TI - Camptothecins. PMID- 1727921 TI - Activity of fludarabine in previously treated non-Hodgkin's low-grade lymphoma: results of an Eastern Cooperative Oncology Group study. AB - PURPOSE: Fludarabine (2-fluoro-arabanoside-monophosphate) is a new antimetabolite chemotherapeutic agent. We performed a multicenter, phase II study of this drug in previously treated patients with refractory or relapsed non-Hodgkin's lymphoma (NHL) to determine its response rate by histologic classification. PATIENTS AND METHODS: Sixty-two assessable patients were given 18 mg/m2 by intravenous (IV) bolus injection daily for 5 days, every 28 days. Forty-eight percent had previously had one chemotherapy regimen, and the remainder had had two regimens; 42% had had radiation. RESULTS: Patients received 273 cycles of fludarabine chemotherapy, with a median of two cycles and ranging up to 25 cycles. Sixty patients were assessable for response, including nine complete responses (CRs; 15%) and nine partial responses (PRs; 15%). The response rate for patients with lower-grade histology was 52% (13 of 25); the greatest response rate was seen in those with follicular small cleaved-cell lymphoma, including seven of 11 treated. Five responders remain in unmaintained remission; the median survival of responders is greater than 30 months. Toxicity included mild neutropenia and a 10% incidence of grade 3 neurologic toxicity with occasional reversible visual and auditory changes. CONCLUSION: Fludarabine is active in patients with previously treated NHL (particularly low-grade histologies). Future studies will examine its activity in combination with other chemotherapeutic agents in previously untreated patients. PMID- 1727923 TI - The use of interleukin-2 and lymphokine-activated killer cells for the treatment of patients with non-Hodgkin's lymphoma. AB - PURPOSE: The study was undertaken to assess whether immunotherapy regimens with bolus high-dose interleukin-2 (IL-2) alone or with lymphokine-activated killer (LAK) cells are active in previously treated, relapsed patients with non Hodgkin's lymphoma. PATIENTS AND METHODS: Nineteen patients with low- or intermediate-grade lymphomas were treated with bolus high-dose IL-2 alone (11 patients) or IL-2 with LAK cells (eight patients). IL-2 was administered by intravenous bolus infusion at 720,000 IU/kg every 8 hours. Eight patients had low grade histologies; 11 patients were intermediate-grade. Eighteen patients had received second- or third-generation combination chemotherapy, and eight had also received radiation. All 19 relapsed after a median of two chemotherapy regimens. RESULTS: Four responses were observed, three partial and one complete, in patients with follicular histologies who received IL-2 with LAK cells. Response durations were 10, 16, 16, and 26 months, and three responders were re-treated after relapse with subsequent disease control for an additional 16, 39+, and 2+ months, respectively. CONCLUSION: High-dose, bolus IL-2-based immunotherapy with LAK cells may be an effective treatment for patients with non-Hodgkin's lymphoma and merits further testing with larger numbers of patients in phase II trials. PMID- 1727924 TI - Bone marrow transplantation versus high-dose cytarabine-based consolidation chemotherapy for acute myelogenous leukemia in first remission. AB - PURPOSE: Despite substantial progress in the treatment of acute myeloid leukemia (AML), fewer than 25% of patients survive free of leukemia for more than 5 years without allogeneic bone marrow transplantation (BMT). In this study we analyzed the results of one or more cycles of high-dose cytarabine-based consolidation chemotherapy as compared with allogeneic BMT in first remission. PATIENTS AND METHODS: The results in 28 adult patients, aged 16 to 45 years, who underwent a closely HLA-matched BMT for AML in first remission were compared with those in 54 consecutive, age-matched, adult patients treated with one or more cycles of high dose, cytarabine-based consolidation chemotherapy. RESULTS: After a median follow up of 4 years, the actuarial risk of leukemic relapse was considerably lower in the transplant group than in the group treated with consolidation chemotherapy (32% +/- 26% v 60% +/- 14%; P = .05). Treatment-related mortality, however, was much higher in the group treated with BMT (32% v 6%, P = .002). The actuarial disease-free survival at 5 years was not significantly different for the two groups (45% +/- 24% v 38% +/- 14%). CONCLUSIONS: Our results show that BMT in first remission AML did not offer a disease-free survival advantage over intensive postremission consolidation chemotherapy. Larger studies are needed to identify patients who might benefit most from BMT. PMID- 1727925 TI - High-risk multiple myeloma treated with high-dose melphalan. AB - PURPOSE: This study was undertaken to evaluate the efficacy and toxicity of high dose melphalan (HDM) 140 mg/m2 in poor-risk multiple myeloma (MM). PATIENTS AND METHODS: Thirteen patients were previously untreated, and 13 had been pretreated with vincristine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), and dexamethasone (VAD) for refractory or relapsed MM. RESULTS: All 11 fully assessed, untreated patients responded, and six achieved a complete response. Remissions were of excellent quality, but response duration--a median of 16 months--was short. This was probably due to the high incidence of unfavorable prognostic signs, like a high beta 2-microglobulin (B2M) and/or a high plasma cell labeling index (LI). None of the nine pretreated patients with a measurable M component had more than 50% reduction of M component after HDM, indicating that intensive treatment has no effect on a residual tumor population. The relapse free period after HDM in this group of patients (median, 9 months) was not better than in a historical control group of patients treated with VAD alone. The major complications due to the prolonged myelosuppression were severe infections. After primary HDM, median time to recovery to greater than 0.5 x 16(9) granulocytes was 30 days; in previously treated patients, the recovery period was even longer. There were three toxic deaths. Fulminant relapses with features of J-chain disease were frequently observed, indicating a dedifferentiated tumor, probably induced or selected by the HDM. CONCLUSIONS: HDM is an effective treatment resulting in good remissions for untreated MM. However, other therapy strategies should be explored first, focusing on the reduction of toxicity and prolongation of the relapse-free period, before HDM can be recommended as first-line treatment for the younger MM patient. PMID- 1727926 TI - High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. AB - PURPOSE: To determine the toxicity and the therapeutic efficacy of the combination of the recombinant tumor necrosis factor alpha (rTNF alpha), recombinant interferon gamma (rIFN-gamma), and melphalan, we designed a protocol using isolation limb perfusion (ILP) with hyperthermia for in-transit metastases of melanoma and recurrent sarcoma. The triple combination was chosen because of the reported synergistic antitumor effect of rTNF alpha with IFN-gamma and of rTNF alpha with alkylating agents. PATIENTS AND METHODS: Twenty-three patients received a total of 25 ILPs with the triple combination. There were 19 females and four males with either multiple progressive in-transit melanoma metastases of the extremities (stage IIIa or IIIab; 19 patients) or recurrent soft tissue sarcoma (five). The rTNF alpha was injected as a bolus in the arterial line, and total dose ranged between 2 and 4 mg, under hyperthermic conditions (40 degrees C to 40.5 degrees C) for 90 minutes. The rIFN-gamma was given subcutaneously (SC) on days -2 and -1 and in the perfusate, with rTNF alpha at the dose of 0.2 mg. Melphalan (Alkeran; Burroughs Wellcome Co, London, England) was administered in the perfusate at 40 micrograms/mL. RESULTS: Toxicity observed during three ILPs in a pilot study with rTNF alpha included only two severe toxicities: one severe hypotension with tachycardia and transient oliguria and one moderate hypotension for 4 hours followed by severe kidney failure with complete recovery on day 29. In all 18 ILPs performed in the triple combination protocol, the patients received continuous infusion dopamine at 3 micrograms/kg/min from the start of ILP and for 72 hours and showed only mild hypotension and transient chills and temperature. Regional toxicity attributable to rTNF alpha was minimal. There have been 11 cases with hematologic toxicity consisting of neutropenia (one grade 4 and one grade 3) and neutropenia with thrombocytopenia (one grade 4 and three grade 2). Twelve patients had been previously treated with melphalan in ILP (11) or with cisplatin (one). The 23 patients are assessable: there have been 21 complete responses (CRs; range, 4 to 29 months; 89%), two partial responses (PRs; range, 2 to 3 months), and no failures. Overall disease-free survival and survival have been 70% and 76%, respectively, at 12 months. In all cases, softening of the nodules was obvious within 3 days after ILP and time to definite response ranged between day 5 and 30. CONCLUSION: This preliminary analysis of a phase II study suggests that high-dose rTNF alpha can be administered with acceptable toxicity by ILP with dopamine and hyperhydration. Tumor responses can be evidenced in melanoma and sarcoma. Furthermore, combination of rTNF alpha, rIFN-gamma, and melphalan seems to achieve high efficacy with minimal toxicity, even after failure of prior therapy with melphalan alone. PMID- 1727927 TI - Diagnosing infection in febrile granulocytopenic patients with indium-111-labeled human immunoglobulin G. AB - PURPOSE: Delineation of focal infection is a major problem in the management of febrile granulocytopenic patients. The utility of indium-111-labeled human nonspecific immunoglobulin G (In-111-IgG), a newly developed radiopharmaceutical for imaging focal inflammation, was reported in patients with adequate WBC counts. In the present study, we investigated whether In-111-IgG scintigraphy could be used to locate infection in granulocytopenic patients. MATERIALS AND METHODS: Granulocytopenic rats with focal infection were imaged after In-111-IgG injection. Thereafter, In-111-IgG scintigraphy was performed in 20 granulocytopenic patients. Images were obtained 4, 24, and 48 hours after injection of 75 mBq In-111-IgG. Scintigraphic findings were compared with clinical, roentgenologic, and ultrasonographic methods and culture results. RESULTS: In the animal model high In-111-IgG accumulation was observed in the infectious focus. In the patients, 13 proven pulmonary, abdominal, joint, and soft tissue infections of both bacterial and fungal origin were detected adequately. In-111-IgG uptake not due to verified inflammation was observed in the large bowel of two patients. A thoracic wall infiltrate showing only mild inflammatory activity was not detected. Small toxoplasmosis lesions in heart, liver, and kidneys were obscured by physiologic In-111-IgG activity in these organs. CONCLUSIONS: In-111-IgG scintigraphy is a useful technique to delineate focal infection in patients with granulocytopenia. Accumulation of the radiopharmaceutical does not appear to be granulocyte-mediated. In-111-IgG is a safe and convenient radiopharmaceutical that probably contributes to the early diagnosis of focal infection in granulocytopenic patients. PMID- 1727928 TI - Enhancement of radiation-induced downstaging of rectal cancer by fluorouracil and high-dose leucovorin chemotherapy. AB - PURPOSE: To determine if fluorouracil (5-FU) plus high-dose leucovorin (LV) enhances local response in patients receiving preoperative radiation therapy (RT) for adenocarcinoma of the rectum, we compared the degree of downstaging in patients receiving preoperative RT with or without chemotherapy. PATIENTS AND METHODS: For this comparison, three groups of patients who were treated with identical doses and techniques of preoperative pelvic RT (total dose of 5,040 cGy) were examined. Group 1 included 20 patients with unresectable disease who received combined RT and LV/5-FU. Group 2 included 11 patients with unresectable disease who received preoperative RT. Group 3 included 21 patients with invasive, resectable, primary disease who received preoperative RT. RESULTS: Patients with unresectable disease who received LV/5-FU had a higher rate of pathologic complete response (20% v 0%) and a lower incidence of positive nodes (30% v 64%) compared with those who did not receive chemotherapy. Even when the most favorable group of patients was included (group 3), patients who received LV/5-FU still had a higher complete response rate (20% v 6%) and a lower incidence of positive nodes (30% v 53%) compared with those who received RT without LV/5-FU. Of those patients with initially unresectable disease, the resectability rate was higher in those who received LV/5-FU compared with those who did not receive LV/5 FU (90% v 64%). Patients who received LV/5-FU experienced slightly more grade 1 to 2 fatigue, stomatitis, nausea, and grade 3 diarrhea, tenesmus, and dysuria. CONCLUSIONS: Despite the fact that patients who received chemotherapy (group 1) had more advanced disease compared with those with resectable disease (group 3), the addition of LV/5-FU increased the resectability and downstaging rates. The ultimate impact of a complete response as well as a decrease in the incidence of pelvic nodes on local control and survival remains to be determined. However, given the enhancement of down-staging in patients with unresectable rectal cancer, we are encouraged by the combined modality approach. PMID- 1727930 TI - Antagonist drugs and bone vascular smooth muscle. AB - An ex vivo canine tibia model was used to quantitate the specific adrenergic subtype contribution in bone vasculature. Tibiae were obtained from mongrel dogs, the nutrient artery was catheterized, and the bone was placed in an ex vivo perfusion apparatus at constant flow. Perfusion was accomplished using oxygenated Krebs-Ringer solution. A norepinephrine dose-response curve was obtained by using incremental single bolus doses. Each bone was perfused with a vasoactive drug at a standard physiologic dosage. After 30 min of perfusion, a second norepinephrine dose-response curve was generated. The degree of attenuation of the norepinephrine dose-response curve, as determined by the total area under the curve, was interpreted as the relaxation effect of the drug on the smooth muscle of the vascular bed. Prazosin (alpha 1-receptor antagonist), rauwolszin (alpha 2 receptor antagonist), propranolol (beta-receptor antagonist), and diltiazem (calcium-entry inhibitor) were evaluated. Our data suggest that alpha 1 and alpha 2 adrenergic receptor antagonism results in a quantitatively similar attenuation of norepinephrine-induced vascular smooth muscle contraction. Calcium-entry antagonism produced less, but significant, attenuation of smooth muscle contractility. Beta-adrenergic receptor blockade yielded only a slight, although consistent, reduction in reactivity. Simple perfusion with Krebs-Ringer solution had no effect. PMID- 1727929 TI - Serum sialyl Tn as an independent predictor of poor prognosis in patients with epithelial ovarian cancer. AB - PURPOSE: Monoclonal antibody (moAB) TKH-2 directed to the tumor-associated O linked sialyl 2-6-alpha-N-acetylgalactosaminyl (sialyl Tn; STN) epitope was generated by immunization with ovine submaxillary mucin (Kjeldsen et al, Cancer Res 48:2214-2220, 1988). We investigated whether circulating serum levels of STN antigen might influence the prognosis of patients with ovarian cancer. PATIENTS AND METHODS: Serum samples were obtained from 126 healthy nonpregnant women, 157 patients with benign gynecologic disease, and 89 patients with histologically proven epithelial ovarian cancer. Circulating serum STN-antigen concentrations (U/mL) were determined by a competitive radioimmunoassay kit (Otsuka Assay Laboratories, Tokushima, Japan) in a one-step procedure. RESULTS: Serum antigen levels were elevated in 48.3% of the patients. The levels of STN antigen were significantly higher in the sera of patients with cancer when compared with levels in benign and healthy controls (P less than .05). The 5-year survival rate for patients with STN-negative (serum STN levels less than 50.0 U/mL) versus STN positive (greater than or equal to 50 U/mL) tumors was 76.9% versus 10.8%, respectively (P less than .05). The progression-free interval (PFI) at 5 years was 51.9% versus 5.4%, respectively (P less than .05). The overall survival probability and PFI were worse in patients with STN-positive sera. Multivariate regression analysis revealed that stage, residual tumor size, positive STN, performance status, and histologic grade were the five important variables for predicting overall survival. CONCLUSION: We conclude that a positive STN-antigen level in sera is an independent predictor of poor prognosis in ovarian cancer. PMID- 1727931 TI - Combined effect of acute denervation and ischemia on the microcirculation of skeletal muscle. AB - Using direct in vivo videomicroscopy and a fluorescein dye technique, reperfusion injury after 3 h of ischemia was studied in the acutely denervated cremaster muscle of the rat. Compared with normally innervated controls, ischemia-induced reperfusion injury was more severe in the denervated group and included a delay of blood flow recovery, vortex formation, edema, hemorrhage, and vessel spasm. Vessel size was reduced at the arteriole and small artery level, and there was a decrease of reactive hyperemia. The injury mechanism may be related to a loss of active vasomotion and vascular response to vasoactive substances after denervation. The results suggest that shortening the ischemia time of denervated tissues may reduce ischemia-induced reperfusion injury. Similarly, given the same ischemia time, improved tissue reperfusion may be expected if the nerve supply is maintained. PMID- 1727932 TI - Angular deformities and forearm function. AB - Angular deformities were created in cadaver forearms at proximal, middle, and distal third levels of the radius and ulna separately, and at middle and distal third levels of both bones, to determine the corresponding limitations of pronation and supination. The ranges of pronation and supination were recorded using a rotational motion measurement apparatus instrumented with a 360 degrees goniometer. These experimental results were compared to data obtained from clinical and radiographic examination of 105 patients with similar residual deformities following treatment of fractures by nonsurgical means, to evaluate the accuracy of the experimental model and to determine if loss of rotational motion could be predicted based on radiographic findings. With cadaver forearms, on the average, angulation of 10 degrees of the radius or ulna in coronal or sagittal planes limited pronation and supination by less than 24 degrees, whereas angulation of 10 degrees of both the radius and the ulna limited pronation and supination by less than 18%. Comparison of experimental results with clinical findings showed that, despite the errors involved in measuring forearm deformities in patients using biplanar radiographs, the experimental results predicted the clinical loss of pronation and supination to within 17% for the fractures of the radius, and within 8% accuracy for the fractures of the ulna. PMID- 1727933 TI - A finite element study of the initiation of failure of fixation in cemented femoral total hip components. AB - In order to study initial mechanisms of failure in cemented femoral total hip components, an anatomically accurate three-dimensional linear finite element model was constructed and verified against experimental strain measurements in the cement mantle. Good agreement was found between predicted and measured strains. The likelihood of failure initiation due to cement-prosthesis debonding and crack initiation at voids was studied for loading conditions simulating both one-legged stance and stair climbing. The "out of plane" forces involved in stair climbing appear to be the greatest threat to the fixation of total hip replacements. In stair climbing, cement-prosthesis debonding and pore crack initiation were probable in the proximal anteromedial region of the cement mantle, and near the distal tip of the implant. The proximal stresses in stair climbing were higher than the distal stresses in either stair climbing or one legged stance. PMID- 1727934 TI - Colony-forming efficiency response of bone marrow stromal cells to acute blood loss. AB - Colony-forming efficiency (CFE) was used to monitor the proliferative response of alkaline phosphatase-positive rabbit bone marrow stromal cells to acute blood loss. The CFE of animals subjected to a 1% blood loss was 0.97 compared with 0.06 (p less than 0.01) in nonbled animals. Sera obtained from animals 10 days after an initial blood loss stimulated the CFE of marrow cultures from nonbled donors to the same degree as osteogenin. Erythropoietin and control sera (from nonbled animals) had no effect. Hence, acute blood loss and sera from bled animals stimulate proliferation of alkaline phosphatase-positive marrow stromal cell colonies. The agent(s) responsible is unknown but it is present in serum in response to blood loss. Confirmation of a specific effect on osteoprogenitor cells may warrant the designation "osteopoietin." PMID- 1727935 TI - Effect of hylan on cartilage and chondrocyte cultures. AB - The protective role of hylan, a hyaluronan [hyaluronic acid (HA)] derivative, was studied in explanted bovine cartilage and isolated chondrocytes. Cartilage and chondrocytes were exposed to degradative enzymes (lysate from activated polymorphonuclear leukocytes), oxygen-derived free radicals (ODFR), conditioned media from mononuclear cells (MCCM), and interleukin-1 (IL-1), in the presence and absence of hylan. The effect of HA was also studied. In cartilage explants susceptibility to pertubation was evaluated in terms of 35S release and proteoglycan depletion and was compared to control cultures; high viscosity hylan was found to reduce 35S release in cartilage explants caused by degradative enzymes, ODFR, MCCM, and IL-1. The hylan effect was reversible and viscosity dependent. In chondrocyte cultures, high viscosity hylan was effective in reducing cell injury caused by degradative enzymes and ODFR. The data suggest that the glycosaminoglycan hylan, as well as native HA, may mediate exposure to and/or response to stimuli associated with initiation of degenerative processes in cartilage tissues. PMID- 1727936 TI - The reliability of the Mankin score for osteoarthritis. AB - For the histopathological classification of the severity of osteoarthritic lesions of cartilage, the Mankin score is frequently used. A necessary constraint on the validity of this scoring system is the consistency with which cartilage lesions are classified. The intra- and interobserver agreement of the Mankin score was determined. The intra- and interobserver agreement of the 14-point Mankin score was adequate. Between observers 95% of differences were less than approximately 7 points. By a more strict definition of the elements of the Mankin score, the intraobserver differences were reduced only for some observers. The interobserver differences were only slightly reduced: between observers 95% of differences were less than approximately 6 points. We found the Mankin score to be an adequate histopathological tool. PMID- 1727937 TI - Human growth plate development in the fetal and neonatal period. AB - The development of the normal human upper tibial growth plate was studied at autopsy in 46 stillborns and 79 newborns of 20-41 weeks gestational age. During this time period, the histology of this plate evolves from a highly cellular structure with relatively poor columnar organization and matrix development to the well known structure seen later in postnatal life. The thickness of the growth plate, assessed in the area surrounding the longitudinal tibial axis, decreases continuously from 1.15 mm on the 20th week to 0.6 mm on the 38th week. This decrease results from losses of both matrix and cellular components, mostly of the latter. However, the relative fraction of area occupied by the matrix significantly increased (12%) and matrix area per cell increased 1.5 times over the last half of gestation, indicating a maturation process of the plate towards a more matrix-oriented structure with age. In this maturation process the number of cells per unit area does not change and the average size of the cells appears to decrease. Plate thickness does not decrease further in the final 3 weeks of pregnancy and increases in early neonatal life; this has no apparent influence on the tibial growth rate. In the period under study the relative anatomical participation of the upper tibial growth plate decreases from approximately 4% of the radiographic length of the tibia on the 20th week to less than 1% at term. Present data will provide fetal and neonatal growth plate standards needed to obtain a better understanding of this structure during both normal and abnormal conditions. PMID- 1727938 TI - Structural consequences of subchondral bone involvement in segmental osteonecrosis of the femoral head. AB - Appearance of a crescent sign usually marks the onset of necrotic femoral head collapse, but very little is known about which local factors contribute most critically to avoiding or postponing fracture of at-risk juxtaarticular cancellous bone. A three-dimensional finite element model was used to test the hypothesis that an initially mechanically uncompromised subchondral plate could provide a substantial degree of stress protection to a weakened underlying segmental infarction. The computational simulation of osteonecrosis showed that the principal stress distribution for an assumption of subchondral plate weakening (given also an underlying, comparably weakened segmental infarction) differed inappreciably from that of a normal femoral head. However, the tendency for local structural failure, as reflected in the ratio of stress to strength, was substantially higher in the former instance. If, instead, the mechanical integrity of the subchondral plate overlying the weakened segmental infarction was assumed to be preserved, computed stress levels in the at-risk subjacent necrotic cancellous bone were still over 70% as high as for the weakened-plate case. The data thus indicate that even a fully normal subchondral plate can provide only modest stress protection of a weakened underlying segmental infarction, whereas weakening of the necrotic cancellous bone throughout the infarction induces marked stress increase in the overlying subchondral plate. These findings suggest that the onset of collapse is probably dominated much more strongly by the degree of structural degradation of the cancellous bone within the main infarct body, than by the degree of structural degradation within the subchondral plate. PMID- 1727939 TI - Ultrastructural morphometry of anterior cruciate and medial collateral ligaments: an experimental study in rabbits. AB - This study presents morphometric analyses of collagen subfascicle area fraction and collagen fibril diameter distributions for the anterior cruciate (ACL) and medial collateral (MCL) knee ligaments from transmission electron micrographs of ligament cross sections of five mature, female New Zealand White rabbits. Statistically significant differences in subfascicular area fractions were found between the ACL and MCL (0.89 +/- 0.02, 0.97 +/- 0.01, respectively; p less than 0.001). Mean fibril diameters for the ACL and MCL were also significantly different (0.059 +/- 0.005, 0.085 +/- 0.011 microns, respectively; p less than 0.025). Fibril eccentricity (a measure of parallel alignment of collagen fibrils within the ligaments, defined as the ratio of minor to major axes of elliptical fibril outlines) was 0.89 +/- 0.03 and 0.85 +/- 0.08, respectively, for the ACL and MCL; these data were not significantly different (p greater than 0.1). The relative amount of variation in the pooled fibril diameter data due to variation between animals, ligaments, locations within ligaments, and among fibrils at individual locations are reported. The variation of fibril diameter distributions between the ACL and MCL was substantially greater than the variation between different locations within each ligament cross section as well as between different animals. The structural differences reported may help explain known differences in the biomechanical properties of the ACL and MCL. PMID- 1727940 TI - Cardiovascular diseases remain nation's leading cause of death. PMID- 1727941 TI - Lipid particles may help solve puzzle regarding genesis of some cardiovascular diseases. PMID- 1727942 TI - Gene scene: a master control switch for myogenesis muscles its way into the clinic. PMID- 1727943 TI - From the Food and Drug Administration. PMID- 1727944 TI - From the Secretary of Health and Human Services. PMID- 1727945 TI - From the Centers for Disease Control. Control of influenza A outbreaks in nursing homes: amantadine as adjunct to vaccine--1989-1990. PMID- 1727946 TI - From the Centers for Disease Control. Update: influenza activity and vaccine availability--United States, 1991 and 1992. PMID- 1727947 TI - From the Centers for Disease Control. Quarterly table reporting alcohol involvement in fatal motor-vehicle crashes. PMID- 1727948 TI - What if Americans ate less fat? PMID- 1727949 TI - What if Americans ate less fat? PMID- 1727950 TI - What if Americans ate less fat? PMID- 1727951 TI - What if Americans ate less fat? PMID- 1727952 TI - Aspirin use and cardiovascular disease in women. PMID- 1727953 TI - Another pound of cure. PMID- 1727954 TI - Another pound of cure. PMID- 1727955 TI - Examination scores fall with time: but so what? PMID- 1727957 TI - Examination scores fall with time: but so what? PMID- 1727956 TI - Examination scores fall with time: but so what? PMID- 1727958 TI - The importance of being empathic. PMID- 1727959 TI - Prevalence of tuberculin positivity and skin test anergy in HIV-1-seropositive and -seronegative intravenous drug users. AB - OBJECTIVES: --To identify differences in purified protein derivative (PPD) tuberculin positivity and skin test anergy rates by human immunodeficiency virus (HIV) serostatus, CD4+ lymphocyte count, and other risk factors in intravenous drug users (IVDUs); and to evaluate the appropriateness of the Centers for Disease Control (CDC)--recommended definition for a positive PPD tuberculin skin test result in HIV-1-seropositive patients. DESIGN: --Nested case-control and cross-sectional analyses. SETTING: --Community-based cohort of IVDUs. PATIENTS: - Two hundred sixty HIV-1-seropositive and -seronegative IVDUs, drawn from an unselected cohort, were skin-tested for sensitivity to PPD tuberculin, mumps, and Candida antigens using the Mantoux method. OUTCOME MEASURES: --Positivity to PPD tuberculin, skin test anergy. RESULTS: --Even using the CDC definition of an induration 5 mm or greater in diameter in HIV-1 seropositives, this group was substantially less likely to be PPD tuberculin positive than HIV-1 seronegatives (13.8% vs 25.2%; P = .02). In the HIV-1 seropositives the relative odds of being PPD positive varied depending on whether 10 mm or greater (odds ratio [OR], 0.3; 95% confidence interval [CI], 0.2 to 0.7), 5 mm or greater (OR, 0.5; 95% CI, 0.2 to 0.9), or 2 mm or greater (OR, 0.7; 95% CI, 0.4 to 1.3) was used to define a positive test result. The mean diameter induration in the HIV-1-seropositive group was 2.6 mm vs 5.4 mm in the seronegative group (P = .005). Skin test anergy (to mumps and Candida) appeared to explain the differential. Anergy was substantially higher in the HIV-1 seropositive group and increased as the CD4+ lymphocyte count fell (chi 2 for linear trend, 24.5; P less than .0001). An inverse linear trend for PPD positivity and CD4+ lymphocyte count was also observed (chi 2 for trend, 6.1; P = .01). In multivariate analyses, being 35 years of age or older and being HIV-1 seronegative were significantly associated with PPD positivity, while history of previous police arrest was of borderline significance. Only HIV-1 seropositivity was significantly associated with anergy. CONCLUSIONS: --These findings show that CDC-recommended definition of an induration 5 mm or greater in diameter for PPD tuberculin positivity in HIV-1 seropositives significantly underestimates the "true" infection rate (using the PPD positivity rate in HIV-1 seronegatives as the criterion standard). A definition of 2 mm or greater would appear to be a better cutoff for reducing misclassification in HIV-1 seropositives. This study also confirms that delayed type hypersensitivity is seriously depressed in HIV-1 seropositive IVDUs and that anergy testing is mandatory to properly assess a negative PPD test result. PMID- 1727960 TI - Factors influencing publication of research results. Follow-up of applications submitted to two institutional review boards. AB - OBJECTIVE: --To investigate factors associated with the publication of research findings, in particular, the association between "significant" results and publication. DESIGN: --Follow-up study. SETTING: --Studies approved in 1980 or prior to 1980 by the two institutional review boards that serve The Johns Hopkins Health Institutions--one that serves the School of Medicine and Hospital and the other that serves the School of Hygiene and Public Health. POPULATION: --A total of 737 studies were followed up. RESULTS: --Of the studies for which analyses had been reported as having been performed at the time of interview, 81% from the School of Medicine and Hospital and 66% from the School of Hygiene and Public Health had been published. Publication was not associated with sample size, presence of a comparison group, or type of study (eg, observational study vs clinical trial). External funding and multiple data collection sites were positively associated with publication. There was evidence of publication bias in that for both institutional review boards there was an association between results reported to be significant and publication (adjusted odds ratio, 2.54; 95% confidence interval, 1.63 to 3.94). Contrary to popular opinion, publication bias originates primarily with investigators, not journal editors: only six of the 124 studies not published were reported to have been rejected for publication. CONCLUSION: --There is a statistically significant association between significant results and publication. PMID- 1727961 TI - Survival from in-hospital cardiac arrest with interposed abdominal counterpulsation during cardiopulmonary resuscitation. AB - OBJECTIVE: --To determine whether interposed abdominal counterpulsation (IAC) during standard cardiopulmonary resuscitation (CPR) improves outcome in patients experiencing in-hospital cardiac arrest. DESIGN AND SETTING: --Randomized controlled trial in a university-affiliated hospital. PATIENTS: --Patients experiencing in-hospital cardiac arrest during a 6-month period. INTERVENTIONS: - Patients were randomized to receive either IAC during CPR or standard CPR in the event of cardiac arrest. Abdominal compressions were performed during the relaxation phase of chest compression, corresponding to CPR diastole, at a rate of 80/min to 100/min. MAIN OUTCOME MEASURES: --The three end points studied were (1) return of spontaneous circulation, (2) survival 24 hours after resuscitation, and (3) survival to hospital discharge. In addition, we examined neurological outcome in those patients surviving to hospital discharge. RESULTS: --During the study period there were 135 resuscitation attempts in 103 patients. Return of spontaneous circulation was significantly greater in the group receiving IAC during CPR than in the group receiving standard CPR (51% vs 27%, P = .007). At hospital discharge, a significantly greater proportion of patients was alive in the IAC group than in the control group (25% vs 7%, P = .02). Eight (17%) of 48 patients who received IAC during CPR survived to hospital discharge neurologically intact, compared with only three (6%) of 55 patients from the standard CPR group (not significant). CONCLUSIONS: --We conclude that the addition of IAC to standard CPR may improve meaningful survival following in hospital cardiac arrest. The optimal use of this technique awaits further clinical trials. PMID- 1727962 TI - Adverse events following pertussis and rubella vaccines. Summary of a report of the Institute of Medicine. AB - In August 1991, the Institute of Medicine released a report entitled Adverse Effects of Pertussis and Rubella Vaccines, which examined 18 adverse events in relation to diphtheria-tetanus-pertussis (DTP) vaccine and four adverse events in relation to the currently used rubella vaccine strain, RA 27/3. The committee spent 20 months reviewing a wide range of information sources, including case series and individual case reports, both published and unpublished, epidemiologic studies, studies in animals, and other laboratory studies. The committee found that the evidence indicates a causal relation between DTP vaccine and anaphylaxis and between the pertussis component of DTP vaccine and extended periods of inconsolable crying or screaming. The committee also reported that the evidence indicates a causal relation between the rubella vaccine and acute arthritis in adult women. The committee found the available evidence weaker but still consistent with a causal relation between DTP vaccine and two conditions--acute encephalopathy and hypotonic, hyporesponsive episodes--and between rubella vaccine and chronic arthritis in adult women. Estimated incidence rates of these adverse events following vaccination are provided, where possible. The committee found that the evidence does not indicate a causal relation between the DTP vaccine and infantile spasms, hypsarrhythmia, Reye's syndrome, and sudden infant death syndrome. The committee found insufficient evidence to indicate either the presence or absence of a causal relation between DTP vaccine and chronic neurologic damage, aseptic meningitis, erythema multiforme or other rash, Guillain-Barre syndrome, hemolytic anemia, juvenile diabetes, learning disabilities and attention-deficit disorder, peripheral mononeuropathy, or thrombocytopenia, and between rubella vaccine and radiculoneuritis and other neuropathies or thrombocytopenic purpura. The committee's evaluative methods are briefly described and a summary of research needs is provided. PMID- 1727963 TI - Sequence of changes in body composition induced by testosterone and reversal of changes after drug is stopped. AB - OBJECTIVE: --To study the changes in body composition produced by large doses of testosterone and reversal of changes when the drug is discontinued. DESIGN: - Weekly injections of testosterone enanthate were given to young adult male volunteers for 12 weeks. Repeated assays of lean body mass (LBM) by potassium 40 counting were made during this period and at intervals during the ensuing 5 to 6 months. PARTICIPANTS AND SETTING: --Subjects who were living on their own, who were known to be free of significant disease, and who volunteered as controls for a study of patients with neuromuscular disease. Assays were done in the Clinical Research Center. MAIN OUTCOME MEASURES: --Changes in body weight, LBM, and (by subtraction) body fat. RESULTS: --Testosterone treatment produced a progressive increase in LBM and a progressive decrease in body fat. Body composition reverted slowly toward normal when the injections were stopped; thus, the effects of testosterone lingered for some time. The magnitude of the observed changes in LBM was in keeping with the change in urinary creatinine excretion reported for these same subjects. CONCLUSION: --Testosterone is a powerful anabolic agent that also serves to reduce body fat content. PMID- 1727964 TI - Chronic Chlamydia trachomatis infections in infants. AB - OBJECTIVE: --To study the natural history of Chlamydia trachomatis infections in infants. DESIGN: --Bacteriologic and serologic study of an inception cohort. SETTING: --University of Washington Medical Center, Seattle. PARTICIPANTS: - Twenty-two infants with C trachomatis infections either not treated early in life or recurring after antimicrobial treatment. MAIN OUTCOME MEASURES: --Persistence of infection in various anatomic sites, antibody responses to specific serovars (serologic variants) of C trachomatis, and serovars of isolates from mothers and infants. RESULTS: --The cumulative proportion of infants still infected at the age of 1 year was 35%. Infection persisted in the conjunctiva, nasopharynx, and oropharynx in one child for as long as 866 days (28.5 months), when she was cured by treatment. In none of the infants did serologic tests suggest acquisition of infection other than at birth. Isolates of C trachomatis from mothers and their respective infants were always of the same serovar. CONCLUSIONS: --Many infants infected with C trachomatis at birth remain infected for months or years in the absence of specific antimicrobial therapy. Such infections may be confused with those acquired by sexual abuse. PMID- 1727965 TI - Balancing incentives. How should physicians be reimbursed? AB - The May 15, 1991, JAMA theme issue presented 14 different proposals for reforming the US medical care system. Though these proposals varied widely, they encompassed only three proposed methods for paying physicians: fee-for-service, salary, and capitation. When examined from a perspective that considers the incentives affecting practicing physicians, each of these methods has serious flaws. I outline these flaws and suggest an alternative method for reimbursing primary care physicians: that of combining capitation and fee-for-service. Specialists would be paid fee-for-service, but the cost of specialty care would be limited by giving each primary care physician a budget for such care. Consistent failure by a physician to stay within the budget would result in ongoing review, but not in direct financial sanctions. This system of reimbursing physicians is intended to balance incentives so that physicians can concentrate on making medical decisions with a minimum of distraction by third parties and by personal financial considerations. PMID- 1727966 TI - The problem of prenatal cocaine exposure. A rush to judgment. PMID- 1727967 TI - Tuberculin skin testing and the HIV epidemic. PMID- 1727968 TI - Publication bias. The triumph of hope over experience. PMID- 1727969 TI - Normal body temperature. PMID- 1727970 TI - Relief of functional bowel obstruction. PMID- 1727971 TI - Sexual behavior among high school students--United States, 1990. AB - Since the 1970s, sexually transmitted diseases (STDs) (including human immunodeficiency virus infection and acquired immunodeficiency syndrome), unintended pregnancies, and other problems that result from sexual activity have increased among adolescents in the United States. For example, approximately 1 million adolescent girls become pregnant each year and 86% of all STDs occur among persons aged 15-29 years. This article presents self-reported data from 1990 about the prevalence of sexual intercourse, contraceptive use, condom use, and STDs among U.S. high school students. PMID- 1727972 TI - Early childhood vaccination levels among urban children--Connecticut, 1990 and 1991. AB - In the United States, the high incidence of measles among urban preschool-aged children who had not received age-appropriate vaccination has focused attention on the adequacy of and barriers to early childhood vaccinations. To assess early childhood vaccination levels of urban Connecticut children, during fall 1990 and spring 1991, the Connecticut Department of Health Services conducted retrospective surveys of first-grade students in Hartford and New Haven, both with populations greater than 100,000 persons. PMID- 1727973 TI - Locally acquired neurocysticercosis--North Carolina, Massachusetts, and South Carolina, 1989-1991. AB - From October 1989 through November 1991, three persons with neurocysticercosis acquired in the eastern United States (North Carolina, Massachusetts, and South Carolina) were reported to CDC. This report summarizes clinical and epidemiologic information for these cases. PMID- 1727974 TI - Hepatitis B and injecting-drug use among American Indians--Montana, 1989-1990. AB - From November 1989 through March 1990, five cases of serologically confirmed acute hepatitis B (HB)* among American Indians from two Montana Indian reservations (combined population: 6300) were reported by Indian Health Service (IHS) clinic staff to the Billings IHS Area Office. In comparison, during 1986 1988, an average of six HB cases among American Indians were reported annually among persons residing both on reservations and statewide (1990 population of American Indians in Montana: 47,679). Four of the five persons with acute HB reported histories of injecting-drug use (IDU). PMID- 1727975 TI - Surgeon General's Conference on Agricultural Safety and Health, 1991. AB - On April 30-May 3, 1991, CDC's National Institute for Occupational Safety and Health (NIOSH) convened the Surgeon General's Conference on Agricultural Safety and Health in Des Moines, Iowa. The theme for this conference was "FarmSafe 2000: A National Coalition for Local Action." Agricultural safety and health professionals, equipment manufacturers, farmer and migrant health organizations, rural health-care professionals, and agricultural youth organizations met to deliberate on methods to reduce health and safety risks in agriculture. More than 700 persons attended and represented 41 states and Puerto Rico. Through small group interactive discussions, the conference addressed surveillance, research, and intervention--three public health activities necessary to meet year 2000 national health objectives (1). This report summarizes the issues considered at the conference and recommendations proposed by attendees. PMID- 1727976 TI - The prevalence of ulcerated plaques in the aortic arch in patients with stroke. AB - BACKGROUND AND METHODS: The cause of cerebral infarction is obscure in up to 40 percent of patients with this disorder who are studied prospectively. In this investigation, we determined the frequency of ulcerated plaques in the aortic arch and explored the part they may play in the formation of cerebral emboli. Using an autopsy data bank, we studied the prevalence of ulcerated plaques in the aortic arch in 500 consecutive patients with cerebrovascular and other neurologic diseases who were studied at autopsy. RESULTS: Ulcerated plaques were present in 26 percent of the 239 patients with cerebrovascular disease but in only 5 percent of the 261 patients with other neurologic diseases (P less than 0.001). After we controlled for age and heart weight, the adjusted rates were 16.9 percent and 5.1 percent, respectively (adjusted odds ratio, 4.0; 95 percent confidence interval, 2.1 to 7.8; P less than 0.001). Among the patients with cerebrovascular disease, the prevalence of ulcerated plaques in the aortic arch was 28 percent in the 183 patients with cerebral infarcts and 20 percent in the 56 patients with brain hemorrhage. The prevalence of ulcerated plaques was 61 percent among the 28 patients with no known cause of cerebral infarction, as compared with 22 percent among the 155 patients with a known cause of cerebral infarction (P less than 0.001). After adjustment for covariates, the prevalence was 57.8 percent among patients with no known cause of cerebral infarction and 20.2 percent among those with a known cause (adjusted odds ratio, 5.7; 95 percent confidence interval, 2.4 to 13.6; P less than 0.001). The presence of ulcerated plaques in the aortic arch was not correlated with the presence of extracranial internal-carotid artery stenosis, suggesting that these were two independent risk factors for stroke. CONCLUSIONS: Ulcerated plaques in the aortic arch may play a part in causing cerebral infarction, especially in patients in whom cerebral infarction has no known cause. PMID- 1727977 TI - The pathogenesis of coronary artery disease and the acute coronary syndromes (1). PMID- 1727978 TI - How far should blood pressure be lowered? PMID- 1727979 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 4-1992. Pancytopenia, splenomegaly, and retinal hemorrhage in a 52-year-old diabetic man. PMID- 1727980 TI - Pituitary-adrenal function during corticosteroid therapy. Learning to live with uncertainty. PMID- 1727981 TI - Transient neutropenia induced by intravenous immune globulin. PMID- 1727982 TI - Alternating morphology of the QRST complex preceding sudden death. PMID- 1727983 TI - Tumor necrosis factor-alpha and disease progression in multiple sclerosis. PMID- 1727984 TI - Perioperative total parenteral nutrition in surgical patients. PMID- 1727985 TI - Are we mortgaging the medical profession? PMID- 1727986 TI - Are we mortgaging the medical profession? PMID- 1727987 TI - Are we mortgaging the medical profession? PMID- 1727988 TI - Are we mortgaging the medical profession? PMID- 1727989 TI - Are we mortgaging the medical profession? PMID- 1727990 TI - Harmonica player's "hemoptysis". PMID- 1727991 TI - Extracorporeal membrane oxygenation and early-onset group B streptococcal sepsis. AB - Recently, extracorporeal membrane oxygenation (ECMO) has been used as rescue therapy for newborns with overwhelming early-onset group B streptococcal sepsis. To determine which clinical factors best predict mortality and to evaluate the outcome of this therapy, a retrospective examination of the clinical course and outcome of ECMO-eligible newborns with early-onset group B streptococcal sepsis was undertaken. The study period was divided into two phases based on when ECMO was initially used at Kosair Children's Hospital as therapy for septic neonates. Phase 1 (pre-ECMO) was the period from January 1, 1982, through June 15, 1986, and phase 2 (ECMO) from June 16, 1986, through December 31, 1989. Newborns with gestational age greater than or equal to 34 weeks, birth weight greater than or equal to 2000 g, and evidence of early-onset group B streptococcal sepsis were eligible for study. Only newborns who received mechanical ventilation were evaluated. Sixteen patients from phase 1 met the above criteria. Of those, 10 exhibited no sign of hypotension and all survived. Of the 6 patients with hypotension, 3 died. Forty patients were identified from phase 2. Seven patients remained normotensive and all survived. Thirty-three patients were hypotensive, of which 15 received ECMO and 13 survived. Of the 18 who did not receive ECMO, 7 died. Regarding all hypotensive newborns, those who did not receive ECMO had a trend toward lower survival (P less than .06) and were more likely to die if they were of lower birth weight, manifested a persistent acidosis (pH less than or equal to 7.25), and had an absolute neutrophil count less than 500 cells/mm3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727992 TI - Drug screening of newborns by meconium analysis: a large-scale, prospective, epidemiologic study. AB - A large-scale, prospective drug screening of newborns by meconium analysis was done to determine more accurately the prevalence and epidemiologic characteristics of drug use in a high-risk urban, obstetric population. Every other neonate delivered in a perinatal center from November 1988 to September 1989 was prospectively enrolled and their meconium was analyzed by radioimmunoassay for the metabolites of three commonly abused drugs--cocaine, morphine (opiates), and cannabinoid. In 3010 subjects studied, 44% were positive for cocaine, morphine, or cannabinoid; 31% were positive for cocaine, 21% for morphine, and 12% for cannabinoid. In contrast, only 335 (11%) mothers admitted to illicit drug use: 52% of their newborns had a positive urine drug screen and 88% had a positive meconium drug screen. Prevalence of drug use among the pregnant women varied per month. A profile of the pregnant addict in the population studied was noted (P less than .001): service patient, single, multigravid (greater than 3), and little or no prenatal care. The major problems associated with drug use during pregnancy were principally noted in the group that was exposed to cocaine and opiates and in the group where the mothers admitted to the use of illicit drugs. On the other hand, a large number of neonates who have been exposed to drugs in utero, particularly those whose mothers denied the use of drugs, appear normal at birth and may not be recognized. Improved detection of these newborns at risk can be achieved with a high index of suspicion and meconium drug analysis. PMID- 1727993 TI - Neuroblastoma represents distinct clinical-biologic entities: a review and perspective from the Quebec Neuroblastoma Screening Project. AB - Data are presented suggesting that neuroblastoma represents at least two distinct clinical-biologic entities. One, favorable neuroblastoma, is associated with young age and early stage at diagnosis, triploid karyotypes, no 1p abnormalities or N-myc gene amplification, more mature catecholamine synthesis and excretion, and excellent clinical outcome despite no or minimal therapy. The other, unfavorable neuroblastoma, is associated with older age and advanced stage, pseudodiploid karyotypes and 1p deletions, N-myc oncogene amplification, less mature catecholamine synthesis and excretion, and poor outcome despite aggressive multimodality therapy including bone marrow transplantation. Favorable neuroblastomas rarely, if ever, evolve into unfavorable disease. Unresolved issues regarding this hypothesis and implications for the efficacy of preclinical detection through mass screening are discussed. PMID- 1727994 TI - Facing tragic decisions with parents in the neonatal intensive care unit: clinical perspectives. PMID- 1727995 TI - Epidemiology of transfusion-associated acquired immunodeficiency syndrome in children in the United States, 1981 through 1989. AB - From 1981 through 1989, 212 cases of transfusion-associated (TA) acquired immunodeficiency syndrome (AIDS) were reported to the Centers for Disease Control. In a study of the epidemiology of pediatric TA AIDS, this group was compared with perinatally acquired (PA) and adult TA AIDS cases. The number of pediatric TA AIDS cases reported each year began to stabilize in 1988 and declined 41% in 1989. Reported adult TA AIDS cases continued to increase by 33% in 1988 and declined by 15% in 1989. The number of reported PA cases has continued to increase each year. Seventy percent of the children with TA AIDS were transfused in their first year of life. The median age at diagnosis was 4 years (range 0.3 to 12.8 years) compared with a median age at diagnosis of 1 year (range 0.1 to 12.9 years) in the PA cases. Using a nonparametric estimation procedure for truncated data, the estimated incubation period from time of infection to diagnosis of AIDS was longer for pediatric TA AIDS cases than PA cases (median, 3.5 years vs 1.75 years) but shorter than for adult TA cases (median, 4.5 years). The median survival after diagnosis of TA AIDS in children did not differ from that in PA cases (13.7 vs 14.3 months) but was longer than in adult TA cases (5.6 months P less than .01). The decline in the reported incidence of pediatric and adult TA AIDS cases reflects the effects of donor deferral and donor screening for human immunodeficiency virus infection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727996 TI - Randomized trial of comprehensive prenatal care for low-income women: effect on infant birth weight. AB - The effect of comprehensive prenatal care on birth weight was examined using a prospective randomized design. A total of 428 pregnant women were randomly assigned to comprehensive prenatal care (n = 217) or standard prenatal care (n = 211). Comprehensive care was provided by a multidisciplinary team of nurse midwives, social workers, a nutritionist, paraprofessional home visitors, and a psychologist. Standard prenatal care consisted of medical care provided by obstetric residents. Multiple regression analysis using behavioral, demographic, and medical variables showed a strong relationship between the set of predictors and birth weight. Comprehensive care was related to higher birth weights for primiparous but not multiparous mothers. Separate analyses of variance for primiparas and multiparas similarly showed a favorable effect of comprehensive care on birth weight for primiparous but not multiparous mothers. PMID- 1727997 TI - Randomized European multicenter trial of surfactant replacement therapy for severe neonatal respiratory distress syndrome: single versus multiple doses of Curosurf. AB - There is now convincing evidence that the severity of neonatal respiratory distress syndrome can be reduced by surfactant replacement therapy; however, the optimal therapeutic regimen has not been defined. This randomized European multicenter trial was designed to determine whether the beneficial effects of a single large dose of Curosurf (200 mg/kg) in babies with severe respiratory distress syndrome (arterial to alveolar oxygen tension ratio approximately 0.10) could be enhanced by using multiple doses of surfactant. Preterm neonates (birth weight 700 to 2000 g) with severe respiratory distress syndrome requiring artificial ventilation with fraction of inspired oxygen greater than or equal to 0.6 were randomized into two groups at an age of 2 to 15 hours. Both groups received the usual dose of Curosurf (200 mg/kg) immediately after randomization. In neonates randomized to receive multiple-dose treatment, two additional doses of Curosurf (100 mg/kg each) were instilled into the airways (12 and 24 hours after the initial dose) provided that the patients still needed artificial ventilation with fraction of inspired oxygen greater than 0.21. In both groups (single dose: n = 176, multiple doses: n = 167) there was a rapid improvement in oxygenation as reflected by a threefold increase in arterial to alveolar oxygen tension ratio within 5 minutes after surfactant instillation (P less than .001), and peak inspiratory pressure and mean airway pressure could be reduced significantly during the first 6 hours after surfactant treatment. In addition, ventilatory requirement (peak inspiratory pressure, ventilatory efficiency index) was reduced in the multiple-dose group 2 to 4 days after randomization (P less than .05 to .01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1727998 TI - Controlled trial of nebulized albuterol in children younger than 2 years of age with acute asthma. AB - To determine the response to nebulized beta 2 agonist, 28 children younger than 2 years of age who visited the emergency department during an episode of acute asthma were studied. Each subject had a previous history of recurrent wheezing episodes. They were randomly assigned to receive two administrations of either nebulized albuterol (0.15 mg/kg per dose) or placebo (normal saline) with oxygen, 1 hour apart. After two nebulizations, the albuterol-treated patients had a greater improvement in clinical status (respiratory rate, degree of wheezing and accessory muscle use, total clinical score, and arterial oxygen saturation) than the placebo group. None of the patients in the albuterol group experienced a decrease of arterial oxygen saturation of greater than or equal to 2%. It is concluded that a trial of nebulized beta 2 agonists is warranted in the treatment of acute asthma in infants and young children. PMID- 1727999 TI - Long-term treatment of severe familial hypercholesterolemia in children: effect of sitosterol and bezafibrate. AB - Seven prepubertal children (age range 5.3 to 10.8 years) with severe heterozygous familial hypercholesterolemia (serum cholesterol concentration 416 +/- 85 mg/dL and low-density lipoprotein [LDL] cholesterol concentration 360 +/- 90 mg/dL) were first treated by dietary intervention, second by sitosterol (3 x 2 g/d), and third by bezafibrate (2 x 200 mg/d). Each treatment period lasted 3 months. Subsequently, a treatment combining half the dose of sitosterol and bezafibrate was administered for the following 24 months. Diet alone reduced total and LDL cholesterol values by 4.5% (not significant) and 6.6% (P less than .05), respectively. Sitosterol lowered total and LDL cholesterol values by 17% (P less than .05) when compared with diet alone. Compared with sitosterol, bezafibrate produced a more pronounced effect on total and LDL cholesterol values (-18% and 28%, P less than .05), and high-density lipoprotein cholesterol concentration increased significantly from 48 mg/dL to 55 mg/dL. Combined treatment with half the dose each of sitosterol and bezafibrate was as effective as the higher dose of bezafibrate, and reduction averaged almost 40% and 50% for total and LDL cholesterol values; this lipid-lowering effect persisted for the next 24 months. Laboratory safety parameters and physical examination revealed no obvious side effects. This study indicates that the combination of sitosterol (3 x 1 g/d) plus bezafibrate (1 x 200 mg/d) is an alternate, acceptable, safe, and effective therapeutic approach for treatment of severe hypercholesterolemia in children with high-risk familial hypercholesterolemia. PMID- 1728000 TI - Infant obesity, weight reduction with normal increase in linear growth and fat free body mass. PMID- 1728001 TI - Exchange transfusion in the treatment of severe theophylline poisoning. PMID- 1728002 TI - Repeated, childhood vaginal bleeding is not always precocious puberty. PMID- 1728003 TI - Influence of portagen and pregestimil on essential fatty acid status in infantile liver disease. PMID- 1728004 TI - Academically talented children: the case for early identification and nurturance. PMID- 1728005 TI - Do you remember E-Ferol? The penalty for selling untested drugs in neonatology: fines and a jail sentence. PMID- 1728006 TI - American Academy of Pediatrics Committee on Infectious Diseases: Chemotherapy for tuberculosis in infants and children. PMID- 1728007 TI - Ibuprofin safety. PMID- 1728008 TI - Location of port-wine stains and likelihood of complications. PMID- 1728009 TI - Allergic reactions to MMR vaccine. PMID- 1728010 TI - Home visitation investment. PMID- 1728011 TI - Psychosocial intervention. PMID- 1728012 TI - Perineal confusion. PMID- 1728013 TI - Questions on ECMO study. PMID- 1728014 TI - A different view of surfactant development. PMID- 1728015 TI - Increased incidence of asthma in children of smoking mothers. AB - The relationship between parental smoking and both subsequent development of asthma and subsequent lung function (before age 12) was studied in more than 700 children enrolled before age 5. Children of mothers with 12 or fewer years of education and who smoked 10 or more cigarettes per day were 2.5 times more likely (95% confidence interval 1.42 to 4.59; P = .0018) to develop asthma and had 15.7% lower maximal midexpiratory flow (P less than .001) than children of mothers with the same education level who did not smoke or smoked fewer than 10 cigarettes per day. These relationships were independent of self-reported respiratory symptoms in parents. There was no association between maternal smoking and subsequent incidence of asthma or maximal midexpiratory flow among children of mothers with more than 12 years of education. It is concluded that children of lower socioeconomic status may be at considerable risk of developing asthma if their mothers smoke 10 or more cigarettes per day. It is speculated that recently reported increases in prevalence of childhood asthma may be in part related to the increased prevalence of smoking among less educated women. PMID- 1728016 TI - Candy cigarettes: do they encourage children's smoking? AB - Candy and bubble gum cigarettes are packaged to resemble cigarette brands, and so they may encourage young children to smoke. Two studies of the role of these products in the development of children's attitudes and behaviors toward smoking were conducted. In the first study, six focus group interviews were conducted with 25 children in three age groups (4 through 5, 6 through 8, and 9 through 11 years old). Children in each group were shown five candy and snack foods and asked about their opinions and experiences with each item. In the second study, 195 seventh-grade students in a southeastern city school system were surveyed about their cigarette smoking and candy cigarette use. In the focus groups, candy cigarettes were recognized by most children. Young children played with the candy cigarettes more than with other candy or snack items and made general references to smoking behaviors. Older children made favorable references to smoking behavior; most knew which stores sold candy cigarettes, and many had chosen to buy and use these items, despite parental disapproval. Candy cigarettes may play a role in the development of children's attitudes toward smoking as an acceptable, favorable, or normative behavior. Elimination of these products should be part of efforts to prevent initiation of smoking by children. PMID- 1728017 TI - Pediatric telephone advice in the emergency department: results of a mock scenario. AB - To assess the appropriateness of pediatric telephone advice given by emergency departments (EDs), a mock scenario simulating a 5-week-old with fever of 102 degrees F and signs compatible with meningitis was used to evaluate the responses of 61 randomly selected EDs, of which half were affiliated with pediatric residency training programs. All EDs were given the identical chief complaint: "My baby has been having a fever all day, and I can't seem to get it down." Calls were made by research technicians and monitored by one or more of the investigators by speakerphone. Fifty-three (87%) programs gave advice by telephone; in 42 (79%) of the 53 respondents the individual giving advice was a nurse. Fourteen programs (26%) gave advice without asking either the age of the child or height of the fever. Few of the respondents took historical information assessing irritability (4 programs), fluid intake (11 programs), urine output (8 programs), or breathing pattern (6 programs). Thirty-eight (71.7%) EDs advised the patient to see a physician, while only 32 (60.4%) suggested same-day evaluation. In several instances the caller was not advised to seek medical attention despite having given a history documenting the infant's fever, irritability, and lethargy. These findings show variability and inadequacies in pediatric telephone advice currently offered by the EDs that were studied. PMID- 1728018 TI - Attitudes of teenagers toward sun exposure and sunscreen use. AB - A survey of 220 adolescents attending a multiphysician pediatric office in Virginia was conducted to determine the frequency with which they used sunscreens. Eighty-one percent of the teenagers in the study stated that they spent most weekends in the sun; however, only 9% always used sunscreen, while 33% never did. Factors found to be associated with increased sunscreen use included female sex (odds ratio = 4.5, P less than .0001), having a best friend who routinely used sunscreen (odds ratio = 3.0, P less than .001), having parents who insisted on sunscreen use when the teenagers were children (odds ratio = 3.0, P = .006), and knowing that the maximum time for safe exposure to the sun is short (odds ratio = 6.2, P less than .0001). Adolescents with a history of skin cancer in the family were not more likely to use sunscreens than other teenagers. Thirty three percent of the girls and 16% of the boys older than 15 years of age reported that they had visited a tanning salon at least once. This survey substantiates poor compliance with sunscreen use by teenagers despite increasing evidence of the dangers of excessive sun exposure. PMID- 1728019 TI - Tissue-type plasminogen activator, plasminogen activator inhibitor, and histidine rich glycoproteins in stressed human newborns. AB - Normal newborns have reduced levels of tissue-type plasminogen activator (tPA). Stressed newborns have an increased prevalence of thrombotic diseases. An impaired release of tPA and/or increased plasminogen activator inhibitor (PAI) is associated with thrombotic risk in the adult patient. The purpose of this study was to assay the plasma levels of tPA, PAI, histidine-rich glycoprotein (HRG), and other fibrinolytic proteins in 15 severely stressed newborns. The stressed babies showed significantly higher (p less than .001) levels of tPA antigen compared with normal newborns. Also, PAI activity and PAI-1 antigen levels were increased. Levels of both HRG and plasminogen were higher in the stressed group but the ratio of HRG to plasminogen was the same as that in the normal control newborns (1:3), suggesting an insignificant effect of HRG. D-dimers were significantly elevated in the stressed newborns. However, 8 patients died and 4 of these were found to have massive thrombotic disease on autopsy. These results show that the newborn when stressed will increase tPA levels and activate the lytic system. However, the activity is suboptimal inasmuch as PAI activity did not decrease and thrombotic disease was observed. PMID- 1728020 TI - Dobutamine pharmacokinetics and cardiovascular responses in critically ill neonates. AB - The pharmacokinetics and pharmacodynamics of dobutamine were studied in 13 critically ill neonates requiring inotropic support. Dobutamine was administered as a constant infusion in increasing doses of 2.5, 5, and 7.5 micrograms/kg per minute. During dobutamine infusions, there were significant increases in cardiac output measurements above perinfusion values. There were no statistically significant changes in systolic or diastolic blood pressure or heart rate during the infusions. The mean calculated threshold value, or the minimum plasma concentration necessary for a change in cardiac output, was 39 +/- 8 ng/mL. The mean plasma clearance rate was 90 +/- 38 mL/min per kilogram and was most consistent with first-order kinetics over the range of dosages studied. Plasma catecholamine levels were unchanged during the dobutamine infusions. These data suggest that dobutamine is an effective but limited inotropic agent in the neonate. Dobutamine may be most beneficial when cardiogenic failure is presented. PMID- 1728021 TI - Prospective randomized comparison of high-frequency oscillatory and conventional ventilation in respiratory distress syndrome. AB - A prospective randomized trial with a crossover design was conducted to compare the efficacy and safety of two distinct strategies of high-frequency oscillatory ventilation (HFOV) to conventional intermittent mandatory ventilation (CV) in the management of respiratory distress syndrome. Only premature neonates with a birth weight less than 1.751 kg were eligible for enrollment into the study. Of 83 patients studied, 26 patients were assigned to CV-only, 27 to HFOV for 72 hours followed by CV (HFOV/CV), and 30 to HFOV-only until extubation. There was no difference among the three groups with respect to the incidence of pulmonary airleak, intraventricular hemorrhage, or death. The highest incidence of chronic lung disease was in the CV-only group. Although both HFOV groups had a lower incidence of chronic lung disease assessed at 30 days and 36 weeks postconception age, the difference was statistically significant only between the CV-only and HFOV-only groups (65% vs 30% at 30 days; P = .008; 38% vs 10% at 36 weeks postconception age, P = .013). These results suggest that use of HFOV as the predominant mode of ventilation in the management of respiratory distress syndrome is as safe as CV and can contribute to a decreased incidence of chronic lung disease. Furthermore, a short (72-hour) period of HFOV support does not provide the same advantage as continuous HFOV. PMID- 1728022 TI - Primary care physicians' knowledge about diphtheria-tetanus-pertussis immunizations in preterm infants. AB - Preterm infants often receive their diphtheria-tetanus-pertussis (DTP) immunizations on a delayed schedule or in reduced dosage. Since primary care physicians (PCPs) immunize many preterm infants, the purpose of this study was to describe PCPs' knowledge about the use of DTP immunizations in preterm infants. Among the 479 PCPs who completed the questionnaire, 84% of pediatricians and 60% of family physicians correctly identified chronologic age as a criterion for initiating DTP immunizations in preterm infants. However, nearly 45% of PCPs linked this with other criteria such as a minimum weight requirement. Family physicians' answers differed from the recommendations more often than pediatricians' answers. The answers of pediatricians and family physicians who completed residency greater than 20 years ago differed from the recommendations more often than those who completed training less than or equal to 20 years ago. The answers of PCPs with fewer than five preterm infants in their practices differed from the recommendations more frequently than the answers of those with five or more preterm infants in their practices. Educational interventions are needed to bring PCPs' knowledge and practices into compliance with the American Academy of Pediatrics recommendations concerning DTP immunizations for preterm infants. PMID- 1728023 TI - Covering the costs of care in neonatal intensive care units. AB - The continued rise of health care costs, despite private and governmental control efforts, has sustained cost containment as a central issue for health care researchers and policy makers. In keeping with these concerns, the Florida Health Care Cost Containment Board conducted a study of neonatal intensive care units (NICUs) in Florida to ascertain the costs, charges, and net revenues associated with NICU services in individual hospitals, to document cost shifting and cross subsidization as a means of financing NICU care for indigent populations, and to assess the fiscal impact of NICUs in state-sponsored vs non-state-sponsored Regional Perinatal Intensive Care Center hospitals providing NICU care. Hospitals in the state-sponsored program reported a loss of approximately $16.5 million in contrast to the non-state-sponsored hospitals, which reported a gain of $1 million. Payment being generated by private-pay patients amounted to almost 60% of total revenues but constituted less than one third of the costs in state sponsored hospitals, indicating a high level of cost shifting. Government support of state-sponsored NICUs, while substantial, has been insufficient; increasing constraints on this funding source would likely worsen the deficit and increase the necessity of cost shifting. PMID- 1728024 TI - Comprehensive computerized neonatal intensive care unit data system including real-time, computer-generated daily progress notes. AB - A neonatal intensive care unit patient data system, NeoData, which was developed using microcomputers connected by a local area network, is described. The system allows for real-time generation of daily progress notes, as well as admission and discharge summaries. It includes two databases: one for daily patient data and one for admission/discharge summary data. Both sets of data are easily accessible for later analysis and report generation. The daily patient data are entered directly into a computer by the neonatal intensive care unit medical and nurse practitioner staff; a progress note is printed immediately thereafter for inclusion in the patient's chart. Data from the previous day are selectively carried forward into the current day's note, minimizing data entry. Several benefits are derived from this progress note system, including legibility, tracking of laboratory and other data, tracking of management plans and procedures due at a later date, and significant time savings. The system has proved to be easy to learn, and the neonatal intensive care unit staff have found it to contribute to the efficient delivery of patient care. PMID- 1728025 TI - Analysis of facial shape in children gestationally exposed to marijuana, alcohol, and/or cocaine. AB - The association between fetal marijuana and/or alcohol exposure and facial features resembling fetal alcohol syndrome was investigated in a sample of 80 children. Standardized lateral and frontal facial photographs were taken of 40 children, 5 to 7 years of age, whose mothers reported frequent use of marijuana during the first trimester of pregnancy and 40 children whose mothers reported no use of marijuana during pregnancy. The marijuana-exposed and unexposed children were group-matched on alcohol exposure prior to and during pregnancy, sex, race, and age at the time of assessment. The photographs were assessed clinically by a study staff dysmorphologist and morphometrically by computerized landmark analysis. Fetal alcohol syndrome-like facial features were not associated with prenatal marijuana exposure in this study sample. No consistent patterns of facial features were identified among the marijuana-exposed group. Maternal consumption of two or more ounces of alcohol per day, on average, in early gestation was found to be associated with fetal alcohol syndrome-like facial features identified both clinically and morphometrically. Cocaine use reported by 13 of the 80 women was independently associated with mild facial dysmorphic features of hypertelorism and midfacial flattening. The results demonstrate the usefulness of this diagnostic technique for quantifying anomalies apparently unique to fetal alcohol syndrome and for targeting clusters of anomalies in new conditions for future evaluation. PMID- 1728026 TI - Trends in bicycle helmet use by children: 1985 to 1990. AB - Research has demonstrated that helmets protect against head injury during bicycle crashes. Several investigators have shown that large-scale, community-wide programs can increase the rate of helmet use by children. The objective of this research was to determine whether helmet use had changed over a 5-year period in a community with no formal programs designed to increase the use of helmets. In 1985 and again in 1990, project staff observed student bicyclists arriving at four elementary schools, three middle schools, three high schools, and one university campus. The same schools were used both years. There was no significant increase in the percentage of students who used helmets at the middle schools (0 both years), the high schools (1.85% vs 1.45%), or the university (10.0% vs 4.0%). At the four elementary schools, helmet use increased from 1.85% in 1985 to 17.1% in 1990. Much of this increase was attributable to one school at which helmet use increased from 4.4% to 21.4%. This school, and no others, had begun teaching about helmet use in the classroom. The results suggest that (1) helmet use will not increase at the middle school level or higher without specific interventions and (2) simple, low-cost, classroom interventions can increase helmet use by elementary school children. PMID- 1728027 TI - Genetic causes of hydrops fetalis. AB - A series of 1790 fetal and neonatal autopsies performed between 1976 and 1988 were retrospectively investigated for the presence of hydrops. Thirty (5.5%) and 35 (2.8%) cases of hydrops were found in the groups of fetal and neonatal autopsies, respectively. Genetic causes accounted for 35%. A careful search for previously reported genetic causes of fetal hydrops indicated 64 different etiologies. Twenty-one of them were not mentioned in the previous reviews: these include 9 skeletal dysplasias, 5 inborn errors of metabolism, 3 autosomal recessive, 3 autosomal dominant conditions, and 1 chromosomal abnormality. PMID- 1728028 TI - Lead intoxication in infancy. AB - Four years of experience in the evaluation and management of lead intoxication in the first year of life were reviewed. This study was conducted in a lead referral program within the state of Massachusetts, whose comprehensive lead laws include extensive (and now mandatory) lead screening of all children. Over the period of study, 50 (14%) of 370 new patients enrolled in the program were infants aged 12 months or younger. Median age of these infants was 11 months (range 1 through 12 months). Mean peak lead level was 39.0 micrograms/dL while the mean peak erythrocyte protoporphyrin concentration was 111.9 micrograms/dL of whole blood. Thirty-two percent of infants were ambulatory at the time lead intoxication was diagnosed; only 24% had a history of pica. Twenty-six percent of parents were welfare dependent. Apparent sources of plumbism included house-hold renovation (n = 20), direct ingestion of paint chips (n = 10), formula preparation with lead contaminated water (n = 9), lead dust importation (n = 1), and congenital exposure to elevated maternal lead level (n = 1). In 9 cases the source was not found. When this profile was compared with that of a randomly selected group of 47 children aged 18 through 30 months, who were seen in the lead program during the same interval, apparent sources of intoxication in the older group were paint chip ingestion (n = 41), household renovation (n = 2), and unknown (n = 4) (P less than .0001). On the basis of these data, it is concluded that lead intoxication in infants is common and has significantly different origins from that in toddlers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728029 TI - Effect of thyroxine and chicken growth hormone on immune function in autoimmune thyroiditis (obese) strain chicks. AB - The effect of thyroxine (T4) and/or recombinant chicken growth hormone (rcGH) supplementation on immune function and on immune cell maturation was examined in Obese strain chickens. Day-old Obese strain chicks received the control treatments or were treated with either T4 (supplemented in the diet), T4-rcGH, or rcGH (by daily injection) in a full factorial design. At 4 weeks of age, the proliferative activity of peripheral blood T cells to either mitogenic or allogenic cell (mixed lymphocyte response) challenge was assessed. At the same time, peripheral blood lymphocytes and thymocytes were collected and prepared for flow cytometry analysis. Proliferative responses to both T cell mitogens and allogeneic splenocytes were significantly increased (P less than 0.05) by rcGH treatment, while the combined T4-rcGH treatment resulted in a significant increase in allogeneic and in concanavalin A responsiveness, but not in the response to phytohemagglutinin. All supplemented groups showed a significant decrease in the mean fluorescent intensity for CT-1a+ thymocytes, while thymocytes from birds receiving either T4 or rcGH alone had higher proportions of CD4+ and CD8+ cells. The monoclonal antibody staining of thymocytes from T4-rcGH supplemented animals more closely resembled that of the unsupplemented controls. Among the peripheral blood lymphocytes, there were no changes in the numbers of CD4+, CD8+, or sIg+ cells as a result of treatment. The mean fluorescent intensity of sIg+ cells was significantly decreased, however, as a result of T4 supplementation when given either alone or in combination with rcGH. Finally, the mean fluorescent intensity ratios of CD4+ to CD8+ cells was significantly increased as a result of rcGH supplementation. These results strongly support a role for both the thyroid hormones and growth hormone in regulating and/or enhancing immune function, with changes in functional responses paralleled by concomitant changes in the T cell populations as expressed by shifts in T cell surface marker expression. PMID- 1728030 TI - Effects of H-2 and vitamin A on eye defects in congenic mice. AB - Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A(2R), B10.A(5R), B10.A(15R), B10.A(1R), B10.A(18R), and B10.OL) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the 18th day of gestation and the fetuses were sexed and examined for defects in eye development. It was found that the frequency of microphthalmia and anophthalmia in the female progeny of mice fed Mouse Chow was 7.4-9.2% in B10.A and B10.BR, 4.0-5.5% in B10.A(18R), B10, B10.A(5R), B10.A(1R), B10.A(15R), and B10.A(2R), and 0.8% and 1.4% in B10.D2 and B10.OL mice, respectively. On average, the frequency of these defects in the female progeny was 6.2 times greater than that in males (P less than 0.001). The right eye was 5.8 times more often affected than the left (P less than 0.001). The addition of vitamin A to the diet increased the frequency of these eye abnormalities in all strains, suggesting that this effect is not mediated by loci associated with H-2, as is the case with vitamin A-enhanced cleft palate. The addition of vitamin A to the diet did not affect the ratios of affected males to females, affected right to left eye, or microphthalmia to anophthalmia. The results suggest that there are two loci on chromosome 17, one centromeric to E beta and one telemeric to C4, that interact to determine to some degree the frequency of microphthalmia and anophthalmia. PMID- 1728032 TI - Bioavailability of bound pesticide residues and potential toxicologic consequences. PMID- 1728031 TI - The effect of a new calcium channel blocker (TA-3090) on lipoprotein profile and intestinal lipid handling in rodents. AB - Recent interest has focused on findings that drugs used to lower blood pressure may adversely modify plasma lipids and lipoprotein metabolism. This observation may explain why pharmacologic control of hypertension has failed to reduce the incidence of morbidity and mortality from coronary artery disease. The present study aims to evaluate the effect of TA-3090, a new calcium channel blocker, on fasting plasma lipids and lipoproteins, as well as on processes of intestinal fat absorption. Rats were treated by gavage with TA-3090 (10 mg/kg twice daily) for 4 days and compared with controls (n = 6 per group). Plasma cholesterol was increased in the treated group to (mean +/- SE) 74 +/- 2 vs 60 +/- 4 mg/dl (P less than 0.01), due mainly to an increased high density lipoprotein-cholesterol level (50 +/- 2 vs 37 +/- 3 mg/dl, P less than 0.005). Notably plasma triglycerides (TG) and low density lipoprotein-cholesterol were not significantly affected. Another group of TA-3090-treated animals was given an intraduodenal fat meal, and the rise in plasma TG and chylomicrons followed over 4 hr. Postprandial hypertriglyceridemia and chylomicronemia were significantly lower at 2 hr (P less than 0.05) and 3 hr (P less than 0.01) compared with controls. In a separate group of animals, the addition of TA-3090 to a 2% intralipid infusion intraduodenally was associated with significantly reduced TG and chylomicron-TG transport into lymph (P less than 0.05). Furthermore, experiments in rats pretreated with TA-3090 intraperitoneally and then given 2% intralipid intraduodenally were shown to have a significant decrease in mean flow rate (27%), TG transport (31%) and chylomicron-TG output (37%), when compared with controls. In vitro studies using jejunal organ culture to examine the effect of TA-3090 on intracellular lipid synthesis and secretion revealed that the addition of the drug to the medium resulted in significantly decreased TG synthesis and secretion. These data suggest that TA-3090 could be effective in increasing HDL cholesterol and reducing postprandial chylomicronemia. Our findings support a role for TA-3090 directly on enterocyte absorption and/or intracellular lipid transport, and thus indicate the importance of intracellular calcium on these processes. PMID- 1728033 TI - Early pathological changes of endothelia in a model using LDL perfusion at physiological LDL-cholesterol concentration. AB - The aim of the present research was to provide further insight into the debated problem of the existence of modified LDL in vivo. For this purpose a novel model was devised for studying LDL injurious effect on endothelial cells (EC) by infusing native cholesterol rich LDL, diluted to physiological LDL cholesterol concentration. Normal rabbits were infused with LDL separated from rabbits previously fed either with standard food (I-LDL Group), 1% cholesterol (II-LDL Group) or 1% cholesterol plus probucol (IV-LDL Group). Cu++ modified II-LDL was infused as well (III-LDL Group). After dilution as above, lipid oxide (LP) significantly increased in III- and II-LDL media, as compared to I- and IV-LDL media. EC of III- and II-LDL Groups showed irregular shape and surface pattern. Further, they showed adhering clusters of monocytes, platelets and erythrocytes. Endocytic vesicles and ruthenium red-positive particles increased too. EC of IV LDL Group were only slightly affected as compared to I-LDL Group. These data suggest that native LDL from hypercholesterolemic rabbits contain an oxidized form which is noxious to EC even when LDL is infused at physiological LDL cholesterol concentration. This early injury is in part LP-associated and actively involves platelets and monocytes. PMID- 1728034 TI - Microenvironmental factors that influence mast cell phenotype and function. PMID- 1728035 TI - Resistance to outflow of cerebrospinal fluid after central infusions of angiotensin. AB - Infusions of artificial cerebrospinal fluid (CSF) into the cerebroventricles of conscious rats can raise CSF pressure (CSFp). This response can be modified by some neuropeptides. One of these, angiotensin, facilitates the rise in CSFp. We measured CSFp in conscious rats with a computerized system and evaluated resistance to CSF outflow during infusion of artificial CSF, with or without angiotensin, from the decay kinetics of superimposed bolus injections. Angiotensin (10 ng/min) raised CSFp (P less than 0.05) compared with solvent, but the resistance to CSF outflow of the two groups was similar (P greater than 0.05). Because CSFp was increased by angiotensin without an increase in the outflow resistance, a change in some volume compartment is likely. Angiotensin may raise CSFp by increasing CSF synthesis; this possibility is supported, since the choroid plexuses contain an intrinsic isorenin-angiotensin system. Alternatively, angiotensin may dilate pial arteries, leading to an increased intracranial blood volume. PMID- 1728036 TI - Effects of bile salt infusion on chlorpromazine-induced cholestasis in the isolated perfused rat liver. AB - The present study has demonstrated that tauroursodeoxycholate (TUDC), but not taurocholate, can reverse chlorpromazine (CPZ)-induced cholestasis in the isolated perfused rat liver. At an infusion rate of 1.5 mumol/min, TUDC led to restoration of bile flow in the perfused rat liver made cholestatic by the addition of 250 microM CPZ. This reversal was accompanied by an increased excretion of CPZ and its metabolites. A higher infusion rate of 5.0 mumols TUDC/min, however, led to only a transient increase in bile flow and to no increase in CPZ excretion. In contrast to the effects of TUDC, infusion of taurocholate led to an exacerbation of CPZ-induced cholestasis. The differences in the efficacy of the two bile salts may be due to their relative detergent (hydrophobic) properties. PMID- 1728037 TI - Drug-induced cholestasis in the perfused rat liver and its reversal by tauroursodeoxycholate: an ultrastructural study. AB - Chlorpromazine at a concentration of 250 microM and estradiol-17 beta-D glucuronide at 17.5 microM on infusion led to a sharp reduction in bile flow by the in vitro perfused rat liver. This was accompanied by fragmentation and a loss of canalicular microvilli, dilatation of canaliculi, and thickening of pericanalicular ectoplasm. Less prominent were the smooth endoplasmic reticulum dilatation, lysosomal lamination, and the appearance of amorphous bile in hepatocyte cytoplasm. The bile flow and electron microscopy appearance were restored to normal by infusion of tauroursodeoxycholate in a concentration of 5 mumols/min for the estradiol-17 beta-D-glucuronide-induced cholestasis and 1.5 mumol/min for the chlorpromazine-induced cholestasis. Changes in ultrastructure paralleled changes in bile flow. These observations demonstrate the feasibility of electron microscopy studies on the perfused liver, and the rapidity with which cholestatic changes appear. PMID- 1728038 TI - Glomerular function in spontaneously diabetic rats. AB - This study was undertaken to determine whether hyperfiltration exists at the single nephron level and whether albumin excretion is increased early in the course of diabetes in Biobreeding rats. Diabetic rats were studied at 8-12 weeks after the onset of diabetes. Control animals were age-matched, diabetes-resistant rats. Urinary and tubular fluid albumin concentrations were measured by polyacrylamide gel electrophoresis. Clearance and micropuncture techniques were used to determine whole kidney and single nephron glomerular filtration rate, renal blood flow, and glomerular capillary pressure. The urinary albumin excretion rate (1.3 +/- 0.1 mg/24 hr) and the tubular fluid albumin concentration (4.7 +/- 0.7 mg/dl) in the diabetic group were significantly elevated when compared with urinary albumin excretion (0.9 +/- 0.1 mg/24 hr) and tubular fluid albumin concentration (2.5 +/- 0.5 mg/dl) in the control group. There were no significant differences in glomerular hemodynamics (whole kidney or single nephron glomerular filtration rate or glomerular capillary pressure) between diabetic and control rats. The kidney weight and kidney weight to body weight ratio were significantly higher in diabetic rats when compared with control rats. Early diabetes in Biobreeding rats is characterized by mild albuminuria and increased kidney size, but not glomerular hyperfiltration. PMID- 1728039 TI - Extracellular volume estimation from ratios of bromide to chloride in urine or saliva. AB - Use of either urine or saliva samples to estimate extracellular water volume was investigated in 10 men using nonradioactive bromide (Br) and in seven newborn piglets using radioactive Br (82Br) and chloride (36Cl). The relation to Br to Cl concentrations in urine enabled an estimation of Br dilution volume from human urine (267 +/- 42 ml/kg, mean +/- SD) that was not significantly different (P = 1.0) from the Br dilution volume calculated from plasma Br concentration (268 +/- 20 ml/kg). Although the Br dilution volume estimated from saliva was not different from that of plasma, the error in the estimates of Br dilution volume from saliva was too large (mean difference, -36 +/- 64 ml/kg) to make its use practical. The data from piglets showed good agreement between 82Br and 36Cl dilution volumes calculated from 4-hr plasma samples (356 +/- 14 ml/kg and 347 +2 12 ml/kg; P greater than 0.1) and between 82Br dilution volumes calculated from urine 82Br:36Cl and plasma 82Br (360 +/- 31 ml/kg and 356 +/- 14 ml/kg; P greater than 0.1). Extracellular water volume can be estimated in both adult and young animals using the Br dilution volume calculated from urine samples. It requires (i) two urine collections: one before and one 4 to 8 hr after administration of Br; (ii) a measurement or estimate of plasma Cl concentration; and (iii) a correction factor that describes the relationship of the ratio of Br to Cl in urine to that ratio in plasma. PMID- 1728040 TI - Effect of short-term fasting/refeeding on epidermal growth factor content in the gastrointestinal tract of suckling rats. AB - Epidermal growth factor (EGF) is trophic for varying regions of the developing gastrointestinal tract (GIT) of suckling rats. The presence of large amounts of EGF in milk from various species, combined with low production of EGF by suckling animals, led to speculation that milk is a major source of EGF for suckling rats. We report that short-term fasting (8 hr) of 12-day-old suckling rats resulted in a significant decrease in the levels of immunoreactive EGF (irEGF) in the GIT. Pups refed by lactating mothers for 1 to 4 hr exhibited an increase in irEGF to original levels, whereas pups fed a rat milk substitute by gastric gavage did not have an increase in irEGF content. The irEGF levels in the GIT of pups that were manually fed normal rat milk, or rat milk substitute supplemented with EGF, returned to the prefasted levels. Fasted suckling rats refed 2 ml of rat milk in 2 h exhibited significantly higher level of irEGF in the GIT than did those refed with 0.5 ml in 45 min. Since rat milk irEGF exists in three distinct forms (A, B, and C; C is equal to authentic submandibular gland EGF, the irEGF forms in the GIT were characterized by native polyacrylamide gel electrophoresis. In the stomach luminal contents of the fed suckling rats, only the larger form, Peak B, was observed. Both the luminal content and the mucosa scrapings of all other segments of all groups contained only Form D (comigrating with desarginyl EGF), a metabolic derivative of EGF. All forms were immunoreactive, exhibited receptor binding, and stimulated DNA synthesis in growth-arrested fibroblasts. The rapid changes in EGF within the GIT of suckling rats suggest the EGF can acutely modify some GIT functions of suckling rats. PMID- 1728041 TI - Rates and tissue sites of noninsulin- and insulin-mediated glucose uptake in diabetic rats. AB - The purpose of the present study was to determine whether streptozotocin-induced diabetes alters the rates and tissue distribution of insulin-mediated glucose uptake (IMGU) and noninsulin-mediated glucose uptake (NIMGU). In vivo glucose disposal was assessed using the tracer [U-14C]-2-deoxyglucose technique in chronically catheterized conscious rats. For nondiabetic animals, rates of NIMGU were determined during severe insulinopenia (less than 5 microU/ml), induced by the infusion of somatostatin, under both euglycemic (6 mM) and hyperglycemic (17 mM) conditions. In diabetic rats, in which a severe insulin deficiency already existed, NIMGU was determined under basal hyperglycemic conditions and during euglycemic conditions produced by inhibiting hepatic glucose output. IMGU was determined in both groups using the euglycemichyperinsulinemic clamp technique. Glucose uptake was consistently higher (50-280%) in all tissues removed from diabetic rats under basal conditions, compared with tissues from control animals in the basal state. When control animals were rendered insulinopenic, glucose uptake by the skeletal muscle, heart, and diaphragm was reduced 30-60%, indicating that the uptake by these tissues occurred by both insulin- and noninsulin-mediated mechanisms. Glucose disposal by the other tissues sampled was entirely due to NIMGU under basal conditions. When blood glucose levels were elevated from 6 to 17 mM in control animals, NIMGU increased in all tissues (60 280%) except the brain. Rates of NIMGU were essentially identical between control and diabetic animals, under either euglycemic or hyperglycemic conditions, when glucose uptake was determined under the same steady-state plasma glucose levels. In contrast to the normal rate of NIMGU by muscle, IMGU by the skeletal muscle and heart from diabetic rats were reduced under mild hyperinsulinemic conditions (100 microU/ml), compared with control animals. Furthermore, in response to a maximal, stimulating dose of insulin (500 microU/ml), IMGU was impaired in the diaphragm, liver, lung, spleen, skin, and kidney removed from diabetic animals. These results indicate that the majority of glucose disposal under basal postabsorptive conditions occurs by NIMGU in both control and diabetic rats. Furthermore, while IMGU was selectively impaired in this model of insulin dependent diabetes, the rates and tissue distribution of NIMGU were unaltered when glucose uptake was determined under similar plasma glucose levels. PMID- 1728042 TI - Effects of ritanserin on transmembrane action potentials in canine Purkinje fibers. AB - Ritanserin has been reported to be a potential antiarrhythmic. We studied the cellular electrophysiologic effects of ritanserin in canine Purkinje fibers. Ritanserin produced significant depressant effects on transmembrane action potentials elicited in canine Purkinje fibers. At concentrations of 10 and 40 mg/liter, ritanserin decreased Vmax (the upstroke velocity) of action potential in a dose-dependent fashion and shortened the duration of fast response action potential. These concentrations of ritanserin also reduced the amplitude and duration of the slow response action potentials induced in Purkinje fibers treated with isoproterenol (10(-5) M) and high K+ (22 mM). These in vitro results suggest that the cellular electrophysiologic actions of ritanserin may be due to its direct actions on cardiac sodium and calcium channels, which, in turn, may account for its antiarrhythmic effects. PMID- 1728043 TI - Measurement of urinary clusterin as an index of nephrotoxicity. AB - Measurements of tissue immunoassayable clusterin, a protein associated with programmed cell death and tissue reorganization, were performed in rats treated with nephrotoxic doses of gentamicin sulfate. Adult Lewis rats were treated with 100 mg/kg/day of gentamicin sulfate for 12 days. Urine, serum, and tissue levels of clusterin protein were measured, as were urinary N-acetyl beta-glucosaminidase (NAG) and serum creatinine levels. Induction of renal injury by gentamicin was detectable within 4 days by increased levels of urinary N-acetyl beta glucosaminidase (from 280 +/- 66 (mean +/- SD) to 910 +/- 210 nmol/mg creatinine), and within 9 days of initiating gentamicin treatment by increased serum creatinine (from 0.5 +/- 0.1 to 1.2 +/- 0.4 mg/dl). Paralleling these changes, renal, urinary, and serum levels of clusterin increased 10-, 116-, and 3 fold (P less than 0.05). Treatment with gentamicin sulfate did not increase clusterin levels in the seminal vesicle, ventral prostate, testis, or epididymis. The measurement of urinary or serum clusterin may play a role in the early detection of renal injury. PMID- 1728044 TI - Lower-extremity surgery for children with cerebral palsy: physical therapy management. AB - The purpose of this article is to discuss physical therapy for children with cerebral palsy who undergo orthopedic surgery. Children with spasticity (increased tone) often undergo surgical procedures to increase the length of the hip, knee, and ankle musculature in an attempt to improve musculoskeletal alignment and functional abilities. Presurgical assessment of posture and movement to determine potential for change in function and postsurgical management are discussed. Intervention immediately following soft tissue surgery at the hips and knees and intervention at the time of cast removal for those children immobilized in a hip spica cast are reviewed. Specific postsurgical management protocols related to immobilization in splints/casts, positioning, and treatment activities are presented. PMID- 1728045 TI - The Ilizarov procedure: limb lengthening and its implications. AB - The purpose of this article is to provide a historical and clinical perspective on the Ilizarov method of external fixation for limb lengthening and deformity correction of the lower extremity. Though relatively new in the United States, the technique has been applied for orthopedic problems with great success for over three decades in Russia and Europe. Physical therapy management is discussed from the preoperative planning phase to removal of the apparatus. PMID- 1728046 TI - Assessment of lower-extremity alignment in the transverse plane: implications for management of children with neuromotor dysfunction. AB - The authors present nine clinical assessment procedures designed to detect factors contributing to transverse-plane structural and joint alignment abnormality in children with neuromotor dysfunction. Where applicable, each assessment is accompanied by a discussion of the normal features of pediatric lower-extremity torsional and rotational alignment; limitations of the assessment procedures as regards reliability, specificity, and age-related normative findings; and clinical management suggestions. The authors urge clinical evaluators to expand the existing knowledge base. PMID- 1728047 TI - Considerations related to weight-bearing programs in children with developmental disabilities. AB - Standing is a common modality used in the management of children with developmental disabilities. The purpose of this article is to examine the scientific basis for standing programs, with specific emphasis on the known effects of weight bearing on bone development. Guidelines for the use of standing programs are presented, and the supporting rationale is discussed. PMID- 1728048 TI - Foot trajectory in human gait: a precise and multifactorial motor control task. AB - The trajectory of the heel and toe during the swing phase of human gait were analyzed on young adults. The magnitude and variability of minimum toe clearance and heel-contact velocity were documented on 10 repeat walking trials on 11 subjects. The energetics that controlled step length resulted from a separate study of 55 walking trials conducted on subjects walking at slow, natural, and fast cadences. A sensitivity analysis of the toe clearance and heel-contact velocity measures revealed the individual changes at each joint in the link segment chain that could be responsible for changes in those measures. Toe clearance was very small (1.29 cm) and had low variability (about 4 mm). Heel contact velocity was negligible vertically and small (0.87 m/s) horizontally. Six joints in the link-segment chain could, with very small changes (+/- 0.86 degrees - +/- 3.3 degrees), independently account for toe clearance variability. Only one muscle group in the chain (swing-phase hamstring muscles) could be responsible for altering the heel-contact velocity prior to heel contact. Four mechanical power phases in gait (ankle push-off, hip pull-off, knee extensor eccentric power at push-off, and knee flexor eccentric power prior to heel contact) could alter step length and cadence. These analyses demonstrate that the safe trajectory of the foot during swing is a precise endpoint control task that is under the multisegment motor control of both the stance and swing limbs. PMID- 1728049 TI - Treatment of limited shoulder motion using an elevation splint. AB - This article describes the management of a patient with limited shoulder range of motion (ROM) by use of an elevation splint. The limited ROM was believed to be due to structural changes in the tissues surrounding the glenohumeral joint following a Magnuson-Stack repair for anterior glenohumeral instability. The patient's ROM plateaued approximately 6 months postoperatively and did not improve with a variety of physical therapy techniques. Use of an inexpensive, easily fabricated elevation splint was begun 8 months postoperatively, and subsequent improvements in ROM were observed. The rationale and suggestions for clinical use of the splint are discussed. PMID- 1728050 TI - Effect of a single 30-minute treatment of high voltage pulsed current on edema formation in frog hind limbs. AB - The purpose of this study was to investigate the effect of a single treatment of high voltage pulsed current (HVPC) on edema formation. Twenty-four frogs were anesthetized, and both hind limbs of each frog were traumatized by impact. Limb volumes were measured by water displacement immediately before and after trauma and at predetermined intervals for 24.0 hours posttrauma. One limb of each frog was randomly selected to receive 30 minutes of continuous, 120-pulse per second, cathodal HVPC at voltages 10% less than motor threshold levels. Data were analyzed by an analysis of variance for repeated measures. Sources of significant differences were determined by paired t tests (probability values determined by Bonferroni adjustment). A single 30-minute application of HVPC significantly curbed edema formation for between 4.0 and 7.5 hours following treatment (ie, volumes of treated limbs were significantly less than those of untreated limbs). These results suggest that regimens currently applied to humans (ie, one treatment per day or three times per week) may be insufficiently aggressive to provide sustained treatment effects. PMID- 1728051 TI - Pentoxifylline does not spare acute radiation reactions in rat lung and skin. AB - Male rats were exposed to single doses (0-30 Gy) of 60Co gamma rays to the right hemithorax. Half of each dose group consumed only control powdered chow after irradiation, and half consumed feed containing 0.10% (w/w) pentoxifylline (50 mg/kg/day). The severity of epilation and desquamation in the field of the radiation port was scored weekly. Two months after irradiation the animals were killed, and pulmonary endothelial function was monitored by the activity of lung angiotensin converting enzyme (ACE) and plasminogen activator (PLA), and by production of prostacyclin (PGI2) and thromboxane (TXA2). The amount of hydroxyproline (HP) in the lung served as an index of pulmonary fibrosis. Radiation produced a dose-dependent decrease in ACE and PLA activity in the right lung and an increase in the production of PGI2 and TXA2. This endothelial dysfunction was accompanied by an increase in wet weight and in protein and HP content in the irradiated lung. Pentoxifylline spared only the increase in lung wet weight and protein content, and actually elevated the radiation-induced hyperproduction of PGI2 and TXA2. The severity of the epilation and desquamation reactions increased with increasing radiation dose and time but was independent of diet. These data indicate that pentoxifylline, despite some promising pharmacological actions, has no beneficial effect on acute radiation reactions in rat lung and skin. PMID- 1728052 TI - Ultraviolet radiation, solar dermatosis, and cutaneous neoplasia in beagle dogs. AB - Beagle dogs that were part of a life span study of the effects of low-level ionizing radiation during development were evaluated for the incidence of skin neoplasia and solar dermatosis. A total of 991 dogs up to 14 years of age were examined. The dogs were housed in gravel-based, outdoor pens with doghouses in a high-altitude, high-sunshine level environment. Solar dermatosis was restricted to the sparsely haired, nonpigmented abdominal skin. Skin neoplasms were either removed surgically or found at necropsy. Solar dermatosis was diagnosed in 363 of the 991 dogs, an incidence of 36.6%. There were 175 hemangiomas, hemangiosarcomas, or squamous cell carcinomas of the skin in the 991 dogs. Of these, 129 tumors occurred in dogs with, and only 46 in dogs without, solar dermatosis. Of the dogs with solar dermatosis, 93 (26%) had at least one of the three tumor types, compared to only 44 (7%) of dogs without solar dermatosis. Thirty-two dogs had multiple tumor types and solar dermatosis, compared to only two dogs with multiple tumor types and no solar dermatosis. There was a highly significant correlation (P less than 0.001) between the occurrence of these tumor types and solar dermatosis in the unpigmented abdominal skin. This correlation was strongest for the malignant neoplasms. Whole-body gamma-radiation exposures were delivered at one of three prenatal or three postnatal ages up to 1 year of age. There appeared to be an increased risk for hemangiosarcomas and squamous cell carcinomas in dogs with solar dermatosis and given gamma-ray exposures at 1 year of age. This suggests an interaction between exposures to ionizing and ultraviolet radiation. PMID- 1728053 TI - Response of the rat Dunning R3327-AT1 prostate tumor to treatment with fractionated fast neutrons. AB - Reports indicate that cancer of the prostate, soft tissue sarcomas, salivary gland tumors, and melanomas respond well to fast-neutron treatment. To better understand the action of fast neutrons on such tumor tissues, we have begun studies with the versatile Dunning rat prostate tumor system. In our initial studies with the R3327-AT1 subline we observed a relative biological effectiveness (RBE) of approximately 3 for single doses of 14-meV fast neutrons. As a continuation of those studies the present report discusses our findings following fractionated treatments with 10 equal fractions of 14-MeV fast neutrons or 60Co gamma rays at several dose levels per fraction. After either fractionated neutron or photon treatment the volume of the tumors continued to increase for 2 weeks and then reached a plateau, the level of which was dose dependent. Tumor growth resumed and no local control was observed. Analysis of the data using growth delay as biological end point yielded an RBE of approximately 4.2 +/- 1.3. PMID- 1728054 TI - The comparative tumorigenic effects of fission neutrons and cobalt-60 gamma rays in the B6CF1 mouse. AB - In the period from 1971 to 1986, both sexes of the B6CF1 (C57BL/6 x BALB/c) mouse were exposed at 110 +/- 7 days of age to single, 24 once-weekly or 60 once-weekly doses of fission neutrons or 60Co gamma rays. A small group of males was also exposed to gamma rays for 22 h/day, 5 days/week, for either 23 or 59 weeks, the elapsed times for the 24 and 60 once-weekly series. All mice were followed for their natural lifetimes. A gross pathology report is available on 32,000 animals, and a histopathology record is available on about 19,000. About 85% died with or from one or more neoplastic diseases. The principal tumors observed at death were of lymphoreticular (45-60%), vascular (20%), or pulmonary (35-50%) origin. From 4 to 10% died with fibrosarcomas, hepatocellular tumors, ovarian tumors, and tumors of the Harderian, adrenal, and pituitary glands. Dose-response equations (linear and linear-quadratic) were fitted to the data for deaths from and occurrences of eight different individual or groups of tumors. Equations were constrained through the control intercepts and fitted separately for the two sexes, the two radiation qualities, and all exposure patterns for the two intervals of 600-799 days and 800-999 days from first exposure. RBE values were derived from the ratios of linear coefficients of dose-response curves. RBE values increased as dose was protracted, largely due to the reduced effectiveness of protracted gamma irradiation; however, about 28% of the increase can be attributed to the increase in neutron-induced injury caused by dose protraction. Highest RBE values were seen for tumors of epithelial tissue origin and the lowest for tumors of connective tissue origin. The range for significant values was from about 2 to over 50. Nonneoplastic diseases accounted for about 5% of all deaths, and 10% were classified as from unknown causes. Neither category responded to differences in radiation quality or exposure patterns. PMID- 1728055 TI - Radiation-induced damage to DNA in drug- and radiation-resistant sublines of a human breast cancer cell line. AB - An Adriamycin-resistant subline of a human breast cancer cell line, MCF-7 ADRR, has been shown to exhibit radioresistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, damage to DNA of MCF-7 sublines WT and ADRR by 60Co gamma radiation was measured by filter elution techniques. The initial amount of DNA damage, measured by both alkaline and neutral filter elution, was lower in ADRR cells, suggesting that these cells are resistant to radiation-induced single- and double-strand DNA breaks. In the case of double-strand breaks the difference between WT and ADRR cells was significant only at the lower radiation doses studied (up to 100 Gy). In cells depleted of glutathione (GSH) by L-buthionine sulfoximine (BSO) treatment, ADRR cells were sensitized to radiation-induced DNA damage, while WT cells were unaffected. The rate of repair of single- and double-strand DNA breaks following radiation was the same for both sublines, and repair of radiation damage was not affected by BSO treatment in either cell line. The relative resistance of ADRR cells to initial DNA damage by radiation is the only difference so far detected at the molecular level which reflects radiation survival, and it is possible that other factors are involved in the resistance of ADRR cells to killing by radiation. Sensitization of ADRR cells to radiation-induced DNA damage by GSH depletion, although not likely to involve inhibition of GSH-dependent detoxification enzymes per se (irradiation was done at 4 degrees C), suggests that at the molecular level radioresponse in this subline is related to maintenance of GSH/GSSG redox equilibrium. PMID- 1728056 TI - A radiation resistance factor in cultured Cloudman S91 mouse melanoma cells. AB - The gamma-ray survival of a radiation-sensitive amelanotic subclone of Cloudman S91 mouse melanoma, S91/amel, is increased by the presence in the tissue culture dishes of heavily irradiated cells from the same cell line (amel-HRCells) and from clonally related melanotic S91/I3 radioresistant cells (I3-HRCells). The D0 of the target S91/amel cells increases from 1.25 to 2.08 Gy in the presence of 60,000 heavily irradiated S91/amel cells per dish. The presence of I3-HRCells in dishes of target S91/I3 cells does not increase their radioresistance. Comparable numbers of I3-HRCells are more effective than amel-HRCells at increasing survival of target S91/amel cells irradiated with 3 Gy of gamma rays. Conditioned medium from the S91 melanoma cells also increases the radioresistance of S91/amel, but is not as effective as the HRCells. Unirradiated cells can condition the medium as effectively as irradiated cells. It is concluded that the radiosensitive mouse melanoma cell line is made significantly more resistant by a diffusible cellular factor(s) elaborated more proficiently by radiation-resistant cells. PMID- 1728057 TI - Interplanetary crew exposure estimates for galactic cosmic rays. AB - Using the Langley Research Center galactic cosmic-ray transport computer code and the Computerized Anatomical Man model, initial estimates of interplanetary exposure of astronauts to galactic cosmic rays, during periods of solar minimum activity, are made for a realistic human geometry shielded by various thickness of spacecraft aluminum shielding. Conventional dose assessment in terms of total absorbed dose and dose equivalent is made for the skin, ocular lens, and bone marrow. Included in the analyses are separate evaluations of the contributions from the incident primary ions, from subsequent-generation fragmentation products, and from target fragments. In all cases considered, the equivalent sphere approximation yielded conservative overestimates for the actual organ exposures. PMID- 1728058 TI - Influence of dose rate on survival time for 239PuO2-induced radiation pneumonitis or pulmonary fibrosis in dogs. AB - Beagle dogs were exposed once or repeatedly to 0.75-microns-diameter monodisperse aerosols of 239PuO2 by pernasal inhalation. The dogs that were exposed once received alveolar depositions (+/- standard deviation) of 3.9 +/- 1.9 kBq/kg body mass and accumulated doses of 23 +/- 8 Gy to the lung before death at 5.4 +/- 1.7 years after exposure. Dogs exposed repeatedly received a total alveolar deposition of 5.3 +/- 0.9 kBq/kg body mass during 7 to 10 semiannual exposures and accumulated doses of 22 +/- 5 Gy to the lung before death at 4.9 +/- 0.7 years after first exposure. Clearance of the plutonium from the lung in the dogs exposed repeatedly was slower than in the dogs exposed once. All dogs in the repeated-exposure study and all but one dog in the single-exposure study died from radiation effects. Pulmonary fibrosis accounted for 72% of the radiation related deaths in the single-exposure study and 87% in the repeated-exposure study. The remaining dogs died with pulmonary cancer. Based on total cumulative radiation dose, the times after exposure to death from radiation pneumonitis and pulmonary fibrosis were not significantly different for single and repeated exposures. Thus dose rate does not appear to be an important factor in predicting death from radiation pneumonitis or pulmonary fibrosis for dogs inhaling 239PuO2. PMID- 1728059 TI - Evaluation of a fluorinated 2-nitroimidazole binding to hypoxic cells in tumor bearing rats by 19F magnetic resonance spectroscopy and immunohistochemistry. AB - We have examined a hexafluorinated 2-nitroimidazole, CCI-103F, as a probe for hypoxic tumor cells by in vivo 19F magnetic resonance spectroscopy (MRS). Following initial intraperitoneal injections of the drug in tumor-bearing (Dunning R3327-AT1-Matlylu) rats, 19F spectra were obtained on an Otsuka 2.0T Vivospec spectrometer using a 1.5-cm surface coil. Signal at 1- and 2-h time points indicated initial biodistribution of drug in the tumor. At 4 and 8 h, a progressive increase in signal intensity was observed, indicating retention of drug within the tumor. Tumor signal remained detectable in 4 of 10 rats at 24 h, indicating possible nitroreductive bioactivation by hypoxic cells. Immunohistochemistry of these tumors revealed a staining pattern consistent with labeling of hypoxic cells. No detectable 19F signal was found at 24 h for the other rats, indicating complete washout of unbound drug. Immunohistochemical assessment of these tumors revealed some staining for bound drug at the periphery of necrotic zones. 31P-MRS of the tumors showed good correlation with the presence or absence of hypoxia as evaluated by 19F-MRS, T1- and T2-weighted images, and immunohistochemistry. These results provide the groundwork for further studies using this misonidazole analog for noninvasive identification of hypoxic tumor cells in vivo by MRS. PMID- 1728060 TI - Modification of intracellular pH and thermosensitivity. AB - The effects of amiloride (an inhibitor of Na+/H+ antiport), DIDS (an inhibitor of Na(+)-coupled and Na(+)-independent HCO3-/Cl- exchange) and nigericin (K+/H+ ionophore) alone and in various combinations on the intracellular pH (pHi) and thermosensitivity of SCK tumor cells were studied. Hyperthermia alone at 43 degrees C for 2 h decreased pHi of SCK cells by 0.15-0.20 pH units, as measured fluorometrically using the pH-sensitive dye BCECF. When the cells were treated with 0.5 mM amiloride at 37 degrees C, the pHi declined by 0.10-0.15 pH units at an extracellular pH (pHe) of both 7.2 and 6.6. Amiloride at 0.5 mM enhanced the thermal damage to SCK cells at pHe 6.6 but not at pHe 7.2. DIDS alone at 0.1 mM exerted no effect on pHi or cellular thermosensitivity. DIDS, however, enhanced the effects of amiloride in decreasing pHi and in increasing the thermoresponse of SCK cells, particularly at pHe 6.6. Treatment of the cells with nigericin at 0.1-1.0 micrograms/ml lowered the pHi and enhanced the thermosensitivity of the cells in a dose-dependent manner. Reductions in pHi and increases in thermosensitivity by nigericin at the lower concentration at pHe 6.6 were far greater than at pHe 7.2. When a mixture of 1.0 micrograms/ml nigericin, 0.5 mM amiloride, and 0.1 mM DIDS was present in the medium, the pHi rapidly decreased by about 0.3 and 0.4 pH units at pHe 7.2 and 6.6, respectively. This drug combination was also extremely effective in sensitizing SCK cells to heat, particularly at pHe 6.6. The fact that the thermosensitization by these drugs at pHe 6.6 is more pronounced than at pHe 7.2 and that intratumor environments are known to be acidic strongly suggested that it may then be possible to enhance the thermal damage with such drugs preferentially in tumors relative to normal tissues. PMID- 1728061 TI - Preimplantation growth delay and micronucleus formation after in vivo exposure of mouse zygotes to fast neutrons. AB - Mouse zygotes were irradiated with fast neutrons (0.06 to 1.00 Gy) 1 h after conception and examined at various intervals (24 to 100 h after conception) for embryonic development and micronucleus formation. The frequency of micronuclei per cell increased linearly with dose in 2-cell embryos observed at 24 h after conception and in 4-cell and 8-cell embryos at 48 h after conception. Compared with X rays, the relative biological effectiveness of neutrons for the induction of micronuclei per embryo was 2.5 at 24 h after conception and 3.5 at 48 h after conception. Neutron-induced micronucleus formation was accompanied by morphological growth delay and a significant decrease in the number of cells in the embryos. An inverse relationship was found between the number of cells in embryos and the number of micronuclei when observed at 48 h after conception following irradiation with 0.12 to 1.00 Gy and at 78 h after conception following exposure to 0.50 Gy. The effect of neutron irradiation on embryonic development was likely to be mediated by cell death, as suggested by a significantly increased dead cell index in blastocysts following irradiation of zygotes. PMID- 1728062 TI - The radiosensitivity of the chromosomes of the cells of human squamous cell carcinoma cell lines. AB - Measurement of the radiation sensitivity of chromosomes was used to address the influence of cell cycle distribution and of DNA content and ploidy on radiation responses in seven human squamous cell carcinoma cell lines. The cell lines varied about twofold in DNA content and chromosome number, and the X-ray sensitivities (D0) of the lines ranged from 1.1 to 2.7 Gy. The more resistant cell lines (D0 greater than 1.8 Gy) had faster growth rates and larger proportions of cells in S phase in asynchronous cultures. Aberration frequencies were measured in cells irradiated in G1 and G2 phase. The more resistant lines had fewer induced aberrations in both phases than did sensitive lines, implying that they were more resistant to radiation in both of these cell cycle phases. Therefore, while the larger S-phase population seen in the resistant cell lines probably contributes to the resistant phenotype, it cannot explain all of the intrinsic differences in radiation sensitivity. There was no relationship between DNA content and radiation sensitivity as measured by the cell survival assay or the induction of chromosome aberrations, although cells with larger DNA contents tended to have more chromosome damage per cell at equitoxic doses. PMID- 1728063 TI - Pneumonia, selective decontamination, and multiple organ failure. PMID- 1728064 TI - Cystic duct remnant fistulization to the gastrointestinal tract. AB - Cystic duct remnant (CDR)-enteric fistulization is a rare entity, with only four recorded cases in the literature. CDRs can be found in at least 30% of patients after cholecystectomy and have been reported in as many as 83% of these patients. Calculous obstruction of the CDR or the common bile duct in a patient with a CDR must be present for fistulization to occur. Patients with a CDR-enteric fistula will have biliary tract symptoms after cholecystectomy and may have biliary sepsis. The septic episode or cholangitis may and can resolve when the CDR decompresses through the fistula. In a patient with persistent biliary tract symptoms, CDR should be considered as a possible cause, and common bile duct stones are often associated with CDRs. Signs of systemic infection in patients with biliary symptoms after cholecystectomy may indicate CDR fistulization. If a CDR is suspected, endoscopic retrograde cholangiopancreatography is the diagnostic and potentially therapeutic test of choice. If the patient cannot be successfully treated with endoscopic retrograde cholangiopancreatography or has recurrent symptoms, operative therapy is indicated, including division of the fistula, excision of the CDR, and common bile duct exploration. There may be an increase in the number of complications associated with CDRs, considering the increasing frequency of laparoscopic cholecystectomy resulting in more lengthy CDRs. PMID- 1728065 TI - Intermittent changes in portal venous flow in relation with spleno-pancreatic collaterals--"the pancreatic siphon"--after a selective shunt in a patient with idiopathic portal hypertension: case report. AB - The case of a patient with idiopathic portal hypertension who was subjected to a selective shunt because of variceal bleeding is reported. Large pancreatic collaterals--the pancreatic siphon--were documented 2 years after the operation with loss of portal flow. At 14 years of follow-up, the pancreatic collaterals have disappeared gradually with normalization of portal venous flow in spite of patency of the shunt. PMID- 1728066 TI - Mucinous pancreatic ductal ectasia of latent malignancy: an emerging clinicopathologic entity. AB - The natural history of classic mucinous cystic neoplasms of the pancreas has been previously defined. In this report, an unusual variant of a pancreatic mucinous cystic neoplasm, termed "mucinous pancreatic duct ectasia of latent malignancy," is described. The lesion is characterized by massive dilatation of the main pancreatic duct and its tributaries. Histologically, the ducts are lined by epithelium, which is indistinguishable from the classic mucinous cystic neoplasms. Until the natural history of classic mucinous cystic neoplasm is better documented, resection appears to be the treatment of choice. PMID- 1728067 TI - Bilateral symptomatic adrenal myelolipoma. AB - Adrenal myelolipomas are rare, nonfunctioning benign tumors that consist of mature fat and bone-marrow elements. In the first half of this century, most adrenal myelolipomas were found incidentally at autopsy. These tumors are usually unilateral and asymptomatic. Today they are detected by ultrasonography, computerized tomography, or magnetic resonance imaging scan, done for other reasons. Adrenal myelolipomas can be diagnosed because of their characteristic images. Thus they are classified as "incidentalomas." We report the case of a 50 year-old man who had bilateral adrenal myelolipomas and whose right-side tumor was symptomatic. To our knowledge it is the third operated case reported in the literature. A right adrenalectomy was performed, keeping the asymptomatic left adrenal myelolipoma to preserve adrenal function. PMID- 1728068 TI - Nonoperative management of clinically occult arterial injuries is not always warranted. PMID- 1728069 TI - Conservative treatment of penetrating vascular wounds: reply. PMID- 1728070 TI - The effect on protein and amino acid metabolism of an intravenous nutrition regimen providing seventy percent of nonprotein calories as lipid. AB - BACKGROUND: The purpose of this study was to determine which peripheral intravenous nutrition (IVN) regimen, one containing 70% nonprotein calories as lipid IVN or one containing all nonprotein calories as glucose IVN, was most effective at reversing some effects of surgery on protein metabolism. METHODS: Twenty patients who required IVN after operation were randomized into two well matched groups that received 36 kcal.kg-1.day-1 glucose IVN or 37 kcal.kg-1.day-1 lipid IVN. RESULTS: Both IVN regimens resulted in similar changes of nitrogen balance, plasma liver enzymes, blood urea, plasma albumin, and plasma prealbumin. Mean plasma transferrin rose significantly after glucose IVN (p less than 0.01), a change greater than that after lipid IVN (p less than 0.05). Lipid IVN resulted in continued net efflux of alanine from peripheral tissues at rates similar to pretreatment values; glucose IVN significantly reduced net alanine efflux (p less than 0.05). Most of the extra alanine produced by peripheral tissues during lipid IVN appeared to derive from an increased uptake of intramuscular glutamate and from an increased uptake of branched chain amino acids. CONCLUSIONS: The results of this study indicate that 38 kcal.kg-1.day-1 lipid IVN was equivalent to glucose IVN, except for the continuing gluconeogenesis of alanine and the delayed recovery of plasma transferrin concentration after surgery. A greater infusion rate of such a regimen may be necessary to provide sufficient glucose to suppress gluconeogenesis. PMID- 1728071 TI - Neonatal aortic thrombosis. AB - Thrombosis of the aorta in the neonate is a potentially catastrophic event. The incidence of this problem has increased concomitantly with the widespread use of umbilical artery catheters in the management of infants who are critically ill. The natural history and appropriate management of this complication has not been well established. This is due in part to the wide spectrum of presentations and lack of consensus regarding its classification. Aortic thrombosis may vary from deposition of a fibrin sheath surrounding the length of an umbilical artery catheter to aggregates of nonocclusive thrombus within the aorta or to complete occlusion of the aorta and concomitant occlusion of its main branches. The reported treatments recommended for this problem have ranged from supportive care only to mandatory surgical intervention in all cases. This spectrum of advocated therapies has resulted in considerable confusion regarding the proper management of this problem. This paper presents two cases of neonatal aortic thrombosis: one case was treated medically and the other case was treated with surgical intervention. We review these cases and the current literature, with specific attention directed towards highlighting the critical elements involved in formulating a reasonable approach to the management of neonatal aortic thrombosis. In addition, we offer an algorithm for management of these patients according to the degree of aortic thrombosis, severity of systemic manifestations, and the general condition of each individual patient. PMID- 1728072 TI - Evaluation and surgical correction of esophagitis after partial gastrectomy. AB - Among 51 patients with refractory symptomatic reflux esophagitis seen during an 18-month period, 8 (16%) had undergone previous partial gastrectomy. Either Billroth II (n = 6) or Billroth I (n = 2) resection had been carried out for peptic ulceration 18 months to 30 years beforehand. Each patients was evaluated by symptom scoring, endoscopy, and 24-hour pH monitoring plus a 16-hour esophageal aspiration study, in which 2-hourly aliquots were measured for acid, pepsin, conjugated and unconjugated bile acids, and trypsin. After conversion to a 45 cm Roux-en-Y gastroenterostomy, symptom scoring and endoscopy were repeated at 6 to 12 months in all eight patients. Pepsin, acid, and unconjugated bile acids were seldom present in esophageal aspirates. Conjugated bile acids in concentrations up to 30 mmol/L and trypsin up to 428 micrograms/ml were found in cases of severe esophagitis, mostly during nocturnal rest. Esophagitis, heartburn, regurgitation, and bilious vomiting were eradicated by Roux-en-Y conversion, but other postgastrectomy symptoms (early satiety, dumping, epigastric pain, and diarrhea) were largely unchanged. Postgastrectomy esophagitis resistant to medical therapy seems likely to be caused by nocturnal exposure to trypsin and conjugated bile acids; it is well controlled by a 45 cm Roux-en-Y conversion. PMID- 1728073 TI - Total abdominal evisceration: an en bloc technique for abdominal organ harvesting. AB - This paper describes an en bloc total abdominal evisceration (TAE) technique that has been used successfully in 81 consecutive multi-organ procurements in donors ranging from 2.5 to 85 kg. Preliminary dissection performed by the surgeon and physician's assistant averaged 30 to 45 minutes before aortic cross-clamping. Removal of all abdominal organs (liver, kidneys, pancreas, bowel) en bloc averaged 16 to 24 minutes after aortic cross-clamping, depending on the speed of the thoracic procurement. Organ grafts were preserved with the University of Wisconsin preservation solution. Total procurement time for the removal of the liver, pancreas, and kidneys averaged 1.5 to 2.25 hours. Because all vascular anomalies were easily recognized ex vivo, vascular reconstruction was possible, so that all donors could potentially provide for combined liver, pancreas, and kidney transplantation. In the TAE group, primary liver graft nonfunction was 1.2% (1/81 grafts), which is less than the non-TAE liver graft nonfunction rate of 7% (7/99 grafts); this is statistically significant (p less than 0.05). Also, the incidence of fresh frozen plasma support after liver transplantation in the TAE group (2/81 transplantations) was lower than the non-TAE group (9/99 transplantations) (p less than 0.05). The overall liver recipient survival rate was 87% (non-TAE; 78/94 recipients; TAE; 65/70 recipients). Kidney-graft initial function has been similar in both the TAE and non-TAE groups. All pancreas tissue was histologically normal, and extraction of viable islet cells (average, 3600 islets per gram pancreas) was possible with yields similar to standard pancreatic (average, 379 islets per gram pancreas) harvest techniques. Preliminary experience with combined liver and whole-organ pancreas transplantations has been encouraging, with immediate discontinuation of intraoperative insulin during transplantation. PMID- 1728074 TI - The surgical applications and implications of cultured human epidermis: a comprehensive review. AB - Replacement of skin has long been the ultimate task for surgeons facing skin resurfacing challenges such as thermal burns and chronic ulcerations. Autologous skin grafts have been the "gold standard" for wound closure, but in patients who are massively burned, the availability of normal skin is the limiting factor. In the past 15 years the technique of in vitro cultivation of human epidermis has been developed in an attempt to deal with the problem of extensive skin loss. Although the technique is costly and arduous, grafting patients who are severely burned with cultured epidermal autografts has proved to be a life-saving measure where few alternatives exist. Cultured allografts have promoted rapid healing and pain relief in patients with chronic ulcers. Although longer follow-up is necessary, recent evidence suggests that cultured epidermis provides a wound cover that is just as durable and esthetically acceptable as conventional split thickness skin grafts. This article reviews the development and applications of epidermal cell culture. PMID- 1728075 TI - Surgical treatment as a principle in patients with advanced abdominal carcinoid tumors. AB - Seventy-five patients with advanced abdominal carcinoid tumors (65 midgut, 10 others) have been examined retrospectively to evaluate the role of surgical treatment as a principle, irrespective of stage of disease. Eighteen of 52 patients (35%) exhibited the carcinoid syndrome. Two or more primaries were found in 39% of patients with midgut lesion, 81% of these patients had regional metastases, 5% of these patients had distant lymph node metastases, and 74% of the patients had liver secondaries. All patients underwent operation, an additional 34% of the patients had a further reoperation, 9% of the patients had a second reoperation, 3% of the patients had a third reoperation, and one patient (2%) had a fourth reoperation. Intraoperative debulking (liver excluded) was performed in 33% of the patients, and 48% of the patients had treatment (resection, hepatic artery ligation, embolization) directed at the liver. The postoperative mortality rate was 2% after the primary operation for midgut lesions. The median survival for midgut tumors was 92 months, compared to 40 months for other lesions (not significant). A significantly higher survival rate was revealed for those patients with midgut lesion who were undergoing intraabdominal debulking procedures (liver excluded); median survival was 139 months versus 69 months without debulking. For those patients with liver metastases, median survival after intervention was 216 months and 48 months without such treatment (p less than 0.001). It is concluded that resection of intraabdominal carcinoid tumor masses can be performed in a high proportion of patients. Despite the retrospective, uncontrolled nature of this study, the difference in survival probabilities in favor of aggressive surgical therapy is so marked that it is not unreasonable to conclude that surgery has played a role in prolonging life in these patients. PMID- 1728076 TI - The long-term effect of jejunoileal autotransplantation on intestinal function. AB - Disturbed intestinal absorption has been demonstrated almost uniformly early after intestinal autotransplantation. Our aim was to study the long-term effects of autotransplantation on intestinal absorptive function. Studies of nutritional status and absorptive function were performed on groups of dogs at three intervals after autotransplantation: I (less than 6 months; n = 4), II (6 to 12 months; n = 4), and III (12 to 18 months; n = 4). At death samples of intestinal fluid were obtained for bacteriologic analysis, and studies of morphology and in vitro absorption were performed on intact and autotransplanted intestine. Similar studies were performed on a group of five control animals. Although body weight and serum albumin levels remained stable in dogs that had undergone autotransplantation and initial diarrhea improved, stool moisture was persistently elevated and late defects in fat and D-xylose absorption developed (4.8% +/- 3.2% stool fat at 12 months vs 2.1% +/- 0.6% before surgery and 3.4 +/- 2.0 x 10(-2) mmol/L xylose/hr at 12 months vs 8.8 +/- 5.4 x 10(-2) mmol/L xylose/hr before surgery; p less than 0.05). In vitro glucose uptake and villus height were similar in autotransplanted and adjacent intact intestine at death. Compared with control animals, animals that had undergone autotransplantation demonstrated significant overgrowth of fecal flora in jejunum and ileum (14/18 segments greater than 10(5) bacteria vs 6/15 segments; p less than 0.05). Thus delayed defects in intestinal absorption of fat and D-xylose occurred more than 12 months after autotransplantation. Because intestinal structure and function of the autotransplanted intestine were similar to those of adjacent intact intestine, this malabsorption may be related to bacterial overgrowth or other in vivo factors. PMID- 1728077 TI - Ranitidine improves postoperative suppression of antibody response to preoperative vaccination. AB - The effect of the histamine-2 receptor antagonist ranitidine (100 mg intravenously every 12 hours for 72 hours) on postoperative serum antibody responses to preoperative immunization with six limit of flocculation tetanus toxoid and six limit of flocculation diphtheria toxoid was assessed in a double blind, placebo-controlled randomized study in 26 patients undergoing major abdominal surgery. The preoperative antitetanus antibody level was less than 0.1 IU/ml in all patients, and they were inoculated with both antigens 48 hours before surgery. Serum samples for analysis of antitetanus toxoid and antidiphtheria toxoid were drawn before skin incision and on postoperative days 1, 3, 5, 7, 10, 14, 21, and 28. Ranitidine significantly increased the postoperative antibody response to tetanus toxoid, (p less than 0.01) and insignificantly increased that to diphtheria toxoid vaccination (p less than 0.2) compared with placebo. PMID- 1728078 TI - Glutaraldehyde affects biocompatibility of bioprosthetic heart valves. AB - A marked release of glutaraldehyde from commercially available pericardial bioprosthetic heart valve (BHV) material in washing solutions was found by high performance liquid chromatography (up to 1.8 ppm of glutaraldehyde per gram of dry tissue). In vitro endothelial cell proliferation rate was impaired dose dependently in the presence of increasing glutaraldehyde concentrations of the cultivation medium (r = 0.9; p less than 0.05). Cultivation of endothelial cells was impossible on the surface of commercially available BHV material, but successful and uninhibited when toxic glutaraldehyde ligands of the BHV material were antagonized by treatment with L-glutamic acid. PMID- 1728079 TI - Effect of dobutamine infusion on endotoxin-induced lipid peroxidation in awake sheep. AB - beta-agonists are known to not only increase oxygen delivery, but also attenuate the inflammatory response. We studied the effect of infusing the beta-agonist, dobutamine, on the oxidant-induced lung and liver tissue lipid peroxidation seen after endotoxemia. Twelve unanesthetized adult sheep with lung and soft tissue (prefemoral) lymph fistulae were given 5 micrograms/kg of Escherichia coli endotoxin intravenously. In six sheep, dobutamine 10 to 15 micrograms/kg/min was infused beginning 3 hours after endotoxin to increase oxygen delivery by 75% above baseline. Animals were killed at 6 hours, and lung and liver lipid peroxidation, measured as malondialdehyde, was obtained. Data were compared to six control sheep. Endotoxin alone produced increased lung and soft tissue vascular permeability as evidenced by a twofold increase in protein-rich lymph flow. Lung and liver malondialdehyde increased to 116 +/- 40 nmol/gm and 202 +/- 64 nmol/gm, respectively, compared to control values of 42 +/- 7 nmol/gm and 110 +/- 20 nmol/gm, respectively. Dobutamine infusion after endotoxin increased oxygen delivery by 75%, although changes in total oxygen consumption were not different from those seen with endotoxin alone. Lung and soft tissue lymph flow did not change with dobutamine. However, lung malondialdehyde was 41 +/- 17 nmol/gm, not different from controls. Liver malondialdehyde remained elevated at 164 +/- 26 nmol/gm. We conclude that dobutamine infusion prevents further oxidant induced lung tissue lipid peroxidation but does not reverse the increased permeability already present. Liver lipid peroxidation was not decreased, suggesting the liver oxidant process may not be caused by the same mechanism as the lung lipid peroxidation. PMID- 1728080 TI - Hemoductal pancreatitis. AB - Gastrointestinal tract hemorrhage from rupture of the splenic artery into the pancreatic duct is unusual. This obscure cause of intermittent gastrointestinal tract bleeding should be suspected when the more common causes of bleeding have been ruled out. Duodenoscopy carried out during active hemorrhage may reveal blood coming from the papilla of Vater. Coeliac arteriography will show the pathognomonic findings and confirm the diagnosis. We have treated three patients who had chronic pancreatitis and who developed pseudocyst formation and pseudoaneurysms of the splenic artery. The pseudoaneurysm ruptured into the duct of Wirsung, causing obscure upper-gastrointestinal bleeding. Treatment was distal pancreatectomy and splenectomy, including the pseudoaneurysm and pseudocyst. A review of the literature suggests that three different types of bleeding into the pancreatic duct can occur. The cause of each is described. PMID- 1728081 TI - Reduction by superoxide dismutase of leukocyte-endothelial adherence after liver transplantation. AB - One hour after orthotopic rat liver transplantation, hepatic microcirculation and adhesion characteristics of leukocytes to the endothelial wall were studied with intravital microscopy. Cold storage for 1 and 8 hours in Euro-Collins solution resulted in reduction of perfused sinusoids to 83% and 48% and a decrease of leukocyte velocity from 417 microns/sec in controls to 311 microns/sec and 269 microns/sec, respectively. Additionally, the number of permanently adherent leukocytes rose from 4% in controls to 33% and 43% after cold storage for 1 and 8 hours. Intravenous injection of the free radical scavenger human recombinant superoxide dismutase (40 mg/kg) during reperfusion resulted in marked improvement of the hepatic microcirculation after both 1 and 8 hours of cold storage (91% and 69% perfused sinusoids; 420 microns/sec and 350 microns/sec leukocyte velocity; p less than 0.05). Furthermore, the percentage of permanently adherent leukocytes decreased to 13% and 28% after 1 and 8 hours of cold ischemia, respectively. These results support the hypothesis that oxygen-derived free radicals contribute to leukocyte adherence and microcirculatory failure after cold liver ischemia and transplantation. Thus, application of oxygen free radical scavengers during liver transplantation procedures might be useful to improve graft function. PMID- 1728082 TI - Mycoplasma hominis infection of perihepatic hematomas in a liver transplant recipient. AB - We report the case of a recipient of liver transplantation in whom postoperative perihepatic hematomas were infected by Mycoplasma hominis. Etiologic diagnosis was delayed because this organism is a rare cause of postoperative infection and usually does not grow on standard bacteriologic media. The role of M. hominis in postoperative infections and the diagnostic problems of this organism are discussed. PMID- 1728083 TI - Diary from a week in practice. PMID- 1728084 TI - Exercise stress testing for the family physician: Part I. Performing the test. AB - Equipment required for exercise stress testing includes a monitor, a recorder and a treadmill or ergometer, and costs from about $13,000 to $18,000. Primary candidates for the procedure include asymptomatic patients at high risk for coronary artery disease and patients with atypical chest pain. Careful pretest patient selection will usually prevent complications such as precipitation of an acute myocardial infarction. The physician performing the test is responsible for selecting the appropriate exercise protocol, monitoring the patient during the test and determining when to terminate the test. PMID- 1728085 TI - Emotional responses to therapeutic abortion. AB - Healthy women who choose to terminate an unintended pregnancy in the first trimester have few serious or negative emotional consequences. Although a few women may have ambivalent feelings or feelings of guilt, most have a sense of relief and other positive reactions. However, the emotional response of a woman and her family to therapeutic abortion is complicated. A number of factors may help identify women at risk of emotional difficulty and depressive symptoms after abortion. Women who terminate their pregnancy during the second trimester, have a history of multiple abortions, have preexisting psychiatric problems or perceive a lack of support at home are more likely to have emotional difficulty. Women who have an abortion for medical or genetic reasons are at increased risk of developing depressive symptoms. PMID- 1728086 TI - The inflammatory response in asthma. AB - Asthma is remarkably underdiagnosed and undertreated. While the clinical hallmark of asthma is reversible airflow obstruction, airway inflammation is the major pathologic feature. Studies have linked airway hyperresponsiveness, the basic physiologic defect in asthma, with airway inflammation, even in patients who have mild asthma. Management is directed toward treating the underlying disease process with anti-inflammatory therapy and alleviating breakthrough symptoms with bronchodilators. PMID- 1728087 TI - Chest manifestations of AIDS. AB - Chest radiographs, computed tomography and gallium scanning are useful in diagnosing the pulmonary manifestations of acquired immunodeficiency syndrome. Most opportunistic infections in patients with AIDS affect the lung as the primary target organ. Bilateral perihilar or basilar interstitial infiltrates, which may progress to the ground-glass appearance of adult respiratory distress syndrome, are commonly seen in cases of Pneumocystis carinii pneumonia. Unilateral or miliary infiltrates and cavitary lesions may be atypical presentations. Diffuse interstitial infiltrates are also seen in mycobacterial, fungal and cytomegalovirus infections. Mycobacterium tuberculosis infection in AIDS patients resembles primary tuberculosis infection rather than secondary tuberculosis reactivation. Intrathoracic adenopathy in AIDS patients suggests neoplastic processes, such as lymphoma and Kaposi's sarcoma, and opportunistic infections such as M. tuberculosis, Mycobacterium avium-intracellulare and fungal infections. Bronchoscopy with bronchoalveolar lavage and transbronchial biopsy are usually necessary for identification of the etiologic agent. PMID- 1728088 TI - Latrodectism: bite of the black widow spider. AB - Latrodectism, the clinical syndrome that follows envenomation by the black widow spider, may easily be confused with more common conditions. Acute manifestations are characterized by agonizing pain and muscle spasm. Prolonged symptoms, primarily related to neurologic dysfunction, may occur. Familiarity with the manifestations of latrodectism is the key to diagnosis. Family physicians should consider latrodectism in patients presenting with severe pain and muscle cramps, particularly if the setting and history are consistent with a possible spider bite. Optimal therapy remains controversial. Early use of specific antivenin in severely envenomated patients may prevent the development of lingering symptoms, usually related to neurologic dysfunction. PMID- 1728089 TI - Wegener's granulomatosis. AB - Wegener's granulomatosis is a rare disease of unknown etiology. Until recently it was considered uniformly fatal. Family physicians should be aware of the variable presentations of this disease and keep the diagnosis in mind when faced with a puzzling, chronic, progressive multisystem process. New laboratory markers can lead to earlier diagnosis, and aggressive treatment can improve the prognosis. PMID- 1728090 TI - Culture and antigen detection tests for streptococcal tonsillopharyngitis. AB - A throat culture is necessary for accurate diagnosis of group A beta-hemolytic streptococcal tonsillopharyngitis. The use of penicillin therapy in every patient with sore throat results in overtreatment of 85 percent of children and 95 percent of adults presenting to family physicians with the complaint of sore throat. Indiscriminate use of penicillin also increases the risk of drug side effects and subjects some patients to unnecessary alterations of microbial ecology. The signs and symptoms of group A beta-hemolytic streptococcal tonsillopharyngitis are nonspecific, and reliable clinical diagnosis is difficult. Throat culture is cost-effective and, if properly obtained and processed, more than 95 percent accurate. Antigen detection tests (rapid strep tests) are a viable laboratory alternative to throat cultures if these tests are properly performed and if negative test results are confirmed with traditional throat culture. PMID- 1728091 TI - Disseminated gonococcal infection. AB - The most frequent systemic complication of acute, untreated gonorrhea is disseminated infection, which develops in 0.5 to 3 percent of the more than 700,000 Americans infected with Neisseria gonorrhoeae each year. The classic triad of features consists of dermatitis, tenosynovitis and migratory polyarthritis. Disseminated gonococcal infection is most common in young women but may develop in sexually active persons of any age. The diagnosis often is not suspected because the initial mucosal infection is frequently asymptomatic, providing no clue to an infectious etiology. Prompt identification and treatment are essential to prevent complications such as endocarditis, meningitis, perihepatitis and permanent joint damage. PMID- 1728092 TI - Indicators of poor nutritional status in older Americans. AB - Physicians need to be alert to indicators of poor nutritional status in older patients. These indicators include physical signs, specific symptoms or sets of symptoms, and measurable parameters that indicate poor nutritional status is already present and is causing some effect (although the effect may not be symptomatic and may not be quantifiable). Indicators of poor nutritional status may be detected by interviewing an elderly patient or his or her caregivers, by observing the patient in his or her environment, or by physically examining the patient and/or making specific measurements, including laboratory tests. Since some of these indicators are sensitive to change in the nutritional status over time, they may be potentially useful in confirming the improvement or deterioration in nutritional status. PMID- 1728093 TI - Pharmacologic management of acute abstinence syndromes. AB - Patients addicted to alcohol and other psychoactive substances are frequently seen by family physicians. When admitted to the hospital, these patients may develop abstinence syndromes that require pharmacologic intervention. The pharmacologic management of acute abstinence syndrome from discontinuation of central nervous system depressants involves substitution of another sedative hypnotic agent and gradual tapering of the dose over a few days. The same principle applies to opioid detoxification. Pharmacologic detoxification is generally not required in the management of abstinence syndromes involving central nervous system stimulants, cannabinoids, hallucinogens, inhalants or phencyclidine. PMID- 1728094 TI - Treatment of otitis media. AB - Amoxicillin is the first-line drug for otitis media. Effective second-line drugs for resistant beta-lactamase-producing bacterial strains include trimethoprim sulfamethoxazole, erythromycin-sulfisoxazole, cefaclor, cefuroxime axetil and cefixime. In choosing an antibiotic, the physician should consider proven efficacy, cost, side effect profile, compliance issues, spectrum of coverage and the age of the child. Children with recurrent infections may benefit from antibiotic prophylaxis. About 10 percent of children with episodes of acute otitis media develop a chronic middle ear effusion that persists beyond three months. Referral for insertion of tympanostomy tubes is most appropriate for patients with documented language delay and/or significant medical complications. PMID- 1728095 TI - Management of NSAID-induced ulcer disease. AB - The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with an increased risk of gastric and duodenal ulcers, especially in patients with previous ulcer disease, heavy smokers, patients who are taking steroids and those with other illnesses. In patients at risk for gastroduodenal complications, prophylactic therapy with misoprostol or an H2-receptor antagonist should be considered. If ulcers occur during NSAID therapy, the anti-inflammatory drug should be discontinued and standard ulcer-healing therapy instituted. If NSAID therapy must be continued, ulcer healing may be prolonged. PMID- 1728096 TI - NIH releases consensus statement on panic disorder. PMID- 1728097 TI - AAFP age charts for periodic health examinations: seven to 12 years. The Commission on Public Health and Scientific Affairs. PMID- 1728098 TI - Monitoring the effects of legislation on patients. PMID- 1728099 TI - Spontaneous abortion. PMID- 1728100 TI - Prompt curettage for inevitable abortion. PMID- 1728101 TI - Gout and uric acid excretion. PMID- 1728102 TI - Intravenous glucagon to dislodge an intestinal foreign body. PMID- 1728103 TI - Treatment of obesity. PMID- 1728104 TI - Ingestion of corrosive substances by adults. AB - Compared with the ingestion of corrosive substances in children, this problem tends to be more serious, in adults, because its intent is often suicidal, rather than accidental. The severity and extent of damage produced to the gastrointestinal tract depends on the morphological form of the caustic agent. In the acute stage, perforation and necrosis may occur. Long-term complications include esophageal stricture, antral stenosis, and the development of esophageal carcinoma. X-rays of the abdomen and chest should be done initially to detect any evidence of perforation. Endoscopy should be performed as soon as possible in all cases to evaluate the extent and severity of damage, unless there is evidence of perforation. A complete examination is feasible in most cases. Stricture formation is more common in patients with second- and third-degree burns. Measures to prevent stricture formation, including the use of steroids, have not been successful. Esophageal carcinoma usually occurs 40 yr after the time of injury. PMID- 1728105 TI - Long-term functional results of colon resection and rectopexy for overt rectal prolapse. AB - We reviewed the long-term functional results of colon resection and suture rectopexy for complete rectal prolapse in 47 patients followed for more than 3 yr (mean 65 months). Thirty-three patients underwent sigmoidectomy, eight patients underwent subtotal colectomy, and four patients underwent sigmoidectomy with subsequent subtotal colectomy. Three patients (6.3%) developed recurrent full thickness prolapse, and four patients (8.5%) developed rectal mucosal prolapse. Twenty patients presented with constipation, 10 (50%) of whom improved after surgery. Constipation improved in seven (70%) patients who underwent subtotal colectomy. Twenty-one patients presented with incontinence, eight (38%) of whom improved. Continence worsened in six patients, and four patients developed significant diarrhea. These complications did not correlate with the extent of bowel resection. Three patients required subsequent stomas. Colon resection and rectopexy provides long-term control of rectal prolapse with an acceptable recurrence rate. Subtotal colon resection is frequently helpful in patients with associated constipation. However, colon resection of any magnitude entails a small risk of chronic diarrhea and/or diminished continence. PMID- 1728106 TI - Evaluation of enzyme-linked immunosorbent assay using mycobacterial saline extracted antigen for the serodiagnosis of abdominal tuberculosis. AB - The efficacy of enzyme-linked immunosorbent assay (ELISA) was evaluated in the sera of 215 individuals as a diagnostic aid in abdominal tuberculosis. The subjects had abdominal tuberculosis (group 1), intestinal disorders other than tuberculosis (group 2), cirrhosis of the liver (group 3), and peritoneal malignancy (group 4). Sera from patients of pulmonary tuberculosis and healthy volunteers (group 5) were also analyzed for enzyme activity and served as positive and negative controls. In patients with abdominal tuberculosis, the absorbance (OD) values were significantly higher than for the other groups and healthy volunteers (p less than 0.001). OD values were similar in abdominal and pulmonary tuberculosis (p greater than 0.05). Level above 0.7 (OD) in serum suggests tuberculosis with a sensitivity of 81%, specificity of 88%, and a diagnostic accuracy of 84%. These results suggest that ELISA may be used for the diagnosis of abdominal tuberculosis and in differentiating it from other nontuberculous abdominal diseases. PMID- 1728107 TI - Effects of alpha-interferon and prednisone on serum-soluble interleukin-2 receptor (sIL-2R) in chronic hepatitis B infection. AB - The level of serum-soluble interleukin-2 receptor (sIL-2R) was measured in 32 patients to investigate the effect of prednisone and alpha-interferon therapy on chronic hepatitis B virus infection. All the patients were seropositive for hepatitis B surface antigen and hepatitis B e antigen, with histological evidence of chronic persistent or chronic active hepatitis. Twenty-six patients received oral prednisone, followed by subcutaneous recombinant alpha-interferon, and six patients received multivitamin tablets and served as controls. After 4 wk of prednisone in reducing dosage, serum sIL-2R fell significantly from 673.6 +/- 52.9 U/ml to 584.8 +/- 39.4 U/ml (mean +/- SE, p less than 0.05). It rose to 733.4 +/- 45.7 U/ml (p less than 0.05) on the 4th wk of interferon, but returned to pretreatment level at completion of interferon. There was a significant correlation between serum sIL-2R and alanine aminotransferase levels (r = 0.36, p less than 0.001). The level of serum sIL-2R before treatment and its response to prednisone and interferon were not useful in predicting seroconversion of hepatitis B e antigen and anti-hepatitis B e. PMID- 1728108 TI - Spontaneous esophageal perforation in herpes simplex esophagitis. AB - A 32-yr-old, previously healthy man with severe chest pain of sudden onset was found to have purulent pericarditis and pleural effusions. Several days later, an esophagogram revealed a perforation of the thoracic esophagus. Endoscopy showed a picture highly suggestive of a late stage of an extensive herpes simplex virus (HSV) esophagitis. Biopsies revealed evidence of massive HSV infection, confirmed by immune microscopy and virus culture. At surgery, a mediastinal abscess was found, and an esophageal perforation was identified. These findings suggest that the etiology of the perforation was an unusually severe herpetic infection. To our knowledge, HSV esophagitis has not previously been implicated as the cause of spontaneous esophageal perforation. PMID- 1728109 TI - Tortilla corn chip-associated esophageal perforation: an unusual presentation of achalasia. AB - Laceration of the esophagus related to ingestion of tortilla corn chips has been described in the past. However, no cases of perforation of the esophagus are known to be associated with tortilla corn chip ingestion. We describe a case of previously undiagnosed achalasia in a patient who presented with an esophageal perforation after ingestion of tortilla corn chips. PMID- 1728110 TI - Retrograde gastroesophageal intussusception. AB - This is an initial report of spontaneous retrograde gastroesophageal intussusception in an adult. The patient is a 72-yr-old women with a history of ovarian cancer and hiatal hernia, who presented with symptoms of upper gastrointestinal obstruction. Retrograde intussusception was diagnosed endoscopically and confirmed radiographically with an upper gastrointestinal series. Heightened awareness of this entity may lead to its more frequent diagnosis. PMID- 1728111 TI - Helicobacter pylori and gastric lymphonodular hyperplasia in children. AB - The association of gastric lymphonodular hyperplasia and Helicobacter pylori infection has been reported only in children. Lymphonodular hyperplasia of the stomach is a well known radiographic and endoscopic entity. Over the past three decades, it has been associated with many conditions, ranging from a normal variant to a premalignant lesion. We have recently encountered five children with gastric lymphonodular hyperplasia, all of whom had H. pylori infection of the antrum. The literature regarding this association is reviewed, and a possible explanation for this age-dependent expression of H. pylori infection is offered. PMID- 1728112 TI - Clostridium cadaveris: an unusual cause of spontaneous bacterial peritonitis. AB - Bacterial peritonitis has been known to complicate severe liver disease. Aerobic organisms are responsible for the vast majority of cases, whereas anaerobic bacteria are responsible for less than 5% of all cases reported in the literature. We now report a case of Clostridium cadaveris anaerobic bacterial peritonitis in a 58-yr-old female, an organism that to our knowledge has not been previously implicated as an infectious agent in this entity. PMID- 1728113 TI - Extensive investigation on colonic motility with pharmacological testing is useful for selecting surgical options in patients with inertia colica. AB - Three women with idiopathic severe chronic constipation and inertia colica, who failed to respond to medical treatment, were extensively investigated for gut motor function, especially that of the colon. Twenty-four-hour manometric recordings disclosed that motility was severely reduced throughout the entire colon and response to eating was minimal. One of the patients also was tested for esophageal, gastric, and small bowel motor activity, which gave normal results. Edrophonium chloride stimulation (10 mg iv) provoked no increase in colonic contractile activity in any patient. On these grounds, the patients were submitted to surgical intervention (total colectomy with ileorectal anastomosis two, and left hemicolectomy the other) with fairly good results at follow-up. These results indicate the wisdom of carrying out extensive functional investigations in severely constipated patients before surgery is contemplated. PMID- 1728114 TI - Sigmoid volvulus presenting as chronic secretory diarrhea responsive to octreotide. AB - Secretory diarrhea can be seen in a variety of pathologic states; however, intermittent colonic obstruction usually is not considered as a possible cause. We report a 68-yr-old patient with chronic secretory diarrhea and hypokalemia due to intermittent sigmoid volvulus. Because the volvulus was not originally diagnosed, the patient was treated with the long-acting somatostatin analogue octreotide for 1 yr, with marked clinical improvement. Surgical resection of the redundant sigmoid responsible for the volvulus resulted in prompt and complete resolution of all signs and symptoms. Detailed macroscopic and microscopic examination of the resected specimen was normal. The patient continues to be asymptomatic 12 months after surgery. Increased colonic fluid and electrolyte secretion was caused by intermittent sigmoid volvulus and resulted in chronic secretory diarrhea. PMID- 1728115 TI - Eosinophilic hepatitis after ingestion of choline magnesium trisalicylate. AB - Choline magnesium trisalicylate is a non-acetylated salicylate used widely as a nonsteroidal anti-inflammatory drug. Although mild transient hepatotoxicity associated with aspirin and other salicylates has been well documented, most commonly with high-dose treatment for rheumatologic disorders 112), we report a case of severe hypersensitivity hepatitis with striking tissue and peripheral eosinophilia after ingestion of choline magnesium trisalicylate. PMID- 1728116 TI - Re: Cost-effective way of removing gastrostomy tubes. PMID- 1728117 TI - Esophageal tuberculosis. PMID- 1728118 TI - Food hypersensitivity and the irritable bowel syndrome. PMID- 1728119 TI - Cytology: a simple, rapid, sensitive method in the diagnosis of Helicobacter pylori. AB - Cytology and the rapid urease test on gastric biopsies may diagnose Helicobacter pylori (H. pylori) infection within an hour. We evaluated the sensitivity and reproducibility of touch cytology (imprints from biopsies). In 19 patients with duodenal ulcer, biopsies were obtained from the antrum, fundus, and bulb. H. pylori was diagnosed in 42 sites on smears: 28 by culture and 23 by histology. H. pylori was present in the antrum and fundus in 16 and in the bulb in 11. Assessment of paired antral biopsies in 29 additional patients with or without touch cytology (imprints) before specimens were sent for histology or culture revealed no difference for the presence of H. pylori. A second reading of the 58 smears by a second observer revealed agreement on the presence or absence of H. pylori in 53 (91%). In conclusion, touch cytology is a simple rapid, sensitive, and reproducible diagnostic method for H. pylori that does not alter the quality of biopsies for subsequent culture or histologic examination. For the first time, diagnostic methods have been compared on the same biopsies, eliminating sampling variation. PMID- 1728120 TI - Helicobacter pylori and duodenal ulcer recurrence. AB - Preliminary evidence suggests that eradication of Helicobacter pylori (H. pylori) may lead to prolonged remission of duodenal ulcer (DU). The aim of this study was to assess the long-term effect of eradication of H. pylori on the natural history of DU. Fifty-one patients with endoscopically proven duodenal ulcers, who were found to have H. pylori infection on histology and culture, and who were successfully eradicated of H. pylori with combination treatment of colloidal bismuth subcitrate and antibiotics, were studied. All patients were endoscoped at entry, 4 wk after cessation of treatment and again at 1 yr or sooner, if symptoms recurred. At each endoscopy, two antral biopsies were taken and assessed histologically and microbiologically for evidence of H. pylori infection. Recurrence of H. pylori infection occurred in 18/51 patients (35.3%) and, of these, 12 patients had evidence of recurrent peptic disease (five DU, seven duodenitis). In contrast, of the 33 who remained negative for H. pylori at 1 yr, none developed evidence of recurrent DU. Overall, DU recurrence occurred in 5/51 patients (11.7%), and occurred only in patients reinfected with H. pylori. This relapse rate compares favorably with patients on maintenance H2-receptor antagonist treatment. These results lend further support to the hypothesis that antral reinfection with H. pylori is associated with relapse of DU. PMID- 1728121 TI - H. pylori, the most common bacterial infection in Africa: a random serological study. AB - This study was carried out to determine the prevalence of antibodies to Helicobacter pylori in northern Nigeria, a region with a low incidence of peptic ulceration. In a random, serological survey of 268 subjects, 228 (85%) of the population studied had IgG antibodies to H. pylori. Fifty-eight of these subjects had experienced dyspepsia in the preceding 6 months. The majority of the population (82%) is infected between the ages of 5 and 10. Despite the high prevalence of antibodies to H. pylori, peptic ulcer is uncommon, suggesting that H. pylori is not important in the etiology of peptic ulcer in this population. Indeed, most patients infected by H. pylori are asymptomatic. The possible reasons for this are discussed. PMID- 1728122 TI - Adenoma and carcinoma of the duodenum and papilla of Vater: a clinicopathologic study. AB - In a retrospective study (1982-1990), 12 adenomas, 35 carcinomas of the papilla of Vater, and 21 duodenal adenomas were examined. All patients had endoscopicbioptic examinations (5-10 forceps biopsies, snare biopsy or forceps biopsies after endoscopic sphincterotomy). Special attention was paid to malignant transformation of adenomas and of residual adenomatous tissue in surgical resected cancer. Follow-up data were gained by reexamination or questionnaires. In papillary adenomas, an adenocarcinoma was found in 30% at operation or by follow-up. In 41.2% of the operated cases, residual adenomatous tissue was found, more often in well-differentiated adenocarcinomas than in other histological types. A transformation from duodenal adenomas to adenocarcinomas was seen less frequently (9.5%). Therefore, the risk of malignancy in ampullary adenomas is greater than elsewhere in the duodenum. In eight of 11 patients (72.7%) with duodenal adenomas, one or more simultaneously developed colonic adenomas were found (in four cases a Gardner syndrome not known before). We conclude that there is strong evidence that most ampullary and duodenal carcinomas develop in preexisting adenomas, with an adenoma-cancer sequence similar to that accepted for colorectal carcinoma. This has to be kept in mind for diagnostic as well as therapeutic reasons. When either an adenoma of the ampulla or duodenum is diagnosed, colonoscopy is mandatory to find or exclude colonic adenomas. In patients with familiar adenomatosis, the duodenum and the papilla of Vater have to be examined endoscopically. PMID- 1728123 TI - Immunohistochemical study of pancreatic cystadenocarcinoma. AB - Seven cases of pancreatic cystadenocarcinoma were studied clinicopathologically and immunohistochemically. Four patients died of the disease or recurrence with metastasis to the liver and peritoneum within 2 yr after surgery. The remaining three patients are well after surgery. To distinguish the cystoadenocarcinoma with excellent prognosis from that with poor prognosis, we performed immunohistochemical staining with monoclonal antibodies directed against tumor markers. Five specimens were strongly and diffusely reactive with tumor marker antibodies, whereas two were reactive with the apical portion of lining epithelial cells. With the findings of the immunohistochemical examination, the diagnosis of two patients could be revised from cystadenocarcinoma to premalignancy. Thus, immunohistochemical examination with tumor markers could correlate with the clinical outcome of the patients and is useful in distinguishing two distinct populations of mucinous cystic tumor. PMID- 1728124 TI - Oral disodium cromoglycate treatment on irritable bowel syndrome: an open study on 101 subjects with diarrheic type. AB - Several studies on the usefulness of oral disodium cromoglycate (DSCG) in the treatment of systemic adverse reaction to foods have been performed, with less attention to gastroenterological symptoms. In the present study, we selected 101 patients with diarrheic-type irritable bowel syndrome which improved after an elimination diet and worsened after a challenge with specific food(s). All patients were then tested for 48 commercial alimentary antigens by skin prick test (SPT) and underwent 8 wk of oral DSCG (500 mg three times a day), and the results were evaluated by means of a semiquantitative subjective and objective score. We observed an improvement of the symptoms in 67% of the 74 SPT-positive patients, whereas only 41% of the 27 SPT-negative patients showed a positive response to DSCG (p less than 0.05). These data confirm the protective role of DSCG in food-dependent diarrheic-type irritable bowel syndrome with food allergy features. PMID- 1728125 TI - Percutaneous endoscopic gastrostomy as an unrecognized source of methicillin resistant Staphylococcus aureus colonization. AB - Methicillin-resistant Staphylococcus aureus (MRSA) caused colonization or infection around the gastrostomy site of seven hospitalized patients, five of whom were in the long-term care unit. All cultures of gastrostomy sites were retrospectively reviewed, and 28% had MRSA. The gastrostomy site was responsible for 6.3% of all MRSA cultures, and 12.5% of all MRSA-positive patients with gastrostomy site cultures had involvement at that site. The implications of MRSA and gastrostomy tubes are discussed. PMID- 1728126 TI - Benefits and risks of an intensive very-low-calorie diet program for severe obesity. AB - Comprehensive very-low-calorie diet (VLCD) programs are the preferred treatment for selected obese individuals. They combine energy intakes of 400-800 kcal/day with medical monitoring and intensive lifestyle education. Typical VLCD patients have median body mass indexes of 36 kg/m2 and have median ages of 40 years. About 70% are female. Commonly associated medical problems include hypertension in 50%, hyperlipidemia in 41%, and diabetes mellitus or glucose intolerance in 14%. Typical weight loss with VLCD is around 21 kg in 16 wk. Reductions of 8-13% in blood pressure, 5-15% in serum total cholesterol, 5-20% in low-density lipoprotein-cholesterol, 15-50% in triglycerides, and decreases in blood glucose and glycohemoglobin in diabetic individuals accompany weight loss. VLCD associated side effects can be managed medically without discontinuing treatment. Lifestyle education promotes long-term weight maintenance of approximately 56% 2 yr after VLCD treatment. Weight losses using comprehensive VLCDs allow moderately to morbidly obese persons to achieve greater benefits than other nonsurgical treatments and should be considered before opting for surgical treatment. PMID- 1728127 TI - A prospective study of bidirectional endoscopy (colonoscopy and upper endoscopy) in the evaluation of patients with occult gastrointestinal bleeding. AB - One hundred patients with occult gastrointestinal bleeding (OGIB) (i.e., guaiac positive stools and/or iron deficiency anemia) were prospectively evaluated with bidirectional endoscopy [upper endoscopy (EGD) and colonoscopy] to determine the origin of occult bleeding. Predetermined criteria were used to prospectively define gastrointestinal bleeding sources. Among the 58 males and 42 females, the median age was 65 yr. Thirty-one percent of the group had gastrointestinal symptoms. Sixty-six percent of the study group were inpatients. Bidirectional endoscopy detected the source of OGIB in 53% of patients, with a positive finding on EGD of 36%, and with colonoscopy, of 26%. In only 9% of patients was a source of OGIB detected on both EGD and colonoscopy. Acid peptic disease accounted for the source of OGIB in 27%, colonic adenomas 14%, angiodysplasia 13%, colorectal carcinoma 6%, and gastric cancer in 1% of patients. The diagnostic yield was significantly higher with EGD than with colonoscopy in patients with anemia and guaiac-positive stools (45% vs. 26%, p less than 0.01). Upper endoscopy directed a change in patient management in 29 patients. IN CONCLUSION: for the patient population described in this study, bidirectional endoscopy determined the source of OGIB in 50%. As expected, colonoscopy resulted in a higher cancer detection rate than EGD--yet EGD detected the origin of OGIB in 68% (36/53) of patients found to have an occult bleeding source, and resulted in a therapeutic initiation or a change in therapy for 30% of all patients. PMID- 1728128 TI - Immunological determination of fecal hemoglobin and transferrin levels: a comparison with other fecal occult blood tests. AB - Immunological determination of fecal hemoglobin and transferrin levels was performed in inpatients on an unrestricted diet, including patients with colon cancer or polyps and a control group. When hemoglobin levels of 5.1 micrograms/g feces and transferrin levels of 0.4 microgram/g feces were designated as positive, 48 of the 60 fecal specimens from colon cancer patients were positive. This result was significantly superior to that for another fecal occult blood immunological test (FECA-EIA) (p less than 0.005), and similar to the results of two chemical tests (guaiac and Hemoccult). Twenty-eight of the 78 fecal specimens from patients with colonic polyps were positive, again a result superior to the FECA-EIA (p less than 0.005) and similar to the chemical tests. Three of the 99 control fecal specimens were positive, which was a similar result to that obtained with the FECA-EIA and significantly superior to the chemical tests (both p less than 0.005). Thus, combined detection of fecal hemoglobin and transferrin levels can be used as a fecal occult blood test in patients without dietary restriction. PMID- 1728129 TI - Colonic pH: a comparison between patients with colostomies due to trauma and colorectal cancer. AB - Colonic pH is important in the regulation of colonic cell growth, control of absorption and secretion, and bile acid degradation, which may be a key step in the development of colon cancer. This study determined the mucosal, luminal, and fecal pH in right and left colons of otherwise healthy patients who underwent colostomies because of trauma. The pH was evaluated while patients consumed a western diet. In addition, the mucosal, luminal, and fecal pH in patients with colostomies carried out for colorectal cancer also was assessed. All patients were blacks--a low-risk group for colorectal cancer. The results showed that mucosal pH was alkaline (pH 8) and was similar on both sides of the colon of healthy colostomates and in colon cancer patients. Luminal pH (7.6) was the same in healthy right colostomates and cancer patients and was significantly lower that mucosal pH. Fecal pH was significantly lower in right colostomates (6.0) than in left (6.5), and in healthy right colostomates compared with colon cancer patients (6.6). In addition, fecal pH was significantly lower than mucosal and luminal pH. PMID- 1728130 TI - Lipohyperplasia of the ileocecal valve. AB - Submucosal lipohyperplasia of the ileocecal valve (ICV) is reportedly a rarely diagnosed lesion of uncertain significance. Eight cases of ICV lipohyperplasia diagnosed in surgical specimens (seven resections, one biopsy) are reviewed: three cases were associated with right lower abdominal quadrant pain and ICV mass on barium enema or operative examination, two were associated with ICV mucosal acute inflammation and necrosis, and three were incidental in resections for cecal, appendiceal, and sigmoid neoplasia. To evaluate the frequency of ICV lipohyperplasia and any associated processes, a series of 51 autopsies was studied. Regarding lipohyperplasia in these valves, 10 (19.6%) were determined to have none, 14 (27.5%) were mild, 20 (39.2%) were moderate, and 7 (13.7%) were marked cases. Degree of lipohyperplasia correlated statistically with degree of cardiac right ventricular fatty infiltration (p = 0.0001), pancreatic fatty infiltration (p = 0.0314), and greater body weight of patient (p = 0.0009). No definite correlation was demonstrated with left ventricular, adrenal, or lymph node fatty infiltration, or with hepatic fatty change, body height, age of patient, or blood glucose. Various gastrointestinal symptoms and lesions accompanied lipohyperplasia, but no definite causal relationship was identified, except for one case of marked lipohyperplasia associated with marked mucosal necrosis and acute inflammation of ICV. In conclusion, ICV lipohyperplasia is a common finding that occasionally may be associated with clinical symptoms and other valve pathology. It correlates to some extent with right ventricular and pancreatic fatty infiltration and with greater body weight. PMID- 1728131 TI - Diversion colitis in children with severe gastrointestinal motility disorders. AB - We found colitis in 11 of 14 children, 4 months to 7 yr after surgical diversion of the colon for chronic intestinal pseudo-obstruction. Colonoscopic examination was incidental during placement of a catheter for colon manometry and transit studies. All 14 children had complained of diffuse, poorly localized abdominal pain, but only three had a history of bloody stools. Diversion colitis had not previously been suspected in six of eight affected children without hematochezia. Biopsies showed a nonspecific acute and chronic inflammation and/or nodular lymphoid hyperplasia. There was no correlation between the duration of the colonic diversion and the severity of the colitis. Diversion colitis may be an indolent inflammatory nidus and a potential cause for repeated bacteremia, abdominal pain, and bleeding. PMID- 1728132 TI - Intravenous immunoglobulin therapy for active, extensive, and medically refractory idiopathic ulcerative or Crohn's colitis. AB - To determine whether intravenous immunoglobulin produces demonstrable clinical improvement in patients with refractory idiopathic inflammatory bowel disease, a pilot, open-label, nonrandomized, safety and therapeutic efficacy study was carried out at a tertiary care referral medical center. Twelve consecutive patients with refractory idiopathic colitis (nine ulcerative colitis, three Crohn's colitis) who were reluctant to receive immunosuppressive therapy or have surgical intervention were referred by physicians not participating as investigators in this study. Eleven patients were symptomatic for at least 6 months, with endoscopically moderate or severe mucosal inflammation despite medical therapy, including systemic corticosteroids in all cases, and one patient was dependent on oral prednisone to remain in clinical remission. Ten patients had extensive colitis, six of whom had pancolitis and four of whom had colitis extending to the hepatic flexure or transverse colon. Nine patients required hospitalization for treatment of colitis. Intravenous immunoglobulin was administered in one or two induction phases (2 g/kg over 2 or 5 days), followed by a maintenance phase (200-500 mg/kg every 2 wk for 12 or 24 wk). Tapering of systemic corticosteroid therapy was attempted, whereas other medications for idiopathic colitis were continued. Treatment response was assessed clinically and by colonoscopy with multiple biopsies whenever possible. Immunoglobulin therapy was well-tolerated and did not produce any biochemical abnormalities. In six patients who completed the treatment protocol, mean reductions +/- SE were achieved in subjective symptoms as quantified by a colitis activity score, 13.3 +/- 1.2 to 4.7 +/- 0.9 (p less than 0.001), and daily mg dose of prednisone, 41.7 +/- 8.0 to 1.9 +/- 1.2 (p less than 0.001). For all 12 patients, statistically significant reductions were achieved in the colitis activity score and daily prednisone dose. Of five patients who completed the treatment protocol and improved clinically, four underwent post-treatment colonoscopic and biopsy evaluations and had unequivocal reductions in the intensity of colonic mucosal inflammation. Three patients who had objective improvement with intravenous immunoglobulin experienced relapses of colitis after discontinuation of this therapy. Six patients did not complete the treatment protocol, two of whom required surgical intervention and four of whom withdrew to undergo colectomy electively. Intravenous immunoglobulin may be beneficial in subsets of patients with idiopathic colitis. The results of our pilot study justify the undertaking of a prospective, randomized controlled trial to determine the efficacy of intravenous immunoglobulin in carefully defined subsets of patients with idiopathic inflammatory bowel disease. PMID- 1728133 TI - Retinal toxicity in human immunodeficiency virus-infected children treated with 2',3'-dideoxyinosine. AB - To assess the safety and antiretroviral activity of 2',3'-dideoxyinosine, we enrolled 43 children with symptomatic (Centers for Disease Control class P-2) human immunodeficiency virus infection in a Phase I-II study and monitored them prospectively for the development of ocular complications secondary to HIV infection or drug toxicity. Follow-up ranged from 12 to 103 weeks with a median follow-up of 71 weeks. Three of 43 children (7.0%) developed peripheral atrophy of the retinal pigment epithelium during treatment with 2',3'-dideoxyinosine. The two children with the most severe retinal atrophy were enrolled in the study at the highest dosage studied (540 mg/m2/day). In contrast to findings in children, no retinal atrophy in HIV-infected adults treated with 2',3'-dideoxyinosine has been evident to date. PMID- 1728134 TI - Delayed bilateral involvement in the acute retinal necrosis syndrome. PMID- 1728135 TI - Locally administered hyperoxic therapy for aphakic cystoid macular edema. PMID- 1728136 TI - Macular edema after carotid endarterectomy in ocular ischemic syndrome. PMID- 1728137 TI - Episodic bilateral corneal edema caused by hair groom gel. PMID- 1728138 TI - Forceps for lens nucleus extraction. PMID- 1728139 TI - Stability of refraction during four years after radial keratotomy in the prospective evaluation of radial keratotomy study. PMID- 1728140 TI - Anterior chamber aspirate cultures after uncomplicated cataract surgery. PMID- 1728141 TI - Isolated neurofibromas of the conjunctiva. PMID- 1728142 TI - Severe periphlebitis, peripheral retinal ischemia, and preretinal neovascularization in patients with multiple sclerosis. AB - Two patients with definite multiple sclerosis and marked retinal periphlebitis developed occlusive peripheral retinal vasculitis, which resulted in peripheral retinal ischemia and peripheral retinal neovascularization. Results of investigation for other causes of peripheral proliferative retinopathies were negative in both patients although one patient had a positive anticardiolipin antibody. Both patients have been followed up for over seven years and have maintained good visual acuity with mild regression of the preretinal neovascularization without laser intervention. An analysis of these two cases and six other reported cases indicates that severe periphlebitis can evolve into occlusive peripheral vasculitis, which results in peripheral retinal neovascularization in patients with multiple sclerosis. PMID- 1728143 TI - HLA-A29.2 subtype associated with birdshot retinochoroidopathy. AB - Birdshot retinochoroidopathy is strongly associated with HLA-A29. This antigen can be divided into two subtypes, A29.1 and A29.2, using an immunoprecipitation method succeeded by one-dimensional electrofocusing gel electrophoresis. We reviewed the HLA typings of 58 white French patients who had birdshot retinochoroidopathy. Of these 58 subjects, 54 (93.1%) had HLA-A29 with a relative risk of 157.30. We further analyzed the HLA-A29 subtypings of 33 patients with birdshot retinochoroidopathy. Evaluation of the results showed that HLA-A29.2 subtype was present in all patients (100%). We concluded that the absence of HLA A29.1 subtype is statistically significant (P less than .01) in this study of HLA A29 subtyping. PMID- 1728144 TI - Retinal oxygenation and laser treatment in patients with diabetic retinopathy. AB - The oxygen tension in the preretinal vitreous cavity was measured in human patients undergoing vitreous operations for proliferative diabetic retinopathy. The oxygen tension was significantly higher (P = .004) over areas of retina that had been treated with panretinal photocoagulation than it was over untreated areas in the same retina. This confirmed previous results in animals that showed that panretinal photocoagulation increases the inner retinal oxygen tension. We concluded that panretinal photocoagulation improves the oxygen supply to the inner retina and thereby minimizes the influence of retinal ischemia in diabetic retinopathy. PMID- 1728145 TI - Pattern reversal visual-evoked response as a prognostic indicator in macular gliosis. AB - We recorded the preoperative pattern reversal visual-evoked responses in 16 subjects (16 eyes) with macular gliosis who underwent membrane-peeling operations. A postoperative visual improvement of one octave or more was observed in 11 of 16 eyes (68.8%). Preoperatively, ten eyes had peak amplitudes greater than or equal to 2 microV, all 11 eyes had either lowpass or bandpass curve shapes, and ten eyes had recordable responses to 20- or 10-minutes of an arc check sizes. Each of the three preoperative criteria was significantly associated with a postoperative visual improvement of one octave or more (P = .01, .02, and .02, respectively). These results demonstrate that the preoperative pattern reversal visual-evoked response can objectively assess the function of the underlying macula in patients with macular gliosis and, consequently, is helpful in determining which patient would most likely benefit from a membrane-peeling operation. PMID- 1728146 TI - Detection of subretinal neovascular membranes with indocyanine green and an infrared scanning laser ophthalmoscope. AB - We studied 80 patients with subretinal neovascular membranes to demonstrate the features and limitations of indocyanine green angiography. Indocyanine green increased the detection of ill-defined membranes or those in larger exudative maculopathies. Each membrane had a characteristic small dark rim that demarcated it from surrounding choroidal tissue. Nevertheless, the complex vascular structures of the choroid and retina, which are displayed at one time, can sometimes render the separation of new vessels difficult when displayed at the same time. The results of our study indicate that indocyanine green angiography may be suggested as an additional diagnostic tool in cases of ill-defined or exudative subretinal neovascular membranes. PMID- 1728147 TI - An atypical fulminant course of choroidal osteoma in two siblings. AB - We studied two cases of bilateral choroidal osteoma in an otherwise healthy 5 year-old boy and his only sibling, a 7-year-old sister. Both children were known to have normal fundus appearances at younger ages. The tumor showed slow growth in all four eyes, but severe visual acuity loss developed in three eyes because of neovascular complications that could not be effectively treated by photocoagulation. Secondary retinal cysts developed in three eyes. PMID- 1728148 TI - Structural and functional studies of the corneal endothelium in diabetes mellitus. AB - We performed specular microscopy, anterior segment ocular fluorophotometry, corneal pachymetry, and tonometry on 14 patients with chronic type I diabetes and nonproliferative retinopathy and on 14 age-matched control subjects. The eyes of patients with diabetes had an increased coefficient of variation of endothelial cell area, a decreased percentage of hexagonal endothelial cells, increased corneal autofluorescence, and increased intraocular pressure, which confirmed previous studies. There was no difference, however, in corneal thickness or endothelial permeability to fluorescein. Thus, we were unable to detect any abnormality in endothelial function in these diabetic corneas in the unstressed state, despite structurally abnormal endothelial cells. PMID- 1728149 TI - Argon laser treatment of trichiasis. AB - The argon laser was used to treat trichiasis in 44 patients over a five-year period. During follow-up intervals of between one month and more than four years (mean, 13 months), ablation of misdirected cilia was accomplished with one treatment in 26 patients (59%). No complications were observed. Laser is less effective than cryotherapy for destroying aberrant eyelashes, but cryotherapy is less precise and incites greater posttreatment inflammation. Argon laser treatment is a useful option when only a few, scattered eyelashes require ablation or in patients with disorders such as ocular pemphigoid, in which the stimulation of inflammation is undesirable. PMID- 1728150 TI - Immune deposits in iris biopsy specimens from patients with Fuchs' heterochromic iridocyclitis. AB - To investigate whether Fuchs' heterochromic iridocyclitis may be an immune complex vasculitis, we used an immunofluorescence technique to detect immunoglobulins and complement in iris biopsy specimens from nine patients with Fuchs' heterochromic iridocyclitis, 12 patients with other types of uveitis, and nine patients with glaucoma but without uveitis. No specific immune deposits were observed in the irises of the patients with Fuchs' heterochromic iridocyclitis. Immunoglobulin G, IgA, IgM, and complement were detected in patients with Fuchs' heterochromic iridocyclitis and patients with uveitis, and these results differed significantly (P less than .05) from the group without uveitis. The immune deposits were found only in the iris vessel walls. No light-microscopic evidence of an inflammatory vascular process could be detected. Further studies are necessary to investigate whether the immune reactants originate from the circulation or result from local formation. PMID- 1728151 TI - Visual dysfunction without retinitis in patients with acquired immunodeficiency syndrome. AB - Patients with human immunodeficiency virus infection may have noninfectious and infectious retinopathies, as well as clinical symptoms consistent with optic nerve dysfunction. Noninfectious acquired immunodeficiency syndrome-related retinopathy is seen in most patients with AIDS. Morphologic studies have shown that the number of retrobulbar optic nerve fibers in patients with AIDS is decreased compared to the number of optic nerve fibers in normal control eyes. To determine whether these patients had a visual dysfunction consistent with damage to the macula and optic nerve, 78 subjects (156 eyes) were studied using color vision and contrast-sensitivity testing. The Farnsworth-Munsell 100-Hue color vision test was performed on all subjects and age-corrected color-vision scores for all groups were compared. A significant decrease in color discrimination was found in the patients with AIDS (P less than .001). Contrast-sensitivity testing disclosed a deficit of contrast threshold in patients with AIDS at four of five spatial frequencies and in patients with AIDS-related complex at three of the five spatial frequencies examined. This study demonstrated a functional visual deficit in eyes without retinitis consistent with dysfunction of the macula or optic nerve in patients with AIDS. PMID- 1728152 TI - Semiconductor diode laser transscleral cyclophotocoagulation in patients with glaucoma. AB - We used the semiconductor diode laser to perform transscleral cyclophotocoagulation in 14 patients with glaucoma. Laser settings used for this procedure were 990 milliseconds, 100-microns spot size, and 1,200 mW of power. Applications were placed 1 mm posterior to the surgical corneoscleral limbus and 1 mm defocused toward the ciliary body. The mean preoperative intraocular pressure was 34.8 +/- 13 mm Hg, and the mean intraocular pressure six months after a single treatment session was 24.3 +/- 18 mm Hg (P greater than .001, paired t-test). The mean number of glaucoma medications decreased from 2.2 preoperatively to 1.4 postoperatively. Complications included conjunctival burns and uveitis in 14 patients, and pain in one patient. These results suggested that semiconductor diode transscleral cyclophotocoagulation may be useful as a treatment to reduce the intraocular pressure in patients with glaucoma. PMID- 1728153 TI - Ocular motility anomalies in developmental misdirection of the optic chiasm. AB - A 35-year-old normally pigmented man underwent monocular hemifield visual-evoked potential examinations that indicated a lack of normal decussation of nasal paramacular retinogeniculate fibers in the optic chiasm. We studied effects of this anomaly on ocular motility using electro-oculography and the magnetic search coil technique. The patient exhibited horizontal congenital nystagmus with a predominantly positive exponential waveform. Horizontal smooth pursuit and optokinetic nystagmus were consistently reversed, independent of eye position in the orbit. Vertical tracking was uniformly normal. Horizontal vestibulo-ocular reflexes recorded in the dark during passive rotation exhibited normal gain and phase, whereas rotation recorded in the light reduced gain. Although active head movements reversed horizontal vestibulo-ocular reflexes, vertical vestibulo ocular reflexes in light and darkness were normal. Our study suggested an association between a lack of normal decussation of retinal fibers in the optic chiasm, and reversed visual tracking and congenital nystagmus. PMID- 1728154 TI - Lens-induced endophthalmitis after Nd:YAG laser iridotomy. PMID- 1728155 TI - Hypertropia after implantation of a Molteno drainage device. PMID- 1728156 TI - Anxiety syndromes as epiphenomena of primary major depression: outcome and familial psychopathology. AB - OBJECTIVE: Anxiety symptoms often appear within depressive episodes, but their significance is uncertain. This study sought to determine whether they indicate the coexistence of a separate disease process and whether they have prognostic significance. METHOD: A series of patients with primary depression who entered a follow-up and family study included 37 who also had obsessions or compulsions, 93 who had panic attacks, 101 who had phobias, and 196 who had none of these anxiety syndromes. Each of the overlapping groups defined by the presence of a specific anxiety syndrome was compared to the group that had none of these syndromes with respect to baseline demographic, phenomenological, and historical features, illness rates among directly interviewed relatives, and diagnostic stability and clinical outcome at semiannual follow-ups over a period of 5 years. RESULTS: Depressive symptoms at intake were more longstanding and severe among patients with specific anxiety symptoms, and these patients went on to experience more depressive morbidity during the ensuing 5 years. The development of autonomous anxiety disorders was rare, however, and specific anxiety syndromes in the probands did not increase risks for the corresponding disorders among relatives. CONCLUSIONS: When restricted to episodes of major depression, anxiety syndromes appear to be prognostically significant epiphenomena rather than indicators of an additional disorder. PMID- 1728157 TI - A double-blind comparison of valproate and lithium in the treatment of acute mania. AB - OBJECTIVE: This study was carried out to compare the efficacy of lithium carbonate with that of valproate in acute mania and to determine whether pretreatment clinical characteristics, such as the presence of a mixed affective state, might predict a differential response to the two drugs. METHOD: Twenty seven patients meeting DSM-III-R criteria for acute manic episodes underwent a 3 week, randomized, double-blind, parallel-groups trial of treatment with lithium carbonate or valproate. Symptom severity was measured by using the Schedule for Affective Disorders and Schizophrenia, change version (SADS-C), the Global Assessment Scale (GAS), and the Brief Psychiatric Rating Scale (BPRS). Drug effects were compared by using repeated measures analysis of variance (ANOVA). RESULTS: At the end of the study, nine of 14 patients treated with valproate and 12 of 13 patients treated with lithium had responded favorably, as measured by changes in the SADS-C mania, BPRS, and GAS scores. Elevated pretreatment SADS-C depression scores were associated with good response to valproate. ANOVA revealed a significant interaction between drug and mixed affective state with respect to treatment response. CONCLUSIONS: Lithium and valproate were both effective in improving manic symptoms, and lithium was slightly more efficacious overall. Unlike the case with lithium, favorable response to valproate was associated with high pretreatment depression scores. Therefore, treatment with valproate alone may be particularly effective in manic patients with mixed affective states. PMID- 1728158 TI - DSM-IV and new diagnostic categories: holding the line on proliferation. AB - The authors discuss aspects of the decision-making process for including "new" diagnostic categories in DSM-IV. They detail the different kinds of new categories proposed for inclusion in DSM-IV and discuss the risks and benefits of incorporating them. The authors comment on whether new diagnostic categories should be included in official nosologies as a stimulus for research or as a culmination of research. They also highlight problems with "sunsetting" diagnoses. The criteria for change in DSM-IV--a way to deal with the expanding array of proposals for additional diagnostic entities--are discussed. The authors also offer a series of specific examples of the different kinds of new categories being considered for inclusion in DSM-IV. PMID- 1728159 TI - Abnormal intracellular calcium ion concentration in platelets and lymphocytes of bipolar patients. AB - The authors measured intracellular Ca2+ concentrations in four manic and five bipolar depressed patients and seven comparison subjects. Platelet and lymphocyte intracellular Ca2+ concentrations were comparable. The patients' mean intracellular Ca2+ concentrations were higher than those of the comparison subjects and demonstrated more interindividual variation. These findings suggest a diffuse abnormality in mechanisms affecting intracellular calcium homeostasis in bipolar disorder. PMID- 1728160 TI - Verapamil versus lithium in acute mania. AB - Twenty acutely manic patients were studied in a double-blind randomized trial comparing verapamil with lithium. The Petterson Mania Scale, the Brief Psychiatric Rating Scale (BPRS), and the Clinical Global Impression (CGI) were administered before treatment and weekly during 4 weeks of treatment to evaluate response to verapamil and lithium. Both treatment groups improved significantly, and there were no significant overall differences between treatments. PMID- 1728161 TI - What is meant by the term "binge"? AB - Systematic examination of the use of the word "binge" by 243 young women revealed discrepancies between the lay and technical uses of the term; the young women placed great emphasis on loss of control and less on the quantity eaten. These discrepancies indicate that the term "binge" should be clearly defined in clinical practice. PMID- 1728162 TI - Cognitive impairment in dramatic personality disorders. PMID- 1728163 TI - Ineffectiveness of clomipramine for obsessive-compulsive symptoms in a patient with schizophrenia. PMID- 1728164 TI - Pharmacotherapy and psychotherapy for major depression in a man with AIDS. PMID- 1728165 TI - Seizure with low doses of clozapine. PMID- 1728166 TI - Flunarizine as maintenance treatment of a patient with bipolar disorder. PMID- 1728167 TI - Methadone treatment of Tourette's disorder. PMID- 1728168 TI - Situational influence on development of delusions of pregnancy in a man. PMID- 1728169 TI - Differentiating paranoia and legitimate fears. PMID- 1728170 TI - Control groups in the study of effects of medication. PMID- 1728171 TI - PTSD and risk of suicide. PMID- 1728172 TI - The Dissociative Experiences Scale. PMID- 1728173 TI - Failure to use ECT in treatment of catatonia. PMID- 1728174 TI - Need for integrating environmental and biological factors in psychiatry. PMID- 1728175 TI - Comorbidity of attention deficit hyperactivity disorder and other disorders. PMID- 1728176 TI - Comorbidity of attention deficit hyperactivity disorder and other disorders. PMID- 1728177 TI - Comorbidity of attention deficit hyperactivity disorder and other disorders. PMID- 1728178 TI - Comorbidity of attention deficit hyperactivity disorder and other disorders. PMID- 1728179 TI - Comorbidity of attention deficit hyperactivity disorder and other disorders. PMID- 1728180 TI - Response of obsessive-compulsive disorder and trichotillomania to serotonin reuptake blockers. PMID- 1728181 TI - Are schizophrenia and affective disorder related? A selective literature review. AB - OBJECTIVE: Although most modern investigators accept the Kraepelinian view that schizophrenia and affective disorder are biologically distinct, others have suggested the psychoses are on a continuum of liability. This article is a selective review of evidence for the continuum model. METHOD: The author focuses on family, twin, and adoption data that do not support the Kraepelinian view of psychosis. Evidence characterized to a lesser extent includes the frequency of intermediate forms of illness (i.e., schizoaffective disorder), the inability to separate psychoses by classical symptoms into well-defined clusters, and the inability of laboratory measures to clearly define psychotic subgroups. RESULTS: The data demonstrate that schizophrenia and affective disorder do co-occur in some families. Whether this co-occurrence reflects true overlap is unclear, and significant pathophysiological heterogeneity may underlie clinical continuity. In some recent studies the inclusion of nonmelancholic depressions in the affective illness category may have masked overlap. CONCLUSIONS: The author suggests that the Kraepelinian view of psychoses may need modification. Future research should focus on factors that may reveal overlap between schizophrenia and affective disorder: severity of schizophrenia and affective disorder in probands, severity of depression in relatives, the effect of the unipolar-bipolar disorder relationship on the co-occurrence of affective disorder and schizophrenia, and the relationship of nongenetic factors that might alter the clinical expression of a shared genotype. Also, investigators should not presume a dichotomy or continuum but should examine pure and mixed pedigrees and look for state- and trait-related endophenotypes, the convergence of which would provide the basis for focused molecular genetic study. PMID- 1728182 TI - Should caffeine abuse, dependence, or withdrawal be added to DSM-IV and ICD-10? AB - OBJECTIVE: The authors reviewed basic science and clinical data on caffeine abuse, dependence, and withdrawal in order to make a conclusion about whether these disorders exist and should be included in DSM-IV and ICD-10. METHOD: Studies were located through computerized searches, reference sections of published articles, and written requests. RESULTS: The studies show that abstinence from caffeine induces a withdrawal syndrome of headache, fatigue, and drowsiness which begins within 12-24 hours and lasts about 1 week. The syndrome can be severe and appears to be one reason for continued use of coffee. The prevalence of this caffeine withdrawal syndrome is unknown. Use of caffeine may aggravate some common behavioral and medical disorders. In double-blind tests, a subset of coffee and soda drinkers reliably self-administered caffeinated beverages in preference to uncaffeinated beverages. Clinical indicators of dependence, such as difficulty stopping use of caffeine and use despite harm, have not been documented. CONCLUSIONS: Caffeine withdrawal but not caffeine abuse or dependence should be included as a diagnosis in DSM-IV and ICD-10. Future research should focus on whether some caffeine users exhibit clinical indicators of drug dependence. PMID- 1728183 TI - Attempted suicide among young adults: progress toward a meaningful estimate of prevalence. AB - OBJECTIVE: The results of epidemiologic surveys on attempted suicide are often difficult to interpret; they compare and provide varying estimates of the prevalence of attempted suicide. The authors sought to estimate the prevalence of attempted suicide in a young adult population and to define more precisely what respondents mean when they report a suicide attempt. METHOD: Survey respondents were a representative sample of all 18-24-year-old freshman students at a major public university. The self-administered, anonymous survey included questions about suicidal thoughts and behaviors and about any injury and need for medical care resulting from reported attempts. RESULTS: Of the 694 respondents, 374 (54%) reported having ever considered suicide and 181 (26%) had considered suicide during the preceding 12 months. Thirteen (2%) students reported having attempted suicide during the preceding 12 months, and 72 (10%) reported ever having attempted suicide. The number of students answering affirmatively to questions about injuries sustained, medical care sought, and hospitalization as a result of attempted suicide decreased progressively: only 18 (3%) students reported having ever sought medical care due to a suicide attempt, and seven (1%) were ever hospitalized. CONCLUSIONS: The prevalence of self-reported attempted suicide is not representative of the prevalence of self-injury and provides little information concerning the seriousness of the attempt. The use of specific questions similar to those used in this study should be considered in future surveys. PMID- 1728184 TI - The relationship between adolescent suicidal behavior and life events in childhood and adolescence. AB - OBJECTIVE: Although the relationship between experience of problematic life events and adolescent suicidal behavior has frequently been recognized during the past decade, few studies of life events have been initiated that discriminated between adolescent suicide attempters and depressed adolescents. Therefore, the authors compared adolescent suicide attempters with both depressed and nondepressed adolescents who never attempted suicide with respect to life events that happened in two periods: childhood (defined as the period up to age 12 years) and adolescence (age 12 and older). METHOD: Using a semistructured interview, the authors gathered life event data about childhood and adolescence from three groups of adolescents: 48 suicide attempters, 66 depressed adolescents who had never made a suicide attempt, and 43 nondepressed adolescents who had never made a suicide attempt. RESULTS: The group of adolescents who attempted suicide differed from both of the other groups in that they had experienced more turmoil in their families, starting in childhood and not stabilizing during adolescence. During adolescence, they were more often sexually abused. During the last year before the attempt, further social instability, such as changes in residence and having to repeat a class, occurred. CONCLUSIONS: For suicidal adolescents, the suicide attempt seems embedded not just in the problems every adolescent has to deal with but in greater turmoil in their families, rooted in childhood and not stabilizing during adolescence, in combination with traumatic events during adolescence and social instability in the year preceding the attempt. PMID- 1728185 TI - Psychiatric correlates of incest in childhood. AB - OBJECTIVE: The purpose of the study was to describe more precisely the type of psychiatric illness associated with incest during childhood. METHOD: The Diagnostic Interview Schedule was administered to 52 adult women who had been victims of incest during childhood and to 23 age- and race-matched comparison subjects from local self-help agencies. RESULTS: The prevalence of 19 psychiatric disorders was higher in the incest group than base population rates. Rates of anxiety disorders (panic disorder, agoraphobia, social and simple phobia), major depression, and alcohol abuse and dependence were significantly higher in the incest group than in the comparison group. More severe types of incestuous abuse were associated with a higher risk for the development of psychiatric disorders. CONCLUSIONS: There was an association between incest and psychiatric disorders in this community-based treatment population. All patients, especially those who present with these specific psychiatric disorders, should be queried about childhood sexual abuse during the history. PMID- 1728186 TI - Congenital malformations and structural developmental anomalies in groups at high risk for psychosis. AB - OBJECTIVE: Early somatic developmental anomalies may be one expression of a genetic influence toward psychosis. The purpose of this study was to investigate whether higher rates of early developmental anomalies are associated with heightened genetic risk for psychosis. METHOD: Rates of congenital malformations and minor structural developmental anomalies were prospectively investigated in 84 high-risk offspring of women with histories of psychosis of nonorganic origin (schizophrenic, schizoaffective, affective, and other psychoses) and in 100 offspring of demographically similar control women with no history of psychosis. Data were collected by means of multiple physical examinations through the first 3-4 years of the offspring's lives. RESULTS: The rates of total congenital malformations were high, but the great majority of these malformations in both the index group and the control group represented minor physical aberrations. Rates of congenital malformations in the offspring of the index women (or any specific diagnostic subgroup of these women) were not different from those in the offspring of the control women. CONCLUSIONS: The inferred genetic risk for psychosis does not appear to be associated with greater rates of early somatic developmental anomalies, suggesting that early developmental anomalies do not represent an expression of genetic influence toward psychosis. PMID- 1728187 TI - Wisconsin Card Sorting Test performance in schizophrenia: remediation of a stubborn deficit. AB - OBJECTIVE: Schizophrenic patients typically perform poorly on the Wisconsin Card Sorting Test, which is a putative index of prefrontal functioning. The authors attempted to remediate the deficits of schizophrenic patients on this measure by giving detailed instructions and monetary reinforcement. METHOD: Forty-six inpatients with chronic schizophrenia and 20 control subjects with other psychiatric illnesses were given the Wisconsin Card Sorting Test under four conditions that varied in monetary reinforcement and level of instructions. The schizophrenic patients were given the Brief Psychiatric Rating Scale (BPRS) and three information processing measures (the Continuous Performance Test, Span of Apprehension, and Pin Test). RESULTS: Schizophrenic patients performed worse than psychiatric control subjects across most conditions. Monetary reinforcement had little effect on performance, but detailed instructions significantly improved the scores for both groups. When instructions were withdrawn and monetary reinforcement was maintained, both groups continued to show improved performance over baseline. Symptoms were not significantly associated with Wisconsin Card Sorting Test performance. One measure (the Pin Test) correlated significantly with performance on the Wisconsin Card Sorting Test. CONCLUSIONS: The results suggest the importance of combining motivational with instructional factors for training psychiatric patients in problem solving. PMID- 1728188 TI - Clozapine-induced weight gain: prevalence and clinical relevance. AB - OBJECTIVE: The aim of this study was to determine the prevalence and clinical relevance of weight gain during clozapine treatment. Previous reports indicated clinically significant weight gain in 13% to 85% of patients and an average gain of 9.0 to 24.7 lb. METHOD: Twenty-one state hospital patients with treatment resistant schizophrenia or schizoaffective disorder were weighed weekly for 12 weeks before clozapine treatment and during the first 16 weeks of treatment. Psychiatric symptoms were rated with a modified version of the Brief Psychiatric Rating Scale (BPRS). RESULTS: The mean weight gain for the entire group was 13.9 lb, or 8.9% of body weight. During the 16 weeks of clozapine treatment, 38% of the patients experienced marked weight gains and 29% had moderate weight gains. The improvements in BPRS total score and composite negative symptom score were significantly greater for the eight patients with marked weight gains than for the other 13 patients. CONCLUSIONS: Clozapine's propensity to induce weight gain may relate to the drug's efficacy and/or its unique neuropharmacologic effects. Increased attention to this phenomenon is important because of the morbidity associated with obesity. PMID- 1728189 TI - Seasonal patterns of bulimia nervosa. AB - OBJECTIVE: The aim of this research was to determine whether a seasonal pattern to symptoms of bulimia nervosa could be identified. METHOD: In study 1, seasonal patterns of binge-purge frequency and mood were compared between 31 patients with bulimia nervosa and 31 age-matched normal comparison subjects, using a modified (to include binge and purge items) version of the Seasonal Pattern Assessment Questionnaire. Study 2 involved a cross-sectional examination of binge and purge frequency and of depressive symptoms in 197 patients with bulimia nervosa assessed at various months of the year over a 4-year period. RESULTS: In both the retrospective and cross-sectional studies, binge behavior was found to be highly associated with photoperiod. According to the modified Seasonal Pattern Assessment Questionnaire, purging behavior and mood also varied seasonally among patients with bulimia nervosa. However, purging behavior and severity of depression did not appear to be related to photoperiod in the cross-sectional study. The rate of seasonal affective disorder (syndromal and subsyndromal) defined by the Seasonal Pattern Assessment Questionnaire was higher among the bulimic group than the comparison subjects, but not as high as has been reported for depression in bulimia nervosa. CONCLUSIONS: The results strongly support the interpretation that symptoms of bulimia nervosa primarily associated with food intake patterns are influenced by seasonal variation, and this effect may be mediated by light availability. PMID- 1728190 TI - Pharmacologic and cognitive-behavioral treatment for bulimia nervosa: a controlled comparison. AB - OBJECTIVE: This study examined the relative effectiveness of desipramine, cognitive-behavioral therapy, and their combination in the treatment of bulimia nervosa, together with the effects of withdrawing medication after two different lengths of treatment. METHOD: Seventy-one patients meeting DSM-III-R criteria for bulimia nervosa, recruited from an eating disorders clinic or by advertisements, were assigned at random to one of five groups: desipramine (withdrawn at 16 or 24 weeks), combined treatment (medication withdrawn at 16 or 24 weeks), and cognitive-behavioral therapy (15 sessions). All treatments were conducted individually in an outpatient clinic. The primary outcome measures were binge eating and purging rates assessed at pretreatment, 16, 24, and 32 weeks. The results were analyzed as three groups (medication, cognitive-behavioral therapy, and combined treatment) at 16 weeks and as five groups at subsequent assessments. RESULTS: At 16 weeks, both cognitive-behavioral therapy and the combined treatment were superior to medication given for 16 weeks in reducing binge eating and purging. At 32 weeks, however, only the combined 24-week treatment was superior to medication given for 16 weeks. The combined treatment was also more effective in reducing dietary preoccupation and hunger. Continuing cognitive behavioral therapy appeared to prevent relapse in patients withdrawn from medication at 16 weeks. CONCLUSIONS: Overall, the results favor the use of a combination of medication and cognitive-behavioral therapy in the treatment of bulimia nervosa, with medication continued for at least 24 weeks. PMID- 1728191 TI - Multiple personality disorder in Switzerland. AB - OBJECTIVE: There are no reliable data on the prevalence of multiple personality disorder. The objective of the study was to determine whether and, if so, how frequently patients with multiple personality disorder are encountered and diagnosed in Switzerland. METHOD: All qualified Swiss psychiatrists were sent a questionnaire on multiple personality disorder along with the DSM-III description of multiple personality disorder and three case examples. A total of 836 psychiatrists (66%) answered after two mailings, and 770 questionnaires qualified for evaluation. In addition, a random sample of nonresponders were contacted by telephone. RESULTS: Three percent of the psychiatrists indicated that, at the time of the inquiry, they were treating or examining one or more patients who met DSM-III criteria for multiple personality disorder, and 10% indicated that they had seen multiple personality disorder at least once during their professional career. The patients were not equally distributed among the psychiatrists; three colleagues reported that they had seen much higher numbers of patients with multiple personality disorder. The point prevalence of multiple personality disorder among patients seen by psychiatrists in Switzerland amounts to 0.05% 0.1%. CONCLUSIONS: Multiple personality disorder appears to be a disorder that genuinely exists, even though it occurs relatively rarely. PMID- 1728192 TI - The meaning of expressed emotion: theoretical issues raised by cross-cultural research. AB - The finding that expressed emotion is associated with the course of psychiatric disorder has generated a great deal of clinical and research interest in expressed emotion as an important risk factor. Theoretical elucidation of the construct of expressed emotion has lagged considerably behind this interest, however. The authors contribute to a dialogue on what is inside the "black box" called expressed emotion. They argue that cross-cultural research can provide an empirical basis for the theoretical grounding of expressed emotion factors. A comparative approach reveals that the construct of expressed emotion is essentially cultural in nature. The constellation of emotions, attitudes, and behaviors that are indexed by the expressed emotion method represent cross culturally variable features of family response to an ill relative. Questions surrounding the cultural validity of the construct of expressed emotion, the qualitative dimensions of expressed emotion, and statistically significant cross cultural variations in expressed emotion profiles are discussed. Finally, the authors provide an outline of diverse (cultural, psychobiological, social ecological) features of expressed emotion. Anthropological analysis of expressed emotion reveals that although expressed emotion indexes a Pandora's box of diverse features, culture provides the context of variation through which these factors are most productively analyzed. PMID- 1728193 TI - Recovery and major depression: factors associated with twelve-month outcome. AB - OBJECTIVE: In spite of the prevalence and chronicity of major depression, there is no consensus regarding which clinical and psychosocial variables are associated with recovery. The authors examined the probability of recovery from a major depressive episode 12 months after hospital discharge, the factors most closely associated with recovery, and the patterns of improvement distinguishing patients who recovered from those who did not. METHOD: Seventy-eight inpatients with a DSM-III diagnosis of major depression were assessed at hospitalization and at monthly intervals for 12 months after discharge on a variety of clinical and psychosocial factors. Recovery status at 12-month follow-up was then used as a basis for comparing acute-phase patient characteristics and change in symptoms over time. RESULTS: By the 12th month of follow-up, 34 (48.6%) of 70 patients met criteria for recovery. The five most important factors related to recovery were shorter length of hospital stay, older age at onset of depression, better family functioning, fewer than two previous hospitalizations, and absence of comorbid illness. The majority of patients who had recovered by 12 months had done so within 6 months of discharge; the average length of time to recovery was 4.9 months. CONCLUSIONS: Patients hospitalized for major depression have less than a 50-50 chance of recovering by 1 year. Some variables associated with nonrecovery (e.g., comorbid illness, poor family functioning) are amenable to clinical intervention; however, findings also suggest that there may be two distinct types of depressive illness with respect to recovery, one that remits quickly and the other with a more prolonged course of illness. PMID- 1728194 TI - Malignant histiocytic neoplasms of the small intestine. AB - Immunologic studies have demonstrated that the vast majority of hematolymphoid neoplasms previously designated as "histiocytic" are lymphoid in origin. Consequently, malignancies of macrophage lineage are considered rare by most authors; indeed, their existence is doubted by some. Herein we report two cases of malignant histiocytic neoplasms (malignancies of macrophage lineage) of the small intestine. Both patients presented in the 7th decade with symptoms related to an abdominal mass. The polypoid tumors protruded into the intestinal lumen, extended through the entire thickness of the bowel wall, and involved regional lymph nodes. Microscopically, sheets of large pleomorphic histiocytic cells infiltrated around crypts and were associated with an admixture of bizarre giant cells and inflammatory cells. Mitotic figures were easily found. Ultrastructurally, the cells lacked desmosomes and had indented or kidney-shaped nuclei and cytoplasm containing mostly lysosomes and dense lipid droplets. In both cases, paraffin section immunohistochemistry revealed reactivity of tumor cells for CD45RB (LCA), CD45RO (A6), CD68 (KP1), CD15 (LeuM1), and lysozyme. Frozen section immunohistochemistry performed in one case further supported the macrophage phenotype. Southern blot studies of this case did not reveal immunoglobulin or T-cell receptor beta chain gene rearrangements. One patient initially treated by surgery only died of disease 3 years after diagnosis. The second patient is alive and disease-free 2 years following postoperative combination chemotherapy. The diagnosis of malignant histiocytic neoplasms requires the use of a panel of immunohistochemical markers and may be supported by electron-microscopic studies. PMID- 1728195 TI - Histiocyte-rich B-cell lymphoma. A distinct clinicopathologic entity possibly related to lymphocyte predominant Hodgkin's disease, paragranuloma subtype. AB - This study reports six non-Hodgkin's lymphoma cases that we called histiocyte rich B-cell lymphoma (BCL) because of the prominent reactive histiocytic infiltrate obscuring the malignant B-cell population. The involved lymph nodes are characterized by a mixed nodular and diffuse infiltrate and occasionally feature prominent sinuses. The infiltrate is composed of reactive lymphocytes and numerous histiocytes obscuring a tumor population composed of variably sized scattered cells with irregular or multilobar vesicular nuclei. Immunostaining of paraffin sections for the B-cell marker recognized by L26 helps in the identification of these neoplastic cells. The clonal nature and further evidence of the B-cell lineage of this condition is shown by immunoglobulin gene rearrangements detected in three cases. The six cases of histiocyte-rich BCL are remarkably similar clinically: all presented with stage IVB disease with splenomegaly and follow an aggressive clinical course. Except for these features, our series show striking similarities to paragranuloma lymphocyte-predominant Hodgkin's disease, including male preponderance (all patients are male), age distribution (mean age, 41 years), propensity to progress to a diffuse, large B cell lymphoma (two cases), as well as morphology of the neoplastic B-cell population and expression of Hodgkin's cell markers (Leu-M1 positivity after neuraminidase digestion in three cases, Leu-M1 positivity without neuraminidase digestion in one case, and additional epithelial membrane antigen [EMA] positivity in two cases). Both morphologically and clinically, the present series can be differentiated from other types of infiltrate-rich BCL, such as T-cell rich BCL. Although additional cases will have to be recognized, histiocyte-rich B cell lymphoma most likely represents a distinct clinicopathological entity. We speculate that it develops from a subset of B cells that also gives rise to the lymphocytic-histiocytic (L/H) cell, the Hodgkin's cell variant of lymphocyte predominant Hodgkin's disease, paragranuloma subtype. PMID- 1728196 TI - A modest proposal. PMID- 1728197 TI - Primary lymphoma of the trachea with morphologic and immunophenotypic characteristics of low-grade B-cell lymphoma of mucosa-associated lymphoid tissue. AB - A patient with primary extranodal lymphoma arising in the trachea presented with severe upper airway obstruction. The tumor was localized at presentation. The patient has remained disease-free for 12 months following surgical resection and local radiation treatment. The tumor had distinctive morphologic features characteristic of low-grade lymphomas of mucosa-associated lymphoid tissue (MALT), including a diffuse infiltrate of small lymphocytes and centrocyte-like cells surrounding reactive follicles, with plasmacytoid differentiation, and lymphoepithelial lesions. The tumor cells expressed monotypic immunoglobulin but not CD5 or CD10. This case provides evidence that primary lymphomas of the trachea in some cases are tumors of mucosa-associated lymphoid tissue. PMID- 1728198 TI - Histiocytic tumor of Meckel's cave. An intracranial equivalent of juvenile xanthogranuloma of the skin. AB - We present the case of a 7-year-old boy who had a solitary mass within Meckel's cave that recurred 6 weeks after the initial resection. The histological, immunohistochemical, electron-microscopical, and molecular genetical features established the lesion's histiocytic nature. Our findings showed that it was closely related to juvenile xanthogranuloma, a benign lesion that usually occurs in the skin but has not yet been histologically confirmed in the brain. The present tumor is different from other intracranial histiocytic and xanthogranulomatous lesions. PMID- 1728199 TI - Multilocular thymic cyst. PMID- 1728200 TI - Sinonasal intestinal-type adenocarcinoma. PMID- 1728201 TI - Malignant lymphoma of the mucosa-associated lymphoid tissue. PMID- 1728202 TI - Granulocyte-macrophage colony-stimulating factor (GM-CSF) as adjunct therapy in relapsed Hodgkin disease. AB - OBJECTIVE: To determine the clinical and economic effects of granulocyte macrophage colony-stimulating factor (GM-CSF) as adjunct therapy in relapsed or refractory Hodgkin disease. DESIGN: A randomized, double-blind, phase III clinical trial. SETTING: A tertiary referral center. PATIENTS: Twenty-four patients (twelve of whom were controls) treated with high-dose chemotherapy and autologous bone marrow transplantation. MAIN RESULTS: The 12 patients treated with GM-CSF, when compared with placebo recipients, had shorter periods of neutropenia (median duration of an absolute neutrophil count of less than 1000 cells/mm3, 16 days compared with 27 days; P = 0.02), shorter periods of platelet transfusion dependency (median duration, 13.5 days compared with 21 days; P = 0.03), and shorter hospitalizations (median hospital stay, 32 days compared with 40.5 days; P = 0.004). Other clinical outcomes, such as frequency and severity of toxicities, development of pneumonia or infection, in-hospital death, and response rate were similar in the two groups. Actuarial long-term disease-free survival was 64% for patients treated with GM-CSF and 58% for patients who received placebo after 32 months of follow-up (P = 0.15). The group treated with GM-CSF had lower total charges after infusion of autologous marrow than the placebo group (median in-hospital charges, $39,800 compared with $62,500; P = 0.005) because of lower post-infusion charges for room and board, antibiotic therapy, transfusions, laboratory tests, and physical therapy visits. CONCLUSIONS: Administration of GM-CSF was associated with acceleration of myeloid and platelet recovery and was cost effective in the treatment of patients with relapsed Hodgkin disease who received intensive chemotherapy. PMID- 1728203 TI - Comparison of adenosine, dipyridamole, and dobutamine in stress echocardiography. AB - OBJECTIVE: To compare adenosine, dipyridamole, and dobutamine in stress echocardiography with regard to sensitivity, specificity, accuracy, and side effects. DESIGN: Crossover, blinded comparison, with coronary angiography serving as the criterion standard. SETTING: U.S. Army tertiary care hospital. PARTICIPANTS: Forty participants, 25 with coronary disease and 15 without coronary disease. Patients were eligible if they had coronary angiography within 6 weeks of stress testing or if they had a risk for coronary disease of less than 5%. MEASUREMENTS: Left ventricular wall motion was recorded after dobutamine (0.38 mg/kg body weight), adenosine (0.84 mg/kg body weight), and dipyridamole (0.84 mg/kg body weight) stress testing. Stress echocardiographic evaluation was considered to be abnormal if the patient developed new or progressive wall motion abnormalities. The rate of side effects for the types of echocardiography and the patient preference were recorded. MAIN RESULTS: The sensitivity of dobutamine stress echocardiography (76%; 95% CI, 59% to 93%) was significantly higher than that of adenosine echocardiography (40%; CI, 21% to 59%; P less than 0.001) and that of dipyridamole echocardiography (56%; CI, 37% to 75%; P = 0.019). The specificity of adenosine testing (93%; CI, 80% to 100%) was significantly higher than that of dobutamine echocardiography (60%; CI, 35% to 85%; P = 0.008) and that of dipyridamole echocardiography (67%; CI, 43% to 91%; P = 0.028). Symptoms were more frequent with adenosine echocardiography (100%) than with dipyridamole (88%; P less than 0.001) or dobutamine (80%; P less than 0.001) echocardiography. Treatment for persistent symptoms was required in more patients after dipyridamole echocardiography (40%) than after dobutamine (12%; P less than 0.001) or adenosine (0%; P less than 0.001) echocardiography. More patients preferred dobutamine (48%) or dipyridamole (40%) echocardiography to adenosine echocardiography (12%; P less than 0.001). CONCLUSIONS: Dobutamine stress echocardiography is more sensitive and is better tolerated than adenosine or dipyridamole stress echocardiography. Adenosine echocardiography is more specific than dobutamine or dipyridamole echocardiography and is less likely to cause persistent symptoms. PMID- 1728204 TI - Urine free cortisol in the high-dose dexamethasone suppression test for the differential diagnosis of the Cushing syndrome. AB - OBJECTIVE: To develop criteria for interpreting the high-dose dexamethasone suppression test using urine free cortisol as an end point for the differential diagnosis of the Cushing syndrome. DESIGN: Retrospective review. SETTING: Inpatient research ward. PATIENTS: Patients (118) with surgically confirmed causes of the Cushing syndrome: 94 with pituitary disease, 14 with primary adrenal disease, and 10 with ectopic adrenocorticotropic hormone (ACTH) secretion. MAIN OUTCOME MEASURES: The sensitivity, specificity, and diagnostic accuracy were determined for the high-dose dexamethasone suppression test using urine free cortisol and using 17-hydroxysteroid excretion. For each analysis, patients with pituitary disease were considered to be "diseased" and patients with nonpituitary disease were considered to be "non-diseased". The level of suppression that gave 100% specificity was determined for each steroid. RESULTS: The accuracy of urine free cortisol when used as an end point in the high-dose dexamethasone suppression test was equivalent to that of 17-hydroxysteroid excretion. At all levels of sensitivity and specificity, however, the degree of suppression of urine free cortisol used for the diagnosis of pituitary disease was greater than that of 17-hydroxysteroid excretion. The likelihood ratios for pituitary disease based on urine free cortisol suppression of greater than 50%, of greater than 80%, and of greater than 90% were 4.2, 10.1, and "infinite," respectively. Suppression of urine free cortisol greater than 90% or suppression of 17-hydroxysteroid excretion greater than 64% was associated with 100% specificity. When these criteria were combined, the percentage of correct predictions (102 of 118 [86%; 95% CI, 78% to 92%]) was higher than that obtained using either steroid alone (89 of 118 [75%; CI, 65% to 83%]) (P = 0.009) and higher than that obtained using the traditional criterion of 50% suppression for 17-hydroxysteroid excretion (95 of 118 [80%; CI, 71% to 87%]) (P = 0.016). CONCLUSIONS: In the high-dose dexamethasone suppression test, the degree of suppression of urine free cortisol used for the diagnosis of pituitary disease is greater than that traditionally used for 17-hydroxysteroid excretion. The diagnostic performance of the test is improved by measuring both urine free cortisol and 17-hydroxysteroid excretion and by requiring greater suppression of both steroids. PMID- 1728205 TI - Myocardial infarction mimicked by acute cholecystitis. PMID- 1728206 TI - The role of risk stratification in the early management of a myocardial infarction. AB - OBJECTIVE: To review the literature on early management of myocardial infarction. DATA SOURCES: Papers published or referenced in major English-language cardiology journals for the last 15 years. STUDY SELECTION: Large recent multicenter studies and the guidelines for early management of patients with acute myocardial infarction (American College of Cardiology/American Heart Association Task Force) were emphasized. DATA SYNTHESIS: A strategy for risk stratification was developed from information available in the emergency department, from the first days of the hospitalization, and before discharge to identify patients in whom intervention might improve prognosis. CONCLUSIONS: Treatment of myocardial infarction requires establishing patency of the infarct-related artery, usually with thrombolysis. Risk stratification begins in the emergency department (phase 1) using clinical (primarily the electrocardiogram) and historical data to identify patients at risk for massive infarction. For patients at highest risk, the efficacy of thrombolytic therapy must be assured and, if not effective, emergency angiography and mechanical reperfusion should be considered. During days 2 to 5, (phase 2), patients with large amounts of ischemic myocardium, postinfarction angina, "flash" pulmonary edema, or anterior non-Q infarctions are identified and studied. Predischarge (phase 3) stratification identifies those patients at risk for early death. A previous infarction, an ejection fraction less than 0.40, pulmonary congestion during the hospitalization, delayed afterpotentials on signal-averaged electrocardiography, symptomatic ventricular ectopic beats, decreased heart rate variability, limited exercise tolerance, or ischemia on exercise testing identifies patients at high risk. Patients with jeopardized myocardium must be identified for revascularization to try to improve survival. More data are needed to determine whether angioplasty or bypass surgery will improve prognosis in these patients. PMID- 1728207 TI - Alcohol and other substance abuse and impairment among physicians in residency training. AB - Substance abuse and impairment are serious societal problems. Physicians have historically had high rates of substance abuse, which has been viewed as an occupational hazard. Most authorities agree that the rate of alcoholism among practicing physicians is similar to that among control populations and that the rates of other substance abuse are greater, although some studies have shown no difference. Data about substance abuse among residents in training are limited but suggest that the use of benzodiazopines is greater than that among age matched peers, whereas the use of alcohol is similar between the two groups. Medical institutions, including those with teaching programs, have legal and ethical responsibilities concerning substance abuse among current and future physicians. Many training programs, however, do not provide educational programs on this subject, do not have faculty trained in substance abuse medicine, and do not have a formal system to address the problem of residents who are suspected or known to be substance abusers. This position paper examines the extent of substance abuse, including alcohol abuse, among physicians in residency training. It outlines approaches to the problem and delineates responsibilities of institutions and residency program directors. Recommendations are made to establish an informational program and a clearly defined, organized process to address the problems of substance abuse among residents. Careful and humane approaches can be used to identify and treat residents with substance abuse problems and thus allowing them to complete their training as competent and drug free professionals. PMID- 1728208 TI - Osler's legacy: the centennial of The Principles and Practice of Medicine. PMID- 1728209 TI - Granulocyte-macrophage colony-stimulating factor (GM-CSF): answers or more questions? PMID- 1728211 TI - Patient-centered and physician-centered approaches to interviewing. PMID- 1728210 TI - Valuing clinical strategies early in their development. PMID- 1728212 TI - Methicillin-resistant Staphylococcus aureus in nursing homes. PMID- 1728213 TI - Methicillin-resistant Staphylococcus aureus in nursing homes. PMID- 1728214 TI - Optimal timing of initial breast cancer surgery. PMID- 1728215 TI - Postmenopausal estrogen and prevention bias. PMID- 1728216 TI - Transplants from the same donor. PMID- 1728217 TI - A study on optimal temperature for isolated lung preservation. AB - Fifty-two heart-lung blocks (grafts) of New Zealand white rabbits were used for determining optimal temperature in lung preservation. Grafts were inflated with room air and preserved by simple surface cooling at arbitrarily determined temperatures for 18 hours. Graft function was assessed by nonrecirculated perfusion with autologous blood. Segmented regression models between functional parameters and preservation temperature (T) were applied for determining optimal temperature. Graft ability was also assessed from the point of view of pulmonary circulation by indocyanine green dilution rate of effluent and histological distribution of carbon particles. Significant segmented regression curves and lines between parameters of effluent oxygen tension (PO2) and wet-dry weight ratio (W/D), and T were obtained as follows: PO2 = 150/(1 + 3208.1[e-1.17T]), p less than 0.01; PO2 = -13.8T + 222.6, p less than 0.05; W/D = 5.0 + 1.5/(1 + 0.0028[e0.94T]), p less than 0.01; W/D = 0.075T + 4.52, p less than 0.05. Optimal temperature for lung preservation by topical cooling was calculated as 8 degrees to 9 degrees C from each intersecting point of regression equations. Analysis of regression curves suggested that the most common hypothermic ischemic injury during preservation by topical cooling is pulmonary vascular obstruction, which might be induced at temperatures lower than the critical temperature of 6 degrees to 7 degrees C. Indocyanine green dilution rate and histological findings supported the results of graft functional parameters. PMID- 1728218 TI - Long-term experience with descending aortic dissection: the complication-specific approach. AB - We analyzed long-term results in 71 patients (45 men and 26 women) treated over 17 years for documented descending aortic dissection. Forty-nine patients were treated medically and 22, surgically. Actuarial survival was 65% at 1 year, 57% at 3 years, 50% at 5 years, and 28% at 10 years for the whole group. For the group treated medically, survival was 73%, 63%, 58%, and 25% at 1 year, 3 years, 5 years, and 10 years, respectively, and for the group treated surgically, 47%, 40%, and 28% at 1 year, 3 years, and 5 years, respectively. Ten (20.4%) of the 49 medically treated patients died early (5 of rupture), and 14 (28.6%) died late (8 of dissection). Five medically treated patients crossed over to surgical management for complications of dissection. Among the surgically treated patients, 6 underwent standard graft replacement of the proximal descending aorta, 8 underwent the fenestration procedure (with a standardized retroperitoneal abdominal approach), and 4 underwent the thromboexclusion operation. Specific analysis of fenestration in 14 patients (including some with persistent descending aortic dissection after replacement of the ascending aorta for dissection) found it to be safe and effective. Actuarial survival after fenestration was 77%, 77%, and 53% at 1 year, 3 years, and 5 years, respectively. Thromboexclusion was found effective, and postoperative studies confirmed thrombosis of the descending aorta with preservation of the lowest intercostal arteries. Fifteen of the 21 surviving medically treated patients agreed to return for follow-up imaging. Nine had thrombosis of the false lumen. An interesting radiographic finding was that 4 of the 15 restudied patients had a saccular aneurysm in the aorta at the level of the left subclavian artery. We recommend a complication-specific approach to the management of descending aortic dissection. Uncomplicated dissection is treated medically, whereas complicated dissection is treated surgically, with realized rupture treated by standard graft replacement, limb ischemia treated by fenestration, and enlargement or impending rupture treated by thromboexclusion. PMID- 1728219 TI - Repeat mediastinoscopy in the assessment of new and recurrent lung neoplasm. AB - From 1976 to 1990, 140 patients (mean age, 66 years; 91% male) underwent repeat mediastinoscopy as a routine staging procedure. The mean interval between first and second mediastinoscopy was 56 months. Owing to adhesions, 26 repeat mediastinoscopies (18%) were considered incomplete. There was no mortality, and 10 complications did not require interventional therapy. The results were positive in 20 patients, thus avoiding an unnecessary thoracotomy. In 7 patients with negative findings, positive lymph nodes were found at thoracotomy or by transcarinal puncture biopsy. The sensitivity of repeat mediastinoscopy in this series is 74%, and the accuracy 94%. We consider repeat mediastinoscopy a safe and reliable preoperative staging procedure in new or recurrent lung cancer. PMID- 1728220 TI - Variation in cryolesion penetration due to probe size and tissue thermal conductivity. AB - The purpose of this study is to present data comparing the penetration of cryolesions created by various sizes and shapes of cryoprobes in human cadaveric myocardium, fat, and tissue of the central fibrous body. Ten cryolesions were made for each combination of tissue and cryoprobe studied. All cryolesions enlarged most rapidly during the first minute of cryothermia (p less than 0.01). Maximal cryothermic penetration into nontrabeculated myocardium was 8.5 +/- 0.5 mm (15-mm flat probe) and 6.1 +/- 1.0 mm (5-mm small probe). Maximal cryothermic penetration into trabeculated myocardium was 9.4 +/- 1.0 mm (10-mm cone-tipped probe) and 7.4 +/- 0.5 mm (10-mm flat probe). Maximal cryothermic penetration into fat was 4.7 +/- 0.7 mm (15-mm flat probe) and 3.9 +/- 0.7 mm (5-mm flat probe). The deeper penetration of cryothermia into myocardium as compared with fat (p less than 0.05) is related to the lower thermal conductivity of fat. Maximal cryothermic penetration of the central fibrous body was similar to that of the myocardium with transmural freezing of the central fibrous body after 4.4 +/- 0.3 minutes of cryothermia. These data can be used when determining the optimal cryothermic exposure for ablation of arrhythmogenic tissue. PMID- 1728221 TI - Left ventricular assistance without thoracotomy: mediastinal and transseptal approaches to the left heart. AB - Two methods to cannulate the left atrium for initiating mechanical left ventricular circulatory assistance using a centrifugal pump were investigated in 25 sheep. A modified Dennis transatrial septal approach produced flow rates of 88.6 +/- 14 mL.kg-1.min-1 through 21F catheters inserted during fluoroscopy through the jugular vein. In 8 animals the septal perforation was plugged after decannulation with a modified Rashkind umbrella plug. Fibroendothelial tissue covered the plug by 4 week. In 7 other animals, the septal defect was not plugged. The septal defect reached pinpoint size by 2 weeks and was completely closed by 4 weeks. In 10 sheep, the left atrium was cannulated from the neck through the mediastinum. Left ventricular assistance flow averaged 71.6 +/- 14 mL.kg-1.min-1. Mean blood loss during 1 hour of left ventricular assistance was 47 mL. In 8 animals, the atrial perforation was plugged with a mean blood loss of 253 +/- 194 mL. In 2 animals, the perforation was intentionally not plugged; mean blood loss was 700 mL. All animals survived. The modified Dennis transatrial method is recommended as a safe, expeditious, cost-effective method to implement left ventricular assistance without thoracotomy. The mediastinal approach, which is technically possible in humans, is more difficult but feasible. Left ventricular assistance has been proven to be the most effective way to rest the failing, ejecting left ventricle. Implementation without thoracotomy potentially expands applications of left ventricular assistance for temporary support of patients with severe manifestations of ischemic heart disease. PMID- 1728222 TI - Recurrent intravenous leiomyomatosis with cardiac extension. AB - A case of recurrent intravenous leiomyomatosis with cardiac extension and a temporally extended presentation is described. Complete excision was achieved employing simultaneous sternotomy and laparotomy and deep hypothermia with circulatory arrest. Coronary revascularization was performed concomitantly with complete tumor resection. Diagnostic, operative, and pathologic considerations are reviewed and a preferred surgical approach discussed. PMID- 1728223 TI - Esophagobronchial fistula associated with corrosive stricture of the esophagus. PMID- 1728224 TI - Unsuspected metastatic choriocarcinoma presenting as unilateral spontaneous pneumothorax. AB - Spontaneous pneumothoraces are usually caused by subpleural apical blebs but may also be secondary to metastasis to the lung. We present the case of a 20-year-old woman with spontaneous pneumothorax secondary to choriocarcinoma metastatic to the lung. PMID- 1728225 TI - Complete thoracic ectopia cordis with double-outlet right ventricle: neonatal repair. AB - A case of total thoracic ectopia cordis with double-outlet right ventricle and ventricular septal defect is presented. Prenatal diagnosis allowed single-stage correction immediately after birth. This approach proved to be technically feasible. Death occurred on the twelfth postoperative day owing to sepsis unrelated to the repair. PMID- 1728226 TI - Successful treatment of acute postoperative pulmonary hypertension with nifedipine. AB - We report the successful use of nifedipine in the treatment of acute pulmonary hypertension in an infant after a cardiac operation. This patient had undergone total surgical correction of his truncus arteriosus malformation. He had signs of severe pulmonary artery hypertension unresponsive to hyperventilation, oxygenation, sedation, and a myriad of vasodilators. Nifedipine, 0.2 mg/kg every 4 hours, effectively treated his pulmonary artery hypertension and allowed for a smooth postoperative course and positive outcome. PMID- 1728227 TI - Angiographic demonstration of no-flow anatomical patency of internal thoracic coronary artery bypass grafts. AB - To clarify the no-flow situation of the stringlike internal thoracic artery graft, we angiographically examined such grafts by temporarily occluding the recipient coronary artery with a percutaneous transluminal coronary angioplasty balloon and were able to reveal anatomical patency of the internal thoracic artery graft in 2 patients 1 year and 3 years after the operations. Thus, there is a possibility that internal thoracic artery grafts may continuously maintain anatomical patency even under no-flow situations just like nonfunctioning collateral vessels and may function properly later as a graft when the native coronary flow decreases. Also, this angiographic technique can be a new method for detecting anatomical patency of no-flow and functionally closed internal thoracic artery grafts. PMID- 1728228 TI - New needle for catheter introduction in left atrium. AB - We describe a new needle for left atrial catheter introduction. It allows catheter introduction through the right superior pulmonary vein or through the interatrial septum. Used in 32 patients (adults and children), the device proved to be highly efficient, simple, and safe. PMID- 1728229 TI - Sheathless insertion of the percutaneous intraaortic balloon pump: an alternate method. AB - A technique is described that allows insertion of a standard percutaneous intraaortic balloon without use of the larger 12F sheath. Standard 9.5-cm. Percor balloons were inserted in a series of patients without using the provided sheath. Elimination of the sheath reduced the potential vascular complications related to the extra bulk placed in the femoral artery by the sheath. No serious complications were noted in more than 25 patients. PMID- 1728230 TI - Methylene blue guidance for simplified resection of a lung lesion. AB - A 60-year-old patient returned 1 year after right pneumonectomy with a new primary squamous cell carcinoma of the left lower lobe. Using fluoroscopic guidance, the lesion and the shortest track to the surface were marked by methylene blue preoperatively. The lesion was easily excised by wedge resection without the need for manipulation or deflation of the lung. PMID- 1728231 TI - Improved technique for hilar vascular stapling. AB - The use of stapling instruments is common in thoracic surgery, but there is a continued reluctance to use them on hilar vascular structures. We have developed a technique that satisfies the major objections to stapler use on hilar vascular structures. PMID- 1728232 TI - Staging: the key to rational management of lung cancer. AB - Staging is the quantitative assessment of malignant disease and allows logical groupings of patients with a similar extent of disease for prognostic, therapeutic, and analytic purposes. In bronchogenic carcinoma a stage is assigned based on size, location, and the extent of invasion of the primary tumor, as well as the presence of any regional or metastatic disease. Selecting the most appropriate treatment for a patient with bronchogenic carcinoma depends on precise staging. The extent of local invasion and presence of metastatic disease will determine the likelihood of complete resection and possible cure. Careful assessment of the history, blood chemistry, radiographic studies, bronchoscopy, mediastinoscopy, and exploration (thoracotomy) are all important staging tools. Routine radionuclide scans have no useful role when there is no clinical or laboratory evidence of metastases. The T status of a tumor is best judged by bronchoscopy and at thoracotomy. Thoracic surgeons must be familiar with the techniques available to determine T status intraoperatively and use this information when planning resection. Computed tomography of the chest has fallen short in predicting direct invasion of the mediastinum and chest wall. Cervical and anterior mediastinoscopy remain important tools in determining operability. Intraoperative assessment of node involvement determines the extent of resection and likelihood of cure. PMID- 1728233 TI - Benign tumors of the lung. PMID- 1728235 TI - Combined superior-transseptal approach to the mitral valve. PMID- 1728234 TI - Nonhemic prime in cardiopulmonary bypass. PMID- 1728236 TI - Bronchoscopic appearance after single-lung transplantation. PMID- 1728237 TI - Fine-needle aspiration biopsy. PMID- 1728238 TI - Operative risks and long-term results of operation for left ventricular aneurysm. AB - A review of 336 consecutive patients who underwent repair of left ventricular aneurysm from 1978 to 1989 disclosed that partial resection of the aneurysm and conventional closure of the ventriculotomy was performed in 281 patients, inverted T closure in 17, and endocardial patch in 38. These two latter techniques were developed in an attempt to restore normal left ventricular geometry. The operative mortality was 6.8% (23 patients). A stepwise logistic regression analysis of various preopeative clinical, hemodynamic, and angiographic variables revealed that left ventricular ejection fraction of 0.20 or less, age greater than 60 years, previous myocardial revascularization, lack of angina, and New York Heart Association functional class IV were independent predictors of operative mortality. The technique of repair was not a predictor of outcome, but when patients with poor left ventricular function were analyzed separately, the operative mortality was reduced from 12.5% to 6.5% when newer techniques were employed. Patients were followed up during a mean of 6.3 years. There have been 51 late deaths, 45 cardiac. Cox regression analysis indicated that poor left ventricular function and left main coronary artery stenosis were the only two predictors of late mortality. The actuarial survival at 10 years was 63% +/- 4%. Most patients (88%) are in New York Heart Association class I or II. These data indicate excellent long-term results after repair of left ventricular aneurysm. Newer techniques of repair are valuable in patients with poor left ventricular function. PMID- 1728239 TI - Evaluation of an extraaortic counterpulsation device in severe cardiac failure. AB - A valveless, single-orifice polyurethane ventricle with a maximum stroke volume of 60 mL was implanted on the brachiocephalic artery just above the aortic arch in sheep (n = 14) to act as an extraaortic counterpulsation device. In parallel, an intraaortic balloon was placed in the descending thoracic aorta. Both devices were pneumatically driven with an intraaortic balloon pump console that was gated by the electrocardiogram to provide aortic diastolic augmentation at a stroke volume of 40 mL. To compare the efficacy of counterpulsation for each device during severe cardiac failure, biventricular block was induced by continuous infusion of esmolol (100 to 600 micrograms.kg-1.min-1), titrated to reduce aortic flow and pressure to less than 75% of baseline. Pulsatile coronary and aortic flows were recorded with ultrasonic flow probes placed around their respective vessels. Aortic root and left ventricular pressures were recorded using micromanometers. The enhancement of hemodynamic variables for both devices were compared for optimal timing conditions, which were defined as inflation set just before the dicrotic notch and deflation bordering on isovolumetric systole. The extraaortic counterpulsation device was able to significantly augment aortic and coronary flows while simultaneously decreasing left ventricular tension time index and aortic end-diastolic pressure (p less than 0.02). The intraarotic balloon pump was able to significantly reduce only tension time index (p less than 0.002) to a lesser extent that the extraaortic counterpulsation device. All analysis was performed with the paired-samples t test. The extraaortic counterpulsation device greatly improves the myocardial oxygen supply-consumption ratio of the left ventricle by increasing diastolic coronary flow and reducing left ventricular wall tension during systole.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728240 TI - Surgical repair of aortic root aneurysms in 280 patients. AB - Bentall's technique for repair of annuloaortic ectasia has been associated with postoperative bleeding and with false aneurysms at the anastomotic site between the coronary orifices and valve-containing graft. To reduce the incidence of these complications, we modified the Bentall procedure, using a simplified technique to implant the graft and to create a fistula between the closed perigraft space and right atrium to control bleeding. A continuous suture of monofilament polypropylene was used to implant the prosthetic valve ring and to anastomose the coronary orifices to the Dacron fabric. In some instances, a brief period of hypothermic circulatory arrest was needed to perform the distal aortic anastomosis. Among 562 patients undergoing operation for aneurysm of the ascending aorta between January 1, 1980, and February 28, 1990, 280 underwent graft replacement with a valve-containing composite conduit. Most (82%) had annuloaortic ectasia. In 267, we performed a classic Bentall procedure with direct anastomosis between the coronary orifices and fabric graft. The remaining 13 patients underwent other procedures for coronary connection. Early mortality was 5.0%. Reoperation for bleeding was needed in 13.2% of patients who underwent operation before we used the right atrial fistula technique and in 4.4% after we began to use the technique (p = 0.044). Actuarial survival was 71% at 5 years and 65% at 7 years. For hospital survivors, it was 76% at 5 years and 70% at 7 years. During follow-up, only 9 patients have required reoperation. A false aneurysm at the coronary anastomosis, which was associated with prosthetic valve endocarditis, developed in 1 patient. No permanent fistulas have developed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728241 TI - Retrograde cerebral perfusion through a superior vena caval cannula protects the brain. AB - Retrograde cerebral perfusion through a superior vena caval cannula is a new technique for protecting the brain during aortic arch operations. In mongrel dogs (n = 10; 13 to 15 kg) we have performed retrograde cerebral perfusion (300 mL/min) by infusing blood through a superior vena caval cannula with aortic and inferior vena caval drainage. We have measured the cerebral tissue blood flow, oxygen consumption, and carbon dioxide exudation during retrograde cerebral perfusion at normothermia (NT, 37 degrees C) and hypothermia (HT, 20 degrees C) and have compared these values with values obtained in dogs during cardiopulmonary bypass (1,200 mL/min). Cerebral tissue blood flow was measured by the hydrogen clearance method. During retrograde cerebral perfusion about 20% of the superior vena caval perfusate was returned through the aorta and the rest drained from the inferior vena cava. Cerebral vascular resistance during retrograde cerebral perfusion was lower than that during cardiopulmonary bypass (NT, 63.8 +/- 52.5 versus 126.9 +/- 58.4; HT, 28.4 +/- 32.8 versus 69.5 +/- 28.7 x 10(3) dynes.s.cm(-5). Retrograde cerebral perfusion provided half the cerebral tissue blood flow of cardiopulmonary bypass (NT, 14.7 +/- 6.4 versus 34.3 +/- 7.8; HT, 17.6 +/- 5.6 versus 37.2 +/- 10.6 mL/min). Retrograde cerebral perfusion also provided a third of the oxygen (NT, 4.4 +/- 2.1 versus 12.3 +/- 7.1; HT, 1.4 +/- 0.8 versus 4.2 +/- 1.3 mL/min) and discharged 20% of the carbon dioxide (NT, 0.24 +/- 0.08 versus 1.19 +/- 0.58; HT, 0.15 +/- 0.06 versus 0.51 +/- 0.17 mmol/min) when compared with cardiopulmonary bypass. Retrograde cerebral perfusion may reduce ischemic damage during interruption of cerebral blood flow. PMID- 1728242 TI - Laryngotracheal resection and reconstruction for subglottic stenosis. AB - Eighty patients with inflammatory stenoses of the subglottic larynx and upper trachea were treated by single-stage laryngotracheal resection and reconstruction. Fifty stenoses originated from postintubation lesions (endotracheal tubes, tracheostomy, cricothyroidostomy), 7 originated from trauma, 19 were idiopathic, and 4 were miscellaneous. Repair consisted of resection of the anterolateral cricoid arch in all patients, plus resection of posterior laryngeal stenosis where present, with salvage of the posterior cricoid plate, appropriate resection and tailoring of the trachea, and primary anastomosis using a posterior membranous tracheal wall flap to resurface the bared cricoid cartilage in 31 patients. One postoperative death resulted from acute myocardial infarction. Long-term results were excellent in 18 patients, good in 48, satisfactory in 8, and failure in 2. Three additional patients had good results at discharge but were followed up for less than 6 months. PMID- 1728243 TI - Neutrophils are not necessary for ischemia-reperfusion lung injury. AB - The role of neutrophils (PMNs) in ischemia-reperfusion injury after lung transplantation is unclear. If PMNs are involved in ischemia-reperfusion injury in the intact rat, then PMNs should sequester in the injured lung and PMN depleted rats should develop less injury. Group A rats were treated with a rabbit anti-rat PMN antibody causing profound neutropenia (less than 100 PMNs/microL) and group B with control serum (greater than 2,000 PMNs/microL). Rats were anesthetized and left lung ischemia was sustained for 90 or 180 minutes by clamping the bronchus and the pulmonary artery and vein. Lung injury was quantified by the accumulation of radiolabeled (125I) albumin in ischemic left and nonischemic right lungs (cpm per gram of lung/cpm per gram of blood). Ischemia caused significant lung injury (p less than 0.05) in both PMN-depleted (albumin leak index: 90 min, 0.208; 180 min, 0.218) and nondepleted (90 min, 0.222; 180 min, 0.241) animals compared with nonischemic controls (depleted: 90 min, 0.050; 180 min, 0.100; nondepleted: 90 min, 0.063; 180 min, 0.101); microscopy also demonstrated lung injury. The injury was not associated with PMN sequestration as shown by light microscopy. Thus, we conclude that PMNs are not necessary for ischemia-reperfusion injury and PMN-depletion does not attenuate ischemia-reperfusion injury. PMID- 1728244 TI - University of Wisconsin versus modified Euro-Collins solution for lung preservation. AB - In a canine model, the quality of lung preservation was assessed using pulmonary artery flush after prostacyclin administration with either modified Euro-Collins solution or University of Wisconsin solution. Twelve combined heterotopic heart and orthotopic left lung allotransplantations were performed after 6 hours of cold ischemia. Myocardial preservation was achieved using St. Thomas Hospital solution. Donor organs were anastomosed parallel to the recipient's heart and right lung, and the superior vena cava inflow was directed into the transplanted heart-left lung block after ligation of the recipient's superior vena cava proximal to the caval anastomosis. Postoperatively, cardiorespiratory function was evaluated separately for donor and recipient organs at an inspired oxygen fraction of 0.4 for a maximum of 12 hours. Significantly improved oxygenation and lower pulmonary vascular resistance index of the donor lung was observed in the University of Wisconsin + prostacyclin group, whereas pulmonary artery pressures showed no significant differences in between both groups. It is concluded that superior results in lung preservation can be achieved with pulmonary artery flush perfusion using University of Wisconsin solution and prostacyclin when compared with Euro-Collins solution and prostacyclin. PMID- 1728245 TI - Normal bronchial healing without bronchial wrapping in canine lung transplantation. AB - The deleterious effect of steroids on bronchial healing in lung transplantation has led to the development of techniques to protect the anastomosis and to the exclusion of steroid-dependent patients from transplantation. The effect of steroids on bronchial healing was tested in a canine single-lung allotransplantation model. Twenty size-matched mongrel dogs (20 to 30 kg) underwent left lung transplantation without anastomotic wrap or direct revascularization. Postoperatively, all received daily doses of cyclosporine (15 mg/kg) and azathioprine (1 mg/kg) and were subdivided into three steroid dosage groups. Group A (n = 10) animals received 1.5 mg/kg of prednisone per day whereas groups B (n = 5) and C (n = 5) received 5.0 mg/kg of prednisone per day for 28 postoperative days. In addition, group C received prednisone (5.0 mg.kg-1.day-1) for 1 month preoperatively. In group A, 8 of 10 dogs survived 28 days without evidence of respiratory compromise, with anastomotic bursting pressure greater than 510 mm Hg. In group B, all 5 dogs survived to 28 days without evidence of respiratory compromise and with intact bronchial anastomoses (bursting pressures greater than 510 mm Hg). In group C, 3 of 5 animals survived to 28 days with intact anastomoses. Histological examination demonstrated normal bronchial healing in all anastomoses. These data suggest that preoperative steroid dependence should not be a contraindication to lung transplantation and that bronchial anastomotic wrapping with vascular tissue may not be essential. PMID- 1728246 TI - Pleuroperitoneal shunts for refractory chylothorax after operation for congenital heart disease. AB - Between 1980 and 1990, 10 of 12 children with a symptomatic chylothorax after operation for congenital heart disease failed to respond to traditional medical therapy (thoracentesis, tube thoracostomy, low-fat diet). All 10 patients underwent placement of a pleuroperitoneal shunt, with complete resolution of the chylothorax in 9 patients (90%). Cardiac catheterization, performed before placement of the pleuroperitoneal shunt in 5 patients, demonstrated elevated right atrial pressure in all patients (range, 10 to 25 mm Hg). The pleuroperitoneal shunt functioned effectively in 4 patients with moderately elevated right atrial pressures (range, 10 to 16 mm Hg; median, 13.5 mm Hg) but not in 1 patient with a right atrial pressure of 25 mm Hg. Pleuroperitoneal shunting as treatment for chylothorax after operation for congenital heart disease is safe and effective, even in the face of moderate elevations in right atrial pressure. PMID- 1728248 TI - Thoracic surgery participation in cooperative group studies of cancer therapy. PMID- 1728247 TI - Bronchial revascularization in double-lung transplantation: a series of 8 patients. Bordeaux Lung and Heart-Lung Transplant Group. AB - Donor airway ischemia is the main cause for defective tracheal or bronchial healing after double-lung transplantation. Anatomical studies and bronchial arteriograms have shown that the right intercostal bronchial artery is constant (95% of instances) and provides an important blood supply to the distal trachea, the carina, and the right bronchial tree as well as to the left side through a subcarinal and periadventitial anastomostic network. To maintain this important bilateral bronchial circulation, it is of capital importance not to mobilize the arteries individually and to avoid large dissections around the carina. Both bronchi can thus be revascularized by indirect aortic reimplantation using a bypass graft to a single aortic patch that includes the origin of the right intercostal bronchial artery. Furthermore, the origin of other vessels (a common trunk and left arteries) can be found within a short distance of the right intercostal bronchial artery and possibly be contained within the same aortic patch. From a series of 56 lung transplantations, 8 patients underwent restoration of the bronchial vascularization using a recipient saphenous vein graft between the donor bronchial arteries and the anterior aspect of the recipient's ascending aorta. A lower tracheal anastomosis was performed. Bronchial arterial blood supply was evaluated both by endoscopy and by arteriography at about the 15th postoperative day. The bronchial circulation was visualized at this time in five of seven arteriographies, and this was associated with excellent tracheal healing in all 8 patients. PMID- 1728249 TI - Serotonergic function in obsessive-compulsive disorder. Behavioral and neuroendocrine responses to oral m-chlorophenylpiperazine and fenfluramine in patients and healthy volunteers. AB - To evaluate serotonergic (5-hydroxytryptamine) function in obsessive-compulsive disorder, behavioral and neuroendocrine responses to m-chlorophenylpiperazine (m CPP; 0.5 mg/kg orally) and fenfluramine hydrochloride (60 mg orally) were examined in 20 patients and 10 healthy controls under double-blind, placebo controlled conditions. Following m-CPP, but not fenfluramine or placebo, 55% (11/20) of the patients with obsessive-compulsive disorder experienced a transient exacerbation of obsessive-compulsive disorder. Prolactin response was blunted in patients following m-CPP but not following fenfluramine. Patients with greater behavioral response to m-CPP had smaller prolactin responses. Cortisol response to m-CPP and fenfluramine did not significantly differ between the groups. Behavioral and neuroendocrine responses appeared divergent. This does not suggest simply upregulation or downregulation of 5-hydroxytryptamine receptors, but rather complex mechanisms involving multiple neurotransmitter and neuromodulator systems. PMID- 1728250 TI - Psychopathologic precursors and sociodemographic risk factors for the schizophrenia syndrome. AB - This article presents a prospective analysis of an antecedent psychopathologic features and sociodemographic risk factors in schizophrenia with data from five community sites in the National Institute of Mental Health Epidemiologic Catchment Area Program. Three nonoverlapping psychotic cases were defined using DSM-III definitions as implemented by the Diagnostic Interview Schedule (DIS): (1) DSM-III Schizophrenia Criterion A; (2) Criterion A and Affective Episode; and (3) full Schizophrenia. In a 1-year follow-up period, the cumulative incidence rate of Criterion A was 0.79 per 100, for Criterion A with Affective Episode it was 0.17 per 100, and for Schizophrenia the rate was 0.20 per 100. In multivariable logistic regression modeling, the patterns of associations between sociodemographic factors and DIS/DSM-III Schizophrenia resembled patterns in clinically based registry data. Male subjects had an earlier peak onset than female subjects, and marital status and employment were strongly related to odds of developing DIS/DSM-III Schizophrenia. An interaction between gender and never marrying showed never-married men at 50 times higher odds of developing DIS/DSM III Schizophrenia, never-married women at 14 times higher odds, and married women at 2.5 times higher odds, relative to married men. Adjusting for sociodemographic factors, DIS/DSM-III Obsessive Compulsive Disorder and Social Phobia were both associated with more than 3.5 times increased odds of developing DIS/DSM-III Schizophrenia. Several other psychopathology items, including panic attacks, were associated with increased odds of developing DIS/DSM-III Schizophrenia. There were both similarities and differences in risk factor structure between DIS/DSM III Schizophrenia and the other two defined categories of case. PMID- 1728251 TI - Chromosome fragility and psychopathology in obligate female carriers of the fragile X chromosome. AB - The relationship between fragility (the percentage of cells exhibiting the fragile X chromosome abnormality) and psychopathological conditions was investigated in a sample of 40 obligate female carriers of the fragile X chromosome. Subjects were categorized by those with positive fragility greater than 0% (n = 19) and those with 0% fragility (n = 21). Compared with women with 0% fragility, it was expected that women with positive fragility would have a higher likelihood of manifesting a spectrum of social and psychological disability previously shown to be associated with fragile X syndrome in women. It was also expected that within the group with positive fragility, degree of fragility would be related to severity of symptoms. Results partially supported the hypotheses: women with fragility over 0% were more likely to be assigned a diagnosis of schizotypal features, were rated higher on symptoms associated with the schizophrenia spectrum, and scored lower on IQ, level of healthiest functioning, education, and socioeconomic status than women with 0% fragility. Subsequent comparisons with a control group indicated that the group with 0% fragility and normal controls did not differ on these variables. Within the group with positive fragility, increasing fragility was related to greater severity of symptoms and lower IQ, education, socioeconomic status, and levels of adaptive functioning, as predicted. Contrary to expectations, positive fragility was not associated with proportion of affective disorder diagnoses or ratings on affective disorder symptoms. The results of the study provide evidence that degree of fragility is a potentially important predictor of psychopathology among women with normal IQ who are carriers of the fragile X chromosome abnormality. PMID- 1728252 TI - The positive-negative distinction in schizophrenia. Review of natural history validators. AB - A review of the interaction between the positive-negative symptom distinction in schizophrenia and multiple measures of illness natural history reveals some redundant and compelling patterns. Negative or deficit symptoms are often associated with inferior social/instrumental functioning premorbidly, more abnormal voluntary/involuntary movements at illness presentation, and poorer long term outcome when present beyond the early phase of illness. Negative symptoms are semi-independent of positive symptoms. They are variable early in the illness but accrue in severity, stability, and prognostic weight with time. The nature of the processes that generate negative symptoms and their specificity to schizophrenia remain to be elucidated. Nevertheless, it is clear that negative symptoms are a common and valid component of schizophrenia and deserve recognition as such in our nosology. PMID- 1728253 TI - Subjective conclusions about schizophrenia. PMID- 1728254 TI - Nervous system Lyme borreliosis--revisited [corrected; erratum to be published]. AB - A great deal of confusion surrounds the diagnosis, clinical phenomenology, and treatment of Lyme borreliosis. Most diagnostic methods currently in use are indirect and do not differentiate between prior exposure and current infection. A critical review of the literature permits the characterization of a distinct set of neurologic disorders that are almost certainly caused by this infection and their differentiation from the plethora of syndromes that have been anecdotally linked to infection, but in which causality has never been established. This article describes the range of clinical disorders associated with Lyme borreliosis, provides an overview of current approaches to diagnosis, and reviews current treatment protocols. PMID- 1728255 TI - Combined trochlear nerve palsy and internuclear ophthalmoplegia. AB - We report a case of left superior oblique palsy combined with a right internuclear ophthalmoplegia. A right mesencephalic lesion involving the trochlear nerve nucleus (or its fibers prior to decussation) and the medial longitudinal fascicle was hypothesized. Magnetic resonance imaging showed a lesion at the suspected level. PMID- 1728256 TI - 'Haw River syndrome' or dentato-rubro-pallido-luysian atrophy? PMID- 1728257 TI - Idiopathic intracranial hypertension without papilledema: related to sleep apnea? PMID- 1728258 TI - Presurgical electroencephalographic patterns and outcome from anterior temporal lobectomy. AB - We reviewed data from 48 patients after anterior temporal lobe resection for medically intractable epilepsy. All had ictal electro-encephalographic (EEG) evidence of unilateral temporal lobe onset. Depth electrodes were used in 19 patients. Successful surgical outcome correlated significantly with factors that suggested a temporal lobe focus, particularly in the interictal scalp EEG. The most successful outcome occurred in patients with well-localized unilateral interictal temporal spikes (100% improved). The group with well-localized bilateral temporal spikes also did well (76% improved). Patients with extratemporal spread of the interictal spike on scalp EEG, either unilaterally or bilaterally, did less well. Only one third improved, despite extensive extracranial and intracranial monitoring, when indicated. The interictal scalp EEG may be the only EEG necessary for the presurgical evaluation of selected patients with intractable temporal lobe epilepsy. PMID- 1728259 TI - Evaluation of cerebral biopsies for the diagnosis of dementia. AB - To identify those patients most likely to benefit from a cerebral biopsy to diagnose dementia, we reviewed a series of 14 unselected biopsies performed during a 9-year period (1980 through 1989) at Duke University Medical Center, Durham, NC. Pathognomonic features allowed a definitive diagnosis in seven specimens. Nondiagnostic abnormalities but not diagnostic neuropathologic changes were seen in five additional specimens, and two specimens were normal. Creutzfeldt-Jakob disease was the most frequent diagnosis. One patient each was diagnosed as having Alzheimer's disease, diffuse Lewy body disease, adult-onset Niemann-Pick disease, and anaplastic astrocytoma. We conclude that a substantial proportion of patients presenting clinically with atypical dementia are likely to receive a definitive diagnosis from a cerebral biopsy. However, in those with coexisting hemiparesis, chorea, athetosis, or lower motor neuron signs, cerebral biopsies are less likely to be diagnostic. PMID- 1728260 TI - Immediate and delayed prose recall among normal and demented adults. AB - The Logical Memory subtest of the Wechsler Memory Scale (Form I) was administered as part of a battery of tests to 64 subjects without dementia and 51 with very mild dementia. The demented group's immediate and delayed recall was significantly impaired relative to the control group. Immediate and delayed scores were highly correlated in both groups. Hierarchical multiple-regression analyses revealed that dementia classification did not significantly predict delayed recall performance above and beyond immediate recall performance. This suggests that, in its early stages, dementia primarily affects the encoding of prose material. PMID- 1728261 TI - Transient unresponsiveness in the elderly. Report of five cases. AB - Five hospitalized elderly patients had one or more self-limited episodes of unresponsiveness that could not be explained by metabolic, structural, convulsive, or psychiatric disorders. They accounted for approximately 2% of cases referred to us for evaluation of coma. Despite some heterogeneity, shared clinical features in these patients suggest an age-related disorder of arousal that may be more common than is generally appreciated. PMID- 1728262 TI - A prospective controlled study of magnetic resonance imaging of the brain in gay men and parenteral drug users with human immunodeficiency virus infection. AB - To detect the earliest structural changes in the brain in human immunodeficiency virus (HIV) infection, 118 gay men and 115 parenteral drug users enrolled in a study of the natural history of HIV infection underwent magnetic resonance imaging evaluations. Routine T2-weighted and heavily T2-weighted scans for quantification of brain water were obtained, blinded to HIV serostatus. Atrophy and foci of increased signal did not correlate with any medical, immunologic, neurologic, or neuropsychologic parameters in the group as a whole, or in the gay men or parenteral drug user subgroups. Three subjects had progressive multifocal leukoencephalopathy and one had central nervous system lymphoma. In a subgroup in whom intracranial water percent was calculated, correlations were found with CD4 counts and CD4/CD8 ratios. We conclude that standard magnetic resonance imaging of the brain does not differentiate asymptomatic and mildly symptomatic HIV positive individuals from HIV-negative individuals, regardless of risk group. However, intracranial water percent may distinguish HIV-positive from HIV negative individuals because it correlates with raw CD4 counts and CD4/CD8 ratios. PMID- 1728263 TI - Event-related potential P300 in multiple sclerosis. Relation to magnetic resonance imaging and cognitive impairment. AB - Cerebral involvement in multiple sclerosis may result not only in sensory and motor symptoms but also in impaired mentation. We hypothesize that cognitive dysfunction occurs due to cortical deafferentation or disconnection arising from subcortical white-matter disease. We examined the P300 event-related potential in 31 patients with multiple sclerosis, correlating it with disease severity ratings based on magnetic resonance imaging signal intensity changes, cognitive dysfunction, and disability status. The patients with multiple sclerosis exhibited significantly prolonged P300 wave latencies compared with 32 control subjects. The P300 latency was strongly correlated with the presence of demyelinative brain lesions seen on magnetic resonance imaging scans and with cognitive impairment, but was only weakly associated with the Kurtzke disability status score, consistent with this scale primarily reflecting spinal rather than cerebral demyelination. Our study results support a relationship between subcortical white-matter lesions and cognitive impairment in multiple sclerosis. PMID- 1728264 TI - Cerebral hemorrhage with biopsy-proved amyloid angiopathy. AB - Clinical, radiological, and immunohistochemical findings in brain biopsy specimens from six patients with cerebral amyloid angiopathy-associated intracerebral hemorrhage were reviewed. Acute clinical presentations included headache, nausea and vomiting, loss of consciousness, and focal neurological deficits such as hemiplegia and blindness. Transient ischemic attacks experienced by one patient and referable to one hemisphere did not indicate impending hemorrhage in that region. Computed tomographic scans revealed acute, irregular, superficial, lobar hemorrhage with occasional ring enhancement. Immunohistochemical studies were performed on biopsy specimens using primary antibodies against portions of the Alzheimer A4 (beta-) peptide or gamma-trace peptide (the vascular amyloid protein in patients with hereditary cerebral hemorrhage with amyloidosis-Icelandic type). In all patients, anti-A4 and anti gamma-trace labeled cerebral microvessels. Immunoreactive senile plaques were few compared with the numbers of stained microvessels. Reactive astrocytes in some patients were labeled by both antiserum samples, suggesting uptake or production of these proteins by the astrocytes. This study demonstrates the heterogeneous clinical and radiological features of cerebral amyloid angiopathy-related brain hemorrhage and the value of anti-A4 and anti-gamma-trace immunohistochemical study of biopsy material from patients with suspected cerebral amyloid angiopathy related intraparenchymal bleeding. PMID- 1728265 TI - Double cortex. A neuronal migration anomaly as a possible cause of Lennox-Gastaut syndrome. AB - Band heterotopia, or "double cortex," is a neuronal migration disorder that consists of a symmetrical subcortical neuronal band. The overlying cortex may be normal or macrogyric. We describe two severely mentally retarded girls, aged 14 and 18 years, who had band heterotopia and Lennox-Gastaut syndrome. Band heterotopia was evident in both hemispheres as a subcortical symmetrical layer isointense with gray matter on magnetic resonance T1- and T2-weighted images. Both patients had atonic seizures, atypical absences, and tonic seizures. The electroencephalograms in both cases showed frequent generalized paroxysms and slow background activity. The association of a Lennox-Gastaut syndrome with double cortex in these two patients and in a previously reported autopsy confirmed case suggests that this malformation may be responsible for other similar cases. PMID- 1728266 TI - Increased adherence of T cells to human endothelial cells in patients with human T-cell lymphotropic virus type I-associated myelopathy. AB - We investigated the adherence of T cells to human umbilical vein endothelial cells in seven patients with human T-lymphotropic virus type I (HTLV-I) associated myelopathy. The adherence of T cells to endothelial cells increased significantly in all the patients with HTLV-I-associated myelopathy when compared with the adherence in the seronegative controls (1.3- to 2.8-fold) and compared with the adherence in the anti-HTLV-I-seropositive non-HTLV-I-associated myelopathy carriers (1.4- to 2.8-fold). Prior treatment of the endothelial cell monolayer with recombinant interferon gamma (50 IU/mL) enhanced the T cell endothelial cell adhesion in both the controls and patients with HTLV-I associated myelopathy. However, values after prior treatment in the patients with HTLV-I-associated myelopathy were significantly higher than those in seronegative controls and carriers. The results suggest that the significantly increased T cell-endothelial cell adherence may be related to the initial stages of lymphocyte migration from the blood to the central nervous system in patients with HTLV-I-associated myelopathy. PMID- 1728267 TI - Headaches in children younger than 7 years of age. AB - Headache in young children is frequently a cause of concern to parents and physicians. We have reviewed our experience with 104 children with onset of headaches prior to 7 years of age seen by age 9 years. Headaches could be classified in more than 90% of cases. The most common headache type in this population referred to a child neurologist was migraine that constituted 75% of the cases. Seventy-two of 78 cases were common migraine. Posttraumatic headaches accounted for an additional 12%. Associated symptoms such as autonomic signs, nausea, and vomiting were common, particularly in the migraine group. Neuroimaging studies when performed did not reveal any significant abnormalities. Other laboratory tests were also generally unhelpful. No child has gone on to develop new neurologic abnormalities or evidence of an intracranial tumor. We conclude that even in young children headaches are generally benign. Even in this population, neuroimaging studies have a very low yield in the absence of other symptoms and findings and are not always indicated. PMID- 1728268 TI - Intra-arterial cisplatin--associated optic and otic toxicity. AB - Over a 22-month period, we investigated optic and otic toxicity accompanying intra-arterial cisplatin therapy. Baseline and serial neurologic and ophthalmologic examinations, visual evoked potentials, and brain-stem auditory evoked potentials were performed in six patients, aged 37 to 53 years. Patients received infraophthalmic intra-arterial cisplatin (60 mg/m2) every month for three to 10 treatments (mean, six treatments). Five of the six patients had progressive optic toxicity. In two patients, the visual evoked potential prolongation preceded acuity loss by at least 4 months. Two patients had evidence of otic toxicity by either brain-stem auditory evoked potential or click threshold and brain-stem auditory evoked potential. Intra-arterial cisplatin neurotoxicity may be significant in patients with already limited survival. Visual evoked potential and brain-stem auditory evoked potential should be used to monitor patients receiving potentially neurotoxic therapy. PMID- 1728269 TI - Establishing the limits of the Mini-Mental State. Examination of 'subtests'. AB - It has been suggested that the Mini-Mental State examination can be used to examine a patient's cognitive profile. We therefore examined the validity of Mini Mental State subtests and individual items. The memory item, attention concentration items, and constructional item had satisfactory sensitivity specificity and correlated significantly with scores on neuropsychological tests. In contrast, four of the five Mini-Mental State language items had very low sensitivity, and three of five failed to correlate with neuropsychological test scores. These findings establish limits with regard to the ability of the Mini Mental State to generate a cognitive profile. Our data also provide information regarding validity, difficulty level, and optimal cutoff scores for widely used mental status tasks. PMID- 1728270 TI - Retinocalcarine function in Alzheimer's disease. A clinical and electrophysiological study. AB - Impaired visual function in Alzheimer's disease (AD) could result from either precortical or cortical lesions, or both. In a parallel psychophysical study of visual function in AD, we found that contrast sensitivity function, color vision, stereoacuity, and backward masking were impaired relative to the performance of age-matched control subjects, whereas performance on a critical flicker fusion test was normal. The intent of the present study was to determine whether abnormalities of the retinocalcarine pathway contribute to visual dysfunction. We performed neuro-ophthalmological examinations on 38 patients with AD; from this group, 25 received additional psychophysical testing and 13 underwent electrophysiological testing. Clinical neuro-ophthalmological examinations, full field electroretinograms, focal electroretinograms, and pattern visual evoked potentials were normal in all patients tested. There was no evidence of retinocalcarine abnormality specific to AD. We conclude that the visual impairment experienced by some patients with AD primarily results from involvement of the visual association cortices rather than from precortical damage, at least before the end stage of the disease. PMID- 1728271 TI - Endoscopic sinus surgery. PMID- 1728272 TI - Photodynamic laser therapy. PMID- 1728273 TI - Analysis and perspective from the infectious disease department. PMID- 1728274 TI - Indirect microlaryngostroboscopic surgery. AB - Detailed preoperative laryngostroboscopic examination is a prerequisite for phonosurgical correction of organic dysphonia. Although suspension microlaryngoscopic surgery has proved its value in the past, it excludes functional control during the removal of vocal fold swellings. Using an indirect microlaryngostroboscopic surgical technique with topical anesthesia, functional control can be achieved during surgery. This enables the removal of vocal fold swellings with a high degree of precision. Postoperative voice evaluation was performed in 31 patients after suspension microlaryngoscopic or indirect microlaryngostroboscopic surgery. The results showed that indirect microlaryngostroboscopic surgery is at least as good as, and in some respects even better than, suspension microlaryngostroboscopic surgery. Large vocal fold swellings, extensive Reinke's edema, and submucosal swellings are considered less suitable for indirect microlaryngostroboscopic surgery, because such lesions require bimanual instrumentation. PMID- 1728275 TI - Study of vibratory pattern of the vocal folds in the excised canine larynx. AB - The effect of simulated thyroarytenoid and cricothyroid muscle contraction on the vibratory pattern of the vocal folds was studied in the excised canine larynx. To simulate the action of the thyroarytenoid muscle, small balloons were inflated in the paraglottic space at the level of the vocal folds. To simulate the action of the cricothyroid muscle, longitudinal tension was applied to the anterior commissure of the vocal folds. The photoglottographic and electroglottographic signals, sound intensity, and airflow rate were measured. This study showed that balloon inflation simulating thyroarytenoid muscle contraction produced an elevation of frequency of vibration with a decrease in open quotient, and that an increase in longitudinal tension simulating cricothyroid muscle contraction produced an elevation of frequency with an increase in open quotient. Vocal resistance decreased with increasing open quotient, amplitude of the photoglottographic waveform, and the frequency of vibration. Vocal efficiency increased with increasing photoglottographic amplitude and decreased with increasing frequency. The vocal efficiency peaked when the open quotient was approximately 0.5. This study suggests that glottographic parameters may be useful in assessing the effect of intrinsic laryngeal muscle activity on vocal efficiency and glottic resistance. PMID- 1728276 TI - The effects of inhalation anesthetic agents on survival in a pig random skin flap model. AB - In recent years a myriad of studies have been performed investigating the effects on flap survival of various pharmacologic agents. One class of agents, however, that has received relatively little attention is the inhalational anesthetics. Yet, they are widely used during reconstructive efforts using skin flaps and they possess several pharmacologic properties shown to affect flap survival. Using a dorsally based random skin flap model in 28 swine, the influence of nitrous oxide and isoflurane on skin flap survival was examined. The mean area of skin flap survival in the isoflurane, nitrous oxide, euoxemic control, and hyperoxygenated control groups was 79.4%, 29.7%, 42.0%, and 28.6%, respectively. A significant improvement in flap viability was seen only in the group using isoflurane as the anesthetic agent. Arterial blood gas content (PO2, PCO2, and HCO3), respiratory rate, acid-base balance, blood pressure, pulse, and temperature were monitored. Improved survival of the isoflurane group was independent of these parameters. These data suggest that the choice of anesthetic agent may effect random skin flap survival with isoflurane providing the greatest benefit of the agents tested in this model. PMID- 1728277 TI - Third-generation cephalosporins in the treatment of acute pneumococcal otitis media. An animal study. AB - There is concern that third-generation cephalosporins may not be effective in the treatment of acute otitis media due to Streptococcus pneumoniae. Using the chinchilla animal model, we compared two third-generation cephalosporins, cefixime (Suprax) and ceftibuten (investigational), with ampicillin and saline controls in an investigator-blinded, randomized trial. Whereas the saline controls performed worse than all other groups, no significant differences were detected among the three antibiotics regarding the time required to sterilize the middle ear cleft, or the prevalence of positive cultures after 10 days of therapy. The statistical power of the comparisons of cefixime and ceftibuten with ampicillin were 98% and 67%, respectively. The results of this in vivo animal study fail to support the contention that the two third-generation cephalosporins investigated are not effective in the treatment of pneumococcal acute otitis media. Caution is advised when extrapolating these results to the general clinical setting. PMID- 1728278 TI - Bacterial tympanogenic labyrinthitis, meningitis, and sensorineural damage. AB - Pathologic changes (sensorineural hearing loss, labyrinthitis, meningitis) can follow otitis media. Various macromolecular substances demonstrably enter the inner ear via the round window membrane, but its permeability to bacteria is less known. We inoculated Streptococcus pneumoniae type 7F bilaterally into the middle ears of two groups of chinchillas, with and without grafted round window membranes. Inner ears of inoculated animals were observed by light and electron microscopy. None with continuous grafts had labyrinthitis. Bacteria penetrated all three layers of nongrafted round window membranes and into all cochlear turns, entering Schuknecht's channels and following neuronal pathways; nerves were often degenerated, hair cells were damaged or missing, and the stria vascularis was edematous and hemorrhagic. The neural damage suggests a mechanism for the hearing loss that can follow otitis media. Absence of labyrinthitis and meningitis in grafted animals suggests a tympanogenic pathway for the bacteria. PMID- 1728279 TI - Anastomosis of the cervical trachea in children. AB - Numerous techniques for the surgical management of laryngotracheal stenosis in children have been described in the literature. These surgical modalities include endoscopic management and open laryngotracheal reconstruction using costal cartilage grafts for expansion of the stenotic subglottic region. Although tracheal resection with primary reanastomosis for the management of tracheal stenosis is reported frequently in the adult population, children rarely have stenotic lesions that are amenable to this particular technique. Laryngotracheal stenosis in children most commonly involves the subglottis. This makes tracheal resection with anastomosis technically difficult to perform, due to the close proximity of the vocal cords. We have found a subpopulation of children at our institution with high tracheal stenoses, with minimal lower subglottic involvement, who were amenable to tracheal resection with primary anastomosis. We review our experience with this technique. The indications for this surgical modality in children are discussed, as well as the surgical technique. PMID- 1728280 TI - An approach to management of keloids. AB - Keloids present a major therapeutic dilemma for the surgeon because of frequent recurrences. We use a new protocol for management of large primary or recalcitrant keloids. The technique employs carbon dioxide laser resection of the keloid and then allows the open wound bed to heal by secondary intention. The open wound is treated as though it were a third-degree skin burn. The wounds invariably orient their long axis parallel to the relaxed skin tension lines. No keloid has ever been noted to recur before epithelial migration is complete. Careful follow-up detects early recurrences that are then treated with injection consisting of 40 mg/mL of triamcinolone acetonide (Kenalog), 150 mg of hyaluronidase (Wydase), and 2% lidocaine via a dermajet. Thirty-seven patients were treated with this protocol and have been followed up for at least 2 years. A control rate of 84% has been achieved with compliant patients. PMID- 1728281 TI - The effects of carbocisplatin and radiation on skin flap survival. AB - Carbocisplatin is used as an inductive chemotherapeutic agent prior to irradiation in the treatment of head and neck cancers. Controversy exists whether carbocisplatin sensitizes normal epithelial tissues, including skin, to radiation. The combined effect of radiation and carbocisplatin on the survival of skin flaps was studied in an experimental model using dorsal flaps in Sprague Dawley rats. Skin flaps were created 6 weeks after exposure to irradiation and carbocisplatin. Flap survival was assessed 7, 14, and 21 days after the flaps were initially created. Exposure of the flaps to irradiation alone, carbocisplatin alone, combined irradiation and carbocisplatin, or combined irradiation and fractionated carbocisplatin did not result in any significant decrease in flap survival when compared with untreated animals. PMID- 1728282 TI - The exacerbation of symptoms in Meniere's disease during the premenstrual period. AB - The pathophysiology of the characteristic episodic symptoms of vertigo, low frequency hearing loss, and tinnitus in Meniere's disease remains poorly understood. It is likely that the manifestation of this condition may be multifactorial and related to elements affecting the inner ear beyond the underlying pathology of endolymphatic hydrops. We have identified a subgroup of female patients with Meniere's disease in which the symptoms of this disorder are correlated with the late luteal phase of the menstrual cycle (premenstrual period). Through audiometric and vestibular testing, we have documented these inner ear effects in six women. Although many hormonal effects occur during the premenstrual period, compartmental fluid redistribution within the body may be the most pertinent. Endolymphatic hydrops represents a fluid imbalance within the inner ear and, when combined with an additional fluid shift, may produce symptomatic dysfunction. Case histories demonstrating the correlation of the symptoms of Meniere's disease and the premenstrual period will be presented along with theoretical mechanisms of pathophysiology. PMID- 1728283 TI - Primary lymphoma of the temporal bone. AB - Involvement of the temporal bone by lymphoreticular neoplasm is rare; all reported cases have been of secondary involvement. This article presents what we believe to be the first two reported cases of primary temporal bone lymphoma. The patients, an elderly man and a boy, both presented with infection of the ear, hearing loss, and facial nerve paresis. In both cases, facial paresis resolved after appropriate chemotherapeutic treatment. Patient presentation and clinical course are discussed in light of published work on temporal bone malignancy. Further investigation, including computed tomography and biopsy, should be considered for patients who present with an apparent middle ear infection unresponsive to medical therapy. The development of facial paralysis in such a patient warrants heightened suspicion of malignancy. PMID- 1728284 TI - Adjuvant hyperbaric oxygen in malignant external otitis. AB - Necrotizing invasive pseudomonal infection of the external auditory canal (malignant external otitis) is an uncommon but important disorder in the elderly. The high morbidity, and even mortality, of this disorder has been reduced by the early and intensive use of combination antipseudomonal antibiotics. However, in severely immunocompromised patients or in infection involving the base of the skull, multiple cranial nerves, or the meninges, conventional therapy has been prolonged, intensive, and relatively ineffective. We treated 16 patients with malignant external otitis with adjuvant hyperbaric oxygen therapy. In six patients, infection was in advanced stages, infections were recurrences after previous treatment, and repeated treatment with antipseudomonal antibiotics had failed. All 16 cases responded promptly when a 30-day course of hyperbaric oxygen was added to the antibiotic regimen, and all patients remained free of infection or neurologic deficit during 1 to 4 years of follow-up. No complications of this treatment modality were noted. Hyperbaric oxygen therapy reverses tissue hypoxia, which enhances phagocytic killing of aerobic microorganisms, and stimulates neomicroangiogenesis. In addition, hyperbaric oxygen augments the action of aminoglycoside antibiotics. Adjuvant hyperbaric oxygen therapy should be considered in advanced or recurrent cases of malignant external otitis. PMID- 1728285 TI - Necrotizing 'malignant' external otitis caused by Staphylococcus epidermidis. AB - Necrotizing "malignant" external otitis is a life-threatening skull base infection that originates in the external auditory canal and is characterized by otalgia and purulent aural discharge with external auditory canal cellulitis and granulation. Necrotizing external otitis, seen almost exclusively in elderly diabetics, is almost always caused by Pseudomonas aeruginosa. To our knowledge, there have been only six nonpseudomonal cases reported to date. We describe a 70 year-old diabetic man with necrotizing external otitis caused by Staphylococcus epidermidis, confirmed by serial cultures. This case was characterized by otalgia, purulent otorrhea, preauricular swelling, bony external auditory canal erosion, and a conductive hearing loss. Despite prolonged intravenous antistaphylococcal antibiotic therapy and frequent local debridement, the patient's symptoms never completely resolved. As demonstrated by the treatment failure, S epidermidis necrotizing external otitis, may represent a more refractory form of this already virulent disease process. We believe this to be the first reported case of necrotizing external malignant otitis caused by S epidermidis. PMID- 1728286 TI - Pathologic quiz case 1. Primary melanoma of the left maxillary sinus. PMID- 1728287 TI - Pathologic quiz case 2. Myxoma of the nasal bone. PMID- 1728288 TI - Mechanisms of endothelial growth control. AB - It is well documented that vascular endothelial cells constitute an unusually quiescent epithelial cell population. These slowly dividing cells are known to undergo more rapid division in association with wound healing as well as a variety of pathologic conditions such as tumor vascularization and diabetic retinopathy. Although the precise mechanisms responsible for vascular quiescence have not been elucidated, some insights into the regulators of vascular growth control are beginning to be gained. Nearly all of the investigations have utilized cultured vascular cells. Regulation of vascular endothelial growth by polypeptide growth regulators, extracellular matrix molecules, cell-cell interactions, and mechanical forces has been demonstrated in these tissue culture systems. PMID- 1728289 TI - Response of human endothelial cell antioxidant enzymes to hyperoxia. AB - To explore the level of regulation of the expression of the major antioxidant enzymes in response to hyperoxia, we exposed human umbilical vein endothelial cells to 95% O2 for 3 and 5 days and measured (1) the steady-state mRNA levels, (2) the activities, and (3) the immunoreactive content of CuZn and Mn superoxide dismutases (SOD), catalase (CAT), and glutathione peroxidase (GP). We found that a 3-day exposure to 95% O2 caused (1) an increase in CuZnSOD mRNA (by 41%), CAT mRNA (by 26%), and GP mRNA (by 173%); (2) an increase in CuZnSOD activity (by 30%), a decrease in CAT activity (by 37%), and an increase in GP activity (by 60%); and (3) an increase in CuZnSOD immunodetectable protein (by 26%) and a loss in CAT immunoreactive protein (by 27%). After a 5-day exposure to 95% O2, there was (1) a 93% increase in CuZnSOD mRNA, a 71% increase in CAT mRNA, and a 127% increase in GP mRNA; (2) a 56% increase in CuZnSOD activity, a 70% decrease in CAT activity, and an 89% increase in GP activity; and (3) a 35% increase in CuZnSOD immunoreactive protein and a 55% loss in CAT immunoreactive protein. There was no change in the steady-state MnSOD mRNA level after 3 days in 95% O2, but a 100% increase was observed on day 5 of oxygen exposure. MnSOD activity was unchanged in cells exposed to hyperoxia for 3 and 5 days. These data suggest that, in human umbilical vein endothelial cells, the regulation of antioxidant enzymes expression in response to O2 is complex and exerted at different levels. PMID- 1728290 TI - Release of mucus glycoconjugates by Pseudomonas aeruginosa rhamnolipid into feline trachea in vivo and human bronchus in vitro. AB - Pseudomonas aeruginosa colonizes the lower respiratory tracts of patients with severe bronchiectasis, including cystic fibrosis, a condition associated with increased airway mucus output. We have shown that an extract containing chloroform-soluble extracellular products of P. aeruginosa releases glycoconjugates into the cat trachea in vivo. This activity was not related to pyocyanin, a major component of the extract, but was associated with the rhamnolipids. Purified monorhamnolipid (100 micrograms/ml) released radiolabeled and periodic acid-Schiff (PAS)-reactive glycoconjugates (delta 3H = +490 +/- 70%, delta 35S = +170 +/- 40%, delta PAS = +8.6 +/- 1.7 micrograms/min; n = 6, P less than 0.02 for each). Dirhamnolipid (200 micrograms/ml) was also effective (delta 3H = +640 +/- 70%, delta 35S = +130 +/- 20%, delta PAS = +9.3 +/- 1.5 micrograms/min; n = 6, P less than 0.02 for each). Monorhamnolipid (100 micrograms/ml) also released 35S-labeled and PAS-reactive glycoconjugates from human bronchial tissue in vitro (delta 35S = +189 +/- 47%, delta PAS = +26.3 +/- 8.5 micrograms/min; n = 7, P less than 0.001 versus control tissues in which no stimulus was given). The cat tracheal glycoconjugates released by the rhamnolipids differed from those released by pilocarpine 50 microM, in having a higher 3H:35S ratio (P less than 0.001). After gel chromatography on a Sepharose CL-4B column, the void volume fractions of the glycoconjugates also had different profiles in a cesium chloride density gradient. Those released by rhamnolipid banded at 1.62 g/ml, while those released by pilocarpine banded mainly at 1.50 g/ml, with some of the higher density material also present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728291 TI - Persistent interleukin-2 activity and molecular evidence for expression of lymphotoxin in the hapten-immune model for pulmonary interstitial fibrosis. AB - The hapten-immune model for pulmonary fibrosis shows that a specific T-cell mediated immune response is essential for the induction of a nonresolving fibrosis. Here, we report results from studies that identify soluble factors released by activated T lymphocytes that might mediate long-lasting fibrosis. Pulmonary fibrosis was induced by priming hamsters for contact hypersensitivity responses with an epicutaneous application of 2,4,6-trinitro-1-chlorobenzene (TNCB) in carrier and challenging intratracheally (IT) 5 days later with a single dose of the soluble form of the immunizing hapten. Bronchoalveolar lavage fluid was harvested at various time points after IT challenge and assayed for tumor necrosis factor (TNF) and interleukin-2 (IL-2) bioactivity. After IT challenge with the sensitizing hapten, only the immune animals contained IL-2 activity in the bronchoalveolar lavage fluid. TNF activity was detected in lungs of both immune and nonimmune animals. Interestingly, the TNF activity was significantly higher (P less than 0.05) in nonimmune challenged than in immune challenged animals on day 5. Molecular hybridization studies showed that a similar amount of TNF-alpha mRNA was expressed in adherent cells from both groups. The nonadherent subpopulation of mononuclear cells harvested from challenged-immune animals expressed TNF-beta (lymphotoxin) mRNA. These data show, for the first time, an association of lymphotoxin with the appearance of pulmonary fibrotic disease in an animal model for pulmonary fibrosis. These observations are consistent with the postulates that lymphotoxin and IL-2 participate in the immunopathogenesis of hapten-immune induced pulmonary fibrosis and that TNF-alpha is associated with the healing of the fibrotic process initiated by toxic lung injury. PMID- 1728292 TI - Epithelial strips: an alternative technique for examining arachidonate metabolism in equine tracheal epithelium. AB - We have developed an alternative method for examining equine tracheal epithelial arachidonic acid (AA) metabolism that utilizes strips of pseudostratified columnar epithelium attached to a layer of elastic tissue 80 to 130 microns thick. We compared the responses of this preparation with those of enzymatically dispersed suspensions of tracheal epithelium obtained from the same animal. Strips incubated with [3H]AA incorporated 40.8 +/- 3.6% of added radioactivity and released 2.55 +/- 0.23% of incorporated radioactivity when stimulated with 5 microM A23187. Values for the cell suspension were 59.6 +/- 1.6% and 1.90 +/- 0.08%, respectively. Stimulation with 50 microM histamine or bradykinin resulted in significant release of free [3H]AA only from the strips. High-performance liquid chromatography radioactivity profiles of eicosanoids released following stimulation with 5 microM A23187 demonstrated peaks that coeluted with free AA, prostaglandin (PG) E2, and PGF2 alpha for the strips, and free AA, leukotriene B4, and 5-HETE for the cell suspensions. The absence of PGE2 production by cell suspensions was confirmed by assaying immunoreactive PGE2 in supernatants from unlabeled strips and suspensions stimulated with 5 microM A23187. Epithelial strips produced 10.3 +/- 1.3 ng PGE2/ml supernatant, whereas 5 x 10(6) cells in suspension produced less than 100 pg/ml. Despite the lack of PG production by the cell suspensions, immunocytochemical staining with an anti-PGH synthase antibody demonstrated the presence of PGH synthase in epithelial cells of both preparations. These data indicate that, in contrast to epithelial cell suspensions, epithelial strips synthesize cyclooxygenase metabolites and respond to peptide agonists.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728293 TI - Epidermal growth factor transcription, translation, and signal transduction by rat type II pneumocytes in culture. AB - Epidermal growth factor (EGF) is known to induce fetal lung maturation and its receptor is present in the lungs of several species. Recently, EGF has been immunolocalized in type II pneumocytes in rat lung. We postulated that EGF is synthesized in type II pneumocytes and that, because of its position-restricted distribution within the alveolus, EGF might act as an autocrine regulator of type II pneumocyte function. Herein, we have tested the hypothesis using adult rat type II pneumocytes in primary culture. In situ hybridization, using an oligonucleotide probe corresponding to amino acid residues 1070 to 1081 of mouse EGF precursor, demonstrated the presence of EGF precursor mRNA. Upon S-200 Sephacryl gel chromatography of type II pneumocyte extracts, EGF-reactive protein eluted as a high-molecular-weight form (greater than 100 kD). EGF immunoreactivity was localized within type II pneumocytes in the periphery of groups of 10 to 15 cells in culture. The type II pneumocytes bound [125I]EGF in a specific manner, indicating the presence of EGF receptors. Scatchard plots gave an apparent affinity constant (Ka) of 1 x 10(9) liters/mol, and the number of receptors was estimated to be 4.8 x 10(11) mg protein (50 per cell). EGF receptor binding specificity was confirmed by the absence of an autoradiographic signal for cells incubated in the presence of a 100-fold excess concentration of transforming growth factor-alpha. Binding of [125I]EGF could also be downregulated 95% by incubation with 0.2 nM transforming growth factor alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728294 TI - A rat alveolar type II cell line developed by adenovirus 12SE1A gene transfer. AB - The regulation of pulmonary alveolar type II cell proliferation and differentiation is poorly understood and has been difficult to study, in part due to lack of proliferation, cellular heterogeneity, and phenotypic instability of type II cells in primary culture. To develop a stable population of homogeneous cells capable of proliferation, we transfected type II cells isolated from the lungs of neonatal rats with an immortalizing oncogene, adenovirus 12SE1A, using a retroviral vector. Individual clones were isolated, screened for cytokeratin expression, and further characterized. One of the 12SE1A expressing clones, E1A T2, has epithelial features such as cytokeratin expression and tight junctions, and coexpresses vimentin. E1A-T2 rapidly proliferate when grown in 10% fetal bovine serum, and slow their growth at confluence. A labeling index of greater than 90% during a 24-h pulse of [3H]thymidine reflects a uniform population of proliferating cells. E1A-T2 can be grown and passed in 0.4% fetal bovine serum, suggesting the production of an autocrine growth factor(s). The type II cell Maclura pomifera agglutinin (MPA)-binding glycoprotein, MPA-gp200, appears to be expressed in an incompletely glycosylated form, whereas other features of differentiated type II cells, such as lamellar bodies, surfactant protein A, and a high percentage of saturated phosphatidylcholine, are absent. Homogeneous, clonally derived type II cell lines, such as E1A-T2 may retain sufficient type II cell features of interest to test new hypotheses relating to cell proliferation and differentiation otherwise not feasible using primary cultures of type II cells. PMID- 1728295 TI - Induction of multinucleated giant cell formation from in vitro culture of human monocytes with interleukin-3 and interferon-gamma: comparison with other stimulating factors. AB - One of the hallmarks of the granulomatous response to infection is the formation of multinucleated giant cells (MGC.) In an effort to study MGC, we examined the fusion-promoting effects of a variety of stimulating factors on human peripheral blood monocytes cultured on plastic surfaces in serum-supplemented media. MGC formation was minimally to moderately enhanced by interferon-gamma (IFN-gamma), interleukin (IL)-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), retinoic acid (RA), and IL-6. IL-4 (which has been reported to promote MGC formation from murine macrophages) had an inhibitory effect. IFN-gamma was not required for MGC formation but it significantly increased the fusion-promoting activity of GM-CSF, 1,25-(OH)2D3, RA, and IL-6, IL-3, a hematopoietic growth factor, has been recently shown to induce osteoclast formation from murine bone marrow mononuclear cells. The most striking effect was seen with the combination of IL-3 and IFN-gamma. Fusion index is defined as a percentage of nuclei found within MGC, and an index of 67% at 1 wk was found. The formation of some very large cells with 50 to 100 nuclei was noted. Both Langhans' and foreign-body type cells were seen. Transmission electron micrographs clearly demonstrate the absence of plasma membrane between nuclei. Induction of MGC from peripheral human blood monocytes by IL-3 and IFN gamma provides an in vitro system for the study of the formation and function of these cells. PMID- 1728296 TI - Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques. AB - We describe a novel method for the isolation and subsequent culture of pulmonary neuroendocrine cells (PNEC) from normal fetal rabbit lung using immunomagnetic techniques with a monoclonal antibody, MOC-1. This surface antigen has originally been identified on small cell carcinoma of the lung. Our immunohistochemical studies have shown that MOC-1 cross-reacts with PNEC of human and rabbit fetal lungs on frozen sections, and in fixed cultures of rabbit fetal lung. Using a combination of mechanical and enzymatic disaggregation, a single-cell suspension of fetal rabbit lung was obtained. These cells were incubated with MOC-1 conjugated to magnetic beads. PNEC were selectively removed from the heterogeneous mixture using a magnet, giving up to 2-fold enrichment compared with our previously reported method. These cells were maintained in culture in a functional state for up to 7 days. The ability to prepare PNEC from rabbit fetal lung offers an opportunity to develop in vitro models to investigate the physiologic and biochemical properties of these cells, and ultimately it may lead to a better understanding of their function in health and disease. PMID- 1728297 TI - Murine hypersensitivity pneumonitis: a study of cellular infiltrates and cytokine production and its modulation by cyclosporin A. AB - The progression of hypersensitivity pneumonitis (HP) was evaluated in mice repeatedly challenged with the actinomycete Faeni rectivirgula (Micropolyspora faeni) (90 or 180 micrograms), at the cellular level and at the mediator level. Instillation of F. rectivirgula by the intranasal route determined a granulomatous inflammation in the lungs of animals correlated with a dramatic increase (5- to 6-fold) in cellularity in the bronchoalveolar space and an increase in the percentage of lymphocytes. Disease in mice was also correlated with high spontaneous release of the cytokines interleukin-1 (IL-1) (60 U/ml), interleukin-6 (IL-6) (72 U/ml), and tumor necrosis factor-alpha (TNF-alpha) (56 U/ml) in the bronchoalveolar lavage (BAL) fluid, as well as by an enhanced capacity for cytokine release by macrophages upon stimulation with F. rectivirgula. It was also found that the pulmonary inflammation was correlated with a 60 to 70% increase in total lung weight after 4 wk and a significant lung fibrosis as seen by a 2-fold increase in lung hydroxyproline levels. Treatment of challenged mice with cyclosporin A (CyA) led to an abrogation of the disease as seen by an abrogation of the increase in lung index, lack of IL-1 and TNF-alpha release in the BAL. CyA did not, however, completely prevent the alveolitis as seen by the cellular infiltrate (2- to 3-fold in BAL cell increase). It also did not prevent the T-lymphocyte recruitment associated with HP, although these cells did not proliferate in response to the F. rectivirgula antigen, in contrast to BAL cells from F. rectivirgula-challenged mice treated with excipient only.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728298 TI - Regulation of human alveolar macrophage- and blood monocyte-derived interleukin-8 by prostaglandin E2 and dexamethasone. AB - Mononuclear phagocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophages produce a vast array of regulatory and chemotactic cytokines. Interleukin-8 (IL-8), a potent neutrophil activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammatory events. In this investigation, we describe the effects of prostaglandin E2 (PGE2) and dexamethasone (Dex) on IL-8 mRNA and protein expression from lipopolysaccharide (LPS)-treated human peripheral blood monocytes (PBM) and alveolar macrophages (AM). We demonstrate the dose-dependent suppression of IL-8 from LPS-stimulated PBM by PGE2. Treatment of stimulated PBM with 10(-6) M PGE2 resulted in maximal inhibition, causing 60% suppression of both IL-8 mRNA and extracellular protein levels. In contrast, PGE2 (10(-6) to 10( 8) M) did not significantly alter IL-8 mRNA or protein expression from LPS treated AM. Treatment of LPS-stimulated PBM and AM with Dex (10(-6) to 10(-8) M) resulted in 75% decline in IL-8 mRNA and extracellular protein from either cell population. Pretreatment of PBM with PGE2 or Dex 1 or 2 h before LPS stimulation caused a significant suppression of steady-state IL-8 mRNA levels; however, administration of either of these modulators 1 or 2 h after LPS stimulation failed to have an inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728299 TI - Insulin-like growth factor I and pulmonary hypertension induced by continuous air embolization in sheep. AB - Chronic pulmonary hypertension is associated with arterial structural remodeling. Insulin-like growth factor I (IGF-I) has been proposed as one of the mediators of vascular change because of its ability to stimulate proliferation in, and elastin production by, cultured vascular smooth muscle cells. We have shown previously that 12 days of continuous air embolization into the pulmonary arterial circulation of sheep results in the functional and structural changes of chronic pulmonary hypertension. In the present study, measurements of IGF-I (by radioimmunoassay) and IGF-I binding protein activity in sheep lung lymph and plasma were made before and during the 12 days of air embolization in six sheep. Two untreated animals served as controls. Baseline lung lymph contained 23.5 +/- 3.6 ng/ml (mean +/- SEM) of IGF-I, and there was a slight increase to 36.7 +/- 9.8 on day 3, but by day 6 levels were back to baseline. The flux of IGF-I from the lung (concentration times lymph flow) increased significantly by day 2 embolization and remained elevated through day 12 (baseline = 37.2 +/- 11.1 ng/15 min; day 2 = 237.7 +/- 55.8; day 5 = 190.2 +/- 53.4; day 6 = 82.6 +/- 21.9; day 12 = 78.7 +/- 12.5). IGF-I binding protein activity was also present in lung lymph at baseline (29.6 +/- 3.0%) and was unchanged during air embolization. Plasma levels of IGF-I and plasma binding protein activity remained at baseline throughout the 12 days of embolization (71.51 +/- 34.48 ng/ml and 36.4 +/- 3.5%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728300 TI - Age-related decrease in respiratory muscle mitochondrial function in rats. AB - The purpose of this study was to elucidate the effects of aging on diaphragmatic mitochondrial function. Diaphragm mitochondria were prepared from specific pathogen-free rats aged 7 wk (n = 7), 35 wk (n = 7), and 55 wk (n = 7). The activities of various portions of the mitochondrial electron transport chain, i.e., complexes I, II, III, and IV, were measured enzymatically. The specific activities of complex I decreased significantly (P less than 0.01) in 35-wk-old rats (726 +/- 90 nmol/min/mg protein) compared with 7-wk-old rats (1,018 +/- 121), and the decrease was more remarkable in 35-wk-old rats (565 +/- 64; P less than 0.01 versus 35-wk-old). The activities of complex IV also decreased significantly (P less than 0.01) in 55-wk-old rats (1,222 +/- 191) compared with 7-wk-old rats (1,797 +/- 208); however, no significant changes in complex IV activities between 7-wk-old rat and 35-wk-old rats were observed. In contrast, the activities of complex II and III were not affected by aging. Limb muscle, heart, and liver mitochondria were also prepared from the same rats. The same tendency was observed in limb muscle mitochondria; however, in heart and liver mitochondria, activities of all four complexes were not changed in rats of all age groups. These results indicate that vulnerability of mitochondrial electron transport chain to aging differs from organ to organ and that it also differs from portion to portion in the electron transport chain and the most vulnerable site was complex I.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728301 TI - Type II alveolar epithelial eicosanoid metabolism: predominance of cyclooxygenase pathways and transcellular lipoxygenase metabolism in co-culture with neutrophils. AB - Arachidonic acid (AA) metabolism was studied in freshly isolated type II alveolar epithelial cells of rabbits. Substantial basal secretion of prostanoids with predominance of prostaglandin (PG) I2 was noted. Challenge with the calcium ionophore A23187 resulted in a time- and dose-dependent increase in the generation of all AA cyclooxygenase products to severalfold values following the rank order of 12-heptadecatrienoic acid (12-HHT) greater than PGI2 greater than PGE2 greater than or equal to thromboxane A2 greater than PGF2 alpha approximately PGD2. Even larger augmentation of prostanoid generation was evoked by challenge with free exogenous AA. Generation of the different AA cyclooxygenase products was inhibited by acetylsalicylic acid with IC50 in the range between 250 and 500 microM. In addition to the prostanoid release, ionophore-challenged type II pneumocytes liberated substantial amounts of AA lipoxygenase products with leukotriene (LT) B4 greater than 15 hydroxyeicosatetraenoic acid (HETE) greater than 12-HETE greater than 5-HETE. Generation of LTs and HETEs was markedly increased upon simultaneous disposal of free exogenous AA. No omega-oxidation of LTB4 was noted, and no evidence for secretion of intact LTA4 was obtained. The epithelial cells displayed avid uptake of exogenously offered LTA4 with subsequent enzymatic conversion to LTB4. Co stimulation of pneumocytes with neutrophils (PMN) resulted in an amplification of LTB4 generation, paralleled by a decrease in nonenzymatic decay products of PMN derived LTA4; both phenomena were dose dependent on the pneumocyte-PMN ratio.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728302 TI - Effect of interferon-gamma on the 5-lipoxygenase pathway of rat lung macrophages. AB - In view of conflicting reports concerning the effect of macrophage activation on arachidonic acid metabolism, we examined the effect of the macrophage activator, interferon-gamma (IFN-gamma), on the 5-lipoxygenase pathway in rat lung macrophages. Rat lung macrophages were conditioned in the presence or absence of 10(2) U/ml IFN-gamma for 4 h before stimulation with 1 microM A23187 for 15 min or 100 micrograms/ml opsonized zymosan for 60 min at 37 degrees C as well as other stimuli. Lipoxygenase products in extracted cell supernatants were identified and analyzed by high-pressure liquid chromatography and ultraviolet spectroscopy. The predominant lipoxygenase products included leukotriene (LT) B4, LTC4, and 5-hydroxyeicosatetraenoic acid (5-HETE). These products were not qualitatively altered by conditioning with IFN-gamma. However, 5-lipoxygenase pathway activity, as measured by LTB4 release, was maximally increased 2-fold after conditioning with IFN-gamma and stimulating with either A23187 or opsonized zymosan. IFN-gamma-conditioned macrophages, stimulated with A23187, released greater quantities of lipoxygenase products in comparison with control cells (307.6 +/- 13.3 versus 167.6 +/- 3.9 pmol LTB4/10(6) cells) (mean +/- SEM) (P less than 0.05). Similar results were obtained with the less potent stimulus, opsonized zymosan. IFN-gamma had no direct stimulatory effect on the 5 lipoxygenase pathway. No effect was observed with a variety of other stimuli with or without IFN-gamma conditioning.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728303 TI - Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor. AB - The interactions between ligands containing the recognition sequence arginine glycine-aspartic acid (RGD) and integrin receptors are important in many cell cell and cell-protein interactions. The platelet contains five integrin receptors and they contribute significantly to platelet adhesion and aggregation. To investigate the RGD binding domains on platelet integrins, we immobilized a series of RGD peptides containing variable numbers of glycine residues [(G)n RGDF] on polyacrylonitrile beads and evaluated the ability of the beads to interact with platelets. With native platelets, virtually no interaction occurred with G1-RGDF beads, but the interactions increased as the number of glycine residues increased, plateauing with the G9-RGDF and G11-RGDF beads. ADP pretreatment enhanced the interactions with all of the beads, whereas prostaglandin E1 pretreatment eliminated the interactions with the shortest peptide beads, but only partially inhibited interactions with the longer peptide beads. Monoclonal antibodies to glycoprotein (GP) IIb/IIIa were most effective in inhibiting the interactions, but antibodies to GPIIb/IIIa with similar inhibitory effects on fibrinogen binding varied dramatically in their ability to inhibit the interaction between platelets and immobilized RGD peptides. Our data indicate that the majority of RGD binding sites on GPIIb/IIIa can be reached by peptides that extend out approximately 11 to 32 A from the surface of the bead, and these results are in accord with the dimensions of integrin receptors deduced from electron microscopy. Activation of GPIIb/IIIa facilitates the interactions, but platelet inhibition fails to eliminate the interactions with the longer peptide beads, suggesting that access to the RGD binding site on at least a fraction of the GPIIb/IIIa receptors is always possible for preferred ligands. Finally, we found that the G3-RGDF peptide beads were uniquely sensitive to the activation state of the GPIIb/IIIa receptor. PMID- 1728304 TI - Monoclonal antibodies to idiotype inhibit in vitro growth of human B-cell lymphomas. AB - The effect of monoclonal antibodies (MoAbs) recognizing idiotype, IgM heavy chain, and IgD heavy chain on the in vitro DNA synthesis of five randomly selected leukemic human low-malignancy B-cell lymphomas was investigated. In three lymphomas of different histologic subtype, low concentrations of anti idiotypic (anti-Id) MoAb completely inhibited spontaneous 3H-thymidine uptake of T-cell-- and monocyte-depleted tumor cells, whereas two other tumors were not affected. Maximal inhibition of DNA synthesis was achieved at MoAb concentrations ranging from 0.5 to 250 micrograms/mL and required crosslinking by bivalent antibody but not Fc-mediated effects. While two anti-IgM MoAbs were similarly efficient as anti-Id MoAb in inhibition of DNA synthesis, two anti-IgD MoAbs had no effect. Thus, surface IgD molecules seemed to be neither able to deliver inhibitory signals themselves nor to antagonize IgM-mediated signals when simultaneously crosslinked by anti-Id MoAb. Leukocyte differentiation antigen expression, IgM density, and IgM/IgD ratio on the surface of lymphoma cells did not distinguish between sensitive and resistant tumors. In vitro tumor cell survival was differently affected by prolonged incubation with anti-Id antibody. In a centrocytic lymphoma and an immunocytoma, but not in a chronic lymphocytic leukemia, suppression of 3H-thymidine uptake persisted after removal of MoAb and tumor cell viability decreased during prolonged incubation with anti-Id MoAb. These results suggest that direct inhibitory signaling via surface IgM may contribute to anti-Id MoAb-mediated tumor regression in certain human B-cell lymphomas. PMID- 1728305 TI - Continuous release of secretory granule proteoglycans from a cell strain derived from the bone marrow of a patient with diffuse cutaneous mastocytosis. AB - A human cell strain (designated HBM-M) that was derived from the bone marrow of a child with diffuse cutaneous mastocytosis was previously found to possess features that suggested it belonged in the mast cell/monocyte lineage. HBM-M cells synthesized approximately 150-Kd Pronase-resistant proteoglycans that were recognized by an antihuman secretory granule proteoglycan peptide core antibody. These cells also contained in relatively high abundance the same sized mRNA transcript that encodes the peptide core of proteoglycans that are normally localized to secretory granules of hematopoietic cells. However, unlike most other hematopoietic cells, HBM-M cells continuously released their newly synthesized 35S-labeled proteoglycans rather than retaining them in an intracellular storage compartment. Chondroitinase ABC, nitrous acid, and heparinase degraded approximately 76%, 17%, and 7%, respectively, of the HBM-M cell-derived 35S-labeled proteoglycans. As assessed by high performance liquid chromatography, 91% of the unsaturated 35S-labeled disaccharides generated by treatment with chondroitinase ABC were delta Di-4S. The remaining chondroitin sulfate 35S-labeled disaccharides appeared to be primarily a complex mixture of disulfated disaccharides. The 35S-labeled glycosaminoglycans that were not degraded by chondroitinase ABC migrated in two-dimensional cellulose acetate electrophoresis as if they were heparan sulfate or under-sulfated heparin. Thus, although the HBM-M cell-derived proteoglycans had some of the features of proteoglycans produced by normal human mast cells, the heparin-like and chondroitin sulfate glycosaminoglycans bound to the HBM-M cell proteoglycans were considerably less sulfated. Because the only human cell types that have so far been shown to synthesize proteoglycans that have heparin-like glycosaminoglycans bound to a protease-resistant peptide core are mast cells and basophilic leukocytes from patients with myelogenous leukemia, it is possible that the HBM-M cell is a mast cell progenitor cell. PMID- 1728306 TI - Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects. AB - We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined. PMID- 1728307 TI - First continuous propagation of B19 parvovirus in a cell line. AB - The pathogenic human parvovirus B19 has extreme tropism for human erythroid progenitor cells and has resisted cultivation in conventional cell lines. We report first propagation of this virus in an erythropoietin-dependent strain of a megakaryoblastic leukemia cell line called UT-7. Virus protein was present in about 5% of cells after 1 week of culture. Appropriate ratios of major and minor capsid proteins were determined by immunoblot, and newly synthesized capsid protein was detected by immunoprecipitation of radioactively labeled cell lysates. High molecular weight monomer and dimer intermediates were detected by Southern analysis, indicating active viral replication. Approximately 1,000 genome copies were present per infected cell, and at the optimal multiplicity of infection 20- to 50-fold more virus was produced than inoculated. Virus propagation only occurred in UT-7 cells that were adapted to growth in erythropoietin; virus signal was not detected in UT-7 cells adapted for growth in granulocyte-macrophage colony-stimulating factor or interleukin-3, even with exposure to erythropoietin for several days. Infectious virus was detected in cultures as long as 3 months after inoculation. Despite persistence, there was no evidence of viral integration on Southern analysis. This cell line may prove useful for the production of infectious virus and in the analysis of B19 parvovirus persistence, cytotoxicity, and permissivity. PMID- 1728308 TI - Interleukin-4 is an autocrine growth factor secreted by the L-428 Reed-Sternberg cell. AB - Recent evidence indicates that Reed-Sternberg (RS) cells from many cases of Hodgkin's disease have features of activated lymphocytes and that lymphokines from activated lymphocytes induce proliferation of L-428 RS cells. It is shown here that a lymphokine similar to a lymphokine secreted by activated lymphocytes is secreted by L-428 cells. This lymphokine has a molecular weight approximately equal to 68,000 daltons, identical to glycosylated recombinant interleukin-4 (rIL 4), and cross-reacts with monoclonal anti-IL-4 in Western immunoblotting. This Hodgkin's cell growth factor (HCGF) is 100% neutralized by polyclonal anti-IL-4 antibodies and competes for the IL-4 receptor. After acid-elution, the L-428 RS cell has been shown to have 3,396 +/- 120 high-affinity receptor sites/cell. HCGF competes with rIL-4 for this receptor and L-428 cells contain mRNA for IL-4. Although all evidence indicates that IL-4 is an important secreted autocrine growth factor for L-428 RS cells, anti-IL-4 has no effect on the sustained serum free growth of these Hodgkin's cells, suggesting that either the IL-4 receptor and the IL-4 receptor-growth factor complex are protected from antibody inhibition or other mechanisms are responsible for the sustained proliferation of L-428 RS cells. PMID- 1728309 TI - A mechanism of resistance to glucocorticoids in multiple myeloma: transient expression of a truncated glucocorticoid receptor mRNA. AB - Despite their widespread use, little is known of either the mechanism of action of glucocorticoids in the treatment of multiple myeloma or why patients ultimately become resistant to their therapeutic effects. Here, we address these issues by examining the direct effects of the glucocorticoid dexamethasone (DEX) on a hormone-sensitive clone (MM.1S) of a human multiple myeloma line and compare them with those of its hormone-resistant counterpart (MM.1R). MM.1S expresses approximately 50,000 glucocorticoid receptors (GR) per cell, the full-length 7.1 kb GR mRNA at high levels, and is lysed by DEX. DEX-induced cytolysis is effectively blocked by the glucocorticoid antagonist, RU 486, indicating the specificity of this response for the GR. In contrast to MM.1S, MM.1R is not lysed by hormone, has little hormone-binding activity, and expresses the 7.1-kb GR mRNA at low levels. Interestingly, we have found that two distinct phenotypes emerge from MM.1R with increasing periods of growth in culture. The first or "early" form, MM.1Re, expresses high levels of a variant GR mRNA of 5.5 kb that has a deletion in its 3' end. With further growth in the presence or absence of selective media, the expression of this transcript is repressed, resulting in the second or "late" phenotype characteristic of MM.1RL. No discernible differences in the organization of the genomic GR sequence in DEX-sensitive and -resistant cells were detectable by Southern analysis, suggesting that no gross deletions, rearrangements, or allelic variations in the genomic sequence account for the resistant phenotypes of MM.1R. PMID- 1728311 TI - Mean corpuscular volume of heterozygotes for beta-thalassemia correlates with the severity of mutations. AB - The relationship between the degree of microcytosis and the type of mutation carried by beta-thalassemia heterozygotes was investigated. In 113 individuals, 18 different mutations were identified, correlated with mean corpuscular volume (MCV) values, and analyzed statistically. Overall, there was a wide range of MCV (56.3-87.3 fL). In almost all cases, carriers of beta(0) mutations had an MCV below 67 fL, whereas all but a few beta(+) heterozygotes had MCVs above this cutpoint. Mean MCV of beta(0) carriers was statistically significantly lower than those of beta(+) heterozygotes. The various beta(+) mutations were associated with significant differences in mean MCV values. In contrast, all the beta(0) (null) mutations had virtually identical ranges of MCV. The results indicate that degree of reduction in MCV is directly related to the severity of the mutation. Deviations, in four cases, were associated with concurrent alpha gene rearrangements, whereas in three other cases, the MCV was not significantly affected by concurrent alpha rearrangements. The MCV of beta-thalassemia heterozygotes is a valuable parameter in planning strategies for rapid identification of mutations in populations with great mutational diversity. Its use can be particularly advantageous in the setting of prenatal diagnosis. The pattern of mean corpuscular hemoglobin (MCH) values was similar to the MCV pattern. However, our results suggest that MCH may be preferred for carrier detection in population screening. PMID- 1728310 TI - VH gene rearrangement events can modify the immunoglobulin heavy chain during progression of B-lineage acute lymphoblastic leukemia. AB - The presence of multiple VHDJH joinings in upwards of 30% of acute lymphoblastic leukemias (ALL) suggests a relative instability of the rearranged immunoglobulin heavy chain (IgH) gene, but the mechanisms involved are not completely understood. An investigation of the structure of the VHDJH joinings using complementarity determining region (CDR)3 polymerase chain reaction (PCR) in 12 leukemias at both diagnosis and relapse indicates that this instability may increase as a function of time. In only one of seven cases in which relapse occurred within 3 years from diagnosis was a new VHDJH joining identified and this coexisted with the original diagnostic joining. Most strikingly, new VHDJH joinings were identified in four of five cases in which relapse occurred more than 5 years from diagnosis. In this latter population, the instability of the joinings was generated from VH----VH gene replacement events in two cases, since the new joinings retained the original DJH sequences and partial N region homology at the VHD junction, and probably in a third case from a VH gene rearrangement to a common DJH precursor. Furthermore, in five of 23 (21.7%) additional cases studied at diagnosis, subclones were identified that had similar modifications of the VH-N region. These data indicate that VH gene replacement events and VH gene rearrangements to a common DJH joining contribute to the instability of the VHDJH joining in ALL. This phenomenon should be taken into consideration in those methodologies that exploit IgH rearrangements for detection of minimal residual disease. PMID- 1728312 TI - Newly identified iron-binding protein in human duodenal mucosa. AB - Studies were undertaken using human duodenal mucosa to determine whether it contained a counterpart to a newly identified iron-binding protein recently isolated from rat duodenum and named mobilferrin. Water-soluble homogenates were prepared from duodena of patients undergoing surgery for pancreatic carcinoma. An iron-binding protein with an approximate molecular mass of 56 Kd was purified to homogeneity using 60% ammonium sulfate and serial chromatographic steps. The protein was biochemically and immunologically distinct from transferrin and ferritin, and competitively bound to zinc, cobalt, and lead. Each molecule bound one molecule of iron with a kd of 8.9 x 10(-5). Human isolates reacted in an enzyme-linked immunosorbent assay with a polyclonal antibody raised in rabbits against a similar duodenal protein isolated from rat duodenum. It is postulated that mobilferrin plays a significant role in the absorption of iron and other metals and may explain partially the competition between certain metals for absorption in the small intestine. PMID- 1728313 TI - Origin and fate of iron mobilized by the 3-hydroxypyridin-4-one oral chelators: studies in hypertransfused rats by selective radioiron probes of reticuloendothelial and hepatocellular iron stores. AB - The mechanism of in vivo iron chelation by 3-hydroxypyridin-4-ones (CP compounds) was studied in hypertransfused rats in which the major storage iron pools in hepatocytes and in the reticuloendothelial (RE) system have been labeled by selective radioiron probes. Both dimethyl-3-hydroxypyridin-4-one (CP 20 or L1) and diethyl-3-hydroxypyridine-4-one (CP 94) have an identical and very high (log beta 3 36) binding constant and selective affinity to iron(III), but the lipid solubility of CP 94 is considerably higher than that of CP 20. Both chelators induced an increase in the fecal excretion of hepatocellular iron with no effect on urinary excretion. In contrast, about one third to one half of the iron mobilized from RE cells was excreted in the urine. The chelating efficiency of CP 20 was comparable with that of deferoxamine (DF), whereas CP 94 was up to eight times more effective than DF. Unlike DF, which had no effect by the oral route, the oral and parenteral effectiveness of both CP compounds was identical. These findings indicate that: (1) lipid solubility is an important determinant of in vivo chelating efficiency; (2) urinary iron excretion induced by the CP compounds is derived from RE cells; (3) part of the iron mobilized from RE cells and all of the iron derived from hepatocytes is excreted through the bile; and (4) contrary to previous observations in cell cultures, there is no in vivo evidence for a diminishing chelating efficiency at the lowest doses used. PMID- 1728314 TI - Characteristics of red blood cell populations fractionated with a combination of counterflow centrifugation and Percoll separation. AB - Red blood cell (RBC) fractions were studied after separation of whole blood by means of counterflow centrifugation, Percoll column (Pharmacia, Uppsala, Sweden), and a combination of both separation techniques. Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and hemoglobin A1c (HbA1c) were measured in each fraction. From the results it was obvious that the combination of both techniques was the best separation technique of these three. MCV had a good correlation with cell age as measured with HbA1c concentration gradient; MCH and MCHC less so. MCV and MCH decreased in parallel to an increase in HbA1c. MCHC increased with increasing HbA1c. From these data it is concluded that there is a steadily ongoing loss of cellular hemoglobin and proportionally more cellular water during the life of the RBC. PMID- 1728315 TI - Bone marrow transplantation for severe aplastic anemia: influence of conditioning and graft-versus-host disease prophylaxis regimens on outcome. AB - Data for 595 patients with severe aplastic anemia receiving HLA-identical sibling bone marrow transplants were analyzed to determine the effect of pretransplant conditioning and graft-versus-host disease (GVHD) prophylaxis on outcome. Transplants were performed between 1980 and 1987 and reported to the International Bone Marrow Transplant Registry. Three conditioning regimens (cyclophosphamide alone, cyclophosphamide plus limited field radiation, and cyclophosphamide plus total body radiation) were studied; none was associated with superior long-term survival. Three GVHD prophylaxis regimens (methotrexate, cyclosporine, and methotrexate plus cyclosporine) were studied. Recipients of cyclosporine with or without methotrexate had a significantly higher probability of 5-year survival (69%, 95% confidence interval 63% to 74%) than patients receiving methotrexate only (56%, 49% to 62%, P less than .003). Higher survival with cyclosporine resulted from decreased risks of interstitial pneumonia (P less than .0002) and chronic GVHD (P less than .005). Additional risk factors adversely associated with survival included infection pretransplant (P less than .004), use of parous or transfused female donors (P less than .005), older patient age (P less than .005), and 20 or more pretransplant transfusions (P less than .006). These data may prove useful in planning randomized clinical trials and in identifying patients at high-risk of treatment failure. PMID- 1728316 TI - Prognostic significance of Philadelphia chromosome-positive cells detected by the polymerase chain reaction after allogeneic bone marrow transplant for chronic myelogenous leukemia. AB - Although rare cells expressing the bcr/abl fusion transcript can be detected by the polymerase chain reaction (PCR) in patient blood or marrow after allogeneic bone marrow transplant (BMT) for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukemia (CML), the prognostic significance of this finding is unknown. This paper reports clinical, cytogenetic, and molecular data derived from 64 CML patients following allogeneic BMT. Nested primer PCR was performed on patient blood and bone marrow samples to detect the presence of residual bcr/abl (+) cells in CML patients considered to be in clinical remission at the time of study. Bcr/abl transcripts were detected in 37 of 64 patients for at least one timepoint post-BMT. Thirteen of these 37 bcr/abl (+) patients have subsequently relapsed, as defined by clinical and/or persistent cytogenetic findings, in contrast to 0 relapses among the 27 bcr/abl (-) patients (P = .0025). The median time from first (+) bcr/abl PCR signal to relapse was 150 days (range 90 to 832). Fifty-four patients were studied at two or more timepoints post-BMT: five of eight patients persistently bcr/abl (+) have relapsed; 5 of 23 patients with both bcr/abl (+) and (-) assays during follow-up have relapsed; and none of 23 patients persistently (-) have relapsed (cumulative actuarial relapse rates 77%, 20%, and 0%, respectively, P = .0017). These data indicate that among CML patients in apparent clinical remission after BMT, nested primer bcr/abl PCR can define subgroups with low, intermediate, and high risk of relapse. The pattern of bcr/abl PCR detection after transplant may aid in the development of trials designed to reduce the risk of relapse, or allow for early intervention in patients who fail to clear the malignant clone. PMID- 1728317 TI - Cumulative incidence of secondary solid malignant tumors in aplastic anemia patients given marrow grafts after conditioning with chemotherapy alone. PMID- 1728318 TI - Subcutaneous erythropoietin for treatment of refractory anemia in hematologic disorders. Results of a phase I/II clinical trial. AB - We have used recombinant human erythropoietin (rHuEPO) in a phase I/II clinical trial to evaluate its ability to reverse refractory anemia in hematologic disorders. rHuEPO was administered subcutaneously 5 days per week at escalating doses (50 to 150 U/kg per day). The aim of treatment was a hemoglobin (Hb) level greater than or equal to 10 g/dL without blood transfusion. Of 25 patients treated, 17 were evaluable, most of them with a regular need for transfusion. Eight of these had lymphoproliferative disorders (three cases of malignant lymphoma and five of monoclonal gammopathy) and were exposed to cytotoxic therapy. The other nine patients had hematopoietic stem cell disorders (four cases of myelodysplastic syndrome, three of idiopathic myelofibrosis, and two of chronic myelogenous leukemia). All patients with lymphoproliferative disorder had serum EPO levels inappropriately low for the degree of anemia, while patients with stem cell disorder showed variable values. Erythroid marrow activity was inadequate in all cases. Seven of eight patients with lymphoproliferative disorder responded to treatment maintaining Hb above 10 g/dL without transfusion. The median dose of rHuEPO required for correction of anemia was 75 U/kg. In four cases response was maintained with 50 U/kg, three times per week. There was no complete response among patients with hematopoietic stem cell disorder, although transfusion requirement was eliminated or reduced in four cases. Four patients developed functional iron deficiency during rHuEPO treatment and required iron supplementation to obtain response. Aggravation of splenomegaly was observed in two cases of myeloproliferative disorder. We conclude that: (1) subcutaneous administration of rHuEPO can be effective and safe in patients with lymphoproliferative disorder exposed to chemotherapy and showing inappropriate EPO response to anemia; (2) this is less likely in hematopoietic stem cell disorders, although favorable responses may be observed in occasional patients; and (3) functional iron deficiency as a cause of nonresponse to rHuEPO is frequent also in nonrenal anemia. PMID- 1728319 TI - Analysis of immunoglobulin heavy chain gene rearrangement using the polymerase chain reaction. PMID- 1728320 TI - c-myc is an erythropoietin early response gene in normal erythroid cells: evidence for a protein kinase C-mediated signal. AB - The proto-oncogene c-myc has been identified as an early response gene for erythropoietin (Epo) in transformed murine erythroleukemia cells. Epo activation of c-myc in these cells requires protein kinase C. We now show the fidelity of this signaling pathway in normal erythroid cells isolated from the spleens of phenylhydrazine-treated mice. Mouse spleen cells rich in erythroid progenitors were washed free of endogenous Epo and then incubated in the absence of Epo. Subsequent addition of Epo for 1 hour led to a dramatic elevation of c-myc transcript. Addition of the protein synthesis inhibitor cycloheximide did not prevent the c-myc response, thus identifying c-myc as an Epo early response gene in normal cells. We used this c-myc response as a reporter for signals initiated by the Epo receptor. Using a series of inhibitors with known specificities and established rank-orders of potency for different kinases, we determined that the c-myc response to Epo was blocked with the following rank order: staurosporine much greater than H7 greater than sangivamycin greater than H8. This sequence is identical to that obtained using transformed cells and is diagnostic of a protein kinase C-dependent signal. Because direct activation of protein kinase by phorbol esters does not induce terminal differentiation of normal cells, the pathway to c myc established by these studies must represent one part of a signal transduction mechanism. PMID- 1728321 TI - Cytochalasin D and E: effects on fibrinogen receptor movement and cytoskeletal reorganization in fully spread, surface-activated platelets: a correlative light and electron microscopic investigation. AB - This study investigates the involvement of actin microfilaments in fibrinogen receptor redistribution and cytoskeletal reorganization that takes place in fully spread, surface-activated platelets. Colloidal gold-labeled fibrinogen (Fgn-Au label) in conjunction with video-enhanced differential interference contrast light microscopy (VDIC) was used to identify fibrinogen binding sites, glycoprotein IIb/IIIb (GPIIb/IIIa), on fully spread platelets. Platelets were treated with cytochalasins D and E (5 x 10(-5) mol/L to 5 x 10(-8) mol/L) for 10 minutes, before or after incubation with Fgn-Au label. Results observed with VDIC were subsequently confirmed by high-voltage transmission and low voltage-high resolution scanning electron microscopic examination of the specimens. Preincubation of activated platelets with cytochalasin D or E (5 x 10(-5) and 5 x 10(-6) mol/L) inhibited fibrinogen receptor redistribution and abolished cytoskeletal reorganization in fully spread platelets. After surface-activated platelets were incubated with Fgn-Au label, treatment with the above concentrations of cytochalasin D or E disrupted cytoskeletal reorganization and caused random movement of previously redistributed receptor-ligand complexes. Incubation of platelets with cytochalasin E 5 x 10(-6) mol/L prevented platelet activation and spreading. Thus, actin filaments appear necessary for platelet spreading from the discoid to the fully spread stage. The ligand-triggered, cytoskeletally directed movement of fibrinogen receptors in fully spread platelets appears to be dependent on the presence of intact, polymerized actin. This movement is distinct from the cytochalasin-insensitive accumulation of GPIIb/IIIa-ligand in the channels of the open canalicular system. PMID- 1728322 TI - On empowering staff through quality programs. PMID- 1728323 TI - Empowering staff nurses through quality assurance. PMID- 1728324 TI - PROSTAR: a recognition and reward program for empowering nurses to improve quality. PMID- 1728325 TI - Empowering the clinical nurse through quality assurance in a shared governance setting. PMID- 1728326 TI - Professional practice actualized through an integrated shared governance and quality assurance model. PMID- 1728327 TI - Empowerment through collaboration: implementing a team quality assurance model. PMID- 1728328 TI - Quality assurance generated by professional nursing practice in long-term care. PMID- 1728329 TI - Improving patient outcomes: the role of the clinical nurse specialist in quality assurance. PMID- 1728330 TI - Development and implications of an interdisciplinary quality assurance monitor on unplanned transfers into the intensive care units. PMID- 1728331 TI - Developing and implementing computer-generated nursing care plans. PMID- 1728332 TI - Quality of care: discovering a modified practice theory. PMID- 1728333 TI - As the nursing QA coordinator, I plan quarterly reports. PMID- 1728334 TI - Empowering staff nurses through quality assurance. PMID- 1728335 TI - Cancer statistics, 1992. PMID- 1728336 TI - Regional differences in surgical management of breast cancer. AB - Data voluntarily submitted to the NCDB by 597 hospitals throughout the US indicate marked variations in the type of surgery used for the treatment of breast cancer. Patients in New England with early stage breast cancer are much more likely to be treated with partial mastectomy than are patients in the East South Central or West North Central regions. PMID- 1728337 TI - Update January 1992: the American Cancer Society guidelines for the cancer related checkup. PMID- 1728338 TI - The autopsy in oncology. PMID- 1728339 TI - Cancer in African Americans. PMID- 1728340 TI - Introduction to "Supectrophotometer: new instrument for ultrarapid cell analysis" by Kamentsky, Melamed, and Derman. PMID- 1728341 TI - Cancer statistics for African Americans. AB - Although cancer remains a major public health burden for African Americans, progress is being achieved. Since 1984, the cancer mortality rate has declined two percent. Stomach and uterine cancer death rates have shown dramatic decreases in the last 30 years. Tobacco use is declining among blacks and is much lower among black adolescents than among their white counterparts. Black women are getting Pap smears more frequently than are any other ethnic group. Evidence is now accumulating that the causes of increased cancer morbidity and mortality in African Americans are related more to poverty and lack of education and access to care than to any inherent racial characteristics. Such observations support a range of opportunities whereby the impact of cancer in African Americans can be diminished through community programs and public health action. PMID- 1728342 TI - The new LMCC. PMID- 1728343 TI - Are general surgeons a dying breed? PMID- 1728344 TI - Are general surgeons a dying breed? PMID- 1728345 TI - Are general surgeons a dying breed? PMID- 1728346 TI - Are general surgeons a dying breed? PMID- 1728347 TI - Asbestos: the turbulent interface between science and policy. PMID- 1728348 TI - Adverse effects of psyllium. PMID- 1728349 TI - Sexuality, birth control and childbirth in orthodox Jewish tradition. AB - This paper examines some of the traditional texts that deal with sexuality, birth control and childbirth in the orthodox Jewish tradition and presents the rules governing these areas. For instance, a married woman should avoid being alone with a male physician unless other people are in earshot and have access to the room. A husband and wife must separate during the woman's menses and for the first 7 days afterward. Contraception is permitted if childbearing would endanger a woman's life or health. Termination of pregnancy is also permitted to preserve a woman's health, including her mental health. During childbirth the health of the mother is primary and supercedes all other rules or laws, including those of Sabbath observance. In general, orthodox Jewish women try to live as much as possible within the framework of Halacha. These customs are examined as examples of the need for sensitivity to cultural norms that affect the behaviour of different ethnic groups. PMID- 1728350 TI - Superwarfarin ingestion. PMID- 1728351 TI - Folic acid and prevention of neural tube defects. PMID- 1728352 TI - Foreword 1926. PMID- 1728353 TI - There's nothing wrong with physician advertising, many observers say. PMID- 1728354 TI - Plastic surgeons take advantage of relaxed rules, launch ad campaigns. PMID- 1728355 TI - Britain's health service reels under reorganization. PMID- 1728356 TI - Health-policy research becoming a growth industry in Canada. PMID- 1728357 TI - Characterization of 21 newly established esophageal cancer cell lines. AB - Twenty-one esophageal cancer cell lines (KYSE series) have been established from the resected specimens of patients with esophageal cancer. Three lines, KYSE-30, KYSE-50, and KYSE-70, were derived from the implanted tumor of nude mice (initial passage); others were derived from resected specimens. Each cell line was morphologically distinct. Detailed cytogenetic analysis indicated that each cell line was karyotypically unique, and DNA fingerprint analysis showed no cross contamination among cells. Doubling time ranged from 13.7 to 75.5 hours, and modal chromosome numbers ranged from 46 to 120. Most cell lines grew in monolayer, but two cell lines (KYSE-50 and KYSE-360) grew as floating cell aggregates. No correlation was demonstrated between the establishment of cell lines and cell differentiation. These cell lines are the first reported to be homogeneous and individually unique and may provide a useful model for the study of human esophageal cancer. PMID- 1728358 TI - Concurrent preoperative chemotherapy and radiation therapy in localized esophageal adenocarcinoma. AB - Twenty-four patients with localized, potentially resectable adenocarcinoma of the esophagus were enrolled in this study to evaluate the use of preoperative chemotherapy and radiation therapy, followed by transhiatal esophagectomy. The patients were newly diagnosed and had received no prior treatment. Radiation therapy consisted of 4900 cGy, administered as 350-cGy fractions 5 days a week for 14 fractions. The chemotherapy consisted of 5-fluorouracil 300 mg/m2/day administered as a continuous 24-hour intravenous infusion for 96 hours each week, concomitantly with the radiation therapy. After a 3-week rest, patients underwent transhiatal esophagectomy. Twenty-two patients could be observed for their responses to the chemotherapy and radiation regimen. Radiographically, 41% showed improvement, 36% had stable disease, and 23% had progression. Nineteen patients underwent surgery; all patients had total gross removal of disease, and two patients had a complete histologic response. All 24 patients could be examined for toxicity assessment. There were three deaths during the treatment period: one patient died of a perioperative complication, one of pneumonia, and one of a myocardial infarction. Eleven patients eventually had pleural and/or pericardial effusions, and six of these were symptomatic. All 24 patients could be examined for survival analysis. The median follow-up for all patients was 12.5 months, with 32.5 months for all surviving patients. Median survival was reached at 11 months. Disease-free survival was 9.5 months. It was concluded that the radiation fractionation schedule in this preoperative regimen was associated with marked toxicity in the form of pleural and pericardial effusions. There was no improvement in survival compared with historic controls. The role of combined preoperative treatment in this patient population has yet to be determined. PMID- 1728359 TI - The intriguing nature of gastric tumors in Carney's triad. Ultrastructural and immunohistochemical observations. AB - The authors describe clinical and pathologic features present in an adolescent girl who had a gastric tumor and mediastinal mass. The latter was shown to be a paraganglioma, and the gastric neoplasm was classified as malignant "leiomyoblastoma," with the use of current histologic criteria. This tumor had metastasized to the liver but not to the lungs. Although the histologic criteria for leiomyoblastoma were fulfilled, no definite evidence of smooth-muscle cell differentiation was present ultrastructurally or by immunostaining methods. Gastric tumors that form part of "Carney's triad" are known to differ clinically and pathologically in important ways from smooth-muscle cell malignant neoplasms that are not part of this syndrome. Some have been classified as gastrointestinal autonomic nerve tumors, but the current study did not confirm this contention. The nature of gastric leiomyoblastomas in Carney's multitumoral association remains undecided. PMID- 1728360 TI - Does endoscopic follow-up improve the outcome of patients with benign gastric ulcers and gastric cancer? AB - This study investigated whether an endoscopic surveillance program for patients with "benign" gastric ulcers and gastric cancer leads to early detection of neoplasms and improves survival. The clinical course of all patients diagnosed between 1977 and 1986 as having either gastric ulcers or gastric cancer was followed for a minimum of 3 years. Of 597 patients with initially benign gastric ulcers, 452 (76%) returned for the recommended endoscopic follow-up examinations. In eight patients (1.8%), repeated biopsies disclosed malignant neoplasms; four of these patients (0.9%) had become asymptomatic. Survival curves were nearly identical in patients who complied and those who did not. Of 241 patients with gastric cancer, 72 underwent partial gastric resection with curative intent and survived the first year. Resectable cancer was detected in 5 of 48 patients who complied (10%); none of these patients died of cancer. However, 5-year actuarial survival rates were similar between the patients who complied and those who did not. Although endoscopic surveillance may detect resectable cancer in selected patients, it remains to be shown that such a strategy improves survival. PMID- 1728361 TI - Superficial flat-type early carcinoma of the stomach. AB - A clinicopathologic study was done on 21 cases of superficial flat-type early gastric carcinoma (IIb type EGC). In one case there was the two IIb type EGC. Nine patients had no symptoms, whereas the other 12 had either epigastralgia, hematemesis, or anorexia. The preoperative diagnosis was accurate in 15 patients; eight were demonstrated by barium study, and 13 by endoscopy. The suspicious finding of IIb type EGC was either the disappearance or irregularity of the areae gastricae by barium study and a mucosal color change by endoscopy. Well differentiated or moderately differentiated adenocarcinomas showed a slight redness of the affected mucosa whereas the poorly differentiated adenocarcinomas were pale in color. Histologically, many well-differentiated or moderately differentiated adenocarcinomas occupied the entire thickness of the mucosal layer whereas most of the poorly differentiated adenocarcinomas spread horizontally with preservation of non-cancerous glands and foveolae. The growth pattern was super type in ten lesions and small mucosal type in 12 and no pen-type growth was seen. Concerning the cell nuclear DNA ploidy pattern, 21 showed a low ploidy pattern and only one had a high ploidy pattern. The IIb type EGC seemed to have a less malignant potential from the viewpoint of growth pattern and DNA ploidy pattern. Care must be taken at the proximal line of excision of the tumor so as not to leave behind residual carcinoma cells. PMID- 1728362 TI - Proliferative activity and malignancy in human gastric cancers. Significance of the proliferation rate and its clinical application. AB - The authors sought useful indicators for predicting the proliferative activity of human gastric cancer and attempted to evaluate its clinical significance. One hundred seventy-two patients with gastric cancer were entered in this study. All patients received bromodeoxyuridine at 200 to 1000 mg/body before laparotomy. Cell kinetics studies using the migration chase method were done for 56 patients, and the DNA synthesis time (Ts) was found to be prolonged in tumors, especially in aneuploid tumors, compared with normal mucosae. Ts correlated with bromodeoxyuridine (BrdUrd) labeling indices (LI) (r = 0.453, P less than 0.0005) and DNA indices (DI) (r = 0.534, P less than 0.0005). Thus, the DNA synthesis time was significantly prolonged in the tumors having a high S-phase fraction or DNA aneuploidy. The result of multivariate analysis indicated that LI/DI was the most potent indicator for predicting the proliferation rate (PR), which was calculated by the formula LI/Ts, and correlated significantly with PR (r = 0.863, P less than 0.0001). As was clear from the result of Cox's proportional hazard model, the predicted proliferation rate (pPR) was the most notable factor for the prognosis because pPR correlated clinically with metastasis, such as that to liver and lymph nodes. The patients with a high pPR (greater than 10%) had a worse prognosis (4-year survival rate: 16.3%) than did those with a low value (less than 10%) (4-year survival rate: 85.1%). In vitro pPR obtained by in vitro BrdUrd labeling of the specimens obtained at biopsy correlated significantly with the in vivo pPR (r = 0.960, P less than 0.0001). The authors concluded that the proliferation rate was the most important factor in judging the malignancy of human gastric cancers and that this rate should be most helpful in determining the treatment and evaluating the prognosis of individual patients. PMID- 1728363 TI - Variables correlated with the risk of lymph node metastasis in early rectal cancer. AB - To ascertain the risk of lymph node metastasis (LNM) from early rectal cancer, the authors retrospectively analyzed 154 patients with pT1 or pT2 rectal cancer treated by radical resection. Gross and microscopic pathologic characteristics of the primary tumor were examined as predictors of LNM. Comparisons were done by Fisher's test; significance was defined as a P value of less than 0.05. The incidence of LNM for T1 and T2 tumors was 3 of 26 (12%) and 28 of 128 (22%), respectively. LNM occurred significantly less often in well-differentiated cancers (0 of 12.0%). The incidence of LNM for T1/T2 tumors without lymphatic vessel invasion (LVI) or blood vessel invasion (BVI) (20 of 119, 17%) was significantly less than that for T1/T2 tumors with LVI or BVI (10 of 32, 31%). None of the T1 tumors without LVI or BVI had LNM. There was a trend toward decreased LNM for sessile nonulcerated tumors compared with nonpolypoid, exophytic, or ulcerated lesions (P = 0.06). Tumor size and colloid histologic characteristics were not significant predictive features for LNM. The data suggest that local excision alone is adequate for well-differentiated or moderately differentiated T1 rectal cancer in the absence of LVI or BVI and for well-differentiated T2 tumors. Radical resection or local excision combined with pelvic radiation therapy may be more appropriate for the remainder of early cancers. PMID- 1728364 TI - Conservative management of extensive low-lying rectal carcinomas with transanal local excision and combined preoperative and postoperative radiation therapy. A report of a phase I-II trial. AB - Between 1986 and 1990, 16 patients were enrolled in a prospective Phase I/II study of transanal local excision and combined preoperative and postoperative radiation therapy (RT). All patients had biopsy-proven adenocarcinoma extending to within 6 cm of the anal verge and involvement of at least one third of the rectal circumference with tumor. Five of 16 patients (32%) had T3 tumors, and only two patients had T1 tumors. Patients received a single 500 cGy fraction of RT to the pelvis within 24 hours before surgery and underwent transanal excision followed by postoperative RT (median dose, 5040 cGy). With a median follow-up of 33 months, overall 3-year actuarial survival was 94%. Two patients had isolated local recurrences (both successfully salvaged), and four had distant metastases but maintained local control. The 3-year actuarial rates of continuous freedom from any relapse, continuous local control, and no evidence of disease at last follow-up were 53%, 80%, and 71%, respectively. Only three of 16 patients required colostomy, resulting in a 3-year actuarial colostomy-free rate of 77%. There was a trend toward a higher rate of relapse (P = 0.066) in patients with T3 tumors than those with T1 and T2 tumors. Sphincter-preserving therapy for low lying rectal carcinomas using local excision and combined preoperative and postoperative RT is feasible, although improved local and adjuvant therapy is needed for patients with T3 lesions. PMID- 1728365 TI - Clinical significance of the epidermal growth factor receptor gene in squamous cell carcinomas of the nasal cavities and paranasal sinuses. AB - The authors retrospectively analyzed epidermal growth factor receptor (EGFR) gene amplification in 49 cases of squamous cell carcinoma (SCC) arising from the nasal cavities (NC) and paranasal sinuses (PS) by using slot-blot analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues. Also, the relationship between the results of gene analysis and the clinical features of the patients was studied to investigate the clinical significance of the EGFR in SCC of the NC and PS. Amplification of the EGFR gene was detected in 5 of the 49 cases (10%). No significant difference was observed between EGFR gene amplification and the presence of lymph node metastases, local recurrence, or prognosis. This suggests that EGFR gene amplification is not related to the local progression or metastasis of the SCC in the NC and PS. In addition, it appears that amplification of the EGFR gene is not a prognostic indicator for SCC in the NC and PS. PMID- 1728366 TI - Sarcomatoid carcinoma of the lung. Immunohistochemical and ultrastructural studies of 14 cases. AB - The authors retrospectively reviewed data regarding 14 patients with sarcomatoid carcinomas of the lung seen and treated at M.D. Anderson Cancer Center from 1955 to 1986. The following were the histologic criteria for inclusion in the study: (1) the concurrent presence of malignant epithelial and sarcomatoid spindle cell components, and (2) positive immunoreactivity for antikeratin antibody or ultrastructural demonstration of epithelial differentiation in sarcomatoid tumors in which the epithelial component was inconspicuous. For the sarcomatoid components, the most frequent pattern was malignant fibrous histiocytoma, which was present in ten tumors. An unclassified sarcomatoid pattern was found in two cases and a fibrosarcomatous pattern in two remaining cases. Clinically, the median patient age was 59 years; 12 patients were male and 2 were female; 13 were smokers and 1 used snuff. Three patients had Stage I, ten had Stage III, and one had Stage IV disease. One patient with Stage I, seven with Stage III, and one with Stage IV disease died of their carcinomas 2 to 26 months after diagnosis (median survival time 12 months). All patients who had lymph node metastases at presentation died of disease. The authors concluded the following: (1) patients with sarcomatoid carcinoma of the lung usually presented at an advanced stage; (2) lymph node metastasis, as with a usual carcinoma of the lung, is an important prognostic factor; and (3) for all lung tumors with a sarcomatoid pattern, especially a malignant fibrous histiocytoma pattern, extensive samples should be obtained and immunoperoxidase or ultrastructural studies done to identify epithelial differentiation. PMID- 1728367 TI - Primary sarcomas of the heart. AB - Seventy-five primary sarcomas of the heart were classified by histologic appearance as angiosarcoma (26 cases), undifferentiated sarcoma (18 cases), osteosarcoma (9 cases), fibrosarcoma (6 cases), malignant fibrous histiocytoma (6 cases), leiomyosarcoma (4 cases), myxosarcoma (3 cases), synovial sarcoma (2 cases), and neurofibrosarcoma (1 case). The ages of the patients ranged from 1 to 75 years at the time of presentation (mean, 39 years). Angiosarcomas were predominantly right-sided and osteosarcomas left-sided. Forty patients treated surgically were examined, and survival correlated with clinical and histologic parameters. the survival rate was poor, with a mean of 11 months and median of 6 months. By univariate analysis, the survival rate was more favorable for patients with tumors located on the left side of the heart, without necrosis, with a low mitotic count, and without metastasis at diagnosis. Survival rates were better in patients receiving chemotherapy and radiation therapy. Age, gender, presence of differentiation, and histologic type did not affect prognosis. By multivariate analysis, a low level of mitotic activity and any therapy were the only significant factors affecting survival rate. Immunostaining with commercially available antisera was useful in the diagnosis of sarcoma but not in subclassification of 19 tumors so tested. Although the prognosis for patients with cardiac sarcomas is dismal, histologic grading is useful in predicting outcome, as has been shown for soft tissue sarcomas of other sites. PMID- 1728368 TI - Second bone marrow transplants for relapsed leukemia. AB - Seventeen patients who had a relapse at a median of 9 months after marrow transplant (14 allogeneic and three syngeneic) received second transplants. Eight patients were in remission when transplanted. Of the nine patients with active disease at the time of transplant, six had complete remissions, and one converted from blastic to chronic phase of chronic myelogenous leukemia. The median survival was 9 months (95% confidence interval, 4 to 17 months). Four patients died within 100 days of transplantation, and three were disease-free. Ten patients died after 100 days, all except two of disease relapse. Five patients had remissions that were greater than 12 months and longer than the remission after their first transplant (inversions). Three patients remain alive and disease-free at 37+, 55+, and 61+ months, the former two despite remissions of less than 1 year after their first transplant. Second transplants with a different cytoreductive regimen can eradicate disease resistant to prior myeloablative treatment; some patients may benefit from second transplants, even if the first transplant only achieves a short remission. PMID- 1728369 TI - Splenic involvement by aggressive malignant lymphomas of B-cell and T-cell types. A morphologic and immunophenotypic study. AB - To determine whether there are any consistent morphologic differences between B cell and T-cell aggressive non-Hodgkin's lymphomas of the spleen, the authors analyzed 16 spleens involved by mixed cell (1 case) or large cell (15 cases) lymphomas. Immunologic data were derived from cell suspensions or frozen tissue in each case. Five cases had a T-cell phenotype, and 11 were B-cell. Morphologic features favoring a T-cell phenotype included epithelioid histiocytic reactions, confinement of the lymphomas to the splenic T-zones (periarteriolar lymphoid sheath and marginal zone), and clear cell or polymorphous cytologic features. Features favoring a B-cell phenotype included multiple discrete nodules in the white pulp, large coalescent tumor nodules in association with small lymphocytic lymphoma, and large non-cleaved or immunoblastic plasmacytoid cytologic characteristics. Four cases were unusual because most neoplastic large cells were distributed diffusely or formed only small aggregates in the red pulp without definite tumor masses or nodules involving the white pulp. Because of this distribution and the frequently encountered erythrophagocytosis by benign appearing histiocytes, these cases resembled malignant histiocytosis. A T-cell phenotype was predicted for all four cases; however, only one case, a lymphoma with polymorphous cytologic characteristics, was of T-cell lineage. The other three cases were of B-cell lineage. The authors' results indicate that in most instances the B-cell or T-cell nature of aggressive splenic lymphomas is predictable from the distributional and cytologic features. As in lymph nodes, there are cases for which the morphologic characteristics of B-cell and T-cell lymphomas are indistinguishable. PMID- 1728370 TI - The effects of surgical treatment on survival and local recurrence of cutaneous malignant melanoma. AB - All patients with a diagnosis of cutaneous malignant melanoma (CMM) in Western Australia from 1980 to 1981 were observed for up to 6 years to determine vital status and to detect the development of local recurrences of the primary lesion. Approximately 35% of all patients had their tumors excised with surgical margins of less than 1 cm. When compared with patients whose tumors were excised with margins of at least 2 cm, the fatality rate in those with narrow margins was slightly less (rate ratio, 0.60; 95% confidence interval [CI], 0.20% to 1.80% for margins of 5 to 9 mm; rate ratio, 0.69; 95% CI, 0.26% to 1.87% for margins of 1 to 4 mm); however, this difference could have been caused by chance alone. The risk of local recurrence within 5 years after diagnosis was 2% (95% CI, 1% to 4%). The risk was strongly related to age and tumor thickness, but did not appear to be influenced by the width of excision (greater than 1 cm versus less than 1 cm: rate ratio, 1.03; 95% CI, 0.25% to 4.34%). The apparent lack of effect could be caused by to chance alone because the number of local recurrences was small. PMID- 1728371 TI - Effective chemotherapy for melanoma after treatment with interleukin-2. AB - Twenty patients with biopsy-proven metastatic malignant melanoma, previously treated with interleukin-2 (IL-2), received combination chemotherapy for progressive disease. Treatment included carmustine, cisplatin, dacarbazine, and tamoxifen (BCDT). Nausea was the most common toxicity (100%) and usually was mild. Persistent thrombocytopenia was the most frequent toxicity limiting further treatment. Eleven patients (55%) had an objective partial response, three patients (15%) had a minor response, and six patients (30%) had no change or progressive disease in response to this treatment. These results were comparable to the high response rates (21 of 40, 53%) achieved with BCDT in previously untreated patients with melanoma. It was concluded that prior therapy using IL-2 does not significantly alter the response rate of metastatic melanoma to BCDT, thus suggesting that immunomodulators (e.g., IL-2) and chemotherapeutic agents are not cross-resistant treatments. PMID- 1728372 TI - Clonal chromosomal abnormalities in desmoid tumors. Implications for histopathogenesis. AB - Desmoid tumors (aggressive fibromatosis) are regarded as lesions of uncertain histopathogenesis. Cytogenetic analyses of 26 desmoid tumor specimens from abdominal or extraabdominal sites of 22 patients with or without Gardner's syndrome (GS) showed clonal karyotypic abnormalities in 7 cases, random abnormalities in 14 cases, and striking telomeric fusion in 5 cases. Loss of chromosome Y, a reported feature of fibromatosis in penile and palmar locations, was detected as a clonal aberration in two patients. Additionally, involvement of 5q was observed in six patients, two of whom had GS. Clonal interstitial deletions of 5q were observed in three patients, one with and two without GS. These findings confirm a clonal and probable neoplastic origin for desmoid tumor and suggest that abnormalities of the Y chromosome and 5q may be important in the genesis of this neoplasm. PMID- 1728374 TI - Lactic acidosis. A presentation of metastatic breast cancer arising in pregnancy. AB - Lactic acidosis B is a rare metabolic complication of malignancy. It usually is associated with advanced and extensive metastatic disease. The authors report a case in which lactic acidosis was the presenting feature of a previously undiagnosed case of metastatic breast cancer in a pregnant woman and that resolved with successful antineoplastic treatment. The authors review the likely cause and management of the condition. PMID- 1728373 TI - Clinical course of patients with breast cancer with ten or more positive nodes who were treated with doxorubicin-containing adjuvant therapy. AB - Between 1974 and 1986, 283 patients with ten or more positive nodes were treated in four prospective trials using doxorubicin-containing adjuvant chemotherapy. At a median follow-up of 92 months, 182 patients had had a recurrence, and 158 died. An estimated 41% and 37% were disease-free at 5 and 7 years, respectively. Patients with ten positive nodes had a significantly better disease-free survival than those with more than ten such nodes (P = 0.04). The disease-free survival rate and overall survival rate were not influenced by the estrogen receptor status of the tumor, patient age, or disease stage. Long-term data on a large number of patients treated at this institute showed the natural history of this subgroup of patients. Approximately 30% of patient survived disease-free at 10 years after treatment with the systemic therapies used in these protocols. Newer approaches are needed to alter the prognosis of this subgroup of patients further. PMID- 1728375 TI - Trends in the diagnosis of in situ breast cancer in the Detroit metropolitan area, 1973 to 1987. AB - The recent increase in incidence in situ breast cancer has been marked by a higher detection rate among white women. Although the increase in incidence may reflect the concomitant uptrend in mammographic screening, the lower proportion of cases among black women is of major public health concern. Time trends in the diagnosis of in situ breast cancer were evaluated in a population-based analysis of data accrued from the Metropolitan Detroit Cancer Surveillance System. The proportions of in situ cases detected among all women with breast cancer were measured annually between 1973 and 1987, and the average interval percentage changes were calculated for eight subgroups of women stratified by race and age at diagnosis. Although the proportions of in situ cancers were generally higher among white than black women, the greatest increase in average interval percentage change was observed in the oldest age category of black women. The disparity seen in younger black and white women suggests possible implications for breast cancer screening. From 1973 through 1987, the largest increase in diagnosis of in situ breast cancer occurred in black women older than 70. PMID- 1728376 TI - Sequential cytopunctures during preoperative chemotherapy for primary breast carcinoma. II. DNA flow cytometry changes during chemotherapy, tumor regression, and short-term follow-up. AB - Between May 1986 and May 1987, 35 primary noninflammatory breast carcinomas (T3N0 N1M0) were studied by means of DNA flow cytometry (FCM-DNA) before and after each of three courses of preoperative chemotherapy (doxorubicin, vincristine, cyclophosphamide, methotrexate, and 5-fluorouracil) to assess initial nuclear DNA content, initial S-phase fraction (SPF), and the impact of chemotherapy on these parameters. Correlations were sought with objective regression and short-term follow-up. Four sequential cytopunctures were performed for cytologic examination and FCM-DNA analyses. Ten tumors were diploid and 25 aneuploid. No significant changes in FCM-DNA parameters during chemotherapy were observed in diploid tumors, and no regression was seen in nine of the ten cases. Among the 25 aneuploid tumors, 10 showed changes in DNA content and/or kinetic parameters. A significant correlation was observed between objective regression and initial FCM DNA content (P = 0.008), initial SPF (P = 0.004), and changes in FCM-DNA patterns observed during chemotherapy (P = 0.00005). During the follow-up period (range, 27 to 41 months), 13 patients had relapses. Patients with aneuploid tumors were more likely to have relapses (n = 11) than patients with diploid tumors (n = 2), and patients with high SPF were more likely to have relapses than patients with low SPF, but the differences were not significant. Similarly, changes in FCM-DNA parameters were observed more often in patients who had relapses, but, again, the difference was not significant. In 5 of the 13 patients who had relapses, FCM-DNA analyses were performed on cytopunctures of the recurrences: patterns were identical to those observed before treatment even when the primary tumor regressed or showed changes in FCM-DNA parameters during chemotherapy. PMID- 1728377 TI - A randomized trial of two dosage schedules of mitomycin C in advanced breast carcinoma. AB - For mitomycin C (MMC), an effective agent in the treatment of metastatic breast cancer, the optimal dosing strategy in responding patients must be defined because of the dose-limiting, long-term hematologic toxic effects. Sixty-seven patients received treatment for metastatic breast cancer (MMC 20 mg/m2, intravenously) and then were selected randomly to receive either "standard doses" (SD) (20 mg/m2, intravenously) or "low doses" (LD) (5 mg/m2, intravenously) of MMC every 6 weeks. The primary objective was to show that the LD regimen would result in fewer toxic effects and at least equal disease control. Response rates in the two arms were similar: there were no complete responses and five partial responses (15%) in the SD group and two complete responses and six partial responses (24%) in the LD group (P = 0.332). In the SD and LD groups, median times to progression (11 versus 12 weeks, respectively), response duration (10 versus 6 1/2 weeks, respectively), and survival (26 versus 26 weeks, respectively) were similar. The hematologic toxicity was significantly less in the LD group. Nine patients in the LD group were treated with SD at disease progression, and one complete response was observed. It is concluded that, in this group of patients, administration of MMC in LD, compared with SD, resulted in fewer hematologic toxic effects and similar disease control. PMID- 1728378 TI - A reappraisal of the International Federation of Gynecology and Obstetrics staging system for cervical cancer. A study of patterns of care. AB - The Patterns of Care Study (PCS) database for squamous cell cancer of the uterine cervix has been used to evaluate the International Federation of Gynecology and Obstetrics (FIGO) staging system, with particular focus on the association of tumor bulk with outcome. The 1558 patients treated in the United States in 1973 and 1978 constitute the data set, and survival and in-field pelvic control were the end point of assessment. In summary, this reappraisal of the FIGO staging system for cancer of the uterine cervix showed that the current FIGO system is not discriminating within stage. This study suggests specific indicators of progressive bulk of cancer that should be considered, because they were associated with significant differences in survival rate and in-field pelvic control within Stage II and III disease. PMID- 1728379 TI - Endometrial adenocarcinoma with squamous cell differentiation. AB - In a histopathologic review of all cases of endometrial carcinoma diagnosed in Norway between 1970 and 1978, 255 cases of adenocarcinoma with squamous cell differentiation were found among the 1985 cases reviewed. One hundred eighty-one (9.1%) were adenoacanthoma and 74 (3.7%) adenosquamous carcinoma. The mean age for patients with adenoacanthoma was 57.7 years (range, 32 to 85 years) and for adenosquamous carcinoma, 62.8 years (range, 43 to 84 years). Five-year and 10 year survival rates for all patients were 83.5% and 71.8%, respectively. For patients with adenosquamous carcinoma, corresponding figures were 64.9% and 52.7%, and for those with adenoacanthoma, the figures were 91.2% and 79.6%, respectively. When stratified for grade and depth of myometrial infiltration, there was no difference in survival rates between patients with adenoacanthoma and adenosquamous carcinoma, provided hysterectomy was part of the primary treatment. In patients who had surgery, myometrial infiltration was the most important single prognostic factor. It is recommended that the terms adenoacanthoma and adenosquamous carcinoma be replaced by the descriptive term adenocarcinoma with squamous cell differentiation. PMID- 1728380 TI - Uterine papillary serous carcinoma after radiation therapy for carcinoma of the cervix. AB - This article describes the clinicopathologic features of six cases of uterine papillary serous carcinoma (UPSC), which developed several years after radiation therapy (RT) for cervical carcinoma. The possible etiologic role of radiation is discussed, and the literature on endometrial carcinomas developing after RT is reviewed. PMID- 1728381 TI - Critical reassessment of second-look exploratory laparotomy for epithelial ovarian carcinoma. Minimal diagnostic and therapeutic value in patients with persistent cancer. AB - From 1979 to 1984, 88 women with epithelial ovarian cancer were treated with surgery and chemotherapy, achieved a clinical complete response, and then had "second-look" exploratory laparotomy to assess the pathologic status of their disease. Persistent cancer was found in 50 (57%) patients: 34 of 50 (68%) had gross tumor, which was larger than 2 cm in 12 (24%) and smaller than 2 cm in 22 (44%), and 16 (32%) had microscopic disease. Salvage therapy was as follows for these patients: whole abdominal irradiation, 29 (58%); chemotherapy, 17 (34%); intraperitoneal chromic phosphate, 1 (2%); and no further therapy, 3 (6%). With a follow-up time of 4 to 8 years, 7 (14%) patients are alive without evidence of cancer, 7 (14%) are alive with disease, 35 (70%) are dead of disease, and 1 (2%) has died of treatment complications. At 5 years, the relapse-free rate was 18% and the survival rate was 25%. Seventy-two parameters of suspected prognostic significance and 64 potential sites of tumor involvement were correlated with survival in a univariate analysis. The factors favorably affecting survival included the following: lower grade; microscopic tumor versus gross disease at second-look laparotomy; removal of the uterus; removal of the omentum; pelvic and paraaortic lymph node biopsy; negative results of a right diaphragm biopsy; and radiation therapy at Stanford University Medical Center, Stanford, California. There was no survival advantage for whole abdomen irradiation compared with chemotherapy or for the patients who had their disease successfully debulked at second-look laparotomy. The above factors and others were evaluated by multivariate regression. The best model (P = 0.000004) for predicting survival included largest tumor mass (P = 0.0002), operative blood loss (P = 0.002), perioperative blood transfusion (P = 0.003), and grade (P = 0.004). The detection of persistent ovarian cancer by second-look exploratory laparotomy should identify a subgroup of patients whose conditions can be salvaged by a second-line therapy. Unfortunately, that subgroup is small (8%) and an effective salvage therapy remains to be identified. PMID- 1728382 TI - Prediction of prognosis in untreated stage A2 prostatic carcinoma. AB - Carcinoma is found unexpectedly in approximately 10% or more of the 400,000 prostatectomies performed annually in the United States. Patients with Stage A2 carcinoma die of their disease in only 35% of the cases. To alter the course of disease in these patients, 65% of Stage A2 patients may be treated unnecessarily by radical prostatectomy, radiation therapy, or hormonal therapy. An accurate method to predict the outcome of patients with Stage A2 carcinoma is needed. Histologic sections from 18 patients with Stage A2 prostatic carcinoma followed without further treatment until progression, or followed without progression, were evaluated by several investigators who did not have knowledge of patient outcomes and who employed standard pathologic grading systems as well as morphometric, cytophotometric, flow cytometric, and immunohistochemical techniques. Outcome was predicted correctly by random sampled absolute (17 of 18 cases) and relative (16 of 18) nuclear roundness factor (NRF), tumor volume expressed as percent of specimen (13 of 16), primary (13 of 18), secondary (14 of 18), sum (15 of 18), and worse (14 of 18) Gleason grades and prostate-specific antigen immunohistochemical findings (13 of 18) that produced statistically significant separation of the two groups. Significant separation was not obtained with Mostofi's pattern, nuclear, sum, and worse grades, Johns Hopkins' grade, absolute tumor volume, nuclear DNA content measured by image cytophotometric study of Feulgen-stained histologic sections and flow cytometric study of propidium iodide-labeled suspensions of nuclei obtained from paraffin blocks, nonrandom sampled NRF of worse and most prevalent neoplastic areas, and prostatic acid phosphatase and peanut agglutinin immunohistochemical study. NRF measured by a random technique best predicted outcome in these patients with A2 prostatic carcinoma and should be evaluated prospectively as a means for selecting patients who require therapy. PMID- 1728383 TI - Morphologic analysis of surgical margins with positive findings in prostatectomy for adenocarcinoma of the prostate. AB - Apical invasion and positive apical margins were assessed in 165 consecutive radical prostatectomies. Apical invasion, defined as cancer in the distal 8 mm of the prostate, was evident in more than 80% of the cases, and apical margins occurred in 16% of the specimens with apical Clinical judgement was not effective in predicting apical cancer. Frequency of apical margins increased in proportion to greater cancer volume, from 9.8% in cancers smaller than 4 cc to 30.7% in cancers larger than 12 cc. However, most positive margins in the group with cancers smaller than 4 cc were caused by inadvertent incision into the prostate during the operation, whereas the vast majority of apical margins in cancers larger than 4 cc were caused by capsule penetration of the tumor. Although margins associated with capsule penetration occurred characteristically in the posterior (rectal) portion of the apex, margins caused by incision into the prostate were distributed over the entire apical surface of the gland. Positive margins at the urethral stump were quite uncommon (occurring in four cases). These findings suggest that modifications of surgical technique might reduce the frequency of this complication. PMID- 1728384 TI - Effects of bacillus Calmette-Guerin on cytotoxic activities of peripheral blood lymphocytes against human T24 lined and freshly isolated autologous urinary bladder transitional carcinoma cells in patients with urinary bladder cancer. AB - The effects of bacillus Calmette-Guerin (BCG) on the cytotoxic activities of peripheral blood lymphocytes against human T24 lined and freshly separated autologous urinary bladder transitional carcinoma cells in patients with urinary bladder cancer were analyzed in a 12-hour chromium 51 (51Cr) release assay. The results of this study indicate that BCG activates the tumor killing system through stimulation of effector cells and elevation of target cell susceptibility in patients with urinary bladder cancer, suggesting that BCG-augmented cytotoxicity may be oriented specifically to urinary bladder cancer cells. This could explain the remarkable clinical benefits of intravesical instillation of BCG against urinary bladder cancer. PMID- 1728385 TI - Congenital genitourinary anomalies. Is there a predilection for multiple primary malignant neoplasms? AB - A case of simultaneous uterine and renal cell carcinoma in an elderly woman who had a septate vagina, double cervix, uterus didelphys, and a single kidney secondary to contralateral renal agenesis is reported. She was treated for a period of 8 months, first with pelvic irradiation followed by total abdominal hysterectomy and bilateral salpingo-oophorectomy and subsequently with heminephrectomy. Her renal function was normal postoperatively. The patient died of congestive heart failure in June 1990 after being free of carcinoma for approximately 18 years. The authors believe that this is the only case of its kind currently reported in the literature. Four of her family members died of either gastric (n = 3) or lung (n = 1) cancer, and one sister is alive with colon cancer. Only 19 proven cases of this constellation of congenital anomalies have been reported in the literature, and none have been associated with genitourinary (GU) carcinomas. There is a 50% to 70% incidence rate of genital tract anomalies in female patients with unilateral renal agenesis, secondary to the intimate association of the mesonephric and mullerian ducts. It has been suggested that the GU tract is prone to multiple primary malignant neoplasms, and there are families genetically predisposed to the development of large bowel and GU carcinomas. No conclusions can be drawn concerning the development of carcinoma in patients with congenital GU anomalies because of the small number of patients and the lack of follow-up in the literature. PMID- 1728386 TI - Regional biologic therapy. Hepatic arterial infusion of recombinant human tumor necrosis factor in patients with liver metastases. AB - Twenty-two chemotherapy-resistant patients with liver metastases received 46 courses of recombinant human tumor necrosis factor (rhTNF) administered by 5-day continuous infusion through percutaneously inserted hepatic arterial catheters. The maximum tolerated daily dose of rhTNF was 150 micrograms/m2. This is six times the maximum tolerated daily dose of rhTNF that could be given systemically (intravenous) on the same schedule. The dose-limiting toxicity resulted in severe, although transient, hypophosphatemia (less than 1.0 mg/dl) associated with myocardial dysfunction. Objective tumor response (partial tumor response or greater) was observed in 2 of 14 patients (14%) with colorectal cancer and lasted as long as 3 months. Three additional minor responses occurred among these patients with colorectal cancer. Plasma carcinoembryonic antigen levels also decreased significantly (greater than 25%) in 7 of the 14 (50%) patients with colorectal cancer. Regional biologic therapy with rhTNF as a sole modality has definite antitumor activity in colorectal cancer metastatic to the liver and warrants additional study in previously untreated patients. PMID- 1728387 TI - Clinical and biologic effects of combination therapy with gamma-interferon and tumor necrosis factor. AB - Tumor necrosis factor (TNF) and gamma-interferon (gamma-IFN) are cytokines with synergistic biologic and antiproliferative effects in vitro and in mouse models. The biologic effects of the combination of TNF and gamma-IFN, however, have not been studied well in humans. A Phase I trial was conducted of TNF and gamma-IFN therapy in 24 patients with advanced malignancies to determine the tolerability of the combination and the biologic effects of TNF and gamma-IFN in vivo. Both TNF and gamma-IFN were administered as 30-minute intravenous infusions three times per week. Doses of TNF ranged from 25 to 100 micrograms/m2; all patients received 100 micrograms/m2 of gamma-IFN. Dose-limiting toxicity consisted primarily of orthostatic hypotension and constitutional symptoms. The maximum tolerated dose level (MTDL) of 50 micrograms/m2 of TNF and 100 micrograms/m2 of IFN-gamma was less than the maximum tolerated dose (MTD) observed in previous Phase I trials of gamma-IFN and TNF alone. Biologic responses were studied in seven patients treated at the MTDL. Serum interleukin-2 receptor levels and neopterin secretion were enhanced significantly 24 hours after therapy (P = 0.002); enhancement of monocyte Fc receptor levels had borderline statistical significance (P = 0.07). With the exception of the mean fluorescent intensity on monocytes positive for histocompatibility antigen HLA-DR (P = 0.03), HLA Class I and II cell surface protein expression was not increased. The combination significantly enhanced indoleamine dioxygenase activity and serum beta 2 microglobulin expression (P less than 0.04) but not 2',5'-oligoadenylate synthetase activity, bactericidal function, or chemiluminescence. These results were compared retrospectively with those observed in previous Phase I trials of gamma-IFN and TNF alone. The combination of TNF and gamma-IFN significantly increased urinary kynurenine levels more than either TNF alone or gamma-IFN alone. Given the limitations inherent in any retrospective analysis, however, the enhancement in the other biologic parameters measured at the MTDL during this trial did not differ significantly from the changes observed at the MTD of either TNF or gamma-IFN alone. It was concluded that the combination of TNF and gamma IFN, when administered at the MTDL of the combination, does not offer any enhancement in biologic responses over either agent alone. PMID- 1728388 TI - Perforation through undiagnosed small bowel involvement in primary thyroid lymphoma during chemotherapy. AB - A case is described of fatal perforation of the small bowel through an area of undiagnosed secondary involvement from primary thyroid lymphoma during treatment by chemotherapy. There is a known association between primary thyroid lymphoma and gastrointestinal metastases. To avoid this lethal complication, a specific search should be made for gastrointestinal involvement before chemotherapy is started in patients with advanced thyroid lymphoma. PMID- 1728389 TI - Home chemotherapy for children with cancer. AB - A program was established in which the parents of children with cancer were trained to administer intravenous chemotherapy to the children in their homes. The main objectives of this program (Home Intravenous Chemotherapy by Parents [HICP]) were to improve the quality of life for children with cancer and their parents and to decrease the cost of medical care for these children by decreasing the number of clinic visits and hospital stays intended solely for the administration of chemotherapy. Twenty-four months of experience with this program indicates that with a close communication network and a good working relationship among a home care organization (HCO), the parents of the patient, a pediatric oncology nurse, and an attending pediatric oncologist, many chemotherapeutic agents can be safely administered to these children by their parents in their homes. The patients and their parents are enthusiastic about this program because of the valuable time saved and the increased participation in care. In addition, this program greatly decreased the cost of medical care for these children. PMID- 1728390 TI - Choroid plexus carcinoma of childhood. AB - The presentation, growth patterns, and response to therapy of 11 consecutive children with choroid plexus carcinomas were analyzed, and the results were compared with the outcome reported in other series. Patients were a median of 26 months of age at diagnosis. Two patients had thalamic tumors, one had a posterior fossa primary, and the rest had ventricular lesions. Five of 11 (45%) children remain in continuous progression-free remission a median of 48 months from diagnosis. Four of the five in continuous remission had a "gross total" surgical resection, and only one received radiation therapy. Five of six patients with subtotal resections relapsed despite postoperative treatment with radiation therapy (three) and chemotherapy (one). The response to treatment with radiation therapy or chemotherapy at relapse was disappointing, with only one child (treated with etoposide) responding. In combination with other series, 11 of 14 children had prolonged progression-free survival after gross total resection (only two of whom received adjuvant therapy) compared with two of 20 after less than total resections, independent of the type of adjuvant therapy given. Adjuvant therapy for children with choroid plexus carcinomas is of unproven benefit, and this must be considered when analyzing innovative treatment trials for such children, especially for those with totally resected tumors. Patients with partially resected lesions fare poorly with present forms of treatment. PMID- 1728391 TI - A health survey of radiologic technologists. AB - A health survey of more than 143,000 radiologic technologists is described. The population was identified from the 1982 computerized files of the American Registry of Radiologic Technologists, which was established in 1926. Inactive members were traced to obtain current addresses or death notifications. More than 6000 technologists were reported to have died. For all registrants who were alive when located, a detailed 16-page questionnaire was sent, covering occupational histories, medical conditions, and other personal and lifestyle characteristics. Nonrespondents were contacted by telephone to complete an abbreviated questionnaire. More than 104,000 responses were obtained. The overall response rate was 79%. Most technologists were female (76%), white (93%), and employed for an average of 12 years; 37% attended college, and approximately 50% never smoked cigarettes. Radiation exposure information was sought from employer records and commercial dosimetry companies. Technologists employed for the longest times had the highest estimated cumulative exposures, with approximately 9% with exposures greater than 5 cGy. There was a high correlation between cumulative occupational exposure and personal exposure to medical radiographs, related, in part, to the association of both factors with attained age. It is interesting that 10% of all technologists allowed others to practice taking radiographs on them during their training. Nearly 4% of the respondents reported having some type of cancer, mainly of the skin (1517), breast (665), and cervix (726). Prospective surveys will monitor cancer mortality rates through use of the National Death Index and cancer incidence through periodic mailings of questionnaires. This is the only occupational study of radiation employees who are primarily women and should provide new information on the possible risks associated with relatively low levels of exposure. PMID- 1728392 TI - Legal perspectives on mammography and self-referral. PMID- 1728393 TI - Human immunodeficiency virus-associated Hodgkin's disease. PMID- 1728394 TI - Hodgkin's disease in children 4 years of age or younger. PMID- 1728395 TI - Mammographic parenchymal patterns and family history of breast cancer. PMID- 1728396 TI - Is electromagnetic fields and cancer an issue worthy of study? PMID- 1728397 TI - The origin of point mutations in human tumor cells. PMID- 1728398 TI - Cigarette smoking and cancers of the renal pelvis and ureter. AB - A population-based case-control study of renal pelvis and ureter cancers (502 cases, 496 controls) conducted in three areas of the United States found cigarette smoking to be associated with a 3.1-fold increase in risk, with long term (greater than 45 years) smokers having a 7.2-fold increased risk. Statistically significant dose-response associations were observed for both cancer sites and in both sexes regardless of the measure used: cigarettes per day, duration of use, or pack years. A significant decreasing trend in risk with increasing years quit smoking was also demonstrated for these cancers. Attributable risk estimates indicate that approximately 7 of 10 cancers of the renal pelvis and ureter among men and almost 4 of 10 among women are caused by smoking. The results of this study, the largest to date, confirm that cigarette smoking is the major cause of cancers of the renal pelvis and ureter, and that cessation of smoking could eliminate a large proportion of these tumors. PMID- 1728399 TI - The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice. AB - We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine. PMID- 1728400 TI - Molecular dosimetry in rat urine of aflatoxin-N7-guanine and other aflatoxin metabolites by multiple monoclonal antibody affinity chromatography and immunoaffinity/high performance liquid chromatography. AB - The development of molecular dosimetry methods will simplify the identification of people at high risk for cancer. A combined monoclonal antibody immunoaffinity chromatography/high performance liquid chromatography method has been devised to isolate and quantify aflatoxin-DNA adducts and other metabolites in rat urine samples. We report the production of 11 different monoclonal antibodies recognizing aflatoxin B1, aflatoxin Q1, aflatoxin G1, aflatoxicol, and aflatoxin M1 and the application of these antibodies to a multiple monoclonal antibody affinity chromatography technique. Using the multiple monoclonal antibody affinity column with rat urines obtained from dosed animals, between 90 and 95% of total aflatoxin metabolites can be bound to the column and isolated. Analytical immunoaffinity chromatography/high performance liquid chromatography analysis of these isolated aflatoxins reveals that more than 55% of the aflatoxins in rat urine are aflatoxin-dihydrodiol, aflatoxin-N7-guanine, aflatoxin Q1, aflatoxin M1, aflatoxin P1, and aflatoxin B1, accounting for 1.5, 9.6, 1.8, 34.5, 8.0, and 1.0% of the total aflatoxins, respectively. Further, a perchloric acid digestion of the aflatoxin-N7-guanine peak was used to confirm its identity by its conversion to guanine. The measurement of aflatoxin-N7 guanine excretion in rat urine was examined to assess its utility as a marker of DNA adduct formation in the liver, and a dose-dependent excretion in urine was found with a correlation coefficient of 0.99. A comparison of the dose-dependent residual levels of aflatoxin binding to liver DNA with the amount of aflatoxin-N7 guanine excreted in urine showed a correlation coefficient of 0.98. Besides the nucleic acid adduct excretion data, aflatoxin M1 and aflatoxin P1 were evaluated as molecular dosimeters in the urine. Aflatoxin M1 was found to be an excellent marker, whereas no linear relationship between dose and aflatoxin P1 excretion in urine was found. PMID- 1728401 TI - Inhibition of colon and breast carcinoma cell growth by interleukin-4. AB - Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB 468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth. PMID- 1728402 TI - Growth kinetics of microscopic hepatocellular neoplasms in carcinogen-resistant and carcinogen-responsive strains of mice. AB - The initiation and growth of microscopic hepatocellular neoplasms in C57BL/6 mice, considered relatively resistant to hepatocarcinogenesis, was compared with that of the more responsive C3H and B6C3F1 (C57BL/6 x C3H) strains. Tumors were induced by giving male mice injections of diethylnitrosamine when they were 15 days old. During the first 18 weeks postinjection, the growth rates of neoplasms in the three strains were almost identical (doubling time of 2.1 to 2.5 weeks). However, after that time, only the growth rates of the C57BL/6 neoplasms slowed; between 30 and 42 weeks the doubling time had increased to 13 weeks. In addition, at all sacrifice times the number of neoplasms in the C3H strain was at least 2.5 times higher than in the C57BL/6 and B6C3F1 strains. These results suggest that the genetic determinant(s) for inhibited tumor growth (expressed only in C57BL/6 mice) are recessive to those for unimpeded tumor growth (expressed in C3H and B6C3F1 mice), while the determinant(s) for large numbers of tumors (expressed only in C3H mice) are recessive to those for small numbers of tumors (expressed in C57BL/6 and B6C3F1 mice). In addition to the interstrain differences in tumor growth, two other types of tumor growth heterogeneity were identified. First, in each of the three strains, the largest tumors were found to grow faster than the smaller tumors. This suggests that the very broad range in tumor size that is seen in this model results from the long-term differences in the growth rates of individual neoplasms. Second, we found that in microscopic hepatic neoplasms in B6C3F1 mice, the thymidine labeling indices were 2.3 times greater in the outer 50-microns shell (2 cells thick) than in the next deeper 50-micron layer cells. This suggests that even in these minute neoplasms, gradients in blood-borne oxygen, nutrients, or growth factors are responsible for heterogeneous growth. PMID- 1728403 TI - 17 beta-hydroxysteroid dehydrogenase gene expression in human breast cancer cells: regulation of expression by a progestin. AB - The expression of the 17 beta-hydroxysteroid dehydrogenase (17-HSD) gene in a series of human breast cancer cell lines was studied by Northern blot hybridization with a cDNA probe and by a time-resolved immunofluorometric assay using polyclonal antibodies against the enzyme protein. The 17-HSD enzyme protein concentration was measured in the 800 x g cell extract. A high concentration was measured in the BT-20 cell line, corresponding to one-fourth of the average concentration in placental tissue. Western blot analysis indicated that the antigen corresponded to a single Mr 35,000 band. In 2 other cell lines (MDA-MB 361 and T-47D), the 17-HSD protein concentration was much lower, but still measurable, whereas in the remaining 5 cell lines (HBL-100, MCF-7, MDA-MB-231, MDA-MB-468, and ZR-75-1) it was below the detection limit of the assay. Treatment of the cells for 5 days with the synthetic progestin, ORG2058, resulted in an increase of the 17-HSD protein concentration only in the T-47D cell line. By Northern blot analysis, a low level of 2.3-kilobase mRNA transcripts was detected in all 8 cell lines. In addition, a 1.3-kilobase 17-HSD mRNA was present in the samples from the 3 cell lines containing measurable amounts of 17-HSD protein in the cell extract, and the band intensities were proportional to the amount of protein measured with the immunofluorometric assay. Only in the T-47D cell line did progestin treatment correspond to an increased amount of the 17-HSD 1.3 kilobase mRNA. These results suggest that the 1.3-kilobase mRNA for 17-HSD is the form most closely associated with protein expression and is also the only form responding to the progestin induction of the 17-HSD gene. PMID- 1728404 TI - A general transcription initiation factor, human transcription factor IID, overexpressed in human lung and breast carcinoma and rapidly induced with serum stimulation. AB - A general transcription factor IID which binds to the TATA box promoter element on RNA polymerase II genes regulates and initiates eukaryotic mRNA synthesis. A quantitative polymerase chain reaction procedure was developed and the human transcription factor IID (hTFIID) transcript was measured in normal human tissues, lung carcinomas, lung carcinoma cell lines, and breast carcinomas. In some normal tissues such as liver, fetal lung, and placenta, relatively low to moderate levels of hTFIID mRNA were detected. In contrast, hTFIID transcript was highly expressed in nearly all solid lung carcinomas and cell lines including both small cell lung cancer and non-small cell lung cancer. hTFIID mRNA was present to a greater extent in small cell lung cancer than non-small cell lung cancer in solid tumors and cell lines. In solid carcinomas of breast, overexpression of hTFIID was also detected. A serum induction study using a serum starved small cell lung cancer cell line, Lu134BS, indicated hTFIID transcription to be rapidly induced at 15 min following stimulation and its response essentially similar to that of protooncogene, c-fos. These results indicate the involvement of the expression of the general transcription factor hTFIID in lung and breast carcinoma, such as being associated with poor differentiation and high mitotic activity. PMID- 1728405 TI - Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues. AB - Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed. PMID- 1728406 TI - Isolation and characterization of a 1-beta-D-arabinofuranosylcytosine-resistant Chinese hamster ovary cell mutant that is also X-ray sensitive and is noncomplementary with ataxia telangiectasia cells. AB - In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-beta-D arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair. Such mutant cells could also be used for the isolation and characterization of human DNA repair genes. In particular, it was predicted that the repair gene defective in individuals with ataxia telangiectasia (AT) might be amenable to study with ara-C-resistant (X-ray sensitive) mutants, since additional studies, presented here, have shown that AT cells are resistant to ara-C. In the long term, it is hoped that determining the specific defect in AT might lead to an understanding of the possible role of defective repair in tumor induction and/or progression. The general approach used to isolate ara-C-resistant Chinese hamster ovary cell mutants was to treat cells with ethyl methanesulfonate and select in increasing concentrations of ara-C. Although several mutants were isolated, one in particular, Ara-CR213, has been studied most extensively. It was selected largely because it shows the greatest sensitivity to X-rays. Ara-CR213 cells were hypersensitive to the killing effect of X-rays with an LD10 of 2.5 Gy as compared to the wild-type cells that had an LD10 of 6 Gy. The mutant showed an increased frequency of X-ray-induced chromosomal aberrations in the G1 and G2 stages of the cell cycle compared to wild-type frequencies. There was no increase in sister chromatid exchange levels. All of these observations in Ara-CR213 are very similar to those made with AT cells in our and other laboratories. Even more important, complementation analysis of Ara-CR213 x AT hybrid cells indicated that the gene responsible for X ray sensitivity of AT is also mutated in Ara-CR213 cells. Thus, Ara-CR213 appears to have a mutant phenotype and probably genotype that is very similar to, if not exactly the same as, those of AT. This makes it quite different from other X-ray sensitive cells that have been isolated in other laboratories. PMID- 1728407 TI - A quantitative analysis of tumor specific monoclonal antibody uptake by human melanoma xenografts: effects of antibody immunological properties and tumor antigen expression levels. AB - The time-dependent (5 min-72 h) localization of 3 radiolabeled anti-melanoma monoclonal antibodies (MAbs 436, IND1, and 9.2.27) was studied in paired label experiments in small (4-12 mg) s.c. human melanoma xenografts (SK-MEL-2 and M21) in athymic nude mice. MAb 436 recognizes a Mr 125,000 cell surface melanoma associated glycoprotein antigen (125 kDa-MAA); MAbs IND1 and 9.2.27 recognize a high molecular weight melanoma-associated antigen, but with equilibrium association constants differing by 2 orders of magnitude (10(8)-10(10) M-1). The two tumors were found to differ in their antigen expression levels and in both interstitial and vascular volumes. Accumulation of MAbs in both tumors was determined primarily by antigen expression levels and also by physiological factors such as vascular permeability and vascular volume; at the dose administered (20 micrograms/mouse), differences in MAb affinity among specific MAbs had minimal effect on accumulation. Quantitative flow cytometry measurements showed that antigen expression in vivo differed from that of cultured tumor cells. In vivo, expression of the Mr 125,000 MAA decreased by a factor of about 2.5 in both tumors. In contrast, the in vivo expression of the high molecular weight MAA decreased in M21 tumors but increased by 2.0-3.5-fold in SK-MEL-2 tumors. Data were analyzed using a three-compartment pharmacokinetic model (C. Sung et al., Cancer Res., 52:377-384, 1992) to provide plasma-to-tissue transport constants (k), the interstitial fluid flow rate (L), and estimates of the in vivo interstitial MAb binding site concentration (B0). For all MAbs, the plasma-to tissue transport constants were consistently greater for M21 tumors (0.44-0.85 microliter/min/g) than for SK-MEL-2 tumors (0.28-0.66 microliter/min/g), and values of k for both tumors were approximately 1 order of magnitude greater than those for skeletal muscle (0.06-0.08 microliter/min/g). The model-estimated binding site concentration of melanoma-specific antibodies was 15-70 times lower than that predicted by experimental measurements of tumor antigen concentrations. Factors that may contribute to this discrepancy include inaccessibility of tumor cell binding sites to MAb and MAb catabolism. In summary, these results indicate that, for the MAb dose used in this study, variables pertaining to the tumor target (i.e., antigen expression levels, vascular volume, and vascular permeability) are the most important for determining MAb accumulation in tumors. PMID- 1728408 TI - Spatial distribution of tumor-specific monoclonal antibodies in human melanoma xenografts. AB - The time-dependent (1-72-h) spatial distribution of three biotinylated anti melanoma monoclonal antibodies (MAbs), a control MAb, and several macromolecular tracers was studied in two small (4-12-mg), well-characterized human melanoma xenografts (SK-MEL-2, M21) growing in the s.c. space of athymic nude mice. The specific MAbs (436, IND1, and 9.2.27) recognize two different melanoma cell surface antigens (Mr 125,000 glycoprotein melanoma-associated antigen and high molecular weight melanoma-associated antigen) and have equilibrium association constants differing by two orders of magnitude (10(8)-10(10) M-1). SK-MEL-2 tumors were poorly vascularized and were composed of one or several collections of tumor cells with few intratumor blood vessels. In contrast, M21 tumors induced a strong angiogenic response and were organized into multiple small tumor cell nests separated from each other by fine blood vessels. Neither tumor developed extensive connective tissue stroma. In both tumors, hyperpermeable blood vessels were concentrated at the tumor-host interface but some intratumor vessels in M21 tumors were also leaky. Macromolecular tracers extravasated extensively from leaky vessels into tumor stroma but penetrated poorly into tumor parenchyma. All three tumor-specific MAbs stained tumor cell surfaces in a time-dependent fashion such that one-half or more of all tumor cells were stained by 24-48 h. Tumor cell staining was favored by increased density of tumor cell antigens but, at the doses studied, was little affected by differences in affinity among tumor specific antibodies. The distribution of MAb staining was nonuniform in two respects: (a) peripherally situated tumor cells were more likely to be stained than centrally placed cells, and only in the smallest tumors did MAb reach centrally placed tumor cells; and (b) staining was nonuniform in different parts of the same tumor. The inhomogeneity of tumor cell staining by tumor-specific MAb was attributable to several factors, including: tumor blood vessel number, distribution, perfusion and permeability; distribution of tumor connective tissue stroma; small volume of the parenchymal interstitial space and relatively impaired diffusion of macromolecules in that space (low effective diffusivity of MAb); and interactions between specific MAbs and tumor cells. Of these factors, those associated with the parenchymal compartment apparently were rate limiting, and strategies that enhance parenchymal penetration are likely to improve solid tumor therapy with MAbs. PMID- 1728409 TI - Predicted and observed effects of antibody affinity and antigen density on monoclonal antibody uptake in solid tumors. AB - The uptake and binding of monoclonal antibodies (MAbs) in solid tumors after a bolus i.v. injection are described using a compartmental pharmacokinetic model. The model assumes that MAb permeates into tumor unidirectionally from plasma across capillaries and clears from tumor by interstitial fluid flow and that interstitial antibody-antigen interactions are characterized by the Langmuir isotherm for reversible, saturable binding. Typical values for plasma clearance and tumor capillary permeability of a MAb and for interstitial fluid flow and interstitial volume fraction of a solid tumor were used to simulate the uptake of MAbs at various values of the binding affinity or antigen density for a range of MAb doses. The model indicates that at low doses, an increase in binding affinity may lead to an increase in MAb uptake. On the other hand, at doses approaching saturation of antigen or when uptake is permeation limited, an increase in the binding affinity from moderate to high affinity will have only a small effect on increasing MAb uptake. The model also predicts that an increase in antigen density will greatly increase MAb uptake when uptake is not permeation limited. Our experiments on MAb uptake in melanoma tumors in athymic mice after injection of 20 micrograms MAb (initial plasma concentration, about 120 nM) are consistent with these model-based conclusions. Two MAbs differing in affinity by more than 2 orders of magnitude (3.8 x 10(8) M-1 and 5 x 10(10) M-1) but with similar in vivo antigen densities in M21 melanoma attained similar concentrations in the tumor. Two MAbs of similar affinity but having a 3-fold difference in in vivo antigen density in SK-MEL-2 melanoma showed that the MAb targeted to the more highly expressed antigen attained a higher MAb concentration. We also discuss the model predictions in relation to other experiments reported in the literature. The theoretical and experimental findings suggest that, for high dose applications, efforts to increase MAb uptake in a tumor should emphasize the identification of an abundantly expressed antigen on tumor cells more than the selection of a very high affinity MAb. PMID- 1728410 TI - Eosinophils, tissue eosinophilia, and eosinophil-derived transforming growth factor alpha in hamster oral carcinogenesis. AB - Eosinophilia in tissues and/or circulating blood is known to be associated with a wide variety of malignancies but the role of the eosinophil in neoplastic conditions is not known. Using the cheek pouch of the Syrian hamster as an experimental model for oral carcinogenesis, it has recently been shown that eosinophils at sites of developing oral cancer express the multifunctional cytokine, transforming growth factor alpha (TGF-alpha). This study investigated the time course of eosinophil infiltration, tissue eosinophilia associated with malignant epithelium, and eosinophil-derived TGF-alpha mRNA during the 16-week 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer development process. The results reveal that the occasional eosinophil is normally present in the lamina propria of hamster oral mucosa. With progressive DMBA treatments, there is an increase of eosinophils infiltrating into the lamina propria. By weeks 12-16, the number of eosinophils is significantly higher in DMBA-treated pouches than in control pouches treated with the vehicle mineral oil alone. Analysis of the infiltrating eosinophils into fully developed hamster oral carcinomas reveals that tissue eosinophilia is associated with 78% of the stromal areas associated with malignant epithelium, while only 7% of sites associated with non-tumor oral epithelium (normal, hyperplastic-dysplastic) exhibited eosinophilia. Furthermore, the majority of the eosinophils associated with malignant epithelium were found to contain TGF-alpha mRNA. The number of TGF-alpha mRNAs containing eosinophils associated with malignant oral epithelium is significantly higher than that associated with nonmalignant oral epithelium. Together, these results suggest that eosinophils are recruited to tumor-developing sites, that they predominantly associate with malignant epithelium, and that most tumor-associated eosinophils express the cytokine TGF-alpha. PMID- 1728411 TI - Human xenograft-nude mouse model of adoptive immunotherapy with human melanoma specific cytotoxic T-cells. AB - We investigated the efficacy of human melanoma-specific cytotoxic T-cells (CTLs) in treating experimental human melanoma metastases in a nude mouse model of adoptive immunotherapy. Hepatic metastases were generated by the intrasplenic injection of 1.5 x 10(6) human melanoma cells. Animals were then randomized to receive saline, interleukin-2 only, or CTLs and interleukin-2. CTLs were effective when administered 3 or 7 days after generation of hepatic metastases, with 96 and 88% of animals disease-free, respectively, when examined at one month. Interleukin-2 alone was not effective. In addition, CTLs were effective when as few as 2.5 x 10(6) T-cells were adoptively transferred. Only 33% of the animals were tumor-free when CTLs were administered on day 10, and CTLs were not effective when given at day 14. Human CTLs that were not cytotoxic for the tumor line used in vivo, when tested in a 51Cr assay, were also not effective in the model of immunotherapy. This suggests that the tumor-specific CTLs maintain their specificity in vivo, and eliminates a nonspecific inflammation directed against the human CTLs as a possible cause of the antitumor effect. These studies lay the foundation for clinical trials of CTLs in the adoptive immunotherapy of patients with metastatic melanoma. PMID- 1728412 TI - Protection from 1-beta-D-arabinofuranosylcytosine-induced alopecia by epidermal growth factor and fibroblast growth factor in the rat model. AB - The present study was designed to examine the effect of epidermal growth factor (EGF) and fibroblast growth factor (FGF) on 1-beta-D-arabinofuranosylcytosine (ARA-C)- and cyclophosphamide-induced alopecia in the young rat model. Seven-day old rats were given ARA-C with or without human or murine EGF daily for 7 days. Alopecia was scored on the 12th day of the experiment. Both human and murine EGF protected rats from ARA-C-induced alopecia. The topical application of murine EFG in dimethyl sulfoxide offered significant protection limited to the treated area. In other experiments the administration of acidic FGF (aFGF) with ARA-C resulted in protection from alopecia limited to the site of FGF injection. Neither EGF nor FGF had any influence on alopecia from cyclophosphamide. It is concluded that both EGF and FGF are effective in protecting against ARA-C-induced alopecia in the rat model. PMID- 1728413 TI - Glycosphingolipids of various human ovarian tumors: a significantly high expression of I3SO3GalCer and Lewis antigen in mucinous cystadenocarcinoma. AB - Among several human ovarian tumors, which include mucinous cystadenocarcinoma, serous cystadenocarcinoma, and clear cell adenocarcinoma, the mucinous cystadenocarcinoma showed a unique glycosphingolipid composition. In particular, more than 90% of the acidic glycosphingolipids in the mucinous cystadenocarcinoma is comprised of sulfolipids, which are hardly detected in normal ovary and are contained in concentrations of less than 40% in the other type of ovarian tumors. By means of negative ion fast atom bombardment mass spectrometry and gas liquid chromatography, the major sulfolipid in mucinous cystadenocarcinoma is confirmed to be I3SO3-GalCer with N-cerebronoyl phytosphingosine, that which contrasts with I3SO3-GalCer with N-nonhydroxy fatty acyl sphingosine as the major molecular species in the other ovarian cancers. In mucinous cystadenocarcinoma, galactosylceramide is found in the relatively high concentration and is also composed of N-cerebronoyl phytosphingosine. In addition, the concentrations of glycolipids with Le(a) and Le(b) antigenicities are significantly higher in mucinous cystadenocarcinoma than those in normal ovary and the other ovarian tumors. PMID- 1728414 TI - Detection of nuclear matrix proteins in serum from cancer patients. AB - Morphological characteristics of the cell nucleus have long been used by pathologists in the clinical diagnosis of cancer. The nuclear matrix of the cell, the structure that serves to organize the chromatin within the nucleus, is known to reflect these morphological characteristics with regard to cell and cancer type. Monoclonal antibodies were developed to extracted nuclear matrix proteins. These antibodies were used in two-site immunometric assays to detect soluble nuclear matrix proteins in the supernatants of two dying cell lines and 15 tumor tissues. Furthermore, nuclear matrix proteins were detected at elevated levels in the sera of cancer patients compared with normal patient sera. In one assay, 63.2% of cancer sera read above 95% of normal sera, and in another assay, 73.7% of cancer sera read above 95% of the normal sera. With the development of monoclonal antibodies with greater cancer specificity, the detection of circulating nuclear matrix proteins may become an important clinical tool in the diagnosis and monitoring of cancer. PMID- 1728415 TI - Increased growth of NIH/3T3 cells by transfection with human p120 complementary DNA and inhibition by a p120 antisense construct. AB - The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody in most human malignant tumors but not in most resting human tissues (J. W. Freeman et al., Cancer Res., 48: 1244-1251, 1988) and has been used as a prognostic tumor marker in breast cancer patients (J. W. Freeman et al., Cancer Res., 51: 1973-1978, 1991). After the complementary DNA and gene for the human p120 protein were isolated and sequenced (review: H. Busch, Cancer Res., 50: 4830 4838, 1990), constructs were prepared to study the expression of the sense p120 and its antisense, p021 message. NIH/3T3 cells were transfected by electroporation with pSVX plasmids containing either the p120 complementary DNA (pSVX120) or the antisense, p021 DNA (pSVX021), and clones containing these constructs were selected. The expression of p120 or p021 in these constructs was regulated by Moloney murine leukemia virus long terminal repeats. In pSVX120 transfected NIH/3T3 cells, the expressed human p120 protein was localized to the nucleoli as shown by anti-p120 monoclonal antibody immunofluorescence. Expression of the p120 message and protein was confirmed by Northern (mRNA) and Western (protein) blots. Transfection of the p120 complementary DNA in sense orientation caused malignant transformation of NIH/3T3 cells in vitro and produced rapidly growing tumors in nude mice. Transfection of the antisense p120 constructs markedly delayed the growth of these tumors in vitro and in vivo (L. Perlaky et al., Proc. Am. Assoc. Cancer Res., 32: 1682, 1991). When transformed 3T3/pSVX120 cells were transfected with an inducible antisense p120 construct (pMSG021), dexamethasone induction decreased the growth rate by 62%, and the cell line returned to its normal phenotype. Northern blot analysis showed a decreased level of p120 mRNA, and the immunofluorescence was also markedly reduced. PMID- 1728416 TI - Prolonged decrease of serum calcium concentration by murine gamma-interferon in hypercalcemic, human tumor (EC-GI)-bearing nude mice. AB - Murine gamma-interferon (MuIFN-gamma) is a potent inhibitor of bone resorption induced by interleukin 1 and parathyroid hormone-related protein in vitro. To investigate whether MuIFN-gamma is also effective in vivo, the cytokine was injected s.c. into hypercalcemic, tumor (EC-GI)-bearing nude mice, in which parathyroid hormone-related protein and interleukin 1 alpha are synergistically responsible for causing humoral hypercalcemia. When MuIFN-gamma was injected s.c. at a dose of 1 to 20 x 10(4) units for 5 days consecutively, serum calcium concentrations in the tumor-bearing mice decreased in a dose-dependent manner. The minimal effective dose was 5 x 10(4) units/mouse. Unlike calcitonin, which decreased the serum calcium concentration for only 1 to 2 days despite continuous daily injections, MuIFN-gamma decreased it for more than 7 days even after the injections had been stopped. Human gamma-interferon was completely ineffective. The decrease in serum calcium concentration was accompanied by a decrease in urinary calcium excretion. Histological examination of the femur revealed a decreased number of osteoclasts in the MuIFN-gamma-treated mice. Furthermore, MuIFN-gamma, when injected into nude mice or normal mice at a dose of 15 x 10(4) units for 3 days, almost completely abolished the formation of multinucleated osteoclast-like cells in vitro. These findings suggest that MuIFN-gamma suppresses the formation and maturation of osteoclasts and inhibits osteoclastic bone resorption, resulting in the prolonged decrease of serum calcium concentration seen in hypercalcemic, tumor-bearing nude mice. Therefore, bone resorption inhibitors like MuIFN-gamma, which ameliorate humoral hypercalcemia without an escape phenomenon, are potentially useful for the treatment of malignancy-associated hypercalcemia. PMID- 1728417 TI - Neutrophil chemotactic factors produced by a cell line from thyroid carcinoma. AB - A neutrophil chemotactic factor (human interleukin 8, human granulocyte macrophage colony-stimulating factor)-producing cell line, named KHM-5M, was established from a patient with an undifferentiated thyroid carcinoma, neutrophilia, and malignant pleurisy with many neutrophils and a few malignant cells. The cell line was transplanted into nude rats, and the infiltration of neutrophils was observed in and around the transplanted tumor tissue. Neutrophil chemotactic activity was predicted from the clinical features and pathological findings in this case. The extreme chemotactic activity of the neutrophils was demonstrated in conditioned medium from KHM-5M cells using the modified Boyden chamber technique. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least two neutrophil chemotactic activities in conditioned medium from the cell line were observed. The levels of these activities derived from KHM-5M cells were screened by measuring conditioned medium from the COS cells, which expressed a complementary DNA library from the KHM-5M cells. Chemotactic activities (human interleukin 8, human granulocyte-macrophage colony stimulating factor) were identified by DNA cloning. These results show that the KHM-5M cells derived from an undifferentiated thyroid carcinoma produce multicytokines and suggest that those cytokines modified some pathological features in this case. PMID- 1728418 TI - Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. AB - Staurosporine, a microbial alkaloid, is a strong inhibitor of protein kinases. The effects of staurosporine on the cell cycle progression and nuclear morphology of normal human lymphocytes stimulated to proliferate by phytohemagglutinin were studied and compared with the effects of this drug on human lymphocytic leukemic MOLT-4 cells. Exposure of normal lymphocytes to either 5-10 or 50-100 ng/ml of staurosporine resulted in the preferential accumulation of cells in G1 or G1 and G2 phases of the cell cycle, respectively. In contrast, regardless of the concentration (5-100 ng/ml), staurosporine arrested MOLT-4 cells initially in G2; these cells then initiated additional rounds of DNA replication, without division. Staurosporine (5-100 ng/ml) induced severe changes in the nuclear morphology of MOLT-4 cells, manifested as nuclear elongation, deep invaginations of the nuclear membrane, extensive fragmentation, and micronucleation. At concentrations of 5-10 ng/ml, staurosporine had no apparent effect on the nuclear morphology of normal lymphocytes and at 50-100 ng/ml it produced minor changes in the nuclear shapes of these cells. The data indicate that the kinase(s) involved in the regulation of cell exit from G1 and G2, respectively, in normal and leukemic lymphocytes may have different sensitivities to staurosporine, which suggests that the mechanisms controlling exit from G1 in these cells may be different. In MOLT-4 cells the staurosporine-sensitive kinase(s) appear to also be involved in phosphorylation of nuclear constituents essential for organization of gross chromatin structure. The different response of normal versus leukemic lymphocytes to staurosporine, if confirmed on clinical material, opens new strategies of tumor treatment. PMID- 1728419 TI - Rhabdomyosarcoma spheroids with central proliferation and differentiation. AB - A novel type of multicellular spheroids was established and characterized with regard to growth behavior, proliferation, and differentiation. The spheroids were grown from clonal rat rhabdomyosarcoma cells using the spinner flask technique. The cell aggregates showed several unique properties that were different from those observed in most of the spheroids investigated to date. These properties include a non-Gompertzian volume growth; the coexistence of undifferentiated mononuclear cells and of differentiated, myotube-like giant cells with numerous nuclei; a relatively homogeneous intraspheroidal distribution of proliferating mononuclear cells with thymidine labeling even in the center of spheroids greater than 1 mm; the absence of necrosis in such large spheroids, and the accumulation of myotube-like cells in the center of these spheroids instead. There was a decrease in the overall proliferative activity of the mononuclear cells with increasing proportion of giant cells in the rhabdomyosarcoma spheroids. PMID- 1728420 TI - Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. AB - It is fairly well accepted that the presence of estrogen receptor (ER) and progesterone receptor (PgR) identifies breast cancer patients with a lower risk of relapse and better overall survival. But patients with discordant receptors, the ER+/PgR- phenotype, are often intermediate in clinical response. We focused upon this group of patients and have identified a truncated ER which is abundant in some ER+/PgR- breast tumors and which inhibits the binding of wild-type ER to its cognate response element. This variant interferes in a dominant negative manner with wild-type ER function and may represent a mechanism for modulation of estrogen responsiveness. PMID- 1728421 TI - Interstitial fluid pressure in solid tumors following hyperthermia: possible correlation with therapeutic response. AB - Elevated interstitial fluid pressure (IFP) of tumors may be a physiological barrier to the delivery of certain therapeutic agents. The objective of this study was to find out if IFP could be lowered using localized hyperthermia and if the reduction in IFP could predict the tumor response to treatment. Amelanotic melanoma (A-Mel-3) implanted into the dorsal skin of Syrian golden hamsters was exposed to hyperthermic treatment after 7 days of tumor growth at tumor volumes of about 100-150 mm3. Hyperthermia was induced by immersing the tumor in a water bath at 43 degrees C for 30 or 60 min. Forty-eight h later the IFP of control and treated tumors was determined by using the wick-in-needle technique. The mean IFP in control tumors was 12.6 mmHg. Hyperthermic treatment for 30 min induced a significant decrease to 2.8 mmHg (P less than 0.001 versus controls), whereas a 60-min immersion of the tumors induced a further decrease to 0.8 mmHg (P less than 0.05 versus 43 degrees C for 30 min). Separate experiments on tumor growth in corresponding groups of animals revealed a significant growth delay of 2.7 days after hyperthermia for 30 min. Enhanced growth delay and partial tumor response in 66% of the tumors were found following 60 min of hyperthermia at 43 degrees C. The thermal dose-dependent decrease in IFP presumably results from the dose-dependent damage to the tumor vasculature. In addition, the association of an enhanced biological effect with a more pronounced reduction of interstitial fluid pressure suggests that the IFP might serve as a quantitative parameter to predict the response of tumors to hyperthermic therapy. PMID- 1728422 TI - Involvement of BCL-2 in glucocorticoid-induced apoptosis of human pre-B leukemias. AB - To determine the role of BCL-2 in the glucocorticoid-induced apoptosis of lymphocytes, we analyzed the effect of glucocorticoid on two human pre-B-cell lines which express different levels of BCL-2. Glucocorticoid treatment of the 380 cell line which expresses high levels of BCL-2 resulted in inhibition of cellular proliferation without induction of apoptosis. On the other hand, glucocorticoid treatment of the 697 cell line which expresses lower levels of the BCL-2 resulted in both inhibition of cellular proliferation and apoptosis with characteristic internucleosomal DNA cleavage. The glucocorticoid-induced inhibition of cellular proliferation in both cell lines was also associated with repression of the c-myc mRNA expression. Taken together, our data suggest that BCL-2 blocks the glucocorticoid-induced apoptosis of the 380 pre-B-lymphocytes by extending their survival when the level of c-myc expression is repressed. Also by repressing the expression of c-myc, glucocorticoid causes apoptosis of the 697 pre-B-lymphocytes in the absence of high level of BCL-2 expression. PMID- 1728423 TI - Nonpharmacologic therapy of ventricular arrhythmias. AB - Since the first attempt at surgical therapy for VT, much progress has been made in developing techniques for both localization of arrhythmogenic regions as well as their removal or alteration. In the setting of previous myocardial infarction, surgery for VT has evolved from an experiment/last resort procedure to the treatment of choice in many cases, yielding complete freedom from arrhythmia recurrence without adjunctive antiarrhythmic drugs in up to three quarters of operative survivors. Remaining issues in this field include (1) further reduction of operative mortality, perhaps by more careful patient selection (and use of alternative forms of therapy in those judged to be too high risk); (2) better and more accurate mapping techniques to enhance the antiarrhythmic efficacy of surgery; and (3) development of more effective procedures to deal with VT in the setting of cardiomyopathy. Judging from the progress made in the last decade and a half in this field, surgery for VT in many pathologic settings may take on a greater role in the future as further refinements in techniques are realized. PMID- 1728424 TI - Early postmyocardial infarction ventricular arrhythmias. PMID- 1728425 TI - Asymptomatic ventricular ectopic activity and chronic ischemic heart disease. PMID- 1728426 TI - Mechanisms of arrhythmias in chronic ischemic heart disease. PMID- 1728428 TI - Post open heart surgery ventricular arrhythmias. PMID- 1728427 TI - Chronic ischemic heart disease: symptomatic ventricular arrhythmias. PMID- 1728429 TI - Ventricular arrhythmias in patients with congenital heart disease. PMID- 1728430 TI - The cardiomyopathies: mortality, sudden death, and ventricular arrhythmias. PMID- 1728431 TI - Mechanisms of ventricular arrhythmias in acute ischemia and reperfusion. AB - Coronary occlusion leading to nearly total absence of myocardial perfusion is the major cause of lethal ischemic arrhythmia in humans. In this setting, intracellular acidosis rapidly develops and leads to accelerated K+ efflux from the myocyte. Other metabolites, including lipid amphiphiles such as LPC, also rapidly accumulate in the ischemic zone. Elevated extracellular K+ and LPC cause membrane depolarization, which leads to slow conduction and increased refractoriness. These electrophysiologic changes contribute to the development of re-entrant rhythms, which predominate during early ischemia (phase 1a). Diffusion of extracellular K+ from the ischemic zone and release of endogenous catecholamines result in improvement in electrophysiologic parameters and are associated with a short arrhythmia-free interval, which occurs approximately 10 minutes after coronary occlusion. A second phase of arrhythmia (1b) then occurs and may be due in part to catecholamine-mediated triggered activity. Irreversible cell injury occurs 15 to 20 minutes after coronary occlusion and is associated with cell Ca++ overload, loss of gap junctions, and impaired cell coupling. This may lead to re-entrant arrhythmias. Reperfusion of ischemic myocardium leads to arrhythmia predominantly mediated by non re-entrant mechanisms. In humans, these reperfusion arrhythmias are usually relatively benign. PMID- 1728432 TI - Contemporary management of ventricular arrhythmias. PMID- 1728433 TI - Ventricular arrhythmias in patients with mitral valve prolapse. PMID- 1728434 TI - Drug-induced ventricular proarrhythmia. PMID- 1728435 TI - Unusual forms of ventricular arrhythmias: arrhythmogenic right ventricular dysplasia and repetitive monomorphic ventricular tachycardia. PMID- 1728436 TI - Signal-averaged electrocardiography and ambulatory monitoring. PMID- 1728437 TI - Programmed electrical stimulation of the heart in patients with ventricular tachyarrhythmias. PMID- 1728438 TI - Blood pressure control by the renin-angiotensin system in normotensive subjects. Assessment by angiotensin converting enzyme and renin inhibition. AB - BACKGROUND: The participation of the renin-angiotensin system in the control of blood pressure in normal, sodium-replete subjects is not clear. The use of a specific inhibitor of human renin should allow a better delineation of the importance of this system. METHODS AND RESULTS: Blood pressure responses were measured 1 hour after randomized, double-blind administration of the renin inhibitor Ro 42-5892 (600 mg p.o.) or the angiotensin converting enzyme inhibitor captopril (50 mg p.o.) in 20 healthy men on an ad libitum sodium diet. Effective inhibition of the renin-angiotensin system by either compound was indicated by increases of immunoreactive renin associated with an increase of angiotensin I production rate of 67.8 +/- 33.6% after captopril and a decrease of 79.5 +/- 16.4% after Ro 42-5892. Furthermore, Ro 42-5892 decreased plasma renin activity by 64%. Whereas intra-arterial diastolic (60 +/- 5.1 to 51.4 +/- 7.2 mm Hg, p less than 0.01) and mean arterial (77.7 +/- 6.0 to 71.4 +/- 8.5 mm Hg, p less than 0.001) pressures decreased after captopril, they remained unchanged after Ro 42-5892. Captopril, but not Ro 42-5892, increased forearm blood flow (2.4 +/- 0.8 versus 1.9 +/- 0.8 ml/min/100 ml, p less than 0.01) and significantly enhanced the increase of forearm blood flow to brachial artery infusions of bradykinin (0.15, 1.5, 5, 15, and 50 ng/min/100 ml; 5 minutes each) from 744 +/- 632% to 1,383 +/- 514% (p less than 0.01). Furthermore, repeat bradykinin infusions resulted in further decreases of blood pressure (from mean pressure of 71.4 +/- 8.5 to 63.2 +/- 7.6 mm Hg, p less than 0.01) only after captopril. Changes of blood pressure after captopril were unrelated to baseline plasma renin activity but correlated with captopril-induced enhancement of vasodilation to bradykinin (r = 0.68, p less than 0.05). CONCLUSIONS: The lack of blood pressure effects of renin inhibition in contrast to angiotensin converting enzyme inhibition suggests that the renin-angiotensin system does not contribute significantly to blood pressure control in normotensive, sodium-replete subjects. The hypotensive activity of angiotensin converting enzyme inhibitors may result from additional hormonal effects, for example, inhibition of bradykinin degradation and/or subsequent increases of vasodilating prostaglandins or endothelium-derived relaxing factor(s). PMID- 1728439 TI - Percutaneous transluminal coronary angioplasty of chronic total occlusions. Primary success, restenosis, and long-term clinical follow-up. AB - BACKGROUND: Angioplasty of chronically totally occluded vessels has been associated with a success rate well below and restenosis rate well above that for angioplasty of stenosed segments. However, long-term clinical outcome after successful revascularization of a chronically totally occluded vessel has not been reported in detail. METHODS AND RESULTS: Accordingly, data for 480 patients undergoing angioplasty for chronic total occlusion at Emory University Hospital, Atlanta, Ga., from 1980 to 1988 were analyzed for predictors of in-hospital procedural and clinical (procedural success and absence of in-hospital complications) success, restenosis, and 4-year clinical follow-up. The study population was grouped by procedural and clinical success and failure. The groups were then compared for outcome, both in hospital and long term. The initial clinical success rate was 66% (317 of 480 patients). Independent correlates of failure were the number of vessels diseased (p less than 0.001), vessel location of the lesion (p = 0.016), and absence of any distal antegrade filling (p = 0.002). Follow-up data revealed 98% cardiac survival and 96% overall survival at 4 years for the group as a whole. Freedom from myocardial infarction or cardiac death was significantly greater in patients with clinical success (93%) than with clinical failure (89%, p = 0.0044). In the successful group, 87% were free from coronary surgery after 4 years compared with 64% in the failure group (p less than 0.0001). Two thirds of the patients were free of angina at last follow-up. The presence of angina at follow-up was the same for patients successfully treated and for those with failed angioplasty, which may be related to the frequent use of coronary surgery in the failure group. CONCLUSIONS: In well selected cases, the success rate for angioplasty of chronic total occlusion is acceptable. Furthermore, long-term clinical benefit is suggested by the high freedom from coronary surgery, myocardial infarction, and death in the patients who underwent successful revascularization. PMID- 1728440 TI - Outcome and assessment after the modified Fontan procedure for hypoplastic left heart syndrome. AB - BACKGROUND: We reviewed the outcome of 76 consecutive patients (age range, 5 months to 6 years; median age, 19 months) who underwent a modified Fontan procedure after initial palliative surgery for hypoplastic left heart syndrome (HLHS) between January 1984 and December 1989. METHODS AND RESULTS: Modifications of the Fontan procedure included transatrial baffle of pulmonary venous return to the tricuspid valve (n = 10) or inferior vena cava baffle within the right atrium to the superior vena caval-pulmonary artery anastomosis, with pulmonary artery augmentation (n = 66). Actuarial survival rates were 74% (1 month), 58% (12 months), 56% (2 years), and 52% (4 years). Of the 43 survivors, 25 patients have returned for postoperative cardiac catheterization at a medium of 13 months after the Fontan procedure. Mean +/- SD hemodynamic values were cardiac index, 2.8 +/- 0.6 l/min/m2; right arterial pressure, 11 +/- 2 mm Hg; pulmonary artery wedge pressure, 6 +/- 3 mm Hg; and arterial oxygen saturation, 94 +/- 3%. No patient had significant tricuspid or native pulmonary valve insufficiency. CONCLUSIONS: Survival after the Fontan procedure in patients with HLHS is comparable to survival after a Fontan procedure in patients with other complex congenital heart lesions. In the subgroup of patients with HLHS who survived both reconstructive surgery and a Fontan procedure and have been evaluated by cardiac catheterization after a Fontan procedure, the use of the right ventricle as the systemic ventricle yielded excellent intermediate results for Fontan physiology. PMID- 1728441 TI - Double-transseptal, double-balloon valvuloplasty for congenital mitral stenosis. AB - BACKGROUND: Eight patients with severe congenital mitral stenosis underwent double transseptal, double-balloon valvuloplasty; two had isolated congenital mitral stenosis, six had additional cardiac defects, and one had previous surgical valvotomy. Ages ranged from 0.6 to 36 years (median, 9 years). METHODS AND RESULTS: All procedures were tolerated well. After valvuloplasty, the left atrial a wave minus the left ventricular end-diastolic pressure (LVEDP) gradient was reduced from 25 +/- 6 mm Hg to 9 +/- 3 mm Hg (p less than 0.001), the mitral valve mean gradient was reduced from 18 +/- 7 mm Hg to 8 +/- 3 mm Hg (p = 0.003), and the LVEDP was unchanged. All patients had marked clinical improvement. Only one patient developed significant mitral regurgitation. Two of the first four patients underwent repeat balloon valvuloplasty 7 months later. Follow-up evaluation on six patients from 4 to 54 months revealed no recurrence of symptoms or increased mitral regurgitation. CONCLUSIONS: Double transseptal, double balloon valvuloplasty is an effective treatment for many forms of congenital mitral stenosis. Mitral regurgitation is uncommon after this procedure. The double transseptal approach results in less trauma to the atrial septum and femoral veins and allows easy assessment of any residual postvalvuloplasty gradient. PMID- 1728442 TI - Two-dimensional echocardiographic phase analysis. Its potential for noninvasive localization of accessory pathways in patients with Wolff-Parkinson-White syndrome. AB - BACKGROUND: In patients with the preexcitation syndrome who are undergoing transcatheter or surgical ablation, accurate localization of accessory pathways is critical. Because preexcitation is known to alter ventricular activation sequence and result in focal areas with presystolic contraction, we investigated whether phase analysis applied to two-dimensional echocardiographic cine loops objectively identifies these focal areas and can be used to localize ventricular insertion sites of accessory pathways. METHODS AND RESULTS: We prospectively obtained phase images in 17 patients (11 males; age range, 11-35 years) during minimal preexcitation in normal sinus rhythm and during maximal preexcitation induced by right atrial pacing. A group of 11 normal subjects (six men; age range, 26-37 years) served as controls. Pathway locations predicted from phase imaging were compared with those predicted from routine 12-lead ECGs, from visual inspection of cine loop images, and from catheter-mounted electrode endocardial mapping. Cross-sectional views in a digital cine loop format were mathematically transformed using a first harmonic Fourier algorithm to obtain the corresponding phase images. Phase angle histograms were derived in eight wall segments. Mean and earliest phase angles were derived by computer analysis to quantitate contraction sequence. We found that during right atrial pacing, phase angles in focal areas markedly deviated from normal--mean phase angles from 33 degrees to 164 degrees, and earliest phase angles from 50 degrees to 180 degrees. Accessory pathways could be precisely localized in 53% of the patients by 12-lead ECG, in 59% by visual inspection of cine loop images, in 82% by phase imaging, and in 94% by a combination of the three methods. CONCLUSIONS: Our results suggest that phase imaging, especially when used in combination with cine loop and 12-lead ECG, can be used to localize ventricular insertion sites of accessory pathways and may be clinically useful as a noninvasive adjunct to endocardial mapping in patients with Wolff-Parkinson-White syndrome. PMID- 1728443 TI - Preventive administration of intravenous N-acetylcysteine and development of tolerance to isosorbide dinitrate in patients with angina pectoris. AB - BACKGROUND: Development of tolerance to organic nitrates may be related to depletion of sulfhydryl groups in vascular smooth muscle. N-Acetylcysteine (NAC), a sulfhydryl donor, has been reported to potentiate the effect of nitroglycerin and reverse tolerance in humans. However, its ability to prevent or delay the development of nitrate tolerance in patients with angina pectoris has not been established. METHODS AND RESULTS: Ten patients with stable angina pectoris were treated with intravenous isosorbide dinitrate (ISDN; 5 mg/hr) combined with NAC (2 g i.v. over 15 minutes followed by 5 mg/kg/hr) or matching placebo for 30 hours in a double-blind, randomized, crossover study with a washout interval of 8 days. Bicycle exercise tests were performed before and at 1 1/2, 8, 20, 24, and 30 hours after start of treatment. After 24 hours of infusion, exercise parameters were not significantly different from pretreatment values (p greater than 0.05) during ISDN plus placebo, indicating development of tolerance to ISDN. In contrast, time to onset of angina, time to 1-mm ST segment depression, and total amount of ST segment depression were still significantly improved after 24 hour infusion of ISDN plus NAC (p less than 0.05). In addition, compared with placebo, a significant difference (p less than 0.05) in favor of NAC was observed regarding time to angina (507 +/- 63 versus 445 +/- 69 seconds, mean +/- SEM), time to 1-mm ST segment depression (435 +/- 43 versus 407 +/- 45 seconds), and total ST segment depression (1.8 +/- 0.9 versus 3.1 +/- 0.4 mm). CONCLUSIONS: These results suggest that infusion of high doses of NAC in combination with ISDN for 30 hours affects and partially prevents the development of tolerance to antianginal effects normally observed during infusion with ISDN. PMID- 1728444 TI - Thrombolysis in unstable angina. Randomized double-blind trial of t-PA and placebo. AB - BACKGROUND: Because coronary thrombosis is important in the pathogenesis of unstable angina and correlates with in-hospital cardiac events, we hypothesized that thrombolytic therapy would decrease cardiac events. METHODS AND RESULTS: We randomized 70 patients with unstable angina to tissue-type plasminogen activator (t-PA) (0.49 MU/kg for 1 hour followed by 0.07 MU/kg per hour for 9 hours) or placebo. All patients received full doses of intravenous heparin for 96 hours and aspirin (325 mg beginning at 72 hours). The primary end points of the study were in-hospital death, myocardial infarction, and urgent revascularization. Three secondary end points were also evaluated. Myocardial perfusion was assessed with resting planar thallium scintigraphy 90 minutes after initiation of therapy. Silent ischemia was assessed with 48-hour Holter monitoring for ST shift beginning at time of initiation of drug therapy. Coronary angiography was performed at 18 +/- 6 hours and analyzed quantitatively to assess the stenosis responsible for unstable angina, the presence of intraluminal filling defects consistent with intracoronary thrombus, and stenosis morphology and severity. There was no difference in total in-hospital cardiac events between patients receiving t-PA (5% or 14%) and those receiving placebo (7% or 20%) (p = 0.83). Resting thallium defects were larger in the patients receiving t-PA than in those receiving placebo (130 +/- 118 versus 76 +/- 84 degrees, p less than 0.04), and this difference persisted when corrected for previous infarction. Although the numbers of patients with ST shift were similar, the duration of ST shift was significantly longer in the patients receiving t-PA than with placebo (20 +/- 46 versus 3 +/- 10 minutes, p less than 0.045). The frequency of intracoronary thrombi in patients with stenoses greater than 50% was significantly less in patients treated with t-PA (11 of 22, 52%) as compared with placebo (23 of 25, 92%) (p = 0.002), but there was no significant difference in minimal lesion cross sectional area (0.49 +/- 0.42 versus 0.57 +/- 1.08 cm2, p = 0.75) or ulceration index (0.79 +/- 0.16 versus 0.77 +/- 0.15, p = 0.71) of the culprit artery. CONCLUSIONS: We conclude that a prolonged infusion of t-PA in unstable angina reduces intracoronary thrombi but does not significantly decrease in-hospital cardiac events. The sample size, however, does not provide sufficient power to rule out a treatment effect. Paradoxically, there appears to be an increase in ST shift and worsening of myocardial perfusion with t-PA compared with therapy with heparin alone. PMID- 1728445 TI - Ventricular tachycardia rate and morphology determine energy and current requirements for transthoracic cardioversion. AB - BACKGROUND: The electrical current and energy required to terminate ventricular tachyarrhythmias are known to vary by arrhythmia: Ventricular tachycardia (VT) is generally considered to require less energy than ventricular fibrillation (VF). The hypothesis of our study was that current requirements for transthoracic termination of VT are further determined by VT rate and QRS complex morphology. METHODS AND RESULTS: We prospectively studied 203 patients who received a total of 569 shocks for VT or VF by following a current-based protocol. This protocol recommended shocks for VT beginning at 18 A (70 +/- 22 J) and shocks for VF beginning at 25 or 30 A (137 +/- 52 J or 221 +/- 70 J). The ventricular tachyarrhythmias were subclassified as monomorphic VT (MVT): uniform QRS complex morphology on surface electrocardiogram and heart rate greater than 100 beats per minute; polymorphic VT (PVT): nonuniform QRS complex morphology and heart rate less than or equal to 300 beats per minute; or VF: nonuniform QRS complex morphology and heart rate greater than 300 beats per minute. We found that shocks of 18 A and 25 A for terminating MVT had success rates of 69% and 82%, respectively, whereas such low-current shocks were less successful for PVT (33% at 18 A) and for VF (19% at 18 A, 53% at 25 A). High-current shocks of 35 A and 40 A were equally successful for the three ventricular tachyarrhythmias. Subdividing MVT revealed that slower MVT (heart rate less than 200 beats per minute) had a significantly better success rate with low-current shocks of 18 A and 25 A than did faster MVT (greater than 200 beats per minute) (89% versus 72% success, p less than 0.01). Bundle branch block morphology, QRS axis, and duration of ventricular tachyarrhythmia did not alter current requirements. CONCLUSIONS: Heart rate and electrocardiographic degree of organization of ventricular tachycardia are important determinants of transthoracic energy and current requirements for cardioversion and defibrillation. Transthoracic termination of MVT requires relatively low current or energy, but PVT behaves more like VF and requires higher electrical current or energy. PMID- 1728446 TI - Frequency domain measures of heart period variability and mortality after myocardial infarction. AB - BACKGROUND: We studied 715 patients 2 weeks after myocardial infarction to establish the associations between six frequency domain measures of heart period variability (HPV) and mortality during 4 years of follow-up. METHODS AND RESULTS: Each measure of HPV had a significant and at least moderately strong univariate association with all-cause mortality, cardiac death, and arrhythmic death. Power in the lower-frequency bands--ultra low frequency (ULF) and very low frequency (VLF) power--had stronger associations with all three mortality end points than power in the higher-frequency bands--low frequency (LF) and high frequency (HF) power. The 24-hour total power also had a significant and strong association with all three mortality end points. VLF power was the only variable that was more strongly associated with arrhythmic death than with cardiac death or all-cause mortality. In multivariate Cox regression models using a step-up approach to evaluate the independent associations between frequency domain measures of heart period variability and death of all causes, ULF power was selected first (i.e., was the single component with the strongest association). Adding VLF or LF power to the Cox regression model significantly improved the prediction of outcome. With both ULF and VLF power in the Cox regression model, the addition of the other two components, LF and HF power, singly or together, did not significantly improve the prediction of all-cause mortality. We explored the relation between the heart period variability measures and all-cause mortality, cardiac death, and arrhythmic death before and after adjusting for five previously established postinfarction risk predictors: age, New York Heart Association functional class, rales in the coronary care unit, left ventricular ejection fraction, and ventricular arrhythmias detected in a 24-hour Holter ECG recording. CONCLUSIONS: After adjustment for the five risk predictors, the association between mortality and total, ULF, and VLF power remained significant and strong, whereas LF and HF power were only moderately strongly associated with mortality. The tendency for VLF power to be more strongly associated with arrhythmic death than with all cause or cardiac death was still evident after adjusting for the five covariates. Adding measures of HPV to previously known predictors of risk after myocardial infarction identifies small subgroups with a 2.5-year mortality risk of approximately 50%. PMID- 1728447 TI - Trends in survival of hospitalized myocardial infarction patients between 1970 and 1985. The Minnesota Heart Survey. AB - BACKGROUND: The Minnesota Heart Survey is a population-based study designed to monitor and explain trends in cardiovascular mortality, morbidity, and risk factors. As part of this effort, a 50% sample of patients hospitalized for myocardial infarction (MI) in the seven-county Twin Cities (Minneapolis and St. Paul) metropolitan area was reviewed in 1970, 1980, and 1985. Those with a validated definite MI were followed for 4-year mortality. The purpose was to determine whether the improved survival observed between 1970 and 1980 was extended to the 1980-1985 period. METHODS AND RESULTS: Crude 28-day mortality in men changed from 18% in 1970 to 12% in 1980 to 13% in 1985; in women it changed from 27% in 1970 to 22% in 1980 to 18% in 1985. After adjustment for severity factors (e.g., age, previous MI, and admission heart rate and systolic blood pressure), 28-day mortality was significantly lower in 1980 than in 1970 in men (RR, 0.66; 95% CI, 0.47, 0.92) and in women (RR, 0.69; 95% CI, 0.46, 1.04), but no change occurred from from 1980 to 1985 (p greater than 0.25). After adjustment for severity indicators, 4-year survival was better in 1980 than in 1970 for men (RR, 0.67; 95% CI, 0.54, 0.83) and for women (RR, 0.72; 95% CI, 0.54, 0.98), but there was no significant change from 1980 to 1985 (p greater than 0.25). CONCLUSIONS: These results suggest that improvements in survival among hospitalized MI patients contributed to the overall decline in coronary heart disease mortality in the Twin Cities area between 1970 and 1980 but not between 1980 and 1985. PMID- 1728448 TI - Effect of valve deformity on results and mitral regurgitation after Inoue balloon commissurotomy. AB - BACKGROUND: The effect of valve deformity and patient age adversely affect the results of percutaneous transvenous mitral commissurotomy (PTMC) with conventional balloons. METHODS AND RESULTS: These factors were characterized after PTMC with the Inoue balloon. The increases in mitral valve area and mitral regurgitation after the procedure were evaluated comparing echocardiographic score of 8 or less versus more than 8, age of less than 60 versus age of 60 years or more, and age of less than 70 versus age of 70 years or more. One hundred sixty-two patients (mean age, 52 +/- 14 years) were studied. For the entire group, mitral valve area increased from 1.0 to 1.8 cm2 (p less than 0.001). Valve area increased from 1.0 +/- 0.3 to 1.8 +/- 0.6 cm2 in patients with echocardiographic score of 8 or less (n = 102) and from 1.0 +/- 0.3 to 1.7 +/- 0.5 cm2 with echocardiographic score of more than 8 (n = 44). Patients less than 60 years old (n = 104) had increases in valve area from 1.0 +/- 0.3 to 1.8 +/- 0.6 cm2 versus 1.0 +/- 0.4 to 1.8 +/- 0.6 cm2 for those 60 years old or older (n = 50) (p = NS). There was no significant difference in resultant valve area when the age division was increased to less than 70 versus 70 years or more. Similarly, the percentage of patients with 2+ or greater increase in mitral regurgitation was not different for those with higher than for those with lower echocardiographic scores (4% versus 12%, p = NS), age of less than 60 versus age of 60 years or more (10% versus 10%, p = NS), or age of less than 70 versus age of 70 or more years (9% versus 18%, p = NS). Valve replacement for mitral regurgitation was performed in four patients (one emergency), all with echocardiographic scores of less than 8. CONCLUSIONS: Age and extent of valve deformity do not have significant effects on acute results of PTMC using the Inoue balloon. Unique balloon geometry or the controlled, stepwise balloon sizing may explain these acceptable acute results in patients with more-deformed valves. PMID- 1728449 TI - Valvular heart disease in four patients with Maroteaux-Lamy syndrome. AB - BACKGROUND: Maroteaux-Lamy syndrome is a lysosomal storage disease of mucopolysaccharide metabolism (MPS type VI) that may involve the mitral and aortic valves. Affected patients have other skeletal and oropharyngeal malformations that complicate anesthetic and surgical management. METHODS AND RESULTS: The present report describes the clinical, echocardiographic, and pathological findings in four patients with Maroteaux-Lamy syndrome. Two of three siblings underwent successful double-valve replacement for aortic and mitral valve stenoses. The third sibling, whose aortic and mitral valves were thick and fibrotic, died from septicemia after hip surgery. A fourth, unrelated patient also had successful double-valve replacement. CONCLUSIONS: Our experience emphasizes the potential difficulties in preoperative assessment and surgical treatment as well as the unique problems related to airway management in patients with this syndrome. PMID- 1728450 TI - A permanent transvenous lead system for an implantable pacemaker cardioverter defibrillator. Nonthoracotomy approach to implantation. AB - A transvenous lead system for implantable defibrillators would obviate a surgical thoracotomy and reduce the morbidity and mortality associated with implantation. We evaluated the clinical performance of a new nonthoracotomy lead system that included a defibrillation lead in the coronary sinus. At the time of defibrillator implantation, transvenous defibrillation leads were inserted percutaneously through the left subclavian vein into the right ventricular apex (RVA), superior vena cava (SVC), and distal coronary sinus (CS) under fluoroscopic guidance. A subcutaneous patch electrode (SQ) was also available if required. The first single- or dual-pathway electrode configuration that successfully terminated three of four ventricular fibrillation episodes using 18 J or less was implanted. Eleven men and three women aged 39-77 years (60.0 +/- 10.1 years) with left ventricular ejection fraction ranging from 16% to 63% (33.4 +/- 13.1%) were evaluated. Nine presented with ventricular tachycardia, three had ventricular fibrillation, and two had both. A totally transvenous lead system (RVA/CS/SVC) was implanted in seven patients (50%) with a mean defibrillation threshold of 15.6 +/- 2.9 J (10-18 J). Four patients received a partial transvenous lead system (RVA/CS/SQ). An effective nonthoracotomy lead system was not found in three patients; they received epicardial electrodes. After cumulative follow-up of 73 patient-months, nine patients remain alive and free of problems related to the implanted nonthoracotomy leads. One patient died of respiratory failure 3 months after defibrillator implant, and the leads from another patient were removed at 9 months because of bacterial infection. A transvenous lead system that includes a defibrillation lead in the coronary sinus is a safe, reliable, and, at least in the short term, effective nonthoracotomy approach for automatic defibrillator implantation. PMID- 1728451 TI - Are aortic aneurysms caused by atherosclerosis? AB - BACKGROUND: The emerging controversy concerning the causal role of atherosclerosis in the development of aortic aneurysms was examined using the accumulated clinical and autopsy data obtained during a 20-year follow-up of a cohort of more than 8,000 men of Japanese ancestry in Hawaii. METHODS AND RESULTS: Analyses of 174 clinical incident events indicated that there were two types of aneurysmal disease, 151 aortic aneurysms and 23 aortic dissections. The baseline risk factors that predicted the clinical aortic aneurysms were the same factors that predicted aortic atherosclerosis in the same cohort, namely, high blood pressure, high serum cholesterol, and cigarette smoking. These same risk factors were also significantly associated with the occurrence of 27 aortic aneurysms among 293 autopsied men. The less common aortic dissections had an age specific incidence pattern indicative of an innate susceptibility precipitated by an exposure to another factor. This pattern was consistent with the findings that the incidence of aortic dissections was predicted mainly by baseline high blood pressure. CONCLUSIONS: From the perspective of prevention, it appears that the risk factors for aortic atherosclerosis and probably atherosclerosis itself are necessary elements in the causal pathway for the great majority of aortic aneurysms in this cohort. PMID- 1728452 TI - Doppler echocardiographic evaluation of pseudoaneurysms complicating composite grafts of the ascending aorta. AB - BACKGROUND: Pseudoaneurysms of the ascending aorta is a rare and serious complication after composite graft surgery for combined disorders of the aortic valve and ascending aorta. METHODS AND RESULTS: Echocardiographic and Doppler findings are described in eight patients (seven men, one woman; mean age, 45 +/- 12 years) with documented pseudoaneurysm of the ascending aorta and are compared with those by aortography and at surgery. The diameter of the ascending aorta ranged from 6 to 14 cm. Pseudoaneurysm was diagnosed by echocardiography in seven cases (six transthoracic, one transesophageal), by aortography in five, and by both methods in all patients. All three patients not diagnosed by aortography had a single dehiscence at the aortic annulus anastomosis. Five patients had more than one site of origin of the pseudoaneurysm. Periannular dehiscence (n = 7) was identified by color flow Doppler in six cases and by aortography in only one, and coronary artery dehiscence (n = 6) was detected by echocardiography in three and by aortography in two arteries. Of the three patients with distal graft dehiscence, one was identified by aortography and none by echocardiography. In cases of dehiscence at the aortic annulus, continuous wave Doppler further supported the diagnosis by demonstrating two distinct jets, one through the prosthetic valve and another with higher velocity through the communication. CONCLUSIONS: Echocardiography with Doppler can diagnose the presence of pseudoaneurysms complicating composite grafts and identify their proximal sites of origin. Furthermore, it complements aortography in the overall evaluation of patients with suspected pseudoaneurysm, particularly in those with single dehiscence of the graft at the aortic annulus anastomosis. PMID- 1728453 TI - Decreased HDL2 and HDL3 cholesterol, Apo A-I and Apo A-II, and increased risk of myocardial infarction. AB - BACKGROUND: A large and consistent body of evidence supports the judgment that elevation of total plasma blood cholesterol is a cause of myocardial infarction (MI) and that high levels of low density lipoprotein (LDL) cholesterol have a positive relation and high levels of high density lipoprotein (HDL) cholesterol an inverse relation with MI. At present, however, the roles, if any, of the major subfractions of HDL, namely, HDL2 and HDL3, have not been clarified. In addition, the relation of plasma apolipoprotein concentrations to MI and whether they provide predictive information over and above their lipoprotein cholesterol associations is unknown. METHODS AND RESULTS: We evaluated these questions in a case-control study of patients hospitalized with a first MI and neighborhood controls of the same age and sex. Cases had significantly lower levels of total HDL (p less than 0.0001) as well as HDL2 (p less than 0.0001) and HDL3 (p less than 0.0001) cholesterol. These differences persisted after controlling for a large number of demographic, medical history, and behavioral risk factors and levels of other lipids. There were significant (p less than 0.0001) inverse dose response relations with odds ratios for those in the highest quartile relative to those in the lowest of 0.15 for total HDL, 0.17 for HDL2, and 0.29 for HDL3 cholesterol levels. Levels of LDL and very low density lipoprotein cholesterol and triglycerides were also higher among cases than controls, but only for triglycerides was the difference statistically significant after adjustment for coronary risk factors and other lipids (p = 0.044). Apolipoproteins A-I and A-II were both significantly (p less than 0.0001) lower in cases, and differences remained even after adjustment for coronary risk factors and lipids. There were significant dose-response relations for both apolipoprotein A-I (p = 0.026) and A II (p = 0.002). Neither apolipoprotein B nor E was significantly related to MI after adjustment for lipids and other coronary risk factors. When all four apolipoproteins were taken together, there was an increased level of prediction of MI over the information provided by the lipids and other coronary risk factors (p = 0.003), but this appeared present only for the individual apolipoproteins A I (p = 0.027) and A-II (p = 0.011). CONCLUSIONS: These data indicate that both HDL2 and HDL3 cholesterol levels are significantly associated with MI. They also raise the possibility that apolipoprotein levels, especially A-I and A-II, may add importantly relevant information to determination of risk of MI. PMID- 1728454 TI - Capillary recruitment and pain relief on leg dependency in patients with severe lower limb ischemia. AB - BACKGROUND: Patients suffering from severe lower limb ischemia may experience pain relief on leg dependency despite the fact that dependency normally results in arteriolar vasoconstriction. To clarify this possible paradox, skin microcirculation of the limb was investigated in 75 patients with different stages of lower limb ischemia and in 12 asymptomatic subjects. METHODS AND RESULTS: Using nailfold capillary video microscopy, red blood cell-perfused capillary density and diameter and red blood cell velocity were assessed in supine and sitting positions. Capillary density increased by changing from the supine to the sitting position, especially in patients with limb-threatening ischemia (showing a 4.5-fold increase versus a 1.5-fold increase in asymptomatic subjects). In subjects without or with mild ischemia, capillary perfusion was two to four times lower in the sitting than in the supine position. In patients with limb-threatening ischemia, perfusion was strongly reduced, being slightly higher in the sitting position. Patients with relief of pain while sitting did not always have a higher capillary perfusion but did have a higher capillary density in the sitting position. CONCLUSIONS: The arteriolar postural vasoconstrictive mechanism at the nutritive level is still intact in subjects without or with mild ischemia but not in patients with severe ischemia. Capillary recruitment rather than disturbed arteriolar vasoconstriction could explain why patients with severe leg ischemia prefer leg dependency. PMID- 1728455 TI - Independent and incremental prognostic value of tests performed in hierarchical order to evaluate patients with suspected coronary artery disease. Validation of models based on these tests. AB - BACKGROUND: The additive prognostic value of tests done in a hierarchical order for the detection of coronary artery disease (CAD) is not always known. The principal goal of this study, therefore, was to assess the incremental prognostic value of data obtained in succession (clinical, exercise stress testing, 201Tl imaging, and coronary angiography) in patients with suspected CAD. A second goal was to develop models for determining prognosis based on results of these tests and to test the clinical validity of these models in unrelated patients. METHODS AND RESULTS: Data from two groups of patients who had undergone such evaluation and had been followed for a mean of 4.4 years were analyzed. There were 204 patients from Massachusetts General Hospital (MGH) and 299 from the University of Virginia (UVA). There were 20 deaths and 21 nonfatal infarctions in the MGH group and 41 deaths and nine infarctions in the UVA group. Both univariate and multivariate Cox regression analyses were performed to assess the individual and incremental prognostic value of these tests. In both groups, 201Tl imaging provided significant additional prognostic information compared with clinical and exercise stress test data (p less than 0.05). At MGH, where the lung/heart 201Tl ratio had been analyzed, coronary angiography did not provide additional prognostic information. In this group of patients, the combination of clinical and exercise 201Tl variables provided greater prognostic information than the combination of clinical and angiographic data (p less than 0.001). In the UVA cohort, in which the lung/heart ratio had not been analyzed, coronary angiography provided incremental prognostic information compared with clinical and exercise 201Tl data alone (p less than 0.05). When models developed using data from either sample were applied to the other unrelated sample, there was often close agreement between the overall observed rates and those predicted by the models. This was also true for the low-risk and high-risk subgroups. Some models, however, did not perform as well as other models, which suggests that models that do well in one sample may not always be generalized to other groups. CONCLUSIONS: Tests performed in hierarchical order for the evaluation of suspected CAD provide additional prognostic information. Models developed using clinically relevant combinations of test results obtained from different patient populations are frequently able to predict absolute levels of survival in unrelated but similar samples. PMID- 1728456 TI - Evidence for the association of unexplained pulmonary hypertension in children with the major histocompatibility complex. AB - BACKGROUND: A link between primary pulmonary hypertension (PPH) and autoimmune disorders has been postulated. To investigate this relation, we performed immunofluorescent antinuclear antibody tests (ANA) and serological human leukocyte antigen (HLA)-A, B, C, DR, and DQ typing on two groups of Caucasian children with unexplained pulmonary hypertension (PHT) and their parents. METHODS AND RESULTS: Group 1 consisted of 17 children with PPH including two patients with familial PPH and three patients with trivial congenital pulmonary to systemic communications. Group 2 consisted of 13 children with advanced PHT and anatomically large congenital pulmonary to systemic communications (PHT + shunt). Both groups had comparably severe pulmonary vascular disease documented by cardiac catheterization. The following statistically significant data (p less than 0.05) were obtained when the study groups were compared with those published for normal controls. Although positive ANAs and varying titers of autoantibodies were found in both groups of children and mothers (not fathers), +ANAs were only significant for the maternal groups. The PPH (group 1) children had increased frequencies of HLA-DR3, DRw52, and DQw2 and decreased DR5, whereas the group 2 (PHT + shunt) children (also their parents) had no statistically significant alterations in any of the DR or DQ alleles. The PPH mothers had decreased DQw3, an allele in linkage disequilibrium with DR5. CONCLUSIONS: These immunogenetic data suggest that childhood PPH appears to be associated with the major histocompatibility complex alleles HLA-DR3, DRw52, and DQw2. This newly found correlation of juvenile PPH with these alleles adds this disease to the DR3+ group of autoimmune diseases. Further studies are needed to determine whether there is also an immunological or autoimmune component in some children with PHT + shunt lesions because this group lacked an HLA association. PMID- 1728457 TI - Structural remodeling of human myocardial tissue after infarction. Quantification with ultrasonic backscatter. AB - BACKGROUND: Remodeling of myocardial tissue after infarction may culminate in the development of either a well-healed scar or a thin, expanded heart wall segment that predisposes to ventricular aneurysm formation, congestive heart failure, or ventricular tachycardia. The three-dimensional architecture of mature human infarct tissue and the mechanisms that determine it have not been elucidated. We have previously shown that quantitative ultrasonic backscatter can be used to define the transmural organization of human myofibers in the normal ventricular wall by measuring the dependence of backscatter on the angle of insonification, or ultrasonic anisotropy. We propose that measurement of ultrasonic anisotropy of backscatter may permit quantitative characterization of the transmural architecture of tissue from areas of myocardial infarction and facilitate identification of fundamental mechanisms of remodeling of the ventricular wall. METHODS AND RESULTS: We measured integrated backscatter in 33 transmural sections from 12 cylindrical biopsy specimens (1.4-cm diameter) sampled from central regions of mature infarction in six explanted fixed human hearts. Tissue samples were insonified in two-degree steps around their entire circumference at successive transmural levels with a 5-MHz broad-band piezoelectric transducer. Backscatter radio frequency data were gated from the center of each specimen, and spectral analysis was performed on the gated radio frequency for the computation of integrated backscatter. Histological morphometric analysis was performed on each specimen for determination of the predominant fiber orientation and the percentage of tissue infarcted at consecutive transmural levels. The average percentage of tissue infarcted for all transmural levels was 49 +/- 3% (range, 13 80%). Histological attributes varied from patchy fibrosis to extensive confluent zones of scar tissue. The angle-averaged integrated backscatter for all transmural levels in infarct tissue was approximately 5 dB greater than that previously measured in normal tissue in our laboratory (-48.3 +/- 0.5 versus 53.4 +/- 0.4 dB, infarct versus normal). Marked anisotropy of backscatter was observed in tissue from areas of infarction and was characterized by a sinusoid like dependence on the angle of insonification at each transmural level. Insonification perpendicular to infarct fibers yielded values for integrated backscatter 14.8 +/- 0.5 dB greater than those for insonification parallel to these fibers. Juxtaposition of the sinusoid-like anisotropy functions from all consecutive transmural levels demonstrated a progressive shift in the orientation of scar tissue elements from epicardial to endocardial levels of 14.6 +/- 1.5 degrees/mm of tissue. The transmural shift in fiber orientation per millimeter of tissue from the area of infarction exceeded that previously measured for normal tissue (9.2 +/- 0.7 degrees/mm) by 59%. This marked augmentation in angular shift per millimeter of tissue results from a generalized structural rearrangement (or reorientation) of fibers across the entire ventricular wall in the infarct zone that we hypothesize is determined in part by dynamic mechanical forces, imposed by the surrounding functional normal tissue, that tether the "infarcted" tissue. CONCLUSIONS: Myocardial tissue from areas of myocardial infarction manifests substantial anisotropy of ultrasonic scattering that may be useful for quantitative characterization of the alignment and overall three-dimensional anatomic organization of mature infarct scars. PMID- 1728458 TI - Treatment of prolonged ventricular fibrillation. Immediate countershock versus high-dose epinephrine and CPR preceding countershock. AB - BACKGROUND: Early countershock of ventricular fibrillation has been shown to improve immediate and long-term outcome of cardiac arrest. However, a number of investigations in the laboratory and in the clinical population indicate that immediate countershock of prolonged ventricular fibrillation most commonly is followed by asystole or a nonperfusing spontaneous cardiac rhythm, neither of which rarely respond to current therapy. The use of epinephrine in doses greater than those currently recommended has recently been shown to improve both cerebral and myocardial perfusion during cardiopulmonary resuscitation (CPR). The purpose of this study was to compare cardiac resuscitation outcome between immediate countershock of prolonged ventricular fibrillation with high-dose epinephrine therapy and conventional CPR before countershock of prolonged ventricular fibrillation in a canine model. METHODS AND RESULTS: After sedation, intubation, induction of anesthesia, and instrumentation, ventricular fibrillation was electrically induced in 28 dogs. After 7.5 minutes of ventricular fibrillation, animals were randomly allocated to two treatment groups: group 1, immediate countershock followed by recommended advanced cardiac life support (ACLS) interventions, or group 2, 0.08 mg/kg epinephrine and manual closed-chest CPR before countershock and ACLS. In both groups, ACLS was continued until a spontaneous perfusing rhythm was restored or for 20 minutes (total arrest time, 27.5 minutes). A spontaneous perfusing rhythm was restored in three of 14 group 1 animals and in nine of 14 group 2 animals (p = 0.014 by sequential analysis method of Whitehead). Coronary perfusion pressure (aortic minus right atrial pressure during CPR diastole) before countershock was significantly greater in group 2 (21 +/- 7 mm Hg) when compared with mean circulatory pressure in group 1 (9 +/- 8, p less than 0.01). CONCLUSIONS: The findings of this study suggest that a brief period of myocardial perfusion before countershock improves cardiac resuscitation outcome from prolonged ventricular fibrillation. PMID- 1728459 TI - Imaging of thrombi with tissue-type plasminogen activator rendered enzymatically inactive and conjugated to a residualizing label. AB - BACKGROUND: Contemporary cardiovascular practice relies increasingly on thrombolysis as a therapeutic modality. Its optimal use requires prompt, noninvasive delineation of thrombotic occlusion in arterial beds and rapid detection of reocclusion after initially successful thrombolysis. METHODS AND RESULTS: We have been developing an approach to noninvasively image thrombi in which plasminogen-activating properties of tissue-type plasminogen activator (t PA) are attenuated by treatment with D-Phe-L-Pro-L-Arg-chloromethyl ketone (PPACK) and have shown that the inactive t-PA avidly and promptly binds to clots in vitro. In the present study, we conjugated this material to a residualizing label, radioiodinated dilactitol tyramine (*I-DLT), and characterized the potential use of the inactivated, conjugated t-PA as a radiopharmaceutical for imaging thrombi in vivo. The approach developed requires not only avid binding of the tracer to thrombi but also rapid clearance from plasma and a lack of prompt release of radiolabeled degradation products from the liver. The rapid clearance of unaltered or PPACK-treated t-PA was not influenced by conjugation to *I-DLT, but the release of radioiodinated degradation products into plasma after injection of *I-DLT-conjugated t-PA was markedly less than release of degradation products of directly radioiodinated t-PA. When 131I-DLT-PPACK-t-PA was infused for 15 minutes intravenously after a bolus injection of 20% in dogs with coronary, pulmonary, or carotid artery thrombi, clearance was rapid. Mean +/- SEM thrombus-to-blood ratios of radioactivity were high, ranging from 37 +/- 9:1 and 2.8 +/- 0.6:1 with carotid thrombi formed concomitantly or approximately 30 minutes before infusion of tracer, respectively, to 35:1 for concomitantly formed coronary thrombi, 42 +/- 7:1 and 8.1 +/- 0.8:1 for concomitantly formed and preformed pulmonary thrombi, respectively, and 18:1 for a preformed femoral artery thrombus. Thrombi were detectable by planar gamma scintigraphy even though image quality was affected adversely by low concentrations of radioactivity that in aggregate composed a relatively large amount of radioactivity in underlying and overlying tissues. This limitation was overcome by tomographic imaging, which was used to detect both femoral and pulmonary thrombi. CONCLUSIONS: Use of enzymatically inactivated t-PA coupled to a residualizing label permits rapid detection and localization of thrombi in vivo. PMID- 1728460 TI - Noninvasive arterial thrombus imaging with 99mTc monoclonal antifibrin antibody. AB - BACKGROUND: The T2G1s monoclonal antifibrin antibody binds specifically to fibrin but not to fibrinogen. METHODS AND RESULTS: In a canine model of acute arterial thrombosis, we determined the feasibility of imaging thrombi using a 99mTc labeled Fab' fragment. In 14 dogs, 10 carotid and 13 femoral artery thrombi were produced using 2-hour temporary occlusion, crush injury, and local thrombin injection methods. A sham-operated carotid artery served as control. Antifibrin antibody was injected intravenously at the end of temporary occlusion. Serial planar radionuclide images were obtained immediately and at 1 and 2 hours. Following killing the dogs at 2 hours, we measured antibody uptake ex vivo in 5 mm-long segments of thrombus, the adjacent injured artery, and a control artery. Antibody was cleared from the blood with a mean +/- SD t1/2 of 121 +/- 23 minutes. The thrombi weighed 218 +/- 140 mg. Antibody uptake in the thrombi was patchy, and the thrombi were closely adherent to the injured arterial wall. In the segment with maximal ex vivo antibody uptake, the ratio of control artery to blood counts/g/sec was 0.65 +/- 0.46, the injured artery-to-blood ratio was 2.35 +/- 1.01 (p less than 0.0001 versus control), and the thrombus-to-blood ratio was 4.24 +/- 2.58 (p less than 0.0001 versus control). In three dogs, an isotype matched ovarian tumor antibody labeled with 111In was injected with T2G1s but was not taken up in the thrombus or the adjacent arterial wall. Visual analysis of the in vivo carotid radionuclide images showed uptake by 2 hours in all 10 carotid thrombi. Quantitative image analysis, measured as the thrombus-to opposite carotid artery ratio, showed increasing uptake over time with ratios of 1.1 +/- 0.3, 1.6 +/- 2.0, and 2.2 +/- 1.3 on the immediate, 1-hour, and 2-hour images, respectively. All quantitative ratios of 1.3 or greater were visually identified. CONCLUSIONS: 99mTc-labeled Fab' fragments of the T2G1s antibody are taken up specifically by acute arterial thrombi after intravenous injection. Uptake is progressive over a 2-hour period, and all thrombi are detected by radionuclide imaging at 2 hours. These results show that it is feasible to noninvasively detect arterial thrombi within 2 hours of formation. PMID- 1728461 TI - Lipoprotein profile in men with peripheral vascular disease. Role of intermediate density lipoproteins and apoprotein E phenotypes. AB - BACKGROUND: The role of lipoprotein disturbances in the development of peripheral vascular disease (PVD) has not been sufficiently clarified. METHODS AND RESULTS: The relations among concentrations of intermediate density lipoproteins (IDL), apoprotein (apo) B, apo E, and other lipoproteins were studied in 102 men with PVD and 100 healthy men who formed the control group. Patients with PVD had significantly higher levels of serum triglycerides, very low density lipoprotein (VLDL) cholesterol, VLDL triglycerides, VLDL proteins, IDL cholesterol, and IDL triglycerides and lower levels of high density lipoproteins (HDL) than controls. Serum cholesterol and triglycerides were normal in 30 patients (cholesterol, less than 5.2 mmol/l; triglycerides, less than 2.3 mmol/l), who had significant increases in IDL triglycerides and significant decreases in HDL cholesterol compared with the 47 controls, who had normal cholesterol and triglyceride levels. Patients with more severe distal involvement showed higher cholesterol and triglycerides carried by IDL and a greater reduction in HDL cholesterol. Smoking patients with PVD showed increased VLDL cholesterol and VLDL triglycerides and lower HDL concentrations. Apo E polymorphism in our study population does not differ from that reported for other European populations. Alleles epsilon 2 and epsilon 4 had a major impact on serum triglycerides and VLDL lipids in our patients with PVD. CONCLUSIONS: Lipoprotein disturbances are a major risk factor for PVD. IDL abnormalities play an important role in the development and severity of PVD and should also be considered a vascular risk factor in normocholesterolemic and normotriglyceridemic patients. PMID- 1728462 TI - Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. AB - BACKGROUND: We investigated the effect of inhibition of plasminogen activator inhibitor-1 (PAI-1) activity by a murine monoclonal anti-human PAI-1 antibody (MAI-12) on in vitro thrombolysis and on in vivo thrombolysis and thrombus extension in an experimental animal model for thrombosis. METHODS AND RESULTS: Thrombolysis, mediated by recombinant tissue-type plasminogen activator (rt-PA), was studied in an in vitro system consisting of fibrinogen, plasminogen, and thrombin. Addition of purified platelets during clot formation inhibited rt-PA mediated thrombolysis in a dose-dependent manner. Platelet-dependent thrombolysis resistance could be completely neutralized by the monoclonal antibody MAI-12, supporting the notion that the observed resistance is due to PAI-1 released from alpha-granules of platelets. Subsequently, we monitored in vivo thrombolysis and thrombus extension of human whole blood thrombi that were allowed to form in rabbit jugular veins. During thrombus formation, either MAI-12 or an irrelevant antibody was incorporated. Thrombolysis and thrombus extension were simultaneously measured during endogenous thrombolysis or upon administration of different dosages of rt-PA. We observed that endogenous thrombolysis was enhanced in the presence of MAI-12 compared with the control antibody. Significantly, thrombus extension was partially prevented by MAI-12 and not by the control antibody irrespective of whether exogenous rt-PA was systematically administered. CONCLUSIONS: These observations further confirm the essential role of PAI-1 in the regulation of the thrombolytic system and support the exploration of adjunctive therapy based on inhibition of PAI-1 activity in thrombolytic strategies. PMID- 1728463 TI - Coronary vasodilator reserve in ischemic myocardium of the exercising dog. AB - BACKGROUND: Previous work has reported that coronary vasodilator reserve may persist in myocardium rendered ischemic by hypoperfusion. This study investigated the presence and extent of residual coronary vasomotor tone in myocardial regions made acutely ischemic by a flow-limiting coronary stenosis during exercise. METHODS AND RESULTS: Studies were done in chronically instrumented dogs undergoing treadmill exercise in the presence of a coronary stenosis that decreased distal left circumflex coronary artery perfusion pressure to approximately 40 mm Hg. Measurements of myocardial blood flow were made with radioactive microspheres during exercise (6.5 km/hr, 6% grade) before and during intracoronary infusion of the potent coronary vasodilator adenosine (40 micrograms/kg/min). Distal coronary perfusion pressure was held equal before and during intracoronary adenosine infusion (43 +/- 5 versus 42 +/- 5 mm Hg) by adjusting the hydraulic coronary occluder. During exercise in the presence of a coronary stenosis, myocardial blood flow (milliliter per minute per gram) was significantly reduced in all layers of the ischemic posterior region compared with the nonischemic anterior region. During intracoronary adenosine infusion, with no change in coronary perfusion pressure, myocardial blood flow was significantly increased compared with preadenosine flows for both the subendocardial layer flow (1.03 +/- 0.74 versus 0.66 +/- 0.50; p less than 0.05) and mean transmural flow (1.54 +/- 0.59 versus 1.16 +/- 0.36; p less than 0.05). In the presence of a coronary stenosis, regional myocardial segment shortening in the ischemic region during exercise fell significantly to 49 +/- 8% of shortening in the absence of a coronary stenosis but improved modestly during adenosine infusion (65 +/- 7 versus 49 +/- 8%; p less than 0.05). CONCLUSIONS: These results indicate that adenosine-responsive coronary vasodilator reserve persists during exercise-induced myocardial ischemia and suggest that residual microvascular vasoconstrictor tone may affect the extent of myocardial hypoperfusion occurring consequent to a flow-limiting coronary stenosis. PMID- 1728464 TI - Color Doppler regurgitant characteristics of normal mechanical mitral valve prostheses in vitro. AB - BACKGROUND: To evaluate normal regurgitant characteristics of St. Jude (SJ) and Medtronic-Hall (MH) mitral valves, four sizes (25-31 mm) of each were studied in a pulsatile flow model. METHODS AND RESULTS: Regurgitant flow was measured by flowmeter at left ventricular pressures of 80, 130, and 180 mm Hg. Peak regurgitant flow rates ranged from 6.2 to 12.7 cm3/sec in SJ valves and from 7.9 to 17.5 cm3/sec in MH valves. Regurgitant orifice areas calculated from the Doppler continuity equation ranged from 1.6 to 2.0 mm2 in SJ valves and from 2.2 to 2.9 mm2 in MH valves. Regurgitant volumes across the closed valve at a left ventricular pressure of 130 mm Hg were normalized to an ejection time of 280 msec and ranged from 1.5 to 1.9 cm3 in SJ valves and from 2.1 to 2.8 cm3 in MH valves. Jets were imaged by color Doppler in six rotational planes, and jet size and morphology were compared with those of regurgitant jets from circular orifices with sizes comparable to the calculated prosthetic valve regurgitant orifices (1.1-3.1 mm2). SJ valves showed two converging jets from the pivot points, one central jet, and a variable number of peripheral jets. The mean color jet area derived from the six image planes ranged from 1.6 to 5.3 cm2. Aliasing occurred only close to the valve (maximal distance 0.5-2.0 cm). MH valves showed a large central jet with a maximal length of aliased flow between 2.0 and 5.5 cm. Depending on valve size, driving pressure, and image plane, one or two small peripheral jets were found. These jets did not show aliasing in any case. The mean color jet area ranged from 5.1 to 11.0 cm2. Jets originating from circular orifices of comparable size showed jet areas from 5.5 to 13.9 cm2 and aliasing distances from 3.3 to 7.3 cm. At similar regurgitant orifice areas, driving pressures, and regurgitant flows, the measured color areas and aliasing distances were smallest in SJ valves, larger in MH valves, and largest in simple circular orifices. CONCLUSIONS: Large, complex regurgitant jets can be found in normal closed SJ and MH valves by color Doppler, although regurgitant flow volume is minimal. Jet size and velocity distribution differs markedly between SJ valves, MH valves, and circular orifices, even with comparable driving pressure, regurgitant orifice area, and regurgitant volume. The characteristic patterns of normal regurgitation must be recognized to avoid incorrect diagnoses of pathological regurgitation in SJ and MH prosthetic valves. MH valves should not be removed solely on the basis of a central regurgitant jet with a long aliasing distance. Peripheral jets in MH valves and all jets in SJ valves should be considered normal as long as no or only minimal aliasing is present. In contrast, peripheral jets with significant aliasing may represent strong evidence of pathological regurgitation. PMID- 1728465 TI - Influence of the Coanda effect on color Doppler jet area and color encoding. In vitro studies using color Doppler flow mapping. AB - We studied surface adherence and its effects on color Doppler jet areas and color encoding in an in vitro model with a noncompliant receiving chamber into which a steady flow jet was directed parallel to either a straight or a curved surface adjacent to and 4 mm away from the inflow orifice (1.50 mm2) with the control condition being a free jet matched for flow rates and driving pressures. Jets were imaged perpendicular to the plane of the surface, the plane in which most clinical images of jet-surface interactions are obtained. Ten different flow rates ranging from 0.13 to 0.30 l/min were used. Surface-adherent jet areas were smaller than control jets for every driving pressure-volume combination (paired t test, p less than 0.01). Computer analysis of color Doppler images showed more green and blue (reverse flow) pixels on the surface side of the adherent jets than the control jets (p less than 0.05), suggesting that viscous energy loss and flow deceleration and reversal play a role in the jet-surface interaction. Analysis of variance demonstrated that linear regression slopes of flow rate versus jet area for surface jets were lower (slopes, 11-21 cm2/l/min; r = 0.95 0.97) than those for the control (slope, 33 cm2/l/min; r = 0.97) (p less than 0.0001). Surface adherence (Coanda effect) influences jet size and color encoding, causing smaller color Doppler jet areas and greater variance and reverse velocity encoding. PMID- 1728466 TI - Response of high-energy phosphates and lactate release during prolonged regional ischemia in vivo. AB - BACKGROUND: The functional impairment of persistently ischemic, or "hibernating," myocardium may serve to maintain myocardial cell viability through a reduction of energy requirements. Although previous studies have, in a variety of experimental models, independently shown variable responses in lactate metabolism and intracellular phosphates during prolonged ischemia, the responses of these metabolites under identical flow conditions have not been adequately described. METHODS AND RESULTS: To examine the responses of high-energy phosphates and lactate metabolism to prolonged ischemia induced by partial coronary artery stenosis, 12 open-chest pigs were studied using 31P nuclear magnetic resonance spectroscopy. Concurrent measurements of blood flow, segment shortening, high energy phosphates, and lactate release (in nine animals) were made during 2 hours of regional ischemia. Subendocardial blood flow and segment shortening were persistently depressed during ischemia, with parallel reductions in ATP, phosphocreatine (PCr), and the ratio of phosphocreatine to inorganic phosphate (PCr/Pi). Pi was persistently elevated during the ischemic period. In contrast, lactate release increased significantly from 0.23 +/- 0.04 to 1.34 +/- 0.28 mumol/ml after 15 minutes of ischemia (p less than 0.05) but then decreased to 0.73 +/- 0.17 mumol/ml at 2 hours (p less than 0.05 versus 15 minutes, p = NS versus control). Similarly, pH increased significantly from a nadir of 6.82 +/- 0.07 at 30 minutes of ischemia to 6.98 +/- 0.05 at 2 hours. CONCLUSIONS: Changes in high-energy phosphates parallel changes in blood flow and function during prolonged ischemia, whereas there is a partial amelioration in lactate production and acidosis. These data support the concept that reduction of myocardial energy requirements during prolonged flow reduction results in signs of reduced ischemia. PMID- 1728467 TI - Endothelins. Myocardial actions of a new class of cytokines. AB - There is growing evidence to support the existence of a dynamic interaction in vivo between cardiac myocytes and adjacent microvascular endothelial cells in the regulation of both cardiac myocyte and possibly endothelial cell phenotype and function. Endothelins may be only one of several endogenous cytokines or autocoids that are released by the cardiac microvascular and/or endocardial endothelium and transported vectorially to adjacent myocytes that could modify cardiac contractile state, perhaps in response to changes in microvascular blood flow. Similarly, cardiac myocytes themselves could release cytokines that could directly affect endothelial cell proliferation or angiogenesis and indirectly elicit or modify the release of endothelium-derived cytokines and autocoids. Thus, in addition to modifying function, endothelial cell-cardiac myocyte interactions may also be of importance in the dynamic events that lead to myocardial wall remodeling and angiogenesis during hypertrophic growth and in the response to cardiac injury. PMID- 1728468 TI - Evaluation of emerging technologies for coronary revascularization. Participants in the National Heart, Lung, and Blood Institute Conference on the Evaluation of Emerging Coronary Revascularization Technologies. AB - Despite significant advances in coronary angioplasty, the problems of restenosis, chronic total occlusion, diffuse disease, and abrupt closure after the procedure remain to be solved. New devices that address these problems continue to evolve. Although a controlled clinical trial is the ultimate test of any device, rapid device design changes and resource limitations require evaluating which of the new devices justify a full clinical trial. A registry mechanism for this initial evaluation is proposed and detailed. Payment for procedures using new devices is a problem for industry, insurers, hospitals, and physicians. It is proposed that reimbursement be based on the service performed rather than the device used to perform it. Costs beyond the clinical costs should be borne by the sponsor of the device. Financial conflict of interest is a recognized problem in new device evaluation. It is not always practical to insist on exclusion from the evaluation process of individuals with a financial stake in the outcome, but disclosure of the presence of such a financial interest is recommended in the reporting of results. PMID- 1728469 TI - Joint lipid risk factors and coronary heart disease. PMID- 1728470 TI - Noninvasive diagnosis of cardiac allograft rejection. Another of many searches for the grail. PMID- 1728472 TI - Fontan procedure for hypoplastic left heart syndrome. PMID- 1728471 TI - Joint effects of serum triglyceride and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study. Implications for treatment. AB - BACKGROUND: We studied the joint effect of baseline triglyceride and lipoprotein cholesterol levels on the incidence of cardiac end points in the trial group (n = 4,081) of the Helsinki Heart Study, a 5-year randomized coronary primary prevention trial among dyslipidemic middle-aged men. The relative risks (RR) were calculated using Cox proportional hazards models with a dummy variable technique that allows simultaneous study of subgroup combinations from the placebo and treatment groups. METHODS AND RESULTS: In the placebo group (n = 2,045), the low density lipoprotein cholesterol (LDL-C)/high density lipoprotein cholesterol (HDL C) ratio was the best single predictor of cardiac events. This ratio in combination with the serum triglyceride level revealed a high-risk subgroup: subjects with LDL-C/HDL-C ratio greater than 5 and triglycerides greater than 2.3 mmol/l had a RR of 3.8 (95% CI, 2.2-6.6) compared with those with LDL-C/HDL-C ratio less than or equal to 5 and triglyceride concentration less than or equal to 2.3 mmol/l. In subjects with triglyceride concentration greater than 2.3 mmol/l and LDL-C/HDL-C ratio less than or equal to 5, RR was close to unity (1.1), whereas in those with triglyceride level less than or equal to 2.3 mmol/l and LDL-C/HDL-C ratio greater than 5, RR was 1.2. The high-risk group with LDL C/HDL-C ratio greater than 5 and triglyceride level greater than 2.3 mmol/l profited most from treatment with gemfibrozil, with a 71% lower incidence of coronary heart disease events than the corresponding placebo subgroup. In all other subgroups, the reduction in CHD incidence was substantially smaller. CONCLUSIONS: Serum triglyceride concentration has prognostic value, both for assessing coronary heart disease risk and in predicting the effect of gemfibrozil treatment, especially when used in combination with HDL-C and LDL-C. PMID- 1728473 TI - Phase image analysis. A battle won, but was the war already over? PMID- 1728474 TI - Thrombolysis in unstable angina. PMID- 1728475 TI - Aortic aneurysms and atherosclerosis. PMID- 1728476 TI - Autoimmune disease and unexplained pulmonary hypertension. PMID- 1728477 TI - Imaging arterial thrombi. An elusive goal. PMID- 1728478 TI - Inhibiting the inhibitor. PMID- 1728479 TI - Diastolic perfusion time and stress-induced myocardial ischemia. PMID- 1728480 TI - Exercise-induced left ventricular wall motion abnormalities in athletes. PMID- 1728481 TI - Increased susceptibility to reentrant arrhythmias by flecainide. PMID- 1728482 TI - Catheter ablation in patients with cardiac arrhythmias. PMID- 1728483 TI - A definition of the intima of human arteries and of its atherosclerosis-prone regions. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. PMID- 1728484 TI - Control of arteriolar resistance in heart failure. Partial attenuation of specific phosphodiesterase inhibitor-mediated vasodilation by digitalis glycosides. AB - BACKGROUND: The vasodilatory response to local specific type III phosphodiesterase inhibition with amrinone was evaluated before and immediately after local administration of digoxin in 14 patients with severe congestive heart failure (CHF). METHODS AND RESULTS: A 3F polyethylene catheter was inserted into the common femoral artery for drug administration and pressure monitoring. Mean blood flow velocity (MBFV) was continuously determined in the superficial femoral artery by transcutaneous Doppler ultrasonography. After intra-arterial administration of 10 mg amrinone, group MBFV increased from 7.7 +/- 1.4 to 16.0 +/- 2.1 cm/sec (p less than 0.05, n = 10). Local administration of 20 micrograms digoxin, which was infused over 20 minutes, did not alter group MBFV (i.e., 8.2 +/- 1.6 versus 7.6 +/- 1.5 cm/sec; p = NS, n = 10). The second administration of 10 mg amrinone, which immediately followed completion of local digoxin infusion, increased group MBFV but to a lesser extent than that produced by the first amrinone administration (i.e., 11.9 +/- 1.9 versus 16.0 +/- 2.1 cm/sec; p less than 0.05, n = 10). When placebo was administered instead of digoxin, group MBFV was similar after the first and second administrations of amrinone (i.e., 15.3 +/ 3.3 versus 15.6 +/- 3.8 cm/sec; p = NS, n = 4). CONCLUSIONS: Although local administration of digoxin did not significantly alter baseline vascular tone in patients with CHF, it substantially decreased the direct vasodilatory effect induced by specific type III phosphodiesterase with amrinone. PMID- 1728485 TI - Early postoperative reduction of monoclonal antimyosin antibody uptake is associated with absent rejection-related complications after heart transplantation. AB - BACKGROUND: Detection and treatment for rejection after transplantation are based on the identification of myocyte damage upon endomyocardial biopsy. Noninvasive detection of such damage is possible with 111In-labeled monoclonal antimyosin antibodies (MAA). Although the presence and degree of MAA uptake parallels the rejection activity detected by biopsy, the relation between the degree of uptake and the occurrence of severe rejection-related complications has not been previously assessed. METHODS AND RESULTS: Two hundred forty-seven MAA studies were performed coinciding with biopsies in 52 patients 1-71 months after transplantation. A heart-to-lung ratio (HLR) was used as a measure of relative MAA uptake, with an HLR of 1.55 discriminating normal from abnormal studies. Of the 247 antimyosin studies, 149 coincided with absent, 38 with mild, and 60 with moderate rejection at biopsy. HLR was 1.68 +/- 0.27, 1.79 +/- 0.22, and 1.91 +/- 0.33 in the three biopsy groups, respectively (p less than 0.0001). Two hundred thirty-eight of 247 antimyosin studies coexisted with absent rejection-related complications; in nine of 247 patients, such complications were detected (five congestive heart failure episodes due to rejection and four episodes of vascular occlusion, which resulted in five deaths), and mean HLR was 1.74 +/- 0.3 and 2.1 +/- 0.16 in the two groups, respectively (p less than 0.0001). No complications were noted in 193 studies of patients with HLR of less than 2.00, whereas in nine of 45 with HRL of 2.00 or greater, complications occurred (p less than 0.0001). None of the 23 patients prospectively followed since surgery who had a gradual decrease in MAA uptake during the first 3 months showed rejection-related complications, whereas persistent uptake was associated with complications in five of nine patients (p less than 0.001). CONCLUSIONS: No rejection-related complications are seen coinciding with HLR of less than 2.00, whereas patients who have complications have an HLR of more than 2.00. The early 3-month pattern of decreasing MAA uptake is associated with a clinical course free of rejection related complications, whereas a persistent pattern is a signal of the possibility of such complications. PMID- 1728486 TI - Nitroglycerin-induced coronary vasodilation in cardiac transplant recipients. Evaluation with in vivo intracoronary ultrasound. AB - BACKGROUND: Coronary artery vasomotion is altered after cardiac transplantation. The impact of accelerated transplant coronary atherosclerosis and myocardial rejection on vasomotion is not well understood. Intravascular ultrasound is a new imaging method with the ability to study real-time changes in coronary artery dimensions. METHODS AND RESULTS: Epicardial coronary artery response to nitroglycerin was studied in 32 cardiac transplant recipients (age, 47 +/- 11 years) 3 weeks to 10 years after transplantation with intracoronary ultrasound. Cross-sectional luminal area and diameter were measured at a fixed position in the left anterior descending artery immediately before and every 30 seconds for 5 minutes after 0.4 mg of sublingual nitroglycerin. Cross-sectional area increased from a baseline of 13.1 +/- 3.9 mm2 to 15.8 +/- 3.9 mm2 at maximal vasodilation; luminal diameter increased from 4.0 +/- 0.6 mm to 4.5 +/- 0.6 mm. This increase reached statistical significance (p less than 0.001) at 1.5 minutes after administration of nitroglycerin; mean maximum increase occurred at 4.5 minutes (24% for cross-sectional area and 11% for luminal diameter). Patients with biopsy proven mild or moderate concurrent rejection had a significantly blunted vasodilatory response versus the nonrejection group (9% versus 27% for cross sectional area, p less than 0.04), although a vasodilatory effect was still present. Nitroglycerin response was well preserved in patients up to 10 years after transplantation; however, there was a trend toward a decreased response in patients studied immediately after transplantation (21% versus 29%, p = 0.37). Coronary intimal thickness, as measured by ultrasound, had no impact on the vasodilatory response (R = 0.23, p = 0.34). CONCLUSIONS: Vasodilatory response to nitroglycerin in cardiac transplant recipients is attenuated during episodes of cardiac rejection. This response is preserved in long-term survivors and is independent of the degree of intimal thickening. Intravascular ultrasound provides a new method to document real-time epicardial coronary vasomotion. PMID- 1728487 TI - Quantitative angiographic morphology of the coronary artery lesions at risk of thrombotic occlusion. AB - BACKGROUND: Coronary angiography in acute myocardial infarction has revealed complicated atherosclerotic plaque and a high rate of thrombotic occlusion. However, the characteristics of lesions at high risk of subsequent occlusion are not well known. METHODS AND RESULTS: In the present study, the qualitative and quantitative angiographic features of 38 coronary artery lesions that occluded within 3 years to cause an acute myocardial infarction were compared with 64 control segments from the same patients that did not occlude. Compared with control lesions, the lesions that occluded were more likely to have a division branch originating within the stenosis (76% versus 52%, p less than 0.05). The percent lumen diameter reduction was more severe (47.5 +/- 17.8% versus 41 +/- 12.5%, p less than 0.05) and the inflow (21 +/- 10 degrees versus 16 +/- 7 degrees, p less than 0.05) and outflow (20 +/- 10 degrees versus 16 +/- 8 degrees, p less than 0.05) angles of the stenosis were steeper. Time to myocardial infarction after the angiogram interacted with the importance of these features (p less than 0.02). Thus, paired analysis of the lesions that occluded within 3 months and of the most severe control lesion from each patient showed percent lumen diameter reduction of 62.1 +/- 11.5% and 46.4 +/- 11.4%, respectively (p less than 0.001). The length of the stenosis, its asymmetry, and the irregularity of the contours did not help differentiate occlusive from control segments. CONCLUSIONS: Coronary artery lesions at high risk of thrombotic occlusion share common characteristics that favor higher shear stress and flow separation. PMID- 1728488 TI - Normalization of coronary vasomotion after percutaneous transluminal coronary angioplasty? AB - BACKGROUND: Coronary vasomotion was evaluated at rest and during bicycle exercise in 33 patients (age, 53 +/- 7 years) with coronary artery disease. In a first group of patients (n = 15), vasomotion was studied before and 4.3 +/- 2.3 months (early) after percutaneous transluminal coronary angioplasty (PTCA), whereas in a second group (n = 18), exercise coronary arteriography was performed 30 +/- 11 months (late) after successful PTCA. Patients with restenosis (percent area stenosis greater than or equal to 75% or percent diameter stenosis greater than or equal to 50%) were excluded. METHODS AND RESULTS: Luminal areas of a normal segment and the stenotic segment were determined at rest, during supine bicycle exercise, and 5 minutes after sublingual nitrate administration by using biplane quantitative coronary arteriography. Work loads before and early after PTCA were identical in group 1 and similar late after PTCA in group 2. Percent area stenosis decreased from 86% to 36% (p less than 0.001) in group 1 and from 93% to 46% (p less than 0.001) in group 2. Normal coronary arteries showed mild vasodilation during exercise before (+3%, NS versus rest), early (+7%, NS versus rest), and late after (+10%, p less than 0.05 versus rest) PTCA. Administration of sublingual nitrate was associated with significant vasodilation of the normal vessel segment before (+27%, p less than 0.001 versus rest), early (+31%, p less than 0.001 versus rest), and late (+21%, p less than 0.001 versus rest) after PTCA. In contrast, the stenotic vessel segments showed coronary vasoconstriction during exercise before PTCA (-25%, p less than 0.001 versus rest), whereas minimal vasomotion was observed early (+2%; NS versus rest) as well as late (+5%; NS versus rest) after PTCA. Individual post-PTCA (early and late) exercise data elicited vasodilation in 19, no vasomotion in four, and vasoconstriction in 10 instances. Sublingual administration of nitrate was associated with a significant increase in minimal luminal area before (+18%, p less than 0.05 versus rest), early (+24%, p less than 0.01 versus rest), and late (+16%, p less than 0.001 versus rest) after PTCA. An inverse linear correlation was found between the percent change in minimal luminal area during peak exercise and percent area stenosis at rest (r = 0.77, p less than 0.001). CONCLUSIONS: Exercise-induced stenosis narrowing is observed before PTCA but normal vasomotion is reestablished in two thirds of all patients early and late after PTCA. In one third, an abnormal reaction to exercise (i.e., vasoconstriction) persisted after PTCA, mainly in those patients with a residual area stenosis of 50% (percent diameter stenosis of 30%) or more. Thus, PTCA appears to have a salutary effect on coronary vasomotion during exercise, which, however, remains dependent on the severity of the residual stenosis. PMID- 1728489 TI - Regional oxidative metabolism in patients after recovery from reperfused anterior myocardial infarction. Relation to regional blood flow and glucose uptake. AB - BACKGROUND: Enhanced uptake of the glucose analogue 18F-fluorodeoxyglucose (FDG) in relation to flow has been proposed as an accurate method of identifying viable myocardium. The evaluation of myocardial oxidative metabolism could be an alternate way to identify reversible injury. The aim of the present study was to investigate in patients with reperfused anterior infarction whether differences in regional oxidative metabolism exist among regions with and without flow metabolism mismatch. METHODS AND RESULTS: Fifteen patients with reperfused anterior myocardial infarction were studied between 2 weeks and 3 months after the acute event. Regional myocardial blood flow (13N-ammonia; three-compartment model), oxidative metabolism (11C-acetate; monoexponential clearance), and glucose uptake (FDG, linear graphic analysis) were evaluated with dynamic positron emission tomography. Flow-metabolism patterns were used to differentiate reversibly (FDG/flow greater than 1.2) from irreversibly injured myocardium (FDG/flow less than 1.2) using circumferential profile technique. Relative 13N ammonia uptake was reduced in 71 of 90 anterior and/or septal segments, including 24 with (seven patients) and 38 without (eight patients) flow-metabolism mismatch. Acetate clearance (k), reflecting oxidative metabolism, was reduced by 51% in the center of the infarct area versus remote segments (27 +/- 12 versus 55 +/- 13 min-1.10(-3), p less than 0.001). Compared with infarct segments without flow-metabolism mismatch, segments exhibiting increased glucose uptake relative to flow had faster acetate clearance (35 +/- 14 versus 23 +/- 9 min-1.10(-3), p less than 0.01). Similarly, myocardial blood flow was better preserved in segments with flow-metabolism mismatch (54 +/- 13 versus 45 +/- 8 ml/min/100 g, p less than 0.01) compared with segments without mismatch. However, at similar levels of hypoperfusion, there was no significant difference in acetate clearance among segments with and those without flow-metabolism mismatch: 37 +/- 14 versus 41 +/- 15 min-1.10(-3), respectively. A positive correlation (r = 0.89, p less than 0.001) was found between absolute myocardial blood flow and acetate clearance, regardless of the flow-metabolism pattern. CONCLUSIONS: In patients with reperfused myocardial infarction studied between 2 weeks and 3 months after the acute event, regional oxidative metabolism is reduced in proportion to residual myocardial blood flow and does not differ significantly among similarly hypoperfused segments with and without flow-metabolism mismatch. PMID- 1728490 TI - Coronary angioplasty performed within the thrombolysis in Myocardial Infarction II study. AB - BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) of the infarct related artery was performed within 42 days of recombinant tissue-type plasminogen activator (rt-PA) administration in 1,414 of the 3,534 patients who participated in the Thrombolysis In Myocardial Infarction (TIMI) II study. Primary angiographic success was obtained in 88.7%, with bypass surgery within 24 hours in 3.3% and death within 24 hours in 0.7% of patients. By 1 year, 25.1% of the 1,414 patients had sustained one or more adverse outcomes including death (3.6%), reinfarction (8.4%), or the need for further revascularization (20%). METHODS AND RESULTS: Despite these generally favorable results, multivariate testing identified several anatomic and clinical subgroups as having an increased risk ratio (RR) for adverse outcome: Unsuccessful PTCA was more common in patients undergoing protocol-assigned PTCA within 2 hours of rt-PA administration (RR, 2.7; p less than 0.001) and in patients over age 70 years (RR, 1.7; p = 0.034). The need for further revascularization within 1 year was increased in the 30.4% of patients with multivessel disease (RR, 2.5; p less than 0.001), patients with prior angina (RR, 1.4; p less than 0.006), or those undergoing ischemia driven PTCA within 15 hours of rt-PA administration (RR, 1.7; p = 0.022). The risk of death or recurrent infarction within 1 year was increased by the presence of multivessel disease (RR, 1.6; p = 0.007) or prior angina (RR, 1.5; p = 0.014). CONCLUSIONS: These observations do not necessarily apply to patients undergoing primary PTCA (or PTCA after other thrombolytic agents); however, they do offer a unique yardstick against which to evaluate the results of PTCA in myocardial infarction. PMID- 1728491 TI - Current perspectives on the problem of sudden cardiac death. Dallas, Texas, September 24-25, 1990. PMID- 1728492 TI - General evaluation of out-of-hospital sudden cardiac death survivors. AB - Survivors of out-of-hospital sudden cardiac death may have any of a number of etiologies as the cause. Careful in-hospital evaluation is necessary for providing adequate therapy for such patients. In the great majority of survivors of out-of-hospital sudden cardiac death, such evaluation necessarily includes invasive electrophysiological testing for both diagnostic and therapeutic indications. Given the high incidence of recurrence, effective therapy, based on individualized evaluation, must be found. PMID- 1728493 TI - Role of myocardial revascularization in sudden cardiac death. AB - Extensive atherosclerotic coronary artery disease is by far the most common pathological finding in patients with sudden cardiac death, and acute myocardial ischemia is often a contributing factor. Clinical trials using beta-blockers in postinfarction patients and bypass coronary artery surgery in patients with stable coronary artery disease have demonstrated a reduction in both sudden and total cardiac mortality after intervention. Information concerning the presence and extent of coronary artery disease, global and regional ventricular function, the presence or absence of ventricular aneurysms, and whether or not ischemia is inducible influences therapeutic management. Myocardial revascularization should be considered a primary therapy in patients with critical coronary artery stenosis, significant regions of myocardium at risk, and no inducible ventricular arrhythmias at electrophysiological testing. In patients with inducible polymorphic ventricular tachycardia or ventricular fibrillation, postoperative testing is essential, since only 50% will be suppressed by coronary artery surgery alone. In patients with inducible sustained monomorphic ventricular tachycardia and scars due to prior myocardial infarction, surgical revascularization alone will usually not be sufficient to prevent postoperative induction of the same arrhythmia. There are no data to support percutaneous transluminal coronary angioplasty as the sole therapy for post-sudden death patients who have inducible ventricular tachycardia or ventricular fibrillation on electrophysiological testing. PMID- 1728494 TI - Antiarrhythmic drug therapy in the management of cardiac arrest survivors. AB - Antiarrhythmic drugs may be used as primary therapy to prevent recurrent cardiac arrest or as adjunctive treatment in patients given an implantable cardioverter defibrillator. In the latter instance, drugs are given to suppress nonlethal arrhythmias that are capable of initiating defibrillator discharge or to slow and/or decrease the number of episodes of sustained ventricular tachyarrhythmias. When used as primary therapy, drug efficacy should be judged by nonempirical methods, preferably serial electrophysiological testing. Although no study has compared noninvasive with invasive testing to determine antiarrhythmic drug effectiveness in a substantial number of cardiac arrest survivors, several investigations have demonstrated that electrophysiological testing is useful for this purpose. One important limitation of serial electrophysiological testing is that nearly 40% of cardiac arrest survivors do not have a sustained ventricular tachyarrhythmia initiated at baseline study; nonpharmacological treatment would appear preferable in these patients. Further, since a relatively high arrhythmic recurrence rate has been noted in individuals with suppressible sustained ventricular tachycardia/fibrillation during drug therapy and a left ventricular ejection fraction less than 30%, we recommend serial electrophysiological/pharmacological testing primarily for patients with inducible sustained ventricular tachyarrhythmias with an ejection fraction greater than or equal to 30%. PMID- 1728495 TI - Management of sudden cardiac death survivors. Role of surgical and catheter ablation. AB - Ventricular tachyarrhythmias are the most common arrhythmias documented at the time of sudden cardiac death. Since pharmacological therapy often does not completely suppress these arrhythmias, surgical procedures have been developed to provide an alternative mode of therapy. Coronary artery revascularization in patients with ischemic heart disease is associated with decreased sudden death mortality in patients with left ventricular dysfunction who have no history of prior cardiac arrest or ventricular tachycardia. In sudden death survivors, coronary revascularization appears effective chiefly for patients without inducible ventricular tachycardia or with inducible ventricular fibrillation. Cardiac arrest survivors with sustained monomorphic ventricular tachycardia at electrophysiological study require an ablative procedure that is usually guided by electrophysiological mapping. Current technique should permit elective operations to be carried out with a less than 10% mortality and a greater than 85% rate for suppressing ventricular tachycardia. Nonsurgical catheter ablation techniques have already shown promise in patients with slower, well-tolerated tachycardias that allow extensive catheter mapping. Application of these techniques in patients with the rapid tachycardias associated with sudden death has, to date, been very limited, but as catheter mapping and energy delivery techniques continue to evolve, they may be feasible even in these patients. PMID- 1728496 TI - Role of implantable cardioverter defibrillator therapy in the management of high risk patients. AB - Cardiovascular mortality from ventricular tachycardia (VT) and ventricular fibrillation (VF) continues to be a major health problem. Several therapeutic approaches are now available to treat patients with known VT/VF. Among the various therapeutic options are antiarrhythmic drugs, catheter or surgical ablation of VT focus, and implantable cardioverter defibrillator (ICD). The overall 2-year cardiovascular mortality is significantly reduced by ICD therapy. The ICD is particularly useful in patients with 1) no inducible but clinical VT/VF, 2) drug refractory VT/VF, and 3) VT/VF in association with left ventricular ejection fraction of less than or equal to 30%. Significant improvements in ICD therapy have already been made; these improvements include tiered antitachycardia therapy, antibradycardia pacing, lower defibrillation threshold, and longer life of generator. Further improvements are expected, including nonthoracotomy approach to defibrillation, pectoral implant, and dual chamber sensing. It is likely that with all of the advances in ICD therapy its acceptance as a therapeutic option will increase. PMID- 1728497 TI - Noninvasive identification of patients at high risk for sudden cardiac death. Signal-averaged electrocardiography. AB - Signal-averaged electrocardiography allows the detection of late potentials, which have been associated with delayed and disorganized ventricular activation. This article reviews the technique, describes the findings recorded from patients with ventricular tachyarrhythmias, and assesses the prognostic value of late potentials for ventricular tachyarrhythmias and sudden cardiac death in patients after an acute myocardial infarction. The role of signal-averaged electrocardiography in the evaluation of patients with syncope and cardiomyopathies is also briefly discussed. PMID- 1728498 TI - Role of invasive electrophysiological testing in the evaluation and treatment of patients at high risk for sudden cardiac death. AB - Invasive electrophysiological testing has contributed importantly to the objective evaluation and management of patients at high risk for sudden cardiac death. The clinical application of the technique is based on the hypothesis that the reproducible induction of ventricular arrhythmias by programmed cardiac stimulation constitutes a marker of risk for spontaneous ventricular arrhythmias and sudden death as well as an objective end point to guide the selection of antiarrhythmic therapy. The value of electrophysiological testing is well established in patients with ischemic heart disease and a history of sustained ventricular tachycardia or fibrillation and in some subsets of patients with unexplained syncope. More recently, the technique has been used by some investigators to identify individuals at high risk for sudden death among patients with recent myocardial infarction and those with left ventricular dysfunction and recurrent nonsustained ventricular tachycardia. The predictive value of the technique in patients with nonischemic heart disease is unknown. In addition to its use as an objective end point in the selection of antiarrhythmic drug therapy, invasive electrophysiological testing has advanced our knowledge of the mechanisms of life-threatening ventricular arrhythmias and contributed importantly to the development of new therapies, such as implantable arrhythmia control devices and catheter ablation techniques. PMID- 1728499 TI - Sudden cardiac death. Future approaches. AB - The purpose of the present review is to suggest future approaches to the problem of sudden cardiac death. Short-term efforts should be directed toward delivering cardiopulmonary resuscitation and electrical therapy as soon as possible after the onset of an arrest. Long-term efforts encompass four interrelated, stepwise objectives that include identification of individuals at risk, mechanisms responsible for the arrhythmias, and interventions to prevent the arrhythmias. This is then followed by large-scale testing of the interventions in high-risk populations. Present funding for such research is inadequate, and the National Heart, Lung, and Blood Institute should be encouraged to consider supporting a major research initiative to combat the problem of sudden cardiac death. PMID- 1728500 TI - Anatomic features in victims of sudden coronary death. Coronary artery pathology. AB - Study of the detailed pathology of the myocardium and coronary arteries in ambulatory subjects dying suddenly of coronary heart disease shows that they can be divided into two groups. In one group, there is atherosclerosis with a new vascular event involving coronary thrombosis, which initiates acute myocardial ischemia. In the other group, there is chronic high-grade stenosis due to atherosclerosis, but there is no recent vascular change; the myocardium in this group shows scarring from a previously healed infarction acting as a substrate for reentrant ventricular arrhythmias. A study of 168 consecutive cases of sudden coronary death in London showed 73.3% to have had a recent coronary thrombotic lesion, giving a ratio of 2.7:1 for patients with versus patients without new acute myocardial ischemia. The widely differing ratios reported in the literature probably reflect the patterns of case selection. Prodromal pain immediately before the onset of ventricular fibrillation in a patient without previous known coronary disease selects for a thrombotic cause and acute myocardial ischemia. Absence of pain in a patient known to have had a previous infarction selects for a primary arrhythmia on the basis of preexisting myocardial hypertrophy and/or scarring. PMID- 1728501 TI - Sudden cardiac death. Structure, function, and time-dependence of risk. AB - Sudden cardiac death (SCD) remains a major unresolved clinical and public health problem, accounting for more than 300,000 of the deaths in the United States annually. The ability to identify potential SCD victims is limited by the large size of the population subgroups that contain the majority of SCD victims and by the apparent time dependence of risk of sudden death. The latter refers to the tendency for SCD to follow other cardiovascular events within a high-risk period of 6-18 months after a primary cardiovascular event, with risk decreasing thereafter. The combination of time dependence and denominator pool size provides a basis for future studies to identify the higher risk individuals. Pathophysiologically, SCD can be viewed as an interaction between structural abnormalities of the heart, transient functional disturbances, and the specific electrophysiological events responsible for fatal arrhythmias. Structural abnormalities provide the anatomic substrate for chronic risk and include the myocardial consequences of coronary artery disease, left ventricular hypertrophy, myopathic ventricles, and specific electrophysiological anatomic abnormalities such as bypass tracts. The functional factors responsible for destabilizing a chronic electrophysiological abnormality include transient ischemia and reperfusion, systemic factors (e.g., electrolyte disturbances, acidosis, and hemodynamic dysfunction), autonomic fluctuations (both systemic and at a tissue level), and myocardial toxic influences such as proarrhythmic effects of various drugs. Each of these changes is able to destabilize myocardial membrane integrity, some regionally and some globally, making the heart susceptible to an electrical triggering event for ventricular tachycardia or fibrillation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728502 TI - Mechanisms of ventricular arrhythmias. AB - Ventricular arrhythmias may result from abnormalities of impulse initiation and/or impulse propagation. The former include automatic arrhythmias, which may occur at high (normal) levels of membrane potential or at low (abnormal) levels of membrane potential. They also include triggered activity, which may result from early (occurring before complete repolarization) or delayed (occurring after complete repolarization) afterdepolarizations. Arrhythmias resulting from abnormal impulse propagation may be reentrant, determined in part by anatomic or functional conduction block, or the result of reflection. The factors determining various arrhythmogenic mechanisms are discussed. PMID- 1728503 TI - Experimental models of ventricular tachycardia and fibrillation caused by ischemia and infarction. AB - Experimental animal models of ventricular arrhythmias have been developed to elucidate mechanisms of arrhythmogenesis in humans. Studies of animal models have enabled the vulnerable period of the ventricles to be discovered and characterized and should eventually lead to improved techniques of defibrillation. Animal models of ischemic heart disease and myocardial infarction have also helped investigators elucidate the mechanisms of arrhythmias occurring at each stage of infarct development and healing. Data obtained from the experimental studies have been shown to apply to the clinical situation. PMID- 1728504 TI - Sudden cardiac death in patients with chronic coronary heart disease. AB - Sudden cardiac death (SCD) is responsible for 300,000-400,000 deaths per year with a recurrence rate of up to 40% in survivors within the first 2 years. SCD often occurs in patients with chronic coronary artery disease, which is manifested by myocardial infarction and left ventricular dysfunction but is infrequently associated with acute infarction. SCD may be the initial symptom of coronary artery disease. Primary or rapid ventricular tachycardia are the most common arrhythmic causes of SCD. Endocardial mapping studies during electrophysiological study have shown areas of slowed conduction with abnormal endocardial electrograms in SCD patients with moderately damaged ventricles. SCD increases with age and occurs more frequently in men with coronary artery disease as a significant risk factor. Complex ventricular ectopy, once thought of as an independent risk factor, is not as good a predictor as poor left ventricular function for recurrence of SCD. While signal-averaged electrocardiograms can identify patients with slowed conduction, their positive predictive value for SCD is poor. Initial evaluation should be aimed at the identification of ischemia, since those patients with SCD and acute myocardial infarction do well when treated for their ischemia. The arrhythmias that are inducible during electrophysiological study are rapid and poorly tolerated. Patients with inducible ventricular tachycardia who are rendered noninducible pharmacologically have a good prognosis, whereas those who are still inducible or have no inducible arrhythmias have a high recurrence rate of SCD and should be considered for subendocardial resection when appropriate or for placement of an implantable defibrillator. PMID- 1728505 TI - Lack of relation between ventricular arrhythmias and sudden death in patients with chronic heart failure. AB - Although both asymptomatic ventricular arrhythmias and sudden death are common in patients with chronic heart failure, there is little evidence that patients who have frequent or complex ventricular arrhythmias are at increased risk of sudden death. Two hypotheses may explain the lack of an arrhythmia-sudden death relation in this disorder. First, complex ventricular arrhythmias may be a nonspecific manifestation of a dying left ventricle rather than an indication of a specific arrhythmogenic substrate. In fact, during long-term follow-up, patients with mild heart failure who have nonsustained ventricular tachycardia are more likely to develop clinical progression of the disease rather than sudden death. Second, sudden death may be related to events other than a malignant ventricular arrhythmia. The most common myocardial ischemia, whereas the terminal event in patients with an idiopathic dilated cardiomyopathy is commonly a severe bradyarrhythmia or electromechanical dissociation. Neither outcome can be predicted by a prior history of ventricular arrhythmias on ambulatory electrocardiographic monitoring. If asymptomatic ventricular arrhythmias do not lead to sudden death, then there would appear to be little reason to expect that antiarrhythmic drugs could prevent cardiac arrest in patients with chronic heart failure. This may explain why drugs that suppress ambulatory arrhythmias do not prevent sudden death in these patients, whereas interventions may reduce the risk of unexpected circulatory collapse in this disorder without suppressing ventricular ectopic activity. To make matters more complicated, the desirable actions of antiarrhythmic drugs are attenuated and their negative inotropic and proarrhythmic actions are enhanced in patients with severe cardiac dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728506 TI - Sudden cardiac death in hypertrophic cardiomyopathy. AB - Sudden death is an important facet of the natural history of hypertrophic cardiomyopathy. Issues related to the nature and etiology of sudden death and the prospective identification of those patients at increased risk have been the subject of intense study, and consequently, our concepts and knowledge have continued to evolve. Occurrence of sudden death has been reported to be about 2 3% per year in a hospital-based referral population and, although described in most age groups, is most common in older children and young adults. The typical profile is that of a young asymptomatic patient with substantially increased left ventricular wall thickness who dies while performing sedentary or modest physical activities; however, a substantial minority die suddenly during or just after severe exertion, including those participating in competitive athletics. Other risk factors that have been identified in patients with hypertrophic cardiomyopathy include nonsustained ventricular tachycardia on ambulatory electrocardiogram, a strong family history of sudden death, and prior occurrence of syncope (or cardiac arrest). Electrophysiological studies have shown that most patients judged at increased risk for sudden death have sinoatrial or His Purkinje conduction disease or inducible supraventricular or ventricular tachycardia; inducibility of a sustained ventricular arrhythmia is associated with prior occurrence of syncope or cardiac arrest. Hemodynamic and electrophysiological studies in patients with hypertrophic cardiomyopathy have demonstrated several potential mechanisms for cardiac arrest or sudden death, including atrial arrhythmias associated with hypotension, bradyarrhythmias, and ventricular tachyarrhythmias, all of which can be exacerbated in the presence of left ventricular outflow tract obstruction or myocardial ischemia. PMID- 1728507 TI - Sudden cardiac death in the pediatric population. AB - Sudden death in children as in adults is usually due to cardiac disease. Sudden death in the pediatric population may be divided into the sudden infant death syndrome, sudden death in previously apparently healthy children, and sudden death in patients with known cardiac disease. The sudden infant death syndrome is not proved to be due to a cardiac cause and may well be due to central nervous system and/or pulmonary causes. However, interest remains in the cardiac hypothesis. Recent work from our laboratory shows that screening for prolonged QT interval in normal infants is not likely to detect those prone to sudden infant death syndrome. In children with apparently normal hearts, symptoms of syncope or palpitation should be given close attention. Detailed electrocardiography and echocardiography will detect many, but not all, children with subtle forms of heart disease. Vigorous treatment may prevent sudden death in many of these children. Some sort of screening program should be devised for varsity athletes. Children with congenital heart defects are now, for the most part, corrected early in life, so that the congenital heart defect itself rarely causes sudden, unexpected death. The residua and sequelae of the heart defect and the surgery to repair it, however, may lead to sudden death. Improvements in surgical technique and earlier repair of congenital cardiac defects will ameliorate this problem. Prospective evaluation of postoperative patients and attention to dysrhythmias can prevent sudden deaths in those who are prone to them. PMID- 1728508 TI - Electrolyte abnormalities underlying lethal and ventricular arrhythmias. AB - It is well known that changes in serum potassium cause ventricular arrhythmias as a result of clearly documented changes in the electrophysiological characteristics of single fibers. Hypopotassemia induced by thiazide and loop diuretics may contribute to the incidence of sudden cardiac death in patients with hypertension and those with congestive heart failure. In addition, hypopotassemia appears to be an independent risk factor for lethal ventricular arrhythmias occurring in the setting of acute myocardial infarction and contributes significantly to arrhythmias associated with starvation and alcoholism. The increase in myocardial extracellular potassium that occurs in the ischemic zone after coronary occlusion is clearly a major factor in the genesis of lethal ventricular arrhythmias that occur in this setting. A decrease in serum magnesium is also believed to be arrhythmogenic, and magnesium depletion is thought to play a role in many of the arrhythmias associated with hypopotassemia. Moreover, the administration of magnesium salts may be effective in the management of life-threatening ventricular arrhythmias. However, definite evidence establishing a causal relation between ventricular arrhythmias and hypomagnesemia or intracellular magnesium depletion is lacking. Changes in intracellular calcium contribute to the arrhythmias associated with acute ischemia and with reperfusion and may be important in the genesis of ventricular tachycardia induced by exercise and by digitalis. Thus, electrolyte and metabolic abnormalities clearly underlie lethal ventricular arrhythmias in a wide variety of clinical situations and should be routinely considered as potential etiologic factors in patients with life-threatening ventricular arrhythmias, particularly those with hypertension and congestive heart failure who are receiving thiazide and loop diuretics. PMID- 1728509 TI - Autonomic nervous system and sudden cardiac death. Experimental basis and clinical observations for post-myocardial infarction risk stratification. AB - The analysis of the autonomic control of the heart, by means of indirect markers, may represent a new approach for identifying patients at higher risk for sudden cardiac death after a myocardial infarction. This possibility is based on the evidence that autonomic responses during acute myocardial ischemia are a major determinant of the outcome (i.e., occurrence of ventricular fibrillation or survival). Specifically, sympathetic activation can trigger malignant arrhythmias, whereas vagal activity may exert a protective effect. Several experimental observations have provided new insights on the relation between sympatho-vagal interactions and the likelihood for the occurrence of ventricular fibrillation. In an established experimental model for sudden death involving conscious dogs with a healed myocardial infarction, either depressed reflex chronotropic responses during a blood pressure rise or reduced variability of heart rate (respectively, markers of reflex and tonic cardiac vagal activity) identify dogs at greater risk to develop malignant arrhythmias during a new ischemic episode. In anesthetized cats, direct neural recording of vagal activity to the heart confirmed that vigorous reflex vagal activation during acute myocardial ischemia is associated with protection from ventricular fibrillation. Furthermore, in these experiments the reflex neural response to acute myocardial ischemia was predicted by the analysis of baroreflex sensitivity. The antifibrillatory effect of vagal activation is confirmed by the prevention of ventricular fibrillation during acute ischemia in dogs susceptible to sudden cardiac death by direct electrical stimulation of the right vagus. The clinical counterpart of these experimental data lies in three separate prospective studies showing a higher cardiac mortality in patients who after a myocardial infarction have a depressed baroreflex sensitivity or a decreased heart rate variability. A definitive answer on the role that the analysis of markers of cardiac vagal activity may play in risk stratification of patients with coronary artery disease should be provided by Autonomic Tone and Reflexes After Myocardial Infarction (ATRAMI), an ongoing prospective study. In ATRAMI, baroreflex sensitivity and heart rate variability will be assessed within 20 days after a myocardial infarction in 1,200 patients enrolled in Europe, U.S.A., and Japan with a minimum follow up of one year. PMID- 1728510 TI - Sudden arrhythmic death without overt heart disease. AB - Nine patients (eight males) are reported with one or more episodes of circulatory collapse in the absence of overt heart disease or other known causes of arrhythmias; sudden arrhythmic death occurred in one of these patients. Age at first episode ranged from 16 to 41 (mean, 28) years. In seven patients, ventricular fibrillation was documented at the time of resuscitation. One patient had ventricular flutter. In the remaining patient, documentation of the arrhythmia during the collapse was not available. Four patients had frequent early ventricular premature beats, and in three of these patients, they were accompanied by episodes of rapid nonsustained polymorphic ventricular tachycardia. Failure to suppress this ectopic activity by drug therapy seems to be of prognostic significance. Of the three patients showing persistence of frequent early ventricular premature beats, one died suddenly, and two had recurrences of symptomatic arrhythmic episodes. The value of noninvasive and invasive tests in the management of these patients is not clear, with the exception of exercise testing in patients with exercise-related arrhythmias and long-term electrocardiographic monitoring in patients with frequent spontaneous ventricular ectopic activity. Follow-up varied from 21 to 192 (mean, 84) months. One patient died suddenly 21 months after his first collapse. Selection of antiarrhythmic drug therapy was largely empirical. In view of the relative rarity of sudden arrhythmic death in the absence of heart disease and the many uncertainties about its mechanism(s) and management, a worldwide registry of these patients is suggested. PMID- 1728511 TI - Community-based interventions for sudden cardiac death. Impact, limitations, and changes. AB - Since 1970, Seattle Fire Department paramedics have treated 5,120 victims of out of-hospital ventricular fibrillation (VF). During the past decade, there was an impressive decline in the annual incidence of VF, probably reflecting a general reduction in age-adjusted mortality attributed to coronary heart disease. Since 1975, annual survival rates to hospital discharge fluctuated between 24% and 33%, averaging 28.9%. In spite of continuing efforts to improve basic and advanced life support, survival rates have not risen concomitantly. Since the early 1970s, average ages of victims have increased from 63.4 to 66.1 years (p less than 0.0001). Additionally, in survivors of VF arrest, habitual cigarette smoking has become much less frequent (48% versus 31%, p less than 0.0001). Longevity of VF survivors has improved in recent years, with 1- and 5-year survival rates increasing from 74% and 44%, respectively, for those resuscitated during 1970 1975 to 83% and 57%, respectively, for those resuscitated during 1982-1987 (p less than 0.0001). It is likely that medical or surgical therapy and improved hygienic measures have contributed to the better outcomes. The vast majority of resuscitated victims have not had symptomatic ventricular arrhythmias before VF. Accordingly, current efforts to control such arrhythmias will not have an important impact on the community incidence of sudden cardiac death. Successful strategies for further containment will likely be those that address the problem of coronary atherogenesis, although medical and surgical therapies may also have a role. Additionally, it is timely to evaluate the widespread use of automated defibrillators by persons other than emergency medical technicians or paramedics. PMID- 1728512 TI - Intestinal manometry--technical advances, clinical limitations. PMID- 1728513 TI - Effect of selective and nonselective muscarinic blockade on cholecystokinin induced gallbladder emptying in man. AB - In this study we investigated the effect of selective (M1) and non-selective (M1 and M2) pharmacologic blockade of muscarinic receptors on cholecystokinin-induced gallbladder emptying. After validating the method of study, the gallbladder function was evaluated in 15 normal volunteers by quantitative biliary scintigraphy, and the effect of intravenous atropine (0.15 mg/10 kg) and pirenzepine (10 mg) was analyzed in each subject. Atropine significantly reduced the ejection period and the ejection fraction of gallbladder evacuation. Pirenzepine reduced the ejection period, but the ejection fraction remained unchanged. We conclude that the effect of cholecystokinin on gallbladder motility is mediated through muscarinic receptors. Our results suggest that M2 receptors, but not M1 receptors, are involved in this response. PMID- 1728514 TI - Altered Na+ and Cl- flux during diet-induced mixed gallstone formation in the prairie dog. AB - Recent studies suggest that altered gallbladder absorptive function may be an important and previously unrecognized factor in the pathogenesis of experimentally induced gallstones. The present study was designed to define the specific changes in gallbladder epithelial ion transport that occur during mixed gallstone formation. Fifteen prairie dogs were fed either control or corn-alfalfa chow for six months. No control animals developed gallstones or crystals. Three of eight corn-alfalfa-fed animals had large black stones, and the remaining five had crystals ("pregallstone" group). Corn-alfalfa-fed animals had significant increases in gallbladder bile cholesterol, phospholipids, and calcium as compared to controls. Gallbladders were removed and mounted in a Ussing chamber for electrophysiologic and ion flux studies. Gallbladders from animals fed corn alfalfa demonstrated significant decreases in short-circuit current and potential difference as compared to controls (P less than 0.05). 22Na and 36Cl were used to determine unidirectional ion fluxes. While net ion fluxes were similar in pregallstone animals and controls, stone-forming animals exhibited a significant decrease in net Na+ flux and a significant reversal in the direction of net Cl- flux (from secretion to absorption) as compared to controls (P less than 0.05). These data indicate that mixed gallstone formation is associated with alterations in gallbladder ion transport. The role of these changes in the pathogenesis of mixed gallstones remains to be determined. PMID- 1728515 TI - Microscopic examination of bile directly collected during endoscopic cannulation of the papilla. Utility in patients with suspected microlithiasis. AB - The usefulness of microscopic examination of pure bile directly collected from the biliary tract during endoscopic retrograde cholangiography and without hormonal simulation was prospectively evaluated in 72 patients. According to clinical, biochemical, ultrasonographic, and radiographic data, the patients were separated into two groups: group 1, patients with proven stones (N = 50), and group 2, patients with suspected microlithiasis presenting symptoms suggestive of cholelithiasis but without evidence of macroscopic stones at echography or cholangiography (N = 22). Cholesterol crystals and/or bilirubinate granules were observed (eg, positive examination) in the bile of 41 of the 50 patients of group 1 (82%). Among patients of group 2, seven (32%) had a positive bile examination: cholecystectomy (N = 2) or endoscopic sphincterotomy (N = 5) disclosed minute stones in all cases. In the 15 patients of group 2 with a negative bile examination, cholecystectomy (N = 3), sphincterotomy (N = 2), and clinical (and/or echographic) 20-month follow-up (N = 9) revealed biliary lithiasis in only one patient, in whom recurrent cholangitis led to disclosure of one bile duct stone. According to these results, microscopic examination of bile samples collected during endoscopic retrograde cholangiography exhibited a sensitivity and a specificity for cholelithiasis recognition of 82.7% and 100%, respectively, with a positive predictive value of 88%. We conclude that the accuracy of this method makes it useful to investigate and manage patients with suspected microlithiasis. PMID- 1728516 TI - Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. AB - A newly isolated strain of a species of Lactobacillus of human origin, designated GG (Lactobacillus GG), has been studied to determine its ability to survive in the human gastrointestinal tract. When fed to 76 volunteers as a frozen concentrate or as a fermented preparation in milk or whey, Lactobacillus GG was recovered in the feces of all subjects receiving the fermented milk or whey and in 86% receiving the frozen concentrate when a single fecal specimen was cultured. The organism was also present in the feces of subjects concurrently receiving ampicillin. After terminating feeding of the organism, Lactobacillus GG persisted in the feces of 87% of volunteers four days later and in 33% of subjects seven days later. Lactobacillus GG lowered fecal bacterial beta glucuronidase activity by approximately 80% in volunteers given the organism for four weeks. These studies demonstrate that Lactobacillus GG can survive and temporarily colonize the human gastrointestinal tract and can affect the metabolic activity of the resident microflora. PMID- 1728517 TI - Effect of yogurt on clindamycin-induced Clostridium difficile colitis in hamsters. AB - Yogurt exhibits in vitro bactericidal activity against a variety of pathogenic microorganisms, including Clostridium difficile. In the present studies, we tested whether yogurt ingestion could prevent or ameliorate antibiotic associated colitis in the clindamycin-treated hamster model. Male golden Syrian hamsters were given 5 mg/kg clindamycin subcutaneously 24 hr before and 6 hr following inoculation with 0.5 ml of less than 10, 10(3), 10(5), or 10(6) CFU/ml of C. difficile. Hamsters in the control group ingested chow and water ad libitum, whereas the experimental group ingested chow and a 1:1 (v/v) mixture of yogurt and water ad libitum, beginning 24 hr before the first injection of clindamycin and continuing throughout the course of the study. Animals were monitored for colonization with C. difficile, pathological evidence of colitis, and death. Mortality was 100% in yogurt-treated animals, and all animals showed histological changes of severe colitis. Fecal and intestinal segment cultures were positive for C. difficile in all animals. Thus, in the hamster model, we found no evidence to support the possible efficacy of yogurt in the prevention of C. difficile colitis. PMID- 1728518 TI - Cecal inflammatory fibroid polyp presenting with chronic diarrhea. A case report and review of the literature. PMID- 1728519 TI - Adenocarcinomas arising in tongues or short segments of Barrett's esophagus. AB - The diagnosis of Barrett's esophagus is established when the esophageal mucosa is lined by 2-3 cm of columnar epithelium or when specialized (intestinal type) columnar epithelium of any length is present. Emphasis is frequently placed on long segments of Barrett's because these patients reportedly are at higher risk of developing adenocarcinoma than patients with shorter segments. We present four cases of adenocarcinoma that arose in tongues or short segments (less than 2 cm) of specialized columnar epithelium near the gastroesophageal junction. We emphasize the need for biopsy of minimal appearing abnormalities in this area, and we suggest that histologic subtype, rather than length of involvement, be the major criterion for establishment of Barrett's esophagus. PMID- 1728520 TI - Maturation of antroduodenal motor activity in preterm and term infants. AB - Previous studies have shown that duodenal motility patterns differ in preterm and term infants, but antral motor activities were not compared. Using a validated, low-compliance, continuous-perfusion, neonatal manometric system, antral and duodenal motility was studied in 19 preterm and nine term infants. Antral motility consisted of isolated single contractions and clustered phasic contractions in term and preterm infants. There were no differences in the occurrence or amplitude of antral activity between the two groups of infants. Thus, there was no change of antral motor activity with advancing gestational age. As has been shown in other previous studies, however, intestinal motor characteristics were more immature in preterm than term infants; clustered phasic contractions occurred more frequently (P less than 0.02) and were of shorter duration (P less than 0.02) and lower amplitude (P less than 0.005). Duodenal clusters were significantly less common, while their amplitudes were significantly increased with increasing gestational age. The proportion of antral clusters that were temporally associated with duodenal activity was significantly lower in preterm infants than in term infants (P less than 0.001). Moreover, the degree of association of antral and duodenal activity increased significantly with gestational age (r = 0.5, P = 0.006). These data show that fasting antral motor activity per se is comparable in preterm and term infants; they also suggest that the temporal association of antral and duodenal activity develops in association with progressive changes in duodenal motor activity in the preterm infant. PMID- 1728521 TI - Dysphagia lusoria caused by persistent right aortic arch with aberrant left subclavian artery and diverticulum of Kommerell. AB - It requires a high index of suspicion to make the diagnosis of dysphagia lusoria. Clinically, these adults will present with symptoms of intermittent solid food dysphagia, and a mediastinal abnormality may be seen on chest x-ray. Noninvasive imaging of the chest with either computerized tomography or magnetic resonance scanning are excellent methods for evaluating the mediastinum for solid tumors or vascular anomalies that can cause extrinsic esophageal compression. Dysphagia lusoria caused by a persistence of the right embryologic aortic arch and diverticulum of Kommerell with an aberrant left subclavian artery may be satisfactorily managed by dietary modification when the symptoms are mild. PMID- 1728522 TI - Granulomatous hepatitis due to combination of amoxicillin and clavulanic acid. AB - We report the case of a patient with amoxicillin-clavulanic acid-induced hepatitis with histologic multiple granulomas. This type of lesion broadens the spectrum of liver injury due to this drug combination, mainly represented by a benign cholestatic syndrome. The association of granulomas and eosinophilia favor an immunoallergic mechanism. As penicillin derivatives and amoxicillin alone are known to induce such types of lesions, the amoxicillin component, with or without a potentiating effect of clavulanic acid, might have a major role. PMID- 1728523 TI - Hemoperitoneum in the setting of metastatic cancer to the liver. A report of two cases with review of the literature. AB - Two cases of hemoperitoneum occurring as a result of hepatic rupture due to metastatic neoplasms are presented. They represent examples of a striking and devastating but fortunately uncommon entity. The variety of primary neoplastic sites is diverse. Several possible mechanisms have been put forward to explain the event of hepatic rupture itself. Finally, it is important to note the uniformly poor survival rates following hepatic rupture despite therapy. PMID- 1728524 TI - Nitroglycerin in situ reduces upper anal canal pressure. PMID- 1728525 TI - Starch digestibility in vivo and in vitro. PMID- 1728526 TI - Antroduodenal manometry. Usefulness and limitations as an outpatient study. AB - We performed fasting and postprandial recordings of antroduodenal manometry in 21 normal volunteers, 13 patients with insulin-dependent diabetes mellitus and gastrointestinal symptoms, and 11 patients with the irritable bowel syndrome. None of the patients or volunteers had previously undergone an intestinal intubation study. Recordings could not be obtained from four of the diabetic patients due to failure to intubate the pylorus. Catheter migration led to incomplete antral data in a further 21% of all recordings. Due to the wide variations demonstrated by the normal volunteers, parameters of either the migrating motor complex (MMC) or the fed response could not differentiate between either of the patient groups and/or the controls. Similarly, while abnormal patterns of either fasting or postprandial motility were common in the diabetic patients, manometry had a sensitivity of only 67% in comparison to the less invasive radionuclide gastric emptying study. Furthermore, manometry failed to identify any diagnostic abnormality in irritable bowel patients; in particular, the incidence of "clustered" contractions was similar in all three groups. We conclude that short duration antroduodenal manometry is of limited diagnostic usefulness due to the difficulties in pyloric intubation in the presence of a dilated stomach and the intrinsic variability in normal motor patterns, perhaps excerbated by the stressful effects of the procedure itself in tube-naive subjects. PMID- 1728527 TI - Placebo-controlled trial of oral 5-ASA in relapse prevention of Crohn's disease. AB - Treatment of Crohn's disease (CD) in clinical remission is still a debated issue. Previous studies have shown a high risk of relapse for patients with CD in clinical remission (CDAI less than 150) but with some abnormally high laboratory parameters as well as a possible beneficial role of low-dosage steroid treatment in this group of patients. Furthermore, good results have been reported on the efficacy of 5-aminosalicylic acid (5-ASA) in moderately active CD. In our study we verified the efficacy of a slow-release oral 5-ASA preparation in preventing relapses in a group of patients in clinical remission but with raised laboratory parameters. Forty-four patients were randomized in a double-blind manner to receive either 5-ASA (2 g/day) or placebo for four months. Location of disease and previous steroid treatment were similar in both groups. One patient in the 5 ASA group discontinued the drug because of uterine bleeding. During the study period, 13 of 22 placebo-treated patients and 11 of 21 5-ASA-treated patients relapsed (corrected chi square = NS). Considering the location of disease, three of 10 patients in the 5-ASA group and six of nine patients in the placebo group with ileal CD relapsed (therapeutic gain with 5-ASA: 36.6%; 95% allowance for error from -6% to 79.2%). Moreover, in seven patients with ileal CD who remained in remission, we found a statistically significant decrease in alpha 1 acid glycoprotein and C-reactive protein from the second month of the study.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728528 TI - Role of T lymphocytes in intestinal mucosal injury. Inflammatory changes in athymic nude rats. AB - To determine the role of T cells versus mast cells in mucosal injury, we documented structural and functional changes in the intestine of congenitally athymic nude rats during infection with the enteric parasite, Nippostrongylus brasiliensis. Studies were conducted at days 4, 7, 10, and 21 postinfection; controls were uninfected. Villus damage was indicated by morphological abnormalities at days 7, 10, and 21 and reduced activities of disaccharidase enzymes at days 10 and 21. The activity of the proliferative enzyme, thymidine kinase, was increased only at day 21, at which time the crypts were elongated. Epithelial permeability increased significantly: 5-hr recovery (in urine and blood) of the probe molecule, [51Cr]EDTA, following injection into ligated jejunal segments, was elevated at days 7 and 10. Uptake of a protein antigen, ovalbumin, from lumen to blood followed a similar pattern. No evidence of functional T cells was demonstrated. However, mucosal mast-cell activation was indicated by elevated serum levels of rat mast-cell protease II at days 7 and 10. We conclude that the absence of thymus-derived T cells does not preclude mucosal damage involving impaired barrier and digestive function. Mucosal mast cells may be involved in causing the injury in this model. PMID- 1728529 TI - Activity distribution of seven digestive enzymes along small intestine in calves during development and weaning. AB - Ten groups of calves were used to study the changes in activity levels and distribution of seven hydrolases in the intestinal mucosa during development and weaning. The calves in the first group were sacrificed at birth while those in the remaining nine groups were either milk-fed until slaughter on days 2, 7, 28, 56, 70, and 119; or weaned between days 28 and 56 and then slaughtered on days 56, 70, and 119, respectively. The small intestine was immediately cut off and divided into five segments, ie, duodenum, proximal jejunum, median jejunum, distal jejunum, and ileum. In the milk-fed animals, the activity levels of aminopeptidases A and N, alkaline phosphatase, lactase, and isomaltase were maximum at 2 days of age, and then declined sharply between days 2 and 7 but did not change significantly thereafter. By contrast, the maltase activity increased between days 7 and 119, while no sucrase activity was detected. Weaning resulted in a decrease in the activity of lactase and an increase in that of aminopeptidase N, maltase, and isomaltase. The distribution of all these enzymes along the small intestine was slightly influenced by age but not at all by weaning. PMID- 1728530 TI - Effect of base precursors on water and electrolyte transport during oral hydration solution perfusion in secreting rat intestine. AB - In situ steady-state, single-pass small intestine perfusions in rats were carried out to compare the effect of the bicarbonate and citrate World Health Organization oral rehydration solutions and a base precursor-free solution on intestinal water and electrolyte transport after inducing intestinal secretion with purified heat-stable Escherichia coli enterotoxin. When toxin was not perfused, the rates of water, sodium, and bicarbonate absorption were significantly greater from the bicarbonate-containing solution than from the citrate or base precursor-free solutions. Chloride absorption was greater from the base precursor-free solution, but this might reflect the higher chloride concentration of the perfusate. When toxin was perfused, there was no significant difference among the solutions in the rates of water, potassium, or chloride absorption. Sodium absorption occurred at significantly greater rates from both the bicarbonate and the base precursor-free solutions than from the citrate solution. Base precursor-containing solutions may not provide any advantage over a base precursor-free solution in stimulating water and sodium absorption in 5' cyclic guanosine monophosphate mediated acute diarrhea. PMID- 1728531 TI - Jejunal secretion of secretory immunoglobulins and gliadin antibodies in celiac disease. AB - The aim of this study was to determine the secretion of secretory immunoglobulins and gliadin antibodies in the small bowel in celiac disease. Twenty-four patients were investigated by perfusion of a defined jejunal segment. Four of the patients studied had a serum IgA deficiency and had no measurable amounts of secretory IgA in the perfusion fluid. The other patients demonstrated a significant increase in the jejunal concentration of secretory IgA (median 28.5 mg/liter) compared with healthy controls (median 16 mg/liter, N = 16) and of IgM, celiac (median 12.3 mg/liter) compared to healthy controls (median 6.8 mg/liter, N = 16). Jejunal IgA gliadin antibodies were detected in all patients except those with an IgA deficiency. All patients had jejunal IgM gliadin antibodies, but none of the patients had measurable jejunal IgG gliadin antibodies. A positive correlation was detected between serum and jejunal IgA gliadin antibody levels in the celiac patients, (P less than 0.01). Calculation of the ratio between gliadin antibodies and total levels of IgA and IgM in serum and jejunal perfusate demonstrated that the jejunal synthesis of gliadin antibodies of IgA and IgM type is both more pronounced and persistent than the systemic humoral immune response to gliadin. PMID- 1728532 TI - Impaired intestinal electrolyte transport in rats infested with the common parasite Syphacia muris. AB - An incidentally discovered infestation with the nematode Syphacia muris of cecum and colon in spontaneously hypertensive (SHR) and normotensive control (WKY) rats was investigated over a two-year period. Infestation rates in WKY were higher than in SHR, while clinical signs as well as histological changes of colonic tissues were absent in both strains. In vivo net water absorption (microliter/hr/cm2) in control worm-free SHR turned into secretion in infested rats, ie, from 74.2 +/- 23.2 to -7.5 +/- 35.0 (P less than 0.001); this corresponded with a decrease in net absorption (mumol/hr/cm2) of Na from 18.5 +/- 2.4 to 9.3 +/- 4.3 (P less than 0.001) and of Cl from 14.0 +/- 3.2 to 3.2 +/- 5.7 (P less than 0.001). In WKY, net water absorption decreased from 112.2 +/- 23.2 to 48.0 +/- 25.1 (P less than 0.001) and Na and Cl absorption from 22.3 +/- 3.1 to 16.0 +/- 4.2 (P less than 0.005) and from 19.4 +/- 2.7 to 10.9 +/- 4.7 (P less than 0.005), respectively. Antihelminthic treatment with 0.007% pyrvinium pamoate in the ration (four weeks on, six months off) eradicated Syphacia muris in both rat strains. Body weight gain of young rats on normal and pyrvinium pamoate substituted diet studied over 18 months was similar, indicating a good tolerance of the treatment. It is concluded that results obtained during comparative intestinal transport studies between SHR and WKY may not only be impaired but also significantly distorted by Syphacia muris infestation as SHR appear to be more susceptible to effects induced by this common parasite than WKY. PMID- 1728533 TI - Functional analysis of peripheral blood lymphocytes isolated from patients with chronic hepatitis type B. AB - Cell-mediated immunity, evaluated by lymphocyte proliferation and expression of the activation antigen interleukin-2 receptor in response to mitogens such as phytohemagglutinin and concanavalin-A, has been reported to be defective in chronic hepatitis B virus carriers. However, no definite conclusion on the functional state of T cells from these patients can be drawn. In the present study, we have investigated the expression of a wide set of lymphoid activation molecules as well as the proliferative response of peripheral blood lymphocytes isolated from patients with chronic hepatitis type B after in vitro stimulation with monoclonal antibodies to both the T-cell receptor-CD3 complex and the CD2 molecule, which are the two main T-cell activation pathways. Our findings show that peripheral T lymphocytes from patients with chronic hepatitis type B express the activation antigens 4F2 molecule, interleukin-2 receptor, and activation inducer molecule (AIM) antigen, and proliferate normally after specific stimulation through either the T-cell receptor-CD3 complex or the CD2 molecule. These results suggest that the peripheral blood T cells of patients with chronic hepatitis B are fully operative and functionally competent in vitro. PMID- 1728534 TI - Exocrine and endocrine functional reserve in the course of chronic pancreatitis as studied by maximal stimulation tests. AB - Thirty patients suffering from chronic alcoholic pancreatitis (18 calcified) were entered into a study of exocrine and endocrine pancreatic function based on two maximal stimulation tests, namely the secretin-cerulein test and the glucagon test with serum assays of C peptide. The glucagon test was also performed in 19 control subjects. In addition, 10 chronic pancreatitis patients and nine controls were subjected to an oral glucose tolerance test (OGTT) with serum insulin determinations. C peptide basal values were decreased only in patients with severe pancreatic exocrine insufficiency (P less than 0.001), while delta C peptide values were also reduced in patients with moderate exocrine insufficiency (P less than 0.001). Lipase output correlated very well with delta C peptide values (P less than 0.001). While serum insulin levels during OGTT and C peptide basal values showed no significant differences between the chronic pancreatitis and control groups, delta C peptide values were significantly reduced in chronic pancreatitis patients (P less than 0.02). Both endocrine and exocrine function are impaired in chronic pancreatitis, as demonstrated by maximal tests, even in early stages of the disease. PMID- 1728535 TI - Methyl tert-butyl ether in the endoscopic treatment of common bile duct radiolucent stones in elderly patients with nasobiliary tube. AB - Methyl tert-butyl ether is an effective dissolution agent for cholesterol stones. The aim of this work was to evaluate the effect of methyl tert-butyl ether on radiolucent common bile duct stones in patients in whom endoscopic extraction has failed. From September 1985 to September 1987, 1374 patients underwent endoscopic retrograde cholangiopancreatography in our Liver Unit. An endoscopic sphincterotomy was indicated in 195 patients with common bile duct (CBD) stones because of an age over 65 years and/or surgical contraindications. Endoscopic sphincterotomy was efficient in 187 patients, allowing complete stone removal in association with conventional endoscopic methods and mechanical lithotripsy in 170 patients. Twelve of the 17 patients with failure of conventional endoscopic treatments were either older than 75 years (11 patients; mean age, 86 +/- 4.5 years) or exhibited a surgical contraindication. Stones completely obstructed CBD in six patients and had a diameter exceeding 25 mm in the six other patients. These subjects were selected for stone dissolution by methyl tert-butyl either (MTBE) according to the following protocol. MTBE was directly infused into CBD through a nasobiliary catheter, twice daily for 4-13 days (mean, seven days). Bile duct opacification, repeated after MTBE treatment, revealed the complete disappearance of CBD stones in one patient, a decrease in stone size in five patients and no change in the six other patients. MTBE treatment was well tolerated except in three patients who complained from transient abdominal pains and nausea. At the second attempt of endoscopic treatment, CBD stones were found to be softened and easily broken up, allowing a complete clearance in six patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728536 TI - Complications of chest thump for termination of supraventricular tachycardia in children. AB - We report on two cases of mechanical termination of supraventricular tachycardia by chest thump which were followed by serious complications. In a 3-year-old boy with an otherwise normal heart, incessant supraventricular tachycardia was converted to sinus rhythm by a single precordial thump. This, however was followed by thrombo-embolic infarction of the left-sided middle cerebral artery. In another case of a 9-year-old girl, recurrent episodes of supraventricular tachycardia were associated with Ebstein anomaly of the tricuspid valve. Chest thump was successful in terminating supraventricular tachycardia but induced a short run of ventricular tachycardia which terminated itself and was then followed by sinus rhythm. It is concluded that even a slight precordial thump implies undetermined risks in the acute management of supraventricular tachycardia in children and should therefore be abandoned in favour of other methods. PMID- 1728537 TI - Is testosterone therapy for boys with constitutional delay of growth and puberty associated with impaired final height and suppression of the hypothalamo pituitary-gonadal axis? AB - We report the treatment of 44 boys with constitutional delay of growth and puberty (CDGP) at a mean chronological age of 14.3 years (range, 12.4-17.1) and bone age of 12.1 years (range, 9.1-15.0). All were below the 3rd height percentile for chronological age. They received monthly intramuscular injections of depot testosterone esters (50 mg) for a mean period of 0.35 years (range, 0.25 0.5). Means (SD) height velocity was 4.5 (1.5) cm/year during a pretreatment period of 0.5 years. During a period of 0.9 years which included the period of treatment with depot testosterone, mean growth velocity increased to 8.8 (1.9) cm/year (P less than 0.001). In the initial 1.8 years following the cessation of treatment growth velocity was sustained at 7.0 (1.7) cm/year. Pretreatment height standard deviation score (SDS) for bone age was -0.89 and this gradually reduced over the next 1.5 years to a minimum of -1.48. Thereafter, height SDS for bone age gradually increased to attain a value of -1.2, 3 years after the commencement of therapy (P less than 0.02). The same pattern of an initial decrease, followed by an increase, in height prediction was also observed when TW2 height prediction was analysed. Sexual maturation progressed during treatment, with mean testicular volume increasing both during and after treatment, confirming the diagnosis of CDGP. The time interval for progress through puberty was shorter in boys with testosterone therapy than in the normal population. The mean duration of puberty was 2.2 years compared to 3.3 years in normal boys. We conclude that low-dose depot testosterone treatment is safe and effective for boys with CDGP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728538 TI - Metabolic pigmentary retinopathies: diagnosis and therapeutic attempts. AB - Retinal degeneration in children occurs in errors of lipid, peroxisomal and mitochondrial (including respiratory chain) metabolism. In this review the most frequent inborn errors of metabolism with retinal degeneration are discussed including abetalipoproteinaemia, classical Refsum disease, neuronal ceroid lipofuscinosis, hydroxydicarboxylic aciduria, Sjogren-Larsson syndrome, infantile Refsum disease, Kearns-Sayre syndrome and gyrate atrophy. These metabolic disorders must be differentiated from those with retinal degeneration but without known metabolic basis. In patients with such a disorder metabolic investigations should be considered whenever atypical manifestations are encountered. PMID- 1728539 TI - Urinary excretion of 17-hydroxypregnanolones in patients with different forms of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. AB - To improve diagnostic criteria in different (classical salt-wasting (SW), classical simple virilizing (SV) and non classical late onset (LO)) forms of congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we investigated the urinary excretion of 17-hydroxypregnanolones (17OH-PO(5 beta) and (5 alpha)), 15 beta-hydroxypregnanolone(15 beta OH-PO), pregnanetriol(PT) and 11-oxo-pregnanetriol (11-O-PT) compared to hydrocortisone metabolities. During the 1st month of life newborn infants with CAH-SW excreted from barely detectable to very large amounts of 17OH-PO(5 beta), 15 beta OH-PO and PT, and, in 12 of 14 cases, also 11-O-PT in their urines. From the 1st to the 28th day of life, cortisol metabolites were virtually absent in urines of CAH-SW infants. This was in contrast of 36 healthy newborn infants. We measured the excretion of 17OH-PO(5 alpha) in children with CAH of whom 19 patients with CAH-SV had a median 17OH PO(5 alpha) excretion of 1110 micrograms/day (range: 152-5515). In 21 patients with CAH-LO, median excretion of 17OH-PO(5 alpha) was 294 micrograms/day (range: 66-1273). Besides the conventional metabolites of 17-hydroxyprogesterone (17OH PO(5 beta), PT and 11-O-PT), no 17OH-PO(5 alpha) was detected in the urines of 14 patients with precocious pubarche, in 14 patients with virilization of unknown origin and in 94 healthy children of comparable age. The ratio of 17OH-PO(5 alpha) to tetrahydrocortisone (THE) discriminated between CAH-SV and CAH-LO from the 1st to the 18th year of age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728540 TI - Insulin-like growth factors in lysosomal storage disease. AB - Recent data indicate that insulin-like growth factor II (IGF II) and lysosomal enzymes bind to a common receptor. We measured serum IGF I and II levels in 16 patients with various lysosomal storage disorders. The IGF serum concentrations were normal as long as no marked liver disease was present. Under these conditions no direct interconnection between the lysosomal system and the serum IGF levels was found. PMID- 1728541 TI - An unusual case of recurrent hypoglycaemia: 10-year follow up of a child with insulin auto-immunity. AB - We report the case of a child with hypoglycaemia due to insulin auto-immunity. Insulin auto-immunity is the third most frequent cause of hypoglycaemia in Japan where the first cases were described. The child has been followed for the past 10 years with recurrence of hypoglycaemic symptoms and high titres of insulin antibodies. PMID- 1728542 TI - Spectrin/band 3 ratio as diagnostic tool in hereditary spherocytosis. AB - Recently it has been clearly established that partial deficiency of spectrin (SP) evaluated by radioimmunoassay is a common feature of many different forms of hereditary spherocytosis (HS). In this paper the determination of SP density (spectrin/band 3 ratio) in 46 HS patients and in 50 normal controls is presented. The comparison between the membrane SP density of HS subjects and controls showed a statistically significant difference (P less than 0.0005). Moreover no overlap between normal and HS subjects was observed. Membrane spectrin/band 3 ratio has been found related to some clinical features: indeed patients with severe HS showed a smaller SP density than those with milder HS. Our results show that the evaluation of membrane SP density permits a prompt diagnosis of HS and avoids extensive and unnecessary studies for other anaemias. PMID- 1728543 TI - Cystic mediastinal lesions in children: evaluation by magnetic resonance and conventional imaging. AB - The signals obtained from mediastinal cystic lesions in children by magnetic resonance imaging (MRI) have been analysed. The advantages and pitfalls in establishing the final diagnosis by MRI are compared to the conventional radiographic technique and to computed tomography. PMID- 1728544 TI - No interference of rheumatoid factor(s) with toxoplasmosis IgM determination in infancy. AB - Sera from 263 newborns and infants suspected of congenital toxoplasmosis were tested for the presence of rheumatoid factor (RF) by the latex agglutination test, of which 40 were also tested by the enzyme-linked immunosorbent assay (ELISA). RF was detected in only one serum sample (0.38%), suggesting that false positive results of the IgM-indirect fluorescent antibody test and the IgM-ELISA due to RF is most unusual in infancy. PMID- 1728545 TI - Kawasaki disease differs from anaphylactoid purpura and measles with regard to tumour necrosis factor-alpha and interleukin 6 in serum. AB - It has been reported that tumour necrosis factor-alpha (TNF-alpha) is capable of inducing vascular injury, and interleukin 6 (IL-6) of inducing production of acute phase proteins and the maturation of megakaryocytes. Kawasaki disease (KD) is a systemic vasculitis with severe inflammation. We investigated whether TNF alpha and IL-6 activities in serum from patients with KD differs from those in anaphylactoid purpura (AP) and measles. Serum TNF-alpha levels were measured by a sandwich enzyme immunoassay and IL-6 activities in serum were assessed by a colorimetric assay. Both KD and AP patients but not patients with measles had increased serum TNF-alpha levels during the acute stage. With respect to IL-6, patients with KD and measles, but not AP, had increased IL-6 activities in serum during the acute stage. IL-6 activities in serum of KD patients correlated with serum C-reactive protein levels and correlated to some extent with maximum platelet counts during the course of illness. These results suggest that KD differs from AP and measles regarding both cytokines. The combination of TNF alpha, which may be responsible for severe vascular injury, and IL-6, which may be responsible for severe inflammation, may play an important role in acute KD. PMID- 1728546 TI - Plasma folate levels in preterm infants, with and without a 1 mg daily folate supplement. AB - One hundred and four preterm infants were studied during the first few months of life in the Special Care Baby Unit of Addenbrooke's Hospital, Cambridge, United Kingdom. Previously, it had been the daily practice within the Unit to give a 1 mg oral supplement of folate (in the form of pteroylglutamic acid), once the infants had commenced full enteral feeding. At least one blood sample was obtained from 70 infants before oral folate supplementation was started. In these, the plasma folate levels fell progressively from a median value of 45 micrograms/l to a median of 12 micrograms/l, by the 2nd-3rd week of life. Once started on the oral supplement, 83 of the infants provided at least one blood sample. The plasma folate level of these infants rose immediately to a median value of 300 micrograms/l and a maximum of 1000 micrograms/l. Within individuals, these plasma folate levels decreased progressively following the introduction of the supplement, despite continuing daily supplementation. In a typical baby this decrease appeared to be explained by an increase in body-size, i.e. dilution of the folate into a larger pool. The implications of this level of supplementation are discussed, and in the light of our observations we suggest that daily supplementation in the range, 0.05-0.2 mg folate may be preferable for well preterm infants. PMID- 1728547 TI - High folate intakes related to zinc status in preterm infants. AB - The former practice of giving 1 mg (2.27 mumoles) oral folic acid daily to premature infants receiving enteral feeds was assessed with respect to zinc status in Cambridge, United Kingdom. A group of 60 preterm infants, 80% of whom were receiving 1 mg oral folic acid daily, were studied for up to the first 16 weeks of life. Plasma folate and plasma zinc were measured for each subject. A significant inverse relationship was found between the maximum attained serum folate level and the minimum attained serum zinc level, (t = 5.0, 58 df, P less than 0.0001). This remained significant after corrections had been made for gestational age at birth, fetal growth retardation, birth weight, sex, diet, assisted ventilation and length of time to full enteral feeding. The hypothesis that very high folate intakes may adversely affect serum zinc levels and, by inference, zinc status in preterm infants could not be rejected. Caution is therefore advised when prescribing such very high folate doses daily for small preterm infants. PMID- 1728548 TI - Connatal rickets following repeated administration of phosphate enemas in pregnancy: a case report. AB - We present a case of antenatal failure of bone growth and mineralisation in a newborn whose anorectic mother repeatedly administered hypertonic phosphate enemas during pregnancy. Phosphate overload in pregnant women appears to impede calcification of the fetal skeleton. PMID- 1728549 TI - A new baby-spacer device for aerosolized bronchodilator administration in infants with bronchopulmonary disease. AB - The response of salbutamol (Ventolin, Glaxo), topically administered from a metered dose inhaler (MDI) through a new baby-spacer-device (Babyhaler, Glaxo) was studied in 14 infants (8 wheezy infants, 3 infants with cystic fibrosis and 3 infants after respiratory distress syndrome), age 2.9-18.8 months. Changes in thoracic gas volume (TGV) as an estimate of pulmonary hyperinflation and changes in airway conductance (Gaw) as an estimate of bronchial obstruction were assessed by whole-body plethysmography. After baseline measurements, 1 puff of 100 micrograms salbutamol was given repeatedly at 5 min intervals until 600 micrograms have been inhaled and TGV and Gaw were measured after each inhalation at 5, 10, 15, 20, 25 and 30 min. Significant improvement in lung function was achieved in 57.1% of infants after 400 micrograms and in 92.9% of infants after 600 micrograms salbutamol. The study shows usefulness of bronchodilator treatment in infants with bronchopulmonary disease by a system with a MDI and baby-spacer device. However a special dose-time relationship must be respected. PMID- 1728550 TI - "Milky" urine--a child with chyluria. AB - A 10-year-old boy with chyluria due to a congenital fistulous communication between the lymphatic system and the bladder is described. Chyluria can be parasitic or non-parasitic. Many causes of non-parasitic chyluria have been reported. Lymphography is the preoperative imaging procedure of choice since it demonstrates the site, the calibre and the number of the fistulous communications. Lymphoscintigraphy shows very well the site of the fistula but is not as precise as lymphography. However, it has the advantage to be less invasive and is an excellent alternative in the non-surgical cases. The prognosis of non parasitic chyluria is usually very good and the treatment is mostly conservative. PMID- 1728551 TI - Periventricular haemorrhagic infarction associated with subependymal germinal matrix haemorrhage in the premature newborn. Report of two cases. AB - Two cases of unilateral subependymal germinal matrix haemorrhage associated with homolateral periventricular haemorrhagic infarction (PVHI) are reported in two premature newborns. This association is rather rare. Indeed, PVHI occurs generally with large intraventricular haemorrhage. Diagnosis is made by brain imaging (ultrasound, MRI) scans and single photon emission computed tomography. PVHI is probably caused by obstruction of periventricular venous drainage by large intraventricular haemorrhage leading to a haemorrhagic venous infarction. From our cases, we conclude that extraventricular haemorrhage leading to a large subependymal haematoma can result in obstruction of periventricular venous drainage, subsequent PVHI and abnormal neuromotor development. PMID- 1728552 TI - Abnormal length of cilia--a cause of primary ciliary dyskinesia--a case report. AB - A 7-year-old Turkish boy had suffered from chronic coughing from early childhood. Severe bronchiectasis in the right lung was confirmed by bronchography. Ciliary beat frequency determined in a bronchial mucosal biopsy was markedly decreased (5.7 Hz). Electron microscopy revealed cilia with a length of 15 microns. No structural abnormality was found. A possible link between the abnormally long, slow beating cilia and the clinical symptoms is discussed. PMID- 1728553 TI - Suprasellar arachnoid cyst as a cause of precocious puberty and bobble-head doll phenomenon. PMID- 1728554 TI - Recovery of sensory function in skin deprived of its innervation by lesion of the peripheral nerve. PMID- 1728555 TI - Tactile function in skin-equivalent grafts. AB - Cultured grafts are excellent wound covers; however, their somatosensory capabilities are unknown. This is a preliminary report of a study which determined whether grafts of cultured skin become innervated and also examined whether seeding grafts with target tissue improved nerve growth or functional recovery. Autologous skin for grafting was generated from adult rat biopsy tissue. Dissociated keratinocytes were seeded on top of fibroblast-contracted collagen gels (skin-equivalents). Some animals received grafts composed entirely of skin-equivalents. Others had grafts with 2-mm punch biopsies (normal skin or touch domes) inserted into them. Prior to sacrifice, whole nerve recordings of the cutaneous nerves supplying the grafts were made following tactile mechanical stimulation of the graft surfaces. Tissue was processed for light and electron microscopy as well as silver stained. Nerve fibers were present in the dermis (generated from the fibroblast contracted collagen gels) of all animals and often extended to the epidermis. Light brushing of the cultured areas of the grafts produced little or no activity in the cutaneous nerves; however, afferent impulses were generated after rubbing the skin with a glass rod or pinching it with fine forceps. The implanted regions within the skin-equivalents varied from this pattern. Lightly brushing their surface resulted in vigorous activity in the nerves. Elements in the skin therefore seemed to enhance nerve regeneration and function. However, the quality of the engraftment was also important. Implanted regions of grafts experiencing poor "takes" had compromised innervation. PMID- 1728556 TI - Neurogenesis and plasticity in the CNS of adult birds. AB - Neurogenesis, typically a developmental phenomenon, continues into adult life in song birds. Cells born in the walls of the lateral ventricle migrate and differentiate throughout the adult telencephalon. I will argue here that birds take advantage of these new neurons as a form of plasticity. Most of the neurons connecting the different song control nuclei are born early in development. One important exception is the central efferent motor pathway for learned vocalization. This pathway is formed by projection neurons born during juvenile and adult life. Recruitment of new projection neurons at different times of the year and in different species correlates with vocal learning. Adult neurogenesis as a form of plasticity may serve learning and it may also teach us how to repair the damaged brain. PMID- 1728557 TI - The specification of sensory cortex: lessons from cortical transplantation. AB - The mammalian neocortex is functionally organized into numerous specialized "areas." The distinct functional properties characteristic of each area are in large part due to connectional and architectural differences among the areas. However, these "area-specific" features which distinguish mature areas are not apparent early in the development of neocortex. We have used heterotopic cortical transplantation to examine whether these area-specific features are prespecified or emerge as a result of epigenetic interactions. Here, we review our studies in which late fetal rat cortex was transplanted heterotopically into the cortex of newborn rats to test its capacity to differentiate features normally unique to other cortical areas. We find that regions of the developing neocortex have similar potentials to differentiate the connectivity and functional architecture that distinguish neocortical areas in the adult. We conclude that the neocortical neuroepithelium generates comparable populations of cells across its extent, and when exposed to the same extrinsic cues, these populations can differentiate in comparable ways. These studies support the concept that the neocortical neuroepithelium generates a "protocortex" (20), specified to have fundamental cortical features but lacking a rigid specification of "area-specific" features. PMID- 1728558 TI - Age-related development of olfactory bulb transplants in rats. AB - We are using the rat olfactory system to study developmental aspects of neurotransplantation (TX). Age-related TX maturation and subsequent establishment of connections are of special concern. Previous studies of deafferentation by olfactory bulb (OB) removal suggested "critical" periods of plasticity in the system. We present here preliminary attempts at relating age of host receiving TX to maturation of the TX and its connections. This investigation used hosts of postnatal age (PN) 13-14 days with fetal donors at Embryonic Day 15; the former having one OB ablated and receiving a fetal donor OB TX immediately placed in the vacated space. The fetal tissue was labeled previously in utero with tritiated thymidine. After 2 months a small coagulation lesion was placed in the OB TX and 2 days later the tissue was taken, serially sectioned, and processed for [3H] autoradiography, degeneration, and olfactory marker protein (OMP). Extensively 3H labeled OB TXs with localized small lesions were studied. The cellular architecture of the TX is less well organized than in normals but substantial OMP reactivity occurs throughout. Degeneration occurs mainly near the lesion and little if any degeneration is seen beyond the 3H-labeled TX tissue. The results show that OB TX survive and develop in the PN 13-14 age group as they do in the younger animals and that primary olfactory neurons likewise reinnervate the TX but that PN 13-14 TX efferent projections are far more limited than those of younger hosts. PMID- 1728559 TI - The structural and functional aspects of hair cell regeneration in the chick as a result of exposure to intense sound. AB - This paper summarizes the structural and functional damage caused by intense sound exposure in neonatal chicks. Scanning electron microscopy has been used to follow the structural changes to the papilla and their subsequent repair. Pure tone exposures produced a localized lesion consisting of tectorial membrane destruction, changes in surface organization of the papilla, and hair cell loss. The papilla underwent significant repair following the exposure and new hair cells could be identified on the sensory surface after 4 days of recovery. In addition, various evoked-potential methods provided an objective assessment of auditory function and demonstrated that the peripheral ear was severely impaired by overstimulation. Auditory function returned to near normal levels within 3 days postexposure. The inescapable conclusion from these observations was that hair cell regeneration had little to do with the functional recovery observed during the first 3 days. Tectorial membrane regeneration and the restoration of cochlear micromechanics were combined to form a hypothesis to account for the restoration of auditory function. PMID- 1728560 TI - The inferior colliculus: calbindin and parvalbumin immunoreactivity in neural grafts. AB - The inferior colliculus was selected as a brain stem site for study of neural grafting and identification of calcium binding proteins. Unilateral ablation sites of eight midbrain inferior colliculus in adult Long-Evans rats were implanted with E17-18 caudal tectum. After 2 to 9 months animals were sacrificed and sections reacted using antibodies for calbindin and parvalbumin. The central nucleus of normal inferior colliculus shows high density of neuronal and fiber staining for parvalbumin. Typical graft cores had similar staining distributions including discoid and stellate neuron populations. Graft cores showed low densities of reactivity for calbindin comparable to central nucleus. In surrounding graft regions there was substantive-neuronal and fiber labeling for calbindin and parvalbumin including stellate neuron populations normally found in the dorsal and lateral nuclei of inferior colliculus. These results demonstrate that the expression of calcium binding proteins in tectal grafts resembles that of inferior colliculus. PMID- 1728561 TI - Anatomical evidence of synaptic plasticity in the cochlear nuclei of white-deaf cats. AB - Synapses are dynamic structures reflecting environmental events. It was observed that densities associated with apposing synaptic membranes were altered in response to changing conditions of auditory stimulation (Gulley et al. 1978. J. Comp. Neurol. 180: 707-742). Synapses also exhibit plasticity during early development of the auditory system (Larsen and Pappas, 1985. Proceedings: 43rd Annual Meeting of the Electron Microscopy Society of America, pp. 493-494). Larsen and Pappas reported that, in young kittens, the end bulb of Held (EBH) terminal was distinguished by excessive density at apposing synaptic membranes which assume gradually the pattern observed in adult cats. The maturation of synapses parallels emerging function and may depend upon auditory stimulation (Larsen and Kirchhoff, 1987. Neurosci. Abstr. 13: 1260). Larsen and Kirchhoff found that EBH synapses on large spherical cells of white-deaf cats resembled the immature synapses found in young kittens (Fig. 1). They also found terminals that had an increased number and length of the membrane densities associated with synapses. Because the number of synaptic vesicles in these presynaptic terminals was equivalent to the number of vesicles found in normal-hearing cats, Larsen and Kirchhoff (1987) suggest that this is evidence of synaptic plasticity. We have completed a quantitative study of the synapses of EBH terminals found in adult cats that had been deaf for at least 3 years. PMID- 1728562 TI - Centrifugal growth in orthotopic grafts of allogeneic dorsal root ganglia in adult rats: evidence for possible central ingrowth? AB - Fetal allogeneic dorsal root ganglion (DRG) transplants from 13-15 day rat embryo's (E13-E15) survived and differentiated when grafted orthopically (within the capsules of the excised 4th and 5th lumbar (L4-L5) ganglia) in adult rats. Survival of grafted neurones was established by prelabeling the grafts with a fluorescent vital dye (DiI) and visualizing the retained fluorescent marker 3 to 9 months later. Simultaneous retrograde tracing using fluorescent tracers applied in the spinal cord and peripheral nerve, respectively, yielded double-labeled dorsal root ganglion neurons, some of which were prelabeled. These findings demonstrate that prelabeled E13-E15 ganglia survive orthopic grafting, organotypically differentiate into mature DRG neurones, and can be double-labeled with fluorescent dyes applied to their peripherally and centrally directed processes. The presence of DiI containing cells which were retrogradely labeled from the spinal cord suggests that fetal (E13-E15) ganglia may have the capability of growing into a mature spinal cord. PMID- 1728563 TI - A "calcium set-point hypothesis" of neuronal dependence on neurotrophic factor. AB - In this commentary, we discuss evidence suggesting that cytoplasmic free calcium concentration determines neurotrophic factor dependence. Developing sympathetic and neural crest-derived sensory neurons require nerve growth factor (NGF) for survival both in vivo and in vitro. Chronic depolarization of these cells in culture causes a modest sustained elevation of cytoplasmic calcium concentration and promotes their survival in the absence of NGF. The amount of calcium increase caused by depolarization is closely correlated with the ability of the cells to survive in NGF-free medium. At an optimal calcium concentration, that we refer to as a "set point," survival is equivalent to that of cells grown in the presence of NGF. PMID- 1728564 TI - Is there capacity for anatomical and functional repair in the adult somatosensory thalamus? AB - The capacity for structural and functional remodeling in damaged adult CNS sensory systems can be studied by replacement of neurons in damaged structures by fetal cells from these anatomical origins. For integration to take place, the replacement paradigm assumes that (a) reconnection of adult host afferent fibers onto developing neurons is possible and (b) that the correct molecular signals exist also in the adult brain for fetal neurons to extend axons and pattern synaptic contacts. We have tried to answer some of these fundamental questions by using neuronal depletion models followed by neuronal replacement in the adult rat CNS (Isacson et al. 1984. Nature (London) 311: 458-460; Isacson et al. 1988. Prog. Brain Res. 78: 13-27; Nothias et al. 1988. Brain Res. 461: 349-354; Peschanski and Isacson. 1988. J. Comp. Neurol. 274: 449-463; Sofroniew et al. 1990. Prog. Brain Res. 82: 313-320). In one such model, kainic acid infusions deplete the ventrobasal complex (VB) of all neurons projecting to the somatosensory cortex, while afferent axons from the lemniscal and monoaminergic systems remain in the area. Direct implantation of fetal neurons (gestation age 15-16) of ventrobasal destination allows reconnection of circuitry to take place at the thalamic level, as studied by anatomical tracers, electron microscopy, and functional 2-deoxyglucose studies, while fetal thalamic VB neurons appear less likely to grow through the internal capsule toward the cortical level. PMID- 1728565 TI - Changes in the acoustic nerve after hair cell regeneration. AB - Hair cells of the avian inner ear have been shown to regenerate following acoustic or ototoxic insult. The consequences of this regeneration on the acoustic nerve have yet to be defined. The purpose of the present study was to use TEM analysis following cochlear damage and hair cell regeneration to describe afferent and efferent neural terminals on hair cells in the newly repopulated sensory epithelium. Following acoustic overstimulation (12 h, 115 dB SPL, 1500 Hz) adult quail were sacrificed immediately (0 day), or at 2, 12, or 24 weeks. Serial thin sections were taken from the embedded papilla in a plane tangential to the basilar membrane in the area consistent with regenerative activity. Immediately following noise exposure very few hair cells could be seen within the epithelia; afferent terminals on remaining cells appeared normal. Two weeks later afferent terminals showed signs of degeneration; efferent terminals were rarely seen on tall hair cells but remained relatively normal on short hair cells. Three to six months later afferent terminals had regained a more normal appearance but were less numerous on tall hair cells; some return of efferent-like terminals was seen often contacting two tall hair cells. Large normal appearing, efferent terminals remained on short hair cells. These results suggest that regenerated hair cells are likely to receive neural innervation. It would appear that some degeneration of afferent terminals takes place prior to final innervation of new hair cells. PMID- 1728566 TI - Oligodendrocyte- and myelin-associated inhibitors of neurite outgrowth: their involvement in the lack of CNS regeneration. AB - Until now central nervous system (CNS) neurites have been thought to have little capacity for regeneration following a lesion. When allowed to grow into peripheral nervous system (PNS) grafts, however, lesioned CNS axons are known to regenerate. Recently, an inhibitory substrate effect of CNS myelin and oligodendrocytes has been discovered which could be directly involved in the lack of regeneration. In culture, neurite growth cones were shown to specifically arrest their movement when contacting oligodendrocyte processes. The inhibitory components were characterized as two proteins of 35 and 250 kDa. A specific monoclonal antibody was generated (IN-1) that could neutralize these inhibitory effects. The role of the inhibitors in CNS regeneration was investigated in young rats receiving lesions of the corticospinal tract and implanted with a source of IN-1 mAB or control mAB. Results showed clear regeneration to over 10 mm in 2-5 weeks in IN-1 mAB-treated animals, while no fibers were detected further than 1 mm caudal to the lesion in controls. A similar, highly significant enhancement of regeneration was also found for the cholinergic septohippocampal pathway and for the optic nerve. These results show that lesioned CNS neurons can regenerate in CNS tissue when specific myelin components are neutralized, thus demonstrating that these inhibitory components play a crucial role in the lack of CNS regeneration. PMID- 1728568 TI - Video-enhanced DIC images of the noise-damaged and regenerated chick tectorial membrane. AB - Exposure of the chick cochlea to acoustic overstimulation results in a loss of hair cells and a disruption of the tectorial membrane. With time, new hair cells are produced to replace those that are lost and, concurrently, a new tectorial membrane is regenerated. Previous studies of tectorial membrane regeneration examined tissues that were fixed and processed for scanning and transmission electron microscopy. This processing results in a considerable shrinkage of the membrane, and, therefore, it was unclear how the noise damage and subsequent regeneration affected the unfixed, in situ structure of the tectorial membrane. We have recently developed techniques for studying the unfixed tectorial membrane with video-enhanced differential-interference-contrast (DIC) light microscopy. Exposure to a 1500-Hz pure tone at 120 dB SPL for 24 h causes localized damage to the hair cells and tectorial membrane in the mid-proximal region of the basilar papilla. Examination of the unfixed membrane immediately after noise exposure shows that the damage to the tectorial membrane is actually caused by the acoustic trauma and is not an artifact of fixation. After 14 days of recovery, a thick, honeycomb of new matrix has grown from the supporting cells in the basilar papilla and has formed new connections with the stereocilia of surviving and regenerating hair cells. Moreover, this new honeycomb has fused with the remainder of the surrounding, undamaged tectorial membrane, thus reestablishing a continuity in the structure of the membrane across both the damaged and undamaged regions of the basilar papilla. PMID- 1728567 TI - Hair cell regeneration in the avian vestibular epithelium. AB - Research conducted in the past 4 years has shown that the avian vestibular system retains the capacity to generate hair cells postnatally. In the present paper we review information on postnatal proliferation and differentiation of hair cells in the avian vestibular system. In addition, we present preliminary accounts of recent experiments regarding regeneration of vestibular hair cells following aminoglycoside toxicity. The overall consensus is that the avian vestibular system is able to regenerate hair cells, both on an ongoing basis and after damage. PMID- 1728569 TI - Degeneration followed by partial regeneration of the organ of Corti in deafness (dn/dn) mice. AB - The deafness mouse is a Mendelian recessive mutant which never hears and has no stimulus-related receptor or neural auditory responses. From birth through 12 days after birth (DAB), the organ of Corti develops normally as seen with light microscopy, except that the space of Nuel does not fully develop. The inner and outer hair cells are degenerating between 12 and 24 DAB and are gone by 45 DAB. As the hair cells degenerate, other cells of the organ of Corti become less recognizable, appear to collapse, and lose their identities as differentiated cells. By 45 DAB, from base to apex, the organ of Corti is composed of a low, roughly cuboidal epithelium with no distinguishing cell types; a hint of a tunnel of Corti remains at the apex. In the basal turn, the organ of Corti remains in this degenerated state through at least 460 DAB (senility for these mice). In the apical organ of Corti, considerable regeneration occurs between 45 and 90 DAB. By 90 DAB the apical turn of the organ of Corti has readily identifiable inner and outer pillar cells, inner and outer supporting cells, Hensen's cells, and Claudius' cells. A tunnel of Corti and space of Nuel are also present in the apex but there are no hair cells. Mechanisms are not known for either the degeneration or the regeneration. PMID- 1728570 TI - Early microfilament reorganization in injured auditory epithelia. AB - Microfilaments (MFs) play an important role in wound healing and other regenerative events. The purpose of this study was to characterize changes in the distribution of MFs in traumatized auditory epithelia and compare these changes between avian (regenerating) and mammalian (nonregenerating) ears. Chicks and guinea pigs were acoustically overstimulated and their auditory epithelia analyzed fluorescence microscopy with phalloidin as a MF-specific marker. Immediately or several hours after overstimulation, we observed a substantial reduction of MFs in stereocilia and the cuticular plate. The circumferential belt of MF which is associated with the adherens junctional complex was constricted in damaged hair cells (HCs) as early as 1 day after the exposure. Concomitant with the junctional constriction, the apical surface area of supporting cells was increased relative to normal, whereas the surface area of HCs was decreased. We conclude the changes in the amount and distribution of MFs which characterize early responses to acoustic damage are similar in avian (regenerating) and mammalian (nonregenerating) auditory epithelia. We hypothesize that changes in MF mediated tensile forces trigger the process of tissue repair in auditory epithelia in response to insult. In mammals the reorganization of MFs may help maintain the integrity of the reticular lamina and thereby prevent further damage. In contrast, early changes in MFs in chicks may play a role in regulating regenerative tissue responses. PMID- 1728571 TI - Explorations of otic transplantation. AB - Embryonic rat inner ears were transplanted to the anterior chamber of the eyes of adult rats. While considerable development was evident, the structures present were limited to the vestibular division. We hypothesized that this selective survival could be due to the rate of vascularization. To test the effects of graft vascularization we made transplants in which the internal structures were exposed by removing the apex and base of the developing cochlea. The transplants were rapidly vascularized by the iris. Many of the soft labyrinthine structures of the cochlea from 1-day-old donors showed considerable development, including the spiral limbus, basilar membrane, and organ of Corti. To test the possibility that the cochlea requires inductive or trophic support beyond Embryonic Day 15 (E15), we cotransplanted the embryonic inner ear with developing brain stem. In these transplants, we observed improved development of the cochlea, with spiral ganglion cells and an organ of Corti possessing hair cells, Deiter's cells, and pillar cells. To further address the effect of developing CNS tissue on the development of grafted inner ear, we transplanted E15 inner ears to either the cortex or the brain stem of neonatal rats. In these experiments we have seen evidence of both vestibular and cochlear sensory surfaces. In the cochlea, an organ of Corti-like structure can be seen. The possibility of neural connections with the host brain has yet to be investigated. PMID- 1728572 TI - Epithelial repair following mechanical injury of the developing organ of Corti in culture: an electron microscopic and autoradiographic study. PMID- 1728573 TI - Ablation of the olfactory bulb up-regulates the rate of neurogenesis and induces precocious cell death in olfactory epithelium. AB - Young adult rats were unilaterally bulbectomized and tritiated thymidine ([3H]TdR) was injected at variable times following surgery to determine the effect of bulbectomy on the rates of cell proliferation and cell death in the olfactory epithelium. Removal of the olfactory bulb elicits a two- to fourfold increase in the proliferation rate of ipsilateral olfactory epithelial cells 7-50 days following surgery. On the contralateral side, there was a temporary twofold increase in the proliferation rate during the second week after surgery, but this returned to control values at 3 weeks. This temporary increase was in parallel with the response on the ipsilateral side so that the ratio between operated and unoperated sides remained at two. Cell death in olfactory epithelium is also up regulated following bulbectomy. Death of cells can occur as early as 1 day following incorporation of [3H]TdR, i.e., well before the sensory neurons become mature. This means there is an over-production of sensory cells, and they die at all stages of their life cycle. The number of cells dying is greater after bulbectomy, indicating that the overproduction of olfactory cells is more pronounced after surgery. PMID- 1728574 TI - Response of the gustatory system to peripheral nerve injury. AB - Peripheral taste nerve damage occurs as a result of disease and surgery. The response depends on the taste field affected and the species. Nerve regeneration is robust after the nerve is crushed or after it is cut if the severed ends are anastomosed. Taste buds, which appear after nerve regeneration, may be derived from dormant stem cells outside of taste buds or from remnants of taste buds that persist following denervation. Sprouting by intact taste nerves into denervated fields apparently does not occur. Regenerated primary afferents have taste response specificity, but it is unknown if neural response types are retained peripherally or centrally. Recent behavioral studies show specific deficits following loss of restricted taste fields in rodents, but little is known about recovery after nerve regeneration. Specific deficits have not been demonstrated in humans, although taste sensitivity has been correlated with numbers of taste buds. Enigmas such as these may be solved once the response of the central nervous system to gustatory nerve injury is defined. PMID- 1728575 TI - Control of vertebrate retinal cell production. AB - Regeneration of vertebrate sensory cells can be seen as an extension and elaboration of the process of cellular repair and to understand repair requires knowledge of how cell division and cell fate are determined. To approach these problems, we have developed a slice culture for the teleost retina. Cells continue to divide in the same pattern in this slice culture as they do in vivo as demonstrated with [3H]thymidine labeling. Moreover, cells which divided in culture became retinal cell phenotypes as identified with monoclonal antibodies. Some presumptive rod progenitors in the outer nuclear layer in the center of the retina were also labeled cone-specific, possibly as a regeneration response. These data add to the evidence that cell fate is determined by the environment. This slice preparation will be a useful model system for analyzing putative environmental cues responsible for guiding cell proliferation and differentiation in the fish retina. PMID- 1728576 TI - Recovery of contrast sensitivity during optic nerve regeneration in fish. AB - Psychophysical experiments on goldfish and sunfish studied the recovery time course of visual contrast detection during optic nerve regeneration. The results showed delayed recovery of detection of positive as compared to negative contrasts, and of high as compared to low spatial frequencies. The findings are related to previous electrophysiological and anatomical results in the fish retinotectal system. PMID- 1728577 TI - Regeneration in the auditory system. AB - The auditory organs of birds and mammals normally stop producing sensory hair cells during embryonic development, so loss of those cells later in life results in hearing deficits that have been considered irreversible. In contrast to this, the ears of some fish and amphibians produce hair cells continuously throughout life and even increase in sensitivity. The lateral line organs in the skin of fish and aquatic amphibians also contain hair cells and have long been known to be replaceable through regeneration. Recently, it was discovered that after acoustic trauma or antibiotic poisoning, injured hair cells in the mature auditory organs of birds also could be replaced through regeneration. This is especially notable because it occurs in populations of cells that are mitotically quiescent in undamaged ears. More recent investigations have focused on identifying the cells that give rise to new hair cells during regeneration. In the lateral line organs of salamanders, time-lapse video microscopy has revealed that surviving supporting cells divide to give rise to progeny that can differentiate either as hair cells or as supporting cells. Definitive identification of the progenitors of regenerated hair cells in the avian cochlea awaits further investigation, but evidence that points to two possible candidate cell types is discussed. PMID- 1728578 TI - Monitoring photoreceptor transplants with nuclear and cytoplasmic markers. AB - Two methods are described for identifying transplanted photoreceptors in a foreign host retina. One involves the use of [3H]thymidine to label the nuclei of photoreceptors which are dividing for 1 week after birth in myomorphic retina. These photoreceptors can be identified by autoradiography. The second involves the use of a transgenic mouse carrying a bovine rhodopsin promoter in tandem with the bacterial LacZ gene. These mice express beta-galactosidase in their rods. X gal reaction allows these rods to be identified by routine light and electron microscopy. These methods have been used to follow photoreceptor transplants in adult Royal College of Surgeons strain rat and C3H mouse mutants which have lost virtually all their photoreceptors. Dissociated photoreceptors transplanted to the subretinal space of these animals survive for at least 3 months. The inner segment, cell body, and synaptic terminal of these transplanted photoreceptors remain morphologically normal; the outer segment, however, becomes rudimentary. PMID- 1728579 TI - Photoreceptor transplantation: anatomic, electrophysiologic, and behavioral evidence for the functional reconstruction of retinas lacking photoreceptors. AB - We have investigated the possibility of using transplantation of immature or mature rodent photoreceptors as well as mature human photoreceptors to reconstruct retinas in which photoreceptor degeneration is either inherited or environmentally induced. To this end, we have devised methods for isolating and transplanting the outer nuclear layer (ONL) (e.g., the photoreceptor layer) to the subretinal space of mature rodents. In addition we found that if portions of the inner retina are transplanted along with the intact photoreceptor sheet, photoreceptor organization is better maintained. In ultrastructural studies of the reconstructed retina an outer plexiform-like layer (OPL) is visible at the interface of the transplanted ONL and the host inner nuclear layer, with invaginating ribbon synapses characteristic of those formed by rod photoreceptors evident within this OPL. Ribbon synapses are found only rarely in unreconstructed retina. These results suggest that synaptic connections between transplanted photoreceptors and host cells may be made. Evidence for the potential recovery of function following photoreceptor transplantation is found in visually evoked cortical responses and behavioral responses (pupillary reflex) to light stimulation of the reconstructed eye. These findings suggest the possibility that neural transplantation can reconstruct a sensory end organ--in this case the retina--to restore evoked activity and an appropriate behavioral response to sensory stimulation. PMID- 1728580 TI - Analysis of the p21 ras system during development of meiotic competence in Xenopus laevis oocytes. AB - The ability of Xenopus oocytes to undergo insulin- or insulin-like growth factor 1-induced meiotic maturation develops during oogenesis, with cells 1.0 mm in diameter or larger responding in a size-dependent manner. Since insulin-induced oocyte maturation was shown previously to be p21 ras-dependent, experiments were performed to test whether a deficiency in the p21 ras system might account for meiotic incompetence in small oocytes (less than or equal to 0.9 mm diameter). Both small and large oocytes were found to contain comparable levels of membrane associated p21, as determined by protein immunoblotting. Treatment of both small and large oocytes with 2 microM insulin for 2 hr increased endogenous levels of membrane-associated p21 by approximately 70%. Stimulation of microinjected p21 membrane association by insulin was observed to be both time- and concentration dependent in large oocytes with an EC50 of 50 nM. In addition, comparable levels of GTPase activating protein were measured in extracts prepared from oocytes ranging from 0.8 to 1.3 mm in diameter. Therefore, the p21 system is apparently not limiting during oogenesis, and expression of some other cellular component must account for development of meiotic competence in Xenopus oocytes. PMID- 1728581 TI - Developmental profiles of epidermal mRNAs during the pupal-adult molt of Tenebrio molitor and isolation of a cDNA clone encoding an adult cuticular protein: effects of a juvenile hormone analogue. AB - Changes in translatable mRNAs from the wing epidermis of the Coleoptera Tenebrio molitor have been investigated during metamorphosis by analysis of in vitro translated products. Striking differences between the patterns obtained from mRNAs extracted during pupal and adult cuticle secretion indicated that a drastic change in gene expression occurs during the pupal-adult transition. In addition to these stage-specific modifications, the mRNA patterns changed within each cuticular synthesis program (pupal or adult), especially at ecdysis. After tritiated leucine incorporation, some of the major radiolabeled cuticular proteins showed similar changes suggesting that the sequential appearance of mRNAs corresponds to sequential deposition of cuticular proteins. In supernumerary pupae obtained after juvenile hormone analogue (JHA) application on newly ecdysed pupae, translatable mRNA were very similar to those of pharate pupae. The JHA seemed, therefore, to prevent the expression of the adult program. By immunoblotting in vitro translated products with a monoclonal antibody recognizing an adult-specific cuticular protein, the developmental profile of the corresponding mRNA was studied. This mRNA was detected in anterior wing epidermis during the first 80 hr of the pharate adult stage. Using the same antibody, a cDNA clone was isolated from epidermal mRNA. The hybrid selected mRNA coded for only one protein with an apparent MW of 22 kDa which was, furthermore, recognized by the antibody. The Northern blot analysis performed with the clone confirmed the Western blot analysis of the in vitro translation products. JHA application at the beginning of the pupal-adult reprograming prevented the appearance of this mRNA; however, this transcript was present during the following molting cycle. This reversibility of the JHA action was confirmed by immunogold labeling of the cuticles formed in treated animals. PMID- 1728582 TI - Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis. AB - A major unsolved problem in developmental biology is to determine when and how time- and position-restricted instructions are signaled and received during secondary embryonic inductions such as branching morphogenesis. The mouse embryonic lung rudiment was used to test the hypothesis that endogenous peptide growth factors, specifically epidermal growth factor (EGF), serve as instructive epigenetic signals for morphogenesis. The presence of EGF precursor mRNA transcripts was detected using the reverse-transcriptase-coupled polymerase chain reaction both in E11-E17-day mouse embryo lung tissues in vivo and in E11-day lung cultured for up to 7 days in vitro under chemically defined, serum-free conditions. Immunolocalization identified a position-restricted distribution of EGF in and around the primitive airways both during in vivo lung morphogenesis and in culture. EGF receptors (EGFR) coimmunolocalized with EGF in the primitive airways. Addition of exogenous EGF to lungs in culture resulted in significant concentration-dependent stimulation of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA. Conversely, the addition of tyrphostin (specific EGF receptor kinase antagonist) to lungs in culture resulted in concentration-dependent inhibition of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA without apparent cytotoxicity. The inhibition of the EGF signal by tyrphostin was confirmed by immunoprecipitation of tyrosine phosphoproteins. We conclude that early mouse embryo lungs express EGF transcripts and corresponding EGF peptides in a specific position-restricted distribution which coimmunolocalizes with EGFR in the primitive airways, while stimulatory and inhibitory studies indicate a functional role for the transduced EGF signal in the epigenetic regulation of lung branching morphogenesis. We speculate that the peptide growth factor EGF serves a function in secondary embryonic morphogenetic inductions, which may be modulated by interaction with other growth factors. PMID- 1728583 TI - Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster. AB - The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero dorsal region of the brain. Dividing neuroblasts increase during the late first late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800 1200 per hemisphere. PMID- 1728584 TI - Partial restriction in the developmental potential of late emigrating avian neural crest cells. AB - Trunk neural crest cells migrate along two major pathways: a ventral pathway through the somites whose cells form neuronal derivatives and dorsolateral pathway underneath the ectoderm whose cells become pigmented. In avian embryos, the latest emigrating neural crest cells move only along the dorsolateral pathway. To test whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells, cultures were prepared from the neural tubes of embryos at various stages of neural crest cell migration. "Early" and "middle" aged neural crest cells differentiated into many derivatives including pigmented cells, neurofilament-immunoreactive cells, and adrenergic cells. In contrast, "late" neural crest cells differentiated into pigment cells and neurofilament-immunoreactive cells, but not into adrenergic cells even after 10-14 days. To further challenge the developmental potential of early and late emigrating neural crest cells, they were transplanted into embryos during the early phases of neural crest cell migration, known to be permissive for adrenergic neuronal differentiation. The cells were labeled with the vital dye, DiI, and injected onto the ventral pathway at stages 14-17. Two and three days after injection, some early neural crest cells were found to express catecholamines, suggesting they were adrenergic neuroblasts. In contrast, DiI labeled late neural crest cells never became catecholamine-positive. These results suggest that the late emigrating neural crest cell population has a more restricted developmental potential than the early migrating neural crest cell population. PMID- 1728585 TI - Stomatal patterning in Tradescantia: an evaluation of the cell lineage theory. AB - The cell lineage theory, which explains stomatal patterning in monocot leaves as a consequence of orderly divisions, was studied in Tradescantia. Data were collected to test the theory at three levels of organization: the individual stoma; stomata distributed in one dimension, in linear fashion along cell files; and stomata apportioned in two dimensions, across the length and breadth of the leaf. In an attempt to watch the patterning process through regeneration, stomata in all visible stages of development were laser ablated. The results showed that the formation of stomatal initials was highly regular, and measurements of stomatal frequency and spacing showed that pattern was determined near the basal meristem when the stomatal initials arose. Following the origin of initials, the pattern was not readjusted by division of epidermal cells. Stomatal initials were not committed when first present and a small percentage of them arrested. The arrested cells, unlike stomata, were consistently positioned in cell files midway between a developed pair of stomata. At the one-dimensional level of pattern, stomata in longitudinal files were separated by a variable number of epidermal cells and the frequency of these separations was not random. The sequential spacing of stomata also was not random, and stomata separated by single epidermal cells were grouped into more short and long series than expected by chance. The stomatal pattern across the width of the leaf resulted from cell files free of stomata which alternated with cell files containing stomata, but not with a recurring periodicity. Files lacking stomata were found only over longitudinal vascular bundles. Laser ablations of developing stomata did not disrupt the pattern in nearby cells or result in stomatal regeneration. We conclude that the cell lineage theory explains pattern as an individual stomatal initial arises from its immediate precursor and satisfactorily accounts for the minimum spacing of stomata in a cell file, i.e., stoma-epidermal cell-stoma. However, the theory does not explain the collective stomatal pattern along the cell files, at the one dimensional level of patterning. Nor does the theory account for the for the two dimensional distribution of stomata in which regions devoid of stomata alternate with regions enriched with stomata, but not in a highly regular nor haphazard manner. We suggest that the grouping of epidermal cells and stomata separated by single epidermal cells in cell files may result from cell lineages at a specific position in the cell cycle as they traverse the zone where stomatal initials form.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1728586 TI - Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro. AB - Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture. PMID- 1728587 TI - Constitutive myc expression impairs hypertrophy and calcification in cartilage. AB - The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage. PMID- 1728588 TI - Induction of manganese superoxide dismutase in cultured human trophoblast during in vitro differentiation. AB - The antioxidant responses of human cell differentiation and membrane fusion are not known and may be important in understanding cellular response to injury in the human placenta. We studied the regulation of antioxidant enzymes in human trophoblasts which differentiate from mononucleated cellular trophoblasts to synctium in vivo and in culture. We characterized morphological and biochemical differentiation of cultured trophoblasts from term placenta in the presence or absence of serum, on different growth surfaces, and with a range of plating densities. Culture of cellular trophoblasts consistently and transiently induced the mRNAs of the mitochondrial antioxidant manganese superoxide dismutase (Mn SOD) but not the mRNAs for the antioxidant enzymes copper zinc SOD or catalase. Fibrin and type I collagen substrates modulated only the expression of the placental specific proteins, human chorionic gonadotropin, and human placental lactogen. Both Mn SOD induction and terminal differentiation, as reflected by human chorionic gonadotropin expression, were dependent on trophoblastic plating density. Increased levels of a smaller Mn SOD mRNA species correlated temporally with an increase in Mn SOD enzyme activity in cultured trophoblasts. These results demonstrate that Mn SOD gene expression and enzyme activity precede or are coordinately regulated with morphological and biochemical trophoblastic differentiation. PMID- 1728589 TI - Hormonal regulation and segmental specificity of motoneuron phenotype during metamorphosis of the tobacco hornworm, Manduca sexta. AB - The abdominal prolegs of Manduca sexta larvae are eliminated at the onset of metamorphosis. Previous work showed that the prepupal peak of ecdysteroids in the hemolymph causes the dendritic arbors of proleg motoneurons to regress and a stereotyped subset of the motoneurons to die. In the present study we investigated the parameters of ecdysteroid exposure that are important for eliciting these responses by directly infusing 20-hydroxyecdysone (20-HE) into the hemolymph of insects deprived of their own endocrine glands. Doses of 20-HE that were near threshold for evoking regression or death were consistently more effective when infused over a longer duration. Theoretical calculations of hemolymph hormone profiles produced by the infusions support a model of ecdysteroid action in which the hormone concentration must remain above a threshold level for a critical duration of time to be physiologically effective. We further found that segmental location can influence both the metamorphic fate and the hormonal sensitivity of Manduca motoneurons. PMID- 1728590 TI - Developmental regulation of alternative splicing in the mRNA encoding Xenopus laevis neural cell adhesion molecule (NCAM). AB - The neural cell adhesion molecule (NCAM) is thought to play a role in the formation of the vertebrate nervous system. In mammals and chicken, it is known that more than 100 different forms of the NCAM protein can be generated by alternative splicing of one primary transcript and it is possible that these different forms have distinct biological functions. A large part of the diversity is generated by alternative mRNA splicing in two regions, called the pi and the muscle specific domain (MSD), that encode portions of the extracellular domain of the NCAM protein. In this report, we describe the tissue and developmental expression of the pi and MSD sequences in the amphibian, Xenopus laevis. Our experiments show that NCAM transcripts are present in all tissues examined including muscle, heart, liver, kidney, and brain. We have identified a 30-base exon, similar to the pi domain observed in mammals, that is not present in maternal NCAM RNA but appears in a subset of the NCAM mRNA population shortly after neural induction. At the predicted location of the MSD we have detected only two alternatively spliced exons, 3 bases and 15 bases in length. In no X. laevis tissue examined did we detect the two additional alternatively spliced exons which are present in the MSD region of mammalian and chicken NCAM RNAs. Finally, the analysis has revealed a dynamic and complex pattern of expression of alternatively spliced NCAM mRNAs during embryogenesis. High levels of expression of specific forms of NCAM RNA correlate with major morphogenic events such as neural tube formation and metamorphosis. PMID- 1728591 TI - Characterization of a cell surface adhesion molecule expressed by a subset of developing chick neurons. AB - We have isolated a 105-kDa membrane glycoprotein expressed by subsets of developing chick neurons. This glycoprotein, identified by the JC7 monoclonal antibody, is present on the surface of axons and cell bodies of developing spinal motor neurons, dorsal root ganglion sensory neurons, sympathetic and parasympathetic neurons, and a small subset of brain neurons. Late in development the JC7 antigen is expressed at high levels on CNS nonneuronal glial-like cells. When attached to latex beads this glycoprotein can mediate homophilic adhesion and when used as a culture substrate stimulates a highly branched pattern of neurite outgrowth from dorsal root ganglion explants. The JC7 antigen appears to be identical to the SC1, BEN, and DM antigens. Its limited distribution, adhesive qualities, and ability to stimulate neurite outgrowth suggest it may play a role in the selective growth of neural processes during development. PMID- 1728592 TI - Actin and myosin genes are transcriptionally regulated during mouse skeletal muscle development. AB - During primary and secondary myotube formation in utero and subsequent maturation of muscle fibers after birth there are complex changes in the pattern of contractile protein gene expression at the RNA and protein levels. In order to determine the degree of transcriptional regulation of actin and myosin genes we have carried out "nuclear run-on" experiments using nuclei prepared from the limb muscle of mice at 14.5, 15.5, 17.5, and 18.5 days in utero and at 10-12 and 12.5 days after birth. We show that transitions in the expression of these genes in vivo are regulated transcriptionally. Transcription of the sarcomeric alpha actins changes from cardiac to predominantly skeletal actin over this time period; transcription of the beta-actin gene is repressed. The myosin heavy chain and myosin light chain genes also undergo transcriptional transitions during muscle development. Notably, transcription from the MLC3F promoter is activated after that of the MLC1F promoter, which is part of the same gene. These results are discussed in the context of published RNA data. PMID- 1728593 TI - Tissue-restricted accumulation of a ribosomal protein mRNA is not coordinated with rRNA transcription and precedes growth of the sea urchin pluteus larva. AB - We have identified an mRNA that encodes a protein, SpS24, of the small ribosomal subunit in the sea urchin, Strongylocentrotus purpuratus. RNA blot and in situ hybridization analyses show that the SpS24 gene is active during early oogenesis, downregulated in the mature egg and during cleavage, and reactivated in the early blastula. The mRNA then increases in abundance at least 100-fold. Later in development, expression of SpS24 mRNA becomes restricted primarily to cells in the oral ectoderm and endoderm of the pluteus larva, and the message is undetectable in aboral ectoderm cells and most mesenchyme cells. To determine whether transcription of the ribosomal RNA genes occurs at a higher rate in oral ectoderm and endoderm tissues, a probe for the transcribed spacer was used in RNase protection and in situ hybridization assays. High concentrations of rRNA processing intermediates were observed in unfertilized eggs and shown to reside primarily, if not exclusively, in the cytoplasm. The spatial and temporal distributions of these sequences strongly suggest that they are associated with heavy bodies. New embryonic rRNA transcripts are first detectable at the very early blastula stage. In later embryos, the content of this transcribed spacer sequence is similar in all but a few cells, which implies that they synthesize rRNA at a similar low rate. Comparison of available estimates of rRNA transcription rate with the potential rate of SpS24 protein synthesis, calculated from SpS24 mRNA prevalence, shows that oral ectoderm and endoderm cells have the capacity to synthesize 15- to 30-fold more SpS24 protein than is required to keep pace with rRNA synthesis in these cells. Because the sea urchin embryo develops from an egg to a pluteus larva in the absence of growth, this stockpiling of SpS24 mRNA anticipates rather than accompanies the onset of growth, which does not begin until after feeding. Upregulation of this gene is therefore part of the developmental program, rather than a physiological response to nutrient availability. PMID- 1728594 TI - The proximal promoter of the aldolase A gene remains active during myogenesis in vitro and muscle development in vivo. AB - The gene for aldolase A in mouse has been shown to be regulated by alternative promoters with attendant alternative first exons. The distal promoter/exon M functions only in muscle while the proximal promoter/exon H is active in early muscle development and in most other tissues. We have analyzed the developmental expression of M and H promoters in mouse throughout myogenesis both in vitro and in vivo. In C2C12 cells RNase protection assays revealed the M promoter is induced within 24 hr of the onset of myogenic differentiation, and both M- and H specific mRNAs accumulate over 5 days in culture. Nuclear run-on transcription and in situ hybridization with an exon-specific probe demonstrate that the H promoter remains transcriptionally active even in differentiated myotubes. The in vitro results were then compared to similar RNase protection studies of M and H expression during muscle development in vivo. These data show a marked similarity between promoter activation and steady-state transcript accumulation in vivo and in vitro, but within a limited developmental time frame (E15 to 1 week postnatal). In situ hybridizations suggest that simultaneous transcription from both promoters may also occur early in muscle development. Furthermore, the M promoter shows no fiber-type restriction until 1 to 3 weeks postnatally, coincident with muscle maturation, while the H promoter remains transcriptionally active at all stages of development and in all fiber types. PMID- 1728595 TI - Purification and characterization of maturation-promoting factor in fish. AB - Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33 kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase. PMID- 1728596 TI - Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. AB - The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg. PMID- 1728597 TI - Multiple roles for cAMP-dependent protein kinase during Dictyostelium development. AB - The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells. PMID- 1728598 TI - Cell kinetic analysis of human brain tumors by in situ double labelling with bromodeoxyuridine and iododeoxyuridine. AB - Fifty-seven patients with brain tumors (29 gliomas, 23 meningiomas, 5 miscellaneous) received infusions of intravenous iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) 1-5 hr apart shortly before tumor removal. Excised tumor specimens were stained sequentially for BUdR and IUdR. The percentage of BUdR labelled cells was determined to establish the labelling index (LI), or S-phase fraction, and the ratio of cells labelled only with IUdR to cells labelled with BUdR or with BUdR and IUdR was determined to calculate the duration of S-phase (Ts) and the potential doubling time (Tp) of each tumor. The BUdR LIs varied from less than 1% to 20%, reflecting the malignancy of each tumor. Despite the difference in LIs, however, Ts was fairly uniform (mean +/- SD, 8.7 +/- 2.0 hr). Tp varied from 2 days to more than 1 month and correlated closely with the BUdR LIs (Tp = 23/LI0.93; r2 = 0.91). Double-labelling studies with IUdR and BUdR allow the S-phase fraction, Ts and Tp to be determined from a single biopsy specimen and thus provide more useful information on the growth characteristics of individual tumors than can be obtained by single-labelling studies with BUdR. PMID- 1728599 TI - Lack of association between in vitro clonogenic growth of human cervical carcinoma and tumour stage, differentiation, patient age, host cell infiltration or patient survival. AB - Biopsies from 117 patients with cervical carcinoma were studied using a clonogenic assay to assess in vitro growth. Successful colony growth was achieved in 84 tumours (72%) with a mean colony-forming efficiency (CFE), based on total viable nucleated cell counts, of 0.18 +/- 0.49% (+/- 1 standard deviation). There was a wide range of values, from 0.003-4.28%, with a coefficient of variation of 272%. The relationship between clinical features of cervical carcinoma and in vitro colony formation was investigated. No significant association was demonstrated between in vitro growth and either clinical stage (r = 0.02), tumour differentiation (r = -0.08) or patient age (r = -0.12). There was no significant difference in tumour grade between the group of tumours which failed to grow in culture and those which grew well (p = 0.70). In addition, there was no correlation between CFE and the degree of macrophage (r = 0.001), lymphocyte (r = 0.12), or granulocyte (r = 0.08) infiltration. There was no significant difference between CFEs of tumours from patients who had died and from those who were alive and well after a minimum of 2 years' follow-up after radiotherapy (p = 0.51). Ability to form colonies in agar was not associated with a worse prognosis (p = 0.49). Although CFE is an independent biological parameter, our results suggest that, for cervical carcinoma, it is not useful as a univariate prognostic factor. PMID- 1728600 TI - Lethal deformation of cancer cells in the microcirculation: a potential rate regulator of hematogenous metastasis. AB - The hypothesis has been advanced that deformation-induced lethal mechanical trauma, resulting in surface-membrane rupture, is inflicted on circulating cancer cells trapped in the microcirculation, and that this rapid cell-killing mechanism is a potentially important rate regulator for hematogenous metastasis. We describe and discuss an in vivo test of this hypothesis. Vital fluorescence microscopy was performed on the microcirculation of cremaster muscle preparations in mice, following retrograde injections into the femoral artery of acridine orange-stained sarcoma cells. Cancer cells having mean diameters of 16.5 microns in suspension, were deformed from spheres into cylinders having a mean length of 53 microns, in 7-microns diameter capillaries. Most of these cells were dead several minutes after injection. It was estimated that sphere-to-cylinder shape transitions of this magnitude required an average increase of 52% in apparent cell surface area. Evidence is presented that most of this apparent increase was achieved by non-lethal surface "unfolding", utilizing membrane "excess". That cancer-cell deformation of the magnitude observed in vivo is the direct cause of lethal, surface-membrane rupture was indicated by the observed loss of membrane integrity in cells deformed from spherical to cylindrical shape in vitro, by aspiration into micropipettes of capillary dimensions. The experimental observations are therefore consistent with the hypothesis. PMID- 1728601 TI - Enhanced induction of colon carcinogenesis by azoxymethane in Wistar rats fed a low-protein diet. AB - The effects of ad libitum feeding of synthetic, low-protein diets on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), on the norepinephrine concentration in the colon wall tissue and on the labelling index of colon mucosa were investigated in Wistar rats. Rats received 10 weekly injections of 7.4 mg/kg body weight of AOM and were given synthetic diets of equal calorie content containing 25% casein (normal-protein diet), 10% casein (low-protein diet) or 5% casein (very-low-protein diet). Administration of the low- and very-low-protein diets resulted in significant increases in the incidence and number of colon tumors at week 30. However, it did not affect the histology of the colon tumors. The low- and very-low-protein diets also resulted in significant increases in norepinephrine concentration in the proximal and distal portions of the colon wall and in the labelling indices of both parts of the colon mucosa. Our findings indicate that low- and very-low-protein diets enhance colon carcinogenesis and that this may be related to their effects in increasing the norepinephrine level in the colon wall and in stimulating proliferation of colon epithelial cells. PMID- 1728602 TI - MCF-7 breast cancer cells grown as multicellular spheroids in vitro: effect of 17 beta-estradiol. AB - To obtain multicellular spheroids from MCF-7 human breast cancer cells we adhered to the following procedure: (a) limiting the adherence of cell to the substratum; (b) seeding more than the minimum number of cells; (c) guaranteeing the presence of estrogens in the culture medium. Charcoal-dextran (CD)-treated sera seemed to inhibit spheroid formation. A reduction in the concentration of CD-human sera (from 10% to 5%) added to phenol-red-free medium facilitated progress from cellular aggregates to multicellular spheroids. Once the spheroids became initiated, size increased at a rate that showed a good fit to a Gompertzian equation (A = 0.368 +/- 0.067 alpha = 0.065 +/- 0.013, r range = 0.890-0.989). Three different patterns of spheroid morphology and proliferative kinetic were defined: (a) spheroids with diameter less than 200 microns had a constant pattern of heterogeneity in the distribution of 3H-TdR-labelled cells and in the expression of estrogen receptors; (b) spheroids 250 to 700 microns in diameter showed a decrease in the proportion of 3H-TdR-labelled cells accompanying inward progression (50% in the outer shell, less than 10% in a cell layer located at a depth of 150 microns) while, at a depth of 170 microns, of signs of concurrent cellular degeneration and death were apparent; and (c) spheroids with a diameter of greater than 750 microns showed a crust of viable cells uniformly labelled with thymidine without impairment of the proportion of labelled cells when progressing inward from the spheroid crust. The larger the spheroid volume, the lower its growth fraction and the longer its volume doubling time. The hormone dependence of MCF-7 cells in forming multicellular spheroids represents a unique experimental model for assessing estrogen action on cell organization and proliferation. PMID- 1728603 TI - Occurrence of tumours in the descendants of CBA male mice prenatally treated with diethylstilbestrol. AB - There is well documented evidence both in humans and in experimental animals that exposure to diethylstilbestrol (DES) during pregnancy results in an increased incidence of tumours in the progeny. The increased cancer risk has been reported to persist in the second generation descendants of DES-exposed pregnant mice. In the present experiment, female mice of the CBA strain were treated at day 17 of pregnancy with 1 microgram/g body weight of DES. The descendants of DES-treated mothers, described as F1DES, were mated among each other or with untreated animals. The F1DES females were found to be sterile when mated with either F1DES or untreated males. F1DES males were successfully mated with untreated females. In the female offspring so obtained, but not in the male, a statistically significant increased incidence of tumours was observed, in particular of uterine sarcomas, and also of benign ovarian tumours and of lymphomas. PMID- 1728604 TI - Elevated activities of protein kinase C and tyrosine kinase correlate to leukemic cell aggressiveness. AB - We report a linkage between cell aggressiveness, protein kinase C (PKC) activity, tyrosine kinase (PTK) activity and serum requirement. We used 2 leukemic cell lines induced by Moloney murine leukemia virus (MLV). One line was highly aggressive (BS-24-1) and required low serum concentrations (3%) for optimal growth in comparison to the less aggressive line (RO2T) that needed 10% serum for optimal growth. The more malignant cells exhibited higher PKC and PTK activity. This activity was independent of serum concentration between 0.01-10%. In contrast, the weakly malignant cells need a high serum concentration (10%) for optimal PKC or PTK activity. Immunoblot analysis revealed a higher level of PKC protein in the BS-24-1 cells than in the RO2T cells. Serum induction of PKC activity did not change the amount of PKC protein in the cytosol or the membrane fractions, indicating post-translational mechanism regulation of PKC. We suggest that the aggressiveness of BS-24-1 resulted from its ability to become independent of growth regulation by serum factors, via autocrine stimulation of PKC and PTK. PMID- 1728605 TI - Mucin synthesis and secretion in relation to spontaneous differentiation of colon cancer cells in vitro. AB - The synthesis and secretion of mucin-like high-molecular glycoprotein was studied in 2 human colon cancer cell lines that spontaneously differentiate in culture (Caco-2 and T84) and in 2 cell lines that do not spontaneously differentiate (LS174T and HT29). Mucin, quantitated by 3H-glucosamine labelling and chromatography on Sepharose CL-4B was found to be produced by all 4 cell lines. The mucinous nature of the labelled high-molecular glycoprotein was verified by enzymatic degradation treatments (heparinase, hyaluronidase, chondroitinase ABC, and N-glycanase), alkaline-borohydride treatment, inhibition of labelling by the glycosylation inhibitor benzyl-alpha-GalNAc, and by CsCl-density-gradient centrifugation. In all 4 cell lines, an inverse correlation of mucin synthesis with cell density was demonstrated. In Caco-2 cells, the spontaneous post confluent enterocytic differentiation with increased brush-border enzyme expression was associated with a decrease in mucin synthesis and in the activities of polypeptidyl GalNAc transferase and beta 1,3-galactosyltransferase activity. Using cDNA probes for 2 distinct human intestinal mucins (MUC2 and MUC3), we found that all 4 colon cancer cell lines expressed mucin message, but the types of mucin mRNA expressed differed. These data indicate that mucin-like glycoproteins can be synthesized by cell lines derived from non-mucinous colon cancer, whether or not they undergo spontaneous differentiation in culture. These cell lines may serve as in vitro models for studying apomucin heterogeneity and control of mucin gene expression. PMID- 1728606 TI - Characterization of a U-937 subline which can be induced to differentiate in serum-free medium. AB - We report the selection and characterization of a U-937 subline which is capable of long-term growth in serum-free medium and can be induced to differentiate. The subline (U-937-1SF) can be maintained in standard RPMI-1640 medium supplemented by antibiotics only. As compared to the serum-dependent U-937 parental cell line, U-937-1SF produced lower amounts of lysozyme and elastase and had a decreased surface expression of complement receptor 1 (CD35) and myeloid antigens CDw17 and CD38. Apart from these alterations, the U-937-1SF cells appear to be morphologically, cytogenetically and phenotypically similar to the parental U-937 clone-1 cells. The capacity of U-937 clone-1 cells to undergo phorbol myristic acid (PMA)-, vitamin D3 (VitD3)- and retinoic-acid (RA)-induced differentiation was retained in the U-937-1SF cells as evidenced by the induced growth arrest, development of a monocyte/macrophage morphology and increased expression of differentiation-associated antigens, e.g. CD11b, CD11c, CD14 and CD18. The growth inhibitory response to cytokines involved in the activation and differentiation of monocytes, IFN-gamma, TNF-alpha, IL-1 beta, IL-6 and GM-CSF, was normal. Our results suggest that the U-937-1SF subline can be used as a serum-free model system for studies on various aspects of monocyte differentiation. PMID- 1728607 TI - Non-contraceptive oestrogens and breast cancer: an update. PMID- 1728608 TI - The time trends of multiple myeloma in Connecticut, 1935-1987. PMID- 1728609 TI - Apparent fusion of basement membranes in colorectal carcinoma. AB - Previous studies on colorectal carcinomas indicate that consistent differences in epithelial basement membrane (EBM) integrity are present between the tumour centre and periphery. We report that within the tumour centre, EBM staining between back-to-back (BTB) neoplastic glands (i.e., adjacent glands in direct contact with no intervening connective tissue) generally follows a pattern different from that of EBM staining at the tumour:stromal interface (TSI). Such distinctions are important, since the factors responsible for EBM deficiencies may vary with intra-tumoural location, as may the prognostic significance of these deficiencies. Analysis of paraffin sections from 130 colorectal carcinoma cases showed that EBM staining between BTB glands is generally weaker and more discontinuous than at the TSI, sometimes appearing as a linear array of immunostained granules on high-resolution light microscopy. By double-labelling immunofluorescence analysis of cryostat sections from 30 cases, a decrease in type-IV collagen:laminin staining intensity ratio was found between BTB glands. Hence, the composition of EBM between BTB glands appears to be abnormal. As much recent evidence indicates that epithelial:mesenchymal interactions play an essential role in EBM formation, the demonstration of immunostained EBM fragments between BTB glands requires an explanation: We suggest that the synthesis of EBM between BTB glands involved previously intervening stromal (mesenchymal) cells, and that EBM fusion and dissolution occur between BTB glands following the displacement of these cells. PMID- 1728610 TI - Oesophageal mucosa in a population at risk of oesophageal cancer: post-mortem studies. AB - We performed post-mortem studies of oesophageal mucosa from 513 consecutive autopsies of cases who died from unnatural causes or from diseases not related to the oesophagus. Of these, 170 cases were rural blacks from endemic high-risk areas, 98 were urban blacks at high risk, 158 were coloureds at moderate risk and 87 whites at low risk for oesophageal carcinoma. Oesophagi were studied macroscopically and histologically to determine malignant and precursor lesions. A prevalence of 1.0 to 1.8% of squamous carcinoma and 7 to 7.5% of mucosal dysplasia was detected in cases older than 30 years from high-risk urban and rural population groups. These results are in accord with our previous cytological and endoscopical screening studies. No significant differences in the prevalence of oesophagitis, glycogenic acanthosis and atrophy were found in the groups studied. A study of oesophageal melanocytes showed a 3% prevalence, unrelated to dysplastic and malignant lesions, and suggested that these cells were of little if any practical significance as precursor lesions. PMID- 1728611 TI - Stefins and lysosomal cathepsins B, L and D in human breast carcinoma. AB - In the study of 50 matched pairs of breast carcinoma and normal breast tissue, the activities of cysteine proteinases (CPs), cathepsin (Cat) B and Cat L in tumors were increased on average by 18.5-fold and 52.5-fold respectively. The differences in activity of cysteine proteinase inhibitors (CPIs) between tumor and control breast tissues was also observed: in approximately two thirds of carcinomas, lowered CPI activity was measured (group-I patients), while similar or higher tumor CPI activity was measured in the remaining samples (group-II patients). Relative increases in specific activity of Cat B and Cat L in group I were significantly higher than in group II. In group I more patients with histopathological tumor grade III and negative estrogen (ER) and progesterone receptor (PR) levels were found, but the metastatic involvement of regional lymph nodes was similar in both groups. A 2-year follow-up study showed a significant inverse correlation between disease-free survival and increased Cat L activity, but the differences in group I and group II patients were not significant in this short time interval. In 20 matched pairs of breast carcinoma and normal breast tissue, the mean activity of Cat D was 5.8-fold higher in tumors compared with controls. The hypothesis that elevated Cat D activity increased CP activity and/or lowered tumor CPI activity due to post-translational proteolytic modification appeared less likely, since no correlations between corresponding activities were observed. We suggested that lowered CPI might rather reflect changes in transcription of intracellular CPIs, the stefins. Immunoassay and Northern blot analysis showed that the average value of stefin A protein and mRNA content respectively in the majority of investigated breast carcinoma samples were lowered, suggesting the possible value of stefin A in diagnosis and/or prognosis of the disease. PMID- 1728612 TI - Intramural metastasis of thoracic esophageal carcinoma. AB - Of 393 patients with squamous-cell carcinoma in the thoracic esophagus, 60 were found by histologic examination to have intramural metastasis. Metastases in 50 of these were identified by gross inspection. There appeared to be no preference for location proximal to the primary lesion. Eighteen patients had metastasis to the gastric wall, which suggested the existence of communicating lymphatic channels between the wall of the esophagus and the stomach. All 60 primary tumors invaded beyond the submucosa. These 60 patients (group A) were compared with a group of matched control patients without intramural metastasis (group B). The tumor size in group A was significantly larger than in group B (p less than 0.01). The number of patients with lymph-node metastasis was significantly higher in group A (p less than 0.01), and the average number of positive nodes in group A was greater than in group B (p less than 0.01). Recurrent disease in the mediastinal lymph nodes and in the liver is characteristic of group A. The survival curve for patients in group A was significantly lower than that for group B (p less than 0.001). Conventional radiotherapy or chemotherapy after surgery were ineffective in improving prognosis. These results indicate that the presence of intramural metastasis is an important factor to consider when evaluating the prognosis of patients with squamous-cell carcinoma of the esophagus. PMID- 1728613 TI - Cancer control programme in India: opportunities for implementation and evaluation. AB - Cancer is now being appreciated in India as an emerging public health problem. Approximately 600,000 new cancer cases occur in India every year, and the absolute number of new cancer patients will increase considerably, due to growth in the size of the population and an increase in the proportion of elderly persons due to improved life expectancy following control of communicable diseases. This emerging problem has received the attention of the Government of India, and some state governments within the Indian Federation have formulated National and State Cancer Control Programmes to deal with the situation. In the event of these programmes being implemented, consideration should be given now to measures of evaluation of the activities, as many of the indices, used to monitor programmes in developed countries, such as 15% reduction from peak cancer mortality, or in peak mortality from specific cancers, are not applicable in developing countries like India. Factors such as the pattern of tobacco habits in the community, knowledge, attitude and practice (KAP) patterns regarding cancer in the general population, referral practices, and the national pattern of extent of disease at presentation, gain considerable importance from the evaluation point of view. Our article mainly deals with the sources and the quality of baseline data for such factors and the realistic quantitative goals which could be set for the above factors. PMID- 1728614 TI - Quantitative dot blot analyses of blood-group-related antigens in paired normal and malignant human breast tissues. AB - Membranes were prepared from 31 breast-cancer specimens and adjacent mammary tissues, dot-blotted to nitrocellulose paper, and reacted with monoclonal antibodies (MAbs) (A, B, Lewis a, Lewis b, sialylated Lewis a, Lewis x, and Lewis y) and lectins (Ulex europaeus, peanut agglutinin) having various blood-group specificities. The expression of epithelial membrane antigen was assayed with MAb MA5. The ratio of breast-cancer to normal mammary membrane preparations (C/N ratios) of these reagents was measured by densitometric scanning. We observed a decrease in the levels of A, B, Lewis a, Lewis b, sialylated Lewis a, and Lewis y antigens and an increase of Lewis x, T, and MA5-reactive determinants in breast cancers. The incidence of incompatible A, as well as A and B, antigens was demonstrated for 2 patients of blood group B and O respectively. When the receptor content was plotted against the C/N ratio of these various reagents, a significant inverse relationship between the C/N ratio of Lewis x antigen and estrogen (ER) and progesterone receptor (PR) content was observed in breast cancers. The mean C/N ratio of Lewis x antigen was significantly higher in the ER negative/PR-negative (ER-/PR-; 2.33 +/- 1.17), as compared with the ER positive/PR-positive (ER+/PR+; 0.97 +/- 0.80). According to these observations, Lewis x antigen expression may be influenced by hormonal stimuli such as estrogen and progesterone. PMID- 1728615 TI - Immunologic aspects of fibrosis in mouse mammary carcinomas. AB - The nature of the fibrosis associated with mammary carcinomas MC2 and MC3 was investigated in syngeneic C3H mice. Accelerated and enhanced peri-tumor cellular and fibrotic responses and retarded tumor growth were observed in actively immunized and in adoptively immunized mice, and in mice treated with IL-2. T lymphocytes and, particularly, macrophages were closely associated with collagen deposition at the tumors. The collagen deposition frequently resulted in the encapsulation and regression of the less invasive tumor MC2. A cellular fibrous response was not observed at tumors implanted into athymic C3Hnu/nu mice. The results suggest that tumor fibrosis may in some circumstances be promoted by an immune response. PMID- 1728616 TI - Blood coagulation changes in nude mice bearing human colon carcinomas. AB - We studied several blood coagulation parameters and tumor tissue procoagulant activity (PCA) in nude mice bearing human colorectal carcinomas (HCC). In a control group of 51 tumor-free nude mice, platelet number was 1.2 +/- 0.03 x 10(6)/microliters, thrombotest activity 90% +/- 2.6 and fibrinogen 172 +/- 11 mg/dl. The same parameters were studied in nude mice (n = 71) bearing 7 different HCC lines subcutaneously (s.c.). The results did not significantly differ from those in control mice but there was broad variability among groups of mice injected with different HCC lines, ranging from 0.36 to 2.55 x 10(6)/microliters for platelets, from 100 to 28% for thrombotest activity and from 42 to 460 mg/dl for fibrinogen. The results were significantly (p less than 0.05) different from those in the tumor-free group when each group of HCC-bearing animals was analyzed individually. A malignant HCC line that grew in the liver of nude mice (n = 24) significantly (p less than 0.001) reduced thrombotest activity (58% +/- 5.9). The PCA of tissue extracts from tumors grown s.c. in nude mice was assayed. All the HCC xenografts expressed PCA which differed significantly for the various tumor lines (from 25.5 +/- 1.9 to 2.8 +/- 0.6 unit/mg in tumor tissue). Cancer procoagulant (CP), a cysteine proteinase with a direct factor-X-activating effect, was present in different amounts (84.7 +/- 4.3 to 59.5 +/- 9.0%) in the tumors. Our results indicates that the nude mouse is a suitable model for evaluating the hemostatic changes induced by human tumors and may represent a tool for investigating the underlying biochemical mechanisms. PMID- 1728617 TI - Insulin dependence of murine lymphoid T-cell leukemia. AB - The in vitro proliferation of the spontaneous lymphoid T-cell leukemia designated LB was enhanced by physiological, intermediate and supraphysiological concentrations of insulin. The enhancing effect was observed in both serum-free medium (SFM) and medium containing low concentrations of serum. Guinea-pig anti insulin serum, but not guinea-pig normal serum, inhibited the proliferation of LB cells incubated either in medium containing serum alone or in medium containing serum and supplemented with insulin. This finding suggests that LB cells use serum insulin as a growth factor. Insulin-like growth factors I (IGF-I) and II (IGF-II) failed to stimulate an appreciable proliferation in LB cells, whereas in the same experiment insulin markedly enhanced the proliferation of this lymphoid leukemia. Furthermore, the concentration of unlabelled insulin required to displace 50% of 125I-insulin bound to LB cells was 3 orders of magnitude lower than the concentration of IGF-I required to achieve the same displacement. Our findings indicate that interaction of insulin with its own receptor, and not with IGF-I receptor, triggers the proliferation of LB cells. Radio-receptor assays revealed that LB cells express approximately 3,200 molecules of high affinity (Kd = 10(-9) M) insulin receptor per cell. None of 7 other tumor cell lines tested responded to insulin. The proliferation of insulin-stimulated LB cells was also inhibited with tyrphostin, a tyrosine kinase blocker analogous to tyrosine, which perhaps blocks the tyrosine kinase activity of the insulin receptor beta-chain. PMID- 1728619 TI - Tissue vitamin A repletion is impaired by exposure to carcinogen. AB - Vitamin A appears to exert a protective effect against certain cancers. Epithelial cancers, such as those of the skin, bladder, oropharynx and respiratory tract, have the strongest association with vitamin A. These same cancers are causally associated with exposure to carcinogens such as benzo(alpha)pyrene (BP), a product of combustion found in cigarette smoke and charbroiled meat. This study was designed to determine whether BP exposure affects tissue vitamin A nutriture. Female Sprague-Dawley rats were randomized to purified diets, sufficient or deficient in vitamin A, and with or without 200 mg BP/kg feed. Rats were killed after 4 or 6 weeks. Serum, liver and lungs were assessed for vitamin A levels; trachea, stomach, small intestine and bladder were examined for histologic change. Lack of dietary vitamin A resulted in a profound decrease in vitamin A in the serum, liver and lungs (p less than .005). No histologic changes were evident in any tissues examined. Serum vitamin A was not affected by dietary BP. In vitamin-A-sufficient rats, dietary BP caused a significant decline in hepatic and lung vitamin A. In rats fed vitamin-A deficient diets, dietary BP had no effect on tissue vitamin A. We conclude that chronic exposure to the carcinogen BP leads to tissue depletion of vitamin A, despite a vitamin-A-sufficient diet. We postulate that BP impairs tissue repletion by metabolizing incoming vitamin A rather than in situ vitamin A, since BP had no effect on tissue vitamin A levels in rats fed a diet devoid of vitamin A. This BP-induced vitamin depletion may eventually have a deleterious effect on epithelial tissue health, and may help to explain the association between vitamin A and cigarette-smoke-related cancers. PMID- 1728618 TI - Altered prolactin response of the lymphocytes of tumor-bearing mice. AB - Interaction of prolactin and glucocorticoid on thymocytes and splenocytes of normal and tumor-bearing mice with reference to their mitogen-responsive blastogenesis has been studied. Prolactin alone had little or no mitogenic effect on thymocytes and splenocytes of normal mice but it was co-stimulatory, with ConA, in inducing blastogenesis in normal splenocytes. Thymocytes and splenocytes of tumor-bearing mice responded differently to prolactin. Hormone alone inhibited growth of thymocytes but at certain concentrations stimulated splenocytes. When cultured with prolactin and ConA, the thymocytes of tumor hosts responded with increased proliferation compared to that induced by ConA alone. Glucocorticoid suppressed ConA-induced lymphocyte proliferation in both normal and tumor-bearing mice. Prolactin reversed the inhibitory effect of glucocorticoid in normal mice but failed to abrogate inhibition in tumor hosts. Altered responsiveness of lymphocytes of tumor hosts to prolactin was not a function of circulating prolactin, as the serum prolactin level was similar in normal and tumor-bearing mice. Prolactin, however, could not reverse estradiol-induced suppression of lymphocyte proliferation. The lactogenic hormone, but not somatogenic hormones, altered the glucocorticoid inhibition of lymphocyte growth. PMID- 1728620 TI - Dietetic educator as mentor. PMID- 1728621 TI - Dietary fat reduction strategies. AB - In this study, we used computer modeling to identify which techniques designed to achieve dietary fat reduction were the most effective in meeting the dietary recommendations of the American Heart Association Step-One diet. Menus were developed for men and nonpregnant, nonlactating women, 25 to 50 years old, according to the Continuing Survey of Food Intakes by Individuals (with 36% and 37% of energy from fat for men and women respectively). The menus were modified realistically using the Minnesota Nutrition Data System. The following five strategies were applied: skim milk replaced whole milk and 2%-fat milk (SKM); medium-fat meat exchanges replaced higher-fat ones (MMtEx); lean meat exchanges replaced higher-fat ones (LMtEx); fat-modified products were used (FMP); and 2% fat milk replaced whole milk (LFM). For men, strategies LMtEx, SKM + LMtEx, SKM + LMtEx + FMP, LMtEx + FMP, LMtEx + FMP + LFM, and LMtEx + LFM reduced energy by 195 to 415 kcal and achieved the targeted level of energy from fat (less than or equal to 30 +/- 1%) and cholesterol (less than 300 mg) while maintaining 67% or more of the Recommended Dietary Allowances for other nutrients. For women, however, no single strategy achieved the goal. Certain combinations of strategies, SKM + LMtEx, SKM + FMP, SKM + MMtEx + FMP, reduced energy by 150 to 268 kcal and achieved the targeted dietary fat and cholesterol goals while maintaining 67% or more of the Recommended Dietary Allowances for other nutrients. All strategies led to a reduction in both saturated fatty acids (to 9% to 10% of energy) and monounsaturated fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728623 TI - Comparison of six microcomputer dietary analysis systems with the USDA Nutrient Data Base for Standard Reference. AB - We compared the general operating features and nutrient databases of six microcomputer dietary analysis systems. A 3-day food record with 73 food items was entered into each program; nutrient averages were compared with the US Department of Agriculture Nutrient Data Base for Standard Reference (USDA NDB), full version, release 9, for microcomputers. The six programs were found to vary widely in cost, number of foods and nutrients in the database, use of non-USDA data and imputation of data for missing values, number of print/export options, time to analyze the 3-day food record, and overall ease of use. Although all of the microcomputer dietary analysis systems were within 7% of the USDA NDB for energy, protein, total fat, and total carbohydrates, the proportion of other nutrients varying more than 15% from the USDA NDB varied considerably between programs. Variance among programs for 3-day food record nutrient values occurred because of differences in the number of food items included in the database (leading to varying degrees of substitution), the recency of the nutrient data (whether or not the most recent USDA releases had been incorporated), and the number of missing values (the degree to which non-USDA sources or estimated calculations were used to fill in the blanks from the USDA standard). Our results demonstrate that it is important for each dietitian to carefully choose a microcomputer dietary analysis system that is suitable to specific and predetermined needs. PMID- 1728622 TI - The Diet Habit Survey: a new method of dietary assessment that relates to plasma cholesterol changes. AB - The Diet Habit Survey was designed to identify eating habits and measure dietary changes made over time by 442 adults in the Family Heart Study, a coronary heart disease prevention project. Reliability was determined by test-retest analysis. Validity was assessed by comparison with 24-hour dietary recalls and by comparing changes in diet with changes in plasma cholesterol levels. At baseline, 89% of the subjects were classified as eating the current American diet (37% fat), 10% reported eating Diet 1 (30% fat), and 1% reported eating Diet 2 (25% fat). After 5 years of dietary intervention, the population's eating habits had shifted; 48% reported eating the current American diet, 37% reported Diet 1, 14% reported Diet 2, and 1% reported Diet 3 (20% fat). Significant plasma cholesterol lowering was associated with changes in Diet Habit Survey scores reflecting lower cholesterol and saturated fat and higher complex carbohydrate intakes. This questionnaire is an inexpensive, reliable, and valid instrument for rapid assessment of eating habits and diet composition and, thus, is an important new tool for dietetics researchers and practitioners. PMID- 1728624 TI - Improved food intake and reduced nausea and vomiting in patients given a restricted diet while receiving cisplatin chemotherapy. AB - Administration of cisplatin alone or in combination with other cytotoxic agents commonly produces intractable nausea and vomiting, which is currently controlled through high-dose antiemetic drugs. However, patients often refuse continued therapy because of suboptimal control of nausea and vomiting and substantial decline in nutritional status. In this pilot study, 19 patients receiving cisplatin were evaluated for nausea and vomiting, amount of food intake, and subjective assessment of well-being. The study group received a colorless, odorless, predetermined meal three times daily; the meal included cottage cheese, apple sauce, vanilla ice cream, and other selected foods. Control-group patients selected their own meal. Study-group patients exhibited higher overall food intake, decreased nausea and vomiting, and a higher scored estimation of well being. The findings of this preliminary study indicate that the study diet helps provide nutrition care to cancer patients receiving cisplatin chemotherapy and helps create an atmosphere where the patient believes he or she has some control in the treatment outcome. PMID- 1728625 TI - Focus group sessions on formats of nutrition labels. AB - Four consumer focus group sessions, with a total of 40 participants, were conducted to gather information on the utility and appropriateness of selected components of nutrition label formats. The formats reviewed were bar graphs, pie charts, numeric listings, and adjectival descriptors such as high and low. Participants were asked to compare food labels using various format types and to discuss the utility and interpretability of the formats. The outcomes suggested that these consumers did not find pie charts useful. They considered bar graphs confusing or unnecessary when numeric values were provided. Participants expressed concern that adjectival descriptors could be misleading. The numeric listing format they considered the most useful consisted of two columns of numbers: one listing the amounts of food components present in a serving of the food, and a second listing either the percentage of the label reference value (eg, the US Recommended Daily Allowance) or the quantity established as the label reference value. Participants repeatedly stressed their interest in a simple label. The results form one component of the Food and Drug Administration's efforts to evaluate nutrition label formats and will be used in conjunction with ongoing experimental and quantitative research studies. PMID- 1728626 TI - Role of food and nutrition in the health perceptions of young children. AB - Sixty healthy children, 4 to 7 years of age, were interviewed to evaluate their health perceptions in general and to determine the degree to which they included food and eating behavior in their perceptions. Individual interviews with children incorporated both closed-ended and open-ended questions. Concept maps were used to analyze interview transcripts. The pretest/posttest experimental design randomly assigned children to experimental and control groups. Children in the experimental group completed a 4-week, home-based, nutrition education program to determine the feasibility of changing children's health perceptions with an educational intervention. Pretest and posttest health perception scores were compared by analysis of covariance. Results indicated that children perceived nutrition as a meaningful concept in relation to their health perceptions at pretest, but that after program participation, children significantly increased their perception that health and nutrition were related concepts. Our findings indicate that young children are cognitively ready to learn more about food, nutrition, and health than previously thought, but closed ended questions may not be sensitive enough to evaluate their learning at this age. PMID- 1728627 TI - Use of mentor programs in dietetic education. AB - Persons entering their first professional position can benefit from the career guidance and role models provided by a mentor program. To determine whether mentor programs were being used in dietetic education programs, surveys were mailed to all 667 program directors listed in The American Dietetic Association (ADA) Directory of Dietetic Programs, 1990-1991. Of the 329 (49%) directors who responded, approximately half (47%) reported that their program had never considered including a mentor program. Nonetheless, directors from each of the six dietetic education options reported that their program included a mentor program. Respondents were generous with their comments. Directors identified limited time as the greatest obstacle for implementing mentor programs. Changes in curricular demands were also cited. Program directors indicated a need for more information and models of mentor programs. Networks linking the dietetic practice groups of the ADA Council on Practice and research on mentor programs' effect on retention of dietitians in professional practice are needed. PMID- 1728628 TI - AIDS: legal implications for managers. AB - As the incidence of acquired immunodeficiency syndrome (AIDS) escalates, its consequences increasingly will affect the workplace. Dietitians with management responsibilities will be called upon to make decisions concerning the rights of both employees and clients or patrons. They must be knowledgeable about AIDS related legislation and its implementation in organizations and businesses. A major legal protection for workers with AIDS is the Vocational Rehabilitation Act, which prohibits discrimination against individuals on the basis of physical or mental disabilities. The Employee Retirement Income Security Act and the Comprehensive Omnibus Budget Reconciliation Act provide legal protections related to employee benefit programs. The National Labor Relations Act provides protection for all employees, including those infected with the AIDS virus. Current medical evidence indicates that AIDS is not transmitted through casual contact. Therefore, the presence of AIDS-infected individuals in the workplace does not violate the provisions of the Occupational Safety and Health Act. More recently, the Americans with Disabilities Act was amended to prohibit employers from removing workers from food-handling positions solely because of infection with AIDS. This review of legislation and litigation indicates that coworker and public fears, as well as management concerns about productivity and costs, are inadequate defenses against discrimination claims. Employees with AIDS are protected legally against adverse employment decisions motivated by their disease. PMID- 1728629 TI - A network model for improving access to food and nutrition data. AB - In this article we propose a network in which existing food composition and consumption databases are linked through a master database of complete and detailed food descriptions. The proposal arises from an analysis of the importance of food data, their descriptive and analytical nature, and their uses. Lack of detail and standardization in food description hinders the retrieval of food and nutrition data from various databases and the integration of such data. Standardized food descriptions can be developed and maintained in the master database, which can then serve as the interface to the many existing databases of analytical data (especially food composition data) and to databases containing data on food production, consumption, and effects, thereby linking these databases in a coordinated system, or network. The ability to link food-related databases by standardized food descriptions offers a powerful tool for scientists and practitioners in the field of food and nutrition. PMID- 1728630 TI - Relationship of self-reported prepregnant weight and weight gain during pregnancy to maternal body habitus and age. PMID- 1728631 TI - Estimation of sodium intake by analyzing food records with augmented nutrition software and by overnight urine collections. PMID- 1728632 TI - Amenorrheic and eumenorrheic adolescent runners: dietary intake and exercise training status. PMID- 1728633 TI - Fat replacements: a new strategy for dietary change. PMID- 1728634 TI - Differential modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin. AB - Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming growth factor-beta 1, revealed increased extracellular and decreased intracellular expression of transforming growth factor-beta 1 in retinoic acid-treated, compared to vehicle treated, skin. Transforming growth factor-beta 1 staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming growth factor-beta 1 expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming growth factor-beta 1 protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-beta 1 and TGF beta 2 immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin deposition, which is induced by transforming growth factor-beta, was elevated in retinoic acid-treated but not sodium lauryl sulfate treated skin. Cultured adult human keratinocytes also expressed predominantly transforming growth factor-beta 1 protein, as measured by ELISA, and mRNA. Treatment of keratinocytes with retinoic acid resulted in a 50% induction of transforming growth factor-beta 1 protein, without any detectable change in transforming growth factor-beta 2. These data demonstrate disassociation of modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming growth factor-beta 1 expression were observed in both retinoic acid- and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid-treated skin. PMID- 1728635 TI - Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors. AB - We examined the altered expression of alpha-smooth muscle actin (alpha-Sm) in human benign, pre-malignant, and malignant pigment cell tumors by immunohistochemical as well as biochemical (Western blot) analysis using anti alpha-Sm monoclonal antibody (anti-alpha-Sm MoAb). The expression of alpha-Sm has been revealed immunohistochemically to be associated with mesodermal cells rather than with pigment cells. Western blot analysis using anti-alpha-Sm MoAb detected alpha-Sm expression as a 43-kD band in the extracts from normal papillary dermis, nevus cell nevus, and metastatic melanoma with stromal tissues, but not from primary melanoma with stromal tissues examined. The above findings of alpha-Sm expression by Western blot analysis were further characterized immunohistochemically in terms of the localization at the cellular level as follows. 1) In normal papillary dermis, pericytes encircling capillary vessels showed only positive staining with anti-alpha-Sm MoAb. 2) In nevus tissues, nevus cells were not shown to be positively stained, despite similar positivity of pericytes in normal papillary dermis. 3) In melanoma tissues, alpha-Sm expression of metastatic melanoma detected by Western blot analysis was found to be derived from fibroblasts with smooth-muscle differentiation (myofibroblasts), but not from melanoma cells. Such myofibroblastic stromal changes could not be found on primary melanoma tissue sections, which showed no reactivity in Western blot analysis. We conclude that the major sources of alpha-Sm in benign and pre malignant pigment cell tumors are capillary pericytes, whereas alpha-Sm found in malignant melanoma tissue is primarily from melanoma-surrounding stromal fibroblasts that were changed to myofibroblasts by some cytokine factor(s), presumably secreted from melanoma cells. PMID- 1728637 TI - Interaction of trichohyalin with intermediate filaments: three immunologically defined stages of trichohyalin maturation. AB - "Trichohyalin" is a 220-kD protein found in trichohyalin granules that are present as major differentiation products in the medulla and inner root sheath cells of human hair follicles. It was unclear whether this protein served as an intermediate filament precursor in the inner root sheath or as an intermediate filament-associated (matrix) protein. We have produced a panel of monoclonal antibodies (AE15-17) to this protein and used them to trace its fate during inner root sheath differentiation. These studies have allowed us to define three immunologically distinct forms of this trichohyalin protein. They are 1) the AE15 positive form, which is found throughout all trichohyalin granules; 2) the AE16 positive form, which is localized as discrete punctae on the surface of trichohyalin granules; and 3) the AE17-positive, intermediate-filament-bound form, which associates with the inner root sheath filaments with a regular, 400 nm periodicity. From these results we suggest that the 220-kD trichohyalin protein is an intermediate-filament-associated protein that may play a role in the lateral aggregation, precise alignment, and stabilization of inner root sheath filament bundles. PMID- 1728636 TI - Human keratinocyte locomotion: the effect of selected cytokines. AB - Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are two powerful mitogens for human keratinocytes that also have been shown to promote the healing of in vivo wounds. Transforming growth factor-beta (TGF-beta) markedly inhibits human keratinocyte proliferation and growth and yet has been shown to promote wound healing. Using a migration assay that evaluates pure cell locomotion independently from cell proliferation, we examined the influence of EGF, bFGF, and TGF-B on human keratinocyte locomotion. Although these agents had profound influences upon the growth potential of keratinocytes in parallel thymidine incorporation assays, they had no significant effect upon keratinocyte locomotion when cells were apposed to either tissue culture plastic or a collagen substratum. In contrast, we found that bovine pituitary extract (BPE), a poorly defined mitogen that is commonly used in keratinocyte cultures, could stimulate keratinocyte locomotion when the cells were apposed to a collagen substrate. These studies demonstrate that i) keratinocyte locomotion and proliferation operate by completely independent mechanisms, ii) the positive effects upon wound healing by EGF, bFGF, and TGF-beta are not due to a direct promotion of keratinocyte locomotion, and iii) that one or more components of BPE are capable of directly promoting keratinocyte locomotion on collagen. PMID- 1728638 TI - Migration of a human keratinocyte cell line (HACAT) to interstitial collagen type I is mediated by the alpha 2 beta 1-integrin receptor. AB - The migratory response of the human keratinocyte cell line HaCaT to collagen type I and the molecular mechanism underlying collagen-mediated migration have been analyzed. The migratory response of HaCaT cells to collagen type I consisted of a dose-dependent migration to insoluble step gradients of substratum-bound collagen (haptotaxis) and to gradients of soluble collagen (chemotaxis). Checkerboard analysis demonstrated a minor chemokinetic component. Denatured collagen type I was less chemoattractive than the native triple-helical form. Pre-treatment of cells with 25-250 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence RGD (Arg-Gly-Asp) resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis, whereas chemotaxis to collagen was not affected. We then investigated the role of VLA/collagen-receptors for collagen type I-induced chemotaxis. Monoclonal antibody (MoAb) 5E8, which selectively blocks function of the alpha 2 subunit of the VLA-2/collagen receptor, dose-dependently inhibited the chemotactic response of HaCaT cells to collagen. This effect was specific for collagen-mediated chemotaxis because the chemotactic response to fibronectin remained unaffected. In contrast, a function blocking MoAb directed to the alpha 3 subunit of the coexpressed VLA-3 receptor, which is also capable of binding collagen, had no effect. However, function blocking MoAb directed to the beta 1-chain of integrins completely inhibited chemotaxis to collagen type I. Based on our results, we propose that the chemotactic migration of the human keratinocyte cell line (HaCaT) to collagen type I is specifically mediated by the RGD independent VLA-2/collagen receptor (alpha 2 beta 1) of the integrin family. PMID- 1728639 TI - Stimulation of cutaneous T-cell lymphoma cells with superantigenic staphylococcal toxins. AB - Stimulation of T cells by superantigenic bacterial toxins is selective for cells bearing particular B chain variable (VB) gene segments of T-cell receptor (TCR). In humans, staphylococcal exfoliating toxin (ExT) and toxic shock syndrome toxin 1 (TSST-1) are known to stimulate VB 2-bearing T cells and staphylococcal enterotoxin B (SEB) does not activate VB 2-bearing T cells. We examined the proliferative response of cutaneous T-cell lymphoma (CTCL) cells to ExT, TSST-1, and SEB. Leukemic VB 2.1-bearing CTCL cells were reactive to ExT and TSST-1, but not SEB. In addition, two leukemia CTCL-VB 2- cell samples (one of which was VB 8) showed no substantial response to ExT. Thus, it was shown that Sezary cells proliferate in response to bacterial superantigens in a manner that is restricted by their VB usage. The addition of interleukin-1 (IL-1) in combination with ExT enhanced the stimulative response of VB 2.1-bearing CTCL cells that were pre cultured with ExT for 7 d, suggesting that IL-1 can be a co-factor for the stimulation. The present study indicates that the superantigen reaction occurs with CTCL cells and implies a possible involvement of bacterial toxins in the pathogenesis of CTCL. PMID- 1728640 TI - Soluble interleukin-2 receptors inhibit interleukin 2-dependent proliferation and cytotoxicity: explanation for diminished natural killer cell activity in cutaneous T-cell lymphomas in vivo? AB - In patients with cutaneous T-cell lymphomas (CTCL), soluble interleukin-2 receptor serum levels (sIL-2R) were determined by ELISA technique, and natural killer cell (NK) activity, by a 4-h chromium-51 release assay. Decrease of NK activity correlated with the augmentation of serum sIL-2R. After a 4-d stimulation with interleukin 2 CTCL patients' peripheral mononuclear cells (PMC) showed an increase of cytotoxic activity similar to that in healthy donors' PMC. Normal donors' PMC demonstrated a diminished IL-2-induced cytotoxic activity in 25% CTCL serum (sIL-2R of 3000, 7330, and 10700 U/ml, respectively) compared to control serum (sIL-2R of 400, 340, and 420 U/ml, respectively). IL-2-dependent proliferation of 2-d phytohemagglutinin (PHA) blasts was lower in CTCL serum than in control serum. sIL-2R was enriched from one CTCL patient's serum by IL-2 affinity chromatography. Transfection of the Tac gene into NIH/3T3 fibroblasts resulted in the production of a recombinant sIL-2R. The presence of enriched native or recombinant sIL-2R inhibited interleukin-2-dependent generation of cytotoxic activity and PHA blast proliferation. We suggest that elevated sIL-2R levels account for diminished NK activity by neutralizing interleukin 2 in CTCL patients. PMID- 1728641 TI - Ultrastructural studies on the invasion of melanomas in initial lymphatics of human skin. AB - The way in which melanoma cells invade the initial lymphatics of the skin was investigated in this study. Samples of sixty melanomas were examined by transmission electron microscopy. Tumor cells invading lymph vessels were demonstrated in 20 specimens. In most cases the melanomas penetrated the subendothelial space as single cells. These fused with the endothelial cytoplasmic membrane and subsequently destroyed the endothelial wall. PMID- 1728642 TI - Increased adhesion of fibroblasts from patients with scleroderma to extracellular matrix components: in vitro modulation by IFN-gamma but not by TGF-beta. AB - A characteristic feature of systemic scleroderma is fibrosis of the skin and eventually of internal organs resulting from an overproduction of collagen and other connective tissue components by the resident fibroblasts. The balance between the cells and the amount of the surrounding extracellular matrix is then altered. Because cellular metabolism depends to a large extent on cellular contacts and communications with connective tissue molecules, we have therefore investigated the interactions with extracellular matrix components of fibroblasts obtained from skin of patients affected with scleroderma. In comparison to fibroblasts from healthy skin, all fibroblasts from scleroderma patients had an increased adhesion capacity to collagens I, IV, VI, fibronectin, and laminin. In addition, whereas adhesion of control fibroblasts was stimulated by a pre treatment with transforming growth factor-beta, adhesion patterns of scleroderma fibroblasts remained unchanged. However, pre-incubation of the cells with interferon-gamma decreased the adhesion of both scleroderma and control fibroblasts. PMID- 1728643 TI - Interleukin-8 immunoreactivity in the skin of healthy subjects and patients with palmoplantar pustulosis and psoriasis. AB - Previous studies have shown that neutrophil-activating peptide 1/interleukin-8 (IL-8) is present in psoriatic scales and to a lesser extent in normal human epidermis. A panel of monoclonal antibodies and polyclonal antisera raised against IL-8 was used to localize IL-8 with immunoperoxidase techniques in non lesional and lesional skin of patients with psoriasis and palmo-plantar pustulosis (PPP), and in corresponding sites from healthy subjects. Intracellular IL-8 immunoreactivity was found in all epidermal cell layers in biopsies of healthy subjects and in non-lesional and lesional skin in both PPP and psoriasis. The most intense immunolabeling was regularly found in the basal cell layer. Intercellular epidermal IL-8 immunolabeling was regularly detected in lesional biopsies in PPP and psoriasis, but not in healthy subjects or non-lesional skin in PPP and psoriasis. No intercellular immunolabeling was detected after successful treatment of lesional skin. The majority of cells along the eccrine sweat glands, dermal mononuclear cell infiltrates, and endothelial cells were IL 8 immunoreactive in all biopsies studied. The present study suggests that IL-8, its precursor form, or, alternatively, a degradation product is present in normal human epidermis. PMID- 1728644 TI - Dog tapeworm (Dipylidium caninum) infestation in a 6-month-old infant. PMID- 1728645 TI - Screening for mood disorders. PMID- 1728646 TI - Treatment of hypercholesterolemia. PMID- 1728647 TI - Labor and birth care. PMID- 1728648 TI - Definition of life. PMID- 1728649 TI - Management of NIDDM. PMID- 1728650 TI - Relationships between family physicians and the pharmaceutical industry. PMID- 1728651 TI - There is no such thing as a free lunch. Developing policies on pharmaceutical industry support. PMID- 1728652 TI - Papanicolaou testing and colposcopic screening. PMID- 1728653 TI - Sample medication dispensing in a residency practice. AB - BACKGROUND: The distribution of sample medications to physicians by pharmaceutical manufacturers has been regulated by Congress and extensively critiqued in the medical literature. Manufacturers distributed 2.4 billion samples in 1988, yet there are no published reports on the clinical use of sample medications. METHODS: A 4-week descriptive study was conducted that catalogued the contents of a sample medication collection in a family practice residency model office, calculated the value of the sample collection (average wholesale price [AWP]), and monitored dispensing of medication samples. RESULTS: The collection initially contained 5546 samples with an AWP of $19,273. A total of 1012 samples worth $4154 was withdrawn from the collection during the study period. Patients received 548 of the sample packages in 105 dispensements ($2583), physicians or their families received 169 samples in 44 dispensements ($603), others received 26 samples in 6 dispensements ($152), and the destination of 269 samples ($816) was unknown. When a prescription was written at the time that a sample was dispensed, it was almost always for the same brand-name medication. CONCLUSIONS: Although a majority of medications dispensed were given to patients, approximately one third of the value of the medications withdrawn either went to physicians and their families or had an unknown destination. The high association of sample dispensing and simultaneous prescribing of the same brand-name drug supports the contention that sampling influences physician prescribing habits. Further research should define how the availability of free sample medications affects physician-prescribing practices. PMID- 1728654 TI - Involvement of pharmacy faculty in the development of policies for pharmaceutical sales representatives. AB - BACKGROUND: Few studies evaluating the impact of the pharmaceutical industry on postgraduate medical education have been done. Recently, position statements and professional guidelines have emerged to ensure the integrity of physician industry relationships in the areas of clinical judgement, research, and medical education. METHODS: The present study surveyed directors of family practice residency programs in the United States to define the level of pharmacotherapy curriculum development and the existence of policies for pharmaceutical sales representatives. RESULTS: Of the 383 directors, 325 (85%) responded to a mailed survey. Nearly one third (32%) of the responding programs had pharmacist faculty, the majority of whom held a doctor of pharmacy degree. Approximately 30% of programs reported that they had printed guidelines for pharmaceutical sales representatives. CONCLUSIONS: Programs with pharmacist faculty are more likely to have a well-developed pharmacotherapy curriculum and printed guidelines for pharmaceutical sales representatives. PMID- 1728655 TI - Policies regulating the activities of pharmaceutical representatives in residency programs. AB - BACKGROUND: Residents frequently interact with pharmaceutical representatives during their training. The purpose of this study was to determine the prevalence of policies restricting or regulating the interactions of pharmaceutical representatives with family medicine residents. METHODS: A descriptive, cross sectional survey was sent to all 386 accredited family practice residency programs. Programs were surveyed for the presence of restrictions or policies regarding the following circumstances and activities through which pharmaceutical representative-resident interactions could occur: (1) contact during working hours, (2) clinic drug samples, (3) personal samples for residents, (4) displays, (5) distribution of literature, (6) gifts and outings, and (7) group presentations. RESULTS: Overall, residency programs tended to allow most of these activities and had only informal guidelines regarding pharmaceutical representative interaction. Written policies were present in 58% of the programs. Prohibitions of some type were present in 41% of the programs. A higher prevalence of written policies was noted in military programs, larger programs, and programs located in hospitals with only family practice residents. CONCLUSIONS: There are wide variations among family practice residency programs regarding the regulation of pharmaceutical representative-resident interactions. In view of the educational mission of residency training programs and the recent concern over the ethics of the relationship between the medical profession and the pharmaceutical industry, it would be prudent for all residencies to develop written policies addressing the activities of pharmaceutical representatives in training sites. PMID- 1728656 TI - Nicotine chewing gum use in the outpatient care setting. AB - BACKGROUND: The purpose of this study was to assess nicotine gum use when prescribed in a nonresearch, routine outpatient setting. Special attention was given to comparing actual use patterns with established guidelines for use based on clinical research. METHODS: A randomly selected group of 612 patients who had received a prescription for nicotine gum during an 18-month period were surveyed regarding their smoking history and use of the gum. RESULTS: Most of the gum prescriptions (75%) were requested by patients rather than recommended by medical care providers. Less than one half of the users were heavy smokers. The reported amount of gum used was small, with more than one half reporting consumption of one box or less, and about one third reporting use of the gum for only 1 week or less. Larger amounts of gum use, however, were associated with abstinence from tobacco. Only one in 20 users attended a structured behavioral treatment program while using the gum. Over one half of the patients reported using nicotine gum to help them cut down on, rather than quit, smoking. CONCLUSIONS: Only a small percentage of the patients used the nicotine gum according to the established guidelines, and most of the patients used the gum in ways that have been shown to be ineffective for smoking cessation. Providers should educate their patients in the techniques that maximize the use and effectiveness of nicotine gum in smoking cessation. PMID- 1728657 TI - Glaucoma screening in primary care: the role of noncontact tonometry. AB - BACKGROUND: Guidelines for glaucoma screening by the primary care physician have not been firmly established. Despite its limitations as a screening test, intraocular pressure measurement by tonometry remains the mainstay of glaucoma monitoring but is not widely used in the primary care setting. The purpose of this study was to compare the effectiveness of noncontact tonometry using the Pulsair instrument with that of conventional tonometry using the Goldmann applanation tonometer as a screening tool for glaucoma. METHODS: Intraocular pressure was measured by non-contact and Goldmann applanation tonometry in both eyes of 50 volunteers who enrolled in a glaucoma screening program at a primary care clinic. RESULTS: Noncontact tonometry correctly identified over 90% of the patients with intraocular pressures greater than 22 mm Hg. CONCLUSIONS: Noncontact tonometry is an easy, practical, and well-tolerated method of intraocular pressure measurement. When combined with direct ophthalmoscopy, noncontact tonometry can easily be used in routine primary care health examinations to detect glaucoma. PMID- 1728658 TI - Support services for family practice residents. AB - BACKGROUND: Internship and residency are stressful experiences for physicians in training. Residency programs vary in their provision of supportive services for residents. METHODS: A random sample of 50% of the nation's family practice residency programs was surveyed to determine the prevalence of 19 support services, 10 of which were assessed a decade previously. Programs were also asked about on-call frequency, vacation benefits, and program size. RESULTS: Approximately 91% of the programs responded. The surveys indicated that residents were on call an average of once every four nights, a 10% decrease from a decade ago. The prevalence of three support services had increased over the last decade: seminars and speakers on the stresses and conflicts of being a physician, support groups for residents, and child care services. "Night-float" rotations and part time residencies are the least offered support services of those studied. CONCLUSIONS: Support for family practice residents is increasing, yet in many cases remains inadequate. PMID- 1728659 TI - Improving and maintaining preventive services. Part 1: Applying the patient path model. AB - Research in the past two decades has made remarkable progress in determining the variables that affect preventive care within primary care practices. The level of preventive care that a patient receives is largely determined by factors within the medical office setting. Many of these factors can be modified by physicians to encourage preventive care. An overview of these factors, presented as the Patient Path Model, can provide a framework for systematic practice evaluation. This model can be applied to almost any office setting to help identify opportunities to enhance and improve preventive care. PMID- 1728660 TI - Improving and maintaining preventive services, Part 2: Practical principles for primary care. AB - Recent research has recognized several themes that have been common to many successful projects for increasing cancer screening and other prevention activities. The most common of these themes have been condensed into "principles for implementation," intended to help physicians and other health care providers to improve the provision of preventive medical care within their practices. PMID- 1728661 TI - Nifedipine: use of an oral antihypertensive agent in the emergency department setting. PMID- 1728662 TI - Cadmium treatment and lead-induced suppression of splenic erythropoiesis. AB - Young adult female mice were injected with lead acetate (d 0). Following injection, determinations were made of the percentages of radioactive iron (59Fe) uptake into the hemoglobin of erythrocytes produced by spleen. Control 59Fe uptake percentage vacillated between 4.2 and 5.5 within the 7-d period of observation. On d 4 following lead treatment, splenic percentages were dramatically reduced below those of the saline-injected controls; by d 6 the splenic 59Fe uptake of lead-treated mice was comparable to that of controls. For rodents injected with cadmium chloride on 0, the 59Fe uptake values showed a statistically significant elevation by d 2, which was extended beyond that of the controls' d 4 value. For those animals receiving both lead and cadmium (d 0), the uptake percentages paralleled those of the controls throughout the 7-d period of observation. These data suggest that the inhibitory effect of lead on erythropoiesis of the spleen is blocked by a concurrent cadmium treatment. Results are interpreted in regard to a possible vulnerable target and competition for the target by lead and cadmium. PMID- 1728663 TI - Biotransformation of butachlor through mercapturic acid pathway in rat tissue homogenates. AB - The metabolism of butachlor was studied in rat liver and kidney homogenates. In vitro incubation of butachlor with liver fractions (S9, microsome, and cytosolic fractions) formed a considerable amount of butachlor glutathione conjugate (BGSC), while the conjugating activity was not efficient for the kidney S9 fraction. There is a sex difference in the distribution of glutathione S transferase in the liver. It seems that more enzyme activity is detected in the female liver microsome, while this is not the case in its cytosolic fraction. Further biotransformation of BGSC to mercapturate was not observed in the liver S9 fraction. This metabolite was further transformed to butachlor acetyl cysteine conjugate (BACC) in the presence of acetyl CoA, but to butachlor cysteine conjugate (BCC) in the absence of acetyl CoA. These findings demonstrated that butachlor is initially conjugated with GSH to form BGSC by the enzyme glutathione S-transferase in the liver. This metabolite is apparently transported to the kidneys, where it is transformed to the mercapturate. PMID- 1728664 TI - Investigation of TCDD half-life heterogeneity in veterans of Operation Ranch Hand. AB - The half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the contaminant of Agent Orange, has been recently estimated in 36 members of Operation Ranch Hand, the Air Force unit responsible for the aerial spraying of Agent Orange in Vietnam, as 7.1 yr with a 95% [corrected] confidence interval of 5.8-9.6 yr. We investigated the variability of TCDD half-life with percent body fat in these 36 Ranch Hand veterans who have two TCDD assay results from serum drawn in 1982 and 1987. Using a repeated measures linear model, we found a marginally significant change in half-life with percentage of body fat (p = .09) and no statistically significant change in half-life with relative changes in percentage of body fat from 1982 to 1987 (p = .60). PMID- 1728665 TI - Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein. AB - The purpose of this study was to determine if Zn pretreatment could protect rat primary hepatocyte cultures from the cytotoxicity of five metals that have little or no affinity for metallothionein (MT). Hepatocytes were grown in monolayer cultures for 22 h and subsequently treated with ZnCl2 (100 microM) for 24 h; which increased the MT concentration 15-fold. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Mn, V, Cr, Se, or Fe for an additional 24 h. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all five metals was significantly reduced in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 h) of Mn or Se. Zn pretreatment also did not increase the distribution of Mn or Se to the cytosol and neither metal was bound to MT, suggesting the protection was not due to their binding to MT. However, Zn pretreatment significantly decreased Mn-, Cr-, and V-induced cellular glutathione depletion. In summary, Zn pretreatment of rat primary hepatocyte cultures protects against Cr-, Mn-, Fe-, Se-, or V-induced hepatotoxicity. This protection does not appear to be related to MT induction but may be due to Zn-induced thiol or membrane stabilization and/or other biological changes produced by Zn. PMID- 1728666 TI - Cigarette smoke exposure does not prevent cadmium-induced alterations in rat lungs. AB - Cigarette smoke has been demonstrated to suppress the biosynthesis of connective tissue in the lung. To further characterize this suppressant effect, we studied the ability of cigarette smoke to prevent or ameliorate cadmium-induced alterations in rat lungs in vivo. The effects of beta-aminopropionitrile (beta APN), an agent that inhibits the cross-linking of elastin, also were studied. Eighty-eight young female Long-Evans rats were randomly divided into seven groups as follows: control, cigarette smoke, sham smoke, beta APN, cadmium, cadmium + cigarette smoke, and cadmium + beta APN. Each animal in the cigarette smoke group was exposed to mainstream smoke generated from University of Kentucky 2R1 reference cigarettes (10 puffs daily for 12 wk). Sham-treated animals received room air in place of cigarette smoke. beta APN (0.5 g/kg) was injected intraperitoneally twice weekly. In cadmium-treated groups, each rat received intermittently three intratracheal instillations of cadmium chloride (0.15 mumol/kg) over a 5-d period. For the cadmium + cigarette smoke group, smoke exposure began 3 d after the first cadmium instillation and was continued for 12 wk. The beta APN administration began 5 d before cadmium instillation and also was continued for 12 wk. After these treatments, pulmonary function and lung morphometry were examined. Neither cigarette smoke, sham smoke, nor beta APN produced significant changes in lung function or morphometry. Cadmium caused significant decreases in total lung capacity, dynamic and static compliance, and carbon monoxide diffusing capacity, as well as significant increases in lung weight and alveolar wall thickness. In addition, the quasistatic deflation pressure-volume curve showed a rightward shift whereas the mean linear intercept of the alveoli did not change significantly. Efforts to prevent or ameliorate the changes through exposure to cigarette smoke or administration of beta APN were unsuccessful. It is concluded that interventions designed to inhibit the biosynthesis of lung connective tissue do not perforce inhibit the development of cadmium-induced pulmonary changes in the rat. PMID- 1728667 TI - Comparison of rat pulmonary and hepatic cytosolic alcohol dehydrogenase activities. AB - The liver is the major organ responsible for ethanol oxidation, and alcohol dehydrogenase (ADH) is the main enzyme involved. There is limited evidence suggesting the involvement of the lung in ethanol metabolism. To determine the degree to which pulmonary ADH plays a role in ethanol metabolism, ADH activity was measured spectrophotometrically using hepatic and pulmonary cytosolic fractions prepared by differential centrifugation and Sephadex G-50 column chromatography. Apparent Km values for hepatic and pulmonary ADHs were determined. Inhibition constants were calculated using 4-methylpyrazole. The ADHs were characterized by examining the influence of pH on enzyme activity. Pulmonary ADH activity was much lower at near neutral pH than at pH 9.0 or 10, whereas hepatic ADH activity was also pH dependent but was significantly higher. Pulmonary ADH is less sensitive to inhibition by 4-methylpyrazole than is hepatic ADH, as evidenced by a 1000-fold higher Ki. Pulmonary ADH would be expected to make only a minor contribution to ethanol metabolism in vivo. PMID- 1728668 TI - Unsuspected preexisting saphenous vein disease: an unrecognized cause of vein bypass failure. AB - Our prior anecdotal experience with unsuspected preexisting saphenous vein disease prompted us to study its incidence, its relation to graft failure, and to identify techniques for its detection. Thick-walled, postphlebitic sclerotic occluded, postphlebitic sclerotic recanalized, calcified, and varicose vein lesions were detected in 63 (12%) of 513 infrainguinal vein bypasses. In 13 (2% to 5%) cases, severe saphenous vein disease precluded use of the vein. In the remaining 50 cases, the entire vein or a portion thereof, with minimal or unsuspected disease, was used for bypass. Early graft failures occurred in 10 (20%) of the 50 cases. The cumulative primary patency rate at 30 months for bypasses performed with diseased veins was 32%. This was significantly less than the 73% cumulative primary patency rate for bypasses with veins without detectable disease (p less than or equal to 0.001). Retrospective evaluation of preoperative duplex ultrasonography (n = 21) originally used to evaluate saphenous vein length and diameter correctly identified thick-walled, occluded, calcified, and varicose veins in 62% of cases. Intraoperative methods of vein evaluation included inspection, palpation, irrigation, catheter or valvulotome insertion to identify obstruction, and intraoperative arteriography. Histologic examination of diseased veins demonstrated a spectrum of disease with thickening of the intima and media, vein wall calcification, and luminal recanalization. We conclude that (1) unsuspected preexisting saphenous vein disease occurs in approximately 12% of cases and results in both early and late graft failures; (2) detection, in some cases, is possible with duplex ultrasonography and intraoperative techniques; and (3) diseased veins that are recanalized, calcified, or thick-walled should not be used if an alternative vein is available. PMID- 1728669 TI - Causes of primary graft failure after in situ saphenous vein bypass grafting. AB - In situ saphenous vein bypass grafts originating in the groin were performed in 455 consecutive patients. Primary failure occurred in 92 grafts during follow-up, including 22 (4.8%) with nonocclusive stenosis and 70 (15.4%) with occlusion. The cause for failure could not be determined in seven grafts; 104 contributory causes were identified in the remaining 85 grafts. Among the 104 likely causes, 66 (63%) were intrinsic to the graft itself and contributed to failure of 55 (12.1%) of 455 grafts. These causes included perianastomotic stenosis (48), vein stricture (14), focal vein stenosis (10), valvulotome injury (9), kink (6), retained valve leaflet (4), intimal flap (3), and residual arteriovenous fistula (2). Among these intrinsic causes, 20 were directly related to the in situ technique, contributing to failure of 15 (3.3%) of 455 grafts. Thirty-eight (37%) of the 104 causes were extrinsic to the graft, including compromised inflow (2) or outflow (19), hypercoagulability (9), systemic hypotension (6), and graft sepsis (2). Hypothetically, improvements in technique, patient selection, and perioperative management might have eliminated 46 (44%) of 104 causes of primary graft failure. Delayed graft and anastomotic stenosis and late progression of outflow disease remain resistant to modern therapy. PMID- 1728670 TI - An evaluation of new methods of expressing aortic aneurysm size: relationship to rupture. AB - The diameter of aortic aneurysms were standardized to measures of patient size and normal aortic size in an effort to define indexes that might be more predictive of aneurysm rupture than raw aneurysm diameter alone. Normal aortic diameters were measured in 100 patients undergoing abdominal CT scans for other reasons, and an average infrarenal aortic diameter of 2.10 +/- 0.05 cm was observed. Normal aortic diameter was dependent on both age and sex, ranging from 1.71 +/- 0.06 cm in women below age 40 years to 2.85 +/- 0.04 cm in men above age 70 years. Overall, 11 (5.1%) of the ruptures occurred in aneurysms less than 5 cm in diameter, and four (1.9%) occurred in aneurysms less than 4.0 cm in diameter. When the CT scans of 100 patients undergoing elective aneurysm resection were compared with those of 36 patients with ruptured aneurysms, no threshold diameter value accurately discriminated between the two groups. However, standardization of the aneurysm diameter to the transverse diameter of the third lumbar vertebral body as an index of patient body size produced an accurate predictor of rupture when a threshold ratio of 1.0 was used. No aneurysm ruptured below this ratio, but 29% of elective aneurysms were smaller than the vertebral body diameter. Receiver operating characteristic curve analysis confirmed the superiority of the aneurysm to vertebral body diameter ratio as a discriminator of ruptured aneurysms. It appears that aneurysm diameter alone is not sufficiently predictive of rupture to be used as the sole indication for elective resection. PMID- 1728671 TI - A blinded comparison of angiography, angioscopy, and duplex scanning in the intraoperative evaluation of in situ saphenous vein bypass grafts. AB - Angiography, angioscopy, and duplex scanning have each been advocated for intraoperative assessment of in situ saphenous vein grafts. We compared these three modalities during operation in a prospective, blinded study during the construction of 20 femoral-infragenicular in situ saphenous vein grafts. Each modality was used and interpreted by a surgeon blinded to the results of the other studies. Abnormalities requiring intervention were defined as (1) patent vein side branches, (2) residual valve cusps, and (3) anastomotic stenoses greater than 30%. Criteria, specific to the modality, corresponding to each category were prospectively defined. Fourteen residual valve cusps, 49 patent vein branches, and 6 anastomotic stenoses were suggested by at least one modality. Nine residual valve cusps, 32 patent vein branches, and no anastomotic stenoses were actually found (and corrected) by direct inspection. Sensitivity of detecting patent side branches for angiography, duplex scanning, and angioscopy was 44%, 12%, and 66%, respectively. Both angiography and angioscopy were significantly more sensitive than duplex scanning for detection of unligated side branches (p less than 0.01). Sensitivity of detecting residual valve cusps was 22% (angiography), 11% (duplex scanning), and 100% (angioscopy). Angioscopy was significantly more sensitive than either duplex scanning or angiography in detection of residual valve cusps (p less than 0.01). Since no anastomotic stenoses were confirmed, the false-positive rates for stenosis detection were 20% for angiography, 10% for duplex scanning, and 0% for angioscopy. Time requirement was 17 to 20 minutes and did not differ among the three modalities. No stenosis or arteriovenous fistula has been detected in any graft by postoperative duplex surveillance (mean, 10-month follow-up).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728672 TI - A twelve-year experience with the popliteal-to-distal artery bypass: the significance and management of proximal disease. AB - The value of the popliteal-to-distal artery bypass in limb salvage is well documented. However, the influence of progression of disease in the superficial femoral artery or proximal popliteal artery, and the role of percutaneous transluminal angioplasty of these vessels before bypass have not been adequately assessed. To evaluate these and other factors, we reviewed our experience with 153 nonsequential popliteal-to-distal artery bypasses performed over a 12-year period. Limb salvage was the indication for all procedures, and 87% of the patients were diabetic. The 5-year primary and secondary graft patency rates were 55% and 60%, respectively, and the limb salvage rate was 73%. Preoperative arteriograms were evaluated for stenosis in the superficial femoral artery or popliteal artery proximal to the graft. Fifty-six grafts with a proximal stenosis 20% or less were identified and had primary graft patency of 77% at 2 years, similar to the 70% patency for the 20 grafts placed distal to a 21% to 35% stenosis. The 18 grafts placed distal to a stenosis greater than 35% had 53% 2 year primary graft patency (p = 0.25). Percutaneous transluminal angioplasty of a superficial femoral artery or popliteal artery stenosis (24% to 85% luminal narrowing) in 19 limbs resulted in 68% 2-year graft patency, not significantly lower than grafts with 35% or less proximal stenosis (75%, p = 0.25). Other factors associated with significant decreases in graft patency included a vein graft diameter less than 3.0 mm, a dorsalis pedis outflow site, and poor quality outflow. Thus the popliteal-to-distal bypass is a durable procedure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728673 TI - The lesser saphenous vein: an underappreciated source of autogenous vein. AB - Use of the ipsilateral greater saphenous vein for arterial bypass procedures is frequently limited by previous stripping, bypass operations, or anatomic unsuitability. In such cases the contralateral greater saphenous vein or arm veins are often used. However, over the past 5 years we have used the lesser saphenous vein as a preferred alternative autogenous vein. Duplex scanning has been used in 311 cases for preoperative mapping and assessment with excellent correlation with actual anatomy found at operation. Harvest of the lesser saphenous vein has been facilitated by the use of a medial subfascial approach not requiring special positioning of the leg. A total of 91 lesser saphenous veins have been used for arterial bypass procedures; 66 of these were repeat cases. Vein use was 90.2%. In 40 of these cases the lesser saphenous vein was used as the entire conduit, including 10 in situ, 20 reversed vein (including 18 for coronary artery bypass), and 10 orthograde vein bypasses. In the remaining 33 cases the lesser saphenous vein was spliced to another vein to complete a bypass procedure. In the entire group, patency was 77% at 2 years. These data suggest that the lesser saphenous vein should be a principal alternative to ipsilateral greater saphenous vein for arterial bypass because of its ready availability, high use rate, ease of harvesting and preparation, and ideal handling characteristics. PMID- 1728674 TI - Vascular disease in the antiphospholipid syndrome: a comparison with the patient population with atherosclerosis. AB - The antiphospholipid syndrome was diagnosed in 19 of 1078 patients treated between 1987 and 1991. All patients with antiphospholipid syndrome had either anticardiolipin antibody (16/19) or lupus anticoagulant (10/19); three patients had thrombocytopenia, eight patients had a prolonged partial thromboplastin time, and 10 patients had an elevated erythrocyte sedimentation rate. The most common site of involvement was the cerebral circulation (nine patients), manifested by transient ischemic attacks or stroke. Eight patients had upper extremity disease, characterized by symptoms of Raynaud's phenomenon, with angiographic lesions involving the brachial, radial, ulnar, and/or digital arteries. Lower extremity disease occurred in seven patients, with clinical presentations similar to those of atherosclerosis and varying angiographic patterns. In comparison with the population having atherosclerosis, patients with arterial manifestations of antiphospholipid syndrome were more likely to be women (13 of 19 versus 411 of 1078, p less than 0.02), were significantly younger (46.2 years versus 63.6 years, p less than 0.0001), did not smoke (1 of 19 patients versus 700 of 1078, p less than 0.0001), had a higher percentage of upper extremity involvement (8 of 18 versus 13 of 1078, p less than 0.0001), and had a higher incidence of early graft failure (9 of 12 grafts versus 13 of 371 grafts, p less than 0.0001). The syndrome is associated with the repetitive failure of vascular reconstructions and occlusion of native vessels. Antiphospholipid syndrome should therefore be suspected in young, female, nonsmokers with vascular disease, especially those with involvement of the upper extremity, cerebrovascular disease with normal findings on extracranial carotid angiography, and premature graft failure. PMID- 1728675 TI - The economics of femorocrural reconstruction for critical leg ischemia with and without autologous vein. AB - It is well established that primary arterial reconstruction, even to crural vessels, is cheaper than amputation. Reintervention increases expenditure and may produce mean costs exceeding those of primary amputation. Furthermore, secondary amputation may eventually become necessary. Femorocrural grafts have the highest average "reconstruction policy" cost (i.e., primary procedure and all further operations necessary during follow-up). We must therefore seek support for this potentially expensive form of treatment. In conjunction with health economists we have compared the average policy cost of 130 reconstructions with grafts exceeding 70 cm in length (89 vein grafts, 41 polytetrafluoroethylene grafts with a distal vein collar) with 67 vascular amputations, at mean follow-up of 3 years. One-month mortality rate after reconstruction was less than 1% but was 10% after amputation. At 3 years, however, 20% of both groups were dead. Overall 3-year patency is 65% (72% for vein grafts, 48% for polytetrafluoroethylene grafts). Ninety-seven percent of irreversible graft occlusions resulted in amputation in these patients. After autologous vein grafting reintervention, our follow-up showed increased mean costs from $6898 to $15,024 per patient. After prosthetic grafting, the higher reintervention rate increased from $6898 to $20,416. These mean costs remained less than amputation, reintervention, and additional mobility costs, which amounted to a mean of $21,726 per patient. Important differences in outcome were observed: 70% of patients undergoing amputation were confined to the home compared with only 9% of patients undergoing reconstruction; 30% of patients undergoing amputation were confined to bed or had to use a wheelchair compared with 1% of patients undergoing reconstruction. PMID- 1728676 TI - Myointimal thickening in experimental vein grafts is dependent on wall tension. AB - This study examines the relative contributions of intraluminal pressure, blood flow, wall tension, and shear stress to the development of myointimal thickening in experimental vein grafts. To study these different hemodynamic parameters, several experimental models were created in 30 New Zealand White rabbits separated into six groups: common carotid interposition vein grafts harvested at 4 weeks (VG-4) or 12 weeks (VG-12), common carotid-linguofacial vein arteriovenous fistulas harvested at 4 weeks (AVF-4) or 12 weeks (AVF-12), AVFs with partial outflow obstruction harvested at 4 weeks (AVFobs), and combination VG-AVFs in series harvested at 4 weeks (VGAVF). Blood pressure and flow in the graft or vein were measured by use of a transducer-tipped pressure catheter and electromagnetic flow meter. At harvest, veins were perfusion-fixed and proximal, middle, and distal sections were subjected to computerized morphometric analysis. Vein grafts were characterized by a high mean pressure (VG-4, 51 +/- 4; VG-12, 62 +/- 3 mm Hg), low mean flow (VG-4, 17 +/- 1; VG-12, 16 +/- 4 ml/min), large luminal area (VG-4, 19.7 +/- 2.4; VG-12, 19.3 +/- 3.9 mm2), high wall tension (VG 4, 17.0 +/- 1.5; VG-12, 19.5 +/- 2.4 x 10(3) dyne/cm), low shear stress (VG-4, 0.75 +/- 0.13; VG-12, 0.96 +/- 0.38 dyne/cm2), and a high degree of myointimal thickening (VG-4, 5.89 +/- 0.90; VG-12, 4.72 +/- 0.83 mm2). Arteriovenous fistulas were characterized by a low mean pressure (AVF-4, 5 +/- 1, AVF-12, 6 +/- 2 mm Hg), elevated blood flow (AVF-4, 82 +/- 16; AVF-12, 82 +/- 17 ml/min), small luminal area (AVF-4, 2.43 +/- 0.58; AVF-12, 7.14 +/- 2.68), low wall tension (AVF 4, 0.62 +/- 0.19; AVF-12, 0.89 +/- 0.24 x 10(3) dyne/cm), elevated shear stress (AVF-4, 108 +/- 32; AVF-12, 71 +/- 50 dyne/cm2), and decreased myointimal area (AVF-4, 1.18 +/- 0.26; AVF-12, 1.90 +/- 0.55 mm2). The addition of outflow obstruction to AVFs (AVFobs) resulted in elevated pressure (48 +/- 2 mm Hg), decreased flow (17 +/- 4 ml/min), larger luminal area (8.71 +/- 2.31 mm2), elevated wall tension (10.3 +/- 1.7 x 10(3) dyne/cm), and a degree of myointimal thickening approaching that of vein grafts (3.79 +/- 0.66 mm2).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1728677 TI - The selective management of small abdominal aortic aneurysms: the Kingston study. AB - The management of small abdominal aortic aneurysms less than 5.0 cm maximum diameter remains controversial particularly in patients who are medically fit. All patients referred with abdominal aortic aneurysms less than 5.0 cm maximum diameter were prospectively followed regardless of their fitness for operation. Two hundred sixty-eight patients had been entered into the study by December 31, 1988, and monitored until December 31, 1990, by at least two aneurysm sizings by ultrasonography, CT scanning, or both. The mean follow-up was 42 months. Operations were performed on 114 patients (if they were fit for operation) when the aneurysm reached 5.0 cm, expanded more than 0.5 cm in a 6-month period, or when the patient had significant occlusive disease requiring repair. In this group the mean annual increase in diameter was 0.9 cm. One hundred fifty-four patients were monitored without operation for a mean period of 42 months. One rupture occurred in this group. The average annual increase in diameter in the group not undergoing operation was 0.24 cm. This study supports a policy of observation for abdominal aortic aneurysms less than 5.0 cm in maximum diameter. PMID- 1728678 TI - Adventitial cystic disease of the femoral vein: a case report and review of the literature. AB - Painless edema of the left leg developed in a 65-year-old man without a history of venous disease, and he was found to have a mass compressing the lumen of the left common femoral vein. The intramural cyst was drained through transvenous exposure and found to contain mucoid material. This is the seventh case of adventitial cystic disease of a vein in the world literature. Analogous to adventitial cystic disease of arteries, it is defined by venography, CT scanning, and duplex ultrasonography. Surgical drainage is the treatment of choice. PMID- 1728679 TI - Disruption of proximal axillobifemoral bypass graft anastomosis. AB - Anastomotic disruption of axillobifemoral bypass grafts is a rare but serious complication. Previously described causes of anastomotic disruption include the following: infection, technical errors, and severe mechanical stress. In this report we describe a proximal anastomotic disruption of an axillobifemoral bypass graft in which the suture line remained intact on the axillary artery and tore through the polytetrafluoroethylene graft. This suggests a possible role of material failure contributing to axillobifemoral anastomotic disruption. PMID- 1728680 TI - Splenic artery dissection: a case report and review of the literature. AB - Spontaneous arterial dissection may occur in a dramatic fashion and is often fatal if treatment is not instituted promptly. Diagnosis may be particularly difficult when cases of arterial dissection appear in unusual locations. We report a rare case of atraumatic dissection of the splenic artery that occurred in a 66-year-old male patient, who was admitted with acute severe epigastric and substernal pain, which worsened on inspiration. On admission, his physical examination was unremarkable, and he was hemodynamically stable. After excluding cardiopulmonary catastrophes and aortic dissection as a cause, a left retroperitoneal mass was found on arteriography to be a contained rupture of a splenic artery dissection. The patient underwent urgent resection of the splenic artery with preservation of the spleen. Splenic artery dissection is a rare condition. Only 11 cases have been previously published in the literature, and all of these cases were diagnosed after death. Successful management depends on consideration of the diagnosis, especially when other more common disease processes have been excluded. PMID- 1728681 TI - Vertical ramus osteotomy for improved exposure of the distal internal carotid artery: a new technique. PMID- 1728682 TI - Notice of duplicate submission. Use of the superficial femoral vein as a replacement for large veins. PMID- 1728683 TI - The need for quality assurance in vascular surgery. PMID- 1728684 TI - New mechanism for disruption of axillofemoral bypass. PMID- 1728685 TI - Interruption of critical aortoiliac circulation during nonvascular operations: a cause of acute limb-threatening ischemia. PMID- 1728686 TI - Mesenteric venous thrombosis. PMID- 1728687 TI - Wound complications of the retroperitoneal approach to the aorta and iliac vessels. AB - Repeated complaints of postoperative wound pain prompted this review of 113 consecutive vascular operations involving a retroperitoneal approach to the aorta or iliac vessels or both. Flank muscle-splitting incisions (n = 53) had been used to approach the terminal aorta or iliac arteries. Two types of muscle-dividing incisions had also been used: incisions through the eleventh intercostal space (n = 41) to approach the infrarenal aorta; and incisions through the eighth, ninth, or tenth intercostal space with division of the diaphragm (n = 19) to approach the suprarenal aorta. Data on incisional pain, lumbosacral neuritic pain, incisional hernia, and deforming abdominal bulge were culled from the records of follow-up examinations conducted on all patients during periods ranging from 2 to 48 months. Both types of muscle-dividing incisions used to expose the aorta were associated with a 23% (14/60) incidence of abdominal bulge, a 7% (4/60) incidence of incisional hernia, and, more important, a 37% (22/60) incidence of prolonged disabling pain. Thus, although retroperitoneal exposure may be the preferred or the safest approach to certain aortic lesions, its routine use via muscle dividing incisions is not recommended when the proposed operation can be carried out equally well by the conventional midline transperitoneal approach. PMID- 1728688 TI - Cefuroxime versus cefazolin as prophylaxis in vascular surgery. AB - Although cefazolin prophylaxis has proven efficacy in vascular surgery, Staphylococcus aureus wound infections are still an important postoperative complication. In cardiac surgery, cefazolin's susceptibility to hydrolysis by staphylococcal beta-lactamase has been proposed to account for some prophylaxis failures. To determine whether the incidence of vascular wound infections can be reduced by administering a more beta-lactamase-stable cephalosporin, we undertook a prospective, randomized trial of cefuroxime versus cefazolin. Cefuroxime was administered as a 1.5 gm dose before operation and 750 mg every 3 hours during operation. Cefazolin was given as 1 gm before operation and 500 mg every 4 hours during operation. Both agents were continued every 6 hours after operation for 24 hours. Deep wound infections developed in seven of 272 (2.6%) cefuroxime and three of 287 (1.0%) cefazolin recipients (p = 0.2). Staphylococcus aureus wound infections occurred in five cefuroxime versus two cefazolin recipients. In vitro evaluation of six of the study isolates plus an additional eight S. aureus strains from vascular wound infections showed greater susceptibility of the strains to cefazolin than cefuroxime (median minimal inhibitory concentrations of 0.5 and 2.0 micrograms/ml, respectively, p less than 0.05). Furthermore, despite its more frequent intraoperative redosing, cefuroxime exhibited lower trough serum concentrations than cefazolin. Among cefuroxime recipients, infection associated procedures were significantly longer than infection-free procedures (p less than 0.05), suggesting that low tissue antibiotic concentrations may have contributed to the pathogenesis of these infections. In contrast, the length of the procedure was not a risk factor for infection among cefazolin recipients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728689 TI - The impact of selective use of dipyridamole-thallium scans and surgical factors on the current morbidity of aortic surgery. AB - Preoperative cardiac testing in patients undergoing vascular surgery remains controversial. We have advocated selective use of dipyridamole-thallium scans based on clinical markers of coronary artery disease before aortic surgery. The present study assessed both the efficacy of this policy and the role of surgical factors in the current morbidity of aortic reconstruction. Two hundred two elective aortic reconstructions (151 abdominal aortic aneurysms, 51 aortoiliac occlusive disease) performed in the period from January 1989 to June 1990 were reviewed. Preoperative dipyridamole-thallium scanning was performed in 29% of all patients, prompting coronary angiograms in 11% and coronary artery bypass grafting/percutaneous transluminal coronary angioplasty in 9% of patients before aortic reconstruction. The overall operative mortality rate was 2%, with one cardiac-related death. Major cardiac (nonfatal myocardial infarction, unstable angina) and pulmonary complications occurred in an additional 4% and 6%, respectively, of patients. Coronary artery disease clinical markers and surgical factors were analyzed with stepwise logistic regression for the prediction of operative mortality rates and major cardiopulmonary complications. Variables retaining significance in predicting postoperative death or cardiopulmonary complications included prolonged (more than 5-hour) operative time (p less than 0.004), operation for aortoiliac occlusive disease (p less than 0.010), and a history of ventricular ectopy (p less than 0.002). Prolonged operative time (p less than 0.006) and the detection of intraoperative myocardial ischemia (p less than 0.030) were predictive of major cardiac complications after univariate analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728690 TI - Passing the torch--the molecular biology of a profession. PMID- 1728691 TI - The incidence of perioperative myocardial infarction in general vascular surgery. AB - In a 1-year period all patients undergoing general vascular surgery (491 patients, 534 procedures) underwent monitoring by creatine phosphokinase isoenzymes and electrocardiograms (ECG) to detect perioperative myocardial infarction. Only those patients with severe symptomatic coronary artery disease (31 patients, 5.8%) characterized by unstable angina pectoris, uncontrolled arrhythmia, or severe congestive heart failure had any testing for coronary artery disease beyond history, physical examination, and ECG. Only three patients (0.5%) had prophylactic coronary artery bypass performed before general vascular procedures. Twenty-one (3.9%) myocardial infarctions (five asymptomatic, detected by enzymes only, and 16 symptomatic, four of which were fatal) were associated with the 534 procedures (aorta 105, carotid 87, infrainguinal bypass 207, extraanatomic 51, other 84). Eight noncardiac perioperative deaths occurred. All operative deaths (12 of 534, 2.2%) including all four fatal myocardial infarctions occurred associated with surgery on an urgent or emergency basis (12 of 249 procedures, urgent/emergent operative mortality rate 4.8%). No operative deaths and no fatal myocardial infarctions associated with the 285 elective procedures occurred. Nine of the 17 nonfatal myocardial infarctions (53%) also occurred after urgent/emergent procedures. The rate of perioperative myocardial infarctions (eight of 285, 2.8%) after elective surgery in this patient series is no different from that reported by multiple recent authors advocating widespread screening for and prophylactic treatment of coronary artery disease before general vascular surgery. Our experience confirms the therapeutic approach that expensive and invasive coronary screening programs in patients to undergo vascular operations should be limited to carefully selected patients with severely symptomatic coronary disease. PMID- 1728692 TI - Selective deep hypothermia of the spinal cord prevents paraplegia after aortic cross-clamping in the dog model. AB - We tested, in the dog, the hypothesis that selective deep hypothermia (19 degrees to 12 degrees C) of the spinal cord protects it from the ischemia that follows double aortic cross-clamping. The extracorporal perfusion system consisted of heat exchanger and a pump, infusing saline solution at 5 degrees C into the subarachnoid space (L-6) and draining it through the cisterna magna. After 30 minutes this system cools a normally perfused spinal cord to a stable temperature gradient of 13 degrees C (inflow) to 18 degrees C (outflow). Proximal and distal intrathecal, proximal and distal aortic, and central venous pressures were continuously recorded. Rectal temperature was maintained between 36.5 degrees C and 38.5 degrees C. Eight control dogs had cross-clamping of the aorta below the left subclavian artery and above the diaphragm without cord hypothermia. Nine experimental dogs had cord hypothermia initiated 50 minutes before systemic heparinization (100 U/kg) and double cross-clamping of the aorta. Cross-clamping was maintained for 45 minutes. The aorta was then unclamped, heparin was reversed, cord cooling was discontinued, and the dura was closed. Hindlimb function of animals was graded by use of Tarlov's scale at recovery and 24 hours later. The dogs were then killed, and the cords were removed and fixed for microscopy. All control animals were paraplegic and had histologic confirmation of spinal cord infarction. All experimental animals had intact hindlimb function and normal appearing cords on histologic examination. A two-tailed Fisher's exact test (chi square) shows this difference to be significant to p = 0.00004. In the dog selective deep hypothermia of the cord avoids the ischemic injury induced by aortic cross-clamping that results in paraplegia. The implications of these findings in thoracoabdominal aortic clamping in humans is discussed. PMID- 1728693 TI - Renal revascularization for recurrent pulmonary edema in patients with poorly controlled hypertension and renal insufficiency: a distinct subgroup of patients with arteriosclerotic renal artery occlusive disease. AB - Recurrent pulmonary edema in patients with poorly controlled hypertension and renal insufficiency appears to be a marker of bilateral renal artery occlusive disease. The effectiveness of renal revascularization to prevent recurrent pulmonary edema in this distinct subgroup with renal artery occlusive disease was analyzed in 17 consecutive patients treated at the University of Michigan Hospital between 1984 and 1990. Their mean preoperative blood pressure was 207/110 mm Hg, and mean serum creatinine clearance was 3.8 mg/dl. Pulmonary edema occurred despite evidence of normal ventricular function in 65% of these patients. Bilateral renal artery occlusive disease affected 94% of the patients, and 54% had an occluded renal artery. Renal revascularization was accomplished by iliorenal bypass (41%), aortorenal bypass (29%), endarterectomy (24%), and transluminal angioplasty (6%). Contralateral nephrectomy (41%) and concomitant aortic reconstruction (24%) were also required frequently. No postoperative deaths occurred, and no patient had early postoperative pulmonary edema. Control of hypertension was improved in all patients, two of whom were discharged from the hospital on no antihypertensive medications. Two of the three patients requiring dialysis before operation were able to discontinue dialysis after operation. Late follow-up (mean, 2.4 years) revealed hypertension to be cured in one patient (6%), and improved in 16 patients (94%). Pulmonary edema occurred in one patient during late follow-up. Late follow-up showed renal function (mean creatinine, 1.7 mg/dl) to be improved in 77%, stable in 12%, and worse in two patients; one required dialysis. A single episode of pulmonary edema in a patient with poorly controlled hypertension and renal insufficiency should prompt consideration of this clinical syndrome and early diagnostic angiography.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728694 TI - Aneurysmal enlargement of the aorta during regression of experimental atherosclerosis. AB - We explored the relationship between regression of diet-induced atherosclerosis and aneurysmal enlargement of the aorta in cynomolgus monkeys. Atherosclerotic plaques were induced in 17 monkeys by feeding them a diet containing 2% cholesterol and 25% peanut oil for 6 months (group I, n = 6; group III, n = 6) or 12 months (group II, n = 5). Regression was induced in group III by feeding a regression diet consisting of 0.25% cholesterol and 15% corn oil in a standard chow diet, for 6 months after the 6-month induction period. Serum cholesterol was 788 +/- 80 mg/dl after 6 months of induction, 508 +/- 53 mg/dl after the 12-month induction period, and 198 +/- 15 mg/dl in the regression group at 12 months. Aortas were fixed in situ under conditions of controlled pressure perfusion, and transverse sections of the unopened vessels were taken at standard levels in the midthoracic and abdominal aortic segments. The area encompassed by the internal elastic lamina was taken as a measure of artery size. Plaques were abundant in abdominal and thoracic sections after the 6- and 12-month induction periods, and no significant difference was observed in lumen area or artery size between the groups. The ratio of abdominal to thoracic aortic plaque area was markedly reduced in the regression group (0.3 +/- 0.2 for regression compared with 0.6 +/- 0.3 for 6-month induction and 1.3 +/- 0.2 for 12-month induction animals; p less than 0.05 for both). A twofold increase was observed in abdominal aortic lumen area in the regression group (10.0 +/- 1.5 mm2 for regression compared with 5.6 +/- 0.7 mm2 for the 6-month and 4.2 +/- 0.7 mm2 for the 12-month induction groups; p less than 0.05 for both) as well as a twofold increase in internal elastic lamina area (10.5 +/- 1.5 mm2 compared with 6.0 +/- 0.7 mm2 for the 6 month and 5.9 +/- 0.8 mm2 for the 12-month induction group; p less than 0.05 for both). Aortic enlargement in the regression group was accompanied by a reduction in media thickness in the abdominal aorta. No significant vessel enlargement or alteration in media thickness occurred in the thoracic aorta. One of six regression animals (17%) had a threefold enlargement of the abdominal aorta and was thought to have a manifest aneurysm.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1728695 TI - Quality of life after myocardial revascularization. Effect of increasing age. AB - The effect of increasing age on quality of life, survival, and risk of reoperation was studied in 2479 patients followed up prospectively 2 to 20 years after myocardial revascularization. Quality of life was determined from annual questionnaires, which we used to calculate a health status index from the patient's symptomatic status and subjective response to the operation, which was graded between zero and 1.00 (asymptomatic). Four age groups were studied: age 49 years or less (AG40), 50 to 59 years (AG50), 60 to 69 years (AG60), and 70 years or older (AG70). Associated problems (left ventricular aneurysm, valve disease, acute myocardial infarction) necessitating treatment were present in 17% (61/361) of AG40 patients, 19% (165 of 859) of AG50 patients, 23% (213/927) of AG60 patients, and 31% (102/332) of AG70 patients. The hospital mortality rate was higher in older patients undergoing combined procedures but not in patients undergoing coronary bypass grafts only. Probability of survival and health status indexes were calculated excluding patients with valve disease and cardiogenic shock. Probability of survival was significantly better (p less than 0.001 by the Wilcoxon test) in patients less than age 60 than in those 60 years or older, but in patients with an ejection fraction greater than or equal to 0.40, probability of survival at 12 years was 0.64 (age less than 60) versus 0.62 (age greater than or equal to 60). The actuarial risk of reoperation, calculated as the difference between probability of survival and probability of survival without reoperation, progressively increased in younger patients but not in patients aged 60 years or older. At 15 years, the reoperation rates were 26% (AG40), 14% (AG50), 5% (AG60), and 7% (AG70). Mean health status index for years 1 to 5 was 0.85 in AG40 patients, 0.84 in AG50 patients, 0.89 in AG60 patients, and 0.90 in AG70 patients; for years 6 to 10, 0.81, 0.80, 0.86, and 0.89; and for years 11 to 15, 0.77, 0.78, 0.84, and 0.84, respectively. Thus quality of life after myocardial revascularization is better, improvement lasts longer, and reoperation rate is less in patients aged 60 years or older. PMID- 1728696 TI - Reactivity of gastroepiploic and internal mammary arteries. Relevance to coronary artery bypass grafting. AB - The gastroepiploic artery is an alternate conduit for coronary artery bypass grafting. To test the hypothesis that its vasoreactive properties are different from those of the internal mammary artery, we obtained gastroepiploic artery segments from human gastrectomy specimens. Trimmed internal mammary artery segments were obtained during coronary artery bypass. Ring segments were mounted on a strain gauge and stretched to optimum resting length (90% of the internal circumference at 100 mm Hg). Potassium chloride, serotonin, and norepinephrine were chosen to simulate physiologic vasospasm induced by depolarization, platelet aggregation, or adrenergic stimulation, respectively. Contractions to potassium and a concentration-response curve to serotonin or norepinephrine were obtained. Sodium nitroprusside was used to assess relaxation. Gastroepiploic artery segments had stronger contractions to the depolarizing agent (potassium chloride), adrenergic stimulation (norepinephrine), and product of platelet aggregation (serotonin). The gastroepiploic and internal mammary arteries showed equal sensitivity, measured by concentration causing half-maximal contraction to norepinephrine and serotonin. There was no difference in relaxation to sodium nitroprusside. These data suggest that prevention of platelet-, adrenergic-, or potassium-induced contraction may be more important when the gastroepiploic artery is used as an alternate conduit for coronary artery bypass, reinforcing consideration of nitrovasodilators and platelet inhibitors in the perioperative interval. PMID- 1728697 TI - Mitral valve repair for bacterial endocarditis. AB - Twenty-two patients with mitral insufficiency resulting from native valve endocarditis underwent mitral valve repair. Six patients had acute endocarditis with positive blood cultures and active valve infection. Sixteen patients were cured of active infection, but mitral insufficiency developed as a result of prior infection. Mean age was 48.5 +/- 21.7 years; 13 (59%) were male. Mean New York Heart Association functional class was 2.6 +/- 1.2. Multiple valve lesions were present in 11 (50%) patients. Valve abnormalities included leaflet perforation in 13 patients, chordal rupture or elongation in 14, vegetations in 5; and annular abscess in 1. In patients with acute endocarditis all macroscopically infected tissue was excised. Multiple techniques were required to achieve valve competence. Suture or patch closure of perforation was done in 14 patients, chordal shortening or transfer in 9, leaflet resection and closure in 4, leaflet resection with pericardial patching in 5, and annuloplasty in 15. Mitral valvuloplasty was combined with other procedures in 11 (50%) patients. There were two (9%) hospital deaths, both occurring in patients with healed endocarditis. There was one (9%) death in a patient undergoing an isolated procedure and one (9%) in a patient undergoing a combined procedure. Mean follow up was 24 +/- 16.8 months and was complete. Seventeen (85%) were in New York Heart Association functional class I, and three (15%) were in class II. There were no late deaths, reoperations, recurrent endocarditis, thromboembolic events, or other valve-related morbidity. We conclude that mitral valve repair for insufficiency resulting from bacterial endocarditis (1) is possible in acute and healed disease, (2) has a low operative mortality, and (3) has resulted in patients free of recurrent infection and valve-related morbidity and mortality. Mitral valve repair is an attractive alternate to valve replacement in bacterial endocarditis. PMID- 1728698 TI - Aortic valve replacement for active infectious endocarditis in 108 patients. A comparison of freehand allograft valves with mechanical prostheses and bioprostheses. AB - A total of 108 patients hospitalized with active (acute) endocarditis on either a native aortic valve (n = 66) or a previously inserted replacement device (n = 42) underwent aortic valve replacement because they were too ill for hospital discharge. A nonstented aortic allograft valve was used in 78 patients and prosthetic (mechanical or bioprosthetic) valves in 30 patients. The survival rate was 82% at 1 months, 73% at 1 year, 64% at 5 years, and 36% at 15 years. It was better in patients with native valve endocarditis than prosthetic valve endocarditis. The incremental risk factors for death in the early phase postoperatively were older age at operation, higher New York Heart Association functional class, and a larger number of previous aortic valve procedures. There were 13 episodes of recurrent endocarditis, giving an actuarial freedom of 80% at 10 years. The hazard function for recurrent endocarditis had only a low constant phase when allograft valves were used, which contrasted with the existence of a high peaking early phase (in addition to the constant phase) when prosthetic devices were used. No risk factors for recurrent endocarditis were found in patients receiving a prosthesis, and "localized" versus "extensive" endocarditis was the only risk factor when an allograft was used. Reoperation was performed in 24 patients for a variety of reasons, and freedom from reoperation was 61% at 10 years. It is concluded that the allograft valve is the valve of choice when aortic valve replacement is required for active endocarditis. PMID- 1728699 TI - Triangle of auscultation thoracotomy for esophageal atresia. AB - A technique of thoracotomy via the triangle of auscultation is described. It avoids the division of latissimus dorsi, serratus anterior, and trapezius muscles, with its consequent morbidity, and provides excellent access to the thoracic cavity and the posterior aspect of the mediastinum. PMID- 1728700 TI - Cardiac fibroma. Long-term fate after excision. AB - Between 1980 and July 1983 three infants and children with cardiac fibromas underwent surgical resection at Kobe Children's Hospital. Two of them survived and have an excellent clinical result 6 years and 7 years postoperatively. The results of late follow-up with the use of 24-hour dynamic electrocardiography, two-dimensional echocardiography, thallium-201 myocardial scintillation scan, and technetium 99m sodium pertechnetate-gated blood pool imaging have proved that the patients are free of arrhythmic episodes, free of recurrence of tumor, have no significant myocardial perfusion defect, and have normal left ventricular function. PMID- 1728701 TI - Improved healing of small-caliber polytetrafluoroethylene vascular prostheses by increased hydrophilicity and by enlarged fibril length. An experimental study in rats. AB - This study was undertaken to test whether increasing the hydrophilicity of small caliber polytetrafluoroethylene vascular prostheses by alcohol pretreatment or increasing their fibril length might improve their healing without affecting their patency. Polytetrafluoroethylene vascular prostheses (length 1 cm, inside diameter 1.2 mm) (1) with a fibril length of 30 microns (control group; n = 18), (2) pretreated with alcohol (n = 18), or (3) with a fibril length of 60 microns (n = 18) were implanted into the abdominal aorta of rats. The prostheses were evaluated by means of routine light and scanning electron microscopy during a 6 week period after implantation. All prostheses were patent at harvesting. On implantation, the control polytetrafluoroethylene vascular prostheses were only scarcely covered with platelets. At 6 weeks they had healed in a small area adjacent to the anastomoses only. In contrast, both the alcohol-pretreated polytetrafluoroethylene prostheses and the polytetrafluoroethylene prostheses with a fibril length of 60 microns were completely covered by a thin clot layer on implantation. At 6 weeks after implantation these prostheses had almost completely healed as a result of organization of the thin clot layer by ingrowth of both endothelial and smooth muscle cells. These results demonstrate that increasing hydrophilicity of polytetrafluoroethylene vascular prostheses by alcohol pretreatment or enlarging their fibril length improves their healing by induction of a thin luminal clot layer. This clot layer provides a suitable matrix for ingrowth of both endothelial and smooth muscle cells and does not lead to thromboembolic complications. PMID- 1728702 TI - Evaluation of cryopreserved allograft venous conduits in dogs. AB - We investigated the effects of cryopreservation, immunosuppression, and antibiotic treatment on the patency and histologic appearance of venous conduits in the arterial circulation. Twenty-eight dogs received arterial replacements with autograft vein, fresh allograft, and two types of cryopreserved allograft vein implanted into both carotid and both femoral arteries. All animals were given aspirin, and half were given cyclosporine. After 3 months the vein grafts were harvested. Patency and light, transmission, and scanning electron microscopic criteria were scored to evaluate quality of preservation of the endothelium, the appearance of rejection, and the effects of cryopreservation with and without antibiotic pretreatment. The results show that patency is not statistically different based on graft type or treatment modality. The histologic appearance among the various vein types was remarkably similar at 3 months, with the exception of a cellular infiltrate present most prominently in the fresh allografts and least in the fresh autografts. Cyclosporine, even at a low dose, decreased the incidence of cellular infiltration. Preservation of endothelium was generally good in the cryopreserved allografts both with and without antibiotic pretreatment. In general, the effects of cryopreservation, cyclosporine, and antibiotics ameliorated the effects of venous allografting into an arterial position. PMID- 1728703 TI - Presence of circulating beta-glucan during cardiopulmonary bypass. PMID- 1728704 TI - Increased need for formal thoracotomies to manage chronic pneumothorax caused by the use of plastic chest tubes: a justification to expand laparoscopic surgery into the thorax. PMID- 1728705 TI - Bronchopleural fistula: the use of tissue glue. PMID- 1728706 TI - Inappropriate dosing of cefuroxime in cardiac surgery. PMID- 1728707 TI - Direct ostioplasty of the left main coronary artery for isolated nonarteriosclerotic ostial stenosis. PMID- 1728708 TI - Nonsteroidal antiinflammatory drugs for postthoracotomy pain. A prospective controlled trial after lateral thoracotomy. AB - Diclofenac (Voltarol) as an adjunct to papaveretum for pain relief was examined by a prospective, randomized trial in 44 patients who had lateral thoracotomies. Patients given diclofenac, 75 mg intramuscularly twice daily, required less papaveretum in the first 3 days after operation (p less than 0.005) and had lower pain scores on a visual analog scale on all 5 postoperative days (p = 0.02 to less than 0.001); their respiratory vital capacity on the first postoperative day was also significantly higher (p less than 0.02). Diclofenac is a useful adjunct in the management of postthoracotomy pain. PMID- 1728709 TI - Subcutaneous heparin and myointimal proliferation in arteriovenous bypass grafts. PMID- 1728710 TI - Late neurologic complication after deep hypothermia and circulatory arrest--a hypothesis. PMID- 1728711 TI - Prostaglandin E1 infusion for right ventricular failure after cardiac transplantation. AB - The infusion of prostaglandin E1, a vasodilating substance with predominant effects on the pulmonary vasculature, has been found effective in the management of pulmonary hypertension associated with various diseases. The reported experience with prostaglandin E1 after cardiac transplantation is, however, limited. We used prostaglandin E1 in 18 patients in whom acute right ventricular failure developed after orthotopic cardiac transplantation. The infusion was started within 24 hours after operation in 16 patients and was continued for up to 7 days. Maximal doses of prostaglandin E1, administered via a central venous catheter, ranged from 30 to 120 ng/kg/min. Norepinephrine was simultaneously infused via a left atrial catheter in 10 patients to prevent a reduction in systemic arterial pressure. The prostaglandin E1 infusion resulted in significant reductions in mean arterial pressure and pulmonary vascular resistance and simultaneous increases in cardiac index and stroke index. Mean arterial pressure was stable and left ventricular stroke work increased. The alveolar oxygen tension/forced inspiratory oxygen index tended to decrease during the infusion. Three patients died, two of right heart failure and one of multiple organ failure associated with cardiac allograft rejection. In patients in whom right ventricular failure associated with pulmonary hypertension develops after cardiac transplantation, prostaglandin E1, combined with norepinephrine whenever the arterial pressure declines, can effectively reduce pulmonary artery pressures and improve global cardiac function without compromising systemic perfusion. PMID- 1728712 TI - A new technique for double lung transplantation. "Bilateral single lung" transplantation. AB - Two techniques are currently practiced to achieve bilateral lung transplantation for the treatment of patients with end-stage pulmonary disease associated with infection: heart-lung transplantation, which is illogical, and double lung transplantation by the Toronto technique, which is difficult and entails tracheal complications. After our short experience with single lung transplantation without any bronchial problems, we have performed three double lung transplantations by a new technique, "bilateral single lung" transplantation. After a sternal bithoracotomy, first one lung was transplanted and then the other, without cardiopulmonary bypass. This bilateral single lung transplantation provides all the advantages of single lung transplantation and is particularly recommended for use in patients with severe pleural adhesions. PMID- 1728713 TI - Growth of composite conduits utilizing longitudinal arterial autograft in growing lambs. AB - We examined the growth potential of a longitudinal strip of autologous aortic wall incorporated in an autologous pericardial conduit in 10 lambs (mean age 26 days, mean weight 10.1 kg). A 15 mm length of descending thoracic aorta (diameter 11.5 +/- 7 mm) was excised and replaced with a composite autograft conduit of autologous pericardium with a longitudinally inserted aortic strip 5 mm in width taken from the excised aortic tissue. Radiopaque markers along all suture lines allowed determination of growth of the aortic strip relative to growth of the composite conduit and descending aorta, in addition to growth assessment by pathologic analysis. Plain x-ray films and aortograms were performed at 7 days (baseline) and at 3, 6, 9, and 12 months. No graft became stenotic or aneurysmal. The diameter of the descending aorta distal to the conduit increased from 11.7 +/ 1.3 mm to 18.7 +/- 2.1 mm. Appropriate growth of the autograft conduit was demonstrated by a minimal change in the diameter ratio of conduit to distal aorta from 1.00 to 1.02 during a period of 12 months. The aortic strip increased to 172% +/- 19%, 148% +/- 15%, and 256% +/- 31% of baseline width, length, and area, respectively (p less than 0.05). Histologic study confirmed the maintenance of normal architecture in the aortic strip and colonization of the pericardial tissue by aortic intimal and medial elements. A clinical implant with an autologous aortic strip in an aortic homograft in a 4-year-old child with tetralogy and pulmonary atresia has also grown, according to angiography, from 15 to 21 mm in diameter at 1 year's follow-up. This study confirms that the incorporation of a free autologous arterial patch graft as part of cardiovascular reconstructive procedures permits growth. PMID- 1728714 TI - Ventricular septal defect with tricuspid pouch with and without transposition. Anatomic and surgical considerations. AB - In a 10-year review, patients operated on for ventricular septal defect and tricuspid valve pouch were divided into two groups, because the effect of the tricuspid valve pouch is influenced by which ventricle has the higher pressure. Group I comprised patients with ventricular septal defect without transposition of the great arteries and group II, ventricular septal defect with transposition. In 72 of 392 group I patients, the septal tricuspid valve leaflet was incised to expose the edges of the hidden ventricular septal defect to accomplish proper anatomic repair. Forty-eight patients had a tricuspid valve pouch, the diagnosis being established by angiography, echocardiography, or at operation. Ages at operation ranged from 5 months to 22 years and the pulmonary-systemic flow ratio ranged from 1 to 3.4, with 16 being less than 1.5. In one patient the pouch produced a 40 mm Hg pressure gradient in the right ventricular outflow tract. At operation, through a transatrial approach, the tricuspid valve pouch was opened radially, the actual ventricular septal defect patched, and the tricuspid valve leaflet repaired. There were no deaths, no significant intraoperative or postoperative morbidity, and no tricuspid valve dysfunction. The average postoperative hospital stay was 4.8 days. In group II, six of 83 patients operated on for transposition with ventricular septal defect had significant left ventricular outflow tract obstruction from the tricuspid valve pouch. Five of six had a Mustard procedure, two requiring a left ventricular-pulmonary artery conduit, and in two of the six the ventricular septal defect was closed through the pulmonary artery. One patient had heart transplantation after a Mustard repair and tricuspid valve replacement. The sixth patient in group II had a successful arterial switch at 9 years of age, after the presence of left ventricular outflow tract obstruction was proved to be due to the pouch. The presence of a tricuspid valve pouch in group I may lead the surgeon to close false small openings produced by the pouch rather than the actual ventricular septal defect. Incising the pouch is safe and essential for proper exposure and secure closure of the true defect. In group II, the systemic right ventricular pressure can push the pouch into the left ventricular outflow tract, causing significant obstruction, and may contribute to tricuspid valve insufficiency after atrial baffle repair. Arterial switch is preferred because it returns the obstructive tricuspid valve pouch and abnormal tricuspid leaflet to the lower pressure pulmonic right ventricle. PMID- 1728715 TI - Age-dependent sensitivity to unprotected cardiac ischemia: the senescent myocardium. AB - Six young, sexually mature sheep and seven senescent sheep (aged 0.75 +/- 0.11 years and 7.1 +/- 0.45 years) were instrumented with sonomicrometric crystals and micromanometers to assess global left ventricular mechanics while preload was varied during right heart bypass both before and 30 minutes after 15 minutes of global normothermic ischemia. Left ventricular weight and end-diastolic volume were not significantly different between age groups when indexed to body weight. Contractility was quantitated by the slope of the linear preload-recruitable stroke work relationship and diastolic mechanics by an exponential end-diastolic pressure versus volume function generated over physiologic cardiac workloads. Postischemic systolic functional recovery was markedly worse in the older group (22.7% +/- 10.7% versus 54.2% +/- 9.5%, old versus young, p less than 0.05). However, diastolic stiffness was not changed in either group postischemically. These data demonstrate that the senescent myocardium is less tolerant of ischemia and may require specific intraoperative myocardial management strategies to preserve global pump function. PMID- 1728716 TI - Controlled reperfusion of the regionally ischemic myocardium with leukocyte depleted blood reduces stunning, the no-reflow phenomenon, and infarct size. AB - Open-chest sheep underwent 90 minutes' occlusion of the diagonal branch of the left anterior descending coronary artery, followed by vented cardiopulmonary bypass. After 30 minutes of cardioplegic arrest, simulating distal anastomoses, the occlusion on the coronary artery branch was released. Controlled reperfusion (40 to 50 mm Hg, 135 to 150 ml/min) for the first 20 minutes was delivered at the aortic root with either unmodified whole blood (control, n = 7) or blood passed through leukocyte filters (filters, n = 7). Serial measurements were made during 3 additional hours reperfusion off cardiopulmonary bypass. During ischemia, the major determinants of infarct size, which include area at risk, collateral myocardial blood flow, and rate-pressure product were not significantly different between groups. Overall, during reperfusion, mean left ventricular stroke work index in the filter group was greater than in the control group (28.7 +/- 5.8 versus 12.6 +/- 6.4 x 10(3) erg/gm, p less than 0.05), as was mean rate of rise of left ventricular pressure (1900 +/- 260 versus 1348 +/- 279 mm Hg/sec, p less than 0.05). Myocardial blood flow to the area at risk at 3 1/2 hours of reperfusion in the filter group was also significantly better than in the control group (0.57 +/- 0.15 versus 0.27 +/- 0.05 ml/min/gm, p less than 0.05), as was necrotic area as a percentage of area at risk (40% +/- 6% versus 70% +/- 5%, p less than 0.05). These results demonstrate amelioration of myocardial stunning and the no-reflow phenomenon, as well as decreased infarct size. We conclude that controlled reperfusion with leukocyte-depleted blood is superior to whole-blood reperfusion for the surgical treatment of acute regional ischemia. PMID- 1728718 TI - Significance of the concentrated red cell and albumin priming method with particular reference to anaphylatoxin generation. AB - Anaphylatoxins generated by cardiopulmonary bypass were observed in basic and clinical studies (n = 120 in the latter). In vitro immunoglobulin fractions denatured by oxygen bubbling produced C4a, C3a, and C5a, but albumin identically treated did not. Therefore concentrated red cells with albumin were used to prime homologous blood for clinical application during cardiopulmonary bypass. Complement levels were compared with type of oxygenator (bubble or membrane) and the ratio of primed homologous blood to circulating autologous blood volume. With the bubble oxygenator at a low ratio of homologous to autologous blood (arbitrarily defined as less than 20%), C3a levels during cardiopulmonary bypass tended to be lower in the concentrated red cells plus albumin priming group than in the ordinary priming group (p less than 0.1, at 60 and 90 minutes of cardiopulmonary bypass). C4a and C3a levels increased less after protamine administration with concentrated red cells plus albumin priming (p less than 0.05, p less than 0.01, respectively, 90 minutes after protamine) than with ordinary priming. Such changes in the membrane oxygenator group were less remarkable. Thus C3a levels were approximately the same in both oxygenator groups primed with concentrated red cells plus albumin. The higher the homologous to autologous ratio, the steeper the C4a and C3a increase from the beginning of cardiopulmonary bypass with the bubble oxygenator. This tendency was less remarkable in the membrane oxygenator group. Early postoperative pulmonary function was improved by concentrated red cells plus albumin priming, especially in the bubble oxygenator group. In conclusion, (1) oxygenator systems primed with concentrated red cells plus albumin produced less anaphylatoxin than those with homologous blood, especially with the bubble oxygenator, and (2) our clinical results support the importance of immunoglobulin denatured by oxygen bubbling in anaphylatoxin generation (by means of the classical pathway), as shown by our in vitro study. PMID- 1728717 TI - Enhanced myocardial protection during global ischemia with 5'-nucleotidase inhibitors. AB - Depletion of adenosine triphosphate precursors, such as myocardial adenosine, during global ischemia results in poor postischemic adenosine triphosphate repletion and functional recovery. Neonatal hearts may be more resistant to this deleterious effect of ischemia, because they are characterized by low 5' nucleotidase activity, which may result in higher sustained endogenous myocardial adenosine triphosphate precursor levels during ischemia. Adult hearts, however, have high levels of 5'-nucleotidase activity leading to depleted precursors during ischemia and poor postischemic functional recovery. Augmenting myocardial adenosine exogenously during ischemia in adult hearts has a beneficial effect on recovery. The present study tested if preservation of nucleotide precursors, better adenosine triphosphate repletion, and enhanced postischemic myocardial recovery in adult hearts could be achieved with a "neonatal" strategy. Therefore 5'-nucleotidase inhibitors were administered to isolated, perfused adult rabbit hearts subjected to 120 minutes of ischemia (at 34 degrees C) to determine if this improved functional recovery. Hearts received St. Thomas' Hospital cardioplegic solution (control hearts) or cardioplegic solution containing 5' nucleotidase inhibitors: pentoxifylline, thioinosine, [s-(p-nitrophenyl)-4 thioinosine], or thioinosine's dimethyl sulfoxide vehicle alone. After ischemia and reperfusion, recovery of systolic function, diastolic function, and myocardial oxygen consumption was significantly better with 5'-nucleotidase inhibition. No changes in coronary flow were noted. We speculate and are pursuing the theory that the mechanism of 5'-nucleotidase inhibition's favorable action is due to preventing the catabolism, transport, and loss of nucleotide precursors during ischemia, maintaining adenosine triphosphate precursor availability. PMID- 1728719 TI - Abnormalities in von Willebrand factor and antithrombin III after cardiopulmonary bypass operations for congenital heart disease. AB - In patients with congenital heart disease two poorly understood postoperative complications are pulmonary hypertensive crises after repair of large atrioventricular or ventricular septal defects and right atrial and pulmonary thrombi after the Fontan operation. In this study we assessed whether cardiopulmonary bypass in these patients is associated with the release of agents that might induce platelet aggregation and vasoconstriction, such as biologically active von Willebrand factor and platelet-activating factor. In addition, we measured levels of anticoagulants such as antithrombin III and proteins C and S. Three groups of patients with congenital heart disease undergoing cardiopulmonary bypass were monitored through the perioperative period for secundum atrial septal defects, large atrioventricular or ventricular septal defects, and tricuspid atresia or univentricular heart (Fontan candidates). Control values were obtained from age-matched patients; patients requiring major noncardiac operations and those with cardiac disease not requiring cardiopulmonary bypass were also studied. After cardiopulmonary bypass in all three groups biologic activity of von Willebrand factor increased markedly in the immediate and early postoperative periods compared with preoperative values, whereas antithrombin III values were decreased. Platelet-activating factor was detected in only two patients with congenital heart disease, both in the early postoperative period. In contrast, patients who did not have cardiopulmonary bypass did not show these abnormalities. All measured parameters normalized at late follow-up (6 to 18 months after operation). Although cardiopulmonary bypass in these patients resulted in increased von Willebrand factor activity and decreased antithrombin III, changes that may predispose the patient to platelet aggregation and thrombus formation, absolute values in individual patients alone were not predictive of pulmonary hypertensive crises or detectable thrombi. This suggests that these hematologic abnormalities may contribute to but are not by themselves a cause of morbidity in the early postoperative period. Moreover, the increased von Willebrand factor biologic activity seen postoperatively in patients with congenital heart disease suggests that use of synthetic vasopressin may be ineffective and potentially detrimental. PMID- 1728720 TI - Effects of insulin on myocardial uptake of branched chain amino acids soon after cardiac operations. AB - Infusion of insulin-glucose-potassium is used to support the failing heart after cardiac operations. Although the effects on myocardial uptake of carbohydrates and lipids have been described, the effects on myocardial extraction of amino acids are unknown. This study was undertaken to clarify the effect of insulin glucose-potassium on the pattern of amino acid uptake/release in myocardial and skeletal muscle after coronary operations. The amino acid uptake/release of the heart and of the leg was studied in 18 patients 1 hour after coronary bypass operations. The patients were randomized to treatment with 25 U of fast-acting insulin as a bolus injection followed by a continuous infusion of 1 U/kg body weight for 1 hour, or to serve as control patients. The hyperinsulinemic "clamp" technique was used to keep blood glucose unchanged during the study. In the insulin-treated group, the arterial concentration of 17 of 22 individual amino acids, including the three branched chain amino acids, decreased, the remainder being unchanged. The amino acid uptake/release of the leg was unchanged. The net myocardial uptake of leucine and isoleucine shifted to a no-uptake/no-release in the insulin-treated group, whereas the no-uptake/no-release of tyrosine and phenylalanine turned into a significant release. A positive correlation was observed between arterial concentration and myocardial uptake/release of the three branched chain amino acids. It is suggested that insulin, by lowering the arterial concentration of leucine and isoleucine, inhibited the myocardial uptake of these amino acids. This may have a negative effect on postoperative myocardial protein balance suggested by the release of tyrosine and phenylalanine. PMID- 1728721 TI - Asymmetrical brain modulation of immune reactivity in mice: a model for studying interindividual differences and physiological population heterogeneity? AB - Functional brain asymmetry is associated with immune reactivity in mice. This association depends on the immunological parameters tested and on the sex and genetic background of animals. Each individual may be characterized by a paw preference score in relation with a particular immune pattern. The interindividual differences in immune reactivity connected with brain asymmetry give a new insight in the relation brain immunity and may represent a new aspect of population polymorphism. PMID- 1728722 TI - In vivo bidirectional regulation of mouse natural killer (NK) cell cytotoxic activities by Leu-enkephalin: reversibility by naloxone. AB - Intraperitoneal injection of Leu-enkephalin (LENK, 10 or 7.5 mg/kg) induced bidirectional modulation of natural cytotoxic activities in spleens of CBA mice (suppression followed by enhancement). NK-cytotoxic activity was more affected than the ADCC. Early suppression of NK activity could be reversed by 4 x M excess of naloxone injected 20 min before LENK, suggesting that the suppression was mediated by opioid receptors. Subsequent increase of NK activity could not be abrogated by naloxone, at least not completely. Naloxone itself decreased NK activity 12 hours after treatment, but enhanced ADCC at 24 and 48 hours. This increase was abrogated by LENK. In addition to functional alterations, LENK also induced phenotypic changes of spleen cells, i.e. a decrease in the percentage of asialo-GM-1+ cells 24 hours posttreatment. There was no correlation between LENK induced alterations of cytotoxic function and the percentage of cells with NK phenotype (GM-1+). Thus, LENK modulates cytolytic functions and the phenotype of NK cells in vivo in a complex way, which besides opioid mechanisms may also include non-opioid ones. PMID- 1728723 TI - Physostigmine: dose-response effects on endurance and thermoregulation during exercise. AB - We previously reported that the administration of 200 micrograms/kg of physostigmine (PH) to rats exercising on a treadmill resulted in decrements in both endurance (decreased running time to exhaustion) and thermoregulation. However, it was necessary to determine the dose-response effects of PH administration before PH-treated exercising rats could be used as a model with which to examine the relative anticholinergic potency of drugs. In the present work saline, 50, 100, or 200 micrograms/kg of physostigmine salicylate (0%, 40%, 50%, and 60% whole blood cholinesterase inhibition) was administered to rats (N = 12/group) prior to treadmill exercise (26 degrees C, 50% rh, 11 m/min, 6 degrees incline). The saline control group ran for 67 +/- 6 min (mean +/- SE) with a rate of rise of core temperature of 0.051 +/- 0.007 degrees C/min. The run times declined (80%, 64% and 48% of control) as rate of rise of core temperature increased (116%, 180%, and 214% of control) in a dose-dependent manner (50, 100, 200 micrograms/kg PH). Cholinergic symptoms such as salivation, tremors, and defecation were also affected in a dose-dependent manner by PH administration. Since cholinergic symptoms, thermoregulatory effects, and endurance decrements all vary in a dose-dependent manner with physostigmine administration, the exercising rat represents a useful model for examining the relative potency of cholinergic therapies. PMID- 1728724 TI - Effects of immobilization stress on tuberoinfundibular dopaminergic (TIDA) neuronal activity and prolactin levels in lactating and non-lactating female rats. AB - The effects of immobilization stress on the prolactin secretory response and on the activity of the tuberoinfundibular dopaminergic (TIDA) neurons were determined in intact, virgin female rats on the morning of diestrus or proestrus and in post-partum, lactating female rats. The virgin females exhibited a significant increase in circulating levels of prolactin which was evident by 1 minute and persisted during the immobilization (5 minutes). In contrast, the prolactin secretory response in lactating females was significantly attenuated compared to non-lactating animals. The activity of the TIDA neurons was not altered by the 5 minutes of stress. Even after 30 minutes of immobilization, TIDA neuronal activity was not affected in either the lactating or cycling females. These data suggest that the cycling female rat is capable of a prolactin secretory response to the stressor without inhibition of TIDA neuronal activity. It seems likely that prolactin releasing factors mediate this response. In contrast, stress did not produce a similar prolactin increase during lactation. It seems likely that, during lactation, the pituitary is not sensitive to releasing factors unless the TIDA neurons are inhibited. There appear to be differences in the sensitivity of the pituitary depending on the physiological state of the model employed. PMID- 1728725 TI - [3H]dopamine uptake by platelet storage granules in schizophrenia. AB - [3H]Dopamine (DA) uptake by platelet storage granules was determined in 26 schizophrenic male patients, paranoid type (14 acute stage; 12 in remission) and 20 age-matched, normal controls. Maximum velocity (Vmax) of DA uptake was significantly higher in acute patients, than patients in remission or controls (p less than 0.05). The apparent Michaelis constant (Km) of DA uptake in acute patients was also significantly different from chronic patients (p less than 0.05). Preincubation with reserpine (10(-4), 10(-5) M) produced a substantial diminution of DA uptake, while haloperidol (10(-4), 10(-5) M) did not affect the assay. Considering that a DA dysequilibrium in schizophrenia may be expressed not only in the brain, but also in the periphery and that an increased amount of DA accumulated in the vesicles, implies that an increased quantity of catecholamine is available for release, our findings suggest additional evidence for the role of DA overactivity in the pathophysiology of this disorder. PMID- 1728726 TI - Effects of chlordiazepoxide and beta-carboline 3-carboxylic acid ethyl ester on non-suppressed and minimally-suppressed responding in the squirrel monkey. AB - Lever-pressing of squirrel monkeys was maintained under a multiple fixed-interval (FI) 5-min schedule of food presentation. In one component, responding was suppressed to various degrees by the presentation of electric shock following each 30th response. When responding was either substantially or minimally suppressed, intermediate doses of chlordiazepoxide (CDAP, 1-30 mg/kg) increased both suppressed and non-suppressed responding. Beta-carboline 3-carboxylic acid ethyl ester (beta-CCE, 0.1-3 mg/kg) had little effect at low to intermediate doses (0.1-0.3 mg/kg) and decreased both minimally-suppressed and non-suppressed responding to a comparable extent at higher doses. Repeated daily dosing with beta-CCE (up to 10 mg/kg) resulted in rapid tolerance to its rate-decreasing effects. As agonists do not typically exhibit rapid tolerance for anxiolytic efficacy, the current results suggest that some behavioral effects of inverse agonists may not be strictly opposite those of benzodiazepines. PMID- 1728727 TI - Pamidronate. PMID- 1728728 TI - Hysterectomy prevalence and death rates for cervical cancer--United States, 1965 1988. AB - Since the 1960s, hysterectomy has been one of the most frequently performed inpatient surgical procedures in the United States, with an estimated 33% of women undergoing a hysterectomy by 60 years of age. However, rates of cervical cancer mortality that do not allow for the proportion of women with hysterectomies in the population will underestimate the rates in the true at-risk population (i.e., women with intact uteri) and may influence apparent secular trends in rates of cervical cancer mortality. This report uses national mortality and hospital-discharge data to compare death rates, corrected and uncorrected for hysterectomy prevalence, for women who died with an underlying diagnosis of cervical cancer (International Classification of Diseases, Ninth Revision [ICD-9] and ICD-9-Clinical Modification, code 180). PMID- 1728729 TI - Death rates of malignant melanoma among white men--United States, 1973-1988. AB - Since 1973, death rates for malignant melanoma (International Classification of Diseases, Ninth Revision, codes 172.0-172.9) have increased in the United States and other countries; this increase has occurred disproportionately among white men. To develop hypotheses on the etiology of this increase, the Boston University Schools of Medicine and Public Health and CDC reviewed data from the Surveillance, Epidemiology, and End Results (SEER) Program for the National Cancer Institute and other existing databases. This report summarizes patterns of malignant melanoma among whites in the United States from 1973 through 1988 and suggests possible causes for these patterns. PMID- 1728730 TI - The second 100,000 cases of acquired immunodeficiency syndrome--United States, June 1981-December 1991. AB - The first cases of acquired immunodeficiency syndrome (AIDS) were reported in June 1981. From 1981 through December 1987, 50,000 AIDS cases had been reported to CDC, and by August 1989, 100,000 cases had been reported. From September 1989 through November 1991, state and territorial health departments reported 100,000 additional cases. By December 31, 1991, a cumulative total of 206,392 cases had been reported (Figure 1), and the cumulative number of reported deaths associated with AIDS was 133,232. This report presents characteristics of the first and second 100,000 persons with AIDS. PMID- 1728731 TI - The timing of prophylactic administration of antibiotics and the risk of surgical wound infection. AB - BACKGROUND: Randomized, controlled trials have shown that prophylactic antibiotics are effective in preventing surgical-wound infections. However, it is uncertain how the timing of antibiotic administration affects the risk of surgical-wound infection in actual clinical practice. METHODS: We prospectively monitored the timing of antibiotic prophylaxis and studied the occurrence of surgical-wound infections in 2847 patients undergoing elective clean or "clean contaminated" surgical procedures at a large community hospital. The administration of antibiotics 2 to 24 hours before the surgical incision was defined as early; that during the 2 hours before the incision, as preoperative; that during the 3 hours after the incision, as perioperative; and that more than 3 but less than 24 hours after the incision, as postoperative. RESULTS: Of the 1708 patients who received the prophylactic antibiotics preoperatively, 10 (0.6 percent) subsequently had surgical-wound infections. Of the 282 patients who received the antibiotics perioperatively, 4 (1.4 percent) had such infections (P = 0.12; relative risk as compared with the preoperatively treated group, 2.4; 95 percent confidence interval, 0.9 to 7.9). Of 488 patients who received the antibiotics postoperatively, 16 (3.3 percent) had wound infections (P less than 0.0001; relative risk, 5.8; 95 percent confidence interval, 2.6 to 12.3). Finally, of 369 patients who had antibiotics administered early, 14 (3.8 percent) had wound infections (P less than 0.0001; relative risk, 6.7; 95 percent confidence interval, 2.9 to 14.7). Stepwise logistic-regression analysis confirmed that the administration of antibiotics in the preoperative period was associated with the lowest risk of surgical-wound infection. CONCLUSIONS: We conclude that in surgical practice there is considerable variation in the timing of prophylactic administration of antibiotics and that administration in the two hours before surgery reduces the risk of wound infection. PMID- 1728732 TI - Angioscopic evaluation of coronary-artery thrombi in acute coronary syndromes. AB - BACKGROUND: Disruption of an atherosclerotic plaque in a coronary artery followed by the formation of a thrombus is believed to be the cause of both unstable angina and acute myocardial infarction. Although thrombolytic therapy is efficacious in patients with acute myocardial infarction, for unknown reasons it is far less effective in patients with unstable angina. We postulated that there might be differences in the composition of the coronary-artery thrombi in unstable angina and acute myocardial infarction. METHODS: To investigate the appearance of coronary-artery thrombi, we performed percutaneous transluminal coronary angioscopy in 15 patients with unstable angina and 16 with acute myocardial infarction. Angioscopy was performed within 48 hours after an episode of pain at rest in the patients with unstable angina and within 8 hours of onset in those with acute myocardial infarction. RESULTS: Angioscopy revealed coronary thrombi in all but two patients (one in each group). Of the 29 patients with thrombi, those with unstable angina were frequently observed to have grayish white thrombi (10 of 14, 71 percent), but none were seen in the 15 patients with acute myocardial infarction (P less than 0.01). By contrast, reddish thrombi were observed in all 15 patients with acute myocardial infarction who had thrombi, but in only 4 of the 14 patients with unstable angina and thrombi (P less than 0.01). As assessed by coronary angiography, occlusive thrombi occurred frequently in patients with acute myocardial infarction (13 of 16 patients) but were not seen in any of the 15 patients with unstable angina (P less than 0.01). CONCLUSIONS: Coronary-artery thrombi play an important part in the pathogenesis of unstable angina and acute myocardial infarction. However, the appearance of the thrombi is different in the two conditions, possibly reflecting differences in the composition of age of the thrombi or the presence or absence of blood flow in the artery. This difference may account for the contrasting results of thrombolytic therapy. PMID- 1728733 TI - Resistance to erythromycin in group A streptococci. AB - BACKGROUND: The use of erythromycin in Finland nearly tripled from 1979 to 1989. In 1988, we observed an unusually high frequency of resistance to erythromycin in group A streptococci in one geographic region. Because routine testing does not detect the sensitivity of these organisms to antibiotics, we initiated a national study to evaluate the extent of this resistance. METHODS: We studied 272 isolates of group A streptococci obtained from blood cultures from 1988 through 1990. In 1990 we collected from six regional laboratories 3087 consecutive isolates from throat swabs and 1349 isolates from pus samples. Resistance was indicated by growth on blood agar containing 2 micrograms of erythromycin per milliliter after incubation in 5 percent carbon dioxide. We also evaluated the clinical importance of erythromycin resistance in a retrospective study of consecutive patients with pharyngitis. RESULTS: The frequency of resistance to erythromycin in group A streptococci from blood cultures increased from 4 percent in 1988 to 24 percent in 1990. From January to December 1990, the frequency of resistance in isolates from throat swabs increased from 7 percent to 20 percent, and resistance in isolates from pus increased from 11 percent to 31 percent. In four communities within 50 km of each other, the frequency of erythromycin resistance ranged from 2 to 5 percent to 26 to 44 percent. Several distinct DNA restriction profiles and serotypes were found among resistant isolates from the same area, suggesting a multiclonal origin. The treatment of pharyngitis with erythromycin failed in 9 of 19 patients infected with erythromycin-resistant group A streptococci, as compared with 1 of 26 patients with erythromycin-susceptible isolates (47 percent vs. 4 percent, P = 0.008). CONCLUSIONS: In Finland since 1988 there has been a rapid and substantial increase in resistance to erythromycin in group A streptococci. The extent of this resistance is particularly serious since there are only a few alternative antibiotics available for peroral treatment of group A streptococcal infections. PMID- 1728734 TI - Cigarette advertising and magazine coverage of the hazards of smoking. A statistical analysis. AB - BACKGROUND: Health professionals have charged that magazines that depend on revenues from cigarette advertising are less likely to publish articles on the dangers of smoking for fear of offending cigarette manufacturers. Special concern has focused on magazines directed to women. Restricted coverage of smoking hazards could lead readers to underestimate the risks of smoking in relation to other health risks. METHODS: Using logistic-regression analysis of a sample of 99 U.S. magazines published during 25 years (1959 through 1969 and 1973 through 1986), we analyzed the probability that the magazines would publish articles on the risks of smoking in relation to whether they carried advertisements for cigarettes and in relation to the proportion of their advertising revenues derived from cigarette advertisements. We controlled for other factors that might influence coverage. RESULTS: The probability of publishing an article on the risks of smoking in a given year was 11.9 percent for magazines that did not carry cigarette advertisements, as compared with 8.3 percent for those that did publish such advertisements (adjusted odds ratio, 0.73; 95 percent confidence interval, 0.42 to 1.30). For women's magazines alone, the probabilities were 11.7 percent and 5.0 percent, respectively (adjusted odds ratio, 0.13; 95 percent confidence interval, 0.02 to 0.69). When the proportion of revenues derived from cigarette advertising was the independent variable, the probability of publishing an article on the risks of smoking in a given year was reduced by 38 percent (95 percent confidence interval, 18 percent to 55 percent) for magazines with the average proportion of total advertising revenues derived from cigarette advertising for the entire sample of magazines (6 percent) as compared with magazines with no cigarette advertising. This relation was particularly strong in the case of women's magazines. An increase of 1 percent in the share of advertising revenue derived from cigarette advertisements decreased the probability of covering the risks of smoking by three times as much as in other magazines. CONCLUSIONS: This study provides strong statistical evidence that cigarette advertising in magazines is associated with diminished coverage of the hazards of smoking. This is particularly true for magazines directed to women. PMID- 1728735 TI - The pathogenesis of coronary artery disease and the acute coronary syndromes (2). PMID- 1728736 TI - Sinusitis in children. PMID- 1728737 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 5-1992. A 20-year-old man with diffuse pulmonary infiltrates and disseminated intravascular coagulation. PMID- 1728738 TI - Preoperative antibiotic prophylaxis. PMID- 1728739 TI - Global surveillance of antibiotic resistance. PMID- 1728740 TI - The Oregon Medicaid Demonstration Project--will it provide adequate medical care? PMID- 1728741 TI - Transdermal nicotine patch for smoking cessation. PMID- 1728742 TI - Transdermal nicotine patch for smoking cessation. PMID- 1728743 TI - Transdermal nicotine patch for smoking cessation. PMID- 1728744 TI - Cutaneous melanoma. PMID- 1728745 TI - Cutaneous melanoma. PMID- 1728746 TI - Cutaneous melanoma. PMID- 1728747 TI - Vaccination with gp160 in HIV. PMID- 1728748 TI - Vaccination with gp160 in HIV. PMID- 1728749 TI - Fetal protection and job discrimination. PMID- 1728750 TI - Therapeutic use of type F botulinum toxin. PMID- 1728751 TI - Advertising baldly to the public. PMID- 1728752 TI - Arctic first aid. PMID- 1728753 TI - Lime disease in the antarctic. PMID- 1728754 TI - [New year, new home]. PMID- 1728755 TI - [Physician and pharmaceutical industry. I. Along the royal road or via Royal Class?]. PMID- 1728756 TI - [Clinical, bacteriological and cost-saving effects of antibiotic prophylaxis in craniotomy]. AB - Objective of the study. To investigate the effect of antibiotic prophylaxis on the incidence of infections, on the bacterial flora of wounds and on the health care costs. A retrospective study disclosed an incidence of infection of 8.1% in patients who underwent craniotomy. Methods. Double-blind, placebo-controlled study of the effects of cloxacillin (4 x 1 gr intravenously during 24 h, perioperatively) in 310 patients who had to undergo a craniotomy. Results. In the cloxacillin group significantly fewer infections occurred than in the placebo group, 6 infections in 156 and 20 infections in 154, respectively. In the cloxacillin group significantly fewer samples contained micro-organisms than in the placebo group. Cloxacillin prophylaxis reduces the cost of patient care by about 20%. Conclusion. Cloxacillin prophylaxis in craniotomy cases reduces the percentage of infections, the percentage of positive cultures of the wound, and the costs of patient care. PMID- 1728757 TI - [Muscular hypotonia as symptom of primary hyperparathyroidism: is surgery worthwhile?]. PMID- 1728758 TI - [Only limited effect of treatment of thyroid gland diseases on symptoms of musculoskeletal system]. AB - In order to assess the long term effect of treatment for thyroid dysfunction on musculoskeletal symptoms, 102 patients were traced by a computer search; these patients visited two outpatient clinics of rheumatology and were known to have thyroid disorders. Of these 102 patients, 58 patients met the study criteria of having abnormal thyroid function tests at first visit to the outpatient clinic; 46 patients participated in the study. They were interviewed with a structured questionnaire about their past and current musculoskeletal complaints. The 46 patients (45 females, 1 male) had a mean age at interview of 58 years (range 21 81); 37 and 9 patients had been treated for hypothyroidism and hyperthyroidism respectively. The thyroid dysfunction was considered the only explanation of the original musculoskeletal symptoms in 24 patients (group I), in 19 patients an additional (rheumatological) diagnosis was made (group II), and in 3 patients no apparent relation between musculoskeletal complaints and thyroid dysfunction was found. After treatment for thyroid dysfunction the original complaints decreased in 52% and 47% of the patients in group I and II, respectively. At the time of the follow-up study (mean follow-up duration 67 months) 91% of the patients had musculoskeletal symptoms, 80% of the patients said their present complaints were similar to their original symptoms. Treatment for thyroid dysfunction resulted in a temporary beneficial effect on musculoskeletal symptoms in 50% of the patients; in 91% symptoms persisted. PMID- 1728759 TI - [Results of intensive care treatment in patients with hematologic malignancies; relation to infections]. AB - In a retrospective study we determined the factors that influence the outcome of patients requiring intensive care (IC) for the complications of the treatment of haematological malignancies. All consecutive patients suffering from haematological malignancy admitted to the IC unit of the University Hospital Nijmegen over a 4 year period (1986-1989) were studied; 39 patients (21 female, 18 male) with a median age of 41 year (range 18-71). Various clinical variables were assessed at admission and related to the outcome of IC treatment in terms of death or survival. Previously, 33 patients had undergone chemotherapy. Eight (21%) survived. One of a subgroup of 13 bone marrow transplantation patients survived. Hypoxaemic respiratory failure requiring mechanical ventilation was the most frequent reason for admission (20 patients of whom four recovered). The second most frequent reason was combination of respiratory failure and septic shock (eight patients, none recovered). Three out of five patients admitted with septic shock survived. The need for mechanical ventilation had the highest predictive power for a poor prognosis (p = 0.002). Mortality increased with increasing APACHE II score (p = 0.03). When more than four major organ systems were affected (seven patients), mortality was 100%. Granulocytopenia (leucocyte count less than 1 x 10(9)/l) at admission turned out to be of no prognostic significance, but absence of leucocyte recovery was a prognostically bad sign (p = 0.05). A considerable number of patients (20) suffered from a non-bacterial (opportunistic) infection, carrying a high mortality (only two patients survived: one with candida, one with a cytomegalovirus infection).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1728760 TI - [The homes of the Nederlands Tijdschrift voor Geneeskunde]. PMID- 1728761 TI - [Physician and pharmaceutical industry. II. Postmarketing surveillance]. PMID- 1728762 TI - [Is usually more often than frequently; or how often is sometimes?]. PMID- 1728763 TI - [2 epidemics of mortality due to 'symptoms and incompletely described diseases']. PMID- 1728764 TI - [The Netherlands and postmarketing surveillance]. PMID- 1728765 TI - [Classification of fibromyalgia: criteria of the American College of Rheumatology]. PMID- 1728766 TI - Chlamydial infection. Screening and management update, 1992. AB - The need for a cost-effective, highly sensitive and specific test for Chlamydia trachomatis infection remains, although progress is being made. Clinicians can minimize the incidence of complications and erroneous test results by being alert to the possibility of this infection, screening appropriate patients, using careful collection techniques, following the most recent treatment recommendations, and knowing the limitations of available tests. PMID- 1728767 TI - Syncope in elderly patients. Why their risk is higher. AB - The elderly are at increased risk for syncope not only because of physiologic changes due to aging but also because they have a higher incidence of chronic illness and use a greater number of medications that may cause orthostatic hypotension. Although evaluation should emphasize cardiovascular and neurologic components, the cause may remain elusive in as many as 50% of patients. PMID- 1728768 TI - Chronic abdominal pain due to periostitis pubis. A new syndrome. AB - Periostitis pubis is a clinical syndrome previously undescribed in the literature. It is characterized by lower abdominal pain that may have persisted for several weeks to several years. Physical findings are limited to tenderness in one of the lower abdominal quadrants and over the os pubis on the affected side. The diagnosis can be confirmed by injecting lidocaine hydrochloride into the area of point tenderness over the os pubis, which should relieve tenderness in both sites. An elaborate laboratory workup is not necessary. The condition can be cured with an injection of prednisolone tebutate at the site of tenderness over the os pubis. PMID- 1728769 TI - Colorectal cancer. Recent developments and continuing controversies. AB - In the last 2 years, much progress has been made in colorectal cancer research. Many of the findings, especially those involving adjuvant therapy, have important clinical applications. For example, fluorouracil (5-FU) (Adrucil) combined with levamisole hydrochloride (Ergamisol) is now recommended as adjuvant therapy for patients with Dukes' stage C (III) disease. Some aspects of colorectal cancer remain controversial, particularly screening recommendations and monitoring of carcinoembryonic antigen levels. PMID- 1728770 TI - Emergency management of cocaine intoxication. Counteracting the effects of today's 'favorite drug'. AB - The toxic effects of cocaine are becoming more widely recognized as the popularity of the drug increases. Although overdose is usually fatal, treatment is available for users who experience agitation, seizure, arrhythmias, severe hypertension, or other side effects. Hospital admission is often necessary to allow monitoring of the acute situation. Initial management involves standard life-support measures. Therapeutic agents are given, as appropriate, both to control acute reactions and to counteract continuing toxicity. PMID- 1728771 TI - A cautionary tale. PMID- 1728772 TI - Esophageal ulceration following doxycycline ingestion. AB - Esophageal ulceration occasionally occurs in patients taking doxycycline capsules or tablets. In the case described here, examination of biopsy specimens obtained at endoscopy showed polarizable matrix material in the ulcer bed, thus confirming the diagnosis. PMID- 1728773 TI - Pharmacologic perfusion imaging. Who needs it and why? AB - Pharmacologic perfusion imaging is an excellent choice for patients who cannot undergo treadmill exercise stress testing. The use of pharmacologic imaging has proved valuable in all branches of medicine and surgery. Patients requiring cardiac evaluation before vascular or orthopedic surgery can now be examined quite completely; there are very few patients today who cannot be "stressed" adequately. Advances in this field are being made every day, and the accuracy of testing will improve further when such new technologies as positron-emission tomography are more widely available. PMID- 1728774 TI - The eyes give the clue. Ocular manifestations of systemic disease. AB - Many systemic diseases have ocular findings that can be seen with flashlight illumination or a direct ophthalmoscope. Many more diseases can be identified with extensive and specialized examination techniques, which are usually not readily available to primary care physicians. However, all physicians can increase their diagnostic accuracy by being aware of and looking for easily observed ocular signs of systemic disease. PMID- 1728775 TI - Deep venous thrombosis and pulmonary embolism. The importance of heightened awareness. AB - Left untreated, deep venous thrombosis and pulmonary embolism have a high rate of mortality and long-term morbidity. Physicians therefore must maintain a high index of suspicion for these conditions. Accurate diagnosis is facilitated by knowing the most common sites of thrombus formation, the likelihood of propagation, which patients are at greatest risk, signs and symptoms, and which tests to order. Prompt administration of anticoagulants and, in some cases, thrombolytic agents can minimize the consequences of these diseases. Interruption of the inferior vena cava, thrombectomy, and thromboembolectomy are other treatment options. PMID- 1728776 TI - Palpitations and arrhythmias. Separating the benign from the dangerous. AB - Palpitations are a nonspecific symptom and do not necessarily imply serious heart disease. The vast majority of palpitations are benign. Goals in evaluation include detecting and identifying an arrhythmia, clarifying symptom severity, and defining the extent of underlying heart disease. If palpitations are infrequent and not accompanied by angina, congestive heart failure, or syncope, outpatient transtelephonic monitoring yields useful clinical information in most patients and is more cost-effective than Holter monitoring. Patients with major symptoms require hospitalization for aggressive cardiac evaluation and, possibly, electrophysiologic testing to guide treatment. PMID- 1728777 TI - Depression in the elderly. How to recognize masked symptoms and choose appropriate therapy. AB - Depression in the elderly is one of the most serious undiagnosed health problems in the United States. All physicians who care for elderly patients need to become aware of the signs of masked depression and treat it vigorously. Dr Yesavage explains how to differentiate depression from dementia, describes useful diagnostic screening tests, and offers recommendations for pharmacologic treatment of geriatric depression. PMID- 1728778 TI - Ambulatory blood pressure monitoring. When is it warranted? AB - Ambulatory blood pressure monitoring allows repetitive and non-invasive measurements to be taken over a 24-hour period or longer in the patient's usual environment. A technically successful recording yields valuable information about circadian blood pressure patterns and the mean, peak, nadir, standard deviation, and variability of both systolic and diastolic blood pressure. Although ambulatory monitoring should not be used in all hypertensive patients, it can aid in the evaluation of specific problems, such as borderline, resistant, and episodic hypertension; transient hypotension; and blood pressure-related target organ damage. It also helps in assessing the efficacy of anti-hypertensive medications and in conducting longitudinal epidemiologic studies of target-organ damage and cardiovascular events. PMID- 1728779 TI - Healthcare reform is coming--but what will it be? PMID- 1728780 TI - Percutaneous endoscopic gastrostomy. What are the benefits, what are the risks? AB - One way to nutritionally support patients who cannot swallow is to administer formula directly into the stomach. Placing a gastrostomy tube percutaneously using endoscopy avoids the risks of general anesthesia and wound healing that accompany surgical gastrostomy. Although certain conditions (eg, sepsis, coagulation disorder, portal hypertension) are contraindications to the procedure, it can be done in patients who have had previous abdominal surgery and in those with severe illness. A commercially available feeding formula is used. The type chosen and the frequency of administration are based on the patient's specific needs. With regular medical monitoring and daily care of the gastrostomy site, appropriately selected patients may be safely maintained with enteral feeding for months. An advantage of the percutaneously inserted tube is that it is easily removed when the patient regains the ability to eat, and the fistula heals rapidly. PMID- 1728781 TI - Painless intussusception. Giving conservative treatment another chance. AB - Treatment options for intussusception in children range from reduction by barium enema to surgical intervention. The authors describe a case in which a conservative option--a fifth attempt to reduce an ileocolic intussusception by barium enema, this time using general anesthesia--successfully resolved the problem. PMID- 1728782 TI - Comparatively speaking. PMID- 1728783 TI - Fine-needle aspiration biopsy. PMID- 1728784 TI - Diagnosing COPD. How to identify patients with irreversible obstruction of the airways. AB - Chronic obstructive pulmonary disease (COPD) is seen often in primary care offices. Its hallmark is dyspnea with obstruction. Smoking is the leading risk factor. Smokers should be counseled at every encounter to quit smoking to prevent development of COPD and lower the rate of decline of pulmonary function. Office spirometry detects the early changes of small airways obstruction. All patients with symptoms should receive baseline spirometry and follow-up spirometry once they have received maximal medical therapy. If evidence of obstruction is found and spirometric abnormality persists after therapy, COPD is present. Treatment early in the clinical course can markedly improve COPD and avoid the severe disability of end-stage disease. PMID- 1728785 TI - Outpatient care of COPD patients. AB - Chronic obstructive pulmonary disease (COPD) is a disorder of airflow limitation that is often progressive. Interventions aimed at slowing this progression include smoking cessation, avoidance of exacerbating factors, and prevention or early treatment of infection. Reversible symptoms can be controlled pharmacologically, with supplemental oxygen prescribed when needed. Finally, quality of life can be improved through education and pulmonary rehabilitation. PMID- 1728786 TI - Thrombolytic therapy in cerebrovascular disorders. AB - The knowledge obtained from the ongoing investigational trials of tPA for acute ischemic stroke will not only help establish the appropriate dose range and complication rates but will also further develop the clearly mandatory rapid, aggressive team approach needed to truly treat acute ischemic strokes successfully. Experimental cerebral ischemia data have pointed to the need to treat acute clinical stroke within only a few hours or less to effectively reduce stroke morbidity and mortality. Specifically, with reversible MCA occlusion models of focal cerebral ischemia (dogs and cats), the animals uniformly survive without neurological deficit if the occlusion is for less than 2 to 3 hours. Similarly in primates, MCA occlusion for 3 hours or less will lead to clinical improvement and a decrease in infarct size, with complete recovery generally associated with less than 2 hours of MCA occlusion. Therefore, it appears unlikely that ischemic brain can be salvaged if vascular occlusion persists longer than 4 to 6 hours (similar to the pathophysiology of myocardial ischemia). Further, at least one third of ischemic stroke patients reperfuse spontaneously (and obviously too late) within 48 hours of stroke onset. Several factors believed to be related to successful outcome after thrombolytic therapy are summarized in Table 16. A schematic approach to determining the response to thrombolytic agents in acute ischemic stroke is outlined in Table 17. Zivin succinctly reviews thrombolysis for stroke, both experimental and clinical, and summarizes some of the difficulties of the early clinical stroke trials with thrombolytic agents and speculates about future prospects. He believes tPA may prove valuable in the treatment of some forms of thromboembolic stroke. Its usefulness may depend in part on how quickly the drug can be initiated and the risk of side effects; factors that will require further study. The currently used doses of tPA may be too low to lyse large cerebral arterial clots and, therefore, if current trials do not show a positive treatment response, further trials with higher doses may be indicated. The implications of a potentially effective treatment for truly acute stroke are enormous: stroke will need to be considered by all (lay public through to caregivers) as a true medical emergency, analogous to MI and trauma.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1728787 TI - Angioplasty/thrombolytic treatment of failing and failed hemodialysis access sites: comparison with surgical treatment. AB - Angioplasty is a valuable alternative to surgical revision of failing hemodialysis access sites and may be the treatment of choice because no further vein is compromised during the revision and because patency rates with repeat dilatations approach or equal those of surgical revision. Thrombolysis/angioplasty is a worthy substitute for surgical thrombectomy/revision in thrombosed access sites because dialysis can be resumed immediately, without the need of placement of a temporary subclavian vein access catheter, and lysis can be performed on an outpatient basis. Long-term secondary patency also approaches that of surgical therapy. Again, future access sites are not compromised. Either with percutaneous catheter or surgical therapy, it must be recognized that repeat treatment will be necessary to maintain patency of the access site after it has thrombosed. Close follow-up of these patients to observe for signs of recurring deterioration is mandatory. Because the number of vascular access sites is limited, the preservation of each site for as long as possible is important for the long-term management of these patients. PMID- 1728788 TI - Monitoring thrombolytic therapy. PMID- 1728789 TI - Stage B (P2/3A/N0) transitional cell carcinoma of bladder highly curable by radical cystectomy. AB - Seventy-one patients with pathologic Stage B (P2/3a/N0) transitional cell carcinoma (TCC) of the bladder underwent radical cystectomy alone without preoperative radiotherapy or perioperative chemotherapy between 1983 and 1987 and have been followed a median of fifty months. The five-year actuarial survival and disease-free survival rates were 82 percent and 77 percent, respectively, and only 13 patients (18%) have relapsed. Histologic parameters were evaluated as to prognostic impact; none correlated with disease-free survival rates although the presence of vessel involvement portended a worse disease-free survival rate (68% versus 80%). During this same period, an additional 15 patients underwent radical cystectomy for pathologic Stage B disease but received adjuvant chemotherapy on the basis of vessel invasion. Their disease-free survival rate at five years was 80 percent, comparable to the disease-free survival rate for patients with vessel invasion treated by surgery alone (68%). Although the role of systemic chemotherapy in the management of invasive bladder cancer remains under investigation, it would appear that patients with Stage B TCC are best treated with radical cystectomy alone. Continued analysis of modern surgical results grouped by current pathologic staging criteria is needed to identify patients who have a relatively low risk of relapse and thus little need for additional therapeutic intervention. These results demonstrate that Stage P2/3a/N0 TCC of the bladder is highly curable by surgery. PMID- 1728790 TI - Brain metastases from transitional cell carcinoma of urinary bladder. AB - Of 293 patients with transitional cell carcinoma of the bladder seen at our institution between April 1977 and December 1987, 9 patients were found to have brain metastasis. Seven of 9 patients were found to have a solitary brain lesion, and in 4 of these, no other site of metastatic disease was identified. Five patients received palliative whole brain irradiation, 3,000 cGy in 10 fractions, due to the presence of multiple lesions of the central nervous system (CNS) or metastases to other sites. The average survival for this group was seven weeks. One patient with a solitary brain metastasis and no other documented metastatic site was hospitalized at another institution, and was managed expectantly receiving only parenteral steroid therapy and survived four weeks. Three patients with solitary lesions and no evidence of other metastatic sites were treated with a combined surgical and radiotherapeutic approach receiving 4,000-5,000 cGy to the lesion site postoperatively. The average survival of that group was twenty nine months, with one five-year survivor and 1 patient with no evidence of disease fourteen months after treatment. It appears that survival is longer in those patients with solitary lesions, perhaps due, at least in part, to a more aggressive therapeutic approach. PMID- 1728791 TI - Carcinoma of male urethra: management of locally advanced disease with combined chemotherapy, radiotherapy, and penile-preserving surgery. AB - Squamous cell carcinoma of the urethra is a rare urologic malignancy. In spite of aggressive management with radical and often times disfiguring surgery and/or radiation therapy, prognosis remains poor. Initial success in treating squamous cell carcinoma of the esophagus and anal canal has been reported with a combined radiation and chemotherapy protocol. In hopes for improving the treatment outcome for patients with squamous cell carcinoma of the urethra, we have applied the new combination chemotherapy/radiation therapy protocol. Herein, we report the successful downstaging (clinical Stage C to pathologic Stage TO) and treatment of squamous cell carcinoma of the anterior urethra with combination chemotherapy/radiation therapy. This was followed with penile-preserving surgery to document local control of disease and to avoid the morbidity of a radical disfiguring operation. PMID- 1728792 TI - Accuracy in staging of renal cell carcinoma involving vena cava. AB - Accurate preoperative staging of renal cell carcinoma is necessary to determine patient prognosis and surgical approach, particularly when tumor thrombus invades the vena cava. The pathologically-confirmed tumor stage was compared with the radiographic preoperative stage in 44 patients undergoing surgery for renal cell carcinoma invading the vena cava (T3cNxMx). Nine patients (20%) were upstaged as the result of extracapsular tumor extension. Twelve patients (27%) were upstaged due to unrecognized regional lymphadenopathy, and 1 patient was downstaged. Only 1 patient was upstaged as the result of unrecognized metastases. The level of tumor thrombus extension for surgical approach was accurately determined in all but 2 patients. Overall, 15 patients (34%) were upstaged as a result of pathologic studies, 28 patients (64%) were correctly staged, and 1 patient was downstaged. Radiographic staging of extracapsular tumor extension and regional lymphadenopathy is unreliable, but current radiographic techniques delineate the level of thrombus extension for surgical approach with high accuracy. PMID- 1728793 TI - Radical prostatectomy: OSU and affiliated hospitals' experience 1985-1989. AB - A total of 528 patients have been treated with radical prostatectomy and node dissection during the last five years. Correlation of clinical and pathologic staging will be presented. Over 85 percent of these patients had Gleason scores of 6 or less. Patients who had nerve-sparing surgery had a potency rate of over 60 percent post surgery. PMID- 1728794 TI - Carcinoma of prostate in men aged fifty and under: therapeutic options. AB - We made a retrospective study of 20 men, aged fifty or under, with adenocarcinoma of the prostate to evaluate presenting symptoms, stage, grade, and therapeutic results. Sixty-five percent were found to have extracapsular spread of disease (Stage C or D). The therapy used was one or a combination of three types: radical prostatectomy, radiation therapy, and hormonal manipulation. Five of 6 patients with Stage B disease and 3 of 6 patients with Stage C disease were treated with radiation therapy. The other Stages B and C patients underwent radical prostatectomy. In all 5 of Stage B patients receiving radiation, therapy failed; the mean time to tumor recurrence was 3.2 years. Two of 3 patients with Stage C disease died of metastatic disease within three years of receiving radiation. The 4 patients (Stages B and C) who underwent radical prostatectomy are free of disease. There was a statistically higher failure rate among the radiation therapy patients with Stages B and C disease than among the surgical patients (X2 = 8.4, p less than 0.1). PMID- 1728795 TI - Surgical approach and outcome in torsion of testis. AB - A total of 31 patients with acute torsion treated over a fifteen-year period are reviewed. The peak incidence of the disease, its seasonal variations, predisposing factors, surgical procedure, and clinical outcome are analyzed. Most of the patients were operated upon via an inguinal incision and underwent detorsion and fixation or untwisting only of the involved testis. The contralateral one was never anchored. All patients were asked to complete a questionnaire, with 2 of 3 responding from one to fifteen years (mean, 6.7 years) after surgery. None underwent recurrent surgery on either testicle. We conclude that fixation of the testes, both detorsioned and normal is not necessary. PMID- 1728796 TI - Hypertension as a complication of multicystic dysplastic kidney. AB - The detection of multicystic dysplastic kidney in utero using prenatal ultrasound is becoming a more frequent occurrence. An accurate diagnosis of multicystic dysplastic kidney usually can be made radiographically, and therefore the main indication for surgery in the asymptomatic patient may be the potential risk of complications developing later in life. We report hypertension as a complication of multicystic dysplastic kidney and review the literature. PMID- 1728797 TI - Adjuvant radiotherapy in patients post-radical prostatectomy with tumor extending through capsule or positive seminal vesicles. AB - Between 1976 and 1989, 115 patients at UCLA had radical retropubic prostatectomy for clinically localized prostate cancer with positive surgical margins, penetration of tumor into or through the capsule, or positive seminal vesicles. Twenty-four of those received adjuvant radiotherapy after having recovered from surgery. Complications of adjuvant treatment were uncommon and included urethral strictures in 3 patients and transient leg edema in 1. No patient in this group has had proved clinical disease progression though 6 have isolated detectable serum prostate-specific antigen (PSA) values. Clinical disease-free survival at five and seven years was 92 percent. If detectable PSA is also considered as evidence of tumor recurrence, the corresponding disease-free survival rates were 75 percent at five years and 54 percent at seven years. The 91 patients who received no postoperative radiotherapy had a clinical disease-free survival of 67 percent at five years and 56 percent at seven years. Disease-free survival drops to 43 percent and 24 percent, respectively, if detectable follow-up PSA is considered an indicator of disease progression. The comparisons of the survivorship curves in this retrospective study for the two treatment groups are statistically significant both for disease-free survival (p = 0.041), and disease free survival with undetectable PSA (p = 0.043). Adjuvant radiotherapy has a beneficial effect after radical prostatectomy in patients with local tumor extension. PMID- 1728798 TI - Sperm hyperactivation as quality control for sperm penetration assay. AB - The sperm penetration assay (SPA) is subject to considerable variation, and controls are needed to verify the accuracy of the results. It is proposed that sperm hyperactivation (HA) can serve as a quality control check for the SPA. The objective was to determine if there was an association between the SPA outcome and sperm HA measured at various times during the SPA procedure. The data showed a significant correlation between percent sperm HA and percent zona-free oocyte penetration by sperm preincubated for three hours prior to sperm-oocyte interaction (short preincubation). Some sperm hyperactivity was observed in liquefied raw semen samples, but this was insignificantly related to SPA results. Low correlation was observed between SPA results and sperm HA determined immediately after centrifuge washing of sperm. The results suggest that it is possible to utilize sperm HA measured immediately after the sperm-oocyte interaction period as a quality control check of SPA results. PMID- 1728799 TI - Lower urinary tract dysfunction in multiple sclerosis. AB - Previous investigators have shown that in multiple sclerosis failure to empty the bladder was secondary to detrusor-distal sphincter dyssynergia or areflexia. However, our urodynamic evaluation of 46 female and 43 male patients with multiple sclerosis revealed that 63 percent of patients failed to empty their bladders because of a hypocontractile detrusor, and only 6 percent had areflexia. Detrusor-distal sphincter dyssynergia (6%) and bladder neck obstruction (6%) were present in only 12 percent of patients. Hyperreflexia was common (78%) and was associated with hypocontractility in 63 percent of patients. Urgency incontinence was significantly more common in females and voiding difficulty significantly more common in males. Sensation was also reduced in 74 percent of female and 77 percent of male patients. In conclusion, failure to empty the bladder in multiple sclerosis is most commonly associated with hypocontractility, and the combination of hyperreflexia and hypocontractility produces the symptoms of urgency and incomplete emptying. PMID- 1728801 TI - Ureteral injury in abdominal vascular reconstructive surgery. AB - Iatrogenic ureteral injuries in vascular reconstructive surgery are rarely reported. We present a case of ureteral transection during repair of an aortic aneurysm in a patient with a previously placed aortobifemoral graft. In reported series of surgical ureteral injuries, 17 of 381 injuries occurred during vascular procedures. A review of the literature and management scheme for ureteral complications in the presence of prosthetic vascular grafts is presented in light of current endourologic materials and techniques. PMID- 1728800 TI - Endoureteropyelotomy in adults. Review of procedure and results. AB - We report on 17 adult patients who underwent antegrade endoureteropyelotomy for primary (7) and secondary (10) ureteropelvic junction obstruction. Complications were limited to failure to relieve the obstruction, which occurred in 2 patients (12%) requiring a subsequent open pyeloplasty. The two failures were due to high insertion of the ureter in one, and lower renal pole vessel in the other case. A new endoureteropyelotomy stent was developed to enhance healing and patient comfort. No failures occurred due to technical difficulties. PMID- 1728802 TI - Renal lobectomy: parenchyma-sparing technique for partial nephrectomy. PMID- 1728803 TI - Penile implant surgery: rear tip extender that stays behind. PMID- 1728804 TI - Use of protective plastic sheath for prostatic biopsy. PMID- 1728805 TI - Sonography of intrascrotal adenomatoid tumor. AB - A case is reported of an adenomatoid tumor of the epididymis. A discussion of the appropriate differential of mass of the epididymis as well as a review of adenomatoid tumors per se and their occurrence in the scrotum are presented. PMID- 1728806 TI - Movement of pseudomonas aeruginosa along catheter surfaces. A mechanism in pathogenesis of catheter-associated infection. AB - The etiologic mechanism involved in the establishment of catheter-associated bacteriuria is suggested in this in vitro study of the movement of Pseudomonas aeruginosa along a catheter surface against a flowing artificial urine milieu in the presence and absence of antibiotics. Following a lag phase, during which a bacterial biofilm becomes firmly established at a site of contamination, the bacteria ascend the surface of the Foley catheters in a rapidly expanding coherent biofilm. The speed of the bacterial ascent is increased as a result of turbulence-associated planktonic saltatory bacterial movement within the urine column. Bacteriocidal concentrations of antibiotics in the urine can slow down the bacterial ascent, but they do not preclude it. PMID- 1728807 TI - Vitamin and mineral status in physically active men: effects of a high-potency supplement. AB - Changes in nutritional status during supplementation with a high-potency multivitamin-mineral supplement were examined in 22 physically active men randomly assigned to take a supplement (n = 11) or placebo (n = 11) for approximately 12 wk. Four-day dietary intakes, blood concentrations, and urinary excretions of selected vitamins and minerals were measured before, during (approximately 6 and 12 wk), and after supplementation. No changes were observed in blood concentrations of vitamins A and C and measures of zinc, magnesium, and calcium status; the supplement provided less than 300% of the recommended dietary allowance (RDA) of these nutrients. In contrast, blood concentrations of thiamin, riboflavin, vitamins B-6 and B-12, pantothenate, and biotin increased significantly (P less than 0.05) by 6 wk to values that were maintained until the end of the supplementation. These vitamins were provided in amounts that ranged from 396% (biotin) to 6250% (vitamin B-6) of the RDA. Urinary excretions of these vitamins also increased during supplementation and both blood and urine values returned to presupplementation concentrations at approximately 13.5 wk postsupplementation. PMID- 1728808 TI - Short-term changes in erythrocyte alpha-tocopherol content of vitamin E-deficient patients with cystic fibrosis. AB - Polyunsaturated fatty acids of biomembranes are a major target of lipid peroxidation. In vitamin E deficiency an efficient delivery of a high oral loading dose of all-rac-alpha-tocopheryl acetate to erythrocyte membranes could provide an early onset antioxidative effect. We investigated short-term changes in erythrocyte alpha-tocopherol after a single oral dose of 100 mg all-rac-alpha tocopheryl acetate/kg in 10 vitamin E-deficient cystic fibrosis (CF) patients. Over 24 h, erythrocyte alpha-tocopherol increased 68% to 420% of preloading concentrations. With two exceptions, peak values were achieved 12 or 24 h after administration, which was 3-18 h later than peak plasma concentrations. Separate median-based curve estimates for the changes in erythrocyte alpha-tocopherol for five patients with and five without associated cholestatic liver disease were obtained. Cross-sectional test results revealed significantly lower erythrocyte alpha-tocopherol for the 9- and 24-h observations for patients with cholestatic liver disease compared with those without. Oral all-rac-alpha-tocopheryl acetate can be rapidly incorporated into erythrocyte membranes in vitamin E-deficient CF patients. PMID- 1728809 TI - Hair chromium content of women with gestational diabetes compared with nondiabetic pregnant women. AB - Hair chromium concentration (HCC) of normal and diabetic pregnant women was determined by atomic-absorption spectroscopy. For nondiabetic pregnant women the value from 68 hair samples was 472 +/- 61 ng/g (mean +/- 95% CI); for gestational diabetics it was 734 +/- 155 ng/g from 42 hair samples. The difference was highly significant (P less than 0.005). Intermediate hair chromium concentrations were observed in 20 pregnant women with pregestational, overt diabetes mellitus (mean: 575 +/- 182 ng/g). Fifty-two women had a second hair sample taken later during pregnancy that showed a significant decrease in HCC (P less than 0.05). However, this decrease was confirmed only for the diabetic pregnant group. Age and parity did not influence the HCC. The data suggest that impaired utilization of chromium may be a possible etiology for gestational diabetes mellitus. PMID- 1728810 TI - Nutritional assessment and management in cystic fibrosis: a consensus report. The Consensus Committee. AB - This report is a summary of a meeting convened by the Cystic Fibrosis Foundation to develop a consensus among nutrition specialists and cystic fibrosis care givers regarding optimal nutritional management of patients with cystic fibrosis. The first section of the report provides a rationale for emphasizing nutritional management of this genetic disorder. The multiple factors causing malnutrition and a negative energy balance are outlined. The second section provides guidelines for routine assessment of nutrition in these patients. Five categories of nutritional status are defined based on ideal weight for height, age, and gender. These categories are used to formulate a graded response for nutritional intervention. Recommendations are provided for routine dietary supplements, vitamin supplements, and pancreatic enzyme replacement. The primary aim of this report is to educate clinicians as to the importance of frequent assessments and early intervention. PMID- 1728811 TI - A critical appraisal of the usefulness of perioperative nutritional support. AB - Preoperative malnutrition is often associated with poor postoperative outcome, yet there is no consensus about whether perioperative nutritional support reduces postoperative complications to the level occurring in well-nourished patients undergoing similar procedures. This is partly because reports evaluating effect of perioperative nutritional support on postoperative outcome vary widely in number of patients studied, primary diagnosis, and duration and quality of perioperative nutritional support. These concerns warrant caution in interpreting reported results, even of randomized studies. However, analysis of published reports suggests that when total parenteral nutrition (TPN) is given to malnourished patients in adequate amounts for greater than or equal to 7-15 d preoperatively, significant improvements in both nutritional status and postoperative clinical outcome are likely to occur. Preoperative total enteral nutrition (TEN) is as effective as TPN in improving postoperative clinical outcome. Postoperative TPN, TEN, and ad libitum oral nutrition are equally effective in reducing postoperative complications. Potential candidates for surgery for whom prompt initiation of preoperative TPN or TEN may reduce operative morbidity and mortality irrespective of nutritional status can be identified on admission. PMID- 1728812 TI - The pathogenesis of homocysteinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. AB - A unified, biochemical hypothesis is proposed to explain the pathogenesis of homocysteinemia. This hypothesis is based on the existence of coordinate regulation by S-adenosylmethionine (SAM) of the partitioning of homocysteine between de novo methionine synthesis and catabolism through cystathionine synthesis. This coordination, which serves to modulate the cellular concentration of homocysteine based on the requirements for methionine, is impaired in homocysteinemia. This hypothesis is evaluated in the context of the conditions known to be associated with homocysteinemia, including enzymatic defects and vitamin deficiencies. The novelty of the hypothesis is the assertion that impairment of one homocysteine metabolic pathway must lead to the impairment of the other homocysteine metabolic pathway to cause homocysteinemia. This extends the simplistic view that a block of only one of the pathways is sufficient to cause homocysteinemia. PMID- 1728813 TI - Energy expenditure under free-living conditions in normal-weight and overweight women. AB - Total energy expenditure under free-living conditions of 12 normal-weight and 26 overweight women was determined with the 2H2(18)O method. Overweight women tended to expend more energy (mean +/- SD, 11.20 +/- 1.79 MJ/d) than normal-weight women (9.46 +/- 0.87 MJ/d, P less than 0.005). Approximately half of this effect was explained by an increase in basal metabolic rate (BMR) in the overweight group compared with the normal-weight group (6.47 +/- 0.74 vs 5.68 +/- 0.39 MJ/d, respectively, P less than 0.005) and the other half by an increase in above-basal energy expenditure (4.73 +/- 1.49 vs 3.78 +/- 0.94 MJ/d, P less than 0.05). Total energy expenditure was approximately 1.7 times the BMR in both groups. After adjusting energy expenditure for weight or lean body mass by analysis of covariance, there was no significant difference between normal-weight and overweight groups. We conclude that most overweight subjects must consume more energy than lean subjects to maintain their excess weight, although some could maintain their obesity without eating more than lean subjects. PMID- 1728814 TI - Dietary fish and serum lipoproteins. PMID- 1728815 TI - Additional erroneous nomograms for BMI. PMID- 1728816 TI - Statistical estimation of dietary parameters: implications of patterns in within subject variation--a case study of sampling strategies. AB - Patterns within intraindividual variation in energy intake were described previously. Using case studies based on the same Beltsville One-Year Dietary Intake Study data set, we examined the interaction between random and nonrandom variation and the choice of sampling strategy in estimation of individuals' usual intakes over 1 y. Mean intake estimates derived from adjacent-day samples were less reliable and more likely to be biased than were those based on randomly selected days. A finite adjacent-day sample fails to encompass longer-term trends. Because adjacent-day samples underestimate true within-subject variation, by customary tests they appear more reliable. This may present an interpretational problem. Comparisons of random weekend and week-day samples confirm that failure to proportionately sample both will bias the estimation of the usual (1-y mean) intake and the within-subject variance. PMID- 1728817 TI - Potential regulators of feeding behavior in anorexia nervosa. AB - We recruited 10 patients with anorexia nervosa and 6 age- and height-matched control subjects. Basal and postprandial concentrations of glucose, insulin, cholesterol, amino acids, gastrin, and pancreatic polypeptide (PP) were measured in response to a standard mixed meal. The only satiety signal that was significantly different between the anorectic group and the control group was PP (P less than 0.001). Tryptophan-LNAA and tyrosine-LNAA ratios were not significantly different in the two groups; however, there was a trend toward a lower tryptophan-LNAA ratio in the anorectic group. Gastrin concentrations were significantly decreased in the anorectic group (P less than 0.001) as were basal insulin concentrations (P less than 0.05). Decreased gastrin concentrations may play a role in the gastric symptoms associated with anorexia nervosa. Previous findings that PP release is diminished in obesity, together with the present findings of PP increase in anorexia nervosa, suggest that this peptide may play a role in appetite control mechanisms. PMID- 1728818 TI - Short-term effects of different amounts of protein, fats, and carbohydrates on satiety. AB - This study investigated the satiating efficiencies of proteins, fats, and carbohydrates (CHOs). Twenty-nine female, normal-weight subjects each received 10 liquid breakfasts, which varied in energy and macronutrient contents. Besides a zero condition [0.3 MJ (8 kcal)], there were three energy levels [0.42, 1.05, and 1.67 MJ (100, 250, and 400 kcal)] combined with three dominant sources of macronutrients (99% of energy from CHO, 92% of energy from fat, and 77% of energy from protein). After breakfast the subjects were not allowed to eat or drink (except water) for 3.5 h. They then recorded their voluntary food intake for the remainder of the day. Subjects also rated their subjective feelings concerning food intake on five different types of appetite. The results showed that neither energy content nor macronutrient composition of the liquid breakfasts had any effect on energy and macronutrient intake during lunch and the remainder of the day. Ratings of different types of appetite showed an increasing satiating effect with increasing energy content of the breakfasts. Proteins, fats, and CHOs had similar effects on appetite. PMID- 1728819 TI - Plasma cholesterol-lowering potential of edible-oil blends suitable for commercial use. AB - We tested semihardened blends of edible oils, suitable for commercial food manufacture, with a lower-than-conventional saturated fatty acid content, for their effects on plasma cholesterol. Twenty-six mildly hypercholesterolemic men took part in a double-blind crossover experiment in which two test blends were compared with two control dietary periods [which resembled the Australian fat intake: proportions of polyunsaturated, monounsaturated, and saturated fatty acids (PMS) 0.4:0.9:1]. PMS in the test diets was approximately 0.8:1.3:1 and resulted in significantly lower LDL-cholesterol concentrations (reductions of less than or equal to 7.7%). HDL cholesterol and plasma triglyceride were unchanged. The trans fatty acid (mainly elaidic) content of the blends was 16%, raising its contribution to energy by 4% but without apparent effect on LDL and HDL concentrations. Provided the overall ratio of linoleic acid to palmitic acid in commercial edible-oil blends exceeds that in the prevailing national diet, partial hydrogenation will not negate the LDL-lowering potential. PMID- 1728820 TI - Comparison between the effects of dietary saturated (16:0), monounsaturated (18:1), and polyunsaturated (18:2) fatty acids on plasma lipoprotein metabolism in cebus and rhesus monkeys fed cholesterol-free diets. AB - Cebus and rhesus monkeys were fed cholesterol-free diets providing 40% of energy as fat for 6-wk periods. The fats were high-linoleic acid safflower oil (HLSO), high-oleic acid safflower oil (HOSO), or palm oil (PO), rich in polyunsaturated (18:2), monounsaturated (18:1), or saturated (16:0) fatty acids, respectively. In cebus monkeys, plasma cholesterol concentrations during HLSO intake were 17-19% lower than those during HOSO or PO intake, attributed to a decrease in high density lipoprotein (HDL). Plasma triglyceride (TG) and low-density-lipoprotein (LDL) cholesterol concentrations were comparable during all dietary treatments. Sixty-eight percent of total LDL catabolism was receptor mediated in all dietary groups and this was associated with similar apolipoprotein B pool sizes and fractional catabolic rates. Rhesus monkeys revealed similar cholesterol concentrations (total, LDL, and HDL) during all dietary treatments. TG concentrations during PO intake were 34% and 63% higher than those during HOSO and HLSO intakes, respectively. Hence, dietary 16:0 and 18:1 produce similar effects on LDL and HDL metabolism in normocholesterolemic primates. PMID- 1728821 TI - Protein turnover and resting energy expenditure in patients with undernutrition and chronic lung disease. AB - Whole-body protein metabolism was studied in 11 undernourished cystic fibrosis (CF) patient (7 female), 12 normally nourished CF patients (3 female), 7 anorexia nervosa (AN) patients (all female), and 15 normal control subjects (9 female). Protein turnover was studied by the single dose [15N]glycine method and the cumulative excretion of labeled urinary urea and ammonia. Energy metabolism was studied by open-circuit indirect calorimetry. Contrary to previous reports, no differences were found between the protein turnover of CF groups and the normal control group. However, patients with AN had a negative net protein deposition. Resting energy expenditure was significantly reduced in AN patients and increased in CF patients. The gender of CF patients did not affect protein and energy metabolism but fat mass was higher and fat-free mass was lower in CF females. PMID- 1728822 TI - Survival of bifidobacteria ingested via fermented milk during their passage through the human small intestine: an in vivo study using intestinal perfusion. AB - The ability of a strain of Bifidobacterium sp to survive passage through the upper gastrointestinal tract when ingested in fermented milk was investigated in six fasting healthy adults by using in vivo ileal perfusion. After ingestion of 10.0 +/- 0.5 log10 bifidobacteria in 400 g fermented milk, ileal flow of bifidobacteria increased significantly and reached a maximum of 8.8 +/- 0.2 log10 bifidobacteria/h 1.7 +/- 0.4 h after ingestion of fermented milk. The average number of bifidobacteria recovered from the terminal ileum during the 8 h after fermented-milk ingestion was 9.0 +/- 0.1 log10 and constituted 23.5 +/- 10.4% of the number ingested. These results indicate that in healthy adults Bifidobacterium sp survive transit through the gastrointestinal tract when ingested in fermented milk. Further studies are needed to investigate the behavior of these exogenous bacteria in the colonic lumen and to explore their effects on the physiology of the human gastrointestinal tract. PMID- 1728823 TI - Differences in skeletal muscle and bone mineral mass between black and white females and their relevance to estimates of body composition. AB - This study tested the hypothesis that black females have an increase in skeletal muscle and bone mineral mass compared with white females matched for age (+/- 5 y), weight (+/- 2 kg), height (+/- 3 cm), and menstrual status. Conventional [underwater weighing, whole body 40K counting (WBC), 3H2O dilution] and newly developed (dual-photon absorptiometry) techniques were used to provide ethnicity independent estimates of body composition in 28 pairs of matched subjects. Black females had greater appendicular skeletal muscle (P less than 0.001), bone mineral (P less than 0.001), and total body potassium (TBK) (P = 0.05) compared with white females. Two classic coefficients used in body composition research [density of fat-free mass (FFM) for underwater weighing and TBK/FFM for WBC] differed significantly (P less than 0.05) between black and white females; currently applied coefficients underestimated fat in black females. This study confirms that black and white females differ in body composition and that errors in fat estimates occur when ethnicity is not accounted for in body composition models. PMID- 1728824 TI - Glycemic and insulinemic responses after ingestion of ethnic foods by NIDDM and healthy subjects. AB - In an attempt to apply the concept of glycemic index (GI) and insulinemic index (II) to local eating habits, we examined the plasma glucose and insulin responses in subjects with non-insulin-dependent diabetes mellitus (NIDDM) and healthy subjects to five mixed meals of different ethnic origins. All meals contained 50 g carbohydrate and were compared with a 50-g glucose load. The GI was highest for the Polish dish and lowest for the Syrian dish (66 +/- 5.5 vs 24 +/- 5.1). However, the II was the highest for the standard meal and lowest again for the Syrian dish (174 +/- 27 vs 66 +/- 25). A high correlation was found between the area under the glucose curve and the predicted GI in both NIDDM and healthy subjects. The GI concept is valid and potentially useful in diet planning and legume foods should be incorporated as a carbohydrate source when diets are being planned for NIDDM subjects or individuals with impaired glucose tolerance. PMID- 1728826 TI - Serotonin and the biology of feeding. AB - There is good evidence that the experimental manipulation of serotonin causes changes in feeding behavior and that adjustments in feeding and in the nutritional supply bring about responses in the level or activity of serotonin. These data suggest that 5-HT systems in the body mediate nutritional input and the drive to feed. In addition, it is known that serotonin is a phylogenetically primitive neurotransmitter, which may therefore occupy a central role in the relationship between food and brain organization. A framework can be developed by considering the interrelationships among feeding processes (operations of the satiety cascade), peripheral physiological mechanisms, and brain serotonin systems. Two key issues are how nutritional information is transcribed onto brain 5-HT systems and the nature of this information. The neuroanatomical distribution of 5-HT neurons occupy an appropriate position in which to coordinate peripheral physiological and metabolic information, environmental features, and the behavioral response. PMID- 1728825 TI - A survey of the opinions of obesity experts on the causes and treatment of obesity. AB - A survey of opinions on the causes and effectiveness of treatment of obesity was carried out on 50 physicians and scientists involved in obesity research. Responses were grouped by region (Europe, North America, and United Kingdom), sex, age (30-50 and greater than 50 y) and degree (MD or PhD). Genetic factors were considered the most important causes of obesity overall. Females viewed lack of physical activity, carbohydrate craving, and weight cycling as significantly more important causes than did their male colleagues and viewed exercise as a more effective treatment. There were regional variations in the assessment of the importance of metabolic defects and weight cycling as causes of obesity and in the usefulness of diet in the treatment of obesity. The older group of respondents rated low-fat diet more highly as a treatment than did their younger colleagues. All groups viewed serotonergic and thermogenic drugs as effective treatments whose usefulness would increase during the next 10 years. PMID- 1728827 TI - Progress report on the anorexia induced by drugs believed to mimic some of the effects of serotonin on the central nervous system. AB - Some agents that increase serotoninergic transmission in the brain show anorectic activity at doses that do not interfere with the behavior of rats and other animal species. These agents reduce food intake by a mechanism that clearly differs from that involved in the anorectic activity of d-amphetamine. d Fenfluramine, fluoxetine, and sertraline are three drugs that have already been tested and are used in man. These compounds accumulate in the brain and are metabolized through N-dealkylation. They affect the uptake and release of serotonin at different concentrations, with mechanisms that do not completely overlap. There is pharmacological evidence that d-fenfluramine and sertraline exert their anorectic activity by enhancing the stimulation of 5-HT1nonA receptors whereas fluoxetine seems to affect at anorectic doses both serotoninergic and dopaminergic systems. The role of serotonin in controlling food intake will be discussed, and the effects of agents that reduce serotoninergic transmission will also be considered. PMID- 1728828 TI - Metabolic consequences of fenfluramine for the control of body weight. AB - The chronic ingestion of fenfluramine results in a sustained depression in body weight despite the return of ad libitum food intake to normal levels. This chronic suppression of body weight is immediately reversed after discontinuation of the drug treatment. Such a phenomenon indicates that the drug must increase metabolic rate. However, studies in both humans and animals have failed to demonstrate an increase in metabolic rate after the administration of the drug. Instead, fenfluramine appears to potentiate the expenditure of energy whenever increases in energy expenditure occur. Fenfluramine potentiates the thermic effect of food (TEF) both in animals as well as in humans. Moreover, the energy cost of locomotor behavior also appears to be potentiated by this drug. Most importantly from a therapeutic perspective, unlike the anorectic effect of fenfluramine, tolerance does not appear to develop to its ability to potentiate energy expenditure. PMID- 1728829 TI - Clinical studies with d-fenfluramine. AB - d-Fenfluramine (dF) (15 mg twice daily) has been studied in controlled trials in human obesity and has been shown to increase adherence to diet, to enhance its efficacy, and most importantly, to prevent weight regain when continued over 1 y. Few side effects, mostly transient, have been observed. A long-term use of dF in the management of some obese patients could be foreseen. Additionally, evidence that dF improves eating symptoms and dysphoric impairments in obese cravers, premenstrual syndrome, seasonal affective disorder, and smoking withdrawal syndrome has been presented. PMID- 1728830 TI - Preclinical pharmacology of fluoxetine, a serotonergic drug for weight loss. AB - Fluoxetine selectively inhibits serotonin uptake in vitro and in vivo and thus enhances serotonergic function, leading to a decrease in food intake beginning with the first dose and a decrease in body weight or in weight gain after multiple doses of fluoxetine. Fluoxetine and other drugs that increase serotonergic function decrease food intake with characteristics that make them attractive for use in weight reduction. In rats, for instance, fluoxetine and other serotonergic drugs suppress stress-induced eating, suppress carbohydrate consumption selectively, and suppress insulin-induced hyperphagia. Fluoxetine and other serotonergic drugs do not cause amphetamine-like behavioral stimulation in animals and have no known abuse or addiction liability. In obese yellow mice and in normal mice, as in rats, fluoxetine causes a sustained decrease in food intake and body weight. The pharmacologic effects of fluoxetine in animals suggest its potential use in weight-reduction programs in obese humans. PMID- 1728831 TI - Clinical studies with fluoxetine in obesity. AB - Fluoxetine is a highly specific serotonin reuptake inhibitor. In studies that used a dose of 60 mg once daily, fluoxetine-treated patients consistently had greater weight loss than placebo-treated patients. In six double-blind, placebo controlled studies of 6-8 wk duration, mean weight changes on fluoxetine were approximately 0.5 kg/wk. Longer term studies have shown maximum mean weight loss to occur at 12-20 wk of therapy. Studies have consistently shown improvements in indices of glycemic control as well as weight loss in obese diabetic patients. Safety analysis has been performed on data from 3491 obese patients in controlled clinical trials of up to 52 wk duration. Adverse events with an incidence of greater than 5%, which were reported significantly more frequently by fluoxetine treated patients, were headache, asthenia, nausea, diarrhea, somnolence, insomnia, nervousness, sweating, and tremor. Fluoxetine is effective, well tolerated, and safe in the treatment of obesity and obese diabetics. PMID- 1728832 TI - Sertraline, a serotonin-uptake inhibitor, reduces food intake and body weight in lean rats and genetically obese mice. AB - Sertraline was found to inhibit weight gain and decrease food intake without affecting locomotion in rats and genetically obese (ob/ob) mice. Doses of 10, 17.8, and 32 mg/kg, administered intraperitoneally, (bid) significantly reduced the time rats spent in contact with their feeders and body weight in a dose related manner. During a 5-d bid treatment regimen, vehicle-treated rats gained 37 +/- 3 g (mean +/- SEM), whereas animals treated with 32 mg sertraline/kg lost 34 +/- 4 g. The effects of sertraline on feeding and body weight in rats appeared to be specific because locomotor activity was not altered. In ob/ob mice, sertraline (44 mg/kg, ip, bid) lowered body weight relative to vehicle-treated controls for the duration of a 12-d study. There was no evidence for tolerance to the hypophagic and weight-loss effects of sertraline during either of the chronic dosing studies. These results suggest a potential role for sertraline in the treatment of human obesity. PMID- 1728833 TI - Appraisal of the clinical value of serotoninergic drugs. AB - Principal objectives in obesity management comprise the prevention of weight gain, the promotion of weight loss, and the treatment of obesity-related complications, including diabetes, hypertension, and depression. Serotonin agonists reduce food intake. The resultant weight loss is variable and there appears to be no way of predicting good responders, nor is there evidence that additional weight loss attributable to drug therapy is sustained once treatment is discontinued, although nonpharmacological strategies for preventing weight regain are worthy of exploration. Serotonin agonists are of clinical value if there is a short-term need for weight reduction or if long-term pharmacotherapy can be justified. This implies that sometimes the dangers of the obese state outweigh the potential hazards of drug treatment. Clearly, if the same agent also improves diabetic control, blood pressure, or depression then a longer term usage is more readily justified. The extent to which this may be achieved by the currently available 5-HT agonists is discussed. PMID- 1728834 TI - Clinical and basic aspects of an anorexiant, mazindol, as an antiobesity agent in Japan. AB - The Japanese Mazindol study group investigated the action of an anorexiant, mazindol, and found that it reduced food intake by directly suppressing neurons in the lateral hypothalamus, inhibited gastric acid secretion, increased motor activity, decreased glucose absorption, and inhibited insulin secretion. It thus appears that the main effect of mazindol is to decrease food intake through suppressing feeding centers in the hypothalamus. A multicenter open study of mazindol in Japan revealed that loss of body weight and relative body weight in 14 wk were 4.6 kg and 9.2%, respectively, with suppression of appetite in the majority of obese patients. A multicenter double-blind study demonstrated that mazindol was superior to the placebo in the treatment of simple obesity. We also suggest that mazindol is effective in the maintenance of reduced body weight after obesity therapy and in the treatment of obesity-related diseases such as diabetes, hypertension, or hyperlipidemia. PMID- 1728835 TI - Phenylpropanolamine and blood pressure: a review of prospective studies. AB - The use of phenylpropanolamine (PPA) as an anorectic has provoked commentary and disagreement. Its use in the last decade has been associated with a series of adverse clinical events. As in all case reports, these associations may be noncausal, particularly in light of PPAs extensive use. We have reviewed prospective clinical trials in which the administration of PPA was planned to assess impact on blood pressure. Many of these employ sedentary, healthy volunteers but also included are studies of overweight, moderately hypertensive, and ambulatory subjects. An analysis of such studies leads us to believe that PPA is an appropriately marketed over-the-counter drug, with an acceptable margin of safety. Further, we have reanalyzed our own earlier published data, which indicate that the margin of safety may actually be increased in subjects with elevated basal sympathetic tone; eg, those who are overweight and those with slight elevations of arterial blood pressure. PMID- 1728836 TI - Centrally acting anorectic drugs: a clinical perspective. AB - This paper reviews the anorectic activity and effectiveness of catecholamine and serotoninergic anorectic drugs in the management of obesity. It discusses the clinical implications of the experimental findings and suggests prescribing strategies for effective long-term therapeutic benefit. The authors advocate that there is a role for the recently developed serotonin-mediated anorectic compounds in the treatment of obesity, particularly in those individuals with abnormal glucose tolerance or who are hypertensive. PMID- 1728837 TI - A brief overview of human energy metabolism and its relationship to essential obesity. AB - Twenty-four hour energy expenditure (24EE) can be measured in a respiratory chamber. 24EE is comprised of the basal metabolic rate, the thermic effect of food, and the energy cost of physical activity. The major determinant of 24EE, fat-free mass, accounts for approximately 80% of the variance observed between individuals. Genetic factors seem to be the cause of the familial aggregation of 24EE in man. The variability of 24EE for a given body size and composition is of importance because a low metabolic rate is a major risk factor for weight gain in man. There is increasing evidence that obesity, often an inherited disorder, cannot always be attributed to gluttony and sloth. Similar to the need to treat essential hypertension, there is a need to treat a disorder perhaps best called essential obesity. PMID- 1728838 TI - Peptides affect the intake of specific nutrients and the sympathetic nervous system. AB - Food intake can be increased or decreased after either central or peripheral administration of peptides. Galanin, neuropeptide Y, opioid peptides, growth hormone-releasing hormone, and desacetyl-melanocyte stimulating hormone increase food intake whereas insulin, glucagon, cholecystokinin, anorectin, corticotropin releasing hormone, neurotensin, bombesin, cyclo-his-pro, and thyrotropin releasing hormone reduce food intake. Many of these peptides have reciprocal effects on food intake and sympathetic activity with those peptides that stimulate food intake reducing sympathetic activity and vice versa. In addition, neuropeptide Y specifically increases carbohydrate intake. Galanin and opioid peptides on the other hand increase fat intake whereas enterostatin reduces fat intake. Glucagon decreases protein intake. The effect of peptides on specific nutrients suggests that peptides may work in part by modulating basic feeding mechanisms to lead to the selection of specific nutrients from the diet. This hypothesis might be called a nutrient-specific model of peptide-induced food intake. PMID- 1728839 TI - Feeding modulation by pentose and hexose analogues. AB - D-Glucosamine (GlcN), N-acetyl-D-glucosamine (GlcNAc) and 2,5-anhydro-D-mannitol (2,5-AM) were infused into the rat third cerebroventricle (icv) to compare their effects on food intake. GlcN (24 mumols/L) accelerated eating, and concomitantly increased plasma glucose, free fatty acids, and glycerol without affecting plasma insulin. GlcN accelerated lateral hypothalamic (LHA), and reciprocally decreased ventromedial hypothalamic (VMH) neuronal activity. Infusion of 12 mumols GlcNAc icv did not affect feeding, but oral administration (1200 mumols/L) induced feeding. The GlcNAc-induced feeding was completely abolished by bilateral truncal vagotomy. Infusion of 2,5-AM dose-dependently induced feeding (P less than 0.01). A maximal dose (24 mumols/L) did not substantially change plasma glucose or insulin. Unilateral 2,5-AM microinfusion (1.2 mumols/L) into the VMH, but not into the LHA, elicited feeding. The characteristic actions of these analogues are useful to clarify central control of food intake and also as probes to examine relations between feeding modulation and energy metabolism in the central nervous system. PMID- 1728840 TI - Are gut peptides a new class of anorectic agents? AB - In the past 20 years, the mechanisms of the satiating effect of food that terminate a meal have been investigated intensively in rodents and in humans. This research has revealed that three peptides, cholecystokinin, pancreatic glucagon, and bombesin, released by ingested food from the gastrointestinal tract decrease meal size in a specific, dose-related manner without signs of acute toxicity or tolerance. In humans, the three peptides decrease meal size without decreasing the reported pleasure or satisfaction of the meal. Although their chemical structure and specific effect justify calling these peptides a new class of anorectic agents, not enough work has been done to evaluate their efficacy for weight loss in obese humans or their safety when administered for months. PMID- 1728841 TI - Potent and sustained satiety actions of a cholecystokinin octapeptide analogue. AB - The relative ability of a norleucine substituted cholecystokinin (CCK) analogue, U-67827E, to interact with CCK receptors and to inhibit food intake was examined across a variety of paradigms. U-67827E and CCK had identical in vitro potencies as demonstrated by their ability to induce pyloric contractions or competitively inhibit [125I]CCK-8 binding to type A and B CCK receptors. However, the in vivo potency of U-67827E was significantly greater than that of CCK-8. In rats, U 67827E inhibited food intake with 10-100 times the potency of CCK. In rhesus monkeys, U-67827E produced significantly greater inhibitions of daily food intake and did so in a dose-dependent manner with no evidence of compensation or tolerance. U-67827E also inhibited gastric emptying for significantly longer durations than CCK. Together these results demonstrate that CCK analogues with increased in vivo bioavailability can affect food intake beyond a single meal. PMID- 1728842 TI - CCK antagonists and CCK-monoamine interactions in the control of satiety. AB - The introduction of potent cholecystokinin (CCK) receptor antagonists, selective for either the CCK-A or the CCK-B subtype, has provided a great impetus to the study of activity of endogenous CCK in relation to the control of feeding. This paper reviews experiments in which devazepide (a selective CCK-A receptor antagonist) and L-365,260 (a selective CCK-B-gastrin receptor antagonist) have been used. Both compounds increase food consumption (under certain conditions) and postpone the onset of satiety. L-365,260 is the more potent, suggesting a role for central CCK-B type receptors in satiety. In addition, use of CCK antagonists permits the study of important functional interactions between CCK and other neurochemical factors that serve to control feeding. Thus, devazepide, but not L-365,260, blocked the anorectic effect of either d-fenfluramine or serotonin. Hence, CCK-A type receptors appear to be involved in the anorectic effect of these drugs. This result serves as an example to illustrate a principle of cooperativity in the satiety-inducing effects of diverse neurochemical signals. PMID- 1728843 TI - A clinical perspective on peptides and food intake. AB - Viewed from a clinical perspective, it is difficult to generate enthusiasm for the likelihood of finding a peptide that could be helpful in the treatment of obesity. A deeper understanding of obesity, as will emerge from molecular biology, is more likely to point the way to a useful peptide than further evaluations of the clinical dilemmas posed by obesity. However, a clinical perspective may be useful in pointing the way to a system that needs to be examined by molecular biology and also to inject caution in the evaluation of early findings when peptides are used in treatment. PMID- 1728844 TI - Physiology and pathophysiology of intestinal absorption. AB - Final digestion and absorption of carbohydrates (CHO) occur after intraluminal hydrolysis by pancreatic alpha-amylase at the surface of the mucosal membrane in close relationship between disaccharide hydrolysis and the glucalogue carrier system. In general, Na(+)-dependent transport is the rate-limiting step of CHO absorption. The rate of absorption is determined by mode of ingestion, chemical composition of the meal, gastric emptying, pancreatic digestion, intestinal digestion and absorption, and intestinal motility. A delay of absorption of CHO may be achieved by dietary fibers, alpha-amylase inhibitors, or alpha-glucosidase inhibitors. Final protein digestion is achieved by a dual mechanism: intact peptide absorption with subsequent intracellular hydrolysis to free amino acids (AA) and membrane hydrolysis of peptides followed by absorption of free AA. More complex is the mechanism of lipid absorption: emulsification, lipolysis, micellar formation, membrane translocation, intracellular resynthesis, chylomicron formation, and lymphatic drainage. The most critical steps in lipid digestion are lipolysis and micellar formation. PMID- 1728845 TI - Initial studies in humans with the novel gastrointestinal lipase inhibitor Ro 18 0647 (tetrahydrolipstatin). AB - Excessive intake of dietary fat contributes to the development and maintenance of both obesity and hyperlipidemia. Inhibition of gastrointestinal lipases could decrease the amount of ingested fat that is absorbed systemically by preventing the hydrolysis of triglycerides. Ro 18-0647, a chemically synthesized derivative of the natural product lipstatin, inhibits the action of gastrointestinal lipases. Initial studies in humans have shown that Ro 18-0647 can reliably increase fecal fat excretion. Ro 18-0647 has also been shown to be well tolerated in the majority of normal volunteers and obese patients studied. Further research must be conducted to determine whether clinical endpoints of weight loss or cholesterol lowering can be produced by using this new pharmacologic principle. PMID- 1728846 TI - Effect of an intestinal disaccharidase inhibitor (AO-128) on obesity and diabetes. AB - A new disaccharidase inhibitor, AO-128, showed 190-3900-fold more potent inhibition of purified rat small intestine sucrase-isomaltase (S-1) complex and 23-33-fold more potent inhibition of semipurified porcine small intestine disaccharidases than acarbose. AO-128 suppressed elevation of the blood glucose concentration after oral sucrose, maltose, and starch, but not after oral glucose, fructose, and lactose. The chronic addition of AO-128 to the diet produced antiobesity and antidiabetic actions in obese and/or diabetic animals. Undesirable side effects, such as diarrhea and soft feces, were observed only for the first 5-7 d and suppression of intestinal disaccharidase activities was observed even at the end of the experiment, suggesting that the suppressive or delaying effect of AO-128 on elevation of the postprandial blood glucose concentrations is involved in reduction in body weight gain and prevention and/or amelioration of the diabetic state. Thus, AO-128 is useful as an adjunct to the dietary management of obesity and diabetes. PMID- 1728847 TI - Pharmacological treatment of obesity: digestion and absorption inhibitors clinical perspective. AB - Dependent on the dosages used, digestion and absorption inhibitors or disaccharidase inhibitors, such as Acarbose, might cause malabsorption of nutrients, and hence, among other effects, affect caloric balances. This negative effect on caloric balance has actually been well documented in animal experimentation. However, in nondiabetic subjects with excessive degrees of obesity, no consistent weight reduction could be induced by disaccharidase inhibitors. Subsequently, Acarbose has been advocated for type 2 diabetic patients in dosages that might reduce postprandial hyperglycemia and insulinemia, whereas significant degrees of malabsorption should be excluded. At these dosages of the drug, there is no clinical perspective with regard to weight-reducing (side) effects of disaccharidase inhibitors. Whether a hypothetical diminution of serum insulin daily profiles during Acarbose treatment in obese type 2 diabetic patients might contribute to a normalization of the metabolic syndrome and to a facilitation of weight-reducing efforts remains speculative. At present, there does not seem to be much rationale in trying to exploit digestion and/or absorption inhibitors for weight-reduction therapies in obesity, unless they are used to enforce a negative caloric balance by malabsorption of nutrients. PMID- 1728848 TI - Toward a unified concept of vascular smooth muscle response to injury. PMID- 1728849 TI - Cost analysis. Bedside blood glucose testing. AB - Cost analysis of the bedside blood glucose testing program at a Veterans Affairs medical center indicated a per-test cost of $11.50, as opposed to the conventional laboratory testing cost for serum glucose of $3.19. Extrapolated to the 172 Veterans Affairs hospitals, the extra cost of the procedure is estimated to be in excess of $3 million each year. PMID- 1728850 TI - Prevalence of non-A,non-B hepatitis/hepatitis C virus antibody in human immunoglobulins. AB - Human intravenous immunoglobulins prepared by the cold ethanol fractionation technique of Cohn are considered safe with respect to infectivity. However, there have been several instances of transmission of both hepatitis B and non-A,non-B hepatitis viruses after administration of intravenous immunoglobulins. To determine the prevalence of hepatitis C virus antibody in intravenous immunoglobulins and protein preparations, 30 commercially available products were tested. Using the Abbott enzyme immunoassay for hepatitis C virus antibody, 27 of 30 (90%) immunoglobulins tested positive. The Ortho immunoassay showed that 28 of 30 (93%) were positive, with one discordant result between the Ortho and Abbott assays. An antigen-blocking or neutralization test (Abbott) confirmed the results of the Ortho assay. Bovine, sheep, goat, and horse sera also were tested before and after isolation of animal immunoglobulins. All results on the animal sera were negative, indicating that the fractionation process did not produce false positive results. The high prevalence rate of hepatitis C virus antibody in intravenous immunoglobulins has important implications for follow-up of recipients, selection of serum donors, and implementation of anti-hepatitis C virus testing. PMID- 1728851 TI - Aspergillus terreus as a cause of septic olecranon bursitis. AB - A 72-year-old, non-insulin-dependent diabetic man with a 2-month history of painful right olecranon bursitis was examined after a fall on the sidewalk that resulted in some abrasion of the skin overlying the elbow. Fluid aspirated from the bursa showed growth of Aspergillus terreus, as did tissue from a bursectomy performed 1 week later. Septic bursitis is an uncommon disease that is nearly always caused by Staphylococcus aureus or hemolytic streptococci. Mycotic bursitis is very rare and this is the first reported instance of any Aspergillus species causing septic bursitis. PMID- 1728852 TI - Treponema pallidum and Helicobacter pylori recovered in a case of chronic active gastritis. AB - A 49-year-old man complaining of epigastric pain underwent endoscopy, during which thickened stomach folds below the fundus were observed. Microscopic examination of gastric tissue biopsy specimens revealed chronic active gastritis. Dieterle stain revealed overwhelming numbers of "corkscrew-like" spirochetes. These were proved to be consistent with Treponema pallidum. A comprehensive study of the tissue revealed the added presence of Helicobacter pylori. This appears to be the first case report describing the involvement of H. pylori and T. pallidum together in a case of chronic active gastritis. PMID- 1728853 TI - Lymph node histopathologic findings in cutaneous T-cell lymphoma. A prognostic classification system based on morphologic assessment. AB - The histopathologic features of 251 lymph nodes obtained from 200 patients with various clinical expressions of cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome) were reviewed retrospectively. Lymphomatous involvement, defined as partial or complete effacement of lymph node architecture by malignant cells, was identified in 89 specimens (35%) and was characterized by morphologic variability from case to case. The involved specimens were classified into four major histologic subtypes according to the morphologic appearance of the malignant cells in a manner analogous to a modified Rappaport classification of diffuse non-Hodgkin's lymphomas. Although lymph node involvement was associated with a poor prognosis regardless of histologic subtype, the survival of patients with small cell (cerebriform) subtype was found to be significantly better (median survival time, 40 months) than other subtypes (median survival time, 20 months), possibly because this type of involvement sometimes preceded the development of the more aggressive mixed and large cell subtypes. Dermatopathic lymphadenopathy compared to other reactive patterns had no special prognostic importance other than its more frequent occurrence in black patients and in patients with more extensive skin involvement. PMID- 1728854 TI - Effect of iron status on reticulocyte mean channel fluorescence. AB - Flow cytometric reticulocyte enumeration measures the fluorescence intensity of the reticulocyte population, the reticulocyte mean channel fluorescence. Reticulocyte mean channel fluorescence, used as an indicator of reticulocyte maturation, is directly proportional to the amount of intracellular RNA. Other factors, such as iron stores, may affect reticulocyte mean channel fluorescence. Iron status in normal controls, patients with anemia of chronic disease, and pregnant women was evaluated by hemoglobin, hematocrit, red blood cell indices, iron, total iron-binding capacity, and ferritin. Reticulocyte mean channel fluorescence was significantly elevated (P less than 0.0001) to 85.6 +/- 4.6 (mean +/- 1 standard deviation) in iron-deficient anemic patients and to 81.1 +/- 8.4 in iron-depleted patients compared to healthy individuals (69.7 +/- 2.6). The reticulocyte mean channel fluorescence in anemia of chronic disease was 71.3 +/- 5.8 and was not significantly different from that of normal controls. Reticulocyte mean channel fluorescence showed significant correlations with total iron-binding capacity (P less than 0.0001, r = 0.62) and ferritin (P less than 0.0001, r = 0.40). A possible explanation for these findings, describing differences in cytoplasmic levels of transferrin receptor mRNA, is discussed. PMID- 1728855 TI - Lymph node interdigitating cell sarcoma. A case report. AB - A 54-year-old man was admitted because of right supraclavicular lymphadenopathy of some weeks duration. Computed axial tomography revealed a large multinodular lesion in a supraclavicular lymph node. The patient then had a supraclavicular lymph node biopsy. Light microscopy showed a tumor whose structure was suggestive of an interdigitating cell sarcoma. Enzyme and immunohistochemical analysis showed that the tumor cells possessed membranous adenosine triphosphatase activity, intracytoplasmic S100 protein, surface CD1a and CD4 antigens, and HLA DR antigen. Ultrastructural examination showed that the cells exhibited many interdigitating cytoplasmic extensions, but no Birbeck granules. DNA content analysis of the tumor cells proved that the cells were malignant. These data are consistent with derivation from a lymph node interdigitating cell. PMID- 1728856 TI - Mitotically active leiomyomas of the uterus. AB - A series of 15 uterine smooth muscle tumors with histologic features of typical leiomyomas, except for the presence of mitotic activity exceeding 4 mitotic figures per 10 high-power fields (HPF), were studied. The patients' ages ranged from 25 to 50 years (median, 37 years). At least 60% of the tumors were submucosal. The maximum dimension of the tumors ranged from 1.3 to 8.0 cm (median, 3.8 cm). On gross examination, the tumors appeared to be generally unremarkable. By definition, none had cytologic atypia. Mitosis counts were performed in the most active areas by two methods. Counts ranged from 5 to 15 mitotic figures per 10 HPFs when the highest count in any single set of 10 HPFs was recorded (method A) and from 4.2 to 10.2 mitotic figures per 10 HPFs when the average count from 50 consecutive HPFs was determined (method B). Treatment included hysterectomy in eight patients, myomectomy followed by hysterectomy in one, and myomectomy only in six. All but one patient with evaluable endometria were in the secretory phase of the menstrual cycle. Follow-up periods ranged from 6 months to 10.5 years (mean, 2.5 years; median, 2.1 years). None developed local recurrences or metastases. The benign clinical behavior of such tumors warrants the designation of mitotically active leiomyoma rather than smooth muscle tumor of uncertain malignant potential or low-grade leiomyosarcoma. PMID- 1728857 TI - Splenic lymphoma with circulating villous lymphocytes. Report of a case with immunologic and ultrastructural studies. AB - A case of splenic lymphoma with circulating villous lymphocytes is reported. Short surface cellular expansions were observed on blood and marrow films and by transmission electron microscopy. The immunophenotype was that of mature B cells without CD5, CD10, CD11c, or CD25 expression or tartrate-resistant acid phosphatase. Despite a basophilic plasmacytoid-like cytoplasm, this case of splenic lymphoma with circulating villous lymphocytes differed from splenic immunocytoma in that immunofluorescence and ultrastructure suggested that the neoplastic cells did not possess high levels of intracytoplasmic immunoglobulin. Treatment of cytopenia was best achieved by splenectomy and the total follow-up thus far (30 months) seems to indicate a case of low-grade malignant lymphoma. PMID- 1728858 TI - Emmerich von Haam, M.D., 1904-1988. PMID- 1728859 TI - Evaluation of culture techniques for GABHS. PMID- 1728860 TI - Histopathologic findings of duodenal biopsy specimens in HIV-infected patients with and without diarrhea and malabsorption. AB - The significance of the human immunodeficiency virus (HIV) in the small intestinal lamina propria in patients with the acquired immune deficiency syndrome or conditions related to that syndrome who have chronic diarrhea and malabsorption is unclear. To investigate this issue, upper endoscopy (after a 12- to 16-hour fast) with duodenal biopsy and aspirate was performed in 20 HIV infected seropositive homosexual men referred for diarrhea of more than 8 weeks duration (Group 2) and in 9 HIV-infected homosexual men referred for dysphagia or dyspepsia with no symptoms of malabsorption (Group 1). All biopsy specimens were examined by light microscopy and immunochemical staining with monoclonal antibody against HIV glycoprotein gp41. Electron microscopy was performed in 18 patients in Group 2 and in all patients in Group 1. Immunogold electron microscopy was used as a confirmatory test for identified HIV particles. In addition, D-xylose absorption was measured in all patients after a 25-g dose of D-xylose with measurement of serum D-xylose concentration 1 hour after the dose and measurement of 5-hour urinary D-xylose excretion. Mean serum D-xylose was 35.4 +/- 4.5 mg/dL in Group 1 and 15.8 +/- 2.3 mg/dL in Group 2 (P less than 0.001), whereas mean urine D-xylose was 5.5 +/- 0.6 g in Group 1 and 2.0 +/- 0.4 g in Group 2 (P less than 0.001). Immunoperoxidase for gp41 was positive in 5 (56%) patients in Group 1 and in 12 (60%) patients in Group 2. Lamina propria HIV viral particles were identified by electron microscopy in both patient groups. Viral particles were seen within and adjacent to the cytoplasm of mononuclear cells and were not present in enterocytes or neuroendocrine cells. There were no significant differences in serum or urine D-xylose tests between patients with and without lamina propria HIV. In addition, lipid accumulation in intercellular spaces near the basolateral membrane of adjacent enterocytes was seen in 33% of patients with chronic diarrhea. These findings suggest that lamina propria HIV is not a direct cause of enteropathy in HIV-infected patients and that lymphatic obstruction may be one pathophysiologic mechanism producing this malabsorptive state. PMID- 1728861 TI - Use of the polymerase chain reaction mismatch technique to identify the HLA-DQw8 allele in patients with insulin-dependent diabetes mellitus. AB - The allelic forms of the HLA-DQB gene have been recognized as susceptibility markers of type 1 diabetes mellitus. One of these alleles, the DQw3.2 (DQw8), accounts for the well-documented association of the DQw3 locus with the disease. This report describes a method using the polymerase chain reaction mismatch technique to amplify the three different DQw3 allele sequences in 26 insulin dependent diabetic patients. Primers were designed that differed only at one base at the growing end of their sequences. Using a common oligonucleotide primer located downstream in the first domain of the DQB gene and three other primers located at the other end of the sequence being amplified, it was possible to identify and distinguish the DQw8 allele from the other two closely related alleles (DQw7, DQw9). This method, which could be useful in excluding HLA-related susceptibility to diabetes mellitus, is rapid and nonisotopic, and indeed could be adapted to investigate any DNA sequence polymorphism. PMID- 1728862 TI - Fourier transform infrared spectroscopic analysis of organic oil mastitis. AB - The prevalent use of "medical grade" silicone oils, gels, and elastomers in medical practice has largely obscured the fact that other (illicit) materials also are still in use. Injection fluids used for tissue augmentations are sometimes composed of adulterated silicone oil formulations containing a variety of organic oils. The differential diagnosis between silicone mastitis and other organic oil mastitis in biopsy and mastectomy specimens cannot be resolved by oral history and histopathologic examination alone. In two of three specimens clinically and histopathologically diagnosed initially as silicone mastitis at the authors' institution, examination by Fourier transform infrared spectroscopy revealed that the principal organic oils were mineral oil and soy or olive oil. Only one specimen of mastitis contained silicone oil. In view of the significant prognostic as well as medicolegal implications of the pathologic diagnosis, the generic term "organic oil mastitis" should be used in the absence of additional objective data. PMID- 1728863 TI - Necrotizing squamous/mucinous metaplasia in oncocytic salivary gland tumors. A potential diagnostic problem. AB - Tumor necrosis and squamous and/or mucinous metaplasia was found in 4 of 26 oncocytic salivary gland tumors (24 Warthin's tumors and 2 oncocytomas). The necrosis was extensive in two cases, producing architectural and cytologic atypia sufficient to simulate a squamous carcinoma. In a third tumor, necrotic and inflammatory debris occurred within dilated tumor spaces exhibiting squamous and mucinous foci, suggesting low-grade mucoepidermoid carcinoma. Adequate sampling revealed Warthin's tumors in all four cases. An additional 13 tumors showed incidental foci of squamous metaplasia, often accompanied by stromal scarring but without necrosis. Four of these tumors also had focal mucinous metaplasia. In the adjacent non-neoplastic salivary gland, oncocytic metaplasia of ducts was seen in 22 glands; there were 7 oncocytic cysts and 3 oncocytic nodules. The tumor necrosis and metaplasia are reminiscent of necrotizing sialometaplasia of the minor salivary gland, thought to be ischemic in origin. The etiology of necrotizing squamous/mucinous metaplasia described here and the extent to which oncocytosis contributes to these changes is unknown. Possibly the extravasation of oncocytic and/or mucinous secretions or cyst contents may result in the reactive changes observed. Necrotizing sialometaplasia and squamous/mucinous metaplasia of oncocytic tumors appear to be related only morphologically, but the shared histologic features may be useful in excluding the diagnosis of salivary gland carcinoma. PMID- 1728864 TI - "Tree-barking" of the ascending aorta. Syphilis or systemic lupus erythematosus? AB - A case of unsuspected classical aortitis with "tree-barking" of the ascending aorta in a young woman with systemic lupus erythematosus and inconclusive syphilitic serologic results is presented. At autopsy, no definite diagnostic clues as to syphilitic or lupic aortitis could be obtained. Although infrequent today, the possibility of complicated cardiovascular syphilis still should be considered. Involvement of the ascending aorta by other systemic diseases is well known and can imitate syphilitic aortitis. Although the possibility of two concomitant diseases cannot be ruled out, the young age of the patient, the weak syphilitic serologic result, and active systemic lupus erythematosus demonstrated in other organs favor a diagnosis of lupic aortitis of the ascending aorta. PMID- 1728865 TI - Atrophoderma of Pasini and Pierini. An immunopathologic case study. AB - Atrophoderma of Pasini and Pierini (APP) is a rare and distinctive form of dermal atrophy of uncertain origin. In only one previous report have immunopathologic methods been used to study a case of atrophoderma of Pasini and Pierini, and on the basis of the results obtained it was concluded that immunologic mechanisms were relevant to the pathogenesis of the condition. A detailed investigation of a case of atrophoderma of Pasini and Pierini was conducted using immunofluorescence and immunoperoxidase techniques. The epidermal Langerhans cells were abundant and expressed polyclonal immunoglobulin M on the cell-surface membrane. Biopsy of the same lesion was repeated 6 months later and revealed staining for immunoglobulins A and M and also for C3. This pattern of staining could not be reproduced in a range of other atrophic or scarring cutaneous lesions. Immunophenotypic analysis of the mild perivascular mononuclear cell infiltrate revealed an aberrant T-cell phenotype of uncertain significance. PMID- 1728866 TI - Intranodal myofibroblastoma in a submandibular lymph node. A case report. AB - A typical case of intranodal myofibroblastoma arising in a submandibular lymph node is reported. It provides proof that this tumor can occur in extrainguinal regions and suggests that the submandibular region is a prevalent site. The combination of benign spindle cells and foci of pale fibrillary matrix and hemosiderin granules were characteristic features in the fine-needle aspiration cytologic findings. PMID- 1728867 TI - New developments in the pathologic diagnosis of adrenal cortical neoplasms. A review. AB - In the past decade, our knowledge of neoplasms arising in the adrenal cortex has been expanded greatly. Histologic criteria for distinguishing benign from malignant adrenal cortical neoplasms have been developed. In this review, three systems useful in making this distinction are reviewed and compared. Pathologic indicators of prognosis for adrenal cortical carcinomas have been proposed and these include mitotic rate, stage, surgical resectability, nuclear grade, and tumor size. Of these, mitotic rate appears to be the best indicator. Adrenal cortical carcinomas with a high mitotic rate behave most aggressively. The role of immunohistochemical and DNA content analysis in the diagnosis of adrenal cortical neoplasms is limited. Neoplastic adrenal cortical cells contain a low density of keratins that is often destroyed by routine fixation and paraffin embedding. Thus, the absence of keratins in adrenal cortical neoplasms, particularly carcinomas, in routinely processed tissue should not dissuade the pathologist from making the diagnosis of an epithelial neoplasm. DNA content analysis has revealed an imperfect correlation between DNA ploidy and histologic diagnosis. Some adrenal cortical adenomas contain aneuploid stem cell lines, whereas some adrenal cortical carcinomas have diploid DNA content. Molecular genetic analyses suggest that one or more tumor suppressor genes may be involved in the pathogenesis of adrenal cortical neoplasms. PMID- 1728868 TI - Interleukin-1 alpha as a factor in occlusive vascular disease. AB - The purpose of this study was to examine the recognized ability of interleukin-1 alpha (IL-1 alpha) to alter the functional properties of endothelial cells and to induce replication of smooth muscle and fibroblasts. Such changes could potentially link IL-1 alpha pathogenetically to the myointimal proliferation of vascular sclerosis. Using a peroxidase-immunoperoxidase immunohistochemical method, saphenous veins and internal mammary arteries were examined for the presence of IL-1 alpha before their implantation as aortocoronary bypass grafts. Occluded saphenous vein grafts requiring replacement because of recurrent angina pectoris also were similarly examined. Interleukin-1 alpha, deposited as a scarlet immunoprecipitate, was seen on the luminal surface, in the subintima, and on the spindle cells and infiltrating macrophages in the media of 13 phlebosclerotic veins before surgical insertion. The remaining 30 unchanged veins did not contain IL-1 alpha. Similarly, IL-1 alpha was not identified in any of the 43 sampled internal mammary arteries that were all considered structurally intact. All the 55 bypass grafts, which were examined by biopsy during revascularization and demonstrated diverse histopathologic abnormalities consisting of reduced luminal patency, myointimal proliferation, mononuclear cell infiltration, mural collagenization, and luminal-mural hemorrhage, also contained widely distributed IL-1 alpha. The observation that IL-1 alpha was absent in all of the internal mammary arteries concomitant with maintenance of normal microanatomic structure may help explain, in part, their recognized resistance to reduction in luminal patency and their improved clinical survival when used as coronary artery bypass grafts. Alternatively, the consistent presence of IL-1 alpha in all vessels with sclerotic histopathologic changes suggests that this cytokine may be an important in situ indicator of and a potential participant in vascular injury. Interleukin-1 alpha may be a pathogenetic factor in the complex processes leading to vascular occlusion. PMID- 1728869 TI - Clinical significance of antibodies to bovine and human thrombin and factor V after surgical use of bovine thrombin. AB - Two patients exposed during surgery to bovine thrombin developed antibodies reacting with both bovine and human thrombin and Factor V. Their thrombin times were markedly prolonged with bovine thrombin and modestly prolonged with human thrombin. High titer anti-bovine Factor V created diagnostic confusion in one patient by neutralizing bovine Factor V in a prothrombin assay substrate. Although weaker, antibody activity against human Factor V led to postoperative factor V deficiency in both patients. Such cross-reacting antibodies, recognizable by their higher titer against bovine than human Factor V, should be suspected when a patient surgically exposed to bovine thrombin develops a Factor V anticoagulant after operation. Crossed immunoelectrophoresis of adsorbed bovine plasma with each patient's plasma as antibody revealed many precipitin arcs indicative of immunization of the patients to additional proteins in a commercial thrombin preparation. PMID- 1728870 TI - Cold agglutinin disease after chicken pox. An uncommon complication of a common disease. AB - Chicken pox, although common, is rarely associated with autoimmune hemolytic anemia. Reported here is the case of an 8-year-old boy who was found to have cold agglutinin disease and severe anemia several days after he contracted chicken pox. The cold agglutinin appeared to be a polyclonal immunoglobulin M antibody with anti-Pr specificity. To our knowledge, this is the fifth reported case of autoimmune hemolytic anemia and the second reported case of cold agglutinin disease with anti-Pr specificity to be associated with chicken pox. PMID- 1728871 TI - Quality assurance and reproducibility of high-resolution two-dimensional electrophoresis and silver staining in polyacrylamide gels. AB - Qualitative data from high-resolution two-dimensional electrophoresis (2DE) have become clinically useful in immunoglobulin clonality analysis, in resolution of ambiguities in immunofixation typing of paraproteins, and in genetic typing of serum proteins. Since 1986, the authors have been evaluating the College of American Pathologists Reference Preparation for Serum Proteins (RPSP) as a quality control material for 2DE because (1) it is prepared exclusively from pooled human sera, (2) the pool yields a reference pattern of mixed heterozygosity for genetic markers, and (3) RPSP is widely available as a lyophilized preparation that currently serves in the authors' laboratory as a qualitative quality control preparation and that may become a quantitative quality control material or external quality assessment material for 2DE. Using the ISO-DALT 2DE system and silver-staining, the peptide patterns were examined in 11 lots of RPSP and compared with fresh serum and with each other. Consistent differences in the 2DE pattern between RPSP and fresh serum included the presence of freeze-thaw peptides, the presence of degradation spots of apolipoprotein A-I, and the diminution of apolipoprotein spot intensities in RPSP. All lots of RPSP yielded clear identification of the eight serum proteins used for quality control calculations. Run-to-run coefficients of variation for a single lot of RPSP for four parameters of 2DE spot location and gradient reproducibility were comparable with band location reproducibility for the one-dimensional procedures of serum protein electrophoresis and lactate dehydrogenase isoenzyme electrophoresis. It is concluded that the reproducibility, that is, imprecision, of 2DE is the same as one-dimensional clinical electrophoresis techniques and that either RPSP or pooled fresh serum can serve as a satisfactory internal quality control material. PMID- 1728872 TI - Kaposi's sarcoma involvement of the bone marrow. PMID- 1728873 TI - Oncogene alterations in rat colon tumors induced by N-methyl-N-nitrosourea. AB - The authors assayed oncogene alterations in rat colon tumors induced by the direct-acting chemical carcinogen, N-methyl-N-nitrosourea (MNU). DNA isolated from 34 adenomas and eight carcinomas, as well as adjacent normal colon, of 11 rats was assayed by Southern blotting for restriction fragment length polymorphisms and gene amplifications and deletions in 13 oncogenes known to be involved in human or other animal tumors. In addition to finding apparent point mutations or other small alterations in the fos and abl genes in individual rat colon tumors, the authors observed what appear to be larger alterations (ie, rearrangements, or intragenic insertions or deletions) in the H-ras and myb loci in several tumors. In contrast, no changes in the K-ras, N-ras, myc, N-myc, neu, raf, fms, met, and hst genes were seen in any of these tumors. The frequency of myb gene alterations was higher in carcinomas than in adenomas, suggesting that these changes occurred relatively late during tumorigenesis and were not direct effects of the carcinogen. In addition, the finding of alterations in two or three oncogenes in several MNU-induced rat colon tumors suggests the possibility of more widespread genomic lesions in this model. PMID- 1728874 TI - The oral cephalosporins--a review. AB - There are currently six cephalosporins available in the United States for oral use. These agents possess a wide spectrum of activity, are relatively safe to use, and are effective for certain infections involving the respiratory tract, skin, and urinary tract. Nonetheless, in general, they offer no advantage over other classes of antimicrobial drugs and are far more expensive. As a result, the oral cephalosporins are not agents of first choice for the treatment of infection but should be reserved for use as alternative therapy. PMID- 1728875 TI - Southwestern Internal Medical Conference: New developments in the diagnosis and treatment of sexual precocity. AB - This article covers considerations in the etiology of various forms of precocious puberty and premature sexual development. The normal pubertal process with maturation of the hypothalamic pituitary gonadal axis is reviewed. The differential diagnosis of precocious puberty is discussed with particular emphasis on the difference between gonadotropin-dependent and gonadotropin independent processes. Established therapies and newer medical treatments with their pathophysiologic rationale are considered in detail. PMID- 1728876 TI - Oral labetalol versus oral clonidine in the emergency treatment of severe hypertension. AB - This study was designed to compare the clinical efficacy and safety of oral clonidine and oral labetalol in the treatment of severe hypertension in an emergency department setting. Thirty-six patients with severely elevated blood pressure (mean baseline blood pressure 199/132 mm Hg) without acute end-organ dysfunction were treated with either oral labetalol or oral clonidine in a randomized double-blind prospective study. Labetalol was administered as an initial dose of 200 mg, followed by hourly 200 mg doses up to 1,200 mg. Clonidine was administered as an initial dose of 0.2 mg, followed by hourly 0.1 mg doses up to 0.7 mg. Labetalol reduced diastolic blood pressure in 94% of the patients within 6 hours, with a mean reduction in blood pressure of 54/37 mm Hg. Clonidine reduced diastolic blood pressure in 83% of the patients within 6 hours, with a mean reduction in blood pressure of 57/32 mm Hg. The authors conclude that oral labetalol was comparable to clonidine in efficacy, had a similar incidence of side effects, and offered the clinician a useful alternative for the treatment of severe hypertension in an emergency department setting. Further studies are indicated to determine appropriate dosing regimens for oral labetalol in the acute treatment of severe hypertension. PMID- 1728877 TI - 12th Annual Meeting of the Society of Perinatal Obstetricians. February 3-8, 1992, Orlando, Florida. Abstracts. PMID- 1728878 TI - Fibrous dysplasia of the sphenoid sinus. PMID- 1728879 TI - "Adenocarcinoma, not otherwise specified": a diminishing group of salivary carcinomas. AB - As refinements in classification with clinicopathologic correlations proceed, the "adenocarcinomas, not otherwise specified" (NOS) of salivary tissue are reduced in number. Clinicopathologic entities such as salivary duct carcinoma, terminal duct carcinoma, and epimyoepithelial carcinoma, formerly in the NOS category, are examples of this process. There remain, however, adenocarcinomas of salivary tissues that cannot be accommodated in conventional classifications. They are the least common of salivary carcinomas and manifest a cytoarchitecture ranging from a well-differentiated, low-grade appearance to high-grade, invasive lesions. This report addresses this group of carcinomas for which the NOS designation is still applicable. PMID- 1728880 TI - Meniere's symptom complex: medical treatment. 1934. PMID- 1728881 TI - Annals memorabilia. 1892-1991. PMID- 1728882 TI - Factors contributing to phoneme recognition ability of users of the 22-channel cochlear implant system. AB - A study was carried out to determine which factors contributed to the vowel and consonant recognition ability of recipients of the 22-channel cochlear implant system. On the basis of the statistical analysis, no isolated factor showed a strong correlation with vowel recognition score. On the other hand, negative correlations were found between patients' consonant recognition scores and postoperative psychophysical percepts such as threshold levels and maximum comfortable loudness levels. However, multiple regression analysis also showed that the combination of lower threshold levels, a larger number of usable electrodes, and wider dynamic ranges contributed to higher consonant recognition scores. PMID- 1728883 TI - Management of flap necrosis in cochlear implantation. AB - Skin flap complications are the most commonly reported problems in cochlear implant surgery when the anteriorly based C-shaped flap is used for the incision. If the prosthesis is exposed by flap necrosis, local skin flaps may be used to obtain coverage. Unfortunately, the long-term viability of such flaps may be compromised by the pressure exerted by the transmitter. Two cases of flap necrosis severe enough to expose the prosthesis have been successfully managed by relocating the device to a position superior to the auricle, under healthy skin. In one case the receiver was removed owing to infection and reimplanted at a later date. In this case, the electrode array was left in place at explantation in order to stent the cochlea. The surgical techniques and flap designs for this procedure are presented. No further surgical complications have developed in either case. The devices are performing well for both patients at this time. We have found relocation of the implant a useful technique in the management of major flap necrosis. This technique may also be useful to prevent flap necrosis should excessive flap thinning occur during the implant operation. PMID- 1728884 TI - Model for understanding the influence of some parameters in cochlear implantation. AB - Considering the recognition performance obtained by an implanted patient, the authors have developed models to explain the decrease in performance when the number of open channels on the prosthesis is increased. The French cochlear implant Chorimac was used in this experiment. Two models have been developed. The first is monodimensional and the second is multidimensional. They respectively represent an increase in information and its superposition. Results suggest that for the patient, the superposition factor prevails and is detrimental to recognition. Its elimination should be a major goal. A good selection of electrodes in a relatively small number seems to be the best policy. This is already done in some cochlear implants. Some other parameters in the signal that seem worth being analyzed are introduced. PMID- 1728885 TI - Complications of percutaneous endoscopic gastrostomy in head and neck cancer patients. AB - Percutaneous endoscopic gastrostomy (PEG) has been shown to benefit patients with resectable carcinoma of the head and neck. In order to determine whether patients with existing tumor or postresection anatomic changes of the upper respiratory tract can undergo this procedure with an acceptably low complication rate, 349 patients with attempted PEG were studied. The PEG procedure was successful in 114 of 122 carcinoma patients, as compared to 220 of 227 patients in a control group (patients with neurologic disease). Intraoperative complications preventing PEG placement included pharyngeal or esophageal obstruction, inadequate transillumination of the abdominal wall, and respiratory distress and occurred in 7% of carcinoma patients and 3% of controls. The incidence of airway obstruction during endoscopy was equal between groups (1%). Postoperative complications related to the gastrostomy tube were more frequent in the non-head and neck cancer group (14% versus 5%). Younger age, fewer concomitant medical problems, and better nutritional status may account for this difference. These findings suggest that preoperative, postoperative, and unresectable head and neck cancer patients are appropriate candidates for PEG, and postgastrostomy performance appears superior to that in other patient populations. PMID- 1728886 TI - Comparison of new telescopic video microlaryngoscopic and standard microlaryngoscopic techniques. AB - A new approach to microlaryngeal surgery using a specially designed video microlaryngoscope with a rigid endoscopic telescope and an attached video camera was introduced by Kantor et al in 1990. The ability to video document and perform surgery of the larynx by viewing a high-resolution television image was demonstrated. This method was recommended over the standard microscopic technique for increased visibility with greater depth of field, unimpeded instrument access, instant documentation, and superior teaching value. The authors tried this new method and the standard microscopic technique at the same sitting on a series of patients. This paper will compare these two different techniques and discuss their advantages and disadvantages. Although the new method has many advantages, the standard microscopic technique remains as a valuable method in laryngeal surgery. PMID- 1728887 TI - Lower complication rates associated with bronchial foreign bodies over the last 20 years. AB - A retrospective comparison of all endoscopic bronchial foreign body (BFB) removals performed at Children's Hospital and Medical Center, Seattle, Washington, during two separate 5-year periods is reported. There were 54 patients between July 1, 1964, and June 30, 1969, and 119 patients between July 1, 1984, and June 30, 1989. Bronchoscopic removal of foreign bodies in the late cohort was performed almost exclusively with Hopkins telescope-guided foreign body graspers as opposed to traditional forceps guided by the naked eye in the first group. There were no differences in the average age, foreign body type, anesthetic technique, operative length, or anatomic distribution between cohorts. There were significantly fewer complications in the late cohort than the early. Complication rates increased with the duration of the BFB in situ. There were significantly fewer missed BFBs at initial bronchoscopy in the late cohort (4) than the early (10). Inability to endoscopically remove the BFB resulted in thoracotomy in 3 patients in the early cohort and 1 patient in the late cohort. There was one instance in which foreign body migration from right to left main stem occurred during the delay between diagnosis and operation, resulting in the necessity for emergent bronchoscopy with the patient in extremis. Prompt endoscopy in patients with suspected BFBs using the Hopkins rod bronchoscopic system will result in fewer complications and fewer missed foreign bodies. PMID- 1728888 TI - Multichannel electromyographic observations in spasmodic dysphonia patients and normal control subjects. AB - Spasmodic dysphonia is primarily a disorder of vocalization. Increasing evidence, however, suggests that individuals with this disorder comprise a heterogeneous population characterized by abnormal motor control throughout the vocal tract. Multichannel simultaneous electromyography was performed on 11 spasmodic dysphonia patients and 10 normal awake subjects to investigate both the distribution of neuromotor abnormality within the vocal tract (eg, intrinsic and extrinsic laryngeal muscles, tongue, and palate) and the contribution of activation of higher central nervous system centers to observed abnormality. Experimental tasks ranged from vegetative (quiet breathing) to simple linguistic (short sentences). Digitized electromyographic signals were analyzed to compute the amplitude envelope and extract a set of parameters that represent amplitude characteristics. Electrode insertions were cross-validated by quantitative analysis of patterns of activation across selected reference tasks and by traditional qualitative methods. Between-group differences were found for measures of normalized median and peak token amplitudes. These differences are both task- and measure-dependent. Results highlight the complex and interactive effects of muscle, task, and quantitative measures on between-group differences. PMID- 1728889 TI - Stable internal fixation of fractures of the partially mineralized thyroid cartilage. AB - Thyroid cartilage fractures due to external blunt trauma have typically been thought to occur in patients over the age of 40. Lack of mineralization of the cartilage has been considered to be the protective mechanism. Our experience with laryngeal injuries has demonstrated that younger persons are indeed at risk for thyroid cartilage fractures, and that these injuries may be easily overlooked. Although these fractures do not lead to laryngeal stenosis if untreated, they may cause noticeable phonatory changes. Fixation of these fractures is difficult because of the usual soft character of the unmineralized cartilage, prompting us to adopt a wire-tube fixation technique. This technique has been uniformly successful in restoring the anatomic contour of the thyroid cartilage, and our results appear to justify open reduction of these moderately displaced or angulated thyroid cartilage fractures. PMID- 1728890 TI - Injection and removal of Teflon for unilateral vocal cord paralysis. AB - For over 70 years, reinnervation attempts have been unsuccessful in restoring motion to paralyzed vocal cords, in spite of occasional claims to the contrary. Fortunately, the major defect of unilateral vocal cord paralysis, a soft and breathy voice, can be eliminated if the edge of the paralyzed vocal cord is moved to the midline. This permits the mobile vocal cord to adduct and therefore to vibrate firmly against the edge of the paralyzed vocal cord during phonation, eliminating the air leak between the vocal cords. Teflon injection of the paralyzed vocal cord does this effectively. It is accomplished most easily and reliably via indirect laryngoscopy under local anesthesia, so the effect on the voice can be monitored during the injection. Teflon can be easily removed from the vocal cord via direct laryngoscopy. The disadvantages of trying to medialize the edge of a paralyzed vocal cord via a window in the thyroid cartilage (laryngeal framework surgery) will be discussed. PMID- 1728891 TI - Effect of prior antibiotic treatment on middle ear disease in children. AB - The effect of prior antibiotic treatment on the course of otitis media was assessed in a group of 62 children who experienced 83 episodes of ear infection during 3 years of observation. Bacterial quantitation in middle ear fluids demonstrated a significantly higher colony count in symptomatic children (3.9 x 10(4) +/- 12 bacteria per milliliter) compared to asymptomatic children (6.3 x 10(3) +/- 10 bacteria per milliliter; p = .05). Bacterial counts similarly tended to be higher in children with Streptococcus pneumoniae (4.0 x 10(6) +/- 16 bacteria per milliliter) and Hemophilus influenzae (2.0 x 10(6) +/- 16 bacteria per milliliter), who were more often symptomatic (73% and 55%, respectively, versus 38%) than children with Moraxella catarrhalis (7.9 x 10(3) +/- 2). Antibiotic therapy between 3 and 30 days prior to bacterial diagnosis was associated with a reduction in symptoms from 70% to 38% (p less than .025). However, prior treatment did not statistically reduce bacterial colony counts, although S pneumoniae decreased 90% in the previously treated group. Resistance to ampicillin occurred in 0% of S pneumoniae, 39% of nontypeable H influenzae, and 80% of M catarrhalis subjects without prior treatment and in 0%, 46%, and 100%, respectively, of subjects previously treated (p less than .025). These data suggest that prior treatment has a significant impact on the subsequent course of otitis media in children. PMID- 1728892 TI - Plasma cell granuloma of the middle ear and mastoid. Case report. AB - We present the case of a 37-year-old man with plasma cell granuloma affecting the middle ear and mastoid. At magnetic resonance imaging scan, the lesion appeared as a homogeneously enhancing mass of soft tissue replacing the majority of the mastoid bone and causing vascular compression. After surgical resection, microscopic examination showed predominantly plasmacytes, and histochemical studies confirmed a polyclonal origin consistent with nonneoplastic plasma cell granuloma. We believe this is the first case report of plasma cell granuloma affecting the middle ear and mastoid. PMID- 1728893 TI - Effects of aerosolized dexamethasone on acute subglottic injury. AB - Aerosolized dexamethasone was used in a two-phase study to determine the possible effects on acute subglottic injury in the ferret animal model. In phase 1, equivalent subglottic injuries were made in 10 animals by using the brush technique, and the animals were divided into two groups. The treatment group received aerosolized dexamethasone at 2, 4, and 6 hours postinjury. All animals were examined 2, 4, 6, and 24 hours after the injury. The clinical condition of each animal was evaluated and their airways were measured. The animals were then painlessly killed and the larynges were frozen, sectioned, and photographed at 1 mm intervals. A computer-linked digitizer pad was used to measure the subglottic dimensions. The results show a trend for the treated animals to have a larger subglottic airway as compared to the untreated (control) group. The phase 1 study suggests that there may be an improvement in the subglottic airway when treated acutely with aerosolized dexamethasone. In phase 2, 20 additional animals were studied by using the same methods of injury and treatment as in phase 1. The subglottic airways of these animals were evaluated with histomorphometric analysis on fixed histologic sections. A statistically significant difference was found between the subglottic airways of the treated and untreated animals favoring treatment with aerosolized dexamethasone. Aerosolized dexamethasone appears to be beneficial in preserving the subglottic airway after injury, possibly secondary to decreasing the edema associated with injury. PMID- 1728894 TI - Antibiotic prophylaxis in clean-contaminated head and neck oncologic surgery. AB - The use of antibiotic prophylaxis in head and neck oncologic surgery has greatly reduced the risk of postoperative wound infection and the corresponding increase in morbidity and health care costs. Conversely, inappropriate perioperative use of antibiotics increases costs and risk to patients. Antibiotic prophylaxis is beneficial only in clean-contaminated head and neck surgery; targets are the bacterial flora that commonly inhabit the skin and upper aerodigestive tract, with antibiotics effective against gram-positive aerobic organisms and anaerobic organisms providing the best coverage. Maximum efficacy is achieved with immediate preoperative and short-term (less than 48 hours) postoperative antimicrobial administration in adequate doses. Optimum benefit from prophylaxis in head and neck oncologic surgery depends on appropriate selection and administration of antibiotics in combination with sound, established surgical principles. PMID- 1728895 TI - Otitis media update: pathogenesis and treatment. AB - Otitis media primarily affects children, but can also lead to lifelong sequelae. Middle ear histopathologic changes and clinical manifestations can represent any part of a disease continuum, from acute to recurrent to chronic otitis media. Acute otitis media is most often caused by an acute respiratory viral infection and secondary replication of bacteria in the middle ear space and tissues, leading to symptoms and signs of infection (ie, fever, pain, tympanic membrane erythema). Antimicrobial therapy is the mainstay of management, and clinical response to different antimicrobial drugs appears to be similar. The bacteriologic efficacy of these drugs, however, is quite variable. Clearly, antimicrobial treatment of acute otitis media, which currently is largely empiric, must be fine-tuned on the basis of patient and disease variation. PMID- 1728896 TI - Adenoidectomy and otitis media. AB - Adenoid enlargement has traditionally been considered a factor in otitis media; adenoid size, however, does not appear to be correlated with otitis media occurrence. Presence of pathogenic bacteria in the adenoids of children with otitis media has been shown, and adenoidectomy appears to affect the middle ear primarily by removal of the source of infection in the nasopharynx. Three recent randomized, controlled studies showed the efficacy of adenoidectomy in the treatment of chronic secretory otitis media. In one study comparing no treatment, adenoidectomy, and adenotonsillectomy, a significant benefit was seen with adenoidectomy that was not enhanced by tonsillectomy. Another study that compared adenoidectomy, tympanostomy tubes, and a combination of the two showed a significant reduction in effusion time and less surgical retreatment over 2 years in the two adenoidectomy groups. The third study demonstrated the effect of adenoidectomy in children with recurrent chronic otitis media with effusion after failure of tympanostomy tube insertion. All three studies showed that the effect of adenoidectomy was independent of adenoid size. This review discusses current concepts of adenoid physiology and pathology, the major adenoidectomy studies, and indications for the procedure. PMID- 1728897 TI - Antimicrobial prophylaxis for recurrent acute otitis media. AB - Antimicrobial prophylaxis for recurrent otitis media was first reported in 1960 in an uncontrolled study using a long-acting sulfonamide in Native American children younger than 11 years of age. In subsequent controlled studies using various antimicrobial drugs (primarily aminopenicillins or sulfonamides) subjects receiving prophylaxis continued to have episodes of acute otitis media, but at rates substantially lower than those of controls. More recently, prophylaxis has appeared effective in reducing the number of acute recurrences, but not the cumulative proportion of time with middle ear effusion that was present independent of such recurrences. Although questions remain about choice of drug, optimal dosage schedules, risk of untoward drug reactions, duration of use, and the risk of encouraging the emergence of resistant strains of pathogenic bacteria, antimicrobial prophylaxis currently appears to be the most logical first approach in the management of the child with recurrent otitis media. PMID- 1728898 TI - Sinusitis in infants and children. AB - The major clinical problem in considering a diagnosis of sinusitis is differentiating uncomplicated upper respiratory tract infection from a secondary bacterial infection of the paranasal sinuses that may benefit from antimicrobial therapy. A diagnosis of sinusitis is suggested by presentation with protracted upper respiratory tract symptoms or a cold that is more severe than usual with fever and purulent nasal discharge. Confirmatory tests of sinus disease are transillumination (useful in adolescents if interpretation is confined to the extremes--normal or absent); radiographic findings of opacification, mucous membrane thickening, or an air-fluid level; and sinus aspiration (indicated for severe pain, clinical failures, or complicated disease). When clinical signs and symptoms are accompanied by abnormal radiographic findings, bacteria in high colony count are recovered from the maxillary sinus aspirate in 70% of patients. The common bacterial species recovered from children with acute maxillary sinusitis are Streptococcus pneumoniae, Moraxella (Branhamella) catarrhalis, and Hemophilus influenzae. PMID- 1728899 TI - Surgical management of sinus disease in children. AB - In the past few years, interest in pediatric sinus surgery has increased in response to the introduction of new endoscopic techniques and to mounting societal pressure to resolve the problem of persistent rhinosinusitis in the day care setting. As yet, there are no prospective controlled studies demonstrating efficacy of sinus surgery in children with uncomplicated acute or recurrent acute sinusitis, and medical management overwhelmingly remains the treatment of choice for these patients. Sinus surgery may be indicated, however, for three groups of pediatric patients: those with true chronic disease, those with serious underlying disease states aggravated by recurrent sinusitis, and those with suppurative complications of sinusitis. PMID- 1728900 TI - Medical and surgical management of sinusitis in adults. AB - Sinusitis may be caused by bacteria, viruses, or trauma and may appear in immunosuppressive settings. Acute sinusitis is most commonly diagnosed on the basis of pain and discharge; endoscopic or fiberoptic examination may be helpful in less obvious cases. Radiography can identify maxillary, frontal, and sphenoid sinusitis; transillumination can be used if radiography is undesirable. Culture and Gram stains may help determine the appropriate antibiotic therapy. Surgery may be necessary if the frontal or sphenoid sinus is involved, or if ethmoiditis is progressing to orbital cellulitis. In chronic sinusitis, endoscopic examination and computed tomographic scanning are useful for diagnosis. Chronic sinusitis may be associated with airway disease, aspirin allergy, and such diseases as cystic fibrosis. Antibiotic therapy that acts against anaerobes and beta-lactamase-producing organisms should be chosen. Surgical treatment includes intranasal and external ethmoidectomy, antrostomy, and, on occasion, obliteration of the involved cavity. PMID- 1728901 TI - Antimicrobial use in otolaryngeal infections: general considerations. AB - With the development of numerous new antimicrobials and the improved efficacy of existing agents, more infections are being treated successfully, but the benefits of one agent over another have become an issue of subtle distinctions. Some clinical studies of new drugs have inherent drawbacks in their design and may not yield a comprehensive picture of antimicrobial characteristics in a wide range of patient types and diseases. Studies should therefore be carefully evaluated to determine whether a real advantage exists for a new agent. At Barnes Hospital (St Louis, Missouri), antimicrobials are chosen for the formulary on the basis of efficacy, toxicity, and cost. One or two agents are selected from a group of "therapeutic equivalents." Nonformulary agents or uses must be approved by the infectious disease staff. Evaluation and discussion of therapy with formulary and nonformulary drugs educates house staff, who can then use approved agents with greater knowledge and skill. PMID- 1728902 TI - Etiology and management of pharyngitis and pharyngotonsillitis in children: a current review. AB - Although viruses are the most common causes of childhood throat infections, interest in the etiology of these infections has primarily focused on whether an individual episode is caused by group A beta-hemolytic streptococcus (GABHS), particularly since the recent outbreaks of rheumatic fever in certain areas of the country. Penicillin remains the cornerstone of treatment in GABHS pharyngitis. Early treatment effects prompt clinical improvement and reduces the risk of transmission. Whether early treatment suppresses immunologic response and results in a higher recurrence rate than does delayed treatment is still unknown, but recent evidence suggests that it does not. The causes of persistent GABHS carriage, its clinical importance, and optimal methods of treatment are all still in question. When penicillin treatment does not eradicate carriage, other drugs may be efficacious. In children severely affected with recurrent throat infection, tonsillectomy is generally effective and is sometimes a desirable option. PMID- 1728903 TI - Current indications for tonsillectomy and adenoidectomy. AB - Tonsillectomy and adenoidectomy are currently the most common pediatric surgical procedures performed in the United States. Tonsillectomy may be effective in recurrent acute throat infection (acute tonsillitis), chronic tonsillitis, tonsillar hypertrophy, and peritonsillar abscess. Antimicrobial therapy may also be beneficial. Clinical trials evaluating children with obstructive adenoids are currently being evaluated; anecdotal evidence points to improvement in development and quality of life after surgery. The efficacy of adenoidectomy in paranasal sinusitis has not been evaluated in clinical trials; antimicrobial therapy or the possibility of upper respiratory tract allergy should be considered in such cases. For acute otitis media, recommendations range from no treatment in cases that will abate with time, to anti-microbial prophylaxis, to myringotomy with tympanostomy tube insertion, adenoidectomy with or without tonsillectomy, or a combination of adenoidectomy with myringotomy and/or tympanostomy tubes. The decision for or against otic and/or pharyngeal surgery should be individualized on the basis of severity, duration, and frequency of illness; previous treatment; and risk. PMID- 1728904 TI - Diagnosis and management of anaerobic infections of the head and neck. AB - Anaerobic bacteria are important pathogens in head and neck infections such as chronic otitis media, chronic sinusitis, chronic mastoiditis, head and neck abscesses, cervical adenitis, parotitis, and postoperative infection. Bacteroides sp (Bacteroides melaninogenicus group, Bacteroides oralis, and Bacteroides fragilis group), Peptostreptococcus sp, and Fusobacterium sp predominate. The observed recent increase in the number of beta-lactamase-producing strains of Bacteroides sp isolated in head and neck infections has been associated with increased failure rates of the penicillins in the management of these infections. The pathogenicity of these organisms is expressed through their ability not only to survive penicillin therapy but also to shield penicillin-susceptible pathogens from the drug. Because of these direct and indirect virulent characteristics of anaerobic bacteria, appropriate antimicrobial therapy must be directed against all pathogens in mixed infections. PMID- 1728905 TI - Influenza vaccination. Knowledge, attitudes, and behavior among high-risk outpatients. AB - The Minneapolis and Pittsburgh Veterans Affairs Medical Centers conduct virtually identical institution-wide influenza vaccination programs that include annual educational and publicity mailings to all outpatients. Despite these efforts, 40% to 50% of high-risk outpatients at both centers fail to receive influenza vaccine each year. To assess differences between high-risk vaccine recipients and nonrecipients, a self-administered questionnaire was mailed to 500 randomly selected outpatients from each site. The questionnaire asked about risk factors, vaccination status, and knowledge and attitudes regarding influenza and "flu shots." Patient risk characteristics and vaccination rates in Minneapolis and Pittsburgh were similar with 75.6% and 76.3% reporting high-risk conditions and 65.6% and 56.1% of high-risk respondents reporting influenza vaccination, respectively. High-risk vaccine recipients and nonrecipients had similar knowledge but different attitudes about influenza and "flu shots." Using stepwise logistic regression, factors positively associated with vaccination behavior were: intention to follow physician or nurse recommendations for "flu shots" (odds ratio [OR] = 7.09); previous vaccination behavior (OR = 6.36); and physician or nurse recommendations for a "flu shot" (OR = 4.29). Factors negatively associated with vaccination behavior were difficulty in coming to the medical center (OR = 0.42) and previous side effects from the vaccine (OR = 0.19). These findings suggest areas in need of additional emphasis if influenza vaccination rates are to be improved. PMID- 1728906 TI - Results of nocturnal penile tumescence studies are abnormal in sexually functional diabetic men. AB - Nocturnal penile tumescence (NPT) studies are commonly used in the assessment of sexual dysfunction in diabetic men. While much of the evidence in favor of its use has come from the observation of markedly abnormal NPT in impotent diabetic men, little research has focused on the quality of nocturnal erections in sexually functional diabetics. Ten diabetic men who reported normal daytime sexual function were studied with 4 nights of polysomnography, including NPT assessment. They had significantly diminished NPT profiles when compared with that of an age-matched, nondiabetic, healthy control group. Without controlling for the effect of diabetes on NPT, between 70% and 90% of sexually functional diabetics had NPT profiles in a range that would be classified as indicative of organic sexual dysfunction for a man presenting for evaluation of sexual dysfunction. The finding of NPT abnormalities in a diabetic man should not be taken as evidence for irreversible sexual dysfunction. Rather, the condition of diabetes appears to result in NPT abnormalities, regardless of the adequacy of daytime sexual function. PMID- 1728907 TI - Serum albumin level on admission as a predictor of death, length of stay, and readmission. AB - We studied the serum albumin level within 48 hours of hospitalization for acute illness to predict in-hospital death, length of stay, and readmission in 15,511 patients older than 40 years. Patients with low serum albumin levels (less than 34 g/L), who made up 21% of the population, were more likely to die, had longer hospital stays, and were readmitted sooner and more frequently than patients with normal albumin levels. The in-hospital mortality was 14% among patients with low albumin levels, as compared with 4% among patients with normal levels. Although the serum albumin level was a nonspecific marker, it was a stronger predictor of death, length of stay, and readmission than age. We conclude that the serum albumin level on admission is an important variable that should be incorporated in severity-of-illness measures based on physiologic indexes. PMID- 1728909 TI - Variability of indirect methods used to determine blood pressure. Office vs mean 24-hour automated blood pressures. AB - Blood pressure is a cardiovascular measurement with dynamic characteristics that can be influenced by a number of internal and external factors. The preferred blood pressure determination method would be one that reduces variability between measurements and that reflects the true blood pressure level. In this article, we present the variability of, and agreement between, the blood pressures collected by two indirect methods on the same patients during a hypertensive research project. Data obtained on patients in a typical clinical setting are also provided. Twenty-four-hour diastolic pressures obtained by the automated method demonstrated no regression to a lower mean, while blood pressures obtained casually in the office exhibited such regression. The 95% confidence interval of repeated measures for casual office blood pressure on a patient in a research setting (35/17 mm Hg) or in typical clinic practice (26/19 mm Hg) were similar, while the range of the mean 24-hour automated blood pressure monitoring (21/11 mm Hg) was smaller and demonstrated less variability. The magnitudes of the differences in blood pressures obtained on separate occasions in the same subjects were significantly lower with automated vs casual blood pressure determination methods (7.9/4.6 vs 13.7/7.4 mm Hg for both systolic and diastolic pressures). The agreement (95% confidence interval) between blood pressures obtained by the two methods (19/12 mm Hg) was found to be similar to the repeatability of automated blood pressure monitoring alone, and superior to that for data recorded casually in the office (35/17 mm Hg). Thus, the variability in mean 24-hour automated blood pressures is less than that for casual office blood pressures. The clinician should understand that the variability of blood pressures measured on an individual may be much greater than that reported for populations of hypertensive patients, and must be considered when applying epidemiologic group data to a specific patient. Moreover, any methodology of indirect blood pressure measurement that may reduce the variability and improve repeatability of casual office blood pressures deserves further consideration. PMID- 1728908 TI - The Trial of Antihypertensive Interventions and Management (TAIM) study. Adequate weight loss, alone and combined with drug therapy in the treatment of mild hypertension. AB - This report examines the effect of weight loss, alone and in combination with drugs, on diastolic blood pressure change in the Trial of Antihypertensive Interventions and Management (TAIM), which is a randomized, multicenter, placebo controlled clinical trial of drug and diet combinations in the treatment of mild hypertension among 787 patients. Diastolic blood pressure drop (11.6 mm Hg) at 6 months among those patients who were randomized to weight reduction and placebo drug treatment was greater among those who lost 4.5 kg or more, than the 7-mm Hg drop for those who lost less than 2.25 kg or for the placebo-treated control group, and it was statistically equivalent to the reduction achieved by 25 mg of chlorthalidone or 50 mg of atenolol (11.1- and 12.4-mm Hg drop, respectively). Weight loss potentiated effects of drugs, with reductions of 18.4 mm Hg, for those patients who were taking atenolol and had a 4.5-kg or more weight loss, and of 15.4 mm Hg, for those patients who were taking chlorthalidone and had at least a 2.25-kg weight loss. We concluded that effective weight loss (greater than or equal to 4.5 kg) lowers blood pressure similarly to low-dose drug therapy and potentiates drug effects, with the apparent 4.5-kg threshold being lowered to 2.25 kg for those patients who receive chlorthalidone. PMID- 1728910 TI - A study of chest compression rates during cardiopulmonary resuscitation in humans. The importance of rate-directed chest compressions. AB - A prospective, cross-over trial was performed comparing two different rates of precordial compression using end-tidal carbon dioxide as an indicator of the efficacy of cardiopulmonary resuscitation in 23 adult patients. A second purpose of this study was to determine the effect of audio-prompted, rate-directed chest compressions on the end-tidal carbon dioxide concentrations during cardiopulmonary resuscitation. Patients with cardiac arrest received external chest compressions, initially in the usual fashion without rate direction and then with rhythmic audiotones for rate direction at either 80 compressions per minute or 120 compressions per minute. Nineteen of 23 patients had higher end tidal carbon dioxide levels at the compression rate of 120 per minute. The mean end-tidal carbon dioxide level during compressions of 120 per minute was 15.0 +/- 1.8 mm Hg, slightly but significantly higher than the mean level of 13.0 +/- 1.8 mm Hg at a compression rate of 80 per minute. However, end-tidal carbon dioxide levels increased rather dramatically when audiotones were used to guide the rate of chest compressions. Mean end-tidal carbon dioxide concentration was 8.7 +/- 1.2 mm Hg during standard cardiopulmonary resuscitation immediately before audio prompted, rate-directed chest compression and increased to 14.0 +/- 1.3 mm Hg after the first 60 seconds of audible tones directing compressions. Using end tidal carbon dioxide as an indicator of cardiopulmonary resuscitation efficacy, we conclude that audible rate guidance during chest compressions may improve cardiopulmonary resuscitation performance. PMID- 1728911 TI - Microalbuminuria in a population-based study of diabetes. AB - The prevalence of microalbuminuria in younger-onset diabetic participants in a large population-based study of diabetic retinopathy was determined, and the relationships of microalbuminuria to blood pressure and other risk factors were investigated. Using an agglutination inhibition test (AlbuScreen), the frequency of microalbuminuria was 21.2%. To evaluate the association of several characteristics with the presence of microalbuminuria, multivariate models based on logistic regression were developed. Microalbuminuria was associated with having higher systolic or diastolic blood pressure and higher glycosylated hemoglobin. These findings give further impetus to efforts to reduce controllable risk factors in younger-onset diabetic persons. PMID- 1728912 TI - Optimal management of suspected lower-extremity deep vein thrombosis. An evaluation with cost assessment of 24 management strategies. AB - Traditionally, patients suspected to have lower-extremity deep vein thrombosis have undergone venography, which is invasive, is expensive, and may cause deep vein thrombosis in healthy individuals. Recent studies have shown the safety and efficacy of alternative noninvasive approaches that employ impedance plethysmography or real-time ultrasonography. We compared these tests using decision analysis to model the consequences of 24 different management strategies for ambulatory patients suspected to have deep vein thrombosis. We also calculated the incremental cost per additional life saved for each strategy. Our analysis revealed that the optimal approach was to perform real-time ultrasonography followed by anticoagulation therapy if deep vein thrombosis is found. This approach was both effective and cost saving compared with no testing or treatment. Serial follow-up studies of patients whose initial study suggested no DVT saved additional lives, but at a cost of $390,000 per each additional life saved for patients with one follow-up study and $3.5 million per each additional life saved for patients with a second follow-up study. Venography should play a limited role in the contemporary evaluation of patients suspected to have deep vein thrombosis. Future research should focus on the determination of clinical predictors of patients who should undergo serial examinations. PMID- 1728913 TI - Reversibility of long-standing urinary tract obstruction requiring long-term dialysis. AB - Urinary tract obstruction of longer than 4 to 6 weeks' duration is usually said to be irreversible. Older reports of unilateral obstruction have documented return of kidney function after longer periods of obstruction. The duration of bilateral obstruction compatible with return of life-sustaining renal function is poorly defined. We report herein three cases of long-standing urinary tract obstruction leading to apparent dialysis-dependent end-stage renal disease, where relief of obstruction eventually led to discontinuation of dialysis. PMID- 1728914 TI - Evidence for interference with the intestinal absorption of levothyroxine sodium by aluminum hydroxide. AB - A patient with hypothyroidism who was euthyroid on a fixed-dosage, long-term maintenance regimen of levothyroxine sodium developed persistently elevated serum thyrotropin levels while receiving an aluminum hydroxide-containing antacid. The thyrotropin levels returned to normal shortly after cessation of the antacid therapy. These observations indicate that aluminum hydroxide may interfere with the bioavailability of thyroxine. The thyroid function of patients who are receiving replacement or suppressive thyroxine therapy should be monitored following the commencement of concurrent treatment with medications containing aluminum hydroxide. PMID- 1728915 TI - Carbamazepine-induced cardiac dysfunction. Characterization of two distinct clinical syndromes. AB - A patient with sinus bradycardia and atrioventricular block, induced by carbamazepine, prompted an extensive literature review of all previously reported cases. From the analysis of these cases, two distinct forms of carbamazepine associated cardiac dysfunction emerged. One patient group developed sinus tachycardias in the setting of a massive carbamazepine overdose. The second group consisted almost exclusively of elderly women who developed potentially life threatening bradyarrhythmias or atrioventricular conduction delay, associated with either therapeutic or modestly elevated carbamazepine serum levels. Because carbamazepine is widely used in the treatment of many neurologic and psychiatric conditions, the recognition of the latter syndrome has important implications for the use of this drug in elderly patients. PMID- 1728916 TI - Some cautions in labeling effects. PMID- 1728917 TI - Site for subcutaneous heparin injection. PMID- 1728918 TI - Does an apple a day keep the cardiologist away? PMID- 1728919 TI - Normal sedimentation rates and giant cell arteritis. PMID- 1728920 TI - Alanine aminotransferase: a nonspecific marker of liver disease. PMID- 1728921 TI - Fever of unknown origin. An old friend revisited. PMID- 1728922 TI - A note of caution on empiric use of ciprofloxacin for diarrhea. PMID- 1728923 TI - Advancing the cause of advance directives. PMID- 1728924 TI - Withdrawing life-sustaining treatment. Lessons from Nancy Cruzan. PMID- 1728925 TI - Two different views of the relationship of hypertriglyceridemia to coronary heart disease. Implications for treatment. AB - Hypertriglyceridemia is commonly found in patients with coronary heart disease. The reason for this connection, however, is not well understood, and two different views have been put forth to explain the link. First, triglyceride-rich lipoproteins, particularly very-low-density lipoproteins, may be directly atherogenic. Or second, the metabolic consequences of hypertriglyceridemia may account for the triglyceride-coronary heart disease relationship. These consequences include an increase in postprandial lipoproteins, large very-low density lipoprotein particles, small, dense low-density lipoprotein particles, low levels of high-density lipoprotein cholesterol, and possibly a procoagulant state. The appropriate treatment of hypertriglyceridemia depends on which of these views is nearer the truth. If triglyceride-rich lipoproteins are directly atherogenic, then the preferred therapy would be hepatic hydroxymethylglutaryl coenzyme A reductase inhibitors, which lower both very-low-density lipoprotein and low-density lipoprotein levels. On the other hand, if the link to atherogenesis is through the metabolic consequences of hypertriglyceridemia, the appropriate therapy would be to directly lower serum triglyceride levels, as with niacin or a fibric acid. Thus, discovery of the mechanism of the connection between triglycerides and coronary heart disease is crucial for developing a rational therapy. PMID- 1728926 TI - Reflections on the impact of antihypertensive medications on mood, sedation, and neuropsychologic functioning. AB - All antihypertensive treatments, including placebo, are associated with cognitive side effects. These side effects are rarely clear-cut and involve mood, quality of sleep, daytime sedation, and various neuropsychologic functions. This review article reflects on the vast literature on this topic and comments on substantive findings, as well as methodologic difficulties in sorting through findings from quality-of-life studies. Hypertension itself is associated with many of the above changes, and some cognitive functions actually improve with treatment. Nonetheless, many medications are accused of causing side effects, despite the fact that side effects with placebo are common, particularly for nonspecific complaints, such as fatigue. Side effects are not limited to sympatholytic antihypertensive agents and are commonly reported even by patients who are treated with diuretics. It is not at all clear that side effects are more commonly reported in patients who are treated with lipophilic beta-blockers. The patient may be a relatively poor observer of these subtle changes. The spouse is an important source of additional information with regard to side effects. The virtual absence of any standardization in quality-of-life measures makes comparisons across studies extremely difficult. PMID- 1728927 TI - Refractory potassium repletion. A consequence of magnesium deficiency. AB - Experimental and clinical observations support the view that uncorrected magnesium (Mg) deficiency impairs repletion of cellular potassium (K). This is consistent with the observed close association between K and Mg depletion. Concomitant Mg deficiency in K-depleted patients ranges from 38% to 42%. Refractory K repletion due to unrecognized concurrent Mg deficiency can be clinically perplexing. Refractory K repletion as a consequence of Mg deficiency may be operative in patients with congestive failure, digitalis toxicity, cisplatin therapy, and in patients receiving potent loop diuretics. Therefore, we recommend that: (1) serum Mg be routinely assessed in any patients in whom serum electrolytes are necessary for clinical management and (2) until serum Mg is routinely performed consideration should be given to treating hypokalemic patients with both Mg as well as K to avoid the problem of refractory K repletion due to coexisting Mg deficiency. PMID- 1728928 TI - Joint report of the Council on Scientific Affairs and the Council on Medical Service. Technology assessment in medicine. AB - The rapid proliferation of health care technology and the increasing concern about cost containment are major converging forces in today's health care system. These forces argue persuasively for the rigorous evaluation of the safety and effectiveness of technologies and for the use of such evaluative information as the foundation of both clinical decision making and public policy formulation. Thus, technology assessment is an essential tool in improving the quality of health care delivered and in maximizing the efficiency of the health care system. The American Medical Association historically has been dedicated to the provision of sound scientific information to enhance the appropriate utilization of health care technology. This objective remains the primary goal of the American Medical Association's activities in technology assessment. Additionally, with the ascendancy of nonphysician segments of the health care community in making policies that affect the availability of technology, the American Medical Association's assessment programs must represent physicians' views and concerns cogently in public policy debates (eg, coverage). The Council on Scientific Affairs and the Council on Medical Service examined the area of technology assessment and its influence on the payment for and the utilization of health care technology. The councils made specific recommendations to ensure that the American Medical Association maintains the sophisticated capabilities and sufficient capacity to meet the needs of physicians and patients alike in today's complex health care environment. PMID- 1728929 TI - Fever of unknown origin in the 1980s. An update of the diagnostic spectrum. AB - OBJECTIVE: To determine the relative proportions of the diagnostic categories in patients with fever of unknown origin who were examined in the 1980s. STUDY DESIGN: Prospective case series. SETTING: General Internal Medicine Service based at University Hospital, Leuven, Belgium. PATIENTS: One hundred ninety-nine consecutive patients meeting the classic criteria of fever of unknown origin who were treated in the 1980s. MAIN OUTCOME MEASUREMENT: The final diagnosis established at discharge or during follow-up. RESULTS: Infections were found in 45 patients (22.6%), tumors were found in 14 (7%), multisystem diseases were found in 42 (21.5%), drug-related fever was found in six (3%), factitious fever was found in seven (3.5%), habitual hyperthermia was found in five (2.5%), miscellaneous diseases were found in 29 (14.5%), and no diagnosis was reached in 51 (25.6%). CONCLUSIONS: Tumors were a less important cause of fever of unknown origin in the 1980s. The same holds true for some infectious diseases, such as abscesses and hepatobiliary disorders. Multisystem diseases were more frequently found, and the number of undiagnosed cases increased. Although these shifts in the disease spectrum in fever of unknown origin most probably resulted from a constellation of factors, we suspect that these changes are mainly due to easy and early diagnosis by new diagnostic modalities, such as ultrasonography and computed tomography, of previously common causes of fever of unknown origin. PMID- 1728930 TI - Serum cholesterol, blood pressure, cigarette smoking, and death from coronary heart disease. Overall findings and differences by age for 316,099 white men. Multiple Risk Factor Intervention Trial Research Group. AB - To assess the combined influence of blood pressure (BP), serum cholesterol level, and cigarette smoking on death from coronary heart disease (CHD) and to describe how these associations vary with age, data on those factors and on mortality for 316,099 men screened for the Multiple Risk Factor Intervention Trial (MRFIT) were examined. Vital status of participants has been determined after an average follow-up of 12 years; 6327 deaths from CHD have been identified. Strong graded relationships between serum cholesterol levels above 4.65 mmol/L (180 mg/dL), systolic BP above 110 mm Hg, and diastolic BP above 70 mm Hg and mortality due to CHD were evident. Smokers with serum cholesterol and systolic BP levels in the highest quintiles had CHD death rates that were approximately 20 times greater than nonsmoking men with systolic BP and cholesterol levels in the lowest quintile. Systolic and diastolic BP, serum cholesterol level, and cigarettes per day were significant predictors of death due to CHD in all age groups. Systolic BP was a stronger predictor than diastolic BP. These results, together with the findings of clinical trials, offer strong support for intensified preventive efforts in all age groups. PMID- 1728931 TI - The automatic implantable cardioverter-defibrillator. Long-term clinical experience and outcome at a hospital without an open-heart surgery program. AB - From November 1982 through April 1989, 111 patients with refractory sustained ventricular tachycardia/fibrillation had the automatic cardioverter-defibrillator implanted at our institution, the first community hospital involved in implantation of such a device. We have reviewed our long-term clinical experience to assess the feasibility, learning curve, and efficacy of device implantation in a facility with cardiac electrophysiology expertise but without open-heart surgery facilities. All patients were considered inoperable or at high risk for other concomitant surgery. Eighty-six patients (77%) underwent uneventful implantation. Nine patients (8%) died prior to hospital discharge. Operative mortality declined from 10.9% to 5.4% during the first half (55 patients; November 1982 through September 1986) and second half (56 patients; October 1986 through April 1989) of the experience. Other postoperative complications occurred in 16 patients (14%), 12 of whom experienced complications during the first half of the experience. At 22 +/- 20 (mean +/- SD) months' follow-up, 78 (76%) of 102 patients discharged were alive, and 24 patients (24%) had died. Fifty patients (49%) had experienced at least one automatic cardioverter-defibrillator discharge associated with hypotensive symptoms. The actuarial incidence of sudden death at 1, 2, and 3 years was 1.2%, 5.5%, and 6.2%, respectively. We concluded that the automatic implantable cardioverter-defibrillator is an effective therapy for refractory ventricular tachycardia/fibrillation and that device implantation at community hospitals with an experienced cardiac electrophysiology team is both feasible and practical. PMID- 1728932 TI - A multivariate model for the prediction of relapse after outpatient treatment of decompensated chronic obstructive pulmonary disease. AB - PURPOSE: To develop and validate a multivariate model for predicting relapses after treatment of decompensated chronic obstructive pulmonary disease in an emergency department. METHODS: A 5-year survey was conducted, including training and validation periods. Stepwise logistic regression was used to develop a multivariate predictive model using clinical data obtained at the time of each visit. A relapse was defined as an unscheduled return to the emergency department within 48 hours. SITE: The study was conducted in the emergency department of the Albuquerque (New Mexico) Veterans Affairs Medical Center. SUBJECTS: The subjects were 289 patients with documented chronic obstructive pulmonary disease. MEASUREMENTS AND MAIN RESULTS: During the first 3 years, there were 705 visits in which the patient was treated and released from the emergency department. Relapse occurred 82 times (11.6%). Logistic regression showed that the following variables had an effect on the risk of relapse: the relapse rate for previous visits, a previous visit within 7 days, long-term home oxygen therapy, the number of doses of nebulized bronchodilators, the administration of aminophylline, and the use of antibiotics and prednisone at the time of discharge from the emergency department. During the next 2 years, the 48-hour relapse rate was 9.9% (47 of 476 discharges). When the model was fitted to these data, all of the original variables contributed to the prediction of relapse except antibiotic use and long term home oxygen therapy. The logistic model was used to categorize each visit during the validation phase. The relapse rate for "high-risk" visits was significantly higher than that for "low-risk" visits (18.4% vs 6.1%). The method identified 57.4% of visits that ended in relapse at 48 hours. CONCLUSIONS: A multivariate model can be used to identify patients with a poor prognosis after the outpatient treatment of decompensated chronic obstructive pulmonary disease. PMID- 1728933 TI - A multivariate model for predicting hospital admissions for patients with decompensated chronic obstructive pulmonary disease. AB - PURPOSE: To develop a method for predicting hospital admissions for patients with decompensated chronic obstructive pulmonary disease treated in an emergency department. METHODS: A 4-year survey including training and validation periods was conducted. Stepwise logistic regression was used to develop a multivariate model using information from the patient's previous visits and results of baseline pulmonary function tests. MEASUREMENTS AND MAIN RESULTS: During the first 2 years, there were 693 visits to the emergency department for decompensated chronic obstructive pulmonary disease. The patient was admitted to the hospital on 210 occasions (30.3%). Logistic regression showed that the probability of admission was related to the following: the admission and relapse rates for previous visits, the proportion of previous discharges from the emergency department in which "conservative therapy" was given, the highest baseline post-bronchodilator forced expiratory volume in 1 second within 3 years of entry, and the highest baseline pre-bronchodilator forced expiratory volume in 1 second-vital capacity ratio. A relapse was defined as an unscheduled return to the emergency department within 48 hours. "Conservative therapy" was any treatment regimen that did not include parenteral medications. During the next 2 years, the model was validated with patients not previously treated at this medical center. Seventy-six (28.3%) of 269 episodes resulted in hospital admission. The logistic model was used to categorize each visit during the validation phase. "High-risk" visits had calculated probabilities of admission greater than .208, while "low-risk" visits had values that were less. The admission rate for 98 low-risk visits (8.2%) was much lower than the rate for 171 high-risk visits (39.8%). CONCLUSIONS: A multivariate model can be used to identify patients with decompensated chronic obstructive pulmonary disease who are unlikely to need hospitalization. This model could be used to select episodes of decompensated chronic obstructive pulmonary disease for treatment at home. PMID- 1728934 TI - Gemfibrozil also corrects dyslipidemia in postmenopausal women and smokers. AB - We compared the efficacy of three formulations and two dosage regimens of gemfibrozil in 322 dyslipidemic (non-high-density lipoprotein [HDL] cholesterol level, greater than or equal to 5.2 mmol/L [greater than or equal to 200 mg/dL]) middle-aged male and postmenopausal female patients in a 1-year open-label trial. Of the patients studied, 109 received the standard 1200-mg dose of gemfibrozil, ie, two 300-mg capsules twice daily; 107 received one 600-mg tablet twice daily; and 106 received a single dose of 900 mg of gemfibrozil in two 450-mg tablets in the evening. The three treatment groups showed equal changes in each lipoprotein measure studied, ie, in serum levels of triglycerides, total cholesterol, low density lipoprotein and HDL cholesterol, HDL subfractions HDL2 and HDL3, and apolipoproteins A-I, A-II, and B. When the therapeutic responses were analyzed separately in men (n = 219) and women (n = 103), significantly greater decreases in serum levels of total triglycerides, low-density lipoprotein cholesterol, and apolipoprotein B, and a significantly greater increase in HDL3 cholesterol level and apolipoprotein A-I/B ratio, were seen in the women. When the study population was divided into smokers (n = 80) and nonsmokers (n = 242), the changes were similar in all lipoprotein measures except HDL3 cholesterol level, in which a significantly greater increase was seen in the non-smokers. This study showed that gemfibrozil is as effective, or more so, in dyslipidemic postmenopausal women as in dyslipidemic middle-aged men, and that smoking does not abolish its lipid-regulating effects. Importantly, a daily dose of 900 mg was found to be as effective as the standard dose of 1200 mg. PMID- 1728936 TI - Recent advances in dermatology. PMID- 1728935 TI - IgG subclass deficiency and sinopulmonary bacterial infections in patients with alcoholic liver disease. AB - Abnormalities in IgG subclass distribution were sought in serum samples and bronchoalveolar lavage fluid from 15 patients with alcoholic liver disease to explain their increased susceptibility to bacterial respiratory infections. Serum IgG4 deficiency alone or in association with low IgG2 levels was revealed in approximately 30% of patients with alcoholic liver disease. This fact prompted us to further investigate the immunoglobulin concentrations in broncho-alveolar lavage fluid, paying special attention to the distribution of IgA and IgG subclasses. IgA levels were found to be normal or slightly elevated. However, there were substantial defects in total IgG and IgG1 concentrations, often associated with reduced IgG2 and IgG4 levels, in approximately 70% of patients with alcoholic liver disease, which proved to be closely correlated with the number and type (pneumonia) of bacterial respiratory infections. A prospective study of intravenous immunoglobulin substitutive therapy involving two patients with recurrent pneumonia and very low serum IgG2 values demonstrated a reduction in the number of respiratory infectious episodes as well as an increase in both serum and, to a lesser extent, bronchoalveolar lavage fluid IgG1 and IgG2 levels. We identified immune defects that may represent an important pathogenetic mechanism that, when considered together with the alcohol-related suppression of alveolar macrophage and ciliary functions and the inhibition of leukocyte migration into the lungs, should help clarify the complex relationships between alcohol and immune defense. PMID- 1728937 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 3-1992. A 40-year-old woman with intermittent hemoptysis and mucosal ulceration found on bronchoscopic examination. PMID- 1728938 TI - The nurse consultant's role in products liability litigation. PMID- 1728939 TI - Will the non-clinical medical-legal consultant perish? PMID- 1728940 TI - Mistakes consultants and experts make. PMID- 1728941 TI - General form for requesting hospital documents. PMID- 1728942 TI - Cytogenetic findings in terminal large cell transformation in a case of Sezary syndrome. AB - A long-lasting case of Sezary syndrome, whose chromosomal pattern had been repeatedly investigated during a follow-up period of several years, was studied in the terminal transforming phase, which took place more than 5 years after the initial diagnosis. To the best of the authors' knowledge, this appears to be the first instance of cytogenetic studies carried out in a large cell transformation of cutaneous T-cell lymphoma. The results clearly indicate that the atypical large cells seen in the transforming phase were clonally derived from the pre existing cerebriform cells. Newly detected relevant cytogenetic findings were: a) drop of tumor cell ploidy from hypotetraploid to hypotriploid, with striking chromosomal imbalance; b) additional structural aberrations of chromosomes 2 and 7, which had been already preferentially involved in the earlier phases, and involvement of the previously unaffected chromosomes 1, 3, and X; and c) presence in 100% of the abnormal metaphases of a large HSR on the long arm of chromosome 17. PMID- 1728943 TI - Chromosome 11 rearrangement at band 11q21 in a patient with essential thrombocythemia. AB - A case of essential thrombocythemia with a partial deletion of the long arm of chromosome 11, del(11)(q21) as a sole chromosomal anomaly is reported. Rearrangement of chromosome 11 at band 11q21 has been reported in six patients with chronic myeloproliferative disorders: four with post-polycythemic myelofibrosis, one with myelofibrosis with myeloid metaplasia, and one with Ph+ chronic myeloid leukemia in blastic phase. Except for the last patient, all patients had been treated with 32P and/or an alkylating agent prior to cytogenetic examination. This is the first report of the 11q21 abnormality in essential thrombocythemia seen at diagnosis. PMID- 1728944 TI - Human benign chondroblastoma with a pseudodiploid stemline characterized by a complex and balanced translocation. AB - The chromosomes of a human benign chondroblastoma of the jaw were studied by in vitro technique. Approximately one-third of the analyzed cells had a normal karyotype. The remaining two-thirds constituted an abnormal monoclonal population with a complex and balanced translocation. The observations are different from those in previously studied benign primary bone tumors. The breakpoint in 22q, however, is similar or very close to that observed in two extraskeletal myxoid chondrosarcomas earlier reported. PMID- 1728945 TI - Cytogenetic findings in secondary acute nonlymphocytic leukemia. AB - We have report the results of cytogenetic studies carried out in eight patients with acute nonlymphocytic leukemia developed after primary neoplasias. In seven of the reported cases, clonal chromosome aberrations were found, some being specific of de novo acute nonlymphocytic leukemia (ANLL). Numerical abnormalities were detected, such as the total monosomy of chromosomes 5, 7, 21, trisomy of chromosomes 8, 11, 15, and duplication of chromosome Y. Structural changes were also observed: a del(12)(p12), a del(16)(q22), the translocations t(3;5)(p21;q35),t(3;7)(p21;q35), and t(12;14)(p12;q32) and other changes involving chromosome 8. The finding of a hypertetraploid karyotype with complex structural chromosome aberrations in a patient with erythroleukemia, developed after non-Hodgkin's lymphoma, is of particular interest. Data reported in this work are discussed with regard to the relationship between secondary and de novo ANLL and the finding of chromosome aberrations other than total or partial monosomy of chromosomes 5 and 7 is emphasized. PMID- 1728946 TI - Giant cell tumor of bone. Chromosomal analysis of 48 specimens and review of the literature. AB - Giant cell tumor of bone (GCT) is a distinct clinical, radiographic, and pathologic benign entity that constitutes 5% of all primary bone tumors. For a 5 year period, 47 benign GCTs and 1 malignant GCT from 34 different patients were cytogenetically characterized. Analysis showed clonal karyotypic abnormalities in 16 specimens. Clonal structural abnormalities detected in more than one patient included translocations involving 11p15, fus(14p;21p), and fus(15p;21p). None of the clonal numerical abnormalities observed occurred in more than one patient. Thirty-seven of the 44 successfully analyzed specimens (84%) demonstrated telomeric fusion, with most frequent involvement of chromosomal telomeres 11p, 13p, 15p, 18p, 19p, and 21p. We also compared the presence or absence of random and/or clonal karyotypic abnormalities with clinical behavior to determine if a relationship existed. Most notably, chromosomal abnormalities were detected in all 13 successfully analyzed recurrent lesions, five of which were clonally aberrant. This study summarizes the cytogenetic findings and their relevance in 48 specimens analyzed at our institution and reviews the findings of the 18 other published cases. PMID- 1728947 TI - Inversion of chromosome 16 with the Philadelphia chromosome in acute myelomonocytic leukemia with eosinophilia. Report of two cases. AB - Two cases are described with the rare combination of inv(16)(p13q22), strongly associated with acute myelomonocytic leukemia with eosinophilia, M4Eo, and the Philadelphia translocation, t(9;22)(q34;q11), hallmark of chronic myeloid leukemia (CML) and rarely found, (less than 1%), in acute nonlymphocytic leukemia. The patients were: case 1, a 9-year-old girl presenting with a white blood cell count (WBC) 42 x 10(9)/L with 32% blasts and bone marrow with blasts and eosinophil precursors consistent with M4Eo, and case 2, a 25-year-old man with WBC 34.7 x 10(9)/L with 13% blasts and bone marrow with features of M4Eo and basophilia. Both patients achieved remission but died following bone marrow transplantation in first remission (case 1) or in relapse (case 2). Cytogenetic findings were: case 1, at diagnosis, 46,XX,inv(16)(p13q22)(21)/46,XX,t(9;22) (q34;q11),inv(16)(8)/46,XX(10), and case 2, at diagnosis, 46,XY,t(9;22) (q34;q11),inv(16)(p13q22) (16) and in remission, 46,XY,t(9;22)(q34;q11) (1)/46,XY (24). Investigation of the breakpoint on 22 in case 1 with Southern blotting and the polymerase chain reaction demonstrated the presence of a p190 mRNA and a breakpoint typical of acute leukemia. Thus a diagnosis of M4Eo was supported by clinical and cytogenetic sequelae in each case; the Ph in case 1 was apparently secondary to inv(16), in case 2 the Ph probably preceded inv(16) in the etiology of the leukemia. PMID- 1728948 TI - Cytogenetic study of B-cell lymphoma of mucosa-associated lymphoid tissue. AB - The recently described B-cell lymphomas arising in mucosa-associated lymphoid tissue (MALT) form a distinct clinico-pathologic group of non-Hodgkin's lymphoma, and therefore would be expected to be characterized by a recurrent chromosomal aberration. We have analyzed the cytogenetics of 23 cases of MALT lymphomas arising in the stomach, small intestine, lung, and lacrimal gland. In each case the presence of an abnormal clonal cell population was confirmed by the identification of rearranged bands when digested tumor DNA was hybridized with a probe to the joining region of the immunoglobulin heavy chain gene. Metaphase spreads were obtained in 14 cases, of which 9 cases showed an abnormal karyotype. Although no unifying aberration was detected, rearrangements of chromosome 1p, and numerical abnormalities of chromosomes 3 and 7, may play a role in the genesis of these tumors. PMID- 1728949 TI - Novel translocation (2;4) with consistent involvement of 2p23 in acute nonlymphocytic leukemia (M2). AB - A translocation involving the short arm of chromosome 2 and the long arm of chromosome 4 is described in three patients, all of whom had acute nonlymphocytic leukemia (M2). One patient had M2 de novo, one progressed from refractory anemia with excess blasts in transformation to M2 over a 4-month period, and one had had sideroblastic anemia 30 years prior to development of M2. In all three patients, the translocation involved the breakpoint p23 on chromosome 2, but the breakpoints on chromosome 4 varied between q25, q31, and q35. Translocations specifically involving 2p23 have not previously been described in leukemia, but more cases are required to identify a specific association with either the FAB type or prognosis. PMID- 1728950 TI - Ring chromosome in a dermatofibrosarcoma protuberans. AB - We report the cytogenetic findings in a case of dermatofibrosarcoma protuberans in a 40-year-old male. Chromosome analysis revealed one clone consisting of +7, +11, +13, +14, +15, and a ring chromosome. This is consistent with two previously reported cases, each of which also had a single ring chromosome. PMID- 1728951 TI - Chromosomal localization of amplified N-myc in neuroblastoma cells using a biotinylated probe. AB - G-banded chromosome analysis of neuroblastoma cells from two children revealed homogeneously staining regions (hsr) in one patient and double minutes (dmin) in the other. Subsequently, both abnormalities were confirmed as areas of N-myc amplification using chromosomal in situ hybridization with a biotinylated N-myc probe. In addition, the first patient's karyotype contained a possible derivative chromosome 17, which was also demonstrated to contain amplified N-myc, indicating the presence of an hsr unidentified by G-banding. Intercellular heterogeneity in the degree of amplification was also identified in the nuclei of interphase cells. This technique provides a quick method for detecting gene amplification, the identification of which may have useful clinical implications. PMID- 1728952 TI - Cytogenetic abnormalities in a rare case of giant cell osteogenic sarcoma. AB - The cytogenetic analysis of a rare, nonirradiated case of giant cell tumor of bone with osteogenic sarcoma transformation is presented for the first time in a 19-year-old female. Telomeric associations involving 4p, 8p, 11p, 14p, 17p, 17q, and 20q were observed. Additionally, monosomy 13, 11p abnormalities and marker chromosomes were identified in tumor cells. Chromosome 11 involvement, particularly 11p translocations and 11p telomeric associations, were frequently observed in the tumor cells obtained from our patient, which suggests that chromosome 11p may play a role in the development of giant cell osteogenic sarcoma. PMID- 1728953 TI - Paternal origin of 11p15 duplications in the Beckwith-Wiedemann syndrome. A new case and review of the literature. AB - A boy suffering from the Beckwith-Wiedemann syndrome (BWS) was found to have partial trisomy of the short arm of chromosome 11 [46,XY,der(5)t(5;11)(p15.2;p14)]. Both his parents were phenotypically normal, but his father carried a balanced translocation between chromosomes 5 and 11 [46,XY,t(5;11)(p15.2;p14)]. DNA analysis of polymorphic markers on 11p15 confirmed the paternal origin of the duplicated material in the child. This case is the sixth report of paternal duplication of 11p15 in BWS. These results are discussed in relation to the possible role of genomic imprinting in BWS and in Wilms' tumor. PMID- 1728955 TI - Translocation (8;21) in two cases of refractory anemia with excess of blasts in transformation. AB - We report two cases of refractory anemia with excess of blasts in transformation (RAEB-T) with the translocation (8;21), which is frequent in ANLL but not in myelodysplastic syndromes (MDS). A review of such cases leads us to conclude that myeloproliferative disorders characterized by the t(8;21) may be preceded by an MDS phase. PMID- 1728954 TI - Amplification of ETS2 oncogene in acute nonlymphoblastic leukemia with t(6;21;18). AB - Cytogenetic and molecular studies in a case of acute nonlymphoblastic leukemia (ANLL) are reported in this paper. Bone marrow blasts carried a hypodiploid karyotype with a complex t(6;18;21)(6qter----6p21::21q22----21qter;18qter --- 18p11::6p22----6pter; 21pter----21q22::6p21----6p22::18p11----18pte r) and other numerical and structural changes. We studied the organization and the expression of the ETS2 gene which is located on chromosome 21 in order to investigate its possible involvement in the disease. DNA analysis showed a 20-fold amplification of ETS2 sequences; an increase of 3- to 4-fold in the mRNAs level compared to normal was shown by Northern hybridization. PMID- 1728956 TI - Cytogenetic studies of eight primary gastric cancers. AB - Cytogenetic analysis was performed on eight primary gastric cancers. Three of them had simple chromosome changes: 47,XX,+X/48,XX,+X,+X; 48,XX,+8,+19,t(3;5) (q21;q31) and 47,XY,+del(7)(q22). The five others had complicated chromosome changes; both 3p- and 7q- were noted in four cases and i(5p) was noted in two cases. PMID- 1728957 TI - Isochromosome (6p) in Waldenstrom's macroglobulinemia. AB - Cytogenetic data on two cases of previously treated Waldenstrom's macroglobulinemia (WM) are presented. An i(6p) was identified in 60 and 56% of bone marrow (BM) metaphases from each patient, respectively. In both cases, i(6p) occurred as part of a complex karyotype but was also observed as the sole abnormality in a proportion of metaphases. The literature on the cytogenetics of WM and the relevance of i(6p) is discussed. PMID- 1728958 TI - Near-triploid myeloblastic transformation of chronic myeloid leukemia with bizarre blast morphology. AB - We describe a case of chronic myeloid leukemia (CML) in myeloblastic transformation in which near-triploidy with complex karyotypic abnormalities occurred together with unusual blast morphology. A review of the literature shows that near-triploidy is extremely unusual in blastic transformation (BT) of CML. PMID- 1728959 TI - Ph-positive chronic myeloid leukemia with t(8;21)(q22;q22) in blastic crisis. AB - A patient diagnosed with chronic myeloid leukemia was studied periodically during his illness. The result showed the presence of a Philadelphia (Ph) chromosome by a 9;22 translocation as a single abnormality to the time of blastic crisis. At that time, the chromosome studies showed a clonal evolution. Furthermore, a second derivated line was added to the Ph line. This new anomaly consisted of a 8;21 translocation, considered as specific of M2 type acute nonlymphoblastic leukemia of French-American-British classification. PMID- 1728960 TI - Estrogen and cardiovascular disease. PMID- 1728961 TI - IFN-gamma enhances the expression of cell surface receptors for monoclonal nonspecific suppressor factor. AB - Monoclonal nonspecific suppressor factor (MNSF) is a lymphokine derived from a murine T cell hybridoma. The action of MNSF is mediated by specific cell-surface receptors. Since IFN-gamma alters the cellular response to MNSF (M. Nakamura, H. Ogawa, and T. Tsunematsu, J. Immunol. 138, 1799, 1987), we investigated whether IFN-gamma has an effect on the expression of MNSF receptor on target cells. IFN gamma enhanced the expression of MNSF receptor on both MOPC-31C cells (a murine plasmacytoma line) and EL4 (a murine T lymphoma line). Incubation with IFN-gamma increased the number of specific MNSF-binding sites by about 50 to 90%, with no significant change in binding affinity. IFN-alpha and IFN-beta also increased MNSF binding, although the effect of the saturating amounts was lower than that seen with IFN-gamma. Maximal enhancement of receptor expression was observed after about 15 hr of incubation with IFN-gamma. No demonstrable change occurred in the kinetics of internalization of 125I-MNSF bound to MOPC-31C cells preincubated without or with IFN-gamma. PMID- 1728962 TI - Characterization of N-terminal amino acid sequence of monoclonal nonspecific suppressor factor. AB - Monoclonal nonspecific suppressor factor (MNSF), a product of a murine T cell hybridoma, suppresses the antibody response to lipopolysaccharide. In an attempt to clarify the N-terminal sequence, MNSF was prepared and purified by affinity chromatography with the use of an anti-MNSF monoclonal antibody (MO6), and reverse-phase high-pressure liquid chromatography. On the SDS-PAGE, the purified MNSF showed a single band with a molecular weight of 12,000. The N-terminal amino acid sequence of the protein was determined and showed no strong homology to any of the sequences of known biologically active proteins. However, the sequence revealed significant (60%) amino acid identity to transforming growth factor beta 2 (TGF beta 2). PMID- 1728963 TI - In vivo lymphocyte responses in the draining lymph nodes of mice exposed to Schistosoma mansoni: preferential proliferation of T cells is central to the induction of protective immunity. AB - The in vivo cellular responses associated with the induction of specific immunity by attenuated larvae of Schistosoma mansoni in mice have been investigated. Using in vivo 5-bromo-2'-deoxyuridine incorporation, the changes in cell proliferation in the skin- and lung-draining lymph nodes (LN) of vaccinated animals were measured. A marked increase in the number of dividing cells was detected in both groups of LN, with a preferential increase in the proportion of proliferating T, relative to B, lymphocytes. Several dynamic components of cell migration have been examined to assess their relative contribution to the overall changes in the LN of immunized mice. It was determined that a significant part of the observed accumulation of cells is due to the effect of hyperaemia. There was no alteration in the affinity of the LN for T and B lymphocytes, but we concluded that the majority of recruited B cells failed to exit the nodes. The results have highlighted the importance of T cell proliferation within the draining LN for the successful immunization of mice with attenuated parasites. PMID- 1728964 TI - In situ detection of autoanti-idiotype antibody-forming cells induced by influenza virus infection. AB - In situ immunocytochemical-staining methods combined with computer-aided image analysis were employed to examine autoanti-idiotype antibody-forming cell expansion in vivo. Autoanti-idiotype antibody-forming cells were demonstrated in the spleens of C57BL/6J (B6) strain mice intranasally infected with the influenza virus A/Hong Kong/168/(H3N2)[R] X-31. Autoanti-idiotype B cells were detected and elevated in spleen tissues after secondary influenza infections compared to normal B6 mice, and were specific for a dominant idiotype antibody called PY206 reactive with the influenza virus hemagglutinin. Influenza virus-infected BALB/c mice, which make little PY206 Id, did not have increased autoanti-Id against PY206 compared to normal mice. Results provide evidence for an idiotype network mediated stimulation of autoanti-idiotype B cell production during influenza virus infection. PMID- 1728965 TI - Immunosuppression by human gangliosides. II. Carbohydrate structure and inhibition of human NK activity. AB - Gangliosides shed by tumors enhance tumor formation, possibly by suppressing host antitumor immune function, and gangliosides purified from animal tissues and cultured cells inhibit human cellular immune function in vitro. Determination of immunosuppressive activity of highly purified gangliosides, to uncover structure activity relationships, is therefore important. Here we have studied a series of gangliosides obtained from human tissue and determined their effects on human natural killer (NK) activity. Total gangliosides from human brain tissue were moderately inhibitory; 100 nmol/ml reduced NK activity of human nonadherent PBMC by 43%. The influence of carbohydrate structure upon inhibitory activity was determined by study of eight highly (HPLC) purified individual gangliosides. Of these, we unexpectedly found that the two minor brain gangliosides with the simplest carbohydrate structures, GM2 and GM3, were very active inhibitors (75 and 47%, respectively, at 50 nmol/ml). In contrast, the structurally more complex major species, GM1, GD1a, GD1b, GT1b, and two other minor gangliosides, GD2 and GD3, were inactive. Reduced effector-target binding in a single-cell binding assay by GM2 but not GM3 suggests different mechanisms of inhibition by these two active gangliosides. Since GM2 and GM3 are present in high concentrations in, and are shed by, several common human tumors (e.g., neuroblastoma, melanoma, and glioma), their ability to inhibit NK cytotoxicity supports the hypothesis of a role of shed tumor gangliosides in the enhancement of tumor formation. PMID- 1728966 TI - Human cytotoxic T lymphocytes and activated peripheral blood lymphocytes enhance cytochalasin B-induced DNA fragmentation of indicator bystander cells. AB - The cytochalasins are known to have multiple effects on cellular function. Not only do they induce secretion from granule compartments but they can induce DNA fragmentation in numerous cells. Evidence is presented which shows that treatment of human cytotoxic T lymphocytes and activated peripheral blood lymphocytes with cytochalasin B induces release of a factor capable of enhancing DNA fragmentation in cytochalasin-susceptible target cells. This activity can be transferred in the supernatants of cytochalasin B-activated CTL. PMID- 1728967 TI - Immunoregulatory effects of transforming growth factor-beta in a prolonged period of culture. AB - It is well known that transforming growth factor-beta (TGF-beta) strikingly inhibits numerous immune functions in short-term cultures. In this study we investigated the effects of TGF-beta on the immune responses of murine spleen cells in a prolonged period of culture. The addition of exogenous TGF-beta (0.1 ng/ml) inhibited the proliferation of Con A- or LPS-stimulated spleen cells, polyclonal IgM and IgG antibody production, and NK cell activity during 4 days of the initial culture and subsequently enhanced their responses on Day 10. The augmented polyclonal IgM and IgG responses in murine spleen cells induced by LPS and TGF-beta on Day 10 were suppressed by the secondary addition of TGF-beta on Day 6. These results suggest that TGF-beta acts as an immunoregulator in prolonged period responses by immunoactivators. PMID- 1728968 TI - Use of antibody/peptide constructs of direct antigenic peptides to T cells: evidence for T cell processing and presentation. AB - Human T cells can express MHC-class II products and were shown to be potential antigen-presenting cells. However, they are unable to capture the antigen and only antigens, which bind to T cell membranes such as the gp120 glycoprotein of HIV, are internalized, processed, and presented by T cells. To better understand the role of T cells as antigen-presenting cells, we established a method which overcomes the lack of antigen capture by T cells. Antigen (tetanus toxoid, TT) or an antigenic peptide of TT (residue 830-843, P2) was coupled to antibodies directed to T cell surface molecules such as CD2, CD4, CD8. Antibody/TT and antibody/P2 constructs stimulated P2-specific T cell clones in the absence of accessory cells, if the antibody recognized a T cell surface structure. Compared to the peptide alone, a 100-500 times lower molar concentration of the antibody/peptide construct was required to achieve a similar proliferative response. T cell stimulation via the constructs involved intracellular processing, as nonspecific, glutaraldehyde fixed T cell lines pulsed with the constructs could present the peptide and processing inhibitors like Leupeptin or Chloroquine inhibited the development of a proliferative response to the constructs. Our data underline the ability of T cells to function as antigen processing and -presenting cells and show that antibody/antigen or antibody/peptide constructs are able to direct a certain antigen or peptide to a T cell. Antibody/peptide constructs may be interesting tools to better understand antigen processing and to study the consequences of antigen presentation by different cells. PMID- 1728969 TI - Generation of MHC-nonrestricted and restricted oncolytic subsets from human bone marrow. AB - We analyzed the cytotoxicity and characterized the phenotype of oncolytic bone marrow (BM) lymphocyte subsets generated in vitro by interleukin-2 (IL-2) and stimulator cells (SC). Two irradiated B-lymphoblastoid cell lines (Daudi and EBV transformed BSM) and fresh human acute myelogenous leukemia (AML) were used as SC. Stimulation with Daudi and IL-2 resulted in a substantial increase in cytotoxic activity (100- to 1000-fold) against a broad range of tumor targets, and total cellular expansion was higher compared to stimulation with IL-2 alone. The most prominent increase was observed in the CD16+ and CD56+/CD3- natural killer (NK) cell subset; however, a significant increase was also observed in CD56+/CD3+ T cells. Functional analysis of Daudi- and IL-2-generated subsets using fluorescence-activated cell sorting (FACS) revealed that most of the lytic activity was mediated by NK cells. Significant potentiation of oncolytic activity and cell growth was also seen in the cultures stimulated with BSM or fresh AML and IL-2. The highest oncolytic activity in the latter cultures was mediated primarily by CD8+, CD3+, and CD56- T cells, although NK cells also participated in cytotoxic activity. The T cell-mediated cytotoxicity was restricted by the major histocompatibility complex (MHC), since most cytotoxicity could be blocked by HLA I antibodies. Additionally, we observed that optimum stimulation of cytotoxicity required effector cell-stimulator cell contact. These data indicate that depending on the tumor used for stimulation, different lymphocyte subsets may be generated in IL-2 cultures. These different approaches may be useful in both specific and nonspecific immunotherapy. PMID- 1728970 TI - Surface mu heavy chain expressed on pre-B lymphomas transduces Ca2+ signals but fails to cause growth arrest of pre-B lymphomas. AB - Little is known about the role of signals transduced by cell surface IgM (sIgM) expressed during early B cell development. A subclone (1.6) of the late pre-B cell lymphoma 70Z/3.12 was used to study signal transduction by surface mu heavy (H) chain before and after transition to the early immature B cell stage, and the functional consequences thereof. Although kappa L chain expression can be induced on 1.6 cells by LPS or cytokines, immunoprecipitations indicated that the non induced 1.6 cells expressed mu H chain with an alternative protein(s) which may be a surrogate light chain(s). Consistent with this, anti-mu but not anti-kappa or anti-lambda antibodies caused transient Ca2+ mobilization in noninduced 1.6 cells. The Ca2+ signal was derived from both intracellular stores and Ca2+ influx in either noninduced cells or in cells that had been preinduced to express kappa L chain. Thus, the ability of mu H chain to mobilize Ca2+ as a second messenger does not depend upon the expression of mature L chains. The immature B lymphomas, WEHI-231 and CH1, express mature forms of IgM and undergo growth arrest when stimulated by anti-mu antibody. In contrast, signals generated by mu H chain on either noninduced or preinduced 1.6 cells or in the sIgM+ pre-B cell transfectant 300-19 mu lambda 36/8 did not cause growth arrest. These results suggest that mu H chain expressed on pre-B cells is capable of mobilizing Ca2+, but that this signal alone is insufficient to induce growth arrest in the pre-B cell. PMID- 1728971 TI - Suppression of experimental autoimmune uveitis in rats by the oral administration of the uveitopathogenic S-antigen fragment or a cross-reactive homologous peptide. AB - The oral administration of S-antigen fragment (a synthetic peptide designated as peptide M and known to be uveitopathogenic for rat, guinea pig, and monkey) to Lewis rats prior to challenge with an emulsion of peptide M and CFA resulted in either a total or partial suppression of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease studied as a model for human uveitis and experimental autoimmune pinealitis (EPA). Both the clinical and histopathologic manifestations of the disease were suppressed in a dose-dependent manner. Pinealitis associated with EAU was also suppressed by the oral administration of peptide M. Additionally, ingestion of a fragment of baker's yeast (Saccharomyces cerevisiae) histone H3, which has five consecutive amino acids identical to peptide M and which has been found to be uveitopathogenic in Lewis rats, induced tolerance to either peptide M or synthetic histone H3 peptide. In addition, the proliferative response to peptide M was inhibited in peptide M-fed rats. The suppression of EAU and in vitro lymphocyte proliferative responses to peptide M were observed to be antigen specific, since oral feeding of a control protein (BSA) exerted no suppressive effect. Furthermore, the T cells isolated from the spleen and lymph nodes of animals rendered tolerant by oral administration of peptide M can transfer protection against EAU adoptively. These results demonstrate that the oral administration of an autoantigen or its homologous peptide initiates an antigen-specific cellular mechanism which may ameliorate EAU. PMID- 1728972 TI - Post-transcriptional regulation of MHC class II expression in human T cells. AB - Human T lymphocytes are among those cells which are cell surface class II- in the resting state, but can be induced to express class II following treatment with appropriate stimulators. Although resting T cells do not express detectable surface class II, cell surface class II can be detected on purified T cells as early as 30 min following stimulation with PHA and PMA, well before the initiation of DNA synthesis, and the percentage of positive cells gradually increases with time. One hypothesis explaining this very rapid surface expression of class II is that the genes can be regulated post-transcriptionally in T cells. To test this, we used nuclear run-on assays to measure the transcriptional rate of diverse class II genes in resting and activated T cells. Our results demonstrated that transcripts for DR, DP, and DQ could be detected in cells which were neither dividing nor transcribing mRNA for another marker of T cell activation, the IL-2 gene. Northern blot analysis demonstrated low to moderate steady-state levels of DR beta mRNA in these cells. Moreover, treatment of activated T cells with cycloheximide resulted in superinduction of class II for DR, DQ, and DP. These results suggest that resting T cells can transcribe mRNA for class II genes, but that they do not express the protein product on the cell surface in a detectable way until following activation. In addition, they suggest that there may be a protein factor which negatively influences class II levels in T cells. Thus, the regulation of class II in T cells is complex and involves post transcriptional regulation, at least in part. PMID- 1728973 TI - T cell receptors, immunoregulation, and autoimmunity. AB - T cell receptor (TCR) peptide vaccines have proven useful in the prevention and treatment of autoimmune disease in animal models. Prospects for developing TCR peptide vaccines for human autoimmune disease are only now being explored. Preliminary indications provide cause for optimism that immunization with TCR peptides eventually will be a viable treatment option for autoimmune pathologies in humans. In the long term, development of this technology may permit reliable manipulation of T cell immunity, leading to treatments for autoimmunity, T lymphoproliferative disorders, and, in the broadest interpretation, any pathogenesis mediated by oligoclonal T cell populations. PMID- 1728974 TI - Clinical manifestations in patients with autoantibodies specific for nuclear lamin proteins. AB - IgG antibodies to nuclear lamin proteins have been found in serum samples from 31 patients using immunofluorescence on HEp-2 cells, Western blotting, and enzyme linked immunosorbent assay, performed against a nuclear lamina preparation from Ehrlich ascites tumor cells. Antilamin antibodies were most prevalent among patients with nonerosive, seronegative polyarthritis, or patients showing serum antiphospholipid reactivity as well. It is possible that anti-lamin antibodies may thus be a marker for a subgroup of polyarthritis patients who have a different prognosis from that of those with seropositive rheumatoid arthritis. The mechanism for the combined occurrence of anti-lamin and antiphospholipid autoantibodies is obscure. Future studies will answer whether these two antibodies represent a distinct antibody profile in patients with antiphospholipid antibody syndrome. PMID- 1728975 TI - Histological detection of c-myb and c-myc proto-oncogene expression in infiltrating cells in cutaneous lupus erythematosus-like lesions of MRL/l mice by in situ hybridization. AB - A relationship between lymphocytic activation and the overexpression of proto oncogenes such as c-myb or c-myc has been demonstrated in human autoimmune disease. In autoimmune-prone MRL/l mice, which spontaneously develop lupus erythematosus (LE)-like lesions on the back, increased expression of myb RNA has been found in the lymphoid organs. We detected the overexpression of c-myb and c myc proto-oncogenes in infiltrating cells in the cutaneous lesions of MRL/l mice by using in situ hybridization. No specific hybridization signals of either of the probes used were seen in the nonlesional skin of MRL/l mice or in the apparently normal skin of aged MRL/n and young MRL/l mice. These results suggest that the increased expression of myb and myc proto-oncogenes in the cutaneous LE like lesions of MRL/l mice is related to a state of activation in the infiltrating cells and is involved in the development of these lesions. PMID- 1728976 TI - The third international symposium on Sjogren's syndrome. PMID- 1728977 TI - Patterns of heavy and light chain utilization in the antibody response to single stranded bacterial DNA in normal human subjects and patients with systemic lupus erythematosus. AB - Although anti-DNA antibodies are generally considered to be specific markers for systemic lupus erythematosus (SLE), antibodies binding DNA from certain bacterial species can be found in the sera of normal subjects. To characterize the immunochemical properties of these antibodies, the IgG subclass and light chain profile of antibodies to single-stranded micrococcal DNA (MC DNA) in the sera of normal subjects and patients with SLE was determined. The anti-MC DNA response in normal sera was predominantly of the IgG2 subclass with a marked predominance of kappa light chains. In contrast, anti-MC DNA antibodies in SLE sera exhibited all IgG subclasses with a predominance of the IgG1 subclass and both kappa and lambda light chains were represented. These results suggest that antibodies to bacterial DNA in the sera of normal subjects and patients with SLE differ in patterns of immunoglobulin gene expression; the restricted response of normal subjects may be related to the binding to a discrete DNA determinant. PMID- 1728978 TI - Comparison of the intestinal and serum antibody response in patients with dermatitis herpetiformis. AB - Dermatitis herpetiformis (DH) is an intensely pruritic, blistering skin disease characterized by cutaneous IgA deposits and an associated, most often asymptomatic, gluten-sensitive enteropathy. When patients with DH are placed on a gluten-free diet both the intestinal abnormality and the cutaneous manifestations of the disease are controlled, suggesting that a mucosal immune response is important in the pathogenesis of DH. Although patients with DH continue to ingest gluten only 40-50% have evidence of an ongoing mucosal immune response in their serum. In order to investigate directly the mucosal immune response in patients with DH the antibody response to dietary antigens was analyzed in intestinal secretions and compared to that found in the serum. Intestinal secretions from six patients with DH and five normal subjects were collected using an intestinal lavage solution and analyzed for total IgA, IgG, IgM, and IgA subclasses, and for IgG, IgA, and IgM antibodies against the dietary antigens bovine beta lactoglobulin and gliadin. Intestinal secretions from patients with DH contained more IgA than those from normal subjects (mean total IgA: DH = 2.3 mg/ml; normal subjects (NL) = 0.143 mg/ml, P = 0.017). This increase in IgA in intestinal secretions from patients with DH was composed primarily of IgA1 (intestinal IgA: 86% IgA1, 14% IgA2; NL gut secretions: IgA1 = 54%; IgA2 = 46%). Increased IgA antibodies directed against beta-lactoglobulin and gliadin were detected in gut secretions of two of six patients with DH and in none of the normal subjects. Serum IgA antibodies against beta-lactoglobulin and gliadin were detected only in the two subjects who had detectable IgA antibodies in their intestinal secretions. Serum and intestinal IgA anti-beta-lactoglobulin antibodies had similar isoelectric spectrotypes (pI 5.0-6.5), IgA subclass composition, and antigenic reactivity by immunoblot analysis, demonstrating the close relationship between the serum and intestinal IgA antibodies. These data demonstrate that in patients with DH an ongoing mucosal immune response is present in the gut as evidenced by a significantly increased concentration of IgA, predominately IgA1. The strong correlation between detectable serum and intestinal IgA antibodies against dietary antigens demonstrates that the lack of serum IgA antibodies against dietary antigens in some patients with DH is not due to the presence of "blocking" IgA anti-dietary antigen antibodies in intestinal secretions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1728979 TI - The contribution of antibody-mediated cytotoxicity and immune-complex formation to tubulointerstitial disease in passive Heymann nephritis. AB - Passive Heymann nephritis (PHN), an experimental model of membranous nephropathy, is produced by Fx1A antiserum, which also reacts with antigens on the brush border (gp 330) and basolateral membrane (gp 90) of proximal tubules. We examined tubulointerstitial disease in PHN, identifying two distinct processes occurring on the luminal and basolateral membranes, respectively. Injected antibody bound diffusely to the tubular brush border from Day 1 to Day 7, followed by sloughing of microvilli and tubular-cell regeneration. Fine granular deposits of Fx1A antibody were present along the basolateral cell membrane by Day 1. These deposits rearranged in situ, enlarged, and became more focally distributed along tubular basement membranes (TBM). Interstitial inflammation, dominated by macrophages (Ia+, ED-1+) in association with a smaller number of T-cytotoxic cells (OX19+, OX8+) began by Day 3, reached peak intensity and persisted throughout the autologous phase (to Day 21). The distribution of focal clusters of interstitial macrophages predominately in association with TBM-immune deposits was demonstrated. Complement depletion prevented proteinuria but TBM deposits developed and the interstitial inflammation was unchanged. All aspects of the tubulointerstitial disease were amplified by a second injection of Fx1A antiserum. In vitro, Fx1A antibody bound to the surface of isolated proximal tubular epithelial cells and redistributed to form clusters of immune aggregates. Anti-Fx1A-induced cytotoxicity of tubular cells was demonstrated by prelabeling cells with 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The degree of cytotoxicity was dependent on complement concentration and the duration of incubation at 37 degrees C. PHN induced by Fx1A antiserum causes tubular-cell injury following interactions with brush-border antigens and TBM immune-complex disease associated with interstitial inflammation. These findings may be relevant to the acute and chronic interstitial disease of human membranous nephropathy. PMID- 1728980 TI - Interleukin-1 enhances the development of type II collagen-induced arthritis only in susceptible and not in resistant mice. AB - The development of type II collagen-induced arthritis (CIA) in DBA/1 mice is readily accelerated by treatments with interleukin-1 beta (IL-1 beta). In an attempt to further characterize this IL-1 beta-mediated enhancement of CIA, we first examined the effects of IL-1 beta treatments in other "CIA-susceptible" strains and "CIA-resistant" mice. It was observed that treatments with IL-1 beta also enhanced the onset of arthritis in two B10 recombinant CIA-susceptible strains, B10.T (6R) and B10.DA, and in the SJL mice which develop CIA with a relatively low and variable incidence. On the other hand, IL-1 beta failed to augment the expression of arthritic disease in several CIA-resistant strains. We also investigated the potentiating effects of IL-1 beta in mice that were depleted of L3T4+ T cells. It was found that the ability of IL-1 beta to accelerate the development of CIA was significantly reduced in DBA/1 mice pretreated with the monoclonal anti-L3T4 antibody. In further studies, we demonstrated that the induction of CIA upon transfer with collagen-primed spleen cells was also augmented by IL-1 beta, and this enhancing effect by IL-1 beta on the adoptive transfer of CIA was associated with a significant increase in the levels of serum anti-collagen antibodies. Moreover, IL-1 beta treatments did not potentiate the induction of CIA in mice that were transferred with either collagen-immune splenic cells that were depleted of L3T4+ T cells or only T cells obtained from collagen-immunized animals. However, IL-1 beta enhanced the development of arthritis in animals that had been transferred with two subpopulations of collagen-immune cells: (i) enriched T cells and (ii) splenic cells that were depleted of L3T4+ T cells. Thus, IL-1 beta potentiated the inflammatory responses in animals that were genetically predisposed to developing arthritis. In contrast, IL-1 beta was incapable of accelerating the development of arthritis in various mouse strains that were genetically resistant to CIA. The administration of IL-1 beta also failed to potentiate the development of CIA in L3T4-deficient mice or in animals transferred with collagen-primed spleen cells that were depleted of L3T4+ T cells. These results indicate that IL-1 beta readily accelerates the induction of arthritis when the disease is present, but that IL-1 beta is incapable of promoting the expression of the arthritis in the absence of underlying disease. PMID- 1728981 TI - Prolonged survival of MRL-lpr/lpr mice treated with Tripterygium Wilfordii Hook F. AB - MRL-lpr/lpr mice were treated with Tripterygium Wilfordii Hook-F (TWHF), a Chinese herbal medicine successfully used for human rheumatoid arthritis. Dramatic prolongation of survival and the reduction of urinary protein were observed in mice treated with a high dose of TWHF (20 mg/kg, three times a week) compared to the control animals receiving solvent alone. A slight but significant reduction was also seen in the degree of lymphadenopathy and arthritis with the high dose treatment. Surprisingly, no significant difference was present in IgM rheumatoid factor and IgG anti-double-stranded DNA antibody levels or in the histopathology of lupus nephritis. These results indicate the dissociation of histopathological renal disease and the degree of proteinuria or the life span. Although further pharmacological analysis is required, TWHF might be potentially useful for diseases such as systemic lupus erythematosus or nephrotic syndrome. PMID- 1728982 TI - Cytotoxicity of fatty acid oxygenase activation in rat basophilic leukemia cells. AB - Apart from the generation of potent inflammatory mediators, the effects of fatty acid oxygenase activation, per se, on the host cell have not been well delineated. Fatty acid oxygenases were activated in rat basophilic leukemia cells (RBL-1) by incubating them for 2-4 hr with 33-300 microM of arachidonic acid (AA) or linoleic acid (LA). As a control, the cells were incubated with one of two analogs of these fatty acids which are not oxygenase substrates: eicosatetraynoic acid or linoelaidic acid. Effects of oxygenase activation on cell viability were monitored by an assay for mitochondrial function. Cytotoxicity occurred in incubations with exogenous AA or LA in direct proportion to the substrate concentration but was not found in the control incubations or in incubations with the principal monohydroxylated AA products, 5-, 15-, and 12-HETE. Nordihydroguaiaretic acid (80 microM) and alpha-tocopherol (100 microM) significantly decreased the cell death observed during incubations with AA or LA. It is concluded that extensive oxygenase activation can result in cell death from intermediates produced proximal to the stable monohydroxylated derivatives. PMID- 1728983 TI - IgG subclass antibodies to dietary antigens in IgA deficiency quantification and correlation with serum IgG subclass levels. AB - IgG subclasses of antibodies to the dietary antigens ovalbumin, beta lactoglobulin, casein, bovine IgM, and glycgli (a gluten component) were quantified in 20 adults and 10 children with IgA deficiency and healthy controls (21 adults and 7 children). In the IgA-deficient subjects the levels of IgG subclasses in serum were determined. Detectable antibody levels were observed in the majority of the subjects in IgG1 and IgG4 for anti-ovalbumin and beta lactoglobulin, and in IgG1, followed by IgG2, IgG3, and IgG4 for antibodies to casein, bovine IgM, and glycgli. Levels of IgG1 and IgG2 antibodies to bovine IgM were higher in the IgA-deficient adults than in controls (P less than 0.00005, P = 0.0007, respectively), whereas the other antibody levels did not differ significantly between the two groups. An analysis of correlation between the IgG subclass antibody levels did not provide evidence for a particular IgG subclass antibody response pattern against different protein antigens within the single individual. Serum IgG4 levels correlated positively with the summed IgG4 antibody levels (Spearman's p = 0.673, P = 0.0051). The IgA-deficient subjects, when compared with healthy controls, did not show a particular IgG subclass pattern or restriction of antibodies to dietary antigens. PMID- 1728984 TI - Enhanced expression of high-affinity interleukin-2 receptors in scleroderma: possible role for IL-6. AB - Scleroderma (systemic sclerosis) is characterized by tissue fibrosis, a distinctive vascular and microvascular disorder, and a perivascular mononuclear cell infiltration of involved organs. The pathogenesis of scleroderma is not known; however, there is evidence for a cell-mediated immune mechanism in the disease. Enhanced IL-2 production has been documented both in vivo and in vitro. In this study, the effect of IL-2 on lymphocyte proliferation in vitro was examined. An enhanced proliferative response to IL-2 was seen in scleroderma lymphocytes over that in matched control lymphocytes. Since high-affinity IL-2 receptors (HIL-2-R) mediate the growth-promoting activity of IL-2, we examined HIL-2-R expression on lymphocytes from 13 scleroderma and 11 matched control subjects by a radioiodinated IL-2 binding assay. Significantly higher numbers of HIL-2-R were noted in scleroderma cells (3054 +/- 618 in scleroderma vs 1721 +/- 181 in control cells, mean +/- SD; P less than 0.001). The addition of IL-6 to control cell cultures 24 hr prior to binding determination led to changes in IL-2 binding that were identical to scleroderma cell binding characteristics, while the addition of neutralizing IL-6 antibody to scleroderma cells led to a reduction in HIL-2-R expression. Other cytokines (IL-1, IL-3, IL-4, IL-5, TNF, LT, IFN-gamma, and TGF-beta) had no effect on IL-2 binding, suggesting that IL-6 may mediate the enhanced expression of HIL-2-R. This conclusion was further supported by the finding that scleroderma lymphocytes released in vitro 10- to 20 fold higher concentrations of IL-6 than control cells. The data demonstrate an amplification of IL-2 binding in scleroderma and suggest IL-6 as the mediator of this phenomenon. PMID- 1728985 TI - Recombinant human hematopoietic growth factors in the treatment of cytopenias. AB - The hemolymphopoietic growth factors, including the colony-stimulating factors (CSF) and interleukins (IL), are described and categorized on the basis of their biological features in laboratory systems. Although these agents are varied and exceptions exist, in general they lack lineage specificity although they may express lineage-predominant activity. They act at multiple levels of hemolymphopoietic cell differentiation, demonstrate additive or synergistic effects when combined in vitro, require surface receptors on target cells to directly express their activity, and may be produced by a variety of cells. This framework of behavioral generalizations, completed by the specifics of each factor's activity, despite the artifactual and simplified nature of in vivo systems, forms the basis for concepts of in vitro activity and for clinical applications. Hemolymphopoietic growth factors studied in the clinic have demonstrated impressive and important activity, validating much of the in vitro data. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have clearly reduced neutropenia and infection rates when administered following conventional chemotherapy and high-dose chemotherapy followed by autologous bone marrow transplantation. To a varying degree, similar results with G-CSF and/or GM-CSF have been described in other diseases including acute myelogenous leukemia (AML) treated following induction chemotherapy, myelodysplastic syndrome, hairy cell leukemia, aplastic anemia, and chronic neutropenias. In preliminary studies IL-3 has been shown to have similar qualitative activities. However, these agents have not demonstrated a reproducible salutary impact on platelet or red cell lineages. Adverse effects on platelet counts and/or platelet recovery have been noted. Additionally, hemolymphopoietic growth factor receptors have been identified on malignant cells, suggesting that these factors could stimulate neoplastic growth. Studies with GM-CSF and IL-3 have demonstrated blast proliferation in some cases of AML and myelodysplasia, underscoring the capacity of these agents to stimulate the growth of myeloid leukemia. No clinically evident impact of these factors upon the growth of solid tumors has been identified but this issue has not been adequately studied. The toxicity of these agents has been surprisingly limited and appears to be related to their biologic activities. Hemolymphopoietic growth factors as single agents have broad clinical applications in cytopenias. Several methods for enhancing the clinical activity of these agents are under study, including the use of combinations of growth factors synergistic in vitro. PMID- 1728986 TI - Inflammatory cytokines. AB - The immune system produces cytokines and other humoral factors to protect the host when threatened by inflammatory agents, microbial invasion, or injury. In some cases this complex defense network successfully restores normal homeostasis, but at other times the overproduction of immunoregulatory mediators may actually prove deleterious to the host. Some examples of immune system-mediated injury have been extensively investigated including anaphylactic shock, autoimmune disease, and immune complex disorders. More recently it has become clear that the cytokine cachectin/tumor necrosis factor (TNF) occupies a key role in the pathophysiology associated with diverse inflammatory states and other serious illnesses including septic shock and cachexia. For example, when cachectin/TNF is produced by resident macrophages during early microbial infection, it mediates an inflammatory response that may alienate and repel the attacking organisms. If the infection spreads, however, the subsequent release of large quantities of cachectin/TNF into the circulation may be catastrophic and trigger a state of lethal shock. These toxic effects occur by direct action of TNF on host cells and by the interaction with a cascade of other endogenous mediators including interleukin-1 and interferon-gamma. The biology of cachectin/TNF will be reviewed, along with the potential for modulating the effects of this pluripotent molecule in a variety of pathologic states. PMID- 1728987 TI - T-cell growth factors and the treatment of patients with cancer. AB - T-cell maturation has traditionally been felt to occur primarily within the thymus but it is now clear that dynamic processes in the periphery govern many functions such as T-cell activation, proliferation, tolerization, and migration into peripheral tissues. Four T-cell growth factors have now been identified. These include: interleukin-2 (IL-2), IL-4, IL-7, and a potent cofactor recently described, IL-10. These factors are believed to work synergistically in the fine regulation of the lymphoid pool. Both IL-2 and IL-4 have entered clinical trials with significant responses in the IL-2-based regimens of up to 40 to 50% in certain tumors. IL-7 and IL-10 are in preclinical studies. Although IL-2, IL-4, and IL-7 have been shown to induce lymphokine-activated killer cell activity from sensitive precursors, such studies have yet to be performed with IL-10. PMID- 1728988 TI - Cytokine regulation of eosinophil function. AB - The development and function of eosinophils are regulated by a number of cytokines. Three cytokines have major effects on eosinophilopoiesis. Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 stimulate the development of eosinophils as well as other leukocytes. Interleukin 5 promotes eosinophil development and terminal differentiation. These three cytokines also effect the functions of mature eosinophils and can prolong their longevity in in vitro culture, enhance their capacity for release of leukotriene C4 (LTC4), augment their capacity for helminthotoxicity and degranulation, and render them less dense ("hypodense") than normal, unactivated eosinophils. GM-CSF can also induce the expression of HLA-DR on mature eosinophils, which can enable eosinophils to serve as antigen-presenting cells in stimulating T-cell responses. A T-cell-derived cytokine, lymphocyte chemoattractant factor (LCF), which stimulates the migration and function of CD4+ lymphocytes and eosinophils, also utilizes CD4 expressed on human eosinophils as its receptor. LCF stimulates eosinophil migration but not degranulation, leukotriene C4 release, or respiratory burst activity. Interleukin-2 is also a potent chemoattractant for eosinophils. Thus, cytokines are involved in both increased production of eosinophils as well as regulation of the functions of mature eosinophils. These functions of mature eosinophils include effector functions and collaborative interactions with lymphocytes and other tissue cellular elements. PMID- 1728989 TI - Interleukin-6 and its relation to inflammation and disease. AB - Interleukin-6 (IL-6) is a multifunctional cytokine that regulates the immune response, acute phase reactions, and hematopoiesis. Its receptor system consists of two molecules: a ligand-binding 80-kDa molecule and a non-ligand-binding signal transducer, gp 130, both of which were found to belong to the cytokine receptor family. Deregulated IL-6 gene expression has been implicated as being involved in the pathogenesis of a number of diseases, especially autoimmune diseases and plasma cell neoplasias. In fact, IL-6 transgenic C57BL/6 mice showed a massive polyclonal plasmacytosis with production of autoantibodies and mesangial proliferative glomerulonephritis, indicating that IL-6 plays a critical role in the development of plasma cell abnormalities and glomerulonephritis. PMID- 1728990 TI - Modulation of autoimmunity by intravenous immune globulin through interaction with the function of the immune/idiotypic network. AB - Infusion of intravenous immune globulin (IVIG) has resulted in clinical improvement and/or a fall in autoantibody titer in a number of autoimmune diseases in which direct or indirect evidence suggests a pathogenic role for autoantibodies. IVIG may react with disease-associated autoantibodies through idiotypic interactions as shown by the following lines of evidence: (1) inhibition of autoantibody activity in F(ab')2 fragments of patients' IgG by F(ab')2 fragments of IVIG; (2) retention of autoantibodies on affinity columns of Sepharose-bound F(ab')2 fragments of IVIG; and (3) recognition of the same idiotypic determinants on autoantibodies by heterologous anti-idiotypic antibodies and by IVIG. IVIG also interacts with idiotypic determinants on natural autoantibodies as indicated by the binding of monoclonal IgM secreted by Epstein-Barr virus-transformed normal human B cells to F(ab')2 fragments of IVIG and by idiotypic interactions between normal IgG antibodies within the IVIG preparations. Infusion of IVIG into patients with autoimmune diseases alters the kinetic behavior of disease-associated and natural autoantibodies of unrelated specificities. It is our view that IVIG is effective in autoimmune diseases not merely by a passive transfer of suppressive anti-idiotypes, but rather by imposing a normal function on the defective network in autoimmune patients. The intrinsic complexity of IVIG would provide a more logical (physiological) rationale for immunoregulatory therapy of autoimmune disease than idiotype specific suppression. PMID- 1728991 TI - The effects of intravenous immune globulin on complement-dependent immune damage of cells and tissues. AB - The mechanism of action of intravenous immune globulin (IVIG) in the therapy of autoimmune disease has been speculated upon for many years. Previous studies have raised the possibility that IVIG acts via an effect on IgG Fc receptors (FcRs) on phagocytic cells and B lymphocytes, on the production of anti-idiotype antibody, or on the control of the immune response. In the course of our studies of complement function we were struck by the fact that complement activation often leads to the binding of complement components to individual immunoglobulin molecules. For example, C3 has been shown to bind to the Fd fragment of IgG in the form of a C3b/IgG one-to-one complex. The C3b/IgG complex has new properties that differ from those of either IgG or C3b alone in that the complex can interact with two receptors on phagocytes: CR1, which recognizes C3b, and FcR, which recognizes the Fc fragment of IgG. Particles opsonized with IgG/C3b interact with both receptors and are phagocytized rapidly. The complex acts as a superopsonin and superlysin. IgG/C3b resists degradation by factors H and I, which also adds to its inflammatory potential. We and others have noted that bacteria coated with immunoglobulin and then incubated in serum have C3 deposited on their surfaces, which in many instances is bound to the IgG molecules. For example, we found that 30% of the C3 deposited on antibody-coated pneumococci is bound not to the pneumococcal surface but rather to the coating immunoglobulin. We reasoned that IVIG may act as a receptor for activated complement components, preventing their attachment to targets. This was tested directly in a number of animal and human models. The results of these tests and their clinical implications are presented. PMID- 1728992 TI - Monoclonal antibodies in the therapy of experimental neonatal group B streptococcal disease. AB - Group B streptococcal (GBS) infections continue to be a major cause of morbidity and mortality in human neonates. This has led a number of investigators to explore the role of immunotherapy in the treatment of neonatal GBS disease. In early studies, we showed that intravenous immune globulin (IVIG) offered some protection against less virulent strains of GBS in a neonatal rat model of disease. Against more virulent strains, which produce an excess of sialic acid containing type-specific antigen, IVIG offered little protection even when given in much higher doses. For this reason, we developed murine monoclonal antibodies (MuMAb) against type III GBS. MuMAb directed against the type III-specific antigen provided excellent protection against virulent (greater than 95%) and less virulent (94-100%) strains of GBS when administered in doses as low as 400 micrograms/kg up to 24 hr after bacterial inoculation. MuMAb IgM antibody was approximately 100-fold more effective than MuMAb IgG2a antibody. Unfortunately, MuMAbs are unlikely to be approved for use in human neonates. For this reason, we have evaluated a human monoclonal antibody (HuMAb) preparation against GBS derived from Epstein-Barr virus-immortalized peripheral blood B lymphocytes. This IgM HuMAb, which appears to be directed against the group B carbohydrate, is extremely active in both opsonic and protective assays against type Ia, II, and III GBS. Optimal immunotherapy of neonatal GBS disease may involve the use of HuMAb preparations, alone or in combination with polyclonal IVIG. PMID- 1728993 TI - Directed immune globulin for the prevention or treatment of neonatal group B streptococcal infections: a review. AB - Intravenous immune globulin (IVIG) is now used in many nurseries to prevent or treat neonatal infections. The most common cause of early-onset neonatal sepsis is the group B streptococcus (GBS). Commercially available IVIG preparations have variable levels of specific antibody directed against GBS. Therefore, to ensure high levels of anti-GBS antibody, we developed a polyvalent IVIG directed against GBS (GBS-IVIG) by immunizing plasma donors. This GBS-IVIG was superior to standard IVIG both in vitro using opsonic studies and in vivo using a lethal suckling rat model of GBS sepsis. GBS-IVIG also protected neonatal rhesus monkeys in a GBS sepsis model. Safety and pharmacokinetic studies have been completed in 20 neonates with suspected sepsis. Fifteen infants were randomized to receive 500, 250, or 100 mg/kg of GBS-IVIG and were compared with 5 infants given 500 mg/kg of standard IVIG. No adverse effects of standard IVIG or GBS-IVIG were observed. While total serum IgG and IgG subclasses reflected the dose administered, the specific GBS antibody reflected both the dose and IVIG preparation utilized. At 500 mg/kg, the GBS-specific antibody rises more than fourfold above baseline in all babies that were observed for greater than 42 days postinfusion, while standard IVIG provided a fourfold rise in less than 20% of babies for less than 1 day. These studies suggest that GBS-IVIG can effectively and reliably elevate GBS-specific antibody levels in neonates. Clinical trials are needed to evaluate the efficacy of GBS-IVIG in preventing or treating neonatal GBS infections. PMID- 1728994 TI - Symposium: 1991 proceedings of the Hip Society. PMID- 1728995 TI - Automated scanning and digitizing of roentgenographs for documentation and research. AB - A comprehensive system has been developed for analyzing and reporting the results of total hip arthroplasty. The personal-computer-based system links patient demographic data with digital storage, retrieval, and analysis of roentgenographs. The system consists of a roentgenograph scanner for converting sheet film to digital data, an optical mark reader for patient data input, an archiving system with optical storage, and a physician display station for preoperative planning and postoperative evaluation. Once a roentgenograph has been digitized and stored, the image can be retrieved and manipulated in a manner not possible with the original sheet film. A selected roentgenograph can be brought to full or enlarged scale, enhanced, and overlaid with templates for preoperative planning or for postoperative measurement of changes. In addition, an intelligent database system has been developed for linking patient demographic information with the roentgenographic data. The database system employs uniform criteria and terminology and allows the retrospective study and statistical analysis of comparable cases. Three machine-readable code sheets are used: Form A, Replacement of the Hip; Form B, Hip Prosthesis Reoperation; and Form C, Follow up. Forms A and B contain information concerning anamnesis, diagnosis, treatment, postoperative course, recovery, and discharge of the patient from the hospital. Form C provides information on physical examination, pain, mobility of the hip, walking ability, and evaluation of the results by the surgeon as well as the patient. PMID- 1728996 TI - Lessons of 30 years of total hip arthroplasty. AB - The following are lessons of 30 years of total hip arthroplasty (THA): Prosthetic components should allow for easy preoperative graphic planning with one or two templates only. Polyethylene is the weak link in THA and ought to be replaced by perfectly concentric metallic sockets and femoral heads of casted Cr/Co/Mo alloys. The operating technique should guarantee large exposure, suitable orientation of both components, and adequate fixation. Loosening is correlated with loss of bone stock and changes of the position of the implants. In case of progressive bone loss, one should reoperate already in presence of slight clinical symptoms to prevent more difficult and dangerous revisions. To evaluate objectively the outcome of THA, a combined data and roentgenographic documentation system with standardized terms and scales is necessary. PMID- 1728997 TI - Will stress shielding limit the longevity of cemented femoral components of total hip replacement? AB - One must acknowledge the speculative nature of evaluating whether stress shielding will limit the longevity of cemented femoral components. One can, however, evaluate the data available from patients studied up to 20 years. Stress shielding does lead to disuse osteoporosis, particularly in the proximal medial cortex, and also more slowly and to a lesser extent elsewhere. Concern also exists that progressive endosteal enlargement will lead to failure of many or all cemented femoral stems. No evidence exists that disuse osteoporosis has limited the longevity of cemented femoral stems up to 20 years. Moreover, new data show that what has been described as the normal process of endosteal enlargement after inserting a cemented femoral component is a misinterpretation. What actually happens is that (1) neocortex develops around the cement mantle, (2) the original cortex thins, (3) the trabeculae in the zone of decreased density that develops between them maintain the structural integrity between those two bony masses, and (4) the implant remains rigidly fixed. PMID- 1728998 TI - The relationship between stress shielding and bone resorption around total hip stems and the effects of flexible materials. AB - Bone resorption around hip stems is a disturbing phenomenon, although its clinical significance and its eventual effects on replacement longevity are as yet uncertain. The relationship between implant flexibility and the extent of bone loss, frequently established in clinical patient series and animal experiments, does suggest that the changes in bone morphology are an effect of stress shielding and a subsequent adaptive remodeling process. This relationship was investigated using strain-adaptive bone-remodeling theory in combination with finite element models to simulate the bone remodeling process. The effects of stem material flexibility, bone flexibility, and bone reactivity on the process and its eventual outcome were studied. Stem flexibility was also related to proximal implant/bone interface stresses. The results sustain the hypothesis that the resorptive processes are an effect of bone adaptation to stress shielding. The effects of stem flexibility are confirmed by the simulation analysis. It was also established that individual differences in bone reactivity and mechanical bone quality (density and stiffness) may account for the individual variations found in patients and animal experiments. Flexible stems reduce stress shielding and bone resorption. However, they increase proximal interface stresses. Hence, the cure against bone resorption they represent may develop into increased loosening rates because of interface debonding and micromotion. The methods presented in this paper can be used to establish optimal stem-design characteristics or check the adequacy of designs in preclinical testing procedures. PMID- 1728999 TI - Imaging of the hip joint. Computed tomography versus magnetic resonance imaging. AB - The authors reviewed the applications and limitations of computed tomography (CT) and magnetic resonance (MR) imaging in the assessment of the most common hip disorders. Magnetic resonance imaging is the most sensitive technique in detecting osteonecrosis of the femoral head. Magnetic resonance reflects the histologic changes associated with osteonecrosis very well, which may ultimately help to improve staging. Computed tomography can more accurately identify subchondral fractures than MR imaging and thus remains important for staging. In congenital dysplasia of the hip, the position of the nonossified femoral head in children less than six months of age can only be inferred by indirect signs on CT. Magnetic resonance imaging demonstrates the cartilaginous femoral head directly without ionizing radiation. Computed tomography remains the imaging modality of choice for evaluating fractures of the hip joint. In some patients, MR imaging demonstrates the fracture even when it is not apparent on radiography. In neoplasm, CT provides better assessment of calcification, ossification, and periosteal reaction than MR imaging. Magnetic resonance imaging, however, represents the most accurate imaging modality for evaluating intramedullary and soft-tissue extent of the tumor and identifying involvement of neurovascular bundles. Magnetic resonance imaging can also be used to monitor response to chemotherapy. In osteoarthrosis and rheumatoid arthritis of the hip, both CT and MR provide more detailed assessment of the severity of disease than conventional radiography because of their tomographic nature. Magnetic resonance imaging is unique in evaluating cartilage degeneration and loss, and in demonstrating soft tissue alterations such as inflammatory synovial proliferation. PMID- 1729000 TI - Use of computed tomographic reconstruction in planning osteotomies of the hip. AB - Certain deformities of the hip joint seem to predispose the hip to the development of osteoarthrosis. Successful surgical correction of these deformities before the onset of osteoarthrosis requires accurate characterization of the anatomic deviations from normal as the first step in planning corrective osteotomy. Three-dimensional computed tomography (CT) reconstruction in planning reconstructive hip osteotomy has most often been employed in developmental dysplasia of the hip. Computed tomography scanning with three-dimensional reconstruction can characterize the often complex deviations from normal in shape and attitude of acetabulum and femoral head in cases with residual hip dysplasia. Three-dimensional reconstruction also allows simulation of redirectional femoral or pelvic osteotomies to facilitate precise application of newer powerful surgical techniques for reorienting the acetabulum. PMID- 1729001 TI - Use of ultrasonography in dysplasia of the immature hip. AB - Real-time ultrasonography reconstruction is an imaging technique of relatively recent origin that has dramatically affected the diagnosis and treatment of patients with hip dysplasia. Ultrasonography is replacing conventional radiography as the primary method of diagnosing hip dysplasia and evaluating its treatment during the first six to nine months of life. While conventional radiography reveals a familiar two-dimensional image of ossified structures and to a much lesser extent, soft-tissue structures, it has disadvantages that include ionizing radiation, a relative inability to evaluate unossified tissues, and an inability to gain three-dimensional information without supplemental techniques. PMID- 1729002 TI - Current status of acetabular fixation in primary total hip arthroplasty. AB - Factors that influence the outcome of acetabular replacement are design materials, means of fixation, operative technique, and patient-related parameters (e.g., etiology of osteoarthrosis). Whereas improved cementing techniques have produced a marked reduction in the rate of femoral component loosening, the incidence of acetabular loosening has been only slightly influenced by such improvements. Presently, uncemented porous-coated acetabular components represent the state of the art in total hip arthroplasty. Experimental and clinical data have shown in histologic, radiologic, clinical, and survivorship studies that hemispheric cups are superior to other designs and that primary stability can be better maintained by creating "intrinsic" stability (e.g., "oversized cup") rather than by screw fixation. Threaded cups have failed to demonstrate any improvement in results and have been virtually abandoned in the United States. The idea of metal backing has some obvious theoretical advantages. However, metal backing has failed to provide any improvement with respect to cemented cups. There are great reservations concerning metal backing in cementless fixation. Although there is some enthusiasm about hydroxyapatite, a "wait-and-see" attitude is justified because of the brittleness of the material, its questionable strength of bonding to substrate, and its unproven long-term behavior in vivo. Polyethylene as a bearing surface remains problematic, and the future will show whether new technologies are able to solve the problems encountered with metal-to metal combinations. For the size of the femoral head, a compromise between smaller (22 mm) and larger (32 mm) components seems to be most effective. PMID- 1729003 TI - Natural factors that affect the shape and strength of the aging human femur. AB - The human skeleton loses bone mass with increasing age. A wide variety of evidence suggests that the skeleton compensates for this decreased bone mass by increasing the second moment of area in the midshaft of long bones. Strain is the mechanical transducer of bone's adaptation to its mechanical environment. In the absence of strain, bone resorption occurs. Contemporary femoral prostheses are stiffer than the cortical bone that serves as their host and, therefore, induce stress shielding and bone resorption that augments the naturally occurring loss of bone mass with age. With the expectation that total hip replacement can endure 15-25 years, one must be aware that a new limit to its longevity may be the biologic failure of the host bone. Particulate debris, stress shielding, and the natural consequences of aging are conspiring to make the proximal femur a diminishing site. The design strategy and materials of fabrication of future total hip prostheses should seek to maximize the preservation of bone. PMID- 1729004 TI - Total hip arthroplasty. PMID- 1729005 TI - Determinants of stress shielding: design versus materials versus interface. AB - Experimental studies of cementless porous-coated total hip arthroplasty indicate that a critical design variable for femoral remodeling is stem stiffness. In the long term (two years) in the canine model, other variables, including the presence, type, and placement of the porous coating, did not significantly affect the pattern of bone remodeling when tested with metallic stems. The basic pattern of bone remodeling was characterized by proximal cortical atrophy, and distal cortical and medullary bone hypertrophy. In the short term (six months), the use of low-stiffness stems altered this pattern, leading to reduced proximal bone loss, increased proximal medullary bone hypertrophy, and no distal cortical hypertrophy, suggesting that stem stiffness had a profound effect on stress shielding. PMID- 1729006 TI - Hip fractures in geriatric patients. Results of an interdisciplinary hospital care program. AB - The care of geriatric patients who sustain hip fractures is difficult because of associated medical comorbidities, the risk of medical and surgical complications, and the functional limitations that are often present before the fracture. The authors developed and used a comprehensive, interdisciplinary care program that has so far treated 431 geriatric hip fracture patients. The results of the program group were compared to a matched nonprogram group of patients (n = 60) cared for before the initiation of the program (and before the initiation of diagnosis-related groups). The program patients had fewer postoperative complications, significantly fewer (p less than .05) intensive care unit transfers (10.2% versus 20%), significantly improved (p less than .001) ambulatory ability at discharge (56.3% independent with assistive devices versus 18.2%), and proportionately fewer discharges to nursing homes (8.1% versus 19.3%). These results support the use of an interdisciplinary approach as a means of improving the inhospital care of geriatric hip fracture patients. PMID- 1729007 TI - Lessons learned in 30 years of total hip arthroplasty. AB - Lessons learned in 30 years of total hip arthroplasty (THA) include the following: Know and understand the evolution of hip arthroplasty. Selection of the prosthesis must be carefully made from sound clinical and scientific data. Before embarking on a prosthesis program, establish an immediate and a prospective protocol for the operation during the neosurgical period and for an indefinite long-term follow-up period. Establish a data bank for easy and complete retrieval of all pertinent material. Be familiar with the basic biomechanical principles of load and stress about the hip. Learn to use polymethylmethacrylate properly. The author's studies, and others, have shown that with newer, improved methods, results are excellent. Do not accept biologic (ingrowth) fixation as the ultimate aim in THA. Many problems are emerging with biologic fixation. Be wary of the complications in THA that relate to technique as well as design and even patient selection. Loosening, infection, continued pain, ion absorption, stress shielding, possible malignancy, and foreign body reaction are all complications that must be addressed. A surgeon should never lose the ability to review his or her own experience with unbiased, objective scrutiny. PMID- 1729008 TI - The influence of anatomic factors in elbow joint dislocation. AB - Twenty-seven patients with elbow joint dislocation were reviewed and treated between 1979 and 1983. For statistical reasons a sample of 27 elbows of healthy adults matched on age and sex were also roentgenographed. A computer-aided analysis of the carrying angle of the arm and the bow angle of the ulnar trochlear notch was made. The stability of the joint was tested clinically. The carrying angle showed no pathologic deviation. Values of the bow angle were at the lower end of the range given in the literature. Referring to these results, only normal anatomic constitutional factors predisposing to dislocation were found. Ligament repair was not routinely performed in this series. Nevertheless, recurrent dislocation did not occur and no relevant instability was detected. Except for injuries with osseous lesions, conservative treatment is advisable in most cases. PMID- 1729009 TI - Laboratory evaluation of alignment and kinematics in a unicompartmental knee arthroplasty inserted with intramedullary instrumentation. AB - The purposes of this study were to evaluate the reliability of intramedullary (IM) instrumentation for unicompartmental total knee replacement and to assess the stability characteristics of the knee after implantation of a relatively unconstrained articular surface. Five adult, human cadaver lower extremities including hip, knee, and ankle were used to evaluate IM alignment. Five adult, fresh-frozen knee specimens were used to evaluate knee kinematics. Long anterioposterior roentgenograms were used to evaluate valgus angle and position of the center of the knee relative to the mechanical axis of the lower extremity. IM instrumentation returned the knee to normal alignment in all cases. The greatest valgus angle change was 3 degrees, and the position of the center of the knee relative to the mechanical axis was not significantly altered. Knee kinematics after unicompartmental knee replacement followed the predicted pattern of normal stability in extension and had slightly less varus-valgus laxity at 30 degrees (p less than 0.01), 45 degrees (p less than 0.01), and 60 degrees (p less than 0.05), and less anteroposterior displacement at 45 degrees (p less than 0.01) and 60 degrees (p less than 0.05). This study offers encouraging evidence that unicompartmental knee replacement with unconstrained components can restore normal knee kinematics, and that alignment can be restored with a high degree of accuracy with an intramedullary alignment system. PMID- 1729010 TI - Preoperative planning for high tibial osteotomy. The effect of lateral tibiofemoral separation and tibiofemoral length. AB - To calculate the tibial wedge size in preoperative planning of high tibial osteotomy, the weight-bearing line (center femoral head to center tibiotalar joint) is first restored to a selected position on the lateral tibial plateau. Ten full-standing roentgenograms were examined and used to derive mathematical formulas for correcting limb alignment. A 3-4-mm shift in the weight-bearing line on the tibia occurred with each degree of tibiofemoral angulation. This showed that the position of the weight-bearing line is sensitive to the lengths of the tibia and femur as well as the surgeon's preoperative calculations. The problem of increased varus angulation due to slack lateral ligament restraints with distraction of the tibiofemoral joint was analyzed. Trigonometric analysis showed that each millimeter of lateral tibiofemoral joint separation caused about 1 degree varus angular deformity, requiring subtraction in preoperative calculations to avoid overcorrection. An algorithm was designed to evaluate complex lower-limb alignments. PMID- 1729011 TI - Patella alta and recurrent dislocation of the patella. AB - Fifteen knees were treated with a surgical technique designed for recurrent patellar dislocations when associated with patella alta. The average number of patellar dislocations preoperatively was 12, and the determination of patella alta was made using the technique described by Insall and Salvati, which is a ratio of the length of the patellar tendon to the longest diagonal length of the patella. The normal value for this ratio is 1.02 plus or minus 20%. The average preoperative ratio in this study was 1.58 (range, 1.2-2.1), which changed to 1.08 (range, 0.99-1.14) by the time of follow-up examination. The surgical technique used involves transposing the patellar tendon insertion distally without any medialization or recessing and allows for good bone contact for healing, secure fixation, and immediate postoperative motion. There were no recurrences of patellar dislocation postoperatively and few complications. Only one patient complained of anterior knee pain in the follow-up period. This technique is thought to give good results when it is used specifically for recurrent patellar dislocations associated with patella alta. PMID- 1729012 TI - Benign metastasizing giant-cell tumor of the hand. Report of a case and review of the literature. AB - Benign metastasizing giant-cell tumor of the hands is an extremely infrequent neoplasm. A 37-year-old man with one of these neoplasms in the second phalanx of his fifth left finger was examined. Microscopically, the tumor was a conventional Grade 1 giant-cell tumor that metastasized to the lung. Although this tumor is most infrequent, its metastatic potential indicates inclusion of benign metastasizing giant-cell tumor in the differential diagnosis with other giant cell neoplasms of the hands. PMID- 1729013 TI - Second cancers in long-term survivors of Ewing's sarcoma. AB - Previous reports suggest an increased risk of a second cancer, primarily osteosarcoma, in survivors of Ewing's sarcoma. In a retrospective review of 25 long-term irradiated survivors of Ewing's sarcoma, the incidence of second cancers was determined. The patients were free of disease for more than three years (except for one patient who developed a second cancer 2.5 years after diagnosis), with a median follow-up period of 7.6 years. All received megavoltage radiation to the primary tumor. Twenty-four of the 25 patients were treated with chemotherapy. Second cancers developed in two patients. Acute myelogenous leukemia (AML) developed in a seven-year-old 15 months after treatment. An osteosarcoma developed within an irradiated field in a 13-year-old three years after treatment. The actuarial risk of developing a second cancer at five years is 8% whereas the actuarial risk of developing a bone sarcoma is 4%. Genetic factors may play a role in the development of AML in patients with Ewing's sarcoma. Megavoltage radiation, particularly doses greater than 60 Gy, as well as alkylating agent chemotherapy may contribute to the risk for bone sarcoma. The risk of a second cancer after successful treatment of Ewing's sarcoma is similar to that expected for survivors of all childhood cancers. PMID- 1729014 TI - Bone graft incorporation around titanium-alloy- and hydroxyapatite-coated implants in dogs. AB - To evaluate cancellous allogenic bone graft incorporation into porous-coated implants, the fixation of titanium alloy-(Ti) and hydroxyapatite-(HA) coated implants with and without bone graft was compared. An unloaded model with unilateral carragheenin-induced osteopenia of the knee was used in 12 mature dogs. Ti- and HA-coated cylinders were implanted in the distal femoral condyles and centralized in 2-mm overreamed drill holes. Allogenic, fresh-frozen (-80 degrees) cancellous bone graft was packed around the implants in six dogs. In a matched group of six other dogs, the implants were left in overreamed canals without bone graft. After six weeks the interface shear strength of grafted Ti coated implants had significantly increased compared to the nongrafted Ti implants. However, HA coating used without bone graft was capable of enhancing the bone-implant interface shear strength to nearly the same degree. The fixation of grafted Ti- and HA-coated implants was equal. No significant difference in implant fixation was found between osteopenic and control bone. Histomorphometric evaluation of mineralizing surfaces in direct contact with the implant confirmed the results from the push-out test. Bone-implant fixation when using allogeneic fresh-frozen cancellous bone graft in osteopenic and control bone was enhanced by hydroxyapatite coating but the HA coating alone appeared to offer almost the same improvement in anchorage in 2-mm defects. Loss of bone stock around loose prosthetic implants often requires bone grafting. However, because of anatomic constraints in joint prosthetic surgery, a complete filling of defects with bone graft is difficult, and areas of gaps between bone and implant will remain. Provided mechanical stability of the prosthesis, the results reported here suggest that these areas will probably be filled early with new mineralizing bone if the prosthesis is coated with a thin layer of hydroxyapatite. PMID- 1729015 TI - Synovial fluid stimulates the proliferation of rabbit ligament. Fibroblasts in vitro. AB - This study was designed to test the hypothesis that synovial fluid may be inhibitory to cell proliferation. The effects of bovine synovial fluid (SF) and hyaluronic acid (HA) on the proliferation of normal rabbit medial collateral ligament (MCL), anterior cruciate ligament (ACL), and MCL scar cells were therefore investigated. Cell lines established from rabbit tissues were plated, incubated, and allowed to attach before treatment with varying concentrations of SF, HA, and a balanced salt solution (BSS). The BSS group was added as a control to observe the effects of media dilution alone on cell proliferation. Cell numbers from each group were quantified at 24, 48, 72, and 96 hours. Results showed that for all cell types, cell proliferation during the log phase of growth was significantly stimulated by SF. Maximum stimulation occurred in 20% SF with stimulation decreasing at higher concentrations of SF. HA had virtually no effect on scar and ACL cells, and only a slight stimulatory effect on MCL cells. Media dilution had no effect on scar cells and began to inhibit cell proliferation of ACL and MCL cells only at high dilutions. These findings suggest that low concentrations of bovine SF stimulate proliferation of rabbit ligament and scar fibroblasts in vitro by a mechanism that appears not to involve HA. Even in high concentrations, SF was not inhibitory to proliferation. The implications of these findings to ligament healing and normal ligament physiology require further investigation. PMID- 1729016 TI - Signs by which to diagnose congenital dislocation of the hip. 1928. PMID- 1729017 TI - Total hip arthroplasties in the elderly. Morbidity, mortality, and cost effectiveness. AB - Forty-two patients older than 80 years were treated with hip arthroplasty from 1980 to 1986. Seventy-five percent experienced a complication. The most common complications were excessive bleeding, postoperative confusion, urinary tract infection, and dislocation. Hospital stay averaged 16 days and was more prolonged in 14 patients. There was one postoperative death. The survival time of the other 41 patients currently ranges from nine months to eight years. At this time, 50% are alive and functional at an average of five-years follow-up evaluation. Comparing the cost of hip arthroplasty to the cost of nursing home placement, the procedure is clearly cost effective. PMID- 1729018 TI - Pathologic fracture associated with amyloid deposition in the bone of a chronic hemodialysis patient. A case report. AB - A 63-year-old male who had been on hemodialysis for 14 years developed extensive amyloid deposition in the femoral head and neck that, after a fall, resulted in a femoral neck fracture. The plasma level of beta 2-microglobulin in this patient was elevated, and bone specimens from the fracture site demonstrated positive Congo-red staining, suggesting an association with amyloid deposition. PMID- 1729019 TI - Nondiaphyseal osteoid osteomas in the pediatric patient. PMID- 1729020 TI - Correction of distal femoral deformity. PMID- 1729021 TI - Intraoperative heparin thromboembolic prophylaxis in primary total hip arthroplasty. A prospective, randomized, controlled, clinical trial. AB - Venous thromboembolic disease remains the most common and potentially fatal complication after total hip arthroplasty (THA). Proximal femoral deep vein thrombosis (DVT) is especially prone to propagate and embolize. The authors' hypothesis was that intraoperative intravenous heparin administration could reduce proximal DVT in THA. There were 286 patients who entered into a prospective, double-blind, randomized clinical trial at the authors' institution between June 1988 and May 1990. All patients had unilateral primary THA under hypotensive epidural anesthesia. The epidural catheter was placed at least 60 minutes before heparin administration. Intravenous heparin was given during surgery only. All patients received aspirin twice daily (650 mg/day) after surgery. Detection of DVT was by contrast venography on Postoperative Day 6 or 7. The study was divided into three phases. There was four groups: control (intraoperative saline), 30 minutes (1000 U heparin at beginning of surgery followed by 500 U every 30 minutes), continuous adjusted (1000 U or 1500 U initial bolus followed by continuous heparin infusion maintaining anticoagulation at 30%-50% elevation from baseline), and fixed dose (1000 U bolus before hip dislocation, and 500 U bolus before femoral canal preparation). Proximal femoral DVT was effectively reduced from 9.1% in the control group to 1.7% in the heparin groups (1.7% in 30 minute, 1.6% in continuous adjusted, 1.7% in fixed dose) (p less than 0.02). The overall DVT rate was also significantly reduced from 24.3% to 10% (p less than 0.01). No adverse effects from heparin administration were noted. Postoperative drainage, hematocrit levels on Postoperative Day 2 and at discharge, and transfusion requirements were not significantly different among the groups. The current recommended protocol is 1000 U bolus five minutes before hip dislocation, followed by 500 U bolus five minutes before femoral preparation. This, in conjunction with hypotensive epidural anesthesia and postoperative aspirin, is effective in reducing proximal DVT to less than 2% in primary THA. PMID- 1729022 TI - A long-term study on defect filling and bone ingrowth using a canine fiber metal total hip model. AB - When performing primary and revision total hip arthroplasty (THA), bone defects are often encountered. At present, grafting osseous defects with autogeneic bone is a common means of treatment. In this study, defects in bone were created in the femora and acetabula of dogs being treated with cementless THA with a fiber metal implant (Group A) or a hydroxyapatite tricalcium phosphate (HA/TCP) sprayed implant (Group B). The following methods of defect filling were compared: (1) leaving defects unfilled, (2) filling with autogeneic bone graft, (3) filling with a 50:50 mixture of autograft and a biphasic ceramic composed of HA/TCP, and (4) filling with a collagen-HA/TCP-bone marrow mixture. Analysis of defect healing and the extent of ingrowth into the overlying fiber metal, at defect sites and sites distant from defects, was made at six, 12, and 24 weeks postimplantation. Defect healing was enhanced at six and 12 weeks in all grafted groups when compared with ungrafted controls. Bone ingrowth into the porous fiber metal overlying the defects was not significantly affected by grafting the defects, compared with the ungrafted defects. The extent of bone ingrowth into the fiber metal acetabular implant at sites away from the defects increased during the entire study. In contrast, the extent of bone ingrowth on the femoral side was maximal at 12 weeks. The HA/TCP coating enhanced ingrowth into the acetabular component at 12 weeks, compared with the uncoated prosthesis, but did not enhance ingrowth on the femoral side. The data from this study demonstrate that defect healing is enhanced with graft materials. However, this does not necessarily result in increased ingrowth into porous surfaces overlying osseous defects. General bone ingrowth and ingrowth at defect sites at 12 weeks postimplantation can be enhanced on the acetabular side with the use of HA/TCP sprayed implants. However, no positive effect is seen with the use of an HA/TCP sprayed femoral implant. PMID- 1729023 TI - The first 32 years of total hip arthroplasty. One surgeon's perspective. AB - During the first 32 years of total hip arthroplasty, high risk of sepsis, improved prevention of infection, problems of loosening, new disease of bone lysis secondary to particulate debris, and the complexities of cementless fixation have taught orthopedic surgeons many lessons. These lessons include the following: (1) In cemented THA, bone cement can be made five-times stronger just by porosity reduction; the critical interface in a cemented femoral stem is the cement-metal interface, not the cement-bone interface; and no one has solved the long-term fixation problem in cemented sockets. (2) In cementless implants, the disuse osteoporosis that occurs around cementless femoral components can be severe; the use of cementless implants does not eliminate bone lysis; only small amounts of bone ingrowth occur in many cementless implants, particularly in revision cases; and little or no bone ingrowth occurs from grafts. Today, however, some of these lessons are ignored by surgeons. If progress is to be made in arthroplasty, these lessons from the past should be learned and warning signs of the present should be heeded. PMID- 1729024 TI - The mechanism of loosening of cemented acetabular components in total hip arthroplasty. Analysis of specimens retrieved at autopsy. AB - Late aseptic loosening of cemented acetabular components is governed by the progressive, three-dimensional resorption of the bone immediately adjacent to the cement mantle. This process begins circumferentially at the intraarticular margin and progresses toward the dome of the implant. Evidence of bone resorption at the cement-bone interface was present even in the most well-fixed implants before the appearance of lucent lines on standard roentgenographic views. The mechanical stability of the implant was determined by the three-dimensional extent of bone resorption and membrane formation at the cement-bone interface. The leading edge of the membrane is a transition zone from regions of membrane interposition between the cement and the bone to regions of intimate cement-bone contact. Histologic analysis revealed that progressive bone resorption is fueled by small particles of high density polyethylene (HDP) migrating along the cement-bone interface and bone resorption occurs as a result of the macrophage inflammatory response to the particulate HDP. Evidence in support of a mechanical basis for failure of fixation was lacking. The mechanism of late aseptic loosening of a cemented acetabular component is therefore biologic in nature, not mechanical. This is exactly opposite to the mechanism of loosening on the femoral side of a cemented total hip replacement, which is mechanical in nature. PMID- 1729025 TI - Producing and avoiding stress shielding. Laboratory and clinical observations of noncemented total hip arthroplasty. AB - Experimental canine model studies of stiff versus flexible, fully porous-coated, metallic femoral stems (differing by three- to fivefold in stiffness characteristics) revealed markedly different resorptive bone remodeling patterns. The flexible stem resulted in about 30% more cortical bone retention adjacent to the implant at one-year postimplantation and larger differences in dogs killed two and three years after surgery. Strain-gauge studies confirmed that there are differences in cortical bone strains with the two stem designs, the flexible stem producing a more uniform and more nearly normal strain distribution medially. Differences in cortical bone remodeling were quantified using dual energy X-ray absorptiometry (DEXA). The bone mineral content in femora with the flexible stem decreased less than 20%, compared to normal. At three years postimplantation, the bone mineral content of the femora with the stiff stem was about 50% that of the femora with the flexible stem. Clinically, DEXA revealed that 5%-15% changes in bone mineral density at various periimplant sites were common within the first two years after surgery; these changes were not usually evident roentgenographically. Serial roentgenographically distinct bone resorption was usually associated with bone mineral density changes of 20%-50%. Five- to 13-year roentgenographic follow-up observations of 213 cases with the Anatomic Medullary Locking prosthesis showed that pronounced bone resorption occurred in 33% of patients. Larger stems (greater than 13 mm in diameter) and stems with extensive porous coating had a significantly higher incidence of pronounced bone resorption than smaller stems and those with proximal coating. The stiffness characteristics of the human femur were established as a function of canal size and compared with those of noncemented hip prostheses. Increased mechanical compatibility was found for stems made of titanium alloy and with design features that reduce cross sectional area and moment of inertia. Clinical data suggest that to reduce the likelihood of pronounced bone resorption, it would be beneficial for the implant to possess a bending stiffness of about one half to one third that of the human femur. PMID- 1729026 TI - Results of implant retrieval from postmortem specimens in patients with well functioning, long-term total hip replacement. AB - The evaluation of postmortem specimens provides a unique opportunity to gain understanding of the interface between host and well-functioning prostheses unavailable from revision specimens that, by nature, are accompanied by the artifacts generated during their removal. The preliminary findings from the relatively limited number of specimens described in this collaborative study demonstrate the value of the effort and are presented to encourage surgeons to participate in this program and to make their patients aware of the value of the information they may provide. PMID- 1729027 TI - Special issues: glucose and the brain. AB - PURPOSE: This review focuses on the neurologic issues concerning the treatment of hypo- or hyperglycemia in the critically ill patient. DATA SOURCES: Articles written in English and identified through the Bibliographic Retrieved Service Colleague database. STUDY SELECTION: Articles chosen on the basis of their relevance to the issue of blood glucose management and its neurologic effects in critically ill patients. DATA EXTRACTION: Data from articles were analyzed to obtain a scientific foundation and rationale for treating abnormal blood glucose levels. DATA SYNTHESIS: Moderate hypoglycemia may evoke a significant stress response, behavioral changes, and alterations in cerebral blood flow and metabolism. It is unclear what effect prolonged or repeated episodes of moderate hypoglycemia may have on patient outcome. However, alterations in cerebral vascular physiology must be addressed when caring for patients with cerebral ischemia or intracranial compliance problems. Depending on its severity, hypoglycemia has varying influences on neurologic damage after ischemia. Hyperglycemia may impair neuronal recovery following cerebral ischemia. However, the detrimental effects of hyperglycemia vary depending on the types of brain ischemia sustained (focal or global). Evidence suggests that hyperglycemia during global and incomplete global ischemia events is detrimental to neurologic outcome. However, the relationship between hyperglycemia and outcome after focal ischemia is controversial. CONCLUSION: Because both hypo- and hyperglycemia may produce neurologic changes, aggressive management of abnormal glucose values is warranted. PMID- 1729028 TI - Immediate postoperative enteral feeding decreases weight loss and improves wound healing after abdominal surgery in rats. AB - OBJECTIVE: To determine the effect of immediate vs. delayed (72 hrs) postoperative enteral feeding on weight loss and wound healing after experimental abdominal surgery. DESIGN: Prospective, randomized, controlled study. SETTING: Laboratory of a large university-affiliated medical school. SUBJECTS: Seventeen male Sprague-Dawley rats, each weighing 350 to 400 g. INTERVENTIONS: Four centimeter longitudinal midabdominal incisions were made and gastroduodenal feeding tubes were inserted in the animals. The abdominal wound was closed in two layers. Immediately after closure, animals were randomized to receive immediate enteral feeding (early-fed group) with a peptide-based enteral formula or 5% dextrose in water at 4 mL/hr. Seventy-two hours after surgery, the 5% dextrose in water group was switched to the peptide formula (late-fed group). Animals were weighed daily. On postoperative day 5, the strength of the abdominal wound was determined using a balloon-bursting pressure technique. Blood was also obtained for measurement of insulin growth factor 1 concentrations. Mucosal protein content of the small bowel was measured. RESULTS: Total body weight loss was less in the early-fed group (26 +/- 4 vs. 46 +/- 5 g/5 days) and wound strength was increased in the early-fed group compared with the late-fed group (6 +/- 0.4 vs. 2.9 +/- 0.8 kPa; 45 +/- 3 vs. 22 +/- 6 mm Hg). There were no differences between groups for circulating insulin growth factor 1 concentrations or small intestinal mucosal protein concentrations. CONCLUSIONS: Immediate postoperative enteral feeding results in decreased weight loss and improved wound healing after abdominal surgery in rats. PMID- 1729029 TI - Longitudinal distribution of pulmonary vascular resistance after endotoxin administration in sheep. AB - BACKGROUND AND METHODS: Pulmonary hypertension may increase pulmonary capillary pressure and exacerbate pulmonary edema in acute respiratory failure. The effects of pulmonary hypertension on pulmonary capillary pressure depend on the longitudinal distribution of pulmonary vascular resistance. Since pulmonary hypertension occurs during acute respiratory failure, we hypothesized that acute respiratory failure may produce time-dependent changes in the longitudinal distribution of pulmonary vascular resistance. Therefore, we measured pulmonary capillary pressure and the longitudinal distribution of pulmonary vascular resistance in an animal model of acute respiratory failure. Escherichia coli endotoxin (2.5 to 5.0 micrograms/kg) was administered over a 1-hr period in eight anesthetized sheep. Pulmonary and systemic hemodynamics, including pulmonary artery occlusion pressure (PAOP), pulmonary capillary pressure, and the longitudinal distribution of pulmonary vascular resistance, were measured over the next 5 hrs. Pulmonary capillary pressure was estimated by analysis of the pressure decay following pulmonary artery balloon inflation. RESULTS: Endotoxin administration resulted in sustained pulmonary hypertension for the subsequent 5 hrs of the study. Pulmonary capillary pressure was increased 7 mm Hg above baseline at 0.5 and 0.75 hrs during the infusion of endotoxin but returned to baseline values at 1.5 hrs. Despite sustained pulmonary hypertension, pulmonary capillary pressure remained at baseline values for the duration of the study. Similar to pulmonary capillary pressure, pulmonary venous (or postcapillary) resistance was increased approximately four-fold over baseline at 0.5 and 0.75 hrs after initiating endotoxin administration, but returned to baseline values by the end of endotoxin administration and remained at baseline values throughout the remainder of the study. In contrast, pulmonary arterial (or precapillary) resistance remained at values approximately three times baseline during the infusion and throughout the duration of the study. CONCLUSIONS: In this experimental model of acute respiratory failure, the effects of endotoxin on the longitudinal distribution of pulmonary vascular resistance are time-dependent. If these data from animals can be extrapolated to humans, we speculate that the importance of pulmonary venoconstriction in exacerbating pulmonary edema may vary over time in patients with acute respiratory failure. PMID- 1729030 TI - Effects of levemopamil on neurologic and histologic outcome after cardiac arrest in cats. AB - BACKGROUND AND METHODS: A study was performed to examine the effects of the calcium-channel blocker levemopamil on neurologic outcome and neuropathology in a clinically relevant model of complete global cerebral ischemia (ventricular fibrillation in cats). Levemopamil was administered to cats starting 5 mins after resuscitation from 14 mins of cardiac arrest. In a "blinded" manner, 46 animals received levemopamil 1 mg/kg over 15 mins followed by 10 micrograms/kg.min for 16 hrs or vehicle. In a nonblinded manner, eight additional animals were pretreated with levemopamil beginning 45 mins before cardiac arrest. After resuscitation, levemopamil was infused at 10 micrograms/kg.min for 16 hrs. Animals in all three groups remained sedated, paralyzed, and mechanically ventilated for 24 to 30 hrs after resuscitation. Neurologic examinations were performed at 2, 4, and 7 days after resuscitation. Thirty-five cats were entered into data analysis (16 levemopamil posttreated, 14 vehicle-treated, and 5 levemopamil pretreated). RESULTS: Neurologic deficit scores and over-all neuropathologic scores did not differ among groups at any interval after resuscitation. However, the occipital cortex and CA1 region of the pretreated animals showed less severe damage than was observed in the animals that received levemopamil or vehicle, starting after resuscitation (p less than .01). CONCLUSIONS: Postarrest administration of levemopamil was not associated with improved neurologic or neuropathologic outcome. However, the data suggest that prearrest administration may result in regionally selective improvement in neuropathology. PMID- 1729031 TI - Regeneration of small bowel mucosa after intestinal ischemia. AB - BACKGROUND AND METHODS: The objective of this study was to evaluate the histologic reconstitution of the small intestinal mucosa after a standardized ischemic injury and to determine if the early repair process takes place by cell renewal or migration of existing mucosal cells. Therefore, male Wistar rats, weighing 190 to 320 g, were subjected to total warm intestinal ischemia by means of a hydrostatic pressure clamp for 45 or 90 mins. These rats were compared to sham-operated controls. Intestinal biopsies were obtained just before reperfusion and at various times up to 48 hrs thereafter. Mucosal injury was evaluated microscopically by a blinded examiner. RESULTS: Variable mucosal reconstitution occurred within 3 hrs, after 45 mins of ischemia, whereas mucosal repair required up to 18 hrs after 90 mins of ischemia. In a second series of experiments, 45 or 90 mins of ischemia and 5 hrs of reperfusion were followed by the iv administration of radioactively labeled thymidine. Intestinal biopsies were taken 1 hr later and prepared for autoradiography. No increase in mucosal mitoses was observed. CONCLUSIONS: The mucosal reconstitution occurred rapidly after 45 mins and 90 mins of total warm intestinal ischemia and primarily through mucosal cell migration. PMID- 1729032 TI - Critical care fellowship graduates--1992. The Society of Critical Care Medicine. PMID- 1729033 TI - Rapidly progressive respiratory failure due to Aspergillus pneumonia: a complication of short-term corticosteroid therapy. PMID- 1729034 TI - Use of plasmapheresis in acute theophylline toxicity. PMID- 1729035 TI - Crystalloid versus colloid: focus the debate. PMID- 1729036 TI - Defining the hypoxic threshold. PMID- 1729037 TI - The Salem sump anti-reflux valve. PMID- 1729038 TI - Comparison of continuous versus intermittent furosemide administration in postoperative pediatric cardiac patients. AB - OBJECTIVE: To compare the effects of furosemide administered by intermittent iv infusion vs. continuous iv infusion on urine output, hemodynamic variables, and serum electrolyte concentrations. DESIGN: Prospective, randomized trial. SETTING: Pediatric ICU. PATIENTS: Postoperative pediatric cardiac patients. INTERVENTIONS: Patients were assigned to either the continuous iv infusion or the intermittent infusion groups. The intermittent group received 1 mg/kg iv of furosemide every 4 hrs to be increased by 0.25 mg/kg iv every 4 hrs to a maximum of 1.5 mg/kg iv if the urine output was less than 1 mL/kg.hr. The continuous infusion group received an initial furosemide dose of 0.1 mg/kg iv (minimum 1 mg) followed by an iv infusion rate of 0.1 mg/kg.hr of furosemide to be doubled every 2 hrs to a maximum of 0.4 mg/kg.hr if the urine output was less than 1 mL/kg.hr. MEASUREMENTS AND MAIN RESULTS: Demographic variables, fluids, electrolyte and inotropic requirements were the same in both groups. A significantly (p = .045) lower daily dose of furosemide (4.90 +/- 1.78 vs. 6.23 +/- 0.62 mg/kg.day) in the continuous iv infusion group produced the same 24-hr urine volume as that of the intermittent group. There was more variability in urine output in the intermittent group as well as more urinary losses of sodium (0.29 +/- 0.15 vs. 0.20 +/- 0.06 mmol/kg.day, p = .0007) and chloride (0.40 +/- 0.20 vs. 0.30 +/- 0.12 mmol/kg.day, p = .045). CONCLUSION: Furosemide administered by continuous iv infusion is advantageous in the post-operative pediatric patient because of a more controlled and predictable urine output with less drug requirement and less urinary loss in sodium and chloride. PMID- 1729039 TI - Pulmonary mechanics in infants after cardiac surgery. AB - OBJECTIVE: To determine pulmonary mechanical characteristics in neonates after cardiac surgery. DESIGN: A prospective study. SETTING: A 23-bed, pediatric ICU in a 280-bed children's hospital. PATIENTS: Twenty-six infants on the first post operative day after cardiac surgery. METHODS: Pulmonary mechanics measurements were made during spontaneous breathing, using the esophageal balloon technique and a pneumotachometer. The least mean square method of analysis was used to calculate mechanics. Infants who tolerated withdrawal of mechanical ventilation (group 1) were compared with those infants with respiratory failure (group 2). RESULTS: Spontaneous respiratory rate, tidal volume, and minute ventilation were similar in groups 1 and 2. Lung compliance was decreased, with no difference between groups. Total lung resistance (34.3 +/- 21.6 cm H2O/L.sec in group 1 vs. 136.8 +/- 105.8 cm H2O/L.sec in group 2, p = .002) and resistive work of breathing (33.4 +/- 29.9 g.cm/kg in group 1 vs. 212.9 +/- 173.8 g.cm/kg in group 2, p = .001) were significantly higher in group 2. All infants with a total lung resistance greater than 75 cm H2O/L.sec exhibited respiratory failure (resistance greater than 75 cm H2O/L.sec correlated with respiratory failure, r2 = .73, odds ratio of 70 [confidence interval, 4.4 to 3240], p less than .001). CONCLUSIONS: Increased lung resistance identifies neonates with respiratory failure after cardiac surgery. Pulmonary mechanics testing may be useful in timing withdrawal of mechanical ventilation. PMID- 1729040 TI - Increased morbidity with increased pulmonary albumin flux in sepsis-related adult respiratory distress syndrome. AB - OBJECTIVE: To determine the feasibility of utilizing a scintigraphic technique to differentiate patients with adult respiratory distress syndrome due to sepsis syndrome from control volunteers and patients with congestive heart failure. Gamma scintigraphy was compared with chest roentgenograms to predict mortality rate and morbidity in adult respiratory distress syndrome (ARDS) patients. DESIGN: Prospective study. SETTING: University hospital ICUs. PATIENTS: Thirty five control volunteers, 19 patients with congestive heart failure, 30 patients with a diagnosis of sepsis. MEASUREMENTS AND MAIN RESULTS: All patients were infused iv with technetium 99m-labeled albumin and underwent computerized gamma scintigraphic analysis with a portable gamma camera. Lung-to-heart ratio of tracer was calculated and expressed as the slope index. Increase in slope index indicated increased pulmonary albumin flux. Slope index was no different in controls compared with congestive heart failure patients, unless the pulmonary artery occlusion pressure (PAOP) was greater than 30 mm Hg. Patients with a diagnosis of sepsis had an overall increased slope index compared with the other groups. A subgroup of patients in the septic group had a normal slope index. Septic patients with an increased slope index had a significantly (p less than .01) longer duration of mechanical ventilation (36 +/- 5 vs. 7 +/- 1 days), spent longer in the ICU (67 +/- 9 vs. 11 +/- 1 days), and had a longer hospital stay (113 +/- 20 vs. 35 +/- 5 days) than septic patients with a normal slope index. CONCLUSIONS: Gamma scintigraphy successfully differentiated between control volunteers and patients with congestive heart failure with PAOP less than 30 mm Hg from patients with sepsis-induced ARDS. Although all of the patients with a clinical diagnosis of septic ARDS had similar impairments in oxygenation and chest roentgenograms, those patients with a significantly increased pulmonary albumin flux (greater than 2 SD above control mean) had a markedly increased morbidity. PMID- 1729041 TI - Frequency of upper gastrointestinal bleeding in a pediatric intensive care unit. AB - OBJECTIVE: To determine the frequency of upper gastrointestinal (GI) bleeding in pediatric ICUs. DESIGN: Prospective, descriptive study. SETTING: Pediatric ICU in a university hospital. PATIENTS: All children admitted to a pediatric ICU over a 55-wk period. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Upper GI bleeding was considered to be present if there was an episode of hematemesis or if any amount of blood was seen in drainage from a nasogastric tube. Sixty-three (6.4%) upper GI bleeds were detected among 984 patients: 5.2% in 698 patients who did not receive upper GI bleeding prophylaxis, and 9.4% in 286 patients who did receive some prophylaxis. Density was defined as the number of events/1000 days.patient. The mean density was 10.8 GI bleeding episodes/1000 days.patient in a pediatric ICU. A multivariate analysis detected four independent risk factors or risk markers for upper GI bleeding: high Pediatric Risk of Mortality score, coagulopathy, pneumonia, and multitrauma. Age, sex, hepatic and respiratory failures were identified as confounding variables. An upper GI bleeding episode was defined as being clinically important if hypotension, death, or transfusion occurred within 24 hrs after the bleeding. There were four clinically important GI bleeding episodes. All were caused, at least in part, by a coagulopathy. The GI bleeding was associated with a need for transfusion in four children, and with hypotension in two. CONCLUSIONS: The frequency of upper GI bleeding is substantial, but the rate of occurrence of clinically important upper GI bleeding is low, even in a pediatric ICU where most patients do not receive any prophylaxis. PMID- 1729042 TI - Patients' preferences for intensive care. AB - OBJECTIVES: To determine patients' preferences for intensive care and to evaluate the influence of a recent ICU experience on preferences for future ICU treatment. DESIGN: Survey of nonrandomized patient sample using structured interviews. SETTING: Large, urban, tertiary academic medical center. PATIENTS: Eighty-four adult inpatients discharged from the medical ICU between June and August 1990. MEASUREMENTS: Agreement with life-supportive care under each of four potential outcome scenarios was assessed on a 5-point scale. An overall preference score was created by summing scores for the four items. Patients were also asked about their recent experiences in the ICU. RESULTS: Patients identified sources of stress associated with their ICU stay, yet most (76%) rated their ICU experience positively. Preferences for future intensive care varied with perceived outcome, and were strongest for health restoration and weakest for persistent vegetative states. No significant relationships were found between ICU preferences and any demographic or clinical variable except race. CONCLUSIONS: Patients tolerate intensive care well and desire it to restore health. Most patients modify their desire for intensive care if less favorable outcomes are likely. Patients' preferences for intensive care cannot be predicted from demographic features or previous ICU experiences. PMID- 1729043 TI - Comparison of reflection and transmission pulse oximetry after open-heart surgery. AB - OBJECTIVE: To determine if there was a difference between reflection and transmission pulse oximeters in their ability to regain data display after hypothermia in patients recovering from open-heart surgery. DESIGN: Prospective, randomized, controlled study. PATIENTS: Nineteen adult patients scheduled for open-heart surgery were studied immediately after surgery in the ICU. INTERVENTIONS: Transmission and reflection sensors were used in random order in two simultaneously monitoring identical oximeters and probes. The time difference at the start of display between the oximeters was measured and the skin temperatures in the region of the probes, cardiac index, systolic BP, pulse pressure, and systemic vascular resistance index were recorded. RESULTS: The mean skin temperatures at the probe sites differed significantly (p = .001) at the moment of data acquisition. The mean forehead, ear lobe, and fingertip temperatures (simultaneously measured) were 33.9 degrees C, 31.8 degrees C, and 28.8 degrees C, respectively. The hemodynamic variables were comparable at the moment when the oximeters resumed display. The reflection probe was the first to resume function in 12 patients and the transmission probe was the first to resume function in four patients (p less than .02). The bias of the reflection probe was 1.4% (SD 2.2) and that of the transmission probe was -0.4% (SD 2.7). All the patients were normoxic throughout the study. CONCLUSION: The forehead reflection probe regained signal detection earlier than the transmission probe on the ear lobe in patients with compromised peripheral blood flow and cool periphery. This finding may be due to higher skin temperature at the reflection probe site, since the systemic hemodynamic conditions were equal at the time of the data acquisition of both sensors. PMID- 1729044 TI - Phagocyte--monokine interactions during intra-abdominal sepsis: potential for immunointervention. PMID- 1729045 TI - Alterations in anion gap following cardiopulmonary bypass. AB - OBJECTIVES: To evaluate the changes in the anion gap and their relation to hyperlactatemia and alterations in plasma proteins after cardiopulmonary bypass. DESIGN: Prospective study. SETTING: Cardiothoracic intensive therapy unit. PATIENTS: One hundred eleven consecutive patients after cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS: Data were collected before cardiopulmonary bypass and every 6 hrs for 24 hrs after cardiopulmonary bypass. Results were analyzed for the entire cohort and for hyperlactatemic subgroups. The major finding of this study was that the anion gap decreased significantly at all sampling periods relative to precardiopulmonary bypass values, despite the presence of clinically important hyperlactatemia. No correlation between the decrease in plasma protein concentrations and the decrease in anion gap could be demonstrated. CONCLUSIONS: The decrease in anion gap after cardiopulmonary bypass appears to represent a balance between the influences of increased serum chloride and lactate concentrations and reduced plasma protein concentrations. This analysis demonstrates the limitations of the anion gap in the evaluation of a metabolic acidosis after cardiopulmonary bypass. PMID- 1729046 TI - Early plasmapheresis in patients with thrombotic thrombocytopenic purpura. AB - OBJECTIVES: To investigate the relationship of thrombotic thrombocytopenic purpura to adult respiratory distress syndrome (ARDS) and study the responses of thrombotic thrombocytopenic purpura patients to early plasmapheresis. DESIGN: Case series. SETTING: ICU of a university hospital. PATIENTS: Twenty-four consecutive patients with thrombotic thrombocytopenic purpura, with various periods of time (1 to 18 days) having elapsed since the onset of this condition. Patients ranged in age from 17 to 66 yrs. INTERVENTIONS: Plasmapheresis, using intermittent flow separators, was instituted soon after the patients' ICU admission. The retinoscopic findings on admission and the relationship of Pao2 to platelet counts before and after plasmapheresis therapy were recorded. Antiplatelet agents were given to the survivors to prevent relapses. MEASUREMENTS AND MAIN RESULTS: Eighteen patients survived and six died. Plasmapheresis was administered for a range of 1 to 5 days (mean 3) and 3 to 18 days (mean 9.8) in survivors and nonsurvivors, respectively (p less than .001). Four patients with confluent fundus hemorrhages died and seven without these fundoscopic findings had easily controlled disease. Increases in Pao2 paralleled increases in platelet counts after plasmapheresis (p less than .001) in this small series of patients. Three of 18 discharged survivors relapsed over a period of 3 to 56 months of follow-up. CONCLUSIONS: Early introduction of plasmapheresis in thrombotic thrombocytopenic purpura seems to increase the survival rate and to halt the development of ARDS. Fundus findings may be a prognostic factor in thrombotic thrombocytopenic purpura. The antiplatelet agents seem to be efficacious in the prevention of relapses. PMID- 1729047 TI - Intestinal epithelial restitution after ischemia. PMID- 1729049 TI - Diabetes insipidus. AB - OBJECTIVES: To review the pathophysiology, diagnosis, and treatment of the syndromes of diabetes insipidus with an emphasis on those situations likely to be encountered in the critical care setting. DATA SOURCES: Extensive clinical experience and relevant publications from the English literature identified via MEDLINE search, citation in reviews, publications of original data, and endocrine texts. STUDY SELECTION AND DATA EXTRACTION: Landmark papers pertaining to all aspects of diabetes insipidus were selected. Reviews, primary articles, and case reports pertaining to diabetes insipidus in the critical care setting were identified and selected according to their content of clinically useful information. DATA SYNTHESIS AND CONCLUSIONS: Diabetes insipidus may result from impaired synthesis and release of vasopressin from the hypothalamic-pituitary unit (neurogenic) or renal insensitivity to circulating vasopressin (nephrogenic). A number of interventions, diseases, and drugs commonly encountered in the critical care setting may result in the development or exacerbation of diabetes insipidus. The diagnosis of diabetes insipidus requires the exclusion of other causes of polyuria and a systematic demonstration of the response of homeostatic mechanisms to controlled dehydration. The treatment of diabetes insipidus depends on many factors, including the clinical setting, degree and pathophysiologic classification, ability of the patient to compensate for free water losses, and expected duration of the abnormality. Underlying disorders should be treated appropriately whenever possible. PMID- 1729048 TI - Esophageal electrodes allow precise assessment of cardiac output by bioimpedance. AB - OBJECTIVES: To analyze the impact of the position of the thoracic external electrodes on the values of cardiac output measured by electrical bioimpedance and to compare the results obtained by bioimpedance with those values determined by thermodilution in critically ill patients. DESIGN: Open, prospective, comparative trial. SETTING: ICU of a teaching hospital. PATIENTS: Twenty healthy volunteers and ten critically ill patients. INTERVENTIONS: Measurements of cardiac output by bioimpedance at rest and after physical activity in normal volunteers and after changing the neck or xiphoid electrodes. Comparisons of cardiac output obtained by thermodilution and bioimpedance with internal and external electrodes in patients. MEASUREMENTS AND MAIN RESULTS: Mean +/- SD values are presented. Cardiac output values at rest and after exercise were 6.7 +/- 1.3 and 10.8 +/- 2.6 L/min at rest and after exercise, respectively (p less than .001). Displacement of the xiphoid electrodes 3 cm in the caudal direction was accompanied by a decrease of the mean cardiac output from 7.1 +/- 1.2 to 5.8 +/- 1.3 L/min (p less than .001) and displacement 3 and 6 cm cranially was accompanied by increases in cardiac output from 7.1 +/- 1.2 to 8.1 +/- 1.4 L/min (p less than .001) and 8.6 +/- 1.5 L/min (p less than .001), respectively. In the ten patients, cardiac output measurements were virtually identical when results obtained by thermodilution (6.7 +/- 3.1 L/min) were compared with those results obtained by bioimpedance using internal esophageal (6.6 +/- 3.1 L/min), but not external (4.7 +/- 1.6 L/min) electrodes. CONCLUSIONS: a) The values of cardiac output derived from measurements obtained by bioimpedance using internal electrodes were comparable with those values derived from thermodilution. b) Values of cardiac output from bioimpedance studies with external electrodes were dependent on the position of the xiphoid electrodes. PMID- 1729050 TI - Metabolic alterations in the critically ill patient. PMID- 1729051 TI - Disorders of sodium metabolism: hypernatremia and hyponatremia. AB - OBJECTIVE: Discussion of abnormal plasma sodium concentrations with an emphasis on the pathogenesis, diagnosis, and treatment. DATA SOURCES: Relevant literature in the English language and the authors' clinical experience. STUDY SELECTION: No special study has been carried out for the present discussion. DATA EXTRACTION: The information from the literature and the data from the authors' clinical experience have been used to illustrate important points in the discussion. DATA SYNTHESIS: A most important aspect in the approach to hypernatremia is determination of the mechanism responsible for impaired water intake. Various mechanisms of abnormal water loss can be determined from measurement of urine osmolality. Hypernatremia is treated by water replacement and measures to reduce abnormal water loss. In most instances, hyponatremia is caused by inappropriate concentration of urine because of either appropriate or inappropriate antidiuretic hormone secretion. The determination of appropriateness of antidiuretic hormone secretion requires the assessment of effective arterial volume. Treatment depends on the pathogenetic mechanism. CONCLUSIONS: Abnormal plasma sodium concentration results from abnormal water intake or water output. Treatment is guided by determining the pathogenetic mechanism. PMID- 1729052 TI - The greatest international killer. PMID- 1729053 TI - Acute pulmonary edema induced by overdosage of phenothiazines. AB - Three schizophrenic adults with previous histories of using phenothiazine derivatives developed acute pulmonary edema after taking large amounts of these drugs. The clinical manifestations included coma (three), hypothermia (two), tachycardia (two), miosis (two) and hypotension (one). All three patients underwent gastric lavage and were treated supportively. The fulminant pulmonary edema in the three cases resolved within 18 to 40 h. The etiology of pulmonary edema following overdosage of phenothiazines remains unknown. The authors hypothesize that the most likely pathogenesis is a drug-induced neurogenic pulmonary edema resulting from a disturbance of hypothalamic function. PMID- 1729054 TI - Negative polysomnogram in patients with obstructive sleep apnea syndrome. AB - We evaluated the possibility that in some patients with obstructive sleep apnea, the initial polysomnogram may be negative. We reviewed polysomnograms performed at the Medical College of Georgia from 1984 to 1990 and found nine patients whose initial polysomnogram was negative but whose repeat polysomnogram confirmed obstructive sleep apnea. All nine patients (five women and four men; average age, 44.2 years) had an apnea index of less than 5 (fewer than five apneic episodes per hour) and had a total of fewer than 20 apneic episodes during the initial overnight polysomnogram. The change in average weight was not significant. Three patients had received short-term oxygen therapy, and two of these three received nasal continuous positive airway pressure prior to the initial study. The time that patients spent supine increased from 101 min in the initial study to 180 min in the second, but this was not significant (p = 0.12). Comparison of the initial and diagnostic polysomnograms showed significantly reduced total sleep time (from 3.75 +/- 1.84 h to 5.32 +/- 1.11 h; p = 0.04) and reduced rapid eye movement (REM) sleep time (from 0.27 +/- 0.27 h to 0.75 +/- 0.58 h; p = 0.037) in the initial study. We conclude that in a small subset of patients with obstructive sleep apnea, the initial polysomnogram may be falsely negative, which could be due to previous therapy, a reduction in total sleep time and REM sleep, or other unidentified factors. PMID- 1729055 TI - "Acquired" subvalvular aortic stenosis after repair of a ventricular septal defect. AB - Of 353 children who underwent surgical repair of a congenital heart defect, including closure of a ventricular septal defect (VSD), 12 patients (four with tetralogy of Fallot, five with a VSD, and three with a double-outlet right ventricle) developed subaortic stenosis, which was diagnosed one to six years after the surgical procedure. Five patients required surgical treatment of the subaortic stenosis, and one required percutaneous balloon angioplasty. Postsurgical subaortic stenosis appears to be an uncommon progressive acquired disease. PMID- 1729056 TI - Functional results of surgery for bullous emphysema. AB - Forty-six patients with bullous emphysema were operated on. Respiratory function was investigated before and immediately after surgery, and during the follow-up to five years. The larger the volume of the bullae, the less disturbances of lung function caused by their removal immediately after operation. Respiratory function improved significantly during the long-term follow-up after removal of the bullae that were more than one third of the hemithorax, but it did not change when the bullae were less than one third of the hemithorax and deteriorated after pulmonary resection for the bullae associated with long-term pneumonia. No new bullae were revealed roentgenographically at five years postoperatively. PMID- 1729057 TI - Color Doppler echocardiography of isolated cleft mitral valve. Roles of the cleft and the accessory chordae. AB - To study the mechanism of altered mitral function in the presence of an isolated cleft mitral valve (ICMV) with regard to the relative roles of the cleft and of the accessory chordae, seven patients with ICMV were studied with color Doppler echocardiography. Mitral insufficiency ranging from mild to severe was demonstrated in six cases. The regurgitant jet originated in each case from the site of the cleft: in five patients the regurgitant jet had a narrow base originating exactly from the cleft; in the sixth patient, the regurgitant flow presented as a broad-based jet suggesting that accessory chordae restricted the motion of the anterior mitral leaflet. Turbulent flow in the left ventricular outflow tract, starting at the level of the accessory chordae, was found in one patient in whom a pressure drop of 44 mm Hg was detected with continuous-wave Doppler imaging. The altered function of the mitral valve cleft stems from two elements, the cleft itself and the accessory chordae. Color Doppler flow imaging showed that the cleft was the main factor causing mitral insufficiency. The accessory chordae played an additional pathogenetic role in two patients by causing restricted mitral motion or left ventricular outflow tract obstruction. PMID- 1729058 TI - Aortic pressure during human cardiac arrest. Identification of pseudo electromechanical dissociation. AB - We measured aortic pressure during clinically apparent cardiac electromechanical dissociation (EMD). Patients with pulse pressures were designated as having pseudo-EMD; those without, as having true EMD. Of the 200 patients studied, 54 presented with EMD, and 40 others developed it during resuscitation. Of the 94 with EMD, 39 were found to have pseudo-EMD. We compared the two types of EMD for electrocardiographic duration, return of palpable pulses, and response to standard- and high-dose epinephrine. The mean resting aortic pressure was 18 +/- 11 mm Hg in patients with true EMD and 28 +/- 11 mm Hg in those with pseudo-EMD. The mean pulse pressure in patients with pseudo-EMD was 6.3 +/- 3.5 mm Hg. Patients with pseudo-EMD had a higher proportion of witnessed arrests, higher PaO2, and lower PaCO2 than patients with true EMD. Patients with pseudo-EMD had shorter QR and QRS durations than patients with true EMD. They had a better response to standard- and high-dose epinephrine than patients with true EMD. Many patients diagnosed clinically to be in EMD have mechanical cardiac activity; this should be considered when interpreting the results of cardiac arrest research. PMID- 1729059 TI - Utility of the peak expiratory flow rate in the differentiation of acute dyspnea. Cardiac vs pulmonary origin. AB - This study examined the utility of a peak expiratory flow rate (PEFR) measurement in the differentiation of acute moderate to severe dyspnea secondary to congestive heart failure or chronic lung disease. A PEFR was determined in 41 episodes of acute respiratory distress in 40 patients prior to emergency department therapy. The mean PEFR +/- SD for the congestive heart failure group (n = 18) was 224 +/- 82 L/min, which was significantly higher (p less than 0.001) than that of the chronic lung disease group (n = 23), which had a mean PEFR of 108 +/- 49 L/min. No single cutoff value allowed 100 percent accurate classification, but the results suggest that the PEFR may be a useful adjunctive tool in the differentiation of acute dyspnea of cardiac vs pulmonary origin. PMID- 1729060 TI - Is an abbreviated bronchial challenge with histamine valid? AB - Investigators have validated an abbreviated protocol for testing nonspecific bronchial reactivity with methacholine. We performed a similar validation study with histamine, another bronchoprovocative agent known to induce airflow obstruction. Histamine is pharmacologically distinct from methacholine and, under some circumstances, may provide specific clinical and investigative advantages to methacholine. Twenty-four patients with a clinical history of asthma underwent bronchoprovocative testing using the standard histamine airway protocol recommended by the American Academy of Allergy, Committee on Standardization of Bronchoprovocation. In addition, two abbreviated histamine challenge protocols were tested using the same administration and testing equipment. The abbreviated protocols involved fewer dilutions and dosages of histamine than the standard histamine protocol but covered the same range of cumulative doses. The two abbreviated protocols differed only in the intervals for determination of FEV1 between doses of histamine (30 s vs 3 min). The sequence of these three protocols was randomized for each study subject and each airway challenge was separated by one week. The two abbreviated protocols took significantly less time to administer than the standard protocol--18 min vs 30 min vs 44 min. Both the provocative dose to cause a 20 percent decline in the FEV1 (PD20 FEV1) and the slope of the dose-response curve were not significantly different between the standard protocol and either of the two abbreviated protocols. Moreover, a high degree of agreement was observed between the two abbreviated protocols and the standard histamine protocol for both the PD20 FEV1 and the slope of the dose response curve. These findings indicate that similar estimates of bronchial reactivity are obtained from either of the abbreviated protocols when compared with the standard histamine protocol. PMID- 1729061 TI - Pneumococcal bacteremia with pneumonia. Mortality in acquired immunodeficiency syndrome. AB - OBJECTIVE: To compare mortality due to bacteremic pneumococcal pneumonia in patients with acquired immunodeficiency syndrome (AIDS) vs (control) patients without human immunodeficiency virus (HIV) infection. Non-AIDS patients with HIV infection were incidentally tabulated as a separate group. DESIGN: A two-year retrospective study. SETTING: Inpatients of St. Clare's Hospital, a community hospital in New York City. PATIENTS: Forty-nine patients had 50 separate episodes with at least one positive blood culture for Streptococcus pneumoniae (all were penicillin-sensitive) and pneumonia on chest roentgenogram. Twenty-four patients had no HIV infection, 14 patients had AIDS, and 11 patients with 12 bacteremic episodes were HIV-positive without AIDS. INTERVENTIONS: Treatment for pneumonia was determined by the patient's individual physician. MEASUREMENTS AND MAIN RESULTS: AIDS patients with pneumonia had a mortality of 57.1 percent (8/14), which was significantly higher than the 25 percent (6/24) seen in patients without HIV infection (p less than 0.025, two-sample test for independent proportions). Septic shock, usually occurring within the first five days of hospitalization, was the primary cause of death, occurring in six of eight AIDS patients and six of six patients without HIV infection. If the mortality in the first five days of hospitalization was excluded, the mortality would drop to 33.3 percent in the AIDS population and 5.3 percent in patients without HIV infection. Eleven HIV-infected patients without AIDS survived 12 episodes of bacteremic pneumococcal pneumonia. CONCLUSIONS: Bacteremic pneumococcal pneumonia in the setting of AIDS has a survival rate of less than 50 percent with septic shock as the usual mode of death. This is the highest pneumococcal pneumonia mortality rate ever reported in a large subgroup of patients in the antibiotic era. On the other hand, HIV-infected patients without progression to AIDS have an excellent chance for survival. This may be related in part to young age, absence of many underlying diseases, and a better humoral immune system. PMID- 1729062 TI - Social nocturnal alcohol consumption and pharmacokinetics of theophylline. AB - Although moderate alcohol consumption is postulated to enhance theophylline metabolism, to our knowledge, the effect of casual nocturnal alcohol consumption has not been assessed. Eight normal volunteers, at the same time, one week apart, were given whiskey, 3 ml/kg of body weight, on one evening and on the alternate evening an equivalent volume of water. Each evening was followed by assessment of the pharmacokinetics of fast-release bibtheophylline, 5 mg/kg, taken the next morning at 9 AM. Alcohol taken the night before caused a statistically significant decrease in plasma clearance together with an increase in elimination half-life of 33 percent with consequent increases in plasma concentrations. These findings have potentially important implications for the accuracy of outpatient monitoring of serum theophylline and might explain intermittent toxic reactions occurring in some patients. PMID- 1729063 TI - Effect of passive smoking on asthmatic children who have and who have not had atopic dermatitis. AB - We studied 240 children with asthma who were themselves nonsmokers and had been referred consecutively to our clinic. They were aged 6 to 17 years. The severity of asthma was assessed by symptom score, by spirometry, and, in those who could perform the test reliably, by histamine bronchial challenge test. Those who reported having had a chronic or chronically relapsing itchy rash in characteristic locations were recorded as having had atopic dermatitis. Multiple analysis of variance revealed that children whose mothers smoked had significantly more severe asthma (p less than 0.001) but that atopic dermatitis had no apparent effect on the severity of asthma, either in its main effect (p = 0.71) or in its interaction with maternal smoking (p = 0.66). Although our previous study indicates that smoking mothers' children are more likely to develop asthma if they have had atopic dermatitis than if they have not, the severity of asthma does not appear to be associated with a history of atopic dermatitis. In smoking mothers' children the asthma was just as severe in those who had not had atopic dermatitis as in those who had. PMID- 1729064 TI - Theophylline and salbutamol improve pulmonary function in patients with irreversible chronic obstructive pulmonary disease. AB - To investigate the efficacy of bronchodilators in patients with irreversible chronic obstructive pulmonary disease (COPD), we conducted a double-blind, randomized, four-phase, crossover comparison between placebo, oral theophylline, inhaled salbutamol, and a combination of both drugs in 12 patients with stable COPD (mean age, 63 years) whose increase in forced expiratory volume in 1 s (FEV1) was less than or equal to 15 percent following 200 micrograms of inhaled salbutamol. Patients received two weeks of therapy with each of the test regimens. Both theophylline and salbutamol resulted in statistically significant improvement in FEV1, forced vital capacity (FVC), slow vital capacity (SVC), residual volume (RV), airway resistance (Raw), and maximum expiratory flow rate at 50 percent of vital capacity (V50). In most instances, there were no significant differences between theophylline and salbutamol. Combination therapy produced significantly greater improvement in FEV1, FVC, V50, Raw, and RV than either agent alone. The two drugs interacted in an additive fashion. Neither of the drugs, used singly, significantly reduced the severity or incidence of symptoms. The reduction in dyspnea and wheeze during combination therapy approached statistical significance (p = 0.06) and patient preference was significantly in favor of the combination regimen. None of the active treatments produced significantly more side effects than placebo. We conclude that theophylline and inhaled salbutamol produce significant, and approximately equal, improvement in pulmonary function in patients traditionally classified as suffering from "irreversible" COPD. The combination of theophylline and inhaled salbutamol generally results in additional improvement over that obtained with either drug used alone and this improvement is reflected by reduced symptomatology and treatment preference. PMID- 1729065 TI - Calcium inhibits the cardiac stimulating properties of dobutamine but not of amrinone. AB - To contrast the effect of increasing blood calcium concentrations on the cardiovascular actions of intravenous beta-adrenergic agonists and phosphodiesterase inhibitors, 46 patients recovering from aortocoronary bypass surgery received either dobutamine or amrinone both in the presence and absence of a calcium infusion. Cardiac output, systemic arterial pressure, pulmonary arterial pressure, central venous pressure, pulmonary artery occlusion pressure, heart rate, and blood ionized calcium concentration were measured before and during infusions of dobutamine (2.5 and 5.0 micrograms/kg/min) and amrinone (0.75 mg/kg bolus + 10 micrograms/kg/min or 2.25 mg/kg bolus + 20 micrograms/kg/min). After the initial dobutamine infusion period, patients were randomly and blindly assigned to receive either a calcium or placebo infusion, and the dobutamine infusions were repeated. Because of the long duration of amrinone's actions, the amrinone maintenance infusion was continued while randomized, blinded infusion of either calcium or placebo was added. Dobutamine (5 micrograms/kg/min) increased cardiac output from 7.1 +/- 0.3 L/min to 9.1 +/- 0.4 L/min, and increased heart rate from 93 +/- 4 beats/min to 107 +/- 4 beats/min. Systemic vascular resistance decreased and stroke volume increased. Dobutamine had no significant effects on other hemodynamic values. Amrinone (2.25 mg/kg bolus + 20 micrograms/kg/min) increased cardiac output from 5.6 +/- 0.4 L/min to 6.9 +/- 0.5 L/min, and increased heart rate from 87 +/- 3 beats/min to 98 +/- 3 beats/min. Amrinone decreased mean arterial pressure, systemic vascular resistance, pulmonary artery occlusion pressure, central venous pressure, and pulmonary artery pressure. Calcium infusion increased arterial pressure (8 to 13 percent) but had no significant effects on any other hemodynamic parameters. Calcium reduced the increase in cardiac output produced by dobutamine by 30 percent, but it did not alter the cardiotonic actions of amrinone. Thus, calcium inhibits the cardiotonic actions of certain beta-adrenergic agonists, most likely by interfering with signal transduction through the beta-adrenergic receptor complex. PMID- 1729066 TI - Effects of long-term treatment with prazosin on left ventricular diastolic function in mild to moderate hypertension. AB - It is not well established if blood pressure control is associated with an improvement in diastolic function, whose impairment represents an early marker of cardiac involvement in systemic hypertension. The purpose of this study was to evaluate whether a prolonged treatment with an alpha 1-blocking agent can lead to a reversal of the abnormalities of left ventricular filling. Eleven never-treated patients with mild to moderate essential hypertension were examined before and after at least six months of treatment with prazosin. Cardiac function and left ventricular mass were measured by means of radionuclide ventriculography and echocardiography. Average blood pressure values significantly decreased during the treatment period: from 163.54 +/- 17.80 mm Hg to 146.81 +/- 13.14 mm Hg for systolic blood pressure and from 106.09 +/- 6.96 mm Hg to 92.90 +/- 8.93 mm Hg for diastolic blood pressure. All the indices of left ventricular mass showed a trend toward reduction, but the differences with respect to the baseline values did not reach statistical significance. Average value of ejection fraction was normal before treatment and did not change significantly after treatment. All indices of diastolic function were significantly lower than normal controls' values at the beginning of the study and tended to worsen at the end of the study. Our findings suggest that diastolic function is not consistently affected by the therapy with alpha 1-adrenoreceptor antagonists despite good blood pressure control. PMID- 1729067 TI - Gastric colonization by gram-negative bacilli and nosocomial pneumonia in the intensive care unit patient. Evidence for causation. AB - The purpose of this article is to assess critically the evidence for a causal relationship between gastric colonization by Gram-negative bacilli and nosocomial pneumonia in the intensive care unit. Articles were found using MEDLINE search and citations in relevant articles. Nine diagnostic tests of causation were applied and analysis showed that the major tests were satisfied. The strongest evidence comes from randomized controlled trials of selective gut decontamination and stress ulcer prophylaxis in intensive care units. These studies confirm that the incidence of nosocomial pneumonia correlates directly with the rate of gastric colonization by Gram-negative bacilli. Further support comes from other tests of causation such as strength and consistency of association, temporal relationship, and dose-response gradient. The data reviewed suggest that gastric colonization with Gram-negative bacilli plays a causal role in the development of nosocomial pneumonia in the intensive care unit patient. This relationship impacts on future studies of pathogenesis and prevention of this potentially lethal infection. PMID- 1729068 TI - Present and past smoking history and other predisposing factors in 100 lung cancer patients. AB - This study assessed the accuracy of obtaining smoking history, relationships between smoking and the histologic subtypes of lung cancer, past and present smoking history, and co-carcinogen history in 100 patients seen between 1982 and 1989. A standard questionnaire filled out by the patients, a data base filled out by the physician, and medical records were abstracted, and detailed information on smoking and co-carcinogen history was obtained. Eleven percent of the patients were nonsmokers and another 41 percent were former smokers who had quit smoking more than one year prior to the diagnosis of lung cancer. Mean ages at onset and cessation of smoking and diagnosis were 17, 59, and 62 years, respectively. The histologic subtypes were as follows: adenocarcinoma, 34; squamous, 18; small cell, 24; adenosquamous, nine; large cell, nine; and bronchioloalveolar carcinoma, six. Mean pack-years of cigarette smoking for the subtypes were as follows: squamous, 82; small cell, 78; large cell, 72; adenocarcinoma, 65; adenosquamous, 48; and bronchioloalveolar carcinoma, 41. The patient and physician questionnaires had comparable data on smoking status in continued smokers and never smokers. Many former smokers filled out the patient questionnaire as a nonsmoker, but on query by the physician admitted to smoking in the past. The physician data set was more accurate in former smokers than questionnaires completed by the patients. Patients with squamous and small cell carcinomas were heavier smokers than patients with adenosquamous and bronchioloalveolar carcinomas. About 50 percent were active smokers until the diagnosis of lung cancer, but only 18 percent of patients continued to smoke after the diagnosis. About 10 percent were never smokers and about 40 percent were former smokers. Most former smokers quit smoking less than five years antecedent to the diagnosis of lung cancer. PMID- 1729069 TI - Recurrent Pseudomonas aeruginosa pneumonia in an intensive care unit. AB - We reviewed the records of all patients in the intensive care unit (ICU) who had Pseudomonas aeruginosa pneumonia over a 2.5-year period. Of patients with P aeruginosa pneumonia, 20 of 34 survived the initial episode of pneumonia. Ten of these 20 developed recurrence. In the nonrecurrent group, nine of ten survived hospitalization, compared to only four of ten in the recurrent group. Comparing the recurrent to the nonrecurrent group, factors associated with recurrence were the APACHE 2 score (12.3 +/- 2.7 vs 8.6 +/- 4.2 [p less than 0.03]), APS score (7.0 +/- 3.5 vs 2.7 +/- 2.1 [p less than 0.01]), and chronic pulmonary disease (8/10 vs 2/10 [p less than 0.05]). The recurrent P aeruginosa group was younger (63 +/- 10 vs 74 +/- 11 years old [p less than 0.03]) and spent more time receiving mechanical ventilation (95 +/- 64 vs 26 +/- 36 days [p less than 0.01]), in the ICU (101 +/- 61 vs 33 +/- 35 days [p less than 0.01]), and in the hospital (144 +/- 77 vs 84 +/- 32 days [p less than 0.03]). Although not statistically significant, in the recurrent group, eight of ten patients had tracheostomy and seven of ten had COPD, vs three of ten and two of ten, respectively, in the nonrecurrent group. Recurrent P aeruginosa pneumonia in the ICU is associated with increased morbidity and mortality and does not appear to be related to the adequacy of antibiotic treatment. Chronic lung disease appears to predispose patients to recurrent P aeruginosa pneumonia. PMID- 1729070 TI - Etiology and diagnosis of pneumonia requiring ICU admission. AB - The spectrum of pathogens and the microbiologic investigations used to obtain a diagnosis in 178 patients with severe pneumonia (88 percent requiring intermittent positive-pressure ventilation) are reviewed. Ninety-five patients had primary pneumonia, 31 had nosocomial pneumonia, 24 were immunocompromised patients, and 28 had aspiration pneumonia. While the spectrum of isolates conformed to the usual patterns for the different types of pneumonia, the incidence of Gram-positive infections (15 percent), predominantly Klebsiella pneumoniae, Staphylococcus aureus, (8 percent), and Legionella pneumophila (5 percent) in primary pneumonia was much higher than in community or general hospital-based studies, and only one case of Mycoplasma pneumoniae was identified. Gram stain of sputum or tracheal aspirate taken on intubation in primary pneumonia was reliably predictive of the causative organisms in both Gram positive and Gram-negative infections when compared with infections proven by blood culture. Serologic studies were valuable in patients in whom no positive microbiologic diagnosis was evident; however, fiberoptic bronchoscopy contributed minimally to the microbiologic diagnosis in this group of patients. The cause of severe primary pneumonia differs from less severe disease, and this should be recognized when selecting empiric antibiotic therapy. PMID- 1729071 TI - Oxygen therapy, exercise, and cystic fibrosis. PMID- 1729072 TI - Determinants of immediate survival among chronic respiratory insufficiency patients admitted to an intensive care unit for acute respiratory failure. A prospective multicenter study. The French Task Group for Acute Respiratory Failure in Chronic Respiratory insufficiency. AB - In this study, 322 patients were evaluated with two aims: determination of identifiable factors at the time of admission to an ICU that predict short-term survival. Application of the SAPS to this population. Characteristics of patients were as follows: age, 65.5 +/- 14.5 years; COLD, 45 percent; restrictive, 13.4 percent; obstructive and restrictive CRI, 13 percent; asthma progressing to CRI, 11.2 percent; diffuse bronchiectasis, 7.2 percent; neuromuscular diseases, 2.2 percent; others, 8 percent. Cachectic patients, those confined to home, those with initial coma or those who required MV had a higher percentage of M. The SAPS at admission was higher in those patients who died; however, there was no link between the SAPS and M. Prognostic factors in ARF complicating CRI and identifiable at the time of admission to an ICU are few and reflect severity of chronic respiratory disease; SAPS appears to be less useful in ARF complicating CRI. PMID- 1729073 TI - Hospital and posthospital survival in patients mechanically ventilated for more than 29 days. AB - Experience with prolonged mechanical ventilation has improved over recent years. Retrospective analysis of the records of 104 patients older than 16 years of age who were mechanically ventilated for more than 29 days over a 29-month period from May 1986 to October 1988 revealed the following findings. The mean patient age was 66.3 +/- 15.7 years (SD). The mean number of in-hospital ventilator days was 59.9 +/- 36.7 days (range, 29 to 247 days). The mean number of days of oral or nasal endotracheal intubation prior to tracheostomy (96 patients) was 21.5 +/- 14.2 days. The mean length of hospital stay for the 104 patients was 79.9 +/- 45.4 days. The majority of the 104 patients (82.6 percent) were surgical patients. Nine patients left the hospital receiving extended mechanical ventilation. Mortality was highest in multiple organ system failure and lowest among the trauma patients. The total days of mechanical ventilation did not appear to be related to mortality if patients older than 16 years survived for seven days. Postdischarge survival of the 53 of 60 patients who survived and whom we were able to contact was 67 percent at one year and 56 percent at three years. PMID- 1729074 TI - Multiple pulmonary masses with air bronchograms. PMID- 1729075 TI - Physician decisions regarding life support in the intensive care unit. PMID- 1729076 TI - Imaging techniques in the evaluation of pulmonary parenchymal neoplasms. AB - Conventional PA and lateral chest radiographs continue to be the initial examination of choice to evaluate patients who are suspected of having a pulmonary parenchymal neoplasm. A lung lesion can be characterized as probably benign or malignant based on its radiographic appearance (size, shape, margins, presence of calcification, cavitation or air bronchograms, growth rate). A spiculated or lobulated lesion greater than 3 cm in size that is noncalcified is highly suspicious for malignancy. A lung lesion less than 3 cm in size with smooth borders that appears noncalcified on conventional radiographs should be examined by CT, including densitometry to detect calcification or fat, which indicates benignity. In patients with known lung cancer, CT can help to stage the tumor by indicating hilar or mediastinal involvement, or distant metastases. Currently, MR imaging has a limited role, but can be used as a "problem solving" modality for selected cases in evaluating pulmonary parenchymal neoplasms. PMID- 1729077 TI - Preparation of the patient for awake flexible fiberoptic bronchoscopy. PMID- 1729078 TI - Improvement in exercise capacity after nocturnal positive pressure ventilation and tracheostomy in a postpoliomyelitis patient. AB - Progressive neuromuscular symptoms years after recovery from acute paralytic poliomyelitis have been termed the PPS. We describe a 52-year-old man who contracted poliomyelitis at age 9 years who fully recovered and 33 years later developed progressive dyspnea. Neurologic evaluation revealed bilateral paralysis of the vocal cords, generalized weakness, and accentuated mouth occlusion pressure and ventilatory responses to hypercapnic, hyperoxic breathing. An EMG and muscle biopsy showed changes consistent with acute and chronic denervation. Cardiopulmonary exercise evaluation demonstrated a pulmonary mechanical limit with excessive ventilation relative to CO2 output. Tracheostomy and nocturnal positive pressure ventilation resulted in increased respiratory muscle strength, normalization of ventilatory drive and marked improvement in exercise capacity. PMID- 1729079 TI - The ventilator-assisted individual. Cost analysis of institutionalization vs rehabilitation and in-home management. AB - The purpose of this article is to present a cost analysis of in-home vs institutionalization for severely physically disabled ventilator-assisted individuals (VAIs). Following rehabilitation and adaptation to noninvasive methods of ventilatory support, 30 VAIs were maintained in the community for 12.9 +/- 1.1 years with personal care attendants organized by a home care vendor reimbursed by New York City Medicaid. The program permitted self-directed severely disabled clients, including these 30 exclusively nontracheostomized VAIs, to live in the community and direct their attendant care and personal affairs. Prior to discharge home, the 30 patients resided in the respiratory unit of a long-term care facility for a mean of 8.9 +/- 10.1 years. The unit is currently reimbursed at a mean rate of $718.80 per patient per day. The current mean total cost of maintaining these VAIs in the community is $235.13 +/- 56.73 per patient per day. The conversion to and/or maintenance on 24-h nontracheostomy ventilatory support permitted discharge to the community by allowing the VAI to be attended by trained but uncredentialed home care attendants, thus avoiding prohibitively expensive in-home nursing for tracheostomy care. This created a savings to the public of 77 percent or $176,137 per year per client. We conclude that conversion to and/or use of noninvasive methods of ventilatory aid can be a reasonable and cost-saving goal. More respiratory rehabilitation centers are needed to free up hospital beds and facilitate discharge of VAIs to the community. There is also evidence that trained attendants should be permitted to suction tracheostomized VAIs in the home. PMID- 1729080 TI - Hypoplastic left heart syndrome associated with congenital right-sided diaphragmatic hernia and omphalocele. AB - Congenital diaphragmatic hernia (CDH) is associated with a variety of cardiac anomalies. However, its association with hypoplastic left heart syndrome (HLHS) is rare. We treated a female newborn with CDH, HLHS, and omphalocele. The operation for omphalocele and the diaphragmatic defect was successful, although the patient died of cardiac failure after Norwood's operation for HLHS. To our knowledge, this is the first reported case with a combination of these three major anomalies: CDH, HLHS, and omphalocele. PMID- 1729081 TI - Fatal respiratory failure caused by pulmonary infiltration by pseudo-Gaucher cells. AB - Pseudo-Gaucher cells are reticuloendothelial cells that are found in several diseases. We report a case of pulmonary tuberculosis in which extensive pulmonary involvement with these cells resulted in fatal respiratory failure. PMID- 1729082 TI - Upper lobe pulmonary parenchymal calcification in a patient with AIDS and Pneumocystis carinii pneumonia receiving aerosolized pentamidine. AB - In patients with AIDS-related Pneumocystis carinii infection occurring during aerosolized pentamidine prophylaxis, roentgenographic findings may be atypical. Pulmonary parenchymal calcification due to P carinii is rare. In this case, extensive upper lobe pulmonary parenchymal, splenic, and nodal calcifications occurred after two years of monthly treatments with aerosolized pentamidine. PMID- 1729083 TI - Drug-induced lupus pleuritis mimicking pleural space infection. AB - A 78-year-old man presented with acute lupus pleuritis due to procainamide. The pleural fluid was a turbid, yellow exudate with a WBC count of 53,200/cu mm (70 percent polymorphonuclear leucocytes), LDH of 4,296 IU/L, and pH of 7.195. Although these fluid characteristics suggested pleural space infection, they were due to pleural inflammation from drug-induced lupus. LE cells were present in the fluid and results of microbiologic studies were negative. Clinical and roentgenographic improvement followed discontinuation of procainamide. PMID- 1729084 TI - Laser resection of granulation tissue secondary to transtracheal oxygen catheter. AB - Oxygen therapy through a transtracheal catheter has been used increasingly for the long-term delivery of continuous oxygen. Compared to nasal cannula it results in significant reduction in oxygen flow requirements. This form of therapy has gained patient acceptance because of several advantages including improved convenience, aesthetics, compliance, and mobility. Reported complications generally have been minor, including subcutaneous emphysema, cough, "mucous ball" formation and mild hemoptysis. In this report, we describe a case of granulation tissue formation at the transtracheal catheter puncture site which was treated with Nd:YAG laser bronchoscopy to reestablish patency of the upper airway. No recurrence was noted after two years of follow-up. PMID- 1729085 TI - Acute myocardial infarction complicating the clinical course of dilated cardiomyopathy in childhood. AB - An acute myocardial infarction occurred in a 6-year-old child with dilated cardiomyopathy. This caused severe hemodynamic deterioration that led to a fatal outcome. Autopsy revealed diffuse myocardial atrophy without cell infiltrate, normal epicardial coronary arteries, and a massive healed anteroapical infarction. Coronary embolism or spasm could not be ruled out as the cause of the infarction. PMID- 1729086 TI - Respiratory tract infection complicating transtracheal oxygen therapy. AB - Transtracheal oxygen is generally well tolerated in patients with chronic hypoxemia. Minor complications are common, but there are few reports of serious respiratory tract infections associated with transtracheal oxygen therapy. We describe four patients with interstitial lung disease who had frequent lower respiratory tract infections requiring hospitalization after initiation of transtracheal oxygen therapy. PMID- 1729087 TI - Evidence of acute inflammatory response in reexpansion pulmonary edema. AB - We analyzed edema fluid in two cases of reexpansion pulmonary edema during thoracotomy. High value of the fluid to plasma protein concentration ratio indicates an increase in pulmonary microvascular permeability. There were marked increases in polymorphonuclear leukocyte (PMN) count and concentration of PMN elastase in edema fluid. There were also increases in concentrations of thromboxane B2 and 6-keto-PGF1-alpha in both edema fluid and plasma. These findings strongly suggest that the mechanism of reexpansion pulmonary edema is an inflammatory response and that PMNs in the reexpanded lung may play a role in the increase in permeability. PMID- 1729088 TI - Hypercoagulopathy induced by chemotherapy in a patient with lung cancer. A possible role for a factor with thrombosis-inducing activity (TIA). AB - We treated a patient with lung cancer in whom a hypercoagulopathy was induced acutely by chemotherapy. He received systemic chemotherapy twice and in both instances developed disseminated intravascular coagulopathy (DIC), accompanied by acute decrements of the peripheral platelet count and plasma fibrinogen, an increment of the fibrin degradation products (FDP), and bleeding tendency with the appearance of skin purpura. In each instance, the plasma thrombosis-inducing activity (TIA) appeared one to three days after chemotherapy and subsided subsequently. PMID- 1729089 TI - Long-term follow-up of tuberculoma of the brain in an AIDS patient. AB - Human immunodeficiency virus infection and extrapulmonary TB are defined as AIDS. The clinical manifestations of the TB are related to the interplay of the organism and the host's immune system. A seven-year follow-up of a woman successfully treated for biopsy- and culture-documented tuberculous brain abscess is described. Antibodies to HIV have been positive on repeated testing, yet CD4 counts remain over 500. Aggressive diagnostic and therapeutic maneuvers for all forms of TB in AIDS are warranted since long-term prognosis may be good. PMID- 1729090 TI - Detection of aortopulmonary window with ventricular septal defect by Doppler color flow imaging. AB - Aortopulmonary window is a rare congenital cardiac anomaly. When it coexists with a ventricular septal defect, the accurate diagnosis of aortopulmonary window on the basis of clinical examination is difficult. We report the case of an infant who had an aortopulmonary window together with a ventricular septal defect. An accurate diagnosis could be attained by visualization of the defect using two dimensional echocardiography and detection of the flow through it by Doppler color flow imaging. PMID- 1729091 TI - Fibrosing alveolitis responsive to corticosteroids following Legionnaires' disease pneumonia. AB - Two male patients ages 54 and 58 years had persisting pneumonia with dry cough, dyspnea, weight loss, and fever up to 39 degrees C that did not respond to erythromycin treatment. There was extensive restrictive impairment of ventilation and loss of diffusing capacity for carbon monoxide. Histologic examination of the basal pulmonary infiltrates showed fibrosing alveolitis. Serologic titers indicated that the patients had suffered from Legionella pneumophila infection. We believe that Legionella had caused the fibrosing alveolitis since there was absence of any other causative agents or factors. Both patients responded to corticosteroid treatment with rapid clinical improvement but delayed radiologic regression. PMID- 1729092 TI - Primary endobronchial actinomycosis in association with foreign body aspiration. AB - A 66-year-old diabetic man presented with a bilobar pneumonia two months after aspiration of a chicken bone. Flexible fiberoptic bronchoscopy demonstrated a mass in the bronchus intermedius. Histologic examination of endobronchial biopsy specimens revealed bone fragments, vegetable matter, and sulfur granules containing Actinomyces organisms. The patient responded to bronchoscopic removal of the foreign body and penicillin therapy. To our knowledge, the association of actinomycotic infection with an aspirated endobronchial foreign body has not previously been reported. PMID- 1729093 TI - The effect of pressure support ventilation on auto-PEEP in a patient with asthma. AB - We report the effect of pressure support ventilation (PSV) on auto-PEEP in a patient with asthma. The patient showed a high level of auto-PEEP during spontaneous breathing through a T-piece. PSV effectively decreased auto-PEEP and inspiratory muscle effort with increasing levels of PSV. PMID- 1729094 TI - Multichamber gunshot wounds of the heart. The utility of transesophageal echocardiography. AB - A patient had a gunshot wound to the heart involving three cardiac chambers. Conventional echocardiography failed to identify the intracardiac injuries. The utility of transesophageal echocardiography in a patient with cardiac trauma is described. PMID- 1729095 TI - Chylothorax in familial lymphedema of the Meige type. PMID- 1729096 TI - Myxedematous pleural effusion. PMID- 1729097 TI - Bronchoscopy with bronchoalveolar lavage in the diagnosis of pulmonary tuberculosis. PMID- 1729098 TI - Usefulness of bronchoscopy in the diagnosis of tuberculosis. PMID- 1729099 TI - The concertina effect in preexcitation. PMID- 1729100 TI - Unrecognized central venous catheter embolus. PMID- 1729101 TI - Occurrence of mitral valve prolapse in nonsmoking patients with spontaneous pneumothorax. PMID- 1729102 TI - Delayed quinidine-induced diarrhea after five years of treatment. PMID- 1729103 TI - Erythromycin for the treatment of bronchial hyperresponsiveness in asthma. PMID- 1729104 TI - Incorrect use of metered dose inhalers by medical personnel. AB - We administered a questionnaire and observed usage of a placebo metered dose inhaler (MDI) among 35 physicians, 14 nurses, and 12 respiratory therapists. Ninety-two percent of the respiratory therapists performed at least four of seven steps correctly, compared with 65 percent of house staff physicians, 57 percent of nurses, and 50 percent of nonpulmonary faculty. Most participants followed package insert instructions, while only 18 percent followed recent recommendations for proper MDI use. We conclude that (1) medical personnel should have additional instruction in proper MDI usage and (2) respiratory therapists and nurses can play a prominent role in instructing patients in their proper use. PMID- 1729105 TI - The alternation between atrial flutter and atrial fibrillation. AB - Atrial fibrillation and atrial flutter share a common reentrant mechanism. However, the relationship between these arrhythmias has not been systemically studied to date. To evaluate the degree to which these arrhythmias may alternate, consecutive Holter monitor recordings which showed fibrillation or flutter in 96 patients were reviewed. One half of the patients were studied after open-heart surgery and the other half for varying indications. One quarter of the patients had atrial flutter in addition to fibrillation, and this alternation with flutter was significantly associated with the use of a type 1A antiarrhythmic drug (p = 0.007), but not with the use of digoxin or beta blockers (p = NS for both). Furthermore, this alternation with flutter was more common in the postoperative group (p = 0.01). A history of embolization was less common in patients who were in the postoperative group (p = 0.003) and patients who had flutter in addition to fibrillation (p = 0.05). PMID- 1729106 TI - Late prosthetic valve endocarditis. Immediate and long-term prognosis. AB - From 1975 to 1989, 307 consecutive episodes of infective endocarditis were diagnosed in our hospital. Of those, 35 were cases of late prosthetic valve endocarditis, defined as those occurring after 12 months of valvular replacement. Blood cultures grew streptococci in 15 patients (43 percent), staphylococci in seven (20 percent), enterococci in five (14 percent), Gram-negative bacilli of HACEK group in four (11.5 percent), and Candida in one. Blood cultures were negative in three cases (prosthetic infection was confirmed at surgery). Heart failure due to prosthetic dysfunction occurred in seven patients (20 percent) and emboli in 12 (34 percent). Early valvular replacement was performed in six patients (17 percent). Complications and mortality were dependent on the infective agent. Overall mortality was 23 percent, no death occurred from streptococcal infection, whereas mortality with endocarditis by organisms of the HACEK group and Staphylococcus was 50 percent and 43 percent, respectively. During a mean follow-up of five years, 11 patients (those with prosthetic leaks diagnosed during the active infection and patients with biologic prostheses) required surgery. There was one relapse in a patient with staphylococcal endocarditis and one recurrence, six years after the initial episode. We conclude that immediate prognosis of late prosthetic valve endocarditis depends on the infective agent. Although the immediate prognosis of streptococcal infections is good, the need for early reoperation during follow-up due to progressive perivalvular leak is high. Also, it appears that deterioration of bioprostheses proceeds swiftly after the cure of infection. PMID- 1729107 TI - The impact of new federal regulations on the blood gas laboratory. PMID- 1729108 TI - Comparison of cardiac output determinants in response to upright and supine exercise in patients with cystic fibrosis. AB - This study characterizes cardiac output response to progressive submaximal upright cycling in CF patients. Thirty-one CF patients as well as 11 aged-matched CF control subjects completed cardiac output determinations (CO2-rebreathing) at rest, and at submaximal exercise corresponding to 30, 50 and 75 percent VO2max, in both upright and supine positions. The VO2max was similar in three of four groups, but lower in those with severe CF. The cardiac output generally increased with exercise intensity in both positions, except in severe CF. The change from upright to supine posture resulted in a significant increase in SI at rest and for every submaximal exercise in control subjects, but not CF patients. These observations may suggest that the abnormal cardiac output response observed in severe CF could be related to a potential limitation in ventricular diastolic reserve found in all CF patients independent of disease severity which becomes more apparent under increased ventricular preload. PMID- 1729109 TI - Tetracycline pleurodesis. Adios, farewell, adieu. PMID- 1729110 TI - Supplemental oxygen and exercise performance in patients with cystic fibrosis with severe pulmonary disease. AB - Patients with cystic fibrosis (CF) and advanced pulmonary disease have pulmonary limitation of exercise, often associated with arterial oxygen desaturation. Improving oxygenation during exercise by providing supplemental oxygen may improve exercise performance in these patients. To test this, we performed graded exercise stress tests in 22 CF patients with severe pulmonary disease (mean PaO2, 64 +/- 2 mm Hg [+/- SE]; PaCO2 46 +/- 2 mm Hg; RV/TLC, 57 +/- 4 percent; FEV1, 38 +/- 4 percent of predicted; FEF25-75%, 13 +/- 2 percent of predicted; median age, 26 years) and compared them to 21 controls (RV/TLC, 27 +/- 4 percent; FEV1, 112 +/- 2 percent of predicted; FEF25-75%, 80 +/- 4 percent of predicted; median age, 29 years). Each subject performed graded exercise stress tests while breathing FIO2 of 0.21 and FIO2 of 0.30. Subjects were blinded to the composition of the inspired gas, and the order of testing was randomized. We found that CF subjects exercised longer, had a higher maximal VO2, higher O2 pulse, and less arterial oxygen desaturation when receiving supplemental O2. Control subjects exercised longer when breathing supplemental O2 but had no significant change in maximal VO2, O2 pulse, or SaO2. Both CF and control subjects had increased end-tidal PCO2 when exercising while breathing supplemental O2. We conclude that CF patients with advanced pulmonary disease have increased exercise tolerance and aerobic capacity when exercising while breathing supplemental O2. PMID- 1729111 TI - Cancer due to asbestos exposure. AB - To determine the relationship between malignancies and asbestos exposure, the number of asbestos bodies in wet lung tissue was counted by light microscopy according to the modified method of Smith and Naylor, and occupational histories were examined. The results revealed that 17 (89 percent) of 19 malignant mesotheliomas, 39 (38 percent) of 104 lung cancers, 23 (37 percent) of 62 gastric cancers, and 13 (28 percent) of 45 colon cancers were shown to be cases with asbestos exposure. These values were significantly higher than those of noncancerous cases (200 cases). It is of interest that five out of ten cases of leukemia were related to asbestos exposure. Nearly all multiple cancers including lung and gastric cancer in this study were also cases with asbestos exposure. Additional research should be conducted on the carcinogenicity of asbestos for multiple cancers. PMID- 1729112 TI - Autologous "blood patch" pleurodesis for persistent pulmonary air leak. AB - A persistent pulmonary air leak, whether as a result of pulmonary surgery or as a result of a traumatic or spontaneous pneumothorax, is a difficult and frustrating problem to manage. Several therapies have been employed, including thoracotomy and repair of the air leak, prolonged tube thoracostomy suction, and chemical pleurodesis. We report two cases in which patients with a prolonged air leak who were not candidates for thoracotomy had immediate successful treatment with an autologous "blood patch" pleurodesis. An autologous blood patch pleurodesis is, in our limited experience, a simple, painless, inexpensive, and effective treatment for patients with a persistent pulmonary air leak. PMID- 1729113 TI - Eflornithine treatment of refractory Pneumocystis carinii pneumonia in patients with acquired immunodeficiency syndrome. AB - Eflornithine was offered as compassionate treatment of 33 episodes of Pneumocystis carinii pneumonia in 31 patients with acquired immunodeficiency syndrome who were intolerant of and/or unresponsive to conventional trimethoprim sulfamethoxazole or pentamidine therapy. A full course of eflornithine consisted of ten days at 400 mg/kg/d but no more than 30 g/d in four divided intravenous doses, four days at 300 mg/kg/d in four divided intravenous doses, and then up to six weeks at 300 mg/kg/d in four divided oral doses where tolerated. Of 33 patient-episodes, 15 patients were discharged from the hospital without need for supplemental oxygen after receiving ten or more days of parenteral therapy and were classified as responders. Of the 16 episodes classified as treatment failures, death occurred within the first 10 days of therapy in 12, and supplemental oxygen could not be withdrawn in 4. The other two patients left the hospital without need of oxygen after receiving one and six days of treatment with eflornithine and were not considered evaluable for efficacy. The most serious adverse effect was thrombocytopenia, which occurred in 12 of 19 patients treated for ten days or more. Serious bleeding associated with thrombocytopenia was observed in two patients. Other common adverse effects were anorexia, nausea, and diarrhea. Prior to receiving eflornithine, 13 of 15 responders had received ten or more days of conventional therapy without demonstrating clinical improvement. Two had improved while receiving conventional therapy but were switched to eflornithine because of a treatment-limiting adverse effect of standard therapy. These results suggest that eflornithine may be useful as an alternative therapeutic agent for Pneumocystis carinii pneumonia. Studies designed to determine proper dosage, duration of therapy, and efficacy as primary therapy are warranted. PMID- 1729114 TI - The pattern of respiratory muscle recruitment during pursed-lip breathing. AB - Data from the present study indicate a change in the pattern of chest wall muscle recruitment and improved ventilation with pursed-lip breathing (PLB) in COPD. Pursed lip breathing led to increased rib cage and accessory muscle recruitment during inspiration and expiration, increased abdominal muscle recruitment during expiration, decreased duty cycle of the inspiratory muscles and respiratory rate, and improved SaO2. In addition, PLB resulted in no change in pressure across the diaphragm and a less fatiguing breathing pattern of the diaphragm. Changes in chest wall muscle recruitment and respiratory temporal parameters concomitant with the increased SaO2 indicate a mechanism of improving ventilation with PLB while protecting the diaphragm from fatigue in COPD. Alterations in the pattern of respiratory muscle recruitment with PLB may be associated also with the amelioration of dyspnea. Further investigation is necessary to explore the relationship between the pattern of respiratory muscle recruitment during PLB and dyspnea. PMID- 1729115 TI - High incidence of bronchospasm with regular administration of aerosolized pentamidine. AB - A systematic evaluation of changes in pulmonary status by objective spirometric assessment and subjective rating using visual analog scale was performed in a cohort of 134 patients receiving aerosolized pentamidine (AP) for the prevention of Pneumocystis carinii pneumonia. Significant bronchospasm defined as greater than or equal to 15 percent reduction in the forced expiratory volume in 1 s was noted in 26 of 100 (26 percent) of patients receiving AP alone. Despite the use of salbutamol (albuterol) as concurrent aerosolized treatment in 34 subjects, bronchospasm developed in 9 of 34 (26 percent) of the patients. The subjective respiratory status rating scale was found to be unreliable in correctly predicting the development of bronchospasm. We conclude that a high incidence of bronchospasm is present in patients receiving regular AP administration using an ultrasonic nebulizer as studied, and concurrent administration of salbutamol is not fully protective of this acute adverse pulmonary reaction. PMID- 1729116 TI - Lung transplantation of ventilator-dependent patients. The Washington University Lung Transplantation Group. AB - During the last few years, lung transplantation has been extended to patients with a variety of end-stage lung diseases, but recipient selection guidelines have remained relatively strict. Ventilator-dependent patients have traditionally been considered poor candidates for transplantation. However, patients who have been thoroughly evaluated and accepted for transplantation and subsequently develop respiratory failure caused by progression and/or exacerbation of their underlying disease and recipients who experience respiratory failure caused by graft failure may be suitable candidates while ventilator-dependent if no other major complications or contraindications arise before a donor organ becomes available. PMID- 1729117 TI - Deposition of aerosolized pentamidine and failure of pneumocystis prophylaxis. AB - OBJECTIVE: To determine if outcome of Pneumocystis carinii prophylaxis is related to total lung dose of aerosolized pentamidine. SETTING: AIDS treatment centers at a VA and University Hospital. PATIENTS: Fifty-eight HIV-infected patients receiving P carinii prophylaxis with aerosolized pentamidine using a nebulizer (CIS-US AeroTech II) were followed up over a 90-week period. Treatment consisted of 60 to 90 mg every two weeks. MEASUREMENTS: In all patients, deposition of pentamidine aerosol was measured using a radioaerosol filter technique. Factors thought to be important in deposition, nebulizer output and breathing pattern were also measured. Six months later, repeated deposition studies were performed in 20 patients. Pentamidine dose to the lung was related to occurrence of P carinii pneumonia and correlated with nebulizer function and breathing parameters. Outcome was assessed in terms of pentamidine deposition and patient characteristics, including demographic, immunologic, physiologic, and medical variables. RESULTS: Ten patients (17.2 percent) had development of P carinii pneumonia. However, pentamidine deposited in the failures (8.18 +/- 4.74 mg) was no different than deposition in protected patients (6.39 +/- 3.07 mg, p = NS). Most of the variability in deposition was accounted for by variability in nebulizer output (r = 0.919, p less than 0.001). Deposition did not significantly correlate with any of the measured breathing parameters. Serial deposition measurements were not significantly different by paired analysis. The incidence of P carinii pneumonia did not correlate with any measured patient characteristic. CONCLUSIONS: Failure of aerosolized pentamidine prophylaxis is not related to total lung dose of pentamidine. Other factors such as inadequate microscopic deposition between alveoli, pentamidine clearance, or drug resistance may be important. In HIV-infected patients, interpatient variability in aerosol deposition can be reduced by reducing variability in nebulizer output rather than control of breathing pattern. PMID- 1729118 TI - Interlaboratory and intralaboratory variability in pulmonary function testing. A 13-year study using a normal biologic control. AB - The purpose of this study was to document the precision with which measurements can be made on a normal subject as a "biologic control" when comparisons are made both on an intralaboratory and interlaboratory basis. One individual had a total of about 300 routine pulmonary function measurements in 22 laboratories during a 13-year period. In this study, significant variation was found in interlaboratory testing, while less variation was found in intralaboratory measurements. This technique of periodic testing of normal individuals as biologic controls is useful for documenting reproducibility and precision; evaluation of accuracy will require a check of the physical calibration of the equipment. The testing (on at least a monthly basis) of a normal nonsmoking subject as a biologic control is recommended as an integral component of quality assurance in the pulmonary function laboratory. PMID- 1729119 TI - Sushi, Salmonella, and safety. PMID- 1729120 TI - Magnesium deficiency and diabetes. PMID- 1729121 TI - Eating away from home: teaching creatively and successfully. PMID- 1729122 TI - Impact of community-based diabetes education on program attenders and nonattenders. AB - This study reports on the differential impact of a moderately intensive diabetes education program on program attenders and dropouts. Sixty-one individuals with diabetes mellitus participated in a diabetes education program to increase knowledge, self-care, and metabolic control. Program attenders demonstrated a significant increase in diabetes knowledge and foot care irrespective of whether they attended the control or education groups. A discriminant analysis suggested that the nonattenders were more poorly educated, had less income, were younger, had had diabetes twice as long, reported more barriers to self-care, and were in poorer health than attenders. These results suggest that patients who attend interventions may be able to benefit from even minimal levels of intervention, while program dropouts may need special assistance to overcome obstacles to program participation. PMID- 1729123 TI - Risk taking among diabetic clients. AB - Diabetic clients must make daily decisions about their health care needs. Observational and anecdotal evidence suggests that vast differences exist between the kinds of choices diabetic clients make and the kinds of chances they are willing to take. The purpose of this investigation was to develop a diabetic risk assessment tool. This instrument, which is based on subjective expected utility theory, measures risk-prone and risk-averse behavior. Initial findings from a pilot study of 18 women clients who are on insulin indicate that patterns of risk behavior exist in the areas of exercise, skin care, and diet. PMID- 1729124 TI - An assessment of computer use, knowledge, and attitudes of diabetes educators. AB - A questionnaire to survey attitudes, use, and knowledge of computers was sent to 816 randomly selected members of AADE to determine the degree to which currently available computer resources are used in diabetes education and to investigate the need for future computing resources designed to support diabetes education. Analysis of the data showed that even diabetes educators who use computers infrequently have a generally favorable attitude toward them. Highest use of computers is in noneducational applications, mostly for word processing and record keeping. Most respondents believe that computers have yet to make a major contribution to the teaching and learning process in diabetes education, and few felt adequately prepared for creative use or development of computer applications. Increasing the role of computers in support of patient education will require encouragement and demonstrations of computer efficacy from health care institutions and professional organizations. PMID- 1729125 TI - The scope of practice for diabetes educators and the standards of practice for diabetes educators. American Association of Diabetes Educators. AB - The American Association of Diabetes Educators is a professional organization dedicated to enhancing the competence of health professionals who teach people with diabetes, advancing the specialty practice of diabetes education, and improving the quality of diabetes education and care. In keeping with this mission, a multidisciplinary task force of health professionals has developed a Scope of Practice for Diabetes Educators and a Standards of Practice for Diabetes Educators. These guidelines will not only foster high professional standards for those who teach people with diabetes, but will also provide a consistent point of reference for developing evaluation tools, quality assurance programs, orientation procedures, and professional appraisal systems. PMID- 1729126 TI - Alternatives to the exchange system for teaching meal planning to persons with diabetes. AB - Although the most well known, exchange lists are not the only meal-planning approach for persons with diabetes. This paper outlines the steps in the nutrition education process, including initial and continued education stages, and presents six alternative approaches for individualizing meal planning. These include the High Carbohydrate-High Fiber Exchange System, Calorie/Fat Counting, Total Available Glucose, the Point System, Month of Meals, and Individualized Sample Menus. The approaches are rated according to emphasis on weight loss, glucose control, and ease of learning and according to type of diabetes. A complete resource guide is provided. PMID- 1729127 TI - Certification: a summary of the first 5 years. PMID- 1729128 TI - 1991 Walter J. Johnson Prize. PMID- 1729129 TI - DNA polymerase alpha in the fission yeast Schizosaccharomyces pombe: identification and tracing of the catalytic subunit during the cell cycle. AB - A recombinant protein was obtained in Escherichia coli by subcloning part of the Schizosaccharomyces pombe POL1 gene at the 3'-end of lacZ. Antibodies raised against this protein were used to identify the POL1 gene product in extracts of exponentially growing S. pombe cells. A major 170-kDa protein, whose structure and properties were typical of the catalytic subunit of eukaryotic DNA polymerases alpha (pol alpha), was detected. The same antibodies were used to trace pol alpha and to quantify its level during the S. pombe cell cycle. We found that pol alpha was present at all stages of the cycle and that its cellular pool was subject to limited (three-fold) increase in G1 and S phases, with a decline to the initial level soon after. In addition, we found that a second form of pol alpha with slightly lower molecular weight (165 kDa) existed only during late G1 and S phases. Moreover, absence of initiation or perturbations in the course of DNA replication induced overproduction of the 165-kDa form. PMID- 1729130 TI - Laminin-induced process outgrowth from isolated fetal rat C-cells. AB - A method for isolation of C-cells from rat fetuses was developed, and the morphological plasticity of the cells in primary culture systems was tested. Thyroid-parathyroid-ultimobranchial body (UB) complexes from 16-day rat fetuses were treated with 0.1% collagenase and 1000 PU/ml Dispase at 37 degrees C for 1 h. After dissociation by pipetting, UBs were obtained as remaining cell aggregates with diameters of 150-200 microns. The isolated UBs were cultured on untreated, fibronectin-coated, or laminin-coated substratum in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) supplemented with 5% fetal calf serum. In some experiments, the medium was changed to serum-free medium after 24 h of incubation, until the UBs had formed cell sheets. At Day 4 in vitro, the cultures were subjected to immunostaining using anti-calcitonin antiserum. On untreated or fibronectin-coated substratum, most of the C-cells exhibited polygonal or ovoid shapes, and 5-8% of them were found to project processes. On laminin-coated substratum, the ratio of process-bearing C-cells to total C-cells was 23% in serum-supplemented medium and 51% in serum-free medium. The longest processes reached 150 microns in length. The processes were intensely reactive with anti-alpha-tubulin antibody and were completely disintegrated by colcemid, suggesting that the microtubule cytoskeleton participated in the maintenance of the processes. Thus it was demonstrated that fetal rat C-cells are still responsive to environmental signals, such as laminin, and extend neuritic processes. PMID- 1729131 TI - Quantitative analysis of autocrine-regulated, matrix-induced, and tumor cell stimulated endothelial cell migration using a silicon template compartmentalization technique. AB - Autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration was quantitatively analyzed using a two-dimensional, two-compartment coculture system. Silicon templates were used to subdivide 35-mm tissue culture dishes into two separate compartments. Endothelial cells were grown to confluence in the inner compartment and released from growth arrest by removal of the silicon template. The distance of endothelial cell outgrowth from the monolayer was measured in 24-h intervals. Endothelial cells from different vascular beds migrated with different migration rates (large vessel endothelial cells greater than hemangioendothelioma cells greater than microvessel endothelial cells). Prior coating of tissue culture wells with fibronectin, type I collagen, or type IV collagen and increasing serum concentrations strongly enhanced endothelial cell migration. Seeding tumor cells into the outer compartment prior to removal of the silicon template permitted the direct coculture analysis of tumor cell induced endothelial cell migration. Microvascular endothelial cell migration was stimulated in a tumor cell number-dependent fashion, whereas large vessel endothelial cells could not consistently be stimulated by coculture with tumor cells. It is concluded that silicon templates offer a useful approach for the quantitative study of migration of anchorage-dependent cells, permitting follow up measurements over several days, the study of matrix effects, and the direct coculture analysis of cell migration. PMID- 1729132 TI - Mammalian vitreous humor contains networks of hyaluronan molecules: electron microscopic analysis using the hyaluronan-binding region (G1) of aggrecan and link protein. AB - Vitreous humor from human, bovine, and chicken eyes was analyzed by rotary shadowing to characterize further the supramolecular organization of the gel-like matrix which forms this tissue. Extensive filamentous networks, distinct from collagen fibrils, were found in both human and bovine vitreous but not in chicken vitreous. The networks consisted of branching structures of various diameters, due to variable numbers of hyaluronan molecules being laterally associated with each other and apparently giving rise to a three-dimensional lattice. These networks could be decorated in a specific and regular manner by the hyaluronan binding region called G1 purified from bovine nasal septum cartilage. The extent of decoration of hyaluronan was dependent on the relative concentration of G1. In the presence of an excess of G1 the networks were destabilized giving rise to individual unbranched hyaluronan chains of varying length that were saturated with G1. One or more globular proteins, as yet uncharacterized, were seen interacting with the hyaluronan networks, often at branch points. These proteins may serve to stabilize the three-dimensional structure of the matrix although highly ordered networks were also observed without globular proteins. Link protein, which also binds to hyaluronan, bound to the networks in a fashion clearly distinct from G1. Neither G1 nor link protein bound directly to human or bovine vitreous collagen fibrils. However, link protein did bind extensively to the glycosaminoglycan coat of chicken vitreous collagen fibrils described previously (D. W. Wright, and R. Mayne J. Ultrastruct. Mol. Struct. Res. 100, 224 234, 1988), while G1 did not. Digestion of the chicken vitreous collagen fibrils with Streptomyces hyaluronidase did not result in the removal of the glycosaminoglycan coat of the collagen fibrils nor did it affect the binding of G1 or link protein to the fibrils, indicating that hyaluronan is not a component of this structure. These studies demonstrate that proteins with specific binding properties can be used as probes to investigate the structure of the native vitreous humor gel from several species and suggest that this method potentially can be used for structural studies of other connective tissue matrices. PMID- 1729133 TI - A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis. AB - The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chromatin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and repressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone H5 as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termination stage-specific protein) is hyperexpressed at the terminal stage of chicken erythropoiesis and is accumulated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It appeared to be a basic polypeptide (pI 9.2) which, however, precipitated at low pH. When reconstituted in vitro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleosomes, thus mimicking the processes occurring in vivo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correlation with their transcriptional activity. No other basic proteins (H5, cytochrome c, RNase A) added to the nuclear preparation at the same level as MENT (protein/DNA = 0.005) caused any effect on nuclear organization. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei having little or no histone H5. We propose that MENT cooperates with histone H5 to complete the nuclear collapse in mature nucleated erythrocytes. PMID- 1729134 TI - A relationship between gap junction-mediated intercellular communication and the in vitro developmental capacity of murine embryonic stem cells. AB - Two metabolic cooperation-deficient variants, 1P9 and 2P2[1], have been isolated from the embryonic stem cell line B2B2. Characterization of these cell lines has shown that both variants are severely restricted with respect to embryoid body differentiation capacity while a revertant isolated from 1P9, designated 2H4, has its differentiation phenotype restored to a level comparable with that of the parent line. These results are interpreted as indicating that the metabolic cooperation deficiency and the restricted differentiation phenotype of 1P9 are causally related. A revertant isolated from 2P2[1], designated H19, remains severely restricted with regard to embryoid body differentiation, suggesting that in these lines a secondary event deleterious to differentiation, but unrelated to metabolic cooperation, has taken place. PMID- 1729135 TI - Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways. AB - Interleukin 1 (IL1), a key mediator in the cytokine network, alters many pathophysiologically important functions of blood vessel wall cells. Vascular cells, such as endothelial cells and smooth muscle cells (SMC) can themselves transcribe IL1 genes, raising the possibility that IL1 regulates blood vessel wall functions by local autocrine or paracrine mechanisms. However, IL1 lacks a recognizable signal sequence and it is still unclear how vascular cells might release IL1 or if IL1 derived from vascular cells can actually produce autocrine or paracrine effects. We explored these issues in human vascular SMC, the most numerous cell type in arteries and veins, using cultured SMC and short term organoid cultures. SMC treated with lipopolysaccharide recombinant tumor necrosis factor (recTNF), or recIL1 itself ("activated SMC") elaborated thymocyte costimulatory activity, a biological activity traditionally ascribed to IL1. However, neutralization experiments with monospecific antibodies disclosed that the more recently recognized cytokine IL6 rather than IL1 accounted for most of the soluble thymocyte costimulatory activity released by activated SMC. Using the D10S assay that distinguishes IL1 from IL6 and TNF we found that the culture supernatant of activated SMC contained little or no IL1, but that the cytosol and surface of these cells did exhibit this activity. Antiserum to recIL1 alpha inhibited stimulation of D10S cells by surface-associated IL1 of activated SMC, while treatment with acid to elute receptor- or nonspecifically bound IL1 did not abrogate this D10S proliferation. Short term organoid cultures of both normal veins and human arteriosclerotic plaque also expressed tissue-associated IL1 activity upon stimulation with LPS but did not release significant soluble IL1 activity. To establish further the biological functions of cell-associated IL1, we incubated stimulated or unstimulated SMC that were fixed with paraformaldehyde and washed extensively (fixed SMC) with overlayered viable SMC of the same donor (responder SMC). Contact with fixed SMC that bore surface IL1 following TNF or IL1 stimulation evoked up to 20-fold higher IL6 release from responder SMC than did exposure to unstimulated SMC (57 vs 1052 ng/ml/day). Addition of anti-IL1 antibody inhibited the release of IL6 from the responder SMC. These results demonstrate that cytokine-activated SMC express biologically active IL1 on their cell surface and illustrate how these cells might actually participate in autocrine and paracrine signaling in the vessel wall. The requirement for direct intercellular contact for IL1 effects could facilitate local information exchange among vascular wall cells and/or infiltrating leukocytes and permit costimulation while limiting undue propagation of inflammatory stimuli. PMID- 1729136 TI - Identification of a structural protein component of rat synaptonemal complexes. AB - Synaptonemal complexes (SCs) are evolutionarily conserved nuclear structures of meiotic cells which form during the zygotene stage of the first meiotic prophase and are responsible for the pairing of homologous chromosomes. Their formation appears to be a prerequisite for crossing-over events and proper chromosome segregation during the first meiotic division. Despite knowledge of their central role in genetic recombination processes very little is known about the molecular composition and the mechanisms governing the assembly of the SCs. In the present study we report on the characterization of a monoclonal antibody (SC14f10) which enabled us to identify a novel SC protein termed SC48. Protein SC48 has a Mr of 48,000 and migrates in two-dimensional gels with a pH value of 6.9. By means of immunogold EM we localized this protein to the central region of the SC. In cell fractionation experiments we recovered protein SC48 together with SC-residual structures in a karyoskeletal fraction of pachytene spermatocytes. Our results indicate that SC48 is a meiosis-specific structural protein component of the SC probably involved in the pairing of homologous chromosomes. PMID- 1729137 TI - Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer. AB - We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite. PMID- 1729138 TI - Culture of human dermal fibroblasts in collagen gels: modulation of interleukin 1 induced prostaglandin E2 synthesis by an extracellular matrix. AB - Human dermal fibroblasts, cultured as suspensions in collagen gels and as monolayers, were stimulated with recombinant human interleukin-1 beta (rIL 1 beta) at 72 h, and prostaglandin E2 (PGE2) was assayed 24 h later. Fibroblasts in gels were less responsive to rIL 1 beta than monolayers, PGE2 synthesis increasing from less than 1 ng/microgram DNA without rIL 1 beta to maxima of 11.3 and 32.9 ng/micrograms DNA, with half maximal release occurring at 7.47 and 0.75 pM rIL 1 beta for the gel and monolayer cultures, respectively. Increased PGE2 was first detected 4 h after addition of rIL 1 beta to gels and was inhibited by 10(-5) M indomethacin. The amount of PGE2 synthesized per fibroblast increased with the time the gels had been in culture when stimulated with rIL 1 beta and was proportional to the number of fibroblasts in the gels, but inversely related to the collagen concentration. A common feature of these experiments was significantly greater induction of PGE2 synthesis at higher cell densities in collagen gels. Exogenous 10(-4) M arachidonic acid further increased PGE2 synthesis by rIL 1 beta-stimulated fibroblasts, but the differential in the amount of PGE2 released from fibroblasts at high and low population densities in the gels was maintained. These results are consistent with interleukin 1 (IL 1) stimulating PGE2 synthesis in dermal fibroblasts by increasing cyclooxygenase activity. Furthermore, the results show that dermal fibroblasts have an additional regulatory mechanism, related to the cell population densities or their interactions with an extracellular matrix, to finely modulate the amount of PGE2 synthesized in response to IL 1. PMID- 1729139 TI - A ceramide analogue (PDMP) inhibits glycolipid synthesis in fish embryos. AB - Glycolipids were depleted from medaka embryos using 1-phenyl-2-decanoylamino-3 morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. Embryos cultured in the presence of 20 microM PDMP exhibited a dramatic decline in glycolipid synthesis and cell surface expression. Metabolic labeling of glucosylceramide declined by 87% on Days 3-6 of development and 72% on Days 7-10 (hatching occurred on Day 10). In parallel, PDMP-treated embryos exhibited a striking loss of several tissue-specific glycolipid antigens, including 9-O acetyl GD3 from brain and retina, GT3/GQ1C from brain, neural tube, and retina, and sulfated glycolipid from skin and gut. Despite these changes in glycolipid expression, PDMP-treated embryos were fully viable with no evidence of developmental abnormality. PDMP appears to provide a useful tool for identifying glycolipid antigens in embryos and investigating their role in development. PMID- 1729140 TI - Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol ester. AB - Chronic lymphocytic leukemia lymphocytes were used to study mechanisms involved in apoptosis (programmed cell death). Apoptosis, which was determined by morphological changes including cell death and by internucleosomal DNA fragmentation, occurred during culture for 1 to 2 days in a portion of the cells from three of the four patients tested. Most of the cells underwent apoptosis and DNA fragmentation was greatly enhanced when cells were cultured in the presence of the microtubule inhibitor colchicine, the topoisomerase II inhibitor etoposide, or the glucocorticoid methylprednisolone. Tumor-promoting phorbol esters inhibited spontaneous DNA fragmentation and cell death including that induced by colchicine, etoposide, and methylprednisolone, indicating that they act on an event common to apoptosis caused by diverse stimuli. Phorbol esters probably act through protein phosphorylation, since they were effective at concentrations which modulated protein kinase C (PKC) and their action was prevented by H-7, which binds to and inactivates the catalytic site of PKC. In the absence of phorbol ester, H-7 itself induced some apoptosis. These findings implicate PKC in the suppression of apoptosis, but its precise role requires systematic investigation. PMID- 1729141 TI - How is the flagellar length of mature sperm determined? III. Comparison of initial growth rate of flagella in spermatids from Cynops and Xenopus in the presence and absence of cycloheximide. AB - Previous studies on flagellar growth in round spermatids from Cynops and Xenopus in vitro have shown that the period and rate of flagellar growth are greater in Cynops than in Xenopus. The present study shows, however, that during the initial phase of flagellar growth (for the first 12 h following the second meiotic division), the growth rate is very similar in both Cynops and Xenopus (0.5-0.6 microns/h at 22 degrees C). The difference in the growth rate between Cynops and Xenopus was observed beyond 12 h following the second meiotic division. When round spermatids in both species were inoculated with 10 microM cycloheximide, flagella grew at the same rate as in the absence of cycloheximide for the first 12 h following the second meiotic division. Beyond 12 h, however, cycloheximide suppressed flagellar growth in round spermatids in both species. These results indicate that the initial flagellar growth in round spermatids is provided for by flagellar protein pools which were present just after the second meiotic division; the growth beyond 12 h in round spermatids is contributed by newly synthesized flagellar proteins. PMID- 1729142 TI - Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser. AB - We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd YAG) laser at a wavelength of 1.06 micron. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage. PMID- 1729143 TI - Geriatrics: a global perspective. PMID- 1729144 TI - Newly-diagnosed Parkinson's disease: a therapeutic update. AB - For patients with newly-diagnosed Parkinson's disease, current research is pointing to new therapeutic approaches. Those that are now available allow the institution of levodopa to be delayed until there is some functional disability requiring treatment. Treating mild symptoms with anticholinergics and other agents may delay the use of levodopa for up to 3 years. And when finally required, levodopa may be used at lower doses when combined with carbidopa and a dopamine agonist. The monoamine oxidase type B inhibitor selegiline is also being used by many physicians to delay the onset of disability. PMID- 1729145 TI - Geriatric primary care: a European perspective, Part I. AB - Europe is considered the parent of geriatric medicine, which was first recognized as a specialty in the United Kingdom. For the benefit of U.S. primary care physicians, GERIATRICS Editor-in Chief Robert N. Butler, MD, convened a panel of leading European geriatricians in Lausanne, Switzerland, for a discussion of the successes and problems they are encountering in providing medical care to the aging world population. In this first installment, the panelists describe the healthcare services available to the elderly, particularly in the United Kingdom and Switzerland. A physician who is a regional adviser for the World Health Organization adds the perspective of other European nations. PMID- 1729146 TI - Maintaining functional independence by mobilizing the aged. AB - Maintaining mobility among the geriatric population is key to preventing unnecessary nursing home placement. Older patients who become bedridden as the result of acute illness or injury are at high risk of morbidity and mortality from the physical and mental effects of deconditioning. The primary care physician plays an important role in encouraging the mobilization of patients who have been confined to bed. Although mobilization is contraindicated in some patients, many others can be gotten 'back on their feet' again with a stepwise mobilization procedure. PMID- 1729147 TI - A physician's guide to early detection of oral cancer. AB - Oral cancer is a disease of the elderly that is more prevalent in patients who drink alcohol, smoke, or are chronically exposed to the sun. An oral examination is recommended annually for all elderly patients and more often for patients with these risk factors. The key to diagnosis and long-term patient survival is recognition of early lesions, which are often asymptomatic. Cancerous lesions occur more frequently at specific oral sites and may be difficult to differentiate from lesions caused by such benign conditions as denture irritation and herpetic ulcers. PMID- 1729148 TI - Life-sustaining treatment: the physician's role in decision-making. PMID- 1729149 TI - Data watch. Study: 21% rise in HIV treatment costs by 1994. PMID- 1729150 TI - Health care reform a priority in the new legislative year. Congressional leaders outline their agendas on today's vital health issues. AB - The issue of health care reform set against the backdrop of an election year promises to make 1992 one of the most interesting and significant years in the history of health care policy. This special issue of Hospitals, which coincides with the annual meeting of the American Hospital Association in Washington, DC, brings together the viewpoints of some of the major players in the legislative arena. Congressional leaders expect the 102nd Congress to take unprecedented steps in terms of action on health care reform. Many in the health care field agree that now is the time for action: Among other groups, the AHA considers the status quo unacceptable and is pushing for change. No one knows what the eventual outcome will be, but the mood is shifting toward immediate action. This may be a benchmark year for health care policy. PMID- 1729151 TI - Local governments cope with Medicaid, work for reform. PMID- 1729152 TI - A decade of competition ends--a new era of cooperation begins. PMID- 1729153 TI - Health care reform tops the AHA's list of priorities. PMID- 1729154 TI - Advocacy through the courts: the AHA's legal strategy. PMID- 1729155 TI - Physicians seek image changes through 'mini-internship' programs. PMID- 1729156 TI - Family-friendly: hospitals begin to modernize their time-off policies. PMID- 1729157 TI - Purchasing: hospitals weigh environment factor. PMID- 1729158 TI - Switching careers: straight thinking, planning. PMID- 1729159 TI - Teaching hospitals to track, prevent drug errors. PMID- 1729160 TI - Hospitals in the Information Age: time to act. PMID- 1729161 TI - What nursing specialty will be right for you. PMID- 1729162 TI - Managing the politics of the workplace. AB - The workplace--usually the hospital--is the laboratory for the new nurse's first year of practice. Lacking experience, the neophyte will need to learn organizational savvy and judgment and gain clinical experience with the help of a preceptor-mentor. Properly managing the first year of professional practice in the workplace will result in career success and satisfaction. PMID- 1729163 TI - The mentoring relationship: what makes it work? PMID- 1729164 TI - Tips on successful interviewing. PMID- 1729165 TI - Tips on resume writing. PMID- 1729166 TI - What to expect from your first year of nursing practice. PMID- 1729167 TI - Imprint career planning guide. PMID- 1729168 TI - NCLEX-RN preparation: three steps for success. PMID- 1729169 TI - C4 genes of the chimpanzee, gorilla, and orang-utan: evidence for extensive homogenization. AB - The human complement component 4 is encoded in two genes, C4A and C4B, residing between the class I and class II genes of the major histocompatibility complex. The C4A and C4B molecules differ in their biological activity, the former binding more efficiently to proteins than to carbohydrates while for the latter, the opposite holds true. To shed light on the origin of the C4 genes we isolated cosmid clones bearing the C4 genes of a chimpanzee, a gorilla, and an orang-utan. From the clones, we isolated the fragments coding for the C4d part of the gene (exons and introns) and sequenced them. Altogether we sequenced eight gene fragments: three chimpanzee (Patr-C4-1*01, Patr-C4-1*02, Patr-C4-2*01), two gorilla (Gogo-C4-1*01, Gogo-C4-2*01), and three orang-utan (Popy-C4-1*01, Popy-C4 2*01, Popy-C4-3*01). Comparison of the sequences with each other and with human C4 sequences revealed that in the region believed to be responsible for the functional difference between the C4A and C4B proteins the C4A genes of the different species fell into one group and the C4B genes fell into another. In the rest of the sequence, however, the C4A and C4B genes of each species resembled each other more than they did C4 genes of other species. These results are interpreted as suggesting extensive homogenization (concerted evolution) of the C4 genes in each species, most likely by repeated unequal, homologous, intragenic crossing-over. PMID- 1729170 TI - Activation of immunoglobulin control elements in transgenic mice. AB - To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EmukCAT, contains the Ig heavy chain enhancer (Emu) and the kappa light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, delta EmukCAT, CAT is under the control of the kappa promoter alone. Emu and kappa relative activity were assessed by CAT assay. In EmukCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In delta EmukCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon-gamma (IFN-gamma) increased CAT expression to varying extents in cells derived from EmukCAT mice but not in spleen cells prepared from delta EmukCAT mice. Thus, the presence of Emu, in addition to the kappa promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EmukCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN-gamma caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters. PMID- 1729171 TI - Molecular definition of an elusive third HLA-A9 molecule: HLA-A9.3. AB - The HLA-A9 family has been characterized as possessing two well defined specificities; HLA-A23 and A24. Serological studies have suggested the presence of a third member of this family HLA-A9.3, however there is doubt surrounding the existence of this specificity. HLA-A23, A24, and the putative A9.3 proteins were analyzed biochemically by immunoprecipitation and isoelectric focusing. Both HLA A24 and A9.3 have identical isoelectric points whereas A23 is different. We have sequenced cDNA encoding HLA-A23, A24, and A9.3. From the observed protein sequences, we found A9.3 to differ from A24 by two amino acid substitutions located in the alpha 2 helix of the class I molecule. These substitutions are expected to significantly change the shape of the peptide binding cleft. PMID- 1729172 TI - HLA-A29 subtypes and birdshot chorioretinopathy. PMID- 1729173 TI - Chromosomal locations of the genes coding for the integrin beta 6 and beta 7 subunits. PMID- 1729174 TI - Pneumocystis carinii induces an oxidative burst in alveolar macrophages. AB - There is evidence that alveolar macrophages (AM) play a role in the clearing of Pneumocystis carinii from the lungs. To investigate the mechanisms involved in this process, we studied in vitro the induction of an oxidative burst by P. carinii in a cell line of macrophages (NR8383) and AM from normal rats. P. carinii was added to macrophage monolayers (10(6) cells), and the H2O2 produced after 4 h of incubation was measured. Both NR8383 macrophages and normal rat AM produced H2O2 in response to P. carinii cysts and trophozoites isolated from dexamethasone-treated rats, although the amount of H2O2 induced in AM from normal rats was larger. NR8383 macrophages bound and phagocytized both P. carinii cysts and trophozoites and produced increasing amounts of H2O2 as a dose-related response to cysts and trophozoites. Opsonization of P. carinii with normal rat serum increased H2O2 production by both types of macrophages; this enhancement was decreased, but not abolished, when the serum was first depleted of complement by heat treatment. These findings demonstrate that NR8383 macrophages and normal rat AM produce an oxidative burst in response to P. carinii and that this response is enhanced by complement. PMID- 1729175 TI - Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli. AB - The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin. PMID- 1729176 TI - Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. AB - The relationship between capsular type and virulence for mice was examined with 69 fresh human isolates of Streptococcus pneumoniae. These isolates represented eight capsular types or groups. Serologic and molecular weight differences in PspA (pneumococcal surface protein A) indicated that the strains were clonally distinct. Mice were infected intravenously with washed bacteria of all 69 isolates in sterile salt solutions. Twenty-eight of the isolates were also injected intraperitoneally to permit comparisons between the intravenous and intraperitoneal routes. With a few exceptions, there was concordance between the ability of strains to cause fatal infections by the two routes. About 30% of the 69 isolates were virulent for mice. The abilities of the isolates to kill mice and the length of time between inoculation and death were strongly associated with capsular type. All type 4 isolates, 40% of type 3 isolates, and 60% of group 6 isolates were virulent for mice; type 1 isolates were marginally virulent; and all type or group 14, 19, and 23 isolates were avirulent. Times to death were generally longer for mice infected with group 6 or type 1 than for those infected with type 3 or 4 pneumococci. There was no relationship between clinical diagnosis or tissue source of the isolates and virulence for mice. PMID- 1729177 TI - Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (O126:H27). AB - The adherence of diarrhea-associated Escherichia coli to the small-bowel mucosa is an important step in the pathogenesis of diarrheal diseases. In tissue culture systems, diarrhea-associated strains show three distinct patterns of adherence: localized adherence, diffuse adherence (DA), and the recently described aggregative adherence. To study the molecular basis of the DA phenotype, we investigated the diarrhea-associated DA strain 2787 (O126:H27), isolated from a case of infantile diarrhea. The DA phenotype is mediated by a 6.0-kb DNA fragment derived from a 100-kb plasmid harbored by the wild-type strain. This fragment codes for a 100-kDa protein which can be released from the bacterial cell into the supernatant by mild heat treatment. Recombinant DA+ strains as well as the isolated 100-kDa protein were used to engender specific antisera in rabbits. As demonstrated by Western blotting (immunoblotting), the antibodies engendered by the recombinant DA+ strain recognized a 100-kDa protein in the wild-type strain 2787 and in all recombinant strains showing DA. Immunogold electron microscopy localized the 100-kDa protein to the bacterial cell surface. Serologically related proteins of similar size were detected by Western blotting in other DA+ diarrhea-associated strains belonging to enteropathogenic E. coli serotypes. The 100-kDa protein denoted AIDA-I (adhesin involved in diffuse adherence) binds in a saturable fashion to HeLa cells. AIDA-I-specific immunoglobulin G antibodies- and, to an even greater extent, Fab fragments derived thereof--inhibited bacterial attachment to HeLa cells. This is direct evidence that the 100-kDa protein is the adhesin mediating the DA phenotype of these diarrhea-associated strains and is representative of a group of serologically related proteins in other DA+ strains. PMID- 1729179 TI - T-cell immune responses in Mycobacterium avium-infected mice. AB - Mycobacterium avium infection was substantially more severe in C57BL/6 (Bcgs) than in (C57BL/6 x DBA/2)F1 hybrid (Bcgr) mice both in terms of bacterial growth in the spleens and lungs and in host survival. Prior Mycobacterium bovis BCG vaccination resulted in increased resistance as well as enhanced tuberculin hypersensitivity to both PPD-S (Mycobacterium tuberculosis) and PPD-A (M. avium). Mice heavily infected with M. avium were used as T-cell donors in an adoptive transfer system. Substantial resistance was observed for both recipient hosts regardless of the genotype of the donor strain. Transfer of resistance was ablated by treatment of the immune spleen cells with anti-Thy 1.2 monoclonal antibody and complement or by cyclophosphamide treatment. Spleen cells which were monodepleted of L3T4+ or Lyt-2+ T cells did not lose their ability to transfer resistance against a subsequent challenge. However, when these cells were doubly deleted, all resistance was ablated in both the BCG-susceptible and -resistant mice. The recipient host expressed a detectable adoptive immune response although the donor had been unable to reduce the growth of the primary M. avium infection in vivo. PMID- 1729178 TI - T-cell-dependent immunity and thrombocytopenia in rats infected with Plasmodium chabaudi. AB - Normal, splenectomized, and athymic Fischer rats were infected with Plasmodium chabaudi. In normal rat infections, acute-phase infection resolved rapidly and completely. In splenectomized rats, infection resulted in high parasitemia and ultimately death. In nude rats, parasite growth was reduced compared with normal rats, and a persistent parasitemia (between 20 and 45%) was observed for several months. Complete resolution of the infection was achieved after adoptive transfer of T lymphocytes, even when transfer occurred during the course of infection. These results indicated that an acquired, T-lymphocyte-dependent immunity was necessary for the complete recovery observed in normal rats. In normal rats, thrombocytopenia and splenomegaly occurred during infection. By contrast, in nude rats, both of these pathological manifestations were only observed after thymus grafting. Thrombocytopenia was also absent in the splenectomized animals. Despite an increase in platelet-associated immunoglobulin levels during the infection, thrombocytopenia was not transferred by injection of infected rat serum to normal recipients. It has been concluded that the nude rat infection can be regarded as a novel and useful model for studying the T-cell-dependent effector and pathological mechanisms and to investigate the anti-P. chabaudi immune response. PMID- 1729180 TI - Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing. AB - The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120- and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickettsii. The 168-kDa putative precursor of the avirulent strain of R. rickettsii was not extracted from the surface by dilute buffers, as is the 120-kDa protein of virulent R. rickettsii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain. PMID- 1729181 TI - Biological activity of interleukin-2 bound to Candida albicans. AB - The lymphokine interleukin-2 (IL-2), which is necessary for the generation of an optimal cell-mediated immune response, has recently been shown to have lectinlike properties, with specificity for high-mannose groups. Therefore, the ability of IL-2 to bind to the mannose-rich fungus Candida albicans was examined. Heat killed fungi preincubated with IL-2 stimulated, in a dose-dependent manner, proliferation of the IL-2-dependent cell line CTLL20. Soluble mannan, which is rich in exposed mannose groups, inhibited binding of IL-2 to C. albicans by approximately 60%, suggesting that the lectinlike properties of IL-2 are partially responsible for its fungal binding capacity. Binding of IL-2 to fungi appeared to be reversible, as C. albicans preincubated with IL-2 stimulated CTLL20 proliferation even when the fungi and cells were separated by an 0.4 microns-pore-size membrane. The lymphoproliferative response of normal human peripheral blood mononuclear cells to C. albicans was augmented when the fungus was preincubated with IL-2. Binding of 125I-IL-2 could not be inhibited by unlabeled IL-2, suggesting the absence of high-affinity receptors on C. albicans for IL-2. While the in vivo relevance remains to be determined, these data demonstrate that IL-2 can bind to C. albicans in vitro and thereby influence the host response to this medically important fungus. PMID- 1729182 TI - Mechanism of YadA-mediated serum resistance of Yersinia enterocolitica serotype O3. AB - Complement activation via the alternative pathway was analyzed with isogenic strains of Yersinia enterocolitica serotype O3 differing in plasmid content (p- or p+ strains) or selective lack of YadA expression (YadA- strain). The p+ strain was serum resistant, even after antibody-enhanced complement activation. Serum sensitivity was observed with the p- and YadA- strains but was more pronounced in the p- strain. The p+ strain deposited less C5b-9(m) complexes on its surface than the p- and YadA- strains. No size difference, however, was detected with solubilized C5b-9(m) complexes obtained from resistant and sensitive strains. At the C3 level, it became evident that surface-bound C3b was degraded faster into iC3b on the p+ strain than on the p- and YadA- strains. Our results demonstrate that YadA inhibits complement activation at the C3 and C9 level. As a result, reduced amounts of C5b-9(m) are generated on the surface of YadA-bearing bacteria. In addition, YadA seems to protect against the lytic action of those C5b-9(m) complexes whose deposition could not be prevented. PMID- 1729183 TI - Complement component 3 binding to Haemophilus influenzae type b in the presence of anticapsular and anti-outer membrane antibodies. AB - Antibodies directed against the capsular polysaccharide (polyribosyl ribitol phosphate [PRP]) or the outer membrane proteins (OMP) of Haemophilus influenzae type b (Hib) promote bactericidal activity, complement 3 (C3) binding, and ingestion by phagocytic cells. To assess the relative contribution of anti-OMP to host defense against Hib, we compared the opsonic activities of anti-PRP and anti OMP as reflected by the amounts of C3 bound to the bacterial surface. Immunoglobulin G (IgG) fractions containing either anti-PRP or anti-OMP were incubated with Hib in the presence of a C5-deficient complement source. C3, total IgG, and IgG subclasses bound to the bacteria were quantified by enzyme-linked immunosorbent assay. The maximum amount of C3 which could be bound to Hib was greater in the presence of anti-PRP than in the presence of anti-OMP. Also, except at low IgG concentrations, the rate of increase in bound C3 as a function of increasing IgG concentration was greater for anti-PRP than for anti-OMP. Hib bound anti-OMP consisted primarily of IgG1 and IgG3, whereas bound anti-PRP was primarily IgG1 and IgG2. Thus, the potential for C3 binding to Hib is greater in the presence of anti-PRP than in the presence of anti-OMP, probably because of the larger number of binding sites available to the former. Nonetheless, OMP appear to provide important targets for opsonic antibody and would be logical components of a PRP-conjugate vaccine or may be efficacious as vaccines against nontypeable H. influenzae. PMID- 1729184 TI - Arg-Gly-Asp (RGD) peptides alter hepatic killing of Candida albicans in the isolated perfused mouse liver model. AB - The isolated perfused mouse liver model was used to study the effect of Arg-Gly Asp (RGD)-containing peptides on hepatic trapping and killing of Candida albicans. After extensive washing, 10(6) C. albicans CFU were infused into mouse livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicates that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/ 2% was killed within the liver. Prior to their infusion into livers, 10(7) CFU of C. albicans were incubated at 37 degrees C for 30 min in the presence of various RGD peptides (0.1 mg/ml). Repeatedly, more than 90% of the infused RGD treated C. albicans was trapped by the perfused liver. In comparison with the 23% killing rate observed in control livers, perfused livers killed approximately 40 to 50% of the infused C. albicans treated either with fibronectin, PepTite 2000, RGD, or RGDS. Hepatic killing of C. albicans treated with PepTite 2000 or fibronectin was dose dependent. Treatment of C. albicans with GRGDTP, GRGDSP, GRADSP, or GRGESP did not alter the ability of the perfused liver to kill C. albicans, suggesting that a degree of specificity for RGD peptides is associated with an increased ability of liver to kill RGD-treated C. albicans. Together, the data suggest that RGD peptides bind to a receptor on the surface of C. albicans, thereby increasing hepatic, and presumably Kupffer cell, killing of C. albicans. Natural or synthetic RGD peptides may serve as opsonins promoting C. albicans killing by Kupffer cells. PMID- 1729185 TI - Shigella flexneri enters human colonic Caco-2 epithelial cells through the basolateral pole. AB - The commonly accepted view that enteroinvasive bacteria enter cells of the intestinal epithelial lining through the apical surface can be challenged in the case of shigellosis. This study is based on in vitro experiments that showed that the invasion of human colonic Caco-2 cells by Shigella flexneri occurred through the basolateral pole of these cells. In these experiments, the few bacteria that interacted with the apical surface either bound to microvilli of the cell dome without causing detectable alteration or bound at the level of intercellular junctions at which they demonstrated a limited capacity for paracellular invasion, which permitted subsequent entry through the lateral domain of the cells. Treatment of Caco-2 cell monolayers with ethylene glycol-bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly enhanced the rate of cell infection. These observations suggest a physiopathological paradox that may have important consequences for the understanding of the process of colonic invasion in vivo during shigellosis. PMID- 1729186 TI - Effect of specific antibody on adherence of Staphylococcus aureus to bovine mammary epithelial cells. AB - Staphylococcus aureus is a major pathogen in the bovine mammary gland. The ability of S. aureus to adhere to epithelial cells in the ductules and alveoli of the bovine mammary gland is believed to add greatly to its virulence and may be necessary for colonization. Two in vitro methods were developed for the purposes of quantifying adherence and of determining the effect which specific antibody may have in inhibiting the adherence of this organism. Both methods utilize bovine mammary epithelial primary cells as targets for labeled bacteria. In one assay, the bacteria are labeled with [methyl-3H]thymidine and incubated on the primary epithelial monolayers. The second assay involves labeling the bacteria with biotin. An enzyme-linked immunosorbent assay is then performed with streptavidin conjugated to horseradish peroxidase. Both methods have proven to be reliable and allow for the testing of many criteria in one assay. Cows were immunized with a whole-cell vaccine, and immune serum and milk were collected. The bacteria were then incubated in the presence of serum or milk as a test for antiadherent capability. By using the methods described, distinct antiadherent activity in both serum and milk was demonstrated. PMID- 1729187 TI - Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells. AB - The sex pheromone system of Enterococcus faecalis is a unique, highly efficient plasmid collection mechanism for this species. A crucial role in this system is played by an adhesin called aggregation substance which enables the cell-cell contact between donor and recipient strains. The existence of the amino acid motif Arg-Gly-Asp-Ser in the adhesin prompted us to look for a possible binding of E. faecalis cells expressing aggregation substance to eucaryotic cells. We were able to show that the adhesin mediated binding to cultured renal tubular cells (porcine cell line LLC-PK1) via light microscopic, electron microscopic, and enzyme-linked immunosorbent assay-based studies. Synthesis of the adhesin was induced by some component(s) of serum. These data are interpreted to mean that aggregation substance is an adhesin mediating not only cell-cell contact between different E. faecalis strains but also binding of E. faecalis to eucaryotic cells, and therefore it might contribute to virulence. PMID- 1729188 TI - In vitro characterization of T cells from Mycobacterium w-vaccinated mice. AB - Tuberculosis caused by the intracellular bacterial pathogen Mycobacterium tuberculosis still represents a major health problem, and its effective control would best be accomplished by active vaccination. Although vaccination with M. bovis BCG has proven highly effective in certain parts of the world, in several developing countries it has been found to confer only marginal protection. Hence, novel vaccination strategies are warranted. Mycobacterium w is a saprophytic cultivable mycobacterium which shares several antigens with M. tuberculosis. In the murine system, vaccination with killed M. w was found to protect against subsequent tuberculosis. In order to characterize the responsible immune mechanisms more precisely, mice were vaccinated with killed M. w and T cells restimulated in vitro with mycobacterial antigens. These T cells produced interleukin 2 and gamma interferon but no detectable interleukin 4 and interleukin 5. Killed M. w induced significantly stronger T-cell responses than killed M. tuberculosis, and both vaccination regimes were markedly improved by administration in a mild adjuvant, i.e., the Ribi adjuvant containing trehalose dimycolate, monophosphoryl lipid A, and mycobacterial cell wall skeleton. Our data suggest that M. w-induced immunity against M. tuberculosis rests primarily on TH1 cells, which are thought to be of major relevance for acquired antituberculosis resistance. Our study therefore provides a further step toward the identification of a novel tuberculosis vaccine. PMID- 1729189 TI - Early hepatic stages of Plasmodium berghei: release of circumsporozoite protein and host cellular inflammatory response. AB - After injection of Plasmodium berghei sporozoites into Norway-Brown rats, we were able to localize these sporozoites and the early hepatic trophozoites developing from them in histological sections of the liver stained with a sensitive immunogold-silver procedure. Sporozoites invading hepatocytes released substantial quantities of circumsporozoite protein into the hepatocyte cytoplasm. This intrahepatic cytoplasmic distribution reached a maximal level at about 4 h post-sporozoite injection. As the hepatic parasites continued to differentiate, circumsporozoite protein became undetectable within the cytoplasm of the hepatocytes and became localized around the periphery of each parasite. There was generalized cellular inflammation within the liver of the host, which first became evident at around 4 h post-sporozoite injection and progressed to the formation of well-defined granulomas by 24 h. Such histopathological changes were not seen in rats injected with killed sporozoites, indicating that the cellular inflammation was induced by viable, infective sporozoites. We did not observe cellular infiltration specifically associated with any of the developing hepatic stages that we observed, even up to 28 h post-sporozoite inoculation. These results indicate that viable sporozoites induced rapid and generalized hepatic inflammation in host rats. However, sporozoites that successfully invaded hepatocytes and then proceeded to develop further did not appear to be the target of inflammatory cells until a period beginning at around 40 h post-sporozoite inoculation. PMID- 1729190 TI - Experimental congenital syphilis: guinea pig model. AB - Neonates born to female guinea pigs of either a highly susceptible (C4D) or a resistant (Albany) strain, infected prior to or during pregnancy with a single dose of Treponema pallidum, showed in their sera from the first day of life immunoglobulin M (IgM) antibodies to T. pallidum, circulating immune complexes consisting of IgM antibodies and treponemal antigens, and IgM rheumatoid factor. Although the animals were asymptomatic for a 6-month observation period, several lines of evidence indicated that they were infected in utero. Molecular analysis of whole sera, purified serum IgM fraction, or dissociated immune complexes demonstrated IgM reactivity against one (47 kDa) or more of several T. pallidum peptides (15, 17, 37, 42, 45, and 87 kDa) recognized as integral membrane components. Sequential analysis of the neonates' sera by immunoblot and enzyme linked immunosorbent assay, using alcohol-treated T. pallidum, T. phagedenis biotype Reiter, and T. vincentii, demonstrated early IgM antibodies followed 3 to 4 months later by IgG2- and IgG1-specific antibodies to T. pallidum. Moreover, an infectivity test done in five rabbits with pooled tissue extracts prepared from liveborn or stillborn animals evoked a seroconversion in two rabbits (reactive Venereal Disease Research Laboratory and fluorescent treponemal antibody tests), suggesting the presence of T. pallidum in the organs. Sera from neonates born to either T. phagedenis biotype Reiter-injected mothers or three normal pregnant females were all serologically negative. The model offers new possibilities for exploration of factors responsible for asymptomatic infection often observed in human congenital syphilis. PMID- 1729191 TI - A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. AB - A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one step growth curve and should be useful to study the molecular basis of the host parasite interaction. PMID- 1729192 TI - Restoration of exocytosis occurs after inactivation of intracellular tetanus toxin. AB - Tetanus toxin blocks carbachol-stimulated release of noradrenaline from bovine adrenal chromaffin cells in culture, provided it can gain access to the cells. This can be achieved by electropermeabilization of the plasma membrane or by enriching the membrane with exogenous gangliosides which serve as carriers of the toxin. The inhibition of noradrenaline release persists for at least 6 days, even in the presence of specific anti-tetanus toxin antibodies in the culture medium. However, the block is preventable, for the most part, when antibodies enter chromaffin cells during electropermeabilization, before the uptake of the toxin is facilitated by inserting exogenous gangliosides into the plasma membrane 2 days later. This indicates that the antibodies pass into the cells through the physically induced pores and that these intracellular antibodies neutralize incoming tetanus toxin. If, on the other hand, exocytosis has been inhibited by tetanus toxin, it will recover within 3 days, provided specific anti-tetanus toxin antibodies are introduced into the cells by electropermeabilization. The recovery is not linked to a specific route of entry of the toxin. It is concluded that the restoration of noradrenaline release requires not only the intracellular neutralization of tetanus toxin but also the reconstitution of the as yet unknown target molecule of the toxin. PMID- 1729193 TI - Cell-free system responsible for internal radiolabeling of glycopeptidolipids of the Mycobacterium avium complex. AB - Internal radiolabeling of serotype-specific glycopeptidolipids with [14C]mannose was accomplished with a cell-free system derived from serotype 20 of the Mycobacterium avium complex. Similar radiolabeling was not apparent with a cell free system derived from the rough colony variant, previously shown to be devoid of glycopeptidolipids. Although a comparative sodium dodecyl sulfate polyacrylamide gel electrophoresis protein analysis of the parent and rough variant strains revealed a close similarity, there were some proteins unique to the parent strain. PMID- 1729194 TI - Adherence of oral streptococci to salivary glycoproteins. AB - We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity. PMID- 1729195 TI - Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera. AB - Previous investigators have demonstrated that a sialic acid residue is added to the terminal galactose moiety of gonococcal lipooligosaccharide (LOS) when incubated with 5'-CMP-N-acetylneuraminic acid. When this in vitro sialylation occurs, gonococci become resistant to the bactericidal activity of normal human serum. This is believed to result because the added sialic acid residue blocks the binding of bactericidal anti-LOS antibodies present in normal human serum. We extend these studies by demonstrating that sialylated gonococci also become resistant to the bactericidal effect of immune sera containing antibodies that recognize exposed components of the outer membrane besides LOS. Prevention of antibody binding to the organism was not the cause, since the same percentage of bactericidal antibodies to the major outer membrane protein, Protein I, can be absorbed with sialylated organisms as with wild-type organisms. In addition, gonococcal sialylation prevents opsonophagocytosis by antigonococcal antisera. The negative effect of sialic acid on the complement pathway might be the reason for the findings in this study. PMID- 1729196 TI - A neutral glycoprotease of Pasteurella haemolytica A1 specifically cleaves O sialoglycoproteins. AB - A neutral metalloprotease with marked specificity for an O-sialoglycoprotein has been isolated from culture supernatants of Pasteurella haemolytica A1. The 35-kDa enzyme cleaves human erythrocyte glycophorin A, which is O glycosylated, but does not cleave N-glycosylated proteins or nonglycosylated proteins. Glycophorin A was cleaved when it was present in situ in erythrocyte ghost plasma membranes or when it was free in solution. The glycoprotease did not hydrolyze glycophorin A from which sialate residues had been removed by neuraminidase treatment. An immobilized preparation of the enzyme cleaved glycophorin A at several positions, with a major site of cleavage at Arg-31-Asp-32. The glycoprotease is inhibited by EDTA, citrate, and ascorbate, but inhibition appears to be due to the masking of metal ion activators rather than to their removal. The enzyme is not inhibited by phosphoramidon, an inhibitor of other bacterial neutral metalloproteases. PMID- 1729197 TI - Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis. AB - Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding. PMID- 1729198 TI - Production, purification, and characterization of botulinolysin, a thiol activated hemolysin of Clostridium botulinum. AB - A hemolysin, botulinolysin, produced by Clostridium botulinum was purified to homogeneity and characterized. First, a strain of C. botulinum type C, strain C 203 Tox, which produced a large amount of hemolysin, was selected, and optimal culture medium and conditions for its production of hemolysin were determined. The hemolysin produced in the culture supernatant of this strain under optimal conditions was purified by a combination of ammonium sulfate precipitation, DEAE Sepharose CL-6B column chromatography, Sephadex G-75 gel permeation chromatography, and SP-Toyopearl 650 M cation-exchange column chromatography, with a recovery of 12%. The purified hemolysin gave a single protein band in polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulfate (SDS). The protein in this band in PAGE with SDS was estimated to have a molecular weight of 58,000 and was immunostained with a neutralizing monoclonal antibody. In PAGE without SDS, the hemolytic activity corresponded in position to the single protein band. The pI of the hemolysin was 8.4. Amino acid analysis of the purified hemolysin indicated the presence of four half-cystine residues per molecule. The purified hemolysin had a specific activity of 2,100 hemolytic units per microgram of protein on rabbit erythrocytes. It was activated by SH compounds, inhibited by cholesterol, and heat labile. The optimum pH for hemolysis was 6.0 to 7.0. Rabbit, human, and guinea pig erythrocytes were the most susceptible to the hemolysin, while sheep, mouse, rat, and chicken erythrocytes were much less susceptible. The purified hemolysin had a lethal effect in mice and was cytotoxic for some cultured cells: its 50% lethal dose in mice was 310 ng, and its 50% cytotoxic dose for Vero cells was 120 ng/ml. PMID- 1729199 TI - In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies. AB - The role(s) of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF alpha), and interleukin-4 (IL-4) in establishment and maintenance of protective immunity to Francisella tularensis LVS in mice (C3H/HeN) was examined by selective removal of these cytokines in vivo with neutralizing antibodies. The 50% lethal dose (LD50) for mice infected intradermally with F. tularensis alone was 136,000 CFU; treatment of mice with anti-IFN-gamma or anti-TNF-alpha at the time of infection significantly reduced (P much less than 0.05) the LD50 to 2 and 5 CFU, respectively. Abrogation of protective immunity, however, was effective only when anti-IFN-gamma or anti-TNF-alpha was administered prior to day 3 postinfection. In contrast, the LD50 for mice treated with anti-IL-4 was repeatedly higher (555,000 CFU) than for controls; this difference, however, was not significant (P greater than 0.05). Thus, IL-4 may be detrimental, while IFN gamma and TNF-alpha were clearly crucial to the establishment of protective immunity to F. tularensis during a primary infection. The importance of IFN-gamma and TNF-alpha during a secondary immune response to F. tularensis was also investigated. Spleen cells from immune mice passively transfer protective immunity to recipient mice in the absence of confounding antibody-mediated immunity. This passive transfer of immunity, however, was abrogated by treatment of recipient mice with anti-IFN-gamma or anti-TNF-alpha at the time of challenge infection. That anticytokines effectively abrogate protective immunity very early in the course of infection with F. tularensis suggests that T-cell-dependent activation of macrophages for microbicidal activity is unlikely. These T-cell independent events early in the course of infection may suppress bacterial replication until a T-cell-dependent response ultimately clears the bacteria. PMID- 1729200 TI - Comparison of irradiated-cercaria schistosome vaccine models that use 15- and 50 kilorad doses: the 15-kilorad dose gives greater protection, smaller liver sizes, and higher gamma interferon levels after challenge. AB - The protection and immune response to infection caused by the parasite Schistosoma mansoni were studied by comparison of two murine irradiated-vaccine models. Mice were exposed from 1 to 4 times to infective-stage cercariae attenuated with either a moderate dose (15 kilorads) or a high dose (50 kilorads) of radiation. Seven weeks after challenge infection, the mice were assessed for resistance, liver size, and lymphokine responses to parasite antigens. Both vaccine models showed high levels of protection, but the moderate-dose model proved superior in that mice in those groups achieved higher levels of resistance in fewer exposures. Additionally, the mice exposed three times and four times to moderately irradiated cercariae all had significantly lower liver weights independent of worm burden. Assessment of lymphokine production by the spleen cells at the time of perfusion showed that gamma interferon was the only lymphokine of those measured that was differentially produced in the two models and correlated with a decrease in size of in vitro granulomas. The findings suggest that a selected vaccine regimen may lead to greater resistance and decreased liver pathology, the latter of which appears to be mediated by induction of gamma interferon. PMID- 1729201 TI - Free versus liposome-encapsulated muramyl tripeptide phosphatidylethanolamide in treatment of experimental Klebsiella pneumoniae infection. AB - The effect of free and liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) on resistance to Klebsiella pneumoniae infection in mice was investigated. It was shown that administration of MTPPE, at 24 h before bacterial inoculation, led to a dose-dependent antibacterial resistance in terms of increased clearance of bacteria from the blood and bacterial killing in various organs. The lowest effective dose of MTPPE was 50 micrograms per mouse. Administration of liposome-encapsulated MTPPE was also effective at a dose of 25 micrograms per mouse. The time of administration of both free and liposome encapsulated MTPPE, with respect to the appearance of bacteria in the blood, was very important and indicated that repeated administration is necessary to obtain protection for a prolonged period. In view of the toxicity of MTPPE, it was an important observation that repeated administration of MTPPE in the liposome encapsulated form also produced antibacterial resistance. Administration of free and liposome-encapsulated MTPPE resulted in increased numbers of granulocytes, monocytes, and lymphocytes in the blood of uninfected mice and prevented leukopenia in infected mice. PMID- 1729203 TI - Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli. AB - We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1 containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp. PMID- 1729204 TI - Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti. AB - Acetobacter aceti has an ability to grow under two different culture conditions, on shaking submerged cultures and on static pellicle-forming cultures. The respiratory chains of A. aceti grown on shaking and static cultures were compared, especially with respect to the terminal oxidase. Little difference was detected in several oxidase activities and in cytochrome b and c contents between the respiratory chains of both types of cells. Furthermore, the results obtained here suggested that the respiratory chains consist of primary dehydrogenases, ubiquinone, and terminal ubiquinol oxidase, regardless of the culture conditions. There was a remarkable difference, however, in the terminal oxidase, which is cytochrome a1 in cells in shaking culture but cytochrome o in cells grown statically. Change of the culture condition from shaking to static caused a change in the terminal oxidase from cytochrome a1 to cytochrome o, which is concomitant with an increase of pellicle on the surface of the static culture. In contrast, reappearance of cytochrome a1 in A. aceti was attained only after serial successive shaking cultures of an original static culture; cytochrome a1 predominated after the culture was repeated five times. In the culture of A. aceti, two different types of cells were observed; one forms a rough-surfaced colony, and the other forms a smooth-surfaced colony. Cells of the former type predominated in the static culture, while the cells of the latter type predominated in the shaking culture. Thus, data suggest that a change of the culture conditions, from static to shaking or vice versa, results in a change of the cell type, which may be related to the change in the terminal oxidase from cytochrome a1 to cytochrome o in A. aceti. PMID- 1729202 TI - Environmental signals controlling expression of virulence determinants in bacteria. PMID- 1729205 TI - Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR. AB - Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calculated molecular weight of 49,225. E. coli ASL is homologous to the corresponding enzymes from Bacillus subtilis and chickens and also to fumarase from B. subtilis. Gene phoP is 232 bp downstream of purB. Gene purB is regulated threefold by the purine pool and purR. Transcriptional regulation of purB involves binding of the purine repressor to the 16-bp conserved pur regulon operator. The purB operator is 224 bp downstream of the transcription start site and overlaps codons 62 to 67 in the protein coding sequence. PMID- 1729206 TI - Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. AB - Two DNA segments, dnrR1 and dnrR2, from the Streptomyces peucetius ATCC 29050 genome were identified by their ability to stimulate secondary metabolite production and resistance. When introduced into the wild-type ATCC 29050 strain, the 2.0-kb dnrR1 segment caused a 10-fold overproduction of epsilon rhodomycinone, a key intermediate of daunorubicin biosynthesis, whereas the 1.9 kb dnrR2 segment increased production of both epsilon-rhodomycinone and daunorubicin 10- and 2-fold, respectively. In addition, the dnrR2 segment restored high-level daunorubicin resistance to strain H6101, a daunorubicin sensitive mutant of S. peucetius subsp. caesius ATCC 27952. Analysis of the sequence of the dnrR1 fragment revealed the presence of two closely situated open reading frames, dnrI and dnrJ, whose deduced products exhibit high similarity to the products of several other Streptomyces genes that have been implicated in the regulation of secondary metabolism. Insertional inactivation of dnrI in the ATCC 29050 strain with the Tn5 kanamycin resistance gene abolished epsilon rhodomycinone and daunorubicin production and markedly decreased resistance to daunorubicin. Sequence comparison between the products of dnrIJ and the products of the Streptomyces coelicolor actII-orf4, afsR, and redD-orf1 genes and of the Streptomyces griseus strS, the Saccharopolyspora erythraea eryC1, and the Bacillus stearothermophilus degT genes reveals two families of putative regulatory genes. The members of the DegT, DnrJ, EryC1, and StrS family exhibit some of the features characteristic of the protein kinase (sensor) component of two-component regulatory systems from other bacteria (even though none of the sequences of these four proteins show a significant overall or regional similarity to such protein kinases) and have a consensus helix-turn-helix motif typical of DNA binding proteins. A helix-turn-helix motif is also present in two of the proteins of the other family, AfsR and RedD-Orf1. Both sets of Streptomyces proteins are likely to be trans-acting factors involved in regulating secondary metabolism. PMID- 1729207 TI - Cloning, sequencing, expression, and functional studies of a 15,000-molecular weight Haemophilus somnus antigen similar to Escherichia coli ribosomal protein S9. AB - Haemophilus somnus is a gram-negative bacterium capable of causing a number of disease syndromes in cattle. This article describes the cloning and characterization of a gene coding for a 15,000-molecular-weight (15K) polypeptide which reacts strongly with antiserum against H. somnus. Analysis of plasmid encoded polypeptides by polyacrylamide gel electrophoresis showed that the corresponding gene is the second in a transcriptional unit. The first gene codes for a protein with a molecular weight of approximately 17,000. Using antiserum against the two recombinant proteins, we could show that the natural proteins are predominantly present in purified ribosomes from H. somnus. The nucleotide sequence of both genes and flanking regions has been determined, and the deduced amino acid sequence of the two polypeptides was used to search for sequence homology in the GenBank data base. The 15K polypeptide showed 89% similarity to the Escherichia coli ribosomal protein S9, and the 17K polypeptide showed 94% similarity to the E. coli ribosomal protein L13. In E. coli, the corresponding genes constitute a bicistronic operon, with the same gene order as that found in H. somnus. A plasmid expressing the 15K protein was found to complement an E. coli rpsI mutation. When a frameshift mutation was introduced into the 15K protein gene, the resulting plasmid failed to complement this rpsI mutation, demonstrating functional homology between the 15K protein and S9 from E. coli. Downstream from the 15K protein gene is located another open reading frame, which could code for a polypeptide with a predicted molecular weight of 24,427. A protein with a similar molecular weight was detected in minicells containing the recombinant clone. This polypeptide is 69% similar to the stringent starvation protein (Ssp) of E. coli. PMID- 1729208 TI - Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan. AB - Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan. PMID- 1729209 TI - Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus. AB - DNA within Escherichia coli colonies carrying cloned Acinetobacter calcoaceticus genes transforms mutant A. calocaceticus cells with high efficiency. Therefore, E. coli colonies containing such cloned genes can be identified by replica plating onto a lawn of A. calcoaceticus mutant cells. Transformation of A. calcoaceticus also facilitates gap repair and thus allows recovery of specified chromosomal segments in recombinant plasmids. These procedures were used to demonstrate the clustering of A. calcoaceticus genes required for utilization of p-hydroxybenzoate. Chromosomal linkage of the bacterial genes, contained in different operons separated by about 10 kbp of DNA, may have been selected on the basis of their physiological interdependence. PMID- 1729210 TI - A zinc finger protein from Candida albicans is involved in sucrose utilization. AB - A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans. PMID- 1729211 TI - Integration host factor is required for positive regulation of the tdc operon of Escherichia coli. AB - A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter. PMID- 1729212 TI - The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium. AB - A detailed deletion map of the CobII and CobIII regions of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2 has been constructed. The CobII region encodes functions needed for the synthesis of lower ligand 5,6 dimethylbenzimidazole (DMB); CobIII encodes functions needed for the synthesis of the nucleotide loop that joins DMB to the corrin macrocycle. The genetic analysis of 117 deletion, insertion, and point mutations indicates that (i) the CobII and CobIII mutations are contiguous--that is, they are grouped according to function; (ii) the CobII region is composed of four complementation groups (cobJKLM); (iii) cobM mutations do not complement mutations in any of the other three CobII groups; and (iv) CobIII mutations include three complementation groups that correspond to the cobU, cobS, and cobT genes. PMID- 1729213 TI - Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts. AB - Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon. PMID- 1729214 TI - Evolutionary relationships among sulfur- and iron-oxidizing eubacteria. AB - Some 37 reverse transcriptase, partial 16S rRNA sequences from sulfur- and/or iron-oxidizing eubacteria, including sequences from species of the genera Thiobacillus, Thiothrix, Thiomicrospira, Acidophilium, "Leptospirillum," Thiovulum, and Chlorobium, have been determined. In addition, 16S sequences from a number of unnamed sulfur- and/or iron-oxidizing bacteria from hydrothermal vent sites, from invertebrate-bacterial endosymbioses, and from various mineral recovery operations also have been determined. The majority of sequences place their bacterial donors in one or another of the subdivisions of the Proteobacteria. However, three unnamed facultatively thermophilic iron-oxidizing isolates, Alv, BC, and TH3, are affiliated with the gram-positive division. One H2S-oxidizer, from the genus Thiovulum, is affiliated with Campylobacter, Wolinella, and other genera in what appears to be a new subdivision of the Proteobacteria. Three "Leptospirillum"-helical vibrioid isolates, BU-1, LfLa, and Z-2, exhibit no clear phylum level affiliation at all, other than their strong relationship to each other. A picture is emerging of an evolutionary widespread capacity for sulfur and/or iron oxidation among the eubacteria. PMID- 1729215 TI - Separable domains define target cell specificities of an RTX hemolysin from Actinobacillus pleuropneumoniae. AB - The leukotoxin (LktA) from Pasteurella haemolytica and the hemolysin (AppA) from Actinobacillus pleuropneumoniae are members of a highly conserved family of cytolytic proteins produced by gram-negative bacteria. Despite the extensive homology between these gene products, LktA is specific for ruminant leukocytes while AppA, like other hemolysins, lyses erythrocytes and a variety of nucleated cells, including ruminant leukocytes. Both proteins require activation facilitated by the product of an accessory repeat toxin (RTX) C gene for optimal biological activity. We have constructed six genes encoding hybrid toxins by recombining domains of ltkA and appA and have examined the target cell specificities of the resulting hybrid proteins. Our results indicate that the leukocytic potential of AppA, like that of LktA, maps to the C-terminal half of the protein and is physically separable from the region specifying erythrocyte lysis. As a consequence, we were able to construct an RTX toxin capable of lysing erythrocytes but not leukocytes. The specificity of one hybrid was found to be dependent upon the RTX C gene used for activation. With appC activation, this hybrid toxin lysed both erythrocytes and leukocytes, while lktC activation produced a toxin which could attack only leukocytes. This is the first demonstration that the specificity of an RTX toxin can be determined by the process of C-mediated activation. PMID- 1729216 TI - Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism. AB - The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found. PMID- 1729217 TI - Transcriptionally active regions in the genome of the archaebacterium Haloferax volcanii. AB - Transcriptionally active regions of the Haloferax volcanii genome were mapped by hybridization of radiolabeled cDNA to Southern blots of our minimal set of overlapping cosmid clones covering 96% of the 4.1-Mbp genome. Transcription during exponential growth occurred in nearly every region of the 2,920-kbp chromosome. Large parts of the 690- and the 86-kbp plasmids were transcribed, but the 440-kbp plasmid showed little expression. Transcription after a 40-min heat shock at 65 degrees C was generally reduced, apart from a small set of strongly expressed loci all situated on the chromosome. PMID- 1729218 TI - Sequence and expression of the Escherichia coli K1 neuC gene product. AB - The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon. PMID- 1729219 TI - In vivo inhibition of TonB-dependent processes by a TonB box consensus pentapeptide. AB - The TonB box, a conserved pentapeptide sequence found in TonB-dependent colicins and receptors, is thought to interact physically with the TonB protein to facilitate TonB-dependent processes. Strains of Escherichia coli were treated in vivo with the synthetic TonB box pentapeptide Glu-Thr-Val-Ile-Val. The pentapeptide inhibited several TonB-dependent processes, including cell growth in low-iron medium, phi 80 infection, and killing by colicins B and Ia. Two unrelated control pentapeptides had no effect on TonB-dependent processes. PMID- 1729220 TI - Localization of alg, opr, phn, pho, 4.5S RNA, 6S RNA, tox, trp, and xcp genes, rrn operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO. AB - The genes encoding the rrn operons, the 4.5S and 6S RNAs, elements of protein secretion, and outer membrane proteins F and I, and regulatory as well as structural genes for exotoxin A, alkaline phosphatase, and alginate and tryptophan biosynthesis, were assigned on the SpeI/DpnI macrorestriction map of the Pseudomonas aeruginosa PAO chromosome. The zero point of the map was relocated to the chromosomal origin of replication. PMID- 1729221 TI - Evidence for differential regulation of genes in the chondroitin sulfate utilization pathway of Bacteroides thetaiotaomicron. AB - Expression of the chondroitin sulfate utilization (csu) genes of Bacterioides thetaiotaomicron is regulated by chondroitin sulfate. We have now found, however, that the csu genes are not all regulated in the same way. In particular, the gene encoding beta-glucuronidase (csuE) is expressed under two different conditions that do not lead to expression of other csu genes. PMID- 1729222 TI - New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways. AB - Proper positioning of division sites in Escherichia coli requires balanced expression of minC, minD, and minE gene products. Previous genetic analysis has shown that either MinD or an apparently unrelated protein, DicB, cooperates with MinC to inhibit division. We have isolated and sequenced minC mutations that suppress division inhibition caused by overproduction of either DicB or MinD proteins. Most missense mutations were located in the amino acid 160 to 200 region of MinC (231 amino acids). Some mutations exhibited preferential resistance to one or the other coinhibitor, suggesting that two distinct proteins, possibly MinD and DicB themselves, interact in slightly different manners with the same region of MinC to promote division inhibition. PMID- 1729223 TI - Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion. AB - Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive. PMID- 1729224 TI - Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. AB - The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division. MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are required for the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor. PMID- 1729225 TI - Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1. AB - The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase. PMID- 1729226 TI - The rcsA gene of Escherichia coli O9:K30:H12 is involved in the expression of the serotype-specific group I K (capsular) antigen. AB - Escherichia coli produces two distinct types of capsular polysaccharide (designated groups I and II), which are distinguished by chemical, physical, and genetic characteristics. The K30 capsular antigen is a member of the group I, or heat-stable, capsules. We have cloned rcsA from E. coli O9:K30 and determined the nucleotide sequence. The rcsAK30 sequence is virtually identical to the rcsAK-12 sequence (V. Stout, A. Torres-Cabassa, M. R. Maurizi, D. Gutnick, and S. Gottesman, J. Bacteriol. 173:1738-1747, 1991). RcsAK-12 is a transcriptional activator involved in expression of the extracellular polysaccharide colanic acid in E. coli K-12. rcsAK30 complemented an rcsAK-12 mutation and activated colanic acid synthesis in E. coli K-12 strains. However, in E. coli K30, increasing the levels of RcsA by introducing multicopy rcsAK30 or a Lon mutation resulted in elevated synthesis of the K30 capsular polysaccharide; no colanic acid was detected. E. coli K-12 strains in which the chromosomal his region was replaced by that from E. coli K30 were able to synthesize K30 capsular polysaccharide. These K-12/K30 hybrid strains did not produce colanic acid, suggesting that the genes for synthesis of colanic acid and the K30 capsular polysaccharide may be allelic. rcsA sequences were also detected in the group II strains E. coli K1 and K5. Introduction of rcsAK30 into group II strains resulted in activation of colanic acid biosynthesis rather than the group II capsule. Given the role of RcsA in other members of the family Enterobacteriaceae, our results provide further evidence that this protein may be a relatively widespread regulatory component for the synthesis of enterobacterial extracellular polysaccharides. PMID- 1729227 TI - The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail. AB - A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, Salmonella minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. We determined the nucleotide sequence of the fragment containing the rck locus. Mutations in the two major open reading frames confirmed that the complement resistance mediated by pADE016 was due to a single 555-bp rck gene encoding a 17-kDa outer membrane protein. Analysis of the rck gene revealed that the Rck outer membrane protein consisted of 185 amino acid residues, with a calculated postcleavage molecular mass of 17.4 kDa. Rck is homologous to a family of outer membrane proteins expressed in gram-negative bacteria, two of which have been associated with virulence-related phenotypes: PagC, required by S. typhimurium for survival in macrophages and for virulence in mice; and Ail, a product of the Yersinia enterocolitica chromosome capable of mediating bacterial adherence to and invasion of epithelial cell lines. Rck, most closely related to PagC, represents the third outer membrane protein in this five member family with a distinct virulence-associated phenotype. PMID- 1729228 TI - Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. AB - It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane. PMID- 1729229 TI - Life after log. PMID- 1729230 TI - Distribution of an L-isoaspartyl protein methyltransferase in eubacteria. AB - A protein carboxyl methyltransferase (EC 2.1.1.77) that recognizes age-damaged proteins for potential repair or degradation reactions has been found in all vertebrate tissues and cells examined to date. This enzyme catalyzes the transfer of methyl groups from S-adenosylmethionine to the carboxyl groups of D-aspartyl or L-isoaspartyl residues that are formed spontaneously from normal L-aspartyl and L-asparaginyl residues. A similar methyltransferase has been found in two bacterial species, Escherichia coli and Salmonella typhimurium, suggesting that this enzyme performs an essential function in all cells. In this study, we show that this enzyme is present in cytosolic extracts of six additional members of the alpha and gamma subdivisions of the purple bacteria: Pseudomonas aeruginosa (gamma), Rhodobacter sphaeroides (alpha), and the gamma enteric species Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Serratia marcescens. DNA probes from the E. coli methyltransferase gene hybridized only to the chromosomal DNA of the enteric species. Interestingly, no activity was found in the plant pathogen Erwinia chrysanthemi, a member of the enteric family, nor in Rhizobium meliloti or Rhodopseudomonas palustris, two members of the alpha subdivision. Additionally, we could not detect activity in the four gram-positive species Bacillus subtilis, B. stearothermophilus, Lactobacillus casei, and Streptomyces griseus. The absence of enzyme activity was not due to the presence of inhibitors in the extracts. These results suggest that many cells may not have the enzymatic machinery to recognize abnormal aspartyl residues by methylation reactions. Since the nonenzymatic degradation reactions that generate these residues occur in all cells, other pathways may be present in nature to ensure that these types of altered proteins do not accumulate and interfere with normal cellular physiology. PMID- 1729231 TI - Developmental regulation of CUP gene expression through DNA methylation in Mucor spp. AB - Inserts which carried the CUP gene from Saccharomyces cerevisiae or Mucor racemosus were used as hybridization probes to measure the methylation state and expression of the CUP gene from Mucor rouxii at different stages of growth. It was observed that the fungus contains a CUP multigene family. All the CUP genes were present in a hypermethylated DNA region in nongrowing and isodiametrically growing spores and were not transcribed at these stages. After germ tube emergence, CUP genes became demethylated and transcriptionally active. Development, demethylation, and transcription of CUP genes were blocked by the ornithine decarboxylase inhibitor 1,4-diaminobutanone. These results suggest that genes that are activated during development became demethylated in this fungus. PMID- 1729232 TI - Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan. AB - By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall. PMID- 1729233 TI - Regulation of the Salmonella typhimurium metA gene by the metR protein and homocysteine. AB - The DNA sequence of the Salmonella typhimurium metA control region is presented. S1 nuclease mapping was used to determine the transcription initiation site. By measuring beta-galactosidase levels in Escherichia coli strains lysogenized with lambda phage carrying a metA-lacZ gene fusion, the MetR protein was shown to activate the metA gene. Homocysteine, an intermediate in methionine biosynthesis, plays a negative role in the MetR-mediated activation mechanism. Gel mobility shift assays and DNase I protection experiments showed that the MetR protein binds to a DNA fragment carrying the metA control region and protects a 26-bp region beginning 9 bp upstream of the -35 promoter sequence. PMID- 1729234 TI - Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases. AB - The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47 amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73 amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit. The evolutionary and functional meanings of these similarities are discussed. PMID- 1729235 TI - cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth. AB - The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5' phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3' phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis. PMID- 1729236 TI - Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography. AB - An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10(-4) M, and the Vmax was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10( 5) M. Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed. PMID- 1729237 TI - Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus. AB - A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined. PMID- 1729238 TI - Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein. AB - To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication. PMID- 1729239 TI - Temporal expression of a membrane-associated protein putatively involved in repression of initiation of DNA replication in Bacillus subtilis. AB - A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990). PMID- 1729240 TI - Molecular analysis of the Escherichia coli phoP-phoQ operon. AB - The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium. PMID- 1729241 TI - Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12. AB - Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene- 4 thiolide] (TLM) is a unique antibiotic structure that inhibits dissociated type II fatty acid synthase systems but not the multifunctional type I fatty acid synthases found in mammals. We screened an Escherichia coli genomic library for recombinant plasmids that impart TLM resistance to a TLM-sensitive strain of E. coli K-12. Nine independent plasmids were isolated, and all possessed a functional beta-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabB15(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented the fabB(Ts) mutation, and directed the overproduction of synthase I activity. TLM selectively inhibited unsaturated fatty acid synthesis in vivo; however, synthase I was not the only TLM target, since supplementation with oleate to circumvent the cellular requirement for an active synthase I did not confer TLM resistance. Overproduction of the FabB protein resulted in TLM resistant fatty acid biosynthesis in vivo and in vitro. These data show that beta ketoacyl-acyl carrier protein synthase I is a major target for TLM and that increased expression of this condensing enzyme is one mechanism for acquiring TLM resistance. However, extracts from a TLM-resistant mutant (strain CDM5) contained normal levels of TLM-sensitive synthase I activity, illustrating that there are other mechanisms of TLM resistance. PMID- 1729242 TI - Characterization of amino acid aminotransferases of Methanococcus aeolicus. AB - Four aminotransferases were identified and characterized from Methanococcus aeolicus. Branched-chain aminotransferase (BcAT, EC 2.6.1.42), aspartate aminotransferase (AspAT, EC 2.6.1.1), and two aromatic aminotransferases (EC 2.6.1.57) were partially purified 175-, 84-, 600-, and 30-fold, respectively. The apparent molecular weight, substrate specificity, and kinetic properties of the BcAT were similar to those of other microbial BcATs. The AspAT had an apparent molecular weight of 162,000, which was unusually high. It had also a broad substrate specificity, which included activity towards alanine, a property which resembled the enzyme from Sulfolobus solfataricus. An additional alanine aminotransferase was not found in M. aeolicus, and this activity of AspAT could be physiologically significant. The apparent molecular weights of the aromatic aminotransferases (ArAT-I and ArAT-II) were 150,000 and 90,000, respectively. The methanococcal ArATs also had different pIs and kinetic constants. ArAT-I may be the major ArAT in methanococci. High concentrations of 2-ketoglutarate strongly inhibited valine, isoleucine, and alanine transaminations but were less inhibitory for leucine and aspartate transaminations. Aromatic amino acid transaminations were not inhibited by 2-ketoglutarate. 2-Ketoglutarate may play an important role in the regulation of amino acid biosynthesis in methanococci. PMID- 1729243 TI - Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni. AB - Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure. PMID- 1729244 TI - NotI genomic cleavage map of Escherichia coli K-12 strain MG1655. AB - Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains. PMID- 1729245 TI - Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. AB - The prfA gene of Listeria monocytogenes encodes a protein that activates transcription of the listeriolysin gene (lisA). In order to explore the role of the prfA gene product in the pathogenesis of listerial infection, we constructed a site-directed insertion mutation in prfA by the chromosomal integration of a novel suicide vector containing a portion of the prfA coding region. This mutation not only transcriptionally silenced the listeriolysin (lisA) gene but also abrogated production of specific RNA transcripts corresponding to the phosphatidylinositol-specific phospholipase C (pic) and metalloprotease (mpl) genes, two further virulence gene products expressed only by pathogenic Listeria strains. The strain was also found to be avirulent when tested in a mouse model of listerial infection. The concomitant loss of multiple characteristics such as production of LisA, Pic, Mpl, and loss of virulence in a mouse infection model is the result of a mutation in a single gene and demonstrates that the prfA gene product is a positive regulator of multiple virulence determinants in L. monocytogenes. PMID- 1729246 TI - Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in Bacillus subtilis. AB - We report the cloning and characterization of the Bacillus subtilis sporulation locus spoIVA, mutations at which cause an unusual defect in spore formation in which the coat misassembles as swirls within the mother cell. We show that spoIVA is a single gene of 492 codons that is capable of encoding a polypeptide of 55 kDa. Transcription of spoIVA is induced at about the second hour of sporulation by the regulatory protein sigma E from two closely spaced promoters designated P1 and P2. Experiments in which the upstream promoter P1 was removed show that transcription of spoIVA from P2 is sufficient for efficient spore formation. Based on these and other findings, we infer that the spoIVA gene product is a morphogenetic protein; we discuss its role in the deposition of coat polypeptides around the developing forespore. PMID- 1729247 TI - Characterization of a sporulation gene, spoIVA, involved in spore coat morphogenesis in Bacillus subtilis. AB - Mutations in the spoIVA locus of Bacillus subtilis abolish cortex synthesis and interfere with the synthesis and assembly of the spore coat. We have characterized the cloned spoIVA locus in terms of its physical structure and regulation during sporulation. The locus contains a single gene capable of encoding an acidic protein of 492 amino acids (molecular weight, 55,174). The gene is transcribed from a sigma E-dependent promoter soon after the formation of the spore septum. A genetic test indicated that expression of spoIVA is only necessary in the mother cell compartment for the formation of a mature spore. This, together with the phenotypic properties of spoIVA mutations, would be in accord with the hypothesis that sigma E is only active after septation and in the mother cell compartment. PMID- 1729248 TI - Gene V protein-mediated translational regulation of the synthesis of gene II protein of the filamentous bacteriophage M13: a dispensable function of the filamentous-phage genome. AB - Introduction of a deletion in the genome of wild-type M13 bacteriophage that eliminates translational repression of M13 gene II by its cognate gene V protein had no effect on phage viability. Furthermore, it was noted that gene V protein of phage IKe, a distant relative of M13, does not function as a translational repressor of its cognate gene II protein. The data strongly indicate that the gene V protein-mediated control of gene II expression in bacteriophage M13 is an evolutionary relic of the ancestral filamentous-phage genome and thus dispensable for proper filamentous-phage replication. PMID- 1729249 TI - Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis. AB - Analysis of the sequence for the gene encoding PspA (pneumococcal surface protein A) of Streptococcus pneumoniae revealed the presence of four distinct domains in the mature protein. The structure of the N-terminal half of PspA was highly consistent with that of an alpha-helical coiled-coil protein. The alpha-helical domain was followed by a proline-rich domain (with two regions in which 18 of 43 and 5 of 11 of the residues are prolines) and a repeat domain consisting of 10 highly conserved 20-amino-acid repeats. A fourth domain consisting of a hydrophobic region too short to serve as a membrane anchor and a poorly charged region followed the repeats and preceded the translation stop codon. The C terminal region of PspA did not possess features conserved among numerous other surface proteins, suggesting that PspA is attached to the cell by a mechanism unique among known surface proteins of gram-positive bacteria. The repeat domain of PspA was found to have significant homology with C-terminal repeat regions of proteins from Streptococcus mutans, Streptococcus downei, Clostridium difficile, and S. pneumoniae. Comparisons of these regions with respect to functions and homologies suggested that, through evolution, the repeat regions may have lost or gained a mechanism for attachment to the bacterial cell. PMID- 1729250 TI - Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene. AB - Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts. PMID- 1729252 TI - Molecular characterization of mutations affecting expression level and growth rate-dependent regulation of the Escherichia coli zwf gene. AB - We characterized three cis dominant mutations which elevate glucose 6-phosphate dehydrogenase level. Growth rate-dependent regulation and oxidative stress control of enzyme level were altered by the mutations. DNA sequencing and transcript mapping showed that the "up" mutations created new promoters whose hyperactive expression overrides the normal regulation of the native promoter. PMID- 1729251 TI - Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. AB - We show that in Escherichia coli at 32 degrees C, the T7 RNA polymerase travels over the lacZ gene about eightfold faster than ribosomes travel over the corresponding mRNA. We discuss how the T7 phage might exploit this high rate in its growth optimization strategy and how it obviates the possible drawbacks of uncoupling transcription from translation. PMID- 1729253 TI - Sensitization of Escherichia coli cells to oxidative stress by deletion of the rpoH gene, which encodes the heat shock sigma factor. AB - A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress. The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal. Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin. sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type. PMID- 1729254 TI - Structural organization of pBC1, a cryptic plasmid from Bacillus coagulans. AB - The complete nucleotide sequence of the Bacillus coagulans plasmid pBC1 was determined. The sequence revealed an open reading frame encoding a polypeptide of 259 amino acids. This open reading frame shows sequence similarity to genes coding for replication-associated proteins in a group of gram-positive bacterial plasmids known to replicate via single-stranded intermediates. A region required for replication in cis, when the intact replicon is supplied in trans, was identified as well. PMID- 1729255 TI - Fumarate or a fumarate metabolite restores switching ability to rotating flagella of bacterial envelopes. AB - Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged. PMID- 1729257 TI - DNA strand breaks and death of thymocytes induced by N-methyl-N-nitrosourea. AB - N-Methyl-N-nitrosourea (MNU) is a potent carcinogen in various sites of experimental animals and induces thymic lymphoma in rats, which has long been hard to induce by any carcinogen. To analyze the action of MNU on thymocytes, DNA strand breaking in thymocytes from the MNU-treated rat and that in MNU-treated cultured thymocytes were assayed. Fluorometric analysis of DNA unwinding (FADU assay), first reported by Birnboim and Jevcak to detect X-ray-induced DNA damage, was modified and applied to detect DNA damage in thymocytes treated with MNU in vitro or in vivo. In the present modified method, cell lysate was admixed with 0.15 M sodium hydroxide, and DNA unwinding was processed at pH 12.0 for up to 2 h at 0 degree C in iced water. Double-stranded DNA remaining after alkaline reaction was detected by binding ethidium bromide and measuring its fluorescence. The severity of DNA damage, both in vivo and in vitro, depended on the MNU concentration. In addition, the sequential survival rate and cell-size distribution of thymocytes treated with MNU in vitro were investigated. A close relationship between the severity of DNA damage and cell death was demonstrated in MNU-treated thymocytes, and DNA damage by a non-cell-killing dose of MNU was detected with this FADU assay. MNU-induced cell death is not programmed as in apoptosis, which is caused in thymocytes physiologically, immunologically and by X-ray irradiation or corticoids. PMID- 1729256 TI - Immunoscintigraphy for cancer detection: "a thousand ills require a thousand cures". PMID- 1729258 TI - Dose/response study of the effects of oestrogens on tumour growth and morphology in the Dunning R3327 prostatic adenocarcinoma. AB - The present study was undertaken to investigate to what extent the oestrogen induced effects on growth and morphology of the Dunning R3327 rat prostatic adenocarcinoma are dose-dependent. Castrated and testosterone-supplemented rats were used in order to study effects of increasing doses of oestrogens on the tumour. It was found that the lowest dose of oestradiol-17 beta that reduced the overall growth, the volume density of the epithelium and epithelial cell area in Dunning R3327 prostatic tumours is 10 micrograms given as daily injections. Higher oestrogen doses (50 micrograms, 200 micrograms, and 500 micrograms), in addition to reducing the volume of tumour epithelium, also induced an increase of the volume density of tumour stroma. The area of stroma cell nuclei was increased by 50 micrograms and 200 micrograms oestradiol-17 beta. These observation, may indicate that the lowest effective oestrogen dose is different in the epithelium and stroma of Dunning tumours and that large doses of oestrogen stimulate the stromal compartment. This stimulatory effect did not influence the inhibitory effects seen on the overall growth of the tumour and on the tumor epithelium. PMID- 1729259 TI - Consideration of the use of 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5- alpha androstan-3-one (4MA), a 5 alpha-reductase inhibitor, in prostate cancer therapy. AB - We investigated the effect of 17 beta-N,N-diethylcarbamoyl-4-methyl-4aza- 5 alpha androstan-3-one (4MA), a 5 alpha-reductase inhibitor, on growth inhibition of androgen-sensitive rat prostatic tumour (R3327-H) and correlated it with changes in weight of normal androgen target tissues and with levels of androgens. Groups of male Copenhagen rats were treated for 28 days with a daily injection of various, increasing doses of 4MA (0.01-4.0 mg/day) and the results were compared with control (vehicle-treated) and with castrated animals. 4MA decreased tumour growth rate in a dose-dependent manner, which was reflected in a decreased incorporation of BrdUrd in DNA of glandular epithelial cells in the tumour. Normal prostate wet weight was also decreased after high-dose 4MA treatment while serum testosterone levels were not affected by 4MA treatment. Contrary to expectations, however, tissue levels of dihydrotestosterone in tumour and ventral prostate were still considerable in 4MA-treated animals. The tumour-inhibiting action of 4MA, therefore, has to be interpreted as not being purely due to 5 alpha-reductase inhibition. On the other hand, it was not possible to demonstrate any direct tumoricidal effect of 4MA in vitro. The relevance of these findings in terms of the endocrine mechanism of action of 4MA on tumour growth is discussed. PMID- 1729260 TI - Effects of tumour necrosis factor alpha on bone marrow aspirates of patients with acute myelogenous leukemia determined by flow-cytometric cell-cycle analysis. AB - Tumour necrosis factor alpha (TNF alpha) exerts cytotoxic and antiproliferative effects on neoplastic cells. It has been used as a therapeutic agent for solid tumours and haematological malignancies. We report on the ex vivo determination of the effect of recombinant human rhuTNF alpha on bone marrow aspirates by a bromodeoxyuridine/propidium iodide method. Cell samples were drawn after 0.5, 2, 4, 6, 8, 10, 22, and 25 h from short-term suspension bone marrow cultures from patients with acute myelogenous leukemia (AML). Flow-cytometric cell-cycle analysis was performed after double DNA staining with propidium iodide and anti BrdU antibodies. By this method the effect of rhuTNF alpha on cell proliferation can be evaluated after only 35 h. In about two-thirds of the bone marrow aspirates of AML an inhibiting effect on rhuTNF alpha can be demonstrated, developing to its full extent after 10 h. PMID- 1729261 TI - Phase I study of doxorubicin, ICRF-187 and granulocyte/macrophage-colony stimulating factor. AB - A group of 16 patients with advanced malignancy were entered on a phase I trial of escalating doses of doxorubicin with ICRF-187 for cardioprotection and granulocyte/macrophage-colony-stimulating factor (GMCSF) for bone marrow protection. Patients received intravenous ICRF-187 (dose ratio 20:1 ICRF 187:doxorubicin) 30 min prior to doxorubicin. GMCSF at a dose of 15 micrograms kg 1 day-1 was self-administered subcutaneously on days 3-14 of the cycle. Doxorubicin was administered every 21 days. Substantial hematological and non hematological toxicity was seen. Fever, malaise, and pulmonary symptoms, thought to be due to GMCSF, were not eliminated by reduction in the GMCSF dose to 10 or 5 micrograms kg-1 day-1. Severe hematological toxicity was seen despite GMCSF administration and it was not possible to escalate the doxorubicin dose above 72 mg/m2 with this combination. Dose escalation of doxorubicin may be more feasible with the use of other growth factors or growth factor combinations. PMID- 1729262 TI - (Sub)periosteal Ewing's sarcoma of bone. AB - Ewing's sarcoma is a small malignant round-cell tumour that arises from mesenchymal cells, predominantly in the medullary cavity of bone. In exceptional cases it originates in the soft tissues and subsequently invades the underlying bone. A (sub) periosteal origin of Ewing's sarcoma is a very rare condition: only a few cases have been published so far. Three cases of (sub)periosteal Ewing's sarcoma, having received neoadjuvant chemotherapy and radiation therapy as well as wide excision, are reported. PMID- 1729263 TI - Mitotic indexes as prognostic predictors in female breast cancer. AB - A series of 688 women with breast cancer were followed-up for a mean of 13 years. Tumour size, axillary lymph node status, histological grade, histological type and two mitotic indexes (M/V; MAI) were assessed and related to disease outcome. Primary tumour size (P less than 0.0001), the volume-corrected mitotic index (M/V) (P less than 0.0001), the mitotic activity index (MAI) (P = 0.0001), and histological grade (P = 0.0074) predicted axillary lymph node status. Recurrence as well as recurrence-free survival was significantly related to the axillary lymph node status (P less than 0.0001), M/V index (P less than 0.0001), MAI (P less than 0.0001), tumour size (P = 0.0031) and histological grade (P = 0.0208). Multivariate analyses disclosed the tumour size and M/V index as independent predictors of axillary metastasis at diagnosis. Recurrence was related independently to M/V index, axillary metastasis and tumour size. Independent predictors of recurrence-free survival in Cox's analysis were M/V index and axillary lymph node status. Axillary lymph node status (P less than 0.0001), tumour size (P less than 0.0001), M/V index (P less than 0.0001), MAI (P less than 0.0001) and histological grade (P = 0.0009) predicted survival in that order. Cox's analysis showed that axillary lymph node status was the most important independent predictor of survival followed by tumour size and M/V index. In a separate Cox's analysis of axillary-lymph-node-negative patients the M/V index and tumour size were independently related to survival. In conclusion the M/V index is an important prognostic factor in breast cancer and also in axillary-lymph-node-negative breast tumours. PMID- 1729264 TI - Differences in glucose recognition by individual rat pancreatic B cells are associated with intercellular differences in glucose-induced biosynthetic activity. AB - In vitro incubated rat islet B cells differ in their individual rates of protein synthesis. The number of cells in biosynthetic activity increases with the glucose concentration. Flow cytometric monitoring of the cellular redox states indicated that islet B cells differ in their individual metabolic responsiveness to glucose. A shift from basal to increased NAD(P)H fluorescence occurred for 18% of the cells at 1 mM glucose, for 43% at 5 mM, and for 70% at 20 mM. The functional significance of this metabolic heterogeneity was assessed by comparing protein synthesis in metabolically responsive and unresponsive subpopulations, shortly after their separation by autofluorescence-activated cell sorting. The glucose-sensitive subpopulation exhibited four- to fivefold higher rates of insulin synthesis during 60-min incubations at 2.5-10 mM glucose. Its higher biosynthetic activity was mainly caused by recruitment of cells into active synthesis and, to a lesser extent, by higher biosynthetic activity per recruited cell. Cells from the glucose-sensitive subpopulation were larger, and presented a threefold higher density of a pale secretory vesicle subtype, which is thought to contain unprocessed proinsulin. It is concluded that intercellular differences in metabolic responsiveness result in functional heterogeneity of the pancreatic B cell population. PMID- 1729265 TI - Histamine and histidine decarboxylase are correlated with mucosal repair in rat small intestine after ischemia-reperfusion. AB - The aim of this experiment was to demonstrate whether histamine and histidine decarboxylase (HDC) contribute to mucosal repair in small intestine subjected to ischemia-reperfusion (I/R). The superior mesenteric artery was occluded for 15 min followed by reperfusion. In jejunal mucosa, histamine content and HDC activity increased after I/R. Histamine output in mesenteric lymph was also elevated after I/R. These increases in HDC activity, and mucosal and lymph histamine levels were suppressed by pretreatment of alpha-fluoromethylhistidine (alpha-FMH), a suicide inhibitor of HDC. alpha-FMH also attenuated the increase of ornithine decarboxylase (ODC) activity normally observed after I/R. Transport of dietary lipid into lymph markedly decreased at 24 h after I/R, yet it was restored to normal at 48 h after I/R. alpha-FMH inhibitor led to a sustained deficit in lipid transport at 48 h after I/R. This sustained functional impairment in alpha-FMH treated animals was associated with blunted responses of HDC activity and histamine content to I/R. Our results suggest that histamine and HDC contribute to the restoration in mucosal function observed at 48 h after I/R. This response may be related, at least in part, to stimulation of ODC activity by histamine. PMID- 1729266 TI - Macrophage functions are regulated by murine decidual and tumor extracellular matrices. AB - Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Understanding the mechanisms which prevent maternal immunological rejection of the fetus remains a fundamental unsolved problem in immunology. We have previously reported that macrophages are inhibited by maternal decidual stromal cells residing at the maternal-fetal interface. In view of the central role of macrophages in cell-mediated immunity, this inhibition may contribute to preventing maternal antifetal responses. We now report that it was the solid phase signals embedded in the extracellular matrix (ECM) made by decidual cells which are responsible for inhibiting macrophage mediated lysis of TNF-alpha-resistant P815 mastocytoma cells. The latter macrophage function is acquired after stimulation by interferon gamma and endotoxin. All these macrophage functions were also inhibited by ECM isolated from the Engelberth-Holm-Swarme (EHS) tumor. This tumor ECM has a similar biochemical composition to decidual ECM. This ECM inhibited the effector, as opposed to the stimulator, phase of macrophage-mediated tumor lysis. Laminin, type IV collagen, and heparan sulfate proteoglycans, the major known components of decidual and EHS ECMs, did not inhibit the above macrophage functions. Altogether these data indicate that macrophages were inhibited by solid phase signals embedded in decidual and EHS ECMs. Whether the solid phase signals in these two ECMs are biochemically identical remains to be determined. To our knowledge, such signals are a novel pathway of inhibiting macrophage functions which may be important in understanding the maternal-fetal immunologic relationship, and the pathogenesis of perinatal infections. Furthermore, the ability of EHS tumor ECM to inhibit macrophage functions may indicate that some tumors may defend themselves against host macrophage responses using solid phase signals. This may be important in understanding some host-tumor relationships. PMID- 1729267 TI - Correlates of aldosterone-induced increases in Cai2+ and Isc suggest that Cai2+ is the second messenger for stimulation of apical membrane conductance. AB - Studies were performed on monolayers of cultured A6 cells, grown on permeable filters, to determine the second messenger system involved in the aldosterone induced increase in electrogenic sodium transport. Addition of aldosterone (1 microM) to the solution bathing the basal surface of cells caused both an increase in Isc and threefold transient rise in intracellular calcium Cai2+ after a delay of approximately 60 min. Because both events were inhibited by actinomycin D and cyclohexamide, they appeared to require transcriptional and translational processes. Addition of BAPTA to the bathing media to chelate Cai2+ reduced Isc and the delayed Cai2+ transient; 50 microM BAPTA inhibited Isc and the rise in Cai2+ by greater than 80%. Further studies suggested that the action of aldosterone to increase Isc may be dependent on a calcium/calmodulin-dependent protein kinase, because W-7 and trifluoperazine reduced the aldosterone-induced Isc in a dose-dependent manner. Taken together, these observations suggest that calcium is a second messenger for the action of aldosterone on sodium transport, and suggest, for the first time, that agonists which bind to intracellular receptors can utilize, via delayed processes dependent on de novo transcription and translation, intracellular second messenger systems to regulate target cell function. PMID- 1729268 TI - Type VII collagen gene expression by cultured human cells and in fetal skin. Abundant mRNA and protein levels in epidermal keratinocytes. AB - Type VII collagen, a genetically distinct member of the collagen family, is present in the cutaneous basement membrane zone as an integral component of the anchoring fibrils. We have recently isolated several cDNAs that correspond to human type VII collagen sequences. One of these cDNAs (clone K-131) was utilized to examine type VII collagen gene expression in cultures of human cells by Northern analyses, in situ hybridizations and indirect immunofluorescence. Northern hybridizations revealed the presence of an approximately 9-kb mRNA transcript, and indicated a high level of expression in epidermal keratinocytes as well as in an oral epidermoid carcinoma cell line (KB), while the expression was considerably lower in skin fibroblasts and in several virally or spontaneously transformed epithelial cell lines. In situ hybridizations of cultured keratinocytes supported the notion of a high level of gene expression. Indirect immunofluorescence of skin from a 19-wk fetus revealed type VII collagen gene expression at the dermal-epidermal basement membrane zone. These results indicate that several different cell types including epidermal keratinocytes and dermal fibroblasts express the type VII collagen gene, but epidermal keratinocytes may be the primary cell source of type VII collagen in developing human skin. PMID- 1729269 TI - Increased lipolysis and its consequences on gluconeogenesis in non-insulin dependent diabetes mellitus. AB - The present studies were undertaken to determine whether lipolysis was increased in non-insulin-dependent diabetes mellitus (NIDDM) and, if so, to assess the influence of increased glycerol availability on its conversion to glucose and its contribution to the increased gluconeogenesis found in this condition. For this purpose, we infused nine subjects with NIDDM and 16 age-, weight-matched nondiabetic volunteers with [2-3H] glucose and [U-14C] glycerol and measured their rates of glucose and glycerol appearance in plasma and their rates of glycerol incorporation into plasma glucose. The rate of glycerol appearance, an index of lipolysis, was increased 1.5-fold in NIDDM subjects (2.85 +/- 0.16 vs. 1.62 +/- 0.08 mumol/kg per min, P less than 0.001). Glycerol incorporation into plasma glucose was increased threefold in NIDDM subjects (1.13 +/- 1.10 vs. 0.36 +/- 0.02 mumol/kg per min, P less than 0.01) and accounted for twice as much of hepatic glucose output (6.0 +/- 0.5 vs. 3.0 +/- 0.2%, P less than 0.001). Moreover, the percent of glycerol turnover used for gluconeogenesis (77 +/- 6 vs. 44 +/- 2, P less than 0.001) was increased in NIDDM subjects and, for a given plasma glycerol concentration, glycerol gluconeogenesis was increased more than two-fold. The only experimental variable significantly correlated with the increased glycerol gluconeogenesis after taking glycerol availability into consideration was the plasma free fatty acid concentration (r = 0.80, P less than 0.01). We, therefore, conclude that lipolysis is increased in NIDDM and, although more glycerol is thus available, increased activity of the intrahepatic pathway for conversion of glycerol into glucose, due at least in part to increased plasma free fatty acids, is the predominant mechanism responsible for enhanced glycerol gluconeogenesis. Finally, although gluconeogenesis from glycerol in NIDDM is comparable to that of alanine and about one-fourth that of lactate is terms of overall flux into glucose, glycerol is probably the most important gluconeogenic precursor in NIDDM in terms of adding new carbons to the glucose pool. PMID- 1729270 TI - Effects of osmolality on bicarbonate absorption by medullary thick ascending limb of the rat. AB - Previously we demonstrated that arginine vasopressin (AVP) directly inhibits bicarbonate absorption (JHCO3, pmol/min per mm) in the medullary thick ascending limb (MTAL) of the rat. To determine whether changes in osmolality also may affect bicarbonate absorption, MTAL were studied in vitro with 25 mM HCO3- solutions. Control osmolality was 290 mosmol/kg H2O. In the absence of AVP, increasing osmolality to 560 in perfusate and bath by addition of 150 mM NaCl reduced JHCO3 from 13.7 to 4.5. With 2 x 10(-10) M AVP in the bath, adding 150 mM NaCl to perfusate and bath reduced JHCO3 from 6.9 to 0.6, while adding NaCl to the bath alone reduced JHCO3 from 7.1 to 0.5. Adding 150 mM NaCl to perfusate and bath caused a similar inhibition of JHCO3 in MTAL perfused with furosemide to inhibit net NaCl absorption. In the presence of AVP, adding 600 mM urea to perfusate and bath inhibited JHCO3 by 55%; adding 300 or 600 mM mannitol to perfusate and bath inhibited JHCO3 by 75%. The effects on JHCO3 were reversible and dissociable from changes in transepithelial voltage. CONCLUSIONS: (1) osmolality is a factor capable of regulating renal tubule bicarbonate absorption; (2) hypertonicity produced with NaCl, urea, or mannitol markedly inhibits bicarbonate absorption in the MTAL; (3) this inhibition occurs independent of, and is additive to, inhibition by vasopressin. Hypertonicity may shift TAL HCO3- absorption from medulla to cortex, thereby limiting delivery of bicarbonate to the medullary interstitium during antidiuresis. PMID- 1729271 TI - Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells. A potential mechanism of self perpetuation in liver fibrogenesis. AB - Rat liver perisinusoidal lipocytes (PL) cultured on uncoated plastic transform spontaneously within 6-10 d to myofibroblastlike cells (MFBlC). Parallel to the transformation the TGF alpha- and TGF beta 1-mRNA expression increased and was highest in MFBlC. Competitive radioligand binding assays demonstrated that in contrast to untransformed PL the MFBlC synthesize and secrete transforming growth factor (TGF)-alpha (15 fmol/cell per 24 h) and predominantly the latent form of TGF beta 1 (0.2 fmol/cell per 24 h). Medium conditioned by MFBlC (MFBcM) significantly stimulated PL proliferation with little effect on PL proteoglycan synthesis. By transient acidification of the MFBcM, known to activate the latent form of TGF beta 1, the stimulatory effect on PL proteoglycan synthesis was enhanced and furthermore PL transformation (measured by expression of iso-alpha smooth muscle actin and loss of retinylpalmitate) was accelerated. Preincubation of this medium with neutralizing antibodies to TGF beta resulted in (a) the growth inhibitory effect was converted to a growth stimulation and (b) the stimulatory effect on proteoglycan synthesis was abolished. In summary our data indicate that progressive activation of PL on plastic (transformation to MFBlC) leads to an enhanced expression of the TGF alpha- and TGF beta 1-mRNAs and secretion of the corresponding proteins. Medium conditioned by MFBIC stimulates proliferation, transformation, and PG synthesis of untransformed PL. These mechanisms are suggested to be relevant in self perpetuation of liver fibrogenesis. PMID- 1729272 TI - Effects of chain length on the immunogenicity in rabbits of group B Streptococcus type III oligosaccharide-tetanus toxoid conjugates. AB - One method to improve the immunogenicity of polysaccharide antigens is the covalent coupling of the native polysaccharide or a derivative oligosaccharide to a carrier protein. In general, T cell-dependent properties are enhanced in conjugates of smaller saccharides, but a conformational epitope of the native polysaccharide may be better expressed in conjugates of larger saccharides. We have reported previously the synthesis and immunogenicity in animals of an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. In this study, we sought to determine the optimal size of group B Streptococcus type III oligosaccharide for use in a conjugate vaccine by evaluating the relative immunogenicity of conjugate vaccines containing oligosaccharides that were twofold smaller (7,000 Mr) or larger (27,000 Mr) than that reported previously (14,500 Mr). All three type III oligosaccharide conjugate vaccines were immunogenic in rabbits, in contrast to native, uncoupled group B Streptococcus type III polysaccharide. However, with respect to eliciting specific antibodies that were protective in vivo, the vaccine containing the intermediate-size oligosaccharide was superior to the smaller or larger conjugate vaccine. Analysis of opsonic activity of vaccine-induced antibodies demonstrated a predominance of IgG antibodies, thought to reflect T cell dependence, in response to shorter chain length conjugates, while the conformational epitope of the native polysaccharide was maximally expressed on longer chain length conjugates. These opposing trends may account for the optimal immunogenicity of an intermediate-size group B Streptococcus type III oligosaccharide conjugate vaccine. PMID- 1729273 TI - Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP). AB - Tumor necrosis factor (TNF)-treated 3T3-L1 adipocytes were used as a model for studying the effects of systemic inflammation on adipose tissue. Lipopolysaccharide-treated monocyte-conditioned medium or recombinant human TNF alpha induced morphological dedifferentiation of the adipocytes and led to loss of adipocyte specific gene expression. Gel shift, Southwestern and Western immunoblot analysis demonstrated that dedifferentiation was preceded by a decrease in the DNA binding activity and protein level of the transcription factor CCAAT/enhancer binding protein (C/EBP). Liver activating protein, a related protein that binds identical DNA sequences, increased during cytokine treatment. Both proteins activate specific enhancer elements located in the promoter region of many genes whose transcription is altered during systemic inflammation. Pulse-chase labeling followed by immunoprecipitation demonstrated that C/EBP is a rapidly turning over protein in adipocytes and that cytokine treatment led to a specific, time dependent decrease in its rate of synthesis. Because C/EBP binding sites have been shown to play an important role in regulating the expression of genes involved in adipocyte metabolism, we propose that the TNF-induced changes in the complement of transcription factors binding those sites may be important in the pathogenesis of inflammation-induced atrophy of adipose tissue. PMID- 1729274 TI - Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. AB - Vascular endothelial growth factor (VEGF) is a secreted heparin-binding mitogen; its growth-promoting activity is limited to vascular endothelial cells in vitro and VEGF also stimulates angiogenesis in vivo. To identify target cells for VEGF and investigate the potential physiological role of this factor, iodinated recombinant human VEGF (125I-rhVEGF) was used for in vitro ligand autoradiography on tissue sections from adult rats. 125I-rhVEGF exhibited saturable, displaceable binding to a single class of sites with high affinity and low capacity in all tissues and organs examined. Colocalization of 125I-rhVEGF binding with Factor VIII-like immunoreactivity demonstrated binding sites associated with vascular endothelial cells of both fenestrated and nonfenestrated microvessels and the endothelium of large vessels, while no displaceable binding was evident on nonendothelial cells. Specific binding was associated with quiescent as well as proliferating vessels. These findings support the hypothesis that VEGF plays a specific role in both the maintenance and in the induction of growth of vascular endothelial cells. PMID- 1729275 TI - Gold-specific T cells in rheumatoid arthritis patients treated with gold. AB - Gold-specific T lymphocyte clones were isolated from a patient with rheumatoid arthritis who developed delayed type hypersensitivity reactions to gold. All of the isolated T cell clones required histocompatible antigen presenting cells as well as gold for induction of proliferation. Using a panel of HLA-homozygous Epstein Barr virus-transformed B (EBV-B) cells and anti-HLA antibodies, the clones were shown to recognize gold in the context of DR1 molecules. Gold recognition did not require active antigen processing since specific proliferation was not affected by glutaraldehyde fixation of the DR1 homozygous antigen presenting cells. Furthermore, we could show that gold salts inhibited peptide-induced responses of a peptide-specific T cell clone. In addition to providing evidence for gold-specific T cells in gold-treated RA patients exhibiting delayed type hypersensitivity responses, these data suggest that gold can alter MHC-peptide complexes. The latter observation may in part explain the mechanism/s responsible for both the therapeutic and the toxic effects of gold. PMID- 1729276 TI - Cloning and characterization of the novel gene for mast cell carboxypeptidase A. AB - No gene for a hematopoietic cell carboxypeptidase has previously been characterized. Mast cell carboxypeptidase A (MC-CPA) is a prominent secretory granule marker of mast cell differentiation and phenotype. The 32-kb human MC-CPA gene was isolated, localized to chromosome 3, and found to contain 11 exons. No significant homology was found between the 5' flanking region of the MC-CPA gene and those of three rat pancreatic carboxypeptidase genes (carboxypeptidase A1 and A2, and carboxypeptidase B [CPB]). In contrast, the intron/exon organization of the MC-CPA gene was conserved, most closely resembling the CPB gene. MC-CPA is unique among carboxypeptidases in having a CPA-like substrate-binding pocket and enzymatic activity despite overall protein and gene structures more similar to CPB. Evolutionary tree analysis of the carboxypeptidase gene family showed that, before the mammalian species radiation, a common MC-CPA/CPB ancestor diverged by gene duplication from the lineage leading to CPA, and then underwent another gene duplication to form separate but similar gene structures for MC-CPA and CPB. MC CPA mRNA was prominent in dispersed lung cells enriched for mast cells but was undetectable in other nontransformed populations of several lineages, demonstrating that transcription of MC-CPA, a novel carboxypeptidase gene, provides a specific molecular marker for mast cells among normal hematopoietic cell populations. PMID- 1729277 TI - Ca-mediated stimulation of Cl secretion by reactive oxygen metabolites in human colonic T84 cells. AB - Monochloramine (NH2Cl), a granulocyte-derived reactive oxygen metabolite (ROM), increases short-circuit current (Isc) in cultured T84 monolayers in a concentration-dependent manner up to nonlethal concentrations of 75 microM. Isc increases slowly after NH2Cl, reaching a peak value of 18 +/- 2 microA/cm2 20 min after addition. The Isc changes are persistent (lasting over 20-30 min), depend on medium Cl, and are inhibitable with bumetanide. 36Cl flux studies demonstrated that NH2Cl increases serosa-to-mucosa flux of Cl without changing mucosa-to serosa flux, consistent with stimulation of electrogenic Cl secretion. Isc responses to NH2Cl, but not PGE2, are dependent on medium calcium. As demonstrated in fura-2-loaded T84 cells, NH2Cl increases free cytosolic calcium by influx of extracellular Ca2+ and by release of Ca2+ from endogenous stores. However, NH2Cl had no effect on phosphatidylinositol metabolism or cyclic nucleotide levels. We conclude that ROM directly stimulate electrolyte secretion, an effect in part mediated by increases in cytosolic Ca2+, possibly through increasing Ca2+ permeability of cellular membranes. PMID- 1729278 TI - An anticoagulant dermatan sulfate proteoglycan circulates in the pregnant woman and her fetus. AB - Investigation of the in vitro ability of plasma from pregnant women to inhibit exogenous thrombin (25 nM) demonstrated that heparin cofactor II inhibited more thrombin (3.0 +/- 0.7 nM, mean +/- SD) than plasma from women 3-5 d postpartum (1.9 +/- 0.5 nM) or plasma from nonpregnant adults (1.5 +/- 0.4 nM). Levels of heparin cofactor II were only slightly increased over normal in both pregnant and postpartum women and did not account for the observed increase in thrombin bound to heparin cofactor II. Assay of pregnancy plasma for dermatan sulfate anticoagulant activity demonstrated the presence of activity equivalent to 0.23 +/- 0.02 micrograms/ml of porcine mucosal dermatan sulfate. This activity could not be demonstrated in normal adult plasma or plasma from women on the contraceptive pill. The mass of dermatan sulfate in pregnancy and umbilical cord plasmas was increased over adult control plasma by 0.20 micrograms/ml (53%) and 0.29 micrograms/ml (76%), respectively. The glycosaminoglycan-containing fraction of plasma was isolated and an assay for anticoagulant dermatan sulfate confirmed its presence in both pregnancy and cord plasmas but minimal activity in adult plasma. Gel chromatography of isolated fractions from both pregnancy and cord plasmas revealed a polydisperse, active species with apparent Mr 150,000 D. Reductive elimination decreased the apparent Mr of the active species on gel chromatography to 31,000 D for cord and 21,000 D for pregnancy products. This confirmed the presence of an anticoagulant active dermatan sulfate proteoglycan circulating in the plasmas of pregnant women at term and fetuses at delivery. PMID- 1729279 TI - Variations in codons 84-101 in the core nucleotide sequence correlate with hepatocellular injury in chronic hepatitis B virus infection. AB - Individuals with chronic hepatitis B virus (HBV) infection are generally divided into asymptomatic healthy carriers and patients with chronic liver disease. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes (CTL). To investigate the possible pressure site from CTL, the entire core region of HBV DNA was sequenced in 30 subjects (10 asymptomatic healthy carriers and 20 patients with chronic liver disease). No significant changes in the nucleotide sequence and deduced amino acid residue were noted in the 10 healthy carriers. In contrast, a cluster of changes in a small segment of 18 amino acids (codons 84-101 from the start of the core gene) was found in 15 of the 20 chronic liver disease patients. All these 15 patients had advanced liver diseases (chronic active hepatitis and cirrhosis), whereas only mild liver disease (chronic persistent hepatitis) was found in the five patients without mutations. These data suggest that the region with mutation clustering is the major target of CTL, and that the mutations evolve under the pressure of immune selection. PMID- 1729280 TI - Interleukin 6. A potential autocrine/paracrine factor in Paget's disease of bone. AB - Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation. Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures. At least part of this activity could be ascribed to interleukin 6 (IL-6). In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC. 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6. Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6. These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts. PMID- 1729281 TI - Collagen-induced release of interleukin 1 from human blood mononuclear cells. Potentiation by fibronectin binding to the alpha 5 beta 1 integrin. AB - PBMC express cell surface receptors for extracellular matrix components known as integrins. We have recently shown that ligand binding to one PBMC integrin, the collagen receptor alpha 2 beta 1, stimulates the secretion of interleukin 1 (IL 1). We have now investigated the role of fibronectin (Fn), an adherence protein that has binding sites for both PBMC and collagen, in the generation of the IL-1 response to collagen. In contrast to collagen, Fn did not stimulate IL-1 release but Fn-depleted serum decreased the release of IL-1 induced by collagen. A polyclonal antiserum directed against Fn also decreased the collagen-induced IL-1 secretion. The IL-1 response to collagen from cells incubated in Fn-depleted serum was restored by the addition of either purified Fn or the 120-kD cell binding fragment of Fn, which contains the cell-binding site but not the collagen binding domain. Smaller Arg-Gly-Asp (RGD) peptides failed to enhance the PBMC response to collagen but inhibited in a concentration-dependent fashion the potentiating effect Fn. As expected, a MAb against the alpha 2 beta 1 collagen receptor decreased collagen-induced IL-1 release. However collagen-induced IL-1 release was also inhibited by a MAb against the alpha 5 beta 1 Fn receptor. The effect of the two MAbs was not additive, suggesting that the occupancy of both receptors by ligands is required in order for collagen to induce an maximal response from PBMC. The mechanism by which Fn exerts its effect remains unknown. However, flow-cytometric analysis revealed that Fn does not alter expression of the alpha2beta1 receptor on PBMC. These data demonstrate a potentiating effect of Fn on the collagen-induced secretion of IL-1 from human PBMC and suggest that this effect is mediated via the integrin alpha5beta1. These findings indicate a complex interactive role for specific integrin receptors in the regulation of the mononuclear cell immune response. PMID- 1729282 TI - Hemodynamic regulation of myosin heavy chain gene expression. Studies in the transplanted rat heart. AB - Cardiac work is a major determinant of heart size and growth. Heterotopic cardiac isografts are hemodynamically unloaded and undergo atrophy. To determine the molecular changes that occur as a result of hemodynamic unloading, we have studied the rate of synthesis of total cardiac proteins and myosin heavy chain (MHC) and the expression of the myosin heavy chain gene as reflected in the messenger RNA levels for alpha- and beta-MHC isoforms. 72 h after transplantation there is a significant decrease in left ventricular size accompanied by a 27% decrease in the rate of total cardiac protein synthesis and a 53% decrease in the rate of myosin heavy chain synthesis. In contrast to isografts 14 d after transplantation which have a decrease in protein synthetic capacity, simultaneous measurements of 18S ribosomal RNA and myosin messenger RNA suggest that after 3 d the decrease in synthesis is due to a change in the efficiency of protein translation. While the working in situ heart expresses primarily alpha-MHC mRNA (97%) hemodynamic unloading leads to a 43% decrease in alpha-MHC mRNA concentration and the de novo expression of the beta-MHC mRNA. Total MHC mRNA (alpha plus beta) concentration analyzed by a quantitative S1 nuclease protection assay was similar in the two groups of hearts. Thus, in association with hemodynamic unloading there are changes in cardiac myosin heavy chain content as a result of both gene transcription and protein translation mechanisms. PMID- 1729283 TI - Estrogen maintains trabecular bone volume in rats not only by suppression of bone resorption but also by stimulation of bone formation. AB - Estrogen is generally considered to maintain bone mass through suppression of bone resorption. We have previously demonstrated that administration of pharmacologic doses of estrogen increases bone formation in ovary-intact rats. To assess the effects of physiological concentrations of estrogen on bone formation, estrogen was administered to ovariectomized rats in which bone resorption was suppressed by the bisphosphonate 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP). Animals receiving exogenous 17 beta-estradiol (E2) (1, 10, and 100 micrograms/kg daily for 17 d) showed a dose-dependent increase in trabecular bone volume of 1.9, 25.8, and 43.6%, respectively, compared with those rats treated with AHPrBP alone. The increase in bone volume was associated with an increase in bone formation in E2-treated animals, in which bone resorption had been almost completely suppressed by AHPrBP. Neither ovariectomy, AHPrBP, nor E2 treatment had a significant effect on the volume or rate of formation of cortical bone. Thus, the increased bone resorption, which is a consequence of estrogen deficiency, entrains increased bone formation, which masks a simultaneous reduction in estrogen-dependent bone formation. Therefore, in addition to the nonspecific effect of estrogen to depress formation via coupling, we have identified a specific effect of estrogen to increase formation independent of coupling. Thus it appears that estrogen maintains bone volume not only through inhibition of bone resorption, but also through stimulation of bone formation. PMID- 1729284 TI - Marfan syndrome: defective synthesis, secretion, and extracellular matrix formation of fibrillin by cultured dermal fibroblasts. AB - We studied the synthesis, secretion, and aggregation into the extracellular matrix of fibrillin by dermal fibroblasts from 26 probands with the Marfan syndrome. Cells from seven probands synthesized approximately half the normal amount of fibrillin when compared with intrafamilial or unrelated controls. Cells from an additional seven probands synthesized a normal amount of fibrillin but secreted the protein less efficiently than control cells. Cells from a further eight probands synthesized and secreted normal amounts of fibrillin but the protein was poorly incorporated into extracellular matrix. Cells from the remaining four probands were indistinguishable from control cells in their synthesis and processing of fibrillin. Cells from 18 family members of 10 of the probands were also studied. Cells from affected individuals in the same family had the same biochemical defect and those from unaffected family members were indistinguishable from controls. These results indicate that mutations in the gene that encodes fibrillin are responsible for the Marfan syndrome in the majority of individuals (confirming recent immunohistochemical and genetic linkage studies) and that a variety of mutations can produce the phenotype associated with the syndrome. PMID- 1729285 TI - Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion. AB - T cell proliferative responses to hepatitis B virus-encoded envelope antigen (S + preS2 + preS1), recombinant core antigen (HBcAg), and natural hepatitis B e antigen (HBeAg) were examined in 22 HBeAg-positive patients with chronic type B hepatitis and 17 healthy hepatitis B surface antigen (HBsAg) carriers. The results showed that HBeAg-positive patients had (a) higher levels of T cell responses to HBcAg/HBeAg than those of healthy HBsAg carriers (P less than 0.001 and P less than 0.01, respectively); (b) a further increase in these T cell responses during acute exacerbations (P less than 0.05 and P less than 0.05, respectively); (c) subsidence in the T cell responses to HBcAg/HBeAg after recovery from acute exacerbations and HBeAg seroconversion, whereas the responses would persist at high levels if the patients did not enter a clinical remission; and (d) low levels of T cell responses to S + preS2 + preS1 either before or after HBeAg seroconversion. The appearance of increasing T cell responses to HBcAg/HBeAg usually occurred in the early phase of acute exacerbations. These findings imply that HBcAg/HBeAg-specific T cells play an important role in the exacerbations of chronic hepatitis B and in HBeAg seroconversion. HBcAg/HBeAg specific precursor T cell frequencies were serially studied in selected cases by limiting dilution assay. Elevation (two- to fourfold) of HBcAg/HBeAg-specific precursor T cell frequencies contributed to the increase of HBcAg/HBeAg-specific T cell proliferation during acute exacerbations. PMID- 1729287 TI - The 1991 Nobel Prize in chemistry awarded to an MRI investigator. PMID- 1729286 TI - In vivo cyclooxygenase expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and streptococcal cell wall arthritis. AB - Cyclooxygenase (COX), or prostaglandin (PG) H synthase, plays a role in inflammatory diseases, but very limited data exist on the regulation of COX in vivo. We, therefore, studied the in vivo expression of COX in synovia from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), as well as joints of rats with streptococcal cell wall (SCW) and adjuvant arthritis. Extensive and intense intracellular COX immunostaining, which correlated with the extent and intensity of mononuclear cell infiltration, was observed in cells throughout RA synovia. Significantly less or equivocal staining was noted in OA and normal human synovia. Similarly, COX immunostaining was equivocal in the joints of normal and arthritis-resistant F344/N rats. In contrast, high level expression developed rapidly in euthymic female Lewis (LEW/N) rats throughout the hindlimb joints and overlying tissues including skin, preceding or paralleling clinically apparent experimental arthritis. COX was expressed in the joints of athymic LEW.rnu/rnu rats 2-4 d after injection of SCW or adjuvant but was not sustained. Physiological doses of antiinflammatory glucocorticoids, but not progesterone, suppressed both arthritis and COX expression in LEW/N rats. These observations suggest that, in vivo, (a) COX expression is upregulated in inflammatory joint diseases, (b) the level of expression is genetically controlled and is a biochemical correlate of disease severity, (c) sustained high level up-regulation is T cell dependent, and (d) expression is down-regulated by antiinflammatory glucocorticoids. PMID- 1729288 TI - Medullary CT enhancement in acute renal artery occlusion. AB - The diagnosis of traumatic renal artery occlusion by CT is based on finding the cortical rim sign, nonopacification of the pelvocalyceal system, and occasionally direct visualization of the thrombosed renal artery. We present four patients with renal artery occlusion who have an unusual pattern of medullary enhancement that has a vermiform and spoke-wheel appearance. It is important to realize that this enhancement does not represent functional renal tissue with a partially occluded renal artery. Instead, it is part of the spectrum of findings seen with complete occlusion of the renal artery. PMID- 1729289 TI - MRI and MRA in treatment planning of subdiaphragmatic radiation therapy. AB - Radiotherapy treatment planning needs optimum definition of target volume in its relative position to normal tissue. The aim of our study was to achieve individual field definition in subdiaphragmatic radiotherapy by visualization of the target volume using fast, breath-held MRI and MR angiography. A modified rapid acquisition SE technique (SE 150/10) was used to obtain a coronal image within a 14 s breath-holding period, displaying kidneys, spleen, and lumbar spine on one slice. Coronal MR angiography acquisition in breath-hold technique was performed using a sequential FLASH-2D sequence (FLASH-2D 30/10/30 degrees). For reconstruction of the MR angiogram in coronal view, we used a maximum intensity projection algorithm. A computer program superimposed the MR angiogram onto the MR image. Correct magnification of the superposition image allowed direct projection onto the simulation film. Problems of distortion and different projection techniques were taken into account and quantified by phantom measurements. The localization error measured in a reference plane was less than 5 mm within a radius of 140 mm. Fourteen cases of Hodgkin disease and non-Hodgkin lymphomas were treated employing the novel technique. By superposition of the MR image and the MR angiogram, demarcation of vascular architecture from parenchymatous organs was achieved. Projection of the MR superposition onto the simulation film yielded accurate and convenient field definition using noninvasive imaging techniques. PMID- 1729290 TI - Characterization of musculoskeletal masses using dynamic Gd-DTPA enhanced spin echo MRI. AB - Early studies evaluating the utility of Gd-diethylenetriamine pentaacetic acid (DTPA) enhanced MR imaging for characterization of musculoskeletal masses have demonstrated inconsistent and often conflicting results. In this study a new method, dynamic Gd-DTPA enhanced rapid acquisition spin echo MR imaging, was implemented in the evaluation of 18 musculoskeletal lesions and the enhancement features of these lesions were analyzed. Lesions were evaluated before, during, and sequentially following bolus Gd-DTPA injection. Analysis of intensity, volume, timing of onset, progression, uniformity, and pattern of enhancement did not demonstrate significant differences between benign (n = 8) and malignant (n = 10) masses. Significant variations in enhancement were noted in different regions within these masses, which limits the utility of previous dynamic contrast enhanced methods that provide only a single imaging slice for analysis, and are therefore subject to sampling error. This pilot study indicates no advantage for using dynamic Gd-DTPA enhanced imaging for qualitative lesion characterization. PMID- 1729291 TI - Wavy pelvis sign in CT of multiple hereditary osteochondromatosis. AB - We report three patients with multiple hereditary osteochondromatosis with pelvic CT findings indicating the presence of multiple small osteochondromata. Despite normal appearance of plain radiography in these cases, a characteristic wavy appearance of pelvic brim, which has not been described to date, was clearly shown in all three cases. The finding of wavy pelvis may indicate that pelvic osteochondromata are not as rare as indicated by plain radiographic studies and that malignant degeneration in pelvic osteochondromata may be related to their high incidence. One of these patients had an intracapsular hip joint loose body, originating from femoral neck osteochondroma. This complication is previously unreported. PMID- 1729292 TI - Shoulder MRI: arthroscopic correlation with emphasis on partial tears. AB - In 28 patients the preoperative MR results and arthroscopic examination findings, in which both sides of the rotator cuff were systematically examined, were compared. The interpretive MR criteria were both sensitive and specific for detection of full thickness tears: 100% (5 of 5) and 100% (23 of 23), respectively. Criteria were insensitive in 71% (10 of 14) but specific in 93% (13 of 14) for detection of all tears; 4 of 9 partial thickness tears were not detected of MR. The potential significance of these tears and the use of fat suppression MR sequences to improve detection and depiction of rotator cuff abnormalities are discussed. PMID- 1729293 TI - MRI of anterior cruciate ligament reconstruction. AB - Eleven asymptomatic patients 1-9 months after arthroscopic assisted anterior cruciate ligament (ACL) reconstruction with autogenous semitendinosus and gracilis tendons as a "neoligament" were studied by MR. Each neoligament was clinically intact. Examinations were performed at 1.5 T with T1- and T2-weighted sagittal and oblique spin echo images in the plane of ACL repair. On MR in 9 of the 11 patients (82%) the ACL neoligament appeared as a smooth well-defined band of low signal intensity along its entire course. In two patients (18%) the integrity of the neoligament could not be determined by MR. Ligaments in which integrity could not be determined demonstrated irregularity or a wavy contour, high signal intensity change within the ligament, or discontinuity of the ligament. We conclude that, contrary to previous reports, MR can demonstrate an intact ACL reconstruction. PMID- 1729294 TI - MR tissue characterization of a right atrial mass: diagnosis of a lipoma. AB - A case of histologically confirmed benign lipoma of the right atrium is presented. Magnetic resonance imaging was successfully used to visualize and characterize the tumor previously detected by echocardiography. T1-weighted MR was superior to echocardiography and was in surgical agreement with examination in identifying the relationship of the lipoma to the right atrial wall, the coronary sinus, and the interatrial septum. Comparison of measurements of the tumor's signal intensities on T1- and T2-weighted images and T2 relaxation time with those of surrounding myocardium and mediastinal fat allowed a preoperative diagnosis of lipoma. Magnetic resonance may obviate surgical intervention in selected asymptomatic cases where the diagnosis of benign lipoma appears likely. PMID- 1729295 TI - Recovery of native liver after heterotopic liver transplantation for fulminant hepatic failure: MR studies. AB - Heterotopic liver transplantation involves the transplantation of an auxiliary liver into the subhepatic space while leaving the native liver intact. This procedure is a viable treatment for select patients with fulminant hepatic failure who fail medical treatment. The MR-pathologic correlation of a patient who developed graft failure and recovered full function of her native liver after heterotopic liver transplantation is presented. Based on the imaging and biopsy findings, immunosuppression was withdrawn and the patient remains asymptomatic with normal liver function. Interpreters of imaging studies in this group of patients should not restrict their attention to the heterotopic graft. The return of the native liver in both a structural and functional sense is a clinically important phenomenon that can be detected with MR imaging. PMID- 1729296 TI - Cocaine-induced hepatic necrosis: CT demonstration. AB - Computed tomography of a man with cocaine-induced hepatic necrosis demonstrated a narrow peripheral zone of decreased attenuation that showed widening on follow-up examination 4 days later. The patient died of fulminant hepatic necrosis 10 days after presentation. PMID- 1729297 TI - MR detection of breast implant rupture. AB - Breast implant rupture can be difficult to diagnosis. Various modalities including direct clinical palpation, ultrasound, CT, and mammography have been used to evaluate for the presence of prosthesis rupture. We report a case in which the presence of breast implant rupture was determined using MR with characterization of the inflammatory reaction in the soft tissues around the implant. The absence of ionizing radiation with MR makes it especially well suited for evaluating implant rupture in younger patients in whom breast irradiation should be minimized. PMID- 1729298 TI - Palatal myoclonus and inferior olivary lesions: MRI-pathologic correlation. AB - In a patient with palatal myoclonus, MR imaging demonstrated bilateral hyperintense lesions in the ventral part of the medulla. Microscopic examination of the inferior olives showed gliosis, enlargement and vacuolation of the neurons, and demyelinization. PMID- 1729299 TI - Myxopapillary ependymoma with extensive sacral destruction: CT and MR findings. AB - There have been few reports documenting primary myxopapillary ependymomas in the sacrococcygeal region that result in extensive involvement of the sacrum. We present a 21-year-old man whose CT and MR findings showed massive bony destruction of the sacrum and a large lobulated soft tissue mass. Myxopapillary ependymoma should be included along with giant cell tumor, chordoma, and aneurysmal bone cyst in the differential diagnosis of a destructive osteolytic sacral lesion. PMID- 1729300 TI - Synergistic enhancement of MRI with Gd-DTPA and magnetization transfer. AB - Magnetization transfer (MT) between protons of macromolecules and protons of water molecules is a recently introduced mechanism for tissue contrast in MR imaging. The MT effect is strong in tissues where there is an efficient cross relaxation between macromolecular protons and water protons and where this interaction is the dominant source of relaxation. Paramagnetic ions shorten relaxation times and decrease the MT effect. These two facts led to the assumption that, in the case of contrast enhanced MRI, the combination of the T1 weighted imaging method and the MT technique may yield increased contrast, compared with standard methods. The synergistic effect is demonstrated in this work with studies of egg white samples and by imaging three patients with different brain pathologies. The lesion-to-white matter contrasts, with standard T1-weighted sequences with and without the MT effect, were compared before and after the introduction of Gd-DTPA. In each case the synergistic effect of T1 weighting and MT improved the contrast enhancement provided with Gd diethylenetriamine pentaacetic acid. PMID- 1729301 TI - Contrast-enhanced MRA of the brain. AB - Most sequences for MR angiography (MRA) used today exploit the macroscopic motion of the blood to differentiate vessels from the stationary tissues. An alternative approach to inflow based MRA is contrast enhanced MRA, in which relaxation agents are used to selectively shorten the T1 of the blood below the T1 value of the stationary tissues. We have evaluated cerebral Gd enhanced MRA, comparing it with conventional angiography and noncontrast inflow based MRA. Contrast/enhanced MRAs were obtained at 1.0 T with a 3D FISP sequence with TR/TE/alpha: 35-40 ms, 7-11 ms/TE/25 degrees. Contrast enhancement was obtained by a biphasic injection of a double dose of Gd-DOTA (0.2 mmol/kg) during image acquisition. With the described technique the conspicuity of both cerebral arteries and veins is improved compared to nonenhanced inflow MRA. PMID- 1729302 TI - Sedation for pediatric patients undergoing CT and MRI. AB - Adequate sedation remains one of the most important parts of performing high quality cross-sectional imaging in children. This is a noncomparative retrospective analysis of existing sedation protocols used in 1,158 children between the ages of 1 day and 18 years, checking for safety and efficacy. The most commonly used drugs were chloral hydrate (60-120 mg/kg) by mouth for infants less than 18 months and intravenous Nembutal (2-6 mg/kg) for older children. Sedation was successful in 97% of patients. PMID- 1729303 TI - Dural sinus occlusion due to calvarial metastases: A CT blind spot. AB - Dural sinus thrombosis can be a difficult diagnosis to establish because it may present with nonspecific signs of raised intracranial pressure. Diagnosis by CT is well documented but signs may be subtle. Angiography is the "gold standard" but is invasive and requires a very high index of clinical suspicion to request. Magnetic resonance offers a method of demonstrating the dural sinuses in multiple planes and, furthermore, flow within the sinuses may be depicted by MR angiography. We report on three cases where the diagnosis of superior sagittal sinus thrombosis due to calvarial metastases was missed by CT, primarily due to their site over the convexity, but was demonstrated accurately using MR with MR angiography. PMID- 1729304 TI - Use of MR angiography for stereotactic planning. AB - With the introduction of MR angiography (MRA) into clinical routine MR protocols, it has become possible now to image flowing as well as stationary tissue with excellent contrast using a single modality. This has opened up new perspectives for planning stereotactic approaches, which are characterized by high risks for damaging intracerebral vessels or vital brain structures. In this article we present an MRA based planning method for the treatment of arteriovenous malformations by stereotactic radiosurgery. It includes flow compensated gradient echo pulse sequences for the acquisition of angiographic MR datasets, a stereotactic MR marker system, an algorithm for the correction of geometric distortion of MR image data, and a three-dimensional workstation system for the creation and evaluation of treatment plans. The latter is based on the concept of simultaneously displaying both MR slice and angiographic projection images. This allows the evaluation of intracerebral vasculature together with brain anatomy. The MRA guided planning approach was tested and compared to a conventional X-ray angiographic technique in a clinical study. Our satisfactory results suggest that MRA is a technique that can be used advantageously for stereotactic planning. PMID- 1729305 TI - Signal-to-noise and contrast in fast spin echo (FSE) and inversion recovery FSE imaging. AB - Fast spin echo (FSE) imaging has recently experienced a renewed enthusiasm in the clinical setting for its ability to provide high contrast T2-weighted images in short imaging times. This article evaluates the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) properties of the FSE sequence, inversion recovery (IR) FSE sequence, and conventional SE imaging. The results indicate that FSE imaging displays similar contrast properties to SE imaging, but that the SNR and CNR are improved secondary to the longer TRs and longer effective TEs that may be used. The SNR per unit time of the FSE sequence, and hence its efficiency, is at least a factor of 8 better than the SE sequence when 16 echoes are acquired for each excitation. The addition of a slice selective inversion pulse in IR-FSE allows rapid generation of IR images with image contrast similar to that of conventional IR sequences. When used with a multicoil array for abdominal, pelvic, and spine imaging, the IR-FSE sequence produces images that are virtually free of motion artifact from the subcutaneous fat immediately adjacent to the coils. Both FSE and IR-FSE, when compared with SE imaging, provide superior image contrast and SNR in reduced imaging time. PMID- 1729306 TI - Cerebrovascular enhancement in spoiled GRASS (SPGR) images: comparison with spin echo technique. AB - The signals and artifacts in cerebral blood vessels were systematically studied using a new three-dimensional Fourier transform (3DFT) technique, spoiled gradient recalled acquisition in steady state (SPGR), with parameters optimized for T1-weighted brain imaging. Twenty consecutive patients referred for routine cranial MR imaging were prospectively studied before and after the intravenous administration of Gd-DTPA using both 3DFT SPGR and conventional 2DFT SE imaging. A significant difference between the two techniques was noted in regard to the appearance of cerebral blood vessels and the degree of vascular pulsation artifact. Precontrast SPGR images demonstrated high signal in all (100%) internal carotid arteries and in nearly all (85-95%) vertebral, basilar, and proximal posterior, middle, and anterior cerebral arteries. High signal was variably present (5-50%) in more distal arterial branches. High signal from venous structures was not seen except in the superior sagittal sinus, which was bright in 75% of cases. Postcontrast SPGR images reliably demonstrated uniform high signal in all (100%) major arterial branches, up to fourth-order branches in the middle cerebral artery, diminishing toward the vertex. All (100%) major deep and superficial venous structures were of uniformly high signal, diminishing slightly as they exited the skull base. Flow related artifacts were found to be significantly (p = 0.001) reduced in contrast enhanced SPGR compared to SE images. As implemented in this study, SPGR and SE images demonstrate significantly different patterns of vascular signal that must be recognized for the proper interpretation of MR images. PMID- 1729307 TI - MR measurement of spinal CSF flow with the RACE technique. AB - The purpose of our study was the application and validation of a phase-sensitive pulse sequence that allowed real time CSF flow measurement without need for electrocardiographic (ECG) synchronization. After excitation of a slice perpendicular to the axis of the spine, projective data were obtained with a gradient echo sequence [contrast enhanced Fourier acquired steady-state technique (CE-FAST)] without spin warp gradient [real time acquisition and evaluation of motion technique (RACE)], allowing one-dimensional spatial resolution across the region of interest with a total sampling time of 20-30 ms. The sequence was calibrated with a spinal CSF phantom with oscillatory fluid motion. The calculated mean pulsation amplitudes of 20 healthy volunteers in the cervical region were 16 mm (range 9-36 mm), in the thoracic region 11 mm (5-21 mm), and in the lumbar region 3 mm (1-6 mm). The technique was capable of demonstrating physiologic alterations of CSF flow during respiratory maneuvers and may provide a tool to evaluate the altered CSF dynamics resulting from spinal block, inflammatory processes, or hemorrhage. PMID- 1729308 TI - FDG-PET in pediatric posterior fossa brain tumors. AB - Seventeen pediatric patients with posterior fossa brain tumors were studied with 2-[18F]fluoro-2-deoxy-D-glucose (FDG) and positron emission tomography (PET). The FDG uptake was ranked by two observers, and the results were correlated with tumor histology. Increased FDG uptake was associated with more malignant and aggressive tumor types. Heterogeneity of FDG uptake was associated with previous therapy, including radiation therapy and chemotherapy. 2-[18F]Fluoro-2-deoxy-D glucose PET will likely be an important adjunct in the management of pediatric posterior fossa tumors, much as in adult patients with brain tumors. PMID- 1729309 TI - Pre- and postcontrast MR studies in tuberous sclerosis. AB - The MR findings in eight patients with intracranial manifestations of tuberous sclerosis are reported. There were subependymal lesions in seven patients and peripheral lesions (i.e., cortical and subcortical) in all patients. All lesions were supratentorial. We emphasize two findings that have not been previously stressed. The signal intensity patterns of the subependymal lesions varied from patient to patient, but in all patients receiving intravenous contrast medium, the majority of these lesions enhanced. Although this finding may signify early breakdown of the blood-brain barrier with potential for lesion growth, the high frequency of enhancement challenges earlier concepts of equating this phenomenon with existence of actively growing giant cell astrocytomas. The peripheral lesions were more numerous than subependymal lesions. These lesions were nearly always hyperintense in the proton density weighted and T2-weighted images. Most notable is the fact that, in half of the lesions, signal intensity was also elevated in the T1-weighted image, an observation that has not been emphasized in previous reports. Although not pathologically confirmed this signal pattern may represent early stages of calcification within these lesions. Finally, unlike subependymal lesions, none of the peripheral lesions showed contrast enhancement. PMID- 1729310 TI - Design and implementation of magnetization transfer pulse sequences for clinical use. AB - The transfer of magnetization between a free and a bound pool of spins is described in terms of the respective longitudinal relaxation times and the life times of spins in each pool. The effect of an off resonance radiofrequency (RF) pulse in producing saturation in the bound pool and a consequent decrease in both the available longitudinal magnetization and the T1 of spins in the free pool is described. The effects of increasing duration of the saturating RF pulse on image pixel signal intensity were used to determine values for the decrease in both T1 and the available magnetization in gray and white matter of the brain as well as in muscle, fat, and CSF. At 0.15 T the available magnetization of muscle was reduced by approximately 60% and its T1 was decreased from 350 to 150 ms. The available magnetization of white and gray matter was reduced by 40% and their values of T1 were reduced by 80-110 ms. The reduction in available magnetization was used to increase contrast on proton density weighted or T2-weighted SE pulse sequences. These changes were also used to design inversion recovery (IR) pulse sequences with particular contrast properties. A short inversion time (TI) magnetization transfer (MT) IR (MT-STIR) pulse sequence was used to reduce the signal from normal muscle to zero to produce an angiographic effect in the leg. Increased tissue contrast was observed with a T2-weighted (MT-SE) sequence in a patient with bilateral cerebral infarction and with an MT-IR pulse sequence in a patient who had an intracranial hematoma. Three patients with cerebral tumors showed high lesion contrast with MT-STIR sequences. Components within two tumors were changed to different degrees by MT and in one case change in the brain attributable to recent radiotherapy treatment was only identified with an MT-STIR sequence. Magnetization transfer can be used to manipulate both the available longitudinal magnetization and the T1 of normal and abnormal tissues. The changes in tissue contrast produced by this can be very substantial and are likely to be of importance in clinical imaging. PMID- 1729311 TI - MRI of ossification of ligamentum flavum. AB - Magnetic resonance imaging of 28 patients with radiological and/or histopathologically proved ossification of the ligamentum flavum (OLF) was reviewed. The locations of OLF were cervical (n = 4), thoracic (n = 22), and lumbar (n = 2). On T1- and T2-weighted images, OLF demonstrated low signal intensity. Areas of high or intermediate signal intensity within the OLF on T1 weighted images were observed in three cases and were interpreted to be due to fat infiltration. In six cases, high intensity areas in the spinal cord caused by compressing OLF were demonstrated on T2-weighted images. Gadolinium diethylenetriamine pentaacetic acid, which was used in four cases, showed cord enhancement at the level of compression by OLF in three cases. PMID- 1729312 TI - MRI, antibody-guided scintigraphy, and glucose metabolism in uveal melanoma. AB - To evaluate the usefulness of structural and biochemical imaging techniques for the diagnosis of uveal melanoma, 12 patients with choroidal melanoma were examined. Magnetic resonance imaging was used in 11 of 12 patients, as one had a metal prosthesis. All the subjects underwent single photon planar scintigraphy (SPPS) and single photon emission computed tomography (SPECT) using the 99mTc labeled F(ab')2 of the anti-melanoma monoclonal antibody 225.28S ([99mTc]MoAb) and positron emission tomography (PET) using [18F]fluorodeoxyglucose ([18F]FDG). Magnetic resonance identified 6 of 11 melanotic lesions (definite melanomas) and 4 of 11 hypomelanotic lesions (probable melanomas), whereas in one case it was inconclusive. [99mTc]MoAb uptake was observed in 5 of 12 lesions using SPPS and 8 of 12 lesions using SPECT. [18F]FDG uptake was observed in 3 of 12 lesions by PET. These results demonstrate that both MR and radioimmunoscintigraphy are sensitive techniques for the diagnosis of choroidal melanomas and suggest that the detection of melanomas by MR, SPPS, and SPECT is largely dependent upon their size. The validity of these conclusions was verified in four subjects in whom the diagnosis was based on MR and/or SPECT findings only and confirmed by histology. The finding that only some of the uveal melanomas of larger size are visualized based on [18F]FDG uptake suggests that melanomas can have either high or low glucose consumption. PMID- 1729313 TI - Aneurysmal bone cysts of the jaws: CT and MR findings. AB - Two cases of aneurysmal bone cyst of the jaws, one in the mandible and the other in the maxilla, are reported in young girls 7 and 15 years old. One was a primary lesion; the other was associated with fibrous dysplasia. In both cases CT and MR demonstrated characteristic fluid-fluid levels. The diagnosis was confirmed after biopsy and surgical resection of the lesions. PMID- 1729314 TI - CT of submucosal and occult laryngeal masses. AB - Computed tomographic examinations were performed on 24 patients with entirely submucosal laryngeal mass lesions. Presenting complaints were hoarseness (17 patients), dysphagia (1 patient), airway obstruction (5 patients), and a cervical nodal metastasis (1 patient). The masses were visible endoscopically as submucosal bulges in 21 patients. Three other patients presenting with hoarseness and vocal cord paresis or paralysis had otherwise negative endoscopy and a mass demonstrated on CT. Thirteen patients were eventually diagnosed as having squamous cell carcinoma, which was the primary working diagnosis following CT in 12 cases. The group of 13 carcinoma patients had a range of two to five endoscopic procedures with one to four negative biopsies and a 6 week to 9 month delay in histologic confirmation of cancer. Other lesions included five laryngoceles, two chondrosarcomas, and one case each of paraganglioma, fibrosarcoma, lymphoma, and tuberculous laryngitis. Computed tomography is an indispensable tool for evaluating submucosal laryngeal masses or otherwise unexplainable symptoms (usually hoarseness) that might herald such a mass. A definite submucosal mass on CT should prompt a deep or wedge biopsy to reach a pathologic diagnosis. This will avoid the delay in diagnosis that frequently occurs in these patients. PMID- 1729315 TI - Significance of bowel wall enhancement on CT following blunt abdominal trauma in childhood. AB - Over a 3-year period, 12 children with blunt abdominal trauma were noted to have intense bowel wall enhancement (BWE) on CT. In four, with fatal CNS injury, there was no evidence of bowel perforation, and the changes may be related to the hypovolemic complex. In the remaining eight patients with a gastrointestinal perforation, BWE was associated with the presence of diffuse or focal bowel wall thickening and free peritoneal fluid. The enhancement and thickening were due to peritonitis secondary to perforation as shown at operation or autopsy. In three further patients with bowel perforation, thickening and enhancement were absent in two with retroperitoneal perforation and thickening, and no enhancement was noted in one patient in whom the study was considered suboptimal in retrospect. Intense BWE, therefore, aids in the diagnosis of the hypovolemic complex: CNS injury and bowel perforation. When combined with bowel thickening and free fluid, perforation and peritonitis should be strongly suggested. Enhancement and thickening should suggest perforation even if other visceral injury is present to account for the free fluid. Surgical intervention should thus be more strongly considered when bowel wall thickening accompanies BWE. PMID- 1729316 TI - Findings in primary hepatic carcinoid tumor: US, CT, MRI, and angiography. AB - Two asymptomatic patients with surgically proven solitary primary hepatic carcinoid tumors are reported. Ultrasonography showed hyperechoic masses containing multiple small cystic areas in both cases. On unenhanced CT, one tumor was of low density and one was isodense with multiple low density foci. One tumor showed marked retention of contrast medium on post-angiographic CT. Magnetic resonance imaging revealed low intensity masses on T1-weighted images and high intensity tumors with multiple areas of higher intensity on T2-weighted imaging. The small low density areas in these masses corresponded histopathologically to multiple vascular lakes. Late enhancement of the mass was presumed to correspond with proliferative fibrous tissue within the mass. PMID- 1729317 TI - Does thrombolysis in myocardial infarction (TIMI) perfusion grade 2 represent a mostly patent artery or a mostly occluded artery? Enzymatic and electrocardiographic evidence from the TEAM-2 study. Second Multicenter Thrombolysis Trial of Eminase in Acute Myocardial Infarction. AB - One measure of the success of thrombolysis is the early patency status of the infarct-related coronary artery. The Thrombolysis in Myocardial Infarction (TIMI) study group designated patency grades 0 (occluded) or 1 (minimal perfusion) as thrombolysis failure and grade 2 (partial perfusion) or 3 (complete perfusion) as success. To evaluate their true functional significance, perfusion grades were compared with enzymatic and electrocardiographic (ECG) indexes of myocardial infarction in 359 patients treated within 4 h with anistreplase (APSAC) or streptokinase. Serum enzymes and ECGs were assessed serially. Patency was determined at 90 to 240 min (median 2.1 h) and graded by an observer who had no knowledge of patient data. Results for the two drug arms were similar and combined. Distribution of patency was grade 0 = 20%, n = 72; grade 1 = 8% n = 27; grade 2 = 16%, n = 58 and grade 3 = 56%, n = 202. Interventions were performed after angiography but within 24 h in 51% (n = 37), 70% (n = 19), 41% (n = 24) and 14% (n = 28) of patients with grades 0, 1, 2 and 3, respectively. Outcomes were compared among the four patency groups by the orthogonal contrast method. Patients with perfusion grade 2 did not differ significantly from those with grade 0 or 1 in enzymatic peaks, time to peak activity and evolution of summed ST segments, Q waves and R waves (contrast 2). Conversely, comparisons of patients with grade 3 perfusion with those with grades 0 to 2 yielded significant differences for enzymatic peaks and time to peak activity for three of the four enzymes (p = 0.02 to 0.0001) and ECG indexes of myocardial infarction (p = 0.02 to 0.0001) (contrast 3). Thus, patients with grade 2 flow have indexes of myocardial infarction similar to those in patients with an occluded artery (grades 0 and 1 flow). Only early grade 3 flow results in a significantly better outcome than that of the other grades. Because early achievement of grade 2 flow does not appear to lead to optimal myocardial salvage, the frequency of achieving grade 3 perfusion alone may best measure the reperfusion success of thrombolytic therapy. PMID- 1729318 TI - Dipyridamole vasodilator response after human orthotopic heart transplantation: quantification by oxygen-15-labeled water and positron emission tomography. AB - To assess coronary vasodilator reserve after orthotopic heart transplantation, regional myocardial perfusion was measured with oxygen-15-labeled water and dynamic positron emission tomography in 14 cardiac allograft recipients who were not experiencing rejection and who had no angiographic evidence of epicardial coronary sclerosis 15 to 73 months (mean +/- SD 43 +/- 19) after transplantation (group I). Twelve normal men with an average age of 31 years (group II) served as a control group. Regional perfusion was measured at rest and after the intravenous administration of 0.6 mg/kg body weight of dipyridamole. Rest regional myocardial blood flow was homogeneously distributed throughout the left ventricle and was significantly higher in transplant recipients (mean 1.16 +/- 0.26 ml/g per min [range 0.8 to 1.73] than in normal subjects (mean 0.85 +/- 0.13 ml/g per min [range 0.57 to 0.99]; p = 0.001) as was rest heart rate-systolic blood pressure product (rate-pressure product 11,255 +/- 2,540 vs. 7,073 +/- 1,306; p less than 0.001). After dipyridamole, perfusion in the transplant recipients was homogeneous and slightly lower (2.73 +/- 1.03 vs. 3.40 +/- 1.09 ml/g per min; p = NS), whereas rate-pressure product was slightly higher (12,179 +/- 2,266 vs. 10,885 +/- 1,895; p = NS) than the value in normal subjects. Dipyridamole vasodilator response (dipyridamole/rest myocardial blood flow) ranged from 1.23 to 4.92 (mean 2.50 +/- 1.13) in group I and from 2.65 to 5.45 (3.97 +/- 0.89) in group II (p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729319 TI - Toward an improved understanding and management of human heart transplant recipients. PMID- 1729320 TI - Exercise-induced ST depression and ST/heart rate index to predict triple-vessel or left main coronary disease: a multicenter analysis. AB - The aim of this investigation was to determine the difference in accuracy between two frequently published noninvasive indicators of severity of coronary artery disease (exercise-induced ST segment depression and heart rate-adjusted ST depression [ST/HR index]). The study was designed as a survey of consecutive patients undergoing exercise electrocardiography and coronary angiography. There were a total of 2,270 patients without prior myocardial infarction or cardiac valvular disease referred for angiography from eight institutions in three countries; 401 of these patients had triple-vessel or left main coronary artery disease. The sensitivities of ST depression and ST/HR index in detecting triple vessel or left main coronary artery disease were, respectively, 75% and 78% (p = 0.08) at cut point values where their specificities were equal (64%). This small increase in the accuracy of the ST/HR index was evident only at peak exercise heart rates below the median value of 132 beats/min, where the sensitivities of ST depression and ST/HR index were 73% and 76% (p = 0.03), respectively, at cut point values corresponding to a specificity of 60%. These results were consistent at all eight participating institutions. The increase in accuracy achieved by dividing exercise-induced ST depression by heart rate is small and confined exclusively to a low exercise heart rate. This lack of superiority cannot be generalized to all methods of heart rate adjustment. PMID- 1729321 TI - Dissociation of termination and prevention of inducibility of sustained ventricular tachycardia with infusion of procainamide: evidence for distinct mechanisms. AB - To determine if termination of hemodynamically tolerated, sustained ventricular tachycardia during intravenous infusion of procainamide predicts the success of procainamide therapy in preventing induction of tachycardia, 15 patients with inducible, sustained ventricular tachycardia in the setting of chronic coronary artery disease were studied. Procainamide was infused at a rate of 50 mg/min during ventricular tachycardia until the arrhythmia terminated spontaneously or a total dose of 15 mg/kg was administered. An infusion (2 to 10 mg/min) was given after the loading dose to maintain constant serum drug concentrations after termination of the tachycardia. The infusion of procainamide was well tolerated and resulted in termination of ventricular tachycardia in 14 (93%) of 15 patients after administration of 100 to 1,080 mg (median dose 600 mg). In all patients, programmed ventricular stimulation was repeated immediately after termination of the arrhythmia until ventricular tachycardia was reinitiated or until the stimulation protocol was completed. Of the 14 patients whose ventricular tachycardia terminated during the infusion of procainamide, 1 patient had no inducible sustained tachycardia with repeated programmed stimulation. In the remaining 13 patients, programmed stimulation resulted in initiation of sustained ventricular tachycardia of the same configuration in 7 patients and of a different configuration in 6. In the former 7 patients, the serum procainamide concentration (7.7 +/- 4 vs. 7.4 +/- 3.3 mg/liter, p = NS) and the observed drug effects on the tachycardia cycle length (449 +/- 78 vs. 450 +/- 81 ms, p = NS) and QRS duration (184 +/- 38 vs. 185 +/- 38 ms, p = NS) were similar at the times of termination and reinitiation of ventricular tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729322 TI - Differential effect of intravenous procainamide on anterograde and retrograde accessory pathway refractoriness. AB - Although procainamide may markedly impair or abolish anterograde conduction over an accessory atrioventricular (AV) pathway, orthodromic AV reentry may remain inducible. This difference may be related to a systemic differential effect of procainamide on anterograde and retrograde accessory pathway refractoriness. To examine this phenomenon, an infusion of procainamide producing five incremental blood levels over 75 min was administered to 15 patients with the Wolff-Parkinson White syndrome. At each procainamide level, accessory pathway effective refractory period and accessory pathway block cycle length were determined in the anterograde and retrograde directions. At baseline, there were no significant differences between anterograde and retrograde accessory pathway effective refractory periods (282 +/- 7 vs. 266 +/- 9 ms, p = 0.08) and block cycle lengths (288 +/- 15 vs. 283 +/- 9 ms, p = 0.66). The concentration of procainamide resulting in 50% prolongation of accessory pathway refractoriness was less in the anterograde direction than in the retrograde direction (27.5 [log concentration 4.56 +/- SE 0.13] vs. 64.6 [-4.19 +/- 0.11] mumol/liter, p = 0.02). Similarly, the concentration of procainamide resulting in 50% prolongation of accessory pathway block cycle length in the anterograde direction (25.1 [-4.60 +/- 0.13] mumol/liter) was less than in the retrograde direction (52.5 [-4.28 +/- 0.07] mumol/liter, p = 0.01). The probability of persistence of accessory pathway conduction in the anterograde direction was less than in the retrograde direction by Kaplan-Meier analysis (p = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729323 TI - Geometrically accurate transaortic mapping of left ventricular endocardial activation during surgery. AB - A device was developed for mapping left ventricular endocardial activation through the aortic valve during surgery. It uses an exploring electrode at the tip of a hand-held probe that is mounted on a mechanical arm with six joints whose movements are digitized by a computer while the position of the probe tip is calculated continuously. The probe is inserted by the surgeon into the left ventricle retrogradely through the aortic valve after the patient is on total cardiopulmonary bypass, the aorta has been opened and the coronary arteries cannulated. The electrode position relative to the aortic valve and left ventricular apex is displayed continuously on a computer screen. When electrograms are recorded from the probe, their positions are displayed on the screen relative to a stylized grid of the left ventricular endocardial surface and are color-coded to indicate the activation sequence. In a patient with nonischemic ventricular tachycardia, the arrhythmia was successfully mapped and cryoablated with use of the device. The device will be developed so that a cryoprobe can be substituted for the exploring electrode and positioned at the source of activation determined by the map. PMID- 1729324 TI - Separate and joint influences of obesity and mild hypertension on left ventricular mass and geometry: the Framingham Heart Study. AB - Increased left ventricular mass has been shown to be a significant independent predictor of cardiovascular risk. The purpose of this study was to assess the separate and combined relations of obesity and hypertension with left ventricular mass and geometry. Echocardiographic findings in subjects in the Framingham Heart Study who were free of cardiopulmonary disease and were not taking cardiovascular medications were examined. M-mode studies that were adequate for estimating left ventricular mass were available in 624 men and 1,209 women. Height and weight measured at the time of echocardiography were used to calculate body mass index (in kg/m2), a measure of obesity. Casual sitting blood pressure measurements were obtained to detect rest hypertension. In subgroup analyses of lean normotensive, obese normotensive, lean hypertensive and obese hypertensive subjects, hypertension and obesity each had significant independent associations with left ventricular mass and wall thickness (all p less than 0.001 in men and women). Obesity was also associated with left ventricular internal diameter (p less than 0.001 in men and women). There were no synergistic influences of hypertension and obesity on any echocardiographic left ventricular variables. It is concluded that obesity and hypertension each have distinct associations with left ventricular mass and geometry. These strengths of association are additive but not synergistic. PMID- 1729325 TI - Patterns of anomalous pulmonary venous connection/drainage in hypoplastic left heart syndrome: diagnostic role of Doppler color flow mapping and surgical implications. AB - Differentiation between anomalous connection and anomalous drainage of the pulmonary veins in hypoplastic left heart syndrome is important before either the Norwood procedure or heart transplantation is performed. To determine the prevalence of echocardiographically detected anomalous connection or drainage, or both, of pulmonary veins in patients with this syndrome, preoperative two dimensional echocardiographic and Doppler color flow mapping studies of 317 patients who underwent the stage I Norwood procedure were reviewed. The term "connection" was used to describe the precise anatomic attachment of the pulmonary veins and the term "drainage" to describe the physiologic end point of pulmonary venous flow. Twenty patients (6.3%) had anomalous connection or drainage, or both, of the pulmonary veins by preoperative echocardiographic and Doppler examination. The subcostal and suprasternal scans best showed the anatomic details of the pulmonary veins. All these patterns were confirmed intraoperatively and could be grouped as follows: 1) partial anomalous connection and drainage (two patients); 2) total anomalous connection and drainage (eight patients); 3) normal connection with total anomalous drainage (eight patients); and 4) normal connection with partial anomalous drainage (two patients). The advantage of adding Doppler color flow mapping to two-dimensional echocardiography and conventional Doppler study was clearly demonstrated in the detection of small accessory vertical veins, their course and the presence or absence of obstruction. Doppler color flow mapping was especially helpful in detecting anomalous drainage of the right pulmonary veins to the right of the superior attachment of the septum primum. PMID- 1729326 TI - Echocardiographic evaluation of atrioventricular orifice anatomy in children with atrioventricular septal defect. AB - In atrioventricular (AV) septal defect, the common AV valve can have a common orifice or can be divided by bridging leaflet tissue into two separate orifices. To determine the accuracy of a two-dimensional echocardiographic technique devised specifically for evaluation of the number of AV valve orifices, all 69 children undergoing surgical repair of AV septal defect from April 1987 to August 1990 were examined prospectively. The presence of bridging leaflet tissue and the number of AV valve orifices were determined with use of a subcostal imaging plane. From a standard subcostal four-chamber view, the plane of sound was rotated 30 degrees to 45 degrees clockwise until the AV valve was seen en face. The plane of sound was then tilted from a superior to an inferior direction so that cross-sectional views of the AV valve were examined from the inferior margin of the atrial septum to the superior margin of the ventricular septum. Of the 69 patients, 6 (9%) were excluded because the appropriate subcostal images were not obtained (in 3 because of obesity and in 3 as a result of operator failure). The remaining 63 children, ranging in age from 1 day to 13.5 years and in weight from 1 to 55 kg, constituted the study group. Echocardiographic results were compared with surgical observations in 62 patients and with autopsy findings in 1 patient. With the two-dimensional echocardiographic technique, 32 of 33 patients with a common orifice and 28 of 30 patients with two separate AV valve orifices were correctly identified.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729327 TI - Maximal voluntary exercise variables in children with postoperative coarctation of the aorta. AB - Thirty-one children with postoperative coarctation of the aorta underwent maximal graded bicycle ergometry using an electronically braked ergometer and the James protocol; 18 also underwent expiratory gas measurement using a mass spectrometer. Twenty-two age- and gender-matched normal subjects were used as a control group. The mean age at operation was 41 months and the mean age at evaluation was 134 months (mean follow-up interval 93 months). The original surgical repair was subclavian flap repair in 8 patients, end to end anastomosis in 21, patch aortoplasty in 1 patient and tubular graft in 1. Patients exercised until exhaustion and maximal exercise variables were obtained. The maximal voluntary peak heart rate was 183 beats/min (94.6% of predicted value), indicating excellent effort. Mean power was 111% of predicted value and, when measured, maximal oxygen consumption was 89% of predicted value with an anaerobic threshold at 63 +/- 3.5% of exercise time. The observed work variables were not different from values in the control group and were not affected by the type of repair. The mean peak systolic blood pressure was 152 +/- 7.6 mm Hg versus 147 +/- 5.7 mm Hg in the control group (p = NS). Patients who had associated intracardiac lesions had significantly lower maximal oxygen consumption (85 +/- 3% vs. 98 +/- 4% of predicted value). The results suggest that adequate cardiopulmonary function, normal or above average work capacity and normal exercise systolic blood pressure can be obtained in children with satisfactory repair of coarctation of the aorta performed before school age. PMID- 1729328 TI - Intracoronary ethanol ablation for the treatment of recurrent sustained ventricular tachycardia. AB - The selective infusion of ethanol into the coronary circulation supplying the site of origin of incessant ventricular tachycardia has been demonstrated to abolish this arrhythmia in selected patients. The present study was designed to evaluate the efficacy and safety of the intracoronary ethanol ablation technique in patients with paroxysmal ventricular tachycardia related to prior myocardial infarction. Twenty-three patients with sustained monomorphic ventricular tachycardia that was refractory to conventional antiarrhythmic drug therapy were prospectively studied. After induction of ventricular tachycardia by programmed electrical stimulation, the response of the arrhythmia to the infusion of radiographic contrast medium or saline solution into the ostia of the native coronary arteries and coronary artery bypass grafts was assessed. If ventricular tachycardia was reliably interrupted by injections into the proximal coronary artery or bypass graft, the vessel was cannulated with a steerable guide wire and 2.7F infusion catheter to determine the smallest arterial branch that would result in termination of the arrhythmia with selective injections. If reliable interruption of ventricular tachycardia was observed with saline or contrast injections, ethanol (2 ml) was then delivered through the infusion catheter. Ventricular tachycardia could be terminated by injections of saline solution or contrast medium in 11 of 21 patients in whom the protocol could be completed. Ethanol was infused in 10 of these patients. Ventricular tachycardia was inducible in only 1 of 10 patients immediately after ethanol infusion. At a follow-up electrophysiologic study performed 5 to 7 days after ablation, ventricular tachycardia became inducible in two other patients, in one of whom the arrhythmia substrate was successfully ablated after three sessions. The mean left ventricular ejection fraction was 0.33 +/- 0.1 before and 0.35 +/- 0.11 after ablation. Complications of the procedure included complete atrioventricular block in four patients and pericarditis in one patient. Thus, intracoronary ethanol ablation is associated with a moderate degree of efficacy but the potential for important complications. Despite these limitations, this technique may provide effective long-term control of ventricular tachycardia for some patients. PMID- 1729329 TI - High dose oral amiodarone loading: electrophysiologic effects and clinical tolerance. AB - Although amiodarone is an effective drug for the treatment of life-threatening ventricular arrhythmias, no standard oral loading dose protocol has been defined, and patients often undergo prolonged hospitalization for amiodarone loading. High dose (greater than 1,800 mg/day) oral loading has usually been reserved for unstable patients with incessant ventricular tachyarrhythmias. The current study was designed to 1) examine the clinical and electrophysiologic effects of a high dose oral amiodarone loading regimen in more stable patients; and 2) ascertain its safety and tolerance, possibly allowing shortened amiodarone loading periods and potentially decreased length of hospital stay. The study group included 16 patients with a history of recurrent ventricular arrhythmias and decreased left ventricular function, who were refractory to prior antiarrhythmic drug therapy. The oral loading protocol was 50 mg/kg per day of amiodarone for 3 days, then 30 mg/kg per day for 2 days, followed by maintenance therapy of 300 to 400 mg twice daily. Electrophysiologic testing was performed at baseline, on days 1 and 5 and during week 6. Amiodarone and desethylamiodarone levels were measured and symptoms monitored. Clinically, the high dose loading protocol was well tolerated in 15 of the 16 patients. Arrhythmias were rendered noninducible by day 1 in three patients and remained noninducible throughout the study period in two of the three. The remaining patients continued to have inducible ventricular tachycardia. Ventricular tachycardia cycle length and right ventricular effective refractory period both progressively increased significantly over baseline, starting on day 1. The 15 patients who remained in the study had no significant side effects during the loading period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729330 TI - Fish oils in the prevention of atherosclerosis. AB - The hypothesis that oils derived from the flesh of fish and marine mammals inhibit the atherosclerotic process is critically reviewed. Populations consuming a diet rich in fish have low rates of coronary heart disease. Dietary fish oil is associated with changes in serum lipids, prostaglandin and leukotriene metabolism, enhanced endothelial function and effects on growth factors released from platelets, leukocytes and endothelial cells. Dietary fish oil supplementation has been associated with inhibition of atherosclerosis experimentally induced by dietary hyperlipidemia and balloon injury. Results of studies of the use of fish oil to inhibit postangioplasty restenosis in human subjects have been inconclusive. PMID- 1729331 TI - Unusual sequelae after percutaneous mitral valvuloplasty: a Doppler echocardiographic study. AB - Percutaneous mitral valvuloplasty is a promising new technique for the treatment of mitral stenosis, with a relatively low complication rate reported to date. To assess the sequelae of this procedure, Doppler echocardiographic studies were prospectively performed before and after percutaneous mitral valvuloplasty in a series of 172 patients (mean age 53 +/- 17 years). After balloon dilation, mitral valve area increased from 0.9 +/- 0.3 to 2 +/- 0.8 cm2 (p less than 0.0001), mean gradient decreased from 16 +/- 6 to 6 +/- 3 mm Hg (p less than 0.0001) and mean left atrial pressure decreased from 24 +/- 7 to 14 +/- 6 mm Hg (p less than 0.0001). Although most patients were symptomatically improved, six (4%) were identified who had unusual sequelae evident on Doppler echocardiographic examination immediately after percutaneous mitral valvuloplasty. These included rupture of a posterior mitral valve leaflet, producing a flail distal leaflet portion with severe mitral regurgitation detected on Doppler color flow mapping (n = 1); asymptomatic rupture of the chordae tendineae attached to the anterior mitral valve leaflet with systolic anterior motion of the ruptured chordae into the left ventricular outflow tract (n = 1); a double-orifice mitral valve (n = 1); and evidence of a tear in the anterior mitral valve leaflet (n = 3), producing on both pulsed Doppler ultrasound and color flow mapping a second discrete jet of mitral regurgitation in addition to regurgitation through the main mitral valve orifice. All six patients made a satisfactory recovery and none has required mitral valve replacement.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729332 TI - Heart rate adjustment of ST segment depression: is the glass half empty or half full? PMID- 1729333 TI - Catheter balloon commissurotomy for mitral stenosis: complications and results. PMID- 1729334 TI - Low doses of superoxide dismutase and a stable prostacyclin analogue protect in myocardial ischemia and reperfusion. AB - The effects of low dose human superoxide dismutase and low dose taprostene, a stable analogue of prostacyclin, were investigated separately and together in a model of myocardial ischemia (1.5 h) with reperfusion (4.5 h) in open chest, anesthetized cats. Taprostene (60 ng/kg per min), human superoxide dismutase (0.25 mg/kg per h), both agents together, or their vehicle, were infused intravenously in cats starting 0.5 h after occlusion of the left anterior descending coronary artery. Neither low dose taprostene nor low dose human superoxide dismutase exerted any endothelial or myocardial protection in this model. However, the two agents together showed a significant endothelial and myocardial protection in cats with myocardial ischemia and reperfusion. Compared with cats that were untreated or received only taprostene or human superoxide dismutase, cats receiving both agents exhibited a lower plasma creatine kinase activity at every time point observed after reperfusion, a reduced area of cardiac necrosis (7 +/- 2% vs. 21 +/- 5% area at risk, p less than 0.001), lower myeloperoxidase activity in the ischemic region (p less than 0.01) and a significant preservation of vasorelaxant responses of left anterior descending coronary rings to endothelium-dependent vasodilators, acetylcholine (p less than 0.001) and A-23187 (p less than 0.001). Taprostene appears to act additively with human superoxide dismutase to inhibit neutrophil adherence and activation and to inactivate superoxide radicals, and thus reduce cellular injury 4.5 h after reperfusion of the ischemic heart. Use of this agent may allow low doses of superoxide dismutase to be used more effectively in early myocardial ischemia. PMID- 1729335 TI - Pharmacologic perturbation of neutrophils by Fluosol results in a sustained reduction in infarct size in the canine model of reperfusion. AB - Previous studies have demonstrated that intravenous administration of large doses of Fluosol, a perfluorochemical preparation, reduced infarct size 24 h after reperfusion, an effect that was associated with reduced neutrophil infiltration. The effect of a clinically tolerable dose of Fluosol on infarct size after a prolonged period of reperfusion and its mechanism of action on neutrophils remain unknown. Twenty-one anesthesized closed chest dogs were subjected to 90 min of proximal left anterior descending coronary artery occlusion and 72 h of reperfusion. An additional five dogs that did not undergo regional myocardial ischemia were utilized to explore the mechanism of action of Fluosol on neutrophil function. In the infarct study, animals were randomized to receive either intravenous Fluosol (n = 10) or an equivalent volume of Ringer's lactate solution (control; n = 11) at 15 ml/kg body weight during the last 30 min of occlusion and for the 1st 30 min of reperfusion. Fluosol significantly reduced infarct size when expressed as percent area at risk 72 h after reperfusion (13.7 +/- 2.7% vs. 38.3 +/- 4.5%, respectively, p less than 0.001). This reduction was associated with significant improvement in regional wall motion (18.4 +/- 2.3% vs. 5.5 +/- 2%, p less than 0.001). Endocardial blood flow in the ischemic bed was significantly higher 3 h after reperfusion in Fluosol-treated dogs (0.63 +/- 0.08 vs. 0.34 +/- 0.07 ml/min per g, p = 0.01). Reduced capillary plugging by neutrophils with relative preservation of endothelial cell structure was observed in Fluosol-treated animals. Infusion of Fluosol produced a marked transient decrease in peripheral neutrophil and platelet counts in both ischemic and nonischemic dogs and was associated with a significant reduction in total hemolytic complement levels. Studies of neutrophil function ex vivo revealed a reduction in chemotaxis and lysozyme degranulation after infusion of Fluosol. In vitro experiments showed that Fluosol produced a rapid and sustained activation of neutrophils determined by superoxide anion production. These data demonstrate that low dose intravenous Fluosol produces a sustained reduction in infarct size in the canine model. The beneficial effect may be in part due to the suppression of various neutrophil functions in the reperfused myocardium subsequent to peripheral activation by Fluosol. Such interventions may offer a novel therapy to enhance myocardial salvage by sequestration of circulating neutrophils during the critical early reperfusion period. PMID- 1729336 TI - Impaired endothelium-dependent cholinergic coronary vasodilation in patients with angina and normal coronary arteriograms. AB - The coronary vasomotor responses to selective infusion of graded concentrations (10(-6) to 10(-4) M) of acetylcholine into the left anterior descending artery were assessed by quantitative coronary arteriography in 24 patients with normal coronary arteriograms (12 patients with atypical symptoms and 12 patients with typical anginal pain) and 36 patients with coronary artery disease with different degrees of atherosclerosis of the left anterior descending artery. In the patients with normal coronary arteries and atypical chest pain, acetylcholine induced predominantly a vasodilator response, which was maximal during a 10(-5) M acetylcholine infusion. In contrast, in patients with coronary artery disease, acetylcholine caused dose-dependent vasoconstriction, which was observed even if the left anterior descending artery itself was smooth. Marked vasoconstriction was also induced in the patients with typical anginal pain and angiographically normal coronary arteries. In nine of these patients, this constrictor response was associated with anginal pain and electrocardiographic evidence of myocardial ischemia. Intracoronary administration of isosorbide dinitrate (1 mg) relieved the anginal pain and dilated all vessels. These data suggest that 1) patients with normal coronary arteriograms and angina pectoris manifest impairment of endothelium-dependent vasodilation similar to that observed in patients with overt coronary atherosclerosis; and 2) abnormal coronary vasoconstrictor responses resulting from this impairment may contribute to the pathogenesis of myocardial ischemia and angina in these patients. PMID- 1729337 TI - JACC: entering its 10th year of scientific excellence. PMID- 1729338 TI - Ode to the electrophysiologist. PMID- 1729339 TI - Overestimation of valve area by the Gorlin formula. PMID- 1729340 TI - Role of the cardiologist in peripheral vascular disease. PMID- 1729341 TI - Hemorrhaging heart patients. PMID- 1729342 TI - Syndrome X: the epicardial view. PMID- 1729343 TI - Myocardial perfusion and regression of coronary artery disease in patients on a regimen of intensive physical exercise and low fat diet. AB - This intervention program tested the applicability and effects of intensive physical exercise and a low fat diet on progression of coronary atherosclerotic lesions and stress-induced myocardial ischemia in patients with stable angina pectoris. Eighteen patients participated in this program for 1 year; they consumed a low fat, low cholesterol diet (less than 20 energy % fat, cholesterol less than 200 mg/day) and exercised for greater than 3 h/week. Change in coronary morphology was assessed by angiography and digital image processing; stress induced myocardial ischemia was measured by thallium-201 scintigraphy. Results were compared with those in patients receiving "usual care." In the intervention group, significant regression of coronary atherosclerotic lesions was noted in 7 of the 18 patients; no change or progression was present in 11 patients. In patients receiving usual care, regression was detected in only 1, with no change or progression in 11 patients (different from intervention, p less than 0.05). There was a significant reduction in stress-induced myocardial ischemia, which was not limited to patients with regression of coronary atherosclerotic lesions. Thus, regular physical exercise and a low fat diet may retard progression of coronary artery disease; however, improvement of myocardial perfusion may be achieved independently from regression of stenotic lesions. PMID- 1729344 TI - Complications of transvenous right ventricular endomyocardial biopsy in adult patients with cardiomyopathy: a seven-year survey of 546 consecutive diagnostic procedures in a tertiary referral center. AB - To determine the incidence, nature and subsequent management of complications occurring during right ventricular endomyocardial biopsy in patients with cardiomyopathy, all events occurring during 546 procedures in 464 consecutive patients were prospectively recorded. The internal jugular vein was the primary site of introduction in 96% of cases. A total of 33 complications (6%) occurred: 15 (2.7%) during catheter insertion including 12 arterial punctures (2%), 2 vasovagal reactions (0.4%) and 1 episode of prolonged bleeding (0.2%), all without sequelae; 18 (3.3%) during biopsy included 6 arrhythmias (1.1%), 5 conduction abnormalities (1%), 4 possible perforations (0.7%) and 3 definite perforations (0.5%) (pericardial fluid). Two (0.4%) of the three patients with a perforation died. There was no secular trend in the complication rate, nor were complications associated with specific clinical or hemodynamic characteristics. It is concluded that the overall rate of endomyocardial biopsy complications (6%) is low, but mortality may occur. PMID- 1729345 TI - Influence of preoperative pulmonary artery pressure on mortality after heart transplantation: testing of potential reversibility of pulmonary hypertension with nitroprusside is useful in defining a high risk group. AB - Patients with pulmonary hypertension are at risk of developing fatal right heart failure after heart transplantation. To evaluate this risk potential, candidates for heart transplantation are screened by measuring rest right heart pressures and the response to nitroprusside. To test the validity of this approach, the influence of pretransplantation right heart catheterization data on outcome after transplantation was analyzed in 293 of 301 consecutive patients. Patients with a pulmonary vascular resistance greater than 2.5 Wood units measured at baseline study had a 3-month mortality rate of 17.9% compared with 6.9% in patients with resistance less than or equal to 2.5 units (p less than 0.02). Patients with a pulmonary vascular resistance greater than 2.5 units at baseline study could be differentiated further according to their hemodynamic response to nitroprusside; those whose resistance could be reduced to less than or equal to 2.5 units with a stable systemic systolic pressure greater than or equal to 85 mm Hg had a 3-month mortality rate of only 3.8%. In contrast, patients whose pulmonary vascular resistance could not be reduced to less than 2.5 units, and those whose resistance could be reduced to less than or equal to 2.5 units but only at the expense of systemic hypotension (systolic pressure less than or equal to 85 mm Hg) had a 3-month mortality rate of 40.6% and 27.5%, respectively. Furthermore, all 10 patients who died of right heart failure belonged to the latter two groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729346 TI - Cardiac adaptation to obesity and hypertension after heart transplantation. AB - Obesity and hypertension frequently develop after heart transplantation. The cardiac adaptation to obesity and hypertension was studied by determining hemodynamic and echocardiographic indexes in 10 obese hypertensive patients (body mass index greater than or equal to 27.8 kg/m2 in men or greater than or equal to 27.3 kg/m2 in women) matched by mean arterial pressure, age and gender with 10 nonobese hypertensive patients 1 year after cardiac transplantation. Cardiac output was 30% greater (p less than 0.02) and systemic vascular resistance 25% lower (p less than 0.01) in the obese than in the nonobese patients. Right ventricular systolic and pulmonary artery systolic, diastolic and mean pressures were also significantly higher (p less than 0.05) in the obese patients. Left ventricular end-diastolic diameter was 25% greater (p less than 0.05), left ventricular mass 28% greater (p less than 0.02) and left ventricular end diastolic volume 20% higher (p less than 0.01) in the obese subjects. Left ventricular ejection fraction was significantly lower in the obese than in the nonobese subjects (34% vs. 51%, p less than 0.05). These results indicate that the cardiac adaptation to obesity and hypertension after heart transplantation consists of left ventricular dilation and an increase in left ventricular mass associated with an increased cardiac output and lower peripheral vascular resistance. These adaptive changes that occur in obese hypertensive patients after heart transplantation might increase the long-term risk of graft failure, as suggested by their lower left ventricular ejection fraction 1 year after transplantation. PMID- 1729347 TI - Serial assessment of left ventricular function and mass after orthotopic heart transplantation: a 4-year longitudinal study. AB - Long-term changes in left ventricular performance and geometry in the transplanted human heart have been incompletely described. Therefore, two dimensional echocardiograms were performed on 22 recipients of an orthotopic heart transplant at 1 month (32 +/- 20 days), 1 year (11 +/- 3 months) and 4 years (54 +/- 9 months) after transplantation. All studies were performed at a time when the patient had no pathologic evidence of rejection. Ten healthy men served as a normal control group. Over 4 years of follow-up, mean systolic blood pressure in the study patients increased from 121 +/- 12 (p = NS vs. values in the control group) to 139 +/- 11 mm Hg (p less than 0.05 vs. both control values and values at 1 month); mean diastolic blood pressure increased from 72 +/- 7 (p = NS vs. normal values in the control group) to 93 +/- 8 mm Hg (p less than 0.05 vs. both control values and values at 1 month). Left ventricular end-systolic volume increased from 42 +/- 10 (p = NS vs. control values) to 51 +/- 14 ml (p less than 0.05 vs. both control values and values at 1 month) and end-diastolic volume increased from 103 +/- 28 (p = NS vs. control values) to 112 +/- 27 ml (p less than 0.05 vs. control values) over 4 years. Left ventricular mass and ejection fraction did not change significantly within the patient cohort and remained similar to that found in the control group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729348 TI - Accuracy and limitations of exercise echocardiography in a routine clinical setting. AB - Despite the high reported accuracy of exercise echocardiography in the detection of coronary artery disease, factors that compromise its sensitivity and specificity are less clear. This study examined the results of 179 post-treadmill stress echocardiograms in 150 consecutive patients who also underwent cardiac catheterization and in 29 normal persons at low risk for coronary artery disease. Of 114 patients who had significant coronary stenoses at angiography, 96 had an abnormal exercise echocardiogram (overall sensitivity 84%). False negative results correlated with the performance of submaximal exercise, single-vessel disease and moderate (50% to 70% diameter) stenoses. After the exclusion of seven patients performing submaximal exercise, the sensitivity was 90%. In 54 patients without previous infarction performing maximal exercise, the sensitivity was 87%, higher in patients with multivessel coronary disease (96%) than in those with single-vessel disease (79%). After the exclusion of patients with nondiagnostic results, due either to the performance of submaximal stress or the presence of electrocardiographic (ECG) changes at rest, exercise echocardiography had a higher sensitivity than did exercise electrocardiography (87% vs. 63%, p = 0.01). In 36 patients without significant coronary disease, exercise echocardiography had an overall specificity of 86%. After the exclusion of patients with a nondiagnostic test, exercise echocardiography had a specificity of 82% compared with 74% specificity for exercise electrocardiography (p = NS). Similarly, of the 29 normal subjects, 93% had a normal exercise echocardiogram and 97% had a normal exercise ECG (p = NS). Similarly, of the 29 normal subjects, 93% had a normal exercise echocardiogram and 97% had a normal exercise ECG (p = NS). Age, gender, body weight and image quality did not significantly influence the accuracy of exercise echocardiography.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729349 TI - Exercise echocardiography: phase II, convincing the skeptics. PMID- 1729350 TI - Doppler echocardiographic demonstration of the differential effects of right ventricular pressure and volume overload on left ventricular geometry and filling. AB - To compare the effects of isolated right ventricular pressure and volume overload on left ventricular diastolic geometry and filling, 11 patients with primary pulmonary hypertension, 11 patients with severe tricuspid regurgitation due to tricuspid valve resection and 11 normal subjects were studied with use of Doppler echocardiographic techniques. Right ventricular systolic overload in primary pulmonary hypertension resulted in substantial leftward ventricular septal shift that was most marked at end-systole and early diastole and decreased substantially by end-diastole. Right ventricular diastolic overload after tricuspid valve resection resulted in maximal leftward ventricular septal shift at end-diastole sparing end-systole and early diastole. The early diastolic distortion of left ventricular geometry associated with right ventricular pressure overload resulted in prolongation of isovolumetric relaxation of the left ventricle (129 +/- 39 ms) and a reduction in early diastolic filling compared with values in normal subjects. Late diastolic distortion of left ventricular geometry associated with right ventricular volume overload had no influence on the duration of left ventricular isovolumetric relaxation (52 +/- 32 ms) but caused a reduction in the atrial systolic contribution to late diastolic filling of the left ventricle compared with values in normal subjects. In patients with right ventricular pressure overload, 52 +/- 16% of left ventricular filling occurred in early diastole compared with 78 +/- 11% in patients with right ventricular volume overload (p less than 0.001). The differential effects of systolic and diastolic right ventricular overload on the pattern of left ventricular filling appear to be related to the timing of leftward ventricular septal displacement. PMID- 1729351 TI - Utility of continuous wave Doppler echocardiography in the noninvasive assessment of left ventricular outflow tract pressure gradient in patients with hypertrophic cardiomyopathy. AB - Subaortic obstruction is an important determinant of the clinical presentation of and therapeutic approach to patients with hypertrophic cardiomyopathy. Therefore, assessment of the presence and magnitude of the intraventricular pressure gradient is paramount in the clinical evaluation of these patients. To establish the utility of continuous wave Doppler echocardiography in assessing the pressure gradient in hypertrophic cardiomyopathy, 28 patients representing the wide hemodynamic spectrum of this disease underwent simultaneous determination of the subaortic gradient by continuous wave Doppler ultrasound and cardiac catheterization. With use of the modified Bernoulli equation, the Doppler estimated gradient showed a strong correlation with the maximal instantaneous pressure difference measured at catheterization, both under basal conditions (r = 0.93; p less than 0.0001) and during provocative maneuvers (r = 0.89; p less than 0.0001). In 26 of the 28 patients, all assessments of the subaortic gradient were in agreement within 15 mm Hg (average difference 5 +/- 3 mm Hg). In the other two patients there were substantial differences between these measurements (under basal conditions in one patient and after provocation in another), although the Doppler technique predicted the presence of marked subaortic obstruction in each. In both patients the erroneous interpretation was due to superimposition of the mitral regurgitation signal on that of left ventricular outflow. Doppler waveforms from the left ventricular outflow tract showed variability in contour among different patients and in individual patients. Hence, continuous wave Doppler echocardiography is a useful noninvasive method for estimating the subaortic gradient in patients with hypertrophic cardiomyopathy. However, technical factors such as contamination of the outflow tract jet with that of mitral regurgitation and variability in waveform configuration may importantly influence such assessments of the subaortic gradient. PMID- 1729352 TI - The effect of avidin-biotin interactions in detection systems for in situ hybridization. AB - The effect of avidin-biotin interactions in several detection systems for the non radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems. PMID- 1729353 TI - Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies. AB - To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types. PMID- 1729354 TI - Localization of calcium changes in stimulated rat mast cells. AB - We studied intracellular free, bound, and sequestered calcium in rat mast cells after various stimulations. The use of a fluorescent probe combined with digitized imaging on individual living cells demonstrated transient increases of free Ca2+ in the micromolar range. The use of histochemical techniques (K pyroantimonate and anhydrous fixation), together with X-ray microanalysis, energy electron-loss spectroscopy, and electron spectroscopic imaging, revealed large amounts of stored calcium within the cells (in the millimolar range). Chelation experiments and stimulations enabled us to identify at least two pools of bound calcium which exhibited different dynamic behaviors. Stimulation in the presence of EGTA did not modify calcium from granules, granule membranes, and heterochromatin, whereas it decreased calcium from other cell compartments. Stimulation triggered variations in the amount of bound calcium but they did not parallel free calcium movements. Hence, whereas free calcium is implicated in exocytosis, bound calcium may be involved in altogether different cell functions. PMID- 1729355 TI - Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes. AB - We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy. PMID- 1729356 TI - Co-localization of an endocytic marker and acid phosphatase in a tubular/reticular compartment in macrophages. AB - Cultured resident murine macrophages are incubated in the continuous presence of the fluorescent endocytic marker Lucifer Yellow and a phorbol ester that activates protein kinase C. Under these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubular/reticular compartment. Enzyme cytochemical localization of acid phosphatase in the same cells showed essentially a one-to-one correlation between the Lucifer Yellow- and acid phosphatase-containing compartments. Procedures for epifluorescence observation and subsequent enzyme cytochemical examination of the same whole mount cells are described. In addition, chemical fixation methods for the preservation of this labile tubular/reticular compartment are presented. PMID- 1729357 TI - T cell responses specific for subregions of allogeneic MHC molecules. AB - The repertoire of C3H (H-2k) CD4+ T cells for I-Ab allopolymorphisms was analyzed by studying the responses of unprimed populations of T cells and of I-Ab-specific T cell clones for recombinant MHC molecules containing combinations of polymorphic subregions of the alpha- and beta-chains from the I-Ab and I-Ak molecules. In this system, polymorphisms in the predicted MHC alpha-helices were more potent than polymorphisms in the beta-strands in stimulating unprimed alloreactive T cells. Similarly, 75% of I-Ab-specific T cell clones responded to recombinants containing b polymorphisms in both the alpha- and beta-chains helices and tolerated the substitution of k polymorphisms in the beta-pleated sheet. Furthermore, 20% of the clones responded to a molecule containing allogeneic b residues in just the beta-chain helix. The results demonstrate that the T cell response to allogeneic MHC molecules consists largely of sets of T cells with overlapping specificities for subregions of the MHC molecule. In addition, they highlight the importance of the alpha-helices in these responses and a diminished role for polymorphisms in the beta-strands when, as in the present case, MHC structure and conformation is tolerant of beta-sheet substitutions. These results sharply contrast with observations made in the analysis of Ag-specific T cells and lead to the suggestion that a subset of alloreactive T cells are not peptide specific and can directly recognize MHC polymorphisms. PMID- 1729358 TI - Immunologic memory to phosphocholine keyhole limpet hemocyanin. Recurrent mutations in the lambda 1 light chain increase affinity for antigen. AB - Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the lambda 1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the lambda 1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three lambda 1 CDR2 positions in Ag binding. Compared to antibodies expressing germline lambda 1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated lambda 1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of lambda 1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints. PMID- 1729359 TI - Anti-IgM antibody-induced cell death in a human B lymphoma cell line, B104, represents a novel programmed cell death. AB - We investigated the mechanisms of anti-IgM antibody-induced cell death in a recently established human surface IgM+ IgD+ B lymphoma cell line, B104, the growth of which is irreversibly inhibited by anti-IgM antibody but not by anti IgD antibody, and compared it with the cell death of T cells via TCR/CD3 complex and with the cell death of a murine anti-IgM antibody-sensitive B lymphoma cell line, WEHI-231. The rapid time course of B104 cell death and its requirements for de novo macromolecular synthesis and Ca2+ influx suggest that anti-IgM antibody induced B104 cell death is an active Ca(2+)-dependent programmed cell death. Moreover, cyclosporin A rescued B104 cells from this lethal signal, via surface IgM, suggesting that the intracellular mechanisms involved are quite similar to those of T cell death. DNA fragmentation, which has been reported in TCR/CD3 complex-mediated T cell death, apoptosis, was not involved in the B104 cell death process, but the possible involvement of DNA single-strand breaks was suggested. Observations under light microscopy and transmission electron microscopy indicated that the morphologic features of dying B104 cells resembled necrosis rather than apoptosis. B104 cell death was shown to be quite distinct from that of WEHI-231 in cell death kinetics, the mode of cell death, and the response to cyclosporin A. These data collectively indicate that the death of B104 cells resulting from surface IgM cross-linking represents a hitherto undefined mode of programmed cell death. PMID- 1729360 TI - Antigen-specific CD8+ T cells switch the immune response induced by antigen from an IgG to a cell-mediated mode. AB - We have developed an in vivo/in vitro immunization procedure with xenogeneic RBC as Ag that results in the generation of a lymphoid population that expresses potent delayed-type hypersensitivity (DTH) and produces little, if any, antibody. This lymphoid population contains Ag-specific CD8+ T cells that can inhibit the induction of a strong IgG response. These CD8+ T cells are shown to not only inhibit the antibody response in an Ag-specific manner but allow the Ag to induce cells of the target population to express DTH. Furthermore, the Ag-specific inhibition of the antibody response and the Ag-specific enhancement of the induction of DTH appear to be coordinately regulated, as the same number of CD8+ T cells cells is required to achieve both effects. Thus these CD8+ T cells are shown to switch the response induced by Ag from a humoral to a cell-mediated mode. These regulatory characteristics are consistent with a physiologic role for these cells of ensuring the absence of antibody production during a strong, cell mediated response. PMID- 1729361 TI - HLA-DR2, [HLA-B7, SC31, DR2], and [HLA-B8, SC01, DR3] haplotypes distinguish subjects with asthma from those with rhinitis only in ragweed pollen allergy. AB - MHC haplotypes were determined for 52 patients with ragweed pollen allergy, with and without asthma, and 27 non-atopic controls. Total IgE levels were unimodally distributed in all study groups and were higher in atopic patients in general compared with non-atopics. There was no difference in total IgE levels in patients with rhinitis only compared with those with rhinitis and asthma. IgE anti-Amb a V was detected (after subtraction of values representing the means + 2 SD of the non-atopics) in 9 of 20 asthmatics but only 3 of 32 patients with only rhinitis and was thus associated with asthma. Mean anti-Amb a V was much higher in the antibody-positive asthmatics (1710 U/ml) than in the positive patients with rhinitis only (469 U/ml). The extended MHC haplotype [HLA-B7, SC31, DR2] and its possible DR-containing fragment (SC31, DR2), were found almost exclusively among the patients with IgE anti-Amb a V and were significantly elevated in patients with asthma. DR2 in general, but not DR2 without SC31, was significantly increased in frequency in patients with anti-Amb a V. In contrast, the extended haplotype [HLA-B8, SC01, DR3] and DR3 in general were increased among patients with rhinitis only and patients without IgE anti-Amb a V compared with general controls. Thus, [HLA-B8, SC01, DR3] and DR3 appear to be "protective" for the production of this antibody and the occurrence of asthma. The findings are consistent with an MHC-linked gene or genes on [HLA-B7, SC31, DR2] (but not necessarily DR2, Dw2, or DQw6 in general) controlling the IgE immune response to Amb a V and associated with asthma in ragweed pollen-sensitive subjects. In patients with rhinitis alone and generally undetectable levels of IgE anti-Amb a V, the increase in [HLA-B8, SC01, DR3] and DR3 may mark a response to an as yet unidentified Ag associated with ragweed allergy and rhinitis only. PMID- 1729362 TI - Induction of IFN-alpha in peripheral blood mononuclear cells by HIV-infected monocytes. Restricted antiviral activity of the HIV-induced IFN. AB - PBMC cocultured with HIV-infected monocytes for 12 to 48 h released high levels of IFN activity. IFN titers were directly dependent upon time after virus infection and level of HIV replication in infected cells. IFN induction in PBMC was evident with HIV-infected monocytes and PBMC and with myeloid and lymphoblastoid cell lines with at least three different HIV strains. In HIV infected cell line pairs in which virus infection occurs in both productive and restricted forms, IFN induction in PBMC occurred only with productive infection. IFN activity was acid stable and completely neutralized by antibodies against IFN alpha. Induction of IFN required cell-cell contact between HIV-infected cells and PBMC, but was independent of MHC compatibility. With PBMC co-cultured with autologous HIV-infected monocytes, IFN induction was highly selective: IL-1 beta, IL-6, or TNF-alpha activity and mRNA were not detected. Cell surface determinants on HIV-infected monocytes that induced IFN in PBMC remained active after fixation in 4% paraformaldehyde. Both adherent and nonadherent PBMC produced IFN after coculture with HIV-infected monocytes. Ability to produce IFN by PBMC was not affected by depletion of T cell, NK cell, B cell, or monocyte subpopulations. The IFN activity produced by PBMC cocultured with HIV-infected cells was about 20 fold less active than equal quantities of rIFN-alpha 2b for inhibition of HIV replication in monocytes and at low concentrations enhanced virus growth. Clinical studies with HIV-infected patients and parallel findings in animal lentivirus disease suggest an adverse role for IFN in disease progression. Conditions for induction of IFN in the culture system described in this report may mimic those in the HIV-infected patient. Defining the molecular basis for IFN induction, the cells that produce IFN, and the altered biologic activity of this important cytokine may provide insight into the pathogenesis of HIV disease. PMID- 1729363 TI - Tetraparental mice reveal complex cellular interactions of the mutant, autoimmunity-inducing lpr gene. AB - Lpr is a murine recessive autosomal mutant gene that, in conjunction with the background genes of different inbred mouse strains, produces varied syndromes of lymphadenopathy and autoantibody production. We have investigated the cellular basis of the lpr determined phenomena by constructing allophenic (tetraparental) chimeras of MRL/Mp-lpr/lpr combined with C57BL/6 and MRL/+ combined with C57BL/6 lpr/lpr. The appearance of lymphadenopathy, lpr atypical T cells, and autoantibodies was found to depend on the relative amounts +/+ and lpr tissue. The failure of these three abnormalities to correlate in certain animals suggested different pathways of lpr gene action. Furthermore, in the lpr predominant animals, both the lymphadenopathy and autoantibodies were essentially entirely contributed by the lpr donor. The expression of the lpr gene within the same cells expressing the MRL or B6 background determined the pathologic and serologic manifestations characteristic of the disease of that lpr strain, despite the presence of +/+ cells of the other background in the same individual. The lpr syndrome thus depends on the intracellular interaction of the lpr and background genes, and on positive signalling between different lpr cell populations. PMID- 1729364 TI - A lipoyl synthetic octadecapeptide of dihydrolipoamide acetyltransferase specifically recognized by anti-M2 autoantibodies in primary biliary cirrhosis. AB - Close to 95% of patients with established clinical, biochemical and histologic features of primary biliary cirrhosis (PBC) possess antimitochondrial M2 antibodies reacting with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We examined the ability of synthetic peptides of E2 to be recognized in ELISA by sera from patients with PBC and autoimmune related disorders. Sera from 14 PBC M2+ patients, 1 PBC M2- patient, 5 non-PBC M2+ patients, and 6 patients with chronic active hepatitis were studied. Among the seven E2 synthetic peptides tested (namely peptides 87-119, 167-184, 169-202, 267-302, 456-477, 498-513 and 530-543), only peptide 167-184 used as OVA conjugate and prepared with lipoic acid (LA) located on lysine 173 (natural inner lipoyl-binding site) was recognized in direct ELISA by PBC M2+ sera. The conjugated peptide 167-184 LA was not recognized in direct ELISA by non-PBC M2+ sera or by sera from patients with chronic active hepatitis. The free peptide 167 184 LA inhibited the ELISA reaction of PBC antibodies to PDH and totally abolished the typical immunofluorescence reaction of PBC sera on rat kidney, stomach and liver, or human HEp-2 cell substrates. No inhibition of ELISA or immunofluorescence reaction was found with the other E2 fragments including peptide 167-184 without LA. Our results show that the lipoyl moiety forms an integral part of a dominant conformational epitope recognized by PBC sera. Inasmuch as the peptide 167-184 LA was not recognized by non-PBC sera in direct ELISA, it could be used as a valuable probe for PBC diagnosis. PMID- 1729365 TI - The melanoma growth stimulatory activity receptor consists of two proteins. Ligand binding results in enhanced tyrosine phosphorylation. AB - The human melanoma growth-stimulatory activities (MGSA alpha, beta, gamma/GRO) are products of immediate early genes coding for cytokines that exhibit sequence similarity to platelet factor-4 and beta-thromboglobulin. MGSA/GRO alpha has been demonstrated to partially complete for binding to the approximately 58-kDa neutrophil receptor for another beta-thromboglobulin-related chemotactic protein, IL-8. We demonstrate that when [125I]MGSA/GRO alpha was cross-linked to receptors/binding proteins from human placenta, there were two major [125I]MGSA cross-linked bands of approximately 64,000 and approximately 84,000 Mr. Because [125I]MGSA exists primarily in monomer and dimer forms at the concentrations used here, it is not clear whether the receptor/binding proteins represented by the cross-linked bands are approximately 50,000 and approximately 70,000 or approximately 58,000 and approximately 78,000 Mr. Ligand binding to the receptor proteins is associated with enhanced tyrosine phosphorylation of a number of substrates, including proteins in the same Mr range as the MGSA/GRO receptor/binding proteins. PMID- 1729366 TI - Cartilage and joint inflammation. Regulation of IL-8 expression by human articular chondrocytes. AB - Injury to cartilage is a recognized sequela of neutrophil activation in arthritic joints. This study examined the possibility that chondrocytes may play a direct role in intraarticular neutrophil activation. We demonstrate that IL-1 beta stimulated primary and subcultured human articular chondrocytes, express the gene for the potent neutrophil chemotactic and activating cytokine, IL-8. Expression of IL-8 mRNA is also inducible by TNF-alpha and LPS and, to a lesser degree, by the chondrocyte growth factor, transforming growth factor-beta, but not by platelet-derived growth factor, acidic and basic fibroblast growth factor, or epidermal growth factor. Analysis of IL-1 beta-stimulated cartilage organ cultures by in situ hybridization demonstrates that chondrocytes in all zones of cartilage are rapidly induced to express the IL-8 gene in high copy number. Metabolically labeled IL-1 beta-stimulated chondrocytes synthesize IL-8 de novo, which comigrates on SDS-PAGE with IL-8 produced by synovial fibroblasts. Furthermore, the conditioned media of IL-1 beta-stimulated chondrocytes and cartilage organ cultures contain neutrophil chemotactic activity which is completely neutralized by a specific antibody to IL-8, establishing that a bioactive form of IL-8 is the major secreted neutrophil chemotactic factor. By using a specific RIA, we demonstrate that not only IL-1 beta, but also TNF-alpha and LPS can induce abundant IL-8 secretion from chondrocytes. In conclusion, articular chondrocytes are readily inducible to express the IL-8 gene and secrete biologically active IL-8 which can promote neutrophil-mediated inflammation and cartilage destruction. PMID- 1729367 TI - Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response. AB - The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA. PMID- 1729368 TI - Human recombinant IL-3 is a growth factor for normal B cells. AB - IL-3 is a well known hemopoietic cell growth and differentiation factor. However, its functional role in normal B cell differentiation has not been established. We have investigated the effect of IL-3 on the growth and differentiation of human B cells. IL-3 enhanced the proliferation of Staphylococcus aureus Cowan 1 strain stimulated B cells. The optimal time of IL-3 to stimulate B cell growth was on day 2 to day 3, suggesting that IL-3 was a B cell growth factor acting in the late stage. IL-3 synergized with IL-2 to enhance B cell proliferation and differentiation. Pretreatment of B cells with IL-3 for more than 3 days increased the expression of IL-2R on B cells. However, pretreatment of B cells with IL-2 did not alter the subsequent response to IL-3, suggesting that the synergy between IL-2 and IL-3 may be attributed to the up-regulation of IL-2 response by IL-3. In addition, pretreatment of B cells with IL-4 decreased subsequent response of B cells to IL-3 as well as IL-2, suggesting that IL-3- and IL-2 responding cells passed a similar way during the early stage of B cell activation. It appears that IL-3 and IL-6 mediate normal B cell differentiation via separate mechanisms. IL-3-induced B cell differentiation was mainly mediated by increasing cell growth, whereas IL-6 induced B cell differentiation without affecting proliferation. PMID- 1729369 TI - Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. AB - To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects. PMID- 1729370 TI - The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin. AB - Systemic release of endotoxin (LPS) after Gram-negative infection initiates a cascade of host cytokines that are thought to be the direct cause of shock, multisystem organ failure, and death. Endogenous LPS-binding proteins may play a role in regulating LPS toxicity in vivo. The human neutrophil granule protein bactericidal/permeability-increasing protein (BPI) shares sequence homology and immunocrossreactivity with an acute phase lipopolysaccharide binding protein (LBP) which has been shown to bind to LPS and accelerate LPS activation of neutrophils and macrophages. Although structurally similar, LBP and BPI are apparently functionally antagonistic. We previously showed that BPI inhibits LPS mediated neutrophil activation in vitro. Here we demonstrate that BPI binds to LPS near the lipid A domain, and formation of the LPS-BPI complex abrogates detrimental host responses to LPS. For example, BPI blocks LPS-stimulated TNF release in vitro and in vivo, and LPS complexed to BPI is not pyrogenic in rabbits. Results demonstrating that BPI is released by stimulated human neutrophils further support the idea that BPI functions extracellularly in vivo to neutralize endotoxin. Taken together, these data argue that BPI neutralizes the toxic effects of LPS in vivo, and that BPI may represent a new therapeutic approach to the treatment of endotoxic shock. PMID- 1729371 TI - Ca2+ and calmodulin selectively regulate lipopolysaccharide-inducible cytokine mRNA expression in murine peritoneal macrophages. AB - The role of Ca2+ and Calmodulin in regulating LPS-induced cytokine gene expression in murine peritoneal macrophages has been investigated. Treatment of macrophages with three structurally distinct antagonists of Calmodulin (Trifluoperazine, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide, and 1,3 dihydro-1-(-((4-mentyl-4H,6H-pyrrolo(1,2-a) (4,1)-benzoxazepin-4-yl)methyl)-4 peperidinyl)-2H-benzimi dazol-2-one) resulted in a characteristic modulation of the expression of three LPS-inducible cytokine genes: IL-1 alpha mRNA levels were only modestly reduced, IL-1 beta mRNA levels were markedly suppressed and IP-10 mRNA levels were increased. The same pattern of modulation was seen when LPS stimulated cells were also treated with two different Ca2+ antagonists (8 (diethylamono)-octyl-3,4,5-trimethoxybenzoate hydrochloride and bis-(o amonophenoxy)-ethane-N,N,N'N'-tetraacetic acid). Although the suppression of IL-1 beta mRNA accumulation by N-(6-amonohexyl)-5-chloro-1-napthalene-sulfonamide or bis-(o-amonophenoxy)ethane-N,N,N'N'-tetraacetic acid occurred even if the antagonist was added after LPS, the potentiation of IP-10 mRNA levels required the use of the agent before or along with the LPS stimulus. Elevation of intracellular Ca2+ using ionomycin did not initaite cytokine gene expression and thus changes in Ca2+ cannot replace the LPS-initiated signal. Furthermore, removal of extracellular Ca2+ did not block the response to LPS. Calmodulin antagonists selectively increased the transcriptional activity of the IP-10 gene but decreased the stability of all three mRNA measured. Thus the mechanisms involved in Ca2+/Calmodulin control of macrophage gene expression are multifactorial and contribute to the diversity of macrophage inflammatory behavior. In concert with previous reports, the present results indicate that Ca2+, acting through Calmodulin may be a necessary but insufficient component of the signalling process that mediates intracellular response to LPS. Agents that alter intracellular Ca2+ levels without inducing cytokine gene expression may thereby indirectly regulate inflammation. PMID- 1729372 TI - Metabolic requirements for macrophage presentation of Listeria monocytogenes to immune CD8 cells. AB - Though ingested Ag are readily degraded into peptides within endocytic vesicles, APC usually cannot present these fragments to CD8 cells. Despite this generalization, some exceptions have been noted. For example, murine macrophage targets readily process heat-killed Listeria monocytogenes (HKLM) into a form recognizable by immune CD8 CTL. Using an assay of Listeria-specific, CD8-mediated cytotoxicity to quantitate Ag presentation by C57Bl/6 macrophage targets, we have examined some of the cellular requirements for this form of Ag processing. To assess whether the physical form of the Ag is an important determinant of processing, we compared the ability of macrophages to present intact HKLM, fractionated L. monocytogenes (LM) membranes, and octyl-beta-d thioglucopyranoside-solubilized extracts of LM membranes. Macrophages presented each Ag form in a similar manner indicating that processing is not critically dependent on the presence of intact bacteria or even on the introduction of Ag in a particulate form. To gain insight into the metabolic requirements for Ag processing, we examined the effects of several inhibitors. As might be expected, listerial Ag presentation was blocked by brefeldin, a known inhibitor of the endogenous pathway of Ag processing. LM Ag presentation, however, was also blocked by inhibitors of endosomal acidification (chloroquine, ammonium chloride, and monensin) and by the acid protease inhibitor pepstatin A, suggesting that endocytic processing may play an essential role in CD8 recognition of this Ag. To formally establish that this pattern of exogenous Ag processing requires the presence of a class I MHC product, we demonstrated that beta-2 microglobulin deficient macrophages, which lack class I MHC product expression, cannot present HKLM to CD8 cells. However, we could not block Ag presentation by incubating macrophages with monoclonal anti-H-2K or H-2D antibodies, suggesting that LM Ag presentation may be mediated by some other class I MHC product. Additional characterization of this pathway of Ag presentation is warranted in view of its possible role in initiating CD8-mediated immunity against microbial Ag. PMID- 1729373 TI - A protective monoclonal antibody specifically recognizes and alters the catalytic activity of schistosome triose-phosphate isomerase. AB - mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner. Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice. Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate. After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced. Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI). Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction. Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI. To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme. PMID- 1729374 TI - IFN-gamma-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-alpha. AB - Activated murine peritoneal macrophages inhibit the intracellular proliferation of Toxoplasma gondii and produce a number of cytokines, such as TNF-alpha and IL 1. Both TNF-alpha and IL-1 have been reported to be involved in the immune response against various microorganisms, but the mechanisms responsible for these effects are not known. In the present study it was investigated whether endogenously produced TNF-alpha and IL-1 are involved in the activation of peritoneal macrophages by rIFN-gamma leading to toxoplasmastatic activity and the production of reactive nitrogen intermediates. The rIFN-gamma-induced toxoplasmastatic activity was inhibited by neutralizing antibodies against mouse TNF-alpha in a dose-dependent and time-dependent way, but neutralizing antibodies against mouse IL-1 alpha and IL-1 beta did not affect this activity. Involvement of TNF-alpha in the induction of toxoplasmastatic activity was confirmed by our finding that rTNF-alpha in combination with a nonactivating concentration of rIFN gamma inhibited the intracellular proliferation of T. gondii. No synergistic activity of rIL-1 and rIFN-gamma on the inhibition of T. gondii proliferation was found. Both rTNF-alpha and rIL-1 alpha alone inhibited the intracellular proliferation of T. gondii only slightly. Because it has been reported recently that activated macrophages produce reactive nitrogen intermediates that are essential in the induction of toxoplasmastatic activity, we investigated whether these intermediates are involved in the TNF-dependent induction of toxoplasmastatic activity. Neutralizing antibodies against mouse TNF-alpha inhibited also the release of NO2- by rIFN-gamma-activated macrophages almost completely. Macrophages incubated with rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma released substantial amounts of NO2-, but rTNF-alpha and rIL-1 alpha alone, and the combination of rIL-1 alpha and a nonactivating concentration of rIFN-gamma induced only little NO2(-)-release by macrophages. To assess whether reactive nitrogen intermediates act directly or indirectly on the intracellular proliferation of T. gondii, macrophages were incubated with the L-arginine analog NG-monomethyl-L-arginine or the NADPH inhibitor diphenylene iodonium, both inhibitors of the generation of reactive nitrogen intermediates. Good correlation was found between toxoplasmastatic activity and the release of NO2- during the 24-h activation period before infection of the macrophages with T. gondii, but no correlation was found between toxoplasmastatic activity and the release of NO2- during infection of the macrophages.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1729375 TI - Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection. AB - Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals. PMID- 1729376 TI - A murine cDNA encodes a pan-epithelial glycoprotein that is also expressed on plasma cells. AB - Using a subtractive cDNA approach, we have identified a number of genes expressed in murine plasmacytomas, but not B or pre-B lymphomas. One of these genes, 289A, expresses a 1.8-kb microsomally localized mRNA that encodes a 314-amino-acid protein containing a signal sequence and a hydrophobic transmembrane domain. Sequence comparison suggests that the predicted protein is the murine homologue of a human cell surface pan-epithelial glycoprotein known variously as EGP, GA733 2, KSA, and KS1/4, recognized by mAb HEA125, GA733, KS1/4, CO17-1A, M74, and 323/A3. The 289A mRNA is highly expressed in normal murine tissues containing epithelial cells, and at a low level in plasma cells induced by LPS stimulation of spleen B lymphocytes. It is expressed in 15 of 16 plasmacytomas, but at a much lower level, if at all, in pre-B or B lymphomas. In human B cell lines, 289A detects a 1.5-kb mRNA in the myeloma cell line 8226, but not in Burkitt's lymphoma or lymphoblastoid cell lines. Subsequent FACS analysis of human cell lines with the mAb GA733 and KS1/4 demonstrated concordant expression of the mRNA and the protein. We conclude that 289A is the murine homologue of EGP, GA733-2, KSA, and KS1/4 Ag. Although its expression was previously thought to be restricted to epithelial cells, it is also expressed in plasma cells and is a B lymphocyte differentiation Ag. Because of the multiplicity of names, we propose calling the human gene hEGP314, and the murine gene mEGP314. PMID- 1729377 TI - Identification of a novel gene expressed in activated natural killer cells and T cells. AB - We have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript (NK4) that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3' untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. PMID- 1729378 TI - Human mb-1 gene: complete cDNA sequence and its expression in B cells bearing membrane Ig of various isotypes. AB - The transmembrane protein, IgM-alpha, a product of mb-1 gene, has been shown to be specifically associated with membrane-bound IgM on the plasma membrane of B lymphocytes. Recent studies have suggested that IgM-alpha may play a role in transducing signals from the Ag receptors during the activation of B cells. A large amount of information has been obtained in the mouse system regarding IgM alpha and other components of the newly conceived B cell Ag receptor complex. Here we report the cloning and the nucleotide sequencing of cDNA clones of human mb-1, covering the entire length of the mRNA. At the amino acid sequence level, human and murine mb-1 share a high homology in their transmembrane and intracytoplasmic segments, suggesting an important biologic function for these regions of mb-1. A major difference, mainly in the 3' untranslated part, exists between our cDNA sequence and the published partial human mb-1 cDNA sequence. It has also been observed that human mb-1 is expressed not only by B cell lines expressing membrane-bound Ig of mu and delta isotypes but also those expressing membrane-bound Ig of alpha and gamma isotypes. PMID- 1729379 TI - Shared human melanoma antigens. Recognition by tumor-infiltrating lymphocytes in HLA-A2.1-transfected melanomas. AB - Three predominantly CD8+ CTL lines, TIL 501, TIL 620, and TIL 660, were generated from three HLA-A2+ melanoma patients by culturing tumor-infiltrating lymphocytes in 1000 U/ml IL-2. These tumor-infiltrating lymphocytes lysed 12 of 18 HLA-A2+ autologous and allogeneic melanomas, but none of 20 HLA-A2-negative melanomas. They also did not lyse the MHC class I negative lymphoma-leukemia cell lines, Daudi, K562, or HLA-A2+ non-melanoma cell lines including PHA or Con A-induced lymphoblast, fibroblast, EBV-transformed B cell, Burkitt's B cell lymphoma, and colon cancer cell lines. Autologous and allogeneic melanoma lysis was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag among melanoma cell lines in a TCR-dependent, HLA-A2 restricted manner. Six HLA-A2-negative melanoma cell lines obtained from five HLA A2-negative patients were co-transfected with the HLA-A2.1 gene and pSV2neo. All 17 cloned transfectants expressing cell surface HLA-A2 molecules, but none of 12 transfectants lacking HLA-A2 expression, were lysed by these three HLA-A2 restricted, melanoma-specific CTL. Lysis of the HLA-A2+ transfectants was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag on transfectants in a TCR-dependent, HLA-A2 restricted manner. These results identify the HLA-A2.1 molecule as an Ag presenting molecule for melanoma Ag. They also suggest that common melanoma Ag are expressed among melanoma patients regardless of HLA type. These findings have implications for the development of melanoma vaccines that would induce antitumor T cell responses. PMID- 1729380 TI - Influence of MHC genes on spontaneous recovery from Friend retrovirus-induced leukemia. AB - Genes influencing the rate of spontaneous recovery from erythroleukemia induced by a low dose of Friend virus complex were located in the right and left portions of the mouse MHC. The right side gene was most likely the previously described Rfv-1 in the H-2D region. Using the B6.C-H-2bm12 mutant mice, the left side gene was mapped to the A beta class II locus. The A beta b was a resistant allele and A beta k and A beta bm12 were susceptible alleles. Genes at this class II locus controlled the responsiveness of Th cells to envelope glycoprotein of Friend murine leukemia helper virus and affected the class switching of virus neutralizing antibodies from IgM to IgG in FV-infected mice. PMID- 1729381 TI - Short-term effects of triiodothyronine on the bowfin, Amia calva (Holostei), and the lake char, Salvelinus namaycush (Teleostei). AB - To assess the role of triiodothyronine (T3) in mediating short-term changes in metabolism, such as those occurring in circadian patterns, we examined the effects of intraperitoneal injection of T3 on the oxidation of substrates by isolated mitochondria from liver of the bowfin, Amia calva, and red muscle and liver of the lake char, Salvelinus namaycush. Selected enzymes were measured in red muscle and liver of the lake char. Three hours after intraperitoneal injection of T3, oxidation of some substrates by mitochondria isolated from the liver of the bowfin was reduced. Similar treatment had no effect on substrate oxidation in liver mitochondria isolated from lake char. Oxidation of substrates by lake char red muscle mitochondria was stimulated by T3 injection. Citrate synthase levels were increased in red muscle suggesting that changes in enzyme activity may be in part responsible for the short-term mitochondrial responses to T3 injection. PMID- 1729382 TI - Embryonic retinal ablation and post-metamorphic optic nerve crush: effects upon the pattern of regenerated retinotectal connections. AB - We examined relationships between healing observed during embryonic Xenopus retinal and optic nerve regeneration and resultant visuotectal pattern formation. Dorsal (D) and nasoventral (NV) 1/3 sized eye fragments were surgically created in stage 32 Xenopus laevis embryos. Gross anatomical healing modes of these fragments were examined 2 days post-surgery (stage 43). Healing was categorized according to the degree of cell movements observed. Animals were reared through metamorphosis and electrophysiologic mapping techniques were employed on those animals whose eyes regenerated. All D 1/3 fragments showed normal (non duplicated) projections to the tectum; most (80%) of the healing observed showed little cell movements (the remaining 20% showed substantial cell movements, yet failed to show duplicated projections). Most NV 1/3 fragments (73%) formed two mirror image projections to the contralateral midbrain optic tectum (pattern duplication). Most (88%) of the healing observed among these animals showed massive cell movements in the ventral retinal region (the remaining 12% showed moderate cell movements). The remaining NV 1/3 fragments (27%) showed moderate cell displacement and failed to show duplicated projections). These data are compatible with a cell-movement:intercalary cell division hypothesis in which duplication is dependent upon specific positional confrontation and subsequent cell division. In additional studies, in adult animals, the optic nerves of eyes with duplicated projections were crushed and allowed to regenerate for 1 year. Duplicated projections were restored, indicating that developmental and maturational factors are probably not responsible for duplicative pattern formation; rather, information intrinsic to the eye, possibly created during healing interactions and/or fiber ingrowth to the tectum, underlies duplicate innervation of the tectum. PMID- 1729383 TI - Prolactin-dependent seasonal changes in pelage: role of the pineal gland and dopamine. AB - The Siberian hamster displays seasonal changes in pelage that are dependent upon fluctuations in circulating prolactin levels. Pinealectomy prevented the decrease in serum prolactin and molt to the winter pelage displayed by castrated males housed under a short-day photoperiod. A dopaminergic antagonist, pimozide, enhanced prolactin levels in both pinealectomized and sham-operated animals under both long and short photoperiods. In the short-day animals, this effect of pimozide was associated with a prevention of the development of winter pelage. These results indicate that seasonal prolactin levels and related pelage changes are dependent upon the integrity of the pineal gland. However, basal prolactin levels under different photoperiod conditions appear to be only partly regulated by the actions of the dopaminergic system. PMID- 1729384 TI - Effects of gonadotropin-releasing hormone variants on plasma and testicular androgen levels in intact and hypophysectomized male frogs, Rana esculenta. AB - The effects of vertebrate gonadotropin-releasing hormone (GnRH) variants on plasma and testicular androgen level in intact and hypophysectomized (PDX) male frogs, Rana esculenta, have been investigated. In intact animals, mammalian (m) GnRH, m-GnRH analog (buserelin), salmon (s)-GnRH, chicken (c) I-GnRH, cII-GnRH, D Arg6-cII-GnRH (cII-GnRHA), and lamprey (l)-GnRH (1.5 micrograms and 6 micrograms, total dose given on alternate days for 5 days) were able to enhance androgen production showing that specificity of pituitary responsiveness to GnRH variants appears to be low. Chicken II-GnRH was more effective than s-GnRH in eliciting testicular and circulatory androgen level increase. Moreover, in animals treated with 6 micrograms of cII-GnRH and s-GnRH in combination, androgens decreased as compared with animal treated with cII-GnRH only, suggesting that GnRH receptors bind preferentially the s-GnRH form. In PDX animals, buserelin (1.5 and 6 micrograms), cII-GnRH, and its analog (6 micrograms) were able to increase plasma androgen levels whereas testis androgen concentrations were increased by cII-GnRH (1.5 and 6 micrograms), D-Arg6-cII-GnRHA, and buserelin (6 micrograms). Since androgen production in PDX animals is influenced especially by peptides sharing cII-GnRH structure, it is suggested that a testicular cII-GnRH-like material play a role as local modulator of the gonadal activity in Rana esculenta. PMID- 1729385 TI - On the convergent cell movements of gastrulation in Fundulus. AB - Mainly because of its transparency, the Fundulus gastrula constitutes ideal material for direct study of morphogenetic cell movements in vivo. Marking studies show that deep cells of the germ ring converge toward and enter the embryonic shield, where they undergo extension. Those close to the shield move faster. Analysis of videotapes reveals that all deep cells of the dorsal germ ring move toward the shield. But none moves in a direct line. All meander considerably. Germ ring cells nearer the shield move toward it at a higher net rate than those farther away because they meander less. This suggests that exogenous factors promote their directionality. Cells in the prospective yolk sac adjacent to the germ ring also show net convergence, but they meander more. Directional forces are apparently stronger in the germ ring. Converging deep cells move both by filolamellipodia and, less frequently, by blebs. However, there is very little individual cell movement; all cells are almost always in adhesive contact with other cells in moving cell clusters. Clusters vary constantly in size, continually aggregating with other cells and other clusters and splitting. Filolamellipodial cells show contact inhibition of cell movement. Nevertheless, they move and do so directionally, presumably in part because, as members of cell clusters, much of their movement is passive. They also show intercalation or invasive activity, but, consistent with their contact-inhibiting properties, only when neighboring cells separate and provide free space. Cells moving by blebbing locomotion are non-contact inhibiting and intercalate readily. Cell division continues during convergence. Although this temporarily arrests their movement, the daughter cells soon join in the mass convergent movement. PMID- 1729386 TI - Evolutionary modification of regenerative capability in vertebrates: a comparative study on teleost pectoral fin regeneration. AB - The regenerative ability of the pectoral fins of 14 species from 6 euteleostean families was tested. Blastema formation and distal outgrowth was observed in all species, indicating the initiation of regeneration in all species tested. Interspecific variation exists with respect to the frequency of malformations and the patterns produced by heteromorphic regeneration. Taking into account published reports on pectoral fin regeneration, the systematic distribution of homo- and heteromorphic regeneration leads to the following conclusions: 1) regenerative ability of pectoral fins is a property inherited from the common ancestor of euteleosteans. Whether it is also the ancestral condition for the whole teleostean group cannot be determined, because reports on more primitive teleosteans like the herring and the osteoglossimorphs are missing. 2) A propensity to produce high frequencies of heteromorphic regenerates originated independently at least three times in Cypriniformes, Scorpaeniformes, and Perciformes. 3) Impaired regeneration is most commonly found in bottom fishes, although not all ground fish groups show heteromorphic regeneration. This suggests that impaired regeneration is not directly related to bottom dwelling, but most probably originated as a side effect of other adaptive changes. Hence, neither the presence nor the loss of faithful regeneration can be associated with particular adaptive scenarios in this group, since regeneration seems to be ancestral to all major euteleost groups and its loss has no clear adaptive significance. Whether there are adaptive reasons to maintain regenerative capability or whether there are cases of reestablishment of regeneration after it was lost cannot be decided on the basis of recent evidence. More observations on phylogenetically closely related species with variable regenerative capability are necessary to assess adaptive explanations of regeneration. PMID- 1729387 TI - Developmental changes in the incidence of chromosome anomalies of bovine embryos fertilized in vitro. AB - In total, 196 two- to 32-cell bovine embryo and 104 blastocysts were obtained by the in vitro fertilization of follicular oocytes matured in vitro, and 15 blastocysts fertilized in vivo were used. Chromosomal anomalies in these embryos and the inner cell mass (ICM) separated immunologically were investigated. Chromosomal anomalies were observed in 12.1% (5/41) of 2-cell embryos, 20.0-36.4% of 4- to 16-cell embryos, 7.1% (1/14) of 17- to 32-cell embryos, 44.2% (15/34) of blastocysts, and 18.6% (13/70) of ICM cells derived from in vitro fertilization. These anomalies were mainly 3N and 4N at 2-cell stage, 1N and 1N/2N at 4- to 32 cell stages, and 2N/4N in blastocysts and in their ICM cells. Chromosomal anomalies of blastocysts from in vivo fertilization and their ICM were observed in 20.0% (1/5) of blastocysts and 33.3% (3/9) of ICM cells and these compositions were mainly 2N/4N. These results indicate that the abnormalities at early and blastocyst stages of embryos derived from in vitro fertilization were caused by abnormal fertilization in vitro and abnormal cleavage, respectively. Furthermore, a definite location of the chromosomal anomalies was observed in the trophectoderm of blastocysts derived from in vitro fertilization. PMID- 1729388 TI - Regulation of ovarian steroidogenesis in vitro in the viviparous shark, Squalus acanthias. AB - We investigated the steroid biosynthetic capabilities of ovarian granulosa and thecal elements of the viviparous dogfish, Squalus acanthias. In this report we present evidence that granulosa cells secrete quantitatively important amounts of progesterone (P), testosterone (T), and estradiol-17 beta (E), while theca has a more limited capacity to synthesize T and E. Ovarian granulosa cells were obtained from animals at each stage of gestation. After collagenase dispersion, an aliquot of 250,000 cells was incubated at 18 degrees C in basal medium, containing Eagle's salts, glutamine, penicillin, streptomycin and adjusted with 136 mM sodium chloride and 350 mM urea. After a 4 hour incubation, the content of P, T, and E in medium was determined by radioimmunoassay. P was not detectable at any time, while E was present throughout the cycle, being maximal when gestation is three quarters complete (Stage C). T gradually increased from Stage B toward late pregnancy. In Stage C granulosa cells, E production increased in the presence of graded doses of T substrate. Also, a homologous pituitary extract (1/25 equivalents) and the calcium ionophore A23187 stimulated production of all 3 steroids. Using radioisotopes, granulosa cells showed a wide range of synthetic capacities. In Stage C thecal tissue, E production also increased in the presence of graded doses of T substrate, while pituitary extract only increased T. When granulosa and theca were recombined, in the presence of pituitary extract, P levels decreased with a corresponding increase in T, when compared to granulosa alone. These data suggest a possible interaction between granulosa and theca for steroid biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729389 TI - Nonopioid effect of morphine on electrically evoked acetylcholine release from Torpedo electromotor neurons. AB - The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed. PMID- 1729390 TI - Rat brain adenosine deaminase after 2'-deoxycoformycin administration: biochemical properties and evidence for reduced enzyme levels detected by 2' [3H]deoxycoformycin ligand binding. AB - Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729391 TI - Synapsin II phosphorylation and catecholamine release in bovine adrenal chromaffin cells: additive effects of histamine and nicotine. AB - Primary cultures of bovine adrenal medullary chromaffin cells can be stimulated with nicotine, which mimics the cholinergic stimulus from the splanchnic nerve. Histamine also stimulates catecholamine release in a time- and dose-dependent manner. We have previously shown that nicotine stimulates incorporation of 32Pi into the vesicle-associated phosphoprotein synapsin II. We report here that histamine, too, stimulates an increase in 32Pi incorporation into synapsin II, which is blocked by the H1-histamine receptor-specific antagonist pyrilamine. The time course of histamine-stimulated synapsin II phosphorylation closely paralleled that of histamine-stimulated catecholamine release. Interestingly, histamine and nicotine produced an additive increase in both catecholamine release and synapsin II phosphorylation, suggesting that these two secretogogues stimulate the phenomena via independent mechanisms. When we investigated the dependence of these two agonists on extracellular calcium, we found that nicotine stimulated release and synapsin II phosphorylation were reduced to basal levels at low calcium concentrations. However, the histamine-stimulated effects remained significantly elevated. This suggests that calcium arising from two separate pools can stimulate catecholamine release and synapsin II phosphorylation in bovine chromaffin cells. Taken together, these data support the hypothesis that synapsin II phosphorylation is a component of the secretory response from these cells. PMID- 1729392 TI - Posttranslational processing of proenkephalin in SK-N-MC cells: evidence for phosphorylation. AB - SK-N-MC cells have recently been shown to be a rich source of proenkephalin and/or the proenkephalin-derived peptide, peptide B. We have investigated the synthesis and the posttranslational processing of proenkephalin in these cells. SK-N-MC cells retain very little of the proenkephalin synthesized; greater than 99% of the immunoreactive enkephalin synthesized within a 48-h period is secreted into the medium rather than contained intracellularly. When medium samples were subjected to gel filtration and assayed for the various enkephalins present within proenkephalin, only two major molecular-weight classes of peptides, with molecular weights and immunoreactive profiles consistent with those of proenkephalin and the 3.6-kDa carboxyl-terminal fragment peptide B, were observed. The proenkephalin-like peptide present in medium samples was shown by western blot procedures to consist of a 32-kDa protein with a slight amount of a higher-molecular-weight immunoreactive component above it. Only proenkephalin sized peptides were present within cell extracts. Radiolabeled proenkephalin added to cell cultures was also cleaved to products similarly sized to those found in medium extracts; radiolabeled proenkephalin incubated in the absence of cells was not cleaved. Cleavage of exogenous proenkephalin thus probably at least partially occurs following secretion. Cell radiolabeling experiments with [32P]orthophosphate demonstrated that SK-N-MC proenkephalin is phosphorylated. Microheterogeneity of proenkephalin was also observed using isoelectric focusing coupled with western blotting. Our results suggest that the SK-N-MC cell line represents a useful model to study the earliest steps of the posttranslational processing of human proenkephalin in a neuronal cell type. PMID- 1729393 TI - Cytokine regulation of neuronal survival. AB - Interleukin-1 is a cytokine involved in the immune response to infection and inflammation as well as a growth promotor for several cell types. Interleukin-1 like immunoreactive material has been found in the nervous system. We now show that antisera, which blocked the T-cell proliferative effects of interleukin-1 alpha, decreased neuronal cell counts (to 40% of control) in dissociated spinal cord cultures derived from fetal mice. This neuronal loss was prevented by addition of interleukin-1 alpha, and to a lesser extent by interleukin-1 beta. Exogenous interleukin-1 alpha increased the survival of neurons when added to cultures in which the electrical activity was blocked with tetrodotoxin, whereas no such cytokine-related increase in neuronal survival was observed in electrically active cultures. The antiserum-induced death could also be prevented by cotreatment of the cultures with 0.1 nM vasoactive intestinal peptide, a substance that induces the secretion of neuronal trophic factors from nonneuronal spinal cord cells and thereby increases neuronal survival in electrically inactive cultures. These studies indicate that the cytokine interleukin-1, or an immunologically cross-reactive protein, can increase neuronal survival. PMID- 1729394 TI - Identification of two distinct populations of protein kinase C in rat brain membranes. AB - The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident. PMID- 1729395 TI - Muscarinic autoreceptors of Torpedo electric organ are of the M1 subtype: evidence by radioligand binding using selective antagonists. AB - The presynaptic muscarinic autoreceptor of Torpedo marmorata electric organ has been characterised by radioligand binding studies using the subtype-selective antagonists pirenzepine, (+)-telenzepine, methoctramine, and AF-DX 116. The presynaptic receptor had relatively high affinity for the M1 antagonists pirenzepine and (+)-telenzepine (Ki = 35 and 7 nM, respectively) and lower affinities for the M2 antagonists AF-DX 116 and methoctramine (Ki = 311 and 277 nM, respectively). Comparison of these binding data with those from an M2 receptor (rat heart membranes) assayed under identical conditions and with data in the recent literature suggests that the Torpedo muscarinic autoreceptor has a pharmacology most similar to the M1 pharmacological subtype of muscarinic acetylcholine receptor. PMID- 1729396 TI - Brain synthesis and cerebrovascular action of epoxygenase metabolites of arachidonic acid. AB - The purpose of this study was to determine if whole brain makes epoxygenase metabolites of arachidonic acid and, if so, whether they are vasoactive on the cerebral microcirculation. Blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, and the extracted metabolites were resolved by HPLC. Metabolite structures were confirmed by gas chromatography/mass spectrometry. In addition to prostaglandins, leukotriene B4, and hydroxyeicosatetraenoic acids, mouse brain metabolized arachidonic acid into several other compounds. Among them, we identified 5,6- and 14,15 epoxyeicosatrienoic acid. Next, we tested the effect of topical application of brain-synthesized 5,6-epoxyeicosatrienoic acid and synthetic epoxyeicosatrienoic acids on in vivo rabbit cerebral arteriolar diameter using the cranial window technique and in vivo microscopy. Brain-synthesized 5,6-epoxyeicosatrienoic acid caused a transient 28% arteriolar dilation, similar to that produced by 5 micrograms/ml of synthetic 5,6-epoxyeicosatrienoic acid. A concentration of synthetic 14,15- and 11,12-epoxyeicosatrienoic acid of 5 micrograms/ml CSF had little or no effect on diameter, whereas 8,9-epoxyeicosatrienoic acid caused a maximum dilation of 8%. These studies suggest that brain-synthesized 5,6 epoxyeicosatrienoic acid may play a role in the normal or pathophysiological regulation of the cerebral microcirculation. PMID- 1729397 TI - Glucose, insulin, and insulin-like growth factor I regulate the glycogen content of astroglia-rich primary cultures. AB - The glycogen content of astroglia-rich primary cultures derived from the brains of newborn rats depends on the concentration of glucose in the culture medium. After administration of culture medium lacking glucose, the glycogen content decreases with a half-time of 7 min. Readdition of glucose results in replenishment of the glycogen stores within 2-3 h, but fully only if glucose is present in a concentration of at least 4 mM. Insulin, or the more potent insulin like growth factor I, increases the content of glycogen approximately 1.7-fold, with the half-maximal effects being attained at concentrations of 10 and 0.5 nM, respectively. These results suggest that (a) glucose or a metabolite of it and (b) insulin-like growth factor I or a closely related peptide, but not insulin, are likely to be physiological regulators of the level of glycogen in astrocytes. PMID- 1729398 TI - Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases. AB - We have recently reported on the differential alterations of various cholinergic markers in cortical and subcortical regions in Alzheimer's disease (AD). The main purpose of the present study was to determine if cholinergic deficits observed in patients with AD are unique to this disorder or can be generalized to others such as idiopathic Parkinson's disease (PD) and PD with Alzheimer-type dementia (PD/AD). Muscarinic M1, M2, and nicotinic receptor binding parameters (KD and Bmax) were determined in various cortical and subcortical areas using selective radioligands ([3H]pirenzepine, [3H]AF-DX 116, and N[3H]methylcarbamylcholine). Choline acetyltransferase activity was also determined as a marker of the integrity of cholinergic innervation. Alterations of cholinergic markers are comparable in cortical areas in AD, PD, and PD/AD brains. In frontal and temporal cortices, as well as in the hippocampus, choline acetyltransferase activity and binding capacities of M2 and nicotinic binding sites are similarly decreased in these three disorders compared with age-matched control values. M1 receptor binding parameters are not significantly modified in cortical areas in patients with these disorders. In contrast, important differences between AD and PD brain tissues are found in subcortical areas such as the striatum and the thalamus. The density of M1 sites is significantly increased in striatal areas only in patients with AD, whereas densities of nicotinic sites are decreased in thalamus and striatum in PD and PD/AD, but not AD, brain tissues. The binding capacity of M2 sites is apparently unchanged in subcortical areas in all three disorders, although tendencies toward reductions are observed in the striatum of PD and PD/AD patients. Thus, although comparable alterations of various cholinergic markers are observed in cortical areas in the three neurological disorders investigated in the present study, important differences are seen in subcortical areas. This may be relevant to the respective etiological and clinical profiles of AD and PD. PMID- 1729399 TI - Aluminum alters the electrophoretic properties of neurofilament proteins: role of phosphorylation state. AB - Exposure of each of the three neurofilament proteins (NFPs) to AlCl3 resulted in their failure to migrate into sodium dodecyl sulfate (SDS)-containing gels. This effect was dependent on length of incubation (minimum, 2 h) and AlCl3 concentrations (minimum, 50 microM) and was not reversed by 20% SDS, 6 M urea, freeze-thawing, boiling, or extensive dialysis. The migration of vimentin and glial fibrillary acidic protein was not affected by AlCl3. The high-molecular weight neurofilament subunit (NF-H) entered SDS-containing gels after exposure to aluminum lactate but migrated aberrantly as a long high-molecular-weight streak. Migration of the 160-kDa alpha-chymotryptic cleavage product of NF-H, which contains the higher phosphorylated tail domain, was also prevented from migrating into SDS-containing gels by AlCl3. Dephosphorylation of NF-H and the middle molecular-weight neurofilament subunit (NF-M) eliminated these effects on gel migration. EDTA, EGTA, MgCl2, CaCl2, or FeCl3 had no effect on NF-H or NF-M migration; furthermore, preincubation with, or simultaneous exposure to, CaCl2 or FeCl3 did not alter the effect of AlCl3. One interpretation of these results is that Al3+ interacts with phosphate groups on extensively phosphorylated C terminal sidearms of NFPs, resulting in intermolecular cross-linking. These findings demonstrate a direct effect of aluminum on NFPs and provide a possible mechanism for neurofilament accumulation in perikarya during aluminum intoxication. PMID- 1729400 TI - Affinity purification of human tau proteins and the construction of a sensitive sandwich enzyme-linked immunosorbent assay for human tau detection. AB - Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml. PMID- 1729401 TI - Protein tyrosine kinases in human brain and gliomas. AB - Tyrosine kinase activity was determined in neonatal and adult human brain, oligodendrogliomas, and astrocytomas. The astrocytomas were divided into low- (grade I and grade II) and high-grade (grade III and grade IV) tumors. We measured the tyrosine kinase activity in the cytosolic and membrane fraction using poly(glutamic acid:tyrosine, 4:1) as an artificial substrate. The cytosolic activity in oligodendrogliomas (n = 7), low-grade astrocytomas (n = 7), and neonatal brain (n = 1) was increased, on average, two- to fourfold compared with that in normal adult brain (n = 14). The cytosolic activities of high-grade astrocytomas (n = 11) were in approximately the same range as found in normal adult brain. The absence of an increase in cytosolic activity in high-grade astrocytomas compared with adult brain is likely due to the occurrence of necrosis in these tumors. In contrast to the cytosolic activity, no differences were found in the membrane-bound activity. By fast protein liquid chromatography, at least three forms of cytosolic protein tyrosine kinase could be separated, which eluted at 0, 115, and 210 mM NaCl. In most cases the highest amount of activity eluted at 210 mM NaCl. However, in oligodendrogliomas, high-grade astrocytomas, and neonatal brain, more activity eluted at 115 mM NaCl than in normal adult brain (p = 0.043). Nevertheless, protein tyrosine kinases from all three peaks contributed to the elevated levels of total cytosolic activity of oligodendrogliomas and low-grade astrocytomas. PMID- 1729402 TI - Involvement of putrescine in the development of kindled seizure in rats. AB - During the development of kindling by daily electrical stimulations applied to the left amygdala of rats, concentrations of the polyamines putrescine, spermidine, and spermine were measured in the left amygdala and the remainder of the cerebrum. A significant increase of putrescine concentration appeared first at the left amygdala in prekindled rats and then propagated to the remainder of the cerebrum with the development of kindling. This increase in putrescine concentration in the left amygdala was higher in prekindled rats than in fully kindled rats and lasted for at least 24 h after the final kindling stimulation. The concentrations of spermidine and spermine were slightly increased in a fully kindled state. To clarify the role of putrescine in kindling, the development of amygdaloid kindling was examined in rats after microinjections of alpha difluoromethylornithine, a specific inhibitor of polyamine synthesis, and putrescine into the ipsilateral amygdala. Pretreatment with alpha difluoromethylornithine (50 nmol) for 10 days accelerated both the development of behavioral kindling and the propagation of the afterdischarge from the left amygdala to the frontal cortex. In contrast, pretreatment with putrescine (200 nmol) for 10 days retarded the development of kindling. These results suggest that the increase in putrescine concentration in the kindled brain has an inhibitory effect on the development of kindling. PMID- 1729403 TI - Characterization of serotonin N-acetyltransferase in the lateral eye of the green frog Rana perezi: protective action of EGTA. AB - The kinetics of serotonin N-acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 mM and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35 degrees C. Km and Vmax estimations for acetyl-CoA at a 10 mM tryptamine concentration were 63.3 microM and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the Km was not affected by the acetyl-CoA concentration (50 or 250 microM), whereas the Vmax was significantly increased at a 250 microM acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin (Km = 20.7 microM) than for tryptamine (Km = 48-60 microM); Vmax, however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (greater than or equal to 4 mM) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue. PMID- 1729404 TI - Characterization of proenkephalin-cleaving proteinases in bovine adrenal chromaffin granules using [35S]proenkephalin copolymerized into sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AB - Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin. PMID- 1729405 TI - Specificity of neurotensin metabolism by regional rat brain slices. AB - Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation. PMID- 1729406 TI - Glutamate decarboxylases in nonneural cells of rat testis and oviduct: differential expression of GAD65 and GAD67. AB - gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission. PMID- 1729407 TI - Regulation of aromatic L-amino acid decarboxylase by dopamine receptors in the rat brain. AB - Decarboxylation of phenylalanine by aromatic L-amino acid decarboxylase (AADC) is the rate-limiting step in the synthesis of 2-phenylethylamine (PE), a putative modulator of dopamine transmission. Because neuroleptics increase the rate of accumulation of striatal PE, these studies were performed to determine whether this effect may be mediated by a change in AADC activity. Administration of the D1 antagonist SCH 23390 at doses of 0.01-1 mg/kg significantly increased rat striatal AADC activity in an in vitro assay (by 16-33%). Pimozide, a D2-receptor antagonist, when given at doses of 0.01-3 mg/kg, also increased AADC activity in the rat striatum (by 25-41%). In addition, pimozide at doses of 0.3 and 1 mg/kg increased AADC activity in the nucleus accumbens (by 33% and 45%) and at doses of 0.1, 0.3, and 1 mg/kg increased AADC activity in the olfactory tubercles (by 23%, 30%, and 28%, respectively). Analysis of the enzyme kinetics indicated that the Vmax increased with little change in the Km with L-3,4-dihydroxyphenylalanine as substrate. The AADC activity in the striatum showed a time-dependent response after the administration of SCH 23390 and pimozide: the activity was increased within 30 min and the increases lasted 2-4 h. Inhibition of protein synthesis by cycloheximide (10 mg/kg, 0.5 h) had no effect on the striatal AADC activity or on the increases in striatal AADC activity produced by pimozide or SCH 23390. The results indicate that the increases in AADC activity induced by dopamine-receptor blockers are not due to de novo synthesis of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729408 TI - Aging produces a specific pattern of striatal dopamine loss: implications for the etiology of idiopathic Parkinson's disease. AB - To examine the possible causal contribution of normal or accelerated aging to the neurodegenerative process of Parkinson's disease, we measured the influence of aging on subregional striatal dopamine and homovanillic acid levels in postmortem brain of 23 neurologically and psychiatrically normal human subjects 14-92 years old. We observed a significant decline in striatal dopamine levels and increase in the homovanillic acid/dopamine molar ratios with increasing age. The dopamine loss, on average, was of the same magnitude in the caudate nucleus and the putamen (-60% in the 84-year-old group as compared with the 22-year-old group), with the caudal component of both nuclei being more affected than the rostral subdivisions. The level of subregional dopamine metabolism, as measured by the homovanillic acid/dopamine ratio, in our young individuals (mean age, 22 years) was found to be inversely correlated to the degree of subregional dopamine loss suffered by the individuals in the older age groups. We conclude the following: (a) Striatal subdivisions with physiologically higher dopamine metabolism are not at a greater risk of suffering dopamine neuronal damage with advancing age, as would seem to be implied by the oxidative stress hypothesis; thus, formation of dopamine-derived oxy radicals in the human striatum appears unlikely to be a primary factor responsible for the age-related striatal dopamine loss. (b) The regional and subregional pattern of striatal dopamine loss in normal aging differs substantially from the pattern typically observed in idiopathic Parkinson's disease; therefore, the cause of idiopathic Parkinson's disease cannot be primarily an age-dependent neurodegenerative process. PMID- 1729409 TI - Development and maintenance of the neuronal cytoskeleton in aggregated cell cultures of fetal rat telencephalon and influence of elevated K+ concentrations. AB - Serum-free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development-dependent expression of several cytoskeletal proteins, including the heavy- and medium sized neurofilament subunits (H-NF and M-NF, respectively); brain spectrin; synapsin I; beta-tubulin; and the microtubule-associated proteins (MAPs) 1, 2, and 5 and tau protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular beta-tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M-NF preceded that of H-NF, with the latter being detectable only after approximately 3 weeks in culture. Furthermore, MAP 2 and tau protein showed a development-dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron-enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M-NF, H-NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145-kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron-enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton. PMID- 1729410 TI - Barium and calcium stimulate secretion from digitonin-permeabilized bovine adrenal chromaffin cells by similar pathways. AB - We compared the characteristics of secretion stimulated by EGTA-buffered Ba(2+)- and Ca(2+)-containing solutions in digitonin-permeabilized bovine adrenal chromaffin cells. Half-maximal secretion occurred at approximately 100 microM Ba2+ or 1 microM Ca2+. Ba(2+)-stimulated release was not due to release of sequestered intracellular Ca2+ because at a constant free Ba2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba2+. The maximal extents of Ba(2+)- and Ca(2+)-dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba(2+)-induced secretion to a lesser extent than Ca(2+)-induced secretion. Half-maximal concentrations of Ba2+ and Ca2+, when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba2+ and Ca2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba(2+)- and Ca(2+)-dependent secretion to a similar extent. Ba2+, unlike Ca2+, did not activate polyphosphoinositide-specific phospholipase C. These data indicate that (1) Ba2+ directly stimulates exocytosis, (2) Ba(2+)-induced secretion is stimulated to a lesser extent than Ca(2+)-dependent secretion by MgATP, (3) Ba2+ and Ca2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide-specific phospholipase C. PMID- 1729411 TI - Differential effect of intrastriatal kainic acid on the modulation of dopamine release by mu- and delta-opioid peptides: a microdialysis study. AB - The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid. PMID- 1729412 TI - Monoamines and their precursors and metabolites in the chicken brain, pineal, and retina: regional distribution and day/night variations. AB - Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5 hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4 hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night. PMID- 1729413 TI - Manganese uptake and efflux in cultured rat astrocytes. AB - Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific high-affinity transport system for Mn2+. Saturation kinetics are clearly indicated by both 1/v versus 1/s plots (Km = 0.30 +/- 0.03 microM; Vmax = 0.30 +/ 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2+, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 microM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn(2+)-free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 +/- 2.2% of 54Mn2+ transported extracellularly, and 37.2 +/- 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system. PMID- 1729414 TI - Transcription-dependent and -independent induction of cerebral ornithine decarboxylase. AB - Ornithine decarboxylase (ODC; EC 4.1.1.17) is a highly inducible, rate-limiting enzyme of the polyamine pathway. We have studied the mechanisms that lead to the induction of ODC activity in response to electrical stimulation in three brain regions. Hippocampal ODC activity was found to exhibit much larger elevations than that of the neocortex and the cerebellum. The levels of ODC gene expression were also followed to examine its relationship to the existing regional differences in ODC activity. In the neocortex, there was an elevation of both the ODC mRNA and enzyme activity. However, the hippocampal ODC mRNA level was not increased by electroconvulsive shock. Furthermore, the effects of hormonal changes and seizures on these regional differences in ODC induction were also examined. Adrenalectomy did not affect ODC activity, but pretreatment with the anticonvulsant MK-801 caused a depression of the induced levels of enzyme activity. Our data suggest that ODC activity in all the brain regions studied is directly elevated by electrically stimulated seizures. However, this induced ODC activity may or may not involve enhanced gene expression. PMID- 1729415 TI - Elevated gamma-aminobutyric acid levels attenuate the metabolic response to bilateral ischemia. AB - Bilateral ischemia has been shown to alter the net brain levels of energy metabolites such as ATP, phosphocreatine, glucose, and glycogen. The amino acid neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on neural activity. The present studies were designed to evaluate the influence of elevated GABA levels on the metabolic sequelae of ischemia. The GABA transaminase inhibitor gamma-vinyl-GABA (GVG; vigabatrin) was administered to Mongolian gerbils before the production of a bilateral ischemic incident. GABA levels were elevated in all regions assayed. Levels of energy metabolites were also increased, an indication of reduced energy utilization. In control animals, in the absence of GVG, 1 min of bilateral ischemia produced decreases in the levels of all metabolites. In animals pretreated with GVG, the effects of 1 min of bilateral ischemia were attenuated. These data suggest that the level of ongoing activity may affect the response to an ischemic insult. Furthermore, GVG may have a clinical indication in reducing the effect of minor ischemic incidents. PMID- 1729416 TI - Potentiation of gamma-aminobutyric acid-mediated chloride flux by pentobarbital and diazepam but not ethanol. AB - The influx of 36Cl- into cerebral cortical and cerebellar microsacs from ICR mice and Sprague-Dawley rats was studied in incubations lasting 3 s, 500 ms, or 21 ms. In the 3-s assay, 10-40 mM ethanol did not affect either basal or gamma aminobutyric acid (GABA)-mediated Cl- flux, at any GABA concentration tested. Only at a concentration of 600 mM did ethanol potentiate Cl- flux in both mouse and rat preparations. Ethanol (20 mM) also did not affect the significant potentiation of GABA-mediated flux produced by 50 microM pentobarbital or 2 microM diazepam in ICR mouse microsacs. In 21- and 500-ms incubations (quench flow method), 50 microM pentobarbital significantly potentiated GABA-mediated Cl- flux in rat cortical microsacs, but 10-50 mM ethanol did not. These studies suggest that some as yet unrecognized factor is essential for ethanol enhancement of GABA-mediated Cl- flux, as reported by others in brain homogenates and in tissue culture. PMID- 1729417 TI - Biochemical correlates of epilepsy in the E1 mouse: analysis of glial fibrillary acidic protein and gangliosides. AB - The E1 (epileptic) mouse is considered a model for complex partial seizures in humans. Seizures in E1 mice begin around 7-8 weeks of age and persist throughout life. To determine if astrocytic gliosis was present in adult seizing E1 mice, the distribution of glial fibrillary acidic protein (GFAP) was studied in the hippocampus using an antibody to GFAP. The mean number of GFAP-positive cells per square millimeter of hippocampus was approximately 15- to 40-fold higher in adult E1 mice than in nonseizing control C57BL/6J (B6) mice or in young nonseizing E1 mice. Relative GFAP concentration (expressed per milligram of total tissue protein) in hippocampus and cerebellum was estimated by densitometric scanning of peroxidase-stained western blots. GFAP concentration was 2.7-fold greater in hippocampus of adult seizing E1 mice than in the control B6 mice. No differences in GFAP content were detected between the strains in the cerebellum. Because gangliosides can serve as cell surface markers for changes in neuronal cytoarchitecture, they were analyzed to determine if the gliotic response in E1 mice was associated with changes in neural composition. Although the total ganglioside concentration of hippocampus, cerebral cortex, and cerebellum was similar in adult E1 and control B6 mice, a synaptic membrane enriched ganglioside, GD1a, was elevated in the adult E1 cerebral cortex and hippocampus. The findings indicate that E1 mice express a type of gliosis that is not accompanied by obvious neuronal loss. PMID- 1729418 TI - Evidence for the action of endogenous adenosine in the rabbit retina: modulation of the light-evoked release of acetylcholine. AB - Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent. The cholinergic amacrine cells of the rabbit retina were labeled with [3H]choline, and the effects of enzymatic adenosine degradation or adenosine antagonists on the light-evoked efflux of acetylcholine were evaluated. When endogenous adenosine was degraded by addition of adenosine deaminase, the light-evoked release of radioactivity derived from [3H]choline was significantly increased compared with control values. A similar response was observed when rabbit eyecups were superfused with a selective adenosine A1 receptor antagonist. The effect elicited by adenosine deaminase could be almost completely reversed by addition of cyclopentyladenosine, a highly selective A1 receptor agonist. These effects were observed in either the presence or the absence of picrotoxin. The results demonstrate a modulation of retinal physiology by adenosine. PMID- 1729419 TI - Phorbol esters cause preferential secretion of norepinephrine from bovine chromaffin cells. AB - Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13 dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine-containing cells or epinephrine-containing cells. In response to 55 mM K+, catecholamine release from norepinephrine-containing cells was two times greater than that from epinephrine-containing cells. In response to phorbol 12,13-dibutyrate, secretion from norepinephrine-containing cells was 13 times greater than that from epinephrine-containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine containing cells. PMID- 1729420 TI - Ca(2+)-dependent release from rat brain of cannabinoid receptor binding activity. AB - As a result of the identification, pharmacological characterization, and localization of the cannabinoid receptor in the CNS, the existence of an endogenous ligand for this receptor can be hypothesized. Following the premise that such a substance could have the properties of a neuromodulator being stored in intracellular vesicles, we tested the ability of increased intracellular Ca2+ levels to stimulate release. We demonstrate here that the Ca2+ ionophore A23187 can induce release of cannabinoid-like binding activity in the presence but not in the absence of Ca2+. The effect of A23187 was maximal at 1.2 microM, consistent with vesicular release. It was necessary to increase the concentration of extracellular free Ca2+ to greater than 60 nM to evoke release. The released cannabinoid-like binding activity displaced [3H]CP-55940 binding to cannabinoid receptors in rat synaptosomal membranes in a concentration-dependent manner. This is the first report of a substance present endogenously in brain that can be released in a Ca(2+)-dependent manner and that binds to the cannabinoid receptor. PMID- 1729421 TI - Irreversible inhibition of mitochondrial complex I by 1-methyl-4 phenylpyridinium: evidence for free radical involvement. AB - Incubation of 10 mM 1-methyl-4-phenylpyridinium (MPP+) with sonicated beef heart mitochondria caused an irreversible time-dependent decrease in NADH-ubiquinone-1 (CoQ1) reductase activity (52% inhibition after 1 h). Inclusion of glutathione, ascorbate, or catalase in the incubation mixture protected the NADH-CoQ1 reductase activity. These results suggest that the interaction of MPP+ with complex I induces free radical generation, which in turn leads to the irreversible inhibition of complex I activity. The generation of free radicals by neurotoxin-induced inhibition of complex I has important implications for our interpretation of the increased oxidative stress observed in Parkinson's disease substantia nigra and for our understanding of the cause(s) of dopaminergic cell death in this disorder. PMID- 1729422 TI - Cardiovascular effects of cocaine. AB - This article reviews the chemical nature of cocaine, an increasingly abused stimulant. Following a description of the clinical manifestations of drug use, cardiovascular effects as well as routes of administration are described. Clinical complications of acute and chronic cocaine abuse include dysrhythmias, acute myocardial ischemia and infarction, sudden death, myocarditis, and cardiomyopathy. Clinical manifestations and assessment protocols are summarized. Treatment protocols remain focused on symptom presentation at this time, as there is no known antidote for cocaine toxicity or overdose. PMID- 1729423 TI - Cardiovascular effects of methamphetamine. AB - Stimulant abuse has grown in popularity, particularly since the advent of crack cocaine. Another commonly abused drug is methamphetamine (MAP) hydrochloride. Known as crank, crystal, ice, crystal meth, and speed, MAP can be produced easily from ephedrine, and it is widely available. This article describes the pharmacology, cardiovascular effects, and toxicology of MAP, as well as the management principles of MAP abuse. PMID- 1729424 TI - Cardiovascular implications of anabolic steroid abuse. AB - Anabolic steroids are synthetic derivatives of the hormone testosterone. Anabolic effects such as anticatabolism, increased skeletal muscle mass, and increased aggressiveness are usually desired; however, androgenic effects also result. Decreases in high-density lipoprotein, increases in low-density lipoprotein, changes in total serum cholesterol, hematocrit, and clotting factors, and the development of hypertensive diseases have all been implicated as resulting from anabolic steroid use. Research does not directly link anabolic steroids with cardiovascular diseases, but steroid use can significantly increase risk factors for atherosclerotic cardiovascular disease. PMID- 1729425 TI - Cocaine-induced cardiovascular dysfunction: a case study. AB - This article focuses on the authors' experience with a 25-year-old female patient exhibiting cocaine-induced cardiovascular dysfunction. A care plan is provided that emphasizes the patient's need for assistance with individual coping. PMID- 1729426 TI - Noninvasive diagnosis of cardiovascular disease using ultrasound imaging. AB - Drug abuse causes a variety of cardiovascular complications that can be diagnosed with cardiac ultrasound imaging, including valvular disorders and ventricular dysfunction. The basic principles of cardiac ultrasound imaging, including fundamental engineering principles applicable to the health care environment, are described in this article. The basic modes of imaging--two-dimensional imaging, M mode, Doppler, and color Doppler--are discussed as are applications related to cardiac ultrasound: transesophageal echocardiography and peripheral vascular imaging. The article closes with examples of technologic developments expected to arrive in this decade. PMID- 1729427 TI - Cocaine abuse. AB - The Addiction Research Center at the National Institute on Drug Abuse is conducting a program of research focusing on both the acute and longer term effects of intravenous cocaine. This column reviews and critiques a pair of studies, conducted at the Center, that explore the direct biochemical effects of intravenous cocaine administration. Implications for nursing practice and research are discussed. PMID- 1729428 TI - Changes in family patterns six months after a myocardial infarction. AB - Descriptive data were gathered from 15 families on which changes in the family were reported as a result of a myocardial infarction in one family member. Persons surviving their first myocardial infarction and their families were interviewed individually and simultaneously by one of five trained interviewers in each subject's home 6 months after myocardial infarction. Data were analyzed by reduction, display, and comparison of responses. Categories of changes that emerged from the data were those in family and social activities, emotions and personal life-style habits. PMID- 1729429 TI - Why me? Causal thinking, affect, and expectations in myocardial infarction patients. AB - This study investigated whether patients who seek an explanation for their heart attack, as compared to those who do not, differ in their affect, in expectations about their future recovery, and in expectations about coping with their future. Forty-two myocardial infarction patients were interviewed, in both the acute and convalescent stages, as to whether they had thought about "Why me?" Approximately half of the patients at each stage reported searching for an answer to that question. The patients remained generally consistent in their self-reported anxiety, depression, and hostility over time; however, patients who had not thought about "Why me?" reported less anxiety than those who had. No significant differences were found in affect in patients who gave a specific cause for their heart attack and in those who could not. Patients were significantly less optimistic about their future recovery at follow-up than when they were in the hospital, but there were no differences in expectations for future recovery or for future coping of those who had and those who had not thought about "Why me?" PMID- 1729430 TI - The effectiveness of teaching a relaxation technique to patients undergoing elective cardiac catheterization. AB - The effects of a relaxation technique (RT) on anxiety, vital signs, procedure length, and amount of diazepam given were examined in patients undergoing cardiac catheterization (CC). Forty patients were randomly assigned to an experimental (RT) or a control (no RT) group. No significant differences were found between groups in pre-CC or post-CC State-Trait Anxiety Inventory (STAI) scores, vital signs, or procedure length. The experimental group received significantly less diazepam, and their STAI scores declined significantly. PMID- 1729431 TI - Intracellular analysis in vivo of different barosensitive bulbospinal neurons in the rat rostral ventrolateral medulla. AB - Neurons located in the rostral ventrolateral medulla (RVLM) with projections to the intermediolateral column (IML) in the spinal cord were electrophysiologically characterized and anatomically identified using an intracellular recording technique in vivo. A group of spontaneously active neurons was antidromically activated by electrical stimulation of the IML in the thoracic spinal cord (T2-T3 level). The axonal conduction velocities ranged from 1.5 m/sec to 11.0 m/sec; mean value, 5.5 +/- 2.6 m/sec (+/- SD). The firing pattern and changes in membrane potential in relation to the cardiac cycle were investigated in these bulbospinal neurons. A first group discharged action potentials with higher frequency at the end of the diastolic/beginning of the systolic period. The average of the neuronal membrane potentials demonstrated depolarizing potentials at the end of the diastolic/beginning of the systolic period. These depolarizing potentials increased in magnitude when the neurons were hyperpolarized. Therefore, they were characterized as EPSPs. The baroreceptor reflex activation produced by the increase in systemic arterial pressure following intravenous injection of phenylephrine elicited hyperpolarization, a decrease in the rate of discharge, and an increase in the membrane input resistance, suggesting that a disfacilitatory effect was produced by the activation of baroreceptor inputs on these bulbospinal neurons. Conversely, the inactivation of the baroreceptor reflex by intravenous injection of sodium nitroprusside produced depolarization and an increase in the firing rate. These neurons were characterized as baroreceptor-sensitive type I neurons. A second group of bulbospinal neuron in the RVLM was differentiated from the first group because it demonstrated a decrease in the frequency of discharge at the end of the diastolic/beginning of the systolic period. The average of the membrane potentials showed hyperpolarizing potentials that decreased in magnitude when the neuron was hyperpolarized. These hyperpolarizing potentials occurred at the end of the diastolic/beginning of the systolic period and were reversed in polarity after intracellular injections of chloride ions for several minutes. Therefore, these potentials were characterized as chloride-dependent IPSPs locked to the cardiac cycle. In some of these neurons, the electrical stimulation of the IML produced, in addition to the antidromic action potential, a monosynaptic EPSP with a shorter latency. Based on these unique characteristics, these neurons were defined as barosensitive type II neurons. During constant baroreceptor inactivation achieved by the hypotension produced by intravenous infusions of sodium nitroprusside, the pattern of discharge of barosensitive type II neurons became very regular, and the IPSPs locked to the cardiac cycle were absent.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1729432 TI - Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons. AB - Fibroblast growth factors (FGFs) are known to stimulate mitogenesis in a variety of non-neuronal cell types and to support the survival in vitro of many neuronal cell types. The physiological role of FGFs in the CNS is currently not known. The present study determined the distribution in the rat CNS of a prominent member of the FGF family, basic FGF (bFGF). Immunohistochemical analysis showed that bFGF immunoreactivity was found predominantly in astrocytes throughout all regions of the CNS. In contrast, only a few neuronal populations were found to contain bFGF immunoreactivity, most prominent among them, neurons in the CA2 area of the hippocampus. This predominant localization of bFGF to astrocytes was confirmed by two other observations: (1) highly enriched cultures of astrocytes contained bFGF immunoreactivity and bioactivity, whereas highly enriched cultures of cerebral cortical neurons contained no detectable bFGF, and (2) neonatal rat cerebral cortex, which contains only a few differentiated astrocytes, also contained no detectable bFGF immunoreactivity and only low amounts of bFGF bioactivity. Immunocytochemical analysis also suggested that bFGF immunoreactivity was present in the nucleus as well as the cytoplasm of astrocytes and CA2 neurons. This nuclear localization was confirmed by EM analysis of the intracellular distribution of the immunoperoxidase reaction product. In addition, preparations of both nuclear and soluble fractions of brain extracts contained bFGF immunoreactivity and bioactivity. These data suggest that bFGF might be involved in mediating astrocytic influences on the late postnatal maturation and plasticity in the CNS, and that the nuclear localization of bFGF within astrocytes may play an important role in the differentiation of these cells. In addition, bFGF may play a similar role in a few specific neuronal populations, such as CA2 hippocampal neurons. PMID- 1729433 TI - Plasticity in the barrel cortex of the adult mouse: effects of chronic stimulation upon deoxyglucose uptake in the behaving animal. AB - We investigated experience-dependent regulation of neuronal activity in the whisker-to-barrel pathway of the adult mouse using the autoradiographic deoxyglucose (DG) method. Animals were placed in the Lausanne whisker stimulator, and three of their whisker follicles were passively stimulated for a period of 1, 2, or 4 d. After this period, mice received a dose of DG and were placed in a cage containing a pile of wooden sticks. Mice that underwent the same procedure except the passive stimulation served as controls. Patterns of stimulus-dependent DG uptake were studied in the somatosensory cortex and in the trigeminal sensory brainstem complex. DG uptake in the barrels corresponding to the passively stimulated whiskers was lower than in controls. This decrease was present throughout the radial extent of a barrel column and was observed in all passively stimulated animals. Quantitative analysis confirmed these observations and, furthermore, showed a statistically significant decrease in DG uptake in barrels neighboring the passively stimulated ones. In half of the animals, the brainstem nuclei showed a decreased DG uptake in the representation of the passively stimulated whiskers, whereas in the other animals the pattern of DG uptake was as in controls. We propose that the signs of cortical plasticity are due to a mechanism that operates in layer IV and functions as a gate for peripheral sensory activity to enter cortical circuitry. PMID- 1729434 TI - Binocular interactions in accommodation control: effects of anisometropic stimuli. AB - In binocular viewing of real targets, the accommodative demand in the two eyes is not in general identical, yet the accommodation response in the two eyes is equal. In order to investigate how the accommodative signals from the two eyes are combined, this study has examined the effects of several forms of dynamic anisometropic stimulation on the accommodation response in both man and the rhesus monkey (Macaca mulatta). All experiments were performed in a computer controlled haploscopic apparatus to allow independent control of the accommodative stimuli to the two eyes and of the vergence stimulus. The vergence stimulus was held constant while the accommodation demand was modulated independently in each eye. Accommodation was monitored continuously with a dynamic infrared optometer. Four anisometropic conditions were used. In two of these conditions, accommodation demand was varied sinusoidally with time in both eyes, but with phases differing by 90 degrees or 180 degrees between the two eyes. In the two remaining conditions, accommodation demand in one eye varied sinusoidally, while the accommodation demand was constant in the other. In all cases, the form of the target pattern was identified in the two eyes. The accommodation responses observed with these stimulus conditions were similar in both man and the monkey. When presented with conflicting stimuli in the two eyes, the accommodation response appeared to be best described as a compromise between the inputs to the two eyes; there were no indications of a purely random alternation of eye dominance of the form seen in binocular contour rivalry. When the accommodation demand was modulated in only one eye, there was a modulated accommodation response of similar phase to the control condition (i.e., both eyes modulated in phase) but with a much smaller gain (mean, 39% of control gain). When the accommodation demand was modulated in both eyes with a phase difference of 180 degrees, no significant modulation was observed in the accommodation response at the stimulation frequency. When the interocular phase difference was 90 degrees, a modulated response was observed that showed a mean phase lag 41 degrees more than that observed in the control condition (both eyes modulated in phase) and an appreciably smaller gain (mean, 55% of control gain). The extent to which the results can be described by a linear vector average of the uniocular inputs is considered. PMID- 1729435 TI - Expression of peptidylglycine alpha-amidating monooxygenase (EC 1.14.17.3) in the rat central nervous system. AB - An important step in the posttranslational modification of many bioactive neuropeptides, the carboxy-terminal amidation of glycine-extended peptides, is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3). The expression of the gene encoding this enzyme was examined in adult rat brain by in situ hybridization histochemistry and immunocytochemistry. PAM mRNA transcripts and PAM-like immunoreactivity were detected in all major brain areas with the exception of the cerebellum. Very high levels of PAM mRNAs were found in the hypothalamic magnocellular neurons, the hippocampal formation, and olfactory cortex. These areas also showed strong PAM-like immunoreactivity. Regions known to contain high levels of amidated neuropeptides also expressed high levels of PAM mRNA. The observed heterogeneous PAM mRNA levels may reflect differences in the peptidergic activity of different neuronal systems. Interestingly, all pyramidal neurons of the hippocampus expressed very high levels of PAM mRNA, although no identified amidated peptide matches this distribution completely. Furthermore, PAM was not expressed exclusively in neuronal tissue but was also present in non-neuronal tissue. PAM transcripts could be localized in certain ventricular ependymal cells, with the highest expression in the lateral ventricle. Localization of PAM to non-neuronal cells and neurons not known to produce alpha-amidated peptides suggests that these cells may be producing as yet unidentified amidated neuropeptides. PMID- 1729437 TI - Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain. AB - The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death. PMID- 1729436 TI - Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity-induced expression of Fos immunoreactivity. AB - The bowel is the only organ of the body in which neural reflexes can be elicited in the absence of input from the brain or spinal cord. This activity is mediated by the enteric nervous system (ENS), which contains primary afferent neurons. Experiments were carried out to locate the primary afferent neurons of the ENS. Two types of stimulation were used to activate neurons in the wall of the gut in vitro: exposure of the mucosa to cholera toxin or delivery of pressure to the mucosal surface with puffs of N2 from a micropipette. Neurons that became active in response to these stimuli were identified by demonstrating the intranuclear immunoreactivity of Fos, the product of the c-fos protooncogene. No Fos immunoreactivity could be detected in the absence of stimulation; however, application of cholera toxin and puffs of N2 each induced the appearance of Fos immunoreactivity in neurons in both the submucosal and myenteric plexuses. With either stimulus, the induction of Fos immunoreactivity was antagonized by TTX and therefore depended on neuronal activity. The appearance of Fos immunoreactivity could also be prevented by the 5-HT1P receptor antagonist N-acetyl-5 hydroxytryptophyl-5-hydroxytryptophan amide. In contrast, the stimulus-induced expression of Fos immunoreactivity was inhibited, but not abolished, by hexamethonium, which limited the spread of activation within the submucosal plexus and completely prevented expression of Fos immunoreactivity by myenteric neurons in response to mucosal puffs of N2. FluoroGold was injected into single ganglia of the myenteric plexus in order to identify submucosal neurons with myenteric projections. Submucosal neurons in which Fos immunoreactivity was induced by the stimuli were doubly labeled by FluoroGold. A subset of the submucosal, but not myenteric, neurons that expressed Fos immunoreactivity was doubly labeled by antibodies to calbindin. Submucosal calbindin-immunoreactive neurons were found to contain substance P immunoreactivity and could also be immunostained by anti-idiotypic antibodies that react with 5-HT1P receptors. A subset of dynorphin1-8-immunoreactive submucosal neurons (which are known to costore vasoactive intestinal peptide and to be secretomotor in function) expressed nuclear Fos immunoreactivity in response to cholera toxin, but not puffs of N2. These data suggest that intrinsic primary afferent neurons are located in the submucosal plexus, project to the myenteric plexus, and are activated by 5-HT acting on the 5-HT1P receptor subtype. These neurons are probably cholinergic and costore calbindin and substance P. PMID- 1729438 TI - F3/F11 cell surface molecule expression in the developing mouse cerebellum is polarized at synaptic sites and within granule cells. AB - The distribution of the F3/F11 neuronal cell surface molecule was investigated in the developing and adult mouse cerebellum by immunocytochemistry at the light and electron microscopic levels. F3/F11 was confined to subsets of neuronal types, since the Purkinje cell body and dendritic arborization as well as the stellate cells were not immunoreactive. In the young developing cerebellum, the granule cell axons strongly express F3/F11 as soon as they begin to grow, consistent with a functional role in promoting directional outgrowth of neuronal processes. In 10 d-old and adult cerebella, the granule cell bodies and dendrites were not immunoreactive whereas the parallel fibers, which are the granule cell axons, were labeled including in their presynaptic varicosities. By contrast, dendrites, cell bodies, and axons of Golgi cells were labeled by anti-F3 antibodies. Hence, F3/F11 can either be expressed throughout the cell or be polarized to the axons. This raises the question of how segregation of the glypiated F3/F11 molecule between different subcellular compartments depending on the type of neuron is achieved. F3/F11 was found to be present at three types of synaptic sites, suggesting that it might play a role in the formation and maintenance of synapses. However, in each type of synpase, F3/F11 was present at only the pre- or postsynaptic site, never at both: the parallel fiber varicosities contained F3/F11 whereas the postsynaptic compartment in contact, that is, the Purkinje cell dendritic spines, did not. The granule cell dendrites were unlabeled while the mossy fiber terminals contacting them were immunoreactive, and finally, the Golgi cell dendrites and dendritic spines were labeled while the presynaptic compartment contacting them was not. If F3/F11 functions as an adhesion molecule in vivo as indicated by in vitro assays, F3/F11-mediated adhesion is likely to be heterophilic. PMID- 1729439 TI - NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog scanning mutagenesis. AB - Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity. PMID- 1729440 TI - Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex. AB - We used a thalamocortical slice preparation to record both spike trains and synaptically evoked responses from neurons of mouse barrel cortex. Cells were classified as regular spiking (RS), intrinsically bursting (IB), or fast spiking (FS) according to their temporal firing patterns when injected with current. RS cells were further separated into two subtypes, RS1 and RS2 cells, the latter encountered only in the infragranular layers. Synaptic responses were elicited by focal electrical stimuli in the ventrobasal nucleus of the thalamus (VB) while holding the cells at different membrane potentials. Postsynaptic potentials were classified as excitatory (EPSPs) or inhibitory (IPSPs), and their latencies were measured from the onset of the extracellularly recorded fiber volley in layer IV. EPSPs fell into three groups, according to latency. Those in the early cluster had latencies shorter than 1 msec and were coincident with the postsynaptic layer IV population response; they were considered monosynaptic. A second group, with latencies between 1.3 and 2.5 msec, were coincident with all IPSPs and were classified as disynaptic. The rest had latencies longer than 5 msec and were considered polysynaptic. The synaptic order of a cell was correlated with its laminar position and its electrophysiological class. Specifically, monosynaptic responses were restricted to infragranular RS cells and to FS cells, while disynaptic EPSPs were found in supragranular RS cells and in IB cells. Disynaptic IPSPs were found in both deep and superficial layers; in the deep layers they nearly always followed monosynaptic EPSPs, while in the superficial layers they were mostly found in isolation. We conclude that the intrinsic spiking characteristics of a neuron are an important determinant of its position in the cortical circuit and may have a substantial role in determining its response properties. PMID- 1729441 TI - A distinct type of GD3+, flat astrocyte in rat CNS cultures. AB - We have identified what is apparently a distinct type of astrocyte in primary cultures from several regions of the neonatal rat CNS. These cells express GD3 ganglioside for long periods in vitro, and are GFAP+, but do not express the oligodendrocyte antigens O4 or galactocerebroside (GC). The majority, but not all, are A2B5+. The cells grow in a flat, highly spread morphology with many thin cytoplasmic processes. Gene transfer with a replication-deficient retrovirus combined with immunostaining for astro- and oligodendroglial markers (antibodies to GFAP, GD3 ganglioside, GC, and the A2B5 and O4 antibodies) demonstrated that in the neonatal rat CNS cultures these cells are clonally separate from oligodendrocytes and from the majority of (GD3-) astrocytes. The clonal analysis suggests a distinct progenitor cell and a distinct developmental sequence for these astrocytes. PMID- 1729442 TI - Modulation of la EPSP amplitude: the effects of chronic synaptic inactivity. AB - In this study, we test the hypothesis that monosynaptic connections between la afferents and spinal motoneurons are strengthened by chronic disuse. Impulse activity along the medial gastrocnemius (MG) nerve was blocked for 2 weeks using TTX delivered by an osmotic minipump to a Silastic cuff placed around the nerve. The duration and specificity of this block were confirmed by chronic EMG recordings from several triceps surae muscles. The effect of TTX-induced inactivity of presynaptic elements on EPSP amplitude was distinguished from the effect of treating the postsynaptic target by comparing the results from heteronymous synaptic connections, where only one or the other element was treated. After 2 weeks of synaptic inactivity, the heteronymous EPSPs generated by MG la afferents in lateral gastrocnemius/soleus (LG-Sol) motoneurons were significantly (p less than 0.005) larger than control values (48%). Sample differences in rheobase current and half-afterhyperpolarization, both of which may covary with EPSP amplitude, did not account for the differences between groups. Segregation of the two samples of motoneurons by rheobase current identified the increase as being confined to those LG-Sol cells whose rheobase fell below 10 nA. In addition, EPSPs generated by untreated LG-Sol la afferents in treated MG motoneurons were significantly enhanced (39%, p less than 0.05). Thus, TTX treatment of either presynaptic or postsynaptic elements increases synaptic strength. This increase in monosynaptic EPSP amplitude following TTX induced inactivity may reflect an alteration intrinsic to the la afferent to motoneuron synapse, but influences from extrinsic sources cannot be discounted. PMID- 1729443 TI - Acetylcholine receptors in extrajunctional regions of innervated muscle have a slow degradation rate. AB - Scanning EM autoradiography was used to determine the degradation rate of extrajunctional ACh receptors (AChRs) in innervated sternomastoid muscles of the mouse. We report that in innervated muscles, extrajunctional AChRs have a slow degradation rate (t1/2, approximately 8 d), similar to that seen at the neuromuscular junction. We conclude that slowly degrading AChRs (Rs) need not be localized at the specialized structure of the nerve-muscle junction. Degradation of extrajunctional as well as junctional AChRs may depend primarily on the state of innervation of the muscle. PMID- 1729444 TI - Pathfinding and target selection by developing geniculocortical axons. AB - During development of the mammalian cerebral cortex, thalamic axons must grow into the telencephalon and select appropriate cortical targets. In order to begin to understand the cellular interactions that are important in cortical target selection by thalamic axons, we have examined the morphology of axons from the lateral geniculate nucleus (LGN) as they navigate their way to the primary visual cortex. The morphology of geniculocortical axons was revealed by placing the lipophilic tracer Dil into the LGN of paraformaldehyde-fixed brains from fetal and neonatal cats between embryonic day 26 (E26; gestation is 65 d) and postnatal day 7 (P7). This morphological approach has led to three major observations. (1) As LGN axons grow within the intermediate zone of the telencephalon toward future visual cortex (E30-40), many give off distinct interstitial axon collaterals that penetrate the subplate of nonvisual cortical areas. These collaterals are transient and are not seen postnatally. (2) There is a prolonged period during which LGN axons are restricted to the visual subplate prior to their ingrowth into the cortical plate; the first LGN axons arrive within visual subplate by E36 but are not detected in layer 6 of visual cortex until about E50. (3) Within the visual subplate, LGN axons extend widespread terminal branches. This represents a marked change in their morphology from the simple growth cones present earlier as LGN axons navigate en route to visual cortex. The presence of interstitial collaterals suggests that there may be ongoing interactions between LGN axons and subplate neurons along the entire intracortical route traversed by the axons. From the extensive branching of LGN axons within the visual subplate during the waiting period, it appears that they are not simply "waiting." Rather, LGN axons may participate in dynamic cellular interactions within the subplate long before they contact their ultimate target neurons in layer 4. These observations confirm the existence of a prolonged waiting period in the development of thalamocortical connections and provide important morphological evidence in support of the previous suggestion that interactions between thalamic axons and subplate neurons are necessary for cortical target selection. PMID- 1729445 TI - Electrical activity increases growth cone calcium but fails to inhibit neurite outgrowth from rat sympathetic neurons. AB - Previous studies have shown that the growth of axons from both mouse dorsal root ganglion neurons and Helisoma neurons is arrested when the cells are electrically stimulated (Cohan and Kater, 1986; Fields et al., 1990a). Furthermore, in the case of Helisoma neurons, this arrest has been attributed to a rise in the calcium concentration in the growth cones (Cohan et al., 1987). To test the generality of these results, we examined the response of cultured rat superior cervical ganglion (SCG) neurons to electrical stimulation and changes in cytoplasmic calcium. Suprathreshold electrical stimulation of SCG neurons at 10 Hz by extracellular patch electrodes for periods of up to 1 hr had no measurable effect on their rate of growth. In agreement with previous studies, electrical stimulation was accompanied by a rise in the internal calcium concentration: when measured by the fluorescence of fura-2, growth cone calcium levels rose from about 100 nM to greater than 500 nM and then settled to a plateau value of about 350 nM. Despite this increase, however, growth of SCG neurons' processes continued. Our results show that electrical activity is not a universal signal for neurons to stop growing and that a rise in internal calcium does not always arrest the migration of growth cones. PMID- 1729446 TI - Nurse administrators in job transition: defining the issues. PMID- 1729447 TI - Facility planning. A blueprint for nurse executives. AB - Nurse executives have a critical role to ensure that facility planning will meet the needs of patients and make effective use of nursing personnel. The authors discuss the stages of the facility planning process. A detailed checklist assists nurse executives involved in facility remodeling, expansion, retrofitting, and new construction projects. PMID- 1729448 TI - How do home health nurses spend their time? AB - Of critical importance to the financial viability of any home health agency are its productivity standards for staff. The authors discuss the results of a study that analyzed staff time spent in direct patient care, documentation of that care, travel, and other activities, and how the resulting data influenced agency operations. PMID- 1729449 TI - Designing a marketing plan that works. AB - As healthcare organizations incorporate marketing strategies into daily operations, nurse executives find themselves involved in the process with varying degrees of comfort and success. The authors discuss the nurse executive's role and responsibilities for the success of a marketing plan and describe the importance of recognizing how far marketing has evolved within an organization in order to design a suitable plan successfully. PMID- 1729450 TI - Clinical research in home care. A report of affiliates of the Visiting Nurse Associations of America. AB - Home care providers rarely initiate research because of limited resources and the perception that research belongs to academic institutions. A survey of the affiliates of the Visiting Nurse Associations of America identified the types of research activities performed and the interest level of nurses in participating in research activities. PMID- 1729451 TI - Developing a nurse recruitment plan. AB - Your hospital's nurse staffing needs will be more effectively served if recruiters develop and implement a formal nurse recruitment plan for each fiscal year. Such an annual planning process proactively manages nurse recruitment by rational anticipation. It systematically determines current nursing needs, predicts future needs, establishes recruitment objectives, identifies matching strategies for realizing these objectives, executes the plan, and evaluates its success. PMID- 1729452 TI - Cost-effective management of the hospital-based hospice program. AB - As hospital-based hospice programs proliferate across the country, most are under the leadership of a nurse administrator. Nurse administrators must be prepared to manage the many components that constitute the broad scope of this role. Cost effective management is the greatest challenge. The author explores this management role, including a discussion of hospice-program reimbursement, hospital-based program advantages, options to increase staff productivity, management of drugs and durable medical equipment, inpatient admissions, volunteer services, and fund-raising. Cost-effective measures are explored throughout the discussion, along with a history and explanation of the hospice concept of care. PMID- 1729453 TI - The career development internship program. Pathway to enhanced nursing practice. AB - Nurse administrators can enhance nursing practice and improve patient care by providing clinical nurses with ways to develop and share their expertise in areas such as education, management, quality assurance, advanced clinical practice, and research. The authors describe the Career Development Internship Program that grew out of the Management Internship Program. This creative program allows for professional growth in specific fields while also developing leadership capability. PMID- 1729454 TI - Administrator bashing. PMID- 1729455 TI - Rural nursing research priorities. AB - The magnitude and variety of clinical concerns in rural healthcare delivery mandate a focused plan for building a substantiated body of knowledge. The author identifies research priority areas to develop a body of knowledge for rural nursing. PMID- 1729456 TI - Marketing nursing beyond the walls. AB - Nurse self-actualization and service to the community are two key concepts that nurse administrators must address in developing the vision for the future direction of the nursing department. A marketing venture at a university teaching hospital demonstrated a cost-effective mechanism to meet these goals. PMID- 1729457 TI - Depressed immune response to tetanus in children with vitamin A deficiency. AB - A randomized, double-masked, placebo-controlled clinical trial was conducted with 236 preschool children, age 3-6 y, in Indonesia to assess immune status in mild vitamin A deficiency. The immune response to tetanus immunization was used as a measure of immune competence. Clinically normal children (n = 118) and children with mild xerophthalmia (n = 118) were randomly assigned to receive oral vitamin A (60,000 micrograms retinol equivalent) or placebo treatment for a total of four study groups. Two weeks after treatment, children were immunized with diphtheria pertussis-tetanus vaccine. The immunoglobulin G (IgG) responses to tetanus at baseline and 3 wk following immunization were measured by ELISA. After adjusting for previous tetanus immunization, clinically normal and xerophthalmic children receiving vitamin A had a significantly greater IgG response to tetanus than clinically normal and xerophthalmic children receiving placebo (P less than 0.05). These results suggest that children with mild vitamin A deficiency have a relative immune depression compared with children who have been supplemented to normal vitamin A levels. PMID- 1729458 TI - Dietary calcium content, calcium balance and mode of uptake in a subterranean mammal, the Damara mole-rat. AB - Calcium flux and mode of uptake were investigated in an underground dwelling mole rat, Cryptomys damarensis, fed diets of varying Ca content. The amount of dietary Ca positively influenced the amounts ingested, absorbed and retained. The linear relationship between ingested and absorbed Ca was significantly (P less than 0.001) correlated, implying that this process is nonsaturable. When mole-rats were fed a diet low in Ca, apparent fractional absorption of Ca was high (85.88%). This increased still further when the diet was changed to a food of greater Ca content (96.13%, carrots; 96.97%, gemsbok cucumber). Mineral homeostasis is regulated at the intestinal level in most mammals. Regardless of dietary Ca content, uptake of 45Ca (examined via the everted gut sac technique) was passive, confirming that absorption is via a nonsaturable process. Plasma Ca concentrations were not tightly regulated, yet when fed the diet with the highest Ca content, mole-rats were not hypercalcemic. Regardless of diets, Ca apparent fractional retention was positive, and approached physiological maxima (greater than 97%). Cryptomys damarensis, in using highly efficient modes of mineral uptake and retention, is therefore capable of fully exploiting the limited food resources of their arid ecotope. PMID- 1729459 TI - Beta-carotene uptake and tissue distribution in ferrets (Mustela putorius furo). AB - Ferrets accumulate beta-carotene in liver and adipose tissue after chronic feeding. This study was designed to further evaluate the time course of uptake and depletion of beta-carotene in ferret serum and tissues. Male ferrets (n = 15; 1000-1200 g) were given a single dose of beta-carotene (10 mg/kg body wt) with a meal. Animals were killed at various time points over an 11-d period. Blood and tissue samples were extracted and analyzed for beta-carotene by HPLC. Peak serum beta-carotene levels (0.68 +/- 0.18 mumol/L) were observed 8 h after the test meal. beta-Carotene was essentially cleared from the blood by 76 h. Peak beta carotene concentrations (nmol/g) were observed between 8 and 16 h after ingestion for liver (1.20 +/- 0.04), lung (0.042 +/- 0.012), kidney (0.090 +/- 0.015) and spleen (0.076 +/- 0.012). Ferret liver also seemed to contain a variety of other polar and nonpolar carotenoids. Ferrets were shown to absorb beta-carotene from a meal and have a consistent serum response pattern. Absorbed beta-carotene is differentially distributed in a variety of tissues. The ferret seems to be a useful model for the study of beta-carotene absorption and metabolism. PMID- 1729460 TI - Postprandial lipoprotein composition in pigs fed diets differing in type and amount of dietary fat. AB - To determine the effects of diet on postprandial lipoprotein composition, growing pigs were fed diets containing 20 or 40% of energy as soybean oil, tallow or a 50:50 blend of soybean oil and tallow. At the end of wk 6, a blood sample was drawn from pigs fasted for 12 h. Pigs were then fed, and blood samples were drawn 1 and 4 h later. In LDL, concentrations of free and total cholesterol were greater in pigs fed 40% of energy as fat than in pigs fed 20% of energy as fat (P less than 0.02). Pigs fasted for 12 h had lesser concentrations of triacylglycerol and greater concentrations of phospholipid in LDL and HDL than did pigs fasted for 1 and 4 h (P less than 0.05). In HDL, total cholesterol and phospholipid concentrations were greater in pigs fed 40% of energy as fat than in pigs fed 20% of energy as fat (P less than 0.01). A greater concentration of triacylglycerol was found in VLDL of pigs fed 40% of energy as fat than in pigs fed 20% of energy as fat (P less than 0.01). Amount of dietary fat had a greater effect than did type of dietary fat on composition of lipoproteins from postprandial pigs. PMID- 1729461 TI - Response of somatomedins (IGF-I and IGF-II) in lactating cows to variations in dietary energy and protein and treatment with recombinant n-methionyl bovine somatotropin. AB - Mid-lactation Holstein cows (n = 4) were assigned to four dietary sequences in a 4 x 4 Latin square to determine energy and protein effects on somatomedins. Diets were designed so that intakes were either high or low for net energy (NE) or crude protein (CP) with the range being representative for a lactation cycle. Each dietary treatment lasted 16 d and consisted of an adjustment period (d 1 to 7), a basal period (d 8 to 12) and a period of bovine somatotropin (bST) (40 mg/d) administration (d 13 to 16). Blood was obtained via jugular catheters every 4 h on d 11 to 16. Basal milk yield was decreased by NE or CP restriction. Milk yield was increased for cows fed all diets with bST, but response was greatest for those fed the high NE/high CP diet (31%, 7.7 kg/d). Plasma insulin-like growth factor (IGF)-I and IGF-II concentrations were not affected by diet. For all diets, bST caused an increase in plasma IGF-I (125%) and IGF-II (21%), with the increase being substantially greater for cows fed the high NE/high CP diet. Basal insulin levels differed among diets and increased with exogenous bST in cows fed the high NE/high CP diet. Results are consistent with a role of somatomedins in the mechanism by which exogenous bST increases milk yield, and variations in somatomedin response due to nutritional status may explain part of the differences in milk yield response to exogenous bST. PMID- 1729462 TI - Energy requirements for space flight. AB - Both the United States and the Soviet Union perform human space research. This paper reviews data available on energy metabolism in the microgravity of space flight. The level of energy utilization in space seems to be similar to that on Earth, as does energy availability. However, despite adequate intake of energy and protein and in-flight exercise, lean body mass was catabolized, as indicated by negative nitrogen balance. Metabolic studies during simulated microgravity (bed rest) and true microgravity in flight have shown changes in blood glucose, fatty acids and insulin concentrations, suggesting that energy metabolism may be altered during space flight. Future research should focus on the interactions of lean body mass, diet and exercise in space, and their roles in energy metabolism during space flight. PMID- 1729463 TI - Bone composition and histology of young growing rats fed diets of varied calcium bioavailability: spinach, nonfat dry milk, or calcium carbonate added to casein. AB - Bone composition and histology were evaluated in young growing rats fed nutritionally complete but calcium-restricted (0.15%) diets in which calcium was derived from spinach, nonfat dry milk (NFDM), or CaCO3 added to casein. Groups of male weanling rats were pair-fed for 28 d. A 0.5% calcium casein-based diet group fed ad libitum was included to provide a comparison of normal bone structure and composition. Bone growth and bone ash were depressed in spinach-fed rats. Total bone tibia calcium in 0.5% calcium casein-based, 0.15% calcium casein-based, NFDM and spinach diet groups were 64.0, 29.2, 30.7 and 13.8 mg, respectively. All other measured bone mineral levels were also lower, except for potassium. Femur hydroxyproline concentrations were 1.2, 1.6, 1.6 and 2.1% in 0.5% calcium casein based, 0.15% calcium caseinbased, NFDM and spinach diet groups, respectively. Bone histomorphometry indicated gross underdevelopment and compromised mineralization of trabecular bone of spinach-fed rats. For the first time, it has been demonstrated with histologic techniques that calcium from the low bioavailable source, spinach, compromises both the quantity and quality of bone. In contrast, when calcium is fed to growing animals at levels below the National Research Council requirement but from a highly bioavailable source (i.e., NFDM and CaCO3), there is only a reduction in bone quantity. PMID- 1729464 TI - Free fatty acids in exercising Arabian horses fed two common diets. AB - Four Arabian geldings were used in a randomized, repeated measure design to study the effect of two different diets on plasma free fatty acids at rest and during exercise. On each of four sampling days, two horses were fed one of two isoenergetic diets, either 100% corn or 100% alfalfa, at 22% of their estimated daily energy requirement. Two hours after the consumption of the diet, each horse participated in a submaximal standard exercise test consisting of three consecutive 10-min runs of increasing intensity at heart rates of 132, 140 and 147 beats/min, respectively. There were no significant (P greater than 0.05) differences between the two groups (corn-fed vs. alfalfa-fed) in pre-meal (0.554 +/- 0.031 vs. 0.629 +/- 0.033 mmol/L), post-meal (0.520 +/- 0.027 vs. 0.609 +/- 0.041 mmol/L), and basal (0.392 +/- 0.036 vs. 0.401 +/- 0.052 mmol/L) free fatty acid concentrations. However, free fatty acids were significantly higher in the horses fed alfalfa (0.608 +/- 0.038 mmol/L) rather than corn (0.484 +/- 0.031 mmol/L) during exercise. PMID- 1729465 TI - Determining optimal heat treatment of soybeans by measuring available lysine chemically and biologically with rats to maximize protein utilization by ruminants. AB - Soybeans were heated in a forced air oven at 120 and 130 degrees C for 60 and 180 min, and at 140, 150 and 160 degrees C for 10, 30, 60, 90 and 120 min. Two types of measurements were used to determine optimal heat treatment of soybeans to maximize protein utilization by ruminants. One was to estimate the rate and extent of protein degradation in the rumen using an in vitro ruminal system. The second was to determine the nutritional availability of lysine. Methods used to determine available lysine were an indirect fluorodinitro-benzene chemical method and a rat growth assay. The product of undegraded intake protein and available lysine content was used to estimate the amount of lysine that would escape ruminal degradation and be available for intestinal absorption. As heat input increased, ruminal undegraded intake protein increased, and protein degradation rates and total and available lysine decreased. As temperature increased, the time required to maximize post-ruminal available lysine decreased. The optimal heat treatment for soybeans heated in a forced air oven was: 140 degrees C for 120 min or greater, 150 degrees C for 60 min or 160 degrees C for 30 min. A loss of 15-22% of chemically determined available lysine was necessary to achieve the heat treatment that resulted in maximal post-ruminal available lysine. PMID- 1729466 TI - Dietary protein, as egg albumen: effects on bone composition, zinc bioavailability and zinc requirements of rats, assessed by a modified broken-line model. AB - The effect of dietary protein concentration on zinc bioavailability, requirements and incorporation into bones was investigated in growing rats. Zinc requirements were determined by the broken-line method. Protein did not affect either absorption or biological half-life of 65Zn added to the diet. Zinc requirements based on weight gain or tibia zinc were generally greater when rats were fed 30% rather than 15% egg white. When fed 30% rather than 15% egg white, zinc-deficient rats gained less weight and tended to incorporate less zinc into bone, whereas zinc-adequate rats gained weight similarly and incorporated more zinc into bone. Dietary protein concentration apparently elevated the maximum amount of zinc incorporated into bones of rats fed adequate zinc. When dietary zinc was adequate (25 mg/kg diet), tibia zinc concentrations increased linearly with dietary concentrations of 15, 25, 35 and 45% egg white. When dietary zinc was adequate, higher protein diets resulted in lower tibia nitrogen, and higher tibia zinc, without substantial changes in tibia size or calcium concentration. These results indicate that high protein diets increase zinc requirements and bone zinc deposition, the latter being a consequence of altered bone zinc metabolism, rather than improved zinc bioavailability. PMID- 1729467 TI - Starch digestion: understanding and potential for improvement-introduction. PMID- 1729468 TI - Starch digestion and absorption in nonruminants. AB - Starch digestion and absorption is augmented appreciably by physical processing of grain or legume and by heating to 100 degrees C for several minutes before its ingestion. Starch, a polysaccharide composed of alpha 1,4-linked glucose units (amylose) and alpha 1,4-1,6-linked branched structure (amylopectin), is cleaved in the duodenal cavity by secreted pancreatic alpha-amylase to a disaccharide (maltose), trisaccharide (maltotriose), and branched alpha-dextrins. These final oligosaccharides are hydrolyzed efficiently by complimentary action of three integral brush border enzymes at the intestinal surface: glucoamylase (maltase glucoamylase, amyloglucosidase), sucrase (maltase-sucrase) and alpha-dextrinase (isomaltase). The final monosaccharide glucose product is then cotransported into the enterocyte along with Na+ by a specific brush border 75-kDa transport protein in the rate-limiting step for overall starch assimilation. By virtue of this sequential luminal and membrane digestion followed by glucose transport, starch is assimilated in a very efficient manner in nonruminants. PMID- 1729469 TI - Thiazolidine-4-carboxylate and 2-phenylthiazolidine-4-carboxylate are active as cysteine precursors but have no effect on growth of a methionine-dependent tumor in rats. AB - Diets with partial replacement of sulfur amino acids by thiazolidine-4 carboxylate or 2-phenylthiazolidine-4-carboxylate were fed to normal and to rhabdomyosarcoma-bearing rats (methionine-dependent tumor) to evaluate their efficacy as cysteine precursors and as antitumor agents. Food intake, weight gain, food efficiency and plasma albumin and plasma sulfur amino acid concentrations were not different when these diets were compared with isosulfurous diets containing either methionine or N-acetylcysteine. 2 Phenylthiazolidine-4-carboxylate induced a lower plasma glutathione (GSH) level than the latter diets. Tumor-bearing rats had lower plasma GSH concentration. A negative linear relationship was found between plasma GSH levels and tumor weight and also the tumor weight: body weight ratio. This could mean that the tumor becomes the most important organ in the uptake of GSH. However, there was also a significant positive correlation between plasma GSH and albumin, suggesting a reduced GSH hepatic synthesis due to amino acid uptake by the tumor. There were no differences in tumor growth among rats receiving diets containing N acetylcysteine, thiazolidine-4-carboxylate or 2-phenylthiazolidine-4-carboxylate. PMID- 1729470 TI - Regulation of pancreatic exocrine secretion in ruminants: a review. AB - Mechanisms regulating ruminant pancreatic exocrine function differ in some respects from those in nonruminants. This may affect the post-ruminal digestion of certain dietary nutrients such as starch. Ruminants do not exhibit clearly defined cephalic and gastric phases of pancreatic regulation, a likely consequence of the continuous nature of digesta flow from the rumen. Local neural reflexes and secretin-mediated exocrine responses may be more important than stimulation by cholecystokinin. Additionally, the ruminant pancreas may be stimulated by short-chain fatty acids produced in the rumen. A "ruminal phase" of pancreatic exocrine regulation has been proposed. The failure of cattle to digest efficiently starch in the small intestine may result from an asynchrony between delivery of starch to the intestines and pancreatic amylase release. PMID- 1729471 TI - Assessing the potential dietary impact of replacing dietary fat with other macronutrients. PMID- 1729472 TI - Iron deficiency alters DMBA-induced tumor burden and natural killer cell cytotoxicity in rats. AB - Natural killer (NK) cell activity is impaired in iron-deficient rats. Natural killer cells destroy tumor cells; therefore, iron-deficient rats may be less able to combat cancer growth. Natural killer cell cytotoxicity, both basal and interferon gamma (IFN gamma)-stimulated, was studied in moderately and severely iron-deficient rats challenged with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Female weanling rats were fed ad libitum semipurified diets containing 8, 13 or 42 mg Fe/kg. A pair-fed group was fed the 42 mg Fe/kg diet at the level consumed by the 8 mg Fe/kg group. Following 6 wk of dietary treatment, DMBA treated rats received a single intragastric dose of DMBA. Dietary treatment was continued. Rats were killed at 1, 4, 8, 14 and 20 wk post-DMBA treatment. Natural killer cell cytotoxicity (both basal and IFN gamma-stimulated) was analyzed. Feeding the 13 mg Fe/kg diet resulted in lower NK cell activity (P = 0.006) and greater tumor burden (P = 0.045) and tumor incidence. Interferon gamma treatment relieved the lower NK cell cytotoxicity observed in moderate iron deficiency. Feeding the 8 mg Fe/kg diet impaired NK cell activity (P = 0.006), but tumor burden and incidence were less than in moderate iron deficiency. In this model, iron deficiency, particularly moderate iron deficiency, contributed to cancer development and compromised NK cell cytotoxicity. PMID- 1729473 TI - Low copper status of rats affects polyunsaturated fatty acid composition of brain phospholipids, unrelated to neuropathology. AB - To determine the molecular basis of the neuropathology of copper deficiency, brain lipid composition was determined in second-generation copper-deficient rats. Non-parous female rats were fed a low copper diet (0.5 mg Cu/kg) or a control diet (10 mg Cu/kg) during gestation and lactation, and the offspring were continued on the respective diets to approximately 8 wk of age. In one experiment the source of carbohydrate was 15% starch and 52.6% sucrose, and in another experiment sucrose and glucose were compared in a factorial design. The source of carbohydrate had no effect on copper status. Myelin concentration was estimated by isolation, by 2',3'-cyclic nucleotide phosphodiesterase activity, and by concentration of 20:1 (n-9). Fatty acid compositions of the phospholipid fractions of brain parts and of myelin were determined. Copper deficiency reduced myelin concentration slightly in the midbrain region and increased the proportion of linoleic acid [18:2(n-6)] in both brain and isolated myelin. In the cerebellum the long-chain polyunsaturated fatty acids, particularly 22:6(n-3), were lower. Approximately one-half of the copper-deficient rats developed Parkinson-like signs and had low striatal dopamine, but neither myelin content nor brain fatty acid composition was associated with the neuropathology. PMID- 1729474 TI - Potentiation of acute acetaminophen lethality by selenium and vitamin E deficiency in mice. AB - The effects of selenium, vitamin E, and DL-methionine deficiency on the acute lethality and hepatotoxicity of acetaminophen in male CD-1 mice were studied. Vitamin E and selenium deficiencies led to an increase in the acute lethality of acetaminophen, with a decrease in the LD50 from 376 to 84 mg/kg. These dietary deficiencies impaired the inducibility of the hepatic microsomal mixed function oxidase system by phenobarbital, but on the basis of the covalent binding of acetaminophen to microsomes, these treatments did not alter the activation of acetaminophen to a reactive intermediate by this system. Addition of methionine to the deficient diet restored hepatic glutathione content to control levels but did little to protect against the acute lethality of acetaminophen. In methionine supplemented animals, the addition of either selenium or vitamin E increased the LD50 of acetaminophen to 167 and 200 mg/kg, respectively. Administration of a sublethal, toxic dose of acetaminophen (LD30) to the methionine-supplemented and selenium- and vitamin E-deficient mice did not produce any hepatic damage as evidenced by a lack of plasma aminotransferase elevation. In view of the known antioxidant effects of vitamin E and selenium, these data suggest the involvement of a reactive radical in the acute lethality of acetaminophen and further suggest that death from acute acetaminophen overdose in chronic selenium- and vitamin E deficient mice may be unrelated to liver necrosis. PMID- 1729475 TI - High dietary taurine effects on feline tissue taurine concentrations and reproductive performance. AB - The reproductive performance and outcome of kittens was determined for female cats fed 0.05, 0.2 or 1% taurine. No adverse effects of high taurine diets were noted in the adults or offspring, and the reproductive performance was slightly better than that of females fed the normal (0.05% taurine) diet. Body weight at birth and brain weight at weaning were significantly greater in the very high taurine group than in the normal taurine group, although the greatest growth rate was achieved by the normal taurine group. The concentration of taurine in milk of lactating females was substantially higher in cats fed the higher taurine diets. Brain of adult cats was resistant to increases in brain taurine concentrations, as was brain of newborn cats. However, brain of juvenile cats responded to higher dietary taurine intake with increased taurine concentrations. These results indicate that the higher taurine content in cat foods recently introduced for prevention of feline dilated cardiomyopathy should have no adverse effects over a prolonged period on health and reproduction of cats. PMID- 1729476 TI - Cysteine-rich intestinal protein and intestinal metallothionein: an inverse relationship as a conceptual model for zinc absorption in rats. AB - Dietary zinc may regulate zinc absorption in part via the inhibitory effect of intestinal metallothionein, but the mechanism is unknown. We recently showed that cysteine-rich intestinal protein (CRIP) binds zinc during transmucosal zinc transport, and that CRIP may function as an intracellular zinc carrier. The present experiments examine the interaction of CRIP and metallothionein with zinc to evaluate their potential roles in the mechanism of zinc absorption. Intestinal metallothionein concentrations were lower and zinc absorption rates from isolated intestinal loops were higher in rats fed a low zinc diet compared with those fed a high zinc diet or given parenteral zinc to induce metallothionein synthesis. Zinc status did not affect the apparent CRIP concentration, but markedly altered the distribution of 65Zn in intestinal cytosol as determined by gel filtration HPLC. More 65Zn was associated with CRIP (40 vs. 14%) and less was bound to metallothionein (4 vs. 52-59%) in rats fed the low zinc diet compared with rats of high zinc status. Luminal zinc concentration also affected the distribution of 65Zn in the cytosol. CRIP bound progressively less (from 42 to 25%) of the 65Zn taken up from the lumen as the luminal zinc concentration was increased from 5 to 300 mumol/L. Collectively these data suggest that CRIP is a saturable, intracellular zinc transport protein, and that metallothionein inhibits zinc absorption by binding zinc in competition with CRIP. A hypothetical model for the mechanism of transcellular zinc absorption involving metallothionein and CRIP is presented and discussed. PMID- 1729477 TI - Plasma carotenoid levels in human subjects fed a low carotenoid diet. AB - The purpose of this study was to investigate the effect of a low carotenoid diet on plasma carotenoid levels in humans. Twelve healthy male subjects were fed a low carotenoid diet under controlled conditions for 13 wk in a live-in metabolic unit, as part of a study of vitamin C requirement. Plasma carotenoids (zeaxanthin/lutein, cryptoxanthin, lycopene, alpha-carotene, beta-carotene) were measured with HPLC on study days 2-3, 14-15, 35-36 and 63-64. The rate of decline was rapid between d 2-3 and d 14-15, when the concentration of each carotenoid decreased significantly (P less than 0.05). Although accurate figures for half life are not possible without more frequent sampling points, mean plasma depletion half-life seemed to be less than 12 d for beta-carotene, alpha-carotene and cryptoxanthin, between 12 and 33 d for lycopene and between 33 and 61 d for zeaxanthin/lutein. Because the decline was not linear over the study period, these data suggest the possibility of at least two body pools of these compounds, with one pool having a more rapid turnover rate. Because there is a significant decline in plasma carotenoid levels within the first 2 wk of a low carotenoid diet, determination of levels of these compounds may be useful only in the assessment of short-term intake. PMID- 1729478 TI - Is legislated killing coming? PMID- 1729479 TI - Supporting Minnie's choice. PMID- 1729480 TI - Anna: fully alive in the face of death. PMID- 1729481 TI - Fear in the Gulf: an Israeli nurse sees God at work. PMID- 1729482 TI - Knowing where to stand. PMID- 1729483 TI - A miracle of sight. PMID- 1729484 TI - Living wills: down the slippery slope? PMID- 1729485 TI - Can people be vegetables? PMID- 1729486 TI - Living wills: down the slippery slope? Help the dying to live. PMID- 1729487 TI - Living wills: down the slippery slope? Not all help is helpful. PMID- 1729488 TI - Living wills: down the slippery slope? Understand your right to make choices. PMID- 1729489 TI - Living wills: down the slippery slope? I had to quit to fight. PMID- 1729490 TI - Extra-adrenal pheochromocytoma. AB - Extra-adrenal pheochromocytomas may arise in any portion of the paraganglion system, although they most commonly occur below the diaphragm. The most common site of occurrence of extra-adrenal pheochromocytoma is the superior para-aortic region between the diaphragm and lower renal poles. Although the traditional teaching has been that 10% of all pheochromocytomas are at extra-adrenal sites, this may be an underestimation. Extra-adrenal pheochromocytomas probably represent at least 15% of adult and 30% of childhood pheochromocytomas. They may be malignant in up to 40% of the cases, although conflicting data add to the uncertainty of this point. Patients with tumors arising at extra-adrenal sites commonly present with headache, palpitations, sweating and hypertension. The diagnosis is most often confirmed by demonstrating increased catecholamine production, usually by measurement of urinary catecholamines and/or their metabolites. CT scanning is presently the imaging procedure of choice for localization. The roles of MRI and 131I-MIBG scintigraphy in the localization process are still being determined. Thorough preoperative pharmacological preparation, attentive intraoperative monitoring and aggressive surgical therapy all have an important role in achieving the safest and most successful outcome. Complete surgical excision is the treatment of choice for primary extra-adrenal pheochromocytoma as well as recurrent or metastatic disease. When residual tumor cannot be resected, medical therapy for symptomatic relief is preferred, since radiotherapy and chemotherapy have limited effectiveness. Extra-adrenal pheochromocytomas are more likely to recur and to metastasize than their adrenal counterparts, making lifelong followup with annual determinations of catecholamine production essential. PMID- 1729491 TI - Bladder inhibition by penile nerve stimulation in spinal cord injury patients. AB - Detrusor hyperreflexia causing voiding dysfunction in spinal cord injury patients is a difficult problem and is not always treated effectively by anticholinergic agents. We have been investigating electrical stimulation methods to inhibit hyperreflexia and dorsal penile nerve stimulation is the most promising. Six chronic suprasacral spinal cord injury men (average age 36 years) underwent stimulation testing with water cystometry before, during and after stimulation. Dorsal penile stimulation was done with carbon rubber butterfly electrodes (Medtronic) with parameters of 5 pulses per second, 0.35 msec. pulse duration, and current at a level above the threshold for pelvic twitching activity and adjusted for optimal bladder effect (range 25 to 70 mamp.). In all 6 patients the cystometrogram during stimulation showed an increase in bladder volume over the prestimulation cystometrogram (range 27 to 150%). In 2 patients there was no detrusor activity after filling to 500 cc. Stimulation was then stopped and a spontaneous contraction occurred. The cystometrogram conducted after the stimulus also had less volume than that performed during stimulation but it was larger than the prestimulation volume. Penile nerve stimulation was painless with no side effects. Penile nerve electrical stimulation is effective for inhibiting bladder hyperreflexia and should be easily adaptable for chronic home use as an alternative to current therapy. PMID- 1729492 TI - Adrenocortical carcinoma in a child with specific pedigree of family associated with cancer aggregation. AB - We report on a 5-year-old boy with functioning adrenocortical carcinoma as a proband of a specific pedigree with several young family members who had cancer. Most of the members who died of cancer had early onset of osteosarcoma, hepatoblastoma or malignant lymphoma. The finding of cancer aggregation in the family corresponded to the criteria for the cancer family syndrome. PMID- 1729493 TI - Chordee without hypospadias: experience with 33 cases. AB - We treated 33 patients 6 months to 19 years old with penile chordee without hypospadias from 1966 to 1990. In all cases the penile shaft was degloved, the urethra was widely mobilized and chordee was resected. The shaft skin then was closed, usually using Byars' flaps to shift some preputial skin ventrally. This method sufficed to straighten the penis in 10 patients. In 3 patients urethral mobilization plus placement of a dermal graft to the shaft accomplished straightening of the penis. A total of 20 patients also required lengthening of the penile urethra. Lengthening was done with a graft taken from the prepuce in 14 patients, bladder in 2 and arm in 4. Four of those patients also required a dermal graft to the shaft. There were 8 complications in 7 patients, including anastomotic stenosis, ballooning of a graft, balanitis xerotica obliterans in a graft, urethral fistula and persistent chordee. Reoperation corrected each complication. All end results were satisfactory. PMID- 1729494 TI - Deoxyribonucleic acid profile and tumor progression in primary carcinoma in situ of the bladder: a study of 63 patients with grade 3 lesions. AB - In 63 patients with primary grade 3 carcinoma in situ of the bladder flow cytometric deoxyribonucleic acid (DNA) analysis was performed at diagnosis and during an average followup of 63 months. The results of DNA measurements were related to disease progression, that is invasive tumor and/or metastatic disease. The DNA histograms were classified as diploid (2 patients) or aneuploid (61). A total of 3 categories of aneuploid tumors with different prognostic significance could be defined: 1) carcinoma in situ with 1 aneuploid cell population at diagnosis and with no change to multiple aneuploid cell populations throughout observation, 2) carcinoma in situ with 1 aneuploid cell population at diagnosis but with a later change to multiple aneuploid cell populations and 3) carcinoma in situ with multiple aneuploid cell populations already at diagnosis. At 5 years the progression-free survival for the 3 categories was 94%, 43% and 20%, respectively. Over-all, of the patients with multiple aneuploid cell populations (categories 2 and 3) 76% had progression, in contrast to 19% of those in category 1 (p less than 0.0005). In category 2 development of multiple aneuploid cell populations preceded progression in 8 of 11 progressive cases by an average of 20 months. Therefore, the occurrence of multiple aneuploid cell populations must be considered as a sign of high aggressiveness. We conclude that flow cytometric DNA analysis is a potent predictor of prognosis in cases of primary carcinoma in situ of the bladder. PMID- 1729495 TI - Cavernous hemangioma of the adrenal gland. AB - Cavernous hemangiomas of the adrenal gland are rare. We report on a patient with a left adrenal hemangioma successfully treated by radical excision. The literature pertaining to this interesting pathological condition is reviewed. PMID- 1729496 TI - A strategy of cystine stone management. AB - Simple mechanical disintegration of cystine calculi by extracorporeal shock wave lithotripsy and/or percutaneous nephrolithotomy is being performed widely around the world. However, it cannot be denied that the cystine calculus is one of the most difficult stones for mechanical disintegration. We previously reported the oral medical chemolysis of cystine calculi in 1982 and a third of the patients became stone-free. In another third of the patients the cystine component was replaced by apatite during the medical treatment and apatite stones formed in this manner are easily disintegrated. In view of the complications of mechanical disintegration, oral medical treatment for chemolysis should be recommended before simple destruction of cystine calculi. PMID- 1729497 TI - Bilateral renal parenchymal malacoplakia: a case report. AB - Histological examination of the radical nephrectomy and renal biopsy specimens in a 49-year-old woman revealed bilateral renal malacoplakia. The literature reports bilateral renal malacoplakia to be uniformly fatal. The preoperative diagnosis is based upon patient presentation and imaging studies. Insights into the bacterial etiology of this systemic disease are presented. PMID- 1729498 TI - Ureteral obstruction and hydronephrosis as a complication of lymphomatoid granulomatosis: a case report and literature review. AB - Lymphomatoid granulomatosis is an angioinvasive proliferation of atypical T lymphocytes, with frequent pulmonary, cutaneous and neurological manifestations. Urological complications are infrequent. We describe the case of a 40-year-old man who presented with typical intrathoracic findings of lymphomatoid granulomatosis. Following a chemotherapy-induced remission he had retroperitoneal recurrence with bilateral ureteral obstruction, hydronephrosis and renal insufficiency. Histological examination revealed, in addition to the characteristic lymphoid infiltrates of lymphomatoid granulomatosis, a sclerosing process similar to idiopathic retroperitoneal fibrosis. PMID- 1729499 TI - Ureteral obstruction following aortobifemoral bypass: management by endoscopic balloon dilation. AB - A 71-year-old man presented with left hydronephrosis 1 year after aortofemoral bypass. Hydronephrosis was due to extrinsic compression of the ureter between the graft anteriorly and the native iliac artery. Treatment by endoscopic transluminal balloon dilation resulted in complete resolution of the hydronephrosis. PMID- 1729500 TI - Ureterocolic fistula: a unique complication of extracorporeal shock wave lithotripsy. AB - A unique case of ureterocolic fistula at the site of stone fragment impaction after piezoelectric shock wave lithotripsy is described. Pathological examination of the nephroureterectomy specimen indicated xanthogranulomatous pyelonephritis with the process extending into the ureter and fistulous tract. PMID- 1729501 TI - Retroperitoneal fibrosis following radiotherapy for stage I testicular seminoma. AB - Retroperitoneal fibrosis, proved by surgical exploration and pathology, was discovered in a patient 13 years after 3,000 rad external beam cobalt retroperitoneal radiation therapy for stage I testicular seminoma. Ureteral obstruction resulted in the loss of 1 kidney, necessitating ipsilateral nephrectomy and contralateral ureterolysis. To my knowledge this is the first reported case of retroperitoneal fibrosis occurring after radiotherapy for testicular cancer. PMID- 1729502 TI - Conversion of an ileal conduit to a continent catheterizable stoma. AB - A technique is described by which a previously constructed ileal conduit is used as an efferent limb of a continent urinary reservoir. The ileal segment is tapered; 1 end is tunneled submucosally into a reconfigured colonic reservoir and the other end is brought to the skin as a catheterizable stoma. This modification of the Mitrofanoff principle provides a highly continent stoma that is easily catheterized, and allows for preservation of the terminal ileum and ileocecal valve within the gastrointestinal tract. PMID- 1729503 TI - Villous adenoma in augmentation colocystoplasty: a case report and discussion of the pathogenesis. AB - Reports of malignant tumors in enterocystoplasties have recently been accumulating. To date no case of benign tumors has been recorded. We present a case of villous adenoma in a sigmoid colocystoplasty. The possible etiological factors and pathogenesis are discussed, and recommendations are made about followup. PMID- 1729504 TI - The use of recombinant human erythropoietin in a Jehovah's Witness requiring major reconstructive surgery. AB - We report on a new approach to the anemic Jehovah's Witness patient requiring a major operation using preoperative and perioperative erythropoietin. The use of recombinant human erythropoietin in this and other clinical situations is discussed. PMID- 1729505 TI - Intravesical migration of intrauterine device. AB - Intrauterine devices have been plagued by many early and late complications, including uterine perforation and migration into adjacent structures. To our knowledge only 18 cases have been reported in the literature of migration of an intrauterine device into the bladder. We report on a 38-year-old woman in whom an intrauterine device eroded from the uterus 3 years after placement. The device remained asymptomatic in the pelvis for an additional 13 years before the patient presented with urinary symptoms. The literature is reviewed. PMID- 1729506 TI - Emphysematous cystitis: a review of the spectrum of disease. AB - Emphysematous cystitis is an uncommon condition in which pockets of gas are formed in and around the bladder wall by gas-forming organisms. Persons with diabetes, neurogenic bladder and chronic urinary infection are predisposed to the disease. Severity of illness ranges from an asymptomatic condition to life threatening cystitis. We present 2 cases of emphysematous cystitis. One case was an incidental finding on evaluation of abdominal discomfort with resolution upon removal of predisposing factors. The other patient presented with an acute abdomen that progressed to severe necrotizing cystitis ultimately requiring cystectomy. The initial involvement of the urologist as a consultant is emphasized. A complete review of the literature describes the incidence, various presentations, associated diseases and organisms, pathogenesis, and available methods for diagnosis and treatment reported for this disease. Successful management depends on early diagnosis with correction of underlying causes, administration of appropriate antibiotics, establishment of adequate bladder drainage and surgical excision of involved tissue when required. Early detection and prompt treatment are encouraged. PMID- 1729507 TI - Bladder involvement in metastatic breast carcinoma. AB - The literature concerning the uncommon findings of bladder involvement of breast carcinoma is suggestive of a recent increase in reported cases. We report on 3 additional women with vesical deposits of metastatic breast carcinoma. The clinical presentation and dire significance of such cases, and their relationship to progesterone and estrogen receptor expression are discussed. We also postulate on the possible increase in the numbers of reported cases. PMID- 1729508 TI - Urethral injuries in female subjects following pelvic fractures. AB - Pelvic fractures resulting from high speed motor vehicle and/or pedestrian-motor vehicle accidents commonly coexist with urethral injuries in the male patient. A review of 130 female patients with pelvic fractures managed at our institution revealed coexisting urethral injuries in 6 (4.6%). Partial urethral disruptions accounted for the majority of morbidity with early removal of the Foley catheter resulting in urinary extravasation, voiding difficulties and vulvar edema. In 3 patients the injury was misdiagnosed, 2 of whom had life-threatening sepsis with necrotizing fascitis as a consequence. Blood at the vaginal introitus was noted in more than 80% of our patients. However, only half of them had a careful vaginal inspection. If this pertinent portion of the physical examination had been performed more than two-thirds of our patients could have been correctly diagnosed. The need for meticulous vaginal examination when blood is located at the vaginal introitus, and the need for careful cystoscopic and/or radiographic evaluations in the female patient with voiding difficulties and/or vulvar edema in the acute post-traumatic phase are stressed. PMID- 1729509 TI - Tapering of intussuscepted ileal nipple valve or ileocecal valve to correct secondary incontinence in patients with urinary reservoir. AB - Malfunction of the outlet mechanism, that is leakage of urine or difficulty in catheterization, has been the main problem in the evolution of continent urinary reservoirs. Urinary leakage in 3 patients with a right colonic reservoir (2 with an intussuscepted ileal nipple valve and 1 with a plicated ileal segment as a continence mechanism) was managed with tapered narrowing of the nipple valve and the ileocecal valve, respectively, using stapling techniques. Continence was thereby reestablished without impeding catheterization. Tapering is easy to perform and, when applicable, can obviate the need for a major surgical procedure. PMID- 1729510 TI - Clozapine-associated priapism: a case report. AB - Priapism is a recognized side effect of antipsychotic therapy. Recently, new agents known as atypical antipsychotics, such as clozapine, have been introduced with the intent of ameliorating psychosis without the extrapyramidal side effects associated with standard antipsychotic therapy. Priapism has not been observed previously with atypical antipsychotic therapy. We report a case of veno occlusive priapism associated with the use of clozapine. This priapistic episode was complicated by the development of recurrent post-ischemic priapism. PMID- 1729511 TI - Intracorporeal needle breakage: an unusual complication of papaverine injection therapy for impotence. AB - We report an unusual complication of papaverine injection therapy for impotence. After 2.5 years of uneventful injections excessive syringe manipulation following needle insertion resulted in needle breakage and a retained intracavernous needle fragment requiring surgical extraction. A synopsis of the presentation, diagnosis and therapy is presented. PMID- 1729512 TI - Rupture of the deep dorsal vein of the penis during sexual intercourse. AB - The sudden onset of pain, swelling and ecchymosis of the penis during sexual intercourse is the typical presentation of traumatic rupture of the corpus cavernosum, commonly termed fracture of the penis. We report a case of isolated traumatic rupture of the deep dorsal vein of the penis. The condition has essentially the same presentation as corpus cavernosal rupture, and should be considered in the evaluation and treatment of the acute penis. PMID- 1729513 TI - Treatment of congenital penile curvature with penile torsion: a new twist. AB - Congenital penile curvature secondary to asymmetry of corpora cavernosal length is an uncommon cause of penile deformity. Although the deformity generally is not severe enough to preclude sexual intercourse it can be a source of great concern to the patient and may cause him to avoid all sexual contact. The Nesbit procedure is a simple, effective surgical technique to correct lateral or ventral curvature. Rarely penile deviation is accompanied by penile torsion. This unique problem requires a novel surgical approach to create a straight, nontwisted erection. We report 2 cases of congenital lateral penile curvature with accompanying penile torsion and describe a simple modification of the Nesbit procedure for surgical correction. PMID- 1729514 TI - Penile reconstruction with de-epithelized superficial external pudendal artery flap. AB - Complete loss of skin, Buck's fascia and tunica albuginea of the corpus cavernosum occurred on both sides of the penile shaft after placement of a penile prosthesis for the treatment of Peyronie's disease. We describe this unusual complication and surgical reconstruction of the penis with a de-epithelized superficial external pudendal artery axial pattern flap (January 1988). A good result was obtained. PMID- 1729515 TI - Penile metastasis secondary to supraglottic squamous cell carcinoma: review of the literature. AB - We report an unusual supraglottic carcinoma metastasis to the penis. Review of the literature revealed more than 300 cases of metastatic lesions to the penis, excluding primary neoplasms from skin, urethra and blood. Of these metastatic neoplasms 16 originated above the diaphragm, only 4 of which were from the head and neck region. The most common neoplastic metastases to the penis in order of frequency were from the bladder, prostate, rectum and rectosigmoid areas, and kidney in 32, 30, 13 and 8% of the cases, respectively. The incidence of other primary tumor sites that metastasize to the penis is extremely rare. PMID- 1729516 TI - Acupuncture in the treatment of renal colic. AB - A prospective randomized study was performed to compare the effect of acupuncture and intramuscular Avafortan injection in the treatment of renal colic. Our results showed that acupuncture is as effective in relieving renal colic as Avafortan but it had a more rapid analgesic onset (3.14 +/- 2.88 minutes versus 15.44 +/- 7.55 minutes, p less than 0.05). Of the patients in the Avafortan group 7 (43.8%) had side effects, including skin rash in 3, tachycardia in 2, drowsiness in 1 and facial flush in 1. No side effects were noted in the acupuncture group. During 2 hours of observation acupuncture and Avafortan seemed to be ineffective in promoting stone passage. However, patients receiving Avafortan treatment were more likely to have paralytic ileus. In summary, acupuncture can be a good alternative for the treatment of renal colic. PMID- 1729517 TI - Arteriovenous malformation of scrotum: a case report. AB - Arteriovenous malformations of the scrotum are rare. We report a case of arteriovenous malformation involving the scrotal vessels. The patient presented with painful scrotal swelling, which was mainly in the skin and the Dartos layers. Vascular lesions of the scrotum are rare. By far the most common condition is varicocele. Our patient was examined by 2 other urologists before us and no one made the diagnosis of arteriovenous malformation on clinical examination. Diagnosis was made by auscultation with the Doppler stethoscope. We suggest that the possibility of a scrotal arteriovenous malformation and hemangiomas should be entertained in patients with unilateral scrotal swelling. PMID- 1729518 TI - Papillary cystadenocarcinoma of the epididymis: a case report and review of the literature. AB - Primary epididymal carcinoma is rare. We report a case of papillary cystadenocarcinoma of the epididymis presenting with a painful scrotal mass. The clinical course and histopathological features are reviewed. PMID- 1729519 TI - Ectopic prostatic tissue of the anal canal. AB - A unique case is reported of ectopic prostatic tissue present within the anal submucosa from a routine ischial decubitus resection specimen. Although ectopic prostatic tissue has been reported at loci adjoining the prostate, most notably within the urethra, this case is noteworthy by merit of its disparate location. PMID- 1729520 TI - Prostatic abscess due to Histoplasma capsulatum in a patient with the acquired immunodeficiency syndrome. AB - Disseminated histoplasmosis is a systemic fungal infection that may occur in previously healthy or immunocompromised patients. The condition is being recognized with increasing frequency in persons infected with the human immunodeficiency virus. The most common organs involved include the lung, bone marrow, lymph nodes, liver, adrenals and central nervous system, with genitourinary involvement being exceedingly unusual. We describe a Histoplasma capsulatum prostatic abscess occurring after therapy for pulmonary histoplasmosis in a patient with the acquired immunodeficiency syndrome. The prostate may be a difficult focus from which to eradicate disseminated fungal infection in immunocompromised patients. PMID- 1729521 TI - Struvite stone formation by Corynebacterium group F1: a case report. AB - Struvite stones are caused by urea-splitting, usually gram-negative, organisms. A case of aggressive struvite stone production caused by Corynebacterium group F1 is reported that responded to the appropriate antibiotic treatment. To our knowledge this organism has never been associated previously with struvite stone formation. PMID- 1729522 TI - Re: Laser treatment of obstruction from incrusted ureteral catheter. PMID- 1729523 TI - Re: Augmentation cystoplasty complicated by postoperative ventriculoperitoneal shunt infection. PMID- 1729524 TI - Re: Primary malignant lymphoma of the bladder. PMID- 1729525 TI - Re: Diagnostic value of the radioisotope erection penogram for vasculogenic impotence. PMID- 1729526 TI - Re: Decreasing the risk of human immunodeficiency virus or hepatitis B virus infection during endoscopic surgery. PMID- 1729527 TI - Re: Decreasing the risk of human immunodeficiency virus or hepatitis B virus infection during endoscopic surgery. PMID- 1729528 TI - The action potential and net membrane currents in isolated human detrusor smooth muscle cells. AB - The basic electrophysiological properties of the human detrusor have been investigated in vitro using isolated single cells obtained by collagenase digestion of bladder biopsy specimens. Recordings were made using the 'whole-cell patch clamp' technique using either a physiological filling solution or one in which cesium was used to block any outward current. Spontaneous and stimulated action potentials have been recorded and we have performed the first voltage clamp analysis of the currents that underlie the action potential in human detrusor. The depolarising phase of the action potential occurs by an inward current of Ca2+ ions which can be shown to be of sufficient magnitude to support the rate of upstroke. Repolarisation occurs due to an outward K+ current that is partially Ca2+ dependent. The techniques described here permit the investigation of the ionic basis for the control of contractility in the human bladder and may permit the characterisation of any underlying abnormality in the overactive detrusor. PMID- 1729529 TI - Effect of time interval and overdistension on repeated urodynamic studies. AB - Repeated urodynamic testing of the lower urinary tract is often needed both clinically and experimentally. The objective of this study is to find out the time interval needed if repeated urodynamic tests are required even if overdistension occurs initially. Three hundred forty urodynamic studies were performed using five female rhesus monkeys (Macaca mulatta). Two groups of experiments (with or without bladder overdistension) were performed at the following time intervals: immediate, 15, 30 and 45 minutes after the initial urodynamic study. All urodynamic parameters (pressure, capacity, compliance and detrusor strength) were reproducible after a 30-minute waiting period. In case of overdistension, a 45-minute period is required. PMID- 1729530 TI - A new technique for bladder washing. AB - We describe a simple adaptation of the Water Pik (Teledyne Water Pik, Fort Collins, Colorado) irrigating device which allows vigorous, direct-vision agitation of the bladder wall. Three groups of mongrel dogs were subjected to cystoscopy and either syringe barbotage, half-speed Water Pik irrigation, or full speed Water Pik irrigation of the bladder wall. Transitional cell counts were then done on centrifuged aliquots of each bladder wash specimen. The average number of transitional cells per high-power field were similar between the control group and the syringe barbotage group (2.5 and 1.5 respectively). However, both the half-speed and the full-speed Water Pik groups demonstrated statistically higher cell counts (5.7 and 13.7) when compared to both the controls and syringe barbotage groups. We conclude that Water Pik irrigation is an effective method to increase cell yield in bladder wash specimens. PMID- 1729531 TI - Suprarenal Greenfield filter placement to prevent pulmonary embolus in patients with vena caval tumor thrombi. AB - The presence of tumor thrombus secondary to inferior vena caval extension from renal carcinoma carries the threat of pulmonary tumor embolus. In theory, safe prophylaxis could be accomplished by placement of a Greenfield filter in the suprarenal vena cava, which has been accomplished without complication. We treated 6 patients with renal call carcinoma and extensive tumor thrombus of the vena cava with suprarenal filter placement as an adjunct to thrombectomy and nephrectomy. Clinically all 6 patients have done well. However, the over-all rate of vena caval thrombosis or occlusion associated with infrarenal filter placement is 3 to 5%. To investigate the potential risk to renal function if a vena caval occlusion occurred above a solitary kidney shortly after unilateral nephrectomy, we performed suprarenal inferior vena caval ligations after unilateral nephrectomy in 10 dogs. A total of 6 dogs suffered persistent loss of renal function and 3 of these 6 died of uremia. Of 4 dogs who underwent suprarenal inferior vena caval ligation only 1 (25%) had persistent compromise of renal function. A total of 2 dogs underwent unilateral nephrectomy only without compromise of normal renal function. We conclude that the risk of total vena caval occlusion after suprarenal Greenfield filter placement is small. However, should it occur in the setting of recent nephrectomy there is potential for significant renal morbidity. In selected patients this risk may be offset by the potential benefits that the filter offers in terms of protection against tumor and/or bland pulmonary embolus. Further clinical experience will be needed to strengthen and clarify the indications and benefits of preoperative or intraoperative filter placement as reported. PMID- 1729532 TI - The urodynamic characteristics of different ileal reservoirs: an experimental study in dogs. AB - Using adult mongrel dogs, the urodynamic characteristics of three types of ileal reservoirs were studied and compared. Segments of ileum of the same length were utilized to construct simple loop pouches (five dogs), DeKlerk pouches (five dogs) and Kock pouches (five dogs). Six to eight weeks after surgery, urodynamic evaluation was carried out. This included determination of the volume/pressure relationship and measurement of the contractions of the circular and longitudinal muscle fibers. Results indicate that the Kock pouch offers the best features in terms of the volume capacity, the volume/pressure relationship and contractile activity. Detubularization abrogated the muscle tone but it did not affect the phasic contractile activity of the circular muscle layer. PMID- 1729533 TI - Short term effects of cis-platinum on male reproduction, fertility and pregnancy outcome. AB - In order to find out the short term effects of cis-platinum treatment on reproductive function of the treated male rats and their progeny, sexually mature male Sprague-Dawley rats were given a single intra-peritoneal injection of either saline or cis-platinum (2, 4, 8 mg./kg.body wt.). One week following the treatment, the animals were mated with proestrus females of proven fertility. The females were scored positive or negative depending upon whether or not spermatozoa were seen in the vaginal smear after mating. However, all the females were watched until day 18, and on day 19, the females that became pregnant were subjected to laparotomy, and the number of corpora lutea, implantation sites and fetuses were counted. The fetuses were weighted and observed for the presence of any morphological abnormalities. Significant pre-implantation loss was seen in the treated groups. The weights of the fetuses were also significantly lower than those from the control group. Analysis of the effects of cis-platinum on the reproductive system of treated males revealed that cis-platinum reduced the reproductive organ weights, sperm counts, sperm motility, fertility and the levels of testosterone, LH and FSH. These results suggest that cis-platinum has a profound deleterious effect on the reproductive system and on the fertility potential of the treated male rats. PMID- 1729534 TI - An investigation of factors influencing the in vitro induction of LAK activity against a variety of human bladder cancer cell lines. AB - We investigated the sensitivity of transitional cell carcinoma cells, derived from the human bladder, to lymphokine activated killer cells. Recombinant interleukin-2 activated peripheral blood mononuclear cells were studied for their ability to mediate the cytolysis of a panel of four established human bladder transitional cell carcinoma cell lines. Lymphokine activated killer activity was assessed using a standard four hour chromium release assay. All four bladder cancer cell lines proved to be susceptible to lymphokine activated killer mediated cytolysis. This was found to be dependent upon the dose of cytokine and upon the duration of the activation period. The four cell lines were differentially susceptible to lysis (specific cytotoxicity at effector to target ratio of 40:1; RT112 = 22.9%, RT4 = 49.2%, MGH-U1 = 49.1%, EJ18 = 62.3%). The varying susceptibility of lymphokine activated killer mediated cytotoxicity was found to be independent of the histological grade of the parent tumour or the donor of effector cells. Both interferon-alpha and tumour necrosis factor-alpha also elicited lymphokine activated killer cell activity, although the maximum specific cytotoxicity achieved was considerably lower than that obtained with interleukin-2 alone. Interleukin-2, at optimal concentration, and tumour necrosis factor-alpha were found to behave synergistically in the generation of lymphokine activated killer effectors. However, concentrations of tumour necrosis factor alpha higher than 100 Uml.-1 resulted in a decrease in specific cytotoxicity. These findings suggest a possible use of adoptive immunotherapy in human bladder cancer and indicate the optimum conditions for the generation of such effector cells. PMID- 1729535 TI - Immunotherapy of murine transitional cell carcinoma of the bladder using alpha and gamma interferon in combination with other forms of immunotherapy. AB - BCG immunotherapy is very effective in the treatment of superficial transitional cell carcinoma of the bladder, but its significant toxicity may limit its use in some patients. In an effort to find less toxic and potentially more effective treatments we investigated the possible immunotherapeutic potential of combinations of Alpha Interferon (1000 IU) and Gamma Interferon (500 IU) with bacillus Calmette Guerin (BCG) (10(7) cfu), Interleukin-2 (4000 IU), and Keyhole Limpet Hemocyanin (50 micrograms.) in the MBT2 murine bladder cancer model. Significant reductions (p less than 0.05) in tumor incidence relative to the saline control, 83%, Day 35) was observed in groups receiving alpha interferon (42%), Keyhole limpet hemocyanin (42%), interleukin-2 (25%), alpha interferon + Keyhole limpet hemocyanin (17%), alpha interferon + interleukin-2 (33%), gamma interferon + BCG (42%), and gamma interferon + interleukin-2 (17%). All treatment groups with the exception of the group receiving gamma interferon had significantly reduced tumor volume (p less than 0.05) relative to the saline control. Combination treatment groups were significantly more effective than single agent treatments (p = 0.0057). The exhibited anti-tumor effect of these immunotherapeutic agents alone and in combination suggest that they may prove to be effective forms of immunotherapy for transitional cell carcinoma of the bladder. PMID- 1729536 TI - Effects of lithotripter-generated high energy shock waves of mammalian cells in vitro. AB - The effects of high energy shock waves on an established human prostatic carcinoma cell line (PC-3) were investigated. HESW were administered to PC-3 cell suspensions using an electrohydraulic lithotripter (Dornier HM3). Experimental variables included the number of shocks to which the cells were exposed, spark generator potential, and the position of the cell sample in the acoustic field. Two types of cellular damage were observed: immediate cell destruction (lysis) as measured by electronic particle counting and the loss of reproductive capacity (viability) among the remaining cells as determined by colony formation assay. Over the range of the experimental variables studied, cell lysis was dependent to a greater extent on the number of shocks administered than the generator potential. Viability was affected less but was also dependent on both the generator potential and shock number. Cell lysis was strongly dependent on the position of the sample in the acoustic field with the extent of damage increasing as the sample was moved along the central axis of the shock wave from the f2 focus towards the electrodes. Possible mechanisms of damage and the relationship of the in vitro effects to the damage observed in normal tissues of patients undergoing extracorporeal lithotripsy for kidney stone disease are discussed. PMID- 1729537 TI - Acute hyperoxaluria, renal injury and calcium oxalate urolithiasis. AB - Single intraperitoneal injections of three, seven, or 10 mg. of sodium oxalate per 100 gm. of rat body weight were administered to male Sprague-Dawley rats. At various times after the injection, urine samples were analyzed for oxalate, and urinary enzymes, alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and N-acetyl-beta-glucosaminidase. The kidneys were processed for light microscopy and renal calcium and oxalate determination. Oxalate administration resulted in an increase in urinary oxalate and formation of calcium oxalate crystals in the kidneys. The amount and duration of urinary excretion of excess oxalate and retention of crystals in the kidneys correlated with the dose of sodium oxalate administered. At a low oxalate dose of three mg./100 gm., crystals moved rapidly down the nephron and cleared the kidneys. At higher doses crystals were retained in kidneys and at a dose of 10 mg./100 gm. were still there seven days post-injection. Crystal retention was associated with enhanced excretion of urinary enzymes indicating renal tubular epithelial injury. PMID- 1729538 TI - The effects of ultrasound-guided shock waves during early pregnancy in Sprague Dawley rats. AB - Pregnancy is a contraindication to extracorporeal shock wave lithotripsy (ESWL) because of its possible harm to the embryo or fetus caused by shock waves or ionizing radiation. In this study timed-pregnant rats were subjected to shock waves early in gestation using a lithotriptor having ultrasound imaging. In a pilot group undergoing immediate laparotomy, it was determined to what extent the pelvic structures were effected. Then a test group was exposed to shock waves and carried to near term pregnancy along with an identical group of pregnant, sham procedure rats. A laparotomy all were inspected for fetal viability, fetal abnormalities, and maternal organ damage. Fetuses located nearest the focal area of maximum shock wave energy showed lower mean weight than controls. There was no recognizable gross or microscopic fetal damage. PMID- 1729539 TI - Monoclonal antibody Due ABC 3 directed against transitional cell carcinoma. I. Production, specificity analysis, and preliminary characterization of the antigen. AB - The development of the hybridoma technology allows the identification of tumor associated antigens with monoclonal antibodies (mAbs). Employing this technology mAb Due ABC 3 was obtained by immunization of a BALB/c mouse with bladder tumor cell line SW 1710 and subsequent cell fusion of spleen cells with P3. X63.Ag8.653 mouse myeloma cells. MAb Due ABC 3, an IgM antibody, was found to recognize an antigen present in the membrane of tumor cells in 25 out of 28 (89%) transitional cell carcinoma specimens but rarely (three out of 25 specimens, 12%) on normal urothelial cells. Cross reactions were seen with proximal tubular epithelium of the kidney and seven out of 12 renal cell carcinomas examined. Furthermore, the antigen was expressed by granulocytes, some gastrointestinal epithelia, ovarian and breast carcinoma. The antigen recognized by mAb Due ABC 3 was stable to fixation with formaldehyde and paraffin emmbedding, different proteases, alkaline treatment and heat exposure up to 70C. Antigenicity was abandoned by incubation with periodate but not with neuraminidase treatment. The antigen could be extracted with chloroform/methanol suggesting the involvement of a glycolipid. Immuno-thin layer chromatography revealed a single lipid band reacting with mAb Due ABC 3 but not with anti-CD15, directed against the Lewis X antigen. Although not tumor-specific, mAbs directed against differentiation antigens may be of value for the investigation of cell transformations as well as for diagnostic use. PMID- 1729540 TI - Immunotherapy with interleukin-2 and alpha-interferon in patients with metastatic renal cell cancer with in situ primary cancers: a pilot study. AB - A total of 12 patients with stage 4 renal cell carcinoma and primary renal tumors in situ was entered into a pilot study using treatment with interleukin-2 and alpha-interferon followed by radical nephrectomy. Of the patients 11 underwent nephrectomy after an initial course of immunotherapy. Ten patients were able to receive a second course of immunotherapy given after nephrectomy. One patient achieved a complete response of lung and mediastinal metastases without any change in the primary renal tumor but after nephrectomy the patient remained in complete remission for greater than 11 months. A total of 3 patients achieved a partial response at some extrarenal sites but they had progression elsewhere. Toxicity was similar to previous experience with this immunotherapy regimen. Therefore, we demonstrated that metastatic tumor regression is possible with primary renal tumors in situ and that aggressive interleukin-2-based immunotherapy can be tolerated in the presence of a large renal tumor. PMID- 1729541 TI - Gastrocystoplasty in dogs: an ulcerating effect of acid urine. AB - Six dogs underwent bladder augmentation using an isolated flap of the body of the stomach after a supratrigonal cystectomy. Each dog was followed for six months with periodic determinations of urine pH, blood chemistry, cystometry and cystography. Postoperatively, urine pH decreased markedly after a meal in most dogs. No persistent hypergastrinemia was found. Autopsy showed that histological erosions developed in the bladder remnant in five dogs, and an ulcer in the bladder remnant in one dog. These results indicate that when an isolated, vagally denervated gastric segment is incorporated into the bladder, acid secretion from the segment may ulcerate the bladder. PMID- 1729542 TI - Electrically-induced, nerve-mediated relaxation of rabbit urethra involves nitric oxide. AB - Isolated smooth muscle preparations from the rabbit urethra precontracted with noradrenaline (10(-5) M), endothelin (10(-7) M), or arginine vasopressin (10(-7) M) responded to electrical field stimulation by frequency-dependent non adrenergic, non-cholinergic relaxations, which could be blocked by tetrodotoxin (10(-6) M). Relaxation was more pronounced in preparations precontracted by endothelin than by noradrenaline or arginine vasopressin. The electrically induced relaxations were reduced in a concentration-dependent manner by pretreatment for 30 minutes with NG-nitro-L-arginine (10(-6) to 10(-4) M) and NG monomethyl-L-arginine (10(-5) to 10(-4) M). At the highest concentration of NG nitro-L-arginine used (10(-4) M), relaxation was abolished and/or changed into a contraction. The effect of NG-nitro-L-arginine was reversible. NG-nitro-D arginine had no effect. Pretreatment for 30 minutes with L-arginine (10(-3) M) slightly, but significantly, enhanced the maximum relaxation to field stimulation in noradrenaline-precontracted preparations. L-arginine pretreatment also prevented the effects of low, but not high, concentrations of NG-nitro-L arginine. In contrast, D-arginine had no effect. Electrically induced relaxations were not significantly affected by methylene blue (10(-5) M) or superoxide dismutase (20 U/ml). Addition of nitric oxide (present in acidified solution of NaNO2) caused transient and concentration-dependent relaxations in preparations precontracted by noradrenaline. At the maximum concentration used (10(-3) M), the relaxant response averaged 67% of the tension induced by noradrenaline. Nitric oxide-induced relaxations were not affected by NG-nitro-L-arginine or L-arginine, but were significantly inhibited by methylene blue. In preliminary experiments, effects similar to those found in rabbit urethra were also observed in isolated urethral preparations obtained from three patients. It is suggested that in the urethra, nitric oxide is involved in the mediation of relaxation evoked by electrical stimulation of nerves. PMID- 1729543 TI - Ability of each lumbar splanchnic nerve and disability of thoracic ones to generate seminal emission in the dog. AB - Control of seminal emission by canine thoracolumbar splanchnic nerves which constitute the caudal mesenteric plexus (inferior mesenteric and superior hypogastric plexuses in human) was investigated. Electrical stimulation of a splanchnic nerve group which branched from sympathetic trunks at thoracic and L1 ganglia and descended on the ventral wall of the aorta between bilateral spermatic arteries via the intermesenteric plexus did not cause seminal emission in all 13 dogs examined. In contrast, electrical stimulation of the other splanchnic nerve group which branched from lumbar sympathetic trunks at ganglia L1-L5 and descended behind bilateral spermatic arteries induced seminal emission regardless of branching levels or sides. The results indicate that efferent signals via the intermesenteric plexus do not generate seminal emission, while those via each lumbar splanchnic nerve have ability to generate seminal emission in the dog. PMID- 1729544 TI - Effects of sensitization on the permeability of urothelium in guinea pig urinary bladder. AB - Permeability of the guinea pig urinary bladder was investigated in a model of experimental cystitis induced by intravesical antigen challenge following sensitization. Guinea pigs were sensitized by intraperitoneal injections of ovalbumin (10 mg./kg.) given on days 1, 3, and 5. The studies described were done four weeks after the last injection. Controls (injected with saline) were used at the same time as sensitized animals. Each group (control and sensitized), was divided into two subgroups; guinea pigs challenged with intravesical ovalbumin (10 mg./ml., one ml.) and those receiving one ml. saline intravesically. Immediately following the antigen (or saline) challenge, one ml. of 14C-urea urea was placed into the bladder for two hours. We examined the peripheral blood concentrations of 14C-urea at periods of time up to 120 minutes. There was a progressive increase in the blood level of 14C-urea with time only in the sensitized group challenged with antigen (ovalbumin). There was no 14C-urea present in the blood of the sensitized group without antigen challenge, or in either unsensitized group. We also measured isotope concentration in the bladders and found a significantly higher concentration of isotope in the bladders from ovalbumin-treated sensitized guinea pigs. We believe that this animal model of cystitis is an improvement over previous models because of its physiological relevance. In this model, cystitis is produced without mechanical or chemical damage to the bladder mucosa. A discussion of the model in relation to interstitial cystitis is presented. PMID- 1729545 TI - Evaluation of epidermal growth factor receptor DNA amplification and mRNA expression in bladder cancer. AB - The epidermal growth factor receptor has been implicated in the malignant transformation of cells because the v-erbB oncogene is a truncated form of the epidermal growth factor receptor. DNA amplification and/or mRNA overexpression of the epidermal growth factor receptor is associated with the malignant potential of several human epithelial tumors. To determine the frequency of epidermal growth factor receptor DNA amplification and mRNA overexpression in bladder cancer, we evaluated 12 bladder tumors for DNA amplification and another 14 bladder tumors for mRNA overexpression. By Southern hybridization, we found no evidence of DNA amplification or gene rearrangements in 12 bladder tumors. Five of 14 bladder tumors overexpressed epidermal growth factor receptor mRNA four to 15 fold compared to normal urothelium from the same bladder by Northern analysis. These findings suggest that epidermal growth factor receptor amplification and/or gene rearrangement occurs infrequently in bladder cancer. Epidermal growth factor receptor mRNA overexpression, however, is found in 36% of bladder tumors and may play a role in bladder tumorogenesis. PMID- 1729546 TI - Phase 1 trial of oral bropirimine in superficial bladder cancer. AB - A total of 34 patients with measurable superficial transitional cell cancer of the bladder entered into a phase 1, nonrandomized, noncomparative trial to assess the toxicity of the oral interferon inducer bropirimine. Of the patients 26 were also evaluable for response. The toxicity of bropirimine was minimal. At the 3 month evaluation 6 patients had experienced complete regression of tumor and had negative cytology studies, and 2 had partial responses. The majority of complete responses were in patients with carcinoma in situ only, with most responses seen at higher dose levels. One patient with papillary tumor and carcinoma in situ had a complete response. Some early responses appear to be durable. Most importantly, a high rate of complete response was noted at higher dose levels among patients who had failed prior therapy with bacillus Calmette-Guerin. Further clinical trials of bropirimine in bladder cancer appear warranted. PMID- 1729547 TI - The prognostic significance of deoxyribonucleic acid flow cytometry in muscle invasive bladder carcinoma treated with preoperative irradiation and cystectomy. AB - Deoxyribonucleic acid (DNA) flow cytometry measurements were performed in nuclear suspensions obtained from paraffin-embedded biopsies from 83 patients with stages T2, T3 and T4a bladder carcinoma. All patients were treated with preoperative radiotherapy and cystectomy from 1976 through 1985. Of the tumors 13 (16%) were diploid, 18 (22%) tetraploid and 52 (63%) aneuploid. A total of 19 tumors (23%) had 2 or 3 stemlines in addition to the diploid cells. Post-radiotherapy stage reduction (absence of muscle infiltration in the cystectomy specimen) occurred more often in tumors with only 1 nondiploid stemline than in diploid tumors or nondiploid tumors with multiple stemlines. The 5-year survival rate was significantly poorer for patients with a diploid (33%) than for those with a nondiploid (66%) tumor (p = 0.05), although this was only marginally retained in a multivariate analysis (p = 0.11). The clinical significance of DNA ploidy in muscle infiltrating bladder cancer seems not to be as evident as has been shown for superficial bladder tumors but it may be of value in selecting patients for preoperative radiotherapy. PMID- 1729548 TI - Management of the bladder outlet in patients requiring enterocystoplasty. AB - We reviewed 30 patients who required augmentation enterocystoplasty and a procedure to modify the bladder outlet for the treatment of intractable incontinence. Of the 30 patients 16 were treated with simultaneous cystoplasty and an outlet procedure, 6 initially underwent an outlet procedure followed by cystoplasty and in 8 cystoplasty was performed first with a subsequent operation to modify the bladder outlet. Continence was achieved in 29 patients. The current methods for evaluation of the bladder and its outlet are reviewed, focusing on the predictive value of preoperative testing to determine which patients require cystoplasty and an outlet modifying procedure. PMID- 1729549 TI - Dual radioisotopic study: a technique for the evaluation of vasculogenic impotence. AB - Arterial and venous systems are the main points for the evaluation of vasculogenic impotence. To evaluate both of these systems in the same study we propose a dual radioisotopic study in which 99mtechnetium (99mTc) and 133xenon (133Xe) were used. The changes in 99mTc and 133Xe radioactivities administered intravenously and intracavernously, respectively, were monitored before and after intracavernous papaverine injection. These changes were determined as time activity curves, which were generated from the region of interest over the penis. A 99mTc penogram index derived from the 99mTc time activity curve was significantly different in the control and arteriogenic impotence groups (131.67 +/- 74.6 versus 62.94 +/- 51.6, p less than 0.01). A meaningful correlation between 99mTc penogram index results and duplex ultrasonographic findings were observed (r = 0.905). 133Xe penogram index, derived from the 133Xe washout curve was significantly different in the control and venogenic impotence groups (-25.65 +/- 24.9 versus -56.09 +/- 13.4, p less than 0.01). Also, a meaningful correlation was obtained between pharmacocavernosometry and 133Xe penogram index results of venogenic impotent patients (r = 0.86). These findings suggest that the dual radioisotopic study will be a useful technique in the evaluation of the entire vascular system of the penis, since it is a noninvasive method. PMID- 1729550 TI - Transcutaneous registration of cavernous smooth muscle electrical activity: noninvasive diagnosis of neurogenic autonomic impotence. AB - Registration of cavernous electrical activity was shown to be a possible method for the evaluation of cavernous autonomic innervation. Recent studies in patients with normal erectile function showed that cavernous electrical activity is synchronous throughout the entire cavernous bodies. Therefore, we examined the feasibility of transcutaneous registration of cavernous electrical activity in 8 normal and 62 impotent patients. In the sitting patient cavernous electrical activity was recorded with a 2-channel electrophysiological unit. Recording was done with a coaxial needle electrode in the proximal left cavernous body and with surface electrodes bilaterally on the penile shaft. In 7 of 8 normal patients swelling of the penile shaft after circumcision resulted in a dramatically decreased amplitude of the potentials. In 41 of 62 impotent patients recordings were similar. In 10 of 62 patients no recording or markedly decreased amplitudes were noted with the surface electrodes and in these patients a small penis or penile retraction with consecutive electrode displacement was found. Careful repositioning of the surface electrodes with the patient in the supine position resulted in similar recordings in 9 (inconsistently in 4). In 11 of the 62 patients more information was obtained with the surface than with the needle electrode. Our results show that recording of cavernous electrical activity can be done in a completely noninvasive manner using surface electrodes with similar or even better information obtained than with needle electrodes. PMID- 1729551 TI - Assessment of penile blood flow by duplex ultrasonography in 44 men with normal erectile potency in different phases of erection. AB - Duplex ultrasonography is important in the diagnosis of vasculogenic erectile dysfunction. We measured the ultrasonographic parameters of cavernous blood flow in different phases of penile erection. We examined 44 volunteers with normal erectile potency. Doppler spectra of the cavernous artery were obtained in a time dependent manner after intracavernous administration of papaverine. Following intracavernous pharmacological stimulation, the Doppler spectrum alters according to a specific pattern indicating the different hemodynamic phases of erection. Peak flow velocity and acceleration time, measured in the early post-injection phase, may be used to grade arterial inflow. The difference between resistance index in the pre-injection and late post-injection phases may be used to estimate veno-occlusive function. References values are defined. PMID- 1729552 TI - The hemodynamics of vacuum constriction erections: assessment by color Doppler ultrasound. AB - Duplex ultrasound with pulsed Doppler and color flow sonography were used to image the penis and conduct blood flow velocity studies in 5 patients. Cavernous body cross sectional area, cavernous artery diameters and peak systolic velocities were measured in the flaccid shaft, after transient exposure to negative pressure in a vacuum constriction device, and with a vacuum constriction device band applied to the tumescent shaft. We found that exposure to vacuum transiently increased central cavernous arterial blood flow velocities compared to baseline values in all patients. After a trial of a vacuum constriction device and application of the constricting band cavernous body cross sectional areas doubled. Despite increased cavernous arterial diameters in 4 of 5 patients and increased blood flow in all patients after vacuum-induced tumescence alone, we could not visualize arterial inflow in the penile shaft once the constricting band was in place. Color Doppler ultrasound can detect cavernous artery systolic flow as low as 2 to 9 cm. per second. Our data suggest that the erectile state maintained distal to the vacuum constriction device band is low flow and relatively ischemic. PMID- 1729553 TI - Patient and partner satisfaction with the AMS 700 penile prosthesis. AB - Patient and partner satisfaction with the use of an AMS 700 inflatable penile prosthesis was evaluated by reviewing the records from 387 patients and questionnaires completed by 272 of these patients. The evaluation demonstrated that 83% of the patients and 70% of the partners were satisfied with use of this device. Couples who reported the lowest levels of satisfaction were characterized by men who required more than 1 procedure for prosthesis implantation. Pain and appearance of the penis were the most common causes for dissatisfaction with the device. Results from this evaluation recognize the importance of careful surgical technique to avoid any surgical complications but, more importantly, they emphasize the need for physician-manufacturer interaction to maximize mechanical reliability of the prosthesis. PMID- 1729554 TI - The G.F.S. Mark II inflatable penile prosthesis. AB - The G.F.S. Mark II inflatable penile prosthesis was implanted in 80 men who were followed for up to 27 months. In this study there have been no mechanical problems. Of the patients 6 required repositioning of the reservoir pump and 4 required postoperative addition of fluid to the reservoir pump. This study indicates that the revised connectorless G.F.S. Mark II inflatable penile prosthesis has eliminated the previous problems with connectors and tubings. The G.F.S. Mark II inflatable penile prosthesis reservoir pump provides a means of postoperative fluid adjustment within the system performed as an office procedure. PMID- 1729555 TI - Functional characteristics of sperm obtained by electroejaculation. AB - Sperm obtained by electroejaculation in 32 anejaculatory men were examined for functional characteristics. Raw specimens showed high sperm counts but motility averaged only 11%. Average viability was 10% for antegrade and 5% for retrograde fractions. Bovine cervical mucus penetration was normal (30 mm. or more in 30 minutes) in only 24% of the electroejaculation samples but it was normal in all of the donor samples tested. Processed sperm motility averaged 30% with 71% forward progression. At 20 hours patient samples retained 46% of the original motility, while donor controls retained 81%. In the hamster egg penetration assay patient sperm penetrated 14% of the oocytes while donor sperm penetrated 40%. Therefore, we identified 4 characteristics of sperm obtained by electroejaculation: 1) low viability, 2) poor survival after overnight incubation, 3) moderately impaired cervical mucus penetration and 4) moderately poor fertilizing capability as measured by the hamster egg penetration assay. Poor sperm survival and impaired function may explain the low pregnancy rates from insemination with electroejaculated sperm. PMID- 1729556 TI - Color Doppler ultrasonography in the evaluation of the acute scrotum. AB - Color Doppler ultrasonography was used to assess 20 patients with the acute onset of scrotal pain. Patients were categorized into 3 groups according to the initial clinical impression of the examining physician: ischemia, inflammation or trauma. Color Doppler ultrasonography correctly predicted the need for surgery in 8 of 9 operated patients (89%) and correctly predicted the outcome in all 11 nonoperated patients (100%). The anatomical resolution possible, as well as information regarding blood flow made color Doppler ultrasonography a useful tool in the assessment of acute scrotal processes. PMID- 1729557 TI - Transitional cell carcinoma of the prostate in patients undergoing radical cystoprostatectomy. AB - To assess the impact of prostatic involvement with transitional cell carcinoma we reviewed the clinical outcome of 49 patients with transitional cell carcinoma of the prostate. In addition, 115 step-sectioned cystoprostatectomy specimens removed for bladder transitional cell carcinoma were studied to determine the true incidence of secondary prostatic involvement by transitional cell carcinoma. Specimens from 300 prostates removed for prostatic adenocarcinoma also were reviewed to investigate the presence of incidental transitional cell carcinoma arising within the prostate. Transitional cell carcinoma was found in 29% of the step-sectioned specimens and in none of the radical prostatectomy specimens. The presence of prostatic invasion either into the stroma or involving prostatic ducts and acini only had no adverse effect on outcome. Lymph node status and bladder stage, and not prostatic invasion were the determining factors of survival. The presence of seminal vesicle involvement or prostatic stromal invasion appeared to predict for lymph node involvement. With a mean followup of more than 3 years 75% of our patients who had negative lymph nodes and low stage bladder lesions are alive without evidence of disease. In our series prostatic involvement by transitional cell carcinoma did not impact on survival when patients were treated aggressively with radical cystoprostatectomy. PMID- 1729558 TI - Local anesthesia for extracorporeal shock wave lithotripsy: a study comparing eutetic mixture of local anesthetics cream and lidocaine infiltration. AB - A study of the anesthetic efficacy of a eutetic mixture of local anesthetics (EMLA cream) versus lidocaine infiltration in extracorporeal shock wave lithotripsy (ESWL) was done. A total of 46 patients had 30 gm. of EMLA cream applied to the skin over the kidney and 45 had subcutaneous infiltration anesthesia with 20 ml. 1% lidocaine with epinephrine. All patients received an intravenous dose of morphine just before ESWL. The patients were comparable with regard to age, sex, weight, morphine dosage, number of shock waves given and duration of treatment. Median pain score and the amount of supplementary analgesics were not significantly different between the 2 groups. There were no significant differences between the groups with regard to post-ESWL skin changes. Therefore, EMLA cream can be recommended for ESWL provided it is applied correctly. PMID- 1729559 TI - Topical anesthesia with eutetic mixture of local anesthetics cream in vasectomy: 2 randomized trials. AB - Two paired randomized trials testing topical anesthesia with a eutetic mixture of local anesthetics (EMLA cream*) in vasectomy were performed. In 1 trial EMLA cream was applied on 1 side of the scrotum, while infiltration anesthesia into the skin and subcutaneous tissue with mepivacaine was used on the contralateral side. All but 1 of the 13 patients (p less than 0.05) preferred infiltration anesthesia because of pain as the incision reached the subcutaneous tissue. In the other trial 29 patients received EMLA cream on 1 side of the scrotum before bilateral mepivacaine infiltration. There was significantly less pain on the sides with the anesthetic cream (p less than 0.001). Many patients would pay the price of the cream. In conclusion, EMLA cream cannot replace but it can supplement infiltration anesthesia during vasectomy. PMID- 1729560 TI - Women's health initiative leads way as research begins to fill gender gaps. PMID- 1729561 TI - Postpolio syndrome may not be progressive. PMID- 1729562 TI - Worldwide antipolio campaign strategy evolving. PMID- 1729563 TI - From the Assistant Secretary for Health, US Public Health Service. PMID- 1729564 TI - From the Centers for Disease Control. Tuberculosis among homeless shelter residents. PMID- 1729565 TI - From the Centers for Disease Control. Deaths among homeless persons--San Francisco. PMID- 1729566 TI - From the Centers for Disease Control. FDA approval of use of diphtheria and tetanus toxoids and acellular pertussis vaccine. PMID- 1729567 TI - Controversial costs of cocaine:. PMID- 1729568 TI - Direct-to-consumer advertising with added inducements. PMID- 1729569 TI - The treatment of AIDS-related lymphoma. French-Italian Cooperative Study Group. PMID- 1729570 TI - Detection of osteomyelitis associated with diabetic foot ulcers. PMID- 1729571 TI - Noncritical aortic stenosis in two men unable to quit running marathons--well, one quit. PMID- 1729572 TI - Transient myopia following metronidazole treatment for Trichomonas vaginalis. PMID- 1729573 TI - Living alone after myocardial infarction. Impact on prognosis. AB - OBJECTIVE: To determine if the presence of a disrupted marriage or living alone would be an independent prognostic risk factor for a subsequent major cardiac event following an initial myocardial infarction. DESIGN: Prospective evaluation in the placebo wing of a randomized, double-blind drug trial in patients with an enzyme-documented acute myocardial infarction who were admitted to a coronary care facility. Data for living alone and/or a marital disruption were entered into a Cox proportional hazards model constructed from important physiologic and nonphysiologic factors in the same database. SETTING: Multicenter trial in a mixture of community and academic hospitals in the United States and Canada. PATIENTS: All consenting patients who were 25 to 75 years of age and without other serious diseases were enrolled (placebo, N = 1234) within 3 to 15 days of the index infarction and followed for a period of 1 to 4 years (mean, 2.1 years). Nine hundred sixty-seven patients were followed for 1.1 years and 530 for 2.2 years. PRIMARY OUTCOME MEASURE: Recurrent major cardiac event (either recurrent nonfatal infarction or cardiac death). RESULTS: Living alone was an independent risk factor, with a hazard ratio of 1.54 (95% confidence interval, 1.04 to 2.29; P less than .03). Using the Kaplan-Meier statistical method for calculation, the recurrent cardiac event rate at 6 months was 15.8% in the group living alone vs 8.8% in the group not living alone. Risk remained significant throughout the follow-up period (P = .001). A disrupted marriage was not an independent risk factor. CONCLUSION: Living alone but not a disrupted marriage is an independent risk factor for prognosis after myocardial infarction when compared with all other known risk factors. PMID- 1729575 TI - Iron supplementation after femoral head replacement for patients with normal iron stores. AB - OBJECTIVE: To assess the efficacy of oral iron therapy in the recovery of patients' hemoglobin levels after major surgery. DESIGN: Randomized controlled trial. SETTING: Private orthopedic practice confined to one large community hospital. PATIENTS: One hundred seventy consecutive elderly patients undergoing hip surgery; 75 failed to meet entry hematologic or medical criteria; 95 were randomized, with 16 withdrawn because of complications. INTERVENTION: Thirty seven patients received ferrous sulfate orally four times a day for the duration of their hospitalization. Forty-two patients who received no iron supplement served as the control group. MAIN OUTCOME MEASURES: Changes in hemoglobin levels and reticulocyte counts over the 2- to 3-week follow-up period. RESULTS: There was no significant difference in mean hemoglobin levels between the treatment and control groups (95% confidence interval [CI] for difference of -6.6 to 5.4 g/L). Corrected reticulocyte fractions increased equally in both groups (95% CI for difference of -9 x 10(3) to 2 x 10(-3). The study was designed to detect a difference in mean hemoglobin levels of 8.5 g/L or greater or a difference in mean reticulocyte fraction of 10 x 10(-3) between the two groups with a power of 0.80 at the .05 (two-sided) level of significance. CONCLUSION: The administration of oral iron supplements to elderly, healthy orthopedic patients postoperatively did not hasten the recovery of hemoglobin levels, provided adequate tissue iron stores were present. PMID- 1729574 TI - Prognostic importance of social and economic resources among medically treated patients with angiographically documented coronary artery disease. AB - OBJECTIVE: To evaluate the hypothesis that diminished social and economic resources impact adversely on cardiovascular mortality in patients with coronary artery disease. DESIGN: Inception cohort study of patients undergoing cardiac catheterization from 1974 through 1980 and followed up through 1989. SETTING: Tertiary care university medical center. PATIENTS: Consecutive sample of 1965 medically treated patients with stenosis 75% or greater of at least one major coronary artery. Five hundred patients were not enrolled due to logistic problems; 33 refused; 64 had missing data on key medical variables. The final study population included 1368 patients, 82% male, with a median age of 52 years. MAIN OUTCOME MEASURE: Survival time until cardiovascular death. RESULTS: Independent of all known baseline invasive and noninvasive medical prognostic factors, patients with annual household incomes of $40,000 or more had an unadjusted 5-year survival of 0.91, compared with 0.76 in patients with incomes of $10,000 or less (Cox model adjusted hazard ratio, 1.9; 95% confidence interval, 1.57 to 2.32; P = .002). Similarly, unmarried patients without a confidant had an unadjusted 5-year survival rate of 0.50, compared with 0.82 in patients who were married, had a confidant, or both (adjusted hazard ratio, 3.34; 95% confidence interval, 1.84 to 6.20; P less than .0001). CONCLUSIONS: Low levels of social and economic resources identify an important high-risk group among medically treated patients with coronary artery disease, independent of important medical prognostic factors. Additional study will be required to see if interventions to increase these resources improve prognosis. PMID- 1729576 TI - Folate deficiency and cervical dysplasia. AB - OBJECTIVE: To test the hypothesis that nutritional deficiency affects the incidence of cervical dysplasia in young women. DESIGN AND SETTING: Case-control study. Participants were derived from community family-planning clinics and referrals to a colposcopy center. PARTICIPANTS: A total of 726 subjects were screened, yielding 294 cases of dysplasia and 170 controls defined by coexistent cytologic and colposcopic evidence. MAIN OUTCOME MEASURES: Planned prior to data collection. Odds ratios were computed using logistic regression models to evaluate association between cervical dysplasia and sociodemographic, sexual, and reproductive factors; smoking; oral contraceptive use; human papillomavirus (HPV) infection; and 12 nutritional indices determined by blind analysis of nonfasting blood specimens. RESULTS: The number of sexual partners, parity, oral contraceptive use, and HPV-16 infection were significantly associated with cervical dysplasia. Plasma nutrient levels were generally not associated with risk. However, red blood cell folate levels at or below 660 nmol/L interacted with HPV-16 infection. The adjusted odds ratio for HPV-16 was 1.1 among women with folate levels above 660 nmol/L but 5.1 (95% confidence interval, 2.3 to 11) among women with lower levels. Interactions of red blood cell folate levels with cigarette smoking and parity were also present but were not statistically significant. CONCLUSION: Low red blood cell folate levels enhance the effect of other risk factors for cervical dysplasia and, in particular, that of HPV-16 infection. PMID- 1729577 TI - Critical care of patients with AIDS. AB - OBJECTIVE: We sought to review the clinical and ethical issues surrounding critical care for patients with the acquired immunodeficiency syndrome (AIDS). DATA SOURCES: We reviewed published studies and abstracts dealing with the outcome of critical care for patients with AIDS, decision making about life sustaining treatments in patients with AIDS, and infection control in the intensive care unit. We also consulted with a number of experts in the field. STUDY SELECTION: We selected outcome studies in which patients with documented AIDS or infection with the human immunodeficiency virus (HIV) were analyzed. We rejected data concerning patients with suspected or presumed AIDS and data concerning presumed cases of Pneumocystis carinii pneumonia (PCP). DATA SYNTHESIS: Most AIDS patients who require critical care do so because of respiratory failure caused by PCP. Although studies early in the epidemic reported survival rates to hospital discharge of 0% to 14%, recent studies demonstrate improved survival rates of 36% to 55%. Treatment for patients with PCP and respiratory failure should include either intravenous trimethoprim-sulfa methoxazole or pentamidine isethionate, as well as adjuvant corticosteroids. Patients with AIDS may require critical care for many other indications, including seizures, sepsis, and hypotension, or reasons unrelated to their immunodeficiency. In general, such patients have a better prognosis than those with respiratory failure. CONCLUSION: The provision of critical care for PCP and respiratory failure specifically or AIDS generally cannot be considered futile. Therefore, decisions about the use of critical care should be guided by the particular clinical situation and the patient's preferences. More research is needed to elucidate the reasons for the improving survival for patients with PCP and respiratory failure and the predictors of such survival. PMID- 1729578 TI - Improvement of depression and triggering of mania by sleep deprivation. PMID- 1729579 TI - Health USA. A national health program for the United States. AB - The Health USA Act of 1991 addresses two fundamental health services financing problems: the more than 30 million uninsured persons and the rising costs for health care and for health insurance. Health USA would provide coverage of the entire resident population for comprehensive medical and preventive health and long-term care services through a universal tax-funded financing system. The federal government would contribute an average of 87% of program costs to each state, which would establish, under federal guidelines, a state health program. Each individual or family may enroll in any health plan approved by the state program, including many private plans, or a plan run by the state program. Through the approved plan of their choice, enrollees would receive covered services and obtain their care from participating physicians and other professional practitioners, hospitals, and other facilities. The state program would pay approved plans a capitation payment for every person enrolled. The plans would pay professional providers fees, as part of an all-payer system of fee schedules and expenditure targets, or capitation payments or salary. Hospitals would be financed through global budgets negotiated by the state program with each hospital. The plan run by the state program would pay the health care costs of any person who does not enroll in an approved plan, making the state plan the payer of last resort and eliminating uncompensated care and cost shifting by providers. Health USA would separate health care coverage from employment, ensuring uninterrupted coverage and eliminating employers' administrative role in providing coverage. Federal and state taxes would replace present methods of financing by private insurance premiums and large out-of pocket expenditures. Building on the present system of health plans, Health USA would offer all persons a wide choice of competing plans in which to enroll and offer professional providers a wide choice of plans in which to practice. It would control costs by increasing financial accountability of providers and health plans, reducing present reliance on intrusive utilization review and on patient cost sharing. By controlling health care and administrative costs, Health USA would cover the entire population and, according to independent cost estimates, reduce national health expenditures by $11.5 billion in 1991. PMID- 1729580 TI - Psychosocial influences on mortality of patients with coronary heart disease. PMID- 1729581 TI - A tradition of testing ironclad practices. PMID- 1729582 TI - Follow-up after coronary artery bypass surgery. PMID- 1729583 TI - Occupational exposure to Freon: an association with severe hypertension? PMID- 1729584 TI - Variation in health service use among HIV-infected patients. AB - The effects of sociodemographic factors on health service use among people with human immunodeficiency virus (HIV) infection are assessed. Data are from a survey of 939 clients of the Robert Wood Johnson Foundation's AIDS Health Services Program in nine communities across the country. Dependent variables are the number of outpatient visits, use of the emergency room, and whether the respondent had been admitted as an inpatient. In the 3 months before the interview, the sample averaged 7.46 outpatient physician/clinic visits: 35.9% reported an emergency room visit, and 29.9% had been hospitalized. The data suggested differential patterns of health service use, such that those who are white, male, and non-intravenous drug users have higher rates of outpatient clinic/physician use, whereas those who are nonwhite, female, and intravenous drug users have higher rates of emergency room use. Whether these observed differences are attributable to the system's response to different socioeconomic groups, or to differences in individual orientations toward use of medical care is discussed. PMID- 1729585 TI - Characteristics of children with high and low usage of physician services. AB - This study examines utilization of physician services using a sample of 17,110 children younger than 18 years from the Child Health Supplement to the 1988 National Health Interview Survey on Child Health. Although children averaged 3 contacts with physicians, 21% of children did not use physician services and 7% had 10 or more contacts and accounted for 37% of all contacts for children during 1988. Age and ethnicity of the child; family income, health insurance status, size, and area of residence; and mother's educational attainment were important sociodemographic correlates of low usage. In contrast, demographic and socioeconomic characteristics were found to be only modest predictors of high usage. Children's health characteristics, especially number of childhood health conditions, were highly predictive of both high and low physician services usage. Compared with children who had no reported conditions, children with multiple conditions were eight times as likely to be high users and were only a fourth as likely to be low users. Implications of these results are discussed. PMID- 1729586 TI - Explaining area variation in the use of Medicare home health services. AB - This study examines the determinants of area-level variation in Medicare home health use in 1985 for the entire United States, using data from Medicare Home Health Bills, the Medicare/Medicaid Automated Certification System, the Medicare Provider Analysis and Review Files, and other sources. Weighted two-stage least squares regression was used to analyze variation in the number of home health users per 1,000 enrollees and the average number of visits received per user. The data were aggregated to the Metropolitan Statistical Area and the rural part of the state, resulting in 343 units of analysis. According to the study's results, higher proportions of Medicare enrollees use home health services in areas with fewer nursing home beds per enrollee, higher hospital discharge rates, and shorter mean lengths of stay, higher Medicare reimbursement ceilings for skilled nursing home health visits, and more home health agencies per enrollee. Other things being equal, beneficiaries in New England are 40% more likely to use home health services than their counterparts in other regions with similar climates. The average number of visits received by home health users appears to be higher in areas where there are more agencies per enrollee and a higher share of agencies that are proprietary. There also appear to be large regional differences in the number of visits received per user. Our results imply that constrained access to nursing home beds is leading to higher levels of Medicare home health use and that there may be further savings from the substitution of home health services for hospital days. The study shows that Medicare reimbursement ceilings may constrain use and that access may be a problem for beneficiaries in areas with fewer agencies per enrollee. This study also points to significant regional variation in the proportion of beneficiaries who use home health services, even with controls for many different explanatory variables. Overall, our results suggest the possibility of serious limitations in access to Medicare home health services. PMID- 1729587 TI - Determinants of physician acceptance of assignment: an examination of Medicare beneficiary characteristics. AB - This study examines the degree to which patient characteristics predict physicians' ad hoc decisions regarding acceptance of Medicare assignment. The study is based on a random sample of Medicare Part B enrollees living independently in the Salem, Oregon metropolitan area. Beneficiary characteristics and beneficiary reports of physician behavior are obtained from an hour long face to-face survey. The findings show that patient characteristics are significant predictors of physician behavior. Those respondents with poor health, no supplementary coverage to Medicare Part B, and who are more sensitive to the cost of health care are significantly more likely to report that their physician accepts assignment than are respondents without these characteristics. Policy and research implications are discussed. PMID- 1729589 TI - Volume-outcome relationships and in-hospital mortality: the effect of changes in volume over time. AB - This study examines whether patient outcomes are affected by changes in volume over time within hospitals and whether such effects are consistent with cross sectional results previously reported in the literature. Investigating the existence of volume-outcome relationships longitudinally for specific groups of patients relates directly to the policy issue of whether, and how, specific inpatient services should be regionalized. The analysis uses up to 8 years of observations from a national sample of nearly 500 community hospitals. Outcomes are measured as inhospital mortality adjusted for case severity. Instrumental variables techniques are used to test and control for the possibility of selective referral. The results suggest that higher volume leads to better outcomes for certain groups of patients. Among the groups studied here, increases in volume lowered adjusted mortality rates for acute myocardial infarction, hernia repair, and respiratory distress syndrome in neonates; correlations were observed between volume and outcome for coronary artery bypass grafts, which seemed to be due primarily to referral patterns; and, no significant findings were found for hip replacements. In general, the effects of volume on outcome appear to be larger when estimated from longitudinal, rather than cross sectional, data. PMID- 1729588 TI - Adequacy and duration of antidepressant treatment in primary care. AB - Among a sample of 119 distressed high-utilizers of primary care, 45% of patients evaluated by a psychiatrist as needing antidepressant treatment had been treated in the year before the examination. However, only 11% of the patients needing antidepressants had received adequate dosage and duration of pharmacotherapy. In the year following the intervention, study patients whose physicians were advised regarding treatment during a psychiatric consultation were more likely to receive antidepressant medications (52.7%) relative to a randomized control group (36.1%). However, the intervention did not significantly increase the provision of adequate antidepressant therapy (37.1% vs 27.9%). Among study patients using antidepressants, patient characteristics did not differentiate patients who received adequate dosage and duration of antidepressant medications from those who did not. Analysis of data on the duration of antidepressant therapy for all health maintenance organization enrollees initiating use of antidepressants showed that only 20% of patients who had been given prescriptions for first generation antidepressants (amitriptyline, imipramine, or doxepin) filled four or more prescriptions in the following six months, compared to 34% of patients who had prescriptions for newer antidepressants (nortriptyline, desipramine, trazodone and fluoxetine). Experimental research evaluating whether these newer medications (with more favorable side effect profiles) improve adherence, and thereby patient outcome, is needed. PMID- 1729590 TI - Antimicrobial prophylaxis in surgery. PMID- 1729591 TI - Interferon induction of gene transcription analyzed by in vivo footprinting. AB - The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription. PMID- 1729592 TI - Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5'-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions. AB - We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S adenosylmethionine-dependent methylation reactions. We further show that the LPS induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway. PMID- 1729593 TI - Test of the double-strand-break repair model of recombination in Xenopus laevis oocytes. AB - A direct test was made of predictions of the double-strand-break repair (DSBR) model of recombination in Xenopus laevis oocytes. The DNA substrate injected into oocytes had two directly repeated copies of a 1.25-kb sequence and was cleaved within one of them. Different products were expected to result from concerted, conservative events, as predicted by the DSBR model, and from nonconservative events. Only very low levels of recombination products, both conservative and nonconservative, were observed. When individual, apparent DSBR products were cloned and characterized, it emerged that the majority of them had arisen by nonconservative recombination through short, terminal homologies and not from the gene conversion events predicted for DSBR. Two cloned products among 44 tested corresponded to the predications of the DSBR model, but these could also have been generated by other processes. The most efficient recombination events in oocytes are nonconservative and are based on long, terminal homologous overlaps; when these are not available, short, imperfect overlaps support a lower level of nonconservative recombination; genuine, conservative DSBR events occur rarely, if at all. PMID- 1729594 TI - A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression. AB - The gene encoding mZP3, the mouse sperm receptor, is expressed exclusively in growing oocytes during oogenesis. To investigate the molecular basis of oocyte specific mZP3 gene expression, we generated several lines of mice harboring a transgene that contains 470 bp of mZP3 gene 5'-flanking sequence (nucleotides 470 to +10) fused to the firefly luciferase gene coding region. Three of four expressing transgenic lines exhibited luciferase activity only in growing oocytes, suggesting that the 470-bp fragment is sufficient to direct Iocyte specific expression of the luciferase gene. Results of DNase I footprinting and gel mobility shift assays suggested the presence of an ovary-specific protein that binds to a small region (nucleotides-99 to -86) within the 470-bp fragment of the mZP3 promoter, with 5'-G(G/A)T(G/A)A-3' representing the minimal sequence required for binding. Southwestern (DNA-protein) gel blots revealed the presence of an oocyte-specific, approximately 60,000-Mr protein, called OSP-1, that binds to the minimal sequence. Changes in levels of OSP-1 during oogenesis and early cleavage are consistent with the pattern of mZP3 gene expression during these developmental stages in mice. Therefore, OSP-1 may be a mammalian oocyte-specific transcription factor involved in regulating oocyte-specific mZP3 gene expression. PMID- 1729595 TI - A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C. AB - Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self phosphorylated EGF receptors and PLC-gamma is necessary for enzyme activation in vivo. We propose that both binding of PLC-gamma to activated EGF receptors and tyrosine phosphorylation of the enzyme are necessary to elicit biological responses. Kinase-active EGF receptors lacking sites of tyrosine phosphorylation are unable to signal increased inositol phosphate formation and increases in cytosolic Ca2+ concentration. PMID- 1729596 TI - Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. AB - At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences. PMID- 1729597 TI - Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. AB - The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1. PMID- 1729598 TI - Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes. AB - Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells. PMID- 1729599 TI - Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs. AB - We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA. PMID- 1729600 TI - GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme. AB - In the yeast Saccharomyces cerevisiae, glycogen serves as a major storage carbohydrate. In a previous study, mutants with altered glycogen metabolism were isolated on the basis of the altered iodine-staining properties of colonies. We found that when glycogen produced by strains carrying the glc-1p (previously called gha1-1) mutation is stained with iodine, the absorption spectrum resembles that of starch rather than that of glycogen, suggesting that this mutation might reduce the level of branching in the glycogen particles. Indeed, glycogen branching activity was undetectable in extracts from a glc3-1p strain but was elevated in strains which expressed GLC3 from a high-copy-number plasmid. These observations suggest that GLC3 encodes the glycogen branching enzyme. In contrast to glc3-1p, the glc3-4 mutation greatly reduces the ability of yeast to accumulate glycogen. These mutations appear to be allelic despite the striking difference in the phenotypes which they produce. The GLC3 clone complemented both glc3-1p and glc3-4. Deletions and transposon insertions in this clone had parallel effects on its ability to complement glc3-1p and glc3-4. Finally, a fragment of the cloned gene was able to direct the repair of both glc3-1p and glc3-4. Disruption of GLC3 yielded the glycogen-deficient phenotype, indicating that glycogen deficiency is the null phenotype. The glc3-1p allele appears to encode a partially functional product, since it is dominant over glc3-4 but recessive to GLC3. These observations suggest that the ability to introduce branches into glycogen greatly increases the ability of the cell to accumulate that polysaccharide. Northern (RNA) blot analysis identified a single mRNA of 2,300 nucleotides that increased in abundance ca. 20-fold as the culture approached stationary phase. It thus appears that the expression of GLC3 is regulated, probably at the level of transcription. PMID- 1729601 TI - Selective activation of a discrete family of endogenous proviral elements in normal BALB/c lymphocytes. AB - Intracisternal A-particle (IAP) proviral elements are abundant and widely dispersed in the mouse genome. IAP-related transcripts have been detected in normal mouse tissues where expression is under genetic control. In this study, we sought to determine whether IAP expression in BALB/c thymus and lipopolysaccharide-stimulated B cells was due to selective or indiscriminate activation of IAP elements. cDNA libraries were prepared from each source. A total of 86 IAP cDNA clones were isolated from both libraries, and 37 of these were sequenced over a common 0.7- to 1.0-kb region of the IAP genome that included the 3' long terminal repeat (LTR). Three highly related families of elements were found to be expressed in the two cell types examined. All of the related elements had a distinctive U3 regulatory region. Thirteen individual IAP proviral elements were distinguished on the basis of sequence differences within the R region of the LTR. Hybridization of genomic DNA with element-specific oligonucleotide probes confirmed the presence of a restricted number of proviral copies in the lymphocyte-specific family of elements. Most of these copies were found to be methylated in the lymphocyte DNA, but at least seven were hypomethylated in their 5' LTRs. This study shows that activation of IAP elements in normal normal mouse lymphocytes is highly selective. Activation is probably a function of both sequence specificity and methylation status of the proviral LTR. PMID- 1729602 TI - The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon. AB - We initiated a genetic reversion analysis at the HIS4 locus to identify components of the translation initiation complex that are important for ribosomal recognition of an initiator codon. Three unlinked suppressor loci, suil, sui2, and SUI3, that restore expression of both HIS4 and HIS4-lacZ in the absence of an AUG initiator codon were identified. In previous studies, it was demonstrated that the sui2 and SUI3 genes encode mutated forms of the alpha and beta subunits, respectively, of eukaryotic translation initiation factor 2 (eIF-2). In this report, we describe the molecular and biochemical characterizations of the sui1 suppressor locus. The DNA sequence of the SUI1+ gene shows that it encodes a protein of 108 amino acids with a calculated Mr of 12,300. The sui1 suppressor genes all contain single base pair changes that alter a single amino acid within this 108-amino-acid sequence. sui1 suppressor strains that are temperature sensitive for growth on enriched medium have altered polysome profiles at the restrictive temperature typical of those caused by alteration of a protein that functions during the translation initiation process. Gene disruption experiments showed that the SUI1+ gene encodes an essential protein, and antibodies directed against the SUI1+ coding region identified a protein with the predicted Mr in a ribosomal salt wash fraction. As observed for sui2 and SUI3 suppression events, protein sequence analysis of His4-beta-galactosidase fusion proteins produced by sui1 suppression events indicated that a UUG codon is used as the site of translation initiation in the absence of an AUG start codon in HIS4. Changing the penultimate proline codon 3' to UUG at his4 to a Phe codon (UUC) blocks aminopeptidase cleavage of the amino-terminal amino acid of the His4-beta galactosidase protein, as noted by the appearance of Met in the first cycle of the Edman degradation reaction. The appearance of Met in the first cycle, as noted, in either a sui1 or a SUI3 suppressor strain showed that the mechanism of suppression is the same for both suppressor genes and allows the initiator tRNA to mismatch base pair with the UUG codon. This suggests that the Sui1 gene product performs a function similar to that of the beta subunit of eIF-2 as encoded by the SUI3 gene. However, the Sui1 gene product does not appear to be a required subunit of eIF-2 on the basis of purification schemes designed to identify the GTP-dependent binding activity of eIF-2 for the initiator tRNA. In addition, suppressor mutations in the sui1 gene, in contrast to suppressor mutations in the sui2 or SUI3 gene, do not alter the GTP-dependent binding activity of the eIF-2. The simplest interpretation of these studies is that the sui1 suppressor gene defines an additional factor that functions in concert with eIF-2 to enable tRNAiMet to establish ribosomal recognition of an AUG initiator codon. PMID- 1729603 TI - Evidence for the involvement of protein kinase activity in transforming growth factor-beta signal transduction. AB - Transforming growth factor-beta 1 (TGF-beta 1) rapidly increases the expression of junB transcription factor and plasminogen activator inhibitor-1 (PAI-1) and prevents the cell cycle-dependent phosphorylation of the RB retinoblastoma susceptibility gene product during late G1 phase in Mv1Lu lung epithelial cells. These responses are shown in this report to be blocked by the potent serine/threonine protein kinase inhibitor, H7, added with TGF-beta 1. Added alone, H7 does not alter the basal junB or PAI-1 mRNA levels, the deposition of PAI-1 into the extracellular matrix, or the phosphorylation of RB in late G1 phase, suggesting that this inhibitor does not have a general nonspecific effect on the cell. The analogs H8 and H9, which are preferential inhibitors of cyclic nucleotide-dependent protein kinases, are fivefold less potent than H7 as inhibitors of the TGF-beta response. The PAI-1 response to TGF-beta 1 is not affected by the simultaneous addition of staurosporine, which is a protein kinase C inhibitor, or by the prolonged preincubation of cells with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C. The results suggest the possibility that H7 and its analogs block various early TGF-beta responses by inhibiting a protein serine/threonine kinase(s). PMID- 1729604 TI - C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae. AB - We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane. PMID- 1729605 TI - MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import. AB - The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae. The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene. The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1. Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures. Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures. The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C. These results indicate a role for a DnaJ cognate protein in mitochondrial protein import. PMID- 1729606 TI - The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA. AB - RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA. PMID- 1729607 TI - Two conserved essential motifs of the murine immunoglobulin lambda enhancers bind B-cell-specific factors. AB - Two highly homologous enhancers associated with the two murine immunoglobulin lambda constant-region clusters were recently identified. In order to better understand the molecular basis for the developmental stage- and cell-type restricted expression of lambda genes, we have undertaken an analysis of the putative regulatory domains of these enhancers. By using a combination of DNase I footprinting, electrophoretic mobility shift assay, and site-specific mutations, four candidate protein binding sites have been identified at analogous positions in both enhancers. A mutation of any of these sites decreases enhancer activity. Two of the sites, lambda A and lambda B, are essential for enhancer function, and both of these sites appear to bind both B-cell-specific and general factors. Nevertheless, isolated lambda A and lambda B sites show no evidence of inherent transactivating potential, alone or together, even when present in up to three copies. We suggest that the generation of transactivating signals from these enhancers may require the complex interaction of multiple B-cell-specific and nonspecific DNA-binding factors. PMID- 1729608 TI - Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays. AB - Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks. PMID- 1729609 TI - Molecular characterization of the lam locus and sequences involved in regulation by the AmdR protein of Aspergillus nidulans. AB - The lam locus of Aspergillus nidulans consists of two divergently transcribed genes, lamA and lamB, involved in the utilization of lactams such as 2 pyrrolidinone. Both genes are under the control of the positive regulatory gene amdR and are subject to carbon and nitrogen metabolite repression. The lamB gene and the region between the two genes have been sequenced, and the start points of transcription have been determined. Within the lam locus are two sequences with homology to elements, required for AmdR regulation, found in the 5' regions of the coregulated genes amdS and gatA. In vitro and in vivo assays were used to investigate the lam and gatA regulatory elements. One of the three gatA elements and one of the two lam elements were shown to bind AmdR protein in vivo and activate transcription. With a gel shift mobility assay, in vitro binding of AmdR protein to the functional gatA element was detected. Both the functional gatA and lam boxes contain within them a CAAT sequence. In vitro binding analysis indicates that a CCAAT-specific factor(s) binds at these sequences, adjacent to or overlapping the AmdR protein-binding site. PMID- 1729610 TI - Gene replacement with one-sided homologous recombination. AB - Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering. PMID- 1729611 TI - PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity. AB - PU.1 is a B-cell- and macrophage-specific transcription factor. By an electrophoretic mobility shift assay and dimethyl sulfate methylation interference assays, we show that PU.1 binds to DNA sequences within the immunoglobulin kappa 3' enhancer (kappa E3'). Binding of PU.1 to the kappa E3' enhancer assists the binding of a second tissue-restricted factor, NF-EM5, to an adjacent site. Binding of NF-EM5 to kappa E3' DNA sequences requires protein protein interaction with PU.1 as well as specific protein-DNA interactions. This is the first known instance of PU.1 interacting with another cellular protein. NF EM5 does not cofractionate with PU.1, suggesting that it is a distinct protein and is not a posttranslational modification of PU.1. UV-crosslinking studies and elution from sodium dodecyl sulfate-polyacrylamide gels indicate that NF-EM5 is a protein of approximately 46 kDa. Site-directed mutagenesis studies of the PU.1- and EM5-binding sites indicate that these sites play important roles in kappa E3' enhancer activity. By using a series of PU.1 deletion constructs, we have identified a region in PU.1 that is necessary for interaction with NF-EM5. This segment encompasses a 43-amino-acid region with PEST sequence homology, i.e., one that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T). PMID- 1729613 TI - Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ. AB - The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence. PMID- 1729612 TI - NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes. AB - We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation. PMID- 1729614 TI - Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice. AB - High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT 2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression. PMID- 1729615 TI - Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo. AB - We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into positioned nucleosomes. Evidence is presented supporting the stable association of transcription factors with the somatic 5S RNA genes. Histone H1 is shown to have a role both in determining the organization of nucleosomes over the oocyte 5S DNA repeat and in repressing transcription of the oocyte 5S RNA genes. PMID- 1729616 TI - Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine. AB - We have determined the sequences and positions of the cis elements required for proper functioning of the ARG3 promoter and proper arginine-specific control. A TATA box located 100 nucleotides upstream of the transcription start was shown to be essential for ARG3 transcription. Two sequences involved in normal arginine mediated repression lie immediately downstream of the TATA box: an essential one (arginine box 1 [AB1]) and a secondary one (arginine box 2 [AB2]). AB1 was defined by saturation mutagenesis and is an asymmetrical sequence. A stringently required CGPu motif in AB1 is conserved in all known target sites of C6 zinc cluster DNA-binding proteins, leading us to propose that AB1 is the binding site of ARGRII, another member of the C6 family. The palindromic AB2 sequence is suggested, on the basis of published data, to be the binding site of ARGRI, possibly in heterodimerization with MCM1. AB2 and AB1 correspond respectively to the 5' and 3' halves of two adjacent similar sequences of 29 bp that appear to constitute tandem operators. Indeed, mutations increasing the similarity of the other halves with AB1 and AB2 cause hyperrepression. To mediate repression, the operator must be located close to the transcription initiation region. It remains functional if the TATA box is moved downstream of it but becomes inoperative in repression when displaced to a far-upstream position where it mediates an arginine and ARGR-dependent induction of gene expression. The ability of the ARG3 operator to act either as an operator or as an upstream activator sequence, depending on its location, and the functional organization of the anabolic and catabolic arginine genes suggest a simple model for arginine regulation in which an activator complex can turn into a repressor when able to interfere sterically with the process of transcription initiation. PMID- 1729617 TI - Treatment of postmenopausal osteoporosis with calcitriol or calcium. AB - BACKGROUND AND METHODS: Osteoporosis is a common problem whose management is controversial. To evaluate the efficacy and safety of calcitriol (1,25 dihydroxyvitamin D3) in the treatment of postmenopausal osteoporosis, we conducted a three-year prospective, multicenter, single-blind study in 622 women who had one or more vertebral compression fractures. The women were randomly assigned to receive treatment with calcitriol (0.25 micrograms twice a day) or supplemental calcium (1 g of elemental calcium daily) for three years. New vertebral fractures were detected by means of lateral roentgenography of the spine each year, and calcium absorption was measured in 392 of the women. RESULTS: The women who received calcitriol had a significant reduction in the rate of new vertebral fractures during the second and third years of treatment, as compared with the women who received calcium (second year, 9.3 vs. 25.0 fractures per 100 patient-years; third year, 9.9 vs. 31.5 fractures per 100 patient-years; P less than 0.001). This effect was evident only in women who had had five or fewer vertebral fractures at base line (second year, 5.2 vs. 25.3 fractures per 100 patient-years; third year, 4.2 vs. 31.0 fractures per 100 patient-years; P less than 0.0001). The groups also differed significantly in the number of peripheral fractures; 11 such fractures occurred in 11 women in the calcitriol group, whereas 24 occurred in 22 women in the calcium group (P less than 0.05). There was no significant difference between the groups in the incidence of side effects requiring withdrawal of treatment (8.6 percent in the calcitriol group vs. 6.5 percent in the calcium group). CONCLUSIONS: Continuous treatment of postmenopausal osteoporosis with calcitriol for three years is safe and significantly reduces the rate of new vertebral fractures in women with this disorder. PMID- 1729618 TI - Risk of cancer in patients with dermatomyositis or polymyositis. A population based study. AB - BACKGROUND: An association between polymyositis and cancer was first proposed in 1916, but the existence of the association has been disputed. An association between dermatomyositis and cancer is better accepted, but its magnitude is not known. METHODS: We undertook a study to provide accurate estimates of the risk of cancer in patients with dermatomyositis or polymyositis. We studied the incidence of cancer and the rate of mortality from cancer in a population-based cohort of 788 patients with dermatomyositis or polymyositis in Sweden from 1963 through 1983. The results were compared with those for the general population. RESULTS: Among the 396 patients with polymyositis, 42 cancers were diagnosed at the same time or after polymyositis was diagnosed in 37 patients (9 percent). The relative risk of cancer was 1.8 (95 percent confidence interval, 1.1 to 2.7) in the male patients and 1.7 (95 percent confidence interval, 1.0 to 2.5) in the female patients. Eighty-four males and 85 females died, and in 24 of these cases (14 percent) cancer was the principal cause of death. The mortality ratio (the rate of mortality from cancer in these patients as compared with that in the general population) was 0.90 (95 percent confidence interval, 0.6 to 1.4). Among the 392 patients with dermatomyositis, 61 cancers were diagnosed at the same time or after dermatomyositis was diagnosed in 59 patients (15 percent). The relative risk of cancer was 2.4 (95 percent confidence interval, 1.6 to 3.6) in the male patients and 3.4 (95 percent confidence interval, 2.4 to 4.7) in the female patients. Fifty-seven males and 110 females died, and in 67 of these cases (40 percent) cancer was the principal cause of death (mortality ratio, 3.8; 95 percent confidence interval, 2.9 to 4.8). CONCLUSIONS: The risk of cancer is increased in patients with polymyositis or dermatomyositis. In patients with dermatomyositis there is also a higher rate of mortality from cancer. PMID- 1729619 TI - Topical tretinoin (retinoic acid) treatment for liver spots associated with photodamage. AB - BACKGROUND: The hyperpigmented lesions commonly called liver spots distress patients, in part because such lesions are associated with aging. We investigated their treatment with topical 0.1 percent tretinoin (retinoic acid). METHODS: Fifty-eight patients completed a 10-month randomized, double-blind study in which they applied either 0.1 percent tretinoin (n = 28) or vehicle (n = 30) cream daily to the face, upper extremities, or both. Fifteen patients who responded well were than randomly assigned to continue tretinoin therapy or use vehicle alone for six more months. Patients were evaluated by physical examination every month and by analysis of biopsy specimens of lesions obtained at base line and at the end of the 10-month trial. RESULTS: After one month of treatment the patients treated with tretinoin had significant lightening of hyperpigmented lesions as compared with the patients who received vehicle (P less than 0.002). After 10 months, 20 (83 percent) of the 24 patients with facial lesions who were treated with tretinoin had lightening of these lesions, as compared with 8 (29 percent) of the 28 patients with facial lesions who received vehicle. The results for lesions of the upper extremities were similar. As compared with vehicle, tretinoin caused a significant decrease in the degree of epidermal pigmentation and increases in the degree of compaction of stratum corneum, thickness of the granular cell layer, and epidermal thickness. Reductions in epidermal pigmentation evident on histologic analysis were significantly correlated with the degree of clinical lightening of lesions (r = -0.53, P less than 0.0001). During the 6-month follow-up study, specifically identified lesions that had disappeared during the first 10 months of tretinoin treatment did not return in any patient, and six of seven patients who continued to use tretinoin had further improvement. CONCLUSIONS: Topical 0.1 percent tretinoin significantly improves both clinical and microscopical manifestations of liver spots; these lesions do not return for at least six months after therapy is discontinued. PMID- 1729620 TI - Seroprevalence of HTLV-1 and HTLV-2 among intravenous drug users and persons in clinics for sexually transmitted diseases. AB - Background. The human T-cell lymphotropic virus Type I (HTLV-I) is associated with adult T-cell leukemia and myelopathy, whereas HTLV-II infection has uncertain clinical consequences. We assessed the seroprevalence of these retroviruses among intravenous drug users and among patients seen at clinics for sexually transmitted diseases (STD clinics). METHODS: We used serum samples that were collected in eight cities in 1988 and 1989 during surveys of human immunodeficiency virus infection among intravenous drug users entering treatment and persons seen in STD clinics. The serum samples were tested for antibodies to HTLV, and positive specimens were tested further by a synthetic peptide-based enzyme-linked immunosorbent assay to differentiate between HTLV-I and HTLV-II. RESULTS: Among 3217 intravenous drug users in 29-drug-treatment centers, the median seroprevalence rates of HTLV varied widely according to city (range, 0.4 percent in Atlanta to 17.6 percent in Los Angeles). Seroprevalence increased sharply with age, to 32 percent in persons over 44 years of age. HTLV infection was more common among blacks (15.5 percent) and Hispanics (10.7 percent) than among whites (4.1 percent), and it was strongly associated with a history of heroin injection (P less than or equal to 0.001). Among 5264 patients in 24 STD clinics, the median rates of HTLV infection were much lower (range, 0.1 percent in Atlanta and Newark to 2.0 percent in Los Angeles). Again, this infection was more common among intravenous drug users (7.6 percent) than among non-drug users (0.7 percent). Eighty-four percent of the seropositive samples from drug treatment centers and 69 percent of those from STD clinics were due to HTLV-II infection (P = 0.03). CONCLUSIONS: HTLV infections are common among intravenous drug users and are primarily caused by HTLV-II. Among patients seen at STD clinics, HTLV is strongly associated with intravenous drug use, but the retrovirus is also prevalent among non-drug users. PMID- 1729622 TI - Where have all the primary care applicants gone? PMID- 1729621 TI - Mortality over a period of 10 years in patients with peripheral arterial disease. AB - BACKGROUND: Previous investigators have observed a doubling of the mortality rate among patients with intermittent claudication, and we have reported a fourfold increase in the overall mortality rate among subjects with large-vessel peripheral arterial disease, as diagnosed by noninvasive testing. In this study, we investigated the association of large-vessel peripheral arterial disease with rates of mortality from all cardiovascular diseases and from coronary heart disease. METHODS: We examined 565 men and women (average age, 66 years) for the presence of large-vessel peripheral arterial disease by means of two noninvasive techniques--measurement of segmental blood pressure and determination of flow velocity by Doppler ultrasound. We identified 67 subjects with the disease (11.9 percent), whom we followed prospectively for 10 years. RESULTS: Twenty-one of the 34 men (61.8 percent) and 11 of the 33 women (33.3 percent) with large-vessel peripheral arterial disease died during follow-up, as compared with 31 of the 183 men (16.9 percent) and 26 of the 225 women (11.6 percent) without evidence of peripheral arterial disease. After multivariate adjustment for age, sex, and other risk factors for cardiovascular disease, the relative risk of dying among subjects with large-vessel peripheral arterial disease as compared with those with no evidence of such disease was 3.1 (95 percent confidence interval, 1.9 to 4.9) for deaths from all causes, 5.9 (95 percent confidence interval, 3.0 to 11.4) for all deaths from cardiovascular disease, and 6.6 (95 percent confidence interval, 2.9 to 14.9) for deaths from coronary heart disease. The relative risk of death from causes other than cardiovascular disease was not significantly increased among the subjects with large-vessel peripheral arterial disease. After the exclusion of subjects who had a history of cardiovascular disease at base line, the relative risks among those with large-vessel peripheral arterial disease remained significantly elevated. Additional analyses revealed a 15-fold increase in rates of mortality due to cardiovascular disease and coronary heart disease among subjects with large-vessel peripheral arterial disease that was both severe and symptomatic. CONCLUSIONS: Patients with large-vessel peripheral arterial disease have a high risk of death from cardiovascular causes. PMID- 1729623 TI - The many pitfalls in the diagnosis of myeloma. PMID- 1729624 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 6-1992. A 67-year-old man with aphasia and memory loss, followed by progressive dementia. PMID- 1729625 TI - Osteoporosis and its treatment. PMID- 1729626 TI - Primary care applicants--they get no respect. PMID- 1729627 TI - Medical evaluation of children adopted from abroad. PMID- 1729628 TI - Medical evaluation of children adopted from abroad. PMID- 1729629 TI - Medical evaluation of children adopted from abroad. PMID- 1729630 TI - Transmission of HCV by organ transplantation. PMID- 1729631 TI - Transmission of HCV by organ transplantation. PMID- 1729632 TI - Limiting access and patient selection in liver transplantation. PMID- 1729633 TI - Long-term effects of a cholesterol-free diet on serum cholesterol levels in Zen monks. PMID- 1729634 TI - Ratio of low-density lipoprotein cholesterol to ubiquinone as a coronary risk factor. PMID- 1729635 TI - GM-CSF and accelerated hemolysis. PMID- 1729636 TI - Using genes to define motherhood--the California solution. PMID- 1729637 TI - Animal experimentation. AFRC lays down the law. PMID- 1729638 TI - Authorship. Writer's cramp. PMID- 1729639 TI - AIDS treatment. Confusion over therapy. PMID- 1729640 TI - Burt files reopened. PMID- 1729641 TI - Consistent policy? PMID- 1729642 TI - Nature of human sexuality. PMID- 1729643 TI - Patenting of genes. PMID- 1729644 TI - Patenting of genes. PMID- 1729645 TI - Malaria. How chloroquine works. PMID- 1729646 TI - Antigen processing. Who needs peptide transporters? PMID- 1729647 TI - Neural networks. Learning from your neighbour. PMID- 1729648 TI - Crystallization by centrifugation. PMID- 1729649 TI - Mirror-script and left-handedness. PMID- 1729650 TI - Self-organizing neural network that discovers surfaces in random-dot stereograms. AB - The standard form of back-propagation learning is implausible as a model of perceptual learning because it requires an external teacher to specify the desired output of the network. We show how the external teacher can be replaced by internally derived teaching signals. These signals are generated by using the assumption that different parts of the perceptual input have common causes in the external world. Small modules that look at separate but related parts of the perceptual input discover these common causes by striving to produce outputs that agree with each other. The modules may look at different modalities (such as vision and touch), or the same modality at different times (for example, the consecutive two-dimensional views of a rotating three-dimensional object), or even spatially adjacent parts of the same image. Our simulations show that when our learning procedure is applied to adjacent patches of two-dimensional images, it allows a neural network that has no prior knowledge of the third dimension to discovery depth in random dot stereograms of curved surfaces. PMID- 1729651 TI - Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. AB - The incidence of human malaria has increased during the past 20 years; 270 million people are now estimated to be infected with the parasite. An important contribution to this increase has been the appearance of malaria organisms resistant to quinoline-containing antimalarials such as chloroquine and quinine. These drugs accumulate in the acid food vacuoles of the intraerythrocytic-stage malaria parasite, although the mechanism of their specific toxicity in this organelle is uncertain. The primary function of the food vacuole is the proteolysis of ingested red cell haemoglobin to provide the growing parasite with essential amino acids. Haemoglobin breakdown in the food vacuole releases haem, which if soluble can damage biological membranes and inhibit a variety of enzymes. Rather than degrading or excreting the haem, the parasite has evolved a novel pathway for its detoxification by incorporating it into an insoluble crystalline material called haemozoin or malaria pigment. These crystals form in the food vacuole of the parasite concomitant with haemoglobin degradation, where they remain until the infected red cell bursts. The structure of haemozoin comprises a polymer of haems linked between the central ferric ion of one haem and a carboxylate side-group oxygen of another. This structure does not form spontaneously from either free haem or haemoglobin under physiological conditions, and the biochemistry of its formation is unclear. Here we report the identification and characterization of a haem polymerase enzyme activity from extracts of Plasmodium falciparum trophozoites, and show that this enzyme is inhibited by quinoline-containing drugs such as chloroquine and quinine. This provides a possible explanation for the highly stage-specific antimalarial properties of these drugs. PMID- 1729652 TI - Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway. AB - The transport of proteins destined for post-endoplasmic reticulum locations in the secretory pathway is mediated by small vesicular carriers. Transport vesicles have been generated in cell-free assays from the yeast Saccharomyces cerevisiae, and mammalian systems. Yeast genes encoding cytosolic components that participate in vesicular traffic were first identified from the collection of conditional lethal sec-(secretory) mutants. Mutations in the yeast SEC7 gene disrupt protein transport in the secretory pathway at the nonpermissive temperature. The SEC7 gene product is a phosphoprotein of relative molecular mass 230,000 that functions from the cytoplasmic aspect of intracellular membranes. We report that in a yeast cell-free transport assay, the introduction of antibodies to Sec7 protein (Sec7p) results in the accumulation of transport vesicles. These vesicles are retrieved with Sec7p-specific antibodies by immuno-isolation for biochemical and electron microscopic characterization. Sec7p on the surface of the accumulated transport vesicles, in combination with previous genetic and biochemical studies, implicate Sec7p as part of a (non-clathrin) vesicle coat. This Sec7p-containing coat structure is proposed to be essential for vesicle budding at multiple stages in the yeast secretory pathway. PMID- 1729654 TI - Paying for research. PMID- 1729655 TI - US government targets indirect cost agreements. PMID- 1729653 TI - New yeast actin-like gene required late in the cell cycle. AB - Actin, a major cytoskeletal component of all eukaryotic cells, is one of the most highly conserved proteins. It is involved in various cellular processes such as motility, cytoplasmic streaming, chromosome segregation and cytokinesis. The actin from the yeast Saccharomyces cerevisiae, encoded by the essential ACT1 gene, is 89% identical to mouse cytoplasmic actin and is involved in the organization and polarized growth of the cell surface. We report here the characterization of ACT2, a previously undescribed yeast split gene encoding a putative protein (391 amino acids, relative molecular mass (Mr) 44,073) that is 47% identical to yeast actin. The requirement of the ACT2 gene for vegetative growth of yeast cells and the existence of related genes in other eukaryotes indicate an important and conserved role for these actin-like proteins. Superimposition of the Act2 polypeptide onto the three-dimensional structure of known actins reveals that most of the divergence occurred in loops involved in actin polymerization, DNase I and myosin binding, leaving the core domain mainly unaffected. To our knowledge, the Act2 protein from S. cerevisiae is the first highly divergent actin molecule described. Structural and physiological data suggest that the Act2 protein might have an important role in cytoskeletal reorganization during the cell cycle. PMID- 1729656 TI - Japanese science. Universities win. PMID- 1729657 TI - Common hand problems. PMID- 1729659 TI - Carpal tunnel syndrome. AB - Attention in the press and in trade publications has created a widespread public awareness of carpal tunnel syndrome, the most frequently encountered peripheral compression neuropathy. Diagnosis and treatment is facilitated by familiarity with its stages of presentation, association with various pathologies, and appropriated use of predictive tests. Controversial issues in operative management are explored based on the introduction of some new techniques and some old ideas. PMID- 1729658 TI - Comminuted distal radius fractures. AB - Comminuted displaced fractures of the distal end of the radius pose a significant treatment challenge. The goal of treatment is to restore functional, painless motion of the wrist and fingers. Although satisfactory results correlate to a large extent with obtaining and maintaining normal anatomy, excessive distraction and a flexed wrist position with external fixation cause more harm than good, even if the anatomy is restored. PMID- 1729660 TI - Cubital tunnel syndrome. AB - Cubital tunnel syndrome is the second most common compressive neuropathy of the upper extremity. Key factors in the history, physical, and differential are outlined to assist the clinician in making an accurate diagnosis. Nonoperative measures and surgical options are reviewed, with medial epicondylectomy being the authors' preferred operative technique. PMID- 1729661 TI - Acute flexor tendon injuries. AB - Current surgical techniques and controlled postoperative mobilization have significantly decreased the amount of intertendinous adhesions that plagued earlier attempts at repair. Nevertheless, the ability to recover normal function after flexor tendon injury remains a challenging and often elusive goal. In the civilian population, good to excellent results may be expected in 69% to 90% of patients. A thorough understanding of tendon anatomy and physiology, attention to atraumatic surgical technique, and a well-designed postoperative therapy regimen will ensure satisfactory results in the majority of cases. Further improvements can be expected as we continue to make advances in the areas of suturing technique, rehabilitation, and pharmacologic intervention. PMID- 1729662 TI - Extensor tendon injuries. AB - A thorough knowledge of anatomy, injury patterns, repair techniques, and evolving rehabilitation methods is necessary to best treat extensor tendon injuries. These injuries are conceptualized as occurring in one of eight zones, which are numbered distally to proximally in the hand and forearm. Even though surgical technique and rehabilitation are specific in each zone, injuries over and proximal to the proximal interphalangeal joint tend to yield less satisfactory results. Dynamic postoperative extension splinting is one factor that is improving long-term results. PMID- 1729663 TI - Fingertip and nailbed injuries. AB - Injuries of the fingertip and nailbed require treatment to minimize pain, speed healing, and shorten the time of functional impairment. Alternatives for therapy include simple dressing changes, graft coverage, and flap transposition. The choice of treatment is based on the location and severity of the injury. PMID- 1729664 TI - Acute management of thermal and electrical burns of the upper extremity. AB - Acute management of upper extremity thermal and electrical injuries requires an aggressive treatment protocol which combines meticulous wound care, intensive hand therapy, and early stable wound coverage to salvage upper extremity function. Electrical injuries inflict severe deep-tissue destruction that frequently results in major limb amputation. PMID- 1729665 TI - Hand infections. AB - The diagnosis and treatment of common hand infections are reviewed. A practical approach to the treatment of these conditions is detailed. This approach emphasizes the anatomic compartments of the hand, the microbiology of infecting organisms, and the patient conditions which modify treatment. Prompt and accurate diagnosis, treatment, and rehabilitation are key for optimal outcome. PMID- 1729666 TI - Metacarpal fractures and dislocations. AB - Metacarpal fractures are common injuries. Most can be treated successfully by closed reduction and cast or splint immobilization. Unstable fractures, however, require internal fixation. Many such techniques are discussed. Metacarpal dislocations are more difficult to diagnose and treat than are metacarpal fractures. Careful attention to detail will help ensure a successful result. PMID- 1729667 TI - Treatment principles for proximal and middle phalangeal fractures. AB - After a proximal phalangeal fracture, optimal results are obtained by methods that permit active interphalangeal joint motion and tendon gliding during fracture healing. Typical apex palmar angulation of proximal phalangeal fractures demonstrates dorsal skeletal shortening and secondary incompetence of the extensor mechanism with PIP joint extensor lag. Apex palmar deformities of the middle phalangeal fractures demonstrate similar problems with skeletal shortening resulting in loss of distal joint extension. Proximal and middle phalangeal shaft fracture deformities rotate about their flexor tendons and their fibro-osseous tunnels. Functional restoration requires accurate skeletal realignment that restores normal skeletal length necessary for extensor tendon competence. A splint that holds the wrist in slight extension and all four finger MP joints in full flexion combined with active interphalangeal joint exercises form the essential elements of postoperative care. PMID- 1729668 TI - Gamekeeper's thumb. AB - The term gamekeeper's thumb is widely used interchangeably with skier's thumb in reference to acute or chronic tears of the ulnar collateral ligament of the thumb MCP joint. The frequency of thumb injury in alpine skiing has not changed with recent changes in pole design. Release of the pole from the hand prior to impact with the ground will minimize injury. Arthrogram may be a useful adjunctive technique to identify the presence of a Stener lesion and strengthen the indications for surgery in borderline cases. The sensitivity and specificity of this test have not been established. Current trends favor acute surgical repair for complete tears of the ulnar collateral ligament of the thumb MCP joint as documented by clinical examination with stress radiography. Good results are obtainable with late reconstruction, and comparable results have been noted with the most commonly used techniques. PMID- 1729669 TI - Chronic wrist pain. AB - Chronic wrist pain is among the most difficult diagnostic and therapeutic challenges faced by hand surgeons. The causes are often elusive, of long duration, and not uncovered easily by routine diagnostic methods. This article outlines a systematic evaluation of the patient with chronic wrist pain to determine which of many potential conditions is the cause of the wrist pain so an effective therapeutic plan can be formulated. PMID- 1729670 TI - Common tendinitis problems in the hand and forearm. AB - Although common and often transitory, tendinitis involving the hand and forearm may be disabling. Most tendinitis conditions will respond to conservative measures. The hand surgeon's best tools to obtain a swift and successful resolution include a careful history and physical examination, coupled with and appreciation of the anatomy of the commonly affected sites. PMID- 1729671 TI - Scaphoid fractures. AB - Scaphoid fractures are often a difficult management problem, most typically affecting young men in their most active and productive years. A systematic approach to diagnosis and treatment can decrease the risk of management error and morbidity. PMID- 1729672 TI - Tennis elbow (lateral epicondylitis). AB - Tennis elbow (lateral epicondylitis) is the pattern of pain most commonly seen at the origin of the wrist extensors from the lateral epicondyle of the humerus and less commonly seen at the origin of the flexor-pronator from the medial epicondyle. This article discusses methods of diagnosis and both conservative and operative treatment techniques. PMID- 1729673 TI - Osteoarthritis at the base of the thumb. AB - Osteoarthritis at the base of the thumb is a functionally disabling condition and, as such, the basal joint complex represents the most commonly operated focus in the osteoarthritic upper extremity. Degenerative disease is predicted on instability of the trapeziometacarpal joint secondary to deterioration of the capsular and ligamentous restraints. Ligament reconstruction in the setting of a symptomatic hypermobile joint with intact cartilage surfaces may retard progression of arthritic disease. Complications related to silicone implant arthroplasty have given way to ligament reconstruction tendon interposition arthroplasty as the preferred procedure for the osteoarthritic thumb basal joint complex. PMID- 1729674 TI - Expression cloning of a Na(+)-independent neutral amino acid transporter from rat kidney. AB - Uptake of long-chain and aromatic neutral amino acids into cells is known to be catalyzed by the Na(+)-independent system L transporter, which is ubiquitous in animal cells and tissues. We have used a Xenopus oocyte expression system to clone the cDNA of a system L transporter from a rat kidney cDNA library. The 2.3 kilobase cDNA codes for a protein of 683 amino acids. The transporter has four putative membrane-spanning domains and bears no sequence or structural homology to any known animal or bacterial transporter. When transcribed and expressed in Xenopus oocytes, the transporter exhibits many, but not all, of the characteristics of L-system transporters, suggesting that this represents one of several related L-system transporters. PMID- 1729675 TI - Construction of a map of chromosome 16 by using radiation hybrids. AB - A human-hamster cell hybrid carrying a single copy of chromosome 16 as the only human genetic material was irradiated with a single dose of gamma-rays (7000 rads; 1 rad = 0.01 Gy) and then fused with a thymidine kinase-deficient hamster cell line (RJKM) to generate radiation hybrids retaining unselected fragments of this human chromosome. In two experiments, 223 hybrids were isolated in hypoxanthine/aminopterine/thymidine (HAT) medium and screened with 38 DNA probes, corresponding to anonymous DNA or gene sequences localized on chromosome 16. The most likely order and location of the 38 DNA sequences were established by multiple pairwise analysis and scaled to estimate physical distance in megabases. The order and the distances thus obtained are mostly consistent with available data on genetic and physical mapping of these markers, illustrating the usefulness of radiation hybrids for mapping. PMID- 1729676 TI - Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins. AB - Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100 amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation. PMID- 1729677 TI - Pertussis toxin has eukaryotic-like carbohydrate recognition domains. AB - Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type. PMID- 1729678 TI - Cellular oxidative modification of low density lipoprotein does not require lipoxygenases. AB - The oxidative modification of low density lipoprotein (LDL) may play an important role in the pathogenesis of atherosclerosis. LDL can be oxidatively modified in vitro by endothelial cells, mouse peritoneal macrophages, or copper ions. Studies using lipoxygenase inhibitors have suggested that lipoxygenase(s) is required for the cellular modification of LDL [Rankin, S. M., Parthasarathy, S. & Steinberg, D. (1991) J. Lipid Res. 32, 449-456]. We have reexamined the effect of lipoxygenase inhibitors on cellular modification and found that (i) inhibitors specific for 5-lipoxygenase do not block LDL modification; (ii) inhibitors that block lipoxygenase by donating one electron to the enzyme (reductive inactivation) prevent LDL modification by cells and also modification mediated by copper ions, implying that they act as general antioxidants; (iii) the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid blocks 15-lipoxygenase activity in intact macrophages at concentrations 100 times less than those required to block LDL modification by macrophages; and (iv) 5,8,11,14 eicosatetraynoic acid is cytotoxic at concentrations about twice those required to prevent modification. Furthermore, macrophages and the RECB4 line of endothelial cells modify LDL with similar efficiencies despite dramatic differences in 15-lipoxygenase activity. Thus we conclude that neither 5 lipoxygenase nor 15-lipoxygenase is required for modification of LDL by cultured cells. PMID- 1729679 TI - Pyridine nucleotide redox state parallels production of aldosterone in potassium stimulated adrenal glomerulosa cells. AB - Extracellular potassium ions (K+) raise the intracellular concentration of free Ca2+ ([Ca2+]i) by gating voltage-dependent Ca2+ channels and stimulate aldosterone production in adrenal glomerulosa cells. The pathway leading from calcium influx to increased steroid synthesis has not been completely elucidated. In the present study we demonstrate that the reduction of pyridine nucleotides known to be required for steroid hydroxylation is enhanced by K+ (4.1-8.4 mM) in single rat glomerulosa cells. The action of K+ was strictly dependent on the presence of extracellular Ca2+. Amytal, a blocker of site I of the mitochondrial respiratory chain, abolished the K+ effect, indicating a mitochondrial origin for the recorded changes. Supraphysiological K+ concentration (18 mM) resulted in a further increase in [Ca2+]i, while steroidogenesis was decreased as measured in cell suspensions. However, a possible explanation for this dichotomy is provided by the finding that the level of reduced pyridine nucleotides also decreased at supraphysiological K+ concentration. PMID- 1729680 TI - Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea. AB - Pheromone biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone biosynthesis in female Helicoverpa (Heliothis) zea. Two oligonucleotide probes representing two overlapping amino acid regions of PBAN were used to screen 2.5 x 10(5) recombinant plaques, and a positive recombinant clone was isolated. Sequence analysis of the isolated clone showed that the PBAN gene is interrupted after the codon encoding amino acid 14 by a 0.63-kilobase (kb) intron. Preceding the PBAN amino acid sequence is a 10-amino acid sequence containing a pentapeptide Phe-Thr-Pro-Arg-Leu, which is followed by a Gly-Arg-Arg processing site. Immediately after the PBAN amino acid sequence is a Gly-Arg processing site and a short stretch of 10 amino acids. This 10-amino acid sequence contains a repeat of the PBAN C-terminal pentapeptide Phe-Ser-Pro-Arg-Leu and is terminated by another Gly-Arg processing site. It is suggested that the PBAN gene in H. zea might carry, besides PBAN, a 7- and an 8-residue amidated peptide, which share with PBAN the core C-terminal pentapeptide Phe-(Ser or Thr)-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence of PBAN represents the minimum sequence required for pheromonotropic activity in H. zea and also bears a high degree of homology to the pyrokinin family of insect peptides with myotropic activity. It is possible that the putative heptapeptide and octapeptide might be new members of the pyrokinin family, with pheromonotropic and/or myotropic activities. Thus, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea. PMID- 1729681 TI - Protein kinase C isozymes epsilon and alpha in murine erythroleukemia cells. AB - Protein kinase C (PKC) has a role in signal transduction during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia cells (MELC). Separation of MELC PKC isozymes by hydroxylapatite chromatography yields a major peak (III) and a minor peak (II) of PKC activity, previously reported to contain the PKC alpha and beta isozymes, respectively. In the present study, we confirm that peak III activity is PKC alpha but show that peak II contains PKC epsilon and little or no PKC beta. Immunoblot analysis with isozyme-specific anti alpha and anti-epsilon PKC antibodies detected PKC alpha in peak III and PKC epsilon in peak II. Peak III activity was markedly enhanced (up to 20-fold) by phosphatidylserine, diolein, and Ca2+, whereas addition of these cofactors to the reaction mixture stimulated peak II activity only 2- to 4-fold. RNase protection analysis of MELC RNA showed that PKC alpha and PKC epsilon RNAs were in a ratio of approximately 2:1, but PKC beta RNA was barely detectable. Taken together, these data indicate that MELC contain PKC alpha and PKC epsilon but little or no PKC beta. PMID- 1729682 TI - Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+) binding site in the protease domain. AB - The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180 310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231 265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa. PMID- 1729683 TI - Transcription factor AP-1 activity is required for initiation of DNA synthesis and is lost during cellular aging. AB - Activation of the AP-1 complex of transcription factors is one of the earliest nuclear responses to mitogenic stimuli. We demonstrate directly that AP-1 activity is required for human cells to proliferate in response to serum. We also find that activity of the AP-1 complex is selectively reduced in old human fibroblasts prior to their entering a fully senescent state. Levels of Fos protein induced through diverse signal transduction pathways, the amount of AP-1 DNA binding activity in vitro, and the activity of an AP-1-dependent reporter gene in vivo are substantially decreased as fibroblasts age. Moreover, the composition of the AP-1 complex changes, so that old cells produce predominantly Jun-Jun homodimers instead of Fos-Jun heterodimers. Changes in AP-1 activity may be due in part to changes in posttranslational modification of Fos protein that impair its ability to form active DNA-binding heterodimers with Jun. These data suggest that changes in AP-1 activity may contribute to the inability of senescent cells to proliferate in response to mitogens. PMID- 1729684 TI - Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge. AB - The signal sequence of secretory proteins and the signal-anchor sequence of type II membrane proteins initiate the translocation of the following polypeptide segments, whereas the signal-anchor sequence of cytochrome P-450-type membrane proteins mediates the membrane insertion of the polypeptide via a signal recognition particle-dependent mechanism but does not lead to the translocation of the following C-terminal sequences. To establish the structural requirements for the function of signal and signal-anchor sequences, we constructed chimeric proteins containing artificial topogenic sequences in which the N-terminal net charge and the length of the hydrophobic segment were systematically altered. Utilizing an in vitro translation-translocation system, we found that hydrophobic segments consisting of 7-10 leucine residues functioned as signal sequences whereas segments with 12-15 leucine residues showed different topogenic functions, behaving as signal sequences or P-450-type signal-anchor sequences, depending on the N-terminal charge. From these observations, we propose that the function of N-terminal topogenic sequences depends on a balance between the N terminal charge and the length of the following hydrophobic segment. PMID- 1729685 TI - Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation. AB - The role of insulin-induced receptor autophosphorylation in its internalization was analyzed by comparing 125I-labeled insulin (125I-insulin) internalization in Chinese hamster ovary (CHO) cell lines transfected with normal (CHO.T) or mutated insulin receptors. In four cell lines with a defect of insulin-induced autophosphorylation, 125I-insulin internalization was impaired. By contrast, in CHO.T cells and in two other CHO cell lines with amino acid deletions or insertions that do not perturb autophosphorylation, 125I-insulin internalization was not affected. A morphological analysis showed that the inhibition is linked to the ligand-specific surface redistribution in which the insulin-receptor complexes leave microvilli and concentrate on nonvillous segments of the membrane where endocytosis occurs. PMID- 1729686 TI - Peripherin: an islet antigen that is cross-reactive with nonobese diabetic mouse class II gene products. AB - The nonobese diabetic (NOD) mouse, in which major histocompatibility complex genes may be involved in the susceptibility to diabetes, has been developed as a model of autoimmune diabetes. The NOD mouse expresses I-A-encoded class II major histocompatibility complex antigens, which differ from those of other mouse haplotypes by the presence of a serine at position 57 of the A beta chain. Identifying islet autoantigens may help elucidate the role of class II antigens in the activation of autoreactive T cells and, thus, in the development of diabetes. We have detected autoantibodies directed against a 58-kDa islet cell antigen in NOD mice but not in other strains, including lupus-prone mice. Apart from insulin-secreting cells, the 58-kDa antigen was only found to be expressed by neuroblastoma cells and was identified as peripherin, an intermediate filament protein previously characterized in well-defined neuronal populations. This autoantigen cross-reacted with I-Anod class II antigens, suggesting that it may contribute to defective self-tolerance of islet beta cells in the NOD mouse. PMID- 1729687 TI - Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26. AB - A successful test is reported to generate long-range contiguous coverage of DNA from a human cytogenetic band in overlapping yeast artificial chromosomes (YACs). Seed YACs in band Xq26 were recovered from a targeted library of clones from Xq24 q28 with 14 probes, including probes for the hypoxanthine guanine phosphoribosyltransferase- and coagulation factor IX-encoding genes and nine probes used in linkage mapping. Neighboring YACs were then identified by 25 "walking" steps with end-clones, and the content of 71 probes in cognate YACs was verified by further hybridization analyses. The resultant contig extends across 8 million base pairs, including most of band Xq26, with an order of markers consistent with linkage data. YAC-based mapping, thus, permits steps toward a fully integrated physical and genetic map and is probably adequate to sustain most of the human genome project. PMID- 1729688 TI - Rhizobium meliloti produces a family of sulfated lipooligosaccharides exhibiting different degrees of plant host specificity. AB - We have shown that a Rhizobium meliloti strain overexpressing nodulation genes excreted high amounts of a family of N-acylated and 6-O-sulfated N-acetyl-beta 1,4-D-glucosamine penta-, tetra-, and trisaccharide Nod factors. Either a C(16:2) or a C(16:3) acyl chain is attached to the nonreducing end subunit, whereas the sulfate group is bound to the reducing glucosamine. One of the tetrasaccharides is identical to the previously described NodRm-1 factor. The two pentasaccharides as well as NodRm-1 were purified and tested for biological activity. In the root hair deformation assay the pentasaccharides show similar activities on the host plants Medicago sativa and Melilotus albus and on the non-host plant Vicia sativa at a dilution of up to 0.01-0.001 microM, in contrast to NodRm-1, which displays a much higher specific activity for Medicago and Melilotus than for Vicia. The active concentration range of the pentasaccharides is more narrow on Medicago than on Melilotus and Vicia. In addition to root hair deformation, the different Nod factors were shown to induce nodule formation on M. sativa. We suggest that the production of a series of active signal molecules with different degrees of specificity might be important in controlling the symbiosis of R. meliloti with several different host plants or under different environmental conditions. PMID- 1729689 TI - Differential expression of two distinct forms of mRNA encoding members of a dipeptidyl aminopeptidase family. AB - We have identified two cDNAs encoding dipeptidyl aminopeptidase-like proteins (DPPXs) in both bovine and rat brains that have different N-terminal cytoplasmic domains but share an identical transmembrane domain and a long C-terminal extracellular domain. In both species, one of the cDNAs encodes a protein (designated DPPX-S) of 803 amino acid residues with a short cytoplasmic domain of 32 amino acids, and the other cDNA encodes a protein (designated DPPX-L) with a longer cytoplasmic domain--the bovine cDNA encodes 92 amino acids and the rat cDNA encodes 88 amino acids. The membrane topology of DPPX-S and -L is similar to that of other transmembrane peptidases, and DPPX-S share approximately 30% identity and 50% similarity with reported yeast and rat liver dipeptidyl aminopeptidase amino acid sequences, suggesting that DPPX is a member of the dipeptidyl aminopeptidase family. DPPX-S mRNA is expressed in brain and some peripheral tissues including kidney, ovary, and testis; in contrast, DPPX-L mRNA is expressed almost exclusively in brain. No transcripts for either form are found in heart, liver, or spleen. In situ hybridization studies show that the two transcripts have different distributions in the brain. DPPX-L mRNA is expressed in limited regions of brain with the highest level of expression in the medial habenula. More widespread expression is seen for DPPX-S mRNA. The differential distribution of mRNAs for the DPPX-S and -L suggests that these proteins are involved in the metabolism of certain localized peptides and that the cytoplasmic domain may play a key role in determining the physiological specificity of DPPX. PMID- 1729690 TI - Levinthal's paradox. AB - Levinthal's paradox is that finding the native folded state of a protein by a random search among all possible configurations can take an enormously long time. Yet proteins can fold in seconds or less. Mathematical analysis of a simple model shows that a small and physically reasonable energy bias against locally unfavorable configurations, of the order of a few kT, can reduce Levinthal's time to a biologically significant size. PMID- 1729691 TI - Magnetic resonance imaging of perfusion using spin inversion of arterial water. AB - A technique has been developed for proton magnetic resonance imaging (MRI) of perfusion, using water as a freely diffusable tracer, and its application to the measurement of cerebral blood flow (CBF) in the rat is demonstrated. The method involves labeling the inflowing water proton spins in the arterial blood by inverting them continuously at the neck region and observing the effects of inversion on the intensity of brain MRI. Solution to the Bloch equations, modified to include the effects of flow, allows regional perfusion rates to be measured from an image with spin inversion, a control image, and a T1 image. Continuous spin inversion labeling the arterial blood water was accomplished, using principles of adiabatic fast passage by applying continuous-wave radiofrequency power in the presence of a magnetic field gradient in the direction of arterial flow. In the detection slice used to measure perfusion, whole brain CBF averaged 1.39 +/- 0.19 ml.g-1.min-1 (mean +/- SEM, n = 5). The technique's sensitivity to changes in CBF was measured by using graded hypercarbia, a condition that is known to increase brain perfusion. CBF vs. pCO2 data yield a best-fit straight line described by CBF (ml.g-1.min-1) = 0.052pCO2 (mm Hg) - 0.173, in excellent agreement with values in the literature. Finally, perfusion images of a freeze-injured rat brain have been obtained, demonstrating the technique's ability to detect regional abnormalities in perfusion. PMID- 1729692 TI - Specific inflammatory cytokines regulate the expression of human monocyte 15 lipoxygenase. AB - Arachidonate 15-lipoxygenase (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33) is a lipid-peroxidating enzyme that is implicated in oxidizing low density lipoprotein to its atherogenic form. Monocyte/macrophage 15-lipoxygenase is present in human atherosclerotic lesions. To pursue a basis for induction of the enzyme, which is not present in blood monocytes, the ability of relevant cytokines to regulate its expression was investigated. Interleukin 4 (IL-4), among 16 factors tested, specifically induced 15-lipoxygenase mRNA and protein in cultured human monocytes. Interferon gamma and hydrocortisone inhibited this induction. High-performance liquid chromatography analysis of lipid extracts from IL-4-treated monocytes detected 15-lipoxygenase products esterified to the cellular membrane lipids, indicating enzymatic action on endogenous substrates. Stimulation of IL-4-treated monocytes with calcium ionophore or opsonized zymosan A enhanced the formation of 15-lipoxygenase products. These data identify IL-4 and interferon gamma as physiological regulators of lipoxygenase expression and suggest an important link between 15-lipoxygenase function and the immune/inflammatory response in atherosclerosis as well as other diseases. PMID- 1729693 TI - Chromosomal context dependence of a eukaryotic recombinational hot spot. AB - The single base-pair mutation M26 in the ade6 gene of the fission yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity. Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting. PMID- 1729694 TI - Generation of plasmacytomas with the chromosomal translocation t(12;15) in interleukin 6 transgenic mice. AB - The mechanisms through which pristane or mineral oil can induce plasmacytomas in BALB/c or NZB mice are not fully understood, but involvement of interleukin 6 (IL 6), a growth factor for plasmacytomas and myelomas, has been strongly suggested. To clarify the role of IL-6 in plasmacytomagenesis, a human IL-6 cDNA was introduced into mouse germ lines under the transcriptional control of the murine major histocompatibility complex class I (H-2Ld) promoter. IL-6 transgenic mice of C57BL/6 origin developed a massive plasmacytosis but not plasmacytomas. However, introduction of BALB/c genetic background into IL-6 transgenic mice could generate monoclonal transplantable plasmacytomas with the chromosomal translocation t(12;15). These results provide firm evidence of the critical role of IL-6 in the plasmacytoma development. PMID- 1729695 TI - Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene. AB - Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair proficient cells into XP cells has provided a means of mapping the genes involved in this disease. We now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells. PMID- 1729696 TI - A conserved retina-specific gene encodes a basic motif/leucine zipper domain. AB - Using subtraction cloning, we have isolated a human cDNA, AS321, which is expressed in retina and retinoblastoma cell lines but not in any other tissue or cell line tested. AS321 mRNA is detected in all cells of neural retina, with a high level of expression in photoreceptors. The polypeptide sequence deduced from the cDNA reveals consensus phosphorylation sites for protein kinase A and proline directed protein kinase. Its C-terminal region contains a basic motif and a leucine zipper domain similar to the DNA binding proteins of the jun and fos oncoprotein family, and it shows a strong similarity to the product of an avian retroviral oncogene, v-maf. The gene for AS321 is conserved during evolution and is expressed in vertebrate retina. We propose to name the gene NRL (neural retina leucine zipper. PMID- 1729697 TI - Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains. AB - A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates. PMID- 1729698 TI - Yeast casein kinase I homologues: an essential gene pair. AB - We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues. PMID- 1729699 TI - The molecular defect of ferrochelatase in a patient with erythropoietic protoporphyria. AB - The molecular basis of an inherited defect of ferrochelatase in a patient with erythropoietic protoporphyria (EPP) was investigated. Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway and catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. In Epstein-Barr virus transformed lymphoblastoid cells from a proband with EPP, enzyme activity, an immunochemically quantifiable protein, and mRNA content of ferrochelatase were about one-half the normal level. In contrast, the rate of transcription of ferrochelatase mRNA in the proband's cells was normal, suggesting that decreased ferrochelatase mRNA is due to an unstable transcript. cDNA clones encoding ferrochelatase in the proband, isolated by amplification using the polymerase chain reaction, were found to be classified either into those encoding the normal protein or into those encoding an abnormal protein that lacked exon 2 of the ferrochelatase gene, indicating that the proband is heterozygous for the ferrochelatase defect. Genomic DNA analysis revealed that the abnormal allele had a point mutation, C----T, near the acceptor site of intron 1. This point mutation appears to be responsible for the post-transcriptional splicing abnormality resulting in an aberrant transcript of ferrochelatase in this patient. PMID- 1729700 TI - Replication of single-stranded plasmid pT181 DNA in vitro. AB - Plasmid pT181 is a 4437-base-pair, multicopy plasmid of Staphylococcus aureus that encodes tetracycline resistance. The replication of the leading strand of pT181 DNA initiates by covalent extension of a site-specific nick generated by the initiator protein at the origin of replication and proceeds by an asymmetric rolling circle mechanism. The origin of the leading strand synthesis also serves as the site for termination of replication. Replication of pT181 DNA in vivo and in vitro has been shown to generate a single-stranded intermediate that corresponds to the leading strand of the DNA. In vivo results have suggested that a palindromic sequence, palA, located near the leading strand termination site acts as the lagging strand origin. In this paper we report the development and characterization of an in vitro system for the replication of single-stranded pT181 DNA. Synthesis of the lagging strand of pT181 proceeded in the absence of the leading strand synthesis and did not require the pT181-encoded initiator protein, RepC. The replication of the lagging strand required RNA polymerase dependent synthesis of an RNA primer. Replication of single-stranded pT181 DNA was found to be greatly stimulated in the presence of the palA sequence. We also show that palA acts as the lagging strand origin and that DNA synthesis initiates within this region. PMID- 1729701 TI - Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini. AB - An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of lambda repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands. PMID- 1729702 TI - Crystallographic detection of a second ligand binding site in influenza virus hemagglutinin. AB - X-ray crystal structures have been determined for several complexes between influenza virus hemagglutinin and derivatives of its cell-surface receptor, sialic acid (Neu5Ac). Difference electron density maps establish the existence of a second binding site in addition to the primary site characterized previously. Three compounds bind to both sites: Neu5Ac(alpha 2-3)Gal(beta 1-4)Glc [(alpha 2 3)sialyllactose], alpha-2-O-(4'-benzylamidocarboxybutyl)-5-N-acetylneuraminic acid, and alpha-2-O-(4'-methylamidocarboxybutyl)-5-N-acetylneuraminic acid; and four other compounds bind only to the primary site: Neu5Ac(alpha 2-6)Gal(beta 1 4)Glc [(alpha 2-6)sialyllactose], alpha-2-O-methyl-5-N-acetylneuraminic acid, 4-] acetyl-alpha-2-O-methyl-5-N-acetylneuraminic acid, and 9-amino-9-deoxy-alpha-2-O methyl-5-N-acetylneuraminic acid. The maps also extend earlier results by showing the location of all three sugar residues of (alpha 2-3)sialyllactose in the primary binding site. The affinity of (alpha 2-3)sialyllactose for the second site was estimated by collecting x-ray diffraction data at various ligand concentrations and was found to be at least four times weaker than its affinity for the primary site. Although it is not yet known whether the second binding site participates in the infection process, it nevertheless offers a potential target for the design of antiviral drugs. PMID- 1729703 TI - Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. AB - Expression of the bacterial gene for cytosine deaminase (CD; EC 3.5.4.1) in mammalian cells was evaluated as a negative selection system or suicide vector for potential use in gene transfer studies and therapies. Mammalian cells, unlike certain bacteria and fungi, do not contain the enzyme CD and do not ordinarily metabolize cytosine to uracil. Nor do they metabolize the innocuous compound 5 fluorocytosine to the highly toxic compound 5-fluorouracil. The Escherichia coli CD gene underwent PCR oligonucleotide-directed mutagenesis to enhance its expression in a eukaryotic system and it was then cloned into an expression vector, pLXSN, that also contains a neomycin-resistance gene. Murine fibroblast lines were transfected with the plasmid and subjected to brief selection in the neomycin analogue G418. Lysates from these cell populations exhibited significant CD activity detected by conversion of radiolabeled cytosine to uracil. In clonogenic assays transfected cells expressing CD were selectively killed by incubation in 5-fluorocytosine, whereas control cell lines were not. Dose response studies evaluating [3H]thymidine incorporation or cloning efficiency demonstrated profound inhibition at and above 65 micrograms of 5-fluorocytosine per ml. Mixed cellular assays showed that CD-positive cells could be eliminated without bystander killing of other cells. Retrovirus-mediated CD gene transfer into various tissues was also demonstrated. Thus CD, with its ability to produce the toxic antimetabolite 5-fluorouracil from 5-fluorocytosine, may be useful as a negative selection system for studies and treatments employing gene transfer techniques. PMID- 1729704 TI - Circulatory half-life but not interaction with the lutropin/chorionic gonadotropin receptor is modulated by sulfation of bovine lutropin oligosaccharides. AB - Certain of the glycoprotein hormones, including bovine lutropin (bLH), bear asparagine-linked oligosaccharides terminating with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2Man alpha. To establish the biologic significance of these sulfate-bearing oligosaccharides we have compared properties of native bLH, desulfated bLH, recombinant bLH produced in Chinese hamster ovary cells that bears asparagine-linked oligosaccharides terminating with sialic acid alpha 2- 3Gal beta 1-4GlcNAc beta 1-2Man alpha rather than sulfated oligosaccharides (bLH/CHO), and desialyzed bLH/CHO. Using cultured MA-10 cells, a Leydig cell tumor line expressing the lutropin/chorionic gonadotropin receptor, we have found no differences in binding, cAMP production, or progesterone production between native and desulfated bLH. Sulfation of bLH oligosaccharides does not, therefore, modulate bLH bioactivity at the level of the lutropin/chorionic gonadotropin receptor. Removal of sulfate from bLH oligosaccharides and sialic acid from bLH/CHO oligosaccharides results in rapid clearance from the circulation by the hepatocyte asialoglycoprotein receptor. Thus sulfate, like sialic acid, prevents clearance from the circulation by the asialoglycoprotein receptor. The rapid removal of desulfated bLH from the circulation causes a 4- to 16-fold increase in the amount of bLH required to stimulate ovulation compared with native bLH. Particularly striking were differences in the metabolic clearance rates for native bLH and bLH/CHO, 7.3% per min and 1.7% per min, respectively. These differences were unexpected because bLH and bLH/CHO do not differ significantly in charge or size. The different metabolic clearance rates obtained for bLH and bLH/CHO indicate that the presence of sulfated rather than sialylated oligosaccharides on bLH results in a shorter circulatory half-life, which has a significant impact on in vivo bioactivity. PMID- 1729705 TI - Complexity and expression patterns of the desmosomal cadherins. AB - Desmosomes are intercellular junctions that contain two major kinds of transmembrane glycoproteins, desmoglein and desmocollins I and II, involved in cell-cell adhesion. Recent sequence analyses have shown that both desmosomal glycoproteins belong to the larger cadherin family of cell adhesion molecules, in which they represent two different subgroups characterized by their specific sequence and topogenesis. In analyses of cDNA sequences and Northern blot experiments we have now found that both desmoglein and desmocollins are not unique gene products but occur in different subtypes produced from different genes. Comparison of the complete amino acid sequences of type 1 and type 2 desmocollins and of two desmoglein subtypes shows considerable divergence. While the desmoglein genes can be differentially expressed in different cell types, both type 1 and type 2 desmocollins can coexist in the same cells of certain stratified epithelia as shown by in situ hybridization. We conclude that the cadherin composition of desmosomes is much more complex than assumed and can differ in the various epithelia. PMID- 1729706 TI - Liposomal malaria vaccine in humans: a safe and potent adjuvant strategy. AB - This study describes the safety and immunogenicity of a liposome-based vaccine injected into human subjects. Thirty healthy adult male volunteers were immunized with a liposome-encapsulated recombinant protein (R32NS181) containing epitopes from the repeat region of the circumsporozoite protein of Plasmodium falciparum. This antigen had previously been found to be poorly immunogenic in humans when it was adsorbed with Al(OH)3. In the present study, R32NS181 was encapsulated in liposomes containing monophosphoryl lipid A that were subsequently adsorbed to Al(OH)3. Increasing doses of liposomes containing antigen and monophosphoryl lipid A were used, but the liposomes were always adsorbed to the same dose of Al(OH)3. R32-specific serum IgG antibody responses to liposome-encapsulated R32NS181 were much higher than levels attained previously in humans with R32NS181 adsorbed to Al(OH)3. Geometric mean specific IgG levels after three doses ranged from 14 to 33 micrograms/ml. Sera from volunteers receiving the two highest doses inhibited P. falciparum sporozoite invasion of cultured hepatoma cells by an average of 92%, a result that was again superior to previously reported vaccines. Moderate but acceptable transient local reactogenicity was noted at high doses of the vaccine formulation, but little or no systemic toxicity was seen despite liposomal monophosphoryl lipid A doses up to 2200 micrograms. We conclude that encapsulation of poorly immunogenic circumsporozoite protein repeat peptides in monophosphoryl lipid A-containing liposomes is a successful adjuvant strategy in humans for inducing high levels of specific antibody production. PMID- 1729707 TI - Mutagenic specificities of four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides. AB - The pS189 shuttle vector carrying a supF target gene was used to compare the mutagenic specificities of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide. One of these isomers is the most tumorigenic dihydrodiol epoxide tested to date and another is essentially inactive as a tumorigen. Overall mutagenicities were not correlated with tumorigenicities, but each configurational isomer induced a unique spectrum of mutational hot spots in the supF target gene, which monitors primarily point mutations. It is suggested that the demonstrated isomer-specific selectivity for mutation targets within the supF gene may be indicative of a similar selectivity for one gene versus another and that such selectivity may be one determinant of relative tumorigenicity. PMID- 1729709 TI - Chemical implementation and thermodynamics of collective neural networks. AB - The chemical implementation of a neuron and connections among neurons described in prior work is used to construct collective neural networks. With stated approximations, these chemical networks are reduced to networks of the Hopfield type. Chemical networks approaching a stationary or equilibrium state provide a Liapunov function with the same extremal properties as Hopfield's energy function. Numerical comparisons of chemical and Hopfield networks with small numbers (2-16) of neurons show agreement on the results of given computations. PMID- 1729708 TI - Molecular characterization of NSCL, a gene encoding a helix-loop-helix protein expressed in the developing nervous system. AB - We report here the molecular cloning and chromosomal localization of an additional member of the helix-loop-helix (HLH) family of transcription factors, NSCL. The NSCL gene was identified based on its hybridization to the previously described hemopoietic HLH gene, SCL. Murine NSCL cDNA clones were obtained from a day 11.5 mouse embryo cDNA library. The coding region is 399 base pairs and encodes a predicted protein of 14.8 kDa. The nucleotide sequence shows 71% identity and the amino acid sequence shows 61% identity to murine SCL in the HLH domain. The NSCL protein-coding region terminates six amino acids beyond the second amphipathic helix of the HLH domain. Expression of NSCL was detected in RNA from mouse embryos between 9.5 and 14.5 days postcoitus, with maximum levels of expression at 10.5-12 days. Examination of 12- and 13-day mouse embryos by in situ hybridization revealed expression of NSCL in the developing nervous system. The NSCL gene was mapped to murine chromosome 1. The very restricted pattern of NSCL expression suggests an important role for this HLH protein in neurological development. PMID- 1729710 TI - Class II (B) general transcription factor (TFIIB) that binds to the template committed preinitiation complex is different from general transcription factor BTF3. AB - A class II (B) general transcription factor of 34 kDa has been purified from HeLa cells to apparent homogeneity. This factor appears to be transcription factor IIB (TFIIB), since it binds in vitro to template-committed preinitiation complexes formed between a template containing the TATA box/cap-site elements of the adenovirus type 2 major late promoter (Ad2MLP) and recombinant human or yeast TFIID (previously called BTF1) expressed in Escherichia coli. DNase I footprint studies show an extended pattern of protection of Ad2MLP TATA box/cap-site sequences when TFIIB is bound to template-committed complexes, even though TFIIB does not bind on its own to the template in the absence of TFIID. We also show that TFIIB is different from BTF3 by a number of criteria. PMID- 1729711 TI - Component H of the DNA-dependent RNA polymerases of Archaea is homologous to a subunit shared by the three eucaryal nuclear RNA polymerases. AB - The gene encoding component H of the DNA-dependent RNA polymerase (RNAP, EC 2.7.7.6) of Sulfolobus acidocaldarius has been identified by comparison of the amino acid sequence with the derived amino acid sequence of an open reading frame (ORF88) in the RNAP operon. Corresponding genes were identified in Halobacterium halobium and were cloned and sequenced from Thermococcus celer and Methanococcus vannielii. All these rpoH genes are situated between the promoters of the RNAP operons and the corresponding rpoB and rpoB2 genes. The archaeal H subunits show high sequence similarity to each other and to the C-terminal portions of the largest of four subunits shared by all three specialized nuclear RNAPs. These correlations are further evidence for the striking similarity between archaeal and eucaryal RNAP structures and transcription systems. PMID- 1729712 TI - Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily. AB - The majority of human urinary stones are primarily composed of calcium salts. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Inhibitors are among the multiple factors that may influence the complex process of urinary stone formation. We have isolated an inhibitor of calcium oxalate crystal growth from human urine by monoclonal antibody immunoaffinity chromatography. The N-terminal amino acid sequence and acidic amino acid content of this aspartic acid-rich protein, uropontin, are similar to those of other pontin proteins from bone, plasma, breast milk, and cells. The inhibitory effect of uropontin on calcium oxalate crystal growth in vitro supports the concept that pontins may have a regulatory role. This function would be analogous to that of other members of the aspartic acid-rich protein superfamily, which stereospecifically regulate the mineralization fronts of calcium-containing crystals. PMID- 1729713 TI - Genetic characterization and transovarial transmission of a typhus-like rickettsia found in cat fleas. AB - The identification of apparently fastidious microorganisms is often problematic. DNA from a rickettsia-like agent (called the ELB agent) present in cat fleas could be amplified by PCR with conserved primers derived from rickettsial 17-kDa common protein antigen and citrate synthase genes but not spotted fever group 190 kDa antigen gene. Alu I sites in both the 17-kDa and citrate synthase PCR products obtained with the rickettsia-like agent and Rickettsia typhi were different even though both agents reacted with monoclonal antibodies previously thought specific for R. typhi. The DNA sequence of a portion of the 17-kDa PCR product of the rickettsia-like agent differed significantly from all known rickettsial sequences and resembled the 17-kDa sequences of typhus more than spotted fever group rickettsiae. The rare stable transovarial maintenance of this rickettsia in cat fleas has important implications for the disease potential of cat fleas. PMID- 1729714 TI - Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture. AB - The ability of neurons in the central nervous system to grow through a lesion and restore conduction has been analyzed in a developing spinal cord. The preparation consists of the entire central nervous system of the newly born opossum (Monodelphis domestica), isolated and maintained in culture. Cell division, cell migration, and reflexes are maintained in such preparations for up to 8 days in culture. In the present experiments, massive lesions were produced by crushing the spinal cord, which abolished all conduction for a day. By 2-3 days after injury, electrical conduction across the crush could be observed. After 4-5 days, clear recovery had occurred: the amplitude of the conducted volley was comparable to that in acute preparations. In such preparations, the spinal cord had largely regained its normal appearance at the crush site. Axons stained by carbocyanine dyes or horseradish peroxidase had, by 4 days, grown in profusion through the lesion and several millimeters beyond it. These experiments demonstrate that neurons in the central nervous system of newly born mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors of myelin. With rapid and reliable regeneration occurring in vitro, it becomes practicable to assay the effects of molecules that promote or inhibit the restoration of functional connections. PMID- 1729715 TI - Long-term potentiation of inhibitory circuits and synapses in the central nervous system. AB - Glycinergic inhibition evoked disynaptically in the teleost Mauthner cell by stimulation of the contralateral eighth nerve exhibits long-term potentiation following classical tetanization of that pathway. This enhancement occurs at the synapses between primary afferents onto second-order interneurons and the connections between these inhibitory cells and the Mauthner neuron. The evidence for modifications of glycinergic transmission is that the slope of the relation between the presynaptic volley and the synaptic conductance can be greater after the tetanus. This increase in gain is still manifest after pharmacological block of potentiation at the excitatory synapse with glutamate antagonists. Inhibitory long-term potentiation is induced by tetani weaker than those required for enhancement of the monosynaptic excitation of the other (ipsilateral) Mauthner cell. Thus, in vivo learning can alter the balance between excitation and inhibition within a network by modifying one or both of them. PMID- 1729716 TI - An rbcL sequence from a Miocene Taxodium (bald cypress). AB - During the past decade, ancient DNAs from both animals and plants have been successfully extracted and analyzed. Recently, the age of DNA that can be recovered and sequenced was increased manyfold by the amplification and sequencing of a DNA fragment from a Magnolia fossil obtained from the Miocene Clarkia deposit (17-20 million yr old). However, the validity of this report has been questioned based on models predicting that DNA should be completely degraded after 4 million yr. We report here the successful amplification, sequencing, and analysis of a 1320-base-pair portion of the chloroplast gene rbcL from a Miocene Taxodium specimen, also from the Clarkia site. These data not only validate the earlier report of sequence data for a Magnolia species from the same site but also suggest that it may be possible to isolate and sequence DNAs routinely from the Clarkia deposit. The ability to recover and sequence DNAs of such age offers enormous research possibilities in the areas of molecular evolution, biogeography, and systematics. PMID- 1729717 TI - parB: an auxin-regulated gene encoding glutathione S-transferase. AB - We have isolated an auxin-regulated cDNA, parB, from the early stage of cultured tobacco mesophyll protoplasts. The expression of parB was observed during transition from G0 to the S phase of tobacco mesophyll protoplasts cultured in vitro. The predicted amino acid sequence of parB cDNA has 213 amino acid residues with a relative molecular weight of 23,965. Nucleotide sequence analysis revealed that parB cDNA has homology to glutathione S-transferase (GST; RX:glutathione R transferase, EC 2.5.1.18) from several sources including plant and animal cells. When we introduced expression vector pKK233-2, which retains parB cDNA, into Escherichia coli, we could detect GST activity in the parB gene product. Accordingly a significant increase of GST activity was detected in the tobacco mesophyll protoplasts cultured in the presence of 2,4-dichlorophenoxyacetic acid. This is an example in which the function of auxin-regulated gene product is shown to be ascribed to a specific enzymatic activity. As GST, and its substrate glutathione, are shown to be related to cell proliferation as well as detoxification of xenobiotics in plant and animal cells, the role of parB is discussed in relation to the induction of proliferative activity in differentiated and nondividing mesophyll protoplasts of tobacco. PMID- 1729718 TI - Psychophysical support for a two-dimensional view interpolation theory of object recognition. AB - Does the human brain represent objects for recognition by storing a series of two dimensional snapshots, or are the object models, in some sense, three-dimensional analogs of the objects they represent? One way to address this question is to explore the ability of the human visual system to generalize recognition from familiar to unfamiliar views of three-dimensional objects. Three recently proposed theories of object recognition--viewpoint normalization or alignment of three-dimensional models [Ullman, S. (1989) Cognition 32, 193-254], linear combination of two-dimensional views [Ullman, S. & Basri, R. (1990) Recognition by Linear Combinations of Models (Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge), A. I. Memo No. 1152], and view approximation [Poggio, T. & Edelman, S. (1990) Nature (London) 343, 263-266]- predict different patterns of generalization to unfamiliar views. We have exploited the conflicting predictions to test the three theories directly in a psychophysical experiment involving computer-generated three-dimensional objects. Our results suggest that the human visual system is better described as recognizing these objects by two-dimensional view interpolation than by alignment or other methods that rely on object-centered three-dimensional models. PMID- 1729719 TI - Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine. AB - RNA hairpin helices whose sequences are based on the acceptor stems of alanine and histidine tRNAs are specifically aminoacylated with their cognate amino acids. In these examples, major determinants for the identities of the respective tRNAs reside in the acceptor stem; the anticodon and other parts of the tRNA are dispensable for aminoacylation. In contrast, the anticodon is a major determinant for the identity of a methionine tRNA. RNA hairpin helices and hybrid duplexes that reconstruct the acceptor-T psi C stem and the acceptor stem, respectively, of methionine tRNA were investigated here for aminoacylation with methionine. Direct visualization of the aminoacylated RNA product on an acidic polyacrylamide gel by phosphor imaging demonstrated specific aminoacylation with substrates that contained as few as 7 base pairs. No aminoacylation with methionine was detected with several analogous RNA substrates whose sequences were based on noncognate tRNAs. While the efficiency of aminoacylation is reduced by orders of magnitude relative to methionine tRNA, the results establish that specific aminoacylation with methionine of small duplex substrates can be achieved without the anticodon or other domains of the tRNA. The results, combined with earlier studies, suggest a highly specific adaptation of the structures of aminoacyl-tRNA synthetases to the acceptor stems of their cognate tRNAs, resulting in a relationship between the nucleotide sequences/structures of small RNA duplexes and specific amino acids. PMID- 1729720 TI - A mutation in the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 dominantly interferes with fusion and infectivity. AB - Several domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been identified that are involved in HIV-1-mediated membrane fusion. One domain that is involved in membrane fusion is the hydrophobic amino terminus of the HIV-1 transmembrane glycoprotein gp41. Here we show that a polar substitution at gp41 amino acid 2 (the 41.2 mutation) results in an envelope glycoprotein that dominantly interferes with both syncytium formation and infection mediated by the wild-type HIV-1 envelope glycoprotein. The interference by the 41.2 mutant is not a result of aberrant envelope glycoprotein synthesis, processing, or transport. The 41.2 mutant elicits a dominant interfering effect even in the presence of excess wild-type glycoprotein, suggesting that a higher order envelope glycoprotein complex is involved in membrane fusion. These results shed light on the process by which the HIV-1 envelope glycoproteins induce membrane fusion reactions and present a possible approach to anti-HIV therapy. PMID- 1729721 TI - Chromophore-protein interactions and the function of the photosynthetic reaction center: a molecular dynamics study. AB - The coupling between electron transfer and protein structure and dynamics in the photosynthetic reaction center of Rhodopseudomonas viridis is investigated. For this purpose molecular dynamics simulations of the essential portions (a segment of 5797 atoms) of this protein complex have been carried out. Electron transfer in the primary event is modeled by altering the charge distributions of the chromophores according to quantum chemical calculations. The simulations show (i) that fluctuations of the protein matrix, which are coupled electrostatically to electron transfer, play an important role in controlling the electron transfer rates and (ii) that the protein matrix stabilizes the separated electron pair state through rapid (200 fs) and temperature-independent dielectric relaxation. The photosynthetic reaction center resembles a polar liquid in that the internal motions of the whole protein complex, rather than only those of specific side groups, contribute to i and ii. The solvent reorganization energy is about 4.5 kcal/mol. The simulations indicate that rather small structural rearrangements and changes in motional amplitudes accompany the primary electron transfer. PMID- 1729722 TI - Identification of act2, an essential gene in the fission yeast Schizosaccharomyces pombe that encodes a protein related to actin. AB - Actins are a family of highly conserved proteins that are ubiquitously found among eukaryotic organisms. All actins that have previously been identified, including those of animals, plants, fungi, and protozoa, are 374-376 amino acids long and exhibit at least 70% amino acid sequence identity when compared with one another. We have cloned a gene from the fission yeast Schizosaccharomyces pombe that encodes a distantly related member of the actin protein family, herein referred to as act2. In contrast to all other actins, the derived amino acid sequence reveals that act2 is 427 residues long and exhibits only 35-40% identity to actins, including act1 from Sch. pombe. Comparison to the known x-ray crystallographic structure of rabbit skeletal muscle actin indicates that the ATP and divalent metal ion binding sites are largely conserved in act2, while regions involved in actin-actin and actin-myosin interactions are relatively divergent. Disruption of the act2 gene demonstrated that this gene encodes a function essential for germination of haploid spores. These findings indicate that while act2 and act1 are related proteins, they appear to have distinct functions. In addition, they demonstrate that the actin protein family is more diverse than was previously thought. PMID- 1729723 TI - A peptide-hormone-inactivating endopeptidase in Xenopus laevis skin secretion. AB - An endopeptidase was isolated from Xenopus laevis skin secretions. This enzyme, which has an apparent molecular mass of 100 kDa, performs a selective cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, or Gly) of a number of peptide hormones, including atrial natriuretic factor, substance P, angiotensin II, bradykinin, somatostatin, neuromedins B and C, and litorin. The peptidase exhibited optimal activity at pH 7.5 and a Km in the micromolar range. No cleavage was produced in vasopressin, ocytocin, minigastrin I, and [Leu5]enkephalin, which include in their sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The endopeptidase activity was inhibited by divalent cation chelators and by phosphoramidon only at high concentrations (IC50 = 50 microM), whereas it was insensitive to classical inhibitors of chymotrypsin, angiotensin convertase, and serine and cysteine peptidases, as well as carboxypeptidases. It is hypothesized that this enzyme, which is distinct from neutral endopeptidase (EC 3.4.24.11), constitutes the prototype of a family of related metalloendopeptidases that inactivate peptide substrates by cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond. PMID- 1729725 TI - Leaders and followers: a psychiatric perspective on religious cults. Committee on Psychiatry and Religion. Group for the Advancement of Psychiatry. PMID- 1729724 TI - Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes. AB - The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 10(9) hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human alpha 1-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had significant human alpha 1-antitrypsin in the serum for 1 month. Although the serum levels of human alpha 1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver. PMID- 1729726 TI - Psychotherapy in the future. Committee on Therapy. Group for the Advancement of Psychiatry. PMID- 1729727 TI - Genital tuberculosis at Tygerberg Hospital--prevalence, clinical presentation and diagnosis. AB - Over a period of 30 months (1 July 1986-31 December 1988) 57 cases of genital tuberculosis were diagnosed at Tygerberg Hospital. Forty of these cases were diagnosed as a result of routine screening in 650 patients who presented with infertility and the other 17 were diagnosed in patients admitted to the gynaecological wards. The prevalence in patients presenting with infertility was 6.15%. The commonest gynaecological presenting symptom was infertility (73.7%). Dysmenorrhoea in 29.8% and deep dyspareunia in 12.3% were the only other frequently occurring gynaecological symptoms. Menstruation was normal in 50 patients (87.7%). Seven per cent of patients were postmenopausal. Abdominal symptoms were only present in 15.8%. These findings re-emphasise that genital tuberculosis is often a disease of absent or few symptoms. General, abdominal and pelvic examinations were normal in 56.1% of patients and even when clinical signs were present they were nonspecific. Menstrual fluid collection and culture proved to be the most reliable diagnostic procedure, since it was positive in 11 patients in whom premenstrual endometrial sample cultures were negative and also in 17 patients in whom histological examination of premenstrual endometrial samples for tuberculosis were negative. The possible reasons for this and its clinical importance are discussed. Other than histological examination of operation and/or biopsy specimens, special investigations proved to be of little help in the diagnosis of genital tuberculosis. PMID- 1729728 TI - Social factors associated with tuberculous meningitis. A study of children and their families in the western Cape. AB - A descriptive study was conducted in order to assess some of the social factors associated with the long-term effects of tuberculous meningitis on children and their families. The specific areas examined were the socio-economic status of the children, maternal employment, the utilisation of health services and the scholastic progress of the children. Recommendations are made. The sample was drawn from a register of children with tuberculous meningitis in the Western Cape Health Region for the period 1985-1987. All the available survivors (16 black and 91 coloured children) were included in the study and the care-giver was interviewed by a researcher. Forty-six subjects lived in an urban area and 61 in a rural area. All the children came from socially deprived families and lived in over-crowded conditions; a significant number of families had incomes below the household subsistence level. Of the mothers previously employed, 35% had stopped working and 19% of the families experienced a financial loss as a result of the child's illness. The majority of urban children utilised public health services and the majority of rural children were dependent on the private sector for health care. School-going children had a high failure rate (53%). PMID- 1729729 TI - The absorption characteristics of six sustained-release theophylline preparations. AB - Dosing intervals for sustained-release theophylline preparations depend on the rate of formulation absorption, the rate of elimination by the patient, and clinically acceptable fluctuations in serum concentration. A comparative study of intersubject variation in fraction absorbed-time profiles, a process-independent method of comparing rates of absorption, was performed with 6 sustained-release preparations in 6 healthy male volunteers. The sulphasalazine/sulphapyridine method of assessing orocaecal transit time was implemented so that upper gastrointestinal and colonic absorption could be estimated. Time until 90% absorption varied from 4.43 hours to 7.46 hours and the mean percentages of theophylline remaining to be absorbed from the colon were limited to between 7.5% and 29.7% with the various formulations. There was a great intersubject variability in the rate of theophylline absorption and also considerable differences among the volunteers in their formulation-to-formulation absorption profiles. Promotional literature depicting mean or group data masks this variability in absorption profiles. Because host factors related to gastro intestinal physiology impose highly variable theophylline absorption profiles on sustained-release formulations, it is technically impossible to formulate a suitable once-a-day product for the majority of patients. PMID- 1729730 TI - Smoking policies in the workplace in the western Cape. AB - A postal survey of workplace smoking restrictions among the member organisations of the Cape Chamber of Industries was carried out in 1989. The response rate was 57.1%. Of the 572 respondent organisations, 66.1% had some smoking restrictions. Large workplaces were more likely to restrict smoking than small workplaces: 42.0% of those with fewer than 10 employees had restrictions, increasing to 90.9% of those with more than 500 employees. Organisations producing manufactured goods (other than engineering) were more likely to have restrictions than non manufacturing concerns. Smoking was commonly restricted on the factory floor (61.3%) and in warehouses (55.8%), but only 7.4% prohibited smoking in shared offices. The reasons for smoking restrictions stated most frequently were the fire hazard (85.3%) and legislation (66.0%). Only 29.1% stated that health care concerns were an important reason for restrictions, while a further 16.3% stated that health was a minor reason for restrictions. Of the respondents, 48.4% expressed a need for guidance in improving their smoking policies. These results indicate that there is considerable potential for intervention to decrease both active and passive smoking in local workplace settings. PMID- 1729731 TI - Mental retardation--who cares? PMID- 1729732 TI - Kawasaki disease manifesting with acute cholangitis. A case report. AB - A 3-year-old boy, who developed the signs and symptoms characteristic of Kawasaki disease, is described. The child also had an 8 cm tender hepatomegaly. Hydrops of the gallbladder could not be shown. Liver biopsy showed marked infiltration of inflammatory cells, including neutrophil and eosinophil leucocytes in the portal tracts involving the periphery of the portal arteries and veins, and acute inflammation of the bile ducts with neutrophil and eosinophil infiltration of the walls. Overt cholangitis has been described only once before in Kawasaki disease, when a viral agent was suggested as being important in the pathogenesis. Although the clinical and laboratory findings in cases of Kawasaki disease clearly suggest an acute infection--as they did in this case--no aetiological agent has yet been incriminated. The possibility of a drug-induced auto-allergic or hypersensitivity state is considered. Evidence for such a state includes a history of drug administration, pathological findings similar to peri-arteritis nodosa--a condition often associated with a hypersensitivity state--the presence of eosinophils in the lesions and a response to treatment with aspirin, a drug known to ameliorate hypersensitivity states. PMID- 1729733 TI - Immunological disorders--the evolution of an hypothesis. AB - In honouring the memory of Dr Alwyn Zoutendyk, a respected member of the staff of the South African Institute for Medical Research, attention is called to the studies of the immunological disorders. While investigating serum hepatitis affecting soldiers of the US army following the administration of yellow fever vaccine, an antigen similar to that later called the Australia antigen, now hepatitis B surface antigen, was found in the acute phase serum and the corresponding antibody was found in convalescence. This finding and subsequent studies suggested there was a group of disease, which we called the hyperreactive auto-allergic disorders, of which examples were to be found in every system. The obverse of these we called the hyporeactive immunologically deficient disorders resulting from defects of the cell or serum components of the immunological reactions, of which many examples have also been found. PMID- 1729734 TI - Management of childhood and adolescent asthma--1991 consensus. South African Childhood Asthma Working Group. PMID- 1729735 TI - Comparative dosage of angiotensin-converting enzyme inhibitors in the treatment of hypertension. PMID- 1729736 TI - Analysis of routine sputum specimens at Hillbrow Hospital. PMID- 1729737 TI - Oxygen masks. PMID- 1729738 TI - Treatment of refractory immune thrombocytopenic purpura with ascorbate. PMID- 1729739 TI - Neuroleptics in alcohol withdrawal. PMID- 1729740 TI - The vertebral arteries and the human brain. PMID- 1729741 TI - Family history as a risk factor for coronary heart disease in South African families. AB - This article presents data on, and applies a procedure for the statistical quantification of, family history as a risk factor for coronary heart disease (CHD) in three sub-samples (groups) of families: I--a healthy control group; II- families with familial hypercholesterolaemia (FH); and III--families identified by an index case with CHD. With regard to the average family history of CHD (calculated as an index for each family, and as a mean index for each group), group II differs significantly from group I and marginally significantly from group III; family groups I and III do not differ from each other statistically. By means of significance tests developed for this purpose, the groups of families are shown to be significantly heterogeneous, by being composed of families highly resistant against and susceptible to CHD. This is illustrated for example in group II, where some FH families can be shown to be highly resistant to CHD, compared with other FH families with a very strong history of CHD. The exact number and proportion of such families at different levels of significance is calculated and the actual families with the highest and lowest calculated family indices, respectively, are then identified (illustrated by examples). The practical significance of the statistical procedure of quantifying and applying family history as a risk factor for CHD, is discussed in terms of epidemiological and preventive health considerations. PMID- 1729742 TI - The natural history of carcinoma of the bile duct in patients less than forty five years of age. AB - Traditionally regarded as a disease of the elderly, the natural history of carcinoma of the bile duct in young patients has not been well defined. Of 186 patients (mean age of 62 years) treated at UCLA (1954 to 1988) for carcinoma of the bile duct, 26 were less than 45 years old. Younger patients had symptoms for an average of 4.5 +/- 0.8 months prior to diagnosis, as compared with 2.3 +/- 0.2 months for patients more than 45 years old (p less than 0.03). Of the younger patients, 96 per cent were managed surgically with either resection, surgical palliative bypass or laparotomy and tube drainage. Among the younger patients who underwent resections, 92 per cent were alive at one year, as compared with 60 per cent of patients who underwent palliative bypass procedures. Two patients who underwent tumor resections survived four years or longer. We conclude that carcinoma of the bile duct is not limited to the elderly and occurs in a significant number of young patients. In the younger population, carcinoma of the bile duct is characterized by delays in diagnosis. Early suspicion and aggressive management of young patients with obstructive jaundice are essential to ensure the best possible outcome for patients with this disease. PMID- 1729743 TI - A modified technique to create a neovagina with an isolated segment of sigmoid colon. AB - The creation of a functional vagina in patients with congenital vaginal aplasia or male transsexualism is a challenging problem. A group of 40 patients, including 23 male transsexuals, in whom a neovagina was created using a sigmoid transplant, is reported. The technique, a modification of Kun's "colocolpopoiesis," is described in detail. Ten patients showed some direct postoperative complications and five were readmitted the first six weeks postoperatively for a variety of reasons. No extensive complication occurred. Thirty-two patients were evaluated at the routine six week postoperative check up. Four patients had had intercourse at that time and an adequate vagina was found in 21 other patients. It is concluded that this modification of Kun's technique, known as colocolponeopoiesis, has had, at short term in the majority of patients, functionally good results and an acceptable complication rate. PMID- 1729744 TI - Arterial emboli of venous origin. AB - In a small but significant group of patients with documented systemic emboli, a source is never determined. It is in this group of patients that an arterial embolus of venous origin should be considered. During the past 20 years, we identified four patients who fulfilled the diagnostic criteria for an arterial embolus of venous origin. In each, the diagnosis was made during life. In addition, we reviewed the 40 additional patient reports in the literature that appeared to meet the criteria for the diagnosis of venous origin arterial emboli. Noninvasive methods were useful in determining the presence of thrombus in the venous system, and right to left shunting across an intracardiac defect. We conclude that treatment with heparin is the mainstay of therapy, and that caval interruption should be used only on a selective basis. PMID- 1729745 TI - Patterns of recurrence after curative resection of carcinoma of the colon and rectum. AB - Data on 818 patients who had undergone curative resection for Dukes' B2 or Dukes' C carcinoma of the colon and rectum were analyzed to determine the timing and patterns of recurrence based on such tumor characteristics as location, Dukes' stage, grade, ploidy and the presence of obstruction, perforation or adherence to adjacent organs or tissues. Three hundred and fifty-three patients (43 per cent) had recurrent disease. There was recurrence in 52 per cent of patients with carcinoma of the rectum and in 40 per cent of patients with carcinoma of the colon. The median time to recurrence for all patients was 16.7 months, with a range from 1 month to 7.5 years. Dukes' C lesions and the presence of adhesion or invasion, or both, or perforation were associated with significantly earlier recurrence. Among patients with recurrence, the most frequent sites were hepatic in 33 per cent, pulmonary in 22 per cent, local or regional, or both, in 21 per cent, intra-abdominal in 18 per cent, retroperitoneal in 10 per cent and peripheral lymph nodes in 4 per cent. Rectal primary sites, when compared with colonic, had proportionally more local or regional, or both, recurrences (p = 0.00003) and fewer involving retroperitoneal nodes (p = 0.022). Both primaries of the rectum and colon at stage C, when compared with stage B, had fewer local or regional recurrences, or both (p = 0.01), but a greater tendency to involve retroperitoneal or peripheral nodes. Primaries of the colon with adhesion to, or invasion of, adjacent organs had a lesser tendency to pulmonary metastasis (p = 0.036). Whereas the grade of anaplasia and ploidy had a strong influence on the rate of recurrence, they did not influence timing or patterns of recurrence. Patterns of recurrence based on the characteristics of the tumor may facilitate selection of the most appropriate adjuvant procedures, particularly those directed toward local or regional recurrence, or both, and also may guide efforts at early recognition of recurrence. PMID- 1729746 TI - Properitoneal synthetic mesh repair of recurrent inguinal hernias. AB - Safe reconstruction of the inguinal floor is the goal of any operation for repair of groin herniation. Operating in the properitoneal space avoids dissection of the scarred cord, and the incidence of testicular complications is markedly lowered. This study reports our experience with placing synthetic mesh between the peritoneum and the deficient inguinal floor for the repair of recurrent hernias of the groin area. During a five year period, 84 men underwent repair of 100 recurrent inguinal hernias using the properitoneal approach. Fifty-four patients had repair of a unilateral recurrent hernia, 16 had repair of a bilateral recurrent hernia and 14 had repair of both a recurrent hernia and a contralateral primary hernia. Postoperative complications occurred in six patients. No testicular complications were observed. Postoperative follow-up study ranged from six months to five years. There were only three recurrent hernias after this repair. All occurred within the first six months postoperatively. The properitoneal approach for repair of recurrent groin hernias using prosthetic mesh safely creates a new "fascia transversalis" with a low rate of recurrence and effectively eliminates testicular complications. PMID- 1729747 TI - The natural history of macroscopic cysts in the breast. AB - Macroscopic cysts of the breast, defined as clinically apparent lesions, are common causes of masses in the breast. In a 14 year study of 4,207 patients, cysts in the breast were diagnosed by percutaneous aspiration in 286 women who had cysts on 561 occasions. These cysts accounted for the presenting problem in 4 per cent of 15,600 visits to the clinic. Patients with cysts were observed for a mean period of 70 months. The patient was aware of a mass 83 per cent of the time, but cysts were discovered in asymptomatic patients in 17 per cent. The majority of cysts (76 per cent) were in premenopausal women (mean age of 48 years), with peak occurrence between age 40 and 50 years. Only 5 per cent were in women who were more than 60 years of age, none of whom had concurrent or subsequent carcinoma. Cysts did not recur in 60 per cent of the patients. There were two to five recurrences in 36 per cent and more than five recurrences in 4 per cent. Patients with more than five recurrences were five years younger than the over-all group when the first cyst appeared and the events developed over a period of time ranging from 51 to 161 months (mean of 96 months) and appeared at intervals averaging 17 months. Only three patients who were postmenopausal had more than one cyst. Carcinoma occurred in three patients (1 per cent) diagnosed between three and five years after the first cyst was discovered. Macrocystic disease seems to be a condition of women who were perimenopausal. An association between these cysts and carcinoma has not been proved. PMID- 1729748 TI - Anastomotic stricture with the EEA stapler after colorectal operation in the dog. AB - Anastomosis of the gastrointestinal tract has been made more secure by the use of the EEA (U. S. Surgical Corp.) stapler. The development of anastomotic strictures after stapling anastomosis is one of the major postoperative complications of this method. This study was done to compare the incidence of anastomotic stricture between stapling anastomosis and layer-to-layer handsewn anastomosis. Twelve dogs were divided into two groups. In each group, two colonic anastomoses were performed. Intestinal contents were not allowed to pass through one of the anastomotic sites created in an isolated segment of the colon, but were allowed to pass through the other site in the remaining colon. By the 28th postoperative day, anastomoses made with the EEA stapler, which had been excluded from contact with feces, had developed significantly more strictures when compared with the other anastomoses (p less than 0.05). The anastomotic strictures were membranous in nature when examined macroscopically and histologically. PMID- 1729749 TI - Early postlaparotomy percutaneous endoscopic gastrostomy. AB - Percutaneous endoscopic gastrostomy (PEG) is a relatively new procedure, the indications for which are evolving. We have recently attempted PEG placement in 19 patients during the early postlaparotomy period (less than 14 days) for initiating enteral feedings or providing decompression of the gastrointestinal tract. All patients had unexpected postoperative complications, including neurologic catastrophe, recurrent obstruction of the small intestine, acute respiratory failure, enterocutaneous fistula or poor oral intake. Of 19 attempts, PEG placement was successful in 18 patients (94.7 per cent). There were no major complications; two minor complications (exit site infection) were associated with catheter placement. All PEG placed for enteral feedings were successfully used for nutritional support, and gastrointestinal decompression was accomplished in seven of eight patients requiring PEG. The results of this study demonstrate that PEG is technically feasible, safe and effective in the early postlaparotomy period in high-risk patients with complicated postoperative courses and the need for long term enteral access. PMID- 1729750 TI - Meticulous attention to foot care improves the prognosis in diabetic ulceration of the foot. AB - Ulceration of the foot is a major cause of morbidity in patients with diabetes, and its treatment has become a significant part of general surgical practice. It is, therefore, important to develop an efficient and effective approach to the care of this complication. We established a clinic dedicated to the care and prevention of foot ulcers in diabetic patients and since its inception in 1985, 343 patients have been seen. We provide regular prophylactic care and education to patients without ulcers, as well as treating those with ulcers. To assess the effectiveness of the clinic, we compared two groups of patients. Group 1 contained those who had ulcers while attending our prophylactic care program. Group 2 comprised those who were referred to us with lesions already present. There were 21 patients in group 1 and 150 in group 2. There were no statistical differences between the two groups with respect to age, sex, type and duration of diabetes, smoking history, prevalence of peripheral neuropathy, peripheral vascular disease, renal impairment and retinopathy. The sites and sizes of lesions were also no different between the groups. In spite of these similarities, however, patients in group 1 had a significantly better prognosis than those in group 2. The over-all number of lesions per patient was lower (1.52 +/- 0.98, compared with 2.06 +/- 1.33, p less than 0.05), the mean time required for lesions to heal was shorter (111.9 +/- 80.5 days compared with 160.5 +/- 151.3 days, p less than 0.05). The major amputation rate was lower and fewer patients required partial foot amputation. Prior to the opening of the clinic, the mean length of inpatient treatment was 30 days. This now has been reduced to 12.9 +/- 12.8 days. We conclude that the improved prognosis for those in group 1 can be attributed to the earlier detection and treatment of both potential and actual foot lesions. These results support the contention that the establishment of a dedicated diabetic foot care clinic and regular patient review can reduce the morbidity associated with diabetic foot ulceration. PMID- 1729751 TI - The judicial use of venous duplex imaging and strain gauge plethysmography (single or combined) in the diagnosis of acute and chronic deep vein thrombosis. AB - Sixty-eight patients (79 limbs) with clinically suspected deep vein thrombosis were evaluated by duplex imaging, strain gauge plethysmography and venography. The diagnostic accuracies were projected over a spectrum of disease incidences ranging from 10 to 90 per cent of the population. The sensitivity, specificity, positive and negative predictive values, and over-all accuracy in detecting acute deep vein thrombosis were 90.9, 87.1, 83.3, 93.1, and 88.7 per cent, respectively, for venous duplex imaging, and 81.8, 69.6, 56.3, 88.9 and 73.5 per cent, respectively, for strain gauge plethysmography. The positive predictive value and over-all accuracy of venous duplex imaging were statistically significantly higher than that of strain gauge plethysmography. When both tests were combined and compared with venous duplex imaging alone, none of these parameters were statistically significant. For chronic deep vein thrombosis, the sensitivity, specificity, positive predictive value, negative predictive value and over-all accuracy for venous duplex imaging were 75, 86, 80, 86 and 82 per cent, respectively. Fourteen per cent had inconclusive results obtained at venous duplex imaging. When strain gauge plethysmography was combined with venous duplex imaging, the over-all accuracy was 82 per cent. As the true incidence of the disease increases, the positive accuracy differences between strain gauge plethysmography and venous duplex imaging decrease to a negligible level. We concluded that over-all, venous duplex imaging is superior. However, the strain gauge plethysmography has reasonable accuracy and may be used in places where venous duplex imaging is not available. Combined use of venous duplex imaging and strain gauge plethysmography would be helpful in patients with inconclusive results obtained at venous duplex imaging and, as the true incidence increases, the positive accuracy rate of strain gauge plethysmography becomes close to that of venous duplex imaging. PMID- 1729752 TI - ABO-incompatible orthotopic liver allografting in urgent indications. AB - The influence of ABO-compatibility was reviewed in 70 emergency orthotopic hepatic transplantations (OHT) performed at our institution in 60 highly urgent recipients between February 1984 and March 1989. Thirty-eight were ABO-identical (Id); 16, compatible (Comp), and 16, incompatible (Inc) transplants, respectively. The three groups did not differ statistically with respect to the indications, the adult/child ratio and the proportions of first OHT and retransplantations. Graft survival rates of ABO-Id, ABO-Comp and ABO-Inc OHT at one year were 47, 38 and 19 per cent, respectively (p less than 0.02). Incidences of perioperative mortality, arterial thrombosis and irreversible rejection were slightly (although not significantly) higher in the ABO-Inc group. Retransplantation rates were 19, 7 and 36 per cent in the ABO-Id, Comp and Inc groups, respectively. Patient survival rates at one year were 59 per cent for the ABO-Id group versus 43 per cent for both ABO-Comp and Inc combinations (NS). The results of this series of highly urgent OHT confirm that graft survival is lower with ABO-Inc livers; their use should be strictly considered as a short term life saving procedure. Improvement of patient survival after a first urgent ABO-Inc OHT may require an aggressive policy of retransplantation. PMID- 1729753 TI - A secure end colostomy technique. AB - A secure end colostomy technique preventing the common complications of intestinal prolapse and paracolostomy hernia is presented. Prosthetic mesh is fitted and secured to the intestine and the underside of the abdominal wall, giving considerable strength to the area and avoiding complications. PMID- 1729754 TI - Exchange of chest tubes using a nasogastric tube. PMID- 1729755 TI - The importance of Glisson's capsule and its sheaths in the intrahepatic approach to resection of the liver. AB - Glisson's capsule extends into the liver as sheaths around the hepatic ducts, hepatic arteries and portal tributaries. Within the hepatic substance, these structures need not be dissected individually, but the sheath can be ligated "en masse." These sheaths can be approached either anteriorly (after division of the main fissure or right fissure or umbilical fissure) or posteriorly from behind the porta hepatis. We recently used these approaches in 70 patients during a 27 month period. The median blood loss was zero units and there was one postoperative death. We believe the technique adds precision and safety to surgical treatment of the liver. PMID- 1729756 TI - Using lidocaine to ease the insertion of the circular stapler. PMID- 1729757 TI - A new portal venous shunt for bypass in orthotopic hepatic grafting. AB - A new heparin bonded portal venous cannula is described for use during orthotopic hepatic transplantation. This new shunt has a "shoulder" 4 centimeters from the caged tip, allowing it to be securely maintained within the portal vein. With a 60 degree curvature 7 centimeters from the tip, the shunt can conveniently be positioned away from the operative field. PMID- 1729758 TI - Cryosurgery in the treatment of cancer. AB - The range of application of cryosurgical techniques to the treatment of cancer is widely diversified and slowly increasing in scope. From these, one may reach the general conclusion that cryosurgical techniques are a standard method of treatment, competitive with other methods of therapy, in cancer located in some sites. For cancers located in other sites, cryosurgery is only useful as an end resort in selected patients. In some areas, especially in the viscera, cryosurgical techniques are only in developmental stages. Cryosurgery is most useful in easily accessible areas of the body. The results of the treatment of most carcinomas of the skin with cryosurgical techniques are as good as any other method of therapy. In carcinoma involving skin, cryosurgery has a special advantage in those situations when malignant tissue overlies bone. Cryosurgery is also useful in the management of dysplastic disease or carcinoma in situ, principally in the oral cavity and the uterine cervix. These applications are sufficiently valuable to be included in the textbooks concerned with those areas. Invasive cancer in other accessible sites, such as the oral cavity or the rectum, can be cured by cryosurgery, but the reports in the medical literature have not led to general use of the technique or descriptions of the technique in textbooks, except for occasional brief mention. Nevertheless, patients who are at high risk for surgical treatment because of coagulopathy or severe cardiopulmonary disease are appropriate candidates for the use of cryosurgical techniques. In the oral cavity, the possibility of preserving the bony structure is an attractive feature that maintains interest in cryosurgery. Unfortunately, there are no control studies to assist in the judgment of merit and in many cited reports, it is not easy to determine the survival rate or compare results with conventional therapy. In these sites, freezing techniques are more often used to achieve palliation of distressing symptoms by tumor bulk reduction, especially when little else can be done, and under these conditions, chemotherapy and radiotherapy are also commonly used. In less accessible sites, which generally require endoscopic or surgical exposure, cryosurgery is not often used. The treatment of carcinoma of the prostate gland by cryosurgery remains viable because of continued interest in the potentiation of immunologic defenses against carcinoma. This possible benefit is most evident in experimental tumors, but clinical evidence of benefit is not as clear.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1729759 TI - Renal metallothionein metabolism after a reduction of renal mass. II. Effect of zinc pretreatment on the renal toxicity and intrarenal accumulation of inorganic mercury. AB - In the present study we examined the effects of zinc pretreatment (to induce the renal synthesis of metallothionein) on the renal accumulation and intrarenal distribution of inorganic mercury in uninephrectomized (NPX) and sham-operated (SO) rats 24 h after the animals were given a 0.75, 1.0 or 1.5 mumol/kg intravenous (i.v.) dose of inorganic mercury. We also examined the effects of zinc pretreatment on the nephropathy induced by the three doses of inorganic mercury. Zinc was administered at a dose of 306 mumol/kg (20 mg/kg) subcutaneously (s.c.) in the form of zinc sulfate once daily for 2 consecutive days prior to the administration of inorganic mercury. Following zinc pretreatment, the renal accumulation of injected inorganic mercury increased in both NPX and SO rats treated with the three doses of inorganic mercury, but the increase was significantly greater in the NPX rats. The enhanced accumulation of mercury was associated with an altered pattern in the intrarenal distribution of mercury, particularly in the NPX rats. The increased renal accumulation of mercury in both the NPX and SO rats was due primarily to its increase in the renal cortex. We have recently found that the synthesis of metallothionein in the renal cortex increases in NPX and SO rats given zinc. Therefore, it appears that there is a relationship between the content of preinduced cellular metallothionein in the cortex and the content of mercury that accumulates in the cortex. Zinc pretreatment also prevented the nephropathy induced by the three doses of inorganic mercury from occurring in both the NPX and SO rats. We propose that some of the protection may be related to the altered intrarenal accumulation and distribution of mercury that occurs after pretreatment with zinc. Hepatic accumulation of mercury also increased in both groups of rats, but the increase again was significantly greater in the NPX rats. Our findings show clearly that a significant reduction in renal mass alters the hepatic and renal accumulation of mercury when zinc pretreatment is used to induce the renal and hepatic synthesis of metallothionein. In addition, our findings show that zinc pretreatment protects both normal and remnant kidneys in rats from the nephrotoxic effects of inorganic mercury. PMID- 1729760 TI - Inhibition of the activity of glutathione peroxidase by tertiary butylhydroperoxide in cultured Chinese hamster cells and the role of cellular glutathione in the recovery of the activity. AB - Treatment of Chinese hamster cells with 1 mM tertiary-butylhydroperoxide (t BuOOH) for 1 h markedly inhibited the activity of glutathione peroxidase (GSH Px). However, the activity returned to pre-inhibition levels within 1-2 h with post-treatment incubation. The inhibition of the activity of GSH-Px by t-BuOOH was enhanced by depletion of levels of cellular GSH with the addition of L buthionine sulfoximine (BSO) or diethylmaleate (DEM) and no subsequent recovery of the activity was observed for at least 4 h. The ratio of levels of GSSG to total GSH increased as a result of treatment with 1 mM t-BuOOH or diamide for 1 h but not as a result of treatment with hydrogen peroxide. After treatment with t BuOOH, the level of GSH rapidly increased to more than twice the control level during 15-40 min of post-treatment incubation. Depletion of GSH by DEM after treatment with t-BuOOH reduced the rate of the recovery of the activity of GSH Px, suggesting a role of cellular GSH in the recovery. However, the decrease in the rate of the recovery caused by DEM was small and in no way equivalent to the extent of depletion of GSH, suggesting that the rapid increase in the level of GSH after treatment with t-BuOOH is not closely related to the rapid recovery of the activity of GSH-Px. PMID- 1729761 TI - Species differences in the hepatotoxicity of coumarin: a comparison of rat and Mongolian gerbil. AB - The acute hepatic effects of coumarin (2H-1-benzopyran-2-one) in male Wistar rats and Mongolian gerbils has been compared. A single dose of coumarin (125 mg/kg, intraperitoneally (i.p.)) was hepatotoxic to rats within 24 h as assessed by its effects on a variety of hepatic parameters. Coumarin-induced hepatotoxicity was associated with significant increases in relative liver weight, plasma alanine and aspartate aminotransferase activities and hepatic non-protein sulphydryl groups. Cytochrome P-450 content and 7-ethoxycoumarin O-deethylase and glucose 6 phosphatase activities were significantly lower in coumarin-treated compared with control rats. Centrilobular necrosis was only observed in two out of six rats at this dose, but was present in all four coumarin-treated rats when the dose was increased to 150 mg/kg. In contrast to the effects observed in the rat, no evidence was found for coumarin-induced hepatotoxicity in gerbils following a single i.p. dose of 125 mg/kg. These data indicate that the gerbil is less sensitive to the hepatotoxic effects of coumarin than the rat. PMID- 1729762 TI - Studies on valproate-induced perturbations of neurulation in the explanted chick embryo. AB - The effect of the anticonvulsant sodium valproate on in vitro neurulation of the chick embryo, explanted after a 25-h in ovo incubation period, is described. Sodium valproate, at concentrations of 0.5-1.5 mM did not appear to have any profound effect on embryo growth when assessed by light microscopy. However scanning electron microscopy revealed a dose-dependent increase in the incidence of open anterior and posterior neuropores after 20 h of in vitro development (Stage 11). Concentrations of sodium valproate which were greater than 1.5 mM markedly increased the number of gross malformations, which were manifested as a complete disruption of the neural tube along its entire length. Failure of neuropore closure could not be attributed to a drug-induced neurodevelopmental delay as these defects were still apparent following 27 h of in vitro culture, a time coincident with the onset of embryo torsion. PMID- 1729763 TI - The effect of hexane on the ventricular fibrillation threshold of the isolated perfused rat heart. AB - This investigation was conducted to determine the influence of hexane on the ventricular fibrillation threshold of the isolated perfused rat heart and myocardial electrolyte levels. Ventricular fibrillation threshold was measured using the Langendorff perfusion apparatus. Heart rate was measured by a universal digital counter and the cardiac flow by collecting the outflow of the heating chamber below the heart into a graduated measuring cylinder. Magnesium and zinc were measured by atomic absorption spectrophotometry and potassium by flame photometry. Two groups of rats were studied; those in the experimental group were given 0.2 ml of hexane and the control group 0.2 ml olive oil subcutaneously for 90 days. Their hearts were removed under anaesthesia. Half of the experimental and control hearts were mounted on the Langendorff perfusion apparatus and the heart rate, coronary flow and ventricular fibrillation threshold were measured. The hearts of the other half were used to measure myocardial electrolyte levels. In the experimental group the ventricular fibrillation threshold decreased (4.72 (S.D. +/- 1.87) vs 9.48 (S.D. +/- 2.98); P less than 0.001). There was no change in the coronary flow and heart rate in between the groups. The mean myocardial potassium levels (2586 (S.D. +/- 162) vs 2968 (S.D. +/- 218) micrograms/g; P less than 0.001), magnesium levels (164 (S.D. +/- 28) vs 208 (S.D. +/- 18) micrograms/g; P less than 0.001) and zinc levels (19.6 (S.D. +/- 4) vs 33.8 (S.D. +/- 6.8) micrograms/g; P less than 0.001) were significantly lower in the hexane treated group compared to controls. Hexane, a constituent of glue and benzine, is cardiotoxic; marked derangement in myocardial electrolytes and a reduced ventricular fibrillation threshold, indicating an increased myocardial vulnerability to arrhythmias, was noted in the experimental animals. PMID- 1729764 TI - Effect of oral administration of 1,3-diphenylguanidine on sperm morphology and male fertility in mice. AB - An oral testicular toxicity and male fertility study was carried out in CD-1 mice with 1,3-diphenylguanidine (99.9% purity). 1,3-Diphenylguanidine was administered to male mice by daily gavage at dose levels of 0, 0.06, 0.25, 1, 4 and 16 mg/kg body wt. per day during an 8-week premating period. Females were not dosed at any time during the study. Sperm abnormality evaluation was performed in approximately half the males, randomly selected from the control and 16-mg/kg dose group on completion of dosing. The remaining males in the control, 4- and 16 mg/kg body wt per day groups were mated with non-dosed females. Reproductive performance, necropsy findings and litter data were recorded. No differences were found between control and dosed groups in body weight gain during the dosing period, macroscopic observations and organ weights at necropsy. Microscopic examination of the testes and determination of the frequency of total sperm abnormalities in the 16-mg/kg body wt per day group, did not show any effect due to 1,3-diphenylguanidine dosing when compared to the control group, except for a slight increase in sperm with folded tails but normal heads. Male and female fertility as well as reproduction performance were comparable in the groups examined (0, 4 and 16 mg/kg body wt per day). Maternal necropsy findings and litter data did not reveal any dose-related effect. It was concluded that under the conditions of the present study, 1,3-diphenylguanidine did not exert any significant adverse effects on fertility, reproductive capacity or embryonic/fetal development in CD-1 mice when administered to males at levels up to 16 mg/kg body wt per day. PMID- 1729765 TI - Nickel-induced hyperglycaemia: the role of insulin and glucagon. AB - Glucagon and insulin changes were measured in acute nickel-treated rats. Also, several parameters related to glucose homeostasis were evaluated. Nickel treatment caused an important and transitory rise in plasma glucose levels. These changes occurred simultaneously to hyperglucagonemia and hypoinsulinemia, leading to a drastic drop in the insulin/glucagon plasma ratio. In such a catabolic situation, hepatic and muscular glycogen levels remained almost unaltered. Hepatic fructose-2,6-bisphosphate (an indicator of gluconeogenic/glycolytic state) was drastically reduced a short time after nickel injection. Such events suggested that it was mainly gluconeogenesis and not glycogenolysis, which contributes to enhanced plasma glucose. Animals treated with large doses of glucagon did not mimic the hyperglycaemic responses induced by nickel, due to counteracting effects of insulin on plasma glucose. When diabetic rats were treated with nickel, the hyperglucagonemic response still remained, but plasma glucose levels did not increase at the same extent as when nickel was applied to control animals. Overall results suggest that both, glucagon and insulin changes are essential in the development of nickel-induced hyperglycaemia. Also, the lack of glycogenolytic response insinuates a direct or indirect inhibition of this process mediated by nickel and will need further investigation. PMID- 1729766 TI - Calcium valproate-induced uterine adenocarcinomas in Wistar rats. AB - Calcium valproate is an anticonvulsant agent with pharmacokinetic properties similar to sodium valproate and valproic acid. Potential carcinogenesis of calcium valproate was evaluated in B6C3F1 mice and Wistar rats given 125, 250 and 500 mg/kg in the diet for 104 weeks. Survival in treated rats increased in a dose related pattern despite a tumorigenic response in females. Adenocarcinomas of the uterus and cervix were increased in treated rats when compared to controls. The incidence of uterine neoplasia was 8, 20, 14 and 32% in the control, 125, 250 and 500 mg/kg groups, respectively. Neoplasia in treated rats were detected against a higher than expected background of adenocarcinomas in concurrent controls, since 8% incidence in controls was substantially above the laboratory historical database value of 0.6%. Tumors varied from epithelial masses confined to the endometrium, to transmural, highly desmoplastic neoplasms that invaded the serosa lining and the peritoneal cavity. These tumors metastasized in treated rats but not in controls. The statistically significant (P less than 0.01) increase in uterine adenocarcinomas found in females given 500 mg/kg of calcium valproate contrasts the absence of this tumor type in a previous rat carcinogenicity bioassay with valproic acid. Subcutaneous fibrosarcomas were significantly increased in valproic acid-treated males, but no uterine tumors were reported in females. It is puzzling that a true carcinogenic potential would be expressed by markedly different target organs as obtained with the acid and calcium salt of this moiety. PMID- 1729768 TI - Effect of the fungicide benomyl on xenobiotic metabolism in rats. AB - The effect of benomyl administered orally (p.o.) and intraperitoneally (i.p.) on the activity of hepatic microsomal mixed-function oxidases (MFOs) was studied in rats. A dose of 100 mg/kg given i.p. reduced the activities of several hepatic drug-metabolizing enzymes 24 h following the treatment. A similar reduction in the activities of the MFOs was also noted 24 h following oral benomyl administration at a dose of 500 mg/kg. Furthermore, in vivo inhibition of drug metabolism by benomyl was demonstrated by increased pentobarbital sleeping-time 24 h after p.o. as well as i.p. dosing. No alterations were found in the serum sorbitol dehydrogenase (SDH) at 24 h after i.p. or oral benomyl indicating a lack of hepatotoxic effect. These results indicate that benomyl shows a route independent effect on MFOs and is not toxic to the liver. PMID- 1729767 TI - Inhibition of mitochondrial respiration and oxygen-dependent hepatotoxicity by six structurally dissimilar peroxisomal proliferating agents. AB - The purpose of this study was to test the hypothesis that a variety of structurally dissimilar peroxisomal proliferators inhibited O2 uptake and caused O2-dependent hepatotoxicity in the perfused rat liver. Aspirin, valproate, ethylhexanol, clofibric acid, ciprofibrate and perfluorooctanoate were selected as a representative group of weak, moderate, and potent peroxisomal proliferators, respectively. All compounds studied inhibited state 3 but not state 4 rates of oxygen uptake in isolated mitochondria (perfluorooctanoate greater than ciprofibrate greater than ethylhexanol greater than clofibric acid greater than aspirin greater than valproate; half maximal inhibition occurred at concentrations ranging from 0.6 to 3.2 mM depending on the compound). Clofibric acid, ethylhexanol and aspirin inhibited oxygen uptake only in upstream, oxygen rich periportal regions of the perfused liver lobule by 30-40%. Perfusion with the six agents studied caused release of lactate dehydrogenase into the effluent perfusate in a dose-dependent manner and caused damage predominantly in periportal regions of the lobule as reflected by trypan blue uptake. A strong correlation between the concentration of compound needed to inhibit respiration in isolated mitochondria and cause hepatotoxicity in the perfused liver was observed. We propose that peroxisomal proliferators accumulate in the liver due to their lipophilicity where they inhibit actively respiring mitochondria in periportal regions of the liver lobule and cause local toxicity. PMID- 1729769 TI - Effect of inhaled methanol on pituitary and testicular hormones in chamber acclimated and non-acclimated rats. AB - Two experiments were conducted in which the acute effects of inhaled methanol on serum hormones associated with reproductive function in the male rat were evaluated. In the first experiment, rats exposed to methanol (0, 200, 5000 and 10,000 ppm) for 6 h were killed at the end of the exposure period (6 h) or the following morning (24 h). Also, because the process of exposure itself could modify neuroendocrine function, the effect of the handling associated with placing the rat in the exposure chamber was evaluated further by dividing the exposed animals into acclimated (2 weeks of prior handling) and non-acclimated groups. At 6 h, an effect of prior handling was noted in the sham-exposed rats, with serum luteinizing hormone (LH) of the non-acclimated group being greater than that of the acclimated group. Serum LH concentrations were altered by methanol exposure, but the direction of change and the exposure level at which an effect was noted differed between the acclimated and non-acclimated rats. Methanol (5000 ppm) reduced serum LH in the non-acclimated animals, while 10,000 ppm increased LH in the acclimated rats. Follicle stimulating hormone (FSH) and testosterone were unchanged by methanol in rats killed at 6 h. Thus, this experiment did not confirm earlier reports that exposure to 200 ppm for 6 h reduced serum testosterone. At 24 h, an effect of prior handling was still present in the hormonal measures, with serum and interstitial fluid testosterone concentrations being greater in the non-acclimated rats. Also, there was a dose x handling interaction with methanol exposure inducing an increase in serum testosterone in the non-acclimated rats (up to 5000 ppm) and a decrease in the acclimated rats (up to 10,000 ppm). In the second experiment, groups of acclimated and non-acclimated rats were exposed to 0 or 5000 ppm methanol for 1, 2 and 6 h and killed immediately after removal from the chamber. Serum LH, testosterone and FSH values were not different in sham- vs methanol-exposed rats at any time point. As in experiment 1, an effect of prior handling was noted. In general, the concentrations of these hormones and serum prolactin in the non acclimated rats were greater than those observed for acclimated rats. Methanol exposure resulted in increased prolactin concentrations under both handling conditions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1729770 TI - A cytosolic oxygenase activity involved in the bioactivation of 2-aminofluorene. AB - The contributions of the hepatic microsomal and cytosolic fractions in the metabolic activation of the promutagen 2-aminofluorene into mutagenic intermediates in the Ames test were investigated. Rat hepatic postmitochondrial, microsomal and cytosolic preparations could convert 2-aminofluorene to mutagens, the postmitochondrial preparation being the most and cytosol the least efficient. Pretreatment of the rats with Aroclor 1254 markedly enhanced the cytosol-mediated mutagenicity of the amine but increased microsomal- and postmitochrondrial mediated mutagenicity only modestly. The cytosol-mediated mutagenicity of 2 aminofluorene was abolished by heat treatment and by incubation with proteinase K, but was unaffected by dialysis emphasising the protein nature of the cytosolic activation system. Oxygen radical generating systems and oxygen radical scavengers did not significantly influence the cytosol-mediated mutagenic response. Similarly incorporation of xanthine or allopurinol into the cytosolic activation system did not modulate the mutagenic response indicating no role for the molybdenum oxygenases. The cytosolic activation of 2-aminofluorene differed from that mediated by the microsomes in cofactor requirement, substrate specificity and sensitivity to DMSO and (+)-catechin. Further centrifugation of the cytosolic fraction to remove any microsomal contamination did not decrease the cytosolic activation of 2-aminofluorene. It is concluded that the hepatic cytosol contains an oxygenase activity capable of activating certain aromatic amines. PMID- 1729771 TI - Renal metallothionein metabolism after a reduction of renal mass. I. Effect of unilateral nephrectomy and compensatory renal growth on basal and metal-induced renal metallothionein metabolism. AB - The effects of unilateral nephrectomy and compensatory renal growth on renal metallothionein metabolism were evaluated in the present study. In rats, the renal content of metallothionein increased in proportion to the increase in renal mass after unilateral nephrectomy and compensatory renal growth. However, when zinc was used to induce the synthesis of renal metallothionein, the remnant kidney in uninephrectomized (NPX) rats produced significantly greater amounts of metallothionein on a per gram kidney basis than a normal kidney in sham-operated (SO) rats. In both NPX and SO rats, zinc pretreatment caused metallothionein synthesis to increase primarily in the renal cortex and renal outer stripe of the outer medulla. Zinc pretreatment also changed the pattern for the intrarenal accumulation of inorganic mercury in NPX rats. After pretreatment with zinc, the accumulation of inorganic mercury predominated in the renal cortex rather than in the outer stripe of the outer medulla in the NPX rats. In addition, both NPX and SO rats were afforded complete protection against the nephrotoxic effects of a low, toxic dose of inorganic mercury when they were pretreated with inorganic zinc. The protection is postulated to be related to the alteration in the pattern of renal accumulation of inorganic mercury. In conclusion, the capacity to synthesize metallothionein increases significantly in rats after they have undergone unilateral nephrectomy and compensatory renal growth. The increased capacity of the remnant kidney to synthesize metallothionein may involve adaptive changes both in transcriptional and/or translational controls of metallothionein synthesis. PMID- 1729772 TI - Theoretical analysis of sound attenuation mechanisms in blood and in erythrocyte suspensions. AB - A theoretical analysis of the effect of the elasticity of cell membranes on sound attenuation in blood and erythrocyte suspensions was carried out. It is shown that the shell model of a cell adequately describes the attenuation in red blood cell suspensions. The contribution of viscous drag losses to the sound attenuation decreases with frequency, and at a frequency of 1 MHz it can be as high as 44% in water suspensions of erythrocytes and 24% in blood. PMID- 1729773 TI - Ultrasonics: a window into biomedical science. AB - Ultrasonics has always been concerned with biomedical research. When the journal started, most applications were in therapy and surgery, but diagnosis soon became dominant. Many key developments received early publication: these included Doppler ultrasound, real-time imaging, tumour neovascularization and, arguably most significant of all, phased array scanning. Now in its thirtieth year of publication, Ultrasonics has contributed greatly to the development of biomedical science and it promises to have an important continuing role. PMID- 1729774 TI - Failure to confirm increase in unscheduled DNA synthesis in sonicated mammalian cells in vitro. AB - Two in vitro mammalian cell lines (HeLa, mouse L5178Y) were exposed/sham exposed to ultrasound (2.3 or 2.6 MHz, I-SPTA 35 W cm-2, 10 microseconds burst duration, 200 Hz pulse repetition frequency) for 20 min and subsequently autoradiographically scored for unscheduled DNA synthesis (UDS). A positive control (20 and 40 J m-2 germicidal ultraviolet at a flux density of 1 J m-2 s-1) yielded statistically significant increases in UDS; no such increase was observed for the ultrasound regimens. The results fail to confirm an earlier report by Liebeskind et al. of ultrasound-induced increases in UDS. PMID- 1729775 TI - Interventional endoscopy of the biliary and pancreatic ducts: current indications and methods. AB - The use of interventional endoscopy of the biliary and pancreatic ducts has increased dramatically in recent years. Although choledocholithiasis is the most common reason for endoscopic treatment, other indications include pancreatolithiasis, cholangitis, biliary pancreatitis, papillary stenosis, sphincter of Oddi dysfunction, and benign or malignant ductal strictures. Endoscopic sphincterotomy is the cornerstone of therapeutic endoscopy and often precedes the use of balloon and basket stone extractors and placement of stents and endoprostheses. Other endoscopic methods include the use of lithotripsy, placement of drainage and infusion catheters, and coupling with percutaneous techniques. Radiologists need to be aware of the expanding indications and variety of endoscopic methods available for treating biliary and pancreatic disorders so that they can understand when the procedures are indicated. PMID- 1729776 TI - Percutaneous gastrostomy and transgastric jejunostomy. AB - Gastrostomy for feeding or decompression of the stomach or small intestine can be performed by using surgical or percutaneous, nonsurgical techniques. Although use of the surgical technique is well established, recent interest has focused on the nonsurgical methods because of their lower rates of morbidity. Percutaneous gastrostomy by either the endoscopic or the fluoroscopically guided Seldinger technique was introduced in the early 1980s. A number of technical modifications have been described, and sufficient clinical data have been accumulated and published to validate the safety of the percutaneous approach. Several published studies compare surgical with nonsurgical gastrostomy, but none compare the two percutaneous techniques. The purpose of this article is to review the current status of the fluoroscopically guided technique, its indications, and its results and to examine the relative merits of the surgical and nonsurgical techniques. PMID- 1729777 TI - Edema and tumor perfusion: characterization by quantitative 1H MR imaging. AB - Tumor perfusion is of central importance to the clinical oncologist because it has a direct effect on the success of cancer therapy. Yet determining whether a tumor is well or poorly perfused is difficult without the use of invasive techniques, because the variables that affect tumor perfusion are poorly understood. Quantitative MR imaging of tumor edema may provide a means of characterizing tumor perfusion, of studying heterogeneity of perfusion within the tumor mass, or of monitoring changes in tumor perfusion after therapy. A combination of factors often results in production of a large amount of edema within cranial or extracranial tumors. Any tumor that is encapsulated, whether by a fibrous tumor capsule or by a structure such as the cranium, will have an elevation in interstitial fluid pressure because dissipation of fluid is hindered. Elevated pressure of interstitial fluid acts to occlude tumor capillaries, so edema can cause a striking reduction of tumor perfusion. Because MR imaging can potentially be used for quantitative imaging of tumor edema, it may provide a means of indirectly measuring tumor perfusion. A review of the literature suggests that diffusion-weighted MR imaging may be better than T1- or T2-weighted MR imaging for quantitative imaging of tumor edema. I do not propose that diffusion-weighted imaging can measure perfusion directly; rather I hypothesize that a diffusion-weighted image can be correlated with tumor edema. Because edema indirectly regulates perfusion through the mechanism of interstitial fluid pressure, I propose an indirect correlation between the diffusion-weighted image and regional tissue perfusion. If the relationship between tumor perfusion and the pharmacokinetics of chemotherapeutic agents is better understood, MR imaging of tumor edema may even aid in predicting the delivery of drugs to a tumor. PMID- 1729778 TI - High-resolution CT appearance of diffuse alveolar septal amyloidosis. PMID- 1729779 TI - National Cancer Institute Breast Imaging Workshop, September 1991. PMID- 1729780 TI - Risk factors for breast cancer in women undergoing mammography. AB - To determine risk factors for carcinoma of the breast, we compared women with cancer on screening and diagnostic mammography with those in whom cancer was not detected. For 39 months, medical histories were collected by mammography technologists on 3492 women having routine screenings or diagnostic mammograms at our institution. Potential risk factors of women with biopsy-proved breast cancer were compared with those in women who had normal findings on mammograms or negative biopsy results (control subjects). Of the 3492 women, 49 had biopsy proved breast cancer. There were 3361 patients in the control group, including those women with normal findings on mammograms (3294) and those with negative biopsy results (67). Eighty-two women had incomplete questionnaires or were lost to follow up. Nearly all of the patients with breast cancer were postmenopausal compared with 68% of the control subjects. The mean length of lactation for breast cancer patients was significantly less than for control subjects: 5.6 vs 7.5 weeks (p = .015). This was true also for the postmenopausal patients: 8.1 vs 6.1 weeks (p = .041). Postmenopausal breast cancer patients had menstruated significantly more years (p = .016) than the postmenopausal control subjects: 34 vs 31 years, although the mean age at menarche was not different. When corrected for age, there was no significant difference in the total duration of menstruation in the postmenopausal cancer patients compared with the postmenopausal control subjects. Postmenopausal breast cancer patients had a significantly greater (p = .021) average body weight than postmenopausal control subjects: 71.7 vs 66.7 kg, although body weight was the same when all patients were considered. Similar results were found when Quetelet's index for obesity (weight in kg/height in cm2) (p = .004) was calculated for postmenopausal patients: 28 for cancer patients and 26 for control subjects. There was no significant difference in height between the cancer patients and control subjects when all patients or just the postmenopausal patients were considered. History of oral contraceptive use was significantly less common among postmenopausal breast cancer patients than among postmenopausal control subjects: 9% vs 20%. Patients with breast cancer had lower parity than the control subjects. In our series of patients, women in whom breast cancer was detected on mammography lactated less, showed no significant difference in years of menstruation when corrected for age, had a greater average body weight, used oral contraceptives less often, and had fewer children than women in whom no cancer was detected on mammography. PMID- 1729781 TI - Functional abnormalities of the gastrointestinal tract in patients with spinal cord injuries: evaluation with imaging procedures. AB - One quarter of patients with spinal cord injuries eventually have severe chronic gastrointestinal symptoms. Because there are about 1.5 million such patients in the United States, major chronic gastrointestinal symptoms will develop in approximately 400,000 patients, all of whom are likely to need the services of radiologists. These gastrointestinal abnormalities, however, are quite different from the gastrointestinal problems that occur in the general population. For this reason, the imaging methods used for diagnosis in these patients are also different from those used with persons who do not have spinal cord injuries. The purpose of this review is to describe the role of diagnostic imaging in patients with severe chronic gastrointestinal symptoms associated with spinal cord injury. PMID- 1729782 TI - Swallowing dysfunction in the postpolio syndrome: a cinefluorographic study. AB - Twenty patients with a remote history of poliomyelitis and recent or progressive dysphagia were evaluated with cinefluorography. Radiographic abnormalities were present in the pharynx in varying degrees in all but one of the patients. Findings included atrophy of the prevertebral soft tissues, unilateral or bilateral weakness of the tongue or soft palate, paresis or paralysis of the pharyngeal constrictor muscle, incomplete or absent epiglottic tilt, poor laryngeal elevation, poor laryngeal closure with laryngeal penetration, aspiration (often without a cough), and luminal narrowing at the cricopharyngeal level. Other structural lesions included a Zenker diverticulum in one patient, bilateral pharyngeal pouches in five, and a unilateral pouch in one. Additional structural lesions contributing to dysphagia were found in two other patients, including a focal stricture in the cervical esophagus in one patient and two stenotic rings in the distal esophagus in another. In four patients (one of whom had the Zenker diverticulum), the inferior constrictor muscle contracted forcibly above a prominent cricopharyngeus muscle, perhaps contributing to the formation of the diverticulum. It is important to examine postpolio patients with dysphagia carefully with dynamic imaging to assess the severity of decompensation and to detect other lesions that may be treatable. The information derived can be used to guide management. PMID- 1729783 TI - Rectal carcinoma treated by preoperative irradiation: MR imaging and histopathologic correlation. AB - We evaluated the role of MR imaging in assessing the effect of preoperative irradiation in 11 patients with primary rectal carcinoma. Findings on MR images obtained before radiotherapy and 5-6 weeks afterward were analyzed and correlated with the histopathological findings (nine patients) or the findings at laparotomy (two patients). Before irradiation, the tumor volumes on MR images were between 3.3 and 51.7 cm3 (mean 19.7 cm3). After irradiation, the volumes were from 0.8 to 33.2 cm3 (mean, 10.4 cm3), representing a decrease in volume of 11% to 88% (mean, 55%). On the MR images obtained before irradiation, the tumors were confined to the bowel wall in four cases (stage A-B1), penetrated the perirectal fat in six cases (stage B2), and involved an adjacent organ in one case (stage B3). After irradiation, no apparent changes were seen in the MR appearance of the local tumor stage in nine of the 11 patients. In one patient, progression of stage was suspected on the postirradiation MR images, but this was not confirmed at histologic examination. In one patient, possible downstaging occurred after irradiation, although this could not be proved. Our findings suggest that MR imaging may be useful for determining the effect of preoperative radiotherapy on rectal carcinomas. PMID- 1729784 TI - Visceral metastases from melanoma: findings on MR imaging. AB - Typical ocular and CNS melanomas are hyperintense on T1-weighted MR images and hypointense on T2-weighted MR images. We performed MR imaging in 48 patients with melanoma metastatic to visceral organs. Images were reviewed retrospectively in order to determine whether there were predominant MR features specific for visceral melanoma and to see if visceral metastases have MR characteristics similar to metastases in the CNS. Eleven patients also were examined after injection of gadopentetate dimeglumine to evaluate the enhancement characteristics of these tumors. Two hundred sixty-one lesions were found. Lesions were classified according to their signal intensities relative to uninvolved liver on T1-weighted, T2-weighted, and short TI inversion recovery (STIR) pulse sequences. Most commonly, lesions were either hypointense or isointense on T1-weighted sequences and hyperintense on T2-weighted and STIR sequences (185 lesions). Less frequently, lesions were hyperintense on T1 weighted sequences and hypointense or isointense on T2-weighted and STIR sequences (59 lesions). A mixed pattern was seen on T1- and T2-weighted sequences in 17 lesions. The patterns did not correlate with lesion size. Of the three sequences studied by subjective comparison, the STIR sequence in our series had the highest sensitivity for lesion detection and yielded the highest lesion conspicuity. Injection of gadopentetate dimeglumine in 11 patients did not increase either the number or the conspicuity of lesions seen. Our results show that visceral metastases from melanoma have a wide variety of appearances on MR images. The STIR sequence appears to be optimal, and the metastases do not enhance with gadopentetate dimeglumine. PMID- 1729785 TI - Delineation of surgical segmental liver anatomy: value of PRISE, an MR fast scanning technique. PMID- 1729786 TI - Who did it first? PMID- 1729787 TI - Dynamic MR imaging of the abdomen with gadopentetate dimeglumine: normal enhancement patterns of the liver, spleen, stomach, and pancreas. AB - To show the normal contrast enhancement patterns of the upper abdominal organs, dynamic gadopentetate dimeglumine-enhanced MR imaging of the upper abdomen was performed in 48 patients. Although all patients were originally examined for focal hepatic lesions, none of them had diffuse parenchymal disease of any of the examined organs. Dynamic gadopentetate dimeglumine-enhanced MR imaging was done by using a heavily T1-weighted gradient-echo sequence (100/5 [TR/TE], 80 degrees flip angle) performed before, and repeatedly for a period of 10 min after, an IV bolus injection of gadopentetate dimeglumine (0.1 mmol/kg). Signal enhancement in each of the organs was calculated by measuring the signal intensity before and after administration of contrast medium. All organs showed signal enhancement within the first 2 min (p less than .001) and a continuous decline thereafter. The enhancement of the pancreas, liver, stomach wall, spleen, and renal cortex reached peaks of 75%, 78%, 96%, 144%, and 216%, respectively, 45 sec after administration of contrast medium. Liver and pancreas showed a homogeneous enhancement pattern throughout the examination. The spleen appeared heterogeneous during the first 60 sec and homogeneous thereafter. Two zones could be distinguished on the contrast-enhanced images of the stomach wall: an enhanced inner zone and an unenhanced outer zone. We conclude that homogeneous enhancement of the liver and pancreas, early heterogeneous enhancement of the spleen, and enhancement of the inner gastric wall are normal patterns on dynamic gadopentetate dimeglumine-enhanced MR images. PMID- 1729788 TI - A simple method to reduce air-bubble artifacts during percutaneous extraction of biliary stones. PMID- 1729789 TI - Macronodular tuberculoma of the liver: CT and MR findings. PMID- 1729790 TI - Imaging of the biliary sump syndrome. AB - Side-to-side choledochoduodenostomy is a safe and effective surgical technique to improve biliary drainage in selected patients. The segment of common bile duct between the anastomosis and the ampulla of Vater may act as a stagnant reservoir or sump. When debris, stones, or infected bile accumulates in the sump, usually because of malfunction of the ampulla of Vater, recurrent abdominal pain or symptoms of cholangitis, pancreatitis, or biliary obstruction may develop. This uncommon (0.14-1.30%) complication is known as the sump syndrome. On imaging studies, diagnostic findings are debris or stone(s) in the common bile duct. Suggestive findings are dilated bile or pancreatic ducts, and changes due to pancreatitis, cholangitis, or liver abscess. Patients with this syndrome frequently have multiple imaging studies before the condition is recognized. The purpose of this essay is to illustrate the imaging findings of this syndrome. PMID- 1729791 TI - Value of lipid- and water-suppression MR images in distinguishing between blood and lipid within ovarian masses. AB - The distinction between blood and lipid in ovarian masses on MR imaging is important in the differential diagnosis of these lesions. However, this is often difficult on routine MR images because both blood and lipid within tumors can have the same signal intensity as subcutaneous fat. Accordingly, we studied the value of lipid- and water-suppression MR images in making this distinction in 16 patients (21 lesions). As proved by surgery (six patients) or laparoscopy (10 patients), there were 16 endometriomas, one hemorrhagic leiomyosarcoma, and four lipid-containing mature cystic teratomas. The signal intensity in all 17 hemorrhagic lesions was greater than that of subcutaneous fat on lipid suppression images and less than that of fat on water-suppression images. This compared with the signal intensity of the four lesions that contained lipid, in which the signal intensity was similar to that of subcutaneous fat on both the lipid- and water-suppression images. Thus, the lipid- and water-suppression MR images allowed an accurate distinction between the two. Our experience suggests that the appearance of blood and lipid in ovarian tumors is sufficiently different on lipid- and water-suppression MR images to allow an accurate distinction between the two. The two techniques should be useful in the differential diagnosis of such lesions by MR imaging. PMID- 1729792 TI - Sonographic features of genitourinary tuberculosis. PMID- 1729793 TI - Idiopathic tumoral calcinosis. PMID- 1729794 TI - Benign giant-cell tumor of bone with pulmonary metastases: clinical findings and radiologic appearance of metastases in 13 cases. AB - Tumors that metastasize are considered "malignant" by definition. However, benign giant-cell tumor of bone is an exception because of the potential for histologically benign pulmonary metastases, a fact seldom emphasized in the radiologic literature. We therefore report our experience with 13 cases of pulmonary metastasis among 475 patients (prevalence, 3%) in whom benign giant cell tumor of bone was diagnosed before 1990 at our institution. Five (38%) of the 13 primary bone tumors were located in the distal radius. Local recurrence at the site of the primary bone tumor tumor occurred in seven patients (54%) before pulmonary metastases developed. The mean interval from the diagnosis of the primary bone tumor to the onset of pulmonary metastasis was 3.8 years, with a maximum of 10.7 years. Fifty-four percent of the patients (7/13) had pulmonary metastases 3 years after diagnosis of the primary bone lesion, and 92% (12/13) had pulmonary metastases 7.5 years after diagnosis. Overall mortality rate directly due to giant-cell tumor and its metastases was 23%. On chest radiographs and CT scans, pulmonary metastases appeared as rounded, nodular opacities of homogeneous density, ranging from 0.5 cm to 8.0 cm in diameter. Peripheral regions of the lungs were involved in 85% of the cases and basilar regions in 62%. Our study shows that benign giant-cell tumor of bone can produce pulmonary metastases, that metastases most often occurred with recurrent local disease and distal radial lesions, that the prognosis was relatively favorable, and that such metastases had no distinguishing radiologic features. PMID- 1729795 TI - Hematopoietic bone marrow hyperplasia: high prevalence on MR images of the knee in asymptomatic marathon runners. AB - In a prior study of marathon runners, we noticed that MR scans of the knee frequently showed hyperplasia of red (i.e., hematopoietic) bone marrow. Because the frequency of this finding in various populations is unknown, the purpose of this study was to determine the relative prevalences of hematopoietic bone marrow hyperplasia on MR examinations of the knees of healthy volunteers (n = 74), patients with symptoms of knee disorders (n = 54), and asymptomatic marathon runners (n = 23). The prevalence of hematopoietic bone marrow hyperplasia was 3% (2/74) for the healthy volunteers, 15% (8/54) for the patients, and 43% (10/23) for the marathon runners. The difference in prevalence between each of the three groups was statistically significant at p less than .05 in each case with hematopoietic bone marrow hyperplasia, the distal femur was the only area affected, while the epiphysis and proximal tibia were uninvolved. This pattern of affected bone marrow with hyperplasia of the hematopoietic marrow may be useful for the differential diagnosis. We postulate that the high prevalence of hematopoietic bone marrow hyperplasia in marathon runners may develop as a response to "sports anemia", which is commonly found in highly conditioned, aerobically trained athletes. Furthermore, this is considered to be a normal variant when found in the pattern described here. PMID- 1729796 TI - Full-thickness tears of the rotator cuff of the shoulder: diagnosis with MR imaging. AB - The purpose of this study was to describe MR findings in full-thickness tears of the rotator cuff. Of 102 shoulders examined by MR imaging, 31 were found to have a full-thickness tendon tear at arthroscopy/bursoscopy (five shoulders) or open surgery (26 shoulders). All shoulders were imaged in oblique coronal and axial planes. MR images of the 102 shoulders were evaluated for (1) the presence of fluid in the subacromial and subdeltoid bursae; (2) abnormal signal of the supraspinatus, subscapularis, infraspinatus, and teres minor tendons; (3) interruption of tendon continuity and thinning of the tendon; and (4) proximal retraction of the junction of the muscle and tendon. The presence or absence of each finding was determined by consensus of two radiologists, who interpreted the images without knowledge of the surgical findings. Results in those 31 shoulders with proved full-thickness tears were: fluid in the subacromial bursae (29 shoulders), interruption of tendinous continuity (22 shoulders), focally increased signal of the tendon equivalent to that of water (27 shoulders), and musculotendinous retraction (24 shoulders). The finding of subacromial fluid was a sensitive indicator (93%) of a full-thickness tear, and interruption of tendinous continuity was a specific finding (96%) in diagnosing a full-thickness tear. Our experience shows interruption of tendon continuity is the most specific MR finding of full-thickness rotator cuff tears, while subacromial fluid is the most common finding. PMID- 1729797 TI - Thoracic complications of extracorporeal membrane oxygenation: findings on chest radiographs and sonograms. AB - Neonates treated with extracorporeal membrane oxygenation (ECMO) for respiratory failure have a high frequency of complications related to systemic anticoagulation, ECMO and other life-support lines and catheters, and the antecedent pulmonary disease. Many of these complications involve the thorax and can be defined on chest radiographs or thoracic sonograms. The purpose of this essay is to illustrate the findings of the various thoracic complications of ECMO on chest radiographs and sonograms. This study is based on a review of the medical records and findings on chest radiographs and sonograms of 150 neonates who were treated with ECMO at our institution. PMID- 1729798 TI - Enlarged amniotic cavity: a new sonographic sign of early embryonic death. AB - In the process of evaluating sonograms to determine the status of early gestations, it was noted that enlargement of the amniotic cavity appeared to correlate with embryonic death. This study tested that hypothesis by comparing the size of the amniotic cavity with the crown-rump length (CRL) and the size of the chorionic cavity in 25 normal gestations and 10 cases of embryonic death. Measurements included diameter of the amniotic cavity (Da), diameter of the chorionic cavity (Dc), and CRL. Normal first-trimester embryos have distinct rates of growth for both chorionic and amniotic cavities. Least-squares linear regression of CRL on Da for normal embryos reveals Da = 1.1 x CRL - 0.07 (r = .988, n = 25, p less than 10(-8)), indicating that Da and CRL are almost equal. An abnormal embryo, especially at 5-6 weeks' gestation, will have an abnormally large amniotic cavity for its CRL and the size of its chorionic cavity. The CRL Da difference of 0.11 +/- 0.20 cm in normal embryos differed significantly from that difference of 0.86 +/- 0.38 cm in abnormal embryos (p less than 10(-8)); however, the chorionic cavity was still appropriate in size for the CRL. These results suggest that an amniotic cavity that is enlarged relative to the CRL and the size of the chorionic cavity is evidence of embryonic death. PMID- 1729799 TI - Congenital disorders of sexual differentiation: MR findings. AB - A broad spectrum of anomalies of sexual differentiation may exist at birth. These include male and female pseudohermaphroditism, gonadal dysgenesis, and true hermaphroditism. When ambiguous genitalia are present, expedient identification of the anomaly is required for proper gender assignment and appropriate surgical or hormonal correction. As the appearance of the external genitalia often is not distinctive enough to make a specific diagnosis, this must be accomplished by clinical findings along with a combination of cytogenetic, biochemical, and radiologic studies. Because the causes of abnormal sexual differentiation are diverse and often exhibit incomplete expression, they produce much anatomic variability. Sonographic and radiographic studies are often used initially to evaluate such conditions. The noninvasive multiplanar nature of MR imaging makes it a useful alternative method with which to characterize the abnormal anatomy in this group of disorders, as we illustrate in this pictorial essay. PMID- 1729800 TI - Fat-suppression MR imaging in neuroradiology: techniques and clinical application. AB - Fat-suppression techniques are useful in MR imaging to eliminate strong signals from fatty tissues that interfere with signals from adjacent areas. Various methods of fat suppression have been devised, but when suppression of fat is used in combination with contrast enhancement employing paramagnetic agents (e.g., gadopentetate dimeglumine), the definition of normal anatomic structures is significantly improved, enhancing lesions become more conspicuous, and lesional margins are better defined in regions of the body with large amounts of fat, whose signal is suppressed. Contrast-enhanced fat-suppressed T1-weighted images provide more information than do conventional MR images. In this review, several types of fat-suppression techniques and their clinical applications in neuroradiology are described. Gadopentetate dimeglumine-enhanced, fat-suppressed T1-weighted images appear to have significant advantages over conventional T1 weighted contrast-enhanced images and should replace them in imaging regions of the body where large amounts of fat are present. PMID- 1729801 TI - Localized fat collection adjacent to the intrahepatic portion of the inferior vena cava: a normal variant on CT. AB - We describe a normal focal collection of fat that has the appearance of a mass on CT scans. The fat is adjacent to the intrahepatic portion of the inferior vena cava and is contiguous to the fat around the subdiaphragmatic portion of the esophagus. This finding occurred in 11 (0.5%) of 2227 patients who had CT scans at our institution. The fat collections did not change in size or shape on follow up CT scans obtained 2 to 29 months later (mean, 14 months). The localized fat collections were at the level of or above the confluence of the hepatic veins and the inferior vena cava, and were medial to the inferior vena cava. They were less than 22 mm in length, oval, and had attenuation values ranging from -113 H to -23 H on unenhanced CT scans. The collections enhanced slightly on scans obtained after contrast administration. T1-weighted (600/15) MR images obtained in three cases showed a high-intensity mass that was contiguous to high-intensity fat around the esophagus, medial to the intrahepatic portion of the inferior vena cava. Our experience suggests that a masslike fat collection adjacent to the intrahepatic portion of the inferior vena cava is a normal variant on CT scans and should not be mistaken for an abnormality. PMID- 1729802 TI - The upper arm approach for placement of peripherally inserted central catheters for protracted venous access. PMID- 1729803 TI - Comparison of the efficacy of digital subtraction and film-screen angiography of the lower limb: prospective study in 50 patients. AB - We prospectively compared current digital subtraction angiography (DSA) with conventional film-screen angiography (FSA) of the lower limb for evaluation of areas of arterial stenosis and degree of arterial visualization. Fifty patients had both DSA and FSA of a single lower limb. Specific anatomic sites (examiner selected sites) throughout the lower limb were marked on each film by an experienced angiographer (examiner). These sites consisted of the common femoral, superficial femoral, popliteal, anterior tibial, posterior tibial, peroneal, and dorsalis pedis arteries and bypass grafts, when present. The films were then reviewed blindly by two different experienced angiographers (observers). All sites were graded for the degree of arterial narrowing based on a standard scale (grade 1 = normal, grade 5 = occluded) that also included grading for nonvisualization (grade 6). Each observer also selected the most stenotic site in each anatomic area (observer-selected sites). The data were analyzed for the entire lower limb and at specific anatomic sites. DSA sites were judged to be slightly more narrowed (p less than .05) in the superficial femoral artery by both observers and in the common femoral artery, bypass graft, and overall by a single observer. No other significant differences were found in grade of stenosis or vessel visualization for examiner-selected sites. For observer-selected sites, observers agreed on the location of the most stenotic site 76% of the time for FSA and 69% of the time for DSA. No significant difference was found in grade of stenosis or vessel visualization for either observer for the entire lower limb or at specific anatomic sites. These findings were present when all sites chosen were considered and when there was agreement between sites chosen on FSA and DSA for each observer. In conclusion, optimal-quality FSA and DSA produced virtually equivalent results for angiography of the lower limb for both grade of stenosis/occlusion and vessel visualization. PMID- 1729804 TI - Diagnostic efficacy of digitized images vs plain films: a study of the joints of the fingers. AB - Four hundred fifteen finger joints from 30 patients were evaluated for the presence of joint-space erosion, narrowing, and degenerative spurring on plain films, low-resolution digitized images (1024 x 840 bytes x 12 bit matrix), and high-resolution digitized images (2048 x 1680 bytes x 12 bit matrix). Three hundred four joints were abnormal. Low- and high-resolution digital images were displayed on a 1K x 1K monitor with the ability to change level, window, orientation, and brightness. Five radiologists interpreted images. The presence or absence of each abnormality was determined by consensus of two skeletal radiologists who did not otherwise participate in the study. Receiver-operating characteristic analysis was used to obtain an area and a true-positive rate at a 0.10 false-positive rate for each interpreter. Randomized block analysis of variance with interpreters as blocks was used to compare areas and true-positive rates among imaging techniques for each type of abnormality; no statistically significant differences were found. In conclusion, the efficacy of display of digitized images on high- and low-resolution modes is not significantly different from that of plain films in the detection of erosions, joint-space narrowing, or degenerative spurring in small joints of the hands. PMID- 1729806 TI - International Skeletal Society: eighteenth annual refresher course, September 1991. PMID- 1729805 TI - Receiver-operating-characteristic study of chest radiographs in children: digital hard-copy film vs 2K x 2K soft-copy images. AB - Two methods are commonly used to visualize digital radiologic imaging data: (1) hard-copy viewing, in which the digital data are used to modulate the intensity of a laser beam that exposes an analog film and (2) soft-copy viewing, in which the digital data are converted to an analog video signal and presented on a CRT monitor. The film method allows new digital imaging systems to be easily integrated into conventional radiologic management and viewing methods. The second method, soft-copy viewing, allows digital imaging data to be managed and viewed electronically in a picture archiving and communication system (PACS). These PACS systems are hypothesized to have improved operational efficiency and enhanced image-analysis capabilities. The quality of soft-copy images is still not widely accepted. This article reports on the results of a large-scale receiver-operating-characteristic study comparing observers' performance in detecting various pediatric chest abnormalities on soft-copy 2048 x 2048K byte displays with their performance with digital laser-printed film from computed radiography. The disease categories studied were pneumothorax, linear atelectasis, air bronchogram, and interstitial disease. The selected data set included 239 images; 77 contained no proved abnormality and 162 contained one or more of the abnormalities mentioned. Seven pediatric radiologists participated in the study, two as judges and five as observers. Our results show no significant difference between viewing images on digital hard copy and soft copy for the detection of pneumothoraces and air bronchograms. A slight performance edge for soft copy was seen for interstitial disease and linear atelectasis. This result indicates that computed chest radiographs in children viewed in a soft-copy PACS environment should result in diagnoses similar to or slightly more accurate than those obtained in a laser-printed film-based environment. PMID- 1729807 TI - Gallbladder contractility in patients with spinal cord injury. PMID- 1729808 TI - Insulinomas: MR imaging with STIR sequences and motion suppression. PMID- 1729809 TI - The azygos lobe: a nonentity? PMID- 1729811 TI - Grid for CT-guided percutaneous fine-needle aspiration. PMID- 1729810 TI - Erosion of spinous process after surgery. PMID- 1729812 TI - MR appearance of a retained surgical sponge. PMID- 1729813 TI - Myocardial salvage with direct coronary angioplasty for acute infarction. AB - To assess the changes in myocardial function following direct coronary angioplasty, we evaluated 323 consecutive patients undergoing coronary angioplasty without antecedent thrombolytic therapy for acute myocardial infarction. Left ventricular function was evaluated using contrast ventriculography immediately preangioplasty and at the time of predismissal follow-up angiography (a mean of 7 days after infarction). The global ejection fraction increased from 52.6% to 58.9% (p less than 0.0005). Multivariate correlates of improved global left ventricular function included baseline ejection fraction less than or equal to 45%, and a patent infarct vessel at the time of predischarge follow-up angiography. Systolic function in the infarct zone improved by a mean of 30%. Logistic regression analysis identified sustained infarct vessel patency and anterior myocardial infarction as multivariate correlates of improved regional function in the infarct zone. In patients presenting with baseline ejection fractions less than or equal to 40%, the mean ejection fraction increased from 28% to 42%. Long-term survival was compromised in patients with global ejection fractions of less than or equal to 40% at the time of dismissal. Thus significant improvement in left ventricular function can be expected in the majority of patients undergoing direct infarct angioplasty. The myocardial salvage appears to be most significant in patients suffering large infarctions, and in those with sustained infarct vessel patency. PMID- 1729814 TI - Metabolic and cardiovascular benefits deriving from beta-adrenergic blockade in chronic congestive heart failure. AB - Ten patients with congestive heart failure were given metoprolol (50 mg/day) or placebo during a double-blind, crossover, randomized study. After a run-in period of 4 weeks, metoprolol and placebo were administered over a period of 3 months, which was separated by a washout period of 4 weeks. At the end of the run-in, metoprolol, and placebo periods, all patients underwent metabolic (oral glucose tolerance and hyperinsulinemic glucose clamp tests) and noninvasive cardiologic (New York Heart Association classification, bimodal echocardiographic left ventricular end-diastolic determination, maximal oxygen consumption, left ventricular radionuclide ejection fraction) tests. Our results show that beta adrenergic blockade significantly enhances insulin-mediated suppression of hepatic glucose output (p less than 0.005) and increase in glucose uptake (p less than 0.01) with a concurrent improvement in New York Heart Association functional class (p less than 0.05) and the multistage exercise treadmill test score (p less than 0.05). After administration of metoprolol all changes in glucose turnover parameters were found to correlate with the decrease in basal plasma free fatty acid levels. In conclusion, our findings confirm the beneficial cardiologic effects of beta-adrenergic blockade in congestive heart failure and demonstrate that metoprolol is also useful for reversing the metabolic damage caused by exaggerated plasma norepinephrine levels. PMID- 1729815 TI - Lymphocytic active myocarditis characterized by numerous clusters of lymphocytes: a chronic variant of myocarditis? AB - We report three patients with a particular form of myocarditis characterized by numerous clusters of lymphocytes. Their common clinical manifestation was progressive and fatal heart failure with a 3- to 6-year duration. Atrioventricular and intraventricular conduction disturbances were observed in two patients. At necropsy, the hearts weighed 480, 530, and 430 gm, respectively, and showed marked dilatation of the bilateral ventricles and atria, with frequent mural thrombi. Histologic examination revealed numerous lymphocytic clusters and scattered foci of acute myocardial cell damage on a background of extensive fibrosis. We propose the term "chronic active myocarditis" to denote clinicopathologic characteristics of the present cases. PMID- 1729816 TI - Ventricular thrombi and thromboembolism in dilated cardiomyopathy: a prospective follow-up study. AB - To determine the prevalence and natural history of left ventricular thrombus in dilated cardiomyopathy, we prospectively performed two-dimensional echocardiograms in 25 patients with nonischemic dilated cardiomyopathy who were not receiving anticoagulation. Eighty-five echocardiograms were performed serially over a 9- to 30-month period (mean follow-up 21.5 months). A left ventricular thrombus was present on initial echocardiogram in 11 (44%) patients, became present during follow-up in an additional four, and disappeared in two. Thrombus was significantly more common in patients with fractional shortening of less than or equal to 10% (12 of 15) than in those with a fractional shortening 11% to 25% (3 of 10) (p less than 0.02). Five embolic events (four cerebral) occurred over the follow-up period, four of which were associated with a previously visualized left ventricular thrombus. Three of five thrombi that protruded into the left ventricular cavity subsequently embolized. We conclude that in nonanticoagulated patients with dilated cardiomyopathy left ventricular thrombus and thromboembolism are common. Echocardiography may be helpful in predicting which patients are at risk of thromboembolism. PMID- 1729817 TI - Diagnosis of pericardial abnormalities by 2D-echo: a pathology-echocardiography correlation in 85 patients. AB - A blinded pericardial echocardiography-pathology correlation was performed using 85 pericardiectomy or autopsy patients. All patients had two-dimensional (2D) echocardiography performed within 6 months of autopsy or surgery. 2D echocardiography was able to detect pericardial abnormalities in 35% of patients with a pathologic pericardium. Obliterative processes, such as fibrosis after open-heart surgery, were particularly not well detected echocardiographically. A specificity of 90% to detect pericardial abnormalities is reported. Acute fibrin strands, malignancies, and chronic fibrous connective tissue involving the pericardium were recognized as abnormal. Interobserver variability does exist, but overall reporting was similar. Specific 2D-echocardiographic signs of pericardial disease require prospective validation including direct pathologic correlation. PMID- 1729818 TI - Preangioplasty complicated coronary stenosis morphology as a predictor of restenosis. AB - To assess whether complicated preangioplasty coronary stenosis morphology is associated with restenosis, 41 patients (47 stenoses) who underwent repeat angiography 6 to 8 months after percutaneous transluminal coronary angioplasty (PTCA) were studied. Stenosis diameter and morphology were assessed by computerized quantitative coronary angiography before and immediately after PTCA and at follow-up angiography. Before PTCA 18 stenoses were concentric (symmetric narrowings with smooth borders), 12 were eccentric (asymmetric narrowings with smooth borders), and 17 were complicated (asymmetric with rough borders and overhanging edges). Restenosis occurred in 18 lesions: two (11%) concentric, four (33%) eccentric, and 12 (70%) complicated (p less than 0.05), whereas 29 lesions remained unchanged. Stenosis diameter before and immediately after PTCA was not significantly different in the 18 patients with and the 23 patients without restenosis. Follow-up angiograms showed that 11 (61%) stenoses in the group with restenosis and 18 (63%) in the group without restenosis had morphology similar to that before PTCA. Restenosis occurred in seven (30%) patients who initially had chronic stable angina and in 11 (61%) who were first seen with unstable angina (p less than 0.05). In patients with stable angina 1 of 13 concentric stenoses, two of eight eccentric stenoses, and four of five complicated lesions restenosed. In patients with unstable angina one of five concentric, two of four eccentric, and 8 of 12 complicated lesions had restenosis. Stenoses that were complicated before PTCA tended to adopt an irregular morphology if they recurred, whereas concentric stenoses rarely occurred.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729819 TI - Hemodynamic evaluation of the CarboMedics prosthetic heart valve in the aortic position: comparison of noninvasive and invasive techniques. AB - Seventy-three patients with a CarboMedics aortic bileaflet valve prosthesis were examined by Doppler ultrasonography, and 27 of them were also assessed by transseptal catheterization. The ultrasonic mean systolic gradient was 17.1 +/- 5.6 mm Hg for valve size 19 mm, falling gradually with increasing valve size to 6.8 +/- 2.5 mm Hg for size 27 mm. The catheter mean systolic gradient was consistently smaller than the ultrasonic gradient (4.3 +/- 4.8 mm Hg), but Tobit regression analysis showed a significant association between the two methods. In all patients both methods revealed negligible to small amounts of retrograde leakage, which is assumed to be a normal finding for this valve. The effective flow areas of the valves calculated from the ultrasonic data were similar to the in vitro calculated flow areas. The hemodynamic potential of this valve is therefore completely utilized in vivo. The effective orifice area corrected for body surface area increased with increasing valve size, which demonstrates a moderate valve-patient mismatch. PMID- 1729820 TI - Effect of viscosity and iodine concentration of nonionic radiographic contrast media on coronary arteriography in patients. AB - To assess the influence of viscosity and iodine concentration, three matched and standardized left coronary arteriograms were obtained in 20 patients using iopamidol (Isovue-370), ioversol (Optiray-320), and iohexol (Omnipaque-350). The order of contrast media was randomized and the administration of contrast was double-blinded. Quantitative densitometric angiographic evaluation of the coronary angiograms was performed in addition to independent operator qualitative assessment. The injection volume of iopamidol (5.4 +/- 1.0 ml) was slightly but significantly less than that of ioversol and iohexol (5.6 +/- 1.0 ml, 5.7 +/- 1.0 ml, both p less than 0.05). The calculated iodine concentration was also lower for ioversol (1.7 +/- 0.32 gm) than for iopamidol (1.98 +/- 0.35 gm) and iohexhol (1.9 +/- 0.35 gm, both p less than 0.05). There were significantly lower contrast syringe injection pressures for ioversol (6.6 +/- 0.8 atm) than for iopamidol (7.5 +/- 0.9 atm) and iohexol (7.2 +/- 1.1 atm, both p less than 0.05). The quantitative densitometric analysis failed to demonstrate significant differences among the contrast media with respect to image density parameters for any individual agent. All coronary angiograms were deemed of diagnostic quality. The data in this study indicated that although differences in iodine concentration exist among the three agents, operator compensation with more rapid contrast delivery (higher volume) and lower viscosity (lower injection pressure) produced equivalent image opacification during coronary angiography. Given the same incidence of adverse hemodynamic and clinical effects, selection of a low viscosity media theoretically provides an advantage during procedures using small diameter catheters or interventional procedures requiring contrast visualization through reduced catheter lumina. PMID- 1729821 TI - Can signal intensity of the continuous wave Doppler regurgitant jet estimate severity of mitral regurgitation? AB - Visual estimates of the intensity of the continuous wave (CW) Doppler regurgitant jet signal have been used to estimate the severity of valvular regurgitation. Theoretically, the strength of the reflected Doppler signal is a function of the number of scatterers. To test this approach quantitatively, free jets were produced in 27 experiments using a power injector and cornstarch suspension varying in concentration from 1% to 3%. Flow volume was varied from 5 to 15 ml, and orifice diameter varied from 2.5 to 10 mm. Machine settings were kept constant. Also, 22 patients with mitral regurgitation (MR)--5 mild, 11 moderate, and 6 severe by angiography--were studied. Average signal intensity under the CW Doppler flow curve was calculated using a computer image processor. In MR patients, average regurgitant flow (RF) intensity was compared with average mitral forward flow (FF) signal intensity. (1) The intensity under the CW flow signal in the free jet experiments correlated well with injection volume (r greater than 0.98). (2) RF average signal intensity did not correlate with angiographic MR severity (r = 0.21), but the ratio of RF to FF average signal intensity did correlate with MR severity (r = 0.73). (3) The sensitivity and the specificity of an RF/FF ratio greater than 0.65 for angiographically severe mitral regurgitation were both 83%. (4) The sensitivity and specificity of an RF/FF ratio less than 0.50 for angiographic mild mitral regurgitation were both 80%. The ratio of regurgitant to forward mitral flow CW Doppler signal intensity appears to be an accurate and clinically applicable method for estimating the severity of mitral regurgitation. PMID- 1729822 TI - Intraoperative determination of cardiac output by transesophageal continuous wave Doppler. AB - A new prototype transesophageal transducer with continuous-wave Doppler and pulsed Doppler capabilities was evaluated to calculate intraoperative cardiac output from the main pulmonary artery. Fifteen consecutive patients undergoing elective coronary artery bypass surgery were studied. The main pulmonary artery diameter above the pulmonic valve was measured with the single horizontal plane transesophageal transducer. The pulmonary artery cross-sectional area was calculated from its diameter using the formula: Area = 1/4 pi (diameter)2. Continuous-wave Doppler and pulsed Doppler spectra were recorded from the main pulmonary artery and their flow velocity integrals were then multiplied by pulmonary artery area and heart rate to yield cardiac output. The main pulmonary artery diameter could not be confidently measured in 2 of 15 patients (13%). In the remaining 13 patients, Doppler cardiac output measurements were correlated with simultaneous thermodilution measurements. The closest correlation with thermodilution cardiac output was with the continuous-wave Doppler cardiac output method: R = 0.91, SEE = 0.2 L/min, and y = 1.1x . 0.2 (p less than 0.001). The correlation of thermodilution with pulsed Doppler cardiac output was R = 0.83, SEE = 0.5 L/min, and and y = 0.86 + 1.0 (p less than 0.001). Transesophageal continuous-wave Doppler is a new technique that may be used in selected patients for accurate determination of intraoperative cardiac output. PMID- 1729823 TI - The prevalence of valvular regurgitation in children with structurally normal hearts: a color Doppler echocardiographic study. AB - To determine the prevalence of valvular regurgitation in children (from birth to 14 years old) with structurally normal hearts, the records of 1360 consecutive patients referred for echocardiographic and Doppler examination were analyzed. A total of 461 (33.9%) patients were found to have structurally normal hearts. Flow patterns across the four valves were examined by pulsed, continuous-wave, and color Doppler imaging techniques. Regurgitation was detected in 124 (26.9%). Pulmonic regurgitation was most commonly found and was detected in 101 (21.9%) patients, tricuspid regurgitation in 29 (6.3%), and mitral regurgitation in 11 (2.4%). Aortic regurgitation was not found. Regurgitation of one valve occurred in 106 (23.0%) patients and of two valves in 18 (3.9%) patients. No patient had regurgitation of more than two valves. The prevalence of pulmonic regurgitation increased significantly with age (p less than 0.0001), whereas the prevalence of mitral, tricuspid, and bivalvular regurgitation did not change with age. Valvular regurgitation was trivial or mild in 87% of patients. Thus mild valvular regurgitation is commonly found in children with structurally normal hearts. PMID- 1729824 TI - Prehospital diagnosis and treatment of acute myocardial infarction: a critical review. PMID- 1729825 TI - Role of "buttoned" double-disc device in the management of atrial septal defects. AB - Sixteen patients seen over a 9-month period ending in August 1990 were offered transcatheter closure of their ASD with a custom-made "buttoned" double-disc device. The study was approved by the Institutional Review Board and informed consent was obtained in each case. The device consists of an occluder, a counteroccluder, and a loading wire and is delivered to the ASD site via an 8F sheath. Parents of two children elected surgical closure. In five children the stretched diameter of the ASD was too large (greater than 20 mm) and transcatheter closure was not attempted. These seven children underwent elective surgical closure without incident. In one child the defect measured 5 mm and the Qp:Qs was 1.4:1 and therefore ASD closure was not recommended. In the remaining eight children transcatheter closure was attempted. In two of the children the occluder pulled through the ASD and was successfully retrieved and the children later underwent uneventful elective surgical closure. The device was implanted across the ASD in six children. In one child the device dislodged from the ASD site within minutes after implantation and the child was sent to emergency surgery, where the device was removed and the ASD was closed. In the remaining five patients, aged 7 months to 45 years (weight 3.6 to 50 kg), with a Qp:Qs range of 1.3 to 2.3 and a stretched diameter of 10 to 19 mm, the ASD closure was successful with 25 to 40 mm size devices. Repeat echo-Doppler studies 2 weeks and 3 months after the procedure in all patients and 6 months later in two children did not reveal any residual shunt. It is concluded that (1) the custom-made "buttoned" double-disc device can be implanted across the ASD safely and effectively via an 8F sheath, thus making transcatheter ASD closure feasible even in very young infants; (2) measurement of stretched diameter of the ASD in the catheterization laboratory is a useful guide for selection of an appropriate sized device; and (3) additional clinical trials are warranted to confirm the efficacy and safety of the device. PMID- 1729826 TI - Failure or success of complex catheter-based interventional procedures assessed by intravascular ultrasound. AB - Intravascular ultrasound provides high resolution cross-sectional images of vessel walls and may help to characterize atherosclerotic plaque morphology subtypes. This new imaging modality may have an important role in assessing the results of standard and investigational interventional therapeutic procedures. Four case histories of patients with coronary artery disease treated with different catheter-based therapies are presented. In each case, intravascular ultrasound added diagnostic information unobtainable from standard radiographic imaging techniques. These cases, involving PTCA (balloon dilatation), directional coronary atherectomy, high-speed rotational ablation, and balloon-expandable stent implantation, each represent an interesting example of procedure success or failure that could not be fully discerned without the use of intravascular ultrasound. Specifically, the distribution of intramural dissection, the presence and magnitude of intracoronary calcification, and morphologic patterns of intimal hyperplasia leading to restenosis, were accurately identified by ultrasound images. Thus intracoronary ultrasound imaging significantly enhances the understanding of failure modes, success, and complications after therapeutic interventions in patients with complex coronary disease. PMID- 1729827 TI - Characterization of ultraviolet laser-induced autofluorescence of ceroid deposits and other structures in atherosclerotic plaques as a potential diagnostic for laser angiosurgery. AB - Unstained frozen sections of normal and atherosclerotic human aorta and coronary artery were examined using histochemical and fluorescence microscopic techniques to identify the structures responsible for autofluorescence under 351 to 364 nm laser excitation. These structures included elastin and collagen in normal and atherosclerotic specimens, calcium deposits in calcified plaques, and granular or ring-shaped deposits histochemically identified as ceroid found in both calcified and non-calcified plaques. Qualitatively, both the color and intensity of ceroid autofluorescence differed greatly from that of elastin or collagen. The emission spectra of elastin, collagen, and ceroid were examined by microscopic spectrofluorimetry, and were found to differ significantly as well. When compared with spectra of elastin and collagen, spectra of ceroid were broader, shifted to the red, and were somewhat resistant to bleaching. We conclude that detection of laser-induced ceroid autofluorescence may aid in identifying plaques for laser ablation. PMID- 1729828 TI - Intraoperative transesophageal echocardiographic diagnosis of left atrial pseudoaneurysm. PMID- 1729829 TI - Coronary artery fistula: diagnosis by transesophageal two-dimensional and Doppler echocardiography. PMID- 1729830 TI - Two-vessel PTCA of single anomalous coronary artery. PMID- 1729831 TI - Surgical treatment of an atherosclerotic aneurysm of the left main coronary artery. PMID- 1729832 TI - The use of transesophageal echocardiography in detecting aortic atherosclerosis in patients with embolic disease. PMID- 1729833 TI - Transesophageal echocardiographic features of left ventricular pseudoaneurysm resulting after mitral valve replacement surgery. PMID- 1729834 TI - Role of transesophageal echocardiography in sinus of Valsalva aneurysm. PMID- 1729835 TI - Transesophageal echocardiographic diagnosis of isolated tricuspid valve prolapse with severe tricuspid regurgitation. PMID- 1729836 TI - Echocardiographic demonstration of mitral block caused by left atrial spindle cell sarcoma. PMID- 1729837 TI - Persistent left superior vena cava in Turner's syndrome: a transesophageal echocardiographic study. PMID- 1729838 TI - Transesophageal echocardiographic evaluation of St. Jude Medical and bioprosthetic valve endocarditis. PMID- 1729839 TI - Development of obstruction to ventricular outflow and impairment of inflow in glycogen storage disease of the heart: serial echocardiographic studies from birth to death at 6 months. PMID- 1729840 TI - The syndrome of right atrial myxoma, spotty skin pigmentation, and acromegaly. PMID- 1729841 TI - Dual ventriculoatrial conduction caused by closely approximated accessory atrioventricular pathways. PMID- 1729842 TI - Catheter fulguration ablation of left atrial ectopic tachycardia in a child. PMID- 1729843 TI - Oral sotalol in pediatric atrial ectopic tachycardia. PMID- 1729844 TI - An attempt at electrical catheter ablation of the arrhythmogenic area in idiopathic ventricular fibrillation. PMID- 1729845 TI - Tricuspid atresia associated with aortopulmonary window: controlling pulmonary blood flow with a fenestrated patch. PMID- 1729846 TI - Pulling it all together: changing the cardiovascular outlook. PMID- 1729847 TI - Treatment of patients following bypass surgery: a dilemma for the 1990s. PMID- 1729848 TI - Hemodynamic effects of the vascular selective calcium antagonist felodipine in patients with impaired left ventricular function. PMID- 1729849 TI - Temporal increase in resting coronary blood flow causes an impairment of coronary flow reserve after coronary angioplasty. AB - Impaired coronary flow reserve immediately after coronary angioplasty may be attributed to an increase in resting coronary blood flow. To test this hypothesis we measured great cardiac venous flow (GCVF) at rest and during rapid atrial pacing before and immediately after angioplasty in 22 patients with significant narrowing of the left anterior descending artery and 12 patients (control group) with minimal narrowing. A follow-up (6 months) study was also done in seven patients. Immediately after angioplasty the coronary flow reserve (peak GCVF during pacing/resting GCVF) was not fully restored (1.5 +/- 0.36 before angioplasty, 1.76 +/- 0.42 after angioplasty, and 2.13 +/- 0.33 in the control group). Resting coronary vascular resistance (2.4 +/- 0.9 mm Hg/ml/min) was significantly decreased after angioplasty (2.0 +/- 0.8 mm Hg/ml/min), whereas coronary vascular resistance during rapid pacing was fully restored to normal. Resting hyperemia was restored 6 months later, whereas coronary vascular resistance during pacing was unaltered. In five patients, however, slight ischemic ST-T changes were observed during rapid pacing, even after successful angioplasty associated with a decrease in the lactate extraction ratio. These results indicate that the impaired coronary flow reserve immediately after angioplasty may be attributed mainly to the temporal but significant increase in resting coronary flow, although impaired coronary vascular response to augmented myocardial oxygen demand may also be partially involved. PMID- 1729850 TI - Silent myocardial ischemia in patients with non-insulin-dependent diabetes mellitus as judged by treadmill exercise testing and coronary angiography. AB - Patients with diabetes were compared with nondiabetic control subjects, with respect to the prevalence of silent myocardial ischemia, by means of treadmill exercise testing and coronary angiography. Results of treadmill exercise testing showed ischemic ST depression in 41 of the 132 diabetic patients (mean age 61 +/- 4 years) and in 42 of the 140 nondiabetic control subjects (mean age 60 +/- 8 years) (31% vs 30%, p = NS). Coronary angiography was performed in 36 of 41 diabetic patients and 34 of 42 nondiabetic control subjects with positive results of treadmill exercise tests, who gave their consent. Among "treadmill-positive" subjects, diabetic patients had a prevalence of silent myocardial ischemia that was 2.2 times higher than that in nondiabetic control subjects (p less than 0.05). Diabetic patients who received insulin had a 2.6 times higher prevalence of silent myocardial ischemia than those who did not (p less than 0.05). Similarly diabetic patients with retinopathy has a 2.5 times higher prevalence of silent myocardial ischemia than those without it (p less than 0.05). PMID- 1729851 TI - The relationship between ECG signs of atrial infarction and the development of supraventricular arrhythmias in patients with acute myocardial infarction. AB - ECGs obtained on arrival at the hospital from 277 patients with acute myocardial infarction were analyzed retrospectively for PR displacements, which were classified as major or minor criteria for atrial infarction and related to the later occurrence of supraventricular arrhythmia in the hospital. Major criteria were (1) PR segment elevation greater than 0.5 mm in leads V5 and V6 with reciprocal PR segment depression in leads V1 and V2, (2) PR segment elevation greater than 0.5 mm in lead I with reciprocal PR segment depression in leads II and III, and (3) PR segment depression greater than 1.5 mm in precordial leads and greater than 1.2 mm in leads I, II, and III. Abnormal P waves were classified as minor criteria. Major and minor criteria were found in 15 (5.4%) and 19 (6.9%) patients, respectively. Eight (53.3%) patients with major and six (31.6%) with minor criteria had supraventricular arrhythmias, giving odds ratios of 9.9 and 3.7, respectively. Enzyme-estimated infarct size, the occurrence of heart failure, and mortality rates did not differ in patients with or without major criteria for atrial infarction. We conclude that the occurrence of PR segment displacements on the admission ECG may predict the risk of developing supraventricular arrhythmias during hospitalization for myocardial infarction. PMID- 1729852 TI - Residual coronary stenoses and calculated transstenotic gradients after intravenous streptokinase versus tissue plasminogen activator. AB - To compare the relative success of intravenous streptokinase (STK) and tissue plasminogen activator (TPA) on the severity of residual infarct-related coronary stenoses, we evaluated 45 patients receiving thrombolytic therapy for acute myocardial infarction. Twenty-three patients (18 men and 5 women) received STK (1.5 million units), while 22 patients (18 men and 4 women) received TPA (100 mg) within 6 hours of chest discomfort. Cardiac catheterization was performed before hospital discharge (8 days) with quantitative coronary arteriography and estimation of transstenotic pressure gradients using fluid dynamic equations. Although angina pectoris was equally common (STK, 7 of 23 [30%] versus TPA, 5 of 22 [23%], p = NS), recurrent infarction (STK, 3 of 23 [13%] versus TPA, 7 of 22 [32%], p less than 0.05) and coronary angioplasty (STK, 2 of 23 [9%] versus TPA, 7 of 22 [32%], p less than 0.05) were more frequent in those receiving TPA. Infarct-related coronary patency was greater in TPA-treated subjects (STK, 15 of 23 [65%] versus TPA, 19 of 22 [86%], p less than 0.05), although minimum stenotic diameter (STK, 0.77 +/- 0.48 mm versus TPA, 0.57 +/- 0.38 mm, p less than 0.05), and calculated transstenotic pressure gradient (STK, 8.7 +/- 17.0 mm Hg versus TPA, 23.7 +/- 30.2 mm Hg, p less than 0.05) suggested severe residual stenosis. These effects were accentuated at elevated coronary flow velocities (8 to 20 cm/sec).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729853 TI - Efficacy and electrophysiologic effects of oral sotalol in patients with sustained ventricular tachycardia caused by coronary artery disease. AB - The efficacy of oral sotalol in preventing sustained ventricular tachycardia induction by invasive electrophysiological testing was assessed in 22 patients (60 +/- 9 years) with prior myocardial infarction. Programmed stimulation consisted of two basic drives followed by up to three extrastimuli at two right ventricular sites. At baseline, sustained monomorphic ventricular tachycardia was inducible in all patients. With sotalol (360 +/- 172 mg/day), it was no longer inducible in 10 patients; in 12 others, it remained inducible and its cycle length was only minimally prolonged (322 +/- 42 to 345 +/- 44 msec, p less than 0.05). Sotalol markedly prolonged sinus cycle length, uncorrected QT interval, and right ventricular effective and functional refractory periods, but had little effect on ventricular conduction time either in sinus rhythm or with right ventricular pacing. There was no significant difference in drug dose or in electrophysiologic effect of drug that related to efficacy, nor was there any correlation between drug-induced prolongation of ventricular tachycardia cycle length and its effects. Six patients received oral sotalol over the long term without spontaneous recurrence of ventricular tachycardia (follow-up: 23 +/- 18 months). These results demonstrate that sotalol is effective (45%) against sustained ventricular tachycardia induction at moderate doses and is well tolerated over a long term in the setting of remote myocardial infarction. However, its electrophysiologic effects as measured at invasive testing are not predictive of efficacy against ventricular tachycardia induction. PMID- 1729854 TI - A multicenter, randomized, double-blind, placebo-controlled trial of pimobendan, a new cardiotonic and vasodilator agent, in patients with severe congestive heart failure. AB - Pimobendan, a new oral cardiotonic and vasodilator agent, increases myocardial contractile force through specific inhibition of phosphodiesterase type III and increased calcium sensitivity of the myocardial contractile elements. The effects of pimobendan on left ventricular performance and maximal exercise capacity were studied in a multicenter, randomized, double-blind, placebo-controlled trial involving 52 patients with severe congestive heart failure despite diuretics, digoxin, and angiotensin-converting enzyme inhibitors. The acute hemodynamic evaluation included three single doses of 2.5, 5.0, and 10.0 mg of oral pimobendan, which was subsequently administered at a daily dose of 5 or 10 mg for 4 weeks. Acute administration of pimobendan significantly increased the resting cardiac index and lowered pulmonary capillary wedge pressure in a dose-dependent manner, whereas heart rate and systemic arterial pressure were not substantially altered. Patients receiving pimobendan, 5 and 10 mg daily, had a significantly greater increase in maximal exercise duration than those receiving placebo, that is, 144 +/- 30 and 124 +/- 33 seconds versus 58 +/- 25 seconds (p = 0.05). Peak oxygen uptake increased by 1.7 +/- 0.8 and 2.2 +/- 1.3 ml/kg/min in patients receiving pimobendan at a daily dose of 5 and 10 mg, respectively, whereas it decreased by 0.1 +/- 0.6 ml/kg/min in patients receiving placebo (p = 0.06). Thus pimobendan acutely improves resting left ventricular performance and chronically increases exercise duration and peak oxygen uptake in patients with severe congestive heart failure concomitantly treated with digoxin, diuretics, and angiotensin-converting enzyme inhibitors. PMID- 1729855 TI - Assessment of global and regional left ventricular performance at rest and during exercise after thrombolytic therapy for acute myocardial infarction: results of the Thrombolysis in Myocardial Infarction (TIMI) II Study. AB - Global and regional left ventricular performances were evaluated with equilibrium radionuclide angiocardiography in patients in the Thrombolysis in Myocardial Infarction (TIMI) II trial at the time of hospital discharge. Studies at rest were available in 1,162 (69%) of the invasive and 1,150 (69%) of the conservative strategy patients, and exercise studies in 1,133 (67%) of the invasive and 1,145 (69%) of the conservative patients. Repeat studies were performed at the time of 6-week follow-up. Global and regional ejection fraction at rest were both comparable in patients assigned to each of the treatment strategies. However, at the time of hospital discharge patients in the invasive strategy had normal exercise responses more frequently (29.7 vs 25.8% p = 0.01), greater peak exercise LV ejection fraction (54.8 +/- 13.8% vs 53.1 +/- 14.1%, p = 0.004), greater exercise--rest change in LV ejection fraction (3.7 +/- 6.7% vs 2.7 +/- 7.2%, p less than 0.001) and greater peak exercise infarct zone regional ejection fraction (53.2 +/- 31.1% vs 50.3 +/- 33.0%, p less than 0.001) than patients assigned to the conservative strategy. At 6-week follow-up these differences between treatment strategies were no longer evident. When data were restricted to those collected at comparable work loads, similar differences in hospital discharge exercise performance between invasive vs conservative strategy patients were observed. Thus, there is a small transient difference in exercise global and regional LV performance associated with an invasive as opposed to conservative strategy after thrombolytic therapy. These differences are noted at the time of hospital discharge but not at 6 weeks, and are unlikely to confer clinical benefit. PMID- 1729856 TI - Influence of collateral filling of the occluded infarct-related coronary artery on prognosis after acute myocardial infarction. AB - Previous studies showed that long-term morbidity and mortality after acute myocardial infarction (AMI) are influenced by the presence or absence of anterograde flow in the infarct artery. In comparison with patients with anterograde flow, those whose infarct artery remains occluded are more likely to have unstable angina, recurrent AMI, congestive heart failure and sudden death. This study was performed to assess the influence of collateral filling of the infarct artery on long-term morbidity and mortality in surviving patients of initial AMI in whom the infarct artery was occluded. Over a 12.5-year period, 146 subjects (108 men and 38 women, aged 25 to 76 years) with AMI, no anterograde flow in the infarct artery, and no disease of other coronary arteries were medically treated and followed for 42 +/- 28 (mean +/- standard deviation) months. Of these subjects, 120 had angiographic evidence of collateral filling of the infarct artery (group I), whereas the remaining 26 did not (group II). The groups were similar in age, sex, cardioactive medications, left ventricular performance and infarct artery. They were also similar in incidence of unstable angina (19% of group I, 31% of group II; p = not significant [NS]), recurrent AMI (12% of group I, 8% of group II; p = NS), congestive heart failure (16% of group I, 12% of group II; p = NS) and cardiac death (16% of group I, 19% of group II; p = NS). Thus, angiographic evidence of collateral filling of the infarct artery in surviving patients of AMI exerts no demonstrable influence (beneficial or detrimental) on long-term morbidity or mortality. PMID- 1729857 TI - Effects of enalapril on long-term mortality in severe congestive heart failure. CONSENSUS Trial Group. AB - All surviving patients in a double-blind study comparing the effects of enalapril and placebo on survival in severe congestive heart failure were recommended to be treated with active drug after stopping the trial. Two-year follow-up from the end of the blinded trial demonstrated that among 77 survivors of 127 patients originally allocated to the group with enalapril, 38 were still alive. Of 126 patients allocated to the group with placebo 58 survived the blinded study, and after 2-year follow-up 26 were still alive. Thus, the difference between the original treatment groups remained, despite that treatment with enalapril was made available to all surviving patients and that those in the group with enalapril were sicker at baseline than those in the group with placebo. If enalapril was prescribed, the mortality was 47% compared with 75% if it was not. Life-table analysis suggests a marked carry-over effect of treatment in the group with enalapril that lasted for up to 15 months before mortality rates became comparable in the 2 treatment groups. This strongly suggests that enalapril confers structural protection to the failing myocardium. PMID- 1729858 TI - Left ventricular filling and ventricular diastolic performance after percutaneous balloon mitral valvotomy. AB - The time course of left ventricular (LV) filling and LV diastolic performance were examined in 27 consecutive patients in sinus rhythm before and acutely after balloon mitral valvotomy (BMV). The mitral valve area acutely increased from 1.1 +/- 0.3 to 2.1 +/- 0.8 cm2. Simultaneous pressure-volume data were obtained using digital subtraction left ventriculography and LV micromanometer pressure before and 10 minutes after BMV. The time constant of LV isovolumic relaxation was unchanged after BMV (50 +/- 10 ms before BMV vs 47 +/- 13 ms after BMV). In addition, values before and after BMV for LV end-diastolic volume (123 +/- 29 vs 125 +/- 36 ml), end-diastolic pressure (11 +/- 4 vs 12 +/- 4 mm Hg) and diastolic filling time (337 +/- 126 vs 338 +/- 152 ms) were not altered by the procedure. After BMV the peak diastolic filling rate (403 +/- 143 vs 469 +/- 302 ml/s) was maintained despite a 36% reduction in left atrial filling pressure. There was a trend toward earlier occurrence of the peak filling rate (196 +/- 127 vs 146 +/- 148 ms, p = 0.08). The percentage of diastolic filling in the first third of diastole, however, was similar (42 +/- 9 vs 48 +/- 16%) before and after the procedure. Thus, the time course of LV filling is not significantly altered acutely after BMV, but is maintained at reduced left atrial filling pressure. Neither LV relaxation or LV chamber compliance are altered acutely after BMV.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729859 TI - Cardiac function after domino-donor heart transplantation. AB - A major limitation in cardiac transplantation is donor availability. A possible way to increase the supply of donor hearts is to use explanted hearts from patients undergoing heart-lung transplantation for primary lung disease. One potential advantage of this approach, termed domino-donor transplantation, is the existence of a donor right ventricle already adapted to pulmonary hypertension, which would therefore theoretically decrease the likelihood of acute donor right heart failure in recipients with preexisting elevation of pulmonary vascular resistance. Potential disadvantages include graft failure secondary to chronic effects of pulmonary hypertension on the right ventricle, arrhythmia and infections. Seven domino-donor transplants were performed at Stanford University Hospital; graft and patient survival to date are 100% at a mean follow-up of 20 months (range 1 to 26). Infection and rejection rates have been comparable to those of the current Stanford experience for conventional orthotopic transplantation. Right ventricular function and size have either improved or remained unchanged in all patients after transplantation. Transient early postoperative donor right ventricular dilation, a characteristic adaptive response seen in nondomino transplants, occurred in 4 patients with pulmonary hypertension before surgery. These data indicate that, with adequate assessment before surgery, domino-donor cardiac transplantation is an appropriate means of augmenting the donor pool. PMID- 1729860 TI - Predictive factors of effectiveness of streptokinase in deep venous thrombosis. AB - In a prospective study, 174 patients (118 women and 56 men, average age 44 years, range 14 to 82) with proximal extensive thrombosis received streptokinase (100,000 U/hour) for an average of 2.8 days (range 0.5 to 7) through the catheter of a temporary caval filter. Twenty-seven of 45 (60%) patients with nonocclusive clots were completely free of clots at the second phlebography versus 17 of 116 (14%) with occlusive clots (p less than 0.001). Among nonocclusive clots, proximal ones (caval, iliac and femoral) were more easily lysed than popliteal clots (88 of 116 [76%] vs 26 of 58 [45%]; p less than 0.001). In 41 of 132 (31%) patients, a daily injection of contrast medium through the filter-carrying catheter enabled the observation of a clot in the filter, which was lysed by streptokinase. Seventy patients with follow-up greater than 2 years (median 34 months) were examined clinically. Nineteen of 22 (86%) patients with venograms free of clots at discharge were free of clinical sequelae versus 16 of 48 (33%) without normal venograms (p less than 0.001). It is concluded that: (1) in the case of occlusive clots, only a few patients were normalized after streptokinase; (2) proximal nonocclusive clots were most effectively lysed; (3) when venograms were free of clots at discharge, the majority of patients did not have venous sequelae at follow-up; and (4) embolic migration seems to occur frequently with streptokinase. PMID- 1729861 TI - Left ventricular function in myasthenia gravis. AB - Myasthenia gravis is an autoimmune disorder with autoantibodies to acetylcholine receptors of skeletal muscle. Left ventricular diastolic function was studied with M-mode and Doppler echocardiography in 25 patients with myasthenia and in a group of age- and heart rate-matched control subjects. In the patients, diastolic peak filling rate was reduced by 37%, and Doppler peak early filling velocity (E) was reduced by 12% compared with the control subjects (2.7 +/- 0.7 vs 4.2 +/- 1.0 s-1, and 76 +/- 8 vs 85 +/- 15 cm/s, respectively; p less than 0.05). Peak atrial filling velocity (A) was increased by 38% (68 +/- 17 vs 48 +/- 9 cm/s; p less than 0.01), and consequently the E:A ratio in the group of patients was reduced by 33% (1.22 +/- 0.40 vs 1.81 +/- 0.33; p less than 0.001). End-diastolic dimension was 5.0 +/- 0.5 cm in both groups, heart rate was 70 +/- 12 vs 68 +/- 16 beats/min (p = not significant [NS]), M-mode ejection fraction was 76 +/- 8 vs 79 +/- 5% (p = NS), M-mode peak ejection rate was -1.9 +/- 0.4 vs -2.1 +/- 0.3 s 1 (p = NS), and peak aortic outflow velocity was 109 +/- 18 vs 98 +/- 13 cm/s (p = NS). Twenty-three patients and 15 control subjects were studied before and after intake of the acetylcholine-esterase inhibitor pyridostigmine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729862 TI - Intravascular ultrasound imaging of saphenous vein graft stenosis. PMID- 1729863 TI - Prognostic significance of silent ischemia in elderly patients with peripheral arterial disease with and without previous myocardial infarction. PMID- 1729864 TI - A comparison between lovastatin and gemfibrozil in the treatment of primary hypercholesterolemia. AB - A randomized, multicenter, double-blind, prospective, 18-week comparison of lovastatin with gemfibrozil was performed to compare their efficacy and tolerability in adults with types IIa and IIb primary hypercholesterolemia. Sixty men and 44 women aged 24 to 78 years participated in the trial. Each treatment group of 52 patients was closely matched by the randomization procedure. All participants met national cholesterol education program guidelines for evaluation and treatment. In all, 94 (90%) completed the 18 weeks of study. After 18 weeks of diet-plus-active treatment, lovastatin decreased serum total cholesterol and low-density lipoprotein (LDL) cholesterol significantly better than gemfibrozil (adjusted mean decreases were 63 vs 35 mg/dl for total cholesterol and 67 vs 28 mg/dl for LDL; p = 0.0001). Gemfibrozil was more effective than lovastatin in increasing high-density lipoprotein (HDL) cholesterol (8 vs 5 mg/dl adjusted mean HDL cholesterol increases; p = 0.0086) after 18 weeks. No significant differences in the adjusted mean ratio of total to HDL cholesterol were noted, but the lovastatin group had a significantly greater adjusted mean reduction in the ratio of LDL to HDL cholesterol (1.8 vs 1.3; p = 0.0013). The gemfibrozil group achieved significantly greater reductions in very low density lipoprotein (VLDL) cholesterol and triglycerides compared with the lovastatin group (adjusted mean decreases were 14 vs 1 mg/dl for VLDL cholesterol and 71 vs 15 mg/dl for triglycerides). After 18 weeks of lovastatin therapy, 49% of patients achieved goal LDL cholesterol, whereas only 9% of those who took gemfibrozil achieved this goal (p = 0.0001). PMID- 1729865 TI - Physiologic responses to recumbent versus upright cycle ergometry, and implications for exercise prescription in patients with coronary artery disease. AB - To clarify the influence of body position on exercise prescription, 14 men (mean age +/- standard deviation 60.0 +/- 6.1 years) with coronary artery disease who underwent randomized recumbent and upright cycle ergometer tests to volitional fatigue were studied. At 100 watts, heart rate (HR), systolic blood pressure, oxygen consumption (VO2), rate pressure product and rating of perceived exertion were greater (p less than 0.05) in the upright than in the recumbent position. At peak exercise, however, these variables were not significantly different. Regressions of relative HR versus VO2 for recumbent and upright cycle ergometry were comparable: y = 1.24x - 32.7 and y = 1.26x - 31.5, respectively, where y = % maximal VO2, and x = % maximal HR. These findings indicate that recumbent exercise prescriptions may be based on the peak HR and VO2 values obtained during upright cycle ergometry, and vice versa. However, differences in the cardiorespiratory responses at submaximal exercise preclude the interchangeability of upright and recumbent training work rates. PMID- 1729866 TI - Prognostic significance of exercise thallium-201 testing in patients aged greater than or equal to 70 years with known or suspected coronary artery disease. AB - The prognostic value of exercise thallium-201 myocardial perfusion imaging has not been studied in an elderly (aged greater than or equal to 70 years) population. Retrospective analysis of 120 consecutive elderly patients undergoing Bruce protocol exercise stress with quantitative planar thallium-201 scintigraphy, followed clinically for a mean of 36 +/- 12 months after testing, revealed a 10% cardiac event rate (6 cardiac deaths from arrhythmia or congestive heart failure, and 5 fatal and 1 nonfatal myocardial infarction). There were no exercise stress-related complications. Survival without cardiac events was associated with greater exercise duration (5.6 +/- 2.4 vs 3.1 +/- 2.4 minutes; p less than 0.0007) and peak exercise heart rate (131 +/- 18 vs 120 +/- 19 beats/min; p less than 0.05). Univariate variables associated with higher cardiac event rates included: (1) peak exercise less than or equal to stage I (18 vs 6%; p = 0.04); (2) maximal ST-segment depression greater than or equal to 2 mm (27 vs 6%; p = 0.003); and (3) presence of a fixed or reversible thallium-201 perfusion defect (18 vs 2%; p = 0.004). Multivariate stepwise logistic regression analysis identified the combination of peak exercise less than or equal to stage I and any thallium-201 perfusion defect as the most powerful predictor of subsequent cardiac events (relative risk = 5.3 at 1 year). Thus, exercise thallium-201 scintigraphy in elderly patients is safe and provides important prognostic information.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729867 TI - Feasibility of high-dose dipyridamole-magnetic resonance imaging for detection of coronary artery disease and comparison with coronary angiography. AB - To assess the feasibility, safety and usefulness of gradient-echo magnetic resonance imaging (MRI) combined with pharmacologic stress testing for the detection of coronary artery disease, 23 patients without previous myocardial infarction but with significant stenosis (greater than 70% diameter stenosis) of greater than or equal to 1 major coronary artery were selected for dipyridamole MRI stress testing. Each patient underwent MRI at rest, and high-dose dipyridamole-MRI (0.75 mg/kg over 10 minutes) of corresponding basal and midventricular short-axis tomograms. Additionally, these patients performed symptom-limited exercise stress tests. All short-axis tomograms were evaluated on a standardized segmental basis by grading each segment as normal, hypokinetic, akinetic or dyskinetic. Dipyridamole-MRI was considered pathologic if segmental wall motion deteriorated by greater than or equal to 1 grade after dipyridamole. For comparison with coronary angiography, segmental wall motion gradings were related to the respective coronary artery territories in the short-axis plane. Pathologic dipyridamole-MRI was obtained in 18 of 23 (78%) patients. For 1- and 2 vessel diseases, sensitivity was 69 and 90%, respectively. Exercise stress tests were pathologic in 14 of 23 (66%) patients. For 1- and 2-vessel diseases, sensitivity of exercise stress test was 58% (7 of 12 patients) and 77% (7 of 9), respectively. Sensitivity/specificity of dipyridamole-MRI for the localization of the stenosed coronary artery was 78/100% for left anterior descending, 73/100% for left circumflex, and 88/87% for right coronary artery stenoses. It is concluded that dipyridamole-MRI is a feasible nonexercise-dependent test for detection and localization of functionally significant coronary artery disease. PMID- 1729868 TI - Intracoronary urokinase as an adjunct to percutaneous transluminal coronary angioplasty in patients with complex coronary narrowings or angioplasty-induced complications. AB - The effectiveness of intracoronary urokinase infusion as an adjunct to percutaneous transluminal coronary angioplasty (PTCA) was studied in 50 patients who underwent angioplasty for complex coronary narrowings or had thromboembolic complications during PTCA (29 [58%] men, 3 [6%] stable and 37 [74%] unstable angina, and 16 [32%] prior coronary bypass surgery). The primary indications for intracoronary urokinase infusion were intracoronary thrombus in 27 patients (54%), distal coronary embolization in 9 (18%), and abrupt reclosure in 14 (28%). Urokinase was infused in a mean (+/- standard deviation) dosage of 399,000 +/- 194,000 IU (range 150,000 to 1,000,000) at an average rate of 5,000 to 20,000 IU/min. Angiographic success was achieved in 43 patients (86%). Complications included the need for urgent bypass surgery in 3 patients, Q-wave myocardial infarction in 2, and non-Q-wave myocardial infarction in 12 (8 of whom had peak creatine kinase less than twice the upper normal limit). The incidence of myocardial infarction was significantly higher in patients with vein grafts (69%) than in those with PTCA of native vessels (14%). Two patients died (1 massive gastrointestinal necrosis 24 hours after angioplasty, and 1 after urgent bypass surgery). Mean (+/- standard deviation) fibrinogen levels were 355 +/- 73 mg/dl before urokinase infusion, and 361 +/- 70, twelve hours afterward. Three patients had local bleeding, but no transfusions were needed. It is concluded that intracoronary urokinase is a safe and effective adjunct to PTCA in patients with associated thrombi and may improve the success rate in angioplasty complicated by thrombus formation. PMID- 1729869 TI - Peripheral vascular complications after conventional and complex percutaneous coronary interventional procedures. AB - To determine whether complex cardiovascular interventional procedures (including coronary stent implantation, directional atherectomy, aortic valvuloplasty, and the use of an intraaortic balloon pump or cardiopulmonary bypass support) are associated with an increased likelihood of vascular access site complications, 2,400 consecutive cardiac catheterization procedures were prospectively screened over a 12-month study period. Complications occurred in 35 patients after 39 procedures (1.6%) and included the need for vascular surgical repair (17 patients), blood transfusion (28 patients) and systemic antibiotic therapy (7 patients). The incidence of complications after 1,519 diagnostic studies was 0.6%, after 698 conventional coronary balloon angioplasties 2.6%, and after 183 complex interventions 6.0% (p less than 0.0001); 43% of the complications occurred after procedures of greater than 2 hours' duration and 14% occurred in patients in whom arterial sheaths remained in situ for greater than 24 hours. Detailed demographic and procedural characteristics were compared between the 35 patients with vascular complications and 150 patients randomly drawn from a computerized database of the uncomplicated procedures performed during the screening period. By univariate analysis with correction for multiple comparisons, variables predicting the likelihood of vascular complications included: periprocedural use of heparin (p less than 0.001) or fibrinolytic therapy (p less than 0.001), arterial sheath size greater than or equal to 8Fr (p less than 0.001), patient age greater than or equal to 65 years (p = 0.01), and the presence of peripheral vascular disease (p = 0.03). The results of this study suggest that the overall incidence of access site complications is low but increases with the use of complex cardiovascular interventional procedures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729870 TI - Results of coronary angioplasty of chronic total occlusions (the National Heart, Lung, and Blood Institute 1985-1986 Percutaneous Transluminal Angioplasty Registry). AB - There has been increasing application of coronary angioplasty to patients with chronic total occlusions. The acute and long-term outcome in 271 patients after coronary angioplasty (142 single and 129 multiple stenoses) of a total occlusion was compared with 1,429 patients undergoing angioplasty of subtotal (less than or equal to 99% stenosis) occlusions (885 single and 544 multilesion) participating in the 1985-1986 National Heart, Lung, and Blood Institute Percutaneous Transluminal Coronary Angioplasty Registry. Baseline characteristics were similar for each lesion group except for a higher incidence of prior myocardial infarction and left ventricular dysfunction (ejection fraction less than 50%) in patients with total occlusion. Major complications (death, myocardial infarction or emergency bypass surgery) were similar (p = not significant) between patients with total and subtotal occlusions for single (6 vs 7%) and multilesion angioplasty (9 vs 6%). At 2 years, after making adjustments for baseline variables, patients with a total occlusion had a significantly increased risk of death compared with those with subtotal occlusion. There were no significant differences in cumulative event rates for myocardial infarction or bypass surgery. Approximately three-fourths of patients in each group were free of angina at 2 years. In conclusion, angioplasty of chronic total occlusions is associated with a similar acute complication rate. Despite similar relief of anginal symptoms, patients in the total occlusion group have a higher 2-year mortality. PMID- 1729871 TI - Establishing comprehensive, quantitative criteria for detection of restenosis and remodeling after percutaneous transluminal coronary angioplasty. AB - To establish comprehensive criteria for detecting restenosis and remodeling, inter- and intraobserver reproducibility of quantitative arteriography in the analysis of 20 lesions immediately after and 6 months after percutaneous transluminal coronary angioplasty (PTCA) were assessed. Geometric single-plane (minimum, maximum, mean diameter and percent diameter stenosis), biplane (absolute and relative cross-sectional area stenosis), relative densitometric area stenosis and the average of densitometric area stenosis in orthogonal views were compared. A high intra- and interobserver reproducibility of all absolute measurements was found, with the highest correlations for minimum diameter and cross-sectional area (interobserver, r = 0.85 and 0.85; intraobserver, r = 0.93, and 0.95 for minimum diameter and cross-sectional area, respectively). Of the relative measurements, biplane geometric percent cross-sectional area stenosis was the most reliable and percent densitometric area stenosis was the most variable (interobserver, r = 0.67; intraobserver, r = 0.71). Only small differences were demonstrated for the absolute measurements between the analysis of lesions immediately after PTCA and after follow-up, whereas a greater variability was found for relative measurements, especially videodensitometry. In both circumstances, a poor correlation between relative densitometric cross sectional area from orthogonal views was found, whereas geometric elliptical cross-sectional area correlated quite well with the average of densitometric percent cross-sectional area in orthogonal views (interobserver, r = 0.86; intraobserver, r = 0.84). Thus, data in this study support the suitability of geometric quantitative analysis for the assessment of PTCA results. Densitometry was the least reliable quantitative parameter. PMID- 1729872 TI - Comparison of ischemic and physiologic responses during exercise tests in men using the standard and modified Bruce protocols. AB - The importance of low-level (warm-up) exercise for reducing exercise-induced myocardial ischemic symptoms in patients with coronary artery disease is well recognized by clinicians. Whether altering the abruptness of exercise, such as that which occurs during different frequently used testing protocols, affects myocardial ischemic variables and maximal exercise capacity has not been resolved. This study seeks to determine the effects of altering the increment of work-rate change per exercise stage on both the ischemic threshold and maximal exercise capacity using 2 frequently used exercise testing protocols. Thirty-two patients with documented coronary artery disease and previously positive exercise tests (ischemic ST depression greater than or equal to 1.0 mm) performed symptom limited exercise tests using both the standard and modified Bruce protocols in random order, 1 hour apart. Exercise electrocardiograms were analyzed to determine the ischemic threshold, defined as heart rate at onset of greater than or equal to 1.0 mm ischemic ST depression. Patients achieved a higher peak heart rate (124 +/- 19 vs 117 +/- 21 beats/min; p less than 0.0001), rate-pressure product (21.4 +/- 3.9 vs 19.8 +/- 4.1 beats/min x mm Hg x 10(3); p less than or equal to 0.0001) and oxygen consumption (VO2) (18.5 +/- 3.7 vs 16.5 +/- 4.4 ml/kg/min; p less than or equal to 0.001) with the standard than with the modified Bruce protocol. At matched submaximal exercise stages there were no differences in VO2, heart rate or oxygen pulse between protocols. Time to ischemic threshold was significantly reduced with the standard compared with the modified Bruce protocol (312 +/- 107 vs 607 +/- 221 seconds; p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729873 TI - Comparison between technetium-99m-teboroxime and thallium-201 dipyridamole planar myocardial perfusion imaging in detection of coronary artery disease. AB - Technetium-99m (TC-99m)-teboroxime is a new myocardial perfusion imaging agent. The purpose of this prospective study was to compare Tc-99m-teboroxime with thallium-201 imaging after the administration of dipyridamole. Thirty patients referred for the evaluation of chest pain were studied with both thallium-201 and Tc-99m-teboroxime dipyridamole scans (mean interval 2 days). Dipyridamole was administered at 0.142 mg/kg/min for 4 minutes. Planar imaging (3 standard views) was obtained at 5 and 240 minutes after the injection of 2.2 mCi of thallium-201. Tc-99m-teboroxime (18 to 25 mCi) was injected after dipyridamole infusion. A second injection, at rest, was repeated 4 hours later. Planar imaging (3 standard views of 1 minute/view for the first 2 views, and 90 seconds for the last view) was obtained 2 minutes after Tc-99m-teboroxime injection. Blinded reading was performed by 3 observers. Thallium-201 showed perfusion defects in 182 myocardial segments corresponding to 33 of 45 (73%) significantly stenosed coronary arteries (greater than or equal to 70% reduction in endoluminal diameter), and Tc-99m teboroxime detected 160 abnormal segments corresponding to 29 of 45 (64%) stenosed arteries. Thallium-201 and Tc-99m-teboroxime studies were normal in 3 patients. In conclusion, this study shows that there is a good correlation in the imaging results found with thallium-201 and Tc-99m-teboroxime using dipyridamole infusion on both a segmental and a diagnostic comparison. PMID- 1729874 TI - Exercise testing and thallium-201 emission computed tomography in patients with intraventricular conduction disturbances. AB - The specificity of exercise thallium-201 emission computed tomography for coronary artery disease was assessed in patients with intraventricular conduction disturbances. Eighty-seven patients were studied: 33 with right bundle branch block (RBBB), 11 with RBBB and left-axis deviation, 11 with left (L)BBB, 12 on right ventricular pacing, and 20 with Wolff-Parkinson-White (WPW) syndrome. A control group of 349 consecutive patients with normal intraventricular conduction was also examined. The specificity of diagnosis of coronary artery disease in patients with LBBB (30%), right ventricular pacing (44%) or RBBB plus left-axis deviation (50%) was significantly lower than in patients with normal intraventricular conduction (94%; p less than 0.01). In contrast, there was no significant difference between specificity in patients with RBBB (86%) or WPW syndrome (90%) and patients with normal intraventricular conduction. Perfusion defects were found in the anterior, septal and inferior segments in patients with LBBB, and in the septal and inferior segments in patients with RBBB plus left axis deviation despite the absence of coronary stenosis. Furthermore, diffuse slow washout was seen more often in patients with WPW syndrome (35%) than in controls who had normal intraventricular conduction (11%; p less than 0.05), despite a good exercise performance in the former group. This study suggests that there is an increased incidence of abnormal perfusion and clearance during exercise thallium-201 emission computed tomography in patients with intraventricular conduction disturbances. PMID- 1729875 TI - Clinical risks of thrombolytic therapy. AB - Understanding the clinical risks of intravenous thrombolytic therapy is critical to appropriate patient selection. The major risks can be classified into 5 major categories: intracranial hemorrhage, systemic hemorrhage, immunologic complications, hypotension, and myocardial rupture. Although theoretical concern exists about thromboembolic complications, they rarely occur. Although cardiac rhythm disturbances are somewhat more likely to occur at the time of reperfusion, the clinical significance of "reperfusion arrhythmias" is minimal. Intracranial hemorrhage, the most devastating complication, occurs in 0.2-1% of patients treated with thrombolytic therapy. Factors associated with incremental risk are now being identified from large clinical trials. Systemic hemorrhage is uncommon in patients without major vascular punctures and seldom leads to serious adverse outcomes. Immunologic complications--including anaphylaxis, which is rare, and immune complex disease, which is more common--occur only with streptokinase or agents with a streptokinase moiety, including anistreplase (anisoylated plasminogen--streptokinase activator complex, APSAC). Hypotension, which can be managed easily in most patients, is also observed much more frequently with streptokinase and anistreplase. Myocardial rupture is increasingly being recognized as a possible complication of late thrombolysis. A proper perspective on clinical risk can only be gained in the context of potential benefit of therapy. In many cases individual patients considered to be at highest risk for complications also stand to gain the most from treatment. Many of the questions raised by currently available data about bleeding risk are being addressed in the ongoing Global Utilization of t-PA and Streptokinase (GUSTO) Trial. A paradigm for considering this decision making problem is presented. PMID- 1729876 TI - Ischemic stroke and intracranial hemorrhage following thrombolytic therapy for acute myocardial infarction: a risk-benefit analysis. AB - Stroke is a potentially serious complication of acute myocardial infarction (AMI). In the prethrombolytic era, most strokes were attributed to cerebral embolism. On the basis of available information, the occurrence of stroke in the thrombolytic era appears to be less than in the prethrombolytic era. In the thrombolytic era, the occurrence of various forms of intracranial hemorrhage has increasingly been documented in addition to cerebral embolism, with intriguing features. In general, however, the delineation of specific stroke subtypes has been imprecise and must take into account factors that are unique to this setting. Age is a risk factor for both ischemic and hemorrhagic stroke. Potential risk factors for intracranial hemorrhage include hypertension, dosage of fibrinolytic agents, and prior neurologic disease. Potential causes of intracranial hemorrhage include combined fibrinolytic/adjunctive therapies, various cerebrovascular lesions, and head trauma. Existing data suggest that mortality related to stroke complicating AMI is on the decline as well. More research is needed in order to quantify precisely the occurrence and proportions of stroke subtypes, risk factors, and causes in order to define mechanisms and preventive measures. PMID- 1729877 TI - Clinical benefits of thrombolytic therapy in acute myocardial infarction. AB - The value of coronary artery reperfusion resulting from pharmacologically induced fibrinolysis in patients with evolving myocardial infarction has been rigorously evaluated. Improved left ventricular function and even more impressive improvements in survival rates have been demonstrated consistently in controlled studies. Benefit is related to the restoration of myocardial blood flow. Maximal benefit is achieved with early and sustained restoration of coronary artery patency. Benefits observed during initial hospitalization are sustained for at least 1 year in the majority of patients, even without subsequent mechanical revascularization. To date, analysis of subgroups has not identified a population of patients with evolving infarction that should routinely be excluded from consideration for thrombolysis. As with many potent pharmacologic agents, activators of the fibrinolytic system are associated with a degree of risk whenever they are administered to a patient. Therefore, patients must be assessed carefully prior to initiating treatment, especially for potential bleeding hazards, and appropriate follow-up evaluation and concomitant therapy needs to be planned. However, given the overwhelming body of data now available regarding its benefits and relative safety, thrombolysis should be considered as conventional therapy for patients with acute evolving myocardial infarction (AMI). PMID- 1729878 TI - Thrombin antagonists and antiplatelet agents. AB - The clinical benefits of thrombolytic therapy for the treatment of myocardial infarction are recognized widely. However, 2 major limiting factors have become evident: (1) 20-25% of coronary arterial thrombi are resistant to lysis; and (2) coronary reocclusion occurs in 10-15% of patients. There is increasing evidence that both phenomena are caused by heightened procoagulant activity localized primarily at the site of atheromatous plaque rupture. Thrombin, the pivotal enzyme in all coagulation processes, is activated, stimulating fibrin formation and platelet aggregation. Platelet activation, by thrombin- and nonthrombin mediated mechanisms, occurs as well, further increasing thrombotic tendency. Thus, a potent and well-localized procoagulant state may be continuously amplified, increasing both during and frequently after thrombolytic therapy. Current treatment strategies are designed to enhance fibrinolytic and anticoagulant activity, while neutralizing the expression of procoagulant factors. Thrombin antagonism and platelet inhibition, primarily with heparin and aspirin, respectively, form the mainstay of conjunctive therapy. Their benefits have been recognized, decreasing thromboembolic events and patient mortality. However, intrinsic limitations suggest that more potent and selective agents will be required to overcome effectively the problems of thrombolytic resistance and coronary reocclusion. In experimental models, specific thrombin antagonists and antiplatelet agents have shown superiority over heparin and aspirin. Further investigation to define the overall safety and efficacy profile of these newer agents will be required, however, prior to their widescale implementation in clinical practice. PMID- 1729879 TI - Fibrinolytic parameters and hemostatic monitoring: identifying and predicting patients at risk for major hemorrhagic events. AB - The use of thrombolytic therapy has dramatically altered the treatment of acute myocardial infarction and is rapidly spreading from large medical centers to community hospitals throughout the country. The widespread use of thrombolytic therapy will benefit a wide range of people, but the potential risks of this form of therapy must be understood. Hemorrhage is one of the major risks of thrombolytic therapy. This review will focus on the data available from a number of recent, large trials of thrombolytic therapy for acute myocardial infarction with respect to laboratory parameters that may predict hemorrhagic complications and/or help with their management. We will discuss both conclusions drawn from currently available data and address future research directions. PMID- 1729880 TI - Thrombolytic therapy: adjuvant mechanical intervention for acute myocardial infarction. AB - Following successful pharmacologic thrombolysis, early coronary angiography frequently shows a tight residual stenosis in the infarct-related artery at the site of recent occlusion. Approaches to the management of the residual stenosis have undergone a gradual evolution from an aggressive strategy of immediate balloon dilation to a more conservative approach. Randomized, controlled trials have indicated that immediate percutaneous transluminal coronary angioplasty (PTCA) is associated with no greater recovery in regional or global left ventricular function, and a tendency toward an increased incidence of complications, including the need for emergency coronary artery surgery and blood transfusion. The role of immediate rescue PTCA for failed thrombolysis has not been as rigorously investigated, but selected patients, including those with evidence of ongoing myocardial ischemia or hemodynamic instability, may benefit from this approach. A major source of current controversy is the value of routine coronary angiography after uncomplicated myocardial infarction. Two carefully conducted trials have indicated that a conservative strategy of clinically indicated, predischarge cardiac catheterization may be associated with an increased need for readmission and late, elective cardiac catheterization when compared with a more invasive strategy of routine coronary angiography, but that the conservative approach is not associated with an increased incidence of death or reinfarction. Provision was not made in these studies, however, for evaluating the positive economic and psychologic impact of early coronary angiography, early hospital discharge, and early return to work of patients with a favorable postinfarction prognosis. It is concluded that early mechanical revascularization following thrombolysis should be considered for ongoing myocardial ischemia, but should otherwise be deferred pending the results of predischarge functional studies. For most patients, routine coronary angiography is likely to remain an important diagnostic tool and an integral component of the management of the convalescent phase of acute myocardial infarction. PMID- 1729881 TI - Designing thrombolytic agents: focus on safety and efficacy. AB - Thrombolytic therapy for evolving acute myocardial infarction (AMI) reduces infarct size, preserves ventricular function, and reduces mortality. Intravenous streptokinase is commonly followed by approximately 50% patency of coronary arteries within 90 minutes and by reduction of mortality by 25%. Recombinant tissue plasminogen activator (rt-PA) is more potent for coronary arterial thrombolysis, producing both more rapid and more frequent recanalization (approximately 75% patency at 90 minutes) with a dose of 100 mg given over 3 hours. Side effects (mainly bleeding) associated with the use of streptokinase and rt-PA are not markedly different. That the higher efficacy of rt-PA would translate into a larger reduction of mortality is suggested by the results of several small trials but remains to be confirmed in well-designed comparative clinical trials. This question has not been adequately answered by the recent International rt-PA/streptokinase mortality trial and the International Study on Infarct Survival (ISIS-3) study, because of concerns with respect to the role of conjunctive intravenous heparin administration and the dose of rt-PA used in ISIS 3. All available thrombolytic agents still have significant shortcomings, including the need for large doses to be maximally efficient, a limited fibrin specificity, and a significant associated bleeding tendency. New developments toward improved efficacy and fibrin-specificity of thrombolytic agents include the use of mutants of rt-PA, chimeric rt-PA or single chain urokinase plasminogen activator molecules, and antibody-targeted thrombolytic agents. Some of these artificial plasminogen activators have a 5- to 10-fold increased potency (thrombolytic activity per unit dose), but whether they are safe enough to be clinically useful remains to be established. The conjunctive use of anticoagulants and antiplatelet agents with thrombolytic agents increases their efficacy to an extent that monotherapy with a plasminogen activator alone is no longer tenable. Heparin and aspirin are only moderately efficient for acceleration of lysis and prevention of reocclusion, but are relatively safe. More selective thrombin inhibitors and antiplatelet agents are more potent, but their safety remains to be confirmed. Continued investigation in this area will provide new insights and promote progress toward the development of the ideal thrombolytic therapy, characterized by maximized stable coronary arterial thrombolysis with minimal bleeding. PMID- 1729883 TI - Y chromosome probe p49a detects complex PvuII haplotypes and many new TaqI haplotypes in southern African populations. AB - Y-specific 49a/TaqI haplotypes were determined for 831 individuals drawn from 21 different southern African populations. A total of 31 new haplotypes were observed, some of which contained new alleles or allelic variants. Duplication, in addition to CpG mutation, is implicated in the generation of certain allelic variants. Cluster analysis of genetic distances between the populations, calculated using the 49a/TaqI haplotype frequencies, revealed a basic split between African and non-African populations. Hybrid groups cluster with the caucasoid groups, indicating that male gene flow has occurred from the latter into the former. Clustering of the negroid and Khoisan groups is not what might have been expected from the known linguistic affinities. It is suggested that the 49a/TaqI haplotype analysis of these populations is not sufficiently sensitive to distinguish between many of the populations. The Y-specific 49a/PvuII polymorphism was studied in 127 individuals from southern African populations, and 17 polymorphic fragments ranging in size from 3.6 kb to greater than 48 kb were identified. A total of 53 PvuII haplotypes were observed, corresponding to only 30 TaqI haplotypes. There appears to be poor correlation between the two polymorphisms. PMID- 1729882 TI - Influence of MHC and MHC-linked genes on reproduction. PMID- 1729884 TI - Biochemical and immunological characterization of serum biotinidase in profound biotinidase deficiency. AB - The biochemical and immunological characterization of biotinidase was performed in sera from 100 normal individuals, 68 children with profound biotinidase deficiency (less than 10% of mean normal activity) who were identified symptomatically and by newborn screening, and 63 of their parents. On isoelectric focusing, serum enzyme from normal individuals exhibits extensive microheterogeneity, consisting of at least four major and five minor isoforms at pH 4.15-4.35. Patients with profound biotinidase deficiency can be classified into at least nine distinct biochemical phenotypes, on the basis of (a) the presence or absence of cross-reacting material (CRM) to biotinidase, (b) the number of isoforms, and (c) the distribution frequency of the isoforms. None of the patients with CRM had an abnormal Km of the substrate for the enzyme. All of the parents had normal isoform patterns. The mean activities, CRM concentrations, and specific activities were not significantly different between parents of CRM positive children and parents of CRM-negative children. There is no relationship between either the age at onset or the severity of symptoms and the isoform patterns or CRM status of the symptomatic children. The isoform patterns of children identified by newborn screening are not different from those of symptomatic children. PMID- 1729885 TI - Caucasian genes in American blacks: new data. AB - Data on 15 polymorphic protein-coding loci are used to estimate the proportion of Caucasian genes in U.S. blacks from the greater-metropolitan area of Pittsburgh. Allele frequencies from U.S. whites of the same region and from a sample of Nigerians are used as representatives of the genetic contributions of the source populations between which admixture has occurred. These materials provide 18 unique variants that occur exclusively in the blacks and 5 variants that are restricted to the Caucasians only. As a result, when all segregating alleles (52) at these 15 loci are considered, the proportion (mean +/- SE) of Caucasian genes in U.S. blacks (25.2% +/- 2.7%) is estimated with a precision much better than that of all other previous estimates. The estimate based on the frequencies of these 18 unique variants of African origin (24.8% +/- 6.2%) is also consistent with the pooled estimate obtained from the 15 loci by the weighted least-square method. The homogeneity of locus-specific estimates of admixture indicates that these loci are appropriate for studying the proportion of black genes in any admixed population involving African admixture. The advantages of employing such loci for genetic-epidemiologic studies in U.S. blacks is discussed in the context of the availability of these large number of unique African variants at these protein loci. PMID- 1729886 TI - Characterization of Robertsonian translocations by using fluorescence in situ hybridization. AB - Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions. PMID- 1729887 TI - Dentin phosphoprotein gene locus is not associated with dentinogenesis imperfecta types II and III. AB - Dentinogenesis imperfecta (DGI) is an autosomal dominant inherited dental disease which affects dentin production and mineralization. Genetic linkage studies have been performed on several multigeneration informative kindreds. These studies determined linkage between DGI type II and III and group-specific component (vitamin D-binding protein). This gene locus has been localized to the long arm of human chromosome 4 in the region 4q11-q21. Although this disease has been mapped to chromosome 4, the defective gene product is yet to be determined. Biochemical studies have suggested abnormal levels of dentin phosphoprotein (DPP) associated with DGI type II. This highly acidic protein is the major noncollagenous component of dentin, being solely expressed by the ectomesenchymal derived odontoblast cells of the tooth. The purpose of the present study was to establish whether DPP is associated with DGI types II and III, by using molecular biology techniques. The strategy was to use a synthetic degenerative DPP oligonucleotide probe to map this sequence to the long arm of human chromosome 4, 4q13-q21, by using somatic cell hybrids. Our results indicated that DPP is not localized to any region of human chromosome 4, thus suggesting that the DPP gene is not directly associated with DGI type II or DGI type III. Our data do not exclude the possibility that other proteins associated with DPP posttranslational modifications might be responsible for this genetic disease. PMID- 1729888 TI - A specific test for transthyretin 122 (Val----Ile), based on PCR-primer introduced restriction analysis (PCR-PIRA): confirmation of the gene frequency in blacks. AB - The variant transthyretin (TTR) allele, TTR (122 Val----Ile), associated with cardiac amyloidosis in blacks, is caused by a G----A transition which destroys a MaeIII site. This variant has previously been detected by PCR around codon 122, followed by MaeIII digestion, but this test is not specific: any of 12 mutations in the MaeIII recognition site, each of which yields a different amino acid change, would also destroy this site. A modification of PCR, termed "PCR-primer introduced restriction analysis," was used to introduce a new FokI site into the PCR products derived from the variant (122 Ile) but not wild-type (122 Val) allele. This test demonstrated that each of six previously identified MaeIII(-) alleles had lost its MaeIII site because of a G----A transition encoding TTR (122 Val----Ile), confirming that the same TTR variant was present both in 4/177 healthy black individuals and as a homozygous variant in an individual with cardiac amyloidosis. PMID- 1729889 TI - Germ-line mosaicism for a valine-to-methionine substitution at residue 553 in the glycoprotein Ib-binding domain of von Willebrand factor, causing type IIB von Willebrand disease. AB - The origin of new single-gene mutations resulting in inherited disease is an issue which may be at least partially resolved by our enhanced ability to detect these changes. In this report we describe the identification of a missense mutation at codon 553 (guanine to adenine) in the von Willebrand factor (vWf) gene in affected members of a family with type IIB von Willebrand's disease (vWd). We found no evidence for this substitution in 190 normal vWf genes. The encoded substitution of a methionine for a valine at this residue is nonconservative in nature and has affected a vWf protein region which has been shown to facilitate binding to the platelet receptor glycoprotein Ib. In patients with type IIB vWd this interaction is characteristically increased in affinity. This mutation has also recently been recorded in four other type IIB vWd families. Thus, there is strong circumstantial evidence to incriminate this substitution as the disease causing mutation in this family. As further supporting evidence for this claim, we have shown by vWf polymorphism analysis that the mutation originated in a vWf gene transmitted from a phenotypically normal grandfather. Analysis of the sperm from this individual showed that approximately 5% of the germ line contained the mutant 553 sequence. These results confirm (1) that the candidate type IIB vWd mutation in this family occurred at some time during the development of the germ line of the grandfather and presumably was related to a mitotic cell division and (2) that, as a result, he is a low-level germ-line mosaic for the mutation. PMID- 1729890 TI - Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. AB - A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations. PMID- 1729891 TI - Interaction of apolipoprotein E genotype and dietary cholesterol in determining plasma cholesterol levels. PMID- 1729892 TI - A novel -32 (C-A) mutant identified in amplified genomic DNA of a Chinese beta thalassemic patient. PMID- 1729893 TI - From molecular variant to disease: initial steps in evaluating the association of transthyretin M119 with disease. AB - Traditionally, clinical research has sought to determine the molecular basis of clinical signs and symptoms. Increasingly, the traditional process will be reversed, as many structural protein variants are elucidated as a result of powerful PCR-based methods. Herein we describe a variant of transthyretin (TTR) found by direct genomic sequencing and illustrate the utility of PASA (PCR amplification of specific alleles) in the initial characterization of such variants. TTR is an intriguing protein of unknown function, but deposition of mutant TTR produces familial amyloidotic polyneuropathy (FAP). We identify a carrier of a variant TTR in which threonine119 is changed to methionine (T119--- M). T119 is invariant in five mammalian species, suggesting that this residue is important for normal protein function. To determine the frequency of the M119 variant, individuals of northern- and western-European descent were rapidly screened by generating a PASA assay for the sequence change. Four additional individuals were found to be heterozygous for the mutation, for a total of five M119 alleles in 1,666 genes (1/333). Clinical records, initial clinical interviews, and family history of these patients hint at a high frequency of early-onset venous insufficiency and perhaps mild renal dysfunction. Haplotype analysis on the heterozygotes could be performed, despite the absence of samples from relatives, by performing "double PASA." The haplotype data suggest that the M119 variant derives from a common ancestor. The putative functional deficiency caused by TTR M119 should be most marked in the homozygotes, who can be calculated to occur in 1/100,000 conceptions. If viable, these individuals may provide important clues about the physiological role of TTR. Although the nature (if any) of disease caused by TTR M119 remains to be defined, the genetic and clinical data indicate that this mutation does not cause FAP. Future family studies can determine whether the heterozygous state for TTR M119 cosegregates with a disease or trait. PMID- 1729894 TI - Multicolor in situ hybridization and linkage analysis order Charcot-Marie-Tooth type I (CMTIA) gene-region markers. AB - This study demonstrates a clear and current role for multicolor in situ hybridization in expediting positional cloning studies of unknown disease genes. Nine polymorphic DNA cosmids have been mapped to eight ordered locations spanning the Charcot-Marie-Tooth type 1 (CMT1A) disease gene region in distal band 17p11.2, by multicolor in situ hybridization. When used with linkage analysis, these methods have generated a fine physical map and have firmly assigned the CMT1A gene to distal band 17p11.2. Linkage analysis with four CMT1A pedigrees mapped the CMT1A gene with respect to two flanking markers (8B10-5 cM[LOD 5.2] CMT1A-3.5 cM[LOD 5.3]-10E4). Additional loci were physically mapped and ordered by in situ hybridization and analysis of phase-known recombinants in CMT1A pedigrees. The order determined by multicolor in situ hybridization was 17cen LEW301-8B10-5H5/6A9-VAW409- 5G7-6G1-4A11-VAW412-10E4-pter. Two ordered probes, 4A11 and 6G1, reside on the same 440-kb partial SfiI restriction fragment. These data demonstrate the ability of in situ hybridization to resolve loci within 0.5 Mb on early-metaphase chromosomes. Multicolor in situ hybridization also excluded the possibility of pericentric inversions in two unrelated patients with CMT1 and neurofibromatosis type 1. When used with pulsed-field gel electrophoresis, multicolor in situ hybridization can establish physical location, order, and distance in closely spaced chromosome loci. PMID- 1729895 TI - Decreased fecundability in Hutterite couples sharing HLA-DR. AB - To study the effects of parental HLA sharing on pregnancy outcome, we initiated population-based studies in the Hutterites. We previously reported longer intervals from marriage to each birth among couples sharing HLA, particularly HLA DR. In the present report, we present the results of a prospective, 5-year study of fecundability and fetal loss rates in this population. Between April 1986 and April 1991, 154 pregnancies were observed in 104 couples. The median number of months of unprotected intercourse to a positive pregnancy test was significantly longer among couples sharing HLA-DR who stopped nursing prior to the first menses as compared with couples not sharing HLA-DR who stopped nursing prior to the first menses (5.1 vs. 2.0 mo, respectively; P = .016). Fetal loss rates were increased among couples sharing HLA-B as compared with couples not sharing HLA-B (.23 vs. .12, respectively; P = .041, adjusted for age, gravidity, and kinship). These data suggest that our earlier observations of increased birth interval lengths among Hutterite couples sharing HLA were predominantly due to longer intervals until a clinical pregnancy among couples sharing HLA-DR and, to a lesser degree, were due to increased fetal loss rates among couples sharing HLA B. PMID- 1729896 TI - The gene for human erythrocyte protein 4.2 maps to chromosome 15q15. AB - Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5' and the 3' coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). Our results demonstrate that the locus of the P4.2 gene is located within 15q15. PMID- 1729897 TI - Aphidicolin-inducible common fragile-site expression: results from a population survey of twins. AB - Common chromosomal fragile sites appear to be ubiquitous in humans and other mammals, and, although the molecular basis and function of these sites remain an enigma, it has been speculated that they may be a cytogenetic expression of gene activity. A population survey of 28 twin pairs was conducted to assess the heritability of common fragile-site expression. Our data yielded a heritability estimate of .88 for total site expression, suggesting that these sites may result from some common process that is under relatively stringent genetic control. An analysis of the expression of individual autosomal sites revealed that expression on both homologues in the same cell occurred more frequently than expected. PMID- 1729898 TI - The economics of clinical genetics services. IV. Financial impact of outpatient genetic services on an academic institution. AB - Those clinical genetic services that do not involve laboratory tests or procedures--i.e., the "cognitive" services such as diagnosis, management, and counseling--are labor-intensive, time-consuming, and not self-supporting. However, as a result of an evaluation at a genetics clinics, a patient will often receive other services at the same medical center. The full economic impact of the genetics clinic may be underappreciated. Therefore, at one medical center we examined (a) three settings that delivered genetics services and (b) two specialty clinics providing services to children with genetics conditions; and we calculated charges and payments for an unselected, consecutive group of outpatients. The results showed that cognitive genetics services accounted for a variable, but generally low, percentage of both the professional (generally physicians') and total charges accumulated by patients as a consequence of their visit to the genetics clinic. With laboratory and procedural charges included, patients seen in general genetics clinics (or their insurance plans) paid up to three times as much to the medical center and to its health professionals as to the genetics professional. These data confirm that clinical genetics services, while not generating enough income to cover their own costs, bring considerable revenue to the medical center. This fact alone should prove useful to the director of clinical genetics programs when they are negotiating finances with institutional administrators. PMID- 1729899 TI - Beating burnout. PMID- 1729900 TI - Must we negotiate? PMID- 1729901 TI - Survey results. Who helps you with your work? PMID- 1729902 TI - Solving the puzzle of chest pain. PMID- 1729904 TI - Books of the year. PMID- 1729903 TI - Upgrading practice with critical pathways. PMID- 1729905 TI - Jobfocus. Your guide to nursing opportunities in the Northeast. PMID- 1729906 TI - Revisit your sources. PMID- 1729907 TI - How do you test a digital sphygmomanometer? PMID- 1729908 TI - Cadillac or Chevrolet nursing? Look under the hood. PMID- 1729909 TI - Melanie's request. PMID- 1729910 TI - Regs put new legal force behind universal precautions. PMID- 1729911 TI - Life support. PMID- 1729912 TI - An application of health services research to anesthesiology. PMID- 1729913 TI - Halothane does not alter Ca2+ affinity of troponin C. AB - Troponin C has been suggested as a possible target for the negative inotropic action of volatile anesthetics. This study has examined the effect of halothane on the structure and response of isolated cardiac troponin C to Ca2+ and the response of skinned soleus and cardiac muscle fibers to Ca2+. The high-affinity Ca(2+)-binding sites of cardiac troponin C were assessed by measurement of the change in intrinsic tyrosine fluorescence and ultraviolet circular dichroism in response to Ca2+ in the presence and absence of halothane. Halothane (0.9 mM, 1.4%) did not alter the 45% enhancement in intrinsic tyrosine fluorescence that occurs with saturation of the high-affinity sites or change the Ca2+ concentration at which half-maximal enhancement occurred. The molar ellipticity in the far ultraviolet region, a measure of the secondary structure, increased to a similar extent with addition of 10(-6) M Ca2+ in the absence and presence of 1.0 mM (1.6%) halothane. The binding rate of the sulfhydryl reagent, 5,5' dithiobis (2-nitrobenzoic acid), to troponin C in response to Ca2+ titration was used as a measure of the integrity of the low-affinity Ca(2+)-binding site in troponin C in the presence and absence of 1.0 mM (1.6%) halothane. The rate of reaction was stimulated twofold, and the half maximal effect was observed at pCa 4.8 +/- 0.2 in both control and halothane-treated samples. Halothane (5 mM; 7.8%) did not change the pCa/tension response of skinned soleus fibers; the data were fit to the Hill equation and yielded dissociation constants of 6.2 x 10(-7) M for control and halothane-treated specimens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729914 TI - Effects of prostaglandin E1 and hydralazine on the longitudinal distribution of pulmonary vascular resistance during vasoconstrictor pulmonary hypertension in sheep. AB - Pulmonary capillary pressure (Ppc) is dependent upon left atrial pressure, pulmonary venous resistance, and cardiac output. The effects of pulmonary vasodilators on Ppc will therefore depend upon any alterations in the longitudinal distribution of pulmonary vascular resistance (precapillary [arterial] and postcapillary [venous] components). We therefore studied the effects of two pulmonary vasodilators (prostaglandin E1 and hydralazine) on Ppc and the longitudinal distribution of pulmonary vascular resistance. Pulmonary hypertension was produced in sheep by the continuous administration of the thromboxane A2-mimetic U46619. Ppc was measured by analysis of pulmonary artery occlusion pressure decay curves. U46619 increased Ppc by 9 mmHg and increased both the arterial and venous components of pulmonary vascular resistance. Subsequent administration of prostaglandin E1 decreased Ppc by 5 mmHg and decreased both the arterial and venous components of pulmonary vascular resistance (by 50 and 69% respectively). Hydralazine produced smaller decreases in the arterial and venous components of pulmonary vascular resistance (by 35 and 49% respectively) and did not significantly reduce Ppc. We conclude that prostaglandin E1 but not hydralazine is effective in decreasing Ppc in this experimental model of pulmonary hypertension. PMID- 1729915 TI - Dose-response relationship of isoflurane and halothane versus coronary perfusion pressures. Effects on flow redistribution in a collateralized chronic swine model. AB - The authors studied the redistribution of myocardial blood flow in a collateral dependent (CD) zone as a function of coronary perfusion pressure (CPP) during isoflurane and halothane anesthesia. A swine model with CD myocardium distal to a chronically occluded left anterior descending coronary artery was developed and studied. Sixteen piglets were allowed to grow for 8-10 weeks after banding of the left anterior descending coronary artery. They were randomly anesthetized with either isoflurane (n = 8) or halothane (n = 8) as the sole anesthetic, which was used to regulate specific CPP. The resultant regional myocardial blood flows were measured using radiolabeled microspheres. Four randomly allocated CPPs, of 30, 40, 45, and 55 mmHg, were studied in each animal. Four additional collateralized animals were anesthetized with alpha-chloralose, and the same CPPs were obtained using an intravenous adenosine infusion (1-5 microM kg-1) to validate this model. There was a proportional decrease in heart rate and blood pressure in both the isoflurane and and the halothane group with CPP. Cardiac output was significantly decreased in the halothane group at 30 mmHg when compared to 55-mmHg CPP, but it was maintained in the isoflurane group. Systemic vascular resistance was significantly lower in the isoflurane group at 30 and 40 mmHg when compared to 55 mmHg CPP. Both the isoflurane and the halothane group showed a proportional and significant decrease in endo-, mid-, and epicardial blood flows at 30-mmHg CPP when compared to baseline. In both CD and normal perfusion zones, isoflurane consistently sustained a higher endocardial blood flow than halothane (5.7 41.1%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729916 TI - Differential effects of halothane, enflurane, and isoflurane on Ca2+ transients and papillary muscle tension in guinea pigs. AB - These studies were designed to examine the effects of inhalational anesthetics on rapid changes in myocardial intracellular Ca2+ and Ca2+ sensitivity of the contractile apparatus. The effects of halothane, enflurane, and isoflurane on rapid changes in intracellular Ca2+ (Ca2+ transients as measured with bioluminescent protein aequorin) and contractile characteristics were compared in guinea pig right ventricular papillary muscles. In addition to examination of their potencies at equianesthetic concentrations, the effects of these agents on alterations in Ca2+ sensitivity at myofilaments were also investigated. The negative inotropic effects of halothane (0.65 and 1.15%) and enflurane (1.0 and 2.2%) were dose-dependent and closely related to a decrease in Ca2+ transients. In the presence of isoflurane (0.77 and 1.6%), the contractile force decreased in a dose-dependent manner, but the decrease was significantly less as compared to that with equianesthetic concentrations of halothane and enflurane. An additional feature observed in the presence of isoflurane was a dissociation between intracellular Ca2+ availability and contractile force. Although the magnitude of the Ca2+ transients did not change when the percentage of isoflurane was increased from 0.77 to 1.6, the contractile force decreased. Because of these findings, the effects of halothane (1.2%), enflurane (2.2%), and isoflurane (1.6%) on the relationship between intracellular Ca2+ and tension developed in the papillary muscle were examined in order to assess myofibrillar responsiveness to Ca2+. The results indicate that only isoflurane slightly but significantly shifted the Ca2+/isometric tension curve toward higher intracellular Ca2+ concentrations; no differences were observed in the absence and presence of equianesthetic concentrations of halothane and enflurane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729917 TI - EU 4093 decreases intracellular [Ca2+] in skeletal muscle fibers from control and malignant hyperthermia-susceptible swine. AB - The mechanisms causing the malignant hyperthermia (MH) syndrome are related to a malfunction of intracellular Ca2+ homeostasis and can be prevented or reversed by dantrolene. EU 4093 (Azumolene, 1-[[[5-(4-bromophenyl)-2-oxyzolyl] methylene]amino]-2-4- imidazolidinedione) is a 30-fold more water-soluble analogue of dantrolene that is believed to have the same effects as dantrolene on the intracellular free Ca2+ concentration [( Ca2+]i) in skeletal muscle and that should have similar efficacy in treating and preventing the clinical manifestations of MH in response to a halothane/succinylcholine challenge. To test this hypothesis, experiments were carried out in four controls (Yorkshire) and eight MH-susceptible crossbreed swine (Poland China X Pietrain). The resting [Ca2+]i in normal muscle fibers measured by Ca(2+)-selective microelectrodes was 111 +/- 12 nM (mean +/- standard deviation, n = 30), whereas in the MH muscles the resting [Ca2+]i was 395 +/- 36 nM, (n = 28) (P = 0.0001). EU 4093 decreased [Ca2+]i in MH-susceptible skeletal muscle in a dose-related fashion from 207 to 38 nM after 0.5 to 2.0 mg/kg, respectively, and had a similar effect in control skeletal muscle (58 to 30 nM) after the same doses. In MH-susceptible swine, a dose of 2.0 mg/kg was successful in preventing any clinical signs of the MH syndrome during a subsequent halothane/succinylcholine challenge. A dose of 0.5 mg/kg was able to attenuate but not reverse the clinical signs of the MH syndrome after a halothane challenge, whereas a dose of 1.0 mg/kg was completely successful in reversing this effect in all subjects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729918 TI - Spinal cutaneous fistula following continuous spinal anesthesia. PMID- 1729919 TI - Atypical response to scopolamine in a patient with type IV hereditary sensory and autonomic neuropathy. PMID- 1729920 TI - Detection of occult hemopericardium using intraoperative transesophageal echocardiography. PMID- 1729921 TI - Pulse oximeter overload. PMID- 1729922 TI - A method for ensuring proper function of multiorifice catheters. PMID- 1729923 TI - Prediction of myocardial oxygen consumption. PMID- 1729924 TI - An armored laryngeal mask airway. PMID- 1729925 TI - When the endotracheal tube will not pass over the flexible fiberoptic bronchoscope. PMID- 1729926 TI - Burn associated with temperature monitoring during magnetic resonance imaging. PMID- 1729927 TI - Naloxone reversal of nystagmus associated with intrathecal morphine administration. PMID- 1729928 TI - An American dentist pioneered anesthesia in Spain. PMID- 1729929 TI - Treatment of pain on the surgical ward using epidural morphine. PMID- 1729930 TI - Jugular venous compression helps to identify the source of venous air embolism during craniectomy in patients in the sitting position. PMID- 1729931 TI - Prediction of malignant hyperthermia susceptibility in low-risk subjects. An epidemiologic investigation of caffeine halothane contracture responses. The North American Malignant Hyperthermia Registry. AB - The most commonly used laboratory test for predicting malignant hyperthermia susceptibility is the caffeine halothane contracture test. However, the specificity and sensitivity of proposed North American diagnostic guidelines for this test have never been evaluated in a large, human study population. Therefore, the authors conducted a multiinstitutional, prospective study of skeletal muscle contracture responses in a subject population at low risk for malignant hyperthermia susceptibility to help determine the specificity of the proposed guidelines. Subjects were selected arbitrarily from a population of patients undergoing surgery unrelated to performance of a diagnostic muscle biopsy. Subjects were admitted to this study and were presumed nonsusceptible if there was no evidence of any of the following malignant hyperthermia risk factors: prior abnormal response to triggering anesthetic agents, myopathy, or family history of malignant hyperthermia susceptibility. The authors suggested rejection of the proposed diagnostic guidelines if an 85% specificity estimate among subjects could not be obtained. The authors analyzed the responses of 1,022 muscle fascicles, derived from 176 subjects, to the following: 1) separate administration of 3% halothane or incremental caffeine concentrations, or 2) the joint administration of 1% halothane and incremental caffeine concentrations. The following contracture results were obtained. First, for individual fascicles, 9.2% exceeded a greater than 0.7 g threshold for 3% halothane, 15.2% exceeded a greater than or equal to 0.2 g threshold for 2 mM caffeine, 32.4% exceeded a 1-g increase for less than 4 mM caffeine, 2.6% had a greater than 7% maximal increase in tension at 2 mM caffeine, and 63.5% had a "halothane caffeine-specific concentration" at less than or equal to 1 mM caffeine. Second, the percentages of subjects with 1 or more fascicles exceeding the proposed threshold were as follows: 45.8% for the four-component, 28.8% for the three-component, and 32.7% for the two-component contracture test. Third, the percentages of subjects with 1 or more fascicles exceeding the proposed threshold for both halothane and caffeine were as follows: 9.5% for 3% halothane and 2 mM caffeine, 2.0% for 3% halothane and 7% maximal increase in tension at 2 mM caffeine, and 11.0% for 1% halothane and 2 mM caffeine. Fourth, center-to-center differences were the major source of variation in the rate that subjects exceeded proposed thresholds. These data demonstrate that proposed diagnostic guidelines must be modified to improve specificity estimates before adoption by diagnostic centers.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1729932 TI - Oral ketamine preanesthetic medication in children. AB - The authors sought to define a dose of oral ketamine that would facilitate induction of anesthesia without causing significant side effects. Forty-five children (ASA Physical Status 1 and 2; aged 1-7 yr) were assigned randomly in a prospective, double-blind fashion to three separate groups that received either 3 mg/kg, 6 mg/kg, or no ketamine mixed in 0.2 ml/kg cola-flavored soft drink. They also were evaluated preoperatively and postoperatively for acceptance of oral ketamine as a premedicant, reaction to separation from parents, emotional state, and emergence phenomena. The authors detected no episodes of respiratory depression, tachycardia, or arterial hemoglobin desaturation before, during, or after surgery. The 6 mg/kg dose was well accepted; provided uniform, predictable sedation within 20-25 min; and allowed calm separation from parents and good induction conditions. The 3 mg/kg dose did not always cause sedation and calm separation from parents. Neither dose of ketamine increased the incidence of laryngospasm, prolonged recovery times, or caused emergence phenomena. The authors conclude that an oral dose of 6 mg/kg ketamine is easily administered and well accepted in young children and provides predictable, satisfactory premedication without significant side effects. PMID- 1729933 TI - Multicenter study of general anesthesia. III. Predictors of severe perioperative adverse outcomes. AB - Little information is available about the incidence of severe adverse outcomes, and even less information is available about the identification and quantification of independent predictors of severe perioperative adverse outcomes. The purpose of this study was to identify and quantitate independent predictors of severe perioperative adverse outcomes in a prospective randomized clinical trial of general anesthesia in 17,201 patients. Twenty-nine prognostic variables for 15 severe outcomes in 847 patients were tested by multiple stepwise logistic regressions from which 20 significant (P less than 0.05) predictors were identified. A history of cardiac failure or myocardial infarction less than or equal to 1 yr; ASA physical status 3 or 4; age greater than 50 yr; cardiovascular, thoracic, abdominal or neurologic surgery; and the study anesthetics were significant predictors of "any severe outcome, including death." There were 17 significant predictors for 10 severe cardiovascular outcomes in 608 patients, including a history of ventricular arrhythmia, hypertension, cardiac failure, myocardial ischemia, myocardial infarction less than or equal to 1 yr or myocardial infarction greater than 1 yr, and smoking; ASA physical status; age; cardiovascular, thoracic, abdominal, eyes-ears-nose-throat/endocrine, neurologic, musculoskeletal, or gynecologic surgery; and the study anesthetics. There were 9 significant predictors for 4 severe respiratory outcomes in 163 patients, including a history of cardiac failure, myocardial ischemia, or chronic obstructive pulmonary disease; obesity; smoking; male gender; ASA physical status; abdominal surgery; and the study anesthetics. Colinearity between related prognostic variables (such as disease and ASA physical status) was assessed using progressively segregated groups of variables in eight stepwise logistic regressions. We conclude that the comprehensive stepwise logistic regression of 29 prognostic variables reported here provides a valid estimate of the risks of severe perioperative outcomes associated with general anesthesia. PMID- 1729934 TI - Accelographic train-of-four at near-threshold currents. AB - The authors evaluated train-of-four (TOF) fade, as quantified by accelography, in response to neurostimulation at currents ranging from 10 to 60 mA. This was done to determine the range of currents over which measurements of fade remain consistent. In 31 patients (ASA Physical Status 1,2, and 3), anesthesia was induced with fentanyl, midazolam, and thiopental and was maintained with isoflurane and 66% nitrous oxide in oxygen. Surface stimulating electrodes were placed over the ulnar nerve, and an acceleration transducer was placed on the thumb. Succinylcholine was administered to facilitate tracheal intubation; after neuromuscular recovery, a bolus of vecuronium (0.01-0.05 mg.kg-1) and an infusion (0.25-1.5 micrograms.kg-1.min-1) were administered. After documentation of a stable TOF ratio, accelographic TOF responses were quantified in response to 200 microseconds stimulation at 10, 15, 20, 30, 40, 50, and 60 mA, in random order. A total of 95 data sets were collected at different depths of blockade. The TOF ratios maintained intercurrent consistency (P = not significant by nonparametric repeated measures analysis of variance), except at currents near the fourth twitch (T4) threshold current. This inconsistency was eliminated by testing at greater than or equal to 10 mA above threshold. TOF ratios obtained at 10 mA above T4 threshold correlated highly with those at 60 mA (Spearman r value = 0.94). The authors conclude that the TOF ratio is consistent over a wide range of stimulating currents and that testing with submaximal currents can be performed reliably at greater than or equal to 10 mA above the T4 threshold.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729935 TI - Oral transmucosal fentanyl citrate for preanesthetic medication of pediatric day surgery patients with and without droperidol as a prophylactic anti-emetic. AB - he safety and efficacy of oral transmucosal fentanyl citrate (OTFC) as a preanesthetic medication and the efficacy of droperidol as a prophylactic anti emetic were evaluated in 100 children aged 2-8 yr undergoing general anesthesia for outpatient surgery. Patients were randomly assigned to one of four groups and managed in a double-blinded manner: 1) placebo lozenge 45 min preoperatively and placebo (normal saline) injected intravenously after induction of anesthesia; 2) placebo lozenge 45 min preoperatively and 50 micrograms/kg droperidol intravenously after induction; 3) 15-20 micrograms/kg OTFC lozenge 45 min preoperatively and placebo intravenously after induction; and 4) 15-20 micrograms/kg OTFC lozenge 45 min preoperatively and droperidol 50 micrograms/kg intravenously after induction. Anesthesia was induced and maintained with halothane and nitrous oxide in oxygen. Heart rate, respiratory rate, blood pressure, and hemoglobin oxygen saturation (SpO2) were monitored throughout the study. Scoring systems were used to evaluate sedation, anxiety, cooperation, and ease and quality of anesthetic induction. Emergence, recovery, and discharge times were recorded. Nausea, vomiting, and adverse effects were noted. Preoperatively, children receiving OTFC had significantly greater sedation, slower respiratory rates, lower SpO2, and less excitement during induction. Postoperative nausea and vomiting occurred significantly more frequently after OTFC than after placebo. Prophylactic droperidol did not significantly reduce the incidence of nausea and vomiting. The authors conclude that, in pediatric surgical outpatients, OTFC reliably induces preoperative sedation and facilitates inhalation induction of anesthesia, but it is associated with significant decreases in respiratory rate and SpO2 and a high incidence of postoperative nausea and vomiting that is not significantly reduced by prophylactic droperidol. PMID- 1729936 TI - Reduction of the MAC of desflurane with fentanyl. AB - Opioids are known to affect the MAC of inhalational anesthetics. We have determined the interaction between fentanyl and desflurane, following a bolus injection of fentanyl at induction in 134 adult patients. Five groups of patients were studied. Four groups received desflurane or isoflurane in oxygen with either fentanyl 3 or 6 micrograms/kg and thiopental 2-5 mg/kg given as a bolus injection at the time of induction. An additional group received desflurane in oxygen alone. Groups were stratified by age. MAC determination, in response to the stimulus of skin incision, was made using the "up-down" method and logistic regression. The MAC desflurane in oxygen was 6.3% (5.3-7.6%, 95% confidence interval [CI]). Fentanyl 3 micrograms/kg produced a fentanyl plasma concentration of 0.78 +/- 0.53 ng/ml at skin incision and resulted in a MAC for desflurane of 2.6% (2.0-3.2%, 95% CI) %. Fentanyl 6 micrograms/kg produced a fentanyl plasma concentration of 1.72 +/- 0.76 ng/ml at skin incision and resulted in a MAC for desflurane of 2.1% (1.5-2.6%, 95% CI). To compare recovery times to eye-opening and response to commands, patients were grouped according to the plasma fentanyl concentrations at the time of awaking. Recovery was faster in patients who received desflurane than in those who received isoflurane. The authors conclude that the MAC of desflurane is significantly reduced 25 min following a single dose of 3 micrograms/kg of fentanyl and that increasing the fentanyl dose to 6 micrograms/kg produces little further decrease in MAC. Desflurane is also associated with faster recovery from anesthesia than is isoflurane. PMID- 1729937 TI - Oxygen uptake during recovery following naloxone. Relationship with intraoperative heat loss. AB - The increased metabolic and respiratory demand during naloxone recovery from opioid-based anesthesia could be related to the return of thermoregulation in hypothermic patients and thus be avoided by preventing intraoperative hypothermia. In this study, we measured O2 uptake (VO2) during naloxone-induced recovery in two groups of patients to determine the effect of intraoperative heat loss on postoperative VO2 changes. In seven patients, intraoperative hypothermia was prevented (normothermic group), whereas hypothermia was allowed to develop in seven other patients (hypothermic group). Core and skin temperatures were measured throughout the study to calculate changes in body heat content. Before naloxone antagonism of fentanyl-supplemented anesthesia, core temperature (mean +/- SEM) was 36.8 +/- 0.1 degrees C in the normothermic group and 34.2 +/- 0.2 degrees C in the hypothermic group (P less than 0.001). After titrated administration of naloxone during recovery, VO2 and minute ventilation (VE) increased in the hypothermic group, by 114 +/- 37% and 97 +/- 52% respectively (P less than 0.05), with a three-fold increase in four patients. In the normothermic group, VO2 increased significantly less (25 +/- 5%), without any significant change in VE. The change in VO2 and VE was significantly greater in patients who were hypothermic. VO2 was integrated throughout the recovery period to calculate recovery energy expenditure. Recovery energy expenditure and intraoperative heat loss were highly correlated (r = 0.88; P less than 0.01). This study demonstrates that the metabolic and respiratory stresses associated with naloxone-induced recovery from opioid-based anesthesia depend on the intraoperative heat loss and can therefore be reduced by preventing intraoperative hypothermia. PMID- 1729938 TI - Pharmacodynamics of alfentanil. The role of plasma protein binding. AB - The role of protein binding in relation to the pharmacodynamics of alfentanil was investigated in 15 female and 13 male patients, aged 21-85 yr, ASA physical status 1 or 2, undergoing upper abdominal surgery. All patients had normal cardiac, hepatic, renal, and pulmonary function. None was receiving medication or had a history of alcohol or other drug abuse. Anesthesia was induced and maintained with 66% nitrous oxide in oxygen and alfentanil. Alfentanil was administered by a computer-controlled infusion pump. If, during surgery, the patient exhibited signs of inadequate anesthesia (i.e., response), the target alfentanil plasma concentration was increased by 50-100 ng/ml. If there was no response during a 15-min period, the target concentration was decreased by 50-100 ng/ml. Arterial blood samples were taken before any change of the target concentration and 4 min after the computer had indicated that the new target concentration had been reached. In addition, blood samples were taken before intubation, skin incision, and in the patients in whom ventilation recovered spontaneously before extubation. In the remaining patients a blood sample was taken before the administration of naloxone. Plasma alfentanil concentrations were determined by capillary gas chromatography. Alfentanil protein binding was determined by equilibrium dialysis in an arterial blood sample taken before induction of anesthesia. Alfentanil concentration-effect data were evaluated by logistic regression, where effect was either response or no response to perioperative stimuli. The average free fraction of alfentanil was 9.3 +/- 3.9% (range 3.7-19.1%). For intubation, skin incision, and postanesthesia ventilation, it was not possible to characterize the concentration-effect curves based on total plasma concentrations with logistic regression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729939 TI - Effects of alfentanil on intracranial pressure in children undergoing ventriculoperitoneal shunt revision. AB - The effects of alfentanil on intracranial pressure in patients with diminished intracranial compliance has not been established. Ten patients with hydrocephalus of varying etiologies, ages 16 months to 20 yr, presenting for ventriculoperitoneal shunt revision were studied. Following induction of anesthesia with thiopental, nitrous oxide/oxygen, and isoflurane, the trachea was intubated and anesthesia was maintained with isoflurane (0.5%), nitrous oxide (70%), and oxygen. After a minimum of 30 min and after the new shunt was placed, alfentanil was administered in increments of 10, 20, and 40 micrograms/kg at 3 min intervals, and intracranial pressure was measured over 12 min via the new shunt. In these unstimulated, normocapnic (PETCO2 32-38 mmHg) patients, heart rate, mean arterial pressure, and cerebral perfusion pressure declined from 110 +/- 26 beats/min, 90 +/- 11 mmHg, and 71 +/- 14 mmHg, to 84 +/- 25 beats/min, 66 +/- 11 mmHg, and 45 +/- 16 mmHg (mean +/- SD), respectively, by 3 min after the third dose (P less than 0.001). Intracranial pressure did not change from baseline (19 +/- 14 mmHg vs. 21 +/- 11) after any dose of alfentanil. Contrary to earlier studies in adult patients with brain tumors, the authors found that alfentanil, in pediatric patients with hydrocephalus anesthetized with oxygen, nitrous oxide, and isoflurane, did not increase intracranial pressure within a 9 min study period. The significant decreases in cerebral perfusion pressure observed merit concern and further study. PMID- 1729940 TI - Brain bioenergetics during cardiopulmonary resuscitation in dogs. AB - Cardiac arrest causes a rapid loss of cerebral adenosine triphosphate [corrected] (ATP) and a decrease in cerebral intracellular pH (pHi). Depending on the efficacy of cardiopulmonary resuscitation (CPR), cerebral blood flow levels (CBF) ranging from near zero to near normal have been reported experimentally. Using 31P magnetic resonance spectroscopy, the authors tested whether experimental CPR with normal levels of cerebral blood flow can rapidly restore cerebral ATP and pHi despite the progressive systemic acidemia associated with CPR. After 6 min of ventricular fibrillation in six dogs anesthetized with fentanyl and pentobarbital, ATP was reduced to undetectable concentrations and pHi decreased from 7.11 +/- 0.02 to 6.28 +/- 0.09 (+/- SE) as measured by 31P magnetic resonance spectroscopy. Application of cyclic chest compression by an inflatable vest placed around the thorax and infusion of epinephrine (40 micrograms/kg bolus plus 8 micrograms/kg/min, intravenously) maintained cerebral perfusion pressure greater than 70 mmHg for 50 min with the dog remaining in the magnet. Prearrest cerebral blood flows were generated. Cerebral pHi recovered to 7.03 +/- 0.03 by 35 min of CPR, whereas arterial pH decreased from 7.41 +/- 0.4 to 7.08 +/- 0.04 and cerebral venous pH decreased from 7.29 +/- 0.03 to 7.01 +/- 0.04. Cerebral ATP levels recovered to 86 +/- 7% (+/- SE) of prearrest concentration by 6 min of CPR. There was no further recovery of ATP, which remained significantly less than control. Therefore, in contrast to hyperemic reperfusion with spontaneous circulation and full ATP recovery, experimental CPR may not be able to restore ATP completely after 6 min of global ischemia despite restoration of CBF and brain pHi to prearrest levels. PMID- 1729941 TI - The effects of sevoflurane, halothane, enflurane, and isoflurane on hepatic blood flow and oxygenation in chronically instrumented greyhound dogs. AB - Inhalational anesthetics produce differential effects on hepatic blood flow and oxygenation that may impact hepatocellular function and drug clearance. In this investigation, the effects of sevoflurane on hepatic blood flow and oxygenation were compared with those of enflurane, halothane, and isoflurane in ten chronically instrumented greyhound dogs. Each dog randomly received enflurane, halothane, isoflurane, and sevoflurane, each at 1.0, 1.5, and 2.0 MAC concentrations. Mean arterial blood pressure and cardiac output decreased in a dose-dependent fashion during all four anesthetics studied. Heart rate increased compared to control during enflurane, isoflurane, and sevoflurane anesthesia and did not change during halothane anesthesia. Hepatic arterial blood flow and portal venous blood flow were measured by chronically implanted electromagnetic flow probes. Hepatic O2 delivery and consumption were calculated after hepatic arterial, portal venous, and hepatic venous blood gas analysis. Hepatic arterial blood flow was maintained with sevoflurane and isoflurane. Halothane and enflurane reduced hepatic arterial blood flow during all anesthetic levels compared to control (P less than 0.05), with marked reductions occurring with 1.5 and 2.0 MAC halothane concomitant with an increase in hepatic arterial vascular resistance. Portal venous blood flow was reduced with isoflurane and sevoflurane at 1.5 and 2.0 MAC. A somewhat greater reduction in portal venous blood flow occurred during 2.0 MAC sevoflurane (P less than 0.05 compared to control and 1.0 MAC values for sevoflurane). Enflurane reduced portal venous blood flow at 1.0, 1.5, and 2.0 MAC compared to control. Halothane produced the greatest reduction in portal venous blood flow (P less than 0.05 compared to sevoflurane).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729942 TI - Antinociceptive synergy between intrathecal morphine and lidocaine during visceral and somatic nociception in the rat. AB - Clinical investigations have suggested a synergistic interaction between the analgesic effects of intrathecal opioids and local anesthetics; however, basic pharmacologic evidence for this observation has not been reported. Therefore, the authors have used models of visceral and somatic nociception to quantify the interaction between intrathecal morphine and lidocaine in a crossover study of 24 rats in four equal groups. Combinations of morphine and lidocaine were administered separately, corresponding to time of peak effect for each drug. Colorectal distention, as a noxious visceral stimulus, was applied to two groups while cardiovascular and visceromotor responses, respectively, were recorded. A third group received hot plate testing as a somatic nociceptive stimulus. Intrathecal morphine and lidocaine both attenuated the cardiovascular and visceromotor responses to colorectal distention and increased hot plate latencies in a dose- and time-dependent manner. With the use of isobolographic analysis, the coadministration of morphine and lidocaine demonstrated a synergistic, supraadditive interaction during visceral nociception (P less than 0.001) and somatic nociception (P less than 0.005). In a fourth group, motor function was evaluated by an inclined screen method. Intrathecal lidocaine in the dosage range tested during isobolographic analysis revealed no motor deficits. These data clearly demonstrate antinociceptive synergy between intrathecal morphine and lidocaine during visceral and somatic nociception at dosages that do not impair motor function. PMID- 1729943 TI - ANA promotes health care reform. PMID- 1729944 TI - OSHA releases final standard on HIV, hepatitis B exposures. PMID- 1729945 TI - Nurses win with statewide strategy. PMID- 1729946 TI - Hatch Act limits some actions. PMID- 1729948 TI - VA commission makes recommendations for veterans health care. PMID- 1729947 TI - New Medicare fee schedule will affect many nurses. PMID- 1729950 TI - School nursing demands creativity. PMID- 1729949 TI - Bill would increase reimbursement for advanced practice nurses. PMID- 1729951 TI - Gaining political skills is easy. As I see it. PMID- 1729952 TI - Get involved in the 1992 elections. PMID- 1729953 TI - Uncharted waters of managed care. PMID- 1729954 TI - Health care reform is a priority. PMID- 1729955 TI - Bills would benefit advanced practice RNs. PMID- 1729956 TI - Nurses to educate for end-of-life decisions. PMID- 1729957 TI - [A case of recurrent endometrial cancer successfully treated with oral administration of etoposide]. AB - A 61-year-old female with recurrent endometrial cancer (serous papillary adenocarcinoma) was treated with etoposide because the pelvic tumor progressively increased in size with external beam irradiation. The etoposide (25 mg/day) was given orally for 10 days; the tumor decreased in size. And after an additional two courses of etoposide for 8 and 4 days, respectively, the tumor disappeared and the serum CA 125 level came to within normal limits. Because of moderate nausea and vomiting the etoposide could not be given for 14 days in the first 3 courses. Myelosuppression was not evident. Ten courses of etoposide (for 14 consecutive days a month) were followed without gastro-intestinal side effects, and the patient is alive with no evidence of recurrence at this writing. This case suggests that oral administration of etoposide may be effective for a patient with recurrent endometrial cancer, and this treatment could be administered on an outpatient basis. PMID- 1729958 TI - [Clinical pharmacology of anticancer agents (Part 3). Plant alkaloids]. PMID- 1729959 TI - [An overview of new prognostic factors for ovarian cancer]. AB - One of the most characteristic features of epithelial ovarian cancer is its inherent heterogeneity with respect to biological behavior ranging from the relatively indolent nature of borderline tumors to highly aggressive malignant diseases. This can be reflected in a wide variety of prognostic factors, among which the stage of disease is by far the most important, followed by histologic subtype, histologic grade, volume of residual disease, age at diagnosis, and performance status (PS). Within a given stage, histologic grade is the most powerful prognostic factor in stage I disease, followed by dense adherence and large volume of ascites. On the other hand, the main prognostic factor in more advanced stage disease (II-IV) includes an effect of chemotherapy and a volume of residuum, and PS. However, these factors alone cannot always predict patients' survival correctly. A number of new and potentially valuable prognostic factors have emerged as a result of the recent technical advances in molecular genetics, flow cytometric analysis, development of monoclonal antibody, immunohistochemical study, and steroid hormone receptor analysis. This article reviews some of these newly developed "investigational" prognostic variables, as well as the "widely accepted" clinicopathological prognostic factors. PMID- 1729960 TI - [A study of combination chemotherapy (BHAC-ACVP) for adult acute lymphocytic leukemia. Hanshin Co-operative Study Group of Hematological Malignancies]. AB - Twenty-nine adult patients with acute lymphocytic leukemia (ALL) were treated with combination chemotherapy consisting of behenoyl-ara-C, adriamycin, cyclophosphamide, vindesine and prednisolone (BHAC-ACVP regimen). Complete remission (CR) was obtained in 7 of 13 (54%) of the previously untreated, and 4 of 16 (25%) of the previously treated patients. Six of 10 (60%) L1 and 5 of 17 (29%) L2 patients achieved CR. Side effects such as nausea, GPT elevation and fever were observed, but these were not severe in most cases. The result indicates that BH-AC is useful for the treatment of adult patients with ALL. PMID- 1729961 TI - [New factors of possible prognostic value in breast cancer]. AB - Over the past 10 years, we have witnessed a variety of potential prognostic factors of breast cancer including proliferative rate, ploidy, growth factor receptors, oncogenes and cathepsin D production. Some of these variables seem to predict the prognosis of the patients but the available data are conflicting and call for carefully conducted quality-control studies to analyze intra- and interlaboratory variations. In this review, we provided a framework from which prognostic factor information can be used directly to make treatment decisions. PMID- 1729962 TI - [Experimental study of protective effect of solcoseryl on cisplatin nephrotoxicity]. AB - Cis-diamminedichloroplatinum (II) (cisplatin) is known to possess nephrotoxicity. Recently, it is said that the nephrotoxicity closely correlated with active oxygen. In the present study, an attempt was made to examine, whether or not solcoseryl, antioxidant like scavenger, decreases the nephrotoxicity. Three groups of Sprague-dawley rats were injected cisplatin (3mg/kg) only (C group), cisplatin and 2 times solcoseryl (8mg/kg), (CS2 group), and cisplatin and 5 times solcoseryl (CS5 group). BUN levels in C and CS2 groups were elevated compared to CS5 group. In the light microscopy, 62.5% to 100% in C and CS2 groups revealed massive necrosis in straight part of the proximal tubules, while the damage in CS5 group was only 25%. In the electron microscopy, these findings were similar to the light microscopic observations. Solcoseryl is known to have several antioxidative effects such as inhibition of superoxide producing and hyperperoxidation. It is suggested that these effects of solcoseryl might decrease the nephrotoxicity. Solcoseryl seems to be a useful drug for reducing nephrotoxicity and also it is a safe drug. Therefore, solcoseryl can be used for the prevention of cisplatin nephrotoxicity. PMID- 1729963 TI - [Comparison between clinical response and in vitro chemosensitivity of solid tumors in the succinic dehydrogenase inhibition test]. AB - We describe our experience with succinic dehydrogenase inhibition (SDI) test for solid tumors as a chemosensitivity test using 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. Specimens were obtained from 76 surgical resected tumors, including 32 colon cancers, 24 stomach cancers and 16 lung cancers. Following enzymatic dissociation of scissors-minced tumors, viable cells were cultured in serum free medium (S-Clone SF-B) for 4 days with eight drug concentrations obtained by 2-fold dilution of drugs. Among 76 specimens tested, 48 specimens including 15 colon cancers, 18 stomach cancers and 15 lung cancers were successfully evaluated. For the purpose of judgement, 50% inhibitory concentration (IC50) was calculated in each case. Tumor specimen was regarded as sensitive to a given agent when the IC50 value was the same or smaller than the cut-off concentrations (1 microgram/ml for mitomycin C, 5 micrograms/ml for cisplatin, 2 micrograms/ml for adriamycin and 50 micrograms/ml for 5 fluorouracil), and was regarded as resistant when it was larger than these levels. In vitro vs in vivo drug sensitivity was successfully evaluated in 23 cases. The overall predicting accuracy rate was 78% (18/23), with one true positive, 5 false positive and 17 true negative cases. This test appeared to be useful to tailor effective agents for patients because of its relatively high successful and predictive rates. PMID- 1729964 TI - Quadriceps muscle activity in women runners with and without patellofemoral pain syndrome. AB - The purpose of this study was to determine whether there was a difference in the electromyographic (EMG) patterns of the quadriceps muscles in women runners diagnosed with patellofemoral pain syndrome (PFPS) compared to the quadriceps activity of women runners free of knee pain and with normal lower extremity alignment. Linear envelope EMGs from vastus medialis, vastus lateralis, and rectus femoris, together with a footswitch signal, were recorded as each subject ran on a treadmill at 80% of their normal running pace and at 12km/h. Each stride period was normalized to 100%, then the linear envelopes for ten trials were ensemble averaged to achieve a mean ensemble for each muscle from each subject. Subsequently, the ensembles for each subject were normalized by dividing by the maximum EMG per cycle; they were then averaged across subjects to obtain the grand mean ensembles of each muscle for each group. Comparisons between the experimental and control groups at both speeds showed that nowhere during the stride cycle did the mean EMG levels of the two groups differ by more than two standard deviations. It was concluded that any changes in the running pattern of the runners with patellofemoral pain syndrome could not be detected by changes in the EMG patterns. PMID- 1729965 TI - The role of allergen immunotherapy in the respiratory complications of quadriplegia. AB - Patients with traumatic quadriplegia have frequent respiratory complications. These complications may be exacerbated by the presence of common and previously well-tolerated allergic disease. Quadriplegic patients are limited in their ability to cope with upper and lower respiratory allergies by a reduced vital capacity and impairment of maneuvers needed to keep the upper and lower respiratory tracts clear. This report describes two patients with traumatic quadriplegia who were treated with allergen immunotherapy. After immunotherapy, both patients had negligible long-term postinjury respiratory complications and an improved quality of life. Allergen immunotherapy should be considered early in the management of allergic patients with traumatic quadriplegia. PMID- 1729966 TI - Krusen Award recipient. Robert C. Darling. PMID- 1729967 TI - Reliability of knee flexor peak torque measurements from a standardized test protocol on a Kin/Com dynamometer. AB - The test-retest reliability of a specific test protocol for the measurement of peak torque of the knee flexors using a Kin/Com dynamometer was evaluated. The maximum voluntary torque generated by the left knee flexors during constant velocity resisted-muscle shortening (RMS) and muscle lengthening (RML) was measured in a sitting position in 11 healthy women with no history of knee pathology. Each subject performed two tests at each of two velocities (30 degrees/sec and 180 degrees/sec) in a single session. All subjects repeated these four tests one week later. A test consisted of four complete RMS/RML cycles through a range of 65 degrees. The peak torque generated from each test was used to measure test-retest reliability. All data were gravity compensated. Intraclass correlation coefficients (ICCs) were calculated from ANOVA tests for RML and RMS at both velocities. The within sessions ICCs ranged from .94 to .98 for 30 degrees/sec and from .92 to .97 at 180 degrees/sec. The ICCs between sessions were generally lower and ranged from .79 to .90 for 30 degrees/sec, and from .75 to .88 for 180 degrees/sec. It is concluded that using these test protocols, peak torques for both RMS and RML can be measured with a high degree of reliability at two commonly used velocities. PMID- 1729968 TI - Skeletal muscle function in patients with hemophilia A and unilateral hemarthrosis of the knee. AB - Acute hemarthrosis is a frequent complication of hemophilia A. To test the hypothesis that recurrent hemarthrosis has an adverse effect on neuromuscular function, ten patients with hemophilia A and history of unilateral hemarthrosis of the knee were studied. The uninvolved (U) side was used as the control. The time since diagnosis, factor level, and the number of bleeding episodes in the involved (I) knee averaged 11.6 +/- 6.6 years, 7.7 +/- 5.1%, and 7.0 +/- 7.4, respectively. Neuromuscular function was evaluated on a Cybex 340 isokinetic dynamometer. Knee extensor strength measured at 60 degrees/sec was significantly (p less than .05) lower in the I side (mean = 58Nm) than in the U side (mean = 85Nm). Similarly, lower (p less than .05) total work (I = 479J, U = 656J) and average power output (I = 59W, U = 83W) values were obtained. Adjusting for thigh circumference did not eliminate any of these differences. Testing the extensors at faster angular velocities (180 degrees/sec and 240 degrees/sec) and the flexor muscle groups at the three speeds revealed no differences between I and U. X-rays showed minimal changes. These data show that patients with hemophilia A and history of unilateral hemarthrosis of the knee have neuromuscular dysfunction in the I extremity that precedes the appearance of radiologic evidence of joint pathology. It is suggested that strength training should be started early in the rehabilitation of hemophiliacs. PMID- 1729969 TI - Osteoarthritis of the knee: effects on gait, strength, and flexibility. AB - This study examined the differences in gait mechanics, isokinetic knee strength, and flexibility between a group of adults with symptomatic osteoarthritis (OA) of the knee (n = 15) and an age-, mass-, and gender-matched group of control subjects (n = 15). Both groups performed under similar environmental conditions. Our results suggest that patients with symptomatic OA of the knee have poorer flexibility in both the affected and unaffected legs and demonstrate significantly less (p less than .05) knee angular velocity and, to a lesser extent, knee range of motion during gait. They have an increased loading rate in the unaffected leg after heel strike, exert less peak vertical force during pushoff, and are significantly weaker in both the dominant and nondominant legs compared to adults with no lower extremity disease. PMID- 1729970 TI - Correlation of creatine kinase and gait measurement in the postpolio population. AB - Measurements of stride length, gait speed, and distance walked during seven days were obtained from 15 postpolio and eight control subjects. Pedometers were used to measure distance walked. Measurements of stride length and speed were performed three times, and there was a high correlation between tests (R = .852 .969). The pedometers failed to record accurately in some postpolio subjects, and these subjects were dropped from analysis when ambulation distance was used as a variable. There were significant differences between the postpolio subjects and controls with respect to gait speed (47.7 +/- 14.0 vs 74.9 +/- 15.9m/min, p less than 0.0005), stride length (55.3 +/- 11.7 vs 69.8 +/- 8.6cm, p = .006), and average kilometers walked per day for seven days (1.97 +/- 1.3 vs 3.89 +/- 1.7, p = .016). The postpolio subjects had their serum creatine kinase (CK) levels measured at the end of the study. Forty percent of subjects had a level above the normal limits of our laboratory. There was a significantly positive correlation between CK levels and the distance walked during the previous 24 hours (R = .75, p = .012). The findings of this study illustrate the impact of gait abnormalities on the ambulatory abilities of the postpolio population. The correlation of CK with ambulation supports the association of exercise as a source of elevated CK levels in the postpolio population. PMID- 1729971 TI - Objective assessment of spasticity, strength, and function with early exhibition of dantrolene sodium after cerebrovascular accident: a randomized double-blind study. AB - A double-blind, placebo-controlled trial was conducted to determine whether early exhibition of Dantrium (Dantrolene Sodium) in patients with cerebrovascular accidents, before the onset of significant spasticity, would enhance the functional outcome of rehabilitation. Thirty-eight patients were enrolled in the trial and 31 satisfactorily completed the study. A modified Cybex isokinetic dynamometer was used to gather information on strength and muscle tone. Clinical, functional, and biochemical data were also collected. It was found that Dantrium reduced strength in the unaffected limbs but did not alter strength in the paretic limbs. Dantrium produced no alteration in clinical tone, functional outcome, or biochemical tests at the dosage (200 mg per day) used in this study. PMID- 1729972 TI - Recovery of zero-grade muscles in the zone of partial preservation in motor complete quadriplegia. AB - This prospective study was designed to demonstrate root level recovery in the zone of preservation by examining muscles with an initial strength of grade 0/5 in 32 motor complete (Frankel A and B) patients who had cervical spinal cord injury at the C4 through C7 levels. The biceps (C5), extensor carpi radialis (C6), triceps (C7), and flexor digitorum profundus (C8) muscles were used as key muscles when their strength at the initial manual muscle test, which was performed between three and seven days postinjury, was grade 0/5 and the muscle innervated by the cord segment directly rostral to that innervating the key muscle (grade 0/5) was grade greater than or equal to 3/5. If the biceps muscle was used, C4 pin sensation was required to be normal. Further manual muscle tests were performed weekly for four weeks, and then at 2, 3, 6, and 12 months postinjury. None, 6%, 17%, 35%, and 43% of the patients recovered to grade greater than or equal to 3/5 by 1, 2, 3, 6, and 12 months postinjury, respectively. It was also noted that in 86% of our patients, improving to grade greater than or equal to 1/5 by one month postinjury was a predictor of recovering to grade greater than or equal to 3/5 by 12 months postinjury (p less than .002). Similarly, in 100% of our patients, improving to grade greater than or equal to 2/5 by three months postinjury was a predictor of recovering to grade greater than or equal to 3/5 by 12 months postinjury (p less than .001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1729973 TI - Upper extremity pain in the postrehabilitation spinal cord injured patient. AB - The purpose of this study was to determine the prevalence of upper extremity (UE) pain in outpatients with chronic spinal cord injury (SCI). A total of 239 SCI outpatients (136 with quadriplegia and 103 with paraplegia) were interviewed for the presence of UE pain at the shoulder, elbow, wrist, and hand. The average age of the subjects at the time of interview was 37.4 years, and the average time since onset was 12.1 years. Subjects who reported pain were referred to SCI clinics to determine the etiology. Fifty-five percent of the patients with quadriplegia reported UE pain, most commonly at the shoulder. Prevalence of reported pain was highest for subjects in the first five years postinjury. Sixty four percent of patients with paraplegia reported UE pain. Complaints related to carpal tunnel syndrome were the most common, followed by those related to shoulder pain. This study documents the prevalence and nature of UE pain in chronic SCI patients and emphasizes the need for further research to develop strategies for prevention and treatment of pain syndromes. PMID- 1729974 TI - Cardiorespiratory responses to upper extremity aerobic training by postpolio subjects. AB - The cardiorespiratory responses of ten postpolio subjects participating in a 16 week upper extremity aerobic exercise program were compared to ten non-exercised controls. The subjects trained three times a week for 20 minutes per session. Exercise intensity was prescribed at 70% to 75% of heart rate reserve plus resting heart rate. Dependent variables were resting heart rate, maximal heart rate, resting and immediate-post-exercise systolic and diastolic blood pressures, maximal oxygen consumption, maximal carbon dioxide production, minute ventilation, respiratory exchange ratio, power, and exercise time. After training, the exercise group was superior to the control group in oxygen consumption, carbon dioxide production, minute ventilation, power, and exercise time. There was no reported loss of muscle strength. It was concluded that postpolio subjects can safely achieve an increase in aerobic capacity with a properly modified upper extremity exercise program. This improvement is comparable to that demonstrated by able-bodied adults. PMID- 1729975 TI - Relation between clinical and instrumented measures of motor coordination in traumatically brain injured persons. AB - Right and left upper extremity motor coordination was evaluated in 40 traumatically brain injured subjects using two different measurement techniques- a conventional clinical evaluation and an instrumented evaluation. The relationship between the results from the clinical evaluation (finger to nose test) and the instrumented evaluation (timed visual-arm coordinated lateral reach) was determined using canonical correlation analysis (Rc). Clinical and instrumented test scores were significantly correlated (Rc = .685, p = .002; Rc = .629, p = .008 for the right and left sides, respectively). The scores obtained by the clinical test had little variance; instrumented scores varied greatly among subjects. Significant differences did not exist between the right and left sides for the instrumented variables of average movement speed, accuracy, and index of coordination (paired t-tests, p = .550, p = .548, p = .627, respectively) or for the clinical variables of time of execution (paired t-test, p = .468) and tremor (Wilcoxon matched-pairs, p = .228). Although meaningful correlations were obtained, they do not conclusively indicate that the two tests measure the same concept of coordination. Therefore, it is suggested that the instrumented evaluation be used to complement the traditional clinical evaluation to increase the degree of objectivity in clinical measurement. PMID- 1729976 TI - Development and clinical evaluation of a computerized limb volume measurement system (CLEMS). AB - Limb edema is a common problem in both rehabilitation and acute care settings. In the past, attempts to determine an optimal management strategy for limb edema have been limited by the lack of accurate, noninvasive, rapid, clinical tools for quantifying limb volumes. The water displacement method is slow and difficult to use in the clinical setting. Furthermore, water displacement requires that the limb be in a dependent position. The tape measure method is unreliable because it is difficult to position the tape measure on a swollen limb. The development and evaluation of a new tool called the computerized limb volume measurement system (CLEMS) is described. The shape and volume of a limb or limb segment can be rapidly measured by CLEMS, independent of limb position. The limb volumes generated by CLEMS were compared to volumes determined by water displacement and by a tape measure. Volumes of eighteen legs (plaster, nonedematous and edematous) were measured using CLEMS, water displacement, and the tape measure. In all cases, the CLEMS and water displacement methods showed close agreement. CLEMS was found to be a reliable and valid new method of determining limb volume; whereas, the tape measure method was found to be invalid. This new tool allows clinicians to measure the efficacy of different treatment strategies in the management of limb edema. PMID- 1729977 TI - Across-tarsal-tunnel motor-nerve conduction technique. AB - Tarsal tunnel syndrome is a commonly considered compression of the tibial nerve and its plantar divisions as the nerve curves behind the medial malleolus underneath the flexor retinaculum. Motor, sensory, and/or mixed-nerve conduction studies are used to confirm or exclude the presence of compression of the posterior tibial nerve and its plantar divisions. In previous studies, stimulation has been done either proximal to the tunnel or distally in the sole of the feet or in the toes. Thus, differentiation between compression of the nerve within the proximal tarsal tunnel, as distinguished from compression of the plantar nerves in the distal tarsal tunnel or distal to the tunnel, has not been feasible. In addition, onset latency is frequently difficult to measure, and peak latencies have not been reported for the motor-evoked action potential. This study reports across-tarsal-tunnel latencies and amplitude decrements for both the medial and the lateral plantar nerves. For the medial plantar nerve with active electrodes placed over the medial head of the flexor pollicis brevis, the calculated mean + 2SD across tunnel onset latency is 3.2msec, peak latency is 2.9msec, and amplitude decrement is 29.3%. For the lateral plantar division, the calculated across-tunnel onset latency is 3.2msec, peak latency is 2.9msec, and amplitude decrement is 27.2%. Medial plantar nerve latency distal to the tarsal tunnel for the mean + 2SD is 5.9msec to onset and 9.5msec to peak, and the lateral plantar nerve latency is onset 5.9msec and peak 9.7msec. PMID- 1729978 TI - Meralgia paresthetica: the diagnostic value of somatosensory evoked potentials. AB - Somatosensory evoked potentials elicited by stimulation of the lateral femoral cutaneous nerve were investigated in 20 able-bodied persons and 22 patients diagnosed clinically to have meralgia paresthetica. There was no statistically significant difference between the right and left sides for P0 and N1 latencies in able-bodies subjects. For all patients, abnormalities were found on the side clinically affected. The mean values were 38.11 msec for P0 and 47.49msec for N1 on the affected side and 32.62msec and 41.44msec on the unaffected side (p less than .01). The mean latency differences between the two sides P0 and N1 were 5.49msec and 6.05msec, respectively (p less than .01). Hence, this technique proved to be a useful objective diagnostic aid in meralgia paresthetica. PMID- 1729979 TI - Influence of caster diameter on the static and dynamic forward stability of occupied wheelchairs. AB - The hypothesis that the static and dynamic forward stability of an occupied wheelchair would increase as a function of the caster diameter was tested in 20 able-bodied subjects. A device was attached to the wheelchair frame so that casters with different diameters could be used. With platform testing of static stability, the occupied wheelchair equipped with casters with diameters of 10.2, 20.3, 25.4, and 33.0cm, tipped at mean (+/- 1SD) angles of 23.8 degrees (+/- 1.3 degrees), 25.2 degrees (+/- 1.4 degrees), 26.1 degrees (+/- 1.4 degrees), and 28.2 degrees (+/- 1.9 degrees), respectively. The relationship between static stability (y, in degrees) and caster diameter (x, in cm) can be expressed by the equation y = 23.5-.0196 chi + .00484 x2 (p = .0001). Dynamic stability was tested by having subjects descend a 5 degree ramp, by gravity alone, from progressively farther up the ramp until a full forward tip occurred (footrests contacted the floor) when the wheelchair struck a 5-cm-high obstruction with sufficient speed. The mean tipping speeds for the dynamic tests were .85 (+/- .08), .85 (+/- .08), .89 (+/-.07), and 1.04 (+/- .14) m/sec for the wheelchair fitted with the caster diameters ranging from smallest to largest, respectively. The relationship between dynamic stability (y, tipping speed in m/sec) and caster diameter (chi, in cm) can be expressed by the equation y = .788 + .0139 chi-.00110 chi 2 + .0000276 chi 3 (p = .0001). The relationship between caster diameter and forward wheelchair stability should be considered in wheelchair design and prescription. PMID- 1729980 TI - Neuromuscular stimulation in spinal cord injury: I: Restoration of functional movement of the extremities. AB - The spinal cord injured patient has been the focus of clinical and research efforts to restore functional movement and obtain therapeutic benefits by electric stimulation of upper-motor-neuron paralyzed muscles. Our review articles treat developments in this field from 1983 to 1990. Efforts have been directed to restoring hand function, standing, and walking (covered in part I), as well as prevention of secondary complications through ventilatory function, bladder function, and achieving therapeutic effects of electric stimulation (covered in Part II). The technology for hand function, standing, and walking is used primarily in the research laboratory, as clinical applications are minimal. Much work remains to be done to solve the difficult problems associated with applying this promising technology to spinal cord injury. PMID- 1729981 TI - Silent cardiac ischemia in cervical spinal cord injury: case study. AB - The management of ischemic heart disease in patients with chronic spinal cord injury (SCI) will become an increasingly major concern as this population ages. Although silent ischemia has become an important topic in the medical literature, the relationship with cervical SCI has not been adequately explored. A literature search revealed no case reports of documented asymptomatic cardiac ischemia in SCI patients. This is a case report of a 65-year-old patient with chronic C7 incomplete SCI who had multiple risk factors for coronary artery disease and an abnormal electrocardiogram. Despite being completely asymptomatic, the patient was found to have significant myocardial ischemia induced by minimal stress using atrial paced thallium scintigraphy. This finding led to the cancellation of an elective surgical procedure. This case illustrates the importance of suspecting silent myocardial ischemia in cervical SCI patients. PMID- 1729982 TI - Management of apraxic gait in a stroke patient. AB - There is little information available regarding management of apraxic gait. We present a 61-year-old man with a five-year history of right-sided cerebrovascular accident, apraxic gait, difficulty in walking, and frequent falls. A CT head scan revealed moderate cerebral atrophy, a small lacunar infarction. The patient was unable to initiate walking, was bed ridden and housebound. Traditional gait training and balance exercises failed to improve his gait. Two straight canes were modified by fixing florescent horizontal projections approximately two inches up from the tip of the cane. The patient was instructed to step over the horizontal projected portion, making use of visual cues from the florescent painted projections. The patient became independent with safe ambulation after practicing for approximately three weeks and was discharged home. PMID- 1729983 TI - The Lambert-Eaton myasthenic syndrome: a cause of delayed recovery from general anesthesia. AB - A 70-year-old man required prolonged ventilation after surgery to remove a rectal neoplasm. The cause of the slow recovery from the effects of neuromuscular blocking agents used during his anesthetic was the Lambert-Eaton myasthenic syndrome (LEMS). Before surgery, he had no neuromuscular symptoms, even in retrospect. LEMS should be considered in the diagnosis of prolonged recovery from neuromuscular blockade, even in previously asymptomatic patients. PMID- 1729984 TI - Protective effect of serum antibody on respiratory infection of influenza C virus in rats. AB - The effects of serum antibody on the replication of influenza C virus in the nose and lung were evaluated in rats challenged with the virus by the intranasal and endotracheal routes, respectively. Convalescent rat serum administered intraperitoneally prior to infection suppressed virus replication significantly in both the nasal and pulmonary tissues. Resistance achieved was however much greater in the lung than in the nose. Rats with a serum neutralizing antibody titer of 1:800 showed almost complete resistance to pulmonary virus infection, and virus yield from the lung was reduced 10- to 100-fold in animals with the antibody titer of 1:80-160 or less. In contrast, significant decrease in virus shedding from the nose was observed only in animals with a serum antibody titer of 1:800 or greater. The effect of adoptive transfer of monoclonal antibodies (MAbs) to haemagglutinin-esterase (HE) glycoprotein and matrix (M) protein on pulmonary virus replication was also examined. Anti-HE MAbs with neutralization activity prevented virus shedding from the lung almost completely whereas non neutralizing anti-HE MAb and anti-M Mab showed no inhibitory effect. PMID- 1729985 TI - Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells. AB - The structure of the N-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo-beta-N acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography. The results demonstrated that the glycoproteins decorating the virus shed from infected cells have N-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells. However, the E1 glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminal N acetylglucosamine. The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at different N-glycosylation sites as in the case of E2, or at the same N glycosylation site as in the case of E1. In the presence of 1 deoxymannojirimycin, no structural heterogeneities in the glycan chains were found. This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes. PMID- 1729986 TI - Processing of dengue virus type 2 structural proteins containing deletions in hydrophobic domains. AB - The 5' end of the genome of the dengue virus type 2 encoding the structural proteins was expressed using recombinant vaccinia virus. Three additional recombinants derived by deletion of selected dengue sequences within the parental construct were also expressed. They were designed to assess the role of hydrophobic domains in the processing of the viral polyprotein in intact cells. The first construct contained a deletion of nucleotides encoding most of the C protein; nucleotides encoding the hydrophobic domain at the carboxy terminus were retained. The second and third constructs contained smaller deletions of 72 bp and 129 bp encoding hydrophobic domains at the carboxy termini of C and prM respectively. Indirect immunofluorescence and radioimmunoprecipitation were used to detect prM and E in cells infected with recombinant viruses. The results showed that deletion of 90% of C had no apparent effect on the processing of prM and E, and that the signal sequence for E at the carboxy terminus of prM was active in the absence of the upstream signal sequence for prM at the carboxy terminus of C. Deletion of the hydrophobic sequences preceding the amino terminus of E prevented cleavage at the prM-E junction. These results obtained using infected cells were consistent with the published findings for the translation of flavivirus RNA in vitro, and indicated the importance of membrane association in the cleavage of structural proteins from the flavivirus polyprotein. In addition, cells infected with the recombinant virus containing the large deletion in the C coding region released the E glycoprotein into the culture medium. PMID- 1729987 TI - Replication complexes associated with the morphogenesis of rubella virus. AB - Thin section electron microscopy was used to investigate cellular changes associated with the replication of rubella virus (RV) in Vero cells and to compare these changes to those of the related alphavirus, Semliki Forest virus (SFV). Conspicuous membrane-bound cytoplasmic vacuoles analogous to the alphavirus replication complexes were observed in RV infected cells but not in mock infected cells. The vacuoles were characterised by membrane-bound vesicles measuring about 60 nm which often displayed an irregular dense core and/or a network of fibres. These vesicles were morphologically distinct from RV particles and were generally located at regular intervals on the inner side of the surrounding membrane of the RV replication complex. Degenerating cellular material was often found in the membrane-bound vacuole of a replication complex. The replication complexes were intimately associated with the rough endoplasmic reticulum (RER), which was localised 45-75 nm from the surrounding membrane of the replication complex. Parallel studies of replication complexes in SFV infected cells did not reveal such an intimate association with the RER. RV replication complexes appeared as early as 8 h post infection (p.i.), before detection of RV particles by electron microscopy, and their peak production at 24 h p.i. coincided with the time of maximum virus titre. PMID- 1729988 TI - The health care system in the year 2000: three scenarios. AB - Because no one can predict the direction that national health policy might take over the next decade, the author outlines three of the more likely scenarios for the year 2000. He first describes seven major forces that have a stake in national health policy: the federal government, the state governments, business, organized labor, the public at large, the health industry, and the opinion makers. he next indicates three ways these forces might come together (the scenarios), and last, gives a fuller description of the scenarios and the issues each would raise. He demonstrates in depth how the motivations of most of these forces are in conflict with one another and states that no national health policy is likely to emerge until there is a broad compromise that brings most of those conflicts to some sort of viable consensus. He urges those involved in health care to prepare for a range of possible policies such as, but not limited to, those outlined in this overview, and concludes by maintaining that no matter what health policy emerges, health professionals will use their common sense and good faith to make it work, as they always have. PMID- 1729989 TI - Computer databases of medical school curricula. AB - As the pace of curriculum reform in medical education has accelerated during the past decade, so too have demands on curriculum managers to supply increasingly detailed information about the curriculum. In response, a number of schools have joined together to begin work on designs for computer databases of the curriculum. The authors describe three of the most mature curriculum database prototypes, developed by groups at the medical schools of the University of North Carolina at Chapel Hill (UNC), The University of Maryland, and the University of Miami. All three groups have employed relational database management systems to organize information about each "instructional unit" in the preclinical curriculum, including a set of keywords defining the major concepts presented. The keywords are indexed to a controlled vocabulary, either the Medical Subject Headings (MeSH) or a MeSH derivative. The UNC database also employs a textfile management system to provide users with an overview of the entire curriculum. Future work will focus on identifying a suitable controlled vocabulary; capturing content in greater contextual detail; incorporating alternative learning formats, such as problem-based learning; creating links between content items and examination questions; and capturing information generated by student-patient interactions in clinical settings. As a result of recent collaboration with the Association of American Medical Colleges, work to define a prototype national database has begun and a consortium of interested schools is addressing further development activities. PMID- 1729990 TI - Teaching students about occupational health issues through worksite visits. PMID- 1729991 TI - Information technology and the academic medical center. PMID- 1729992 TI - Medical honesty and other oxymorons. PMID- 1729993 TI - M.D.-Ph.D. training at the Johns Hopkins University School of Medicine, 1962 1991. AB - From 1962 to 1991, 150 students earned both the M.D. and the Ph.D. degrees in a combined program of study at The Johns Hopkins University School of Medicine (JHUSM). Seventy-five of these individuals were supported by the Medical Scientist Training Program (MSTP) of the National Institutes of Health. The authors analyzed the professional development of these dual-degree recipients, focusing particularly on the 109 M.D.-Ph.D.s who graduated since 1980, when the first MSTP-supported student received both degrees. Of the 109 graduates since 1980, 42 are now in career positions. Thirty-four of the 42 graduates (81%) obtained clinical housestaff training, and 21 of the 34 also had postdoctoral science training. Nearly all of the 42 M.D.-Ph.D.s are in full-time academic posts (81%) or positions in research institutes (14%); the remaining 5% hold research positions in biotechnology firms. All 42 graduates are actively involved in research, and 67% have regular and well-defined clinical responsibilities. Analysis of the representation of M.D.-Ph.D.s on the JHUSM faculty from 1962 to 1991 shows a striking increase with time in the percentage of M.D.-Ph.D.s among the full-time faculty, particularly at the level of assistant professor. These findings suggest that M.D.-Ph.D. graduates nationwide are being recruited in increasing numbers to medical school faculties and are pursuing medical careers encompassing both research and clinical practice. PMID- 1729994 TI - Six years of comprehensive, clinical, performance-based assessment using standardized patients at the Southern Illinois University School of Medicine. AB - By the end of 1990-91, the Southern Illinois University School of Medicine had had six years of experience with comprehensive, performance-based examinations of senior medical students' levels of clinical competence; this report assesses the psychometric aspects of the six examinations given during that period. The examinations were aimed at determining the students' readiness for postgraduate training. Compared with other clinical performance-based assessments that use standardized patients (SPs), these examinations had two important and unique features: (1) the examinations assessed a comprehensive range of clinical skills and reasoning; and (2) they approximated the challenges of real clinical practice wherein a practitioner's skills need to be orchestrated and prioritized in order to meet the challenges of the case encountered. Each year, the performance-based assessment given was an intensive clinical examination requiring each student to work up 13 to 18 SP problems over a three-day period. To administer an examination to an entire class of students took three weeks. Because all students after the first year of administration (1986) were required to pass these examinations, the fairness of test design and scoring and the setting of performance standards for the examinations became important issues for the faculty. The results, accumulated over six years and based on a total of 6,804 student-patient encounters involving 405 students, indicate that this kind of clinical performance-based examination can discriminate a wide range of students' clinical performances. The results provide evidence for the examinations' test security, content validity, construct validity, and reliability. PMID- 1729995 TI - A model for faculty practice teaching clinics developed at the Oregon Health Sciences University. AB - In 1988 the Oregon Health Sciences University established its first faculty practice teaching clinic wherein physicians in training were incorporated into a faculty private practice clinic; this pilot project proved very successful and has been subsequently adopted as the model for essentially all outpatient clinics (both medical and surgery) in the university system. The model encourages efficiency, overhead control, and appropriate staffing; it also compensates faculty members for their additional time spent teaching. The authors conclude this model may help other academic training centers adapt to the changing demands of medical education. PMID- 1729996 TI - Comparing postgraduate medical education at university and non-university hospitals in Japan. AB - In 1988 the authors surveyed all the teaching hospitals in Japan to evaluate the present status of postgraduate medical education (PGME); they received responses from 67 (84%) of the university and 172 (89%) of the non-university teaching hospitals. It was found that a large proportion of residents had spent two years in a residency without having had a single experience of some of the basic clinical skills. Consequently the residents' confidence in their abilities to perform these skills was low. The residents at the university hospitals, in particular, had had fewer experiences and were less confident about their clinical skills than were the residents at the non-university hospitals. The lack of standard and minimum requirements for PGME in Japan may be the cause of the poor level of acquisition of clinical skills of residents during PGME. Other possible causes are the tendency in Japanese medical society to attach greater importance to academic attainment than to clinical competence and the excessive gravitation of residents toward university hospitals. The authors suggest their results show the necessity to improve the training in basic clinical skills in PGME in Japan, especially in university hospitals. PMID- 1729997 TI - Women's attitudes toward careers in academic medicine at the University of California, San Francisco. AB - In order to identify the concerns and possible barriers for women considering careers in academic medicine, in 1990 the authors surveyed both men and women medical students, housestaff, postdoctoral students, and junior faculty at The University of California, San Francisco (UCSF). The authors achieved a 58% response rate from students and faculty, a 21% response rate from postdoctoral students, and a 15% response rate from housestaff. Results indicated that women at all levels were less interested in academic careers than were their male colleagues. Concerns about balancing family responsibilities, clinical practice, and teaching in addition to the research required of an academic career were mentioned most frequently. Women, especially those among the housestaff and junior faculty, reported fewer mentor relationships and role models. The authors discuss these findings in relation to other studies and describe what they are doing to foster women's interest and success in academic medicine at UCSF. PMID- 1729998 TI - Paternalistic attitudes and moral reasoning among physicians at a large teaching hospital. PMID- 1729999 TI - Resistance to peer evaluation in an internal medicine residency. PMID- 1730000 TI - Teaching medicolegal issues using a standardized patient and mock trial. PMID- 1730001 TI - Teaching surgical knot-tying skills. PMID- 1730002 TI - A national, interdisciplinary consortium of primary care organizations to promote the education of generalist physicians. AB - This essay begins with the history from 1989 through late 1991 of the Primary Care Organization's Consortium (PCOC), a group of representatives from nine major academic and professional organizations for primary care specialties. The PCOC was formed to discuss what might be done to reverse the alarming decrease in the number of medical students who choose primary care specialties. The article reviews some of the conditions that many believe have caused the continuing move away from primary care careers, and concludes with a description of the PCOC's program to encourage medical students to choose primary care careers, and the new opportunities for collaborative planning of such programs that are now available to medical schools. The PCOC's success in defining its program is due to a process of interdisciplinary planning and collaboration at the national level that hopefully will facilitate similar collaboration among medical school departments. PMID- 1730003 TI - Rapid effects of laminin on the growth cone. AB - To gain insight into how laminin promotes neurite growth, high resolution video microscopy was used to determine the rapid effects of laminin on growth cone structure. Sympathetic growth cones in serum-free medium on polylysine substrate displayed extensive motility and protrusive activity and often had large lamellipodia. However, their neurites grew slowly because membranous organelles from the central region advanced into the lamellipodium only slowly. Acute addition of laminin accelerated growth severalfold and had visible effects on the growth cone within minutes. Laminin dramatically accelerated the advance of membranous organelles, which, with microtubules, rapidly filled the lamellipodium. Retraction of individual protrusions (filopodia and veils) was rapidly reduced. These effects of laminin are important in accelerating growth and suggest a mechanism for pathway selection by growing neurites. PMID- 1730004 TI - fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system. AB - A transgenic mouse line containing a fos-lacZ fusion gene was derived in which beta-galactosidase activity identified cell populations expressing fos either constitutively or after stimulation. Seizures and light pulses induced nuclear lacZ activity in defined populations of neurons in vivo, and an array of neurotransmitters, including glutamate, induced the transgene in primary brain cultures. In unstimulated mice, the major sites of fos-lacZ expression were skin, hair follicle, and bone. fos-lacZ mice provide a new avenue for activity mapping studies based on gene expression. PMID- 1730005 TI - Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization. AB - We have generated a polyclonal antibody against myosin II from a neuronally derived cell line in order to assess potential roles for myosin II in growth cone movement and synaptic transmission. The distribution of neuronal myosin II, in isolated cells as well as in tissues of the adult rat brain and spinal cord, was examined at the light microscopic and ultrastructural levels. In isolated neuroblastoma cells and dorsal root ganglion neurons, myosin II was found at the leading edge of growth cones, within neuritic processes and cell soma, and adjacent to the plasma membrane. The subcellular distribution of myosin II overlapped significantly with that of both actin and single-headed myosin I. These results implicate both myosin I and myosin II as molecular motors required for neurite elongation and growth cone motility. An exclusive postsynaptic distribution of myosin II in neurons of the mature central nervous system suggests that myosin II cannot play a role in the mobilization of synaptic vesicles, but could participate in synaptic plasticity. PMID- 1730006 TI - Bioenergetics. Dedicated to Professor E.C. Slater on the occasion of his 75th birthday. PMID- 1730007 TI - Effect of Ca2+ ions on the slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase. AB - Slow active/inactive transition of the membrane-bound mitochondrial NADH ubiquinone reductase (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) is sensitive to Ca2+ and other divalent cations. Millimolar concentrations of Ca2+ drastically reduce the rate of the turnover-dependent activation of NADH-ubiquinone reductase. When NADH oxidase, the rotenone sensitive NADH-ubiquinone reductase or the succinate-supported delta mu H+ dependent NAD+ reduction were initiated by the deactivated enzyme preparations all the three activities were strongly inhibited by Ca2+; no sensitivity of these reactions to Ca2+ was observed when the assays were started by the activated enzyme preparations. The affinity of the deactivated enzyme to polyvalent cations was in the following order: Ni2+ greater than Co2+ greater than La3+ greater than Mn2+ greater than Ca2+ approximately Mg2+ greater than Ba2+. Monovalent metal cations had no effect on the slow turnover-dependent enzyme activation. The apparent affinity of the deactivated enzyme to Ca2+ was strongly pH-dependent. The KCa2+ values of 5.7 mM and 0.6 mM at pH 7.5 and 8.5 were determined from the presteady-state kinetics parameters. The spontaneous temperature-dependent deactivation of the enzyme was insensitive to Ca2+. Ca2+ increases the reactivity of the enzyme sulfhydryl group in the deactivated preparations towards N ethylmaleimide. This effect was also used to quantitate Ca2+ affinity for the enzyme. The KCa2+ values of 1.2 mM and 0.4 mM at pH 8.0 and 9.0, respectively, were determined. The data obtained suggest that Ca2+ content in the mitochondrial matrix may play an important role in the control of NADH oxidation by the respiratory chain. PMID- 1730008 TI - Identification of type 1 and type 2 light-harvesting chlorophyll a/b-binding proteins using monospecific antibodies. AB - The amino acid sequences of more than 40 apoproteins of the light-harvesting complex associated with Photosystem II (LHC II) of various plants have been deduced by sequencing their corresponding genes. These highly conserved sequences fall into two major categories, type 1 and type 2, that differ mainly in a small number of domains close to the N-terminus. We have made polyclonal, monospecific antibodies against synthetic peptides corresponding to the most unique sequence domains of the N-terminal regions of type 1 and type 2 LHC II apoproteins, using sequences derived from petunia genes. On Western blots our anti-type 1 and 2 antibodies crossreact with light-harvesting proteins of petunia, tomato, spinach and several other plants. By using a new gel-system based on ammediol (2-amino-2 methyl-1,3-propanediol), we are able to resolve up to eight LHC II apoproteins. On petunia, tomato and spinach blots the anti type 1 antibodies bind to two or more of the higher molecular weight LHC II polypeptides, whereas the anti type 2 antibodies recognize very specifically only one or two of the lower molecular weight LHC-proteins. In all plants studied, the type 1 LHC II apoproteins are more numerous and span a greater size range than the type 2 apoproteins. This is consistent with the smaller number of type 2 LHC II CAB genes that have been discovered to date. PMID- 1730009 TI - Some effects of different extracellular proteins on oxygen consumption and heat production in isolated rat hepatocytes. AB - When hepatocytes prepared from 24-h-fasted rats were washed, suspended and incubated in Krebs-Henseleit bicarbonate-buffered saline, the endogenous rates of O2 consumption and heat production were 2.13 +/- 0.13 mumol/min per g wet wt. and 1.00 +/- 0.05 J/min per g wet wt. respectively. The inclusion of 2.5% (w/v) defatted and dialysed bovine serum albumin in either the cell suspension (washing) buffer or the cell incubation buffer produced a 20-25% increase in O2 consumption and heat production: these rates were increased by an additional 20 25% when the albumin (2.5%) was present in both the cell suspension and the cell incubation buffers. There was an inverse relationship between the increases in O2 consumption and heat production and the leakage of lactate dehydrogenase from the isolated hepatocytes: the inclusion of purified bovine serum albumin decreased lactate dehydrogenase leakage from 40% to 15% of total enzyme content. The calorimetric-respirometric ratios for hepatocytes incubated both in the absence ( 461 +/- 19 kJ/mol O2) and presence (-477 +/- 8 kJ/mol O2) of the purified protein are very similar to the theoretical, thermochemically derived oxycaloric equivalents. PMID- 1730010 TI - Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC. AB - Mitochondrial functions depend not only on the properties of the particular enzyme systems, but also on the continual flux of metabolites between the cytoplasm and mitochondrial spaces. We report the results of experiments that strongly indicate that a soluble mitochondrial protein can regulate mitochondrial respiration by reducing the permeability of the outer membrane. This protein is known as the VDAC modulator because it induces the outer mitochondrial membrane channel, VDAC, to close. When added to intact mitochondria, the modulator reduces the ADP-stimulated respiration. This inhibition can be prevented by damaging the outer membrane prior to modulator addition. Another mitochondrial activity, adenylate kinase, is reduced by 40% by the addition of the VDAC modulator to intact mitochondria. Again, damaging the outer membrane removed the modulator effect. Dextran sulfate, an artificial polyanion that acts on VDAC channels in a similar way to the VDAC modulator, has the same effects on intact mitochondria. The findings correlate well with observations of the actions of the VDAC modulator on reconstituted VDAC channels, in which the modulator induces the channel to enter a very low conductive state. The ability of a mitochondrial protein to regulate mitochondrial activities by reducing the permeability of the outer membrane further fuels the hypothesis that this membrane participates in the overall regulation of mitochondrial functions. PMID- 1730011 TI - Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia. AB - We present for the first time a method for isolation of the membranes of extrusive organelles (trichocysts) from sterile culture of different strains of Paramecium tetraurelia. First, trichocysts are isolated according to a new method (Glas-Albrecht, R. and Plattner, H. (1990) Eur. J. Cell Biol. 53, 164-172) with high purity and yield. Then the organelles are subjected to osmotic swelling. Since trichocysts then easily 'decondense' and entangle membranes, these cannot be isolated directly by centrifugation, but only by passage through a filter and subsequent centrifugation. Purity of membrane fractions is analysed by electron microscopy and SDS-PAGE, combined with silver staining or, after biotinylation, by avidin-peroxidase labelling. Molecular masses resolved in our gels are in a range from less than or equal to 15 to greater than or equal to 105 kDa. Main bands obtained with nd9-28 degrees C trichocyst membranes (most bands also being common to wild type trichocysts) are of about 16.5, 19-21, 27-29, 33-34, 44-45 (strong), 47-48 (strong), 57, 61, 65 (strong), 68-71, 75, 81, 94-95 (strong), 104 and greater than or equal to 110 kDa, from a total of approx. 23 bands resolved. There is no remarkable occurrence of dominant protein bands from trichocyst contents ('trichynins'), though these might represent up to 10(3)-times more of the total trichocyst proteins. The ratio of phospholipid/protein is approx. 0.2 mg/mg. The methodology developed might also be valuable for the isolation of extrusome membranes from some other protozoan species. PMID- 1730012 TI - Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles. AB - The shared transport system for uptake of L-cystine and L-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na+. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K+ out of the vesicle. Membranes prepared by Mg aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods. PMID- 1730013 TI - Low- and high-Km transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytes. AB - Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio 3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high affinity transporter for DNP-SG and GSSG in erythrocytes is suggested. PMID- 1730014 TI - Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hysteresis, stability. AB - Surface tension-area relations from pulmonary surfactant were obtained with a new apparatus that contains a leak free captive bubble of controllable size. Rat pulmonary surfactant was studied at phospholipid concentrations of 50, 200 and 400 micrograms/ml. At the highest concentration, adsorption was rapid, reaching surface tensions below 30 mN/m within 1 s, while at the lowest concentration, approximately 3 min were required. Upon a first quasi static or dynamic compression, stable surface tensions below 1 mN/m could be obtained by a film area reduction of approximately 50%. After three to four cycles the surface tension-area relations became stationary, and the tension fell from 25-30 to approximately 1 mN/m for a film area reduction of less than 20%. Hysteresis became negligible, provided the films were not collapsed by further area reduction. Under these conditions, the films could be cycled for more than 20 min without any noticeable loss in surface activity. After only three to four consecutive cycles, surfactant films exhibited the low surface tensions, collapse rates and compressibilities characteristic of alveolar surfaces in situ. Remarkably, surface tension and area are interrelated in the captive bubble which may promote low and stable surface tensions. If the surface tension of the captive bubble suddenly increases ('click') because of mechanical vibration or unstable surfactant, the bubble shape changes from flat to more spherical. The associated isovolumetric decrease in surface area prevents the surface tension from rising as much as it would have in a constant-area situation. This feedback mechanism may also have a favorable effect in stabilizing alveolar surface tension at low lung volumes. PMID- 1730015 TI - Liposomes as a model for the study of the mechanism of fish toxicity of sodium dodecyl sulfate in sea water. AB - The mechanism underlying the shark repellency of SDS was studied by comparing it with the shark nonrepelling detergent, Triton X-100. The findings can be summarized as follows: (1) The effective concentration of SDS for termination of shark tonic immobility (an immediate and fast response) was close to its critical micellar concentration in sea water (70 microM). The fish lethal concentrations (LD50) were far below the CMC value for SDS, and at CMC level for Triton X-100. (2) In sea water SDS possesses a strong affinity for lipid membranes, expressed in a lipid sea water partition coefficient (Kp) of about 3000. (3) In liposomal systems examined by assays of turbidity, fluorescence resonance energy transfer and kinetics of carboxyfluorescein (CF) release, the pattern of SDS induced changes in the phospholipid bilayer suggests: (a) absence of vesicle-vesicle fusion; (b) occurrence of vesicle size increase, and (c) nonlytic gradual release of CF above and below its CMC values. In contrast, Triton X-100 above its CMC induces membrane solubilization. (4) Assays coupling CF release from liposomes to potassium diffusion potential induced by valinomycin indicate that SDS related CF release can also be attributed to a specific mechanism such as cation pore formation and not only to membrane solubilization. The hypothesis of pore formation by SDS is discussed. PMID- 1730016 TI - Effect of amphipathic peptides with different alpha-helical contents on liposome fusion. AB - The peptide-induced fusion of neutral and acidic liposomes was studied in relation to the amphiphilicities evaluated by alpha-helical contents of peptides by means of a carboxyfluorescein leakage assay, light scattering, a membrane intermixing assay and electron microscopy. An amphipathic mother peptide, Ac-(Leu Ala-Arg-Leu)3-NHCH3 (4(3], and its derivatives, [Pro6]4(3) (1), [Pro2,6]4(3) (2), and [Pro2,6,10]4(3) (3), which have very similar hydrophobic moments, caused a leakage of contents from small unilamellar vesicles composed of egg yolk phosphatidylcholine and egg yolk phosphatidic acid (3:1). The abilities of the peptides to induce the fusion of the acidic liposomes increased with increasing alpha-helical content: in acidic liposomes the helical contents were in the order of 4(3) greater than 1 greater than 2 greater than 3 (Lee et al. (1989) Chem. Lett., 599-602). Electron microscopic data showed that 1 caused a transformation of the small unilamellar vesicles (20-50 nm in diameter) to large ones (100-300 nm). Based on the fact that these peptides have very similar hydrophobic moments despite of decreasing in the mean residue hydrophobicities to some extent, it was concluded that the abilities of the peptides to induce the fusion of liposomes depend on the extent of amphiphilic conformation evaluated by alpha-helical contents of the peptides in the presence of liposomes. For neutral liposomes of egg yolk phosphatidylcholine, all the proline-containing peptides showed no fusogenic ability but weak leakage abilities, suggesting that the charge interaction between the basic peptides and acidic phospholipid is an important factor to induce the perturbation and fusion of the bilayer. PMID- 1730017 TI - Bilayer stabilization of phosphatidylethanolamine by N biotinylphosphatidylethanolamine. AB - We have examined the ability of biotinylated phosphatidylethanolamine and similar lipids to stabilize the bilayer phase of polymorphic dioleoylphosphatidylethanolamine (DOPE). Sonicated lipid mixtures were characterized in terms of their aggregation state, size and ability to encapsulate and retain the fluorescent dye, calcein. Titration of DOPE with N biotinyl-PE indicated that stable liposomes could be produced by sonication of DOPE based dispersions containing N-biotinyl-PE at concentrations greater than 8 mol%. These liposomes were relatively small, could efficiently encapsulate calcein, and showed minimal leakage upon prolonged storage at 4 degrees C. Maleimido-4-(p-phenylbutyrate)-PE (MPB-PE) was equally effective at stabilizing the bilayer phase of DOPE whereas N-dinitrophenyl-PE and N-(dinitrophenyl caproyl)-PE were relatively poor stabilizers, requiring at least 15 mol% for stabilization at pH 7.4. Differential scanning calorimetry of dielaidoylphosphatidylethanolamine (DEPE)/N-biotinyl-PE mixtures indicated that stabilizer concentrations as low as 2 mol% could abolish the L alpha/HII phase transition of DEPE. PMID- 1730018 TI - Isolation of plasma membrane exovesicles from BHK cells using merocyanine 540. AB - Treatment of cultured BHK cells with merocyanine 540 caused the non-lytic release of vesicular material having the phospholipid composition characteristic of plasma membrane. The protein composition of the vesicles closely resembled that of the soluble fraction of the cell, as expected for exovesicles budding from the cell surface. Vesicles prepared from cells surface-iodinated with 125I contained no obvious iodinated membrane polypeptides, suggesting that no major proteins in the plasma membrane of the BHK cell are free to diffuse with lipids. The procedure described should represent a general method, applicable to a wide range of cell types, for isolating plasma membrane vesicles. PMID- 1730019 TI - Glycosylation of the rabbit intestinal brush border Na+/glucose cotransporter. AB - The glycosylation of the mature form of the rabbit intestinal Na+/glucose cotransporter was investigated by using both glycosidases and chemical treatment. The protein was identified on Western blots using polyclonal antibodies directed against peptide sequences from the cloned transporter as a Mr 68,000 polypeptide. The effect of these treatments on the size of the transporter is consistent with the major post-translational processing being a single N-linked glycosylation of either the tri- or tetra-antennary complex type. Either method of deglycosylation reduced the SDS-PAGE size by 11,000 to Mr 57,000. These results also suggest that O-linked glycosylation, if present, contributes little to the apparent size of the transporter. The relative size of the deglycosylated mature protein appears to be greater than that of the in vitro primary transcript (Mr 45,000), suggesting either a difference in a stable conformational state insensitive to reduction and denaturation by SDS or an additional post-translational modification. In addition, deglycosylation of the native transporter does not affect transport activity in brush border membrane vesicles. The transporter, an integral membrane protein having several membrane-spanning regions, has an anomalous mobility in SDS-PAGE as shown by Ferguson analysis. We estimate that the actual size of the mature Na+/glucose cotransporter is 86,000, and that N linked glycosylation contributes about 15,000 to the mass. PMID- 1730020 TI - Cl-/base exchange and cellular pH regulation in enterocytes isolated from chick small intestine. AB - Intracellular pH (pHi) and Cl-/base exchange activity have been examined in isolated chicken enterocytes, both in the presence and absence of 25 mM HCO3-/5% CO2. Intracellular pH was measured with BCECF, a pH-sensitive carboxyfluorescein derivative. Under resting conditions pHi was 7.17 in Hepes and 7.12 in HCO3(-) buffered solutions. Cells became more alkaline upon withdrawal of Cl-. Cells depleted of Cl- acidified upon reinstatement of Cl-. These changes were faster in the presence of HCO3- than in its absence. After an alkaline load (removal of HCO3- from the medium) pHi decreases towards base line in the presence of Cl-, but not in its absence. The Cl(-)-dependent pHi changes were prevented by H2DIDS and were unaffected by Na+. The Cl(-)-induced recovery from an alkaline load exhibited simple saturation kinetics, with an apparent Km of 12.5 mM Cl- and maximum velocity of approximately 0.20 pH units min-1. The Cl-/base exchange is functional under resting conditions, as shown by cell alkalinization on exposure to 0.5 mM H2DIDS, both in the presence and in the absence of HCO3-. It is concluded that Cl-/base exchange participates in setting the resting intracellular pH in isolated chicken enterocytes and helps recover from alkaline loads. The exchange operates both in the presence and in the absence of bicarbonate. PMID- 1730021 TI - Relationship of hepatic cholate transport to regulation of intracellular pH and potassium. AB - Modulation of hepatic cholate transport by transmembrane pH-gradients and during interferences with the homeostatic regulation of intracellular pH and K+ was studied in the isolated perfused rat liver. Within the concentration range studied uptake into the liver was saturable and appeared to be associated with release of OH- and uptake of K+. Perfusate acidification ineffectually stimulated uptake. Application of NH4Cl caused intracellular alkalinization, release of K+ and stimulation of cholate uptake, withdrawal of NH4Cl resulted in intracellular acidification, regain of K+ and inhibition of cholate uptake. Inhibition of Na+/H(+)-exchange with amiloride reduced basal release of acid equivalents into the perfusate, initiated K(+)-release, and inhibited both, control cholate uptake and its recovery following intracellular acidification. K(+)-free perfusion caused K(+)-release and inhibited cholate uptake. K(+)-readmission resulted in brisk K(+)-uptake and recovery of cholate transport. Both effects were inhibited by amiloride. Interference with cholate transport through modulation of pH homeostasis by diisothiocyanostilbenedisulfonate (DIDS) could not be demonstrated because DIDS affected bile acid transport directly. Biliary bile acid secretion was stimulated by intracellular alkalinization and by activation of K(+) transport. Uncoupling of the mutual interference between pH-dependent cholate uptake and K(+)-transport by amiloride indicates tertiary active transport of cholate. In this, Na+/K(+)-ATPase provides the transmembrane Na(+)-gradient to sustain Na+/H(+)-exchange which maintains the transmembrane pH-gradient and thus supports cholate uptake. Effects of canalicular bile acid secretion are consistent with a saturable, electrogenic transport. PMID- 1730022 TI - Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study. AB - Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase. PMID- 1730023 TI - The ability and inability of ATP to stop aluminum from reducing the sodium efflux in unpoisoned barnacle muscle fibers. AB - A study has been made in single barnacle muscle fibers with the object of determining whether ATP is able to protect the resting Na efflux from the effects of injected aluminum (Al) and whether Al is able to reduce or abolish the stimulatory action of ATP on the efflux. The results of the experiments show that neither ATPMg nor ATPNa2 preinjection stops Al from reducing the basal Na efflux in unpoisoned fibers which undergo a large fall (hypersensitive fibers). Preinjection of Al into such fibers reduces or abolishes the stimulatory response of the Na efflux to ATP injection. In less hypersensitive fibers, however, ATPMg is protective. This is also true of ATPNa2 preinjection in both classes of fibers showing stimulation. Injection of a mixture of AlCl3-ATPNa2 into unpoisoned fibers causes less inhibition than AlCl3 injection. The hypothesis that both ATPMg and ATPNa2 are protective is also supported by the results obtained with ouabain-poisoned fibers: (i) Al injection after ATP fails to reverse the stimulatory response to ATP, while ATP injection after Al exerts only a small or no effect. (ii) Mg2+ injection fails to reverse the stimulatory response to Al injection in poisoned fibers. And (iii) Anti-proteolysis agents e.g. leupeptin and pepstatin, upon preinjection, do not alter the kinetic results obtained by injecting Al into unpoisoned and ouabain-poisoned fibers. PMID- 1730024 TI - The thermal behaviour of phosphatidylcholine-glycophorin monolayers in relation to monolayer and bilayer internal pressure. AB - A thermodynamic relationship which allows calculation of the internal pressure (Pi) of a monolayer has been derived, viz: Pi = T(delta pi/delta T)A-pi. Surface pressure (pi)-area (A) isotherms were determined for dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and a mixture of the latter with 0.28 mol% glycophorin, an intrinsic membrane protein. The isotherms (10 degrees C 42 degrees C) were used to obtain values of (delta pi/delta T)A. Under conditions in which a monolayer is believed to most closely model a bilayer membrane (i.e. 37 degrees C and A = 0.6 nm2/molecule) internal pressures were: DOPC = 0.32 N m 1; DPPC = 0.13 N m-1 and glycophorin/DPPC = 0.36 N m-1. The results do not support some measurements on amphiphile solubility in natural membranes which had been interpreted as evidence of a large increase in internal pressure, due to the intrinsic membrane proteins. PMID- 1730025 TI - Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli. AB - Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom. PMID- 1730026 TI - Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase. AB - Purified rat liver plasma membranes were incubated for 0-60 min with [gamma 32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2 methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established. PMID- 1730027 TI - Stability properties of Aplysia oxymyoglobin: effect of esterification of the heme propionates. AB - When compared with sperm whale MbO2 as a reference Aplysia MbO2 is extremely susceptible to autoxidation, and its pH dependence for the reaction is also unusual. Kinetic and thermodynamic analyses have shown that two types of carboxyl group with pKa = 4.3 and 6.1 at 25 degrees C are involved in the stability property of Aplysia MbO2, the protonation of these groups being responsible for an increase in its autoxidation rate in the acidic pH range. Since protoheme has two carboxyl groups of the propionic acid side-chains, we have prepared Aplysia myoglobin containing the dimethylester of protohemin. The autoxidation of this derivative was found to be described by the involvement of only one type of carboxyl group with pKa = 4.4. A possible candidate for the protein residue was therefore discussed on the basis of the known amino acid sequences of three Aplysia myoglobins: it is suggested that Glu-94, next to the proximal His-95, is the most likely candidate. PMID- 1730028 TI - Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties. AB - Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P. PMID- 1730029 TI - Phenoloxidase catalyzed coupling of catechols. Identification of novel coupling products. AB - Phenoloxidases from insect cuticle as well as from other sources oxidize catechols resulting in the formation of various coupling products. The two dominating products from 4-methylcatechol and the main product from N acetyldopamine were purified and identified by means of plasma desorption and electron impact mass spectrometry and by 1H- and 13C-NMR spectroscopy. The main product from both catechols has a quinoid trihydroxybiphenyl structure, indicating oxidative coupling between a catechol and the corresponding trihydroxy derivative. The second product from 4-methylcatechol is a biphenyltetrol derivative, indicating oxidative coupling between two catechols. PMID- 1730030 TI - Halogenated alcohols as solvents for proteins: FTIR spectroscopic studies. AB - Fourier transform infrared (FTIR) spectroscopy has been applied to investigate the secondary structure of proteins and polypeptides in halogenated alcohols. Each alcohol studied was able, as a pure liquid, to induce conversion of the beta sheet protein concanavalin A into a predominantly alpha-helical configuration. In 2H2O/alcohol mixtures, helicogenisis was also apparent, decreasing in the order dichloroethanol greater than bromoethanol greater than trifluoroethanol greater than chloroethanol greater than fluoroethanol. At concentrations below those found to be helicogenic, disruption of the protein secondary structure by the alcohols resulted in pronounced aggregation. At concentrations insufficient to cause noticeable disruptions of the secondary structure at room temperature, the thermal stability of the protein was greatly reduced. We suggest the helicogenic effect exhibited by halogenated alcohols to be related to a combination of a relatively low dielectric constant and a high dipole moment, the latter causing disruption of the internal hydrogen bond networks and the former causing refolding to a helical configuration. The results presented here highlight the risk of using halogenated alcohols, both as solvents for proteins and as a test of the intrinsic capacity of proteins and peptides to adopt helical secondary structures. PMID- 1730031 TI - On the molecular interactions between plasminogen-staphylokinase, alpha 2 antiplasmin and fibrin. AB - The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2 antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma. PMID- 1730032 TI - Tyrosine protein kinase in boar spermatozoa: identification and partial characterization. AB - A protein-tyrosine kinase has been isolated from a detergent-soluble extract of boar spermatozoa, using poly(Glu, Tyr)4:1 as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, polyamino acid affinity and Sephadex G-100 molecular sieving, and results in more than a 1200-fold enrichment. Analysis of the most purified preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major Coomassie blue-stained band of molecular mass 42 kDa. The Tyr-protein kinase does not seem to be autophosphorylable. The Km value for poly(Glu, Tyr)4:1 is relatively low, 2.3 microM, and the tyrosine-polymer phosphorylating activity is apparently inhibited by tyrphostin. The characteristics shown by this new tyrosine kinase- the first to be described in mature male germ cells--support the hypothesis that it belongs to the group of non-receptor-associated tyrosine kinases. PMID- 1730033 TI - Inactivation of propanediol oxidoreductase of Escherichia coli by metal-catalyzed oxidation. AB - 1,2-Propanediol oxidoreductase, which reduces the L-lactaldehyde formed in the fermentation of L-fucose or L-rhamnose to L-1,2-propanediol in E. coli, was inactivated by a component of E. coli cell extracts in the presence of oxygen. Pure propanediol oxidoreductase preparations were shown to be inactivated in vitro by aerobic incubations in the presence of Fe3+ and ascorbate. The Fe3+ ascorbate-mediated inactivation reaction was inhibited by catalase, although not by superoxide dismutase. Under anaerobic conditions, the presence of H2O2 strongly inactivated the enzyme. Propanediol oxidoreductase was rapidly degraded in the presence of oxygen, while the native enzyme displayed high stability as long as no oxygen was present. PMID- 1730034 TI - Mutations affecting nitrogenase switch-off in Rhodobacter capsulatus. AB - In vivo 'switch-off' and subsequent reactivation of nitrogenase activity in Rhodobacter capsulatus or Rhodospirillum rubrum in response to a variety of environmental stimuli, including the addition of fixed nitrogen, is thought to be due to the action of two nitrogenase Fe protein modifying activities; DRAT (dinitrogenase reductase ADP-ribosyl transferase) and DRAG (dinitrogenase reductase-activating glycohydrolase). Here it is demonstrated that strains, including one mutated in glnB, that constitutively express nif in the presence of fixed nitrogen are never-the-less capable of Fe protein modification. Thus the regulation of Fe protein modification is separate from that of its expression. The observations that Mn-deficient cultures are unable to fix nitrogen and that DRAG activity requires a divalent metal cation, most notably Mn2+, prompted the search for mutants (pseudo-prototrophs) capable of in vivo nitrogen fixation under Mn-deficient conditions. In the present study the isolation and partial characterization of several putative mutants is described. One, AF1, was shown to be altered in the in vivo regulation of N2ase activity in response to fixed nitrogen and to have an altered in vitro activity in glutamate grown cells. However, this strain was shown to possess in vitro DRAT activity and to have a modifiable Fe protein. Two-dimensional gel analysis indicates that this strain is altered in the synthesis of a 48 kDa protein of as yet unknown function. Thus, the mutation in this strain must affect, in an as yet undetermined manner, the response of the modifying system to fixed nitrogen. PMID- 1730035 TI - Detritiation of L-[3-3H]alanine in the glutamate-pyruvate transaminase reaction. AB - The detritiation of L-[3-3H]alanine in the reaction catalyzed by pig heart glutamate-pyruvate transaminase was monitored in the absence or presence of lactate dehydrogenase. The results indicated that each monodirectional conversion of L-[3-3H]alanine to [3-3H]pyruvate resulted in the generation of 3HOH at a rate representing one-third of the total 3H flux. No isotopic discrimination in reaction velocity between tritiated and 14C-labelled L-alanine was observed. The mathematical modelling of the reaction revealed that, as a consequence of the detritiation process, the steady-state ratio in L-[3-3H]alanine/[3-3H]pyruvate does not inform on either the absolute or relative size of the amino acid and 2 keto acid pools. PMID- 1730036 TI - Oxidized alpha 1-proteinase inhibitor: a fast-acting inhibitor of human pancreatic elastase. AB - Unlike human neutrophil elastase or porcine and rat pancreatic elastases, human pancreatic elastase is rapidly inhibited by oxidized alpha 1-proteinase inhibitor. The second-order association-rate constant for the reaction of the oxidized inhibitor with this enzyme (kass = 10(5) M-1 s-1) is only 8-fold lower than that measured with native alpha 1-proteinase inhibitor. Elastase releases faster from its complex with the oxidized inhibitor (t1/2 approximately 0.7 days) than from its complex with the native inhibitor (t1/2 approximately 5 days). Oxidized alpha 1-proteinase inhibitor is as efficient as the native inhibitor in inhibiting the elastolytic activity of elastase. Oxidized alpha 1-proteinase inhibitor may thus be considered as a physiological inhibitor of human pancreatic elastase which may prevent degradation of blood vessel elastin during acute hemorrhagic pancreatis. PMID- 1730037 TI - Role of an evolutionarily invariant serine for the stability of human carbonic anhydrase II. AB - There are several evolutionarily invariant amino acids in the primary structures of all known isoenzymes of carbonic anhydrase. One of these is Ser-29 which is situated in the peripheral part of the active site interacting by hydrogen bonds with amino acids located nearby in the tertiary structure. Furthermore, the neighbourhood of Ser-29, composed of Gln-28, Pro-30, Tyr-194, Ser-197 and Trp 209, has a totally invariant structure. The structural role of Ser-29 was investigated by site-directed mutagenesis. The stability of two enzyme mutants, where Ser-29 was replaced by alanine and cysteine, towards denaturation by guanidine-HCl was studied. Changing Ser-29 to Ala resulted in a destabilization by 2.6 kcal/mol, corresponding to the loss of 2-3 hydrogen bonds. Interestingly, Ser-29 is within hydrogen bond distance to Tyr-194, Ser-197 and Trp-209 in the tertiary structure. Therefore, rupture of these interactions caused by the Ser-29 ---Ala substitution could explain the observed destabilization of this enzyme variant. Substituting cysteine for Ser-29 gives rise to a drastic decrease in the stability of the protein (change in midpoint concentration of denaturation from 0.96 M to less than 0.1 M guanidine-HCl) despite the minor structural change (O-- -S atom). This destabilization corresponds to approx. 7-8 kcal/mol and cannot be explained by changes in hydrogen bond pattern only, but must also include unfavourable conformational changes to avoid van der Waals collisions originating from the somewhat larger thiol group. PMID- 1730038 TI - Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study. AB - Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme. PMID- 1730039 TI - Structure model of core proteins in photosystem I inferred from the comparison with those in photosystem II and bacteria; an application of principal component analysis to detect the similar regions between distantly related families of proteins. AB - A principal component analysis based on the physico-chemical properties of amino acid residues is developed to assign similar regions between distantly related families of proteins, taking account of the species diversities in respective families. The most important advantage of this analysis should be that it reflects different physico-chemical properties and thus can predict more detailed structural properties, including the transmembrane helices, than the hydropathy analysis. Its first application reconfirms the similarity between the core proteins of photosynthetic reaction center in purple bacteria and those of photosystem II, indicating that the low percentage of identical amino acid residues estimated previously between them is due to much allowance for amino acid substitutions in purple bacteria. The application of this analysis to the core proteins of photosystem I reveals that any of these proteins includes two domains, each showing high similarity to the amino acid sequences of core proteins in photosystem II and purple bacteria. A core structure model of A1 and A2 proteins folded into four layers of sheets of transmembrane helices is proposed to provide a molecular basis for the electron pathway suggested by spectroscopic experiments as well as for the interaction sites with plastocyanin, 9 kDa protein and LHC proteins. PMID- 1730040 TI - Structure and functional properties of lipoprotein lipase. PMID- 1730041 TI - Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats. AB - Exogenous hypercholesterolemic (ExHC) rats, that develop hypercholesterolemia for exogenous cholesterol, are an established strain Isolated from Sprague-Dawley (SD) rats by Imai and Matsumura ((1973) Atherosclerosis, 18, 59-64). The present study was carried out to clarify the cause of hyperresponsivity in ExHC rats to dietary cholesterol. As early as one day after feeding a high cholesterol diet (1%) serum cholesterol level was doubled in ExHC rats, while the level of hepatic cholesterol was two-thirds of SD rats. The elevation of serum cholesterol was mainly attributed to the d less than 1.006 g/ml fractions. Cholesterol feeding increased fecal bile acid excretion in both strains, but to a more greater extent in SD rats. Absorption of dietary cholesterol and synthesis of cholesterol in vivo were similar between the strains. The uptake of beta-very-low-density lipoproteins (beta-VLDL) in vivo and the primary cultured hepatocytes was lower in ExHC rats, when a high-cholesterol diet was fed. Even without feeding of a high-cholesterol diet, preincubation with cholesterol-rich lipoproteins caused a lower association and degradation of beta-VLDL by the hepatocytes from ExHC rats. Incubation of hepatocytes with cholesterol-rich lipoproteins did not affect the secretion of [14C]cholesterol into the density less than 1.006 g/ml fraction, but suppressed the secretion into the medium density greater than 1.006 g/ml fractions. These results suggest that ExHC rats, as compared to SD rats, are defective of hepatic uptake and processing cholesterol to bile acids. PMID- 1730042 TI - Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes. AB - The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihydroxyeicosatetraenoic acids generated by omega/omega-1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B4 in double-bond geometry alone. The other two differed from leukotriene B4 in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids. PMID- 1730043 TI - Solubilization and modulation of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity in rat liver microsomes. AB - The acylation of 1-acyl-glycerophosphocholine is an important mechanism for the maintenance of the asymmetrical distribution of acyl groups in phosphatidylcholine. The majority of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase is located in the microsomal fraction. In this study, the rat liver microsomes were incubated with various detergents, and the solubilized enzyme was separated from the remainder by centrifugation. Sodium cholate, sodium deoxycholate and octylglucopyranoside caused the solubilization of 14-25% of the enzyme activity. The acyl specificity of the solubilized enzyme was similar to the insoluble enzyme, indicating that there was no selective solubilization of any acyl specific acyltransferase. The solubilized enzyme did not display any lipid requirement, and its activity was inhibited by phosphatidylcholine, phosphatidylethanolamine and 1,2-diacylglycerol. Kinetic studies with varying concentrations of acyl-CoAs revealed that the inhibition by 1,2-diacylglycerol was essentially uncompetitive. The modulation of acyltransferase activity by 1,2 diacylglycerol may be an important mechanism for controlling the acylation of lysophosphatidylcholine. PMID- 1730044 TI - Transport of poly-beta-hydroxybutyrate in human plasma. AB - Poly-beta-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20-30% of total plasma PHB; 6-14% in the very low density lipoproteins (VLDL), 8-16% in the low density lipoproteins (LDL), and less than 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70-80%) was found in protein fractions of density greater than 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')2 identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of 14C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects. PMID- 1730045 TI - Apparent relative retention of the phosphatidylethanolamine molecular species 18:0-20:5(n-3), 16:0-22:6(n-3) and the sum 16:0-20:4(n-6) plus 16:0-20:3(n-9) in the liver microsomes of pig on an essential fatty acid deficient diet. AB - Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of chromatographic techniques and fast-atom bombardment-mass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn-1 position and (iii) fatty acids at the sn-2 position. The highest apparent relative retentions were displayed by the 18:0-20:5(n-3)-PE and 16:0-22:6(n-3)-PE. It is noteworthy that the behavior of 20:3 n-9--which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n-6--was very similar to that of 20:4 n-6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n-6) and 20:3(n-9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum. PMID- 1730046 TI - Synthesis of beta-hydroxy fatty acids and beta-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics. AB - The iturinic antibiotics, which contain long chain beta-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing beta hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its beta-hydroxy fatty acids was n C16, iso C16, iso C15 or anteiso C15. The percentages of each beta-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain alpha-amino acids to the culture medium. These results demonstrate for the first time that iso C14 beta-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive beta-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [14C]acetate seems also characterize the different strains producing iturinic antibiotics. PMID- 1730047 TI - Substrate specificities of lipases A and B from Geotrichum candidum CMICC 335426. AB - The mould Geotrichum candidum produces extracellular lipases with different substrate specificities according to strains. We purified two lipases - termed lipase A and lipase B - from Geotrichum candidum CMICC 335426. The specificity of the two lipases was investigated using hydrolysis assays on emulsions of pure acylglycerols and a wide range of fatty acid esters. Lipase B was very highly specific for hydrolysis of esters of cis-delta 9-fatty acids. Lipase A did not show such strict specificity, because it hydrolysed a wider variety of fatty acid esters, in particular those of palmitic acid and isomers of oleic acid. We think that differences in specificity previously observed for crude lipases from various strains of G. candidum can be explained by the presence of different levels of specific (lipase B) and non-specific (lipase A) lipases. As lipases A and B are structurally related proteins, a minor variation in structure may be responsible for the differing specificities. PMID- 1730048 TI - Effects of cholestyramine on lipoprotein levels and metabolism in Syrian hamsters. AB - Oral administration of cholestyramine to adult male hamsters not only induced a marked decrease in plasma concentrations of cholesterol and LDL but had a similar lowering effect on plasma triacyglycerol and VLDL concentrations. The hypotriglyceridaemic effects of resin administration were not due to an increase in lipoprotein lipase, as post-heparin plasma lipoprotein lipase activities were unchanged, but rather to a 35% decrease in VLDL synthesis. Measurement of the disappearance rate of apolipoprotein B from VLDL after i.v. injection of 125I labelled hamster or human VLDL into control and cholestyramine-fed recipient animals showed a 2-times lower T1/2 in the drug-treated animals. The fraction of VLDL apolipoprotein B, recovered at any time after injection in the LDL, was equal or higher in cholestyramine-fed animals as compared to controls. These data indicate that the lowering in plasma LDL by cholestyramine in male hamsters is due not only to LDL receptor up-regulation but also to a lower rate of VLDL synthesis. No indications were found for a decreased efficiency of VLDL to LDL conversion in cholestyramine-fed animals. PMID- 1730049 TI - Hepatic uptake and processing of cholesterol and cholesteryl ester from chylomicron remnants: an in vivo study in the rat. AB - The metabolic fate of cholesterol in chylomicron remnants was studied after intravenous infusion into biliary drained rats. The remnants were radiolabelled with [3H]cholesterol in vivo, so that radioactivity was incorporated into both the unesterified (27% of label) and esterified (73% of label) cholesterol fractions, or with 14C-labelled unesterified cholesterol after exchange in vitro. Blood and bile samples were collected at intervals for 180 min, after which the animals were killed for analysis. The total amount of radioactivity found in the liver (46%) and bile (4.5%) after infusion of [3H] remnants was higher than that found when the label was 14C (33 and 2.7%, respectively). Radioactivity from 14C labelled unesterified cholesterol was cleared more rapidly from the blood, but the distribution of the label between the lipoprotein fractions VLDL + LDL, HDL2 and HDL3 at the end of the experiment was similar to that found when total [3H]cholesterol was used. In experiments with both types of label, approximately 94% of the total radioactivity secreted into bile was associated with the bile acid, with only about 6% in biliary unesterified cholesterol, and these proportions did not change during 180 min. When the chylomicron remnants were labelled with total [3H]cholesterol the specific radioactivity of the bile acid, taurochenodeoxycholic acid, in the bile was approximately twice that observed when the label was unesterified [14C]cholesterol. The specific radioactivity of unesterified biliary cholesterol was very low in the latter case, but higher and more comparable to that of taurochenodeoxycholic acid in the former. Thus, the metabolic fate of chylomicron remnant cholesterol differs, depending on whether it is in the esterified or unesterified form, suggesting that hepatic cholesterol originating from the two fractions may mix to a different extent with the various intracellular pools. In addition, the experiments with 14C indicate that the behaviour of chylomicron remnant unesterified cholesterol resembles that exhibited by cholesterol in HDL more than that carried in VLDL or LDL. PMID- 1730050 TI - Competitive inhibition of lipolytic enzymes. V. A monolayer study using enantiomeric acylamino analogues of phospholipids as potent competitive inhibitors of porcine pancreatic phospholipase A2. AB - For the first time, we have shown that a stereospecific interaction occurs between porcine pancreatic phospholipase A2 and a monomolecular film of amidophospholipid used as inhibitor. Direct binding experiments, using radiolabelled phospholipase A2, showed that 13 times more enzyme was bound to phospholipid films of the L series by comparison with films of the D series. These results were confirmed by indirect binding studies using re-spreading experiments. Kinetic studies of the porcine pancreatic PLA2, using enantiomeric acyl-amino phospholipid analogues, have shown that: (1) inhibitors of the L series are more potent than inhibitors of the D series, (2) inhibitors having a negative charge are more potent than zwitterionic inhibitors, (3) inhibitory power values are greater when evaluated in micellar system than in a the monolayer system, (4) the inhibitory power increases continuously with surface pressure. PMID- 1730051 TI - An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3? AB - An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-[35S]Met-tRNAfMet to ribosomes. In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-[35S]Met tRNAfMET/poly(A,U,G)/ribosome complexes. Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet. Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3. The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor. Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits. PMID- 1730052 TI - Expression of enzymatically active rat liver and human placental catechol-O methyltransferase in Escherichia coli; purification and partial characterization of the enzyme. AB - To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14. Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside. Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting. Both enzymes were purified from E. coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI. PMID- 1730053 TI - The function of DNA polymerases in DNA repair synthesis of ultraviolet-irradiated human fibroblasts. AB - Ultraviolet-induced DNA repair synthesis was measured in saponin-permeabilized normal human fibroblasts by the incorporation of [alpha-32P]dTMP into DNA. The involvement of DNA polymerases alpha, beta, delta, and epsilon in excision repair of pyrimidine dimers was examined using specific inhibitors. Dose-response curves resulting from experiments with up to 12 different inhibitor concentrations were analyzed by linear regression. Inhibitor concentrations at which repair activity was reduced to 50% were calculated. The following K50 values were found: aphidicolin, 0.2 microM; ddTTP, 12.5 microM; butylphenyl-dGTP, 7.6 microM; butylanilino-dATP, 6.0 microM. Comparison of K50 values with in vitro Ki values of DNA polymerases revealed that in permeabilized human fibroblasts reparative DNA synthesis is catalyzed by DNA polymerase delta and by DNA polymerase epsilon. PMID- 1730054 TI - Enhancement of DNA transfection efficiency by heat treatment of cultured mammalian cells. AB - The expression of genes introduced into various mammalian cell lines was enhanced by raising the temperature of the cells to 42 degrees C for a few hours after DNA transfection. This heat treatment resulted in an up to 10-fold increase in the frequency of the cells that transiently expressed a foreign gene such as that of beta-galactosidase, whereas it had only a limited enhancing effect on the development of stable transformants. By immunotitration analysis, it was confirmed that the enhanced expression of beta-galactosidase activity correlated well with the increase of the enzyme protein. This procedure may have an applicability for augmenting the frequency of transient gene expression in many cell types. PMID- 1730055 TI - The structure and complete nucleotide sequence of the avian lipoprotein lipase gene. AB - The entire gene for chicken lipoprotein lipase (LPL) has been isolated and characterized by primer extension and sequence analysis. The gene is 17 kilobase pairs long and comprises 10 exons and 9 introns. As determined by primer extension analysis the start sites of transcription map 176, 204 and 218 nucleotides upstream of the initiator methionine codon. The 1947 base pairs of 5' flanking sequence contains several putative regulatory elements including two adjacent Oct I binding elements, four glucocorticoid regulatory elements and a sequence very homologous to the previously described fat specific element at- 1402 nt. The first intron is very large (6433 bp) and contains four consensus SpI binding-site sequences. Five polyadenylation signals are found in the 3' untranslated region, the last three of which give predicted mRNA species identical in size to those determined by Northern blot. The 5' flanking sequences of the LPL, pancreatic lipase and hepatic lipase genes do not show homology, however. This may account for the homologous amino acid sequences but dissimilar gene expression of these enzymes. PMID- 1730056 TI - Selective inhibition of the polypeptide chain elongation in eukaryotic cells. AB - The effect of Cephalotaxus alkaloids--homoharringtonine and cephalotaxine--on translation in a cell-free system from rabbit reticulocytes and on phenylalanine polymerisation by human ribosomes was studied. The effect of the alkaloids on the nonenzymatic and the eEF-1-dependent Phe-tRNA(Phe) binding to poly(U)-programmed 80S ribosomes, diphenylalanine synthesis accompanying nonenzymatic Phe-tRNA(Phe) binding and acetylphenylalanyl-puromycin formation was examined. Homoharringtonine was shown to inhibit the formation of diphenylalanine and acetylphenylalanyl-puromycin catalysed by human and rat liver ribosomes, but was inactive as an inhibitor on the E. coli elongation system. Neither nonenzymatic nor enzymatic Phe-tRNA(Phe) binding was noticeably affected by the alkaloid. It has been proposed that the site of homoharringtonine binding to 80S ribosomes should overlap or coincide with the acceptor site of the ribosomal peptidyl transferase centre. The association constant of homoharringtonine for 80S human ribosomes was estimated to be (2.57 +/- 0.33).10(7) M-1 in the presence of puromycin. Cephalotaxine did not exert a significant influence on the polypeptide chain elongation. PMID- 1730057 TI - Succinylation of histone amino groups facilitates transcription of nucleosomal cores. AB - Treatment of nucleosomal cores with succinic anhydride, which modifies preferentially the amino-terminal domains of core histones, takes place without dissociation of the particles. Low levels of modification, which cause small structural effects, are accompanied by substantial increases in the efficiency of the nucleosomal cores as in vitro transcription templates for RNA polymerase II. The transcriptional properties of the succinylated nucleosomal cores are similar to those of the acetylated particles (Pineiro et al., (1991) Biochem. Biophys. Res. Commun. 177, 370-376), indicating that no specific blocking by acetyl residues is required to facilitate in vitro transcription. Moreover, to obtain a certain level of stimulation, a smaller number of groups has to be modified by succinic than by acetic anhydride. PMID- 1730058 TI - Differential translatability of antifreeze protein mRNAs in a transgenic host. AB - The expression of fusion gene constructs containing Drosophila regulatory sequences and the structural portions of fish antifreeze protein genes have been examined by transfer into Drosophila melanogaster using P elements. A fusion gene, containing the enhancer, promoter, and cap site of the yolk polypeptide 1 gene, joined in the 5'-untranslated region to the structural portion of the winter flounder type I antifreeze gene, was transcribed in mature female transformants to give an mRNA of the predicted size, but no antifreeze protein was detected by Western blotting. When the same antifreeze protein gene was fused to a Drosophila hsp 70 gene regulatory region and placed downstream of the yolk polypeptide gene enhancer, appropriate expression of mRNA was directed by both gene regulatory elements. However, a translation product from this mRNA was only observed under heat shock conditions and was present at low levels. It is suggested that type I antifreeze mRNA, with its high content of alanine codons and their grouping into clusters of up to seven in a row, is poorly translated when in competition with other host mRNAs. In agreement with this hypothesis, a fusion gene construct between the yolk protein gene regulatory region and two type III antifreeze protein genes produced sub-mmolar concentrations of antifreeze protein in mature females from each of several transgenic lines analysed. The type III antifreeze protein does not have an imbalanced amino acid composition or sequence irregularities, and may be an appropriate choice for conferring freeze protection to frost-susceptible hosts by gene transfer. PMID- 1730059 TI - Differential mechanisms of induction of ornithine decarboxylase in rat intestine by L- and D-amino acids. AB - A single intragastric administration of glycine, L- and D-alanine, and L-and D serine into rats resulted in a more than 20-fold stimulation of intestinal mucosal ornithine decarboxylase (ODC) within 4 h. The stimulation of ODC activity was accompanied by an increase in the amount of immunoreactive ODC protein. The induction of ODC by D-amino acids was in all likelihood attributable to an enhanced accumulation of ODC-specific mRNA species as revealed by Northern blot and dot-blot hybridization analyses. However, the induction by glycine and L amino acids was not explainable by changes of mRNA since the changes in mRNA contents were only marginal. Since the turnover rates of L-serine-induced and D serine-induced intestinal ODC protein were the same as the non-induced control, we concluded that the induction by glycine and L-amino acids was brought about by an increased efficiency of translation of the ODC message. PMID- 1730060 TI - Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene. AB - The human gene encoding pituitary adenylate cyclase activating polypeptide (PACAP) was isolated and its nucleotide sequence was determined. By comparison with a human PACAP cDNA, the exon/intron organization of PACAP gene was determined. The last exon encoded the longer form of PACAP, PACAP38 and 3' untranslated sequences, suggesting that the shorter form of PACAP, PACAP27 is not generated by alternative splicing mechanisms. The 5'-flanking region of the PACAP gene contains several sequence motifs homologous to CRE, TRE, and GHF-1. On the basis of DNA isolated from mouse A9 microcell hybrid clone containing a single human chromosome, the PACAP gene was assigned to human chromosome 18. Furthermore, we determined the locus of the gene to be 18p11 by the chromosomal in situ hybridization technique. PMID- 1730061 TI - Completion of the nucleotide sequence of the 'maltose B' region in Salmonella typhimurium: the high conservation of the malM gene suggests a selected physiological role for its product. AB - We have subcloned and sequenced the genes malF and malM of Salmonella typhimurium, thereby completing the determination of the nucleotide sequence of its 'maltose B' regulon. The malM gene, encoding a periplasmic protein of unknown function in Escherichia coli, is a highly conserved as genes encoding proteins of known function from the same region. PMID- 1730062 TI - The nucleotide sequence of leuB from Salmonella typhimurium. AB - The nucleotide sequence and deduced polypeptide sequence of the Salmonella typhimurium leuB are reported, as well as a conserved region that might bind the enzyme substrate. PMID- 1730063 TI - Molecular cloning of a cDNA encoding a novel protein related to the neuronal vesicle protein synaptophysin. AB - The structure of synaptophysin, an integral membrane protein present in synaptic vesicles, is highly conserved. We report the sequence analysis of a clone, HL-5, isolated from a human erythroleukemia cell cDNA library, that is similar to synaptophysin in DNA and amino acid sequence. The predicted protein product derived from this clone is truncated, and the tissue distribution of HL-5 is different from that of synaptophysin. Thus, HL-5 appears to be a member of a previously undescribed family of synaptophysin-like genes. PMID- 1730064 TI - Unique use of alternative polyadenylation signals in the mouse aldolase B gene. AB - We isolated and sequenced two mouse aldolase B cDNAs. They differ only in the length of the 3' untranslated region. This is consistent with Northern blot analysis of liver RNA which shows two transcripts differing by 400 nucleotides. We also isolated and sequenced the corresponding 3' genomic region and found four polyadenylation signals in the final exon. RNase protection studies demonstrate that all four of these signals are utilized, but not equally. This is unique to the mouse aldolase B gene. PMID- 1730065 TI - Multiple forms of rat calpastatin cDNA in the coding region of functionally unknown amino-terminal domain. AB - Partial mouse and rat calpastatin cDNAs containing functionally unknown amino terminal regions were cloned by the polymerase chain reaction (PCR) method. Two types of clones were obtained for the rat calpastatin; one had a sequence similar to mouse and human calpastatins, while the other had a deletion of 38 amino acid residues in this region. Both types of the rat sequence differed from the previously reported rat calpastatin which had additional deletions. The occurrence of multiple forms of calpastatin suggests alternative splicing in the functionally unknown domain. PMID- 1730066 TI - Growth and morphogenetic factors in bone induction: role of osteogenin and related bone morphogenetic proteins in craniofacial and periodontal bone repair. AB - Bone has considerable potential for repair as illustrated by the phenomenon of fracture healing. Repair and regeneration of bone recapitulate the sequential stages of development. It is well known that demineralized bone matrix has the potential to induce new bone formation locally at a heterotopic site of implantation. The sequential development of bone is reminiscent of endochondral bone differentiation during bone development. The collagenous matrix-induced bone formation is a prototype model for matrix-cell interactions in vivo. The developmental cascade includes migration of progenitor cells by chemotaxis, attachment of cells through fibronectin, proliferation of mesenchymal cells, and differentiation of bone. The bone inductive protein, osteogenin, was isolated by heparin affinity chromatography. Osteogenin initiates new bone formation and is promoted by other growth factors. Recently, the genes for osteogenin and related bone morphogenetic proteins were cloned and expressed. Recombinant osteogenin is osteogenic in vivo. The future prospects for bone induction are bright, and this is an exciting frontier with applications in oral and orthopaedic surgery. PMID- 1730067 TI - Ontogeny of immunity to oral microbiota in humans. AB - This article reviews the ontogeny of immune systems in the human oral cavity that may influence the colonization, accumulation, or pathogenesis of oral microbiota. The prenatal development of cellular components associated with the secretory immune system reveals that the initial organization of tissue into Peyer's patches can first be detected immunohistologically at 11 weeks gestation. Epithelial cells positive for secretory component and immunocytes positive for IgM can be detected in salivary gland tissue by 19 to 20 weeks and continue to predominate during gestation. After birth, immunocytes containing IgA begin to dominate. Essentially, no IgA can be detected in saliva at birth. However, salivary IgA and IgM often appear soon thereafter, presumably in response to environmental antigenic and mitogenic challenges. Salivary IgA in young infants has molecular characteristics of secretory IgA and becomes the quantitatively predominate Ig in saliva. Both IgA subclasses are present in proportions characteristic of adult pure glandular salivas in many 1- to 2-month-old infants, although the appearance of IgA2 is delayed in some subjects. Many innate, antibody, and cellular immune components are found in maternal colostrum and breast milk. The antibacterial properties of these maternal factors are diverse and can exert multifaceted protective effects on the infant's alimentary tract. The infant apparently can mount mucosal immune responses quite early in life. For example, salivary antibody activity to organisms that originally colonize the gut (e.g., E. coli) or the oral cavity (e.g., S. mitis, S. salivarius) can be detected by 1 to 2 months of age. Most of this antibody activity has characteristics of secretory IgA, although some IgM antibody can also be initially detected. Salivary IgA1 and IgA2 antibody specificities to S. mitis and S. salivarius components increase qualitatively and quantitatively during the first few years of life. Salivary IgA antibody to components of streptococci that require hard surfaces for colonization (e.g., S. sanguis and mutans streptococci) generally appear after tooth eruption. The loss of placentally derived maternal IgG antibody specificities to these microbiota in the circulation is replaced by de novo synthesis, presumably as a result of the teething process. These IgG antibodies can enter the oral cavity in the gingival crevicular fluid and by the process of teething.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730068 TI - Proteoglycans of oral tissues. PMID- 1730069 TI - In vitro studies on the regulation of endochondral ossification by vitamin D. AB - The research described in this article has focused on the complex autocrine, paracrine, and endocrine regulation of endochondral ossification using vitamin D metabolites and TGF-beta as models. By comparing results from a number of laboratories utilizing a diverse array of in vivo and in vitro systems, a coherent picture is beginning to emerge. Vitamin D metabolites influence cell differentiation and maturation and have direct effects on cell function. Differentiation of the mesenchymal cells into chondroblasts is regulated by both 1,25-(OH)2D3 and 24,25-(OH)2D3, as well as by TGF-beta. The resting zone chondrocytes respond primarily to 24,25-(OH)2D3 in terms of matrix synthesis and matrix vesicle biochemistry. They synthesize both metabolites and other factors that stabilize matrix vesicle enzymes like AHSG. In addition to the paracrine role these factors may play in regulating the matrix, it is possible that they may influence the cells in the growth plate itself. Growth zone chondrocytes also synthesize both metabolites, but respond primarily to 1,25-(OH)2D3 for the parameters measured in the studies described. These cells also synthesize TGF beta which further increases alkaline phosphatase activity, perhaps via an autocrine stimulation of the cell. While cells from the calcified zone have not yet been studied directly in culture, it is likely that they respond to paracrine signals from the avascular cartilage as well as to serum-derived factors. How the signals are transferred among the cells is unknown. Certainly one can postulate information flow in both upward and downward directions. The signal transduction mechanisms for the factors at the cellular level are complex. While it is known that 1,25-(OH)2D3 stimulates gene transcription and stabilization of mRNA for proteins like alkaline phosphatase, its nongenomic effects are only beginning to emerge. Membrane effects of this metabolite have been shown in intestine and kidney in conjunction with studies on Ca flux. It is becoming increasingly evident that other steroid hormones may operate in similar ways. Studies with the rat costochondral chondrocytes are the first to show that there are specific membrane effects for at least two vitamin D metabolites and that membrane enzymes, including those involved in phospholipid metabolism, can be differentially regulated by them. Furthermore, these experiments have provided for the first time a clear hypothesis for how cells can regulate events in the extracellular matrix after the matrix vesicles are produced and incorporated into the matrix. PMID- 1730070 TI - Host mediators in gingival crevicular fluid: implications for the pathogenesis of periodontal disease. AB - During the past few years, a considerable number of studies have examined different aspects of the host response in gingival crevicular fluid (GCF), including the relationship of specific markers to the active phases of periodontal disease. Various indicators of the acute inflammatory response (the lysosomal enzymes beta-glucuronidase and collagenase, the cytoplasmic enzyme aspartate aminotransferase, and the arachidonic acid metabolite PGE2) have been shown to be associated with clinical attachment loss in chronic adult periodontitis in man and experimental periodontitis in animal models. In contrast, the relationship of indicators of the humoral immune response in GCF to active periodontal disease is equivocal. Furthermore, a number of indicators of the cellular immune response have been identified recently in GCF (i.e., Interleukin-1 alpha, IL-1 beta, tumor necrosis factor-alpha), but their relationship to active phases of periodontal disease have not been studied. The polymorphonuclear leukocyte (PMN) is the cellular hallmark of acute inflammation. Evidence from the GCF studies suggests that hyperreactivity of these cells plays a critical role in the active phases of some forms of periodontal disease. Metabolic activation of PMN can be associated with a number of potentially destructive reactions. The major effector mechanism for tissue destruction that can be specifically identified with the PMN is the synergistic effect of the release of PMN proteases and the generation of reactive oxygen metabolites by these cells. Priming of the PMN, where the PMN response is enhanced by agents that do not initiate the response, may be an important mechanism for PMN activation in the crevicular environment; for example, cytokines such as IL-1 beta and TNF-alpha, and lipopolysaccharides released from subgingival Gram negative bacteria, can serve this function. The hypothesis proposed here argues that in addition to the severe forms of periodontal disease that have been associated with qualitative or quantitative PMN defects, tissue destruction in the periodontum can be observed with hyperreactivity of these cells. These differing conclusions do not create a dilemma, but may represent opposite ends of a balance that is no longer in equilibrium. PMID- 1730071 TI - The role of brushite and octacalcium phosphate in apatite formation. AB - Studies of apatite mineral formation are complicated by the possibility of forming several calcium phosphate phases. The least soluble, hydroxyapatite (HAP), is preferentially formed under neutral or basic conditions. In more acidic solutions phases such as dicalcium phosphate dihydrate (Brushite, DCPD) and octacalcium phosphate (OCP) are often found. Even under ideal HAP precipitation conditions the precipitates are generally nonstoichiometric, suggesting the formation of calcium-deficient apatites. Both DCPD and OCP have been implicated as possible precursors to the formation of apatite. This may occur by the initial precipitation of DCPD and/or OCP followed by transformation to a more apatitic phase. Although DCPD and OCP are often detected during in vitro crystallization, in vivo studies of bone formation rarely show the presence of these acidic calcium phosphate phases. In the latter case the situation is more complicated, since a large number of ions and molecules are present that can be incorporated into the crystal lattice or adsorbed at the crystallite surfaces. In biological apatite, DCPD and OCP are usually detected only during pathological calcification where the pH is often relatively low. In normal in vivo calcifications these phases have not been found, suggesting the involvement of other precursors or the formation of an initial amorphous calcium phosphate phase (ACP) followed by transformation to apatite. PMID- 1730072 TI - Signal transduction mechanisms involved in salivary gland regulated exocytosis. PMID- 1730073 TI - Immunoelectron microscopy of enzymes, multienzyme complexes, and selected other oligomeric proteins. AB - The collective term "immunoelectron microscopy" subsumes a number of techniques in which the biological material is decorated with specific antibodies, prior to being visualized in the electron microscope. In this article, we have reviewed literature on immunoelectron microscopy that focusses on the analysis of the molecular architecture of proteins, in particular of enzymes and of multienzyme complexes. Molecular immunoelectron microscopy has been remarkably successful with multi-subunit enzymes of complex quaternary structures, and in many cases the data have been the basis for the eventual development of detailed three dimensional molecular models. The elucidation of subunit composition and juxtaposition of a given enzyme, an important accomplishment in itself, has in turn stimulated and guided discussions on the catalytic mechanism; illustrative examples include F1 ATPase and citrate lyase, among others. Here we have chosen a variety of enzymes, multienzyme complexes, and non-enzymatic proteins to demonstrate the versatility of immunoelectron microscopy, to illustrate methodological prerequisites and limitations, and to discuss significance and implications of individual immunoelectron microscopy studies. PMID- 1730074 TI - Ultrastructure appearance of atherosclerosis in human and experimentally-induced animal models. AB - We describe here the basic structure of the aorta, the changes with aging and ultrastructural appearance of atherosclerosis of human and animal models. The architecture of the aortic wall is highly organized, for adaptation to changes of blood pressure. The main cells composing the vessel are endothelial cells and smooth muscle cells. They maintain the integrity and homeostasis of the aorta along with the extracellular matrix of collagen fibrils, elastic fibers and glycosaminoglycans. The structural changes with aging and atherogenesis are a compensative or degenerative phenomenon caused by many factors. Three major cells are the endothelial cell, smooth muscle cell and monocyte-derived macrophages (as well as platelets) all of which are involved in atherogenesis. Foam cells in atheromatous lesions are derived from macrophages and smooth muscle cells. Recently, the molecular biological nature and function of these cells and their derived-factors have been thoroughly investigated in cell culture and in experimental animal models caused by a mechanical injury of the endothelium or by a dietary induced hypercholesterolemia. However, the mechanism of the endothelial injury in vivo as well as formation of atheromatous cores of human atherosclerosis is not exactly understood. Some structural and functional changes inherent to the arterial wall during aging may play an important role in initiation or progression of human atherosclerosis. PMID- 1730075 TI - Cryo-electron microscopy of vitrified muscle samples. AB - A great deal of information on the 3-dimensional structure of the protein assemblies involved in muscle contraction has been obtained using conventional transmission electron microscopy. In recent years, developments in cryo-electron microscopy have facilitated work with fully hydrated, non-chemically fixed specimens. It is shown how this technique can be used to visualize muscle sarcomere filaments in quasi-native conditions, to access hitherto inaccessible states of the crossbridge cycle, and to obtain new high resolution structural information on their 3-dimensional protein structure. A short introduction to the crossbridge cycle and its biochemically accessible states illustrates the problems amenable to studies using the electron microscope, as well as the possibilities offered by cryo-microscopy on vitrified samples. Work on vitrified cryo-sections and myosin filament suspensions demonstrates the accessibility of crossbridge states and gives implications on the gross structural features of myosin filaments. Recent studies on actin filaments and myosin (S1) decorated actin filaments provide the first high resolution data on vitrified samples. The use of photolabile nucleotide precursors allows the trapping of short lived states in the millisecond time range, thereby visualizing intermediate states of the crossbridge cycle. PMID- 1730076 TI - Electron spectroscopic imaging of chromatin and other nucleoprotein complexes. AB - Electron spectroscopic imaging (ESI) is a microanalytical technique which is being used to determine elemental distributions at the resolution limit of the electron microscope. Detection and mapping of phosphorus by energy filtered imaging makes it possible to determine the organization of the nucleic acid component in nucleoprotein complexes, because phosphorus is present at much higher levels in nucleic acids than in the associating proteins. ESI has provided a method for approaching numerous questions related to chromatin structure at the level of the specific protein-DNA interactions, at the nucleosome level and at higher organizational levels of chromosome structure. PMID- 1730077 TI - Freeze-substitution studies of bacteria. AB - Typically, models of bacterial structure combine biochemical data obtained from bulk analyses of cell populations with electron microscopic observation of individual cells. Recent development of a battery of cryotechniques specific for biological electron microscopy have begun to supercede routine procedures such as conventional thin sectioning. One of these cryotechniques, freeze-substitution, combines the advantages of ultrarapid freezing with standard microtomy methods. This technique is particularly well suited to the examination of bacterial structure and has yielded additional ultrastructural information consistent with biochemical data but often challenging models of cell structure obtained from conventional microscopical methods. In addition to retaining more accurately the spatial distribution of cell components, freeze-substitution has been successfully combined with immunochemical labelling techniques and has enabled identification and localization of specific molecules both within the cell and on the cell surface. In this review, I describe current ideas on bacterial ultrastructure, modified in accordance with new data obtained from recent freeze substitution studies. PMID- 1730078 TI - Expression of activin A/erythroid differentiation factor in murine bone marrow stromal cells. AB - Accumulating evidence suggests that activin A/erythroid differentiation factor (EDF), a homodimer of beta A chain, is a physiologic hematopoietic factor, particularly of erythroid lineage. Media conditioned by phorbol myristate acetate (PMA)-stimulated murine bone marrow stromal cell lines, MC3T3-G2/PA6 and ST2, contained activin A/EDF, assayed as the activity inducing erythroid differentiation of an activin A/EDF-responsive murine erythroleukemia cell clone F5. Follistatin, a protein specifically binding to activin A/EDF, abolished this erythroid differentiation. Northern blot analysis showed that PMA rapidly increased beta A chain messenger RNA levels in MC3T3-G2/PA6 and ST2 cells. In a search for natural stimulators, we found that tumor necrosis factor-alpha, in itself and synergistically with interleukin-1 beta, induced activin A/EDF production in both cell lines. These results indicate that stromal cells produce activin A/EDF in bone marrow. PMID- 1730079 TI - Iron chelation with desferrioxamine B in adults with asymptomatic Plasmodium falciparum parasitemia. AB - To determine if iron chelation therapy has activity against human malaria, we administered desferrioxamine B in amounts of 100 mg/kg per day by continuous 72 hour subcutaneous infusions to 28 volunteers with asymptomatic Plasmodium falciparum infection in a randomized, double-blind, placebo-controlled crossover trial. Peripheral blood concentrations of P falciparum ring forms were determined at 12-hour intervals in all subjects and serum concentrations of desferrioxamine B + ferrioxamine (the iron complex of desferrioxamine B) were measured in 26 subjects. Geometric mean concentrations of asexual intraerythrocytic parasites decreased with both chelator and placebo treatment, but the decrement with desferrioxamine B was significantly greater than that with placebo (P less than .006) during both the initial and crossover periods. Compared with placebo, desferrioxamine B treatment was associated with an almost 10-fold enhancement of the rate of parasite clearance during both phases of the trial (P less than .007). Mean +/- SEM steady state concentrations of desferrioxamine B + ferrioxamine were 6.90 +/- 0.60 mumol/L at 36 hours and 7.72 +/- 0.68 mumol/L at 72 hours; in vitro, the ID50 has been reported to be approximately 4 to 20 mumol/L. No drug toxicity was detected. Parasitemia recurred in 19 of 24 participants followed-up over 1 to 6 months. We conclude that desferrioxamine B enhances the clearance of P falciparum parasitemia and that iron chelation may provide a new strategy to be developed for the treatment of malaria. PMID- 1730080 TI - Cytarabine plus idarubicin or daunorubicin as induction and consolidation therapy for previously untreated adult patients with acute myeloid leukemia. AB - The purpose of this study was to determine the relative merits of idarubicin and daunorubicin in acute myeloid leukemia (AML) therapy. Thirty-two sites provided 214 previously untreated adults with AML aged 15 years or more who were randomized to receive for induction therapy cytarabine 100 mg/m2/d as a continuous 7-day infusion plus either daunorubicin 45 mg/m2/d (A + D) or idarubicin 13 mg/m2/d (A + I), daily on the first three days of treatment. Postremission therapy consisted of two courses of the induction regimen at the same daily doses, with the anthracycline administered for 2 days and cytarabine for 5. The complete response (CR) rates for evaluable patients were 70% (A + I) and 59% (A + D) (P = .08). The difference in CR rates was significant in patients aged 18 to 50 years (88% for A + I, 70% for A + D, P = .035). Resistant disease was a significantly more frequent cause of induction therapy failure with A + D than with A + I. Hyperleukocytosis (white blood cell count greater than 50,000/microL) unfavorably affected the attainment of CR with A + D but not with A + I. CR duration was significantly greater after A + I. CR duration was significantly greater after A + I treatment, and the survival of all randomized patients treated with A + I was significantly better than that observed after A + D treatment (median 12.9 months v 8.7 months, respectively, P = .038). Toxicity of the two treatments was similar, although A + I patients experienced more prolonged myelosuppression during consolidation therapy, and a greater incidence of mild chemical hepatitis was observed in the A + I group. It is concluded that, at the doses and schedule used in this study, A + I is superior to A + D for induction therapy of AML in adults. PMID- 1730081 TI - Dexamethasone recruitment of self-renewing osteoprogenitor cells in chick bone marrow stromal cell cultures. AB - Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis. PMID- 1730082 TI - Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP 33) that is expressed on the surface of activated platelets. AB - To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before. PMID- 1730083 TI - Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome. AB - The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites. PMID- 1730084 TI - A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage. AB - Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site-directed DNA-mediated mutagenesis. Thrombin-activated human recombinant-derived factor VIII was isolated in an active form by passage over Mono-S fast protein liquid chromatography. The peak fractions had a specific activity of 60,000 U/mg. The subunit composition in the peak fraction contained the 50-Kd A1 domain from the heavy chain, the 73-Kd light chain fragment, and trace amounts of the 43-Kd A2 domain. The requirement of domain A2 for functional activity was shown in several ways. First, the addition of an inhibitory monoclonal antibody that recognizes domain A2 destroyed factor VIIIa activity. Second, addition of a Mono-S FPLC fraction that contained the A2 domain polypeptide back to the peak activity fraction increased activity of the factor VIIIa by 22-fold. The maximum specific activity achieved was 180,000 U/mg. Finally, expression of an A2 domain deletion mutant did not yield procoagulant activity, although the mutant was effectively secreted from the cell, exhibited appropriate heavy and light chain association, and was susceptible to thrombin cleavage. Cotransfection of this A2 domain deletion mutant with an A2 domain expression vector yielded a secreted complex and restored procoagulant activity in the conditioned medium. This result shows that the A2 domain can fold and assemble with A2-deleted factor VIII to yield a functional molecule. We conclude that the A2 domain is required for functional factor VIIIa activity and loss of activity in activated factor VIII may result from dissociation of A2 from the thrombin-activated heterotrimer. PMID- 1730085 TI - Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation. AB - To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/ 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high affinity Ca2+ binding sites in the Gla domain of factor IX. PMID- 1730086 TI - Endotoxin enhances the expression of monocyte prothrombinase activity. AB - Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner. Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles. Endotoxin was without effect on the expression of lymphocyte prothrombinase activity. Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes. Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold. The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time. In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin. The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses. The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter. 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin. The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles. In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells. These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process. Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms. PMID- 1730087 TI - Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1. AB - A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296-G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296 G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296-G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296 G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730088 TI - Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ib alpha leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease. AB - The primary sequences of the three individual glycoprotein (GP) chains, GPIb alpha, GPIb beta, and GPIX, comprising the normal platelet GPIb/IX receptor for von Willebrand factor (vWF) have recently been determined, opening the possibility for characterization of disorders of this receptor at the molecular level. The presence of a leucine tandem repeat in each of these chains is of particular interest, because such repeats may be involved in associations between polypeptide segments. We now report an autosomal dominant variant of Bernard Soulier disease associated with the heterozygous substitution of phenylalanine for a highly conserved leucine residue within the GPIb alpha leucine tandem repeat. Affected individuals experienced a moderate bleeding tendency, thrombocytopenia, and an increased mean platelet volume. Platelet aggregation was decreased only in response to ristocetin or to asialo-vWF. The kd for 125I-vWF binding to patients platelets was significantly increased over control values at 0.5 mg/mL ristocetin, but was normal at 1.0 or 1.5 mg/mL ristocetin. While sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an essentially normal complement of all components of the GPIb/IX complex, a minor amount of a putative proteolytic fragment was identified that migrated faster than GPIb and was immunoreactive with polyclonal anti-GPIb alpha antibody, but not with a monoclonal antibody directed against the 45-Kd amino-terminal region of GPIb alpha. However, because the great majority of patient GPIb alpha comigrates with normal GPIb alpha, the major functional abnormalities of the patient platelets are most likely a consequence of the altered structure of the nonproteolyzed protein. Full concordance within the studied family between phenotypic expression and a heterozygous single nucleotide substitution in genomic DNA coding for a phenylalanine in place of the wild-type leucine at residue 57 of the mature GPIb alpha, absence of this substitution in 266 alleles from the normal population, and the lack of any other abnormality of patient DNA throughout the entire coding sequence for GPIb alpha provide strong support that this substitution may constitute a pathologic point mutation responsible for the observed phenotypic abnormalities. While the roles that leucine tandem repeats may normally play within the GPIb/IX complex are not yet known, the perturbation of such a repeat in GPIb alpha may impair interaction with other components of the complex and/or with the binding of vWF. PMID- 1730089 TI - A human monoclonal autoantibody to platelet glycoprotein IIb derived from normal human lymphocytes. AB - Tonsillar lymphocytes from an otherwise healthy nonthrombocytopenic male child were fused with the lymphoblastoid cell line GM 4672. Twenty of 472 (4%) hybridomas had antiplatelet reactivity detected using intact platelets in an enzyme-linked immunosorbent assay. One hybridoma (STO 171) reacted to platelet glycoprotein IIb (integrin alpha IIb) as determined by radioimmunoprecipitation and immunoblotting. Antibody specificity was confirmed using immunodepletion experiments with isotypic antibodies derived from a mutlitransfused Glanzmann's thrombasthenic patient. The antibody reactivity was restricted to platelets and did not react with other integrin alpha-chain proteins expressed on granulocytes or cultured human brain-derived microvascular endothelial cells. These studies indicate that lymphocytes of normal, nonthrombocytopenic individuals have the genetic potential to produce antiplatelet autoantibodies. PMID- 1730090 TI - Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. AB - NF-IL6 was originally identified as a DNA binding protein regulating interleukin 1 (IL-1)-stimulated IL-6 expression. Direct cloning of NF-IL6 showed its homology with C/EBP, a hepatocyte- and adipocyte-specific transcription factor. This study showed that the expression of NF-IL6 messenger RNA (mRNA) increased markedly during the differentiation to a (mRNA) increased markedly during the differentiation to a macrophage lineage in mouse myeloid leukemia cells M1, human histiocytic leukemia cells U937, promyelocytic leukemia cells HL-60, and human peripheral monocytes. Particularly in HL-60 cells that undergo granulocyte or macrophage differentiation depending on inducers, NF-IL6 mRNA was specifically upregulated during macrophage differentiation but not granulocyte differentiation. It was also shown that the functional NF-IL6 protein increased during the differentiation of U937 cells. Furthermore, recombinant NF-IL6 was found to bind to the regulatory regions of the IL-1, tumor necrosis factor, granulocyte colony-stimulating factor, and lysozyme genes, which are expressed in mature macrophages. These results suggest that NF-IL6 may possibly be involved as an important transcription factor in the process of activation and/or differentiation of macrophages. PMID- 1730091 TI - Effect of recombinant human growth hormone on acute and chronic human immunodeficiency virus infection in vitro. AB - Growth hormone (somatotropin) is a potent anabolic protein currently being evaluated clinically in cachexia associated with malignancy and human immunodeficiency virus (HIV) disease. Growth hormone can also lead to enhancement of lectin-mediated cellular proliferation, macrophage activation, and cytokine induction, events linked to induction of latent HIV in vitro. We thus explored the ability of recombinant human growth hormone (rhGH) to affect viral replication in acute and chronic HIV infection, and to alter transcription at the HIV-1 long terminal repeat (LTR). A clone of promonocytic cells, chronically infected with HIV-1 and susceptible to viral induction by a variety of cytokines and protein kinase C activators, was unperturbed by rhGH used over broad concentrations (10 to 500 ng/mL) and time intervals. This unresponsiveness paralleled the lack of effect of rhGH on HIV-associated trans-activation in both monocytic and CD4+ T-cell lines. In contrast, rhGH enhanced viral replication in acutely infected peripheral blood mononuclear cells (PBMC) by twofold to 20-fold, albeit having no adverse effect on the antiviral efficacy of zidovudine (AZT). Augmentation of HIV growth correlated with stimulation of cellular DNA synthetic responses and an increase in tumor necrosis factor-alpha (TNF-alpha) secretion. These data are discussed in the context of ongoing clinical trials of rhGH in HIV seropositive individuals with wasting syndromes. PMID- 1730092 TI - Mutations of the p53 gene in adult T-cell leukemia. AB - The p53 tumor suppressor gene was examined by direct sequencing of polymerase chain reaction-amplified DNA from fresh tumor cells of 10 patients with adult T cell leukemia (ATL). Samples included nine patients with acute or lymphomatous ATL, and one patient in whom samples were examined in both his acute and chronic stages of ATL. Four missense mutations and one silent point mutation in the coding region of the p53 gene were found in cells from five patients with either acute or lymphomatous ATL. The missense mutations were homozygous and occurred in evolutionarily highly conserved regions of p53. One patient had no p53 mutation in his leukemic cells during chronic phase of ATL, but had a homozygous point mutation at codon 273 (Arg to His) when he progressed to acute ATL. In summary, we show that p53 is frequently mutated in the acute phase of ATL and one informative case suggests that p53 mutations may be associated with the transition from chronic to acute ATL. PMID- 1730093 TI - Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells. AB - The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the Ig loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bcl-2 locus were evident in only one of the myeloma cell lines, U-266-1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266-1984), suggesting that bcl-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast to previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bcl-2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells. PMID- 1730094 TI - Clinical and immunologic effects of prolonged infusion of low-dose recombinant interleukin-2 after autologous and T-cell-depleted allogeneic bone marrow transplantation. AB - Bone marrow transplantation (BMT) can produce prolonged clinical remission in some patients with hematologic malignancies. Unfortunately, disease relapse may occur despite BMT. Studies in animal models and clinical experience have provided evidence that immunologic factors play an important role in preventing relapse post-BMT. To stimulate immunologic activity in patients post-BMT, we administered prolonged uninterrupted continuous infusions of low-dose recombinant interleukin 2 (rIL-2). Thirteen marrow recipients (seven autologous BMT, six CD6 T-depleted allogeneic BMT) received rIL-2 at a dose of 2 x 10(5) U/m2/d for a scheduled period of 90 days. rIL-2 was administered through a Hickman catheter with a portable pump beginning a median of 85 days after BMT. Toxicity was minimal and all treatment could be undertaken in the outpatient setting. No patient developed any signs of graft-versus-host disease, hypotension, or pulmonary capillary leak syndrome. Treatment did not affect the absolute neutrophil count or hemoglobin level, but eosinophils increased substantially in most patients. Platelet counts decreased by 20% in 10 of 13 individuals within 2 weeks, but stabilized thereafter. Despite the low dose of rIL-2 administered, significant immunologic changes were noted. Specifically, all 13 patients experienced a marked increase (fivefold to 40-fold) in natural killer (NK) cell number. Phenotypic characterization showed that the majority of NK cells were CD56bright+ CD16+ CD3 . In contrast, a minor increase in T-cell number was noted in only 4 of 13 patients. Low-dose rIL-2 treatment resulted in augmentation of in vitro cytotoxicity against K562 and COLO tumor targets. This cytotoxic activity could be dramatically enhanced by incubation with additional rIL-2 in vitro. The immunologic effects of rIL-2 treatment were similar in both autologous and allogeneic marrow recipients. Our data suggest that prolonged infusion of rIL-2 at low doses is safe and can selectively increase NK cell number and activity after BMT. Further studies to assess the impact these changes may have on disease relapse post-BMT will be undertaken. PMID- 1730095 TI - Selection of histocompatible apheresis platelet donors by cross-matching random donor platelet concentrates. AB - It can be impossible to identify compatible platelet donors for alloimmunized patients whose HLA type cannot be determined or who have uncommon HLA types. We have previously shown that histocompatible donors can be rapidly identified by "mass screening" of platelet concentrates (PC), which are readily available in all blood banks, using a solid-phase adherence platelet cross-matching technique. Compatible PC were given to five alloimmunized patients with multispecific HLA antibodies refractory to random donor (RD) PC and selected single-donor platelet transfusions. After transfusions which produced satisfactory responses, we identified the original whole blood donors to serve as apheresis donors. Thus, the donors selected were compatible in vitro by cross-matching, and in vivo by transfusion. Only 3% to 13% of PC cross-matched for these alloimmunized patients were potentially compatible and it was necessary to screen large numbers (65 to 205 U) of PC per patient. Eighteen of 22 PC-selected transfusions produced satisfactory increments, allowing selection of 12 donors, all of whom were willing to undergo apheresis. Ten of 12 of these single-donor transfusions were successful; the two unsuccessful transfusions were infused 2 weeks after the initial PC cross-match and were still compatible with the original serum, but incompatible with more recent serum, demonstrating a change in antibody reactivity. The HLA types of the successful single donors selected by PC cross matching differed widely from the patients' HLA types and, therefore, these donors would not have been selected by standard approaches using HLA typing. Cross-matching large numbers of RD PC for the identification of apheresis donors is helpful in the management of the alloimmunized patient and may be of particular utility for blood centers that do not have access to HLA-typed donor pools. PMID- 1730096 TI - Anemic Gaucher patients with elevated endogenous erythropoietin levels may not respond to recombinant erythropoietin therapy. PMID- 1730097 TI - Erythropoietin and decreased erythropoiesis in pregnancy. PMID- 1730098 TI - All-trans retinoic acid: not only a differentiating agent, but also an inducer of thromboembolic events in patients with M3 leukemia. PMID- 1730099 TI - Rheumatoid arthritis in thyroid disease positive and negative same-sexed sibships. AB - A total of 249 rheumatoid arthritis (RA) same-sexed sibships were clinically documented, HLA-typed and had auto-antibody screens performed. Sibships with a history of thyroid disease (TD) or significant thyroid antibodies were categorized as 'thyroid' sibships and the rest 'non-thyroid'. TD was more common in the female RA sibships than controls, particularly in the RA probands. The presence of thyroid microsomal antibody, but not thyroglobulin antibody, was significantly higher in all members of the female sibships, and in the probands and non-RA siblings of the male sibships. Comparing the thyroid and non-thyroid sibships, there was no significant difference in the distribution of HLA haplotypes in RA-RA sibling pairs, and HLA-DR status, clinical or immunological characteristics of the probands. These data do not support the concept that predisposition to RA in thyroid sibships might be non-DR4 or non-HLA linked. PMID- 1730100 TI - IL-1 secreting cell assay and its application to cells from patients with rheumatoid arthritis. AB - The cells within a population that were secreting interleukin-1 (IL-1) were enumerated and visualized by an ELISA-SPOT assay. Initial experiments designed to validate the assay revealed that the number of IL-1 beta spot forming cells was increased by exposing normal blood monocytes to LPS and that spot formation was prevented by incubating the cells with cycloheximide. Normal blood polymorphonuclear leucocytes (PMNs) produced IL-1 alpha and IL-1 beta in response to recombinant granulocyte monocyte colony stimulating factor (rhGMCSF) but not to cytochalasin B, calcium ionophore or LPS. Monocytes and PMN were isolated from the synovial fluid (SF) and blood of patients with rheumatoid arthritis (RA) and the ability of these cells to secrete IL-1 alpha and IL-1 beta compared. A higher proportion of SF derived monocytes were found to secrete IL-1 beta spontaneously compared to the corresponding blood cells. IL-1 alpha secreting monocytes were not detected although high numbers of IL-1 alpha secreting cells were found among cells isolated from rheumatoid synovium. By contrast SF PMNs did not produce IL-1 alpha or IL-1 beta whereas blood PMNs from some (3/8) RA patients produced IL-1 alpha and/or IL-1 beta. It is considered that the IL-1 ELISA-SPOT is a highly sensitive technique for detecting IL-1 secreting cells. PMID- 1730101 TI - Salmonella specific antibodies in serum and synovial fluid in patients with reactive arthritis. AB - The occurrence of Salmonella specific antibodies was analysed in paired serum and synovial fluid samples from 12 patients with Salmonella triggered reactive arthritis. The antibody concentration of IgA2 subclass in synovial fluid exceeded that of serum in 8 of the 12 pairs studied, compared to 0-3 in the other immunoglobulin classes and subclasses. This finding indicates intra-articular production of IgA2 class antibodies against the triggering microbe. PMID- 1730102 TI - T-cell receptors and rheumatic disease: approaches to repertoire analysis. AB - T-lymphocytes are thought to play a major role in the pathogenesis of a number of autoimmune rheumatic diseases. Techniques have recently been developed to study the T-cell receptor (TCR) usage of individual T-cells, and are likely to give major insight into the pathogenesis of rheumatic diseases. We describe the development of the TCR repertoire and the techniques available to study it. There is evidence that germline TCR complex polymorphisms may contribute to genetic susceptibility to rheumatoid arthritis. A number of studies have looked at rheumatoid synovial T-lymphocytes, some of which have found restricted TCR usage. PMID- 1730103 TI - A search for Chlamydia trachomatis in synovial fluids from patients with reactive arthritis using the polymerase chain reaction and antigen detection methods. AB - A polymerase chain reaction (PCR) technique to detect Chlamydia trachomatis DNA was used to examine synovial specimens from patients with reactive arthritis. We were able to detect C. trachomatis DNA in synovial specimens which had been seeded with intact elementary bodies or chlamydial DNA. However, we were unable to detect chlamydial DNA in unseeded synovial specimens from 10 patients with sexually acquired reactive arthritis, 17 patients with reactive arthritis and 11 control patients with other arthropathies. In addition, using a monoclonal antibody technique, we were unable to detect chlamydial antigen in any of the synovial cell deposits examined. We conclude that C. trachomatis DNA was not present in the joints of these patients at the time of synovial fluid collection, and suggest that either DNA degradation occurred rapidly after viable chlamydiae had entered the joint or that chlamydial DNA was not present at any stage of the reactive response. PMID- 1730104 TI - The effect of nasal hCT on bone turnover in Paget's disease of bone--implications for the treatment of other metabolic bone diseases. AB - Thirty pagetic patients were treated for 6 months with a daily nasal application of 2 mg of synthetic human calcitonin (hCT). Serum alkaline phosphatases (SAP) and urinary hydroxyproline/creatinine ratio (OH/Cr), reflecting bone turnover, were significantly reduced from the first month of treatment (mean +/- SEM: SAP, 13.9 +/- 2.2%; OH/Cr, -22.2 +/- 5.8%; both P less than 0.01) and until the end of the 6-month course (mean +/- SEM: SAP, -29.7 +/- 4.6%; OH/Cr, -22.5 +/- 5.9%; both P less than 0.01). Nasal hCT was perfectly tolerated both locally and systemically. These results allow us to consider nasal hCT for long-term trials in metabolic bone diseases characterized by a relative increase of bone resorption. PMID- 1730105 TI - Treatment of fixed flexion deformities of the knee in rheumatoid arthritis using the Flowtron intermittent compression stocking. AB - Thirty established fixed flexion deformities of the knee in 18 rheumatoid arthritis patients were studied prospectively to assess the results of 8 weeks of out-patient treatment using the Flowtron intermittent compression stocking. A 44% mean correction of the fixed flexion deformity was obtained from an initial mean of 29 +/- 1 degrees to 16 +/- 1 degrees (P less than 0.001). More than 85% of this correction was obtained in the first 4 weeks of treatment. There was a significant correlation between the correction obtained and the inflation pressure (P less than 0.05), but the time of daily use (minimum 60 min) was not a significant factor. The modified Stanford Health Assessment Disability Index fell from an initial mean of 2.17 to 2.03 (P less than 0.01). PMID- 1730106 TI - Non-steroidal anti-inflammatory drug usage and requirement in elderly acute hospital admissions. AB - There is a strong feeling that despite the increasing awareness of their adverse effects, non-steroidal anti-inflammatory drugs (NSAIDs) are not always used appropriately. To examine this problem amongst elderly patients who may be at particular risk, 500 acute admissions to Health Care of the Elderly wards were studied prospectively. Sixty-five patients were currently receiving NSAIDs; 56 had medical conditions possibly caused by, or aggravated by NSAIDs. Indications for NSAIDs were often no longer apparent, and these drugs were successfully discontinued in 56; in 22 no alternative therapy was required. Importantly, following discharge the majority of patients remained off NSAIDs. These data support the need for continual review of NSAID requirements and regular monitoring for adverse effects, particularly in this high risk group. PMID- 1730107 TI - Test/inform/retest: a useful technique in undergraduate rheumatology teaching. AB - Twenty final year medical students had their ability to interpret rheumatological physical signs tested by 10 multiple choice questions (MCQ) on days 1 and 4 of an educational study. Ten theory questions were used as controls. They were then given an information sheet on the interpretation of physical signs, and repeated the MCQ on day 26. There was a progressive improvement in the theory scores but no improvement in clinical scores until after the information sheet was available. The improvement in clinical scores was significantly greater than the improvement in theory scores (P less than 0.001). We suggest that the use of an information sheet in conjunction with an MCQ paper can improve undergraduates' ability to interpret physical signs when used in the pattern test/inform/retest. PMID- 1730108 TI - Complement C4 null alleles as a marker of gold or D-penicillamine toxicity in the treatment of rheumatoid arthritis. AB - C4 null alleles and HLA-DR antigens were defined in 48 rheumatoid arthritis (RA) subjects who had developed renal or heamatological side effects to gold or penicillamine, as compared to 33 RA subjects who had received the drugs for similar time periods without developing side effects. A C4A null allele was found in 56% of subjects with and 31% of those without side effects (P = 0.027, relative risk 2.8). A similar but statistically non-significant trend was observed with the C4B null allele (P = 0.64) resulting in a higher risk of drug toxicity in rheumatoid patients bearing either a C4A or C4B null allele (relative risk 5.7). Frequencies of DR3 and DR4 were similar in the two groups. PMID- 1730109 TI - Gout due to renal disease. AB - From 120 patients attending a referral gout clinic, 12 patients were found to have primary renal disease at the time of, or prior to, their first attack of acute gouty arthritis. This number excluded those with chronic lead nephropathy, polycystic kidneys or who were receiving diuretics. The nature of the renal disease was usually of the tubulointerstitial variety rather than of glomerular origin. The renal clearance of urate per unit of glomerular filtration rate, which usually increases with renal disease, was generally reduced, suggesting impairment of renal excretion of urate. Nine of the patients were female (four premenopausal) and only three were males. The degree of renal impairment was only mild to moderate. Other common associations with gout, such as obesity, hypertension and regular alcohol consumption, were not prominent. The intrinsic renal disease in these patients was considered to be the major contributor to their development of hyperuricaemia and gout. PMID- 1730110 TI - Total knee arthroplasty infection due to Gemella haemolysans. AB - Gemella haemolysans, a relatively unknown commensal of the upper respiratory tract, rarely causes clinically important infections. This report deals with an infection of a total knee arthroplasty due to Gemella haemolysans in a patient with rheumatoid arthritis. The microbiology of this bacterium is discussed and the clinical features of previously reported cases of Gemella infections are briefly reviewed. PMID- 1730111 TI - Calcinosis and the anticentromere antibody: its clinical, radiological and immunogenetic aspects. AB - The relationship between calcinosis and the anticentromere antibody (ACA) was studied from a clinical, radiological and immunogenetic standpoint. Ten ACA positive scleroderma patients, 34 ACA-negative scleroderma patients, 31 ACA positive patients without sclerodermatous skin changes and 140 ACA-negative patients with various rheumatic diseases were compared with regard to the incidence of calcinosis as measured by radiographs of hand and/or foot. Calcinosis was found in 10 (100%), 12 (35%), 13 (42%) and eight (6%) patients in each group respectively. Frequent sites for calcinosis in the ACA-positive patients were foot (24%), hand (21%) and leg (19%). The forearm was not usually involved (6%). During the follow-up term (1.0-9.5 years; mean 4.5 years) of 11 ACA-positive patients without calcinosis, four (36%) developed new calcinosis and this incidence was significantly higher (P less than 0.02) than that in the ACA negative control group (2/42; 4.8%). As a whole, 23 (59%) of 39 patients with ACA showed calcinosis. In the ACA-positive patients with calcinosis, sclerodactyly (P less than 0.005) and CREST syndrome (P less than 0.001) were found more frequently than in ACA-positive patients without calcinosis. HLA-A2 was found more frequently (67%) in the ACA-positive patients with calcinosis when compared to normal subjects (41%) (P less than 0.02). We concluded that calcinosis seems closely related to scleroderma, especially those with ACA, and that the development of calcinosis requires a certain genetic background. PMID- 1730112 TI - Home study CE: epidural and intrathecal pain management [continuing education credit]. PMID- 1730113 TI - The indications for elective treatment of the neck in cancer of the major salivary glands. AB - To define the indications for elective neck treatment, the cases of 474 previously untreated patients were reviewed who had locally confined major salivary gland cancers treated between 1939 and 1982. Clinically positive nodes were present in 14% (67 of 474). Overall, clinically occult, pathologically positive nodes occurred in 12% (47 of 407). By univariate analysis, several factors appeared to predict the risk of occult metastases; however, multivariate analysis revealed that only size and grade were significant risk factors. Tumors 4 cm or more in size had a 20% (32 of 164) risk of occult metastases compared with a 4% (nine of 220) risk for smaller tumors (P less than 0.00001). High-grade tumors (regardless of histologic type) had a 49% (29 of 59) risk of occult metastases compared with a 7% (15 of 221) risk for intermediate-grade or low grade tumors (P less than 0.00001). In view of the low frequency of occult metastases in the entire group, routine elective treatment of the neck is not recommended. High-grade tumors and larger tumors have a high rate of occult neck metastases, and treatment should be considered in this group. PMID- 1730114 TI - Preoperative imaging of colorectal cancers. Targeting the epithelial membrane antigen with a radiation-labeled monoclonal antibody. AB - The epithelial membrane antigen (EMA) is expressed by the majority of colorectal cancers but has not previously been investigated as a target for radiation labeled monoclonal antibodies (MoAb) in the imaging of patients with colorectal cancer. A rat IgG2a MoAb that recognizes EMA, ICR2, was labeled with Indium-111 (100 megabecquerel per milligram [MBq/mg]MoAb) using the bicyclic anhydride of the chelating agent diethylene triamine pentacetic acid (ccDTPA) and was administered intravenously to 22 patients known to have or thought to have colorectal cancer. Daily gamma camera imaging was performed for 3 days during the time between the administration of the radiation-labeled antibody and surgical procedure. At operation, the biopsies were done of the tumors and the normal colon, and the uptake of radiation-labeled MoAb was measured in a gamma well counter. Immunocytochemistry for EMA expression also was done on resected tumors. Independent unblinded and blinded reporting was done on all scans. The sensitivity of 111In-ICR2 for detecting cancers preoperatively was 80% and 60%, respectively, on unblinded and blinded reporting, and the corresponding specificity 20% and 60%. The low unblinded specificity was attributable to a false-positive localization in severely dysplastic benign tumors (n = 2) and inflammatory tissue (n = 2). Liver metastases present in three patients were cold relative to normal liver. Lymph node metastases were localized in 1 of 6 patients preoperatively. The mean absolute uptake of 111In-ICR2 in tumor tissue was 7.75 +/- 3.77 x 10(-3) percent of injected dose per gram, and the ratio to normal colon was 2.10 +/- 0.92:1. On immunohistochemistry, EMA was expressed by 16 of the 17 primary cancers, both dysplastic adenomas, and all nodal metastatic deposits. EMA-negative tumors (1 cancer + 1 colonic lipoma) had negative antibody scans, and patients whose tumor was negative or only focally positive for EMA expression had lower tumor/normal colon ratios of radioactivity (1.30 +/- 0.26 versus 2.45 +/- 0.65, P = 0.005) on gamma well-counting of excised specimens. These results suggest a possible role for 111In-ICR2 in the detection of colorectal cancer and metastases but not its liver deposits. PMID- 1730115 TI - Clinical and pathologic prognostic indicators in colorectal cancer. A population based study. AB - The institution of a colorectal Cancer Register in a health care district of Northern Italy gave the authors the opportunity to evaluate the prognostic relevance of several morphologic and clinical variables by univariate and multivariate analyses. Of the 134 patients registered in 1984, 132 were followed up until the end of 1989. Overall 5-year survival was 37%, but the figure increased to 43% when only colorectal cancer-related deaths were considered. Univariate analysis for clinical variables showed that TNM staging and age at diagnosis were significantly related to prognosis, whereas none of the other parameters were indicative of the clinical outcome. With a similar analysis, among the various morphologic variables, pattern of growth (infiltrating versus expanding) and extent of fibrosis (extensive versus little or absent) appeared to be indicators of prognosis. When the variables that were significant (stage, age, pattern of growth, and fibrosis) in the univariate analysis were entered into the Cox model of multivariate analysis, TNM staging was the only parameter that maintained an independent prognostic importance. The authors state that their results confirm the importance of stage in predicting survival for cancer of the large bowel and suggest that the possible prognostic value of clinical and morphologic variables should be investigated within each of the major TNM or Dukes' classes. PMID- 1730116 TI - Management of advanced squamous cell carcinomas of the maxillary sinus. AB - From 1970 to 1988, 41 cases of advanced maxillary sinus cancers were treated at the University of Kansas Medical Center. Local control for the 37 evaluable patients was achieved in 21 (57%). Local control by radiation therapy alone was achieved in ten of 19 (53%) patients compared with eight of 14 (57%) treated with a combination of surgery and radiation therapy. A dose greater than 6500 cGy correlated with better local control in patients treated with radiation therapy alone. Neck node failure occurred in three of 35 (8%) patients when not electively treated. Neck metastasis either at presentation or at a later stage reduced survival. The overall absolute survival for the entire group at 5 years was 35%. A combination of preoperative radiation therapy and surgery is recommended for patients with advanced-stage maxillary sinus cancer. Radiation therapy is an equally good alternative for those who are not surgical candidates or refuse surgery. PMID- 1730117 TI - Five-year survival after pulmonary metastasectomy for adult soft tissue sarcoma. AB - Determinants of 5-year survival were evaluated after complete resection of pulmonary metastases from adult soft-tissue sarcomas. Fifty-eight patients had complete resection (median survival 25 months, P = 0.0002), with a 25.8% absolute 5-year survival (15 of 58 patients); six patients had unresectable disease (median survival 6 months) and were excluded from additional analysis. Eleven patients remain disease free, with a median follow-up of 76 months. Significant independent prognostic indicators associated with improved survival (P less than 0.05) included metastasis doubling time of 40 days or greater (median survival 37 months versus 15 months if less than 40 days); unilateral disease on preoperative radiography (33 months versus 15 months if bilateral disease); three or fewer nodules on preoperative computed tomography (40 months versus 14 months if 4 or more nodules); two nodules or fewer resected (40 months versus 17 months if 3 or more nodules resected), and tumor histology (33 months for malignant fibrous histiocytoma versus 17 months for all others). Multivariate analysis identified the number of nodules detected by computed tomography preoperatively as having significant prognostic value. PMID- 1730118 TI - The leukocyte count and risk of lung cancer. AB - The association between leukocyte count and subsequent risk of lung cancer was evaluated in three large cohorts from the United States and Britain. A total of 309 lung cancer events occurred among 28,181 men whose cases were followed-up for 7 to 12 years. In all three cohorts, there was a marked increase in risk of lung cancer with increasing leukocyte count, after adjustment for age and the number of cigarettes smoked per day. The adjusted relative odds in the three cohorts, for a 2000/microliters difference in leukocyte count, were 1.58 (P = 0.0001), 1.29 (P = 0.003) and 1.20 (P = 0.02). These relative odds persisted when current smokers were considered alone, when serum markers of cigarette smoking exposure were adjusted for, and when men with lung cancer events during the first 5 years of follow-up were excluded. The leukocyte count appears to be linked to the pathogenesis of smoking-related lung cancer. PMID- 1730119 TI - Survival for clinical stage I lung cancer not surgically treated. Comparison between screen-detected and symptom-detected cases. The Japanese Lung Cancer Screening Research Group. AB - To assess the extent of overdiagnosis bias in lung cancer screening, clinical Stage I lung cancer cases detected by chest radiograph examination, with histologic or cytologic evidence of malignancy and not treated by surgical operation, were followed up for more than 10 years. Of 1297 screen-detected and 1297 symptom-detected cases collected from 20 institutions, 42 screen-detected and 27 symptom-detected cases satisfied the study criteria. In about half of the cases, the patients had no contraindication for surgical treatment, but they refused surgical procedure. All such patients from the screen-detected and symptom-detected groups died within 122 and 67 months, respectively, of diagnosis. Among the screen-detected and symptom-detected cases, 80% and 81%, respectively, of the patients died of lung cancer. The median survival time was 25 and 13 months for those in the screen-detected and symptom-detected groups, respectively. The difference in survival was statistically significant between the two groups, which indicated the effect of lead time and length-biased sampling. Analysis of the causes of death other than lung cancer showed that there was no difference in the observed cumulative rates of deaths of other causes between the two groups, and these figures were almost the same as those expected from the general population. This indicates that overdiagnosis bias would be minimal in screen-detected lung cancer cases detected by chest radiograph examination. PMID- 1730120 TI - Survival, prognosis, and therapeutic response in osteogenic sarcoma. The Memorial Hospital experience. AB - Two hundred seventy-nine consecutive patients with Stage II osteogenic sarcoma of the appendicular skeleton treated between 1976 and 1986 were studied to identify predictors of long-term survival. Survival was 77% and 73% at 5 and 10 years, respectively, with continuously disease-free survival being 70% and 69%. On univariate analysis, the most significant predictors of survival were the location of the primary lesion, local control of the tumor, and the degree of necrosis in the primary tumor after intravenous neoadjuvant chemotherapy (histologic response). On initial multivariate analysis, similarly, only location and histologic response to chemotherapy predicted disease-free outcome. After statistical control for local recurrence, only histologic response to chemotherapy was retained as an independent predictor, suggesting that in this data set, the location of primary lesion exerted its effect only secondarily through its association with the ability to provide local control. The risk of local recurrence was almost fivefold higher in tumors of the femur than in tumors of other locations (relative risk, 4.6) and, within the femur, was more than threefold higher in the proximal femur than in the distal femur (relative risk, 3.4). None of the other primary tumor or patient characteristics studied yielded independent predictive significance for survival. The rate of failure was almost fivefold as high in those with an incomplete response to chemotherapy compared with those with a complete response to chemotherapy (relative risk, 4.9; 95% confidence interval, 2.2 to 11). Even in those patients with minimal or no necrosis in the primary tumor, ultimately 62% and 54% were disease-free at 5 and 10 years, respectively. PMID- 1730121 TI - Treatment of relapsed or refractory adult acute lymphocytic leukemia. AB - Sixty-six adult patients were treated for relapsing or refractory acute lymphocytic leukemia (ALL). The induction treatment consisted in a (1) first phase with vindesine 3 mg/m2 intravenously (IV) on days 1, 8, and 15; daunorubicin 45 mg/m2 IV on days 1, 8, and 15; erwinia-asparaginase 10,000 U/m2 IV on days 7, 8, 14, and 15; and prednisone 60 mg/m2 orally on days 1 to 21 and a (2) second phase with cytarabine 3000 mg/m2 as a 3-hour infusion two times a day on days 1 to 4 (in patients greater than 50 years of age we used 1000 mg/m2), and etoposide 100 mg/m2 IV on days 1 to 5. Side effects of induction Phase I were predominantly hematologic with subsequent infections. In Phase II, some patients additionally had gastrointestinal, cutaneous, ocular, and hepatic toxicity. Five patients died during Phase I and another died during Phase II. Five of these patients had T-cell ALL. Thirty-four (64%) of 54 patients in their first relapse had a complete remission (CR) with a median disease-free survival (DFS) of 2.9 months. The median overall survival (OAS) was 6.6 months. Seven of 12 patients with primary refractory disease, a second relapse, or relapse after bone marrow transplantation (BMT) had a CR. The CR rate and survival after first relapse was significantly better in patients with a preceding CR of more than 18 months compared with those with a shorter preceding remission. The leukocyte count was a second significant but not independent risk factor. There was a negative correlation between the leukocyte count and the duration of the preceding CR. The duration of the preceding CR was the major prognostic factor for survival in multivariate analysis. Twenty-two patients received BMT. None of nine patients with autologous BMT is alive and disease-free; 5 of 13 who underwent allogeneic BMT are. It was concluded that this treatment efficiently induced remission with tolerable toxicity. The remission duration should be improved by optimized consolidation treatment. PMID- 1730122 TI - Cutaneous pseudo-T-cell lymphomas. A clinicopathologic study of 20 patients. AB - The histologic features of 20 patients with cutaneous pseudo-T-cell lymphomas other than actinic reticuloid and lymphomatoid papulosis were investigated. Two histologic types of cutaneous pseudo-T-cell lymphomas were designated. The band like (MF-like) pattern that simulated mycosis fungoides (MF) and a nodular pattern that mimicked cutaneous T-cell lymphomas (CTCL) other than MF. Both patterns showed histologic features that generally are not found in CTCL and thus may be helpful in the differential diagnosis from CTCL. However, at present the differential diagnosis between pseudo-T-cell lymphomas and CTCL should be based on a combination of clinical and histologic data. PMID- 1730123 TI - Primary non-Hodgkin's lymphomas of the female breast. AB - The charts of 35 women with primary malignant non-Hodgkin's lymphomas (NHL) of the breast were retrieved from the files of the Istituto Nazionale Tumori, Milan, over a 30-year period (1957 to 1986). These cases represented 0.1% of the more than 25,000 primary malignant tumors of the breast treated during the same period. The median age of these patients was 57 years (range, 28 to 81 years). In most cases, the clinical diagnosis was carcinoma. The tumors were either Stage IE(48%) or IIE(52%) at presentation, and only two patients had B symptoms. The right breast was involved in 17 patients, the left breast in 14, and both breasts in two. According to the updated Kiel classification and the Working Formulation (WF) for Clinical Usage, three cases were lymphoplasmacytoid (immunocytoma) NHL (WF, A); three, centroblastic-centrocytic, follicular NHL (WF, B); four, centroblastic-centrocytic, diffuse NHL (WF, F); 17 centroblastic NHL (WF, G); three immunoblastic NHL (WF, H); two B-lymphoblastic NHL (WF, I); and one, a Burkitt-like NHL (WF, J). Treatment consisted either of a combination of surgery, radiation therapy, and chemotherapy or radiation therapy and chemotherapy. The follow-up period for 32 patients ranged from 6 to 161 months (mean, 45 months); 17 patients died of their disease. The prognosis appeared to be related to the histologic type and stage of the disease. Median survival periods were 63, 52, 42, and 47 months for centroblastic-centrocytic follicular, centroblastic centrocytic diffuse, centroblastic, and immunoblastic NHL, respectively. The overall 5-year survival rate was 43%; the 5-year survival rate and the probability of freedom from progression at 5 years were, respectively, 61% and 50% for Stage I and 27% and 26% for Stage II disease. PMID- 1730124 TI - Randomized trial comparing cisplatin with radioactive phosphorus or whole-abdomen irradiation as adjuvant treatment of ovarian cancer. AB - In this study, 347 patients with epithelial ovarian cancer without residual tumor after primary laparotomy, were assigned randomly to receive either intraperitoneal instillation of radioactive phosphorus (32P) or six courses of cisplatin (50 mg/m2). Patients randomized to receive 32P with extensive intraperitoneal adhesions were treated with whole-abdomen irradiation instead of 32P (n = 28). The median follow-up was 62 months. Crude and disease-free survival were similar in all groups. Late bowel complications occurred more often in patients treated with 32P compared with the cisplatin group. The estimated 5-year crude survival rate was as high as 95% in patients with borderline or well differentiated tumors in Stage I. It is suggested that these patients can be treated adequately by operation alone. Patients with moderately or poorly differentiated cancers in Stage I disease had a 5-year crude survival rate of 75%. In these patients, the relapse risk was high enough to warrant postoperative treatment. The efficacy of adjuvant treatment in this subgroup of patients can only be established in a prospective randomized study comparing postoperative adjuvant treatment with a no-treatment arm. Because of the high number of late bowel complications after 32P treatment, it was recommended that cisplatin be used as standard adjuvant treatment for subsequent controlled studies. PMID- 1730125 TI - Correlation between side of palpable tumor and side of pelvic lymph node metastasis in clinically localized prostate cancer. AB - Of 411 patients with palpable but clinically localized (Stages B or C) adenocarcinoma of the prostate, 100 (24.3%) were found at complete bilateral pelvic lymphadenectomy to have one or more lymph nodes positive for metastasis. These patients were divided into five subgroups on the basis of the location of the palpable tumor at digital rectal examination: left side only, left predominantly, both sides, right side predominantly, or right side only. Among 35 patients with positive nodes and a palpable tumor limited to one side of the prostate (clinically unilobar), metastases were found in the ipsilateral pelvic lymph nodes in 29 (83%). Only 6 (17%) of the patients had contralateral metastasis alone. A unilateral pelvic lymphadenectomy (ipsilateral to the side of the largest palpable tumor, or on either side if the tumor were bilateral) would have detected 80% of the patients with positive lymph nodes, with a positive predictive value of 100% and a negative predictive value of 94%. Lymph node metastases in patients with clinically localized palpable prostate cancer are most likely to be found on the same side as the palpable tumor and are considerably less likely to be found on the contralateral side alone. If frozen section examination of lymph nodes or laparoscopic lymph node dissection is planned before definitive therapy for prostate cancer, the pelvic lymph nodes ipsilateral to the side of the palpable tumor should be removed first. PMID- 1730126 TI - Prostatic embryonal rhabdomyosarcoma in adults. A clinicopathologic review. AB - Embryonal rhabdomyosarcoma of the prostate is a rare, highly malignant tumor that occurs predominantly in male infants and children, in whom it is the most common prostatic sarcoma. Six cases occurring in adults have been published, and the authors report three additional cases. The natural history is characterized by rapid growth, with the typical formation of large pelvic or abdominal masses, often leading renal failure due to bilateral ureteric obstruction. The tumor eventually disseminates widely, mainly to the lungs, bone, liver, and serosal surfaces, and unlike most other sarcomas, regional lymph node metastases are common. Combined modality therapy has resulted in marked improvement in survival rates and reduced surgical morbidity for children with these tumors. However, in adults the prognosis remains poor, with all patients dying of disseminated disease within 16 months of histologic diagnosis (mean survival, 8 months). PMID- 1730127 TI - Lymphoid irradiation results in long-term increases in natural killer cells in patients treated for Hodgkin's disease. AB - Therapeutic lymphoid irradiation has been shown to produce profound long-term alterations in lymphocyte subpopulations and immunologic responsiveness. Dual immunofluorescence flow cytometry and functional cytolytic assays were used to investigate the effects of lymphoid irradiation either alone or in combination with chemotherapy on T-cell and natural killer (NK) cell populations in the blood of patients treated for Hodgkin's disease. Patients treated with mantle and paraaortic lymphoid irradiation show significant increases in the proportion of cells bearing the NK cell phenotypic marker Leu-11 (CD16). These patients also display proportionately increased cytotoxicity against K562 tumor targets in vitro. A sizable number of these NK cells label dimly with Leu-2 (CD8) although they lack the pan-T-cell marker Leu-4 (CD3). The emergence after lymphoid irradiation of this population of Leu-11+2+ NK cells may lead to an apparent decrease in the ratio of helper to suppressor T-cells, although the actual ratio of these T-cell subsets generally is normal. These changes persist for years after the completion of radiation therapy. It was concluded that lymphoid irradiation may produce profound changes in NK cell populations in patients treated for Hodgkin's disease; the clinical significance of these changes is unclear. PMID- 1730128 TI - Long-term sequelae of autologous bone marrow or peripheral stem cell transplantation for lymphoid malignancies. AB - The study was made to evaluate the long-term physical and psychosocial changes after high-dose therapy and autologous bone marrow or peripheral stem transplantation for recurrent lymphoid malignancies. Patients who had undergone high dose therapy and autologous bone marrow or peripheral stem cell transplantation for recurrent lymphoid malignancies at least 1 year previously were contacted by phone interview regarding their status after the transplant. The patients' comments were confirmed by checking medical records when possible. Fifty patients who had undergone transplantation at the University of Nebraska Medical Center at least 1 year before the interview were available for interview and willing to answer questions. After transplant, many patients noticed temporary changes in their appearance, which usually returned to normal within 1 year. Few patients reported remarkable cardiovascular, gastrointestinal, or pulmonary changes after transplantation. However, up to one-third of the patients reported changes in sexual function or desire. The most common infectious problem after transplant was Herpes zoster, which occurred in 25% of the patients. Overall, the patients had a positive outlook after high-dose therapy and transplantation, with most being able to return to work and enjoy a normal life style. Ninety-six percent of the patients stated that they would be willing to undergo high-dose therapy and transplantation again under the same circumstances. PMID- 1730129 TI - A histologic and flow cytometric study of Kaposi's sarcoma. AB - One hundred forty-three biopsies of Kaposi's sarcoma (KS) from 96 patients were assessed histopathologically, and mitoses were counted. Ninety-seven samples from 66 patients were analyzed by flow cytometry. Six of 97 (5.8%) KS lesions were DNA aneuploid with a clustering around a DNA index of 1.5 (range, 1.4 to 1.6). The median percentage of S-phase plus G2-phase cells (%S + G2) was 16.7%. Increasing mitotic counts and %S + G2 were seen with progression of the phase and pattern of disease. Nodular KS and spindle cell predominant KS had the highest mitotic counts and %S + G2, with nodular KS larger than 4 mm having a higher mitotic count than those smaller than 4 mm. These findings suggest a low level of DNA aneuploidy in KS and important changes in the level of cell proliferation with the phase and pattern of the disease. However, flow cytometry does not solve the dilemma of whether KS is a hyperplastic or neoplastic process. PMID- 1730130 TI - X;6 translocation in a child with congenital acute lymphocytic leukemia. AB - A case of congenital acute lymphoblastic leukemia (ALL) displayed an X;6 translocation. This is the third reported case of ALL with an X;6 translocation. In addition, two of the three ALL cases occurred during infancy, at ages 2 months and newborn, and both translocations involved the band q15-16 region of chromosome 6. Anomalies of the long arm of chromosome 6, mainly interstitial and terminal deletions, have been reported as a recurrent karyotypic event in a significant number of ALL cases. The molecular basis and propensity of an X;6 rearrangement in this case of congenital ALL is unclear and merits further investigation. The similarities in this case and the other infant ALL case cited suggest that an X;6 rearrangement with a breakpoint in bands q15-16 of chromosome 6 is characteristic of a form of congenital ALL. PMID- 1730131 TI - Coping with cancer during the first year after diagnosis. Assessment and intervention. AB - The emotional coping of 205 patients newly diagnosed with cancer was evaluated every 4 months during a 1-year period. Patients received a psychosocial intervention either immediately (early intervention, EI), or after a 4-month delay (later intervention, LI). No significant differences were found between the two groups, except at 8 months, when the LI group was significantly less depressed, anxious, and worried, and felt more in control than the EI group. The LI group continued to have less worry related to illness at 12 months. Patients with high ego strength had low levels of distress at baseline and may not have needed the intervention. The emotional coping of patients with breast cancer improved during the year regardless of the intervention timing. Patients with other diagnoses appeared to benefit most from the IL. It was concluded that patients with low ego strength and diagnoses other than breast cancer might be at higher risk for psychosocial complications and could benefit from the intervention. PMID- 1730132 TI - The changing needs of patients with cancer at home. A longitudinal view. AB - Changes in the daily living needs of 629 patients with advanced cancer were investigated (1) during and (2) 3 to 6 months after a course of outpatient chemotherapy and/or radiation treatment. The analytic sample consisted of patients completing both baseline and follow-up interviews (n = 434). At both times, the point prevalence of need and unmet need for assistance with personal care, instrumental activities, transportation, and home health tasks was calculated. In addition, the prevalence of new need and unmet need at follow-up was determined as were the rates of resolution of baseline need. The prevalence of need for assistance with personal care increased from 7% at baseline to 16% at follow-up; the dynamic of need acquisition and resolution resulted in relatively constant prevalence rates in other task areas. Acquisition of need at follow-up was associated primarily with disease and treatment-related characteristics. Approximately one third of patients reporting need for assistance during at least one interview did not have enough help. New unmet need at follow-up was associated most strongly with patients' mobility and the ability of their informal support system to provide care. The apparently rapid fluctuation in patients' experience of need and unmet need suggests the necessity for ongoing appraisal of patients' physical condition and social situation. PMID- 1730133 TI - Protection against 7,12-dimethylbenzanthracene-induced tumour initiation by protein A in mouse skin. AB - Protein A is an immunostimulating glycoprotein obtained from Staphylococcus aureus Cowan I. Its antitumour activity is proven in various tumour models. Its ability to provide protection against tumour initiation by the chemical carcinogen 7,12-dimethylbenzanthracene (DMBA) has been investigated in the present study using a mouse skin model of two-stage carcinogenesis. Protein A was administered intraperitoneally (1 microgram/animal 20 g body wt.) twice a week for 2 weeks, prior to initiation by DMBA. The promotion was performed by twice weekly applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (3 or 5 micrograms/animal in 100 microliters acetone). Protein A provided significant protection to animals from DMBA-induced tumour initiation as was observed by the decrease in cumulative number of tumours, percent of animals developing tumours, number of tumours per animal and rate of tumour growth. Our data indicate that protein A has anticarcinogenic properties. PMID- 1730134 TI - Detection of transforming oncogenes in rat colon tumors induced by direct perfusion with N-methyl-N-nitrosourea. AB - We assayed rat colon tumors induced by N-methyl-N-nitrosourea (MNU) for transforming oncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA from 3 of 3 adenomas and 3 of 5 carcinomas induced transformed foci on NIH 3T3 cells. DNA from 2 of 3 primary foci also possessed focus-forming activity, and rat-specific sequences were observed in secondary focus DNAs. Furthermore, NIH 3T3 cells transfected with DNA from a carcinoma and from a primary focus derived from it, both positive in the focus-forming assay, induced tumors in nude mice. We found no evidence for rat H-ras, K-ras, or N-ras sequences in the DNA of any of 16 primary foci derived from 6 rat tumors; thus, in contrast to other animal tumor models induced by MNU, activation of the ras genes does not appear to predominantly occur in MNU-induced rat colon tumors. We also did not observe, in any of these foci, sequences corresponding to the rat neu, raf, fms, met, or hst genes, thus indicating that none of these is the transforming oncogene in our model. These results suggest that an as yet unidentified transforming oncogene may be activated in rat colon tumors induced by MNU. PMID- 1730135 TI - The effect of ellagic acid on xenobiotic metabolism by cytochrome P-450IIE1 and nitrosodimethylamine mutagenicity. AB - Ellagic acid (EA) is an inhibitor of the in vitro mutagenicity of N nitrosodimethylamine (NDMA) in Salmonella typhimurium strain TA100 using pyrazole induced rat liver 9000 x g supernatant (S-9). In order to understand this activity, the effect of EA on the metabolic hydroxylation of 4-nitrophenol, a substrate, as is NDMA, for cytochrome P-450IIE1 was studied using pyrazole induced rat S-9 and microsomal protein. It is shown that EA has an inhibitory effect on 4-nitrophenol hydroxylase with both enzyme preparations. This effect on cytochrome P-450IIE1 may be responsible, at least in part, for the inhibition of NDMA mutagenicity by EA. PMID- 1730136 TI - Systemic modulation by ultraviolet irradiation of cutaneous N-methyl-N'-nitro-N nitrosoguanidine-induced carcinogenesis. AB - Ultraviolet irradiation can systemically enhance subsequent skin cancer induction by benzo[a]pyrene, methylcholanthrene, or UV radiation. The present study was designed to determine whether UVB irradiation influences host susceptibility to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Female C3H/HeJ mice were exposed dorsally to UVB radiation from banks of 6 Westinghouse FS40 sun lamps. The mice received a total UV dose of approximately 8.1 x 10(5) J m-2 over a 15-week period. After termination of UVB treatments, ventral tumors were induced by 4 applications of 30 mumol of MNNG at 8-day intervals. At 20 weeks after the first MNNG treatment, UVB-irradiated mice had 7-fold more MNNG-induced, ventral tumors than did the unirradiated control mice (P = 0.026, Wilcoxon rank sum test). Ventral application of MNNG after cessation of dorsal UVB exposure, but before UV tumor appearance, did not influence photocarcinogenesis. These results demonstrate that UV irradiation can systemically decrease host resistance to tumor induction by the methylating agent, MNNG. PMID- 1730137 TI - Milk cream does not enhance 2,7-dimethylbenz[a]anthracene-induced mammary tumorigenesis. AB - We have previously reported that a diet enriched with butter showed an inhibitory effect on the development of mammary tumors in mice and rats. To solve the problem of whether the inhibitory effect of butter was caused by lipids of cow's milk, we have studied the effects of dried milk (WM), skim milk (SM) and milk cream (CR) on mammary tumorigenesis in rats. The lowest incidence of mammary tumors was observed in the CR group, although the difference from other groups was statistically not significant. However, the number of papillary carcinomas in the CR group was significantly lower than the WM group. The result indicates that milk lipids have no enhancing effect on mammary tumorigenesis. PMID- 1730138 TI - Circumvention of adriamycin resistance: effect of 2-methyl-1,4-naphthoquinone (vitamin K3) on drug cytotoxicity in sensitive and MDR P388 leukemia cells. AB - The effect of vitamin K3 (2-methyl-1,4-naphthoquinone) on Adriamycin (ADR) induced growth inhibition of drug sensitive and multidrug resistant P388 leukemia cells was evaluated. Exposure to ADR concentrations of 100-5000 ng simultaneously with 1 microM vitamin K3 elicited an enhanced inhibition of tumor cell survival. The effect of treatment with ADR alone, or in combination with vitamin K3 on DNA and RNA biosynthesis in the sensitive and resistant tumor cells, was also assessed. DNA and RNA biosynthesis inhibition was increased in P388/S (the parental cell line) and P388/ADR cells (the ADR resistant cell line which exhibits the multidrug resistant (MDR) phenotype) exposed to ADR after pretreatment for 3 h with vitamin K3. Concurrent administration in vivo of vitamin K3 and ADR illustrated a therapeutically significant increase (P less than 0.05) in the life span of sensitive and resistant tumor cell bearing animals. Vitamin K3 caused a depletion of the intracellular glutathione (GSH) levels in P388/S and P388/ADR leukemia cells but at concentrations greater than those that enhanced ADR cytotoxicity. Pretreatment of the tumor cells with 1 microM vitamin K3 induced a 35-50% (P less than 0.001) elevation in the intracellular ADR accumulation in MDR P388 leukemia cells, while such an effect was absent in P388/S tumor cells. DNA binding studies performed utilizing calf thymus DNA, indicated that vitamin K3 enhanced the intercalation potential of ADR and also altered the equilibrium between the free and bound form of ADR in a cell free system. These factors and their possible effects on the potentiation of ADR cytotoxicity and the therapeutic significance of utilizing vitamin K3 as an adjuvant in the chemotherapy of MDR tumors is discussed. PMID- 1730139 TI - Flow cytometric analysis of tumour infiltrating lymphocyte activation and tumour cell MHC class I and II expression in breast cancer patients. AB - The primary tumour cells and tumour infiltrating lymphocytes (TILs) of 31 breast cancer patients have been analysed by dual colour flow cytometry to determine whether the phenotype and/or activation status of the TILs bears any relationship to the expression of MHC antigens on the tumour cells. The phenotype and activation status of 5000 TILs were studied using Mabs to CD4, CD8, HLA DR, CD25 (the low affinity inducible IL-2 receptor) and the transferrin receptor and related to Class I and Class II MHC expression on 5000 primary tumour cells. On the tumour cells, Class I MHC expression ranged from 1-74%, averaging 12.9%. HLA DR expression ranged from 1-69% averaging 14.3%. When the phenotypic proportions of the lymphocytic infiltrate were analysed there was found to be a correlation between tumour expression of Class I MHC and the proportion of both CD4+ (P less than 0.05) and CD8+ (P less than 0.02) T cells within the tumour. No such relationship was found with the MHC Class II antigen. When TIL activation markers were analysed, the percentage of CD8+ TILs positive for HLA DR expression correlated strongly with the expression of Class I (P less than 0.001) and Class II (P less than 0.001) antigens on the tumour cells. The percentage of CD4+ TILs positive for HLA DR expression also correlated significantly, but less strongly with the expression of Class I (P less than 0.01) and Class II (P less than 0.02) antigen expression on the tumour cells. The percentage of CD4+ TILs positive for CD25 expression correlated with both Class I (P less than 0.05) and Class II (P less than 0.03) expression on the tumour cells while the percentage of CD8+ TILs positive for CD25 did not. The percentage of TILs bearing the transferrin receptor showed no measurable correlation with the expression of either class of MHC antigen on the tumour. The data suggest that MHC expression on the tumour cells has a selective effect on the response capacity of different parts of the immune system. PMID- 1730140 TI - Methylation of rat and mouse DNA by the mushroom poison gyromitrin and its metabolite monomethylhydrazine. AB - Consumption of false morel (Gyromitra esculenta Fr.) has been associated not only with acute poisoning, but also with a carcinogenic risk. The hydrolysis of acetaldehyde-N-methyl-N-formylhydrazone (gyromitrin, the main toxic component of false morel) results in the formation of the methylating agents N-methyl-N formylhydrazine (MFH) and N-methylhydrazine (MMH) (by further hydrolysis of MFH). This study reports traces of N-7-methylguanine (N7MeGu) in liver DNA from mice and a rat treated with gyromitrin. After repeated administration of MMH, N7MeGu was identified in rat liver DNA. In mice exposed to MMH according to a dosing scheme identical to that reported to induce tumours in this species, O6 methylguanine was present in liver and kidney DNA. The results indicate that a relatively low carcinogenic risk is associated with false morel consumption. The risk may be greater in individuals with a decreased detoxification rate (acetylation) of MFH, in whom larger amounts of MMH are formed from gyromitrin. PMID- 1730141 TI - Inhibitory effect of prostaglandin oligomeric derivatives on 9,10-dimethyl-1,2 benzanthracene-induced hamster lingual carcinomas. AB - The inhibitory effects of prostaglandin oligomeric derivatives OC-3186 and OC 5186 were examined in hamster lingual carcinoma induced by 9,10-dimethyl-1,2 benzanthracene (DMBA). These compounds caused a regression of 40-90% in the size of lingual carcinomas in the hamster within several days after systemic or local administration. PMID- 1730142 TI - Induction of colorectal cancer in rats by 20-methylcholanthrene. AB - Colorectal carcinoma was induced in Sprague-Dawley rats by 20-methylcholanthrene. Macroscopical studies revealed that the tumors, either sessile type or semi pedunculated polyp, were generally observed after 32 weeks of the carcinogen treatment. In the distal colon 46.9% tumors appeared, whereas 20.4% and 32.6% tumors were found in the rectum and proximal colon, respectively. Sequential histopathological studies indicated that hyperplasia of goblet cells was common in early stages, which was reduced thereafter. Carcinogenesis progressed with the appearance of the different grades of dysplasia in colorectal mucosa with first incidence of the severe dysplasia in rats at the 20th week and in situ carcinoma at the close of 28th week. Most of the carcinomas were multifocal in origin and were well differentiated adenocarcinoma with primary invasion at the submucosa. In immunohistological studies, this carcinoma was also reactive with monoclonal antibody 660, prepared against a colorectal carcinoma associated mucin antigen. PMID- 1730143 TI - Relative stabilities of nitrenium ions derived from polycyclic aromatic amines. Relationship to mutagenicity. AB - The relative energetics of arylamine N-hydroxylation and N-O heterolysis (ArNH2-- -ArNHOH----ArNH+) for condensed systems of two, three and four rings were calculated using semiempirical AM1 molecular orbital theory. The overall thermodynamics of N-hydroxylation were almost insensitive to the structure of the amine while differences in the energetics of nitrenium ion formation varied from 0 to 35 kcal mol-1. Limited correlations between the latter and the experimental TA98 and TA100 mutagenicities of the amines are presented. PMID- 1730144 TI - Comparative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by mouse and rat liver microsomes. AB - The comparative metabolism of the carcinogenic pollutants 7H-dibenzo[c,g] carbazole (DBC) and dibenz[a,j]acridine (DBA) was investigated in vitro using 3 methylcholanthrene (3MC) induced Sprague-Dawley rat and Hsd:ICR(Br) mouse liver microsomal preparations with benzo[a]pyrene (BaP) as the positive control. Metabolites were isolated and separated by HPLC and identified by spectroscopic and co-chromatographic techniques using synthetic standards. The major metabolites of DBC were the phenols: the 5-OH-DBC, 3-OH-DBC, and 2-OH-DBC. Traces of 1-OH-DBC were also found yet no dihydrodiols were identified. The major metabolites of DBA were the 3,4-diol-DBA and 5,6-diol-DBA, 1,2-diol-DBA, DBA-5,6 oxide and 4-OH-DBA. Treatment of both mice and rats with 3MC resulted in significant (P less than or equal to 0.05) increases relative to control in the microsomal metabolism of DBA to dihydrodiol and phenol metabolites, similar to that observed for BaP. 3MC-induced rat liver microsomes significantly (P less than or equal to 0.05) increased DBC metabolism relative to control microsomes whereas DBC metabolism was not increased with 3MC-induced mouse liver microsomes. These data indicate that different enzymatic pathways are involved in the metabolic activation of DBC in the Hsd:ICR(Br) mouse and Sprague-Dawley rat. PMID- 1730145 TI - The structure-activity relationship of skin carcinogenicity of aromatic hydrocarbons and heterocycles. AB - From a study of 239 aromatic and heteroaromatic compounds causing skin cancer in mice, a quantitative structure-activity relationship has been derived. Carcinogenicity depends heavily on the relative hydrophobicity of the chemicals as defined by octanol/water partition coefficients (log P). It is also correlated with the energy of the highest occupied molecular orbital and the presence of substituents on the L and K regions of the carcinogen. The results are discussed in terms of the bay region concept for carcinogenic activity. PMID- 1730146 TI - Relative stabilities of nitrenium ions derived from heterocyclic amine food carcinogens: relationship to mutagenicity. AB - The bacterial mutagenicities of a wide variety of complex heteroaromatic amine mutagens and carcinogens present in cooked foods are approximately related to the stabilities of the corresponding nitrenium ions through equations of the kind: log(m) = a delta delta H + b. The stabilities of the nitrenium ions (delta delta H) were computed using the semiempirical AM1 molecular orbital procedure. Parallel calculations of the energies, charge distributions and geometries of simple model compounds provides a qualitative framework within which the stabilities of the nitrenium ions derived from the food carcinogens can be easily understood. PMID- 1730147 TI - Evaluation of the cytotoxic mechanisms mediated by the broad-spectrum antitumor alkaloid acronycine and selected semisynthetic derivatives. AB - Acronycine (I) is a broad-spectrum antitumor agent whose development as a clinically useful agent has been hindered, in part, due to its poor solubility characteristics. With the goal of acquiring information that may prove of value in the development of structurally related compounds of greater clinical utility, mechanistic studies were performed with acronycine (I) and two semisynthetic derivatives, 2-nitroacronycine (II) and acronycine azine (III). These three substances demonstrated cytotoxic activity with several human tumor cell lines (breast, colon, lung, melanoma, KB-3, and drug-resistant KB-V1). Compounds II and III demonstrated greater activity than I, and more detailed studies were performed with cultured human breast cancer cells (UISO-BCA-1). Acronycine azine (III) induced the cells to accumulate in the G0/G1 phase of the cell cycle. It effectively inhibited the in vitro catalytic activities of partially purified DNA and RNA polymerases in a manner that was competitive with respect to DNA substrate. As judged by spectrophotometric titration, compound III interacted with calf thymus DNA, calf liver RNA, and a variety of single- and double stranded (deoxy)ribonucleotides. Although no nucleic acid base specificity was discernable, this interaction appeared to be related to the cytotoxic mechanism of this dimeric substance. Monomeric compounds I and II did not interact with nucleic acids, but were effective inhibitors of DNA and RNA synthesis as judged by in vitro systems comprised of cultured UISO-BCA-1 cells or homogenates derived from these cells. The relative inhibitory activities of compounds I and II correlated with their cytotoxic activities suggesting a causal relationship. In addition, these two compounds induced cultured cells to accumulate in the phase of the cell cycle wherein the DNA content ranged from 2n-4n (S + G2/M), and inhibited in vitro DNA and RNA synthesis in a manner that was competitive with respect to nucleotide (TTP or UTP) substrate. Compounds I and II demonstrated greater cytotoxic activity with drug-resistant KB-V1 cells as compared with the parent (drug-sensitive) cell line, whereas this was not the case with compound III. Based on these results and previous literature reports, compounds I, II and III are likely to function by multiple mechanisms of action. However, it appears that alteration of nucleic acid metabolism is key to the activity of each of the substances. PMID- 1730148 TI - NADH-dependent reductive stress and ferritin-bound iron in allyl alcohol-induced lipid peroxidation in vivo: the protective effect of vitamin E. AB - The role of iron in allyl alcohol-induced lipid peroxidation and hepatic necrosis was investigated in male NMRI mice in vivo. Ferrous sulfate (0.36 mmol/kg) or a low dose of ally alcohol (0.6 mmol/kg) itself caused only minor lipid peroxidation and injury to the liver within 1 h. When FeSO4 was administered before allyl alcohol, lipid peroxidation and liver injury were potentiated 50-100 fold. Pretreatment with DL-tocopherol acetate 5 h before allyl alcohol protected dose-dependently against allyl alcohol-induced lipid peroxidation and liver injury in vivo. Products of allyl alcohol metabolism, i.e. NADH and acrolein, both mobilized trace amounts of iron from ferritin in vitro. Catalytic concentrations of FMN greatly facilitated the NADH-induced reductive release of ferritin-bound iron. NADH effectively reduced ferric iron in solution. Consequently, a mixture of NADH and Fe3+ or NADH and ferritin induced lipid peroxidation in mouse liver microsomes in vitro. Our results suggest that the reductive stress (excessive NADH formation) during allyl alcohol metabolism can release ferrous iron from ferritin and can reduce chelated ferric iron. These findings provide a rationale for the strict iron-dependency of allyl alcohol induced lipid peroxidation and hepatotoxicity in mice in vivo and document iron mobilization and reduction as one of several essential steps in the pathogenesis. PMID- 1730149 TI - Plasma protein binding of APD: role of calcium and transferrin. AB - The bisphosphonate drug APD (pamidronate, 3-amino-1-hydroxypropylidene-1,1 bisphosphonate) has been shown to bind to human plasma proteins. This was an unexpected observation since this hydrophilic, anionic drug is not typical of molecules that exhibit this characteristic. At a concentration of 5 micrograms/ml the extent of binding of APD to fresh human plasma in vitro was variable between subjects 30.2% +/- 8.5% (mean +/- S.D., n = 10). Binding was not influenced by the time or concentration of APD over the range 0.05-10.0 micrograms/ml. At 20 and 50 micrograms/ml some precipitation of APD occurred. Both calcium and iron play a role in the binding of APD to plasma proteins, addition of calcium to plasma increased the degree of binding of APD, whereas the calcium chelators EDTA and EGTA reduced the binding of APD. Similarly, addition of iron to plasma increased the binding and the inclusion of the iron chelator desferrioxamine diminished the binding of the drug. The effects of iron and desferrioxamine were less pronounced than those of calcium and EDTA, indicating that the majority of the binding involves calcium ions and a smaller contribution is made by ferric ions. The equilibrium dissociation constants (Kd) for APD binding to calcium and iron binding sites on plasma proteins were estimated to be 852 microM and 29 microM, respectively. Calcium binding sites were of high capacity but low affinity and the iron binding sites were of lower capacity and higher affinity. Electrophoresis of plasma proteins following incubation with [14C]APD revealed binding to the transferrin and globulin fractions. However, there was some dissociation of protein bound APD during the electrophoresis. The consequences of hypercalcaemia on the pharmacokinetics of APD are discussed. PMID- 1730150 TI - Hemoglobin binding of monocyclic aromatic amines: molecular dosimetry and quantitative structure activity relationships for the N-oxidation. AB - Aromatic amines are important intermediates in industrial manufacturing. N oxidation to the N-hydroxyarylamines is a key step determining the genotoxic properties of aromatic amines. N-hydroxyarylamines can form adducts with DNA, with tissue proteins and with the blood proteins albumin and hemoglobin in a dose dependent manner. The determination of hemoglobin adducts is a useful tool for biomonitoring exposed populations. We have established the hemoglobin binding index (HBI) [(mmol compound/mol Hb)/(mmol compound/kg body wt)] of several aromatic amines in female Wistar rats. Including the values obtained by other researchers in the same rat strain, the logarithm of hemoglobin binding (log HBI) was plotted against the following parameters: the sum of the Hammett constants (sigma sigma = sigma p + sigma m), pKa, log P (octanol/water), the half wave oxidation potential (E1/2) and the electronic descriptors of the amines and their corresponding nitrenium ions obtained by semiempirical calculations (MNDO, AM1 and PM3), such as atomic charge densities, energies of the HOMO and LUMO and their coefficients, the C-N bond order, the dipole moments and the 'reaction enthalpy' [MNDOHF, AM1HF or PM3HF = Hf(nitrenium) - Hf(amine)]. The correlation coefficients were determined from the plots of all parameters against log HBI for all amines by means of linear regression analysis. The amines were classified into three groups: group 1, all para-substituted amines, group 2, all amines with halogens and group 3, all amines with alkyl groups. For the amines of group 1, log HBI correlates with sigma sigma, MNDOHF, E1/2, the pKa and the log P with r = 0.84, 0.71, 0.73, - 0.69 and 0.50, respectively. For the amines of group 2, log HBI correlates with pKa, sigma sigma, MNDOHF, E1/2 and log P with r = 0.81, 0.76, -0.55, -0.46 and -0.20, respectively. For the amines of group 3, log HBI correlates with the E1/2, PM3HF, sigma sigma, pKa and log P with r = 0.92, 0.89, 0.76, 0.19 and 0.12, respectively. The apparent Michaelis-Menten constants Km and Vmax of the N-acetyltransferase of liver cytosol were determined for several amines. Km and Vmax do not correlate with any of the electronic descriptors. Female Wistar rats were dosed with nitroarenes. Hemoglobin binding of nitroarenes correlates with the energy levels of the LUMO. This investigation determines for a large variety of aromatic amines the bioavailability of the N-hydroxyarylamine- the genotoxic metabolite--and the utility of electronic descriptors for prediction of the N-oxidation. PMID- 1730151 TI - A different outlook at the phenotype-function relationships of T cell subpopulations: fundamental and clinical implications. PMID- 1730152 TI - Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3. AB - The present study was undertaken to examine and compare the direct effect of two Pseudomonas enzymes, elastase and alkaline protease, on the serum hemolytic complement as a whole, and on the two recognition molecules of complement, C1q and C3 in particular. The results of our study show that incubation of serum with 0-50 micrograms/ml elastase or protease (60 min, 37 degrees C) resulted in a dose dependent depletion of hemolytic complement with the protease being 3-4 times more efficient than elastase. Incubation of highly purified C3 (20 hr, 37 degrees C) with protease (2% w/w) resulted in the conversion of the 190-kDa molecule to a 120-kDa fragment. When analyzed by SDS-PAGE under reducing conditions, the 120 kDa piece yielded three distinct bands: an intact 75-kDa beta-chain and two alpha chain pieces of approximately 41- and 26-kDa. NH2-terminal end sequence analysis localized the 26-kDa fragment within the cysteine-rich 41-kDa, COOH-terminal piece. This in turn suggests that the 70-kDa fragment which is not accounted for on SDS-PAGE is derived from the NH2-terminal end of the alpha-chain molecule which is completely degraded into small fragments. While the degradation pattern obtained with elastase is similar to that of protease, the latter enzyme was found to be more efficient. Exposure of C1q (0-5 hr, 37 degrees C) to protease or elastase on the other hand appears to reveal preferential sensitivity of the 28 kDa A-chain and 24-kDa C-chain, of the C1q molecule, with the protease being more potent than the elastase. Since both C1q and physiologic fragments of C3 (C3b, iC3b, and C3dg) are important opsonins of varying efficiencies, degradation of these molecules by Pseudomonas enzymes may, in part, facilitate the survival and proliferation of the organism in plasma. Furthermore, degradation of the key recognition molecules of complement, C1q and C3, would enhance the virulence of this organism by aborting complement-mediated bacterial killing. In addition the results imply that during Pseudomonas bacteremia, PaAP may be a much more destructive enzyme than PaE with regards to C3 and C1q but combined, the synergistic effect may overwhelm not only the proteins of the complement system, but other proteins of the humoral immune defense system as well. PMID- 1730153 TI - Cellular composition of germinal centers in lymph nodes after HIV-1 infection: evidence for an inadequate support of germinal center B lymphocytes by follicular dendritic cells. AB - Germinal centers in lymph nodes with follicular hyperplasia from 15 patients with HIV-1 infection were analyzed by qualitative and quantitative electron microscopical methods and compared with control follicular hyperplasia (FH). Using a pattern recognition method, two main clusters were recognized within the germinal centers of HIV and FH lymph nodes on the basis of the relative frequencies of small centroblast and centrocytes. All FH lymph nodes and 6 HIV-1 lymph nodes (HIV-Clu-1) were placed in cluster 1; 9 HIV-1 lymph nodes (HIV-Clu-2) formed cluster 2. Germinal centers in the HIV-Clu-2 lymph nodes were characterized by a cell composition of predominantly lymphoid blasts and decreased numbers of centrocytes, but without altered numbers of mitotic figures. The frequency distribution of ultrastructurally distinct FDC subtypes differed between these clusters. In HIV-Clu-2 the frequencies of FDC types with an undifferentiated and regressive morphology occurred at a higher frequency, whereas FDC types with a highly differentiated morphology had a lower frequency. We conclude that 9 out of 15 lymph nodes with HIV-1 associated follicular hyperplasia show changes in FDC morphology indicative of a less differentiated functional stage of FDC. The changes in FDC morphology are closely associated with changes in the germinal center B-cell population resulting in an inverted blast to the centrocyte ratio. PMID- 1730154 TI - Autoantibodies and circulating immune complexes in sera from patients with hepatitis B virus-related chronic liver disease. AB - Sera from 56 patients with hepatitis B virus-related chronic liver disease (CLD) and 30 normal individuals as controls were examined by enzyme immunoassay (EIA) for the presence of autoantibodies directed against actin, tubulin, myosin, double-stranded (ds) DNA, polymerized human albumin, thyroglobulin, and trinitrophenyl coupled to bovine serum albumin (TNP-BSA). Patients with CLD had consistently elevated levels of IgG, IgA, and IgM antibodies directed against all the panel antigens. The percentage of patients with autoantibodies of the IgA class was particularly high: respectively, 88 and 78% of the patients had strikingly high levels of anti-actin and anti-TNP-BSA IgA autoantibodies. High amounts of IgA and IgG antibodies to polymerized albumin as well as IgG to thyroglobulin were also detected. Circulating immune complexes (CIC) were isolated from patients' sera and their autoantibody activities were tested on the same antigen panel. The autoantibodies thus detected were of the same class and possessed the same activities, although at higher values than those present in the homologous sera. These results indicate that, regardless of their origin, autoantibodies are present in high amounts in the sera of these patients. Moreover, autoantibodies participating in the formation of CIC might play a pathological role. PMID- 1730155 TI - Monocyte-derived macrophage function in HIV-infected subjects: in vitro modulation by rIFN-gamma and rGM-CSF. AB - We investigated monocyte-derived macrophage function in 25 HIV-positive patients, 19 in the CDC class III and 6 class IV; 17 were intravenous drug abusers (IVDA) and 8 were homosexual men. Macrophages from HIV-positive patients behaved normally in assays of superoxide anion (O2-) production and candidacidal activity. After 3 days' treatment with 200 U/ml recombinant interferon-gamma (rIFN-gamma) or 250 U/ml recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF), both control and HIV-positive patients' phagocytes expressed the activated state, as indicated by the increased O2- production in response to phagocytable or soluble stimuli; however, these cytokines did not enhance candidacidal activity. Compared to appropriate HIV-negative controls (18 healthy heterosexuals, 4 homosexuals and 4 IVDA), macrophages from 19 of the 25 HIV positive patients presented a significant defect in their Fc receptor (FcR) dependent phagocytosis, independently from the CDC stage, AZT therapy, or life style. Treatment of macrophages with rIFN-gamma impaired their capacity to ingest IgG-coated erythrocytes, both in controls and HIV-positive subjects. Treatment of phagocytes with rGM-CSF significantly increased their FcR-dependent phagocytosis in controls, whereas in HIV-positive patients and in HIV-negative homosexuals and IVDA only an upward tendency was observed. Although the mechanism of the impaired FcR-dependent phagocytosis in HIV-positive patients remain to be clarified, our results suggest that this functional defect may be secondary to phagocyte priming by circulating IFN-gamma in vivo. This macrophage alteration may be implicated in the immunodeficiency of HIV-positive patients. However, considering the potential role of FcRs in HIV infection enhancement, the defective FcR function might even be a protective mechanism against FcR-mediated HIV dissemination. In the light of these findings, the immunotherapeutic potential of IFN-gamma and GM-CSF in HIV infection merits further investigation. PMID- 1730156 TI - Restricted tissue reactivity of autoantibodies to a 64-kDa eye muscle membrane antigen in thyroid-associated ophthalmopathy. AB - We studied the tissue specificity of eye muscle (EM) membrane-reactive autoantibodies detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In preliminary studies, such antibodies were shown to react, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, with human thyroid (THY) and other human skeletal muscle (HSM) membrane antigens. We carried out absorption with human EM (HEM), THY, and HSM membranes of sera from patients with TAO and autoimmune thyroid disease without ophthalmopathy which reacted with one or more of 55-, 64-, and 95-kDa antigens in pig eye muscle (PEM) membrane in immunoblotting, the majority of which were also cytotoxic to HEM cells in an antibody-dependent cell-mediated cytotoxicity assay. In Western blotting, serum antibodies reactive with PEM membrane antigens of 55, 64, and 95 kDa were cross-absorbed by HEM, THY, and HSM but not by spleen or brain membranes and showed some species specificity, being absorbed by pig and human, but not bovine, EM membranes. When incubated with cultured HEM, THY, and HSM cells in vitro, autoantibodies in TAO sera immunoprecipitated a 64-kDa antigen from the first two tissues, but not from HSM, suggesting a specific binding to autoantigenic epitopes in HEM and THY. Sera from patients with TAO as well as those from patients with thyroid autoimmunity without ophthalmopathy immunoprecipitated a approximately 66-kDa protein, shown to be distinct from the 64-kDa antigen. The restricted immunological cross-reactivity of antibodies to a THY and HEM 64-kDa membrane antigen is discussed in the context of the association of ophthalmopathy with thyroid autoimmunity. Further experiments are needed to show whether autoantibodies to the 64-kDa eye muscle and thyroid shared antigen are cytotoxic, and thus likely to play a major role in the pathogenesis of the eye disease, or just markers of the orbital autoimmune process. PMID- 1730157 TI - Follow-up of soluble interleukin-2 receptor levels after thymectomy in patients with myasthenia gravis. AB - Soluble interleukin-2 receptor (sIL-2R) levels were followed up after thymectomy by a quantitative immunoradiometric assay in 59 patients with myasthenia gravis (MG). Increased levels of sIL-2R were found in 30.5% of the patients before thymectomy. Serum levels were significantly higher in severely affected patients. Sequential sampling after thymectomy indicated a significant and progressive decline of sIL-2R levels within 2 years after surgery, which was well associated with clinical improvement or remission. The sIL-2R purified from sera of patients with MG had a molecular mass of 45 kDa as the normal sIL-2R. The decline after thymectomy of sIL-2R titers suggests a possible role of the thymus in the occurrence of sIL-2R in the periphery. Soluble IL-2R levels may represent a marker of disease severity in MG, which might be useful in the follow-up of individual patients. PMID- 1730159 TI - Inhibition of a novel model of murine experimental autoimmune orchitis by intravenous administration with a soluble testicular antigen: participation of CD8+ regulatory T cells. AB - Recently, we established a novel murine model of experimental autoimmune orchitis (EAO) in C3H/He mice by means of two sc injections of 1 x 10(7) viable syngeneic testicular germ cells (TC) without the use of any adjuvants. Using this model, an effective and reproducible system of immunoregulation in EAO was developed. The induction of this EAO was suppressed by pretreatment with five iv injections of a soluble (deaggregated) form of murine testicular antigen (mTA). The antigen, mTA, was prepared by acid extraction and ammonium sulfate precipitation of defatted testes and epididymides. The development of EAO and relevant delayed-type hypersensitivity was suppressed in an antigen-specific fashion, but anti-TC antibody formation was not affected. A single dose of cyclophosphamide at 2 days after the tolerogenic regime abrogated the unresponsiveness to EAO. Three doses of recombinant interleukin 2 at every other day starting on the next day of the last pretreatment did not overcome the unresponsiveness to EAO. CD8+ T cells isolated from the spleen of deaggregated mTA-pretreated animals could adoptively transfer suppression against EAO into naive recipients, whereas CD4+ T cells failed to transfer the suppression. PMID- 1730158 TI - Acetylcholine receptor-reactive antibodies in experimental autoimmune myasthenia gravis differing in disease-causing potential: subsetting of serum antibodies by preparative isoelectric focusing. AB - Antibodies obtained from the sera of Lewis rats demonstrating impaired neuromuscular function following immunization with purified acetylcholine receptor (AChR) were fractionated by preparative isoelectric focusing. Upon passive transfer of fractionated anti-receptor antibodies into immunologically naive, healthy recipient rats it was observed that two main subsets of AChR specific antibody could be identified. One subset, representing about one-third of the expressed clonotypic antibody repertoire, was capable of directly perturbing AChR-dependent neuromuscular function following transfer. A second subset, demonstrated no detectable ability to induce disease symptoms following transfer. Although the anti-AChR antibodies were produced by immunization with Torpedo AChR, the inability of some antibody fractions to perturb AChR function was not explained by their inability to react with AChR of mammalian origin. Furthermore, the ability to transfer symptoms did not correspond with a particular antibody isotype (although the response was dominated by IgG2a) and did not depend solely on high relative binding avidity (benign reactivities of high relative binding avidity were also observed). Nonetheless, an anti-AChR antibody subset can be directly identified and purified from immune serum that is likely to contain reactivities that are most directly responsible for neuromuscular disease symptoms demonstrated by rats with experimental autoimmune myasthenia gravis. PMID- 1730160 TI - Effects of swimming exercise on the pathogenesis of acute murine Toxoplasma gondii Me49 infection. AB - The effects of swimming exercise on the pathogenesis of acute murine toxoplasma infection were studied. Swimming (45 min/day) initiated on the day of inoculation with the avirulent Me49 strain of Toxoplasma gondii did not alter survival of infected mice. At a later stage of infection, daily swimming appeared to promote the recovery of appetite and weight gain. Immune activation was apparent in toxoplasma-infected mice, and swimming blunted splenic enlargement but not the respiratory burst activity of peritoneal exudate cells. Infection caused a significant elevation of serum tumor necrosis factor (TNF) levels which was attenuated by a daily swimming program. These data show that swimming exercise is not deleterious to mice acutely infected with T. gondii Me49 and that the more rapid recovery in exercised mice is associated with reduced serum TNF levels. PMID- 1730161 TI - Background genes mediate the development of autoimmunity in (NZB x PL/J)F1 or (NZB x BIO.PL)F1 mice. AB - The (NZB x NZW)F1 mouse develops a lupus-like disease including anti-double stranded DNA antibodies and renal disease. It has been demonstrated that genes linked to the H-2 locus contributed by NZW correlate with development of this disease. We investigated whether mice with identical class II molecules but different background genes could contribute to autoimmunity when crossed with NZB. We report that two strains, PL/J and BIO.PL, when crossed to NZB, do not result in F1 with autoimmunity. Therefore, background genes present in NZW but not in PL/J or BIO.PL contribute to the development of disease. PMID- 1730162 TI - I-A alpha k transgene pairs with I-A beta b gene and protects C57BL10 mice from developing autoimmune myasthenia gravis. AB - The I-A beta gene has been implicated in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), with amino acids at positions 67, 70, and 71 of the I-A beta b chain that play a crucial role. In addition, the cell surface expression of the I-E molecule in C57BL10.E alpha k transgenic mice was associated with the partially suppressed serum anti-acetylcholine receptor antibody and clinical expression of EAMG. In this study, the contribution of the I-A alpha gene and the A beta b:A alpha k transpair in EAMG pathogenesis is assessed. The I-A alpha k transgene was introduced into the B10 (A beta b:A alpha b) strain, and the I-A alpha k chain was paired with I-A beta b; therefore, the A beta b:A alpha k complex was expressed on the cell surface. Expression of the A beta b:A alpha k transpair in C57BL10 transgenic mice suppressed the cellular and humoral autoimmune responses to acetylcholine receptors (AChR), reduced the amount of muscle AChR bound with antibody, and significantly reduced the incidence of muscle weakness and its associated abnormal electrophysiological response. PMID- 1730163 TI - Concentration of dietary N-6 polyunsaturated fatty acids and the human immune status. AB - We examined the effect of the dietary concentration of total fat and n-6 polyunsaturated fatty acids (PUFA) on the immune status of seven healthy women (age 30-65 years) who lived at our metabolic suite. During the first 20 days all subjects consumed a stabilization diet that contained 5.2 energy percent (en%) PUFA and 41.1 en% fat. For the next 40 days, three subjects consumed a diet with 3.2 en% PUFA and 26.1 en% fat, while the remaining four subjects consumed a diet with 9.1 en% PUFA and 31.1 en% fat. For the next 40 days, the diets of the two groups were crossed over. Blastogenesis of peripheral blood mononuclear cells cultured with phytohemagglutinin, concanavalin A, protein A, and pokeweed, and the serum concentrations of complement fractions C3 and C4 were significantly increased upon the feeding of both low fat (26.1 or 31.1 en%) diets compared to the values when the high fat (41.1 en%) diet was fed. None of the indices tested were different when the high PUFA (9.1 en%) and low PUFA (3.1 en%) diets were compared. Our results indicate that low fat diets improve some of the indices of human immune status and that a moderate increase in the level of n-6 PUFA in an otherwise low fat diet does not suppress the human immune system. PMID- 1730164 TI - Collagen vascular disease. AB - The iatrogenic L-tryptophan-induced eosinophilia-myalgia syndrome, often considered to be a "new" disease, has proven to be a remarkable mimic of the classic sclerosing rheumatologic disorders. Although subacute cutaneous lupus erythematosus remains a clinically defined entity, supportive histologic and immunopathologic findings have recently been proposed. Rheumatoid neutrophilic dermatitis needs to be added to our usual differential diagnosis of a neutrophilic dermatosis without leukocytoclastic vasculitis. The antiphospholipid syndrome is associated with noninflammatory vascular thrombosis and often has recognizable cutaneous findings. Finally, ANCA are a valuable adjunct in the systemic evaluation of patients with vasculitis syndromes and suggest a common pathogenesis for several of the systemic vasculitides. PMID- 1730165 TI - New developments in dermatopathology. PMID- 1730166 TI - The recognition of recently described and potentially problematic melanocytic lesions of the skin. AB - Our attempt has been to examine unusual or problematic melanocytic lesions to enable the reader to recognize them histologically and to enhance the understanding of their biologic significance. An awareness of these lesions by the clinical dermatologist will, it is hoped, result in optimal patient management. The proper identification of melanocytic neoplasms is, and will continue to be, of prime importance to all concerned: the clinician, the dermatopathologist, and especially the patient. PMID- 1730167 TI - Dysplastic nevi and the dysplastic nevus syndrome. AB - Patients with all the clinical features of FDNS but no family history of multiple abnormal nevi or melanoma can be compared with patients with neurofibromatosis due to a spontaneous mutation of the gene in utero. Whether or not such patients are in fact genetically identical to patients with FDNS and share their high risk of malignant melanoma remains to be determined. An isolated dysplastic nevus alone is not an adequate definition of SDNS, because current data are insufficient to show that its presence correlates with a uniquely high risk of melanoma when compared with other known risk factors. Until more specific tests for the FDNS gene become available, the diagnosis of SDNS must be made on the basis of close clinical and histopathologic resemblance to FDNS. Patients who present as adults with one or a few dysplastic nevi are best not labeled as having SDNS, because that label implies a genetic identity with FDNS that is probably not true and that deflects attention from other risk factors that are at least as important in estimating individual risks of developing melanoma. PMID- 1730168 TI - Unusual benign fibrous and fibrohistiocytic tumors of the skin. AB - This article reviews recently reported unusual benign fibrous and fibrohistiocytic tumors of the skin. Some of these lesions are variants of common and well-known entities. Because some of these variants have cytologic atypia, they could be confused with malignant neoplasms. PMID- 1730169 TI - Newly recognized neural neoplasms relevant to the dermatopathologist. AB - Several forms of neural neoplasia have recently been described and are still not well known. Entities such as nerve sheath myxoma, cellular schwannoma, plexiform schwannoma, and palisaded encapsulated neuroma have special clinicopathologic relevance and should be diagnosed correctly by the dermatopathologist. Familiarity with these new entities and with their differential diagnosis contributes to better patient management. PMID- 1730170 TI - Lymphomas. AB - The diagnosis of lymphoma currently relies on a combination of clinical, routine histopathologic, and immunohistochemical studies, sometimes supplemented by other special techniques such as flow cytometry, DNA analysis, and gene rearrangement study. This review considers the practical diagnosis of cutaneous lymphoma, with particular emphasis on three areas: Hodgkin's disease, non-Hodgkin's lymphomas and their differentiation from lymphocytoma cutis, and mycosis fungoides and other T-cell lymphomas. PMID- 1730171 TI - Therapy for genital warts. AB - This article presents the spectrum of genital wart infection, including possible subclinical infection and reinfection from partners. Present therapy is effective in warts of recent onset but usually requires multiple treatments and is painful, and warts are frequently recurrent. Trials of combination therapy appear to have greater efficacy than does single-agent treatment. Problems for the future include the best management of warts in the sexually abused child, the immunocompromised host, and the pregnant patient. Genital warts are increasingly linked with the potential development of carcinoma, mandating more effective treatment regimens in the future. PMID- 1730172 TI - Panniculitis. AB - An accurate assessment of subcutaneous lesions can be made by joining the differential diagnoses generated by clinical and histologic features. Anatomic location of lesions, presence or absence of ulceration, occurrence of lipoatrophy, history of trauma, association with immunologic or metabolic disorders, and age of the patient are important clinical data to consider in conjunction with the microscopic features. Individual histologic features such as calcification, cytophagic histiocytes, pseudocysts, lymphoid nodules, and crystals should be evaluated in conjunction with the inflammatory infiltrate (i.e., granulomatous, neutrophilic, lymphocytic, or combined); presence and type of necrosis (e.g., basophilic, hyaline, lobular, or septal); and presence and extent of sclerosis. For optimal management of patients, it is important to consider the differential diagnosis generated by the dominant histologic features as well as the clinical and histologic abnormalities. PMID- 1730173 TI - Dermatopathologic findings in patients infected with HIV. AB - Several inflammatory, infectious, and neoplastic conditions in HIV-infected patients are distinctive or require a biopsy for diagnosis. Some differ subtly from similar conditions seen in noninfected patients. The exanthem of acute HIV infection cannot be diagnosed specifically on biopsy as its histologic appearance is similar to that of other viral exanthemata. A condition that closely resembles seborrheic dermatitis occurs in HIV-infected patients. Plasma cells, necrotic keratinocytes, and leukocytoclasis may be present, in contrast to findings in sporadic seborrheic dermatitis. Psoriasis and Reiter's disease also occur in HIV infected patients and can be specifically diagnosed as such. The category "psoriasiform dermatitis of AIDS" thus seems to include several distinct entities and not to be a single disease. Bacillary angiomatosis is a treatable infection caused by a rickettsialike organism similar to Rochalimaea quintana, the agent of trench fever. Cutaneous lesions are characterized by lobules of capillaries with protuberant endothelial cells, neutrophils and their debris, and purplish staining clumps of organisms, which can be demonstrated with silver stains or electron microscopy. An unusual reaction to atypical mycobacterial infection, in which spindle-shaped macrophages are seen, resembles histoid leprosy. Viral skin diseases that may challenge the dermatopathologist include unusual verrucous reactions to chronic varicella-zoster infection and flat warts caused by the human papillomavirus associated with epidermodysplasia verruciformis. Keratinocytes with foamy basophilic cytoplasm may be a marker for one of these viruses, human papillomavirus type 5. Neoplastic complications of HIV disease include Kaposi's sarcoma and mycosis fungoides. The earliest lesions of the patch stage of Kaposi's sarcoma show a slightly increased number of cells with small ovoid nuclei around preexistent structures, accompanied, in some cases, by sparse infiltrates of lymphocytes and plasma cells. Staining with antisera to type IV collagen may highlight the vascular spaces in these early lesions. Later lesions that resemble hemangiomas may also prove challenging and require level sections to demonstrate the presence of spindle cells and eosinophilic globules. Although HIV is cytotoxic to helper T cells, neoplastic proliferations of them may be seen in HIV-infected patients. These cases of mycosis fungoides do not seem to differ from sporadically occurring ones and occur in patients who seem not to be infected by HTLV-I. PMID- 1730174 TI - Leprosy. AB - Growing out of the successful transmission of leprosy to armadillos, making available large quantities of M. leprae, there have been remarkable recent advances in the knowledge of the leprosy bacillus. These bacilli and their isolated chemical constituents provide organisms for in vitro testing of new drugs, reagents for the study of the immunologic dysfunction in leprosy patients, development of early diagnostic methods, and the preparation of candidate vaccines. Leprosy is usually transmitted by the nasorespiratory route, but occasionally, there is transplacental infection. There are reports suggesting that patients have acquired leprosy by contact with wild M. leprae-infected armadillos in Louisiana and Texas. Perturbations in lymphocyte-macrophage interaction appear to be most closely related to the defective CMI in leprosy. The helper T/suppressor T cell populations vary markedly in lesions of the various forms of leprosy, with enhanced suppression of T-cell activity in lepromatous disease. Infiltration of IL-2 and gamma-interferon seems to stimulate CMI in situ in lesions of lepromatous leprosy. Vaccination of lepromatous patients with a killed M. leprae-plus-BCG preparation stimulates CMI and clears tissues of leprosy bacilli, providing an immunotherapeutic approach to the management of leprosy. Immunoprophylactic vaccine trials are in progress, and initial results should be available in 1991. Because of drug resistance, dapsone monotherapy of leprosy is no longer recommended. Multidrug regimens, composed of dapsone, rifampin, and clofazimine or a thioamide, are now required and appear to reduce the incidence of leprosy when applied assiduously. Newer experimental drugs that may eventually be included in these regimens include the fluoroquinolones, minocycline, and clarithromycin. There is no clear evidence that the early serologic diagnosis of leprosy is generally applicable. Favorable response to therapy in multibacillary patients, however, may be assessed by noting drops in levels of M. leprae-specific antigens in blood and urine and, to a lesser extent, levels of specific antibodies in serum. There are conflicting reports on the influence of AIDS on leprosy. There are no convincing data showing that AIDS and leprosy affect each other. Although chemotherapy offers the best current hope for the control of leprosy, effective immunoprophylaxis and improved socioeconomic conditions in endemic areas are thought to be essential in programs for the eradication of leprosy. PMID- 1730175 TI - Acquired benign and "borderline" vascular lesions. AB - In recent years, the classification of vascular lesions has been expanded and modified with the addition of several newly described entities, the redefinition of others, the recognition of lesions of borderline biologic behavior, and the need to avoid misdiagnosis with early Kaposi's sarcoma. This review clarifies the nomenclature, updates information on previously known lesions, and summarizes data on several recently discovered, lesser-known entities such as glomeruloid hemangioma, microvenular hemangioma, and multinucleate cell angiohistiocytoma. Clinicopathologic features and differential diagnosis are emphasized. PMID- 1730176 TI - Transdermally administered fentanyl for pain management. AB - The physicochemical properties, pharmacology, pharmacokinetics, serum concentrations and clinical effects, adverse effects and contraindications, and dosage of transdermally administered fentanyl are described, and clinical studies evaluating the use of a transdermal fentanyl system in the treatment of postoperative pain and chronic cancer-associated pain are reviewed. After application of a transdermal system, fentanyl is absorbed into the skin beneath the patch, where a depot forms in the upper skin layers. Plasma fentanyl concentrations are barely detectable for about two hours after patch placement. Eight to 12 hours after patch placement, concentrations approximate those achieved with equivalent i.v. doses of fentanyl. Some studies comparing transdermally administered fentanyl with placebo in postoperative patients showed that the patients who received fentanyl required fewer supplementary analgesics and reported less pain than the patients who received placebo. However, the overall efficacy and safety of the transdermal fentanyl system for the treatment of postoperative pain have not been adequately evaluated. Studies of cancer patients showed that transdermally administered fentanyl appears to be effective in the management of chronic, cancer-related pain. Dermatological reactions to the fentanyl patch are generally transient and mild. Other adverse effects are those that are commonly associated with narcotic analgesics. The 25-micrograms/hr patch should be used for initial treatment in patients not previously treated with narcotics. The dosage may be gradually increased until effective analgesia is obtained. Although experience with the product is limited, transdermally administered fentanyl appears to be effective for the long-term management of cancer-related pain. PMID- 1730177 TI - Features and treatment of rabies. AB - The epidemiology, virology, transmission, pathology, clinical manifestations, diagnosis, and treatment of rabies infection are described. The incidence of rabies is increasing. The virus is usually transmitted through the bite of an infected animal; however, corneal transplantation from an infected donor and viral inhalation may also result in infection. The clinical course of rabies occurs in five stages. Stage I is the incubation period, stage II is the prodromal period, stage III is the neurological period, stage IV is coma, and stage V, which occurs infrequently, is recovery. Early in the disease, constitutional symptoms and signs of local wound healing may be present. During the later stages, a wide array of clinical manifestations may occur, including hydrophobia and aerophobia, which are pathognomonic for rabies. Once inoculation with the rabies virus is suspected, prompt therapy, including appropriate wound care and rabies vaccination, is imperative, even in previously immunized individuals. Human diploid-cell vaccine and rabies vaccine adsorbed, which stimulate the production of antibodies, and human rabies immune globulin, which provides protective antibodies, are nearly 100% effective in preventing progression from stage I disease. If left untreated, rabies is usually fatal. However, treatment with human diploid-cell vaccine or rabies vaccine adsorbed and with human rabies immune globulin is nearly always curative if initiated early in the incubation period. PMID- 1730178 TI - Interference by antiarrhythmic agents with function of electrical cardiac devices. AB - The possible interference of drugs with the function of implanted electrical cardiac devices in two patients is described, and the literature on the interaction between these drugs and devices is reviewed. A 59-year-old woman had a permanent pacemaker implanted after diagnosis of tachycardia-bradycardia syndrome, and her drug regimen of digoxin, verapamil, and warfarin was supplemented with flecainide to prevent paroxysmal atrial fibrillation. A Holter monitor recording performed after five days of flecainide therapy showed the pacemaker was sensing and pacing normally. Approximately three weeks after pacemaker implantation and initiation of flecainide therapy, a Holter monitor recording showed high-grade atrioventricular block and failure of the pacemaker to capture. A chest roentgenogram showed that the pacemaker lead was properly placed. The pacemaker's pulse amplitude and pulse width were increased, and the device again functioned reliably. Six months later the pacing threshold was noted to have returned to normal. A 64-year-old man had a history of aborted sudden cardiac death from ventricular tachyarrhythmias. The patient received an automatic implanted cardioverter-defibrillator (AICD); at that time, a 15-J discharge was required to terminate induced ventricular fibrillation (VF). Three years later, the AICD was replaced; the energy required for VF termination with the new unit was 16 J. Seven months after implantation of the new unit, the patient had several episodes of ventricular tachycardia (VT). Moricizine therapy was initiated. An electrophysiologic study (EPS) three days later showed that a larger shock (28 J) was required to terminate VF and that the pacing threshold had increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730179 TI - Muscle spasms associated with intrathecal morphine therapy: treatment with midazolam. PMID- 1730180 TI - Working group issues report on aluminum contamination of parenteral nutrient solutions. PMID- 1730181 TI - Criteria for use of zidovudine in adult and pediatric inpatients and outpatients. PMID- 1730182 TI - Clinical evaluations of the Wellcolex Colour Shigella and Salmonella tests. AB - The Wellcolex Colour Salmonella and Shigella tests are rapid latex agglutination procedures for grouping Salmonella and Shigella using an antibody attached to multicolored latex particles. The tests correctly grouped 46 (100%) of 46 Salmonella clinical isolates in groups A through E and G, 42 (100%) of 42 Shigella clinical isolates, and seven (88%) of eight Shigella stock cultures. The stock culture missed had been passed through many transfers and may have lost some of its antigenicity. The Wellcolex Colour Salmonella test was also found useful in detecting and grouping Salmonella spp. directly from blood culture bottles. PMID- 1730183 TI - Quantitative tip culture methods and the diagnosis of central venous catheter related infections. AB - The diagnostic usefulness of two quantitative catheter culture methods was compared in a prospective study of central venous arterial catheters. The roll plate method followed by sonication was used to culture 177 catheters from 85 patients, and the sonication method was used to culture 136 catheters from 68 patients. All patients were evaluated for catheter-related infections. Catheter related infections were associated with greater than or equal to 100 colony forming units (CFU) isolated from catheter tips by either roll plate (p = 0.01) or sonication (p less than 0.001). The sensitivity, specificity, and positive and negative predictive values of greater than or equal to 10(3) CFU by roll plate for catheter-related septicemia were 56%, 97%, 63%, and 96% compared with 93%, 95%, 76%, and 99%, respectively, for the same level by sonication. For central venous and arterial catheters, the sonication method can distinguish infection from contamination and is superior to the roll-plate method in that it may offer a more sensitive and predictive alternative in the diagnosis of catheter-related septicemia. PMID- 1730184 TI - Rapid detection of influenza-B virus in respiratory secretions by immunofluorescence during an epidemic. AB - A commercially available indirect immunofluorescent assay for detection of respiratory viruses in nasal secretions was compared with cell culture during an influenza-B epidemic. This assay had poor sensitivity in the detection of influenza-B virus when used directly patients specimens. PMID- 1730185 TI - The impact of converting to a biphasic blood-culture system on the overall cost and the incidence of pseudobacteremia. AB - After converting from a conventional broth (CB) system to a biphasic (BP) agar slide blood-culture system (Septi-Chek), our laboratory noted an increase in positive blood cultures in general, and in coagulase-negative staphylococci (CNS) in particular. To investigate these findings, we compared all blood cultures collected over a 21-month period using CB and then BP systems, totaling 28,199 blood cultures. The frequency of positive blood cultures increased from 9.2% to 12.7% (p less than 0.0001), whereas CNS isolation increased from 2.6% to 5.2% (p less than 0.0001). There was no significant change in the incidence of true primary or secondary bacteremia due to CNS (p = 0.9). The isolation of other pathogens, including Staphylococcus aureus, Candida albicans, Bacteroides species, and Gram-negative bacilli increased from 6.5% to 7.1% (p less than 0.05). We estimated the cost of processing 28,000 blood cultures by both CB and BP systems, using positivity rates of 9.2% and 12.7%, respectively, and standards provided by the College of American Pathologists (CAP, 1991) for workload hours of technologist time. We calculated a higher overall cost for the BP system. However, the use of this system eliminated the use of needles and syringes for subculture of bottles showing no growth, thus decreasing the risk of technologist exposure to body fluids. Despite the increased cost and more frequent occurrence of pseudobacteremia, the enhanced sensitivity and increased safety of the BP system justified its use in the prompt identification of patients with true bacteremia. PMID- 1730186 TI - Pneumonia. Patient profiles, choice of empiric therapy, and the place of third generation cephalosporins. AB - Choosing appropriate antimicrobial therapy for patients with pneumonia requires knowledge of the etiologic agents seen in specific kinds of patients at specific times and places. For community-acquired pneumonia, there is an important difference in the agents seen in the normal and the compromised host. The normal host most often presents with viral, mycoplasmal, or pneumococcal pneumonia. The exact place of Chlamydia pneumoniae is still under study. A normal host who aspirates is at risk of anaerobic pneumonia. Normal hosts with influenza may acquire superinfection with Streptococcus pneumoniae, Haemophilus influenzae, or Staphylococcus aureus. Under specific epidemiologic conditions, community acquired pneumonia may be due to Legionella species, Yersinia pestis, Francisella tularensis, Coxiella burnetii, Chlamydia psittaci, a mycotic agent, or tuberculosis. Patients with chronic bronchitis and emphysema are predisposed to H. influenzae, Moraxella catarrhalis, and S. pneumoniae infections. HIV-infected patients are likely to have Pneumocystis carinii pneumonia and pneumonia due to cytomegalovirus, S. pneumoniae, and H. influenzae. Patients with diabetes, nursing-home patients, hospitalized patients, immuno-compromised patients, and patients with recent antibiotic therapy are predisposed to pneumonia due to Gram negative aerobic bacilli of enteric and environmental origin. Initial therapy should be directed at the likely organism or organisms based on hospital susceptibility surveillance. In the normal host with community-acquired pneumonia, the therapy will often be penicillin G or erythromycin. In the patient predisposed to Gram-negative pneumonia, a third-generation cephalosporin with or without an aminoglycoside is the usual choice. PMID- 1730187 TI - Current status of bacterial resistance to third-generation cephalosporins. AB - A review of the evolution of bacterial resistance to third-generation cephalosporins is presented, focusing mainly on the prototypical member of this group-cefotaxime. Third-generation cephalosporins generally remain highly active against most Enterobacteriaceae, staphylococci, streptococci, Haemophilus, and Neisseriaceae. Only enterobacteria with a high frequency of mutant derepressed strains that hyperproduce chromosomally mediated beta-lactamase, Pseudomonas spp., and some glucose nonfermenter Gram-negative bacilli have demonstrated increased levels of resistance. The significance of derepressed strains and of the recently described extended-spectrum, plasmid-mediated beta-lactamases to the usefulness of the third-generation cephalosporins is discussed. PMID- 1730188 TI - Comparison of cefotaxime with ceftriaxone given intramuscularly 12-hourly for community-acquired pneumonia. AB - Cefotaxime 1 g intramuscularly (i.m.) 12-hourly was compared with ceftriaxone 1 g i.m. 12-hourly in adult patients requiring hospitalization with uncomplicated community-acquired pneumonia. Fifty-two patients were enrolled and two were subsequently withdrawn, leaving 50 patients who completed the study; 23 received cefotaxime and 27 received ceftriaxone. Clinical cure was achieved in 49 of the 50 patients (98%). One treatment failure occurred in a patient who received ceftriaxone. The only significant pathogen isolated from the pretreatment sputum cultures was Streptococcus pneumoniae (50%). All isolates were sensitive to both drugs. Cefotaxime 1 g i.m. 12-hourly was as effective as ceftriaxone in the treatment of patients with uncomplicated community-acquired pneumonia requiring hospital admission. PMID- 1730189 TI - A multicenter, open comparative study of parenteral cefotaxime and ceftriaxone in the treatment of nosocomial lower respiratory tract infections. AB - A multicenter Canadian study enrolled 74 persons to compare low-dose cefotaxime at 1 g every 8 hr to ceftriaxone 1 g every 12 hr in patients with nosocomial pneumonia. Of 57 evaluable patients (30 cefotaxime and 27 ceftriaxone) in this preliminary report, 93% responded to therapy in both groups. Ceftriaxone patients tended to have more side effects (14.2%). This study is continuing to accrue patients to achieve 100 evaluable patients. Interim data, however, support the continued use of low-dose cefotaxime as an appropriate alternative for clinically effective and cost-effective management of nosocomially acquired pneumonia. PMID- 1730190 TI - Comparison of the efficacy and adverse effect profile of cefotaxime and ceftriaxone in ICU patients with susceptible infections. AB - In a prospective randomized double-blind trial, the efficacy and safety of cefotaxime and ceftriaxone were compared in intensive care unit (ICU) patients with serious infections requiring systemic antimicrobial therapy. Patients were randomly assigned to receive either cefotaxime 1 g i.v. t.i.d. or ceftriaxone 2 g every 24 hr. Clinical and bacteriologic assessments were made before treatment, at 48 hr and 5 days during treatment, and 48 hr after treatment. At the time of reporting, a total of 34 patients had been entered into the trial, 27 of whom were evaluable; 23 patients (85%) completed a minimum of 5 days antibiotic treatment. At the end of treatment, using current statistics 67% of cefotaxime and ceftriaxone patients demonstrated clinical cure or improvement. Bacteriologic responses appeared greater in the cefotaxime group (55% vs 42%). The incidence of adverse effects, which were usually minor, was similar in each group. From these preliminary results, it would appear that at the doses used in this study, cefotaxime and ceftriaxone are equally effective in the treatment of infections in the ICU. PMID- 1730191 TI - Evaluating the cost-effectiveness of treatment with third-generation cephalosporins. AB - Spreadsheet computer software was used to compare the estimated global treatment costs of the third-generation cephalosporins, cefotaxime and ceftriaxone, in the management of pneumonia using treatment schedules taken from current studies. Included in the analysis were not only acquisition costs, but also costs that contribute to total expenses for a course of treatment, such as those of (a) preparation and administration (disposable supplies, nursing, and pharmacy time), (b) projected laboratory costs to monitor for hypoprothrombinemia, and (c) complication costs (diarrhea, superinfection, pseudocholelithiasis, and so on). The cost analysis was performed using United States trial-derived factors. Where published cost factors were not available, reasonable estimates were sought. Our analysis indicates that cefotaxime therapy may be less costly than ceftriaxone therapy in the dosage schedules used in these clinical studies and routine clinical practice. PMID- 1730192 TI - [Rhesus(D)-IgG-antibodies in the treatment of autoimmune thrombocytopenia]. AB - 25 patients with confirmed chronic autoimmune thrombocytopenia (AITP) (20 women, 5 men; mean age 42 [17-75] years) were treated with anti-erythrocytic rhesus (D) antibodies. In the course of 5 days they each received three doses of 1200 micrograms of anti-D IgG antiserum by short-duration infusion. No adverse effects were noted. In 19 out of 25 (76%) the platelet counts (determined on the 6th day after the last infusion) rose by more than 30,000/microliters, in some by over 200,000/microliters. In 6 patients the maximal rise in platelets was below 30,000/microliters. There was little or no fall in haemoglobin concentration in 24 of the patients, although the direct antiglobulin test was clearly positive in all cases. Moderate transitory anaemia occurred in one case only (haemoglobin fell from 12.4 to 8.4 g/dl). The platelet counts gradually declined after treatment, taking several weeks or months to reach their original levels. The mechanism of anti-D therapy in AITP is not fully understood, but it probably depends on competitive inhibition of Fc receptors on phagocytic cells in the reticuloendothelial system. PMID- 1730193 TI - [Toxic epidermal necrolysis (Lyell's syndrome) after rubella]. AB - Rubella was confirmed clinically and serologically in a 14-year-old boy who had been admitted with mild general symptoms and an exanthematous rash presenting with medium size maculae. Within only three days the exanthema had faded. Five days later the boy again became ill with high fever (40 degrees C) and renewed erythema with at first irregular then rosette-like and widely confluent spots. On admission several nonspecific inflammatory signs were markedly elevated (erythrocyte sedimentation rate, C-reactive protein, alpha-globulin). There were mild abnormalities of left-ventricular repolarization at the electrocardiography. The skin changes were progressive and at first interpreted as erythema exudativum multiforme. Fever persisted, despite antibiotic treatment, and signs of epidermolysis appeared. After the diagnosis of non-staphylogenic Lyell's disease was made high-dosage prednisolone administration was begun (initially 3 mg/kg in 6 hours, then 2.5 mg/kg daily for 8 days). The patient's condition and the electrocardiographic findings returned to normal within 2 days. The epidermolysis was arrested within a few hours and the blisters dried out within 24 hours. There were no complications. Since no drugs were given before the second day of epidermolysis being noted, it is highly likely that rubella precipitated Lyell's disease. PMID- 1730194 TI - [Malignant lymphomatous polyposis of the gastrointestinal tract]. AB - Two years after endoscopic excision of several polyps of the colon (histological examination revealing infiltration by a malignant non-Hodgkin lymphoma) a 67-year old man was again found to have blood and mucus in his stool. Because local recurrence was suspected a coloscopy was performed. This revealed multiple polyps throughout the entire colon, predominantly in the sigmoid and rectum. Biopsy showed focal infiltration of the submucosa and mucosa with centrocytic non Hodgkin lymphoma, compatible with the diagnosis of malignant lymphomatous polyposis of the gastrointestinal tract. Gastroduodenoscopy then demonstrated duodenal polyps and massive coarsening of the gastric folds due to infiltration. For the first time tumour masses were also found in the epipharynx. After 6 chemotherapy cycles according to the CHOP scheme (day 1: cyclophosphamide, 1500 mg, adriblastin, 100 mg and vincristine, 2 mg; days 1-5: prednisolone, 100 mg) all lymphoma infiltrates regressed completely. The patient has been symptom-free for 9 months. PMID- 1730195 TI - [Echocardiography in the diagnosis of pulmonary embolism]. PMID- 1730196 TI - [Pancreas--shock organ and shock mediator]. PMID- 1730197 TI - [Heparin therapy in coronary disease]. PMID- 1730198 TI - [Meningitis--exclusion by uric test strips]. PMID- 1730199 TI - [Alcohol consumption and coronary sclerosis]. PMID- 1730200 TI - [Postoperative complications of elective resection of the colon in diverticulitis]. AB - Data on 142 patients (57 men, 85 women; mean age 64.2 [37-91] years) who had elective colon resection for diverticulitis were retrospectively analysed with respect to postoperative morbidity and mortality. Associated systemic disease was present in 47.9% of patients; intraabdominal complications were found preoperatively in 55.6%. Resection with primary anastomosis was performed in 97.9% of patients. Local postoperative complications were noted in 14.1%, general complications in 31.7%. Two patients died postoperatively of the consequences of local complications. In both cases diverticulitis had been complicated by fistulas (mortality rate 1.4%). With careful preparation, standardized operative technique and intensive perioperative supervision the risk of elective colon resection for diverticulitis is low. It can thus be undertaken, even after the first severe attack of diverticulitis, in patients without associated disease. PMID- 1730201 TI - [Ultrasonographic findings in varicose phlebitis of the great saphenous vein. Evidence of thrombus growth and detachment with asymptomatic lung embolism]. AB - In five women (mean age 58 [31-74] years) with clinically diagnosed varicose phlebitis of the long saphenous vein appositional thrombus growth was demonstrated by serial ultrasonography (B-mode compression and colour-coded duplex sonography). In one patient the appositional thrombus was found to be free floating as far as the common femoral vein, but this was not seen by phlebography. During therapeutic heparinization there was ultrasonographic evidence of softening and partial liquefaction of thrombus material in a cranial direction. There were no clinical signs of pulmonary embolism, but pulmonary perfusion scintigraphy demonstrated perfusion deficit characteristic of emboli. Four of the five patients had a surgical crossectomy and partial saphenous vein resection. The congruence between ultrasonographic and macro- as well as micropathological findings demonstrates the great value of B-mode and duplex ultrasound examination in the area of the epifascial veins. Ultrasonography should be performed in every case of varicose phlebitis of the large veins to exclude the presence of apposition thrombi. PMID- 1730202 TI - [Successful multiple resuscitation in flecainide poisoning]. AB - After a family quarrel a 37-year-old woman swallowed, with suicidal intent, a large number of flecainide tablets (exact amount unknown) together with alcohol. On admission to hospital some hours later her pupils were fully dilated, fixed and of irregular outline; she was unconscious and in cardiorespiratory failure. Nine hours after admission several episodes of ventricular fibrillation and asystole occurred, two of them lasting for 2 and 3 hours, respectively, before successful resuscitation (after defibrillation). The highest plasma flecainide level, between 3 and 10 hours after swallowing the drug, was 6160 ng/ml, i.e. six times the maximal therapeutic level. Under the influence of flecainide the ECG of the previously healthy woman had shown idioventricular rhythm with marked QRS widening and Q-T prolongation. The tachyarrhythmias, at times torsades de pointes, were successfully treated with high doses of lidocaine (4 g daily) after repeated defibrillations. As a late complication the patient went into acute left ventricular failure with pulmonary edema and pneumonia. There were no recognizable permanent sequelae on discharge 37 days after admission. PMID- 1730203 TI - [Color-coded Doppler echocardiography in the diagnosis of myocardial rupture after myocardial infarct]. PMID- 1730204 TI - [Carotid endarterectomy. A critical analysis of current studies]. PMID- 1730205 TI - [Unclear cerebral fits]. PMID- 1730206 TI - [Reserpine and reserpine-containing preparations in hypertension?]. PMID- 1730207 TI - [Converting enzyme inhibitors in preterminal renal failure]. PMID- 1730208 TI - [Stent bridging of malignant esophagojejunal anastomosis recurrence]. PMID- 1730209 TI - [Chromosomal dosimetry]. PMID- 1730210 TI - [Thoracic fluoroscopy]. PMID- 1730211 TI - [Adjuvant therapy in colonic carcinoma]. PMID- 1730212 TI - [Chronic intermittent urokinase therapy in therapy-refractory angina pectoris]. AB - To assess the effect of urokinase-induced reduction of fibrinogen concentration on myocardial perfusion, urokinase infusions were administered for 3 months to 24 men (mean age 59 +/- 10 years) in the inoperable end-stage of coronary heart disease with treatment-resistant angina pectoris. Initially 500,000 IU urokinase were infused i.v. daily until a fibrinogen concentration of 150-200 mg/dl was reached. Treatment was then continued as out-patients at a dosage of 500,000 IU two to four times per week. After 12 weeks the fibrinogen concentration had fallen from 348 +/- 88 to 211 +/- 52 mg/dl and plasma viscosity from 1.44 +/- 0.08 to 1.33 +/- 0.09 mPa.s (P for each less than 0.01). Up to the end of 12 weeks after the end of treatment the frequency of anginal attacks fell significantly from 3.2 +/- 1.6 to 0.7 +/- 0.4 daily (P less than 0.01), while ergometric exercise capacity increased by 76%. Thallium myocardial scintigraphy demonstrated an increased perfusion in all but three of 19 patients, global in 10, regional in 6. These results indicate that in patients with treatment resistant angina due to coronary heart disease chronic intermittent urokinase infusion provides a promising treatment alternative. PMID- 1730213 TI - [Needle tract metastasis following sonographically guided puncture of a mesenteric lymph node metastasis in Pancoast's tumor]. AB - Peripheral bronchial carcinoma with infiltration of the right lateral thoracic wall (Pancoast tumour) was demonstrated in a 60-year-old man with breathing related pain in the right thoracic wall of two months' duration. As part of tumour staging needle puncture of an intra-abdominal space-occupying lesion was performed, guided by ultrasonography. Histological examination confirmed it as a bronchial carcinoma metastasis. Combined radio- and chemotherapy hardly influenced tumour growth. Three months later a subcutaneous lesion became palpable in the area of the previous needle puncture which on excision proved to be a metastasis. The patient died 10 months later from the bronchial carcinoma. Percutaneous puncture of potentially malignant space-occupying lesions must be strictly indicated. The frequency of needle tract seeding is not exactly known. PMID- 1730214 TI - [A lethal course in pseudomembranous enterocolitis during the parenteral administration of vancomycin and imipenem]. AB - A 48-year-old woman required mechanical ventilation after aortic valve replacement for decompensated aortic valve stenosis when bleeding complications developed and rethoracotomy had to be performed. Acute renal failure necessitated haemodialysis. Septic fever of unknown aetiology failed to respond to oxacillin, cefotaxim and tobramycin. The endotracheal cannula and central venous catheter were changed on the 24th postoperative day and the antibiotic treatment altered to 250 mg imipenem and 125 mg vancomycin three times daily intravenously. The fever soon subsided, but recurred on the 32nd postoperative day, accompanied by increasing leucocytosis. The patient was obstipated but had no intraabdominal signs. Four days later ultrasonography demonstrated thickening of the intestinal wall and coloscopy showed typical pseudomembranous colitis. Intestinal contents were positive for Clostridium difficile toxin. Despite immediate rectal and intragastric administration of 250 mg vancomycin four times daily the patient died of pseudomembranous colitis, confirmed at autopsy. The case demonstrates that vancomycin cannot always prevent the development of pseudomembranous colitis. PMID- 1730215 TI - [Diagnostic and therapeutic problems in Lamblia infections]. AB - A 28-year-old woman complained of chronic diarrhoea of about one year's duration, made worse by taking food. As a lactose tolerance test showed evidence of severe intolerance she was given a lactose-free diet, but this brought no improvement. Duodenal biopsy showed villous atrophy, and in the duodenal juice there were numerous flagellated lamblia. After a single oral dose of 2 g tinidazole the diarrhoea stopped, the lamblia disappeared, and the lactose intolerance and villous atrophy cleared up. Lamblia were also detected in the duodenal juice of a 50-year-old woman, but her infection was much more difficult to treat. Tinidazole (single dose of 2 g), metronidazole (800 mg twice daily for 6 days), ornidazole orally 500 mg twice daily for 10 days and ornidazole intravenously 500 mg twice daily for four days all proved ineffective. However, after a 5-day course of epsilon-9-aminacridine (100 mg three times daily by mouth) the diarrhoea ceased and both vegetative forms and lamblia cysts disappeared. These cases emphasize that lambliasis should be considered as a possible cause of severe chronic diarrhoea even when there is no history of travel abroad and when the symptoms are atypical. Conventional chemotherapeutic agents may be ineffective. PMID- 1730216 TI - Changes of the human liver GM3 ganglioside molecular species during aging. AB - Sialosyl-lactosylceramide, GM3, is the major ganglioside of human liver, where it constitutes more than 90% of the total lipid-bound sialic acid. When analyzed by thin-layer chromatography, human liver GM3 migrates as two main spots. They are representative of ganglioside molecular species which differ in the acyl moiety. The faster running spot is mainly composed of molecular species with non hydroxylated C22-C24 acyl chains; the other contains mainly molecular species bearing non-hydroxylated C16-C18 and alpha-hydroxylated C16-C24 acyl chains. In this study the content of the two GM3 molecular species groups was investigated in 31 subjects ranging from 19 to 85 years of age. By thin-layer chromatography we observed that the group of molecular species containing non-hydroxylated C22 C24 acyl chains, decreased linearly with subject age, while that of non hydroxylated C16-C18 acyl chains and hydroxylated C16-C24 acyl chains increased linearly. Fast-atom-bombardment mass spectrometry performed on seven samples from subjects ranging from 21 to 78 years of age demonstrated that the age-dependent increase of the lower spot is caused by an increase in the hydroxylated fatty acid form of GM3, the content of non-hydroxylated C16-C18 fatty acid species remaining constant with age. PMID- 1730217 TI - Inhibition of factor X, factor V and prothrombin activation by the bis(lactobionic acid amide) LW10082. AB - The minimum concentrations of heparin, dermatan sulfate, hirudin, and D-Phe-Pro ArgCH2Cl required to delay the onset of prothrombin activation in contact activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin III is an octadecasaccharide with high affinity for antithrombin III. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor II can catalyze thrombin inhibition by heparin cofactor II. A highly sulfated bis(lactobionic acid amide), LW10082 (Mr 2288), which catalyzes thrombin inhibition by heparin cofactor II and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the onset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition. PMID- 1730218 TI - Structural investigation on the lipopolysaccharide of Escherichia coli rough mutant F653 representing the R3 core type. AB - The chemical structure of the core oligosaccharide of the lipopolysaccharide isolated from Escherichia coli rough mutant strain F653, representing the enterobacterial R3 core type, was investigated by quantitative and methylation analyses, nuclear magnetic resonance spectroscopy, gas-liquid chromatography/mass spectrometry, and determined as [formula: see text] All sugars are present as alpha-pyranosides but the anomeric configurations of the 3-deoxy-D-manno octulopyranosonic acid (Kdo) residues could not be determined. The third Kdo and the heptose-linked GlcN residue are present in nonstoichiometric amounts; the GlcN residues may be, at least partially, N-acetylated. PMID- 1730219 TI - Glycosylation of interleukin-6 purified from normal human blood mononuclear cells. AB - Interleukin 6 (IL-6) is a glycosylated cytokine which is important in exerting cell-specific growth-inducing, growth-inhibiting and differentiation-inducing effects. IL-6 produced in mammalian cell lines is heterogeneous, reflecting specific cell-type-dependent post-translational modifications. Native IL-6 was purified from human blood mononuclear cells and the oligosaccharides released, radiolabelled and sequenced by a combination of sequential exoglycosidase digestion using Bio-Gel P-4 high-resolution gel chromatography and acetolysis. N- and O-linked glycans were found. The N-linked glycans were sialylated di- and tri antennary complex-type and oligomannose-type structures. However, the most predominant N-linked oligosaccharide was a small tetrasaccharide with the sequence Man alpha 6Man beta 4GlcNAc beta 4GlcNAc. This is the first report of this structure on a circulating glycoprotein. This structure has only previously been reported to be present on the syncytiotrophoblast of human placenta. The presence of the oligomannose structures and the mannose-terminating tetrasaccharide on IL-6 may be important in maintaining a high local concentration of the cytokine while limiting its systemic serum level via interaction with soluble mannose-binding serum lectins. PMID- 1730220 TI - Immobilization and single-step purification of fusion proteins using DEAE cellulose. AB - We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding domain--beta-galactosidase chimera, which can be purified by this procedure and shows a high beta-galactosidase activity when immobilized in the column. A vector plasmid, pCUZ1, containing the lppp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-beta-D-galactoside procedure. A chimera between the choline-binding domain and the pneumococcal hemolysin was also constructed and purified using pCUZ1. PMID- 1730221 TI - Expression of a pheromone-binding protein in insect cells using a baculovirus vector. AB - A cDNA encoding a pheromone-binding protein from the male silkmoth Antheraea pernyi has been integrated into the genome of the Autographa californica multiple nuclear polyhydrosis virus such that the transcription was under the control of the strong polyhedrin promoter. Recombinant pheromone-binding protein was expressed in a baculovirus-infected insect cell line (Sf9) and secreted from the cells into the culture medium. Using a two-step protocol, recombinant pheromone binding protein has been isolated and purified to homogeneity. Pheromone binding of recombinant protein has been demonstrated using a tritiated analog of (E,Z) 6,11-hexadecadienyl acetate. PMID- 1730222 TI - Nucleic acid binding properties of the 92-kDa replicase subunit of alfalfa mosaic virus produced in yeast. AB - The 92-kDa non-structural protein of alfalfa mosaic virus (one of the replicase subunits) was synthesized by Saccharomyces cerevisiae transformed with a recombinant expression vector. The yeast-expressed protein had the immunological and size characteristics of the naturally made viral protein. It was partially purified and its nucleic acid binding properties were tested by gel-retardation electrophoresis and nitrocellulose adsorption. The protein interacted with single stranded RNA, double-stranded RNA and double-stranded DNA in a salt-dependent manner, with a slight preference for RNA. These properties may be related to its putative function as a core RNA polymerase. PMID- 1730223 TI - X-ray diffraction study of the interaction between carboxypeptidase A and (S)-(+) 1-amino-2-phenylethyl phosphonic acid. AB - The structure of the carboxypeptidase A complex with the inhibitor (S)-(+)-1 amino-2-phenylethylphosphonic acid has been determined at 0.23 nm resolution. The delta F map shows electron-density peaks both in the S1 and S'1 sites, where the inhibitor molecule can be modeled in two different orientations with approximate 50% occupancy. In the proposed model, the phosphonate group binds to the zinc ion in a monodentate fashion. Other anchoring groups for the inhibitor molecule are Arg127 (hydrogen bonds with the phosphonate oxygen atoms) and Glu270 (hydrogen bond with the amino group in one of the two orientations). A recent spectroscopic investigation of the complex between cobalt(II) carboxypeptidase A and (S)-(+)-1 amino-2-phenylethylphosphonic acid is essentially in agreement with our results. PMID- 1730224 TI - Characterization of heterotrimeric collagen molecules in a sea-pen (Cnidaria, Octocorallia). AB - The collagen of a primitive invertebrate, the sea-pen Veretillum Cnidaria, Octocorallia), was studied with respect to its molecular-chain composition. The soft extracellular tissues (mesoglea) were solubilized by limited pepsin proteolysis and the collagen was isolated by selective precipitation at 0.7 M NaCl under acidic conditions. The pepsinized molecules were 260 nm in length, as demonstrated by electron microscope studies of rotary-shadowed molecules and of the segment-long-spacing crystallites obtained by dialysis against ATP. SDS/PAGE of the extract produced two main bands susceptible to bacterial collagenase, designated as the alpha 1 and alpha 2 chain, which were differentiated clearly by their CNBr cleavage products and the higher glycosylation rate of the alpha 2 chain. The latter finding corresponds with the high hydroxylysine content of the alpha 2 chain. The alpha 1/alpha 2 chain ratio observed in SDS/PAGE and the fact that only one peak was obtained by concanavalin-A affinity chromatography of a non-denatured 0.7 M NaCl extract demonstrate the alpha 1 [alpha 2]2 molecular structure of this collagen. These results contrast with data on the structure of other coelenterates (i.e. [alpha]3 for sea anemone collagen molecules and alpha 1 alpha 2 alpha 3 for jellyfish collagen molecules). They are discussed in relation to the evolution of collagen. PMID- 1730225 TI - Amphibian lutropin and follitropin from the bullfrog Rana catesbeiana. Complete amino acid sequence of the alpha subunit. AB - The amino acid sequence of the alpha-subunit of the gonadotropins, lutropin and follitropin from bullfrog, Rana catesbeiana, has been determined. The alpha subunit was identified in both hormones by the amino acid composition and ovulation activity of lutropin in the Xenopus ovary, by means of reconstituted hormones in various combinations. The amino acid sequences of two identical alpha subunits from lutropin and follitropin were determined or deduced by different strategies. The alpha-subunit of those gonadotropins have 97 amino acid residues, the longest among the known alpha-subunits of gonadotropins, and one arginine insertion at position 29. Ten cysteine residues and two sugar-chain binding sites at Asn57 and Asn83 are completely conserved among the species. The molecular mass of this subunit is 11,026 Da not including the sugar chains. The bullfrog alpha subunit has approximately 70% sequence identity with mammalian alpha-subunits. PMID- 1730226 TI - Structure and orientation of the surfactant-associated protein C in a lipid bilayer. AB - The secondary structure of native and depalmitoylated porcine surfactant associated protein C (SP-C) was studied by attenuated total reflection Fourier transform infrared spectroscopy. Both forms of porcine SP-C adopt mainly an alpha helical conformation. These two forms of the protein were reconstituted in a lipid bilayer. The insertion of the protein in a membrane is associated with an increase of the alpha-helical content. Dichroic measurements show that, in both cases, the long axis of the alpha-helix is oriented parallel to the lipid acyl chains. PMID- 1730227 TI - The identification of cation-binding domains on the surface of microsomal cytochrome b5 using 1H-NMR paramagnetic difference spectroscopy. AB - One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations. PMID- 1730228 TI - Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase. AB - Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme. PMID- 1730229 TI - Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii. AB - A new lipase from Penicillium camembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin-A-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalin-A-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase. PMID- 1730230 TI - Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775. AB - In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions. PMID- 1730231 TI - Enzyme function in organic solvents. AB - Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry. PMID- 1730232 TI - Neutral oligosaccharides of bovine submaxillary mucin. A combined mass spectrometry and 1H-NMR study. AB - Twenty-two neutral O-linked oligosaccharides ranging from monosaccharides to octasaccharides were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3-linked to N acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(beta 1-6) [Gal(beta 1-3)]GalNAcol or GlcNAc(beta 1-6)[GlcNAc(beta 1 3)]GalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAc alpha 1-6GalNAcol (GalNAc, N acetylgalactosamine and the hexasaccharide GlcNAc(beta 1-6) [GalNAc(alpha 1-3)( Fuc (alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-3)]GalNAcol (Fuc, L-fucose). Oligosaccharides of the blood-group-A type have not been detected previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric mucosal glycoproteins has been established by classical immunochemical studies. PMID- 1730233 TI - Fluorescent CMP-sialic acids as a tool to study the specificity of the CMP-sialic acid carrier and the glycoconjugate sialylation in permeabilized cells. AB - The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CMP-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialic-acids to gain access to cellular organelles. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMP glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N fluoresceinylaminoacetylneuraminic acid (5-FTIUNeuAc; 5-N fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminic acid analogues can be produced and used for the characterization of the Golgi CMP sialic-acid carrier. PMID- 1730234 TI - Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding. AB - Using a concanavalin-A-based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37 degrees C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin-A-retained forms of alpha 1 acid glycoprotein, T-kininogen, alpha 1 protease inhibitor and alpha 1 inhibitor III was slower than that of the concanavalin-A-non-retained forms. When hepatocytes were incubated at 20 degrees C, secretion was blocked with the accumulation of mainly endoglycosidase-H sensitive forms. The secretion kinetics of the concanavalin-A-differentiated forms were still different when the temperature was shifted back to 37 degrees C. The divergence between the secretion rates of the concanavalin-A-differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins. PMID- 1730235 TI - N-linked glycopeptides with blood group determinants lacking neuraminic acid from the epithelial cells of rat small and large intestine. AB - The N-linked type of glycans were prepared as their glycopeptides after pronase digestion of the epithelial cells from the small and large intestine of two inbred strains of rat. These glycopeptides were analysed for sugar composition, for blood-group activity, by 1H-NMR spectroscopy, and after permethylation by electron-impact mass spectrometry. The glycopeptides were of the triantennary and tetraantennary types with intersected GlcNAc. The terminal parts were, in contrast to most N-linked glycans, devoid of neuraminic acid residues. Instead they contained blood-group determinants. Blood-group-H types 1 (Fuc alpha 1-2Gal beta 1-3GlcNAc) and 2(Fuc alpha 1-2Gal beta 1-4GlcNAc) were found in the small and large intestines of both strains, although type-1 predominated. One rat strain (GOT-W) did not express blood-group-A glycopeptides in the small intestine, but the large intestine from the same strain did. The other strain (GOT-BW) expressed blood-group-A determinants in the small intestine. The lack of neuraminic acid residues in the small and large intestine and of blood-group-B activity in the large intestine differed from that found in glycosphingolipids obtained from the same organs. PMID- 1730236 TI - Photoaffinity labeling of the nucleotide-binding site of the uncoupling protein from hamster brown adipose tissue. AB - The nucleotide binding center of the uncoupling protein from brown adipose tissue (UCP) was probed by photoaffinity labeling with 8-azido-ATP. The isolated dimeric UCP in non-ionic detergent was used. 8-azido-ATP binds to UCP with a Kd = 3 microM, i.e. with an only threefold lower affinity than ATP and a maximum number of binding sites of about 12 mumol/g protein corresponding to about 1 mol/mol dimer UCP. UCP is rapidly degraded by ultraviolet radiation, and therefore only near ultraviolet and visible light can be used for photoaffinity labeling. The total covalent incorporation is shown to be dependent on the concentration of azido-ATP and on competing phospholipids. The specific, i.e. ATP-sensitive incorporation only to the binding site depends on the presence of cysteine. With CNBr cleavage the 8-azido-[gamma-32P]ATP insertion within the primary structure was located by identifying ATP-sensitive labeled peptides in SDS/PAGE. A major specific 8-azido-ATP incorporation was found by autoradiography in the smallest CNBr fragments. Identification of the radioactive peptides was difficult since 8 azido-ATP insertion causes a distinct shift in the gels from the stained peptides. Identification was possible by specific disulfide formation at the C terminal within the UCP dimer which only removed the CB7 (CB, CNBr fragment) portion of the low-molecular-mass peptides but did not move the radioactive band. This excludes the C-terminal CB7 and identifies the labeled peptide as CB6. Also, limited tryptic cleavage of intact UCP at Lys293 did not remove the radioactivity. Cleavage of tryptophanes support localization of 8-azido-ATP between residues 173-280 which includes CB6. Solid-phase sequencing of the labeled CB6 both after serine lactone and carboxyl coupling suggest incorporation into Thr260. These results indicate that the adenine-binding site is within the third domain of the tripartite UCP structure at a putative hydrophilic channel which can be assessed both from the cytosol and matrix of mitochondria. PMID- 1730237 TI - Hormonal regulation of malic enzyme and glucose-6-phosphate-dehydrogenase expression in fetal brown-adipocyte primary cultures under non-proliferative conditions. AB - The expression of malic enzyme and glucose-6-phosphate (Glc6P) dehydrogenase was investigated in primary cultures of fetal brown adipocytes after the prolonged presence (6 d or 10 d) of various hormones under non-proliferative conditions. The presence of triiodothyronine for 6 d and 10 d resulted in maturation of the triiodothyronine regulatory mechanism of malic-enzyme expression at the mRNA level. However, triiodothyronine had no effect on Glc6P dehydrogenase expression. Insulin increased malic-enzyme and Glc6P dehydrogenase expression at the mRNA and protein level after 6 d and 10 d of culture. The joint presence of triiodothyronine and insulin produced an additive effect on malic-enzyme expression at the mRNA and protein level after 6 d and 10 d of culture, by two independent mechanisms. Noradrenaline prevented the effect at the protein level after 6 d, but not after 10 d, probably due to loss of the beta-adrenergic response of brown adipocytes after prolonged culture. Triiodothyronine overexpressed the Glc6P dehydrogenase mRNA induced by the presence of insulin at 6 d and 10 d of culture. There was no adrenergic regulation of Glc6P dehydrogenase expression in cultured fetal brown adipocytes, regardless of the time of culture. PMID- 1730238 TI - Effect of protein kinase C activators on the phosphorylation and the surface expression of the CDw50 leukocyte antigen. AB - The CDw50 antigen is a constitutively non-phosphorylated leukocyte surface molecule which becomes highly phosphorylated in all the normal and lymphoblastoid cells analyzed (peripheral blood mononuclear cells, Molt 4, CEM, 8402, Namalwa), after stimulation with tumor promoter agents (phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, mezerein). This phosphorylation is rapid (within 1-5 min), dose-dependent and results in the incorporation of PO(3-)4 groups on serine residues. Furthermore, the level of CDw50 phosphorylation induced by tumor promoter agents is decreased by the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Activation of peripheral lymphocytes with concanavalin A, phytohemagglutinin and cross-linking of CD3 molecules also induces CDw50 phosphorylation, but the response is delayed and less intense than when tumor promoting agents are used. Treatment with any of the aforementioned agents is not accompanied by quantitative changes in the CDw50 surface expression. We therefore conclude that protein-kinase-C-mediated mechanisms are involved in phosphorylation, but not in regulation of the surface expression of the CDw50 leukocyte antigen. PMID- 1730239 TI - Protein interaction with ice. AB - Many organisms have evolved novel mechanisms to minimize freezing injury due to extracellular ice formation. This article reviews our present knowledge on the structure and mode of action of two types of proteins capable of ice interaction. The antifreeze proteins inhibit ice crystal formation and alter ice growth habits. The ice nucleation proteins, on the other hand, provide a proper template to stimulate ice growth. The potential applications of these proteins in different industries are discussed. PMID- 1730240 TI - Rat liver DNA ligases. Catalytic properties of a novel form of DNA ligase. AB - A novel form of rat liver DNA ligase (molecular mass 100 kDa) can be differentiated from DNA ligase I by several biochemical parameters. It is a more heat-labile enzyme and unable to join blunt-ended DNA, even in the presence of poly(ethylene glycol) concentrations which stimulate such joining by DNA ligase I and T4 DNA ligase. It also lacks the AMP-dependent nicking/closing reaction, which is a property of all other DNA ligases tested so far, including DNA ligase I from rat liver. Both rat liver DNA ligases were inhibited by deoxyadenosinetriphosphate, however this inhibition was competitive with respect to ATP, for DNA ligase I (Ki 22 microM) and non-competitive for the 100-kDa DNA ligase (Ki 170 microM). These results support the idea that, when compared with other DNA ligases, the novel form of DNA ligase has a unique AMP-binding site, may have an absolute requirement for single-strand breaks and, furthermore, may have an altered reaction mechanism to that which is conserved from bacteriophage to mammalian DNA ligase I. PMID- 1730241 TI - Effect of dexamethasone on mRNA levels for 5-aminolevulinate synthase in different rat tissues. AB - 5-Aminolevulinate synthase mRNA levels from different tissues were quantitated by Northern blot hybridization analysis utilizing the rat liver 5-aminolevulinate synthase cDNA clone as probe. A 5-aminolevulinate synthase mRNA species of size 2.3 kb was seen in all the tissues examined. Densitometric scanning of the autoradiographs demonstrated that the adrenal gland contained the largest amount of 5-aminolevulinate synthase mRNA. Levels corresponding to approximately 50% of this amount were found in the small intestine, lung, heart, muscle and testes. In the liver and kidney the level was approximately 25% of that found in the adrenal gland. These results demonstrate the housekeeping role of this gene. Dexamethasone treatment for 1 day or 5 days dramatically induced 5 aminolevulinate synthase mRNA levels in the liver and small intestine, and to a lesser extent in lung, heart, kidney and muscle. Nuclear run-off experiments suggest that a post-transcriptional mechanism predominantly contributes to the dexamethasone-induced increase in 5-aminolevulinate synthase mRNA levels observed in the liver. Interestingly, in the steroidogenic tissues of the adrenal gland and testes, there was a substantial decrease in 5-aminolevulinate synthase mRNA levels after dexamethasone administration but the mechanism of this control remains to be investigated. PMID- 1730242 TI - Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus. AB - Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed. PMID- 1730243 TI - Unresolved rearrangement of thiazoline form of glutathione. AB - The thiazoline form of glutathione was investigated with regard to its unresolved instability under neutral conditions. A simple method was developed for production of the thiazoline in stable solid form, thereby facilitating preparation of its neutral solution without using excess base and enabling isolation of the reaction product in quantity by ion-exchange chromatography. Analysis of the product by HPLC, infrared and ultraviolet absorption spectroscopy, mass spectrometry and proton magnetic resonance led to the identification of a cyclic amidine form of glutathione. The instability of the thiazoline is, therefore, due to an intramolecular rearrangement reaction, rather than hydrolysis. Once formed, the amidine is stable at pH 5-7 and in concentrated HCl, showing no tendency to rearrange back to either the original thiazoline or glutathione under these conditions. PMID- 1730244 TI - The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium. Is the presence of N-epsilon-methyllysine related to thermostability? AB - The complete amino acid sequence of glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage by trypsin, cyanogen bromide, Staphylococcus aureus V8 protease and pepsin. The enzyme subunit is composed of 421 amino acid residues yielding a molecular mass of 46.078 kDa. The presence of N-epsilon-methyllysine in six positions of the sequence was observed. Comparison of the sequence of glutamate dehydrogenase from S. solfataricus with the other known primary structures of the corresponding enzyme from different sources, gives an overall identity of 9.2% and shows a symmetrical evolutionary distance of this archaebacterial protein from the two groups of vertebrate on one side and eubacterial and low eucaryote enzymes on the other side. The occurrence of specific substitutions and a possible role for N-epsilon-methylation of lysine residues are discussed in view of current hypotheses on the molecular basis of thermal adaptation of proteins. PMID- 1730245 TI - Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide. AB - Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2 tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site. PMID- 1730246 TI - The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. PMID- 1730247 TI - Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803. AB - NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2 oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme. PMID- 1730248 TI - CD4 binding to major histocompatibility complex class II antigens induces LFA-1 dependent and -independent homotypic adhesion of B lymphocytes. AB - T helper cells recognize processed antigen (Ag) in the context of major histocompatibility complex (MHC) class II antigens present on the surface of B cells and other Ag-presenting cells. This interaction is mediated through the T cell receptor complex with associate recognition of class II molecules by the CD4 molecule. In this study, the binding of a soluble recombinant CD4/Ig heavy chain fusion protein (CD4-gamma 3) or monoclonal antibody (mAb) to class II antigens on human B cells was shown to induce rapid and specific homotypic adhesion of B cells and most B lymphoblastoid cell lines. mAb reactive with CD4 inhibited CD4 gamma 3-induced adhesion and a mutant B lymphoblastoid cell line deficient in class II antigens failed to respond. Induction of homotypic adhesion was dependent on energy metabolism and a functional cytoskeleton, and class II+ pre-B cells did not exhibit adhesion in response to these stimuli, suggesting that cross-linking of class II molecules generated a transmembrane signal and did not simply aggregate cells. In addition, MHC class II-induced adhesion was Fc receptor independent, as 15 mAb of different Ig isotypes reactive with HLA-D or HLA-DQ gene products induced adhesion. Anti-class II mAb and CD4-gamma 3 were able to induce adhesion at concentrations as low as 10 ng/ml and 100 ng/ml, respectively. Suboptimal stimulation of B cell lines through HLA-D antigens induced homotypic adhesion that was dependent on the activation of LFA-1 (CD11a/CD18), and which could be blocked by specific mAb. However, at greater signal strengths, adhesion was not blocked by mAb against the known adhesion receptors, suggesting the induction of a novel adhesion pathway. Consistent with this, homotypic adhesion induced by engagement of MHC class II antigens was observed with LFA-1-deficient B cell lines, and was independent of CD49d or CD18 expression. Thus, the direct engagement of B cell class II antigens by CD4 is likely to generate transmembrane signals which trigger both LFA-1-dependent and LFA-1-independent adhesion pathways. PMID- 1730249 TI - Acute human vs. mouse graft vs. host disease in normal and immunodeficient mice. AB - Recent reports of persistent engraftment of human lymphocytes and myeloid cells in hereditary immunodeficient severe combined immunodeficient mice (SCID) and beige athymic nude X-linked immunodeficiency (Bg/Nu/XID) mice have raised the question of why attempts to graft human cells into artificially immunosuppressed normal mice have failed so far. In the present study we provide evidence that this difference is due to the absence of natural antibodies in the mutant mice. We demonstrate that human PBL can be grafted in normal mice immunosuppressed by heavy doses of total body irradiation, provided the transplant is performed when the recipients lack natural antibodies in their serum, e.g. as in newborn normal mice, in mice treated with anti-mouse IgM antibody from birth, and in 3-week-old B cell-deficient CBA/N mice. In all cases, large numbers of human PBL were required. Under these conditions an acute and fatal graft vs. host disease (GVHD) developed in the recipients, regardless of whether these were artificially immunosuppressed or hereditary immunodeficient. The clinical manifestations and the histopathology of this xenogeneic acute GVHD are quite different from those of allogeneic GVHD. The former is primarily confined to the hematolymphoid tissues and locations close to accumulations of proliferating lymphoblasts, such as the peritoneal cavity in case of i.p. transplantation. The discordant xenogeneic GVHD is induced by human T lymphocytes and can be abrogated by treatment with anti-human T cell serum. PMID- 1730250 TI - Tolerance of self induced in thymus organ culture. AB - F liver protein occurs in serum at low concentration, and therefore induces tolerance of self only in T cells. T cells which mature in cultured thymus lobes in the absence of this protein become reactive towards it but can be prevented from doing so by exposure to the protein while in culture. The threshold of tolerance induction for this soluble antigen is estimated in this way at approximately 1 microgram/ml, which is slightly less than the threshold of response of primed T cells in a proliferation assay. Freshly isolated thymocytes do not display reactivity to self-F protein, indicating that T cells normally become tolerant while still within the thymus. PMID- 1730251 TI - The human cord blood antibody repertoire. Frequent usage of the VH7 gene family. AB - We have previously demonstrated a consistent preferential usage of a small set of VH, DH and JH gene segments in second-trimester fetal liver. To examine the extent of heavy chain repertoire diversification in newborns, we generated an unrestricted cDNA library from cord blood mononuclear cells and sequenced the variable domains of randomly isolated Cmu+ transcripts. We found this set of transcripts to be enriched for JH4, 5 and 6, whereas previously reported fetal transcripts preferentially expressed JH3 and 4. The cord blood transcripts used a number of different DH gene segments, whereas fetal transcripts were enriched for DHQ52. Of the thirteen cord blood sequenced, three were members of the newly described VH7 family which to date has not been detected in fetal liver. Indeed, only one of the isolated VH gene segments had been previously observed in fetal transcripts. As a result of enhanced N-region addition and use of longer DH and JH gene segments, both the sequence diversity and range of potential antigen binding structures of cord blood complementarity-determining region 3 domains was vastly expanded. Thus, the repertoire bias evident in fetal liver was no longer apparent in this more mature population of cord blood B cells. PMID- 1730252 TI - Restricted utilization of germ-line VH3 genes and short diverse third complementarity-determining regions (CDR3) in human fetal B lymphocyte immunoglobulin heavy chain rearrangements. AB - Twenty-one independent immunoglobulin heavy chain VH3DJH rearrangements were cloned and sequenced from livers of human fetuses at 7, 13 and 18 weeks of gestation. The VH elements expressed were not somatically mutated. Eight out of the estimated 30 VH3 elements were utilized with a preference for five of them. One of these VH3 sequences, designated FL13-28, represented a thus-far unknown VH3 gene segment. From the six functional JH elements the JH3 and JH4 segments were utilized preferentially and from the estimated 30 D segments the DQ52 element and the Dxp family were found to rearrange frequently. D elements were utilized both in normal and inverted orientation, as single copies or in D to D fusions. Addition of N nucleotides, removal of nucleotides from the coding sequences and utilization of DIR elements (D genes with irregular recombination signals) further expanded the third complementarity-determining region (CDR3) diversity. One fourth of the fetal CDR3 regions lacked N regions. Due to utilization of DQ52, the relative absence of N regions and extensive exonuclease activity operating on the D elements, the fetal CDR3 regions were significantly shorter than those found in adult B lymphocytes. PMID- 1730253 TI - Cell kinetics of the germinal center reaction--a stathmokinetic study. AB - The changes in splenic germinal center (GC) cell proliferation were measured during primary and secondary responses to a T-dependent antigen in vivo to examine the regulation of the GC reaction. Adult C3H/HeN mice were immunized with sheep red blood cells and boosted 7 or 21 days later. GC cell proliferation was assessed by measurement of GC cell birth rates using a stathmokinetic technique. Actual GC growth and regression were assessed in terms of total splenic volume and number. Pre-existing GC had a mean cell birth rate of 33 cells/1000 cells/h. The GC reactions following each immunization showed a biphasic pattern of changes in cell birth rate, comprising an initial fall immediately succeeded by a transient, but significant, increase. These fluctuations occurred earlier in secondary compared to primary responses. Significant increases in total GC volumes succeeded the peaks of cell birth rate following both primary and early secondary immunization. However, there was a substantially smaller increase following later secondary immunization. We propose that the initial cell birth rate reduction is due to inhibition of pre-existing GC clones and represents one component of the phenomenon of GC dissociation. The succeeding peak birth rate represents early, massive proliferation of newly activated antigen-specific clones. The different patterns of GC expansion, despite similar proliferative responses, may reflect different pathways of differentiation dependent on the timing of antigenic stimulation. PMID- 1730254 TI - The human intraepithelial lymphocyte marker HML-1 is an integrin consisting of a beta 7 subunit associated with a distinctive alpha chain. AB - The membrane antigen defined by the monoclonal antibody (mAb) HML-1 is abundantly expressed on, and largely restricted to, the T cells which populate the intestinal epithelium. We show that the mature form of the antigen is a heterodimer comprising a 150-kDa alpha chain and a 120-kDa beta chain. Direct sequencing of tryptic peptides cleaved from the purified beta chain identified this polypeptide with the integrin beta 7 isotype. cDNA clones coding for the beta 7 chain have recently been isolated from T cell cDNA libraries, but the beta 7 chain had not been identified at the protein level. No information is available concerning the primary structure of the HML-1 alpha chain. We show that this subunit is synthesized as a precursor form that undergoes, like several other integrin alpha subunits, a post-translational cleavage of a peptide bond. Among the 11 human integrin alpha chains previously identified, 10 have biochemical features and/or a distribution different from those of HML-1 alpha. One, VLA alpha 4 (CD49d), has a molecular mass of 150 kDa and is expressed on HML-1+ cells but is not recognized by HML-1 mAb. We conclude that HML-1 is a novel member of the integrin family made of the beta 7 chain and of an as-yet-undescribed human alpha chain characterized by the post-translational cleavage of a 10-kDa peptide. HML-1 is, thus, probably the human counterpart of the mouse antigen M290. PMID- 1730255 TI - Liposome-mediated delivery stimulates a class I-restricted cytotoxic T cell response to soluble antigen. AB - Most soluble protein antigens are poor at priming class I-restricted cytotoxic cell activity. In this study we show that incorporation of one protein, ovalbumin, into liposomes converts this antigen into an effective stimulator of antigen-specific cytotoxicity. The mechanisms responsible for this phenomenon are discussed. PMID- 1730256 TI - Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern. PMID- 1730257 TI - Pre-B lymphocyte-specific transcriptional control of the mouse VpreB gene. AB - The VpreB genes, which encode surrogate immunoglobulin light chain molecules, are expressed as RNA almost exclusively in pre-B cells. We have investigated the transcriptional control mechanisms which are responsible for the pre-B cell specific RNA expression of the mouse VpreB1 and VpreB2 genes. Nuclear run-on analyses demonstrate that the pre-B cell-specific expression of both VpreB genes is controlled primarily at the level of initiation of transcription. S1 nuclease protection-mapping defined two or three major start sites of transcription for the VpreB genes. To find a promoter and other potential cis-acting regulatory elements, a 700-bp fragment 5' of the transcription start sites of the VpreB1 gene was used in gene transfer experiments and found to act as a promoter in pre B lymphocytes. Deletion experiments showed that 191 bp upstream of the most 5' transcription start site is required for the pre-B cell promoter activity. DNA sequence analysis of the 5' region of the mouse VpreB1, VpreB2 and human VpreB genes reveal that this region of approximately 200 bp is strongly conserved. This 200-bp promoter region contains several conserved nucleotide sequence motifs which may act to mediate the pre-B cell-specific transcription of the VpreB genes. PMID- 1730258 TI - A pre-B- and B cell-specific DNA-binding protein, EBB-1, which binds to the promoter of the VpreB1 gene. AB - The VpreB1 protein is thought to be expressed on the surface of pre-B cells in association with lambda 5 and mu heavy chain, and to play an important role on B cell differentiation. The expression of VpreB1 and lambda 5 is pre-B cell specific, and regulated at the initiation of transcription. We have identified at least two sequence-specific DNA-binding proteins which bind to the region -191 to -74 of the promoter of the mouse VpreB1 gene. These DNA-binding proteins also bind to the promoter of the mouse lambda 5 gene. One of the two DNA-binding proteins, called EBB-1, is restricted to pre-B and B cells, but not detected in plasma cells, T cells and cells of other lineages. Transient transfection analysis of reporter constructs revealed that the binding sites of these proteins play a significant role in the activity of the promoter, especially the binding site of EBB-1. Taken together these results suggest that EBB-1 might be one of the crucial factors which regulates a series of intracellular events in B cell differentiation. PMID- 1730259 TI - Treatment of rats with monoclonal anti-CD4 induces long-term resistance to streptococcal cell wall-induced arthritis. AB - To investigate the role of CD4+ cells in the induction and maintenance of streptococcal cell wall (SCW)-induced arthritis, Lewis rats were treated with a monoclonal antibody against rat CD4 (W3/25). Injection before onset of the arthritis resulted in resistance to SCW arthritis. Treatment with anti-CD4 during ongoing arthritis induced an amelioration of the arthritis, demonstrating that CD4+ cells are involved in both the induction and effector phases of the chronic arthritis. After return of CD4+ cells to normal levels in the circulation, no arthritis occurred in protected rats, despite the continued presence of SCW in the body. Even reinjection of SCW could not induce arthritis in these rats, suggesting that tolerance to SCW had occurred. In addition, these tolerized rats were refractory to actively induced adjuvant arthritis (AA), but were susceptible to passively transferred AA. Our data imply, that (a) treatment with anti-CD4 plus SCW induces a long-term resistance to SCW-induced arthritis and adjuvant arthritis, (b) SCW and M. tuberculosis may use similar mechanisms of regulation of arthritis and (c) active peripheral suppression is not the mechanism of this nonresponsiveness. PMID- 1730260 TI - Genetic correction of cultured T cells from an adenosine deaminase-deficient patient: characteristics of non-transduced and transduced T cells. AB - T lymphocytes derived from peripheral blood of a patient with adenosine deaminase (ADA) deficiency were expanded in vitro. The human ADA (hADA) gene was introduced into these replicating ADA- T cells with the use of an amphotropic recombinant retrovirus carrying the hADA gene. Subsequently, infected T cells were selected on the basis of their ADA expression, by exposure to a combination of the toxic agent xylofuranosyl-adenine and the specific ADA inhibitor 2'-deoxycoformycin. CD4+ and CD8+ T cells could be infected and selected with equal efficiencies. The genetically modified T cells were shown to contain intact copies of the provirus and to express normal levels of hADA. As expected, uninfected T cells from the ADA-deficient patient displayed an increased sensitivity to 2'-deoxyadenosine. Following genetic modification, however, this sensitivity was restored to normal levels in both CD4+ and CD8+ T cells. The introduction of the hADA gene into the genome of the in vitro expanded T cells did not alter their phenotype, proliferative capacity or cytotoxic potential. These characteristics were identical to those of T cells derived from healthy individuals. These findings are of critical importance for the clinical application of hADA gene-transducted T cells. PMID- 1730261 TI - T lymphocyte adhesion to the fibronectin and laminin components of the extracellular matrix is regulated by the CD4 molecule. AB - The adhesion of T cells to components of the extracellular matrix (ECM) is mediated by the beta 1 subfamily of integrin receptors, designated VLA. It has been recently demonstrated that the binding of VLA receptors to protein components of the ECM is rapidly augmented by the activation of the T cells without, however, any actual change in the level of expression of the VLA receptors for fibronectin (FN) or laminin (LN). Thus, it is likely that activation of existing VLA receptors is required for binding. The activation must be regulated by T cell surface molecules capable of transducing signals into the cell. We studied the role of the CD4 molecule in the binding of rat CD4+ T cells to the FN and LN components of the ECM. We now report that the CD4 molecule appears to play a major role in regulating T cell interactions with ECM. This conclusion is based on the following observations: (a) monoclonal antibodies directed against the CD4 molecule inhibited T cell adhesion to both FN and LN; (b) down-regulation of the CD4 molecule resulted in partial loss of the ability of CD4+ T cells to adhere to FN and LN; (c) a CD4+ T cell clone adhered to both FN and LN while a CD4-CD8- clone expressing an identical T cell receptor bound weakly to both proteins and (d) treatment of the CD4+ T cells with an inhibitor of the CD4-associated tyrosine protein kinase activity inhibited T cell adhesion to both ECM proteins. PMID- 1730262 TI - Developmentally controlled selection of antibody genes: characterization of individual VH7183 genes and evidence for stage-specific somatic diversification. AB - Nucleotide sequence analysis of a large number of rearranged immunoglobulin heavy chain V region genes allowed the identification of six new members of the VH7183 gene family. These six new genes plus the eight previously defined genes agrees with the previously estimated complexity of this gene family. Twelve of these genes were represented among the isolated clones. A comparison of the clones, derived from 1-day- and 14-week-old BALB/c mice, suggested a biased and developmentally controlled VH7183 gene utilization. Furthermore, a developmentally controlled, non-random distribution of the functional vs. non functional VHDJH rearrangements was observed among clones utilizing genes of this family, suggesting unsuspected regulatory aspects of Ig rearrangements in the process of B cell differentiation. Finally, a limited junctional diversity was revealed among the neonatal clones as the result of a low frequency of N-sequence addition. A similar discrepancy was also observed between neonatal and adult VHJ558 clones. In conclusion, these data suggest a programmed generation of B cell diversity similar to what has been observed for the establishment of gamma/delta T cell repertoires. PMID- 1730263 TI - Thymic nurse cell lymphocytes react against self major histocompatibility complex. AB - It has been postulated that thymic nurse cells (TNC), lymphoid-epithelial complexes composed of thymocytes enclosed within major histocompatibility complex (MHC) class I+ and class II+ cortical epithelial cells, may provide an optimal microenvironment for the process of T cell selection. By transplanting single TNC in the avian chorionallantoic membrane assay we demonstrate that a significant portion of intra-TNC lymphocytes (TNC-L) possess reactivity against self-MHC molecules. The frequency of these autoreactive cells among TNC-L exceeds by far that of thymocytes or peripheral blood lymphocytes of the same donor. These results indicate that TNC-L constitute a T cell population enriched for self-MHC reactivity, i.e. cells that have undergone positive selection, but not yet deletion and/or deactivation. PMID- 1730264 TI - Plasmodium berghei: in vivo generation and selection of karyotype mutants and non gametocyte producer mutants. AB - We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events. PMID- 1730265 TI - Sarcocystis muris (Apicomplexa): a thiol protease from the dense granules. AB - Homogenates of Sarcocystis muris merozoites (cyst form) and the subcellular fraction of dense granules were assayed for protease activity with substrate impregnated SDS-polyacrylamide gels. Four acidic and several basic proteases were detected in the merozoites. One of the basic proteases was further characterized as a thiol protease (EC 3.4.22). The activity of this protease was enriched in the dense granule fraction. PMID- 1730266 TI - Toxoplasma gondii: the role of a 30-kDa surface protein in host cell invasion. AB - C1E3, a monoclonal antibody recognizing protein P30, a major surface antigen of Toxoplasma gondii tachyzoites, was shown to have a consistent effect on invasion in adult bovine kidney cells. In 10 replicate assays, the overall invasion was reduced to 37% of control values (P less than 0.0001). These results support the role of a functional role for P30 in mediating invasion. PMID- 1730267 TI - Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum. AB - The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing. The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli. The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites. In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte. This is the first time a P. falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol. Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway. PMID- 1730268 TI - Amblyomma americanum: characterization of salivary prostaglandins E2 and F2 alpha by RP-HPLC/bioassay and gas chromatography-mass spectrometry. AB - Secretagogue-induced saliva of the tick, Amblyomma americanum (L.) was fractionated by reversed-phase-high-performance liquid chromatography (RP-HPLC) and bioassayed in smooth muscle preparations. Material with retention times of authentic prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) were found to cause contraction of preparations of rat colon and rat stomach strips. Gas chromatography-mass spectra of selected ions of both HPLC-purified fractions confirmed the existence of PGE2 and PGF2 alpha. Bioassay of individual samples obtained from ticks stimulated to salivate with pilocarpine, dopamine + theophiline, or dopamine + theophiline + GABA indicated that all these secretagogues induced similar amounts of prostaglandin secretion, averaging 469 ng PGE2/ml. These pharmacological doses of prostaglandin are hypothesized to assist in tick feeding by inducing vasodilation and/or other pharmacological events in their hosts. PMID- 1730269 TI - Babesia bigemina: quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe. AB - Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia bigemina specific DNA probe. DNA from progeny of female Boophilus microplus infected with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3+) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microplus demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately 10(6) infective forms. This number of infective forms is suitable for isolation and further characterization. PMID- 1730270 TI - Effect of nifedipine treatment on oxidative metabolism of peritoneal macrophages and neutrophils of Plasmodium berghei-infected mice. AB - The oxidative metabolism of peritoneal macrophages (PM) and neutrophils from nifedipine (calcium channel blocker)-treated, Plasmodium berghei (NK 65)-infected and normal infected Swiss Albino mice was studied. A significant fall in oxidative metabolism as evidenced by decreased chemiluminescence (CL) response (P less than 0.001) was recorded both in PM and neutrophils from nifedipine-treated mice compared to the control animals. When the oxidative metabolism of these phagocytes was studied after infection of the host, higher CL response was recorded from both PM and neutrophils isolated during the early course of infection (0-1 and 5-10% parasitaemia) when compared to uninfected mice (P less than 0.001). A similar pattern was observed in the case of nifedipine-treated and infected mice even though the CL response was much lower. The increasing parasite load not only resulted in subnormal CL response but also prolonged the time required for the phagocytes to exhibit peak oxidative activity both in normal infected and CCB-treated infected mice, but the time taken to show peak CL response was shortened following drug administration compared to controls. These observations revealed the profound in vivo effect of CCB on the functioning of phagocytic leucocytes and thereby questions the use of CCB in combination with chloroquine for reversal of drug resistance. PMID- 1730271 TI - Trypanosoma cruzi: predominance of IgG2a in nonspecific humoral response during experimental Chagas' disease. AB - The kinetic and isotypic pattern of hypergammaglobulinemia has been investigated in C3H/HeJ infected with Trypanosoma cruzi. Hypergammaglobulinemia appeared 14 days postinfection, increased until Day 28 postinfection, and persisted throughout the chronic phase (greater than 60 days of infection). The main isotype secreted was IgG2a, reaching 10-fold the control level. High titers of autoantibodies were found of IgM and IgG subclasses. Isotypic characterization of antibodies against myosin, myelin, and keratin, was performed and determined to be IgG2a subclass in the chronic stage of infection. Specific responses against T. cruzi took place 2 weeks postinfection when the parasitemia was high. Interestingly, parasite-specific response was maximal after 4 weeks of infection and plateaued during the chronic phase when parasites were rare. In contrast to the humoral polyclonal response in the chronic stage, showing a preferential IgG2a pattern, the anti-T. cruzi response consisted of all the different isotypes: IgM, IgG1, IgG3, IgG2a, and IgG2b, throughout the infection. Identical patterns of parasite antigens were recognized by IgG2a and IgG2b antibodies. Few different antigens were identified by the IgG3. Some antigens were recognized by several isotypes, others by only one isotype. With regard to the existence of antigenic cross-reactivities between host and parasite, we designed absorption experiments on parasite-specific immunoadsorbent showing that specific antibodies eluted from the column failed to recognize the natural antigens. These studies suggest that nonspecific and antiparasite-specific responses may be maintained by different regulatory pathways. PMID- 1730272 TI - Brugia malayi: patterns of expression of surface-associated antigens. AB - Patterns of expression of surface-associated antigens were analyzed in the filarial nematode Brugia malayi immediately prior, and during development in the vertebrate host. Two surface-associated protein molecules, i.e., accessible to surface radioiodination and soluble in aqueous buffers, were investigated: Mrs 29 30,000 and 16,000, both of which are antigenic in infected animals. The Mr 29 30,000 glycoprotein is expressed in a surface-associated manner by adult worms and by fourth-stage larvae, but is not detectable in preparasitic third-stage larvae. The 16,000 component, which appears not to be glycosylated, is surface associated in adult worms and fourth-stage larvae. In contrast to the 29-30,000 glycoprotein, the 16,000 protein is also expressed both by pre- and postparastic third-stage larvae. However, it becomes surface-associated only after infection. Thus, immediately prior, and during development within the vertebrate host, B. malayi displays at least two different patterns of expression of surface associated antigens: (i) de novo, intiated either immediately after infection (phase specific) or during genesis of the fourth-stage larva (stage specific); (ii) continuous, but with phase-dependent surface exposure of previously cryptic antigens, during the transition from intermediate to definitive host. PMID- 1730273 TI - Trypanosoma brucei brucei: antigenic stability of its LDL receptor and immunological cross-reactivity with the LDL receptor of the mammalian host. AB - The rapid growth of Trypanosoma brucei brucei in the blood and tissue fluids of vertebrates requires the receptor-mediated endocytosis of LDL from the host (Coppens et al. 1987; Gillett and Owen 1987) and is slowed by a monospecific rabbit antiserum against the purified LDL receptor of the parasite. We have used this antiserum in combination with several well-characterized antigenic variants (originating from stock 427: MITat 1.1a, 1.3a, 1.4a, 1.5a, 1.5d, 1.8b) to examine whether the LDL receptor of T. b. brucei is a stable surface antigen, common to all parasite variants despite antigenic variation of the major surface glycoprotein, and whether it is immunologically distinct from the LDL receptor of the host. At low concentrations, binding at 4 degrees C of rat LDL to several variants of T. b. brucei and to isolated rat hepatocytes was inhibited to a similar extent by the antiserum. In double immunodiffusion, a single precipitation line was observed, showing continuity between the extracts of all variants as well as between that of trypanosomes and of mammalian tissues. In Western blots of trypanosome extracts, the LDL receptor was strongly labeled as a single band of Mr 145,000, whereas with a rat liver extract, a single band of similar electrophoretic mobility was weakly labeled at a high concentration of the antiserum. In conclusion, the LDL receptor occurred in all variants of T. b. brucei, was a stable surface antigen despite variation of the major surface glycoprotein, and displayed biochemical and immunological similarities with the LDL receptor of the rat host. PMID- 1730274 TI - Primary sequence heterogeneity and tissue expression of glutathione S transferases of Fasciola hepatica. AB - Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date. PMID- 1730275 TI - The receptors for regulatory molecules of hematopoiesis. AB - Proliferation and differentiation of hematopoietic cells are controlled by pleiotropic regulatory molecules. While the sequences of these factors are not related, their membrane receptors are restricted to two gene families with homologous domains. The members of the hematopoietic (or cytokine) receptor family (for erythropoietin, interleukins-2, -3, -4, -6 andu-7, granulocyte macrophage and granulocyte colony-stimulating factor) are composed of multiple subunits necessary for high-affinity binding and cell signalling. Signal transducing mechanisms are largely unknown. The occurrence of variant signal transducers in different tissues could explain the pleiotropy of these regulatory molecules. Members of the receptor tyrosine kinase family bind dimeric forms of macrophage colony-stimulating factor, stem cell factor and platelet-derived growth factor leading to kinase activation and phosphorylation of many substrates involved in production of second messengers. Soluble forms (binding proteins) exist for members of both families. These may be proteolytic cleavage products of transmembrane receptors or naturally secreted products. Such binding proteins can potentially function as inhibitors in feedback regulation and in protection and transport of cytokines and would provide a rational therapy when cytokines are produced in excess. Knowledge of signal transduction mechanisms and of the three dimensional structure of ligands and receptors can lead to the design of drugs with cell-specific effects. PMID- 1730276 TI - Incidence of polycythemia vera in a defined population. AB - In this retrospective investigation from Malmo, a city well-suited for epidemiologic studies, 177 patients (88 males and 89 females) with polycythemia vera (PV) were identified between 1950 and 1984. The incidence rate (number of cases/100,000/yr) in both sexes increased significantly, being 1.0 in 1950-1959 and 2.6 in 1980-1984 (adjusted to the European age-standardized population). This is the highest rate reported to date. In 1970-1984 the highest age-specific incidence rates (number of cases/100,000/yr) were found in males greater than or equal to 80 yr and females 70-79 yr of age, being 18.3 and 14.6, respectively. A subgroup of 12 (7%) was identified where the PV diagnosis was not obvious on entry into the study but where it became clear during follow-up. 16 PV patients (9%) had verified or suspected arterial hypoxemia caused by a concomitant condition. We conclude that the increasing PV incidence rates, mainly confined to older age groups, are probably due to better case ascertainment. PMID- 1730277 TI - Remission may continue after termination of rIFN alpha-2b treatment for essential thrombocythemia. AB - Essential thrombocythemia, a myeloproliferative disorder of clonal origin, is often associated with various clinical manifestations resulting from thromboembolic or hemorrhagic complications. The long-established successful method of treatment with cytotoxic agents or radioactive phosphorus has recently been superseded by interferon alpha. We treated 14 symptomatic patients with 5 x 10(6) IU recombinant interferon alpha-2b s.c. daily. 12/14 pts responded within 14-75 days. When platelet counts decreased to below 450 g/l the frequency of administration was reduced stepwise. 7 patients remained in CR during this reduction phase and treatment was stopped in 5 pts after 12-32 months. Until now, 3 of them are still in continuous good PR without any drug therapy and free of symptoms for 3+, 19+ and 36+ months. Continuous response during maintenance was associated with age, initial platelet count and time required to reduce platelet counts to less than 450 g/l. PMID- 1730278 TI - Combination chemotherapy MOCCA in resistant and relapsing multiple myeloma. Finnish Leukaemia Group. AB - 80 patients with resistant or relapsing multiple myeloma received a combination of vincristine, cyclophosphamide, lomustine, melphalan and methylprednisolone (MOCCA) as a second-line chemotherapy. 27 of them were resistant to primary chemotherapy with alkylating agents, and 53 had relapsed after initially responding to these drugs. An objective response was achieved in 39 patients (49%): in 14 patients who were primarily resistant (52%) and in 25 patients who had a relapse (47%). Of 41 patients relapsing during maintenance chemotherapy 14 (34%) responded, while 11 of 12 patients (92%) treated for a relapse off-therapy responded. The median duration of response was 22 months. Severe complications, in most cases infections, occurred in 30% of patients, and were fatal in 9% of the cases. According to our experience the five-drug combination MOCCA is an effective second-line chemotherapy for myeloma patients primarily resistant to or relapsing after therapy with single alkylating agents. PMID- 1730279 TI - Bilineage response in refractory aplastic anemia patients following long-term administration of recombinant human granulocyte colony-stimulating factor. AB - 5 patients with refractory aplastic anemia (AA) received long-term administration (2-11 + months) of recombinant human G-CSF (rhG-CSF) in doses from 250-500 micrograms/body/day by intravenous infusion or 75-300 micrograms/body/d by subcutaneous injection. All 5 evaluable patients showed a substantial increase in absolute neutrophil count (ANC) with a recovery of myeloid components in the bone marrow after 1 to 2 months of treatment. Interestingly, 2 out of the 5 patients showed a dramatic improvement in severe anemia after 2 to 4 months of treatment accompanying a recovery of erythroid components in the bone marrow. In addition, there was no serious infection before or during therapy. Long-term administration of rhG-CSF was well tolerated because of its minimal toxicity. Clonal assay revealed a recovery of myeloid progenitors in all patients and a recovery of erythroid progenitors in 3 out of the 5 patients. These results suggest that long term administration of rhG-CSF at least mobilizes residual myeloid as well as erythroid progenitor cells and induces a bilineage response in severe refractory AA. PMID- 1730280 TI - Comparison of poly- and monoclonal antibodies for determination of B-cell clonal excess in an routine clinical laboratory. AB - Flow cytometry (FCM) has gained wide use in the determination of clonality in B cell lymphoproliferative diseases and many methodological variations exist. We have compared the suitability of a) dual fluorochrome (FITC/PE)-labelled monoclonal antibodies, b) single fluorochrome (FITC)-labelled monoclonal antibodies and c) F(ab')2 fragments of FITC-labelled polyclonal antibodies for flow cytometric determination of clonality using commercially available software and a short sample preparation protocol. The FCM method was validated by analysis of immunoglobulin heavy chain and light chain gene rearrangements. We recommend the use of FITC-labelled monoclonals to obtain three parameters, the kappa/lambda ratio, D and D/S(n) values (Kolmogorov-Smirnov statistics) instead of the commonly used kappa/lambda ratio and D values only. This allows the use of a rapid sample preparation protocol to blood and bone marrow aspirates without sacrificing sensitivity or specificity obtained by the usual FCM method. PMID- 1730281 TI - Interactions between erythropoietin and iron metabolism in anaemia of chronic disorders. PMID- 1730282 TI - Reticulocyte analysis by flow cytometry using a modified gating procedure. PMID- 1730283 TI - Surgical restaging of advanced Hodgkin's disease after first line chemotherapy. PMID- 1730284 TI - Ascorbate for the treatment of ITP. PMID- 1730285 TI - Structural determinants of Cys2His2 zinc fingers. AB - Two mutants of the zinc finger peptide Xfin-31 (Ac-YKCGLCERSFVEKSALSRHQRVHKN CONH2) containing alterations to the conserved hydrophobic core have been constructed and their zinc-bound structures investigated by 1H NMR techniques. In the first (Xfin-31B) a double mutation R8F/F10G places the conserved core aromatic residue at position 8 rather than position 10. In the second (Xfin-31C), Phe-10 is replaced by Leu. A qualitative analysis of 1H chemical shifts, NOE connectivities and coupling constants indicates that the global folds of both mutants are similar to that of the wild-type protein. However, amide exchange rates suggest that the F10L mutant is much less stable than either the wild-type or the R8F/F10G mutant. PMID- 1730286 TI - Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid. AB - The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with Mr 28,500 on SDS-PAGE, while the Mr of TIMP 2 was 21,000. The inhibitory activity was stable under heat and acid pH. N terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting. PMID- 1730287 TI - Immunologic and structural relatedness of the integrin beta 7 complex and the human intraepithelial lymphocyte antigen HML-1. AB - We recently cloned the newest human integrin beta subunit, termed beta 7, from a cDNA library constructed from SEA-activated T lymphocytes. In this communication, we report on the structure of the human integrin beta 7 protein complex determined using a rabbit anti-beta 7 peptide antibody raised to an N-terminal 22 amino acid residue sequence deduced from the human beta 7 subunit cDNA. The beta 7 subunit (Mr 116,000) expressed on PHA lymphoblasts associates with a single major alpha subunit (alpha H) that is distinct from the prominent T cell marker, integrin alpha 4. The alpha H subunit (Mr 180,000 nonreduced) displays a distinctive shift in size on reduction to an apparent Mr of 150,000. We show that these structural properties of the integrin beta 7 complex are shared with the cell surface antigen HML-1 found highly expressed on T cells which populate the intestinal epithelium and are proposed to be involved in mucosal immunity. Sequential immunoprecipitation and Western blotting demonstrate identity or close homology between the alpha H beta 7 and HML-1 proteins. PMID- 1730288 TI - Analysis of the structure of photosystem I in cyanobacterial thylakoid membranes. AB - Using electron microscopy of negatively stained specimens, we have investigated the shape and degree of association of photosystem (PS) I complexes in cyanobacterial thylakoid membranes. When incubated at high concentrations of magnesium chloride (greater than 0.15 M), the PSI complexes form small ordered arrays in the membrane composed of monomeric complexes in a P1 square lattice of dimensions a = b = 11 nm. Averaged projections of the complex resemble those found for the purified PSI reaction centre after reconstitution (Ford, R.C, Hefti, A. and Engel, A. (1990) EMBO J. 9, 3067-3075). Some small differences in its shape are discussed, with particular reference to the differences in the polypeptide composition of the 2 preparations. We find that the complex remains in the monomeric form in the thylakoid membrane under all the conditions tested. PMID- 1730289 TI - The presence of free D-serine in rat brain. AB - Free amino acid enantiomers in adult rat brain extracts were analyzed as their N,O-pentafluoropropionyl isopropyl derivatives by gas chromatography on a capillary column of Chirasil-L-Val. A peak X, which exhibited the same retention time as the N,O-pentafluoropropionyl isopropyl derivative of authentic D-serine, was detected in the brain extracts. Electron impact and positive chemical ionization mass spectra of the peak X of the brain extracts were identical to those of authentic D-serine. The concentration of free D-serine and the ratio of D-serine/total serine in the brain were estimated to be 0.27 and 0.23 mumol/g of wet weight, respectively. These data provide the first evidence that substantial quantities of free D-serine are present in mammalian brain tissues. PMID- 1730290 TI - A substantial proportion of cardiac Gs is not associated with the plasma membrane. AB - The precise interactions between the subunits of Gs (alpha s, beta, gamma) and the plasma membrane remain to be established. If alpha s is associated loosely with the inner membrane, is labile during activation, or is always present to some extent in the cytoplasm, then it should fractionate to the supernatant of a high-speed centrifugation. We identified abundant alpha s (52-66% of total cellular) in the supernatant fraction of right atrial and left ventricular membrane preparations of porcine heart as shown by two distinct measures of alpha s (immunoblotting and ADP ribosylation by cholera toxin). However, functional assays utilizing reconstitution of cardiac alpha s with cyc- S49 membranes revealed that the supernatant fraction contained approximately 16% of total cellular alpha s activity. The alpha s present in the supernatant fraction did not result from contamination by sarcolemmal membrane fragments. We conclude that traditional methods for quantifying alpha s which utilize only detergent extracts from high-speed pellets do not account for a sizable proportion of total cellular alpha s, but that the majority of this population of cardiac alpha s may not be functional, at least with respect to adenylyl cyclase activation. PMID- 1730291 TI - Production of inositol phosphates and reactive oxygen metabolites in quartz-dust stimulated human polymorphonuclear leukocytes. AB - The present paper explores phosphoinositide turnover in quartz-stimulated human polymorphonuclear leukocytes. Separation of inositol phosphates was carried out with a new ion-pair, reverse-phase high performance liquid chromatographic method applying a gentle tetrabutyl ammonium phosphate buffer gradient. The method separates inositol monophosphates, inositol 1,4-bisphosphate, inositol trisphosphates and inositol 1,3,4,5-tetrakisphosphate. Reactive oxygen metabolites, indices for leukocyte activation, were measured with a luminometric assay. Quartz increased the production of reactive oxygen metabolites, preceded by facilitated inositol phosphate turnover. This finding provides evidence that inositol phosphate second messengers may be involved in quartz-induced leukocyte activation and subsequent production of reactive oxygen metabolites. PMID- 1730292 TI - Primary structure of rat pancreatic lipase mRNA. AB - The sequence of rat pancreatic lipase mRNA was determined. The data have been assigned the following accession number, X61925, in the EMBL data library. The total length of the messenger is 1531 nucleotides, plus a poly(A) stretch of about 60 nucleotides. A 72-nucleotides 5'-noncoding region is followed by a 1419 nucleotides open reading frame which encodes a protein of 473 amino acids, including the 17 amino acid signal peptide. The mature enzyme (456 residues) has 6 additional C-terminal amino acids, as compared with the amino acid sequence of pig (direct amino acid sequence), dog, man and rat isoenzyme from Genbank, M58369 (all deduced from the nucleotide sequence). A higher degree of homology exists between the amino acid sequence of rat mature enzyme with those of dog (88%), pig (75%) and man (75%) than with that of rat isolipase (74%). PMID- 1730293 TI - Arachidonic acid induces phosphorylation of an 18 kDa protein in electrically permeabilised rat islets of Langerhans. AB - Arachidonic acid (AA) was shown to induce concentration-dependent, calcium independent, in situ phosphorylation of a protein of approximate molecular weight 18 kDa in electrically permeabilised rat islets of Langerhans. This protein did not appear to be a substrate for protein kinase C (PKC) since stimulation of PKC by 4 beta phorbol myristate acetate (4 beta PMA) did not result in 32P incorporation into an 18 kDa protein, and since AA-induced phosphorylation was observed in islets in which PKC had been down-regulated by prolonged exposure of islets to 4 beta PMA. These results suggest that AA stimulates protein phosphorylation by a mechanism other than PKC activation. PMID- 1730294 TI - Isolation and some properties of a 34-kDa-membrane protein that may be responsible for ribosome binding in rat liver rough microsomes. AB - We have isolated, by hydroxyapatite chromatography with a non ionic detergent and a high salt concentration, a non-glycosylated, membrane protein with a relative molecular weight of 34 kDa that had previously been found to be a major constituent of the membrane protein fraction showing ribosome-binding activity derived from rat liver rough microsomes (RM). The isolated 34 kDa protein (p34), when incorporated into a liposome model membrane, exhibited significant binding activity toward ribosomes, its binding properties being similar to those observed with intact RM. Immunochemical analyses using antibodies directed against p34 suggested that it is a membrane-embedded RM surface protein, which is specifically localized in ribosome-attached organelles and widely distributed among mammalian tissues. These results would constitute evidence that p34 is a likely candidate for an RM ribosome-binding protein. PMID- 1730295 TI - 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid by cytochrome P-450 in pig liver microsomes. AB - Pig liver microsomes were found to catalyze the 7 alpha-hydroxylation of several potential bile acid precursors besides cholesterol. 26-Hydroxycholesterol, 3 beta hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid were all efficiently converted into the 7 alpha-hydroxylated products. Two cytochrome P 450 fractions showing 7 alpha-hydroxylase activity could be isolated. One fraction catalyzed 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy 5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid but was inactive towards cholesterol. The other fraction catalyzed 7 alpha-hydroxylation of cholesterol in addition to the other substrates. 26-Hydroxycholesterol in equimolar concentration did not inhibit the cholesterol 7 alpha-hydroxylase activity of this fraction. It is concluded that liver microsomes contain a cytochrome P-450 catalyzing 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5 cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid. The results indicate that this cytochrome P-450 is different from that catalyzing 7 alpha-hydroxylation of cholesterol. PMID- 1730296 TI - Appearance of the Na(+)-motive terminal oxidase in Bacillus FTU grown under three different conditions lowering the delta mu H+ level. AB - The terminal oxidases and coupled Na+ transport have been studied in intact cells and inside-out subcellucar vesicles of alkalo- and halotolerant Bacillus FTU grown under different conditions. Cells grown at pH 7.5 are shown to possess a system of respiration-dependent Na+ transport which is (i) inhibited by protonophorous uncoupler and (ii) activated by the delta psi-discharging agent valinomycin, suggesting that the Na+ transport is due to cooperation of the H(+) motive oxidase and Na+/H+ antiporter. On the other hand, growth under conditions lowering the delta mu H+ level, namely (i) pH 8.6, (ii) pH 7.5 in the presence of protonophore, and (iii) pH 7.5 in the presence of low cyanide concentrations results in appearance of terminal oxidase-supported Na+ transport which is stimulated by protonophores (the Na(+)-motive oxidase). In all three cases, the appearance of ascorbate (+ TMPD) oxidation resistant to low and sensitive to high cyanide concentrations was found to occur. It is concluded that not only alkaline pH but also other conditions which lower delta mu H+ can cause substitution of Na+ for H+ as a coupling ion. PMID- 1730297 TI - Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum. AB - DEAE-Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases. A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B. Two histone deacetylases, HD1 and HD2, could be discriminated. They differ with respect to substrate specificity and pH dependence. For the first time the substrate specificity of histone deacetylases was determined using HPLC-purified individual core histone species. The order of acetylated substrate preference is H2A much greater than H3 greater than or equal to H4 greater than H2B for HD1 and H3 greater than H2A greater than H4 for HD2, respectively; HD2 is inactive with H2B as substrate. Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity. PMID- 1730298 TI - Cis-trans isomerization is rate-determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase. AB - The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t1/2 = 9 min). Kinetic data from 'double-jump' measurements indicate that proline isomerization might be rate determining in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics: the presence of isomerase during reactivation lowers the half-time of the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis peptidyl-Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) were constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPIase the P202N mutant behaved exactly like the unmutated enzyme. Thus, cis-trans isomerization of the Pro202 cis-peptidyl bond is not rate determining in the reactivation process. The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied. PMID- 1730299 TI - Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis. AB - A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate dependent enzymes, completely abolishes enzyme activity. PMID- 1730300 TI - Recognition of transition metal ions by peptides. Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of flight mass spectrometry. AB - Metal-binding peptides in proteolytic digest maps have been identified by matrix assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine-rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF-MS with dihydroxybenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal-binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide-metal ion interactions in protein digest maps. The ability to evaluate peptide-metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal-binding proteins. PMID- 1730301 TI - Ethical and legal issues in preimplantation genetic screening. PMID- 1730302 TI - The secretory endometrial protein, placental protein 14, in women with ectopic gestation. AB - OBJECTIVE: To determine the serum level of the secretory endometrial protein, placental protein 14 (PP14) and progesterone (P) in women with ectopic gestation. DESIGN: Blood samples were collected prospectively and preoperatively. Reference range was determined from a prospective population of 98 women with uncomplicated pregnancies and normal outcome. SETTING: The women were admitted to a university hospital. PATIENTS: Fifty-nine women with laparoscopically verified ectopic pregnancy entered the study. INTERVENTION: At the time of diagnosis PP14 and P were measured. MAIN OUTCOME MEASURE: After observing the low serum levels of PP14 and P, a correlation analysis was made and compared with the findings in normally pregnant women. RESULTS: A significant positive correlation was found between the level of PP14 and P (P less than 0.00002), not found in normal intrauterine pregnancies. CONCLUSIONS: These findings suggest that the regulation of the PP14 production involves either a control mechanism from the ovary or is mediated by paracrine secretion. PMID- 1730303 TI - The effects of gender-specific diagnosis on men's and women's response to infertility. AB - OBJECTIVE: To determine if differences could be distinguished between men's and women's emotional response to infertility based on the assignment of a gender specific diagnosis. DESIGN: Gender-specific diagnoses were examined in relation to stigma, perception of loss, role failure, and self-esteem, using structured interviews. SETTING: Tertiary clinical care in private practice settings. PARTICIPANTS: Thirty-six self-selected volunteer couples undergoing infertility treatment. MAIN OUTCOME MEASURES: Stigma, perception of loss, role failure, and lowered self-esteem emerged from content analysis of structured interview data. RESULTS: No differences were found among women in their emotional response to infertility regardless of whether a female or male infertility factor was present, whereas men with a male factor experienced more negative emotional response to infertility than men without a male factor. CONCLUSIONS: Although both women and men are affected by infertility, their emotional response is significantly influenced by a gender-specific diagnosis. Men's response to infertility closely approximates that of women if the infertility has been attributed to a male factor but differs considerably if a male factor is not found. PMID- 1730304 TI - Growth hormone: revisited. PMID- 1730305 TI - Psychosocial, treatment, and demographic predictors of the stress associated with infertility. AB - OBJECTIVE: To determine which psychosocial, treatment, and demographic factors relate to the amount of perceived stress that infertile women and men experience. DESIGN: A cross-sectional, structured interview research design was used. SETTING: In-person interviews were conducted in study participants' homes. PARTICIPANTS: Wives and husbands from 185 couples in Southeastern Michigan with primary infertility were studied. MAIN OUTCOME MEASURES: A nine-item rating scale of perceived stress associated with infertility was the outcome measure. RESULTS: For both women and men, stress was significantly positively correlated with treatment costs and number of tests and treatments received; stress was significantly negatively correlated with confidence that one will have a child and perceived control. For women only, attitudes about infertility treatments, importance of children, attributions of responsibility to physicians, and social support also significantly related to perceived stress. For men only, income, number of physicians seen, and self attributions of responsibility also significantly related to perceived stress. CONCLUSIONS: As hypothesized, a variety of treatment characteristics and psychosocial factors were related to experienced stress. Contrary to expectation, demographic factors such as age and number of years married were not related to experienced stress. This study's results suggest that attempts by health care providers to increase patients' sense of control, optimism (within realistic limits), and social support should reduce stress. PMID- 1730306 TI - Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm. AB - OBJECTIVE: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. RESULTS: Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). CONCLUSIONS: One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients. PMID- 1730307 TI - An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program. AB - OBJECTIVE: To select in vitro fertilization (IVF) patients with a low sperm penetration assay (SPA) value and to determine if the penetration rate, fertilization rate, and the pregnancy rate (PR) can be improved in these patients by treating sperm with refrigeration, calcium ionophore A23187, prostaglandin E1, prostaglandin E2, or heparin. DESIGN: The study consists of three parts: identification of patients with poor SPA values, analysis of treatments to improve the SPA value, and evaluation of the treatments to improve fertilization and PRs. RESULTS: The data indicate that treatment of sperm with refrigeration can improve fertilization and PRs during IVF in selected patients previously shown to have an improved SPA value with the treatment. CONCLUSIONS: Three points are emphasized: (1) the treatments analyzed in this study can improve SPA values in some of the patients with low sperm penetration capacity; (2) of the treatments studied, sperm refrigeration resulted in the largest improvement in sperm penetration capacity; and (3) sperm refrigeration can increase fertilization and PRs during IVF in this select group of patients. PMID- 1730308 TI - Evidence for maternal predisposition to chromosome aneuploidy in multiple oocytes of some in vitro fertilization patients. AB - OBJECTIVES: To assess the rate of chromosome aneuploidy (e.g., extra or missing chromosomes) in oocytes remaining unfertilized in our in vitro fertilization (IVF) program. To determine whether two parameters of the IVF technique, advanced maternal age and hormonal follicle stimulation, affect this rate. DESIGN: Data on oocyte retrieval, fertilization, and aneuploidy rates are analyzed to test for possible relations with maternal age and two hormonal stimulation regimens. SETTING: Patients of our IVF program from 119 stimulated cycles over 8 months. PATIENTS, PARTICIPANTS: In vitro fertilization patients selected for having oocytes (1 to 18) remaining unfertilized after insemination in vitro. RESULTS: Advanced maternal age decreases both the number of retrieved oocytes and the fertilization rate, but hormonal treatments have no effect. Aneuploidy (rate 27%), involving group G most frequently, appears associated with advanced age. Patients who were previously parous produced significantly reduced numbers of aneuploid oocytes compared with the nonparous group. A significant excess (P = 0.01) of patients had multiple oocytes all alike (all haploid or all aneuploid), showing correlation among multiple oocytes of a patient in chromosome status. CONCLUSIONS: Maternal age affects reproductive performance and is related to specific chromosomal aneuploidy. Women who were previously parous are more likely to produce normal oocytes than nonparous women; oocyte normality therefore may improve the chance for a future successive pregnancy. Nonrandomness in chromosome abnormality of some patients' multiple oocytes is evidence for maternal predisposition to meiotic nondisjunction. Consequently, these patients are at risk for failed IVF cycles. PMID- 1730309 TI - In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1990 results from the IVF-ET Registry. Medical Research International. Society for Assisted Reproductive Technology (SART), The American Fertility Society. AB - OBJECTIVE: To summarize the procedures and outcomes of assisted reproductive technology (ART) initiated in the United States in 1990. DESIGN: The registry follows a prospective study format. PARTICIPANTS: One hundred eighty clinics submitted data on their ART treatments to the In Vitro Fertilization (IVF) Registry. MAIN OUTCOME MEASURES: The outcomes measured included clinical pregnancy, ectopic pregnancy, abortion, stillbirth, delivery, chromosomal abnormalities, and congenital defects. RESULTS: During 1990, the clinics reported performing 25,744 ovarian stimulation cycles. From all ART treatments there were 5,150 clinical pregnancies and 3,951 live deliveries. The overall live delivery rates were 14% for IVF (based on 16,405 retrievals), 22% for gamete intrafallopian transfer (based on 3,750 retrievals), and 16% for zygote intrafallopian transfer and related practices (based on 1,370 retrievals). The delivery rates for frozen embryo transfer cycles and IVF with donor egg cycles were 9% and 22%, respectively. CONCLUSIONS: The 1990 results indicate an increase in the use of gonadotropin-releasing hormone analogs and an increase in the number of embryos being transferred per cycle, along with no important changes in clinical pregnancy and delivery rates. In addition, there was an increase in both numbers of frozen embryo transfers and IVF cycles with donor oocytes. PMID- 1730310 TI - Hormonal secretions in singleton pregnancies arising from the implantation of fresh or frozen embryos after oocyte donation in women with ovarian failure. AB - OBJECTIVE: To determine whether levels of human chorionic gonadotropin (hCG), 17 beta-estradiol (E2), and progesterone (P) are different in the peri-implantation phase of fresh versus frozen embryos. DESIGN: Hormonal secretions were measured on days 9 and 11 after implantation and at 4, 5, and 6 weeks gestation. PATIENTS: Thirty-one pregnancies were achieved in 65 patients with ovarian failure. Seventeen singleton pregnancies developed after implantation of 4 frozen and 13 fresh embryos. RESULTS: Human chorionic gonadotropin and E2, contrary to P, were higher in cases of fresh embryos from the 9th day after transfer to the 5th week at which time they become statistically significant (respectively, for hCG and E2, 5,800.3 +/- 332.3 versus 2,027.3 +/- 916.3 [mean +/- SD] mIU/mL for hCG and 562.3 +/- 215 versus 291 +/- 152 pg/mL for E2). CONCLUSIONS: This difference might be explained by either the higher number of fresh embryo replaced or by the fact that the number of blastomeres and also their metabolic activity could be reduced after freezing and thawing. PMID- 1730311 TI - Mock embryo transfer in early luteal phase, the cycle before in vitro fertilization and embryo transfer: a descriptive study. AB - OBJECTIVE: To investigate visually the uterine retention or sequestration of boluses of radiopaque dye, mimicking embryo transfer (ET). DESIGN: During the cycle before in vitro fertilization (IVF) and ET, patients underwent a mock ET of 40 microL of radiopaque dye into the uterine cavity. The patient was positioned supine for retroverted or axial and knee chest for anteverted uteri. The position of the dye at injection, during and after catheter removal, and during patient roll over and standing was monitored. SETTING: Treatment of infertility in a private practice. PATIENTS: Thirty-four IVF patients. INTERVENTIONS: During the cycle before IVF/ET, patients underwent a mock ET using a bolus of 40 microL of radiopaque dye. MAIN OUTCOME MEASURES: Planned before visual observation began. RESULTS: The dye remained primarily in the uterine cavity in only 68% (optimum ET position is knee chest for anteverted and supine for retroverted or axial uterus) and 48% (nonoptimum position is supine for anteverted uterus) at mock ET; in those groups, a 33% clinical pregnancy rate (PR) per retrieval resulted. Dye motility into the fallopian tube(s), cervix, and/or vagina is 38.2%, 8.8%, and 11.8%, respectively. CONCLUSIONS: If the mock ET had been the actual ET, 32% (optimum ET position) and 52% (nonoptimum ET position) of all patients would have lost their opportunity for pregnancy as a result of the ET procedure. Our 33% PR per retrieval among those patients who retained the dye in utero is more consistent with our expectations, given the advanced technologies of IVF/ET today. PMID- 1730312 TI - Cost-effectiveness of gamete intrafallopian transfer in comparison with induction of ovulation with gonadotropins in the treatment of female infertility: a clinical trial. AB - OBJECTIVE: To compare the cost-effectiveness of gamete intrafallopian transfer (GIFT) with that of conventional infertility treatment in couples with female infertility, excluding tubal factors. DESIGN: Patients were randomly divided in two groups: receiving GIFT or conventional infertility treatment. For a period of 2 years, GIFT was compared with conventional infertility treatment in couples with endometriosis, anovulation, idiopathic infertility, cervical mucus factor, female immunologic factor, or multifactorial causes of infertility in a randomized clinical trial. SETTING: The study was performed in the Unit for Human Reproduction, Department of Obstetrics and Gynaecology, Faculty of Medicine, University of the Orange Free State, Bloemfontein, Republic of South Africa. PATIENTS: One hundred seventy-four successive couples with female infertility were selected for the study. All couples were from the higher socioeconomic bracket. INTERVENTIONS: One group received GIFT and the other received conventional infertility treatment consisting of induction of ovulation with gonadotropins followed by intrauterine artificial insemination or normal intercourse. MAIN OUTCOME MEASURES: The results were stratified according to the specific cause of infertility. Outcome was measured by the success rate per treatment cycle, as well as the cost per pregnancy. RESULTS: Overall, GIFT proved to be successful in 26.7% of treatment cycles compared with 9.7% with conventional therapy. CONCLUSIONS: After careful analysis, the authors came to the conclusion that GIFT is more cost-effective than conventional infertility treatment in patients with endometriosis and anovulation. In patients with idiopathic infertility, immunologic infertility, a cervical mucus factor, and multifactorial infertility, induction of ovulation followed by intrauterine artificial insemination or normal intercourse proved to be more cost-effective. PMID- 1730313 TI - Deterioration of semen parameters over time in men with untreated varicocele: evidence of progressive testicular damage. AB - OBJECTIVE: To report observations of semen parameters in men with untreated varicocele over time. DESIGN: Semen parameters in 13 men with varicoceles were obtained at initial presentation, and re-evaluated at a 9 to 96-month interval because of persistent infertility. SETTING: Outpatient male fertility clinic. PATIENTS, PARTICIPANTS: Thirteen men who presented for fertility evaluation and who were found to have varicocele(s) and normal semen parameters. Re-evaluation was performed because of continued fertility problems. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Semen parameters, especially sperm density, sperm mobility, and total sperm. RESULTS: Statistically significant deterioration was noted from normal to abnormal. CONCLUSION: In males with varicocele(s), normal semen parameters should not routinely be expected to remain normal over time. PMID- 1730314 TI - The effect of cocaine on sperm motility characteristics and bovine cervical mucus penetration. AB - OBJECTIVES: To determine the in vitro effects of cocaine on sperm motility and bovine mucus penetration because cocaine abuse is associated with decreased sperm motility, and related compounds, such as procaine, are known to decrease sperm motility. DESIGN: Human semen samples were exposed to a range of cocaine concentrations and the effects quantified using computer-assisted sperm analysis and the bovine mucus penetration test. SETTING: University research laboratory. PATIENTS, PARTICIPANTS: Samples were obtained from 18 healthy volunteers. INTERVENTIONS: Normal semen samples were exposed to concentrations of cocaine ranging from 10(-11) to 10(-4) M. Motility characteristics were evaluated after 2 hours, and bovine mucus penetration was evaluated after 30 minutes, 1 hour, and 2 hours. Mucus penetration by washed sperm was also evaluated. MAIN OUTCOME MEASURES: Motility characteristics were evaluated using computer-assisted sperm analysis, and functional sperm motility was evaluated using the bovine mucus penetration test. RESULTS: Cocaine exposure decreased the percentage of motile sperm in a concentration-dependent manner with a maximum decrease of 23% at 10( 4) M but had no effect on other motility characteristics. Cocaine decreased bovine mucus penetration by 12% at high cocaine concentrations (10(-4) M), but increased penetration by 69% at low concentrations (10(-9) M). Washing sperm before cocaine exposure attenuated the increased sperm penetration. CONCLUSION: The ability of cocaine to decrease the percentage of motile sperm at high concentrations may explain the decreased sperm motility associated with cocaine use. Cocaine's ability to augment sperm penetration at low concentrations suggests an interaction of cocaine with the sperm adrenergic system. PMID- 1730315 TI - Testicular origin of immunobead-reacting antigens in human sperm. AB - OBJECTIVE: To investigate, by means of immunobinding technique, the antigenic surface expression of human vasa efferentia sperm as compared with that of ejaculated sperm. DESIGN: Briefly, vasa efferentia sperm, retrieved microsurgically, and donors' ejaculated sperm (controls) were used. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens, undergoing sperm aspiration as part of their infertility treatment, and controls who were donors of our sperm bank were first assayed by direct immunobead test and, if found negative, were exposed to sera containing known high titers of antisperm antibodies and then retested by indirect immunobead test. RESULTS: The data showed no difference in binding titers and class of immunoglobulins between vasa efferentia and ejaculated sperm. CONCLUSIONS: These findings suggest that, in humans, sperm acquire those surface antigens commonly detected by immunobinding test within the testes before their transit through the epididymis. PMID- 1730316 TI - Platelet-activating factor acetylhydrolase in human seminal plasma. AB - OBJECTIVE: To search for the presence of platelet-activating factor (PAF) acetylhydrolase activity in human seminal plasma. DESIGN: Experimental. SETTING: Reproductive laboratory in a university-affiliated hospital research center. PARTICIPANTS: Human male volunteers were selected on the basis of apparent normal health. RESULTS: Human seminal plasma contained significant levels of PAF acetylhydrolase activity. Enzymatic hydrolysis of PAF displayed typical kinetics and was a calcium-independent process. Platelet-activating factor acetylhydrolase in seminal plasma was associated with a very high-density lipoprotein fraction. Enzymatic activity was independent of sperm count and motility. CONCLUSION: Human seminal plasma contains significant levels of PAF acetylhydrolase. This enzyme may be an important factor in the regulation of PAF concentration and activity. PMID- 1730317 TI - Effects of Ringer's lactate, Interceed(TC7) and Gore-Tex Surgical Membrane on postsurgical adhesion formation. AB - OBJECTIVE: To evaluate the effects of Ringer's lactate instillation, Interceed(TC7) (Johnson and Johnson Medical, Inc., New Brunswick NJ), and Gore Tex Surgical Membrane (W. L. Gore and Associates, Inc., Flagstaff, AZ) in a rat uterine horn model. SETTING: Rats were in a conventional laboratory setting. PARTICIPANTS: Sprague-Dawley white rats, weighing 225 to 250 g. INTERVENTIONS: The left uterine horn was subjected to a standardized lesion by serosal denudation and devascularization. The rats were randomly assigned into control group, Interceed(TC7) group, Gore-Tex group, and Ringer's lactate group. MAIN OUTCOME MEASURES: Degree of adhesions was evaluated 2 weeks after the initial surgery. RESULTS: Adhesion score after Ringer's lactate instillation was significantly lower than those of control, Interceed(TC7), and Gore-Tex groups. Gore-Tex was associated with less adhesion formation than control. No difference was found in the adhesion formation between the Interceed(TC7) group and the control group. CONCLUSION: Confirming our previous observations, Ringer's lactate instillation is effective in decreasing adhesion formation. Gore-Tex reduces adhesion formation, but its efficacy is inferior to those of Ringer's lactate. Contrary to previous reports, Interceed(TC7) is ineffective in our animal model. PMID- 1730318 TI - Murine peritoneal injury and de novo adhesion formation caused by oxidized regenerated cellulose (Interceed [TC7]) but not expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane). AB - STUDY OBJECTIVE: To evaluate the impact of the materials contained in the available adhesion prevention barriers on the peritoneum. STUDY DESIGN, SETTING, PATIENTS: A murine paradigm was used, placing oxidized-regenerated cellulose (Interceed [TC7]) and expanded polytetrafluoroethylene (PTFE; Gore-Tex Surgical Membrane) in the peritoneal cavity for intervals up to 14 days. INTERVENTIONS AND MAIN OUTCOME MEASURES: The appearance of the peritoneum on scanning and transmission electron microscopy and the presence of de novo adhesions were the end-points used. RESULTS: Oxidized-regenerated cellulose caused localized sloughing of the mesothelial cell layer and leukocyte infiltration of the deeper tissue leading to the formation of adhesions to the bowel and liver in 58% of the animals. The surface of the oxidized-regenerated cellulose-injured peritoneum healed in 5 to 7 days. Neither peritoneal injury nor adhesions were noted in sham operated animals or animals with PTFE. CONCLUSIONS: Oxidized-regenerated cellulose but not PTFE has a localized injurious effect on the peritoneum of the mouse, resulting in de novo adhesions. The impact of the barrier material itself on normal peritoneum may be an important consideration in designing surgical barriers for the prevention of postoperative adhesions. PMID- 1730319 TI - Flexible protocol for administration of human follicle-stimulating hormone with gonadotropin-releasing hormone antagonist. AB - OBJECTIVE: To test, using a primate model, a new approach for achieving individualized pituitary-ovarian responses to controlled ovarian hyperstimulation. DESIGN: Normal ovulatory adult monkeys were selected and randomly assigned to one of the three groups according to onset of spontaneous menses. They had no prior exposure to gonadotropin-releasing hormone (GnRH) antagonist or exogenous gonadotropin therapies. SETTING: The laboratories of The Jones Institute for Reproductive Medicine were used. PATIENTS, PARTICIPANTS: Normal adult macaque females were studied. INTERVENTIONS: Monkeys received hormonal therapies of gonadotropins in combination with GnRH antagonist. MAIN OUTCOME MEASURE(S): Pituitary luteinizing hormone (LH) secretion and ovarian estrogen and progesterone production were monitored. RESULTS: Adding GnRH antagonist to ongoing human follicle-stimulating hormone (hFSH) stimulation can prevent unwanted LH surges, whether begun at early, mid, or late points in the stimulation protocol. CONCLUSIONS: The flexible protocol for administration of hFSH with GnRH antagonist yielded satisfactory results, with apparent advantages of economy, convenience, and individuality of treatment compared with GnRH agonist plus gonadotropin regimens used currently. PMID- 1730320 TI - Intramural pregnancy after in vitro fertilization and embryo transfer. AB - A case of intramural but not interstitial pregnancy, established after IVF/ET, is described. The etiologic, diagnostic, and therapeutic aspects of this clinical dilemma are discussed. PMID- 1730321 TI - A prospective study to investigate the value of flushing follicles during transvaginal ultrasound-directed follicle aspiration. AB - In this study, 50 transvaginal US-directed follicle aspiration procedures were performed with follicle flushing using a double-channel needle. The origin of each oocyte was established according to whether it had been obtained in the initial part of the aspirate, in the dead space aspirate, in the first to third flushes, or in the fourth to sixth flushes. Seventeen percent of total oocytes were found in follicle flushes. Only 3.2% of total oocytes were found in the fourth to sixth flushes, and their fertilization rate was reduced. Flushing follicles with a double-channel needle may result in the recovery of 20% more oocytes than would be obtained by aspiration alone. PMID- 1730322 TI - Ectopic pregnancies after induction of ovulation and in vitro fertilization. PMID- 1730323 TI - Sperm antibodies and steroid treatment. PMID- 1730324 TI - Rubella vaccination. PMID- 1730325 TI - Subclinical pregnancy loss in clomiphene citrate-treated women. AB - OBJECTIVE: To ascertain the subclinical pregnancy loss rate in clomiphene citrate (CC)-treated infertile women compared with women of normal fertility. DESIGN: Following a prospective format, serum samples were taken during the luteal phase of 92 menstrual cycles associated with CC treatment and 47 cycles in normal women. Human chorionic gonadotropin (hCG) assay sensitivity was 0.25 IU/L. Human chorionic gonadotropin assay was validated against 95 nonpregnant cycles. Criterion for pregnancy was a single serum sample greater than or equal to 0.5 IU/L. SETTING: All subjects were under clinical management at the University of Virginia Health Sciences Center. PATIENTS AND PARTICIPANTS: Patients undergoing CC induction of ovulation with satisfactory ovulatory response were candidates for the study group (n = 34). Control subjects of proven normal fertility were recruited (n = 22). Nonpregnant control subjects were sexually abstinent or had been surgically sterilized (n = 89). INTERVENTION: A serum sample was taken during the late luteal phase of all subjects. RESULTS: Thirteen percent of CC treated cycles and 4.3% of normal control cycles were subclinical losses (P = 0.09). Fifty percent of CC-induced pregnancies were subclinical losses compared with 16.6% of normal control pregnancies (P = 0.05). Of CC patients who had at least one subclinical loss 47.6% later conceived a term pregnancy compared with 15.3% who did not have a subclinical loss (P = 0.06). CONCLUSION: Subclinical pregnancy loss is more common in CC-treated women than normal women and may be a predictor of subsequent normal conception. PMID- 1730326 TI - Histology of midluteal corpus luteum and endometrium from clomiphene citrate induced cycles. AB - OBJECTIVE: To determine the histologic development of midluteal corpus luteum (CL) and endometrium in normal fertile women after induction of ovulation with clomiphene citrate (CC). DESIGN, PATIENTS, INTERVENTIONS: Twelve normally cycling women planning to undergo an elective tubal ligation were treated with 50 to 150 mg of CC daily on days 5 through 9 of the cycle. Luteectomy and endometrial biopsy were performed simultaneously 7 days after the urinary luteinizing hormone surge. RESULTS: Because polyovulation occurred in 10 of the 12 women, 22 CL and 12 endometrial biopsies were studied. Ten women had luteal and endometrial histology that were within 2 days of the ovulation to biopsy interval. The 2 remaining women had endometrial histology that lagged 3 days behind the chronological postovulatory date. In these women, out-of-phase endometrium occurred despite polyovulatory cycles in which two and three histologically normal CL lutea were present and associated with elevated progesterone concentrations. CONCLUSIONS: In CC-induced ovulatory cycles: (1) midluteal CL histology is normal and (2) apparently out-of-phase preimplantation endometrium occurs in midluteal phase. PMID- 1730327 TI - The effect of ethinyl estradiol on endometrial thickness and uterine volume during ovulation induction by clomiphene citrate. AB - OBJECTIVE: To assess the deleterious effect of clomiphene citrate (CC) on the development of the endometrium and its improvement by the addition of ethinyl estradiol (E2). PARTICIPATING PATIENTS: Infertility-treated patients, monitored for induction of ovulation or timing of insemination (control group). DESIGN: We studied four groups of women during an ovulatory cycle with various treatment schedules. Group 1: untreated patients; group 2: patients treated by CC; group 3: patients treated by CC + ethinyl E2; group 4: patients treated by human menopausal gonadotropin. Follow-up of the patients was done by vaginal ultrasonography and measurements of blood E2. RESULTS: In the group treated by CC, both endometrial thickness and uterine volume growth during the follicular phase were lower as compared with untreated controls and menotropin-treated patients. The addition of ethinyl E2 to these patients reversed this deleterious effect of CC without interfering with ovulation. CONCLUSION: Ethinyl E2 may reverse the deleterious effect of CC on endometrial development during the follicular phase. PMID- 1730328 TI - Hypergonadotropic hypogonadic amenorrhea (World Health Organization III) and osteoporosis. AB - OBJECTIVE: To assess the bone mineral density in World Health Organization (WHO) III women after hormone replacement therapy. DESIGN: We studied the bone mineral density of 41 women with premature ovarian failure (hypergonadotropic hypogonadic amenorrhea--WHO III) before and during hormone replacement therapy. SETTING: All WHO III women were recruited from the Outpatient Department of the First Department of Gynecology and Obstetrics, University of Vienna Medical School, Austria, a public University Hospital. PATIENTS, PARTICIPANTS: Forty-one patients, 30 healthy women. INTERVENTIONS: Twenty-eight of 41 WHO III women received cyclic hormone replacement therapy consisting of 0.625 mg conjugated estrogen (days 1 to 30) and 5 mg medrogeston (days 20 to 30) in addition, with a 7-day interval. MAIN OUTCOME MEASURE: The bone mineral density was evaluated by single photon absorptiometry and dual energy x ray absorptiometry every 6 months (single photon absorptiometry six times, dual energy x ray absorptiometry four times). RESULTS: The bone mineral density in young women with hypergonadotropic hypogonadic amenorrhea (WHO III) was lower than in age-matched controls. Hormone replacement therapy produced an increase in bone mineral density in 28 WHO III women, whereas bone mineral density remained quite constant in the women without therapy. CONCLUSION: Hormone replacement therapy increases the bone mineral density of women with hypergonadotropic hypogonadic amenorrhea. Hormones should be substituted early and consistently in affected patients. PMID- 1730329 TI - Gonadotropin-releasing hormone, follicle-stimulating hormone beta, luteinizing hormone beta gene structure in idiopathic hypogonadotropic hypogonadism. AB - OBJECTIVE: To determine if the genes for gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone beta (FSH beta), and luteinizing hormone beta (LH beta) are present, and if so, whether gene structure is normal in patients with idiopathic hypogonadotropic hypogonadism (IHH). DESIGN: Patients with clinical and laboratory characteristics of IHH were studied at the deoxyribonucleic acid (DNA) level to assess gene structure. SETTING: This study took place in an academic setting. PATIENTS: Human volunteers with documented IHH and fertile controls were studied. INTERVENTIONS: Genomic DNAs were extracted from each patient, Southern blots were constructed and hybridized to DNA probes for GnRH, FSH beta, and LH beta. DNA samples were also subjected to polymerase chain reaction analysis. MAIN OUTCOME MEASURES: Gene structure was assessed by analysis of autoradiographs and gel electrophoresis of polymerase chain reaction products in both the study patients and controls. RESULTS: Each analysis for FSH beta, LH beta, and GnRH demonstrated the same sized fragments in both the study group and control group. A 1.2-kilobase fragment containing the coding region for GnRH was present in all patients with IHH and controls by polymerase chain reaction. CONCLUSIONS: The genes for GnRH, LH beta, and FSH beta are present in patients with IHH. No large deletions or rearrangements of any of these genes were identified in any of these patients. PMID- 1730330 TI - Gonadotropin suppression for the treatment of karyotypically normal spontaneous premature ovarian failure: a controlled trial. AB - OBJECTIVE: To determine if gonadotropin suppression improves ovarian follicle function or ovulation rates in patients with karyotypically normal spontaneous premature ovarian failure. DESIGN: Prospective, double-blind, placebo-controlled, crossover trial. SETTING: Tertiary care research institution. INTERVENTIONS: Two intervention phases lasting 4 months each: one placebo phase, and one treatment phase during which each patient received daily subcutaneous injections of 300 micrograms of the gonadotropin-releasing hormone agonist (GnRH-a) deslorelin. During both phases, patients took a standardized estrogen (E) replacement regimen. PATIENTS, PARTICIPANTS: Twenty-six patients with karyotypically normal spontaneous premature ovarian failure ranging in age from 18 to 39 years. MAIN OUTCOME MEASURES: We measured serum estradiol (E2) and progesterone (P) levels weekly during the 2 months after each intervention. We defined a serum E2 greater than 50 pg/mL (184 pmol/L) as evidence for ovarian follicle function and a serum P greater than 3.0 ng/mL (9.5 nmol/L) as evidence for ovulation. RESULTS: The GnRH-a therapy did not significantly enhance recovery of ovarian follicle function or the chance of ovulation. The power to detect a 40% and a 33% ovulation success rate with therapy was 0.95 and 0.83, respectively. We found evidence for ovarian follicle function in 11 of 23 women (48%), and 4 women (17%) ovulated. CONCLUSIONS: Patients with karyotypically normal spontaneous premature ovarian failure treated with E replacement did not benefit from the additional gonadotropin suppression achieved with GnRH-a. Because these patients have a significant possibility of spontaneous remission, attempts to induce ovulation should be limited to controlled trials designed to determine safety and effectiveness. PMID- 1730331 TI - Noninvasive diagnosis of resistant ovary syndrome by ultrasonography. AB - OBJECTIVE: To compare transvaginal ultrasonographic features of the ovaries and endometrium of patients with premature ovarian failure to normally cycling women on oral contraceptives (OCs), menopausal women with an equivalent duration of amenorrhea to the premature ovarian failure group, and patients with Turner's syndrome. DESIGN: Transvaginal ultrasonography in groups of women with premature ovarian failure, OC, and menopause. SETTING: All subjects were studied in an academic tertiary care center. PATIENTS/PARTICIPANTS: Seventeen women with premature ovarian failure, 20 volunteers on OC, 20 with menopause, and 4 patients with Turner's syndrome were studied. INTERVENTION: None, except for OCs in the OC group only. MAIN OUTCOME MEASURES: Frequency of ovarian, ovarian follicle, and endometrial visualization and their respective measurement in the three groups. RESULTS: At least one ovary was visualized in 84% of the premature ovarian failure patients as compared with 95% of the OC and menopause groups and 25% of the Turner patients. Mean ovarian volume was smaller in the premature ovarian failure group as compared with the OC group but equal to that of the menopause group. In the premature ovarian failure group, 41% had follicles in their ovaries as compared with 95% in the OC group, 5% in the menopause group, and none in the Turner group. The number of follicles per ovary was significantly lower in the premature ovarian failure as compared with the OC group, whereas only one subject had a single follicle in the menopause group. Premature ovarian failure subjects with follicles had larger ovaries than those without follicles. Endometrial thickness was not different among the groups. CONCLUSION: Ultrasonography may serve to identify a substantial subset (approximately 40%) of premature ovarian failure patients with ovarian follicles and potential for fertility consistent with the diagnosis of resistant ovary syndrome. PMID- 1730332 TI - Transvaginal hysterosalpingo-contrast sonography for the assessment of tubal patency with gray scale imaging and additional use of pulsed wave Doppler. AB - OBJECTIVE: To determine whether the additional use of pulsed wave (PW) Doppler can improve the tubal diagnosis reached with gray scale imaging in doubtful cases. DESIGN: The study is an open, uncontrolled clinical trial of women of childbearing age. SETTING: Clinical environment. PATIENTS: Seventeen female patients (23 to 37 years of age) with diagnosed sterility problems. INTERVENTIONS: The contrast agent SH U 454 was administered transcervically during transvaginal gray scale and PW Doppler sonography. MAIN OUTCOME MEASURES: Hysterosalpingo-contrast sonography by gray scale and by PW Doppler and follow-up chromolaparoscopy (n = 16) or hysterosalpingography (HSG, n = 1) were performed. The diagnostic efficacy of gray scale and PW Doppler were compared with each other and against a conventional control procedure (chromolaparoscopy or HSG). RESULTS: The gray scale findings were confirmed by PW Doppler in 5 cases on one side; confirmed by PW Doppler in 7 cases on both sides; corrected by PW Doppler in 4 cases on one side; and corrected by PW Doppler in 1 case on one side and confirmed on the other side by PW Doppler. In all 17 cases, the tubal findings after PW Doppler were confirmed by chromolaparoscopy or HSG. CONCLUSION: The additional use of PW Doppler in hysterosalpingo-contrast sonography is recommended as a supplement to gray scale imaging (1) in cases of suspected tubal occlusion and (2) in the event of intratubal flow demonstrable only over a short distance. PMID- 1730333 TI - Transvaginal color Doppler study of blood flow in ectopic pregnancies. AB - OBJECTIVE: To compare Doppler indices of blood flow in the uterine and spiral arteries and the corpus luteum (CL), in ectopic and intrauterine pregnancies (IUPs). DESIGN: A prospective study of women with an ectopic or singleton IUP at the corresponding stage of gestation. SETTING: The Gynaecological Ultrasound Clinic, King's College Hospital. PATIENTS: Fifty-two women, 19 with an ectopic pregnancy (EP) and 33 with an IUP. INTERVENTIONS: All women were examined by transvaginal ultrasonography with color Doppler immediately before surgery. MAIN OUTCOME MEASURES: The resistance index from the left and right uterine arteries, the spiral arteries, and the CL. The peak blood velocity (cm/sec) from the uterine arteries. The length of gestation. RESULTS: Fetal heart activity was observed in all cases of IUP at 5 weeks' gestation. Three women had an EP with a live embryo, 5 had an embryo with no heart activity, 5 contained only a yolk sac, and 2 had an empty sac. A hematocele was seen in 3 women, and 1 had tubal thickening. The mean uterine and spiral arterial resistance index decreased with the gestational age of IUPs but remained constant during EPs. Peak blood velocity in the uterine arteries increased with the gestational age of IUPs, and the values were significantly higher than in EPs. A CL was seen in 88% of women with an IUP and in 100% of women with an EP. The resistance index was similar in CL associated with both types of pregnancy, and the values did not change with gestational age. CONCLUSION: These data show that: (1) blood flow impedance in the uterine and spiral arteries, and CL, is similar in IUPs and EPs and (2) peak flow velocity in the uterine arteries reflects a decreased blood supply to EPs. PMID- 1730334 TI - A long-acting repeatable form of bromocriptine as long-term treatment of prolactin-secreting macroadenomas: a multicenter study. AB - OBJECTIVE: To assess the efficacy and tolerability of monthly injections of the long-acting repeatable bromocriptine in patients with macroprolactinomas. DESIGN: Open and prospective trial. SETTING: This multicenter trial was carried out in seven university hospitals. PATIENTS: Forty-two patients with prolactin (PRL) secreting macroadenomas. INTERVENTIONS: Fifty to 200 mg of the drug were administered intragluteally every 28 days for 6 to 24 months. MAIN OUTCOME MEASURES: The efficacy was assessed by repetitive plasma PRL measurements, visual field determinations, and computed tomography scan examinations. RESULTS: After the first 50-mg injection, the mean percentage decrease of PRL levels was 71% from baseline on day 14; between days 1 and 28, PRL levels were suppressed to normal in nine cases, and a clear tumor shrinkage was documented in 21% of the patients. Normalization of PRL secretion was obtained in 62%, and a clear-cut reduction in tumor size in 50% of the patients after 6 months of treatment. CONCLUSIONS: The long-acting repeatable form of bromocriptine was a well tolerated and quickly effective treatment in most of these patients with macroprolactinomas. PMID- 1730335 TI - Hysteroscopic treatment of septate uterus with Neodymium-YAG laser. AB - OBJECTIVE: To determine the effectiveness of Neodymium-YAG (Nd-YAG) laser for hysteroscopic transection of the septate uterus to improve pregnancy outcome. DESIGN: Patients treated for recurrent pregnancy loss and/or infertility were evaluated for before versus after treatment pregnancy outcomes. SETTING: All patients were referred to a University Reproductive Endocrine and Infertility practice from 1986 through 1990. PATIENTS, PARTICIPANTS: Nineteen patients underwent hysteroscopic transection of uterine septa after exclusion of other factors that may cause recurrent fetal wastages and/or infertility. They were allowed to conceive 8 weeks after surgery after a postoperative hysterosalpingogram. Fourteen women attempted conception during a time span of 11 to 42 months; 3 patients declined to conceive, and 2 were lost to follow-up. INTERVENTIONS: Hysteroscopic transection of the uterine septum with a Nd-YAG laser was performed in all patients. The Nd-YAG laser delivered via a 600-microns bare fiber or an 800-microns sculpted fiber through operative hysteroscopy. MAIN OUTCOME MEASURES: To evaluate the success and complications of this new laser technique. RESULTS: (1) Thirteen patients conceived; 10 delivered a live infant at term; (2) 87% of the postoperative pregnancies were considered successful as compared with 11% preoperative; (3) complications included a small perforation of the uterus (no treatment needed) and development of uterine adhesions (1 case only). CONCLUSIONS: Hysteroscopic metroplasty with the Nd-YAG laser is a valuable alternative new technique for the treatment of uterine septum. PMID- 1730336 TI - Convenient numerical procedures for estimating cumulative pregnancy curves. AB - OBJECTIVE: To develop a simple estimation procedure that will always converge on a solution for a two-parameter model of cumulative pregnancy curves. DESIGN: Utilize raw data from a previous study in which estimation of the two-parameter model did not converge on a solution, and re-estimate the model with the new estimation method. PATIENTS, SETTING, TREATMENT: Infertile women with endometriosis treated as outpatients with laser laparoscopy. MAIN OUTCOME MEASURES: Pregnancy and ability of the new computer program to converge on a solution. RESULTS: The new estimation procedure converged on a solution. Over all stages of endometriosis, the cure rate from laser laparoscopy was 56%, and the monthly probability of pregnancy among those cured was 9.7%. CONCLUSIONS: The new estimation procedure, written for a personal computer, is easy to use and will virtually always converge on a solution for the two-parameter model of cumulative pregnancy after infertility treatment. PMID- 1730337 TI - Human growth hormone enhances progesterone production by human luteal cells in vitro: evidence of a synergistic effect with human chorionic gonadotropin. AB - OBJECTIVE: To examine the possible direct effect of human growth hormone (hGH) on basal and human chorionic gonadodotropin (hCG)-stimulated progesterone (P) production by cultured human luteal cells. DESIGN: Cultures of human luteal cells from early and midluteal phase. SETTING: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Catholic University, a public care center. PATIENTS, PARTICIPANTS: Twelve nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders such as leiomyomatosis. INTERVENTIONS: Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURE: Luteal cells were incubated with or without hCG and/or hGH at different concentrations. RESULTS: Human growth hormone neither at 250 nor at 500 ng/mL increased basal P production, whereas from 1,000 ng/mL P concentration in media was significantly increased (P less than 0.05). The concomitant treatment with uneffective doses of hCG (6 and 12 ng/mL) and hGH (250 and 500 ng/mL) enhanced P production similarly to that obtained with the highest doses of hGH (1,000 ng/mL or more) or hCG (25 to 50 ng/mL) alone. CONCLUSIONS: These results indicate a direct effect of hGH on the luteal steroidogenesis in vitro. PMID- 1730338 TI - Growth hormone secretory capacity and serum insulin-like growth factor I levels in primary infertile, anovulatory women with regular menses. AB - OBJECTIVE: To test the hypothesis that anovulation and infertility in women is associated with an impaired secretory capacity for growth hormone (GH). DESIGN: Comparison of the hormonal and metabolic response to two GH stimulation tests in a patient group and in a control group. SETTING: Outpatients and healthy volunteers studied at a clinical research unit of a university hospital. PATIENTS, PARTICIPANTS: Eight infertile, anovulatory women (luteal phase serum progesterone [P] less than 25 nmol/L) with regular cyclic bleeding. Eight age- and body mass index-matched healthy volunteers with luteal phase serum P levels greater than 25 nmol/L. INTERVENTIONS: After an overnight fast, each subject underwent a standardized GH stimulation test composed of sequential arginine infusion and heat exposure on days 5 to 8 of the menstrual cycle. MAIN OUTCOME MEASURES: Serum GH, insulin-like growth factor I (IGF-I), insulin and non esterified fatty acids (NEFA). RESULTS: Serum GH increased in both groups but was significantly lower in the study group (P less than 0.03). No difference was found in the circulating levels of IGF-I, insulin, and NEFA. CONCLUSIONS: Relative GH insufficiency seems to be present in these patients, but the clinical significance of this finding remains to be elucidated. PMID- 1730339 TI - Opening address: intermediate uveitis. PMID- 1730340 TI - Intermediate uveitis and sarcoidosis. PMID- 1730341 TI - Intermediate uveitis and multiple sclerosis: considerations and necessary consequences for treatment. PMID- 1730342 TI - Serological evidence of an association between Lyme borreliosis and intermediate uveitis. PMID- 1730343 TI - Intermediate uveitis and focal infection. PMID- 1730344 TI - Diagnostic vitrectomy in intermediate uveitis. PMID- 1730345 TI - Chorioretinal biopsy for diagnostic purposes in cases of intraocular inflammatory disease. PMID- 1730346 TI - Fluorescein angiography in intermediate uveitis. PMID- 1730347 TI - Cellular phenotype of vitreous cells in intermediate uveitis. PMID- 1730348 TI - Intermediate uveitis: do HLA antigens play a role? PMID- 1730349 TI - T cell activation within different intraocular compartments during experimental uveitis. PMID- 1730350 TI - Medical treatment of intermediate uveitis. PMID- 1730351 TI - Nonsteroid anti-inflammatory drugs in the treatment of intermediate uveitis. PMID- 1730352 TI - Corticosteroids in the treatment of intermediate uveitis. PMID- 1730353 TI - Cytotoxic drugs in intermediate uveitis. PMID- 1730354 TI - Ciclosporin (Sandimmun) therapy: experience in the treatment of pars planitis and present therapeutic guidelines. PMID- 1730355 TI - Surgical treatment of intermediate uveitis. PMID- 1730356 TI - Further observations on cryotherapy of the vitreous base in the management of peripheral uveitis. PMID- 1730357 TI - Pars plana vitrectomy in the treatment of chronic uveitis. PMID- 1730358 TI - Clinical picture of intermediate uveitis. PMID- 1730359 TI - Cataract extraction in intermediate uveitis. PMID- 1730360 TI - Cataract surgery and intraocular lens implantation in patients with intermediate uveitis. PMID- 1730361 TI - Cryotherapy in uveitis. PMID- 1730362 TI - Role of the vitreous in experimental uveitis. PMID- 1730363 TI - Vitrectomy in intermediate uveitis. PMID- 1730364 TI - Vitreous surgery in the management of peripheral uveitis. PMID- 1730365 TI - Laser photocoagulation of retinal neovascularization in intermediate uveitis. PMID- 1730366 TI - Immune modulation by ultraviolet light. PMID- 1730367 TI - Light as alternative treatment of intermediate uveitis and chronic iridocyclitis. PMID- 1730368 TI - Plasma exchange and immunoglobulins in the treatment of intermediate uveitis. PMID- 1730369 TI - Biomicroscopy in intermediate uveitis. PMID- 1730370 TI - Intermediate uveitis: history, terminology, definition pars planitis: systemic disease associations. PMID- 1730371 TI - Epidemiology of intermediate uveitis: a prospective study in Savoy. PMID- 1730372 TI - HLA in intermediate uveitis. PMID- 1730373 TI - Diseases masquerading intermediate uveitis. PMID- 1730374 TI - Intermediate uveitis: what is the significance of the pars plana exudate in 'pars planitis'? A summary. PMID- 1730375 TI - Morphology of the pars plana region. PMID- 1730376 TI - Pathology of intermediate uveitis. PMID- 1730377 TI - Immunology of intermediate uveitis. PMID- 1730378 TI - Role of anterior-chamber-associated immune deviation in the pathogenesis of uveitis. PMID- 1730379 TI - Intermediate uveitis: epidemiology, age and sex distribution. PMID- 1730380 TI - Antiretinal autoantibodies in intermediate uveitis. PMID- 1730381 TI - Association between intermediate uveitis and multiple sclerosis. PMID- 1730382 TI - G-actin pool and actin messenger RNA during development of the apical processes of the retinal pigment epithelial cells of the chick. AB - It has been suggested that during development an increase in the pool of G-actin may drive the elongation of actin-containing processes which occur in several types of epithelial cells. The apical processes of chick retinal pigment epithelial (RPE) cells elongate during the last 7 days of embryonic life (E15 E21) reaching lengths of 20 microns or more by hatching (E21). F-actin bundles form the cores of these processes. We followed the elongation by measuring F actin in the cells and cytoskeletons. In correlation with this, we studied by DNAse assay the levels of monomeric actin in supernatants of cell extracts from E13, before elongation starts, to E17, when elongation is well underway. Total F actin increased 1.9-fold over this time period and cytoskeletal actin increased 2.5-fold. In supernatants from extracts of E13 RPE the monomeric actin concentration was 51 +/- 0.5 micrograms/ml. From estimates of cell volume we calculated the cellular monomeric actin concentration at E13 as at least 510 micrograms/ml (13 microM). We compared this with monomeric actin levels in extracts from RPE at E15 and E17. Allowing for the estimated increase in cell volume, our data show little overall change in cellular monomeric actin concentration at these times. Changes in the level of actin mRNA were measured over the same time period. Normalized to equal RNA, we found a twofold increase in beta actin mRNA and a four- to fivefold increase in message for gamma actin at E17 as compared to E13. In summary, we show that (1) there is a substantial pool of monomeric actin in these epithelial cells before elongation starts; (2) process elongation is not associated with a significant change in the size of this pool; and (3) process elongation is associated with a significant increase in actin mRNA. PMID- 1730383 TI - Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development. AB - Epidermal growth factor receptors (EGF-Rs) are expressed at increasing levels on mouse preimplantation embryos. Immunofluorescence assays were used to show that unfertilized eggs and 2-cell embryos have a very low level of reactivity to antimouse EGF-R antibodies, but by the 4-cell stage and later the reactivity increases. The synthesis of EGF-R protein was verified at the blastocyst stage by immunoprecipitation of a 170-kDa metabolically labeled protein. The EGF-R protein is expressed on cell plasma membrane surfaces, but after compaction at the 8-cell stage concentrates on the apical cell surfaces. We also find a low level of expression of EGF-R protein on inner cell mass cells; thus, all cell lineages express receptors from the beginning of gestation. The receptor protein synthesized by the 8-cell embryo and later is probably translated from embryonic transcription, since reverse transcription-polymerase chain reaction indicates increasing levels of mRNA starting after the 4-cell stage. However, we also detected maternal mRNA in zygotes and 2-cell embryos. The pervasive nature of EGF R expression throughout development suggests important roles for these receptors which could include autocrine and paracrine stimulation. PMID- 1730384 TI - Increases in pericellular proteolysis at developing neuromuscular junctions in culture. AB - To determine whether localized changes in pericellular proteolysis contribute to synapse formation, we examined the degradative actions of developing Xenopus laevis nerve and muscle cells on films of extracellular matrix proteins adsorbed to the glass surface of a tissue culture chamber. Skeletal myocytes, growing neurites, and fibroblasts all removed fluorescent fibronectin and laminin from the culture substratum at regions of close cell-surface contact. In addition, however, motor neurites also displayed a particularly enhanced rate of gelatin elimination at developing neuromuscular junctions. It has already been shown (a) that there is a similar remodeling of organized muscle basal lamina proteoglycan accumulations along the path of nerve-muscle contact and (b) that this is the earliest detectable biochemical change specific to developing neuromuscular junctions. Our observations thus suggest that the establishment of motoneuron muscle contact leads to a further activation of pericellular proteinases along both the pre- and the postsynaptic surfaces of the developing junction. We therefore consider whether site-specific proteinase-activation cascades could contribute to the inductive signals that direct synaptic differentiation. PMID- 1730385 TI - Target-derived astroglia regulate axonal outgrowth in a region-specific manner. AB - The potential neuroanatomical specificity of astrocyte influence on neurite outgrowth was studied using an in vitro coculture system in which neurons from embryonic rat spinal cord or hippocampus were grown for 4 days in the presence of, but not in direct contact with, astrocytes derived either from the same region (homotopic coculture) or from different regions (heterotopic coculture) of the rat central nervous system. The results showed that axonal outgrowth was greatly enhanced in heterotopic cocultures in which spinal cord or hippocampal neurons were grown with astrocytes derived from their appropriate CNS target regions. This effect was remarkably specific, because the astroglia harvested from spinal or hippocampal target regions were not effective in promoting axon growth of nonafferent neuronal populations. Dendritic outgrowth was similar under all coculture conditions. These data suggest that diffusible signals, produced by astrocytes, can regulate neurite extension in vitro in a neuroanatomically specific manner and that axons are more sensitive than dendrites to the regional astrocyte environment. PMID- 1730386 TI - Identification of early neurogenic cells in the neural crest lineage. AB - Pluripotent neural crest cells are restricted progressively during development. The sequence of restrictions and the time(s) in early development at which such restrictions are imposed on crest-derived cells are largely unknown. We have used a human autoantibody (Anti-Hu) to characterize neurogenic populations of avian neural crest-derived cells. Anti-Hu binds specifically to neurons and neuroendocrine cells in older (greater than E4) quail embryos. Early in development, Anti-Hu also binds a subpopulation of neural crest-derived cells that lack neuronal morphology and do not express other neuronal traits. These cells may represent a putative neurogenic precursor subpopulation within the early crest cell lineage. To test this hypothesis, we have characterized Anti-Hu immunoreactivity within crest-derived populations known to have, or to lack, the ability to give rise to new neurons. We report that the presence of Anti-Hu+ nonneuronal cells is correlated with the neurogenic ability of a given cell population. Moreover, Anti-Hu+ nonneuronal cells are transient and appear to be replaced by Anti-Hu+ neuronal cells. We conclude that Anti-Hu is a very early indicator of neurogenesis among crest-derived cells and that Anti-Hu+ nonneuronal cells are either neurogenic precursors or immature neurons. PMID- 1730387 TI - Uterine stromal cell chondroitin sulfate proteoglycans bind to collagen type I and inhibit embryo outgrowth in vitro. AB - Chondroitin sulfate proteoglycans (CSPGs) are the major class of proteoglycans synthesized by mouse uterine stroma in vitro (Jacobs, A. L., and Carson, D. D. (1991). J. Biol. Chem. 266, 15,464-15,473). In the present study, stromal CSPGs were isolated and examined with regard to their ability to bind to specific extracellular matrix (ECM) components. Of a variety of ECM components tested, only collagen type I formed stable complexes with stromal CSPGs in both solid phase and solution binding assays. Proteolytic digestion of the CSPGs did not affect binding and suggested that the protein cores did not participate directly in binding. Furthermore, free chondroitin sulfate polysaccharides do not compete effectively in the binding assays. Therefore, interactions with multiple CS chains and/or the higher charge density afforded by intact CSPGs appear to be required for retention by collagen type I. Intact CSPGs were examined for their ability to modulate embryo attachment and outgrowth in vitro on fibronectin- or collagen type I-coated surfaces. In both cases, intact CSPGs, but not their constituent protein cores or polysaccharides, inhibited both the rate and the extent of outgrowth formation. In addition, embryo outgrowth on stromal ECM was enhanced by predigestion with chondroitinase. Addition of exogenous CSPG markedly retarded embryo outgrowth on stromal matrix. Collectively, these data indicate that stromal cell-derived CSPGs are retained by collagen type I in the stromal interstitial ECM where these molecules may attenuate trophoblast invasive behavior. PMID- 1730388 TI - Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum. AB - In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division. PMID- 1730389 TI - Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement. AB - Formation, accumulation, and storage of two components of the Oryzias latipes hatching enzyme, high and low choriolytic enzymes (HCE and LCE), were examined by immunocytochemical and immunoblotting methods. Both of the enzymes were found to be formed specifically in the hatching gland cells at the stages of lens formation to eye pigmentation and their accumulation proceeded markedly and concurrently up to Day 5.5 embryos (the stage just before hatching). The amount of HCE formed was more abundant than that of LCE. In the hatching gland cells, HCE and LCE were found to be packaged in the same secretory granules but in distinct arrangement; HCE is localized to the inside of granules whereas LCE is situated at the periphery of the same granules. Their segregated arrangement is compatible with their relative quantities formed per embryo. The results provide not only the cellular and developmental basis for a view that this hatching enzyme is an enzyme system composed of HCE and LCE but also a clue to the regulatory mechanism of concurrent syntheses of two different specific proteins in the same embryonic cell. PMID- 1730390 TI - Identification and characterization of alternatively spliced fibronectin mRNAs expressed in early Xenopus embryos. AB - Sequence analysis of cDNA clones encoding fibronectin (FN) from Xenopus laevis reveals extensive amino acid identities with other vertebrate FNs, including the presence of the Arg-Gly-Asp (RGD) cell attachment site in type III-10 and of a second, cell-binding site (EILDV) in the alternative spliced V region of the protein. These cDNAs have been used to study the expression of FN mRNAs during early development. Overall, levels of maternal FN mRNA remain constant until the mid- to late-gastrula stage when the accumulation of new FN transcripts is first apparent. RNase protection analyses reveal that the pattern of FN alternative splicing is similar to that reported for other species and does not change with the shift from maternal to zygotic mRNA expression. The cellular forms of the FN protein predominate in the early embryo with the EIIIA and EIIIB exons included in most mRNAs at this time. A comparison of V-region alternative splicing between embryonic and adult liver RNAs indicates a segment of 345 nucleotides that can be either completely excluded or included in mature FN transcripts but there is no evidence for additional V-region variants. Maternal mRNAs encoding alternatively spliced forms of FN can be specifically eliminated from Xenopus oocytes following the injection of antisense oligodeoxynucleotides into the cytoplasm, thereby making it possible to analyze the structure, composition, and function of FN mRNAs in early embryos. PMID- 1730391 TI - Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs. AB - Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo 3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves. PMID- 1730392 TI - Modulation of sea urchin actin mRNA prevalence during embryogenesis: nuclear synthesis and decay rate measurements of transcripts from five different genes. AB - The parameters determining the prevalence of the five actin gene transcripts that are differentially expressed during embryogenesis in the sea urchin Strongylocentrotus purpuratus were measured in vivo. These results and previous studies show that the developmental appearance of the cytoskeletal actin mRNA, CyI, CyIIa, CyIIb, and CyIIIa, and the muscle-specific actin message M, is transcriptionally regulated. The cytoskeletal actin genes are activated at the 64 cell stage or shortly thereafter. At this stage the specification of the early embryonic lineages has just completed. M gene transcription was detected only after muscle cells appear in the late embryo. The CyI, CyIIa, and CyIIb genes are transcribed at a moderate rate that does not vary significantly during development. In contrast, during late cleavage CyIIIa transcripts are produced at the maximum rate observed for structural genes in this embryo. In later stages, CyIIIa transcription is reduced at least 30-fold. The rate at which new actin transcripts enter the cytoplasm was also measured. The data show that essentially all primary actin gene transcripts are processed into mature messages. Actin message stability does not change during development. The mRNA half-life of the various messages was found to range from 4 hr to greater than 14 hr. PMID- 1730393 TI - Cellular mechanism for norepinephrine suppression of pineal photoreceptor-like cell differentiation in rat pineal cultures. AB - Although the rat pineal is an endocrine organ and has no photoreceptor activity, pineals from neonatal rats contain cells that can differentiate into rod-like cells with rhodopsin immunoreactivity (Rho-I), when cultured in vitro. Norepinephrine (NE) reduces the number of Rho-I cells in a dose-dependent manner and has a considerable effect even at 20 nM. When cultured in vitro, pineals removed up to Postnatal Day 4 differentiated into Rho-I cells to the same extent as did those removed at Day 1 (neonatal), but those removed at Day 5 showed a sharp reduction in the number of differentiated Rho-I cells. This suggests that either pineal cells in situ lose their potential to differentiate by Day 5 or the subpopulation of cells involved normally disappears in pineals older than Day 5. The effect of NE was examined in cultures of neonatal pineals by administering it for 1 or 2 days at different stages during a 9-day culture period. NE was most effective when present in the culture medium at an early culture phase and was not efficacious if present only later than Culture Day 7. This indicates that presumptive pineal photoreceptors may become sensitive to NE only for a limited period and that once they are exposed to NE within this period they are irreversibly affected, possibly to degenerate. These cells are similarly and severely affected by potassium ion concentrations as low as 15 mM, suggesting that NE may act at the adrenoreceptor to modify the membrane properties. Serotonin-immunoreactive cells, another cell type (endocrine) found in the cultures, appeared to be regulated by NE by a separate mechanism. NE suppresses process extension by serotonin cells in a reversible manner, and KCl does not have this effect. These findings further evidence that neurotransmitters may have essential roles, other than the transmission of signals, in modulating the developing nervous system. PMID- 1730394 TI - Localization of DER and the pattern of cell divisions in wild-type and Ellipse eye imaginal discs. AB - The compound eye of Drosophila develops from a uniform layer of epithelial cells in the eye imaginal disc. One intriguing aspect of eye development is the establishment of the correct number and spacing of the photoreceptor clusters which give rise to the mature ommatidia. Ellipse (Elp) has been implicated as playing a role in this process because the Elp dominant gain of function mutation dramatically reduces the number of photoreceptor clusters in the compound eye without affecting the morphology of individual clusters that are formed (Baker and Rubin, 1989). Since Elp represents an allele of the Drosophila EGF receptor (DER) locus, it encodes a protein which is structurally capable of mediating inductive cell-cell interactions. In an effort to better understand the role of the DER locus in ommatidial patterning, we compared the localization of DER protein in eye imaginal discs of wild-type and Elp larvae. The distribution of this receptor is consistent with the notion of its mediating interactions between cells at the initial stages of photoreceptor precluster positioning and differentiation. However, the basis of the Elp gain of function mutation is not ectopic or increased expression of the DER protein. Rather, expression of the Elp form of the EGF receptor homolog in the normal localization leads to changes in the proliferative pattern of cells dividing posterior to the morphogenetic furrow. PMID- 1730395 TI - HIV counseling and testing of psychiatric patients: time to reexamine policy and practices. PMID- 1730396 TI - Reducing unnecessary psychiatric consultations for informed consent by liaison with administration. AB - The frequency of a psychiatric consultation being requested to assess a patients' capacity to give informed consent varies among institutions, with most recent surveys reporting a frequency of between 3% and 8% of all consultations. At Montefiore Medical Center, a hospital policy was interpreted as mandating such consultations for all patients with possible or even definite lack of decisional capacity. From 1987 to 1988, 55% of all psychiatric consultations in the institution were for consent. Only 9% of the consent patients seen had an Axis I diagnosis other than organic mental syndrome (OMS). Because many of these consultations were believed to be unnecessary, with patient clearly able or unable to give consent, the consultation service worked first with administration to modify the guidelines, and then educated the medical and nursing staff as to when consultation was indicated. With this program, the number of consent consultations fell from 958 in 1988 to 177 in 1990, representing a major saving of staff time and third-party billings. In this era of cost containment and outside review of professional practices, psychiatrists must take responsibility for identifying areas where patient services and billings for them are not justified by clinical indications. PMID- 1730397 TI - Dyspnea, anxiety, and depression in chronic respiratory impairment. AB - In order to examine the relationship of dyspnea to anxiety and depression, the authors rated dyspnea using several methods in 50 patients with chronic respiratory impairment. Anxiety and depression were measured by the Symptom Checklist-90 and the Symptom Questionnaire. Results varied with the method of assessing dyspnea. Physician-rated dyspnea was significantly associated with patients' self-ratings of breathlessness as well as with pulmonary function tests, but not with any of the self-rating scales of emotions. Self-rated breathlessness was significantly associated with self-rated depression. In multiple regression analyses, depression was predictive of breathlessness. When the sample was limited to patients with chronic obstructive pulmonary disease, the results remained the same. The patients were significantly more depressed and anxious than matched family practice patients. In the study of the complex relationship of dyspnea to physical and emotional factors, it is desirable to use more than one measure of dyspnea because the results depend in part on the method of assessment. PMID- 1730398 TI - Psychiatrists and the General Hospital Ethics Committee. AB - General Hospital Ethics Committees (GHECs) have emerged as institutional forums for addressing bioethical dilemmas. Hospital psychiatrists have important roles to play on these committees. Their skills in group process assessment, mental status examination, and character assessment have diverse applications. Psychiatrists can facilitate communication, both on the committee and as GHEC based clinical ethics consultants. Ethics committees must be concerned with how they arrive at ethical decisions, guarding against political influence or individual monopolization. Psychiatrists can assist these efforts as organizational consultants to GHECs. The perception of psychiatrists as reflective, tolerant of ambiguity, humanizing, and approachable about moral aspects of health care suggests they would make excellent committee leaders. Hospital psychiatrists also have important committee roles to play as ethics educators and policy-makers. More demographic research is needed to investigate psychiatrists' participation on GHECs. Studies of how they are perceived by their ethics committee colleagues may reveal new roles and potential pitfalls for GHEC psychiatrists. PMID- 1730399 TI - Assessing HIV risk in the general hospital psychiatric clinic. AB - Of 101 new admissions to an urban outpatient psychiatric clinic, 17 (16.8%) were known to be at high risk and 11 (10.9%) at suspected high risk for developing or transmitting HIV infection, but apparently few were appropriately counseled. This suggests a need for implementing specific policies and procedures for HIV assessment and counseling. PMID- 1730400 TI - Pharmacotherapy of depression in the medically ill: directions for future research. AB - A common problem facing the psychiatric consultant and the medical practitioner is evaluating depression in patients with concurrent medical illnesses. Depression is difficult to recognize in the medically ill, often presenting with "masked" symptoms and organized into unique syndromes. A primary concern of the clinician is identifying those patients who are likely to benefit from an antidepressant trial. Although antidepressants have been shown to sometimes be of benefit in medical populations, the symptoms predicting antidepressant response remain poorly defined. Important directions for future research include 1) evaluating the safety and efficacy of newer antidepressants in the medically ill and 2) identifying those depressive syndromes that may be responsive to pharmacotherapy. PMID- 1730401 TI - Long-term psychosexual adjustment of acute leukemia survivors: impact of marrow transplantation versus conventional chemotherapy. AB - Psychosexual sequelae associated with surviving acute leukemia treated with conventional chemotherapy or with chemotherapy followed by bone marrow transplantation (BMT) were investigated in 70 patients who were off treatment for at least 1 year. Assessment of psychosexual function included frequency of sexual activity, satisfaction, body image, gender role identity, and adjustment in sexual relations. No differences between BMT and conventional chemotherapy survivors were found on any of these measures, despite the high probability of gonadal impairment with BMT. Compared with physically healthy norms, women survivors generally reported decreased sexual frequency and satisfaction, whereas both men and women survivors reported poorer body image. Longer time since completing cancer treatment predicted greater frequency of sexual activity in women but poorer body image for both men and women. Those survivors who reported decreased sexual frequency, satisfaction, and poorer body image reported greater psychological distress and decreased energy. Results indicate that psychosexual sequelae in survivors of leukemia occur frequently and warrant intensive investigation, particularly to address the need for an intervention in those most distressed. PMID- 1730402 TI - Electroconvulsive therapy and the chronic use of pseudocholinesterase-inhibitor (echothiophate iodide) eye drops for glaucoma. A case report. AB - A case is presented in which a patient who required treatment with electroconvulsive therapy had a history of being treated with pseudocholinesterase-inhibitor eye drops (echothiophate iodide) for glaucoma. As treatment with this antiglaucoma agent contraindicated the use of succinylcholine for a minimum of 10-14 days, the short-acting nondepolarizing agent atracurium was employed instead. The anesthetic management of this patient is described as a guide for clinicians facing similar clinical situations. PMID- 1730403 TI - Physical consequences of depression in the stroke patient. AB - The past literature suggests the hypothesis that depression is associated with decreased physical functional ability in stroke patients. On a medical rehabilitation ward, 21 stroke patients were evaluated for depression by psychiatric interview and self-report, and were also rated on the Barthel's Functional Index (BFI). The hypothesis was supported: Patients scoring 17 or higher on the Beck Depression Inventory (BDI) (N = 7) had lower initial scores on the BFI than patients with lower BDI scores. There was a trend for these seven depressed patients to improve more slowly as ascertained by the BFI. Depression was suggested to lower functional ability by increasing fatigue, hopelessness, and decreasing motivation. PMID- 1730404 TI - Funding consultation-liaison psychiatry via Medicare screening. AB - Despite offering many benefits to patients, the hospital, and the hospital staff, an academic psychiatric consultation service is difficult to fund. By screening Medicare patients for psychiatric complications and comorbid conditions, the consultation-liaison (C-L) service can generate incremental revenue for the hospital by moving patients from lower-paying to higher-paying Diagnostic Related Groups (DRGs). The C-L service chief can negotiate with the hospital to obtain a portion of these incremental funds to support the C-L service. Concurrent psychiatric disorders that move patients to more complex DRGs include substance abuse, substance dependence, drug-induced delirium, drug-induced organic affective syndrome, and psychotic depression. This paper presents a method of calculating the incremental hospital revenue generated by such screening along with the results of applying the method to selected DRGs at a west coast teaching hospital. Implementing this program at that hospital in fiscal year 1989 would have resulted in screening 142 Medicare patients (2.2% of Medicare admissions), discovering an estimated 25 patients with comorbid psychiatric conditions, and generating $51,800 in incremental hospital revenue. In creating a screening program, a C-L service chief must be prepared to negotiate issues with the medical records department, referring physicians, and the hospital administration. PMID- 1730405 TI - Of fin and fur: mutational analysis of vertebrate embryonic development. PMID- 1730406 TI - In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. AB - In vitro selection methods provide rapid and extremely powerful tools for elucidating interactions within and between macromolecules. Here, we describe the development of an in vitro selection procedure that permits the rapid isolation and evaluation of functional hairpin ribozymes from a complex pool of sequence variants containing an extremely low frequency of catalytically proficient molecules. We have used this method to analyze the sequence requirements of two regions of the ribozyme-substrate complex: a 7-nucleotide internal loop within the ribozyme that is essential for catalytic function and substrate sequences surrounding the cleavage-ligation site. Results indicate that only 3 of the 16,384 internal loop variants examined have high cleavage and ligation activity and that the ribozyme has a strong requirement for guanosine immediately 3' to the cleavage-ligation site. PMID- 1730407 TI - The chicken limb deformity gene encodes nuclear proteins expressed in specific cell types during morphogenesis. AB - The chicken limb deformity (ld) mutation affects morphogenesis of both limbs and kidneys and is one of few murine mutations for which the affected gene has been isolated. Analysis of the chicken homolog reveals evolutionary conservation of large parts of the encoded ld gene products. This is the first study of these proteins, their intracellular localization, and their temporal and spatial distribution during embryogenesis. A major 180-kD protein is expressed in chicken embryos and certain adult tissues. The proteins are localized in the nuclei of different embryonic cell types in a characteristic punctate pattern. In the developing chicken limb bud, they are expressed in the newly differentiated apical ectodermal ridge and the mesenchymal compartment, where an unequal distribution along the anteroposterior and, subsequently, the dorsoventral axes, is observed. During kidney morphogenesis, expression is initially restricted to the epithelial compartment of the pronephros and mesonephros. These results correlate well with the previous analysis of the murine ld phenotype and imply determinative roles for ld gene products during the morphogenesis of limbs and kidneys. Unexpected expression in the notochord, floor plate, and ventral horns suggests an involvement of the ld gene products in establishment of the dorsoventral polarity of the neural tube. PMID- 1730408 TI - Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site. AB - Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure. PMID- 1730409 TI - Analysis of G alpha 4, a G-protein subunit required for multicellular development in Dictyostelium. AB - The Dictyostelium G alpha 4 gene encodes a G-protein alpha subunit that is primarily expressed during the multicellular stages of development. g alpha 4 null mutants, created by gene disruption, show aberrant morphological differentiation, reduced levels of prespore gene expression, and a loss of the ability to produce spores. These developmental phenotypes can be rescued by complementation with the wild-type gene. Cells that overexpress the G alpha 4 gene (G alpha 4HC) also show reduced spore production but display an aberrant morphological phenotype distinct from that of g alpha 4 cells. The g alpha 4 phenotype can be partially rescued by the presence of wild-type or G alpha 4HC cells in chimeric organisms, suggesting that G alpha 4-expressing cells produce an intercellular signal that is essential for multicellular development. PMID- 1730410 TI - Interleukin-7 induces N-myc and c-myc expression in normal precursor B lymphocytes. AB - Expression of both N-myc and c-myc is induced rapidly and dramatically in normal pre-B cells after stimulation with interleukin-7 (IL-7), a pre-B cell-specific growth factor. These IL-7-induced increases in N-myc and c-myc expression are mediated at the transcriptional level; for N-myc, a major portion of the induction results from release of an attenuation block between exons 1 and 2. Although c-myc expression has been shown to be regulated by many factors, these studies provide the first demonstration of N-myc regulation by a growth factor. Furthermore, mitogen-induced proliferation of more mature B-lineage cells is accompanied by induction of c-myc expression in the complete absence of N-myc expression. Thus, N-myc expression in normal B-lineage cells, as in transformed cells, is limited to cells that represent the precursor stages of this differentiation pathway. Together, these findings imply a specific function for N myc in the early stages of B-cell development. PMID- 1730411 TI - Myc and Max associate in vivo. AB - Max is a helix-loop-helix zipper protein that associates in vitro with Myc family proteins to form a sequence-specific DNA-binding complex. We show here, by means of a coimmunoprecipitation assay with anti-Myc and anti-Max antibodies, that Myc and Max are associated in vivo and essentially all of the newly synthesized Myc can be detected in a complex with Max. This complex possesses specific DNA binding activity for CACGTG-containing oligonucleotides. Although Max itself is a highly stable protein, Myc is rapidly degraded during or after its association with Max. In vivo Max is shown to be a nuclear protein phosphorylated by casein kinase II, and alternatively spliced forms of Max are expressed in cells. Furthermore, the levels of Max expression are equivalent in quiescent, mitogen stimulated, and cycling cells. We conclude that the highly regulated rate of Myc biosynthesis is likely to be a limiting step in the formation of Myc:Max complexes. PMID- 1730412 TI - Max: functional domains and interaction with c-Myc. AB - The product of the c-myc proto-oncogene is a DNA-binding protein, the deregulated expression of which is associated with a variety of malignant neoplasms. The cDNA for the max gene was recently cloned as a result of the ability of its protein product to interact with the c-Myc protein. We studied bacterially produced Max, c-Myc, and a series of truncated c-Myc proteins. Full-length c-Myc alone cannot bind DNA. However, a truncated c-Myc protein comprising the basic, helix-loop helix, and leucine zipper regions can bind specifically to DNA bearing the sequence GGGCAC(G/A)TGCCC. Max protein, either alone or in a heteromeric complex with full-length c-Myc, binds to the same core sequence. Using a novel combination of chemical and photo-cross-linking analysis, we demonstrate that either Max or a c-Myc/Max heteromeric complex binds to DNA virtually exclusively in a dimeric structure. Using fusion proteins in cultured cells, we establish a number of functional characteristics of Max. First, we show that Max can interact with c-Myc intracellularly in a manner dependent on the integrity of the helix loop-helix and leucine zipper motifs. Second, a nuclear localization domain that contains the sequence PQSRKKLR is mapped to the carboxy-terminal region of Max. Third, Max lacks a transcriptional activation domain that is functional in Chinese hamster ovary cells when fused to a heterologous DNA-binding domain. These data suggest that Max may serve as a cofactor for c-Myc in transcriptional activation or, by itself, as a transcriptional repressor. PMID- 1730413 TI - Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase. AB - Two independent pathways of transcriptional regulation that show functional homology have been identified in yeast. It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated. We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle regulated transcription and nuclear localization similar to those seen previously for SWI5. Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation. SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not. In contrast, ACE2 is a transcriptional activator of the CTS1 gene (which encodes chitinase), whereas SWI5 is not. An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTS1 both are cell cycle regulated in the same way, and HO and CTS1 both require the SWI4 and SWI6 transcriptional activators. Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate. Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain. PMID- 1730414 TI - Second-look versus second-nature. PMID- 1730415 TI - Analysis of ascites from patients with ovarian carcinoma by cell flow cytometry. AB - Cell flow cytometry offers the opportunity to analyze cytopathological samples with regards to DNA content and proliferative activity. To investigate whether this modality can quantitate certain aspects of ovarian carcinoma by analyzing ascites, 43 samples from patients with advanced papillary serous adenocarcinoma of the ovary were studied. In 28 samples (65%) ploidy and the percentage of cells in S phase (%S phase) could be analyzed. Fifteen samples could not be analyzed because of overlapping cell populations distorting distinct cell cycle phases. Of the 28 samples studied, 8 (29%) were diploid and 20 (71%) were aneuploid. The DNA in aneuploid samples ranged from 1.23 to 2.65. The %S phase for aneuploid was greater than that for diploid samples. Patients with diploid samples survived longer. Cytometric analysis of cells from ascites in 4 patients in whom disease progressed after they received chemotherapy showed that the percentage of cells in S phase increased. Cells from ascites established in vitro showed that ploidy and proliferative activity changed as cells were passed in culture. In conclusion, the analysis of ascites by cell flow cytometry may be a prognosticator in patients with advanced ovarian carcinoma. In addition, conclusions extrapolated from in vitro data to the in vivo situation should be done cautiously since late-passaged cells may not always be representative of the initial tumor sample. PMID- 1730416 TI - Persistent gestational trophoblastic disease following evacuation of a tetraploid partial hydatidiform mole. AB - A 31-year-old woman at 11 weeks gestation with ultrasonographic demonstration of partial mole had markedly elevated serum bHCG levels (458,000 mIU/ml). The patient underwent a vacuum curettage with pathological confirmation of the diagnosis, and cytogenetic analysis revealed a tetraploid karyotype (92,XXXX). The patient developed persistent gestational trophoblastic disease and was successfully treated with two courses of actinomycin D. Persistent trophoblastic disease after evacuation of a tetraploid partial mole was not reported previously. PMID- 1730417 TI - Chemotherapy in perineal leiomyosarcoma. AB - A case report of a woman with perineal leiomyosarcoma who was cured by combination chemotherapy is presented, and the literature on chemotherapy of soft tissue is reviewed. PMID- 1730418 TI - Primary vaginal tuberculosis after vaginal carcinoma. AB - A case of primary vaginal tuberculosis is reported. A 62-year-old woman presented with a slightly eroded, granular vaginal lesion 10 months after radiation therapy for Stage II vaginal squamous cell carcinoma. She had previously undergone a total abdominal hysterectomy and left salpingo-oophorectomy at age 37 for abnormal uterine bleeding. This presentation of genital tract tuberculosis is unusual because of the location of the primary lesion, the age at presentation, the occurrence after radiation therapy, and the possible means of infection. We emphasize the need to maintain a high index of suspicion and to biopsy any suspicious vaginal lesions. PMID- 1730419 TI - Retrograde seeding of endometrial carcinoma during hysteroscopy. AB - Fractional dilatation and curettage remain the most reliable methods in the diagnosis of endometrial carcinoma in the symptomatic patient. In the past few years hysteroscopy has become a helpful method, improving the specificity of the diagnosis of this pathology. We report a case of clinical stage IA grade 2 endometrial adenocarcinoma diagnosed by hysteroscopy and endometrial biopsy. Surgical staging revealed positive cytology. We suggest that irrigation of the endometrial cavity during the hysteroscopic procedure with saline may disseminate the disease to the abdominal cavity and may change the prognosis and the course of treatment. PMID- 1730420 TI - DNA ploidy of ovarian dysgerminomas: correlation with clinical outcome. AB - Abnormal nuclear DNA content, as determined by flow cytometry, when combined with conventional prognostic variables such as tumor grade or stage at diagnosis, appears to identify patients who are at increased risk for recurrence of disease. The DNA content of ovarian dysgerminoma, a tumor that is homologous to testicular seminoma and is found in young women of childbearing age, was studied to determine if there is a correlation between DNA content and outcome. Such information would be useful in selecting treatment regimens and making possible the preservation of childbearing potential in women who are likely to have a good outcome. The specimens from 23 cases of ovarian dysgerminoma seen at our institution between 1950 and 1985 were analyzed by DNA flow cytometry. Five of the tumors were diploid (21%) and nineteen were nondiploid (79%). Patient outcome was not predicted any better by nuclear DNA content than by conventional prognostic variables. PMID- 1730421 TI - Clinical stage I adenocarcinoma of the endometrium--analysis of recurrences and the potential benefit of staging lymphadenectomy. AB - Two hundred forty-eight consecutive patients with clinical Stage I adenocarcinoma of the endometrium were seen between 8/77 and 8/88. Twenty-one were medically not operable and eleven others had papillary serous tumors. The remaining 216 were managed by a consistent operative protocol except that routine preoperative cesium was discontinued after 12/83. Patients received postoperative pelvic radiation on the basis of the depth of invasion, extrauterine pelvic disease, and/or cervix involvement. No patient underwent a pelvic lymphadenectomy. Only palpably suspicious nodes were removed. Twenty-one of these two hundred sixteen patients developed a recurrence. These 21 cases are analyzed for the probability of a staging lymphadenectomy having prevented their recurrence. Median follow-up of all 216 patients is 61 months with a mean time to recurrence of 26.5 months. No patient was lost to follow-up. Patients who recurred are analyzed by grade, depth of invasion, surgical stage, time to recurrence, site of recurrence, survival, protocol breaks, and frozen section discrepancies. No patient recurred on the pelvic side-wall. All patients found to have positive para-aortic nodes have died. No patient who received vaginal and/or pelvic radiation recurred in the pelvis. We conclude that staging lymphadenectomy would not have improved the outcome for these patients. PMID- 1730422 TI - Long-term survival and sequelae after surgical management of invasive cervical carcinoma diagnosed at the time of simple hysterectomy. AB - From 1956 to 1988, 27 women (median age, 60 years) found to have occult invasive carcinoma of the cervix at total hysterectomy underwent radical reoperation consisting of radical parametriectomy, upper vaginectomy, and pelvic lymphadenectomy. Residual disease was present at reexploration in 4 (15%) of the 27 patients: in the pelvic lymph nodes in 2, in the parametrium in 1, and in the vagina and a para-aortic node in 1. All patients were followed a minimum of 18 months; there were no deaths within 3 months of operation. However, 2 (7%) of the 27 patients developed ureterovaginal fistulas. Recurrent disease was observed in 6 (22%) of the patients: 2 had successful salvage procedures, and 4 died of disease, all within 4 years of reoperation. Recurrence correlated with the presence of residual disease at reoperation and with nonsquamous histologic findings. At a median follow-up of 8.4 years, 23 of the 27 patients were alive and disease-free. The 5-year absolute survival estimate (Kaplan-Meier) was 82%. Radical reoperation can be performed safely in selected patients who have early stage invasive carcinoma of the cervix at the time of total hysterectomy with the expectation of an acceptable rate of long-term disease-free survival. PMID- 1730423 TI - Pathologic evaluation of gynecologic specimens obtained with the cavitron ultrasonic surgical aspirator (CUSA). AB - Eighty consecutive gynecologic specimens obtained with the Cavitron Ultrasonic Surgical Aspirator (CUSA) were evaluated pathologically. Fifty specimens were obtained during intraabdominal tumor debulking and thirty resulted from ablation of lower genital tract lesions. In 98% of intraabdominal specimens and in 93% of patients with a history of lower genital tract lesions, the CUSA material permitted an accurate diagnosis. Although artifacts related to cellular thermal injury were ubiquitous, nearly all cases were interpretable with a combination of cytologic (smear, Cytospin, Millipore filter) and histologic (cell block) preparations. Squamous intraepithelial lesions (SILs) of the lower genital tract were better preserved in cell block preparations, whereas intraabdominal adenocarcinomas were readily diagnosed by both cytologic and histologic techniques. Accurate grading of SILs and exclusion of invasion were difficult in some cell blocks due to the fragmented and superficial nature of the samples. Cytologic preparations of lower genital tract lesions often consisted of thick uninterpretable fragments and degenerated single cells that contributed little to the evaluation of SILs. We conclude that examination of CUSA specimens confirmed the surgical removal of pathologic tissue in 96% of cases, but an exact diagnosis of SILs was not possible in all cases. PMID- 1730424 TI - Infectious complications after gastrointestinal surgery in patients with ovarian carcinoma and malignant ascites. AB - One hundred four patients with ovarian cancer underwent intestinal reconstruction as part of a cytoreductive effort or for relief of intestinal obstruction from July 1980 to June 1990. Twenty-four percent of patients were obstructed preoperatively, while the remaining seventy-six percent had bowel resections performed in concert with a debulking procedure. The overall infectious complication rate was 14.4%. No statistical association was found between the presence of ascites at the time of laparotomy and infectious morbidity (P = 0.58). The use of a preoperative mechanical bowel preparation was associated with a significant reduction in infectious morbidity (P = 0.01). Additionally, patients considered in adequate nutritional condition experienced significantly less infectious complications than those patients in poor nutritional condition (P = 0.03). Intestinal procedures involving the large bowel were marginally associated with increased infectious complications (P = 0.13). Neither preoperative radiotherapy, the presence of preoperative obstruction, disease presence, extent of debulking, number of intestinal procedures, or hand versus stapled anastomosis was found to be significantly associated with infectious complications. It is concluded that the presence of ascites does not increase the infectious complication rate in ovarian cancer patients who undergo small or large bowel reconstructive procedures. Additionally, patients with preoperative bowel obstruction or previous abdominal radiation therapy were not found to experience a significant increase in the infectious complication rate in the current series. PMID- 1730425 TI - Results of the combination of external-beam and high-dose-rate intracavitary irradiation for patients with cervical carcinoma. AB - We retrospectively analyzed 220 patients with squamous cell carcinoma of the uterine cervix treated by the combination of external-beam and high-dose-rate intracavitary brachytherapy between 1978 and 1986. Five-year survivals for Stage Ib (n = 15), IIa (n = 18), IIb (n = 107), IIIb (n = 50), and IVa (n = 18) were 72.3, 88.9, 68.9, 64.0, and 16.7%, respectively. The incidences of recurrence outside the pelvis in Stage IIb and IIIb patients (15.0 and 24.0%) were higher than those of local recurrences (10.3 and 12.0%). Eighteen patients (8.2% of the total) had serious intestinal complications. These serious complications were closely correlated with the external-beam dosages. External-beam therapy was performed at 1.8-2.0 Gy/day, and high-dose-rate brachytherapy using a remotely controlled afterloading system (RALS) was performed once a week with 6.0-7.5 Gy at points A/fraction. Treatment dosages to the whole pelvis (WP), with central shieldings (CS), and with RALS according to FIGO stage were as follows: Stages Ib, IIa, 30-40 Gy (WP) plus 24-30 Gy (RALS); Stages IIb, IIIb, 40 Gy (WP) plus 10 20 Gy (CS) plus 24-30 Gy (RALS); Stage IVa, 40-50 Gy (WP) plus 10-20 Gy (CS) plus 20-30 Gy (RALS). From these data, the combination of external-beam and high-dose rate intracavitary irradiation is an effective therapy for cervical cancer. It is also suggested that an additional combined chemotherapy is necessary to control metastic lesions outside the pelvis for Stages IIb and IIIb. PMID- 1730426 TI - Para-aortic node sampling in small (3-cm or less) stage IB invasive cervical cancer. AB - Only 2 of 125 patients with FIGO stage IB invasive squamous or adenocarcinoma of the cervix 3 cm or less in diameter who underwent exploration for radical hysterectomy, bilateral pelvic lymphadenectomy, and para-aortic node sampling had metastases to the para-aortic nodes. No patient had gross para-aortic nodal involvement, and both patients with microscopic para-aortic nodal metastases had grossly positive pelvic nodal involvement. Para-aortic node sampling in patients with small stage IB cervical cancers undergoing radical hysterectomy may be restricted to patients with suspicious pelvic or para-aortic nodes. PMID- 1730427 TI - Stage II carcinoma of the ovary: an analysis of survival after comprehensive surgical staging and adjuvant therapy. AB - Ninety-three women with FIGO stage II epithelial ovarian carcinoma underwent comprehensive surgical staging and were randomized prospectively to therapy consisting of either intraperitoneal radioactive phosphorus or oral melphalan. No patient had gross residual disease at the time of randomization. Ten of the forty five women treated with melphalan experienced severe bone marrow depression at some time during therapy and two women expired from leukemia. Four of the forty eight women treated with intraperitoneal phosphorus required surgical reexploration for intestinal obstruction or bowel injury. Twenty-one women died of their disease. Survival was not statistically different between the two treatment arms. The 5-year actuarial survival was 78%. PMID- 1730428 TI - SWOG 8825: melphalan GM-CSF: a phase I study. AB - The use of intravenous melphalan at higher doses is limited by severe myelosuppression. It was postulated that GM-CSF would permit the use of higher dose melphalan with only moderate myelosuppression easily manageable in an outpatient setting. Therefore, a phase I study of intravenous melphalan utilizing GM-CSF (recombinant granulocyte-macrophage colony-stimulating factor) support was initiated. Intravenous melphalan at doses of 15-45 mg/m2 was administered every 28 days. GM-CSF was utilized at doses of 10-20 micrograms/kg/day subcutaneously Days 2-21 on a 28-day cycle. Twenty-five patients received 53 courses of therapy. The dose-limiting toxicities were severe or life-threatening granulocytopenia and thrombocytopenia. Utilizing 20 micrograms/kg/day GM-CSF, the maximum tolerated dose (MTD) of melphalan is 30 mg/m2 and, with 10 mg/kg/day GM-CSF, the maximum tolerated melphalan dose is only 20 mg/m2. One patient with ovarian cancer achieved a partial response. Because the reported MTD of intravenous melphalan without GM-CSF is 30 mg/m2, GM-CSF has not allowed sufficient escalation of the intravenous melphalan dose for routine outpatient use. PMID- 1730429 TI - Clinical presentation and management of stage I cervical adenocarcinoma: a 25 year experience. AB - In this study, we review the clinical presentation, treatment, and prognosis of 89 patients with stage I cervical adenocarcinoma treated at Strong Memorial Hospital over the past 25 years. In the past decade, the mean age of patients with stage I cervical adenocarcinoma was 44 years, in contrast to a mean of 58 years in the prior interval (P less than 0.001). Prior to 1980 only 4% of patients were of childbearing age, whereas in the past decade 27% were under 35 years old (P = 0.02). The difference in age at presentation cannot be explained by earlier detection, as the fraction of stage I patients, the mean tumor size, and the percentage of clinically occult tumors have not changed. There were no ovarian metastases in 41 patients who underwent oophorectomy. Adenosquamous tumours did not differ in prognosis from pure adenocarcinoma. Grade and lymph node status were significant predictors of outcome. Treatment results have not improved over the past 25 years, and combined therapy with radiation and surgery offered no advantage over radiation alone. Because this tumor is more frequently seen in younger patients, the management of occult adenocarcinoma with early stromal invasion has become problematic. Ovarian conservation has been questioned, and the lack of generally accepted criteria for microinvasive adenocarcinoma has led to radical therapy in patients who might have been adequately treated with local excision. Further study is necessary to guide our recommendations regarding preservation of ovarian function or even childbearing potential in young women. PMID- 1730430 TI - High-dose platinum chemotherapy in advanced ovarian cancer: a phase II study. AB - From September 1986 to November 1988, 38 patients with previously untreated ovarian cancer, FIGO stages III and IV, were allocated to a study testing the efficacy of high-dose platinum delivered as combined carboplatin and cisplatin. The regimen was as follows: Day 1, 250-350 mg/m2 carboplatin and 500 mg/m2 cyclophosphamide, Day 15, 100 mg/m2 cisplatin. Overall clinical response was 60%, and complete pathologic response, verified at second-look laparotomy, was 24%. Median survival was 17 months and 3-year survival 27%. The results are similar to those obtained by a standard dose of cisplatin, cyclophosphamide, and doxorubicin. Myelotoxicity was dose limiting. After receiving four cycles, 44% of patients had grade 3 or 4 leucopenia and 65% had grade 3 or 4 thrombopenia. Despite this, dose intensity of combined cisplatin and carboplatin was between 30 and 45 mg/m2/week, expressed as cisplatin by assuming that 1 mg cisplatin equals 4 mg of carboplatin. A correlation between dose intensity and response was not found. In conclusion the study showed that the regimen is feasible, but very myelotoxic. The results were not superior to those obtained by combination chemotherapy using standard doses of cisplatin. PMID- 1730431 TI - Nongenital cancers metastatic to the ovary. AB - We review our experience with 82 patients with nongenital cancers metastatic to the ovary. All patients were referred for evaluation of an ovarian mass. The patients had primary carcinoma of the breast (n = 28), colon (n = 23), stomach (n = 22), pancreas (n = 7), or gallbladder (n = 2). The overall actuarial 5-year survival rate was 10%. Five-year survival in patients with metastatic colon cancer was significantly higher (23%) than that in patients with metastatic cancer of the breast, stomach, gallbladder, or pancreas, all of whom died within 58 months (P less than 0.05). Patients with unilateral metastatic ovarian involvement had a 5-year survival significantly better than that of those with bilateral involvement (28% vs 5%; p = 0.003). Five-year survival in patients with disease limited to the pelvis was significantly higher than that in those with abdominal spread (22% vs 6%; P less than 0.04). The 5-year survival of patients with residual disease less than 2 cm or greater than 2 cm in diameter was 18% or 4%, respectively (P = 0.002). This pattern applied mainly to differences in patients with primary cancer of the breast or colon (P less than 0.008). These data suggest that an aggressive surgical effort seems to be indicated in colon cancer metastatic to the ovary, as some of these patients may survive 5 years. PMID- 1730432 TI - Lymphomas of the cervix and upper vagina: a report of five cases and a review of the literature. AB - Five cases of non-Hodgkin's lymphoma of the cervix or upper vagina presenting over the last 20 years are described. The international literature has been reviewed for similar cases and a further 72 found. In 37 of these cases the pathology had been described according to one of the modern lymphoma classifications and details of clinical presentation, staging, treatment, and outcome were adequately described. The management and outcome of these patients have been critically reviewed and recommendations for the management of patients presenting with this disease have been made. PMID- 1730433 TI - Carcinoma in episiotomy scars. AB - The finding of primary or metastatic carcinoma in an episiotomy scar is a rare event; we report three cases. The first patient presented with an abnormal cervical smear and was found to have a primary squamous cell carcinoma of the vulva in an old, healed episiotomy scar. A second patient, diagnosed as having cervical carcinoma 6 months postpartum, was found to have a metastatic deposit in the episiotomy scar during the staging of her disease. The third patient developed adenocarcinoma metastatic from an endocervical primary in an episiotomy scar that presented as a small nodule at the introitus. These cases exemplify the need for careful inspection and biopsy of any nodular lesions in episiotomy scars as part of the initial assessment and follow-up of patients with premalignant or malignant lesions of the lower genital tract. PMID- 1730434 TI - Diminished phospholipase C activation by dopamine in spontaneously hypertensive rats. AB - It is reported that a defect in dopamine-1 (DA-1) receptor adenylate cyclase coupling in the proximal convoluted tubule in the spontaneously hypertensive rat may contribute to the diminished natriuretic response to DA-1 receptor agonists. Since the tubular DA-1 receptor is also coupled to phospholipase C, and both of these cellular signaling processes are involved in DA-1 receptor-mediated diuresis and natriuresis, it is important to know whether a similar defect is also present in DA-1 receptor-coupled phospholipase C pathway. The present study was therefore designed to determine the functional status of DA-1 receptor phospholipase C coupling system of adult spontaneously hypertensive rats using a renal cortical slice preparation. In addition, the renal response to exogenously administered dopamine (1 microgram/kg/min i.v.) was also determined. We found that basal phospholipase C activity was significantly higher in hypertensive rats than in age-matched Wistar-Kyoto rats (7.36 +/- 0.32% versus 5.61 +/- 0.27%, p less than 0.05). However, compared with the normotensive controls, dopamine induced increases in phospholipase C activity were significantly attenuated in the preparations of hypertensive rats in a concentration-dependent manner (13 +/- 6% versus 38 +/- 6% for 1 mM dopamine, p less than 0.05; 49 +/- 6% versus 71 +/- 9% for 3 mM dopamine, p less than 0.05; 50 +/- 16% versus 106 +/- 22%, p less than 0.05 for 10 mM dopamine).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730435 TI - Potentiation of inositol trisphosphate production by dexamethasone. AB - One of the mechanisms of glucocorticoid-induced hypertension has been thought to be the enhancement of vascular responsiveness to vasoconstrictors. In this regard, the effects of glucocorticoids on inositol trisphosphate production in vascular smooth muscle cells were studied. Angiotensin II and arginine vasopressin transiently increased inositol trisphosphate formation in a dose dependent manner. Pretreatment with dexamethasone for 48 hours shifted the dose response trisphosphate curves of angiotensin II- and arginine vasopressin-induced inositol trisphosphate production to the left, that is, it significantly reduced the half-maximal effective concentrations of angiotensin II (from 25 nM to 5 nM) and arginine vasopressin (from 50 nM to 25 nM). These effects of dexamethasone required a minimum of 12 hours of incubation; maximum effect was observed after 24 hours of treatment. A glucocorticoid antagonist, RU 38486, completely blocked these effects. To elucidate the interaction with prostaglandin, we used indomethacin, a potent inhibitor of prostaglandin synthesis. Treatment with indomethacin shifted the dose-response curves of angiotensin II- and arginine vasopressin-induced inositol trisphosphate production to the left. However, this shift was less than that seen after dexamethasone treatment. Indomethacin alone did not completely reproduce dexamethasone effects, and no additive effect between indomethacin and dexamethasone was observed. These results suggest, at least in part but not entirely, that the effects of dexamethasone depended on prostaglandin synthesis inhibition. We concluded that glucocorticoids altered the responsiveness of vascular smooth muscle cells to angiotensin II and arginine vasopressin through a glucocorticoid-specific receptor. These actions strongly support the mechanism by which the glucocorticoid induced hypertension through the increased sensitivity to vasoconstrictors. PMID- 1730436 TI - Changes in the aortic wall oxygen tensions of hypertensive rabbits. Hypertension and aortic wall oxygen. AB - Hypertension is a known risk factor for atherosclerosis. We hypothesize that hypertension causes artery wall hypoxia that contributes to the formation of atherosclerotic lesions. Therefore, we examined the effect of hypertension on the transarterial wall oxygen gradient of the rabbit aorta. Hypertensive rabbits were created by unilateral nephrectomy and contralateral renal artery narrowing. Transarterial wall oxygen gradients of the infrarenal aorta were measured using an oxygen microelectrode 14-16 weeks (short-term hypertension) and 56-58 weeks (long-term hypertension) after the rabbits were made hypertensive. The transarterial wall oxygen gradients showed significant differences among the groups. Short-term hypertension caused significantly higher oxygen tensions in the outer 30% of the artery wall and significant thinning of the artery wall when compared with long-term hypertension and control groups. Long-term hypertension caused significantly lower oxygen tensions in the inner 40% of the artery wall and significant thickening of the artery wall when compared with short-term hypertension and control groups. These changes were noted despite no difference in the partial pressure of oxygen in arterial blood or visual evidence of atherosclerotic lesion formation in the three groups. These findings suggest that hypertension alters the transarterial wall oxygen gradient. This altered transarterial wall oxygen gradient may contribute to the formation of atherosclerotic lesions. PMID- 1730437 TI - Effect of MK-801 on focal brain infarction in normotensive and hypertensive rats. AB - The effects of the noncompetitive N-methyl-D-aspartate antagonist MK-801 on infarct size and systemic variables after middle cerebral artery occlusion in spontaneously hypertensive and Fischer-344 rats were investigated. Two doses (0.5 and 5 mg/kg) administered before the induction of ischemia were studied. MK-801 significantly reduced the neocortical volume of infarction (by about 32% at both doses) in Fischer-344 rats and had no neuroprotective effects in the striatum. In contrast, MK-801 had no significant influence on either cortical or striatal infarcted volume in spontaneously hypertensive rats. The reduction or lack of MK 801-induced neuroprotection in spontaneously hypertensive rats, as compared with Fischer-344 rats, could be attributed to a reduced collateral supply in the marginal area due to difference in the morphology of the pial anastomoses and/or in the effects of ischemia and treatment on arterial pressure. The results may have major clinical implications since a great proportion of human strokes are associated with hypertension. PMID- 1730438 TI - The hypertensive rat and predisposition to cerebral infarction. PMID- 1730439 TI - Normalization of pressure-natriuresis by nisoldipine in spontaneously hypertensive rats. AB - This study examined whether the calcium antagonist nisoldipine can shift the relations between sodium excretion, papillary blood flow, renal interstitial pressure, and renal perfusion pressure toward lower pressures in spontaneously hypertensive rats. Mean arterial pressure decreased similarly by 9% and 12% in Wistar-Kyoto and spontaneously hypertensive rats after nisoldipine (0.5 microgram/kg bolus + 0.017 microgram/kg/min). Urine flow and sodium excretion increased by 35% and 24% in Wistar-Kyoto rats after nisoldipine. In contrast, urine flow and sodium excretion rose by 121% and 132% in spontaneously hypertensive rats, and fractional sodium excretion rose from 1.9 +/- 0.3 to 4.2 +/- 0.4%. Control sodium excretion, papillary blood flow, and renal interstitial pressure were significantly lower in spontaneously hypertensive rats than in Wistar-Kyoto rats when compared at similar renal perfusion pressures. Sodium excretion, papillary blood flow, and renal interstitial pressure all increased in spontaneously hypertensive rats after nisoldipine, whereas it had no effect on papillary blood flow or renal interstitial pressure in Wistar-Kyoto rats. The relations among sodium excretion, papillary blood flow, renal interstitial pressure, and renal perfusion pressure were shifted toward lower pressures in spontaneously hypertensive rats given nisoldipine and became similar to those seen in Wistar-Kyoto rats. These results indicate that nisoldipine normalizes the relations among sodium excretion, renal interstitial pressure, papillary blood flow, and renal perfusion pressure in spontaneously hypertensive rats perhaps by correcting the defect in renal medullary perfusion associated with resetting of pressure natriuresis in this model of hypertension. PMID- 1730440 TI - Arterial baroreceptor reflex function in borderline hypertensive rats. AB - With increased dietary NaCl intake (8% NaCl), the borderline hypertensive rat develops hypertension, thus expressing the phenotype of the spontaneously hypertensive parent. Since arterial baroreceptor reflex function is impaired in the spontaneously hypertensive parent, it was the objective of this study to examine arterial baroreceptor reflex function in the borderline hypertensive rat made hypertensive by increased dietary NaCl intake. Borderline hypertensive rats were fed either 1% or 8% NaCl from age 4 to 16 weeks. Borderline hypertensive rats fed 8% NaCl (n = 10) were hypertensive compared with borderline hypertensive rats fed 1% NaCl (n = 11) (141 +/- 3 versus 120 +/- 4 mm Hg, p less than 0.01). They were chronically instrumented for the recording of arterial pressure, heart rate, and renal sympathetic nerve activity. The percent change from control in heart rate and renal sympathetic nerve activity resulting from increases (phenylephrine) and decreases (nitroglycerine) in arterial pressure were measured in conscious freely moving animals. With respect to arterial baroreceptor reflex control of heart rate, 8% NaCl borderline hypertensive rats had a similar range (75 +/- 4%) and maximal gain (-2.72 +/- 0.24%/mm Hg) as 1% NaCl borderline hypertensive rats (70 +/- 4%; -2.78 +/- 0.50%/mm Hg). With respect to arterial baroreceptor reflex control of renal sympathetic nerve activity, 8% NaCl borderline hypertensive rats had values for range (205 +/- 22%) and maximal gain (-3.92 +/- 0.93%/mm Hg) that were not significantly different from those for 1% NaCl borderline hypertensive rats (167 +/- 33%, -2.76 +/- 0.62%/mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730441 TI - Increased norepinephrine spillover into the jugular veins in essential hypertension. AB - In essential hypertension sympathetic nerve firing is commonly increased. A central nervous system origin has been presumed but not tested directly. To estimate cerebral norepinephrine release in essential hypertension, spillover of norepinephrine into the cerebrovascular circulation was measured by isotope dilution, with high internal jugular venous sampling. Norepinephrine was released into the cerebrovascular circulation in both hypertensive patients and healthy volunteers and was present after administration of the ganglion blocker trimethaphan and in patients with sympathetic nervous failure, indicating that brain neurons and not cerebrovascular sympathetic nerves were the probable source. Although differing among hypertensive patients, norepinephrine spillover on average was higher in the hypertensive patients (153 +/- 41 pmol/min) than in healthy subjects (59 +/- 12 pmol/min; p less than 0.05), and was elevated in six of 17 patients, in whom the accompanying whole body norepinephrine spillover rate was higher than in the remaining 11 patients (p less than 0.01). To test for a possible link between brain norepinephrine release and human sympathetic nervous function, the effect of the tricyclic antidepressant desipramine (0.3 mg/kg i.v.) on both brain and whole body norepinephrine spillover was measured in healthy volunteers. Desipramine lowered the cerebrovascular spillover of norepinephrine, its precursor dihydroxyphenylalanine, and its metabolite dihydroxyphenylglycol by 50-80% and produced a mean fall of 35% in whole body norepinephrine spillover. One interpretation of these results is that human sympathetic nerve firing is dependent on norepinephrine release within the brain and that increased cerebral norepinephrine release may possibly be present in some patients with essential hypertension, underlying their higher sympathetic nerve firing rates. PMID- 1730442 TI - Renin and angiotensinogen expression during the evolution of diabetes. AB - The expression of renin and angiotensinogen genes and their proteins were studied during the progression of diabetes using adult BioBreeding spontaneously diabetic rats at 1 day and 2-12 months of diabetes. The number of renin-stained cells per juxtaglomerular apparatus was determined by immunocytochemistry. Initially, at 2 months of diabetes the number of renin-stained cells per juxtaglomerular apparatus increased significantly (p less than 0.0001, 2 months versus resistant groups) and was followed by a decrease in the number and intensity of renin stained cells after 12 months of diabetes (p = 0.007, 2 months versus 12 months). A significant negative correlation was observed between the number of renin containing cells and the duration of diabetes (r = 0.99, p = 0.014). Immunoreactive angiotensinogen was restricted to the proximal tubule and appeared increased after 4 and 8 months of diabetes as compared with the 2- and 12-month diabetic groups. Renin messenger RNA (mRNA) levels increased with the onset of diabetes and decreased markedly during chronic diabetes. At 1 day of diabetes, renin mRNA levels were 700% higher than at 12 months of diabetes. Angiotensinogen mRNA levels were unchanged. We conclude that diabetes results in an initial increase in renin gene expression, and as the duration of diabetes lengthens, there is a progressive decrease in renin gene expression and in the number of cells containing renin. These findings suggest that as the duration of diabetes and the age of the animal lengthens, there is a decrease in the number of cells expressing the renin gene. PMID- 1730443 TI - High calcium diet augments vascular potassium relaxation in hypertensive rats. AB - The effects of increased dietary calcium on the development of hypertension and vascular smooth muscle responses were studied in spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. Both hypertensive and normotensive animals were divided into two groups; the calcium content of the normal diet was 1.1% and that of the high calcium diet 3.1%. During the 12-week study, calcium supplementation significantly attenuated the increase in systolic blood pressure in the hypertensive rats but did not affect blood pressure in the normotensive rats. The contractile responses of endothelium-denuded mesenteric arterial rings to potassium chloride were similar in all study groups. The contractions to norepinephrine were not altered by the high calcium diet either, but smooth muscle sensitivity to this agonist was lower in the normotensive than in the hypertensive rats. Potassium relaxation was used to evaluate the activity of vascular smooth muscle Na+,K(+)-ATPase. The maximal rate of potassium relaxation was fastest in the normotensive groups but was also clearly faster in calcium treated hypertensive rats when compared with hypertensive rats on a normal diet. Platelets were used as a cell model for the analysis of intracellular free calcium concentration, which was measured by the fluorescent indicator quin-2. Intracellular free calcium was significantly reduced in the hypertensive rats by calcium supplementation and was not affected in the normotensive rats. In conclusion, a reduction of intracellular free calcium concentration indicating improved calcium regulation and a concomitant alteration in vascular relaxation probably reflecting increased activity of smooth muscle Na+,K(+)-ATPase may contribute to the blood pressure-lowering effect of a high calcium diet. PMID- 1730444 TI - Cumulative sums in quantifying circadian blood pressure patterns. AB - The plotting of cumulative sums (cusums), a technique of proven value in the detection of trends in data collected at intervals of time, may be modified to analyze circadian blood pressure patterns quantitatively. Mean 24-hour ambulatory blood pressure is taken as the reference value and is subtracted from each pressure value. The products of the remainders and the corresponding time intervals are summed in sequence and are plotted against time to form a modified cusum plot. The slope of the plot over any given time period equals the difference between mean blood pressure during that period and mean 24-hour blood pressure. Crest and trough blood pressures (the mean blood pressures of the 6 hour periods of highest and lowest pressures) may be identified as the 6-hour periods where plot slopes are most steeply ascending and descending, respectively. The magnitude of the circadian blood pressure change, defined as the difference between crest and trough blood pressure, is calculated from the difference between crest and trough plot slopes. The height of the cusum plot, which reflects pressure alteration extent and duration, may also be used as a measure of circadian pattern. The modified cusums technique and cusum-derived statistics are illustrated using ambulatory blood pressure profiles of hypothetical and actual hypertensive subjects. Independence from fixed time periods improves precision and reproducibility. Cusum-derived statistics are simply calculated from raw ambulatory data and should prove useful in the quantitative analysis of circadian blood pressure profiles. PMID- 1730445 TI - The kidney, obesity, and essential hypertension symposium. Jackson, Mississippi, March 15-16, 1991. PMID- 1730446 TI - Hyperamylinemia, hyperinsulinemia, and insulin resistance in genetically obese LA/N-cp rats. AB - The experimental evidence supporting a direct role for hyperinsulinemia as a cause of insulin resistance remains equivocal. Amylin, an islet beta-cell peptide cosecreted with insulin in response to nutrient stimuli, causes insulin resistance when infused into intact animals or applied to isolated skeletal muscles. We compared measures of amylin and insulin gene expression between control and genetically obese, insulin-resistant Lister Albany/NIH-(LA/N-cp) rats. Pancreatic amylin messenger RNA levels were increased 7.8 +/- 0.7-fold (mean +/- SEM), and plasma amylin-like immunoreactive material was increased 10.9 +/- 1.1-fold (LA/N-lean, 14 +/- 4 pM; LA/N-cp, 153 +/- 16 pM; p less than 0.0001) in obese rats. Pancreatic insulin I mRNA levels were increased 7.4 +/- 0.5-fold, and plasma insulin levels 20.0 +/- 5.0-fold, in these rats (LA/N-lean, 308 +/- 84 pM; LA/N-cp 6,120 +/- 1,540 pM; p less than 0.0001). The EC50 for insulin stimulated incorporation of glucose into glycogen was about fourfold higher in muscles isolated from obese rats. The present results, coupled with previous observations, support the hypothesis that hyperamylinemia, rather than hyperinsulinemia per se, could have directly caused the insulin resistance in the obese LA/N-cp rats. Hyperamylinemia needs to be considered in future experimental studies probing the relation between hyperinsulinemia and insulin resistance. PMID- 1730447 TI - The Zucker rat model of obesity, insulin resistance, hyperlipidemia, and renal injury. AB - Although the pathogenesis of obesity in OZR is unknown, the association among hyperinsulinemia, insulin resistance, and hyperlipidemia suggests that investigations using OZR may help define how a number of vascular disease risk factors interact to cause end-organ damage. Like other rat strains, OZR do not develop atherosclerosis spontaneously. Nevertheless, in an endothelial injury model, atherosclerosis was worse in OZR than in LZR. Perhaps more intriguing is the fact that OZR develop spontaneous glomerular injury. Although the mechanisms important in the development and progression of glomerular injury in OZR remain to be clarified, both lipid abnormalities and glomerular hemodynamic alterations could play a role. PMID- 1730448 TI - Obesity hypertension. Converting enzyme inhibitors and calcium antagonists. AB - Exogenous obesity is characterized hemodynamically by expanded intravascular (plasma) volume associated with an increased cardiopulmonary volume and cardiac output. In contrast, essential hypertension is related to an increased total peripheral resistance that is more or less uniformly distributed throughout the component organ circulations associated with a contracted plasma volume in proportion to the height of arterial pressure. Thus, both cardiac output and total peripheral resistance are elevated in obesity hypertension, and both impose a load on the left ventricle, resulting in both a volume and a pressure overload left ventricular hypertrophy. Although renal vascular resistance is not as increased as it is in lean hypertensive patients, these patients are subjected to hyperfiltration and proteinuria. Additionally, these hemodynamic alterations coexist with carbohydrate intolerance, hyperinsulinemia, hyperlipidemia, and hyperuricemia. With weight reduction and associated pressure reduction, the hemodynamic and metabolic changes reverse toward normal. However, should this not be achievable, the angiotensin converting enzyme inhibitors and calcium antagonists provide rational physiological approaches to drug therapy. With these agents pressure reduction is achieved through a fall in vascular resistance without intravascular volume expansion, and this is associated with reduced left ventricular mass and preserved cardiac and renal function, and without exacerbation of preexisting metabolic perturbations. Hence, these two classes of antihypertensive agents may provide a rational and physiological means for reversing the pathophysiological alterations of hypertensive disease in those obese patients in whom weight control is not possible. PMID- 1730449 TI - Antihypertensive therapy and cardiovascular risk. Are all antihypertensives equal? AB - The lack of success of antihypertensive drug therapy in decreasing cardiovascular events has caused close examination of the influence of antihypertensive drugs on cardiac risk factors. Striking differences exist in the pharmacological profiles of antihypertensive drug classes and subclasses as well as in the influences of the drugs on electrolyte, lipid, and glucose metabolism. Differences also exist in the effects of the drugs on left ventricular hypertrophy. These differences in the effects of antihypertensive drugs on cardiac risk factors may assist in explaining the lack of a favorable effect on cardiovascular events in previous clinical trials. However, prospective trials are necessary to demonstrate that treatment of hypertension with drugs that have a more favorable effect on cardiac risk factors will reduce cardiac events (i.e., myocardial infarction, heart failure, and sudden death). PMID- 1730450 TI - Hypertensinogenic mechanisms mediated by renal actions of renin-angiotensin system. PMID- 1730451 TI - Kidneys and fluids in pressure regulation. Small volume but large pressure changes. AB - The human body has multiple blood pressure control mechanisms, each of which serves a special and usually different role in pressure regulation. The nervous pressure controllers usually react within seconds and prevent major rapid changes in pressure when acute extraneous forces act on the circulatory system. Then, within minutes to hours, several intermediately acting pressure controllers become activated. Among the more important of these are the renin-angiotensin vasoconstriction system and the shift of fluid volume between the blood and interstitial fluids. Finally, after several hours to days, the kidneys readjust body fluid volumes, especially the extracellular fluid and blood volumes, to bring the pressure to a very precise level. This final adjustment usually requires little change in body fluid volume for two reasons. First, the other pressure controllers often have already made most of the needed pressure adjustments. Second, the increase in fluid volume required to cause a major increase in blood pressure is usually surprisingly small; this is true because the whole body blood flow autoregulation mechanism causes a secondary increase in total peripheral resistance. PMID- 1730452 TI - Sympathetic neural control of the kidney in hypertension. AB - Efferent renal sympathetic nerve activity is elevated in human essential hypertension as well as in several forms of experimental hypertension in animals. In addition, bilateral complete renal denervation delays the development and/or attenuates the magnitude of the hypertension in several different forms of experimental hypertension in animals. Efferent renal sympathetic nerve activity is known to have dose-dependent effects on renal blood flow, the glomerular filtration rate, renal tubular sodium and water reabsorption, and the renin secretion rate, which are capable of contributing, singly or in combination, to the development, maintenance, and exacerbation of the hypertensive state. Of the many factors known to influence the central nervous system integrative regulation of efferent renal sympathetic nerve activity, two environmental factors, a high dietary sodium intake and environmental stress, are capable of significant interaction. This resultant increase in efferent renal sympathetic nerve activity and subsequent renal functional alterations can participate in the hypertensive process. This is especially evident in the presence of an underlying genetic predisposition to the development of hypertension. Thus, interactions between environmental and genetic influences can produce alterations in the sympathetic neural control of renal function that play an important role in hypertension. PMID- 1730453 TI - The immune system and hypertension. AB - Primary or secondary activation of immune mechanisms has been implicated in the pathogenesis of many forms of hypertension. Changes in serum immunoglobulin levels, alterations in both humoral and cellular immune functions, and inherited abnormalities of the complement system have been identified in patients with essential hypertension. In addition, many models of spontaneous hypertension (such as the Okamoto and Lyon strains of hypertensive rats and the hypertensive New Zealand Black mouse) have identifiable abnormalities in immune function that are associated with their hypertensive disease. Other models (such as partial renal infarct hypertension, post-mineralocorticoid-salt hypertension, and hypertension induced by repeated injections of angiotensin II) also may have primary or secondary immunologic factors contributing to their etiology. Although there is a strong association between alterations in immune function and hypertension, the specific immunologic mechanisms that contribute to the pathogenesis of hypertension are not known. Therefore, further investigation will be necessary to elucidate these mechanisms. PMID- 1730454 TI - Obesity-associated hypertension. Hyperinsulinemia and renal mechanisms. AB - Hyperinsulinemia and insulin resistance have been postulated to link obesity and hypertension. Evidence supporting this concept derives mainly from epidemiological studies showing a correlation between insulin resistance, hyperinsulinemia, and blood pressure and from short-term studies suggesting that insulin has renal and cardiovascular actions that, if sustained, could elevate blood pressure. However, a cause-and-effect relation between insulin and hypertension has not been clearly established. Recent studies indicate that chronic hyperinsulinemia, similar to that found in obese hypertensive patients, did not raise blood pressure in normal dogs, even when renal excretory capability was reduced by prior removal of kidney mass. Chronic insulin infusion also failed to elevate blood pressure in dogs maintained on a high sodium intake and did not potentiate the long-term blood pressure responses to angiotensin II or norepinephrine. The presence or absence of insulin resistance may not be a major factor in determining the blood pressure response to hyperinsulinemia since chronic insulin infusion also failed to cause hypertension in obese, insulin resistant dogs. Although hyperinsulinemia causes transient sodium retention, sustained decreases in renal excretory capability sufficient to cause chronic hypertension did not occur in dogs. In rats, insulin infusion causes small increases in blood pressure, although several characteristics of the hypertension (e.g., salt-sensitivity) differ from those observed in obese human hypertensive patients. Whether humans more closely resemble dogs or rats with respect to their long-term cardiovascular responses to insulin remains to be determined. However, very high insulin levels in humans with insulinoma do not cause hypertension, and several studies suggest that there is only a weak correlation between plasma insulin concentration and blood pressure in normal humans. Therefore, additional factors besides hyperinsulinemia per se may be responsible for a major component of obesity-associated hypertension. PMID- 1730455 TI - Cardiovascular regulation in obesity-induced hypertension. PMID- 1730456 TI - Hyperinsulinemia: possible role in obesity-induced hypertension. AB - Data in support of the hypothesis that insulin-mediated sympathetic stimulation contributes to the hypertension that occurs in association with obesity are presented. The relation between insulin and the sympathetic nervous system derives from the effect of diet on sympathetic activity. Fasting suppresses but overfeeding stimulates the sympathetic nervous system. Insulin-mediated glucose metabolism within central neurons sensitive to insulin and glucose appears to be one important signal in this relation between diet and sympathetic activity. In the obese, hyperinsulinemia and hypertension track together in epidemiological studies. Evidence from a human population-based study (the Normative Aging Study) indicates that the abdominal form of obesity is associated with both hyperinsulinemia and increased urinary norepinephrine excretion. Elevations in urinary norepinephrine excretion, moreover, were highest in those with the highest fasting levels of insulin and glucose. These observations are consistent with the hypothesis that insulin-mediated sympathetic stimulation is a mechanism recruited in the obese to increase metabolic rate and restore energy balance; concomitant increases in sympathetic stimulation of the heart, vasculature, and kidney result in hypertension, which, according to this formulation, is an unfortunate by-product of the cardiovascular response to a metabolic adaptation. PMID- 1730457 TI - Obesity, the sympathetic nervous system, and essential hypertension. AB - There is ample evidence that the sympathetic nervous system is important in the etiology of essential hypertension. Plasma catecholamines such as norepinephrine and epinephrine are the most common indexes of sympathetic function used in studies of essential hypertension. Plasma norepinephrine is higher in young essential hypertensive patients than in normotensive subjects. Other methods to examine sympathetic activity, such as blood pressure response to sympatholytic agents, measurement of regional sympathetic activity, vascular reactivity to sympathetic agonists, power spectral analysis, and microneurography, have all provided further evidence for enhanced sympathetic activity in essential hypertension, especially in younger subjects. Certain groups that make up a substantial part of the essential hypertensive population, such as obese subjects, have heightened sympathetic activity that could contribute to hypertension. Plasma norepinephrine levels are significantly higher in obese compared with nonobese subjects, and the remarkable fall in blood pressure with weight loss in obese subjects is correlated with reductions in plasma norepinephrine. Antihypertensive agents have variable effects on sympathetic activity; some agents (diuretics and direct vasodilators) have elevating effects, some agents (centrally acting agents and alpha-antagonists) have lowering effects, and others (converting enzyme inhibitors, calcium blockers, and beta blockers) have mixed effects. Tailoring therapy toward agents that reduce sympathetic activity for specific groups perceived as having neurogenic hypertension, such as obese subjects, is a goal yet to be attained. PMID- 1730458 TI - Effects of insulin on renal sodium excretion. AB - The ability of insulin to decrease urinary sodium excretion has been recognized for more than 30 years. While most investigators agree that this occurs predominantly through increased tubular sodium reabsorption, the nephron segments at which insulin exerts this effect in vivo remain controversial. Additionally, little information is available in mammalian systems on the mechanism of the insulin response or its relation to other hormonal systems important in the regulation of tubular sodium transport. Data from amphibian transporting epithelia suggest a potential for interactions between insulin and several other peptide hormones in the regulation of sodium transport. The following discussion attempts to review our knowledge of the effects of insulin on renal sodium reabsorption and describes new data suggesting that insulin's antinatriuretic response is dependent on antidiuretic hormone but independent of the angiotensin and prostaglandin systems. PMID- 1730459 TI - Hypertension during chronic hyperinsulinemia in rats is not salt-sensitive. AB - The goal of this study was to examine the chronic blood pressure and renal actions of insulin in conscious rats and to determine whether the blood pressure response to insulin is salt-sensitive. The effects of chronic hyperinsulinemia were examined in three groups of Sprague-Dawley rats given low sodium (LS rats, 0.6 meq/day), normal sodium (NS rats, 3.0 meq/day), or high sodium (HS rats, 11.4 meq/day) intakes. After 5-7 days of acclimation and 4 days of control measurements, insulin was infused 24 hr/day (1.5 milliunit/kg/min i.v.) for 7 days, and euglycemia was maintained by infusion of glucose (22 mg/kg/min i.v.). Mean arterial pressure was recorded continuously 19 hr/day, using computerized techniques, from chronically implanted aortic catheters. Chronic insulin infusion increased arterial pressure similarly in the three groups of rats, from 91 +/- 2 to 104 +/- 4 mm Hg in LS rats (n = 6), from 86 +/- 2 to 104 +/- 4 mm Hg in NS rats (n = 5), and from 91 +/- 2 to 105 +/- 8 mm Hg in HS rats (n = 5). There were no significant changes in plasma renin activity or glucose concentration in any group during insulin infusion. Control sodium excretions were 0.5 +/- 0.1, 2.3 +/ 0.1, and 9.3 +/- 0.6 meq/day in LS, NS, and HS rats, respectively, and there were no significant changes in urinary sodium excretion or cumulative sodium balance during 7 days of insulin infusion in any of the groups. These observations indicate that chronic hyperinsulinemia in rats produced hypertension that was not salt-sensitive and not dependent on sodium retention or increased renin secretion. Moreover, insulin-induced hypertension was associated with a shift of renal pressure natriuresis, since sodium balance was maintained at elevated arterial pressures. PMID- 1730460 TI - Pressure natriuresis. Role of renal interstitial hydrostatic pressure. AB - The kidneys play a major role in the long-term regulation of extracellular fluid volume and arterial pressure. A central component of the feedback system for long term control of arterial pressure is the pressure-natriuresis mechanism, whereby increases in renal perfusion pressure lead to decreases in sodium reabsorption and increases in sodium excretion. The specific intrarenal mechanism for the decrease in tubular reabsorption in response to increases in renal perfusion pressure appears to be related to increases in renal interstitial hydrostatic pressure (RIHP). Increases in renal perfusion pressure are associated with significant increases in RIHP. The mechanism whereby RIHP increases in the absence of discernible changes in whole kidney renal blood flow and peritubular capillary hydrostatic and/or oncotic pressures may be related to alterations in renal medullary hemodynamics. Several lines of investigation support an important quantitative role for RIHP in mediating pressure natriuresis. Preventing RIHP from increasing in response to increases in renal perfusion pressure markedly attenuates pressure natriuresis. Furthermore, direct increases in RIHP, comparable to increases measured in response to increases in renal perfusion pressure, have been shown to significantly decrease tubular reabsorption of sodium in the proximal tubule and increase sodium excretion. The exact mechanism whereby RIHP influences tubular reabsorption is unknown but may be related to alterations in tight junctional permeability to sodium in proximal tubules and/or release of renal autacoids such as prostaglandins. PMID- 1730461 TI - Obese Zucker rats are normotensive on normal and increased sodium intake. AB - The purpose of this study was to determine whether genetically obese Zucker rats have higher arterial pressures than lean littermates on normal and high sodium intakes. Mean arterial pressure was directly measured in chronically instrumented Zucker rats (six lean [weight, 345.8 +/- 8.0 g] and five obese [529.0 +/- 6.2 g]) for 2 weeks on both a normal (2 meq sodium/day) and high (6 meq sodium/day) sodium intake (7 days each). In addition, daily heart rate, water intake, urine output, urinary sodium excretion, urinary potassium excretion, and weekly fasting plasma insulin levels were measured. Obese rats exhibited significantly lower heart rate and greater water intake and urine output compared with lean rats whether maintained on control or high sodium intakes. Urinary sodium excretion, however, was identical in lean and obese rats throughout the experiment. Fasting plasma insulin levels in obese rats were seven times greater than those in lean rats. When the rats were maintained on a 2 meq/day sodium intake, mean arterial pressures obtained from the two groups were similar: 103 +/- 1 versus 106 +/- 1 mm Hg (lean versus obese). An increase in sodium intake did not significantly affect mean arterial pressure in either group: 101 +/- 1 versus 105 +/- 1 mm Hg (lean versus obese). These results indicate that at 12-14 weeks of age, male obese Zucker rats do not exhibit higher resting arterial pressures than lean littermates when maintained on normal or high sodium intake. PMID- 1730462 TI - Blunted natriuretic response to an acute sodium load in obese hypertensive dogs. AB - Several studies support the premise that there is a strong relation between obesity and high blood pressure. Although the mechanism for obesity-related hypertension has not yet been fully elucidated, recent studies have suggested that abnormalities in renal sodium handling may be involved in the pathogenesis of obesity-induced hypertension. The purpose of the present study was to determine the effects of an acute saline load on renal excretory function in dogs with obesity-induced hypertension and in normotensive lean dogs. Experiments were performed in two groups of conscious, chronically instrumented dogs. One group of dogs (obese) was fed a high-fat diet for 5-6 weeks, and the other group (lean) ate a normal diet. The body weight of the obese dog group (26.3 +/- 0.7 kg) was 45% higher than the lean dog group (18.1 +/- 0.3 kg). Mean arterial pressure averaged 126 +/- 2 mm Hg in the obese dogs and 100 +/- 1 mm Hg in the lean dogs. The lean dogs had an average heart rate of 104 +/- 7 beats per minute, whereas the obese dogs averaged 134 +/- 8 beats per minute. Plasma renin activity was also significantly higher in the obese dogs. Both groups of dogs were given 135 meq sodium chloride over 60 minutes via an intravenous infusion of isotonic saline. Sodium and water excretion increased significantly in response to the acute saline load. However, the natriuresis and diuresis was markedly attenuated in the obese hypertensive dogs. During the first 40 minutes of saline loading, the increase in sodium and water excretion was 50-70% lower in the obese hypertensive dogs. The results of the present study indicate that obese hypertensive dogs have a reduced capability to excrete an acute sodium load. This abnormality in renal sodium handling may play a role in the pathogenesis of obesity-induced hypertension. PMID- 1730463 TI - Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin dramatically alter their morphology. AB - Bacteroides fragilis has been associated with causation of diarrheal disease in livestock and humans. To date, conventional tissue culture and animal assays used to detect the biologic activity of bacterial enterotoxins have failed with enterotoxigenic B. fragilis. Although enterotoxigenic B. fragilis stimulates intestinal secretion in lamb and calf ligated intestinal loops, infant rabbits, and adult rabbits with ligated ceca, these animal systems are costly and complicated, which limits their usefulness for identification of enterotoxigenic B. fragilis strains. Using the cloned human colonic-epithelial-cell line HT29/C1, we have developed an in vitro assay that is 89% sensitive and 100% specific in detecting enterotoxigenic B. fragilis strains as defined by the lamb ligated intestinal-loop assay. Subconfluent HT29/C1 cells treated with concentrated bacterium-free culture supernatants of enterotoxigenic B. fragilis strains develop specific and striking morphologic changes including loss of cell-to-cell attachments, rounding, swelling, and, in some cases, pyknosis. These morphologic changes are initially visible at 1 h after treatment and progress over at least the first 24 h. This tissue culture assay should prove useful in epidemiologic studies of enterotoxigenic B. fragilis and may facilitate basic studies to identify the B. fragilis toxin(s) and its mechanism of action. PMID- 1730464 TI - Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase. AB - Pseudomonas aeruginosa produces a blue pigment, pyocyanin. Pyocyanin is a redox active phenazine compound that kills mammalian and bacterial cells through the generation of reactive oxygen intermediates. We examined the mechanisms by which P. aeruginosa resists pyocyanin. [14C]pyocyanin was taken up by both Escherichia coli and P. aeruginosa, though more slowly by the latter. Cyanide-insensitive respiration, used as an indicator of intracellular superoxide and/or hydrogen peroxide production, was 50-fold less in pyocyanin-treated P. aeruginosa than in E. coli. P. aeruginosa showed less cyanide-insensitive respiration than E. coli upon exposure to other redox-active compounds (paraquat, streptonigrin, and plumbagin). Electron paramagnetic resonance spectrometry and spin trapping showed that P. aeruginosa generated less pyocyanin radical and superoxide than E. coli. Cell extracts from E. coli contained an NADPH:pyocyanin oxidoreductase which increased the rate of reduction of pyocyanin by NADPH. Conversely, cell extracts from P. aeruginosa contained no NADPH:pyocyanin oxidoreductase activity and actually decreased the rate of pyocyanin-mediated NADPH oxidation. Antioxidant defenses could also reduce the sensitivity of P. aeruginosa to pyocyanin. Under culture conditions of limited phosphate, both pyocyanin production and catalase activity were enhanced. Superoxide dismutase activity was also increased under low-phosphate conditions. When cells were grown in a high-phosphate succinate medium, P. aeruginosa formed a previously described iron-superoxide dismutase as well as a manganese-cofactored superoxide dismutase. These results demonstrate that P. aeruginosa resists pyocyanin because of limited redox cycling of this compound and that under conditions favoring pyocyanin production, catalase and superoxide dismutase activities increase. PMID- 1730465 TI - Secretory immune responses to Mycoplasma pulmonis. AB - Formalinized Mycoplasma pulmonis, along with aluminum hydroxide as an adjuvant, was used to subcutaneously immunize rats in the vicinity of the salivary gland to examine the characteristics of the secretory immune response to this pathogen. The induction of specific antibody to this microorganism was detected in serum and the exocrine fluids, namely, saliva and lung lavage fluid. Both immunoglobulin G (IgG) and IgA isotype antibodies were detected in each of these fluids after primary and secondary local immunizations. Serum responses from immunized animals were significantly greater than in the control group, but a dose response was not observed in either IgG or IgA antibody at the dosages selected for immunization. Salivary IgG antibody responses peaked early after both the primary and secondary immunizations, exhibiting a clear dose response. Salivary IgA in immunized groups was significantly greater than that in the control group but displayed little dose-dependent kinetics, and, at the termination of the experiment, this response had not yet peaked. Lung lavage IgG and IgA were minimal after the primary immunization when the antibody was normalized to total protein but displayed dose-dependent kinetics after a secondary challenge. IgG peaked immediately after a secondary challenge, while IgA peak responses were observed only after 20 days. A positive correlation was noted between the serum, saliva, and lung lavage fluid IgGs after both primary and secondary immunizations and only after a secondary challenge for IgA. In this study we were able to elicit a secretory immune response, consisting of both IgG and IgA, which exhibited a dose-dependent characteristic in lung lavage fluid to this immunogen. Additionally, a positive correlation of antibody levels between saliva and lung lavage fluid suggests that saliva could be used as an indicator for monitoring specific antibody to M. pulmonis in lung lavage secretions without requiring invasive, deleterious procedures. PMID- 1730466 TI - Characterization of inhibition of Mycobacterium avium replication in macrophages by normal human serum. AB - Serum from some AIDS patients permits the rapid multiplication of Mycobacterium avium in cultured human macrophages. Serum from human immunodeficiency virus negative individuals inhibits replication. The characteristics of the serum induced inhibition were examined here. M. avium 7497 serovar 4 grew exponentially in macrophages when they were cultured in serumless medium. Growth was measured by determining the CFU after infected macrophages were lysed at 0 to 7 days after infection. Normal AB serum (5 to 10%) added to infected macrophages resulted in an initial 4-day lag of bacterial growth followed by rapid replication from 4 to 7 days. Serum also inhibited bacterial replication in medium without macrophages. This inhibition was not biphasic but was sustained over 7 days. Macrophage associated M. avium became less responsive to serum inhibitor within 24 h after infection of macrophages. Within 2 days of culture, M. avium no longer responded to inhibitor. Replication of macrophage-derived M. avium (Vi) was in some instances serum inhibitable and at other times was enhanced by serum, when it was used to infect fresh macrophages. The Vi phenotype remained serum inhibitable without macrophages. Preinfection of macrophages with heat-killed M. avium did not alter serum-induced bacterial inhibition or escape from inhibition. PMID- 1730467 TI - Influence of calcium on secretion and activity of the cytolysins of Actinobacillus pleuropneumoniae. AB - In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media. This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells. Because the cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that calcium plays in the secretion and activity of A. pleuropneumoniae cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium. These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II. It was found that both cytolysins required calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III. Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium. Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I. Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium. When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A. pleuropneumoniae cytolysins. PMID- 1730468 TI - Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin. AB - Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii) Inv+ Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and IIa. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin. PMID- 1730469 TI - Circulating and localized immune complexes in experimental mycoplasma-induced arthritis-associated ocular inflammation. AB - Ocular deposits of immune complexes are believed to contribute to the anterior segment inflammations observed in association with the human arthritides. Arthritis-related ocular inflammations may be reproduced in animals by infection with certain species of mycoplasma. To evaluate the role of immune complexes in the production of ocular lesions, we studied their involvement in the rodent model of experimental arthritis-associated ocular inflammation induced by Mycoplasma arthritidis. Sprague-Dawley rats were infected with viable concentrates of M. arthritidis and monitored for the production of related circulating and intraocular immune complexes. Circulating immune complexes were monitored by antigen capture systems, and localized intraocular complexes were identified by indirect immunohistochemistry. Polyacrylamide gel immunoblot analysis of captured complexes confirmed the antigen(s) involved as proteins derived from M. arthritidis. Indirect immunofluorescence revealed localized complexes containing mycoplasma antigens within the ciliary-iris vasculature. Concentrations of the generated complexes diminished rapidly over a 30-day period. While complex deposits within ocular tissues could represent a contributing cause to the localized anterior segment inflammation reported in this rodent model, secondary challenge with viable M. arthritidis, which reproduced high concentrations of intraocular and circulating immune complexes, failed to elicit any ocular response. PMID- 1730470 TI - Role of Vibrio cholerae neuraminidase in the function of cholera toxin. AB - Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ filtrates increased five- and eightfold, respectively, relative to that with NANase- filtrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase- filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 10(4) to 10(8) CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 10(9) CFU than NANase- strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions. PMID- 1730471 TI - Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis. AB - A chymotrypsinlike serine proteinase of Coccidioides immitis with an estimated molecular size of 34 kDa has been shown by immunoelectron microscopy to be associated with the walls of the parasitic cells of this human respiratory pathogen. The proteinase has been suggested to play a role in spherule development. We report the isolation of a 1.2-kb cDNA from an expression library of C. immitis constructed in the lambda ZAP II phage vector. The cDNA is suggested to encode the 34-kDa protein. We demonstrate identity between segments of the deduced amino acid sequence of the open reading frame of the 1.2-kb cDNA and three distinct sequences obtained from cyanogen bromide cleavage peptides of the purified proteinase. The occurrence of N-glycosyl linkage sites in the deduced sequence of 309 amino acids of the open reading frame (ORF) correlates with our identification of such linkage sites in the native glycosylated proteinase. A protein encoded by an 800-bp fragment of the 1.2-kb cDNA, which was produced by transformed Escherichia coli XL1-Blue, was recognized by the anti-34 kDa protein antibody in a Western blot (immunoblot). Northern (RNA) hybridization of total poly(A)-containing RNA of C. immitis with the labeled 1.2-kb cDNA clone revealed a single band of approximately 1.75 kb. Partial homology was demonstrated between the deduced amino acid sequence of the ORF (927 bp) and reported sequences of alpha-chymotrypsin and chymotrypsinogens. Expression of the proteinase gene was examined by Northern dot blot analysis of total RNA from different stages of parasitic cell development in C. immitis. Maximum levels of specific mRNA were detected during early endospore wall differentiation. The 34 kDa proteinase appears to be concentrated in walls of the parasitic cells at stages of active growth. We suggest that the enzyme may participate in wall plasticization and/or intussusception or in cell wall turnover. PMID- 1730472 TI - Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae. AB - Live oral candidate cholera vaccines have previously been constructed by deletion of Vibrio cholerae sequences encoding the enzymatically active A subunit of the cholera toxin. However, volunteer studies have shown that these non-cholera toxin producing strains still provoke mild to moderate diarrhea in some individuals. We recently reported the identification of a second toxin produced by V. cholerae which may be responsible for this residual diarrhea (A. Fasano, B. Baudry, D. W. Pumplin, S. S. Wasserman, B. D. Tall, J. M. Ketley, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 88:5242-5246, 1991). This new toxigenic factor increases the permeability of rabbit ileal mucosa by affecting the structure of the intercellular tight junctions (zonula occludens). We now report the identification and cloning of the gene encoding this new toxin. This gene, named zot (for zonula occludens toxin), consists of a 1.3-kb open reading frame which could potentially encode a 44.8-kDa polypeptide. The location of the zot gene encoding the new toxin is immediately upstream of the ctx operon encoding cholera toxin. PMID- 1730473 TI - Non-O1 Vibrio cholerae intestinal pathology and invasion in the removable intestinal tie adult rabbit diarrhea model. AB - A modified removable intestinal tie adult rabbit diarrhea (RITARD) model was used to investigate the intestinal pathology, intestinal bacterial colonization, intestinal fluid volume, and onset of diarrhea caused by non-O1 Vibrio cholerae. Three strains of non-O1 V. cholerae were studied. RITARD rabbits challenged with 10(3) CFU of strain NRT36S (a strain previously shown to cause diarrhea in volunteers) developed grade 3 diarrhea at 48 to 72 h. The mean counts of non-O1 V. cholerae isolated were 9.3 +/- 0.07 and 8.7 +/- 0.7 CFU/g from the small and large intestines, respectively. Histologic examination showed necrosis of the luminal epithelium in the colon and mild inflammatory cell infiltration in the adjacent lamina propria. The severity and extent of intestinal damage by strain NRT36S was dose dependent. Higher doses of strain NRT36S caused severe necrotizing colitis and enteritis, with bacteremia and mortality at less than 24 h in RITARD rabbits challenged with 10(9) CFU and at less than 48 h in RITARD rabbits challenged with 10(4) CFU. Electron and light microscopy demonstrated invasion of NRT36S into the luminal epithelial cells of the intestine. Challenge of RITARD rabbits with non-O1 V. cholerae A-5 and 2076-79 (strains which did not cause diarrhea in volunteers) did not cause diarrhea or intestinal pathology. Intestinal colonization was transient: at 72 h postchallenge, animals inoculated with strain A-5 were culture negative, while only low numbers of strain 2076-79 were detectable (approximately 0.4 to 0.8 CFU/g). Our data highlight the utility of the RITARD model, when combined with appropriate pathologic and bacteriologic studies, for obtaining insights into pathophysiologic mechanisms of enteric disease by non-O1 V. cholerae. In agreement with volunteer studies, non-O1 V. cholerae NRT36S is clearly pathogenic in this model; direct cell invasion may play a role in its ability to cause illness. PMID- 1730474 TI - Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro. AB - Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies. PMID- 1730475 TI - Role of gamma interferon and tumor necrosis factor alpha in resistance to Salmonella typhimurium infection. AB - In mice infected with a sublethal dose of Salmonella typhimurium, the injection of an anti-gamma interferon (IFN-gamma) monoclonal antibody increased bacterial proliferation in the spleen and led to death on day 7 or 8. Depletion of both CD4+ and CD8+ T cells with monoclonal antibodies in vivo had a much less marked effect during the first week of infection than the administration of anti-IFN gamma antibodies, suggesting that cells other than T lymphocytes participate in the production of IFN-gamma at this time. Administration of anti-tumor necrosis factor alpha (TNF-alpha) antibodies to mice infected with a sublethal dose of S. typhimurium induced the same effect as anti-IFN-gamma antibodies, while the administration of both antibodies resulted in a synergistic interaction. When mice were infected with an avirulent strain of S. typhimurium and challenged on day 7 either with a virulent strain of S. typhimurium or with Listeria monocytogenes, their resistance to reinfection was slightly depressed by anti-IFN gamma or anti-TNF-alpha antibodies given 1 day before challenge and much more strongly depressed by the simultaneous administration of both antibodies. Taken together, these results indicate that IFN-gamma and TNF-alpha play an essential role in acquired resistance during the early phase of S. typhimurium infection. PMID- 1730476 TI - Demonstration of a B-lymphocyte mitogen produced by the Lyme disease pathogen, Borrelia burgdorferi. AB - Lyme disease refers to the multisymptomatic illness in humans which results from infection with the tick-borne spirochete Borrelia burgdorferi. The white-footed mouse is the major reservoir for B. burgdorferi and, upon infection, certain inbred mice develop symptoms similar to those reported in human disease. Sonicated preparations of washed spirochetes were found to have potent mitogenic activity when cultured with lymphocytes from naive C57BL/6, C3H/HeJ, or BALB/c mice. The activity of the B. burgdorferi sonicate was approximately fourfold greater than that of a similarly prepared Escherichia coli sonicate. Polymyxin B efficiently inhibited the mitogenic activity of the E. coli sonicate but only slightly inhibited that of the B. burgdorferi sonicate, suggesting that a lipid A containing lipopolysaccharide was not responsible for the B. burgdorferi activity. Kinetic analysis indicated peak proliferation at 2 to 3 days of culturing, suggesting polyclonal activation. B- and T-lymphocyte depletion experiments indicated that the major cell type responding to the B. burgdorferi mitogen was the B lymphocyte. This mitogen stimulated murine B cells not only to proliferate but also to differentiate into antibody-secreting cells, as demonstrated by the production of immunoglobulin by stimulated splenocytes. Furthermore, the sonicated preparation stimulated the B-cell tumor line CH12.LX to secrete immunoglobulin in the absence of accessory cells. B. burgdorferi also stimulated interleukin-6 production in splenocyte cultures. The observation that B. burgdorferi can stimulate activation of and immunoglobulin production by normal B lymphocytes may directly reflect on the development of arthritis associated with persistent infection by this organism. PMID- 1730477 TI - Effect of parenteral immunization on the intestinal immune response to Salmonella typhi Ty21a. AB - The effects of parenteral administration of a killed typhoid vaccine on the intestinal immune response to live orally administered Salmonella typhi Ty21a in human subjects was evaluated. Priming with parenteral vaccination neither enhanced nor suppressed the subsequent specific serum and intestinal immunoglobulin A (IgA) immune responses to a booster course of live oral vaccine. Neither a single oral dose of live vaccine nor a single dose of parenteral vaccine had any measurable booster effect on the observed primary intestinal IgA response to the live oral vaccine. Two booster doses of subcutaneously administered killed typhoid vaccine did result in a significant increase in the specific intestinal IgA antibody in those subjects primed with the oral live vaccine. This response was comparable in magnitude to the primary intestinal response. No evidence of this response could be found in serum IgA, although nonsignificant rises in serum IgG were evident. Previous parenteral priming had no effect on secondary immune responses to a live oral vaccine in humans. Serum immune responses were generally found to be of little value as indicators of local intestinal immunity. This study confirmed that parenteral vaccination was only able to induce an intestinal immune response following priming with live, orally administered organisms and that multiple parenteral booster doses were necessary to induce a measurable effect on intestinal immune responses. PMID- 1730478 TI - Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment. AB - The structural gene, hap, for the secreted hemagglutinin/protease (HA/protease), a putative virulence factor of Vibrio cholerae, has recently been cloned and sequenced (C. C. Hase and R. A. Finkelstein, J. Bacteriol. 173:3311-3317, 1991). The availability of the null mutant, HAP-1, and HAP-1 complemented with pCH2 (which expresses HA/protease), enabled an examination of the role of HA/protease in the virulence of V. cholerae in an animal model. However, the mutants exhibited reversible colonial variation similar but not identical to that which was previously associated with dramatic changes in virulence of parental strain 3083. Regardless of colonial morphology, the mutants were found to be fully virulent in infant rabbits. Thus, the HA/protease is not a primary virulence factor (for infant rabbits). Observations using cultured human intestinal cells indicated, instead, that the HA/protease is responsible for detachment of the vibrios from the cultured cells by digestion of several putative receptors for V. cholerae adhesins. PMID- 1730479 TI - Protective local and systemic antibody responses of swine exposed to an aerosol of Actinobacillus pleuropneumoniae serotype 1. AB - The isotype-specific antibody responses in serum and in nasal and pulmonary lavage fluids of swine following aerosol immunization with an attenuated strain of Actinobacillus pleuropneumoniae serotype 1, strain CM5A, was investigated. The presence of immunoglobulin G (IgG), IgA, and IgM with specificities for capsular polysaccharide, lipopolysaccharide, and hemolysin was determined by enzyme-linked immunosorbent assay by using purified antigens. Strain CM5A induced serum antibodies of each isotype to the three antigens. The serum antibody response was sustained and typical of persistent antigenic stimulation. The specific IgM response decreased and the specific IgG response increased after challenge with strain CM5. IgA specific for the three antigens was detected in nasal secretions from all immune pigs, whereas specific IgG could only be detected in samples contaminated with blood. Both IgA and IgG specific for each of the antigens were detected in pulmonary lavage samples. There was no significant increase in specific IgA in nasal secretions; however, levels of lipopolysaccharide-specific and hemolysin-specific IgG and IgA in pulmonary secretions rose after aerosol challenge with strain CM5. Passive transfer of immune swine serum resulted in protection against pleuropneumonia and in levels of specific serum IgG which were similar to those in actively immunized pigs. It is concluded that specific serum IgG antibodies are important in protection from porcine pleuropneumonia. PMID- 1730480 TI - An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine. AB - Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv). Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight. Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity. Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity. Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response. Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions. The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form. An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv. Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K). These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively. The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT IIvE167D. When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold. Pigs that were vaccinated with purified, mutant toxin designated SLT IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions. These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED. PMID- 1730481 TI - Characterization of a cellular protease that cleaves Pseudomonas exotoxin. AB - Pseudomonas exotoxin (PE) is a 66-kDa bacterial toxin that is proteolytically cleaved by cells to produce an N-terminal fragment of 28 kDa and a C-terminal 37 kDa fragment which translocates to the cytosol and inhibits protein synthesis (M. Ogata, V.K. Chaudhary, I. Pastan, and D.J. FitzGerald, J. Biol. Chem. 265:20678 20685, 1990). When cells were broken by homogenization, the appropriate proteolytic activity was found associated with cellular membranes and not in a soluble fraction. Proteolysis of PE by crude membranes was stimulated by divalent cations, was ATP independent, and had a pH optimum of 5.5. When cells were disrupted by nitrogen cavitation and fractionated on Percoll gradients, proteolytic activity was present in fractions corresponding to the density of plasma membranes or endosomes but not in fractions containing lysosomes. Proteolytic activity was recovered in detergent extracts after crude membranes were treated with Nonidet P-40 or octylglucoside. Proteolysis of PE by either crude membranes or detergent extracts generated fragments of 28 and 37 kDa. The sizes of these fragments resembled those produced by intact cells. However, when the nontoxic mutant, PEgly276, which cannot be cleaved appropriately by intact cells, was incubated with membranes or extracts there was no production of the 28 and 37-kDa fragments. PMID- 1730482 TI - Enhancement of mucosal antibody responses to Salmonella typhimurium and the microbial hapten phosphorylcholine in mice with X-linked immunodeficiency by B cell precursors from the peritoneal cavity. AB - The observation that approximately half of the B cells in the murine intestinal lamina propria are derived from peritoneal CD5 B-cell precursors raises the question of their contribution to mucosal protection. Using mice with X-linked immunodeficiency which are deficient in CD5+ B cells, we showed that they mount little serum and virtually no intestinal immunoglobulin M (IgM), IgG, and IgA antibody responses following oral inoculation with live Salmonella typhimurium. Nonresponsive Xid mice were reconstituted with responsive CBA/Ca donor cell preparations which were constitutively enriched or depleted of CD5 B-cell precursors. Reconstitution of irradiated Xid mice with CD5 B-cell-deficient bone marrow from CBA/Ca donors marginally improved IgM responses in the intestinal mucosa but had no effect on IgG or IgA in response to oral immunization with live S. typhimurium. Whenever Xid mice were reconstituted with donor cells from the peritoneal cavity, which are enriched for CD5 B-cell precursors, strong IgA and in some cases IgG responses in the intestinal mucosa were stimulated in response to oral immunization. When mucosal and serum antibody responses were compared, the peritoneal donor cells again reinstated maximal serum antibody responses to S. typhimurium. Serum and mucosal responses to the bacterial hapten phosphorylcholine could be induced in Xid mice after immunization with S. typhimurium or hapten-carrier conjugates but only following reconstitution with donor cells containing CD5 B-cell precursors. These observations suggest that different lymphoid compartments are enriched for regulatory or effector cells which vary in their contributions to the mucosal antibody response against epitopes on S. typhimurium. PMID- 1730484 TI - Characterization of the neurotoxin isolated from a Clostridium baratii strain implicated in infant botulism. AB - Botulism is widely known to result from ingestion of food containing botulinum neurotoxin produced in situ by certain strains of Clostridium botulinum. Infant botulism caused by C. botulinum, unlike the food-borne intoxication, is the toxicoinfectious form of botulism (S. S. Arnon, p. 331-345, in G. E. Lewis, ed., Biomedical Aspects of Botulism, 1981). The strain of Clostridium baratii implicated in infant botulism produced a neurotoxin that was neutralized with antiserum for botulinum neurotoxin serotype F (J. D. Hall, L. M. McCroskey, B. J. Pincomb, and C. L. Hatheway, J. Clin. Microbiol. 21:654-655, 1985). We developed a procedure to culture the toxigenic C. baratii (strain 6341) in dialysis bags and a simple purification scheme (precipitation of 900-ml culture supernatant with ammonium sulfate and two anion-exchange chromatographic steps at pH 5.5 and 8.0) that yielded up to 150 micrograms of purified neurotoxin. It is an approximately 140-kDa single-chain protein and has the following sequence of amino acid residues at the N terminus: Pro-Val-Asn-Ile-Asn-Asn-Phe-Asn-Tyr-Asn Asp-Pro-Ile-Asn-Asn-Thr-Thr-Ile- Leu. Comparison of this amino acid sequence with those of the botulinum neurotoxin serotypes A, B, and E showed 40 to 50% identical residues in comparable positions. The specific toxicity of the neurotoxin, approximately 2 x 10(6) 50% lethal doses for mice per mg of protein injected, was not enhanced significantly by mild trypsinization, although the protease cleaved the neurotoxin within a disulfide loop that generated at least two primary fragments, approximately 47 and approximately 86 kDa, that remained linked by an interchain disulfide. These two fragments resembled the light and heavy chains of the well-characterized neurotoxin serotypes A, B, C, D, E, and F produced by C. botulinum. PMID- 1730483 TI - Isolation and characterization of toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1. AB - We have isolated and characterized four toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1. Similar to previously described mutants (B. Wretlind and O. R. Pavlovskis, J. Bacteriol. 158:801-808, 1984), the mutants appear to have a pleiotropic defect in the excretion of several extracellular products, including toxin A, elastase, alkaline phosphatase, and phospholipase C. However, the mutants are not defective in the excretion of either alkaline protease or exoenzyme S. We also examined the localization and processing of toxin A in these mutants by using pulse-labeling experiments. Mature toxin A was found to be localized to the membranes only. Our results suggest that toxin A is localized to the outer membrane but is not exposed to the extracellular surfaces of the outer membranes. The results also suggest that toxin A obtained from the excretion deficient mutants has intact disulfide bonds. PMID- 1730485 TI - Endogenous tumor necrosis factor, interleukin-6, and gamma interferon levels during Listeria monocytogenes infection in mice. AB - Mice were infected intravenously with a sublethal dose of Listeria monocytogenes cells and then levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), and gamma interferon (IFN-gamma) in the bloodstreams, spleens, and livers were monitored. The maximum level of TNF was detected at 72 h in the spleens and livers, but TNF was never detected in the bloodstreams. IL-6 appeared in the bloodstreams and spleens and peaked at 48 h. The maximum level of IFN-gamma could be detected in all three specimens, and the highest titer was shown in the spleens. Endogenous TNF production was suppressed by in vivo administration of anti-CD4 monoclonal antibody (MAb) or anti-asialo GM1 antibody but not by anti CD8 MAb, whereas none of these antibodies suppressed endogenous IL-6 production. Endogenous production of neither IL-6 nor IFN-gamma was inhibited in rabbit anti recombinant mouse TNF-alpha antibody-treated mice. Similarly, production of TNF and IL-6 did not decrease in anti-mouse IFN-gamma MAb-treated animals, but TNF production was augmented in these animals. These results suggest that the these endogenous cytokines are produced by different mechanisms in L. monocytogenes infection. PMID- 1730486 TI - Cloning and expression of a Serpula (Treponema) hyodysenteriae hemolysin gene. AB - Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5 alpha, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens. PMID- 1730487 TI - Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers. AB - Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 delta aroC delta aroD), CVD 908 (Ty2 delta aroC delta aroD), and chi 3927 (Ty2 delta cya delta crp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 10(4) and 5 x 10(5) CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature greater than or equal to 38.2 degrees C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received chi 3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received chi 3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and chi 3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens. PMID- 1730488 TI - Molecular and cellular characterization of the 29-kilodalton peripheral membrane protein of Entamoeba histolytica: differentiation between pathogenic and nonpathogenic isolates. AB - To further characterize the 29-kDa surface antigen of Entamoeba histolytica, we analyzed the complete nucleotide sequence and compared the immunoreactivity of this antigen in pathogenic and nonpathogenic strains. Five cDNA clones (one 1.0 kb full-length clone, designated p13, and four partial-length clones) encoding the antigen were analyzed for allelic variation. Comparison of the nucleotide sequences revealed several single-nucleotide substitutions in all five cDNAs, two of which resulted in amino acid differences. Localization of the antigen to the amebic surface in a previous report (B. E. Torian, B. M. Flores, V. L. Stroeher, F. S. Hagen, and W. E. Stamm, Proc. Natl. Acad. Sci. USA 87:6358-6362, 1990) was corroborated by transmission electron microscopy showing colloidal gold particles on the surface of the trophozoites. Computer analysis of the deduced amino acid sequence predicted that the protein encoded by p13 was a hydrophilic peripheral membrane protein, and these data were confirmed by a Triton X-114 membrane extraction showing the presence of the 29-kDa antigen primarily in the aqueous phase of the detergent partition. Monoclonal antibodies to a fusion peptide differentiated between pathogenic and nonpathogenic clinical strains of E. histolytica in immunoblots. Although no immunoreactive epitopes were detected on nonpathogenic strains, Northern (RNA) analysis and DNA-DNA hybridization with a 700-bp cDNA probe demonstrated that mRNA and the gene encoding the 29-kDa surface antigen were present in both pathogenic and nonpathogenic clinical isolates. PMID- 1730489 TI - Virulence of Streptococcus suis type 2 strains in newborn germfree pigs depends on phenotype. AB - To determine whether the virulence of Streptococcus suis type 2 is associated with the phenotype of the strain, we infected newborn germfree pigs with 10 strains of S. suis type 2 categorized by three phenotypes. In an earlier study, the phenotypes were distinguished by the presence or absence of the muramidase released protein (MRP) and an extracellular factor (EF) and were designated MRP+ EF+, MRP+ EF- and MRP- EF-. Pigs were first inoculated with Bordetella bronchiseptica to predispose them to infection and were then intranasally inoculated with the streptococci. Strains of the MRP+ EF+ phenotype induced fever and increased the number of polymorphonuclear leukocytes in blood. Specific clinical signs of disease such as nervous disorders and lameness were also observed. At necropsy bacteriologic and pathologic examination disclosed meningoencephalitis, polyserositis, and polyarthritis. Strains of the MRP+ EF- phenotype induced only nonspecific clinical signs of disease such as recumbency, lack of appetite, and fever; only slight pathologic changes were detected in the serosae. The four strains of the MRP- EF- phenotype induced no signs of disease. These findings indicate that the 110-kDa EF and, to a lesser degree, the 136-kDa MRP may be associated with the virulence of the bacterium. The results demonstrated that S. suis type 2 strains producing both MRP and EF are pathogenic for pigs. PMID- 1730490 TI - Molecular chimerization of Pasteurella haemolytica leukotoxin to interleukin-2: effects on cytokine and antigen function. AB - A chimeric recombinant protein composed of the lktA gene product from Pasteurella haemolytica fused to bovine interleukin-2 (IL-2) was made. The LKT-IL-2 chimera was compared with recombinant bovine IL-2 with regard to the ability to induce proliferative responses and LAK cell activity in bovine peripheral blood mononuclear cells in vitro. In both instances, chimerization had no effect on IL 2 activity. Similarly, the LKT component was unaffected in its ability to induce an effective immune response after immunization. The adjuvant properties of IL-2 have been established in a number of models, and this effect was tested by using the chimera. A multiple-injection protocol of LKT-IL-2 was compared with single dose administration of LKT. The results obtained indicate that while there was no increase in specific antibody production, the IL-2 component of the chimera may be able to affect antigen-specific proliferation, as assessed by limiting dilution analysis. Use of cytokine-antigen chimeras may provide a valuable antigen-adjuvant formulation that is simple to produce and purify and thus have economic advantages over conventional preparations. Furthermore, chimerization will also ensure that the adjuvant acts at the same site as the antigen, thus optimizing immunostimulatory activity. PMID- 1730491 TI - A vir-repressed gene of Bordetella pertussis is required for virulence. AB - Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS. In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal. We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli. DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441. The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats. Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions. In order to assess the role of vrg gene products in B. pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection. Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus. This is the first evidence that a vir-repressed gene may play an important role in the virulence of B. pertussis and the pathogenesis of whooping cough. PMID- 1730492 TI - Comparative immunogenicity of conjugates composed of the Staphylococcus aureus type 8 capsular polysaccharide bound to carrier proteins by adipic acid dihydrazide or N-succinimidyl-3-(2-pyridyldithio)propionate. AB - Staphylococcus aureus type 8 capsular polysaccharide (CP) was conjugated either to diphtheria toxoid or to Pseudomonas aeruginosa recombinant exoprotein A by using adipic acid dihydrazide (ADH) or N-succinimidyl-3-(2 pyridyldithio)propionate (SPDP) as the joining reagent. The polysaccharide/protein ratios of these two pairs of conjugates were similar. The two synthetic schemes bound the linker to the carboxyls of the type 8 CP by carbodiimide-mediated condensation. ADH was bound to the carboxyls of the protein, whereas SPDP reacted with the amino groups of the protein. Intermolecular linking of the carrier protein, caused by the carbodiimide during the conjugation reaction with the type 8 CP derivative, probably accounts for the larger size of the conjugates formed with ADH compared with those formed with SPDP. Both conjugates synthesized with ADH elicited higher levels of CP antibodies, especially after the first immunization, than did those prepared with SPDP. Similar levels of exoprotein A antibodies were elicited by both conjugates. Higher levels of diphtheria toxoid antibodies were elicited by the conjugate prepared with SPDP than by the one prepared with ADH. The basis for the differences in the immunogenicities of these two pairs of S. aureus type 8 CP conjugates is discussed. PMID- 1730493 TI - Antigenic diversity in Haemophilus ducreyi as shown by western blot (immunoblot) analysis. AB - The antigenic diversity within a panel of 63 Haemophilus ducreyi isolates was examined by Western blot (immunoblot) analysis with a pool of 238 well characterized human antisera. When a serum pool adsorbed on a mixture of Haemophilus influenzae, H. parainfluenzae, and H. parahaemolyticus was used, the immunoprofiles suggested that prominent antigenic proteins involved in the human immune response have apparent molecular masses of 63, 42, 34 to 30, and 28.5 to 28 kDa. Preliminary subcellular localization revealed that these antigens are associated with the cellular membrane. Two subsets of antigens were discriminated by detergent extraction. There was no evidence that the antigen composition is altered by changing the growth conditions. With a serum pool adsorbed on the Haemophilus spp. mixture supplemented with Actinobacillus actinomycetemcomitans, Pasteurella ureae, Neisseria gonorrhoeae, and Escherichia coli, antigenic determinants more specific for H. ducreyi were identified. An immunodominant 28.5 to 28-kDa protein was expressed by all H. ducreyi isolates. In the range from 34 to 30 kDa, 56 isolates revealed a dominant protein with variable molecular mass. By using both proteins (28.5 to 28 kDa and 34 to 30 kDa) as immunotypic markers, seven different immunopatterns were identified. Antigenic diversity among isolates from different geographical origins as well as from a single area was observed. PMID- 1730494 TI - In vitro inactivation of bacterial endotoxin by human lipoproteins and apolipoproteins. AB - A chromogenic Limulus amebocyte lysate assay was used to measure the recovery of 1 endotoxin unit of endotoxin per ml. Purified human high-density lipoprotein, low-density lipoprotein, and apolipoprotein A1 (apo A1) at a maximum concentration of 1 g of protein per liter reduced the recovery to less than 40% of baseline in a both dose- and time-dependent manner and in the absence of other serum components. Furthermore, the lapine fever response to a dose of 1 ml of 5 ng/ml endotoxin per kg was reduced by greater than 0.5 degrees C (P less than 0.005) when the solution was preincubated in vitro with 0.5 g of apo A1 per liter. By the Limulus test, a maximum concentration of 0.01 g of apolipoprotein B (apo B) per liter (which contained deoxycholate, a known endotoxin-disaggregating agent) reduced recovery to 0% in a dose- but not time-dependent manner. In heat inactivated (56 degrees C, 1 h) normal human serum, high-density lipoprotein cholesterol (P less than 0.005) and apo A1 (P less than 0.05) correlated inversely with endotoxin recovery, but, paradoxically, apo B correlated directly with endotoxin recovery (P less than 0.05), while low-density lipoprotein cholesterol showed no significant correlation. INTRALIPID alone had no effect on endotoxin recovery. Addition of a maximum of 10 g of INTRALIPID per liter to 0.0042 g of apo B per liter increased endotoxin recovery from approximately 30 to 80% (P less than 0.001), but addition of INTRALIPID to 0.25 g of apo A1 per liter decreased recovery from approximately 30 to 20% (P less than 0.001). We conclude that (i) lipoproteins are endotoxin inactivators; (ii) this ability of lipoproteins may be modulated by their lipid component (lipid-endotoxin interaction); (iii) apo A1 is capable of directly inactivating endotoxin (protein endotoxin interaction). PMID- 1730495 TI - Genetic association of mating types and virulence in Cryptococcus neoformans. AB - A pair of congenic Cryptococcus neoformans var. neoformans strains, B-4476 (a mating type) and B-4500 (alpha mating type), that presumably differ only in mating type was constructed. This pair and their progeny, five alpha type and five a type, were tested for virulence in mice. In the parent strains as well as the progeny, alpha type was clearly more virulent than a type. In addition, death tended to occur earlier among the alpha-strain-infected mice that died than among the mice that died by infection caused by a strains. These data strongly suggest the genetic association of virulence with mating type in this human fungal pathogen. PMID- 1730496 TI - Identification of the protective 44-kilodalton recombinant antigen of Ehrlichia risticii. AB - Protective studies were conducted with mice by using recombinantly produced antigens, polyacrylamide gel electrophoresis-fractionated antigens, and a monoclonal antibody specific to the 28-kDa antigen of Ehrlichia risticii. Analysis of E. risticii-infected cell culture used as the challenge inoculum indicated an inverse relationship between the progression of cell culture infection and the infective capability of E. risticii for mice. A recombinant 44 kDa antigen was found to protect mice considerably against challenge infection, while the monoclonal antibody and fractionated antigens were not protective. A potentiation of protection was observed when the recombinant 44-kDa antigen was combined with the recombinant 70-kDa antigen and used for mouse immunization. PMID- 1730497 TI - Human secretory and serum antibodies recognize environmentally induced antigens of Giardia lamblia. AB - The variability in duration and severity of infection with Giardia lamblia is likely to be due to trophozoite interactions with immune and nonimmune components of the small intestinal milieu. Despite its potential importance, nothing is known of the isotype or the specificity of the secretory antibody response to G. lamblia. In the present study, we show that serum and secretory antibodies recognize many Giardia antigens whose expression is induced by exposure to selected intestinal conditions. Isotype-specific immunoblots of antigens from trophozoites grown at pH 7.0 without bile or at the intestinal pH of 7.8 with bile were reacted with milk or serum antibodies from subjects with or without histories of giardiasis. While the results were complex, several key observations emerged. Serum and secretory immunoglobulin A (IgA), IgM, and IgG antibodies reacted with many regulated antigens. Antigen recognition patterns varied with isotype and between milk and serum antibodies of the same isotype. Antigen recognition also differed among subjects. Antibodies from virtually every patient recognized some G. lamblia antigens. Furthermore, milk and/or serum samples from putative controls without histories of giardiasis were positive more frequently than would be predicted from published prevalence studies, suggesting either that these antibodies may be cross-reactive or that undiagnosed infections with G. lamblia may be more common than previously thought. Thus, recognition of neoantigens induced by host conditions may be due to conserved or cross-reactive epitopes which could constitute a form of immune evasion by G. lamblia. PMID- 1730498 TI - Induction of proliferative responses of T cells from Babesia bovis-immune cattle with a recombinant 77-kilodalton merozoite protein (Bb-1). AB - A major portion of a Babesia bovis-specific gene encoding a 77-kDa merozoite protein (Bb-1) produced during natural infection in cattle and in microaerophilous culture was subcloned into the pGEX1N expression vector. Recombinant Bb-1 protein fused to glutathione S-transferase (Bb-1-GST) was used to examine cellular immune responses in B. bovis-immune cattle. Sera from rabbits immunized with Bb-1-GST reacted with fusion protein and with the native antigen present in crude B. bovis but not with B. bigemina merozoites. Bb-1-GST but not GST induced strong proliferation of T lymphocytes from these immune cattle, and Bb-1-reactive T-cell lines which consisted of a mixed population of either CD4+ and CD8+ cells or CD4+, CD8+, and "null" (gamma delta T) cells were established by in vitro stimulation of peripheral blood mononuclear cells with the recombinant fusion protein. Three CD4+ CD8- and three CD4- CD8+ Bb-1-specific T cell clones were identified after limiting-dilution cloning of the cell lines. The studies described here demonstrate that the 77-kDa protein of B. bovis contains T-cell epitopes capable of eliciting proliferation of two types of T cells in immune cattle, an important consideration for the design of a recombinant subunit vaccine. PMID- 1730499 TI - Topographic mapping of Helicobacter pylori colonization in long-term-infected pigs. AB - Four barrier-born pigs were inoculated with Helicobacter pylori during gastroscopy. Infection in all pigs was established after 3 weeks, and the animals were kept isolated from other pigs in ordinary experimental sites. The pigs were sacrificed and examined 3, 5, 6, and 6.5 months postinoculation. A detailed urease mapping of the pig stomachs showed a patchy distribution of H. pylori. The bacteria colonized in all pigs, with a concentration of H. pylori-positive areas in the antrum and fundus. Furthermore, the number of colonized areas tended to increase with time, and some of these areas showed a strong urease reaction, indicating a heavy colonization with H. pylori. Biopsies from these areas contained 10(2) to 10(5) CFU per 2-mm-wide biopsy. We conclude that persistence of H. pylori infection in barrier-born pigs can be demonstrated for at least 6.5 months. The patchy distribution and the variability of viable bacteria were similar to those described for humans. PMID- 1730500 TI - Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice. AB - Vaccination with recombinant outer surface protein A (OspA) has been shown to protect mice from infection with Borrelia burgdorferi, the Lyme disease agent. To determine whether antibodies to B. burgdorferi proteins other than OspA are involved in protective immunity, antibodies to OspA were removed from protective anti-B. burgdorferi serum; the residual serum was still protective. Absorption of OspA and OspB antibodies from anti-B. burgdorferi serum eliminated the protective effect. Therefore, active immunization experiments were performed to determine the roles of OspB and flagellin in protective immunity and to determine whether protective immunity induced by OspA is dose dependent. Active immunization with recombinant OspA protected mice from infection with an inoculum of 10(4) spirochetes, but this protection could be overcome with a challenge of 10(7) spirochetes; OspB protected mice from infection with an inoculum of 10(3) spirochetes but was insufficient to fully protect against 10(4) organisms; and immunization with flagellin had no protective effect. These studies suggest that OspA and OspB, but not flagellin, play roles in protective immunity to spirochete infection. PMID- 1730501 TI - Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. AB - The protective efficacy of immunization against anthrax with Bacillus anthracis protective antigen (PA) combined with different adjuvants was tested in Hartley guinea pigs and CBA/J and A/J mice. Adjuvant components derived from microbial products that were tested included threonyl-muramyl dipeptide (threonyl-MDP); monophosphoryl lipid A (MPL); trehalose dimycolate (TDM); and the delipidated, deproteinized, cell wall skeleton (CWS) from either Mycobacterium phlei or the BCG strain of Mycobacterium bovis. Non-microbially derived adjuvants tested included aluminum hydroxide and the lipid amine CP-20,961. In guinea pigs, all adjuvants and adjuvant mixtures enhanced antibody titers to PA as well as survival after a parenteral challenge of virulent B. anthracis Ames spores. In contrast, PA alone or combined with either aluminum hydroxide or CP-20,961 failed to protect mice. Vaccines containing PA combined with threonyl-MDP or MPL-TDM-CWS protected a majority of female CBA/J mice. Statistical analysis of survival data in the guinea pigs indicated that PA-MPL-CWS and PA-MPL-TDM-CWS were more efficacious than the currently licensed human anthrax vaccine. PMID- 1730502 TI - Mycoplasma pulmonis possesses a novel chemoattractant for B lymphocytes. AB - Mycoplasma pulmonis causes chronic murine respiratory mycoplasmosis, which is characterized by extensive peribronchial and perivascular infiltration of mononuclear cells, including B lymphocytes. B-lymphocyte recruitment into sites of inflammation is presently poorly understood but must involve directed chemotaxis of these cells in response to some external recruitment stimulus. In these studies, picogram amounts of M. pulmonis membrane protein were found to possess potent chemoattractant activity for resting rat B lymphocytes. This report is the first description of a bacterially derived chemoattractant for B lymphocytes and offers a unique opportunity to study regulation of B-lymphocyte recruitment to a site of chronic pulmonary inflammation. Furthermore, M. pulmonis membrane activation of fresh rat serum was found to produce a potent stimulus for recruitment of peritoneal and alveolar macrophages. M. pulmonis-mediated recruitment of lymphocytes and macrophages may play a significant role in the pathogenesis of murine respiratory mycoplasmosis, a role in which organisms on the bronchiolar epithelial surfaces may release proteins which can directly or indirectly promote chemotaxis of inflammatory cells from the circulation. PMID- 1730504 TI - Binding of cultured mammalian cells to immobilized bacteria. AB - The invasin protein of Yersinia pseudotuberculosis binds to integrin receptors on mammalian cells and promotes cellular penetration. We demonstrate here that the cell attachment activity of invasin can be detected in bacterial colonies that have been immobilized on filter membranes. Invasin expressed in either Escherichia coli K-12 or Y. pseudotuberculosis mediated binding to membranes, and as few as 10(5) Y. pseudotuberculosis resulted in detectable attachment of cultured epithelial cells. A similar binding activity was detected in clinical isolates of the related pathogen Y. enterocolitica but not in environmental isolates. Although there exist multiple mechanisms for the binding of wild-type organisms to host cells, efficient mammalian cell binding to immobilized Y. pseudotuberculosis required expression of a functional invasin protein. Several pathogens that are known to bind or penetrate mammalian cells were also tested, and only one of these bound cultured mammalian cells efficiently. PMID- 1730503 TI - Geographically restricted heterogeneity of the Plasmodium falciparum circumsporozoite protein: relevance for vaccine development. AB - The design of a subunit vaccine against the malaria parasite relies on the epitopes recognized by T cells being identified and polymorphisms therein being defined. Here we present the analysis of a 354-bp fragment of the circumsporozoite (CS) protein encompassing defined proliferative and cytotoxic T cell recognition regions. We reveal that the polymorphism of CS protein T-cell sites appears to be very limited among Plasmodium falciparum isolates prevalent in certain geographical regions, in particular Papua New Guinea. Furthermore, the more extensive polymorphism noted in other areas appears to be restricted. Although the extent of variation observed for the T-cell recognition domains suggests that any vaccine designed to stimulate this form of immunity will need to be polyvalent, this variation appears to be finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways to increase immune responsiveness can be found, then a vaccine designed to stimulate CS protein-specific T-cell activity may prevent malaria. PMID- 1730505 TI - Clustering of an outer membrane adhesin of Haemophilus parainfluenzae. AB - Haemophilus parainfluenzae synthesizes an outer membrane protein adhesin which mediates binding to oral streptococci, salivary pellicle, and neuraminidase treated erythrocytes. An indirect gold labeling technique and immunoelectron microscopy verified the location of this outer membrane protein. Further, a clustering of gold particles was observed in irregular patches at the cell surface. PMID- 1730506 TI - Intracellular growth inhibition of Histoplasma capsulatum induced in murine macrophages by recombinant gamma interferon is not due to a limitation of the supply of methionine or cysteine to the fungus. AB - Recombinant murine gamma interferon (rMuIFN-gamma) stimulates mouse peritoneal macrophages to inhibit the intracellular growth of the zoopathogenic fungus Histoplasma capsulatum. In some systems, the inhibition of growth of an intracellular parasite by rIFN-gamma has been related to nutritional constraints induced in the host cells by the lymphokine. Such an explanation might apply to H. capsulatum because the fungus is a functional methionine-cysteine (Met-Cys) auxotroph at 37 degrees C; its sulfite reductase is repressed at that temperature. For this reason, we set about to examine whether or not the antihistoplasma state induced in rMuIFN-gamma is due to a restriction in the availability of Met-Cys. Omission of Met-Cys from the medium in which macrophages were cultivated prevented H. capsulatum from growing within them. Addition of Met or Cys to the macrophage cultures did not antagonize the inhibitory effect induced in the cells by rMuIFN-gamma. Thus, there was no evidence from our work that rMuIFN-gamma evokes the antihistoplasma effect in mouse peritoneal macrophages by limiting the supply of Met-Cys to the fungus. PMID- 1730507 TI - Modified lymphocyte response to mitogens induced by the lipopeptide fragment derived from Mycobacterium avium serovar-specific glycopeptidolipids. AB - The beta-elimination procedure was used to obtain two major fragments of Mycobacterium avium glycopeptidolipid antigens. The lipopeptide fragment, not the oligosaccharide, diminished the mitogen-induced blastogenic response of spleen cells at concentrations lower than those which affected viability. Electron microscopy revealed an internalization of lipopeptide and disruption of intracellular organization. PMID- 1730508 TI - Polyclonal cell activity of a repeat peptide derived from the sequence of an 85 kilodalton surface protein of Trypanosoma cruzi trypomastigotes. AB - Some in vitro and in vivo biological activities of an octadecapeptide derived from an 85-kDa surface protein of Trypanosoma cruzi trypomastigote were studied. The peptide coupled to a carrier protein induced the proliferative response of lymph node cells from mice immunized with various antigens. Moreover, sera from mice immunized with the coupled peptide were found to contain antibodies against a number of self and nonself antigens: fibronectin, bovine serum albumin, myosin, tetanus toxoid, ovalbumin, keyhole limpet hemocyanin, and DNA. These results are discussed in the context of Chagas' disease immunopathology. PMID- 1730509 TI - Localized formation of micronuclei in the oral mucosa and tobacco-specific nitrosamines in the saliva of "reverse" smokers, Khaini-tobacco chewers and gudakhu users. AB - "Reverse"-cigar smokers (who hold the burning end of cigars within the mouth), dippers (who place a mixture of Khaini-tobacco and slaked lime into the lower gingival groove) and users of tobacco-containing toothpaste (gudakhu) in Orissa, India, were examined for precancerous oral lesions, the frequency of micronucleated cells at 3 different intra-oral sites, and levels of tobacco specific nitrosamines (TSNA) in the saliva. Among reverse-cigar smokers, a high incidence of leukokeratosis nicotina palati, an elevated frequency of micronucleated cells in the palate (2.5% as compared to 0.6% in non-smokers and non-chewers of tobacco) and tongue (2.1%) from which carcinomas preferentially develop, and up to 5890 ppb nitrosonornicotine and up to 1880 ppb N nitrosoanatabine in the saliva were found. Among Khaini-tobacco chewers, the frequency of micronucleated cells was elevated to 2.1% in the gingival groove, and up to 1580 ng N-nitrosonornicotine, 690 ng N-nitrosoanatabine, 90 ng N nitrosoanabasine, and 180 ng 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone) per ml of saliva were observed. The localized elevation of the frequency of micronuclei and cancer development is probably due to a synergistic effect of hyperthermia and tobacco-related carcinogens among reverse-cigar smokers, and to the close, prolonged contact between the mucosa and tobacco among Khaini tobacco/slaked lime dippers. Neither pre-cancerous lesions nor an elevated frequency of micronuclei were seen in the oral mucosa of users of gudakhu, a tobacco-containing toothpaste, which may be due to the low amount of TSNA released from the gudakhu and the short exposure time, which is restricted to the period of tooth brushing. PMID- 1730510 TI - Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA). AB - An automated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein was employed for chemotherapeutic-drug-sensitivity testing of tumor-cell suspensions from patients with leukemia. Fluorescence was linearly related to cell number, and reproducible measurements of drug sensitivity could be performed using fresh or cryopreserved leukemia cells. A marked heterogeneity with respect to chemotherapeutic drug sensitivity was observed for a panel of cytotoxic drugs tested in 43 samples from 35 patients with treated or untreated acute and chronic leukemia. For samples obtained from patients with chronic lymphocytic and acute myelocytic leukemia, sensitivity profiles for standard drugs corresponded to known clinical activity and the assay detected primary and acquired drug resistance. Individual in vitro/in vivo correlations indicated high specificity with respect to the identification of drug resistance. The results suggest that the FMCA may be a simple and rapid method for in vivo-representative determinations of chemotherapeutic drug resistance in tumor cells obtained from patients with leukemia. PMID- 1730511 TI - HTLV-I-positive and HTLV-I-negative peripheral T-cell lymphomas in Taiwan Chinese. AB - The clinico-pathologic features of 107 adult Chinese patients with peripheral T cell lymphoma (excluding primary cutaneous lymphoma) are described and a comparison between HTLVI+ and HTLV-I- patients is made. There were 27 HTLV-I+ and 80 HTLV-I- patients. The virus-positive and -negative groups both had a male predominance and an identical median age of 48. Most patients in both groups presented with stage-IV disease, B symptoms, lymphadenopathy and hepatosplenomegaly. The HTLV-I+ group had a significantly higher incidence of skin and pulmonary lesions, bone marrow and peripheral blood involvement, hypercalcemia, and elevated LDH level compared to the HTLV-I- group. Sinonasal lesions (10), mediastinal mass (5), and GI tract involvement (6) were only seen in the HTLV-I- group. Leukocytosis with the presence of circulating pleomorphic lymphoid cells was characteristic of HTLV-I+ cases, while cytopenia was more frequently present in HTLV-I- cases. All of the 24 HTLV-I+ patients tested were CD4+CD8-; of the 67 HTLV-I- patients tested, 46 were CD4+CD8-, 9 were CD4-CD8 , 5 were CD4-CD8- and 7 were CD4+CD8+. Phenotypic studies revealed significant differences in the expression of CD7 and CD25 between virus-positive and negative groups. Both groups responded poorly to therapy. The median survival of HTLVI+ and HTLV-I- patients was 4 months and 13.5 months, respectively. Apart from the presence of more than 3 extranodal lesions, none of the other clinical features or histologic subtypes had prognostic significance in the entire group or either of the subgroups. This series of peripheral T-cell lymphomas in Taiwan indicate that HTLV-I+ and HTLV-I- patients had many features in common, but presented several distinct differences. PMID- 1730512 TI - Analysis of regulation of T-cell responses by soluble inhibitory factors from the sera of patients with Hodgkin's disease. AB - Soluble inhibitory factors (SIF) have been demonstrated in the sera of cancer patients, which interfere with the T-cell activation process. We have shown that the major contributory factor to the inhibitory effect of sera from patients with Hodgkin's disease (HD) could be the soluble form of Interleukin-2 receptors (sIL 2R). The parameters studied to show the presence of SIF include (i) inhibiton of mitogen-induced proliferation; (ii) status of high- and low-affinity IL-2R; and (iii) internalization of IL-2-IL-2R complex, by lymphocytes from healthy donors activated with mitogen in presence of HD sera. Parameters studied to show the inhibitory role of sIL2R include (i) quantitation of sIL-2R in HD sera; (ii) effect of high-sIL-2R-containing sera on mitogen-induced proliferation and detection of IL-2 in activated lymphocyte culture supernatants; (iii) effect of exogenous IL-2 supplementation; and (iv) abrogation of inhibitory activity of sIL 2R-containing sera after passing them through IL-2 affinity columns. Our results show that 6/23 HD sera tested had high inhibitory activity (greater than 50% inhibition of mitogen-induced proliferation). The SIF did not affect expression of high- and low-affinity IL-2 receptors, or internalization of the complex by activated lymphocytes. Ten of the 15 sera tested showed significantly high levels of sIL-2R. Pooled sera with high sIL-2R content inhibited mitogen-induced proliferation of normal lymphocytes with a concomitant reduction in IL-2 activity in the lymphocyte culture supernatants. When supplemented with exogenous IL-2, there was a partial recovery of the inhibitory effect. When sIL-2R containing serum pool was passed on IL-2 affinity columns, the inhibitory effect was reduced. The eluted "sIL-2R" adsorbed on the IL-2 column showed anti proliferative effect. PMID- 1730513 TI - A novel O-acetylated ganglioside detected by anti-GD2 monoclonal antibodies. AB - Murine monoclonal antibody (MAb) 3F8 was previously shown to react with disialoganglioside GD2, but not with GD3, GT1b, GD1b, GD1a, GM1, GM3 and GM4. However, when the base-treatment step was ommitted from the standard neuroblastoma ganglioside extraction procedure, immuno-thin-layer-chromatography (ITLC) using 3F8 and other anti-GD2 MAbs revealed a new ganglioside band, abbreviated as NG (Rf 0.342) besides GD2 (R 0.183). It migrated below GD3 (Rf 0.358) on high-performance (HP) TLC plate and its binding to 3F8 on ITLC could be inhibited by rat anti-3F8 idiotypic antibody Idio-2, while the binding of GD2 to MAb 3F8 was not affected. Immunochemical analysis showed that this new neuroblastoma ganglioside contained alkali-sensitive O-acetylated sialic-acid residues recognized by MAb DI.I. After base treatment, its subsequent identity on ITLC was confirmed to be GD2. Lactonization of GD2 yielded 2 major bands, with Rf values (0.401, 0.583) distinct from that of the new ganglioside band. In addition, MAb DI.I did not bind to any of these GD2 lactones. Of 15 anti-GD2 MAbs studied, 13 reacted strongly with the novel ganglioside NG. By ITLC, this NG was found in ganglioside extracts of fresh surgical tumor specimens (4/4 neuroblastomas, I/I schwannoma and I/I anaplastic astrocytoma), and nude mice/rat xenografts (2/2 neuroblastomas, 2/2 osteogenic sarcomas). These data provided the first evidence that O-acetylated GD2 is a naturally occurring ganglioside derivative in human tumors and that it could cross-react with most anti-GD2 antibodies. PMID- 1730514 TI - Tumor-cell dissociation at the invasion front: a new prognostic parameter in gastric cancer patients. AB - Tumor-cell dissociation (TCD) is a well-known feature at the invasion front of malignant tumors, but little attention has so far been paid to its biological significance. Therefore, the aim of the present study was to score the degree of TCD at the invasion front (TCD 0-3) in a retrospective series of 445 gastric cancer patients and to compare its prognostic significance with the parameters T stage, tumor size, grade of tumor differentiation, lymph-node involvement and vascular invasion. We could show that highly significant differences (p less than 0.0001, log-rank test) existed between the 5-year survival rates of patients with different degrees of TCD (TCD 0: 92.3%; TCD 1: 62.1%; TCD 2: 41.2%. TCD 3:38.5%) and that these differences were independent of T-stage 2, tumor size and grade of tumor differentiation. Univariate Cox regression analysis showed that in intestinal-type carcinomas, T-stage (Chi-square: 45.2) followed by blood vessel and lymphatic vessel invasion (Chi-square: 43.6 and 40.0) had the highest prognostic value, whereas TCD (Chi-square: 31.5) revealed a prognostic value quite similar to that of lymph-node involvement (Chi-square: 35.1). This prognostic relevance was still present in multivariate Cox regression analysis, showing that the determination of TCD provided an increase in prognostic information which significantly (P = 0.026) exceeded the information obtained by the combination of T-stage and lymph-node involvement. Our data suggest that TCD is a new prognostic parameter, especially in those gastric carcinomas in which lymph-node involvement or vascular invasion are not present. PMID- 1730515 TI - Type-1 plasminogen activator inhibitor in human breast carcinomas. AB - We have investigated the occurrence of type-1 inhibitor of plasminogen activators (PAI-1) in human breast tumors. PAI-1 levels, measured by enzyme-linked immunosorbent assay, were significantly higher in malignant breast carcinomas (n = 178) than in benign breast tumors (n = 25). The levels of PAI-1 were found to be correlated with those of urokinase-type plasminogen activator (u-PA). The presence of PAI-1 in tumor extracts was also demonstrated by immunoblotting analysis. Immunohistochemical investigations by the use of monoclonal and polyclonal antibodies showed that PAI-1 was mostly localized in the tumor islands, associated with the tumor cells; in addition, it was present in vessel walls and in normal duct epithelia, but absent from the stroma. Analysis of RNA extracted from tumors by polymerase chain reaction revealed the presence of PAI-1 mRNA. We conclude that PAI-1 is present in human breast carcinoma cells, and that it is--at least partially-- produced locally, either by the cancer cells or by other cells in the tumors. We have previously demonstrated that a high level of u PA in human breast carcinomas is associated with poor prognosis. These results, combined with our present findings, present 2 possibilities: either the cancer cells need PAI-1 in order to utilize the u-PA-mediated pathway of plasminogen activation for invasion and metastasis; or PAI-1 represents a defense mechanism against tumor invasion. PMID- 1730516 TI - Food consumption and cancer of the colon and rectum in north-eastern Italy. AB - The relation between dietary factors and the risk of colorectal cancer was investigated in a case-control study conducted in Pordenone province, North eastern Italy, on 123 cases of colon cancer, 125 of rectal cancer and 699 controls admitted to hospital for acute, non-neoplastic or digestive disorders. Consistent positive associations were observed with more frequent consumption of bread (odds ratio, OR = 2.1 for colon and 2.2 for rectum for highest vs. lowest tertile), polenta (OR = 2.1 for colon, 1.9 for rectum), cheese (OR = 1.7 for colon, 1.8 for rectum) and eggs (2.5 for colon, 1.9 for rectum), whereas reduced ORs were observed in subjects reporting more frequent consumption of tomatoes (OR = 0.5 for colon and 0.4 for rectum). High consumption of margarine exerted a significant protection against cancer of the colon whereas high consumption of carrot spinach, whole-grain bread and pasta (favorably) and red meat (unfavorably) affected rectal cancer risk in particular. Thus the present study gives support for a protective effect associated with a fiber-rich or vegetable rich diet, while it indicates that frequent consumption of refined starchy foods, eggs and fat-rich foods such as cheese and red meat is a risk factor for colo rectal cancer. PMID- 1730517 TI - A case-control study for evaluating lung-cancer screening in Japan. Japanese Lung Cancer-Screening Research Group. AB - In order to evaluate the efficacy of lung-cancer screening, a case-control study was conducted using the data from 50 areas where population-based lung-cancer screening programmes have been operated by local municipalities. In most areas, chest X-ray examinations for all participants and sputum cytology for high-risk participants were offered annually. Case series consisted of 273 deceased lung cancer cases. For each case, 2 to 5 controls (a total of 1,269 controls) were collected from those who were alive at the time of diagnosis of the corresponding case, matched by sex, age, smoking status and type of health insurance. Cases and controls were limited to a high-risk group for males and a non-high-risk group for females. Screening histories, which were obtained from the list of screenees, were compared between case and matched controls for the identical calendar period before the time of diagnosis of the case. The odds ratio of dying from lung cancer for those screened within 12 months vs. those not screened was 0.72 (95% confidence interval 0.50-1.03; p = 0.07). The odds ratio increased towards unity, as the length of time in which screening histories were compared increased. After adjusting for some other variables, which appeared to be associated with the opportunities of chest X-ray examination, the estimated odds ratio did not change. These results suggest some benefits from lung-cancer screening in terms of reduction of lung-cancer mortality and should be subject to further research. PMID- 1730518 TI - Effects of sandostatin and castration on pancreatic carcinogenesis in rats and hamsters. AB - The effects of treatment with the somatostatin analogue Sandostatin, separately and in combination with surgical castration, on the development of azaserine induced lesions in rat pancreas and N-nitrosobis(2-oxopropyl)amine (BOP)-induced lesions in hamster pancreas were investigated. The animals were divided in 4 groups and treated as follows: (a) controls, injected s.c. with saline solution (0.9% NaCl); (b) orchiectomy directly after the last treatment with carcinogen; (c) Sandostatin (SMS 201-995) subcutaneously; (d) orchiectomy followed by treatment with Sandostatin. No significant suppressive effects on plasma EGF or IGF-I concentrations were noted after Sandostatin treatment, but plasma gastrin levels decreased slightly in the rats, not in the hamsters. In rats, Sandostatin treatment enhanced rather than inhibited growth of acidophilic atypical acinar cell nodules. In hamster pancreas, by contrast, Sandostatin inhibited the development of putative pre-neoplastic ductular lesions. There was no interaction between treatment with Sandostatin and surgical castration. It was concluded that Sandostatin, when administered prophylactically, has an inhibitory effect on the growth of putative pre-neoplastic ductular, but not acinar, lesions. PMID- 1730519 TI - Elevated p53 RNA expression correlates with incomplete osteogenic differentiation of radiation-induced murine osteosarcomas. AB - An important role for the p53 gene in osteogenic sarcomas has been imputed by identification of somatically acquired gene alterations in human osteosarcomas and by the development of osteosarcomas in p53 transgenic mice. To study the involvement of p53 in radiation-induced osteosarcomagenesis, we have investigated gene alterations and expression of p53 in radiation-induced murine osteosarcomas and tumor-derived cell lines. Eighteen of 31 tumors and 8 of 9 cells lines showed alterations in the p53 gene region, or elevated levels of p53 RNA. Expression of the osteoblast marker gene bone gla protein was substantially reduced in tumors which simultaneously showed high steady-state levels of p53 RNA. Our data indicate that p53, in addition to its function in regulating DNA synthesis, may be involved in the control of osteogenic differentiation in osteosarcomagenesis. PMID- 1730520 TI - Establishment and characterization of a thymic carcinoma cell line (Ty-82) carrying t(15;19)(q15;p13) chromosome abnormality. AB - A new human cell line, designated Ty-82, was established from the pleural effusion of a 22-year-old woman with undifferentiated thymic carcinoma. This cell line consisted of primitive cells that were positive for alpha-naphthyl butyrate esterase and acid phosphatase. The cells were shown to express epithelial membrane antigen, but were completely negative for cytokeratin, carcinoembryonic antigen, glial fibrillary acidic protein, desmin, S-100 protein, lysozyme, Leu-7, HLA-DR (Ia), leukocyte common antigen, Ki-I antigen, T-cell antigens, B-cell antigens, myelomonocyte antigens, and Epstein-Barr-virus nuclear antigen. Electron microscopy showed that the cells were highly anaplastic, with no sign of cellular differentiation to any lineages. The Ty-82 cell line was found to have a karyotype of 46,XX,t(15;19)(q15;p13), being identical to that of the patient's tumor cells. Four of 5 nude mice inoculated sub-cutaneously with Ty-82 cells developed tumors which displayed a histological picture similar to the original tumor. Thymic carcinoma is a recently recognized entity, and its cellular and clinical behavior are poorly understood. The newly established thymic carcinoma cell line would provide a useful tool for the better understanding of this rare disease. PMID- 1730521 TI - Growth-factor-independence and invasive properties of colorectal carcinoma cells. AB - During serial passage of the colorectal carcinoma cell line SW1116 in athymic nude mice, we selected 2 variants that metastasized to the lungs and liver. The metastatic capacity of these in vivo variant cell lines was associated with their ability to (1) grow under growth-factor-deprived conditions, (2) invade and transgress a reconstructed basement membrane with high effectiveness, and (3) produce higher activities of the substrate-degrading enzymes collagenase and plasminogen activator as compared to parental cells. To assess the relative contribution of growth-factor-independence and high levels of invasiveness/motility to the metastatic phenotype, variants of 6 colorectal carcinomas were selected in vitro by adaptation to a growth-factor-free culture medium followed by selection of highly invasive cells in chemoinvasion assays. Four out of 6 cell lines selected for growth-factor-independence showed significantly higher levels of invasiveness through reconstructed membranes, suggesting co-segregation of growth-factor-independence and high levels of invasiveness in vitro. Using an in vitro chemoinvasion assay, 2 poorly and 1 highly invasive cell line were further selected for invasiveness. After 6 selection passages, all cell lines were highly invasive and showed high motility rates. However, when injected s.c. into athymic nude mice to test their metastatic capacity in vivo, double-selected variant cell lines did not form spontaneous metastases. Our results indicate that growth-factor-independence and high levels of invasiveness, although associated with the metastatic phenotype, are not sufficient for experimental metastasis formation of colorectal carcinoma cells in vivo. PMID- 1730522 TI - Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells. AB - Limiting numbers of peripheral-blood mononuclear cells (PBMC) from melanoma patients were stimulated with irradiated autologous tumor cells in the presence of interleukins-2 and -4 and in the absence of feeder cells. The responder cells were restimulated every week. After 2 to 4 weeks, the microcultures were tested for their lytic activity against the autologous tumor cells. Significant lysis of the tumor cells was observed with a fraction of these microcultures, whereas no lysis was observed with control microcultures seeded without stimulator melanoma cells. Because our aim was to measure the precursor frequency of CTL showing specificity for the tumor, and not that of NK-like effectors that were also capable of lysing the melanoma cells, we used cold-target inhibition with an excess of NK target K562 to inhibit the NK-like activity. Microcultures whose lysis on the tumor cells was not abolished by K562 competition were observed. The specificity of these CTL clones was confirmed by the absence of lytic activity on autologous T-cell blasts. The numbers of microcultures with anti-tumor CTL activity fitted the zero-order of the Poisson distribution equation, indicating that they resulted from the activity of single T-cell clones. The frequency of anti-tumor CTL precursor cells (CTL-P) of 7 melanoma patients ranged from 1/900 to 1/33,000. Frequencies of anti-tumoral CTL-P were higher and NK-like effectors were less frequent when sorted CD8+ T lymphocytes were used as responder cells. PMID- 1730523 TI - Inhibition of azoxymethane-induced experimental colon carcinogenesis in Wistar rats by 6-hydroxydopamine. AB - The effects of 6-hydroxydopamine (6-OHDA) on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), and on norepinephrine (NE) concentration in the colon wall and the labelling index of the colon mucosa were investigated in Wistar rats. Rats received sub-cutaneous (s.c.) injections of AOM once a week for 10 weeks, and throughout 35 weeks were also given intraperitoneal (i.p.) injections of 6-OHDA. Prolonged administration of 6-OHDA was found to cause a significant reduction in the incidence and number of colon tumors. However, it had no influence on the histological features or depths of involvement of colon tumors and/or adenocarcinomas. Its administration also caused significant decreases in the NE concentration in the colon wall and in the labelling index of the colon mucosa. Our findings indicate that 6-OHDA has a protective effect against colon carcinogenesis, and that the activity of the sympathetic nervous system may have an important influence on colon carcinogenesis. PMID- 1730524 TI - An immunohistochemical survey of pS2 expression in human epithelial cancers. AB - The oestrogen-inducible pS2 protein isolated from human breast-cancer cells appears to have prognostic significance in breast-cancer patients. Using both monoclonal and polyclonal pS2 anti-sera, we have carried out an immunocytochemical survey of 9 epithelial cancers. Some degree of specific tumour staining was observed in 4 tissues, the extent varying greatly between specimens. Most (11/13) of the colorectal cancers showed immunoreactivity, as did 4/5 pancreatic carcinomas. In some of these cases, patchy staining was also observed in adjacent non-tumour cells. Two bronchio-alveolar carcinomas showed positivity, but 5 poorly differentiated adenocarcinomas of the lung did not. In the ovary, staining was observed in 3/3 mucinous cystadenocarcinomas but only in 1/8 of the serous type. No reactivity was seen in specimens of salivary gland, kidney, liver, prostate or uterus. This pilot study indicates that pS2 may be a useful marker of adenocarcinomas in human neoplasms apart from those in the breast, and suggests that more extensive surveys might be worthwhile. PMID- 1730525 TI - Attenuating effect of ornithine decarboxylase inhibitor (1,3-diaminopropane) on bombesin enhancement of gastric carcinogenesis induced by N-methyl-N'-nitro-N nitrosoguanidine. AB - The effects of combined administration of bombesin and the ornithine decarboxylase (ODC) inhibitor 1,3-diaminopropane (DAP) on the incidence and number of gastric tumors induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the ODC activity of the gastric wall and the labelling index of the gastric mucosa were investigated in inbred Wistar rats. Rats were given drinking water containing MNNG (50 micrograms/ml) for 25 weeks and then drinking water containing DAP (2.5 g/l) and/or injections of 40 micrograms/kg body weight of bombesin in depot form every other day. Administration of bombesin alone resulted in significant increases in the incidence of gastric cancers, the ODC activity of the antral portion of the gastric wall and the labelling index of the antral mucosa. Administration of DAP with bombesin significantly reduced enhancement by the latter of gastric carcinogenesis, ODC activity of the antral portion of the gastric wall and the labelling index of the antral mucosa. Our results suggest that ODC inhibition attenuated the enhancement of gastric carcinogenesis by bombesin, and that this enhancement by bombesin was mediated by polyamine biosynthesis. PMID- 1730526 TI - Identification and characterization of a rat protein (p 105) auto-antigenic in rats bearing a progressive syngeneic colon carcinoma. AB - Sera from BDIX rats inoculated with 2 tumor clones derived from a single syngeneic colon carcinoma were assayed by Western blotting for the presence of antibodies against the grafted tumor. The PROb clone is progressive and produces metastases. We observed that rats bearing this tumor developed antibodies against an unglycosylated water-soluble protein of 105 kDa. The magnitude of this humoral response, as assessed by the intensity of the signal on immunoblots, was inversely correlated with survival of the rats. Furthermore, rats inoculated with the REGb clone, which is immunologically rejected, never developed detectable antibodies against the tumor. Antisera from rats injected with PROb tumor detected p105 antigen in cellular extracts from the REGb clone and from a series of rat and human cell lines. This protein was also detected in variable amounts in some normal adult and fetal tissues. Treatment of PROb or REGb cells by either interferon-gamma or heat shock did not significantly alter the expression of the p105 auto-antigen. PMID- 1730527 TI - Comparison of spontaneous mutagenesis in early-passage human mammary cells from normal and malignant tissues. AB - Spontaneous mutant frequencies were determined in early-passage epithelial-cell strains derived from either normal or malignant human breast tissues, as well as a non-tumorigenic immortalized human mammary epithelial cell line (184B5) derived from normal cells. Mutations at the hypoxanthine-guanine phophoribosyltransferase (HPRT) locus were quantified by determining the 6-thioguanine resistance. Mutant frequencies in human mammary epithelial cells (HMEC) from 4 normal and 5 carcinoma tissue samples did not differ significantly. In contrast, the mutant frequency in the immortalized HMEC was approximately 10 times higher than in average normal HMEC and normal non-immortalized cells from early-passage cultures of the same cell lineage. Our data suggest that the progression of normal breast cells to invasive carcinoma cells does not necessarily involve the establishment of a general genetic instability during this progression. PMID- 1730528 TI - Enhanced CMP-NeuAc:Gal beta 1,4GlcNAc-R alpha 2,6 sialyltransferase activity of human colon cancer xenografts in athymic nude mice and of xenograft-derived cell lines. AB - The activity of an alpha 2,6 sialyltransferase acting on N-acetyllactosaminic sequences (alpha 2,6 ST E.C. 2.4.99.1) has previously been found to be increased in 90% of human colon cancer specimens. In the present study, the alpha 2,6 ST activity of 6 human colon cancer cell lines grown in culture was compared with that expressed by the corresponding nude mice xenografts and by the cell lines derived from the xenografts. We found that xenografts of COLO 205, HT-29, SW 620, SW 948 and SW 948 FL (a non-adherent sub-line of SW 948) cells express an alpha 2,6 ST activity much higher than that of the in vitro-grown cells. SW 48 cells grown either in culture or as xenografts lack the enzyme activity. All the xenograft-derived cell lines except HT-29 retained the increased alpha 2,6 ST activity at least for the first 6 passages. Those derived from SW 948 xenografts showed an enrichment of round, non-adherent cells, strongly reactive with the NeuAc alpha 2,6 Gal/GalNAc-specific lectin from Sambucus nigra (SNA), thus indicating that a selection of these cells has occurred. PMID- 1730529 TI - Tumor-cell motility and invasion within tumors determined by applying computer simulation to histologic patterns. AB - Proliferation and motility are crucial prerequisites for tumor-cell invasion in vivo. While proliferation can be assessed in situ by a variety of methods, the measurement of motility is largely restricted to in vitro models. In previous studies, computer simulations of tumor growth strongly indicated that a close relationship exists between tumor-cell motility on the one hand and the resulting morphological pattern on the other. Moreover, estimates of motility parameters can be based on image analysis of the particular pattern. The objective of the present study was to examine whether tumor-cell populations differing in their in vitro motility produce particular patterns in vivo similar to those predicted by the computer simulations, and whether the motility estimates derived from these patterns are consistent with the in vitro motility results. This was done using murine fibrosarcomas grown in syngeneic animals from various cell lines which had been selected for and confirmed to show greatly increased speeds of motility in vitro relative to the unselected parent cell population. Computer simulations coupled with image analysis of the various variant tumors showed that the calculated motility of tumor cells within the different tumors agreed well with the relative levels of tumor-cell motility observed in vitro. Our results thus show that the computer simulation method, along with histological analysis, produced reliable estimates of tumor-cell motility within tumors. PMID- 1730530 TI - Aging of the human retina. Differential loss of neurons and retinal pigment epithelial cells. AB - The impact of aging on cell loss in the human retina was examined in foveal and temporal equatorial regions in eyes from 35 donors with ages spanning a 78-yr period from the second to the ninth decade of life. Equatorial cones and retinal pigment epithelial cells (RPE) decreased at uniform rates from the second to the ninth decade, 16 and 14 cells/mm2/yr, respectively. Equatorial rods and cells in the ganglion cell layer (GCL) showed nonuniform rate decreases with age. The rates of rod and GCL cell loss were faster between the second and fourth decades (970 and 9 cells/mm2/yr, respectively) than between the fourth and ninth decades (570-330 and 6-3 cells/mm2/yr). The rod and GCL cell densities at the temporal equator maintained a constant ratio (rods-GCL cell ratio = 103 +/- 0.4, mean +/- standard deviation) and the same reduction slope ratio at different times during aging. Thus, the equatorial rod and GCL cell losses were correlated statistically. The ratio of equatorial photoreceptors to RPE cells showed no significant change with age, suggesting parallel loss of these closely apposed cells. At the foveal center, the variability of cone density between individuals in each decade grouping was large (1.7- to threefold). No significant differences were found in cone or RPE cell densities at the foveal center from the second to ninth decade, suggesting that the densities of foveal cones and RPE cells were stable throughout this period. Foveal RPE density was significantly higher than equatorial RPE density in each age group. No significant difference was found between the equatorial photoreceptor-RPE ratio and foveal cone-RPE ratio in any age group. Cells in the GCL in the fovea decreased by approximately 16% from the second to the sixth decade. These results indicated that (1) rod photoreceptors and cells in the GCL were more vulnerable to loss during aging than cones; (2) photoreceptors and RPE cells showed parallel changes during aging; and (3) the photoreceptor loss accompanying aging was less pronounced in the fovea than in the peripheral retina. PMID- 1730531 TI - Susceptibility of corneas from various animal species to in vitro binding and invasion by Acanthamoeba castellanii [corrected]. AB - A crucial requirement for establishing corneal infection by the extracellular protozoal parasite, Acanthamoeba, is the ability of the parasite to bind to the corneal surface. In a series of in vitro studies, we examined the ability of Acanthamoeba castellanii [corrected] to adhere, invade, and damage normal, intact corneas of 11 mammalian and one avian species. A. castellanii [corrected] (80-90% trophozoites and 10-20% cysts) were incubated with corneas for 24 hours in vitro and examined by scanning electron microscopy (SEM). Results of several independent SEM experiments revealed that parasites not only failed to produce cytopathic effects but did not even bind to the corneal epithelium of mice, rats, cotton rats, horses, guinea pigs, cows, chickens, dogs, and rabbits. However, parasites adhered, invaded, and produced severe damage to human, pig, and Chinese hamster corneas during the 24-hour in vitro incubation period. Additional in vitro experiments quantified the binding of A. castellanii [corrected] to the corneas of selected susceptible and nonsusceptible species. In vitro binding assays revealed scant binding of parasites to mouse, rat, and rabbit (range = 5 20 parasites/7.07 mm2 corneal button). In contrast, extensive binding was observed on Chinese hamster, pig, and human corneas (range = 100-200 parasites/7.07 mm2 button). The results indicate that A. castellanii [corrected] exercises rigid host specificity at the host cell surface. PMID- 1730532 TI - Analysis of glycoprotein deposits on disposable soft contact lenses. AB - By using gel electrophoresis, as well as Western blotting with specific antibodies or with the lectin concanavalin A, we characterized the types and amounts of proteins that are deposited on 58% ionic and 38% nonionic water content disposable soft contact lenses (DSCLs) worn for 1 to 21 days by asymptomatic subjects with mild to moderate myopic refractive errors. The total amounts of protein eluted from the lenses ranged from 0.1 to 80 micrograms/lens. The amount of protein deposited on 58% water-content lenses was greater than that on 38% water-content DSCLs. We did not find a strict correlation between the amount of protein deposited and the duration of wear for either type of lens. The major polypeptide fractions detected had apparent molecular weights of 14, 17, 21, 30, and 60 kD. The fractions at 14 kD-bound antibodies specific for human lysozyme, and those at 17 kD corresponded to prealbumin. The 60 kD fraction included IgG heavy chains. The identity of the fractions at 21 kD and 30 kD is unknown. Because oligosaccharide side chains on the proteins attract microbes and facilitate their adherence, knowledge about the types of carbohydrate moieties in lens deposits can provide a rational approach to inhibiting or reversing microbial infection. PMID- 1730533 TI - A pig model of Acanthamoeba keratitis: transmission via contaminated contact lenses. AB - A model of contact lens-induced Acanthamoeba keratitis was developed in Yucatan micropigs. Pigs fitted with parasite-laden soft contact lenses developed corneal infections that clinically and histopathologically mimicked the human counterpart. Three distinct stages of disease became apparent and were categorized as: acute, condensed infiltrate, and resolution stages. Viable parasites were isolated from corneal scrapings and smears were taken during the acute and condensed infiltrate stages. In addition, cysts could be identified deep within the stroma of histological specimens taken during the resolution stages. The characteristic dense, white ring-like infiltrates, stroma edema, keratic precipitates, and the chronic nature of the infections were similar to those observed in human Acanthamoeba keratitis. Histopathological examination of infected corneas revealed extensive neutrophilic infiltrates, stromal necrosis, and disorganization of the collagen lamellae. The strong correlation between the clinical and histopathologic features of contact lens-induced Acanthamoeba keratitis in the pig as well as the anatomical similarity of the pig eye with the human eye make the porcine model a valuable tool for investigations of the immunology, cell biology, and therapy for Acanthamoeba keratitis. PMID- 1730534 TI - Stromal acidosis affects corneal hydration control. AB - An estimate of overall corneal hydration control can be obtained by measuring the rate of thickness recovery following induced corneal swelling; it is expressed as the percent recovery per hour (PRPH). This recovery is nearly, but not exactly, exponential, because there appears to be an initial slower recovery phase lasting about 30-40 minutes. This 30-40 minute period of slower recovery corresponds to the time when corneal pH is reduced secondary to the contact lens-induced swelling, suggesting the possibility that stromal acidosis may retard the corneal deswelling process. In this study, we explored the effects of corneal acidosis on hydration control by monitoring corneal recovery under normal and reduced pH conditions. Corneal pH was controlled by having subjects were goggles and exposing their eyes to air (normal pH) or a gas mixture providing 21% O2 and 7% CO2 (low pH). Relative corneal pH levels were monitored by measuring fluorescence intensity (FI) ratios, which showed that the average (+/- standard deviation) FI ratio was significantly lower under 7% CO2 (0.838 +/- .024) vs air (0.985 +/- .025; P = 0.0001), corresponding to approximate pH values of 7.25 vs 7.50. Under these reduced pH conditions, open-eye steady-state (OESS) corneal thickness was not substantially affected. For 10 subjects, mean (+/- SD) corneal thickness decreased 0.93 +/- 3.7 microns vs 1.10 +/- 4.50 microns after exposures to 60 minutes of 7% CO2 and 40 minutes of air (P greater than or equal to 0.45), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730535 TI - Characterization of a potential marker of corneal epithelial stem cells. AB - Corneal epithelial stem cells are thought to be localized in the basal cell layer of the limbus. We developed a monoclonal antibody designated 4G10.3 that immunolocalized to limbal basal cells in rat corneas. Western blot analysis demonstrated that 4G10.3 reacted with a single band of 50,000 molecular weight in rat and rabbit corneal epithelium and 48,000-49,000 in human epithelium. Following extraction of corneal epithelium in 20 mM Tris-HCl (pH 6.8) and ultracentrifugation at 100,000 x g, the 50-kD protein was detected in the soluble fraction. 4G10.3 also was used to examine the response of the limbal basal cells to epithelial debridement and thermal burn wounds in the rat. In unwounded control corneas, 40.8 +/- 12.0 (mean +/- standard deviation) cell per limbal area bound 4G10.3. Following a 3 mm debridement wound, the number of cells binding 4G10.3 increased to 77.0 +/- 16.9 two days post-injury and returned to control levels by three days. Following thermal burn, 36.2 +/- 11.2, 68.8 +/- 15.8, 85.4 +/- 15.4, 104.6 +/- 13.8, and 88.0 +/- 40.2 cells per limbal area bound 4G10.3 18 hours, and 1, 2, 3, and 7 days post-injury, respectively. The 50 kD protein and 4G10.3 antibody provided a biochemical and immunological marker of limbal basal cells, hypothesized to be corneal epithelial stem cells. PMID- 1730536 TI - Discrimination between normal and glaucomatous eyes. AB - Discriminant analysis of quantifiable optic nerve, nerve fiber layer, and visual field measurements were used to assign eye to normal or glaucomatous groups. A database of 185 glaucoma patient with early visual field loss and 54 normal controls was used to develop and test the discriminant function. Parameters that discriminated best between normal and glaucoma were relative nerve fiber layer height and visual field mean defect. Cup-disc ratio, an estimate of optic nerve structure most commonly used by practitioners, was the weakest of the structural parameters to discriminate between normal and glaucoma. The combination of structural and functional measurements performed better than structural or functional measurements alone. When the discriminant function was applied to a group of 124 age-matched ocular hypertensives, 20% were assigned to the glaucoma group. Discriminant analysis of structural and functional measurements increases precision in identification of early glaucomatous damage, provides a probability that glaucomatous damage is present, and may help identify those ocular hypertensives who actually may have early damage. PMID- 1730537 TI - A study of aqueous humor formation in patients with cystic fibrosis. AB - The circadian pattern of aqueous formation and the effect of timolol on aqueous flow was studied in 12 patients with cystic fibrosis. Cystic fibrosis is a disease characterized by a defect in a chloride channel-associated regulatory protein found in epithelial cells. Improper regulation of these chloride channels, causes abnormal composition of exocrine secretions, including respiratory tract, gastrointestinal tract, exocrine pancreas, and sweat glands. Ocular findings previously reported include abnormal endothelial cell permeability, decreased tear secretion, and abnormal tear composition. In this study, aqueous humor flow was measured by fluorophotometry. No statistically significant difference was found when flow rates measured during the morning, during the afternoon, at night, and after topical timolol treatment were compared to normal values. The conclusion is that the beta adrenergically regulated chloride selective channels defective in patients in cystic fibrosis do not play a major role in the formation of aqueous humor or they are not regulated by the cystic fibrosis transmembrane conductance regulator (CFTR). PMID- 1730538 TI - Effect of paracentesis upon the blood-aqueous barrier of cynomolgus monkeys. AB - Anterior chamber paracentesis disrupts the blood aqueous barrier (BAB) of rabbits and nonhuman primates, but the magnitude and duration of breakdown in monkeys has not been clarified. We have studied anterior chamber paracentesis in cynomolgus monkeys as a potential model of postoperative BAB breakdown. The effect of a single paracentesis upon fluorescein sodium concentration in the anterior chamber after an intravenous injection was measured in 16 eyes of 8 animals. In an additional 10 eyes of 5 animals, aqueous humor was withdrawn for analysis 24 hours and one week following paracentesis. Anterior chamber fluorescein concentration was 57 +/- 22 ng/ml (mean +/- standard deviation) before paracentesis, rose to 81 +/- 47 ng/ml 24 hrs after paracentesis, and was 60 +/- 36 ng/ml at 72-96 hours. Twenty-four hours after paracentesis, total protein concentration was elevated, but ascorbic acid and transforming growth factor-beta levels were not. Paracentesis in monkeys has only a small and short lasting effect upon BAB integrity and is therefore unlikely to be a good model for assessing the effect of agents designed to stabilize the BAB. However, the short lived effect of paracentesis may permit the repetitive collection of "primary aqueous" for physiologic and biochemical studies. PMID- 1730539 TI - Immunocytochemical localization of cyclooxygenase in the rat lens. AB - The localization of rat lens fatty acid cyclooxygenase (prostaglandin synthase) was studied using indirect immunofluorescent and indirect streptavidin-biotin immunoperoxidase staining techniques. Both methods employed monoclonal and polyclonal antibodies directed against fatty acid cyclooxygenase, the key enzyme in the conversion of arachidonic acid to prostaglandins. The immunocytochemical studies demonstrate that (1) fatty acid cyclooxygenase is present in the rat lens epithelial cell layer; (2) the enzyme appears predominantly in the cytoplasm; (3) there is an apparent higher concentration of the enzyme in the region designated as germinative and transitional zones, and meridional rows; and (4) the enzyme appears to be absent in the lens capsule and in the nucleus of the lens. The presence of the cyclooxygenase enzyme in the lens epithelium, especially the relative intense staining in the epithelial mitogenic region, suggests that oxygenation of polyunsaturated fatty acids by the fatty acid cyclooxygenase may have an important role in cellular differentiation. PMID- 1730540 TI - The acute effect of topical epinephrine on macular blood flow in humans. AB - The acute effect of topical epinephrine HCl 2% on macular capillary blood flow was studied in 18 healthy human volunteers using the blue-field simulation technique. This technique provides a method for quantifying the velocity of leukocytes flowing in macular capillaries. Subjects adjusted the velocity of simulated leukocytes on a computer screen to match that of their own entoptically perceived leukocytes before instilling the drug and 2 hr thereafter. An artificial tear solution was instilled into the fellow eye for a control. Epinephrine instillation resulted in a 8% increase in macular leukocyte velocity and presumably blood flow (P less than 0.03, paired student t-test). PMID- 1730541 TI - Beneficial effects of a retinoic acid analog, CBS-211 A, on an experimental model of keratoconjunctivitis sicca. AB - We report the effects of CBS-211 A, a synthetic retinoic acid analog, on a previously described experimental model of keratoconjunctivitis sicca (KCS) in the rabbit. A 9-week topical treatment with 0.02% CBS-211 A in aqueous vehicle significantly increased the conjunctival goblet cell density (P less than 0.01, impression cytology counting), stopped the evolution of the corneo-conjunctival surface alteration (P less than 0.05, rose bengal test), and restored a basically normal mucosecretory product quality in goblet cells (lectin histochemistry) compared to vehicle treatment. The results assess the efficacy of this compound in reversing KCS pathology in a relevant model different from general vitamin A deficiency models, and strongly support the rationale for using such a well tolerated retinoid in dry eye treatment. PMID- 1730542 TI - Catch-up saccade amplitude is related to square wave jerk rate. AB - Mean catch-up saccade (CUS) amplitude and square wave jerk (SWJ) rate during pursuit were recorded in 20 normal controls, 23 patients with schizophrenia, and 15 patients with affective disorder, using infrared oculography. Target speed during pursuit was 5 degrees/sec. An especially robust correlation was noted in normal controls between SWJ rate during pursuit and mean CUS amplitude (Spearman's rs = 0.87, P less than 0.0001). This correlation also was present in the psychiatric patients (rs = 0.53, P = 0.0006), although it was significantly weaker than in normal controls (P less than 0.02). There were no significant differences between the patient groups regarding the strength of the relationship. Furthermore, similar strong correlations between SWJ rate during fixation and mean CUS amplitude also were found for normals (rs = 0.73, P = .0002) and both patient groups combined (rs = 0.52, P = 0.0009). The results suggest that saccadic intrusions during tracking tax the saccade correcting system, delaying correction for the position error that accumulates when gain is less than 1.0. PMID- 1730543 TI - Lowering the calcium concentration in the subretinal space in vivo loosens retinal adhesion. AB - Retinal adhesiveness in vitro is reduced by lowering the external calcium (Ca2+) concentration. The effects of lowering subretinal Ca2+ concentration in living rabbit eyes was investigated by making experimental retinal detachments (blebs) filled with Ca(2+)-free disodium edetate solution. Unlike blebs made with Hanks' solution, these low-Ca2+ blebs enlarged progressively after they were formed, and they were surrounded by a wide whitish halo. This halo region had weak adhesion (shown by the rapid spread of fluorescein solution into the halo and by the measurement of local adhesiveness after enucleation). The retinal pigment epithelial microvilli in the halo appeared stretched toward the center of the blebs as if there had been retinal traction or movement. Measurements of retinal adhesiveness in vivo showed it to be decreased to about 30% of normal by use of this solution. PMID- 1730544 TI - A photographic technique for measuring horizontal and vertical eye alignment throughout the field of gaze. AB - We present a photographic method based upon corneal light reflections for the measurement of binocular misalignment. Our procedures allow for the measurement of eye alignment errors to fixation targets presented at any distance throughout the subject's field of gaze, and allow for the measurement of errors in the horizontal and vertical directions. Furthermore, estimates of the alignment state can be made simultaneously from both eyes while fixation targets are presented monocularly or binocularly. This photographic method represents an enhancement of typical clinical prism and cover methods because: (1) it can provide extensive information efficiently about patterns of misalignment across a large number of fixation locations; (2) it also can provide information about the scatter in addition to the magnitude of convergence error; and (3) it can be easily applied to noncooperative subjects such as animals and young children. Furthermore, the procedure requires relatively inexpensive equipment and technical expertise that are readily available in most clinical or animal research laboratory settings. The method is validated by comparing the results obtained photographically to standard prism and cover assessments of macaque monkeys with strabismus. This comparison demonstrates that results obtained by the two methods are in good agreement and that the degree of accuracy is similar for the two methods. An estimate of the angle of deviation based on 10 photographs has a 95% confidence interval of about two degrees. PMID- 1730545 TI - Superior cervical ganglionectomy in monkeys: surgical technique. AB - Seven cynomolgus monkeys underwent a histologically confirmed left superior cervical ganglionectomy (SCGx). Unilateral ocular sympathetic denervation persisting for at least 2 yr was confirmed by ipsilateral ptosis, miosis, supersensitivity of pupillary dilation to topical phenylephrine, and profound pupillary hyporesponsiveness to topical hydroxyamphetamine. Intraocular pressure 8-9 and 23-27 months postoperatively were identical in the denervated and contralateral control eyes. This model should facilitate studies of aqueous humor physiology and pharmacology. PMID- 1730546 TI - Passive administration of antibody against retinal S-antigen induces electroretinographic supernormality. AB - Electroretinographic supernormality, affecting both the a- and b-waves of the electroretinogram (ERG), occurs consistently before the onset of experimental autoimmune uveoretinitis (EAU) in rabbits and rats. To investigate the possible role of antibody to S-antigen (S-ab) in this phenomenon, affinity-purified polyclonal rat S-ab was injected intravenously into normal rats and administered to isolated rat eyecup preparations by bolus perfusion. In both situations, ERG supernormality was observed. The effect in vivo peaked 90 min after injection, and ERG changes in vitro were observed within 15 sec. The ERG response in vivo and in vitro was dose dependent and was abolished in vivo by pretreatment with cyproheptadine (a serotonin antagonist). The ERG was not affected in either system by a control rat antibody (antiovalbumin) or by murine monoclonal or rabbit polyclonal antibodies to S-antigen. The induction of ERG supernormality in vivo and in vitro by homologous S-ab indicates the operation of species-specific mechanisms both involving and bypassing the blood-retinal barrier and highlights a significant role for humoral autoimmunity in the pathogenesis of S-antigen induced EAU in the rat. PMID- 1730547 TI - Alterations in endothelial superoxide dismutase levels as a function of growth state in vitro. AB - Damage to the retinal vasculature in retinopathy of prematurity is primarily at the level of the growing neovascular front. This clinical observation in combination with experimental observations that correlate the induction of superoxide dismutase (SOD) activity with differentiation led to the hypothesis that the basis for the relative vulnerability to oxygen lies in the relative "undifferentiated" nature of proliferating endothelial cells (EC). To test this hypothesis, an in vitro model of microvascular EC was used and levels of SOD activity were assayed as a function of growth state and differentiation. The SOD activity was elevated significantly in EC that stopped growing as a contact inhibited monolayer compared with its activity in cells in their log phase of growth. In addition, SOD levels were elevated in microvascular cells that stopped growing and were organized into a network of capillary-like tubes, the ultimate differentiated state of microvascular EC. To understand the mechanism of increases in SOD activity with differentiation, the effect of extracellular matrix synthesized by a confluent monolayer of EC was examined as was artificial growth arrest caused by mitomycin. Both treatments led to an increase in SOD activity over control cells. Thus, it is possible that SOD activity in cells is modulated by information provided from the extracellular matrix and "intracellular" signals that indicate cessation of cell growth. These data support the hypothesis that the growing front of retinal microvessels is more vulnerable to effects of oxygen-induced damage because of their relatively undifferentiated state with respect to the oxygen radical-scavenging enzyme system of SOD. PMID- 1730548 TI - Vascular permeability during the early and late phases of ocular anaphylaxis. AB - The role of enhanced vascular permeability in a model of ocular anaphylaxis was investigated during both early- and late-phase reactions (EPR and LPR). Vascular permeability was assessed by measuring the extravascular retention of 125I-bovine serum albumin (125I-BSA) in ocular tissues. Ten groups of guinea pigs (n = 5-12 per group) were injected with dinitrophenylated (DNP) bovine gamma globulin emulsified in Freund's adjuvants and challenged after a 4-week interval by topical application of di-DNP-lysine to one eye and phosphate-buffered saline to the other eye. Thereafter, the eyes were examined and the animals were killed at different intervals after topical challenge. They were injected intravenously with 125I-BSA 0.5 hr before death. Retained radioactivity was measured separately in four tissues. The EPR (period between 0.5-1.5 hr after challenge) was characterized by enhanced retention of radioactivity in lids, conjunctiva, and orbital content. There was no significant retention of extravascular radioactivity in the globe and lacrimal gland. Thereafter (period between 2-3.5 hr after challenge), retained radioactivity was significantly diminished. The subsequent period, between 4.5-6.5 hr (LPR), was characterized by a smaller, although significant, increment of radioactivity retained in lids and conjunctiva but not in the other tissues examined. These findings indicate that enhanced microvascular permeability occurs during two phases in actively immunized guinea pigs challenged topically with di-DNP-lysine and that these phases correspond to the clinical signs that constitute the EPR and LPR of ocular anaphylaxis. PMID- 1730549 TI - RPE transplants stabilize retinal vasculature and prevent neovascularization in the RCS rat. AB - Previous reports indicate that in the Royal College of Surgeons (RCS) rat a decline in the retinal vessel density accompanies the loss of the normal architecture of the deep bed. This begins at about three months with neovascularization that originates in the deep vessel bed and develops in the direction of the retinal pigment epithelial (RPE) cells by four months. A surgical technique has been developed recently for the transplantation of healthy RPE cells into the subretinal space of the RCS rat, resulting in the rescue of photoreceptor cells. This permits evaluation of the possibility that such transplants also protect the retinal vessels. We report for the first time: (1) the stabilization of the normal retinal vasculature by maintenance of the density and architecture of the deep vessel bed; and (2) prevention of neovascularization of the RPE by the surgical transplantation of healthy RPE cells into the subretinal space of the RCS rat. More specifically, we show a maintenance of the deep vessel bed density under the transplant in contrast to a significant reduction in the vessel density that had taken place in corresponding areas in nongrafted and sham injected controls at four months of age. The vessel density in the transplanted group is statistically different from the nongrafted and the sham injected groups. We also report a significant decline in the number of neovascularization profiles around the transplant site of the RPE-grafted RCS retina. We also note that the pathological changes in the vasculature of the RCS rat occur in a predictable central to peripheral gradient. PMID- 1730550 TI - Corneal autofluorescence: an indicator of diabetic retinopathy. AB - The metabolic disorder in diabetics often results in progressive retinopathy with severe visual impairment. Changes in metabolism can influence corneal autofluorescence. This has led to speculation that diabetic retinopathy might be associated with changes in corneal autofluorescence. Corneal autofluorescence of both eyes was determined by fluorophotometry in 94 insulin-dependent diabetes mellitus patients and in 46 healthy controls to evaluate its correlation with diabetic retinopathy. The modified Airlie House classification was used for grading diabetic retinopathy: (1) no or negligible retinopathy; (2) minimal background retinopathy; (3) background retinopathy; and (4) (pre-) proliferative retinopathy. The corneal autofluorescence values of grade 1 retinopathy patients did not differ significantly from those of the healthy controls (mean +/- standard deviation in ng equivalent fluorescein/ml: 11.6 +/- 3.0 and 11.4 +/- 2.8, respectively; P = 0.8). The means of grade 2, 3, and 4 retinopathy patients (mean +/- standard deviation in ngEq fluorescein/ml: 16.2 +/- 4.4, 16.7 +/- 4.3, 20.9 +/- 5.4, respectively) were significantly higher than the means of grade 1 patients and healthy controls (P less than 0.004). The mean values of patients with grade 4 were significantly higher than those of patients with grades 2 and 3 (P less than 0.01). The sensitivity and specificity of corneal autofluorescence as a screening test for diabetic retinopathy were 80% and 76%, respectively; the positive predictive value for the presence of retinopathy was 90%. The values for screening on (pre-) proliferative diabetic retinopathy were 68%, 72%, and 58%, respectively. These data show corneal autofluorescence to be an adequate indicator of diabetic retinopathy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730551 TI - Measurement of corneal thickness by laser Doppler interferometry. AB - The laser Doppler interferometry (LDI) technique, which was recently developed for axial eye length measurement, has been modified to measure the corneal thickness of the human eye in vivo. High accuracy is achieved. The standard deviation of the technique is about 7 microns, and improvement by a factor of 5 is possible. First comparisons with a usual slit lamp pachometer show a general agreement but a systematic difference of about 20 microns. Possible reasons for this discrepancy are discussed. Finally, the new method is compared to standard optical and ultrasound pachometry from a theoretical point of view, and advantages and drawbacks of the various techniques are discussed. PMID- 1730552 TI - Incineration of biomedical low-level radioactive waste. PMID- 1730553 TI - Biodosimetry for a radiation worker using multiple assays. AB - Four state-of-the-art biodosimeters--GPA mutations, chromosome translocations, micronuclei, and dicentrics--were used to evaluate a radiation worker who believed that the official dosimetry records substantially underestimated his actual dose. Dosimetry records indicated that the worker received 0.56 Sv during a 36-y employment history, always within the dose limits. In contrast, the worker believed that his dose equivalent may have been more than 2.5 Sv because much of the exposure was received during the early days of health physics when dosimetry capabilities and practices were not as good as they are today. Because there are no biodosimetric assays that have been fully validated for the long-term low level exposures received by the worker, we did not expect to obtain particularly useful point-estimates of dose. However, because the discrepancy between the dosimetry records and the worker's belief was so large, we believed that biodosimetry using multiple assays together with probabilistic assessment of the uncertainties would provide useful insight. Results showed that the frequencies of chromosome translocations and GPA mutations (stable biodosimeters) were significantly elevated when compared with those for unexposed controls. Our analysis suggests that dose-equivalent estimates in the approximately 0.4 to approximately 2 Sv range (which include the value in the dosimetry records) cannot be confidently excluded at this time based on biodosimetry; however, a value greater than 2.5 Sv appears unlikely. Important new information on the temporal stability of chromosome translocations is also presented. PMID- 1730554 TI - Mammography quality assurance in Washington State. AB - Poor mammography quality control results in the loss of important diagnostic information. Several organizations and agencies recommend or require that mammography quality assurance procedures include the periodic evaluation of equipment. The objective of this study was to assess compliance with published guidelines pertaining to the monitoring of mammography equipment. One hundred eighty-four Washington state mammography facilities were surveyed by mail during late 1989 and early 1990. The response rate was 71%. A large proportion of facilities was not complying with published guidelines concerning the frequency of evaluation of processor parameters and phantom image quality. Results suggest that many facilities were not in compliance with American College of Radiology recommendations concerning annual system evaluation by a physicist. The results of this study reinforce the importance of establishing minimum quality assurance standards and indicate a need for more mammography quality assurance technologist training. PMID- 1730555 TI - Site-specific parameter values for the Nuclear Regulatory Commission's food pathway dose model. AB - Routine operations at the Savannah River Site (SRS) in Western South Carolina result in radionuclide releases to the atmosphere and to the Savannah River. The resulting radiation doses to the off-site maximum individual and the off-site population within 80 km of the SRS are estimated on a yearly basis. These estimates are currently generated using dose models prescribed for the commercial nuclear power industry by the Nuclear Regulatory Commission (NRC). The NRC provides default values for dose-model parameters for facilities without resources to develop site-specific values. A survey of land- and water-use characteristics for the Savannah River area has been conducted to determine site specific values for water recreation, consumption, and agricultural parameters used in the NRC Regulatory Guide 1.109 (1977) dosimetric models. These site parameters include local characteristics of meat, milk, and vegetable production; recreational and commercial activities on the Savannah River; and meat, milk, vegetable, and seafood consumption rates. This paper describes how parameter data were obtained at the Savannah River Site and the impacts of such data on off-site dose. Dose estimates using site-specific parameter values are compared to estimates using the NRC default values. PMID- 1730556 TI - Modeling the behavior of tritiated water vapor in a research reactor containment building. AB - Mathematical models were developed to predict the changes in tritiated water (HTO) concentrations in water pools and of HTO vapor in the Kyoto University Reactor (KUR) containment building in which approximately 3.4 x 10(2) GBq of HTO vapor had leaked from a heavy water facility for more than 1 y. Models reveal that the mechanism of HTO vapor transfer between air and water is controlled by two key parameters: the kinetic constant for HTO exchange and the evaporation rate constant from water to air. A model was constructed based on laboratory experiments using small glass dishes containing various volumes of HTO and was validated by comparing estimates to actual measurements for HTO concentration in water pools of various depths in the containment building. After the leakage from the heavy water facility had been stopped, the decrease in HTO concentration in the sub-pool could be described by this model with a half-life of 15 wk. A mathematical model was also developed to estimate the average HTO vapor concentration in air, which is strongly dependent on the ventilation system's operation even after the removal of the HTO sources. This is due to the continued release of HTO from the concrete material and is analogous to the dynamics of radon emanation. The amount of HTO that has emerged from the concrete was estimated using a model developed for HTO concentration changes in the containment building air, based on long-term monitoring. PMID- 1730557 TI - A beta spectrometer for monitoring environmental matrices. AB - In environmental matrices, pure beta emitters are often present with other beta gamma emitters. This work describes a beta spectrometer that allows measurement of pure beta-emitter activities of about 10(-2) Bq. The detection system consists of a plastic scintillator surrounded in a 4 pi geometry by large anticoincidence NaI(Tl) scintillators. Solid, liquid, and gaseous samples can be analyzed, and no chemical separation is necessary. The system is particularly useful for 90Sr measurement. PMID- 1730559 TI - Contribution of 222Rn in domestic water supplies to 222Rn in indoor air in Colorado homes. AB - The contribution of 222Rn from domestic water wells to indoor air was investigated in a study of 28 houses near Conifer, CO. Air concentrations determined by alpha-track detectors (ATDs) and continuous radon monitors were compared with the predictions of a single-cell model. In many of the houses, the water supply was shown to contribute significantly to levels of indoor 222Rn. The data from the ATD study were augmented with a continuous monitoring study of a house near Lyons, CO. The well water in that house has the highest known concentration of 222Rn in water yet reported (93 MBq m-3). The temporal pattern in the indoor 222Rn concentration corresponds to water-use records. In general, it is difficult to quantify the proportion of indoor radon attributable to water use. Several lines of evidence suggest that the single-cell model underestimates this proportion. Continuous-monitoring data, although useful, are impractical due to the cost of the equipment. We propose a protocol for 222Rn measurement based on three simultaneous integrating radon detectors that may help estimate the proportion of indoor 222Rn derived from the water supply. PMID- 1730558 TI - Measurement of the biotransfer and time constant of radon from ingested water by human breath analysis. AB - Forty-one tests were performed on 38 volunteers to measure elimination rates of 222Rn in expired breath. Participants ranged from ages 9 to 85 y, with 16 males and 22 females. The levels of physical activity of the subjects ranged from very inactive to marathon level. Calibration of our flow-through scintillation cell was accomplished using a medical ventilator and 222Rn reservoir for 5-15 L min-1 flow rates. We found a wide range of percent elimination (12-68%) in 30 min. The percent elimination has a mild correlation with the predicted forced expiratory volume in 1 s and with time passed since eating. Our observations of bio retention half-times range from 17-400 min. The whole-body dose calculations yield a mean of 2.70 +/- 3.43 nGy Bq-1, and the stomach dose calculations yield a mean of 276 +/- 186 nGy Bq-1. These means range beyond those previously reported. PMID- 1730560 TI - Promotion of radiation-induced liver neoplasia by ethanol. AB - The risk of americium-induced liver cancer in beagle dogs that received long-term dietary ethanol was two to three times that of their nonalcoholic cohorts, even though the radionuclide retention time in hepatic tissue was shortened by the alcohol treatment. Liver malignancies did not occur in the ethanol-treated, nonirradiated controls. An ethanol-induced tumor-promoting effect was not observed in organs or tissues other than the liver. PMID- 1730561 TI - Characterization of low-level radioactive waste generated by a large university/hospital complex. AB - Descriptions of the physical characteristics and radioactive contents of waste generated by a large university/hospital complex were recorded in a computerized data base for 1 y. The data were summarized to create a waste stream profile so that major waste sources could be identified and minimization efforts quantified. The resultant profile provides a comprehensive description of the low-level radioactive waste produced by a large biomedical research and patient care facility, and projects the methods that will be used to process and ultimately dispose of the material. PMID- 1730562 TI - Detecting removable surface contamination. AB - Although surveying for radioactive contamination by wiping surfaces is the norm, this practice can be highly variable and may be inefficient for detecting low energy beta emitters. Relying on wipe testing may likewise be an inefficient use of personnel and may seriously underrepresent the amount of contamination present. In general practice, it is better to clean and, where applicable, renew surfaces regularly as part of standard operating protocols and work practices. PMID- 1730563 TI - An intercomparison of neutron dosimeters and detectors for in-containment dosimetry. AB - To improve the methodology for assessing neutron dose at Union Electric's Callaway Nuclear Power Plant, an intercomparison of neutron detectors and dosimeters was performed. Seven different neutron detectors and dosimeters were tested in four different neutron fields utilizing facilities at the Missouri University Research Reactor and at the Southwest Radiation Calibration Center at the University of Arkansas. In general, all results agree within a factor of 2 in predicting the neutron dose equivalent. It was concluded that measurements of dose in containment should utilize the Tissue-Equivalent Proportional Counter (TEPC), the Bonner-sphere system, and the proton recoil spectrometer to accurately assess the neutron dose. These data can then be used to provide correction factors for more traditionally used dosimeters in containment, such as thermoluminescent dosimeters and survey meters. PMID- 1730564 TI - Cytostatic activity of dialyzed SDS-page eluates. PMID- 1730565 TI - An inhibitor of tumor cell growth from normal horse serum. AB - During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines such as African green monkey kidney (Vero) and Madin-Darby bovine kidney cells and normal mammary cells of the dog and goat were inhibited only at high concentrations. The active component is a protein with an Rf value of 0.09 upon electrophoresis in native 7.5% polyacrylamide gels. The eluate from the native gel could be further separated into three polypeptides with molecular weights of approximately 67, 65, and 55 kDa under reduced conditions in sodium dodecyl sulfate-polyacrylamide gels. PMID- 1730566 TI - An in vitro model giving access to adhesion plaques. AB - A new approach was investigated to study the interaction between integrins and actin via intracytoplasmic proteins. Because intracellular processes are hampered by the limiting plasma membrane, we developed an in vitro model with cells perforated by a bacterial toxin, streptolysin O. The specific conditions for the use of permeabilized cells to study the intramolecular associations occurring at adhesion plaques are described. The two cell types used, HUVEC and CHO, showed that the choice of the perforation method is of great importance. After perforation of cells in a monolayer, 75 +/- 10% of the cells remained adherent to a fibronectin substrate; after perforation of cells in suspension, only 25 +/- 10% of the cells readhered. Specific conditions were required however to maintain these adhesive properties up to 4 h: the presence of 1 mM Mg++ in the medium was crucial, and it was necessary to layer the cells on a specific coat rather than a substitute such as gelatin. Immunofluorescence investigations of actin, talin and vinculin, and Normarsky differential interference contrast microscopy showed retention of focal adhesion plaques in perforated cells. Moreover, in perforated cells antibodies directed against actin led to actin disorganization, showing that our model of perforated cells in a monolayer can give new insight to adhesion study. PMID- 1730567 TI - A new DNA profiling system for cell line identification for use in cell banks in Japan. AB - Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and lambda TM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique. PMID- 1730568 TI - Characterization of a fetal urogenital sinus mesenchymal cell line U4F: secretion of a negative growth regulatory activity. AB - Mesenchymal cell lines derived from fetal rat urogenital sinus organ cultures have been characterized to establish an in vitro system for addressing growth and differentiation regulatory factors involved in mesenchymal-epithelial interactions during prostate morphogenesis. A continuous cell line was developed and designated U4F. Immunocytochemical analysis showed vimentin intermediate filament content confirming a mesenchymal origin. Previous studies with urogenital sinus organ cultures have reported the expression of a negative growth activity, which is stimulatory to protein synthesis and secretion and alters phenotypic morphology of NBT-II bladder epithelial cells. Subconfluent and confluent U4F monolayers did not produce this growth inhibitory activity. Foci of stacked cells were observed 3 wk postconfluency, which evolved into multicellular spheroids. The negative growth activity was expressed in the conditioned medium coordinate with spheroid formation. Transplanted spheroids continued to express the growth inhibitory activity. Morphologic analysis of spheroids showed a cellular capsule and a core of extracellular matrix. A continuous cell strain (U4F1) with altered phenotypic properties, arose spontaneously from long-term U4F cultures. The U4F1 cell strain did not form spheroids, yet expressed the negative growth activity constitutively in monolayer culture. Analyses of physicochemical, immunological, and biological properties showed the activity is identical in conditioned media from urogenital sinus organ cultures, U4F spheroids, and U4F1 monolayers. Based on the combined properties, this activity cannot be ascribed to previously characterized negative growth factors. The establishment of this mesenchymal cell culture system will aid in the further identification of paracrine-acting growth and differentiation regulatory factors secreted by fetal mesenchyme. PMID- 1730569 TI - A transport/separation system for clinical specimens. PMID- 1730570 TI - Serum-free culture of fractionated bovine bronchial epithelial cells. AB - Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factor beta 1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1 microM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum. PMID- 1730571 TI - Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels. AB - A new low shear stress microcarrier culture system has been developed at NASA's Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue. PMID- 1730572 TI - Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium. AB - Growth hormone (GH) production by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T3). As measured by radioimmunoassay, apoTf plus T3 induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T3 arrested GH secretion. ApoTf/T3 defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T3 defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T3 defined medium. Even under these conditions, the response to dexamethasone remained T3 dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the response to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions. PMID- 1730573 TI - Human colon carcinoma cell lines from the primary tumor and a lymph node metastasis. PMID- 1730574 TI - Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. PMID- 1730575 TI - Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities. AB - Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters. PMID- 1730576 TI - Bradykinin-induced Ca2+ entry, release, and refilling of intracellular Ca2+ stores. Relationships revealed by image analysis of individual human fibroblasts. AB - Lys-Bradykinin (BK), a mitogen for human foreskin fibroblasts (HSWP cells) (Owen, N. E., and Villereal, M. L. (1983) Cell 32, 979-985), elicits a rapid, transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) in these cells. We have used image analysis of fura-2-loaded HSWP cells to examine the BK-induced [Ca2+]i changes in individual cells. BK-stimulated Ca2+ entry and release of intracellular Ca2+ stores can be distinguished by stimulating cells in the presence or absence of extracellular Ca2+, or by inhibiting Ca2+ entry with 5 mM NiCl2. BK-sensitive intracellular Ca2+ stores can be depleted by exposure of the cells to BK in Ca(2+)-free medium; refilling of the stores requires extracellular Ca2+. A component of BK-stimulated Ca2+ entry persists after removal of agonist, but inactivates with a t1/2 of approximately 5 min. Although previous studies have attributed the Ca2+ entry which persists after agonist removal to a "capacitative Ca2+ entry" pathway activated by the depletion of the intracellular Ca2+ stores, we find that a large component of this BK-stimulated Ca2+ entry is not due to capacitative Ca2+ entry since (1) ionomycin can deplete the BK sensitive intracellular Ca2+ stores without appreciably stimulating Ca2+ entry and without inhibiting the BK-stimulated Ca2+ entry and (2) this Ca2+ entry pathway inactivates at a time when the Ca2+ pools are still empty and a capacitance entry pathway should still be open. On the other hand, refilling of the intracellular Ca2+ stores can occur after the noncapacitative Ca2+ entry component has inactivated or when it is inhibited by Ni2+; in these cases refilling occurs without a detectable elevation of [Ca2+]i suggesting that refilling of internal Ca2+ pools might occur by a capacitative route. PMID- 1730577 TI - Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids. AB - 3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept. PMID- 1730578 TI - Transport of phagosomal components to an endosomal compartment. AB - The participation of phagosomes in interorganellar protein and membrane exchange is important to the maturation of phagosomes into phagolysosomes. To investigate this process, we have developed an assay to measure protein transport from phagosomes to other vesicle populations. J774-E clone macrophages phagocytosed 125I-anti-dinitrophenol IgG-coated Staphylococcus aureus for 3 min followed by chase for intervals of 0-30 min. Following cell fractionation, the intracellular distribution of radioiodinated protein was assayed. We observed a time-dependent increase radioiodinated protein in a non-phagosome vesicle fraction which displayed endosome characteristics. Concomitantly, radioiodinated protein within phagosomes decreased over the chase period. As assessed via Percoll density gradient fractionation, the phagocytosed radioiodinated protein migrated to both heavy (lysosome density) and light (endosome density) vesicle populations. Characterization of the fusogenic properties of the transport vesicles demonstrated that they are capable of in vitro fusion with early endosomes. Furthermore, this fusion event shares many of the biochemical requirements identified for phagosome-endosome and endosome-endosome fusion. Morphological analysis of phagosome maturation provides additional evidence for phagosome to endosome transport. These results suggest phagocytosed material is transferred from phagosomes to endosomes and then recycled out of the cell. PMID- 1730579 TI - K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. AB - The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation. PMID- 1730580 TI - Inhibition of N-linked glycosylation affects organic cation transport across the brush border membrane of opossum kidney (OK) cells. AB - Many organic cations are transported across the apical membrane of the proximal tubule by specific saturable mechanisms. The goal of this study was to determine if the transporter for tetraethylammonium (TEA) in the brush border membrane of an established opossum kidney (OK) cell line is glycosylated and to elucidate the function of this glycosylation. The uptake of TEA was determined in OK cell monolayers treated with tunicamycin (TM), a compound that prevents synthesis of the core oligosaccharide precursor molecules. TM exposure significantly decreased the incorporation of [3H]mannose in OK cell proteins and significantly reduced TEA uptake in a time and a concentration dependent manner. No effect of TM exposure on cellular protein synthesis, DNA content, cell viability, or on [3H]proline uptake was observed. The transport of TEA in control cells was characterized by a Km of 26.9 +/- 16.4 microM and a Vmax of 378 +/- 39 pmol/mg of protein/min. TM treatment (1 microgram/ml for 21 h) significantly increased the Km by over 4-fold to 111.5 +/- 18.4 microM while not affecting the Vmax. The apparent KI values of other organic cations known to interact with this transport system were also significantly increased by TM exposure. Estimated KI values of N1-methylnicotinamide, cimetidine, and mepiperphenidol increased by 6-fold, 4 fold, and 2-fold, respectively, after exposure of OK cells to TM. An increased KI for protons was also observed. Additional inhibitors of the N-linked glycosylation pathway, castanospermine, deoxynojirimycin, and deoxymannojirimycin significantly decreased TEA transport, whereas swainsonine had no effect. Our results suggest that the organic cation transporter is glycosylated. The N-linked oligosaccharide side chain appears to be of the hybrid type, and it either directly or indirectly affects the binding site of the transporter for both organic cations and protons. This is the first report describing the importance of glycosylation in the function of the organic cation transporter in the apical membrane of OK cells. PMID- 1730581 TI - Active site labeling of a receptor-like protein tyrosine phosphatase. AB - The inactivation of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase (PTPase), by iodoacetate and not by iodoacetamide suggested that iodoacetate interacts in a highly selective manner with the enzyme. The data indicate that iodoacetate binds at the active site of the enzyme with a stoichiometry of 0.8 mol of iodoacetate bound per mol of rat LAR. A single [14C]iodoacetate-labeled peptide was isolated following endoproteinase Lys-C digestion of the radiolabeled PTPase. Sequence analysis of the active site labeled peptide demonstrates that Cys-1522 contains the radiolabel. This residue has been shown by site-directed mutagenesis to be essential for rat LAR activity (Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688-19696). Iodoacetate reacts only with the first domain of this double domain PTPase. These results, for the first time, directly identify the highly reactive cysteine residue at the active site of a PTPase and highlight the ability of this residue to participate as a nucleophile in the hydrolysis of phosphate from tyrosine. PMID- 1730582 TI - Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites. AB - Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2 pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor. PMID- 1730583 TI - 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A-- -C substitutions. AB - Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G----T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A----C substitutions, and two were G----T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G----T substitutions and misincorporation of oh8Gua as substrate causing A----C substitutions, both caused by oh8Gua.A mispairs. PMID- 1730584 TI - Heat shock induces translocation to the nucleus of the unliganded glucocorticoid receptor. AB - There have been many reports demonstrating the specific association of several heat shock proteins with unliganded steroid hormone receptors. However, little evidence to date has been proposed to link steroid receptor action with the heat shock response in cells. In this paper, we demonstrate the effect of heat and chemical stress on glucocorticoid receptor subcellular localization in mouse L929 cells and in a stably transfected Chinese hamster ovary cell line (WCL2) which over-expresses the mouse glucocorticoid receptor. When WCL2 cells are exposed to 43 degrees C, there is a time-dependent decrease in glucocorticoid receptor hormone-binding capacity in the cytosolic fraction of these cells that correlates with a decrease in amount of glucocorticoid receptor protein. Analysis of both cytosolic and nuclear fractions for glucocorticoid receptor protein via quantitative Western blotting reveals that the unliganded glucocorticoid receptor of non-shocked L929 and WCL2 cells is localized primarily in the cytosolic fraction, whereas unliganded receptor of heat-shocked cells is found almost exclusively in the nuclear fraction. A similar shift to nuclear localization for unliganded glucocorticoid receptor is noted in L929 and WCL2 cells subjected to chemical shock (sodium arsenite). As treatment of these cells with glucocorticoid hormone also results in glucocorticoid receptor that is tightly bound within the nuclear fraction, it is speculated that heat and chemical stress provide a hormone-independent mechanism by which glucocorticoid receptor is transformed to the high affinity nuclear-binding state characteristic of the hormone-bound, transcriptionally active receptor. PMID- 1730585 TI - Kinetics of plasmin activation of single chain urinary-type plasminogen activator (scu-PA) and demonstration of a high affinity interaction between scu-PA and plasminogen. AB - The activation kinetics of single chain urinary-type plasminogen activator (scu PA) by plasmin have been studied in detail. Nonstandard Michaelis-Menten kinetics were observed. To explain our results, we propose a model in which plasmin can exist in two conformations of lower activity (kcat/Km = 1.4 x 10(6) M-1 s-1) or higher activity (kcat/Km = 16.7 x 10(6) M-1 s-1) depending on whether a lysine binding site is occupied or free, respectively. These kinetic studies demonstrate that scu-PA interacts at this binding site (KD approximately 30 nM) and so is able to act as both a substrate and effector in this reaction. Binding was also demonstrated between scu-PA and Glu- or Lys-plasminogen at a high affinity site (KD approximately 65 nM), sensitive to the presence of lysine analogs. This suggests that scu-PA may be almost completely bound to plasminogen in plasma under normal physiological conditions and provides a possible explanation for the fibrin specificity of this activator, as discussed. PMID- 1730586 TI - Topology of cytochrome b558 in neutrophil membrane analyzed by anti-peptide antibodies and proteolysis. AB - Cytochrome b558 is an important constituent of the superoxide-generating system in neutrophils and B lymphocytes. In this paper, the topology of the cytochrome in human neutrophil membrane was studied using antibodies raised in rabbits against synthetic peptides corresponding to various regions in the large and small subunits of the cytochrome. The antibodies recognized the cytochrome subunits in immunoblots and the cytochrome in situ. An antibody raised against residues 150-172 in the large subunit (anti-L123) bound to intact neutrophils, indicating that this region is exposed to the outside of the cells. In contrast, antibodies raised against any of the carboxyl-terminal regions of the large and small subunits (anti-LC and anti-SC, respectively) or the amino-terminal region of the small subunit (anti-SN), bound to neutrophils only after the cells were made permeable by freezing and thawing. The region close to the carboxyl terminus of the large subunit was digested by extracellularly added papain and, as a result, an 18-kDa carboxyl-terminal fragment was detected. Thus the carboxyl terminal region of the large subunit is cytoplasmic and/or buried in the membrane, and the region around residues 369-398 is exposed on the cell surface. In contrast to the large subunits, the small subunit in neutrophils was resistant to any of the proteinases tested, although the subunit in membrane or Triton solubilized preparation was digestible with papain. These results indicate that the large subunit of cytochrome b558 is a transmembrane protein with at least two regions exposed on the cell surface and that the carboxyl terminus of this subunit and both termini of the small subunit are exposed to the cytoplasmic side. PMID- 1730587 TI - Zonal distribution of cysteine uptake in the perfused rat liver. AB - When in situ perfused rat livers were administered tracer or physiologic concentrations of [35S]cysteine, a zone III (perivenous) predominance of uptake was observed in either antegrade or retrograde single-pass perfusion, as determined by quantitative densitometry of autoradiographs of liver section. This pattern remained unchanged from 30 s to 5 min observed. At higher supraphysiologic doses a more uniform acinar distribution of cysteine uptake was observed. Uptake rates of cysteine in antegrade perfusion indicated an apparent saturable component at low but physiologic cysteine concentrations. That uptake rather than metabolic trapping accounts for this perivenular pattern was supported by finding identical zonal distribution under conditions in which GSH and protein synthesis were markedly inhibited. Furthermore, increasing or decreasing hepatic cysteine pool sizes did not affect the extraction or zonation. These results suggest that a low Km transport system for cysteine is localized in zone III of the hepatic acinus. PMID- 1730588 TI - X-ray structure of a biantennary octasaccharide-lectin complex refined at 2.3-A resolution. AB - The structure and flexibility of saccharides have a profound and specific influence in several biological processes such as protein protection and the maintenance of conformational integrity, and in recognition events involving viruses, enzymes, and lectins. To establish the structural bases of these phenomena, we describe herein the extensively refined 2.3-A resolution x-ray structure of a biantennary octasaccharide of the N-acetyllactosamine type, complexed to isolectin I from Lathyrus ochrus. The two octasaccharides are located in clefts at each end of the long axis of the lectin. The conformations of both the lectin and the saccharide are slightly modified upon binding. The complex is stabilized by numerous hydrogen bonds, many of them involving water molecules. It is also stabilized by van der Waals interactions, including some with aromatic residues. A more general model of a possible lectin-glycoprotein interaction is also proposed. PMID- 1730589 TI - Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol. AB - The alterations in the absorption and fluorescence spectra observed for the polyene antibiotics filipin and nystatin in the presence of cholesterol are due to an exciton interaction (polyene aggregates) and cannot be attributed to a specific sterol-antibiotic complex. Filipin and nystatin molecules partition into the sterol aggregates, these structures being very efficient to induce exciton interaction; the observed splitting profile indicates that the chromophores are in a stacked arrangement (parallel transition dipoles). For filipin incorporated in lipid bilayers, the sterol is able to induce the same type of aggregate, at variance with nystatin. PMID- 1730590 TI - Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR. AB - The reversible conversion between D-glucose 6-phosphate and D-fructose 6 phosphate catalyzed by yeast phosphoglucoisomerase was studied by phase sensitive two-dimensional 13C-[1H] EXSY NMR spectroscopy at 150.869 and 125.759 MHz, using 13C-enriched substrates in the C2 position of the D-hexose 6-phosphates. The shape of the build-up curves of the cross-peaks associated with the 13C2 resonances of the alpha- and beta-anomers of both D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between alpha-D-[2-13C]glucose 6-phosphate and beta-D-[2-13C]fructose 6-phosphate. Quantitative analysis of the build-up curves by three different methods allowed us to conclude that phosphoglucoisomerase not only selectively channels the latter isomerization but also considerably accelerates the anomerization of both D-hexose 6-phosphates. The rate constants of anomerization were indeed much higher in the presence than in the absence of enzyme. The major finding in the present study consists in the anomeric specificity of phosphoglucoisomerase toward the beta-anomer of D fructose 6-phosphate both as a substrate and a product, contrary to previous proposals. This finding supports recent evidence suggesting the direct channelling of beta-D-fructose 6-phosphate from phosphoglucoisomerase to phosphofructokinase. PMID- 1730591 TI - Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells. AB - Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730592 TI - Phorbol ester-induced down-regulation of the 80-kDa myristoylated alanine-rich C kinase substrate-related protein in Swiss 3T3 fibroblasts. Inhibition by staurosporine. AB - A polyclonal antiserum raised against an oligopeptide with an amino acid sequence corresponding to a sequence of the myristoylated alanine-rich C-kinase substrate (MARCKS) from mouse macrophages and rat brain recognizes the 80-kDa C-kinase substrate from Swiss 3T3 fibroblasts. Using this antiserum for quantitative determination of the 80-kDa MARCKS-related protein, we found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid down-regulation of this protein in the fibroblasts. In accordance with earlier reports, TPA causes phosphorylation of the 80-kDa protein which can be inhibited by staurosporine. Staurosporine also suppresses the TPA-induced down-regulation, possibly indicating that the down-regulation of the MARCKS-related protein is dependent on its phosphorylation by protein kinase C. PMID- 1730593 TI - Structural changes that accompany the reduced catalytic efficiency of two semisynthetic ribonuclease analogs. AB - The structures of two catalytically defective semi-synthetic RNases obtained by replacing aspartic acid 121 with asparagine or alanine have been determined and refined at a resolution of 2.0 A (R = 0.186 and 0.172, respectively). When these structures are compared with the refined 1.8-A structure (R = 0.204) of the fully active aspartic acid-containing enzyme (Martin, P.D., Doscher, M.S., and Edwards, B. F. P. (1987) J. Biol. Chem. 262, 15930-15938), numerous and widespread changes, much greater in number and magnitude than the small structural variations noted previously between the semisynthetic complex and RNase A, are found to have occurred. These changes include the movement of the loop containing residues 65-72 away from the active site, a more or less generalized relocation of crystallographically bound water molecules, and a number of rearrangements in the hydrogen bonding network at the active site. Most changes are far removed from the immediate site of the modifications and are distributed essentially throughout the molecule. The details of many of these changes are unique to each analog. In the asparagine analog, a destabilization in the positioning of active site residue His-119 also appears to have occurred. PMID- 1730594 TI - Metabolism of 4'-azidothymidine. A compound with potent and selective activity against the human immunodeficiency virus. AB - 4'-Azidothymidine (ADRT) is a novel nucleoside analog, that selectively inhibits human immunodeficiency virus replication in human lymphocytes. Unlike the dideoxyribonucleoside analogs and 3'-azido-2',3'-dideoxythymidine (AZT), ADRT retains the 3'-hydroxy group. The pathways of ADRT metabolism were elucidated by determining: (i) the kinetics of the interactions of ADRT and its metabolites with enzymes of thymidine metabolic pathways, (ii) the pool sizes of phosphorylated metabolites, and (iii) the nature of ADRT incorporation into human DNA. ADRT is not a substrate for thymidine phosphorylase, but is metabolized by kinases. Thymidine kinase phosphorylates ADRT to ADRT monophosphate (ADRT-MP). For this enzyme, ADRT has a Ki value of 5.2 microM, in comparison to a Km value of 0.7 microM for thymidine. The Km value of ADRT toward thymidine kinase is 8.3 microM and the rate of ADRT phosphorylation is 1.4% that of thymidine phosphorylation. ADRT-MP has a low affinity toward thymidylate kinase (a Ki value of 28.9 microM versus a Km value of 0.56 microM for thymidylate), and toward thymidylate synthase (a Ki value of 180 microM versus a Km value of 8 microM for deoxyuridylate). The results suggest that ADRT can be activated effectively by cellular kinases without significant interference of normal thymidine metabolism. In cultured human lymphocytes (A3.01, H9, and U937 cells), ADRT was phosphorylated efficiently to ADRT 5'-triphosphate (ADRT-TP), which is the major metabolite of ADRT. The intracellular concentrations of ADRT-TP ranged from 1 to 3.3 microM after 24 h of incubation with 2 microM of ADRT and the half-life of ADRT-TP varied from 3 to 6 h. Although ADRT-TP is a poor competitive inhibitor against dTTP toward DNA polymerases alpha and beta with Ki values of 62.5 and 150 microM, respectively. ADRT-MP was found to be internally incorporated into cellular DNA. The extent of ADRT-MP substitution for dTMP in DNA was 1 in 6979 for A3.01 cells incubated with 2.9 microM ADRT for 24 h. Internal incorporation of ADRT-MP contrasts with the mechanism of other 2',3'-dideoxynucleoside analogs (i.e. AZT, ddC, ddI, d4T...), which are DNA chain terminators. This finding indicates that a 3'-deoxy structure in a nucleoside analog is not a prerequisite for anti-human immunodeficiency virus activity. PMID- 1730596 TI - Secretion of alpha-1-proteinase inhibitor requires an almost full length molecule. AB - In the human disease alpha-1-proteinase inhibitor deficiency, some variants of human alpha-1-proteinase inhibitor are not secreted. These secretory variants contain frameshift mutations leading to products with normal amino acid sequences to the points of the mutations followed by short, aberrant C-terminal sequences and then premature termination (Nukiwa, T., Takahashi, H., Brantly, M., Courtney, M., and Crystal, R. (1987) J. Biol. Chem. 262, 11999-12004; Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335; Curiel, D., Brantly, M., Curiel, E., Stier, L., and Crystal, R. G. (1989) J. Clin. Invest. 83, 1144-1152). To examine possible causes for lack of secretion of these null variants, we have altered the alpha-1 proteinase inhibitor cDNA to encode a series of abbreviated forms of this protein that retain authentic sequences to the points of truncation. Examination of the fates of these shortened proteins in transiently transfected Cos 1 cells indicates that the aberrant C-terminal sequences in the naturally occurring variants are not responsible for their lack of secretion and show that truncation prior to Pro391 prevents movement from the endoplasmic reticulum to the Golgi apparatus and therefore secretion. These truncated forms of alpha-1-proteinase inhibitor do not form inclusion bodies in the endoplasmic reticulum, rather they are degraded, probably by the pre-Golgi pathway. Our results support the idea that a sequence of at least 391 of the normal 394 residues is essential for the secretion of alpha-1-proteinase inhibitor and suggest that residue 391 plays an especially important role, perhaps in allowing or directing proper folding or as part of a transport signal, in the secretion of this protein. PMID- 1730595 TI - Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors. AB - To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110 fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors. PMID- 1730597 TI - Bradyrhizobium japonicum nodD1 can be specifically induced by soybean flavonoids that do not induce the nodYABCSUIJ operon. AB - Besides genistein and daidzein, which are active inducers of the nodYABCSUIJ operon in Bradyrhizobium japonicum, soybean seeds also excrete compounds that are not inducers of the nodYABCSUIJ genes but enhance induction of this operon in the presence of a suboptimal genistein concentration. This synergism was studied in detail, and specific compounds were identified in seed exudate which specifically induce the nodD1 gene but not the nodYABCSUIJ operon. Therefore, our current hypothesis is that the observed synergism is caused by a specific induction of nodD1. The specific nodD1 inducers from soybean seed extract have been purified and characterized chemically. They appear to be derivatives of genistein, glycitein, and daidzein with glucose, malonyl, and acetyl groups attached. Both root and seed exudate appear to contain these compounds, with the seed being the major source. No hydrolysis of these compounds to their aglycone forms was detected in the presence of B. japonicum. A model for nod gene induction in B. japonicum is discussed. PMID- 1730598 TI - Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha. AB - Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF. PMID- 1730599 TI - Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA. AB - The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro. Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA. We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex. In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa. Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA. To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay. When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen. Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits. DNA binding was not reconstituted by mixing the individual subunits together. These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein. These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein. PMID- 1730600 TI - Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation. In vitro dephosphorylation of UBF reduces its transactivation properties. AB - We have analyzed the expression, phosphorylation, and localization of the ribosomal DNA transcription factors UBF1 and UBF2 in Chinese hamster ovary cells in response to serum deprivation. In vivo labeling experiments demonstrate that UBF1 and UBF2 are phosphoproteins. Phosphoamino acid analysis of the in vivo labeled proteins demonstrate that UBF is phosphorylated on serine residues. Following serum deprivation there is no alteration in the cellular levels of UBF1 and UBF2 as determined by Western blotting, but there is an 80% reduction in the level of phosphorylation of UBF compared with logarithmically growing cells. Following serum deprivation there is a redistribution of UBF between the nucleolus, the nucleus, and the cytoplasm. Phosphatase-treated UBF demonstrated a reduced ability to rescue transcription by RNA polymerase I from the rDNA spacer promoter in vitro. These findings suggest that phosphorylation of UBF is a prerequisite for transactivation of RNA polymerase I. PMID- 1730601 TI - Mechanism of molecular recognition. Structural aspects of 3,3'-diiodo-L-thyronine binding to human serum transthyretin. AB - The three-dimensional structure of the thyroid hormone metabolite, 3,3'-diiodo-L thyronine (3,3'-T2), complex with human serum transthyretin (TTR) has been refined to R = 18.5% for 8-2 A resolution data. This is the first detailed description of a thyroid hormone metabolite binding to a thyroid transport protein. The four TTR monomeric subunits form a tetramer in the same manner as the native transthyretin reported earlier (Blake, C. C. F., Geisow, M. J., Oatley, S. J., Rerat, B., and Rerat, C. (1978) J. Mol. Biol. 121, 339-356). The two hormone binding sites of the TTR tetramer are occupied by 3,3'-T2. A statistical disorder model for the ligand was applied with a 50% occupancy to account for the discrepancy between the crystallographic 2-fold symmetry of the binding sites and the lack of such symmetry for 3,3'-T2. The bound metabolite has an overall transoid conformation with the either bridge intermediate between skewed and perpendicular. The hormone metabolite is bound 3.5 A deeper and with a different orientation in the channel than observed for thyroxine (T4), thereby revealing the presence of another set of halogen binding sites close to the center of the tetramer. When compared with the binding of T4, these data show that the 3-iodine of 3,3'-T2 occupies the same site as the 3'-iodine of T4, and the metabolite 3'-iodine occupies the water site observed in the T4 complex. The binding affinity of 3,3'-T2, which is 100-fold lower than that of T4, reflects the lack of the second pair of iodine atoms interacting in the channel. In order to understand the tighter binding of T4 observed in the Ala-109----Thr mutant, modeling studies were carried out that indicate that this modification could shorten the contacts between thyroid hormone iodines and residues 108-110 of the binding site. PMID- 1730602 TI - Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. AB - Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor. PMID- 1730603 TI - Isolation, characterization, and cDNA sequence of two fatty acid-binding proteins from the midgut of Manduca sexta larvae. AB - Two abundant fatty acid-binding proteins (MFB1 and MFB2) were isolated from the midgut cytosol of larval Manduca sexta. As isolated, MFB1 and MFB2 were found to contain bound fatty acids in a 1:1 molar stoichiometric ratio. Immunological screening demonstrated that MFB1 and MFB2 were restricted to the midgut in a gradient distribution, with MFB1 more concentrated in the anterior two-thirds of the midgut and MFB2 more concentrated in the posterior two-thirds of the midgut. MFB1 exchanged fatty acid more readily than did MFB2. MFB1 was about 2% and MFB2 about 12% of the cytosolic protein in the midgut. cDNA clones for MFB1 and MFB2 both encode proteins of 131 amino acids that are rich in lysine and acidic residues. Analysis of the amino acid sequence alignment of the MFBs with six mammalian fatty acid-binding proteins revealed a number of shared features: 9 conserved glycines, presumably important in turns of the beta-strands; a basic amino acid in a position corresponding to the residue reported to participate in binding the carboxyl group of the fatty acid (Arg in MFB1 and Lys in MFB2); and conservation of many of the residues important in binding the aliphatic portion of the fatty acid. PMID- 1730604 TI - Inhibitors of transcription and translation act synergistically with tumor necrosis factor to cause the activation of phospholipase A2. AB - In this report we have examined the ability of tumor necrosis factor-alpha (TNF) to induce the activity of phospholipase A2 (PLA2) in the cell line C3HA, a murine 3T3-like cell line which is normally resistant to TNF-induced cytolysis but can be sensitized with inhibitors of transcription and translation. Our results show that TNF is normally unable to induce the activity of PLA2 in this cell, as measured by the release of [3H]arachidonic acid. We find, however, that in the presence of either actinomycin D (Act D) or cycloheximide (CHI), TNF is indeed able to induce phospholipase activity and that the TNF-induced activation of PLA2 occurs 2-4 h before the onset of 51Cr release. The release of [3H]arachidonic acid was inhibited by 40-50% by pretreatment with 1 microM dexamethasone. Treatment with dexamethasone also inhibited cytolysis by 40-50% indicating that the CHI-dependent, TNF-induced activation of PLA2 is a cause, not an effect of cytolysis. The ability of TNF to induce the activity of PLA2 was also tested in two other cell types which are resistant to TNF except in the presence of Act D or CHI: SK-MEL-28, a human melanoma-derived cell line, and pVBETK-1cl15.2, an SV40-transformed murine L cell line. Our results were the same, treatment with a combination of Act D and TNF or CHI and TNF was required to cause activation of PLA2. PMID- 1730605 TI - In vitro transcription faithfully reflecting T-cell activation requirements. AB - T-cell activation is a complex process mediated by cell membrane molecules including the T-cell antigen receptor (TCR), adhesive molecules, and cytokine receptors that collectively produce an increase in intracellular Ca2+, and activation of protein kinase C that initiate a genetic program resulting in immunologic function and irreversible differentiation. To understand how these cell membrane events are translated into a genetic regulatory cascade resulting in T-cell function, we have developed an in vitro transcription system, derived from Jurkat T-cells, which demonstrates inducible, cell-type-specific transcription following T-cell stimulation. Nuclear extracts from cells stimulated with phorbol 12-myristate 13-acetate and ionomycin, which activate protein kinase C and mimic physiological activation through the T-cell antigen receptor, transcribe an interleukin-2 (IL-2) enhancer (-326 to +24) template 5 fold more efficient than nuclear extracts from resting T-cells and severalfold more efficient than extracts from Jurkat cells treated with phorbol 12-myristate 13-acetate or ionomycin alone. Further results demonstrate that in vitro transcription of the IL-2 enhancer is T-cell specific since nuclear extracts from rat liver and stimulated HeLa cells are unable to induce IL-2 transcription. The activation-dependent, T-cell-specific in vitro transcription system described here should facilitate the dissection of signals that emanate from the T-cell surface resulting in IL-2 transcription and T-cell activation. PMID- 1730606 TI - (A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin. AB - The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin. PMID- 1730607 TI - Binding of laminin and fibronectin by the trypsin-resistant major structural domain of the crystalline virulence surface array protein of Aeromonas salmonicida. AB - The surface of Aeromonas salmonicida is covered by a tetragonal paracrystalline array (A-layer) composed of a single protein (A-protein, Mr = 50,778). This array is a virulence factor. Cells containing A-layer and isolated A-layer sheets specifically bound laminin and fibronectin with high affinity. Binding by cells was inactivated by selective removal of A-layer at pH 2.2, and neither isogenic A layer-deficient A. salmonicida mutants nor tetragonal paracrystalline array producing Aeromonas hydrophila and Aeromonas sobria strains bound either matrix protein. Laminin binding was by a single class of high affinity interactions (cell Kd = 1.52 nM), whereas fibronectin bound via two classes of interactions, one being similar to that of laminin (cell Class 2 interaction Kd = 6.6 nM). This interaction with both proteins was partly hydrophobic. The Class 1 fibronectin interaction was of lower affinity (cell Kd = 218 nM) and distinct. Purified A protein inhibited binding of both matrix proteins to A-layer, and trypsin cleavage localized the matrix-protein binding region to the N-terminal major trypsin-resistant structural domain of A-protein. Monoclonal antibody inhibition studies showed that A-protein was folded such that Fabs of only one of two antibodies with epitopes mapping C-terminal to this trypsin-resistant peptide was capable of blocking binding. PMID- 1730608 TI - The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. AB - Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes. PMID- 1730610 TI - Identification of novel blood proteins specific for mammalian hibernation. AB - Mammalian hibernation is a unique physiological adaptation that allows the sustainment of life under extremely low body temperatures. In the chipmunk, we found four proteins related specifically to hibernation. These proteins started to diminish in concentration in the blood before and disappeared during hibernation. These proteins reappeared in the blood as hibernation ceased and remained during nonhibernation. The complete or partial amino acid sequences of the four proteins showed that three (27-, 25-, and 20-kDa) were previously unknown, whereas another (55-kDa) is highly homologous with alpha 1-antitrypsin. The three novel proteins are homologous, indicating that they are a family. In the NH2-terminal regions of these proteins, a collagen-like amino acid sequence is present, whereas in their COOH-terminal regions, two sequences, Ser-Ala-Phe Ala-Val-Lys and Val-Trp-Leu-Glu, are conserved. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and gel permeation chromatography under denaturating conditions revealed that the four proteins form a 140-kDa complex in the plasma fraction. The novel proteins were detected in blood of another hibernator, the ground squirrel, but not in rodent nonhibernators, namely tree squirrels and rats. The present finding is the first identification of a hibernation-specific protein. The presence of specific proteins in hibernators suggests the involvement of genetic factors in the control of hibernation. These proteins provide valuable tools for understanding molecular mechanisms of mammalian hibernation. PMID- 1730609 TI - Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization. AB - Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter. PMID- 1730611 TI - Structural and functional analysis of the 5'-flanking region of the rat ceruloplasmin gene. AB - To characterize the mechanisms determining tissue-specific ceruloplasmin gene expression during development, the rat ceruloplasmin gene was isolated in a series of overlapping phage clones. The 5'-flanking region was characterized and the transcription initiation site was identified by primer extension and RNase protection. Nucleotide sequence analysis of this region revealed a typical eukaryotic promotor structure, but no obvious homology with cis-acting elements previously characterized as determining tissue-specific gene expression. Transient expression of chimeric ceruloplasmin-reporter gene constructs containing up to 5200 base pairs (bp) of the 5'-flanking region revealed that sequences 732 bp upstream of the start nucleotide were sufficient to confer hepatocyte-specific expression. The region from -393 to -348 was determined by deletion analysis to contain a positive-acting element, and includes sequence partially homologous to the rat albumin D site. Mobility shift analysis revealed that this region specifically binds a heat-labile nuclear protein from rat liver and from newborn but not adult rat lung. Binding to this region was competed by oligonucleotides corresponding to the albumin D site, but not by oligonucleotides corresponding to binding sites for the hepatocyte transcription factors HNF-1, HNF-3, HNF-4, and C/EBP. These data indicate that ceruloplasmin gene expression is determined in part by a cis-acting region 393 bp upstream of the transcription start site, which binds a previously uncharacterized nuclear protein. The tissue distribution of this nuclear protein suggests that it plays a role in directing ceruloplasmin gene expression in lung and liver during development. PMID- 1730612 TI - Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA. AB - A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase. PMID- 1730613 TI - Endoplasmic reticulum degradation of a subunit of the asialoglycoprotein receptor in vitro. Vesicular transport from endoplasmic reticulum is unnecessary. AB - The H2a subunit of the human asialoglycoprotein receptor is rapidly degraded from the endoplasmic reticulum (ER) when expressed in CHO15B cells. We have reconstituted ER degradation of H2a in semipermeable cells. At least the initial step in degradation (a proteolytic cleavage inhibited by N alpha-p-tosyl-L-lysine chloromethyl ketone and L-1-tosylamido-2-phenylethyl chloromethyl ketone) can occur in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate or in the absence of ATP and postnuclear supernatant, conditions that do not allow vesicular transport of subunit H1 from the ER to the Golgi. We conclude that vesicular transport from the ER is not required for ER degradation of H2a to occur and thus that it takes place in the ER itself. PMID- 1730614 TI - Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen. AB - The effects of the steroid hormones estrogen and progesterone on peptidylarginine deiminase protein-L-arginine iminohydrolase, EC 3.5.3.15) levels in adult ovariectomized mouse uterus were studied. The amount of the enzyme in the uterus was considerably diminished by ovariectomy. When the mice were injected with a variety of estrogenic compounds, 17 beta-estradiol-3-benzoate, which was the most potent stimulator of uterine cell proliferation among the estrogens tested, dramatically elevated the enzyme formation of the uterus in a dose- and time dependent fashion. Results of immunohistochemistry with the antiserum against mouse peptidylarginine deiminase demonstrated that the induction of the enzyme by the estradiol exclusively occurred at the luminal and glandular epithelia, corresponding with the previous findings in the normal estrous cycle. Furthermore, administration of the estradiol significantly increased the content of mRNA coding for peptidylarginine deiminase in uterus, indicating the evidence of regulation in pretranslation. On the other hand, progesterone alone did not restore the enzyme level of the ovariectomized mouse, but moderated the action of estrogen when given in concert with estrogen. Thus, the expression of peptidylarginine deiminase in luminal and glandular epithelia of mouse uterus is controlled by the amount of the steroid hormones estrogen and progesterone. PMID- 1730615 TI - GTPase-mediated activation of ATP sulfurylase. AB - GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism. The activation of the enzyme, purified from Escherichia coli, is titratable with GTP. The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP. The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis. The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins. This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase. PMID- 1730616 TI - Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs. AB - The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition. PMID- 1730617 TI - Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine. AB - Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin. PMID- 1730618 TI - Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain. AB - Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane. PMID- 1730619 TI - Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals. AB - Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI 1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/ 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells. PMID- 1730620 TI - Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition. AB - Oxidized (ox-) low density lipoproteins (LDL) is characterized by the formation of lipid peroxides and their decomposition to reactive aldehydes which covalently link to apoB in LDL. These chemical changes are believed to be responsible for the enhanced recognition of ox-LDL by receptors on macrophages in culture. When oxidation is extensive, particle aggregation also occurs. The aim of this study was to characterize aggregation formation and how this influences the interaction of ox-LDL with macrophages in culture. When LDL was oxidized by incubating at 500 micrograms of protein/ml with 10 microM Cu2+ at 20 degrees C for up to 25 h, time dependent increases in thiobarbituric acid reactive substances, conjugated diene content, electrophoretic mobility, and fluorescence at 360 excitation/430 emission were found. Particle aggregation increased in parallel with several parameters of oxidation and increased with increasing incubation temperatures and LDL concentrations used. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apoB fragments of reproducible sizes and higher molecular weight species appeared after mild oxidation of LDL. The percent of total apoB remaining aggregated in sodium dodecyl sulfate was 50-80% at high degrees of oxidation, whereas it was far less in LDL that had been aggregated without chemical modification. This suggested that intermolecular cross-linking of apoB had occurred during oxidation of LDL at high concentrations. Degradation of ox LDL in mouse peritoneal macrophages (MPM) increased in parallel with the degree of oxidation and with particle aggregation but reached a plateau after 12 h. Results from cross-competition studies in MPM with soluble and insoluble portions of extensively ox-LDL and with acetyl-LDL were consistent with uptake of soluble ox-LDL via both the scavenger receptor and another receptor on MPM, and uptake of the insoluble ox-LDL by an alternative mechanism. PMID- 1730621 TI - Negative and positive cis-acting elements in the promoter of the mouse gene that encodes the serine/glycine-rich peptide core of secretory granule proteoglycans. AB - The gene that encodes a proteoglycan peptide core rich in serine and glycine (SG PG) is selectively expressed by hematopoietic cells that store in their cytoplasmic granules negatively charged proteoglycans bound ionically to numerous positively charged proteins. With deletion analysis, a negative transcription regulatory element was located between residues -250 and -190 of the 5'-flanking region of the mouse SG-PG gene, and a positive regulatory element was located between residues -118 and -81. The negative regulatory element was dominantly active in fibroblasts that do not express the SG-PG gene whereas the positive regulatory element was dominantly active in hematopoietic cells that do express the SG-PG gene. Site-directed mutagenesis was used to demonstrate that the proximal element within the gene's atypical promoter resided between residues -40 and -20. As assessed by gel mobility shift analyses, the nuclei of rat basophilic leukemia-1 cells and rat-1 fibroblasts contain a number of trans-acting factors that interact with the positive and negative cis-acting regulatory elements of the SG-PG gene. Furthermore, some of these trans-acting factors appear to be different for the two cell types. These studies on cell types that do and do not express the SG-PG gene indicate that transcription of this proteoglycan peptide core gene is regulated constitutively by both positive and negative cis-acting elements located 5' of an atypical promoter. PMID- 1730622 TI - Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting. AB - Cell division requires an accurate partitioning of cytoplasmic organelles. The segregation of vacuoles in the budding yeast Saccharomyces cerevisaie occurs at a specific time in the cell cycle and is spatially targeted to the small bud. Several yeast vac mutants have been isolated which are defective in this process. We have now cloned the VAC1 gene, corresponding to the first of these mutants, vac1-1. This gene encodes a protein of 515 amino acids, without homolog in the current data bases. It contains neither long hydrophobic stretches nor a classical leader peptide. The most notable aspect of the sequence is the presence of three zinc fingers. Yeast in which the VAC1 gene has been entirely deleted are viable. However, they grow more slowly than wild-type cells and only form microcolonies when grown on glycerol at 37 degrees C. These yeast are defective in vacuole segregation at both the permissive and nonpermissive temperatures. The vac1 mutant was previously shown to mislocalize carboxypeptidase Y to the cell surface, suggesting that Vac1p is involved in more than one vesicular traffic pathway. PMID- 1730623 TI - The tetrameric structure of NF-mu NR provides a mechanism for cooperative binding to the immunoglobulin heavy chain mu enhancer. AB - We have analyzed the structure and DNA-binding characteristics of the IgH enhancer-binding regulatory protein NF-mu NR. We estimate that there are at least 5000 molecules of NF-mu NR protein/nucleus of non-B cells, based on the yield of active protein following purification. Purified NF-mu NR exists as a tetramer of 40-kDa subunits, even in the absence of its DNA-binding substrate. The protein complex binds to four binding sites flanking the immunoglobulin heavy chain mu enhancer core. Separately, individual sites bind to the tetrameric complex with affinities varying over a 100-fold range. However, when a low affinity site and a high affinity site are present on the same DNA molecule, both are occupied at the same NF-mu NR concentration; thus, there is a strong cooperative interaction between binding sites. The tetrameric structure provides a mechanism for binding cooperativity in which initial binding is mediated through a high affinity site on the DNA molecule followed by the engagement of the low affinity site juxtaposed to adjacent protein subunits. The presence of multiple binding sites flanking the mu enhancer core may reflect the influence of NF-mu NR binding on enhancer three-dimensional structure, transcription factor binding, and/or nuclear matrix interactions for cell type-specific enhancer function. PMID- 1730624 TI - Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18). AB - The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323 1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues. PMID- 1730625 TI - An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport. AB - In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited. PMID- 1730626 TI - Free fatty acid transfer from rat liver fatty acid-binding protein to phospholipid vesicles. Effect of ligand and solution properties. AB - Fatty acid binding proteins (FABP) are a family of 14-15-kDa proteins found in many mammalian cell types in high abundance. Although their precise physiological role remains hypothetical, the transfer of free fatty acids (ffa) to intracellular membrane sites is believed to be an important function of FABP. To better understand the role of FABP in this process, we have examined how the rate of ffa transfer from liver FABP (L-FABP) to model membranes is influenced by variations in ffa structure and properties of the aqueous phase. The rate of transfer of fluorescent anthroyloxy ffa to model acceptor membranes was monitored using a resonance energy transfer assay. The results show that a monounsaturated ffa transfers 2-fold more rapidly than a saturated ffa of equivalent chain length, and a two-carbon increase in acyl chain length results in a 3-fold decrease in transfer rate. The transfer rate decreases logarithmically with increasing ionic strength, suggesting that the aqueous solubility of the ffa is an important determinant of its dissociation rate from L-FABP. Fatty acid binding and the relative partition of n-(9-anthroloxy) ffa to L-FABP as compared with phospholipid membranes both decrease as pH decreases, indicating that ionized but not protonated ffa bind to L-FABP. The rate of ffa transfer from L-FABP to membranes increases approximately 4-fold with increasing pH, suggesting that ionization of the ffa carboxyl group is also an important determinant of the transfer process. Analysis of the dependence of the transfer rate on temperature demonstrates that the delta G++ of the activated state for ffa transfer arises from both enthalpic and entropic processes. These studies demonstrate that the rate of transfer of long chain ffa from L-FABP to membranes is substantially affected by aqueous phase variables as well as properties of the ffa ligand itself. PMID- 1730627 TI - Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. AB - The substrate recognition regions in cytochrome P450 family 2 (CYP2) proteins were inferred by group-to-group alignment of CYP2 sequences and those of bacterial P450s, including Pseudomonas putida P450 101A (P450cam), whose substrate-binding residues have been definitely identified by x-ray crystallography of a substrate-bound form (Poulos T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700). The six putative substrate recognition sites, SRSs, thus identified are dispersively located along the primary structure and constitute about 16% of the total residues. All the reported point mutations and chimeric fragments that significantly affect the substrate specificities of the parental CYP2 enzymes fell within or overlapped some of the six SRSs. Analysis of nucleotide substitution patterns in closely related members in four subfamilies, CYP2A, 2B, 2C, and 2D, consistently indicated that the SRSs have accumulated more nonsynonymous (amino acid-changing) substitutions than the rest of the sequence. This observation supports the idea that diversification of duplicate genes of drug-metabolizing P450s occurs primarily in substrate recognition regions to cope with an increasing number of foreign compounds. PMID- 1730628 TI - Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli. AB - Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli. Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation. Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate. Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity. The photomodified enzyme was labeled by reduction with NaB[3H]4 in the presence and absence of the substrates succinate plus glyoxylate. Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase. These residues are highly conserved in all five known sequences of this enzyme. PMID- 1730629 TI - Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain. AB - Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa. Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium binding sites. To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed. The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901. The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments. The results demonstrate that the four binding sites can be occupied by Ca2+. Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns. Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb. Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions. To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay. The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb. PMID- 1730630 TI - Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. AB - Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ. PMID- 1730631 TI - Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation. AB - The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors. PMID- 1730632 TI - Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase. AB - Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme. PMID- 1730633 TI - Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein. AB - Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3 ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein. PMID- 1730634 TI - Two novel patterns of transforming growth factor beta (TGF-beta) binding to cell surface proteins are dependent upon the binding of TGF-beta 1 and indicate a mechanism of positive cooperativity. AB - Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex. PMID- 1730635 TI - Maxadilan. Cloning and functional expression of the gene encoding this potent vasodilator peptide. AB - Maxadilan is a potent vasodilator peptide released into the skin when the sand fly Lutzomyia longipalpis, an important vector of leishmania, probes for a blood meal. As several lines of evidence suggest that this peptide may play a critical role in the enhancement of leishmania infectivity attributed to sand fly saliva, the peptide has been proposed as a candidate antigen for a leishmanial vaccine. Although maxadilan is the most potent vasodilator peptide known and shares several properties with calcitonin gene-related peptide (CGRP), studies of its structure, physiological effects, and biological roles have been limited by the miniscule quantities available. Here we report the isolation of cDNA and genomic DNA clones that encode maxadilan. The predicted translation product shows no significant homology with any previously isolated proteins. The coding DNA has been expressed in Escherichia coli and the purified recombinant peptide is biologically active with a specific activity comparable to the natural peptide. Recombinant maxadilan will be useful in studies of vascular biology and could lead to novel therapeutic and prophylactic agents. PMID- 1730636 TI - Post-translational regulation of macrophage apoprotein E production. AB - We have transfected the murine macrophage cell line, J774, which does not express its endogenous apoE gene, with a constitutively expressed human apoE cDNA in order to study post-transcriptional and post-translational control of apoE production in macrophages. Loading cells with cholesterol using preincubations in acetylated low density lipoprotein, previously shown to enhance macrophage apoE gene transcription and apoE synthesis, did not increase apoE synthesis or secretion in constitutively expressing transfected cells, suggesting that sterol control of macrophage apoE production occurs predominantly at a transcriptional locus. However, incubation in human high density lipoprotein (HDL3) stimulated apoE secretion and appeared to inhibit degradation of newly synthesized apoE. This effect could be entirely reproduced by incubation with phosphatidylcholine vesicles which increased apoE accumulation in the medium by 2-6-fold. Pulse-chase experiments indicated that the effect of HDL3 or phospholipid vesicles was very rapid (occurring within 15 min) and was independent of changes in apoE synthesis. Furthermore, the increased apoE which accumulated in the medium in the presence of phospholipid vesicles or HDL3 was not due to altered rates of reuptake of labeled apoE since this difference was completely preserved in the absence of extracellular calcium. These results indicate that alteration of sterol content does not regulate macrophage apoE production at a translational or post translational locus but that incubation with HDL3 or phospholipid vesicles can enhance apoprotein E production independent of changes in apoE gene transcription or apoE synthesis. The nature of the signal generated by the phospholipid vesicles which leads to inhibition of intracellular apoE degradation and enhanced apoE secretion will require further investigation. PMID- 1730637 TI - Evidence that extracellular signal-regulated kinases are the insulin-activated Raf-1 kinase kinases. AB - The Raf-1 proto-oncogene protein kinase can be phosphorylated and activated after stimulation of cells with insulin and a variety of other growth factors and mitogens. We recently presented evidence that insulin and certain other growth factors activated one or more Raf-1 kinase kinase activities (Lee, R.M., Rapp, U. R., and Blackshear, P.J. (1991) J. Biol. Chem. 266, 10351-10357). In the present study, four peaks of Raf-1 kinase kinase activity were identified after anion exchange chromatography of cell lysates, and two of these were activated by insulin. Further chromatographic characterization of these two peaks of insulin activated kinase activity indicated that they contained three apparently distinct kinase activities. Two of these activities comigrated with immunoreactive extracellular signal-regulated kinases (ERK) 1 and 2 (mitogen-activated protein kinase) through three different chromatographic separations. Both ERK1 and ERK2 phosphorylated Raf-1 with reasonably high affinity (Km for ERK1 = 90 nM; Km for ERK2 = 120 nM), and produced similar, complex phosphopeptide maps; both kinases also phosphorylated myelin basic protein. The third kinase activity also phosphorylated Raf-1 and myelin basic protein but did not comigrate exactly with either immunoreactive ERK1 or ERK2. We conclude that two and possibly three insulin-activated Raf-1 kinase kinases are members of the ERK family. PMID- 1730638 TI - Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973. AB - Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation of proteins was examined in cells expressing wild-type (WT-EGFR) EGF receptors or EGF receptors truncated at residue 973 (973-EGFR). A much broader spectrum of tyrosine phosphorylated proteins was found following EGF treatment of 973-EGFR expressing cells compared with cells expressing wild-type receptors. Several additional ras GTPase activating protein-associated tyrosine phosphorylated proteins were found in EGF-treated 973-EGFR cells relative to WT-EGFR cells. Additional tyrosine phosphorylated proteins were also found to co-immunoprecipitate with phospholipase C gamma 1 (PLC gamma 1) following EGF treatment of cells expressing 973-EGFR relative to cells expressing WT-EGFR. EGF-stimulated tyrosine phosphorylation of PLC gamma 1 was found in cells expressing WT-EGFR, but not in cells expressing 973-EGFR. WT-EGF receptor from EGF-treated cells bound well to bacterially expressed src homology (SH) regions of PLC gamma 1 and to a lesser extent to bacterially expressed GTPase activating protein SH regions. No binding of 973-EGF receptor to SH regions of either protein could be detected. EGF treatment greatly reduced the half-life of WT-EGFR, but had relatively little effect on the half-life of 973-EGFR. EGF induced internalization of 973-EGFR at a slower rate than WT-EGFR and caused the appearance of discrete receptor degradation products for both cell types. The data indicate that truncation of the EGF receptor at residue 973 alters receptor substrate specificity, decreases the rate of receptor internalization, and has an inhibitory effect on receptor degradation. PMID- 1730639 TI - Extensive editing of both processed and preprocessed maxicircle CR6 transcripts in Trypanosoma brucei. AB - Transcripts from several genes encoded in the Trypanosoma brucei maxicircle genome are altered by posttranscriptional uridine insertion and deletion through a process called RNA editing. We find that transcripts from the CR6 gene are extensively edited by addition of 132 uridines and deletion of 28 uridines to produce a fully edited mRNA 47% larger than unedited mRNA. Two open reading frames (ORFs) and their initiation and termination codons are created by editing of CR6 mRNA. Both ORFs specify small, hydrophobic proteins with no homology to proteins in three databases. Both unedited and edited CR6 transcripts are more abundant in bloodstream form than in procyclic form parasites. cDNA clones spanning both CR6 and the downstream NADH dehydrogenase subunit 5 (ND5) gene were isolated, indicating that mature CR6 and ND5 transcripts arise from a common precursor. Sequencing of these cDNAs revealed 37 nucleotides of overlap between the 3' end of CR6 and the 5' end of ND5. In addition, the CR6 portion of many of these molecules was extensively edited, indicating that RNA editing can precede precursor processing. These results provide the first clear demonstration of polycistronic transcription of maxicircle genes, and suggest new mechanisms by which both RNA editing and precursor processing may regulate maxicircle gene expression. PMID- 1730640 TI - Cis-acting sequences in the 5'-untranslated region of the ribosomal protein A1 mRNA mediate its translational regulation during early embryogenesis of Drosophila. AB - The rate of ribosomal (r)-protein synthesis in the early Drosophila embryo is low despite the presence of abundant, maternally supplied r-protein mRNAs. This low rate is due to specific repression of r-protein mRNA translation. In contrast to r-protein mRNAs, most other mRNAs are efficiently translated in the early embryo. Here we report on the identification of cis-acting sequences that mediate translational repression of the r-protein A1 (rpA1) mRNA. Chimeric genes containing sequences from the translationally regulated rpA1 mRNA fused to the constitutively translated alpha-tubulin mRNA were constructed and transformed into the Drosophila germ line. Translation of the corresponding hybrid mRNAs was measured in ovaries and embryos of the transgenic flies. The results indicated that a 89-nucleotide sequence in the untranslated rpA1 mRNA leader is by itself sufficient to confer full translational regulation to a heterologous mRNA. PMID- 1730641 TI - Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs. AB - Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins. PMID- 1730642 TI - Deoxygenation-linked association of a tetrameric component of chicken hemoglobin. AB - Deoxygenation-dependent association of hemoglobin tetramers appears to be widespread among amphibians, reptiles, and possibly all or most birds. The evidence for this conclusion depends largely on oxygen equilibria of whole blood which have Hill coefficients that reach values as high as 5-7 at 80-90% oxygenation. Computer simulation of the sedimentation velocity behavior of the major components A and D of chicken hemoglobin shows that component D but not A self-associates to form dimers of tetramers. The gradient profiles at pH 7.5 were satisfactorily fitted with an association constant of 1.26 x 10(4) M-1 and sedimentation coefficients of 4.63 and 7.35 S for tetramer and (tetramer)2, respectively. Since components A and D share common beta chains we conclude that tetramer-tetramer contacts must depend on surface residues of the alpha chains. Comparison of the amino acid sequences of the alpha D and alpha A chains of the hemoglobins from 12 avian species ranging from sparrow to ostrich shows that 20 residues are conserved in the alpha D chains but not in the alpha A chains. Nine of these (45%) are clustered between positions E20 and FG2. Four of the latter, Lys71 (E20), Asn75 (EF4), Gln78 (EF7), and Glu82 (F3) are conserved in all alpha D chains even though they do not appear to participate in intratetramer contacts. Molecular modeling indicates that residues Lys71, Gln78, and Glu82 of the alpha chain are strong candidates for the primary tetramer-tetramer contacts. PMID- 1730643 TI - Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling. AB - The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin. Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical. Genetic deletion of each gene revealed that at least one functional copy of either EFT gene is required for cell viability. Messenger RNA levels of yeast EF-2 parallel cellular growth and peak in mid-log phase cultures. The EF-2 protein sequence is strikingly conserved through evolution. Yeast EF-2 is 66% identical to, and shares over 85% homology with, human EF-2. In addition, yeast and mammalian EF-2 share identical sequences at two critical functional sites: (i) the domain containing the histidine residue that is modified to diphthamide and (ii) the threonine residue that is specifically phosphorylated in vivo in mammalian cells by calmodulin-dependent protein kinase III, also known as EF-2 kinase. Furthermore, yeast EF-2 also contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors, and its GTP-binding domain exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members. Based upon these observations, we have modeled the G-domain of the deduced EF-2 protein sequence to the solved crystallographic structure for EF-Tu. PMID- 1730644 TI - The G226A mutant of Gs alpha highlights the requirement for dissociation of G protein subunits. AB - Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form Gs alpha instead of the wild-type protein. The mutant Gs alpha protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein alpha subunit from the beta gamma subunit complex (Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B., and Bourne, H.R. (1988) Nature 334, 712-715). G226A Gs alpha was synthesized in Escherichia coli, purified, and characterized. Examination of the kinetics of dissociation of guanosine 5'-3-O (thio)triphosphate (GTP gamma S) suggests that G226A Gs alpha is incapable of assuming the conformation necessary for high affinity binding of Mg2+ to the alpha subunit-GTP gamma S complex. Associated changes include the failure of Mg2+ and GTP gamma S to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of alpha from beta gamma. However, the GTPase activity of the mutant protein is near normal (at high Mg2+ concentrations), and the protein is capable of activating adenylylcyclase. A similar defect is present in G49V Gs alpha. Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A Gs alpha, and this mutation thus highlights the crucial nature of this reaction as a component of G protein action. PMID- 1730645 TI - Transcriptional and post-transcriptional control of apolipoprotein E gene expression in differentiating human monocytes. AB - The present studies examined the mechanisms responsible for the regulation of apolipoprotein (apo) E gene expression during human monocytic differentiation. Levels of apoE mRNA were low in undifferentiated THP1 cells, a human monocytic cell line. Addition of 12-O-tetradecanylphorbol-13-acetate (PMA) induced differentiation of these cells to a macrophage-like phenotype and was associated with increased apoE mRNA abundance in a time-dependent fashion, up to 10-11-fold within 32 h. Results of nuclear run-on transcription assays demonstrated that the apoE gene was transcriptionally active in undifferentiated THP1 cells and that differentiation of monocytes with PMA was associated with a maximal increase of apoE gene transcription rate of only 2-3-fold at 6-12 h. Using actinomycin D as an inhibitor of new transcription, we could demonstrate a more rapid degradation of mature apoE mRNA in undifferentiated compared to differentiated cells, suggesting that the apoE mRNA species was more stable in differentiated THP1 cells. Primer extension assays performed using RNA extracts from undifferentiated and differentiated THP1 cells confirmed the increase of apoE mRNA abundance in the latter but failed to disclose heterogeneity in apoE gene transcription start site between these two phenotypes. These findings indicate that apoE gene expression is controlled at both transcriptional and post-transcriptional loci during human monocyte-macrophage differentiation. PMID- 1730646 TI - DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase. AB - In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation. PMID- 1730647 TI - Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo. AB - The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase. No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase). A maltose binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E. coli cells. Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II. Cell growth also was inhibited following induction of MBP27-P synthesis. Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors. Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site. Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site. When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity. Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region. PMID- 1730648 TI - Proteoglycan-Lb, a small dermatan sulfate proteoglycan expressed in embryonic chick epiphyseal cartilage, is structurally related to osteoinductive factor. AB - We have isolated cDNA clones encoding the core protein of PG-Lb, proteoglycan which has been shown to be preferentially expressed in the zone of flattened chondrocytes of the developing chick limb cartilage (Shinomura, T., Kimata, K., Oike, Y., Yano, S., and Suzuki, S. (1984) Dev. Biol. 103, 211-220). The deduced amino acid sequence from the cDNA analysis indicates the presence of consensus leucine-rich repeats which are present in other small proteoglycans, decorin, biglycan, and fibromodulin. However, the homology analysis revealed that chick PG Lb showed a higher homology (about 50% in the region containing leucine-rich repeats) to human osteoinductive factor, OIF, rather than to the other small proteoglycans. Furthermore, 6 cysteine residues are detected in both PG-Lb and OIF with invariant relative positions. Therefore, such an evolutionarily conserved structure in the PG-Lb core protein might be involved in some important biological functions of this molecule. In close relation to the structural similarity to OIF, the unique expression of PG-Lb in the ossifying area of cartilage suggested the possible participation of this proteoglycan in osteogenic processes. PMID- 1730649 TI - Expression and purification of functional G protein alpha subunits using a baculovirus expression system. AB - The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S. PMID- 1730650 TI - Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen. AB - By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen. Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen. PMID- 1730651 TI - Affinity purification of insulin-degrading enzyme and its endogenous inhibitor from rat liver. AB - A metallothiol protease called insulin-degrading enzyme (IDE) seems to be implicated in insulin metabolism to terminate the response of cells to hormone, as well as in other biological functions, including muscle differentiation, regulation of growth factor levels, and antigen processing. In order to obtain highly pure and biologically active IDE, we have developed an immunoaffinity method using a monoclonal antibody to this enzyme (9B12). When the cytosolic fraction of rat liver was first applied to a 9B12-coupled Affi-Gel 10 column, more than 97% of the insulin-degrading activity was absorbed. Among various kinds of buffers successfully eluting the enzyme, only the buffer with a high pH (pH 11) could retain the full biological activity of this enzyme. IDE was further purified via two steps of chromatography using Mono Q anion exchange and Superose 12 molecular sieve columns. The final preparation showed a single band at 110 kDa on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the eluate from the immunoaffinity column, the inhibitory activity associated with the enzyme was also observed. To better recover this endogenous inhibitor, heat-treated cytosolic fraction was fractionated by ammonium sulfate precipitation and applied to the immunoaffinity column on which IDE had been adsorbed. Then, IDE and its inhibitor could be co-eluted with pH 11 as a complex form. After heat treatment of this fraction, the inhibitor was further purified using the same series of chromatography as IDE to more than 20,000-fold; it showed a 14 kDa band on SDS-PAGE. It inhibited both the insulin degradation by IDE in a competitive manner and the cross-linking of 125I-insulin to IDE. Highly purified IDE and the endogenous inhibitor will be useful tools for better understanding the various biological functions of this enzyme. PMID- 1730652 TI - Enhancement of protein kinase C in murine lymphokine-activated killer cells by retinoic acid. AB - We previously reported that retinoic acid (RA) augmented mouse (BALB/c) lymphokine (interleukin-2)-activated killer (LAK) cell activity in a dose and time dependent manner. As evidence available has suggested the role of protein kinase C (PKC) in the regulation of cell mediated cytotoxicity, the present work was to investigate whether or not PKC may mediate the enhancement of LAK cell activity by RA. Accompanied with an augmented LAK cell activity, RA increased total PKC enzyme activity, [3H]phorbol 12,13-dibutyrate binding activity, and the amount of immunoreactive PKC. A prolonged treatment (18 h) of LAK cells with 12-O tetradecanoylphorbol-13-acetate resulted in the loss of both PKC and LAK cell activity. PKC inhibitors, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride and staurosporine, also drastically reduced LAK cell activity. Although most of the total PKC activity (97%) was detected in the cytosol fraction, the increase in PKC activity was attributed to an increased enzyme activity in both cytosol and membrane fractions, and shown to be RA dose dependent. Kinetics study revealed that the increase in PKC was a time-dependent process and the enhancement was detectable as early as 8 h after the addition of RA to LAK cell culture. By immunoblotting, the cytosol PKC of LAK cells was shown to contain alpha and beta isoforms, but not gamma. RA further increased the expression of PKC alpha. The enhanced expression of alpha isozyme of PKC by RA was also in a dose and time dependent manner. Taken together, these results indicate that the mechanism of the augmentation of LAK cell activity by RA may in part result from the increase in PKC, especially PKC alpha isozyme. PMID- 1730653 TI - Direct mobilization of retinol from hepatic perisinusoidal stellate cells to plasma. AB - We have studied the mechanism for mobilization of retinol from stellate cells. Our data show that perisinusoidal stellate cells isolated from liver contained retinol-binding protein (RBP) mRNA. By Western blot analysis we found that cultivated liver stellate cells secreted RBP into the medium. Cultivated stellate cells loaded in vitro with [3H]retinyl ester mobilized radioactive retinol as a complex with RBP. Furthermore, exogenous RBP added to the medium of cultured stellate cells increased the secretion of retinol to the medium. These data suggest that liver stellate cells in vivo mobilize retinol directly to the blood and that a transfer to parenchymal cells for secretion as holo-RBP is not required. The direct mobilization of retinol from liver stellate cells as retinol RBP to blood is indirectly supported by the demonstration of RBP mRNA production and RBP secretion by lung stellate cells. The data suggest that the same mechanism for retinol mobilization may exist in hepatic and extrahepatic stellate cells. This is, vitamin A-storing stellate cells in liver, lungs, and probably also in other organs may synthesize their own RBP (or alternatively use exogenous RBP) and mobilize holo-RBP directly to the blood. PMID- 1730654 TI - Regulation of the Egr-1 gene by tumor necrosis factor and interferons in primary human fibroblasts. AB - Treatment of quiescent primary human fibroblasts with tumor necrosis factor (TNF) alpha, TNF-beta, interleukin-1, interferon (IFN) alpha, IFN beta, or IFN gamma induced Egr-1 mRNA. In primary human fibroblasts TNF-alpha and TNF-beta were mildly mitogenic and IFN alpha and IFN gamma were growth inhibitory. However, in HeLa cells TNF but not IFN induced the expression of Egr-1 mRNA, while both cytokines inhibited HeLa cell division. Kinetic measurements of Egr-1 gene expression showed that TNF-alpha, TNF-beta, and IFN gamma increased the cellular concentration of Egr-1 mRNA within 30 min. A maximum induction of Egr-1 mRNA was detected at approximately 60 min which dropped to basal level by 180 min. Induction was inhibited by H7 and staurosporine but not by HA1004, indicating the involvement of a functional protein kinase C. The Egr-1 message was translated and the cellular Egr-1 protein detected within 60 min of cytokine treatment. Despite similar Egr-1 mRNA induction, the amount of Egr-1 protein translated in IFN alpha- and IFN gamma-treated cells was lower than in those treated with TNF alpha and TNF-beta, and highest in the EGF-treated primary human fibroblasts. Indeed, the level of Egr-1 protein translated in these cells correlated proportionally with both the phosphorylation of cap-binding protein (eukaryotic initiation factor) and the amount of cellular DNA synthesis in the variously treated fibroblasts. These results suggest that both growth stimulatory and inhibitory cytokines can regulate Egr-1 gene expression at the transcriptional and translational level. However, the combination of these regulatory controls may determine the cellular concentration of the Egr-1 gene product and hence, its effect on cell proliferation. PMID- 1730655 TI - Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events. AB - To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors. PMID- 1730656 TI - Identification of glucose response proteins in two biological models of beta-cell adaptation to chronic high glucose exposure. AB - High resolution 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with computerized analysis of gel images was used to search for proteins whose biosynthesis was induced or repressed in pancreatic islet cells chronically exposed to high glucose in an in situ and a tissue culture model of islet cell adaptation to excessive fuel load. The in situ model involved a 4-day intravenous infusion of either 50% glucose or 0.45% saline solution, followed by islet isolation, [35S]methionine labeling at 3 and 18 mM glucose for both groups, and protein analysis by 2-dimensional SDS-PAGE. The tissue culture model involved a 7-day culture of isolated rat islets in RPMI 1640 with 10% fetal calf serum containing either 3 or 30 mM glucose, followed by radiolabeling and 2-dimensional PAGE of proteins as in the in situ model. A small fraction of about 1.5% of the approximately 2000 identifiable proteins can be characterized as adaptive proteins. Of these altogether 58 proteins in the two models, 5 proteins were demonstrable in both models and two of these (proteins 1526 and 7622) are particularly noteworthy. Protein 1526 (Mr 57,000; pI 5.09) showed the same response pattern in both models and its expression was most enhanced when islets from chronically glucose-infused animals or those cultured for 7 days at 30 mM were radiolabeled at 18 mM glucose. Protein 7622 (Mr 68,000; pI 6.50) (also known as GSP-65; Collins, H.W., Buettger, C., and Matschinsky, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5494-5498) showed a different labeling pattern in the two models: stimulation of [35S]methionine incorporation by 18 mM glucose both in control and experimental islets from the infusion study, but lack of such stimulation of radiolabeling in islets cultured for 7 days at 30 mM glucose in contrast to islets cultured at 3 mM. The experimental strategy and the methodology are evaluated and the significance of the results is discussed. Potentials of the approach and plans for future experiments are considered. PMID- 1730657 TI - Structure function relationships in the ribosomal stalk proteins of archaebacteria. AB - The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli. Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region. The mutant protein genes were overexpressed in E. coli and the products purified. The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation. The SsoL12 protein was selectively removed from entire S. solfataricus ribosomes by an ethanol wash. The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity. S. solfataricus L12 protein overexpressed in E. coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity. Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles. Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes. In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome. None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity. Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation. Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed. This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome. PMID- 1730658 TI - Roles of TATA and initiator elements in determining the start site location and direction of RNA polymerase II transcription. AB - Transcriptional initiator elements and TATA elements are functionally similar, in that both can act in concert with a Sp1-dependent activator element to direct high levels of accurately initiated transcription. In this study, we have used in vitro transcription experiments to elucidate the relative activities of TATA and initiator. By varying the locations of TATA and initiator elements within a simple promoter, we compared their abilities to localize transcription start sites and to mediate transcriptional activation by Sp1. In addition, we addressed the contributions of initiator and TATA towards determining the direction of transcription from a promoter. Finally, we addressed the prevalence of initiator elements by analyzing the start site regions of several genes. We found that many of these regions possess initiator activity, although an initiator consensus sequence could not be defined. Taken together, the data presented suggest that an initiator is a common element that can influence the direction of transcription as well as the location of a transcription start site. However, in general, an initiator was incapable of altering these activities of a TATA box within a promoter containing both core elements. PMID- 1730659 TI - Diverse transcriptional functions of the multisubunit eukaryotic TFIID complex. PMID- 1730660 TI - Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies. AB - Heterotrimeric guanine nucleotide binding proteins function in the coupling of a diverse span of cell surface receptors to a variety of intracellular signaling pathways, some of which stimulate cellular proliferation. With the recent discovery that mutated forms of G proteins are present in specific tumors, there has been an increased interest in the determination of the role of specific subtypes of G proteins in the regulation of cellular growth. We have attempted to determine which subtypes of G proteins are directly involved in serum-stimulated DNA synthesis through microinjection of inhibitory antibodies into living cells. Inhibitory rabbit polyclonal antibodies directed against specific Gi alpha subunits were introduced into living Balb/c 3T3 fibroblasts by microinjection, and the effect upon serum-stimulated DNA synthesis was examined. Results of these experiments indicate that Gi2 plays a direct role in serum-stimulated DNA synthesis in living cells and suggest that G proteins may function in a variety of mitogenic signaling pathways initiated by serum growth factors. PMID- 1730661 TI - Characterization of the statin-like S1 and rat elongation factor 1 alpha as two distinctly expressed messages in rat. AB - Previously, we reported a rat S1 protein that is antigenically related to statin, a nonproliferating cell-specific marker; however, it shares high homology with the known human elongation factor-1 alpha (EF-1 alpha). To differentiate S1 from rat EF-1 alpha and to study their respective regulation for expression, a rat EF 1 alpha cDNA clone was isolated and characterized. The nucleotide and deduced amino acid sequences of this partial rat EF-1 alpha cDNA are compared with that of human and mouse as well as with rat S1. Both their messages were detected in rat brain by EF-1 alpha- or S1-specific probes. However, the mRNA encoding EF-1 alpha is more abundant than that encoding S1. S1 and EF-1 alpha expression were investigated in the parotid and submandibular glands of untreated rats and those treated with isoproterenol, a proliferation-inducing catecholamine. Quantitative solution hybridization demonstrated a dramatic reduction (approximately 68%) in the S1 mRNA following isoproterenol injection in proliferation-responsive parotid glands and a mild reduction (approximately 20%) of S1 steady-state messages in the proliferation-refractile submandibular glands. A slight increase or no changes of EF-1 alpha levels in both parotid and submandibular glands following isoproterenol treatment are also observed. Therefore, the EF-1 alpha and S1 genes are different genes, both expressed and regulated in vivo, but in differential quantitative and qualitative patterns. PMID- 1730662 TI - Carbohydrate binding specificity of immobilized Psathyrella velutina lectin. AB - The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1-- -4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue. PMID- 1730663 TI - Purification and characterization of a novel protease from solid substrate cultures of Phanerochaete chrysosporium. AB - The white-rot basidiomycete Phanerochaete chrysosporium is mostly known for its extracellular ligninolytic enzymes. In this paper, the purification and characterization of a novel extracellular protease secreted by this fungus in solid substrate cultures under ligninolytic conditions are described. The purification steps included extraction of enzymes from the wood substrate, concanavalin A-Sepharose chromatography, anion-exchange chromatography (Mono Q), and size-exclusion chromatography (Superose 12). The purified protein migrates with Mr = 40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has an isoelectric point of 5.6 and a sharp pH optimum at 4.0. The protease is totally inhibited by Hg2+, p-hydroxymercuribenzoic acid, and N-bromosuccinimide, but is insensitive to phenylmethanesulfonyl fluoride, pepstatin A, and EDTA. Its amino acid composition and NH2-terminal sequence have also been determined. The sequence data and the binding of the protease to concanavalin A indicate that the protease is a glycoprotein. The protease differs in its physicochemical properties and its response to inhibitors from other extracellular proteases previously found in another strain of P. chrysosporium. The data suggest that it has properties of both aspartate-type and thiol-type proteases. PMID- 1730664 TI - Genomic organization and alternative promoter usage of the two thyroid hormone receptor beta genes in Xenopus laevis. AB - Each of the two Xenopus laevis thyroid hormone receptor beta genes is at least 70 kilobases in length with similar intron-exon organization. There are up to eight alternatively spliced exons in the 5'-untranslated region. Excluding the extreme amino terminus, each receptor is encoded by six exons spanning about 6 kilobases of the genome, in which each of the two zinc fingers that comprise the DNA binding domain is encoded by a separate exon and the hormone-binding domain is split into three exons. The last exon of the coding region also contains at least 600 base pairs of the 3'-untranslated region, which is about 8 kilobases. Each of the receptor genes has two promoters and just one of them is up-regulated in tadpoles by the administration of thyroid hormone. PMID- 1730665 TI - The regulation of thyroid hormone receptor beta genes by thyroid hormone in Xenopus laevis. AB - The mRNA level of the two Xenopus laevis thyroid hormone receptor beta (TR beta) genes is up-regulated in tadpoles and cultured cells by the addition of thyroid hormone (TH). Up-regulation of transcripts of the 5' most exon is detectable 2-3 h before that of full-length TR beta mRNA, strongly suggesting that up-regulation is under transcriptional control. The TH-induced up-regulation in cultured cells is inhibited by cycloheximide when measured either by a transcription initiation assay that measures transcripts of the 5' most exon or by synthesis of full length TR beta mRNA. From this we conclude that in cultured cells protein synthesis is required for up-regulation of TR beta mRNA. The up-regulation of TR beta mRNA by TH in tadpoles is only partially inhibited by protein synthesis inhibitors. A survey of "thyroid response" genes in the literature reveals that the reported change in gene expression is, in most cases, sensitive to protein synthesis inhibition. This is true of genes with demonstrable thyroid hormone response elements. If an unknown protein must be synthesized to effect up regulation of a thyroid hormone response gene, it calls into question assays that only take into account the binary reaction between thyroid hormone receptor and a putative thyroid hormone response element. PMID- 1730666 TI - Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster. AB - The genetic determinants of enterobacterial common antigen (ECA) include the rfe and rff genes located between ilv and cya near min 85 on the Escherichia coli chromosome. The rfe-rff gene cluster of E. coli K-12 was cloned in the cosmid pHC79. The cosmid clone complemented mutants defective in the synthesis of ECA due to lesions in the rfe, rffE, rffD, rffA, rffC, rffT, and rffM genes. Restriction endonuclease mapping combined with complementation studies of the original cosmid clone and six subclones revealed the order of genes in this region to be rfe-rffD/rffE-rffA/rffC-rffT-rffM . The rfe gene was localized to a 2.54-kilobase ClaI fragment of DNA, and the complete nucleotide sequence of this fragment was determined. The nucleotide sequencing data revealed two open reading frames, ORF-1 and ORF-2, located on the same strand of DNA. The putative initiation codon of ORF-1 was found to be 570 nucleotides downstream from the termination codon of rho. ORF-1 and ORF-2 specify putative proteins of 257 and 348 amino acids with calculated Mr values of 29,010 and 39,771, respectively. ORF 1 was identified as the rfe gene since ORF-1 alone was able to complement defects in the synthesis of ECA and 08-side chain synthesis in rfe mutants of E. coli. Data are also presented which suggest the possibility that the rfe gene is the structural gene for the tunicamycin sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis. PMID- 1730667 TI - A site-directed mutagenesis study of human placental aromatase. AB - Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Using the x-ray structure of cytochrome P-450cam as the model, seven mutants of human aromatase were designed and expressed in Chinese hamster ovary cells by a stable expression method. They are His-128--- Gln, His-128----Ala, Cys-299----Ala, Glu-302----Leu, Asp-309----Asn, Asp-309--- Ala, and Ser-312----Cys. The presence of the aromatase mutants in the transfected Chinese hamster ovary cells were confirmed by immunoprecipitation analysis. The kinetic parameters of these mutants using [1 beta,2 beta-3H] androstenedione (or [1 beta-3H]androstenedione), and [1 beta,2 beta-3H]testosterone as substrates were determined. In addition, inhibition profiles for these mutants with two aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide were obtained. Furthermore, the reactions catalyzed by these mutants were examined by evaluating the levels of the product estrone, and two intermediates, 19 hydroxyandrostenedione and 19-oxoandrostenedione by reverse phase high performance liquid chromatography using [7-3H]androstenedione as the substrate. Our results indicate that among the positions we modified, Asp-309 appears to be very important for the enzyme catalysis. PMID- 1730668 TI - Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase. AB - A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme. PMID- 1730669 TI - Primary structure of human thromboxane synthase determined from the cDNA sequence. AB - Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450. PMID- 1730670 TI - Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen activated kinases that recognize the same amino acid motif as S6 kinase II. AB - The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled phosphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82 Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78 Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress. PMID- 1730671 TI - N-acetyl-p-benzoquinone imine induces Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis. AB - The mechanism of N-acetyl-p-benzoquinone imine (NAPQI)-induced release of Ca2+ from rat liver mitochondria was investigated. The addition of NAPQI or 3,5-Me2 NAPQI (a dimethylated analogue of NAPQI with only oxidizing properties) to mitochondria resulted in the rapid and extensive oxidation of NADH and NADPH. High-performance liquid chromatographic analysis of mitochondrial pyridine nucleotides revealed that the formation of NAD+ and NADP+ was followed by a time dependent net loss of total pyridine nucleotides as a result of their hydrolysis, with the formation of nicotinamide. Preincubation of the mitochondria with cyclosporin A completely prevented the quinone imine-stimulated release of sequestered Ca2+ from mitochondria. Cyclosporin A did not affect the ability of NAPQI or 3,5-Me2-NAPQI to oxidize NAD(P)H but prevented the quinone imine-induced hydrolysis of the pyridine nucleotides. Although there was no detectable change in total protein-bound ADP-ribose content during quinone imine-induced Ca2+ release from mitochondria, meta-iodobenzylguanidine, a competitive inhibitor of protein mono(ADP-ribosylation), prevented Ca2+ release by NAPQI and 3,5-Me2 NAPQI; meta-iodobenzylguanidine did not inhibit the quinone imine-induced NAD(P)H oxidation and only partially blocked hydrolysis of the oxidized pyridine nucleotides. It is concluded that NAPQI causes the oxidation of mitochondrial NADH and NADPH, and stimulates Ca2+ release as a result of the further hydrolysis of the oxidized pyridine nucleotides and protein mono(ADP-ribosylation). PMID- 1730672 TI - The orotidine-5'-monophosphate decarboxylase gene of Myxococcus xanthus. Comparison to the OMP decarboxylase gene family. AB - The nucleotide sequence of the Myxococcus xanthus orotidine-5'-monophosphate decarboxylase (OMP DCase) gene was determined. The derived protein sequence is not closely related to other prokaryotic OMP DCase sequences; nor is it closely related to any eukaryotic OMP DCase sequences. Progressive multiple alignment of the M. xanthus OMP DCase protein sequence with 19 other OMP DCase sequences revealed four conserved regions present in all 20 sequences. Ten entirely conserved residues were found in these four regions and one region contains a tight cluster of 5 conserved residues, certain of which may be catalytically active residues. A second open reading frame was found upstream of uraA and oriented in the same direction as uraA. A stretch of 21 consecutive pyrimidine (C or T) residues were found in the intercistronic region between the potential ribosome-binding site of uraA and the UGA stop codon of the upstream open reading frame. RNA directly upstream of the pyrimidine run, including the UGA stop codon of the upstream open reading frame, could be folded into a stable hairpin structure resembling Rho-independent terminators of Escherichia coli. Expression of the uraA gene may be regulated by an intercistronic transcription termination mechanism. PMID- 1730673 TI - Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase. AB - The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5' exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3' azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate. PMID- 1730674 TI - Site-directed deletion mutants of a carboxyl-terminal region of human dihydrofolate reductase. AB - Substrate and inhibitor binding to dihydrofolate reductase (DHFR) primarily involves residues in the amino-terminal half of the enzyme; however, antibody binding studies performed in this laboratory suggested that the loop region located in the carboxyl terminus of human DHFR (hDHFR; residues 140-186) is involved in conformational changes that occur upon ligand binding and affect enzyme function (Ratnam, M., Tan, X., Prendergast, N.J., Smith, P.L. & Freisheim, J.H. (1988) Biochemistry 27, 4800-4804). To investigate this observation further, site-directed mutagenesis was used to construct deletion mutants of hDHFR missing 1 (del-1), 2 (del-2), 4 (del-4), and 6 (del-6) residues from loops in the carboxyl terminus of the enzyme. The del-1 mutant enzyme has a two-amino acid substitution in addition to the one-amino acid deletion. Deletion of only one amino acid resulted in a 35% decrease in the specific activity of the enzyme. The del-6 mutant enzyme was inactive. Surprisingly, the del-4 mutant enzyme retained a specific activity almost 33% that of the wild type. The specific activity of the del-2 mutant enzyme was slightly higher (38% wild-type activity) than that of the del-4 mutant. All three active deletion mutants were much less stable than the wild-type enzyme, and all three showed at least a 10-fold increase in Km values for both substrates. The del-1 and del-2 mutants exhibited a similar increase in KD values for both substrate and cofactor. The three active deletion mutants lost activity at concentrations of activating agents such as KCl, urea, and p-hydroxymercuribenzoate that continued to stimulate the wild-type enzyme. Antibody binding studies revealed conformational differences between the wild type and mutant enzymes both in the absence and presence of bound folate. Thus, although the loops near the carboxyl terminus are far removed from the active site, small deletions of this region significantly affect DHFR function, indicating that the loop structure in mammalian DHFR plays an important functional role in its conformation and catalysis. PMID- 1730675 TI - Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae. AB - Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. We cloned a human pyrroline-5 carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100. Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy. The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy. The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable. The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein. The derived amino acid sequence is 32% identical with that of S. cerevisiae. By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17. PMID- 1730676 TI - Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa tissue factor complexes in the absence of factor Xa. AB - Extrinsic pathway inhibitor plays a key role in modulating tissue factor dependent blood coagulation. We have studied binding of radioiodinated recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. rEPI in the absence of added reactants bound to a limited extent to three cell lines studied. Binding of rEPI to two cell lines possessing surface tissue factor, but not to a cell line lacking surface tissue factor, was markedly increased in the presence of both factor VIIa and factor Xa, and calcium ions. Moreover, some increased tissue factor-dependent binding was also demonstrated with factor VIIa alone. Binding isotherms of rEPI to factor VIIa-tissue factor obtained with an ovarian carcinoma cell line were hyperbolic. Scatchard plots indicated the following: a Kd value of 4.5 +/- 1.5 nM and 335,000 +/- 84,000 sites/cell when factor Xa was present; a Kd value of 11.9 +/- 3.5 nM and 236,000 +/- 68,000 sites/cell when factor Xa was absent. In functional studies, high concentrations of rEPI, e.g. 27-67.5 nM, were found to inhibit factor VIIa-tissue factor catalyzed release of activation peptide from tritiated factor IX in the absence of factor Xa. Whereas factor Xa was thus shown not to be required for rEPI to inhibit factor VIIa-tissue factor catalytic activity, its presence markedly enhanced rEPI's inhibitory function. Since the local concentration of extrinsic pathway inhibitor achieved at a site of tissue injury is unknown, the physiologic significance of the observation of extrinsic pathway inhibitor-induced inhibition of factor VIIa-tissue factor activity in the absence of factor Xa is not clear. However, factor Xa-independent inhibition could play a significant role when large doses of rEPI are administered in experimental studies of thrombosis. PMID- 1730677 TI - Phenylalanine-induced phosphorylation and activation of rat hepatic phenylalanine hydroxylase in vivo. AB - Rats were given intraperitoneal injections of 2 mCi of carrier-free 32Pi and substances known to activate liver phenylalanine hydroxylase. After 30 min, these animals were anesthetized and their livers removed for analysis of enzyme activity, 32Pi incorporation into immunoprecipitated phenylalanine hydroxylase and [gamma-32P]ATP specific activity. Following glucagon treatment, rat liver phenylalanine hydroxylase activity was stimulated more than 6-fold when assayed in the presence of the natural cofactor, tetrahydrobiopterin (BH4). Glucagon injection also resulted in an incorporation of 0.41 mol of 32Pi/mol of hydroxylase subunit (approximately 50,000 Da). In vivo stimulation of phenylalanine hydroxylase activity and 32Pi incorporation by glucagon had been previously observed in this laboratory (Donlon, J., and Kaufman, S. (1978) J. Biol. Chem. 253, 6657-6659). However, we show for the first time in the present study that in vivo treatment with phenylalanine alone results in a 4-fold increase in the BH4-dependent activity of phenylalanine hydroxylase concomitant with a significant incorporation of phosphate into phenylalanine hydroxylase (0.51 mol of 32Pi/mol of hydroxylase subunit). It is further demonstrated in vivo that the combined treatment with phenylalanine and glucagon results in a greater than 10-fold stimulation of BH4-dependent activity and the greatest level of 32Pi incorporation (0.75 mol of 32Pi/mol of hydroxylase subunit). Phenylalanine did not produce an elevation in plasma glucagon in these animals. A model is, thereby, proposed with respect to the ligand binding effects of phenylalanine on the state of phosphorylation and activation of phenylalanine hydroxylase. The significance of these regulatory roles are considered in light of the probable physiological environment of the enzyme. PMID- 1730678 TI - Interaction of ferredoxin with carbon monoxide dehydrogenase from Clostridium thermoaceticum. AB - Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18). Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA. Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G. (1985) J. Biol. Chem. 260, 3970-3977). In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex. In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide. The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex. The native CODH and cross linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage. By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH ferredoxin cross-linked complex. The peptide was identified with residues 229-239 of the alpha-subunit of CODH. PMID- 1730679 TI - Purification and initial characterization of the lymphocyte-specific protein tyrosyl kinase p56lck from a baculovirus expression system. AB - A baculovirus expression system has been used to express large quantities of the lymphocyte-specific protein-tyrosyl kinase p56lck. A series of chromatographic steps, including the novel application of metalchelate affinity chromatography for protein kinase purification, were employed to obtain p56lck in a highly active form. Recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species, both with apparent molecular masses close to 56 kDa. p56lck phosphorylated all assayed substrates exclusively on tyrosyl residues, and underwent autophosphorylation at one principal site, also on a tyrosyl residue. p56lck displayed a high affinity for a synthetic peptide corresponding to the cytoplasmic domain (residues 52-164) of the T-cell receptor zeta-chain (TCR-zeta) (Km approximately 6.5 microM) but a low affinity for a peptide corresponding to its own autophosphorylation site (Km approximately 900 microM). p56lck was also found to be highly active for a purified protein-tyrosyl kinase (Vmax greater than 400 pmol.min-1.micrograms-1 using the TCR-zeta (52-164) as a substrate). A variety of agents were tested for their ability to inhibit p56lck, with zinc ions (I50 approximately 1.7 mM) and staurosporine (I50 approximately 500 nM) proving the most potent. PMID- 1730680 TI - Thyroid hormone responsiveness in human growth hormone-related genes. Possible correlation with receptor-induced DNA conformational changes. AB - Triiodothyronine (T3) induces the transcription of the human chorionic somatomammotropin (hCS) promoter transfected into rat pituitary (GC) cells, but does not stimulate the homologous human growth hormone (hGH) promoter. As demonstrated by forward and reverse mutagenesis, this differential T3 responsiveness is due to subtle structural differences in a T3 response element located between nucleotides -64 and -44 of the 5'-flanking DNA of the hGH and hCS promoters. Synthetic hCS(-70/-40) DNA binds thyroid hormone receptors with a 4 fold higher affinity than the corresponding hGH T3 response element, indicating that small differences in receptor binding properties are reflected by major differences in T3 responsiveness. Analysis of circular permutation fragments containing the native hGH and hCS or mutated hCS(-70/-40) sequences demonstrates that the thyroid hormone receptor induces DNA bending. The extent of bending shows a possible correlation with the function of these sequences, suggesting that the receptor-induced changes in DNA conformation may be required for thyroid hormone receptor action. PMID- 1730681 TI - Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis. AB - Three different oligosaccharides were identified following mild acid hydrolysis of the lipopolysaccharide obtained from Neisseria meningitidis serotype 2 and their structures elucidated by combined chemical and physical techniques. The use of 500 MHz 1H nmr in both one- and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The oligosaccharides were all triantennary nonasaccharides in which the longest antenna terminates in lacto-N-neotetraose. Two of the nonasaccharides (major components), not separable by column chromatography, were distinguishable only by their different patterns of phosphorylethanolamine substitution and the third minor component by the absence of this substituent. PMID- 1730682 TI - Transforming and c-fos promoter/enhancer-stimulating activities of a stimulatory GDP/GTP exchange protein for small GTP-binding proteins. AB - smg GDP dissociation stimulator (GDS) is a stimulatory GDP/GTP exchange protein for a group of ras p21-like small GTP-binding proteins (G proteins) including c Ki-ras p21, smg p21A, smg p21B, and rhoA p21. smg GDS converts the GDP-bound inactive form to the GTP-bound active form of each small G protein by stimulating their GDP/GTP exchange reaction in a cell-free system. The point-mutated c-Ki-ras p21 (c-Ki-rasval12 p21) is known to strongly transform NIH/3T3 cells and to markedly stimulate the c-fos promoter/enhancer in this cell line, whereas the normal c-Ki-ras p21 is weak in these activities. In the present study, we examined the effect of smg GDS on these activities to explore its physiological function. Overexpression of both smg GDS and c-Ki-ras p21 strongly transformed NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly transformed the cells. Furthermore, overexpression of both smg GDS and c Ki-ras p21 markedly stimulated the c-fos promoter/enhancer in NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly stimulated it. These results indicate that smg GDS transforms NIH/3T3 cells and stimulates the c-fos promoter/enhancer in this cell line in cooperation with c-Ki-ras p21. PMID- 1730683 TI - Ligand-independent internalization and recycling of the insulin receptor. Effects of chronic treatment of 3T3-C2 fibroblasts with insulin and dexamethasone. AB - Upon binding insulin at the plasma membrane, the insulin receptor internalizes into the endosomal compartment of the cell with a half-time of approximately 10 min. Our earlier work demonstrated that receptor inactivation (loss of insulin binding capacity) is a regulated process. Long term treatment of cultured cells with insulin or the glucocorticoid dexamethasone increases or decreases, respectively, the rate constant for insulin receptor inactivation (Knutson, V. P., Ronnett, G. V., and Lane, M. D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822 2826). In these studies, monolayer cultures of 3T3-C2 fibroblasts were chronically treated with insulin or dexamethasone. Subsequently, the surface receptors were labeled with the photoactivatable cross-linking agent 125I-labeled 2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionate -insulin. Following equilibration of the radiolabeled receptor between the plasma membrane and internal pools, the steady-state rate constant for receptor recycling was determined by quantitating the rate at which internal radiolabeled receptor was inserted into the plasma membrane. The steady-state rate constant for this recycling process was the same in control, insulin-treated, or steroid-treated cells (t1/2 = 2h). In contrast, the rate constant for receptor internalization was regulated; the half-times were 10 h for control cells, 5 h for insulin treated cells, and 19 h for dexamethasone-treated cells. These changes in rate constants for internalization and inactivation lead to changes in the relative numbers of receptor molecules undergoing recycling versus inactivation. Therefore, whereas the recycling of the insulin receptor is not a regulated process, the internalization of surface receptor in the absence of bound ligand is a metabolically controlled step in receptor processing. PMID- 1730684 TI - Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. AB - Nuclear transport, phosphorylation, ligand binding, and degradation rate of the recombinant androgen receptor (AR) were analyzed in transfected COS cells in the presence of various steroids and antiandrogens. Transcriptional activation was assessed in CV1 cells by cotransfection with an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter vector. Hormone binding specificity of recombinant AR was essentially identical to endogenous AR. AR localized in the nucleus in the presence of methyltrienolone (R1881, a synthetic androgen), dihydrotestosterone, testosterone, hydroxyflutamide, cyproterone acetate, estradiol, progesterone, and RU486. In the absence of hormone or with the antiandrogen, flutamide, AR remained largely in the cytoplasm with a perinuclear distribution. AR was degraded rapidly (t1/2 = 1 h) except in the presence of androgen (t1/2 = 6 h) which accounted for an apparent 2-4-fold androgen-induced increase in AR phosphorylation, indicating that AR phosphorylation was not enhanced by androgen. CAT activity was stimulated by R1881, dihydrotestosterone, testosterone, cyproterone acetate, estradiol, progesterone, and RU486 in a dose-dependent manner. The antiandrogens, flutamide and hydroxyflutamide, lacked agonist activity and inhibited R1881-induced activation of CAT and androgen stabilization of AR. Steroids and antiandrogens with moderate to low affinity for AR promoted both nuclear transport and transcriptional activation but only at high hormone concentrations. Hydroxyflutamide acted as a true antiandrogen since it lacked agonist activity and was an inhibitor of androgen-induced transcriptional activation. PMID- 1730685 TI - Sequence of a cDNA clone encoding pig heart mitochondrial CoA transferase. AB - We have isolated a full-length cDNA clone encoding the cytoplasmic precursor to pig heart mitochondrial CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase (3-oxoacid CoA transferase, EC 2.8.3.5], a key enzyme for ketone body catabolism. The deduced amino acid sequence indicates the presence of a 39 residue mitochondrial signal sequence and a 481-residue mature protein of molecular weight 52,197. CoA transferase is known to be susceptible to proteolytic cleavage to produce a nicked but active enzyme. We have identified the site of proteolysis, and analysis of the sequence in its vicinity suggests that the polypeptide may fold into two domains connected by a highly hydrophilic bridge. PMID- 1730686 TI - Structure of mouse interleukin 3 (IL-3) binding protein (AIC2A). Amino acid residues critical for IL-3 binding. AB - Despite the high degree of sequence homology between two mouse proteins AIC2A and AIC2B (91% at the amino acid level), only the AIC2A protein binds interleukin 3 (IL-3). Soluble AIC2A protein bound IL-3 with affinity similar to the membrane bound AIC2A protein, indicating that binding of IL-3 to AIC2A was mediated by the external domain alone. The extracellular domain of the AIC2A protein has two repeats of the common motif shared by members of the cytokine receptor family. Neither one of these repeats alone bound IL-3. Hybrids of AIC2A and AIC2B revealed that the first domain of the cytokine receptor motif could be replaced with the AIC2B sequence without an affinity change, suggesting the importance of the second domain. By changing individual amino acid residues of AIC2A in the second domain which differ from those of AIC2B, we identified several amino acid residues critical for IL-3 binding. All these residues are located at the putative hinge region within the second domain. PMID- 1730687 TI - Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation. AB - To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions -151 and -257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-actin gene was isolated, and the 5'-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position -214 in the chicken gene and at position -213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-acting elements than other cell types to transcriptionally regulate this gene. PMID- 1730688 TI - A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus. AB - The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus. PMID- 1730689 TI - Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli. AB - Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells. PMID- 1730690 TI - Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity. AB - Ras and Rap1 proteins are related GTP-dependent signal transducers which require Gly-12, the effector domain (residues 32-40), and Ala-59 for stimulation of their GTPase activities by GAP1 and GAP3, respectively. The replacement of Gly-12 by Val or Ala-59 by Thr potentiates the Ras oncogenicity and Rap1A antioncogenicity. However, the mutations in the effector domain, in particular the replacement of Thr-35 by Ala, abolish both Ras oncogenicity and Rap1A antioncogenicity, indicating that the effector domain is involved in interactions of these signal transducers with their targets as well as the GAPs. In this paper, we demonstrate that (i) replacement of Tyr-64 of the Ha-Ras protein or Phe-64 of the Rap1A protein by Glu or other non-hydrophobic amino acids reduces their intrinsic GTPase activities and abolishes their stimulation by GAP1 or GAP3, respectively, (ii) replacement of Tyr-64 by Gly and other non-hydrophobic amino acids results in complete loss of the oncogenicity of the v-Ha-Ras protein, indicating that the hydrophobic residue 64, in addition to the known effector domain, is essential for the Ras protein to interact with its target as well as GAP1. In addition we have found that Asn-26, Glu-31, and Val-45 of the v-Ha-Ras protein are required for its oncogenicity. Replacement of the Ras residues at either positions 26, 31, or 45 by the corresponding Rap1A residues abolishes the Ras oncogenicity. PMID- 1730691 TI - X-ray structural evidence for a local helix-loop transition in alpha-lactalbumin. AB - The three-dimensional structure of human alpha-lactalbumin for two crystal forms has been determined by x-ray analysis. One crystal (the form LT) was obtained at pH 4.2 and room temperature, while the other crystal (the form HT) was grown at pH 6.5 and 37 degrees C. The backbone structure for Lys1-Ile95 residues is almost conserved between the two structures as indicated by the root mean square difference of 0.30 A for the superposition of equivalent C alpha atoms. The calcium ion is surrounded by seven oxygen atoms of three carboxyl groups, two carbonyl groups, and two water molecules, which form a distorted pentagonal bipyramid in both structures. A large difference in polypeptide folding is found in the region of Leu96-Leu123 residues. Especially in the region of Trp104-Cys111 residues, a distorted alpha-helix is observed in the form HT while a loop structure is formed in the other crystal. The fact that the crystals of both forms appeared in the same batch at pH 6.5 and room temperature indicates that the human alpha-lactalbumin structure is highly fluctuated in solution and the folding and unfolding of the alpha-helix of Trp104-Cys111 residues are in equilibrium. Since the crystal of the form HT exclusively appeared around the physiological temperature, the structure of this form can be considered as the native structure. The partially unfolded structure in the form LT indicates that the local denaturation occurs even at room temperature. PMID- 1730692 TI - Isoprenylation of a protein kinase. Requirement of farnesylation/alpha-carboxyl methylation for full enzymatic activity of rhodopsin kinase. AB - The primary structure of bovine rhodopsin kinase (RK), which phosphorylates light activated rhodopsin (Rho*), terminates with the amino acid sequence Cys558-Val Leu-Ser561, a motif that has been shown to direct the isoprenylation and alpha carboxyl methylation of many proteins (e.g. p21Ha-ras). Transient expression of RK in COS-7 cells revealed the presence of two immunoreactive protein species. Consistent with RK being modified by isoprenylation, interconversion of these two species was dependent upon isoprenoid biosynthesis in the cells. Moreover, a serine substitution for Cys558 resulted in a single RK species whose migration on sodium dodecyl sulfate-polyacrylamide gels was identical to that of RK from cells treated with mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and, thus, of isoprenoid biosynthesis. This finding indicates that isoprenylation of RK requires Cys558. The electrophoretic mobility of isoprenylated RK synthesized in COS-7 cells was identical to that of RK from bovine rod outer segments, suggesting that RK is isoprenylated in vivo. RK was determined to be modified by a farnesyl moiety and alpha-carboxyl-methylated. A time course of Rho* phosphorylation revealed that non-processed RK is approximately 4-fold less active than wild-type RK. This is the first demonstration of isoprenylation/alpha-carboxyl methylation of a protein kinase, and suggests that these modifications markedly influence enzymatic activity in vivo. PMID- 1730693 TI - Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link. AB - In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed. PMID- 1730694 TI - Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils. AB - We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function. PMID- 1730695 TI - Adrenal P-450scc modulates activity of P-45011 beta in liposomal and mitochondrial membranes. Implication of P-450scc in zone specificity of aldosterone biosynthesis in bovine adrenal. AB - Bovine adrenal P-45011 beta catalyzes the 11 beta- and 18-hydroxylation of corticosteroids as well as aldosterone synthesis. These activities of P-45011 beta were found to be modulated by another mitochondrial cytochrome P-450 species, P-450scc. The presence together of P-45011 beta and P-450scc in liposomal membranes was found to remarkably stimulate the 11 beta-hydroxylase activity of P-45011 beta and also stimulate the cholesterol desmolase activity of P-450scc. The stimulative effect of P-450scc on 11 beta-hydroxylase activity diminished by the addition of protein-free liposomes to proteoliposomes containing P-45011 beta and P-450scc, thus showing P-450scc to interact with P 45011 beta in the same membranes. Kinetic analysis of this effect indicated the formation of an equimolar complex between P-45011 beta and P-450scc on liposomal membranes. P-45011 beta in the complex had not only stimulated activity for 11 beta- and 18-hydroxylation of 11-deoxycorticosterone but also suppressed activity for production of 18-hydroxycorticosterone and aldosterone. When the inner mitochondrial membranes of zona fasciculata-reticularis from bovine adrenal were treated with anti-P-450scc IgG, aldosterone formation was stimulated to a greater extent than that of zona glomerulosa. This indicates the aldosterone synthesizing activity of P-45011 beta in the zona fasciculata-reticularis to be suppressed by interaction with P-450scc. The zone-specific aldosterone synthesis of P-45011 beta in bovine adrenal may possibly be induced by differences in interactions with P-450scc of mitochondrial membranes in each zone. PMID- 1730696 TI - Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli. AB - The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity. PMID- 1730697 TI - Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase. AB - The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants. These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R. (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids. Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids. Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids. This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity. PMID- 1730698 TI - Purification and characterization of alpha-L-fucosidase from Streptomyces species. AB - Streptomyces sp. 142, isolated from a soil sample, produced alpha-fucosidase when cultured in the presence of L-fucose. The enzyme was purified 700-fold with an overall recovery of 17% from a cell-free extract by cation exchange chromatography and gel filtration chromatography. The apparent molecular weight of the purified enzyme was 40,000 by gel filtration chromatography. The enzyme had a pH optimum of 6.0 and was stable at pH 4.5-7.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine as the substrate showed that the enzyme specifically hydrolyzed terminal alpha 1-3 and alpha 1-4 fucosidic linkages in the oligosaccharides but did not hydrolyze alpha 1-2 or alpha 1-6 fucosidic linkages or a synthetic substrate, p-nitro-phenyl alpha-L fucoside. The purified enzyme released L-fucose from a fucosylated glycoprotein, alpha 1-acid glycoprotein. Thus, the substrate specificities of the Streptomyces alpha-fucosidase resembled those of alpha-fucosidases I and III isolated from almond emulsin rather than those of other microbial alpha-fucosidases. PMID- 1730699 TI - A novel sulfated structure in the carbohydrate-protein linkage region isolated from porcine intestinal heparin. AB - A preparation of porcine stage 14 intestinal heparin, which contains Ser as a predominant amino acid, was used for isolation of the carbohydrate-protein linkage region of heparin. Two glycoserines were isolated in a molar ratio of 96:4 after an exhaustive digestion with a mixture of bacterial heparinase and heparitinases. Their structures were determined by composition analysis, heparitinase digestion, co-chromatography with an authentic glycoserine on high performance liquid chromatography, and by 500-MHz one- and two-dimensional 1H NMR spectroscopy. The structure of the major one is delta GlcA beta 1-3Gal beta 1 3Gal beta 1-4Xyl beta 1-O-Ser and that of the minor is delta GlcA beta 1 4GlcNAc(6-O-sulfate) alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O Ser. The novel 6-O-sulfated GlcNAc residue was demonstrated to occur in the vicinity of the carbohydrate-protein linkage region. The Gal residues were nonsulfated, in contrast to the sulfated Gal structures recently discovered in the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans. The structural features are discussed in relation to biosynthetic mechanisms of the heparin glycosaminoglycans. PMID- 1730700 TI - 14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase. AB - Incubation of cells of the nitrifying bacterium Nitrosomonas europaea with 14C2H2 results in the covalent attachment of 14C label to a membrane-bound polypeptide of an approximate Mr of 28,000 (Hyman, M.R., and Wood, P.M. (1985) Biochem. J. 227, 719-725). A labeling procedure using 14C2H2 generated from Ba14CO3 has been used to investigate the correlation between the extent of covalent modification of this polypeptide by 14C from 14C2H2 and the level of ammonia oxidizing activity in whole cells. The time-dependent inactivation of ammonia monooxygenase by 14C2H2 resulted in a progressive and saturable incorporation of 14C into a 27 kDa polypeptide. In contrast, the specific, time-dependent and complete inactivation of ammonia monooxygenase by light resulted in concomitant decrease in the ability of cells to incorporate 14C from 14C2H2 into this polypeptide. The 14C2H2 labeling procedure was also used to investigate the recovery of ammonia monooxygenase activity after complete inactivation of pre-existing ammonia monooxygenase by either C2H2 or light. The recovery of ammonia monooxygenase activity was closely correlated with a recovery of ability of cells to incorporate 14C label from 14C2H2 into the 27-kDa polypeptide. This recovery process was energy (NH4+)-dependent and was inhibited by chloramphenicol and rifampicin, implying that de novo protein synthesis was required. Additional polypeptides labeled with 14C from 14CO2 were also identified during recovery from C2H2 or light inactivation of ammonia monooxygenase. PMID- 1730702 TI - Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain. AB - The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease. PMID- 1730701 TI - Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. AB - The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome-mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5' to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that involve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor mediated stimulation of complex formation by a combination of eIF-4A, -4B, and 4F was observed, and was one which required the presence of ATP. This factor mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5'-p-fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested. PMID- 1730703 TI - Mechanism of glutamate semialdehyde aminotransferase. Roles of diamino- and dioxo intermediates in the synthesis of aminolevulinate. AB - Glutamate semialdehyde aminotransferase was purified to homogeneity from pea leaves. The enzyme has an absorption spectrum with maxima at 345 and 416 nm. These chromophores were attributed to pyridoxamine phosphate and to pyridoxal phosphate bound as an aldimine respectively. Treatment of the enzyme with increasing concentrations of diaminovalerate produced a rapid fall in the 416 nm chromophore and a simultaneous increase in the 345 nm chromophore in a manner that indicated a stoichiometric reaction between 4,5-diaminovalerate and the pyridoxaldimine form of the enzyme. Treatment with 4,5-dioxovalerate produced the reverse reaction. The enzyme catalyzed the formation of aminolevulinate when dioxovalerate and diaminovalerate were present together and the maximal rate was 40% of that observed when glutamate semialdehyde itself was used as substrate. Conversion of the enzyme from its pyridoxaldimine to pyridoxamine form produced a proportional increase in activity towards glutamate semialdehyde, whereas reduction of the pyridoxaldimine form with sodium borohydride produced no change in this catalytic activity. It was concluded, therefore, that only the pyridoxamine form of the enzyme is active in catalyzing conversion of glutamate semialdehyde to aminolevulinate and that the catalytic mechanism includes enzyme bound diaminovalerate as a central intermediate. PMID- 1730704 TI - In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells. AB - The plasma clearance and the interaction of high (HMW) and low (LMW) molecular weight single-chain urokinase-type plasminogen activator (scu-PA) with rat liver cells was determined. 125I-Labeled HMW- and LMW-scu-PA were rapidly cleared from plasma with a half-life of 0.45 min and a maximal liver uptake of 55% of the injected dose. Liver uptake of scu-PA was mediated by parenchymal cells. Excess of unlabeled HMW-scu-PA reduced the liver uptake of 125I-HMW-scu-PA strongly. In vivo liver degradation of scu-PA was reduced by inhibitors of the lysosomal pathway. A high affinity binding site (Kd 45 nM, Bmax 1.7 pmol/mg cell protein) for both HMW- and LMW-scu-PA was determined on isolated parenchymal liver cells. Cross-competition binding studies showed that LMW- and HMW-scu-PA bind to the same site. Tissue-type plasminogen activator, mannose- or galactose-terminated glycoproteins did not affect the scu-PA binding to parenchymal liver cells. It is concluded that LMW- and HMW-scu-PA are taken up in the liver by a common, newly identified recognition site on parenchymal liver cells and are subsequently degraded in the lysosomes. It is suggested that this site is important for the regulation of the turnover of scu-PA. PMID- 1730705 TI - Exposure of rat peritoneal macrophages to acetylated low density lipoprotein results in release of plasma membrane cholesterol. An efficient substrate for esterification by acyl-CoA:cholesterol acyltransferase. AB - In J774 macrophages and murine macrophages stimulated with acetylated low density lipoprotein (acetyl-LDL), the plasma membrane free cholesterol (FC) became accessible to acyl-CoA:cholesterol acyltransferase (ACAT) as substrate, the result being an accumulation of cholesteryl esters (CE) (Tabas, I., Rosoff, W. J., and Boykow, G. C (1988) J. Biol. Chem. 263, 1266-1272). As the route of delivery of FC to ACAT was not well characterized, we examined this route in the present study. In foam cells derived from rat peritoneal macrophages by preincubation with acetyl-LDL, esterification of the exogenously labeled [3H]FC was low (1.3% of total labeled cholesterol). In contrast, when cells were first labeled with exogenous [3H]FC and then chased with acetyl-LDL, the esterification was more extensive (9.2% of the total labeled cholesterol). During this experiment a significant portion of cellular [3H]FC was released into the medium (up to 33.4% of the total labeled cholesterol). In experiments using a two compartment chamber in which cells in the lower and upper chambers were separated by filter paper yet the cells in both compartments could communicate without direct contact, [3H]FC released into the medium was biologically active and could serve as an efficient substrate for ACAT. Thus, when acetyl-LDL is not included in culture medium, FC delivery from the macrophage plasma membrane to ACAT is not enhanced, whereas in the presence of acetyl-LDL, plasma membrane FC released and bound to acetyl-LDL may re-enter the cells, possibly through the scavenger receptor. This would provide a significant route for CE synthesis in macrophages. PMID- 1730706 TI - Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds. AB - The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp 96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin. PMID- 1730707 TI - A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha. AB - Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available. PMID- 1730708 TI - Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom. AB - A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel. PMID- 1730709 TI - Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae. AB - Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A. PMID- 1730710 TI - Purification of the testicular galactolipid: 3'-phosphoadenosine 5' phosphosulfate sulfotransferase. AB - The rat testicular galactolipid sulfotransferase has been purified by affinity chromatography using 3'5'-adenosine diphosphate-agarose affinity chromatography. Both galactosyl glycerolipid and galactosyl ceramide were effective substrates with Km values of 4.8 and 1.1 microM respectively. A single protein of molecular mass 56 kDa was present in the purified sulfotransferase preparation as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Specific photoaffinity substrate labeling, using an azido derivative of galactosyl ceramide, was used to identify this protein, both in crude extracts and when purified. The protein was also selectively phosphorylated in the presence of the rat testicular galactolipid sulfotransferase stimulatory protein kinase. PMID- 1730711 TI - Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. AB - We have characterized the transport of GSH and the mechanism for impaired GSH transport in mutant Eisai hyperbilirubinemic rats (EHBR) using isolated canalicular membrane-enriched vesicles (cLPM). In control animals, the transport of GSH is an electrogenic process and is trans-stimulated by preloading the vesicles with GSH and is not enhanced in the presence of ATP. GSH transport in cLPM is saturable with a single component having a Km of approximately 16 mM and a Vmax of 6.7 nmol/mg/15 s. EHBR is a Sprague-Dawley rat with hyperbilirubinemia due to impaired bile secretion of organic anions by the ATP-dependent organic anion/GSH-conjugate transporter. In cLPM from EHBR we confirmed the defective stimulation by ATP of the transport of LTC4 and GSSG. In the mutant cLPM, the characteristics and kinetics of GSH transport were the same as in the controls. 2,4-(dinitrophenyl)-glutathione (DNP-GSH), which is a substrate for the ATP dependent canalicular organic anion carrier, in the absence of ATP, cis-inhibited the transport of GSH into cLPM vesicles; however, when the vesicles were preloaded with DNP-GSH, there was a dose-dependent trans-stimulation of GSH transport. In contrast, in the presence of ATP, DNP-GSH enhanced GSH transport in cLPM vesicles; at 0.25 mM DNP-GSH, a concentration which did not cis-inhibit GSH, addition of ATP resulted in accelerated GSH transport; at 1.0 mM DNP-GSH, cis inhibition was completely reversed by the addition of ATP despite a negligible fall in the medium DNP-GSH. Interestingly, sulfobromophthalein-glutathione (BSP GSH) neither cis-inhibited nor trans-stimulated GSH transport in cLPM. This contrasts with bLPM where BSP-GSH interacts with the GSH carrier. Therefore, GSH is transported into bile by a multispecific low affinity electrogenic carrier which is distinct from the multispecific high affinity ATP-driven organic anion transporter. Although both carriers have overlapping specificities, BSP-GSH and GSH are uniquely specific for only one of the carriers. The near absence of GSH in the bile of mutant rats can be best explained as a secondary defect due to cis inhibition from retained substrates for the defective carrier and/or loss of trans-stimulation by these same substrates which normally are concentratively transported into the bile. Other possibilities such as change in GSH carrier activity upon isolation or loss of a negative protein regulator during membrane isolation, although theoretical alternatives are less easily reconciled with the defect in the ATP-driven organic anion transporter. PMID- 1730712 TI - Post-translational modifications of Drosophila acetylcholinesterase. In vitro mutagenesis and expression in Xenopus oocytes. AB - Drosophila acetylcholinesterase (EC 3.1.1.7) is a 150-kDa glycoprotein anchored in plasmic membranes via a glycolipid. It is composed of two active subunits which are themselves made of two noncovalently linked polypeptides of 18 and 55 kDa resulting from the proteolysis of a single precursor of 75 kDa. Active Drosophila acetylcholinesterase can be expressed in Xenopus oocytes as an excreted protein. We have identified some of the amino acids essential in post translational modifications of the protein by site-directed mutagenesis and expression of mutants in this system. The intersubunit disulfide bond involves cysteine at position 615. Cleavage of the 75-kDa precursor, as observed in Drosophila, originates from a hydrophilic peptide (in position 148 to 180) which does not exist in cholinesterase sequences from vertebrates. This cleavage is associated with excretion out of the cell. Drosophila acetylcholinesterase exhibits four effective sites of asparagine-linked glycosylation in positions 126, 174, 331, and 531. We show that glycosylations and dimerization protect the protein against proteolytic digestion. In contrast, none of these post translational modifications significantly affects the activity of acetylcholinesterase or affinity for its substrate. PMID- 1730713 TI - Cholesterol is converted to 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in liver mitochondria. Evidence for a mitochondrial sterol 7 alpha-hydroxylase. AB - The metabolism of cholesterol in isolated intact pig liver mitochondria has been investigated. Six major cholesterol metabolites were identified by gas-liquid chromatography-mass spectrometry, the metabolic end product being 7 alpha-hydroxy 3-oxo-4-cholestenoic acid. Incubations with the synthesized intermediates suggested that the major pathway from cholesterol to this acid proceeds via the sequence of 26-hydroxylation, 7 alpha-hydroxylation, further oxidation of the side chain and oxidation/isomerization in the A-ring. The observed reactions prove that in addition to a sterol 26-hydroxylase, pig liver mitochondria contain significant amounts of a 7 alpha-hydroxylase active on side chain oxygenated 3 beta-hydroxy-delta 5-C27 steroids, an oxidoreductase active in the side chain of 26-hydroxylated steroids and a 3 beta-hydroxy-delta 5 steroid oxidoreductase active on 7 alpha-hydroxylated C27 steroids. Since 7 alpha-hydroxy-3-oxo-4 cholestenoic acid is believed to be an important precursor of chenodeoxycholic acid, this study shows that the first reactions in the biosynthesis of bile acids can be exclusively mitochondrial and thereby bypass microsomal cholesterol 7 alpha-hydroxylase as the rate-limiting enzyme. PMID- 1730714 TI - Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor. AB - The extracellular portion of the macrophage mannose receptor is composed of several cysteine-rich domains, including a fibronectin type II repeat and eight segments related in sequence to Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of animal lectins. Expression of portions of the receptor in vitro, in fibroblasts and in bacteria, has been used to determine which of the extracellular domains are involved in binding and endocytosis of ligand. The NH2 terminal cysteine-rich domain and the fibronectin type II repeat are not necessary for endocytosis of mannose-terminated glycoproteins. CRDs 1-3 have at most very weak affinity for carbohydrate, so the carbohydrate binding activity of the receptor resides in CRDs 4-8. CRD 4 shows the highest affinity binding and has multispecificity for a variety of monosaccharides. However, CRD 4 alone cannot account for the binding of the receptor to glycoproteins. At least 3 CRDs (4, 5, and 7) are required for high affinity binding and endocytosis of multivalent glycoconjugates. In this respect, the mannose receptor is like other carbohydrate-binding proteins, in which several CRDs, each with weak affinity for single sugars, are clustered to achieve high affinity binding to oligosaccharides. In the mannose receptor, these multiple weak interactions are achieved through several active CRDs in a single polypeptide chain rather than by oligomerization of polypeptides each containing a single CRD. PMID- 1730715 TI - Alteration by site-directed mutagenesis of the conserved lysine residue in the ATP-binding consensus sequence of the RecD subunit of the Escherichia coli RecBCD enzyme. AB - The RecD subunit of the RecBCD enzyme from Escherichia coli contains an amino acid sequence common to many enzymes which bind ATP or GTP (Gly-X-X-Gly-X-Gly-Lys Thr). We have changed the conserved lysine residue (amino acid number 177) in the RecD protein to glutamine to investigate the role of RecD, and ATP-binding to RecD, in the enzymatic activities of RecBCD. The mutant RecD protein assembles with the RecB and RecC subunits and the mutant enzyme, designated RecBCD-K177Q, can be purified in the same way as the wild-type RecBCD enzyme. The mutant RecD subunit in RecBCD-K177Q is photolabeled to a lesser extent by the ATP analogue 8 azido-adenosine-5'-triphosphate than is the wild-type RecD subunit in RecBCD, suggesting that the mutation has reduced the affinity of RecD for ATP. PMID- 1730716 TI - Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very long-chain fatty acids in mammalian testes and spermatozoa. AB - Polyenoic very-long-chain fatty acids (VLCFA) have been shown to be localized in unusual molecular species of sphingomyelin in the testes and spermatozoa of the ram, bull, rat, and boar and in the spermatozoa of man. The composition of polyenoic VLCFA-sphingomyelin was comparable in the testes and spermatozoa of each mammalian species; however, the sphingolipid was more concentrated in spermatozoa. The composition of testicular and spermatozoan polyenoic VLCFA sphingomyelin differed considerably between animal types. Human spermatozoa mainly contained n-6 polyenoic VLCFA with two to four double bonds and even carbon chain lengths up to 32. In ram and bull testes and spermatozoa, n-3 and n 6, tetra-, penta-, and hexaenoic VLCFA with even-carbon chain lengths up to 34 predominated. In rat and boar testes and spermatozoa, the polyenoic VLCFA were mainly n-6 derivatives with three to five double bonds and even- and odd-carbon chain lengths up to 34. The testes and spermatozoa of the latter two animal species contained 2-hydroxylated, in addition to non-hydroxylated, polyenoic VLCFA in sphingomyelin. This is the first time that 2-hydroxylated polyenoic VLCFA have been recognized in biological systems. Non-hydroxylated polyenoic VLCFA were initially observed in the sphingomyelin of rat testes 25 days after birth, followed by 2-hydroxylated derivatives at 30 days. The total amount of polyenoic VLCFA associated with rat testicular sphingomyelin increased dramatically from 25 to 40 days of postnatal life and then remained constant to 60 days (sexual maturity). The ratio of 2-hydroxylated to non-hydroxylated polyenoic VLCFA increased during this period. Polyenoic VLCFA-sphingomyelin seems to occur exclusively in the testes and spermatozoa of mammals, and it is postulated that this lipid plays a role in reproduction. PMID- 1730717 TI - Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA. AB - The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling. We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification. We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength. The plasmids fall into a number of different classes, in terms of the conformational behavior. As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence. The two transitions are coupled through the topology of the molecule but are otherwise independent. When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment. Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions. Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences. These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules. PMID- 1730718 TI - Functional and structural analysis of VLA-4 integrin alpha 4 subunit cleavage. AB - The cell surface heterodimer VLA-4 (alpha 4 beta 1), a member of the integrin family of adhesion receptors, is involved in both cell-extracellular matrix and cell-cell adhesion. Unlike any other integrin alpha subunit, the intact (150 kDa) alpha 4 subunit of VLA-4 can sometimes be cleaved into two noncovalently associated fragments (80 and 70 kDa). Using biosynthetic and mixing experiments, we found that human alpha 4 cleavage is a regulated, compartmentalized event, occurring soon after maturation of the beta 1-associated alpha 4 subunit. Cleavage of alpha 4, which is increased following T cell activation, has been suggested to correlate with altered VLA-4 functions. To address directly the functional importance of alpha 4 cleavage, we have studied VLA-4-mediated adhesion functions in cells expressing intact alpha 4 in comparison with cells expressing cleaved alpha 4. For this purpose, we first sequenced the N terminus of the endogenously produced 70-kDa alpha 4 fragment and identified the alpha 4 cleavage site between Lys557-Arg558 and Ser559. To abolish cleavage, we converted Arg558 to Leu or Lys557 to Gln by site-directed mutagenesis of the alpha 4 cDNA and then transfected both mutant and wild type alpha 4 cDNAs into VLA-4-negative K562 cells. Whereas transfection with wild type alpha 4 cDNA yielded predominantly cleaved alpha 4 subunit, the Leu558-alpha 4 yielded only intact alpha 4 subunit, and Gln557-alpha 4 yielded mostly intact alpha 4 subunit. Transfectants with the intact or the cleaved alpha 4 were equally capable of engaging in VLA-4-dependent adhesion to vascular cell adhesion molecule-1 and to the Hep II fragment of fibronectin (40 kDa) and aggregated equally well in response to anti-alpha 4 antibodies. Thus, cleavage of the alpha 4 subunit in these transfectants did not alter any of the known VLA-4-mediated adhesion functions. PMID- 1730719 TI - Membrane topology of the melibiose carrier of Escherichia coli. AB - The minimum structural information necessary to formulate and assess mechanistic models of integral membrane protein function is that of membrane topology. This paper characterizes the topological structure of the melibiose carrier of Escherichia coli based on constraints provided by genetic fusions to the compartment-specific reporter protein alkaline phosphatase. Twenty-eight unique chimeras exhibiting either low alkaline phosphatase activity (cytoplasmic location of the fusion joint) or high alkaline phosphatase activity (periplasmic location of the fusion joint) were characterized and used in conjunction with Goldman-Engelman-Steitz hydropathy analysis to model topological structure. The melibiose carrier is predicted to have a cytoplasmic amino terminus, two sets of six transmembrane domains separated by an unusually large cytoplasmic loop ("six loop-six" arrangement), and a 45-residue cytoplasmic carboxyl tail. Remarkably, the identical six-loop-six arrangement is predicted from the hydrophobicity plots of the H(+)-coupled lactose, arabinose, xylose, and citrate cotransporters of E. coli, the glucose transporter from rat brain, the family of glucose transporters isolated from various human tissues and cell lines, and the human, mouse, and hamster multidrug resistance transporters (Henderson, P.J.F. (1990) Res. Microbiol. 141, 316-328; Maloney, P.C. (1990) Res. Microbiol. 141, 374-383). Such a broad degree of conservation (or convergence) suggests a distinct structural and/or mechanistic advantage associated with the six-loop-six motif. The nature of this advantage is as yet unknown. PMID- 1730720 TI - DNA allosterically modulates the steroid binding domain of the estrogen receptor. AB - The ability of DNA to allosterically alter the conformation of the estrogen receptor's (ER) steroid binding domain was investigated. Using dissociation kinetics we observed that when DNA was bound to the DNA binding domain of the rat uterine ER the rate of estrogen dissociation from the steroid binding domain increased almost 2-fold. This change in the rate of estrogen dissociation depended on the concentration of DNA used and correlated with the thermodynamic binding affinities (Kd) of the ER for two different DNA sequences. We were unable to detect a DNA-induced change in the trypsin cleavage pattern of the amino terminal end of the ER. Using a whole cell dissociation kinetic assay with MCF-7 breast cancer cells we observed a 7-fold slower rate of estrogen dissociation from the ER within the cell than from the ER in vitro. This suggests that additional factors, other than DNA binding, may modify the steroid binding domain within the cell. We conclude that DNA can allosterically modulate the structure of the steroid binding domain of the ER, and we hypothesize that this conformational change may be necessary for the full transcriptional activity of the ER. PMID- 1730721 TI - In vitro activity of the transcription activation functions of the progesterone receptor. Evidence for intermediary factors. AB - The human progesterone receptor (hPR) is a ligand-dependent transcription factor which contains two distinct transcription activation functions (TAFs). The full length hPR and its individual TAFs were overexpressed in the baculovirus system and tested in a HeLa cell-derived in vitro transcription system. hPR stimulated transcription in a ligand-independent manner. When the two TAFs fused to the DNA binding domain of GAL4 were tested, only the constitutive TAF-1 was functional in vitro, strongly suggesting that the transcriptional activity of baculovirus expressed hPR comes solely from TAF-1. The GAL-TAF-1 activator was found to self squelch without affecting basal transcription. A partially purified fraction relieved this self-squelching and, moreover, stimulated transcriptional activation by GAL-TAF-1, while having no influence on basal transcription. These results strongly suggest that the transcriptional activity of GAL-TAF-1 requires a factor(s) distinct from the general transcription factors. PMID- 1730722 TI - Insect glutathione S-transferases. Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides. AB - Two classes of glutathione transferases have been identified and purified from Musca domestica. The first, designated as GST1, migrates as a single band of 28 kDa in SDS-gel electrophoresis, and the second, designated as GST2, migrates as a 32-kDa band. Antisera prepared against each class have no immunological cross reactivity, and heterodimeric associations between the two classes have not been detected. Each class is composed of several isoforms: GST1 is composed of forms with isoelectric points from 4 to 9, whereas all the forms of GST2 have acidic pI values. Screening of cDNA libraries yielded clones coding for GST1, and the gene was sequenced and expressed in Escherichia coli. The high activity found in an insecticide-resistant strain (Cornell R) is correlated with high level of GST1 transcript. PMID- 1730723 TI - A T cell nuclear factor resembling NF-AT binds to an NF-kappa B site and to the conserved lymphokine promoter sequence "cytokine-1". AB - Nuclear extracts from a nontransformed murine T lymphocyte clone contained two inducible factors that bound to a nuclear factor kappa B (NF-kappa B) site. One factor was NF-kappa B, and the other was differentiated from NF-kappa B by its mobility in the electrophoretic mobility shift assay and its lack of sensitivity to protein kinase C depletion. Competition and methylation interference assays showed that the binding site for the novel factor was limited to nucleotides in the 3' half of the kappa B site. This part of the kappa B site resembled sequences in the binding site for a second inducible nuclear factor of T cells, NF-AT, as well as a conserved sequence found in several lymphokine genes, termed "cytokine-1" (CK-1). Competition and methylation interference analysis showed that both NF-AT and CK-1 sequences bound a factor similar to the novel kappa B binding factor and that binding involved a four-nucleotide sequence (TTCC) that the kappa B, CK-1, and NF-AT sites have in common. The complexes that form with each site have characteristics of NF-AT: they are induced upon T cell receptor stimulation, are sensitive to protein synthesis inhibitors and cyclosporin A, and are not sensitive to protein kinase C depletion. Thus, a factor or factors similar to NF-AT can bind to three distinct promoter sequences which occur commonly in several T cell activation genes. These results raise the possibility that related factors binding to kappa B, CK-1, and NF-AT sequences could play a role in the coordinate induction of T cell activation genes. In addition, our results suggest that kappa B and CK-1 sites represent potential cyclosporin sensitive promoter elements by virtue of their ability to bind an NF-AT-like factor. PMID- 1730725 TI - Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum. AB - An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O (thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway. PMID- 1730724 TI - Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex. Mapping of the E1 beta-binding region on E2. AB - A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha keto acid dehydrogenase complex was isolated from a lambda ZAP expression library. The bovine E1 beta cDNA is 1,393 base pairs in length. It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues. The calculated molecular mass of the mature bovine E1 beta subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04. The mature bovine E1 beta subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl beta-D thiogalactopyranoside (IPTG). When expression was induced by IPTG at 37 degrees C, the soluble recombinant E1 beta subunit existed as a single high molecular weight form (Mr congruent to 3.5 x 10(5)), which sedimented during sucrose gradient ultracentrifugation at 2 x 10(5) x g. However, lowering the induction temperature to 25 degrees C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1 beta protein. The low molecular weight form (Mr congruent to 9.1 x 10(4)) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins. The results showed that the E1 beta subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain. In contrast, the recombinant E1 alpha subunit did not bind the E2 component. The data suggest an apparent binding order of E2-E1 beta-E1 alpha, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M.L., Xu, W.-X., Oliver, R.M., Wall, J.S., Hainfeld, J.F., Mullinax, T.R., and Reed, L.J. (1989) Biochemistry 28, 6816-6821). The relatively inaccessible topology of E1 beta may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex. PMID- 1730726 TI - Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. AB - Three ribozymes targeted against different sites of the long terminal repeat RNA (LTR RNA) of human immunodeficiency virus type 1 cleaved a LTR RNA transcript 1,000 x less efficiently than corresponding short synthetic oligoribonucleotide substrates. Varying the stem lengths of the ribozyme resulted in changes of the catalytic efficiency. Almost no cleavage was observed for a ribozyme forming only 10 base pairs instead of 14 with the LTR RNA. Increasing the base pairs to 16 or elongation of the stem formed within the ribozyme revealed only small changes in kcat/Km. The influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA was also examined. 2'-Fluorocytidine substitutions as well as four terminal phosphorothioate internucleotidic linkages influenced the catalytic efficiency of ribozymes only negligibly. However, substitution of uridine by 2'-fluorouridine resulted in a 5-fold decrease of kcat/Km. A ribozyme containing all these modifications revealed only a 7-fold lower catalytic efficiency but a markedly increased stability in cell culture supernatant. These results demonstrate that it is possible to increase the stability of ribozymes toward nucleases without a serious loss in catalytic efficiency. PMID- 1730727 TI - Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis. AB - We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function. PMID- 1730728 TI - The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia. AB - Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia. PMID- 1730729 TI - Conversion of antithrombin from an inhibitor of thrombin to a substrate with reduced heparin affinity and enhanced conformational stability by binding of a tetradecapeptide corresponding to the P1 to P14 region of the putative reactive bond loop of the inhibitor. AB - A synthetic tetradecapeptide having the sequence of the region of the antithrombin chain amino-terminal to the reactive bond, i.e. comprising residues P1 to P14, was shown to form a tight equimolar complex with antithrombin. A similar complex has previously been demonstrated between alpha 1-proteinase inhibitor and the analogous peptide of this inhibitor (Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R. and Laurell, C.-B. (1990) Eur. J. Biochem. 194, 51-56). The antithrombin-peptide complex had a conformation similar to that of reactive bond-cleaved antithrombin and, like the cleaved inhibitor, also had a higher conformational stability and lower heparin affinity than intact antithrombin. These properties suggest that the peptide bound to intact antithrombin at the same site that the P1 to P14 segment of the inhibitor occupies in reactive-bond-cleaved antithrombin, i.e. was incorporated as a sixth strand in the middle of the major beta-sheet, the A sheet. The extent of complex formation was reduced in the presence of heparin with high affinity for antithrombin, which is consistent with heparin binding and peptide incorporation being linked. Antithrombin in the complex with the tetradecapeptide had lost its ability to inactivate thrombin, but the reactive bond of the inhibitor was cleaved as in a normal substrate. These observations suggest a model, analogous to that proposed for alpha 1-proteinase inhibitor (Engh, R.A., Wright, H.T., and Huber, R. (1990) Protein Eng. 3, 469-477) for the structure of intact antithrombin, in which the A sheet contains only five strands and the P1 to P14 segment of the chain forms part of an exposed loop of the protein. The results further support a reaction model for serpins in which partial insertion of this loop into the A sheet is required for trapping of a proteinase in a stable complex, and complete insertion is responsible for the conformational change accompanying cleavage of the reactive bond of the inhibitor. PMID- 1730730 TI - Reactive sites of an anticarcinogenic Bowman-Birk proteinase inhibitor are similar to other trypsin inhibitors. AB - The three-dimensional structure of the Bowman-Birk type proteinase inhibitor (PI II) has been determined by x-ray crystallography and refined at 2.5-A resolution. This protein is a specific inhibitor of trypsin. Two reactive site loops, one at each end of the PI-II molecule, are structurally similar to each other and to reactive-site loops of pancreatic secretory trypsin inhibitor (Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., and Huber, R. (1982) J. Mol. Biol. 162, 839-869) and bovine pancreatic trypsin inhibitor (Deisenhofer, J., and Steigemann, W. (1975) Acta Crystallogr. B31, 238-250). PI II is the first reported Bowman-Birk type inhibitor structure to be refined at high resolution, providing further insight into inhibitor mechanisms. PMID- 1730731 TI - TATA box-mediated polymerase III transcription in vitro. AB - We have defined conditions whereby a functional TATA box can mediate efficient in vitro transcription by RNA polymerase III. A TATA box is absolutely required for this reaction as a single-point mutation in this sequence completely abolishes transcription. Two protein components are also required: a HeLa cell phosphocellulose fraction (fraction B) and at least one other factor that can be supplied by various crude nuclear extracts or by HeLa cell phosphocellulose fractions C and D. The order of addition is critical; fraction B must be preincubated with the template DNA for TATA box-dependent polymerase III transcription to occur. Various TATA sequences are quite similar in their ability to mediate transcription by polymerases II and III. Despite the similarity in sequence requirements, fraction B does not appear to contain any detectable transcription factor (TF) IID activity, and TATA box-mediated polymerase III transcription does not appear to require TFIID in the form contained in phosphocellulose fraction D. It was recently reported that TFIID is required TFIID in the form contained in phosphocellulose fraction D. It was recently reported that TFIID is required for polymerase III transcription of the yeast and human U6 genes (Margottin, F., Dujardin, G., Gerard, M., Egly, J.-M., Huet, J., and Sentenac, A. (1991) Science 251, 424-426; Simmen, K. A., Bernues, J., Parry, H. D., Stunnenberg, H. G., Berkenstam, A., Cavallini, B., Egly, J.-M., and Mattaj, I. W. (1991) EMBO J. 10, 1853-1862). We propose that fraction B may contain TFIID in a modified form that is not functional for polymerase II transcription. PMID- 1730732 TI - Synthesis and assembly of the synaptic cleft protein S-laminin by cultured cells. AB - S-laminin, a homologue of the B1 chain of laminin, is concentrated in a subset of basal laminae (BLs), including the BL at the skeletal neuromuscular junction and bears an adhesive site for motoneuron-like cells. Here, we have begun to characterize the native form of the protein. We show that several muscle- and glia-like cell lines synthesize and secrete S-laminin as well as the A, B1, and B2 subunits of the conventional laminin trimer. Experiments using subunit specific antibodies showed that S-laminin is complexed with the A and B2 subunits of laminin but not with B1, suggesting that S-laminin replaces B1 to form a novel laminin-like trimer. Comparison of material precipitated by different antibodies provided evidence for two immunochemically distinct forms of S-laminin, both of which associate with B2 and A-like subunits. Analysis of tunicamycin-treated cells indicated that N-linked glycosylation is required neither for the selective association of S-laminin with B2 and A subunits nor for the distinction between two forms of S-laminin. Finally, a full-length S-laminin cDNA was constructed and transfected into muscle and non-muscle cells. S-laminin was detected intracellularly in both cell types, in extracellular matrix of muscle cells, and in two immunochemically distinct forms. Thus, the cDNA contains sufficient information to permit assembly, secretion, and post-translational modification of S-laminin. PMID- 1730733 TI - Location of the ATP gamma-phosphate-binding sites on rat liver carbamoyl phosphate synthetase I. Studies with the ATP analog 5'-p fluorosulfonylbenzoyladenosine. AB - The gamma-phosphate subsites of the MgATP sites of rat liver carbamoyl-phosphate synthetase I have been defined by use of the ATP analog 5'-p fluorosulfonylbenzoyladenosine (FSBA). The synthetase utilizes two molecules of MgATP, apparently in mechanistically discrete steps and at separate MgATP sites. Sequence analysis has revealed internal duplication within the synthetase molecule (Nyunoya, H., Broglie, K.E., Widgren, E.E., and Lusty, C.J. (1985) J. Biol. Chem. 260, 9346-9356) and, based on sequence similarity with other nucleotide-binding proteins, potential ATP sites have been predicted for each of the duplicated sequences. The present FSBA studies have identified four peptides within carbamoyl-phosphate synthetase I that are involved in binding MgATP. Differential effects of N-acetylglutamate, a required allosteric activator, on the interaction of FSBA with the peptides were utilized to develop the following model for two distinct MgATP sites. Peptides 631-638 and 1327-1348 (with Cys1327 and/or Cys1337 modified by FSBA) apparently form part of the binding site for the MgATP involved in bicarbonate activation. Peptides 1310-1317 and 1445-1454 (with Tyr1450 modified by FSBA) apparently form part of the binding site for the MgATP involved in phosphorylation of enzyme-bound carbamate. Each of these MgATP sites contains a peptide from one of the internal duplicated regions of the enzyme molecule, which have previously been suggested as containing MgATP sites (Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260, 9346-9356; Powers-Lee, S. G., and Corina, K. (1987) J. Biol. Chem. 262, 9052-9056), as well as a peptide from the flexible C-terminal region. PMID- 1730734 TI - A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability. AB - Cytochrome P450g is polymorphic in the male rat. This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes. Sequencing of cDNAs from high (+g) and low (-g) phenotype rats has shown that the low phenotype is due to a defective mRNA containing nine base mutations encoding 7 amino acid substitutions. To determine the role of these structural changes in the phenotypic expression of P450g, we altered each of these residues by site-directed mutagenesis in the present studies and expressed the normal and mutant cDNAs in Saccharomyces cerevisiae. P450+g protein was expressed at a level 4-6-fold higher than that of P450-g in yeast cells, despite the presence of identical mRNA levels. This difference in protein expression approaches the difference seen in the rat. A single amino acid change from Ser180 in P450+g to Cys in P450-g, in a highly conserved region in the P4502C subfamily, was found to be solely responsible for the phenotypic differences in expression of P450g. Protein half-life studies demonstrated that this mutation increases the degradation of P450g. This is the first example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability. PMID- 1730735 TI - Multidegenerate DNA recognition by the OxyR transcriptional regulator. AB - The Escherichia coli OxyR protein, a regulator of hydrogen peroxide-inducible genes, is a potent stimulator of transcription in its oxidized form but not in its reduced form. OxyR protein purified in its oxidized form was found to bind four of its non-homologous, functional DNA-binding sites with over 10(6)-fold higher affinity than random DNA sequences. A similarly high DNA binding specificity was observed for the reduced (transcriptionally inactive) form of OxyR, consistent with a model in which the OxyR protein is bound to its recognition sequences even in the absence of an oxidative stress. Alignment of five functional OxyR-binding sites revealed a marked lack of perfectly conserved positions, yet an unusually high number of degenerate homologies (positions at which only two of the four possible base pairs are represented). Methylation interference assays on two OxyR-binding sites showed that OxyR contacts its recognition sequences predominantly at positions of degenerate homology. These results suggest that the OxyR protein specifically recognizes seemingly dissimilar sequences through the use of a multidegenerate recognition code. The chemical basis for a plausible degenerate recognition system is discussed. PMID- 1730737 TI - Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells. AB - HL60 and EL4 cells incubated with tumor necrosis factor-alpha (TNF-alpha) plus staurosporin, a potent inhibitor of protein kinases, showed at least 2-fold increased levels of nuclear factor-kappa B (NF-kappa B) activity compared with TNF-alpha alone both during rapid NF-kappa B activation from the cytosolic pool and protein synthesis-dependent NF-kappa B activation. NF-kappa B activation by phorbol 12-myristate 13-acetate (PMA) and interleukin-1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-alpha-induced increase in mRNA for the p51 subunit of NF-kappa B but interfered with any phorbol ester (PMA)-induced increase in p51 mRNA. Thus, induction of NF-kappa B and p51 mRNA by TNF-alpha was not mediated by a staurosporin-sensitive factor, but NF-kappa B activation by TNF-alpha was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-R alpha (TNF receptor type alpha) after staurosporin-treatment correlated with increased induction of NF-kappa B by TNF-alpha. Staurosporin-treatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-kappa B activation by TNF-alpha, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-R alpha by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-kappa B activation by TNF-alpha. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-R alpha phosphorylation. PMID- 1730736 TI - Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11). AB - The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11. PMID- 1730738 TI - The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3' AMP terminus of susceptible tRNA substrates. AB - The preceding paper (Gross, M., Starn, T.K., Rundquist, C., Crow, P., White, J., Olin, A., and Wagner, T. (1992) J. Biol. Chem. 267, 2073-2079) reported the purification and partial characterization of rabbit reticulocyte peptidyl-tRNA hydrolase. In this article we demonstrate that, unlike bacterial and yeast peptidyl-tRNA hydrolase which act by deacylation, the reticulocyte enzyme hydrolyzes N-acylaminoacyl-tRNA to N-acylaminoacyl-AMP. Reticulocyte lysate has a separate enzyme, that we have isolated and termed aminoacyl-AMP deacylase, which hydrolyzes N-acylaminoacyl-AMP and aminoacyl-AMP, recycling the amino acid and nucleotide components. The action of this enzyme is relatively specific for the N acylaminoacyl-AMP generated by peptidyl-tRNA hydrolase, since it is much less active with N-acylaminoacyl-adenosine and inactive with N-acylaminoacyl-ACCAC, N acylaminoacyl-tRNA, or aminoacyl-tRNA. The tRNA product of peptidyl-tRNA hydrolase action is tRNA missing only its 3'-AMP terminus (tRNA(c-c)), since reaminoacylation requires tRNA nucleotidyltransferase but not CTP. The 3' exonucleolytic action of reticulocyte peptidyl-tRNA hydrolase is specific to susceptible tRNA substrates, since it does not hydrolyze CACCA, CACCA-N-acylamino acid, polyuridylic acid, or the 3' polyadenylate tail of globin mRNA, and, since its ability to hydrolyze Escherichia coli f[3H]Met-tRNA(fMet) is not reduced by excess 5 S or 28 S ribosomal RNA and is reduced only slightly by excess tRNA(c c). Reticulocyte peptidyl-tRNA hydrolase also hydrolyzes th 3'-AMP terminus of deacylated tRNA. This property may explain why the 3'-terminal AMP of tRNA undergoes turnover in reticulocytes and reticulocyte lysate, since we find that such turnover in gel-filtered reticulocyte lysate is increased under conditions where aminoacylation is reduced. PMID- 1730739 TI - Localization of the nucleolar protein NO38 in amphibian oocytes. AB - To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes. We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products. The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein. We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations. When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli. To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA. Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation. We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38. This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule. Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain. Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus. We suggest that nucleolar accumulation of NO38 requires more than a single linear domain. PMID- 1730740 TI - Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein. AB - Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis. PMID- 1730741 TI - In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells. AB - We have been studying the differing characteristics of oligodendrocyte-type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O-2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life. PMID- 1730742 TI - Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation. AB - Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro. PMID- 1730743 TI - TGF-beta 1 is an autocrine-negative growth regulator of human colon carcinoma FET cells in vivo as revealed by transfection of an antisense expression vector. AB - Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression. PMID- 1730744 TI - The invasin protein of Yersinia enterocolitica: internalization of invasin bearing bacteria by eukaryotic cells is associated with reorganization of the cytoskeleton. AB - Yersinia enterocolitica, a facultative intracellular pathogen of mammals, readily enters (i.e., invades) cultured eukaryotic cells, a process that can be conferred by the cloned inv locus of the species. We have studied the mechanism by which the product of inv, a microbial outer membrane protein termed "invasin," mediates the internalization of bacteria by HEp-2 cells and chicken embryo fibroblasts. Invasin-bearing bacteria initially bound the filopodia and the leading edges of cultured cells. Multiple points of contact between the bacterial surface and the surface of the cell ensued and led to the internalization of the bacterium within an endocytic vacuole; the same multi-step process could be induced by an inert particle coated with invasin-containing membranes. Both adherence and internalization were blocked by an antisera directed against the beta 1 integrin cell-adherence molecule. Ultrastructural studies of detergent-insoluble cytoskeletons from infected cells and immunofluorescence microscopy of phalloidin labeled cells showed alterations in the structure of the cytoskeleton during the internalization process including the accumulation of polymerized actin around entering bacteria. Bacterial entry was prevented by cytochalasin D indicating that the internalization process requires actin microfilament function. Possible linkages between beta 1 containing integrins and the cytoskeleton were examined during the internalization process through the use of protein-specific antibodies and immunofluorescence microscopy. Like actin, the actin-associated proteins filamin, talin and the beta 1 integrin subunit were also found to accumulate around entering bacteria. These findings suggest that the invasin-mediated internalization process is associated with cytoskeletal reorganization. PMID- 1730745 TI - Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins. AB - Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread. PMID- 1730746 TI - Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells. AB - Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells. PMID- 1730747 TI - Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription. AB - Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1 mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1 mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression. PMID- 1730748 TI - Casein kinase II is a predominantly nuclear enzyme. AB - Casein kinase II (CK II) has been implicated in regulating multiple processes related to cell growth, proliferation, and differentiation. To better understand the function(s) and regulation of this ubiquitous kinase, it is important to know its subcellular distribution. However, this issue has been the subject of contradictory reports. In this study, we have used indirect immunofluorescence microscopy and cell fractionation to study the subcellular distribution of all three subunits of chicken CK II, alpha, alpha', and beta. We examined primary chick embryo fibroblasts, virally transformed chicken hepatoma cells, as well as HeLa cells transiently transfected with cDNAs encoding chicken CK II subunits. We found that each of the three CK II subunits was located predominantly in the cell nucleus, irrespective of the cell type analyzed or the procedure used for cell fixation. No major differences were detected in the subcellular distributions of individual CK II subunits, and no evidence was obtained for subunit redistributions during interphase of the cell cycle. During mitosis, the bulk of the enzyme was dispersed throughout the cell, though a fraction of all three subunits was associated with the mitotic spindle. Biochemical studies based on mechanical enucleation of chicken cells confirmed the predominantly nuclear location of all three CK II subunits. Finally, immunoblotting experiments were carried out to study the expression of CK II subunits. A survey of different adult chicken tissues revealed substantial tissue-specific differences in the levels of CK II protein, but no evidence was obtained for pronounced tissue specificity in the expression of individual CK II subunits. These results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions. PMID- 1730749 TI - Carboxy terminally truncated forms of ribophorin I are degraded in pre-Golgi compartments by a calcium-dependent process. AB - Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the protein-translocating apparatus of this organelle, were expressed in permanent HeLa cell transformants. Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RL467) and the other consisting of the luminal 332 NH2-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (t 1/2 = 25 h), were rapidly degraded (t 1/2 less than 50 min) by a nonlysosomal mechanism. The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of RI467 was monophasic (t 1/2 = 50 min) but the rate of degradation of RI332 molecules increased about threefold approximately 50 min after their synthesis. Several pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of RI332 molecules that are not degraded during the first phase to a second degradative compartment. Thus, when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of RI332. On the other hand, when CCCP was added after the second phase of degradation of RI332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of RI332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded. One is the ER itself and the second is a non-lysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of RI332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca2+ concentration is necessary for the degradation of the luminally truncated ribophorin. PMID- 1730750 TI - Assembly and disassembly of the Golgi complex: two processes arranged in a cis trans direction. AB - We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt 1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans-Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans-Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae. PMID- 1730751 TI - Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. AB - Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC. PMID- 1730752 TI - Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. AB - We have used endocytic and phagocytic tracers in an EM immunocytochemical study to define the compartments of the phagocytic and endocytic pathways in mouse peritoneal macrophages. Endocytosed BSA-gold appeared successively in early endosomes, spherical endosomal vesicles, a late endosomal tubuloreticular compartment (TC), and terminal lysosomes. The TC appeared as an elaborate structure enriched for the lysosomal membrane glycoproteins Lamp 1 and Lamp 2, and expressing significant levels of rab7, a late endosome-specific GTP-binding protein. The cation-independent mannose-6-phosphate receptor was restricted to specialized regions of the TC that were predominantly adjacent to the Golgi complex. Both the early endosome and the TC had coated bud structures whose composition and function are presently unknown. Phagolysosomes containing latex beads expressed the same membrane antigens and received endocytic tracers simultaneously with the TC. Since the membrane surrounding both organelles was also in direct continuity, we assume that both structures form one functional compartment. Macrosialin, an antigen confined to macrophages and dendritic cells, was heavily expressed in TC and phagolysosomal membranes with low levels being detected in other endosomal compartments and on the cell surface. Treatment of cells with wheat germ agglutinin drastically altered the morphology of the TC, giving rise to sheets of tightly adherent membrane and greatly expanded vesicles, in which cell-associated wheat germ agglutinin was concentrated. The spherical endosomal carrier vesicles loaded with internalized gold tracers clustered nearby, often making contact without fusing. Since the delivery of endocytic tracer to the TC was significantly delayed these experiments suggest that the lectin is somehow preventing the endosome vesicles from fusing with the TC. Collectively, our data argue first that the PLC is equivalent to the "tubular lysosomes" commonly described in macrophages, and second that the meeting of the phagocytic and endocytic pathway occurs in this compartment. PMID- 1730753 TI - Isolation of hnRNP complexes from Drosophila melanogaster. AB - Nascent RNA polymerase II transcripts, heterogeneous nuclear RNAs (hnRNAs), become associated with nuclear proteins (hnRNP Proteins), and their processing into mRNAs takes place in these hnRNP complexes. hnRNP complexes have previously been purified from vertebrate cells. Here we report the isolation of hnRNP complexes from an invertebrate organism, the fruitfly Drosophila melanogaster. Candidate hnRNP proteins were purified from D. melanogaster embryos by ssDNA affinity chromatography, and mAbs were produced to many of the major proteins. Genuine hnRNP proteins were identified by several criteria, including nucleoplasmic localization, association with nascent transcripts, crosslinking to poly(A)-containing RNA in living cells, and amino acid sequence. In addition, mAbs that cross-react between the fruitfly and human hnRNP proteins were obtained. Most importantly, using hnRNP-specific mAbs we have purified the hnRNP complexes from D. melanogaster cells. These RNAase-sensitive complexes contain at least 10 major proteins designated hrps, the most abundant proteins having apparent molecular masses of 36, 38, 39, 40, 44, 48, 54, 62, 70, and 75 kD. cDNAs and complete sequences for several of these proteins have been obtained and are presented in the accompanying paper (Matunis, E. L., M. J. Matunis, and G. Dreyfuss. 1992. J. Cell Biol. 116:257-269). The purification of D. melanogaster hnRNP complexes will facilitate genetic and cytological studies on the function of hnRNA-binding proteins and on the posttranscriptional regulation of gene expression. PMID- 1730754 TI - Characterization of the major hnRNP proteins from Drosophila melanogaster. AB - To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster. cDNA clones of several D. melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes. These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D. melanogaster (Matunis et al., 1992, accompanying paper). All of the proteins described here contain two amino terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain. We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly. The sequences of the D. melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD containing proteins has been conserved independently and has a different function. Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins. A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man. These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes. This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins. PMID- 1730755 TI - O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import. AB - Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component. PMID- 1730756 TI - GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. AB - Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles. PMID- 1730757 TI - Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. AB - At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins. PMID- 1730758 TI - Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding. AB - A cDNA encoding a cytoplasmic oxysterol binding protein was expressed at high levels by transfection in animal cells. This protein binds oxysterols such as 25 hydroxycholesterol that regulate sterol metabolism by transcriptional and posttranscriptional effects. In the transfected cells, some of the oxysterol binding protein (OSBP) was distributed diffusely in the cytoplasm, and some was bound to small vesicles near the nucleus, as revealed by indirect immunofluorescence. Upon addition of 25-hydroxycholesterol, most of the OSBP became concentrated in large perinuclear structures that stained with lentil lectin, a protein that stains the Golgi apparatus. The structures that contained OSBP were disrupted by brefeldin A, confirming their identification as Golgi. A mutant OSBP lacking the COOH-terminal oxysterol binding domain localized to the Golgi spontaneously, suggesting that this domain normally occludes the domain that binds to the Golgi and that sterols relieve this occlusion. The previously noted potential leucine zipper sequence in OSBP was not required for Golgi localization, nor was it essential for homodimer formation. We conclude that OSBP is triggered to bind extrinsically to Golgi membranes when it binds oxysterols and speculate that this translocation may play a role in the transport, metabolism, or regulatory actions of oxysterols. PMID- 1730759 TI - Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry. AB - The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions. PMID- 1730760 TI - Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes. AB - We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals. PMID- 1730761 TI - Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits. AB - We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1 M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1 M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular. PMID- 1730762 TI - Myelin sheath survival after guanethidine-induced axonal degeneration. AB - Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration. PMID- 1730763 TI - The HB-6, CDw75, and CD76 differentiation antigens are unique cell-surface carbohydrate determinants generated by the beta-galactoside alpha 2,6 sialyltransferase. AB - Expression of the beta-galactoside alpha 2,6-sialyltransferase (alpha 2,6-ST) was shown to regulate the generation of multiple cell-surface differentiation antigens (Ags) that may be necessary for lymphocyte function. A new mAb was produced, termed HB-6, that was shown to identify a novel neuraminidase-sensitive cell-surface Ag expressed by subpopulations of human lymphocytes and erythrocytes. In attempting to isolate a cDNA encoding the HB-6 antigen by expression cloning, a cDNA encoding the alpha 2,6-ST (EC 2.4.99.1) was obtained. Since expression of the alpha 2,6-ST protein was shown to be limited to the Golgi apparatus, the cell-surface HB-6 Ag was demonstrated to be the product of alpha 2,6-ST activity. Interestingly, alpha 2,6-ST expression also generated two other neuraminidase-sensitive lymphocyte cell-surface differentiation Ags, CDw75, and CD76. The HB-6, CDw75, and CD76 mAb identified distinct Ags that were differentially expressed by different B cell lines and exhibited different patterns of expression in tissue sections. These results indicate that alpha 2,6 ST expression is a critical regulatory step in the formation of the Ags that are recognized by these mAb, and that an alpha 2,6-linked sialic acid residue is an essential component of each Ag. Thus, expression of a single ST can result in the generation of multiple distinct antigenic determinants on the cell surface which can be distinguished by mAb and may have regulatory roles in lymphocyte function. PMID- 1730764 TI - Clusters of neural cell adhesion molecule at sites of cell-cell contact. AB - I have examined the distribution of neural cell adhesion molecule (N-CAM) in cultured C2 myogenic cells and other cell lines to determine if N-CAM accumulates at sites of cell-cell contact. C2 cells growing in log phase display large clusters of neural cell adhesion molecule where they contact each other. These clusters are remarkably stable, do not form at cell-substrate contacts, and appear not to be enriched in a number of other cytoskeletal, membrane, or extracellular proteins. Thus, N-CAM clusters form preferentially in response to cell-cell contact and are specifically enriched in N-CAM. As C2 cultures mature and differentiate, clusters persist at contacts between aligning myoblasts and between myotubes, consistent with a role in myogenesis. N-CAM is also enriched at cell-cell contacts in cultures of PC12, NRK, and CHO cells. These cells have significant amounts of N-CAM as detected on immunoblots. Clusters are not seen in L929 cells, which do not have detectable amounts of N-CAM. Coculture of these cells with C2 cells results in the clustering of N-CAM at heterologous contacts between C2 cells and NRK, CHO, or PC12 cells, but not between C2 cells and L929 cells. These results suggest that N-CAM specifically accumulates where N-CAM bearing cells contact one another. Clustering of N-CAM may be an important step in strengthening intercellular adhesion. PMID- 1730765 TI - Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18). AB - Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac 1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b. PMID- 1730766 TI - A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion. AB - The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect. PMID- 1730767 TI - A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44. AB - TSG-6 cDNA was isolated by differential screening of a lambda cDNA library prepared from tumor necrosis factor (TNF)-treated human diploid FS-4 fibroblasts. We show that TSG-6 mRNA was not detectable in untreated cells, but became readily induced by TNF in normal human fibroblast lines and in peripheral blood mononuclear cells. In contrast, TSG-6 mRNA was undetectable in either control or TNF-treated human vascular endothelial cells and a variety of tumor-derived or virus-transformed cell lines. The sequence of full-length TSG-6 cDNA revealed one major open reading frame predicting a polypeptide of 277 amino acids, including a typical cleavable signal peptide. The NH2-terminal half of the predicted TSG-6 protein sequence shows a significant homology with a region implicated in hyaluronate binding, present in cartilage link protein, proteoglycan core proteins, and the adhesion receptor CD44. The most extensive sequence homology exists between the predicted TSG-6 protein and CD44. Western blot analysis with an antiserum raised against a TSG-6 fusion protein detected a 39-kD glycoprotein in the supernatants of TNF-treated FS-4 cells and of cells transfected with TSG-6 cDNA. Binding of the TSG-6 protein to hyaluronate was demonstrated by coprecipitation. Our data indicate that the inflammatory cytokine (TNF or IL-1) inducible, secretory TSG-6 protein is a novel member of the family of hyaluronate binding proteins, possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis. PMID- 1730768 TI - Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. AB - The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379 16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL metabolism, basement membrane assembly, calcium binding, and growth- and neurite promoting activities. PMID- 1730769 TI - Plasma membrane protein sorting in polarized epithelial cells. PMID- 1730770 TI - Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box. AB - We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B. PMID- 1730771 TI - Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae. AB - We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID- 1730772 TI - Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration. AB - The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells. PMID- 1730773 TI - Analysis of Drosophila paramyosin: identification of a novel isoform which is restricted to a subset of adult muscles. AB - In this report we show that Drosophila melanogaster muscles contain the standard form of the thick filament protein paramyosin, as well as a novel paramyosin isoform, which we call miniparamyosin. We have isolated Drosophila paramyosin using previously established methods. This protein is approximately 105 kD and cross-reacts with polyclonal antibodies made against Caenorhabditis elegans or Heliocopris dilloni paramyosin. The Heliocopris antibody also cross-reacts with a approximately 55-kD protein which may be miniparamyosin. We have cloned and sequenced cDNA's encoding both Drosophila isoforms. Standard paramyosin has short nonhelical regions at each terminus flanking the expected alpha-helical heptad repeat seen in other paramyosins and in myosin heavy chains. The COOH-terminal 363 amino acids are identical in standard and miniparamyosin. However, the smaller isoform has 114 residues at the NH2 terminus that are unique as compared to the current protein sequence data base. The paramyosin gene is located at chromosome position 66E1. It appears to use two promoters to generate mRNA's that have either of two different 5' coding sequences joined to common 3' exons. Each protein isoform is encoded by two transcripts that differ only in the usage of polyadenylation signals. This results in four size classes of paramyosin mRNA which are expressed in a developmentally regulated pattern consistent with that observed for other muscle-specific RNA's in Drosophila. In situ hybridization to Drosophila tissue sections shows that standard paramyosin is expressed in all larval and adult muscle tissues whereas miniparamyosin is restricted to a subset of the adult musculature. Thus miniparamyosin is a novel muscle-specific protein that likely plays a role in thick filament structure or function in some adult muscles of Drosophila. PMID- 1730774 TI - Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells. AB - The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo transduction. PMID- 1730775 TI - Thin-section ratiometric Ca2+ images obtained by optical sectioning of fura-2 loaded mast cells. AB - The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion. PMID- 1730776 TI - Synaptic vesicle membrane proteins interact to form a multimeric complex. AB - Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal. PMID- 1730777 TI - Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein. AB - Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein. PMID- 1730778 TI - Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. AB - The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615 1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin. PMID- 1730779 TI - Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6. AB - In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration. PMID- 1730780 TI - Short-term suramin treatment followed by the removal of the drug induces terminal differentiation of HT29-D4 cells. AB - Suramin is an anti-cancer drug which induces the differentiation of the human colon cancer clone HT29-D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurrence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29-D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin-free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29-D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post-differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29-D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation. PMID- 1730781 TI - Plasma membrane water permeabilities of human oocytes: the temperature dependence of water movement in individual cells. AB - Membrane water permeability values were measured in individual fresh human pre ovulatory oocytes using real time microscopy in a microscope diffusion chamber. The cells were exposed to anisosmotic conditions, their volume responses measured, and from these data the Lp values were computed employing the Kedem Katchalsky analyses of irreversible thermodynamics. Lp values were measured at four temperatures for each oocyte between 37 degrees C and 10 degrees C, and the temperature-related Arrhenius activation energy (Ea) calculated. It was apparent that individual oocytes exhibited a wide range of Lp values; at 37 degrees C Lp values ranged between 0.33 and 1.80 microns/atm/min. However, each oocyte exhibited the expected inverse linear correlation between Lp and temperature, with high linear correlations (R2 values between 0.73 and 0.96). A mean value for Ea of 8.61 +/- 5.11 Kcal/mol was computed. It is apparent that pre-ovulatory human oocytes express a range of biological diversity in terms of membrane water transport, and this fact needs to be considered when attempting to formulate cryopreservation protocols for storage of these oocytes. PMID- 1730782 TI - Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity. AB - The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps. PMID- 1730783 TI - Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. AB - Nuclear factor kappa-B (NF-kappa B) has been shown to play an important role in LPS-mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP-dependent protein kinase in the regulation of NF-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B element was transfected into the RAW264 macrophage-like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8-bromo cAMP. To further study NF-kappa B induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF-kappa B binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA-binding activity confirmed these results and indicated that LPS induced a 55 KD DNA-binding protein. Induction of this NF kappa B binding activity was not inhibited by pretreatment with the PKC inhibitor H-7. H-7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down-regulates PKC, had no effect on LPS induction of NF-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC. PMID- 1730785 TI - Sensitivity of cultured and skinned chick myotube to calcium, strontium, and barium ions examined by recording isometric contractions. AB - Sensitivity of cultured chick myotubes to alkaline earth metal ions was investigated by recording contractile isometric tension through a semiconductor transducer. The myotubes were obtained by culturing myoblasts of chick embryo breast muscles, and skinned chemically before physiological experiments. Contractions developed in response to Ca2+ in a bathing medium higher than 3 x 10(-7) M and reached maximum at 1 x 10(-5) M. Sr2+ was less effective than Ca2+; the threshold concentration was 1 x 10(-5) M and the tension reached maximum at 1 x 10(-3) M. Ba2+ was the least effective among the three alkaline earth metal ions; only one fifth of the Ca(2+)-induced maximum tension was attained at 1 x 10(-3) M. The sensitivity was similar to that of the mature pectoral muscle fiber, a fast twitch muscle fiber, rather than that of the anterior latissimus dorsi, a slow tonic muscle fiber. The sensitivity was shown to be dependent on its troponin C by replacing it with troponin C from the mature pectoral or cardiac muscle. This indicates that TnC of a fast-muscle type is expressed in the cultured chick myotube as in the mature pectoral muscle. The contractile apparatus was thus shown to be well developed in the cultured myotube with characteristics similar to the mature fast twitch muscle fiber. PMID- 1730784 TI - Regulation of prostaglandin H synthase activity in dog urothelial cells. AB - TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 microM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 microM TPA or 1.0 microM A23187, arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 microM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells. PMID- 1730786 TI - Effects of growth factors, hormones, bacterial lipopolysaccharides, and lipotechoic acids on the clonal growth of normal ureteral epithelial cells in serum-free culture. AB - In vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F-12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F-12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F-12* medium could be replaced by three orders of magnitude less IGF-1. Further clonal growth experiments demonstrated that PGE1 is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP-dependent mitogenic pathway. Seven gram-negative bacterial lipopolysaccharides (LPS) and three gram-positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived from Escherichia coli (strains 055:B5, 0128:B12, and 0127:B8), LPS from Klebsiella pneumoniae, and LPS from Pseudomonas aeruginosa all showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 micrograms/ml. LPS derived from E. coli strain (0111:B4) had no growth effects at the highest concentration tested (100 micrograms/ml). In contrast, LT derived from Streptococcus pyogenes, S. faecalis, Staphylococcus aureas, and Bacillus subtilis all markedly enhanced clonal growth at concentrations ranging from 1 microgram/ml less than [LT] less than 50 micrograms/ml. LT from Strep. pyogenes was inhibitory to clonal growth at 100 micrograms/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation-competent uroepithelial cells to growth inhibition by LPS produced by gram-negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth-stimulating activity of LTs produced by gram-positive bacteria may be due to their ability to bind to cell-associated fibronectin and to activate the fibronectin receptor as part of ligand receptor-induced mitogenic transmembrane signalling pathway. PMID- 1730787 TI - Inhibition of human erythroid colony-forming units by interleukin-1 is mediated by gamma interferon. AB - IL-1 inhibits erythropoiesis in vivo and in vitro. This inhibition was studied by comparing the effect of recombinant human IL-1 (rhIL-1) on highly purified CFU erythroid (E) generated from peripheral blood burst-forming units-erythroid (BFU E) (mean purity 44.4%) with its effect on unpurified marrow CFU-E (mean purity 0.36%). Colony formation by marrow CFU-E was significantly inhibited by rhIL-1, while colony formation by highly purified CFU-E was not inhibited. However, purified CFU-E colonies were inhibited by rhIL-1 in the presence of autologous T lymphocytes, and also by cell-free conditioned medium prepared from T-lymphocytes stimulated by rhIL-1. This inhibitory effect was ablated by neutralizing antibodies to gamma interferon (IFN), but not by antibodies to human IL-1, tumor necrosis factor, or beta IFN. Colony formation by highly purified CFU-E was also inhibited by recombinant human gamma IFN (rh gamma IFN). IL-1 and gamma IFN play significant roles in the pathogenesis of the anemia of chronic disease. These studies indicate that rhIL-1 inhibits CFU-E colony formation by an indirect mechanism involving T-lymphocytes and requiring gamma IFN and that gamma IFN itself is most probably the direct mediator of this effect. PMID- 1730788 TI - Heparin inhibits the attachment and growth of Balb/c-3T3 fibroblasts on collagen substrata. AB - In investigating the role of cell-extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell-collagen interactions were examined. Exponentially growing Balb/c-3T3 fibroblasts were radiolabelled with 3H-thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70-90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10-50% when heparin was present from 0.1-100 micrograms/ml. Cell-collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell-collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (greater than 40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr less than or equal to 6KD) with type I collagen was analyzed by affinity co-electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell-collagen and heparin-collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative activity in vivo and in vitro. PMID- 1730789 TI - Low-amplitude, low-frequency electric field-stimulated bone cell proliferation may in part be mediated by increased IGF-II release. AB - We have developed an in vitro model incorporating a low-amplitude (10(-7) V/cm), low frequency (f less than 100 Hz), capacitively coupled electric field in order to study the mechanism through which an electric field may increase bone cell proliferation. Utilizing this model we have previously shown that electric field stimulated bone cell proliferation was dependent on release of mitogen activity into the culture medium from exposed cells. The current studies were intended to characterize this mitogen activity. In these studies we found that electric field stimulated human bone cell proliferation was associated with increased IGF-II mRNA accumulation and IGF-II secretion suggesting that IGF-II may in part mediate the increase in bone cell proliferation following electric field exposure. PMID- 1730790 TI - Effects of pH-variations on the kinetics of growth and energy metabolism in cultured T-lymphoma cells: a microcalorimetric study. AB - The progression of T-lymphoma cells (CCRF-CEM) growing in suspension has been monitored during long term (12-28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH, p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 10(6) cells per ml). The lowering of p(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmol/l, respectively, did not influence Pcell or the growth pattern. No "crowding effect" was observed for the cells in the investigated concentration range (0.6-1.3) x 10(6) cells per ml. PMID- 1730791 TI - ACE inhibitors in the diagnosis of renovascular hypertension. AB - Effective therapy is available but the diagnosis of renovascular hypertension is often elusive. Those affected constitute a minority of the hypertensive population, and traditional screening tests have poor sensitivity and specificity. The merits and limitations of the captopril challenge test and the captopril renogram, alone or in combination, are discussed. PMID- 1730792 TI - Tachycardias in young children. PMID- 1730793 TI - Unusual rhythm after AV nodal ablation. PMID- 1730794 TI - Recent developments in STDs: II. Viral and other syndromes. AB - It is likely that up to 50% of the U.S. population will acquire a sexually transmitted disease by age 30 to 35. In industrialized countries, herpes simplex virus is by far the most common pathogen. The major public health impact of HSV infection is its brutal legacy to newborns. Half of the affected infants die at birth, and many of the survivors have severe neurodevelopmental disabilities. PMID- 1730795 TI - Tuberculous peritonitis: an overlooked diagnosis. AB - Contrary to widespread belief, abdominal tuberculosis is not rare, nor is it confined to the poor or patients with active pulmonary tuberculosis. The disease must be considered in the differential diagnosis of abdominal pain. PMID- 1730796 TI - Killer cheese. PMID- 1730797 TI - The Patient Self-Determination Act: implications for physicians. PMID- 1730798 TI - Dysarthria and a numb hand in an elderly woman. PMID- 1730799 TI - Giant cell arteritis and polymyalgia rheumatica. PMID- 1730800 TI - New perspectives on the coagulation cascade. AB - The view that clotting is basically a succession of zymogen activations remains valid but is in need of some modifications, in part because the intrinsic and extrinsic coagulation pathways are more closely interrelated than had been thought. The new knowledge is beginning to influence treatment of the hereditary bleeding disorders--and to point the way toward genetic cures. PMID- 1730801 TI - Clinical review 30: Clinician's evaluation of a solitary thyroid nodule. PMID- 1730802 TI - Premature adrenarche: a normal variant of puberty? PMID- 1730803 TI - Natural history of premature pubarche: an auxological study. AB - The natural history of girls with premature pubarche is reported to be normal, but the effects on puberty and on final height are not well documented. We assessed the outcome of a group of girls with premature pubarche from two Latin populations in whom 21-hydroxylase deficiency had been ruled out by an ACTH test. Patients comprised 127 girls (70 Northern-Italian and 57 Northern-Spanish), of whom 69 had entered puberty and 38 had attained adult height. Height, bone age, onset and progression of puberty, height prognosis, adult height, and baseline plasma androgen levels were evaluated. Advanced skeletal maturation and tall stature were constant features during the first years of follow-up and subsequently declined. Puberty began at 9.7 +/- 0.9 yr, and age at menarche (12.0 +/- 1.0 yr) was comparable to maternal and population menarcheal ages. The appearance and chronology of pubertal milestones in both populations were very similar. Adult heights correlated with the height prognosis at diagnosis and at onset of puberty, and were above midparental heights. Premature pubarche produces a transient acceleration in growth and bone maturation with no negative effects on the onset and progression of puberty and final height. PMID- 1730804 TI - Role of human chorionic gonadotropin as a thyroid stimulator. PMID- 1730805 TI - Human chorionic gonadotropin may not be responsible for thyroid-stimulating activity in normal pregnancy serum. AB - Although hCG can activate thyroid cells in culture there is considerable doubt as to whether it is responsible for the changes in thyroid function which occur during normal pregnancy. Thyroid-stimulating activity (TSA), measured using iodide uptake into FRTL-5 cells, was demonstrated in 32/51 (63%) first and 15/29 (52%) third trimester sera. Free T3 was increased (P less than 0.001) and TSH decreased (P less than 0.01) in first trimester. In the early pregnancy group there was a positive correlation between hCG and TSA (r = 0.594, P less than 0.001) and a negative correlation between hCG and TSH (r = -0.329, P less than 0.02). In third trimester hCG concentration was often below that required to produce TSA in vitro and TSA could not be neutralized by antibodies to hCG. There was no correlation between hCG and TSH or thyroid hormones in the third trimester. In 26 women TSA decreased in parallel with serum hCG concentration after termination of pregnancy (P less than 0.001). Free T3 also decreased (P less than 0.01) and TSH increased (P less than 0.05) after termination. TSA persisted in many patients even after hCG was either very low or undetectable. Purified hCG stimulated iodide uptake in a concentration-dependent manner. Stimulation of iodide uptake by TSH was inhibited by the simultaneous presence of low concentrations of hCG while activity was restored with high concentrations. hCG may contribute to the thyroid changes in early pregnancy. However the poor correlation between TSA and thyroid tests suggests that other factors may be involved. The partial agonist activity of hCG may account for some of the inconsistencies observed but TSA in serum from late pregnancy or after termination of pregnancy is almost certainly due to another hormone or growth factor. PMID- 1730806 TI - Calcium-regulated secretion of tissue plasminogen activator and parathyroid hormone from human parathyroid cells. AB - The effects of Ca and other agents on secretion of plasminogen activator (PA) and PTH have been examined and compared, using parathyroid cells obtained from the glands of chronic renal patients. During 2 weeks culture at different [Ca], the secretory rates of PA activity and PTH were parallel; steady-state secretion over 24-h periods was maximal at 0.5-0.9 mM Ca, minimal at 1.5-2.5 mM Ca, and the [Ca] at 50% suppression was 1.1 mM. At 2.5 mM Ca, two inhibitors of cellular proteolysis, 3-methyladenine and chloroquine, stimulated secretion of both PA activity and PTH. The results indicated that secretion of PA from human parathyroid cells is regulated similarly to that of PTH. The characteristics of human parathyroid PA were also examined using human parathyroid adenoma tissue. In homogenates, the highest specific activity of PA was in microsomal fractions. The Mr of PA from tissue and from culture media was 70 kilodalton by sodium dodecyl sulfate gel electrophoresis followed by zymography, or by Western blotting using antisera to human tissue PA (tPA). Enzyme activity was inhibited by incubation with antisera to tPA but not to urokinase. In contrast to bovine parathyroid cells that secrete a urokinase, human parathyroids apparently contain and secrete tPA. PMID- 1730807 TI - The growth hormone-releasing activity of a synthetic hexapeptide in normal men and short statured children after oral administration. AB - It has been shown that iv GH-releasing peptide (GHRP; His-DTrp-Ala-Trp-DPhe-Lys NH2) specifically releases GH in man. Because of the clinical value of having an orally active peptide to release GH, five normal men were given GHRP orally at dosages of 100 and 300 micrograms/kg. At the 300 micrograms/kg dosage, the GH rise was detectable at 30 min, peaked between 60-75 min, and gradually declined to the baseline level between 150-180 min. Peak GH levels rose 63- and 202-fold above the baseline after administration of 100 and 300 micrograms/kg GHRP, respectively. Nine short stature children with varying degrees of GH deficiency were also included in this study. All children had short stature, slow growth, and delayed bone age and were being treated with biosynthetic human GH. The studies were performed after stopping GH treatment for 2-3 weeks. The oral GHRP dose administered to all of the children was 300 micrograms/kg, because this dosage was found to consistently increase GH levels in adults. The magnitude and pattern of the GH response to oral GHRP in four of the nine children were essentially the same as those in the five normal men. In the other five children, the GH responses were low but still measurable in three and undetectable in two of the children. Serum immunoreactive GHRP (irGHRP) levels were measured before and at 15- to 30-min intervals 3-5 h after oral as well as iv bolus GHRP administration. For comparison of serum irGHRP and GH levels, results were included after iv bolus administration of 0.1, 0.3, and 1.0 micrograms/kg GHRP to normal men. Since 300 micrograms/kg oral GHRP released about the same amount of GH as 1 microgram/kg, iv, in normal men, it was calculated that oral GHRP has about 0.3% the activity of iv GHRP. After iv GHRP, the peak serum irGHRP levels were immediate and proportional to the dosage, and the disappearance rate decreased exponentially. Between 100-240 min, the mean serum irGHRP level was essentially the same and remained slightly elevated. After administration of 300 micrograms/kg GHRP orally to normal men, serum irGHRP was measurable within 15 min, the peak serum irGHRP level coincided with the GH rise at 60 min, and serum irGHRP levels fell more slowly than serum GH levels. The serum half-life was 20 min, and the distribution volume was 2.5 L after both oral and iv administration.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730808 TI - The source of pulsatile secretion of progesterone during the human follicular phase. AB - This study was designed to establish the normal pattern of serum progesterone and the origin of its secretion during the follicular phase of the normal menstrual cycle. In the first study, 12 normal women were studied on 3 occasions each at different times during a single follicular phase. Serum samples were collected every 10 min over 8 h, for 6 h before and 2 h after an injection of naloxone (5 mg iv). The mean serum progesterone remained constant (0.9 nmol/L) across the follicular phase until just before ovulation; individual subjects showed pulsatility of progesterone (1-6 pulses/6 h) but there was no relationship of this to LH pulsatility and no variation of progesterone pulsatility across the follicular phase. Naloxone caused an increase in the mean serum progesterone in the early follicular phase to 1.9 +/- 0.7 nmol/L and in the mid and late follicular phase to 2.1 +/- 0.7 nmol/L and 3.4 +/- 2.5 nmol/L, respectively. The second study was performed to assess the contribution of the residual corpus luteum and the developing follicle to the pulsatile secretion of progesterone. Seven anovulatory women with low levels of serum LH and absent LH pulsatility were studied before and after clomiphene (100 mg/day for 5 days) by collecting blood samples every 15 min for 6 h before GnRH (10 mg iv) and for 2 h afterwards. The anovulatory women had comparable mean serum concentration of progesterone (0.9 +/- 0.5 nmol/L) to normal women and similar frequency of progesterone pulsatility (2.1 +/- 1.1 pulses/6 h). After administration of clomiphene, there was no significant change in progesterone pulsatility (1.7 +/- 1.0 pulses/6 h) despite a substantial increase in LH pulsatility (from none to 3.0 +/- 1.0 pulses/6 h). There was no significant increase in serum progesterone after clomiphene or GnRH which both caused a substantial increase in serum LH. The third study involved eight normal women studied before and after treatment with dexamethasone (2 mg/day for 2 days) to assess the adrenal component of progesterone secretion. Blood samples were collected every 10 min for 6 h before and 2 h after naloxone (5 mg iv). Dexamethasone reduced serum progesterone to below assay sensitivity (less than 0.2 nmol/L) and obliterated progesterone pulsatility. The increase in serum progesterone and cortisol induced by naloxone was blocked by dexamethasone; the naloxone-induced rise of serum LH was not affected by dexamethasone. We conclude that neither the preceding corpus luteum nor the developing follicle are important contributors to the serum concentration of progesterone during the normal follicular phase.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730809 TI - Paracrine actions of oxytocin, prostaglandin F2 alpha, and estradiol within the human corpus luteum. AB - Human luteal cells are known to interact in an auto- and paracrine fashion using a variety of substances, including prostaglandins (PGs), steroids, and peptides. In cultures of dispersed luteal cells obtained from several animal species prostaglandin F2 alpha (PGF2 alpha) and oxytocin (OXT) inhibit progesterone (P) secretion, indicating a luteolytic effect of these substances. The disadvantage of luteal cell cultures is that the different luteal cell types do not communicate with each other, i.e. auto- and paracrine effects cannot be studied. Therefore, we used a microdialysis tubing, which is implanted in human corpora lutea (CL) kept under short term organ culture conditions. Ringer's solution is pumped through the dialysis tubing, and substances secreted by the luteal tissue can be determined in the effluent fractions. This system also allows topical application of substances with putative intraluteal effects. In the present report we used PGF2 alpha, OXT, and estradiol (E2) to examine the effects of these substances on the respective other hormones and on P release from young human CL. Intraluteal application of PGF2 alpha stimulated OXT, E2, and P release. OXT was stimulatory to E2 and P secretion, an effect that can be blocked by a specific OXT antagonist and by tamoxifen. Elevation of intraluteal E2 concentrations also had marked stimulatory effects on P secretion. From luteal cell culture experiments it is known that PGF2 alpha and OXT have direct inhibitory effects on P production, but both substances stimulate E2 release. It was also shown that E2 counteracts the inhibitory effects on P release. Therefore, the PGF2 alpha- and OXT-induced E2 release may be responsible for the increased P release. This assumption is further substantiated by the observation that intraluteally applied E2 stimulates P secretion, and preexposure of human CL to tamoxifen prevents the OXT-induced stimulation of P, but not E2, secretion. We conclude that in young human CL, PGF2 alpha and OXT have dual effects: direct inhibitory effects on P release and E2-mediated stimulatory effects, which in young CL result in a net stimulation of P secretion. PMID- 1730810 TI - Effects of luteal estradiol on the secretory transformation of human endometrium and plasma gonadotropins. AB - To study the role of luteal estradiol (E2), we interrupted the supply of E2 during the luteal phase of E2 and progesterone (P) replacement cycles. Thirty-one women, aged 26-37 yr, with absent or inactive ovaries received three different treatment regimens: group I (n = 11) received transdermal E2 and vaginal P according to a protocol designed to approximate levels of estrone (E1), E2, and P seen during the menstrual cycle. Groups II (n = 11) and III (n = 9) received identical treatments, except that in group II no E2, and in group III no E2 or P, was administered after day 15. Endometrial biopsies were obtained on days 20 and 24 in groups I and II, and on days 14 and 20 in group III. In group I, plasma E1 and E2 reached menstrual cycle levels, whereas in groups II and III, discontinuation of the E2 supply on day 15 resulted in a prompt decrease to castrate levels of plasma E1 and E2. In groups I and II, menopausal FSH and LH levels decreased to 26 +/- 6 and 30 +/- 7 IU/L, respectively, on day 13 (mean +/- SEM). In group I, administration of E2 and P starting on day 15 further lowered plasma gonadotropin levels. In group II, administration of P only failed to induce a similar decrease in plasma FSH and LH. No uterine bleeding occurred before day 25 in women of groups I or II, while women of group III bled within 2 days of E2 withdrawal. Endometrial biopsies were similar in groups I and II. Histological features were characteristic of early and late luteal phases on days 20 and 24, respectively. Endometrial maturation assessed by estrogen and progesterone receptors identified by immunocytochemistry showed the typical distribution seen on day 24 of the menstrual cycle with no difference between groups I and II. We conclude that in women deprived of ovarian function, administration of P only after 14 days of E2 priming prevented uterine bleeding and induced normal secretory transformations of the endometrium, but failed to suppress plasma gonadotropins. PMID- 1730811 TI - Effect of testosterone on metabolic rate and body composition in normal men and men with muscular dystrophy. AB - We have examined the effect of testosterone enanthate injections (3 mg/kg.week, im) on the basal metabolic rate (BMR) estimated by indirect calorimetry and on lean body mass (LBM) estimated by 40K counting in four normal men and nine men with muscular dystrophy. Testosterone treatment increased plasma testosterone levels in all subjects (3-fold mean elevation). BMR increased significantly after 3 months of testosterone treatment (mean, 10%; P less than 0.01; 13% mean increase in the men with muscular dystrophy and 7% mean increase in the normal subjects). BMR remained elevated (mean increase, 9%) after 12 months of testosterone treatment in four men with muscular dystrophy. LBM also was significantly higher after 3 months of treatment (mean, 10%; P less than 0.01) and remained elevated at 12 months. The percent increase in LBM was similar in men with muscular dystrophy (+10%) and normal men (+11%). When BMR was adjusted for the increase in LBM by linear regression, the men with muscular dystrophy had an increase in adjusted BMR after 3 months of testosterone treatment (mean increase, 7%), but not after 12 months. The normal men did not have an increase in adjusted BMR. Testosterone treatment for 12 months slightly reduced body fat, whereas there was an increase in body fat in subjects with muscular dystrophy who were treated with placebo for 12 months. We conclude that there is a significant increase in BMR associated with pharmacological testosterone treatment, which for the most part is explained by the increase in LBM. However, in men with muscular dystrophy, there is a small hypermetabolic effect of testosterone beyond that explained by increased LBM. PMID- 1730812 TI - Endogenous growth hormone secretion and clearance rates in normal boys, as determined by deconvolution analysis: relationship to age, pubertal status, and body mass. AB - Mean plasma GH concentrations increase in normal boys during mid- to late puberty. To investigate the nature of the pituitary secretory events and/or altered metabolic clearance responsible for these serum GH concentration changes, we performed multiple-parameter deconvolution analysis of 46 24-h serum GH concentration-time series obtained from normal boys at various stages of puberty and young adulthood. The subjects ranged in chronological age from 7-27 yr. The height and weight of each subject were between the 5th and 95th percentile for age. The calculated daily mass of GH secreted was greatest (P less than 0.001) in late pubertal boys (mean +/- SE, 1810 +/- 250 micrograms/24 h) and was triple the value in prepubertal boys (610 +/- 65 micrograms/24 h). When the values were normalized and expressed as mass of GH secreted per unit (m2) body surface area or per L distribution volume, GH secretion in late pubertal boys was still significantly greater than that in any other group (P less than 0.05). These values for late pubertal boys were nearly double the corresponding values for prepubertal boys (1160 +/- 160 vs. 600 +/- 58 micrograms GH/m2.24 h and 440 +/- 63 vs. 270 +/- 25 micrograms GH/L vol.24 h, respectively). When the effect of clearance mechanics on serum GH concentrations was removed mathematically, the primary change in predicted GH secretory burst parameters during pubertal development was an increase in GH mass released per burst resulting from an increase in the maximal rate of GH secretion attained within the bursts. These changes in the amplitude of GH release events were specific, in that they were largely independent of any accompanying alterations in duration or frequency of the GH secretory bursts or in serum GH half-life. Correlation analysis revealed that the 24-h GH secretion rate varied inversely with the subjects' body mass index SD score (r = -0.65; P less than 0.01), suggesting that differences in body mass, even within the normal range, contribute to the wide variability in daily GH secretion rates among normally growing children. The plasma insulin-like growth factor-I concentrations of all subjects correlated positively with the calculated 24-h GH secretion rate (r = 0.51; P less than 0.001). In summary, the primary neuroendocrine alteration responsible for the augmented serum GH concentrations characterizing mid- to late-puberty in boys is an increased mass of GH released per pituitary secretory episode resulting from an increased maximal rate of GH secretion within each burst.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730813 TI - Hip mineral density in females with a recent hip fracture. AB - To evaluate the role of local bone mineral density (BMD) in the etiology of hip fractures, we measured the hip BMD using dual photon absorptiometry in 29 females who had recently suffered a hip fracture associated with minimal or moderate, but not major, trauma and compared their BMD to those of 14 young normal females, 58 early postmenopausal normal females, 13 age-matched normal females, and 114 spinal osteoporotic females without a hip fracture. Hip-fractured patients had a BMD significantly lower (P less than 0.001) than that of all other studied groups, suggesting that a low hip BMD is associated with hip fracture risk. A femoral neck BMD below 0.75 g/cm2 suggests an increased likelihood for developing a hip fracture. Peak BMD was measured at 1.03 g/cm2, a value comparable to published normative data. Thus, a loss in hip BMD of approximately 30% from peak mineral density appears necessary before a hip fracture may occur after moderate trauma. PMID- 1730814 TI - Expression of growth hormone (GH)-releasing factor gene in GH-producing pituitary adenoma. AB - Pituitary cells synthesize various neuropeptides that influence pituitary hormone secretion. GH-releasing factor (GRF) may also be produced by normal or pituitary tumor cells. We examined GRF gene expression in pituitary tumors. Standard techniques for the analysis of GRF gene expression did not appear to be suitable. Highly sensitive reverse transcription coupled to polymerase chain reaction was used. Specimens of pituitary adenoma were obtained by transsphenoidal adenomectomy from six patients with acromegaly and three patients with no clinical evidence of pituitary hormone overproduction; non-functioning adenoma. Pituitary glands were collected at autopsy from three patients who died from nonendocrine disorders. A specific GRF gene transcript was detected in five out of six GH-producing pituitary adenomas, whereas this was not found in three separate specimens of nonfunctioning pituitary adenoma or anterior and posterior pituitary tissue. The data suggest that GRF is synthesized as an intrinsic product in human GH-producing pituitary adenoma. PMID- 1730815 TI - Insulin inhibits adrenal 17,20-lyase activity in man. AB - Experimentally induced hyperinsulinemia reduces serum adrenal androgen levels in man, but does not alter cortisol secretion. To determine whether insulin might selectively inhibit adrenal androgen production by suppressing 17,20-lyase activity, ACTH-stimulated androgen secretion was assessed in 10 normal men after an insulin infusion (hyperinsulinemic-euglycemic clamp) or a control saline infusion. For the insulin clamp study, each man received a 2-U (14.4-nmol) insulin bolus dose, followed by a 2.0-mU/kg.min (14.4-pmol/kg.min) insulin infusion for 5 h. An average insulin level of 746 +/- 35 (+/- SE) pmol/L was achieved; serum glucose was maintained at 4.96 +/- 0.03 mmol/L. At the end of the insulin infusion, an ACTH stimulation test was performed, and serum steroid levels were determined 30 and 60 min later. Subjects returned 1-3 weeks later for control studies, during which 0.45% saline was infused at rates matched exactly to the rates of the dextrose and insulin infusions during the insulin clamp studies, and an ACTH stimulation test was performed after 5 h of saline infusion. After the insulin infusion, stimulation by ACTH resulted in a significant rise in the serum molar ratio of 17 alpha-hydroxyprogesterone to androstenedione (from 0.914 +/- 0.110 at zero time to 1.388 +/- 0.278 60 min after ACTH; P less than 0.05), whereas no change occurred in the ACTH-stimulated ratio of these steroids after the saline infusion (1.067 +/- 0.109 at zero time to 1.060 +/- 0.109 60 min after ACTH; P = NS). The insulin-induced change in this steroid ratio was due to a relative increase in precursor (17 alpha-hydroxyprogesterone) and decrease in product (androstenedione) responsiveness to ACTH. Similarly, insulin treatment resulted in a greater than 100% rise in the difference from baseline in the serum molar ratio of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone 30-60 min after ACTH (P less than 0.004), whereas no change in this difference was observed after the saline infusion (P = 0.71). Again, the insulin-induced change in this steroid ratio was due to a relative increase in precursor (17 alpha hydroxypregnenolone) and decrease in product (dehydroepiandrosterone) responsiveness to ACTH. Of note, insulin treatment altered neither cortisol responsiveness to ACTH nor 17 alpha-hydroxylase activity, as indicated by similar ACTH-stimulated responses in the serum molar ratio of progesterone to 17 alpha hydroxyprogesterone after the insulin and saline infusions (P = 0.71). Hence, the results of this study indicate that the acute elevation of serum insulin levels into the high physiological range selectively inhibits adrenal 17,20-lyase activity in man. PMID- 1730816 TI - Progesterone stimulates luteinizing hormone secretion by acting directly on the pituitary. AB - To determine if progesterone (P) does affect gonadotropin secretion by acting directly on the pituitary, six women with hypothalamic gonadotropin deficiency were studied. They were treated with 17 beta-estradiol (E2; 2 mg/day, orally) to induce P receptors and maintain constant plasma E2 levels during two 15-day periods separated by 1 month. GnRH was administered iv at a dose of 10 microgram/pulse every 90 min during the last 5 days of E2 treatment. Either P (400 mg/day) or a placebo was administered intravaginally in a cross-over randomized design during the 5 days of pulsatile GnRH therapy. A baseline study of pulsatile LH secretion was performed, with sampling performed every 10 min for 8 h. The sampling was then repeated on day 15 of each study period at the end of pulsatile GnRH administration. Plasma levels of E2 and P were measured every day during the 5 days of either GnRH and P or GnRH and placebo treatment. In the six patients, the observed apulsatile pattern of LH during the baseline study confirmed the diagnosis of complete gonadotropin deficiency. Plasma E2 levels were not significantly different at the time of each pulse analysis (288 +/- 61 vs. 252 +/- 77 pmol/L). The plasma P level achieved with the vaginal pessaries was 22 +/- 5 nmol/L. P treatment resulted in all cases in a significant increase in the mean plasma LH level (5.2 +/- 0.9 vs. 3.6 +/- 0.7 IU/L after GnRH plus placebo; P less than 0.001). Furthermore, LH pulse amplitude was significantly increased by P compared to placebo (3.1 +/- 0.3 vs. 1.4 +/- 0.1 IU/L, respectively; P less than 0.01). Mean plasma FSH levels were significantly increased by GnRH regardless of whether P or placebo was present. In conclusion, these data indicate that a short exposure to physiological levels of P in the range of early luteal phase levels has a stimulatory effect on LH secretion by acting directly at the pituitary level. PMID- 1730817 TI - All products of proglucagon are elevated in plasma from uremic patients. AB - We measured and characterized the proglucagon (PG) products in plasma obtained from nine uremic patients and six control subjects in the fasting state. The concentrations of PG products measured by direct RIAs were significantly higher in the uremic patients than in the normal subjects; the concentration of total glucagon immunoreactivity in plasma was 209 +/- 20 vs. 70 +/- 11 pmol/L, the glucagon-like peptide-1 immunoreactivity was 154 +/- 33 vs. 41 +/- 13 pmol/L, and the concentration of pancreatic-type glucagon immunoreactivity was 53 +/- 6 vs. 30 +/- 7 pmol/L. By chromatography, the predominating PG products in both uremic and normal plasma were shown to be glicentin [corresponding to PG-(1-69)] and the major PG fragment [presumably corresponding to PG-(72-158)]. In addition, glucagon-like peptide-1 [presumably corresponding to PG-(72-107) amide] was a major product in uremic plasma. Our results suggest that the kidneys play an important role in the removal from plasma of these products of PG. PMID- 1730818 TI - Comparative studies of the erythroid-potentiating effects of biosynthetic human insulin-like growth factors-I and -II. AB - The erythroid-potentiating effects of biosynthetic human insulin-like growth factor-I (IGF-I) and IGF-II were evaluated and compared in serum-free cultures of human marrow erythroid progenitors. IGF-I and IGF-II enhanced the in vitro growth of relatively mature (CFU-E) and primitive (BFU-E) erythroid progenitors at similar dose-dependent magnitudes. Significant elevations in erythroid colony counts were detected at 0.2-0.6 ng/mL of both peptides, with a maximal increase in CFU-E and BFU-E numbers detected at 2-6 ng/mL. Similar enhancement of erythroid progenitor cell growth by both peptides was also detected in cultures of marrow cells that had been depleted of accessory cells or in cultures of highly enriched hemopoietic progenitors. IGF-I and IGF-II enhanced erythroid progenitor cell growth at both limiting and saturating concentrations of recombinant human erythropoietin or granulocyte-macrophage colony-stimulating factor, but did not alter the sensitivity of CFU-E and BFU-E to their respective hemopoietic regulators. The erythroid-potentiating effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against IGF-I membrane receptors. IGF-I and IGF-II thus appear to exert their effects on human marrow erythroid progenitors via a direct mechanism involving the type I IGF receptor. PMID- 1730819 TI - Partial reversibility during late postpartum of thyroid abnormalities associated with pregnancy. AB - The aim of the present work was to assess during late postpartum the reversibility of thyroidal alterations associated with pregnancy. Thyroid function was reinvestigated 6 months after delivery in 100 randomly selected healthy women and thyroid volume was reevaluated 12 months after delivery in 10 other selected women. The subjects had previously been carefully followed during gestation as they were included in a prospective cohort investigation of the regulation of the thyroid during pregnancy, in an area with a limited dietary iodine intake (less than 100 micrograms/day in 85% of the women). Six months after delivery, an overall normalization of thyroid function was observed. However, an increase in the T3/T4 ratio, which was present in half the cases at delivery, was still evident 6 months postpartum, suggesting the persistence of relative iodine deficiency, probably prolonged in some women through breast feeding. Furthermore, serum thyroglobulin levels, which were increased in half the women at delivery, remained abnormally high in 40% of them 6 months later. Twelve months after delivery thyroid volume, which had increased in average by 54% during pregnancy, had not reverted to the values found during early gestation. Moreover a goiter was still evident in 2/4 cases in whom it had developed during pregnancy. In conclusion, the present study indicates that pregnancy may constitute a prolonged stimulus for the thyroid and shows for the first time that the alterations associated with gestation are not limited to the period of pregnancy, being only partially reversible during late postpartum. In conditions with a limited iodine intake, pregnancy constitutes a risk for the maternal thyroid: goitrogenesis does occur and may be maintained after delivery. The glandular stress of pregnancy may therefore provide a clue to understanding the high prevalence of thyroid disorders in women. The present study provides additional arguments to suggest that iodine supply be increased during pregnancy but also after parturition, in particular in breast-feeding mothers. PMID- 1730820 TI - Enhancing quality critical care education: establishing a consortium. AB - The demand for expanded knowledge and accountability in critical care nursing has increased the need for qualified critical care nurses and is tempered by requirements that their preparation be cost-effective. The Emory University School of Nursing (EUSN) has pioneered in meeting this challenge. Since June 1988, a Critical Care Consortium consisting of the EUSN and Emory-affiliated hospitals has been operational, providing uniform critical care education to multiple hospital nursing staffs. Instructional resources are being economically utilized, eliminating duplication of efforts. This collaboration between academic and service settings enhances productivity, quality patient care, professional growth, and promotes significantly greater interinstitutional cohesiveness. PMID- 1730821 TI - Teaching a new approach to quality improvement. AB - Whatever the educator's role in teaching quality methods, rapid, industry-wide changes in quality practices are having a profound impact on the education department. Familiar, comfortable teaching methods for monitoring and evaluation have disappeared. Clinical practice models, long the mainstay of nursing education, have become obsolete in today's multidisciplined environment. This article describes a new approach to quality improvement that utilizes a model expanded beyond clinical practice: a tri-focus of patient, staff, and system, integrated with the four broad-based categories of standards (structure, outcome, process, and evaluation) to achieve quality improvement within the organization. PMID- 1730822 TI - Research application: teaching staff nurses to use library search strategies. AB - Journal reading is a major mechanism for disseminating research but much goes unread by practicing nurses. A survey revealed the 10 most frequently read journals are primarily of a clinical/technical nature. To encourage reading of research, we organized a series of library sessions to familiarize nurses with the library and modern search strategies. Over half of the 77 nurse participants indicated they had not visited the library in six months and were less than confident in their library skills. Hands-on experiences, including computerized literature search, gave nurses an opportunity to learn techniques for locating research articles. PMID- 1730824 TI - State and association continuing education requirements. PMID- 1730823 TI - Assessment of learning needs of nurse educators: continuing education implications. AB - Because of current structure, content, and outcomes of master's nursing curricula, administrators responsible for hiring nurse educators often have no choice but to employ clinical experts as teachers. This article reports on the results of an investigation designed to ascertain the self-reported learning needs of nurse educators in a southern state. This investigation confirmed that nurse educators have numerous learning needs specific to fulfillment of the nurse educator role. Findings suggest that learning needs differed according to the educator's level of academic preparation and type of employment setting. The results provide useful insights for individuals planning continuing educational offerings for nurse educators with varied backgrounds. PMID- 1730825 TI - The professional issues forum for primary nurses: a method for professional development. AB - This article describes a formal mechanism for facilitating a theory-based practice through professional development of primary nurses in a large teaching hospital. The use of the theoretical concepts in clinical decision-making by primary nurses is illustrated by a case presentation. The continuing nurse educator's role in planning and conducting the forums is discussed. PMID- 1730826 TI - Field-dependence/independence: considerations in staff development. AB - Clinical nurse preceptors, staff development specialists, and staff nurses are becoming more involved with the clinical learning experiences of new graduates, nursing school students, and the highly mobile, experienced nurse. Assessment of learning styles and the cognitive process is necessary for the implementation of successful orientation programs. Consideration of the bipolar learning style of field-dependence/independence can be useful for preceptors involved with teaching in the clinical setting. This article reviews the development of the concept of field-dependence/independence, identifies associated characteristics, and discusses assessment of learning style. Specific teaching strategies for both field-dependent and field-independent learners are discussed. PMID- 1730827 TI - Evaluation of a critical care nursing internship program. AB - Many hospitals have developed internship programs to assist in the movement of new graduates into the critical care unit. The purpose of this study was to evaluate an existing critical care nurse internship program. Five research questions were developed to guide the evaluation process. Results suggest that the goals of the program were met. Specific weaknesses in preparation of preceptors and knowledge base of interns were identified. High burnout scores were also identified, suggesting that new graduates may find the experience of working in critical care very stressful. Implications for revisions of the existing program and for others involved in similar programs are discussed. PMID- 1730828 TI - Mandatory continuing education: what the research tells us. AB - The development of continuing education in nursing closely parallels that of adult education. The primary models researchers have used in studying nursing have been the paradigms of adult education. In today's society, rapid development of technology and expansion of knowledge have made necessary a frequent update of nursing skills. This need in turn has led to mandatory continuing education (CE). PMID- 1730829 TI - Allergic and immunologic disorders of the eye. PMID- 1730830 TI - Conjunctival hyperresponsiveness to ocular histamine challenge in patients with vernal conjunctivitis. AB - We compared the conjunctival responsiveness to histamine diphosphate in patients with vernal conjunctivitis and in healthy control subjects. Fourteen asymptomatic patients with vernal conjunctivitis and 10 healthy volunteers were challenged in one eye with 10 microliters of increasing doses (0.01, 0.05, 0.5, and 1 mg/ml in phosphate-buffered saline) of histamine diphosphate. The contralateral eye was challenged with the diluent only. Photographs of each eye were taken for evaluation of conjunctival redness by two masked investigators. Both patients with vernal conjunctivitis and control subjects reacted to histamine with a dose dependent conjunctival redness 2 to 5 minutes after ocular challenge. Conjunctival redness was more intense in patients with vernal conjunctivitis than in control subjects after ocular challenge with 0.01 and 0.05 mg/ml of histamine diphosphate solution (p less than 0.05). Moreover, the threshold concentration of histamine diphosphate, extrapolated from each individual dose-response curve, was significantly (p less than 0.02) lower in patients with vernal conjunctivitis than in control subjects. Our findings suggest that patients with vernal conjunctivitis demonstrate conjunctival hyperresponsiveness to a nonspecific challenging agent. Nonspecific conjunctival hyperreactivity, a novel concept in allergic eye disease, may be relevant for a better understanding of the pathogenesis and clinical variability of vernal conjunctivitis. PMID- 1730831 TI - Occupational asthma caused by alpha-amylase inhalation: clinical and immunologic findings and bronchial response patterns. AB - Inhalation of dust from different enzymes can be the cause of occupational asthma in exposed workers. alpha-Amylase, derived from Aspergillus oryzae, is one of these enzymes, although there are few studies in the medical literature that refer to its allergologic properties and to clinical studies in sensitized patients. The results obtained in a study performed in 83 pharmaceutical-industry workers exposed to powdered alpha-amylase are described in this article. The existence of sensitization to this enzyme was demonstrated in 26 of the workers by positive skin tests. Specific IgE values were significantly higher in workers with positive skin tests than in workers with negative skin tests (p less than 0.001). The bronchial provocation test with alpha-amylase was positive in six of the 14 patients challenged, and only immediate bronchial responses were observed; the same type of response was obtained by nasal provocation. One of the workers had a positive response to oral provocation with this enzyme, presenting abdominal, skin, and respiratory symptoms a few minutes after ingestion. Consequently, we consider that the bronchial asthma presented by the workers was due to an immediate-type, IgE-dependent, immunologic mechanism. PMID- 1730832 TI - The distribution of cat and dust mite allergens on wall surfaces. AB - This study was undertaken to determine the distribution of cat and dust mite allergens on wall surfaces and to assess the value of wall-wipe samples as a measure of allergen exposure. Paired samples were collected from 31 homes, 20 homes with cats and 11 homes without, by vacuuming 1 m2 of carpet and by wiping 1 square foot of an adjacent wall. Felis domesticus allergen I (Fel d I) was detected in 30 of 31 settled dust samples (range, not detectable to 832,000 mU/gm; median, 10,250 mU/gm) and in 27 of 31 wall-wipe samples (range, not detectable to 113.7 mU per filter; median, 1.2 mU per filter). There was a significant correlation between Fel d I content in settled dust and wall-wipe samples (rs = 0.73; p less than 0.001). To assess the reproducibility of the wall wipe method, multiple wipe samples were obtained from 20 homes, revealing a mean coefficient of variation of 110%. In contrast to Fel d I, although Dermatophagoides farinae allergen I was detected in 23 of 25 settled dust samples (range, not detectable to 11,888 ng/gm; median, 1178 ng/gm), it was detected in only four of 25 wall-wipe samples. We conclude that cat allergen, but not dust mite allergen, is widely distributed on wall surfaces and that wipe samples provide a simple and effective means of assessing household exposure to cat allergen. PMID- 1730833 TI - Report of the Committee on the Role and Care of Animals in Research. PMID- 1730834 TI - Asthma relieved by aspirin: clinical studies, platelet function, and arachidonate metabolism. PMID- 1730835 TI - Reproducibility of skin prick test reactions to common allergens in patients with atopic eczema. PMID- 1730836 TI - Airway hyperresponsiveness and the clinical diagnosis of asthma: histamine or history? PMID- 1730837 TI - Sensitivity and specificity of histamine PC20 determination in a random selection of young college students. AB - Histamine provocative concentration causing a 20% drop in FEV1 (PC20) was measured in 500, randomly selected, young (20 to 29 years) university students. Participation was 500/619, or 81%. In this population, by a research assistant administered questionnaire, we identified 17 subjects with current asthma, 16 with asthma only on allergen exposure, 19 with past (more than 1 year ago) asthma, 158 with rhinitis (77 atopic and 81 nonatopic subjects), and 290 subjects with neither asthma nor rhinitis. Histamine airway hyperresponsiveness (PC20 less than or equal to 8 mg/ml) was observed in 58 subjects and included the 17 subjects with current asthma, 6/16 with asthma only on allergen exposure, 2/19 subjects with previous asthma, 20/158 with rhinitis, and 13/290 subjects with neither asthma nor rhinitis. With "current symptomatic asthma" as the diagnosis and PC20 less than or equal to 8 mg/ml as the positive test, the sensitivity was 100%, the specificity was 93%, and the negative predictive value was 100%; the positive predictive value (for current symptoms of asthma) was only 29%. The strength of this test with a cutoff of 8 mg/ml is the high sensitivity, indicating that a PC20 greater than 8 mg/ml is likely to indicate that current asthma is not present. The weakness is the failure to predict current symptoms of asthma. As the cutoff is lowered, for example, to PC20, 1 mg/ml, the sensitivity falls to less than 50% and the positive predictive value approaches 100%. These data indicate that a PC20 greater than 8 (or 16) mg/ml rules out current asthma in most instances, whereas a PC20 less than 1 mg/ml is almost diagnostic of current asthma. Values between 1 and 8 mg/ml are intermediate in these regards. PMID- 1730838 TI - Anti-IgE autoantibodies mistaken for specific IgG. AB - During isolation of specific IgE by affinity chromatography, we obtained IgG acting as "specific IgG" in dot immunoassay and RAST. Further analysis of this fraction revealed that this specific IgG was indeed an immune complex consisting of IgG anti-IgE autoantibodies and specific IgE that falsely appeared as specific IgG in the immunoassays. After disruption of complexes, more IgE became accessible, resulting in increased counts per minute in the RAST. Thus, anti-IgE autoantibodies may hinder determination of specific IgE in in vitro immunoassays, since they hide specific IgE immunologically. To demonstrate the isotope specificity of such immune complex-derived IgG, the purified anti-IgE autoantibodies were radiolabeled and used as detection antibodies in immunoblotting. These autoantibodies recognized different IgE preparations, including a recombinant IgE peptide. PMID- 1730839 TI - Atopic dermatitis of the face, scalp, and neck: type I reaction to the yeast Pityrosporum ovale? AB - We have previously reported that a lipophilic yeast, Pityrosporum ovale (P. ovale) produced a high frequency of positive skin prick tests and in vitro histamine-release (HR) tests in patients suffering from atopic dermatitis (AD) of the face, scalp, and neck. In the present study, our aim was to confirm the involvement of P. ovale-specific IgE and to produce a standardized extract for diagnostic tests; 7/20 sera from patients with a positive HR test were positive in RAST. Several IgE-binding proteins could be detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Comparison of different extraction methods demonstrated that allergens were not released from P. ovale until after mechanical destruction of the yeast cells. Extraction of cultured P. ovale, obtained from the skin of various individuals suffering from AD of the face, scalp, and neck, resulted in very heterogenous patterns of constituents in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis. Nevertheless, all extracts were recognized by patients' IgE. P. ovale may express varying protein patterns, depending on either source or stage of differentiation. Thus, at present, skin and HR tests may be superior to other available assays for diagnosing type I reactions to P. ovale in AD of the face, neck, and scalp. PMID- 1730840 TI - Skin eosinophilia in patients with allergic and nonallergic asthma and atopic dermatitis. AB - Skin eosinophilia induced by grass pollen or platelet-activating factor (PAF) was investigated in patients with allergic and nonallergic asthma and atopic dermatitis (AD) and in normal control subjects. Intracutaneous injection of PAF resulted in positive immediate skin reactions in all tested individuals. Since most of the patients with allergic and nonallergic asthma and eczema under investigation were characterized by a peripheral blood eosinophilia, we investigated whether PAF should be capable of mobilizing blood eosinophils to the skin of patients with nonallergic asthma and AD using the Rebuck skin window technique. Our results demonstrated that, in allergic individuals, allergen induced a much stronger skin eosinophilia than PAF and in a larger percentage of the patients (77% versus 45%). There was no correlation between skin eosinophilia induced by PAF and the occurrence of late skin reactions to allergen. Furthermore, the PAF-induced and the allergen-induced skin eosinophilia differed histologically. In nonallergic individuals, neither allergen nor PAF was capable of mobilizing eosinophils in the skin window, in spite of the presence of peripheral blood eosinophilia. Our results indicate that the allergen- and PAF induced skin eosinophilia are different and that in patients with nonallergic asthma and AD, another chemoattractant than PAF must be responsible for eosinophil mobilization in the target organ. PMID- 1730841 TI - Exercise-induced asthma is not associated with mast cell activation or airway inflammation. AB - Exercise-induced asthma (EIA) may affect up to 90% of patients with asthma. Hyperpnea associated with exercise leads to increased airway water and heat loss, which contributes to the development of EIA. Measurement of circulating mediators has suggested that mast cells may participate in the development of EIA via release of histamine and neutrophil chemotactic factor. To evaluate further the contribution of pulmonary mast cell-mediator release in the pathogenesis of EIA and to determine whether EIA is associated with enhancement of airway inflammation, we studied 11 subjects with mild stable asthma (FEV1, 93% +/- 3% predicted; mean +/- SEM) with significant EIA (after exercise fall in FEV1, 41% +/- 5%). Bronchoalveolar lavage (BAL) was performed immediately (less than 1 hour) after exercise challenge (EC) and repeated 24 hours later (exercise studies). On another occasion, paired BALs were done 24 hours apart (control studies). A minimum of 2 weeks separated the exercise and control pairs. No changes were observed in BAL cell counts, differentials, or reactive oxygen species metabolism after EC. Neither BAL histamine nor BAL tryptase levels increased, either shortly (less than 1 hour) or 24 hours after EC. We conclude that EC in subjects with asthma is not associated with cellular influx to airspace and that mechanisms other than histamine release by pulmonary mast cells may be responsible for EIA. PMID- 1730842 TI - High-frequency binding of IgE to the Der p allergen expressed in yeast. AB - The production of allergens from cDNA clones will provide a clonally pure source of material for experimental and perhaps clinical studies. Attempts to produce the major mite allergen, Der p I, in a highly antigenic form in bacteria have, to date, had limited success. In this study, a high level of production of Der p I from a Cup1 gene cassette from pYELC5-13T in Saccharomyces cerevisiae is described. Although the protein was insoluble, it could be readily solubilized in a urea solution and remained in solution when it was returned to more physiologic buffers. An amount equivalent to about 1 mg/L of yeast culture could then be isolated by affinity chromatography with an immobilized monoclonal antibody. This product reacted strongly with IgE in 9/11 sera from mite-allergic patients compared to the 50% reactivity achieved for Der p I previously produced as a fusion by bacteria. Similarly, the intensity of binding and ability to absorb out Der p I specificities were much greater for the yeast, pYELC5-13T, product. Studies with monoclonal antibodies also demonstrated the yeast, Der p I, had a high degree of antigenicity, although clear differences with the native allergen were demonstrated. The high frequency of reactivity with IgE of the pYELC5-13T formally demonstrates that a single gene product of Der p I is a major allergen and demonstrates that even for Der p I, which is synthesized from a proenzyme, considerable antigenicity can be obtained by expressing the mature protein. PMID- 1730843 TI - The American Academy of Allergy and Immunology, 48th annual meeting. Orlando, Florida, March 6-11, 1992. PMID- 1730844 TI - Changes in HS mouse erythrocyte mass and area with respect to donor age. AB - Interferometric photographs of blood smears from heterogeneous stock (HS) mice were analyzed for age-related changes in erythrocyte mass using quantitative cytophotometry. Blood smears were obtained from 96 mice (half male/half female) sacrificed at 172, 272, 374, 482, 581, and 664 days of age as part of a larger biomarkers of aging study. Blood smears from each animal were photographed with a Leitz Mach-Zehnder interferometer, and the erythrocyte images on these negatives were measured for dry mass using a Vickers M85a microdensitometer. Cell area measurements were made from a video-microscope image traced with a computer digitizer. Results indicated that animals from middle-age groups (272, 374, and 482 days) have higher red blood cell mass than animals at either young (172 days) or older (581 and 664 days) age groups. Mass changes with respect to age followed similar trends when data were further examined with regard to sex of the animals. Erythrocyte area measurements showed a general age-related decrease in cell size. PMID- 1730845 TI - Decreased gray matter in normal aging: an in vivo magnetic resonance study. AB - Using in vivo magnetic resonance (MR) images, the percentages of gray matter, white matter, and cerebrospinal fluid (CSF) in the brains of 8 young and 6 elderly normal male community volunteers were quantified. Compared with the young men, the elderly men had a significantly lower percentage of gray matter (p less than .01) and higher percentage of CSF (p less than .01). The percentage of white matter was not significantly different between the two groups. This finding suggests that the age-related decrease in brain tissue is chiefly due to loss of gray matter. PMID- 1730846 TI - Effect of dietary restriction on hepatic vitamin A content in aging rats. AB - The size and number of fat-storing cells (FSC), considered to be the main liver storage site of vitamin A, as well as hepatic vitamin A content, were studied in aging female Sprague-Dawley rats subjected soon after weaning to dietary restriction (R), that is, 60% of food consumed by their ad libitum-fed controls (A). In A or R rats, the FSC index (number of cells per 1000 hepatocytes) and volume density (% of hepatic volume) were increased significantly at 24-27 months compared with the younger age groups. The lipocyte index and volume density were also found to be significantly higher, after the first year, in R rats when compared to corresponding age-matched A controls. An increase in total vitamin A content was also noted with age in both groups. R rats exhibited higher retinol, retinyl ester, and total retinoid content than their corresponding controls, but the differences were statistically significant only at 12-14 and 24-27 months. These results indicate that, during aging, dietary restriction markedly increases vitamin A content in liver tissue, a change that may be relevant to the beneficial effect of this dietary manipulation on liver function. PMID- 1730847 TI - Plasma concentrations of glucose, insulin, and percent glycosylated hemoglobin are unaltered by food restriction in rhesus and squirrel monkeys. AB - Plasma concentrations of glucose and percentage of glycosylated hemoglobin have been reported to be reduced in food-restricted rats when compared with ad libitum fed controls (Masoro et al., 1989). A similar experiment in primates, in which we also measured plasma insulin, is reported in this article. Rhesus and squirrel monkeys were fed either ad libitum or 30% less than weight-matched controls for up to 36 months. No significant age or diet effects on plasma concentration of glucose, insulin, or percentage glycosylated hemoglobin were observed, except that glucose concentration decreased with age in ad libitum-fed rhesus monkeys. Moreover, no correlations between glucose levels and the percentage of glycosylated hemoglobin could be established within any group of animals. These results suggest possible differences in glucose-related metabolism in ad libitum and food-restricted primates as compared to observations made in rats. PMID- 1730848 TI - Age and sex effects on mobility of the human ankle. AB - We investigated whether values for passive resistive torque, dorsiflexion strength, and active mobility of the ankle joint varied with age in a randomly selected sample of middle-aged and elderly males and females. The main effects of sex and age group were significant, without an interaction effect, on values for passive resistive torque and voluntary strength. The trends indicated that females had lower values for both variables and that passive torque increased and strength decreased with respect to age. Dorsiflexion range of motion also showed a highly significant trend to decrease across age groups, and more so for females than males. Mean values for middle-aged men vs old men went from 20.0 to 13.5 degrees, while corresponding values for women decreased from 20.7 degrees of dorsiflexion to 10.1. Functional ankle movement thus becomes limited with age, but could be improved by strengthening weak dorsiflexor muscles. PMID- 1730849 TI - Beta adrenergically mediated cardiac chronotropic and vascular smooth muscle responses during propranolol therapy and withdrawal in young and elderly persons. AB - Aging is associated with diminished beta-adrenergic responsiveness in a variety of tissues. Desensitization of tissues secondary to the age-associated increase in sympathetic nervous system activity has been proposed as a potential explanation for diminished beta-adrenergic responsiveness. If this hypothesis is correct, then beta-blockade in older people might be expected to reverse the blunted beta-adrenergic responses of tissues having diminished responsiveness. Cardiac chronotropic responses to bolus isoproterenol (ISO) doses and ISO-induced venous smooth muscle dilatation in superficial hand veins were examined in 8 young (26.2 +/- 2.6 yrs) and 9 elderly (68.0 +/- 2.2 yrs) healthy subjects before, during, and 3 and 7 days following 2 weeks of treatment with propranolol. Baseline cardiac chronotropic responsiveness, CD25, was 1.55 +/- 0.77 mcg in the young and 5.97 +/- 2.77 mcg in the elderly subjects (p less than .01), increasing to 84 +/- 56 mcg and 194 +/- 172 mcg during treatment with propranolol. At 3 and 7 days following withdrawal of propranolol, CD25s were respectively 1.24 +/- 0.79 (p = .14) and 1.10 +/- 0.42 (p = .04) in the young and 5.63 +/- 2.34 (p = .31) and 4.85 +/- 2.37 (p = .05) in the elderly subjects. In contrast, there was no decrease in the ED50 or increase in Emax for ISO-induced venodilation of hand veins following propranolol withdrawal. These results demonstrate that both young and elderly subjects have similar increases in cardiac chronotropic responsiveness following discontinuation of beta-blockade and do not support the concept that desensitization is responsible for the diminished beta-adrenergic responsiveness seen with aging. PMID- 1730850 TI - The effect of influenza vaccination on IL2 production in healthy elderly: implications for current vaccination practices. AB - Age-related senescence of T-cell mediated responses is well recognized. This study was designed to determine how aging affects the T-cell mediated Interleukin 2 (IL2) response to influenza vaccination. A group of healthy elderly individuals were compared to a control group of healthy young adults for their response to the 1990 influenza vaccine. Cultures of peripheral blood mononuclear cells (PBMC) were prepared from venous blood samples taken prevaccination (pre) and 8 and 12 weeks post-vaccination (post). PBMC cultures stimulated with inactivated A/Shanghai/16/89 (contained in the 1990 vaccine) and A/Philippine/2/82 (not contained in the vaccine) were assayed for peak IL2 activity. We find that after influenza vaccination, there was an insignificant increase in IL2 activity when PBMC from the young control group were stimulated with A/Shanghai/16/89 (pre, 5.14 U/mL/10(6) PBMC; post, 6.64 U/mL/10(6) PBMC) but there was a significant increase in IL2 activity when stimulated with A/Phillippine/2/82 (pre, 1.5 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). In similar cultures of PBMC from the elderly group, there was a significant increase in IL2 response to both A/Shanghai/16/89 (pre, 1.6 U/mL/10(6) PBMC; post, 3.5 U/mL/10(6) PBMC) and A/Philippine/2/82 (pre, 0.86 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). Measurements of CD4+/CD8+ populations were not affected by vaccination and were not significantly different in the two groups. Subgroup analysis of the elderly group revealed that previous influenza vaccination in 1989 did not significantly affect IL2 levels measured in the present study. This study shows that in healthy elderly, influenza vaccination effectively restores IL2 activity to normal. There appears to be an age-related decrease in the duration of T-cell memory.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730851 TI - The Nursing Home Behavior Problem Scale. AB - Nursing home patients frequently have serious disturbances of behavior that can lead to use of chemical or physical restraints. To support research into better management of these problems, we developed the Nursing Home Behavior Problem Scale (NHBPS), a 29-item inventory of serious behavior problems designed to be completed by nurses and nursing assistants. NHBPS scores were obtained for two samples of nursing home residents: 431 in Tennessee and 122 in Texas. The interrater correlation was .754 in the Tennessee sample and .827 in the Texas sample. The NHBPS had a correlation of -.747 with the NOSIE scale and .911 with the CMAI. There was a pronounced association of increased NHBPS scores with mental impairment and use of sedative drugs or restraints. These data suggest the NHBPS is a useful research instrument for measuring serious behavior problems in nursing home residents. PMID- 1730852 TI - Salient life events in three-generation families. AB - A short, simple procedure for eliciting salient life events is reported in this article. Respondents from three-generation families were asked to list events that had had an impact on them and their families. Responses were examined for content, criterion, and construct validity. The 10 events listed by 10% of at least one generation were major life cycle markers: Six (marriage, childbirth, divorce, retirement, widowhood, and ill health) pertained to the individuals, and four (marriage, birth, divorce, and ill health) pertained to their extended family networks. The expected relationship between events and depression (CES-D) was observed: The importance of low frequency events was reflected in their relationship to depression in the middle-aged and youngest generations. The importance of network events was reflected both in their presence on the lists of all age groups, and in their relationship to depression in the youngest generation. PMID- 1730853 TI - Social provisions in adulthood: concept and measurement in close relationships. AB - Social gerontologists are increasingly concerned about examining the nature of close relationships among the elderly. Theoretically grounded and empirically validated instruments are needed to advance research in this area. We analyzed the psychometric properties of the Social Provisions Scale using data from a probability sample of 494 community residents aged 65 or older. The theoretical foundation of this scale is Weiss's delineation of social support functions of close relationships. Confirmatory factor analysis revealed a pattern in the data that corresponds to the theoretical definition of relational provisions: Factor 1 was Intimacy; Factor 2 was Social Integration; Factor 3 was Reassurance of Worth; and Factor 4 was Opportunity for Nurturance. Alphas for the four scales ranged from .83 to .94. Convergent validity was supported by significant correlations between the Social Provisions Scale (SPS) and respondent's morale, frequency of contact with friends, feelings of closeness with an adult child, relationship control, and relationship conflict. Discriminant validity was supported by nonsignificant correlations between the SPS and the Eysenck Lie Scale. PMID- 1730854 TI - Coping strategies of older women with hip fractures: resources and outcomes. AB - This study examined the strategies used by 101 older women, aged 65 to 94, to cope with their hip fractures. During an interview, the women described their health, income, ability to perform tasks of daily living, cognitive functioning, locus of control, levels of depression, informal network, and use of coping strategies. Results indicate that the women used a variety of different coping strategies, with "seeking social support" being the most frequent. A strong belief in external control was the only resource predictive of the coping strategies employed. The use of several emotional-focused strategies was associated with poorer functional recovery. PMID- 1730855 TI - Vision, aging, and driving: the problems of older drivers. AB - Although there are well-recognized declines in visual functioning with age, their contribution to the problems of older persons on tasks in the natural environment, including driving, are largely unknown. Adults ranging in age from 22-92 years were surveyed in regard to their visual difficulties when driving and performing everyday tasks. The visual problems of drivers increased with age along five different visual dimensions: unexpected vehicles, vehicle speed, dim displays, windshield problems, and sign reading. Several of the age-related visual problems that were reported appear to be related to the types of automobile accidents more common among older drivers. The study also replicated the findings from an earlier investigation of non-driving tasks that showed visual declines with age on five dimensions: visual processing speed, light sensitivity, dynamic vision, near vision and visual search. These findings indicate promising areas of research regarding the effects of visual aging on tasks in the natural environment. PMID- 1730856 TI - Depression and mortality among institutionalized aged. AB - This study examined the association between depression and mortality among a group of nursing home and congregate apartment residents (initial n = 898) over a 30-month period. Baseline [Time 1 (T1)] and 1-year follow-up [Time 2 (T2)] assessments yielded research-based diagnoses of possible major, minor, or no depression, along with measures of functional disability, cognitive status, and physician-rated health. Event history analyses were used to assess differential mortality as a function of level of depression after T1 and of change in depressive status from T1 to T2. Significant effects for T1 depression at 6, 12, and 18 months after the interview reflected an increased death rate among possible major depressives as compared with other respondents. An effect of change in depressive status from T1 to T2 appeared to be caused by long-term negative effects of T1 depression. Finally, none of the observed associations remained significant when controlled for effects of physical health, functional disability, and cognitive status. Thus, the effects of depression on mortality among this sample appeared to be attributable strictly to the correlation of depression with ill health. However, cautious interpretation is recommended inasmuch as causal paths between depression, ill health, and death remain unclear. PMID- 1730857 TI - Aging and spatial acuity of touch. AB - Spatial acuity of the skin of the fingertip deteriorates markedly with age, as assessed by 2-point thresholds measured with a forced-choice method in 80 subjects between the ages of 18 and 91 years. This loss of "minimum separable" acuity appears to be pervasive, demonstrably affecting many middle-aged and a great majority of the elderly persons tested. Mean threshold was 1.95 mm for young (18-33 years), 2.68 mm for middle-age (41-63 years), and 5.03 mm for elderly (66-91 years) subjects. Over half of the variance in 2-point threshold was attributable to age (r = .74). Threshold on the arm, measured in subsets of 15 young and 15 elderly, was also age-dependent, but (reckoned as a percentage) the difference is minor compared to the finger. Analysis implies that the perception of tactile patterns, in general, and tactile reading (braille, Optacon) by the sensory handicapped, in particular, could run into age-related difficulty by reason of impairment of spatial acuity at the most basic level. PMID- 1730858 TI - Beliefs about memory changes across the adult life span. AB - Beliefs about age-related differences in memory were examined with an adaptation of the Short Inventory of Memory Experiences. In Experiment 1, 142 adults (mean age = 36 years) reported significantly more positive expectations for memory in everyday life for persons aged 25 years than for those aged 70 years. In Experiment 2, a between-subjects design with 189 adults (mean age = 34 years) was employed to examine the generality of memory beliefs about age-related change and the anticipated slope. Beliefs about the memory of 25-year-olds were significantly more positive than for 45- and 65-year-olds, which were correspondingly higher than for 85-year-olds. Secondary regression analyses revealed that participants with good memory self-perceptions anticipated better memory performance for others overall. In addition, older respondents exhibited more differentiated memory beliefs across age groups than younger respondents, especially at the two younger target ages. Examination of age-based memory beliefs with this type of instrument provides a new opportunity to integrate cognitive and social psychological approaches to the study of memory in aging. PMID- 1730859 TI - Age and ideal chunk size. AB - We tested young, middle-aged, and older adults for ability to organize six-letter sequences when these subjects were not externally induced to chunk the letter sets. In order to determine whether subjects would use optimal chunk sizes of two, three, or six letters, a serial recall task on which subjects were cued using digits was employed. From these data, the total number of correctly recalled sequences was computed, in addition to global and stop transitional error probabilities (TEPs). The results indicated that older adults recalled fewer correct sequences than did the young adults. However, both the global and stop TEP analyses demonstrated that all three age groups were chunking the six letter sequences into two sets of three letters each. The present study (along with Allen & Coyne, 1988a, 1989) suggests that there are no appreciable age differences in functional chunk capacity, but that older adults exhibit poorer serial recall than younger adults. PMID- 1730860 TI - Schema-based retrieval processes in young and older adults. AB - Through two experiments we examined the extent to which the memory performance of different-aged adults was dependent upon the conceptual relationship between the to-be-remembered information and the perspective adopted at retrieval. In both studies, young and old adults read a story from one of two perspectives. They then recalled the story twice: first from the perspective taken at reading and then again from the alternative perspective. It was found that memory performance in both age groups was related to the relevance of the story information to the recall perspective. In addition, the interaction between recall perspective and the relevance of the story units was similar across age groups. This suggests that young and older adults are similarly dependent upon schema-based relations in aiding recall, at least when the memory task has a heavy emphasis on conceptually driven retrieval processes. PMID- 1730861 TI - Retirement reason versus retirement process: examining the reasons for retirement typology. AB - Studies have often used reason for retirement as an indicator of the pathway leading to retirement. We discuss the conceptual basis for the retirement-reason typology and evaluate the distinctiveness of various reasons for labor force exit by predicting them in a standard model-based analysis. Data are from the 1982 Social Security New Beneficiary Study, and the analysis is limited to men. A number of factors in the model-based analysis have distinctive effects on exit for particular retirement reasons, but health limits increase the likelihood of all types of retirement. We conclude that reasons for retirement only partially capture distinctive retirement processes. PMID- 1730862 TI - Future caregivers: projected family structures of older persons. AB - Elderly persons depend upon spouses and children for emotional, physical, and financial support. In particular, the use of institutional long-term care has been shown to vary by family status. Changes in past and future levels of mortality, fertility, marriage, and divorce will influence the probability that elderly persons of the future have surviving spouses and children. This research uses multiple decrement life tables and component projection methods to project the future family status of elderly persons until the year 2020. The high level of fertility among women during the 1950s will result in greater proportions of future elderly persons having surviving children. Declines in mortality, coupled with increases in rates of marriage, increase the probability that both men and women will have spouses surviving in their old age. PMID- 1730863 TI - How changes in components of growth affect the population aging of states. AB - We analyzed the demographic determinants of population aging for states of the United States between 1950 and 1980. Using the factorial projections method, we estimated the effects of population momentum and changes in mortality, fertility, and migration on changes in the proportion of persons age 65+. Declining mortality rates caused the population to age in virtually every state in every decade between 1950 and 1980, but the effects were very small. The effects of changes in fertility rates were considerably greater. Population momentum generally had a greater effect on population aging than changes in either fertility or mortality rates. Net migration was by far the most volatile component of population aging, both in terms of changes over time and state-to state differences at a given point in time. We expect this trend to continue in coming decades. PMID- 1730864 TI - Regional differences in the characteristics of elderly return migrants. AB - We examined return migrants age 60+ and argue from a regional analysis of their population characteristics that they fall into two primary types of movers: provincial return migrants and counterstream return migrants. When profiled as a whole, using 1980 census microdata, return migrants are older and more residentially dependent than nonreturn migrants. However, when regional variations are considered, this generalization breaks down. Perhaps return to one's state of birth is overemphasized in discussions of counterstream migration. Provincial return migration seems strongest in the South, with an interesting racial twist, and counterstream return migration seems strongest in the Northeast. Conceivably it is not a return to one's state of birth that is at issue among counterstream migrants, but rather a return from a Sunbelt retirement move to an earlier place of residence, regardless of whether one was born there. PMID- 1730865 TI - Widowhood, health status, and the use of health services by older adults: a cross sectional and prospective approach. AB - Using data from the LSOA, we examined the relationship between widowhood, health status, and the use of health services. Controlling for the characteristics specified in the behavioral model, a cross-sectional assessment of the 2,354 respondents widowed at baseline showed that regardless of how the recency of widowhood is modeled, it is not related to any measure of health status, and it is only marginally associated with two measures of health services utilization: nursing home placement and death. A prospective assessment of the 4,113 respondents reinterviewed at follow-up produced similar results. Becoming widowed did not alter previous reports of health status, nor prior patterns of physician or hospital utilization. Being widowed did, however, significantly increase the likelihood of being placed in a nursing home. For the 14 respondents who were both widowed and placed in a nursing home after baseline, the sequence is always widowhood first, and then nursing home placement. PMID- 1730866 TI - Tyrosine protein kinase is involved in anti-IgM-mediated signaling in BAL17 B lymphoma cells. AB - BAL17 B lymphoma cells, representing mature B lymphocytes, were used to analyze the role of tyrosine kinase in B cell activation. Anti-IgM-induced tyrosine phosphorylation was inhibited by preincubation of cells with tyrosine kinase inhibitor herbimycin A. Enzymatic activity of lyn protein was also inhibited by this drug, accompanied by down-regulation of p53lyn and p56lyn. However, a protein kinase C-mediated event was intact in the herbimycin A-pretreated cells, suggesting that the inhibitor acts selectively on tyrosine kinase. Anti-IgM failed to stimulate herbimycin A-pretreated cells to induce increases in inositol phospholipid metabolism or increased [Ca2+]i, whereas aluminum fluoride-induced metabolism was not altered. Moreover, membrane IgM density as revealed by flow cytometry was not changed by herbimycin A. These results indicate that tyrosine kinase(s) participates in the coupling of an Ag receptor cross-linkage to phospholipase C activation through a phosphorylation event in B lymphoma cells. PMID- 1730867 TI - Regulation of expression of the leukocyte integrin CD11a (LFA-1) molecule during differentiation of HL-60 cells along the monocyte/macrophage pathway. AB - The CD11a/CD18 (LFA-1) leukocyte integrin receptor mediates homotypic and heterotypic leukocyte adhesion by binding to one of two defined ligands, ICAM-1 or 2, on the conjugate cell. In this study we investigated the molecular regulation of expression of the CD11a subunit during myeloid differentiation of HL-60 cells. Induction of monocyte/macrophage differentiation of HL-60 promyelocytic leukemia cells with PMA results in an increase in CD11a surface Ag expression and the acquisition of CD11a/CD18-mediated homotypic adherence. These changes are accompanied by a 40-fold increase in CD11a mRNA levels. Nuclear run on transcription assays indicate that the increase in CD11a mRNA in PMA-induced HL-60 cells is not caused by an increase in CD11a RNA transcription. We assessed the posttranscriptional regulation of CD11a using two methods. By using actinomycin D to block RNA transcription, we demonstrate that the CD11a mRNA half life in HL-60 cells is prolonged after PMA treatment. Inhibition of protein synthesis with cycloheximide also results in enhanced expression of CD11a mRNA in HL-60 cells without increasing CD11a transcription. These findings indicate that, in HL-60 cells induced with PMA to differentiate along the monocyte/macrophage pathway, CD11a expression is regulated primarily at the posttranscriptional level by a labile protein. Identification of the specific CD11a RNA sequences, and the proteins that bind to these sequences may provide insight into lineage commitment during human monocyte/macrophage differentiation. PMID- 1730868 TI - In vivo T cell activation, in vitro defective IL-2 secretion, and response to influenza vaccination in elderly women. AB - IL-2 secretion in response to mitogenic stimulation, assayed in vitro, is significantly reduced in circulating T lymphocytes isolated from healthy old people, but the significance of this abnormality and how it relates to in vivo IL 2 secretion remain unclear. We found that IL-2 secretion in response to PHA plus PMA by peripheral blood T cells isolated from 10 out of 32 (31%) healthy old individuals (mean age 86 yr, range 74-97) was significantly decreased compared with results obtained in 23 younger individuals (mean age 34 yr, range 23-46). This IL-2 secretion defect in vitro was reversible after a 3-day incubation in the absence of activators. The 10 healthy old individuals who had defective IL-2 secretion in vitro also showed increased levels of serum IL-2. T cells from 22 healthy old and 22 young individuals, who had normal IL-2 secretion (geometric mean +/- log of 1 SD: 139 +/- 0.3 U/ml and 212 +/- 0.31 U/ml, respectively) in vitro, showed a remarkable transient T cell defect in IL-2 secretion (15 +/- 0.47 U/ml for the old, 54 +/- 0.28 U/ml for the young) 15 days after influenza vaccination. IL-2 secretion became normal again 30 days after vaccination. The T cell-IL-2 activity, expressed as a T cell-IL-2 activity score (calculated as the logarithm of the serum IL-2 U/ml divided by the logarithm of the IL-2 secretion U/ml, in vitro) was significantly increased in elderly non-responders after influenza vaccination (mean +/- 1 SD: 1.4 +/- 0.51) compared with elderly (0.44 +/- 0.13) and younger responders (0.3 +/- 0.2). Our data suggest that in vitro defective IL-2 secretion is a consequence of T cell activation which seems to occur in a significant proportion of healthy elderly individuals and may be clinically relevant inasmuch as it appears to prevent the normal vaccine-induced antibody response. PMID- 1730869 TI - Glycosylation-inhibiting factor from human T cell hybridomas constructed from peripheral blood lymphocytes of a bee venom-sensitive allergic patient. AB - Human T cell hybridomas, which constitutively secrete glycosylation inhibiting factor (GIF), were constructed from PBL of an allergic individual who was sensitive to honey bee venom. PBMC of the patient were stimulated with either denatured or cyanogen bromide-treated bee venom phospholipase A2 (PLA2), and Ag activated cells were propagated by IL-2 in the presence of human recombinant lipocortin I. T cells obtained in the cultures were fused with a HAT-sensitive mutant of the human lymphoblastoid cell line CEM. Approximately one-third of hybridoma clones constitutively secreted GIF. The GIF-producing hybridomas were CD3+ and bore TCR-alpha beta. GIF formed by unstimulated hybridomas lacked affinity for bee venom PLA2. Upon cross-linking of CD3, however, a majority of the GIF-producing hybridomas formed IgE-binding factors and GIF, the latter of which had affinity for bee venom PLA2. Both nonspecific GIF and Ag-binding GIF from the hybridomas bound to an immunosorbent coupled with the anti-lipomodulin mAb 141-B9. Using an affinity-purified GIF as an immunogen, we established mouse B cell hybridomas that secreted monoclonal anti-human GIF. In order to characterize human nonspecific GIF, one of the GIF-producing hybridomas was adapted to a serum-free medium, and culture supernatant was fractionated by DEAE Sepharose column chromatography and by gel filtration. The majority of nonspecific GIF in the culture supernatant was recovered from DEAE-Sepharose by elution of the column with 10 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl. Affinity-purification of GIF in the DEAE Sepharose fraction by using anti-GIF coupled Affigel, and analysis of the purified GIF by SDS-PAGE revealed that human GIF is a single polypeptide chain of 14 to 15 kDa. Gel filtration of both crude and affinity-purified GIF preparations confirmed the molecular size of the cytokine. PMID- 1730870 TI - Murine recombinant IL-4 is a bifunctional regulator of macrophage growth induced by colony-stimulating factors. AB - Murine peritoneal exudate macrophage (PEM) coexpress receptors for both granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) and can be induced by both factors, either alone or in combination, to undergo extensive proliferation in vitro. In this study the effect of murine rIL-4 (MurIL-4) on the proliferation of PEM was examined. MurIL-4 alone did not support macrophage proliferation but prolonged their survival in vitro. When MurIL-4 was combined with human (Hu)rM-CSF, it enhanced the proliferative response of PEM to rHuM-CSF in a dose-dependent manner, reaching a maximum at approximately 10 ng/ml. Contrarily, MurIL-4 suppressed the proliferative response of PEM to MurGM-CSF. Receptor binding assays using radiolabeled ligands showed that MurIL-4 selectively enhanced the expression of M-CSF receptors; suggesting that at least part of the synergistic effect of MurIL-4 is mediated at the receptor level. Of relevance to this effect is the finding that MurIL-4 greatly promoted the responsiveness of PEM to low concentrations of HurM-CSF. Unlike M-CSF receptors, however, MurIL-4 treatment failed to modulate the levels of GM-CSF receptors in PEM. The proliferative responses of PEM to both MurGM-CSF and HurM-CSF could be inhibited by MurIFN-gamma with similar sensitivity. This inhibitory effect of MurIFN-gamma was partially neutralized by MurIL-4 in cultures containing HurM-CSF but not those containing MurGM-CSF. This study demonstrates that IL-4 is involved directly in the regulation of macrophage production by modulating their responsiveness to various cytokines. PMID- 1730871 TI - Release of a transforming growth factor (TGF)-beta 2-related suppressor factor from postimplantation murine decidual tissue can be correlated with the detection of a subpopulation of cells containing RNA for TGF-beta 2. AB - Postimplantation murine decidual tissue from allopregnant C3H mice has been shown to release in vitro a potent immunosuppressive factor closely related to transforming growth factor (TGF)-beta 2 but slightly lower in apparent molecular weight. Decidual suppressor factor (DSF) activity was first detected in decidual tissue supernatant at day 9.5 of gestation and reached a plateau by day 10.5 to 12.5. By Northern analysis of decidual and placental tissue with a simian TGF beta 2 probe, two characteristic TGF-beta 2 mRNA transcripts were detected in decidual tissue. In situ hybridization analysis of C3H implant sites, with the simian (pGEM-G1G2) TGF-beta 2 riboprobe, revealed a small population of TGF-beta 2+ cells localized to postimplantation decidua basalis and metrial gland cell area after day 8.5. On and before day 8.5, when DSF was not detectable, few TGF beta 2 mRNA+ cells were detected. To test for TGF-beta release in situ, sections of uterine tissue were stained with antibody specific for TGF-beta 2, that identified DSF in Western blots. In postimplantation tissues (day 9.5, 12.5) patchy anti-TGF-beta 2 staining was seen over decidual tissue. Before day 9.5, slight and diffuse staining over decidual tissue was present with more marked staining of extradecidual tissue. Very little staining was noted over day 9.5 decidual tissue by using anti-TGF-beta 1 antibody as a control; however, some staining was seen over postimplantation fetal trophoblast and myometrial tissue. Fractionation of disaggregated postimplantation decidua by velocity sedimentation revealed that TGF-beta 2 mRNA+ cells were predominantly small and sedimented in the same fraction(s) as those cells previously shown to release DSF in vitro. Thus, the release of TGF-beta 2 related DSF correlates with the in situ detection of TGF-beta 2 mRNA and the in situ release of TGF-beta 2 peptide. These studies suggest that DSF may be a form of TGF-beta 2 released by a population of small lymphocytic decidual suppressor cells. PMID- 1730872 TI - IL-2 induces IL-6 production in human monocytes. AB - IL-2 is a potent activator of effector and secretory activities of human monocytes. Since monocytes are an important source of IL-6, we investigated whether IL-2 can induce IL-6 production and whether regulatory circuits can modulate this process. We found that stimulation of monocytes with IL-2 induced expression of IL-6 mRNA and bioactivity in a dose-dependent manner. Production of IL-6 in monocytes can be induced by other cytokines such as IL-1 beta. By using mAb alpha-IL-1 beta we showed that IL-2-induced IL-6 production is not mediated by the autocrine stimulation of IL-1 beta elicited by IL-2. IL-6 induction by monocytes is not a common response to activating signals because IFN-gamma did not induce IL-6 expression under conditions in which it elicits tumoricidal activity. In contrast, IFN-gamma could completely abrogate the induction of IL-6 expression by IL-1 beta but did not affect the levels of mRNA and the secretion of IL-2-elicited IL-6. We have previously reported that transforming growth factor-beta inhibits IL-6 production in response to IL-1 beta. Studies on the inhibitory activity of transforming growth factor-beta demonstrated that this cytokine differs from IFN-gamma because it inhibited both IL-1- and IL-2-induced IL-6 expression. These data demonstrate that, in human monocytes, both IL-1 and IL-2 stimulate IL-6 expression by independent mechanisms that can be dissociated by the susceptibility to the inhibitory effect of IFN-gamma. IL-6 production is also down-regulated by TGF-beta, whose inhibitory activity is stimulus-unrelated. PMID- 1730873 TI - Modulation of interferon consensus sequence binding protein mRNA in murine peritoneal macrophages. Induction by IFN-gamma and down-regulation by IFN-alpha, dexamethasone, and protein kinase inhibitors. AB - IFN play a central role in the activation of macrophages by inducing the expression of several proteins which, in turn, result in increased functional capabilities. Homologous interferon responsive sequences have been found in many IFN-inducible genes, and the gene for a protein that binds these sequences (interferon consensus sequence binding protein, ICSBP) has recently been cloned. In this study, the regulation of ICSBP mRNA induction by IFN-gamma was characterized in murine thioglycolate-elicited peritoneal macrophages. Northern blot analysis revealed two ICSBP mRNA species from these cells. Steady-state levels of both of these species were elevated by IFN-gamma at doses consistent with many IFN-gamma-induced macrophage functional responses. ICSBP mRNA levels increased within 1 h of IFN-gamma treatment, peaked between 4 and 6 h, and subsequently declined to approach baseline levels by approximately 24 h. IFN alpha, at a concentration shown previously to modulate macrophage surface markers and functions, had no effect on ICSBP message levels alone, but antagonized the IFN-gamma-induction of ICSBP mRNA. IFN-gamma-induction of ICSBP mRNA is resistant to cycloheximide but sensitive to protein kinase inhibitors (H7, H8, HA-1004, staurosporine) at doses that suggest that protein kinase C is a likely target. ICSBP mRNA induction is also inhibited by dexamethasone, a synthetic glucocorticoid, well known as an anti-inflammatory drug capable of influencing gene expression in macrophages. The characterization of ICSBP mRNA regulation should help identify functions for this putative IFN trans-acting factor in macrophage activation. PMID- 1730874 TI - IL-10, T lymphocyte inhibitor of human blood cell production of IL-1 and tumor necrosis factor. AB - We have identified and purified a factor that inhibits the production of IL-1 beta and TNF by stimulated human mononuclear cells. The activity is produced by the T cell lines Hut-78 and Mo constitutively under serum-free conditions. Crude conditioned media have titers of up to 100 U/ml (one unit defined as the reciprocal of the dilution producing 50% inhibition). The activity resides mainly in a single size peak of 30 to 35 kDa and an isoelectric point around 8. Other cytokines in this size range that have been reported to be inhibitory for IL-1 and TNF production include TGF-beta, IL-4, and IL-6; these factors were excluded by lack of detection, neutralizing antibody, and low activity compared with our factor. Another factor with these size and charge properties is IL-10, which inhibits T cell cytokine production. By polymerase chain reaction analysis, Mo and HuT-78 lines contain IL-10 transcripts whereas JURKAT is negative; this correlates with inhibitor bioactivity from the three lines. Use of mAb specifically showed the inhibitor to be IL-10. PMID- 1730875 TI - Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance. AB - Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form. PMID- 1730876 TI - Bactericidal activity of C9-deficient human serum. AB - Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes. PMID- 1730877 TI - Effect of pH on MHC class II-peptide interactions. AB - The effect of pH on class II-peptide interactions has been analyzed using several mouse (IAd, IAk, IEd, IEk) and human (DR1, DR5, DR7) MHC specificities, and eight different class II-restricted determinants. In direct binding assays, acidic conditions led to increased binding capacity for many class II-peptide combinations. IE molecules seemed to bind optimally around pH 4.5, whereas IA molecules displayed binding optima in the 5.5 to 6.5 range. In contrast, the DR molecules studied were, in most cases, affected only marginally by pH changes in the 4.5 to 7.0 range. Despite these apparent isotype-specific trends, no general rule could be formulated, because even for the same class II molecules, the binding capacity could be increased for many peptides when the binding was performed under acidic conditions, was unaffected for some, and even decreased for others. The mechanisms responsible for this complex behavior were analyzed in more detail by kinetic and equilibrium analysis of three different class II peptide combinations (IAd/OVA 323-339, IAk/HEL 46-61, and DR1/HA 307-319). It was found that acidic pH conditions could affect both on and off rates for class II peptide complexes. Depending on the net balance of these effects, either increases, decreases, or no effect on overall affinities at equilibrium were detected. In the case of IAd/OVA 323-339, it was also found that acidic conditions influenced the binding capacity of class II molecules by increasing the fraction of sites available for peptide binding, presumably by favoring dissociation of endogenously bound, acid-sensitive peptides. PMID- 1730879 TI - Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contribution of PAF releasing factor in serum. AB - The behavior of platelet-activating factor (PAF) produced in stimulated human polymorphonuclear leukocytes (PMN) was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase (PAF-AH) deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treated with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellular PAF was similar to that of intracellular PAF produced in stimulated PMN in the presence of PAF-AH-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As these data suggested the occurrence of PAF-releasing factor(s) in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 240 kDa, which was different from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10(-9) M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s) releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators. PMID- 1730878 TI - Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages. AB - IgE-binding protein (epsilon BP) is a galactoside-specific lectin containing an S type carbohydrate-recognition domain. It was originally identified in rat basophilic leukemia cells and is now known to be identical to a macrophage surface Ag, Mac-2, and lectins designated as CBP 35/L-34/RL-29. It has also been related to a nonintegrin laminin-binding protein isolated from mouse macrophages. In this report we have shown the following: epsilon BP is present in variable amounts in several mast cell lines, and the surface expression of epsilon BP in these cell lines is quite variable and does not correlate with the total amount of epsilon BP in the cell. epsilon BP is displayed on the cell surface in a manner that is reversible by lactose, most likely through attachment to yet unidentified glycoconjugates. The putative epsilon BP binding sites on the cell surface can be readily demonstrated by using radiolabeled epsilon BP, and the sites are present in comparable amounts in various cell lines. Expression of epsilon BP on the cell surface can be regulated; the most notable example is the upregulation of surface epsilon BP on RBL cells activated through the high affinity IgE receptor by IgE immune complexes. Cell-surface epsilon BP is functional as measured by its ability to promote adhesion of trypsinized rabbit erythrocytes to mast cells and macrophages. On the basis of these results and reported properties of related lectins, we propose that the lectin represented by epsilon BP is a new class of cell-adhesion protein. PMID- 1730880 TI - Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors. AB - The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development. PMID- 1730881 TI - Conservation of the HLA-DQB2 locus in nonhuman primates. AB - The evolutionary history of MHC class II genes is characterized by several examples of gene duplication, leading both to the creation of distinct subregions such as DR, DQ, and DP, as well as to duplicated loci within each of these subregions. In the human MHC, a prominent example of this diversification occurs within the HLA-DQ subregion, where the nonpolymorphic and transcriptionally "silent" DQB2 locus is highly homologous to the polymorphic expressed DQB1 locus. In order to gain some insight into the mechanisms constraining polymorphism at the DQB2 locus, the second exons of five nonhuman primate DQB2 alleles were sequenced. Six nonhuman primate DQB2 analogous sequences were obtained, two each from chimpanzee and owl monkey cell lines, and one each from gorilla and gibbon cell lines. Notably, the DQB2 sequences from the gibbon, gorilla, and one of the two chimpanzee sequences, although containing some silent nucleotide changes, encode a predicted DQB2 protein with 100% homology to the human DQB2 sequence. The owl monkey DQB2 allelic sequences and the other chimpanzee DQB2 sequence contain additional polymorphisms, but maintain approximately 95% nucleotide sequence identity with human and the other primate sequences. Identification of the owl monkey DQB2 locus indicates that the ancestral DQ gene duplication event occurred at least 40 million years ago, rather than 10 million years, as previously thought. Remarkably, nucleotide sequences from amplified cDNA indicate that the DQB2 gene, and not the DQB1 gene, may be transcribed in the owl monkey line. Substitutions occur at sites comparable to codons of well-recognized allelic variation in the functional DQB1 genes, implying that variation within the DQB2 locus operates under similar selection constraints to the DQB1 locus, with an extremely high degree of conservation through primate evolution. PMID- 1730882 TI - Franklin's disease: Ig gamma 2 H chain mutant BUR. AB - The complete sequence of a gamma 2-H chain disease protein BUR is presented. This mutant, a dimer of a 348-residue chain, linked by four disulfide bridges, is composed of a complete V region, hinge, CH2, and CH3 domains. There is one deletion, the CH1 domain, which includes the cysteine residue bridging the H to L chain. Although the V region is encoded by the VHI and JHIII genes, it has several distinctions: methionine at position 11, two unique cysteine residues in the second complementarity determining region (CDR2), and three glycosylation sites, two of which are located in the CDR2 and CDR3 regions. These distinctive characteristics of BUR VH within the framework of a normal VHI may be affected by extensive somatic mutation or by a rare and previously unanalyzed VH gene. PMID- 1730883 TI - Structural diversity of the classical H-2 genes: K, D, and L. AB - Twenty-three class I DNA sequences, representing alleles of the H-2K, D, and L loci, were analyzed to assess patterns of nucleotide and amino acid diversity. Comparisons of the allelic and nonallelic sequences revealed locus specificity in regions encoding the leader peptides and the carboxyl-terminal segments of the Ag presenting molecules. Analyses focusing on the sequences that determine the Ag binding domains revealed weak or insignificant allelic associations, a finding that is in sharp contrast to previously observed relationships among the homologous human sequences. The amino acid positions exhibiting high diversity in the encoded glycoproteins in both mice and humans are localized primarily to the Ag binding site. In the mouse, diverse amino acids were positioned similarly in the K and D/L glycoproteins, although in humans, the A and B glycoproteins exhibit distinctive differences in their locations within the Ag binding site. The absence of locus specificity among the sequences that determine the Ag binding domains of the mouse is consistent with the hypothesis that ectopic gene conversion leads to interlocus exchange of class I sequences. Comparable interlocus exchanges among human class I genes have not played a similar role in shaping human A and B sequences. The basis of this difference between mice and humans is not clear. The nature of amino acid substitutions distinguishing class I loci in mice and humans are comparable, and the role of natural selection in determining diversity appears to be similar in the two species. PMID- 1730884 TI - Exon encoding the antigen-binding site of MHC class II beta-chains is divided into two subregions with different evolutionary histories. AB - The evolution of the Ag-binding site of polymorphic class II molecules was investigated by comparing the pattern of silent and replacement substitutions in the first domain exon of DQB and DRB alleles in two distantly related mammalian species, man and cattle. We show that the first domain can be subdivided into two regions, corresponding to the beta-strand and alpha-helical regions, with distinct evolutionary histories. The data for the alpha-helical region are in conflict with the standard phylogeny of mammalian class II genes. In this region, there is only weak locus divergence at the protein level and the frequency of silent substitutions is extremely low between nonorthologous genes (i.e., DQB DRB) within species. We propose that the major underlying cause for the observed sequence similarity in the alpha-helical region is due to a selective constraint restricting the sequence divergence at the protein level. This selective constraint may be related to the interaction between different isotypic forms of polymorphic class II beta-chains and a common ligand. The extremely low frequencies of silent substitutions between nonorthologous genes within species are most likely due to the transfer of sequence information between loci by the occurrence of gene conversion-like events in the particular gene segment. PMID- 1730885 TI - Immune response to Moloney murine leukemia virus nonviral, tumor-associated antigens fails to provide in vivo tumor protection. AB - Two distinct populations of CTL have previously been shown to be generated in lymphocyte cultures derived from the spleens of C57BL/6 mice that have rejected Moloney murine leukemia virus:Moloney sarcoma virus (MoMuLV:MSV)-induced tumors. One population is specific for MoMuLV viral Ag whereas the other appears to be directed against a nonviral, tumor-associated Ag (TAA). Using a virus-negative variant of the MoMuLV-induced lymphoma MBL-2 that has retained the expression of the MuLV:TAA, we attempted to further characterize the MuLV:TAA-specific CTL population. First, this same pattern of CTL reactivity was observed using a variety of immunization protocols indicating that the TAA-specific CTL population was not an artifact of the original immunization protocol but was a reproducible component of the MoMuLV CTL response. Moreover, CTL precursor frequency analysis indicates that the MuLV:TAA-specific CTL represent approximately 60% of the CTL detected in in vitro cytotoxicity assays. However, when the role of MuLV:TAA CTL in the in vivo rejection of MoMuLV-induced tumors was examined, no role for the MuLV:TAA-specific CTL response could be determined. Immunization protocols that had been shown to give rise to both CTL populations were capable of protecting mice from tumor development after a challenge with the parental MBL-2 tumor cell line but not the virus-negative variant MBLv cell line. In addition, immunization with the variant, shown to give rise to only MuLV:TAA-specific CTL capable of lysing both MBL-2 and MBLv in vitro, failed to protect mice from a tumor challenge of either cell type. PMID- 1730886 TI - Whither infectious diseases? Some data at last. PMID- 1730887 TI - Infectious disease manpower in the United States--1986. 1. Description of infectious disease physicians. Manpower and Training Committee, Infectious Diseases Society of America. AB - A survey designed to assess the number, type, and current practice patterns of all infectious disease (ID) physicians active in the United States in 1986 was carried out in early 1987. Of 4328 mailed questionnaires, 48.3% were returned. One-third of respondents were in private practice, one-third in academics, and the rest in industry or government. Women accounted for 12.4% of the total; they were younger and as a group spent a greater proportion of total effort in ID. Sixty-five percent of all respondents had greater than or equal to 2 years training in ID. Overall, private practitioners worked longer hours than academicians but spent slightly less effort devoted solely to ID. The proportion of total effort devoted to ID has increased among physicians newly entering practice. Seventy-five percent of all respondents held a teaching appointment. Older ID physicians worked less than 50 h/week and tended to have more administrative than patient care responsibilities. In 1986, there were the equivalent of 1792 full-time ID physicians in the United States or 1:134,000 population. PMID- 1730888 TI - Infectious disease manpower in the United States--1986. 2. Changes in practice patterns over time and training needs. Manpower and Training Committee, Infectious Diseases Society of America. AB - Infectious disease-trained internal medicine physicians responding to a questionnaire survey (n = 1802) reported minor differences in time spent in patient care versus laboratory-based research whether they subsequently became practitioners or academicians. Both practitioners and academicians ranked hospital epidemiology first, followed by knowledge of hospital antibiotic policies in order of importance for new trainees to be taught. Internists with greater than 12 months of training in infectious diseases were divided into private practice versus academically based groups, and their distribution of time spent in various professional activities was analyzed by 5-year intervals for each cohort. These studies confirmed an increasing proportion of time spent in infectious disease-related patient care for new practitioners. Over time, patient care activities decreased and administrative activities increased in all groups. These data are important for estimating future manpower needs. PMID- 1730889 TI - The occurrence and distribution of burnout among infectious diseases physicians. AB - The occurrence and distribution of the three dimensions of the burnout syndrome (emotional exhaustion, depersonalization, and lowered feelings of personal accomplishment) were studied among infectious diseases physicians. A written survey was mailed to the entire identified US population of infectious diseases physicians (n = 4328); a 46.3% response rate provided 1840 usable surveys. Statistical analyses of the data demonstrated that 43.5% of the physician sample reported high scores on emotional exhaustion, and 40.3% scored high on depersonalization. Personal accomplishment scores remained high, despite burnout levels, with 91.8% reporting high personal accomplishment. The highest percentage of burnout occurred among physicians in private practice settings (55%), followed by government settings (39%), and academia (37%). The high percentage of infectious diseases physicians experiencing burnout suggests the need for further research to establish trends, to determine if other types of physicians experience similar levels of burnout, to identify casual factors, and to develop avenues to reduce stress and facilitate coping. PMID- 1730890 TI - 29th annual meeting of the Infectious Diseases Society of America. PMID- 1730891 TI - Epidemiologic classics of Carter, Maxcy, Trudeau, and Smith. AB - Four great epidemiologists whose work so concisely linked clinical observations, epidemiologic clues, and logical preventive measures are discussed. Henry Rose Carter set the stage for the Walter Reed successes in Cuba by showing that 9-16 days must elapse after contact before yellow fever develops. This provided the link for the Reed group to allow the "virus" to incubate in the mosquito before becoming infectious. Kenneth Maxcy clarified the controversy between endemic typhus fever and Brill's disease in the southeastern United States. His clinical, epidemiologic, and laboratory findings led him to propose that the causative organism (Rickettsia typhi) was in rodents and the probable vector, fleas. When confirmed, effective control measures were applied. Two other American investigators, Edward L. Trudeau and Theobald Smith, helped prove Robert Koch wrong on three counts: (1) Tuberculin is not an effective therapeutic agent for tuberculosis; (2) there are two distinct types of tubercle bacillus, human and bovine; and (3) the bovine form of Mycobacterium tuberculosis is remarkably pathogenic for humans. The significance of these findings is unlimited. PMID- 1730892 TI - AIDS--the second decade: a global perspective. AB - The human immunodeficiency virus (HIV) and AIDS epidemic merits its designation as a pandemic: AIDS cases are reported to the World Health Organization from 163 countries, and at least 10 million adults have been infected with HIV. The pandemic is a relatively new phenomenon, and therefore it remains dynamic, unstable, and volatile. Transmission continues in all already-affected countries; HIV is spreading, sometimes quite rapidly, to previously unaffected or little affected areas of the world; and the epidemic becomes more complex and differentiated. The major impact of the pandemic is yet to come: In the 1990s, a 10-fold increase is anticipated in the numbers of adults (to 10 million) and children (to 5 million) developing AIDS. The social, cultural, economic, and political impacts of the pandemic are also increasing. The community, national, and international approach to control of the pandemic must continue to evolve, taking into account the specific conditions of the modern world, of which the global interdependence of health has become the major new factor. PMID- 1730894 TI - Amikacin, ceftazidime, and flucloxacillin against suspended and adherent Pseudomonas aeruginosa and Staphylococcus epidermidis in an in vitro model of infection. AB - Bacterial inocula were exposed as suspended cultures or as adherent biofilms on glass beads in a novel in vitro model of infection to oscillating drug concentrations mimicking human serum kinetics during clinical treatment. Amikacin was given once or thrice daily alone or in combination with ceftazidime or flucloxacillin against Pseudomonas aeruginosa or Staphylococcus epidermidis. Killing of adherent bacteria was significantly reduced during single-drug treatment compared with suspended bacteria (P less than .001), and beta-lactams were more active than amikacin against both suspended and adherent bacteria (P less than .01). Amikacin-beta-lactam combinations killed the inocula more rapidly and were consistently bactericidal against both suspended and adherent pathogens (P less than .05). Once-daily dosing of amikacin produced greater initial killing than thrice daily dosing (P less than .05), but both regimens were similarly effective after 48 h. The differences in antibiotic activity against suspended and adherent bacteria may relate to clinical failures in the treatment of foreign body infections by bacteria sensitive to the administered antibiotics, as determined by standard susceptibility tests. PMID- 1730893 TI - Increased risk of early measles in infants of human immunodeficiency virus type 1 seropositive mothers. AB - An increase in illness due to measles is one of the potential consequences of the human immunodeficiency virus (HIV) epidemic in Africa. During a study of perinatal HIV transmission conducted in Kenya, the risk of acquiring measles before vaccination (9 months of age) was found to be 3.8 times higher in infants born to HIV-seropositive mothers than in control infants (10 [9%] of 109 vs. 5 [3%] of 194 infants; P = .02; odds ratio, 3.8; 95% confidence interval, 1.2 13.2). The majority of infants who developed measles in this study had significant sequelae related to their measles infection. The increased risk of measles appeared to be related to relatively lower anti-measles antibody titers detected in cord blood samples of affected infants born to HIV-seropositive mothers. However, 94% of all infants were susceptible to measles on the basis of ELISA testing at age 6 months regardless of maternal HIV serology. These observations highlight the need for improved measles vaccination strategies in Africa and for studies to delineate the effects of HIV infection on the incidence, presentation, and sequelae of childhood infectious illnesses. PMID- 1730895 TI - Inefficient bacteriolysis of Escherichia coli by serum from human neonates. AB - To assess bacteriolysis in human neonates, Escherichia coli O7w:K1:NM were incubated with sera from eight healthy neonates, serum pooled from the eight neonates, and serum pooled from healthy adults. The adult serum killed E. coli. In contrast, the bacteria were not killed during incubation with sera from the eight neonates, the pooled neonatal serum, or with heat-inactivated adult serum. However, the combination of pooled neonatal serum and heat-inactivated adult serum killed the bacteria. Supplemental IgG-containing antibodies that bound to E. coli did not enhance the bactericidal activity of the neonatal serum. Ten of 12 blood isolates of E. coli from septic neonates but only 8 of 15 isolates from septic adults were serum-sensitive (killed during incubation with adult serum) (P less than .05). Therefore, neonatal serum killed E. coli inefficiently and was deficient in non-IgG heat-stabile component(s) required for bacterial killing. Compared with adults, neonates were more frequently septic with serum-sensitive strains of E. coli. PMID- 1730896 TI - Histopathologic-microbiologic correlates of invasiveness in a mouse model of ascending unobstructed urinary tract infection. AB - To clarify the usefulness of histopathology in evaluating invasiveness during acute cystitis and pyelonephritis in a mouse model of urinary tract infection, findings from bladder and kidney sections of mice inoculated transurethrally with Escherichia coli were compared with results of bladder, kidney, spleen, and blood cultures and with changes in peripheral blood leukocyte counts. All of the 14 bladder histopathologic abnormalities evaluated were significantly associated with a positive bladder culture, and 7 were associated with splenic infection. Histopathologic features of cystitis were present in some culture-negative bladders. Eleven of 12 renal histopathologic abnormalities evaluated were significantly associated both with a positive kidney culture and with splenic infection, and two correlated with the development of peripheral leukocytosis. Histopathologic features of pyelitis and nephritis permitted culture-positive kidneys to be categorized as exhibiting colonization only, pyelitis only, or pyelitis plus frank nephritis and demonstrated that some culture-negative kidneys exhibit signs of pyelitis and nephritis. These findings suggest that detailed, semiquantitative histopathologic evaluation can add to quantitative cultures in the assessment of bacterial urovirulence in the mouse model of ascending urinary tract infection. PMID- 1730897 TI - The effect of type-specific polysaccharide capsule on the clearance of group B streptococci from the lungs of infant and adult rats. AB - To study the importance of the type-specific polysaccharide capsule of group B streptococci (GBS) in the pathogenesis of lung infections, bacterial clearance rates and lung cellular responses to encapsulated and unencapsulated variants of types III and Ia GBS strains were investigated in rats. Bacteria were instilled by direct intratracheal inoculation to simulate aspiration during parturition. Neonates failed to eliminate encapsulated or unencapsulated GBS strains within 6 h of inoculation, whereas adults cleared greater than 90% of each strain within 6 h. Neutrophils accumulated rapidly in the lungs of neonates and adults in response to all GBS strains. Immediately after inoculation, neonatal alveolar macrophages contained fewer encapsulated GBS than did adult alveolar macrophages and fewer encapsulated than unencapsulated GBS, suggesting that the capsule impairs the initial phagocytosis of GBS in the lungs of neonates. Animal age was a more important determinant of bacterial elimination from the lung than the type specific GBS capsule. However, both age and the bacterial capsule were important determinants of systemic dissemination. PMID- 1730898 TI - Bacterial evasion of the antibody response: human IgG antibodies neutralize soluble but not bacteria-associated group B streptococcal C5a-ase. AB - Most strains of group B streptococci (GBS) possess an enzyme that inactivates the human anaphylatoxin C5a by cleaving a heptapeptide from the carboxyl terminus of C5a. This enzyme, called GBS C5a-ase, has been purified to homogeneity and cleaves and inactivates C5a in physiologic buffer. The enzymatic activity of soluble C5a-ase is completely inhibited, however, in the presence of plasma or serum from normal human adults. The neutralization of soluble C5a-ase by plasma and serum results largely from naturally occurring IgG antibodies directed against C5a-ase. IgG does not neutralize C5a-ase present on intact encapsulated type III GBS but does neutralize the C5a-ase activity associated with a transposon-induced mutant strain of type III GBS that lacks capsule. The location of GBS C5a-ase on the surface of encapsulated type III GBS permits the C5a-ase to inactivate C5a while evading neutralization by IgG antibodies. PMID- 1730899 TI - J774 macrophages secrete antibiotics via organic anion transporters. AB - Mouse macrophages and J774 macrophage-like cells express probenecid-inhibitable organic anion transporters that remove anionic dyes from the cells' cytoplasmic matrix and secrete these dyes into the extracellular medium. The present studies show that these transporters also secrete antibiotics from J774 macrophages. Penicillin G permeates J774 cells poorly, but after it was introduced into the cell cytoplasm, it was secreted in a probenecid-inhibitable fashion. The quinolone norfloxacin enters macrophages readily. Probenecid retarded the secretion of intracellular norfloxacin by J774 cells and enhanced norfloxacin accumulation three- to fourfold. Thus the intracellular accumulation of norfloxacin is regulated in part by organic anion transporters that secrete norfloxacin (and penicillin G) from J774 cells. This transport process may have clinical significance, as fluoroquinolones inhibit growth of intracellular pathogens such as mycobacteria and Brucella organisms in vitro but fail to arrest infections with these organisms in vivo. PMID- 1730900 TI - Host species-specific antigenic variation of a mannosylated surface glycoprotein of Pneumocystis carinii. AB - All Pneumocystis carinii, irrespective of their host of origin, express an abundant mannosylated surface glycoprotein. While this molecule is commonly referred to as gp120, some variation in the size of this molecule has been noted. Monoclonal antibodies produced to this molecule from ferret, mouse, rat, and human P. carinii made it possible to distinguish the glycoprotein from P. carinii of each host by immunoblot and indirect immunofluorescence assays. The glycoprotein varied in size (approximately 95-140 kDa) depending on the host and method of preparation. One shared epitope involving the mannose part of the glycoprotein was identified, suggesting that despite size and antigenic differences these are homologous molecules. These results indicate the existence of host species-specific serotypes of P. carinii. Referring to this molecule by its molecular mass may be confusing; therefore, it is suggested that this glycoprotein be called surface glycoprotein A, as this was the initial molecule of P. carinii to be isolated and partially characterized. PMID- 1730901 TI - Inhibiting cholesterol synthesis reduces the binding and toxicity of amphotericin B against rabbit renal tubular cells in primary culture. AB - Renal tubular cells are a target of amphotericin B (AmB) toxicity, but the mechanisms involved in the tubular cell-AmB interactions are unknown. Ketoconazole was selected to lower the cholesterol content of rabbit renal tubular cells in primary culture. The consequences of cholesterol depletion on AmB nephrotoxicity was investigated in vitro as the inhibition of Na(+)-dependent phosphate uptake. After 1 h of exposure, AmB decreased phosphate uptake (49%, 77%, and 82% inhibition with 5, 10, and 20 microM of AmB, respectively). Pretreatment of cells with ketoconazole (10 microM for 24 h) reduced by 50% (P less than .01) the phosphate uptake inhibition induced by AmB, decreased cellular cholesterol synthesis (greater than 80% inhibition), and decreased AmB binding to cell membrane by 50%, as measured by the fluorescence extinction of a probe bound to tubular cell membrane. Incubation with exogenous exchangeable cholesterol again increased AmB binding to plasma membrane and restored AmB toxicity. These results demonstrate that the first step of AmB renal tubular toxicity is mediated by cellular cholesterol content and is parallel to the binding of AmB to cell membrane. PMID- 1730902 TI - Passive immunotherapy in AIDS: a randomized trial of serial human immunodeficiency virus-positive transfusions of plasma rich in p24 antibodies versus transfusions of seronegative plasma. AB - To assess the place of passive immunotherapy in the treatment of AIDS, a randomized study was conducted that evaluated the safety and short-term efficacy of serial transfusions of human immunodeficiency virus type 1 (HIV-1) seropositive plasma in 18 patients. Heat-inactivated anti-HIV antibody-rich plasma was compared with seronegative fresh-frozen seronegative plasma given in addition to zidovudine and other conventional prophylactic treatments. Seven transfusions every 2 weeks of immune plasma significantly reduced (2 vs. 8, P = .016) the number of opportunistic infections. Antigenemia became undetectable. When transfusions were stopped, positive p24 antigenemia returned at a level higher than before treatment and was correlated with a severe clinical deterioration, suggesting a rebound effect. This trial suggests that passive immunotherapy is promising in AIDS treatment. It confirms also that plasma donation does not affect donors' CD4 cell count over a 1-year period. In patients with severe immunodeficiency, special attention should be paid to withdrawal of an effective therapy as virologic relapse may be explosive and poorly tolerated. PMID- 1730903 TI - Human T lymphotropic virus (HTLV) type I and II DNA amplification in HTLV-I/II seropositive blood donors of the French West Indies. AB - To confirm the presence of DNA from human T lymphotropic virus type I (HTLV-I), HTLV-II, or both in individuals found HTLV-I/II-positive through systematic screening of blood donations in Guadeloupe (French West Indies), 42 blood donors repeatedly positive for HTLV-I/II by ELISA were studied by polymerase chain reaction (PCR). Three primer pairs (env, pol, tax) targeted on conserved regions of HTLV-I or -II sequences (or both) and six probes (two generic, two HTLV-I specific, two HTLV-II-specific) were used in a multiplex PCR. HTLV-I sequences were detected in 31 individuals (74%). All 31 subjects positive by Western blot (WB) harbored HTLV-I sequences. Fifteen individuals (48%) were positive with the three primer pairs used, 10 (32%) with two, and 6 (20%) with one. Subjects indeterminate or negative by WB were all negative by PCR. No HTLV-II sequences were detected with specific probes. The results indicate the absence of HTLV-I and -II infection in individuals with indeterminate WB, the presence of HTLV-I DNA in individuals positive for WB in the French West Indies, and the absence of HTLV-II infection in the cohort. PMID- 1730904 TI - Impact of influenza virus infection as a cause of pediatric hospitalization. AB - From winter 1989 to spring 1990, a severe epidemic caused by influenza A (H3N2) and B viruses developed in Japan. During the epidemic (December 1989 to February 1990), 244 children were admitted to the pediatric ward of Nippon Kokan Hospital: 53 (21.7%) were hospitalized with influenza virus infection, 22 (9.0%) with rotavirus gastroenteritis, and 17 (7.0%) with respiratory syncytial virus infection. Among those with influenza, 24 had type A and 29 had type B. Most were young healthy children without underlying illnesses (mean age, 4.8 +/- 3.4 years). The impact of the influenza epidemic on pediatric hospitalization is probably much greater than generally thought when a severe epidemic occurs. PMID- 1730905 TI - Seasonal variation in etiology of travelers' diarrhea. Finnish-Moroccan Study Group. AB - The etiology of travelers' diarrhea was studied in 579 adult Finnish tourists participating in two packaged tours to Morocco in the winter (n = 233) and fall (n = 346) of 1989. A research team accompanied the travelers, and a laboratory for enteric pathogens was established in Agadir. At least one pathogen was found in 62% of the 60 diarrhea cases in winter and in 58% of the 111 diarrhea cases in fall. Multiple pathogens were found less often in winter (8%) than in fall (21%, P less than .05). Campylobacter strains were the leading cause of travelers' diarrhea in winter, found alone or with other pathogens in 28% of the cases (but in only 7% in fall), whereas enterotoxigenic Escherichia coli (ETEC) was the most common pathogen in fall, present in 32% of the cases (8% in winter). Both differences are highly significant (P less than .001). Salmonella enterica was almost as common as ETEC in fall (25% of diarrhea cases) but rare in winter (10%, P less than .05). Thus, the etiology of travelers' diarrhea varied according to the season in the same tourist destination. This finding has relevance to both antimicrobial treatment and prophylaxis. PMID- 1730906 TI - Prevalence of esophageal Candida colonization in a Danish population: special reference to esophageal symptoms, benign esophageal disorders, and pulmonary disease. AB - A population sample selected at random after stratification for the presence of pulmonary disease was screened for benign esophageal disease; 175 subjects agreed to participate in the invasive investigation, 86 without pulmonary disease and 89 with chronic obstructive pulmonary disease (COPD). Of these, 169 underwent endoscopy of the upper gastrointestinal tract, 164 had mucosal brushings for the presence of Candida albicans in the esophagus, 169 had esophageal pressure measurements, and 113 had 12-h pH measurements. One hundred fourteen subjects with benign esophageal disease were found. The prevalence of C. albicans in the esophagus (greater than or equal to 50 colonies) in subjects with and without COPD was 12.3% and 25.1%, respectively. C. albicans occurred equally in subjects with and without esophageal symptoms. There was no relation between the presence of C. albicans and benign esophageal disease and no significant clinical correlation between esophageal plaques and colony counts of C. albicans. PMID- 1730907 TI - Early diagnosis of human immunodeficiency virus infection in infants by detection of free and complexed p24 antigen. PMID- 1730908 TI - Evaluation of aqueous penicillin G and ceftriaxone for experimental neurosyphilis. PMID- 1730909 TI - Impact of washing sputum on Streptococcus pneumoniae and other microorganisms. PMID- 1730910 TI - Small calcified nodule: an undescribed radiologic manifestation of human pulmonary dirofilariasis. PMID- 1730911 TI - Immunity to Trichinella spiralis infection in vitamin A-deficient mice. AB - Vitamin A-deficient (A-) mice make strikingly poor IgG responses when they are immunized with purified protein antigens. Previously, we showed that A- T cells overproduce interferon gamma (IFN-gamma), which then could inhibit interleukin 4 (IL-4)-stimulated B cell IgG responses. To determine if the altered IFN-gamma regulation pattern and its immunological consequences would extend to a natural infection, we studied mice infected with the parasitic helminth Trichinella spiralis. The course of the infection was similar in A- and A-sufficient (A+) mice. These mice did not differ with respect to newborn larvae/female/hour produced in the intestine, or muscle larvae burden 5 wk postinfection. They also did not differ in the intestinal worm expulsion rate until day 15, when A- mice still harbored parasites, whereas A+ mice had cleared intestinal worms. Vitamin A deficiency reduced both the frequency of B lymphocytes secreting IgG1 antibodies to parasite antigens, and the bone marrow eosinophilia associated with helminth infection. The cytokine secretion patterns in infected mice were consistent with these observations and with previous studies. Mesenteric lymph node cells from infected A- mice secreted significantly more IFN-gamma, and significantly less IL 2, IL-4, and IL-5 than infected A+ controls. A- splenocytes secreted significantly more IFN-gamma, and equivalent amounts of IL-2, IL-4, and IL-5 compared with A+ controls. Interestingly, CD4-CD8- cells secreted the majority of the IL-4 produced in the spleen. The IL-2, IL-4, and IL-5 steady-state transcript levels correlated with secreted protein levels, but IFN-gamma transcripts did not. Although they secreted more protein, A- cells contained fewer IFN-gamma transcripts than A+ cells. These results suggest two vitamin A-mediated regulation steps in IFN-gamma gene expression: positive regulation of IFN-gamma transcript levels, and negative regulation posttranscriptionally. The essentially unaltered outcome of T. spiralis infection in vitamin A-deficient mice probably reflects a balance between cellular and humoral responses. The IFN-gamma overproduction might have a positive effect on the gut inflammatory response, but the decrease eosinophilia, cytokine production in mesenteric lymph node, and IgG1 secreting cell frequency might have a negative effect on T. spiralis immunity. PMID- 1730912 TI - Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1. AB - The relationship between the characterized mouse regulators of complement activation (RCA) genes and the 190-kD mouse complement receptor 1 (MCR1), 155-kD mouse complement receptor 2 (MCR2), and mouse p65 is unclear. One mouse RCA gene, designated MCR2 (or Cr2), encodes alternatively spliced 21 and 15 short consensus repeat (SCR)-containing transcripts that crosshybridize with cDNAs of both human CR2 and CR1, or CR2 alone, respectively. A five SCR-containing transcript derived from a second unique gene, designated Crry, also crosshybridizes with human CR1. We have previously shown that the 155-kD MCR2 is encoded by the 15 SCR-containing transcript. To analyze the protein products of the other transcripts, which are considered the genetic homologues of human CR1, we have expressed the 21 and the 5 SCR-containing cDNAs in the human K562 erythroleukemia cell line. We demonstrate that cells expressing the 21 SCR transcript express the 190-kD MCR1 protein. These cells react with five unique rat anti-MCR1 monoclonal antibodies, including the 8C12 antibody considered to be monospecific for MCR1. In addition, these cells efficiently form rosettes with mouse C3b-bearing sheep erythrocytes. In contrast, cells expressing the five SCR-containing Crry transcript are strongly recognized by an anti-human CR1 antibody that also defines the mouse p65 protein. Using a functional assay that measures the surface deposition of C3 activated via the classical complement pathway, we show that Crry/p65-expressing cells have a markedly decreased amount of C3 deposited on them as compared with control cells expressing the antisense construct or cells expressing MCR1 or MCR2. This suggests that Crry has intrinsic complement regulatory activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730913 TI - Small B cells as antigen-presenting cells in the induction of tolerance to soluble protein antigens. AB - We have investigated the ability of resting B cells, acting as antigen-presenting cells, to induce tolerance to soluble protein antigens in mice, using an antigen targeted specifically to B cells. We inject mice intravenously with ultracentrifuged Fab fragments of rabbit anti-mouse immunoglobulin D (IgD) (Fab anti-delta). Treatment with Fab anti-delta results in profound tolerance to challenge with 100 micrograms Fab nonimmune rabbit Ig (Fab NRG), precipitated in alum, as measured by antibody production. Tolerance to rabbit Fab is antigen specific, since the treated mice make normal antibody responses to a control antigen, chicken Ig. Tolerance is dependent on antigen presentation by B cells, since intravenous injection of soluble Fab NRG, which is not targeted to B cells, results in a much lower frequency and degree of tolerance, especially at lower doses. T cell help in this system is affected, since T cells from Fab anti-delta treated mice fail to provide help for an adoptive primary antibody response to Fab NRG when transferred together with normal B cells into severe combined immunodeficient (SCID) mice. The antigen-specific B cell compartment is also affected during tolerance induction, since B cells from treated animals make less antibody than normal B cells when transferred into SCID mice with normal T cells. Although the mechanism of nonresponsiveness in the helper T cell compartment remains to be determined, we think it is likely that the precursors of helper T cells are inactivated or deleted by encountering antigen presented by small, resting B cells, which lack accessory signals necessary to induce helper T cell proliferation and differentiation to effector function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1730914 TI - Presentation of antigen by mixed isotype class II molecules in normal H-2d mice. AB - A panel of DBA/2 T cell hybridomas specific for the sperm whale myoglobin epitope 110-121 was found to recognize antigen presented by the mixed isotype class II molecule E alpha dA beta d. The response was blocked by monoclonal antibodies specific for E alpha and A beta d chains; in addition, the hybridomas responded to antigen presented by L cells expressing E alpha A beta d molecules, and made no response with L cells expressing I-Ad or I-Ed molecules. Two more groups of hybridomas isolated from DBA/2 and B10.D2 mice immunized with myoglobin also recognized peptide 110-121 presented by E alpha d A beta d. Thus, although it is expressed at biochemically undetectable levels on spleen cells, the E alpha d A beta d molecule is an important presenting element in normal H-2d mice making a conventional immune response to a protein antigen. These results suggest that high levels of class II expression are not a prerequisite for T cell activation. PMID- 1730915 TI - Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection. AB - Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease. PMID- 1730916 TI - Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes. AB - Recent studies suggest that some T and B lymphocyte cell lines bind to the integrin lymphocyte function-associated molecule 1 (LFA-1) chiefly through a pathway independent of its two known counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. A monoclonal antibody (mAb) was raised that, in combination with blocking mAb to ICAM-1 and ICAM-2, can completely inhibit binding of these cell lines to purified LFA-1. This third ligand, designated ICAM 3 based on its functional relatedness to ICAM-1 and -2, is a highly glycosylated protein of 124,000 Mr. It is well expressed on all leukocytes and absent from endothelial cells. In assays of adhesion of resting lymphocytes to purified LFA 1, ICAM-3 is by far the most functionally important ICAM, implying an important role for ICAM-3 in the generation of immune responses. PMID- 1730917 TI - Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface. AB - To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding. PMID- 1730918 TI - Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody. AB - The CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact cDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to select transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than cells transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IRAWB 14, which reacts with CD44 on all CD44+ cells dramatically induced HA binding by some CD44+ cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution. Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IRAWB 14 mAb, virtually all CD44+ splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4 degrees C, indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface. PMID- 1730919 TI - Dendritic cells are potent antigen-presenting cells for microbial superantigens. AB - Dendritic cells are a small subset of human blood mononuclear cells that are potent stimulators of several T cell functions. Here we show they are 10-50-fold more potent than monocytes or B cells in inducing T cell responses to a panel of superantigens. Furthermore, dendritic cells can present femtomolar concentrations of superantigen to T cells even at numbers where other antigen-presenting cells (APCs) are inactive. Although dendritic cells express very high levels of the major histocompatibility complex products that are required to present superantigens, it is only necessary to pulse these APCs for 1 hour with picomolar levels of one superantigen, staphylococcal enterotoxin B, to maximally activate T cells. Our results suggest that very small amounts of superantigen will be immunogenic in vivo if presented on dendritic cells. PMID- 1730920 TI - A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein. AB - Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection. PMID- 1730921 TI - Anti-CD4 abrogates rejection and reestablishes long-term tolerance to syngeneic newborn hearts grafted in mice chronically infected with Trypanosoma cruzi. AB - The contribution of autoimmunity in the genesis of chronic Chagas' heart pathology is not clear. In the present study, we show that: (a) BALB/c mice chronically infected with Trypanosoma cruzi reject syngeneic newborn hearts; (b) in vivo treatment with anti-CD4 but not anti-CD8 monoclonal antibodies (mAbs) abrogates rejection; (c) CD4+ T cells from chronically infected mice proliferate in vitro to syngeneic myocardium antigens and induce heart graft destruction when injected in situ; (d) anti-CD4 treatment of chronically infected mice establishes long-term tolerance to syngeneic heart grafts; and (e) the state of tolerance is related to in vitro and in vivo unresponsiveness of the CD4+ T cells. These findings allow us to suggest that autoimmunity is the major mechanism implicated in the rejection of syngeneic heart tissues grafted into the pinna of the ear of mice chronically infected with T. cruzi. The similarity of the lesions to those found in humans suggests that autoimmunity is involved in the pathogenesis of chagasic cardiomyopathy in humans. Moreover, this could imply therapeutic strategies by reestablishing long-term tissue-specific tolerance with anti-CD4 mAb treatment, mediating anergy, or deleting the responder CD4+ T cells to heart tissue antigens. PMID- 1730922 TI - Interleukin 5 messenger RNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease. AB - Interleukin 5 (IL-5), the major factor involved in eosinophil differentiation, is produced by T cells or mast cells. In the present study, we found that eosinophils infiltrating the mucosa of four patients with active coeliac disease also express the IL-5 mRNA. No positive signal was obtained in normal duodenum tissues and in the cell infiltrate from patients submitted to gluten restriction. The identification of labeled mucosal cells as eosinophils relied on their typical morphology. Moreover, highly purified blood eosinophils from three out of four patients with eosinophilia were also strongly labeled with the IL-5 antisense but not with the corresponding sense probe. Together, these results suggest that eosinophils have the capacity to synthesize IL-5, which could contribute to paracrine interactions with T and B cells and, in autocrine fashion, locally participate, through binding to the IL-5 receptor, to eosinophil differentiation and activation. These data might have implications not only in the pathology of coeliac disease but also in other diseases associated with eosinophil infiltration. PMID- 1730923 TI - Assessment of in vivo frequency of mutated T cells in patients with systemic lupus erythematosus. AB - The frequency of mutant T cells (FMC) in blood lymphocytes from patients with systemic lupus erythematosus (SLE) was measured by growing cells in the presence and in the absence of 6-thioguanine. Patients with SLE had a spectrum of FMC ranging from normal to about 100 times normal. This high FMC among cells from SLE patients appears to reflect excessive in vivo activation and proliferation during the course of the disease. This represents the first demonstration of such a T cell abnormality in SLE; it supports the hypothesis that SLE T cells demonstrate increased in vivo division and/or survival. PMID- 1730924 TI - Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4. AB - The human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1. PMID- 1730925 TI - Donor major histocompatibility complex (MHC) peptides are presented by recipient MHC molecules during graft rejection. AB - Peptides from donor major histocompatibility complex (MHC) molecules were examined for their activation of allogeneically primed T cells. After immunization with either allogeneic spleen cells or a skin allograft, primed T cells proliferate in response to peptides derived from polymorphic regions of alpha and beta chains of class II allo-MHC molecules. The results demonstrate that presentation of donor-MHC peptides by host-derived antigen-presenting cells is a common event in vivo. Thus, self-restricted T cell recognition of processed alloantigens may play a critical role in transplantation. An in-depth understanding of this response may result in the development of additional molecular therapies to combat allograft rejection. PMID- 1730926 TI - Oligoclonality of human intestinal intraepithelial T cells. AB - T cells bearing the T cell receptor alpha/beta (TCR-alpha/beta) are the predominant lymphocyte population in the human intestinal epithelium. To examine if normal intestinal intraepithelial lymphocytes (IEL) have a TCR repertoire distinct from the TCR-alpha/beta repertoire in peripheral blood lymphocytes (PBL), comparative analysis of relative V beta gene usage in IEL and PBL was performed by quantitative polymerase chain reaction. In each of the six individuals examined, one to three V beta families made up more than 40% of the total V beta transcripts detected in the IEL, whereas there was a more even distribution of V beta gene usage in the paired PBL. The predominant V beta families, especially V beta 1, V beta 2, V beta 3, and V beta 6, were frequently shared among IEL of different individuals. PCR cloning and sequence analysis of the predominant V beta 6 family in two individuals revealed an identical V-D-J-C sequence in 13 of 21 clones obtained from one donor, and a different repeated sequence in 18 of 27 clones examined in the second donor. These data suggest that the V beta skewing in IEL is due to an oligoclonal T cell expansion and may reflect the response of the intestinal mucosal immune system to a restricted set of as yet undefined antigens present in the gut. PMID- 1730927 TI - Mechanism of self-tolerance of gamma/delta T cells in epithelial tissue. AB - The present study examined mechanisms of tolerance for T cell receptor gamma/delta (TCR-gamma/delta) cells. Using a transgenic (Tg) model, we demonstrate that although alloantigen (Ag)-specific TCR-gamma/delta cells are deleted in the thymus and spleen of Ag-bearing mice, intraepithelial lymphocytes (IELs) expressing normal levels of the Tg TCR were present. However, Tg+ IELs from Ag-bearing mice were unresponsive to activation. Furthermore, self-reactive Tg+ IELs decreased in number over time. Thus, in epithelial tissue, Tg TCR gamma/delta cells are eliminated subsequent to and most likely as a result of the induction of clonal anergy. PMID- 1730928 TI - A transgenic model of autoimmune hemolytic anemia. AB - We made double transgenic mice bearing immunoglobulin heavy and light chain genes encoding an autoantibody against the mouse erythrocyte by the cross of C57BL/6 mice carrying the transgene for each chain of the immunoglobulin. Although no obvious disorders were found in the single-chain transgenic mice, severely anemic symptoms were found in some of the double transgenic mice, in which most B cells express, at least on their surface, the autoantibody reactive to self-antigens on the erythrocyte. Individual double-transgenic mice showed a wide variation of phenotypes between severe anemia and no symptoms. Both deletion and anergy of autoreactive B cells were seen in each individual mouse, but their relative contribution to self-tolerance was variable and not directly related to the severity of anemia or the amount of the autoantibody produced. This transgenic system provides a good autoimmune disease model for exploring its onset mechanism, and means of its treatment and prevention. PMID- 1730929 TI - T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. AB - Because mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine- (D-Gal) sensitized mice as a model system to evaluate potential toxic shock symptoms triggered by the superantigen staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 micrograms SEB, respectively. The lethal shock triggered by the superantigen SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T cell-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL-2). Serum-borne lymphokines were detected well in advance of signs of T cell activation, as assessed by IL-2 receptor expression of SEB-reactive V beta 8+ T cells. Passive immunization with anti-TNF-alpha/beta-neutralizing monoclonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)- and exotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T cells. Yet the pathogenesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock. PMID- 1730931 TI - Highlights and prospects of potyvirus molecular biology. PMID- 1730930 TI - Cell-cell interaction in graft rejection responses: induction of anti-allo-class I H-2 tolerance is prevented by immune responses against allo-class II H-2 antigens coexpressed on tolerogen. AB - The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6 C-H-2bm1 (bm1) spleen cells results in almost complete abrogation of anti-bm1 CD8+ helper (proliferative and interleukin 2-producing) T cell (Th) activities. Although an appreciable portion of CD8+ cytotoxic T lymphocyte (CTL) precursors themselves remained after this regimen, such a residual CTL activity was eliminated after the engrafting of bm1 grafts, and these grafts exhibited prolonged survival. In contrast, the intravenous sensitization with (bm1 x B6-C-H 2bm12 [bm12])F1 cells instead of bm1 cells failed to induce the prolongation of bm1 graft survival as well as bm12 and (bm1 x bm12)F1 graft survival. In the (bm1 x bm12)F1-presensitized B6 mice before as well as after the engrafting of bm1 grafts, anti-bm1 CTL responses that were comparable to or slightly stronger than those observed in unpresensitized mice were induced in the absence of anti-bm1 Th activities. bm1 graft survival was also prolonged by intravenous presensitization with a mixture of bm1 and bm12 cells but not with a mixture of bm1 and (bm1 x bm12)F1 cells. The capacity of CD4+ T cells to reject bm12 grafts was eliminated by intravenous presensitization with antigen-presenting cell (APC)-depleted bm12 spleen cells. However, intravenous presensitization with APC-depleted (bm1 x bm12)F1 cells failed to induce the prolongation of bm1 graft survival under conditions in which appreciably prolonged bm12 graft survival was induced. More surprisingly, bm1 graft survival was not prolonged even when the (bm1 x bm12)F1 cell presensitization was performed in CD4+ T cell-depleted B6 mice. This contrasted with the fact that conventional class I-disparate grafts capable of activating self Ia-restricted CD4+ as well as allo-class I-reactive CD8+ Th exhibited prolonged survival in CD4+ T cell-depleted, class I-disparate cell presensitized mice. These results indicate that: (a) intravenous presensitization with class I- and II-disparate cells fails to reduce anti-allo-class I rejection responses that would otherwise be eliminated using only class I-disparate cells; (b) such failure is generated according to the coexpression of both classes of alloantigens on a single cell as tolerogen; and (c) allo-class II antigens coexpressed on tolerogen function to activate CD4+ as well as non-CD4+ Th leading to the generation of anti-class I effector T cell responses. PMID- 1730932 TI - Demonstration of woodchuck hepatitis virus infection of peripheral blood mononuclear cells by flow cytometry and polymerase chain reaction. AB - Peripheral blood mononuclear cells (PBMCs) from 10 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of WHV surface (WHs) and core (WHc) antigens (WHsAg and WHcAg) by cytofluorometry using fluorescein isothiocyanate-conjugated anti-WHs and anti-HBc-purified immunoglobulins from woodchuck and human sera. The presence of viral DNA and RNA was detected in the serum and PBMCs from the same blood samples by polymerase chain reaction (PCR) with two primer sets located in the S and C genes of the WHV genome. Seven animals were found positive for both WHsAg and WHcAg on the surface of PBMCs: four WHV-chronic carriers, two WHsAg-positive animals with acute WHV infection, and one woodchuck which was bled during the incubation phase of WHV infection and which became WHsAg-positive only 1 month later. Sixteen to 71% of the studied leukocyte population expressed WHsAg with a low density of expression whereas 7 to 72% expressed WHcAg with a high density of expression. Only two cases were positive for WHsAg without WHcAg on PBMCs, one WHV chronic carrier and one anti WHs-positive animal. All woodchucks positive for WHcAg and/or WHsAg by cytofluorometry were positive also for WHV DNA and RNA in PBMCs by PCR. The tenth animal was found negative for both viral antigens as well as for WHV DNA and RNA in PBMCs despite the presence of persistent viral DNA in the serum as detected by PCR. Five healthy woodchucks devoid of WHV serological markers served as negative controls. These results obtained with a novel approach further confirm, in the woodchuck model, that a significant proportion of PBMCs are probably permissive for WHV replication. The possible immunopathogenic implications of the phenomenon are discussed. PMID- 1730933 TI - Repression of the hepatitis B virus enhancer by a cellular factor. AB - The enhancer element of hepatitis B virus (HBV) regulates the transcription of all HBV mRNA, including the pregenomic mRNA used during replication. This pregenomic mRNA is transcribed from the core gene promoter which is located 500 bp downstream from the HBV enhancer element. To examine the effect of the HBV enhancer on the activity of the core gene promoter, we constructed various plasmids containing different combinations of HBV enhancer and core gene promoter sequences regulating the expression of the chloramphenicol acetyltransferase gene. When the HBV enhancer was positioned immediately adjacent to the core gene promoter, the enhancer increased the activity of the core gene promoter nearly 30 fold. In contrast, the HBV enhancer only modestly stimulated the core gene promoter (less than threefold) at its native position in the HBV genome. This weak HBV enhancer activity was due to a DNA sequence located between the enhancer and the core gene promoter and not due to the increased distance between the enhancer and the core gene promoter. Competition experiments demonstrated that a trans-acting factor(s) bound this sequence and repressed the enhancer. This DNA sequence contains the C/EBP, AP-1 and NF-1 regulatory sites. No inhibition of enhancer activity was observed when only the AP-1 and C/EBP sites were present. Repression of the HBV enhancer was not detected when the NF-1 site was disrupted, indicating that the NF-1 site was involved in the suppression of the HBV enhancer. PMID- 1730934 TI - Translational modulation in hepatitis B virus preS-S open reading frame expression. AB - A series of hepatitis B virus open reading frame (ORF) preS-S variants, including mutants in which the relative order of the in-frame start codons (AUG1, AUG2 and AUG3) and nearby sequences had been altered, was expressed both in vivo (in HepG2 hepatoblastoma cells) and in vitro (by T7 promoter-driven transcription followed by translation in a rabbit reticulocyte lysate). The ratio of the synthesis of the large, middle (M) and major (S) proteins or their chimeric counterparts was analysed to study the translational regulation of ORF expression. As expected on the basis of the ribosome scanning model, the AUG sequence context was found to be a prominent factor in determining the different translational behaviour of the two preS-S-specific mRNAs of 2.4 kb (predominantly translated from AUG1) and 2.1 kb (which includes AUG2 and/or AUG3 and can be translated from either). Results from both experimental systems suggested that initiation at internal AUGs in the 2.4 kb RNA is possible. In experiments in vitro, preS-S mutants bearing lesions in a region 5' to AUG2, which has been implicated in AUG2/AUG3 cis repression, showed no increase in the utilization of internal AUGs. In addition, the chimeric envelope polypeptides produced in transfected HepG2 cells in this study were informative with respect to preS-mediated endoplasmic retention: replacement of the preS2 N terminus with that from preS1 generated a chimeric M protein that was glycosylated within the putative preS1 retention sequence ad was not secreted. Thus, the preS1 retention sequence most likely acts inside the lumen of endoplasmic reticulum and its function is insensitive to glycosylation. A similar element might be active at the N terminus of M protein. PMID- 1730935 TI - Cleavage profiles of tobacco etch virus (TEV)-derived substrates mediated by precursor and processed forms of the TEV NIa proteinase. AB - Nucleotide sequences coding for proteins containing the tobacco etch virus (TEV) NIa proteinase were generated by polymerase chain reaction amplification and/or site-directed mutagenesis. These coding regions contained sequences for the proteinase alone or as part of higher Mr precursors. Following transcription and translation of these sequences in a cell-free system, the various polyproteins, all containing an active small nuclear inclusion protein (NIa) proteinase, were used to process a TEV substrate series. Most substrates were processed in a similar fashion by all proteolytic forms. However, one substrate which contained the TEV 50K/71K protein junction was differently processed by several of the polyproteins containing NIa proteinase. Substrates which previously had no identified TEV NIa proteinase cleavage sites also were tested and were not cleaved by any of the proteinase-containing polyprotein forms. PMID- 1730936 TI - Expression of cowpea mosaic virus coat protein precursor in transgenic tobacco plants. AB - Tobacco, Nicotiana tabacum L., supports cowpea mosaic virus (CPMV) replication and cell-to-cell movement, and thus may serve as a model system to study coat protein-mediated protection against CPMV. A chimeric gene consisting of the cauliflower mosaic virus 35S promoter, CPMV 60K coat proteins-precursor (CP-P) coding region, and the nopaline synthase polyadenylation signal was transferred to tobacco cv. Burley 21 via the Agrobacterium tumefaciens binary vector system. Gene integration and expression in the transgenic tobacco plants were confirmed by Southern and RNA dot blot analyses. Accumulation of CPMV 60K CP-P in transgenic plants, up to 2 micrograms/g of wet weight tissue, was detected by ELISA and Western blots. The results of Western blots and immunosorbent electron microscopy further indicated that CPMV CP-P neither undergoes autoproteolysis to generate the mature viral coat proteins nor assembles into virus-like capsids, suggesting that processing of the CP-P may be required for virus assembly. Because CPMV neither induces symptoms in tobacco nor moves systemically, evaluation of the reactions of the transgenic plants to virus inoculation was based on virus accumulation in the inoculated leaves. Results from such infectivity experiments did not differentiate between CP-P expressers and vector transformed plants. The transgenic tobacco plants expressing CP-P should provide valuable material for investigating comovirus polyprotein processing and capsid assembly in vivo. PMID- 1730937 TI - Nucleotide sequence analysis of the movement genes of resistance breaking strains of tomato mosaic virus. AB - The nucleotide sequences were determined for the movement genes, encoding 30K proteins, of two resistance breaking strains of tomato mosaic virus (ToMV), LII ToMV and LIIA-ToMV. The putative amino acid sequences of the encoded proteins were compared with L-ToMV and with two previously published resistance breaking strains, Ltb1-ToMV and C32-ToMV. LII-ToMV, a Type 2 strain able to infect tomatoes containing the Tm-2 gene, has four amino acid changes relative to L ToMV. One of these substitutions, Glu-Lys133, is also found in the other Type 2 strains, Ltb1-ToMV and C32-ToMV. LIIA-ToMV, a Type 2(2) strain able to overcome the resistance conferred by the tomato Tm-2(2) gene, has four amino acid differences from L-ToMV. The Type 2(2) strain has no substitutions in common with any of the Type 2 strains; however the predominance of amino acid substitutions that involve a change in the local charge (six out of the eight) suggests an electrostatic interaction between a host factor and the 30K protein may be intrinsic to the function of the movement gene and/or resistance breakage. PMID- 1730938 TI - Expression of brome mosaic virus-encoded replicase genes in transgenic tobacco plants. AB - We introduced replicase genes of brome mosaic virus (BMV) to Nicotiana tabacum cv. Petit Habana (SR1) using two different types of transformation vectors containing cDNAs of BMV RNA 1 and RNA 2. One type (V type) contains cDNA from which complete viral RNAs are transcribed. These RNAs can function as templates for viral replicase. The other type (M type) contains cDNA from which viral RNAs without their 3' non-coding regions are transcribed; these RNAs can only function as mRNA. Viral replicase expressed from the integrated cDNAs in both V and M type transgenic plants can complement an infection by BMV RNA 3. PMID- 1730939 TI - Human non-hepatocytes support hepadnaviral replication and virion production. AB - The competence of non-hepatocytes to support hepatitis B virus (HBV) gene expression and replication was studied by transient transfection of various human cell lines with a head-to-tail dimer of HBV DNA. Independent of their neuroectodermal, mesenchymal or epithelial origin, all non-hepatocyte cell lines tested synthesized and secreted hepatitis B surface antigen (HBsAg) and hepatitis B core/e antigen (HBc/eAg). Further analyses of two of these cell lines (LS 180 and COLO 320) identified the two major HBV transcripts of 3.6 and 2.2/2.4 kb length, respectively. LS 180 cells were permissive for HBV and duck hepatitis B virus (DHBV) DNA replication and secretion of infectious virions. COLO 320 cells also supported HBV DNA replication, but did not appear to export complete viral particles. These findings provide direct evidence that both HBV and DHBV can replicate in non-hepatic tumour cell lines, one of which is shown also to produce infectious virions. PMID- 1730940 TI - Immunoblot analysis demonstrates that the large and small forms of hepatitis delta virus antigen have different C-terminal amino acid sequences. AB - Antisera to a peptide representing the extreme carboxy terminus of the hepatitis delta virus antigen (HDAg) open reading frame (residues 197 to 211) recognized only the large (p27 delta) and not the small (p24 delta) form of HDAg in immunoblots of infected liver extracts, thereby providing direct proof that p27 delta and p24 delta differ in their carboxyl-terminal sequence and that p27 delta results from mutation within the stop codon terminating translation of p24 delta. Reactions with other peptide antisera demonstrated that multiple smaller virus specified proteins were carboxy-terminally truncated forms of HDAg, and immunoprecipitation studies suggested that different forms of HDAg were present as heterologous complexes within the liver extract. PMID- 1730941 TI - Proteins of bovine herpesvirus type 4 released into the culture medium of productively infected cells: identification of a 135K glycoprotein involved in viral attachment. AB - Three bovine herpesvirus type 4 (BHV-4) proteins released into the culture medium of infected cells were identified, with Mr values of 135K, 16K and 14.5K. Among these three proteins, two were precipitated by the monoclonal antibodies characterized in this work. One is a glycoprotein of 135K (gp8) which does not seem to be involved in BHV-4 neutralization. Moreover, this 135K glycoprotein adsorbed onto uninfected susceptible cells. The attachment of gp8 to cells was totally inhibited by the prior adsorption of unlabelled viral proteins. Moreover, anti-gp8 monoclonal antibodies were effective in inhibiting the adsorption of gp8. These results indicate that gp8 could be involved in BHV-4 attachment. PMID- 1730942 TI - Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells. AB - The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma (S49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4.9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5.4, different from that of the virus preincubated at low pH without target membranes. PMID- 1730943 TI - Cells surviving infection by human immunodeficiency virus type 1: vif or vpu mutants produce non-infectious or markedly less cytopathic viruses. AB - Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non infectious or less cytopathic virus. PMID- 1730944 TI - Synthesis and toxicity of full-length and truncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells using a baculovirus vector. AB - Full-length (72K) and truncated (61K) CryIVD mosquitocidal proteins of Bacillus thuringiensis (Bt) were expressed in Spodoptera frugiperda cells and larvae of Trichoplusia ni using a baculovirus vector to investigate the role of CryIVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The cryIVD genes were inserted into the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) under control of the polyhedrin promoter by recombination in S. frugiperda cells between a transfer vector carrying the Bt genes and vDA26Z, a recombinant AcMNPV carrying the Escherichia coli beta-galactosidase gene under control of the DA26 promoter. Recombinant AcMNPVs carrying the genes were detected as blue occlusion body-negative plaques in monolayers of S. frugiperda cells grown in the presence of X-Gal. Infection of S. frugiperda cells and T. ni larvae with plaque-purified recombinant virus, expressing either the full-length or truncated CryIVD protein, resulted in the synthesis of proteins of the expected size, as confirmed by immunoblot analyses, and their crystallization into cuboidal inclusions in the cytoplasm. Infected cells and purified inclusions from the virus (AcCryIVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCryIVD-C) expressing the truncated protein with a 9.6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CryIVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic full-length protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action. PMID- 1730945 TI - Selection bias, survival, and brachytherapy for glioma. AB - Interstitial irradiation is a promising treatment for malignant glioma. Longer than expected survival periods following treatment of recurrent tumor have led to the use of brachytherapy as an adjuvant treatment. The impact of patient selection on survival data was studied among candidates for this therapy. Consecutive, conventionally treated adults with newly diagnosed supratentorial tumors were identified retrospectively at a center where experience with glioma is population-based. Based on imaging and performance status, two surgeons and a radiation oncologist designated each patient as either eligible or ineligible for adjuvant brachytherapy. The survival and prognostic factors in the eligible and ineligible groups were analyzed. Overall, the patients eligible for brachytherapy (32% of the series) lived significantly longer than the ineligible patients (16.57 vs. 9.30 months), were younger, and had larger resections and better function. For glioblastoma, 40% of patients were eligible, and lived much longer than those who were ineligible (13.90 vs. 5.80 months). It is concluded that better outcome following adjuvant brachytherapy for glioma is at least in part the result of patient selection. Randomized trials of comparably selected patients will be necessary to demonstrate conclusively that longer survival is also a result of treatment. PMID- 1730946 TI - A 1- to 4-year follow-up review of treatment of sciatica using chemonucleolysis or laminectomy. AB - To help clarify the comparative effects of chemonucleolysis and discectomy, the author studied 178 consecutive patients with sciatica who did not respond to conservative treatment. None had previously undergone laminectomy or chemonucleolysis or had spinal stenosis. All received postmyelography computerized tomography (CT) and, if the radiological interpretation was that of an extruded migrated disc, a laminectomy was performed; otherwise, the patient was given a choice of the two procedures. Of the 178 patients, 106 underwent chemonucleolysis and 72 laminectomy. Workers' compensation was being paid to 21.6% of the chemonucleolysis patients and 20.8% of the laminectomy patients. Postoperatively, substantial improvement was noted in 82.7% of the chemonucleolysis patients and 92.5% of the laminectomy patients at 6 weeks and in 92.8% of the chemonucleolysis patients and 89.7% of the laminectomy patients at 6 months. The majority of patients in both groups had improved neurological signs. Follow-up questionnaires at 1 to 4 years postoperatively revealed an overall success rate of 86.5% for chemonucleolysis patients and 83.8% for laminectomy patients. In patients not receiving workers' compensation, 90.1% of the chemonucleolysis patients and 88.6% of the laminectomy patients had a successful outcome; in those receiving workers' compensation, 69.6% of the chemonucleolysis patients and 60.0% of the laminectomy patients had a successful outcome. No statistically significant differences in improvement rate in neurological symptoms or signs were identified between the two procedures. Overall, 85.1% of the chemonucleolysis patients and 78.5% of the laminectomy patients were employed at follow-up review. To achieve optimum results and eliminate noncandidates for chemonucleolysis, routine use of postmyelography CT is recommended. When properly used, chymopapain chemonucleolysis is an acceptable alternative to surgical discectomy. PMID- 1730947 TI - Nerve transfer in brachial plexus traction injuries. AB - Brachial plexus palsy due to traction injury, especially spinal nerve-root avulsion, represents a severe handicap for the patient. Despite recent progress in diagnosis and microsurgical repair, the prognosis in such cases remains unfavorable. Nerve transfer is the only possibility for repair in cases of spinal nerve-root avulsion. This technique was analyzed in 37 patients with 64 reinnervation procedures of the musculocutaneous and/or axillary nerve using upper intercostal, spinal accessory, and regional nerves as donors. The most favorable results, with an 83.8% overall rate of useful functional recovery, were obtained in patients with upper brachial plexus palsy in which regional donor nerves, such as the medial pectoral, thoracodorsal, long thoracic, and subscapular nerves, had been used. The overall rates of recovery for the spinal accessory and upper intercostal nerves were 64.3% and 55.5%, respectively, which are significantly lower. The authors evaluate the results of nerve transfer and analyze different donor nerves as factors influencing the prognosis of surgical repair. PMID- 1730948 TI - The extended frontal approach to tumors of the anterior, middle, and posterior skull base. AB - The extended frontal approach is a modification of the transbasal approach of Derome. The addition of a bilateral orbitofrontal or orbitofrontoethmoidal osteotomy improves the exposure of midline lesions of the anterior, middle, and posterior skull base, while minimizing the need for frontal lobe retraction. The authors present a 5-year experience with 49 patients operated on via the extended frontal approach. In seven patients, the extended frontal approach was used alone; in the remaining 42, it was combined with other skull base approaches. Highly malignant tumors were removed en bloc, whereas benign tumors and low-grade malignancies were removed either en bloc or piecemeal. Reconstruction was usually performed using fascia lata, a pericranial flap, and/or autologous fat. A temporalis muscle flap or a distant microvascular free flap was required for some patients. One patient died 1 month postoperatively due to superior mesenteric artery thrombosis. Three patients had postoperative infections, two had cerebrospinal fluid leaks requiring reoperation, and four had brain contusions or hematomas. All but two patients recovered to their preoperative functional level. After an average follow-up period of 26 months (range 6 to 56 months), 64% of patients with benign lesions, 64% of patients with low-grade malignancies, and 44% of patients with high-grade lesions were alive with no evidence of disease. PMID- 1730949 TI - Effect of head elevation on intracranial pressure, cerebral perfusion pressure, and cerebral blood flow in head-injured patients. AB - The traditional practice of elevating the head in order to lower intracranial pressure (ICP) in head-injured patients has been challenged in recent years. Some investigators argue that patients with intracranial hypertension should be placed in a horizontal position, the rationale being that this will increase the cerebral perfusion pressure (CPP) and thereby improve cerebral blood flow (CBF). However, ICP is generally significantly higher when the patient is in the horizontal position. This study was undertaken to clarify the issue of optimal head position in the care of head-injured patients. The effect of 0 degree and 30 degrees head elevation on ICP, CPP, CBF, mean carotid pressure, and other cerebral and systemic physiological parameters was studied in 22 head-injured patients. The mean carotid pressure was significantly lower when the patient's head was elevated at 30 degrees than at 0 degrees (84.3 +/- 14.5 mm Hg vs. 89.5 +/- 14.6 mm Hg), as was the mean ICP (14.1 +/- 6.7 mm Hg vs. 19.7 +/- 8.3 mm Hg). There was no statistically significant change in CPP, CBF, cerebral metabolic rate of oxygen, arteriovenous difference of lactate, or cerebrovascular resistance associated with the change in head position. The data indicate that head elevation to 30 degrees significantly reduced ICP in the majority of the 22 patients without reducing CPP or CBF. PMID- 1730950 TI - Prognostic significance of magnetic resonance imaging in the acute phase of cervical spine injury. AB - Fifty-seven patients with acute cervical spine injuries and associated major neurological deficit were examined within 2 weeks of injury by magnetic resonance (MR) imaging. All patients had abnormal scans, indicating intramedullary lesions. This study was undertaken to determine if the early MR imaging pattern had a prognostic relationship to the eventual neurological outcome. Three different MR imaging patterns were observed in these patients: 21 patients had patterns characteristic of intramedullary hematoma (Group 1); 17 had intramedullary edema over more than one spinal segment, but no hemorrhage (Group 2); and 19 had restricted zones of intramedullary edema involving one spinal segment or less (Group 3). The neurological state was determined using standard motor index scores at admission and at follow-up examination. Characteristically, the patients in Group 1 had admission motor scores significantly lower than the other two groups. At follow-up examination, the median percent motor recovery was 9% for Group 1, 41% for Group 2, and 72% for Group 3. These studies suggest that the MR imaging pattern observed in the acutely injured human spinal cord has a prognostic significance in the final outcome of the motor system. It is only when an accurate prognosis can be given at the outset that useful treatment data might be collected for homogeneous injury groups, and accurately based long-term planning made for the best patient care. PMID- 1730951 TI - Giant interhemispheric cysts associated with agenesis of the corpus callosum. AB - Interhemispheric cysts, often associated with agenesis of the corpus callosum, are rare lesions demonstrating little uniformity of pathogenesis. Four large interhemispheric cystic lesions with several unique features are reviewed. Magnetic resonance imaging clearly showed agenesis of the corpus callosum and was useful in the diagnosis of interhemispheric cysts. The differential diagnosis of these lesions is discussed, along with therapeutic considerations. PMID- 1730952 TI - Lobar intracerebral hemorrhage. A clinical, radiographic, and pathological study of 29 consecutive operated cases with negative angiography. AB - The authors operated consecutively on 50 patients with lobar intracerebral hemorrhage during a prospectively designed study period from January, 1986, to March, 1990. They investigated the correlations between the underlying causes and the clinicoradiographic features in 29 patients who showed no angiographic vascular abnormalities, in order to elucidate the operative indication for such cases. Patients with ruptured saccular aneurysm or trauma were not included in this study. There were 15 males and 14 females, ranging in age from 7 to 76 years (mean 52.4 years). Histological diagnoses of the surgical specimens were as follows: vascular malformation in nine cases (arteriovenous malformation (AVM) in six and cavernous malformation in three), microaneurysm in 11, cerebral amyloid angiopathy in six, and brain tumor in two; in the remaining case the cause was not verified histologically. The underlying cause was determined in 96.5% of cases. The mean patient age was lowest in the cavernous malformation group (27.0 years), followed by the AVM (45.8 years), microaneurysm (59.8 years), and cerebral amyloid angiopathy (70.0 years) groups. Four patients with vascular malformation (three AVM's and one cavernous malformation) had previous episodes of bleeding at the same site, whereas none of those with microaneurysms or cerebral amyloid angiopathy had such episodes. On computerized tomography (CT) scans, the round to oval hematoma was related to the presence of an AVM or cavernous malformation in contrast to microaneurysms and cerebral amyloid angiopathy. Upon infusion of contrast material, variable enhancement was seen in five (two AVM's and three cavernous malformations) of the nine vascular malformations while no enhancement was noted in any patient with microaneurysm or cerebral amyloid angiopathy at the acute stage. Subarachnoid extension of the hematoma was associated with cerebral amyloid angiopathy significantly more frequently than with AVM's (p less than 0.05) and microaneurysms (p less than 0.01). The results suggest that clinicoradiographic pictures in cases with negative angiography are quite different among the three major pathological categories; namely, vascular malformation (AVM and cavernous malformation), microaneurysm, and cerebral amyloid angiopathy. It is suggested that the underlying etiology of a given lobar intracerebral hemorrhage with negative angiography may be predicted by a combination of patient age, history of previous bleeding at the same site, hematoma shape, and subarachnoid extension of the hematoma on CT scans. Based upon these findings, the authors discuss operative indications for such cases. PMID- 1730953 TI - An analysis of the venous drainage system as a factor in hemorrhage from arteriovenous malformations. AB - The authors studied the venous drainage system and its impairment in relation to risk of hemorrhage in 108 cases of supratentorial arteriovenous malformation (AVM). The proportion of AVM's undergoing hemorrhage (hemorrhagic rate) was calculated in relation to: 1) the number of draining veins (one, two, or three or more); 2) the presence or absence of impairment in venous drainage (severe stenosis or occlusion in draining veins); and 3) the location of draining veins (deep venous drainage alone, superficial venous drainage alone, or a combination of the two). Statistical analysis demonstrated that AVM's with the following characteristics had a high risk of hemorrhage: 1) one draining vein (hemorrhagic rate 89% in 54 patients); 2) severely impaired venous drainage (hemorrhagic rate 94% in 18 patients); and 3) deep venous drainage alone (hemorrhagic rate 94% in 32 patients). The present study suggests that the venous drainage system of AVM's is significantly associated with the risk of hemorrhage of these lesions. Therefore, careful preoperative angiographic evaluation of the venous drainage system is mandatory for decision making in the management of patients with AVM's. PMID- 1730954 TI - In vitro analysis of the proliferative potential of T cells from patients with brain tumor: glioma-associated immunosuppression unrelated to intrinsic cellular defect. AB - Patients harboring a malignant brain tumor have been described as being highly immunosuppressed, as evidenced by reduced numbers of T cells and the decreased ability of their lymphocytes to produce interleukin-2 (IL-2). In order to determine whether an intrinsic abnormality exists in the T lymphocytes of glioma patients and to evaluate what role corticosteroids may play in glioma-associated immunosuppression, in vitro T cell proliferative function in the presence of recombinant IL-2 (rIL-2) was examined in age-matched groups of normal control subjects, steroid-free patients with glial tumors, steroid-dependent patients with glial tumors, and steroid-dependent patients with nonglial cerebral tumors. The results demonstrated that, when enriched T cell populations of all brain tumor patients were stimulated with rIL-2 and phytohemagglutinin (PHA), there were no statistically significant differences between any groups. In contrast, when T cell populations were stimulated with mitogenic combinations of phorbol ester, calcium ionophore, and rIL-2, those from steroid-dependent patients with glial tumors had a significantly lower response than those from normal control subjects, suggesting that a population of T cells capable of responding to phorbol ester/ionomycin and not PHA stimulation is inhibited by corticosteroid therapy in glioma patients. In addition, T cells of four brain-tumor patient/age matched control subject pairs were stimulated with either phorbol ester/ionomycin or PHA for 24 hours; three of the four patients expressed low-affinity IL-2 receptor levels as high or higher than their respective control subjects, suggesting that IL-2 receptor expression in these patients may be quantitatively normal once the T cell number is corrected. Taken together, these results show that the decreased PHA responsiveness that has been previously reported in lymphocytes of glioma patients is not due to a cellular abnormality within the potentially responsive cells, but rather reflects the reduced proportion of T cells within their peripheral blood which, as a consequence, reduces the level of IL-2 production attained upon activation. PMID- 1730955 TI - In vivo magnetic resonance imaging of fetal cat neural tissue transplants in the adult cat spinal cord. AB - Magnetic resonance (MR) imaging was evaluated for its possible diagnostic application in determining the survival of fetal central nervous system tissue grafts in the injured spinal cord. Hemisection cavities were made at the T11-L1 level of eight adult female cats. Immediately thereafter, several pieces of tissue, either obtained from the fetal cat brain stem on embryonic Day 37 (E-37), from the fetal neocortex on E-37, or from the fetal spinal cord on E-23, were implanted into the cavities made in seven cats. The eighth cat served as a control for the effect of the lesion only. In another group of four animals, a static-load compression injury was made at the L-2 level. Seven weeks later, the lesion was resected in three cases and fragments of either fetal brain-stem or spinal cord tissue were introduced. A small cyst was observed in a fourth cat in the compression injury group and a suspension of dissociated E-23 brain-stem cells was injected into this region of cavitation without disturbing the surrounding leptomeninges. Five months to 2 years posttransplantation, MR imaging was performed with a 2.0-tesla VIS imaging spectrometer by acquiring multislice spin-echo images (TR 1000 msec, TE 30 msec) in both the transverse and sagittal planes. Collectively, these intermediate-weighted images revealed homogeneous, slightly hyperintense signals at the graft site relative to the neighboring host tissue in seven of the 11 graft recipients. Two of the remaining four cats exhibited signals from the graft site that were approximately isointense with the adjacent host spinal cord, and the final two cats and the lesion-only control presented with very hypointense transplant/resection regions. The hyperintense and isointense images were tentatively interpreted as representing viable graft tissue, whereas the hypointense transplant/resection sites were considered to be indicative of a lack of transplant survival or the absence of tissue in the lesion-only control animal. Postmortem gross inspection of fixed specimens and light microscopy verified the MR findings in the control animal in 10 of the 11 graft recipients by showing either transplants and/or cysts corresponding to the MR images obtained. In one cat in the hemisection group, histological analysis revealed a very small piece of graft tissue that was not detected on the MR images. Therefore, it is suggested that within certain spatial- and contrast resolution limits, MR imaging can reliably detect the presence of transplanted neural tissue in both the hemisected and compression-injured spinal cord of living animals.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1730956 TI - Regeneration of calvarial defects by a composite of bioerodible polyorthoester and demineralized bone in rats. AB - A study was performed to evaluate regeneration of defects in rat calvaria either unfilled or filled with a bioerodible polyorthoester only, demineralized bone only, or a composite of both. At 4 weeks, histological and radiographic studies showed that defects filled with a composite of bioerodible polyorthoester and demineralized bone or demineralized bone alone were bridged by bone. Unfilled defects or defects filled with polyorthoester only did not heal. The polyorthoester caused slight inflammation that subsided by 3 weeks, and only traces of the filler could be detected at 4 weeks. The polyorthoester provided local hemostasis when used either alone or in composites with demineralized bone. The composite implant was moldable, easily contoured, and technically easier to use than demineralized bone alone. PMID- 1730957 TI - Evoked potentials from direct cerebellar stimulation for monitoring of the rodent spinal cord. AB - Although the assessment of spinal cord function by electrophysiological techniques has become important in both clinical and research environments, current monitoring methods do not completely evaluate all tracts in the spinal cord. Somatosensory and motor evoked potentials primarily reflect dorsal column and pyramidal tract integrity, respectively, but do not directly assess the status of the ventral funiculus. The present study was undertaken to evaluate the use of evoked potentials, elicited by direct cerebellar stimulation, in monitoring the ventral component of the rodent spinal cord. Twenty-nine rats underwent epidural anodal stimulation directly over the cerebellar cortex, with recording of evoked responses from the lower thoracic spinal cord, both sciatic nerves, and/or both gastrocnemius muscles. Stimulation parameters were varied to establish normative characteristics. The pathways conducting these "posterior fossa evoked potentials" were determined after creation of various lesions of the cervical spinal cord. The evoked potential recorded from the thoracic spinal cord consisted of five positive (P1 to P5) and five negative (N1 to N5) peaks. The average conduction velocity (+/- standard deviation) of the earliest wave (P1) was 53 +/- 4 m/sec, with a latency of 1.24 +/- 0.10 msec. The other components followed within 4 msec from stimulus onset. Unilateral cerebellar stimulation resulted in bilateral sciatic nerve and gastrocnemius muscle responses; there were no significant differences (p greater than 0.05) in the thresholds, amplitudes, or latencies of these responses elicited by right- versus left-sided stimulation. Recordings performed following creation of selective lesions of the cervical cord indicated that the thoracic response was carried primarily in the ventral funiculus while the sciatic and gastrocnemius responses were mediated through the dorsal half of the spinal cord. It is concluded that the posterior fossa evoked potential has research value as a method of monitoring pathways within the ventral spinal cord of the rat, and should be useful in the study of spinal cord injury. PMID- 1730958 TI - Tension pneumocephalus associated with rupture of a middle fossa encephalocele. Case report. AB - Acquired nontraumatic (spontaneous) encephaloceles of the middle fossa are not common. Rupture of an encephalocele frequently leads to a cerebrospinal fluid fistula. Tension pneumocephalus consequent to rupture of an encephalocele has not been previously reported, but conceivably occurs by means of a ball-valve mechanism in the ensuing fistulous tract. An unusual case is presented of an elderly man who suffered acute life-threatening neurological symptoms from a tension pneumocephalus that likely developed from rupture of an acquired nontraumatic encephalocele of the left middle fossa. The symptoms correlated with the location of the intracranial abnormalities. The literature is reviewed and the pathophysiology of the lesion is discussed. PMID- 1730959 TI - Stenosis of the axis and cervical myelopathy. Case report. AB - A rare case is described of marked segmental stenosis of the axis secondary to developmental hypertrophy of the posterior neural arch causing cervical myelopathy. The patient made a remarkable recovery following decompressive laminectomy. PMID- 1730960 TI - Multilevel anterior cervical fusion using skull bone grafts. Case report. AB - The successful use of autogenous skull bone grafts for a C3-7 anterior cervical fusion is reported and compared with results using other bone grafts. A 51-year old man with C4-7 anterior cord compression due to spurs and ossified posterior longitudinal ligaments developed progressive tetraparesis following a minor head injury. He underwent anterior decompression and fusion. On two occasions an iliac graft had failed; however, a graft of autogenous skull bone was successful. The skull bone was found superior to bone from other sites, such as the iliac crest, rib, tibia, and fibula, showing sufficient strength and less morbidity. The skull may be a better source of graft material for multilevel anterior cervical fusion, which requires long and strong grafts. PMID- 1730961 TI - Skull base amyloidoma. Case report. AB - A mass lesion of amyloid involving the central nervous system is a rare finding. A 64-year-old woman presented with a large amyloidoma at the skull base causing neural tissue compression. The only accompanying disease was an asymptomatic renal cyst. The mass had caused destruction of the bone elements and pathological calcification as seen on x-ray films, computerized tomography (CT) scans, and magnetic resonance (MR) images, and was enhanced after injection of contrast medium on both CT scans and MR imaging. PMID- 1730962 TI - Lipoma involving the skull. Case report. AB - The case of an intraosseous lipoma involving the left frontal bone is reported. Lipomas of the bone are rare; only three cases of lipomas involving the skull have previously been reported. The differential diagnosis includes a healing bone infarction or fracture, meningioma, hemangioma, and fibrous dysplasia. Diagnosis prior to surgery is difficult. PMID- 1730963 TI - Intracerebral penetration of infrared light. Technical note. AB - Near infrared transmission spectroscopy of the human cerebrum may allow noninvasive evaluation of cerebral hemoglobin saturation in humans. The emerging spectroscopy configuration for this application is a side-by-side source-receiver construct. The ability of this spectroscopy paradigm to detect changes in intracerebral attenuation by selective injection of the infrared tracer indocyanine green into the internal and external carotid arteries during endarterectomy is evaluated in five adult patients. In all five, simultaneous two channel infrared transmission spectroscopy over the ipsilateral hemisphere documented tracer bolus transit with a signal-to-noise ratio greater than 100:1. In addition, the two channels could be configured to achieve depth resolution of the collected spectra. PMID- 1730964 TI - Percivall Pott: an 18th century neurosurgeon. AB - This paper examines neurosurgery in the 18th century and suggests that the origins of the specialty can be recognized at the time when surgeons began to use the neurological status of the patient as a guide for surgical intervention. Percivall Pott (1714-1788) was one of the leading surgeons in London in the 18th century. He is remembered through eponyms of Pott's puffy tumor, Pott's fracture, and Pott's disease. A review of his writings and those of his contemporaries indicates that these surgeons were aware of the importance of changes in level of consciousness after head injury. The recognition and significance of the lucid interval was described and understood as a neurosurgical sign in the 18th century. Because of Pott's pre-eminence in the surgery of his time through his writings and lectures, he should be considered one of the founders of neurosurgery as a separate surgical discipline. PMID- 1730965 TI - Neurosurgical liability: the expert witness. PMID- 1730966 TI - Reconstruction of the temporalis muscle. PMID- 1730967 TI - Intraventricular octreotide for cancer pain. PMID- 1730968 TI - Leukotrienes and cerebral edema. PMID- 1730969 TI - Cavernous sinus exploration. PMID- 1730970 TI - Lateral disc herniations. PMID- 1730971 TI - Brain tolerance to radiosurgery: correction. PMID- 1730972 TI - Cyclic oral phosphate and etidronate increase femoral and lumbar bone mineral density and reduce lumbar spine fracture rate over three years. AB - We have performed a study of the safety and efficacy of cyclic sequential oral phosphate, diphosphonate and calcium carbonate. Forty-two postmenopausal women with osteoporosis diagnosed by dual-photon absorptiometry were treated with a sequential cyclic regimen of oral phosphate for 3 days, etidronate for 2 wk, and then a calcium salt for 12 wk. This was repeated cyclically for 3 yr. They were rescanned after every two 101-day cycles. A control group of 20 patient receiving only the calcium salt was matched for age, time since menopause, race and sex. The group treated with cyclic phosphate, etidronate, and calcium regimen had 80% fewer fractures than the control group over 3 yr of follow-up. Significant response in halting bone mineral loss and increasing bone mineral density was seen in none of the controls but in 90% of treated patients' lumbar spine and 70% 80% of the three regions of the femoral neck examined. PMID- 1730973 TI - Uptake mechanism of technetium-99m-d, 1-HMPAO in cell cultures of the dissociated postnatal rat cerebellum. AB - The accumulation and retention mechanisms of 99mTc-d, 1-hexamethylpropyleneamine oxime (99mTc-d, 1-HMPAO) were investigated in cultures of the dissociated rat cerebellum. Our experiments indicate a linear dependency of the uptake on incubation time and on the concentration of the radioligand. Upon chloroform extraction and distribution between the lipophilic and the hydrophilic phases, we located 69.1% of the retained radioactivity in the hydrophilic phase, 24.1% in a bound state and 6.8% in the lipophilic phase. The water-soluble, unbound radioactive contents of the cultures were identified as 99mTcO4- by HPLC analysis. Treatment of cultures with diethyl maleate (DEM) inhibited the accumulation of radioactivity along with a reduction of the GSH contents of the cultures. However, even in the absence of GSH, significant amounts of radioactivity were accumulated. DEM reduced the radioactive contents of cultures predominantly by diminishing the aqueous phase of the chloroform-extracted material. By contrast, the metabolic state, manipulated by treating the cultures with oligomycin B or 2,4-dinitrophenol, had no significant effect on the accumulation of radioactivity. Our experiments suggest two major mechanisms for the retention of radioactivity following the exposure of neuronal tissue to 99mTc d, 1-HMPAO: Conversion of the lipophilic complex to the hydrophilic product, 99mTcO4-, and binding to non-diffusible cell components. PMID- 1730974 TI - Unique scintigraphic findings of bile extravasation in the presence of ascites: a complication of hepatic transplantation. AB - A 99mTc-HIDA scan was performed on a 4-mo-old female, six days after hepatic transplantation. Gradually, a diffuse increase in activity was seen over the peritoneal region, consistent with a slow bile leak into ascitic fluid. Although the scintigraphic appearance of a bile leak has been previously described, it is usually seen as a focal area of extrabiliary activity. In this case, we report a pattern identified when the leak occurs in conjunction with ascites. PMID- 1730975 TI - Reversible increased technetium-99m-HMPAO cerebral cortical activity: a scintigraphic reflection of luxuriant hyperperfusion. AB - A hemiparetic and aphasic patient, 3 days after acute traumatic transection of the left internal carotid artery requiring life-saving total embolic occlusion, revealed ipsilateral increased peripheral hemispheric 99mTc-HMPAO activity. Ten days postocclusion, HMPAO peripheral cortical flow normalized as hemiparesis and aphasia significantly cleared. The initial lateralized HMPAO hyperactivity pattern may reflect reactive hyperemia, a sign previously identified by contrast angiography and often associated with a better prognosis in evolving CVA. Evanescent peripheral cerebral hyperemia may represent beneficial cortical collateralization of the periinfarct area of a deeper lacunar (white matter) CVA. PMID- 1730976 TI - A thallium scan goes to court. PMID- 1730977 TI - Comparison of left anterior oblique, anterior and geometric mean methods for determining gastric emptying times. AB - The accurate determination of gastric emptying time requires correction or compensation for tissue attenuation. The gold standard for tissue attenuation correction for gastric emptying is the geometric mean of the gastric counts from the anterior and posterior views. For reasons of efficiency, many community hospitals acquire only the anterior projection. This study addressed the hypothesis that, using the left anterior oblique view alone, one can minimize the effect of variation in attenuation as the meal moves from the fundus to the stomach to the more anterior antrum to a degree equal to that of the geometric mean technique. We studied 42 consecutive patients using a standardized 300-g meal labeled with 650 muCi of 99mTc-sulfur colloid. The patients were imaged in the anterior (ANT), posterior (POST) and left anterior oblique (LAO) views every 15 min for 90 min. Linear regressions were obtained using the ANT, LAO and GM data. Cross-correlation of the T1/2 for 35 cases showed an R value for the GM versus LAO of 0.95 and GM versus ANT of 0.84. The p value greater than 0.49, for the paired two-tailed t-test of the LAO and GM methods. The p value for the ANT and GM methods is 0.0058 indicating a significant difference between these methods. The cross-correlation, F-test p and t-test p values support the hypothesis that there is no significant difference between the geometric mean and left anterior oblique gastric emptying times. It is therefore reasonable to substitute the left anterior oblique for routine GET when using a solid meal in patients with normal gastric anatomy, albeit altered physiology. PMID- 1730978 TI - Simultaneous dual-isotope SPECT imaging for the detection and characterization of parathyroid pathology. AB - A technique is described involving simultaneous, dual-isotope SPECT imaging. Using 123I and 201TI, a patient study is presented, demonstrating the clinical utility of this technique in the detection and characterization of parathyroid pathology. The use of this technique in other clinical situations is discussed. PMID- 1730979 TI - Kinetic behavior of technetium-99m-HMPAO in the human brain and quantification of cerebral blood flow using dynamic SPECT. AB - The kinetic behavior of 99mTc-hexamethylpropyleneamine oxime (HMPAO) in the human brain was investigated in 11 patients with various brain diseases (176 regions), using dynamic SPECT and a four-compartment model with five parameters (K1: rate constant for the transport of HMPAO from blood to brain, K2: backdiffusion from brain to blood, K3: conversion to a hydrophilic form in the brain, K5: conversion to a nondiffusible form in the blood, and fa: fraction of radioactivity attributable to the vascular compartment). Although K1 correlated well with cerebral blood flow (CBF) measured by the 133Xe inhalation method (Xe-CBF) (r = 0.888), its value was underestimated by 28.9% +/- 11.9%, indicating a low extraction fraction (E) for HMPAO. From E = K1/CBF, a regression equation of E = 0.857 - 0.00335. Xe-CBF was obtained. Significant correlations were observed for K2 versus Xe-CBF (r = 0.679), for K3 versus Xe-CBF (r = 0.483), for K3/(K2 + K3) versus Xe-CBF (r = -0.487), for K3/K2 versus Xe-CBF (r = -0.501), and for K2 + K3 versus Xe-CBF (r = 0.655), but not for K1/K2 versus Xe-CBF (r = 0.005). Thus, this model was useful for elucidating the kinetic behavior of HMPAO in the human brain, and correction for E appears to be indispensable for accurate CBF quantification using HMPAO. PMID- 1730980 TI - Safety and role of repeated administrations of Indium-111-labeled anti carcinoembryonic antigen monoclonal antibody ZCE 025 in the postoperative follow up of colorectal carcinoma patients. AB - The safety and clinical utility of repeated administrations of 111In-ZCE 025 were evaluated in 25 patients who have undergone colorectal carcinoma resection. Fifteen patients were clinically and radiologically free of recurrences and asymptomatic while 10 had rising CEA and/or symptoms. We repeatedly imaged the patients following intravenous administrations of 40 mg ZCE 025, every 4 to 6 mo. Side effects occurred in 16% of patients who received two or more infusions. Sixteen lesions were detected by immunoscintigraphy in 11 patients who were free of disease by CT scans or other imaging modalities. Ten recurrences were surgically confirmed in seven patients. Radiographic and clinical follow-up confirmed the remaining 6 Mab-positive lesions. Elevated human anti-mouse antibody (HAMA) titers were detectable in the sera of 30% and 64% of patients following the 1st and 2nd Mab injection respectively, but did not interfere with successful immunoscintigraphy nor correlated with the occurrence of side effects. This study suggests that a negative Mab scan indicates that a patient will remain free of recurrence; conversely, a positive scan was associated with recurrences of disease. PMID- 1730981 TI - An instant kit method for labeling antimyosin Fab' with technetium-99m: evaluation in an experimental myocardial infarct model. AB - An instant kit method for labeling antibody Fab' fragments was developed. The method utilizes a ligand exchange reaction between the intermediate complex 99mTc D-glucarate and the free sulfhydryl groups on the antibody Fab' fragment. Radiolabeling of the Fab' using generator eluate achieves quantitative 99mTc incorporation in less than 30 min at room temperature. The radiolabel is stable in human plasma for at least 24 hr and stable to incubation with 10 mM diethylene triaminepentaacetic acid (24 hr) and 1 mM diaminodithiol agent (up to 3 hr). Mouse biodistribution of 99mTc-antimyosin shows faster blood clearance and lower uptake in the lungs, liver, and spleen in comparison to 111In-antimyosin. Technetium-99m-antimyosin and 111In-antimyosin showed equivalent ability to detect myocardial infarct in a canine model. PMID- 1730982 TI - Federal deadline looms as activists and politicians challenge California site for low-level radioactive waste. PMID- 1730983 TI - Imaging of the human torso using cone-beam transmission CT implemented on a rotating gamma camera. AB - Radionuclide transmission CT generated on a rotating gamma camera can improve SPECT imaging by providing attenuation maps for attenuation compensation and for anatomical correlation. This paper demonstrates the feasibility and high quality of cone-beam transmission CT (CB-CT) of human subjects, in comparison to conventional parallel-ray CT, and evaluates some possible imaging protocols. Two CB-CT implementation modes, with a cone-beam collimator and without any collimator, were evaluated. Three human subjects of different dimensions were imaged. For the two smaller subjects, the CB-CT images were dramatically superior, in terms of noise and resolution, to those obtained with a parallel-ray geometry. The image noise was less by a factor of 6. CB-CT linear attenuation coefficients were found to be in close agreement with published values for various tissues. For the largest subject, image truncation produced a ring artifact at the edge, but inside the artifact, the image quality was still very good. Cone-beam images obtained without any collimator were acceptable, but photon scatter degraded the image contrast. PMID- 1730985 TI - Radiation safety for beginners. PMID- 1730984 TI - Bremsstrahlung imaging using the gamma camera: factors affecting attenuation. AB - Quantitative imaging of bremsstrahlung from pure beta emitters is proposed as a means for in vivo management of antibody therapy. The method involves the use of high-energy collimation, an empirically selected broad photon energy window to enhance detector sensitivity, and a Wiener restoration filter to compensate for system blur. The measured and filtered data were obtained for an idealized scattering medium and isolated spherical sources. An effective linear attenuation coefficient of about 0.13 cm-1 was determined from the raw image data of 32P. A coefficient of 0.14 cm-1 was determined after the images were restored using the Wiener filter. The measured attenuation was not significantly dependent on the size of the region of interest or the size of the source. Its variation was within the experimental error of measurement (+/- 5%). The measured sensitivity (6 x 10(-6) cps/Bq) was sufficient for imaging therapy doses of 32P or 90Y. PMID- 1730986 TI - The utility of single-photon absorptiometry and dual-energy x-ray absorptiometry. PMID- 1730987 TI - Quantification of myocardial blood flow. PMID- 1730988 TI - Single plasma GFR in children. PMID- 1730990 TI - SNM and ACNP respond to Medicare fee schedule. PMID- 1730989 TI - Procedures of choice in renal nuclear medicine. PMID- 1730992 TI - Radiolabeled monoclonal antibodies for cancer therapy and diagnosis: is it really a chimera? PMID- 1730991 TI - Phase I trial of iodine-131-chimeric B72.3 (human IgG4) in metastatic colorectal cancer. AB - Twelve patients with metastatic colorectal cancer participated in a Phase I trial of 131I-labeled chimeric B72.3 (human IgG4). Consecutive groups of patients received 18 mCi/m2, 27 mCi/m2 and 36 mCi/m2. No acute side effects related to antibody administration were noted. Bone marrow suppression was the only side effect; it was dose-dependent and correlated with whole-body radiation dose estimates. The lowest dose level produced no marrow suppression, whereas 27 mCi/m2 resulted in Grade 1 and 2 marrow suppression in two of three patients. The maximum tolerated dose was 36 mCi/m2 with all six patients at this dose level having at least Grade 1 and two patients with Grade 3 and 4 marrow suppression. Eight of 12 patients had radioimmune imaging of tumor sites at 5-22 days. Seven patients had an antibody response to initial infusion. On retreatment, whole-body kinetics and imaging were altered for patients with a high anti-ch-B72.3 response. Thus, chimeric B72.3 (IgG4) has limited utility as a means of delivering multiple therapeutic doses of 131I in the majority of patients; alternative strategies including second generation anti-TAG-72 monoclonal antibodies, other radioisotopes and other chimeric human isotypes will need to be pursued. PMID- 1730993 TI - A dual-radioisotope technique for the evaluation of penile blood flow during tumescence. AB - A technique is described for concomitant study of both arterial and venous penile blood flow during tumescence. Dual-isotope acquisition is started after labeling red cells in vivo with 99mTc. Xenon-133 in saline is then injected into the corpus cavernosum followed with vasoactive drugs to induce an erection. The resulting xenon and technetium time-activity curves are inputs for a one compartment model. In 14 subjects, the average peak arterial flow rate (PAF) for normal males was calculated as 13.0 +/- 1.28 ml/min (avg +/- s.d.) compared to 16.1 +/- 5.14 and 5.02 +/- 1.78 ml/min for patients with venous leak (VL) or arterial insufficiency (AI), respectively. Peak venous flows (PVF) were 4.25 +/- 1.17, 12.1 +/- 3.75, and 3.78 +/- 1.00 ml/min for normal, VL and AL respectively. Al patients have significantly lower PAF than normal (p = 0.002) or VL patients (p = 0.018), and VL patients had significantly higher PVF than normal (p = 0.012) or Al (p = 0.018). The technique may be helpful in the study of impotence. PMID- 1730994 TI - Vascular testing for impotence. PMID- 1730995 TI - Radionuclide assessment of penile corporal venous leak using technetium-99m labeled red blood cells. AB - In an attempt to evaluate penile corporal venous outflow, a method that utilizes intracorporal injection of Tc-RBC was developed and used in 20 patients with erectile dysfunction. Seven patients showed venous leak and 13 had normal venous outflow. Technetium-labeled RBC corporal clearance in the flaccid state and after intracorporal injection of papaverine (30 mg) and regitine (1 mg) were assessed in sequence by two separate injections of 18.5 MBq of Tc-RBC each. The time for 50% corporal clearance (T50%) was determined from the time activity curves obtained in flaccid state and after intracorporal injection of the vasoactive agent. There were no statistically significant differences in T50% measured in the flaccid state between normal venous outflow (202 +/- 139 sec) and venous leak (92 +/- 35 sec, p = 0.1). However, after intracorporal injection of papaverine and regitine a significant increase in the T50% was noted in normal venous outflow (2892 +/- 1899 sec) as compared to venous leak (213 +/- 123 sec, p less than 0.001). The results suggest that measurement of corporal clearance of Tc-RBC after intracorporal injection of papaverine may be a useful method in detecting venous leak, and could be used as a screening test in patients with erectile dysfunction. PMID- 1730996 TI - Technetium-99m-HMPAO SPECT in the evaluation of patients with a remote history of traumatic brain injury: a comparison with x-ray computed tomography. AB - The functional imaging modality has potential for demonstrating parenchymal abnormalities not detectable by traditional morphological imaging. Fifty-three patients with a remote history of traumatic brain injury (TBI) were studied with SPECT using 99mTc-hexamethylpropyleneamineoxime (HMPAO) and x-ray computed tomography (CT). Overall, 42 patients (80%) showed regional cerebral blood flow (rCBF) deficits by HMPAO SPECT, whereas 29 patients (55%) showed morphological abnormalities by CT. Out of 20 patients with minor head injury, 12 patients (60%) showed rCBF deficits and 5 patients (25%) showed CT abnormalities. Of 33 patients with major head injury, 30 patients (90%) showed rCBF deficits and 24 patients (72%) showed CT abnormalities. Thus, HMPAO SPECT was more sensitive than CT in detecting abnormalities in patients with a history of TBI, particularly in the minor head injury group. In the major head injury group, three patients showed localized cortical atrophy by CT and normal rCBF by HMPAO SPECT. In the evaluation of TBI patients, HMPAO SPECT is a useful technique to demonstrate regional brain dysfunction in the presence of morphological integrity as assessed by CT. PMID- 1730997 TI - Diagnosis of sternal wound infection by technetium-99m-leukocyte imaging. AB - An imaging study is needed that can detect sternal wound infections and distinguish between superficial and deep sternal wound infection when a clinical diagnosis is uncertain and a decision regarding surgical intervention must be made. We retrospectively reviewed the 99mTc-leukocyte scans of 29 patients referred to rule out sternal wound infection. The presence or absence of deep or superficial sternal wound infection was determined by microbiology and long-term follow-up. Images obtained 4 and 20 hr after injection were reviewed by two nuclear physicians who were blinded to the clinical history. Findings were categorized as normal or abnormal. Abnormal images were further defined as having intense uptake at 4 and 20 hr, increasing uptake between 4 and 20 hr, or other patterns such as focal cold regions, irregular uptake at 4 and 20 hr or increasing uptake between 4 and 20 hr were 100% sensitive and 89% specific for the detection of deep sternal wound infection. The images were also useful for determining the extent of infection. Superficial sternal wound infection could not be reliably detected. The results indicate that 99mTc-leukocyte imaging is useful for the diagnosis of deep sternal wound infection. PMID- 1730998 TI - Imaging inflammation: current role of labeled autologous leukocytes. PMID- 1730999 TI - Left ventricular diastolic function in systemic sclerosis: assessment by radionuclide angiography. AB - The aim of this study was to assess left ventricular function in subjects with systemic sclerosis. Twenty-four women with systemic sclerosis (mean age 48 +/- 11 yr) and 14 age- and sex-matched normal subjects were studied by radionuclide angiography performed at rest with a temporal resolution of 20 msec/frame. Left ventricular volume curves were generated and indices of systolic and diastolic function were computed. Left ventricular diastolic asynchrony was evaluated by dividing the left ventricle into five regions and then computing the time-to-peak filling rate for each region. After excluding the valvular region, the coefficient of variation of this index was obtained. The isovolumic relaxation period was prolonged in systemic sclerosis patients in comparison to normal subjects (127 +/- 39 msec versus 87 +/- 44 msec, p less than 0.05). Moreover, 38% of the systemic sclerosis patients had a subnormal peak filling rate. Left ventricular diastolic asynchrony was increased in the systemic sclerosis group, as expressed by a higher coefficient of variation of the regional time to peak filling rate (27.9% +/- 11.5% versus 14.5% +/- 8.6%, p less than 0.05). Our results indicate an impaired relaxation and an increased diastolic asynchrony in patients with systemic sclerosis. PMID- 1731000 TI - Influence of ureteral status on kidney washout during technetium-99m-DTPA diuresis renography in children. AB - To assess the influence of the ureter on renal washout during 99mTc-DTPA diuresis renography, ureteral images were reviewed in 42 children (median age: 5 mo) referred for hydronephrosis. Sixty-minute acquisitions were obtained in hydrated patients under bladder drainage. Furosemide was injected at 30 min. An abnormal ureter was defined as an intense and continuous image of greater than 10 min. A washout index was determined on renal (KT1/2) and ureteral (UT1/2) curves. Curve patterns corresponding to normal (type I), obstructive (II) and nonobstructive (III) cases were described. Compared with the x-ray data, diuresis renography was highly sensitive (91%) and specific (98%) for detecting any abnormality. Despite an obstructive KT1/2 (greater than 20 min), no patient with an abnormal ureter underwent therapy at the ureteropelvic junction. After surgery at the lower level, hydronephrosis regressed. Our data indicate that abnormal ureter findings at diuresis renography have to be recognized before planning therapy for children with hydronephrosis. PMID- 1731001 TI - Effects of ureteral function on assessment of hydronephrosis. PMID- 1731002 TI - Technetium-99m-DTPA aerosol and gallium-67 scanning in pulmonary complications of human immunodeficiency virus infection. AB - We retrospectively compared the results of 67Ga chest scans and 99mTc-DTPA aerosol clearance measurements with those of fiberoptic bronchoscopy in 88 patients infected with the human immunodeficiency virus. Of 100 investigations, a pulmonary infection was diagnosed in 39, mainly Pneumocystis carinii pneumonia and a noninfectious disorder was found in 42, mainly Kaposi's sarcoma and lymphocytic alveolitis. Gallium scans and DTPA clearance were abnormal respectively in 74% and 92% of infectious complications, and in 12% and 60% of noninfectious disorders. In 10 cases, DTPA clearance was accelerated, while chest x-ray, arterial blood gases and even gallium scanning were normal. A value of DTPA clearance greater than 4.5%.min-1 was both sensitive and specific for the diagnosis of Pneumocystis carinii pneumonia. The gallium scan was always normal in bronchopulmonary Kaposi's sarcoma. We conclude that in symptomatic patients: (1) DTPA clearance measurements are useful for detecting lung disease when chest x-ray and/or PaO2 are normal and (2) a gallium scan is indicated to distinguish progressive Kaposi's sarcoma from a superimposed second process when radiological abnormalities of pulmonary Kaposi's sarcoma are present. PMID- 1731003 TI - Absorbed radiation dose to humans from technetium-99m-teboroxime. AB - Tissue distribution data obtained in nine normal volunteers were used to estimate the radiation dose to humans after intravenous administration of 99mTc-teboroxime (Cardiotec). Organ uptake as percent of injected dose was measured using quantitative SPECT. Non-linear regression analysis was performed on the organ time-activity data using SYSTAT software. Cumulative activities in these organs were determined by calculating the area under the respective curves after accounting for the physical decay of the radionuclide. The absorbed dose to individual organs was estimated using the MIRDOSE 2 program. The gallbladder and the upper large intestine (ULI) are the target organs and will receive respectively 26.5 and 33.2 muGy/MBq (98 and 123 mrad/mCi) 99mTc-teboroxime under the assumption that the gallbladder empties every 6 hr. The dose to the gallbladder decreases at shorter emptying intervals; with intervals of 3, 4, and 5 hr, the respective doses to the gallbladder are 18.2, 21.0 and 23.7 muGy/MBq (67.4, 77.8, and 87.9 mrad/mCi) 99mTc-teboroxime. However, the dose to ULI remains almost constant at 123 mrad/mCi and will be the limiting factor. PMID- 1731004 TI - Diaphragm pacing in infants and children. PMID- 1731005 TI - A prospective, multicenter, randomized study of high versus low positive end expiratory pressure during extracorporeal membrane oxygenation. AB - To test the hypothesis that increased positive end-expiratory pressure (PEEP) could prevent deterioration of pulmonary function and lead to more rapid recovery of lung function, we randomly assigned 74 patients undergoing extracorporeal membrane oxygenation (ECMO) at four centers to receive either high (12 to 14 cm H2O) or low (3 to 5 cm H2O) PEEP. The two groups were similar in terms of weight, gestational age, diagnosis, and pre-ECMO course. All other aspects of care were identical. Dynamic lung compliance was measured at baseline and every 12 hours. Radiographs of the chest were obtained daily. Survival rates were similar in the two groups: 36 of 40 for low PEEP and 34 of 34 for high PEEP. The duration of ECMO therapy was 97.4 +/- 36.3 hours in the high-PEEP group and 131.8 +/- 54.5 hours in the low-PEEP group (p less than 0.01). Dynamic lung compliance throughout the first 72 hours of ECMO was significantly higher in patients receiving high PEEP. Radiographic appearance of the lungs correlated well with lung compliance: patients receiving high PEEP had significant deterioration of the radiographic score less frequently than those receiving low PEEP. High PEEP also was associated with significantly fewer complications. We conclude that PEEP of 12 to 14 cm H2O safely prevents deterioration of pulmonary function during ECMO and results in more rapid lung recovery than traditional lung management with low PEEP. PMID- 1731006 TI - Left ventricular mechanics in the preterm infant and their effect on the measurement of cardiac performance. AB - The effects of the transition from fetal to postnatal circulation on left ventricular geometry, wall motion, and echocardiographic measurements of function in the human preterm infant are largely unknown. To determine whether abnormalities in left ventricular geometry are present in the normal preterm infant after birth and, if so, for how long, and to examine possible contributing factors and their effect on the measurement of cardiac performance, we obtained serial echocardiograms of 14 healthy preterm infants (gestational age, 33 +/- 2 weeks; birth weight, 1940 +/- 470 gm) at 9.5 +/- 3.5 days of age (time 1) and again at 51 +/- 16 days (time 2). Left ventricular shape and wall motion were measured and estimates of wall stress and mass were made. Performance was assessed by standard M-mode shortening fraction and by transverse two-dimensional area shortening. At time 1 septal flattening caused distortion of left ventricular shape. As the patients grew older, septal flattening resolved and the left ventricle tended to assume a circular cross-sectional shape. Wall-motion analysis demonstrated poor motion of the midseptum and anterior free wall at time 1, which improved at time 2 (p = 0.06). Left ventricular mass increased from 24 +/- 5 to 41 +/- 7 gm/m2 (p = 0.0001) and wall stress decreased from 49 +/- 21 to 38 +/- 13 gm/cm2 (p = 0.005) between time 1 and time 2. Shortening fraction was lower at time 1 than at time 2 (18% +/- 7% vs 28% +/- 8%; p = 0.001; normal limit = 28% to 45%); however, there was no significant difference in area shortening between time 1 and time 2 (49% +/- 10% vs 53% +/- 8%; normal limit = 45% to 65%). We conclude that the preterm newborn infant has distorted left ventricular shape and abnormal wall motion, which alter measurements of shortening fraction and persist for the first weeks of life. Area shortening may be necessary to assess left ventricular performance during the first weeks of life in the premature infant. PMID- 1731007 TI - Comparison of blood cultures with corresponding venipuncture site cultures of specimens from hospitalized premature neonates. AB - We compared the presence and identities of isolates from blood culture samples obtained by percutaneous venipuncture with those of commensal skin organisms cultured from respective venipuncture sites after skin cleansing; 677 blood and skin site culture pairs from 488 infants were compared. Organisms grew in 58 blood cultures; nine of these cultures had corresponding venipuncture site cultures that also grew organisms. Forty-two blood culture isolates were coagulase-negative staphylococci; five of these were associated with similar venipuncture site cultures. According to restriction-endonuclease fingerprinting of chromosomal DNA and plasmid analysis, three pairs of blood and venipuncture site cultures were identical and two pairs were different. Thus only 7% (3/42) of coagulase-negative staphylococcal blood isolates were associated with identical contamination at the venipuncture site. We conclude that, if the venipuncture site has been carefully cleansed, the growth of coagulase-negative staphylococci in blood cultures of specimens from premature neonates indicates bacteremia rather than skin contamination in the vast majority of cases. PMID- 1731008 TI - Congenital nasal stenosis in newborn infants. AB - Five newborn infants with evidence of nasal obstruction were shown to have congenital nasal stenosis. Conservative treatment, including temporary nasopharyngeal intubation in three, eventually resulted in symptomatic relief, so that surgery could be avoided. PMID- 1731009 TI - Upper airway obstruction in association with perinatally acquired herpes simplex virus infection. AB - Two cases of neonatal upper respiratory tract obstruction caused by herpes simplex virus are described. Infection of the upper respiratory tract with this virus should be included in the differential diagnosis of fever and stridor during the neonatal period. PMID- 1731010 TI - Pseudohypoaldosteronism in a preterm infant: intrauterine presentation as hydramnios. AB - After a pregnancy complicated by severe hydramnios, a preterm infant had clinical and biochemical evidence of pseudohypoaldosteronism. Fetal polyuria probably caused the hydramnios. PMID- 1731011 TI - Intravenously administered labetalol for treatment of hypertension in children. AB - Thirteen children (ages 9.2 +/- 3.7 years, mean +/- SD) received intravenous doses of labetalol, an alpha 1- and beta-adrenergic blocker, on 15 separate occasions for treatment of hypertension. In 12 of 15 episodes an initial dose of 0.55 +/- 0.34 mg/kg was given; in all 15 a continuous infusion of 0.78 +/- 0.39 mg/kg per hour was utilized for 67.3 +/- 57.1 hours. A significant decrease in systemic blood pressure occurred in all episodes (143/99.1 +/- 17.7/11.1 vs 115.6/72.4 +/- 7.7/9.5; p less than 0.01). A clinically unimportant yet statistically significant decrease in heart rate occurred during labetalol infusion (116.3 +/- 19.8 vs 107.8 +/- 11 beats/min; p less than 0.01). The episodes in children with creatinine clearances greater than 50 (n = 6) were compared with those with creatine clearances less than 20 ml/min per 1.73 m2 (n = 9); similar doses of labetalol were required for control of blood pressure. We conclude that infusion of labetalol is effective for control of blood pressure in children with hypertension, regardless of renal function. PMID- 1731012 TI - Tetracycline-induced esophagitis in adolescent patients. AB - Ulcerative esophagitis may be caused by corrosive agents and by commonly prescribed medications. We report severe esophagitis in five adolescents after ingestion of tetracycline preparations with minimal water immediately before going to bed. PMID- 1731013 TI - Pure red cell aplasia and hepatitis in a child receiving isoniazid therapy. AB - A 7-year-old boy had pure red cell aplasia and clinically significant hepatitis during isoniazid therapy. The former complication had been reported only in adults, and the latter is rare in children. PMID- 1731014 TI - Pulmonary hypertension in a seventeen-year-old boy. PMID- 1731015 TI - Deficiency of IgG4 in children: association of isolated IgG4 deficiency with recurrent respiratory tract infection. AB - To study the relationship between serum IgG subclass deficiency and clinical host defense impairment, we reviewed the clinical and immunologic features of 123 patients with a history of recurrent infection who had been examined for immunodeficiency in our laboratory (group 1). We then compared immunoglobulin isotype levels with those in sera from 127 age-matched control subjects without recurrent infection from whom blood had been drawn for evaluation of atopy (group 2). There was a significantly higher prevalence of IgG4 deficiencies among patients with recurrent infections (17% vs 7%; p less than 0.02), solely because of a higher prevalence of isolated IgG4 deficiency (n = 9; 7.3%) than in atopic control subjects (n = 1; 0.8%; p less than 0.05); there was a comparable prevalence of multiple isotype deficiencies that included low levels of IgG4 (9.8% and 6.3%, respectively). All nine group 1 patients with isolated IgG4 deficiency had severe recurrent respiratory tract infections requiring multiple hospitalizations; in addition, five were atopic, five had asthma, and one had chronic diahrrea. Antibody responses to bacterial polysaccharide antigens were normal for age in all patients with isolated IgG4 deficiency; two had defective antibody responses to protein antigens. Isolated IgG4 deficiency appears to be associated with impaired respiratory tract defenses and may occur in the absence of an easily definable antibody deficiency state. This association suggests a physiologic defense role for mucosal IgG4. PMID- 1731016 TI - Acute splenic sequestration in a five-week-old infant with sickle cell disease. PMID- 1731017 TI - Cost-effectiveness of neonatal screening for sickle cell disease. PMID- 1731018 TI - Hyperphosphatasemia in GM1 gangliosidosis. PMID- 1731019 TI - Outpatient treatment of febrile infants 28 to 89 days of age with intramuscular administration of ceftriaxone. AB - STUDY OBJECTIVE: To determine the outcome of outpatient treatment of febrile infants 28 to 89 days of age with intramuscular administration of ceftriaxone. DESIGN: Prospective consecutive cohort study. SETTING: Urban emergency department. PATIENTS: Five hundred three infants 28 to 89 days of age with temperatures greater than or equal to 38 degrees C who did not appear ill, had no source of fever detected on physical examination, had a peripheral leukocyte count less than 20 x 10(9) cells/L, had a cerebrospinal fluid leukocyte count less than 10 x 10(6)/L, did not have measurable urinary leukocyte esterase, and had a caretaker available by telephone. Follow-up was obtained for all but one patient (99.8%). INTERVENTION: After blood, urine, and cerebrospinal fluid cultures had been obtained, the infants received 50 mg/kg intramuscularly administered ceftriaxone and were discharged home. The infants returned for evaluation and further intramuscular administration of ceftriaxone 24 hours later; telephone follow-up was conducted 2 and 7 days later. RESULTS: Twenty seven patients (5.4%) had a serious bacterial infection identified during follow up; 476 (94.6%) did not. Of the 27 infants with serious bacterial infections, 9 (1.8%) had bacteremia (8 of these had occult bacteremia and 1 had bacteremia with a urinary tract infection), 8 (1.6%) had urinary tract infections without bacteremia, and 10 (2.0%) had bacterial gastroenteritis without bacteremia. Clinical screening criteria did not enable discrimination between infants with and those without serious bacterial infections. All infants with serious bacterial infections received an appropriate course of antimicrobial therapy and were well at follow-up. One infant had osteomyelitis diagnosed 1 week after entry into the study, received an appropriate course of intravenous antimicrobial therapy, and recovered fully. CONCLUSIONS: After a full evaluation for sepsis, outpatient treatment of febrile infants with intramuscular administration of ceftriaxone pending culture results and adherence to a strict follow-up protocol is a successful alternative to hospital admission. PMID- 1731020 TI - Eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection. AB - Eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, has been suggested as having an important role in the pathogenesis of asthma. To determine whether ECP plays a similar role in bronchiolitis, we tested samples of nasopharyngeal secretions, obtained from a group of 47 children with various forms of illness related to respiratory syncytial virus (RSV) and from 26 children with non-RSV upper respiratory tract illness or bacterial pneumonia, for the presence of ECP by means of a double antibody radioimmunoassay. Concentrations of ECP in children with RSV bronchiolitis were significantly higher (166.8 ng/ml) than the mean concentration of ECP in both groups of children with RSV upper respiratory tract illness (43.5 ng/ml, p less than 0.002) and RSV lower respiratory tract disease without wheezing (29.1 ng/ml; p less than 0.0002). Children with non-RSV upper respiratory tract illness or bacterial pneumonia had levels of ECP in nasopharyngeal secretions similar to those of children with RSV upper respiratory tract illness or RSV pneumonia. High ECP levels in nasopharyngeal secretions (greater than 50 ng/ml) were predictive of the development of bronchiolitis at the time of RSV infection (p less than 0.001), and the individual ECP levels correlated with severity of the disease as determined by the initial PaO2 concentrations (p less than 0.05). These data suggest that eosinophil degranulation in the respiratory tract occurs during RSV bronchiolitis and may play a significant role in the development of virus-induced airway obstruction. PMID- 1731021 TI - Acute adenovirus hepatitis in liver transplant recipients. AB - An acute or fulminant adenovirus hepatitis developed in 5 of 224 pediatric patients who were recipients of orthotopic liver transplants. All had received prednisolone, azathioprine, and cyclosporine as basal immunosuppression, and four received monoclonal (OKT3) or polyclonal (antithymocyte globulin) antibodies for steroid-resistant rejection episodes. These patients initially had high fever and a worsening condition for a mean of 73 days after transplantation (range 44 to 140 days). Results of biochemical tests showed very high serum levels of lactate dehydrogenase. Aspartate aminotransferase values were always markedly more elevated than those of alanine aminotransferase. Two patients had severe leukopenia. Results of histologic studies of the liver showed extensive areas of confluent necrosis and targetlike hepatocyte nuclei. Typical intranuclear viral inclusions were observed on electron microscopy. Adenovirus was cultured in all patients and in two relatives. Two patients died of liver failure; others recovered after cessation of immunosuppression. We conclude that adenovirus hepatitis can be fatal in liver transplant recipients. There is no specific treatment, and immunosuppression must be discontinued. PMID- 1731022 TI - Use of calcium excretion values to distinguish two forms of primary renal tubular hypokalemic alkalosis: Bartter and Gitelman syndromes. AB - Clinical or biochemical findings were reevaluated in 34 pediatric patients with primary renal tubular hypokalemic metabolic alkalosis. The patients were subdivided into two groups. Bartter syndrome (primary renal tubular hypokalemic metabolic alkalosis with normocalciuria or hypercalciuria) was diagnosed in 18 patients with molar urinary calcium/creatinine ratios greater than 0.20, and Gitelman syndrome (primary renal tubular hypokalemic metabolic alkalosis with magnesium deficiency and hypocalciuria) was diagnosed in 16 patients with molar urinary calcium/creatinine ratios less than or equal to 0.20 and plasma magnesium levels less than 0.75 mmol/L. Some clinically important differences between the groups were observed. Patients with Bartter syndrome were often born after pregnancies complicated by polyhydramnios (8/18) or premature delivery (7/18) and had short stature (11/18) or polyuria, polydipsia, and a tendency to dehydration (16/18) during infancy (12/18) or before school age (18/18). Patients with Gitelman syndrome had tetanic episodes (12/16) or short stature (3/16) at school age (14/16). We conclude that the Bartter and Gitelman syndromes represent two distinct variants of primary renal tubular hypokalemic metabolic alkalosis and are easily distinguished on the basis of urinary calcium levels. PMID- 1731023 TI - Factors limiting the erythropoietin response in rapidly growing infants with congenital nephrosis on a peritoneal dialysis regimen after nephrectomy. AB - To prevent anemia in seven small children with congenital nephrotic syndrome of the Finnish type (age range 1 to 4 years), we gave recombinant human erythropoietin in a dose up to 150 IU/kg/per week. We then studied the limiting factors during 14 weeks. On a peritoneal dialysis regimen after nephrectomy, the patients grew considerably (range +0.1 to 2.2 kg/14 wk; mean + 1.3 kg/14 wk). The amount of blood taken for laboratory studies was estimated. Although the estimated erythrocyte volume increased, the improvement was masked in most patients by enhanced growth. In two patients the target hemoglobin value of 10 gm/dl was reached, and in three patients transfusions were avoided. The reticulocyte count rose in dose-dependent fashion. In five patients protein malnutrition was not prevented, although intake of protein was as recommended. The gradual decrease in serum ferritin values indicated that mobilization of iron stores was adequate. Serum iron values decreased, although in general remaining within normal limits. In six patients the serum copper concentration was low and in two the serum aluminum concentration was slightly elevated. Two patients had several episodes of infection. We conclude that in rapidly growing infants and small children receiving peritoneal dialysis after nephrectomy, the maintenance or elevation of the hemoglobin concentration depends on several limiting and coinciding factors. We speculate that, when protein is limited, body growth has priority over erythropoiesis. A higher dose of erythropoietin might have evoked a better response in hemoglobin concentration but might also have resulted in progression of the protein deficit. PMID- 1731024 TI - Chronic relapsing thrombotic thrombocytopenic purpura in infants with large von Willebrand factor multimers during remission. AB - We studied two children with recurrent schistocytic hemolytic anemia and thrombocytopenia beginning in the neonatal period. One patient had a stroke during one of the episodes of thrombotic thrombocytopenic purpura. The presence of unusually large von Willebrand factor multimers was demonstrated in both children during clinical and hematologic remissions. Treatment with corticosteroids and intravenous injections of immune globulin was unsuccessful in the one patient who received it. Immediate improvement occurred in both patients after the infusion of fresh-frozen plasma. Symptoms of thrombocytopenia continue to recur at regular intervals in the absence of periodic fresh-frozen plasma infusions. One of these children apparently has chronic relapsing thrombotic thrombocytopenic purpura; the second has a chronic relapsing disorder similar to thrombotic thrombocytopenic purpura. PMID- 1731025 TI - Less intensive long-term transfusion therapy for sickle cell anemia and cerebrovascular accident. AB - To determine the efficacy of a less intensive transfusion regimen in preventing recurrent cerebrovascular accidents and reducing transfusion requirements in patients with sickle cell anemia, we offered to 14 patients who had been undergoing aggressive transfusion therapy (sickle hemoglobin concentration kept less than 30% of total) for a mean of 9 years the option of either diminishing or stopping transfusion therapy. Thirteen patients chose to continue a modified transfusion regimen to maintain sickle hemoglobin concentration less than 60%; 10 of these patients have now been followed for 1 year or more (12 to 27 months, mean 15.5 months). There have been no recurrent neurologic events, although two patients have died of complications of hemochromatosis. All patients had a reduction in donor exposure, and there was a mean reduction in net transfusion requirement of 31.4% during the first year after modification. The greatest reduction was achieved in the single patient managed by small-volume (5 ml/kg) simple transfusion rather than partial packed cell exchange. We conclude that although long-term consequences of less aggressive transfusion therapy are unknown, the use of such a regimen may be reasonable, particularly in patients with significant transfusional hemochromatosis. PMID- 1731026 TI - Accuracy of central venous pressure monitoring in the intraabdominal inferior vena cava: a canine study. AB - STUDY OBJECTIVE: To test the hypotheses that in multiple pathophysiologic settings (1) end-expiratory central venous pressure measurements in the intraabdominal inferior vena cava accurately reflect those in the superior vena cava and (2) mean central venous pressure monitoring is as reliable in the inferior vena cava as it is in the superior vena cava. DESIGN: Simultaneous inferior vena caval and superior vena caval pressures were measured during five ventilatory phases: apnea, end-expiratory mechanical ventilation, maximal inspiratory mechanical ventilation, end-expiratory spontaneous ventilation, and maximal inspiratory spontaneous ventilation. Measurements were repeated after progressive intravascular volume depletion. SUBJECTS: Eight puppies. MEASUREMENTS AND RESULTS: Simultaneous inferior vena caval and superior vena caval end expiratory pressures did not differ significantly (mean differences 0 to 0.1 mm Hg) and the limits of agreement of these measurements were within 2 mm Hg. Differences between mean maximal inspiratory pressures in the inferior vena cava and superior vena cava during mechanical and spontaneous ventilation were -0.7 and 3.6 mm Hg, respectively (p less than 0.01), and the limits of agreement extended beyond 2 mm Hg. Furthermore, mean maximal inspiratory pressures in the superior vena cava differed from end-expiratory pressures in the superior vena cava (1.1 and -3.6 mm Hg, p less than 0.01), whereas those in the inferior vena cava did not differ from end-expiratory superior vena caval pressures. CONCLUSIONS: Under the experimental conditions studied (1) end-expiratory intraabdominal inferior vena caval pressures accurately reflected end-expiratory superior vena caval pressures and (2) mean central venous pressure monitoring was as reliable in the inferior vena cava as in the superior vena cava. PMID- 1731027 TI - Measuring the comparative efficacy of antibacterial agents for acute otitis media: the "Pollyanna phenomenon". AB - In randomized, double-blind trials of antibiotic therapy for acute otitis media that determined both clinical and bacteriologic outcomes, clinical success rates were (93%) 236 of 253 for patients with bacteriologic success, (62%) 25 of 40 for those with bacteriologic failure, and (80%) 124 of 155 for those with nonbacterial acute otitis media. These rates were used to calculate the effectiveness of three strategies for assessing drug efficacy: (1) tympanocentesis and culture before and during therapy (bacteriologic efficacy), (2) tympanocentesis before therapy and assessment of clinical efficacy in bacterial acute otitis media, and (3) no tympanocentesis and assessment of clinical efficacy in clinical (total) acute otitis media. For a drug with a bacteriologic efficacy of 100%, calculated clinical efficacy was 93% for bacterial acute otitis media and 89% for clinical acute otitis media. For a drug with bacteriologic efficacy of 27%, a rate consistent with no antibacterial therapy, efficacy was 71% for bacterial acute otitis media and 74% for clinical acute otitis media. We conclude that if efficacy is measured by symptomatic response, drugs with excellent antibacterial activity will appear less efficacious than they really are and drugs with poor antibacterial activity will appear more efficacious than they really are. The predominant phenomenon is that drugs with poor antibacterial activity will appear to be clinically effective in the treatment of acute otitis media. PMID- 1731028 TI - Hypokalemia, hypomagnesemia, and alkalosis: a rose is a rose--or is it? PMID- 1731029 TI - Bacteriology of acute otitis media: a new perspective. AB - Pathogenic bacteria were isolated from 90% of patients with acute otitis media. This higher-than-expected rate of positive cultures was probably related to the meticulous bacteriologic techniques used. PMID- 1731030 TI - Safety and immunogenicity of acellular diphtheria-tetanus-pertussis and Haemophilus conjugate vaccines given in combination or at separate injection sites. AB - This prospective, double-blind, randomized trial compared the immunogenicity and reactogenicity of acellular diphtheria-tetanus-pertussis vaccine and Haemophilus influenzae type b conjugate vaccine-diphtheria toxoid conjugate, given at separate injection sites or at a single site, in 79 children 18 months of age who had received three prior immunizing doses of whole-cell diphtheria-tetanus pertussis vaccine. No significant differences were observed. PMID- 1731031 TI - Breast-feeding and urinary tract infection. AB - A case-control study was conducted to study the association between breast feeding and urinary tract infection. Case patients were 128 infants aged birth to 6 months with urinary tract infection. Control infants were 128 infants admitted to the same ward with an acute illness. The results support the hypothesis that breast-feeding protects infants against urinary tract infection. PMID- 1731033 TI - Meningoencephalitis in a neonate congenitally infected with human immunodeficiency virus type 1. AB - A newborn infant born to a mother infected with human immunodeficiency virus type 1 had acute meningoencephalitis on the second day of life. Human immunodeficiency virus type 1 was isolated from the plasma, cerebrospinal fluid, and peripheral blood mononuclear cells. Specific IgM for human immunodeficiency virus type 1 was detected by an enzyme-linked immunosorbent assay antibody-capture technique in cord blood and in serum obtained 3 weeks later. We believe that the meningoencephalitis was caused by human immunodeficiency virus type 1 acquired in utero. PMID- 1731032 TI - Perceived exertion and exercise intensity in children with or without structural heart defects. AB - To assess the ability of children with cardiac disease to quantify their effort during exercise with the Borg perceived exertion scale, and to determine the validity of the scale for use with children by comparing the ratings with direct measurements of exercise intensity, we exercised 36 children with various cardiac defects and 15 normal children to exhaustion with the Bruce treadmill protocol. The subjects were able to quantify exercise intensity, so perceived exertion ratings can be used to predetermine the level of exercise intensity in an unmonitored setting and may be useful in defining appropriate exercise programs. PMID- 1731034 TI - Sodium restriction versus daily maintenance replacement in very low birth weight premature neonates: a randomized, blind therapeutic trial. AB - To test the hypothesis that restriction of sodium intake during the first 3 to 5 days of life will prevent the occurrence of hypernatremia and the need for administration of large fluid volumes, we prospectively and randomly assigned 17 babies (mean +/- SD: 850 +/- 120 gm; 27 +/- 1 weeks of gestation) to receive in blind fashion either daily maintenance sodium or salt restriction with physician prescribed parenteral fluid intake. Maintenance-group infants received 3 to 4 mEq of sodium per kilogram per day; restricted infants received no sodium supplement other than with such treatments as transfusion. Sodium balance studies conducted for 5 days demonstrated that maintenance salt intake resulted in a daily sodium balance near zero, whereas sodium-restricted infants continued to excrete urinary sodium at a high rate, which promoted a more negative balance (average daily sodium balance -0.30 +/- 1.78 SD in maintenance group vs -3.71 +/- 1.47 mEq/kg per day in restriction group; p less than 0.001). Care givers tended to prescribe daily increases in parenteral fluids for the salt-supplemented infants, perhaps because serum sodium concentrations were elevated in these infants after the first day of the study (p less than 0.001). Hypernatremia developed in two sodium supplemented infants (greater than 150 mEq/L), and hyponatremia developed in two sodium-restricted infants (less than 130 mEq/L); however, the restricted infants were more likely to have normal serum osmolality (p less than 0.05). Both groups of infants produced urine that was neither concentrated nor dilute, with a high fractional excretion of sodium; renal failure was not observed. The mortality rate was not affected, but the incidence of bronchopulmonary dysplasia was significantly less in the sodium-restricted babies (p less than 0.02). We conclude that in tiny premature infants, a fluid regimen that restricts sodium may simplify parenteral fluid therapy targeted to prevent hypernatremia and excessive administration of parenteral fluids. PMID- 1731035 TI - Cyclooxygenase dependency of the renovascular actions of cytochrome P450-derived arachidonate metabolites. AB - The renovascular effects of cytochrome P450-dependent arachidonic acid (P450-AA) metabolites synthesized by rat and rabbit kidneys were studied in the rabbit isolated kidney under conditions of constant flow and examined for their dependency on cyclooxygenase relative to their expression of vasoactivity. Kidneys were perfused with Krebs-Henseleit solution, and perfusion pressure was raised to levels of 90 to 110 mm Hg with the addition of 2 to 3 microM phenylephrine to the perfusate. Close arterial injection of 1 to 20 micrograms of 5,6-, 8,9- and 11,12-epoxyeicosatrienoic acid (EET) dose-dependently decreased perfusion pressure. The 5,6-EET was the most potent and the only epoxide dependent on cyclooxygenase for expression of vasoactivity, being inhibited by indomethacin (2.8 microM). In contrast, 14,15-EET resulted in dose-dependent increases in perfusion pressure. The vasodilator effects of the omega- and omega 1 oxidation products, 20-hydroxyeicosatetraenoic acid (HETE) and the stereoisomers of 19-HETE, were also inhibited by indomethacin. Furthermore, the renal vasodilator responses to 5,6-EET were not inhibited by either superoxide dismutase (10 U) or catalase (40 U) and, therefore, were unrelated to the formation of oxygen radicals generated during transformation of the epoxide by cyclooxygenase. As 5,6-EET and 19- and 20-HETE are synthesized by the renal tubules and can affect movement of salt and water, expression of vasoactivity by P450-dependent arachidonic acid metabolites, and after release from a nephron segment, may represent a mechanism that couples altered renal tubular function to appropriate changes in local blood flow. PMID- 1731036 TI - Paradoxical effect of chronic fentanyl treatment on naltrexone-induced supersensitivity and upregulation. AB - The present study sought to evaluate the influence of chronic opioid antagonist treatment upon the discriminative stimulus and analgesic effects of the opioid receptor agonist fentanyl. Male Wistar rats were trained to discriminate fentanyl (0.04 mg/kg) from saline in a two-lever food reinforced paradigm. After acquisition of the discrimination, they were implanted with osmotic minipumps which delivered either naltrexone (0.07 mg/h) or distilled water, and the sensitivity of discrimination was assessed at various times after pump removal. The influence of chronic naltrexone treatment upon the antinociceptive effects of fentanyl was assessed in drug-naive (control) rats and in rats which had received fentanyl in the same dosage schedule as those in drug discrimination experiments. Chronic infusion of naltrexone for 7 days did not modify the dose-response curve for the fentanyl vs. saline discrimination. Algesiometric tests revealed a significant increase in the antinociceptive effect of fentanyl in control rats after naltrexone treatment. In contrast, such supersensitivity was not observed in rats which had previously received fentanyl injections. Autoradiographic data revealed a naltrexone-induced upregulation of mu opioid receptors in control animals. Paradoxically, this effect was significantly increased in fentanyl pretreated rats. These data suggest that prior drug experience can affect the development of antagonist-induced supersensitivity to the behavioral actions of opioid agonists. Furthermore, it would appear that after chronic agonist treatment the phenomena of opioid receptor upregulation and functional supersensitivity are dissociated. PMID- 1731037 TI - After GM1 ganglioside treatment of sensory neurons naloxone paradoxically prolongs the action potential but still antagonizes opioid inhibition. AB - Low (nanomolar) concentrations of opioid agonists prolong the calcium-dependent component of the action potential duration (APD) of many dorsal root ganglion (DRG) neurons, whereas higher (micromolar) levels shorten the APD. Both effects are blocked by naloxone (1-10 nM). Opioid-induced APD prolongation appears to be mediated by excitatory opioid receptors that are positively coupled via a cholera toxin-A-sensitive Gs protein to adenylate cyclase/cyclic AMP-dependent ion conductances, whereas opioid-induced APD shortening is mediated by inhibitory receptors linked via pertussis toxin-sensitive Gi/Go proteins. Cholera toxin-B subunit, which binds to GM1 ganglioside, also selectively blocks opioid-induced APD prolongation. After brief treatment with GM1 ganglioside, the opioid agonists, dynorphin (1-13) or morphine, prolong the APD at femtomolar vs. the usual nanomolar concentrations, whereas no significant alterations were observed in the sensitivity of these GM1-treated cells to opioid inhibitory effects elicited by higher opioid concentrations. The present study shows that the opioid antagonists, naloxone or diprenorphine (1-30 nM), did not alter the APD of naive DRG neurons. In contrast, after GM1 treatment (1 microM, greater than 10 min), both opioid antagonists (but not (+)naloxone) unexpectedly prolonged the APD of most of the GM1-treated cells, but still continued to antagonize opioid-induced APD shortening. These results suggest that the supersensitivity of GM1-treated DRG neurons to the excitatory effects of opioid agonists and antagonists is due primarily to a remarkably increased efficacy of excitatory Gs-coupled opioid receptor functions, similar to the opioid excitatory supersensitivity that we have recently observed in chronic opioid-treated DRG neurons. PMID- 1731038 TI - Chronic marijuana smoke exposure in the rhesus monkey. II: Effects on progressive ratio and conditioned position responding. AB - Sixty-two male rhesus monkeys were trained to respond in an operant test battery that included tasks thought to allow measurement of aspects of motivation and color and position discrimination. Subjects were assigned to eight treatment groups (n = 7-8) based upon behavioral performance. There were two behavioral groups: ACTIVE = behavior assessed throughout the 365 days of active exposure and beyond, and RESIDUAL = behavior assessed beginning 2 months after the last exposure. Each behavioral group had four dose groups: HI = smoke from one marijuana (MJ) cigarette/day 7 days/week; LO = MJ smoke only on weekends; EX = smoke from one extracted MJ (placebo) cigarette/day 7 days/week; SH = sham exposure 7 days/week. For the motivation task, both HI and LO ACTIVE groups earned significantly fewer reinforcers than did both ACTIVE control groups during the last several months of exposure. These effects disappeared within 2 to 3 months of cessation of treatment, and no similar effect was present when RESIDUAL groups were tested. Performance of the color and position discrimination task was adversely affected in one of eight HI ACTIVE subjects throughout most of the chronic exposure, and there was a trend toward residual deficits in performance of this task in the HI RESIDUAL group compared to both SH and EX RESIDUAL controls. These data could be interpreted to mean that during periods of chronic use, MJ produces an amotivational-like syndrome in rhesus monkeys and that this syndrome disappears only several weeks to months after the last exposure. PMID- 1731039 TI - In vivo evaluation of the antagonist activity of angiotensin I analogs. AB - The objectives of this study were to identify angiotensin I (ANGI) analogs that antagonize angiotensin II (ANGII) receptors and/or block angiotensin converting enzyme (ACE) in vivo, to determine whether any receptor antagonist activity was direct (i.e., mediated by the intact peptide) vs. indirect (i.e., mediated by conversion of the intact peptide to corresponding smaller peptide fragments via ACE) and to determine whether the potency of a given ANGI analog was influenced by circulation through a tissue (lungs) rich in ACE. The effects of six ANGI analogs on mesenteric vascular responses to intramesenteric artery (IMA) infusions of ANGI and ANGII were examined in nephrectomized rats in the absence and presence of captopril. ANGI analogs were infused either intravenously (i.v.) or IMA. It is concluded that: 1) the ANGI analogs did not inhibit ACE sufficiently to attenuate responses to ANGI; 2) Thr8-ANGI does not block ANGII receptors, either directly or indirectly; 3) infused IMA Ile8-ANGI is a weak direct antagonist; however, given i.v., its potency is increased 30-fold due to conversion by pulmonary ACE; 4) Phe4-Tyr8-ANGI is a moderately potent indirect antagonist infused either IMA or i.v.; 5) infused IMA Cys8-ANGI is a moderately potent direct antagonist, and given i.v., its potency is increased 20-fold due to conversion by pulmonary ACE; 6) Val8-ANGI is a highly potent antagonist infused either IMA or i.v. due to both direct and indirect actions; and 7) Leu8-ANGI is a highly potent antagonist infused either IMA or i.v., and its antagonist properties do not require ACE. Possible applications of these findings are discussed. PMID- 1731040 TI - Effect of long-acting calcium entry blocker (anipamil) on blood pressure, renal function and survival of uremic rats. AB - To assess more completely the long-term effect of a long-acting calcium channel blocker, anipamil was given p.o. to rats with subtotal (five-sixths) nephrectomy. The mortality rates in anipamil-treated and control groups were 5% and 20%, respectively, at 6 weeks after separation (P less than .01) and 10% and 55%, respectively, at 10 weeks after separation (P less than .01). Mean arterial blood pressure was also more well controlled in the anipamil-treated group (144 +/- 36 vs. 192 +/- 35 mm Hg; n = 20; P less than .05 at week 5). In paired experiments, the degree of renal impairment in the placebo- and anipamil-treated groups, just before the onset of preterminal azotemia, was determined. Rats that were treated with anipamil had lower serum creatinine concentrations, compared with the placebo controls, at 4 to 6 weeks after separation (0.55 +/- 0.02 vs. 0.87 +/- 0.02 mg/100 ml; P less than .05). To dissociate this beneficial effect of anipamil from mean arterial blood pressure control, experiments were also performed to assess the effects of hydralazine on the remnant kidney model of chronic renal failure. Rats with remnant kidneys were divided into three groups and treated with anipamil (2 mg/kg/day), hydralazine (80 mg/liter in drinking water) or control. The anipamil-treated group exhibited significantly greater protection of renal function than did the hydralazine-treated group for the same level of blood pressure control. Thus, a long-acting calcium channel entry blocker such as anipamil may afford an additional cytoprotective effect in the prevention of progression of chronic renal failure beyond the antihypertensive effects of the agent. PMID- 1731041 TI - Different mechanisms of relaxation of pig coronary artery to bradykinin and cromakalim are distinguished by potassium channel blockers. AB - Bradykinin relaxes porcine coronary artery in an endothelium-dependent manner that is not dependent on release of nitric oxide or cyclic GMP accumulation. The mechanism of this relaxation was investigated in rings of porcine coronary artery by comparing bradykinin-induced relaxation with that induced by cromakalim, an agent know to cause hyperpolarization mediated by potassium channels. Relaxation to bradykinin was determined in rings treated with methylene blue, indomethacin and captopril to inhibit cyclic GMP accumulation, prostaglandin formation and bradykinin degradation, respectively. Relaxation to cromakalim was inhibited by the potassium channel blockers glybenclamide (10(-6) M), tetraethylammonium (10( 2) M), quinine (3 x 10(-5) M) and procaine (5 x 10(-3) M), whereas barium (10(-4) M) and 4-amino-pyridine (10(-3) M) were without effect. None of these potassium channel blockers had any effect on the relaxation to bradykinin. These results suggest that relaxation of pig coronary artery to cromakalim is mediated by a mechanism sensitive to potassium channel blockers. Also, the mechanism of nitric oxide-independent relaxation to bradykinin is distinct from that of cromakalim. PMID- 1731042 TI - Cromakalim effects of acetylcholine-induced changes in cytosolic calcium and tension in swine trachealis. AB - The effects of cromakalim, an ATP-sensitive K+ channel activator, on changes in cytosolic calcium concentration [( Ca++]i) and tension induced by acetylcholine (ACh; 0.1-10 microM) were examined in swine tracheal smooth muscle. Cromakalim (10 microM) hyperpolarized muscle cells by approximately 18 mV from -58 mV (resting membrane potential) to -76 mV. Cromakalim relaxed muscle contractions evoked by ACh at a concentration of 0.1 microM, but not at higher concentrations. Measurement of [Ca++]i using Fura-2 demonstrated that except at 0.1 microM ACh, cromakalim did not alter peak increases in [Ca++]i. At 0.1 microM ACh, the peak transient was decreased, but not eliminated. Cromakalim reduced steady-state increases in [Ca++]i at ACh less than or equal to 1 microM, but not 10 microM ACh. Tension was similarly affected. These data suggest that ACh-induced increases in steady-state [Ca++]i and tension are inhibited by cromakalim-induced hyperpolarization. The initial ACh-induced transient increase in [Ca++]i is not greatly altered. Cromakalim did not alter the transient peak tension and [Ca++]i relationship. The relationship between steady-state [Ca++]i/tension (EC50 = 321 nM) obtained for control, cromakalim inhibition and after glibenclamide reversal of cromakalim inhibition falls to the left of the peak transient [Ca++]i/tension relationship (EC50 = 587 nM). Thus, the Ca++ sensitivity of the contractile proteins during steady-state stimulation by ACh was increased from that at rest. We conclude that electromechanical coupling is important in ACh-induced contraction at concentrations less than 1 microM. Pharmacomechanical coupling with little or no sensitivity to changes in potential is important at higher ACh concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731043 TI - Lack of tolerance to a 24-hour infusion of S-nitroso N-acetylpenicillamine (SNAP) in conscious rabbits. AB - In order to design a means to preclude the development of nitrate tolerance, an in vivo model of nitroglycerin (GTN) tolerance was developed in the rabbit to examine the tolerance potential of S-nitroso vasodilators. Three separate studies were performed with 54 male New Zealand white rabbits which were instrumented for chronic infusion of vehicle or of either S-nitroso N-acetylpenicillamine (SNAP) or GTN (n = 6/group). Each vasodilator was infused for 24 hr at a dose rate of 60 nmol/kg/min. Three and 24 hr after the end of the infusion, each rabbit was anesthetized with pentobarbital and instrumented to monitor blood pressure, and dose-response curves were performed using SNAP or GTN. The dose-response curves were analyzed to determine the degree of displacement of the treated rabbit's dose-response curves compared to that of the vehicle-treated rabbit. Relative to the vehicle group, the GTN dose-response curve for systolic blood pressure in the GTN-infused rabbits was dextrally shifted 25.0-fold (P less than .05); 24 hr later the dextral shift was 0.9. The corresponding shifts in the SNAP dose response curves were 2.7- and 0.5-fold, respectively. When SNAP was infused for 24 hr, the corresponding 3- and 24-hr dextral shifts for GTN were: 7.8- (P less than .05) and 0.7-fold; for SNAP they were: 1.9- and 0.6-fold. In a third study, N-acetylcysteine was coinfused (0.14 mg/kg/min, 200 mg/kg cumulative dose) with GTN over 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731044 TI - Effects of captopril on blood pressure, placental blood flow and uterine oxygen consumption in pregnant rabbits. AB - Uterine renin may regulate uteroplacental blood flow locally through changes in vascular resistance or systemically by supporting arterial blood pressure. Captopril (5 mg/kg) was given i.v. to 14 conscious pregnant rabbits at day 27.5 +/- 0.3 of gestation for the purpose of investigating the effects of angiotensin converting enzyme inhibition on uteroplacental blood flow and oxygen consumption. Control measurements (mean +/- S.E.M.) were compared to measurements made at 1 hr (n = 14) and at 3 to 4 hr (n = 7). Arterial blood pressure decreased from 80 +/- 3 to 66 +/- 3 mm Hg, P less than .01, and then declined further to 56 +/- 4 mm Hg, P less than .01. Cardiac output was unchanged at 1 hr, 799 +/- 79 vs. 705 +/- 61 ml/min, but was decreased to 634 +/- 29 ml/min by 3 to 4 hr, P less than .01. There was no change in renal blood flow from 102 +/- 13 ml/min. Total uterine blood flow decreased from 37 +/- 5 to 29 +/- 5 ml/min, P less than .01, and then to 23 +/- 1 ml/min, P less than .01, whereas placental blood flow decreased from 25 +/- 4 to 19 +/- 3 to 15 +/- 3 ml/min, P less than .01; there was no significant change in myoendometrial flow. Oxygen delivery per uterine horn decreased from 2.4 +/- 0.3 to 1.8 +/- 0.4 to 1.6 +/- 0.2 ml/min, P less than .005. Oxygen consumption per horn decreased from 1.31 +/- 0.14 to 1.05 +/- 0.15 ml/min by 1 hr, P less than .05, and there was no further decrease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731045 TI - Misoprostol accelerates colonic mucosal repair in acetic acid-induced colitis. AB - The objectives of this study were 1) to determine whether misoprostol (MISO) (prostaglandin E1 analog) pretreatment protects the colonic mucosa from the injurious effects of acetic acid by attenuating the initial injury or by enhancing the rate of repair and 2) to assess the relationship between the protective effect of MISO pretreatment and mucosal ornithine decarboxylase activity in the inflamed colon. We found that the intrarectal administration of acetic acid caused rapid and extensive injury to the colonic mucosa, such that mucosal permeability increased 88-, 75-, 26-, 7.5- and 9.3-fold at 1, 2, 6, 24 and 48 hr after the enema, respectively. Intrarectal pretreatment with 50 micrograms of MISO for 30 min did not attenuate the increase in mucosal permeability at 1 hr after enema; however, it did significantly reduce mucosal permeability by 50 to 60% at 2, 6 and 48 hr after enema. We also demonstrated that acetic acid produced an 8.4-fold increase in colonic myeloperoxidase activity and a 1.8-fold increase in colonic weight at 48 hr after enema. MISO significantly reduced the increases in both myeloperoxidase activity and colon weight. Ornithine decarboxylase activity in the descending colon of vehicle pretreated animals increased significantly only at 24 hr after the acetic acid enema. In addition, MISO pretreatment followed by acetic acid enema resulted in significantly higher ornithine decarboxylase activities in the descending colon at 2 and 6 hr, compared with the vehicle plus acetic acid and MISO plus saline groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731046 TI - Clinical characteristics of naloxone-precipitated withdrawal in human opioid dependent subjects. AB - Studies were conducted to investigate the clinical characteristics of naloxone precipitated withdrawal in human opioid-dependent subjects. Each of 20 male patients stabilized on 24 mg of methadone daily received two i.v. pharmacological challenges: one with naloxone (0.05, 0.10, 0.15 and 0.20 mg; five patients each dose), and one with saline placebo. Measures of opioid withdrawal, affective state, cognitive performance and changes in autonomic parameters were assessed after each pharmacological challenge. Naloxone produced dose-dependent increases in opiate withdrawal scale scores and in symptoms of dysphoria as measured by the Profile of Mood States. Differences within subjects between naloxone and placebo infusions in Profile of Mood States scores were highly correlated with differences in opioid withdrawal as assessed by both subjective and objective rating scales. Naloxone also produced substantial increases in pulse, systolic and diastolic blood pressure and respiratory rate, as well as a small decrease in temperature. However, naloxone-induced changes from base-line values in these autonomic parameters correlated only modestly with other measures of opioid withdrawal. No differences between infusions were observed in two measures of cognitive performance (Stroop Color and Word Test, Digit Span Test). The results indicate that dysphoric mood states reflecting a broad range of affective experience must be considered as integral components of the naloxone-precipitated opioid withdrawal syndrome. PMID- 1731047 TI - Use-, concentration- and voltage-dependent limitation by MK-801 of action potential firing frequency in mouse central neurons in cell culture. AB - The anticonvulsant, (+/-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10 imine (MK-801), blocked single postsynaptic responses of mouse spinal cord neurons in cell culture to N-methyl-D-aspartate (NMDA). The block was concentration dependent with IC50 = 10(-7) M against 10(-5) M NMDA and 2 x 10(-7) M against 10(-3) M NMDA. Serial responses were blocked in use-dependent manner by 10 times lower doses of MK-801, depending on rate of NMDA application. MK-801 (approximate IC50, 8 x 10(-8) M) also limited sustained high-frequency repetitive firing of sodium-dependent action potentials (AP) elicited by long (400 msec) depolarizing pulses. Without changing resting membrane potential, single AP elicited by short (0.5-1 msec) depolarizing current pulses were blocked in voltage-, use- and frequency-dependent manner. Maximal rate of rise of individual AP elicited by short or long pulses decreased progressively until failure to fire. The time constant of recovery of AP from inactivation in a paired pulse protocol was prolonged from about 1 msec in control solution to 12 msec in solution containing 3 x 10(-7) M MK-801. These characteristics suggest that MK 801 blocks voltage-sensitive sodium current, which generates the upstroke of the AP. Overlap of concentrations blocking NMDA responses and sustained repetitive firing suggests that both actions may contribute to anticonvulsant efficacy of MK 801. PMID- 1731048 TI - Methimazole protection of rats against chemically induced kidney damage in vivo. AB - Because methimazole has antioxidant properties, the effects of methimazole treatment on cephaloridine, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), 2 bromohydroquinone (2-BHQ) and cis-diaminedichloroplatinum (II) (cisplatin) induced nephrotoxicity were investigated. Rats given cephaloridine (1 g/kg), cisplatin (5 mg/kg), DCVC (100 mg/kg) or 2-BHQ (157 mg/kg) i.p. exhibited significant elevations in blood urea nitrogen concentrations, which correlated with appearance of distinct renal histopathological changes. Cephaloridine, DCVC or 2-BHQ-induced nephrotoxicity was reduced only when methimazole (20-40 mg/kg) was given 30 min before the nephrotoxicant, whereas cisplatin-induced nephrotoxicity was reduced when methimazole was given 30 min before and up to 4 hr after cisplatin. Because the renal organic acid transport system plays an important role in the nephrotoxicity of cephaloridine, cisplatin and DCVC, the role of the organic acid transport system in the renal uptake of methimazole was investigated. With rat kidney cortical slices, methimazole uptake was time- and concentration-dependent; however, the organic acid transport substrates, probenecid (1 mM) and p-aminohippuric acid (7.5 mM), were ineffective in blocking methimazole uptake. Furthermore, cephaloridine (1 mM) uptake by kidney cortical slices was not affected by methimazole (5 mM). Rats given methimazole (40 mg/kg) 30 min before cephaloridine (2 g/kg) had serum and kidney cephaloridine concentrations similar to rats given cephaloridine only, but the methimazole pretreated rats were significantly protected against cephaloridine-induced oxidation of renal nonprotein thiols. These results show that methimazole does not inhibit the transport of cephaloridine into the kidneys, but may protect against cephaloridine-induced renal damage by acting as an antioxidant within the kidneys. PMID- 1731049 TI - [3H]nisoxetine: a radioligand for quantitation of norepinephrine uptake sites by autoradiography or by homogenate binding. AB - The uptake sites for norepinephrine (NE) in brain have not been studied in much detail, probably due to the absence of an adequate radioligand for labeling these sites. This study describes the binding properties of [3H]nisoxetine to uptake sites for NE in rat brain homogenates and in tissue slices analyzed by quantitative autoradiography. The binding of [3H]nisoxetine was found to be saturable and sodium-dependent to a single class of binding sites (Kd = 0.8 nM). The potencies of drugs to inhibit the uptake of NE correlated highly with their potencies to inhibit the binding of [3H]nisoxetine. Studies using [3H]nisoxetine for mapping of sites associated with uptake of NE by quantitative autoradiography indicated that the pattern of binding of [3H] nisoxetine is consistent with the pattern of noradrenergic innervation. Destruction of central noradrenergic neurons by 6-OH-dopamine or DSP-4 resulted in large decreases in the binding of [3H]nisoxetine in almost all areas of the brain regions examined. [3H]Nisoxetine should prove to be a useful tool to study the regulation of uptake sites for NE as well as a useful marker for noradrenergic innervation in the study of various neurological diseases. PMID- 1731050 TI - The sympathetic nervous system facilitates endothelin-1 effects on venous tone. AB - The effects of endothelin-1 (ET-1) or normal saline (0.9% NaCl) on mean arterial pressure (MAP), heart rate (HR) and mean circulatory filling pressure (MCFP), an index of body venous tone, were studied in 10 groups (n = 6 each) of conscious, unrestrained rats continuously i.v. infused with vehicle, verapamil, both hexamethonium and verapamil, phentolamine or, both phentolamine and verapamil. Infusion (i.v.) of normal saline into the five control groups did not significantly alter MAP, HR or MCFP. Cumulative i.v. bolus of ET-1 (0.8, 1.6, 3.2, 6.4, 12.8 and 19.4 x 10(-10) mol/kg) reduced HR in all five treatment groups and dose-dependently increased MAP in the presence of either vehicle or phentolamine, but did not affect MAP in the groups treated with verapamil. The ability of ET-1 to reduce HR in verapamil-treated rats, despite the absence of a pressor response, suggests that ET-1 is negatively chronotropic. ET-1 alone slightly increased MCFP and it did not alter MCFP in the presence of phentolamine. In the presence of verapamil, ET-1 markedly raised MCFP, and this was abolished by concurrent treatment with either hexamethonium or phentolamine. Therefore, ET-1 markedly elevates venous tone in the presence of verapamil via reflex-mediated increase in sympathetic nerve activity and the activation of alpha adrenoceptors. PMID- 1731051 TI - Preclinical studies on LY237733, a potent and selective serotonergic antagonist. AB - 8B-N-cyclohexyl-6-methyl-1(1-methylethyl)ergoline-8-carboxamide (LY237733) is an ergoline with potent and highly selective 5-hydroxytryptamine (5-HT) antagonist activity. The in vitro radioligand displacement studies showed that LY237733 has a preferential affinity for 5-HT1c and 5-HT2 receptors compared to other monoaminergic receptors. This characteristic is shared with other previously described ergoline 5-HT antagonists, such as LY53857 and sergolexole. In parallel ligand displacement assays, LY237733 had a similar potency to sergolexole. LY237733 was equipotent to sergolexole, but slightly less potent than LY53857 in the antagonism of 5-HT-induced elevation in blood pressure and quipazine-induced elevation in corticosterone levels, which are considered to be measures of 5-HT2 and possibly 5-HT1c antagonist activity. LY237733 failed to antagonize pergolide or 8-hydroxy-2-(di-n-propylamino)tetralin-induced elevations in serum corticosterone levels, indicating selectivity for the 5-HT1c/2 receptor, relative to 5-HT1a and D2 dopaminergic receptors. The only in vivo response that could be detected after administration of LY237733 alone in doses less than 1 mg/kg was the amplification of male rat sexual behavior. LY237733 was 10 to 100 times more potent than LY53857 or sergolexole in augmenting sexual responses of male rats with different levels of sexual response capacity. LY237733 has a much longer serum half-life than sergolexole. These studies have provided the pre-clinical rationale to evaluate the effects of this compound in the treatment of sexual disorders such as psychogenic erectile dysfunction, and other therapeutic indications for a 5-HT2 antagonist, including depression, anxiety, schizophrenia and migraine. PMID- 1731052 TI - Comparison of the thrombolytic activity of the novel plasminogen activator, LY210825, to anisoylated plasminogen-streptokinase activator complex in a canine model of coronary artery thrombolysis. AB - We have compared the thrombolytic efficacy of a novel single-chain, recombinant tissue-type plasminogen activator variant, LY210825, containing the second kringle and serine protease domains of native tissue-type plasminogen activator, with anisoylated plasminogen-streptokinase activator complex (APSAC). Male hounds (16-22 kg) were anesthetized, the left circumflex coronary artery was isolated and an electromagnetic flow probe was placed around the artery proximal to the first main branch for the measurement of coronary blood flow. An occlusive thrombus was formed after electrolytic injury of the intima of the coronary artery. After an occlusion period of 1 hr, either LY210825 (n = 8) or APSAC (n = 6) was administered as a single i.v. injection of 0.45 mg/kg. Blood was drawn (3.8% citrate) for determination of plasma fibrinogen, plasminogen and alpha-2 antiplasmin. Time to reperfusion was significantly faster with LY210825 than with APSAC, 20 +/- 2 vs. 54 +/- 8 min, respectively. The incidence of reocclusion was similar for both agents. APSAC produced significant depletion of alpha-2 antiplasmin, plasminogen and circulating fibrinogen, whereas LY210825 caused only slight consumption of plasminogen and only small decreases in fibrinogen. After a single injection of LY210825, thrombolytic concentrations of plasminogen activator were available immediately, whereas there was a significant delay in lytic concentrations of active streptokinase-plasmin complex. Consequently, LY210825 reperfused the coronary artery faster than did APSAC. In addition, LY210825 spared plasma fibrinogen, plasminogen and alpha-2 antiplasmin and therefore, could potentially minimize the risk of bleeding complications. PMID- 1731053 TI - Differences in teniposide disposition and pharmacodynamics in patients with newly diagnosed and relapsed acute lymphocytic leukemia. AB - Teniposide, a widely used investigational anticancer drug, is extensively bound to plasma proteins (greater than 95%). The present study evaluated the clearance and pharmacodynamics of total and unbound teniposide in patients with acute lymphocytic leukemia who were either in first complete remission or who had relapsed and achieved a subsequent complete remission. When compared to values of patients in first remission, the mean total systemic clearance of teniposide in relapsed patients was significantly lower at the time remission reinduction therapy was initiated, but increased to values greater than first remission patients after a subsequent remission was achieved. However, the mean clearance of unbound teniposide (ml/min/m2) was 3-fold lower in relapsed patients during reinduction therapy (1224 vs. 4261, P less than .0001), and improved but remained low after these patients achieved a subsequent remission (1965, P = .025). Changes in plasma protein binding accounted for the increase in total clearance when unbound clearance decreased. Continuous therapy with L-asparaginase was the major treatment difference in those patients with hypoalbuminemia and lower clearance of unbound teniposide. In 15 evaluable patients in complete remission, there was a statistically significant (P = .039) linear correlation between the percentage decrease in white blood cell count and the systemic exposure (AUC) to unbound teniposide, with higher exposure associated with a greater decrease in white blood cell count. There was not a significant correlation between the percent decrease in white blood cell count and the dosage given or the systemic exposure to total teniposide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731054 TI - Effects of the serotonin releasers 3,4-methylenedioxymethamphetamine (MDMA), 4 chloroamphetamine (PCA) and fenfluramine on acoustic and tactile startle reflexes in rats. AB - The substituted amphetamines 4-chloroamphetamine (PCA), 3,4 methylenedioxymethamphetamine (MDMA) and fenfluramine (FEN) share the common neurochemical action of acutely releasing central serotonin (5-HT), and yet their behavioral effects are quite different. The present study evaluated the effects of these compounds on acoustic and tactile startle reflexes. PCA and MDMA were qualitatively similar in producing dose-related increases in acoustic and tactile startle reflexes that were slow in onset, but sustained throughout the 3.5-hr test session. Changes in motor activity did not account for the observed excitation of startle. In marked contrast to MDMA and PCA, FEN did not alter tactile startle and tended to depress acoustic startle. The excitatory effect of 20 mg/kg of MDMA was prevented by the 5-HT uptake blockers MDL 27,777A and fluoxetine. MDMA excitation was not affected by a dose of the dopamine antagonist haloperidol that attenuated the startle-enhancing effect of d-amphetamine. MDMA excitation was greatly attenuated by a general depletion of central 5-HT produced by prior intraventricular injection of the 5-HT neurotoxin 5,7 dihydroxytryptamine. PCA and MDMA excitations of startle were attenuated in rats specifically depleted of spinal 5-HT or in rats with radio frequency lesions of the dorsal raphe nucleus. Thus, PCA and MDMA have similar prolonged excitatory effects on startle reflexes that are mediated by ascending (dorsal raphe) and descending (spinal) pathways, whereas FEN differs in its lack of excitation of startle. Differences in the neurochemical properties of these compounds or their patterns of 5-HT release may underlie their different behavioral profiles. PMID- 1731055 TI - Classification of plasmid-encoded dihydrofolate reductases conferring trimethoprim resistance. PMID- 1731057 TI - Antibiotic resistance in bacteria. PMID- 1731056 TI - A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh. AB - We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation. PMID- 1731058 TI - Specific antibodies in serum of patients with hydatidosis recognised by immunoblotting. AB - Hydatid fluids from sheep, goat, pig and man, after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions, revealed at least 15 discrete polypeptide bands of 8-116 Kda. By ELISA, sera from all 20 cases of hydatidosis showed anti-hydatid antibody, but so did 11 (73%) of 15 sera samples from cysticercosis patients, eight (67%) of 12 sera from patients with other parasitic infections (amoebic liver abscess or hymenolepiasis) and one (4%) of 25 sera from healthy controls. Antibody to cysticercus antigen was found in 14 (93%) of 15 sera from cysticercosis patients, 17 (85%) of 20 sera from hydatid patients, six (50%) of 12 sera from patients with other parasitic infections and one (4%) of 25 sera from healthy controls. Sera from 17 (85%) of 20 hydatid patients, 11 (73%) of 15 cysticercosis patients and five (42%) of 12 patients with other parasitic infections had antibodies to both hydatid and cysticercus antigens. Sera from 20 surgically confirmed cases of hydatidosis reacted with 12 polypeptides of 8-116 Kda in Western immunoblot with hydatid antigens. Polypeptides of 16, 24, 38, 45 and 58 Kda were recognised by all hydatidosis sera but also by many sera from patients with other infections. However, polypeptides of 8 and 116 Kda were recognised by all hydatidosis sera but not by any sera from patients with cysticercosis, other parasitic infections or viral hepatitis, or from healthy controls. Thus, recognition of 8- and 116-Kda hydatid antigens by a patient's serum appears to be a specific test confirming a clinical diagnosis in an individual case of hydatidosis. PMID- 1731059 TI - Aggregation of human granulocytes by Staphylococcus aureus lipase. AB - The effects of purified extracellular lipase from Staphylococcus aureus on human granulocytes were studied in vitro with a turbidimetric technique. Within the concentration range 0.6-4.4 micrograms/ml, lipase caused monophasic aggregation accompanied by the release of lactoferrin; the corresponding concentrations of the solvent in which it was suspended, Triton X100, had no effect. Lipase-induced aggregation did not occur in the presence of autologous plasma. PMID- 1731060 TI - Resistance to desiccation and skin fatty acids in outbreak strains of methicillin resistant Staphylococcus aureus. AB - Resistance to desiccation and to skin fatty acids was measured in three groups of methicillin-resistant Staphylococcus aureus (MRSA) strains and a group of control strains. Organisms from a large outbreak on a special care baby unit (SCBU), where MRSA had been isolated from staff hands but not from the environment, were significantly more sensitive to drying than strains from a burns unit where extensive environmental contamination had been demonstrated. MRSA from other wards, in the same hospital but not associated with large outbreaks, gave heterogeneous results. Fatty-acid resistance, determined by an agar dilution method, was not associated with strain origin. Some epidemic strains of MRSA were relatively sensitive to desiccation, and the abilities of such strains to spread widely on a SCBU by the hand-borne route could not be explained by enhanced resistance to skin fatty acids. PMID- 1731061 TI - Analysis of Aspergillus fumigatus catalases possessing antigenic activity. AB - Analysis of Aspergillus fumigatus water soluble fractions by electrophoresis on non-denaturing polyacrylamide gels (PAGE) showed the presence of at least three catalase bands. They were designated F, S1 and S2 in order of descending electrophoretic mobility with respect to the anode. The multiple enzyme forms appear to be distinct in their physicochemical properties. Enzyme bands S1 and S2 were simple catalases; the F band had an additional peroxidase function. All of the components were antigenic and differed in their binding to specific antibodies raised in rabbits with separate fractions of A. fumigatus mycelium. When serum from patients with aspergilloma, allergic bronchopulmonary aspergillosis, cystic fibrosis and chronic asthma were pre-incubated with A. fumigatus antigens and analysed by PAGE, 17 of 26 samples either abolished or reduced catalase activity. Enzyme F was a non-Concanavalin A (ConA)-binding antigen; the S1 and S2 enzymes were ConA-binding glycoprotein antigens. The major catalase band present in A. niger preparations represented only a minor component in A. fumigatus. PMID- 1731063 TI - Inhibitory effects of N- and C-terminal truncated Escherichia coli recA gene products on functions of the wild-type recA gene. AB - The effects of the expression of Escherichia coli truncated RecA protein on the host recA functions were examined. The recA gene on a multicopy plasmid was manipulated to express the truncated RecA protein from its carboxyl (C) and amino (N) terminal ends where a maximum of four extra amino acid residues was added. The regulatory part of the recA gene was substituted by the lacUV5 promoter in the plasmid to facilitate the artificial control of recA expression. Enzyme linked immunosorbent assay and Western blot analyses revealed great differences in accumulation of the truncated RecA proteins in the cell, depending on the location of the site of truncation. The expression of truncated proteins lacking 62, 77, 93 or 149 amino acid residues from the C-terminal end caused the host recA+ wild-type cell to become sensitive to ultraviolet irradiation and interfered with chromosomal recombination but did not interfere with the induction of lambda prophage. The expression of truncated RecA protein with 25 amino acid residues deleted from the C-terminal end caused the host cell to induce SOS functions constitutively. Truncated RecA proteins with 15 or 28 amino acid residues missing from the N-terminal end severely interfered with all of the host recA functions examined here. The effect of the loss of 41 amino acid residues from the N-terminal end of RecA was significant but less than the effect of proteins lacking 15 or 28 amino acid residues from the N-terminal end. A protein lacking 59 amino acid residues from the N-terminal end showed little interference with any measured recA functions, suggesting that the deletion of the region from around residues 41 to 59, which is rich in hydrophobic side chains, influenced the ability of the truncated protein to interfere with the functions of wild-type RecA protein. We also constructed a mutant gene with an internal deletion whose product was missing a region from residues 184 to 204. That mutant RecA protein was stably accumulated in the cell. This protein had little effect on the function of host wild-type recA gene product. The possible function of the regions at the N and C termini are discussed. PMID- 1731064 TI - C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs. AB - RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein. A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein. Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind. The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein. The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein. The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed. PMID- 1731062 TI - Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome. AB - Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level. PMID- 1731065 TI - Direct evidence for the effect of transcription on local DNA supercoiling in vivo. AB - The B-to-Z structural transition of varying lengths (74 to 14 base-pairs) of (CG) tracts has been used as a superhelicity probe to examine the local topological changes induced by transcription at defined genetic loci in vivo. The local topology reporter sequences indicate that under steady-state transcription the region upstream from the promoter experiences an increase in negative supercoiling whereas the region downstream from the terminator displays a decrease in negative superhelicity. This result provides direct in vivo evidence for the notion that the translocation of an RNA polymerase elongation complex along the double-helical DNA generates positive supercoils in front of it and negative supercoils behind it. Also, this twin-supercoiled domain model was tested inside a transcribed region where a high degree of negative supercoiling generated by the passage of each individual RNA polymerase was detected. Hence, these data indicate that the induced supercoils are confined to the vicinity of each RNA polymerase complex in a multipolymerase system. PMID- 1731066 TI - Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element. AB - We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin. PMID- 1731067 TI - Expectation maximization algorithm for identifying protein-binding sites with variable lengths from unaligned DNA fragments. AB - An Expectation Maximization algorithm for identification of DNA binding sites is presented. The approach predicts the location of binding regions while allowing variable length spacers within the sites. In addition to predicting the most likely spacer length for a set of DNA fragments, the method identifies individual sites that differ in spacer size. No alignment of DNA sequences is necessary. The method is illustrated by application to 231 Escherichia coli DNA fragments known to contain promoters with variable spacings between their consensus regions. Maximum-likelihood tests of the differences between the spacing classes indicate that the consensus regions of the spacing classes are not distinct. Further tests suggest that several positions within the spacing region may contribute to promoter specificity. PMID- 1731068 TI - Minigene rescues acetylcholinesterase lethal mutations in Drosophila melanogaster. AB - The gene encoding acetylcholinesterase in Drosophila melanogaster is over 34,000 base-pairs long. We have constructed a 5800 base-pair minigene containing 1500 base-pairs of genomic sequence upstream from the transcription start spliced to the coding sequence, but lacking the nine introns. After germline genetic transformation, this minigene rescues acetylcholinesterase lethal mutants. Tissue specific distribution appears normal. This allows us to test site-directed mutations of acetylcholinesterase. In a first effort, deletion of most of the unusual 1000 bases leader and its intriguing short open reading frames showed no effect on gene expression. The way is open to study in vivo the structure function relationships of acetylcholinesterase and insecticide resistance. PMID- 1731069 TI - Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA. AB - In Moloney murine leukemia virus, the encapsidation Psi element was shown to be necessary and sufficient to promote packaging of viral RNA, and to be required for dimerization. The conformation of the Psi domain (nucleotides 215 to 565) was investigated in solution by chemical probing. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate at cytosine N3 and adenosine N1, and with a carbodiimide derivative at guanosine N1 and uridine N3. Position N7 of adenine residues was probed with diethylpyrocarbonate. The analyses were conducted on in vitro transcribed fragments corresponding either to the isolated Psi domain or to the 5'-terminal 725 nucleotides. The RNA fragments were analyzed in their monomeric and dimeric forms. A secondary structure model was derived from probing data, computer prediction and sequence analysis of related murine retroviruses. One major result is that Psi forms an independent and highly structured domain. Dimerization induces an extensive reduction of reactivity in region 278 to 309 that can be interpreted as the result of intermolecular interactions and/or intramolecular conformational rearrangements. A second region (around position 215) was shown to display discrete reactivity changes upon dimerization. These two regions represent likely elements of dimerization. More unexpectedly, reactivity changes (essentially enhancement of reactivity) were also detected in another part of Psi (around position 480) not believed to contain elements of dimerization. These reactivity changes could be interpreted as dimerization-induced allosteric transitions. PMID- 1731070 TI - Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability. AB - Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase. PMID- 1731071 TI - GTPase activity of bacteriophage T4 sheath protein. AB - We show by nuclear magnetic resonance studies that, following GTP hydrolysis during phage T4 sheath contraction, GDP remains bound to the sheath protein (gp18), whereas orthophosphate is released. gp18 in the contracted state has GTPase activity and can hydrolyse exogenous GTP; the reaction is calcium dependent and displays high substrate specificity. The process comprises two steps: (1) displacement of GDP from gp18 by exogenous GTP, and (2) GTP hydrolysis proper. The first step appears to be rate-limiting and to be accelerated when the nucleotide-protein interaction is mechanically disrupted by sonication. PMID- 1731072 TI - Sequence and structure of the catalytic RNA of hepatitis delta virus genomic RNA. AB - Human hepatitis delta virus (HDV) RNA has been shown to contain a self-catalyzed cleavage activity. The sequence requirement for its catalytic activity appears to be different from that of other known ribozymes. In this paper, we define the minimum contiguous sequence and secondary structure of the HDV genomic RNA required for the catalytic activity. By using nested-set deletion mutants, we have determined that the essential sequence for the catalytic activity is contained within no more than 85 nucleotides of HDV RNA. These results are in close agreement with the previous determinations and confirmed the relative insignificance of the sequence at the 5' side of the cleavage site. The smallest catalytic RNA, representing HDV genomic RNA nucleotide positions 683 to 770, was used as the basis for studying the secondary structure requirements for catalytic activity. Analysis of the RNA structure, using RNase V1, nuclease S1 and diethylpyrocarbonate treatments showed that this RNA contains at least two stem and-loop structures. Other larger HDV RNA subfragments containing the catalytic activity also have a very similar secondary structure. By performing site specific mutagenesis studies, it was shown that one of the stem-and-loop structures could be deleted to half of its original size without affecting the catalytic activity. In addition, the other stem-and-loop contained a six base pair helix, and the structure, rather than the sequence, of this helix was required for the catalytic activity. However, the structure of a portion of the stem-and-loop remains uncertain. We also report that this RNA can be divided into two separate molecules, which alone did not have cleavage activity but, when mixed, one of the RNAs could be cleaved in trans. This study thus reveals some features of the secondary structure of the HDV genomic RNA involved in self catalyzed cleavage. A model of this RNA structure is presented. PMID- 1731075 TI - Computing the geometry of a molecule in dihedral angle space using n.m.r.-derived constraints. A new algorithm based on optimal filtering. AB - We have developed a method based on optimal filtering to determine the three dimensional structure of a protein from n.m.r.-derived constraints, using the dihedral angle internal representation of the molecule. It differs from currently proposed methods in that it directly produces estimates of errors on the parameters that are refined, hence providing an image of the minimum that has been found. A similar algorithm had already been proposed using cartesian co ordinates as independent parameters, encoded in PROTEAN2. We found that using dihedral angles significantly reduces the computational burden of the technique, and provides better control over a priori informations that can be used, such as geometric restrictions for proline residues and informations from vicinal coupling constants. Performance of the method, encoded in FILMAN, is demonstrated by application to the folding of a ten-residue alanine polypeptide, to the geometric cyclization of an 11-residue peptide, as well as on the folding of a medium size protein, i.e. tendamistat. The validity of the error estimates on the dihedral angles produced by FILMAN is discussed. PMID- 1731074 TI - Refined solution structure and ligand-binding properties of PDC-109 domain b. A collagen-binding type II domain. AB - We have determined, via 1H-n.m.r., the solution conformation of the collagen binding b-domain of the bovine seminal fluid protein PDC-109 (PDC-109/b). The structure determination is based on 341 interproton distance estimates and 42 dihedral angle estimates: a set of 24 initial structures were computed; 12 using the variable target function program DIANA, and 12 using the metric matrix program DISGEO. These structures were optimized by restrained energy minimization and dynamic simulated annealing using the CHARMM and X-PLOR programs. The average pairwise root-mean-square difference (r.m.s.d) between the optimized DIANA (DISGEO) structures is 0.71 A (0.82 A) for the backbone atoms, and 1.73 A (2.03 A) for all atoms. Both sets of structures exhibit the same global fold, secondary structure and placement of most non-polar side-chains. Two central antiparallel beta-sheets, which lie roughly perpendicular to each other, and two irregular loops support a large, partially exposed, hydrophobic surface that defines a putative binding site. A test of a hybrid relaxation matrix-based distance refinement protocol (MIDGE program) was performed using a normalized 250 millisecond NOESY spectrum. The resulting distances were input to the molecular mechanics/dynamics procedures mentioned above in order to optimize the DIANA structures. Our results indicate that relaxation matrix refinement of distances is most useful when used conservatively for identifying underestimated distance constraints. 1H-n.m.r. monitored ligand titration experiments revealed definite, albeit weak, binding interactions for phenethylamine and leucine analogs (Ka less than or equal to 25 M-1). Residues perturbed by ligand binding include Tyr7, Trp26, Tyr33, Asp34 and Trp39. These results suggest that PDC-109/b may recognize specific leucine and/or isoleucine-containing sequences within collagen. PMID- 1731073 TI - Structure refinement of the chromomycin dimer-DNA oligomer complex in solution. AB - We have refined the initial docking model of the Mg(II)-co-ordinated chromomycin d(A2G2C2T2) complex (2 drug equivalents per duplex) by a complete relaxation matrix analysis simulation of the two-dimensional nuclear Overhauser effect (NOESY) spectrum of the complex in 2H2O solution. This relaxation matrix refined structure of the complex exhibits the following characteristics. (1) We observe an unwound and elongated duplex that exhibits characteristics distinct from the A and B-DNA family of helices at the central (G-G-C-C).(G-G-C-C) chromomycin dimer binding and flanking sites. On the other hand sugar puckers, glycosidic torsion angles, displacement of the base-pairs from the helix axis and the minor groove width for this central tetranucleotide segment all fall within the A-family of helical parameters. (2) The chromomycin monomers are aligned in a head-to-tail orientation in the Mg(II)-co-ordinated dimer in the complex. The chromophores are aligned with a slight tilt relative to each other and make an angle of 75 degrees between their planes. The C-D-E trisaccharide segments from individual monomers adopt an extended conformation that projects in opposite directions in the dimer. The divalent metal cation is co-ordinated to the O(1) carbonyl and O(9) enolate atoms of the chromophores and aligns them such that the O(9)-Mg-O(9) angle is 170 degrees while all other O-Mg-O angles are in the 95(+/- 15)degrees range. (3) The sequence specificity of the chromomycin dimer for the widened and shallower (G3 G4-C5-C6).(G3-G4-C5-C6) minor groove binding site is associated with intermolecular hydrogen bonds formed between the OH group at C(8) of the chromophore and the minor groove NH2 group at position 2 and N(3) groups of G4 and between the O(1) oxygen of the E-sugar and the minor groove NH2 group at position 2 of G3 in the complex. (4) Additional intermolecular interactions are primarily van der Waals contacts between anomeric and adjacent CH2 protons on each sugar in the C-D-E trisaccharide segments of the chromomycin dimer and the minor groove surface of the DNA. These results provide insights into the induced conformational transitions required to generate a complementary match between the drug dimer and its DNA binding site on complex formation. PMID- 1731076 TI - Leftward ribosome frameshifting at a hungry codon. AB - Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame. PMID- 1731077 TI - Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 A resolution. AB - We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate). PMID- 1731078 TI - Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues. AB - Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family. PMID- 1731080 TI - Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3' splice site. AB - The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3' terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3' splice site. This circle is formed at the phosphodiester bond of the 5' terminal guanosine residue of the upstream exon via nucleophilic attack by the 3' terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3' splice site, restoring a guanosine to the 3' terminus and re-establishing P9.0 pairing. The new 3' terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3'-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening. PMID- 1731079 TI - Energetic contribution of solvent-exposed ion pairs to alpha-helix structure. AB - Understanding the role of amino acid side-chain interactions in forming secondary structure in proteins is useful for deciphering how proteins fold and for predicting folded structures of proteins from their sequence. Analysis of the secondary structure as a function of pH in two designed synthetic peptides with identical composition but different sequences, affords a quantitative estimate of the free energy contribution of a single ion pair to the stability of an isolated alpha-helix. One peptide contains repeated blocks of Glu4Lys4. The second has repeated blocks of Glu2Lys2. The former contains significant helical structure at neutral pH while the latter has none, based on ultraviolet light circular dichroism measurements and 1H nuclear magnetic resonance spectroscopy. The difference is attributed to formation of helix-stabilizing salt-bridges between Glu- and Lys+ spaced at i, i + 4 intervals in the former peptide. The free energy of formation of a single Glu(-)-Lys+ salt-bridge can be evaluated by using a statistical model of the helix-coil transition that explicitly includes salt bridges: the result is -0.50(+/- 0.05) kcal/mol at 4 degrees C and neutral pH in 10 mM salt, in agreement with a value derived for a single salt-bridge in a helix on the surface of a globular protein. PMID- 1731081 TI - Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui. AB - The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu. PMID- 1731082 TI - Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin type acid proteinase from Aspergillus niger var. macrosporus. AB - Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution. PMID- 1731084 TI - Crystallization of the Fab fragments of anti-peptide monoclonal antibodies and a complex with peptide. AB - The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen. PMID- 1731083 TI - Crystallization and preliminary crystallographic data on Escherichia coli TEM1 beta-lactamase. AB - Two crystal forms of Gram- bacteria TEM beta-lactamase have been obtained. The tetragonal form has a very large unit cell and diffracts to 3.0 A resolution. Orthorhombic crystals, grown using ammonium sulfate and a small amount of acetone as precipitating agents, belong to space group P2(1)2(1)2(1) with cell parameters a = 43.1 A, b = 64.4 A, c = 91.2 A and diffract to 1.7 A resolution. A seeding procedure has been designed that ensures reproducibility of the crystal properties. Molecular replacement, using a model reconstructed from the C alpha co-ordinates from Staphylococcus aureus PC1 beta-lactamase, gives a solution that satisfies crystal packing constraints. PMID- 1731086 TI - Direct interaction between two Escherichia coli transcription antitermination factors, NusB and ribosomal protein S10. AB - The Escherichia coli proteins NusB and ribosomal protein S10 are important for transcription antitermination by the bacteriophage lambda N protein. We have used sucrose gradient co-sedimentation and affinity chromatography with immobilized ribosomal protein S10, a glutathione S-transferase-S10 fusion protein, and NusB to show that NusB binds directly and very selectively to S10. The interaction is non-ionic and has an estimated Kd value of 10(-7) M. We hypothesize that NusB binds to N-modified transcription complexes primarily by interacting with S10. PMID- 1731085 TI - Gene conversions within the skeletal myosin multigene family. AB - Comparisons of the nucleotide sequences of the light meromyosin (LMM) region of developmentally regulated fast chicken myosin heavy chain (MHC) isoforms indicates that chicken MHC isoforms are more similar to each other than to MHC isoforms in other species. The sequence data provide evidence that gene conversion events have occurred recently among the isoforms. An embryonic (Cemb1) isoform and neonatal isoform have the most extensive regions of sequence identity. Similar gene conversion events are present in the rat alpha- and beta cardiac MHCs, but were not obvious in the LMM of developmentally regulated fast human MHC isoforms. The data suggest that gene conversion events can play a significant role in the evolution of the MHC multigene families and that concerted evolution of the chicken multigene family occurred after the divergence of mammals and avians. PMID- 1731087 TI - Nucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7 RNA polymerase. AB - We have examined the effects of nucleosome cores on the initiation and elongation of RNA transcripts by phage T7 RNA polymerase in vitro. A transcription template, pT207-18, was constructed containing tandemly repeated 207 base-pair (bp) nucleosome positioning sequences from a sea urchin (Lytechinus variegatus) 5 S RNA gene inserted between the T7 and SP6 transcription promoters of pGEM-3Z. Nucleosome cores were reconstituted onto supercoiled, closed circular pT207-18 DNA and double label transcription experiments were performed to determine the effects of nucleosome cores on the initiation and elongation of transcripts by T7 RNA polymerase. Both transcript initiation and elongation were inhibited, the extent of the inhibition being directly proportional to the number of nucleosome cores reconstituted onto the pT207-18 DNA templates. Time course transcription experiments indicated that nucleosome cores caused a reduction in the equilibrium length of transcripts and not mere retardation of elongation rates. Continuous regularly spaced linear arrays of nucleosomes were obtained by digesting reconstituted nucleosomel pT207-18 templates with DraI, for which a unique restriction site lies within the nucleosome positioning region of the 207 bp 5 S rDNA repeat sequence. After in vitro transcription with T7 RNA polymerase an RNA ladder with 207 nucleotide spacing was obtained, indicating that transcription can occur through continuous arrays of positioned nucleosome cores. It is demonstrated that nucleosome cores partially inhibit the elongation of transcripts by T7 RNA polymerase, while allowing passage of the transcribing polymerase through each nucleosome core at an upper limit efficiency of 85%. Hence, complete transcripts are produced with high efficiency from short nucleosomal templates, while the production of full-length transcripts from long nucleosomal arrays is relatively inefficient. The results indicate that nucleosome cores have significant inhibitory effects in vitro not only on transcription initiation but on transcription elongation as well, and that special mechanisms may exist to overcome these inhibitory effects in vivo. PMID- 1731088 TI - Small single-copy region of plastid DNA in the non-photosynthetic angiosperm Epifagus virginiana contains only two genes. Differences among dicots, monocots and bryophytes in gene organization at a non-bioenergetic locus. AB - We have determined the nucleotide sequence of a 7 kb (1 kb = 10(3) base-pairs) region that includes the entire small single-copy region (SSC) of the plastid genome of Epifagus virginiana, a non-photosynthetic, parasitic flowering plant. The SSC (4.8 kb) is considerably smaller than those of photosynthetic plants due to the complete deletion of all photosynthetic, chlororespiratory and ribosomal protein genes. This leaves only two genes: a protein gene of 1738 codons whose product is unlikely to be involved in bioenergetic processes and a leucine tRNA gene (trn(LUAG)). Both genes span junctions between the inverted repeat and the SSC, with the consequence that the terminal 20 base-pairs of the repeat is transcribed in both directions and functions both as the 3' end of the tRNA gene and as an internal segment of orf1738. We find that the region of tobacco plastid DNA homologous to Epifagus orf1738 contains a single open reading frame (ORF) of 1901 codons rather than the three ORFs of 1244, 273 and 228 codons originally reported. However, we confirm that the equivalent region of the bryophyte Marchantia contains two genes (1068 and 464 codons) corresponding to the N and C terminal portions of the dicot protein. In contrast, rice plastid DNA contains a severely truncated pseudogene at this locus. PMID- 1731089 TI - Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA. AB - Forty-four nucleotides at the 5' terminus of the genomic RNA of Sindbis virus can form a stable stem-loop structure and have been shown previously to be important for viral replication. The structure formed by the complement of this sequence at the 3' end of the minus-strand RNA has been proposed to be a promoter for RNA replication and as such might be bound in a specific fashion by proteins of either cellular or viral origin. Short oligonucleotide probes (either 62 or 132 nucleotides) representing the 3'-terminal sequence of the minus strand were prepared. When added to extracts from infected or uninfected cells, these probes were bound by cellular proteins, as evidenced by a shift in the electrophoretic mobility of the (labeled) oligonucleotide. Competition experiments confirmed the specificity of the interaction. Proteins of apparent molecular sizes 42 and 44 kDa, and to a lesser extent 52 kDa, could be cross-linked to the minus-sense probes by UV irradiation. A mutant minus-strand probe identical to the longer probe except for a single-nucleotide deletion corresponding to nucleotide 5 in the genomic RNA, which is lethal for the virus, was also found to bind the same proteins as the wild-type probe. The half-life of the mutant probe-cellular protein complex was threefold longer than that of the wild-type complex, however, indicating that the mutant probe was bound more tightly than the wild-type probe. We hypothesize that the binding of cellular factors may be transiently required for initiation of transcription of plus-strand RNA from the minus-strand template and that overly tight binding of such factors is deleterious for RNA replication. PMID- 1731090 TI - Complex formation of human T-cell leukemia virus type I p40tax transactivator with cellular polypeptides. AB - We examined cellular components which associate with p40tax, the viral transactivation molecule of human T-cell leukemia virus type I. Such molecules were searched by immunoprecipitation with polyclonal and monoclonal antibodies specific for p40tax. Two cellular proteins with molecular masses of 95 kDa (p95) and 60 kDa (p60) were specifically coprecipitated with p40tax from extracts of all p40tax-producing cell lines but not from p40tax-negative cell lines. The p60 component was also shown to associate with p40tax in vitro, by using radiolabel chase experiments. Rabbit antisera specific for p60 and p95 were prepared by immunization with electrophoretically purified molecules. While anti-p95 antiserum coprecipitated p40tax, no p40tax could be identified in immunoprecipitates by using a polyclonal anti-p60 antiserum. The partial amino acid sequence of p60 demonstrated that p60 is identical to the human 60-kDa heat shock protein (a member of the chaperonin family of proteins). Although the biological significance of the complex formation of p40tax with p95 and p60 has yet to be determined, it may be that the complex formation is one of the mechanisms by which the biological activity of p40tax can be regulated. PMID- 1731091 TI - Hemagglutinin mutations related to antigenic variation in H1 swine influenza viruses. AB - The hemagglutinin (HA) of a recent swine influenza virus, A/Sw/IN/1726/88 (H1N1), was shown previously to have four antigenic sites, as determined from analysis of monoclonal antibody (MAb)-selected escape mutants. To define the HA mutations related to these antigenic sites, we cloned and sequenced the HA genes amplified by polymerase chain reaction of parent virus and MAb-selected escape mutants. The genetic data indicated the presence of four amino acid changes. After alignment with the three-dimensional structure of H3 HA, three changes were located on the distal tip of the HA, and the fourth was located within the loop on the HA. We then compared our antigenic sites, as defined by the changed amino acids, with the well-defined sites on the H1 HA of A/PR/8/34. The four amino acid residues corresponded with three antigenic sites on the HA of A/PR/8/34. This finding, in conjunction with our previous antigenic data, indicated that two of the four antigenic sites were overlapping. In addition, our previous studies indicated that one MAb-selected mutant and a recent, naturally occurring swine isolate reacted similarly with the MAb panel. However, their amino acid changes were different and also distant on the primary sequence but close topographically. This finding indicates that changes outside the antigenic site may also affect the site. A comparison of the HA amino acid sequences of early and recent swine isolates showed striking conservation of genetic sequences as well as of the antigenic sites. Thus, swine influenza viruses evolve more slowly than human viruses, possibly because they are not subjected to the same degree of immune selection. PMID- 1731092 TI - Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. AB - The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans. PMID- 1731094 TI - A novel particulate influenza vaccine induces long-term and broad-based immunity in mice after oral immunization. AB - The immunogenicity of a novel particulate oral influenza vaccine was examined in terms of antibody response and protection in mice. Oral immunization with chicken erythrocytes (CRBC) adsorbed with gamma-irradiated influenza A virus induced high levels of immunoglobulin G antibodies and protection in the lung compared with gamma-irradiated virus administered alone or CRBC. Immunoglobulin A antibodies were the predominant antibodies in nasal washings, and their presence did not correlate with protection as well as immunoglobulin G antibodies. Immunity was not specific for the immunizing virus subtype, as antibodies and enhanced lung clearance of virus were demonstrated with different virus subtypes. However, mice were not protected when challenged with live influenza B virus. The antibody response and the degree of protection were dependent on both the concentration of virus adsorbed to CRBC and number of CRBC adsorbed to virus. Virus-adsorbed CRBC given subcutaneously failed to induce antibodies or protection. Oral immunization with A/Qld/6/72 (H3N2) virus gave a high level of protection over 12 weeks, which could be demonstrated with different subtypes. Protection correlated with antibody levels in the lung determined by both enzyme-linked immunosorbent and hemagglutination inhibition assays, although the levels detected by the latter declined over time. PMID- 1731095 TI - Pathogenesis of classical swine fever: B-lymphocyte deficiency caused by hog cholera virus. AB - Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus. It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues. Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure. A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera. PMID- 1731096 TI - Differential susceptibilities of U-937 cell clones to infection by human immunodeficiency virus type 1. AB - Single-cell clones derived from the U-937 monocytic cell line were studied for susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). Of four such clones, we found that three (UC12, UC14, and UC18) supported replication of HIV-1 more efficiently than parental U-937 cells, as measured by reverse transcriptase activity and p24 core antigen production. In contrast, another clone (UC11) showed only baseline infection throughout an 8-week culture period, before finally becoming positive for expression of viral antigen. This differential susceptibility to infection directly correlated with accumulation of intracellular viral DNA. Furthermore, the UC11 clone expressed lower levels of Sendai virus-inducible tumor necrosis factor alpha mRNA than did the UC12 or UC18 clones. Susceptibility to infection did not correlate with expression of cell surface CD4, since all clones expressed similar levels of CD4 mRNA and surface membrane CD4 protein. Prior exposure of both susceptible UC18 and resistant UC11 clones to Leu3a antibody completely blocked infection by HIV-1, suggesting that no other independent receptors were recognized by the virus. PMID- 1731093 TI - Extensive sequence-specific information throughout the CAR/RRE, the target sequence of the human immunodeficiency virus type 1 Rev protein. AB - The significance and location of sequence-specific information in the CAR/RRE, the target sequence for the Rev protein of the human immunodeficiency virus type 1 (HIV-1), have been controversial. We present here a comprehensive experimental and computational approach combining mutational analysis, phylogenetic comparison, and thermodynamic structure calculations with a systematic strategy for distinguishing sequence-specific information from secondary structural information. A target sequence analog was designed to have a secondary structure identical to that of the wild type but a sequence that differs from that of the wild type at every position. This analog was inactive. By exchanging fragments between the wild-type sequence and the inactive analog, we were able to detect an unexpectedly extensive distribution of sequence specificity throughout the CAR/RRE. The analysis enabled us to identify a critically important sequence specific region, region IIb in the Rev-binding domain, strongly supports a proposed base-pairing interaction in this location, and places forceful constraints on mechanisms of Rev action. The generalized approach presented can be applied to other systems. PMID- 1731097 TI - Defective interfering influenza virus inhibits immunopathological effects of infectious virus in the mouse. AB - Mice inoculated intranasally with a lethal dose of standard influenza virus die with an immune-mediated pneumonia but are protected by coinoculation with defective interfering (DI) virus. Here we show that recruitment of immune cells into the infected lung is halved by treatment with DI virus although the CD4+/CD8+ cell ratio is not affected. Responsiveness of lung T and B cells to lectins is inhibited by standard virus, but coinoculation of mice with DI virus causes a 13-fold increase in T-cell proliferation and up to a 100-fold increase in immunoglobulin production. This effect appears to be due to lymphocyte specific DI virus-mediated interference, since there is no inhibition of virus multiplication in the lungs. The net result is a shift from a lethal to a beneficial immune response. PMID- 1731099 TI - Conserved cysteine residues in the human immunodeficiency virus type 1 transmembrane envelope protein are essential for precursor envelope cleavage. AB - The transmembrane (TM) protein of human immunodeficiency virus type 1 has been demonstrated to be involved in viral infectivity and syncytium formation. Two highly conserved cysteine residues in the extracellular region of the TM protein are shown to be essential for processing the 160-kDa envelope precursor into the active 120- and 41-kDa mature forms. PMID- 1731098 TI - Hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response in humans: characterization of HLA class II-restricted CTLs that recognize endogenously synthesized HBV envelope antigens. AB - In this study, we show that CD4+, hepatitis B virus (HBV) envelope-specific T cell clones produced by stimulation with a particulate antigen preparation are able to recognize and kill not only autologous antigen-presenting cells incubated with exogenous HBV envelope antigens but also autologous HLA class II-positive cells expressing endogenously synthesized HBV envelope antigens following infection with recombinant vaccinia viruses or transfection with recombinant Epstein-Barr virus expression vectors. Experiments with lysosomotropic agents and brefeldin A suggest that the endosomal compartment is likely involved in the processing of endogenously synthesized viral proteins for recognition by CD4+ T cells. Our study indicates that HBV envelope-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes can potentially participate in the immune clearance of HBV-infected cells and the pathogenesis of hepatocellular injury in hepatitis B. PMID- 1731100 TI - Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy. AB - Glycosylated, membrane-associated E1 (58-kDa) and E2 (47- to 49-kDa) rubella virus proteins and unglycosylated nucleoprotein C (33 kDa), from separately expressed vaccinia virus recombinants, were injected into golden Syrian hamsters. Rubella virus E1 and E2 glycoproteins consistently induced an organ-specific autoimmune disease, autoimmune lymphocytic hypophysitis, which was evidenced by the induction of autoantibodies against pituitary cells and by lymphocytic infiltration of the pituitary. Neonatal thymectomy prevented the disease. In contrast, rubella virus nucleoprotein C did not induce either autoantibodies against pituitary cells or lymphocytic infiltration of the pituitary. This finding raises the possibility that virus-specific protein itself can induce an organ-specific autoimmune disease in certain circumstances. PMID- 1731101 TI - Hepatitis B virus X protein is not central to the viral life cycle in vitro. AB - The hepatitis B x (HBx) gene is the smallest open reading frame of the hepatitis B virus (HBV) genome. It is conserved among all mammalian hepadnaviruses and is expressed during viral infection. While the HBx protein (pX) has been shown to trans-activate the transcription of a wide range of viral and cellular genes and to induce liver cancer in transgenic mice, the significance of pX for the life cycle of HBV itself has not been elucidated. To assess the function of pX in viral replication and virion export, we designed an X-minus mutant by introduction of a stop codon at the beginning of the HBx gene without affecting the viral polymerase gene product. Transient transfection analyses using different cell lines revealed that this X-minus mutant directs the synthesis of wild-type levels of viral proteins, replicative intermediates, and virion export. These data suggest that the expression of the highly conserved HBx gene is not central for the life cycle of HBV in vitro but may be involved in the pathogenicity of hepadnavirus infection, including liver cancer development. PMID- 1731102 TI - Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes. AB - Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar. PMID- 1731103 TI - Paralysis of street rabies virus-infected mice is dependent on T lymphocytes. AB - Street rabies virus (SRV)-infected T-lymphocyte-deficient (nude) mice, in contrast to euthymic mice, did not develop hindlimb paralysis prior to death. To document the role of T lymphocytes in rabies virus-associated paralysis, 10(8) spleen cells from normal immunocompetent euthymic mice were transferred to nude mice and the recipient mice were challenged with SRV. One hundred percent of the reconstituted mice developed paralysis and died. Depletion of T cells from the donor spleen suspension prior to transfer abrogated the development of paralysis but did not prevent the deaths of the recipient animals. Mice receiving 10(8) rabies virus-immune spleen cells did not become paralyzed and did not die. Nude mice inoculated with either rabies virus-immune or normal mouse serum prior to and following SRV inoculation did not develop paralysis. Immune serum protected the mice, whereas animals inoculated with normal serum died. Central nervous system inflammatory responses in nude mice immunologically reconstituted with normal spleen cells were characterized by diffuse cellular infiltrates in the parenchyma and extensive perivascular cuffing. Perivascular infiltrates included CD8+ and CD4+ T lymphocytes and Mac-1+ macrophage-microglial cells. Inflammatory cells in the parenchyma were limited to CD8+ lymphocytes and Mac-1+ cells. These observations indicate that paralysis of SRV-infected mice is dependent on T lymphocytes. Whether injury leading to paralysis is mediated by T lymphocytes or by an influence of T lymphocytes on macrophage-microglial cells or other cells remains to be determined. PMID- 1731104 TI - Replication of parvovirus B19 in hematopoietic progenitor cells generated in vitro from normal human peripheral blood. AB - Erythroid progenitor cells generated in vitro from peripheral human blood in the presence of interleukin-3 and erythropoietin were infected with human parvovirus B19. B19 virus DNA replication was highest 48 to 72 h after infection, and maximum levels of B19 virus proteins were detected in culture supernatants at 72 to 96 h after infection. B19 virus propagated in vitro was infectious. This cell culture system with peripheral blood cells facilitates studies in vitro of B19 virus replication. PMID- 1731105 TI - Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies. AB - It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance. PMID- 1731106 TI - Transcription analysis of the EcoRI D region of the baculovirus Autographa californica nuclear polyhedrosis virus identifies an early 4-kilobase RNA encoding the essential p143 gene. AB - We have investigated the transcriptional activity of the 60.1- to 68.3-map-unit region of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Twelve transcripts mapping to this region were expressed at various times during infection. An early 4.0-kb transcript, potentially coding for a 143 kDa peptide essential for viral DNA replication, was maximally abundant at 6 h postinfection (p.i.). Transcripts of 0.5, 1.1, 1.4, 2.1, and 3.1 kb were most abundant at 12 h p.i., while two large transcripts of 5.2 and 6.8 kb were expressed maximally at 24 h p.i. In the presence of cycloheximide, and in ts8 infected cells at the nonpermissive temperature, only the 4.0-kb RNA was expressed. Northern (RNA) blot analysis using DNA subfragments from the EcoRI D fragment as probes suggested that many of the transcripts overlapped. Strand specific cRNA probes revealed that the majority of the RNAs were transcribed in the counterclockwise direction. S1 nuclease and primer extension analysis were used to map the 5' ends of transcripts coded within the 60.1- to 64.8-map-unit region. Mapping of the 3' ends of the 1.1-, 4.0-, 5.2-, and 6.8-kb transcripts suggested that these RNAs were all coterminal at their 3' ends. A minicistron was found between the early 4.0-kb transcription start site and the predicted ATG start codon of the p143 gene. Several similar sequence motifs were identified in the promoter regions of the p143 gene and the AcMNPV DNA polymerase gene. PMID- 1731107 TI - The requirement for a 5' stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation. AB - Sequences with strong similarity to internal control regions 1 and 2 (ICR1 and 2; A and B boxes) of tRNA genes are found at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other plant viruses. The functionality of these motifs was studied by introducing point mutations into the ICR2-like sequence of pRNA-2 M/S, a BMV RNA-2 deletion mutant that replicates in the presence of RNAs-1 and -2 but does not encode a functional viral protein. The accumulation of positive-strand progeny from pRNAs bearing single and double base substitutions was 70 to 91% lower than that of the wild type, while the addition of single bases at position 8 of this motif reduced replication by 80%. These dramatic decreases in positive-strand synthesis paralleled decreases in transcription seen (C. Traboni, G. Ciliberto, and R. Cortese, Cell 36:179-187, 1984) from a tRNAPro gene containing similar mutations, suggesting comparable functions for the ICR regions in protein factor binding and demonstrating that a wild-type composition of the virus ICR2-like motif is required for proper RNA replication. Substitutions introduced at bases surrounding the ICR2 motif yielded levels of pRNA replication that differed, depending on the maintenance of a putative 5' stem-loop structure in the positive strand of the viral genome. Mutations disrupting this positive-strand stem-loop while maintaining the integrity of the complementary negative-strand structure reduced pRNA replication by 85 to 97%. In contrast, disruption of the negative-strand structure while maintaining the positive-strand stem-loop did not reduce pRNA replication. Similar positive strand structures can be predicted to form from 5' sequences of cucumber mosaic virus (strain Q) and cowpea chlorotic mottle virus RNAs-1 and -2, supporting the concept that such structures comprise an integral part of virus genomic positive strand promoters. The requirement of a stem-loop structure present on the positive-strand provided the basis for a new model describing how these sequence and structural elements act in the production of virus positive-strand RNA. PMID- 1731108 TI - Germiston virus transcriptase requires active 40S ribosomal subunits and utilizes capped cellular RNAs. AB - The transcriptase associated with Germiston virus was assayed in an in vitro reaction in which transcription was coupled to translation by adding reticulocyte lysate under the appropriate salt conditions. When analyzed in polyacrylamide gels, the major transcripts migrated like authentic S mRNAs and possessed 12- to 18-base-long nontemplated 5' extensions similar to the 5' end of viral mRNAs. These transcripts were functional for the synthesis of at least proteins N and NSS. When translation was inhibited by adding protein synthesis inhibitors such as puromycin, cycloheximide, and anisomycin, a drastic inhibitory effect was observed on the synthesis of the complete S mRNA transcripts. However, initiation and part of the elongation process were still active, since short and incomplete RNA molecules with RNA primers at their 5' ends were synthesized. On the other hand, we found that edeine, another inhibitor of protein synthesis, stimulated not only synthesis of S mRNAs but also that of the full-length S cRNAs. Taking into account the mode of action of this antibiotic, we discuss the results, which emphasize the crucial role of active ribosomes during bunyavirus transcription and confirm the observations reported on La Crosse virions. Moreover, we showed that the RNA transcripts synthesized in a transcription-translation reaction were capped and that most of them have acquired the 5' terminal sequences of the alpha or beta-globin mRNA. PMID- 1731109 TI - cis and trans requirements for the selective packaging of adenovirus type 5 DNA. AB - Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DNA. By introducing site-directed point mutations into specific A repeat sequences, we demonstrate that the A repeats represent cis-acting functional components of the packaging signal. Additional elements, located outside the originally defined packaging domain boundaries and that resemble the A repeat consensus sequence, also are capable of promoting the packaging of viral DNA. The cis-acting components of the packaging signal appear to be subject to certain spatial constraints for function, possibly reflecting a necessity for the coordinate binding of packaging proteins to these sites. In agreement with this idea, we present evidence that the interaction of a limiting trans-acting factor(s) with the packaging domain in vivo is required for efficient encapsidation of the Ad5 genome. PMID- 1731110 TI - Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity. AB - Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1. PMID- 1731111 TI - Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses. AB - Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses. PMID- 1731112 TI - Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro. AB - Nested-primer polymerase chain reaction (PCR) has been applied to the molecular cloning of 4.6-kb half-genome fragments of human immunodeficiency virus type 1 (HIV-1) taken directly from the peripheral blood mononuclear cells (PBMC) of an individual with neurological symptoms of HIV-1 infection. In a similar manner, gp120-coding portions of the envelope gene were cloned after PBMC from the same blood sample were cocultivated with uninfected PBMC for 28 days. The complete 1.6 kb nucleotide sequence of the gp120 gene was determined from each of 35 clones examined. Two of 13 (15%) PBMC-derived gp120 genes and 3 of 22 (14%) coculture derived gp120 genes were defective as a result of frameshifts and an in-frame stop codon(s). Mean diversity between individual gp120-coding sequences in PBMC was fivefold greater (3.24%) than after coculture (0.65%). A predominant sequence of "strain" was found after coculture that was distinct from the diverse viral genotypes detected in vivo and therefore was selectively amplified during in vitro propagation. Multiple distinct third variable (V3) regions encoding the principal neutralizing domain of the envelope protein were detected in PBMC derived genes, suggesting the presence of immunologic diversity of HIV env genes in vivo not reflected in the cocultured virus sample. The large size of the HIV fragments generated in this study will permit analysis of the diversity of immunologic reactivity, gene function, and pathogenicity of HIV genomes present within infected individuals, including the functional significance of the loss of diversity that occurs upon coculture. PMID- 1731113 TI - Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex. AB - Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex. PMID- 1731114 TI - Unusual structure of the human immunodeficiency virus type 1 trans-activation response element. AB - The trans-activation response element (TAR) of human immunodeficiency virus type 1 is a structured RNA consisting of the first 60 nucleotides of all human immunodeficiency virus type 1 RNAs. Computer analyses and limited structural analyses indicated that TAR consists of a stem-bulge-loop structure. Mutational analyses showed that sequences in the bulge are required for Tat binding, whereas sequences in both the bulge and the loop are required for trans activation. In this study, we probed the structures of TAR and various mutants of TAR with chemical probes and RNases and used these methods to footprint a Tat peptide on TAR. Our data show that the structure of wild-type TAR is different from previously published models. The bulge, a Tat-binding site, consists of four nucleotides. The loop is structured, rather than simply single stranded, in a fashion reminiscent of the structures of the tetraloop 5'-UUCG-3' and the GNRA loop (C. Cheong, G. Varani, and I. Tinoco, Jr., Nature [London] 346:680-682, 1990; H.A. Heus and A. Pardi, Science 253:191-193, 1991). RNA footprint data indicate that three bases in the bulge are protected and suggest that a conformational change occurs upon Tat binding. PMID- 1731115 TI - gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3. AB - The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins. PMID- 1731116 TI - Identification and expression of rpo19, a vaccinia virus gene encoding a 19 kilodalton DNA-dependent RNA polymerase subunit. AB - The vaccinia virus DNA-dependent RNA polymerase subunit gene rpo19 was identified, and its expression was examined at RNA and protein levels. Antibody to the multisubunit RNA polymerase purified from virions reacted with a polypeptide with an apparent Mr of 21,000 that was synthesized in reticulocyte lysates programmed with (i) mRNA from infected cells that was isolated by hybridization to DNA subclones of the viral genomic HindIII A fragment and (ii) mRNA made in vitro by transcription of the viral open reading frame A6R. Polyclonal antiserum, raised to a recombinant protein product of the A6R open reading frame which could encode an 18,996-Da protein with an acidic N terminus, reacted with Mr-21,000 and -22,000 polypeptides that cosedimented with purified RNA polymerase. Internal sequencing of the two polypeptides confirmed that both were encoded by A6R, and the gene was named rpo19 to indicate the predicted molecular mass of the polypeptide in kilodaltons. Immunoblotting and metabolic labeling of infected cell proteins indicated that synthesis of the Mr-21,000 polypeptide started early and continued throughout virus infection, whereas the Mr-22,000 form appeared late following DNA replication. RNA analyses suggested that the rpo19 mRNA was expressed from a dual early/late promoter and that the protein-coding region of the mRNA was directly preceded by a short 5' poly(A) leader, apparently initiated within the TAAATG motif at the beginning of the open reading frame. PMID- 1731119 TI - For better hip replacement results, surgeon's best friend may be a robot. PMID- 1731118 TI - Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus. AB - The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting. PMID- 1731117 TI - Kinetics of virus spread and changes in levels of several cytokine mRNAs in the brain after intranasal infection of rats with Borna disease virus. AB - We have used the reverse transcriptase-polymerase chain reaction technique to gain insight into the pathogenesis of encephalitis caused by Borna disease virus (BDV). RNA specific for BDV was first detected in the olfactory bulb of intranasally infected rats at 6 days postinfection (p.i.). At 14 days p.i., high levels of BDV RNA were found in all brain regions, and at 26 days p.i., BDV specific RNA was also present in the eye, nasal mucosa, and facial skin. In the chronic phase of the disease, BDV RNA was identified in many peripheral organs but not in blood. Analysis of brain tissue for the presence of cytokine mRNAs revealed that the mRNA levels of interleukin-6 (IL-6), tumor necrosis factor alpha, and IL-1 alpha had increased sharply at 14 and 26 days p.i. These cytokine mRNAs reached maximum levels at the peak of inflammatory reactions and decreased drastically in the chronic phase of the disease. Although IL-2 mRNA was also found in normal brain, it was markedly increased in BDV-infected brain at 14 days p.i. Expression of gamma interferon (IFN-gamma) mRNA, which was not observed in normal rat brain, was detected at 14 days p.i. and reached a maximum level at 38 days p.i. IL-2 and IFN-gamma mRNA expression correlated with expression of CD4 and CD8 mRNAs, indicating that both CD4+ and CD8+ T lymphocytes are induced in the early stages of BDV infection. Since IFN-gamma and CD8 mRNA levels were still highly elevated in the chronic phase of Borna disease, it is likely that CD8+ T lymphocytes act to reduce inflammation and to ameliorate neurological signs during the chronic phase of infection. PMID- 1731120 TI - A lean, mean cyclotron revs up neutron therapy. PMID- 1731121 TI - Pondering how to squeeze fitness programs into schools' budgetary, time constraints. PMID- 1731122 TI - Cardiologists say cut out fat when they're young, but (usually) don't cut in advanced age. PMID- 1731123 TI - From the Centers for Disease Control. Sexual behavior among high school students. PMID- 1731124 TI - From the Centers for Disease Control. Early childhood vaccination levels. PMID- 1731125 TI - The human genome project. PMID- 1731126 TI - See one, do one, teach one? PMID- 1731127 TI - See one, do one, teach one? PMID- 1731128 TI - Physicians and disaster preparedness. PMID- 1731129 TI - Physicians and disaster preparedness. PMID- 1731130 TI - The costs of preventing measles. PMID- 1731131 TI - The costs of preventing measles. PMID- 1731132 TI - The impact of a physician's warning on recovery after alcoholism treatment. AB - OBJECTIVE: To study whether alcoholic workers had seen physicians during the year they were identified by their company, whether they recalled physicians' warnings about drinking, and whether such warnings affected outcomes 2 years later. DESIGN: Workers were interviewed at intake and 2 years later: subgroups who did and did not see physicians and who did and did not recall warnings were compared. SETTING: A company-union employee assistance program. PARTICIPANTS: Two hundred problem drinkers, newly identified on the job, predominantly male, blue-collar workers. OUTCOMES: Drinking, drunkenness, average daily alcohol consumption, and impairment score. RESULTS: Among the 200 participants, 74% saw physicians in the index year; only 22% recalled warnings. Recall of a warning was associated with liver disease, continued drinking while ill, supervisors' job warnings, older age, and marijuana use. Two years later, those warned were more likely to be abstaining, and sober, and were less impaired. CONCLUSIONS: Recalling a physician's warning at intake into alcoholism treatment was associated with better prognosis 2 years later. However, among this group of employees whose drinking was serious enough to be identified on the job, fewer than a quarter recalled physicians' warnings, even though more than three quarters had seen physicians in the year preceding intake. PMID- 1731133 TI - Reduced mortality risk in alcoholics who achieve long-term abstinence. AB - OBJECTIVES: To determine if alcoholic men who achieved stable abstinence experienced fewer deaths than those who relapsed and to develop a model predictive of premature mortality. DESIGN: A cohort of alcoholic men recruited into a prospective study of neurocognitive effects of alcoholism was followed up from 1 through 11 years. A demographically equated group of nonalcoholic men was also followed up. Alcoholics were classified as stable abstainers or relapsers. SETTING: Alcoholics were patients or ex-patients from a Department of Veterans Affairs Alcoholism Treatment Program and/or members of local chapters of Alcoholics Anonymous. PARTICIPANTS: There were 234 alcoholic men who met the Diagnostic and Statistical Manual of Mental Disorders, Third Edition, criteria for alcohol dependence. Follow-up status regarding relapse and mortality was obtained for 199 alcoholic subjects (85%). Of these, 101 had relapsed and 98 had abstained. Ninety-eight nonalcoholic controls equated for age, education, and sex also participated. Mortality status was obtained for 92 subjects in this group (94%). EXCLUSIONS: Major medical and psychiatric illness and history of nonalcoholic drug abuse. MAIN OUTCOME MEASURE: Death during a follow-up period of 1 through 11 years. Death was ascertained through the National Death Index, the California State Department of Health and Vital Statistics, the State Department of Motor Vehicles, and through personal contact with informants, relatives, and significant others of the subjects. RESULTS: There were 19 deaths among relapsed alcoholics compared with the expected number of 3.83 (99% confidence interval (CI), 9.64 to 33.38). Among abstinent alcoholics there were four deaths (expected = 3.21; 99% CI, 0.67 to 12.59). The standardized mortality ratio for relapsed alcoholics was 4.96, which significantly exceeded the expected ratio (P less than .001), whereas the standardized mortality ratio for abstinent alcoholics (1.25) was indistinguishable from the expected. Cox proportional hazards analysis was used to determine if any of several demographic, medical, cognitive, or drinking history variables (in addition to relapse) helped predict mortality among alcoholics. Only relapse was significantly related to increased mortality (chi 2 = 9.15, P = .003). CONCLUSIONS: Alcoholic men who achieve stable abstinence do not differ from nonalcoholic men in mortality experience; however, alcoholics who relapse die at a rate 4.96 times that of an age-, sex-, and race-matched representative sample from the US Bureau of the Census. PMID- 1731134 TI - Impact of Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine on responses to concurrently administered diphtheria-tetanus-pertussis vaccine. AB - OBJECTIVE: To assess whether serum antibody responses to diphtheria-tetanus pertussis (DTP) vaccine were affected by coadministration of Haemophilus influenzae type b capsular polyribosylribitol phosphate polysaccharide-tetanus protein (PRP-T) conjugate vaccine when given to patients at 2, 4, and 6 months of age. DESIGN: Randomized, double-blind clinical trial. SETTING: Urban Santiago, Chile. PATIENTS: Healthy infants assembled from health centers. Two hundred seventy-eight (74%) of 375 eligible infants participated; 222, who complied with the complete protocol, constituted the primary group under analysis. INTERVENTIONS: One of three vaccine regimens was given to study participants at 2, 4, and 6 months of age, either DTP mixed in the same syringe as PRP-T (group 1); DTP and PRP-T given at separate injection sites (group 2); or DTP without PRP T (group 3). PRIMARY OUTCOME MEASURES: Titers of serum antidiphtheria toxoid, antitetanus toxoid, and pertussis agglutinin antibodies were measured in blood samples taken from patients 2 months after each dose. RESULTS: Serum antidiphtheria toxoid and antitetanus toxoid responses showed no important depressions in the patients receiving PRP-T. In contrast, geometric mean titers (GMTs) of pertussis agglutinins, expressed as reciprocal serum dilutions, after both the second and third doses (GMT2, GMT3) were lowest in group 1 (GMT2 = 89; GMT3 = 1230), intermediate in group 2 (GMT2 = 123; GMT3 = 1995), and highest in group 3 (GMT2 = 210; GMT3 = 3090; P less than .05 for trend group 1 less than group 2 less than group 3 after each dose). Antipertussis toxin and antipertussis filamentous hemagglutinin antibody titers also were depressed in patients who received PRP-T. Follow-up of a subset at 18 months revealed an expected decline of pertussis agglutinin titers to near baseline levels in each group. CONCLUSIONS: Concurrent administration of PRP-T vaccine with DTP vaccine, either in the same syringe or at different sites, interfered with antipertussis responses to a primary series of immunizations. Although the clinical significance of this antagonism is uncertain, these data underscore the caution required in decisions to add new vaccines to existing immunization regimens. PMID- 1731135 TI - The quality of mercy. Caring for patients with 'do not resuscitate' orders. AB - OBJECTIVE: To assess (1) the effect of an ethics education intervention for medical house officers on practices surrounding "Do Not Resuscitate" (DNR) orders and (2) the association of DNR care with patient diagnosis and demographic variables. DESIGN: A 1-year randomized, controlled trial. SETTING: An urban, university teaching hospital. PARTICIPANTS: Eighty-eight internal medicine house officers. INTERVENTION: House officers were arbitrarily assigned to four "firms." One firm was randomized to an extensive ethics education intervention (EI), one to a limited intervention, and two served as controls. MAIN OUTCOME MEASURES: Charts of patients with DNR orders were reviewed for compliance with the hospital's DNR policy, which instructs that when DNR orders are written there should be (1) an attending signature, (2) documentation of reasons, (3) appropriate consent, and (4) attention to 11 concurrent care concerns (CCCs) (eg, the appropriateness of intubation, tube feedings, hospice). RESULTS: Thirty-nine charts were reviewed before the intervention and 57 after. The number of CCCs per DNR order fell among patients cared for by controls (1.9 to 1.0, P less than .05) and rose among patients cared for by the EI group (0.9 to 3.8, P less than .05). Compliance with the DNR policy varied among patients with differing diagnoses. "Do Not Resuscitate" orders were signed less frequently (P = .01) for patients with the acquired immunodeficiency syndrome (AIDS) (65%) compared with patients who had other diagnoses (85%) or malignancy (91%). Similarly, appropriate consent was recorded for 59% of patients with AIDS, 83% of others, and 85% of those with malignancy (P less than .05). The number of CCCs per DNR was 0.7 for AIDS, 1.4 for others, and 2.4 for malignancy (P less than .05). In multivariate regression analysis, house officer ethics education and patient diagnosis, but not patient gender, age, race, or insurance status, were predictors of the number of CCCs per DNR. CONCLUSIONS: (1) An extensive ethics education intervention can improve care for DNR patients, especially with respect to CCCs. (2) In this setting, quality of care for DNR patients varied systematically with diagnosis. These results have implications for the design and implementation of ethics education programs. PMID- 1731136 TI - Sources of the growth in Medicare physician expenditures. PMID- 1731138 TI - Insensitivity of rapid antigen detection methods and single blood agar plate culture for diagnosing streptococcal pharyngitis. AB - OBJECTIVE: To compare the sensitivity of five group A streptococcal antigen detection systems and single blood agar plate culture with a two-plate culture method for diagnosis of streptococcal pharyngitis. DESIGN: Two simultaneous throat swabs were obtained from consecutive patients with suspected streptococcal pharyngitis. One swab was tested for streptococcal antigen by physicians' office nurses and the other was cultured on both aerobic blood agar and anaerobic trimethoprim-sulfamethoxazole blood agar plates. SETTING: Community office practice and community hospital laboratory. PARTICIPANTS: Consecutive outpatients seen by one of four pediatricians or a family practice physician. MAIN OUTCOME MEASURES: Results of rapid streptococcal antigen tests were compared with culture results either on a single aerobic blood agar plate or on the two-plate culture method. RESULTS: On throat swabs from 755 consecutive outpatients, the two-plate culture method detected 261 cases (defined as 100%) of group A streptococcal pharyngitis. The anaerobic trimethoprim-sulfamethoxazole plate alone, read at 1 and 2 days, detected 245 cases (94%). The blood agar plate used alone detected 189 cases (72%) at 2 days and 151 cases (58%) at 1 day. Antigen detection test results were positive for 106 throat specimens (41%), with individual kit sensitivity ranging from 31% to 50% compared with the two-plate culture method. Antigen detection test sensitivity decreased with decreasing colony counts. Antigen kit false-positivity rates varied from 0 to 28%. CONCLUSIONS: We conclude that the single blood agar plate culture and the antigen detection tests are insensitive, possibly leading the physician toward undertreatment and risking immunologic, local, or distant sequelae. The two-plate culture method should be the standard of practice to rule out streptococcal pharyngitis. PMID- 1731137 TI - Mental health consequences and correlates of reported medical student abuse. AB - OBJECTIVE: To assess the prevalence, correlates, and mental health consequences of reported medical training-related abuses. DESIGN: A longitudinal cohort study of 137 students surveyed from medical school entrance (time 1) through the winter of the fourth training year (time 4). SETTING: A state college of medicine. OUTCOME MEASURES: Reported training-related abuses (measured at time 4) and mental health status measured at times 1 and 4 (depressive and anxiety symptoms, hostility, problem drinking, escapist drinking, alcohol consumption levels, and gender role orientations). RESULTS: Seventy-two percent of students reported at least one abusive experience during medical training. Females were significantly more likely than males to report gender discrimination, exclusion from informal settings, discomfort from sexual humor, and unwanted sexual advances. Abuse was significantly related to most psychopathological outcomes, controlling for pre existing psychopathology. Males low in masculinity and females low in femininity were most likely to report abuse. CONCLUSIONS: The data provide further support for the assumption that a high proportion of medical students not only experience the training process as abusive in nature but also suffer measurable psychopathological consequences. Efforts should be made to reform medical education, with a prominent focus on gender role-related issues. PMID- 1731139 TI - Patient care and professional staffing patterns in McKinney Act clinics providing primary care to the homeless. AB - OBJECTIVE: To describe the patient care and staffing patterns of the 157 clinics that receive federal funding to provide health care to the homeless. DATA SOURCES: Telephone interviews with clinic medical directors. RESULTS: Clinics treated a mean of 96 homeless patients per week, approximately 50% of the estimated homeless population. Three quarters treated homeless patients only, the others integrated homeless patients into an existing setting. One third of the clinics had no physician more than 5 hours per week, 10% had no physician staff at all, and 80% employed a nurse practitioner. The proportion of patients initially examined by a nurse practitioner and the proportion subsequently referred to a physician ranged between 10% and 100%. Clinic directors reported that in over 50% of clinics, physician recruitment was hampered by poor working conditions, inadequate salaries, physician biases against working with the homeless, and the lack of respect this work receives from the medical profession. CONCLUSIONS: Current financial constraints may be impeding the ability of clinics serving the homeless to ensure adequate access to high quality care. Additional research should evaluate the impact various staffing patterns have on access and quality of care and develop methods to improve physician recruitment. PMID- 1731140 TI - Patients who drink too much. Where are their doctors? PMID- 1731141 TI - The sure Super Bowl bet--injured players are penalized for life. PMID- 1731142 TI - Elucidating and eradicating medical student abuse. PMID- 1731143 TI - . . . And, ladies of the club. PMID- 1731144 TI - Parental leave and medical careers. PMID- 1731145 TI - A warm gesture. PMID- 1731146 TI - Automated prescreening of conventionally prepared cervical smears: a feasibility study. AB - The feasibility of an automated screening device that will analyze conventionally prepared cervical smears was examined. Consecutive normal and abnormal cervical smears were selected retrospectively and analyzed on a customized microscope imaging workstation system. Specialized image processing, feature extraction, and object classification algorithms were integrated on the imaging workstation to provide an Analysis Score for each slide analyzed. A threshold was set for the Analysis Score, and all slides with a score less than the threshold were classified as normal. Examination of the distribution of the Analysis Score for normal and abnormal cervical smears provided an estimate that 50% of all slides will be sorted as normal, and consequently need no cytologist review. With the Analysis Score threshold set for a 50% sort rate, zero false-negative slides were found in this slide set. This study has shown that a large percentage of slides can be correctly classified as normal while maintaining a high sensitivity to abnormal slides. Given the current workload problems in cytology laboratories, reducing the screening workload by one half will be beneficial. This study has shown that it is now feasible with current technology to provide a clinically useful device to automate screening of conventional cervical smears. PMID- 1731147 TI - Fourier transform infrared microscopic identification of foreign materials in tissue sections. AB - Fourier-transform infrared microspectroscopy was used to identify polymeric microparticles, such as polyethylene, polysulfone, polytetrafluoroethylene, polyurethane, silicon-based, and nylon-based plastics, in tissue sections. Foreign particles as small as 30 microns in diameter were identified. In addition to being a nondestructive technique, infrared microscopy allows for more precise structural identification of polymeric materials than do X-ray or scanning electron microscopy. PMID- 1731148 TI - Quantitative analysis of nuclear size for objective malignancy grading: a review with emphasis on new, unbiased stereologic methods. PMID- 1731149 TI - In situ demonstration of proliferating cells in the rat central nervous system during experimental autoimmune encephalomyelitis. Evidence suggesting that most infiltrating T cells do not proliferate in the target organ. AB - Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats and the bromodeoxyuridine (BrdU)-incorporating cells were demonstrated immunohistochemically in lesions in the central nervous system (CNS) to assess the extent of T cell proliferation during EAE. In active EAE, BrdU+ cells were most numerous on day 12 postimmunization, when both clinical signs and inflammation detected by histologic examination were most severe; they declined thereafter although a considerable number of inflammatory foci remained in some rats. In passive EAE, BrdU+ cells were most numerous 2 days before the full-blown EAE and then rapidly decreased in number. On day 6 post-transfer, the CNS showed the most severe histologic changes but virtually no inflammatory cells in the lesions were labeled with BrdU. Double immunofluorescence staining with T cell (OX52) and macrophage/microglia (OX42) markers showed that about half of the BrdU+ cells were labeled with OX52 at the peak of EAE. The proportion of BrdU+OX52+ T cells at later stages was about 20%. BrdU+OX42+ cells ranged between 50 and 80% throughout the course of the disease. Furthermore, serial pulsing experiments during passive EAE revealed that inflammatory cells that had been labeled with BrdU outside the CNS and found later in the CNS were 15 times more numerous than those labeled in situ in the CNS. Taken together, these findings indicate that the majority of T cells involved in EAE undergo DNA synthesis outside the CNS and then infiltrate into the CNS, and that T cells labeled in situ in the CNS are few, and decrease rapidly in number. Since interleukin-2 receptor-positive cells detected by mAb OX39 outnumbered BrdU+ T cells at these later stages, we postulate that an unresponsive state instead of T-cell proliferation was induced in the CNS. PMID- 1731150 TI - Kinetics and fate of IgA-IgG aggregates as a model of naturally occurring immune complexes in IgA nephropathy. AB - The characteristic granular IgA immunofluorescent pattern in the kidneys of IgA nephropathy patients is consistent with immune complex pathogenesis. The possibility of a delayed clearance of IgA-containing immune complexes from circulation in IgA nephropathy patients is still under discussion. Since pure IgA immune complexes are probably nonphlogistic, (in contrast to IgG-containing IgA immune complexes), the in vivo clearance of a mixture of heat-aggregated IgA/G purified from pooled human sera was analyzed. The test probe was efficiently labeled with 123I and the time course of radioactivity was measured by a gamma camera. Both the liver and the spleen were found to be involved in the disappearance of IgA/G complexes. Liver accumulation, which was markedly predominant, closely approximates a gamma-variate function which allowed determination of a mean transit time of 34.37 minutes, range 29.8 to 42.2, in 8 normal and 37.54 minutes, range 30.9 to 50.7 in 17 patients (p less than 0.04). At 2 hours, segmental gut accumulation was found, which demonstrated removal by hepatobiliary system as well. Compartmental analysis in patients indicated 3 major compartments represented by vascular bed, hepatobiliary and reticuloendothelial systems (including both liver and spleen phagocytes). Blood clearance rate, representing the final result of multiorgan removal of the test probe from the blood stream, was found to be significantly delayed in IgA nephropathy patients with a slope (0.035 min-1, range 0.019 to 0.052) significantly less negative compared with controls (0.047 min-1, range 0.038 to 0.053, p less than 0.01). This test probe was able to reproduce both removal routes (macrophages cells and hepatobiliary system) hypothesized for IgA containing immune complexes in humans. PMID- 1731151 TI - Caustic ingestion injuries of the upper aerodigestive tract. AB - Few reports have described in detail the injuries that occur to the oral cavity, pharynx, and larynx following caustic ingestion. The role of dynamic radiographic studies to delineate the extent of damage has been minimized. In-depth radiographic analysis of such cases has not, to our knowledge, been previously reported. In order to examine the injuries and functional abnormalities of these sites following caustic ingestion, the records of The Johns Hopkins Swallowing Center were reviewed. Five patients were identified as having significant upper aerodigestive tract caustic injuries. All patients had dysphagia, epiglottis injuries, and incomplete laryngeal protection with aspiration. Four of five had sustained some degree of esophageal stenosis. Also noted were pharyngeal muscle dysfunction, nasopharyngeal regurgitation, tongue fixation, and hypopharyngeal stenosis. Roentgenographic findings are described and illustrated. The multidisciplinary approach to the management and rehabilitation of these patients is discussed. PMID- 1731152 TI - The retrolabyrinthine transsigmoid approach to midbasilar artery aneurysms. PMID- 1731153 TI - Physiologic motion after vocal cord reinnervation: a preliminary study. AB - This study attempted to reestablish physiologic vocal cord motion, rather than synkinesis, to a reinnervated vocal cord. One mongrel dog underwent a division and reanastomosis of the anterior branch of the right recurrent laryngeal nerve and simultaneous separation and reimplantation of a posterior division nerve muscle pedicle into the posterior cricoarytenoid muscle. After 21 weeks, spontaneous physiologic vocal cord movement and electromyographic (EMG) activity were recorded during respiratory obstruction and laryngeal mechanical stimulation. Acoustic measures and histologic data are also presented from the reinnervated and normal vocalis muscle and from the recurrent laryngeal nerve. This study demonstrated that physiologic vocal cord motion can be achieved after laryngeal reinnervation using this technique. PMID- 1731154 TI - Feasibility of multichannel human cochlear nucleus stimulation. AB - Bipolar electrical stimulation of the brainstem cochlear nucleus (CN) following acoustic tumor removal in an only-hearing ear can provide beneficial hearing. However, the benefits of multichannel stimulation have yet to be defined. Following removal of a second acoustic tumor in a patient with neurofibromatosis 2, a Nucleus mini-22 channel implant device was inserted with the electrode array tip from the foramen of Luschka cephalad along the root entry zone of the eighth nerve, secured by a single suture superficially in the brain stem. Initial stimulation on the sixth postoperative day indicated that electrodes 18 to 22 were capable of CN stimulation without seventh nerve stimulation. Presumed electrode migration precluded further CN stimulation 1 month later. This report illustrates the feasibility of brainstem CN stimulation with an existing multichannel system. PMID- 1731155 TI - Hearing results in retrolabyrinthine vestibular neurectomy. AB - Patients having retrolabyrinthine vestibular neurectomy (RLVN) may have complications that compromise hearing. While most reviews have emphasized sensorineural loss, less attention has been given to conductive hearing loss, which may complicate RLVN. Hearing results of 25 consecutive cases of RLVN performed for Meniere's disease with incapacitating vertigo were tabulated according to 1985 American Academy of Otolaryngology (AAO) guidelines. Nine patients (36%) had improved hearing postoperatively, 5 (20%) had no change in hearing, and 11 (44%) had worse hearing postoperatively. The most commonly observed audiometric change was low-frequency conductive hearing loss, presumably secondary to partial ossicular fixation by bone dust or fat fibrosis in the attic and antrum. Five patients (20%) had low-frequency conductive hearing losses that increased by 10 dB or greater over preoperative levels. An additional 7 patients had lesser losses at low frequencies. One patient had a flat conductive hearing loss. Six (24%) of the patients had a decrease in bone levels of greater than 10 dB. Overall hearing results in this study are comparable to those of other series in the literature. Causes and prevention of conductive hearing loss in RLVN are discussed, and a format for presentation of hearing data that will highlight conductive hearing loss after surgery for Meniere's disease is presented. PMID- 1731156 TI - Analysis of risk factors for postoperative pulmonary complications in head and neck surgery. AB - Preoperative pulmonary function tests (PFTs) are unproven in their predictive value for postoperative pulmonary complications. There is a lack of prospective outcome studies upon which to form an opinion, particularly regarding noncavitary surgery. Seventy-three head and neck surgery patients were prospectively evaluated with preoperative PFTs, arterial blood gas analysis (ABG), medical history, and physical examination. Age, anesthesia duration, forced expiratory volume in 1 second (FEV1), peak flow (PF), PaO2, Roizen class, and pack years of smoking were significantly correlated with postoperative pulmonary complications. As similar studies in head and neck surgery patients have not been previously taken, it is hoped that these results will serve as a basis for future endeavors. PMID- 1731157 TI - Hyperbaric oxygen therapy: effect on middle ear and eustachian tube function. AB - Hyperbaric oxygen therapy (HBO) involves intermittent inhalation of 100% oxygen under a pressure greater than 1 atm. It is an important mode of adjuvant therapy for disease processes such as decompression sickness, osteomyelitis, carbon monoxide poisoning, and poorly healing wounds. Patients undergoing this therapy often complain of ear pain and/or fullness which can be transient or long standing. This prospective study objectively measured the changes in eustachian tube function before and after HBO treatment in 33 adult patients by the 9-step inflation-deflation test described by Bluestone. The results show 15 of the 33 patients (45%) had evidence of eustachian tube dysfunction after treatment was initiated. Of these, 15 (100%) developed the sensation of fullness, 13 (87%) developed serous otitis media, and 7 (47%) required tympanostomy tubes. The overall incidence of middle ear problems was 27 patients (82%) experiencing a sensation of fullness, 17 (52%) developing serous otitis media, and 8 (24%) requiring tympanostomy tubes. The middle ear complications reported in this study are much higher than those in previous reports in the literature. Twelve of 33 patients presented with a subjective history of eustachian tube dysfunction, and all 12 (100%) developed fullness in their ears and serous otitis media during the course of the treatment. The findings reveal that patients manifesting eustachian tube dysfunction after their first HBO treatment were at significantly greater risk toward developing symptoms of fullness and serous otitis media, often requiring tympanostomy tube placement. In addition, a history of eustachian tube dysfunction accurately predicted the development of fullness and serous otitis media. PMID- 1731158 TI - Nuclear morphometry and stereology in nasopharyngeal carcinoma. AB - The prognostic value of nuclear morphometry in nasopharyngeal carcinoma (NPC) was investigated in 28 patients. Seven morphometric nuclear variables were measured on 100 randomly selected tumor cells in nasopharyngeal biopsies from 18 patients with NPC confined to the nasopharynx. The same variables were measured in 6 patients with metastatic NPC, as well as in lymph node biopsies from 4 patients with metastatic NPC. Nuclear area, nuclear perimeter, long and short nuclear axes, nuclear form factor, nucleolar area, and the ratio of nucleolar area to nuclear area were all measured. Volume-weighted mean nuclear volume was also obtained. Tumor cells from patients with NPC confined to the nasopharynx had significantly larger mean nuclear areas, perimeters, and volume-weighted mean nuclear volumes but significantly smaller nucleolar to nuclear area ratios than tumor cells from patients with nodal metastases. Assessment of nuclear form factor and diameters did not differentiate the two groups. PMID- 1731159 TI - Contemporary management of cervical tuberculosis. AB - Although excisional biopsy has traditionally been required to diagnose cervical tuberculosis (TB), fine needle aspiration biopsy (FNAB) has also been found to be useful. The presentation and management of 47 patients diagnosed with cervical TB between 1984 and 1988 were retrospectively reviewed. Chest x-rays were normal in 58% of the patients, and purified protein derivative (PPD) skin testing was positive in 96%. When FNAB was used, TB could be suspected in 83% of cases and definitively established in 62%. Open biopsy correctly diagnosed cervical TB in all masses excised. Medical therapy alone resulted in resolution of disease in 94% of patients diagnosed by FNAB, and subsequent excisional biopsy was necessary in only one patient. The results suggest that FNAB is a useful initial procedure in the diagnosis of cervical TB. Excisional biopsy should be reserved for cases where no diagnosis by FNAB can be made, or for persistent cervical disease despite full-course antituberculous chemotherapy. PMID- 1731160 TI - The controversial treatment of anterior commissure carcinoma of the larynx. AB - There are two basic approaches to the appropriate therapy for carcinoma of the anterior commissure. The dilemma of whether to treat by primary irradiation or by conservative surgery is not yet solved. In this study, 67 patients were treated between 1967 and 1987 for anterior commissure carcinoma of the larynx. Radiation was used with 47 patients and conservation surgery with 20 patients. Initial lesion control was achieved with 72% of the patients treated by primary irradiation. Conservation surgery, when used as a primary treatment modality, achieved local control in 90% of the patients. The new techniques of reconstruction of the larynx enhance the surgeon's ability, strengthen his conviction to proceed to enlarged partial laryngectomies, and thus improve the oncologic control of the anterior commissure carcinoma as well. PMID- 1731161 TI - Mandibular osteotomies for tumor extirpation: the advantages of rigid fixation. AB - Adequate exposure of intraoral tumors occupying the posterior oral cavity, base of tongue, tonsil, and superior hypopharynx for wide-field primary surgical resection is critical to precise tumor ablation. The exposure resulting from a mandibular osteotomy has greatly assisted the tumor ablation of these areas. This procedure has also been beneficial in providing exposure to the anterior skull base, pterygomaxillary, and infratemporal space, clivus, and nasopharynx. In evaluating various osteotomy sites and methods of fixation, we reviewed 26 patients treated for benign or malignant neoplasia of the head and neck requiring mandibulotomy. The osteotomy complication rate was 2 (29%) of 7 for wire osteosynthesis and 1 (5.3%) of 19 for plate osteosynthesis. All patients with osteotomy complications had received preoperative radiation therapy. The one complication in the plated group was associated with a lateral stairstep osteotomy and two screws on either side of the osteotomy. This study suggests advantages of absolute rigid internal fixation of mandibular osteotomies used for tumor ablation. It is also concluded that a midline osteotomy reapproximated with rigid internal fixation has the benefits of 1. primary bone healing by means of plating counteracting the balanced forces acting on the symphysis; 2. improved reapproximation with minimal bony loss, improving occlusion; 3. decreased incidence of osteoradionecrosis as the symphysis lies outside the usual radiation ports; 4. preservation of the neurovascular bundle; and 5. maintenance of osteotomy-site immobility in an infected field. This review helped identify surgical techniques that decrease the complications that are commonly associated with mandibular osteotomies for precise tumor ablation. PMID- 1731162 TI - Laser dyes for experimental phototherapy of human cancer: comparison of three rhodamines. AB - The mitochondrial dye Rhodamine 123 (Rh-123) has been shown to be an effective photosensitizer for argon-laser irradiation of some types of human cancer cells in vitro. We reported that 514.5-nm laser illumination of Rh-123 sensitized human melanoma, and squamous carcinoma cells strongly inhibited tumor-cell proliferation as measured by decreased 3H-thymidine (3H-T) uptake in vitro and may eradicate some tumors when grown as transplants in nude mice. However, several other human tumors were resistant to Rh-123 laser therapy in vitro and in vivo. In the current study, it was possible to obtain 100- to 1000-fold increased sensitivity to 514.5-nm laser illumination by replacement of Rh-123 with the cationic rhodamine dyes Rh-3G and Rh-6G. Cell viability was decreased over 95% and 3H-T incorporation reduced at least 80% by laser phototherapy after sensitizing tumor cells with 1 micrograms/mL Rh-123, 0.01 microgram/mL Rh-3G, or 0.001 microgram/mL Rh-6G. However, Rh-123 alone did not decrease 3H-T uptake significantly unless present at over 10- to 100-fold higher levels than Rh-3G, respectively. The tumor cell dye uptake level was measured by N-butanol extraction and absorption scans at 400 to 600 nm. The results revealed that dye uptake was more rapid, and retention of Rh-3G and Rh-6G was 5- to 10-fold higher than for Rh-123 in the human tumor cells. The data suggest that Rh-3G and Rh-6G may be highly sensitive chromophores for laser phototherapy of human cancer cells. PMID- 1731163 TI - Changes in the microbial flora of airline headset devices after their use. AB - The bacterial flora of 20 headset devices were evaluated before and after they were worn for 1 hour. Bacteria were recovered from all headsets, and their number increased from a mean (+/- standard deviation) of 60 +/- 5 organisms per device to 650 +/- 51 organisms per device (P less than .001). The predominant organisms recovered were Staphylococcus spp, Streptococcus spp, Propionibacterium spp, and Peptostreptococcus spp. This study demonstrates the presence of potential pathogens in headset devices, as well as the increase in the number of these pathogens after the headsets have been worn for 1 hour. PMID- 1731164 TI - Male soprano voice: a rare complication of thyroidectomy. AB - A soprano voice from cricothyroid fusion is a rare complication following thyroidectomy. Thyroid surgeons should be aware of this possibility and recognize it early if voice pitch rises following thyroid surgery. This patient's unfortunate complication may prove fortuitous for phonosurgeons and their patients. Cricothyroid fusion may provide a better long-term retention of frequency elevation than traditional cricothyroid approximation. It is also a reversible procedure. Cricothyroid fusion should be investigated as an alternative to cricothyroid approximation for pitch modification. PMID- 1731165 TI - Surgical management of massive benign symmetric lipomatosis. PMID- 1731166 TI - Seasonal changes in the response of fast and slow mammalian skeletal muscle fibers to zero potassium. AB - While investigating the decline in resting membrane potential (RMP) of rat skeletal muscle fibers in zero potassium solution, we discovered that there is seasonal variation in the response of the extensor digitorum longus muscle (EDL). In January, most EDL fibers hyperpolarize in zero K+; in September, most depolarize; the distribution of RMPs recorded in May is bimodal, with some fibers hyperpolarizing and some depolarizing. Fibers from the soleus muscle depolarize in zero K+ irrespective of the season. The ability of EDL fibers to hyperpolarize appears during the 7th and 8th weeks postpartum, and is dependent upon the presence of a functional nerve, since denervation abolished the response. As possible explanations for these findings, inactivation of K(+)-channels and inhibition of the Na-K pump by zero K+ are discussed. PMID- 1731167 TI - Identification and characterization of melatonin binding sites in the gastrointestinal tract of ducks. AB - Utilizing 2-[125I]iodomelatonin as the radioligand, melatonin binding sites were identified and characterized in the jejunum of ducks. These sites were found to be reversible, saturable, specific and exhibited high affinity for melatonin. Scatchard analyses have established the equilibrium dissociation constant (Kd) for tissues collected during mid-photophase to be 40.9 +/- 7 pM and the maximum quantity of binding sites (Bmax) to be 2.0 +/- 0.4 fmol/mg protein while Kd of samples collected during mid-scotophase was found to be 54.1 +/- 10 pM with a corresponding Bmax of 1.5 +/- 0.3 fmol/mg protein. These Kd values are in good proximity to the kinetically derived equilibrium dissociation constant of 47.3 +/ 20 pM. No significant difference in Kd or Bmax was detected between the mid light and mid-dark samples. Pharmacological profile of these binding sites, developed by their interactions with other indoles and compounds, indicated that these binding sites are highly specific for melatonin. Subcellularly, different densities of binding sites were localized to various fractions in the following order: nuclear greater than microsomal greater than mitochondrial greater than cytosolic. These binding sites in the jejunum might be the receptors accountable for promoting paracrine activities for the locally synthesized gastrointestinal melatonin and/or responsible for eliciting hormonal actions via interactions with melatonin of pineal origin. PMID- 1731168 TI - Direct membrane effects of morphine and endorphins on Amoeba proteus. AB - Morphine, leu-enkephalinamide, met-enkephalin, alpha-neoendorphin and its Arg8 1 8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 microM. Significant effects of leu-enkephalin and naloxone are obtained at 180 microM. All compounds have reached their maximal activity at 720 microM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds. PMID- 1731169 TI - Radioprotective effects of (-)-epigallocatechin 3-O-gallate (green-tea tannin) in mice. AB - Long-term administration of (-)-epigallocatechin 3-O-gallate (EGCG) to mice through drinking water prevented radiation-induced increase of lipid peroxides in liver and significantly prolonged life span after lethal whole-body X irradiation. The result indicates validity of this green-tea component as an orally active radio-protector of very low toxicity. PMID- 1731170 TI - Splenocyte blastogenesis suppressed in rats implanted with an osmotic pump. AB - Rats implanted subcutaneously with an empty osmotic pump connected by a polyethylene catheter to a jugular vein for 5 to 10 days evinced a decreased splenocyte responsiveness to blastogenic stimulation in vitro to bacterial lipopolysaccharide, a known B cell stimulator, as well as to the plant mitogens pokeweed mitogen (PWM), a known stimulator of T and B cells, and Concanavilan A, a known T cell stimulator. The surface of the implanted pumps became infiltrated with lymphoid cells, especially macrophages. Suppression of blastogenic responsiveness after implantation for 10 days with an empty pump or even a pump dispensing pyrogen free saline only in a continuous manner was nearly as marked as that which occurred at 2-5 days after continuous infusion with endotoxin. These depressed blastogenic responses, although less, were also evident when rats were implanted with a catheter into a jugular vein connected by means of a swivel to either an empty pump or one dispensing pyrogen free saline. Suppression of blastogenic responsiveness was not related to alteration in serum complement or corticosteroid levels. Since administration of immunomodulatory substances systemically to individuals often involves implantation of an osmotic pump, investigation into the mechanisms of lymphoid cell suppression associated with an implanted pump itself has potential significance. PMID- 1731171 TI - Antinociceptive activity of metapramine in mice. Relationship with its pharmacokinetic properties. AB - The antinociceptive effect of acutely and chronically (every brain elimination half-life time) administered metapramine, a tricyclic antidepressant without anticholinergic or cardiotoxic effects, was studied in three different pain tests. In the hot plate test, its action was more potent when jumping was used as a pain parameter (acute ED50 = 19 +/- 3 mg/kg, i.p.) than when pain was assessed by licking of forepaws (only 20 mg/kg, i.p. was weakly active). Five chronic doses of 15 mg/kg were as active in the tail-flick test as an acute dose of 20 mg/kg (only active dose). Metapramine was more effective in the PBQ-induced writhing test after acute (ED50 = 9.9 +/- 0.1 mg/kg, i.p.) and chronic administration. A significant linear correlation was found between the effect in this test and plasma and overall brain levels of metapramine. No correlation was observed with levels of its three desmethylated metabolites. The usefullness of using a well-defined pattern of administration based on pharmacokinetic parameters and the involvement of monoaminergic mechanisms and of some metabolites of metapramine are discussed. PMID- 1731172 TI - Fructosamine in human and bovine semen. AB - We detected the presence of fructosamine in human and bovine semen. In seminal plasma of healthy normozoospermic men (N = 17) fructosamine was found in 53% of the cases (fru+). In fru+ semen samples the concentration of fructosamine was (mean +/- S.E.M., N = 9) 0.45 +/- 0.09 mmol/L and varied from 0.15 to 0.75 mmol/L. It was 3-12 times lower than in blood serum of healthy men. In semen of infertile men (N = 57) fructosamine was present only in 21% of the cases and its concentration was lower than in fertile men i.e. (mean +/- S.E.M., N = 12) 0.27 +/- 0.007 mmol/L. In bulls (N = 98) fructosamine was found in semen of 82% of animals. In fru+ semen samples the concentration of fructosamine was (mean +/- S.E.M., N = 80) 0.77 +/- 0.12 mmol/L and varied from 0.30 to 1.15 mmol/L. We did not find any correlation between the concentration of fructosamine on one hand, and that of fructose and glucose on the other hand, in either human or bull semen. The difference in the frequency of fructosamine appearance in semen of fertile and infertile men suggests that fructosamine may be in some way involved in the process of fertilisation. PMID- 1731173 TI - Catecholamine induced growth of gram negative bacteria. AB - The addition of various catecholamines to cultures of gram negative bacteria resulted in dramatic increases in growth. The ability of norepinephrine, epinephrine, dopamine and dopa to enhance the growth of Escherichia coli, Yersinia enterocolitica and Pseudomonas aeruginosa was observed to be dependent on the bacterium employed with each strain showing marked preference for one or more of the catecholamines. Catecholamine induced increases in growth were confirmed by one or more of the following methods: uptake of tritiated thymidine into newly synthesized DNA, changes in optical density or pour plate analysis. None of the catecholamine metabolites resulting from either oxidative deamination or catechol-O-methylation were able to effect similar increases in bacterial growth as compared to either norepinephrine, epinephrine or dopamine. Norepinephrine was consistently observed to effect the greatest increase in bacterial growth for all strains tested. PMID- 1731174 TI - Effects of cocaine on oxygen consumption and mitochondrial respiration in normoxic and hypoxic mice. AB - The administration of cocaine hydrochloride intraperitoneally (25 mg/kg) produces a drop in VO2 in both normoxic and hypoxic mice. The critical PO2 is also decreased and so is the body temperature. The mitochondrial respiration shows a large fall in ST3 and RCR. The addition of cocaine in-vitro to the incubating medium induces changes in the mitochondrial respiration similar to those found after in-vivo administration. This report shows that in addition to its in-vivo actions cocaine alters the respiratory function of the isolated liver mitochondria. PMID- 1731175 TI - Lithium administration modulates platelet Gi in humans. AB - Platelet G proteins were assessed in 7 normal volunteers before and after 14 days of lithium administration at therapeutic plasma levels. Cholera and pertussis toxin catalyzed ADP-ribosylation of platelet membrane proteins were measured by SDS-PAGE. Immunoblotting with specific antibodies was used to measure platelet membrane alpha i content. There was a statistically significant 37% increase in pertussis toxin mediated ADP-ribosylation of a 40,000 Mr protein in platelet membranes after lithium administration, but cholera toxin mediated ADP ribosylation of a 45,000 Mr protein and alpha i immunoblotting were unchanged by lithium. Increased pertussis toxin stimulated ADP-ribosylation in the absence of changes in alpha i content could be explained by a shift in platelet Gi in favor of its undissociated, inactive form. This would be consistent with increased platelet adenylyl cyclase activity found in these same subjects after lithium. PMID- 1731176 TI - The effects of estradiol on pituitary responsiveness to dopamine in vitro: a comparison of ovariectomized Fischer 344 and Holtzman rats. AB - The objective of this study was to determine if the effectiveness of dopamine as an inhibitor of prolactin is altered by estradiol in strains of rats which show marked differences in estrogen-induced pituitary hyperplasia. Groups of Fischer 344 and Holtzman Sprague-Dawley rats were ovariectomized and implanted with Silastic capsules of estradiol. Rats were sacrificed by rapid decapitation following a brief period of ether anesthesia at 2, 4, 6, 8 weeks (F-344) or at 2 and 8 weeks (Holtzman) of estradiol treatment. The pituitary was removed and cut into fragments which were either snap frozen for initial prolactin content measurements or incubated for 60 min in the presence or absence of dopamine (1 x 10(-6) M). Prolactin was measured in the plasma, in sonicates of the pituitary and in the incubation medium by double antibody radioimmunoassay. Pituitary weight and plasma levels of prolactin were significantly less in Holtzman rats compared to Fischer 344 females at 2 or 8 weeks of estradiol treatment but pituitary concentrations of prolactin were not different between the two strains. Pituitary fragments from Fischer 344 rats studied at 2 and 4 weeks of estradiol treatment did not respond to the removal of dopamine in vitro whereas pituitary fragments from Holtzman rats obtained at 2 weeks of estradiol treatment did release significantly more prolactin in the absence than in the presence of dopamine. Pituitary fragments taken from Fischer 344 rats at 6 and 8 weeks were responsive to dopamine whereas pituitary tissue from Holtzman rats was not responsive at 8 weeks. The data indicate that temporal differences in responsiveness to the inhibitory effects of dopamine occur in strains which are susceptible or resistant to the formation of pituitary tumors following prolonged estradiol treatment. PMID- 1731177 TI - Patient outcomes research: a report of an NCNR conference. PMID- 1731178 TI - Vigorous physical activity among high school students--United States, 1990. AB - Vigorous physical activity can improve the health of both adults and children. Among adults, regular physical activity can reduce risk for chronic diseases such as coronary heart disease, hypertension, noninsulin-dependent diabetes mellitus, colon cancer, and depression, as well as lower all-cause death rates (1,2). Among children, regular physical activity can reduce chronic disease risk factors such as obesity, elevated cholesterol, and hypertension (3). Physical activity patterns established during childhood may extend into adulthood (4). This report examines the prevalence of vigorous physical activity among U.S. students in grades 9-12. PMID- 1731179 TI - Bacterial contamination of platelet pools--Ohio, 1991. AB - From June 27 through July 30, 1991, four episodes of bacterial contamination of platelet pools occurred in an Ohio hospital and were reported by the hospital through the Food and Drug Administration (FDA) to CDC. This report summarizes the results of the epidemiologic investigation of these episodes. PMID- 1731180 TI - Iguana-associated salmonellosis--Indiana, 1990. AB - During 1990, the Indiana State Board of Health's Disease Control Laboratory reported isolates of a rare Salmonella serotype, S. marina, from two infants residing in different Indiana counties. This report summarizes epidemiologic and clinical information about these two cases. PMID- 1731181 TI - Update: International Task Force for Disease Eradication, 1990 and 1991. AB - In 1988, a decade after the successful eradication of smallpox, the International Task Force for Disease Eradication (ITFDE) was formed to systematically evaluate the potential for global eradicability of candidate diseases, identify specific barriers to their eradication that might be surmountable, and promote eradication efforts. In its first two meetings in April and October 1989, the ITFDE determined that two of eight diseases examined were eradicable and three others were candidates for elimination of transmission or clinical symptoms (1). In its third and fourth meetings in August 1990 and June 1991, the ITFDE evaluated the potential eradicability of seven other diseases. This report summarizes the results of the third and fourth meetings. PMID- 1731182 TI - Toxic hypoglycemic syndrome--Jamaica, 1989-1991. AB - In January and February 1991, the health officer in the parish of St. Ann, Jamaica, received reports of eight persons with toxic hypoglycemic syndrome (THS), an illness associated with consumption of unripe ackee fruit and, possibly, renta yam; two cases were fatal. On July 25, the Jamaican Ministry of Health (JMH) contacted CDC for assistance in investigating the continued occurrence of THS; the collaborative JMH and CDC epidemiologic investigation focused on characterizing the epidemiology of THS in Jamaica and assessing the role of ackee fruit, renta yams, and other factors. PMID- 1731183 TI - Worksite and Community Health Promotion/Risk Reduction Project--Virginia, 1987 1991. AB - Because cardiovascular disease (CVD) is a leading cause of premature disability and death in the United States, approaches are needed to prevent this problem at the community level. In the Mount Rogers Health District (MRHD) (1990 population: 178,000), a six-county area in southwestern Virginia, age-adjusted death rates for CVD substantially exceed state and national averages. In August 1987, the MRHD implemented the Worksite and Community Health Promotion/Risk Reduction Project to help residents of this large, rural area improve their health by adopting healthy lifestyles and, consequently, reduce their risks for CVD and cancer. This report summarizes activities associated with the project and changes in health indicators for participants from April 1989 through May 1990. PMID- 1731184 TI - Update: influenza activity--United States, 1991-92 season. AB - From October 23, 1991, through January 18, 1992, 46 state health departments reported regional or widespread influenza activity for 1 or more weeks. For the week ending January 4, 34 states reported regional or widespread activity, the most during any single week this season. From late October through mid-December, influenza outbreaks reported from 11 states involved primarily school children. Reports of outbreaks among adults began in mid-November and continued through January and involved persons in a variety of settings (e.g., a business in California; a hospital in Ohio; a county jail in Tennessee; and nursing homes in Maryland, Missouri, New York, Ohio, Utah, and Wisconsin). PMID- 1731185 TI - Pulmonary fibrosis associated with occupational exposure to hard metal at a metal coating plant--Connecticut, 1989. PMID- 1731186 TI - IgG2b or IgE molecules can be co-expressed with those of the IgM and IgD isotypes of the membrane of normal rat B cells. AB - Expression of Ig isotypes other than IgD together with IgM on the membranes of single B cells has been reported in different experimental models. This paper describes the co-expression of IgG2b or IgE with IgM-IgD on the surface of single B cell subpopulations from normal rats. Their expression was demonstrated with anti-IgE or IgG2b monoclonal antibodies and their F(ab')2 fragments. After pronase digestion, the re-expression of these isotypes together with IgM-IgD was observed in vitro and was inhibited by cycloheximide. These observations imply that mechanisms other than class switching may participate in the expression of membrane isotypes in vivo. The role of these membrane isotypes is still to be established, but could be important as IgG2b molecules are found on a large B cell subpopulation. PMID- 1731187 TI - A chimeric mouse/human anti-IL-2 receptor antibody with enhanced biological activities. AB - A chimeric mouse/human MAb against the human p55 IL-2R was constructed from Ig genes isolated from a mouse hybridoma cell line, designated AHT107. AHT107 binds to a different epitope on p55 than IL-2, and similar to observations made for other rodent anti-IL-2R antibodies that do not recognize the same or spatially related epitope as IL-2, murine AHT107 did not efficiently inhibit proliferation of T-lymphocytes in mitogen and MLR PBMC stimulation assays. In contrast, the chimeric AHT107 antibodies containing a human IgG-1 constant region had substantially more anti-proliferative activity than their murine IgG-I counterparts. Our results indicated that the human constant region of the chimeric antibodies interacted more efficiently than the murine constant region with effector components present in the PBMC cultures. This conclusion was supported by our observation that F(ab')2 generated from the chimeric antibodies did not efficiently inhibit proliferation in the PBMC assays, and the chimeric antibodies did not inhibit proliferation of an antigen specific, IL-2 dependent human T-cell clone stimulated in the absence of PBMC. PMID- 1731188 TI - Cloning and expression of the variable regions of mouse myeloma protein MOPC315 in E. coli: recovery of active FV fragments. AB - Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150 200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV. Chimeric FVs composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sFV was shown to be the same as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria. PMID- 1731189 TI - Binding of secretory component to protein 511, a pIgA mouse protein lacking 36 amino acid residues of the C alpha 3 domain. AB - Protein 511, a murine IgA protein described previously by Robinson and Appella [Proc. natn. Acad. Sci. U.S.A. 77, 4909-4913 (1980)] which lacks 36 amino acids in the C alpha 3 domain, was tested for its ability to bind to radiolabelled secretory component (125I-rat SC) and to be transported from blood to bile in the rat, a function described previously to be mediated by the poly Ig receptor (pIg R). When compared to other mouse pIgA proteins, the naturally occurring mutant protein 511 bound 125I-rat SC and was transported from blood to bile in a manner indistinguishable from wild-type pIgA protein. We conclude that the region of Fc alpha which is missing in protein 511, is not involved in mediating the binding of pIgA to the pIg R. PMID- 1731190 TI - Kinetics of inter-heavy chain disulfide bond formation of liganded and unliganded human immunoglobulin G by radioimmunoassay. AB - Monoclonal antibodies (mAbs) to the hinge region of human IgG1 immunoglobulins were prepared by immunization with a proteolytic fragment of hinge segment coupled to keyhole limpet hemocyanin. A mAb, 4G3, was obtained capable of binding to intact IgG but not to partially reduced IgG. Using this mAb, inter-heavy (H) chain disulfide bond formation from partially reduced anti-tetanus antibodies (Abs) in the presence or absence of antigens (Ags) was studied by solid phase radioimmunoassay. When the Abs were partially reduced in solution and then coated to plastic plates, only 10% regeneration of inter-H chain disulfide bonds occurred after reoxidation, although 100% formation occurred in solution [Kishida et al., J. Biochem. (Tokyo) 79, 91-105 (1976)]. This difference in the extent of disulfide bond formation can be explained by the fact that there are two convertible isomers in solution, Conformer I and II, one of which (Conformer I) can form disulfide bonds but is present as a minor component. Since the motion of IgG molecules on plates is restricted by hydrophobic interactions, the two conformers are not convertible as in solution. Therefore only Conformer I which existed before coating formed inter-H chain disulfide bonds. Similar kinetic measurements were performed using plates coated with tetanus toxoid. Abs, partially reduced in solution and then allowed to react with Ags on the plate were able to completely regenerate their inter-H chain disulfide bonds, although the rate of reaction was slow. These results can also be explained by the fact that the two isomers are convertible since Ag-Ab complexes are dissociable. Ag binding therefore did not significantly perturb conformation of the hinge segments. In addition, no difference between liganded and unliganded Abs was observed in the binding of anti-hinge mAbs. These results imply little or no contribution of the hinge region to transmission of the signal produced by Ag binding to Fc. PMID- 1731191 TI - Dynamic equilibria between subcomponents of C1, the first component of human complement. AB - C1r and C1s, the serine protease components of activated C1, form a tetramer in the presence of Ca2+. The stability of this tetramer is sufficient that its association with the third component, C1q, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53-66 (1983)]. We have used the fluorescence anisotropy (A) of fluorescein labeled C1s (s*) to monitor assembly and subcomponent exchange in 0.15 mol/l NaCl, 0.001 mol/l Ca2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r2s*2 causes a small but measurable delta A of 0.01-0.02. The response is too fast to measure at 37 degrees but can be readily followed at 4 degrees where t 1/2 = 0.6 min when [q] = [r2s*2] = 0.5 mumol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled C1s. Slow incremental addition of q to a solution of r2s*2 produces a dose-dependent delta A from which stoichiometry and dissociation constants can be derived. Measurements of Kd as a function of temperature establish an inverse temperature dependence with delta H = -15 kcal/mol and a value of Kd = 0.031 mumol/l at 37 degrees (delta G = + 11, T delta S = -26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex. PMID- 1731192 TI - Nucleotide sequence analysis of H-2Df and the spontaneous in vivo H-2Dfm2 mutation. AB - The nucleotide sequence of the standard H-2Df allele and the spontaneous in vivo H-2Dfm2 mutation are reported here. Locus-specific sequences in the 5' and 3' untranslated regions of the mouse MHC class I H-2D-region genes were used to design primers for the specific amplification and cloning of H-2D-region cDNA from standard B10.M/Sn H-2f and mutant B10.M-H-2fm2/Mob mice. A partial Df genomic clone and direct Df and Dfm2 mRNA sequence analysis confirmed the authenticity of the cDNA clones. Interestingly, H-2Df contains a proline in the alpha-helix of the alpha 1 domain at amino acid position 62; no other known class I molecule has a proline at this position. The H-2Dfm2 mutation, however, replaces this unique proline in Df with the H-2 and HLA consensus arginine at position 62. Although a point mutation cannot be ruled out, the single nucleotide change in the H-2Dfm2 mutation is flanked by a stretch of 47 nucleotide bases with an identical counterpart in H-2Kf, a finding consistent with a recombinatorial event between H-2Kf and H-2Df. PMID- 1731193 TI - The role of amino functions in recombinant human tumor necrosis factor in expression of biological activity. AB - The role of amino functions in the expression of the biological activity of recombinant human TNF (rHuTNF) was studied by chemical modification. rHuTNF is a homotrimer of 17 kD subunits, each of which contains an N-terminal valine and six lysyl residues: two of these lysyl residues are known to be involved in intra- or intersubunit interactions. Chemically reactive amino functions were modified with the N-hydroxysuccinimide ester of acetic acid; modification of amino groups to amide, and the concomitant loss of charge, was monitored by native PAGE. When rHuTNF was reacted with the active ester at increasing mole ratios, up to 12 amino groups per trimer could be modified. When the biological activity of acetylated rHuTNF was determined, a strong correlation between the extent of modification and loss of biological activity was observed. One to three amino groups per trimer could be modified with nearly complete retention (approximately 80-95%) of biological activity; activity was essentially completely destroyed at the highest levels of modification. These results reveal important functions for the amino groups of rHuTNF and significant constraints on strategies involving their modification in development of second-generation-TNF variants. PMID- 1731194 TI - A tyrosine sulfated human glycoprotein with an unusual cell distribution. AB - We describe a human integral cell surface glycoprotein (Mr 142 kD), recognized by monoclonal antibody M2B3. This glycoprotein is absent from resting peripheral blood lymphocytes, but becomes expressed in significant levels with mitogen activation. The M2B3 glycoprotein is present on epithelial cells in the basal layer of epidermal and esophageal tissue as well as in several fresh tumors examined. In addition, it is present on smooth muscle tissue throughout the gastrointestinal tract, but is absent from smooth muscle from several other tissue sources, skeletal muscle and cardiac muscle. The M2B3 glycoprotein is similar, but not identical, in apparent Mr to a transformation-associated glycoprotein, Q14. Further, the M2B3 and Q14 species are related antigenically. Nonetheless, M2B3 and Q14 are distinct glycoproteins based on clear differences in cell distribution and in partial peptide mapping. The M2B3 antigen described herein is sulfated on tyrosine, and represents one of the few cell surface proteins described to date that is sulfated on tyrosine residues. Our studies suggest the function of the M2B3 glycoprotein is likely to be associated with cell proliferation of cell adhesion. PMID- 1731195 TI - Olympic row over sex testing. PMID- 1731196 TI - Cellular biophysics. Proteins as machines. PMID- 1731197 TI - Will women soon outrun men? PMID- 1731199 TI - Human genome project. Still room for HUGO? PMID- 1731198 TI - Protein folding in the cell. AB - In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution. PMID- 1731200 TI - Scientific misconduct. A final frenzy for landmark cases? PMID- 1731201 TI - Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. AB - Positive control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition, alterations in DNA loop structures or optimal positioning of the activator for interaction with polymerase. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a repressor and an Hg(II)-responsive activator of bacterial mercury-resistance genes. Escherichia coli RNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the repressor, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33 degrees relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex. PMID- 1731202 TI - DNA twisting and the effects of non-contacted bases on affinity of 434 operator for 434 repressor. AB - The bacteriophage 434 repressor regulates gene expression by binding with differing affinities to the six operator sites on the phage chromosome. The symmetrically arrayed outer eight base pairs (four in each half-site) of these 14 base-pair operators are highly conserved but the middle four bases are divergent. Although these four base pairs are not in contact with repressor, operators with A.T or T.A base pairs at these positions bind repressor more strongly than those bearing C.G or G.C, suggesting that these bases are important for the repressor's ability to discriminate between operators. There is evidence that the central base pairs influence operator function by constraining the twisting and/or bending of DNA. Here we show that there is a relationship between the intrinsic twist of an operator, as determined by the sequence of its central bases, and its affinity for repressor; an operator with a lower affinity is undertwisted relative to an operator with higher affinity. In complex with repressor, the twist of both high- and low-affinity operators is the same. These results indicate that the intrinsic twist of DNA and its twisting flexibility both affect the affinity of 434 operator for repressor. PMID- 1731203 TI - Battle lines form over fetal tissue research. PMID- 1731204 TI - Gene therapy. Britain gives the green light. PMID- 1731205 TI - Animal research. Court favours mice, rats, birds. PMID- 1731206 TI - Genetic information. Challenge to British forensic database. PMID- 1731207 TI - Biotechnology. Indian centre receives funds. PMID- 1731208 TI - Combating counterfeit drugs. PMID- 1731209 TI - NIH wasted millions on computers. PMID- 1731210 TI - Dirty habits. PMID- 1731211 TI - The grant racket. PMID- 1731212 TI - Handicaps to British 'innovation'. AB - Britain urgently needs to return to traditional, but now abandoned, values in science. Scientific administrators and EC directorates should revise their policies in the light of Japan's experience. PMID- 1731213 TI - Is molecular biology yet a science? PMID- 1731214 TI - Evolutionary physiology. The red flag of optimality. PMID- 1731215 TI - DNA profiling. Law and probabilities. PMID- 1731216 TI - Database contamination. PMID- 1731217 TI - Death by superantigen. PMID- 1731218 TI - Patterns of population spread. PMID- 1731219 TI - Chromatin as an essential part of the transcriptional mechanism. AB - The increasingly detailed biochemical definition of the protein complexes that regulate gene transcription has led to the re-emergence of questions about the role of histones. Much recent evidence suggests that transcriptional activation requires that transcription factors successfully compete with histones for binding to promoters, and that there may be more than one mechanism by which this is achieved. PMID- 1731220 TI - Subtractive and divisive adaptation in the human visual system. AB - Sensory systems can adapt to the conditions imposed on them. In the visual system, adapting to a pattern increases the threshold of the ability to see that pattern, and reduces the perceived contrast of the pattern above threshold. Most neurons of the striate cortex reduce their responsiveness after being stimulated for some time by a high-contrast pattern. Such an effect may lie behind these psychophysical adaptation phenomena. These adaptation effects have been reported to be confined to patterns of similar orientation, which is understandable in that the visual neurons that adapt are only excited by a small range of orientations. Neurophysiological evidence suggests that neurons with different orientation preferences have inhibitory interconnections. It is therefore of interest to explore the possible effects of these connections on perception. Here we show that adapting to a horizontal pattern can reduce the perceived contrast of a vertical test pattern more than a horizontal test pattern. These 'cross orientation' effects are modelled by a division-like process, whereas the more normal 'similar-orientation' effects are modelled by a subtractive process. PMID- 1731221 TI - Regulation of the G1-S transition in postembryonic neuronal precursors by axon ingrowth. AB - In the newly cellularized Drosophila embryo, progress through the cell cycle is regulated at the G2-M transition. We have examined cell-cycle regulation later in Drosophila development, in a group of postembryonic neuronal precursors. The S phase precursor cells, which generate photoreceptor target neurons (lamina neurons) in the central nervous system, are not present in the absence of photoreceptor innervation. Here we report that axons selectively approach G1 phase precursors. Without axon ingrowth, lamina precursors do not enter their final S phase and by several criteria, arrest in the preceding G1 phase. These findings provide evidence that at this stage in development the control of cell division can occur at the G1-S transition. PMID- 1731222 TI - Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries. AB - Antibodies are usually prepared from recently boosted animals and reflect ongoing immune responses. In humans, this is restrictive as ethical constraints generally prevent antigen-boosting. Therefore the rich memory compartment of human antibody responses remains largely untapped. Severe combined immune deficiency (SCID) mice populated with human cells allow the stimulation of human antibody memory without the usual constraints. Here we show how peripheral blood lymphocytes can be stimulated by antigen to produce large secondary responses after transfer to SCID mice. Specific monoclonal human Fab fragments can then be isolated from the mice by repertoire cloning even when the human donor's last contact with antigen was more than 17 years ago. PMID- 1731223 TI - A chimaeric 11 beta-hydroxylase/aldosterone synthase gene causes glucocorticoid remediable aldosteronism and human hypertension. AB - Glucocorticoid-remediable aldosteronism (GRA), an autosomal dominant disorder, is characterized by hypertension with variable hyperaldosteronism and by high levels of the abnormal adrenal steroids 18-oxocortisol and 18-hydroxycortisol, which are all under control of adrenocorticotropic hormone and suppressible by glucocorticoids. These abnormalities could result from ectopic expression of aldosterone synthase, which is normally expressed only in adrenal glomerulosa, in the adrenal fasciculata. Genes encoding aldosterone synthase and steroid 11 beta hydroxylase (expressed in both adrenal fasciculata and glomerulosa), which are 95% identical and lie on chromosome 8q (refs 7, 10), are therefore candidate genes for GRA. Here we demonstrate complete linkage of GRA in a large kindred to a gene duplication arising from unequal crossing over, fusing the 5' regulatory region of 11 beta-hydroxylase to the coding sequences of aldosterone synthase (maximum lod score 5.23 for complete linkage, odds ratio of 170,000:1). This mutation can account for all the physiological abnormalities of GRA. Our result represents the demonstration of a mutation causing hypertension in otherwise phenotypically normal animals or humans. PMID- 1731224 TI - Multiple evolutionary origins of prochlorophytes, the chlorophyll b-containing prokaryotes. AB - Prochlorophytes are prokaryotes that carry out oxygenic photosynthesis using chlorophylls a and b, but lack phycobiliproteins as light-harvesting pigments. These characteristics distinguish them from cyanobacteria, which contain phycobiliproteins, but no chlorophyll b. Three prochlorophyte genera have been described: Prochloron, Prochlorothrix and Prochlorococcus. The prochlorophytes share their pigment characteristics with green plant and euglenoid chloroplasts, which has led to a debate on whether these chloroplasts may have arisen from an endosymbiotic prochlorophyte rather than a cyanobacterium. Molecular sequence data, including those presented here based on a fragment of the rpoC1 gene encoding a subunit of DNA-dependent RNA polymerase, indicate that the known prochlorophyte lineages do not include the direct ancestor of chloroplasts. We also show that the prochlorophytes are a highly diverged polyphyletic group. Thus the use of chlorophyll b as a light-harvesting pigment has developed independently several times in evolution. Similar conclusions have been reached in parallel studies using 16S ribosomal RNA sequences. PMID- 1731225 TI - Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation. AB - The taxonomic group Prochlorales (Lewin 1977) Burger-Wiersma, Stal and Mur 1989 was established to accommodate a set of prokaryotic oxygenic phototrophs which, like plant, green algal and euglenoid chloroplasts, contain chlorophyll b instead of phycobiliproteins. Prochlorophytes were originally proposed (with concomitant scepticism) to be a monophyletic group sharing a common ancestry with these 'green' chloroplasts. Results from molecular sequence phylogenies, however, have suggested that Prochlorothrix hollandica is not on a lineage that leads to plastids. Our results from 16S ribosomal RNA sequence comparisons, which include new sequences from the marine picoplankter Prochlorococcus marinus and the Lissoclinum patella symbiont Prochloron sp., indicate that prochlorophytes are polyphyletic within the cyanobacterial radiation, and suggest that none of the known species is specifically related to chloroplasts. This implies that the three prochlorophytes and the green chloroplast ancestor acquired chlorophyll b and its associated structural proteins in convergent evolutionary events. We report further that the 16S rRNA gene sequence from Prochlorococcus is very similar to those of open ocean Synechococcus strains (marine cluster A), and to a family of 16S rRNA genes shotgun-cloned from plankton in the north Atlantic and Pacific Oceans. PMID- 1731226 TI - Structural basis of latency in plasminogen activator inhibitor-1. AB - Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features. PMID- 1731227 TI - Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography. AB - Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 A resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few angstroms by fitting atomic models of CPMV4 and Fab (Kol)5 to reconstructed density maps. PMID- 1731228 TI - Mixed micelles in drug delivery. AB - Large disk-like mixed micelles composed of a drug and biological lipid are thermodynamically stable and represent a novel drug delivery system. Their unique physical properties are reflected in a significantly improved therapeutic index. PMID- 1731229 TI - Gene technology gems. PMID- 1731230 TI - 'IQ pills' land in trouble. PMID- 1731231 TI - Call for UK gene therapy. PMID- 1731233 TI - Will EC policy boost European health studies? PMID- 1731232 TI - Daniel Zagury cleared of misconduct charges. PMID- 1731234 TI - Drugs tested for women. PMID- 1731235 TI - FDA panel backs interleukin-2. PMID- 1731236 TI - Three share Jeantet award in medicine. PMID- 1731237 TI - Biotech lobby pressure EC. PMID- 1731238 TI - Air pollution and health. PMID- 1731239 TI - Population crisis. PMID- 1731240 TI - Schizophrenia. PMID- 1731241 TI - Needed: fetal tissue research. PMID- 1731242 TI - Cell biology. Centrosome and cell division. PMID- 1731243 TI - Molecular biology. recA: from locus to lattice. PMID- 1731244 TI - AIDS and malaria experiments. PMID- 1731245 TI - AIDS and malaria experiments. PMID- 1731246 TI - The structure of the E. coli recA protein monomer and polymer. AB - The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double stranded DNA. Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles. This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy. Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo. PMID- 1731247 TI - Spatial filtering precedes motion detection. AB - When we perceive motion on a television or cinema screen, there must be some process that allows us to track moving objects over time: if not, the result would be a conflicting mass of motion signals in all directions. A possible mechanism, suggested by studies of motion displacement in spatially random patterns, is that low-level motion detectors have a limited spatial range, which ensures that they tend to be stimulated over time by the same object. This model predicts that the direction of displacement of random patterns cannot be detected reliably above a critical absolute displacement value (Dmax) that is independent of the size or density of elements in the display. It has been inferred that Dmax is a measure of the size of motion detectors in the visual pathway. Other studies, however, have shown that Dmax increases with element size, in which case the most likely interpretation is that Dmax depends on the probability of false matches between pattern elements following a displacement. These conflicting accounts are reconciled here by showing that Dmax is indeed determined by the spacing between the elements in the pattern, but only after fine detail has been removed by a physiological prefiltering stage: the filter required to explain the data has a similar size to the receptive field of neurons in the primate magnocellular pathway. The model explains why Dmax can be increased by removing high spatial frequencies from random patterns, and simplifies our view of early motion detection. PMID- 1731248 TI - Stationary and drifting spiral waves of excitation in isolated cardiac muscle. AB - Excitable media can support spiral waves rotating around an organizing centre. Spiral waves have been discovered in different types of autocatalytic chemical reactions and in biological systems. The so-called 're-entrant excitation' of myocardial cells, causing the most dangerous cardiac arrhythmias, including ventricular tachycardia and fibrillation, could be the result of spiral waves. Here we use a potentiometric dye in combination with CCD (charge-coupled device) imaging technology to demonstrate spiral waves in the heart muscle. The spirals were elongated and the rotation period, Ts, was about 180 ms (3-5 times faster than normal heart rate). In most episodes, the spiral was anchored to small arteries or bands of connective tissue, and gave rise to stationary rotations. In some cases, the core drifted away from its site of origin and dissipated at a tissue border. Drift was associated with a Doppler shift in the local excitation period, T, with T ahead of the core being about 20% shorter than T behind the core. PMID- 1731249 TI - Limbs generated at site of tail amputation in marbled balloon frog after vitamin A treatment. AB - Niazi and Saxena first observed that vitamin A has an inhibitory and modifying influence on tail regeneration in Bufo andersonii tadpoles. A positive relationship was later found between the inhibiting influence of vitamin A and the developmental stage of the regenerating tail in the same species. There have been several subsequent reports on the effects of vitamin A and its derivatives on limb development and regeneration. Thus in regenerating amphibian limbs, application of retinoids produces pattern duplication in the proximodistal and anteroposterior axes of the limb, and local application of retinoic acid to the anterior side of developing chick limbs causes duplications in the anteroposterior axis of limb. Here we show that vitamin A can cause limb development when applied to amputated tail stumps of the tadpoles of the marbled balloon frog Uperodon systoma (Anura Microhylidae). This is the first report of homeotic transformation mediated through vitamin A in vertebrates. PMID- 1731250 TI - S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28. AB - In somatic cells, entry into mitosis depends on the completion of DNA synthesis. This dependency is established by S-phase feedback controls that arrest cell division when damaged or unreplicated DNA is present. In the fission yeast Schizosaccharomyces pombe, mutations that interfere with the phosphorylation of tyrosine 15 (Y15) of p34cdc2, the protein kinase subunit of maturation promoting factor, accelerate the entry into mitosis and abolish the ability of unreplicated DNA to arrest cells in G2. Because the tyrosine phosphorylation of p34cdc2 is conserved in S. pombe, Xenopus, chicken and human cells, the regulation of p34cdc2-Y15 phosphorylation could be a universal mechanism mediating the S-phase feedback control and regulating the initiation of mitosis. We have investigated these phenomena in the budding yeast Saccharomyces cerevisiae. We report here that the CDC28 gene product (the S. cerevisiae homologue of cdc2) is phosphorylated on the equivalent tyrosine (Y19) during S phase but that mutations that prevent tyrosine phosphorylation do not lead to premature mitosis and do not abolish feedback controls. We have therefore demonstrated a mechanism that does not involve tyrosine phosphorylation of p34 by which cells arrest their division in response to the presence of unreplicated or damaged DNA. We speculate that this mechanism may not involve the inactivation of p34 catalytic activity. PMID- 1731251 TI - Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae. AB - Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins. In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein. Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19). However, in contrast to previous observations in S. pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase. Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage. Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined. We propose that there are other unidentified controls that regulate entry into mitosis. PMID- 1731252 TI - A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. AB - The hydrophobic cores of proteins are generally well packed, with few cavities. Mutations in which a bulky buried residue such as leucine or phenylalanine is replaced with a small residue such as alanine can create cavities in the core of a protein (our unpublished results). The sizes and shapes of such cavities can vary substantially depending on factors such as local geometry, whether or not a cavity already exists at the site of substitution, and the degree to which the protein structure relaxes to occupy the space vacated by the substituted residue. We show by crystallographic and thermodynamic analysis that the cavity created by the replacement Leu 99----Ala in T4 lysozyme is large enough to bind benzene and that ligand binding increases the melting temperature of the protein by 6.0 degrees C at pH 3.0. Benzene does not, however, bind to the cavity created by the Phe 153----Ala replacement. The results show that cavities can be engineered in proteins and suggest that such cavities might be tailored to bind specific ligands. The binding of benzene at an internal site 7 A from the molecular surface also illustrates the dynamic nature of proteins, even in crystals. PMID- 1731253 TI - Structure of the recA protein-ADP complex. AB - The recA protein catalyses the ATP-driven homologous pairing and strand exchange of DNA molecules. It is an allosteric enzyme: the ATPase activity is DNA dependent, and ATP-bound recA protein has a high affinity for DNA, whereas the ADP-bound form has a low affinity. In the absence of ATP hydrolysis, recA protein can still promote homologous pairing, apparently through the formation of a triple-stranded intermediate. The exact role of ATP hydrolysis is not clear, but it presumably drives the triplex intermediate towards products. Here we determine the position of bound ADP diffused into the recA crystal. We show that only the phosphates are bound in the same way as in other NTPases containing the G/AXXXXGKT/S motif. We propose that recA protein may change its conformation upon ATP hydrolysis in a manner analogous to one such protein, the p21 protein from the ras oncogene. A model is presented to account for the allosteric stimulation of DNA binding by ATP. The mechanism by which nucleoside triphosphate hydrolysis is coupled to the binding of another ligand in recA protein and p21 may be typical of the large class of NTPases containing this conserved motif. PMID- 1731254 TI - HIV testing: promote a rational approach... PMID- 1731255 TI - Our compliments to the authors. PMID- 1731256 TI - What was the source? PMID- 1731257 TI - Community partnerships in health professions education. PMID- 1731258 TI - Liability issues for the office nurse. PMID- 1731259 TI - Bandwagons revisited. PMID- 1731260 TI - How to bring nursing and system vendors closer together. PMID- 1731261 TI - An introduction to AONE. American Organization of Nurse Executives. PMID- 1731262 TI - Work designs: sociotechnical systems for patient care delivery. PMID- 1731263 TI - Profiling a chief nursing officer. PMID- 1731264 TI - Nursing supervisors: reduction-in-force or redesign roles? PMID- 1731265 TI - Transition: from clinician to administrator. PMID- 1731266 TI - Assistant head nurse: today's catalytic manager. PMID- 1731267 TI - Cross-training: a richer staff for leaner budgets. PMID- 1731268 TI - An admission assessment coordinator. PMID- 1731269 TI - LPN: a monitor nurse for telemetry units. PMID- 1731270 TI - Associate infection control nurse: answers when you need them. PMID- 1731271 TI - Ethical issues: a Washington perspective. PMID- 1731272 TI - The complexity of an authority role. PMID- 1731273 TI - Healthcare workers and occupational exposure to AIDS. PMID- 1731274 TI - Situational leadership. PMID- 1731275 TI - Emergency department clinical indicators. PMID- 1731276 TI - Automated information systems for OR management. PMID- 1731277 TI - Of square eggs and sore hens. PMID- 1731278 TI - Differentiating malignant from benign ovarian tumors with transvaginal color flow imaging. AB - The impedance to blood flow was examined by transvaginal color flow imaging in 53 ovarian masses before exploratory laparotomy. Serum CA 125 levels were measured in all subjects. Thirty-six had benign ovarian tumors and 17 had malignant ovarian tumors confirmed by histopathologic examination. Intratumoral blood vessels, detected in 16 of the malignant tumors, consistently demonstrated low impedance to flow, with a pulsatility index (PI) always below 1. The PI of the intraovarian or intratumoral blood vessels was greater than 1 in 35 of the 36 benign tumors, although 11 had suspicious sonographic findings (P less than .01) and 14 had elevated CA 125 levels (P less than .001). The sensitivity and specificity of the preoperative PI in detecting malignant ovarian tumors were 94 and 97%, respectively. The sensitivity and specificity of preoperative suspicious sonographic findings in detecting malignant ovarian tumors were 94 and 69%, and those of elevated preoperative serum CA 125 levels were 82 and 61%, respectively. Our results suggest that transvaginal color flow imaging may be a useful clinical tool in the preoperative evaluation of ovarian masses. PMID- 1731279 TI - Transvaginal Doppler ultrasound with color flow imaging in the diagnosis of ovarian cancer. AB - Transvaginal Doppler ultrasound with color flow imaging is a new technique for the evaluation of gynecologic diseases. This method was used for the diagnosis of ovarian tumors in 24 women treated in the Department of Obstetrics and Gynecology, Nagoya University Hospital, Japan. Waveforms of the parenchymal tumor arteries or tumor surface arteries were compared using values of the pulsatility index (PI). The value for 1/PI was 0.69 +/- 0.05 in benign tumors and 1.87 +/- 0.65 in malignant tumors (P less than .01). When the cutoff value of 1/PI was set at 0.8 (cutoff value of PI was 1.25), the accuracy of diagnosis was 95.8% (23 of 24). Among 11 tumors with low CA 125 (less than 35 U/mL), all five malignant tumors had high 1/PI (above 0.8), and all benign tumors had low 1/PI (less than 0.8) or nondetectable waveforms. Based on the findings of this study, color flow Doppler proved to be useful for diagnosis of ovarian cancer. PMID- 1731280 TI - Morbidity and mortality associated with primary and repeat operations for ovarian cancer. AB - Aggressive surgery may improve the outcome of patients with ovarian cancer. To assess the risk of operative complications, we analyzed 472 primary and 299 repeat operations for ovarian cancer in 536 women between 14-91 years of age. Intraoperative bleeding estimated to be over 1000 mL (N = 107) was more common after primary (21%) than repeat surgery (3%). Urinary tract infection accompanied 113 operations (18% of primary and 9% of repeat), bowel complication 51 operations (7% of primary and 6% of repeat), fever 23 operations (4% of primary and 1% of repeat), wound complication 17 operations (3% of primary and 1% of repeat), and thromboembolism 11 operations (2% of primary and 0.3% of repeat). Patient age had no effect on the rate of complications. Pelvic and para-aortic lymphadenectomy in connection with primary operations caused substantial blood loss. Five subjects (1%) died after primary operations. On the whole, the surgical procedures, especially repeat operations, were well tolerated and should not be avoided for fear of complications. PMID- 1731281 TI - Treatment of cervical intraepithelial neoplasia using the loop electrosurgical excision procedure. AB - In selected patients with cervical intraepithelial neoplasia (CIN), outpatient ablative procedures represent a readily accepted and highly effective treatment modality. The recently introduced loop electrosurgical excision procedure offers a quick and simple alternative to cryotherapy and laser ablation for treating CIN, and has the distinct advantage of allowing both diagnosis and treatment of selected patients at a single visit. This report presents our clinical experience treating 432 patients with CIN using the loop electrosurgical excision procedure on an outpatient basis. Small loop electrodes were used to excise CIN lesions in 275 patients, and large loop electrodes were used in 157. When performed on an outpatient basis under local anesthesia, loop excision was well tolerated by patients with only minimal discomfort. Post-treatment bleeding occurred in less than 2% of the subjects and responded to either recauterization or packing of the cervix. Post-treatment stenosis occurred in less than 1%. The success rate of the loop electrosurgical excision procedure, as defined by absence of cytologic, histologic, or colposcopic lesions 4-48 months after therapy, was 80% for women treated using the small loop electrodes. Ninety percent of all patients treated using the large loop electrodes were free of disease during 6-12 months of follow up. For women being treated for primary (as opposed to recurrent) disease, the success rate with large loop electrodes was 94%. PMID- 1731282 TI - Why patients fail antibiotic prophylaxis at cesarean delivery: histologic evidence for incipient infection. AB - A prospective, blinded study was conducted to test the hypothesis that antimicrobial prophylaxis failure after cesarean delivery is associated with incipient infection of the uterus, as determined by histologic evaluation of bacterial invasion and acute inflammatory cell response. One hundred nineteen patients undergoing cesarean delivery and receiving antibiotic prophylaxis were included in this study. At the time of the operation, a hysterotomy biopsy was obtained for hematoxylin and eosin staining. Marked histologic differences were noted in decidual inflammation, myometrial inflammation, and myometrial polymorphonuclear cell invasion in those patients who subsequently developed endometritis (N = 7) compared with subjects without postpartum endometritis. Using two techniques for in situ identification of bacteria within myometrial tissue (acridine orange and fluorescein DNA probe to bacterial ribosomal RNA), all clinically infected parturients demonstrated large numbers of organisms in the myometrial layer of the biopsy specimen, compared with few organisms seen in a matched subset of noninfected controls. These data support the concept that incipient infection at the time of cesarean delivery may limit the effectiveness of antimicrobial prophylaxis. Use of rapid-diagnosis methodologies may allow timely identification of these at-risk patients so that therapeutic antibiotics can be initiated. PMID- 1731283 TI - A persistent clinical problem: profile of the term infant with significant respiratory complications. AB - A group of 72 term infants with significant respiratory complications were compared with 11,428 term infants delivered during the same time period and without respiratory morbidity. Compared with controls, the study group had a higher incidence of postdatism (36 versus 7%), intrauterine growth retardation (33 versus 8%), meconium-stained amniotic fluid (AF) (90 versus 9%), fetal heart rate (FHR) abnormalities upon admission to labor and delivery (58 versus 7%), and low 5-minute Apgar scores (46 versus 1.4%). Even in the presence of normal intrapartum FHR and 5-minute Apgar scores, infants with meconium-stained AF had an incidence of respiratory complications 100 times higher than those with clear AF. Of infants with a low 5-minute Apgar score at birth, only 20% went on to develop respiratory complications. The remaining 80% had a significantly lower incidence of postdatism, intrauterine growth retardation, and meconium-stained AF. PMID- 1731284 TI - Reproductive technology: drawing the line. AB - An anonymous survey was administered to clinical clerks rotating through obstetrics and gynecology during weekly ethics rounds. Students were asked to judge the moral acceptability as participants and as referring physicians of 11 reproductive technology scenarios ranging from artificial insemination using a husband's sperm in a conjugal relationship to instances involving in vitro fertilization, uterine donation, surrogacy contracts, self-insemination, and single or lesbian parenthood. Positive judgments regarding personal moral acceptability ranged from 30-100% and as a referring physician from 36-99%. Though most students were consistent in their judgments about personal and professional moral acceptability, some (1-15%) could see themselves acting as referring physicians for something they personally found morally unacceptable. Those students who were opposed to contracting for children (more women than men) were more likely to find the reproductive scenarios morally unacceptable. This survey seemed to be a useful tool for discussing where students draw the line morally and why, and whether they would distance themselves from actions they found morally unacceptable. This technique for teaching applied analytic ethics is especially applicable to obstetrics and gynecology because it addresses fundamental questions involving day-to-day practice in the specialty. PMID- 1731285 TI - Long-term transdermal estradiol therapy: effects on endometrial histology and bleeding patterns. AB - Sixty postmenopausal women were enrolled in a 2-year randomized unmasked trial to determine the long-term safety of estradiol (E2) administration by a transdermal therapeutic system. Group I subjects received 0.1 mg of transdermal E2 for 24.5 days of each 28-day cycle for 96 weeks. Group II subjects received the same dosage of transdermal E2 plus 10 mg of medroxyprogesterone acetate, given orally from days 13-25 of each cycle. Vaginal bleeding patterns and endometrial histology were characterized. The subjects recorded bleeding patterns daily. Endometrial biopsies were performed during scheduled follow-up visits at 48 and 96 weeks or as needed to evaluate abnormal bleeding. Data were analyzed by intention to treat. Ten and four subjects dropped out of the study from groups I and II, respectively. A total of 575 and 627 treatment cycles were observed in the same respective groups. Vaginal bleeding was observed in 980 cycles: 381 of 575 cycles in group I (66.3%) and 599 of 627 cycles in group II (95.5%). Bleeding onset, duration, and quantity were similar for both groups. The incidence of hyperplasia was 42 and 4% for groups I and II, respectively, over the 96-week study period. All cases of hyperplasia in group I were treated with sequential medroxyprogesterone acetate for 12 weeks, followed by rebiopsy. In ten of 11 cases, the progestin therapy converted the hyperplasia to a normal endometrium. In one case, the endometrium became hyperplastic again at 96 weeks, but reverted to normal with 12 weeks of medroxyprogesterone acetate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731286 TI - Prevalence of active Chlamydia trachomatis infection at the time of exploratory laparotomy for ectopic pregnancy. AB - Cultures of the fallopian tube, cul-de-sac, and cervix were obtained from 50 patients undergoing exploratory laparotomy for ectopic pregnancy. Chlamydia trachomatis was cultured from the cervix in four patients (8%). All the cultures from the fallopian tube or cul-de-sac were negative. Immunofluorescent stain of deparaffinized cross-sections of fallopian tube at the site of tubal involvement with the ectopic pregnancy failed to reveal any C trachomatis inclusions. PMID- 1731287 TI - The accuracy of digital examination and ultrasound in the evaluation of cervical length. AB - Assessment of cervical status is an important component of the management of patients at risk for preterm delivery. Although digital examination is the most common method of cervical assessment, there has been recent interest in sonographic cervical examination. To compare the accuracy of digital examination and ultrasound, 20 nongravid women undergoing total hysterectomy for gynecologic indications had measurements of cervical length performed digitally and with both transabdominal and transvaginal ultrasound before surgery. These measurements were then compared with measurements made with a ruler immediately after hysterectomy. Separate examiners performed the digital, ultrasound, and ruler measurements, and each was blinded to the results of the others. Digital examination underestimated cervical length by an average of 13.6 mm and was significantly shorter than ruler measurement (P = .0001). Neither ultrasound method differed significantly from ruler measurement (P greater than .9 for each), and measurements were similar between the sonographic techniques (P greater than .9). These results validate the accuracy of sonographic estimation of cervical length. In addition, they suggest that sonographic measurement is more accurate than digital examination in predicting true cervical length. Finally, in the nonpregnant women, neither ultrasound technique seems superior to the other. PMID- 1731288 TI - Effect of routine weekly cervical examinations at term on premature rupture of the membranes: a randomized controlled trial. AB - OBJECTIVE: The purpose of this study was to determine whether routine antepartum cervical examinations at term are associated with premature rupture of membranes (PROM). METHODS: This was a randomized controlled trial conducted at a health maintenance organization in metropolitan Denver. The subjects were 604 term gravidas randomized to a no examination or examination group. Exclusions included preterm labor, third-trimester bleeding, cerclage, multiple pregnancy, history of PROM (rupture before the onset of labor), and planned induction or cesarean. In the no examination group, routine examinations (without clinical indication) were not performed. In the examination group, weekly examinations were performed from 37 weeks until delivery. RESULTS: No statistically significant difference in PROM or prolonged PROM (more than 6 hours) was observed between those unexposed and those exposed to routine cervical examinations at term. In addition, there were no differences between the groups in other relevant outcomes including cesarean delivery, induction, augmentation, chorioaminionitis, or neonatal infectious morbidity. CONCLUSION: In our population, there is no association between routine weekly antepartum cervical examinations at term and PROM or other study end points. PMID- 1731289 TI - Randomized comparative trial of indomethacin and sulindac for the treatment of refractory preterm labor. AB - This study was designed to investigate the efficacy and safety of sulindac in the treatment of preterm labor. Thirty-six women in preterm labor who had failed initial attempts at tocolysis with magnesium sulfate were randomized to receive either oral indomethacin or oral sulindac for 48 hours. The mean gestational ages at admission were 29 and 30 weeks for the sulindac and indomethacin groups, respectively. There was a significantly greater hourly fetal urine output, deepest amniotic fluid pocket, and amniotic fluid index in the sulindac group. A trend toward higher fetal ductus arteriosus flow velocities noted in the indomethacin group was not seen in the sulindac group. The drugs had similar success in delaying delivery for 48 hours or 7 days. The mean birth weights were 2000 and 2323 g for the sulindac and indomethacin groups, respectively. Sulindac appears to be as effective as indomethacin for refractory preterm labor but with fewer fetal side effects. PMID- 1731290 TI - Longitudinal study of the amniotic fluid index in post-dates pregnancy. AB - Amniotic fluid (AF) was measured in 511 post-dates pregnancies (at least 41 weeks of gestational age) with the use of the AF index. Ultrasonographic evaluations were conducted on a semiweekly basis. Only patients with reliable gestational ages calculated from certain last menstrual period and confirmed by early sonographic estimates participated in the study. Oligohydramnios (AF index of 5.0 cm or less) was detected in 11.5% of the study population. Longitudinal data were available from 121 patients who demonstrated a mean 25% decrease in AF index per week beyond 41 weeks' gestation. The longitudinal change in AF index was statistically significant (P less than .0005). Amniotic fluid index measurements ranged from 1.7-24.6 cm, with a mean of 12.4 at 41 weeks' gestation. Compared with previous cross-sectional studies, this longitudinal study provides a more accurate estimate of changes in AF levels as a function of gestational age. PMID- 1731291 TI - Risk of chromosomal abnormalities in patients with idiopathic polyhydramnios. AB - This prospective investigation was designed to assess the incidence of chromosomal abnormalities in patients with idiopathic polyhydramnios. Polyhydramnios was defined as 25 cm or greater in total vertical height in all four quadrants (amniotic fluid index) in any nonreferral patient (ie, primary care population) undergoing sonographic examination with a singleton pregnancy, normal fetal anatomical survey, normal glucose screening, and negative antibody screen. During the 2-year period from May 1, 1988 through April 30, 1990, 5038 gravidas delivered at Madigan Army Hospital Center. Unexplained polyhydramnios was detected sonographically in 125 patients, an incidence of 2.5%. After obtaining informed written consent, amniocentesis was performed in all patients. Within this group (N = 125), four chromosomal abnormalities (incidence of 3.2%) were detected. There were two trisomy 18 and two trisomy 21 fetuses. None of the four patients had maternal serum alpha-fetoprotein screening performed. The incidence of aneuploidy in patients with idiopathic polyhydramnios (3.2%) is much higher than the reported incidence of major karyotype abnormalities in live births (0.59%). We conclude that fetal chromosomal analysis should be considered in all obstetric patients with sonographic evidence of idiopathic polyhydramnios. PMID- 1731292 TI - Maternal Kell blood group alloimmunization. AB - BACKGROUND: Two recent paper have provided conflicting views regarding the severity of Kell hemolytic disease of the newborn. METHODS: We reviewed our experience during 1944-1990 with pregnant Kell-alloimmunized Manitoban women and similar women referred from outside of Manitoba. RESULTS: Between 1944-1990, 311 Kell-immunized Manitoban women had 459 pregnancies, of which 63 ended in abortion or stillbirth unrelated to anti-Kell. Of the infants born, 376 were unaffected and 20 were affected. Twelve did not require treatment; two needed phototherapy, one required a simple transfusion, and one an exchange transfusion. One died of kernicterus and three were hydropic and died; all four deaths occurred between 1948-1954. Fourteen Kell-immunized women with 16 pregnancies were referred from outside Manitoba. Eleven had a history of Kell hydropic fetuses and ten had hydropic fetuses at referral. Five of the hydropic fetuses survived and five died. Five women had Kell-negative infants correctly predicted by amniocentesis (two) and by fetal blood sampling (three). Serial amniotic fluid delta OD 450 readings were 83-89% accurate in predicting the presence and severity of Kell hemolytic disease. Life-threatening inaccuracies occurred, primarily in the early and middle second trimester. CONCLUSIONS: Kell hemolytic disease, although rare, may be as severe as Rh(D) hemolytic disease when it does occur. When there is a history of hydrops or the father is Kell-positive and the maternal anti-Kell indirect antiglobulin titer is 8 or greater, amniocentesis should be performed at 16-20 weeks' gestation. Fetal blood sampling followed by fetal intravascular transfusion is indicated if delta OD 450 readings approach the 65% level in modified zone 2 of Liley or if amniocentesis is precluded because of an anterior placenta and there is a history of hydrops or ultrasound evidence of fetal hemolytic disease. PMID- 1731293 TI - Substance abuse during pregnancy in a rural population. AB - Drug abuse during pregnancy in rural populations has received less attention than that in urban populations. Urban studies have reported alarming rates, but it is unknown whether the situation is the same in rural areas. To investigate this, urine samples were collected anonymously from 181 pregnant women who presented to the University of Missouri clinics for care and who resided in communities of less than 25,000. Each urine specimen was tested for cocaine, marijuana, amphetamines, barbiturates, opiates, phencyclidine, benzodiazepines, ethanol, and nicotine. Of the 181 specimens, 83 (46%) contained nicotine, 17 (9.4%) contained marijuana, and one each (0.6%) tested positive for cocaine, barbiturates, ethanol, and benzodiazepines. No other tested substances were detected. Excluding nicotine and ethanol, 20 (11%) of the urine samples tested positive for the screened substances. Review of the prenatal records revealed that 46% of the women reported using tobacco, 15% reported using alcohol, and 8.3% reported illicit drug use during pregnancy. This study indicates that there is a substantial drug abuse problem in rural populations, and that the profile of abuse differs from that of urban populations. Tobacco, ethanol, and marijuana were the most prevalent substances abused during pregnancy, but cocaine was a minor problem. This information may help in directing resources to reduce drug abuse during pregnancy. PMID- 1731294 TI - Approximate entropy: a regularity measure for fetal heart rate analysis. AB - Approximate entropy (ApEn), a recently developed mathematical formula quantifying regularity, was applied to fetal heart rate (FHR) data. Three groups were analyzed: 1) 19 women had normal labors (uncomplicated course of labor, vaginal delivery, no unusual FHR tracings, and 1- and 5-minute Apgar scores of at least 7 and 9, respectively; 2) 15 women had presumed fetal distress (severe cord or late decelerations, bradycardia, or tachycardia; delivery by cesarean with both arterial and venous cord pH above 7.20); and 3) 20 women had acidotic fetuses (both venous and arterial cord pH less than 7.20). Hourly mean (+/- SD) ApEn values for the three groups were: acidotic fetuses, 0.924 +/- 0.235, 102 hours; normal fetuses, 1.051 +/- 0.145, 97 hours; and nonacidotic "distressed" fetuses, 1.043 +/- 0.147, 74 hours. The ApEn values for nonacidotic, presumed distressed fetuses were not significantly different from those of normal fetuses (P greater than .75). Acidotic fetuses had many more instances of ApEn hourly values less than 0.8 (28%, 29 of 102) than did the normal and the nonacidotic, presumed distressed fetuses combined (5%, nine of 171). The probability that ApEn was less than 0.8 was larger for acidotic fetuses than for the other groups (P less than .00003), supporting the hypothesis that extremely regular FHR patterns imply a greater likelihood of acidosis. Significant hourly deviations in ApEn generally corresponded to drug administration or to physiologic changes such as cord compression and its relief. Thus ApEn, a major departure from variability statistics, appears to be able to detect subtle and possibly important differences in heart rate that are not visually apparent. PMID- 1731295 TI - Antenatal classification of hydrops fetalis. AB - Among 12,572 pregnant women referred for ultrasound examination from 1985-1990, 76 fetuses had ultrasonographic findings of hydrops fetalis, ten immune and 66 nonimmune. Fetuses with cystic hygroma (20), heart defects or arrhythmias (13), or other congenital anomalies (15) accounted for the majority of the nonimmune cases. Antenatal chromosomal studies were available in 42 fetuses with nonimmune hydrops, of which 14 (34%) were abnormal with seven monosomes and six trisomies. Seventeen cases of hydrops (22%) were classified as idiopathic because they had no recognizable etiology. It is concluded that: 1) The ultrasonographic incidence of fetal hydrops in referral centers can be as high as one in 165 pregnancies; 2) most cases of fetal hydrops are of the nonimmune type, which can occur in a low risk population and can be detected with early second-trimester ultrasound screening; and 3) the complexity of this condition and the high rate of chromosomal abnormalities require referral to a high-risk center for evaluation and pregnancy management. PMID- 1731296 TI - Delayed delivery of second twin: report of four cases and review of the literature. AB - In four cases of delayed delivery of a twin pregnancy with survival of the second twin, the interval ranged at 41-143 days. Review of the literature and our cases supports the following approach: high ligation of the umbilical cord with an absorbable suture, cervical suture in the presence of cervical dilatation, and serial monitoring of fetal growth and maternal coagulation indices. Disseminated intravascular coagulation has not been reported in such cases. PMID- 1731297 TI - Maternal renal artery Doppler velocimetry in normotensive pregnancies and pregnancies complicated by hypertensive disorders. AB - Hypertensive disorders in pregnancy contribute to substantial maternal and perinatal morbidity and mortality. Clinically, these disorders are characterized by hypertension and proteinuria. However, these signs appear some time after the physiologic derangements have been initiated. The primary objectives of this study were as follows: 1) to establish baseline values for the maternal renal artery systolic-diastolic ratio (S/D) as a function of gestational age in normal pregnancies, and 2) to determine whether renal artery blood flow indices can accurately identify those pregnancies complicated by, or destined to develop, hypertensive disorders. Using a pulsed Doppler scanner, maternal renal artery duplex evaluation was performed in four groups of women: normotensive nonpregnant, normotensive pregnant, chronic hypertensive pregnant, and preeclamptic. In 30 normotensive pregnant women followed longitudinally, no change was noted in the renal artery S/D as gestational age advanced, with mean (+/- SD) values of 2.5 +/- 0.20 and 2.6 +/- 0.21 for the left and right sides, respectively. No clinically meaningful discriminations were detected when the four groups were compared. We conclude that maternal renal artery Doppler waveforms are not significantly altered by either pregnancy or hypertensive complications in pregnancy. PMID- 1731298 TI - Changing obstetric practice and 2-year outcome of the fetus of birth weight under 1000 g. AB - The aim of this study was to assess the outcome up to 2 years of age for the fetus of birth weight 500-999 g, over time and in association with changes in obstetric care. Two consecutive cohorts of infants of birth weight 500-999 g were compared from two eras, 1977-1982 and 1985-1987, and their outcome up to 2 years of age was determined with particular emphasis on the effect of various obstetric interventions at the time of birth, such as cesarean delivery, electronic fetal monitoring, antenatal steroid therapy, and tocolytic therapy. The outcome to 2 years was analyzed by logistic function regression to adjust for imbalances in confounding perinatal variables. In the latter era, the survival rate to 2 years increased significantly by almost 50%, and only 7% of the survivors were severely disabled. The rates of delivery by cesarean and of electronic fetal monitoring both increased significantly in the latter era, but neither was associated with the improved outcome. The only variable associated with an improved outcome that was amenable to obstetric intervention at the time of birth was antenatal steroid therapy, which was used equally in both eras. The obstetrician may aid the fetus of birth weight 500-999 g by giving the mother steroids to accelerate fetal lung maturity, but cesarean cannot be recommended as the routine mode of delivery unless there are recognized maternal or fetal indications. PMID- 1731299 TI - The influence of previous low birth weight on birth weight, gestational age, and anthropometric measurements in the current pregnancy. AB - The effect of a previous low birth weight birth (less than 2750 g) was examined using a series of regression analyses. Effects on birth weight were partitioned into those associated with preterm delivery (128 g) and term delivery (178 g). Among term births, a mean difference of 107 g was associated with a previous birth of less than 2750 g, even after controlling for other risk factors including smoking, drug and alcohol use, maternal race, size, and hypertension. The pattern of measurements seen after a previous birth of less than 2750 g included significantly smaller head, chest, abdomen, arm, and thigh circumferences, but an insignificant impact on skinfold thicknesses and no significant effect on length measurements. PMID- 1731300 TI - A story of two miracles: the impact of the discovery of insulin on pregnancy in women with diabetes mellitus. AB - Before the discovery of insulin in 1921, pregnancies in women with diabetes mellitus were a rarity because most reproductive-age patients died soon after diagnosis of this illness. In the limited number of pregnancies reported in the pre-insulin era, both perinatal and maternal mortality were approximately 50%, with stillbirths being the primary cause of perinatal deaths. Insulin treatment restored the fertility of women with diabetes and was associated with a marked reduction in maternal mortality. Women with more severe disease had the opportunity to become pregnant; however, their pregnancies frequently resulted in neonatal death due to prematurity. Therefore, perinatal mortality was not substantially reduced. PMID- 1731301 TI - Ethanol labeling: detection of early fluid absorption in endometrial resection. AB - A study is presented of ethanol labeling of irrigation fluid in endometrial resection. The introduction of ethanol labeling and intraoperative breath ethanol analysis provided an inexpensive and potentially useful means of detecting early fluid absorption during uterine surgery. The breath ethanol analyzer used was a hand-held meter; the irrigant solution was 5% dextrose with 1% ethanol. Simultaneous breath and venous samples were taken from women undergoing endometrial resection. An increase in breath ethanol was positively correlated with fluid absorption, blood ethanol, and serum glucose. This technique may prove valuable in preventing fluid overload during endometrial resection. PMID- 1731302 TI - New technique for genetic amniocentesis in twins. AB - A new technique is described for amniocentesis in twins. Using a curvilinear or linear transducer, the separating membrane and an adjacent pocket of amniotic fluid in each cavity are simultaneously visualized. An amniocentesis needle is advanced into the first cavity. A second needle is introduced into the other amniotic cavity without altering the position of the transducer. This technique permits visualization of both needles simultaneously, providing proof of proper placement of the needle in each cavity. The advantages are unambiguous evidence of correct sampling of each cavity and lack of necessity for injection of a foreign substance into the amniotic sac. Finally, hard-copy documentation of proper needle placement is generated for the records. This procedure has been performed in seven twin sets with good results and patient acceptance. PMID- 1731303 TI - Individual growth curve standards in triplets: prediction of third-trimester growth and birth characteristics. PMID- 1731304 TI - Vaginal cuff closure at abdominal hysterectomy: comparing sutures with absorbable staples. PMID- 1731305 TI - Interpreting the literature in obstetrics and gynecology: I. Key concepts in epidemiology and biostatistics. PMID- 1731306 TI - Maternal and neonatal effects of outlet forceps delivery compared with spontaneous vaginal delivery in term pregnancies. PMID- 1731307 TI - Ovarian cancer in women with prior hysterectomy: a 14-year experience at the University of Miami. PMID- 1731308 TI - Controlled ovarian hyperstimulation as a risk factor for ectopic pregnancy. PMID- 1731309 TI - Nursing moves forward with agenda for health care reform. Interview by Sue Kelly. PMID- 1731310 TI - OSHA final bloodborne standard expected to prevent more than 9,200 infections and 200 deaths per year. Occupational Safety and Health Administration. PMID- 1731311 TI - Innovative strategies for nursing education. PMID- 1731312 TI - Deputy Surgeon General Dr. O. Marie Henry calls for a healthier future. PMID- 1731313 TI - RNs working with unlicensed assistive personnel. PMID- 1731314 TI - Potential hazards with protective restraint devices. PMID- 1731315 TI - Men in nursing: small in number--large in commitment. PMID- 1731316 TI - Human high molecular weight melanoma-associated antigen (HMW-MAA) mimicry by mouse anti-idiotypic monoclonal antibody MK2-23: induction of humoral anti-HMW MAA immunity and prolongation of survival in patients with stage IV melanoma. AB - Twenty-five patients with stage IV melanoma were immunized with the mouse anti idiotypic monoclonal antibody (mAb) MK2-23 (2 mg per injection), which bears the internal image of the determinant defined by anti-HMW-MAA mAb 763.74. Two patients were inevaluable, since they did not complete 4 weeks of therapy. Only 14 patients developed antibodies that were shown by serological and immunochemical assays to recognize the same or spatially close determinant as the anti-HMW-MAA mAb 763.74 and to express the idiotope defined by mAb MK2-23 in their antigen-combining sites. Side effects that are likely to be caused by bacillus Calmette-Guerin present in the immunogen consisted of erythema, induration, and ulceration at the sites of the injections. Occasionally, patients complained of flu-like symptoms, arthralgias, and myalgias. Three of the patients who developed anti-HMW-MAA antibodies achieved a partial response. It consisted of a decrease in the size of metastatic lesions and lasted 52 weeks in 1 patient and 93 weeks in the other 2 patients. Survival of the 14 patients who developed anti-HMW-MAA antibodies was significantly (P = 0.0003) longer than that of the 9 patients without detectable humoral anti-HMW-MAA immunity development. In the multivariate analysis, such an association between development of anti-HMW-MAA antibodies and survival prolongation was still significant (P = 0.001) after adjustment for difference in performance status, the only confounding factor found to be significantly related to survival. Lastly, a significant (P = 0.03 by likelihood ratio test) interaction between anti-HMW-MAA antibodies and patients' performance status was found, since the prolongation of survival associated with anti-HMW-MAA antibodies was more marked in patients with a performance status of less than or equal to 70% than in those with a higher one. These results suggest that anti-idiotypic mAb MK2-23 may represent a useful immunogen to implement active specific immunotherapy in patients with melanoma. PMID- 1731317 TI - Mutagenesis of some conserved residues in human 5-lipoxygenase: effects on enzyme activity. AB - Recombinant human 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) was expressed in Escherichia coli. In incubations of E. coli supernatants with arachidonic acid, 5-hydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene A4 were formed, while incubation with 8,11,14-eicosatrienoic acid gave 8-hydroxy-9,11,14-eicosatrienoic acid. Six conserved histidine residues in 5 lipoxygenase were subjected to site-directed mutagenesis. Exchanges of His-367, 372, or -551 gave mutants for which no enzyme activities were detectable. On the other hand, exchanges of His-362, -390, or -399 gave mutants that were enzymatically active, but less so than the nonmutated control. For two of these (exchanges of His-390 or -399), the activities of the mutants were dependent on the expression temperature. Thus, the histidines in the first group (His-367, 372, -551) were crucial for 5-lipoxygenase activity, possibly because of a function of these residues as metal ligands. Mutagenesis aimed at two other conserved elements in 5-lipoxygenase, Gln-558 and the C terminus, gave mutated proteins with only a small residual activity (substitution of Gln-558), or with no detectable activity (deletion of six C-terminal amino acids), indicating that these regions are important for the function of 5-lipoxygenase. PMID- 1731318 TI - Sequence-specific DNA purification by triplex affinity capture. AB - A DNA isolation procedure was developed by using triple-helix formation and magnetic separation. In this procedure, target DNA is captured by a biotinylated oligonucleotide via intermolecular triplex formation, bound to streptavidin coated magnetic beads, and recovered in double-stranded form by elution with a mild alkaline buffer that destabilizes the triple helix. The effectiveness of the procedure was demonstrated by a model experiment with an artificially reconstructed library and, also, by the isolation of (dT-dC)n.(dG-dA)n dinucleotide repeats from a human genomic library. This procedure provides a prototype for other triplex-mediated DNA isolation technologies. PMID- 1731319 TI - Cloning and characterization of a mouse cDNA encoding a cytoplasmic protein tyrosine-phosphatase. AB - A mouse cDNA encoding a non-receptor-type phosphotyrosine phosphatase (PTP; EC 3.1.3.48) has been isolated. The 1570-base-pair cDNA contains a single open reading frame that predicts a 382-amino acid protein with Mr 44,640. The nucleic acid and amino acid sequences are homologous to those of a previously described human T-cell PTP [Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A., Fischer, E. H. & Krebs, E. G. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261]; however, the mouse and human 3' sequences diverge and predict markedly different protein carboxyl termini. The mouse PTP gene is expressed as a 1.9-kilobase message in several stages of murine embryonic development and in a variety of adult tissues. An additional 1.3-kilobase message was found to be expressed specifically in testes. Finally, we report the isolation of a human T-cell PTP cDNA containing a 3' end sequence homologous to the mouse PTP. PMID- 1731320 TI - Specific inhibition of transcription by triple helix-forming oligonucleotides. AB - Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm. PMID- 1731321 TI - Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+. AB - Receptor-mediated changes in cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the plasma membrane, allowing influx of calcium from the extracellular fluid. Carbachol, a muscarinic receptor agonist, stimulated both calcium influx and inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular calcium release in A9 fibroblast cells expressing a m3 muscarinic receptor clone. The calcium influx persisted even after pretreatment of cells with phorbol 12-myristate 13-acetate, which completely prevented the rise in inositol phosphates and intracellular calcium levels. The calcium influx was blocked by divalent cations but was not affected by inhibitors of voltage-dependent calcium channels or high potassium depolarization, indicating the presence of a receptor-operated and voltage insensitive calcium channel in these cells. Calcium influx was not stimulated by the addition of cAMP analogs or arachidonic acid. To examine the possible involvement of G proteins in m3 receptor-activated calcium influx, two chimeric m2 and m3 muscarinic receptors were expressed in A9 cells in which the third cytoplasmic loop (the primary structural determinant in G protein coupling selectivity of muscarinic receptors) had been exchanged between the m2 receptor, which has no effect on calcium influx, and the m3 receptor. Calcium influx was found to be associated with a structural component of the m3 muscarinic receptor other than the third cytoplasmic loop. PMID- 1731322 TI - A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle. AB - At present immunization against Theileria parva is by infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has major limitations in that live organisms are used and the immunity engendered is parasite stock specific. In an attempt to develop an alternative immunization procedure, the gene encoding p67, a major surface antigen of sporozoites, has been expressed by using the plasmid expression vector pMG1. The gene, which has been characterized previously, encodes 709 amino acid residues, contains a single intron of 29 base pairs, and is only transcribed during sporogony. The recombinant p67 sequences were fused to the first 85 amino acid residues derived from a nonstructural gene (NS1) of influenza virus A. Immunization with a partially purified recombinant antigen emulsified in 3% saponin induced sporozoite neutralizing antibodies in cattle and provided protection in six of nine animals on homologous challenge with T. parva sporozoites. This recombinant antigen is therefore a candidate for development of a vaccine against T. parva. PMID- 1731323 TI - Classification of fungal chitin synthases. AB - Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHS1, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings. PMID- 1731324 TI - Alteration of the carbohydrate binding specificity of verotoxins from Gal alpha 1 4Gal to GalNAc beta 1-3Gal alpha 1-4Gal and vice versa by site-directed mutagenesis of the binding subunit. AB - Verotoxin 1 (VT-1) and Shiga-like toxin II (SLT-II) bind to the glycosphingolipid (GSL), globotriaosylceramide (Gb3), whereas pig edema disease toxin (VTE) binds to globotetraosylceramide (Gb4) and to a lesser degree Gb3. Amino acids important in the GSL binding specificity of VT-1 and VTE have been identified by site directed mutagenesis. One mutation, Asp-18----Asn, in VT-1 resulted in binding to Gb4 in addition to Gb3 in a manner similar to VTE. Several mutations in VTE resulted in the complete loss of GSL binding; however, one mutation resulted in a change in the GSL binding specificity of the VTE B subunit. The double mutation Gln-64----Glu and Lys-66----Gln (designated GT3) caused a selective loss of Gb4 binding, effectively changing the binding phenotype from VTE to VT-1. Both wild type VTE and GT3 were purified to homogeneity and binding kinetics in vitro were determined with purified GSLs from human kidney. The cell cytotoxicity spectrum of the mutant toxin was also found to be altered in comparison with VTE. These changes were consistent with the GSL content of the target cells. PMID- 1731325 TI - Molecular structure of the human desmoplakin I and II amino terminus. AB - Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a full-length 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical-rich bundles. Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-I-specific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site. PMID- 1731326 TI - Perforin and granzyme A expression identifying cytolytic lymphocytes in rheumatoid arthritis. AB - Lymphocytes from the synovial fluid of patients with rheumatoid arthritis were examined for the expression of granzyme A and perforin. Previous studies have demonstrated that the expression of these proteins, which are implicated as mediators of cytotoxicity, can be used to identify putative cytolytic lymphocytes in vivo. Twenty-two synovial fluid samples were analyzed by in situ hybridization and immunohistochemistry. In six patients receiving low doses of immunosuppressant, a population of granzyme A- and perforin-expressing lymphocytes could be identified. In contrast, lymphocytes from patients who were receiving high doses of immunosuppressant did not contain any granzyme A- or perforin-expressing lymphocytes. Synovial fluid lymphocytes from patients with osteoarthritis did not express either marker. The expression of these markers demonstrates the presence of potentially functional cytolytic lymphocytes, expressing proteins required to mediate killing, in the synovial fluid of patients with rheumatoid arthritis. This suggests that cytolytic lymphocytes may be involved in the pathogenesis of rheumatoid arthritis. PMID- 1731327 TI - Characterization of a prototype strain of hepatitis E virus. AB - A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia. PMID- 1731328 TI - Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis. AB - Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process. PMID- 1731329 TI - Methotrexate induces differentiation of human keratinocytes. AB - Terminal differentiation is a key element in the maintenance of tissue homeostasis in the epidermis. We show here that methotrexate (MTX) induces differentiation of human epidermal keratinocytes in vitro. MTX inhibits proliferation of keratinocytes and also induces several markers of differentiation: a change in cell morphology, a marked increase in cell size, an increase in the proportion of cells that express involucrin, and an increase in the amount of cornified envelope protein. These effects of MTX are dose- and exposure-time-dependent and become irreversible after 24 hr, approximately one population doubling time. These effects of MTX cannot be attributed to cytotoxicity since keratinocytes not only remain viable but also actively synthesize proteins. MTX causes reproducible changes in the SDS/PAGE profiles of newly synthesized proteins and, in particular, increases the amount of involucrin synthesis. Thymidine completely prevents these effects of MTX, suggesting that they are caused by a depletion of thymine deoxyribonucleotides. The effect of MTX on keratinocytes may provide a model for studying the relationship between deoxyribonucleotide metabolism and differentiation in normal cells. In addition, the ability of MTX to induce differentiation in keratinocytes suggests a mechanism to explain its therapeutic action in psoriasis. PMID- 1731330 TI - Discrimination between related DNA sites by a single amino acid residue of Myc related basic-helix-loop-helix proteins. AB - A yeast genetic system was developed to study how the basic regions of basic helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites. PMID- 1731331 TI - Femtosecond spectral evolution of the excited state of bacterial reaction centers at 10 K. AB - The femtosecond spectral evolution of reaction centers of Rhodobacter sphaeroides R-26 was studied at 10 K. Transient spectra in the near infrared region, obtained with 45-fs pulses (pump pulses centered at 870 nm and continuum probe pulses), were analyzed with associated kinetics at specific wavelengths. The t = 0-fs transient spectrum is very rich in structure; it contains separate induced bands at 807 and 796 nm and a bleaching near 760 nm, reflecting strong changes in interaction between all pigments upon formation of the excited state. A complex spectral evolution in the 800-nm region, most notably the bleaching of the 796-nm band, takes place within a few hundred femtosecond--i.e., on a time scale much faster than electron transfer from the primary donor P to the bacteriopheophytin acceptor HL. The remarkable initial spectral features and their evolution are presumably related to the presence of HL, as they were not observed in the DLL mutant of Rhodobacter capsulatus, which lacks this pigment. A simple linear reaction scheme with an intermediate state cannot account for our data; the initial spectral evolution must reflect relaxation processes within the excited state. The importance for primary photochemistry of long distance interactions in the reaction center is discussed. PMID- 1731332 TI - Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy. AB - Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, we propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1. PMID- 1731333 TI - Vaccinia virus complement-control protein prevents antibody-dependent complement enhanced neutralization of infectivity and contributes to virulence. AB - The role of a viral gene product in evasion of the host immune response was investigated. The antibody-dependent complement-enhanced neutralization of vaccinia virus infectivity was prevented by the culture medium from vaccinia virus-infected cells. The vaccinia virus complement-control protein (VCP) was identified as the secreted product of vaccinia virus gene C21L and has homology to a group of eukaryotic genes encoding regulators of complement activation. Thus, the culture medium from cells infected with a C21L deletion mutant was VCP deficient and had little or no effect on antibody-dependent complement-enhanced neutralization. In addition, the anticomplement effect was associated with the C21L-encoded protein partially purified from the medium of cells infected with wild-type virus. Antibody-dependent, complement-enhanced neutralization of vaccinia virus occurred with a complement source that was deficient in the classical pathway complement component C4 and required the alternative pathway complement factor B. Furthermore, the presence of VCP abrogated the complement enhanced neutralization in C4-deficient serum. Together with previous hemolysis data, the present result suggests that VCP can inhibit both the classical and alternative pathways of complement activation. Skin lesions caused by the C21L deletion mutant were smaller than those caused by wild-type virus, demonstrating an important role for VCP in virulence. The C21L deletion mutant also was attenuated in C4-deficient guinea pigs, consistent with in vitro studies. Vaccinia virus appears to have acquired the ability to regulate the complement cascade for the purpose of evading the host immune response. PMID- 1731334 TI - B29 gene products complex with immunoglobulins on B lymphocytes. AB - B29 is a B-lineage-specific gene predicted from sequence information to be a transmembrane member of the immunoglobulin (Ig) superfamily, with a single extracellular Ig-like domain. Its presumptive cytoplasmic region contains a peptide motif present in CD3 and other molecules involved in lymphocyte activation. Affinity-purified goat antibodies were prepared to a TrpE fusion protein of B29 and used to study B29 expression on lymphoid cells. The antiserum precipitated surface-labeled heterodimers from B lymphoma cells. One was 65-88 kDa (unreduced) or 36-47 plus 32-34 kDa (reduced) by SDS/PAGE analysis, regardless of detergent. A smaller heterodimer was detected only with Triton detergent extraction. IgM molecules were coprecipitated by the B29 antiserum when the weak detergent digitonin was used. In addition, cocapping experiments revealed that most B29 molecules codistribute with Ig on the cell surface. Although early B-lineage cells and plasma cells contain B29 mRNA, surface expression was detectable only on B cells that had significant amounts of surface Ig. The surface expression was B-lineage-specific and included cells from mutant xid mice and B-cell lines representing mu, delta, gamma, and alpha heavy-chain isotypes and both kappa and lambda light-chain types. The density of surface B29 protein correlated directly with surface mu heavy-chain density on subclones of a B-cell lymphoma and lipopolysaccharide-stimulated pre-B cells. These findings show that B29 is covalently linked in a heterodimer and are consistent with a recently proposed model of surface Ig complexes. PMID- 1731335 TI - Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor. AB - The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera. PMID- 1731337 TI - A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean. AB - The assembly of 11S globulin seed storage proteins in plants is regulated in part by the activity of a protease that cleaves between asparagine and glycine residues. Post-translational cleavage of subunit precursors into acidic and basic polypeptides is associated with the ability of subunits in trimers to aggregate into hexamers in vitro. An activity is present in extracts from immature soybean seeds that specifically cleaves immature 11S seed storage proteins of soybean and Vicia faba into the polypeptides of the mature proteins. Sequence microanalysis has been used to demonstrate that proglycinin and prolegumin are cut at the legitimate site when proteins synthesized in vitro are used as substrates. A single amino acid change in the cleavage site renders the substrate uncleavable. The protease responsible for this activity also hydrolyzes a synthetic octapeptide whose sequence reproduces four amino acids on either side of the glycinin subunit G4 cleavage site. This assay permitted the purification and characterization of the protease. It is a glycosylated enzyme with an acidic pH optimum and a molecular mass of about 45 kDa in solution. PMID- 1731336 TI - Differential regulation of mRNAs for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 in the adult rat brain following cerebral ischemia and hypoglycemic coma. AB - In situ hybridization was used to study expression of mRNAs for members of the nerve growth factor (NGF) family in the rat brain after 2 and 10 min of forebrain ischemia and 1 and 30 min of insulin-induced hypoglycemic coma. Two hours after the ischemic insults, the level of brain-derived neurotrophic factor (BDNF) mRNA was markedly increased in the granule cells of the dentate gyrus, and at 24 h it was still significantly elevated. NGF mRNA showed a pronounced increase 4 h after 2 min of ischemia but had returned to a control level at 24 h. Both 2 and 10 min of ischemia caused a clear reduction of the level of mRNA for neurotrophin 3 (NT 3) in the dentate granule cells and in regions CA2 and medial CA1 of the hippocampus 2 and 4 h after the insults. The increase of BDNF mRNA could be partially blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist NBQX but was not influenced by the N-methyl-D aspartate (NMDA) receptor antagonist MK-801. Both NBQX and MK-801 attenuated the decrease of NT-3 mRNA after ischemia. One and 30 min of hypoglycemic coma also induced marked increases in BDNF and NGF mRNA in dentate granule cells with maximal levels at 2 h. If the changes of mRNA expression lead to alterations in the relative availability of neurotrophic factors, this could influence functional outcome and neuronal necrosis following ischemic and hypoglycemic insults. PMID- 1731338 TI - Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin). AB - The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets. PMID- 1731339 TI - Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene. AB - An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13 acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, beta-naphthoflavone, and PMA. Single AP-1 like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility. Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme. PMID- 1731340 TI - Constitutive activity of the tumor necrosis factor promoter is canceled by the 3' untranslated region in nonmacrophage cell lines; a trans-dominant factor overcomes this suppressive effect. AB - The role of the mouse tumor necrosis factor (TNF) promoter, 5' untranslated region (UTR), and 3' UTR in TNF gene expression has been examined in three nonmacrophage cell lines (HeLa, NIH 3T3, and L-929). The TNF promoter is not macrophage-specific. On the contrary, it constitutively drives reporter gene expression in all three cell lines. Not only the full-length promoter but also truncated versions of the promoter, lacking NF-kappa B binding motifs, are active in each type of cell. The TNF 3' UTR effectively cancels reporter gene expression in HeLa cells and in NIH 3T3 cells but fails to block expression in L-929 cells. L-929 cells contain a factor that overcomes the inhibitory influence of the TNF 3' UTR. Its action depends upon the presence of sequences found in the TNF 5' UTR. Cell-fusion experiments reveal that this activator is trans-dominant. These studies highlight the essential role played by the TNF 3' UTR, which silences the TNF gene in cells that might otherwise express TNF. They also reveal the existence of an escape mechanism whereby inappropriate synthesis of TNF might occur. PMID- 1731341 TI - Cloning of Drosophila transcription factor Adf-1 reveals homology to Myb oncoproteins. AB - The Drosophila sequence-specific DNA binding protein, Adf-1, is capable of activating transcription of the alcohol dehydrogenase gene, Adh, and is implicated in the transcriptional control of other developmentally regulated genes. We have cloned the cDNA encoding Adf-1 by generating specific DNA probes deduced from partial amino acid sequence of the protein. Several cDNA clones encoding an extended open reading frame were isolated from a phage lambda library. The complete amino acid sequence of Adf-1 deduced from the longest cDNA reveals structural similarities to the putative helix-turn-helix DNA binding motif of Myb and Myb-related proteins. DNA sequence analysis of genomic clones and Northern blot analysis of mRNA suggest that Adf-1 is a single-copy gene encoding a 1.9-kb transcript. Purified recombinant Adf-1 expressed in Escherichia coli binds specifically to Adf-1 recognition sites and activates transcription of a synthetic Adh promoter in vitro in a manner indistinguishable from the protein purified from Drosophila. Temporally staged Drosophila embryos immunochemically stained with affinity-purified anti-Adf-1 antibodies indicate that Adf-1 protein is not detectable in very early embryos and does not appear to be maternally inherited. During later stages of embryogenesis, Adf-1 appears to be expressed in the nucleus of most somatic cells in the embryo with possibly higher concentrations found in some tissues. PMID- 1731342 TI - A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. AB - Recombinant retroviruses encoding the histochemically detectable enzyme beta galactosidase have been used to investigate lineage in the vertebrate nervous system. Identification of the descendants of individual progenitors is straightforward when progeny cells are arranged in a reproducible, clustered pattern, but difficulties in interpretation arise when progeny migrate extensively and/or in an irregular pattern. To better resolve clonal boundaries, additional histochemical marker viruses that engender distinctive reaction products can be used in combination with lacZ-bearing viruses. To this end, we have created a retrovirus vector, DAP, encoding an easily assayable enzyme, human placental alkaline phosphatase. DAP was found to be at least as useful as a lacZ encoding retrovirus (e.g., BAG) with respect to high viral titer, stability of expression, and in identification of infected cells in vivo. Moreover, it was found to be neutral with respect to postnatal rodent retinal development and offered superior staining characteristics relative to lacZ. Coinfection of rodent retina with DAP and BAG allowed an examination of the clonal nature of radial arrays of labeled retinal cells that previously had been described as products of a single infected progenitor. Of 1100 radial arrays examined for the presence of both DAP- and BAG-infected cells, only 1.2% were the result of infection with more than one virus. PMID- 1731343 TI - Factors involved in regulation of the RT7 promoter in a male germ cell-derived in vitro transcription system. AB - We recently cloned and characterized a rat male germ cell-specific gene, RT7. The RT7 promoter contains a TATA box as well as sequences with homology to binding sites for a number of transcription factors. To investigate the regulation of the RT7 promoter we developed an active in vitro transcription system derived from rat seminiferous epithelium, which, in contrast to total testis, consists mostly of male germ cells. Also, DNase I footprinting analysis and gel retardation experiments were performed to analyze RT7 promoter-protein interaction. The experiments demonstrate that nuclear extracts prepared from rat male germ cells support in vitro transcription and that the RT7 promoter is positively regulated by a testis-specific transcription factor, TTF-D, by a factor similar to the transcription factor CREB, and by a nuclear factor that binds immediately upstream of the RT7 transcription start site. PMID- 1731344 TI - Linkage of atherogenic lipoprotein phenotype to the low density lipoprotein receptor locus on the short arm of chromosome 19. AB - The atherogenic lipoprotein phenotype (ALP) is a common heritable trait characterized by a predominance of small, dense low density lipoprotein (LDL) particles (subclass pattern B), increased levels of triglyceride-rich lipoproteins, reductions in high density lipoprotein, and a 3-fold increased risk of myocardial infarction. Significant two-point linkage was found between ALP and the LDL receptor locus on the short arm of chromosome 19 in 51 relatives of nine probands with ALP pattern B. The maximum logarithm of odds (LOD) score of 4.07 was observed at a recombination fraction of 0.04, assuming 100% penetrance of ALP pattern B, and 4.27 at a recombination fraction of zero, assuming 90% penetrance of pattern B. Haplotyping data and multipoint linkage analysis suggest that the gene [named ATHS for atherosclerosis susceptibility (lipoprotein-associated)] responsible for ALP is located distal to D19S76 near or at the LDL receptor locus. This result suggests the possibility that genetic variation at the LDL receptor locus or a closely linked locus on chromosome 19 may be responsible for metabolic alterations in ALP pattern B that account for a substantial proportion of the familial predisposition to coronary artery disease in the general population. PMID- 1731345 TI - Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. AB - Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that receive phosphoryl groups from the histidine kinase, CheA. Here we show that the response regulators catalyze their own phosphorylation in that both CheY and CheB can be phosphorylated in the complete absence of any auxiliary protein. Both CheY and CheB use the N phosphoryl group in phosphoramidate (NH2PO3(2-)) as a phospho-donor. This enzymatic activity probably reflects the general ability of response regulators to accept phosphoryl groups from phosphohistidines in their associated kinases. It provides a general method for the study of activated response regulators in the absence of kinase proteins. CheY can also use intermediary metabolites such as acetyl phosphate and carbamoyl phosphate as phospho-donors. These reactions may provide a mechanism to modulate cell behavior in response to altered metabolic states. PMID- 1731346 TI - Treatment of premalignancy: prevention of lymphoma in radiation leukemia virus inoculated mice by cyclosporin A and immunotoxin. AB - Radiation leukemia virus (RadLV)-induced preleukemic (PL) latency is characterized by the appearance of virus-infected PL cells in the thymus. The survival of these PL cells is dependent upon autostimulation with interleukin 4 (IL-4). We have intervened prophylactically in RadLV-induced preleukemia by using cyclosporin-A (CSA), which inhibits IL-4 production, and an immunotoxin (ITx) that kills PL cells. CSA efficiently inhibited IL-4 secretion from RadLV-induced PL and leukemic cells, and its administration to PL mice caused a significant delay in their death. An ITx consisting of anti-RadLV glycoprotein-70 (gp70) antibody coupled to ricin A chain efficiently inhibited protein synthesis in virus-infected cells in vitro and, when injected into PL mice, also delayed their death. Combined treatment with CSA and ITx prevented 75% of the treated PL mice from developing lymphoma. These results show that the development of malignancy from a premalignant state can be averted by a combination of therapeutic modalities that decrease the size and growth rate of the premalignant cell population. PMID- 1731347 TI - Neurons and microvessels express the brain glucose transporter protein GLUT3. AB - To elucidate glucose transport mechanisms in brain and to demonstrate the cellular expression of the brain-type glucose transporter (GLUT3), antisera to a synthetic peptide corresponding to the C terminus were prepared and used as probes for this isoform of the facilitative glucose transporter family. Immunocytochemistry of frozen sections of dog and rat brain demonstrated GLUT3 antigen in pyramidal cell bodies and processes, in microvessels, and in intima pia or glia limitans. Immunoanalysis of Western blots identified a protein (Mr, 45,000) that was present in both neuron/neuropil and microvessel fractions. The presence of the GLUT3 message in brain was confirmed by Northern blot analysis and by amplifying and partially sequencing GLUT3 cDNA by PCR. These findings demonstrate a neuron glucose transporter in tissue and suggest that GLUT3 may play an important role in brain metabolism under physiological and pathophysiological conditions. PMID- 1731348 TI - gamma-Aminobutyric acid-containing basal forebrain neurons innervate inhibitory interneurons in the neocortex. AB - The basal forebrain-neocortex pathway--involved in higher cognitive processing, selective attention, and arousal--is considered one of the functionally most important ascending subcortical projections. The mechanism by which this relatively sparse subcortical pathway can control neuronal activity patterns in the entire cortical mantle is still unknown. The present study in the cat provides evidence that gamma-aminobutyric acid-containing basal forebrain neurons participate in the neocortical projection and establish multiple synaptic connections with gamma-aminobutyric acid-releasing interneurons containing somatostatin or parvalbumin. We propose that a mechanism by which the numerically small ascending pathways can exert a powerful global effect in the neocortex is by the selective innervation of gamma-aminobutyric acid-releasing interneurons, which, in turn, control the activity of large populations of pyramidal cells through their extensive axon arborizations. Finally, these results demonstrate a direct anatomical link between two cell populations implicated in Alzheimer disease pathology: basal forebrain neurons and cortical somatostatin cells. PMID- 1731349 TI - Serotonin hyperpolarizes cholinergic low-threshold burst neurons in the rat laterodorsal tegmental nucleus in vitro. AB - Serotonergic suppression of cholinergic neuronal activity implicated in the regulation of rapid eye movement sleep and its associated phenomenon, pontogeniculooccipital waves, has long been postulated, but no direct proof has been available. In this study, intracellular and whole-cell patch-clamp recording techniques were combined with enzyme histochemistry to examine the intrinsic electrophysiological properties and response to serotonin (5-HT) of identified cholinergic rat laterodorsal tegmental nucleus neurons in vitro. Sixty-five percent of the recorded neurons demonstrated a prominent low-threshold burst, and of these, 83% were cholinergic. In current-clamp recordings 64% of the bursting cholinergic neurons tested responded to the application of 5-HT with a membrane hyperpolarization and decrease in input resistance. This effect was mimicked by application of the selective 5-HT type 1 receptor agonist carboxamidotryptamine maleate. Whole-cell patch-clamp recordings revealed that the hyperpolarizing response was mediated by an inwardly rectifying K+ current. Application of 5-HT decreased excitability and markedly modulated the discharge pattern of cholinergic bursting neurons: during a 5-HT-induced hyperpolarization these neurons exhibited no rebound burst after hyperpolarizing current input and a burst in response to depolarizing current input. In the absence of 5-HT, the relatively depolarized cholinergic bursting neurons responded to an identical hyperpolarizing current input with a burst and did not produce a burst after depolarizing current input. These data provide a cellular and molecular basis for the hypothesis that 5-HT modulates rapid eye movement sleep phenomenology by altering the firing pattern of bursting cholinergic neurons. PMID- 1731350 TI - Truncated staphylococcal nuclease is compact but disordered. AB - Deletion of 13 amino acids from the carboxyl terminus of the 149-amino acid staphylococcal nuclease molecule results in a denatured, partly unfolded molecule that lacks persistent secondary structure but is compact under physiological conditions. Since the modification is a carboxyl-terminal deletion, it is argued that the state resembles a peptide emerging from the ribosome just before the complete folding pathway is initiated. In this paper, we characterize the molecule by nuclear magnetic resonance, circular dichroism, and small-angle x-ray scattering measurements. The truncated nuclease shows wild-type levels of activity in the presence of calcium and is found to fold into a native-like conformation in the presence of 3',5'-bisphospho-2'-deoxythymidine, a potent inhibitor. Thus, the truncated molecule retains the capacity to fold. Our results suggest that extensive solvent exclusion generates a compact polypeptide chain prior to the development of persistent secondary structural features as a protein folds during biosynthesis. PMID- 1731351 TI - Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein. AB - Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem IIB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions. PMID- 1731352 TI - A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion. AB - In this study we examined whether the arrest of DNA polymerase alpha (pol alpha) catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation. An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites. A radioactive 5' end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol alpha. Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified. These DNA chains were elongated by pol alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain. Owing to the high preference of pol alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied. Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains. We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome. pol alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues. A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC. These results suggest that some but not all sites at which pol alpha pauses may constitute loci of mutagenesis. PMID- 1731353 TI - Testis-/embryo-expressed genes are clustered in the mouse H-2K region. AB - The major histocompatibility complex (MHC) of the mouse is located on chromosome 17 in the distal inversion of the t complex. In addition to genes playing major roles in the immune response, it contains a diversity of genes. In humans, numerous diseases are known to be associated with the MHC loci. Moreover, at least three recessive embryonic t-lethal mutations have been mapped to the MHC. Here a molecular genetic approach was used to study the detailed genomic structure of 240 kilobases (kb) surrounding the H-2K gene and 150 kb of a partly homologous region located in the distal inversion of the t complex. Combined with previous findings, the H-2K region was found to contain an impressively high density of genes--12 transcription units in 240 kb. Surprisingly, virtually all of these genes are expressed in testis and/or embryos. The genomic organization of this region is contrasted with the 150 kb of the homologous area where only three genes and an endogenous retrovirus reside. PMID- 1731355 TI - Services should bend to fit their clients. PMID- 1731356 TI - An adequate response to a cry for help? Parasuicide patients' perceptions of their nursing care. AB - People admitted to hospital after a failed suicide attempt present a real challenge to nurses. A survey of parasuicide patients revealed that most felt isolated and ignored during their hospital stay. By sparing time to talk, nurses can help motivate patients to make a new start. PMID- 1731354 TI - Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family. AB - We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes. PMID- 1731357 TI - Can we care to the end? Do nurses have the skills for terminal care? AB - Nurses working with terminally ill patients are subject to stress and anxiety. Inadequate preparation from basic and post-basic education and lack of confidence in advocating patients' rights to doctors are shown to be significant contributory factors. PMID- 1731358 TI - A support line that restores confidence: crisis intervention after a major disaster. AB - People involved in a major disaster can be left with strong feelings of anxiety and helplessness. By developing crisis intervention skills, nurses can help both their patients and themselves place these feelings into context. PMID- 1731359 TI - Every little detail counts. Infection control in i.v. therapy. AB - Much controversy still surrounds the issue of catheter-related infection, and evidence suggests that nurses' practice varies widely in this field. An understanding of the main routes of transmission and the maintenance of standard protocols will help reduce the incidence of infection. PMID- 1731360 TI - Routine assessment will identify problems. Urinary assessment following colorectal and stoma forming surgery. AB - Urinary problems following colorectal surgery, particularly rectal excision, are often accepted as a matter of course by health professionals and patients alike. However, routine urinary assessment of these patients, plus a sound knowledge of the causes and nature of the urinary problems, allows effective helping strategies to be implemented. PMID- 1731361 TI - Herbalism: an overview of an ancient art. Origins and development of herbal therapies. PMID- 1731362 TI - Positive attitude will aid treatment. A guide to rheumatoid arthritis. AB - Rheumatoid arthritis is a chronic destructive disease which can result in deformity and disability. While treatment can do much to aid sufferers, support and encouragement are equally vital to instil a positive attitude. PMID- 1731363 TI - Do you believe I'm in pain? Nurses' assessment of patients' pain. AB - Expression of pain is influenced by physical, psychological and cultural background. It is important, therefore, that assessment is based on individual needs and not value judgements. PMID- 1731364 TI - But does it feel like home? Accommodation needs in later life. AB - A much loved family home can become difficult to look after and expensive to heat for an elderly person living alone. The decision of whether to move belongs to the individual alone, but personal needs must be balanced with health and safety requirements. PMID- 1731365 TI - Look beyond the ulcer itself: Assessment of leg ulcers. AB - Accurate assessment of leg ulcers involves identifying their predisposing, precipitating and perpetuating causes. Once these have been established, effective treatment can be instigated or even, in some cases, recurrence avoided. PMID- 1731366 TI - Fulfilling the need for a coordinated approach. Case management and community nursing. AB - Case management represents an alternative to the multidisciplinary team in coordinating community care. The case manager role is open to community nurses, among other disciplines, and offers an opportunity to combine management skills with clinical care. PMID- 1731367 TI - Who says they're inappropriate? PMID- 1731368 TI - Potassium channels in renal epithelial transport regulation. PMID- 1731369 TI - Physiological actions of taurine. PMID- 1731370 TI - Structure and function of the brain serotonin system. PMID- 1731371 TI - Long-term control of arterial blood pressure. AB - Two concepts for the long-term regulation of arterial pressure were considered in this review, the neural control hypothesis and the volume regulation hypothesis. The role of the nervous system and fluid volume regulation are intertwined in a way that has made it difficult to experimentally evaluate their separate contributions in the long-term regulation of arterial pressure. Nevertheless, from a substantial body of work related to the neural control of cardiovascular function, it appears that the ability of the nervous system to control arterial pressure is limited to the detection and correction of rapid short-term changes of arterial pressure. A long and exhaustive search has yet yielded no new neural mechanisms beyond the classic sinoaortic baroreceptors that can detect changes of arterial pressure. The baroreceptor mechanisms are of great importance for the moment-to-moment stabilization of arterial pressure, but because they do not possess sufficient strength and because they reset in time to the prevailing level of arterial pressure, they cannot provide a sustained negative feedback signal to provide long-term regulation of arterial pressure in face of sustained stimuli. This is not to say that the nervous system cannot affect the long-term level of arterial pressure. A distinction is made here between the many factors that can influence the long-term level of pressure and those that actually serve to detect changes of pressure and serve to maintain the level of pressure within a narrow range over the period of our adult lifetime. In this sense, there is evidence that in genetically susceptible individuals, environmental stresses can influence the long-term level of arterial pressure via the central and peripheral neural autonomic pathways. It is inappropriate, however, to view the nervous system as a long-term controller of arterial pressure because there is yet no evidence that the CNS can detect changes of arterial pressure nor changes in total body sodium and water content over sustained periods whereby it could provide an adequate long-term normalization of such error signals. In contrast, evidence has grown in support of the renal pressure-diuresis volume regulation hypothesis for the long-term control of arterial pressure over the past decade. An enhanced understanding of the mechanisms of pressure diuresis-natriuresis coupled with studies exploring how changes of vascular volume can influence vascular smooth muscle tone provide a compelling basis for this hypothesis of long-term arterial pressure regulation. This overall concept is represented and summarized in Figure 12.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1731372 TI - Human neuronal control of automatic functional movements: interaction between central programs and afferent input. PMID- 1731373 TI - Relation between structure and function in information transfer in spinal monosynaptic reflex. PMID- 1731374 TI - [What is your roentgen diagnosis? Vascular lung hamartoma in Osler disease (= congenital arteriovenous pulmonary aneurysm, cavernous hemangioma)]. PMID- 1731375 TI - [Possibilities in the home nursing care of tumor patients with dyspnea]. AB - Comprehensive care of oncologic patients in the region of Basel-Stadt includes also oncologic care outside of the Hospital (SEOP BS) as a special service of the cantonal federation for domestic and community care of Basel-Stadt (KVHG). Services of the SEOP are prescribed by the family doctor and arranged by the community care organisation. Next to an education in general medical care the attendant has been educated additionally in oncologic nursing. In this article first the working hypothesis of the Basel model is presented followed by a list of those nursing measures that can easily be executed at home. General recommendations for dyspneic patients are derived from the booklet "Ateminstruktion", edited by the Bernese "Hohenklinik Heiligenschwendi". Finally some important addresses providing diverse inhalation facilities and similar means for treatment at home are mentioned. PMID- 1731376 TI - [Terminal dyspnea: morphine may be useful]. PMID- 1731377 TI - Intestinal ischemia. PMID- 1731378 TI - Anatomy of the splanchnic circulation. AB - The blood supply to the intestines is a complex one, including branches of the three main splanchnic arteries as well as a vast collateral circulation. The variant anatomy and collateral pathways are described, based on anatomic dissections and angiographic studies, to focus attention on anatomically based explanations for clinical entities. PMID- 1731379 TI - Radiology in intestinal ischemia. Plain film, contrast, and other imaging studies. AB - Because plain films are usually normal or nonspecific in both colonic and acute mesenteric ischemia, they are not diagnostically helpful. The barium enema is the most useful radiographic examination in the diagnosis of colonic ischemia, and a double-contrast study will show abnormalities in almost all cases. Findings specific for colonic ischemia characteristically change with time. Thumbprinting is the most diagnostic finding; it is seen early in the course of the disease and usually resolves or is replaced within 1 or 2 weeks by an acute ulcerating colitis pattern. The latter may heal over months or go on to stricture formation or a persistent colitis. Nonspecific abnormalities can also be identified on CT and ultrasound, but the incidence of the findings with colonic ischemia is not known. Plain film findings occur late in the course of acute mesenteric ischemia and thus cannot be relied on for the diagnosis, although they may be useful in excluding other conditions. When acute mesenteric ischemia is suspected, angiography should be performed, but CT, ultrasound, and, perhaps, MR imaging may contribute to the diagnosis. PMID- 1731380 TI - Radiology in intestinal ischemia. Angiographic diagnosis and management. AB - Angiography is an essential component of the diagnosis and treatment of patients with acute and chronic intestinal ischemia. Aortography and selective angiography permit identification of the cause and precise anatomy of intestinal ischemic syndromes, and also help plan their potential correction. Direct intra-arterial infusion of pharmacologic agents into splanchnic vessels has now become part of the therapy of these conditions. This article reviews angiographic techniques and their applications in the management of intestinal ischemic syndromes. PMID- 1731381 TI - Operative assessment of intestinal viability. AB - Acute intestinal ischemia and infarction remain serious clinical problems despite early operative intervention. Accurate intraoperative assessment of intestinal viability is essential in determining the limits of resection in patients with intestinal infarction. Clinical features of bowel viability such as color and peristalsis do not correlate uniformly with bowel survival, and as a result, several techniques have been developed to assess intestinal blood flow at the time of operation. The requirements of an ideal viability test are: 1. The technique must have ready availability, preferably in every operating theater dealing with abdominal emergencies. 2. The necessary equipment must not be cumbersome or require specialized personnel. 3. The method must be accurate with a minimum of false-negative results and, more importantly, few false positives. A false-negative results leaves in situ nonviable bowel, which may lead to early perforation and late stricturing. This situation may be recoverable with further surgical intervention, however. On the other hand, a false-positive assessment of bowel viability results in the resection of potentially recoverable intestine, which is lost forever and may represent a vital difference for morbidity mortality and long-term nutrition. 4. The technique must be objective and be reproducible. 5. The method must be cost effective. To date, only two tests have found widespread acceptance and clinical applicability: fluorescein assessment and Doppler studies either with ultrasound or as refined in laser velocimetry. Although other techniques may be of some value today or in the future, the most practical approach would appear to be to use fluorescein assessment under a modified Wood's lamp as the initial method of evaluating intestinal viability and either Doppler ultrasound or perfusion fluorometry for any areas of particularly doubtful viability. PMID- 1731382 TI - Aggressive approach to acute mesenteric ischemia. AB - An aggressive diagnostic and therapeutic approach to acute mesenteric ischemia can dramatically lower the mortality of this lethal disease. The cornerstones of this approach are the earlier and more liberal use of angiography and the use of intra-arterial infusions of vasodilators in the treatment of both nonocclusive and occlusive mesenteric ischemia. PMID- 1731383 TI - Mesenteric venous thrombosis. AB - Mesenteric venous occlusion produces a spectrum of clinical presentations, the most common of which is the acute onset of abdominal pain with progressive signs and symptoms of bowel infarction. This acute form of mesenteric venous thrombosis, compared with other forms of acute mesenteric infarction, occurs in younger patients, typically has a more indolent and nonspecific course, involves shorter segments of bowel, and has a lower mortality rate. In contradistinction to our recommended therapy in other forms of acute mesenteric infarction, immediate anticoagulation is indicated for mesenteric venous thrombosis. Second look operations are used, as in other forms of acute mesenteric infarction, whenever portions of bowel of questionable viability are not resected at the primary operation. Chronic mesenteric venous thrombosis may produce no symptoms or may cause gastrointestinal bleeding from portal hypertension. Newer imaging techniques have increased the ability to diagnose and define the extent of all forms of mesenteric venous thrombosis and have added to the therapeutic options available to manage them. PMID- 1731384 TI - Colonic ischemia. AB - Our understanding of ischemia and the importance of this disease in the elderly have increased dramatically over the past 30 years. Moreover, the number of patients found to have colonic ischemia can be expected to increase over the next several decades as that portion of our population older than 65 years grows and the many forms of ischemic injury to the colon are better appreciated. Earlier diagnosis should follow the wider use of colonoscopy, and recognition of the ischemic nature of the varied disease manifestations and identification of patients at high risk for this disease should result in better management. PMID- 1731385 TI - Chronic visceral ischemia. AB - Visceral ischemic syndromes constitute a spectrum of diseases that may, if not detected and treated, culminate in fatal intestinal gangrene. Symptomatic chronic visceral ischemia may run a prolonged course before diagnosis and effective surgical revascularization is employed. The internist or general practitioner plays an important role in the management of all patients with chronic visceral ischemia, usually being the first physician to evaluate the patient and often being able to direct the efforts to diagnose the problem effectively. An awareness of the disease and a high index of suspicion will lead to the confirmatory arteriogram, after which appropriate consultation will ensure that treatment which becomes readily apparent. The vascular surgeon who possesses the skill to execute transaortic extraction endarterectomy and antegrade aortovisceral bypass can realistically meet the therapeutic challenge of this disease, relieving the patients' symptoms and, more importantly, preventing fatal intestinal gangrene. PMID- 1731386 TI - Systemic diseases affecting the mesenteric circulation. AB - The mesenteric circulation is acutely sensitive to processes that affect the entire body. Such systemic diseases and syndromes are reviewed with particular emphasis on the mechanisms by which they influence the mesenteric vasculature and blood flow. PMID- 1731387 TI - Necrotizing enterocolitis in infancy. AB - The most common gastrointestinal emergency in the newborn is necrotizing enterocolitis. Premature babies are the most likely victims, but it also occurs in full-term infants. Although great strides have been made in elucidating some of the factors responsible for necrotizing enterocolitis, such as intestinal ischemia, bacterial overgrowth, and feeding dysfunction, the exact etiology is as yet unclear. The timing and indications for surgery differ from institution to institution, but the long-term outcome is similar in most large series. The overall mortality rate remains about 20% to 40%, and of the survivors, about one half seem to have no sequelae, the remaining infants having neurologic and gastrointestinal deficits of various degrees of significance. PMID- 1731388 TI - Pathophysiology of mesenteric ischemia. AB - Intestinal ischemia can result from a host of pathophysiologic disturbances and, in turn, may produce a variety of adverse local and systemic consequences. Mechanisms of ischemic injury and the central role of vasoconstriction are discussed. PMID- 1731389 TI - Pathology of intestinal ischemia. AB - Intestinal ischemia produces a spectrum of gross and microscopic pathologic changes that range from transmural necrosis to reversible mucosal injury. Depending on the stage at which tissue is examined, findings may reflect the acute injury or reparative changes. This article reviews the diagnosis of the pathologic findings in differential intestinal ischemia. PMID- 1731390 TI - Reperfusion injury. AB - In conclusion, a large body of evidence demonstrates that reperfusion of ischemic intestine results in significant microvascular and parenchymal cell injury. Reperfusion injury appears to be mediated by both reactive oxygen metabolites and activated polymorphonuclear leukocytes. Xanthine oxidase-derived oxidants initiate the production and release of proinflammatory agents, which in turn lead to polymorphonuclear leukocyte adherence and emigration. The adherent leukocytes mediate microvascular injury by either release of proteases, physical disruption of the endothelial barrier, or both. PMID- 1731391 TI - Diagnostic tests for intestinal ischemia. AB - Mesenteric ischemia is a devastating disease. Without early diagnosis and intervention, the process proceeds to intestinal gangrene with its associated high morbidity and mortality rates. Although newer operative techniques and better intensive care unit management may improve patient outcome, it is only by obtaining an earlier diagnosis that greater patient survival rates will be possible. In an attempt to improve diagnostic accuracy, many modalities have been explored. These include serum biochemical markers, peritoneal fluid analysis, tonometry, radionuclide imaging, laparoscopy, and endoscopic techniques. At present, no single test has enabled the clinician to improve the patient's outcome. We are hopeful that the newer techniques, including radionuclide-labeled antibodies, tonometry, and reflectance spectrophotometry, may in the future be of assistance in improving the results for patients sustaining intestinal ischemia. PMID- 1731392 TI - Pregnancy outcomes after weekly oral administration of ethanol during gestation in the pig-tailed macaque: comparing early gestational exposure to full gestational exposure. AB - An oral dose of 1.8 g/kg ethanol given once per week throughout gestation to gravid pig-tailed macaques (Macaca nemestrina) has been established previously as teratogenic. This study was designed to use this nonhuman primate model to mimic a common problematic human circumstance in which women intermittently abuse alcohol into early pregnancy, realize that they are in fact pregnant, and then want to know the chance that the conceptus is harmed. In order to evaluate this situation, gravid macaques were assigned to one of four dosing cohorts. Animals were given the 1.8 gm/kg dose of ethanol once per week for the first 3, 6, or 24 weeks (full gestation) of pregnancy. Control animals received an isocaloric, isovolemic sucrose solution once per week for 24 weeks. The pregnancies were carefully monitored and the infants were comprehensively evaluated for the first 24 months of life. This paper describes the pregnancies while subsequent papers will describe the infants. Peak plasma ethanol levels ranged from 175 to 250 mg/dl. Weekly maternal exposure to this intoxicating dose of ethanol, starting early in pregnancy, did not influence risk of pregnancy failure during the first 30 days of gestation but appeared to be associated with an increased risk of abortion occurring between gestational days 30 and 160. Of the pregnancies that were successfully carried to full term, the potentially teratogenic dose of ethanol did not alter pregnancy outcome in any clinically significant way. PMID- 1731393 TI - Aberrant convergence of the neural folds in the mouse mutant vl. AB - Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds. PMID- 1731394 TI - In vitro embryotoxicity of a series of para-substituted phenols: structure, activity, and correlation with in vivo data. AB - The embryotoxicity of phenol and twelve para-substituted congeners on mid gestation rat embryos was evaluated in vitro. Through application of correlative procedures and stepwise regression, equations describing the relationship between physical-chemical properties and various measures of activity were developed. Embryotoxicity was quantified by the log of the reciprocal of the potency estimates for reduction in selected growth parameters and induction of four morphological defects. In general, co-cultured hepatocytes ameliorated embryotoxicity; only phenol-induced embryotoxicity was enhanced by the presence of hepatocytes. In the absence of hepatocytes, measures of growth retardation were positively correlated with molar refractivity of the phenols. With hepatocytes, lipophilicity became important for describing the potential to induce growth deficits. The structural defects had varying correlation patterns in both culture systems. Potencies of these congeners in vitro were also compared to maternal and developmental potencies observed in vivo (Kavlock, Teratology, 41:43-59, '90). Two of the congeners were very toxic in both systems. For the remaining congeners, one maternal toxicity measure was found to be positively correlated with embryotoxicity for growth and development in vitro without hepatocytes. With hepatocytes, a broad spectrum of correlations, both positive and negative, were observed between in vivo developmental toxicity endpoints and in vitro embryotoxicity. Data from preliminary dosimetry studies suggest that phenol congeners may accumulate in embryos exposed in vitro more readily than with in vivo exposure. Potency calculations based on dosimetry information may demonstrate better correlations between data and allow additional relationships between chemical structure and activity to be developed. PMID- 1731395 TI - Comparisons of the effects of TCDD and hydrocortisone on growth factor expression provide insight into their interaction in the embryonic mouse palate. AB - Cleft palate (CP) can be induced in embryonic mice by a wide range of compounds, including glucocorticoids and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hydrocortisone (HC), a glucocorticoid, retards embryonic growth producing small palatal shelves, while TCDD exposure blocks the fusion of normally sized shelves. TCDD induction of CP involves altered differentiation of the medial epithelial cells. Recent studies indicate that growth factors such as EGF, TGF-alpha, TGF beta 1, and TGF-beta 2 are involved in palatogenesis, regulating proliferation, differentiation, and extracellular matrix production. A synergism has been observed between HC and TCDD in which doses too low to induce CP alone are able to produce greater than 90% incidence when coadministered. In the present study a standard teratology protocol was performed in C57BL/6N mice to examine the synergism at doses lower than those previously published. Data from this study indicate synergistic interactions at doses as low as 3 micrograms TCDD/kg + 1 mg HC/kg. This extreme sensitivity suggests the involvement of a receptor-mediated mechanism possibly resulting in altered regulation of gene expression. Mechanisms of interaction were further studied by comparing growth of the shelves, fate of the medial epithelium, and expression of growth factor mRNAs and peptides. Pregnant mice were dosed on GDs 10-13 with HC (100 mg/kg sc) or with HC (25 mg/kg sc) + TCDD (3 micrograms/kg orally), doses producing 30% and 99% CP, respectively. The interaction between HC and TCDD results in a small HC-like palate, rather than the morphology typical of TCDD-induced clefting. Both compounds inhibited programmed cell death of the medial epithelium, which instead differentiated into an oral-like epithelium. The alterations in growth factor expression after HC or HC + TCDD were similar. Expression of EGF, TGF-beta 1, TGF beta 2, and EGF receptor increased in specific palatal regions. Increased levels of mRNA were observed only for TGF-beta 1. The effect of TCDD alone on growth factor expression differ from those seen with HC or HC + TCDD. These divergent effects on growth factor expression may contribute to the differences in shelf size and thus to the different mechanisms of HC and TCDD clefting. Thus the synergism between HC and TCDD may involve similar and potentially additive effects on regulators of proliferation and differentiation in the palate, but additional contributing factors cannot be excluded. PMID- 1731396 TI - Inhibition of isotretinoin teratogenicity by acetylsalicylic acid pretreatment in mice. AB - Although isotretinoin (ITR) has been suggested to cause malformations via cytopathic effects on embryonic cells, the molecular mechanisms of ITR cytotoxicity in teratogenesis are not clear. Since ITR undergoes metabolism by prostaglandin synthase to a potentially cytotoxic peroxyl free radical, the possible role of prostaglandin synthase metabolism as a modulator of ITR teratogenicity was evaluated. Craniofacial and limb abnormalities were noted in fetuses on day 18.5 of gestation following administration of ITR to pregnant CD-1 mice in a three dose regimen of 100 mg/kg at 4 hr intervals on day 10.5 of gestation (plug day = day 0.5 of gestation). Mice were also treated with acetylsalicylic acid (ASA), an irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase, at doses of 20 and 60 mg/kg body weight 2 hr prior to each ITR dose. ASA pretreatment of mice receiving ITR treatment showed a dose-dependent decrease in the overall incidence of malformations, number of defects per fetus, and the incidence of specific craniofacial and limb defects. Equivalent doses of ASA given to control mice did not cause malformations or alter the incidence of resorptions. These results demonstrate that ASA is able to ameliorate the teratogenic effects of ITR observed in fetal mice near term and indicate that prostaglandin metabolism could play a mechanistic role in ITR teratogenicity. PMID- 1731397 TI - Teratogenicity of all-trans retinoic acid during early embryonic development in the cynomolgus monkey (Macaca fascicularis). AB - The embryotoxic and teratogenic potential of all-trans retinoic acid was assessed following exposure prior to and during early organogenesis in the cynomolgus monkey (Macaca fascicularis). Sixteen pregnant females were orally administered all-trans retinoic acid (Tretinoin, Hoffmann-La Roche) once daily from GD 10-20 and twice daily from GD 21-24 at three different dosages, 5 (n = 9), 10 (n = 6) and 20 mg/kg (n = 1). Adverse clinical signs resembling hypervitaminosis A were observed in one animal at 5 mg/kg, in three animals at 10 mg/kg, and in the animal treated with 20 mg/kg all-trans retinoic acid. Maternal weight loss was observed in the 10- and 20-mg/kg groups. A dose-dependent increase in embryolethality was observed, with 22% (2/9), 50% (3/6), and 100% (1/1) occurring at 5, 10, and 20 mg/kg, respectively. The majority of embryonic deaths occurred between GD 16 and 20; the incidence of these early losses was higher than in historical and concurrent controls. No malformations, but a single growth retarded fetus, was observed in the 5-mg/kg group. Craniofacial malformations, consisting of external ear defects, mandibular hypoplasia, cleft palate, and temporal bone abnormalities, were seen in three viable fetuses in the 10-mg/kg group. Skeletal variations were common to the majority (70%, 7/10) of viable fetuses in both dose groups and were increased relative to historical controls (32%, 25/77). Unlike previous studies with 13-cis-retinoic acid during the pre- and early organogenic stages of development (Hummler et al., Teratology 42:263 272, 1990), no thymic hypo- or aplasia or heart anomalies were observed, which may be attributable to the slightly longer 13-cis retinoic acid treatment period, i.e., GD 10-27. However, external ear and temporal bone defects were common to both all-trans and 13-cis retinoic acid. The similarity observed in the malformation syndrome induced by both all-trans and 13-cis retinoic acid in the cynomolgus monkey and 13-cis retinoic acid embryopathy in humans supports this macaque species as a model for further developmental toxicity studies of vitamin A-related compounds. PMID- 1731398 TI - The functional importance of the oviduct in neonatally estrogenized mouse females for early embryo survival. AB - Eight-week-old virgin untreated female mice were induced to ovulate using equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG), and were then caged with males overnight. Females with a vaginal plug on the following morning were killed 24 hours later and 2-cell embryos were flushed from the oviduct. These embryos were transferred to the oviduct of 8-week-old control females, to females of the same age treated with 5 micrograms diethylstilbestrol (DES) sc in olive oil for the first 5 days after birth, or to females treated with 1 microgram estradiol-17 beta for 2 days before and 2 days after transfer (estrogen dominated/ED/females). Two days after transfer, a significantly lower number of embryos were recovered from oviducts of DES females compared to control females and a still lower number from ED females. The recovered embryos were cultured in vitro for 4 days testing trophoblast outgrowth ("implantation stage"). The incidence of embryos reaching this stage after development in DES exposed oviducts was only half of that for embryos passing control oviducts or ED oviducts. It is concluded that the adult oviductal environment in neonatally DES treated females significantly decreases early embryo developmental potential. The oviductal factor(s) harmful to the embryo may be related to a persistent and possibly increased level of circulating estrogen level in DES females. PMID- 1731399 TI - A quantitative and morphological study of ectodermal microvilli in ten areas in the control and experimental prenatal rat. AB - This study provides a baseline of mainly quantitative morphologic information in relation to the ectodermal microvilli in ten areas in the control and experimental prenatal rat with special reference to the period of neural tube closure. Embryos from six litters were used and ten areas were examined mainly with the scanning electron microscope. Statistical analysis showed no significant difference between litters nor between the ten areas examined. In the 376 hr (day 15.6) fetus only the ectodermal cells of the nostril region demonstrated a rich population of microvilli, a fact possibly associated with late maturation of that region. Some evidence is provided to show that there is an increase in the population of microvilli in the 276 hr (day 11.5) embryo following experimentally induced zinc deficiency and introduction of nicotine in the culture medium. The possible mechanisms underlying the increase in ectodermal microvilli are i) an attempt by ectodermal cells to absorb nutrients, ii) a reflection of cells under stress, iii) failure of early embryonic ectodermal cells to shed microvilli normally associated with developmental changes, and iv) a generalized developmental delay rather than some cellular response to a trace element nutritional deficiency and a teratogen. PMID- 1731401 TI - Leadership through empowerment. PMID- 1731400 TI - Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse. AB - The viability of early mouse embryos is acutely sensitive to (R)-deoxycoformycin (pentostatin), a tight-binding inhibitor of adenosine deaminase (ADA). Previous studies have shown that a single 5-mg/kg dose on day 7 (plug = day 0) of gestation fully inhibits uteroplacental ADA activity within 0.5 h; causes massive cell death in the neural plate and primary mesenchyme by 6 h, major craniofacial anomalies by day 10, and resorption by day 12 (Knudsen et al., '89; Airhart et al., '91). The present study has examined further the developmental toxicity and early effects of this inhibitor on ADA metabolism. (R)-Deoxycoformycin was administered to pregnant CD-1 (ICR) mice as a single intraperitoneal dose of 0.5 10 mg/kg total body weight on days 6-11 of gestation. The major adverse effect, early resorption, was dose dependent and specific to day 7-8 exposure. Treatment with 5 mg/kg on day 7 resulted in 85% resorptions, 15% malformations, and a 24% reduction in mean fetal weight, whereas the same dose of (S)-deoxycoformycin had no effect. Levels of adenosine and 2'-deoxyadenosine, which are the endogenous substrates of ADA, were monitored in the embryo/decidual unit (E/D) by reversed phase high-performance liquid chromatography (RP-HPLC). In response to the inhibitor, both nucleosides increased transiently in the antimesometrial compartment (antimesometrial decidua + embryo). Peak levels (Cmax) of adenosine and 2'-deoxyadenosine were dose dependent over the range tested (0.05-10 mg/kg). Exposure to 5 mg/kg on day 7 raised adenosine levels within 0.5 h to 42-fold over the basal level of 0.06 nmol/mg protein. There was an even stronger effect on 2' deoxyadenosine levels, which were elevated 674-fold over the detection limit of 0.0005 nmol/mg protein. Direct exposure to the inhibitor in serum-free E/D culture produced similar results: 50 microM (R)-deoxycoformycin within 1 h raised adenosine levels 26-fold and 2'-deoxyadenosine levels 410-fold. In vivo studies also showed a general correlation between embryolethality and the length of adenine nucleoside pool expansion, apparent for exposure on day 7, 8, or 9 but not on day 6, suggesting that the embryo becomes sensitive to adenosine or 2' deoxyadenosine once the neural plate has formed. PMID- 1731402 TI - [A case of death in fattening pigs caused by formation of hydrogen sulfide in the manure cellar]. PMID- 1731404 TI - [Law on the practice of veterinary medicine]. PMID- 1731403 TI - [The ethicus as 'someone calling in the desert'?]. PMID- 1731405 TI - [The significance of ovariectomy and progestagens in the development of mammary carcinoma in cats]. AB - Mammary carcinoma in the cat has a poor prognosis. Because of metastasis and local malignity, these tumours are of relevance not only to veterinary practice but also to comparative research as a model to study possible therapies and the aetiology and pathogenesis of the disease. Little is known about the development of mammary tumours in humans and companion animals. A case-control study was carried out to learn more about the role of endogenous and exogenous sex hormones. In the cat, ovariectomy (RR 0.36) had a clear protective effect against the development of mammary carcinoma, whereas regular administration of progestogens clearly increased the risk of carcinoma development (RR 2.81). Our results show that with regard to the evidence of mammary carcinoma, oestrus prevention by ovariectomy is to preferred to the administration of progestogens. PMID- 1731406 TI - [Cecum amputation in a blond d'Aquitaine bull]. AB - A caecal torsion of 720 degrees was diagnosed in a one-and-a-half-year-old blonde d'Aquitaine bull. A partial amputation of the caecum had to be performed. The diagnosis and surgery of this case are described against the background of the usual dilatations and rotations of the caecum and ansa proximalis. PMID- 1731407 TI - [Diagnostic imaging in cardiological patients]. PMID- 1731408 TI - Hypothermia reduces 72-kDa heat-shock protein induction in rat brain after transient forebrain ischemia. AB - BACKGROUND AND PURPOSE: We examined the influence of concurrent moderate hypothermia (30 degrees C) and transient forebrain ischemia on the induction of 72-kDa heat-shock protein and neuronal damage in male Wistar rats. SUMMARY OF REPORT: Experimental groups included: normothermic with 8 minutes of transient forebrain ischemia (group 1, n = 7), hypothermic without ischemia (group 2, n = 9), and hypothermic (30 degrees C) with 8 minutes of transient forebrain ischemia (group 3, n = 5). Intense 72-kDa heat-shock protein immunoreactivity was demonstrated in rat forebrain 48 hours after induction of normothermic forebrain ischemia (group 1); it was not detected in the brain of animals subjected to hypothermia without ischemia (group 2), and hypothermia during ischemia (group 3) significantly inhibited its expression compared with that in normothermic ischemia animals (group 1). CONCLUSIONS: These observations suggest that 72-kDa heat-shock protein induction is not the mechanism by which moderate hypothermia protects against ischemic cell damage. PMID- 1731409 TI - Cerebral infarction associated with protein C deficiency. AB - BACKGROUND AND PURPOSE: A deficiency of plasma protein C, both the hereditary and acquired types, is one cause of thromboembolic disease. Several antineoplastic agents have been reported to decrease the production of protein C in the liver by impairing either the absorption or metabolism of vitamin K, leading to acquired protein C deficiency. CASE DESCRIPTION: We treated a young woman with protein C deficiency, who had developed a cerebral infarction of the right parietal cortex of sudden onset. On admission, the antigenic level of plasma protein C was 38%. Serial cerebral angiography revealed occlusion of the right middle cerebral artery, which subsequently recanalized completely. This patient had taken fluorouracil derivatives orally for as long as 3 years following a left mastectomy for stage II breast cancer. Tests revealed that the patient's mother had only one-half the normal activity of plasma protein C despite a normal antigenic level. CONCLUSIONS: We speculate that the etiology of the cerebral infarction in this patient might involve an embolic mechanism associated with protein C deficiency induced by an interaction between inherited and acquired factors. PMID- 1731410 TI - Hypoglycemia presenting as basilar artery thrombosis. AB - BACKGROUND AND PURPOSE: Following our treatment of a patient with hypoglycemia induced brain stem symptoms, we examined the possible mechanisms for hypoglycemia presenting as basilar artery disease. CASE DESCRIPTION: We describe a patient who had progressive brain stem symptoms due to a diet-induced hypoglycemia initially diagnosed as basilar artery thrombosis. Symptoms ceased immediately after intravenous glucose. CONCLUSIONS: Before invasive diagnostic and thrombolytic strategies are considered, hypoglycemia as a rare but important cause of acute brain stem dysfunction must be considered in patients suspected to suffer from basilar artery thrombosis. PMID- 1731411 TI - Postpartum dissecting aneurysm of the basilar artery. AB - BACKGROUND AND PURPOSE: Dissecting aneurysms arising from the vertebrobasilar complex are rare and difficult to manage. More of their natural history needs to be known before treatment can be optimized. CASE DESCRIPTION: We report a postpartum dissecting aneurysm of the right vertebrobasilar artery in a 31-year old woman that was confirmed by angiographic identification of a double lumen. The intracranial segment of the right vertebral artery was thrombosed proximal to the aneurysm. The patient, managed conservatively, recovered well and, when reexamined 2 months later, was found to be neurologically intact. A repeat angiographic study at that time demonstrated that the aneurysm had resolved. CONCLUSIONS: Proximal occlusion may have protected the aneurysm from rupture and further dissection, thereby making surgery unnecessary. PMID- 1731412 TI - Caffeine and stroke. PMID- 1731413 TI - Ischemic stroke as the sole manifestation of human immunodeficiency virus infection. PMID- 1731414 TI - A comparison of transcranial Doppler and cerebral blood flow studies to assess cerebral vasoreactivity. AB - BACKGROUND AND PURPOSE: The aim of this study was to determine the ability of transcranial Doppler ultrasonography when used to assess cerebral vasoreactivity. The results of this method were compared with regional cerebral blood flow measurements. METHODS: Forty-three patients with symptoms suggesting cerebrovascular disease took part. Transcranial Doppler findings in the middle cerebral arteries were compared with regional cerebral blood flow in the corresponding perfusion territories before and after acetazolamide administration. RESULTS: There was a significant positive correlation between the absolute increase in cerebral blood flow in milliliters per 100 g per minute and the percent increase in velocity (r = 0.63). The right-left, side-to-side difference of the acetazolamide response obtained by the two methods also showed a positive correlation (r = 0.80). Control limits obtained from healthy subjects were used for both the blood flow increase (absolute values and asymmetry in absolute values) and the velocity increase (percent increase and asymmetry in percent increase). The two methods then agreed in their evaluation of vasoreactivity in 74 (86%) of the 86 middle cerebral artery perfusion territories; 20 (23%) were assessed by both methods as having a reduced vasodilatory reserve. Eleven hemispheres with a slightly reduced regional cerebral blood flow response to acetazolamide were not detected by transcranial Doppler, whereas all territories with a marked reduction were identified by Doppler. Only one hemisphere with a normal cerebral blood flow increase after acetazolamide administration was assessed by Doppler as having reduced vasoreactivity. CONCLUSIONS: Transcranial Doppler and the acetazolamide test may be used in clinical situations to assess cerebral vasoreactivity. PMID- 1731415 TI - Early computed tomographic findings for thrombolytic therapy in patients with acute brain embolism. AB - BACKGROUND AND PURPOSE: The benefits and safety of thrombolytic therapy was studied in patients with acute brain embolism. METHODS: We intravenously administered recombinant tissue plasminogen activator (20-30 MU for 1 hour) to 10 patients with acute (less than 6 hours) brain embolism and examined the neurological outcomes in relation to the findings on computed tomography and angiography. RESULTS: The symptoms ameliorated in four patients within 24 hours after onset, and reopening of the occluded arteries occurred in two of the patients immediately after recombinant tissue plasminogen activator infusion. On the initial computed tomographic scan (less than 3 hours), four patients had already demonstrated early indications of brain ischemia, that is, an obscure margin of the lentiform nuclei, reduced tissue attenuation, or effacement of cortical sulci. These patients failed to demonstrate reopening of the occluded arteries, and one developed a massive brain hemorrhage with clinical deterioration. Of the remaining six patients, two obtained clinical improvement with recanalization soon after the therapy and demonstrated little to slight hemorrhagic complications. The outcomes at 1 month were favorable in five patients and poor in three; death occurred in two. CONCLUSIONS: Thrombolytic therapy with recombinant tissue plasminogen activator may be safe and effective when there are no early computed tomographic findings within 3 hours from the onset of embolic stroke. PMID- 1731416 TI - Seasonal variation of cerebral hemorrhage in 236 consecutive cases in Brussels. AB - BACKGROUND AND PURPOSE: Seasonal variation in the incidence of cerebral hemorrhage has been previously demonstrated. In this study, we sought to identify the climatological data best correlated with this seasonal variation. METHODS: In a retrospectively studied sequential series of 236 patients with nontraumatic cerebral hemorrhage observed in Brussels over a period of 8 years, we cumulatively grouped the dates of stroke occurrence into a single calendar year. RESULTS: We found marked seasonal variation in incidence, with the highest value (23%) observed in November-December and the lowest (10%) in July-August. Seasonal variations in incidence of cerebral hemorrhage were shown to be correlated not only with the inverse of ambient temperature, but also with the inverse of hours of sunshine and with ambient humidity. We found no difference between hypertensive and normotensive patients. CONCLUSIONS: Our study fails to bear out the hypothesis that the higher incidence of cerebral hemorrhage in late autumn and winter is due to the influence of low ambient temperature on blood pressure. PMID- 1731417 TI - Stroke incidence in Copenhagen, 1976-1988. AB - BACKGROUND AND PURPOSE: Temporal trends in stroke incidence in Denmark have not been previously reported. The Copenhagen City Heart Study is a prospective study based on a randomly selected sample of an urban population of, initially, 19,698 participants followed since 1976. Over a period of 12 years, we studied three important aspects of stroke incidence in 848 identified cases: temporal trends, dependence on age and sex, and comparison of responders and nonresponders. METHODS: The participants were invited to two health examinations at 5-year intervals. The participants who attended at least one of the two examinations are termed responders and those who attended none nonresponders. The cases of first ever stroke were collected from responders, the National Patient Register, and the National Register of Deaths and were verified by study of hospital records and death certificates. RESULTS: For responders aged 35-64 years and greater than or equal to 65 years, there were no significant changes in the weighted rates in four consecutive 3-year periods. There was a tendency toward decreasing rates among younger women, but not in older women or men. The age- and sex-adjusted rates per 1,000 (based on the Danish population in 1982) in responders in the entire 12-year follow-up period were 1.61 in women, 2.67 in men, and 2.14 in both sexes combined. Stroke incidence rates increased exponentially with age in both sexes, with rates in men generally twice those in women, even in the greater than or equal to 75 years of age group. Age-adjusted rates were higher in nonresponders than in responders. For women, this ratio was 1.7; for men, 1.1. CONCLUSIONS: The stroke incidence in Copenhagen is relatively high and has shown no decreasing tendency over the period 1976-1988. PMID- 1731418 TI - Clinical trial of nimodipine in acute ischemic stroke. The American Nimodipine Study Group. AB - BACKGROUND AND PURPOSE: A randomized, double-blind, multicenter clinical trial of placebo versus nimodipine was conducted to test the hypothesis that nimodipine would reduce the frequency of death and of worsening by 30% compared with placebo. METHODS: Nimodipine was used in doses of 60 mg, 120 mg, and 240 mg daily in 1,064 patients treated for 21 days. Treatment was begun within 48 hours of stroke due to infarction as inferred by initial computed tomographic scan findings. The Toronto and motor scales were analyzed by analysis of covariance, using covariance-adjusted means, the last-value-carried-forward, to compare the baseline value with the 3 assessment days (days 4, 10, and 21). RESULTS: No difference in mortality or neurological outcome was found with any of the rating scales for the overall cohort. Planned but post hoc subgroup analysis showed a reduction in worsening frequency of 30% compared with placebo and significantly better outcome scores with 120 mg nimodipine daily started within 18 hours of stroke as measured by the Toronto scale (p less than 0.005) and when the pretreatment computed tomographic scan was negative (p less than 0.003). CONCLUSIONS: Nimodipine had no overall effect when treatment was begun within 48 hours. Confirmation of the benefits suggested by post hoc analyses for the subgroup treated with 120 mg nimodipine within 18 hours, and who had negative computed tomographic scans, would require a separate trial. PMID- 1731419 TI - A model of acute focal ischemia in the territory of the anterior cerebral artery in baboons. AB - BACKGROUND AND PURPOSE: We developed a model of acute focal ischemia in the territory of the anterior cerebral artery in baboons to study the ischemic pattern following occlusion and changes in regional cerebral blood flow. METHODS: In nine anesthetized animals, a Scoville clip was placed on the proximal segment of the common anterior cerebral artery via a unilateral transorbital approach. Regional cerebral blood flow was measured by hydrogen clearance in the cortex and corpus callosum. Postexperimentally, arteries were selectively injected. RESULTS: The resulting ischemia involved both hemispheres symmetrically and the corpus callosum. Cortical flows were significantly reduced within a region 15 mm from the midline on each side (p less than 0.01). A gradient of cortical flow reduction was produced between 10 and 25 mm from the midline. This area defines the boundary region between the territories of the anterior and middle cerebral arteries, and is identified as the "penumbra" of the ischemic core, which itself lies within 10 mm of the midline. Blood flows in the corpus callosum decreased from an average of 21.0 to 6.7 ml/100 g/min in the body (p less than 0.01) and from 22.5 to 10.7 ml/100 g/min in the genu (p less than 0.05). CONCLUSIONS: This ischemic model has close physiological and morphological relevance to stroke related clinical circumstances, in particular the acute conditions of focal cerebral ischemia associated with vascular surgery. It also provides a new framework for experimental investigation of the ischemic penumbra. PMID- 1731420 TI - Hypertension with hemodilution prevents multifocal cerebral hypoperfusion after cardiac arrest in dogs. AB - BACKGROUND: Improved neurological outcome with postarrest hypertensive hemodilution in an earlier study could be the result of more homogeneous cerebral perfusion and improved O2 delivery. We explored global, regional, and local cerebral blood flow by stable xenon-enhanced computed tomography and global cerebral metabolism in our dog cardiac arrest model. METHODS: Ventricular fibrillation cardiac arrest of 12.5 minutes was reversed by brief cardiopulmonary bypass, followed by life support to 4 hours postarrest. We compared control group I (n = 5; mean arterial blood pressure, 100 mm Hg; hematocrit, greater than or equal to 35%) with immediately postarrest reflow-promoted group II (n = 5; mean arterial blood pressure, 140-110 mm Hg; hypervolemic hemodilution with plasma substitute to hematocrit, 20-25%). RESULTS: After initial hyperemia in both groups, during the "delayed hypoperfusion phase" at 1-4 hours postarrest, global cerebral blood flow was 51-60% of baseline in group I versus 85-100% of baseline in group II (p less than 0.01). Percentages of brain tissue voxels with no flow, trickle flow, or low flow were lower (p less than 0.01) and mean regional cerebral blood flow values were higher in group II (p less than 0.01). Global cerebral oxygen uptake recovered to near baseline values at 3-4 hours postarrest in both groups. Postarrest arterial O2 content, however, in hemodiluted group II was 40-50% of that in group I. Thus, the O2 uptake/delivery ratio was increased (worsened) in both groups at 2-4 hours postarrest. CONCLUSIONS: After prolonged cardiac arrest, immediately induced moderate hypertensive hemodilution to hematocrit 20-25% can normalize cerebral blood flow patterns (improve homogeneity of cerebral perfusion), but does not improve cerebral O2 delivery, since the flow benefit is offset by decreased arterial O2 content. Individualized titration of hematocrit or hemodilution with acellular O2 carrying blood substitute (stroma free hemoglobin or fluorocarbon solution) would be required to improve O2 uptake/delivery ratio. PMID- 1731421 TI - Sustained damage to energy metabolism of brain regions after microsphere embolism in rats. AB - BACKGROUND AND PURPOSE: Information on sustained damage to cerebral function and metabolism after cerebral ischemia is useful for prophylaxis and therapeutics of cerebral infarction. The purpose of the present study was to induce sustained damage to brain regions after cerebral ischemia in experimental animals. For this purpose, we examined animal behavior and cerebral energy metabolism following microsphere embolism in rats. METHODS: We injected 900 microspheres (48 microns in diameter) into the right internal carotid artery of 110 rats and determined the time course of changes in the rats' behavior and the energy metabolism of the cortex, striatum, and hippocampus of both hemispheres. We injected the same volume of vehicle, without microspheres, into 28 sham-operated rats; there were 14 nonoperated control rats. RESULTS: Peak increase in lactate content and decrease in adenosine triphosphate and creatine phosphate of these brain regions of the right hemisphere were seen on the first day after microsphere embolism, whereas peak increases in glucose and glycogen contents of these regions were observed on the third day. Most of the metabolic alterations in all these regions continued for up to 28 days after operation, although they recovered toward control levels with time after the operation. The extent and trend of metabolite changes of the right hemisphere after microsphere embolism were similar in the three brain regions. In the left hemisphere, similar metabolic changes were observed, but to a lesser degree. The time course of changes in behavioral scores following microsphere embolism revealed marked stroke-like symptoms on the first day and relatively rapid disappearance of the symptoms with time after embolism. CONCLUSIONS: Microsphere embolism is capable of inducing widespread, sustained damage to energy metabolism of brain regions. PMID- 1731422 TI - Effect of 33% xenon inhalation on whole-brain blood flow and metabolism in awake and fentanyl-anesthetized monkeys. AB - BACKGROUND AND PURPOSE: Despite the documented diagnostic value of local cerebral blood flow maps by xenon-enhanced computed tomography, reports of cerebral blood flow activation by inhaled 33% Xe raised concerns about the method's safety and accuracy. We evaluated the effect of 33% Xe inhalation on cerebral blood flow and cerebral metabolic rates for oxygen and glucose in four awake and six fentanyl anesthetized rhesus monkeys. METHODS: Platinum microelectrodes and catheters in the torcular Herophili were used to measure cerebral blood flow by hydrogen clearance, and oxygen and glucose concentrations. Cerebral variables were measured after 5 and 35 minutes of exposure to room air followed randomly by 67% O2 in 33% N2 or Xe. Five- and 35-minute measurements were combined because the duration of exposure had no effect. RESULTS: In awake monkeys, 33% Xe compared with 33% N2 reduced (p less than 0.05) cerebral blood flow from 75 +/- 12 to 66 +/- 9 (mean +/- SD) ml.100 g-1.min-1 and oxygen consumption from 6.1 +/- 0.7 to 5.1 +/- 0.6 ml.100 g-1.min-1. In fentanyl-anesthetized monkeys, cerebral variables during 33% N2 versus 33% Xe were cerebral blood flow, 84 +/- 26 versus 79 +/- 23 ml.100 g-1.min-1; oxygen consumption, 5.0 +/- 0.7 versus 4.9 +/- 0.5 ml.100 g-1.min-1; and glucose consumption, 8.4 +/- 1.9 versus 7.9 +/- 2.0 mg.100 g-1.min-1. CONCLUSIONS: In awake monkeys, 33% Xe reduced rather than activated cerebral blood flow and oxygen consumption by 12% and 16%, respectively; it had no effect in fentanyl-anesthetized monkeys. PMID- 1731423 TI - Protective effects of human recombinant superoxide dismutase on transient ischemic injury of CA1 neurons in gerbils. AB - BACKGROUND AND PURPOSE: It has been postulated that oxygen-derived free radicals are produced in significant quantities upon reperfusion of ischemic brain and that the free radicals play a pivotal role in triggering the ischemic neuronal damage causing delayed neuronal death. This study was undertaken to examine the effects of human recombinant superoxide dismutase on the delayed neuronal death of CA1 neurons and on the change in the expression of messenger ribonucleic acid for endogenous copper-zinc superoxide dismutase after transient ischemia. METHODS: Human recombinant superoxide dismutase (8 x 10(5) units/kg) or apo superoxide dismutase was administered intravenously 1 minute before bilateral carotid artery occlusion in gerbils divided among four experimental groups. Endogenous copper-zinc superoxide dismutase messenger ribonucleic acid was analyzed by in situ hybridization histochemistry using a sulfur-35-labeled oligonucleotide probe. Immunohistochemical localizations of administered human recombinant superoxide dismutase were investigated. RESULTS: All gerbils receiving apo-superoxide dismutase exhibited almost complete destruction of CA1 neurons 7 days after 5 minutes of ischemia. The gerbils treated with human recombinant superoxide dismutase showed mild lesions (p less than 0.01). Discrete localizations were observed for endogenous copper-zinc superoxide dismutase messenger ribonucleic acid. Transient ischemia increased labeling throughout the hippocampus after 30 minutes and 24 hours of reperfusion. This increase was abolished by treatment with human recombinant superoxide dismutase. This phenomenon was confirmed by Northern blot analysis. The interneurons in CA3 and cells in the hilus were mainly stained against administered superoxide dismutase at 5 and 30 minutes, and these reactions had disappeared at 20 hours after the administration. CONCLUSIONS: Our data demonstrate protective effects of human recombinant superoxide dismutase against ischemic neuronal damage and support the hypothesis that the generated free radicals induce a vicious cycle leading to delayed neuronal death. PMID- 1731424 TI - Combined treatment with MK-801 and nicardipine reduces global ischemic damage in the gerbil. AB - BACKGROUND AND PURPOSE: Excessive activation of the N-methyl-D-aspartate receptor by glutamate produces an influx of Ca2+, which in turn is thought to lead to ischemic cell death. In this study we evaluated the combined treatment of the N methyl-D-aspartate antagonist dizocilpine (MK-801) and the dihydropyridine Ca2+ channel blocker nicardipine for the reduction of hippocampal CA1 neuronal loss. METHODS: Global ischemia was induced by bilateral carotid artery occlusion in the gerbil. Body temperature was maintained between 36.5 degrees C and 37.5 degrees C during surgery. MK-801 (5.0 mg/kg) was injected 15 minutes after occlusion whereas nicardipine was given by injection and via a micro-osmotic pump (1.0 mg/kg/day) for 3 days. RESULTS: Postischemic treatment with MK-801 reduced CA1 cell loss by 27.0%, whereas nicardipine reduced CA1 cell loss by 13.3%. Combined postischemic treatment with these drugs yielded an additive, protective effect (44.5% reduction of CA1 loss) that did not appear to result from postischemic hypothermia as assessed by skull and rectal temperature recordings. CONCLUSIONS: Our results demonstrate that MK-801 plus nicardipine significantly attenuates CA1 cell death after forebrain ischemia in the gerbil. Excitatory amino acid antagonists in combination with Ca2+ channel antagonists may be an effective therapy in patients exposed to global ischemic insult. PMID- 1731425 TI - Effect of calcium antagonists on postischemic protein biosynthesis in gerbil brain. AB - BACKGROUND AND PURPOSE: Prolonged inhibition of protein synthesis precedes delayed neuronal death in the CA1 sector of the hippocampus after transient cerebral ischemia. Organic calcium antagonists have been recommended for alleviation of ischemic neuronal damage. The present study was undertaken to investigate whether these drugs improve the recovery of protein biosynthesis after interruption of cerebral blood flow. METHODS: Cerebral protein synthesis was measured biochemically and autoradiographically in gerbils submitted to 5 minutes of bilateral occlusion of the common carotid arteries followed by 2 hours or 2 days of recirculation. Flunarizine (25 mg/kg) or nimodipine (1.5 mg/kg) were applied intraperitoneally shortly after ischemia. RESULTS: Treatment with either calcium antagonist did not markedly influence postischemic recovery of protein synthesis in the resistant regions of the brain and did not prevent the persisting inhibition in the vulnerable stratum pyramidale of the CA1 sector of the hippocampus. CONCLUSIONS: The postischemic application of the organic calcium antagonists nimodipine and flunarizine does not promote postischemic recovery of protein synthesis. The beneficial effects of these drugs must, therefore, be based on other mechanisms. PMID- 1731426 TI - Transcranial Doppler assessment of cerebral flow velocity during cognitive tasks. AB - BACKGROUND AND PURPOSE: The purpose of this study was to assess the ability of transcranial Doppler ultrasonography to detect selective circulatory changes during cognitive activity. METHODS: We measured cerebral artery flow velocity in 21 normal volunteers by transcranial Doppler ultrasonography during rest followed by cerebral activation. Mean and peak systolic flow velocities of the anterior, middle, and posterior cerebral arteries were measured during the performance of a commercial video game. We also measured flow velocity of the anterior cerebral arteries in 18 subjects during a mental arithmetic task. Serial measurements of the right and left sides were made with a headband with two probes. RESULTS: We observed a global increase in the flow velocity above baseline measurements during task performance. During the video game, both middle cerebral arteries (t = 2.6, p = 0.02 for the left; t = 3.3, p = 0.004 for the right) and the left posterior cerebral artery (t = 2.2, p = 0.004) had selective increase in mean flow velocity compared with the ipsilateral anterior cerebral artery. This selective activation was most prominent in the right middle cerebral artery, which had a greater degree of activation than the right posterior cerebral artery (t = 2.8, p = 0.013). We did not observe a statistically significant difference between the right and left middle cerebral arteries, but there was a trend toward a greater activation on the right for both the mean velocity (t = 1.7, p = 0.098) and the peak velocity (t = 1.9, p = 0.079). CONCLUSIONS: Our preliminary investigation suggests that this noninvasive technique has the potential to correlate selective cerebral artery flow dynamics with cognitive activity. PMID- 1731427 TI - Reduction in ischemic brain injury in rabbits by the anion transport inhibitor L 644,711. AB - BACKGROUND AND PURPOSE: We studied the anion transport inhibitor L-644,711, which is known to reduce astrocyte swelling and excitotoxin release in primary astrocyte culture, in two models of thromboembolic stroke to assess its capacity to influence ischemic brain injury. METHODS: New Zealand White rabbits were used in this study. The two models include autologous clot embolized to the brain via the carotid artery, with one model using a transient period of systemic hypotension. Cerebral blood flow was determined by the hydrogen clearance method, intracranial pressure was measured with a fiberoptic transducer, and infarct size was assessed with triphenyltetrazolium chloride staining of the coronally sectioned brain. Both models received a 2-hour infusion of L-644,711 (total dose, 12 mg/kg) beginning 20 minutes before embolization. RESULTS: In both the normotensive (p less than 0.01) and the hypotensive (p less than 0.05) model, treatment with L-644,711 resulted in a significant reduction in infarct size and a significant improvement in regional cerebral blood flow (p less than 0.03, normotensive model, and p less than 0.05, hypotensive model). Raised intracranial pressure, unique to the hypotensive model, was abolished by the administration of L-644,711 (p less than 0.05). A hyperglycemic response associated with embolization, also unique to the hypotensive model, was significantly reduced by the administration of L-644,711 (p less than 0.05). CONCLUSIONS: The ability of L 644,711 to limit brain injury in two related models of thromboembolic stroke suggests a potential therapeutic role for anion channel blockers in cerebral ischemia. PMID- 1731428 TI - Infarct reduction by the platelet activating factor antagonist apafant in rats. AB - BACKGROUND AND PURPOSE: Recent findings suggest a key role for platelet activating factor in neuroinjury. For this reason we evaluated the effects of the platelet activating factor antagonist apafant (4-(2-chlorophenyl)-9-methyl-2[3(4 morpholinyl)-3-propanol-1- yl[6H-thieno[3.2-f[[1.2.4]triazolo]4,3 1]]1.4]diazepine on farct volume and local cerebral blood flow following irreversible occlusion of the left middle cerebral artery in rats to assess the direct and vascular components of apafant's action. METHODS: We measured infarct volume 48 hours after middle cerebral artery occlusion. The effect of multiple doses of apafant (30 mg/kg p.o.) was tested in both pretreatment (n = 8) and posttreatment (n = 8) groups. In the pretreatment group apafant was given 30 minutes before and 2, 6, and 18 hours after occlusion. Rats of the posttreatment group received apafant 1, 6, and 18 hours after middle cerebral artery occlusion. We also examined the effect of a single dose of apafant given 30 minutes prior to occlusion (n = 9) on local cerebral blood flow determined 2 hours after middle cerebral artery occlusion. RESULTS: Both regimens of apafant effectively decreased infarct volume. The reduction in cortical infarct volume was 59% (p less than 0.01; H test, U test) when the rats were treated before and after vessel occlusion whereas the decrease was 47% (p less than 0.05; H test, U test) when treatment began 1 hour after occlusion. Apafant did not change local cerebral blood flow after occlusion compared with controls. CONCLUSIONS: We suggest that the cytoprotection afforded by apafant occurs mainly via a direct effect on brain tissue and has no major vascular component. PMID- 1731429 TI - Preoperative autologous blood donations by high-risk patients. PMID- 1731430 TI - Autologous blood donation: hemodynamics in a high-risk patient population. AB - Transfusion practices have changed dramatically in recent years as a result of the acquired immune deficiency syndrome crisis. The use of preoperative donation of autologous blood units has gained popularity. Healthy individuals tolerate phlebotomy well, experiencing a 2- to 5-percent incidence of vasovagal reactions. However, no systematic study of hemodynamic function during phlebotomy exists for patients who have major cardiovascular or multiple organ disease (high-risk patients). The following protocol examines blood pressure, heart rate, cardiac output, lead II electrocardiogram, and pulse oximetry in 123 patients donating a total of 224 units of blood in a new high-risk autologous blood donation program that was conducted in a postanesthesia care unit over the past 18 months. Changes in hemodynamic variables during phlebotomy were consistent with mild volume depletion; the changes did not result in overall differences in the courses of the groups. Significant numbers of patients exhibited systolic or diastolic hypotension, dysrhythmias, syncope, and tachycardia. Because tolerance for hypotension (vasovagal reactions) is decreased in patients with coronary artery disease, appropriate monitoring may be warranted to maximize the safety of elective phlebotomy. Further study is required to stratify risks and to determine which hemodynamic variables are predictive of adverse events. PMID- 1731431 TI - Moderate and severe reactions in blood donors. AB - During the period April 1985 to March 1986, 217 blood donors were found to have moderate (syncopal) to severe (convulsive) reactions. This population was compared to 5630 randomly selected donors who did not have reactions. An examination of demographic, physical, and societal/emotional factors was conducted to determine if any were predictive of reactions in donors. The results of the research supported the hypothesis that first-time donors have a higher frequency of reactions (1.7%) than do repeat donors (0.19%). A review of the above predictive factors documented that, with regard to demographic factors, 1) the number of prior donations was inversely proportional to the risk of reaction; 2) the gender of the donor was not predictive; and 3) youth was a predictor of reactions. An analysis of the physical factors revealed that donors who reacted were of lower weight (mean, 153.7 lb) than those who did not (mean, 166.4 lb) and that systolic blood pressure was slightly lower in the group with reactions. Although the difference was significant (3 torr), it was not thought to be significant clinically. In a comparison of a group with systolic blood pressure ranging from 80 to 100 torr and a group with systolic blood pressure ranging from 120 to 140 torr, the first group had a 70-percent higher risk of reaction. Finally, with regard to the last category of societal or emotional factors, the research demonstrates 1) that the ingestion of caffeinated beverages was associated with a reduced risk of reactions; 2) that the food intake of donors who reacred was significantly different from that of those who had no reaction, but this difference was not thought to be clinically significant; and 3) that the duration between registration and the onset of phlebotomy was directly predictive of reaction status. The research indicates that first-time donor status and several specific demographic, physical, and societal or emotional factors are predictors of donor reactions. PMID- 1731432 TI - Postoperative infections following autologous and homologous blood transfusions. AB - Perioperative homologous blood transfusion has been linked to immune suppression and increased risk of postoperative infection. Autologous blood transfusion may not be associated with increased risk of infection because it presumably is not immunosuppressive. Fifty recipients of preoperatively donated autologous blood were matched to 50 recipients of homologous blood who underwent the same procedure, and the hospital course was reviewed for evidence of postoperative infection in both groups. Postoperative leukocytosis and febrile episodes were more common in homologous blood recipients (17 and 6 vs. 12 and 4, respectively). Sixteen percent of the 50 homologous blood recipients had positive cultures, as compared to 4 percent of the 50 autologous blood recipients (p less than 0.05). This study suggests that the association of blood transfusion with infection may be partially abrogated by the use of autologous blood. PMID- 1731433 TI - New trends in the preparation and storage of platelets. PMID- 1731434 TI - In vitro and in vivo activity of murine lymphokine-activated killer cells after cryopreservation. AB - The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine-activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin-2 (rIL-2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL-2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre-B cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival greater than 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival greater than 120 days, 50% survival greater than 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy. PMID- 1731435 TI - Amphotericin B-induced injury in stored human platelets. AB - Administration of intravenous amphotericin B is a major factor in the reduction of the recovery of transfused platelet concentrates (PCs). For the study of possible drug-induced platelet lesions, fresh platelets and stored PCs were incubated with therapeutic concentrations of amphotericin B (10-50 micrograms/mL) and examined for membrane changes by scanning electron microscopy. Stored platelets demonstrated the formation of "pits" on the surface membrane, which were maximal immediately after preparation and gradually decreased with storage. The number of pits was significantly higher after exposure to amphotericin B. No increase was detected in fresh platelets following exposure to amphotericin B. The maximal effect of the drug was seen after 5 days of storage as PCs. Amphotericin B did not affect platelet shape or the number of pseudopodia. There was no correlation between pit formation and the pH, pO2, or pCO2 of the concentrates. Amphotericin B did not release 51Cr from prelabeled stored platelets after 2 hours' incubation at 37 degrees C. Thus, amphotericin B appears to exacerbate a membrane lesion induced by the preparation and storage of PCs. PMID- 1731436 TI - Investigation of the effect of long-term whole blood donation on immunologic parameters. AB - Few studies addressing possible immune sequelae of long-term whole blood donation have been published. The purpose of this study was to determine if there were any differences in lymphocyte subsets, monocyte and neutrophil receptors, and antigens important to host defense in committed whole blood donors and in nondonor controls. Blood samples were obtained from 27 whole blood donors who had been donating on a regular basis for at least 4 years and from 21 nondonor controls. A panel of single- and dual-labeled monoclonal antibodies was used to characterize peripheral white cells, and then the cells were analyzed by flow cytometry. Lymphocyte subsets included T (CD3) cells, helper T (CD4) cells, suppressor T (CD8) cells, B (CD19) cells, natural killer (NK) (CD56) cells, and subpopulations of T cells defined by the coexpression of markers for CD3/HLA-DR, CD3/CD56, and CD8/CD11b. Monocyte and neutrophil analysis included quantitation of receptors for C5a, formyl-met-leu-phe, and C3bi (CR3). Monocytes were also analyzed for expression of HLA-DR and CD14 antigens. No significant differences were observed in the whole blood donors and nondonor controls for any of these factors used to assess immunologic status, except for an increase in C3bi receptors on both neutrophils and monocytes from whole blood donors. These findings indicate that the lymphocyte parameters analyzed in this study are unaltered by long-term whole blood donation. Further research is necessary to determine the significance of complement receptor upregulation in whole blood donors and to identify any changes in the functional characteristics of peripheral white cells from whole blood donors. PMID- 1731437 TI - The perioperative use of blood components during heart and heart-lung transplantation. AB - Perioperative blood usage during 172 heart transplants (HTs) and 100 heart-lung transplants (HTLs) performed at the same center during 1986 and 1989 was reviewed. In 1986, 79 HTs were performed with a median (interquartile range) blood component use of 12 (range, 8-20) units, of which 6 (range, 4-8) constituted red cell components. The use of blood components was significantly reduced (p less than 0.05 by the Mann-Whitney U test) in 1989 to 10 (range, 8-20) units, of which red cell components constituted 5 (range, 3-7) units. Fifty HLTs in 1986 used a median (interquartile range) of 25.5 (range, 11.5-53) units of blood components. This use was significantly reduced (p less than 0.05) to a median of 18 (range, 9-29) units in 1989, of which 8 (range, 5-11) were red cell components, 4 (range, 2-8) were units of fresh-frozen plasma, and 5 (range, 0-10) were units of platelets. More blood components were used in patients who had previous cardiothoracic surgery and/or previous transplants; this reached significance in the 1986 HTs (p less than 0.001). Fresh blood was used in only one HT but in 12 (24%) of 1086 HLTs and 4 (8%) of 1989 HLTs. As the indication for fresh blood was excessive bleeding, there was a significantly greater use of blood components in 1989 HLT patients receiving fresh blood than in those not receiving fresh blood (median [range] of 163.5 [62-251] units compared to 15.5 [3 126]).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731438 TI - Blood loss and replacement in total hip arthroplasty: a multicenter study. The Preoperative Autologous Blood Donation Study Group. AB - To determine blood loss, the number of transfusions, and the hemoglobin levels achieved in patients via transfusion in the course of total hip arthroplasty, 324 patient records from 1987 through 1989 were reviewed at three university and three community hospitals. Calculated blood loss was 3.2 +/- 1.3 units in primary procedures and 4.0 +/- 2.1 units in revision procedures (mean +/- SD). Of 777 red cell units transfused, 455 (59%) were autologous units. Transfused patients received 2.0 +/- 1.8 units for primary procedures and 2.9 +/- 2.3 units for revision procedures (mean +/- SD). The maximum number of units given to 95 percent of the transfused patients was 4 for primary procedures and 6 for revision procedures. The mean postoperative hemoglobin level after all transfusions was 103 to 110 g per L, regardless of patient age group of physical status, autologous donor status, or hospital. No difference in length of hospital stay was observed for patients less than 65 years old with hemoglobin concentrations of 80 to 139 g per L at discharge. PMID- 1731439 TI - Transmission of human T-lymphotropic virus type I by blood components from a donor lacking anti-p24: a case report. The Transfusion Safety Study Group. AB - The present criteria for confirmation of human T-lymphotrophic virus types I and II (HTLV-I/II) infection in blood donors are based on seroreactivity to p24 (gag) and gp46 and/or gp61 (env) on Western blot (WB) and radioimmunoprecipitation assays (WB/RIPA). Any single band and other combinations are classified as indeterminate. This case report documents infection in a donor with a repeatedly indeterminate pattern. The blood donor was anti-HTLV-I/II positive on enzyme linked immunoassay, and two sera taken 5 years apart were WB/RIPA-indeterminate (p19 and gp68 only). His donations in the interval were associated with transmission of HTLV-I to four of the six recipients available for study. Other recipients of blood from donors whose WB/RIPA results were indeterminate by present criteria should be examined to determine if additional patterns are at least occasionally associated with transmission. The likelihood that such donors are infected is important to those who are counseling them and making decisions concerning recipients of their bloody. PMID- 1731440 TI - Amphotericin B and platelet transfusion. PMID- 1731441 TI - Intravenous immunoglobulin in combination with other prophylactic and therapeutic measures. PMID- 1731442 TI - False-negative results by polymerase chain reaction due to contamination by glove powder. AB - The polymerase chain reaction (PCR) technique has become an important, widely employed method for the detection and quantitation of the nucleic acid sequences used in the diagnosis and monitoring of genetic and infectious diseases. Much attention has been directed at the problem of false-positive PCR results, which are generally attributed to low-level laboratory contamination of amplified sequences ("carryover"). In contrast, few investigators have commented on the somewhat less frequent, but equally problematic, false-negative PCR results. Investigation of the source of sporadic false-negative PCR reactions found that glove powder, inadvertently introduced into tubes when gloves are changed in an effort to reduce false-positive results, can nonspecifically inhibit each of the major steps in the PCR detection process. Methodologic precautions are recommended to minimize this problem. PMID- 1731443 TI - Enterobacter agglomerans as a contaminant of blood. PMID- 1731444 TI - A proposal to standardize terminology for weak D antigen. PMID- 1731445 TI - The incidence of hemolytic disease of the newborn attributable to anti-Wra. PMID- 1731446 TI - On proper terminology. PMID- 1731447 TI - Human T-lymphotropic virus type I infection among blood donors in the Solomon Islands. PMID- 1731448 TI - Platelet quality after 15-day storage of platelet concentrates prepared from buffy coats and stored in a glucose-free crystalloid medium. AB - It has been reported previously that platelet concentrates (PCs) prepared from buffy coat pools diluted in a simple, glucose-free medium (BC-PCs) are effective in thrombocytopenic patients after 4 to 12 days of storage. Such preparations produce platelet increments similar to those of traditional PCs prepared from platelet-rich plasma (PRP-PCs) stored for 1 to 3 days. The purpose of this study was to obtain a series of in vitro measurements during storage to allow a more detailed characterization of BC-PCs and a more detailed comparison of BC-PCs with PRP-PCs. At the beginning of storage, the level of (alpha granule membrane protein-140 (GMP-140), a marker of platelet activation, was significantly higher on PRP-PC platelets, and BC-PCs were superior in measurements reflecting platelet function, such as osmotic reversal, ATP release, and aggregation with collagen. Compared to PRP-PCs, BC-PCs were superior in the percentage of discs, total ATP, and glycoprotein lb expression by Day 7. This superiority became more striking on Day 11. Overall, 15-day-old BC-PCs compared favorably to 7-day old PRP-PCs: BC PCs were superior in ATP release and aggregation with collagen, but they were not significantly different for all other measurements reflecting platelet quality and function. Thus, the quality of platelets in BC-PCs was superior on Day 1, and this superiority progressed as storage continued. In addition, the metabolism of BC-PCs was favorable.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731449 TI - Long-term survival of emergency department patients with acute chest pain. AB - To evaluate the long-term prognosis of patients with acute chest pain, prospective clinical data and long-term follow-up data (mean 30.1 +/- 9.4 months) were collected for 1,956 patients who presented to the emergency department of an urban teaching hospital with this chief complaint. During follow-up of the 1,915 patients who were discharged alive from the emergency department or hospital, there were 113 (6%) cardiovascular deaths. No differences were detected in the post-discharge cardiovascular survival rates after 3 years of experience with patients who were discharged from the emergency department with a known prior diagnosis of angina or myocardial infarction (89%) and patients who had been admitted and found to have acute myocardial infarction (85%), angina (87%), or other cardiovascular diagnoses (87%). Patients who were discharged from either the hospital or the emergency department without cardiovascular diagnoses had an excellent prognosis. Multivariate Cox regression analysis identified 5 independent correlates of cardiovascular mortality after discharge: age, prior history of coronary disease, ischemic changes on the emergency department electrocardiogram, congestive heart failure and cardiogenic shock. These findings indicate that the postdischarge cardiovascular mortality of patients with chest pain who are discharged from the emergency department with a known history of coronary disease is similar to that of admitted patients with angina or myocardial infarction. These data suggest that the same types of prognostic evaluation strategies that have been developed for admitted patients with ischemic heart disease should also be considered when such patients present to the emergency department but are not admitted. PMID- 1731450 TI - Holter recording of ventricular arrhythmias during intravenous thrombolysis for acute myocardial infarction. AB - Ventricular arrhythmias during thrombolysis for acute myocardial infarction and their relation to coronary artery patency were examined. Twenty-four-hour Holter monitoring was begun 3.1 +/- 0.2 hours after onset of pain in 40 patients (age 54 +/- 1.6 years; anterior infarction 42.5%) treated with streptokinase (42.5%) or recombinant tissue-type plasminogen activator (57.5%) (delay from pain 3.3 +/- 0.2 hours). A Marquette 8000 computer was used for Holter analysis. The infarct related artery was considered as patent (72.5%) or non-patent (27.5%) according to coronary angiography (delay from pain 26.7 +/- 2.5 hours; 60% less than 24 hours). Ventricular arrhythmias were present in all patients. Tolerance was good (1 cardioversion for ventricular fibrillation). The incidence of accelerated idioventricular rhythm was not different between patients with a patent and nonpatent artery (90 vs 82%), nor for ventricular tachycardia (VT) (83 vs 73%). Coronary artery patency was associated with a 14-, 13- and 32-fold increase of ventricular premature complexes, VT and accelerated idioventricular rhythms, respectively. The increased incidence of sustained VT (patent 38%; nonpatent 0%; p less than 0.05) and early (before the first 6 hours) accelerated idioventricular rhythm (patent 76%; nonpatent 18%; p less than 0.01) associated with artery patency suggests that these arrhythmias may be noninvasive diagnostic criteria for reperfusion (sensitivity 38 vs 76%, and specificity 100 vs 82%). A positive correlation was found between the frequency of ventricular premature complexes and VT, and peak creatine kinase. PMID- 1731451 TI - Psychosocial adjustment of patients arriving early at the emergency department after acute myocardial infarction. AB - The psychosocial functioning of patients arriving at the emergency department with an acute myocardial infarction early enough to be candidates for treatment with thrombolytic agents was compared with that of those arriving later. Patients who arrived within 3 hours were significantly more anxious when assessed 1 week after admission and had a consistently worse pattern of psychosocial adjustment 3 months after hospital discharge than did those who arrived later. The implications of these findings for efforts to improve early arrival at the emergency department, as well as for medical and psychosocial outcomes after acute myocardial infarction, were considered. PMID- 1731452 TI - Effect of timolol on cardiopulmonary exercise performance in men after myocardial infarction. AB - The effect of the nonselective beta blocker timolol on maximal cardiopulmonary exercise performance was evaluated in 28 men with previous myocardial infarction without effort angina (mean age 63 +/- 8 years). Patients were randomized to placebo or timolol (10 mg twice daily) for 4 weeks and then crossed over to the alternative therapy in a double-blind manner. At the completion of each treatment period, patients underwent symptom-limited maximal cardiopulmonary exercise on a cycle ergometer. Exercise time, heart rate, oxygen consumption (VO2), oxygen (O2) pulse and respiratory exchange ratio were measured at peak exercise and at a submaximal exercise level defined at a respiratory exchange ratio of 1.00. Timolol treatment reduced peak heart rate from 153 +/- 11 to 102 +/- 14 beats/min (-33%, p less than 0.001). Exercise time decreased from 680 +/- 91 to 633 +/- 78 seconds (-7%, p less than 0.001). Peak VO2 decreased from 25.3 +/- 4.7 to 21.4 +/ 3.5 ml/min/kg (-15%, p less than 0.001). O2 pulse increased from 12.9 +/- 1.9 to 16.7 +/- 2.3 ml/beat (+29%, p less than 0.001). Peak respiratory exchange ratio did not change significantly, indicating comparable effort. At submaximal exercise, defined at a respiratory exchange ratio of 1.00, there was no difference in exercise time between placebo and timolol. Heart rate decreased with timolol compared with placebo, from 126 +/- 16 beats/min by 31% (p less than 0.001), VO2 decreased from 18.5 +/- 4.3 ml/min/kg by 10% (p less than 0.001), O2 pulse increased from 11.5 +/- 2.0 ml/beat by 30% (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731453 TI - Effects of acute K-strophantidin administration on left ventricular relaxation and filling phase in coronary artery disease. AB - In 10 patients with coronary artery disease, preserved left ventricular (LV) performance and absence of previous myocardial infarction, the effects of an acute intravenous administration of k-strophantidin (0.005 mg/kg over 10 minutes) on selected parameters of both LV systolic and diastolic function, including relaxation, were evaluated. An increase in positive first derivative of LV pressure (dP/dt) and in the ratio between dP/dt and the pressure developed (dP/dt/P) (1,530 +/- 287) 1,600 +/- 329 mm Hg/s [p less than 0.05], and 30 +/- 6 to 34 +/- 8 s-1 [p less than 0.05], respectively) demonstrated the inotropic effect of k-strophantidin, whereas volumetric parameters of systolic function (end-systolic and stroke volume indexes, and ejection fraction) did not show any significant change. However, LV relaxation was impaired by k-strophantidin injection; in fact, mean values of T constant were significantly increased from 50 +/- 12 to 55 +/- 13 ms (p less than 0.01). Lowest LV and end-diastolic pressures increased from 8 +/- 4 to 11 +/- 4 mm Hg (p less than 0.05) and from 17 +/- 6 to 20 +/- 8 mm Hg (p less than 0.05), respectively. The end-diastolic volume and maximal rate of volumetric increase during the early and late filling phases were not modified by k-strophantidin. Mean aortic pressure increased from 110 +/- 10 to 120 +/- 12 mm Hg (p less than 0.001). Therefore, in patients with coronary artery disease and LV preserved performance, an acute intravenous administration of k-strophantidin appears to stimulate contractility and to worsen relaxation, and minimal LV and end-diastolic pressures. PMID- 1731454 TI - Tissue plasminogen activator using a rapid-infusion low-dose regimen for unstable angina. AB - The clinical effect of a low-dose rapid-infusion intravenous regimen was assessed using human recombinant tissue-type plasminogen activator (rt-PA) in unstable angina. Fifty patients with unstable angina pectoris were randomly assigned to blinded treatment with either placebo (24 patients) or low-dose (20 mg bolus, 30 mg infusion over 1 hour) intravenous rt-PA (26 patients). Before randomization, all patients were treated with aspirin, twice-daily subcutaneous heparin, and maximally tolerated antianginal therapy. Of the 50 patients assigned, 26 received rt-PA and the outcome was successful in 15 (58%) (angina settled, no myocardial infarction or urgent intervention) compared with 9 (38%) successful outcomes in the 24 who received placebo (0.5 greater than p greater than 0.1). Angina remained refractory in 8 (31%) of the rt-PA group and in 13 (54%) of the placebo group (0.1 greater than p greater than 0.05). Urgent interventions were required in 6 patients (23%) who received rt-PA and in 11 patients (46%) who received placebo. Three patients in each group sustained a myocardial infarction within 72 hours of entering the trial and there were 3 deaths (1 in the active treatment group, 2 in placebo group) within 2 weeks of the trial (p = not significant). Administration of intravenous rt-PA was not associated with any complications. Low-dose rt-PA administration in patients with unstable angina was associated with a tendency to stabilization of anginal symptoms and a reduction in the need for urgent intervention. However, these trends did not achieve statistical significance. PMID- 1731455 TI - Effects of estrogen replacement therapy on serum lipid values and angiographically defined coronary artery disease in postmenopausal women. AB - To examine the effects of estrogen replacement on lipids and angiographically defined coronary artery disease (CAD) in postmenopausal women, lipid profiles were obtained in 90 consecutive postmenopausal women undergoing diagnostic coronary angiography. Eighteen women (20%) were receiving estrogen and 72 (80%) were not. CAD (defined as greater than or equal to 25% luminal diameter narrowing in a major coronary artery) was present in only 22% of women (4 of 18) receiving estrogen and in 68% (49 of 72) who were not (p less than 0.001), with an odds ratio of 0.13. Mean high-density lipoprotein (HDL) cholesterol level was significantly higher (63 +/- 6 vs 48 +/- 2; p less than 0.01) and mean total/HDL cholesterol ratio significantly lower in women receiving estrogen than in those who were not (4.2 +/- 0.5 vs 5.1 +/- 0.2; p less than 0.05). The other lipid values were similar in both groups. On multiple logistic regression analysis, absence of estrogen use was the most powerful independent predictor of the presence of CAD (p less than 0.001), with total/HDL cholesterol ratio as the only other variable selected (p less than 0.01). Thus, among 90 consecutive postmenopausal women undergoing diagnostic coronary angiography, estrogen replacement therapy was associated with an 87% reduction in the prevalence of CAD, and those receiving estrogen had a significantly higher mean HDL cholesterol level and lower mean total/HDL cholesterol ratio. PMID- 1731456 TI - Patterns of referral and recovery in women and men undergoing coronary artery bypass grafting. AB - This study compares women and men undergoing coronary artery bypass grafting. Factors before and after coronary surgery were examined to identify variables related to mortality and morbidity. The study population included 465 women and 465 men matched for age (mean age 64.2 years) who underwent first time isolated coronary artery bypass grafting between 1983 and 1988. There were higher incidences of systemic hypertension, diabetes mellitus, postmyocardial infarction angina, thyroid gland disease, arthritis (p less than 0.001 for all), acute myocardial infarction (p = 0.03), congestive heart failure (p = 0.03), and emergency surgery (p = 0.02) in women, whereas more men had peptic ulcer disease (p less than 0.001). The in-hospital death rate was not significantly different (women 4.3% vs men 3.7%). For all subjects, emergency surgery (p less than 0.001), significant left main narrowing (p less than 0.05) and renal disease (p less than 0.001) were related to death, whereas history of myocardial infarction (p less than 0.05) and diabetes (p less than 0.05) were related to death only in men. Age and body surface area were not related to death. After surgery men had a higher incidence of atrial arrhythmia (p less than 0.001), and women had a higher incidence of congestive heart failure (p less than 0.001). Although women did not have a higher mortality rate, the data suggest that women and men do not share all the same predictors of mortality after surgery. PMID- 1731458 TI - Effectiveness of decremental diameter balloon catheters (tapered balloon). AB - Natural tapering of coronary arteries from larger proximal to smaller distal diameters often creates a dilemma for optimal balloon sizing during percutaneous transluminal coronary angioplasty (PTCA). To demonstrate the need for new dilating catheters suitable for tapered coronary anatomy, 100 consecutive coronary arteries were measured by videodensitometry, 1 cm proximal and distal to the stenosis. In 23 arteries there was a 1 mm or greater taper and 19 arteries showed a 0.5 to 0.99 mm taper. Only 50 arteries showed a nearly uniform diameter at the site of the stenosis, and 8 arteries demonstrated reverse taper, i.e., distal was greater than proximal diameter. To avoid balloon size mismatch with significant tapering, decremental diameter balloon catheters were developed. Series I tapers from 3.5 to 3.0 mm and series II from 3.0 to 2.5 mm over a balloon length of 25 mm. Tapered balloons were used in 80 patients with 94 tapered coronary arteries. Before PTCA, proximal, stenotic and distal mean diameters measured 3.6, 1.1 and 2.6 mm, respectively; after PTCA, proximal, stenotic and distal diameters measured 3.6, 2.8 and 2.5 mm, respectively, thus maintaining the natural tapering after effective dilatation. Only 2 arteries (2.1%) showed significant dissection, with no abrupt occlusions, and none requiring bypass surgery. In summary, decremental diameter balloon catheters provide optimal dilation in tapered arterial segments with few complications and offer a new approach to balloon sizing. PMID- 1731457 TI - Comparison between thallium-201 and technetium-99m methoxyisobutyl isonitrile defect size in single-photon emission computed tomography at rest, exercise and redistribution in coronary artery disease. AB - Defect size on myocardial tomograms was measured in 30 patients who underwent 2 separate studies, 1 with thallium-201 (TI-201), the other with technetium-99m methoxyisobutyl isonitrile (MIBI). A group of 15 patients with myocardial infarction was studied at rest and received both tracers on the same day. The other 15 patients had documented coronary artery disease. They were were given injections of TI-201 at peak exercise and underwent imaging immediately after exercise and again 4 hours later. They then received a dose of MIBI for imaging at rest. A week later they underwent a second exercise test with the same work load and received a second dose of MIBI. Defect size on single-photon emission computed tomographic images was measured and repeated twice. Results were expressed in percentage of the volume of the whole myocardium. Reproducibility of the defect size measurement was high for TI-201 (r = 0.978; SEE = 1.59) as well as for MIBI (r = 0.981; SEE = 0.80). In patients with coronary artery disease the mean size of the defects was significantly larger with TI-201 than with MIBI at exercise (6.7 +/- 5.2 vs 4.6 +/- 5.2%, respectively, p less than 0.05) and at redistribution (5.1 +/- 4.4 vs 2.8 +/- 3.2%, respectively, p less than 0.05), where no difference was seen in patients with myocardial infarction studied only at rest (11.2 +/- 10.4 vs 12.0 +/- 11.5%, respectively, p = not significant). Smaller MIBI defect sizes, when compared with TI-201, in the exercise and redistribution studies were not due to technical artefacts since there was no difference when they were compared at rest. PMID- 1731459 TI - Postangioplasty restenosis rate between segments of the major coronary arteries. AB - Conflicting data have been published regarding the rate of postangioplasty restenosis observed in diverse segments of the coronary tree. However, these studies may be criticized for their biased selection of patients, methods of analysis, and definitions of restenosis. In the present study, 1,353 patients underwent a successful coronary dilatation of greater than or equal to 1 site. In all, 1,234 patients (91%) had a follow-up angiogram after 6 months, or earlier when indicated by symptoms. All films were processed and analyzed at the thoraxcenter core laboratory with the coronary angiography analysis system (automated contour detection). Restenosis was considered present if the diameter stenosis at follow-up was greater than 50%. No differences in restenosis rates were observed between coronary segments using this categorical definition. A continuous approach was also used; absolute changes in minimal luminal diameter adjusted for vessel size were used in order to allow comparison between vessels of different sizes (relative loss). No significant differences were observed between the coronary segments with this continuous approach. These results suggest that restenosis is a ubiquitous phenomenon without any predilection for a particular site in the coronary tree. PMID- 1731460 TI - Spectral analysis of heart rate dynamics in elderly persons with postprandial hypotension. AB - Prior studies suggest that postprandial hypotension in elderly persons may be due to defective sympathetic nervous system activation. We examined autonomic control of heart rate (HR) after a meal using spectral analysis of HR data in 13 old (89 +/- 6 years) and 7 young (24 +/- 4 years) subjects. Total spectral power, an index of overall HR variability, was calculated for the frequency band between 0.01 and 0.40 Hz. Relatively low-frequency power, associated with sympathetic nervous system and baroreflex activation, was calculated for the 0.01 to 0.15 Hz band. High-frequency power, representing parasympathetic influences on HR, was calculated for the 0.15 to 0.40 Hz band. Mean arterial blood pressure declined 27 +/- 8 mm Hg by 60 minutes after the meal in elderly subjects, compared with 9 +/- 8 mm Hg in young subjects (p less than or equal to 0.0001, young vs old). The mean change in low-frequency HR power from 30 to 50 minutes after the meal was +19.4 +/- 25.3 U in young subjects versus -0.1 +/- 1.5 U in old subjects (p less than or equal to 0.02). Mean change in total power was also greater in young (19.0 +/- 26.6 U) subjects compared with old subjects (0.0 +/- 1.6 U, p greater than or equal to 0.02). Mean ratio of low:high-frequency power increased 3.1 +/- 3.3 U in young subjects vs 0.5 +/- 2.7 U in old subjects (p less than or equal to 0.01). The increase in low-frequency HR power and in the low:high frequency band ratio in young subjects is consistent with sympathetic activation in the postprandial period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731461 TI - Pharmacology of the class III antiarrhythmic agent sematilide in patients with arrhythmias. AB - Sematilide, a close structural analog of N-acetylprocainamide, prolongs cardiac action potentials in vitro, whereas it does not depress maximum action potential upstroke slope, a "class III" action. This report outlines an evaluation of the clinical pharmacologic actions of sematilide in 14 patients with chronic high frequency nonsustained ventricular arrhythmias. In all, 36 intravenous infusions (range 0.15 to 1.5 mg/kg over 15 minutes) were administered in a dose-ranging, placebo-controlled study design. Sematilide prolonged rate-corrected QT (QTc) in a dose- and concentration-related fashion, did not alter PR or QRS, and slowed heart rate at high concentrations (greater than or equal to 2 micrograms/ml). The relations between dose and total area under the time-concentration curve, dose and peak plasma concentration, and peak plasma concentration and increase in QTc were linear (r = 0.66 to 0.92; p less than 0.001). QTc increases of approximately equal to 25% were seen at plasma concentrations of approximately equal to 2.0 micrograms/ml. The mean elimination half-life (+/- SD) was 3.6 +/- 0.8 hours, and most of a dose (77 +/- 13%) was recovered unchanged in the urine. Plasma concentrations greater than or equal to 0.8 micrograms/ml suppressed arrhythmias (5 patients) or aggravated them (3), including 1 patient who needed cardioversion for an episode of torsades de pointes (2.7 micrograms/ml). Thus, sematilide exerts class III actions in patients. Further studies to evaluate the role of this antiarrhythmic mode of action should be conducted at doses designed to limit QTc increases. PMID- 1731462 TI - Epidemiologic aspects of isolated systolic hypertension and implications for future research. AB - Isolated systolic hypertension represents an important public health issue in the 1990s because of its prevalence in the elderly and its importance as a risk factor for cardiovascular morbidity and mortality. Methodologic differences may account for the wide variation between prevalence rates in studies reported. With the advent of newer methods of blood pressure (BP) assessment, such as noninvasive ambulatory BP monitoring, it may be possible to define more accurately the true population at risk. Recent data from the Systolic Hypertension in the Elderly Program has indicated a clear benefit of treatment with a reduction in total stroke of 36%, and a reduction of 25 and 32% in the combined end points of coronary heart disease and cardiovascular disease, respectively. Further studies are now required to elucidate what treatment regimens are most effective in preferentially reducing both systolic BP, without producing undesirable effects such as diastolic hypotension, and fatal and nonfatal events. One such trial is underway in Europe using a drug regimen different from that in the Systolic Hypertension in the Elderly Program. The cost implications associated with treating the population at risk are potentially large but these are now based on firm scientific evidence. PMID- 1731463 TI - Hemodynamic and metabolic effects of intravenous clentiazem in hypertensive patients. AB - To determine the hemodynamic and certain metabolic effects of clentiazem, a diltiazem congener, 10 untreated essential hypertensive patients were given the calcium antagonist in 3 successive doses totaling 1.0 mg/kg intravenously. Mean arterial pressure and total peripheral resistance progressively declined from 121 +/- 3 mm Hg and 47 +/- 2 U (mean) to 110 +/- 3 mm Hg and 33 +/- 1 U, respectively (p less than 0.05); heart rate remained unchanged. Cardiac output increased as a result of augmented cardiopulmonary volume (p less than 0.05) produced by peripheral venoconstriction and norepinephrine release (from 258 +/- 41 to 319 +/ 42 pg/ml; p less than 0.01). Surprisingly, there was an immediate reduction in plasma aldosterone (10.4 +/- 1.2 to 6.5 +/- 1.0 ng/dl; p less than 0.01), serum potassium (4.3 +/- 0.1 to 3.6 +/- 0.1 mEq/dl; p less than 0.001) and calcium (9.5 +/- 0.1 to 8.8 +/- 0.1 mg/dl; p less than 0.001) concentrations, whereas epinephrine increased (21.2 +/- 3.3 to 45.8 +/- 5.9 pg/ml; p less than 0.002). Previous studies with diltiazem, conducted similarly, did not show these changes. Therefore, clentiazem reduced mean arterial pressure through a decrease in total peripheral resistance, and released epinephrine was associated with intracellular potassium influx (urinary potassium did not change). The inhibited aldosterone release was not compensated by altered renal blood flow, glomerular filtration or increased plasma renin activity. These findings underscore the concept that calcium antagonists are a remarkably heterogeneous antihypertensive group. PMID- 1731464 TI - Left ventricular ejection performance in mitral stenosis, and effects of successful percutaneous transvenous mitral commissurotomy. AB - Impaired left ventricular ejection performance was reported in pure mitral stenosis. The speculative mechanisms included insufficient preload, increased wall stress, high right ventricular pressure and unknown myocardial factors, but no definitive mechanism has been established. Fifteen patients with tight mitral stenosis who underwent successful percutaneous transvenous mitral commissurotomy were studied to ascertain whether ejection performance would improve with sufficient blood filling. The indexes of preload (end-diastolic volume) and ejection performance (stroke volume, ejection fraction, and mean systolic and mean normalized ejection rates) were calculated angiographically before and immediately after mitral commissurotomy. Improved blood filling (the result of successful mitral commissurotomy) produced an increase in end-diastolic volume (mean +/- SD 99.0 +/- 30.2 to 112.1 +/- 30.1 ml/m2; p less than 0.05). All 4 indexes of ejection performance also improved. There was good correlation between end-diastolic and stroke volumes before intervention (stroke volume = 0.476 x end diastolic volume + 16.77; r = 0.76), and the relation between them showed no change even after mitral commissurotomy. It is concluded that both left ventricular preload and ejection performance improved after successful percutaneous transvenous mitral commissurotomy. Insufficient preload could affect ejection performance in patients with tight mitral stenosis. PMID- 1731465 TI - Findings on Doppler echocardiography in asymptomatic intravenous heroin users. AB - To detect potential cardiac abnormalities induced by intravenous heroin use, 68 persons without a previous episode of infective endocarditis were studied by Doppler echocardiography. A control group of 41 normal subjects was studied for comparison. The following measurements were considered: (1) diameter of heart chambers, (2) systolic left ventricular function, (3) morphologic valvular abnormalities, (4) presence of valve regurgitations, (5) Doppler indexes of diastolic function, and (6) estimation of pulmonary arterial resistances. Results showed no significant differences regarding the size of the heart chambers or systolic left ventricular function. A significantly higher incidence of valvular abnormalities (focal thickening or valve prolapse) was found in drug addicts (p = 0.0009) at the mitral and tricuspid valves, as was valvular regurgitation detected by Doppler (p = 0.04). Also, a significantly prolonged deceleration time of mitral and tricuspid early diastolic Doppler flow was found in the study group (p = 0.0001 and 0.027, respectively) although a different hemodynamic condition in the study group (pharmacologically reduced preload) precluded these findings to be attributable to an actual diastolic dysfunction. No differences were observed in pulmonary arterial resistances. It is concluded that mitral and tricuspid valve abnormalities can be detected by echocardiography in asymptomatic intravenous heroin users, whereas no apparent effects are observed in morphologic or functional parameters of cardiac structures other than the valves. PMID- 1731466 TI - Differences in myocardial fluoro-18 2-deoxyglucose uptake in young versus older patients with hypertrophic cardiomyopathy. AB - The purpose of this study was to determine whether regional myocardial glucose use in patients diagnosed as having hypertrophic cardiomyopathy (HC) at a younger age differs from that in those diagnosed at middle to old age. Sixteen patients with HC (group 1 aged less than 40 years (n = 8); group 2 aged greater than 40 (n = 8) were studied using positron emission tomography and fluoro-18 2-deoxyglucose (FDG). All patients were diagnosed as having HC within 6 years of the study. Contiguous regions of interest were selected circumferentially on each cross sectional image of the left ventricular wall. In each region of interest, % FDG fractional uptake was calculated. In each patient, % left ventricular FDG fractional uptake was determined as a mean value of % FDG fractional uptake in each region of interest. Moreover, as a measure of nonhomogeneity, the % interregional coefficient of variation in FDG fractional uptake was calculated in each patient. Whereas % left ventricular FDG fractional uptake did not differ between the 2 groups, the % interregional coefficient of variation in FDG fractional uptake was increased in group 1 compared with that in group 2 (11.5 +/ 3.6 vs 7.4 +/- 1.6%; p less than 0.02). Interventricular septum/left ventricular posterior wall thickness ratio and total counts in cross-sectional image did not differ between the 2 groups. These data suggest that patients diagnosed as having HC at a younger age have more nonhomogeneous myocardial metabolic characteristics than do patients diagnosed at middle or old age, and support the notion that HC in the young may be different from that in the middle-aged or elderly. PMID- 1731467 TI - Cholinergic baroreflex vasodilatation: defect in heart transplant recipients due to denervation of the ventricular baroreceptor. AB - Afferent denervation of the ventricular baroreceptor may impair reflex vasodilatation after heart transplantation. This may alter the regulation of blood pressure and contribute to arterial hypertension. A baroreceptor-loading procedure was performed in 23 heart transplant recipients with cyclosporine A immunosuppression and 11 control subjects using a continuous infusion of increasing doses of angiotensin II (15 and 30 ng/kg.min). After m-cholinoceptor blockade the procedure was repeated in order to study the contribution of cholinergic effects on vasodilatation in humans. Instantaneous vascular resistance was calculated as the ratio of mean blood pressure to stroke volume as evaluated by echocardiography. When heart transplant recipients were compared with control subjects, infusion of 30 ng/kg.min of angiotensin II resulted in an increase in mean blood pressure of 43 +/- 20 vs 31 +/- 13 mm Hg (p less than 0.05) and an increase in instantaneous resistance of 1.21 +/- 0.61 vs 0.65 +/- 0.38 mm Hg/ml (p less than 0.01), respectively. M-cholinoceptor blockade with atropine (0.015 mg/kg) did not produce any change in blood pressure or resistance response to angiotensin II in heart transplant recipients. However, m cholinoceptor blockade resulted in a significantly increased blood pressure and resistance response to angiotensin II in control subjects, which was similar to the response in heart transplant recipients to angiotensin II alone: The increase in mean blood pressure during administration of 30 ng/kg.min angiotensin II amounted to 47 +/- 11 mm Hg, and the increase in instantaneous resistance to 1.13 +/- 0.48 mm Hg/ml (for both, p less than 0.001 vs control subjects without atropine; p = not significant vs heart transplant recipients).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731468 TI - Evaluation of changes in standard electrocardiographic QRS waveforms recorded from activity-compatible proximal limb lead positions. AB - Proximal limb lead positions are currently used for activity-compatible electrocardiographic monitoring of myocardial ischemia. Two previously described systems for alternate limb lead placement were studied in patients with and without QRS evidence of healed anterior or inferior myocardial infarction. An innovative method was used to simultaneously record 6 standard and 6 modified limb leads, and 3 standard and 3 modified precordial leads on a standard digital electrocardiograph. Both alternate lead placement systems showed rightward frontal plane axis shift and diminished Q-wave durations in lead aVF compared with those of their simultaneous standard controls. Furthermore, potential differences between the standard distal limb lead sites and 5 more proximal sites were explored along each limb. Differences along the left arm were accentuated relative to those along the right arm owing to differences in proximity of the arms to the myocardium. Along the lower limb, and anterior site showed less deviation from standard than did a more lateral site. It is imperative that recordings from alternate sites be labeled accordingly so that their output cannot be confused with that obtained from standard sites. PMID- 1731469 TI - Rapidity and duration of platelet suppression by enteric-coated aspirin in healthy young men. AB - The recent demonstration of aspirin's ability to prevent and reduce the severity of myocardial infarction has led to a marked increase in its use and to a need for information regarding the time-course of onset and offset of its antiplatelet effect. A study of healthy men was conducted to determine (1) the rapidity of onset of inhibition of platelet aggregation in response to adenosine diphosphate, and thromboxane A2 production after chewed enteric-coated aspirin (325 mg, n = 10); and (2) the duration of platelet inhibition after cessation of enteric coated aspirin (325 mg) every other day for 14 days (n = 10). When chewed, enteric-coated aspirin greatly inhibited platelet aggregation response to adenosine diphosphate and thromboxane A2 production within 15 minutes. Complete recovery of platelet aggregation occurred in half of the subjects by day 3, and in 80% of the subjects by day 4; the platelet response was not affected by exercise. This study demonstrates a rapid onset of aspirin's antiplatelet effect and provides information relevant for optimal timing of initiation of aspirin for acute conditions such as myocardial infarction and unstable angina, and cessation of aspirin before surgery. PMID- 1731470 TI - Late, out-of-laboratory, abrupt closure after angiographically successful directional coronary atherectomy. PMID- 1731471 TI - Acute myocardial infarction and day of the week. PMID- 1731472 TI - Effectiveness of indapamide versus enalapril as second-step therapy of systemic hypertension. PMID- 1731473 TI - Efficacy of once-daily felodipine monotherapy in systemic hypertension. PMID- 1731474 TI - Transesophageal echocardiography for study of bioprostheses in the aortic valve position. PMID- 1731475 TI - Localization of mitral periprosthetic leaks by transesophageal echocardiography. PMID- 1731476 TI - Transesophageal echocardiographic detection of atrial septal defect in adults. PMID- 1731477 TI - Location, localization and surgical treatment of cardiac pheochromocytoma. PMID- 1731478 TI - Decreased heart rate variability in congestive heart failure. PMID- 1731479 TI - Comment on the implantable defibrillator backlash. PMID- 1731480 TI - TMJ research: responsibility and risk. PMID- 1731481 TI - Orthodontic risk factors for temporomandibular disorders (TMD). I: Premolar extractions. AB - Concern about claims that premolar extractions may put patients at risk for temporomandibular disorders (TMD) led to this study. We report first findings from a longitudinal study of orthodontic patients begun in 1983. By using the methods of Helkimo, we collected TMD data before initiation of orthodontic treatment, between 0 and 12 months after debanding, and 12 to 24 months after debanding. Analyses related Helkimo scores with premolar extractions in 65 patients for whom orthodontic treatment had been completed. Twenty-six patients were treated without premolar extractions, 25 had four premolars extracted, and 14 had two upper premolars extracted. Tests for significance of differences between mean Helkimo scores were conducted for the nonextraction group compared with the extraction groups, and between pretreatment and posttreatment Helkimo scores for each group. Results included: (1) no significant intergroup differences between mean pretreatment or posttreatment scores, and (2) small but statistically significant (p less than 0.05) differences (in the direction of improvement) between mean pretreatment and posttreatment scores for both the nonextraction group and for the four premolar extraction group. PMID- 1731482 TI - Orthodontics as a risk factor for temporomandibular disorders (TMD). II. AB - Debate about orthodontic treatment as a risk factor for temporomandibular disorders (TMD) led to this study. This report, the second in a series, concerns findings from a longitudinal study in which 30 new orthodontic patients have been enrolled annually since 1983. The method of Helkimo was used to collect TMD data before initiation of orthodontic treatment, and at annual intervals after debanding. Treatment was by fixed edgewise appliances. Data from a pretreatment and at least one posttreatment Helkimo examination were available for 109 patients. Follow-up data were available for 92 patients in the first year after debanding, with the corresponding sample sizes declining to 56, 33, 19, 11, and 7 for the second through the sixth posttreatment years, respectively. Primary analyses involved comparison of mean scores from the Helkimo 25-point dysfunction index scale. There were no significant differences between mean pretreatment and posttreatment Helkimo scores for any of the various groupings except for small, clinically unimportant improvements seen in the 12 to 24 month subgroup of 55 patients and in the 48 to 60 month subgroup of 11 patients. With average follow up time of about 2 years for the 109 patients, 90% had Helkimo scores that stayed the same or improved, and 10% had scores that increased or worsened from 2 to 5 Helkimo points. We conclude that the orthodontic treatment experienced by our sample was not an important etiologic factor for TMD. PMID- 1731483 TI - Comment on legal aspects article. PMID- 1731484 TI - Craniomandibular disorders with special reference to orthodontic treatment: an evaluation from childhood to adulthood. AB - The purpose of the present study was to reexamine a group of children and adolescents with respect to signs and symptoms of craniomandibular disorders (CMD) and to evaluate whether any differences could be found between persons who had received orthodontic treatment earlier and those who had not. A total of 402 children in three age groups (7, 11, and 15 years) had participated in a cross sectional study on the relationship between malocclusion and signs and symptoms of CMD. Ten years later they were asked to answer a questionnaire. In the youngest age groups (now 17 and 21 years old) 190 (76%) subjects answered the questionnaire. In the oldest age group (now 25 years old) completed questionnaires were received from 103 (84%) subjects, and 83 (62%) of those subjects appeared for a clinical examination. Subjects with a history of orthodontic treatment had a lower prevalence of subjective symptoms of CMD (TMJ sounds included) than those without any experience of orthodontics. Although the differences were small, it was more evident for the oldest age group. The clinical examination has shown that persons who had undergone orthodontic treatment had a significantly lower clinical dysfunction index than those who had not. PMID- 1731485 TI - Longitudinal study of signs of temporomandibular disorders (TMD) in orthodontically treated and nontreated groups. AB - This study measured the prevalence and incidence of signs of temporomandibular (TM) disorders in both a group undergoing orthodontic treatment in the University of Florida graduate orthodontic program and a control group. A questionnaire pertaining to the patients' reports of signs and symptoms of TM disorder and a clinical examination were administered by a trained dental examiner. Data collection sessions occurred at baseline (before treatment) and at 12-month intervals to 24 months. Data were also collected for the control group at the same time intervals. There were 102 patients (43 boys, 59 girls) mean age 15.3 years. An untreated control group of 41 nonorthodontically treated subjects mean age 16.2 years was used. The incidence of TM signs for the treatment group and control group were not significantly different. Preliminary results are in agreement with the contention that orthodontically treated patients are not more likely to develop TM signs while undergoing treatment. Results underscore the changing, inconstant, and ephemeral nature of TM signs in many persons over the course of time. PMID- 1731486 TI - The effect of maxillary first premolar extraction and incisor retraction on mandibular position: testing the central dogma of "functional orthodontics". AB - It has been argued by a vocal coterie of disaffected dentists that premolar extraction, incisor retraction, and "backward-pulling" mechanics conspire to "distalize" the condyles and, pari passu, to produce craniomandibular dysfunction. Given the gravity of this conjecture, it seemed appropriate to test the predictions it generates in a sample of patients of the type most often said to be at risk: 42 "edgewise" patients with Class II, Division 1 malocclusions, treated in conjunction with the extraction of two maxillary first premolars. Regional and anterior cranial-base cephalometric superimpositions were used to quantify the individual components of the molar and overjet corrections, to measure both at the chin and condyles the mandibular displacement seen during treatment, and to examine the extent to which this displacement is related to the correction of maxillary incisor protrusion. Although the present patients underwent marked upper incisor retraction (on average, about 5 mm), lip retraction was much less pronounced, and 70% of the sample showed a net forward displacement of mandibular basal bone. Significantly, changes in condylar position were not correlated with incisor retraction, as the "functional orthodontists" would have it, but rather with the changes in the buccal occlusion and the growth of the maxilla. Thus, 30% of the patients who showed evidence of distal displacement were generally nongrowing patients who underwent more than average anchorage loss in the mandible and less than average loss in the maxilla.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731487 TI - Prevalence of temporomandibular joint internal derangement in patients with craniomandibular disorders. AB - To determine the prevalence of temporomandibular joint internal derangement in patients with signs and symptoms of craniomandibular disorders, bilateral imaging was performed in a consecutive series of 115 patients with signs and symptoms of craniomandibular disorders. Ninety patients (78%) had different stages of unilateral or bilateral internal derangement, and 25 patients (22%) had normal temporomandibular joints bilaterally. Out of 230 joints, 60 showed disk displacement with reduction, 8 showed disk displacement without reduction, and 29 showed disk displacement without reduction associated with anthrosis. The study indicates that almost 80% of patients with signs and symptoms of craniomandibular disorders have different forms of internal derangement. PMID- 1731488 TI - Relationship between orthodontic treatment, condylar position, and internal derangement in the temporomandibular joint. AB - The purpose of this study was to test the hypothesis that retraction of maxillary front teeth may lock the mandible in a posterior position, and to evaluate any relationship between condylar position and signs and symptoms of internal derangements in the temporomandibular joint. A total of 29 female patients treated for Angle Class II, Division 1 malocclusion with extraction of maxillary first premolars and 34 female patients treated for Angle Class I malocclusion without tooth extraction consented to participate in a radiographic and clinical follow-up examination. The mean ages of the patients were 16.9 (SD 3.0) and 16.6 (SD 2.6) years, and the mean times after treatment were 1.6 (SD 1.0) and 1.5 (SD 0.9) years, respectively. Condylar position was measured in percent anterior and posterior displacement from absolute concentricity on lateral, central, and medial tomographic sections of each joint. Mean condylar position was more posterior at right central (P less than 0.05) and medial (P less than 0.01) tomographic sections in patients treated with extraction. The difference was due to a higher frequency of anteriorly positioned condyles in the nonextraction cases. No intergroup differences in the sagittal occlusal slide from CR to CO and the number of patients with clicking were found. However, the condyles were located more posteriorly in all tomographic sections (P less than 0.05 for lateral, P less than 0.001 for central and medial) in patients with clicking than in those without. PMID- 1731489 TI - A comparison of clinical examination, history, and magnetic resonance imaging for identifying orthodontic patients with temporomandibular joint disorders. AB - The temporomandibular joint (TMJ) status of 51 juvenile orthodontic patients was assessed with magnetic resonance imaging (MRI), clinical examination, and questionnaire data. The results of this study demonstrated that the prevalence of anterior displacement of the meniscus was 11.8% (6 of 51) as assessed by MRI. Clicking or pain in the TMJ area was found in 9.8% (5 of 51) of the subjects by clinical exam, and 19.8% (10 of 51) of the subjects had a history of pain or clicking of the TMJ. Three subjects had a positive MRI and a negative history and clinical examination. However, all subjects with positive MRI findings had a history of other risk factors known to be associated with TMJ internal derangement (TMJ-ID). Therefore practitioners should use a history form and a clinical examination technique that includes a broad range of signs and symptoms of temporomandibular disorders (TMD) to identify patients who may have abnormal condyle disk relationships and be at risk for TMD. Clicking and pain in the TMJ helped identify only one half of the patients with abnormal condyle-disk relationships in this study population. Future cephalometric studies will monitor the effects of abnormal condyle disk relationships on facial growth during orthodontic treatment. PMID- 1731490 TI - Temporomandibular joint sounds: correlation to joint structure in fresh autopsy specimens. AB - In an attempt to better understand the cause of different types of temporomandibular joint (TMJ) sounds, we recorded joint sounds from 27 fresh autopsy specimens, displayed the time frequency distribution of the sound as a three-dimensional graph, and correlated the sound character to morphologic observations at subsequent dissection. Eleven joints elicited sounds, and 16 joints were silent. All joints with sounds had different degrees of intraarticular changes. These ranged from disk displacement with reduction to displacement without reduction and arthrosis of the articular surfaces. Reciprocal clicking occurred both in joints with disk displacement with and without reduction, as well as in joints with arthrotic changes. Crepitation only occurred in joints with arthrosis and perforation. The sample was too small to demonstrate any statistically significant association between the joint sound classified as clicking or crepitation and joint structure types of joint pathosis in this small sample. A high frequency component to the sound appeared to be associated with arthrosis of the articular surfaces. It was concluded that joint sounds indicate joint abnormality but that the absence of joint sound does not exclude intraarticular pathosis. PMID- 1731491 TI - Temporomandibular joint sounds and condyle/disk relations on magnetic resonance images. AB - This study compared the condyle/disk relationships on magnetic resonance images (MRIs) in a group of subjects with completely silent temporomandibular joints (TMJ) when tested clinically with those in subjects with readily discernible TMJ sounds. The sounds were recorded with an accelerometer as the transducer. Selected degrees of jaw separation were electronically determined and recorded with interocclusal wafers for use with the imaging process. Of the "silent joints" 89% were found to have sounds when tested with the accelerometer. These "subclinical" sounds tended to be of shorter duration and occurred at a greater degree of vertical opening than the clinically discernable sounds. The MRIs of the group with clinically discernable sounds tended to show a change in the relationship between the head of the condyle and the intermediate zone of the disk, at the degree of jaw separation of the sound occurrence, whereas no condyle/disk change occurred in the group with "clinically silent joints." It is likely that all joints create sound during function. The different characteristics of the subclinical sounds versus the clinical sounds may indicate differing sound origins. PMID- 1731492 TI - The risk of orthodontic treatment for producing temporomandibular mandibular disorders: a literature overview. PMID- 1731493 TI - Orthodontic treatment and temporomandibular joint disorders. AB - The overall objective of this project was to study the relationship between orthodontic treatment and temporomandibular joint (TMJ) disorders. This relationship has been and remains an important and complex issue in orthodontics. The objectives of the study were to determine the incidence of TMJ pain and dysfunction in a group of orthodontic patients who were symptom-free on entering treatment, and to assess and characterize the level of pain and dysfunction in patients with symptoms, and track changes in these parameters during the course of orthodontic treatment. Standardized functional indices and physical measurements were used to describe and assess TMJ pain and dysfunction. The results of this study showed that of 451 patients without symptoms undergoing treatment at our university clinic during the 18-month project, no patient developed signs and symptoms of TMJ disorders during that time. In addition, for the 11 patients who presented with signs and symptoms of TMJ disorders at the time of their entry into the treatment program, no clear or consistent changes in levels of pain and dysfunction occurred longitudinally during the treatment period followed in this study. On the basis of these findings, a relationship between either the onset of TMJ pain and dysfunction and the course of orthodontic treatment or the change in TMJ pain and dysfunction and the course of orthodontic treatment could not be established in this particular patient population. PMID- 1731494 TI - An analytic method for evaluating condylar position in the TMJ and its application to orthodontic patients with painful clicking. AB - The purpose of this study was to develop a new method for evaluating the three dimensional position of the mandibular condyle relative to the glenoid fossa and further to investigate its clinical application to orthodontic patients with temporomandibular disorders (TMD). A three-dimensional configuration of the temporomandibular joint was constructed by 108 triangles for the condyle and 180 triangles for the glenoid fossa. The shortest distance between the condyle and glenoid fossa (CGFD) was calculated in the model along a line perpendicular to the center of gravity of a triangle on the condyle. The CGFD was determined in the anterior, posterior, middle, lateral, and medial areas on the condyle. Preliminary investigation revealed that the present technique is accurate, regardless of condylar rotation and/or inclination to the tomographic table. The technique was applied to the diagnosis of orthodontic patients with painful clicking and TMD. It is shown that the present approach provides a method for evaluating the positional relationship between the mandibular condyle and glenoid fossa in patients with TMD. PMID- 1731495 TI - Orthodontic TMJ litigation in the 1990s: an ounce of prevention is worth a pound of cure. PMID- 1731496 TI - Age as a predictor of outcome: what role does it play? PMID- 1731497 TI - Spinal cord syphilis associated with human immunodeficiency virus infection: a treatable myelopathy. AB - A 33-year-old woman, seropositive for human immunodeficiency virus type 1 (HIV 1), presented with progressive weakness and numbness of the lower extremities, gait difficulties, and urinary frequency. Physical examination revealed bilateral lower extremity weakness, a left-sided Babinski reflex, and a thoracic sensory level to pinprick at T8. Serum rapid plasma reagin was 1:64, and fluorescent treponemal antibody-absorption (FTA-ABS) was 4+. Examination of the cerebrospinal fluid showed a mononuclear pleocytosis and reactive FTA-ABS. The myelopathy responded promptly to high-dose intravenous aqueous penicillin. Syphilis needs to be considered in the differential diagnosis of any patient who develops a myelopathy in association with HIV-1 infection. Because of the diverse nature in which syphilis may affect the spinal cord, treatment with intravenous aqueous penicillin, 12 to 24 million units daily, for a minimum of 10 days, should be considered in any HIV-1-seropositive patient with a progressive, unexplained myelopathy and positive serologic studies for syphilis. PMID- 1731498 TI - Necrotizing cellulitis caused by Legionella micdadei. AB - Legionella micdadei is primarily considered a pathogen of the pulmonary tract of immunocompromised patients, the majority of whom have been renal transplant recipients. We report the case of a necrotizing soft tissue infection in a cadaveric renal transplant recipient resulting in amputation of the left arm. Only one other extrathoracic bacteriologically documented L. micdadei infection has been reported in the literature. PMID- 1731499 TI - Drug fever induced by heparin. PMID- 1731500 TI - Response of pancreatic carcinoma to 5-fluorouracil and leucovorin. PMID- 1731501 TI - Severe recurrent lupus laryngitis. PMID- 1731502 TI - An IgG inhibitor against coagulation factor XIII: resolution of bleeding after plasma immunoadsorption with staphylococcal protein A. PMID- 1731503 TI - Polyagglutination: a rare mechanism for intravascular hemolysis. PMID- 1731504 TI - Methadone and immune function. PMID- 1731505 TI - Occupational exposure to HIV. PMID- 1731506 TI - One measurement of serum total cholesterol is enough to predict future levels in healthy postmenopausal women. AB - PURPOSE: The purpose of this study was to investigate how well a single or double measurement of serum total cholesterol represents the spontaneous, future level in a particular person. SUBJECTS AND METHODS: The spontaneous fluctuations in serum total cholesterol levels in 169 healthy early postmenopausal women were followed during the course of 12 years. Serum total cholesterol was measured enzymatically. RESULTS: The initial measurement and the long-term level of serum total cholesterol were highly related (p less than 0.0001). The long-term level (calculated for each woman as the area under the curve of serum total cholesterol versus time) was not statistically significantly different from the initial level (mean difference: 0.036 +/- 0.046 mmol/L [mean +/- SEM], NS). The initial serum total cholesterol level was then used to classify each woman into a high or a low cholesterol group, according to the current recommendations. The predictive value of an initial total cholesterol value in the high level (greater than or equal to 6.2 mmol/L) group was 84%, when compared with the long-term level. The predictive value of an initial total cholesterol level below 6.2 mmol/L was 80%. No improvement in these parameters was found when the average of the initial two (or three, when the difference exceeded 0.9 mmol/L) measurements were used as the baseline value. The fluctuations in serum total cholesterol levels were mainly due to short-term variations. CONCLUSION: For screening purposes, one measurement of serum total cholesterol in a woman gives a good estimate of the long-term level. The current data indicate that repeated measurements of serum total cholesterol do not improve the predictability of future cholesterol levels. The data also suggest that, at least in postmenopausal women with an elevated level of serum total cholesterol, one should proceed immediately to lipoprotein analysis for further risk assessment. PMID- 1731507 TI - Plasma concentration and urinary excretion of erythropoietin in adult nephrotic syndrome. AB - PURPOSE: Nephrotic syndrome (NS) is associated with a significant alteration of protein metabolism. While lowering the plasma concentrations of certain proteins, the disease often raises the level of certain other proteins. The current study was undertaken to determine the effect of NS on erythropoietin (EPO) metabolism. PATIENTS AND METHODS: We measured the EPO concentration in plasma and urine of 26 patients with NS by an immunologic assay using a rabbit antiserum against recombinant human EPO. The results were compared with those obtained in a group of 12 normal control subjects. RESULTS: Despite a significant reduction in the hemoglobin concentration in the NS group compared with the control group (125 +/- 25 g/L versus 148 +/- 11 g/L, p less than 0.05), the plasma EPO concentration in the NS group was not significantly different from that seen in the control group (6.2 +/- 4.5 mU/mL versus 6.7 +/- 2.4 mU/mL, p = NS). No significant correlations were found between plasma EPO and hemoglobin concentration, serum creatinine, serum albumin, or urinary albumin excretion rate. Moreover, comparison of the NS patients with serum creatinine concentrations less than or equal to 1.5 mg/dL (133 mumol/L) with those exhibiting creatinine concentrations exceeding 1.5 mg/dL did not reveal a significant difference in mean plasma EPO concentration. Significant amounts of EPO were found in the urine of the patients with NS, while none was detected in the urine of the control subjects. CONCLUSION: We conclude that plasma EPO is inappropriately low in patients with NS. This is due, at least in part, to the urinary/renal losses of this protein and can potentially contribute to anemia in NS patients or compound the problem in those with concurrent renal insufficiency and diminished EPO production. PMID- 1731508 TI - Barium enemas in the frail elderly. AB - PURPOSE: The objective of this investigation was to determine the frequency of and predictors for inadequate barium enemas in the frail elderly. PATIENTS AND METHODS: The medical and radiologic records of 171 elderly institutionalized patients (mean age = 85.3 years), who underwent barium enema examinations, were retrospectively reviewed. The study outcome of primary interest was the radiologist's report of the adequacy of examination as indicated in the written summary of the results of the barium enema procedure. RESULTS: Eighty-eight (51.5%) of the 171 studies were deemed inadequate, with poor bowel preparation a primary or contributing factor in 89.7% of the inadequate studies. Among a variety of demographic and clinical factors, only long-term laxative and/or cathartic use was associated with an inadequate study (odds ratio = 7.0; 95% confidence interval 2.7 to 18.0). CONCLUSION: These results demonstrate a very high frequency of inadequate barium enema examinations in the very old and suggest a need for improved methods of bowel preparation in this patient population, especially in those who are long-term users of laxatives and cathartics. PMID- 1731509 TI - The risks of blood transfusion: the relative influence of acquired immunodeficiency syndrome and non-A, non-B hepatitis. AB - PURPOSE: The acquired immunodeficiency syndrome epidemic has greatly increased concern about the risk of blood transfusion. Many transfusions are now autologous, and when these are not available, both physicians and patients are more likely to question the advisability of transfusion. We evaluate the risk of preoperative blood transfusion and the contribution of human immunodeficiency virus (HIV) infection to that risk. METHODS: We used decision analysis to characterize the risk associated with HIV infection in days of life lost. The contributions to risk of acute transfusion reaction, hepatitis B, and non-A, non B hepatitis are also estimated. Sensitivity analyses show the implications for transfusion risk of recent information about HIV infection in the blood supply and a new test for hepatitis C. RESULTS: The analysis shows that the contribution of HIV infection to the risk of death from transfusion, expressed in days of life expectancy lost, has become extremely small over the last several years. Currently, HIV infection accounts for less than 1% of the risk of death, while non-A, non-B hepatitis accounts for 97% to 98%. Further reductions in the risk of HIV infection, even to zero, will make relatively little difference in the safety of transfusion. The analysis also shows that the remaining risk from transfusion should decrease sharply, by more than two thirds, with the adoption of the test for hepatitis C. CONCLUSIONS: Efforts to improve the safety of blood should focus on reducing the risk of non-A, non-B hepatitis. The remaining risk of HIV infection is very small. PMID- 1731510 TI - Anaerobic bacteremia: incidence, patient characteristics, and clinical significance. AB - PURPOSE: In the 1970s, blood culture for obligate anaerobic bacteria became routine in most United States hospitals. Since then, various authorities have reported isolation of obligate anaerobes in 5% to 25% of blood cultures. Our experience suggests a much lower frequency; therefore, we retrospectively assessed the occurrence and significance of these cultures at our institutions. PATIENTS AND METHODS: Sixty-six patients at the University of Michigan Hospitals (UMH) and nine patients at the Ann Arbor Veteran's Administration Medical Center (AAVAMC) had one or more blood cultures positive for an obligate anaerobe between July 1, 1987, and December 31, 1988. Their medical records were reviewed retrospectively. RESULTS: The proportion of positive blood cultures yielding obligate anaerobes was 3.2% at the UMH and 1.8% at the AAVAMC. The incidences of clinically significant anaerobic bacteremia at the two hospitals were 0.68 and 0.54 cases per 1,000 patient admissions. Among the 40 patients from whom significant isolates were obtained, 15 (38%) had a fatal outcome. Bacteroides and Clostridium species accounted for 90% of the isolates and all of the fatal cases. The source for anaerobic bacteremia was usually obvious; 30 of the 40 patients were given empiric antibiotic therapy for anaerobes. The gastrointestinal tract was the source in two thirds of the cases and was clearly implicated as the source of 80% of the fatal bacteremias. CONCLUSIONS: The frequency of anaerobic bacteremia in our hospitals is much lower than was suggested in several large studies during the 1970s, probably reflecting a real decline in the incidence. The clinical features of our cases are similar to those of previous studies, and the mortality is still high despite the use of antibiotics effective against anaerobes. Since most patients were thought to have anaerobic infections at the time that cultures were obtained, they were usually treated empirically. Subsequent blood cultures positive for anaerobes infrequently influenced clinical management. PMID- 1731511 TI - Erythromycin ototoxicity: prospective assessment with serum concentrations and audiograms in a study of patients with pneumonia. AB - BACKGROUND AND METHODS: The incidence and risk factors for erythromycin-induced ototoxicity are unknown. We conducted a prospective, nested case-control study of assessment of auditory function in patients receiving erythromycin versus other antibiotics (control group) for community-acquired pneumonia. Sequential audiograms were performed during antibiotic therapy for both cases and controls by an audiologist unaware of the identity of the therapy administered. Erythromycin serum concentrations were obtained for all patients receiving erythromycin. RESULTS: Symptomatic ototoxicity (tinnitus or hearing loss) confirmed by audiograms was documented in five of 30 patients receiving erythromycin and none of 15 receiving other antibiotics. Ototoxicity was significantly related to high peak concentration and high AUC 0-infinity as a function of decreased total systemic clearance. Ototoxicity occurred only in those patients who received 4 g/day versus 2 g/day or no erythromycin (p = 0.05). Ototoxicity resolved in all patients within 6 to 14 days after discontinuation of therapy. CONCLUSIONS: Erythromycin ototoxicity is dose- and serum concentration dependent. Patients receiving erythromycin, especially at a total daily dose of 4 g, should be monitored regularly for subjective evidence of sensorineural hearing dysfunction. Ototoxicity is reversible if the diagnosis is made early in the course. PMID- 1731512 TI - Anagrelide, a therapy for thrombocythemic states: experience in 577 patients. Anagrelide Study Group. AB - PURPOSE: To evaluate the safety and efficacy of anagrelide when used to reduce platelet counts in patients with thrombocytosis. PATIENTS AND METHODS: Five hundred seventy-seven patients were treated with anagrelide to control the thrombocytosis of chronic myeloproliferative diseases. Three hundred thirty-five patients had primary thrombocythemia, 114 chronic granulocytic leukemia, 68 polycythemia vera, and 60 undifferentiated myeloproliferative diseases. Of the 577 patients, 504 were known to have been previously treated unsuccessfully with other modalities. RESULTS: Of the 577 patients treated, 424 were evaluable for response. Anagrelide in doses of 0.5 mg to 1.0 mg four times a day reduced the platelet count by 50%, or to less than 600,000/mm3, for at least 28 days in 396 of the 424 (93%) evaluable patients. Acquired resistance to the drug was not observed. Major side effects were neurologic, gastrointestinal, and cardiac. In more than 5 years, 16% of patients discontinued treatment because of side effects. CONCLUSIONS: Our experience suggests that anagrelide should become a useful agent in controlling the thrombocythemia seen in chronic myeloproliferative diseases and can be effective in patients in whom treatment with currently available agents has failed. PMID- 1731513 TI - Is age an independent predictor of early and late mortality in patients with acute myocardial infarction? AB - PURPOSE: To determine whether advancing age is an independent predictor of increased mortality following acute myocardial infarction or simply a marker for more extensive cardiac disease, a higher prevalence of comorbid conditions, and/or differences in therapeutic approach. PATIENTS: A total of 261 consecutive patients with documented acute myocardial infarction admitted to a university teaching hospital during a 1-year interval. METHODS: Seventy-four variables were analyzed to determine univariate predictors of inhospital and 1-year post discharge mortality. Multiple linear regression models were constructed to determine independent predictors of early and late mortality after adjusting for baseline and therapeutic differences between younger and older patients. RESULTS: Compared with patients less than 70 years (n = 124), patients greater than or equal to 70 years (n = 137) were more likely (all p less than 0.05) to be female and have a prior history of ischemic heart disease. New York Heart Association functional class and Killip class on admission were higher in older patients, as were the admission serum creatinine and blood urea nitrogen levels. Serum albumin and peak creatine kinase levels were lower in older patients, but older patients were more likely to exhibit left ventricular hypertrophy or atrioventricular block on the initial electrocardiogram. Finally, younger patients were three times as likely to receive a thrombolytic agent and 66% more likely to receive intravenous beta-blockade than older patients, and younger patients were also more likely to receive heparin and intravenous nitroglycerin. Hospital mortality was 5.6% in patients less than 70 years versus 16.1% in patients greater than or equal to 70 years (p = 0.013). After adjusting for baseline and therapeutic differences, independent predictors of hospital mortality were systolic blood pressure on admission (inverse correlation, p = 0.0095), beta-blocker therapy (inverse correlation, p = 0.01), age (p = 0.014), peak creatine kinase level (p = 0.015), and Killip class (p = 0.035). Among hospital survivors, 1-year post discharge mortality was 6.8% in patients less than 70 years versus 19.1% in those greater than or equal to 70 years (p = 0.001). Independent predictors of post discharge mortality after adjusting for age-related baseline and therapeutic differences were admission heart rate (p = 0.0004), age (p = 0.011), left ventricular ejection fraction (inverse correlation, p = 0.012), initial non-Q wave myocardial infarction (p = 0.026), and the blood urea nitrogen level (p = 0.036). CONCLUSION: After adjusting for multiple baseline and therapeutic differences between older and younger patients, age per se remains a strong independent predictor of both inhospital and 1-year post-discharge mortality rates in patients with acute myocardial infarction. PMID- 1731514 TI - Hepatic toxicity of unmodified and time-release preparations of niacin. AB - Niacin (nicotinic acid) is used frequently in the treatment of hypercholesteremia. It is available in both unmodified and time-release preparations. The latter were developed in attempts to minimize the skin-flushing reaction that affects virtually all users and may limit acceptance. Adverse effects on the liver from both unmodified and time-release preparations have been recognized for many years. We reviewed the literature on the hepatic toxicity of both types of niacin preparations. Adverse reactions in six patients resulted from the exclusive use of unmodified niacin and in two patients from the exclusive use of time-release preparations. In 10 additional patients, adverse reactions developed after an abrupt change from unmodified to time-release preparations. Many of these patients were ingesting time-release niacin at doses well above the usual therapeutic doses currently recommended. Signs of liver toxicity developed in less than 7 days in four of these 10 patients. In doses that achieve equivalent reductions in serum lipids, hepatic toxicity occurred more frequently with time-release preparations than with unmodified preparations. An awareness of toxicity associated with ingestion of high doses of time-release niacin preparations is important because of their widespread availability and the potential for self-prescribed, unmonitored use. PMID- 1731515 TI - Postmortem management of patient affairs: the resident's perspective. AB - The role of residents continues to evolve, and they are finding themselves increasingly being asked to request an autopsy from a grieving family. Although much has been written previously concerning the need for residents to be more aggressive in obtaining autopsy, this article suggests that structural problems must be addressed if the current low autopsy rate is to be improved. PMID- 1731516 TI - Magic bullets. PMID- 1731517 TI - Fatigue, fevers, and gastrointestinal bleeding in a 62-year-old woman. PMID- 1731518 TI - A fatal case of acute splenic sequestration in a 53-year-old woman with sickle hemoglobin C disease. AB - Acute splenic sequestration crisis (ASSC) is a common complication in infants and young children with homozygous sickle cell disease, but it is infrequent in patients with sickle-hemoglobin C (SC) disease. When it does occur in such patients, it is often associated with exposure to high altitude, either by air travel or mountainous environment. Since 1970, only 15 cases of ASSC have been reported in patients with SC disease who were not exposed to a high altitude. Nine of these were children and adolescents aged 11 to 18 years, while six were adults aged 21 to 44 years. A review of these cases shows only two fatalities from ASSC: a 12-year-old West Indian girl living in England and a 13-year-old black girl in the United States. In this report, we describe the sudden death from ASSC of a 53-year-old black woman with SC disease at low altitude. To our knowledge, death from ASSC has not been previously reported in an adult patient with SC disease. PMID- 1731519 TI - Diversity of T-cell antigen receptor variable genes used by mycosis fungoides cells. AB - The expression of T-cell antigen receptor beta-chain variable genes (V beta) was evaluated in 28 cases of mycosis fungoides. A novel polymerase chain reaction (PCR) technique was used to associate expression of particular V beta genes with monoclonal T-cell populations. In addition, the same biopsies used for PCR analysis were also examined for reactivity with a panel of seven monoclonal antibodies that specifically recognized V beta proteins from four different families. Only three cases clearly stained with the antibodies, a result consistent with a diverse set of V beta genes being used. This was confirmed by PCR analysis, which indicated that V beta genes from many different families were expressed by these tumors. Preferential use of the V beta 8 family, which had been previously use of the V beta 8 family, which had been previously reported for this disease, was not evident among the cases analyzed. PMID- 1731520 TI - Low levels of human immunodeficiency virus replication in the brain tissue of children with severe acquired immunodeficiency syndrome encephalopathy. AB - The authors examined the autopsy brain samples of nine children infected with human immunodeficiency virus (HIV) at birth by histology, immunologic staining, and in situ hybridization. Surprisingly, although seven of these children presented with typical AIDS encephalopathy, the authors could detect a multifocal HIV infection in the brains of only three of these patients. The authors could not detect any significant HIV replication in the brain of four other children despite severe neurologic disease. However, HIV DNA was detected by polymerase chain reaction (PCR) in the central nervous system (CNS) of all patients. In addition, the authors found associated lesions in the brains of three of these four patients. This study shows that severe AIDS encephalopathy exists in children and therefore might exist in adults with few signs or without any signs of HIV replication or inflammation in the CNS. Understanding the pathogenesis of this neurologic disease and the kinetics of HIV replication in brain tissue of children with AIDS encephalopathy is essential to determine the best therapeutic strategy. PMID- 1731521 TI - Widespread p53 overexpression in human malignant tumors. An immunohistochemical study using methacarn-fixed, embedded tissue. AB - p53 is a nuclear protein believed to play an important role, through mutation and overexpression, in the progression of human malignant tumors. The authors employed a monoclonal antibody, 1801, and investigated overexpression of p53 in a series of 255 malignant and benign tumors, using deparaffinized sections of methacarn-fixed tissue. Overall, immunohistochemically detected p53 overexpression was found in 39% of malignant tumors, with considerable variation within individual tumor types (34% of breast carcinomas, 92% of ovarian carcinomas, 33% of soft tissue sarcomas). Homogenous, heterogenous, and focal immunostaining patterns were noted. With rare exceptions, no immunostaining of any benign tumors was noted. No immunostaining was found in adjacent, benign tissues, or in a series of fetal tissues. This is the first demonstration of widespread p53 overexpression in alcohol-fixed, embedded tissue and confirms the major role played by p53 in human malignancies. PMID- 1731522 TI - Correlation of c-fos/c-jun expression with histiocytic differentiation in Hodgkin's Reed-Sternberg cells. Examination in HDLM-1 subclones with spontaneous differentiation. AB - The c-fos proto-oncogene, which is the normal homolog of the transforming gene carried by murine osteogenic sarcoma viruses, interacts with the protein product of another proto-oncogene, c-jun, to form a heterodimer that can recognize and bind to a specific sequence of nucleotides in the DNA. The expression of c-fos and c-jun is linked to the proliferation of certain cells and the differentiation of others, including those of monomyelocyte lineage. The authors used two cultured Hodgkin's Reed-Sternberg (H-RS) cell lines, KM-H2 and HDLM-1, and their single-cell clones to study the correlation of c-fos/c-jun expression with cell differentiation in H-RS cells. Within 48 hours after induction with phorbol ester (TPA), both parent lines exhibited markedly increased expression of c-fos/c-jun. The expression returned to the preinduction level after 96 hours, however, and the cells retained their differentiated status. The transitory increase in c fos/c-jun expression suggests that binding of these proteins to a specific promoter in the nucleus triggers a cascade of events that result in cell differentiation. Expression of these proteins may not be required for the cells to maintain their differentiation. The authors selected three groups of sublines of HDLM-1 cells based on their degree of spontaneous cytologic differentiation. The first group, without obvious differentiation, showed a c-fos/c-jun expression pattern similar to that of the parent line. The second group, with moderate differentiation, had a high degree of expression, which decreased on treatment with TPA. The third group, which had morphologic features resembling those of histiocytes, expressed minimal amounts of c-fos/c-jun, irrespective of TPA treatment. These findings provide further evidence that c-fos/c-jun expression is related to differentiation of H-RS cells, and that these proteins are not byproducts of TPA induction. Expression of c-fos/c-jun also was noted in a subpopulation of H-RS cells in tissues; and this expression also was enhanced when these cells were treated with TPA in culture. These findings indicate that H RS cells can differentiate to become mature-appearing cells in tissues. PMID- 1731523 TI - Ultrastructural localization of endogenous albumin in human aortic tissue by protein A-gold immunocytochemistry. AB - The presence of endogenous plasma albumin in human aortas was demonstrated by immunocytochemical procedures. The protein A-gold approach, combining high resolution and specificity, was applied at the light and electron microscope levels to determine the in situ cellular and extracellular localization of albumin on tissue sections derived from normal and atherosclerotic human aortas. The distribution of albumin across the aortic wall interstitium was found to be uneven, with low to moderate staining intensities in the aortic subendothelial space, low intensities in the media, and high intensities around the vasa vasorum in the adventitia. Albumin was associated with collagen fibers as well as with the electron-dense material bordering the elastic laminae in both normal and pathologic tissues. Extracellular multilamellar structures were found to be characteristic of the necrotic areas of atherosclerotic aortas. These structures were intensely labeled for albumin, with the labeling being closely associated with the membranes. Numerous smooth muscle cell-derived and monocyte-derived foam cells were present in pathologic tissues, and some of their lysosomal compartments were labeled for albumin, suggesting an internalization and degradation of interstitial albumin by these cells. PMID- 1731524 TI - Cell surface heparan sulfate proteoglycan and chondroitin sulfate proteoglycan of arterial smooth muscle cells. AB - Cell surface proteoglycans of aortic smooth muscle cells of atherosclerosis susceptible White Carneau (WC) and atherosclerosis-resistant Show Racer (SR) pigeons were compared to determine differences that may be involved in the greater proliferative properties of cultured WC cells. Using [35S]-sodium sulfate and [3H]-glucosamine as labeling precursors, chondroitin sulfate-proteoglycan (CS PG) and heparin sulfate-proteoglycan (HS-PG) were identified as distinct molecules associated with the plasma membrane. Heparan sulfate-proteoglycan was reduced up to 50% in WC compared with SR cells, and, based on interaction with ion-exchange resin, had a lower charge density. These differences were not observed for the CS-PG from the two cell types. The mode of association of the cell surface PG with the plasma membrane was examined. Dissociation with 1 mol/l (molar) sodium chloride indicated that less than 10% of total cell surface PG were ironically associated with the cells. The remainder required detergent extraction, suggesting hydrophobic interactions with the plasma membrane. Both CS PG and HS-PG displayed affinity for octyl sepharose and both were identified in isolated plasma membranes. These data present the first description of a hydrophobic CS-PG that is a significant and distinct cell-associated PG in arterial smooth muscle cells. The observation of decreased and structurally altered HS-PG in WC compared with SR cells is consistent with a potential growth regulatory function for this molecule. PMID- 1731525 TI - The effects of estrogen on prolactin gene methylation in normal and neoplastic rat pituitary tissues. AB - The effects of estrogen treatment on rat prolactin (PRL) gene methylation were analyzed in normal pituitaries and in three transplantable rat pituitary tumors. Northern analysis showed increased PRL mRNA expression in estrogen-treated pituitary and in MtT/F4 and MtT/F-DMBA tumors. Prolactin mRNA amounts in MtT/W15 tumor were decreased by estrogen treatment. There was an inverse relationship between changes in PRL mRNA expression and changes in gene methylation in the coding regions of the PRL gene after estrogen treatment. The amounts of the 4.6 Kb and 1.8-Kb restriction fragments generated by HpaII digestion in pituitary tissues were influenced by estrogen, with an increase in these fragments in normal pituitary, MtT/F4, and MtT/F-DMBA tumors after estrogen treatment. In contrast, the amounts of the 4.6-Kb and 1.8-Kb fragments were decreased in MtT/W15 tumors after estrogen treatment. Most of the internal -CCGG- sites in the PRL gene were methylated in the liver, and the PRL gene was not expressed in the liver. These data suggest that there is a tissue-specific pattern of DNA methylation of the PRL gene and that PRL gene methylation is influenced by estrogen in vivo in normal and tumorous pituitary tissues. These results also suggest that estrogen may influence PRL expression by multiple mechanisms, including changes in the level of DNA methylation. PMID- 1731527 TI - Histopathologic changes of the nasal mucosa in southwest Metropolitan Mexico City inhabitants. AB - Metropolitan Mexico City (MMC) is one of the most polluted urban areas in the world. The authors characterized the morphologic nasal mucosal changes in short term (less than 30 days) and long-term (more than 60 days) exposures to the polluted southwest MMC atmosphere with high levels of ozone and other contaminants versus a control group of subjects living in a nonpolluted, low ozone Mexican port. Seventy-six inferior turbinate biopsies were examined. The control group showed normal mucociliary epithelium, whereas the short-exposure group displayed loss of normal epithelium, basal cell hyperplasia, and mild dysplasia (17.64%). In the long-term exposure group, 78.72% of dysplasias were found (59.45% mild and 40.54% moderate) together with severe loss of normal respiratory epithelium, prominent basal cell hyperplasia, squamous metaplasia, and submucosal vascular proliferation. Our findings suggest that southwest metropolitan Mexico City inhabitants develop histopathologic changes in their nasal mucosa on exposure to the polluted city atmosphere. PMID- 1731526 TI - Loss of heterozygosity on chromosome 17p13 in breast carcinomas identifies tumors with high proliferation index. AB - The capacity of breast tumor cells to proliferate is considered a potential prognostic factor together with other histopathologic parameters. The authors determined the proliferation index on a large panel of human primary breast tumors by measuring the levels of incorporation of bromodeoxyuridine (BrdU) by fresh tumor specimens in culture. Previous analysis showed that the percentage of cells entering the S-phase of the cell cycle strongly correlates with tumor grade, tumor size, and estrogen and progesterone receptor status. The capacity of tumor cells to proliferate might be associated with specific genetic mutations in primary tumors. To test this hypothesis, a panel of 96 human breast carcinomas, for which the BrdU labeling index (LI) was known, were tested for loss of heterozygosity (LOH) or increased copy number (ICN) at chromosomes 1q, 3p, 13q, 17p, and 18q. On chromosome 17p, LOH and ICN were observed in 27% and 12%, respectively, of the informative breast tumors. The LOH on chromosome 17p was significantly associated with tumors having an elevated BrdU proliferation index (P = 0.022). No association (P = 0.45) was observed between BrdU LI and tumor size (T2 + T3 compared with T1), tumor grade, and lymph node status. Increased copy number on chromosome 17p, LOH or ICN on 1q, and LOH on 13q14, 18q, and 3p also showed no significant correlation with cell kinetic parameters. These data are consistent with the presence of a gene or genes on chromosome 17p13 near the YNZ22.1 locus whose normal functioning is necessary for controlling breast tumor cells proliferation in vivo. PMID- 1731528 TI - Cytolytic T lymphocytes and antibodies to myocytes in adriamycin-treated BALB/c mice. Evidence for immunity to drug-induced antigens. AB - Female BALB/c mice were given a single intravenous injection of between 0.1 and 10 mg adriamycin/kg body weight and were killed between 2 and 16 days later. Natural killer (NK) cell activity in the spleen was measured using YAC cell targets. Natural killer cell activity was slightly elevated 2 to 5 days after drug injection and significantly depressed by day 9 compared with spleen cells from untreated animals. Adriamycin-treated mice developed both cytolytic T lymphocytes (CTL) and antibodies to drug-treated myocytes. Peak CTL response occurred between days 9 and 13, whereas antibody reactivity continued to increase throughout the observation period. The effector cell belonged to the CD8+ T lymphocyte subpopulation, because cytolytic activity could be reduced by treating the cells with anti-Lyt 2 antibody and complement, whereas anti-L3T4 (CD4+ cell specific) treatment either had no effect or increased cytotoxicity. Both CTL and antibody reactivity could be absorbed with adriamycin-treated myocyte monolayers but not by non-drug-treated myocytes. Furthermore CTL reactivity could be only partly removed by adriamycin-treated skin fibroblasts. Adriamycin concentrations in the heart were measured by flourometry and demonstrated only a gradual decrease in the drug over the 16-day period. Immunofluorescent staining of myocardial sections demonstrated increased numbers of both T lymphocytes and macrophages in the hearts of adriamycin-treated mice compared with untreated controls. PMID- 1731529 TI - Coexpression in humans by kidney and fetal envelopes of a 280 kDa-coated pit restricted protein. Similarity with the murine target of teratogenic antibodies. AB - Experimental studies performed in the rat over the last three decades have shown that antibodies raised against kidney or yolk sac, which, in the rat, surrounds the embryo and serves as a placenta during the major part of pregnancy, induced fetal resorptions or malformations. It is generally considered that the teratogenic antibodies decrease internalization and degradation of maternal proteins by yolk sac epithelial cells leading to an inadequate supply of nutriments to the embryo. These observations demonstrating the pathogenic role of antibodies to fetal envelopes are of great potential interest in clinical pathology since most cases of fetal malformations in humans are of unknown cause. The authors have recently shown that the key teratogenic antibodies in the murine system were directed against a 280 kDa-coated pit protein (gp280) specific for the brush border of epithelial cells lining the renal proximal tubule and the yolk sac. This observation allows for the unique opportunity to search for a similar system in humans. In this study, the presence in humans of a protein closely related to murine gp280 is shown, as indicated by extensive immunologic crossreactivity, close apparent molecular weights, strong homology of bidimensional peptide maps, and restricted distribution at the organ and subcellular level. In addition to kidney and yolk sac, human gp280 was also detected within the coated pits of the placental syncytiotrophoblastic cells. When introduced in an in vitro rat embryo culture system, antibodies to human gp280-induced developmental anomalies in a dose-dependent manner. These observations indicate that the antigenic component of the murine model is present in humans and can give rise to heterologous antibodies that cause developmental anomalies, suggesting that the experimental model might be of significance in human pathology. PMID- 1731530 TI - Effects of transforming growth factor-beta on collagen synthesis by normal rat kidney epithelial cells. AB - The effects of transforming growth factor-beta (TGF-beta) on the growth of and collagen production by NRK52E cells, a clonal line established from normal rat kidney epithelial cells, have been characterized. NRK52E cells were grown in the absence or presence of TGF-beta for 4 days followed by incubation for 24 hours in serum-free medium containing [3H]proline. The secreted and cell-associated collagens produced by control and experimental cultures were isolated by limited pepsin digestion and differential salt fractionation. TGF-beta inhibited proliferation by about 50% but did not affect overall culture morphology. Both protein and collagen synthesis were increased in experimental cultures, but the increase in total collagen production exceeded that of total protein synthesis. Although NRK52E cells grown in the presence of TGF-beta continued to produce the same types of collagen (types I, III, IV, and V), their relative amounts were changed. In the experimental cultures, type I collagen production was increased eightfold, types III and V collagen levels were increased two-fold, but type IV production was only slightly enhanced. In addition to increasing total collagen production by about fivefold, TGF-beta increased the ratio of type I to type III about threefold but minimally affected the ratio of secreted to cell-associated molecules. These findings establish that TGF-beta specifically affects collagen production in NRK52E cells and that these alterations differ in many ways from the affects of epidermal growth factor. Because TGF-beta increased total collagen expression, these results provide additional evidence implicating this growth factor as a positive mediator of matrix accumulation in renal disease. PMID- 1731531 TI - The effects of continuous exposure to epidermal growth factor on the spontaneous transformation of cultured rat liver epithelial cells. AB - A long-term continuous exposure to epidermal growth factor (EGF) enhanced the tumorigenicity of spontaneously transformed cells arising in a clonal population of normal cultured rat liver epithelial cells propagated in a selective growth condition. Lengthy EGF exposure also induced the expression of several phenotypes that differed from the phenotypes of rat liver epithelial cells transformed spontaneously in the absence of EGF. Epidermal growth factor treatment caused consistently an enhancement of the constitutive mRNA expression of transforming growth factor-alpha (TGF-alpha), but not of the EGF receptor and transforming growth factor-beta. The overexpression of TGF-alpha persisted in cell lines derived from tumors formed by the EGF-treated transformed cells. These tumors also exhibited high metastatic incidence and ductal cell differentiation. In contrast, untreated spontaneously transformed cells formed non-metastatic tumors with hepatocellular differentiation. These results suggest that long-term, continuous exposure to EGF/TGF-alpha may modulate the phenotypic expressions of neoplastic transformation. PMID- 1731532 TI - Expression of monocyte chemotactic protein-1 in human melanoma in vivo. AB - A common feature of human melanoma is infiltration by monocytes at early stages of tumorigenesis. This infiltration may be highly significant since macrophages have the capacity to alter the behavior of tumor cells. The authors previously demonstrated that the predominant monocyte chemoattractant produced by tumor cells in vitro was monocyte chemotactic protein-1 (MCP-1). The authors identify the expression of MCP-1 in pathologic specimens of both primary and metastatic human melanoma but not in normal skin. The finding that MCP-1 is produced by malignant melanoma suggests that specific genes are expressed in tumor cells that can induce the recruitment of monocytes in vivo. PMID- 1731533 TI - Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells. AB - A murine mesangial cell line (MMC) was established from the glomeruli of SJL mice to study the influence of angiotensin II (ANG II) on their growth and function in a serum-free culture. Murine mesangial cells exhibit the phenotypic characteristics of mesangial cells, including staining for desmin, vimentin, Thy 1, and types I and IV collagen by immunofluorescence. The addition of daily doses of 10(-6) to 10(-11) mol/l ANG II to MMCs also induced their proliferation in serum-free media. This effect on growth was independent of the presence of insulin in the media, and was receptor mediated, because the specific ANG II receptor antagonist DuP 753 abolished proliferative growth. Angiotensin II also stimulated mainly the biosynthesis of type I collagen in our MMCs. Transfection of MMCs with chimeric genes containing enhancer/promoter elements for alpha 2(I) and alpha 1(IV) collagens linked to a chloramphenicol acetyltransferase reporter demonstrated that the stimulatory effect of ANG II for type I depends, at least to some extent, on an increase in transcription. These findings indicate collectively that ANG II in serum-free cultures can be a paracrine catalyst for the growth and biosynthesis of type I collagen in mesangial cells. PMID- 1731534 TI - Pulse oximetry: would further technical alterations improve patient outcome? PMID- 1731535 TI - Effect of pulse oximetry, age, and ASA physical status on the frequency of patients admitted unexpectedly to a postoperative intensive care unit and the severity of their anesthesia-related complications. AB - Unanticipated intensive care unit admission (UIA) associated with anesthesia served as an outcome measure to assess the quality of anesthesia care in a large teaching hospital. We characterized the patient population and types of problems associated with UIAs, attempted to identify patterns of care that could have led to specific adverse outcomes, and determined if a specific intervention, pulse oximetry, reduced UIAs. During a consecutive 65-wk period (July 1985-September 1986), 17,093 surgical patients were expected to enter the recovery room and then return to floor care. Seventy-one patients (0.42%) experienced a UIA from either the recovery room or operating room, and the circumstances of their admissions were analyzed in detail. After introduction of pulse oximetry in all anesthetizing locations (not including the recovery room) in the 29th week, the overall rate of UIAs and, specifically, the rate of UIA to rule out myocardial infarction, decreased significantly. Increasing ASA physical status (ASA-PS) and age significantly increased the probability of UIA. UIA patients with ASA-PS III/IV had a significantly higher acuity in the intensive care unit and were far more likely to die during their hospitalization than ASA-PS I/II patients. Retrospective review of UIAs alone did not identify patterns of care requiring remediation, which leads us to question the utility of UIAs as a generic screen for quality assurance. PMID- 1731536 TI - Response time of pulse oximeters assessed using acute decompression. AB - In human volunteers, the response times of 11 pulse oximeters to a 10% step reduction in arterial oxygen saturation were measured using an acute decompression technique. When finger probes were used, nine oximeters had similar response times and two were significantly slower (P less than 0.05). The ear probe response time was similar on six oximeters assessed, and faster than the finger probes. The response times of the oximeters to an acute increase in arterial saturation were tested by suddenly changing the inspired gas from air to 100% oxygen at an ambient pressure of 380 mm Hg. For ear probes, the response times were similar for all oximeters; for finger probes, three fast-responding and three slow-responding oximeters were identified (P less than 0.05). A faster response could be elicited by placing the probes on the thumb (P less than 0.05). We conclude that if a rapid indication of changes in arterial saturation is required, pulse oximeters with ear probes should be used. If finger probes are used, they should be placed on the thumb. The oximeter used will influence the response time if finger probes are used, but it will have little effect if ear probes are used. PMID- 1731537 TI - Can pulse oximetry be used to measure systolic blood pressure? AB - This study evaluates the use of pulse oximetry to accurately monitor systolic arterial blood pressure in 100 healthy volunteers. Determination of arterial blood pressure using oximetry was made at the disappearance of visual display upon blood pressure cuff inflation, at the reappearance of visual display upon cuff deflation, and by averaging the two. The blood pressures obtained by pulse oximetry were compared with the arterial blood pressures obtained by Korotokoff sounds and noninvasive blood pressure equipment. Good agreement was obtained when the average of oximetry-based systolic blood pressure estimates at the disappearance and reappearance of the waveform were compared with Korotokoff sound pressures and noninvasive equipment pressures. Thus pulse oximetry can be used to measure systolic arterial blood pressure. This technique is specifically important for patients with Takayasu's syndrome (pulseless disease) where conventional techniques often fail to monitor systolic arterial blood pressure. PMID- 1731538 TI - Heparinase in the activated clotting time assay: monitoring heparin-independent alterations in coagulation function. AB - The activated clotting time (ACT) is routinely used to monitor heparin during cardiopulmonary bypass surgery. Activated clotting times may be influenced by a number of factors other than heparin. The presence of heparin in blood samples disguises the occurrence of non-heparin-related changes in coagulation function. During cardiopulmonary bypass, it is difficult to ascertain baseline clotting time fluctuations with ACT alone. Previous attempts to establish accurate baseline data were imprecise and involved extensive sample handling. In this study, we present data obtained using a modified (ACT) assay that incorporates heparinase. The heparinase test cartridge (HTC) instantaneously, specifically, and completely removes heparin in the blood sample at the initiation of the test. In conjunction with standard ACT techniques, the clinician is provided with heparin-independent (baseline) and functional clotting data. The HTC/ACT assay was used in a case study involving 19 patients undergoing cardiopulmonary bypass surgery. The data gathered indicate the usefulness of this assay in monitoring incidents of baseline drift, hemodilution, and hypercoagulation and the efficacy of protamine reversal. PMID- 1731539 TI - Acute colloid administration increases ischemia in the myocardium supplied by a stenotic coronary artery. AB - The effect of acute colloid administration was evaluated in the pig heart in which an external coronary artery stenosis was applied. Seven pigs received thiopentone and halothane anesthesia. Ultrasonic crystals were inserted in the myocardium supplied by the left anterior descending (LAD) and circumflex coronary arteries. Left ventricular pressure was measured and regional myocardial function was quantified with the pressure-length loop and the end-systolic pressure-length ratio. A significant stenosis was applied to the LAD artery, after which the animal received fixed colloids to increase the left ventricular end-diastolic pressure. Regional myocardial ischemia was defined with reference to postsystolic shortening and lactate production from the region supplied by the LAD artery. The application of the stenosis caused an increase in postsystolic shortening from 9.62% +/- 4.24% to 26.12% +/- 5.81% (mean +/- SEM; P less than 0.05), and lactate extraction changed from 15.88% +/- 2.38% to -9.56% +/- 5.32% (P less than 0.05). Acute colloid administration increased the left ventricular end-diastolic pressure from 9.71 +/- 1.77 to 15.93 +/- 2.07 mm Hg (P less than 0.05), and lactate extraction further decreased to -76.63% +/- 19.19% (P less than 0.05). Postsystolic shortening increased to 36.22% +/- 5.10% (P less than 0.05). The oxygen tension in the venous blood draining the LAD region decreased from 36.74 +/- 9.37 to 17.34 +/- 1.23 mm Hg (P less than 0.05). We conclude that in the acute pig model, augmentation of the preload worsens regional myocardial ischemia in an area supplied by a stenotic coronary artery. PMID- 1731540 TI - Influence of five different priming solutions on platelet function in patients undergoing cardiac surgery. AB - The ideal choice of a priming solution of the cardiopulmonary bypass (CPB) and its influence on the hemostatic system are not clear. Addition of albumin was reported to inhibit platelet damaging by blood-surface interactions ("coating"). To explore this possibility in 60 consecutive male patients undergoing elective aortocoronary bypass grafting, five different priming solutions were randomly used: (1) 1000 mL of 5% dextrose + 1000 mL of Ringer's solution (RS) + 250 mL of 5% human albumin (HA); (2) 1850 mL of RS + 400 mL of 20% HA; (3) 1750 mL of RS + 500 mL of 10% low molecular weight hydroxyethyl starch (molecular weight average: 200,000; molar substitution ratio: 0.5); (4) 1750 mL of RS + 500 mL of 3.5% gelatin; (5) 2250 mL of RS. Platelet function was studied by aggregometry (= turbidometric technique; 1.0 and 2.0 mumol/L of adenosine diphosphate (ADP), 4 microL/mL of collagen, 25 mumol/L of epinephrine) before, during, and after CPB until the first postoperative day. Blood loss and need for homologous blood was not different between the groups. During CPB, maximum platelet aggregation induced by ADP was least compromised in group 1 and group 4. At the end of the operation ADP-induced aggregation increased in group 1 (+27%), whereas aggregation was decreased in the other priming solution groups. A significant increase in maximum aggregation was found in group 1 even on the first postoperative day (+132% +/- 16%). Collagen-induced aggregation was also least compromised in group 1. Epinephrine-induced platelet aggregation did not change and was similar for all groups. Maximum gradient of aggregation was influenced in an identical way as maximum aggregation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731541 TI - Epidural patient-controlled analgesia after cesarean section: buprenorphine 0.015% bupivacaine with epinephrine versus fentanyl-0.015% bupivacaine with and without epinephrine. AB - We compared the analgesia, side effects, and plasma concentrations of buprenorphine and fentanyl in a double-blind study of 78 parturients receiving one of these drugs by patient-controlled epidural infusion after elective cesarean section with epidural anesthesia. Patients were randomized to three epidural infusion groups: group 1 (n = 26), 3 micrograms/mL buprenorphine with 0.015% bupivacaine and 1 microgram/mL epinephrine; group 2 (n = 26), 3 micrograms/mL fentanyl with 0.015% bupivacaine and 1 microgram/mL epinephrine; and group 3 (n = 26), 3 micrograms/mL fentanyl with 0.015% bupivacaine. Plasma for determination of opioid concentrations was obtained in some subjects in each group at intervals up to 48 h during the infusion and in some subjects from each group at intervals after the infusion was stopped. Pain relief was similar and satisfactory in all three groups. The median overall satisfaction scores were high for all three groups. Pruritus was more common in the fentanyl groups (P less than 0.05). However, vomiting was more disturbing to the patients and seen only with buprenorphine. No patient had a respiratory rate less than 12 breaths/min. Epinephrine use was associated with a slower infusion rate (P less than 0.05, group 2 vs 3). All patients were able to ambulate without difficulty. Mean opioid plasma concentrations did not exceed 1.5 ng/mL. Thus, epidural patient-controlled analgesia in all three groups provided excellent analgesia, permitted ambulation, and was without serious side effects. Epidural buprenorphine offered no advantages over epidural fentanyl. PMID- 1731542 TI - Plasma inorganic fluoride with sevoflurane anesthesia: correlation with indices of hepatic and renal function. AB - The biotransformation and plasma inorganic fluoride ion production of sevoflurane (the new volatile anesthetic) during and after surgical anesthesia was studied in 50 ASA I or II surgical patients. Twenty-five additional patients served as controls by receiving isoflurane. Sevoflurane or isoflurane was administered with a semiclosed (total gas flow, 2 L/min O2) circle absorption system for durations of 1.0 to greater than 7.0 minimal alveolar concentration (MAC) hours for surgical anesthesia (sevoflurane MAC, 2.05%; isoflurane MAC, 1.15%). Preoperative and postoperative blood urea nitrogen and creatinine concentrations were determined. Blood samples were obtained during and after anesthesia in both groups for determining anesthetic blood concentration analysis and plasma fluoride level. Plasma fluoride concentrations did not significantly increase during isoflurane anesthesia. Sevoflurane biotransformation produced a mean peak plasma inorganic fluoride concentration of 29.3 +/- 1.8 mumol/L, 2 h after anesthesia, which decreased to 18 mumol/L concentration by 8 h after anesthesia. The peak plasma inorganic fluoride ion concentration correlated with duration of sevoflurane anesthetic exposure. Five patients given sevoflurane had peak levels transiently exceeding 50 mumol/L, and one of these had a history of ingesting drugs potentially producing hepatic enzyme induction. No increases in postoperative levels of creatinine, blood urea nitrogen, direct bilirubin, or hepatic transaminase and no changes in serum electrolyte level occurred in either anesthetic group. Indirect bilirubin concentration increased significantly after sevoflurane anesthesia, but the increase was not of clinical significance (from 0.30 +/- 0.03 to 0.38 +/- 0.06 mg/dL). Indirect bilirubin concentrations did not increase after isoflurane anesthesia; the concentrations reached 0.31 +/- 0.04 mg/dL and did not differ significantly from those found with sevoflurane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731543 TI - Plasma fluoride concentrations during and after prolonged anesthesia: a comparison of halothane and isoflurane. AB - Serum inorganic fluoride concentrations were studied in 19 adult patients undergoing prolonged head and neck surgery with either halothane or isoflurane anesthesia (mean 19.5 and 19.2 MAC-hours, respectively). In the group of nine patients anesthetized with isoflurane, plasma inorganic fluoride increased from a mean concentration of 3.5 mumols/L (baseline) to a peak of 43.2 mumols/L. Forty percent of the patients in the isoflurane group had peak plasma inorganic fluoride concentrations of more than 50 mumol/L. In the group that received halothane, plasma inorganic fluoride concentrations increased from a mean of 3.8 mumols/L to a peak of 12.6 mumols/L. Despite similar exposures to both anesthetics, the differences in serum inorganic fluoride concentrations between the two groups were significant at 10, 24, and 48 h after induction of anesthesia (P = 0.035, P = 0.003, and P = 0.003, respectively). Serum electrolyte, urea, and creatinine concentrations and urine output rates during and after surgery were similar in both groups. We conclude that, after anesthesia of up to 20 MAC-hours, metabolism of isoflurane to inorganic fluoride may be of a greater magnitude than has previously been realized. Although no clinical or biochemical evidence was found to suggest postoperative renal dysfunction, we recommend caution using isoflurane when prolonged anesthesia and surgery are planned. PMID- 1731544 TI - Clinical comparison of sevoflurane and isoflurane in healthy patients. AB - We compared blood pressure and heart rate changes in healthy patients during anesthesia with sevoflurane (n = 50) versus isoflurane (n = 25) and the rate of recovery after such anesthesia. After premedication with intravenous administration of midazolam, induction of anesthesia with thiopental, and intubation of the trachea facilitated with succinylcholine or vecuronium, anesthesia was maintained with approximately 1 MAC (sevoflurane, 2.05%; isoflurane, 1.15%) of the volatile anesthetic in oxygen for the duration of the operation. Anesthetic concentration was varied as indicated to maintain arterial blood pressure at +/- 20% of baseline values. Sevoflurane and isoflurane produced similar systolic and diastolic arterial blood pressures, but heart rate after incision was faster in patients given isoflurane. Recovery of response to command was shorter in patients given sevoflurane than that in patients given isoflurane (7.5 +/- 0.5 min versus 18.6 +/- 2.0 min). Consistent with this finding, venous blood drawn after anesthesia showed a more rapid initial decay with sevoflurane. Nausea and vomiting were comparable in both groups. We conclude that sevoflurane anesthesia, as compared with isoflurane, is associated with possible advantageous effects on heart rate and recovery. PMID- 1731545 TI - Lidocaine for the prevention of pain due to injection of propofol. AB - Propofol has a high incidence of pain with injection, particularly into small veins. We sought to determine whether concomitant administration of lidocaine could prevent this pain. In a randomized double-blind trial, 368 women were allocated to one of four groups to receive 19 mL of propofol mixed with either 1 mL of 0.9% saline, 1 mL of 0.5% (5 mg) lidocaine, 1 mL of 1% (10 mg) lidocaine, or 1 mL of 2% (20 mg) lidocaine. The pain of injection was scored as none, mild, moderate, or severe. There was a significant reduction in the overall incidence of pain from 73% with saline to 32% with 20 mg lidocaine. A highly significant negative dose-response relationship between the dose of lidocaine and the severity of pain was demonstrable, both at induction of anesthesia and as recalled in the recovery room (P less than 0.001 for both). Lidocaine (20 mg IV) will significantly reduce the incidence and severity of pain with propofol injection, but about 6% of patients will still suffer unpleasant pain if the dorsum of the hand is used. PMID- 1731546 TI - Tourniquet at 50 mm Hg followed by intravenous lidocaine diminishes hand pain associated with propofol injection. AB - We evaluated the efficacy of intravenous lidocaine, with and without a tourniquet, to decrease the intensity of pain during intravenous propofol injection in 82 patients undergoing general anesthesia. Patients in group A (n = 20) received propofol (2 mg/kg IV); patients in group B (n = 22) received 2% lidocaine (100 mg IV) followed 1 min later by propofol (2 mg/kg). Patients in group C (n = 21, saline placebo) and D (n = 19, 2% lidocaine) had an arm tourniquet inflated to 50 mm Hg applied for 1 min after gravity drainage of venous blood. The intensity of pain along the forearm was marked on a 0-100-mm visual analogue scale. Pain intensity was less in group B (21 +/- 19 mm) than in group A (75 +/- 28 mm; P less than 0.05). Pain intensity was significantly less in group D (1 +/- 2 mm) compared with group B (21 +/- 19 mm; P less than 0.001). We conclude that intravenous lidocaine before propofol injection attenuates the painful response; whereas, lidocaine administered after a tourniquet inflated to 50 mm Hg for 1 min virtually abolishes the pain associated with intravenous propofol. PMID- 1731547 TI - Comparison of induction, maintenance, and recovery characteristics of sevoflurane N2O and propofol-sevoflurane-N2O with propofol-isoflurane-N2O anesthesia. AB - Induction of, maintenance of, and recovery from sevoflurane anesthesia were compared with propofol and isoflurane anesthesia when administered with nitrous oxide to patients undergoing gynecologic surgery. Seventy-five healthy (ASA I or II), consenting patients were randomly assigned to receive either (I) propofol for induction of anesthesia and isoflurane-nitrous oxide for maintenance (control), (II) propofol for induction and sevoflurane-nitrous oxide for maintenance, or (III) sevoflurane-nitrous oxide for induction and maintenance of anesthesia. Inhaled induction of anesthesia with sevoflurane-nitrous oxide was rapid (109 +/- 25 s to loss of consciousness) and without any untoward hemodynamic changes or episodes of coughing and laryngospasm. Mean arterial blood pressure after induction of anesthesia with propofol (71 +/- 11, 73 +/- 12 mm Hg for groups I and II, respectively) was lower than when sevoflurane (80 +/- 14 mm Hg) was used. The emergence time after discontinuation of isoflurane-nitrous oxide (6.7 +/- 2.2 min) was significantly longer than after propofol-sevoflurane nitrous oxide or sevoflurane-nitrous oxide alone (4.1 +/- 2.2 and 4.0 +/- 2.0 min for groups II and III, respectively). However, later recovery events did not differ between groups. Serum fluoride levels increased after administration of sevoflurane but not isoflurane. The levels of fluoride ions correlated with the degree of exposure to sevoflurane in MAC-hours. In conclusion, induction of anesthesia with either propofol or sevoflurane-nitrous oxide was rapid and without significant side effects. Emergence and early recovery after maintenance of anesthesia with sevoflurane-nitrous oxide was significantly faster than that after an isoflurane-nitrous oxide combination. PMID- 1731548 TI - Slow injection does not prevent midazolam-induced ventilatory depression. AB - To determine whether the risk of midazolam-induced ventilatory depression is related to the rate of midazolam administration, we compared the effect of rapid (over 15 s) and slow (over 5 min) administration of midazolam (0.1 mg/kg IV) on the hypercarbic ventilatory response of 10 healthy volunteers. During the first 5 min after the start of midazolam injection, the slope of the ventilatory response to CO2 was significantly lower when the subjects received midazolam rapidly (P less than 0.001). However, after completion of the infusion (between 5 and 20 min), depression of the CO2 response curve slope was independent of the rate of midazolam administration. Similarly, although minute ventilation and tidal volume measured at an end-tidal CO2 tension of approximately 46 mm Hg decreased more quickly after rapid administration of midazolam (P less than 0.001), these variables did not differ significantly between the two rates of administration once the slow infusion was complete. These results suggest that slow administration of midazolam provides no independent protection from respiratory depression. PMID- 1731549 TI - Sedation and recovery of psychomotor function after intravenous administration of various doses of midazolam and diazepam. AB - A placebo-controlled, double-blind, crossover trial in 11 healthy male volunteers compared clinical sedation and psychomotor function after intravenous injection of midazolam (0.05, 0.1, or 0.15 mg/kg), diazepam (0.15 or 0.3 mg/kg), or placebo (saline). The depth of sedation was estimated at 5-10-min intervals during the first hour after injection. A comprehensive battery of psychomotor tests was used to collect objective data of psychomotor performance before drug injection and 1, 3, 5, and 7 h after injection. Midazolam (0.15 mg/kg) produced the highest scores of sedation and most impairment of psychomotor performance. In most tests, the maximal psychomotor effects seen after 0.3 mg/kg of diazepam did not reach those of 0.1 mg/kg of midazolam. Although the strongest psychomotor effects were induced by midazolam, these effects disappeared sooner than those of diazepam. By 5 h after injection, 0.3 mg/kg of diazepam showed the highest scores of psychomotor impairment. The authors conclude that at least four times as much diazepam as midazolam is needed to produce equally severe psychomotor impairment. That the residual effects of midazolam terminate sooner than those of diazepam probably accounts for the occasional underestimation of the potency of midazolam in clinical practice. PMID- 1731550 TI - Airway considerations in the management of patients requiring long-term endotracheal intubation. PMID- 1731551 TI - Effect of cardiopulmonary bypass on plasma cyclosporin A levels in a renal transplant patient. PMID- 1731552 TI - Unilateral bronchospasm after interpleural analgesia. PMID- 1731553 TI - Bilateral analgesia and unilateral paresis after lumbar epidural blockade. PMID- 1731554 TI - A retained esophageal stethoscope as a potential cause of small bowel obstruction. PMID- 1731555 TI - Guide-wire retention after right atrial catheter insertion. PMID- 1731556 TI - Opioid-induced spasm of the sphincter of Oddi apparently reversed by nalbuphine. PMID- 1731557 TI - Horner's syndrome associated with brachial plexus anesthesia using an axillary catheter. PMID- 1731558 TI - Spinal anesthesia and Friedreich's ataxia. PMID- 1731559 TI - Reviewer gets reviewed. PMID- 1731560 TI - Use of the laryngeal mask airway to facilitate fiberscope-aided tracheal intubation. PMID- 1731561 TI - A technique for reducing pain associated with propofol administration. PMID- 1731562 TI - Empty the stomach by endoscopy before pyloromyotomy. PMID- 1731563 TI - Continuous arterial pressure monitoring during magnetic resonance imaging--an alternative method. PMID- 1731564 TI - Droperidol prevents serotonin-induced bronchospasm, pulmonary hypertension, and intrapulmonary shunt. PMID- 1731565 TI - Propofol and pharmacokinetic modeling. PMID- 1731566 TI - The gum-elastic bougie: a life saver. PMID- 1731567 TI - Oliguria due to obstruction of urine bag filter (Terumo Uroguard) PMID- 1731568 TI - A potential defect in an Ohmeda Sevotec III vaporizer. PMID- 1731569 TI - Controversies in hypersensitivity pneumonitis. PMID- 1731570 TI - Maximal exercise testing in single and double lung transplant recipients. AB - Patients with end-stage pulmonary and pulmonary vascular disease can now be offered single-lung (SLT), double-lung (DLT), or heart-lung (HLT) transplantation. Long-term survival with greatly improved pulmonary function has been reported with all three procedures. Little has been reported of the exercise capacity after transplantation. This report documents Stage 1 exercise test results in six SLT (five males and one female; age 50.7 +/- 4.4; FEV1.0 75.5 +/- 6.7% of predicted) and seven DLT (three males and four females; age 37.3 +/- 6.7; FEV1.0 85.1 +/- 10.3% of predicted) recipients, early (3 months) and late (1 to 2 yr) following transplantation. The results show low work rates and VO2max in both SLT and DLT recipients at 3 months after transplant. Heart rate and minute ventilation did not appear limiting. There was no significant improvement when retested 1 to 2 yr after transplant. At 3 months, VO2max was 46% of predicted for SLT and 50% of predicted for DLT recipients. It is concluded that considerable exercise limitation persists after transplantation and does not appear to improve with time. These limitations are similar for both SLT and DLT and are not related to ventilation. The results may suggest chronic muscle deconditioning after long term pretransplant debilitation. PMID- 1731571 TI - Observer agreement for respiratory signs and oximetry in infants hospitalized with lower respiratory infections. AB - To determine observer agreement for a clinical score and oximetry in lower respiratory infection in children less than 2 yr of age, a convenience sample of 56 infants hospitalized with bronchiolitis or pneumonia was assessed independently by two observers. A total of 12 infants had chronic lung disease of prematurity or congenital heart disease. Infants in whom oxygen supplementation could not be discontinued for at least 5 min were excluded. A severity score was assigned for each of four categories (respiratory rate, retractions, wheeze, and general appearance). A total for each patient was obtained by summing the score for each category. Oxygen saturation was measured using a Nellcor oximeter. Agreement beyond chance was measured using the kappa statistic. The relationship between observers for total score and oximetry and the mean total score and mean oximetry value for each patient was expressed as a Pearson correlation coefficient. A total of 56 infants and children were studied: 2 had pneumonia, 11 had an exacerbation of pulmonary signs and symptoms with their underlying cardiac or pulmonary disease, and 43 had bronchiolitis. Kappa was 0.48 for general assessment, 0.38 for respiratory rate, 0.31 for wheeze, and 0.25 for retractions. All values were statistically significantly greater than 0 at p less than 0.01. Correlations for total score and for oximetry were 0.68 and 0.88, respectively. The median difference between oximetry readings was 1. The correlation coefficient between total score and oximetry was -0.04. The limited agreement for clinical signs makes comparison of patient illness severity between studies difficult.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731572 TI - Lung, chest wall, and total respiratory system resistances and elastances in the normal range of breathing. AB - We measured total respiratory system and lung and chest wall resistances (Rrs, Rl, and Rcw) and elastances (Ers, El, and Ecw) in awake, relaxed human subjects during sinusoidal volume forcing at the mouth from 0.2 to 0.6 Hz with tidal volumes (VT) of 6 to 18% VC at constant mean airway pressure. In addition, we repeated measurements with the lowest VT at a lower airway pressure and therefore at a lower mean lung volume (Vl). Rrs and Rcw decreased with increasing respiratory frequency (f) and VT, but Rl was independent of f and VT. All resistances were higher at the lower Vl. Ers and Ecw increased with increasing f and decreased with increasing VT. El increased slightly with increasing f but was not affected by VT. All elastances tended to increase at the lower Vl. We conclude that in the normal range of breathing amplitude and frequency, (1) lung properties are nearly constant if mean lung volume does not change, and (2) f and VT dependencies of total respiratory system properties are caused by the chest wall. PMID- 1731573 TI - Proportional assist ventilation, a new approach to ventilatory support. Theory. AB - The relation between inspiratory effort and ventilatory return (flow and volume) is usually abnormal in patients who require ventilatory support because of respiratory distress. Although all available support methods provide the patient with greater ventilation than would obtain with the same effort while unsupported, the relation between instantaneous effort and ventilatory consequences is not normalized. We describe an approach with which the ventilator simply amplifies patient instantaneous effort throughout inspiration while leaving the patient with complete control over all aspects of breathing pattern (tidal volume, inspiratory and expiratory durations, and flow patterns). This approach is implemented by monitoring the instantaneous rate (V) and volume (V) of gas flow from ventilator to patient and causing applied pressure (P) to change according to the equation of motion [P = f1(V) + f2(V)], where f1 and f2 are appropriately selected functions for the relation between pressure and volume (elastic assist) and pressure and flow (resistive assist). There are several potential advantages to this approach: (1) greater comfort; (2) reduction of peak airway pressure required to sustain ventilation and, hence, the potential for avoiding intubation; (3) less likelihood of overventilation; (4) preservation and enhancement of patient's own reflex, behavioral, and homeostatic control mechanisms since the ventilator essentially becomes an extension of the patient's own muscles; and (5) improved efficiency of negative pressure ventilation. PMID- 1731574 TI - Proportional assist ventilation. Results of an initial clinical trial. AB - The response to proportional assist ventilation (PAV) was tested in four normal subjects during heavy exercise and in five ventilator-dependent patients recovering from assorted medical disorders. The apparatus consisted of a rolling seal piston coupled to a motor that generated pressure in proportion to inspired flow and inspired volume, with the gains adjusted such that the proportionality between airway pressure (Paw) and instantaneous patient-generated pressure (Pmus) was approximately 1:1 (i.e., machine-amplified patient effort by a factor of 2). Normal subjects responded to PAV by decreasing their own effort, as judged from esophageal pressure, such that the changes in ventilation and breathing pattern were rather small (VE: 64.8 +/- 3.6 during PAV versus 56.0 +/- 4.3, p less than 0.01; VT: 2.39 +/- 0.24 versus 2.02 +/- 0.17, p less than 0.05; f: 27.5 +/- 1.9 versus 28.0 +/- 2.2, NS). In patients, elastance ranged from 20 to 35 cm H2O cm/L, resistance ranged from 5 to 10 cm H2O/L/s, and maximal inspiratory pressure ranged from -16 to -65 cm H2O. After a period of observation during synchronized intermittent mechanical ventilation (SIMV) the patient was switched to PAV and maintained on it for 1 to 3 h. No patient had to be replaced on SIMV because of discomfort or deterioration in any of the monitored variables. During PAV peak airway pressure was less than half the value observed with the IMV breaths (16.6 +/- 2.4 versus 35.4 +/- 3.4 cm H2O, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731575 TI - Differing effects of nitrogen mustard and hydroxyurea on lung O2 toxicity in adult sheep. AB - We measured the effects of leukocyte depletion on the pulmonary response to breathing 100% O2 in adult sheep, using two dissimilar agents. A group of eight sheep received 4.0 g/day of hydroxyurea for 4 to 6 days, and six sheep received two to four doses of 0.4 mg/kg of nitrogen mustard before exposure to 100% O2. A group of seven sheep breathed 100% O2 with no drug treatment. A group of five control sheep breathed compressed air for 96 h. Hydroxyurea selectively reduced circulating neutrophils (602 +/- 245 neutrophils, 2,537 +/- 394 lymphocytes per mm3), and nitrogen mustard decreased both circulating neutrophils (633 +/- 326) and lymphocytes (521 +/- 129). In untreated sheep breathing 100% O2, survival time was 92.6 +/- 3.4 h and postmortem blood-free lung water to dry lung ratio was 4.4 +/- 0.2. Hydroxyurea significantly delayed the onset of oxygen toxicity as measured by changes in lung lymph flow, PaO2, PaCO2, and time to respiratory failure (111.8 +/- 7.7 h), although the degree of final lung injury was unchanged and postmortem lung water was elevated (5.3 +/- 0.3). Nitrogen mustard shortened the time to hypoxemia, and CO2 retention and decreased time to respiratory failure (84.0 +/- 4.3 h). Neutrophils were markedly reduced but not absent in the lungs of both groups of leukocyte-depleted sheep. We conclude that neutrophils are not essential for the full expression of lung O2 toxicity in adult sheep. Secondary effects of hydroxyurea and nitrogen mustard appear to influence the rate of development, but not the outcome of oxygen toxicity, by effects independent of leukocyte depletion. PMID- 1731576 TI - Influence of cardiac output on oxygen exchange in acute pulmonary embolism. AB - We investigated interactions between cardiac output, VA/Q distribution pattern, pulmonary gas exchange, O2 transport, and tissue oxygenation in 16 patients during the acute phase of pulmonary embolism (PE). The effects of breathing room air, O2 therapy (FIO2 = 0.40) (11 patients), and dobutamine (four patients) were studied after right catheterization using the multiple inert gas elimination technique. The pattern of VA/Q ratio distributions was found to depend essentially on cardiac output level. The individual blood flow perfusing ventilated areas was found to be inversely related to the mean VA/Q ratio of blood flow distribution. PVO2 was directly related to cardiac index (p less than 0.02), and negatively related to the mean VA/Q of blood flow distribution. In view of the influence of low VA/Q ratios and PVO2 on arterial hypoxemia, our results showed that the heart's response to PE conditioned the strategy of pulmonary gas exchange and O2 transport. Oxygen breathing led to a slight but consistent fall in cardiac output (-0.6 +/- 0.5 L/min, p less than 0.01). However, although PaO2 remained normal and PVO2 was slightly improved, we found no evidence for a role of hypoxic pulmonary vasoconstriction in the pulmonary hypertension observed during the acute phase of PE. Administration of dobutamine improved O2 transport and tissue oxygenation, although PaO2 remained constant or even fell in some cases because of increased VA/Q mismatch. PMID- 1731577 TI - Lack of effect of external warming on sleep architecture in sleep apnea/hypopnea syndrome. AB - Sleep apnea/hypopnea syndrome (SAHS) is characterized by nocturnal apneas (A) and/or hypopneas (H) occurring in various sleep stages. However, these disordered breathing events (DBE) occur rarely in slow-wave sleep (SWS) and are most severe in rapid eye movement (REM) sleep when severe hypoxemia results. Several studies have shown that a rise in body temperature by external warming, induced according to a specific protocol, affects normal human sleep architecture by diminishing the time spent in REM and increasing the time spent in SWS. The purpose of this study was to determine if external warming is as effective in sleep apneic patients in decreasing REM and increasing SWS, hoping that DBE and hypoxemia may diminish. Seven newly diagnosed patients were studied two more nights, on one of which (selected randomly) the sleep study was preceded by sitting in a warm bathtub with temperature of 41 degrees C for 1/2 h, 2 1/2 h before the start of sleep study. The mean maximum rise in oral temperature with warm bath was 2.0 +/- 0.4 degrees C. Total bed time (min) and sleep efficiency, REM, and SWS (%) were, respectively, 320 +/- 17 (SD), 81 +/- 8, 19 +/- 7, and 1.2 +/- 1.1 for the control (C) and 339 +/- 33, 79 +/- 11, 19 +/- 5, and 2.4 +/- 1.6 for the bath (B) night. In both C and B, the lowest O2 saturation (%) occurred in REM sleep and was 78 +/- 7 and 77 +/- 9, respectively. Comparing respective paired values between C and B, no significance was found.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731578 TI - The effect of snoring on mean arterial blood pressure during non-REM sleep. AB - The purpose of this study was to examine the relationship between snoring and mean arterial blood pressure during sleep. This was accomplished by performing continuous, all-night, simultaneous measurements of snoring, oxygen saturation, sleep stages, and arterial blood pressure in a group of eight snorers and five nonsnoring control subjects. The results were analyzed to determine whether changes in mean arterial blood pressure during non-rapid-eye-movement (non-REM) sleep are different in snorers from those in nonsnorers and whether they are related to nocturnal hypoxemia. Both groups were similar with respect to their anthropometric parameters and sleep architecture. Oxygen saturations during different stages of non-REM sleep were similiar within each group. However, the analysis of variance revealed that among snorers mean arterial blood pressure increased slightly during slow-wave sleep, whereas the nonsnorers reduced their blood pressure by 17.4 +/- 3.7% compared with wakefulness values. We also performed multiple linear regression analysis for the entire group of 13 subjects using the change in mean arterial blood pressure relative to wakefulness as the dependent variable and snoring frequency and mean arterial oxygen saturation as the independent variables; the results demonstrated that only snoring frequency, and not oxygen saturation, correlated significantly with the change in mean arterial blood pressure. We conclude that snoring may influence variation of blood pressure during sleep, preventing the normally observed reduction of arterial blood pressure associated with slow-wave sleep. PMID- 1731579 TI - Bronchial clearance of DTPA is increased in acute asthma but not in chronic asthma. AB - To investigate bronchial permeability in asthma, we measured the bronchial clearance of 113mIn-DTPA in seven asthmatics during and after an acute attack of asthma, seven asthmatics with chronic airflow limitation, and seven asthmatics without airflow limitation but with bronchial hyperresponsiveness to methacholine. We compared these results with those from seven normal subjects, seven patients with chronic bronchitis and bronchial infection, and seven patients with emphysema. An aerosol of 113mIn-DTPA was produced with a spinning disc to ensure a predominantly bronchial deposition of inhaled particles (6.3 microns MMAD). Radioactivity over the chest was recorded with a gamma-camera for 10 min after the subject inhaled the aerosol. Central regions of interest were selected, and the logarithm of the radioactivity was plotted against time; bronchial clearance of 113mIn-DTPA was calculated as the negative slope of the regression line. Clearance was substantially higher in asthmatics during their acute attacks than in all other groups (p less than 0.0001), and it decreased toward normal levels after recovery from the acute episode. The bronchial clearance of 113mIn-DTPA in all other groups did not differ from normal. We conclude that the bronchial clearance of 113mIn-DTPA is increased in asthmatics during attacks of asthma but in the stable state is not related either to bronchial hyperresponsiveness or to airflow limitation. Our findings are best explained by an increase in permeability of the bronchial mucosa of asthmatics during acute attacks. PMID- 1731580 TI - Glutathione localization and distribution after intratracheal instillation. Implications for treatment. AB - To gain insight into how glutathione given directly into the lung protects fasted mice against hyperoxic lung damage and to provide a framework for developing treatment strategies in patients, we determined the lung distribution and retention of intratracheally administered glutathione (GSH) and its fate after leaving the lung. Mice received an intratracheal injection of [3H]GSH with or without cold GSH and with or without liposomes. The distribution of the 3H label was equal in both lungs, but the left lung had a higher concentration because of its smaller size. The 3H label was cleared rapidly from the lung: only 1% remained at 24 h, and more than 50% of the label at that time was no longer attached to the GSH. Administration of GSH with liposomes increased the retention of GSH by 20 to 50%, but the amount remaining at 24 h was still only 1%. The increase associated with the liposomes was due to enhanced retention of the GSH encapsulated in the liposomes, not the much larger amount present free in the GSH liposome mixture. Fasting and exposure to 100% oxygen had little effect on GSH retention. Some of the 3H label leaving the lung was excreted by the kidneys, a small amount was retained in the liver, and a large amount accumulated in the blood. Of the amount in the blood, about 60% was in red blood cells (RBC) and the rest in plasma. Much of the 3H label in RBC and lung at 24 h was no longer attached to the GSH, whereas most in the plasma and liver was.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731581 TI - Morphometric analysis of airways in idiopathic pulmonary fibrosis patients with mucous hypersecretion. AB - Five idiopathic pulmonary fibrosis (IPF) patients with sputum since the initial period of the disease (IPF SP+, more than 15 ml/day) were compared with five IPF patients without sputum throughout the course of the disease (IPF SP-) and four control subjects without pulmonary disease matched for age and sex. No significant differences in the duration of symptoms, pulmonary functions, or glucocorticoid therapy were observed between the two IPF groups. Autopsied lungs fixed by immersion into formaldehyde were used for morphometry by digitizing computer. The volume proportion of glands to bronchial wall thickness (gland%), volume proportion of goblet cells to total epithelial layer (goblet%), and luminal mucous volume were measured in central and peripheral airways. The gland percentage in the central airways of the IPF SP+ group was 18 +/- 1% (mean +/- SE), which was significantly greater than 7 +/- 0.6% of the IPF SP- group (p less than 0.001), similar to the 6 +/- 1% of control subjects. Luminal mucous volume in the peripheral airways of the IPF SP+ group was 11 +/- 2%, which was significantly greater than 3 +/- 1% of the IPF SP- group (p less than 0.05) or 0.6 +/- 0.3% of the control subjects (p less than 0.01). Furthermore, luminal mucous volume in both the central and peripheral airways significantly correlated with gland% (p less than 0.01, each). No significant difference in other parameters such as goblet% and cell infiltration between the IPF SP+ group and IPF SP- group was observed. These findings suggest that IPF with hypersecretion is associated with mucous glandular hypertrophy and the accumulation of mucus in the airways. PMID- 1731582 TI - Lung-restricted activation of the alveolar macrophage/monocyte system in pulmonary sarcoidosis. AB - An activation of T-cells that is restricted to the lung has been demonstrated in pulmonary sarcoidosis. The role of blood monocytes (MO) and alveolar macrophages (AM) in this concept of compartmentalized inflammation has not yet been evaluated. In order to elucidate this question, we measured the release of tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) by peripheral blood mononuclear cells (PBMNC) and AM in 43 patients with sarcoidosis (32 with active, 11 with inactive disease) without therapy and correlated the spontaneous monokine release to parameters of the T-cell alveolitis and the course of the disease. TNF alpha as well as IL-1 were spontaneously released by AM of the active group, i.e., 2,385 +/- 735 pg/ml/10(8) cells/24 h and 7/12 (IL-1+/total), respectively. Autologous PBMNC were quiescent, releasing only baseline levels of any monokine. AM were not activated in the inactive group, releasing 500 +/- 212 pg/ml/10(6) cells/24 h TNF alpha, whereas 1/5 were IL-1-positive (p less than 0.05 in both comparisons), which is within the range of the control group. Kinetic experiments revealed that the TNF alpha gene of AM is activated in vivo, resulting in TNF alpha mRNA-positive, TNF alpha-releasing cells that, cultured in vitro, regulate the TNF alpha gene transcription down and cease to release TNF alpha. Interestingly, there is no stringent correlation between the spontaneous release of TNF alpha by AM and signs of T-cell activation as soluble interleukin-2 (IL-2) receptor serum concentration, release of IL-2, and expression of IL-2 receptor by alveolar T-cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731583 TI - Metabolism of exogenously administered surfactant in the acutely injured lungs of adult rabbits. AB - Acute lung injury was induced in adult rabbits with a subcutaneous injection of N nitro-so-N-methylurethane (NNNMU). Clearance of saturated phosphatidylcholine (Sat.PC) from a treatment dose of exogenous surfactant (100 mg/kg) in the injured lungs of these rabbits was similar to normal, control rabbits when measured 24 h after treatment. However, total Sat.PC pool sizes in both the alveolar wash and total lung at this time point were significantly lower for the injured lungs than for the control lungs (p less than 0.05), implying altered endogenous surfactant metabolism in response to surfactant treatment in the injured animals. Although both injured and control animals had comparable ratios of small to large surfactant aggregates, as measured by differential centrifugation of alveolar wash 5 min after treatment, by 24 h this ratio had increased 5-fold in the control animals and remained unchanged in the injured animals. This indicated diminished conversion of large surfactant aggregates to the smaller forms in lung injury. In vivo functional studies of these aggregates were performed by intratracheal injection into surfactant-deficient preterm rabbits. Large aggregates from normal adult rabbits given surfactant had superior functional properties than did the surfactant used for treatment alone, which in turn was better than large aggregates isolated from NNNMU-injured rabbits treated with surfactant. This indicates that the alveolar environment influenced the function of the exogenously administered surfactant differently in normal and injured rabbits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731584 TI - Partial reversal of hypoxic pulmonary hypertension by heparin. AB - Chronic hypoxia produces pulmonary hypertension and an increase in medial thickness of pulmonary arteries that reach maximal values after 10 days of hypoxia. We previously showed that heparin given during the first 10 days of hypoxia reduced the development of both pulmonary hypertension and vascular remodeling in the guinea pig. To determine if heparin could reverse established hypoxic pulmonary hypertension and vascular remodeling, we administered heparin by continuous subcutaneous infusion (20 U/kg/h) for the last 7 days of a 21-day exposure to hypoxia (10% O2, balance N2) and compared these animals with normal saline-infused hypoxic control and room air-exposed animals. Hypoxia increased pulmonary artery pressure from 11 +/- 1 mm Hg (mean +/- SEM) in room air animals to 20 +/- 2 mm Hg (p less than 0.05) in saline-treated hypoxic control animals. Heparin reduced pulmonary artery pressure to 16 +/- 1 mm Hg (p less than 0.05 versus hypoxic control and room air control animals). Total pulmonary resistance (TPR) increased with hypoxia from 0.043 +/- 0.003 mm Hg x min x kg-1 x ml-1 in room air to 0.090 +/- 0.004 in hypoxia (p less than 0.05), and in the rise in TPR was also partially reversed by heparin to 0.068 +/- 0.0003 (p less than 0.05). The percentage of medial thickness of alveolar duct arteries increased from 5.8 +/- 0.6% in room air to 9.5 +/- 0.1% (p less than 0.05) after 3 wk of hypoxia, and heparin therapy partially reversed the increase in medial thickness to 7.2 +/ 0.7% (p less than 0.05 versus both hypoxia control and room air).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731585 TI - Immunocytometry and gene rearrangement analysis in the diagnosis of lymphoma in an idiopathic pleural effusion. AB - We report a patient with an idiopathic pleural effusion in whom the diagnosis of non-Hodgkin's lymphoma was established by immunocytometry of pleural fluid and confirmed by the detection of B-cell immunoglobulin gene rearrangement. Immunocytometry is a rapid, semi-automated laboratory method for phenotyping lymphoid cells by determining immunoglobulin and other cell surface antigen expression. This method defines the cell lineage (T or B cells) and the clonality (monoclonal or polyclonal) of a population of lymphocytes. The presence of a monoclonal population of lymphocytes can also be confirmed by recently developed molecular biologic techniques (e.g., Southern blotting) that provide the ability to detect rearrangements of the genes that encode either B-cell immunoglobulin proteins or T-cell antigen receptor proteins. To our knowledge, this case represents the first reported application of immunophenotypic and gene rearrangement analysis in a previously undiagnosed pleural effusion to establish the diagnosis of lymphoma. These relatively new laboratory methods may have a role in the evaluation of idiopathic lymphocytic pleural effusions. PMID- 1731586 TI - Aerosolized atropine sulfate: influence of inhalation pattern on effective blockade of vagal airway tone. AB - The influence of aerosolized atropine sulfate on airway tone was evaluated in nine healthy adult subjects using three modes of inhalation and a dosimeter to deliver equal doses of aerosol. For six of the subjects additional studies with radioaerosols and scintillation scans were accomplished to qualify lung distributions of deposited aerosol. The three breathing patterns, identified as Tidal, IC, and VC, had average inspiratory volumes of 0.66 +/- 0.1, 2.10 +/- 0.4, and 4.31 +/- 0.9 (SD) L and were initiated from the rest position of the lung for the first two patterns, and residual volume for the third pattern. Total nebulization time and concentration inhaled were identical for each pattern at an atropine dose of 0.025 mg/kg body weight. Average inspiratory flow rates had means of 0.40 +/- 0.1, 0.64 +/- 0.2, and 0.82 +/- 0.2 (SD) L/s for the respective inhalations. Functional indices of FEV1, MMF, and Vmax50 and anticholinergic side effects were assessed for a 4-h period after aerosol administration. Functional improvement and duration of effect were maximal with the IC pattern. Within the first hour, absolute increases in FEV1 averaged 240 ml above baseline (6.2% increase). Increases for MMF and Vmax50 were on average greater than 23% above baseline (airflow benefit exceeded baseline by 0.91 +/- 0.4 L/s for MMF and 1.14 +/- 0.4 L/s for Vmax50). Except for xerostomia, which was present after all patterns, systemic side effects (tachycardia, blurred vision, and urinary retention) occurred only with VC pattern.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731587 TI - Noninvasive assessment of blood gases. PMID- 1731588 TI - Flexible endoscopy of the pediatric airway. PMID- 1731589 TI - Spirometry: quality control and reproducibility criteria. PMID- 1731590 TI - Antiasthma drugs and airway hyperreactivity. PMID- 1731591 TI - Changes in mucociliary clearance during acute exacerbations of asthma. PMID- 1731592 TI - Elevated bronchoalveolar lavage histamine in allergic asthmatics associated with increased airway obstruction. PMID- 1731593 TI - Importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants. AB - The biophysical properties and physiologic effects of a series of clinical exogenous pulmonary surfactants was compared to determine the importance of the hydrophobic apoproteins (SP-B and C) as constituents of these preparations. The three exogenous surfactants studied, calf lung surfactant extract (CLSE), Survanta (Surfactant-TA), and Exosurf, all contain dipalmitoyl phosphatidylcholine (DPPC) as their major constituent. CLSE and Survanta also contain 1 to 2% of SP-B,C but Exosurf has the additives hexadecanol and tyloxapol instead to enhance the activity of DPPC. In adsorption experiments, CLSE reached a final surface tension of 22 mN/m, and Survanta and Exosurf reached 28 and 38 mN/m, respectively. Addition of 1% by weight of an apoprotein isolate containing both SP-B and C to Exosurf slightly improved its adsorption. In oscillating bubble studies, CLSE and Survanta decreased surface tension to low values of less than 1 and 2 mN/m, respectively, but Exosurf achieved a minimum value of only 29 mN/m. Addition of SP-B,C to Exosurf improved this minimum to 1 mN/m and approached the behavior of mixtures of synthetic DPPC with SP-B,C. In both adsorption and pulsating bubble experiments, the minimum surface tensions found for Exosurf were almost identical to those generated by tyloxapol alone. In studies of physiologic activity, 20 mg of CLSE or Survanta restored the pressure volume mechanics of lavaged, surfactant-deficient excised rat lungs to 95 and 50%, respectively, of normal prelavage levels. Instillation of Exosurf (37.5 mg) produced a minimal improvement of only 10% compared to 70% for mixtures containing 1% SP-B,C with either Exosurf or DPPC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731594 TI - Effect of corticosteroid treatment on the recovery of pulmonary function in farmer's lung. AB - The effect of corticosteroid treatment on the course of farmer's lung (FL) was studied in 36 patients randomly allocated in a double-blind placebo-controlled study. All patients were in the acute stage of the disease and had had the first diagnosed attack of FL. Twenty patients were given prednisolone treatment for 8 wk. Sixteen patients received an 8-wk placebo treatment. One patient was withdrawn from the analysis when she terminated corticosteroid treatment because of side effects. After 1 month of treatment there was a significant difference (p = 0.03) in DLCO between the treatment groups. After a follow-up of 5 yr no statistically significant differences were found between the treatment groups in FVC, FEV1, or DLCO. FL recurred in six patients during the follow-up in the corticosteroid group and in one patient in the placebo group, but the difference was not statistically significant. In conclusion, in the corticosteroid group the improvement of pulmonary function was more rapid than in the placebo group, but no influence on the long-term result was found. The possibility that corticosteroid treatment may favor the occurrence of recurrent attacks of FL needs attention. PMID- 1731595 TI - Parenteral followed by oral ofloxacin for nosocomial pneumonia and community acquired pneumonia requiring hospitalization. AB - We report a multicentric, open trial of intravenous followed by oral ofloxacin, 400 mg every 12 h, as therapy for 100 cases of nosocomial pneumonia and community acquired pneumonia requiring hospitalization. The typical subject was 57 yr old, and underlying diseases, such as chronic obstructive pulmonary diseases (COPD), diabetes mellitus, and congestive heart failure, were common. For 10 subjects previous therapy had failed. There were 118 pathogens isolated in blood or sputum; S. pneumoniae was the most common (42), followed by H. influenzae (13), Klebsiella spp. (11), and S. aureus (10). Ofloxacin was administered for an average of 5.7 days intravenously followed by 6.9 days orally. Response to therapy was judged to be cure in 71 subjects, improvement in 24, and failure in 5. Among the more seriously ill subjects, ofloxacin therapy was successful for four of five immunocompromised subjects, for 12 of 12 subjects with nosocomial pneumonia, three of whom were on the ventilator, and for nine of 10 subjects with community-acquired pneumonia and bacteremia, including seven of eight cases due to S. pneumoniae. Univariate risk factor analysis revealed underlying COPD and/or tachypnea upon admission to be associated with failure of ofloxacin therapy, with bacteremia suggestive of failure. Conversely, ofloxacin was equally effective in cases in whom previous therapy failed and in cases of nosocomial pneumonia, multilobar pneumonia, and/or pneumonia due to S. pneumoniae. Results for P. aeruginosa were inconclusive. Intravenous followed by oral ofloxacin was highly effective in many difficult cases of pneumonia. PMID- 1731596 TI - A double-blind placebo-controlled clinical trial of three antituberculosis chemoprophylaxis regimens in patients with silicosis in Hong Kong. Hong Kong Chest Service/Tuberculosis Research Centre, Madras/British Medical Research Council. AB - A double-blind placebo-controlled trial of antituberculosis chemoprophylaxis was undertaken in sillicotic subjects in Hong Kong where there is a high prevalence of both silicosis and tuberculosis. During 1981 to 1987, 679 Chinese men with silicosis, with no history of previous antituberculosis chemotherapy and no evidence of active tuberculosis, were admitted to the trial and have been studied for between 2 and 5 yr. They were allocated at random to four series-rifampin for 12 wk (R3), isoniazid and rifampin for 12 wk (HR3), isoniazid alone for 24 wk (H6), or placebo (Pl)--in a double-blind design with matching placebos for isoniazid and rifampin as appropriate. Active pulmonary tuberculosis developed more frequently during the 5 yr in the placebo series than in the three chemoprophylaxis series (p less than 0.01, log-rank test), but there were no significant differences between the chemoprophylaxis series. The estimated proportions of patients with active pulmonary disease in the placebo series were 9% at 2 yr, 15% at 3 yr, 20% at 4 yr, and 27% at 5 yr. In contrast, in the three chemoprophylaxis series combined they were 5, 8, 10, and 13%, respectively. Thus, although chemoprophylaxis halved the proportion of patients in whom tuberculosis developed, this proportion was still substantial. There was no evidence that chemoprophylaxis led to the selection of drug-resistant strains of bacilli. Adverse effects were reported with a similar frequency in all four series, suggesting that few were drug related. During the first 12 wk, hepatic toxicity was reported in 8 (1%) patients (3 HR3, 3 H6, and 2 Pl), but only 1 (H6) had symptomatic hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731597 TI - Air pollution and respiratory symptoms in preschool children. AB - A diary study on a random sample of 625 Swiss children aged 0 to 5 yr was conducted in two cities in Switzerland to investigate the association between air pollution and respiratory symptoms. Total suspended particulates (TSP), SO2 and NO2 were measured by city monitor. In addition, passive samplers inside and outside the home measured NO2 concentration during the 6 wk each child was on the diary. Diaries were filled out by parents, and 20% were validated with the attending pediatrician's case notes. Incidence and duration of symptom episodes were examined separately. The study included any episode, episodes of coughing without runny nose, upper respiratory episodes, and episodes of breathing difficulty. In regressions using 6-wk average pollution that controlled for medical history, NO2 measured outdoors but not indoors was associated with the duration of any symptom. Total suspended particulates were a more significant predictor of duration of any symptom than NO2. The 6-wk average TSP was significantly associated with incidence of coughing episodes and marginally significant as a predictor of upper respiratory episodes. Previous day's TSP was a significant predictor of incidence of upper respiratory symptoms. Annual average of NO2 was associated with the duration of any episode and of upper respiratory episodes. We conclude that the incidence and duration of respiratory symptom episodes are likely associated with particulate concentrations and duration may be associated with NO2. PMID- 1731598 TI - Spirometric lung function. Distribution and determinants of test failure in a young adult population. AB - Spirometric test failure has been defined as failure by a subject to meet the acceptability and/or reproducibility criteria laid down by the American Thoracic Society for measurements derived from forced expiratory maneuvers. The prevalence and determinants of spirometric test failure were examined in 416 men and women aged 20 to 45 yr working in an office environment. In this study population, 11.5% (28 men and 20 women) exhibited test failure for forced expiratory volume in one second (FEV1). The main determinant of test failure in men was bronchial hyperresponsiveness to methacholine challenge (odds ratio 6.7; confidence interval 1.7, 27.1) and in women being a current smoker (odds ratio 4.02; confidence interval 1.13, 14.33). There was also a relationship to eczema in both men and women, but not at a statistically significant level. When FEV1 variability was defined as the difference (in milliliters) between the two best FEV1 values and the results of men and women combined for analysis, significant predictors were a history of eczema, recurrent chest illness in the past 3 yr, and level of bronchial responsiveness to inhaled methacholine. These findings contribute to the gathering evidence that test failure may be of itself an indicator of impaired respiratory health, and its association with bronchial hyperresponsiveness to methacholine in men suggests that in them test failure is related to airway lability, but in women the relationship to smoking suggests an irritative mechanism. PMID- 1731599 TI - Misclassification of smoking status by self-reported cigarette consumption. AB - To evaluate possible misclassification of smokers and nonsmokers, we compared self-reported cigarette consumption and serum cotinine levels in a sample of 743 Mexican American participants in the Hispanic Health and Nutrition Examination Survey (HHANES). The study sample was stratified by sex and self-reported cigarettes consumed per day (0, 1 to 9, 10 to 19, and greater than or equal to 20) and selected from those with available serum. We defined biochemical smokers as persons with serum cotinine levels greater than or equal to 0.084 microM/L (14 ng/ml). Misclassification was defined as a discrepancy between self-reported smoking and the serum cotinine level used to define a biochemical smoker. Of 189 self-reported nonsmokers, 12 (6.3%) were defined as biochemical smokers and possibly misclassified by self-report. Among 124 never smokers only 5 (4%) were biochemical smokers compared with 7 of 65 (10.8%) self-reported former smokers. Only 1 of the 12 misclassified nonsmokers reported living with a current smoker. In 9 of the 12 misclassified nonsmokers, serum cotinine levels were consistent with light smoking. Among the 547 self-reported smokers, 66 (12.1%) were found to have serum cotinine levels less than or equal to 0.084 microM/L (14 ng/ml) and possibly misclassified by self-report. Of these, one person reported 20 or more cigarettes per day. We conclude that self-reported cigarette consumption may be an insufficient measure of the risks associated with tobacco use and measurement of serum cotinine may be important to assess the magnitude of misclassification of smoking status in epidemiologic studies. PMID- 1731600 TI - Effects of asthma on pulmonary function in children. A longitudinal population based study. AB - Data from a longitudinal study of childhood factors influencing the development of chronic obstructive lung disease were used to assess the effects of asthma on lung function development in male and female children. A population-based cohort of 602 white children, initially aged 5 to 9 yr, was observed prospectively for 13 yr. Spirometry was performed and a standardized respiratory and illness questionnaire was administered by trained interviewers on a yearly basis. Forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and forced expiratory flow between 25 and 75% of vital capacity (FEF25-75) were used as measures of lung function. The total number of children reporting asthma over the course of the study was 67. Male asthmatic subjects (n = 42) had larger average percentage of predicted FVC than nonasthmatic males (n = 277). Female asthmatic subjects (n = 23) had a lower average percentage of predicted FEV1 than nonasthmatic females (n = 260). In a multivariate analysis of the individual lung function measures, adjusting for previous level of pulmonary function, age, height, change in height, and personal and maternal smoking, males reporting active asthma had a significantly larger FVC than males with no history of asthma. In contrast, females with active asthma had a significantly smaller FEV1 than females with no history of asthma. Both males and females with active asthma had decreased FEF25-75. From our analysis, we would predict that a female who develops asthma at age 7 would experience a 5% reduction in FEV1 by age 10 and a 7% deficit by age 15.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731602 TI - Dysfunction of nonadrenergic noncholinergic inhibitory system after antigen inhalation in actively sensitized cat airways. AB - We have investigated whether proteases released during antigen inhalation cause dysfunction of the nonadrenergic noncholinergic inhibitory nervous system (NANCIS). Frequency-response (F-R) studies of NANCIS were performed before and after Ascaris antigen (ASC) inhalation using actively sensitized cats. NANC dilatatory effects were obtained by stimulating bilateral cervical vagi under cholinergic and beta-adrenergic blockade and serotonin-induced bronchoconstriction, and assessed by maximal percent relaxation (rmax) and the frequency causing 50% of maximal relaxation (EF50). ASC inhalation caused a transient increase in pulmonary resistance in all animals. One hour after ASC inhalation, pulmonary resistance returned to the baseline value, but ASC inhalation significantly attenuated NANC inhibitory activities: rmax decreased from 82.2 +/- 4.7 (mean +/- SE) to 64.3 +/- 11.2% (p less than 0.05), and the geometric mean of EF50 increased from 1.7 to 4.3 Hz (p less than 0.05). Dilatatory effects of infused VIP, a possible neurotransmitter of NANCIS, was also attenuated after ASC inhalation. Pretreatment with leupeptin (3 mg/kg) abolished ASC-induced impairment of NANC inhibitory activities. By contrast, dilatatory effects of adrenergic nerve stimulation were not affected by ASC inhalation. These results suggest that NANC inhibitory activities can be impaired after ASC inhalation, and that this impairment of NANCIS may be due to effects of proteases released during allergic reaction. PMID- 1731601 TI - Primary immunization in the canine lung. Soluble antigen induces a localized response. AB - Primary immunization of the dog by intralobar instillation of particulate antigen induces an intense, localized pulmonary antibody response. In contrast, although soluble antigen can also induce local antibody responses after repeated deposition in the canine respiratory tract, its ability to induce local responses after primary immunization has not been well characterized. To document such responses, we immunized five beagle dogs using a bronchoscope to instill 10 mg keyhole limpet hemocyanin (KLH) into a single lung lobe (immunized) and saline into a contralateral lung lobe (control). Over the next 3 wk, we monitored specific immune responses in blood and bronchoalveolar lavage (BAL) fluids obtained from the immunized and control lung lobes. Primary intrapulmonary immunization of dogs with KLH resulted in anti-KLH antibody responses both in blood and in immunized and control BAL fluids. However, immunoglobulin class specific expression of response differed between the immunized and control lung lobes. Specific IgM and IgA responses were significantly greater in the immunized lobes. In contrast, specific IgG, and cells producing specific IgG, were quantitatively similar in lavage fluids derived from immunized and control lung lobes. These studies demonstrate that primary immunization of the dog by intralobar instillation of soluble antigen stimulates a local IgM and IgA response and an IgG response that distributes to both immunized and unimmunized lung. This pattern of immunoglobulin class-specific pulmonary antibody response has the potential to importantly influence regional responses to intrapulmonary antigen. PMID- 1731603 TI - The effect of chlorothiazide on neurally mediated contraction of rabbit bronchial smooth muscle. AB - The neuromodulatory action of chlorothiazide (CTZ) was investigated in isolated rabbit bronchial smooth muscle (BSM) segments contracted with electrical field stimulation (ES). The tissues were placed in organ baths and stimulated with ES frequencies ranging from 1 to 75 Hz. CTZ (10(-4) to 10(-3) M) produced dose dependent increases in ES-induced contractions. In the presence of 10(-3) M CTZ, the mean +/- SEM maximal tension (Tmax) induced by ES increased significantly (p less than 0.03) from 292.8 +/- 39.5 to 363.0 +/- 58.5 g/g tissue. BSM sensitivity to ES, expressed as the log ES frequency producing 50% of Tmax (i.e., log ES50) was also increased (p less than 0.001) in the presence of CTZ as indicated by a fall in the mean +/- SEM log ES50 from 1.05 +/- 0.05 to 0.804 +/- 0.09 Hz. The potentiating effect of CTZ on ES-induced contractions was independently blocked by either the neurotoxin, tetrodotoxin (4 x 10(-6) M), or the cholinergic antagonist, atropine (10(-5) M). In the presence of CTZ, the mean Tmax response to acetylcholine (ACh) was unaffected, whereas BSM sensitivity to the agonist increased significantly (p less than 0.001). On the other hand, the dose-response relationship to carbachol, a cholinergic agonist resistant to cholinesterase degradation, was unaffected by CTZ. In tissues pretreated with 10(-5) M neostigmine, an acetylcholinesterase (AChase) inhibitor, CTZ did not further augment either ES- or ACh-induced contractions. Taken together, these findings suggested that CTZ might be acting as an AChase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731604 TI - Airway hypocapnia increases microvascular leakage in the guinea pig trachea. AB - We have previously shown that airway hypocapnia induced bronchoconstriction in the guinea pig lung by releasing tachykinins. To examine whether airway hypocapnia could also cause an increase in airway microvascular leakage, a tracheal segment was isolated in vivo in anesthetized guinea pigs and unidirectionally ventilated (200 ml/min) for 1 h with fully conditioned air (0% CO2) or isocapnic gas (5% CO2). The lungs were ventilated through a distally placed tracheal cannula. Microvascular leakage was quantitated by the injection of Evans blue (EB) and its extraction from the tracheal segment. EB extravasation was increased in tracheae exposed to 0% CO2 (52.3 +/- 2.0 micrograms/g wet tissue) compared with tracheae exposed to 5% CO2 (26.4 +/- 2.9 micrograms/g; p less than 0.05) and to tracheae from spontaneously breathing guinea pigs (25.2 +/ 2.3 micrograms/g; p less than 0.05). Groups of animals in which trachea were unidirectionally ventilated with 0% CO2 were then pretreated with a range of drugs in an attempt to determine the mediators responsible for the microvascular leakage with 0% CO2. Capsaicin and morphine pretreatment did not significantly alter 0% CO2-induced EB extravasation, and phosphoramidon prevented rather than increased extravasation, suggesting that tachykinins did not play a role. The hypocapnia-induced increase in microvascular leakage was, however, prevented by indomethacin pretreatment and significantly attenuated by dazmegrel, a thromboxane synthetase inhibitor. We conclude that airway hypocapnia causes microvascular leakage in the guinea pig trachea and that this effect is mediated by prostaglandins and/or thromboxane. PMID- 1731605 TI - Pulmonary status of habitual cocaine smokers. AB - We determined the prevalence of respiratory symptoms and lung dysfunction in a large sample of habitual smokers of freebase cocaine ("crack") alone and in combination with tobacco and/or marijuana. In addition, we compared these findings with those in an age- and race-matched sample of nonusers of crack who did or did not smoke tobacco and/or marijuana. A detailed respiratory and drug use questionnaire and a battery of lung function tests were administered to (1) a convenience sample of 202 habitual smokers of cocaine (cases) who denied intravenous drug abuse and (2) a reference sample of 99 nonusers of cocaine (control subjects). The cocaine smokers (85% black) included the following: 68 never-smokers of marijuana, of whom 43 currently smoked tobacco and 25 did not, and 134 ever-smokers of marijuana (42 current and 92 former), of whom 92 currently smoked tobacco and 42 did not. The control subjects (96% black) included the following: 69 never-smokers of marijuana, of whom 26 currently smoked tobacco and 43 did not, and 30 ever-smokers of marijuana (18 current and 12 former), of whom 21 currently smoked tobacco and 9 did not. Cases smoked an average of 6.5 g cocaine per week for a mean of 53 months. The median time of the most recent use of crack prior to study was 19 days (range less than 1 to 180 days). After controlling for the use of other smoked substances, frequent crack use was associated with: (1) a high prevalence of at least occasional occurrences of acute cardiorespiratory symptoms within 1 to 12 h after smoking cocaine (cough productive of black sputum [43.7%], hemoptysis [5.7%], chest pain [38.5%], usually worse with deep breathing, and cardiac palpitations [52.6%]) and (2) a mild but significant impairment in the diffusing capacity of the lung.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731606 TI - Distribution of ADPase activity in the lactating rat mammary gland and its possible role in an ATP cycle in the Golgi apparatus. AB - A Ca2+/Mg(2+)-stimulated ADPase has been found to occur in the lactating rat mammary gland. The enzyme is membrane associated and occurs in mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 6.3 and one at pH 7.4. Studies with inhibitors and activators indicate that the enzyme is similar to ADPases found in other tissues and is distinct from the uridine nucleoside diphosphatase previously reported in the mammary Golgi apparatus. The occurrence of ADPase in the Golgi apparatus indicates a possible role for this enzyme in the milk secretory process, while the microsomal enzyme could be involved in extracellular activities. PMID- 1731607 TI - Inositol 1,4,5-trisphosphate compartmentalization in tracheal smooth muscle. AB - Pool sizes of inositol phosphate species in myo-[3H]inositol-labeled porcine tracheal smooth muscle were determined under three conditions: (a) unstimulated; (b) stimulated with carbachol; (c) atropine-relaxed from a carbachol contraction. In unstimulated muscle, the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content was 14 pmol/100 nmol lipid P1. This is equivalent to a mean [Ins(1,4,5)P3] of about 3 microM (in total cellular water), a level about 30-fold in excess of that required for Ca2+ release from Ins(1,4,5)P3-sensitive sarcoplasmic reticulum (SR). Pool sizes of breakdown products of Ins(1,4,5)P3 were relatively small or absent in unstimulated muscle, suggesting that, under this condition, Ins(1,4,5)P3 was sequestered and had limited access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. During carbachol stimulation, the Ins(1,4,5)P3 pool did not increase while those of other mono-, di-, and trisphosphate isomers increased over 10-fold. Subsequent atropine-induced relaxation resulted in a partial depletion (40%) of total tissue Ins(1,4,5)P3. Decreases in Ins(1,4,5)P3 were paralleled by decreases in Ins(1,4)P2 and Ins(1,3,4)P3. During contraction a portion of total tissue Ins(1,4,5)P3 has access to Ins(1,4,5)P3 3-kinase and 5 phosphatase and to Ins(1,4,5)P3-sensitive SR, though during antagonist-induced relaxation access to Ins(1,4,5)P3-sensitive SR for Ca2+ release is restricted. Data are consistent with a mechanism by which a large pool of Ins(1,4,5)P3 present in the unstimulated state in a sequestered compartment can contribute in activated muscle to increases in [Ins(1,4,5)P3] in a nonsequestered compartment, controlling SR Ca2+ release. PMID- 1731608 TI - Identification of human placental leucine aminopeptidase as oxytocinase. AB - Human placental leucine aminopeptidase (P-LAP) was purified from retroplacental serum for the first time by serial chromatography on columns of Matrex Blue A, DEAE-Sepharose CL-6B, phenyl-Sepharose 4B, chelating-Sepharose, and Sepharose CL 6B. The purified P-LAP was apparently homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis and the apparent molecular weight (Mr) was estimated to be 210,000. By comparing P-LAP activity with cystine aminopeptidase activity, we concluded that both activities were shared by the same molecule. We also examined the hydrolytic activity of P-LAP using naturally occurring peptide hormones and found that the enzyme hydrolyzed oxytocin, vasopressin, and angiotensin III. These results suggest that P-LAP shows oxytocinase activity and plays an important role in the regulation of the plasma level of these hormones during pregnancy. PMID- 1731609 TI - Influence of histidine on lipid peroxidation in sarcoplasmic reticulum. AB - The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo. PMID- 1731610 TI - Cleavage of proteoglycan aggregate by leucocyte elastase. AB - The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains. PMID- 1731611 TI - Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals. AB - Thiyl free radicals have been shown to react with polyunsaturated fatty acids via abstraction of bisallylic hydrogen, forming pentadienyl radicals, and via addition to the double bonds. In the absence of oxygen, the latter pathway leads to regeneration of thiyl radicals through beta-elimination or "repair" of the adduct radicals by thiols. In the presence of oxygen, fixation of thiyl-induced damage occurs through reaction of O2 with the pentadienyl radical (yielding conjugated dienyl peroxyl radicals) and also with the thiyl-to-double bond adduct radical. A quantitative reaction scheme evaluated from these data considers abstraction, addition, rearrangement, and repair reactions, and the evaluation of rate constants for the individual steps. Absolute rate constants have been measured, in particular, for reactions of thiyl free radicals from glutathione, cysteine, homocysteine, N-acetylcysteine, cysteine ethyl ester, penicillamine, captopril, mercaptoethanol, and dithiothreitol with polyunsaturated fatty acids (PUFAs) ranging from 18:2 to 22:6, and the lipids trilinolein and trilinolenin. The rate constants for hydrogen abstraction were found to be typically of the order of 10(7) mol-1 dm3 s-1 and to increase with increasing lipophilicity of the attacking thiyl radical. Thioperoxyl radicals, RSOO., were found to be rather unreactive toward PUFAs, in contrast to the isomer sulfonyl radicals, RSO2., which not only abstract hydrogen from the bisallylic methylene groups of the PUFAs (although only at relatively small yield) but also readily add to the PUFA double bonds (major pathway). Specific information was obtained on the optical properties of the thiyl radical derived from the ACE inhibitor captopril, CpS. (lambda max = 340 nm, epsilon = 460 +/- 50 mol-1 dm3 cm-1), and its conjugate disulfide radical anion (CpS:.SCp) (lambda max = 420 nm). PMID- 1731612 TI - Purification and characterization of two basic beta-1,3-glucanases induced in Colletotrichum lindemuthianum-infected bean seedlings. AB - Two beta-1,3-glucanases which are rapidly induced in the incompatible interaction between bean (cv. Processor) and Colletotrichum lindemuthianum race beta were purified to homogeneity. Characterization of the two enzymes, GE1 and GE2, showed that they both had a basic isolectric point and a similar molecular weight (36,500 for GE1 and 36,000 for GE2), but differed in their pH optimum, thermal stability, and specific activity. GE2 was present in higher amounts but was shown to be less active than GE1 against laminarin and fungal cell walls isolated from race beta of the fungus. Both enzymes were specific for beta-1,3 linkages and showed a strict endolytic mode of action. Further characterization of GE2 was achieved by amino acid sequence analysis of tryptic peptides; the degree of homology shared with other basic beta-1,3-glucanases depended on the plant source. A time-course study showed that GE1 and GE2 were increased during infection. They were also induced by fungal elicitors, thereby indicating that they originate from the host. PMID- 1731613 TI - Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity. AB - Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1. The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine. No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged. Studies with [14C]diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase. These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine. PMID- 1731614 TI - Comparative characterization of human and rat liver glycogen synthase. AB - Glycogen synthase from human liver was studied, and its properties were compared with those of rat liver glycogen synthase. The rat and human liver glycogen synthases were similar in their pH profile, in their kinetic constants for the substrate UDP-glucose and the activator glucose 6-phosphate, and in their elution profiles from Q-Sepharose. The apparent molecular weight of the human synthase subunit was 80,000-85,000 by gel electrophoresis, which is similar to that of the rat enzyme. In addition, antibodies to rat liver glycogen synthase recognized human liver glycogen synthase, indicating that the enzymes of these two species share antigenic determinants. However, there were significant differences between the two enzymes. In particular, the total activity of the human enzyme was higher than that of the rat. The human enzyme, purified to near homogeneity, had a specific activity of 40 U/mg protein compared with 20 U/mg protein for the rat enzyme. The active forms of the rat enzyme had greater thermal stability than those of the human enzyme, but the human enzyme was more stable on storage in various buffers. Last, amino acid analysis indicated differences between the enzymes of the two species. Since glycogen synthase is an important enzyme in liver glycogen synthesis, the characterization of this enzyme in the human will help provide insight regarding human liver glycogen synthesis. PMID- 1731615 TI - Decreased formation of inositol trisphosphate in Madin-Darby canine kidney cells under conditions of beta-glucosidase inhibition. AB - Recent work has demonstrated the enhancement of hormone-stimulated inositol trisphosphate formation in renal epithelial cells under conditions of glucosylceramide depletion. The role of glucosylceramide metabolism was explored further by exposing Madin-Darby canine kidney (MDCK) cells to the beta glucosidase inhibitor conduritol B epoxide, which produced time-dependent and concentration-dependent increases in glucosylceramide levels and decreased bradykinin-stimulated inositol trisphosphate formation from isolated MDCK cell membranes. These data provide further support for an association between glucosylceramide levels and hormone-stimulated inositol trisphosphate formation. PMID- 1731616 TI - Purification and characterization of pig lung carbonyl reductase. AB - A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction. PMID- 1731617 TI - Activation of carbonyl reductase from pig lung by fatty acids. AB - The NADPH-linked reductase activity of pig lung carbonyl reductase was activated two- to fivefold by fatty acids with a carbon chain length greater than nine at pH 7.0. cis-Unsaturated fatty acids of C:18 and C:20 were potent activators, showing Ka values of 2-14 microM which were lower than the values of 21-125 microM for saturated fatty acids (C:9 to C:16). Of the fatty acids arachidonic acid (C20:4) gave the highest activation. No significant stimulatory effect was observed with acyl CoAs, fatty alcohols, phospholipids, and nonionic detergents. Anionic detergents (sodium dodecyl sulfate and sarkosyl) stimulated the enzyme activity more than ninefold, but the Ka values for them were much higher than those for the cis-unsaturated fatty acids. Although no change in molecular weight or in subunit composition was observed in the enzyme activated by C20:4, the activation led to a decrease in thermal stability of the enzyme. The binding of C20:4 to the enzyme was instantaneous and reversible, shifted the pH optimum of the activity from 5.8 to 6.5, and changed the inhibitor sensitivity. In addition, C20:4 acted as an allosteric effector abolishing the negative interaction of the enzyme with carbonyl substrates which was seen without the fatty acid, but the activation increased both Vmax and [S]0.5 values for the substrates. Kinetic analysis with respect to NADPH concentration, in which no cooperativity was detected with or without C20:4, indicated that C20:4 was a nonessential activator of mixed type showing a binding constant of 10 microM. These results suggest that cis-unsaturated fatty acids may be potential modulators of pulmonary carbonyl reductase. PMID- 1731618 TI - Isolation and characterization of a calcium-binding protein derived from mRNA termed p9Ka, pEL-98, 18A2, or 42A by the newly synthesized vasorelaxant W-66 affinity chromatography. AB - W-66 (N-(2-aminoethyl)-N-[2-(4-chlorocinnamylamino) ethyl]-5 isoquinolinesulfonamide), a newly synthesized isoquinolinesulfonamide, was shown to have a potent vasodilatory action and calmodulin (CaM)-antagonizing action. Using the W-66 affinity chromatographic technique, we purified two Ca(2+)-binding proteins from the EGTA-soluble fraction of bovine aorta. One was CaM and the other was an acidic protein with a molecular mass of 11 kDa. It was tentatively named "calvasculin." Calvasculin was a dimeric protein. Equilibrium dialysis showed that 1 mol of calvasculin (dimer) bound to 1.98 +/- 0.30 mol of Ca2+ in the presence of 10(-3) M Ca2+. Calvasculin failed to activate Ca2+/CaM-dependent enzymes such as myosin light chain kinase, Ca2+/CaM-dependent phosphodiesterase, or Ca2+/CaM-dependent protein kinase II and to inhibit the CaM stimulation of these enzymes. The partial amino acid sequence of calvasculin revealed a high homology to the predicted protein derived from mRNA, named pEL-98, 18A2, 42A, or p9Ka. We also examined the physicochemical and biochemical properties of calvasculin. Using the antibody specific for calvasculin, we obtained evidence that calvasculin is present in abundance in bovine aorta but not in brain, lung, heart, or testis. PMID- 1731619 TI - Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae. AB - Klebsiella pneumoniae M5a1 has been shown to possess an inducible transport system for 4-hydroxyphenylacetate (4-HPA). This transport system has a Kt of 16.3 microM and a maximal velocity of 31.2 nmol/min (milligrams dry weight). The transport system has been inhibited by inhibitors of energy metabolism with a concomitant decrease in cellular ATP concentrations, and the 4-HPA binding activity has been detected in the crude shock extracts. All these observations indicate that 4-HPA uptake is an active transport which involves a periplasmic binding protein and it seems to be energized by phosphate bond energy. PMID- 1731620 TI - Isolation and characterization of a human glutathione S-transferase Ha1 subunit gene. AB - We have isolated and characterized a human liver glutathione S-transferase Ha1 subunit gene. The gene spans approximately 12 kilobases and comprises seven exons separated by six introns. The transcription initiation site has been determined by primer extension analysis. A TATA box is located 26 nucleotides upstream from the transcription initiation site, an adenine residue. RNA blot analysis reveals that the gene is expressed at significantly higher levels in human liver than in HepG2 cells. The isolation and characterization of a human glutathione S transferase Ha1 subunit gene should facilitate a detailed analysis of its transcriptional regulation. PMID- 1731621 TI - A new form of mammalian electron-transferring flavoprotein. AB - Mammalian electron-transferring flavoproteins have previously been reported to form the red anionic semiquinone on 1-electron reduction. This work describes a new form of electron-transferring flavoprotein (ETFB) from pig kidney which yields the blue neutral semiquinone upon photochemical, dithionite, or enzymatic reduction. ETFB appears in varying amounts as part of an established purification scheme for ETF. Both the normal form of ETF (ETFR) and ETFB show small differences in the spectra of their oxidized flavins, but no detectable differences in molecular weight or subunit composition. The catalytic activities of ETFR and ETFB are comparable when they mediate the transfer of reducing equivalents between medium chain acyl-CoA dehydrogenase and 2,6 dichlorophenolindophenol. ETFB can be converted into a form showing the characteristic red semiquinone of ETFR by full reduction at pH 6.5 or by preparation of the apoprotein and reconstitution with FAD. In contrast, no conditions for the conversion of red to blue forms of ETF have been found. ETFB contains substoichiometric levels of an unusual FAD analogue which yields a pink flavin species on photochemical or dithionite reduction. The evidence presented suggests that ETFB contains a labile factor or protein modification which is irreversibly lost on conversion to ETFR. The possible physiological significance of these data is discussed. PMID- 1731622 TI - Inhibitory effects of interleukin 6 on prostaglandin I2 production in cultured bovine vascular endothelial cells. AB - The effect of interleukin 6 (IL-6) upon arachidonic acid (AA) metabolism was studied in cultured bovine vascular endothelial cells (BVECs). Although, in those BVECs pretreated with IL-6 for 48 h, there was no effect upon cell proliferation, it was discovered that IL-6 suppressed the conversion of AA to prostaglandin I2 (PGI2) in cellular homogenates. First signs of the suppression occurred 3 h after incubation, and thereafter the suppression increased with time until it leveled off at 12 h. This effect of IL-6 was concentration-dependent, ranging from 20 ng/ml to a maximum of 100 ng/ml with IL-6 at 47% of control. IL-6 had no effect upon AA conversion when exogenously added to the BVEC homogenates. To investigate the mechanism of the suppression induced by IL-6, we studied its effect upon PGI2 synthesizing enzymes. We found that IL-6 had no effect upon the release of AA from phospholipids, but inhibited cyclooxygenase, the key enzyme for PGI2 production from AA. To amplify PGI2 production, AA was added exogenously to both control and IL-6-treated BVECs, and in this case also, the conversion activity in IL-6-treated BVEC's was suppressed. Through immunoblot analysis with an antibody to cyclooxygenase, it was determined that the level of cyclooxygenase protein was lowered in IL-6-treated cells. This is the first report to confirm down expression of cyclooxygenase in addition to the first observation as to the effect of IL-6 on endothelial cells. PMID- 1731623 TI - Protein organization in mouse liver peroxisomes. AB - Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment. PMID- 1731624 TI - Arsenate-induced fluorescence changes in the Ca(2+)-ATPase of sarcoplasmic reticulum membranes. AB - Arsenate, an analogue of inorganic phosphate, causes an increase in the intrinsic fluorescence of the Ca(2+)-ATPase of sarcoplasmic reticulum membranes. This increase in fluorescence is observed regardless of whether Ca(2+)-loaded or leaky vesicles are assayed. The maximal fluorescence change (2-3%) is observed at pH 6.0 in the presence of Mg2+ and is abolished by the addition of micromolar Ca2+ concentrations. Dimethyl sulfoxide (20% v/v) increases the enzyme's affinity for arsenate one order of magnitude. It is concluded that arsenate, after binding, promotes the same conformational change of the enzyme as that produced by Pi. PMID- 1731625 TI - Turmerin: a water soluble antioxidant peptide from turmeric [Curcuma longa]. AB - Dietary spice components have been screened for their protective effect against reactive oxygen species (ROS)-induced, lipid peroxide-mediated membrane and DNA damage and mutagenecity. A new, water soluble, 5-kDa peptide--Turmerin--from turmeric (Curcuma longa) has been found to be an efficient antioxidant/DNA protectant/antimutagen. Turmerin forms 0.1% of the dry weight of turmeric and is obtained in a crystalline form. It is a heat stable, noncyclic peptide containing 40 amino acid residues, with a blocked N-terminal and leucine at the C-terminal. It is insensitive to trypsin and pepsin, heat, and uv radiation. Turmerin contains three residues of methionine which are partly responsible for the antioxidant activity. Turmerin at 183 nM offers 80% protection to membranes and DNA against oxidative injury. ROS-induced arachidonate release and the mutagenic activity of t-butyl hydroperoxide are substantially inhibited by Turmerin. Tumerin is noncytotoxic up to milligram concentrations, as tested by Ames assay and in human lymphocytes. PMID- 1731626 TI - Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes. AB - The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4 hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH mediated protection of microsomes against lipid peroxidation. PMID- 1731627 TI - Removing the two C-terminal residues of actin affects the filament structure. AB - We define conditions under which the two C-terminal residues of actin, Cys-374 and Phe-375, can be selectively removed by proteolysis with trypsin. This modification had little effect on the secondary structure of actin detected by Fourier-transform infrared spectroscopy. However, removing these residues caused small but significant decreases in the critical concentration of actin, in its ability to activate myosin ATPase, and in its interaction with tropomyosin and troponin. Removing residues 374-375 caused dramatic changes in the actin filament as seen by electron microscopy. The filaments had a much greater and more irregular curvature and were intertwined into disordered multifilament bundles. Removing 374-375 also significantly lowered the flow viscosity of filamentous actin solutions. These data suggest an increase in the flexibility and fragility of the filament, supporting the idea that the C-terminus forms one of the major intermonomer contacts in the filament. PMID- 1731628 TI - Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae. AB - The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields. PMID- 1731629 TI - Thiol depletion induces lethal cell injury in cultured cardiomyocytes. AB - Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3 phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion. PMID- 1731630 TI - Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin. AB - The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene) diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2 dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes. PMID- 1731631 TI - Cloning and characterization of a novel CYP3A1 allelic variant: analysis of CYP3A1 and CYP3A2 sex-hormone-dependent expression reveals that the CYP3A2 gene is regulated by testosterone. AB - A clone was isolated from a cDNA library constructed from phenobarbital-treated Wistar rat liver and proven to correspond to the full-length mRNA of a polymorphic variant of Sprague-Dawley CYP3A1. Eight nucleotide differences were detected in a single 76-nucleotide stretch and confirmed to be present in the genomic clone. They are seated in a region implicated in the definition of a substrate binding domain of the native P450. Three out of the eight nucleotide changes are nonconservative, implicating the replacement of Thr/Ala 207, Phe/Ile 213, and Ile/Val 232. This is the first report of an allelic variant of CYP3A1, a new example of interstrain P450 variability. The CYP3A subfamily is composed of several genes coding for active testosterone 6 beta-hydroxylases which are expressed in the liver. CYP3A genes are under strong and distinct developmental regulation. Conversely to CYP3A1, transiently expressed in immature animals, CYP3A2 is constitutively expressed in the liver early after birth and characterized by an extinction in the adult females. Castration of 90-day-old male rats causes a drastic reduction (80%) of CYP3A2 mRNA relative abundance. Administration of testosterone propionate restores the physiological levels of CYP3A2 mRNA characteristic of the male rat liver. Our results demonstrate the existence of a direct relationship between the male hormonal status and the constitutive expression of rat liver CYP3A2. PMID- 1731632 TI - Identification, purification, and characterization of S-adenosyl-L-methionine: isoliquiritigenin 2'-O-methyltransferase from alfalfa (Medicago sativa L.). AB - An O-methyltransferase (OMT) which methylates the 2'-hydroxyl of isoliquiritigenin (2',4,4'-trihydroxychalcone) was identified in alfalfa (Medicago sativa L.) seedlings and cell cultures. The OMT activity increased during early stages of seedling development and was predominantly located in roots. Treatment of alfalfa cell cultures with an elicitor from yeast resulted in a fivefold increase in chalcone OMT activity, whereas treatment of seedlings with CuCl2 caused a reduction in activity. The chalcone OMT was purified to near homogeneity from elicited alfalfa cell cultures. Only one form of the enzyme was found. It consisted of an active monomer of subunit Mr 43,000 which could be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The purified OMT had a pH optimum of 9.0, pI of 4.7, and was highly specific for the 2'-hydroxyl of 2',4,4'-trihydroxychalcone, with essentially no activity toward narigenin chalcone, caffeic acid, or daidzein. Kinetic analysis indicated a sequential bi bi mechanism with Km values of 2.2 and 17.7 microM for 2',4,4'-trihydroxychalcone and S-adenosyl-L-methionine, respectively. S-Adenosyl-L-homocysteine was a potent inhibitor. The chalcone OMT represents the third distinct OMT isolated from alfalfa cell cultures. PMID- 1731633 TI - Wound-inducible pinene cyclase from grand fir: purification, characterization, and renaturation after SDS-PAGE. AB - The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor. PMID- 1731634 TI - Parathyroid-like regulation of parathyroid-hormone-related protein release and cytoplasmic calcium in cytotrophoblast cells of human placenta. AB - Immunohistochemical staining of human placenta revealed intense reactivity for amino terminal and midregional parathyroid-hormone-related protein (PTHrp) in the cytotrophoblast cells and weaker staining in the syncytiotrophoblasts. The cytotrophoblasts also displayed conspicuous surface staining with the monoclonal antibodies E11 and G11, which recognize a Ca2+ receptor mechanism regulating hormone release of parathyroid cells. Cytotrophoblasts enriched on Percoll gradients or by linking surface-bound E11 to magnetic beads revealed biphasic elevation of cytoplasmic Ca2+ ([Ca2+]i) upon a stepwise rise of external Ca2+ from 0.5 to 3.0 mM, with a half-maximal effect at 1.75 mM. Individual cytotrophoblasts identified by their E11 reactivity disclosed a temporary increase of [Ca2+]i upon elevation of external Mg2+, while Mn2+ triggered both a [Ca2+]i transient and an influx of itself. These effects were efficiently blocked by the G11 antibody. Depolarization with K+ or addition of the voltage-dependent Ca2+ channel blocker verapamil had only marginal effects on [Ca2+]i. Raised extracellular calcium inhibited release of PTHrp from the cells, and this inhibition was blocked by the G11 antibody. The virtually parathyroid-identical Ca2+ regulation of [Ca2+]i may mediate feedback control of PTHrp release from the cytotrophoblasts and thereby participate in the regulation of placental Ca2+ transport. PMID- 1731635 TI - Further characterization of cytochrome P450 involved in phytoalexin synthesis in soybean: cytochrome P450 cinnamate 4-hydroxylase and 3,9-dihydroxypterocarpan 6a hydroxylase. AB - Two cytochrome P450 enzymes, cinnamate 4-hydroxylase (C4H) and 3,9 dihydroxypterocarpan 6a-hydroxylase (D6aH), were isolated from elicitor challenged soybean (Glycine max) cell cultures (G. Kochs and H. Grisebach, 1989, Arch. Biochem. Biophys. 273, 543-553). An earlier purification protocol was improved by the use of new chromatographic media, leading to a higher yield of enzymatic activity. After separation of C4H from D6aH on hydroxyapatite, the C4H was identified using anti-C4H antibody from Jerusalem artichoke (Helianthus tuberosus) (B. Gabriac et al., 1991, Arch. Biochem. Biophys. 288, 302-309). The two proteins show molecular weights of about 58,000 for C4H and about 55,000 for D6aH on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzyme activities are dependent on NADPH:cytochrome P450 reductase and cross-react with their respective antibodies. Both cytochrome P450 subspecies show substrate binding and CO-difference spectra typical for cytochrome P450 and were found to be glycoproteins by their cross-reaction with biotinylated lectins in Western blot. The N-terminal sequence of C4H from soybean shows high similarity to the N terminus of C4H from Jerusalem artichoke. PMID- 1731636 TI - Characterization of an endopeptidase of Trypanosoma brucei brucei. AB - A soluble 80-kDa endopeptidase has been isolated from Trypanosoma brucei brucei. The enzyme, which has a pI 5.1, is optimally active at about pH 8.2 and has apparent pKa values of 6.0 and greater than or equal to 10. It is inhibited by the serine protease inhibitor diisopropylfluorophosphate and by the serine protease mechanism-based inhibitor 3,4-dichloroisocoumarin. Unexpectedly, the enzyme is inhibited by the cysteine protease inhibitor benzyloxycarbonyl-Leu-Lys CHN2 but not by the related diazomethane, butoxycarbonyl-Val-Leu-Gly-Lys-CHN2, nor by other cysteine protease specific compounds. Specificity studies with a variety of amidomethylcoumaryl (AMC) derivatives of small peptides show that the enzyme has a highly restricted trypsin-like specificity. The best substrate, based on the magnitude of kcat/Km, was benzyloxycarbonyl-Arg-Arg-AMC; other good substrates were benzyloxycarbonyl-Phe-Arg-AMC, benzoyl-Arg-AMC, and compounds with Arg at P1 and Ala or Gly at P2. The hydrolysis of most substrates obeyed classical Michaelis-Menton kinetics but several exhibited pronounced substrate inhibition. The enzyme did not activate plasminogen nor decrease blood clotting time; it was inhibited by aprotinin but not by chicken ovomucoid. We conclude that the enzyme is a trypsin-like serine endopeptidase with unusually restricted subsite specificities. PMID- 1731637 TI - Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver. AB - A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes. PMID- 1731638 TI - An investigation of the thermal unfolding of swine pepsinogen using circular dichroism. AB - The thermal unfolding of swine pepsinogen is investigated using circular dichroism (CD) in the pH range 6-9. CD spectra and single wavelength melting curves are analyzed to show the presence of two resolvable transitions. Analysis of difference CD spectra by the method of singular value decomposition indicates that the changes in conformation are distinct in the two transitions. Single wavelength melting curves show that only one of the transitions has a strong pH dependence. The results are discussed in terms of earlier kinetic and calorimetric data to suggest the presence of one or more intermediates in the reaction. PMID- 1731639 TI - Depolymerization of heparin by complexed ferrous ions. AB - Treatment of porcine heparin with the ferrous-EDTA complex and ascorbic acid for 24 h at 37 degrees C results in the degradation of most of the glycosaminoglycan to smaller fragments. About 65% of the products comprise oligosaccharides composed of less than 30 sugar units. The extent of depolymerization is decreased significantly if ascorbate or EDTA is not included in the reaction mixture. Gel filtration of the reaction products yielded fractions with narrow chain length ranges. The sulfate content of the fractions and their electrophoretic mobilities on cellulose acetate indicate that the components have equivalent charge densities. Depolymerization products with 20 or more sugar units retain significant anticoagulant potencies as measured by their effect in accelerating the neutralization of factor Xa by antithrombin. PMID- 1731640 TI - Purification and properties of versiconal cyclase from Aspergillus parasiticus. AB - Versiconal cyclase catalyzes the dehydration of versiconal to versicolorin B or versicolorin C [versicolorin B(C)]. The enzyme was purified from mycelia of Aspergillus parasiticus by DEAE-cellulose, hydroxylapatite, and Mono Q column chromatography. The protein contains two identical subunits of molecular weight 72,000 per molecule of native protein. The pI of the enzyme is 3.95. The pH activity curve had a broad maximum with a peak at 5.5. The Km and Vmax for versiconal at 30 degrees C and pH 6.0 are 3.1 microM and 0.15 mumol min-1mg-1, respectively. Most of the formation of versicolorin B(C) in the cell is attributed to the action of versiconal cyclase. PMID- 1731641 TI - Depletion of rat hepatic glutathione and inhibition of microsomal trans-2-enoyl CoA reductase activity following administration of a dec-2-ynol and dec-2-ynoic acid. AB - The effects of administration of dec-2-ynol and dec-2-ynoic acid on the hepatic glutathione (GSH) content and hepatic microsomal trans-2-enoyl-CoA reductase activity were examined in rat. Both compounds, when administered ip, caused a marked depletion of GSH levels and a corresponding inactivation of trans-2-enoyl CoA reductase activity in both a time- and dose-dependent manner. The dec-2-ynoic acid caused greater hepatotoxicity than dec-2-ynol based on serum alanine transaminase activity. Based on the observations that (a) the alcohol did not interact with GSH in the presence or absence of cytosol, (b) the spectral manifestation of the interaction between GSH and the alcohol occurred only when NAD+ was added to the reaction mixture containing the cytosol and reactants, and (c) a similar absorbance spectrum was obtained following the interaction between aldehyde and GSH, it was concluded that dec-2-ynol is converted to an electrophile, dec-2-ynal, which causes depletion of GSH. The decrease in GSH content following administration of the acid appears to be due to activation of the acid to the electrophile, dec-2-ynoyl CoA, which then interacts with GSH, resulting in its depletion, based on the in vitro observations that (a) the acid did not interact with GSH in the presence or absence of cytosol, and (b) the spectral manifestation of interaction between GSH and dec-2-ynoyl CoA occurred both nonenzymatically and enzymatically in the presence of rat liver glutathione S-transferase (Sigma). Bovine serum albumin stimulated the enzymatic reaction. Comparable to the effects on GSH were the effects of dec-2-ynol, dec-2-ynal, dec 2-ynoic acid, and dec-2-ynoyl CoA on the microsomal trans-2-enoyl-CoA reductase activity in vitro. While the alcohol had no effect on the enzyme activity, its electrophilic product, the aldehyde, was a potent inhibitor. Similarly, the acid did not inhibit the enzyme activity unless the acid was present at high concentration; however, its electrophilic product, the CoA thioester, was a very potent inhibitor at very low concentration. PMID- 1731642 TI - The interaction between retinoic acid and the transforming growth factors-beta in calf articular cartilage organ cultures. AB - In calf articular cartilage organ cultures, retinoic acid depressed proteoglycan anabolism to levels approximately 10% of control values and increased their catabolism approximately 14-fold at concentrations of 1 x 10(-8) and 1 x 10(-6) M, respectively, leading to a severe depletion of this component from the extracellular matrix (95% loss in 3 weeks). These effects were powerfully antagonized by maximal levels of transforming growth factors-beta (TGF-beta s) 1, 2, and 3, leading to preservation of matrix components. At a concentration of 1 x 10(-8) M retinoic acid, the TGF-beta s restored anabolism to control levels and lowered catabolic rates greater than 3-fold. While the TGF-beta s increased protein synthesis 2- to 3-fold over controls, retinoic acid alone did not change protein synthesis, as determined by incorporation of [3H]serine. Nevertheless, retinoic acid effectively antagonized the stimulation of protein synthesis by TGF beta and restored control levels of synthesis at 1 x 10(-7) M. Analysis of proteins, labeled using [3H]serine and [35S]sulfate as precursors, by SDS-PAGE revealed that large molecular weight proteins (greater than 100 kDa) were not detectable in retinoic-acid-treated cultures, but treatment with the TGF-beta s restored these components in coincubation cultures, again supporting the antagonistic role of the polypeptide effectors on retinoid action. Treatment of the cultures with retinoic acid elevated levels of TGF-beta 2 synthesis, but not TGF-beta 1. While the role of the newly synthesized TGF-beta 2 in the set of events elicited by retinoic acid in articular cartilage is unclear, the results establish an intrinsic metabolic link between the isoprenoid and TGF-beta in articular cartilage. We propose that the retinoids and TGF-beta s are integral parts of a regulatory network that controls homeostasis, resorption, or growth, depending on their relative contributions. PMID- 1731643 TI - Analysis of stable protein methylation in cultured cells. AB - It was demonstrated recently that substrates for protein N-methyltransferases (J. Najbauer and D. W. Aswad, 1990, J. Biol. Chem. 265, 12,717-12,721) and protein carboxyl methyltransferases (J. Najbauer, B. A. Johnson, and D. W. Aswad, 1991, Anal. Biochem. 197, 412-420) accumulate when rat PC12 cells are cultured in the presence of the methylation inhibitor, adenosine dialdehyde. In the present report, we have further characterized this phenomenon in PC12 cells and in two other, widely used cell types. Adenosine dialdehyde was found to increase the methyl-accepting capacity of proteins in human skin fibroblasts and mouse Sp2/0 myeloma cells. However, both the level of methyl incorporation in untreated cells and the amount of stimulation afforded by inhibitor treatment were substantially lower in these cells than in PC12 cells. All three cell lines accumulated methyl acceptor(s) at 17-21 kDa. The PC12 cells and the fibroblasts also exhibited stimulation of three apparently similar proteins in the 33- to 38-kDa region, where several arginine-methylated proteins involved in RNA processing would be expected. The optimal conditions for methylation of PC12 cell extracts with regard to pH, time of methylation, and S-[methyl-3H]adenosyl-L-methionine concentration were characterized. Increased methyl incorporation was detected after adenosine dialdehyde treatments as short as 2 h, and methylation of most substrates continued to increase as the time of treatment was extended to 72 h. The kinetics of accumulation varied from substrate to substrate. Fluorograms of two-dimensional gels of extracts from untreated PC12 cells incubated in the presence of S-[methyl-3H]adenosyl-L-methionine revealed patterns of methyl incorporation similar to those of treated cells, but longer exposure times were necessary (e.g., 35 days vs 7 days). These findings suggest that the inhibitor treatment works mainly by inhibiting the post- or cotranslational methylation of a "normal" array of cellular proteins. PMID- 1731644 TI - Expression and function of heterologous forms of malate dehydrogenase in yeast. AB - The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene. PMID- 1731645 TI - Is there a field of wound pharmacology? PMID- 1731646 TI - Gallbladder sludge and stone formation in relation to contractile function after gastrectomy. A prospective study. AB - In a prospective trial to determine whether gastric surgery induces gallbladder sludge and stone formation, 48 patients with gastric cancer were ultrasonographically examined with simultaneous observation on changes in gallbladder contractile function before and serially for 5 years after gastrectomy. Gallbladder sludge formation was induced with a high frequency of 42% 1 month after gastrectomy, with corresponding significant lowering of gallbladder contractile function. Most of gallbladder sludges, however, disappeared within 12 months in relation to the gradual recovery of gallbladder contractile function. Conversely, gallstone developed in nine patients (18.8%), mostly more than 6 months after gastrectomy. Interestingly, gallstone formation was induced in seven patients who were sludge negative. An evolvement of gallbladder sludge into stone was observed in only two patients, who were, however, treated with intravenous hyperalimentation. This study first provides evidence for the relationship between gastrectomy and a considerably high frequency of incidence of gallbladder sludge and stone in relation to changes in gallbladder kinetics after gastrectomy. PMID- 1731647 TI - Scarless fetal healing. Therapeutic implications. AB - The purpose of this report is to call attention to the fetal wound healing process as a blueprint for ideal tissue repair. Wound healing in the fetus is fundamentally different from healing in the adult. Fetal tissue repair occurs rapidly and in the absence of scar formation. Because scarring and fibrosis dominate some diseases in every area of medicine, an understanding of fetal wound healing should help develop therapeutic strategies to avert the devastating consequences of excessive scar formation. PMID- 1731648 TI - The surgical spectrum of hereditary pancreatitis in adults. AB - The role of operative intervention for hereditary pancreatitis, a rare form of chronic parenchymal destruction, is unclear. To determine whether surgical therapy is safe and provides prolonged symptomatic relief, the authors reviewed the management of 22 adults (11 men, 11 women) with hereditary pancreatitis treated surgically between 1950 and 1989. Hereditary pancreatitis was defined as a family history of two or more relatives with pancreatitis and clinical, biochemical, or radiologic evidence of pancreatitis. The mean ages at onset of symptoms and at operation were 15 years (range, 3 to 52 years) and 31 years (range, 18 to 54 years), respectively. Pain was the primary indication for operation in all patients. Additional symptoms included nausea, vomiting (73%), weight loss (55%), and diarrhea (41%). Ductal dilatation was present in 68%, pancreatic parenchymal calcifications in 73%, pseudocysts in 36%, and splenic vein thrombosis in 18%. Primary operations included ductal drainage in 10 patients, pancreatic resection alone in three, resection with drainage in three, cholecystectomy plus sphincteroplasty in two, cholecystectomy with or without common bile duct exploration in two, pancreatic abscess drainage in one, and pseudocyst drainage in one. There were no perioperative deaths, and the morbidity rate was 14% (intra-abdominal abscess, wound infection, and urinary tract infection). Symptoms recurred in nine patients. Severity prompted reoperation in five. Secondary operations included pancreatic resection in three, pseudocyst excision in one, and pancreaticolithotomy in one. Follow-up to date is complete and extends for a median of 85 months. Eighteen patients (82%) are clinically improved or asymptomatic. Symptoms have persisted in four patients, and two patients have died of pancreatic carcinoma. Two patients died of unrelated causes. Surgical therapy for patients with hereditary pancreatitis selected on the basis of the traditional indications for surgical treatment of chronic pancreatitis is safe and efficacious. PMID- 1731649 TI - A better model of acute pancreatitis for evaluating therapy. AB - Existing models of acute pancreatitis have limitations to studying novel therapy. Whereas some produce mild self-limited pancreatitis, others result in sudden necrotizing injury. The authors developed an improved model providing homogeneous moderately severe injury by superimposing secretory hyperstimulation on minimal intraductal bile acid exposure. Sprague-Dawley rats (n = 231) received low pressure intraductal glycodeoxycholic acid (GDOC) at very low (5 or 10 mmol/L) concentrations followed by intravenous cerulein. Cerulein or GDOC alone caused only very mild inflammation. However, GDOC combined with cerulein was uniformly associated with more edema (p less than 0.0005), acinar necrosis (p less than 0.01), inflammation (p less than 0.006), and hemorrhage (p less than 0.01). Pancreatic injury was further increased and death was potentiated by increasing volume and duration of intraductal low-dose GDOC infusion. There was significant morphologic progression between 6 and 24 hours. The authors conclude that (1) combining minimal intraductal bile acid exposure with intravenous hyperstimulation produces homogeneous pancreatitis of intermediate severity that can be modulated at will; (2) the injury is progressive over at least 24 hours with finite mortality rate; (3) the model provides superior opportunity to study innovative therapy. PMID- 1731650 TI - Morbidity and mortality after pelvic exenteration for colorectal adenocarcinoma. AB - A retrospective analysis was made of the complications from pelvic exenterations performed over the past 30 years for colorectal adenocarcinoma at the Roswell Park Cancer Institute. Seventy-five patients underwent exenteration, 51 for primary disease (PD) and 24 for recurrent disease (RD). Both total and posterior exenterations were included. Twenty of the fifty-one patients (39%) undergoing exenteration for PD developed severe complications, with an operative mortality rate of 6%. The most common complications were injuries to the ureter or bladder, intra-abdominal abscesses, and anastomotic leaks from the urinary diversion. After exenteration for RD, 12 of 24 patients (50%) developed severe complications, with an operative mortality rate of 4%. The most common major complication was an anastomotic leak from the urinary diversion; this occurred in 33% of all patients with RD (8/24). The authors conclude that, although exenteration for colorectal adenocarcinoma may be performed with a low operative mortality rate, patients must be carefully selected because the associated morbidity rate remains high. PMID- 1731651 TI - Prognostic factors influencing survival in gastrointestinal leiomyosarcomas. Implications for surgical management and staging. AB - The appropriate surgical therapeutic options for either localized or more advanced disease in patients with gastrointestinal leiomyosarcomas remain unclear. A staging classification for this disease has not been adopted nor risk factors identifying patients at risk for recurrence defined. To address these issues, this study evaluated the influence of various clinicopathologic variables on overall and disease-free survival. In an univariate analysis of overall survival involving 191 patients, the Cox proportional hazards model identified four factors that were associated with a significantly better outcome: complete resection without tumor rupture (p less than 0.001), localized lesions (p less than 0.001), low grade of tumor (p = 0.02), and tumors smaller than 5 cm (p = 0.03). When interactive effects of these factors were taken into account, however, type of resection of the tumor was selected as the only significant prognostic factor in a multivariate analysis. Complete resection without tumor rupture improved overall survival of patients with localized disease (median, 46 months) as well as those with contiguous organ invasion (median, 36 months) or peritoneal implants (median, 36 months). In contrast, patients with incomplete resections survived for a median of 21 months. Patients with tumor rupture, despite removal of all gross disease, behaved similarly to those with incomplete resections; median survival was only 17 months. For disease-free survival, important determinants selected from a multivariate analysis were tumor rupture (p = 0.002), contiguous organ invasion (p = 0.02) and high tumor grade (p = 0.02). A staging classification incorporating these prognostic factors of significance was evaluated using a TGM system: T1 (less than 5 cm), T2 (greater than or equal to 5 cm), T3 (contiguous organ invasion or peritoneal implants), T4 (tumor rupture); G: G1 (low grade), G2 (high grade); M: M0 (no metastases), M1 (metastases present). The corresponding 5-year overall survivals for stages I, II, III, IVA, and IVB were 75%, 52%, 28%, 12%, and 7%. Disease-free survival at 2 years after surgery was 89%, 57%, and 47% for stages I, II, and III, respectively. In conclusion, surgery remains the primary modality of treatment for patients with gastrointestinal leiomyosarcomas, and complete resection of all disease without tumor rupture, even of locally advanced disease, improves overall and disease-free survival. A staging classification appears feasible and is recommended to determine outcome in patients with leiomyosarcomas arising from the gastrointestinal tract. PMID- 1731652 TI - The efficacy of gallium scintigraphy in detecting malignant soft tissue neoplasms. AB - A prospective study was conducted on 55 consecutive patients to evaluate the efficacy of gallium scintigraphy in detecting malignancy in any soft tissue mass. It was determined that gallium scintigraphy could detect malignancy with a sensitivity of 96% and a specificity of 87%. Large and small sarcomas, irrespective of their fascial location, were identifiable by gallium imaging. Occult, nonpulmonary sites of disseminated disease were detected in 13%. Gallium scintigraphy proved to be a reliable predictor of malignancy for all soft tissue masses, but because of its cost, it must be used judiciously. PMID- 1731653 TI - Flow volume loops in patients with goiters. AB - Plain radiology is the standard means of assessing upper airway obstruction in patients with goiters. Flow volume loop curves will provide additional information, because they allow a quantitative assessment of airflow dynamics in the respiratory cycle. Fifty-one patients had flow volume loops performed before and after thyroidectomy. There was a significant increase in the maximum inspiratory flow rate (3.9 +/- 0.2 versus 4.9 +/- 0.2 L/second, p less than 0.01) after thyroidectomy. Eight of twelve patients with normal tracheal radiology had improved airflow dynamics in the postoperative period. The flow volume loop curve is a simple noninvasive means of assessing airflow dynamics in patients with goiters and may be superior to conventional radiology. PMID- 1731654 TI - Nonpalpable invasive breast cancer. PMID- 1731655 TI - Survival following locoregional recurrence after breast conservation therapy for cancer. PMID- 1731656 TI - Survival following locoregional recurrence after breast conservation therapy for cancer. PMID- 1731657 TI - The different drummer, the double agent, and future dilemmas in bioethics. PMID- 1731658 TI - Selective surgical approach for atrioventricular reentrant tachycardia. AB - From September 1986 through September 1990, 60 operations were performed in 55 patients (32 male and 23 female; age, 1 to 76 years) for ablation of accessory pathways of atrioventricular reentrant tachycardia; 6 patients had additional cardiac procedures. Between September 1986 and August 1988 the initial surgical approach was exclusively epicardial with adjuvant cryoablation (EPI) in 23 patients (group 1) for a left free wall (LFW) pathway in 11, right free wall (RFW) in 3, posteroseptal (PS) in 7, and anteroseptal in 2. During September 1988 through September 1990, 32 patients (group 2) had the initial surgical approach tailored to the location of the mapped accessory pathway: endocardial approach (ENDO) for LFW in 17 and for juxtanodal pathway in 2, EPI for RFW in 3 and for PS in 9, and combined ENDO and EPI for AS in 1. There was no early or late death in either group. In group 1, 2 patients with LFW pathway had development of recurrent preexcitation in the same compartment requiring ENDO reoperation 10 and 11 months later, 1 with anteroseptal pathway needed immediate ENDO and EPI reoperation, and another with LFW, who required pericardial patch repair of a left atrial tear, had a thromboembolic stroke 2 days later. No serious complications occurred in group 2, but 2 patients with PS required reoperation before discharge for a second accessory pathway in another compartment (1 RFW and 1 LFW). Additionally, 4 patients (2 in each group) had from the beginning ablation of two pathways in different compartments. On complete late follow-up (mean, 28 months) all patients are back to preoperative levels of activity and are free of preexcitation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731659 TI - Calcification of porcine valves: a successful new method of antimineralization. AB - Despite distinct advantages over mechanical cardiac valve prostheses, the use of bioprosthetic valves remains limited due to poor long-term durability, primarily as a result of tissue calcification. A novel anticalcification process, based on treatment of porcine bioprostheses with a derivative of oleic acid, has been developed by one of us (J.M.G.) (US Patent Number 4,976,733). This process employing 2-aminooleic acid (AOA) was tested in a juvenile sheep model. Terminal studies after a 20-week interval included hemodynamic, radiographic, morphologic, and quantitative tissue calcium analyses. All control valves (n = 4) had thickened, immobile, heavily calcified leaflets, whereas all AOA-treated valves (n = 8) were pliable and free of calcium deposits. Calculated valve orifice areas for controls (0.9 +/- 0.2 cm2) (mean +/- standard error of the mean) was less than for AOA-treated valves (2.0 +/- 0.3 cm2) (p less than 0.05). Radiographic calcification scores were greatly elevated in the control (25.5 +/- 5.6) versus AOA-treated valves (0.5 +/- 0.5) (p less than 0.002). In quantitative mineralization studies, the mean calcium content of the control leaflets was 129 +/- 21 milligrams per gram dry weight cusp tissue versus 7.7 +/- 5.8 mg/g for AOA treated valves (p less than 0.001). Pathologic examination confirmed heavy calcification in the control leaflets, which was essentially absent in the AOA treated leaflets. However, cuspal hematomas in areas of structural loosening and surface roughening were noted in AOA-treated valves. This anticalcification process dramatically reduced mineralization of porcine valve prostheses in this model. PMID- 1731660 TI - Granulocyte sequestration and early failure in the autoperfused heart-lung preparation. AB - We investigated the role of pulmonary granulocyte sequestration in the development of early failure of the autoperfused working heart-lung preparation. A significant decline in the total circulating leukocyte count in 21 preparations at 60 minutes of perfusion (5.0 to 1.4 x 10(3)/microL; 28% of baseline; p less than 0.001) was observed. Differential cell counts in 14 of these preparations revealed a predominant decrease in granulocyte count (8.7% of baseline) and a moderate decline in lymphocyte count (46% of baseline). In study I, indium 111 labeled autologous granulocytes were injected intravenously into 10 adult New Zealand White rabbits. In group I (n = 5), an autoperfused working heart-lung preparation was harvested and perfused for 60 minutes. In group II (controls, n = 5), the heart-lung block was harvested following 60 minutes of in situ perfusion. Organ blocks were imaged before and after saline flush. There was a significant decline in granulocyte counts at 60 minutes of perfusion in group I versus no change in group II (I, 2.3 +/- 0.4 to 0.3 +/- 0.1; p less than 0.01; II, 1.7 +/- 0.2 to 2.3 +/- 0.5; not significant; x 10(3)/microL +/- standard error of the mean). Postflush lung activity was significantly increased in group I versus group II (I, 3,751 +/- 566; II, 1,867 +/- 532; p less than 0.05; counts +/- standard error of the mean). In study II, 15 autoperfused preparations were divided into two groups. Group I (n = 10) preparations were controls. Group II (n = 5) animals were depleted of leukocytes by pretreating with nitrogen mustard. Group I (controls) produced a bimodal survival distribution (Ia, 8.2 +/- 1.0; Ib, 26.4 +/- 2.0; hours +/- standard error of the mean). Group II survival was significantly longer than that of group Ia and similar to that of group Ib (II, 25.3 +/- 2.2; p less than 0.01 versus group Ia, not significant versus group Ib; hours +/- standard error of the mean). Hemodynamic profiles for group II closely paralleled those of group Ib. In conclusion, pulmonary sequestration of granulocytes occurs early in the autoperfused working heart-lung preparation (within 60 minutes of autoperfusion), and preoperative leukocyte depletion prolongs survival of the autoperfused working heart-lung preparation by eliminating the subset group Ia (short survivors) seen in untreated preparations. PMID- 1731661 TI - Advances in aortic arch surgery. AB - From 1980 to January 1991, 130 patients (89 men and 41 women, aged 22 to 76 years; mean age, 52 years) underwent 133 interventions on the aortic arch. Aneurysm was diagnosed in 57 patients, whereas 29 had chronic and 44 acute aortic dissection. In 67 instances a partial and in 35 instances a total arch replacement was performed. The distal arch was approached through a left thoracotomy in 14 patients. Local interventions (n = 17) included surgical reconstruction and glue procedures. Additionally, 55 patients required aortic valve replacement, preferably with composite grafts (n = 46), whereas the valve was reconstructed in 14. Procedures were performed using hypothermia (nasopharyngeal temperature, 11 degrees to 25 degrees C) and circulatory arrest (mean time, 27 minutes). Early mortality was 13.9% at the first operation on the aortic arch. Early deaths included 7 of 57 patients with aortic aneurysm (12.3%), 2 of 29 patients with chronic dissection (6.9%), and 9 of 44 patients with acute dissection (20.5%). Neurological (n = 6) and cardiac events (n = 5) were the most common causes of early death. Since 1987, 7 of 88 patients have died for an overall mortality of 8.0%. With growing experience, proper indication, and adequate operative strategy including the use of circulatory arrest in hypothermia, operation on the aortic arch can be performed with an acceptable risk. PMID- 1731662 TI - Blunt injuries of the thoracic aorta. AB - We managed 51 patients with thoracic aortic injuries caused by blunt trauma between 1977 and 1990. Forty-nine injuries were located in the upper descending aorta and one each in the ascending aorta and aortic arch. Three patients arrived moribund and underwent thoracotomy for resuscitation, and all died. The diagnosis was confirmed by aortography in 48. One patient died of aortic rupture, 1 died of hypoxemia, and 1 refused operation and died. Forty-four patients had aortic repair, 42 with graft insertion. Gott shunts were placed in 23 with 3 cases of paraplegia (13%). Simple cross-clamping was used in 19 with 1 case of paraplegia (5.2%). We found statistically significant differences between the cross-clamp times of patients without paraplegia compared with those in whom paraplegia developed in both the shunt and no-shunt groups. Logistic regression analysis showed that the only factor significantly associated with paraplegia was cross clamp time. There were two postoperative deaths (4.4%). Seven patients had medical therapy initially and aortic repair was delayed to allow other injuries to stabilize. Before aortic repair, 18 patients had intraarterial pressure monitoring and 34 received beta-blockers or antihypertensive drugs. We conclude that aortic repair with graft insertion is usually successful in nonmoribund patients, simple cross-clamping is associated with a relatively low risk of paraplegia, the incidence of paraplegia is directly associated with the duration of cross-clamp time, and selected patients can be managed medically while awaiting aortic repair. PMID- 1731663 TI - Activated neutrophils impair rabbit heart recovery after hypothermic global ischemia. AB - Cardiopulmonary bypass is known to cause neutrophil activation, and activated neutrophils appear to be of importance in myocardial reperfusion injury. This study examined the effect of a preischemic infusion of activated neutrophils on the recovery of myocardial function after 40 minutes of hypothermic global ischemia. Studies were carried out in three groups of Langendorff-perfused rabbit hearts: control, control (unactivated) neutrophil infusion, and phorbol myristate acetate-activated neutrophil infusion. The activated neutrophil group showed significant deterioration in function during the activated neutrophil infusion. All three groups demonstrated significant depression of function initially after reperfusion, but the two control groups subsequently recovered to baseline levels. The activated neutrophil group, however, showed a persistent significant depression in ventricular force, rate of ventricular tension development, and rate of ventricular relaxation as well as a significant increase in coronary vascular resistance. It is concluded that activated neutrophils depress myocardial function and contribute to impaired recovery of function after global hypothermic ischemia. PMID- 1731664 TI - Methods of implantable cardioverter-defibrillator-pacemaker insertion to avoid interactions. AB - Experience with combined transvenous pacemaker and implantable cardioverter defibrillator insertion in 21 patients is described. Special techniques are needed to avoid potentially lethal pacemaker-implantable cardioverter defibrillator interaction. Separation between leads for the two devices should be maximized. The electrophysiologic criteria for successful device function can be met, even when some leads for both devices must be placed by a transvenous approach. PMID- 1731665 TI - Twenty-year follow-up of saphenous vein aortocoronary artery bypass grafting. AB - The clinical records of our first 100 patients to undergo saphenous vein aortocoronary bypass grafting were reviewed. The procedures were performed between March 19, 1970, and March 30, 1972. The patient population included 84 men, and the mean age was 51.4 years. There were 12 patients with single-vessel disease, 36 with double-vessel disease, and 52 with triple-vessel disease, for an average of 2.4 involved vessels per patient. Forty-eight patients were judged to have diffuse atherosclerotic disease. Twelve patients had left main coronary artery stenoses. Each patient received an average of 1.8 saphenous vein grafts. Thirty-six patients underwent repeat coronary artery bypass grafting after an average of 132.8 months and received an average of 3.5 grafts. This resulted in cumulative reoperative rates of 5%, 14%, 27%, and 36% at 5, 10, 15, and 20 years, respectively. The 5-, 10-, 15-, and 20-year survival rates were 89.8%, 68.4%, 53.1%, and 40.8%, respectively. Survival was not significantly related to the cause of death, cardiac-related causes being predominant. There were no significant relationships between the length of survival and sex, the number of grafts received, or the presence of left main stenosis. Survival was inversely related to age at initial operation (p = 0.046) as well as initial left ventricular end-diastolic pressure (p = 0.033). Survival positively correlated with the occurrence of triple-vessel disease (p = 0.031) and the presence of diffuse disease (p = 0.0077).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731666 TI - Reoperation for prosthetic valve endocarditis in the third trimester of pregnancy. AB - A pregnant 29-year-old woman underwent emergency aortic valve re-replacement for prosthetic valve endocarditis. Cesarean section was performed with the chest open ready for cannulation. Fears of uterine hemorrhage during systemic heparinization for cardiopulmonary bypass were unfounded, and both mother and the 28-week gestation newborn recovered uneventfully. PMID- 1731667 TI - Availability of the in situ right gastroepiploic artery for coronary artery bypass. AB - The right gastroepiploic artery (GEA) has been successfully used as a coronary bypass graft recently. We examined the in situ GEA graft length required from the pyloric portion to the site of coronary anastomosis at the time of operation. Measured GEA length was 17.0 +/- 1.7 cm for the posterior descending artery anastomosis in 17 patients, 17.8 +/- 1.7 cm for the main right coronary artery anastomosis in 13 patients, 22.0 +/- 2.3 cm for the posterolateral branch anastomosis in 7 patients, and 21.0 cm for the left anterior descending artery anastomosis in 1 patient. We examined 228 randomly selected abdominal angiograms and measured the internal diameter of the right GEA at every 2-cm interval from its origin. Probability of availability of the in situ GEA graft for each site of anastomosis was 97% to the right coronary artery and 88% to the anterior descending or the circumflex artery when the internal diameter of GEA was 1.5 mm or greater. From an anatomical standpoint, we concluded that the GEA can be assumed available without preoperative angiography. PMID- 1731668 TI - Platelet-leukocyte plasmapheresis attenuates the deleterious effects of cardiopulmonary bypass. AB - A method of harvesting a high yield of concentrated platelet- and leukocyte-rich plasma was developed with the goal of attenuating some of the deleterious effects of cardiopulmonary bypass. The study involved 32 patients who underwent coronary artery bypass grafting with plasmapheresis before cardiopulmonary bypass and a control group of 32 patients who did not have plasmapheresis. A volume of 857 +/- 359 mL of platelet- and leukocyte-rich plasma was concentrated from 4.6 +/- 1.5 L of blood, and red cells and plasma were returned to the patient. The platelet- and leukocyte-rich plasma contained yields of 3.5 +/- 1.4 x 10(11) platelets and 3.4 +/- 1.9 x 10(9) leukocytes. There were no differences in age, sex, duration of cardiopulmonary bypass, and major risk factors between groups. However, total mediastinal chest tube drainage was 788 +/- 542 mL in the controls and 425 +/- 207 mL in the plasmapheresis group (p less than 0.01). Homologous units transfused were 3.9 +/- 2 in controls and 1.6 +/- 2 in the plasmapheresis group (p less than 0.01). Arterial oxygen tension on extubation was 94 +/- 32 mm Hg in controls and 119 +/- 25 mm Hg in the plasmapheresis group (p less than 0.01). This technique of platelet and leukocyte protection results in reduced postoperative bleeding, a decreased need for homologous blood products, and improved pulmonary function. PMID- 1731669 TI - New method for describing the performance of cardiac surgery cannulas. AB - Cardiac surgery cannulas are characterized by external diameter only, which provides little information about the pressure-flow characteristics of a cannula. A system has been developed to describe pressure-flow characteristics with a single, unitless number, M, which is patterned after a Reynolds friction factor correlation. A cannula with a lower M number has a more favorable pressure-flow relationship. The M number was determined for 16 arterials cannulas ranging in size from 10F to 26F and 27 venous cannulas sized 12F to 36F. Pressure-flow characteristics vary considerably among cannulas from different manufacturers despite having similar French sizes. Clinical decisions regarding choice of cannula can be simplified by using the M number, which gives a more accurate description of the performance characteristics of a cannula than the French size designation. PMID- 1731670 TI - Experience with the symbion total artificial heart as a bridge to transplantation. AB - From December 1985 through January 1991, 9 patients underwent bridging to transplantation using a Symbion J-7-70 total artificial heart. There were 4 female and 5 male patients aged 31 +/- 14 years (range, 15 to 52 years). Five patients were supported on an intraaortic balloon pump before total artificial heart support, and 2 had biventricular assist devices as well. Total artificial heart support was maintained for 17 +/- 12 days (range, 4 to 44 days); all patients underwent transplantation. Three patients died after transplantation on day 0 (primary donor organ failure), 25 (acute rejection), and 256 (multiorgan failure). The remainder were discharged at 41 +/- 32 days (range, 13 to 101 days). One patient died 28 months after transplantation of late acute rejection. Actuarial 1-year and 3-year survival is 67% and 55%. There were no surgical wound infections. Problems encountered in the J-7-70 period and the period after transplantation were for the most part related to patient condition in the period before implantation. The Symbion J-7-70 total artificial heart is an effective device for total circulatory support in patients with end-stage cardiogenic shock when an organ donor is not available. Organ system failure and infection before implantation may persist into the transplantation period resulting in long-term complications, increased mortality, and prolonged hospital stay; therefore, early implantation of the device when indicated should be applied. PMID- 1731671 TI - Gastric substitution for resectable carcinoma of the esophagus: an analysis of 368 cases. AB - Between 1974 and 1984, 1,188 patients with esophageal malignancies were treated in the Division of Thoracic Surgery of Veterans General Hospital, Taipei. The rate of resectability was 42.6%. Since 1974, the stomach has been used as esophageal substitute, and through 1984, a total of 368 patients were collected. The routes of reconstruction included retrosternal (77.2%), posteromediastinal (7.1%), and intrathoracic (15.7%). The rates of postoperative complications and surgical mortality in these 368 patients were 26.3% and 6.5%, respectively. Leakage of anastomosis was the most frequent complication. The incidence of stricture of esophagogastrostomy was 25.5%. All strictures were relieved by esophageal dilations. An average of 3.9 esophageal dilations were performed per patient (range, 1 to 15). Radical lymph node dissection was not routinely performed in our series. The actuarial 2-year and 5-year survival rates were 26.4% and 7.6%, respectively. Among 76 patients undergoing cervical esophagogastrostomy and surviving for more than 1 year, late complications occurred as follows: acid/bile regurgitation, 46.1%; postprandial fullness of abdomen, 38.2%; dumping syndrome, 13.2%; distended stomach with dyspnea, 11.8%; aspiration pneumonia, 6.6%; and gastric ulcer, 6.6%. Moreover, compared with patients without pyloroplasty, those with pyloroplasty were found to have a higher incidence of bile regurgitation (55.5% versus 8.6%), dumping syndrome (33.3% versus 6.9%), aspiration pneumonia (16.7% versus 3.4%), and gastric ulcer (22.2% versus 1.7%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731672 TI - Effects of shaving methods and intraoperative irrigation on suppurative mediastinitis after bypass operations. AB - To investigate the effects of the hair removal methods and intraoperative irrigation on suppurative mediastinitis after cardiopulmonary bypass operations, 1,980 consecutive adult patients over a 2-year period in our institution were prospectively randomized to manual shaving versus electrical clipping of hair before the skin incision, and to povidone-iodine solution (0.5%) versus saline solution mediastinal and subcutaneous irrigation before wound closure. The overall incidence of suppurative mediastinitis was 0.86% (17/1,980). The infectious rate was significantly higher in the manually shaven (13/990) than in the electrically clipped patients (4/990) with an odds ratio of 3.25 (95% confidence interval, 1.11 to 9.32; p = 0.024). It was also higher in the povidone iodine group (11/990) than in the saline group (6/990), although the difference was not statistically significant (p = 0.16). Fourteen patients were treated with operative debridement with closed tube irrigation, with one failure requiring a conversion to an open wound. Two patients were successfully treated with primary open wound procedures followed by delayed muscular flap closures, and 1 patient succumbed to rapid and profound sepsis soon after open drainage. We conclude that electrical clipping is superior to manual shaving in the prevention of suppurative mediastinitis. The routine use of povidone-iodine (0.5%) irrigation was of no benefit in this study and may increase the incidence of infection due to its known suppressive effects on local leukocytes and fibroblasts. Furthermore, operative debridement with closed tube irrigation was successful in treating the majority of cases in this series. PMID- 1731673 TI - Heart, heart-lung, and lung transplantation in the first year of life. AB - Seventeen infants less than 1 year of age have undergone heart (12), heart-lung (3), and lung (2) transplantation for end-stage cardiopulmonary disease. The infants undergoing heart transplantation had a mean age of 4.5 months (range, 19 days to 12 months) with the diagnosis of cardiomyopathy in 4 and congenital heart disease in 8. Four of the 8 patients (50%) had hypoplastic left heart syndrome. Actuarial survival at 1 and 2 years was 74% and compared favorably with the survival of older children at 1 and 2 years of 82% and 69%. The linearized rejection rate was less in infants as compared with children more than 1 year of age (0.61 versus 1.48 episodes per 100 patient days). In intermediate follow-up, no graft atherosclerosis has been noted. Immunosuppression has included a three drug protocol of cyclosporine, azathioprine, and prednisone. A steroid taper to alternate day steroids or off completely by 6 months has been the goal and has been accomplished in 6 of 12 infants. Heart-lung and lung transplantation has been performed in 5 infants. One infant in each group died: 1 infant secondary to airway complications and sepsis and another due to pulmonary sepsis. A pulmonary lobe from a larger and older donor was transplanted into a 4-week-old infant as a single-lung transplant with good outcome. The 3 surviving infants are well 24, 18, and 2 months after transplantation. Obliterative bronchiolitis has not been clinically apparent in this group. These data support the clinical efficacy of heart, heart-lung, and lung transplantation in the first year of life. PMID- 1731674 TI - Coronary artery endothelial cell and smooth muscle dysfunction after global myocardial ischemia. AB - We hypothesized that coronary artery endothelial cell function and smooth muscle function are modified by global myocardial ischemia and used bradykinin-induced secretion of endothelium-derived relaxing factor as a marker of endothelial cell function. Bradykinin and sodium nitroprusside together determined maximum smooth muscle relaxation. Potassium chloride-induced contraction determined smooth muscle contractility. Endothelium-mediated smooth muscle relaxation expressed as a ratio of total coronary smooth muscle relaxation before and after ischemia quantified endothelial cell function. The effect of global normothermic ischemia on in situ coronary arteries from 7 swine hearts was studied. Coronary arterial rings taken from 0 to 220 minutes of ischemia at 20-minute intervals were studied in vitro. The data revealed unexpected tolerance of endothelium-mediated relaxation to ischemia. Endothelium-derived relaxing factor function was maintained to 160 minutes and smooth muscle function, to 120 minutes of ischemia. Coronary artery dysfunction seen in other studies after less ischemia may be the result of injury introduced during reperfusion, may be the consequence of myocardial injury, or may be due to events operative at the level of small arterioles. PMID- 1731675 TI - Imaged thoracoscopic surgery: a new thoracic technique for resection of mediastinal cysts. AB - Previously, intrathoracic organs have been approached by either thoracotomy or thoracoscopy. A technique, imaged thoracoscopic surgery, using video optics and projection of images on a screen provides another option for the thoracic surgeon. Two patients with mediastinal cysts, one bronchogenic and one esophageal, underwent surgical removal using imaged thoracoscopic surgery. Postoperative pain was markedly reduced, hospitalization shortened, and recovery accelerated. Numerous complex surgical procedures can be performed using imaged thoracoscopic surgery. PMID- 1731676 TI - Surgical management of posteroseptal accessory atrioventricular pathways. AB - From October 1988 through January 1991, 22 consecutive patients with Wolff Parkinson-White syndrome underwent surgical ablation of symptomatic accessory posteroseptal atrioventricular pathways at our institution. As our experience with posteroseptal tracts accumulated, we found that surgical technique was logically dictated by the presence of free wall tracts and the exact location of the posteroseptal tract. Accordingly, we developed an operative approach that involves the selective use of endocardial, epicardial, and cryoablation techniques depending on the anatomic location of accessory tracts. This selective approach allows one to exploit the advantages of each technique while minimizing associated disadvantages. There were 14 men and 8 women with an average age of 25 years (range, 19 to 39 years). All patients had symptomatic tachyarrhythmias caused by accessory atrioventricular pathway(s). Most required several antiarrhythmic medications and 17 (77%) had poor arrhythmia control despite maximal medical therapy. Twelve patients had two accessory pathways and 3 also had dual atrioventricular nodal pathways. There were no early or late deaths. In 2 patients, a delta wave associated with a free wall tract reappeared 3 to 5 days after the initial operation, necessitating a second operation which successfully eliminated the accessory tract. All posteroseptal tracts were successfully eliminated during the initial operation. All patients were relieved of symptoms and are now free of medical therapy. Each patient has undergone a postoperative electrophysiologic study which confirms the absence of posteroseptal accessory conduction. The selective approach has been totally successful in our hands and should prove useful to those interested in optimizing the efficiency of surgical procedures for Wolff-Parkinson-White syndrome. PMID- 1731677 TI - Cyclosporine-associated central neurotoxicity after heart transplantation. AB - Cyclosporine central neurotoxicity has been described after bone marrow, kidney, and liver transplantation but has not been well documented after heart transplantation. This case illustrates severe reversible neurotoxicity after heart transplantation with characteristic radiographic changes in magnetic resonance imaging. PMID- 1731678 TI - Cardiac echinococcosis: cyst removal in a beating heart. AB - A 17-year-old asymptomatic boy from a sheep farm had a systolic murmur on routine examination. The diagnosis of Echinococcus cyst in the right ventricular outflow tract was made by echocardiography. The cyst was removed with success with the patient on cardiopulmonary bypass with a beating heart. PMID- 1731679 TI - Homograft root replacement for juvenile rheumatoid aortic valve incompetence. AB - We report a case of severe, crippling juvenile rheumatoid arthritis and aortic insufficiency in a young woman. Homograft replacement of the aortic root offered both long-term durability and the freedom from thromboembolism that her systemic illness required. PMID- 1731680 TI - Long-term palliation of pulmonary artery sarcoma by radical excision and adjuvant therapy. AB - The case of an extensive pulmonary artery sarcoma managed by radical excision and homograft reconstruction followed by aggressive chemotherapy and irradiation with prolonged survival is presented. Pulmonary artery sarcomas are reviewed with emphasis on the diagnosis and management of these usually fatal tumors. PMID- 1731681 TI - Prolonged intraaortic balloon support for septal rupture after myocardial infarction. AB - Timing of surgical repair of ventricular septal defect developing after acute myocardial infarction remains controversial. We report the case of a 75-year-old man in whom a ventricular septal defect developed 7 days after myocardial infarction. The patient was maintained on intraaortic balloon pump support for 33 days before successful surgical closure. In the present era of aggressive preoperative hemodynamic management, the strategy of prolonged support before definitive operation on patients with myocardial infarction in whom a ventricular septal defect develops must be reconsidered. PMID- 1731682 TI - Systemic recombinant tissue plasminogen activator lysis for left atrial thrombus formation after single-lung retransplantation. AB - This report describes a recipient of single-lung transplantation surviving extraordinary complications: (1) early graft failure mandating retransplantation; (2) left atrial thrombus formation, which resolved by recombinant tissue plasminogen activator lysis; (3) and development of a "locked-in-syndrome." Possible underlying mechanisms are discussed. PMID- 1731683 TI - Mitral valve repair in patients on chronic hemodialysis. AB - Two cases of successful mitral valve repair in patients on chronic hemodialysis are presented. We stress that valve repair is preferable to valve replacement whenever feasible because of improved left ventricular function, reduced complication rate, and freedom from anticoagulation. This especially applies to patients on chronic hemodialysis as they have impaired immunological function, are subjected to repeated fistula punctures with possible bacteremia, and are more susceptible to early calcification and degeneration of tissue valves. PMID- 1731684 TI - Benign schwannoma of the esophagus presenting as a giant fibrovascular polyp. AB - The case of a 62-year-old man with benign schwannoma associated with a giant polyp of the esophagus is presented. His initial symptom was dysphagia. The polyp was removed through cervical esophagotomy. He had no recurrence of symptoms 5 years after this procedure. Pathologic examination showed a rare histology. PMID- 1731685 TI - Vascularized rib strut technique for repair of pectus excavatum. AB - A vascularized rib strut based on the anterior intercostal branch of the internal mammary artery was applied to provide rigid internal fixation of the chest wall after correction of pectus excavatum. The procedure is simple and has substantial advantages when compared with techniques using metallic struts or nonvascularized free rib grafts. PMID- 1731686 TI - Preoperative and postoperative Hemopump support for patients undergoing orthotopic heart transplantation. AB - The Hemopump (Johnson & Johnson Interventional Systems, Rancho Cordova, CA) can be used successfully as a bridge to cardiac transplantation in patients with advanced cardiogenic shock that proves to be irreversible. Some patients, however, may also benefit from maintaining Hemopump support for a period of time after transplantation. A technique has been developed for continuing Hemopump support after transplantation that does not require repositioning of the device. PMID- 1731687 TI - Rapid transfusion after aortic decannulation. AB - A technique for rapid transfusion after aortic decannulation is presented. This technique facilitates the repair of the aortic cannulation site in difficult circumstances. PMID- 1731688 TI - Retrograde cardioplegia: current methods. AB - Retrograde coronary sinus cardioplegia is being used for myocardial protection with ever-increasing frequency during complex cardiac operations. New methods for introducing cardioplegia into the coronary sinus have been facilitated by improved balloon-tipped catheters. Moreover, various solutions are being infused under both hypothermic and normothermic conditions with good results. This report provides background into the application of this technique and illustrates several methods for its daily use in clinical cardiac surgery. PMID- 1731689 TI - Cardiac pheochromocytomas. AB - Cardiac pheochromocytomas are rare. Thirty cases have been reported in the literature. We report the cases of 2 more patients in whom the diagnosis was established using coronary angiography and who underwent surgical resection using cardiopulmonary bypass. We also review the literature on the subject. PMID- 1731690 TI - Retrograde cerebral perfusion. PMID- 1731691 TI - Single venous cannulation for valve operations. PMID- 1731692 TI - Penetrating injury of the innominate artery. PMID- 1731694 TI - Emergency: an urgent problem emergent? PMID- 1731693 TI - Retrograde continuous warm blood cardioplegia. PMID- 1731695 TI - Identification and sequence determination of the capsid protein gene of feline calicivirus. AB - We have determined 4380 bases of the sequence from a cDNA clone containing the 3' end of feline calicivirus strain F9. We find four candidate open reading frames of which three are complete and comprise 245, 317 and 2012 nucleotides. The fourth continues toward the 5' end. We have expressed the largest complete open reading frame in E. coli. Sera raised to this antigen react specifically with the capsid protein and its intracellular precursor molecule. N-terminal sequence analysis of purified, mature capsid protein confirms this assignment and has identified the position at which precursor is cleaved. PMID- 1731696 TI - Bean yellow mosaic, clover yellow vein, and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3' non-coding regions. AB - The sequences of the 3' 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3' 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3' non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3' untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3' untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3' non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3' non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses. PMID- 1731697 TI - The haemagglutinin-neuraminidase glycoprotein of the porcine paramyxovirus LPMV: comparison with other paramyxoviruses revealed the closest relationship to simian virus 5 and mumps virus. AB - The complete nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of the porcine paramyxovirus LPMV, was determined from cDNA derived from viral genomic RNA. The gene was 1906 nucleotides long including a putative gene end and poly A signal. One long open reading frame was found encoding a protein of 576 amino acids with a calculated molecular weight of 63,324. The protein contains four potential N-glycosylation sites and a major hydrophobic region near the N terminal, suggesting a membrane anchor domain. Comparison of the deduced amino acid sequence of the LPMV HN protein with that of other paramyxovirus HN proteins, revealed the highest amino acid identity to simian virus 5 of 43% and mumps virus of 41%. PMID- 1731699 TI - Expression of large hepatitis B envelope protein mutants using a new expression vector. AB - Aminoterminal deletion mutants of the gene encoding the large hepatitis B surface protein were expressed in COS cells using a new expression vector. The truncated protein showed the same intracellular retention like the wild type protein. The findings show that the secretion block of the protein is not due to its aminoterminal myristylation. PMID- 1731698 TI - The distribution of viral antigens and Langerhans cells during zosteriform spread of herpes simplex virus to the skin of the mouse. PMID- 1731700 TI - Reactivity of a recombinant rubella E1 antigen expressed in E. coli. AB - The E1 nucleic acid sequence of rubella virus strain Judith (RJ) has been cloned into an E. coli expression vector LB03. The reactivity of the expressed unglycosylated antigen (E1J) was compared with its glycosylated counterpart in native virus (RJ) using rabbit and human sera. Rabbit antisera raised against RJ and E1J reacted differently with wild type, RJ (laboratory strain) and RA27/3 (vaccine virus) strains in a kinetic neutralisation test. Reciprocally, human post RA27/3 vaccination sera were also found to differ from post infection or post re-infection sera in their reactivity with RJ and E1J antigens. Our observations suggest that E1, in the conformation adopted in the RA27/3 virion may have unique antigenic properties. PMID- 1731701 TI - Ethacrynic acid increases facility of outflow in the human eye in vitro. AB - Anterior segments of human donor eyes were perfused with culture medium at a perfusion pressure of 15 mm Hg in a 5% carbon dioxide environment at 37 degrees C. After determination of a baseline facility of outflow, the perfusion chamber contents were exchanged with either drug vehicle or ethacrynic acid, at concentrations ranging from 0.01 to 0.25 mmol/L, after which postdrug facility was measured in the continuous presence of drug vehicle or ethacrynic acid. Ethacrynic acid increased facility of outflow from 28% to 105% at ethacrynic acid concentrations of 0.01 to 0.25 mmol/L, respectively. No morphologic correlate of the facility increase was observed with 0.01-mmol/L ethacrynic acid, nor were there any signs of cellular toxic effects. At 0.1 mmol/L, separations between trabecular meshwork cells and breaks between inner-wall cells were observed. At 0.25 mmol/L, focal areas of cell swelling and necrosis were noted. This study demonstrated that ethacrynic acid increases outflow facility in the aged human eye at concentrations that produce no apparent toxic effects. Therefore, ethacrynic acid may potentially prove useful in the treatment of glaucoma. PMID- 1731702 TI - Effects of intracameral injection of chondroitinase ABC in vivo. AB - A glycosaminoglycan-degrading enzyme, chondroitinase ABC, was introduced into the anterior chamber of three cynomolgus monkeys. Following the injection, the intraocular pressure decreased in the experimental eyes. Depending on the monkey, the intraocular pressure was lowered for 5, 7, or 14 days before it returned to the normal level. Repeated injections produced a similar response. Structurally, the intertrabecular spaces appeared widened, and marked ballooning of the juxtacanalicular tissue was observed. The outer trabecular beams and the inner wall of Schlemm's canal were greatly disorganized. Considerable loss of the juxtacanalicular tissue was noted even 2 months after the enzyme was injected into the anterior chamber. These observations suggest that chondroitinase ABC digested the trabecular glycosaminoglycans, triggering intraocular pressure reduction and causing disorganization of the extracellular matrices of the disorganized trabecular beams, especially in areas near Schlemm's canal. PMID- 1731703 TI - Comparative anti-inflammatory efficacy of topical corticosteroids with low glaucoma-inducing potential. AB - Fluorometholone and clobetasone butyrate have been developed as ophthalmic corticosteroids because of their lesser potential to elevate intraocular pressure. Nevertheless, their primary use is the inhibition of an inflammatory response. Quantification of their anti-inflammatory effect in the rabbit cornea indicates that 0.1% fluorometholone and 0.1% clobetasone butyrate are effective, but weak, anti-inflammatory agents. An increase in concentration of fluorometholone to 0.25% failed to enhance its anti-inflammatory effectiveness significantly, while an increase in concentration of clobetasone butyrate to 0.5% did significantly increase its anti-inflammatory effect. As with all other corticosteroid bases studied to date, formulation of fluorometholone as an acetate derivative significantly increased its effectiveness, rendering it as effective as 1.0% prednisolone acetate, the most effective of commercially available ophthalmic corticosteroids. PMID- 1731705 TI - Corneal welding using hydrogen fluoride lasers. PMID- 1731704 TI - Long-term complications of the MAI hydrogel intrascleral buckling implant. PMID- 1731706 TI - Distribution of lymphocytes and cell adhesion molecules in iris biopsy specimens from patients with uveitis. AB - To investigate the mechanisms responsible for lymphocyte accumulation in the eye in uveitis, we examined iris biopsy specimens that were obtained from 10 patients with uveitis and from 12 patients with cataract for the presence of adhesion molecules on vascular endothelium, uveal cells, and infiltrating inflammatory cells. Immunoperoxidase staining of iris biopsy specimens that were obtained from patients with uveitis revealed an increased expression of intercellular adhesion molecule 1 (CD54) on endothelial cells, lymphocytes, fibroblasts, and iris epithelial cells. Seven of the 10 iris biopsy specimens that were obtained from patients with uveitis had a significant inflammatory cell infiltrate. Lymphocytes (CD2 positive) that infiltrated the iris were predominantly helper T cells (70%) and strongly expressed the lymphocyte function-associated antigen 1 (CD11a, CD18) molecule, the ligand for intercellular adhesion molecule 1, in four of the seven biopsy specimens. In contrast, small numbers of lymphocytes were evident in only three (25%) of the iris biopsy specimens that were obtained from patients with cataract. Vascular endothelium from the latter group did not express intercellular adhesion molecule 1 or endothelial leukocyte adhesion molecule 1. The results of this study revealed the enhanced expression of vascular endothelial cell and lymphocyte adhesion molecules in the iris biopsy specimens that were obtained from patients with uveitis. The presence of these receptors and their presumed ligands may have important implications for the role of these molecules in the pathogenesis of uveitis. PMID- 1731707 TI - Chlorpromazine-induced anterior segment changes. PMID- 1731708 TI - Posterior chamber intraocular lens implantation combined with lensectomy vitrectomy and intraretinal foreign-body removal. AB - Two patients with intraretinal foreign bodies and traumatic cataracts were treated with pars plana lensectomy, vitrectomy, and removal of the foreign body. In each case, it was technically possible to preserve the anterior capsule despite small but obvious rents caused by the foreign body. This allowed support for the placement of a posterior chamber intraocular lens at the time of the initial repair. Posterior chamber intraocular lens implantation may be a useful adjunctive step in the treatment of selected patients with intraocular foreign bodies. PMID- 1731709 TI - Enzyme deficiency for type 1 primary hyperoxaluria. PMID- 1731710 TI - Peripapillary subretinal neovascularization associated with multiple evanescent white-dot syndrome. PMID- 1731711 TI - The steamroller maneuver and proliferative vitreoretinopathy. PMID- 1731712 TI - Is basal laminar deposit unique for age-related macular degeneration? PMID- 1731713 TI - Excimer laser ablation of infectious crystalline keratopathy. PMID- 1731714 TI - Follicular thyroid carcinoma metastatic to the iris: a solitary lesion treated with iridocyclectomy. PMID- 1731715 TI - Corneal toxicity with an antibiotic/steroid-soaked collagen shield. PMID- 1731717 TI - Ophthalmologists lose big under RBRVS. PMID- 1731716 TI - Acquired arterial collateral vessels at the optic disc. PMID- 1731718 TI - Diagnosing open angle glaucoma. PMID- 1731719 TI - Indications for postoperative fluorouracil therapy. PMID- 1731720 TI - Pharmacologic trabeculocanalotomy. Facilitating aqueous outflow by assaulting the meshwork cytoskeleton, junctional complexes, and extracellular matrix. PMID- 1731721 TI - Ophthalmology. The resident's perspective. AB - To determine how and why current residents in ophthalmology chose their medical specialty, we formulated and distributed a questionnaire to all ophthalmology residents in accredited programs. The results of the survey gave insight into not only their decision-making processes in choosing ophthalmology, but also their backgrounds, the factors that were important in choosing their residency program, and their future plans in ophthalmology. A wide variety of factors was involved in the decision to pursue a career in ophthalmology. In addition, we found current residents to be well qualified academically and generally satisfied with their decision to enter ophthalmology. PMID- 1731722 TI - Treatment of retinopathy of prematurity with argon laser photocoagulation. AB - Fifteen eyes of nine infants were treated for retinopathy of prematurity by confluent photocoagulation of the avascular retina using an argon laser indirect ophthalmoscope. All treated eyes presented at or beyond threshold of stage 3 retinopathy of prematurity with "plus" disease. Three treated eyes had retinopathy of prematurity in zone I. Early complete regression of extraretinal proliferation was seen in 13 eyes. At 6 month's [corrected] minimum follow-up, 11 (73%) of the treated eyes demonstrated a favorable outcome, while four eyes (27%) progressed to an unfavorable outcome. No intraocular hemorrhages occurred during any laser treatment. PMID- 1731723 TI - Ocular findings associated with rhodopsin gene codon 17 and codon 182 transition mutations in dominant retinitis pigmentosa. AB - Six members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-thymine transition mutation in the second nucleotide of codon 17 in the rhodopsin gene that resulted in a threonine to methionine change. Three members from another family with autosomal dominant retinitis pigmentosa showed a guanine-to-adenine transition mutation in the first nucleotide of codon 182 in the rhodopsin gene that resulted in a glycine to serine change. Each of these two mutations presented with a similar phenotype because both showed a regional predilection for pigmentary changes to occur in the inferior part of the retina as well as field impairment predominantly in the superior hemisphere. Electroretinographic amplitudes were more substantial than usually encountered in other forms of retinitis pigmentosa, a finding consistent with the better visual prognosis in patients with either of these two mutations. This article documents the association of two similar phenotypes of autosomal dominant retinitis pigmentosa with specific gene defects at a molecular level. PMID- 1731724 TI - Visual field loss in primary gaze and reading gaze due to acquired blepharoptosis and visual field improvement following ptosis surgery. AB - Acquired blepharoptosis has been associated with loss of the superior visual field (SVF) in primary gaze. Because many patients with acquired blepharoptosis complained of difficulty reading or performing other visual functions in reading gaze, a prospective study was undertaken to determine if acquired blepharoptosis was the cause of these visual dysfunctions. Preoperative and postoperative SVFs were tested in primary gaze and reading gaze in 19 patients with unilateral or bilateral acquired blepharoptosis totaling 30 eyes. Preoperative testing revealed a marked loss of the SVF in both primary gaze and reading gaze. All patients underwent levator aponeurosis defect repair. Postoperative results showed a significant improvement in both primary and reading gaze SVFs. Therefore, patients with good visual acuity complaining of difficulty reading or carrying out other visual functions in reading gaze should be examined for the presence of acquired blepharoptosis. Blepharoptosis repair can be expected to improve the SVF in both primary gaze and reading gaze. PMID- 1731725 TI - Abnormal eye movements in Gerstmann-Straussler-Scheinker disease. AB - Gerstmann-Straussler-Scheinker disease is a familial disorder of progressive ataxia and dementia in adulthood with extrapyramidal signs in some families. Neuro-ophthalmic examinations and eye movement recordings were performed in members of a large Indiana kindred. Five affected members and 11 members at risk were studied. Eye movements were recorded with videotape, electro-oculography, and/or magnetic scleral search coil. All affected members had abnormal eye movements characteristic of extrapyramidal diseases and cerebellar disorders. Nine members at risk had normal eye movements, but two others had slightly abnormal eye movements. Neuro-ophthalmic examination and eye movement recordings might be helpful in detecting early signs of Gerstmann-Straussler-Scheinker disease in persons at risk. PMID- 1731726 TI - The pattern visual evoked potential and pattern electroretinogram in drusen associated optic neuropathy. AB - Sixteen patients (29 eyes) with optic disc drusen were studied prospectively for clinical and electrophysiologic evidence of impaired optic nerve conduction. Abnormalities were detected in the following areas: visual acuity, eight (28%) of 29 eyes; kinetic visual field, 22 (76%) of 29 eyes; results of Farnsworth-Munsell 100-Hue test, 12 (41%) of 29 eyes; and flash visual evoked potential, 13 (54%) of 24 eyes. Simultaneous pattern visual evoked potentials and results of pattern electroretinograms were recorded. The P100 latency of the pattern visual evoked potential was prolonged in 41% of eyes. The P50 and N95 components of the pattern electroretinogram were also analyzed. The P50 amplitude was reduced in only four (17%) of 24 eyes. The most common abnormality was a reduction in amplitude or the absence of the N95 component in 19 (79%) of 24 eyes, reflecting ganglion cell dysfunction. The data support mounting evidence that the P50 and N95 components of the pattern electroretinogram have different retinal origins. PMID- 1731727 TI - Microbial contamination of in-use ocular medications. AB - Two hundred twenty in-use medications from 101 patients with nonmicrobial ocular surface disease were studied by culturing the bottle caps, a drop produced by simple inversion, and the interior contents removed sterilely. Conjunctival cultures were taken from these patients and 50 age-matched controls. Pathogenic organisms were harvested from conjunctivae significantly more frequently (P less than .01) from cases (34 of 101) than from controls (five of 50). Sixty-four medications (29%) had microorganisms cultured from at least one medication site. Gram-negative organisms were significantly more likely (P less than .00001) to be isolated from all medication sites than gram-positive organisms. Additionally, when isolated from medication sites, the gram-negative organisms were highly likely to be cultured from the conjunctiva as well. This was not true for pathogenic gram-positive organisms. We conclude that a cycle of contamination between in-use medications and conjunctivae may represent an important risk factor for microbial keratitis in patients with ocular surface disease. PMID- 1731729 TI - Side-view analysis of the lens. I. The crystalline lens and the evacuated bag. AB - A new technique for studying the anatomy of the lens and other anterior chamber structures in human eyes obtained post mortem is described. An oblique or side view is achieved by creating a uveoscleral window. This provides a clear three dimensional view of such structures as the crystalline lens, zonular apparatus, and ciliary body. The crystalline lens is shown to be approximately 4.5 mm thick and 9.5 mm in diameter. The equator of the lens is 0.2 to 0.3 mm from the center of the ciliary body. After removal of lens substance, the capsular bag collapses, its thickness approaches zero, and the total diameter increases to 10.5 mm. Filling of the capsular bag with a viscoelastic material restores the configuration of the lens to its original state. This technique is also useful for demonstrating the dynamics of surgical procedures during cataract operation. PMID- 1731728 TI - Long-term complications of the MAI hydrogel intrascleral buckling implant. AB - Seven cases in which long-term complications developed from swelling of the MAI hydrogel intrascleral buckling implant are reported herein. Micro-Fourier transform infrared spectroscopic analysis of two recovered implants demonstrated the occurrence of chemical changes leading to increased swelling. Clinical problems with the implants appeared 7 to 11 years after surgery, suggesting the need for periodic, long-term follow-up. It is possible that the present-day MIRAgel implant, which has the same chemical composition as the MAI implant, may require similar precautions. PMID- 1731730 TI - Side-view analysis of the lens. II. Positioning of intraocular lenses. AB - The uveoscleral window technique is useful for studying the position and fixation of posterior chamber intraocular lenses (IOLs). Studies using this technique show that the configuration of an IOL in the capsular bag largely depends on three factors: size, shape, and relative rigidity of the IOL. The general configuration of the capsular bag is that of a flattened saucer created by radial expansion of the haptic. Zonules at the fixation site are relaxed, eliminating the possibility of accommodation with thin IOLs. Downsized IOLs (total diameter of 12.0 mm) provide stable fixation with less tension on the capsule than larger IOLs. PMID- 1731731 TI - The long-term effects of visible light on the eye. AB - The relationship between exposure to sunlight and senile cataract, age-related macular degeneration, pterygium, and climatic droplet keratopathy was examined in 838 watermen who work on the Chesapeake Bay. The presence and severity of lenticular, corneal, and macular changes were assessed by either clinical examination or from stereo macular photographs. From detailed exposure histories, ocular exposure was estimated for three bands of visible radiation-violet (400 to 450 nm), blue (400 to 500 nm), or all visible (400 to 700 nm)-as well as for UV-A (320 to 340 nm) and UV-B (290 to 320 nm). The results with each band of visible radiation were similar. Neither cortical nor nuclear cataract was associated with ocular exposure to blue or all visible radiation, but pterygium and climatic droplet keratopathy were more common with higher exposures. Compared with age matched controls, patients with advanced age-related macular degeneration (geographic atrophy or disciform scarring) had significantly higher exposure to blue or visible light over the preceding 20 years (odds ratio, 1.36 [1.00 to 1.85]) but were not different in respect to exposure to UV-A or UV-B. These data suggest that high levels of exposure to blue or visible light may cause ocular damage, especially later in life, and may be related to the development of age related macular degeneration. PMID- 1731732 TI - Orthopaedic training and education in Australia. PMID- 1731733 TI - Paget's disease: current concepts. AB - The curious geographic variations in the prevalence of Paget's disease remain unexplained and the viral hypothesis remains unproved. An association between transient hyperphosphataemia of infancy and subsequent Paget's disease has not been established. Some 5% of Pagetic patients have hyperparathyroidism although the mechanism of this apparent association remains unknown. Several agents are available for the medical treatment of Paget's disease but there is a lack of consensus concerning therapeutic aims. PMID- 1731734 TI - The diagnosis of Paget's disease of bone. AB - Paget's disease of the bone is a common disorder and usually presents no diagnostic difficulty. However, atypical cases may cause problems in diagnosis, particularly in younger patients, and this paper describes some of the more unusual modes of presentation mainly related to the osteolytic phase and to accentuation of normal anatomical features. The role of various imaging techniques is discussed, emphasizing that recognition of the plain radiographic changes of Paget's disease remains the key to diagnosis in most cases. PMID- 1731735 TI - Orthopaedic surgery in Australia: an international perspective. PMID- 1731736 TI - Outcome after fractures of the femur in Paget's disease. AB - We reviewed ninety-three patients who had one hundred and seven complete fractures of the femur between them and were treated over a twenty-five year period in Western Australia and South Australia. Operative surgical management of complete fractures in the middle and distal thirds was usually successful. In contrast, fractures proximal to the middle third were regularly associated with non-union, implant failure and requirement for revision surgery. In view of this significant difference in outcome after fracture of the pagetic femur in different sites, a comprehensive surgical management strategy is recommended in order to avoid complications. PMID- 1731737 TI - Osteosarcoma: then and now. A fifty year review at Royal Prince Alfred Hospital. AB - The purpose of this study is to review survival, treatment methods and criteria for diagnosis of osteosarcoma at Royal Prince Alfred Hospital during two periods in the last 50 years. The records of 22 patients diagnosed with osteosarcoma and nine with osteosarcoma in Paget's disease (from 1939 to 1950) were reviewed. All but one had died within three years. One patient survived six years. The second series was taken from 1983 to 1990. Forty-nine patients, including three with Paget's sarcoma, were studied. Probability of survival was estimated by actuarial analysis using Kaplan-Meier curves. Overall survival was estimated at 45%. Those patients who were free of metastatic disease at the conclusion of their treatment were estimated to have a probability of survival of 85%. PMID- 1731738 TI - Porous coated anatomic non-cemented total hip arthroplasty. AB - Between November 1984 and December 1989, 318 non-cemented Porous Coated Anatomic (PCA; How-medica, Rutherford, New Jersey) total hip replacements were performed by the authors. A follow-up of 1 to 6 years was allowed. The average age was 53.1 years (from 17 to 71 years). The distribution of right-to-left was approximately equal. There were 192 hip replacements for primary and post-traumatic osteo arthritis, 42 for rheumatoid arthritis, 40 for avascular necrosis, 29 for congenital dislocation or hip dysplasia with secondary osteo-arthritis, 6 for Perthes disease, 5 for previous sepsis, 2 of whom had had a Girdlestone procedure, 2 for revision of a painful cup arthroplasty, and 1 for conversion of a previously fused hip. All patients were evaluated on a one hundred point Harrington Arthritis Research Centre Scale. Points were awarded for pain (0-35), function (0-35), motion (0-10), deformity (0-10) and gait (0-10). Pre-operative total scores averaged 45.5 (9-71) and postoperative scores averaged 83.9 (55-98). The overall results were excellent 20.5% (90-100), good 59.8% (80-90), fair 16.4% (70-80), and poor 3.3%. Postoperative radiographs were evaluated using zonal analysis. There was no deterioration on the radiographs after two years. PMID- 1731739 TI - Frederic Wood Jones, artist and illustrator. PMID- 1731740 TI - The history of orthopaedic surgery at Royal Prince Alfred Hospital, Sydney. PMID- 1731741 TI - The common bilio-pancreatic channel syndrome in childhood. AB - An abnormally long common bilio-pancreatic channel has been found in association with choledochal cysts and biliary strictures in childhood. It may also present with recurrent abdominal pain, vomiting, hyperamylasaemia and jaundice. This has been termed the common channel syndrome (CCS). Two cases with the CCS presenting early in childhood are reported together with a review of the literature. Open sphincteroplasty was performed in both cases with a satisfactory outcome. PMID- 1731742 TI - Thymolipoma. AB - Although the diagnosis of thymolipoma has improved with the introduction of computerized tomography (CT), variations in CT appearance are still being described; this paper describes a case which resembled a lipoma. The CT features of thymolipoma are discussed with special reference to the differential diagnosis. Surgical excision should be considered for patients who are found to exhibit a fatty intrathoracic mass on CT. PMID- 1731743 TI - Infarcation of a jejunal leiomyoma presenting as an acute abdomen. AB - Leiomyomata of the small intestine are rare benign tumours of the small intestine that usually present with gastrointestinal bleeding, vague abdominal pains or small bowel obstruction. A case is reported of a patient who presented with an acute abdomen due to infarction of a leiomyoma of the proximal jejunum. PMID- 1731744 TI - Open lung biopsy without thoracotomy: technique and possible indications. AB - Lung biopsy material is still required in the diagnosis of lung disease, especially in diffuse parenchymal diseases or pulmonary infiltrates in the immunosuppressed patient. This often requires open thoracotomy which is not without morbidity or mortality especially in immunocompromised patients. We report a new technique of lung biopsy using laparoscopic instrumentation in conjunction with a new stapling instrument, the EndoGIA 30 (Auto Suture Aust. N.Z.). This allows removal of a section of lung tissue without air leakage through three small incisions. PMID- 1731745 TI - Strategy for AIDS. PMID- 1731746 TI - Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter. AB - Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane. PMID- 1731747 TI - The effects of the exogenous provision of lactate and the endogenous production of lactate on protein synthesis in the heart. AB - We have investigated the effects of exogenous addition of lactate and of the stimulation of endogenous production of lactate on protein synthesis in the anterogradely perfused rat heart. In the absence of exogenous lactate, hearts release lactate into the perfusate. At lactate concentrations of 0.2 mM and greater, the heart takes up lactate. The best fit for lactate uptake plotted against exogenous lactate concentration is a rectangular hyperbola with a maximal rate of 220 mumol/2 h per heart (wet wt. about 1 g). Uptake is half-maximal at about 1.3 mM-lactate. The stimulation of protein synthesis also exhibits a rectangular-hyperbolic dependence on exogenous lactate concentration, with maximal stimulation being about 38%. Half-maximal stimulation occurs at about 0.9 mM-lactate. We stimulated endogenous lactate production by perfusion with 2 cyanocinnamate (an inhibitor of mitochondrial pyruvate transport) at concentrations up to 70 microM. Cardiac outputs, intracellular pH and the concentrations of phosphocreatine and the adenine nucleotides were not altered. Atrial protein-synthesis rates were unchanged, but ventricular rates were decreased. We conclude that endogenous lactate production is unlikely to stimulate protein synthesis and that the stimulation of protein synthesis by exogenous lactate is related to its uptake. PMID- 1731748 TI - Purification of an Arg-Gly-Asp selective matrix receptor from brain synaptic plasma membranes. AB - Brain synaptic plasma membranes specifically associated with matrix protein monolayers containing the Arg-Gly-Asp sequence recognized by integrin-type adhesion receptors. Experiments using fibronectin affinity chromatography to identify the synaptosomal receptors responsible for this interaction led to the purification of a 55 kDa Arg-Gly-Asp recognition protein that is labelled by antibodies against the alpha 5 beta 1 integrin. PMID- 1731749 TI - Expression of c-myc and c-fos in rat skeletal muscle. Evidence for increased levels of c-myc mRNA during hypertrophy. AB - The levels of c-myc and c-fos mRNA were investigated in rat skeletal muscle by Northern hybridization. During post-natal development in the rat, c-myc mRNA levels were similar at birth and at 7 and 21 days of age, but then declined at 90 days and were barely detectable at 1 year. c-fos mRNA levels followed this pattern of expression until 90 days, but showed a large increase at 1 year. Hypertrophy of soleus and plantaris muscles was induced either by severance of the tendon to the synergistic gastrocnemius (tenotomy) or by administration of the beta-adrenoceptor agonist clenbuterol. In both cases hypertrophy was associated with a rapid increase in c-myc mRNA levels. Following tenotomy the increase was both greater (8-fold) and more rapid (3 h) in soleus than in plantaris (2-3 fold, 12 h). Similar effects were observed during clenbuterol administration. Neither treatment caused any alteration in c-fos mRNA levels in the plantaris muscle. The results show that increased c-myc mRNA levels are an early event in the response of skeletal muscle to hypertrophic stimuli; it is argued that this occurs within the differentiated skeletal muscle fibres. PMID- 1731750 TI - Chlorine-containing compounds produced during Dictyostelium development. Detection by labelling with 36Cl. AB - DIF-1 [Differentiation-Inducing Factor 1; 1-(3,5-dichloro-2,6-dihydroxy-4 methoxyphenyl)hexan-1-one] is a novel chlorinated signal molecule that induces stalk-cell differentiation during development of Dictyostelium discoideum. Here we introduce the use of the radioisotope 36Cl to label DIF-1 and other low-Mr chlorinated compounds produced during development. H.p.l.c. and t.l.c. were used to resolve the labelled compounds. We find the following. (1) At least 14 dialysable 36Cl-labelled compounds are released into the medium by cells labelled continuously through development with Na36Cl. (2) The compounds can be classified into two major groups according to their times of accumulation in development. The early group of compounds starts accumulating at the end of aggregation, co ordinately with DIF-1; the late group is only made at the end of development, by mature fruiting bodies. There may also be an intermediate group made during culmination. (3) The early group of compounds has been identified as comprising DIF-1 and seven of its metabolites by co-chromatography with the authentic compounds. These metabolites had previously only been recognized in suspensions of living cells incubated with exogenous DIF-1. Their detection here, from cells undergoing normal development, suggests that endogenous DIF-1 is metabolized in normal development in much the same way as is DIF-1 added to cells in suspension. (4) The intermediate and late groups of compounds are not obvious DIF-1 metabolites. They may have some role unconnected with DIF signalling. (5) A group of 36Cl-labelled late compounds remain cell-associated after washing of the fruiting bodies, and these are greatly enriched in stalk, compared with spore, cells. (6) Other slime-mould species were labelled with 36Cl. All three tested, namely D. mucoroides, D. vinaceo-fuscum and P. violaceum, also produced chloro compounds. D. mucoroides produced DIF-1 by the criterion of h.p.l.c. co-elution with authentic DIF-1. A developmentally regulated metabolism of chlorinated compounds may therefore be widespread amongst slime moulds. To our knowledge, labelling with 36Cl in vivo has not been reported before and provides a powerful general method for investigating chlorinated compounds in diverse organisms. PMID- 1731751 TI - Mitogenic action of lysophosphatidic acid and phosphatidic acid on fibroblasts. Dependence on acyl-chain length and inhibition by suramin. AB - Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with growth factor-like activities [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 45, 45-54]. We have examined various structural analogues of LPA for their ability to stimulate DNA synthesis in quiescent fibroblasts. When the acyl chain length is varied, the rank order of mitogenic potency is: 1-oleoyl LPA congruent to 1-palmitoyl LPA greater than 1-myristoyl LPA greater than 1-lauroyl LPA greater than 1-decanoyl LPA; the last compound shows almost no activity over the concentration range tested (1-100 microM). An ether-linked LPA (1-O hexadecylglycerol 3-phosphate) has much decreased mitogenic activity as compared with the ester-linked analogue at concentrations less than 25 microM, and becomes cytotoxic at higher concentrations. Hexadecylphosphate, which lacks a glycerol backbone, has negligible activity. On a molar basis, diacyl phosphatidic acid (PA) is about equally potent as the corresponding LPA analogue, showing similar acyl-chain-length dependence; the data argue against the possibility that the mitogenic action of PA is due to contaminating traces of LPA. Although the short chain analogues of LPA and PA fail to antagonize the action of long-chain (L)PAs, the polyanionic drug suramin inhibits LPA- and PA-induced, DNA synthesis in a reversible and dose-dependent manner, at concentrations [IC50 (concn. giving 50% inhibition) approximately 70 microM] that do not affect epidermal-growth-factor induced DNA synthesis. Suramin appears to act in the early G0/G1 phase of the cell cycle, blocking immediate responses to LPA such as phosphoinositide hydrolysis. We conclude that both LPA and PA can function as growth-promoting phospholipids, with the fatty acid chain length being a major determinant of mitogenic potency. PMID- 1731752 TI - Mitochondrial metabolism in different thyroid states. AB - The protonmotive force, as well as the mitochondrial and cytosolic concentrations of malate, 2-oxoglutarate, glutamate and aspartate, were determined in livers from hypo-, eu- and hyper-thyroid rats, by density-gradient centrifugation of freeze-clamped livers in non-aqueous solvents [Soboll, Akerboom, Schwenke, Haase & Sies (1980) Biochem. J. 192, 951-954]. The mitochondrial/cytosolic pH difference and the membrane potential were significantly enhanced in hyperthyroid livers compared with the hypothyroid state, resulting in an increased protonmotive force in the presence of thyroid hormones [Soboll & Sies (1989) Methods Enzymol. 174, 118-130]. The mitochondrial concentrations of 2 oxoglutarate, glutamate and aspartate were significantly higher in the euthyroid than in the hypothyroid state, but only slightly higher in the hyperthyroid state. Mitochondrial malate, on the other hand, increased significantly from the hypothyroid to the hyperthyroid state. The mitochondrial/cytosolic concentration gradients were significantly increased in the presence of thyroid hormones only for malate. The changes in steady-state metabolite concentrations reflect a higher substrate supply and a stimulation of mitochondrial metabolism. However, a clear relationship between the increased protonmotive force, as the driving force for mitochondrial metabolite transport, and the subcellular metabolite concentrations is not observable in different thyroid states. PMID- 1731753 TI - Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases. AB - Inactivators of cysteine endopeptidases were tested as inhibitors of the cytokine stimulated release of proteoglycan from cartilage. The test system consisted of bovine nasal septum cartilage maintained in organ culture, and the stimulus was provided by recombinant human interleukin 1 alpha. L-3-Carboxy-2,3-trans epoxypropionyl-leucylamido-(4-guanidin o)butane (E64) and L-3-carboxy-2,3-trans epoxypropionyl-leucylamido-(3-methyl)b utane (Ep475) showed no inhibition at concentrations up to 100 microM. In contrast, trans-epoxysuccinyl-leucylamido-(3 methyl)butane ethyl ester (Ep453), a 'prodrug' of Ep475, was an effective inhibitor. The LL-, LD- and DL-isomers gave significant inhibition at 10 microM, and the DD-isomer was inhibitory at 100 microM. None of the isomers had any detectable effect on protein synthesis or glycolysis, and their inhibitory effects were reversible. Iodoacetate inhibited proteoglycan release by a general toxic effect. Our results suggest that cysteine endopeptidase(s) play a part in cytokine-stimulated cartilage breakdown, but that effective inhibitors must pass through membranes. PMID- 1731754 TI - Epoxyeicosatrienoic acid stimulates ADP-ribosylation of a 52 kDa protein in rat liver cytosol. AB - In rat liver cytosol, rapid ADP-ribosylation of a 52 kDa protein by endogenous ADP-ribosyltransferase(s) was observed. This ADP-ribosylation was stimulated dose dependently by 14,15-epoxyeicosatrienoic acid (14,15-EET), one of the metabolites of arachidonic acid by NADPH-dependent cytochrome P-450 mono-oxygenase. This stimulatory effect required the presence of GTP or its non-hydrolysable analogues, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma thio]triphosphate. Of four regioisomeric EETs, 14,15-EET was the most potent. No stimulatory effect was observed with addition of 14,15-dihydroxyeicosatrienoic acid, a stable metabolite of 14,15-EET. The 52 kDa protein was not ADP ribosylated by cholera toxin A subunit and pertussis toxin, and was not recognized by anti-Gs alpha and anti-Gi alpha antibodies. However, the 52 kDa protein could be photoaffinity-labelled with 8-azidoguanosine 5'-[alpha 32P]triphosphate. These results suggest that the 52 kDa protein is neither Gs nor Gi, though it may have a GTP-binding site. These results contribute to the understanding of the role of mono-oxygenase metabolites of arachidonic acid in intracellular signal transduction. PMID- 1731755 TI - Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of beta-lactamase. AB - Nafcillin was shown to reversibly inhibit beta-lactamase from Staphylococcus aureus PC1 with characteristics indicative of a type A inhibitor [Citri, Samuni & Zyk (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052]. At nafcillin concentrations above 80 mM, complete inactivation occurred within 200 s. Upon removal of the excess nafcillin the inhibited enzyme was re-activated completely, with a rate constant of 2.0 x 10(-3) s-1 (25 degrees C). The inhibited enzyme was shown to be in the form of a covalent acyl-enzyme intermediate. Digestion by pepsin and trypsin yielded a single nafcillin-labelled peptide fragment which was isolated, sequenced and shown to be: Ala-Tyr-Ala-Ser-Thr-Ser-Lys. This sequence corresponds to the region surrounding the active-site serine residue, Ser-70, indicating that the inhibitor is covalently attached to the same residue as productive substrates. PMID- 1731756 TI - Structure and expression of the rat epididymal secretory protein I gene. An androgen-regulated member of the lipocalin superfamily with a rare splice donor site. AB - The complete rat epididymal secretory protein I (ESP I) gene was isolated from a genomic library constructed in bacteriophage lambda Charon 4A. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined. Interesting features include the presence of a rare, but functional, splice donor site (...GC) and the presence of a putative androgen receptor-binding element. A detailed analysis of ESP I regulation was carried out after castration and subsequent testosterone treatment, demonstrating the requirement for androgens. Efferent-duct ligation and cryptorchism, on the other hand, had no effect on the steady-state concentrations of ESP I transcripts. Comparison of the exon/intron organization of the ESP I gene with those of members of the lipocalin superfamily provides strong support for a common ancestral origin. PMID- 1731758 TI - Mechanistic and active-site studies on D(--)-mandelate dehydrogenase from Rhodotorula graminis. AB - D(--)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(--) mandelate to phenylglyoxylate. D(--)-2-(Bromoethanoyloxy)-2-phenylethanoic acid ['D(--)-bromoacetylmandelic acid'], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(--)-Mandelate dehydrogenase was inactivated by D(--) bromoacetylmandelate in a psuedo-first-order process. D(--)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D( -)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(--) bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(--)-bromoacetylmandelate reacts. D(--)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(--)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway. PMID- 1731759 TI - Increased levels of glutathione S-transferase pi transcript as a mechanism of resistance to ethacrynic acid. AB - Subpopulations of HT 29 human colon carcinoma cells (HT/M and HT/S) were selected for resistance to the glutathione S-transferase (GST) inhibitor ethacrynic acid (EA). Both clones displayed a 2-fold resistance to the selection agent and required its constant presence for the maintenance of the resistant phenotype. Purification and characterization of GST isoforms showed similar profiles in the wild-type (WT) and EA-resistant clones, with microheterogeneous forms of the pi isoenzyme detected in each case. Metabolism of EA in vitro in the presence of GSH and the isolated GST from each cell line was characterized by a biphasic disappearance of the parent drug; the initial rate at which each of these enzymes metabolized EA was similar. These enzymes also displayed similar Km values for 1 chloro-2,4-dinitrobenzene. However, the amount of GST isolated per total cellular protein was 3.0-fold in HT/M and 1.6-fold in HT/S relative to WT in the continuous presence of EA. Under these conditions GST activity was increased by 2.3-fold in HT/M and 3.2-fold in HT/S as were GSH levels (2.7- and 4.1-fold for HT/M and HT/S respectively). When EA was removed, enzyme activity and GSH concentrations decreased to values similar to those of the WT. Slot-blot and Southern analyses of the DNA gave no evidence of GST-pi-gene amplification or rearrangement. However, RNA analyses by both slot-blot and Northern studies indicate a 2.5-3.5-fold elevation in the GST pi transcript in the EA-resistant population. Results from these studies indicate that: (1) maintenance of the EA resistant phenotype requires constant presence of the agent; (2) the 2-fold resistance to EA can be quantitatively related to a 2-3-fold increase in GST activity and amount which appears to be the result of a 2.5-3.5-fold elevation in GST transcript; (3) EA, a Michael-reaction acceptor, can induce GST at the transcriptional level. PMID- 1731760 TI - Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens. AB - Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3'-hydroxy and 5'-phosphate termini, and degraded circular DNA. PMID- 1731761 TI - Immunosuppressive activity of corticotrophin-releasing factor. Inhibition of interleukin-1 and interleukin-6 production by human mononuclear cells. AB - Corticotrophin-releasing factor (CRF) from the hypothalamus stimulates corticotrophin (ACTH) secretion. Concentrations of CRF in the peripheral circulation are normally low, and increase during pregnancy due to CRF secretion by the placenta [Cunnah, Jessop, Besser & Rees (1987) J. Endocrinol. 113, 123 131], although CRF in maternal blood does not appear to stimulate the hypothalamo pituitary-adrenal (HPA) axis [Potter, Behan, Fischer, Linton, Lowry & Vale (1991) Nature (London) 349, 423-426]. We have examined the possibility that the placental. CRF might contribute to the suppression of the maternal immune system, which is necessary to prevent rejection of the foetus, by studying endotoxin evoked cytokine production by monocytes as a model for activation of the immune response. CRF inhibited endotoxin-evoked cytokine production from human mononuclear cells (MNCs), the fraction of peripheral blood containing monocytes. The effects of CRF were reversed by a specific CRF receptor antagonist, and were additive with glucocorticoid inhibition of cytokine secretion. Anti-interleukin-1 (IL-1) antisera inhibited endotoxin-evoked IL-6 production; however, the CRF effect was not additive, suggesting that CRF inhibition of IL-6 production may be secondary to CRF inhibition of IL-1. These results suggest a role for CRF as an immunosuppressant during pregnancy. PMID- 1731757 TI - Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis. PMID- 1731762 TI - Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system. Strong synergistic effects with protein disulphide-isomerase. AB - It has been shown previously that the thioredoxin system (thioredoxin + thioredoxin reductase + NADPH) may replace dithiothreitol (DTT) as a cofactor for vitamin KO and K reductase in salt-washed detergent-solubilized bovine liver microsomes. Here we demonstrate that the system can be improved further by adding protein disulphide-isomerase (PDI) to the components mentioned above. Moreover, NADPH may be replaced by reduced RNAase as a hydrogen donor. In our in vitro system the various protein cofactors were required at concentrations 2-5 orders of magnitude lower than that of DDT, whereas the maximal reaction rate was about 3-fold higher. PDI stimulated the thioredoxin-driven reaction about 10-fold, with an apparent Km value of 8 microM. These data suggest that in the vitro system the formation of disulphide bonds is somehow linked to the vitamin K-dependent carboxylation of glutamate residues. In vivo, both disulphide formation and vitamin K-dependent carboxylation are post-translational modifications taking place at the luminal side of the endoplasmic reticulum of mammalian secretory cells. The possibility that the reactions are also coupled in vivo is discussed. PMID- 1731763 TI - Chain-length dependency of interactions of medium-chain fatty acids with glucose metabolism in acini isolated from lactating rat mammary glands. A putative feed back to control milk lipid synthesis from glucose. AB - The effects of a series of medium-chain fatty acids (C6-C12) on glucose metabolism in isolated acini from lactating rat mammary glands have been studied. Hexanoate (C6) octanoate (C8) and decanoate (C10), but not laurate (C12), decreased [1-14C]glucose conversion into [14C]lipid and the production of 14CO2 (an index of the pentose phosphate pathway). With hexanoate and octanoate, glucose utilization was decreased, whereas decanoate had a slight stimulatory effect on glucose utilization, but there was a large accumulation of lactate. Addition of dichloroacetate (an inhibitor of pyruvate dehydrogenase kinase) decreased this accumulation of lactate and stimulated the conversion of [1 14C]glucose into [14C]lipid and 14CO2. Insulin had no effect on the rate of glucose utilization in the presence of hexanoate. It stimulated the rate in the presence of octanoate and laurate and increased the conversion of [1-14C]glucose into [14C]lipid in the presence of octanoate, decanoate or laurate. The major fate of 1-14C-labelled medium-chain fatty acids (C6, C8 and C12) was conversion into [14C]lipid. The proportion converted into 14CO2 decreased with increasing chain length, whereas the rate of [14C]lipid formation increased. It is concluded that the interactions between medium-chain fatty acids and glucose metabolism represent a feed-back mechanism to control milk lipid synthesis, and this may be important when milk accumulates in the gland. PMID- 1731764 TI - Monomerization of tetrameric bovine caudate nucleus acetylcholinesterase. Implications for hydrophobic assembly and membrane anchor attachment site. AB - Tetrameric detergent-soluble bovine caudate nucleus acetylcholinesterase (AChE) was reduced and alkylated under conditions in which at least 95% of initial activity is retained. This treatment alone did not result in monomerization of AChE, nor did it create a hydrophilic enzyme. However, in the presence of SDS the enzyme became monomerized. Incubation of AChE with trypsin in the presence of the reversible inhibitor edrophonium rendered the enzyme hydrophilic and led to catalytically active monomers being produced. SDS/PAGE of this preparation in non reducing conditions revealed only a small decrease in the subunit molecular mass. N-Terminal sequencing of the enzyme, before and after trypsin treatment, yielded identical N-termini showing that the enzyme was monomerized subsequent to C terminal tryptic cleavage. From our results, we conclude that the most C-terminal cysteine residue is involved in inter-subunit disulphide bonding as well as in the attachment of AChE to the membrane anchor. Furthermore, the C-terminal region in the primary structure provides an area for hydrophobic contacts between the different subunits and also between the subunits and the membrane anchor. PMID- 1731765 TI - Kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline. AB - The kinetic theory of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] has been applied to a study on the kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline. Upon dilution of the enzyme that had been incubated with 1,10-phenanthroline into the reaction mixture, the activity of the inhibited enzyme gradually increased, indicating dissociation of a reversible enzyme--1,10-phenanthroline complex. The kinetics of the substrate reaction with different concentrations of the substrate chloroacetyl-L-alanine and the inactivator suggest a complexing mechanism for inactivation by, and substrate competition with, 1,10-phenanthroline at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-Zn(2+)-1,10-phenanthroline complex is a relatively rapid reaction, followed by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity. PMID- 1731766 TI - Macrophages can convert citrulline into arginine. AB - Rat peritoneal macrophages were incubated in the presence of 0.05-1.0 mM [14C]citrulline. The synthesis of [14C]arginine from 0.1 mM-[14C]citrulline was about 300 pmol/h per 10(6) cells in macrophages from saline-injected (control) rats. Both arginine synthesis from citrulline and nitrate production (an indicator of NO generation) were increased about 3-fold in the cells from lipopolysaccharide (LPS)-treated animals. The arginine synthesis was very sensitive to extracellular citrulline concentration in the range found in plasma (0.05-0.1 mM). The rate of arginine synthesis from citrulline was inhibited by about 20% by 0.5 mM-L-glutamine in both control and LPS-treated rat cells, but was inhibited by 0.5 mM-L-arginine only in control cells. Our results demonstrate that citrulline, produced by NO synthetase, can be recycled to arginine in macrophages. The citrulline-arginine cycle may contribute to the regulation of intracellular availability of arginine and thus the prolonged production of NO by macrophages. PMID- 1731767 TI - Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin. AB - The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s 1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine proteinase inhibitors and their target enzymes. The model for the proteinase inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C. PMID- 1731769 TI - Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae. AB - A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB. PMID- 1731768 TI - Purification and analysis of proteinase-resistant mutants of recombinant platelet derived growth factor-BB exhibiting improved biological activity. AB - Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen. A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB. Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32. Substitution of this arginine residue, or arginine 28 [a potential KEX2 (lysine-arginine endopeptidase) cleavage site], prevents or reduces cleavage of PDGF-BB respectively. These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF BB. These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson [(1990) Mol. Cell Biol. 10, 5496-5501] suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding. PMID- 1731770 TI - Thyroid hormone concentrative uptake in rat erythrocytes. Involvement of the tryptophan transport system T in countertransport of tri-iodothyronine and aromatic amino acids. AB - The kinetic properties of transport system T, which is specific for uptake of aromatic amino acids, were studied in rat erythrocytes in the presence of leucine in order to block the neutral amino acid transport system L. Since the triiodothyronine (T3) transport system and system T are closely related, the trans effect of T3 and tryptophan on [3H]tryptophan transport and the trans effects of aromatic amino acids on [125I]T3 transport were studied. Equilibrium exchange, zero-trans and infinite-trans studies of [3H]tryptophan transport indicated that system T in rat erythrocytes is a simple carrier with exchanging properties resulting in trans-acceleration of influx and trans-inhibition of efflux when tryptophan was present at the trans side of the membrane. In erythrocytes preloaded with unlabelled tryptophan, countertransport resulted in a 7-fold accumulation of labelled substrate inside the cells. T3 on the trans side of the membrane inhibited both influx and efflux of tryptophan, with Ki values similar to the Km values of the T3 transport system. Extracellular tryptophan trans-inhibited [125I]T3 efflux in a manner similar to [3H]tryptophan efflux. Preloading erythrocytes with tryptophan resulted in trans-acceleration of T3 uptake and a transient 5-fold accumulation of free T3 into erythrocytes. Phenylalanine and tyrosine (but not the D-isomer of tryptophan or non-aromatic amino acids) also produced trans-acceleration for T3 uptake and T3 countertransport. These results are compatible with a kinetic model assuming a common simple carrier of T3 and tryptophan transport and point to a countertransport pathway driving the uphill uptake of T3 by hetero-exchange with intracellular aromatic amino acids. PMID- 1731771 TI - Deficiencies in DNA replication and cell-cycle progression in polyamine-depleted HeLa cells. AB - Synchronized HeLa cells depleted of polyamines by alpha-difluoromethylornithine exhibited substantially decreased DNA synthesis, and proliferation ceased after the release of the cells into S phase. Nuclei from these cells synthesized 70-80% less DNA than did nuclei from control cells. Extraction of isolated nuclei with 0.3 M-KCl decreased DNA synthesis by about 60%, which was recovered almost completely in control cell nuclei by reconstitution with the salt extracts of these nuclei. On the other hand, salt extracts of polyamine-depleted nuclei restored only 50% of DNA synthesis in extracted control nuclei. Salt extracts of control cell nuclei contained twice the DNA polymerase alpha activity of polyamine-depleted nuclear extracts. Extracts of cell lysates of both control and polyamine-depleted HeLa cells exhibited similar DNA polymerase alpha activity, suggesting that uptake of the enzyme or its retention by the nuclei of polyamine depleted cells was decreased. Polyamine-depleted nuclei also showed altered phosphorylation of a 31 kDa protein as compared with control nuclei. Almost normal DNA synthesis, cell proliferation, DNA polymerase alpha activity and nuclear protein phosphorylation were restored in polyamine-depleted cells grown in medium supplemented with 20 microM-spermidine at least 10-12 h before S phase. Cultures in which proliferation was blocked by alpha-difluoromethylornithine did not exhibit synchronous growth after the block was removed. Thus it may be concluded that HeLa cells depleted of polyamines are not inhibited at a single control point in the cell cycle, but are arrested at diverse sites throughout G1 phase. PMID- 1731772 TI - Reconstitution and identification of the major Na(+)-dependent neutral amino acid transport protein from bovine renal brush-border membrane vesicles. AB - Amino acid transport activity from bovine renal brush-border membrane vesicles (BBMV) was reconstituted into phospholipid vesicles composed of phosphatidylcholine/5% stearylamine. Reconstitutable transport activity was enhanced in protein fractions binding to various lectins. When solubilized BBMV were fractionated on peanut lectin, a single protein band of average molecular mass 132 kDa was obtained. When this protein fraction was reconstituted into phospholipid membrane vesicles, amino acid transport activity was obtained with properties similar to those in native BBMV with regard to amino acid specificity, although the cation specificity was different. A monoclonal antibody which reacted with the same protein removed reconstitutable amino acid transport activity from solubilized BBMV. These findings may provide the first identification of a renal amino acid-transporting protein, although confirmation of this identification by other approaches will be required. PMID- 1731773 TI - Isolation and characterization of a 22 kDa protein with antifungal properties from maize seeds. AB - We have purified a 22 kDa protein from maize seeds to homogeneity by ammonium sulfate precipitation, chitin extraction and Mono-S column chromatography. The purified protein inhibited the growth of the agronomically important pathogens of potato wilt (Fusarium oxysporum) and tomato early blight (Alternaria solani). Sequence analysis of the purified protein showed that it has 52% homology with the sweet protein thaumatin (Edens, L., Hselinga, L., Klok, R., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. (1982) Gene 18, 1-12), 57% homology with the pathogenesis-related protein (Cornelissen, B. J. C., Huijsduijnen, R. A. M., and Bol, J. F. (1986) Nature 321, 531-532) and 99% homology with the 22 kDa trypsin/alpha-amylase inhibitor (Richardson, M., Valdes Rodriguez, S., and Blanco-Labra, A. (1987) Nature 327, 432-434). PMID- 1731774 TI - Critical micelle concentration and hemolytic activity--a correlation suggested by the marine sterol, halistanol trisulfate. AB - The marine natural product, halistanol trisulfate, has a relatively low critical micelle concentration of 0.001% m/v (14.5 microM) and strong hemolytic potency with an EC50 of 0.00046% m/v (6.67 microM). As expected of a detergent, it inhibits the growth of gram-positive but not gram-negative bacteria. The hemolytic activity of halistanol trisulfate and other detergents has been shown to correlate with critical micelle concentration. This correlation may have important implications in the mechanism of membranolytic bioactivity. PMID- 1731775 TI - In vitro reconstitution of an erythropoietin gene transcription system using its 5'-flanking sequence and a nuclear extract from anemic kidney. AB - We have developed an in vitro transcription system for the erythropoietin (Epo) gene. This system uses a plasmid carrying 0.2 kb of 5'-flanking sequence from the human Epo gene, rNTPs and a nuclear extract from mouse kidney. The transcribed RNA was assayed by primer extension with an end-labeled primer complementary to the sequence of the plasmid, dNTPs and reverse transcriptase. The primer extension product corresponding to the transcript was detected on a sequencing gel. The in vitro promoter activity of the Epo 5'-flanking sequence was observed with a nuclear extract from anemic kidney but not with that from normal kidney. PMID- 1731776 TI - Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301. AB - A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively. PMID- 1731777 TI - Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity. AB - The recent discovery that point mutations in the beta/A4 amyloid precursor protein may be the cause of certain forms of familial Alzheimer's disease provides strong support for the view that a thorough understanding of the metabolism of this protein may elucidate the pathogenesis of most forms of the disease and thus serve as a basis for rational prevention and therapy. Here we show that overexpression of a portion of the amyloid precursor protein molecule produces at least four distinct fragments of the COOH-terminus of amyloid precursor protein, suggesting altered proteolysis of amyloid precursor protein, and that such overexpression is associated with cytotoxicity. The degree of toxicity in the P19 cell culture model (differentiating mouse embryonal carcinoma cells) is shown to be related to the two larger novel COOH-terminal protein fragments (16 and 14 kilodalton), as well as to levels of expression of these two fragments. The toxicity is manifested in several differentiated cell lineages, including neuronal cells. PMID- 1731778 TI - Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells. AB - Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types. PMID- 1731779 TI - Molecular cloning of the cDNA for the major hemoglobin component from Paramecium caudatum. AB - Nucleotide sequence of the cDNA for the major hemoglobin component of Paramecium caudatum was determined. An oligonucleotide was synthesized on the basis of the amino acid sequence, and the Paramecium cDNA library constructed in phage lambda gt11 was screened with it. Three positive clones, of which insert sizes were 0.4, 0.6, and 0.9 kbp, were obtained. Sequence analysis made clear that the 0.4-kbp cDNA retains a full length of the nucleotides encoding 116 amino acid residues, and that in the coding region it contains four TAA codons which are known to encode glutamine. PMID- 1731780 TI - Cisplatin induced alterations in oriented fibers of DNA studied by atomic force microscopy. AB - Oriented fibers of DNA, prepared by the wet spin method, were imaged by atomic force microscopy. It was found that an oriented fiber's substructure is an array of assemblages of DNA. For native DNA, the assemblages exhibit a characteristic width of 76 nm, a thickness of 20 nm, and appear to carry a right handed twist. Treatment with the anticancer drug cisplatin prior to wet spinning induces geometric irregularities, in the form of kinks and width distortions, into the assemblages of DNA. PMID- 1731781 TI - The phospholipase A2 of human spermatozoa; purification and partial sequence. AB - In view of its proposed key role in the acrosome reaction, phospholipase A2 has been isolated and purified from human spermatozoa. Following SDS-PAGE, a single major band was obtained with an estimated molecular mass of 16.7 kDa. Sequence analysis of the N-terminal portion of the molecule revealed the identity of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. From this partial analysis it is evident that the phospholipase A2 of human spermatozoa represents a new sequence. Of interest is the location of glutamine-4, phenylalanine-5, methionine-8 and isoleucine-9; this sequence appears to be highly conserved throughout evolution. PMID- 1731782 TI - The effects of novel cathepsin E inhibitors on the big endothelin pressor response in conscious rats. AB - The aspartic protease, cathepsin E, has been shown to specifically cleave big endothelin (big ET-1) at the Trp21-Val22 bond to produce endothelin (ET-1) and the corresponding C-terminal fragment. To determine whether cathepsin E is a physiologically relevant endothelin converting enzyme (ECE), three novel and potent inhibitors of cathepsin E were administered to conscious rats prior to a pressor challenge with big ET-1. One of the inhibitors of cathepsin E, SQ 32,056 (3 mg/kg i.v.), blocked the big ET-1 response. However, this dose of SQ 32,056 also blocked the pressor response to ET-1. Phosphoramidon specifically inhibited the Big ET-1 pressor response. These results suggest that ECE is not cathepsin E. PMID- 1731783 TI - Characterization and partial purification of a membrane protein from rabbit skeletal muscle which inhibits the cAMP-dependent protein kinase. AB - Rabbit skeletal muscle membranes contain a protein which inhibits the cAMP dependent protein kinase. The activity of the partially purified membrane protein is characterized by an IC-50 of 10 to 30 nM with respect to the inhibition of the activity of the catalytic subunit of the cAMP-dependent protein kinase and is sensitive to treatment with heat, acid, alkali and trypsin. The active fractions contain proteins ranging from 40 to 120 kDa, analysed by SDS-gel electrophoresis. PMID- 1731784 TI - Therapy with coenzyme Q10 of patients in heart failure who are eligible or ineligible for a transplant. AB - Twenty years of international open and seven double blind trials established the efficacy and safety of coenzyme Q10 (CoQ10) to treat patients in heart failure. In the U.S., ca. 20,000 patients under 65 years are eligible for transplants, but donors are less than 1/10th of those eligible, and there are many more such patients over 65, both eligible and ineligible. We treated eleven exemplary transplant candidates with CoQ10; all improved; three improved from Class IV to Class I; four improved from Classes III-IV to Class II; and two improved from Class III to Class I or II. After CoQ10, some patients required no conventional drugs and had no limitation in lifestyle. The marked improvement is based upon correcting myocardial deficiencies of CoQ10 which improve mitochondrial bioenergetics and cardiac performance. These case histories, and very substantial background proof of efficacy and safety, justify treating with CoQ10 patients in failure awaiting transplantation. PMID- 1731785 TI - Greater importance of Ca(2+)-calmodulin in maintenance of ang II- and K(+) mediated aldosterone secretion: lesser role of protein kinase C. AB - In this study we have investigated various components of the stimulus-secretion coupling process leading to aldosterone secretion from the calf adrenal glomerulosa cells as evoked by angiotensin II (AII) and potassium (K+). The roles of Ca2+, calmodulin and protein kinase C in the sustained phase rather than initiation of aldosterone secretion were of special interest. Our investigations revealed that the reduction of extracellular Ca2+ by EGTA or interruption of Ca2+ influx by nitrendipine at various time points after stimulation with either AII or K+ markedly compromised aldosterone secretion. Calmodulin inhibitors, calmidazolium and W-7 reduced aldosterone secretion profoundly. Agonists of protein kinase C, phorbol ester or diacylglycerol analogues failed to stimulate aldosterone secretion while the protein kinase C inhibitor, H-7, only partially inhibited aldosterone secretion at a concentration which completely inhibited protein kinase C activity. Calmodulin inhibitors produced significantly greater inhibition of aldosterone secretion than inhibitors of protein kinase C. PMID- 1731787 TI - The N-terminal segment of protein AA determines its fibrillogenic property. AB - The amyloid fibril protein AA consists of a varying long N-terminal part of the precursor protein serum AA. By using synthetic peptides corresponding to human and murine protein AA segments and cyanogen bromide fragments of human protein AA, we show evidence that the amyloidogenic part of the molecule is the first 10 15 amino acid long segment. Amino acid substitutions in this part of the molecule may explain why only one of the two mouse SAA isoforms is amyloidogenic. PMID- 1731786 TI - A rationale for the prophylactic use of monophosphoryl lipid A in sepsis and septic shock. AB - Monophosphoryl lipid A (MLA), a substructure of bacterial lipopolysaccharide (LPS), is being developed as a prophylactic for sepsis and septic shock. In the present study it was shown that MLA induced a rapid accumulation of IFN-gamma in mice that correlated with an in vivo priming of macrophages. Primed macrophages could be induced in vitro to synthesize nitric oxide, a key mediator of macrophage cytotoxicity. Due to its rapid clearance, MLA was not present in circulation at the time when IFN-gamma accumulated, suggesting that MLA could not synergize with IFN-gamma to systemically activate macrophages in vivo. MLA treatment tolerized mice against the IFN-gamma response--ie., treatment of mice with MLA on day 1 blocked LPS from inducing IFN-gamma on days 2-4. The significance of these results in relation to MLA's ability to enhance non specific resistance and block LPS lethality in animals is discussed. PMID- 1731788 TI - Comparison between synthetic nuclear localization signal peptides from the steroid/thyroid hormone receptors superfamily. AB - The main objective of the study is to demonstrate that short basic peptides from the steroid/thyroid hormone receptors superfamily act as Nuclear Localization Signals out of receptors context. Such synthesized peptides, chemically coupled to Bovine Serum Albumin, were shown to enable the corresponding BSA-conjugate to be transported to the nucleus. A second objective is to demonstrate the utility of viral cointernalization as a good method for rapid quantitation, comparison and competition in nuclear entry. PMID- 1731789 TI - Structural features of carbohydrate moieties in snake venom glycoproteins. AB - The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds. PMID- 1731790 TI - Nucleoside triphosphate binding and hydrolysis by histone H1. AB - We present here further evidence supporting that histone H1 contains a nucleotide binding site interacting e.g. with ADP, ATP, GDP and GTP. The finding is in accordance with the previous observation that nucleotides modulate recognition of DNA by H1. Most interestingly, H1 appears to be capable of hydrolyzing NTPs and incorporating phosphate to exogenous proteins. The mode of nucleotide action on H1 may be considered highly analogous to that of GTPases. Nuclear receptors may thus act through mechanisms similar to those for receptors on the plasma membrane. PMID- 1731791 TI - Interleukin-1 beta induces nitric oxide production and inhibits the activity of aconitase without decreasing glucose oxidation rates in isolated mouse pancreatic islets. AB - The aim of this investigation was to further characterize the process of interleukin-1 beta (IL-1 beta) induced nitric oxide production in isolated pancreatic islets. It was found that both IL-1 beta and nitroprusside increased islet nitrite production. This effect was paralleled by inhibition of islet aconitase activity and glucose oxidation rates. Neither trifluoroperazinen or aminopterin could prevent the IL-1 beta induced increase in nitrite production, aconitase inhibition and decrease in glucose oxidation rates. In a second series of experiments, isolated mouse pancreatic islets were exposed to IL-1 beta for 24 h and subsequently used for nitrite production, aconitase activity and glucose oxidation determinations. The islets responded to IL-1 beta with an increased nitrite production and a decreased activity of aconitase, whereas the islet glucose oxidation rates were not decreased. It is concluded that IL-1 beta in both rat and mouse islets induces nitric oxide formation and that this induction leads to the inhibition of the Krebs cycle enzyme aconitase. In rat islets this probably leads to an inhibited insulin secretion, whereas IL-1 beta in mouse islets suppresses insulin secretion by a non-mitochondrial mechanism. PMID- 1731792 TI - Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli. AB - The Drosophila glutathione S-transferase D genes encode a family of isozymes. We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone. The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids. This sequence has significant homology to the E. coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits. The two proteins have very similar overall amino acid composition as well. It is possible that SSP may be a glutathione S-transferase(s) in E. coli or is evolutionarily related to glutathione S-transferases. Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E. coli RNA polymerase holoenzyme. PMID- 1731793 TI - Glutathione is the reducing agent for the reductive dehalogenation of tetrachloro p-hydroquinone by extracts from a Flavobacterium sp. AB - Tetrachloro-p-hydroquinone is the first intermediate during pentachlorophenol degradation by Flavobacterium sp. strain ATCC 39723, a strict aerobe. We report here that tetrachlorohydroquinone was reductively dehalogenated to 2,3,6 trichloro-p-hydroquinone and subsequently to 2,6-dichloro-p-hydroquinone under anaerobic conditions by the cell extract from Flavobacterium. The reducing agent was identified to be the reduced form of glutathione. This is the first time glutathione has been identified as the reducing agent for reductive dehalogenation. PMID- 1731794 TI - Phosphorylation of Acinetobacter isocitrate lyase. AB - During growth on succinate, Acinetobacter calcoaceticus contains two forms of the enzyme isocitrate dehydrogenase. Addition of acetate to a lag-phase culture grown on succinate causes a dramatic increase in activity of form II of isocitrate dehydrogenase and in isocitrate lyase. Form II of isocitrate dehydrogenase may be responsible for the partition of isocitrate between the TCA cycle and the glyoxylate by-pass. This report describes the phosphorylation of the enzyme isocitrate lyase from A. calcoaceticus. This phosphorylation may be a regulatory mechanism for the glyoxylate by-pass. PMID- 1731795 TI - A novel compound, depudecin, induces production of transformation to the flat phenotype of NIH3T3 cells transformed by ras-oncogene. AB - A novel compound, depudecin, induced production of the flat phenotype of Ki-ras transformed NIH3T3 cells at the low concentration of 1 microgram/ml. This effect was reversible. Actin stress fiber was detected in these cells after depudecin treatment. Almost complete reversion to the flat phenotype was observed at 6 h after depudecin addition. The synthesis of ras-mRNA did not decrease enough with depudecin treatment at the concentration of 10 micrograms/ml to reverse the transformed morphology. PMID- 1731796 TI - Identification and characterization of a new binding site for angiotensin II in mouse neuroblastoma neuro-2A cells. AB - Specific binding site for 125I-angiotensin II (Ang II), with unique pharmacological properties uncommon to the hitherto recognized receptor subtypes, was observed in mouse neuroblastoma cells (Neuro-2A). Differentiation of the cells with 100 nM PGE1 resulted in a 10-fold increase in the number of Ang II binding sites without changing the binding affinity (Kd value: 12.0 nM). 125I-Ang II binding to membranes of differentiated Neuro-2A was inhibited by unlabeled Ang II with a Ki value of 7.06 +/- 1.09 nM but not by Ang III (1 microM). Both AT1 antagonist, Dup753, and AT2 antagonist, PD123319, failed to inhibit 125I-Ang II binding at 1 microM. 125I-Ang II binding was not affected by GTP analogs such as GTP gamma S and Gpp(NH)p. These results suggest that Neuro-2A cells possess a binding site for Ang II which is different from the presently known subtypes of Ang II receptors, and that the number of the binding site is regulated by cell differentiation. PMID- 1731797 TI - Actin molecules promote neurite outgrowth of chick telencephalic neurons in vitro. AB - Chick brain extract contains protein factors for neurite outgrowth from embryonic chick telencephalic neurons in dissociated cell cultures. In the course of purification of the factors using a bioassay system, a 41 kDa protein was detected to be one of the factors. Immunoblot analysis showed that this protein is identical to actin. In addition, G-actin purified from rabbit skeletal muscle exhibits neurite outgrowth activity in the same dose-dependent manner as the 41 kDa protein. The same bioassay carried with other purified proteins did not show such a remarkable activity. PMID- 1731798 TI - Cloning and characterization of a highly conserved HMG-like protein (PF16) gene from Plasmodium falciparum. AB - A novel gene encoding a protein of 147 amino acids (Pf16) has been cloned from Plasmodium falciparum and expressed in E. coli. The protein contains 19 methionines, all of which are localized in the NH2-terminal 35 amino acid residues, and it is also rich in lysine. Pf16 is highly basic, contains a polyacidic domain consisting of aspartic acid and is related to the non-histone high mobility group proteins of higher eukaryotes. The gene is conserved among eight different species of Plasmodium so far examined, suggesting an important function for this gene product in the parasite's life cycle. PMID- 1731799 TI - Ribulose-1,5-bisphosphate carboxylase of thermophilic hydrogen-oxidizing microorganism Bacillus schlegelii. AB - Ribulose-1,5-bisphosphate carboxylase was isolated from thermophilic hydrogen oxidizing Bacillus schlegelii. Molecular mass of the native enzyme is 560,000 and optimal reaction temperature is 70 degrees C. Km value for ribulose 1,5 bisphosphate is 0.27 mM. The carboxylase activity of the enzyme is dependent on Mg2+ with the optimum at 10 mM. The enzyme is an oligomer of L8S8 type with Mr of large subunits and small subunits of 56,000 and 14,000, respectively. Negatively stained enzyme has regular polygonal shape in top view, 12 nm in diameter, with central electron dense patch. PMID- 1731800 TI - Reversible beta-pleated sheet formation of a phosphorylated synthetic tau peptide. AB - Serine416 of human tau protein is believed to be phosphorylated in Alzheimer neurofibrillary tangles. We synthesized a fragment of tau, consisting of amino acids 408-421 in both non-phosphorylated and serine416-phosphorylated forms. Circular dichroism in a trifluoroethanol-water mixture indicated a beta-turn--- beta-pleated sheet conformational transition upon phosphorylation. The beta structure formation is intermolecular and can be inhibited by addition of Ca2+ ions or a phosphorylated tripeptide, but not with its non-phosphorylated analog. The presence of the phosphorylated tau peptide did not facilitate the formation of beta-pleated sheets of a phosphorylated neurofilament fragment. Multivalent cations induced a conformational transition of this phosphorylated neurofilament peptide, but the effect was less specific than the transition induced in the tau fragment, and it could also be reversed with the competing phosphorylated tripeptide. PMID- 1731801 TI - A heterozygous mutation (the codon for Ser447----a stop codon) in lipoprotein lipase contributes to a defect in lipid interface recognition in a case with type I hyperlipidemia. AB - Previously, we reported a case with type I hyperlipidemia due to a lipid interface recognition deficiency in lipoprotein lipase (LPL) (1). The LPL from postheparin plasma of this patient did not hydrolyze TritonX-100-triolein or very low density lipoprotein-triolein but did hydrolyze tributyrin and LysoPC-triolein substrates. Sequence analysis of the probands DNA revealed a heterozygous nucleotide change: a C----G transversion at position of 1595, resulting in changing the codon for Ser447 to a stop codon. Expression studies of this mutant LPLcDNA in Cos-1 cells produced and secreted considerable amounts of LPL mass in the culture media. The mutated LPL hydrolyzed much less TritonX-100-triolein than wild type LPL, whereas hydrolysis of tributyrin and LysoPC--triolein was the same with both the mutant and wild type LPL. These results suggest that this mutation might be responsible for the property of the LPL with a defect in lipid interface recognition in the type I patient we reported. PMID- 1731802 TI - Matrix attachment sites in the murine alpha-globin gene. AB - DNA sequences with a high affinity for nuclear matrix proteins have been identified and localized in the mouse alpha-globin gene. These matrix association regions (MARs) are adjacent, covering the first intron and part of the 5'-coding sequence. The binding sites are in close proximity to DNase I hypersensitive sites and other important signal sequences. The proteins of the nuclear lamina do not bind the alpha-globin gene MARs in the in vitro binding assay. The finding of MARs in the mouse alpha-globin gene creates an apparent paradox, since works from other authors and our results presented here indicate that this gene is not bound to the nuclear matrix in vivo. This contradiction is difficult to explain at present but different possibilities are accounted for in the text. PMID- 1731803 TI - Antileishmanial activity of hamycin: a polyene antibiotic. AB - Hamycin, a polyene antibiotic, now in extensive use in the treatment of candidiasis and otomycosis, is found to be remarkably effective in killing Leishmania donovani promastigotes in a liquid medium at a concentration of 0.2 microgram/ml. The glucose stimulated respiration and the uptake of 2-deoxy-D[U 14C]-glucose was inhibited in cells treated with the drug at a growth inhibitory concentration. An immediate release of isotopic glucose from preloaded cells could be demonstrated after exposure to hamycin. All the above effects could be effectively prevented in the presence of ergosterol. The primary site of action of hamycin on L. donovani promastigote cells appears to be membrane sterols that result in the loss of the permeability barrier to small metabolites. The lower minimum inhibitory concentration of hamycin compared to other established drugs warrants further study in the context of increasing reports of clinical resistance to pentavalent antimonials. PMID- 1731804 TI - Inactivation of purified phenylalanine hydroxylase by dithiothreitol. AB - Purified rat liver phenylalanine hydroxylase is inactivated in vitro by ascorbate and thiol compounds, dithiothreitol being the most effective inhibitor, with a second order rate constant for the inactivation of 0.066 +/- 0.002 mM-1.min-1 at 20 degrees C and pH 7.2. Anaerobic conditions and catalase protected the enzyme from inactivation by dithiothreitol. This suggests that hydrogen peroxide, produced by oxidation of the thiol, is involved in the inactivation. The substrate, L-phenylalanine, also partially protected the enzyme from this inactivation. It is shown that incubation of the enzyme with dithiothreitol at aerobic conditions, followed by gel filtration, causes the release of iron from the active site. The inactivation by dithiothreitol was reversed by incubation of the iron-depleted enzyme with Fe(II). PMID- 1731805 TI - Genetic analysis of a Japanese family with normotriglyceridemic abetalipoproteinemia indicates a lack of linkage to the apolipoprotein B gene. AB - Normotriglyceridemic abetalipoproteinemia is a rare familial disorder characterized by an isolated deficiency of apoB-100. We have previously reported a patient with this disease, who had normal apoB-48 but no apoB-100. To elucidate the genetic abnormalities in this family, we studied the linkage of apoB gene using three genetic markers. The proband and her affected brother showed completely different apoB gene alleles, suggesting that the apoB gene itself is not related to this disorder in this family. By contrast, an American case had a point substitution in the apoB gene generating an in-frame stop codon. These results indicate that this disorder can be caused by defect(s) of either an apoB gene or other genes. PMID- 1731806 TI - AIMS2. The content and properties of a revised and expanded Arthritis Impact Measurement Scales Health Status Questionnaire. AB - OBJECTIVE: The goal of this project was to develop a more comprehensive and sensitive version of the Arthritis Impact Measurement Scales (AIMS). METHODS: AIMS scale items were revised, and 3 new scales were added to evaluate arm function, work, and social support. Sections were also added to assess satisfaction with function, attribution of problems to arthritis, and self designation of priority areas for improvement. The new instrument was designated the AIMS2. A pilot test of format and content and a performance test of reliability and validity were carried out. RESULTS: Questionnaire completion times in a pilot study of 24 subjects averaged 23 minutes, and evaluations were positive regarding the instrument's length and ease of completion, and the subjects' willingness to complete serial forms and return them by mail. Measurement performance was tested in 408 subjects: 299 with rheumatoid arthritis (RA) and 109 with osteoarthritis (OA); 45 of these subjects completed a second AIMS2 within 3 weeks. Internal consistency coefficients for the 12 scales were 0.72-0.91 in the RA group and 0.74-0.96 in the OA group. Test-retest reliability was 0.78-0.94. All within-scale factor analyses produced single factors, except for mobility level in OA. Validity analyses in both the RA and the OA groups showed that patient designation of an area as a problem or as a priority for improvement was significantly associated with a poorer AIMS2 scale score in that area. Reliability, factor analysis, and validity results were consistent in age, sex, and education subgroups. Satisfaction was moderately correlated with level of function in the same health status area, and the satisfaction items formed a reliable scale. Responses to the arthritis attribution items showed that most dysfunction in this sample was due to arthritis. CONCLUSION: The AIMS2 is a revised and expanded health status questionnaire with excellent measurement properties that should be useful in arthritis clinical trials and in outcomes research. PMID- 1731807 TI - Generation and characterization of two monoclonal self-associating IgG rheumatoid factors from a rheumatoid synovium. AB - OBJECTIVE: To study IgG rheumatoid factor (RF) from rheumatoid synovium. METHODS: We fused the K6H6/B5 human-mouse heterohybridoma with unstimulated rheumatoid synovial B cells to generate IgG-RF-secreting hybridomas. RESULTS: The RFs from 2 such hybridomas bound specifically to the Fc fragment of human IgG and self associated to form immune complexes. Such immune complexes are a major characteristic of the pathogenic IgG-RFs in rheumatoid synovium. CONCLUSION: IgG RF-secreting hybridomas have been obtained. Analyses may reveal the underlying mechanisms of the induction of IgG-RF. PMID- 1731808 TI - VH4-21, a human VH gene segment overrepresented in the autoimmune repertoire. PMID- 1731809 TI - Radiologic Vignette. Acute osteomyelitis and septic arthritis of the great toe IP joint. PMID- 1731810 TI - Major histocompatibility complex associations with primary antiphospholipid syndrome. PMID- 1731811 TI - Potential interpretational errors in the use of the polymerase chain reaction to detect Yersinia in clinical specimens. PMID- 1731812 TI - Variable-constant segment genotype of immunoglobulin kappa is associated with increased risk for rheumatoid arthritis. AB - OBJECTIVE: To further investigate the association of rheumatoid arthritis (RA) with a particular genotype identified by a restriction site polymorphism near the constant segment of immunoglobulin kappa (C kappa). METHODS: The frequencies of genomic DNA polymorphisms detected within or near C kappa (the most C kappa proximal variable segment [V kappa] B3 and a T lymphocyte marker [CD8A]) were determined by Southern blotting and hybridization. The frequencies of coding region polymorphisms of C kappa (Km allotypes) were determined by amplification by polymerase chain reaction followed by restriction enzyme digestion. RESULTS: Although the frequencies of B3, Km, and CD8A genotypes were not different between RA and normal control populations, more individuals were homozygous for both C kappa and B3 in the RA group (relative risk 2.2, P less than 0.01), especially in the DR4-negative RA subgroup (relative risk 3.9, P less than 0.001). CONCLUSION: The homozygous genotype of an approximately 30,000-base region including the C kappa segment confers an elevated risk for RA, particularly in the DR4-negative subgroup. PMID- 1731813 TI - Biannual radiographic assessments of hands and feet in a three-year prospective followup of patients with early rheumatoid arthritis. AB - In a prospective followup study of 147 patients with rheumatoid arthritis of recent onset, we assessed the progression of radiographic evidence of joint damage on films of the patients' hands and feet obtained biannually. Patients were receiving first-line and second-line treatment. Ninety patients were followed up for 3 years, and 57 were followed up for only 2 years. Radiographic damage was determined by a modification of the method described by Sharp, and to ensure comparability of findings, we determined the percentage of damage per joint group (actual score divided by the maximum possible score). After 3 years, radiographic damage was present in 70% of the patients, all of whom could be identified after 1 year of study. Overall, 18-20% of the joints of the hands and feet were affected after 3 years, with relatively little abnormality per joint (approximately 8% of maximum possible score). During the entire followup, more foot joints than hand joints were affected. The rate of progression in the first year was significantly higher than in the second and third years of study, indicating a flattening of the curve of radiographic progression of joint damage. PMID- 1731814 TI - Activation of synovial fluid T lymphocytes by 60-kd heat-shock proteins in patients with inflammatory synovitis. AB - OBJECTIVE: Synovial fluid lymphocytes from patients with rheumatoid arthritis and with other forms of inflammatory synovitis demonstrate enhanced proliferative responses to Mycobacterium tuberculosis antigens, in particular, the 65-kd heat shock protein. There is a high degree of homology between the human and the mycobacterial 60-kd family of heat-shock proteins. These studies were performed to determine if the enhanced response to the mycobacterial 65-kd heat-shock protein was due to cross-reactivity of an immune response generated against the human homolog. METHODS: These studies were performed by in vitro culture of isolated synovial fluid mononuclear cells with crude and purified antigens. RESULTS: The synovial fluid lymphocytes of a majority of patients with rheumatoid arthritis recognized the mycobacterial 65-kd heat-shock protein, as evidenced by T cell proliferation. In contrast, only 18% of all samples tested responded to a highly purified recombinant human 60-kd heat-shock protein. With only one exception, proliferative responses to the mycobacterial antigen were stronger than those to the human homolog. The proliferative responses generated against mycobacterial 65-kd heat-shock proteins from different sources were highly correlated. CONCLUSION: The findings suggest that the enhanced proliferative response to the mycobacterial 65-kd heat-shock protein noted in most patients with rheumatoid arthritis and other forms of inflammatory synovitis is not due to cross-reactivity of an immune response directed against the human heat-shock protein. PMID- 1731815 TI - Activation of the alternative complement pathway accompanies disease flares in systemic lupus erythematosus during pregnancy. AB - OBJECTIVE: To assess the activity of systemic lupus erythematosus (SLE) during pregnancy and to distinguish it from preeclampsia. METHODS: We prospectively measured the complement activation products Ba, Bb, SC5b-9, and C4d, as well as the conventional complement determinants C3, C4, and CH50, during pregnancy in 14 patients with SLE and 10 women with preeclampsia. RESULTS: Four of the 14 SLE patients were considered to have disease flares, 3 occurring in the second trimester and 1 postpartum. In these patients, significant abnormalities of Ba, Bb, SC5b-9, and CH50 were noted. In contrast, measures of C4d did not distinguish between pregnant patients who had flares and those whose SLE remained stable. Although decreased values of C3 were rarely seen in the patients with stable disease, normal values of C3 during lupus pregnancy were not reliably associated with stable disease. Three of 10 non-SLE patients with preeclampsia had elevated levels of Ba; however, in each case, the CH50 level was close to or within the normal range. This was in sharp contrast to the findings observed in the 4 patients with active SLE, in whom high levels of plasma Ba were always associated with low CH50 values. Moreover, the ratio of CH50 to Ba was significantly lower in the patients with lupus flares than in the non-SLE patients with preeclampsia. CONCLUSION: While a decline in the CH50 level alone could otherwise be attributed to decreased synthesis of complement components, these data demonstrate that ongoing activation of the alternative complement pathway can accompany disease flares in pregnant women with SLE. PMID- 1731816 TI - Interleukin-1, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor alpha, and interferon-gamma levels in sera from patients with scleroderma. AB - OBJECTIVE: To determine whether interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL 2, IL-4, interferon-gamma (IFN gamma), IL-6, and tumor necrosis factor alpha (TNF alpha) are detected more frequently in sera from scleroderma patients than in sera from controls. METHODS: Serum concentrations of these cytokines were measured in 78 scleroderma patients and 73 controls, using enzyme-linked immunosorbent assay, radioimmunoassay, and bioassay techniques. RESULTS: IL-2, IL 4, and IL-6 were each detected more frequently in sera from scleroderma patients than in sera from controls. TNF alpha and IL-1 alpha were found with equal frequency in patient and control sera. IL-1 beta and IFN gamma were not detected in any sera. CONCLUSION: IL-2, IL-4, and IL-6 may be among the cytokines that contribute to the disease process in scleroderma patients. To our knowledge, this is the first report of elevated serum IL-4 levels in human disease. PMID- 1731817 TI - Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis. AB - To determine the clinical significance of serum antibodies to the U3 small nuclear ribonucleoprotein particle ([U3]snRNP), we studied sera from 416 patients with systemic sclerosis (SSc) and 264 controls, using immunofluorescence and immunoprecipitation assays. The presence of serum anti-(U3)snRNP was highly specific to SSc, was found more frequently in blacks, and was associated with skeletal muscle disease and primary pulmonary arterial hypertension. These antibodies may identify one or more unusual clinical subsets of SSc. PMID- 1731818 TI - Ethical and psychosocial considerations of wound management. AB - Ethical and psychosocial considerations in caring for patients with pressure ulcers are explored in relation to the consequential and the nonconsequential schools of ethical thought. The principles of ethical decision making are related to wound management in terms of beneficence, nonmaleficence, justice, veracity and confidentiality, autonomy and liberty. Allocation of scarce resources and the conduct of research influence ethical decision making in wound management. PMID- 1731819 TI - Nurse validation of pressure ulcer risk factors for a nursing diagnosis. AB - The nursing diagnosis "Potential Impaired Skin Integrity: Pressure Ulcer" provides a model for determining pressure ulcer risk. This article describes assessment parameters and risk factors that may be useful in the assessment of pressure ulcer risk. Results of a diagnostic content validity study of this diagnosis conducted by the author (Sparks, 1990) are presented. PMID- 1731820 TI - A retrospective study of the use of specialty beds in the medical and surgical intensive care units of a tertiary care facility. AB - A retrospective chart audit of 55 patients placed on specialty beds in the medical and surgical intensive care units of a tertiary care hospital during 1989 was performed to establish criteria for placement on specialty beds. A modified Knoll Assessment of Pressure Ulcer Potential tool was used to determine which risk factors were common among the patients. Mean total risk scores was 21.15 with a standard deviation of 4.74. Significant correlations were found between seven of the eight risk factors and the total risk score. A high total score on the modified Knoll tool indicates a need for placement on specialty beds. PMID- 1731821 TI - Adoption of research-based practice for treatment of pressure ulcers in long-term care. AB - Implementation of a clinical trial to evaluate the effectiveness of electrotherapy on pressure ulcer healing provided the stimulus for adoption of research-based innovations for pressure ulcer treatment in one long-term care facility. A five-year retrospective study conducted prior to introduction of the clinical trial revealed that 72 different treatments were applied to pressure ulcers. Forty-two percent of the pressure ulcers were left open to the air or covered with a dry gauze dressing and 64% were treated with some type of antiseptic solution. Since implementation of the clinical trial and the accompanying access to wound healing research knowledge it provided in this setting, the prevailing treatment for pressure ulcers has become moist physiologic dressings. PMID- 1731822 TI - What causes pressure ulcers to heal? PMID- 1731823 TI - [The significance of boiled-egg fibers in biopsied muscles of neuromuscular disorders]. AB - It is believed that one muscle fiber consists of one fiber type determined by its innervating neuron. In biopsied muscles of Duchenne muscular dystrophy (DMD), however, the author has incidentally found a double-typed fiber which is divided into inner and outer parts. The author termed it a "boiled-egg fiber". The author has examined the appearance rate of the boiled-egg fibers on 682 biopsied muscles obtained from patients with various neuromuscular disorders, and classified the types of the inner and outer parts of the boiled-egg fibers by ATPase staining. Boiled-egg fibers were recognized in 17 cases out of 60 with DMD, 5 out of 146 with other types of muscular dystrophy and 6 out of 94 with myositis. No boiled egg fiber was found in the remaining 382 cases with other disorders which did not represent necrosis with regeneration of muscle fibers. The total number of boiled egg fibers was 235 with 192 in DMD and 43 in other disorders. 197 of 235 (83.8%) had the same type for both inner and outer parts and remaining 38 (16.2%) had different types for their inner parts. In 133 of 235 (56.6%), the inner parts were type 2C fibers. Boiled-egg fibers were segmentally found with the length of several hundred micrometers. The above findings suggest that boiled-egg fibers reflect an abnormal regenerating process. It remains to be clarified whether or not inner and outer parts of boiled-egg fibers are double-innervated respectively. PMID- 1731824 TI - [Ergometric and pathologic study of a family with complex I deficiency]. AB - We studied a family with a myopathic form of complex I deficiency with regard to the clinical symptoms, usefulness of the exercise tolerance test with an ergometer for screening of mitochondrial abnormalities, pathological findings in biopsied muscles and genetics. In this family, none of the members had disorders of the central nervous system, such as convulsions, mental deterioration or stroke-like episodes. In the two affected generations, three mothers and three children had mitochondrial abnormalities. Two children were diagnosed as having complex I deficiency. One of them, an 8-year-old girl with normal psychomotor development during infancy, began to experience easy fatigability at about 3 years of age. At the age of 5 years, she experienced respiratory distress and became unconscious. Thereafter, she had similar episodic respiratory problems with lactic acidosis. Ragged-red fibers and respiratory chain enzyme defects were detected in the biopsied muscle. Another child, a 15-year-old boy with easy fatigability but no muscle weakness, had normal respiratory chain enzyme activities and a normal oxysogram: oxygen consumption showed a normal responses when malate and pyruvate were added as substrates for the isolated mitochondria. His muscle pathology revealed rare ragged-red fibers and abnormal subsarcolemmal mitochondrial aggregation. An investigation with an ergometer showed elevated serum lactate and pyruvate levels. Only one mother had muscle weakness and hyper lactic acidemia. The other two mothers had no muscle symptoms, but abnormal results were obtained with the ergometer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731825 TI - [Survey of Fukuyama type congenital muscular dystrophy in Tokyo]. AB - On the 1st of April in 1988, we identified 26 children with Fukuyama type congenital muscular dystrophy (FCMD) among 1,227,000 children in Tokyo whose ages ranged from 6 to 14 years. The prevalence rate of FCMD was 2.1 per 100,000. All affected children attended special school for crippled children. Mean absence from school on account of illness was 33.9 days in a year. Fifteen among 26 affected children had ability of verbal communication. The loss of gross motor function started at ages 6-7 years but muscle weakness might have occurred earlier. Ten affected children were treated with antiepileptic drugs. Six affected children needed to be hospitalized for the treatment of vomiting with dehydration, acute bronchitis, or pertussis infection during one year until the 1st of April in 1989. Two cases among 26 with FCMD died of respiratory complications shortly after admission. PMID- 1731826 TI - [A longitudinal study of children with language delay at 3 years of age; later WPPSI and school attendance]. AB - Ninety-four children (eighty-three boys and eleven girls), who were delayed in verbal expression (expressive language less than two-thirds of the standard for their chronological ages) at the age of three years, were evaluated for school attendance and intelligence at the age of six. The Yamada's Check List for Language Development and Wechsler Preschool and Primary Scale of Intelligence (WPPSI) were used to assess language problems and the intelligence quotient (IQ). Thirty children (32%) were delayed in verbal expression only, and sixty-four (68%) were delayed in both: verbal expression and comprehension were less than two-thirds of the standard for their chronological ages. Of children with delay in verbal expression only, 36% of them had low full-scale IQs (less than 70), and 27% had required special tutoring. Of children with delayed development of verbal expression and comprehension, 85% had low full-scale IQs, and 89% had required special tutoring or had attended schools for mentally retarded children. In expressive and comprehensive language delay, forty-one children avoided personal relations at the age of three years. Later thirty of these children were diagnosed as suffering from infantile autism. Poor mental outcome could be predicted by the delay of expressive and comprehensive language at the age of three years. The high prevalence of developmental disorders at later stages for the children in this study suggests the need for close monitoring of children with delayed language. PMID- 1731827 TI - [Mu rhythm: clinical assessment of the atypical type]. AB - Of 5,218 patients who received EEG examination at our laboratory during a 9-month period in 1989, 241 showed the 7-13 Hz arch-shaped activity originating from over the Rolandic area known as mu rhythm. These subjects were divided into two groups as follows: Group 1, 171 subjects showing typical mu rhythm, i.e., recorded during wakefulness and not affected by visual stimulation but blocked voluntary movements or tactile stimuli; and, Group 2, 70 subjects showing atypical mu rhythm, i.e., accentuated or activated by drowsiness, photic stimuli, or hyperventilation. No difference between the two groups was found with regard to frequency, amplitude or origin of the mu rhythm. Age distribution for Group 1 showed a peak between the ages of 6 and 15 (67.5%), while that for Group 2 peaked between the ages of 11 and 15 (35.7%) considering high incidence in older age range. There was no significant difference between the two groups in regard to gender. Although both groups showed a high incidence of epilepsy, Group 2 showed higher incidence of intractable epilepsy (p less than 0.05), as well as of severe intracranial trauma and of organic brain disease. On EEG recorded among epileptic patients, paroxysmal discharge was more frequent in Group 2 (p less than 0.01), although no other difference between the two groups was observed. Atypical mu rhythm may indicate more severe epilepsy, and careful observation of patients with atypical mu rhythm is recommended. PMID- 1731828 TI - [Deep white matter hyperintensity in the occipital lobe on T2-weighted MRI in children. II. Classification based on the signal intensity]. AB - Twenty-seven children, who had deep white matter hyperintensity in the occipital lobe (DWMH) on T2-weighted MRI, were classified into two groups, mild and severe, based on the signal intensity. The frequency of mild DWMH, which was iso-or hyperintense relative to the gray matter but hypointense relative to cerebrospinal fluid (CSF), decreased with aging; mild DWMH might result from a delayed myelination in the central nervous system. However, the frequency of severe DWMH, which was iso-or hyperintense relative to CSF, was not related to aging and was significantly high in severely retarded children. Therefore, severe DWMH might be a new indicator of mental retardation in children. PMID- 1731829 TI - [A case of subacute sclerosing panencephalitis developing 8 years after immunosuppressive treatment for acute lymphocytic leukemia]. AB - A 13-year-old girl developed subacute sclerosing panencephalitis (SSPE) with atypical absence attacks as an initial symptom. Eight years earlier she had been treated for acute lymphocytic leukemia with cytotoxic treatment and radiotherapy, which had resulted in complete remission. She was first treated with an anticonvulsant because the atypical absence attacks and the presence of epileptic discharges on an EEG suggested epilepsy. However, with this mode of treatment the epileptic discharges did not disappear, but periodic high-voltage slow-wave complex discharges were revealed on subsequent EEGs. The antibody titer for measles virus in the cerebrospinal fluid and serum was elevated, confirming the diagnosis of SSPE. SSPE may arise, though rarely, in an individual in an immunosuppressive state due to congenital immunodeficiency or various kinds of malignancies, and also may arise several years after the contraction of measles infection. Our patient, however, lacked a past history of measles infection or immunization, suggesting the possibility that she had contracted measles during or shortly after the course of treatment for ALL. PMID- 1731830 TI - [Wyburn-Mason syndrome--a case report]. AB - A 5-year-old girl with Wyburn-Mason syndrome was reported. A vascular nevus on the right cheek was noticed since early infancy. External strabismus and impaired vision of the right eye were noticed at 5 years of age. Fluorescein angiography showed an arteriovenous malformation on the right retina. Brain CT, MR, and right carotid angiography demonstrated an arteriovenous malformation from the orbita to the hypothalamic region along the optic nerve. This congenital vascular disorder is extremely rare. PMID- 1731831 TI - [Two cases of hemorrhagic shock and encephalopathy syndrome]. AB - A 10-month-old male infant (case 1) and another male infant aged 1 year and 11 months (case 2) were admitted to our department because of fever, watery diarrhea and convulsion. On admission, they were unconscious and showed rigidity of the limbs. Laboratory examination revealed a marked increase in GOT and GPT, a decrease in platelet and antithrombin III and an increase in FDP. Metabolic acidosis was found by blood gas analysis. Brain CT showed an extensive area of low density in case 1, and low density centering on the cerebral basal ganglia and brainstem in case 2. Rotavirus was detected in case 2 by fecal examination. The clinical pictures in these cases closely resembled those of hemorrhagic shock and encephalopathy (HSE) reported by Levin et al. in 1983. The etiology of this disease is currently unknown, and its prognosis is poor. The relationship between this disease and rotavirus should be examined in future studies. PMID- 1731832 TI - [Secondary carnitine deficiency due to antibiotics therapy using pivaloxyl methyl cephem]. PMID- 1731833 TI - [Multicore disease with pulmonary and heart failure caused by acute pneumonia--a case report]. PMID- 1731834 TI - [Four familial cases including monozygotic twin of Friedreich's ataxia with various clinical course]. PMID- 1731835 TI - Epicardial fat causing pitfalls in CT and MR imaging of the pericardium. AB - To study the complex anatomy of the pericardium and the pericardial recesses, notably the transverse sinus and the recess behind and under the common pulmonary artery, cryomicrotomy sections of 4 frozen cadaver specimens were correlated with CT and MR imaging in multiple planes. In addition, CT chest studies of 254 patients and MR chest studies from 78 patients were reviewed. Epicardial fat interposed between the transverse sinus of the pericardium and the ascending aorta was a normal finding confirmed by cryomicrotomy studies and seen by CT in 23 of 245 patients and in MR imaging in 3 of 78 patients. Epicardial fat indenting the pericardial sac below the common pulmonary artery caused an inhomogeneous signal, mimicking lymphadenopathy on coronal T1 weighted MR images in 4 patients. PMID- 1731836 TI - Decreased perfusion in myocardial region of normal donor artery secondary to collateral development. Stress 201Tl myocardial emission CT performed in patients with single vessel exertional angina having collaterals. AB - Thirty-one patients suffering from single vessel exertional angina with collaterals (Group A) were evaluated by stress 201T1 myocardial emission CT (Tl SPECT) with 16 controls of severely stenotic single vessel exertional angina without collaterals (Group B). Group A included 21 patients (68%) who showed an extensive perfusion defect in double artery myocardial regions, including the normal donor artery myocardial region (DMR). However, there were no such cases in Group B, giving a significant difference between these 2 groups (p less than 0.001). Four patients in Group A, having a perfusion defect both in DMR and in the collateral dependent myocardial region (CMR) underwent a successful percutaneous transluminal coronary angioplasty (PTCA) with disappearance of collaterals. Tl-SPECT findings after PTCA showed no perfusion defect either in CMR or in DMR. This has been explained on the basis that the coronary collaterals stole blood and produced perfusion defect in DMR. PMID- 1731837 TI - "Chess-board pattern" spatial modulation of magnetization. Assessment of myocardial function. AB - Heart motion is a complex combination of translation, rotation, and concentric contraction. Evaluation of these complex motions has been difficult using conventional slice-selective methods. Noninvasive tagging of the heart has been obtained by the use of slice-selective radiofrequency pulses. Through spatial modulation of the magnetization the entire image can be labeled in different patterns. Two new pulse sequences are presented, giving a chess-board like spatial modulation. These pulse sequences have several advantages compared with the previously published methods, as the modulation time is half that required to obtain a 2-dimensional grid, the area in the image with high signal intensity was significantly larger, and the radiofrequency power deposition was substantially decreased. By labeling the heart at diastole the chess-board pattern tagging of the heart wall could be followed through systole. Using this method the complex motions of the heart can be mapped. PMID- 1731838 TI - Phlebography as the gold standard in thromboprophylactic studies? A multicenter interobserver variation study. AB - In 241 patients with total hip arthroplasty and entering a study on thrombosis prophylaxis, phlebography was adequately performed in 451 legs 7 to 11 days after surgery. The phlebograms were primary evaluated by 4 independent observers, and finally a consensus of the images in which disagreement primarily occurred was obtained. The diagnosis of thrombosis in the 4 primary observations varied between 65% and 83% (mean 70%) and the agreement on a negative diagnosis between 97% and 99% (mean 98%). Taking into account agreement by chance, kappa-values varied from 0.60 to 0.83 when the 6 different pairs of observations were compared. When comparing the primary evaluations with the final consensus, agreements on positive diagnosis varied between 70% and 90% (mean 80%) and on negative diagnosis between 97% and 99% (mean 98%). Kappa-values varied from 0.68 to 0.90. The factor of uncertainty in evaluation of phlebography may have to be considered when studies on postsurgical thromboprophylaxis are planned. PMID- 1731839 TI - CT of carcinoma of the renal pelvis. AB - CT in 28 histologically proven carcinomas of the renal pelvis (pTa-2, n = 12; pT3 4, n = 16) in 26 patients was evaluated retrospectively. Twenty-four of 28 tumors could be identified at CT, 17/28 at urography, and 12/14 at retrograde pyelography. Nineteen tumors appeared as a discrete intrapelvic mass with an attenuation close to that of the kidney on noncontrast scans. There was slight to moderate enhancement of the tumors following i.v. contrast medium injection but they appeared hypodense relative to the renal parenchyma. Five tumors caused only a diffuse obliteration of the renal sinus. Criteria to define peripelvic tumor growth are proposed, i.e. tumors obliterating fat planes or abutting of renal parenchyma should not be regarded as signs of extrapelvic extension, while inhomogeneous attenuation of peripelvic fat and renal parenchyma (in the absence of other explanation) should, or if the tumor mass is seen interdigitizing with surrounding structures. Thickening of Gerota's fascia or septa in the perirenal space are unspecific findings. With CT we were able to differentiate tumors confined to the renal pelvic wall from those with more advanced disease including metastases in 22 of 26 patients. PMID- 1731840 TI - CT analysis of metastatic neoplasms of the kidney. Comparison with primary renal cell carcinoma. AB - The CT findings in 32 patients with pathologically proven metastases to the kidney were compared to findings in 74 patients with renal cell carcinoma. Fourteen CT criteria were chosen to describe and characterize the lesions and 2 radiologists evaluated the CT images retrospectively according to these criteria. Renal metastases were characterized as small, multiple, bilateral, wedge-shaped, less exophytic, and located within the renal capsule. Renal cell carcinomas were single, unilateral, nonwedge-shaped, and exophytic, and easily transgressed the renal capsule. The sensitivity of CT to discriminate renal cell carcinoma from renal metastasis was 93.2% for renal cell carcinoma, and to discriminate renal metastasis from renal cell carcinoma was 75.0% for renal metastases by computer posterior probabilities. This study indicates that CT is useful for distinguishing these clinically important tumors. By using posterior probability, some unnecessary biopsies may be avoided. PMID- 1731841 TI - CT and angiography in adrenocortical carcinoma. AB - CT and angiography were performed in 15 patients with adrenocortical carcinoma. The tumors had a mean diameter of 11 cm (range 4-20 cm). At CT, the 8 largest tumors were ill-defined, and in these, the organ of tumor origin could not be established. Angiographically the correct organ of tumor origin was established in all but one patient. It is concluded that CT is excellent in showing the extent of an adrenal tumor, but is often unable to predict the organ of origin in large tumors. Angiography is still of great value in the preoperative work-up in patients with large adrenocortical carcinomas for correct identification of tumor origin and for vascular mapping. PMID- 1731842 TI - Lithotripsy of urinary bladder stones with a mechanical lithotriptor inserted through a narrow introducer. An experimental study in pigs. AB - Mechanical lithotripsy of urinary bladder stones was performed with the RotoLith lithotriptor in 8 pigs with implanted human stones or artificial stones after open cystotomy. The effect of the treatment on the urinary bladder was investigated macro- and microscopically immediately after the lithotripsy in 4 animals and 4 weeks after the lithotripsy in the other 4 animals. The stones were fragmented into very small pieces which would have been possible to pass with the urine. The histopathologic examination immediately after the procedure showed slight mucosal edema, patches of erosions in the mucosa, and minor submucosal bleeding. Microscopic examination of the bladders removed 4 weeks after lithotripsy showed slight chronic inflammation. The experimental results have encouraged us to plan to use this lithotripsy procedure in selected patients with bladder stones. PMID- 1731843 TI - Ultrasonography, CT, and ERCP in the diagnosis of choledochal stones. AB - A prospective study of jaundiced (n = 187) and nonjaundiced (n = 33) cholestatic patients was carried out to evaluate the sensitivity of ultrasonography (US), CT, and endoscopic retrograde cholangiopancreatography (ERCP) in the detection of choledochal stone disease. Altogether 83 patients had the final diagnosis of choledocholithiasis. In the jaundiced patients, the sensitivity of US, CT, and ERCP was 22.5%, 23.2%, and 80.6%, respectively. In cases of cholestasis without jaundice, the values were 20%, 37.5%, and 66.7%. In patients in whom all 3 imaging studies were done (n = 64), the differences between US and ERCP and between CT and ERCP were statistically significant (p less than 0.0001). In most false-negative ERCP studies (10/15), the clinical course of the disease strongly suggested a passed choledochal stone. On the basis of this study, we recommend prompt ERCP to be performed if choledochal stone disease is suspected on clinical grounds. PMID- 1731844 TI - Emergent embolization for control of massive hemorrhage from a splanchnic artery with a new coaxial catheter system. AB - Emergent superselective embolization with a 3.0 F (1 mm) coaxial catheter and a steerable guidewire was performed in 27 patients with massive hemorrhage from a small-caliber splanchnic artery. Eight patients had intraperitoneal hemorrhage, 3 had hemobilia, 9 had gastric hemorrhage, and 7 had intestinal hemorrhage. Out of 27 patients, 7 had hemorrhage from a splanchnic artery pseudoaneurysm. Complete cessation of bleeding was obtained in all patients initially, but in 3 patients gastric hemorrhage recurred later. Otherwise, there was no rebleeding nor any major complication such as marked infarction of tissue or misplacement of embolic materials. This coaxial catheter system was highly reliable for achieving superselective catheterization in small-caliber arteries, minimizing the volume of infarcted tissue and allowing maximal preservation of splanchnic organic function. We conclude that this system represents a major advance in interventional radiology. PMID- 1731845 TI - Computer-aided interpretation of coronary cineangiograms. Accuracy of automatic detection of stenotic lesions. AB - To accurately diagnose stenotic lesions on coronary cineangiograms, an automatic detection method using computer image processing was developed. We evaluated its accuracy by comparing the results of computer-aided interpretation (CAI) with those obtained independently by 3 observers. Evaluation was performed on 129 segments from 27 arteries visualized on angiograms obtained in 18 patients. The detection rates of stenosis of the 3 observers by pure visual interpretation were 7.0%, 27.9%, and 17.1%, and using CAI 40.0%, 42.6%, and 47.3%. By computer recognition alone, a detection rate of 51.9% was achieved. The agreement by at least 2 observers (consensus) on the sites with lesions was 41.1% while the consensus of computer recognition regarding the sites with lesion was 40.3%. Therefore, our findings indicated that computer recognition of cineangiograms is likely to result in overdetection of lesions. However, all 3 observers detected stenotic lesions better with CAI than with pure visual interpretation. Accordingly, CAI may improve the reliability of cineangiographic diagnosis. PMID- 1731846 TI - Relation between lightscanning and the histologic and mammographic appearance of malignant breast tumors. AB - The relation between real-time transillumination (lightscanning) and the histologic appearance of 243 breast carcinomas was evaluated. Lightscanning mainly failed in identifying ductal and lobular carcinomas in situ. The result of lightscanning was also poor regarding small, invasive carcinomas. The absorption patterns in elastosis and scar tissue associated with carcinoma played no important role in the ability of lightscanning to identify a cancer. The relation between the lightscanning and mammographic appearance of 85 breast cancers from the same material was also evaluated. Lightscanning performed poorly in identifying tumors characterized by classifications as compared to tumors with other mammographic appearances. However, the difference was not significant. PMID- 1731847 TI - Diagnostic accuracy of lightscanning and mammography in women with dense breasts. AB - The diagnostic accuracy of lightscanning and mammography in 610 breasts with mammographically dense parenchymal patterns was investigated. Lightscanning identified 31 out of 36 cancers and mammography 32. Lightscanning and mammography were in agreement in 28 cases of cancer. One noninvasive lobular carcinoma was not identified by either modality. Four cancers were not correctly identified with lightscanning alone and 3 cancers with mammography alone. Of the 574 breasts without cancer, lightscanning falsely denoted 101 (18%) as possibly being cancerous (false-positives). The corresponding figure for mammography was 25 (4%). Thus, lightscanning, as performed in this study, has the same sensitivity as mammography in detecting cancer in mammographically dense breasts. However, its usefulness is limited by a low predictive value of a positive test (high rate of false-positives). PMID- 1731848 TI - Diagnostic imaging in fracture of lumbar vertebral ring apophyses. AB - The findings at plain radiography, myelography, CT, and MR imaging in 3 cases of fracture of the lumbar vertebral ring apophysis are presented. Familiarity with this entity is important in evaluating low back pain in children and young adults. Conventional radiographs and/or MR imaging may suggest ring apophysis fracture; CT will confirm and classify the diagnosis. PMID- 1731849 TI - Rhabdomyosarcoma of the petrous ridge. CT and MR imaging in an atypical case with multiple cranial nerve palsy. AB - The CT and MR findings are reported in a case of biopsy proven rhabdomyosarcoma of the skull base. The tumor presumably originated in a pneumatized petrous ridge and had an atypical presentation of multiple cranial nerve palsy. The lesion exhibited a soft tissue density and a nonexpansile bone destruction on unenhanced CT. On MR imaging the lesion showed homogeneous intermediate signal intensity on T1 weighted images and a high signal intensity on proton density and T2 weighted images. The scanty literature on CT and MR features of rhabdomyosarcoma of the head and neck is reviewed. PMID- 1731850 TI - Interobserver variability in the radiographic diagnosis of adult outpatient pneumonia. AB - Acute chest radiographs were obtained from 319 adult patients with acute respiratory infections. Where a lower respiratory infection was diagnosed, follow up chest radiographs were obtained in most patients. A radiologic panel diagnosed pneumonia in 21 patients. The agreements between the panel and 3 independent interpreters, 2 residents in radiology, and one senior chest physician, were assessed. Also the reports given by the specialist in radiology at the Department of Radiology were compared with the panel's evaluation. While the kappa agreements between the panel's interpretations and those by the Department of Radiology and the consultant in chest medicine was 0.71 and 0.72, respectively, the corresponding kappa-values between the residents and the panel was only 0.50. The proportion of agreement when pneumonia was diagnosed was 0.56 between the panel and the Department of Radiology, and 0.59 between the panel and the chest consultant, compared to 0.36 between the panel and the residents. The study demonstrates the difficulty of diagnosing outpatient pneumonia and the importance of experience. PMID- 1731851 TI - Agenesis of the right lobe of the liver with supra-hepatic gallbladder. PMID- 1731852 TI - Prediction of mechanical properties of human atherosclerotic tissue by high frequency intravascular ultrasound imaging. An in vitro study. AB - Intravascular ultrasound may be useful for studying the natural history of atherosclerotic lesions of different morphologies and for guiding interventional strategies. This study was designed to test the hypothesis that tissue appearance by intravascular ultrasound is related to the biomechanical properties of atheroma components. Forty-three atheroma caps were obtained from the abdominal aortas of 22 patients at autopsy and studied with an ultrasensitive, servo controlled spectrometer. By measuring the static strain caused by increasing levels of compressive stress from 30 to 90 mm Hg, the uniaxial unconfined compression stiffness (ratio of stress to strain) was determined. After mechanical testing, specimens were imaged with a 6F, 20-MHz intravascular ultrasound transducer, and images were interpreted by an investigator who was unaware of the mechanical measurements. Specimens were classified as nonfibrous (n = 14), fibrous (n = 18), or calcified (n = 11) based on intravascular ultrasound appearance. The static stiffnesses of the nonfibrous, fibrous, and calcified ultrasound classes were 41.2 +/- 18.8 kPa, 81.7 +/- 33.2 kPa, and 354.5 +/- 245.4 kPa, respectively (p = 0.0002 by analysis of variance). The times to reach static equilibrium (creep time) for the nonfibrous, fibrous, and calcified classes were 79.6 +/- 26.5 minutes, 50.2 +/- 20.0 minutes, and 19.4 +/- 8.1 minutes, respectively (p = 0.0007). Intravascular ultrasound appearance was most significantly related to biomechanical behavior when calcium deposits were noted; the differences in biomechanical behavior between nonfibrous and fibrous tissue appearances were less apparent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731853 TI - Cholesterol and triglyceride fatty acid synthesis in apolipoprotein E2-associated hyperlipidemia. AB - To investigate whether increased endogenous lipogenesis contributes to elevated plasma lipid levels in individuals with apolipoprotein (apo) E2-associated hyperlipidemia (E2-HL), plasma pool cholesterol and triglyceride fatty acid syntheses were measured in subjects with E2-HL and in those with normal lipid levels. Subjects were given a priming dose of deuterium oxide (D2O) followed by maintenance doses over 48 hours. During the first 24 hours, subjects consumed prepared meals, whereas during the 24-48 hour interval, they consumed water only. Blood samples were drawn every 12 hours, and cholesterol and triglyceride fatty acid formation rates were determined from the change in deuterium enrichment. The free cholesterol fractional synthesis rate over 0-24 hours of E2-HL subjects (0.057 +/- 0.010 day-1, mean +/- SEM) was not significantly different from that of normolipidemics (0.075 +/- 0.005 day-1). Calculated cholesterol net synthesis was not different between the two groups (0.56 +/- 0.07 and 0.75 +/- 0.05 g/day, respectively). Mean free cholesterol synthesis for all subjects was higher in the fed (0-24 hour) compared with the fasted (24-48-hour) condition. Initial 12-hour triglyceride fatty acid fractional synthesis was significantly (p less than 0.01) increased in E2-HL subjects (0.143 +/- 0.012 day-1) compared with controls (0.082 +/- 0.0013 day-1). These findings suggest that in E2-HL, elevated plasma cholesterol levels are due to factors other than increased sterol synthesis, while higher de novo fatty acid synthesis contributes to the observed hypertriglyceridemia. PMID- 1731854 TI - Frontiers in cardiovascular science. Quantitative measurements of atherosclerotic manifestations in humans. PMID- 1731855 TI - A definition of the intima of human arteries and of its atherosclerosis-prone regions. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. PMID- 1731856 TI - Role of apolipoprotein E on cholesteryl ester-enriched low density lipoprotein particles in coronary artery atherosclerosis of hypercholesterolemic nonhuman primates. AB - Significant differences among individuals occur in the lipoprotein response to atherogenic diets in cynomolgus and African green monkeys. The range of concentrations of total plasma cholesterol (TPC) was 100-600 mg/dl and of apolipoprotein (apo) E (quantified by enzyme-linked immunosorbent assay) was 3-20 mg/dl in the animal groups of this study. The correlation between the concentrations of TPC and of apo E was r = 0.89 in these animals. To determine which lipoprotein classes contained the majority of apo E, agarose gel-filtration chromatography was used to subfractionate whole plasma. In hypercholesterolemic monkeys, the majority of the apo E and apo B-100 coeluted within the region of low density lipoprotein (LDL). In normocholesterolemic monkeys, the majority of apo E coeluted with apo A-I and high density lipoproteins. A strong positive correlation was seen between the concentrations of plasma apo E and LDL cholesterol (r = 0.9), but there was no significant correlation between high density lipoprotein apo E and either TPC or plasma apo E concentrations. A positive correlation of r = 0.8 was found between the LDL apo E to apo B-100 molar ratio and the average LDL particle size, suggesting an increase in the number of apo E molecules on the larger LDL particles. Within individual animals, LDL were heterogeneous and the LDL subfractions of lower density (1.02 less than d less than 1.03 g/ml) had the highest proportion of apo E, although apo E was present on LDL of all densities. A strong positive correlation between plasma apo E concentration and coronary artery atherosclerosis was identified, and in stepwise regression analysis, plasma apo B concentration and the apo E to apo B molar ratio of LDL together accounted for more than 90% of the variation in the atherosclerosis end point of coronary artery intimal area. These data strongly suggest that the enrichment of LDL with cholesteryl esters and apo E, which occurs in hypercholesterolemic primates, is an atherogenic feature of the plasma lipoproteins. PMID- 1731857 TI - Recognition sites on rat liver cells for oxidatively modified beta-very low density lipoproteins. AB - The in vivo fate of beta-very low density lipoproteins (beta-VLDLs) was investigated after Cu(2+)-mediated oxidative modification (Ox-beta-VLDL). Ox-beta VLDL may be physiologically relevant under conditions of defective VLDL removal by the liver (type III hyperlipoproteinemia) or overloading of the remnant receptor (high cholesterol feeding). On oxidation of beta-VLDL, the kinetics of its removal from the blood and uptake by the liver are unchanged. However, in contrast to beta-VLDL, which is recognized by the remnant receptor of parenchymal cells, liver uptake of Ox-beta-VLDL is mediated mainly by Kupffer cells (65% of liver-associated radioactivity). In vitro competition studies show that the cell association and degradation of iodine-125-labeled Ox-beta-VLDL by both liver endothelial and Kupffer cells are only marginally competed for by acetylated LDL (10-20%), while an efficient blockade is noted with Ox-beta-VLDL, oxidized low density lipoproteins, or polyinosinic acid (80-90%). The capacity of Kupffer cells to associate with and degrade 125I-Ox-beta-VLDL appears to be twofold higher than for endothelial cells. It is concluded that on oxidation of beta VLDL, the recognition system responsible for the uptake of beta-VLDL from the blood circulation is shifted from the remnant receptor to a specific oxidized lipoprotein receptor. The efficiency of the scavenger activity on Kupffer cells will then form the protection system against the prolonged circulation of these atherogenic lipoproteins in the blood. PMID- 1731858 TI - Effects of a monounsaturated rapeseed oil and a polyunsaturated sunflower oil diet on lipoprotein levels in humans. AB - The effects of high oleic acid rapeseed oil compared with polyunsaturated fats on serum lipoprotein levels are largely unknown. Therefore, we fed 30 women and 29 men a baseline diet rich in saturated fat, which was followed by a diet rich in high oleic and low erucic acid rapeseed oil (total energy content of fat, 38%; saturates, 12.4%; monounsaturates, 16%; n-6 polyunsaturates, 6%; and n-3 polyunsaturates, 2%) and one rich in sunflower oil (total energy content of fat, 38%; saturates, 12.7%; monounsaturates, 10%; n-6 polyunsaturates, 13%; and n-3 polyunsaturates, 0%). The oils were incorporated into mixed natural diets that were dispensed in a random order for 3.5 weeks each in a blinded crossover design. The diet composition was confirmed by analysis of duplicate diets. Both test diets reduced serum total cholesterol (TC) and low density lipoprotein (LDL) cholesterol levels from baseline, the monounsaturated rapeseed oil diet more than the polyunsaturated sunflower oil diet (TC: -15% versus -12%, p less than 0.01; LDL cholesterol: -23% versus -17%, p less than 0.01). Very low density lipoprotein (VLDL) cholesterol and total, VLDL, and LDL triglyceride levels were lower during the sunflower oil diet compared with the rapeseed oil diet. Total high density lipoprotein (HDL) cholesterol levels remained unchanged by both diets. The consumption of rapeseed oil resulted in a more favorable HDL2 to LDL cholesterol ratio (0.43 +/- 0.19 versus 0.39 +/- 0.18, p less than 0.01) and an apolipoprotein A-I to B ratio (3.0 +/- 1.4 versus 2.4 +/- 1.6, p less than 0.001) than did the sunflower oil.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731859 TI - Influx in vivo of low density, intermediate density, and very low density lipoproteins into aortic intimas of genetically hyperlipidemic rabbits. Roles of plasma concentrations, extent of aortic lesion, and lipoprotein particle size as determinants. AB - To compare the atherogenic potential of low density lipoprotein (LDL), intermediate density lipoprotein (IDL), and very low density lipoprotein (VLDL) under conditions where plasma levels of these lipoproteins are elevated, the influx of cholesterol in these lipoproteins into the aortic intima was measured in vivo in genetically hyperlipidemic rabbits from the St. Thomas's Hospital strain, an animal model that shares many of the features of the human disorder familial combined hyperlipidemia. Univariate linear regression showed that the arterial influx of LDL cholesterol (n = 25), IDL cholesterol (n = 14), and VLDL cholesterol (n = 10) was positively and linearly associated with plasma concentrations of LDL cholesterol in the range 0.2-6.4 mmol/l, of IDL cholesterol in the range 0.1-7.0 mmol/l, and of VLDL cholesterol in the range 0.7-8.5 mmol/l, respectively, and also with the extent of lesions in the arterial intima in the range 0-100% of the surface area. Multiple linear regression suggested that the arterial influx of LDL, IDL, and VLDL cholesterol was linearly dependent on plasma concentration, independent of lesion size. Furthermore, it appeared that the arterial influx of the three lipoproteins was linearly dependent on the extent of the lesions, independent of lipoprotein concentration. When influx was normalized for plasma concentration (intimal clearance) and for lesion size (compared within the same aorta), the intimal clearance of the larger IDL and VLDL particles was 15-35% less than that of the smaller LDL particles. These findings suggest that the quantitatively most important mechanism for transfer of plasma lipoproteins into the arterial intima involves nonspecific molecular sieving and that at elevated plasma levels, IDL and VLDL share with LDL the potential for causing atherosclerosis. PMID- 1731860 TI - Arterial wall thickness in familial hypercholesterolemia. Ultrasound measurement of intima-media thickness in the common carotid artery. AB - B-mode ultrasound was used to noninvasively determine wall thickness and lumen diameter in the common carotid artery in patients with familial hypercholesterolemia (n = 53) and in a control group (n = 53). The controls were matched for sex, age, height, and weight, and all had a serum cholesterol level below 6.5 mmol/l. The study was performed to evaluate whether the patients had a thicker arterial wall compared with that of the control group. Wall thickness was determined as the combined intima-media thickness of the far wall and is presented as the mean and maximum thickness of a 10-mm-long section of the common carotid artery. The difference between the groups was 0.13 mm in mean wall thickness (p less than 0.001; 95% confidence interval, 0.07-0.18 mm) and 0.20 mm in maximum wall thickness (p less than 0.001; 95% confidence interval, 0.09-0.23 mm). Fifty of the subjects were examined twice to estimate the interobserver variability. The coefficients of variation for mean and maximum wall thickness were 10.2% and 8.9%, respectively. The two study groups were well matched and differed only in lipid levels. Thus, there is reason to believe that the difference in wall thickness can be explained by the background of familial hypercholesterolemia and the increased cholesterol levels. PMID- 1731861 TI - Monocytes may amplify their recruitment into inflammatory lesions by inducing monocyte chemotactic protein. AB - By Northern analysis, freshly isolated monocytes contained no detectable mRNA for monocyte chemotactic protein-1 (MCP-1). However, after 4 hours of incubation at 37 degrees C, MCP-1 mRNA was clearly induced in the monocytes and was found to be highly dependent and directly proportional to the monocyte density. The level of MCP-1 mRNA continued to increase, reaching a peak after 22 hours of incubation. After 3 days in culture, MCP-1 mRNA levels had declined substantially and after 8 days were undetectable in the monocytes/macrophages. The amount of MCP-1 protein secreted correlated with the density-dependent increase in MCP-1 message. We hypothesize that the migration of monocytes into inflammatory lesions may be amplified by the density and time-dependent induction of MCP-1. PMID- 1731862 TI - Demonstration of a keratan sulfate-containing proteoglycan in atherosclerotic aorta. AB - Proteoglycans were isolated from either grossly normal or atherosclerotic pigeon aortas after extraction with 4 M guanidine hydrochloride and purification by ion exchange and size-exclusion chromatography. The small-size proteoglycans (Kav 0.4, on Sepharose CL-4B) from both normal and atherosclerotic tissue contained primarily a dermatan sulfate proteoglycan with an intact molecular size of 220 330 kd and a 45-kd core protein. In addition to the dermatan sulfate proteoglycan, the preparation contained a proteoglycan recognized by monoclonal antibody (MAb) 5-D-4, indicating the presence of sulfated poly-N acetyllactosamine sequences common to corneal and cartilage keratan sulfate. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed a polydisperse proteoglycan of 60-150 kd that was recognized by MAb 5-D-4. Significantly greater immunoreactivity with MAb 5-D-4 was observed for atherosclerotic compared with normal artery. After endo-beta-D-galactosidase treatment of the proteoglycan from atherosclerotic aorta, diminished MAb 5-D-4 reactivity observed by both Western blot analysis and enzyme-linked immunosorbent assay demonstrated that the material was keratan sulfate. Endo-beta-D galactosidase treatment of the intact proteoglycan generated core proteins of 28 and 38 kd. These studies suggest the presence of one or more keratan sulfate proteoglycans in grossly normal and atherosclerotic arteries. Immunochemical data suggest that sulfation of the keratan sulfate proteoglycan may be greater in atherosclerotic aorta. PMID- 1731863 TI - Glutathione-related enzyme activities and lipoperoxide levels in human internal mammary artery and ascending aorta. Relations with serum lipids. AB - The relation among glutathione-related enzyme activities, thiobarbituric acid reactive substances of the human aorta and internal mammary artery, and serum lipids was studied in 40 male patients undergoing coronary revascularization. Glutathione peroxidase and glutathione reductase activities were significantly higher in the internal mammary artery, whereas glutathione transferase activity was elevated in the aortic wall. Moreover, non-selenium-dependent glutathione peroxidase activity was detectable only in the aorta. The levels of thiobarbituric acid-reactive substances were significantly higher in the aorta. A positive correlation was found among the activity of glutathione peroxidase, glutathione reductase, and thiobarbituric acid-reactive substances in the internal mammary artery and total cholesterol, low density lipoprotein cholesterol, and triglycerides. In the aortic wall, a positive correlation among the activity of glutathione peroxidase, glutathione transferase, thiobarbituric acid-reactive substances, and the previously mentioned serum lipids was evident. In contrast, high density lipoprotein cholesterol was inversely related to enzymatic activities and thiobarbituric acid-reactive substances in both the internal mammary artery and aorta. In conclusion, significant differences in the levels of glutathione-related enzyme activities and thiobarbituric acid-reactive substances in the internal mammary artery and aorta were found, suggesting a different ability of the two tissues to counteract oxidative stress: the glutathione-related antioxidant properties and the level of lipid peroxidation in the arterial tissue seem to be specifically influenced by serum lipids. PMID- 1731864 TI - Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization. AB - We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) [Boiteux, S., O'Connor, T. R., Lederer, F., Gouyette, A., & Laval, J. (1990) J. Biol. Chem. 265, 3916-3922]. DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB). The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples. Thirteen products resulting from pyrimidines and purines were detected in gamma irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5 formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA. Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8 OH-Gua. The excision of a small but detectable amount of 8-hydroxyadenine was also observed. The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme. Nine pyrimidine-derived lesions were not excised. The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA. The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731865 TI - Interpretation of the effect of an oscillating electric field on membrane enzymes. AB - Theoretical expressions for the frequency and amplitude dependence of the rate of a catalyzed reaction are fitted to the data of Graziana et al. (1990) [Graziana, A., Ranjeva, R., & Teissie, J. (1990) Biochemistry 29, 8313-8318] for Ca2+ uptake by carrot protoplasts in an oscillating electric field. This uptake is a direct (linear) measure of the rate of increase of ATP caused by a plasma membrane enzyme in the oscillating field. The fit gives 20 ms and 33 microseconds for the relaxation times of the enzyme and roughly 3 for the effective number of elementary changes displaced across the membrane by a conformational change of the enzyme in its catalytic cycle. Additional experiments are suggested to define further the mechanism of the enzymatic reaction. PMID- 1731866 TI - Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties. AB - The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5 fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer. PMID- 1731867 TI - Serine hydroxymethyltransferase: origin of substrate specificity. AB - All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate. For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue. The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties. The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme. The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme. The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme. The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme. The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm. Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process. This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731868 TI - Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence. AB - A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group. PMID- 1731869 TI - Effects of high pressure on the catalytic and regulatory properties of UDP glucuronosyltransferase in intact microsomes. AB - The effects of high pressure on the kinetic properties of microsomal UDP glucuronosyltransferase (assayed with 1-naphthol as aglycon) were studied in the range of 0.001-2.2 kbar to clarify further the basis for regulating this enzyme in untreated microsomes. Activity changed in a discontinuous manner as a function of pressure. Activation occurred at pressure as low as 0.1 kbar, reaching one of two maxima at 0.2 kbar. As pressure was increased above 0.2 kbar, activity decreased, reaching a minimum at about 1.4 kbar followed by a second activation. The pathway for activation at pressure greater than 1.4 kbar was complex. The immediate effect of 2.2 kbar was nearly complete inhibition of activity. The inhibited state relaxed, however, over about 10 min (at 10 degrees C), to a state that was activated as compared with enzyme at 0.001 kbar or enzyme at pressures between 1.4 and 2.2 kbar, which was the highest pressure we could test. Examination of the detailed kinetic properties of UDP-glucuronosyltransferase indicated that the effects of pressure were due to selective stabilization of unique functional states of the enzyme at 0.2 and 2.2 kbar. Activation at 0.2 kbar was reversible when pressure was released. This was true as well as for activation at pressure greater than 1.4 kbar, but after prolonged treatment at 2.2 kbar, UDP-glucuronosyltransferase became activated irreversibly on release of pressure. The process by which prolonged treatment at 2.2 kbar led to permanent activation of UDP-glucuronosyltransferase after release of pressure was not reflected, however, by time-dependent changes in the functional state of UDP glucuronosyltransferase at this pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731870 TI - Regulation of biological functions by an insulin receptor monoclonal antibody in insulin receptor beta-subunit mutants. AB - We investigated the effects of MA-5, a human-specific monoclonal antibody to the insulin receptor alpha-subunit, on transmembrane signaling in cell lines transfected with and expressing both normal human insulin receptors and receptors mutated in their beta-subunit tyrosine kinase domains. In cell lines expressing normal human insulin receptors, MA-5 stimulated three biological functions: aminoisobutyric acid (AIB) uptake, thymidine incorporation, and S6 kinase activation. Under conditions where these biological functions were stimulated, there was no detectable stimulation of receptor tyrosine kinase. We then combined the use of this monoclonal antibody with cells expressing insulin receptors with mutations in the beta-subunit tyrosine kinase domain; two of ATP binding site mutants V1008 (Gly----Val) and M1030 (Lys----Met) and one triple-tyrosine autophosphorylation site mutant F3 (Tyr----Phe at 1158, 1162, and 1163). In cells expressing V1008 receptors, none of the three biological functions of insulin was stimulated. In cells expressing M1030 receptors, AIB uptake was stimulated to a small, but significant, extent whereas the other two functions were not. In cells expressing F3 receptors, AIB uptake and S6 kinase activation, but not thymidine incorporation, were fully stimulated. The data suggest, therefore, that (1) activation of insulin receptor tyrosine kinase may not be a prerequisite for signaling of all the actions of insulin and (2) there may be multiple signal transduction pathways to account for the biological actions of insulin. PMID- 1731871 TI - Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution. AB - Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand enzyme complex in solution. PMID- 1731872 TI - 1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin. AB - A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2) half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model. PMID- 1731873 TI - Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints. AB - The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226 5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF. PMID- 1731874 TI - The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase. AB - In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration. PMID- 1731875 TI - Modulation of thrombin-fibrinogen interaction by specific ion effects. AB - Steady-state measurements of synthetic substrate hydrolysis by human alpha thrombin in the presence of human fibrinogen, under experimental conditions where light scattering due to the formation of fibrin aggregates is negligible, have allowed for a quantitative evaluation of Km for fibrinogen. Measurements of Km for fibrinogen carried out at pH 7.5 and 37 degrees C as a function of NaCl, NaBr, KCl, and KBr concentration, from 50 to 500 mM, show that the derivative d ln Km/d ln a +/-, where a +/- is the mean ion activity, is constant over the entire range of salt concentrations and is strictly dependent on the particular salt present in solution. The values of d ln Km/d ln a +/- are found to be equal to 0.75 +/- 0.03 (NaCl), 0.90 +/- 0.01 (NaBr), 0.62 +/- 0.07 (KCl), and 0.60 +/- 0.03 (KBr). Measurements of Km for two synthetic amide substrates, under identical solution conditions, reveal practically no change in Km with salt concentration, while they show a significant decrease in kcat when Na+ salts are replaced by K+ salts. The drastic difference in the salt dependence of Km between fibrinogen and the synthetic amide substrate points out that a significant role may be played by the fibrinogen recognition site in the energetics of thrombin fibrinogen interaction. The sensitivity of Km for fibrinogen to different salts unequivocally demonstrates that specific ion effects, rather than nonspecific ionic strength effects, modulate thrombin-fibrinogen interaction under experimental conditions of physiological relevance. Analysis of ion effects on clotting curves obtained at pH 7.5 and 37 degrees C also shows a drastic differential effect of cations and anions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731876 TI - Secondary structural analysis of two recombinant murine proteins, interleukins 1 alpha and 1 beta: is infrared spectroscopy sufficient to assign structure? AB - The secondary structure for two murine recombinant proteins, interleukins 1 alpha and 1 beta (rmIL-1 alpha and -1 beta), has been analyzed by Fourier transform infrared (IR) spectroscopy and then compared to results obtained by X-ray diffraction, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. The IR results obtained here for rmIL-1 alpha and -1 beta suggested that their secondary structures consisted predominantly of beta-sheets or strands. However, the analysis also revealed a significant absorption band near 1656 cm-1, which is typically assigned to alpha-helical or random structures. When these same murine polypeptides were analyzed by CD, no evidence of alpha helical structures was observed. Further, published X-ray diffraction and NMR studies characterizing the human forms of IL-1 alpha and -1 beta indicate the absence of alpha-helices and that the human proteins are composed mainly of beta strands (i.e., greater than 55%), with approximately 24% of the amino acids involved in large loops connecting the strands. The murine IL-1 proteins, when compared to their respective human counterparts, each show greater than 80% sequence homology. Given this fact, the CD analyses, and the result that this IR band amounted to 21% of the overall integrated area, the absorption peak at 1656 cm-1 was attributed to the presence of large loops rather than to alpha-helical or random structures. Such a structural assignment appears reasonable and is totally consistent with the established existence of large loops in the human forms as well as in other proteins found to fold similarly (viz., human bFGF).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731877 TI - Lipid polymorphism: a correction. The structure of the cubic phase of extinction symbol Fd-- consists of two types of disjointed reverse micelles embedded in a three-dimensional hydrocarbon matrix. AB - The X-ray scattering study of a cubic phase of extinction symbol Fd--, recently performed on a lipid extract (PFL) from Pseudomonas fluorescens [Mariani et al. (1990) Biochemistry 29, 6799-6810] has been extended to several other systems, all consisting of mixtures of water-miscible (MO, PC, PE, oleate) and of water immiscible (FA, DG) lipids, plus water. In all of these systems the cubic phase was observed in the presence of excess water. Some inconsistencies observed between PFL and the other systems, the fact that in PFL one of the reflections of the cubic phase happened to coincide with the strongest reflection of the hexagonal phase, and the finding, in one of the original cubic samples of PFL kept in the cold for more than 3 years, that the intensity of one of the reflections had decreased dramatically all indicated that a nonnegligible amount of a hexagonal impurity was in fact present in the samples of PFL originally thought to contain a pure cubic phase. The intensities were corrected for that impurity and analyzed again using a pattern recognition approach based upon the axiom that the histogram of the electron density maps is invariant with respect to physical structure, when different phases are compared whose chemical composition is the same. The hexagonal phase provided the reference phase for the comparison. The moments mean value of (delta rho)n were used to compare the histograms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731878 TI - Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast. AB - The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6 branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins. PMID- 1731879 TI - Characterization of the interaction of wheat germ protein synthesis initiation factor eIF-3 with mRNA oligonucleotide and cap analogues. AB - Direct fluorescence titration experiments of wheat germ protein synthesis initiation factor eIF-3 with mRNA cap and oligoribonucleotide analogues were performed in order to determine the equilibrium association constants (Keq) for the eIF-3.mRNA interaction as a function of pH and temperature. These data suggest that (i) the eIF-3.mRNA interaction is not cap-specific (i.e., m7G specific), (ii) ATP hydrolysis is not involved in the interaction, and (iii) the interaction is primarily ionic in nature. Competition experiments between a rabbit alpha-globin mRNA oligoribonucleotide analogue and either mRNA cap analogues or nucleoside triphosphates (NTPs) are also reported; these experiments indicate that NTPs act as both activators and competitive inhibitors of the mRNA.eIF-3 association. The results are consistent with a partially uncompetitive binding mechanism, whereby at low NTP concentrations (less than or equal to 10 microM) the bound NTP enhances subsequent mRNA binding to eIF-3, perhaps by inducing a conformational change, and at higher NTP concentrations, the NTP acts as a competitive inhibitor for the mRNA binding site on eIF-3. PMID- 1731880 TI - Tyrosyl radicals and their role in hydroperoxide-dependent activation and inactivation of prostaglandin endoperoxide synthase. PMID- 1731881 TI - Covalent modification of G-actin by pyridoxal 5'-phosphate: polymerization properties and interaction with DNase I and myosin subfragment 1. AB - Pyridoxal 5'-phosphate (PLP), a lysine-specific reagent, has been used to modify G-actin. At pH 7.5, PLP reacted with 1.7-2 lysines on G-actin. Limited proteolytic digestion experiments indicated that, in agreement with previous works, essentially lysine-61 was modified in a 1:1 fashion by PLP, other lysines being much less reactive. A PLP-derivatized affinity label of ATP binding sites, AMPPLP, reacted with two additional lysines that do not appear to be located in the ATP site on G-actin. PLP-G-actin did not polymerize spontaneously up to 30 microM; however, it retained other essential native properties of G-actin. PLP actin bound to the barbed ends of actin filaments with an equilibrium dissociation constant of 4 microM and prevented dilution-induced depolymerization like a capping protein. PLP-actin copolymerized with unmodified actin. The stability of F-actin copolymers decreased with the fraction of PLP-actin incorporated, consistent with a model within which the actin-PLP-actin interactions in the copolymer are 50-fold weaker, and PLP-actin-PLP-actin interactions are 200-fold weaker than regular actin-actin interactions. PLP-actin bound DNase I with an equilibrium association constant of 2 nM-1, i.e., 10-fold lower than that of unmodified actin. PLP modification did not affect the binding of G-actin to myosin subfragment 1. However, polymerization of PLP-actin by myosin subfragment 1 was not observed in low ionic strength buffers, whereas PLP F-actin-S1 filaments, in which the stoichiometry PLP-actin:S1 is 1:1, were formed with an apparent critical concentration of 4.5 microM in the presence of 0.1 M KCl. PMID- 1731882 TI - Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization. AB - Bovine brain microtubule protein, containing both tubulin and microtubule associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP ribosylation, and incubation of assembled steady-state microtubules with ADP ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly. PMID- 1731883 TI - Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase. Preparation and properties of a cysteine-free enzyme. AB - The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A). Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S). A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed. The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation. The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry. The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT. The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea. Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C. Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential. The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines. We consider the evolutionary implications of these findings. PMID- 1731884 TI - The rate of formation of transition-state analogues in the active site of adenosine deaminase is encounter-controlled: implications for the mechanism. AB - We have previously shown that purine riboside, when bound to adenosine deaminase, forms a complex in which C-6 of the purine is tetrahedral [Kurz, L. C., & Frieden, C. (1987) Biochemistry 26, 8450]. We now report the rates of formation of enzyme-inhibitor complexes of two types, those which do and those which do not form such tetrahedral intermediates. In both cases, the rates are encounter controlled since the progress curves for formation of the complexes are well described by a simple second-order approach to equilibrium and the rate constants show an inverse solvent viscosity dependence. Assuming that the formation of the intermediate-analogue complex is preceded by an initial ground-state analogue complex, the lifetime of that ground-state complex must be less than approximately 20 microseconds. All of the enzyme-inhibitor complexes studied share three characteristics: (1) the complexes generate large UV-difference spectra; (2) a substantial solvent isotope effect is found on the enzyme's affinity for the inhibitors; and (3) a new signal appears in the CD spectra of the complexes. Two of the nucleosides studied, 1-deazapurine riboside and 1-deaza adenosine, form complexes which appear to mimic a ground-state rather than a reactive intermediate when bound to adenosine deaminase. We find that the values for the association rate constants for those inhibitors which form intermediate analogues are very similar to that for adenosine. The presence of a significant solvent isotope effect on the affinity of all inhibitors is attributable in part to a large transfer isotope effect on the free ligand and in part to an effect on the bound ligand. This complicates use of the solvent isotope effect in applications of the multiple isotope method for estimating intrinsic isotope effects and commitment factors. PMID- 1731885 TI - Novel DNA superstructures formed by telomere-like oligomers. AB - DNA oligomers containing three or more contiguous guanines form tetrastranded parallel complexes, G4-DNA, in the presence of alkali cations. However, oligomers that have a single multi-guanine motif at their 3' or 5' end, with a guanine as the terminal base, also form higher order products. Thus, the oligomer T8G3T forms a unique G4-DNA product at neutral pH in the presence of Na+, K+, or Rb+; however, its isomeric counterpart T9G3 in K+ or Rb+ generates an additional ladder of products of substantially lower gel mobility. We show that these larger complexes contain, respectively, 8, 12, or 16 distinct strands of oligomer. The octamer structure formed by T9G3 assembles in moderate salt at room temperature and melts around 60 degrees C in 100 mM KCl. Methylation protection experiments suggest a nested head-to-tail superstructure containing two tetraplexes bonded front-to-back via G quartets formed by out-of-register guanines. Naturally occurring chromosomal telomeres, which all have guanines at their 3' termini, may be able to form these superstructures. PMID- 1731886 TI - Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system. AB - A site-specific triple-helical DNA complex has previously been shown to inhibit DNA binding by eukaryotic transcription factor Sp1. To examine the functional consequences of such inhibition, homopurine target sequences for oligonucleotide directed triple-helix formation were inserted in various configurations relative to Sp1 transcription activator binding sites, upstream of the TATA element of recombinant eukaryotic promoters. The resulting promoters were tested for activity in the presence or absence of recombinant human Sp1 in a Drosophila in vitro transcription system lacking endogenous Sp1. When triple-helical complexes were assembled on the promoters by incubation with specific oligodeoxyribonucleotides, promoter-specific repression of basal transcription was observed in the absence of Sp1. Transcriptional repression required the preassembly of triple-helical complexes before addition of nuclear extract. The degree of basal repression was a function of the number and proximity of triple helical complexes relative to the basal promoter complex. Repression did not result from triple-helix-induced template degradation. Addition of recombinant Sp1 did not cause derepression. These results suggest that triple-helical complexes can repress transcription primarily by blocking promoter DNA assembly into initiation complexes rather than by occluding Sp1 binding. One of several plausible mechanisms for triple-helix-induced repression involves changes in DNA flexibility. Evidence in favor of this model is provided by a permutation dependent gel mobility assay in which formation of site-specific triple-helical complexes is shown to stiffen double-helical DNA. PMID- 1731887 TI - Structural organization and regulatory regions of the human medium-chain acyl-CoA dehydrogenase gene. AB - Medium-chain acyl-CoA dehydrogenase (MCAD) is a highly regulated mitochondrial flavo-enzyme that catalyzes the initial reaction in fatty acid beta-oxidation. Deficiency of MCAD is a common inherited defect in energy metabolism. We have previously shown that the mRNA encoding MCAD in an MCAD-deficient child is homozygous for the point mutation A985 to G [Kelly et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9236-9420]. To define the molecular basis of MCAD deficiency and as an initial step in the study of the regulation of MCAD gene expression, we determined the structure and organization of the human MCAD gene. The gene is comprised of 12 exons which span 44 kb of DNA. Comparison of the MCAD gene to MCAD mRNAs from the MCAD-deficient child revealed that missplicing was common, resulting in a variety of exon deletions and intron insertions. The MCAD gene promoter region is extremely GC-rich and lacks prototypical TATA and CAAT boxes. Several regions upstream of the promoter are homologous with mitochondrial enhancers purportedly involved in coordinate expression of nuclear genes encoding mitochondrial proteins. Transfection of chimeric plasmid constructs with 299 bp of upstream sequence into HepG2 cells revealed high-level transcriptional activity. We conclude that the precursor MCAD mRNA is misspliced to a high degree and complexity in association with the G985 mutation and the MCAD gene contains a strong promoter which shares some structural features with other "housekeeping" genes encoding mitochondrial proteins. PMID- 1731888 TI - The activity of the EcoRV restriction endonuclease is influenced by flanking DNA sequences both inside and outside the DNA-protein complex. AB - The EcoRV restriction endonuclease cleaves DNA not only at its recognition sequence but also at most other sequences that differ from the recognition site by one base pair. Compared to the reaction at the recognition site, the reactions at noncognate sites are slow but 1 out of the 12 noncognate sites on the plasmid pAT153 is cleaved more than 50 times faster than any other. The increase in the reaction rate at the preferred noncognate site, relative to other sites, was caused by the DNA sequences in the 4 base pairs from either side of the site. For enhanced activity by EcoRV, particular bases were needed immediately adjacent to the site, inside the DNA-protein complex. At these loci, the protein interacts with the phosphate groups in the DNA and the flanking sequence may control the activity of the enzyme by determining the conformation of the DNA, thus aligning the phosphate contacts. But the preferential cleavage also depended on sequences further away from the site, at loci outside the complex. At external positions, beyond the reach of the protein, the EcoRV enzyme required flanking sequences that give rise to flexibility in DNA conformation. These may facilitate the distortion of the DNA required for catalysis by EcoRV. PMID- 1731889 TI - Low-barrier hydrogen bonds and low fractionation factor bases in enzymatic reactions. PMID- 1731890 TI - Intramolecular triple-helix formation at (PunPyn).(PunPyn) tracts: recognition of alternate strands via Pu.PuPy and Py.PuPy base triplets. AB - Triple-helical DNA shows increasing potential for applications in the control of gene expression (including therapeutics) and the development of sequence-specific DNA-cleaving agents. The major limitation in this technology has been the requirement of homopurine sequences for triplex formation. We describe a simple approach that relaxes this requirement, by utilizing both Pu.PuPy and Py.PuPy base triplets to form a continuous DNA triple helix at tandem oligopurine and oligopyrimidine tracts. [Triplex formation at such a sequence has been previously demonstrated only with the use of a special 3'-3' linkage in the third strand [Horne, D. A., & Dervan, P. B. (1990) J. Am. Chem. Soc. 112, 2435-2437].] Supporting evidence is from chemical probing experiments performed on several oligonucleotides designed to form 3-stranded fold-back structures. The third strand, consisting of both purine and pyrimidine blocks, pairs with purines in the Watson-Crick duplex, switching strands at the junction between the oligopurine and oligopyrimidine blocks but maintaining the required strand polarity without any special linkage. Although Mg2+ ions are not required for the formation of Pu.PuPy base triplets, they show enhanced stability in the presence of Mg2+. In the sequences observed. A.AT triplets appear to be more stable than G.GC triplets. As expected, triplex formation is largely independent of pH unless C+.GC base triplets are required. PMID- 1731891 TI - Influence of enzyme-substrate contacts located outside the EcoRI recognition site on cleavage of duplex oligodeoxyribonucleotide substrates by EcoRI endonuclease. AB - A complete understanding of the sequence-specific interaction between the EcoRI restriction endonuclease and its DNA substrate requires identification of all contacts between the enzyme and substrate, and evaluation of their significance. We have searched for possible contacts adjacent to the recognition site, GAATTC, by using a series of substrates with differing lengths of flanking sequence. Each substrate is a duplex of non-self-complementary oligodeoxyribonucleotides in which the recognition site is flanked by six base pairs on one side and from zero to three base pairs on the other. Steady-state kinetic values were determined for the cleavage of each strand of these duplexes. A series of substrates in which the length of flanking sequence was varied on both sides of the hexamer was also examined. The enzyme cleaved both strands of each of the substrates. Decreasing the flanking sequence to fewer than three base pairs on one side of the recognition site induced an asymmetry in the rates of cleavage of the two strands. The scissile bond nearest the shortening sequence was hydrolyzed with increasing rapidity as base pairs were successively removed. Taken together, the KM and kcat values obtained may be interpreted to indicate the relative importance of several likely enzyme-substrate contacts located outside the canonical hexameric recognition site. PMID- 1731892 TI - Proton NMR studies of [N-MeCys3,N-MeCys7]TANDEM binding to DNA oligonucleotides: sequence-specific binding at the TpA site. AB - [N-MeCys3,N-MeCys7]TANDEM, an undermethylated analogue of Triostin A, contains two N-methyl groups on the cysteine residues only. Footprinting results showed that [N-MeCys3,N-MeCys7]TANDEM binds strongly to DNA rich in A.T residues [Low, C. M. L., Fox, K. R., Olsen, R. K., & Waring, M. J. (1986) Nucleic Acids Res. 14, 2015-2033]. However, it was not known whether specific binding of [N-MeCys3,N MeCys7]TANDEM requires a TpA step or an ApT step. In 1:1 saturated complexes with the octamers [d(GGATATCC)]2 and [d(GGTTAACC)]2, [N-MeCys3,N-MeCys7]TANDEM binds to each octamer as a bis-intercalator bracketing the TpA step. The octadepsipeptide ring binds in the minor groove of the DNA. Analysis of sugar coupling constants from the phase-sensitive COSY data indicates that the sugar of the thymine in the TpA binding site adopts predominantly an N-type sugar conformation, while the remaining sugars on the DNA adopt an S-type conformation, as has been observed in other Triostin A and echinomycin complexes. The drug does not bind to the octamer [d(GGAATTCC)]2 as a bis-intercalator. Only weak nonintercalative binding is observed to this DNA octamer. These results show unambiguously that [N-MeCys3,N-MeCys7]TANDEM binds sequence specifically at TpA sites in DNA. The factors underlying the sequence specificity of [N-MeCys3,N MeCys7]TANDEM binding to DNA are discussed. PMID- 1731893 TI - HMG proteins 14 and 17 become cross-linked to the globular domain of histone H3 near the nucleosome core particle dyad. AB - HMG proteins were derivatized with the photoactivatable cross-linker N succinimidyl 3-((4-azidophenyl)dithio)propionate and then allowed to associate with nucleosome core particles. Following photolysis, peptide mapping of the principal dimeric adducts was carried out. Cross-linking occurred primarily from a central location in the HMGs to a central location in H3. The positions of these cross-links, considered along with other data from the literature, show that HMG proteins 14 and 17 make important contacts to H3 near the front face of the nucleosome. This raises the possibility that HMGs 14 and 17 participate in the reported conformational transition which exposes the H3 sulfhydryls of active nucleosomes. PMID- 1731894 TI - Compartmentalized ATP synthesis in skeletal muscle triads. AB - Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6 bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3 phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads. PMID- 1731895 TI - Electrostatic changes at the actomyosin-subfragment 1 interface during force generating reactions. AB - The ionic strength dependence of the binding of rabbit skeletal muscle myosin subfragment 1, S1, to F-actin in the presence of saturating concentrations of MgATP or MgADP was analyzed in order to determine the association constants at zero ionic strength [K(0)] and the products of the net effective electric charges (magnitude of zMzA) at the binding interfaces. K(0) and magnitude of zM A were 1 x 10(6) M-1 and 17 esu2 for S1-MgADP,P, and 5 x 10(7) M-1 and 7 esu2 for S1 MgADP, respectively, for binding to F-actin at 25 degrees C. At ionic strengths near physiological, the increase in affinity is close to 10(4)-fold for this transition that may correspond to force generation in muscle fibers. The large, from 17 to 7 esu2, decrease in the electrostatic contribution to binding appears to be correlated with a much larger increase in nonelectrostatic interactions, unlike the simpler transition of actin-bound S1-MgADP to S1, which appears to be due entirely to electrostatic changes [Highsmith, S. (1990) Biochemistry 29, 10690-10694]. These results for acto-S1-MgADP,P to acto-S1-MgADP suggest that a substantial transformation of the actin binding site on S1 occurs even if there is a translocation to a new interface. PMID- 1731896 TI - Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F actin and G-actin. AB - Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. Here, we have readily converted the MBS-G-actin into MBS-F-actin in the presence of phalloidin and salts. The binding of S-1 to the two actin derivatives carrying on their surface free reactive maleimidobenzoyl groups was investigated comparatively in cross linking experiments performed under various conditions to probe further the molecular structure of the actin-heavy chain complex before and after the polymerization process. Like MBS-G-actin, the isolated MBS-F-actin, which did not undergo any intersubunit cross-linking, bound stoichiometrically to S-1, generating two kinds of actin-heavy chain covalent complexes migrating on electrophoretic gels at 180 and 140 kDa. The relative extent of their production was essentially dependent on pH for both G-and F-actins. At pH 8.0, the 180-kDa species was predominant, and at pH 7.0, the amount of the 140-kDa adduct increased at the expense of the 180-kDa entity. The cross-linking of MBS-F-actin to S-1 led to the superactivation of the MgATPase substantiating the ability of this derivative to stimulate the S-1 ATPase as the native protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731897 TI - Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde. AB - Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal. PMID- 1731898 TI - Isolation from opossum serum of a metalloproteinase inhibitor homologous to human alpha 1B-glycoprotein. AB - Fractionation of opossum (Didelphis virginiana) serum with (NH4)2SO4, followed by chromatography on DEAE-Sepharose, phenyl-Sepharose, and Mono Q HR 5/5, has resulted in the isolation in homogeneous condition of a metalloproteinase inhibitor designated oprin (opossum proteinase inhibitor). Oprin is a single chain glycoprotein (26% carbohydrate) with an estimated Mr = 52,000, pI = 3.5, and E(1%/1 cm) = 11. Oprin inhibited snake venom metalloproteinases, but showed no activity on venom serine proteinases or on bacterial metalloproteinases. Incubation of Crotalus atrox alpha-proteinase (EC 3.4.24.1) with oprin, and analysis of the reaction products by chromatography on Mono Q HR 5/5 and by electrophoresis under nondenaturing conditions, indicated formation of an inactive enzyme/inhibitor complex. The complex dissociated during SDS/polyacrylamide gel electrophoresis. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for part of the open reading frame for oprin were isolated. The cDNA inserts contained nucleotide sequences corresponding to two internal amino acid sequences of oprin which had been separately determined by protein sequence analysis. Protein database screening using a 211 amino acid sequence deduced from one of the cDNA inserts showed no significant homology to known proteinase inhibitors. There was, however, a 36% identity with human alpha 1B-glycoprotein, a plasma protein of unknown function related to the immunoglobulin supergene family. In addition, the amino-terminal sequence of oprin showed 46% identity with human alpha 1B-glycoprotein in a 26 amino acid residue overlap.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731899 TI - A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators. AB - The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731900 TI - Interactions of factor XIII with fibrin as substrate and cofactor. AB - Factor XIIIa (a2') is a homodimeric transglutaminase that is formed via limited alpha-thrombin-catalyzed proteolysis of the platelet (a2) or plasma (a2b2) factor XIII zymogen in a reaction that results in proteolytic removal of a 37-aminoacyl residue peptide from the N-terminus of the a chains and exposure of the active site thiol group in the resulting a' chains of factor XIIIa. In this study, we characterized interactions of factor XIII and factor XIIIa with fibrin, a natural substrate for factor XIIIa and a cofactor for the alpha-thrombin-catalyzed activation of plasma factor XIII. The carbamylmethyl derivatives of the active site thiol group of platelet factor XIII (CMa2) and factor XIIIa (CMa2') were prepared, and their interactions with fibrin were measured. The enzyme-like derivative (CMa2') which contained nicked a' chains bound more tightly to fibrin (Kd = 2.1 microM) than did CMa2 (Kd = 14 microM), the platelet zymogen-like derivative with intact a chains, but the binding of each was weaker than the binding of plasma factor XIII zymogen (a2b2) to fibrin (Kd = 0.20 microM) under the same conditions. Saturation of fibrin with plasma factor XIII zymogen (a2b2) did not affect the binding of CMa2' to fibrin, suggesting that the plasma factor XIII zymogen (a2b2) and the active-site-modified form of factor XIIIa (CMa2') bind to separate, noninteracting sites of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731901 TI - Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta lactamases. AB - By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge. PMID- 1731902 TI - Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering. AB - Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data. PMID- 1731903 TI - Experimental and computational infrared CD studies of prototypical peptide conformations. AB - The infrared vibrational circular dichroism (VCD) spectral features of prototypical peptide secondary structures were reported previously by Yasui and Keiderling [Yasui, S.C., & Keiderling, T.A. (1986) Biopolymers 25, 5]. These results demonstrated that the "random coil" peptide conformation exhibits VCD signals which are approximately mirror-image features of those exhibited by alpha helical conformers. We report here a comparison of observed VCD spectra with those computed for several secondary structures, using the extended coupled oscillator formalism employed previously to compute VCD spectra of model DNA [Zhong et al. (1990) Biochemistry 29, 7485]. These studies suggest that the so called random-coil peptide conformation has distinct short-range order and appears to be a left-handed, helical structure. PMID- 1731904 TI - High-resolution solid state 13C NMR of bacteriorhodopsin: characterization of [4 13C]Asp resonances. AB - Solid state 13C nuclear magnetic resonance measurements of bacteriorhodopsin labeled with [4-13C]Asp show that resonances of single amino acids can be resolved. In order to assign and characterize the resonances of specific Asp residues, three different approaches were used. (1) Determination of the chemical shift anisotropy from side-band intensities provides information about the protonation state of Asp residues. (2) Relaxation studies and T1 filtering allow one to discriminate between resonances with different mobility. (3) A comparison of the spectra of light- and dark-adapted bacteriorhodopsin provides evidence for resonances from aspartic acid residues in close neighborhood of the chromophore. In agreement with other investigations, four resonances are assigned to internal residues. Two of them are protonated in the ground state up to pH 10 (Asp96 and Asp115). All other detected resonances, including Asp85 and Asp212, are due to deprotonated aspartic acid. Two lines due to the two internal deprotonated groups change upon dark and light adaptation, whereas the protonated Asp residues are unaffected. PMID- 1731905 TI - Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy. AB - The Fourier transform infrared difference spectra between light-adapted bacteriorhodopsin (BR) and its photointermediates, L and M, were analyzed for the 3750-3450-cm-1 region. The O-H stretching vibrational bands were identified from spectra upon substitution with 2H2O. Among them, the 3642-cm-1 band of BR was assigned to water by substitution with H2(18)O. By a comparison with the published infrared spectra of the water in model systems [Mohr, S.C., Wilk, W.D., & Barrow, G.M. (1965) J. Am. Chem. Soc. 87, 3048-3052], it is shown that the O-H bonds of the water in BR interact very weakly. Upon formation of L, the interaction becomes stronger. The O-H bonds of the protein side chain undergo similar changes. On the other hand, M formation further weakens the interaction of the same water molecules in BR. The appearance of a sharp band at 3486 cm-1, which was assigned tentatively to the N-H stretching vibration of the peptide bond, is unique to L. The results suggest that the water molecules are involved in the perturbation of Asp-96 in the L intermediate and that they are exerted from the protonated Schiff base which changes position upon the light-induced reaction. PMID- 1731906 TI - Purification, characterization, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Escherichia coli: a class III alcohol dehydrogenase. AB - The glutathione-dependent formaldehyde dehydrogenase from Escherichia coli has been purified to homogeneity and characterized. It is a 83,000-kDa homodimer containing 4 g-atom of zinc per dimer with a specific activity of 60 units/mg toward S-(hydroxymethyl)glutathione and NAD+ as substrates. Its isoelectric point, 4.4, is consistent with both its amino acid composition and chromatographic behavior on DEAE HPLC. The N-terminus is unblocked, and 47 residues from the N-terminus were sequenced. A computer search of the Swiss-Prot protein sequence data bank shows that the N-terminal sequence, [sequence; see text], is homologous with the mammalian class III alcohol dehydrogenases with 27 identities when compared to the human enzyme. Like the human, rat, and rabbit enzymes, it has high formaldehyde dehydrogenase activity in the presence of glutathione and catalyzes the oxidation of normal alcohols (ethanol, octanol, 12 hydroxydodecanoate) in a reaction that is not GSH-dependent. In addition, hemithiolacetals other than those formed from GSH, including omega-thiol fatty acids, also are substrates. The wide distribution and high degree of similarity of this enzyme to the plant and animal alcohol dehydrogenases suggest that the E. coli enzyme is closely related to the ancestor of the plant and animal dimeric zinc alcohol dehydrogenases. PMID- 1731907 TI - Purification and characterization of protein kinase C epsilon from rabbit brain. AB - Protein kinase C epsilon was chromatographically purified from rabbit brain to electrophoretic homogeneity. We identified the enzyme as the epsilon species of novel-type protein kinase C (nPKC epsilon), originally discovered and defined by cDNA cloning [Ohno, S., et al. (1988) Cell 53, 731-741], on the basis of the following observations: (i) the enzyme reacts specifically with an antipeptidic antiserum to nPKC epsilon but not with antisera to any of the other molecular species of PKC thus far known; (ii) it exhibits enzymatic behavior essentially identical to that of a recombinant nPKC epsilon purified from transfected COS cells [Konno, Y., et al. (1989) J. Biochem. 106, 673-678] and distinct from that of conventional PKC (alpha, beta I/II, and gamma) in its dependence on magnesium concentration and cofactors such as phospholipids, calcium, and phorbol ester; and (iii) it has an apparent molecular weight of 95.7K +/- 0.4K on SDS-PAGE, significantly greater than the other conventional and novel PKCs thus far identified. Notably, calcium exhibits a complex effect, both positive and negative, on the kinase activity of epsilon depending on the kind of substrate and the coexisting phospholipid, calling for a modification of the current notion that epsilon is a kinase unresponsive to calcium. The amount of epsilon species in the brain was estimated to be comparable to that of each conventional species, indicating that epsilon stands as one of the major PKC family members in brain. Furthermore, the enzyme shows a broader substrate spectrum than conventional PKC when examined with endogenous substrates, implying that it may cover a wider or different range of physiological functions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731908 TI - Vanadate catalyzes photocleavage of adenylate kinase at proline-17 in the phosphate-binding loop. AB - Irradiation of adenylate kinase (AK) from chicken muscle with 300-400-nm light in the presence of 0.25 mM vanadate ion first inactivated the enzyme and then cleaved the polypeptide chain near the NH2 terminus. The addition of the multisubstrate analogue, P1,P5-bis(5'-adenosyl) pentaphosphate, prevented both effects. ATP, but not AMP, blocked both inactivation and cleavage in a saturable manner, suggesting that both effects were due to modification at the ATP-binding site. The polypeptide products of the photocleavage were isolated by HPLC and characterized by amino acid composition, peptide sequencing, and mass spectral analyses. The predominant (greater than 90%) small peptide fragment contained the first 16 amino acids from the amino terminus of the enzyme. The amino terminus of this peptide contained an acetylated serine, and the "carboxy" terminus was modified by a cyclized gamma-aminobutyric acid which originated from photooxidation and decarboxylation of proline-17 by vanadate. Edman sequencing indicated that the majority of the large peptide fragment (Mr approximately 19,500) was amino-terminal blocked, but a small portion was sequenceable starting at either glycine-18 (7%) or serine-19 (2%). These studies indicate that in the ATP-AK complex proline-17 is close to the phosphate chain of ATP but not AMP, consistent with the latest evaluation of nucleotide-binding sites on mitochondrial matrix AK by X-ray crystallography [Diederichs, K., & Schulz, G.E. (1991) J. Mol. Biol. 217, 541-549]. Furthermore, this is the first report that an amino acid other than serine can be involved in vanadate-promoted photocleavage reactions. PMID- 1731909 TI - Amino acid substitutions in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that influence catalytic activity of the holoenzyme. AB - Four unique amino acid substitutions were introduced by site-directed mutagenesis into the third conserved region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans (Synechococcus sp., PCC6301), resulting in the formation of four mutant enzymes, I87V, R88K, G91V, and F92L. Wild-type and mutant proteins were purified after synthesis in Escherichia coli. These single amino acid substitutions do not appear to perturb intersubunit interactions or induce any gross conformational changes; purified mutant proteins are stable, for the most part like the wild-type holoenzyme, and exhibit nearly identical CD spectra. Three of the four mutants, however, are severely deficient in carboxylase activity, with kcat less than or equal to 35% of the wild-type enzyme. While the substrate specificity factors were the same for the mutant and wild-type enzymes, significant alterations in some kinetic parameters were observed, particularly in the Michaelis constants for CO2, O2, and RuBP. All four mutant proteins exhibited lower KCO2 values, ranging from 37 to 88% of the wild-type enzyme. Two of the mutants, in addition, exhibited significantly lower KRuBP values, and one mutant showed a substantial decrease in KO2. The effects of the single-site mutations in rbcS of this study strengthen the hypothesis that small subunits may not contribute directly to substrate specificity; however, individual residues of the small subunit substantially influence catalysis by large subunits. PMID- 1731910 TI - Improvement by benzoquinones of the quantum yield of photoactivation of photosynthetic oxygen evolution: direct evidence for the two-quantum mechanism. AB - Effects of eight differently substituted 1,4-benzoquinones (BQs) on the quantum yield of photoactivation of oxygen evolution (reconstitution of the Mn cluster) were examined with wheat photosystem II (PSII) membranes depleted of the Mn cluster by treatment with 1.0 mM NH2OH. Illumination with 10 flashes at 0.25-s intervals of the PSII membranes in the presence of 2.0 mM Mn2+, 20 mM Ca2+, and 1.2 M Cl- restored 14% of oxygen-evolving activity destroyed by the NH2OH treatment. Among the benzoquinones tested, DBMIB (2,5-dibromo-3-methyl-6 isopropyl-BQ) and tetramethyl-BQ did not enhance the activity recovery, but all the others doubled the recovery when present at their optimal concentrations during illumination. The order of effectiveness was tetrabromo-, phenyl-, and 2,6 dichloro-BQs greater than or equal to 2,5-dichloro-BQ greater than tetrachloro-BQ greater than 2,5-dimethyl-BQ, though the differences were small. This order reflects their efficiencies as electron acceptors of PSII. This finding, together with others, suggests that the enhancement of activity recovery results from rapid oxidation by the benzoquinones of the reduced form of the quinone acceptors in PSII, QA- and QB-, which cause loss of an oxidized intermediate through charge recombination reaction with Mn3+. The flash-number dependence of the recovery of oxygen-evolving activity indicated that the activity was not restored after one flash but recovered significantly after illumination with two flashes and then further increased upon additional flashes. This provides direct evidence that the minimum quantum requirement for photoactivation is two. PMID- 1731911 TI - Cholesterol heterogeneity in the plasma membrane of epithelial cells. AB - The distribution of cholesterol in the plasma membrane of epithelial cells has been determined using renal brush border vesicles as a model. In brush borders treated with Brevibacterium sp. or Nocardia erythropolis cholesterol oxidases, a significant fraction of the free cholesterol was oxidized rapidly, without glutaraldehyde fixation, and the remaining cholesterol was oxidized at a slower rate. The size of the readily accessible cholesterol pool, however, depended on the enzyme used, varying from 16% of the total in membranes treated with N. erythropolis oxidase, to 27% using the Brevibacterium sp. enzyme. The slowly accessible pool detected by the Brevibacterium oxidase was suppressed upon sphingomyelinase addition. On the other hand, the restricted activity of the Nocardia oxidase might depend on phosphatidylcholine/cholesterol interactions. These results indicate that cholesterol distribution is heterogeneous in intact renal brush border vesicles. They suggest that, as proposed for model system [Demel, R.A. Jansen, J.W.C.M., van Dijck, P.W.M., & van Deenen, L.L.M. (1977) Biochim. Biophys. Acta 465, 1-10], preferential interactions between some classes of phospholipids and cholesterol define cholesterol pools in the plasma membrane of epithelial cells. PMID- 1731912 TI - Interactions of acyl-coenzyme A with phosphatidylcholine bilayers and serum albumin. AB - Interactions of oleoyl- and octanoyl-coenzyme A (CoA) with phosphatidylcholine (PC) vesicles and bovine serum albumin (BSA) were investigated by NMR spectroscopy. Binding of acyl-CoA to small unilamellar PC vesicles and to BSA was detected by changes in 13C and 31P chemical shifts relative to the chemical shifts for aqueous acyl-CoA. When oleoyl-CoA (less than or equal to 15 mol %) was added to preformed vesicles, the 13C thioester signal (200.1 ppm) was upfield from the signal for micellar oleoyl-CoA (201.7 ppm), suggesting decreased H bonding (partial dehydration) at the carbonyl group upon binding to the bilayer. When vesicles were prepared by cosonication of oleoyl-CoA and PC, a second peak (199.8 ppm) was seen. The major peak at 200.1 ppm broadened and shifted after addition of Dy(NO3)3 and was not seen after addition of BSA, while the peak at 199.8 ppm was unaffected by either perturbation. Thus, oleoyl-CoA in each bilayer leaflet was distinguished, and transbilayer movement was shown to be slow (t 1/2 greater than or equal to hours). PC vesicles remained intact with less than or equal to 15 mol % oleoyl-CoA, while higher oleoyl-CoA proportions produced mixed micelles. In contrast, 13C spectra revealed rapid exchange (ms) of octanoyl-CoA between the aqueous phase and PC vesicles and a low affinity for the bilayer. Thus, the binding affinity of acyl-CoA for PC bilayers is dependent on the acyl chain length. Oleoyl-CoA in the presence of BSA (1 mol/mol) gave rise to three carbonyl signals at 197.2-203.6 ppm. With 2-5 mol of oleoyl-CoA/BSA, 1-2 additional signals were observed. None of the signals corresponded to unbound oleoyl-CoA. Addition of [13C]carboxyl-enriched oleic acid to oleoyl-CoA/BSA mixtures revealed simultaneous binding of oleic acid and oleoyl-CoA to BSA, with some perturbation of binding interactions. Thus, BSA contains multiple binding sites for oleoyl-CoA and can bind fatty acid and acyl-CoA simultaneously. PMID- 1731913 TI - Hapten conformation in the combining site of antibodies that bind phenylphosphocholine. AB - We have shown previously that anti-phenylphosphocholine antibodies elicited by phosphocholine-keyhole limpet hemocyanin can be divided into two populations according to their ability to recognize the two hapten analogues p nitrophenylphosphocholine (NPPC) and p-nitrophenyl 3,3-dimethylbutyl phosphate (NPDBP). These analogues differ from each other in that NPPC has a positively charged nitrogen in the choline moiety, whereas NPDBP lacks the positively charged nitrogen. Group II-A antibodies bind only NPPC, whereas group II-B antibodies bind both ligands. Here, by infrared and nuclear magnetic resonance spectroscopic investigations, we find that when free in solution NPPC has a predominantly fixed structure in which the termini approach each other, probably due to electrostatic interactions within the molecule; this "bent" structural feature is retained when the ligand is bound by antibody. In contrast, the structure of unbound NPDBP is less fixed, being characterized by rapidly interchanging conformations corresponding to an open chain structure with less overall proximity of the termini compared to NPPC. The overall shape of NPPC is essentially unaltered by binding, whereas in the case of NPDBP what was a minor conformation in the unbound state becomes the predominate conformation of the bound ligand. Thus, our results are consistent with these antibodies providing a molecular template for stabilizing the conformation of the bound ligand. PMID- 1731914 TI - Biphasic changes in the level and composition of Dunaliella salina plasma membrane diacylglycerols following hypoosmotic shock. AB - Hypoosmotic shock has been shown to trigger an immediate and selective increase of plasma membrane diacylglycerols (DAG) in the green alga Dunaliella salina, coinciding with an approximately equivalent loss of phosphatidylinositol 4,5 bisphosphate from this membrane [Ha, K.S., & Thompson, G.A., Jr. (1991) Plant Physiol. 97, 921-927]. Following a slight decline in amount, DAG levels of the plasma membrane resumed their rise by 2 min after the shock and by 40 min had achieved a maximum concentration equivalent to 230% of DAG levels in unstressed cells. This second, more sustained increase of plasma membrane DAG was matched by a DAG increase in the microsome-enriched cytoplasmic membrane fraction, commencing at 2 min and peaking at 140% of control values. The changing pattern of DAG molecular species produced in the plasma membrane during the early phases of hypoosmotic stress was compatible with their derivation from phospholipase C hydrolysis of inositol phospholipids and phosphatidylcholine. From 8 min following hypoosmotic shock, as relatively larger scale DAG accumulations developed in the cytoplasmic membranes, the molecular species composition changed to reflect a marked increase in de novo synthesis of sn-1-oleoyl, sn-2 palmitoylglycerol, and dioleoylglycerol. The former molecular species appears to be synthesized in the chloroplast while the latter is produced in the endoplasmic reticulum. The radioisotope labeling data with Na2(14)CO3 confirmed that the biphasic formation of DAG triggered by hypoosmotic shock culminates in a large scale de novo synthesis of DAG. This is the first clear evidence for de novo synthesis as a source of DAG following PIP2-mediated signaling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731915 TI - Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B12 biosynthesis. AB - The trimethylated intermediate of vitamin B12 (corrin) biosynthesis, precorrin-3, was produced from various 13C-enriched isotopomers of 5-aminolevulinic acid (ALA), using a multiple-enzyme system containing ALA dehydratase, porphobilinogen deaminase, uro'gen III synthetase, and the S-adenosyl-L-methionine-(SAM) dependent uro'gen III methyltransferase (M-1) and precorrin-2 methyltransferase (M-2) in the presence of [13C]SAM. Structural analysis of the resulting product, precorrin-3, reveals a close similarity to precorrin-2 but with several subtle differences in the conjugated array of C = C and C = N bonds which reflect the presence of the new C-methyl group at C20 and its influence on the electronic distribution in the dipyrrocorphin chromophore. The implications of this structure for corrin biosynthesis are discussed. PMID- 1731916 TI - Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines. AB - A mammary-derived growth factor, MDGF1, which stimulates collagen synthesis and proliferation in mammary epithelial cells was previously detected and purified from human milk and primary human breast tumors. MDGF1 binds to putative cell surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. Partial protein sequence (N-terminal 18 amino acid sequence) shows that MDGF1 has no homology to any other known growth promoting peptides. Polyclonal antiserum raised against this synthetic peptide recognizes native milk-derived MDGF1. We hypothesize that MDGF1 might be an autocrine or paracrine factor produced by and acting on normal and malignant human breast epithelial cells possessing MDGF1 receptors. As a first step in testing this possibility, we examined whether human breast epithelial cells in culture produce the growth factor. A protein with the size of MDGF1 was immunologically detected in the concentrated conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but nontumorigenic HBL-100 line, and the normal reduction mammoplasty-derived, nonimmortalized 184 cell strain. A competitive radioreceptor assay (RRA) was used to estimate the level of MDGF1 in the conditioned medium. MDGF1 was present in the nanogram range per 1 million cells. A 62-kDa protein was detected in the above cell lysates by Western immunoblotting or by immunoprecipitation of metabolically labeled cell conditioned media. The polyclonal antisera directed against the 18 amino acid peptide sequence from milk-derived MDGF1 could adsorb MDGF1 biological activity from conditioned medium. In vitro translation of cell mRNA yielded a protein of 55 kDa which was immunoprecipitated by anti-MDGF1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731917 TI - Interactions of saturated diacylglycerols with phosphatidylcholine bilayers: A 2H NMR study. AB - The interactions of a series of saturated diacylglycerols (DAGs) with fatty acid side chain lengths of 6-14 carbons with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts were studied by 2H NMR spectrometry. We found that the perturbation induced by the DAGs into the bilayer structure strongly depends on the length of the DAG fatty acid side chain. Shorter chain 1,2-sn-dihexanoylglycerol and, to a larger degree, 1,2-sn dioctanoylglycerol (diC8) induce transverse perturbation of the bilayer structure: the order parameters of the phospholipid side chains are increased by the intercalating DAG molecules in the region adjacent to the phospholipid headgroups and decreased toward the terminal methyls, corresponding to the bilayer interior. The longer chain DAGs (C greater than or equal to 12) studied in this and previous [De Boeck & Zidovetzki (1989) Biochemistry 28, 7439] work induce lateral phase separation of the lipids into DAG-enriched gellike domains and relatively DAG-free regions in the liquid-crystalline phase. Each of the DAGs studied induces a decrease in the area per phospholipid molecule, and a corresponding increase in the lateral surface pressure of the bilayers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731918 TI - Polymyxin B induces transient permeability fluctuations in asymmetric planar lipopolysaccharide/phospholipid bilayers. AB - The interaction of the polycationic decapeptide polymyxin B with asymmetric planar bilayers from lipopolysaccharide and phospholipid monolayers, which resemble the lipid matrix of the outer membrane of Gram-negative bacteria, was investigated. The addition of polymyxin B in micromolar amounts to the lipopolysaccharide side of the asymmetric bilayers resulted, under voltage-clamp conditions, in a fast macroscopic increase of their ionic conductance, whereas the polymyxin B nonapeptide induced no significant conductance changes. The polymyxin B induced macroscopic conductance exhibited large fluctuations and was strongly dependent on the amplitude and polarity of the transmembrane potential. The temporal pattern and amplitudes of the fluctuations were characterized by power spectra of the membrane currents and their variances, respectively. In the initial phase following peptide addition, the conductance changes appeared to be channellike discrete fluctuations. The lifetimes of the fluctuations were exponentially distributed, and the mean lifetimes were strongly voltage dependent, ranging from approximately 30 ms at +80 mV (positive at the side opposite to peptide addition) to less than 5 ms at reverse polarity. The conductance amplitudes of the single fluctuations exhibited a broad distribution with a mean of 2 nS. A comparison of the features of the macroscopic conductance and of the discrete fluctuations showed that the former can basically be understood as a superposition of a large number of the latter. From the amplitudes of the fluctuations, the diameter of the polymyxin-induced lesions was estimated to about 3 nm. The experimental findings can be understood by assuming a detergent-like action of polymyxin B. PMID- 1731920 TI - Solution structure of the lipophosphoglycan of Leishmania donovani. AB - The three-dimensional solution structure of the repeating -PO4-6Gal beta 1-4Man alpha 1- disaccharide fragment of the lipophosphoglycan (LPG) derived from Leishmania donovani has been determined by use of a combination of homo- and heteronuclear NMR spin coupling constant measurements together with restrained molecular mechanical minimization and molecular dynamics simulations. The fragment exists with limited mobility in solution about the Gal beta 1-4Man linkages, whereas in contrast a variety of stable rotamers exist about the Man alpha 1-PO4-6Gal linkages. These rotamers define several major stable conformers in solution, which are discussed in terms of the proposed biological role of LPG. PMID- 1731919 TI - Scyllatoxin, a blocker of Ca(2+)-activated K+ channels: structure-function relationships and brain localization of the binding sites. AB - Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca(2+)-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of lysine residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca(2+)-activated K+ channels. High-affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin. PMID- 1731921 TI - Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium. AB - The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn 1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731922 TI - Hydrophobic lipid additives affect membrane stability and phase behavior of N monomethyldioleoylphosphatidylethanolamine. AB - The rate of formation of high-curvature intermediates or disordered cubic phases in N-methyldioleoylphosphatidylethanolamine (N-methyl-DOPE) dispersions with or without additives was studied by 31P NMR spectroscopy. In N-methyl-DOPE dispersions, both the L alpha liquid-crystalline phase and the hexagonal HII phase convert into phases of high curvature giving rise to isotropic 31P NMR resonances. Addition of the bilayer destabilizers 1,2-diolein, 1,3-diolein, or eicosane lowers the threshold temperature of the isotropic phase. The isotropic threshold temperature is strongly correlated with the L alpha-HII phase transition temperature (TH). The addition of hexagonal phase promoters does not change the rate of formation of the isotropic phase at a temperature shifted by a fixed amount below TH. However, the formation of "isotropic" phases from the additive-stabilized hexagonal phase is slow compared to that observed in pure N methyl-DOPE lipid dispersions. Membrane leakage and fusion are promoted by the dioleins and well as by eicosane, but changes in the rates of these processes do not correlate well with the extent of formation of isotropic phases. All three additives have similar effects on phase behavior and on vesicle leakage and fusion. These similarities occur despite the fact that eicosane is believed to partition differently into the membrane than diolein. In addition to the general similarities in the effects of the two diolein isomers, 1,2-diolein is somewhat more potent in promoting the hexagonal phase and in increasing rates of leakage and fusion than is 1,3-diolein. PMID- 1731923 TI - Structure of epidermal growth factor bound to perdeuterated dodecylphosphocholine micelles determined by two-dimensional NMR and simulated annealing calculations. AB - The interaction of mouse epidermal growth factor (mEGF) with micelles of a phospholipid analogue, perdeuterated dodecylphosphocholine (DPC), was investigated by two-dimensional 1H NMR. Sequence-specific resonance assignments of the micelle-bound mEGF have been made, and the chemical shifts were compared with those in the absence of DPC. DPC induced large chemical shift changes of the resonances from the residues in the C-terminal tail (residues 46-53) but little perturbation on the residues in the main core (residues 1-45). Starting from the three-dimensional structure in the absence of DPC, micelle-bound structures were calculated using the program XPLOR with interproton distance data obtained from NOESY spectra recorded in the presence of DPC. The C-terminal tail of mEGF was found to change conformation to form an amphiphilic structure when bound to the micelles. It is possible that induced fit in the C-terminal tail of mEGF occurs upon binding to a putative hydrophobic pocket of the EGF receptor. PMID- 1731924 TI - Photoinduced destabilization of liposomes. AB - The stability of two-component liposomes composed of the polymerizable 1,2-bis [10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents. PMID- 1731925 TI - Investigation of heme distortions and heme-protein coupling in the isolated subunits of oxygenated human hemoglobin by resonance Raman dispersion spectroscopy. AB - To probe the distortions of the heme groups resulting from heme-apoprotein interaction in the isolated subunits of oxygenated human hemoglobin (i.e., alpha SH-oxyHbA and beta SH-oxyHbA), the dispersion of the depolarization ratio of the Raman lines at 1375 cm-1 (nu 4) and 1638 cm-1 (nu 10) was measured at various pHs. The data were analyzed in terms of vibronic coupling parameters which depend on symmetry-classified normal distortions of the heme groups. In the alpha-chain the nu 10 mode is not affected by symmetry-lowering distortions. In the beta chain, however, this mode is significantly influenced by asymmetric B1g and B2g distortions. This was interpreted in terms of different interactions between the peripheral substituents and the porphyrin macrocycle in the respective chains. The nu 4 mode of both chains is subject to B1g (B2g) and A2g distortions, which are more pronounced in beta SH-oxyHbA. This is most probably due to differences in the repulsive interactions between the proximal imidazole and the pyrrole. While the depolarization ratio of both lines investigated is pH-independent in alpha SH-oxyHbA, it exhibits a significant pH dependence in beta SH-oxyHbA. This parallels the finding that the isolated beta-chains exhibit a Bohr effect whereas the alpha-chains do not. Consequently, the pH dependence of the coupling parameters and the Bohr effect of beta SH-oxyHbA could be rationalized in terms of the very same proton binding processes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731926 TI - Luminescence quenching by nitroxide spin labels in aqueous solution: studies on the mechanism of quenching. AB - The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 10(5) M-1 s-1) than those of the singlet-state probes; (II) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42 degrees C) and viscosity (up to 60 mPa.s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731927 TI - Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants. AB - A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Forster [Forster, T. (1948) Ann. Phys. (Leipzig) 2, 55 75].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731928 TI - Isolation and characterization of the triply oxidized derivative of a cross linked hemoglobin. AB - Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731929 TI - Contribution of hydrogen bonding to the conformational stability of ribonuclease T1. AB - For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1). When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed. Twelve mutants of RNase T1 [Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)] have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A. On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond. This estimate is in good agreement with results from several related systems. Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect. Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides. PMID- 1731930 TI - Mechanism of the conformational transition of melittin. AB - It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational transition of melittin, we examined in detail the conformation of melittin under various conditions by far-UV circular dichroism at 20 degrees C. We found that the helical conformation is also stabilized by strong acids such as perchloric acid. The effects of various acids varied largely and were similar to those of the corresponding salts, indicating that the anions are responsible for the salt- or acid-induced transitions. The order of effectiveness of various monovalent anions was consistent with the electroselectivity series of anions toward anion exchange resins, indicating that the anion binding is responsible for the salt- or acid-induced transitions. From the NaCl-, HCl-, and alkaline pH-induced conformational transitions, we constructed a phase diagram of the anion- and pH dependent conformational transition. The phase diagram was similar in shape to that of acid-denatured apomyoglobin [Goto, Y., & Fink, A.L. (1990) J. Mol. Biol. 214, 803-805] or that of the amphiphilic Lys, Leu model polypeptide [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396], suggesting a common mechanism of the conformational transition. The anion-, pH-, and peptide concentration dependent conformational transition of melittin was explained on the basis of an equation in which the conformational transition is linked to proton and anion binding to the titratable groups. PMID- 1731931 TI - Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes. AB - The tubulin-colchicine binding reaction appears to involve a number of intermediate steps beginning with rapid formation of a transient preequilibrium complex that is followed by one or more slow steps in which conformational changes in tubulin and colchicine lead to formation of a poorly reversible final state complex. In the present study, we investigated the relative ability of unliganded colchicine and preformed final-stage tubulin-colchicine complex to incorporate at microtubule ends and to inhibit addition of tubulin at the net assembly ends of bovine brain microtubules in vitro. Addition of 0.1 microM final stage tubulin-colchicine complex to suspensions of microtubules at polymer-mass steady-state resulted in rapid incorporation of one to two molecules of tubulin colchicine complex per microtubule net assembly end concomitant with approximately 50-60% inhibition of tubulin addition. Incorporation of colchicine tubulin complex continued slowly with time, without significant additional change in the rate of tubulin addition. In contrast, addition of unliganded colchicine to microtubule suspensions resulted in incorporation of small numbers of colchicine molecules at microtubule ends and inhibition of tubulin addition only after periods of time that varied from several minutes to approximately 20 min depending upon the concentration of colchicine. Inhibition of tubulin addition beginning with unliganded colchicine increased slowly with time, concomitant with increases in the concentration of final-state tubulin-colchicine complex and the amount of colchicine bound per microtubule end. The results indicate that inhibition of tubulin incorporation at microtubule ends is caused by colchicine liganded tubulin in the form of a final-state complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731932 TI - Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering. AB - C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers. PMID- 1731933 TI - Zn(II) coordination domain mutants of T4 gene 32 protein. AB - Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A+B) (residues 22-253). Optical absorption studies with the Co(II)-substituted protein and 113Cd NMR spectroscopy of 113Cd(II) substituted g32P-(A+B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90. These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling [Giedroc, D.P., Johnson, B.A., Armitage, I.M., & Coleman, J.E. (1989) Biochemistry 28, 2410-2418]. To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins. We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form [less than or equal to 0.03 g.atom Zn(II)]. All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT). All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein. These structural features parallel those found for the known metal ligand mutant Cys87----Ser87 (C87S) g32P. In contrast, g32P-(A+B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A+B). Spin echo 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-His g32P-(A+B) [Pan, T., Giedroc, D.P., & Coleman, J.E. (1989) Biochemistry 28, 8828 8832]. These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P. Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1731934 TI - 13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli. AB - The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP NeuAc) synthetase was investigated by NMR using 13C-labeled N-acetylneuraminic acid (NeuAc). Consumption of the beta-anomer of [2-13C]N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid. Inhibition by substrate analogues the anomeric oxygen was determined in a similar manner using [2-13C,(50 atom %)18O]N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the [13C]NeuAc substrate and CMP-[13C]NeuAc product was observed due to the 18O substitution. This result implies conservation of the NeuAc oxygen. Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate. Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP. PMID- 1731935 TI - Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase. AB - The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+) H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10 CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10( 2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731936 TI - Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis. AB - The effector binding site of Escherichia coli aspartate transcarbamoylase, composed of the triphosphate and ribose-base subsites, is located on the regulatory (r) chains of the enzyme. In order to probe the function of amino acid side chains at this nucleotide triphosphate site, site-specific mutagenesis was used to create three mutant versions of the enzyme. On the basis of the three dimensional structure of the enzyme with CTP bound, three residues were selected. Specifically, Arg-96r was replaced with Gln, and His-20r and Tyr-89r were both replaced with Ala. Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme. However, the mutations at His-20r and Tyr-89r produced altered response to the regulatory nucleotides. For the His-20r----Ala enzyme, the affinities of the enzyme for ATP and CTP are reduced 40-fold and 10-fold, respectively, when compared with the wild-type enzyme. Furthermore, CTP is able to inhibit the His 20r----Ala enzyme 40% more than the wild-type enzyme. In the case of the Tyr-89r- --Ala enzyme. ATP can increase the mutant enzyme's activity 181% compared to 157% for the wild-type enzyme, while simultaneously the affinity of this enzyme for ATP decreases about 70%. These results suggest that Tyr-89r does have an indirect role in the discrimination between ATP and CTP. The His-20r----Ala enzyme shows no UTP synergistic inhibition in the presence of CTP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731937 TI - Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli. AB - The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway. PMID- 1731938 TI - Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase. AB - Schistosomiasis is a trematode infection of some 200 million people. The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the major etiologic agent, Schistosoma mansoni, has been proposed as a potential target for antischistosomal chemotherapy [Dovey, H. F., McKerrow, J. H., & Wang, C. C. (1984) Mol. Biochem. Parasitol, 11, 157-167]. The steady-state kinetic mechanism for the schistosomal HGPRTase has been determined by including both hypoxanthine and guanine in the forward and reverse reactions under identical conditions. Double-reciprocal plots of initial velocity versus the concentration of one substrate, at a series of fixed concentrations of the other, give groups of intersecting straight lines indicating a sequential mechanism for the schistosomal HGPRTase-catalyzed reactions. In product inhibition studies, the results show that magnesium pyrophosphate (MgPPi) is a noncompetitive inhibitor with respect to dimagnesium phosphoribose pyrophosphate (Mg2PRPP), hypoxanthine, and guanine. Also, magnesium inosine monophosphate (MgIMP) and magnesium guanosine monophosphate (MgGMP) are noncompetitive inhibitors with respect to hypoxanthine or guanine, respectively, but are competitive inhibitors to Mg2PRPP. Furthermore, Mg2PRPP is a competitive inhibitor with respect to MgIMP and MgGMP but is a non-competitive inhibitor to MgPPi. The minimum kinetic model which fits the experimental data is an ordered bi-bi mechanism, where the substrates bind to the enzyme in a defined order (first Mg2PRPP followed by the purine bases), while products are released in sequence (first MgPPi followed by MgIMP or MgGMP).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731939 TI - Interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates: kinetics, mechanism, and analogy to the acylenzyme mechanism. AB - The interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates [PNPOC(=O)-NHCnH2n+1; n = 4, 8, and 12] proceeds by the three-stage mechanism shown below. After reversible [formula: see text] formation of the enzyme carbamate complex (EC), rapid carbamylation (kc) precedes slow decarbamylation. Therefore, in short-term assays (less than or equal to 30 min) of lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl butyrate, activity is rapidly lost. The inhibition by p-nitrophenyl N-butylcarbamate follows saturation kinetics, which allows determination of Kc = 5.4 +/- 0.9 microM and kc = (4.9 +/- 0.7) x 10(-2)s-1. Saturation kinetics are not observed for the longer inhibitors p nitrophenyl N-octylcarbamate and p-nitrophenyl N-dodecylcarbamate. Rather, plots of the pseudo-first-order rate constant for activity loss versus inhibitor concentration are concave upward, consistent with inhibitor binding to two sites on the enzyme. The inhibition phase is sufficiently rapid that p-nitrophenyl N octylcarbamate can be used to titrate enzyme active sites. On the other hand, long-term assays (greater than 5 h) show sequential inhibition and activity return phases, and from the activity return phase kd is calculated. The long-term activity time course is accurately simulated by Runge-Kutta integration of the differential equations for the three-stage mechanism. These approaches have been used to characterize the kinetics of interaction of the enzyme with the carbamate inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731940 TI - Inhibition of NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue and related anthraquinone dyes: a structure-activity study. AB - Cibacron Blue, a widely used ligand for affinity chromatography, is a potent inhibitor of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) (quinone reductase). This property has been exploited to purify quinone reductase, to identify its nucleotide-binding site, and to obtain diffraction-grade crystals of this enzyme [Prochaska, H. J. (1988) Arch. Biochem. Biophys. 267, 529-538; Ysern, X., & Prochaska, H. J. (1989) J. Biol. Chem. 264, 7765-7767]. To define the structural region(s) of the dye responsible for its inhibitory potency, Cibacron Blue was synthesized and the dye, its synthetic intermediates, and some analogues of these intermediates were crystallized as novel trialkylamine or choline salts. These compounds were characterized by proton NMR and mass spectrometry, and their inhibitory potencies were measured. Only two of the four ring systems of the Cibacron Blue molecule are required for potent inhibition. Acid Blue 25 [1-amino 4-(phenylamino)anthraquinone-2-sulfonic acid] is an inhibitor (Ki = 22 nM) almost as potent as Cibacron Blue (Ki = 6.2 nM). However, removal of any of the three substituents on the anthraquinone ring of Acid Blue 25 markedly reduced inhibitory potency. These results are consistent with the proposal that Cibacron Blue is primarily a mimic for the ADP fragment of mono- and dinucleotides. The difference absorption spectrum of the Acid Blue 25-quinone reductase complex was very different from that of the complex with Cibacron Blue. In contrast to other compounds tested, Procion Blue M-3GS, the electrophilic dichlorotriazine precursor of Cibacron Blue, was an irreversible inhibitor of quinone reductase (KD = 16 nM, k3 = 0.03 min-1), and the inactivation was blocked by Cibacron Blue, a monochlorotriazine. PMID- 1731941 TI - Cation-dependent transition between the quadruplex and Watson-Crick hairpin forms of d(CGCG3GCG). AB - The DNA oligonucleotide d(CGCG3GCG) can form either a Watson-Crick (WC) hairpin or a parallel-stranded quadruplex structure containing six G-quartet base pair assemblies. The exchange between these forms and single strands can be monitored using circular dichroism (CD). NMR results verified the assignment of specific CD bands to quadruplex and hairpin species, respectively. Cations stabilize the quadruplex in the order K+ greater than Ca2+ greater than Na+ greater than Mg2+ greater than Li+ and K+ greater than Rb+ greater than Cs+, indicating that K+ has an optimum ionic radius for complex formation and that ionic charge affects the extent of ion-induced stabilization. The quadruplex is stable in the presence of 40 mM K+ at micromolar DNA concentration and can be kinetically trapped as a metastable form when prepared at millimolar DNA concentration and then diluted into buffer containing 40 mM Na+. The concentration of K+ required to reverse the equilibrium from the hairpin to the quadruplex decreases sharply with increased DNA concentration. The quadruplex has an unusual pKa of ca. 6.8, indicating that C.C+ base pairs are probably forming. This system provides insights into some of the detailed structural characteristics of a ["G4-DNA".ion] complex and an experimental model for the recently proposed "sodium-potassium conformational switch" [Sen, D., & Gilbert, W. (1988) Nature 334, 364-366; Sen, D., & Gilbert, W. (1990) Nature 344, 410-414]. These results may help to explain the lack of cytidine residues in G-rich telomeric DNAs and suggest that methylation of GC rich duplex DNAs in "GpC islands" may induce quadruplex formation within heterochromatin domains, resulting in reversible chromosomal condensation. PMID- 1731942 TI - Bisintercalators of DNA with a rigid linker in an extended configuration. AB - A new class of DNA bisintercalators is reported in which phenanthridinium or acridinium rings are connected by rigid and extended linkers of varied length. Cross-linking of DNA by bisintercalation is inferred from the unwinding and folding of linear DNA induced by the compound; after ligation and removal of the bisintercalator, superhelical circles, catenanes, and knots that bear an imprint of the bisintercalator are observed. These novel bisintercalators are of interest because they can be used to probe the organization of DNA in three-dimensional space, especially near sites of replication, recombination, or topoisomerase action, where two duplexes must be in close proximity. PMID- 1731943 TI - Recognition of foldback DNA by the human DNA (cytosine-5-)-methyltransferase. AB - In order to specify the recognition requirements of the human DNA (cytosine-5-) methyltransferase, two isomeric 48mers were synthesized so as to link a long block of DNA with a shorter complementary block of DNA through a tether consisting of five thymidine residues. These isomeric foldback molecules, differing only in the location of the 5-methyldeoxycytosine, were shown to be unimolecular, to contain a region of duplex DNA, and to contain a region of single-stranded DNA. When used as substrates for the DNA methyltransferase, only one of the isomers was methylated. A comparison of the structures of the two isomers allows us to begin to define the potential sites of interaction between the enzyme and the three nucleotides forming a structural motif consisting of 5 methyldeoxycytosine, its base-paired deoxyguanosine, and a deoxycytosine 5' to the paired deoxyguanosine. PMID- 1731944 TI - Proton and electron transfer in the acceptor quinone complex of Rhodobacter sphaeroides reaction centers: characterization of site-directed mutants of the two ionizable residues, GluL212 and AspL213, in the QB binding site. AB - Proton and electron transfer events in reaction centers (RCs) from Rhodobacter sphaeroides were investigated by site-directed mutagenesis of glutamic acid at position 212 and aspartic acid at 213 in the secondary quinone (QB) binding domain of the L subunit. These residues were mutated singly to the corresponding amides (mutants L212EQ and L213DN) and together to give the double mutant (L212EQ/L213DN). In the double mutant RCs, the rate of electron transfer from the primary (QA) to the secondary (QB) acceptor quinones is fast (tau approximately 300 microseconds) and is pH independent from pH 5 to 11. The rate of recombination between the oxidized primary donor, P+, and QB- is also pH independent and much slower (tau approximately 10 s) than in the wild type (Wt), indicating a significant stabilization of the QB- semiquinone. In the double mutant, and in L213DN mutant RCs at low pH, the P+QB- decay is suggested to occur significantly via a direct recombination rather than by repopulating the P+QA- state, as in the Wt. Comparison of the behavior of Wt and the three mutant RC types leads to the following conclusions: the pK of AspL213 in the Wt is approximately 4 for the QAQB state (pKQB) and approximately 5 for the QAQB-state (pKQB-); for GluL212, pKQB approximately 9.5 and pKQB- approximately 11. In L213DN mutant RCs, pKQB of GluL212 is less than or equal to 7, indicating that the high pK values of GluL212 in the Wt are due largely to electrostatic interaction with the ionized AspL213 which contributes a shift of at least 2.5 pH units. Transfer of the second electron and all associated proton uptake to form QBH2 is drastically inhibited in double mutant and L213DN mutant RCs. At pH greater than or equal to 8, the rates are at least 10(4)-fold slower than in Wt RCs. In L212EQ mutant RCs the second electron transfer and proton uptake are biphasic. The fast phase of the electron transfer is similar to that of the Wt, but the extent of rapid transfer is pH dependent, revealing the pH dependence of the equilibrium QA(-)QB- in equilibrium with QAQBH-. The estimated limits on the pK values--pKQA-QB-less than or equal to 7.3, pKQAQB2- greater than or equal to 10.4--are similar to those derived earlier for Wt RCs [Kleinfeld et al. (1985) Biochim. Biophys. Acta 809, 291-310] and may pertain to the quinone head group, per se.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1731945 TI - Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate. AB - Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs. PMID- 1731946 TI - Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III. AB - The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI III* revealed significant changes for residues located far away from the reactive site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731947 TI - Proton NMR studies of Cucurbita maxima trypsin inhibitors: evidence for pH dependent conformational change and His25-Tyr27 interaction. AB - A pH-dependent His25-Tyr27 interaction was demonstrated in the case of Cucurbita maxima trypsin inhibitors (CMTI-I and CMTI-III) by means of nuclear magnetic resonance (NMR) spectroscopy. pH titration, line widths, peak shapes, deuterium exchange kinetics, and two-dimensional nuclear Overhauser effect spectroscopy (NOESY) were employed to characterize a conformational change involving Tyr27, which was shown to be triggered by deprotonation of His25 around pH 6. A hydrogen bond is proposed to be formed between N epsilon of His25 and OH of Tyr27, as a distance between the atoms, His25 N epsilon and Tyr27 OH, of 3.02 A is consistent with a model built with NOE-derived distance constraints. Both the X-ray [Bode, W., Greyling, J.H., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 282-292] and NMR [Holak, T.A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] structures of CMTI-I at low pH (4.7-5.3) rule out such an interaction between the two aromatic rings, as the ring planes are oriented about 10 A away from each other. The presently characterized relative orientations of His25 and Tyr27 are of functional significance, as these residues make contact with the enzyme in the enzyme-inhibitor complex. Furthermore, trypsin assay and inhibitor-binding studies showed that conformations of trypsin and the squash inhibitor were functionally relevant only in the pH range 6-8. The pKa of His25 was determined and found to be influenced by Glu9/Lys substitution and by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731948 TI - Solution studies of staphylococcal nuclease H124L. 1. Backbone 1H and 15N resonances and secondary structure of the unligated enzyme as identified by three dimensional NMR spectroscopy. AB - The backbone 1H and 15N resonances of unligated staphylococcal nuclease H124L (recombinant protein produced in Escherichia coli whose sequence is identical to the nuclease produced by the V8 strain of Staphylococcus aureus) have been assigned by three-dimensional (3D) 1H-15N NOESY-HMQC NMR spectroscopy at 14.1 tesla. The protein sample used in this study was labeled uniformly with 15N to a level greater than 95% by growing the E. coli host on a medium containing [99% 15N]ammonium sulfate as the sole nitrogen source. The assignments include 82% of the backbone 1HN and 1H alpha resonances as well as the 15N resonances of non proline residues. Secondary structural elements (alpha-helices, beta-sheets, reverse turns, and loops) were determined by analysis of patterns of NOE connectivities present in the 3D spectrum. PMID- 1731949 TI - Solution studies of staphylococcal nuclease H124L. 2. 1H, 13C, and 15N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex. AB - Accurate 1H, 15N, and 13C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone 1H and 15N assignments, obtained by analysis of three-dimensional 1H-15N HMQC-NOESY data [Wang, J., Mooberry, E.S., Walkenhorst, W.F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear 1H NMR studies were at natural isotopic abundance or labeled randomly with 2H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with 15N (to an isotope level greater than 95%) or uniformly with 13C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single 15N- or 13C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [Wang, J., LeMaster, D. M., & Markley, J.L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A.P., Loh, S. N., & Markley, J.L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A.P., Loh, S.N., & Markley, J.L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds. PMID- 1731950 TI - Factors affecting gamma-chain multimer formation in cross-linked fibrin. AB - The major covalently linked multimolecular D fragments found in plasmic digests of factor XIIIa cross-linked fibrin formed under physiological pH and ionic strength conditions consist of D dimers, D trimers, and D tetramers. These fragments are linked by epsilon-amino-gamma-glutamyllysine bonds in the carboxy terminal regions of their gamma chains, which had originated in the cross-linked fibrin as gamma dimers, gamma trimers, and gamma tetramers, respectively. In this study, factors affecting the degree and rate of formation of these three classes of cross-linked gamma chains were determined by analyzing the D-fragment content of plasmic digests of cross-linked fibrin that had been sampled after all gamma chain monomers had been consumed in the cross-linking process. D trimers and D tetramers, expressed as a proportion of the total D-fragment content, both increased at the expense of the D-dimer population as a function of increasing factor XIII concentration, the time of cross-linking, or the CaCl2 concentration. Their levels decreased as the ionic strength was raised by NaCl addition. However, the ionic strength effect could be reversed by concomitantly raising the CaCl2 concentration. Digests of clots prepared from recalcified fresh citrated plasma also contained each type of cross-linked D fragment, and the proportion of D trimers and D tetramers in the digest increased with increasing clot incubation time. These results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1731951 TI - Aspartate 170 of the photosystem II reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster. AB - Eleven site-directed mutations were constructed at aspartate 170 of the D1 polypeptide of the photosystem II (PSII) reaction center of the cyanobacterium Synechocystis sp. PCC 6803. The light-saturated rates of O2 evolution (VO2) measured in whole cells range from close to that of wild-type for Asp170Glu to zero for Asp170Ser and Ala. Those mutant strains that are best able to evolve O2 are also those that show the lowest Km in PSII core complexes for the oxidation of Mn2+ by oxidized Tyr161, the normal oxidant of the Mn cluster responsible for O2 evolution. To a first approximation, the lower the pKa of the residue at position 170, the higher the VO2 and the lower the Km. D1-Asp170 appears to participate in the early steps associated with the assembly of the Mn cluster. It is also the first reported example of an amino acid residue critical to the function and assembly of the oxygen-evolving complex. PMID- 1731952 TI - The Society for Adolescent Medicine, annual meeting. Abstracts and program outline for presentations. March 18-22, 1992, Washington, D.C. PMID- 1731953 TI - Determination of free creatine and phosphocreatine concentrations in the isolated perfused rat heart by 1H- and 31P-NMR. AB - To measure free creatine in the isolated perfused rat heart, the concentration of phosphocreatine, and phosphocreatine plus creatine (sigma Cr) were measured by 31P- and 1H-NMR, respectively. Quantification was performed in the presence and absence of an intraventricular balloon filled with a known amount of PCr, which acted as an external standard. Total (free plus bound) phosphocreatine and creatine were measured by HPLC analysis of extracts from the same hearts, freeze clamped at the end of the perfusions. A greater concentration of creatine (mumol/g dry wt.) in the perfused rat heart was measured by HPLC analysis (40.3 +/- 2.38 (11)) as compared to NMR (34.6 +/- 1.95 (11)), whilst no significant difference was observed in the measurement of phosphocreatine between the two assay methods. Consequently, a greater sigma Cr was measured by HPLC. This work suggests that the majority of Cr in the heart is NMR visible and unbound, so available to interact with creatine kinase. The lower free ADP concentration calculated from NMR measurements (53.3 +/- 3.80 microM (9)) was not significantly different from that determined by HPLC analysis (56.9 +/- 5.90 microM (9)). This suggests that the concentration of free ADP in the heart is higher than values where it can regulate oxidative phosphorylation most effectively. PMID- 1731954 TI - Sex-specific differences in rabbit fetal lung maturation in response to epidermal growth factor. AB - Males and females exhibit different stages of lung development at the same gestation with males lagging behind. We hypothesized that one of the mechanisms responsible for the sex-specific difference in fetal lung maturation is a delay in the onset of epidermal growth factor (EGF) activity in the male fetal lung. EGF influences growth and differentiation during development. We studied the effects of EGF on the incorporation of glycerol into lamellar body disaturated phosphatidylcholine (DSPC) in sex-specific fetal rabbit lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + 10% stripped fetal calf serum with or without EGF (10 ng/ml). The incorporation of [1,3-14C]glycerol into lamellar body DSPC was assessed after 3, 5, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation of glycerol into DSPC over time in response to EGF treatment. Male lung explants prepared at 21 days did not respond to EGF treatment. In explants prepared at 24 days gestation, baseline glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants. EGF-responsiveness was also sex-specific in these more mature explants, with the male explants now responding to EGF with a consistent increase in the incorporation of glycerol into lamellar body DSPC. We conclude that one of the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity. PMID- 1731955 TI - Vascular oxidative metabolism under different metabolic conditions. AB - Control of respiration in vascular smooth muscle was examined while the metabolic state of the tissue was manipulated. During KCl-induced contractures in the presence of 5 mM glucose, oxygen consumption increased by 10 nmol/per min g without any decrease in phosphocreatine (PCr) or ATP as determined by 31P-NMR indicating a control of respiration which does not involve changes in high-energy phosphates (e.g., ADP, phosphorylation potential). However, when aortae with resting tone in the absence of substrate were then provided with 5 mM 2 deoxyglucose as the sole substrate, oxygen consumption increased 7.4 nmol/min per g while PCr decreased by more than 50% (resulting in a 2-fold increase in the calculated free ADP) with no change in tension from resting tone. During a subsequent KCl induced contracture in the presence of 2-deoxyglucose, oxygen consumption increased an additional 7.2 nmol/min per g while PCr continued to decline. Therefore, at least two mechanisms of respiratory control may exist in sheep aorta, one dependent and the other independent of changes in high-energy phosphates. PMID- 1731956 TI - Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation. AB - This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12 HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z) eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production. PMID- 1731957 TI - Uptake and intracellular handling of iron from transferrin and iron chelates by mitogen stimulated mouse lymphocytes. AB - The ability of lymphocytes to utilise iron from different sources has been investigated. Iron uptake from transferrin by proliferating lymphocytes gradually increased as saturation of the protein with iron was increased up to 100%, but rose sharply when addition of further iron resulted in the presence of non transferrin bound iron. Increasing the saturation of transferrin with iron caused an increased rate of proliferation up to about 100% saturation but when the level of iron present exceeded the binding capacity of the protein, proliferation decreased and at high levels of iron it was reduced below that seen in the absence of transferrin. Comparison of the degree of iron uptake from transferrin and from iron chelators showed that the hydrophilic chelator ferric nitrilotriacetate (FeNTA) donated larger amounts of iron to cells than did transferrin or the lipophilic chelator ferric-pyridoxal isonicotinoyl hydrazone (FePIH), but did not promote proliferation, and when present in high amounts caused inhibition. In contrast, FePIH supported proliferation as efficiently as transferrin. In cells cultured with FeNTA, iron was found predominantly in an insoluble form while in the cells cultured with Fe-transferrin or FePIH the largest proportion of iron was found in the non-ferritin high molecular weight fraction, which probably represents iron in enzymes and other metabolically important proteins. In no case did iron associated with ferritin exceed 15% of the total uptake, and the cells showed no marked increase in synthesis of ferritin in response to any of the forms of iron. These results indicate that different forms of iron are handled in different ways by lymphocytes, and that iron delivered from hydrophilic chelates may be toxic and not readily available for metabolic use. Lymphocytes appear to be poorly equipped to sequester excess iron in ferritin, and this may account for abnormalities in the immune system reported in patients with iron overload. PMID- 1731958 TI - Possible roles of arachidonic acid and its metabolites in induction of tissue plasminogen activator (t-PA) production in human fibroblast, IMR-90 cells by proteose peptone. AB - Proteose peptone (p.peptone) had an ability to induce tissue plasminogen activator (t-PA) production by human embryonic lung fibroblast, IMR-90 cells. We previously demonstrated that the induction was closely related to the activation of phospholipase A2 in the cells stimulated by p.peptone. In this report, we describe the involvement of arachidonate metabolism in the induction. The induction was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraenoic acid (ETYA), an inhibitor of both cycloxygenase and lypoxygenase, and also by nordihydroguaiaretic acid (NDGA), which in low concentrations selectively inhibits lipoxygenase. However, indomethacin, a specific inhibitor of cycloxygenase, had no effect on the induction. 5-hydroxyeicosatetraenoic acid (5 HETE), which is an arachidonate metabolite derived from lipoxygenase pathway, had an inductive effect, but prostaglandin E1 (PGE1), which is a metabolite from cycloxygenase pathway, had no effect on t-PA production by the cells. These results suggest that arachidonate metabolism is involved in the induction of t-PA production in IMR-90 cells by p.peptone, and that arachidonate metabolite(s) from lipoxygenase pathway is responsible for the induction. PMID- 1731959 TI - Hormonal regulation of glycosylation process in rat small intestine: responsiveness of fucosyl-transferase activity to hydrocortisone during the suckling period, unresponsiveness after weaning. AB - The aim of this study was to examine the possible role of certain hormones (especially hydrocortisone) in the developmental variations of intestinal fucosyl transferase activity in rats. Thyroxine and insulin, injected into suckling rats, did not induce significant modifications of the fucosyl-transferase activity, under the conditions used, whereas this enzyme activity was highly enhanced after administration of glucocorticoids (cortisone and hydrocortisone). Hydrocortisone administration to suckling rats induced a precocious and progressive activation of the fucosyl-transferase activity up to adult level as a function of the duration of treatment. The responsiveness of suckling rats to hydrocortisone, as shown by increased fucosyl-transferase activity, disappeared at the end of the third week (corresponding to the weaning time). These physiological periods of responsiveness and unresponsiveness to hydrocortisone could be related to the binding of the hormones to receptors since the antiglucocorticoid RU 38486 counteracted the effect of hydrocortisone in suckling rats but did not prevent the developmental rise of the fucosyl-transferase activity, when administered in the third week of life. These results suggest that the normal developmental rise of the fucosyl-transferase activity is independent of glucocorticoids. PMID- 1731960 TI - Differential effect of a new thyromimetic on triiodothyronine transport into myoblasts and hepatoma and neuroblastoma cells. AB - 3,5-Dibromo-3'-pyridazinone-L-thyronine (L-94901), a member of a novel class of thyromimetics, reduces cholesterol plasma levels with little effect on cardiac function in rats. Because receptor binding of L-94901 in isolated heart and liver nuclei is similar but binding in liver nuclei in vivo was 50-fold higher than in cardiac nuclei, we studied its effect on triiodothyronine (T3) transport across the plasma membrane of myoblasts, hepatoma cells and neuroblasts. Previously, we had demonstrated saturable, stereospecific and energy dependent transport of T3 into the three cell lines. After equilibrium of intact cells with hormone, nuclear binding of T3 was decreased by L-94901 in all three cell lines. While whole cell uptake and whole cell binding of T3 was only slightly affected by L 94901, kinetic analysis of the initial rate of uptake showed uncompetitive or noncompetitive inhibition and a differential decrease in Vmax. Furthermore, the Ki for the liver and brain derived cells was 10-fold lower than for the muscle derived cells. This effect on the plasma membrane transport of T3 may explain the differential effect reported in the intact animal. PMID- 1731961 TI - Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression. AB - Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a lambda gt10 library representing poly(A+) RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome. PMID- 1731962 TI - Specific transcription and reinitiation of 2,4-D-induced genes in tobacco nuclei. AB - In order to study the effects of trans-acting factors on cis-acting elements in plant genes an in vitro reinitiating nuclear transcription system is needed. Here we report that run-on experiments with nuclei isolated from 2,4-D-treated auxin starved early-stationary-phase cells of tobacco clearly show reitintiation of transcription of specific 2,4-D-induced genes. Using gamma-thio nucleotides and [alpha-32P]-UTP we were able to demonstrate the presence of reinitiated labelled specific RNAs after isolation on a mercury-agarose affinity column. Addition of heparin as an inhibitor blocked this reinitiation. In a primer extension assay we found that the new transcripts initiate at approximately the same site as used in vivo. PMID- 1731963 TI - cDNA and derived amino acid sequence of a cytosolic Cu,Zn superoxide dismutase from Arabidopsis thaliana (L.) Heyhn. PMID- 1731964 TI - cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana. AB - The DNA sequences of cDNAs for two cor (cold-regulated) genes of Arabidopsis thaliana L. (Heyn) were determined. One cDNA (approximately 70% full-length) corresponds to a cor gene, designated cor47, that encodes a 47 kDa hydrophilic polypeptide. The data indicate that COR47 has amino acid sequence homology with Group II LEA (late embryogenesis abundant) proteins, a class of proteins that accumulate late in embryo development. DNA sequence analysis of a second cDNA (containing the complete protein coding sequence) indicates that it represents a cor gene, designated cor6.6, that encodes an alanine-rich 6.6 kDa hydrophilic polypeptide. COR6.6 is almost identical to KIN1, a cold-regulated Arabidopsis gene that has been suggested to have amino acid sequence similarities with type I fish antifreeze proteins (S. Kurkela, M. Franck, Plant Mol Biol 15: 137-144, 1990). Northern analysis indicated that transcripts for cor47 and cor6.6 do not accumulate to high levels in late-developing embryos or fresh mature seeds as is typical of lea gene transcripts. The similarities and differences between COR and LEA proteins are discussed as are their possible roles in freezing and drought tolerance. PMID- 1731965 TI - Nucleotide sequence and expression of the ndhH gene of the cyanobacterium Synechocystis sp. PCC6803. PMID- 1731966 TI - Two GC-rich DNA elements of Chlamydomonas reinhardtii with complex arrangements of directly repeated sequence motifs. PMID- 1731967 TI - PCR cloning of the full-length cDNA for the seed protein canavalin from the jack bean plant, Canavalis ensiformis. PMID- 1731968 TI - A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice. AB - A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds. PMID- 1731969 TI - Analysis of cis-regulatory elements involved in induction of a tobacco PR-5 gene by virus infection. AB - cis-Regulatory elements involved in tobacco mosaic virus (TMV)-inducible expression were identified in a tobacco PR-5 gene, encoding an acidic thaumatin like protein. By fusing upstream sequences of the PR-5 gene to the GUS reporter gene and analysing transgenic plants containing these fusions for local and systemic induction of GUS activity by TMV, it was found that sequences between 1364 and -718 are involved in TMV induction of PR-5 gene expression. PMID- 1731970 TI - Isolation and transcriptional competence of three tRNA(Trp) genes from Arabidopsis thaliana L. PMID- 1731971 TI - Structure and expression of a root-specific rice gene. AB - Two rice cDNA clones (COS6 and COS9) were isolated, corresponding to genes that were highly expressed in roots from seedlings and mature plants. A genomic clone (GOS9) corresponding to cDNA clone COS9 was isolated and the intron/exon structure was determined by comparing the nucleotide sequences of the mRNA and the genomic clone. 5' ends and 3' ends of the mRNA were determined by primer extension and S1-nuclease mapping respectively. The open reading frame present in GOS9 potentially encodes a protein (14 kDa) that does not show any significant homology to other proteins in databases. PMID- 1731972 TI - Cloning of a low-temperature-induced gene lti2 from the cyanobacterium Anabaena variabilis M3 that is homologous to alpha-amylases. AB - A gene called lti2 which is induced by a shift to a low temperature was isolated from the cyanobacterium Anabaena variabilis strain M3. This gene contained an open reading frame of 552 amino acid residues which exhibited homology to known alpha-amylases. The level of lti2 transcript increased 40-fold within an hour after a temperature shift from 38 to 22 degrees C and then slowly decreased to a low steady-state level. A less extensive expression of the lti2 gene is also induced by a temperature shift from 22 to 38 degrees C. PMID- 1731973 TI - Cloning and characterization of a cDNA encoding a sulfur-rich coixin. AB - A full-length cDNA clone encoding a sulfur-rich Coix prolamin was isolated using a cDNA library constructed from polysomal mRNA prepared from immature Coix endosperm. The deduced amino acid sequence of the cDNA clone predicted a polypeptide of 194 residues, which shared 64% homology with the 17 kDa beta-zein. The mature protein contains the familiar composition of the prolamins and an unusually high content of the sulfur-containing amino acids methionine (11.6%) and cysteine (5.2%). In vitro transcription followed by in vitro translation of the coding region of the pBCX17.9S clone gave rise to a polypeptide with an apparent molecular weight corresponding to the C4 alpha-coixin. Hydropathy analysis showed that C4 alpha-coixin is slightly more hydrophobic than beta-zein. PMID- 1731974 TI - The nucleotide sequence of the rDNA intergenic spacer region in a wild species of the genus Vicia, V. angustifolia. PMID- 1731975 TI - Characterisation of PHSP1, a cDNA encoding a mitochondrial HSP70 from Pisum sativum. AB - A pea cDNA clone, PHSP1, encoding a member of the HSP70 gene family has been isolated. DNA sequence analysis indicates that the protein encoded by PHSP1 is a homologue of the mitochondrial HSP70 proteins, SSP1 from Schizosaccharomyces pombe and SSC1 from S. cerevisiae. It contains an amino-terminal extension of 50 amino acids, rich in basic and hydroxyl amino acids, similar to other plant mitochondrial leader sequences. Western blot analysis indicates that the PHSP1 protein is associated only with mitochondria and not with any other sub-cellular organelle or cytoplasm. Further confirmation of its location within mitochondria was obtained from in vitro protein translocation experiments into purified Pisum sativum mitochondria. It was observed that the precursor protein was efficiently imported and that it is processed to produce a protein with an Mr of the anticipated size of the mature protein. Results are discussed with respect to the structure and function of the mitochondrial HSP70 protein. PMID- 1731977 TI - A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco. AB - When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PR1b protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells. PMID- 1731976 TI - Structure of a rice beta-glucanase gene regulated by ethylene, cytokinin, wounding, salicylic acid and fungal elicitors. AB - A rice beta-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots express Gns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogen Sclerotium oryzae or from the non-pathogen Saccharomyces cereviseae. Shoots also express Gns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The beta glucanase shows 82% amino acid similarity to the barley 1,3;1,4-beta-D glucanases, and from hybridization studies it is the beta-glucanase gene in the rice genome closest to the barley 1,3;1,4-beta-glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G + C in the third position of the codon in the mature peptide coding region, but only 61% G + C in the signal peptide region. PMID- 1731978 TI - Nitrate reductase transcript is expressed in the primary response of maize to environmental nitrate. AB - The nitrate induction of NADH:nitrate reductase mRNA in maize roots, scutella and leaves was investigated in the presence and absence of inhibitors of protein synthesis. In the absence of inhibitors, nitrate treatment caused a fairly rapid (2 to 3 h) increase in the level of the nitrate reductase transcript in all tissues. When cytoplasmic protein synthesis was inhibited by cycloheximide, nitrate reductase mRNA was induced by nitrate in all tissues to levels equal to or greater than those found with nitrate treatment alone. Treatment of maize tissues with cycloheximide in the absence of nitrate had only a small effect on the accumulation of the nitrate reductase mRNA. Inhibition of organellar protein synthesis with chloramphenicol also had little or no effect on nitrate-induced nitrate reductase mRNA accumulation in roots and scutella, but did appear to partially inhibit appearance of transcript in leaves. Excision of scutella in the absence of nitrate was sufficient to cause some accumulation of the nitrate reductase transcript. Since cytoplasmic protein synthesis was not required for expression of nitrate reductase transcripts, induction of these transcripts by nitrate is a primary response of maize to this environmental signal. Thus, it appears that the signal transduction system mediating this response is constitutively expressed in roots, scutella and leaves of maize. PMID- 1731979 TI - Activation of a truncated PR-1 promoter by endogenous enhancers in transgenic plants. AB - PR-1 genes are induced by various environmental stimuli such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5' flanking regions of the PR-la gene and of two PR-1 pseudogenes were joined by a transcriptional fusion to the Escherichia coli beta glucuronidase (GUS) gene. These constructs were stably integrated into the tobacco genome and independent primary transformants were monitored for the expression of the reporter gene. Unexpectedly, out of 55 transformants analysed, four plants exhibited considerable GUS activities without any inductive treatment of the plants. Expression of the endogenous PR-1 genes, however, could not be detected in these plants. Primer extension analyses revealed correct initiation of the PR1/GUS hybrid transcripts from the PR-1a TATA box. When the plants were analysed at the cellular level, clear differences regarding the tissue specificity of expression of the reporter gene were observed. These results strongly suggest that the PR1/GUS hybrid promoter expression cassettes may be activated when integrated in the vicinity of heterologous enhancer elements dispersed in the tobacco genome. In order to support this hypothesis, domain B of the enhancer of the 35S RNA promoter from cauliflower mosaic virus (CaMV) was fused to various PR1/GUS hybrid genes upstream as well as downstream from the RNA start site. These constructs were stably introduced into the tobacco genome. In any primary transformant analysed, strong GUS activities were observed with the PR1/GUS hybrid RNAs originating from the normal transcription start site of the PR-1a gene. The tissue specificity of gene expression was identical to that described previously for the CaMV 35S domain B enhancer element. Thus, modulations of the transcriptional activity of the PR-1 promoter can be achieved by heterologous enhancers in transgenic plants and may be encountered upon random integration of PR-1 promoter constructs into the tobacco genome. PMID- 1731980 TI - Nucleotide sequence of atpB, rbcL, trnR, dedB and psaI chloroplast genes from a fern Angiopteris lygodiifolia: a possible emergence of Spermatophyta lineage before the separation of Bryophyta and Pteridophyta. AB - To elucidate the evolutionary relationship between the Spermatophyta, Pteridophyta and Bryophyta, we cloned a fragment of chloroplast DNA from the fern Angiopteris lygodiifolia (Pteridophyta) and determined its nucleotide sequence. The fragment contained the atpB, rbcL, trnR-CCG, dedB and psaI genes. Comparisons of the deduced amino acid and nucleotide sequences of these genes from the three plant groups indicate that Angiopteris sequences are more closely related to those of Bryophyta species (85% identity on average) than to those of seed plants (76% identity on average), supporting a hypothesis that the Bryophyta and Pteridophyta diverged more recently from one another than their common progenitor diverged from that of the Spermatophyta. PMID- 1731981 TI - Structural organization of the chloroplast genome of the chromophytic alga Vaucheria bursata. AB - The chloroplast genome of the chromophytic alga Vaucheria bursata has been characterized by restriction site and gene mapping analysis. It is represented by a circular molecule 124.6 kb in size. An inverted sequence duplication (IR) not larger than 5.85 kb carries the rRNA genes and separates two single-copy regions of 64.6 kb and 48.3 kb from one another. The Vaucheria plastid genome exists in two equimolar isomers which is due to intramolecular flip-flop recombination within the IR sequences. The coding sites for 21 structural and soluble proteins have been mapped on both single-copy regions using heterologous gene sequences as probes. Although the overall gene order is found to be rearranged when compared with other chromophytic algal and land plant chloroplast genomes, most of the transcriptional units of cyanobacteria and land plant chloroplast genomes appear to be conserved. The phylogenetic implications of these findings are further discussed. PMID- 1731982 TI - Existence of two histone H3 variants in dicotyledonous plants and correlation between their acetylation and plant genome size. AB - Histone H3 proteins were purified to near homogeneity from callus cultures of dicotyledonous plants alfalfa, soybean, Arabidopsis, carrot and tobacco to determine the number of histone H3 variants. In every species two histone H3 variants were identified by gradient gel electrophoresis and reversed-phase chromatography. They were named H3.1 and H3.2 in order of increasing mobility in acid-urea-Triton gels. Co-electrophoresis of histone H3.2 proteins of all species in this gel system and HPLC co-chromatography suggest that all histone H3.2 variants have a primary protein sequence identical to alfalfa H3.2. Two distinct H3.1 variant forms were identified, represented by alfalfa and Arabidopsis H3.1 proteins which differ only at residue 90. Soybean H3.1 resembles H3.1 of alfalfa. Carrot and tobacco H3.1 appear identical to the Arabidopsis H3.1 histone variant. All H3 proteins were acetylated to multiple levels and in each plant the histone H3.2 forms were more highly acetylated. An inverse relationship was observed between plant genome size and the relative abundance of histone variant H3.2 and also with the level of acetylation of both histone H3 variants. This correlation matches the general tendency that in plants with smaller genomes a larger fraction of the genome is transcriptionally active. PMID- 1731983 TI - Segregation of transgenes in maize. AB - Progeny recovered from backcrossed transgenic maize tissue culture regenerants (R0) were analyzed to determine the segregation, expression, and stability of the introduced genes. Transgenic A188 x B73 R0 plants (regenerated from embryogenic suspension culture cells transformed by microprojectile bombardment; see [9]) were pollinated with nontransformed B73 pollen. Inheritance of a selectable marker gene, bar, and a nonselectable marker gene, uidA, was analyzed in progeny (R1) representing four independent transformation events. Activity of the bar gene product, phosphinothricin acetyltransferase (PAT), was assessed in plants comprising the four R1 populations. The number of R1 plants containing PAT activity per total number of R1 plants recovered for each population was 2/7, 19/34, 3/14 and 73/73. Molecular analysis confirmed the segregation of bar in three R1 populations and the lack of segregation in one R1 population. Cosegregation analysis indicated genetic linkage of bar and uidA in all four R1 populations. Analysis of numerous R2 plants derived from crossing transformed R1 plants with nontransformed inbreds revealed 1:1 segregation of PAT activity in three of four lines, including the line that failed to segregate in the R1 generation. Integrated copies of bar in one line appeared to be unstable or poorly transmitted. PMID- 1731984 TI - Dissection of a pollen-specific promoter from maize by transient transformation assays. AB - We have previously reported the isolation and characterization of a gene (Zm13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm13 5' flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5' regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the beta-glucuronidase (GUS) gene under the control of various sized fragments of the Zm 13 5' flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from -100 to -54, while other sequences which amplify that expression reside between -260 and -100. The replacement of the normal terminator with a portion of the Zm13 3' region containing the putative polyadenylation signal and site also increased GUS expression. While the -260 to -100 region contains sequences similar to other protein-binding domains reported for plants, the -100 to -54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression. PMID- 1731985 TI - In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5 bisphosphate carboxylase small subunit RNAs. AB - A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo. The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5 bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules. PMID- 1731986 TI - Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. AB - We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations. PMID- 1731987 TI - Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A. AB - Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile. PMID- 1731988 TI - Synthesis and assembly of soybean beta-conglycinin in vitro. AB - The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2 terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta conglycinin subunits. The results also show that structural changes at the NH2 terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality. PMID- 1731989 TI - An aberrant plastid ribosomal RNA gene cluster in the root parasite Conopholis americana. AB - The plastid ribisomal RNA (rRNA) operon of the achlorophyllous root parasite Conopholis americana was completely sequenced. Full-length rRNA genes are retained in the gene cluster, but significant divergence has occurred in the 16S, 23S and 5S genes. Both the 16S-23S intergenic spacer and the 4.5S-5S intergenic spacer have suffered substantial deletions, including the two tRNA genes typically found in prokaryotic and plastid 16S-23S spacers. PMID- 1731991 TI - Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.). AB - A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/ ) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F2 progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F2 progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut. PMID- 1731990 TI - Characterization of a Euglena gene encoding a polyprotein precursor to the light harvesting chlorophyll a/b-binding protein of photosystem II. AB - A 7.4 kb segment of a Euglena nuclear gene encoding a portion of the polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) has been isolated and sequenced. Nine exons can be assembled into a continuous open reading frame encoding 113 amino acids of the C-terminus of an LHCPII, followed by 4 complete LHCPIIs. The intron-exon junctions required to maintain maximum amino acid sequence homology between the LHCPIIs encoded by the Euglena genomic clone and Arabidopsis LHCPII do not conform to the consensus splice site sequences present in most eukaryotic organisms. Three types of LHCPIIs are contained within the polyprotein precursor. There is greater than 90% sequence identity at both the protein and nucleic acid level within a type while between types, there is less than 65% sequence identity. All Euglena LHCPIIs contain three hydrophobic membrane-spanning domains in the same positions as found in other LHCPIIs. Hybridization of genomic Southern blots with a probe encoding LHCPII suggests that the Euglena genome encodes a large number (30-50) of LHCPIIs. A probe derived from the 3' end of the sequenced genomic clone hybridizes with equal intensity to 2-3 genomic fragments on Southern blots. The probe encoding LHCPII hybridizes to RNAs of 9.5 and 6.6 kb on northern blots of total RNA while the 3'-end probe hybridizes only to the 6.6 kb RNA. The 6.6 kb LHCPII band detected on northern blots appears to be composed of transcription products derived from 2-3 LHCPII genes. Euglena appears to be unique in that a large number of LHCPIIs are encoded by a small number (3-5) of polyprotein genes. PMID- 1731992 TI - Organization and expression of the nuclear gene coding for the plastid-specific S22 ribosomal protein from spinach. AB - We report here on the genomic organization and expression of a nuclear gene coding for a plastid ribosomal protein. The gene encodes the plastid-specific ribosomal protein S22 (formerly named CS-S5). Southern blot analysis suggests that the gene is present in one copy in the spinach genome. The gene consists of 5 exons of sizes ranging from 108 to 273 bp and of 4 introns of 1410, 92, 386 and 82 bp. The exon-intron splice junctions and intron branch sites fit well the consensus sequences for plant introns. The major transcription start site has been determined 29 bp upstream of the AUG initiation codon by primer extension and S1 nuclease mapping. No canonical TATA box is found but some other possible promoter motifs are observed. Transcripts are detected in leaves, etiolated leaves, roots and seeds suggesting that the rps22 gene is expressed constitutively. During germination a marked increase in the relative steady-state level of the mRNA can be seen as soon as 24 h after imbibition of the seeds. PMID- 1731993 TI - Reduced PAL gene suppression in Verticillium-infected resistant tomatoes. AB - In tomato, resistance to the wilt fungus Verticillium albo-atrum is determined primarily by the Ve locus. When two tomato near-isolines which differ at this locus and in their susceptibility to the pathogen were compared, more rapid suberin coating in the xylem of resistant plants correlated closely with a more rapid increase in the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), an enzyme which is essential to the suberization process. In contrast, levels of mRNA did not increase proportionally to the measured enzyme activities; rather, there was a substantial suppression of mRNA levels in the susceptible tomato line, consistent with a much lower elevation of PAL activity and significantly less vascular coating. The suppression was absent or substantially reduced in the resistant line. The results indicate that the pathogen can suppress defense genes in susceptible plants but suggest that their expression is altered in resistant hosts and that post-transcriptional regulation plays a significant role. PMID- 1731994 TI - The tomato nia gene complements a Nicotiana plumbaginifolia nitrate reductase deficient mutant and is properly regulated. AB - A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of functional nia transcript and protein was restored for NR activity by transformation with a cloned tomato nia gene. The transgenic plants expressed from undetectable to 17% of the control NR activity in their leaves. Restoration of growth rates comparable to the wild type was obtained for transgenic plants expressing as little as 10% of the wild-type activity showing that nitrate reduction is not a growth-limiting factor in the wild-type plant. The analysis of the transgene expression showed that the tomato nia gene transcription was regulated by light, nitrate and a circadian rhythm as in tomato plants. These results suggest that all the cis-acting sequences involved in these regulations are contained in the 3 kb upstream region of the tomato nia gene and are still functional in transgenic N. plumbaginifolia plants. The amount of NR transcript synthesized from the tomato nia gene was reduced when a functional N. plumbaginifolia nia locus was introduced by sexual crosses. These data support the hypothesis that nitrate reduction is regulated by nitrate-derived metabolites as demonstrated in fungi. PMID- 1731995 TI - Molecular cloning of an 1-aminocyclopropane-1-carboxylate synthase from senescing carnation flower petals. AB - Synthetic oligonucleotides based on the sequence of 1-aminocyclopropane-1 carboxylate (ACC) synthase from tomato were used to prime the synthesis and amplification of a 337 bp tomato ACC synthase cDNA by polymerase chain reaction (PCR). This PCR product was used to screen a cDNA library prepared from mRNA isolated from senescing carnation flower petals. Two cDNA clones were isolated which represented the same mRNA. The longer of the two clones (CARACC3) contained a 1950 bp insert with a single open reading frame of 516 amino acids encoding a protein of 58 kDa. The predicted protein from the carnation ACC synthase cDNA was 61%, 61%, 64%, and 51% identical to the deduced proteins from zucchini squash, winter squash, tomato, and apple, respectively. Genomic DNA gel blot analysis indicated the presence of at least a second gene in carnation which hybridized to CARACC3 under conditions of low stringency. ACC synthase mRNA accumulates during senescence of carnation flower petals concomitant with the increase in ethylene production and ACC synthase enzyme activity. Ethylene induced the accumulation of ACC synthase mRNA in presenescent petals. Wound-induced ethylene production in leaves was not associated with an increase in ACC synthase mRNA represented by CARACC3. These results indicate that CARACC3 represents an ACC synthase transcript involved in autocatalytic ethylene production in senescing flower petals. PMID- 1731996 TI - The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family. AB - A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb. PMID- 1731997 TI - Nucleotide sequence of a high-pI rice (Oryza sativa) -amylase gene. PMID- 1731998 TI - Genomic RNA sequence of turnip yellow mosaic virus isolate TYMC, a cDNA-based clone with verified infectivity. PMID- 1731999 TI - DNA sequence of the tomato fruit expressed proline-rich protein gene TPRP-F1 reveals an intron within the 3 untranslated transcript. PMID- 1732000 TI - Nucleotide sequence of a region of maize chloroplast DNA containing the 3' end of clpP, exon 1 of rps12 and rpl20 and their cotranscription. PMID- 1732001 TI - Nucleotide sequence, map position and transcript pattern of the intron-containing gene for maize chloroplast ribosomal protein S16. PMID- 1732002 TI - Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect alpha-amylase. AB - The primary structure of the insect alpha-amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal peptide of 25 amino acids. Ala and Leu account for 55% of the signal peptide. CMa is 60-85% identical with alpha-amylase inhibitors of wheat, but shows less than 50% identity to trypsin inhibitors of barley and wheat. The 10 Cys residues are located in identical positions compared to the cereal inhibitor family with a Pro-X-Cys motif present in all. PMID- 1732003 TI - Reassessing the hemostatic disorder associated with acute promyelocytic leukemia. PMID- 1732004 TI - The mutation Arg (53)----Trp causes von Willebrand disease Normandy by abolishing binding to factor VIII. Studies with recombinant von Willebrand factor. AB - von Willebrand factor (vWF) and factor VIII (FVIII) circulate in plasma as a noncovalently linked protein complex. The FVIII/vWF interaction is required for the stabilization of procoagulant FVIII activity. Recently, we reported a new variant of von Willebrand disease (vWD) tentatively named "Normandy," characterized by plasma vWF that appears to be structurally and functionally normal except that it does not bind FVIII. Three patients from one family were found to be homozygous for a C----T transition at codon 816 converting Arg 53 to Trp in the mature vWF subunit. To firmly establish a causal relationship between this missense mutation and vWD Normandy phenotype, we have characterized the corresponding recombinant mutant vWF(R53W). Expressed in COS-7 cells or CHO cell lines, normal vWF and vWF(R53W) were processed and formed multimers with equal efficiency. However, vWF(R53W) exhibited the same defect in FVIII binding as did plasma vWF from patients with vWD Normandy, confirming that this mutation is responsible for the vWD Normandy phenotype. These results illustrate the importance of Arg 53 of the mature vWF subunit for the binding of FVIII to vWF, and identify an amino acid residue within a disulfide loop not previously known to be involved in this interaction. PMID- 1732005 TI - Blood flow to bone marrow during development of anemia or polycythemia in the rat. AB - We applied the radioactive microsphere method to follow the magnitude and time course (0 to 96 hours) of blood flow changes during development and recovery from anemia in awake rats. Blood flow was also monitored during a 96-hour period after polycythemia was induced (erythropoietin administered subcutaneously [SC]). The possible influence of innervation was also examined. After a blood loss of approximately 50% (hypovolemia), blood flow to the femoral marrow tripled within 12 hours and remained elevated for the entire 96-hour period. The relative increase in blood flow to the femoral bone was even greater. Similar findings were obtained in rats with phenylhydrazine (PHZ) hemolytic anemia (normovolemia). Denervation had no detectable effect on the increased blood flow to either marrow or bone. The augmented blood flow during hemolytic anemia was accompanied by a doubling of the oxygen consumption rate by the marrow, while the glucose uptake was not detectably altered. Erythropoietin supplements (3 x 1,000 IU/kg, SC, 6 hour intervals) increased blood flow to the marrow by approximately 25% after 48 hours, and at 72 hours the blood flow had reached a value twice that obtained under control conditions. These results indicate that blood flow to bone marrow is highly variable and hormonally and/or locally regulated. This may have practical consequences for marrow transplantation technology and for administration of drug therapy to patients with insufficient bone marrow hematopoiesis. PMID- 1732006 TI - A-T-rich scaffold attachment regions flank the hematopoietic serine protease genes clustered on chromosome 14q11.2. AB - We have analyzed approximately 70 kb of the chromosome 14q11.2 hematopoietic serine protease gene cluster for the presence of nuclear scaffold attachment regions (SARs). At least 12 potential attachment sites were identified. SARs are present on both sides of the CGL-1/CSP-B and CGL-2/CCP-X genes and upstream from the cathepsin G (CG) gene. We have further characterized the SARs immediately flanking the cytotoxic lymphocyte-specific CGL-1/CSP-B gene. These 5' and 3' SARs are highly A-T-rich, contain multiple attachment sites, and are associated with the scaffolds of nuclei derived from both lymphoid and erythroid cell lines. These SARs contain multiple consensus elements frequently associated with A-T rich sequences, including the vertebrate topoisomerase II (topo II) consensus sequence, the A-box and T-box elements, and the yeast autonomous replicating sequence (ARS). The potential role for the nuclear scaffold in the transcriptional regulation of CGL-1/CSP-B expression is discussed. PMID- 1732007 TI - Transforming growth factor beta inhibits megakaryocyte growth and endomitosis. AB - Using a rat bone marrow culture system, the effect of transforming growth factor beta 1 (TGF beta 1) on megakaryocyte growth and endoreduplication has been studied. Purified human platelet TGF beta 1 inhibited the number of megakaryocytes that appeared in culture at a half-maximal concentration of 0.66 +/- 0.21 ng/mL and inhibited megakaryocyte endoreduplication at a half-maximal concentration of 0.14 +/- 0.08 ng/mL. Under identical conditions, growth of erythroid precursors was half-maximally inhibited at a concentration of 0.125 ng/mL while myeloid growth was not inhibited at concentrations of TGF beta 1 up to 25 ng/mL. These profound inhibitory effects on megakaryocyte growth and endomitosis suggested that TGF beta might play a role in megakaryocytopoiesis. Therefore, we explored the effect of TGF beta in three different experimental situations by using a neutralizing antibody to TGF beta: (1) Serum but not plasma was found to inhibit the number and ploidy of megakaryocytes that grew in vitro. This inhibitory activity was completely neutralized by antibody to TGF beta or on treatment with dithiothreitol. (2) Plasma from thrombocytotic rats was observed to decrease megakaryocyte ploidy on culture but this effect was not prevented by the addition of antibody to TGF beta. (3) Plasma from thrombocytopenic but not normal rats increased megakaryocyte ploidy on culture. Addition of antibody to TGF beta did not alter these results. Therefore, TGF beta is a potent inhibitor of the number and ploidy of megakaryocytes and accounts for all the inhibition seen when megakaryocytes are cultured in serum. However, the differences in effect on megakaryocyte growth that we observe between normal, thrombocytopenic, and thrombocytotic plasmas are not due to variations in the amount of TGF beta. Furthermore, our results show that release of TGF beta from megakaryocytes during culture does not act as an autocrine regulator of megakaryocyte ploidy in vitro. PMID- 1732008 TI - Induced differentiation of HL-60 promyelocytic leukemia cells to monocyte/macrophages is inhibited by hydroquinone, a hematotoxic metabolite of benzene. AB - Chronic exposure of humans to benzene has been shown to have a cytotoxic effect on hematopoietic progenitor cells in intermediate stages of differentiation, which can lead to aplastic anemia and acute myelogenous leukemia. We studied the effect of hydroquinone (HQ), a toxic metabolite of benzene found in the bone marrow, on the human promyelocytic leukemia cell line (HL-60), which can be induced to differentiate to both monocyte and myeloid cells, and thus has been used as a surrogate for a granulocyte/macrophage progenitor cell. Exposure of HL 60 cells to noncytotoxic concentrations of HQ for 3 hours before induction with phorbol myristate acetate (TPA) caused a dose-dependent inhibition of the acquisition of characteristics of monocytic differentiation, such as adherence, nonspecific esterase (NSE) activity, and phagocytosis, but had no effect on cell proliferation. HQ appeared to be affecting maturation beyond the monoblast/promonocyte stages. HQ also prevented differentiation induced by 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3]; however, the block occurred after the acquisition of adherence. HQ at concentrations that inhibited monocytic differentiation had no effect on differentiation to granulocytes, suggesting that the block in the differentiation of these bipotential cells is a step unique to the monocytic pathway. HQ was unable to prevent differentiation induced by the macrophage-derived cytokine, interleukin (IL)-1, a differentiation factor for cells of the monocytic lineage. PMID- 1732009 TI - Tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator mediated proteolysis of extracellular matrix and its antagonism by gamma-interferon. AB - Tumor necrosis factor (TNF) has a profound capacity to alter the endothelial cell phenotype that includes morphologic and functional changes that may be central for proinflammatory processes. Recent observations have indicated that TNF can promote the synthesis and secretion of urokinase plasminogen activator (uPA) in low passage human endothelial cells that normally release little uPA. In this report we have confirmed and expanded upon these initial observations in human endothelial cells and describe the ability of gamma-interferon (gamma-IFN) to inhibit TNF-induced uPA synthesis and secretion in a dose-dependent manner (0.025 to 25 ng/mL). Analysis of cell-free conditioned medium derived from gamma-IFN treated cultures by micro-enzyme-linked immunosorbent assay (ELISA) methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies (MoAbs) indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography is a direct result of a decrease in extracellular uPA antigen and is not a consequence of increased PAI-1 antigen. These findings are supported by Northern blot analyses that indicate that gamma-IFN treatment of endothelial cells resulted in a decreased steady state level of uPA messenger RNA (mRNA) with no measurable change in PAI-1 mRNA. This inhibitory response is specific for gamma-IFN because alpha-IFN fails to elicit a similar inhibitory response. In addition, TNF augmented extracellular proteolysis of radiolabeled subendothelial extracellular matrix (ECM) in a dose-dependent manner. The observed increase in ECM degradation mediated by TNF treatment of endothelial cells was dependent on the presence of plasminogen and could be inhibited by an anticatalytic uPA MoAb implying the requirement of proteolytically active uPA in this process. gamma-IFN (25 ng/mL) treatment of endothelial cells antagonized TNF-promoted degradation of radiolabeled ECM at a concentration that completely inhibited TNF-mediated uPA expression and activity. In addition, endothelial cells treated with TNF (18 hours) displayed the ability to invade ECM and reorganize individual cells into tube-like structures that were not evident in untreated control cultures when grown on Matrigel-coated culture dishes. gamma-IFN treatment of endothelial cells propagated on Matrigel was observed to inhibit TNF-mediated ECM invasion and tube formation at concentrations that were analogous to those required for the inhibition of uPA expression and activity. In summary, these observations suggest a novel homeostatic control mechanism for endothelial cell regulation of subendothelial ECM degradation promoted by TNF and inhibited by gamma IFN.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732010 TI - Elevated serum levels of interleukin-5 in patients with the syndrome of episodic angioedema and eosinophilia. AB - The syndrome of episodic angioedema and eosinophilia is characterized by cyclic edema, marked peripheral blood eosinophilia, and eosinophil degranulation in the dermis. Using a sensitive immunoenzymetric method, we measured serum interleukin (IL)-5 levels in four patients with this syndrome. We also determined the percentage of activated T cells in the peripheral blood of a new patient before and during an attack. In the patient presented, IL-5 levels peaked several days before maximal eosinophilia and then declined. This patient's lymphocytes showed an increased percentage, 28% (normal 2% to 3%), of activated T cells staining for both CD3 and HLA-DR 10 days before maximal eosinophilia, but no increase at the time of peak eosinophilia. In serum from three previously reported cases, elevated serum IL-5 levels were found during attacks. After glucocorticoid administration, IL-5 levels became undetectable in three of the four patients. Production of IL-5 is likely an important determinant of the pathophysiology of this syndrome. PMID- 1732011 TI - Effects on leukocytes after injection of tumor necrosis factor into healthy humans. AB - Tumor necrosis factor (TNF) has been implicated as a proximal mediator of the septic syndrome. To evaluate the possible role of TNF in leukocyte activation in septicemia, we performed a cross-over saline-controlled study in six healthy men who were intravenously injected with recombinant human TNF (50 micrograms/m2), and analyzed changes in circulating white blood cells and parameters for neutrophil and monocyte activation. TNF elicited a very rapid neutropenia, reaching a nadir after 15 minutes, followed by a neutrophilia. Lymphocytes showed a sustained decrease, whereas monocytes declined transiently. TNF injection was also associated with neutrophil activation, as reflected by a mean fivefold increase in the plasma concentrations of elastase-alpha 1-antitrypsin complexes and a mean sevenfold increase in plasma lactoferrin levels. Serum neopterin, a marker of monocyte activation, was significantly increased 24 hours after the administration of TNF. These changes occurred in the absence of detectable complement activation, as indicated by unchanged C3a-desarg plasma values. Serum interleukin-6 showed a nearly 40-fold increase after TNF injection, whereas interleukin-1 remained undetectable throughout. We conclude that the systemic release of TNF, triggered early after invasive infection, may be involved in the alterations in circulating leukocyte numbers and in the activation of leukocytes, during the development of the septic syndrome. PMID- 1732012 TI - Correlation of cytogenetic patterns and clinicobiological features in adult acute myeloid leukemia expressing lymphoid markers. AB - Cytogenetic, biomolecular, and clinicopathologic features were retrospectively studied in 34 adult patients with acute myelogenous leukemia expressing one or more of the following lymphoid-associated markers (LMs): CD7, CD2, CD10, CD19, CD22, TdT. Six patients showed 11q23 rearrangements (group I); three patients had the classic Ph chromosome (group II); 15 patients had aberrations of the myeloid type (group III), including four patients with structural aberrations of 13q or trisomy 13, three patients with 7q and 1q anomalies, and two patients with trisomy 11q. Ten patients had a normal karyotype (group IV). Anomalies exclusively associated with lymphoid malignancies were not seen. Ig H and/or T cell receptor genes were found to be rearranged in 50% and 66% of patients in cytogenetic groups I and II, respectively, versus 8% in group III and 12% in group IV. Likewise, more than one LM was more frequently detected in groups I and II. In group III, two of four patients with aberrations of chromosome 13 expressed two or more lymphoid features. Clinically, patients belonging to cytogenetic groups I and II were generally young, presented with a high white blood cell (WBC) count, and had a low complete remission rate. Survival in Ph chromosome-positive cases was uniformly short. We conclude that although there is no cytogenetic anomaly specifically associated with acute myelogenous leukemia expressing LM, a Morphologic, Immunologic, and Cytogenetic classification may constitute a working basis for further studies aimed at a better definition of clinicopathologic features and optimal treatment strategies for these leukemias. PMID- 1732013 TI - Immunovisualization of high (HK) and low (LK) molecular weight kininogens on isolated human neutrophils. AB - An immunocytochemical study was performed to examine the cellular localization and the subcellular distribution of kininogens in human blood cells. Kininogens were visualised using the immunogold-silver staining method and confocal scanning laser microscopy. We confirmed the existence of high molecular weight kininogen in human neutrophils and describe for the first time the presence of low molecular weight kininogen on these cells. Both high and low molecular weight kininogens were restricted to the neutrophils where they localized as clusters of immunogold particles on the cell membrane. No labeling was observed intracellularly in organelles such as mitochondria, endoplasmic reticulum, and azurophilic or specific granules after permeabilization of the neutrophils with Triton X-100, a procedure that permitted the visualization of elastase in the azurophilic granules. Clusters of high molecular weight kininogen molecules attached to the neutrophil surface could serve as receptors for plasma kallikrein and/or be the source of substrate for a discrete and circumscribed formation of kinins that may in turn facilitate the local diapedesis of neutrophils and the transudation of plasma constituents during acute inflammation. PMID- 1732014 TI - Specific detection of monocytic lysozyme within normal and leukemic cells. AB - A murine monoclonal antibody against human lysozyme (AHL MoAb) was produced and tested on normal and leukemic monocytes using flow cytometry. The antibody gave a positive reactivity on normal monocytes permeabilized by saponin (82% to 98% of positive cells) and a negative reactivity on normal permeabilized neutrophils. This monocyte-specific reactivity had not been observed using a polyclonal antibody. Nevertheless, immunoblotting detected lysozyme in both monocyte and polymorphonuclear leukocyte (PMNL) lysates. The AHL MoAb, in the presence of lysozyme substrate (Micrococcus lysodeikticus cell walls), strongly inhibited the enzymatic activity. Flow cytometric analysis of leukemic cells isolated from patients suffering from different subtypes of acute myeloid leukemia (French American-British [FAB] classification FAB M1-5) showed a highly significant positivity of FAB M5 for lysozyme compared with the other subtypes. The present results were consistent with the detection of a lysozyme epitope by AHL MoAb located near the catalytic site in monocytes. The same epitope was probably masked in PMNL granules. PMID- 1732015 TI - Regulation of globin gene expression in human K562 cells by recombinant activin A. AB - Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot-blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells. PMID- 1732016 TI - Mechanisms of amphipath-induced stomatocytosis in human erythrocytes. AB - We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin-labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration-dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet. PMID- 1732017 TI - Hemoglobin variants and activity of the (K+Cl-) cotransport system in human erythrocytes. AB - To determine if the activation of the (K+Cl-) cotransport system observed in hemoglobin (Hb) S- or C-containing erythrocytes is related either to a global change of isoelectric point of the Hb molecule or to the specific location of these mutations on the position 6 of the beta chain of Hb, we studied the (K+Cl-) cotransport system in erythrocytes containing beta chain variants exhibiting either the Glu----Lys substitution observed in position beta 6 in Hb C (Hb E: beta 26 Glu----Lys; Hb O-Arab: beta 121 Glu----Lys; Hb Siriraj:beta 7 Glu----Lys) or the Glu----neutral residue substitution observed in position beta 6 in Hb S (Hb G-San Jose: beta 7 Glu----Gly; Hb D Punjab or D-Los Angeles: beta 121 Glu--- Gln). The K transport mediated by the (K+Cl-) cotransport was increased in AC, AS and A-Siriraj and A-San Jose red blood cells and was similar to AA control in the other variants. These results indicate that an enhanced (K+Cl-) cotransport is not a property of all positively charged Hb variants, but it is mainly associated with mutations occurring at the beta 6 or beta 7 residues. An interaction of Hb with the cell membrane mediated by the disappearance of one of the negative charged residues (Glu) at this site of the A helix of the beta chain is the most likely candidate for the persistent activation of the (K+Cl-) cotransport system in these Hb variants. PMID- 1732018 TI - Rheological analysis of the adhesive interactions of red blood cells parasitized by Plasmodium falciparum. AB - Adhesion of parasitized red blood cells (RBCs) to vascular endothelium is thought to be a key factor in the pathology of falciparum malaria. However, quantitative analyses of the intercellular forces and of the effects of flow on adhesion have been lacking. We have characterized cytoadhesion of RBCs parasitized by the strains ITO4 (which can bind to receptors ICAM-1 or CD36) and FCR3A2 (which can bind to CD36 only) using micropipette manipulation and flow chamber techniques. Target cells were unfixed or glutaraldehyde-fixed human umbilical vein endothelial cells (HUVEC, bearing ICAM-1 only) or human amelanotic melanoma cells (C32, bearing CD36 and ICAM-1). In the static, micropipette assay, 60% to 70% of parasitized cells would adhere when tested at up to three successive sites. The percentage of cells adhering and the force required for their detachment (approximately 10(-10) N) were similar for each combination of parasite strain and adhesion target (ITO4/HUVEC, ITO4/C32, FCR3A2/C32). In the flow chamber, efficiency of initial adhesion of parasitized cells was essentially constant (at about 1%) up to a stress of 0.1 Pa, and then decreased rapidly with increasing stress. Either receptor (ICAM-1 or CD36) could immobilize flowing cells at a physiologic flow stress (0.1 Pa), but the numbers of cells adhering varied for the different combinations (ITO4/C32 greater than ITO4/HUVEC greater than FCR3A2/C32). When flow was increased in steps, adhered cells were gradually washed off but many could withstand stresses at which they would not initially adhere. The force for detachment estimated in this way was similar to the pipette value, and again, was similar for the different combinations of strains and targets. Adhesion from flow depends on the affinity between surfaces being above a critical level, and once adhesion is established, the fracture energy determines resistance to disruption of adhesion. The results show that the fracture energy is greater than the affinity (ie, that adhesion becomes stabilized after it is initially established) and that the ratio of affinity to fracture energy is different for different receptor/ligand pairs, with ICAM-1 appearing to be the more efficient immobilizing receptor. Also, static and flow based assays of adhesion clearly differ; the affinity is less critical in the static situation, so that most parasitized cells were capable of adhering in a static assay, but fewer did so under flow. Adhesiveness varied markedly from cell to cell, both for targets and parasitized cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732019 TI - Spontaneous delta- to beta-globin switching in K562 human leukemia cells. AB - Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta-globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching. PMID- 1732020 TI - Detection of bone marrow involvement in Hodgkin's disease by quantitative magnetic resonance imaging techniques. PMID- 1732021 TI - In vitro assays for primitive hematopoietic cells. PMID- 1732022 TI - BCR breakpoint subregions and blast crisis lineage in CML patients. PMID- 1732024 TI - Progress in pediatric critical care. PMID- 1732023 TI - The historical evolution, current status, and prospective development of pediatric critical care. AB - This article describes the multiple historic origins of pediatric critical care medicine and the evolution of the subspecialty, from the late 1950s with the first units dedicated to the care of critically ill and injured infants and children, to the present status as a recognized medical subspecialty. Also discussed are current major issues and future challenges that flow from both this historic background and a commitment to critically ill children. Presented are specific considerations for physicians in this subspecialty to address as they plan for the future. PMID- 1732025 TI - Near-drowning in the pediatric population. AB - Near-drowning is a frequently preventable accident that has significant morbidity and mortality for a previously healthy population. It causes an hypoxic-ischemic insult and multisystem organ dysfunction. Effective and aggressive CPR at the scene is the most important therapy presently available. Children needing CPR in an emergency room setting have poor outcome unless the submersion incident occurred in ice-water and the patient is hypothermic upon arrival in the emergency room. Attempts at prevention through parent education, requiring of CPR certification for pool owners, and legislation of barriers around the pool are critical because treatment to improve the outcome of the neurologic insult has proved ineffective. PMID- 1732026 TI - Simultaneous independent lung ventilation in pediatric patients. AB - Current methods of ventilation do not allow adequate ventilation of the affected lungs in the presence of unilateral disease, e.g., unilateral atelectasis, diaphragmatic hernia, or lobar emphysema. Using a bilumen endotracheal tube and two independent ventilators, synchronized simultaneous independent lung ventilation (SILV) can be achieved. This technique provides a method of treating unilobar, unilateral, or multifocal lung disease effectively. This article describes the author's methodology and clinical experience with SILV. PMID- 1732027 TI - Management of reactive airway disease. AB - The diagnosis of lower airway obstruction is one of the most frequent causes of admission to pediatric intensive care units. Morbidity and mortality have noticeably increased during the past decade. Although the exact causes of the increased morbidity and mortality are not clear, factors that have been implicated include underdiagnosis, delay in referral to the hospital, and inadequate treatment, particularly excessive reliance on bronchodilators and too little use of systemic steroids. A thorough understanding of the pathophysiology underlying the disease should lead to more effective management and decreased mortality and morbidity. PMID- 1732028 TI - Inflammatory host responses in sepsis. AB - Although microbes and their associated toxins initiate sepsis, it is the subsequent host inflammatory response that defines most of what we characterize as clinical sepsis. This article considers the various cellular as well as humoral mediators involved in this response in addition to the complex networking that may result in both augmentation and modulation of the inflammatory response in sepsis. PMID- 1732029 TI - Progress in pediatric extracorporeal membrane oxygenation. AB - Prolonged complete support of the circulation and of gas exchange can be achieved by extracorporeal membrane oxygenation (ECMO) in infants and children with potentially reversible, albeit life-threatening, disease. This allows lung rest or cardiac rest at times when dependence in those organs would be physiologically expensive. Although ECMO has no intrinsic healing powers, pediatric hearts and lungs exhibit tremendous recuperative power once the cycle of injury, inefficient performance, abuse, and secondary injury can be broken. Recent advances in technology, although impressive, do not explain the rapid growth of clinical interest in ECMO. Most recent progress in ECMO derives from refinement of clinical practices and the application of this technology to new patient populations. ECMO is not itself an experiment. It is the application of ECMO that is experimental. PMID- 1732030 TI - Ethical dilemmas in pediatric critical care. AB - Advances in the area of pediatric medicine during the past few years have presented ethical dilemmas for the physician to consider. This article discusses the ethical principles upon which clinical reasoning and judgments can be made. PMID- 1732031 TI - Regionalization of pediatric critical care. AB - It is evident that the field of pediatric critical care is evolving rapidly, but the prime issue remains the proper delivery of scarce and expensive resources to the most patients. In order to do so it is necessary to determine where current resources exist. This has been difficult to accomplish accurately on a national basis, although it may be possible on a regional level. To achieve the final goal of building organized systems of care, the special needs of the critically ill and injured child must be recognized. Models of regionalization will provide the basic structure for the development of these systems. The Model for a Pediatric Critical Care System proposed by the California Critical Care Coalition and District IX of the American Academy of Pediatrics should be readily applicable to any region. It is hoped that the information and examples provided in this article will provide some guidelines for those interested in promoting regionalization of pediatric critical care across the nation. PMID- 1732032 TI - Triage and transport of the critically ill child. AB - The aim of critical care transport services is the provision of care prior to and during transportation, similar to that offered in the tertiary care intensive care unit. This can only be achieved by a well prepared and equipped team dedicated to provision of this care. Appreciation of the disease conditions and adverse physiologic events likely to be encountered is necessary for the success of the team. Patient demographics and diseases in various geographic areas have been reported in the past few years. But, at the present time, team composition, responsibilities, and training requirements have not been well defined. In addition, there are no validated scoring systems to assist in team composition, triage of patients, or in the meaningful evaluation of mortality statistics. Within the next few years, one can expect to see genuine attempts made to address some of these issues. PMID- 1732033 TI - Management of the child with severe brain injury. AB - The pathophysiology and clinical management of acute brain injury in infancy and childhood are presented using acute traumatic brain injury as a model. The principles of stabilization, transport, and intensive care management are critically reviewed. PMID- 1732034 TI - Doppler assessment of the cerebral circulation in pediatric intensive care. AB - This article gives an interim overview of the potentials of TCD as a monitoring instrument in pediatric intensive care. In the near future, typical TCD flow patterns associated with adverse neurologic outcomes must be defined so they can promptly be recognized during intensive care surveillance or intraoperatively, before permanent damage occurs. Further applications of monitoring will deliver new and exciting insights into the physiology and pathophysiology of cerebral circulation in the critically ill child. Continuous recording of the Doppler waveforms and ICP may make it possible to determine the critical CPP and to improve the control of the therapy of cerebral edema. PMID- 1732035 TI - Cranial Doppler applications in neonatal critical care. AB - Although Doppler methods have been validated, it must be stressed that, at their best, Doppler results reflect qualitative circulatory changes, and suggest the direction of change. Because the large conduit arteries may also be controlling changes in flow volume, a direct extrapolation of the numeric findings from Doppler studies may lead to incorrect conclusions. Doppler methods have not made their way into routine neonatal critical care until now, but several studies have concluded that Doppler-derived information can be used as adjunct to clinical management in cases with shock, asphyxia, brain death, vascular malformations, and increased intracranial pressure. As with any physiologic variable, serial measurements may be of greater value than a single measurement. The Doppler techniques already are powerful investigative tools, but with continued improvements in technology, they hold promise of becoming important adjuncts in the monitoring of cerebral hemodynamics in perinatal-neonatal critical care units. PMID- 1732036 TI - Many growth factors may not be growth factors. AB - Both organ growth and tumor growth are dependent upon a biased ratio of cell births to cell deaths; this ratio is independent of the frequency of mitosis. Thus, so-called growth factors that affect only the frequency of mitosis are not really growth factors. I shall advance two hypotheses: that the normal adult ratio of cell births to cell deaths is maintained by the activity of a factor or factors produced by the stem cells; and that the differentiating cells, as well as the stem cells, produce a different factor that limits the frequency of mitosis. PMID- 1732037 TI - Prolonged inhibition by X-rays of DNA synthesis in cells obtained by transformation of primary rat embryo fibroblasts with oncogenes H-ras and v-myc. AB - Transfection of primary rat embryo fibroblasts with the H-ras oncogene plus the cooperating oncogene v-myc results in the development of foci of morphologically altered tumorigenic cells. We examined radiation (X-rays) induced inhibition of DNA synthesis in cell lines derived from such transformed clones and compared the results to those obtained with the nontransformed parental cells, rat embryo fibroblasts, as well as with cells immortalized either spontaneously, or after transfection with nuclear oncogenes (v-myc, E1A). Inhibition by X-rays of DNA synthesis was higher and persisted for longer periods of time in the H-ras- plus v-myc-transformed cell lines as compared to their nontrasformed counterparts. When the rate of DNA synthesis was measured as a function of dose 3 h after irradiation, biphasic curves were observed in all cell lines tested with a radiation sensitive and a radiation resistant component, known to correspond to inhibition of replicon initiation and chain elongation, respectively. A substantially larger inhibition of DNA synthesis was observed between 0 and 30 Gy in H-ras- plus v-myc-transformed cell lines, as compared to their nontransformed counterparts, presumably caused by sustained inhibition of replicon initiation. Hypersensitive DNA synthesis to X-rays was also observed in a transformed cell line obtained by transfection of rat embryo fibroblasts with H-ras in cooperation with the oncogene E1A, but normosensitive DNA synthesis in a rare transformant obtained by transfection with H-ras alone. These results suggest a direct or indirect involvement of the oncogene H-ras in cooperation with the oncogene v-myc (or other nuclear oncogenes such as E1A) in the control of DNA synthesis in irradiated cells. This control of DNA synthesis may be mediated via a trans acting mechanism that involves the production of a diffusible factor in response to the radiation insult, or, by a cis-acting mechanism that directly affects the replication machinery. Circumstantial evidence for possible involvement of oncogenes of the ras and myc families in DNA synthesis support this hypothesis. There was an inverse correlation between sensitivity to radiation-induced killing and prolonged inhibition by radiation of DNA synthesis, with radioresistant cell lines displaying longer inhibition of DNA synthesis. However, inhibition by radiation of DNA synthesis was similar in normal human fibroblasts (W138) and cells derived from a radiation-resistant human carcinoma cell line (SQ-20B) suspected to carry an abnormal c-raf-1 oncogene.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732038 TI - Pediatric phase I trial, pharmacokinetic study, and limited sampling strategy for piritrexim administered on a low-dose, intermittent schedule. AB - Piritrexim, an orally administered, lipid-soluble antifolate, was evaluated in a multi-institutional phase I trial in children. The starting dose was 10 mg/m2/dose administered every 8 h daily for 5 days for 3 consecutive weeks, with dose escalations in increments of 5 mg/m2/dose. Eighteen patients (16 with metastatic sarcoma, 1 with acute lymphoblastic leukemia, and 1 with a brainstem glioma), 3.5-20 years of age, with malignancy refractory to therapy, were entered into the study. The dose-limiting toxicities (DLTs), which were myelosuppression and mucositis, occurred in 4 of 4 patients treated at the 25-mg/m2/dose level but in none of the patients treated at the 15- and 20-mg/m2/dose levels. The recommended dose for phase II trials is 20 mg/m2/dose. Pharmacokinetic monitoring was performed in 15 of the 18 children. The area under the concentration-time curve (AUC) was linearly related to the dose administered. Piritrexim was rapidly absorbed, with the median time to peak level occurring 1.5 h after an oral dose. The terminal half-life of piritrexim ranged from 1.5 to 4.5 h. A limited sampling strategy developed earlier, capable of predicting the AUC based on the plasma concentrations at 3 and 6 h after an oral dose, was prospectively tested in this trial and proved to be highly predictive of the AUC (r = 0.98, P = 0.0001). Pharmacodynamic-pharmacokinetic correlations were obtained after combining data from this and the prior phase I pediatric trial. Trough plasma piritrexim concentration strongly correlated with DLT (P = 0.0016). A trough plasma piritrexim concentration greater than 0.5 microM appeared to be predictive of toxicity. Eleven of 15 patients with trough concentrations exceeding this threshold experienced DLTs. Therapeutic drug monitoring may thus play an important role in adjusting the dose and schedule of piritrexim in future trials. PMID- 1732039 TI - Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a mechanism of self-potentiation. AB - 2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity. PMID- 1732040 TI - Platinum anticancer drugs modulate P-450 mRNA levels and differentially alter hepatic drug and steroid hormone metabolism in male and female rats. AB - Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochrome P-450 and other microsomal enzymes involved in steroid hormone and drug metabolism (G.A. LeBlanc, and D.J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). The present study uses the rat model to evaluate the differential effects of cisplatin treatment on liver microsomal enzymes between genders, and also examines whether the modulation of enzyme activities by cisplatin and its analogues involves changes in P-450 gene expression. While cisplatin treatment of male rats caused a severalfold increase in female predominant hepatic enzymes, including testosterone 5 alpha-reductase and testosterone 7 alpha-hydroxylase (P-450 form 2A1), it partially decreased the expression of these enzymes in females. The reduced expression of these estrogen dependent enzymes in females may derive from the loss of circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed that the effects of cisplatin on P-450 catalyzed steroid hydroxylase activities in both male and female rats are primarily operative through the drug's effects on P-450 mRNA expression. P-450 dependent cyclophosphamide activation was significantly compromised in male rats after cisplatin administration; however, this activity was not altered in cisplatin-treated females. This sex-dependent effect of cisplatin was due to its suppression of P-450 form 2C11, a male-specific P-450 that is a major contributor to microsomal cyclophosphamide bioactivation in male rat liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did not have significant effects on hepatic P-450 expression. Together, these findings demonstrate that the response of rat liver to cisplatin-induced changes in hepatic P-450 enzyme profiles and cyclophosphamide bioactivation capacity differs between the sexes, and in addition, these effects can be minimized by use of carboplatin in place of cisplatin. PMID- 1732041 TI - Activation and growth of murine tumor-specific T-cells which have in vivo activity with bryostatin 1. AB - We examined the ability of bryostatin 1 (Bryo), a novel protein kinase C activator, plus ionomycin (Io), a calcium ionophore, to activate T-cells with specific antitumor activity. Lymphocytes from the draining lymph nodes (DLN) of MCA-105 tumor-bearing host mice were stimulated with Bryo/Io, either fresh or after in vitro stimulation with autologous tumor, and then were incubated in interleukin-2 at 20 units/ml. Lymphocytes sensitized with tumor cells in vitro and then stimulated with Bryo/Io exhibited significant expansion (12-fold) after a total of 3 weeks in culture and moderate cytolytic activity (40% at an effector:tumor cell ratio of (80:1) and were exclusively CD8+ T-cells. DLN cells activated immediately with Bryo/Io, without tumor antigen sensitization in vitro, displayed marked growth (130-fold expansion) over 3 weeks in culture, had weak cytolytic activity (8% at an effector:tumor ratio of 80:1), and were a mixed population of CD8+ and CD4+ cells. Despite the differences in phenotypes and in cytotoxicity, both groups of DLN cells were highly effective in vivo against MCA 105 pulmonary metastases. Bryo/Io-activated DLN cells from MCA-105 tumor-bearing hosts had no therapeutic efficacy against B16 melanoma or MCA-203 sarcoma metastases. Lymph node cells from normal mice and non-draining lymph node cells from tumor-bearing hosts could be expanded with Bryo/Io to a degree similar to that of DLN cells but had no antitumor activity. Phenotypic analyses and in vitro and in vivo depletion studies demonstrate that CD8+ cells mediated tumor regression. PMID- 1732042 TI - Endothelin-1 gene expression and biosynthesis in human endometrial HEC-1A cancer cells. AB - In this study, evidence was obtained that endothelin-1 (ET-1) is produced by an established endometrial cancer (HEC-1A) cell line. PreproET-1 mRNA is present in HEC-1A cells, and immunoreactive endothelin is secreted into the medium of these cells maintained in culture. Cycloheximide treatment of these cells caused superinduction of preproET-1 mRNA. Transforming growth factor-beta acts in these cells to increase the levels of preproET-1 mRNA. This effect of transforming growth factor-beta on preproET-1 mRNA accumulation was accompanied by an increase in the amount of immunoreactive endothelin secreted into the culture medium. ET 1, added to the culture medium, did not act as a mitogen in HEC-1A cells. We speculate that ET-1 (which is known to stimulate fibroblast proliferation) produced by endometrial adenocarcinoma cells may participate in the angiogenic process that occurs during the establishment of this carcinoma in vivo. PMID- 1732043 TI - Mutation(s) of the thymidylate synthase gene of human adenocarcinoma cells causes a thymidylate synthase-negative phenotype that can be attenuated by exogenous folates. AB - Biological characterization of a human colon adenocarcinoma cell line deficient in thymidylate synthase (TS-) is described. The clone, designated TS-C1/C1, was derived from the parental line GC3/C1 by selection in medium containing aminopterin, thymidine (dThd), and low concentrations of 5-formyltetrahydrofolate (5-CHO-H4PteGlu), and was subsequently reselected by single-step cloning in 500 microM methotrexate in the presence of dThd. This clone retained its TS- phenotype, was highly resistant to methotrexate (greater than 100,000-fold), and remained tumorigenic in mice (P.J. Houghton, et al., Proc. Natl. Acad. Sci. USA, 86: 1377-1381, 1989). In studies reported, it is shown that high levels of exogenous folate can support the growth of the TS- C1/C1 clone in the absence of dThd. Activation of dTMP biosynthesis de novo was demonstrated within 6 h of exposing cells to 20 microM [6R,S]5-CHO-H4PteGlu, and greater than or equal to 80% of activity was lost within 24 h of removing this folate from the medium. The labeling index was determined by autoradiographic techniques using [6-3H]2' deoxyuridine. None of the greater than 6,000 cells radiolabeled in the absence of [6R,S]5-CHO-H4PteGlu, whereas 33.5% labeled in the presence of 20 microM exogenous folate. Relative to the parental (TS+) clone, there was a greater than 87,500-, an 8,182-, and a 425-fold higher requirement for 5 methyltetrahydrofolate ([6R,S]5-CH3-H4PteGlu), PteGlu, and [6R,S]5-CH3-H4PteGlu to support 50% maximal colony formation in the absence of dThd. Quantitative analysis of the combined pools of 5,10-methylenetetrahydrofolate (CH2-H4PteGlun) and H4PteGlun showed that parental GC3/C1 cells had higher endogenous folate pools compared to TS-C1/C1 cells [168 +/- 40 (SD) and 10.9 +/- 0.3 fmol/10(6) cells, respectively]. Qualitatively the distribution of polyglutamate species and their redistribution in cells exposed to 20 microM [6R,S]5-CHO-H4PteGlu were similar in the two lines. Analysis of pools in a second, independently derived, TS- clone (TS-C3/C3, a transcription-negative mutant) demonstrated undetectable levels of CH2-H4PteGlun and H4PteGlun. This line cannot be rescued by exogenous folate. The data thus suggest that deletion of dTMP synthase activity may cause redistribution of reduced folate pools. In cytosolic extracts from parental GC3/C1 (TS+) cells, [6R]CH2-H4PteGlu1 acted as a cofactor in the release of 3H2O from [5-3H]dUMP, whereas no activity was detected in cytosols from TS-C1/C1. In contrast dTMP synthase activity was detected in cytosols from TS- C1/C1 cells in the presence of [6R]CH2-H4PteGlu5.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732044 TI - Surface immunoglobulin-mediated signal transduction involves rapid phosphorylation and activation of the protooncogene product Raf-1 in human B cells. AB - The protooncogene product, Raf-1, is a serine/threonine kinase and has been implicated as an intermediate in signal transduction mechanisms. We examined neoplastic and normal B cells for phosphorylation and activation of Raf-1 protein in response to anti-immunoglobulin antibody (anti-Ig). Anti-Ig induced rapid phosphorylation of Raf-1 protein in both neoplastic B-cells of hairy cell leukemia and normal tonsillar B-cells which proliferated well in response to anti Ig. The increase in phosphorylation was due primarily to an increase in phosphoserine. The immune complex kinase assay using Histone V-S as an exogenous substrate also showed an increase in Raf-1-associated kinase activity. An inhibitor of protein kinase C, H7, inhibited the proliferation as well as the Raf 1 phosphorylation in response to the proliferative signal of anti-Ig. Further, downregulation of protein kinase C by the treatment with 12-phorbol 13-myristic acid significantly abrogated the induction of Raf-1 phosphorylation. These results suggest that, in human B-cells, Raf-1 protein may be involved in the signal transduction pathway mediated by surface immunoglobulin, and that it may be, at least partially, phosphorylated by activated PKC. PMID- 1732045 TI - Basic fibroblast growth factor in human prostate cancer cells. AB - To increase our understanding of the potential role of basic fibroblast growth factor (bFGF) in malignant progression of prostate cancer, we determined the production of bFGF, the expression of FGF receptor (flg), and the response to exogenous bFGF in LNCaP, DU 145, and PC 3 cells. We observed that these three prostate cancer cell lines, which differed in their dependence on androgens for growth in vitro and in their in vivo behavior in nude mice, could be distinguished as follows: (a) androgen-sensitive LNCaP cells, which do not metastasize in nude mice, did not produce measurable amounts of bFGF, expressed small but measurable amounts of FGF receptor mRNA, and did respond to exogeneous bFGF; (b) androgen-insensitive, moderately metastatic DU 145 cells did produce measurable amounts of biologically active bFGF, expressed large amounts of FGF receptor mRNA, and responded to exogeneous bFGF and the heparin-binding fractions from DU 145 cell extracts; (c) androgen-insensitive and highly metastatic PC3 cells also produced measurable amounts of bFGF but did not demonstrate a growth response to either the heparin-binding fractions from PC3 cell extracts or exogenous bFGF, even though large amounts of FGF receptor mRNA were expressed in PC 3 cells. These results suggest the possibility that differences in production of, and response to, bFGF may be associated with different biological behavior. PMID- 1732046 TI - Blood flow in six human melanoma xenograft lines with different growth characteristics. AB - Blood flow in six human melanoma xenograft lines grown s.c. in BALB/c-nu/nu mice was studied and analyzed in relation to tumor growth characteristics. Two different methods were used to measure blood flow, i.e., uptake of 86Rb and clearance of 133Xe. The percentage of the injected 86Rb taken up per g of tumor tissue and the 133Xe clearance rate were used as parameters for blood flow. The results achieved with these two methods were consistent. Blood flow differed significantly among individual tumors of the same line, even for tumors of similar size. All lines showed a decrease in blood flow with increasing tumor volume. This was due to an increase in necrotic fraction as well as a decrease in blood supply per viable tumor cell. Blood flow also differed significantly among the xenograft lines. All lines showed a lower blood flow than the kidney, spleen, liver, and foot. The blood flow was generally lower in the xenograft lines than in the EMT6 and Lewis lung murine tumor lines. There was no correlation between tumor blood flow and volumetric growth rate. The xenograft lines could be divided into two distinct groups of three lines each with respect to blood supply per viable tumor cell. The three lines showing a high blood supply also showed a high fraction of cells in S phase (23-31%), whereas the three lines showing a low blood supply had a low fraction of S-phase cells (11-13%). Thus, blood supply per viable tumor cell was probably decisive for the cell proliferation activity in the tumors. Moreover, necrotic fraction increased with increasing tumor volume, and the magnitude of this increase was largest for the three lines showing the lowest blood supply per viable tumor cell. These observations were possibly consequences of basic differences in vascular architecture between the two groups of xenograft lines. PMID- 1732047 TI - Heterogeneity of progesterone receptor content and remodeling by tamoxifen characterize subpopulations of cultured human breast cancer cells: analysis by quantitative dual parameter flow cytometry. AB - Breast cancers treated with the antiestrogen tamoxifen invariably become resistant. To analyze the behavior of cell subpopulations within a tumor following tamoxifen treatment, we have used a new flow cytometry-based immunoassay and software that simultaneously quantitate PR levels and the DNA indices of ploidy and cell cycle stage in total cell populations or any subset thereof. The human breast cancer cell line T47D and its clonal derivatives were used as models of stage IV breast cancer, and growth and PR were measured as markers of antiestrogen responsiveness. We demonstrate and quantitate a remarkable heterogeneity in PR content and show the existence of distinct subpopulations with large differences in their PR levels and DNA indices, even among T47D sublines that are clonally derived. Following chronic tamoxifen treatment, an overall decrease in PR levels and growth masks an extensive heterogeneity in the response of cell subpopulations. PR levels decrease in some cells but increase in others; populations having a growth advantage expand while others contract. We find little evidence that cells, unaffected by the hormone, gain a growth advantage. Rather than exhibiting autonomy, we propose that under the influence of tamoxifen, tumors become remodeled as selected subpopulations emerge that are stimulated by the hormone, explaining the paradoxical recurrence of disease in patients undergoing endocrine therapy. PMID- 1732048 TI - Clinical pharmacokinetics of the anthrapyrazole CI-941: factors compromising the implementation of a pharmacokinetically guided dose escalation scheme. AB - The pharmacokinetics of the anthrapyrazole CI-941 has been investigated in conjunction with the Phase I evaluation of the drug with the intent of applying a pharmacokinetically guided dose escalation strategy. A starting dose of 5 mg/m2 was chosen, based on one-tenth the 10% lethal dose in mice. Due to the steep dose lethality relationship and nonlinear pharmacokinetics in mice, a target area under the CI-941 plasma concentration x time curve (AUC) of 110 microM x min (i.e., 40% of the mouse 10% lethal dose AUC) was chosen. This AUC was achieved in mice at 40 mg/m2. A total of 37 patients received 74 courses of CI-941 (5 to 55 mg/m2), with 26 patients consenting to pharmacokinetic monitoring. CI-941 was rapidly cleared from plasma, and a triexponential open model could be fitted to the data (t1/2 alpha = 7.6 +/- 2 min, t1/2 beta = 65 +/- 27 min, t1/2 zeta = 21 +/- 9 h). CI-941 was subjected to only limited urinary elimination, accounting for 5.2 +/- 2.8% of the administered dose. Wide interpatient variability in plasma CI-941 clearance at the starting dose and subsequent doses precluded the implementation of a pharmacokinetically guided dose escalation scheme, and the dose was escalated in 5-mg/m2 increments until the maximally tolerated dose was achieved. A number of investigations were performed to study potential reasons for variability in CI-941 clearance. However, CI-941 plasma protein binding (95 +/- 1%) and measures of pretreatment renal (51Cr-EDTA clearance), hepatic (plasma alanine transaminase and alkaline phosphatase levels), or cardiac function (left ventricular ejection fractions) did not relate strongly to CI-941 clearance. In patients treated at 40 mg/m2, the AUC values (156 to 415 microM x min) approximated or exceeded the target AUC. Fifty mg/m2 was the Phase II recommended dose. Further prospective studies are warranted to assess the utility of pharmacokinetically guided dose escalation strategies and to determine whether or not the variability encountered in clearance is unique to CI-941. PMID- 1732049 TI - Examination of the oncogenic potential of a tumor-associated antigen, intestinal alkaline phosphatase, in HeLa x fibroblast cell hybrids. AB - An exclusive correlation exists between the ectopic expression of the cell surface marker, intestinal alkaline phosphatase (IAP), and the tumorigenic phenotype of segregants derived from suppressed, nontumorigenic HeLa x fibroblast cell hybrids. This specific association suggests that loss of tumor suppressor function is closely linked to the re-expression of IAP and, therefore, that IAP may be a critical oncogenic factor in these cells. To address this directly, we have used a HeLa IAP cDNA expression vector (pHIAP) to introduce constitutive IAP expression into a nontumorigenic HeLa x fibroblast cell hybrid. Sequence analysis of the HeLa IAP cDNA revealed 5 separate nucleotide alterations when compared to the native IAP cDNA sequence; however, none of these resulted in amino acid substitutions. Four pHIAP transfectants were analyzed for the presence of the intact integrated IAP cDNA and their relative expression levels of exogenous IAP mRNA and protein. The functional integrity of the cDNA-derived IAP product was confirmed by demonstrating proper enzymatic activity and localization to the extracellular membrane. The tumorigenic potentials of the pHIAP transfectants were assayed by s.c. injection into athymic nude mice. No tumors were observed, even after an 11-week incubation in animals. Therefore, expression of IAP in the nontumorigenic HeLa x fibroblast cell hybrid is not sufficient to confer the tumorigenic phenotype. Although ectopic IAP expression is unlikely to be functionally relevant to tumorigenicity in these hybrids, the significance of IAP as a tumor marker is still evident from its apparent strong association with a tumor suppressor locus. PMID- 1732050 TI - Blocking effect of human serum but not of cerebrospinal fluid on ricin A chain immunotoxin potentiation by monensin or carrier protein-monensin conjugates. AB - The potentiation of monoclonal antibody/ligand toxin (immunotoxin) cytotoxicity by the ionophore monensin (Mo) or by human serum albumin-monensin (HSA-Mo) conjugates was investigated. Since disulfide cross-linked HSA-Mo (HSA-SPDP-Mo) is rapidly inactivated by human serum (M. Colombatti et al., Cancer Res., 50: 1385 1391, 1990), we synthesized thioether cross-linked HSA-Mo conjugates (HSA-SIA Mo). HSA-SIA-Mo is resistant to treatment with reducing agents (e.g., glutathione, dithiothreitol) and shows potentiating activity identical to that of Mo or of HSA-SPDP-Mo, enhancing immunotoxin (IT) cytotoxicity 45-35,000-fold. Human leukemic and tumor cell lines are highly sensitive to treatment with IT in combination with Mo, HSA-SPDP-Mo, or HSA-SIA-Mo (concentration required to inhibit protein synthesis by 50%, 10(-10)-2.5 x 10(-13) M). IT potentiation by both types of HSA-Mo conjugates, however, is inhibited by whole human serum. In contrast, human cerebrospinal fluid has no effect on the potentiation of IT by Mo or HSA-Mo conjugates. The serum blocking factors reside mostly in a Mr 40,000 90,000 protein fraction. Serum components of low molecular weight (less than 10,000) show no detectable effect upon the stability of HSA-Mo conjugates. The toxicity of HSA-SIA-Mo in vivo was investigated by intrathecal injections in rats. Concentrations of up to 60 micrograms/kg can be injected into the brain with only transient neurological sequelae. We therefore conclude that if the systemic delivery of HSA-Mo conjugates for the potentiation of ricin A chain-IT presents some limitations due to the blocking effect of serum, the application of HSA-Mo conjugates in combination with ricin A chain-IT for regional tumor therapy in the brain appears more promising. PMID- 1732051 TI - The chlorinated pesticide mirex is a novel nonphorbol ester-type tumor promoter in mouse skin. AB - The hepatocarcinogenic organochlorine pesticide, mirex, was examined as a tumor promoter in the mouse skin initiation-promotion model. Female CD-1 mice were initiated with 200 nmol 7,12-dimethylbenz[a] anthracene and topically promoted three times weekly for 20 weeks with doses of 25, 50, 100, or 200 nmol mirex. Mirex promoted tumors at all dose levels in a dose-dependent manner. At 20 weeks, mice promoted with 25, 50, 100, and 200 nmol mirex developed an average of 0.2, 4, 10, and 16 tumors per mouse with a 10, 60, 93, and 96% incidence of tumor bearing mice, respectively. With continued treatment to 34 weeks, mice promoted with 25, 50, and 100 nmol mirex developed an average of 0.7, 7, and 12 tumors per mouse with a 27, 85, and 100% incidence of tumor-bearing mice, respectively. These results demonstrate that mirex is a very effective tumor promoter in mouse skin. The effect of mirex on several biochemical and morphological events associated with tumor promotion was then investigated. Mirex did not stimulate epidermal protein kinase C activity in vitro. Unlike the phorbol ester, 12-O tetradecanoylphorbol-13-acetate, a single topical application of mirex (200 nmol) did not increase [3H]thymidine incorporation into epidermal DNA up to 108 h after application. Furthermore, multiple applications of 200 nmol mirex (3 times weekly for 4 weeks) resulted in only a very weak proliferative response; mirex increased the number of nucleated epidermal cell layers from 1 to 2 in acetone-treated controls to 2 to 3 while 2 nmol 12-O-tetradecanoylphorbol-13-acetate produced 6 to 7 nucleated cell layers. Mirex (200 nmol) did not induce ornithine decarboxylase activity up to 56 h after a single topical application. Collectively, these data indicate that mirex is a novel nonphorbol ester-type tumor promoter in mouse skin. PMID- 1732052 TI - Improved delivery of radiolabeled anti-B1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled anti-B1 monoclonal antibody. AB - A human B-cell lymphoma xenograft model was used to test whether the administration of unlabeled MoAb prior to injection of radiolabeled monoclonal antibody (MoAb) improves delivery of the radiolabeled MoAb to tumor prior to testing in clinical radioimmunotherapy trials. The anti-B1/CD20 pan-B-cell MoAb reactive with human B-cell lymphomas and leukemias but not reactive with mouse B cells was used in this study. Athymic nude mice bearing human Raji Burkitt lymphoma xenografts were given injections of 2.5 muCi (0.3 microgram) 131I labeled anti-B1 with or without a 2-h prior single injection of 100 micrograms of unlabeled anti-B1 antibody. Four days later the animals given injections of 131I labeled anti-B1 and the unlabeled anti-B1 predose had a tumor uptake of 12.72 +/- 1.17% (SEM) of injected dose/g which was 44% greater than the animals receiving the 131I-labeled anti-B1 alone (P = 0.014). The uptake in most normal tissues was unchanged, although the blood level of 131I-labeled anti-B1 appeared to be greater following unlabeled anti-B1 predosing (P = 0.067). Predosing with isotype matched irrelevant MoAb did not result in a greater tumor uptake or blood concentration of 131I-labeled anti-B1 compared to the administration of 131I labeled anti-B1 alone. In studies using 111In-labeled anti-B1, the effect of unlabeled antibody predosing was more pronounced. For animals given injections of 4.5 muCi (0.4 microgram) 111In-labeled anti-B1 and the unlabeled anti-B1 predose, the uptake in tumor was 12.37 +/- 2.07% of injected dose/g which was 162% greater than the animals receiving the 111In-labeled anti-B1 alone (P = 0.009). Predosing decreased 111In-labeled anti-B1 uptake in spleen, while the blood level was significantly greater. Predosing was more effective than simultaneous injection in improving tumor delivery. When tumor-bearing mice were either simultaneously given injections of 36 micrograms of unlabeled anti-B1 and 4 micrograms 111In labeled anti-B1 or were given preinjections of 36 micrograms unlabeled anti-B1 3 h prior to injection of 4 micrograms 111In-labeled anti-B1, tumor uptake 3 days later was 1.3-fold higher in the animals which received the preinjection of unlabeled antibody (P = 0.011). As the quantity of unlabeled anti-B1 was increased (36, 96, 996 micrograms) in the predose, significantly greater uptake in tumor was observed, although this uptake appeared to plateau at the highest predoses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732053 TI - In vivo targeting of OV-TL 3 immunoliposomes to ascitic ovarian carcinoma cells (OVCAR-3) in athymic nude mice. AB - Specific binding of immunoliposomes to target tumor cells was investigated in a xenograft model (athymic nude mice) of i.p. growing human ovarian carcinoma (OVCAR-3). For the first time, quantitative evidence is presented that attachment of a tumor-specific antibody (OV-TL 3) dramatically enhances the association of liposomes with i.p. growing OVCAR-3 cells. The OV-TL 3-mediated binding of liposomes to the OVCAR-3 cells was rapid; 30 min after i.p. injection approximately 70% of the injected dose of OV-TL 3 immunoliposomes was associated with the OVCAR-3 cells while for unconjugated liposomes a value of only approximately 3% was obtained. At 2 h after injection, a maximal binding level of 84% was achieved in case of the OV-TL 3 immunoliposomes whereas the binding level of unconjugated liposomes was still about 3%. Twenty-four h after injection about 83% of the injected dose OV-TL 3 immunoliposomes still was associated with the OVCAR-3 cells, compared to about 10% of the injected dose of unconjugated liposomes. Accordingly, unconjugated liposomes disappeared from the peritoneal cavity much faster than the OV-TL 3 immunoliposomes. By comparison with immunoliposomes bearing irrelevant antibody, the specificity of the binding of the OV-TL 3 immunoliposomes to the OVCAR-3 cells was demonstrated. In addition, it was observed that the sustained high OV-TL 3 immunoliposome levels found in the peritoneal cavity are the result of both reduced particle clearance from the peritoneal cavity and the tenacious binding of the immunoliposomes to the tumor cells. Finally, data are presented showing that the degree of binding of OV-TL 3 immunoliposomes to OVCAR-3 cells in vitro and in vivo correlates positively with the antibody (Fab') density on the liposomes. PMID- 1732054 TI - Failure of RB1 to reverse the malignant phenotype of human tumor cell lines. AB - In addition to retinoblastoma and osteosarcoma, mutation of both alleles of the RB1 gene occurs frequently in several other types of tumors. In order to evaluate the role of RB1 in cancer, the wild type RB1 gene was introduced into the RB1 deleted breast cancer cell line MDA-468-S4 and retinoblastoma cell lines WERI-Rb1 and Y-79. The RB1 complementary DNA was under control of the inducible murine metallothionein promoter in MDA-468-S4 and the thymidine kinase promoter in the retinoblastoma lines. The protein, p110RB1, produced from the exogenously introduced gene appeared normal by immunoprecipitation, Western blot analysis, and nuclear localization and also showed normal cell cycle-dependent phosphorylation and an ability to bind to E1a protein. No changes in growth rate or morphology were observed in either of the reconstituted cell types. Expression of p110RB1 in MDA-468-S4 did not affect anchorage-independent growth when measured by colony formation in soft agar. Although the ability of WERI-Rb1 cells expressing p110RB1 to form colonies in methylcellulose was reduced, the reconstituted retinoblastoma cell lines formed intraocular tumors in immunodeficient mice with the same efficiency as the RB1-negative parent cell lines and the tumors produced by the RB1-reconstituted cells continued to express p110RB1. These experimental results suggest that the malignant phenotype is little affected by the replacement of p110RB1 and that RB1 is a relatively weak tumor suppressor gene. PMID- 1732055 TI - The effect of dietary fat on the rapid development of mammary tumors induced by 7,12-dimethylbenz(a)anthracene in SENCAR mice. AB - We recently reported (J. Leyton et al., Cancer Res., 51: 907-915, 1991) an inverse correlation between skin tumor number and level of dietary linoleic acid (LA) in SENCAR mice following an initiation-promotion protocol. These results differed from the reported (C. Ip et al., Cancer Res., 45: 1997-2001, 1985) positive correlation between dietary LA and tumor incidence for the rat mammary gland. The goal of the study reported here was to determine whether this dissimilarity was due to organ site or species differences. Female SENCAR mice were fed 1 of 3 15% fat diets containing LA at levels of 0.8, 4.5, and 8.4% before, during, and after intragastric administration of 6 mg (1 mg/week) 7,12 dimethylbenz(a)anthracene. A positive correlation between level of dietary LA and mammary tumor incidence was observed such that for the first 15 weeks, the incidence was greatest in the 8.4% LA diet group, followed by the 4.5% and then the 0.8% LA groups. Distinct dietary effects on latency were also noted in that 15, 12, and 8 weeks after cessation of 7,12-dimethylbenz(a)anthracene were required for a 40% carcinoma incidence in the 0.8, 4.5, and 8.4% LA diet groups, respectively. A histopathological analysis of all tumors revealed that the predominant type was the adenosquamous carcinoma, which comprised 46.6, 54.1, and 77.7% of all mammary tumors for diets containing 0.8, 4.5, and 8.4% LA, respectively. The second most common tumor was the adenocarcinoma type B, which was found with a frequency of 33% in the 0.8% and 4.5% LA diet groups and 22% in the 8.4% LA diet group. These results indicate that SENCAR mice have a short latency period for 7,12-dimethylbenz(a)anthracene-induced mammary tumor development and that rat and mouse mammary tumor development is modified by dietary LA in a similar manner, although in the SENCAR mouse dietary LA did not have a saturating effect. In addition, high dietary LA was found to be associated specifically with an increased incidence of adenosquamous carcinomas but not of other types of mammary tumors. PMID- 1732056 TI - Individual transforming events in long-term cell culture of NIH 3T3 cells as products of epigenetic induction. AB - The NIH 3T3 line of mouse fibroblasts undergoes "spontaneous" transformation in culture, exhibited in the development of foci of transformed cells overgrowing the confluent monolayer. Evidence is provided here to support the proposition that the spontaneous generation of individual transformed variants is a product of epigenetic induction by various types of growth inhibition. Novel transformed variants generally arise after prolonged confluence and cessation of net growth, with these new types of foci appearing during a second round of confluence, although not in the first round. Few or no transformed variants exist in the cultures prior to growth constraint. The susceptibility of NIH 3T3 cells to induction of transformation is itself shown to be subject to epigenetic influence, with capacity for transformation reflecting passage histories. Cell sublines maintained in long-term subconfluent passage that allows unimpeded growth become more refractory with time to spontaneous transformation, while sublines passaged in more growth-restricting conditions maintain their sensitivity. The differing capacities of the sublines are reflected in the degree of growth inhibition required to induce individual events of spontaneous transformation, and in the frequency at which such new variants arise. Thus growth inhibition not only induces individual transforming events, but even increases cell susceptibility to further induction of transformation. These phenomena are consistent with the progressive state selection model of heritable change, which postulates a self-regulating selection of better adapted states from among those made available to a biological system during heterogeneous fluctuations in its total pattern of chemical equilibria. PMID- 1732057 TI - Reduced growth rate of dimethylhydrazine-induced colon tumors in rats. AB - alpha-Difluoromethylornithine (DFMO) treatment has been shown to modify carcinogenesis in many experimental tumor models, including breast, urinary bladder, and colon. This study was designed to determine whether DFMO treatment can inhibit tumor growth on chemical-induced colon cancer in rats. Effectiveness of DFMO in combination with mitomycin C (MMC) was also evaluated. Forty-two Sprague-Dawley rats received dimethylhydrazine (20 mg/kg) s.c. once weekly for 20 wk to induce colon cancer. Then a double-contrast barium enema was performed, and colon tumors were detected. The animals were divided into four groups that were subjected to the following treatment: none; DFMO alone; MMC alone; and a combination of DFMO plus MMC. After 5 wk of treatment, the barium enema was repeated. For the evaluation of treatment efficacy, tumor doubling time was adopted. The mean tumor doubling time in the control group was 20.7 +/- 9.1 days (SD). "Response" was judged as effective when tumor doubling time in treatment groups was more than 38.9 days, calculated from the mean + 2 SDs in the control group. Response rates in the DFMO, MMC, and DFMO plus MMC groups were 40.0%, 10.0%, and 82.3%, respectively. DFMO was a more effective inhibitor of tumor growth than MMC, and DFMO in combination with MMC resulted in a synergic diminution of tumor growth. The double-contrast barium enema is useful to observe sequential tumor growth and may be appropriate for the evaluation of new treatment on experimental colon cancer in rats. PMID- 1732058 TI - Inhibition of invasion and metastasis in cells transfected with an inhibitor of metalloproteinases. AB - The balance between levels of metalloproteinases and their corresponding inhibitors is a critical factor in tumor invasion and metastasis. Down-regulation of the activity of these proteases was achieved by transfection of invasive and metastatic rat cells with the complementary DNA for metalloproteinase inhibitor/tissue inhibitor of metalloproteinase 2 (MI/TIMP-2), a novel inhibitor of metalloproteinases recently described. (Y. A. DeClerck et al., J. Biol. Chem., 264: 17445-17453, 1989; W. G. Stetler-Stevenson et al., J. Biol. Chem., 264: 17374-17378, 1989). Secretion of functional MI/TIMP-2 protein in stably transfected cells resulted in a marked decrease in metalloproteinase activity. Partial suppression of the formation of lung colonies after i.v. injection in nude mice was observed in a transfected clone expressing high levels of MI/TIMP 2. Production of MI/TIMP-2 in four clones markedly reduced tumor growth rate in vivo after s.c. injection and completely suppressed local tissue invasion. Thus, down-regulation of metalloproteinase activity has a striking effect on local invasion and partially suppresses hematogenous metastasis. PMID- 1732059 TI - Measurement of thymidine replacement in patients with high grade gliomas, head and neck tumors, and high grade sarcomas after continuous intravenous infusions of 5-iododeoxyuridine. AB - Based upon the radiation sensitization properties of the halogenated pyrimidines, 5-iododeoxyuridine (IdUrd) and 5-bromodeoxyuridine, long term i.v. infusions of halogenated pyrimidines in conjunction with fractionated radiation therapy have been evaluated in the treatment of a variety of human malignancies. While clinical studies have attempted to measure the halogenated pyrimidine incorporation, few have successfully related tumor response to the incorporation of IdUrd by the tumor. The present study reports the continuous IdUrd labeling index (number of cells labeled) and the IdUrd corrected replacement (percentage of thymidine replacement in the labeled cells of the population) from the tumors of 17 patients who received continuous infusions of IdUrd (1000 mg/m2/24 h). The tumors treated included four high grade gliomas, five head and neck tumors, four high grade sarcomas, and five other tumors of varying types. Less than 25% of the cells in three of four gliomas incorporated IdUrd after 5-7-day IdUrd infusion time. Corrected replacement for the gliomas ranged from 0 to 4%. In contrast, 63 85% of the cells in the head and neck biopsies were labeled with IdUrd after 3-7 day IdUrd infusions suggesting that these large tumors (3-12 cm diameter) have a high fraction of dividing cells. Corrected replacements values for the head and neck tumor patients ranged from 2.9 to 26.3%. The high grade sarcomas also demonstrated a high percentage of IdUrd labeled cells (57-79%) with three patients having corrected replacements of 7.5-14.2%. The continuous labeling and thymidine replacement data for four patients from whom serial biopsies were taken during IdUrd infusion demonstrated both an increasing IdUrd replacement and continuous labeling index with an increasing duration of IdUrd infusion. The clinical response of both the high grade glioma and head and neck tumor patients indicate that the IdUrd replacement and labeling data may provide some important predictive information with regard to the successful use of the halogenated pyrimidines in clinical radiation trials. PMID- 1732060 TI - Recombinant interleukin-2 and lymphokine-activated killer cell treatment of advanced bladder cancer: clinical results and immunological effects. AB - The purpose of this study was to evaluate the efficacy of treatment with recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells in patients with advanced bladder cancer and to study the induced changes in the distribution of leukocyte subsets in blood and tumor. Nine patients with metastatic transitional cell cancer of the bladder were treated with a continuous infusion of rIL-2 combined with lymphocytes stimulated in vitro with rIL-2. None of the patients responded to the therapy despite substantial changes observed in the immunological cells, both in tumor and blood. The rIL-2 infusion induced migration of leukocytes to the tumors, which was related to increased expression of the adhesion molecule VLA-1 on both peripheral blood mononuclear cells and the endothelial cells of small tumor vessels. Only T-cells, predominately expressing IL-2 receptors, and macrophages infiltrated the tumors. Natural killer cells remained few or absent in the tumors, even though the natural killer cells in peripheral blood were activated by the treatment. This study shows that the present technique of rIL-2 and lymphokine-activated killer cell therapy is able to induce substantial changes in the immune system of patients with metastatic bladder cancer. However, this treatment did not induce tumor regression, which may be due to the advanced stage of disease. PMID- 1732061 TI - The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. AB - Here we show that the VirD2 protein of A. tumefaciens functions as a nuclear localizing protein in plant cells. The nuclear localization signal of VirD2 consists of two regions containing 4-5 basic amino acids (KRPR and RKRER), located within the C-terminal 34 amino acids. These regions conform to the KR/KXR/K motif required for numerous nuclear localized nonplant eukaryotic proteins. Each region independently directs a beta-glucuronidase reporter protein to the nucleus; however, both regions are necessary for maximum efficiency. VirD2 has been shown to be tightly bound to the 5' end of the single-stranded DNA transfer intermediate, T-strand, transferred from Agrobacterium to the plant cell genome. The present results imply that T-strand transport to the plant nucleus is mediated by the tightly attached VirD2 protein via an import pathway common to higher eukaryotes. PMID- 1732062 TI - A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer. AB - DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type. Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell. PMID- 1732063 TI - Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. AB - Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells. PMID- 1732064 TI - Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins. AB - We generated by gene disruption Dictyostelium cells that lacked both the F-actin cross-linking proteins, alpha-actinin and gelation factor. Several major cell functions, such as growth, chemotaxis, phagocytosis, and pinocytosis, were apparently unaltered. However, in all double mutants, development was greatly impaired. After formation of aggregates, cells were very rarely able to form fruiting bodies. This ability was rescued when mutant and wild-type strains were mixed in a ratio of 70 to 30. The developmental program in the mutant was not arrested, since the expression pattern of early and late genes remained unchanged. Development of the mutant was rendered normal when a functional alpha actinin gene was introduced and expressed, showing the morphogenetic defect to be due to the absence of the two F-actin cross-linking proteins. These findings suggest the existence of a functional network allowing mutual complementation of certain actin-binding proteins. PMID- 1732065 TI - Killer system interactions. PMID- 1732066 TI - Allylamine antifungal drugs. PMID- 1732067 TI - Molecular approach to the toxic action of quinone mycotoxins--chemical structure and biochemistry. PMID- 1732068 TI - Fusarium-caused hyalohyphomycosis: an overview. PMID- 1732069 TI - Teaching medical mycology in Latin America. PMID- 1732070 TI - The need for a mycoses reporting system. PMID- 1732071 TI - The use of molecular techniques for epidemiologic typing of Candida species. AB - The availability of an epidemiologic typing system for Candida species that is sensitive, rapid, inexpensive, and easy to perform would clearly be an advantage to the mycologist, microbiologist, and epidemiologist in the ongoing struggle to understand the epidemiology and pathogenesis of candidiasis. This is particularly true given the increasing prominence of organisms such as C. albicans and C. tropicalis which are ubiquitous members of the normal flora yet are also important causes of nosocomial bloodstream infection. Unfortunately, the ideal epidemiologic typing system does not yet exist. Current data suggest that the molecular typing methods of restriction endonuclease digestion of genomic DNA with ethidium bromide staining (DEtBr typing) and electrophoretic karyotyping using pulsed-field electrophoresis offer rapid, simple, and sensitive means of discriminating strains of Candida species. These methods appear at present to be the most practical typing methods for both large- and small-scale epidemiologic studies. Other typing methods using specific DNA probes provide a powerful means of identifying strains and will undoubtedly be applied more broadly in the future. Thus far, studies employing molecular typing methods have documented that (1) most patients are colonized by one strain of Candida species, (2) isolates of Candida species recovered from blood or deep tissue sites are generally identical to those obtained from colonization sites before infection developed, and (3) nosocomial transmission of a single strain of C. albicans may occur, particularly in an intensive care unit setting. Given the limitations of the available typing methods and the complex nature of the patients at risk for candidiasis, both the epidemiologist and laboratory scientist must use these methods with clear epidemiologic objectives in mind. Whenever possible, all organisms to be typed should be typed by the same person on the same day, and typing should always include unrelated as well as epidemiologically related isolates. Additional studies, based upon sound epidemiologic principles, will be necessary to clarify the role of the various molecular typing methods as epidemiologic markers of Candida species and to further our understanding of the epidemiology and pathogenesis of candidiasis. PMID- 1732072 TI - Bronchopulmonary aspergillosis: diagnostic and therapeutic considerations. PMID- 1732073 TI - Japanese-originated pharmaceutical products in the United States from 1960 to 1989: an assessment of innovation. PMID- 1732074 TI - Heterogeneity of CYP3A isoforms metabolizing erythromycin and cortisol. AB - The N-demethylation of erythromycin and 6 beta-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6 beta-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6 beta-hydroxycortisol/cortisol ratios, a measure of cortisol 6 beta-hydroxylase activity, in nine patients. Erythromycin N demethylation varied fourfold and 6 beta-hydroxycortisol/cortisol ratios varied sevenfold among the subjects; no correlation was found between these activities (r2 = 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6 beta hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms. PMID- 1732075 TI - Interethnic difference in thiopurine methyltransferase activity. AB - A number of metabolic pathways are subject to both genetic polymorphism and interethnic differences. A catabolic pathway of 6-mercaptopurine, red blood cell (RBC) thiopurine methyltransferase (TPMT) activity showed genetic polymorphism in Caucasians, but variation according to ethnicity has not been studied. We investigated if red blood cell thiopurine methyltransferase was subject to interethnic variation in a Saami (Lappish; n = 36) and a Caucasian population (n = 50). The Saami population sample had 29% higher thiopurine methyltransferase activity, 17.0 +/- 3.3 U/ml red blood cell compared with the Caucasian population sample, 13.1 +/- 2.9 U/ml red blood cell (p much less than 0.001). Probit plots and frequency distribution histograms supported bimodality consistent with genetic polymorphism in both study populations. Differences in chronic diseases, drug consumption, age, or gender could not explain the interethnic difference in red blood cell thiopurine methyltransferase activity. The higher red blood cell thiopurine methyltransferase activity in the Saami population group indicates that these subjects may require higher dosages of thiopurine drugs than Caucasians. PMID- 1732076 TI - Pharmacokinetics of metronidazole in severely malnourished and nutritionally rehabilitated children. AB - A comparison of the pharmacokinetics of oral metronidazole, after a single dose of 30 mg/kg body weight, was done in two groups of subjects: group I consisted of 10 severely malnourished children, aged 4 to 43 months; group II consisted of 10 children, aged 3 to 25 months, who were studied after nutritional rehabilitation. The biologic half-life of elimination was significantly longer (p less than 0.01) in severely malnourished children (median, 10.21 hours; range, 4.89 to 22.93 hours) than in rehabilitated children (median, 5.09 hours; range, 2.61 to 8.75 hours). Metabolic clearance of metronidazole was significantly lower in group I (p less than 0.01; median, 0.077 L/kg/hr; range, 0.033 to 0.192 L/kg/hr) than in nutritionally rehabilitated children (median, 0.166 L/kg/hr; range, 0.105 to 0.300 L/kg/hr). Volume of distribution was not different between groups I and II, although both showed higher values than the values reported for children who were not malnourished. These findings suggest that the dose of metronidazole should be reduced in malnourished children, and the therapeutic regimen should be individualized for each patient. PMID- 1732077 TI - Drug-induced hypothyroidism: the thyroid as a target organ in hypersensitivity reactions to anticonvulsants and sulfonamides. AB - Inherited defects in detoxification of reactive metabolites of drugs predispose patients to "hypersensitivity" reactions. Covalent interaction of metabolites with cell macromolecules leads to cytotoxic and immunologic outcomes, manifested clinically by multisystem syndromes with variable organ involvement. Hypothyroidism developed in 5 of 202 patients (age range, 1 to 81 years) we investigated for hypersensitivity reactions to anticonvulsants or sulfonamides shortly after their reaction. None had previous personal or family histories of autoimmune disease. All had low thyroxine levels, elevated levels of thyroid stimulating hormone, and autoantibodies including antimicrosomal antibodies. Patients were 2 to 18 years of age at presentation, and two were male. All returned to a euthyroid state within a year of presentation, and all remain well. The demographics, clinical presentation, and course of the patients is atypical of idiopathic lymphocytic thyroiditis. We investigated the pathogenesis of thyroid toxicity using the hydroxylamine metabolite of sulfamethoxazole as a model. The hydroxyalmine was toxic to thyroid cells in vitro, which did or did not express thyroid peroxidase activity, whereas the parent sulfonamide was toxic only to cells with active thyroid peroxidase. The purified enzyme converted sulfamethoxazole to the hydroxylamine. Formation of reactive drug metabolites by thyroid peroxidase in a host who is genetically unable to detoxify the metabolites may lead directly to cytotoxicity. Covalent binding to macromolecules, including thyroid peroxidase, also may lead to expression of neoantigens and formation of autoantibodies. Patients who have sustained hypersensitivity reactions to drugs should be investigated for possible involvement of the thyroid. PMID- 1732078 TI - Vascular reactivity to phenylephrine and angiotensin II: comparison of direct venous and systemic vascular responses. AB - The objective of this study was to determine the relationship between peripheral venous responsiveness (use of the dorsal hand vein compliance technique) and systemic vascular responsiveness (measurement of blood pressure changes) to phenylephrine and angiotensin II in humans. There was a significant correlation (r = 0.70, p less than 0.02) between the dose causing a 20 mm increase in mean arterial pressure and the dose producing half-maximal response in the hand vein (log ED50) for phenylephrine but not for angiotensin II. There was no correlation between the systemic responses to angiotensin II and phenylephrine, but there was a significant correlation (r = 0.70, p less than 0.02) between the log ED50 measurements for phenylephrine and angiotensin II in the hand vein experiments. These findings suggest that systemic and hand vein responsiveness to phenylephrine are similar. Consequently, in evaluating alpha-adrenergic receptor mediated responses, the dorsal hand vein compliance approach offers a satisfactory alternative to the use of systemic hemodynamic changes. PMID- 1732080 TI - Most elderly go gently. PMID- 1732079 TI - A pharmacodynamic model of erythropoietin therapy for uremic anemia. AB - Fifty-seven patients receiving chronic high-flux hemodialysis began receiving recombinant alpha-human erythropoietin (rHuEPO). The mean initial rHuEPO dose used in 54 evaluable patients was 9963 +/- 4364 U/week; the final dose was 8972 +/- 4058 U/week. Treatment over a mean period of 154 +/- 40 days (84 to 224 days) resulted in an average increase in hematocrit from 24.7% +/- 3.7% to 32.5% +/- 4.4%. We present a model for these data that describes changes in hematocrit during rHuEPO therapy and that allows simultaneous estimation of red blood cell lifespan and rHuEPO-induced increases in red blood cell production rate. Analysis of the hematocrit values of the patients with the model, by use of NONMEM, a computer program for analysis of population data, reveals a nonlinear dose response relationship with large interindividual variability (coefficient of variation) of about 50%. The estimated mean red blood cell lifespan is 64 days, with interindividual variability of about 30% (coefficient of variation). The intraindividual random variability in hematocrit about its prediction is +/- 5% of the prediction. For clinical dose adjustment, we present a method that uses only simple calculations. PMID- 1732081 TI - Automatic cardioverter-defibrillator. PMID- 1732082 TI - Fever: pathology and treatment. PMID- 1732083 TI - Self-scheduling in critical care. PMID- 1732084 TI - Postoperative GI symptoms in cardiac surgery patients. PMID- 1732085 TI - Hyperglycemia during nutrition support. PMID- 1732086 TI - Students' knowledge and perceptions of critical care nursing. PMID- 1732087 TI - Critical incident stress management for care providers in the pediatric emergency department. AB - Critical Incident Stress (CIS) is any event that evokes a critically high level of stress and makes usual coping skills ineffective. Programs to manage this stress have been developed. Due to the unique stressors encountered in the pediatric emergency department, this article proposes a CIS management program for these healthcare providers. PMID- 1732088 TI - Geriatric urinary incontinence. AB - Urinary incontinence (UI) is now recognized as a prevalent, physically and emotionally disruptive, and costly health problem in the geriatric population. Because incontinence may be a manifestation of a subacute or reversible process within or outside of the lower urinary tract, and because effective treatment is available, it is important for primary care physicians to identify and appropriately assess incontinence in their geriatric patients. The initial evaluation of an incontinent geriatric patients. The initial evaluation of an incontinent geriatric patient includes a targeted history and physical examination, urinalysis, and simple tests of lower urinary tract function. Potentially reversible conditions that may be causing or contributing to the incontinence, such as delirium and urinary tract infection (UTI), should be identified and managed. Patients who may benefit from further testing, including urologic or gynecologic examination and/or complex urodynamic tests, should be identified and referred. Several therapeutic modalities can be used to treat geriatric UI. Behavioral therapies are noninvasive and effective, both in functional community-dwelling geriatric patients and in functionally impaired nursing home residents. Behavioral therapies include bladder training, pelvic muscle exercises, biofeedback, scheduled toileting, habit training, and prompted voiding. Pharmacologic therapy is often used in conjunction with behavioral therapy. For stress incontinence, alpha-adrenergic drugs are used and can be combined with topical or oral estrogen therapy in women. For urge incontinence, pharmacologic treatment involves drugs with anticholinergic and direct bladder muscle relaxant properties. Pharmacologic therapy for overflow incontinence is generally not effective on a long-term basis. Surgical treatment is indicated when a pathologic lesion such as a tumor is diagnosed, or when anatomic obstruction is believed to be the cause of the patient's symptoms. Surgical treatment of stress incontinence can be highly effective in properly selected women. Nonspecific, supportive treatments are also important in managing geriatric UI. Education for patients and caregivers is critical for the success of most therapies. Environmental manipulations and the appropriate use of toilet substitutes are especially important in frail, functionally impaired patients. Highly absorbent adult undergarments are helpful for managing many patients, but should not be used as the initial response to incontinence, and are best used in conjunction with more specific treatment whenever possible. Chronic indwelling catheterization should only be used to manage incontinence when it is associated with clinically significant urinary retention, skin conditions that cannot heal because of incontinence, or severe illness that makes the catheter the most comfortable method of management.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732089 TI - Focus on radiology: Part II. PMID- 1732090 TI - Ultrasonography in the emergency setting. AB - US is the imaging modality of choice in many situations encountered in the Emergency Department. It is particularly useful in evaluating renal colic, pain or vaginal bleeding in the pregnant patient, and pelvic pain in the nonpregnant woman; and in diagnosing gallbladder disease, appendicitis, proximal lower extremity DVT, and pericardial effusion. The information presented in each section, including sonographic findings and the role of US, should be helpful in choosing the most appropriate test in the evaluation process. PMID- 1732091 TI - Imaging for deep venous thrombosis. AB - The diagnosis of DVT is a perplexing clinical challenge for the emergency physician. The algorithm depicted in Figure 9 from Hobson et al represents a logical strategy for the application of noninvasive studies. Positive studies in the proper setting that are reliably interpreted can dictate anticoagulant therapy, but equivocal or uninterpretable tests must be followed by invasive testing to exclude clot. Negative tests require close follow-up and serial evaluation to detect potentially silent early clot formation. PMID- 1732092 TI - Fractures and dislocations of the wrist. AB - Wrist injuries are and will continue to be one of the most difficult and interesting orthopedic problems encountered by emergency physicians. With a knowledge of basic wrist anatomy, the gathering of a detailed history of the mechanism of injury, thorough physical examination, and appropriate radiography, the physician will be able to make an accurate early diagnosis. This will prevent the development of serious functional problems such as osteonecrosis or malunion with all their attendant pain and disability, which is especially serious for the young active worker or athlete. A thorough radiographic examination should be done, the six-view series being the most revealing initial study, particularly for possible scaphoid fractures. All injured wrists should be properly immobilized and re-evaluated if a fracture or serious ligamentous injury is at all suspected. An appreciation of the simplicity of design and remarkable functional complexity of the wrist together with a respect for the subtlety of presentation of injury will help the emergency physician to ensure that patients have highest standard of care and an excellent outcome. PMID- 1732093 TI - Commonly missed orthopedic problems. AB - Orthopedic problems that are often "missed" when the patient presents to the Emergency Department are discussed. Treatment suggestions for these conditions are made. PMID- 1732094 TI - Radiologic evaluation of foreign bodies. AB - The correct selection of a radiologic imaging modality along with knowledge of indirect radiologic findings can help determine the presence and location of a foreign body. Plain radiographs should be the initial screening modality for a suspected foreign body. Whereas most metal and glass foreign bodies are detectable on radiographs, many foreign bodies, including wood, are not. We do not advocate using xeroradiography for the detection of foreign bodies. When a suspected superficial foreign body is not delineated on radiographs, ultrasonography should be the next modality of choice. CT should be reserved for deep foreign bodies or when foreign bodies are not seen on radiographs or ultrasonography but are suspected. PMID- 1732095 TI - Legal issues in emergency radiology. Practical strategies to reduce risk. AB - Various joint commission and individual state standards affect emergency radiology practice and have legal implications. The ACEP has entered the burgeoning field of practice guidelines; fortunately, their practice guideline preparation system is arguably the most thorough in medicine at this time. This is of great importance to emergency physicians, because practice guidelines are not without their own potential legal, educational, and compliance problems. Trends toward cost-reduction through reduced radiograph utilization are a real phenomenon in all of medicine. It remains to be seen if costs can be reduced without increasing legal exposure. Excellent clinical guidelines are helpful to emergency physicians. The Massachusetts ACEP model, which appears to effectively blend closed claim data into practice guidelines, has built-in educational support and compliance incentives. This model may become a national standard in the future. Pediatric radiology risk management issues, such as the statute of limitations, have been discussed. Emergency physicians should be knowledgeable about the most commonly missed pediatric emergency radiologic diagnoses. Some aspects of common emergency radiology practice patterns are of relevance to a discussion of risk management in emergency radiology. Issues such as emergency physician radiograph interpretation responsibilities and follow-up systems emerge repeatedly in malpractice claims. The use of CQI strategies may prove helpful in improving practice patterns. Communication between emergency physician and radiologists is critical. Good communication requires the development of good rapport and should pay dividends in improved patient care. PMID- 1732096 TI - Ultrasonography in emergency medicine. AB - The use of ultrasonography in emergency medicine is an area of rapid growth and controversy. This article reviews the current and future applications of emergency ultrasonography with particular emphasis on the role of bedside scanning by the emergency practitioner. Abdominal, pelvic, and cardiac ultrasonographic applications are reviewed, as are the uses of ultrasonography as an adjunct to the performance of procedures in the Emergency Department. PMID- 1732097 TI - Emergency echocardiography. AB - Emergency physicians need an understanding of the utility of echocardiography in the Emergency Department. With the recent emphasis of emergency department use of portable ultrasonography, emergency physicians will have the opportunity to gain proficiency in using echocardiography to diagnose certain conditions. Echocardiography may aid in the diagnosis of acute MI, pericardial effusion and tamponade, acute valvular dysfunction, acute aortic dissection, and post traumatic cardiac disorders. An understanding of the potential limitations of echocardiography, combined with experience in its techniques, will ultimately help the emergency physician with its use in daily patient care. PMID- 1732098 TI - Emergency imaging of the urinary tract. AB - Conventional radiography remains the mainstay of emergency imaging of the urinary tract; however, computed tomography in trauma and ultrasonography in medical conditions have greatly improved the accuracy and speed with which diagnoses can be made and treatment instituted. This article reviews the imaging modalities available to the emergency department physician and their application in the most common emergency situations. PMID- 1732099 TI - Scrotal scintigraphy in testicular torsion. AB - Testicular torsion is a surgical emergency that requires prompt diagnosis and treatment to maximize testicular survival. When the clinical diagnosis is uncertain, testicular scintigraphy can be performed to evaluate testicular perfusion. This noninvasive imaging study is widely validated and highly accurate in predicting nontorsion, which excludes the need for exploration. PMID- 1732100 TI - Centromere detection in vinblastine- and radiation-induced micronuclei of cytokinesis-blocked mouse cells by using in situ hybridization with a mouse gamma (major) satellite DNA probe. AB - Non-isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis-blocked micronucleus assay a dose-dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere-positive hybridization signals in an order of magnitude of 70 90%, but after radiation exposure the magnitude was only 10-20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis-blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy-inducing and clastogenic agents. PMID- 1732101 TI - Direct-acting mutagenic activity in white, rose, and red wines with the Ara test of Salmonella typhimurium. AB - Thirty-two commercially produced white, rose, and red wines from Spain were assayed for genotoxicity. The Ara forward mutagenicity assay with Salmonella typhimurium served as the test system. All the wines were mutagenic in the absence of mammalian microsomal activation (S9 mix) and/or glycosidase activities with the exception of one rose wine which gave a clear dose-response relationship, although its mutagenic potency was considered statistically nonsignificant. The mutagenic activity covered nearly a 30-fold range. Compared to white and rose wines, red wines showed the highest levels of mutagenicity; this wine type included four "very potent" (greater than 3,000 AraR mutants/ml) mutagenic wines. The level of wine mutagenicity did not correlate with either the region or the year of production (vintage). Individual winery methods are suggested as primarily responsible for variations in mutagenic activity. The present study with the Ara test supports the possibility that wine components other than the flavonols quercetin and rutin are the major putative mutagens: (1) white wines, as well as rose or red wines, were detected as being mutagenic; (2) in no case was activation required for the detection of mutagenicity; (3) mutagen(s) were detected mainly (red wine) when not exclusively (white and rose wine) in the polar fraction from XAD-2 chromatography. The high sensitivity of the Ara test has allowed the screening of the mutagenicity of a variety of wines with no previous process of extraction or concentration. The comparison of the mutagenic activity of the entire complex mixture to that of its lyophilized residue has revealed a positive synergistic role for ethanol in the mutagenicity of certain wines. Finally, this work suggests that the Ara test is a useful tool for mutagenicity screening in wines. Thus, this test might play an important role in elucidating the genotoxic mechanism of action of alcoholic beverages, and for studying optional production methods to decrease the mutagenicity of commercial wines. PMID- 1732102 TI - Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes. AB - The human lymphocyte micronucleus (MN) assay is relatively insensitive to genotoxic agents that predominantly induce excision-repairable lesions such as adducts and abasic sites. In this study we have explored the possibility of using cytosine arabinoside (ARA) to convert excision-repairable DNA lesions to micronuclei (MN) within one cell cycle. The system consisted of human lymphocytes as target cells, the cytokinesis-block (CB) method for identifying cells that had completed one nuclear division only, and X-rays, methylnitrosourea (MNU), and ultraviolet light (UV) as mutagens. With each mutagen we have observed significant increments in induced MN in the cultures that had also been treated with ARA during G1. The slope of the dose-response curves for induction of MN was increased by a factor of approximately 1.8 for X-rays and 10.3 for UV and significant MNU induction of MN was only achieved in the cultures treated with ARA. Furthermore, a 24-hr gap between mutagen exposure and the start of the assay did not abolish the increased sensitivity in the cultures treated with ARA. These observations suggested that the combined ARA and cytokinesis-block micronucleus (CBMN) method may enhance the detection of exposure to genotoxic agents that predominantly induce excision-repairable lesions. PMID- 1732103 TI - A QSAR investigation of the role of hydrophobicity in regulating mutagenicity in the Ames test: 1. Mutagenicity of aromatic and heteroaromatic amines in Salmonella typhimurium TA98 and TA100. AB - Quantitative structure-activity relationships (QSAR) have been derived for the mutagenic activity of 88 aromatic and heteroaromatic amines acting on Salmonella typhimurium TA98 + S9 and 67 amines acting on TA100 + S9. Mutagenic activity is linearly dependent on hydrophobicity, the energy of the highest occupied molecular orbital, and the energy of the lowest unoccupied molecular orbital of the amine. The dependence of mutagenic activity on hydrophobicity and electronic effects is nearly identical for TA98 and TA100. Mutagenic activity in TA98 is also found to depend on the size of the aromatic ring system. Different QSARs are derived for the mutagenic activity of hydrophilic amines (log P less than 1) acting on either TA98 or TA100. The mechanism of amine activation and reaction with DNA is considered in light of these findings. PMID- 1732104 TI - Quantitative structure-activity relationship investigation of the role of hydrophobicity in regulating mutagenicity in the Ames test: 2. Mutagenicity of aromatic and heteroaromatic nitro compounds in Salmonella Typhimurium TA100. AB - A quantitative structure-activity relationship (QSAR) has been derived for the mutagenic activity of 117 aromatic and heteroaromatic nitro compounds acting on Salmonella typhimurium TA100. Relative mutagenic activity is bilin-early dependent on hydrophobicity, with an optimal log P of 5.44, and is linearly dependent on the energy of the lowest unoccupied molecular orbital of the nitro compound. The dependence of mutagenic activity on hydrophobicity and electronic effects is very similar for TA98 and TA100. Mutagenic activity in TA100 does not depend on the size of the aromatic ring system, as its does in TA9. The effect of the choice of assay organism, TA98 versus TA100, on nitroarene QSAR is seen to be similar to the effect previously found for aminoarenes. Lateral verification of QSARs is presented as a tool for establishing the significance of a new QSAR. PMID- 1732105 TI - Molecular analysis of mutations affecting hprt mRNA splicing in human T lymphocytes in vivo. AB - Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, we observed i) complete loss of one or more exons, ii) partial loss of one exon, or iii) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns. Furthermore, genomic deletions encompassing entire exons were found. In some mutants, the alteration responsible for incorrect splicing could not be identified, suggesting that the target sequence for splice mutations is larger than merely the splice junctions. Molecular characterization of hprt splice mutations will lead to the identification of specific sequences regulating splicing of hprt mRNA and will reveal whether the mutational spectrum in splice mutants is similar to that found in the hprt coding region. PMID- 1732106 TI - In vivo genotoxicity studies with p-benzoquinone dioxime. AB - P-Benzoquinone dioxime (BQD) appears to be a sex-specific rat carcinogen inducing tumours of the urinary bladder in female rats. The present paper shows that BQD is a direct-acting mutagen in Salmonella typhimurium TA98, confirming published data. In contrast to this in vitro data, negative results were obtained after oral administration of BQD to female rats in both the bone marrow micronucleus test and the in vivo liver UDS test. BQD did, however, induce a marked effect upon S-phase synthesis in the livers of female rats between 14 and 48 hr after a single oral dose of 250 mg/kg. A similar effect was also observed in the livers of male rats. There was no evidence of hepatotoxicity (in terms of elevated liver enzyme levels) after treatment of female rats with the compound indicating that the increase in cell proliferation was due to a direct mitogenic effect of BQD in this organ. Some liver mitogens have been found to be liver carcinogens; this does not appear to be the case for BQD. Nevertheless, the mitogenic activity of this compound might play a contributory role to the induction of bladder cancer in rats if it also acted as a mitogen in this tissue. Further studies are indicated, measuring genotoxicity and cell-proliferative activity in the bladder in order to further elucidate the mechanism of action of this compound as a rodent carcinogen. PMID- 1732107 TI - Cytogenetic effects of alachlor and/or atrazine in vivo and in vitro. AB - The purpose of this study was to assess the cytogenetic effects of two commonly used herbicides, alachlor and atrazine, which are often found together in groundwater. Chromosome damage was examined in bone marrow cells of mice drinking water containing 20 ppm alachlor and/or 20 ppm atrazine, with an immunosuppressive dose of cyclophosphamide used as a positive control. Chromosome damage was also quantified in human lymphocytes exposed in culture to 1.0, 0.1, or 0.01 microgram/ml alachlor and/or atrazine. The in vitro study demonstrated dose related cytogenetic damage not associated with mitotic inhibition or cell death, with damage due to the alachlor-atrazine combination suggesting an additive model. The in vivo study also suggested additive damage due to the alachloratrazine combination after 30 days of treatment, but, unexpectedly, demonstrated less cytogenetic damage and fewer cells with multiple aberrations after 90 days. Also, at 90 days, all treated mice had elevated mitotic indices compared to controls. The fact that the elevated mitotic index was associated with immune suppression in the cyclophosphamide group suggests that death of cells with accumulated chromosomal aberrations resulted in increased bone marrow proliferation, so a higher fraction of cells examined were newer with less damage. Since the alachlor-atrazine combination treated mice showed little systemic toxicity despite bone marrow mitotic indices similar to the cyclophosphamide treated animals, as well as a similar decrease in cytogenetic damage at 90 days compared to 30 days, cell death and replacement must also be involved but cannot completely explain the results. PMID- 1732108 TI - Rational approach to the quantification of genotoxicity. AB - The question of how many and which short-term tests (STT) are necessary for a satisfactory characterization of the genotoxic properties of chemicals is still open. The answer is important for both basic mutagenicity research and risk assessment. This paper, aimed at giving a rational answer to the problem, analyzes with multivariate statistical methods the data generated by the International Program for the Evaluation of Short-Term Tests for Carcinogens (IPESTTC). Although it has been found that this data base has a limited reliability for assessing the ability of STTs to predict carcinogenicity, the IPESTTC results are an important source of information on the relationships among different assays, and their ability to identify genotoxic chemicals. A scale of genotoxicity of the chemicals was established by studying with factor analysis their results in 20 IPESTTC tests. The next step of the analysis consisted in the identification of the STT batteries which are the most able to reproduce the genotoxicity scale based on the entire set of STTs. Different batteries were ranked according to their ability to quantify genotoxicity. As a general conclusion, this study indicated that an articulated range of STTs is necessary, and it is not possible to use only one assay (e.g., Salmonella) as an exhaustive indicator of genotoxicity. PMID- 1732109 TI - Impact of a quality assurance program on gastrointestinal endoscopy. AB - The impact of a quality assurance committee on documentation and use of gastrointestinal endoscopy was assessed. The committee, fulfilling Joint Commission on Accreditation of Healthcare Organizations criteria, performed retrospective (1984-1985) and prospective (1986-1988) reviews of all endoscopies. Criteria were developed from American College of Physicians and American Society for Gastrointestinal Endoscopy guidelines. All reviews of procedures that were questioned were returned to physicians for clarification. After reconsideration of the response, procedures were judged either justified or unjustified. There has been significant improvement in the quality of endoscopy reporting and documentation. The rate of questioned procedures decreased from 21.6% (95% confidence interval (CI), 20.1-23.1) in 1984-1985 to 9.2% (95% CI, 7.9-10.4) (P less than 0.01) in 1988. Improvement in use was reflected in the significant decrease in the rate of unjustified procedures from 8.2% (95% CI, 7.2-9.2) in 1984-1985 to 1.5% (95% CI, 1.0-2.0) (P less than 0.01) in 1988. Most importantly, this process curtailed the previously noted 10% annual increase in the number of endoscopic procedures (P less than 0.01). PMID- 1732110 TI - Effects of 2-camphanone on canine portal vein blood flow and rat smooth muscle. AB - 2-Camphanone is under clinical evaluation for alleviation of hemorrhoidal bleeding and inflammation. Reduced portal venous blood flow may distend, whereas improved portal venous blood flow may alleviate, hemorrhoidal vein distention. The effects of 2-camphanone on canine portal venous blood flow were investigated using pulsed Doppler flow techniques and on the spontaneous contractions of the isolated rat portal vein. Both intravenous (0.06, 0.2, and 0.6 mg/kg) and transdermal (6 mg/dog on the thigh) administration of 2-camphanone to dogs anesthetized with pentobarbital sodium increased portal venous flow velocity by 20%-30% without affecting femoral arterial blood flow, heart rate, or arterial blood pressure compared with vehicle-treated animals. Transdermal administration of 0.6, 2, and 6 mg of 2-camphanone, in a volume of 0.1 mL, to rats decreased the spontaneous contractions of the isolated rat portal vein in vitro. The data suggest that 2-camphanone exhibits a relatively selective effect on portal venous smooth muscle to reduce venous congestion and increase blood flow velocity. 2 Camphanone may be useful in the treatment not only of hemorrhoids, but also of esophageal reflux and portal hypertension. PMID- 1732111 TI - Involvement of the L-arginine-nitric oxide pathway in internal anal sphincter relaxation. AB - The purpose of this study was to examine the role of the L-arginine-nitric oxide pathway in neurogenic relaxation of the internal anal sphincter. Muscle strips representing the internal anal sphincter were prepared from 17 adult opossums. The preparations were mounted in organ baths for recording of isometric tension. N omega-nitro-L-arginine, an agent known to inhibit the L-arginine-nitric oxide pathway, concentration-dependently reduced relaxations induced by transmural field stimulation. At the highest concentration of N omega-nitro-L-arginine (10( 4) mol/L), no relaxation was evoked at any frequency tested (0.5-40 Hz). The inhibitory response to exogenous vasoactive intestinal polypeptide was unaffected by N omega-nitro-L-arginine pretreatment, indicating that vasoactive intestinal polypeptide relaxation does not use the L-arginine-nitric oxide pathway. In addition, responses to forskolin and sodium nitroprusside were not influenced by N omega-nitro-L-arginine preincubation, suggesting that the effect observed was not caused by a direct influence on the adenylate or the guanylate cyclases. It is concluded that the nonadrenergic, noncholinergic innervation of the internal anal sphincter involves an inhibitory substance generated from the L-arginine nitric oxide pathway. Whether this substance is nitric oxide or a related nitroso compound remains to be settled. PMID- 1732112 TI - Clostridium difficile toxin B disrupts the barrier function of T84 monolayers. AB - The contribution of toxin B to Clostridium difficile-associated infection is undefined. Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood. The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin. It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence. To address these questions, we examined the effect of C. difficile toxin B on the phenotype and barrier function of T84 cell monolayers. Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84). In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters. Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function. Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques. Flux studies confirm that the permeability defect is at the level of the tight junction. We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function. There was no evidence of cell death as determined by lactate dehydrogenase release assays. PMID- 1732113 TI - Lewis antigen alterations in gastric cancer precursors. AB - To explore the dynamics of the progressive loss of cell differentiation observed in the gastric precancerous process, the abnormal expression of Lea antigen in the gastric epithelium was investigated. Gastric biopsy specimens of 122 subjects with Le(a-b+) phenotype who had intestinal metaplasia of the gastric mucosa were studied. The subjects are residents of a rural area in the Colombian Andes with very high risk of gastric cancer. The abnormality was detected with increasing frequency in lesions with other markers of progression of the precancerous process, namely, colonic-type of morphology of the metaplastic cells, expression of sulfomucins, and dysplastic changes. The concomitant expression of the abnormal Lea antigen and sulfomucins was found to be a more reliable marker of more advanced lesions such as colonic metaplasia and dysplasia than either marker alone. PMID- 1732114 TI - Cellular localization of procollagen gene transcripts in inflammatory bowel diseases. AB - The cellular localization of procollagen types I, III, IV, and V gene transcripts was determined in tissues from 12 patients with either Crohn's disease (CD) or ulcerative colitis (UC) and nine controls by in situ hybridization with 35S labeled RNA probes. In CD, the signal intensity and number of labeled cells were significantly increased, particularly in deeper intestinal layers. In contrast, the labeled cells in UC were concentrated in the subepithelial intestinal layers, with an overexpression of procollagen III RNA transcripts. Immunohistological stainings for procollagen types I, III, and IV showed a weaker staining in UC than in CD, indicating that increased transcript levels in UC are unrelated to the enhanced collagen protein deposition, although the increase of procollagen messenger RNA levels correlated with the density of the inflammatory infiltrate. It was concluded that both CD and UC show highly increased procollagen RNA transcript levels but differ in collagen deposition. Thus, different posttranscriptional or posttranslational regulatory mechanisms, such as collagen degradation, may account for the observed differences. PMID- 1732115 TI - Transport of glucose polymer-derived glucose by rabbit jejunum. AB - Mechanisms for the assimilation of glucose polymers have been inferred from perfusion studies. To further define these mechanisms, the results of measurements of unidirectional glucose fluxes across short-circuited rabbit jejunal segments in vitro are reported. Glucose polymer-stimulated short-circuit current was similar to that of glucose [19 +/- 6.0 microA/cm2 (n = 7) and 26 +/- 5.7 microA/cm2 (n = 13), respectively] and was inhibited by both acarbose and phlorizin. Acarbose, an alpha-glucosidase inhibitor with no effects of glucose transport, was used to uncouple digestion from absorption. Mucosal-to-serosal flux of glucose polymer-derived glucose was lower than that of an equal weight/volume of glucose [124 +/- 62 nmol.h-1.cm-2 (n = 4) vs. 452 +/- 121 nmol.h 1.cm-2 (n = 6); P less than 0.05] and was inhibited by both phlorizin and acarbose. No glucose polymers were detected in the serosal bath solutions by thin layer chromatography. It is concluded that glucose polymer-derived glucose is transported by a phlorizin-inhibitable process at a rate slower than that of free glucose, a finding that suggests that hydrolysis limits glucose polymer assimilation. PMID- 1732116 TI - Acute noncardiac chest pain in a coronary care unit. Evaluation by 24-hour pressure and pH recording of the esophagus. AB - Twenty-four-hour recording of esophageal pressure and pH was performed successfully in 41 patients admitted to the coronary care unit of a general hospital who had an episode of acute, prolonged retrosternal chest pain and who were initially suspected of suffering from coronary artery disease (severe angina pectoris, myocardial infarction), but in whom the pain was subsequently shown not to be of cardiac origin. The recordings were analyzed with fully automated techniques. A pain episode was considered to be related to abnormal esophageal motility when contraction amplitudes or durations in the pain episode exceeded the patient's upper limit of normal (97.5th percentile) or when the proportion of abnormal propagated contractions (simultaneous, nontransmitted) in the pain episode was significantly increased (chi 2 test). Thirty patients (73%) had one or more pain episodes (in total 63 pain episodes) during the 24-hour recording. Forty-three percent of the pain episodes was related to abnormal motility and 30% to reflux, and 27% was not related to esophageal function disturbance. Using the criterium that the symptom index had to be greater than or equal to 75%, it was found that the pain was related to reflux in 13 patients (43%) and to motor abnormalities in 10 patients (33%). It is concluded that in the majority of patients acutely admitted with noncardiac chest pain, esophageal motor abnormalities and reflux can be shown to be the likely cause of the symptoms. PMID- 1732117 TI - Acute exposure of small intestine to ethanol induces mucosal leakage and prostaglandin E2 synthesis. AB - The small intestines of healthy volunteers were challenged with ethanol during regional perfusion of a defined jejunal segment. Infusion of 30 mL of 5000 mmol/L ethanol to the perfused jejunal segment gave a maximum ethanol concentration of 973 +/- 98 (SEM) mmol/L in the jejunum lumen. This ethanol challenge induced within 20-30 minutes a 10-fold increase in albumin (P less than 0.001) and a two fold increase in the glycosaminoglycan hyaluronic acid (P less than 0.05) in the perfusion fluid. Later during the challenge and simultaneously with a decreased jejunal loss of albumin, the jejunal recovery of prostaglandin E2 increased fourfold (P less than 0.01). The jejunal fluid concentrations of histamine and eosinophil cationic protein remained stable during the ethanol challenge. No changes in the jejunal appearance of albumin or other measured substances were seen when the maximum jejunal fluid concentrations of ethanol were less than 400 mmol/L achieved during challenge with smaller amounts of ethanol. The increased jejunal fluid appearance of hyaluronic acid after ethanol challenge indicates increased leakage from the interstitial/lymph fluid of the gut wall due to altered mucosal permeability. The relatively larger jejunal losses of albumin suggest that ethanol induces increased microvascular permeability of the jejunum as well. PMID- 1732118 TI - Autonomous proliferation of colon cancer cells that coexpress transforming growth factor alpha and its receptor. Variable effects of receptor-blocking antibody. AB - Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions. PMID- 1732119 TI - Influence of meal composition on canine jejunal water and electrolyte absorption. AB - The absorption of water and electrolytes from the proximal jejunal lumen increases immediately after a meal. This meal-induced jejunal absorption occurs in jejunal segments out of normal gastrointestinal continuity. This study was designed to characterize the jejunal absorptive response to a series of isovolumetric gavage-delivered stimuli. Twenty-five-centimeter canine proximal jejunal Thiry-Vella fistulas were constructed, and jejunal absorption studies (n = 66) were performed by luminal perfusion of the jejunal segments with an isotonic buffer containing 14C-labeled polyethylene glycol. Each study consisted of a 1-hour basal period, followed by a 3-hour experimental period. Nine groups were studied, each receiving one of the following isovolumetric stimuli delivered via the gavage route: water, 0.9% saline, mixed meal, protein, lipid, carbohydrate, and mannitol (150 mmol/L, 300 mmol/L, and 600 mmol/L). The water and 0.9% saline gavage groups showed no significant changes in integrated postprandial water and electrolyte absorption above basal. The isocaloric mixed meal, protein, lipid, carbohydrate, and mannitol groups all had significantly increased integrated postprandial jejunal water and electrolyte absorption above basal (P less than 0.05). These results indicate that a proabsorptive signal for meal-induced jejunal absorption originates from or distal to the stomach. Meal induced jejunal absorption occurs in response to nutrients of diverse composition and is also responsive to nonnutritive solutes such as mannitol. These findings support a new role for gastric or intestinal chemo- or osmo-receptors in stimulating the neurohumoral mechanisms that mediate meal-induced jejunal absorption. PMID- 1732120 TI - Factors influencing the eradication of Helicobacter pylori with triple therapy. AB - Helicobacter pylori infection has been associated with gastritis, duodenal ulcer, gastric ulcer, and the epidemic form of gastric carcinoma. Eradication of H. pylori infection has proven to be difficult. Recently, combinations of antimicrobial drugs have been shown to eradicate greater than 50% of infections; however, the results have proven variable, and the factors influencing effectiveness of therapy are unclear. In the present study, the effectiveness of a triple therapy for eradication of H. pylori infection was evaluated. Triple therapy consisted of 2 g tetracycline, 750 mg metronidazole, and five or eight tablets of bismuth subsalicylate daily in 93 patients (70 with duodenal ulcer, 17 with gastric ulcer, and 6 with simple H. pylori gastritis). Combinations of a sensitive urea breath test, serology, culture, and histology were used to confirm the presence of infection, eradication, or relapse. Eradication was defined as inability to show H. pylori greater than or equal to 1 month after ending therapy. The overall eradication rate was 87%. The factors evaluated for their effect on predicting eradication included age, gender, type of disease, duration of therapy, amount of bismuth subsalicylate [five or eight Pepto-Bismol tablets daily (Procter & Gamble, Cincinnati, OH)], and compliance with the prescribed medications. Stepwise regression showed that compliance was the most important factor predicting success; the success rate was 96% for patients who took greater than 60% of the prescribed medications and 69% for patients who took less. For those taking greater than 60% of the prescribed therapy, the eradication rates were similar (a) for patients receiving therapy for 14 days or when tetracycline and bismuth subsalicylate were taken for an additional 14 days; (b) for patients with duodenal ulcer, gastric ulcer, and simple H. pylori gastritis; and (c) whether five or eight bismuth subsalicylate tablets were taken. It is concluded that triple therapy is effective for eradication of H. pylori and that future studies need to take compliance into account for comparisons between regimens. PMID- 1732121 TI - Ion transport in the experimental short bowel syndrome of the rat. AB - The adaptational changes of epithelial ion transport in the short bowel syndrome were studied. Ileal remnants of rats were investigated 8 weeks after 70% proximal small intestinal resection. Pure epithelial resistance measured by impedance analysis decreased from 27 +/- 1 to 21 +/- 1 omega.cm2, and polyethylene glycol 4000 fluxes increased from 2.5 +/- 0.3 to 3.6 +/- 0.3 nmol.h-1.cm-2, indicating increased permeability of the short bowel. Unidirectional flux measurements in control ileum showed absorptive net fluxes of Na+ and Cl- that were assigned to electroneutral NaCl absorption and a short-circuit current that was accounted for by the residual flux (HCO3- secretion). Neither NaCl absorption nor HCO3- secretion were altered in the short bowel. Also, electrogenic Cl- secretion, defined after maximal stimulation by theophylline and prostaglandin E1 was not changed in the short bowel. In contrast, electrogenic Na+/glucose cotransport increased in Vmax from 2.0 +/- 0.3 in controls to 5.0 +/- 1.0 mumol.h-1.cm-2 in the short bowel. Tight junction structure was studied by freeze-fracture electron microscopy. The number of horizontal strands was unchanged, whereas tight junction depth was slightly increased in the short bowel. Microvillus area of short bowels was increased by 20% in villus regions. Under the light microscope, villus height was increased by 30%. In conclusion, the short bowel mucosa undergoes adaptive responses to reduced overall absorptive area by increasing glucose-dependent electrogenic Na+ absorption to 250%, which is partly caused by increased villus and microvillus surface area. Electrogenic Cl- and HCO3- secretion and electroneutral NaCl absorption remained unchanged. The decreased epithelial resistance is caused by mucosal surface amplification. PMID- 1732122 TI - Pattern of gastrointestinal and somatic symptoms across the menstrual cycle. AB - The pattern of gastrointestinal symptoms and select mood and somatic symptoms was examined across two menstrual cycles in women with (n = 19) and without (n = 39) functional bowel distress (FBD). The women (a) rated their gastrointestinal, perimenstrual, mood, and other symptoms and stool frequency and consistency daily; (b) completed the Menstrual Distress Questionnaire-T; and (c) had serum levels of estrogen and progesterone measured during the menses, follicular, and luteal phases. Stomach pain, nausea, and diarrhea were rated higher at menses in the group with FBD than in the group without FBD. Stomach pain was higher during the remaining days as well. The group with FBD reported higher levels of perimenstrual symptoms also on six of the eight Menstrual Distress Questionnaire T subscales (P less than 0.01). Other complaints, e.g., poor work/school performance, were higher in women with FBD, but somatic symptoms that were expected to vary over the cycle did not differ between groups, except cramping pain. There were no significant group differences in ovarian hormone levels or stool consistency/frequency scores. PMID- 1732123 TI - Comparison of triplicated (S) and quadruplicated (W) pelvic ileal reservoirs. Studies on manovolumetry, fecal bacteriology, fecal volatile fatty acids, mucosal morphology, and functional results. AB - Capacity and compliance, efficiency of evacuation, fecal bacteriology, fecal volatile fatty acids, mucosal morphology, and functional outcome were studied in 20 patients with triplicated (S) and 20 patients with quadruplicated (W) reservoirs after ileal pouch-anal anastomosis. Compared with patients with S reservoirs, patients with W reservoirs were found to have greater efficiency of evacuation of radiolabeled synthetic stool [97% (91%-98%) vs. 74% (62%-89%); P less than 0.05], and their reservoirs were more capacious [350 mL (320-400 mL) vs. 228 mL (175-290 mL); P less than 0.01] and compliant [16.0 mL/cm H2O (13.8 19.0 mL/cm H2O) vs. 12.3 mL/cm H2O (7.4-14.6 mL/cm H2O); P less than 0.01]. Effluent from S reservoirs contained significantly greater numbers of bacteroides (P less than 0.05) and concentrations of acetic and propionic acids (P less than 0.05) than effluent from W reservoirs. The degree of mucosal inflammation and villous atrophy in each design of reservoir was not significantly different. The ratio of anaerobes to aerobes in pouch effluent was significantly correlated with the degree of mucosal inflammation (rs = 0.433; P = 0.035). Fecal volatile fatty acids were significantly correlated with the percentage of stool retained after defecation and degree of mucosal inflammation. The frequency of bowel action was significantly less in patients with W reservoirs than in patients with S reservoirs [3.5/day (3-4/day) vs. 6.0/day (4-7/day); P less than 0.01]. The results indicate marked differences between these two ileal reservoir designs. PMID- 1732124 TI - Precore mutant hepatitis B virus infection and liver disease. AB - The type of hepatitis B virus ("wild-type" or precore mutant) in anti-e antigen antibody-positive carriers, viral DNA levels in the serum, and core and e antigen expression in the liver were investigated to search for a possible correlation of these factors with the severity of liver damage. Two major groups of patients were found: the patients in group A were predominantly infected with precore mutant virus and had chronic active hepatitis, expressed nuclear/cytoplasmic core and e antigens in liver biopsy specimens, and usually had high levels of viral DNA in their serum; patients in group B were infected with a mixture of wild-type and mutant viruses, had predominantly chronic persistent hepatitis, showed weaker expression of nuclear core antigen with no cytoplasmic core or e antigen, and had low viremia. A few patients were infected with viruses without precore stop-codon mutation. These data indicate a high prevalence of precore mutant viruses in anti e carriers with chronic liver disease and suggest that monitoring of virus sequence type and DNA level may be of prognostic value for liver disease sequelae. PMID- 1732125 TI - A controlled trial of naloxone infusions for the pruritus of chronic cholestasis. AB - To test the hypothesis that opioid agonist activity contributes to the pruritus of cholestasis, a placebo-controlled single-blinded trial of naloxone, an opioid antagonist, was conducted in eight patients with primary biliary cirrhosis. After discontinuation of all conventional antipruritic medications, one or two continuous (24-hour) IV infusions of naloxone (0.2 micrograms.kg-1.min-1) and placebo solution were administered consecutively in an order that was not predetermined. Pruritus was assessed subjectively by means of four hourly recordings of a visual analogue score. In addition, objective measurements of scratching activity that were independent of gross body movements were continuously recorded using an apparatus specifically designed to measure the frequencies associated with this activity. No side effects associated with naloxone infusions were observed. Only scratching activity data obtained for the same periods of day and night during both naloxone and placebo infusions were compared. Naloxone infusions were consistently associated with a decrease in values of the scratching activity index. In addition, in 50% of the patients the infusions were associated with a decrease in visual analogue score. The mean decrease in scratching activity ranged from 29% to 96% (mean, 50%; P less than 0.001). These findings imply that increased opioid agonist activity contributes to scratching activity in cholestatic patients. PMID- 1732126 TI - Sugar absorption by the biliary ductular epithelium of the rat: evidence for two transport systems. AB - Sugar absorption by the biliary ductular epithelium under steady-state conditions was examined using isolated perfused rat liver. The test sugar and mannitol (as a putative marker of paracellular entry) were added to the glucose-free recirculating perfusate each at a concentration of 5 mmol/L, and apparent active biliary ductular absorption equated with the change in concentration of the test sugar relative to that of mannitol. A metabolizable hexose (D-glucose), pentose (D-xylose), and three nonmetabolizable hexoses (alpha-methyl-glucoside, 3-o methyl-glucose, and L-glucose) were used. All five monosaccharides were well absorbed at constant rates for 2 hours with apparent rates of absorption (mumol.kg body weight-1.min-1, mean +/- SE) of D-glucose, 0.24 +/- 0.01; L glucose, 0.20 +/- 0.02; 3-o-methyl-glucose, 0.19 +/- 0.02; alpha-methyl glucoside, 0.16 +/- 0.03; and D-xylose, 0.10 +/- 0.04. The addition of phloridzin to the perfusate inhibited D-glucose absorption in part but did not inhibit L glucose absorption. When perfusate Na+ was replaced by N-methylglucamine, the bile-plasma ratio of mannitol remained unchanged, as did the apparent absorption rate of D-glucose and 3-o-methyl-glucose. In contrast, absorption of L-glucose and alpha-methyl-D-glucoside gradually ceased. The addition of 15 mmol/L glucose to the perfusate caused decreased bile flow and increased taurocholate concentration in bile, suggesting that glucose absorption by the biliary ductules induced water reabsorption. It is concluded that sugars are absorbed by the biliary ductular system by Na(+)-dependent and Na(+)-independent transport systems, the substrate affinities of which differ from those reported for apical membrane hexose transport systems in renal tubular and intestinal epithelia. Ductular absorption of solutes such as glucose that enter bile passively may have biological use, because ductular absorption decreases the concentration of substrates for bacterial growth in gallbladder bile. On the other hand, ductular absorption of solutes induces reabsorption of biliary water, resulting in decreased bile flow; this might contribute to cholestasis during prolonged hyperalimentation with solutions containing glucose. PMID- 1732127 TI - Decreased glucuronidation and increased bioactivation of acetaminophen in Gilbert's syndrome. AB - Gilbert's syndrome occurs in 5%-7% of the human population and is caused by an inherited deficiency in the glucuronidation of endogenous bilirubin, resulting in its accumulation and jaundice. The authors of the present study have previously shown that rats with a similar deficiency in bilirubin glucuronidation (Gunn rats) had reduced glucuronidation and enhanced susceptibility to the toxicity of the widely used analgesic, acetaminophen. Acetaminophen is eliminated primarily by glucuronidation, which prevents its cytochrome P-450-catalysed bioactivation to a hepatotoxic reactive intermediate. The purpose of this study was to determine whether people with Gilbert's syndrome had reduced glucuronidation and enhanced bioactivation of acetaminophen. Therefore, the biotransformation of acetaminophen, 20 mg/kg IV, was investigated in six subjects with Gilbert's syndrome (total bilirubin, 41 +/- 6 mumol/L; mean +/- SE) and six normal controls (total bilirubin, 11 +/- 2 mumol/L; P less than 0.01). Formation of the acetaminophen glucuronide conjugate measured by high-performance liquid chromatography was quantified by the ratio of the area under the plasma concentration-time curve (AUC) from 0 to 2 hours for the acetaminophen glucuronide divided by the AUC for acetaminophen. Acetaminophen bioactivation was quantified by the molar percentage of acetaminophen excreted in the urine during 24 hours as glutathione-derived conjugates (cysteine and mercapturic acid). Acetaminophen glucuronide formation in subjects with Gilbert's syndrome was 31% lower than that in normal controls (0.27 +/- 0.05 vs. 0.39 +/- 0.03; P less than 0.05), and bioactivation was 1.7-fold higher (3.5% +/- 0.4% vs. 2.1% +/- 0.3%; P less than 0.05). One control subject with normal bilirubin glucuronidation had substantially decreased acetaminophen glucuronide formation (0.20) and enhanced bioactivation (4.8%). Among all subjects, glucuronidation correlated inversely with bioactivation (r = -0.84; P less than 0.001), indicating that a decrease in a major pathway of elimination can shunt more drug through the toxifying route. Thus, a deficiency in bilirubin UDP-glucuronosyltransferase, evidenced by jaundice, can be paralleled by a deficiency in glucuronidation of other compounds. In these cases, jaundice can be a phenotypic determinant of enhanced acetaminophen bioactivation. On the other hand, some people with normal bilirubin glucuronidation may have a deficiency in the glucuronidation of acetaminophen; these people are not easily recognized.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732128 TI - Hepatocellular transplantation into the lung for temporary support of acute liver failure in the rat. AB - Because hepatocyte transplantation into the spleen or the peritoneal cavity, although successful in rats, is more difficult and less successful in larger animals, the lung was chosen for its accessibility and its high oxygen content as a new site for hepatocyte implantation for treatment of acute hepatic failure. Acute hepatic failure was induced by a combination by a portocaval side-to-side shunt and an 80% liver resection, which was associated with a greater than 90% mortality. Hepatocyte transplantation was performed either by injection of 1 x 10(7) cells via the jugular vein (100% mortality) or 5-7 x 10(7) cells transcutaneously into the right lung (92% survival). After injection of the cell free supernatant into the lung, 53.3% of the animals survived. If more than 90% of the liver was resected, none of the animals survived despite hepatocyte or supernatant injection. From these findings, it is concluded that the lung is a suitable home for hepatocytes. However, the hepatocytes survived only in cases of acute hepatic failure with some remaining vital liver parenchyma. PMID- 1732129 TI - Extracorporeal shock-wave lithotripsy of pancreatic calculi. AB - Extracorporeal shock-wave lithotripsy (ESWL) has been used to disintegrate pancreatic stones located in the main pancreatic duct for 123 patients with severe chronic pancreatitis. Endoscopic management following ESWL is aimed at restoring the pancreatic flow to the duodenum. Stone disintegration was achieved in 122 patients, whereas a decrease in the main pancreatic duct diameter resulted in 111, and complete clearance of the main pancreatic duct was obtained in 72. Pain relief, complete (40/88) or partial (35/88), correlated significantly with the results of the endoscopic drainage of the main pancreatic duct (e.g., decrease in main pancreatic duct diameter). Relapsing pain was most often related to recurrent pancreatic duct obstruction. Of 76 patients whose body weight had decreased before ESWL, 54 gained weight. Improvement of the exocrine function, evaluated by the [14C]triolein breath test before and 11 months, on the average, after ESWL, was observed in 12 patients among 22 for whom this test was performed before and after treatment. Improvement of the endocrine function after relief of obstruction of the main pancreatic duct was less frequently recorded (4/41). ESWL of pancreatic stones is a new, safe, and highly effective method of facilitating the endoscopic procedures for relief of pancreatic duct obstruction in severe chronic pancreatitis. PMID- 1732130 TI - Mediation of trypsin inhibitor-induced pancreatic hypersecretion by secretin and cholecystokinin in rats. AB - We investigated a hormonal mechanism in a trypsin inhibitor-induced pancreatic hypersecretion in rats. Intraduodenal administration of a synthetic trypsin inhibitor, camostat, resulted in significant increases in plasma concentration of both secretin and cholecystokinin in a dose-related manner that paralleled a significant increase in exocrine pancreatic secretion. To eliminate the effect of circulating secretin in rats, a rabbit antisecretin serum was given IV that resulted in a 77% reduction in bicarbonate secretion stimulated by intraduodenal camostat. A cholecystokinin receptor antagonist, MK-329, also inhibited significantly the camostat-induced increase in pancreatic secretion; volume and bicarbonate output were reduced by 35% each and amylase output by 73%. The combined administration of antisecretin serum and MK-329 completely abolished the pancreatic exocrine secretion stimulated by camostat. These observations indicate that the camostat-stimulated pancreatic exocrine secretion is mediated by the increased release of both secretin and cholecystokinin in rats. PMID- 1732131 TI - Complications of percutaneous liver biopsy in children. AB - To determine the frequency and nature of complications after liver biopsy and whether risk factors could be identified to predict these complications, the medical records of all patients (age, 1 week to 28 years) who underwent a percutaneous liver biopsy at Children's Hospital over a 6-year period (1981-1986) were reviewed. Data were collected from 469 (97%) of 483 eligible charts. Twenty one patients (4.5%) experienced major complications including bile leak (n = 3, 0.6%), prolonged drainage of ascitic fluid (n = 1, 0.2%), pneumothorax (n = 1, 0.2%), bleeding requiring transfusion (n = 13, 2.8%), and death (n = 3, 0.6%). A subgroup of patients (n = 37) with cancer or bone marrow transplantation was found to be at a nearly fivefold greater risk for transfusion than patients with other diagnoses (P = 0.02). All three deaths in previously stable patients occurred in this same high-risk group of patients with cancer or bone marrow transplantation (P less than 0.001). Two deaths resulted from disseminated intravascular coagulation and one from bleeding. Diagnosis, age, number of percutaneous passes, and prebiopsy coagulation studies were not predictive of subsequent complications. It is concluded that bleeding that requires transfusion is the most common liver biopsy complication and that it occurs more frequently in children than previously reported. Children with cancer or those who have undergone bone marrow transplantation are at a greater risk for bleeding and death following percutaneous liver biopsy. PMID- 1732132 TI - Opposite effects of cholestyramine and loxiglumide on gallbladder dynamics in humans. AB - The aim of this study was to conduct dose-response studies of the effect of cholestyramine, alone or in combination with a test meal, on gallbladder emptying studied by ultrasonography in 31 healthy volunteers. The role of cholecystokinin in mediating the effects of cholestyramine was studied using the cholecystokinin receptor antagonist loxiglumide. In the absence of a test meal, cholestyramine produced a dose-dependent decrease in gallbladder volume; minimal residual volumes were 30% (2 g), 55% (4 g), and 110% (12 g) of the minimal volume produced by a test meal alone. Gallbladder volume was still only 50% of the initial volume, 24 hours after ingestion of 12 g cholestyramine. Cholestyramine also enhanced gallbladder emptying in response to the test meal (n = 7). Oral loxiglumide strongly inhibited gallbladder evacuation in response to either test meal (n = 6) or cholestyramine alone (n = 6) but partially blocked gallbladder emptying when the resin was added to the test meal (n = 12). It is concluded that (a) cholestyramine alone or in combination with a test meal produces a marked decrease in gallbladder volume and (b) the action of the resin itself on gallbladder motor function appears to be mainly, but not solely, mediated by cholecystokinin through the disinhibition of the luminal feedback mechanism between bile salts and the endogenous release of cholecystokinin. PMID- 1732133 TI - Pain in extracorporeal shock-wave lithotripsy: a comparison of different lithotripters in volunteers. AB - The aim of the present study was to investigate pain sensations experienced during extracorporeal shock-wave application, comparing an electrohydraulic (MPL 9000; Dornier Medizintechnik, Germering, Germany), an electromagnetic (Lithostar Plus; Siemens, Erlangen, Germany), and a piezoelectric (Piezolith 2300; Wolf, Knittlingen, Germany) shock-wave system. In nine healty volunteers, three therapeutically used intensities were applied in a randomized order with each lithotripter (MPL 9000: 16, 20, and 24 kV; Lithostar Plus: settings 5, 7, and 9; and Piezolith 2300: settings 2, 3, and 4). The subjects received nine series of 20 shock waves amounting to a total of 180 shock waves per session. The treatment was performed under clinical conditions, and no premedication was given. A visual analog scale and the McGill Pain Questionnaire were used for assessment of pain. In addition, somatosensory evoked potentials caused by shock-wave stimulation were recorded. Some of the volunteers were unable to bear the pain caused by the highest shock-wave intensity of the electrohydraulic (n = 3) and the electromagnetic system (n = 4). Estimates using the visual analogue scale showed increased pain sensations with increasing energy settings for each lithotripter. The amplitudes of the somatosensory evoked potentials became larger, and latencies shortened with increasing stimulus intensities (P less than 0.05). Subjective estimates by means of the visual analogue scale (P less than 0.01) as well as the McGill Pain Questionnaire (NS) and the somatosensory evoked potentials (P less than 0.05) showed that stimulation by the piezoelectric lithotripter was less painful than stimulation by the two other generators. PMID- 1732134 TI - Cholesterol malabsorption in pancreatic insufficiency: effects of enzyme substitution. AB - Defective lipolysis, steatorrhea, and hypocholesterolemia characterize pancreatic insufficiency. Lipid metabolism in pancreatic insufficiency was studied by measuring serum lipoproteins, cholesterol absorption with double labels and serum plant sterols, and bile acid and cholesterol synthesis with fecal and dietary steroid analysis and cholesterol precursor sterols before and during exogenous pancreatic enzyme substitution. Baseline fecal fat, masses, bile acids and neutral steroids, and cholesterol synthesis were increased, whereas cholesterol absorption was markedly reduced. In fact, the present data suggest that sterol absorption may be disturbed more sensitively than fat absorption in pancreatic insufficiency. Enzyme substitution significantly reduced fecal fat, masses, bile acids and neutral steroids, and synthesis of cholesterol and improved cholesterol absorption in relation to serum cholesterol, although normal values were not obtained. Serum level of high-density lipoprotein cholesterol was significantly elevated by exogenous enzymes, whereas levels of cholesterol or triglycerides in other lipoproteins remained unchanged. Improved sterol absorption increased also serum levels of plant sterols and reduced levels of cholesterol precursors and cholesterol synthesis and precursor sterol-plant sterol ratios. Thus, reduced intestinal lipolysis with expanded oil phase appears to be a major reason for impaired cholesterol absorption, causing enhanced cholesterol and, consequently, bile acid synthesis and reduced serum cholesterol level. Exogenous enzyme substitution seems partly to correct these abnormalities, improvements of which can be monitored by the gas-liquid chromatographic determination of serum plant sterols or cholesterol precursor-plant sterol ratios. PMID- 1732135 TI - Persistence of a hyperdynamic circulation in cirrhotic rats following removal of the sympathetic nervous system. AB - The sympathetic nervous system is thought to play a role in the pathogenesis of the hyperdynamic circulation associated with portal hypertension. However, the extent of this role is unknown. After elimination of all neurological control by pithing, systemic and regional hemodynamics were studied in rats with portal hypertension caused by either portal vein stenosis or biliary cirrhosis. In normal rats, pithing induced a two-thirds decrease in mean arterial pressure and cardiac index. Compared with pithed normal rats, pithed portal vein-stenosed rats showed similar values for mean arterial pressure, cardiac index, and portal tributary blood flow. In contrast, pithed cirrhotic rats still showed hyperdynamic circulation with increased cardiac index and portal tributary blood flow. Although pithing dramatically reduced portal pressure in all groups, portal pressure remained significantly higher in portal hypertensive rats than in normal rats. These results indicate that in rats with portal vein stenosis, the sympathetic nervous system plays a major role in hemodynamic alterations, whereas in rats with cirrhosis, nonneurogenic factors participate in the pathogenesis of the hyperdynamic circulation. PMID- 1732136 TI - Appearance of exogenous epidermal growth factor in liver, bile, and intestinal lumen of suckling rats. AB - This study was performed to investigate the distribution and the degradation of IV administered [125I]rat epidermal growth factor (rEGF) in the liver and gastrointestinal tract of suckling rats. The bile duct of anesthetized rats was cannulated, and [125I]rEGF was injected (with or without 2500-fold excess unlabeled rEGF) into the femoral vein. After 5, 30, 60, and 120 minutes, the radioactivity in the liver, stomach, small intestine, blood, kidney, bile, and luminal contents of the stomach and small intestine was measured. The extracted radioactivity was then analyzed by immunoaffinity chromatography and binding to EGF-specific receptors. High levels of radioactivity were found in the liver (57% of total administered) and small intestine (10%) at 5 minutes, which gradually decreased. On the contrary, radioactivity secreted in the bile and luminal contents of the small intestine increased with time. The radioactivity in the bile represented 2.4% and 4.5% of the total administered at 60 and 120 minutes, respectively. During the first 60 minutes, more than 90% of the radioactivity in the liver, small intestine, bile, and intestinal contents was immunoreactive. Thirty-four to seventy percent of the radio-activity in the bile and liver and 20%-41% of radioactivity in the small intestinal wall and contents were capable of binding to EGF-specific receptors. Radioactivity detected in the liver, bile, small intestine, and intestinal contents was profoundly reduced by the coinjection excess of unlabeled EGF. These studies show that IV administered [125I]rEGF is rapidly taken up by the liver and the gastrointestinal tract and secreted into the bile and intestinal luminal contents of suckling rats in form(s) capable of binding to anti-EGF antibody and EGF-specific receptors. The uptake and secretion by the liver and the small intestine appear to be receptor mediated. PMID- 1732137 TI - A familial form of incomplete septal cirrhosis. AB - The clinical features and hepatic histology of a disorder resembling idiopathic portal hypertension and nodular regenerative hyperplasia but most consistent with incomplete septal cirrhosis, occurring in four family members, are described. This represents the first description of the familial occurrence of this entity. Features common to incomplete septal cirrhosis and the noncirrhotic nodular conditions of the liver that may present with complications of portal hypertension are discussed. PMID- 1732138 TI - Ng-nitro-L-arginine reduces nonadrenergic, noncholinergic relaxations of human gut. AB - The aim of this study was to determine whether nonadrenergic, noncholinergic inhibitory neurotransmission in human circular sigmoid colonic and internal anal sphincter muscle involves release of a nitric oxide-like substance. Colonic and sphincter muscle respond to electrical field stimulation by giving nonadrenergic, noncholinergic relaxations. After-contractions always occur in colonic muscle but only sometimes in sphincter muscle. Ng-Nitro-L-arginine abolished relaxations of sphincter muscle and partially reduced those of colonic muscle. After contractions were undiminished as were relaxations of sphincter muscle to sodium nitroprusside. The effects of Ng-nitro-L-arginine were reversed by L-arginine. The results suggest that nitric oxide is possibly the neurotransmitter mediating nonadrenergic, noncholinergic relaxations of the human internal anal sphincter muscle. PMID- 1732139 TI - Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats. AB - Expression of epidermal growth factor receptor (EGFR) was studied immunohistochemically in rat gastric mucosa during healing of acetic acid-induced ulcers. In normal control gastric oxyntic mucosa, EGFR was expressed in proliferative zone cells and in some parietal cells. In mucosa of the ulcer margin, at 3, 7, and 16 days after ulcer induction, there was a 75-fold increase (over controls) in the number of cells expressing EGFR. Seventy percent of ulcers healed by the 16th day, and all were healed by the 25th day. The mucosal scar that replaced the ulcer was composed of dilated glands lined with poorly or aberrantly differentiated cells showing persistence of increased EGFR expression. An increased EGFR expression indicates an important role of EGF in ulcer healing and scar formation. PMID- 1732140 TI - Genetic variation in hepatitis B virus. PMID- 1732141 TI - Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation. AB - Although Helicobacter pylori is now recognized as playing an etiologic role in chronic gastritis and peptic ulcer disease, information on the pathogenesis and natural history of infection is limited. A model is proposed in which luminal H. pylori secrete substances that mediate inflammation that is beneficial to the organism but ultimately deleterious for the host; in addition to tissue damage, inflammation also affects gastric secretory function. In this model, the host may attempt to suppress the inflammatory response, and the adequacy of this postulated down-regulation determines pathological and clinical outcome. The effects of the inflammatory process on gastrin-hydrochloric acid homeostasis may be of critical importance in the pathogenesis of peptic ulcer disease. Because the long-term consequences of H. pylori colonization reflect the continued presence of the organism in the host over years or decades, it may be useful to consider this as a "slow" bacterial infection. PMID- 1732142 TI - Morphine-augmented cholescintigraphy: what exactly is being augmented? PMID- 1732143 TI - Primary sclerosing cholangitis: is medical therapy on the way? PMID- 1732144 TI - NO and NO accommodation. PMID- 1732145 TI - Tagged red blood cell imaging to localize gastrointestinal bleeding: is it really that helpful? PMID- 1732146 TI - The endoscopic Doppler and ulcer rebleeding risk: probing the source. PMID- 1732147 TI - Hepatitis associated with primary HIV infection. PMID- 1732148 TI - Psychological treatment for the irritable bowel syndrome. PMID- 1732149 TI - Reproducibility of measurements of trace gas concentrations in expired air. PMID- 1732150 TI - Gallbladder contractility. PMID- 1732151 TI - Acalculous abdominal pain in patients with abnormal cholescintigraphy: is cholecystectomy the answer? PMID- 1732152 TI - Reconsidering small tubular adenomas: are they really markers? PMID- 1732153 TI - Helicobacter pylori gastric atrophy and pernicious anemia. PMID- 1732154 TI - Cost-effectiveness of symptomatic gallstone management: what exactly are we measuring? PMID- 1732155 TI - Centennial: J. B. S. Haldane, 1892-1964. PMID- 1732156 TI - Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans. AB - Dauer larva formation in Caenorhabditis elegans is controlled by chemosensory cells that respond to environmental cues. Genetic interactions among mutations in 23 genes that affect dauer larva formation were investigated. Mutations in seven genes that cause constitutive dauer formation, and mutations in 16 genes that either block dauer formation or result in the formation of abnormal dauers, were analyzed. Double mutants between dauer-constitutive and dauer-defective mutations were constructed and characterized for their capacity to form dauer larvae. Many of the genes could be interpreted to lie in a simple linear epistasis pathway. Three genes, daf-16, daf-18 and daf-20, may affect downstream steps in a branched part of the pathway. Three other genes, daf-2, daf-3 and daf-5, displayed partial or complex epistasis interactions that were difficult to interpret as part of a simple linear pathway. Dauer-defective mutations in nine genes cause structurally defective chemosensory cilia, thereby blocking chemosensation. Mutations in all nine of these genes appear to fall at a single step in the epistasis pathway. Dauer-constitutive mutations in one gene, daf-11, were strongly suppressed for dauer formation by mutations in the nine cilium-structure genes. Mutations in the other six dauer-constitutive genes caused dauer formation despite the absence of functional chemosensory endings. These results suggest that daf-11 is directly involved in chemosensory transduction essential for dauer formation, while the other Daf-c genes play roles downstream of the chemosensory step. PMID- 1732157 TI - An unusual genomic position effect on Drosophila white gene expression: pairing dependence, interactions with zeste, and molecular analysis of revertants. AB - The white gene in the AR4-24 P[white,rosy] insertion on chromosome 2 has a novel expression pattern, in which it is repressed in the dorsal half of the eye. X-ray mutagenesis led to the isolation of six revertants mapping to chromosome 2, which are wild type in a zeste+ background, and three extreme derivatives, in which white gene expression is repressed in ventral regions of the eye as well. By Southern blot analyses the breakpoints of five of the revertants and one of the extreme derivatives were mapped in the flanking DNA bordering each side of the AR4-24 insertion. The revertants show some dorsal repression of white in the presence of z1, and by this criterion each is only a partial revertant. The extreme derivatives act not only in cis, but also in trans to repress expression of AR4-24 and its various derivatives. We provide evidence that these trans effects are proximity-dependent effects, possibly mediated by pairing of gene copies, as they do not extend to copies of the white gene located elsewhere in the genome. We show that one extreme derivative, E1, is a small deletion spanning the insertion site at the 5' end of the white gene, and propose that the distance between a negative regulatory element in the 5' flanking DNA and the white promoter influences the degree of the repression. PMID- 1732158 TI - Mitochondrial DNA complex I and III mutations associated with Leber's hereditary optic neuropathy. AB - Four new missense mutations have been identified through restriction analysis and sequencing of the mitochondrial DNAs (mtDNA) from Leber's hereditary optic neuropathy (LHON) patients who lacked the previously identified 11778 mutation. Each altered a conserved amino acid and correlated with the LHON phenotype in population and phylogenetic analyses. The nucleotide pair (np) 13708 mutation (G to A, ND5 gene) changed an alanine to a threonine and was found in 6/25 (24%) of non-11778 LHON pedigrees and in 5.0% of controls, the np 15257 mutation (G to A, cytochrome b gene) changed an aspartate to an asparagine and was found in 4 of the 13708-positive pedigrees and 0.3% of controls, the np 15812 mutation (G to A, cytochrome b gene) changed a valine to a methionine and was detected in two of the 15257-positive pedigrees and 0.1% of controls and the np 5244 mutation (G to A, ND2 gene) changed a glycine to a serine and was found in one of the 15812 positive patients and none of 2103 controls. The 15257 mutation altered a highly conserved amino acid in an extramembrane domain of cytochrome b that is associated with the ligation of the low potential b566 heme and the 5244 mutation altered a strongly evolutionarily conserved region of the ND2 polypeptide. The 13708 and 15812 mutations changed moderately conserved amino acids. Haplotype and phylogenetic analysis of the four np 15257 mtDNAs revealed that all harbored the same rare Caucasian haplotype and that the np 13708, np 15257, np 15812 and np 5244 mutations were added sequentially along this mtDNA lineage. Since the percentage of sighted controls decreases as these mutations accumulate, it appears that they interact synergistically, each increasing the probability of blindness. The involvement of both mitochondrial complex I (np 5244, 11778, 13708) and complex III (np 15257, 15812) mutations in LHON indicates that the clinical manifestations of this disease are the product of an overall decrease in mitochondrial energy production rather than a defect in a specific mitochondrial enzyme. PMID- 1732159 TI - Association between rDNA alleles and quantitative traits in doubled haploid populations of barley. AB - Doubled haploids (DH) were generated from reciprocal F1 hybrids which were heterozygous for alleles at the Nor-H3 locus on chromosome 5H of barley. The r DNA alleles did not deviate significantly from the expected 1:1 ratio and the DH progenies were classified into two groups based on the allelic constitution of the Nor-H3 locus. The DHs were grown in a randomized, replicated field experiment and a range of agronomic and quality traits were recorded. The Nor-H3 locus was associated with a significant portion of the genetic variation for: yield, thousand corn weight, water sensitivity and milling energy requirement of the grain. However, the magnitude of the differences between groups was dependent on the direction of the cross. The milling energy requirement of the grain was consistently associated with alleles at the Nor-H3 locus. These results are presented in relation to the dynamics of rDNA evolution and variability. The potential of molecular markers in conjunction with doubled haploids to map quantitative traits in barley is also discussed. PMID- 1732160 TI - Comparing evolvability and variability of quantitative traits. AB - There are two distinct reasons for making comparisons of genetic variation for quantitative characters. The first is to compare evolvabilities, or ability to respond to selection, and the second is to make inferences about the forces that maintain genetic variability. Measures of variation that are standardized by the trait mean, such as the additive genetic coefficient of variation, are appropriate for both purposes. Variation has usually been compared as narrow sense heritabilities, but this is almost always an inappropriate comparative measure of evolvability and variability. Coefficients of variation were calculated from 842 estimates of trait means, variances and heritabilities in the literature. Traits closely related to fitness have higher additive genetic and nongenetic variability by the coefficient of variation criterion than characters under weak selection. This is the reverse of the accepted conclusion based on comparisons of heritability. The low heritability of fitness components is best explained by their high residual variation. The high additive genetic and residual variability of fitness traits might be explained by the great number of genetic and environmental events they are affected by, or by a lack of stabilizing selection to reduce their phenotypic variance. Over one-third of the quantitative genetics papers reviewed did not report trait means or variances. Researchers should always report these statistics, so that measures of variation appropriate to a variety of situations may be calculated. PMID- 1732161 TI - The theoretical distribution of lengths of intact chromosome segments around a locus held heterozygous with backcrossing in a diploid species. AB - When two different isogenic lines of a diploid species (or two different species) are crossed, the resulting F1 individuals should be heterozygous at all the loci fixed for different alleles in the two strains (in the limit, at all the loci of the genome). If one of these loci is then held heterozygous for several generations of repeated backcrossing to the same strain, the average length of intact chromosome segments (with reference to the original parental chromosome) on both sides of the selected locus, or, equivalently, the average length of segments surrounding that locus which are still heterozygous (with reference to the fully heterozygous F1 chromosome), may diminish, but cannot increase. Several authors have derived equations to predict this average. We show that the most widely used criterion, developed by R.A. Fisher, leads to serious overestimations of the true parametric values, when applied to early generation analyses, with the corresponding errors in the interpretation of experimental results. We then derive the exact equations both for the average and standard deviation of the lengths of intact chromosome segments surrounding a locus held heterozygous after any number of generations of backcrossing. Our results are in close agreement with those found by a former author, although involving a rather different approach. PMID- 1732162 TI - The maintenance of single-locus polymorphism. IV. Models with mutation from existing alleles. AB - The ability of viability selection to maintain allelic polymorphism is investigated using a constructionist approach. In extensions to the models we have previously proposed, a population is bombarded with a series of mutations whose fitnesses in conjunction with other alleles are functions of the corresponding fitnesses with a particular allele, the parent allele, already in the population. Allele frequencies are iterated simultaneously, thus allowing alleles to be driven to extinction by selection. Such models allow very high levels of polymorphism to evolve: up to 38 alleles in one case. Alleles that are lethal as homozygotes can evolve to surprisingly high frequencies. The joint evolution of allele frequencies and viabilities highlights the necessity to consider more than the current morphology of a population. Comparisons are made with the neutral theory of evolution and it is suggested that failure to reject neutrality using the Ewens-Watterson test cannot be regarded as evidence for the neutral theory. PMID- 1732163 TI - Pleiotropy and multilocus polymorphisms. AB - It is demonstrated that systems of two pleiotropically related characters controlled by additive diallelic loci can maintain under Gaussian stabilizing selection a stable polymorphism in more than two loci. It is also shown that such systems may have multiple stable polymorphic equilibria. Stabilizing selection generates negative linkage disequilibrium, as a result of which the equilibrium phenotypic variances are quite low, even though the level of allelic polymorphisms can be very high. Consequently, large amounts of additive genetic variation can be hidden in populations at equilibrium under stabilizing selection on pleiotropically related characters. PMID- 1732164 TI - Is Stellate a relict meiotic driver? PMID- 1732165 TI - Multiple alleles and estimation of genetic parameters: computational equations showing involvement of all alleles. PMID- 1732166 TI - Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria. AB - A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined. PMID- 1732167 TI - Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli. AB - To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction. PMID- 1732168 TI - Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. AB - We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine. The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity. This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine. The phenotype induced by this mutation implies that, in S. cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine. This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S. cerevisiae. The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1 1 mutation undergoes a particularly high frequency of meiotic gene conversion. PMID- 1732169 TI - The rec102 mutant of yeast is defective in meiotic recombination and chromosome synapsis. AB - A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11. PMID- 1732170 TI - Multiple effects of mutation on expression of alternative cell surface protein genes in Tetrahymena thermophila. AB - Genes at the SerH locus of the ciliated protist Tetrahymena thermophila specify the major (H) surface protein on cells grown at 20-36 degrees. Alternative proteins L, T, S and I are expressed under different conditions of temperature and culture media. Mutants unable to express SerH genes were examined for expression of these proteins, also called immobilization or i-antigens, at both H and non-H conditions. In all instances, one or more i-antigens were expressed in the absence of H, and, in most instances, expression of i-antigens under non-H conditions was also affected. Examples of the latter include both the continued expression of H-replacement antigens and the inability to express certain other i antigens. Such multiple effects were observed in mutants with trans-acting (rseA, rseB, rseC, RseD) and cis-acting (H1-1 and H1-2) mutations, but not in mutants in which SerH is affected developmentally (B2092, B2101, B2103, B2107). These interactions suggest that the wild-type genes identified by mutation exert both positive and negative effects in the regulation of i-antigen gene expression. PMID- 1732171 TI - Hospital CEOs outline plans to control costs. PMID- 1732172 TI - Dynamic diversification: hospitals pursue physician alliances, 'seamless' care. AB - In the 1980s, "diversification" in health care meant creating new corporate entities to boost revenues. In the 1990s, in places like New Ulm, MN, Long Beach, CA, and Boston, hospitals and physicians are exploring diversifications that utilize their core patient care strengths. This is creating new entities that benefit patient care and help prepare for health care delivery in the future. In fact, despite predictions that RBRVS would drive physicians into competition with hospitals, the opposite is taking place, as the new "seamless" delivery systems take advantage of pooled resources and economies of scale. PMID- 1732173 TI - Hospitals offer extensive financial counseling. PMID- 1732174 TI - Survey: employee, MD issues top list of D&O insurance claims. AB - A new survey finds that the number of directors' and officers' liability insurance claims in health care has increased since last year. Wrongful employee termination and physician credentialing topped the list of claims. PMID- 1732175 TI - Inpatient utilization falls sharply; growth in outpatient visits slows. PMID- 1732176 TI - Hospitals begin to see benefits of MD access to data. PMID- 1732177 TI - Hospitals with the best information systems. PMID- 1732178 TI - Hospitals confront loss of EKG reading payments. PMID- 1732179 TI - How to build partnerships with MD trustees. PMID- 1732180 TI - Improving family dynamics. PMID- 1732181 TI - Health promotion--evaluating a 'well man' clinic. PMID- 1732183 TI - Health education. Sex and teenagers. PMID- 1732182 TI - Stepfamilies--stepping into family life. PMID- 1732184 TI - Child support. Maintenance penalty hits mothers and children. PMID- 1732185 TI - Family trends and public policy. PMID- 1732186 TI - Poverty--benefits, healthy eating and the 'p' word. PMID- 1732187 TI - School health--pathway round the health service. PMID- 1732188 TI - Labour relations--local heroines. PMID- 1732189 TI - Preconception. Stop the cycle of deprivation. PMID- 1732190 TI - Structural modifications and kinetic studies of the substrates involved in the final step of methane formation in Methanobacterium thermoautotrophicum. AB - The 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) reductase catalyzes the final methane-yielding reaction in fastidiously anaerobic methanogenic archaebacteria. This step involves the reductive demethylation of CH3-S-CoM with reducing equivalents from N-7-(mercaptoheptanoyl)-L-threonine O3-phosphate (HS-HTP) to yield methane and the nonsymmetrical disulfide of 2-mercaptoethanesulfonic acid and HS-HTP. We chemically synthesized modified analogs of CH3-S-CoM (which has two carbons in the ethylene bridge) and of HS-HTP (which has seven carbons in the side chain); analog pairs possessed an overall correct number of side chain carbons (i.e., a total of nine in combination). They were simultaneously added to anaerobic cell extracts of Methanobacterium thermoautotrophicum delta H. The ability of the extracts to reductively demethylate the modified substrates was tested by gas chromatography. We also describe here previously unknown inhibitors of methanogenesis, 6-(methylthio)hexanoyl-L-threonine O3-phosphate (a structural analog of HS-HTP) and sodium bromomethanesulfonic acid (a structural analog of CH3-S-CoM). Both analogs were found to be effective competitive inhibitors with respect to HS-HTP. These substrate analogs were also found to inhibit a recently described photoactivation of homogeneous inactive reductase (K. D. Olson, C. W. McMahon, and R. S. Wolfe, Proc. Natl. Acad. Sci. USA 88:4099-4103, 1991). In addition, we probed the mechanism of action of a potent inhibitor of the enzyme, 2-bromoethanesulfonic acid, a structural analog of CH3-S-CoM. PMID- 1732191 TI - Sugar-glycerol cofermentations in lactobacilli: the fate of lactate. AB - The simultaneous fermentation of glycerol and sugar by lactobacillus brevis B22 and Lactobacillus buchneri B190 increases both the growth rate and total growth. The reduction of glycerol to 1,3-propanediol by the lactobacilli was found to influence the metabolism of the sugar cofermented by channelling some of the intermediate metabolites (e.g., pyruvate) towards NADH-producing (rather than NADH-consuming) reactions. Ultimately, the absolute requirement for NADH to prevent the accumulation of 3-hydroxypropionaldehyde leads to a novel lactate glycerol cofermentation. As a result, additional ATP can be made not only by (i) converting pyruvate to acetate via acetyl phosphate rather than to the ethanol usually found and (ii) oxidizing part of the intermediate pyruvate to acetate instead of the usual reduction to lactate but also by (iii) reoxidation of accumulated lactate to acetate via pyruvate. The conversion of lactate to pyruvate is probably catalyzed by NAD-independent lactate dehydrogenases that are found only in the cultures oxidizing lactate and producing 1,3-propanediol, suggesting a correlation between the expression of these enzymes and a raised intracellular NAD/NADH ratio. The enzymes metabolizing glycerol (glycerol dehydratase and 1,3-propanediol dehydrogenase) were expressed in concert without necessary induction by added glycerol, although their expression may also be influenced by the intracellular NAD/NADH ratio set by the different carbohydrates fermented. PMID- 1732192 TI - A lipoprotein of Yersinia enterocolitica facilitates ferrioxamine uptake in Escherichia coli. AB - A cloned fragment of Yersinia enterocolitica DNA complemented the defect in ferrioxamine B uptake of an Escherichia coli fhuE mutant lacking the outer membrane high-affinity transport protein FhuE. Subcloning revealed that a 13.7 kDa outer membrane protein was required for complementation. The amino acid sequence deduced from the nucleotide sequence showed extensive homology to PCPHi, an outer membrane lipoprotein of Haemophilus influenzae. We therefore termed this protein PCPYe. Plasmid-encoded pcpY mediated a low-affinity uptake of ferrioxamine B which may be caused by changes in the permeability of the outer membrane due to an overexpression of this outer membrane protein. A transposon insertion mutant in the plasmid-encoded pcpY gene was transferred into the chromosome of Y. enterocolitica. The resulting mutation had no effect on the high affinity uptake of ferrioxamine B in Yersinia cells. Using the antibiotic ferrimycin we were able to isolate a Y. enterocolitica mutant lacking the high affinity outer membrane receptor for ferrioxamine uptake, termed FoxA. PMID- 1732193 TI - Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene. AB - Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand. PMID- 1732194 TI - The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate. AB - The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Muller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12. PMID- 1732195 TI - Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product. AB - A protein catalyzing methylation at C-5 and C-15 and decarboxylation of the acetic acid side chain at C-12 on precorrin-6y to yield precorrin-8x was purified to homogeneity from a recombinant strain of Pseudomonas denitrificans. It was sequenced at the N terminus and shown to be encoded by the cobL gene. PMID- 1732196 TI - Characterization of the Bacillus subtilis sporulation gene spoVK. AB - The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK. PMID- 1732197 TI - Biomimics of fungal cell-cell recognition by use of lectin-coated nylon fibers. AB - When the mycoparasitic, biocontrol fungus Trichoderma harzianum was allowed to grow on nylon fibers treated with concanavalin A or Sclerotium rolfsii lectin, it coiled around the nylon fibers and produced hooks in a pattern similar to that observed with the real host hyphae. The incidence of interaction between T. harzianum and S. rolfsii lectin-treated fibers was significantly higher than that of the controls (untreated or blocked activated fibers). These findings provide direct evidence for the role of lectins in mycoparasitism. PMID- 1732198 TI - Replication of the R6K plasmid during the Escherichia coli cell cycle. AB - The cell-cycle replication pattern of the R6K plasmid has been investigated by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The high-copy plasmid R6K replicates exponentially in a cell-cycle-independent manner. A mini-R6K plasmid deleted for the ori alpha origin of replication also replicates, exponentially in a cell-cycle-independent manner. PMID- 1732199 TI - Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12. AB - Escherichia coli K-12 harboring a part of the structural genes for the Klebsiella K2 capsular polysaccharide (cpsK*) expresses a large amount of K2 capsular polysaccharide as a thick capsule in the presence of plasmids carrying rmpA and rcsB. We have previously shown that expression of the Klebsiella K2 capsule in E. coli HB101 harboring cpsK* depends on the presence of rmpA, a regulatory gene from a large plasmid of Klebsiella pneumoniae Chedid (O1:K2). E. coli K-12 JM109, however, produces only a small amount of K2 capsular polysaccharide, even in the presence of plasmids carrying rmpA as well as the cpsK* structural genes. Introduction of the rcsB gene, a positive regulator of colanic acid capsule synthesis in E. coli K-12 which was cloned from HB101 on a plasmid, into JM109 cells carrying cpsK* and rmpA, results in the expression of a thick K2 capsule. By Northern (RNA) hybridization analysis, rcsB has been found to enhance transcription of a long strand of mRNA (longer than 14 kb) from cpsK*. These E. coli transformants which produce a thick K2 capsule also express colanic acid production at high levels. Therefore, rcsB can act as a positive regulator of Klebsiella K2 capsule production and two capsular polysaccharides can be expressed in E. coli simultaneously. With a somewhat different strain background, we have found that both of the colanic acid regulators, rcsA and rcsB, contribute to the basal level of Klebsiella K2 capsule expression but that the presence of multicopy rcsB in either an rcsB or an rcsA mutant of E. coli is sufficient to increase the expression of K2 capsular polysaccharide. These results suggest further parallels between the regulation of colanic acid synthesis in E. coli and the regulation of Klebsiella K2 capsule synthesis. PMID- 1732200 TI - Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum. AB - Several Pseudomonas solanacearum strains which produced no detectable extracellular polysaccharide (EPS) in planta had been reported to remain highly virulent when tested at high inoculum concentrations (P. Xu, M. Iwata, S. Leong, and L. Sequeira, J. Bacteriol. 172:3946-3951, 1990; P. Xu, S. Leong, and L. Sequeira, J. Bacteriol. 170:617-622, 1988). Two of these mutants, KD700 and KD710, have now been molecularly and genetically mapped to the EPSI gene cluster described by Denny and Baek (Mol. Plant-Microbe Interact. 4:198-206, 1991). When a range of inoculum concentrations was used, these two mutants and all other EPS defective mutants tested were found to be reduced in virulence to eggplants and tobacco relative to the wild-type strain. Thus, EPS consistently is required for the wild-type level of virulence in P. solanacearum. PMID- 1732201 TI - Enzymatic basis of thiol-stimulated secretion of porphyrins by Escherichia coli. AB - 1-Thioglycerol (TG) stimulates the synthesis of porphyrin in aerobically growing Escherichia coli. Here the levels of delta-aminolevulinate biosynthetic enzymes in untreated and TG-treated E. coli THU and PUC2 (a mutant of THU which overproduces porphyrins in the presence of thiols) cells were determined. TG treatment elevated the activity of glutamyl-tRNA reductase in both strains. The increased activity was not caused by activation of preexisting enzymes by thiols or by oxidizing agents but was dependent on new protein synthesis. PMID- 1732202 TI - Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons. AB - We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea. PMID- 1732203 TI - Membrane properties of a plant-pathogenic mycoplasmalike organism. AB - In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions. PMID- 1732204 TI - Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell. AB - Caulobacter crescentus firmly adheres to surfaces with a structure known as the holdfast, which is located at the flagellar pole of swarmer cells and at the stalk tip in stalked cells. A three-gene cluster (hfaAB and hfaC) is involved in attachment of the holdfast to the cell. Deletion and complementation analysis of the hfaAB locus revealed two genes in a single operon; both were required for holdfast attachment to the cell. Sequence analysis of the hfaAB locus showed two open reading frames with the potential to encode proteins of 15,000 and 26,000 Da, respectively. A protein migrating with an apparent size of 21 kDa in gel electrophoresis was encoded by the hfaA region when expressed in Escherichia coli under the control of the lac promoter, but no protein synthesis could be detected from the hfaB region. S1 nuclease analysis indicated that transcription of the hfaAB locus was initiated from a region containing a sequence nearly identical to the consensus for C. crescentus sigma 54-dependent promoters. In addition, a sequence with some similarity to ftr sequences (a consensus sequence associated with other Caulobacter sigma 54-dependent genes) was identified upstream of the hypothesized sigma 54 promoter. At least one of the hfaAB gene products was required for maximal transcription of hfaC. The sequence of hfaB showed some similarity to that of transcriptional activators of other bacteria. The C terminal region of the putative gene product HfaA was found to be homologous to PapG and SmfG, which are adhesin molecules of enteropathogenic E. coli and Serratia marcescens, respectively. This information suggests that the protein encoded by the hfaA locus may have a direct role in the attachment of the holdfast to the cell, whereas hfaB may be involved in the positive regulation of hfaC. PMID- 1732205 TI - Lysyl-tRNA synthetase gene of Campylobacter jejuni. AB - We report the cloning and complete nucleotide sequence of the Campylobacter jejuni lysyl-tRNA synthetase gene (lysS). The C. jejuni lysS gene sequence shows high homology to the two Escherichia coli lysyl-tRNA synthetase genes, lysS and lysU. The Campylobacter lysyl-tRNA synthetase protein (LysRS) shows 47.9 and 46.6% sequence identity to the E. coli enzymes encoded by the lysS and lysU genes, respectively. The LysRS encoded by the C. jejuni gene is a polypeptide of 501 amino acids with a deduced molecular weight of 57,867. The enzyme is active in E. coli. The gene is expressed from its own promoter, and the transcription start site has been mapped. The carboxyl-terminal codon of the C. jejuni lysS gene overlaps by 1 bp with the Met initiation codon of the glyA gene, which has been shown to have a promoter which is functional in E. coli (V.L. Chan and H.L. Bingham, Gene 101:51-58, 1991). C. jejuni, unlike E. coli, has only one lysyl tRNA synthetase gene. PMID- 1732206 TI - Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB. AB - Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function. PMID- 1732207 TI - Nucleotide sequence and functional analysis of the complete phenol/3,4 dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. AB - The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta cleavage pathway enzyme is discussed. PMID- 1732208 TI - Characterization of Saccharopolyspora erythraea cytochrome P-450 genes and enzymes, including 6-deoxyerythronolide B hydroxylase. AB - Previous studies of erythromycin biosynthesis have indicated that a cytochrome P 450 monooxygenase system is responsible for hydroxylation of 6-deoxyerythronolide B to erythronolide B as part of erythromycin biosynthesis in Saccharopolyspora erythraea (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210 1987). The enzyme was previously purified to apparent homogeneity and found to have a catalytic turnover number of approximately 10(-3) min-1. More recently, disruption of a P-450-encoding sequence (eryF) in the region of ermE, the erythromycin resistance gene of S. erythraea, produced a 6-deoxyerythronolide B hydroxylation-deficient mutant (J. M. Weber, J. O. Leung, S. J. Swanson, K. B. Idler, and J. B. McAlpine, Science 252:114-116, 1991). In this study we purified the catalytically active cytochrome P-450 fraction from S. erythraea and found by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis that it consists of a major and a minor P-450 species. The gene encoding the major species (orf405) was cloned from genomic DNA and found to be distinct from eryF. Both the orf405 and eryF genes were expressed in Escherichia coli, and the properties of the proteins were compared. Heterologously expressed EryF and Orf405 both reacted with antisera prepared against the 6-deoxyerythronolide B hydroxylase described by Shafiee and Hutchinson (1987), and the EryF polypeptide comigrated with the minor P-450 species from S. erythraea on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. In comparisons of enzymatic activity, EryF hydroxylated a substrate with a turnover number of 53 min-1, whereas Orf405 showed no detectable activity with a 6-deoxyerythronolide B analog. Both enzymes showed weak activity in the O-dealkylation of 7-ethoxycoumarin. We conclude that the previously isolated 6-deoxyerythronolide B hydroxylase was a mixture of two P-450 enzymes and that only the minor form shows 6-deoxyerythronolide B hydroxylase activity. PMID- 1732209 TI - Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii. AB - The gene of a halophilic alkaline serine protease, halolysin, from an unidentified halophilic archaea (archaebacterium) was cloned and its nucleotide sequence was determined. The deduced amino acid sequence showed that halolysin consists of 411 amino acids, with a molecular weight of 41,963. The highest homology was found with thermitase from Thermoactinomyces vulgaris. Halolysin has a long C-terminal extension of approximately 120 amino acids which has not been found in other extracellular subtilisin type serine proteases. The gene, hly, was expressed in another halophilic archaea, Haloferax volcanii, in a medium containing 18% salts by using a plasmid shuttle vector which has a novobiocin resistance determinant as a selectable marker. PMID- 1732210 TI - Role of the heat shock response in stability of mRNA in Escherichia coli K-12. AB - The heat shock response in Escherichia coli involves extensive induction of the heat shock proteins, with the concomitant suppression of the synthesis of the non heat shock proteins. While the induction of the heat shock proteins has been shown to occur primarily at the transcriptional level, the suppression of non heat shock proteins is poorly understood. We have investigated the possibility that an increased decay of non-heat shock mRNAs is a means of decreasing the synthesis of non-heat shock proteins during the heat shock response. Heat shock response-defective strains were compared with wild-type controls by several criteria to evaluate both mRNA stability and the induction of enzymes known to be involved in mRNA turnover. Our results indicate that increased mRNA decay is not a mechanism used to regulate the synthesis of non-heat shock proteins. PMID- 1732211 TI - Characterization of a regulatory network that controls sigma B expression in Bacillus subtilis. AB - The sigB operon of Bacillus subtilis encodes sigma B and three additional open reading frames (orfV, orfW, and orfX). Having previously mapped several mutations that alter the induction pattern of a sigma B-dependent promoter (ctc) to regions of cloned B. subtilis DNA which contain these three open reading frames, we directly tested the regulatory potential of orfV, orfW, and orfX by creating null alleles of each of these genes and examining the effects of the mutations, either singly or in pairs, on transcription of ctc and the sigB operon. Using lacZ reporter gene fusions and Northern (RNA) blot analyses, we have determined that all three genes modulate the activation of the sigma B-dependent promoters at both the sigB operon and ctc. Our data are consistent with the three gene products participating in a single pathway of negative control. orfW and orfX single-mutant strains have high levels of sigB and ctc transcription. sigB and ctc transcription in an orfV strain is similar to that found in mutant strains which lack sigma B itself. The orfV mutation is dominant to orfX but recessive to orfW. These results suggest that OrfW is the primary inhibitor of sigma B dependent transcription and that OrfV is capable of counteracting the negative control of OrfW but is prevented from doing this by the orfX gene product. PMID- 1732212 TI - Nucleotide sequences of the arb genes, which control beta-glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new beta-glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans. AB - The phytopathogenic bacterium Erwinia chrysanthemi, unlike other members of the family Enterobacteriaceae, is able to metabolize the beta-glucosides, arbutin, and salicin. A previous genetic analysis of the E. chrysanthemi arb genes, which mediate beta-glucoside metabolism, suggested that they were homologous to the Escherichia coli K-12 bgl genes. We have now determined the nucleotide sequence of a 5,065-bp DNA fragment containing three genes, arbG, arbF, and arbB. Deletion analysis, expression in minicell systems, and comparison with sequences of other proteins suggest that arbF and arbB encode a beta-glucoside-specific phosphotransferase system-dependent permease and a phospho-beta-glucosidase, respectively. The ArbF amino acid sequence shares 55% identity with that of the E. coli BglF permease and contains most residues thought to be important for a phosphotransferase. One change, however, was noted, since BglF Arg-625, presumably involved in phosphoryl transfer, was replaced by a Cys residue in ArbF. An analysis of the ArbB sequence led to the definition of a protein family which contained enzymes classified as phospho-beta-glucosidases, phospho-beta galactosidases, beta-glucosidases, and beta-galactosidases and originating from gram-positive and gram-negative bacteria, archebacteria, and mammals, including humans. An analysis of this family allowed us (i) to speculate on the ways that these enzymes evolved, (ii) to identify a glutamate residue likely to be a key amino acid in the catalytic activity of each protein, and (iii) to predict that domain II of the human lactate-phlorizin hydrolase, which is involved in lactose intolerance, is catalytically nonactive. A comparison between the untranslated regions of the E. chrysanthemi arb cluster and the E. coli bgl operon revealed the conservation of two regions which, in the latter, are known to terminate transcription under noninducing conditions and be the target of the BglG transcriptional antiterminator under inducing conditions. ArbG was found to share a high level of similarity with the BglG antiterminator as well as with Bacillus subtilis SacT and SacY antiterminators, suggesting that ArbG functions as an antiterminator in regulating the expression of the E. chrysanthemi arb genes. PMID- 1732213 TI - Cloning and DNA sequence of a mycoplasmal recA gene. AB - Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein. PMID- 1732214 TI - Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium. AB - Defects in the chemotaxis proteins CheY and CheZ of Salmonella typhimurium can be suppressed by mutations in the flagellar switch, such that swarming of a pseudorevertant on semisolid plates is significantly better than that of its parent. cheY suppressors contribute to a clockwise switch bias, and cheZ suppressors contribute to a counterclockwise bias. Among the three known switch genes, fliM contributes most examples of such suppressor mutations. We have investigated the changes in FliM that are responsible for suppression, as well as the changes in CheY or CheZ that are being compensated for. Ten independently isolated parental cheY mutations represented nine distinct mutations, one an amino acid duplication and the rest missense mutations. Several of the altered amino acids lie on one face of the three-dimensional structure of CheY (A. M. Stock, J. M. Mottonen, J. B. Stock, and C. E. Schutt, Nature (London) 337:745 749, 1989; K. Volz and P. Matsumura, J. Biol. Chem. 266:15511-15519, 1991); this face may constitute the binding site for the switch. All 10 cheZ mutations were distinct, with several of them resulting in premature termination. cheY and cheZ suppressors in FliM occurred in clusters, which in general did not overlap. A few cheZ suppressors and one cheY suppressor involved changes near the N terminus of FliM, but neither cheY nor cheZ suppressors involved changes near the C terminus. Among the strongest cheY suppressors were changes from Arg to a neutral amino acid or from Val to Glu, suggesting that electrostatic interactions may play an important role in switching. A given cheY or cheZ mutation could be suppressed by many different fliM mutations; conversely, a given fliM mutation was often encountered as a suppressor of more than one cheY or cheZ mutation. The data suggest that an important factor in suppression is a balancing of the shift in switch bias introduced by alteration of CheY or CheZ with an appropriate opposing shift introduced by alteration of FliM. For strains with a severe parental mutation, such as the cheZ null mutations, adjustment of switch bias is essentially the only factor in suppression, since the attractant L-aspartate caused at most a slight further enhancement of the swarming rate over that occurring in the absence of a chemotactic stimulus. We discuss a model for switching in which there are distinct interactions for the counterclockwise and clockwise states, with suppression occurring by impairment of one of the states and hence by relative enhancement of the other state. FliM can also undergo amino acid changes that result in a paralyzed (Mot-) phenotype; these changes were confined to a very few residues in the protein. PMID- 1732215 TI - Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination. AB - During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth. PMID- 1732216 TI - Immunocytochemical analysis of AlgP (Hp1), a histonelike element participating in control of mucoidy in Pseudomonas aeruginosa. AB - AlgP, a protein with an unusual carboxy-terminal domain resembling the tails of eukaryotic H1 histones, was detected in whole-cell extracts and within the cells of Pseudomonas aeruginosa by using immunoblotting and immunoelectron microscopy analyses. One known function of AlgP is its participation in the transcriptional activation of the algD gene. This is a pivotal step in the establishment of mucoidy in P. aeruginosa; mucoidy is a critical virulence factor expressed during respiratory infections in patients with cystic fibrosis. Polyclonal and monoclonal antibodies were raised against a synthetic 50-mer peptide containing two sets of six tandem repeats of the motif Lys-Pro-Ala-Ala (and its single-amino acid substitution variants), based on the sequence of the algP gene from the standard genetic strain PAO. Western immunoblots with these antibodies and total protein extracts from P. aeruginosa revealed two polypeptides that reacted with the antibodies in all of the P. aeruginosa strains tested. The detected polypeptides displayed strain-dependent variability in their electrophoretic mobility, in accordance with the previously noted variability of the algP repeats at the DNA level. In strain PAO, the recognized polypeptides had apparent masses of 46.4 and 41.6 kDa. Immunoelectron microscopy revealed that AlgP is an intracellular protein with a wide distribution suggestive of its more general role. To indicate that fact, AlgP is given here an alternative name, Hp1. Since AlgP (Hp1) is a eubacterial histonelike element displaying sequence and domanial similarity with eukaryotic H1 histones, these findings may have implications on the understanding of the organization of the prokaryotic nucleoid and its role in the control of gene expression and bacterial virulence. PMID- 1732217 TI - Morphology and dynamics of protruding spirochete periplasmic flagella. AB - We recently characterized the three-dimensional shape of Treponema phagedenis periplasmic flagella (PFs). In the course of these studies, we observed protrusions on swimming cells that resembled PFs. Here we present a detailed characterization of the shape, structure, and motion of these protrusions. Although protrusion formation occurred primarily in wild-type cells during the stationary phase, a large fraction of exponential-phase cells of cell cylinder helicity mutants (greater than 90% of mutant T-52) had protrusions. These results suggest that cells bearing protrusions can still participate in cell division. T. phagedenis protrusions had the identical helix handedness, pitch, and diameter to those of purified PFs. Protrusions were not present on mutants unable to synthesize PFs, but were present in all motile revertants which regained PFs. These results, taken together with electron microscope observations, suggest that protrusions consist of PFs surrounded by an outer membrane sheath. To analyze protrusion movements, we held cells against a coverglass surface with optical tweezers and observed the motion of protrusions by video-enhanced differential interference contrast light microscopy. Protrusions were found to gyrate in both clockwise and counterclockwise directions, and direct evidence was obtained that protrusions rotate. Protrusions were also observed on Treponema denticola and Borrelia burgdorferi. These were also left-handed and had the same helix handedness, pitch, and diameter as purified PFs from their respective species. The PFs from T. denticola had a helix diameter of 0.26 microns and a helix pitch of 0.78 micron; PFs from B. burgdorferi had a helix diameter of 0.28 micron and a helix pitch of 1.48 microns. Protrusions from these spirochete species had similar structures and motion to those of T. phagedenis. Our results present direct evidence that PFs rotate and support previously proposed models of spirochete motility. PMID- 1732218 TI - Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens. AB - The occ and noc regions of octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of octopine and nopaline, respectively. Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regions contained additional DNA stretches which were under the control of opine inducible promoters, but the functions were unknown. We investigated these stretches by DNA sequence and functional analyses. The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction. The occ and noc region genes are related, but the arrangement of the genes is different. The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria. The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein. We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled octopine showed that the putative transport genes in the occ region code for octopine uptake proteins. The corresponding studies with 3H labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region. PMID- 1732219 TI - Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid. AB - We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of the flaO operon and the adjacent flbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of the flaO operon transcript by using a nuclease S1 protection assay, and the 5' end of the flbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by sigma 28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from the flbF promoter region, that was predicted to be the flbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYVO3 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990). PMID- 1732220 TI - The narJ gene product is required for biogenesis of respiratory nitrate reductase in Escherichia coli. AB - Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, alpha, beta, and gamma, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon. The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme. To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::Tn10 insertion mutant. The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene. In both cases, when nar operon expression was induced, the alpha and beta subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an alpha-beta complex separated from the gamma subunit is known to catalyze full MVH-linked activity but not the formate linked activity associated with the membrane-bound complex. The low-activity forms of the alpha and beta subunits also accumulated in the absence of the NarJ protein when the gamma subunit (NarI) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex. When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed. When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the alpha and beta subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the alpha subunit appeared to be protected from the proteolytic clipping which occurred in the absence of NarJ. We conclude that NarJ is indispensible for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active alpha-beta complex or in facilitating the interaction of the complex with the membrane-bound gamma subunit. PMID- 1732221 TI - Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression. AB - The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with beta-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90 degrees C), thermostable, and resistant to common protein denaturants and proteases. Expression of the gene in mesophilic hosts is needed to uncover the molecular nature of these features. We have obtained expression of beta galactosidase in Saccharomyces cerevisiae under the control of the galactose inducible upstream activating sequence of the yeast genes GAL1 and GAL10. The expressed enzyme is identical in molecular mass, thermostability, and thermophilicity to the native enzyme, showing that these features are intrinsic to the primary structure of the enzyme. We also present a new promoter for the expression of thermostable proteins in S. cerevisiae. This promoter contains a sequence isolated from the nematode Caenorhabditis elegans that works as a strong, heat-inducible upstream activating sequence in S. cerevisiae. Transcription of the lacS gene under the control of this sequence is rapidly and efficiently induced by heat shock. The availability of a plate assay for monitoring beta-galactosidase activity in S. cerevisiae may allow screening for mutants affecting the efficiency and activity of the enzyme. PMID- 1732222 TI - Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus. AB - The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanol-grown cells of Alcaligenes eutrophus was determined. By using oligonucleotides deduced from this sequence the structural gene for AcDH II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). A 2.8-kbp PstI subfragment of D, which harbored acoD, was sequenced. It revealed an open reading frame of 1,518 bp, encoding a protein with a relative molecular weight of 54,819. The insertions of Tn5::mob of two transposon-induced mutants of A. eutrophus, which were impaired in the catabolism of acetoin, were mapped 483 or 1,359 bp downstream from the translational start codon of acoD. The structural gene was preceded by a putative Shine-Dalgarno sequence. The transcriptional start site 57 bp upstream of acoD was identified and was preceded by a sequence which exhibited a striking homology to the enterobacterial sigma 54-dependent promoter consensus sequence. This was in accordance with the observation that the expression of acoD and of other acetoin-catabolic genes depended on the presence of an intact rpoN-like gene. Alignments of the amino acid sequence deduced from acoD with the primary structures of aldehyde dehydrogenases from other sources revealed high degrees of homology, amounting to 46.5% identical amino acids. PMID- 1732223 TI - Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila. AB - The major outer membrane protein of Legionella pneumophila is composed of 28- and 31-kDa subunits cross-linked by interchain disulfide bonds. The oligomer is covalently anchored to the underlying peptidoglycan via the 31-kDa subunit. We have cloned the structural gene ompS encoding both proteins. Oligonucleotide probes synthesized from the codons of the N-terminal amino acid sequence of purified 28- and 31-kDa subunits were used to identify cloned sequences. A 2.9-kb HindIII fragment cloned into pBluescript (clone H151) contained the ompS gene. Nucleotide sequence analysis revealed an open reading frame of 891 bp encoding a polypeptide of 297 amino acids. A leader sequence of 21 amino acids was identified, and the mature protein contained 276 amino acids. The deduced amino acid sequence of OmpS matched the experimentally determined amino acid sequence (32 amino acids), with the exception of two cysteine residues. The deduced amino acid sequence was rich in glycine and aromatic amino acids and contained four cysteine residues, two in the amino terminus and two in the carboxy region. Primer extension analysis (total RNA from L. pneumophila) identified the transcription start at 96 to 98 bp upstream of the translation start, but no Escherichia coli-like promoter sequences were evident. While an mRNA transcript from clone H151 was detected, no cross-reactive protein was detected by immunoblotting with either monoclonal or polyclonal antibody. Attempts to subclone the gene in the absence of the putative promoter region (i.e., under the control of the lac promoter) proved unsuccessful, possibly because of overproduction lethality in E. coli. The ompS DNA sequence was highly conserved among the serogroups of L. pneumophila, and related species also exhibited homology in Southern blot analysis at a moderately high stringency. Evidence is presented to suggest that this gene may be environmentally regulated in L. pneumophila. PMID- 1732224 TI - FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions. AB - In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is independent of nutritional factors. The well-known growth rate-dependent control, displayed by exponentially growing cells studied under various nutritional conditions, is governed by two regulatory mechanisms: repression, presumably by ribosome feedback inhibition, and stimulation by trans activation. FIS allows very fast bacterial growth. PMID- 1732225 TI - Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12. AB - The rfa locus of Escherichia coli K-12 includes a block of about 10 closely spaced genes transcribed in the same direction which are involved in synthesis and modification of the hexose region of the lipopolysaccharide core. We have sequenced the first three genes in this block. The function of the first of these genes is unknown, but we have designated it rfaQ on the basis of its location and similarity to other rfa genes. Complementation of Salmonella typhimurium rfa mutants with E. coli rfa restriction fragments indicated that the second and third genes in the block were rfaG and rfaP. The deduced sizes of the RfaQ, RfaG, and RfaP proteins are 36,298, 42,284, and 30,872 Da, respectively, and the proteins are basic and lack extensive hydrophobic domains. RfaQ shares regions of homology with proteins RfaC and RfaF, which are involved in synthesis of the heptose region of the core. Proteins RfaB, RfaG, and RfaK share a region of homology, which suggests that they belong to a second family of Rfa proteins which are thought to be hexose transferases. PMID- 1732226 TI - Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1. AB - Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material). PMID- 1732227 TI - The expression of virulence genes in Listeria monocytogenes is thermoregulated. AB - The expression of listeriolysin, a major virulence factor of the gram-positive facultative intracellular pathogen Listeria monocytogenes, is positively regulated by a transcriptional activator, the prfA gene product. We had previously shown that mutations within the prfA gene lead to loss of listeriolysin production. In this communication, the regulation of expression of listeriolysin by a specific environmental condition, namely, temperature, was studied in wild-type strains of Listeria monocytogenes. We found that expression of the hemolysis phenotype was thermoregulated. A lisA::lacZ fusion was constructed, and its expression in the wild-type strain was studied at various growth temperatures. The results showed that the fusion beta-galactosidase activity was expressed only when cultures were grown at temperatures above 30 degrees C. This activity could be either specifically repressed or induced, depending on growth temperature. No change in activity was detected in a strain harboring a control beta-galactosidase fusion at the various growth temperatures tested. Northern (RNA) blot analysis of lisA-specific RNA transcripts showed that thermoregulation is manifested at the level of transcription. We also found that the transcription of other PrfA-regulated virulence genes in L. monocytogenes was similarly affected by growth temperature. Hence, as in other facultative intracellular pathogens, Shigella and Yersinia spp., temperature is an important cue in the induction of expression of virulence genes in L. monocytogenes. Our studies revealed that a higher level of regulation is imposed on the PrfA mediated activation of virulence genes in pathogenic L. monocytogenes. PMID- 1732228 TI - Suppression of oxidative envelope damage by pseudoreversion of a superoxide dismutase-deficient mutant of Escherichia coli. AB - Mutants of Escherichia coli that are devoid of superoxide dismutase (SOD) fail to grow in aerobic minimal medium. This is largely because of the O2- sensitivities of several amino acid biosynthetic pathways, since amino acid supplements can restore growth, albeit at a slow rate. We now report that growth in amino acid supplemented medium can be further stimulated by the presence of extracellular osmolytes. Osmolytes also partially suppress the amino acid requirements of the SOD mutant. These data suggest that the combination of oxidative injury and turgor pressure permeabilizes the cell envelope and that critical metabolites, including the limiting products of damaged biosynthetic pathways, escape from the cell. External osmolytes may offer protection by countervailing the usual turgor pressure and thus stabilizing the damaged envelope. This model is consistent with the previous observation that deficiency of cell wall components is lethal to SOD mutants. A pseudorevertant that can grow at a moderate rate in normosmotic medium without amino acid supplementation has been obtained (J. A. Imlay and I. Fridovich, Mol. Gen. Genet. 228:410-416, 1991). Analysis suggests that the suppressor mutation allows the envelope either to resist or to tolerate oxidative lesions. Study of the pseudorevertant may illuminate the molecular basis of this oxidative envelope injury. PMID- 1732229 TI - Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp. strain NS671. AB - Pseudomonas sp. strain NS671, which produces L-amino acids asymmetrically from the corresponding racemic 5-substituted hydantoins, harbored a plasmid of 172 kb. Curing experiments suggest that this plasmid, designated pHN671, is responsible for the conversion of 5-substituted hydantoins to their corresponding L-amino acids by strain NS671. DNA fragments containing the genes involved in this conversion were cloned from pHN671 in Escherichia coli by using pUC18 as a cloning vector. The smallest recombinant plasmid, designated pHPB12, contained a 7.5-kb insert DNA. The nucleotide sequence of the insert DNA was determined, and three closely spaced open reading frames predicted to encode peptides with molecular masses of 75.6, 64.9, and 45.7 kDa were found. These open reading frames were designated hyuA, hyuB, and hyuC, respectively. Cell extracts from E. coli carrying deletion derivatives of pHPB12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gene products of hyuA, hyuB, and hyuC were identified. The functions of these gene products were also examined with the deletion derivatives. The results indicate that both hyuA and hyuB are involved in the conversions of D- and L-5-substituted hydantoins to corresponding N-carbamyl-D- and N-carbamyl-L-amino acids, respectively, and that hyuC is involved in the conversion of N-carbamyl-L-amino acids to L-amino acids. PMID- 1732230 TI - Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene. AB - The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals. Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature. We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci. Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events. The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B. pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions. The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature. Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active. In B. pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects. In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations. PMID- 1732231 TI - Characterization of the Agrobacterium tumefaciens heat shock response: evidence for a sigma 32-like sigma factor. AB - We have characterized the heat shock response of Agrobacterium tumefaciens and compared it with the well-characterized Escherichia coli heat shock response. Four major heat shock proteins with apparent molecular masses of 98, 75, 65, and 20 kDa were identified by pulse-labelling cultures after temperature upshift. The three largest proteins comigrated with proteins that were antigenically related to the E. coli heat shock proteins sigma 70, DnaK, and GroEL, respectively. The heat shock proteins were also strongly induced by ethanol and cadmium chloride and were mildly induced by mitomycin C. To determine whether the A. tumefaciens heat shock regulatory system was similar to that of E. coli, we introduced the E. coli dnaK gene into A. tumefaciens. The E. coli DnK protein was expressed in A. tumefaciens, and its synthesis was induced after heat shock. Primer extension analysis of the E. coli dnaK gene in A. tumefaciens indicated that transcription initiated from one or possibly both of the E. coli heat shock promoters. We conclude that A. tumefaciens has a heat shock response similar to that of E. coli, in that (i) similar proteins are induced by heat shock, (ii) synthesis of these proteins is induced in response to similar stimuli, and (iii) A. tumefaciens can recognize an E. coli heat shock promoter, suggesting that A. tumefaciens has a sigma factor similar to sigma 32. PMID- 1732233 TI - Knee prostheses--one step forward, two steps back. PMID- 1732232 TI - Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase. AB - The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions. PMID- 1732234 TI - High calcitonin levels in unconscious polytrauma patients. AB - We measured levels of calcitonin and other markers of calcium and phosphorus metabolism in both unconscious and conscious patients after multiple trauma. We found dramatic increases in calcitonin levels in unconscious patients, and smaller increases in conscious patients. In two cases, very high levels, more than 100 x normal, appeared to be related to more rapid healing of bone. Calcitonin levels were highest immediately after admission and decreased over the ensuing two weeks. The possible relationship between unconsciousness and the increased rate of healing of fractures is discussed. PMID- 1732235 TI - Fresh osteochondral allografts for post-traumatic defects in the knee. A survivorship analysis. AB - Fresh osteochondral allografts were used to repair post-traumatic osteoarticular defects in 92 knees. At the time of grafting, varus or valgus deformities were corrected by upper tibial or supracondylar femoral osteotomies. A survivorship analysis was performed in which failure was defined as the need for a revision operation or the persistence of the pre-operative symptoms. There was a 75% success rate at five years, 64% at ten years and 63% at 14 years. The failure rate was higher for bipolar grafts than for unipolar and the results in patients over the age of 60 years were poor. The outcome did not depend on the sex of the patient and the results of allografts in the medial and lateral compartments of the knee were similar. Careful patient selection, correction of joint malalignment by osteotomy, and rigid fixation of the graft are all mandatory requirements for success. We recommend this method for the treatment of post traumatic osteochondral defects in the knees of relatively young and active patients. PMID- 1732236 TI - Diagnosis and assessment of pectoralis major rupture by dynamometry. AB - Four patients with pectoralis major ruptures underwent clinical and dynamometric assessment and one patient underwent late surgical repair. The operation is described. Dynamometry proved a useful and objective method of estimating the loss of strength and indicating patients who might benefit from surgical repair. PMID- 1732237 TI - Repair of the calcaneal tendon. An improved technique. AB - We report the use of a simple new technique for the repair of recent and old, neglected disruptions of the calcaneal tendon, which can be used where there is a wide gap. We reviewed 13 patients from one to six years after operation; all had good objective and subjective results. Four had minor skin complications. PMID- 1732238 TI - Partial versus total meniscectomy. A prospective, randomised study with long-term follow-up. AB - Two hundred patients with a meniscal lesion were peroperatively allocated to partial or total meniscectomy in a random manner. The results were compared at one year and at 6.3 to 9.8 years (median 7.8). After one year more patients with partial meniscectomy (90%) than with total meniscectomy (80%) had no complaints. At the later review these figures were 62% and 52%, respectively (p = 0.18). However, patients with partial meniscectomy had higher functional scores. The deterioration in function between the first review and the second showed no significant difference in the two treatment groups. The incidence of mediolateral instability rose from 8% to 47% and was more frequent after total than after partial meniscectomy. Between the two reviews the radiological signs of knee degeneration increased with no difference between the two treatment groups. PMID- 1732239 TI - Fractures of the distal femur treated with the AO dynamic condylar screw. AB - We report the results of treatment with the dynamic condylar screw of 21 cases of supracondylar and intercondylar fractures of the femur in patients aged 22 to 91 years. There were two nonunions and no deaths. We found the device easy to use and the good fixation allowed early patient mobilisation. PMID- 1732240 TI - Experimental stretch neuropathy. Changes in nerve conduction under tension. AB - We developed an animal model of stretch injury to nerve in order to study in vivo conduction changes as a function of nerve strain. In 24 rabbits, the tibial nerve was exposed and stretched by 0%, 6% or 12% of its length. The strain was maintained for one hour. Nerve conduction was monitored during the period of stretch and for a one-hour recovery period. At 6% strain, the amplitude of the action potential had decreased by 70% at one hour and returned to normal during the recovery period. At 12% strain, conduction was completely blocked by one hour, and showed minimal recovery. These findings have clinical implications in nerve repair, limb trauma, and limb lengthening. PMID- 1732241 TI - Ultrasonographic monitoring of limb lengthening. AB - Limb lengthening in nine patients was monitored by radiographs and by ultrasound scans. The distraction gap appeared as a sonolucent area within which echogenic foci developed soon after distraction commenced. By seven weeks a new cortex was detected, and medullary canal began to develop between seven and eight weeks. Ultrasound scanning can be used to measure distraction, but it was not as useful as radiographs in detecting angulation. Its use in patients undergoing limb lengthening could reduce their exposure to radiation. PMID- 1732242 TI - Neuropathic foot ulceration treated by total contact casts. AB - Ulceration of the insensitive foot continues to cause great morbidity in diabetic patients. We treated 46 patients with neuropathic ulceration by applying total contact casts. Most neuropathic ulcers healed within six weeks but ischaemic ulcers did not heal. One patient developed gangrene and required partial amputation of the foot. PMID- 1732243 TI - Mitchell's osteotomy using internal fixation and early mobilisation. AB - We present the results of a prospective trial of osteotomy of the metatarsal neck for hallux valgus in 31 feet of 23 women, using a new stapling device with no plaster splintage and early weight-bearing. Surgery was performed for pain (29 feet) and difficulty with footwear (nine feet). The average time for return to light work was 3.3 weeks, and to full work 8.3 weeks after operation. Seventeen patients had full recovery within three months and 21 of the 23 patients had complete relief of pain. Shoes were more comfortable in 17 feet and 9 patients could wear narrower shoes. Only two patients were unsatisfied with the appearance of the foot. All the osteotomies united, and the average hallux valgus angle was improved on radiographs from 35 degrees to 23 degrees. The mean first metatarsal angle was reduced from 16 degrees to 11 degrees. The new technique allows more accurate surgery and easier postoperative management. PMID- 1732244 TI - The natural history of recurrent dislocation of the patella. Long-term results of conservative and operative treatment. AB - From 1970 to 1978, 29 patients had a unilateral operation for bilateral recurrent dislocation of the patella. We examined 21 of them at a mean of 14 years postoperatively. Eighteen of these patients had evidence of generalised joint laxity. Six of the operated knees and four of the unoperated knees still had recurrent dislocations. The operated knees were clinically worse, with a significantly higher incidence of osteoarthritis. We concluded that the operations used to treat recurrent dislocation of the patella may have had short term benefits, but did not cure the patients in the long term. PMID- 1732245 TI - The unstable hip and mid-lumbar myelomeningocele. AB - We reviewed 55 patients with mid-lumbar myelomeningocele (L3 and L4) first seen over a 17-year period from 1970 to 1986 and followed up for an average of ten years. We assessed a number of factors which might affect hip stability and ability to walk, recording the natural history of clinical and radiological hip deformity. Two-thirds of the hips had become dislocated or subluxed by the end of the first year of life, involving 86% of hips in patients with an L3 level and 45% of those with an L4 level. All the hips that developed instability secondary to muscle imbalance did so within the first year. The neurological level was the most significant determinant of walking ability: all patients with L4 neurological levels could walk but only one-third of those with L3 lesions could do so. Hip stability, intelligence quotient and fixed deformity did not influence walking ability. PMID- 1732246 TI - Can burst fractures be predicted from plain radiographs? AB - Plain radiographs of 67 acute spinal compression fractures in 49 patients were analysed by subjective and objective criteria, using CT scans as the diagnostic standard for the diagnosis of burst fracture. Discriminant analysis correctly predicted the type of fracture in 88% of cases. Burst fractures, however, were almost as frequently misdiagnosed as being wedge compression fractures using this technique, compared with the reading of 25 films from patients without previous information. A quarter of the injuries would have been misdiagnosed had reliance been placed solely on the plain radiographs. CT scans of all patients with acute spinal compression fractures should be considered to decrease this potentially serious diagnostic error. PMID- 1732247 TI - A quantitative histological study of the vascularity of the rotator cuff tendon. AB - Previous perfusion studies of the rotator cuff have demonstrated an area of hypovascularity in the distal part of the supraspinatus tendon. This has been implicated in the pathogenesis of its rupture. We performed a quantitative histological analysis of the vascularity of the tendons of supraspinatus and infraspinatus. Vessel number, size and the percentage of the tendon occupied by vessels were measured at 5 mm intervals from the humeral insertions to the muscle bellies. Both tendons were hypovascular in their distal 15 mm. No significant difference was demonstrated between the vascularity of supraspinatus and infraspinatus. We conclude that factors other than vascularity are important in the pathogenesis of supraspinatus rupture. PMID- 1732248 TI - Complex clavicular injury in childhood. PMID- 1732249 TI - Changes in the ankle reflex related to posture. PMID- 1732250 TI - Tissue reaction and loosening of carbon-reinforced polyethylene arthroplasties. PMID- 1732251 TI - A flexion adduction method for the reduction of posterior dislocation of the hip. PMID- 1732252 TI - Pre-operative planning for intramedullary nailing. PMID- 1732253 TI - Dislocation of a total hip prosthesis by a false aneurysm. PMID- 1732254 TI - Peroneus longus tendon calcification. PMID- 1732255 TI - Condensing osteitis of the clavicle. PMID- 1732256 TI - Polyethylene wear in uncemented knee replacements. AB - Isolated wear of the polyethylene tibial component led to failure in five of a series of 108 uncemented porous-coated knee replacements. The clinical features included pain, effusion and instability with progressive varus deformity. In all cases there was extensive wear on the medial side of the polyethylene surface of the prosthesis. The mechanism of such wear is complex, being due in part to the unconstrained nature of the joint and the incongruity of its surfaces. Other design characteristics may have contributed. PMID- 1732257 TI - Recurrent dislocation of the patella. PMID- 1732258 TI - Aggressive granulomatosis from polyethylene failure in an uncemented knee replacement. PMID- 1732259 TI - Failure of acetabular cups influenced by marker wire anchorage. AB - Failure of an acetabular cup is uncommon and has been attributed to wear or creep, trauma or bony irregularities in the acetabulum. We report ten cases in which fracture of the cup occurred at the site of drill holes used to anchor the marker wire. The role of such indentations as stress raisers has not been previously reported; we suggest that deep indentations or grooves should not be placed in the most highly stressed areas and that the cup thickness should allow for predicted wear rates. PMID- 1732260 TI - The morphology of the proximal femur. A three-dimensional radiographic analysis. AB - Biological fixation of cementless femoral implants requires primary stability by optimal fit in the proximal femur. The anatomy of the bone must then be known precisely. We analysed in vitro the accuracy of bone measurements of 32 femurs and compared the dimensions obtained from radiographs and CT scans with the true anatomical dimensions. Standard radiographs gave only a rough approximation of femoral geometry (mean difference: 2.4 +/- 1.4 mm) insufficiently accurate to allow selection of the best fitting prosthesis from a range of sizes and altogether inadequate to design a custom-made prosthesis. CT scans give greater accuracy (mean difference: 0.8 +/- 0.7 mm) in our experimental conditions, but in clinical practice additional sources of error exist. PMID- 1732261 TI - The clones of osteoarthritic cartilage. AB - We studied the early cartilage changes in osteoarthritis, examining the most normal appearing articular cartilage from the hips of 17 patients. Normal appearing cartilage from five patients treated for fractures was used as control material. Two different types of clone were found. The first had increased staining for proteoglycan and was thought to have been engaged in the synthesis of matrix. The other type was associated with a severe deficiency of proteoglycan, matrix streaks and evidence of degradation and phagocytosis of matrix components. Immunohistochemistry demonstrated large amounts of chondroitin 4 and 6 sulphate about the synthetic-type clones, and little or no reactivity about the degenerative clones which lay more superficially and were associated with matrix destruction. Clones appeared to be engaged in either matrix synthesis or its destruction. The disease process of osteoarthritis appeared to begin at the surface of the articular cartilage. PMID- 1732262 TI - Histology of a lengthened human tibia. AB - We describe the histology of a specimen taken from an amputated leg seven months after a 15 cm bone gap in the tibia had been closed by bone transport. Lengthening appeared to have occurred by repeated minor trauma to the bone, with the fractured trabeculae in sufficiently close contact for the repair process to proceed. Osteogenesis did not occur through a cartilage phase, but the fracture gaps were bridged by collagen fibres, around which new bone formed. Microfractures had repaired by primary healing with woven bone and with no microcallus. Small regions of bone were necrotic. Resorption of the necrotic bone and remodelling of the immature bundle and woven bone were still at an early stage, suggesting that complete remodelling in man may take years rather than months. PMID- 1732263 TI - Fit and fill: fashionable fact or fantasy? PMID- 1732264 TI - A venous foot pump reduces thrombosis after total hip replacement. AB - In a prospective, randomised controlled trial, the efficacy of the A-V Impulse System in the prevention of deep-vein thrombosis was investigated in 84 patients who had undergone total hip replacement. The incidence of venographically proven, and clinically significant postoperative deep-vein thrombosis was 40% in the control group and 5% in the treatment group (p less than 0.001). No adverse reactions were recorded. PMID- 1732265 TI - Thrombo-embolic prophylaxis in total knee replacement. Evaluation of the A-V Impulse System. AB - We performed a prospective randomised controlled trial of a new mechanical method of prophylaxis for venous thrombo-embolism in 60 patients undergoing knee replacement surgery. The method uses the A-V Impulse System to produce cyclical compression of the venous reservoir of the foot. The overall incidence of deep vein thrombosis was 68.7% in patients receiving no prophylaxis and 50% in those using the device. The difference was not significant. There was, however, a reduction of the extent of thrombosis in the treated group. There were 13 major calf-vein thrombi and six proximal-vein thrombi in the control group compared with only five major calf-vein thrombi in the treated group. This difference was significant (p = 0.014). No patient developed clinical features of a pulmonary embolism. PMID- 1732266 TI - Walking ability after total hip replacement. A comparison of gait analysis in unilateral and bilateral cases. AB - We studied 50 patients before and after unilateral total hip replacement, and compared them, using gait analysis, with 22 having staged bilateral operations. The average age of the patients was 65 years at the first operation. The mean follow-up was 53 months for the unilateral cases and 27 months, after the second THR, for the bilateral cases. The average interval between first and second THR was 24 months. Patients with bilateral hip disease did not gain optimal function, even on the first side, until both hips had been replaced. Unilateral replacement gave better gait analysis results than did either side after bilateral procedures. PMID- 1732267 TI - Resorption of bone by inflammatory cells derived from the joint capsule of hip arthroplasties. AB - The role of inflammatory cells in aseptic loosening and failure of cemented joint replacements is unclear. Inflammatory cells from the revision joint capsule of four failed hip arthroplasties were examined to determine their nature and resorptive capacity. The capsules contained numerous macrophages and abundant foreign-body macrophage polykaryons, distinguished from osteoclasts by their antigenic phenotype and lack of response to calcitonin. When cultured on cortical bone slices in vitro, both macrophages and macrophage polykaryons produced small resorption pits and were associated with areas of superficial resorption of the bone surface. These results indicate that foreign-body induced macrophages and macrophage polykaryons are capable of a type of low-grade bone resorption which may be of pathogenic significance in the loosening of cemented joint prosthetic components. PMID- 1732268 TI - Thigh pain after cementless hip arthroplasty. Annoyance or ill omen. AB - A retrospective review of 148 consecutive porous-coated hip arthroplasties (PCA) showed an incidence of thigh pain of 13% one year after surgery, and 22% at two years. Positive correlations were made with femoral stem subsidence (greater than 2 mm) and with distal periosteal and endosteal bone formation. No positive correlations were made with parameters of bone quality or component fit. Resolution of pain occurred in one-third and an anti-inflammatory agent produced partial relief in two-thirds of the patients. We conclude that thigh pain is secondary to stem instability with distal stress transfer in the absence of stable proximal fixation. PMID- 1732269 TI - The histology of the radiolucent line. AB - The radiographic and histological features of radiolucent areas at the cement bone interface were correlated in 15 specimens retrieved at post-mortem from patients who had undergone cemented total hip arthroplasty, two weeks to 15 years prior to death. All but one of the components were securely fixed, as demonstrated by direct measurements of micromotion. Extensive radiolucencies were present in all but one case. In 11 of the 14 specimens with radiolucencies, histological examination showed that the radiolucent areas represented regions of osteoporosis and bone remodelling. The remodelling changes were characterised by osteoporosis, cancellisation and thinning of the endosteal cortex, and osteopenia of the trabecular bone. In two specimens the appearance of radiolucency was found to be due to fibrous tissue at the cement-bone interface and in one specimen there was a mixed picture of osteolysis and fibrosis. The study demonstrates that radiolucent lines can occur with well-fixed components and that they may commonly represent osteoporosis rather than the presence of a fibrous membrane at the cement-bone interface. PMID- 1732270 TI - Pelvic underpinning: eight years' experience. AB - A new method to reconstruct major acetabular floor defects is described. It relies on the placement of special nails into each of the three bones of the hemipelvis. Curved lugs attached to the nails are coalesced using bone cement forming a platform onto which a standard acetabular prosthesis is located. Forty seven cases are reported with a mean follow-up of 4.4 years (1 to 8). No loosening of an acetabular cup or migration of the device has occurred. PMID- 1732271 TI - One-stage reimplantation for infected total knee arthroplasty. AB - One-stage reimplantation for the salvage of infected total knee arthroplasty in 18 patients was reviewed at an average follow-up of five years. There had been one recurrence and one new infection, both in rheumatoid patients with another focus of infection. In four other patients the clinical result was impaired by pain after walking (2) and limited flexion (2). Our results suggest that one stage reimplantation is a reasonably reliable procedure for the management of a loose infected prosthesis. PMID- 1732272 TI - Results of the Harris-Galante cementless hip prosthesis. AB - We reviewed 82 primary arthroplasties (in 71 patients) in which cementless porous coated hip prostheses were used. The mean age of the patients at operation was 52 years (24 to 86); they were followed up for an average of 62.1 months (60 to 66). The diagnosis was avascular necrosis of the femoral head in 35%, fracture of the femoral neck in 24%, primary osteoarthritis in 16% and miscellaneous in 25%. The average preoperative Harris hip score was 56.7 points and the average postoperative score was 83.3 points. Eight hips (10%) had component loosening; four had been revised and four were awaiting revision. In 27 hips (33%) there was a radiolucent line wider than 2 mm in zones 1 and 7. In 55 hips (67%) there was calcar resorption of more than 10 mm. Twenty patients (28%) complained of thigh pain although they had no radiographic evidence of loosening of a component. Factors that may have contributed to the poor clinical and radiographic results were: 1) inadequate surface area for bone ingrowth, particularly on the lateral aspect of the upper part of the prosthesis, 2) poor initial fit of the stem in the metaphysis, which resulted in cantilever motion of the proximal part of the stem about the well-fixed distal stem, and 3) the collar of the prosthesis, which prevented it from subsiding to a naturally stable position and caused damage to the calcar. PMID- 1732273 TI - Carbon-fibre reinforced plates for problem fractures. AB - We report our experience with carbon-fibre reinforced plastic (CFRP) plates in the management of 19 problem fractures complicated by either infection, nonunion, comminution or contamination. The combination offers secure fixation without inhibition of callus formation. PMID- 1732274 TI - Polyethylene wear of metal-backed tibial components in total and unicompartmental knee prostheses. AB - We examined 86 polyethylene inserts, retrieved from total and unicompartmental knee prostheses after an average of 39.5 months in situ, grading them from 0 to 3 for seven modes of polyethylene degradation. Severe wear, with delamination or deformation, was observed in 51% of the implants, and was associated with time in situ, lack of congruency, thin polyethylene, third-body wear debris, and heat pressed polyethylene. Significant under-surface cold flow was identified in some areas of unsupported polyethylene, and was associated with delamination in the load-bearing areas of thin inserts above screw holes in the underlying metal tray. We recommend the use of thicker polyethylene inserts, particularly in young, active patients and in designs with screw holes in the tibial baseplate. Thin polyethylene inserts which are at risk for accelerated wear and premature failure should be monitored radiographically at annual intervals. PMID- 1732275 TI - The floating hip. Ipsilateral pelvic and femoral fractures. AB - A consecutive series is reported of 17 patients who underwent early surgical treatment for acetabular or unstable pelvic fractures associated with ipsilateral fractures of the femur. Treatment included external and internal fixation, and required careful consideration of the surgical approach and the positioning of the patient. The multiple injuries sustained by these patients required simultaneous procedures by several surgical teams. All the femoral fractures were internally fixed at the initial operation and eight patients had primary definitive treatment of all their other fractures as well. In nine patients the definitive treatment of their other fractures was delayed for an average of 11 days. There were no deaths, and no serious infections. The long-term morbidity resulted from the associated injuries and not from the pelvic or femoral fractures. PMID- 1732276 TI - Effect of anti-interferon-gamma and anti-interleukin-2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse. AB - SJL/J mice challenged with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) developed only mild chronic-relapsing experimental allergic encephalomyelitis (EAE) with very low incidence. However, treatment of challenged mice with anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb) determined severe disease in all cases. Similarly, in passive EAE, the addition of anti-IFN gamma to the in vitro MBP-activated cells at the time of transfer led to significant disease exacerbation in all recipients. The disease enhancing effect was observed only when the mAb was given at the time of active challenge or of passive transfer, but not at later times. Anti-interleukin-2 (IL-2) antibody had only a marginal effect in the active induction, but drastically reduced the manifestations of passive EAE, even when mixed with a disease-enhancing dose of anti-IFN-gamma. These findings support the notion that IL-2 is required for disease induction whereas IFN-gamma plays a disease-limiting role early in the development of EAE. PMID- 1732277 TI - Role of acetylcholine receptor antibody complexes in muscle in experimental autoimmune myasthenia gravis. AB - In experimental autoimmune myasthenia gravis anti-rat nicotinic acetylcholine receptor (AChR) antibody titers correlated significantly with the AChR-antibody complexes found in muscle. It was shown that at least a large part of the AChR antibody complexes are formed in vitro, which can be prevented by washing of the muscle homogenate. Using a modified assay, no differences in AChR-antibody complexes could be detected between rats with and without symptoms of experimental autoimmune myasthenia gravis. Also no difference in AChR loss nor in inhibition of alpha-bungarotoxin binding to AChR was found between these groups of rats. However, a significant difference in the reduction of AChR function was found, using an assay measuring agonist-induced 22Na+ flux into the TE671 cell line. PMID- 1732278 TI - Different patterns of glycolipid antibody reactivity: lower motor neuron syndromes vs. immunization. AB - High titers of serum antibodies against GM1 ganglioside occur frequently in patients with lower motor neuron (LMN) syndromes. We compared the specificities of the antiganglioside antibody reactivities in LMN patients with those arising after immunization of Lewis rats with several ganglioside containing preparations including purified GM1, human central nervous system (CNS) grey matter and white matter. Serums with high titers of anti-GM1 antibodies from patients with LMN syndrome usually showed limited cross-reactivity to other glycolipids but often bound to a Gal(beta 1-3)GalNAc-containing neoglycoprotein. In contrast, serums with anti-GM1 antibody arising after immunization showed broad cross-reactivity with other glycolipids but did not bind to the neoglycoprotein. We conclude that the serum patterns of antiganglioside antibody reactivity secondary to immunization with gangliosides and CNS components are different from the natural autoantibodies found in LMN patients. The antiganglioside antibodies seen in LMN patients are unlikely to be a result of autoreactivity to gangliosides after nervous tissue damage. PMID- 1732279 TI - Transforming growth factor-beta 1 (TGF-beta 1) expression and regulation in rat cortical astrocytes. AB - Transforming growth factor-beta 1 (TGF-beta 1) is a potent modulator of immune and glial cells' functions and thus, could play an important role in neuro-immune interaction. However, published reports disagree on whether or not TGF-beta 1 is expressed in normal brain. We demonstrate here the constitutive expression of TGF beta 1 mRNA but not protein in both cerebral cortex and primary rat cortical astrocytes. Steady-state TGF-beta 1 mRNA level increased 2-fold in adult compared to neonatal cortex and during proliferation and differentiation of astrocytes in primary culture. This response was not accompanied by the appearance of detectable TGF-beta protein either in vivo or in vitro. However, both intracellular immunoreactive TGF-beta and extracellular TGF-beta 1 activity were detected upon in vitro stimulation of astrocytes with interleukin-1 (IL-1). The extracellular TGF-beta 1 increased with time of exposure to and concentration of IL-1. In contrast, the amount of TGF-beta 1 mRNA remained unchanged during stimulation of astrocytes with IL-1. These results suggest that the production of TGF-beta 1 in astrocytes is regulated at both mRNA and protein levels. The former may occur during astrocytic development, and the latter during astrocytic response to injury in association with elevation of IL-1. PMID- 1732280 TI - Interleukin-1 beta induction of tumor necrosis factor-alpha gene expression in human astroglioma cells. AB - Cells that produce tumor necrosis factor-alpha (TNF-alpha) require the presence of signaling molecules since this cytokine is not normally expressed in a constitutive manner. It has been demonstrated that glial cells can produce TNF alpha; however, the specific inducing molecules and their mechanism(s) of action have not been clearly defined. In this study, we examined the effect of human recombinant interleukin-1 beta (IL-1 beta) on the expression of TNF-alpha by CH235-MG human malignant glioma cells. CH235-MG cells do not constitutively express TNF-alpha mRNA or protein; however, upon stimulation with IL-1 beta, these cells synthesize and secrete biologically active TNF-alpha. IL-1 beta induces the expression of a 1.9 kb TNF-alpha mRNA species. Kinetic analysis demonstrated optimum TNF-alpha mRNA expression after a 4 h exposure to IL-1 beta, and peak TNF-alpha protein production at 18 h. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased expression of TNF-alpha mRNA in IL-1 beta stimulated CH235-MG cells, indicating that de novo protein synthesis is not required for astroglioma TNF-alpha gene expression. Nuclear run-off analysis demonstrates that IL-1 beta causes transcriptional activation of the TNF-alpha gene, and CHX enhances IL-1 beta-induced TNF-alpha transcription. Studies of TNF alpha mRNA stability using actinomycin D show that IL-1 beta-induced TNF-alpha mRNA has a half-life of approximately 30 min, and CHX increases the half-life of IL-1 beta-induced TNF-alpha mRNA to approximately 210 min. These results indicate that IL-1 beta, a cytokine present in the central nervous system during some pathological disease states, is a potent inducer of TNF-alpha in human malignant glioma cells. PMID- 1732281 TI - The expression of major histocompatibility complex (MHC) class I antigens in the brain differs markedly in acute and persistent infections with lymphocytic choriomeningitis virus (LCMV). AB - Intracranial inoculation of immunocompetent mice with lymphocytic choriomeningitis virus (LCMV) induces a fatal neurologic illness. In this disease a marked increase in MHC class I expression was found, closely associated with viral antigens and inflammatory infiltrates, in meninges, choroid plexus and ventricular ependyma but not within the brain parenchyma. Immunosuppression prevented MHC induction. Mice inoculated at birth had persistent infections, with LCMV antigens found primarily in neurons, but no inflammatory cells or focal increase in MHC class I. Failure of infected neurons to express MHC class I allows them to escape destruction by cytotoxic T cells (CTL) but may increase their susceptibility to be persistently infected by non-lytic viruses. PMID- 1732282 TI - HLA type is not indicative for the effect of thymectomy in myasthenia gravis. AB - The frequency of HLA types in a selected group of 40 patients with myasthenia gravis in relation to the effect of thymectomy and also to gender, and thymus histology was studied. As generally described we found a significant increase in the frequency of HLA-A1, HLA-B8, HLA-DR3 and HLA-DQ2 in the total group. There were no further differences between subgroups of patients, which demonstrates that HLA type is not indicative for the effect of thymectomy in myasthenia gravis. PMID- 1732283 TI - A study of the kinetics of the interaction of spiperone with binding sites on human mononuclear cells: existence of a heterogeneous population of spiperone binding sites. AB - The kinetics and equilibrium of the interaction of [3H]spiperone with binding sites on human mononuclear cells were studied using a competitive displacement of spiperone by ketanserin, sulpiride, haloperidol, (+)- and (-)-butaclamol. [3H]Spiperone binding sites on human mononuclear cells were shown to be not of the D2, but most probably of the 5-HT2 type. The process of [3H]spiperone binding to these sites conforms to the model of ligand interaction with two independent binding sites (the high-affinity site, Kd1 = 3 nM and the low-affinity site, Kd2 = 20 nM), thus suggesting heterogeneity of spiperone sites on mononuclear cells. We found that in contrast to high-affinity binding sites, low-affinity sites show an essentially lower stability during storage of mononuclear cells. PMID- 1732284 TI - Cerebrospinal fluid anti-cerebellar soluble lectin antibodies in human immunodeficiency virus type 1 infection. AB - Cerebrospinal fluid samples from 14 human immunodeficiency type 1 (HIV-1) seropositive patients in various stages of HIV infection were tested for the presence of autoantibodies to an endogenous manose-binding protein, the cerebellar soluble lectin (CSL), which has recently been found to be detected in a high proportion of patients with multiple sclerosis. An immunoblotting test was used with rat CSL as antigen. Seven patients were positive for anti-CSL and seven were negative. The seven anti-CSL-positive patients had signs of intrathecal immunoglobulin G production measured as an elevated IgG index, while the seven anti-CSL-negative patients had a normal IgG index. There was no apparent relation between infectious stage and the presence of anti-CSL. Immunological reactions such as anti-CSL autoantibodies may be a similar pathogenic mechanism in HIV and multiple sclerosis brain disease. PMID- 1732285 TI - Aggressive fibromatosis. AB - Ten patients with aggressive fibromatosis of the extremities were prospectively followed for 2-6 years. Results of treatment methods were compared. Five patients underwent three-dimensional imaging with and without intravenous contrast, and the images were compared with the anatomic extent of the resected lesion. Pathologic specimens and control tissue were tested for the presence of estrogen and progesterone receptors and for expression of the c-sis oncogene and platelet derived growth factor (PDGF), potent mitogens for fibrocytes. Wider surgical resection resulted in a lower recurrence rate, but current chemotherapeutic agents were not effective in eradicating the tumors. Intravenous contrast enhanced the lesions. Two thirds of the tumors tested had estrogen or progesterone receptors. All tumors tested had inappropriate expression of c-sis and PDGF. This inappropriate expression may be responsible for the underlying pathobiology and deregulation of control of growth in aggressive fibromatosis. PMID- 1732286 TI - Lymphangioma of the upper extremity. AB - Lymphangioma of the upper extremity is rare; its treatment is unstandardized. We reviewed five female and one male patient with cavernous lymphangioma of the hand and forearm. Each of them underwent at least one surgical procedure. Five patients had satisfactory results with cosmesis and hand function. Satisfactory results are expected in those treated initially in early childhood, as most lymphangiomas tend to increase gradually in size and infiltrate previously uninvolved normal tissues. PMID- 1732287 TI - Proteus syndrome: musculoskeletal manifestations and management: a report of two cases. AB - The literature on clinical and roentgenographic findings of Proteus syndrome is reviewed. Two patients with Proteus syndrome who exhibited skeletal deformities and characteristic features such as limb length discrepancy, skin lesions, and soft tissue tumors are reported. Persistent difficulties in treating malformed spines and unequal legs, as well as unpredictable tissue reaction to trauma, are discussed. PMID- 1732288 TI - Difficult supracondylar elbow fractures in children: analysis of percutaneous pinning technique. AB - After treatment with two lateral percutaneous pins, 80 children with grade II-III supracondylar elbow fractures were reviewed. The result was good in 55 patients (68%) and was the same for extension- and flexion-type injuries. These patients had less than 10 degrees change in carrying angle and less than 20 degrees total change in range of motion. This method eliminates risk of iatrogenic injury to the ulnar nerve but is technically very demanding. Redisplacement, joint penetration, and damage to the capitellar side of growth plate can be avoided only by positioning the pins divergently. PMID- 1732289 TI - Bone mineral density in the femoral neck and lumbar spine. PMID- 1732290 TI - Fractures after Wagner limb lengthening. AB - A retrospective review of our experience with fractures after 27 Wagner limb lengthenings (23 patients) was undertaken. Ten fractures occurred after lengthening in eight patients (six spontaneous, four traumatic). Seven patients fractured despite a "staged" removal of the fixation plate. Seven of 10 fracture patients required open reduction/internal fixation (ORIF) to obtain union and/or satisfactory alignment. Our results indicate that patients with a congenital limb length inequality are prone to spontaneous fracture after Wagner limb lengthening. These results raise serious questions regarding the quality of bone healing after a Wagner-type lengthening for congenital limb length inequality. PMID- 1732291 TI - Peroneal nerve palsy after early cast application for femoral fractures in children. AB - The incidence and contributing factors associated with post-casting peroneal nerve palsy were examined in a series of 110 consecutive pediatric femoral shaft fractures treated with early hip spica cast application. Four patients with peroneal nerve palsy were identified. All four had 90 degrees/90 degrees casts placed and underwent cast wedging for alignment. All palsies resolved with immediate cast removal. Other treatment options for certain femur fractures with significant initial shortening should be considered. We advise pre- and post-cast neurologic examination and avoidance of forceful distraction. Fracture manipulation, through wedging, should be delayed. PMID- 1732292 TI - Compression (Salter-Harris Type V) physeal fracture: an experimental model in the rat. AB - Histologic evidence of damage to the proximal tibial physis of immature rats was documented in greater than 30% of hind limbs subjected to a valgus and compressive force. The lesion was consistent with that theorized for the Salter Harris Type V fracture. Two additional groups of immature rats were subjected to the same insult and followed to maturity. One of the groups sustained an intentional concomitant ipsilateral femoral shaft fracture. No tibia demonstrated a growth disturbance in the absence of the femoral fracture, while similar angular deformities were noted in four tibiae (13%) in the presence of a fractured femur. Previously theorized traumatic physeal injuries, implicated in subsequent growth disturbances and seen clinically in association with ipsilateral long bone fractures, were identified in this animal study. PMID- 1732293 TI - Efficiency of the bone scan for occult limping toddlers. AB - Fifty consecutive occult limping toddlers were prospectively evaluated by acute triphase 99mTc MDP scintigraphy (TTS) at Arkansas Children's Hospital from 1984 through 1989. Only patients with a limp that could not be diagnosed by an orthopaedist were included. TTS proved essential in localizing the lesion in 27 patients (54%). With only two false negatives and one false positive, this test was shown to be highly sensitive, specific, efficient, and predictive, especially as compared with temperature, white blood cell (WBC) count, erythrocyte sedimentation rate (ESR), and plain radiography. Because no infections were missed by TTS, patients with a normal TTS could be safely observed as outpatients, saving thousands of health care dollars in this small series. PMID- 1732294 TI - Hip involvement in juvenile rheumatoid arthritis. AB - We followed 386 children who met the criteria for juvenile rheumatoid arthritis (JRA) an average of 89 months. Hip involvement in JRA results in poor functional capacity. The prognosis for the pauciarticular group is good, but patients with onset at age greater than 6 years appear to do worse than those aged less than 6 years. In the polyarticular group, age of onset did not change the prognosis, whereas the systemic-onset group aged less than 6 years had a worse prognosis and more frequent radiographic changes than the older group. PMID- 1732295 TI - Closed reduction guided by dynamic ultrasound in late-diagnosed hip dislocation. AB - The potential for dynamic ultrasound in the primary treatment of late-diagnosed congenital hip dislocation was assessed in 21 patients. The study showed that dynamic ultrasound was a useful technique in guiding closed reduction and for assessing the effects of positioning on the stability of children less than 2 years of age. In older children, however, this assessment was less reliable. Although dynamic ultrasound evaluation can be done without sedation, general anesthesia provides optimal conditions for such evaluation. The benefits of ultrasound are that it does not involve exposure to radiation, that the need for elaborate radiographic methods like arthrography and computed tomography is reduced, and that closed reduction is more reliably achieved when guided by direct visualization. Because a thorough knowledge of the dynamics of hip dislocation is essential, the ultrasound evaluation should be performed by orthopedic surgeons. PMID- 1732296 TI - Ossific nucleus eccentricity in congenital dislocation of the hip. AB - We studied plain films, arthrograms, and clinical courses of patients with congenital dislocation of the hip to examine the relationship of ossific nucleus eccentricity (ONE) of the femoral head to the shape of the cartilaginous femoral head and the prognosis of these findings. No statistically significant correlation of ONE and cartilaginous femoral head shape was noted, nor was correlation noted for early femoral head shape and subsequent clinical course. Only the association of ONE in an older subgroup of patients with a complicated clinical course was statistically significant (p less than 0.05). PMID- 1732297 TI - Hip stabilization in severely involved cerebral palsy patients. AB - Thirty-two patients (48 hips) with total body involved cerebral palsy (CP) underwent medial release and proximal femoral osteotomy for hip subluxation or dislocation. Twenty-eight hips were rated good, 15 were rated fair, and five were rated poor at follow-up. The better located the hip preoperatively and the better the reduction obtained at operation, the better the final result. The major factor that correlated with a good result was early operation, performed before significant deformity had occurred. PMID- 1732298 TI - Slipped capital femoral epiphysis in black children. AB - We reviewed 74 black children with 97 slipped capital femoral epiphyses (SCFEs) with an average follow-up of 3.7 years. There were 24 bilateral, 22 right, and 27 left SCFEs. The average age at operation was 12 years 6 months. The average slip severity was 28 degrees with 61 mild, 21 moderate, and 14 severe slips. Group I slips (54) were treated with multiple pins, and group II slips (43) were treated with a single central screw. Satisfactory results were achieved in 74% of the group I and 91% of the group II slips, with only three cases of chondrolysis. We conclude that single central screw fixation of SCFE in black children can lead to excellent results with a low incidence of chondrolysis. PMID- 1732299 TI - Chondrolysis: detection by bone scintigraphy. AB - Premature closure of the physis of the greater trochanter has been reported to be a predictive sign of chondrolysis in hips with slipped capital femoral epiphysis (SCFE). In the present series, the physis of the greater trochanter showed decreased activity on bone scintigraphy in 16 patients with SCFE and concurrent or developing chondrolysis. In five of these patients, the scintigraphic pattern (decreased activity of the physis of the greater trochanter) preceded radiographic changes of chondrolysis. In 13 patients with SCFE without chondrolysis, the physis of the greater trochanter appeared normal on scintigrams and open on radiographs. Use of scintigraphy in patients with SCFE permitted earlier recognition of chondrolysis, increasing the potential of altering the course of the disease. PMID- 1732300 TI - Observer variability in the radiographic measurement and classification of metatarsus adductus. AB - The classification system of Berg was evaluated using four observers and the radiographs of 42 feet from patients with metatarsus adductus. Interobserver disagreement in diagnosis was 36%. Intraobserver inconsistency averaged 26%. The error range for the lateral and anteroposterior talocalcaneal angle measurement was 13.6 and 15.1 degrees intraobserver and 19.8 and 25.2 degrees interobserver, respectively. There was no correlation between classification and the length of time required for cast correction. The irregularity of hindfoot ossification centers makes measurements inconsistent and seriously reduces the usefulness of classification based on such measurements. PMID- 1732301 TI - Iselin's disease. AB - Iselin's disease (traction apophysitis of the tuberosity of the fifth metatarsal) has been reported rarely, but is probably more common than appreciated. It appears to be more common in athletically active older children and adolescents. Four patients with this condition were treated conservatively and all had resolution or improvement of symptoms. In one, however, nonunion developed and continues to cause intermittent pain at age 20 years. Early recognition and treatment may prevent long-term complications. PMID- 1732302 TI - Foot deformities and occult spinal abnormalities in children: a review of 16 cases. AB - A retrospective clinical and radiologic study was made of 16 children with foot deformities and associated occult spinal abnormalities in a 3-year period. Eleven children had bilateral foot deformities; the deformities were unilateral in five. Midline cutaneous lesions of the back were noted in 13 children; the most common dermal sign was a hairy patch. All children had radiologic features of spinal dysraphism on combined computed tomography (CT) scan and myelogram. Spinal dysraphism was not considered in the initial assessment of four children. Children with foot deformity should therefore have a careful assessment of the spine, including a neurologic evaluation. PMID- 1732303 TI - Cyclosporin and the gingival tissues. AB - Cyclosporin is a selective immunosuppressant that has a variety of applications in medical practice. Like phenytoin and the calcium channel blockers, the drug is associated with gingival overgrowth. This review considers the pharmacokinetics, pharmacodynamics, uses and unwanted effects of cyclosporin, in particular the action of the drug on the gingival tissues. Clinical and cell culture studies suggest that the mechanism of gingival overgrowth is a result of an interaction between the drug and its metabolites with susceptible gingival fibroblasts. Plaque-induced gingival inflammation appears to enhance this interaction. PMID- 1732304 TI - Nifedipine-induced gingival overgrowth. Report of a case treated by controlling plaque. AB - A review of the literature reveals little evidence that controlling plaque reduces and controls nifedipine-induced gingival overgrowth. The case presented suggests that significant reduction and control of nifedipine-induced gingival overgrowth can be achieved by thorough scaling and root planing along with meticulous plaque control. PMID- 1732305 TI - Observations and concepts of the oral health consequences of tobacco use of Finnish periodontists and dentists. AB - The observations of Finnish periodontists and other dentists, and their concepts of the oral health consequences of tobacco use as well as their counseling on tobacco use, were surveyed from November 1987 to January 1988. A questionnaire was mailed to all 61 Finnish periodontists and to 535 other dentists; 37 periodontists (61%) and 432 of the other dentists (80%) responded. The periodontists enquired about and advised on smoking significantly more frequently than did the other dentists; 71% of the periodontists often or always enquired about, and 62% advised their patients on smoking. 31% of all dentists had patients who were users of smokeless tobacco, and 62% of those dentists had often or always advised the users to quit. Nearly all dentists had seen some tobacco caused effects. Periodontists reported more frequently than the other dentists that they had observed more periodontitis, impaired healing of periodontitis and more changes in the oral mucosa in smokers compared with non-smokers. The majority of those who had seen users of smokeless tobacco had noticed changes in the oral mucosa and in the color of the gingiva. The majority of all dentists believed that heavy smoking may impair the host response in the periodontium, with periodontists believing in this more strongly than other dentists. PMID- 1732306 TI - Periodontal conditions in insulin-dependent diabetes mellitus. AB - In the present investigation, the frequency and severity of periodontal disease was assessed in a group of 71 patients with a mean duration of 16.5 years of insulin-dependent diabetes mellitus (IDD). The diabetics, aged 17-63 years, were under treatment at the diabetic outpatient clinic of the III Department of Medicine, University Central Hospital of Helsinki and at two clinics of the Helsinki Health Centre. Based upon their long-term medical records, 44 individuals were assessed to have a poorly controlled insulin-dependent diabetes mellitus (PIDD). At baseline of the present study, the PIDD group had a mean blood glucose level of 11.8 mmol/l and a mean glycosylated hemoglobin (HBA1) level of 10.7%. 27 subjects were classified as having a controlled insulin dependent diabetes mellitus (CIDD). For each individual, site-specific recordings were made for the plaque index, gingival index, pocket depth, loss of attachment, bleeding after probing, gingival recession and radiographic loss of alveolar bone. Under similar plaque conditions, adult subjects with a long-term PIDD were found to have lost more approximal attachment and bone than subjects with a CIDD (P = 0.046, P = 0.019). These differences were not equally obvious when the subjects were classified according to the history of medical complications, such as retinopathies, neuropathies and nephropathies. PMID- 1732307 TI - The effect of tensile strength on the clinical effectiveness and patient acceptance of dental floss. AB - This study compared the clinical effectiveness and subjective approval of 2 waxed dental flosses that differed significantly in tensile strength and wax content. At the initial appointment, subjects (20 1st-year dental students) were instructed to stop interproximal cleaning on 2 contralateral quadrants in order to allow plaque to accumulate on these surfaces for 1 week. 1 week later, subjects were instructed to begin flossing these 2 contralateral quadrants with 1 of the 2 types of floss for the next 1-week period, while withdrawing interproximal cleaning on the opposite 2 contralateral quadrants. After flossing these 2 quadrants for 1 week, the subjects began flossing the opposite 2 contralateral quadrants with the same floss. After 2 weeks of flossing contralateral quadrants, the 1st floss was withdrawn and replaced with the alternative floss for another similar 2-week trial period. At the end of each 2 week trial period, subjects completed subjective questionnaires concerning the floss they had used during the previous 2-week period. Pre- and post-flossing plaque indices were calculated for each week for both flosses, and compared statistically by a repeated measures analysis of variance. The results showed that both flosses significantly reduced interproximal plaque deposits, and had equal subjective approval. However, neither the greater-strength nor the lower wax content of the experimental floss was associated with an increase in clinical effectiveness or with a change in subjective approval. PMID- 1732308 TI - Diagnostic characteristics of crevicular fluid aspartate aminotransferase (AST) levels associated with periodontal disease activity. AB - During a 2-year period pocket depth, probing attachment level, gingival index, and crevicular fluid aspartate aminotransferase (AST) levels were monitored in 25 previously treated periodontitis patients. Probing attachment level change was used retrospectively to identify sites where active periodontal destruction had occurred. The ability of crevicular fluid AST activities at 600, 800, 1000, and 1200 microIU levels to recognize active disease was investigated. Probing attachment level changes observed support the concept that the pattern of periodontal disease activity is episodic and infrequent. A loss of greater than or equal to 2 mm was found at 11% of all studied sites, whereas a gain of greater than or equal to 2 mm was noticed for 15% of sites. 2 subjects had 3 teeth that lost greater than or equal to 2 mm of attachment, whereas 15 subjects demonstrated no teeth with disease activity. The remaining 8 subjects had 1 or 2 sites that lost greater than or equal to 2 mm of attachment. Bayes's theorem and ROC curves were used to exemplify the sensitivity and the specificity of AST assessments. The AST 800 microIU demonstrated a sensitivity of 0.93 and specificity 0.68 and an odds ratio of 15.4 for attachment loss greater than or equal to 2 mm. Under the conditional probability of either 50%, 25% or 10% active disease prevalence, AST 800 microIU has a predictability of 73%, 50% and 24% respectively. PMID- 1732309 TI - Location of the mucogingival junction 18 years after apically repositioned flap surgery. AB - The apically repositioned flap procedure, by definition, implies that the mucogingival junction (MGJ) is shifted into an apical location. That this actually would be the case has never been shown in long-term studies. The 13 subjects in the present study had during the years 1964-1965 received treatment of moderately advanced periodontal disease (probing pocket depths less than or equal to 5 mm) in the lower jaw. An apically repositioned flap (ARF) procedure was applied in the left or right half of the mandible and a gingivectomy (GE) was performed in the contralateral side. Starting in December 1981, the patients were recalled for clinical and radiographic determination of long-term results. The width of the band of keratinized gingiva was measured clinically and the distance from the MGJ to the lower border of the mandible (LBM) was measured from orthopantomograms. Slightly less keratinized gingiva was observed on the sides where GE had been used. There was no statistically significant difference in the orthopantomographic distance from the MGJ to the LBM between ARF and GE operations. The results indicate that the apically repositioned flap procedure does not result in a permanent apical shift of the MGJ. PMID- 1732310 TI - Interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid from adults with previous evidence of destructive periodontitis. A cross sectional study. AB - We have estimated the levels of Interleukin-1 beta (IL-1 beta) by ELISA in gingival crevicular fluid (GCF) at 58 sites from 37 patients with adult periodontitis. GCF was collected for 5 s on filter papers and a 2nd sample was collected for 30 s 1 min later. 68/116 strips yielded detectable levels of IL-1 beta. IL-1 beta was present in both the 1st and 2nd samples at 28 sites, in the 1st only at 4 sites and in the 2nd only at 8 sites; 18 sites were below the level of detection for the assay. When the concentrations of IL-1 beta were calculated in the original volume of GCF on each strip, the mean value for positive strips was 34.16 +/- 29.45 (SD) pg/microliters with a range from 1.75 to 97.13 pg/microliters. There were no statistically significant correlations with the plaque index, bleeding index or probable crevice depth (pocket depth). The results indicate that IL-1 is present in the GCF from a proportion of sites with evidence of previous periodontal destruction. PMID- 1732311 TI - Surgical lengthening of the clinical crown. AB - The aim of the present study was to assess the changes in the periodontal tissue levels as an immediate result of the surgical crown lengthening procedure and over a 6-months healing period. 25 patients ranging between 20 to 81 years of age were included in the study. A total of 85 teeth (43 test and 42 control teeth not exposed to surgery) were evaluated over 6 months. After initial therapy, the indication for crown lengthening comprised need for increased retention and accessibility to deep subgingival preparation margins hampering impression taking. During surgery, the alveolar crest was reduced, thereby creating a distance of 3 mm to the future reconstruction margin. The results of this study demonstrated that the mean probable changes in the levels of the periodontal tissues from those defined after surgery were minimal, resulting in changes comparable to the shifts observed at control teeth not exposed to any surgical procedures. Frequency analysis of the number of sites with dislocation of the free gingival margin demonstrated that 12% of the sites with crown lengthening procedure showed 2-4 mm recession of the free gingival margin between 6 weeks and 6 months postoperatively. In esthetically critical, visible areas of the dentition, recessions must be closely observed in the healing period after surgical crown lengthening, when prosthetic reconstructions are planned on such teeth. PMID- 1732312 TI - Wegener's granulomatosis. A distinct gingival lesion. AB - A hyperplastic (strawberry) gingivitis is a feature of Wegener's granulomatosis. A further case is described in which the only manifestations to date have been the gingival lesion. The diagnostic value of the ANCA test is discussed for patients who present with an unusual hyperplastic gingivitis. PMID- 1732313 TI - Biology of cutaneous squamous cell carcinoma. AB - Nonmelanoma skin cancer is the leading cause of cancer in the United States. Cutaneous squamous cell carcinoma is second only to basal cell carcinoma in prevalence and its incidence is increasing. The biology of squamous cell carcinoma is reviewed under the broad areas of etiology, immunobiology, biochemistry, metastatic potential, and therapy, with emphasis on prevention, diagnosis, and management. PMID- 1732314 TI - Malignant proliferative angioendotheliomatosis or angiotropic lymphoma associated with a soft-tissue lymphoma. AB - We report a 63-year-old man with violaceous nummular patches on the trunk. Histopathologic studies were consistent with a diagnosis of malignant angioendotheliomatosis or angiotropic lymphoma. Immunohistochemical study of skin was positive for UCHL-1 antigen and leukocyte common antigen and negative for L 26, Ulex europaeus lectin I, vimentin, cytokeratin, and epithelial membrane antigen. Ultrastructural study ruled out an endothelial origin of the neoplastic cells. These data confirmed the diagnosis of malignant proliferative angioendotheliomatosis. Five years before, a soft tissue lymphoma had been excised. This is an unusual case of malignant angioendotheliomatosis for the following two reasons: (1) a previous association with a soft tissue lymphoma and (2) the rarely described T immunophenotype of neoplastic lymphoid cells. PMID- 1732315 TI - Reversible plane xanthoma, vasculitis, and peliosis hepatis in giant lymph node hyperplasia (Castleman's disease): a case report and review of the cutaneous manifestations of giant lymph node hyperplasia. AB - A patient is described who had generalized plane xanthomas, cutaneous vasculitis, peliosis hepatis, and intraabdominal giant lymph node hyperplasia of the plasma cell type. After excision of the abdominal mass, the xanthomas resolved and the liver returned to its normal size, but the patient continued to develop skin lesions. A review is presented of the cutaneous manifestations of giant lymph node hyperplasia. PMID- 1732316 TI - Linear IgA bullous dermatosis associated with rheumatoid arthritis. AB - A case of linear IgA bullous dermatosis associated with rheumatoid arthritis is described. The eruption consisted of multiple irregular erythematous plaques and small numbers of tense vesicles mainly on the trunk. An immunofluorescence study showed linear IgA and IgG deposition along the basement membrane zone, whereas C3 deposition was not found. IgA or IgG anti-basement membrane zone antibody was not detected in the serum. Treatment with dapsone resulted in good control of the eruption. Coexistence of linear IgA bullous dermatosis and rheumatoid arthritis has not been reported previously. PMID- 1732317 TI - Guidelines of care for basal cell carcinoma. The American Academy of Dermatology Committee on Guidelines of Care. PMID- 1732318 TI - Erythema gyratum repens in a healthy woman. PMID- 1732319 TI - Polymorphous cutaneous cryptococcosis: nodular, herpes-like, and molluscum-like lesions in a patient with the acquired immunodeficiency syndrome. PMID- 1732320 TI - Lichen planus pemphigoides in a 10-year-old girl. PMID- 1732321 TI - Double-blind comparison of naftifine cream and clotrimazole/betamethasone dipropionate cream in the treatment of tinea pedis. PMID- 1732322 TI - Multiple eruptive benign keratoses associated with cyclosporine therapy for psoriasis. PMID- 1732323 TI - Pulsed high-dose corticosteroids and melphalan as an alternative therapy for refractory cutaneous T-cell lymphoma. PMID- 1732324 TI - Two patients with simultaneous, unusually located primary cutaneous nocardiosis. PMID- 1732325 TI - Follicular keratosis of the chin. PMID- 1732326 TI - Lithium-induced psoriasis of the fingernails. PMID- 1732327 TI - Therapeutic effect of anthralin. PMID- 1732328 TI - Antiphospholipid antibody syndrome. PMID- 1732329 TI - Sebaceous hyperplasia and basal cell carcinoma in a renal transplant patient receiving cyclosporine. PMID- 1732330 TI - Pityriasis rubra pilaris: the problem of its classification. PMID- 1732331 TI - Intralesional interferon therapy for basal cell carcinoma. PMID- 1732332 TI - Leiomyosarcoma of the buttock. PMID- 1732333 TI - Allergic contact dermatitis to latex glove antioxidants: an update. PMID- 1732334 TI - Erythema elevatum diutinum: a clinical and histopathologic study of 13 patients. AB - BACKGROUND: Erythema elevatum diutinum is a rare condition representing a chronic leukocytoclastic vasculitis. OBJECTIVE: Clinical and laboratory features of the disease were reviewed to better understand the disease. METHODS: The medical records and histopathologic slides of 13 patients with erythema elevatum diutinum were studied. RESULTS: The lesions were violaceous, deep red, or brown and typically were papules or plaques. Lesions were most often located on the extensor surfaces of the extremities. Associated medical problems included hematologic abnormalities in six patients: IgA clonal gammopathies (four), multiple myeloma (one), and myelodysplasia (one). Erythema elevatum diutinum preceded the myeloproliferative disorders by an average of 7.8 years. All patients showed vasculitis. Leukocytoclasia was present in 27 of 35 specimens. The predominant cell type in the inflammatory infiltrate was polymorphonuclear leukocytes or a mixture of polymorphonuclear leukocytes and lymphocytes. CONCLUSION: The most significant finding of this study is the association of erythema elevatum diutinum with hematologic disease, most frequently an IgA monoclonal gammopathy. PMID- 1732335 TI - Sclerodermatous chronic graft-versus-host disease. Analysis of seven cases. AB - BACKGROUND: Sclerodermatous chronic graft-versus-host disease is a disabling complication after allogeneic bone marrow transplantation from HLA-identical sibling donors. Only a few series of patients have been reported and the dermatologic features have never been extensively described. OBJECTIVE: The purpose of the study was to describe clinical and biologic features of chronic sclerodermatous graft-versus-host disease and to compare them with scleroderma. METHODS: We reviewed 196 patients grafted between April 1973 and July 1987 with survival times sufficient to be at risk of chronic graft-versus-host disease. Seven had the sclerodermatous form. RESULTS: Most patients had disseminated sclerosis of the trunk and the proximal portions of the limbs. In two cases, atrophy of the skin was predominant, corresponding with a severe clinical evolution. Periorbital pigmentation was observed as an initial manifestation in three cases. Visceral manifestations resembled those observed in scleroderma but histologic and immunologic studies demonstrated clear differences. Response to therapy was variable. CONCLUSION: Chronic sclerodermatous graft-versus-host disease may realize two different patterns. Major atrophy is associated with a more severe progression. PMID- 1732336 TI - Demonstration by S-100 protein staining of increased numbers of nerves in the papillary dermis of patients with prurigo nodularis. AB - BACKGROUND: We hypothesized that hyperplasia of papillary dermal nerves was a constant feature of prurigo nodularis. OBJECTIVE: We tested this hypothesis by examining sections from 25 cases of prurigo nodularis, 25 cases of skin lesions characterized by epidermal hyperplasia without clinical pruritus, and 22 cases of clinically pruritic dermatoses with variable degrees of epidermal response for the presence of papillary dermal nerves. METHODS: We used a standard immunohistochemical assay with an antibody to S-100 protein as a means of identification of nerves. RESULTS: In 24 of 25 cases of prurigo nodularis, papillary dermal nerves were identified by immunostaining. Cutaneous nerves were present in 1 of 22 cases of epidermal hyperplasia with pruritus and were absent in the papillary dermis in nonpruritic cases. CONCLUSION: We conclude that hypertrophy of cutaneous papillary dermal nerves is a relatively constant feature of prurigo nodularis. The presence of papillary dermal nerves suggests a neurocutaneous component in the pathogenesis of prurigo nodularis. PMID- 1732337 TI - Level of education and the risk of malignant melanoma. AB - BACKGROUND: The risk for the development of malignant melanoma has been reported to be higher in persons with more formal education than in individuals with less. OBJECTIVE: To study whether those with more formal education are indeed at more risk for malignant melanoma than those with less formal education. METHODS: This case-control study explores the relation between education and melanoma risk by analyzing data collected by the American Cancer Society. A total of 1.2 million people were surveyed for a history of cancer and followed up for 6 years for the development of any cancer. In total, 2780 white persons had a history of malignant melanoma or developed malignant melanoma during the study period. The controls were age-, sex-, and geographically matched white persons selected from the remaining people enrolled. RESULTS: Both men and women were shown to have a statistically significant increase in the relative risk for malignant melanoma with increasing education level (p less than 0.001 and p = 0.001, respectively). This relation was more striking in men when the relative risk with 95% confidence interval was calculated by sex for each education level. CONCLUSION: Americans with more formal education are at greater risk for malignant melanoma than those with less education. PMID- 1732338 TI - Fetal risks in herpes gestationis. AB - BACKGROUND: The potential risks to the fetus in herpes gestationis have long been a controversial subject, but because of the rarity of the disease, have only occasionally been studied. OBJECTIVE: The purpose of this study was to determine the incidence of fetal complications in herpes gestationis. METHODS: We collected and analyzed the obstetric histories of 74 patients and compared their involved pregnancies with their uninvolved pregnancies. RESULTS: There was an obvious tendency for premature delivery associated with herpes gestations. A slight tendency toward small-for-gestational-age newborns associated with herpes gestationis was confirmed, but, perhaps surprisingly, no increase in spontaneous abortions or stillbirths was noted. The use of systemic steroids did not appear to influence risk. CONCLUSION: Herpes gestationis is associated with an increase in prematurity and small-for-gestational-age infants. PMID- 1732339 TI - Severe sun sensitivity and the presence of antinuclear antibodies in patients with polymorphous light eruption-like lesions. A form fruste of photosensitive lupus erythematosus? AB - BACKGROUND: Initial lesions seen in some cases of lupus erythematosus may simulate the clinical features of polymorphous light eruption. OBJECTIVE: To evaluate the significance of antinuclear antibody screening in patients with polymorphous light eruption or to find better methods to define patients at risk to have lupus erythematosus, we analyzed the data of 198 patients with polymorphous light eruption. METHODS: History, type, and persistence of skin lesions were reviewed, and serologic variables, the minimal erythema dose, and the ultraviolet action spectrum for inducing skin lesions were determined. RESULTS: The morphologic features of skin lesions and results of phototesting were consistent with previously published characteristics of patients with polymorphous light eruption. Twenty-eight (14%) had high titers of antinuclear antibody (greater than or equal to 1:80). We found a highly significant correlation between severe sun sensitivity and the presence of high antinuclear antibody titers. Of these patients with severe sun sensitivity and high titers of antinuclear antibody, in three the diagnosis of systemic lupus erythematosus could be established according to American Rheumatism Association criteria. CONCLUSION: These results suggest that a combination of severe sun sensitivity and high titers of antinuclear antibody may characterize a subset of sun sensitive patients with some features of lupus erythematosus. PMID- 1732340 TI - Xeroderma pigmentosum. A clinical study of 24 Libyan cases. AB - BACKGROUND: Despite a high incidence of xeroderma pigmentosum, there is no previous publication from Libya. OBJECTIVE: The purpose was to study the clinical profile of Libyan cases of xeroderma pigmentosum. METHODS: With the help of a special protocol, 24 cases of xeroderma pigmentosum treated between 1981 and 1990 were subjected to detailed analysis. RESULTS: The age of onset of initial manifestations ranged between 6 and 18 months whereas that of malignant lesions ranged from 2 to 10 years. Malignant lesions observed were squamous cell carcinoma in 15 patients, basal cell carcinoma in 12, and basosquamous carcinoma in 2 patients; squamous cell carcinoma of the tongue, carcinoma of the thyroid, and lymphatic leukemia affected individual cases. None of our patients developed malignant melanoma. Six patients have died; the age at death ranged between 9 and 18 years. A history of consanguinity in the parents of patients was recorded in all but two patients. CONCLUSION: We observed early onset of severe ophthalmic lesions affecting a higher percentage of the patients. PMID- 1732341 TI - Clinicopathologic spectrum of specific cutaneous lesions of disseminated coccidioidomycosis. AB - BACKGROUND: Disseminated coccidioidomycosis merits greater attention because the number of persons living and traveling in endemic areas is increasing. OBJECTIVE: Our purpose was to study the clinical and histopathologic findings in patients with specific cutaneous disseminated coccidioidomycosis. METHODS: In six patients with specific skin lesions of disseminated coccidioidomycosis, the diagnosis was confirmed by identification of the organism in tissue or by positive results of tissue culturing. RESULTS: Clinical lesions included solitary granulomatous plaques in two patients and multiple papular, nodular, or pustular lesions in four patients, two of whom also had subcutaneous abscesses. Identifying organisms directly in tissue was possible in only 8 of 17 biopsy specimens and in five of six patients. The histopathologic features showed various degrees of three primary patterns: (1) abscess formation with necrosis, (2) epithelial hyperplasia and granuloma formation with microabscesses, and (3) vascular and perivascular proliferative and inflammatory cell reactions at times suggesting vasculitis. Tissue eosinophilia, present in all patients, was striking in two (eosinophilic abscess formation) and notable in another (vascular inflammation with eosinophilia). CONCLUSION: Cutaneous manifestations of disseminated coccidioidomycosis may be more common and varied than usually recognized. PMID- 1732342 TI - Cyclosporine in psoriasis: a multicenter dose-finding study in severe plaque psoriasis. The German Multicenter Study. AB - Interim results of a randomized, controlled, dose-finding study conducted in 24 dermatology centers on 217 patients with severe chronic plaque psoriasis (entry psoriasis area and severity index of at least 15) are presented. Patients were first treated with cyclosporine either 1.25 or 2.5 mg/kg/day (Sandimmune); in case of inadequate response the dosage was increased to a maximum of 5 mg/kg/day. Cyclosporine was given for 12 to 36 weeks. Treatment was classified as "successful" if the psoriasis area and severity index was reduced to 25% or less of the baseline value or below 8. At the end of the treatment period 18% of patients had improved their psoriasis area and severity index "successfully" with the initial dose of 1.25 mg/kg/day. "Successful" improvement with the initial dose of 2.5 mg/kg/day was achieved in 56% of the cases. In the 1.25 mg group, 44 patients had to increase their dose to 2.5 mg/kg/day and 31 patients to 5 mg/kg/day. In 29 patients whose dosages were started at 2.5 mg/kg/day the dosage was subsequently increased to 5 mg/kg/day. Although 1.25 mg/kg/day proved to be effective in some cases, 2.5 mg/kg/day of cyclosporine is the optimal starting dose. Adverse events were observed in 10% of the patients taking 1.25 mg/kg/day, in 20% taking 2.5 mg/kg/day, and in 32% taking 5 mg/kg/day. The most common were gastrointestinal complaints, followed by the common cold and other viral infections. An increase of serum creatinine level above 130 mumols/L occurred in five patients who could continue therapy after reducing the dose.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732343 TI - Topical capsaicin for treatment of hemodialysis-related pruritus. AB - Pruritus is a significant problem for many patients undergoing long-term hemodialysis. Topical capsaicin depletes and prevents the reaccumulation of substance P in peripheral sensory neurons. Substance P functions in the transmission of pain and probably itch sensations. An open-label, uncontrolled trial and a double-blind, vehicle-controlled trial were conducted to evaluate the efficacy and safety of capsaicin 0.025% cream in the treatment of localized areas of pruritus in patients undergoing long-term hemodialysis. Eight of nine evaluable patients in the open-label trial reported marked relief or complete resolution of itching during the study period, and two of five evaluable patients in the double-blind trial reported complete resolution of itching in the capsaicin-treated arm with no or minimal improvement in the vehicle-treated arm. Twelve patients in the open-label trial and two in the double-blind trial were unevaluable. No serious treatment-related adverse reactions occurred. PMID- 1732344 TI - A histopathologic comparison of Shulman's syndrome (diffuse fasciitis with eosinophilia) and the fasciitis associated with the eosinophilia-myalgia syndrome. AB - A comparison of the histopathologic features of Shulman's syndrome (diffuse fasciitis with eosinophilia) and the fasciitis associated with the eosinophilia myalgia syndrome is presented. The study population consisted of eight biopsy specimens of seven patients with Shulman's syndrome and 11 specimens from 10 patients with eosinophilia-myalgia syndrome. Both groups exhibited inflammatory changes in the subcutaneous fat, septa, and fascia; cutaneous changes were more prominent in cases of eosinophilia-myalgia syndrome. Eosinophils and plasma cells were not consistently present in either condition; mast cells and factor XIIIa positive cells were consistently present in the inflammatory infiltrates. Although there was overlap in the histopathologic findings, Shulman's syndrome tends to involve the subcutis alone and the eosinophilia-myalgia syndrome tends to be a pancutaneous-subcutaneous process. PMID- 1732345 TI - 21st annual meeting and exhibition, American Association for Dental Research. March 11-14, 1992, Boston, Massachusetts. Abstracts. PMID- 1732346 TI - 69th AADS annual session. American Association of Dental Schools. March 8-11, 1992, Boston, Massachusetts. Abstracts. PMID- 1732347 TI - Effects of intravenous urokinase versus alteplase on total pulmonary resistance in acute massive pulmonary embolism: a European multicenter double-blind trial. The European Cooperative Study Group for Pulmonary Embolism. AB - Twelve centers participated in a double-blind study in which 63 patients with angiographically documented acute massive pulmonary embolism were randomly assigned to treatment with either urokinase (4,400 U/kg as an intravenous bolus infusion, then 4,400 U/kg per h over 12 h; n = 29) or alteplase (10 mg as an intravenous bolus infusion, then 90 mg over 2 h) followed by heparin (n = 34). The primary objective was to compare the resolution of pulmonary embolism as judged by the change in total pulmonary resistance over the initial 2 h. Further objectives were to evaluate the changes in total pulmonary resistance over the next 10 h and the degree of angiographic resolution at 12 to 18 h. At 2 h, total pulmonary resistance decreased by 18 +/- 22% in the urokinase group and by 36 +/- 17% in the alteplase group (p = 0.0009). Continuous monitoring of pulmonary artery mean pressure, cardiac index and total pulmonary resistance revealed that these variables improved faster in the alteplase group, with consistently significant intergroup differences from 30 min up to 3 to 4 h. After 12 h, the decrease in total pulmonary resistance was 53 +/- 19% in the urokinase group compared with 48 +/- 17% in the alteplase group and the reduction in the angiographic severity score was 30 +/- 25% compared with 24 +/- 18%, respectively, with no significant intergroup differences. Bleeding was equally frequent in the two treatment groups, except that more urokinase-treated patients experienced hematomas at puncture sites. PMID- 1732348 TI - Pulmonary embolism thrombolysis: a clarion call for international collaboration. PMID- 1732349 TI - Comparison of adenosine and exercise thallium-201 single-photon emission computed tomography (SPECT) myocardial perfusion imaging. The GE SPECT Multicenter Adenosine Study Group. AB - Pharmacologic stress with dipyridamole has provided useful diagnostic, as well as prognostic, information in patients undergoing thallium-201 myocardial perfusion imaging. With its ultrashort half-life and a potent and consistent vasodilator effect, adenosine may be the coronary vasodilator of choice with myocardial perfusion imaging. Fifty-one healthy subjects and 93 patients with suspected coronary artery disease constituted the study group. In this multicenter study the comparative safety and diagnostic efficacy of single-photon emission computed tomography (SPECT) thallium imaging during adenosine-induced coronary hyperemia was compared with exercise treadmill stress. There was a mean increase in heart rate of 37% and a mean decrease in diastolic blood pressure of 5% during the adenosine infusion of 140 micrograms/kg per min for 6 min. Adenosine infusion was well tolerated in 95% of the subjects. Side effects requiring intervention occurred in seven subjects (5%). None of the subjects experienced a life threatening complication. The sensitivity, specificity and predictive accuracy for detection of coronary artery disease with use of quantitative analysis was 83%, 87% and 84% for adenosine SPECT and 82%, 80% and 81% for exercise SPECT studies, respectively. Most false negative results with adenosine, as well as exercise SPECT studies, occurred in patients with single-vessel disease. The first-order concordance (no defect vs. defect) and second-order concordance (no defect vs. irreversible vs. reversible defect) was 89% and 78% between the two studies, respectively. Thus, the results of adenosine SPECT imaging are highly concordant with exercise SPECT thallium imaging. Adenosine SPECT thallium imaging provides a safe and highly accurate imaging mode for the detection of coronary artery disease. PMID- 1732350 TI - Restenosis after coronary angioplasty: the paradox of increased lumen diameter and restenosis. AB - Restenosis after coronary angioplasty is the single complication that most limits this revascularization procedure in clinical practice. The process is largely unpredictable and the lesion-related factors predisposing to restenosis are poorly understood, with little consensus in published reports. In this study using detailed quantitative angiographic measurements to assess 490 lesions, the simple lesion characteristics associated with restenosis were defined and the relation to the restenosis process documented. Restenosis was defined as an absolute deterioration in the minimal lumen diameter by greater than or equal to 0.72 mm, a criterion based on the 95% confidence intervals for repeat angiographic measurements. This was chosen in an attempt to separate spurious changes due to a poor angiographic result and the variability of angiographic measurements from significant changes due to the restenosis process. The principal determinants of restenosis were found to be a large improvement in the minimal lumen diameter at the time of dilation (1.13 mm for the restenosis group compared with 0.86 mm for the no restenosis group [p less than 0.0001]) and an optimal postangioplasty result (minimal lumen diameter 2.28 mm in the restenosis group compared with 2.05 mm [p less than 0.001] in the no restenosis group, corresponding to a 25% and a 30% diameter stenosis, respectively [p less than 0.0001]). These observations reported for the first time suggest that the distinction needs to be made between a "clinical restenosis" of greater than or equal to 50% diameter stenosis and the "restenosis process" as measured by the absolute changes occurring during and after angioplasty.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732351 TI - Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. AB - Restenosis is a reparative response to arterial injury occurring with percutaneous coronary revascularization. However, the quantitative characteristics of the relation between vessel injury and the magnitude of restenotic response remain unknown. This study was thus performed to determine the relation between severity of vessel wall injury and the thickness of resulting neointimal proliferation in a porcine model of coronary restenosis. Twenty-six porcine coronary artery segments in 24 pigs were subjected to deep arterial injury with use of overexpanded, percutaneously delivered tantalum wire coils. The vessels were studied microscopically 4 weeks after coil implantation to measure the relation between the extent of injury and the resulting neointimal thickness. For each wire site, a histopathologic score proportional to injury depth and the neointimal thicknesses at that site were determined. Mean injury scores were compared with both mean neointimal thickness and planimetry-derived area percent lumen stenosis. The severity of vessel injury strongly correlated with neointimal thickness and percent diameter stenosis (p less than 0.001). Neointimal proliferation resulting from a given wire was related to injury severity in adjacent wires, suggesting an interaction among effects at injured sites. If the results in this model apply to human coronary arteries, restenosis may depend on the degree of vessel injury sustained during angioplasty. PMID- 1732352 TI - Arterial injury and the enigma of coronary restenosis. PMID- 1732353 TI - Association of aortic dilation with regurgitant, stenotic and functionally normal bicuspid aortic valves. AB - To determine whether aortic root dilation associated with a bicuspid aortic valve occurs independently of valvular hemodynamic abnormality, aortic root dimensions were measured by two-dimensional echocardiography in 83 adults with a functionally normal (n = 19), mildly regurgitant (n = 26), severely regurgitant (n = 27) or stenotic (n = 11) bicuspid aortic valve and compared with findings in normal subjects matched for age and gender. Aortic root measurements were made at four levels: anulus, sinuses of Valsalva, supraaortic ridge and proximal ascending aorta. Seventy-one percent of patients with a bicuspid aortic valve were men. When compared with control subjects, all hemodynamic subgroups showed a significantly larger aortic root size at three levels: sinuses of Valsalva, supraaortic ridge and proximal ascending aorta (p less than 0.05 to p less than 0.001). The prevalence of aortic root enlargement among all hemodynamic subgroups ranged from 9% to 59% at the level of the anulus, 36% to 78% at the sinuses, 47% to 79% at the supraaortic ridge and 50% to 64% in the ascending aorta. Thus, there is a high prevalence of aortic root enlargement in patients with a bicuspid aortic valve that occurs irrespective of altered hemodynamics or age. These findings support the hypothesis that bicuspid aortic valve and aortic root dilation may reflect a common developmental defect. PMID- 1732354 TI - Intracranial hemorrhage in association with thrombolytic therapy: incidence and clinical predictive factors. AB - In a period of 18 months, 2,469 patients with acute myocardial infarction treated with a thrombolytic agent were prospectively registered in 61 hospitals. Most patients (73%) were treated with streptokinase. Intracranial hemorrhage was observed in 24 patients, corresponding to an incidence rate of 1% (95% confidence interval 0.6% to 1.3%). The median time interval between the start of thrombolytic therapy and the first clinical signs of intracranial bleeding was 16 h (range 3 to 36). In total, 16 (66%) of the 24 patients died as a result of cerebral hematoma. To determine clinical predictive factors, a case-control study was conducted. For every patient with intracranial hemorrhage, two control patients who received thrombolytic therapy because of acute infarction in the same hospital and in the same period were selected. Detailed clinical characteristics of 22 of the 24 patients as well as of 7 other patients with documented intracerebral bleeding from the European Cooperative Study Group and of 2 patients who sustained intracranial hemorrhage outside the registry period were compared with 62 control patients. The results of multivariate logistic regression analysis indicate that patients taking an oral anticoagulant before admission, patients with a body weight less than 70 kg and those greater than 65 years old are at higher risk for intracranial hemorrhage during thrombolytic therapy. PMID- 1732355 TI - Intracranial hemorrhage after thrombolytic therapy: a therapeutic conflict. PMID- 1732356 TI - Quantification of left ventricular performance during transient coronary occlusion at various anatomic sites in humans: a study using tantalum-178 and a multiwire gamma camera. AB - To study the functional significance of transient coronary occlusion on systolic and diastolic left ventricular function relative to the anatomic site of occlusion, first-pass radionuclide angiography with a mobile multiwire gamma camera using tantalum-178 (dose activity less than or equal to 84 mCi/elution) was performed in 46 patients undergoing balloon coronary angioplasty. First-pass images were acquired immediately before angioplasty and during the last 30 s of a 60-s balloon inflation in 23 left anterior descending arteries, 18 right coronary arteries, 8 circumflex arteries and 3 diagonal coronary arteries. Occlusion of the left anterior descending artery resulted in significant decreases in left ventricular ejection fraction (54.6 +/- 12.7% to 32.3 +/- 10.6%, p = 0.0001) and peak filling rate (2.48 +/- 0.68 to 1.75 +/- 0.64 end-diastolic volumes/s, p = 0.0001), accompanied by severe abnormalities in regional function and left ventricular dilation. Right coronary artery occlusion caused inferior hypokinesia, but did not significantly change left ventricular ejection fraction (48.5 +/- 12.4% vs. 45.8 +/- 12.5%, p = NS) or peak filling rate (2.05 +/- 0.81 vs. 2.09 +/- 0.81 end-diastolic volumes/s, p = NS). Circumflex artery occlusion resulted in mild wall motion deterioration and a borderline decrease in ejection fraction (54.7 +/- 11.4% to 50.5 +/- 12%, p = 0.057). Diagonal artery occlusion did not cause significant changes in left ventricular ejection fraction or filling rate. The decrease in left ventricular ejection fraction during coronary occlusion was 9 +/- 25% and 27 +/- 22%, respectively, in those arteries with and without collateral supply (p = 0.052). These data provide strong evidence for the critical importance of the left anterior descending artery and the secondary role of the other coronary arteries in maintaining global systolic and diastolic left ventricular function and suggest a protective role of collateral vessels during coronary occlusion. PMID- 1732357 TI - Tomographic myocardial perfusion imaging with technetium-99m teboroxime during adenosine-induced coronary hyperemia: correlation with thallium-201 imaging. AB - Single-photon emission computed tomographic imaging with technetium-99m teboroxime during exercise has been found to be feasible and the results correlate with those obtained with thallium-201. This study examined the feasibility of this technique and compared tomographic imaging with technetium 99m teboroxime during adenosine-induced coronary hyperemia with thallium-201 imaging. With the patient positioned on the imaging table, adenosine was infused at a rate of 140 micrograms/kg per min for 6 min. At 4 min, 20 to 25 mCi (740 to 925 MBq) of technetium-99m teboroxime was injected intravenously and imaging was started as soon as the infusion was completed with use of a 180 degrees anterior arc and 32 stops at 10 s/stop (total imaging time 7.8 min). Rest images were obtained 60 to 90 min later with use of a similar dose of technetium-99m teboroxime. Exercise tomographic thallium images were obtained within 2 weeks of the teboroxime studies. In the 20 patients studied, the teboroxime images were normal in 2 (50%) of 4 normal subjects and abnormal in 15 (94%) of 16 patients with coronary artery disease; 4 of the 15 had a fixed defect and 11 a reversible defect. There was agreement between teboroxime and thallium studies in 16 patients (80%), in 319 (80%) of 400 segments and in 50 (83%) of 60 vascular segments (p less than 0.05). In two normal subjects, an apparent fixed defect involving the inferior wall was seen on the teboroxime but not the thallium images and was thought to be due to an attenuation artifact secondary to extracardiac activity in the left lobe of the liver.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732358 TI - On-line assessment of ventricular function by automatic boundary detection and ultrasonic backscatter imaging. AB - To provide an approach suitable for on-line analysis of ventricular function, a conventional two-dimensional ultrasound imaging system was modified to detect and track blood-tissue interfaces in real time based on their quantitative acoustic properties. This modification permitted on-line display of the left ventricular cavity area, fractional area change, volumes and ejection fraction on a beat by beat basis. Images were obtained from 54 patients and 12 normal subjects with broad ranges of ventricular dimensions and systolic function. On-line measurements of cavity areas were compared with off-line measurements of cavity areas (analysis of videotaped conventional images). Left ventricular cavity areas measured on-line from short-axis views correlated closely with off-line views as did areas from apical views. On-line fractional area change correlated well with ejection fraction calculated off-line. More than 70% of patients could be studied adequately with the approach developed. Thus, automatic boundary detection based on quantitative assessment of tissue acoustic properties permits on-line quantitation of ventricular cavity areas and indexes of function. PMID- 1732359 TI - Real time ultrasound quantification of ventricular function: has the eyeball been replaced or will the subjective become objective. PMID- 1732360 TI - Value of acceleration flow signals proximal to the leaking orifice in assessing the severity of prosthetic mitral valve regurgitation. AB - To test the value of acceleration flow signals proximal to the leaking orifice in assessing the severity of prosthetic mitral valve regurgitation, 39 consecutive patients undergoing left ventriculography were examined by Doppler color flow imaging. Acceleration flow signals proximal to the regurgitant orifice were detected in 27 of the 31 patients who had prosthetic mitral regurgitation by left ventriculography (sensitivity 87%). All four patients without acceleration flow signals had mild prosthetic mitral regurgitation by angiography. No acceleration flow signals were detected in any patient without prosthetic regurgitation by left ventriculography (specificity 100%). Individual values of the maximal area of acceleration flow signals obtained from three orthogonal planes in seven patients with mild prosthetic mitral regurgitation by angiography ranged from 0 to 17 mm2 (mean 4 +/- 6). In 8 patients with moderate prosthetic mitral regurgitation by angiography, the maximal area of acceleration flow signals ranged from 21 to 58 mm2 (mean 33 +/- 15), whereas the maximal area of acceleration flow signals in 16 patients with severe prosthetic regurgitation ranged from 20 to 173 mm2 (mean 102 +/- 41). The maximal area of the acceleration flow signals from three planes correlated well with the angiographic grade of prosthetic mitral regurgitation. There was a significant difference in the maximal area of acceleration flow signals between mild and moderate (p less than 0.001), moderate and severe (p less than 0.001) and mild and severe (p less than 0.001) prosthetic mitral regurgitation. Thus, measurement of acceleration flow signals by Doppler color flow imaging is useful in assessing the severity of prosthetic mitral regurgitation. PMID- 1732361 TI - What exactly is 2+ to 3+ mitral regurgitation? PMID- 1732362 TI - Performance of the automated complete Selvester QRS scoring system in normal subjects and patients with single and multiple myocardial infarctions. AB - The automated version of the complete Selvester QRS scoring system for estimation of myocardial infarct size was evaluated in 1,344 normal subjects, 706 patients with a single myocardial infarction (366 with inferior infarction, 277 with anterior infarction and 63 with posterolateral infarction) and 131 patients with combined inferior and anterior infarction. The presence and location were determined by angiographic and ventriculographic criteria. The performance of the overall 32-point system, each of the 19 criteria and the 13 criteria sets and each of the 35 criteria within the 13 sets was examined. The mean point scores were 1.7 for normal subjects, 3.7 for posterolateral infarction, 4.1 for inferior infarction, 6.3 for anterior infarction and 6.9 for multiple infarcts. A score greater than 4 yielded a sensitivity of 67% for anterior infarction, 41% for inferior infarction, 32% for posterolateral infarction and 72% for multiple infarcts. However, 7 of 32 criteria failed to achieve 95% specificity and 10 of 35 criteria in criteria sets had a sensitivity that was even lower than their false positive rate. The automated Selvester QRS scoring system currently has limitations that are attributable to development of the original system, which used manual scoring techniques and established criteria limits from middle-aged men. Future automated analysis should use gender- and age-dependent criteria limits. PMID- 1732363 TI - Reversal of antiarrhythmic drug effects by epinephrine: quinidine versus amiodarone. AB - Although previous studies have demonstrated that the electrophysiologic effects of many antiarrhythmic agents can be reversed by catecholamines, the susceptibility of amiodarone to such reversal is unknown. The objective of this study was to compare the relative degree of reversal of the electrophysiologic effects of quinidine and amiodarone by epinephrine infusions that result in plasma epinephrine levels similar to those achieved during various physiologic stresses. Twenty-nine patients who had inducible sustained monomorphic ventricular tachycardia and underwent electropharmacologic testing with quinidine and amiodarone were enrolled in the study. The variables measured before and during an epinephrine infusion (25 or 50 ng/kg per min) included the sinus cycle length, mean arterial pressure, QT interval and effective refractory period at drive train cycle lengths of 600 and 400 ms. The effective refractory period measured at a drive train cycle length of 600 ms shortened less during amiodarone therapy (2 +/- 2%) than during quinidine therapy (6 +/- 4%) or than in the baseline state (6 +/- 4%; p less than 0.01). Similar results were obtained during evaluation of the effective refractory period at a cycle length of 400 ms. Epinephrine infusion, at both 25 and 50 ng/kg per min, completely reversed the effects of quinidine and partially reversed the effects of amiodarone on the effective refractory period. The effects of epinephrine on the sinus cycle length and QT interval were similar in the baseline state and in conjunction with quinidine and amiodarone. Twenty-four patients underwent programmed ventricular stimulation during amiodarone therapy alone and in conjunction with either a 25- or a 50-ng/kg per min infusion of epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732364 TI - Epinephrine infusions during antiarrhythmic drug evaluations: toward a better understanding of drug activity. PMID- 1732365 TI - Sinus automaticity and sinoatrial conduction in severe symptomatic sick sinus syndrome. AB - Electrophysiologic studies with recordings of sinus node electrograms were performed in 38 patients with severe symptomatic sick sinus syndrome. Thirty-two of the 38 patients had episodic tachyarrhythmias and 17 presented with syncope. The clinically documented sinus or atrial pause was 5.6 +/- 2.8 s (mean +/- SD). Patients were divided into three groups according to electrophysiologic findings. Group I consisted of nine patients with complete sinoatrial block. Sinus node electrograms were recorded during the episodes of long pauses. Seven patients had unidirectional exit block, with the atrial impulse being capable of retrograde penetration to the sinus node causing suppression of sinus automaticity; two had bidirectional sinoatrial block. Group II consisted of 22 patients with either 1:1 sinoatrial conduction (group IIa = 13 patients) or second degree sinoatrial exit block (group IIb = 9 patients) during spontaneous sinus rhythm. Sinoatrial exit block, ranging from 1 to greater than 14 sinus beats, was observed during postpacing pauses that ranged from 1,650 to 37,000 ms (mean 7,286 +/- 6,989). The maximal sinus node recovery time ranged from 770 to 5,580 ms (mean 3,004 +/- 1,686) and was normal in 5 patients and prolonged in 17. Group III consisted of seven patients with no recordable sinus node electrogram, reflecting either a technical failure or a quiescence of sinus activity. The sinus node recovery time in these seven patients ranged from 1,190 to 4,260 ms (mean 2,949 +/- 1,121). Thus, abnormalities in both sinus node automaticity and sinoatrial conduction are responsible for the long sinus or atrial pauses in the sick sinus syndrome. However, complete sinoatrial exit block can occur and cause severe bradycardia with escape rhythm; repetitive sinoatrial exit block plays a major role in producing posttachycardia pauses. PMID- 1732366 TI - Severe mitral or aortic valve regurgitation, or both, requiring valve replacement for infective endocarditis complicating hypertrophic cardiomyopathy. AB - Certain clinical and morphologic findings are described in 11 patients with hypertrophic cardiomyopathy complicated by infective endocarditis that produced severe mitral or aortic valve regurgitation, or both, necessitating valve replacement. All 11 patients had changes in the operatively excised valve or valves characteristic of healed infective endocarditis. The infection involved only the mitral valve in seven patients, only the aortic valve in three patients and both valves in one patient. Study of the operatively excised mitral valves indicated that the healed vegetations were located most commonly on the left ventricular aspects of the anterior mitral leaflet, indicating that vegetation had formed at contact points of this leaflet with mural endocardium of the left ventricular outflow tract. In all 11 patients, the infective endocarditis either worsened preexisting valve regurgitation or initiated valve regurgitation and led to worsened signs and symptoms of cardiac dysfunction, necessitating valve replacement. Functional class improved in the nine patients who survived 7 to 101 months after valve replacement. Hypertrophic cardiomyopathy appears to be a factor predisposing to infective endocarditis. Patients with hypertrophic cardiomyopathy should receive prophylactic antibiotic therapy during procedures that predispose to infective endocarditis. PMID- 1732367 TI - Vascular pathology of balloon-expandable flexible coil stents in humans. AB - The morphologic changes in atherosclerotic coronary arteries and saphenous vein bypass grafts after placement of a balloon-expandable flexible coil stent (Cook) are described. In each case, the vessels were patent despite morphologic evidence of injury and dissection in the vessel wall. The stented region was reendothelialized and the tissue overlying the stent wires consisted primarily of smooth muscle cells. There was minimal inflammatory reaction to the stent wires. These findings suggest that the balloon-expandable flexible coil stent can effectively maintain vessel patency even in the setting of postangioplasty lumen disruption. In addition, the vessels tolerate the metal prosthesis with little evidence of tissue inflammatory reaction. PMID- 1732368 TI - Lung perfusion scans in patients with congenital heart defects. AB - In 63 patients with various congenital heart defects, lung perfusion was evaluated with technetium-99mm macroaggregated albumin. Right lung perfusion abnormalities were documented in 34 patients (54%). A particularly high incidence occurred in patients who had undergone a systemic to pulmonary artery shunt operation as an initial palliative procedure or who had had right ventricular outflow reconstruction and in those with bilateral pulmonary artery stenosis. Serial studies were helpful in evaluating the functional results of different transcatheter interventions for optimizing pulmonary blood flow. The quantitative relative perfusion radionuclide method was a more sensitive means of detecting cases of abnormal lung perfusion than was chest radiology. PMID- 1732369 TI - Dobutamine stress echocardiography: a sensitive indicator of diminished myocardial function in asymptomatic doxorubicin-treated long-term survivors of childhood cancer. AB - Doxorubicin is an effective anticancer chemotherapeutic agent known to cause acute and chronic cardiomyopathy. To develop a more sensitive echocardiographic screening test for cardiac damage due to doxorubicin, a cohort study was performed using dobutamine infusion to differentiate asymptomatic long-term survivors of childhood cancer treated with doxorubicin from healthy control subjects. Echocardiographic data from the experimental group of 21 patients (mean age 16 +/- 5 years) treated from 1.6 to 14.3 years (median 5.3) before this study with 27 to 532 mg/m2 of doxorubicin (mean 196) were compared with echocardiographic data from 12 normal age-matched control subjects. Graded dobutamine infusions of 0.5, 2.5, 5 and 10 micrograms/kg per min were administered. Echocardiographic Doppler studies were performed before infusion and after 15 min of infusion at each rate. Dobutamine infusion at 10 micrograms/kg per min was discontinued after six studies secondary to a 50% incidence rate of adverse symptoms. The most important findings were that compared with values in control subjects, end-systolic left ventricular posterior wall dimension and percent of left ventricular posterior wall thickening in doxorubicin-treated patients were decreased at baseline study and these findings were more clearly delineated with dobutamine stimulation. End-systolic left ventricular posterior wall dimension at baseline for the doxorubicin-treated group was 11 +/- 1.9 mm versus 13.1 +/- 1.5 mm for control subjects (p less than 0.01). End-systolic left ventricular posterior wall dimension at the 5 micrograms/kg per min dobutamine infusion for the doxorubicin-treated group was 14.1 +/- 2.4 mm versus 19.3 +/- 2.6 mm for control subjects (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732370 TI - Response of angina and ischemia to long-term treatment in patients with chronic stable angina: a double-blind randomized individualized dosing trial of nifedipine, propranolol and their combination. AB - Seventy-four patients with chronic stable mild angina, mild coronary artery disease (83% had one- or two-vessel disease) and normal left ventricular function were studied to measure the response of treadmill exercise performance and painful and silent ischemia in the ambulatory setting to randomly assigned treatment with nifedipine or propranolol and their combination; titration to maximal tolerated dosages was performed in double-blind manner. At 3 months both nifedipine and propranolol reduced the weekly angina rate (p less than 0.05); during treadmill exercise testing, increases (p less than 0.05) were noted in time to angina and total exercise time and decreases in maximal ST depression at the end of exercise. There were no differences between the responses to nifedipine and propranolol and no significant additional changes were seen after another 3 months of therapy. The combination of nifedipine and propranolol reduced the number of patients with angina on exercise treadmill testing from 64% to 38% (p less than 0.05). During ambulatory electrocardiographic monitoring before treatment, there were 1.4 +/- 2.4 (mean +/- SD) episodes/24 h of painful ischemia and a very low silent ischemia frequency: mean 1.1 +/- 2.7 episodes/24 h, mean duration 16 +/- 25 min/24 h. Treatment with propranolol and nifedipine resulted in reduction of episodes and duration of painful and painless ischemia; approximately 77% of patients were free of all ischemic episodes. It is concluded that patients with chronic stable mild angina have a low incidence of silent ischemia. Nifedipine or propranolol alone, titrated to individualized maximally tolerated dosages, are equally effective in long-term control of painful and painless ischemia, anginal episodes and exercise-induced ischemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732371 TI - Experimental models of coronary artery restenosis. AB - The study of potentially effective drug therapies and mechanical devices for the prevention of restenosis after percutaneous coronary revascularization has relied heavily on the use of experimental animal models. To date, greater than 50 experimental studies have been reported and have suggested that at least nine different classes of pharmacologic agents inhibit the intimal proliferative response to arterial injury. However, no pharmacologic intervention has yet been shown to reproducibly reduce the incidence of restenosis after coronary balloon angioplasty in humans. To identify the reasons for the apparent nonspecificity of the animal models and to determine which model should most reliably predict the efficacy of individual therapies in humans, the distinguishing characteristics of the experimental models were compared. Particular attention was paid to the size and morphologic structure of the treated artery, the susceptibility of the species to spontaneous and diet-induced arterial disease, the nature of the stimulus to intimal proliferation and several practical and logistic considerations. Finally, the reported efficacies of specific drug therapies in the respective animal models and in humans were compared. This review suggests that significant interspecies and occasionally intraspecies differences do exist among the respective animal models, particularly in the extent and composition of the neointimal thickening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732372 TI - Evaluation of thrombolytic and systemic effects of the novel recombinant plasminogen activator BM 06.022 compared with alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis. AB - The thrombolytic and systemic effects of BM 06.022 were evaluated and compared with those of alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis. BM 06.022 consists of the kringle-2 and protease domains of human tissue plasminogen activator (t-PA) and is unglycosylated because of its expression in Escherichia coli cells. Thrombus formation in anesthetized open chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery at a high level site of obstruction. In heparinized dogs, none of six vehicle-treated animals exhibited reperfusion. Reperfusion was achieved in four of six dogs at 18.3 +/- 6 min after intravenous bolus injection of 140 kU/kg (0.24 mg/kg) of BM 06.022, whereas four of six dogs exhibited reperfusion later (p less than 0.05) at 76.5 +/- 16.1 min during infusion of 1.33 mg/kg of alteplase (0.13 mg/kg as initial bolus injection, followed by 0.66 mg/kg over 1 h and 0.53 mg/kg over 2 h). Significantly later (p less than 0.05) reperfusion than that achieved with BM 06.022 was achieved in five of six dogs at 57.8 +/- 12.1 min after intravenous injection of 0.4 U/kg of anistreplase. Streptokinase (21,000 IU/kg over 60 min) and urokinase (20,000 IU/kg as an intravenous bolus injection, followed by 20,000 IU/kg over 89 min) each induced reperfusion in three of six dogs but at 67 +/- 12 and 84.3 +/- 17.1 min (p less than 0.05 vs. BM 06.022), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732373 TI - Electrophysiologic substrate associated with pacing-induced heart failure in dogs: potential value of programmed stimulation in predicting sudden death. AB - To investigate the possible mechanisms of sudden death and the potential role of electrophysiologic testing in congestive heart failure, this study evaluated the electrophysiologic substrate in a model of heart failure induced by rapid pacing. Seventeen mongrel dogs underwent cardiac pacing at 220 to 240 beats/min for 5 weeks (paced group) and 11 other dogs served as a sham-operated control group. Rapid pacing of the right ventricle produced clinical and hemodynamic features of congestive heart failure. Dogs in the paced group had prolonged cardiac conduction time as reflected by longer epicardial activation time (36.1 +/- 2.4 vs. 30.8 +/- 0.8 ms, p less than 0.05). The ventricular effective refractory period was significantly prolonged after the development of heart failure (141 +/ 4 vs. 177 +/- 5 ms, p less than 0.01, at a basic pacing cycle length of 300 ms), whereas no significant change was found in the control group (140 +/- 4 vs. 145 +/- 4 ms, p = NS). The prolongation of the ventricular effective refractory period correlated with an increase in left ventricular end-diastolic pressure (r = 0.55, p less than 0.001) and the ventricular effective refractory period correlated inversely with cardiac index (r = -0.49, p less than 0.025). The rest membrane potential of ventricular muscle was less negative in the paced group compared with the control group (-80.7 +/- 2.2 vs. -85.6 +/- 2.2 mV, p less than 0.05). Intracellularly recorded action potential duration of ventricular muscle was longer in the paced than in the control group (236 +/- 9.8 vs. 198.9 +/- 2.6 ms, p less than 0.01), action potential duration at 90% repolarization).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732374 TI - Mechanical correlates of the third heart sound. AB - In seven chronically instrumented conscious dogs, micromanometers measured left ventricular pressure, and ultrasonic dimension transducers measured left ventricular minor-axis diameter; the latter recording was filtered to examine data between 20 and 100 Hz. Acceptable external heart sounds were recorded with a phonocardiographic microphone in four of the seven dogs. With each dog sedated, intubated and mechanically ventilated, data were obtained during hemodynamic alterations produced by volume loading, phenylephrine, calcium infusion and vena caval occlusion. Damped oscillations were noted consistently in the left ventricular diameter waveform toward the end of rapid ventricular filling. These wall vibrations, assessed by the filtered diameter, correlated well with the third heart sound (S3) on the phonocardiogram. The peak frequency of the wall vibrations increased with increased diastolic pressure (p = 0.004), probably reflecting an increase in myocardial wall stiffness. In contrast, the amplitude of the vibrations varied directly with left ventricular filling rate (p = 0.0001). Thus, S3 seemed to be related specifically to ventricular wall vibrations during rapid filling, and the spectra of the amplitude-frequency relation shifted toward the audible range with increases in diastolic pressure, wall stiffness or filling rate. Spectral analysis of S3 may be useful in assessing pathologic changes in myocardial wall properties. PMID- 1732375 TI - Evaluation of century-old physical signs, S3 and S4, by modern technology. PMID- 1732376 TI - Hemodynamic, left ventricular structural and hormonal changes after discrete myocardial damage in the dog. AB - Transmyocardial direct-current (DC) shock produces localized left ventricular myocardial necrosis without obstruction to coronary blood flow. In 43 dogs sequential measurements of hemodynamic, neuroendocrine and myocardial structural changes were made at baseline and for 16 weeks after DC shock. Six dogs (14%) died in the peri-shock period. By 1 week after shock, left ventricular mass, as measured by nuclear magnetic resonance imaging, had increased from a mean value +/- SD of 67.9 +/- 10.1 to 82.5 +/- 12.9 g (p = 0.0001). Left ventricular end diastolic volume was unchanged at 1 week but increased at 16 weeks from 56.1 +/- 10.3 to 70.3 +/- 10.7 ml (p = 0.0003). Left ventricular mass demonstrated a further increase at 12 months (107.8 +/- 14.8 g). Rest cardiac output was significantly decreased at 4 months (3.67 +/- 1.23 to 3.18 +/- 0.81 liters/min, p less than 0.01) as was stroke volume (43 +/- 9 to 37 +/- 7 ml, p less than or equal to 0.01). Left ventricular ejection fraction decreased progressively from 73% to 38% at 1 year. At 4 months there were increases in mean pulmonary artery pressure (18 +/- 4 to 23 +/- 4 mm Hg, p less than 0.01), pulmonary capillary wedge pressure (9 +/- 3 to 15 +/- 3 mm Hg, p less than 0.01) and right atrial pressure (5 +/- 4 to 9 +/- 3 mm Hg, p less than 0.01). Plasma norepinephrine was increased at 4 months (318 +/- 190 to 523 +/- 221 pg/ml, p = 0.0003), whereas plasma renin activity was not significantly changed (4.3 +/- 2.6 vs. 5.2 +/- 3.4 ng/ml per h). Microsphere regional blood flow studies demonstrated a 50% reduction in skeletal muscle blood flow at 4 months (0.06 +/- 0.06 ml/min per g compared with 0.12 +/- 0.09 in normal dogs, p = 0.05), and a reduction in the endocardial/epicardial blood flow ratio (1.11 +/- 0.13 compared with 1.24 +/- 0.13 in normal dogs, p = 0.02). Therefore, in this model of acute left ventricular damage, left ventricular hypertrophy precedes progressive left ventricular dilation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732377 TI - Does it really make a difference? PMID- 1732378 TI - Factors influencing the pursuit of careers in academic medicine: a survey of MD PhD residents in dermatology programs in the United States. AB - A survey of all MD-PhD residents in dermatology in the United States was conducted to evaluate the factors important to them in making career pathway decisions. Twenty-six MD-PhDs responded to the survey representing a 90% response rate. Although essentially all entered dermatology with the intention of pursuing a career in academic medicine, 77% percent thought they would be in academic medicine 5 years post-residency. Factors explored for a negative influence on entrance into academic medicine post-residency were 1) financial concerns, 2) length of training, 3) research hiatus due to clinical training, and 4) eligibility for existing funding mechanisms. PMID- 1732379 TI - Dapsone suppresses integrin-mediated neutrophil adherence function. AB - The anti-inflammatory influence of dapsone may involve suppression of neutrophil chemotaxis to selected attractants, but other actions of the drug are likely also involved. We have discovered that dapsone may suppress migration of neutrophils to extravascular sites through inhibition of adherence functions required for neutrophil recruitment. Neutrophil adherence mediated by integrins (CD11/CD18 or Mac-1 family receptors) was measured in vitro in terms of binding of stimulated cells to albumin-coated wells of microtiter plates, using phorbol myristate acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (FMLP) as stimuli. Adherence was assessed by staining attached cells with crystal violet dye and measuring the dye concentration at OD590 using an automated plate reader. The role of integrins in this assay was confirmed by the ability of anti-integrin antibody to suppress stimulated neutrophil adherence. The OD590 value for cells adhering to albumin in the absence of stimulus and dapsone averaged 0.2 +/- 0.04 (SEM) over five experiments. In the presence of 0.1 microM PMA or 10(-6) M FMLP, the OD590 values averaged 0.88 +/- 0.1 and 0.75 +/- 0.12, respectively. Dapsone did not affect unstimulated neutrophil adherence but, when present with stimulus, produced a dose-related inhibitory effect on adherence. Fifty percent inhibitory doses were approximately 150 micrograms/ml dapsone for both stimuli. Sulfapyridine reproduced the inhibitory effect of dapsone, but two structurally related compounds, hydrochlorothiazide and furosamide, did not. The observed ability of dapsone to inhibit neutrophil chemotaxis under agarose to FMLP and interleukin-8 may also be explained by interference with integrin-mediated adherence required for motility in this assay system. To consider if dapsone might have a similar inhibitory influence on neutrophil adherence in vivo, we tested the stimulated adherence function of neutrophils isolated from three individuals on dapsone therapy for dermatitis herpetiformis. Stimulated adherence of patients' cells averaged less than 40 percent of the control value. Suppression of leukocyte integrin function may therefore also contribute to the ability of dapsone to inhibit neutrophil infiltration in neutrophilic dermatoses. PMID- 1732380 TI - Evidence for a structural abnormality of collagen VII in a patient with dystrophic epidermolysis bullosa inversa. AB - Recent studies indicate that in skin of patients with dystrophic epidermolysis bullosa (EB) inversa, anchoring fibrils have an abnormal ultrastructure, but the major protein of these fibrils, collagen VII, is expressed and detectable with antibodies at the dermo-epidermal junction. For molecular characterization of this rare EB phenotype, skin biopsies from a patient with dystrophic EB inversa were investigated with indirect immunofluorescence, immunoelectron microscopy, and immunoblotting. Ultrastructural analysis of clinically uninvolved skin showed sublamina densa splitting. In unblistered areas, focal groups of anchoring fibrils that appeared loosely polymerized and without a distinct crossbanding pattern were observed. Indirect immunofluorescence staining with antibodies to collagen VII exhibited a linear fluorescence at the dermo-epidermal junction and at the roof of a spontaneous blister. Immunoelectron microscopy demonstrated staining of the poorly assembled anchoring fibrils, but no significant reaction in areas where no fibrillar structures could be discerned. In contrast to normal control skin, immunoblotting showed immunoreactive collagen VII in both epidermal and dermal extracts. Moreover, the dermis-associated collagen VII appeared as a distinct doubleband that contained the tissue form of collagen VII (250-300 kD) and an additional band with a slightly smaller molecular weight. In epidermal extracts one band, of the size of the tissue form, was detected. The studies on the present patient suggest that a structural abnormality of collagen VII that prevents its aggregation to stable dimers or polymerization to distinct anchoring fibrils may contribute to the etiopathogenesis of dystrophic EB inversa. PMID- 1732381 TI - Effects of chloroquine on antigen-presenting functions of epidermal cells from normal and psoriatic skin. AB - The lysosomotropic drug chloroquine has been added to cultures containing peripheral blood mononuclear cells (PBMC) and allogeneic antigen-presenting cells obtained from the epidermis of normal human skin or from skin of patients with psoriasis. We found that in the presence of chloroquine, the allostimulatory properties of freshly obtained, normal epidermal antigen-presenting cells (EAPC) were profoundly impaired. By contrast, normal EAPC (cultured for 72 h prior to exposure to alloreactive T cells), as well as fresh EAPC from psoriatic skin were not impaired by chloroquine. In fact, in some experiments, cultured EAPC and psoriatic EAPC in the presence of chloroquine displayed significantly enhanced abilities to activate allogeneic T cells. Moreover, chloroquine partially reversed the inhibitory effect of transforming growth factor-beta (TGF beta) on T cell activation induced by cultured normal EAPC. Fresh normal EAPC, which are normally impervious to the effects of TGF beta, were not protected by TGF beta from chloroquine inhibition. We conclude that the addition of chloroquine to tissue culture medium unmasks important differences in antigen processing and presenting properties of fresh, normal EAPC, on the one hand, and cultured normal EAPC and psoriatic EAPC on the other. The ability of chloroquine to exaggerate the accessory cell function of the latter cells may relate to the capacity of this drug to cause the secretion of acid hydrolases into their immediate microenvironment. Moreover, the capacity of chloroquine to enhance the accessory cell functions of freshly obtained psoriatic EAPC emphasizes an abnormality that psoriatic cells in the epidermis constitutively express. We postulate that this abnormality may be related to the clinical observations that psoriatic skin lesions may be induced or aggravated by chloroquine therapy. PMID- 1732382 TI - Evaluating the UVA photoprotection of sunscreens with murine skin edema. AB - The acute and chronic deleterious effects of UVA on skin have prompted a growing interest in developing effective UVA-photoprotective sunscreens. The quantification of their UVA photoprotection remains, however, a major problem. In the present study, murine skin edema induced by 8-methoxypsoralen plus UVA (PUVA) is evaluated as a screening method for quantifying the UVA photoprotection of commercially available sunscreens. The PUVA-induced murine skin edema is provoked on the dorsa of female hairless albino mice and measured with a hand-held micrometer. A clear time course and a well-defined dose-response relationship are demonstrated. Therefore, a UVA-photoprotection factor (UVA-PF) could be defined by dividing the minimal edema dose with sunscreen by the minimal edema dose without sunscreen. The UVA-PF values obtained with this method were quantitatively and qualitatively very similar to those obtained in 8 methoxypsoralen-photosensitized murine skin by using the number of sunburn cells as the biologic end point and were qualitatively similar to UVA-PF values obtained in human skin using phototoxic erythema and UVASUN-induced tanning as the parameter. It is concluded that PUVA-induced murine skin edema offers an objective, reproducible, and easily applicable screening method for quantifying the degree of UVA photoprotection of a sunscreen. PMID- 1732383 TI - Regulation of parathyroid hormonelike protein production in cultured normal and malignant keratinocytes. AB - We have recently demonstrated that parathyroid hormone-like protein (PLP) production by cultured human squamous carcinoma cells (SCC) can be modulated by co-culture with fibroblasts. The interaction of SCC with fibroblasts, possibly occurring during the invasive phase of SCC, may be the stimulus for enhanced PLP production, thus contributing to the genesis of humoral hypercalcemia of malignancy in this type of cancer (Cancer Res 50:3589-3594, 1990). In the present study we show that the fibroblast-induced increase in PLP level in the medium of SCC-4 cells is paralleled by an increase in PLP messenger ribonucleic acid (mRNA) expression in these cells. We also found that the inhibition of secretion of PLP by monensin for 2 h resulted in a marked increase in immunodetectable PLP intracellularly, suggesting that secretion of PLP was a fast process. The modulation of the production of PLP by calcium and hydrocortisone was further examined in SCC-4 cells and was compared to that in normal keratinocytes and in SCC-9 cells. PLP levels in conditioned media were highest in poorly differentiating SCC-4 cells, intermediate in moderately differentiating SCC-9 cells, and lowest in normal keratinocytes showing high differentiating capacity. Furthermore, in each of the cell types used, PLP production was highest in cultures grown under low calcium conditions; at both calcium concentrations used, the presence of hydrocortisone reduced the PLP release into the medium. This reduction was probably due to a direct effect of hydrocortisone on PLP synthesis because the expression of PLP mRNA was also reduced in the presence of hydrocortisone when tested in SCC-4 cells. In conclusion, our findings indicate that the induction of differentiation in both normal and malignant keratinocytes is associated with the inhibition of PLP production. PMID- 1732384 TI - Expression of the alpha 6 beta 4 integrin in lesional skin differentiates bullous pemphigoid (BP) from epidermolysis bullosa acquisita (EBA). AB - The integrin alpha 6 beta 4 complex is a protein of the membrane of basal keratinocytes, localized at the surface of cells in contact with the basement membrane zone in normal skin. The expression of alpha 6 beta 4 was investigated in several autoimmune blistering skin diseases including bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA), bullous systemic lupus erythematosus (BSLE), and pemphigus vulgaris (PV) by an indirect immunofluorescence technique. In lesional bullous skin of BP, alpha 6 beta 4 expression was either absent, or in some cases represented an unusual irregular patchy staining. In contrast, in lesional bullous skin from EBA, BSLE, and PV, alpha 6 beta 4 expression was comparable to that observed in normal skin, i.e., a linear staining of the BMZ. Thus, analysis of the alpha 6 beta 4 integrin reactivity on lesional skin, in conjunction with the typical localization of collagen IV, allows a rapid and accurate distinction between BP and EBA. PMID- 1732385 TI - Structural basis for the barrier abnormality following inhibition of HMG CoA reductase in murine epidermis. AB - Recent studies have shown that increased epidermal 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase activity is crucial for the barrier recovery response that follows solvent-induced barrier perturbation. Upregulation of this enzyme leads to increased cholesterologenesis, formation and secretion of cholesterol-enriched lamellar bodies, and barrier repair. Topical lovastatin induced inhibition of HMG CoA reductase activity both delays the acute barrier repair response, as well as leading to a chronic barrier abnormality when applied repeatedly to intact skin. Presently, we assessed the effects of repeated topical applications of two different specific inhibitors of HMG CoA reductase on barrier function, the lamellar body-secretory system, and stratum corneum intercellular domains, with functional and morphologic parameters. Once-daily applications of lovastatin or fluindostatin (XU62-320; Sandoz) for 4-8 d to intact hairless mouse epidermis produced a progressive abnormality in barrier function (transepidermal water loss greater than 2.0-5.0 in treated versus less than 0.25 mg/cm2/h for weakly active analogues or vehicle controls). The barrier defect was preceded by alterations in lamellar body internal structure and a partial failure of lamellar body secretion into the stratum corneum interstices, further confirmed by enzyme cytochemistry. Moreover, the deposition of abnormal lamellar body contents resulted in the formation of clefts in the intercellular spaces at the stratum granulosum-stratum corneum interface, resulting in increased permeability through these domains shown by lanthanum perfusion. Applications of irritants, even when producing a barrier abnormality, did not alter the lamellar body secretory system. Co-applications of cholesterol with the inhibitors reversed both the barrier abnormality and the abnormalities in the lamellar body secretory system that occur with the inhibitor alone. Finally, membrane bilayer structures in the mid-to-outer stratum corneum of inhibitor-treated specimens appeared normal, but the intercellular domains displayed enormously expanded lacunae. However, because similar dilatations also occurred in vehicle-treated samples, they can be attributed to the vehicle alone. These studies provide further evidence that the inhibitor-induced defect in barrier function a) is initiated by inhibition of HMG CoA reductase; b) can be attributed to defects in both lamellar body structure and deposition with resultant abnormalities in intercellular membrane domains in the lower stratum corneum; and c) is further enhanced by permissive effects of the vehicle on the permeability of the outer stratum corneum. PMID- 1732386 TI - A new method to measure type I and III collagen synthesis in human skin in vivo: demonstration of decreased collagen synthesis after topical glucocorticoid treatment. AB - Collagen is synthesized as procollagen and large extra domains known as propeptides are cleaved off enzymatically. In the present study we have measured the carboxyterminal propeptide of type I collagen (PICP) and the aminoterminal propeptide of type III collagen (PIIINP) in blister fluids of human skin. High concentrations of PICP were found in the spontaneous blisters of patients with bullous pemphigoid, erysipelas, or erythema multiforme. Detectable amounts were also found in suction blisters induced on healthy skin. Because the concentrations in suction blisters were several times higher than in corresponding serum, most of PICP and PIIINP was derived from the underlying dermis. This method was used for assessing type I and type III collagen synthesis after topical glucocorticoid treatment. Clobetasol-17-propionate (CP) decreased the concentrations of PICP by 75% after 1 d of treatment, the maximum inhibition (92%) being found after 2 d treatment. PIIINP was also affected. Hydrocortisone and hydrocortisone-17-butyrate also decreased the concentrations of PICP and PIIINP, but less markedly than CP. Partial recovery was seen 3 d after stopping the treatment. Thus measurement of collagen type specific propeptides in suction blisters can be used as an estimate of collagen synthesis in vivo, avoiding both local anesthesia and skin biopsing. With radioimmunoassays for PICP and PIIINP a large number of samples can also be processed simultaneously. PMID- 1732387 TI - Alteration in keratinocyte ganglioside content in basal cell carcinomas. AB - We examined the ganglioside content of normal human keratinocytes and basal cell carcinomas (BCC). The total ganglioside content of the epidermis was 0.098 +/- 0.01 microgram lipid-bound sialic acid/mg dry weight. GM3 was the predominant ganglioside of epidermis. GM2 and GD3 were also found in significant amounts. Polysialylated gangliosides were identified in only small amounts. In contrast to all other body locations, breast epidermis showed large amounts of GM1. The total ganglioside content of nodular and sclerosing facial BCC was approximately 3.5 times that of normal facial epidermis. This marked elevation of total ganglioside was not affected by dermal ganglioside contamination, because the total ganglioside content of the dermis was similar to that of the epidermis. The relative percentage of GM2 was significantly decreased, whereas the relative percentage of GM3 was slightly decreased in BCC. 9-O-acetyl-GD3 was present in the BCC, but not in normal epidermis or dermis. 9-O-acetyl-GD3 may be a surface marker for BCC. Furthermore, the alterations in amount and composition of individual gangliosides on neoplastic membranes may lead to novel therapeutic interventions. PMID- 1732388 TI - Purification and molecular characterization of beta-naphthoflavone-inducible cytochrome P-450 from rat epidermis. AB - This study was designed to characterize epidermal cytochrome P-450 (P-450) induced by skin application of beta-naphthoflavone (beta-NF). Topical application of beta-NF (40 mg/kg) to rats resulted in a 2.6-times increase in epidermal P-450 content and a 3--14-times increase in epidermal monooxygenase activities. The purified epidermal P-450 showed a major band at 54 kDa on SDS-PAGE, which comigrated with hepatic P-4501A1 and cross-reacted with monoclonal and polyclonal antibodies specific to P-4501A1. The specific content of purified epidermal P-450 was 1.53 nmol/mg protein, representing 42-times purification. HPLC analysis of the purified epidermal P-450 showed similar elution profile and retention time as that of hepatic P-4501A1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation when reconstituted with purified NADPH--P-450 reductase and phospholipid. Peptide fingerprint analysis of the purified epidermal P-450 and hepatic P-4501A1 showed similar monoclonal antibody 1-7-1 reacting epitopes. Partial N-terminal amino acid sequence analysis of purified epidermal P-450 showed complete homology with the known sequence of P-4501A1. Similarly, HPLC analysis of tryptic digest of purified epidermal P-450 and hepatic P-4501A1 showed identical peptide peaks with comparable retention times. N-terminal amino acid sequence analysis of three randomly selected tryptic peptides showed complete homology with the known sequence of P-4501A1. These results indicate that rat epidermal P-450 induced by beta-NF is similar to hepatic P-4501A1. PMID- 1732389 TI - Neutrophil-activating proteins in psoriasis. AB - Neutrophil accumulation in the epidermis is a histologic characteristic of psoriasis. We addressed the question: What is the major protein-like chemotactic principle responsible for neutrophil accumulation? Purification of proteinaceous neutrophil chemoattractants from extracts obtained from psoriatic scales by multistep high-performance liquid chromatography (HPLC) yielded three biochemically distinct polypeptides with potent neutrophil chemotactic activity. Aminoterminal amino acid sequence analysis of the quantitatively major neutrophil attractant revealed the sequence ELRXQXIKTYSK, which is identical to that of a 69 residue form of neutrophil-activating peptide-1/interleukin 8 (NAP-1/IL-8). The second major attractant showed the sequence XXVATELRXQXL . . ., which is identical to that of the gene product of the oncogene "gro" as well as "melanoma growth stimulatory activity, MGSA," whereas the third and minor neutrophil chemotaxin has an NH2-terminal sequence identical with NAP-1/IL-8. Estimation of NAP-1/IL-8-related proteins and gro/MGSA by HPLC combined with bioassay revealed a mean of 3.3 +/- 1.7 ng NAP-1/IL-8-related proteins (n = 11) and 3.2 +/- 1.9 ng gro/MGSA (n = 11) per 1 mg psoriatic scales. In normal heel callus (n = 8), these neutrophil attractants were found at concentrations below 0.02 +/- 0.01 ng/mg. The finding of more than 150-times increased amounts of both NAP-1/IL-8 and gro/MGSA in lesional psoriasis material suggest that these mitogenic as well as neutrophil- and lymphocyte-chemotactic compounds may play an important role in the pathogenesis of psoriasis. PMID- 1732390 TI - Effects of all-trans retinoic acid on UVB-irradiated and non-irradiated hairless mouse skin. AB - The effects of all-trans retinoic acid (t-RA) on photodamaged and normal non irradiated skin were examined in hairless mice (Skh:HR-1). After being exposed to increasing doses of UVB for 10 weeks (total dose = 1.4 J/cm2), the animals were then treated with 0.1% t-RA in ethanol (50 microliters, five times per week) for another 10 weeks. Several animals (the follow-up group) were further observed after the termination of the t-RA treatment to investigate if the t-RA effect was reversible. Wrinkle effacement induced by t-RA was compared with three other parameters: a) de novo collagen synthesis, b) width of the dermal repair zone, and c) epidermal thickening. Interestingly, t-RA did not stimulate collagen synthesis in animals not exposed to UVB. In the irradiated animals, the time course of wrinkle reduction correlated with the stimulation of collagen synthesis. After a synchronous initial lag phase of 4-6 weeks, the wrinkling decreased from the maximum grade of 4 to a mean grade of 1.3, whereas collagen synthesis was enhanced to 245% of the control at week 10 of t-RA treatment. In contrast, a similar lag phase was not observed for either the appearance of the dermal repair zone or epidermal thickening. In the follow-up group, upon termination of t-RA treatment, collagen synthesis returned to the control level. Wrinkle effacement and thickening of the dermal repair zone, however, did not regress, suggesting the anti-photoaging effect of t-RA was not reversible over this time frame. The correlation between the length of the lag phases for collagen synthesis and wrinkle reduction points to the possibility that collagen plays an important role in tRA-induced wrinkle effacement. Both parameters are thus important endpoints for investigating the mechanism of RA-induced repair of photodamaged skin. PMID- 1732391 TI - Langerhans cell sensitivity to in vitro versus in vivo loading with cyclosporine A. PMID- 1732392 TI - Generatio- and topocentricity--widespread contemporary attitudes to clinical research. PMID- 1732393 TI - Radioiodine treatment of recurrent hyperthyroidism in patients previously treated for Graves' disease by subtotal thyroidectomy. AB - Radioiodine therapy is often employed for treatment of patients with relapse of hyperthyroidism due to Graves' disease, after previous thyroid surgery. Little is known about the outcome of this treatment compared to patients with no previous surgery. A total of 20 patients who had received surgical treatment for Graves' hyperthyroidism 1-46 years previously and with relapse of the hyperthyroidism, and 25 patients with hyperthyroidism due to Graves' disease and no previous thyroid surgery were treated with radioiodine, following the same protocol. Early after treatment the previously operated patients showed a higher sensitivity to radioiodine, with more cases of early hypothyroidism, than non-operated patients. However, after 50 months of follow-up the outcome was identical. The results indicate that frequent assessment is necessary after radioiodine treatment of previously operated patients, since some patients develop early hypothyroidism. PMID- 1732394 TI - Hyperleukocytic effects on skin capillary circulation in patients with leukaemia. AB - A decrease in capillary flow due to hyperleucocytosis may cause impairment of organ perfusion in patients with leukaemia. In order to evaluate the effects of different cell types of leukaemic cell origin on skin capillary circulation we have studied patients with (a) chronic lymphocytic leukaemia (CLL) (n = 6) and (b) acute non-lymphocytic leukaemia (ANLL) (n = 6) or chronic granulocytic leukaemia (CGL) (n = 5). Capillary blood cell velocity (CBV) in fingernail-fold capillaries was measured by videophotometric capillaroscopy. After a 1-min arterial occlusion at the finger base, the post-occlusive reactive hyperaemia was evaluated by measuring the peak (p) CBV and the time to pCBV. In patients with ANLL/GGL, both resting CBV and pCBV were lower than in healthy control subjects. In the CLL patients these values were not significantly different compared to controls. In a few patients the time to pCBV was markedly prolonged. Symptoms attributable to hyperviscosity were more common in patients with ANLL/CGL and low CBV values. It is suggested that the cell size of leucocytes may be an important factor in the development of hyperviscosity syndromes and organ malperfusion in leukaemic patients. PMID- 1732395 TI - Evidence for an independent relationship between insulin resistance and fasting plasma HDL-cholesterol, triglyceride and insulin concentrations. AB - Elevated plasma insulin and triglyceride (TG) and decreased high-density lipoprotein (HDL)-cholesterol concentrations have been shown to be risk factors for coronary heart disease (CHD). It has been suggested that these metabolic abnormalities are all secondary to resistance to insulin-stimulated glucose uptake. To examine this in more detail, we divided 18 non-diabetic, moderately overweight, sedentary men aged 25-50 years into three groups on the basis of their steady-state plasma glucose levels (SSPG): a low group, (n = 7; SSPG less than 8.3 mmol l-1), a middle group, (n = 6; SSPG 8.3-11.1 mmol l-1), and a high group (n = 5; SSPG greater than 11.1 mmol l-1). The high group had significantly higher fasting (P less than 0.05) and post-oral glucose challenge (P less than 0.01) insulin concentrations, higher fasting TG (P less than 0.05) and lower fasting HDL-cholesterol (P less than 0.05) concentrations than the other two groups. However, there were no statistically significant differences between the groups with regard to body mass index, waist-to-hip ratio or physical endurance capacity as determined by maximal oxygen consumption during a treadmill test. The data suggest that insulin resistance has an effect on the modulation of plasma insulin, TG and HDL-cholesterol concentrations, independent of generalized, abdominal or physical endurance capacity. PMID- 1732396 TI - Gene transfer and gene therapy. PMID- 1732397 TI - Time course of some effects of cigarette smoking on platelets. AB - Eight male habitual smokers smoked two cigarettes over a 20-min period following a 12-h period of abstinence. Antecubital venipuncture was performed immediately before, immediately after, and 55 min and 2 h after smoking had ceased. At these times, the mean values (+/- SD) of collagen-induced platelet aggregation were 45 +/- 5, 68 +/- 5, 59 +/- 6 and 52 +/- 5 chart units, respectively, while the corresponding values for the mean platelet aggregate ratio were 0.91 +/- 0.01, 0.82 +/- 0.03, 0.87 +/- 0.02 and 0.90 +/- 0.02, respectively. Mean collagen induced platelet aggregation was significantly (P less than 0.005) higher immediately after, and 55 min and 2 h after smoking. The mean platelet aggregate ratio was significantly (P less than 0.001) lower immediately after and 55 min after smoking. Correlation coefficients between the concentration of nicotine in each of the 24 plasma samples obtained after smoking and the corresponding values of collagen-induced platelet aggregation and the platelet aggregate ratio were 0.41 (P less than 0.05) and -0.50 (P less than 0.02), respectively. It is concluded that when habitual smokers abstain from smoking overnight, a 20-min period of cigarette smoking may enhance platelet aggregability for as long as 2 h. PMID- 1732398 TI - Mortality and recurrences during eight years following stroke. AB - A total of 388 patients, of mean age 73 years, with acute cerebrovascular disease (CVD) evaluated in a non-intensive Stroke Unit, and a sample of 209 age- and sex matched similarly acutely admitted patients with surgical diseases were followed up for 5-8 years. The CVD patients had a 21-day hospital mortality of 13%, and 66% mortality during the entire study period, compared to 2% and 48%, respectively, in controls. Old age had only a minor effect on the initial mortality. However, long-term mortality increased markedly with age. The initial mortality in 120 stroke recurrences was 50%. In CVD patients heart diseases were common causes of death, and circulatory diseases were most predominant of all (86%), with an accumulation during the first months after the occurrence of the initial CVD event. These figures clearly show that stroke patients constitute a group with high risk of stroke recurrence and death. Despite declining figures for stroke mortality, and most probably also for case fatality rate after first stroke episodes, much work remains to be done within the field of secondary prevention after stroke. PMID- 1732399 TI - Lack of association between the apolipoprotein B gene 3' hypervariable region alleles and coronary artery disease in Finnish patients with angiographically documented coronary artery disease. AB - Previous studies have suggested that some apolipoprotein B (apoB) 3' variable number of tandem repeats (3'VNTR) locus alleles are associated with coronary artery disease (CAD). We examined the possible association between the apoB 3'VNTR alleles and CAD in 387 Finnish subjects. Using the polymerase chain reaction and polyacrylamide gel electrophoresis, the 3'VNTR genotype was determined in 187 individuals with severe CAD confirmed by coronary angiography (patients), in 121 individuals with normal coronary angiograms (controls), and in 79 apparently healthy subjects (normals). In contrast to previous reports from other populations, the larger apoB 3'VNTR alleles were not significantly more frequent among CAD patients than among controls or normals. In addition, there was no significant association between the 3'VNTR alleles and serum lipid levels in this Finnish population. PMID- 1732400 TI - Five-year follow-up of young adults visiting an emergency unit because of atypical chest pain. AB - A five-year follow-up, by means of a personal interview, was performed on patients below the age of 40 years with acute chest pain without obvious organic cause (n = 64). They had been consecutively admitted to the emergency unit over a period of 8 weeks, and had all been subjected to a thorough medical and psychosocial investigation with feedback soon after the initial consultation (investigation patient group, IP). For comparison, a non-investigation patient (NIP) group (n = 51) was recruited over a period of 8 separate weeks. Half of the patients in each group reported at the follow-up that they continued to suffer from chest pain. Compared to normal subjects, they reported more depression. This means that the initial research programme performed in the investigation group had no sustained therapeutic effect compared to routine care at the emergency unit. Tension, anxiety and number of consultations with a physician, as reported in the initial investigation, were negatively correlated with the outcome at follow-up. We conclude that acute chest pain without obvious organic cause in young adults is a condition with an excellent prognosis in strictly physical terms. However, a high proportion of the patients continue to suffer from chest pain for several years, and many of them continue to be consumers of medical resources. We therefore suggest that therapeutic programmes should be developed, particularly for those who report psychological symptoms and those with a history of many consultations with physicians. PMID- 1732401 TI - Systemic lupus erythematosus and related systemic diseases in a nationwide twin cohort: an increased prevalence of disease in MZ twins and concordance of disease features. AB - Concordance rates for systemic rheumatic diseases among monozygotic (MZ) and same sex dizygotic (DZ) twin pairs ascertained from the older (twins born before 1958) and younger (twins born during the period 1958-1986) parts of the Finnish Twin Cohort were studied. Nine MZ pairs and 10 DZ pairs with at least one member affected by systemic lupus erythematosus (SLE) were identified. Only one MZ pair was concordant for definite SLE by American Rheumatism Association (ARA) criteria. In addition, two more MZ pairs that fulfilled three ARA criteria may have been concordant. None of the DZ pairs were concordant for SLE. MZ twins also had a higher concordance rate for tests of autoantibodies, but the numbers were small. Other systemic rheumatic diseases were infrequent. The cumulative incidence of systemic rheumatic diseases among MZ twin individuals (1.7/1000) was significantly higher than that among DZ twin individuals (0.7/1000). PMID- 1732402 TI - Radionuclide assessment of left ventricular function in patients with myocardial infarction and diabetes mellitus. AB - The aim of this study was to compare left ventricular function, assessed by radionuclide angiocardiography, in 54 diabetics and 194 non-diabetics with acute myocardial infarction (AMI). The most meaningful results concern the inferior AMI group, whose left ventricular ejection fraction (LVEF) and regional wall motion were significantly lower in diabetics than in non-diabetics (LVEF was 44.2 +/- 11 vs. 51.6 +/- 9%, P less than 0.005; the regional wall motion score was 0.46 +/- 1 vs. 1.56 +/- 1, P less than 0.01, respectively), while no significant difference was observed in the anterior AMI group. However, in the group as a whole, the LVEF was 41 +/- 13% in diabetics and 47 +/- 13% in non-diabetics (P less than 0.01), the number of abnormally contracting segments was 2.0 +/- 0.9 and 1.5 +/- 1, respectively, and the wall motion score was 0.2 +/- 1.1 and 1.0 +/- 1.4, respectively (P less than 0.01). These data could be explained by an underlying cardiac dysfunction in diabetes, in addition to AMI. The more marked difference between diabetics and non-diabetics in inferior AMI might be related to the smaller infarct size in this group. PMID- 1732403 TI - Intrafamilial outbreak of Mediterranean spotted fever. AB - The existence of intrafamilial outbreaks of spotted-fever group rickettsioses has seldom been reported, and their true incidence is not known. We here report the occurrence of Mediterranean spotted fever (MSF) in three members of a family: a boy, his grandmother and his grandfather. The infection resolved uneventfully in the boy, and followed a more protracted course in the two older patients, who suffered complications including drowsiness, clotting defects and acute renal failure. This finding highlights the importance of conditioning factors such as age and associated illnesses in the prognosis of infection due to Rickettsia conorii, and suggests that strict surveillance of individuals at risk of infection is necessary for prompt recognition of the onset of the illness and initiation of antibiotic treatment without delay. PMID- 1732404 TI - Progressive necrotic myelopathy as a paraneoplastic syndrome: report of a case and some pathogenetic considerations. AB - Progressive necrotic myelopathy is a syndrome characterized by a spotty necrotic degeneration of the whole spinal cord in both anterior and posterior horns. This syndrome was recorded in a man suffering from a lymphoplasmatocytoid lymphoma. Whilst the usual evolution of this neurological syndrome is inexorably fatal, our case had a better outcome, due to the good response of the neoplasm to therapy. Progressive necrotic myelopathy is an uncommon complication of cancer, but it is probably incorrectly recognized in a number of cases. Two possible pathogenetic hypotheses are suggested: an autoimmune or an infective mechanism. PMID- 1732405 TI - Acute haematogenous osteomyelitis of the patella revealed by a pathological fracture in multiple myeloma. AB - A 57-year-old man was followed up for multiple myeloma with no lytic bone lesions. During a septicaema, he presented with a pathological fracture of the patella. Despite a poor haematological status, he received surgical treatment, which allowed diagnosis of haematogenous osteomyelitis of the patella. The outcome was favourable, with a 6-week plaster immobilization after cerclage and 4 months of intravenous antibiotic therapy. This case emphasizes the fact that one well-known cause of bone lesions may lead to an erroneous diagnosis. The successful clinical course suggests that accurate therapeutic management may be discussed in haematological malignancies when intensive-care support is available. PMID- 1732406 TI - Coronary heart disease in the absence of hypercholesterolaemia. PMID- 1732407 TI - Response to editorial on antioxidant therapy for chronic heart failure. PMID- 1732408 TI - Response to editorial on antioxidant therapy for chronic heart failure. PMID- 1732409 TI - Molecular cloning and expression of the human interleukin 5 receptor. AB - Human interleukin 5 (IL-5) plays an important role in proliferation and differentiation of human eosinophils. We report the isolation of cDNA clones from cDNA libraries of human eosinophils by using murine IL-5 receptor alpha chain cDNA as a probe. Analysis of the predicted amino acid sequence indicated that the human IL-5 receptor has approximately 70% amino acid sequence homology with the murine IL-5 receptor and retains features common to the cytokine receptor superfamily. One cDNA clone encodes a glycoprotein of 420 amino acids (Mr 47,670) with an NH2-terminal hydrophobic region (20 amino acids), a glycosylated extracellular domain (324 amino acids), a transmembrane domain (21 amino acids), and a cytoplasmic domain (55 amino acids). Another cDNA encodes only the extracellular domain of this receptor molecule. Other cDNA clones encode molecules having diversified cytoplasmic domains. COS7 cells transfected with the cDNA expressed a approximately 60-kD protein and bound IL-5 with a single class of affinity (Kd = 250-590 pM). The Kd values were similar to that observed in normal human eosinophils. In contrast to the murine 60-kD alpha chain, which binds IL-5 with low affinity (Kd = approximately 10 nM), the human alpha chain homologue can bind IL-5 with much higher affinity by itself. RNA blot analysis of human cells demonstrated two transcripts (approximately 5.3 and 1.4 kb). Both of them were expressed in normal human eosinophils and in erythroleukemic cell line TF-1, which responds to IL-5. The human IL-5 receptor characterized in this paper is essential for signal transduction, because expression of this molecule in murine IL-3-dependent cell line FDC-P1 allowed these cells to proliferate in response to IL-5. PMID- 1732410 TI - Interleukin 3 is a growth factor for human follicular B cell lymphoma. AB - More than one-half of adults with non-Hodgkin's B cell lymphomas present with low grade follicular lymphomas. These tumor cells are found in close association with follicular T lymphocytes and dendritic cells, suggesting that the surrounding cells may play a role in the support of follicular tumors. Supernatants from activated human peripheral blood lymphocytes were found to promote the in vitro proliferation of follicular tumor cells. This effect was entirely due to interleukin 3 (IL-3), a factor generally thought to cause the growth and differentiation of immature hematopoietic cells. IL-3 receptors were detected on fresh isolates of all primary follicular cell tumors examined. These findings suggest that follicular cell tumors may be dependent in vivo on IL-3 and that therapies directed against IL-3, its receptor, or the T cells that produce it may be effective treatment for follicular lymphoma. PMID- 1732411 TI - Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease. AB - In this report we have investigated macrophage (M phi) activity and tumor necrosis factor alpha (TNF-alpha) production during graft-vs.-host disease (GVHD). TNF-alpha production by M phi requires two signals: priming of M phi by interferon followed by triggering of TNF-alpha production and release by lipopolysaccharide (LPS). The state of M phi activation was examined in nonirradiated B6AF1 recipient mice injected with either 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) parental B6 lymphoid cells. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS-triggered release of significant amounts of TNF-alpha into the serum resulting in death of the animals within 36 h. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-alpha. In vitro studies demonstrated that M phi were primed during GVHD. The level of M phi priming was greater during acute GVHD than nonlethal GVHD since 100-fold less LPS was required to trigger killing of a TNF-alpha-sensitive cell line by M phi from acute GVHD animals. The amount of TNF-alpha released into the serum after LPS injection increased during the course of the GVHD and was significantly greater in acute GVH-reactive mice. Endogenous LPS was detected in the serum of acute GVH reactive animals coincident with the onset of mortality. The data provide evidence that during GVHD M phi are primed as a result of the allogeneic reaction and that endogenous LPS therefore triggers M phi production of TNF-alpha resulting in the symptoms characteristic of acute GVHD. PMID- 1732412 TI - Search for hepatitis B virus cell receptors reveals binding sites for interleukin 6 on the virus envelope protein. AB - The major target organ for hepatitis B virus (HBV) is the liver. However, cells other than hepatocytes, including peripheral blood lymphocytes and monocytes, may become infected with HBV. The cell receptor binding site was assigned to the preS(21-47) segment of the HBV envelope protein. HBV receptors were detected on human liver and hepatoma cells, on B lymphocytes, and, as shown here, on monocytes, and T cell lines, activated by Escherichia coli lipopolysaccharide and concanavalin A, respectively. The cell receptors for HBV have not been characterized until now. The detection of HBV receptors and their "activation antigen" characteristic on distinct cells suggested paths for identification of the receptors with already defined cell surface proteins. This search revealed that interleukin 6 contains recognition sites for the preS(21-47) sequence and mediates HBV-cell interactions. Thus, HBV belongs to a group of viruses utilizing cytokines or cytokine receptors for replication and interference with the host immune system. PMID- 1732413 TI - Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. AB - Cytotoxic T lymphocytes (CTL) recognize class I major histocompatibility complex molecules complexed to peptides of eight to nine residues generated from cytosolic proteins. We find that CTL recognize, in vitro and in vivo, cells synthesizing a 10-residue peptide consisting of an initiating methionine followed by nine residues corresponding to a naturally processed determinant from influenza virus nucleoprotein (NP) (residues 147-155). Addition of two COOH terminal residues corresponding to NP residues 157 and 158 severely reduced presentation of the endogenously produced peptide to CTL in vitro and in vivo. Extension of NH2 and COOH terminal flanking residues to include residues corresponding to NP residues 137-146 and 159-168 failed to increase the antigenicity of this peptide. Its presentation was greatly enhanced, however, by further extending the NH2 and COOH termini to include all of the additional residues of NP. These findings indicate first, that a naturally processed viral ligand (with an NH2-terminal Met) of a class I molecule contains sufficient information to access intracellular class I molecules, and second, that flanking residues can influence the processing and presentation of antigens to CTL. PMID- 1732414 TI - Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa. AB - The mechanism by which Helicobacter pylori, a noninvasive bacterium, initiates chronic antral gastritis in humans is unknown. We now show that H. pylori releases products with chemotactic activity for monocytes and neutrophils. This chemotactic activity was inhibited by antisera to either H. pylori whole bacteria or H. pylori-derived urease. Moreover, surface proteins extracted from H. pylori and purified H. pylori urease (a major component of the surface proteins) exhibited dose-dependent, antibody-inhibitable chemotactic activity. In addition, a synthetic 20-amino acid peptide from the NH2-terminal portion of the 61-kD subunit, but not the 30-kD subunit, of urease exhibited chemotactic activity for monocytes and neutrophils, localizing the chemotactic activity, at least in part, to the NH2 terminus of the 61-kD subunit of urease. The ability of leukocytes to chemotax to H. pylori surface proteins despite formyl-methionyl-leucyl phenylalanine (FMLP) receptor saturation, selective inhibition of FMLP-mediated chemotaxis, or preincubation of the surface proteins with antiserum to FMLP indicated that the chemotaxis was not FMLP mediated. Finally, we identified H. pylori surface proteins and urease in the lamina propria of gastric antra from patients with H. pylori-associated gastritis but not from uninfected subjects. These findings suggest that H. pylori gastritis is initiated by mucosal absorption of urease, which expresses chemotactic activity for leukocytes by a mechanism not involving N-formylated oligopeptides. PMID- 1732415 TI - Structural differences between the two human complement C4 isotypes affect the humoral immune response. AB - An animal model has been used to address the question of the biological importance of the known structural difference between the two isotypes of human C4, i.e., C4A and C4B. Guinea pigs deficient in C4 were reconstituted transiently with either human C4A or C4B protein and immunized with the bacteriophage phi X174. Results from this study showed that C4A-reconstituted animals made a secondary response, i.e., switch from IgM to IgG; whereas the C4B-reconstituted animals did not. PMID- 1732417 TI - Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes. AB - Trans-sialidase and neuraminidase activities have been detected on the surface membrane of trypomastigotes of Trypanosoma cruzi, and both have been implicated in the parasite's invasion of host cells. We show here that these enzymes are structurally related. They are recognized by two independently derived monoclonal antibodies, are anchored to the membrane by glycosylphosphatidylinositol, copurify by ion exchange, molecular sieving, and hydrophobic chromatography, have maximal activities between pH 6.5 and 7.5, and are inactivated by heating at 56 degrees C. Furthermore, the neuraminidase and trans-sialidase reactions are coupled. An increase of the concentration of acceptors of the transfer reaction decreases the amount of free sialic acid released through the neuraminidase reaction. We conclude that a single enzyme can catalyze the transfer or the hydrolysis of macromolecular-bound sialic acid. The predominant direction of the reaction will depend on the availability of appropriate oligosaccharide acceptors of sialic acid. PMID- 1732416 TI - A natural killer cell granule protein that induces DNA fragmentation and apoptosis. AB - We report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called "fragmentin," was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe chromatin condensation within 1 h. Amino acid sequence of tryptic peptides indicated that fragmentin was highly homologous to the NK and T cell granule serine proteases RNK protease 1 and mouse cytotoxic T cell protease I (CCPI)/granzyme B. Preincubation with the serine esterase inhibitor 3,4-dichloroisocoumarin blocked fragmentin-induced DNA damage, but had no effect on cytolysin. Fragmentin activity against four lymphoma target cells was completely dependent on the presence of cytolysin. Fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses, suggesting that fragmentin activity was not limited to its effects on the nucleus. Fragmentin and cytolysin activity were completely inhibited by EGTA, indicating the process was Ca2+ dependent. A role for cytolysin in endocytosis of fragmentin was suggested by the observation that treatment of YAC-1 with cytochalasin B or sodium azide and 2-deoxyglucose blocked DNA fragmentation but not cytolysin activity. A 30-kD N alpha-CBZ-L-lysine thiobenzyl esterase, which copurified with fragmentin, was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin. Fragmentin activity was not dependent on protein synthesis, as cycloheximide treatment of YAC-1 cells did not prevent DNA damage. We postulate that fragmentin is the molecular mediator of NK cell-mediated DNA fragmentation and apoptosis. PMID- 1732418 TI - An antisense oligonucleotide complementary to a sequence in I gamma 2b increases gamma 2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion. AB - An antisense phosphorothioate (S)-oligonucleotide to a sequence in the intervening (I) region of the gamma 2b immunoglobulin (Ig) heavy chain gene inhibits Ig secretion by B cells stimulated with lipopolysaccharide (LPS) or LPS plus interleukin 4. It is also a striking stimulant of DNA synthesis by resting B cells. The antisense S-oligonucleotide causes a 10-20-fold increase in the expression of the gamma 2b germline transcript. Among mutants of the antisense S oligonucleotide, some show all the effects whereas others are inactive. A similar hierarchy exists in the quantitative biological activities of mutant S oligonucleotides and in their capacity to hybridize to the sense oligonucleotide, strongly suggesting that an I gamma 2b sequence in the RNA transcript or in the noncoding strand of the DNA is the target of the antisense S-oligonucleotide. The possible relationship of the overexpression of the germline gamma 2b transcript to the biological functions of the I gamma 2b antisense S-oligonucleotide is discussed. PMID- 1732420 TI - The second chance for advanced Hodgkin's disease. PMID- 1732419 TI - Engagement of major histocompatibility complex class II molecules by superantigen induces inflammatory cytokine gene expression in human rheumatoid fibroblast-like synoviocytes. AB - Cells in the rheumatoid synovium express high levels of major histocompatibility complex (MHC) class II molecules in vivo. We have therefore examined the ability of engagement of MHC class II molecules by the superantigen Staphylococcal enterotoxin A (SEA) to activate interleukin 6 (IL-6) and IL-8 gene expression in type B synoviocytes isolated from patients with rheumatoid arthritis. SEA had a minimal or undetectable effect on the expression of either gene in resting synoviocytes, as determined by Northern blot and specific enzyme-linked immunosorbent assay. However, induction of MHC class II molecule expression after treatment of synoviocytes with interferon gamma (IFN-gamma) enabled the cells to respond to SEA in a dose-dependent manner, resulting in an increase in both the level of steady-state mRNA for IL-6 and IL-8, and the release of these cytokines into the supernatant. IFN-gamma by itself had no effect on the expression of either cytokine. Pretreatment of the cells with the transcription inhibitor actinomycin D prevented the increase in cytokine mRNA induced by SEA, whereas cycloheximide superinduced mRNA for both cytokines after stimulation by SEA. Taken together, these results indicate that signaling through MHC class II molecules may represent a novel mechanism by which inflammatory cytokine production is regulated in type B rheumatoid synoviocytes, and potentially provides insight into the manner by which superantigens may initiate and/or propagate autoimmune diseases. PMID- 1732421 TI - Karnofsky Memorial Lecture. The immunotherapy and gene therapy of cancer. PMID- 1732422 TI - Conventional-dose salvage combination chemotherapy in patients relapsing with Hodgkin's disease after combination chemotherapy: the low probability for cure. AB - PURPOSE: The study was undertaken to evaluate clinical prognostic factors, probability of response to therapy, duration of response, and overall survival of patients with Hodgkin's disease relapsing from a chemotherapy-induced complete remission. PATIENTS AND METHODS: Study population comprised 107 patients with Hodgkin's disease treated with combination chemotherapy at the National Cancer Institute who relapsed after achieving a complete remission. RESULTS: Half of the relapses occurred within the first year of achieving complete remission; among patients in remission 5 years or longer, only 4% relapsed. The overall survival of the relapsed patients is projected to be 17% at 20 years, calculated from the date of relapse. Primary treatment regimen, presence of B symptoms, stage, sex, liver involvement, pleural involvement, marrow involvement, and histologic subtype did not affect the survival of relapsed patients. Only age at diagnosis (older or younger than 30 years) and length of initial remission (shorter or longer than 1 year) made a significant impact on survival. Patients whose initial remission was longer than 1 year had significantly higher complete response rates to salvage therapy, significantly more durable second remissions, and significantly longer survival than patients whose initial remission was shorter than 1 year. Survival beyond 11 years from relapse of patients with long initial remissions was 24%; for those with short initial remissions, 11% (P2 = .027). Despite the fact that with salvage therapy, patients with long initial remission had an 85% complete response rate to mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) with a disease-free survival of 45% at 20 years, acute leukemia and other treatment-related complications combined to lower the survival rate of this more favorable subset. CONCLUSIONS: These data with conventional dose salvage therapy provide results for comparison with novel salvage approaches including myeloablative therapy with autologous marrow or peripheral-blood stem cell support. PMID- 1732423 TI - A prospective study of a new combination chemotherapy regimen in patients older than 70 years with unfavorable non-Hodgkin's lymphoma. AB - PURPOSE: A prospective trial with a new combination of etoposide, mitoxantrone, and prednimustine (VMP), specifically devised for elderly patients with non Hodgkin's lymphoma (NHL), was undertaken. PATIENTS AND METHODS: Between January 1987 and April 1990, 52 consecutive unselected patients older than 70 years (median age, 75.6 years) with stage I to IV intermediate- and high-grade NHL, or with stage III to IV low-grade malignancy with symptomatic disease received etoposide and prednimustine 80 mg/m2 orally for 5 days and mitoxantrone 8 to 10 mg/m2 day 1 intravenously (IV), every 21 days. Fourteen patients were previously treated. RESULTS: Among the 48 assessable patients, the objective response rate was 81%; 46% of the patients achieved a complete response (CR). The overall toxicity seemed to be acceptable, with 15 (7%) episodes of grade 4 leukopenia and 41 (18%) episodes of grade 3, over a total of 226 administered cycles. The median survival was 12 months. The patients who obtained CR have a longer survival than those who did not (34 v 8 months; P less than .001). Fifty-eight percent of patients achieving CR were free from relapse at 24 months; up to 36 months from the start of therapy, 25% were free from relapse. As far as patients affected by diffuse histiocytic lymphoma, 66% of previously untreated patients obtained a CR, and 55% of them were still disease-free at 24 months from the start of therapy. CONCLUSION: We conclude that VMP is effective, well tolerated, and feasible on an outpatient basis in an unselected, elderly population affected by unfavorable NHL. PMID- 1732424 TI - Radiation-free preparation for allogeneic bone marrow transplantation in adults with acute lymphoblastic leukemia. AB - PURPOSE: The study was undertaken to investigate the effectiveness of allogeneic bone marrow transplantation from HLA-identical siblings after preparation with busulfan and cyclophosphamide in adults with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Thirty-nine patients aged 15 to 42 years underwent transplantation at three different centers from November 1984 through November 1990. All patients received 16 mg/kg busulfan and 120 mg/kg cyclophosphamide as preparative therapy. Cyclosporine plus methotrexate or cyclosporine plus corticosteroids with or without methotrexate were given for prevention of graft versus-host disease (GVHD). RESULTS: Twelve patients died of treatment-related complications, 12 patients relapsed, and 15 patients are leukemia-free survivors. For 27 patients in group 1 (first remission, second remission, first relapse), the estimated leukemia-free survival (LFS) rate is 42.3% (95% confidence interval [CI], 22.9% to 71.7%) at 3 years. For 12 patients with more advanced disease (group 2), the 1-year LFS rate is 13.5% (95% CI, 0% to 37.1%). Chronic GVHD occurred at an estimated incidence of 63.3% and developed significantly more frequently among patients who received corticosteroids for prevention of acute GVHD. Chronic GVHD was associated with a significantly lower incidence of relapse and with improved LFS rates. CONCLUSION: LFS rate in this study is comparable to that obtained with radiation-containing regimens; however, the effectiveness of this preparative regimen in ALL requires further study. PMID- 1732425 TI - Ifosfamide and mesna in previously treated advanced epithelial ovarian cancer: activity in platinum-resistant disease. AB - PURPOSE: There is a critical need to find new antineoplastic drugs that are active in platinum-refractory ovarian cancer. We conducted a phase II trial of single-agent ifosfamide with mesna uroprotection in patients with ovarian cancer previously treated with an organoplatinum compound to assess its activity in this clinical setting. PATIENTS AND METHODS: Ifosfamide (1.0 or 1.2 g/m2/d for 5 days, delivered on a monthly schedule) was administered to the 57 patients entered onto this trial. Dose reductions were permitted for unacceptable toxicities. RESULTS: Toxicity included severe bone marrow suppression (WBC count less than 1,000/microL and/or platelet count less than 50,000/microL), renal dysfunction (serum creatinine level greater than 2.0 mg/dL), and reversible CNS dysfunction (disorientation, hallucinations, somnolence, and agitation), which occurred in 20%, 14%, and 12% of patients, respectively. Of 41 patients with strictly defined platinum-refractory ovarian cancer, five (12%) demonstrated a partial (four) or complete (one) response to this treatment program. CONCLUSION: Single-agent ifosfamide has modest but unequivocal activity in platinum-resistant ovarian cancer. Further studies of this drug used as a front-line agent along with an organoplatinum compound or as part of a dose-intensification program with bone marrow, peripheral stem cell, or colony-stimulating factor support are indicated. In addition, single-agent ifosfamide is a reasonable standard second-line treatment strategy in appropriately selected patients with platinum-refractory ovarian cancer. PMID- 1732426 TI - Treatment of children with progressive or recurrent brain tumors with carboplatin or iproplatin: a Pediatric Oncology Group randomized phase II study. AB - PURPOSE: The Pediatric Oncology Group (POG) conducted a randomized phase II study to evaluate the activity of carboplatin and iproplatin in children with progressive or recurrent brain tumors. PATIENTS AND METHODS: The study was designed to evaluate the activity of these agents and to compare the toxicities associated with their use. Treatment consisted of carboplatin 560 mg/m2 at 4-week intervals or iproplatin 270 mg/m2 at 3-week intervals. RESULTS: The major toxicity observed was myelosuppression, particularly thrombocytopenia, for both agents. Ototoxicity (grade 1 or 2) was seen in 2.5% of patients treated with carboplatin and 1.3% of patients treated with iproplatin. The majority of patients with low-grade astrocytic neoplasms treated with carboplatin (nine of 12 patients) or iproplatin (eight of 12 patients) demonstrated tumor response or prolonged stable disease that persisted off-therapy. The duration of stable disease produced by carboplatin was particularly striking, ranging from 2 months to 68 + months (median, 40 + months). Neither drug demonstrated appreciable activity in the treatment of medulloblastoma (two of 26 responses to carboplatin, one of 14 responses to iproplatin), ependymoma (two of 17 responses to carboplatin, none of seven responses to iproplatin), high-grade glioma (two of 19 responses to carboplatin, one of 14 responses to iproplatin), or brain-stem tumors (one of 23 responses to carboplatin, none of 14 responses to iproplatin). CONCLUSION: Carboplatin is active against low-grade gliomas. Further evaluation of the role of carboplatin in the preirradiation treatment of children with low grade gliomas of the optic pathway is currently underway in a clinical trial. PMID- 1732427 TI - A phase III randomized study comparing cisplatin and fluorouracil as single agents and in combination for advanced squamous cell carcinoma of the head and neck. AB - PURPOSE: To determine whether combination chemotherapy is superior to single agents for recurrent/metastatic head and neck cancer, we compared the efficacy and toxicity of cisplatin (CP) and fluorouracil (5-FU), alone and in combination in a phase III trial. PATIENTS AND METHODS: Two hundred forty-nine patients with recurrent head and neck cancer were randomized to one of three treatments: CP (100 mg/m2) and 5-FU (1 g/m2 x 4), CP, or 5-FU every 3 weeks. RESULTS: The overall response rate to the combination (32%) was superior to that of CP (17%) or 5-FU (13%) (P = .035). Response was associated with good performance status (PS) but not with primary site, site of recurrence, histology, prior irradiation, or relative dose intensity. Median time to progression was less than 2.5 months, and there was no significant difference in median survival (5.7 months) among the groups. By multivariate analysis, patients with better PS and poorly differentiated tumors had superior survival. Hematologic toxicity and alopecia were worse in the combination arm. CONCLUSION: Although the response rate to the combination of CP plus 5-FU was superior to that achieved with single agents, survival did not improve. PMID- 1732428 TI - Interferon alfa-2a and fluorouracil in the treatment of patients with advanced esophageal cancer. AB - PURPOSE: The trial was undertaken to determine the response rate to and toxicities from the combination of interferon alfa-2a (IFN) and fluorouracil (FU) in patients with advanced esophageal cancer. MATERIALS AND METHODS: In this prospective phase II trial conducted at a large tertiary referral cancer center and university hospital, 40 patients with advanced locoregional, metastatic epidermoid, or adenocarcinoma of the esophagus were given FU 750 mg/m2 by 24-hour continuous intravenous infusion days 1 to 5, followed by weekly outpatient bolus FU 750 mg/m2 and IFN 9 x 10(6) U three times per week from day 1. Dose was attenuated for fatigue, neurotoxicity, gastrointestinal toxicity, and myelosuppression. RESULTS: Complete and partial responses were seen in 10 of 37 assessable patients with esophageal cancer (27%; 95% confidence interval, 0.13 to 0.41). Although substantial, toxicity was tolerable and primarily involved fatigue and mild myelosuppression. The median duration of response was 6.4 months (range, 2.8 to 14+ months). CONCLUSION: The combination of IFN and FU is an active regimen in the treatment of advanced esophageal cancer with a response rate similar to that reported for cisplatin-containing combinations in similar patient populations. Further studies based on this combination are indicated. PMID- 1732429 TI - A randomized phase II trial of continuous infusion interleukin-2 or bolus injection interleukin-2 plus lymphokine-activated killer cells for advanced renal cell carcinoma. AB - PURPOSE: Since 1985, multiple centers have demonstrated that interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells produce durable anticancer responses in patients with metastatic renal cell carcinoma. High-dose recombinant IL-2 (rIL 2) has been administered by intravenous bolus injection (Rosenberg SA, et al: N Engl J Med 313:1485-1492, 1985) and by continuous intravenous infusion (West WH, et al: N Engl J Med 316:898-905, 1987) combined with lymphokine-activated killer (LAK) cells, with both methods producing responses in patients with advanced renal cell carcinoma. The Extramural IL-2/LAK Working Group has conducted a randomized phase II trial of two intravenous high-dose rIL-2 regimens (bolus three times daily or 24-hour continuous infusion) to determine if either one manifests greater anticancer activity or a more acceptable toxicity profile. PATIENTS AND METHODS: Ninety-four patients with measurable advanced renal cell carcinoma were enrolled on this study: 46 to the bolus injection arm and 48 to the continuous infusion arm. On both arms, patients underwent a priming phase of rIL-2 administration, four daily lymphocytaphereses to harvest mononuclear cells that were placed in 3- to 4-day culture for generation of LAK cells, and an rIL 2/LAK coadministration phase. Patients were then observed monthly for evidence of response to this therapy and were offered up to two additional courses of treatment every 3 months if evidence of response was detected. RESULTS: Twenty percent of patients on the bolus injection arm experienced objective responses (three complete responses and six partial responses); 15% of patients on the continuous infusion arm responded (two complete responses and five partial responses). Complete responses were durable, persisting for 310+ to 700+ days. The incidence of severe life-threatening toxicities typical of high-dose rIL-2 therapy was similar in both arms (eg, patients with hypotension requiring pressors: bolus 71%, continuous 63%; oliguria less than or equal to 200 mL/8 hours: bolus 65%, continuous 71%). More episodes of fever, infection, and serum alkaline phosphatase elevation were associated with the continuous infusion arm, while more thrombocytopenia occurred on the bolus injection arm. Four patients (three bolus injection, one continuous infusion) died of respiratory and circulatory failure while under treatment. No clinical or laboratory parameter accompanying treatment on either arm was, by univariate or multivariate analysis, associated with an increased likelihood of response. CONCLUSIONS: Both methods of high-dose rIL-2/LAK cell administration produce nearly equivalent anticancer activity and toxicity in the treatment of renal cell carcinoma. The ability to predict responding patients based on patient or treatment characteristics is not possible. PMID- 1732430 TI - Tamoxifen and the isomers of 4-hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients. AB - PURPOSE: The antiestrogen tamoxifen is effective in therapy for breast cancer. However, its use is limited by the eventual development of acquired tamoxifen resistance in many patients. The mechanisms responsible for tamoxifen resistance remain unknown; loss of estrogen receptor (ER), selection of hormone-independent breast cancer clones, or alterations in serum tamoxifen levels after long-term use do not explain acquired resistance in most patients. Using an experimental model in which human breast cancer cells develop resistance in athymic mice treated with tamoxifen, we have recently shown that acquired resistance is associated with markedly reduced cellular concentrations of tamoxifen and by isomerization of the trans-4-hydroxy metabolite to the less potent cis isomer. MATERIALS AND METHODS: Using a sensitive high-performance liquid chromatography (HPLC) assay, we have now measured levels of tamoxifen and its major metabolites in a series of 14 tumors from patients treated with tamoxifen. The duration of therapy ranged from 1 month to 6 years. RESULTS: Tumor tamoxifen levels varied over a wide range. Low concentrations were observed in tumors from eight patients, all demonstrating progressive disease at the time of biopsy after a minimum duration of treatment of 6 months. Six tumors had moderate to high tamoxifen levels, two from patients responding to tamoxifen, one from a patient with stable disease, and three from patients with disease progression. Both the cis and trans isomers of the potent antiestrogenic metabolite 4-hydroxy-tamoxifen were detected in 11 tumors. Six tumors had high ratios of the cis to trans isomer (1.10:2.06), all from patients not responding to tamoxifen. The five tumors with low cis:trans ratios included the two tumors from responding patients and three from patients with progression. All but one of the 11 nonresponding patients had either a low tumor tamoxifen level, a high cis:trans ratio, or both. CONCLUSION: This study clearly demonstrates a wide range of tumor tamoxifen levels and accumulation of the less antiestrogenic cis isomer of 4-hydroxytamoxifen in some patients on tamoxifen therapy. Additional study is necessary to determine if these metabolic profiles are related to the development of tamoxifen resistance. PMID- 1732431 TI - Increased teniposide clearance with concomitant anticonvulsant therapy. AB - PURPOSE: A possible pharmacokinetic interaction between teniposide and anticonvulsant medications was evaluated in pediatric patients. PATIENTS AND METHODS: The systemic clearance of teniposide was determined in six pediatric patients with acute lymphocytic leukemia receiving concomitant therapy with anticonvulsants. Clearance was then compared with a control group of patients treated with the same protocol therapy and matched for age at diagnosis, sex, and race but not receiving anticonvulsants or other agents known to induce hepatic metabolism or alter protein binding of drugs. Eight blood samples were obtained during and after 4-hour infusions of teniposide, and plasma concentrations were measured by a specific high-performance liquid chromatography (HPLC) assay. A two compartment model was fitted to each subject's data. RESULTS: The mean systemic clearance (range) for the six anticonvulsant-treated patients studied during 22 courses of therapy was 32 mL/min/m2 (range, 21 to 54 mL/min/m2), significantly higher (P less than .001) than the mean value of 13 mL/min/m2 (range, 7 to 17 mL/min/m2) for the control patients studied during 26 courses of therapy. Clearance estimates for control patients were similar to previously published values for pediatric patients. CONCLUSION: These data indicate that the systemic clearance of teniposide is consistently increased two- to three-fold by concomitant phenobarbital or phenytoin therapy. The consequent substantial reduction in systemic exposure may reduce teniposide's efficacy. PMID- 1732432 TI - Risk assessment in cancer patients with fever and neutropenia: a prospective, two center validation of a prediction rule. AB - PURPOSE: The study was undertaken to validate a clinical model for predicting the medical risk of cancer patients with fever and neutropenia. PATIENTS AND METHODS: A consecutive sample of 444 cancer patients with fever and neutropenia (granulocyte count less than 500/microL) at two hospitals, a specialized cancer referral center and a university-affiliated general medical hospital, was studied to identify clinical characteristics in the first 24 hours that predict subsequent serious medical complications during the hospital stay. To control for bias, major risk factors and complications were subject to blinded review. RESULTS: Serious medical complications occurred in 34% of patients with risk factors identified in a prior study, including prior inpatient status (group I), outpatients with a serious independent comorbidity (group II), or uncontrolled cancer (group III), compared with 5% of the remaining patients (group IV) (P less than .000001). Two of the complications in group IV patients were transient asymptomatic hypotension, and the remaining three complications occurred after at least 1 week of progressive medical deterioration. These risk groups were independently significant in stepwise logistic regression analysis. Multiple complications (17%) and death (10%) were common among patients in groups I through III but did not occur in group IV patients. CONCLUSIONS: This risk assessment model accurately stratified the medical risk of these patients using only clinical information available on the first day of their course. Low-risk patients are an appropriate population in which to study less intensive treatment strategies. PMID- 1732433 TI - A population-based study of neuroblastoma incidence, survival, and mortality in North America. AB - PURPOSE: The purpose of this study was twofold: (1) to provide a population-based estimate of neuroblastoma incidence, disease stage and age distribution, and survival and mortality rates in North America; and (2) to compare these figures in the province of Quebec at a time shortly before the institution of province wide screening with those in a population-based control group, the Greater Delaware Valley (GDV) Pediatric Tumor Registry. MATERIALS AND METHODS: In Quebec, the four major pediatric teaching hospital records were searched for children with a diagnosis of neuroblastoma. Tumor board registry data and information supplied to the Division of Vital Statistics were also reviewed. Birth statistics were obtained from the population registry. The GDV Pediatric Tumor Registry is a population-based registry of pediatric cancer covering all of Delaware and parts of New Jersey, Pennsylvania, and Maryland. Age, stage of disease, and follow-up data were obtained through December 31, 1989, with Evans neuroblastoma staging data used for all comparisons. RESULTS: One hundred thirty children with neuroblastoma were identified in Quebec and 165 in the GDV, in a combined population of 3,178,736 children. The annual incidence of neuroblastoma was 10.95/10(6) under the age of 15 years and 27.75/10(6) between the ages of 0 and 4 years. The annual mortality rate due to neuroblastoma was 4.89/10(6) and 9.10/10(6) for the age groups 0 to 14 and 0 to 4, respectively. The overall 10 year survival rate for the 295 cases of neuroblastoma was 55%. The 10-year survival rates for patients with Evans stage I-IV and IVS disease were 88%, 90%, 63%, 21%, and 81%. There was no significant difference observed in the incidence, mortality, or survival in the two populations. CONCLUSION: These data represent the first large, population-based description of the clinical presentation and outcome of patients with neuroblastoma in North America, with no significant differences noted between Quebec patients and the GDV patients. PMID- 1732434 TI - Telephone counseling improves adherence to colposcopy among lower-income minority women. AB - PURPOSE: A randomized trial was conducted to evaluate the impact of a telephone counseling intervention to improve patient adherence to colposcopic examination for suspected cervical intraepithelial neoplasia (CIN). METHODS: Subjects were lower-income, minority women who missed a scheduled initial appointment for colposcopy at an urban medical clinic. Patients were randomly assigned to either a control condition (n = 42) or a telephone counseling condition (n = 48). The 15 minute, structured telephone counseling intervention protocol addressed educational, psychosocial, and practical barriers to colposcopy adherence. RESULTS: The most common patient-reported barriers to colposcopy adherence included a lack of understanding of the purpose of colposcopy (50%), worry about or fear of cancer (25%), and forgetting (23%). Telephone counseling was found to be highly effective in addressing these barriers and improving adherence to diagnostic follow-up and treatment. Of patients in the control condition, 43% complied with a rescheduled colposcopy appointment, compared with 67% in the telephone counseling condition. Logistic regression analysis indicated that the effect of telephone counseling was independent of sociodemographic confounder variables (odds ratio = 2.6; P less than .003). Additionally, 74% of patients who received the initial telephone counseling adhered to recommended treatment, compared with 53% of patients in the control condition. CONCLUSION: Brief, structured telephone contact may be a cost-effective mechanism for improving adherence to diagnostic follow-up and treatment for a variety of cancer screening tests. PMID- 1732435 TI - Neoadjuvant chemotherapy in head and neck cancer: no way to preserve a larynx. PMID- 1732436 TI - American Joint Committee on Cancer Classification for Melanoma. PMID- 1732437 TI - Burnout syndrome among oncologists. PMID- 1732438 TI - The scintigraphic appearance of Alzheimer's disease: a prospective study using technetium-99m-HMPAO SPECT. AB - Alzheimer's disease produces regional abnormalities in brain blood flow and metabolism that may result in recognizable scintigraphic patterns. We determined the predictive value of 99mTc-HMPAO SPECT for the presence of Alzheimer's disease based on a prospective study of 132 consecutive patients coming to our nuclear medicine clinical unit for evaluation of their memory loss or cognitive abnormalities. During clinical follow-up averaging 10.1 mo, a final diagnosis was established in 113 patients, 52 of which had Alzheimer's disease. The probability of Alzheimer's disease was determined for seven scintigraphic patterns. The probability was 19% that patients with memory loss and normal perfusion had Alzheimer's disease. For abnormal perfusion patterns, the probability of Alzheimer's disease was 82% with bilateral temporoparietal defects, 77% with bilateral temporoparietal defects with additional defects, 57% with unilateral temporoparietal defects, 43% with frontal defects only, 18% with other large defects and 0% with multiple small cortical defects. We conclude that for 99mTc HMPAO SPECT the predictive value of bilateral temporoparietal defects for Alzheimer's disease is high, while the perfusion patterns of unilateral temporoparietal perfusion defects and frontal defects only, which occur in 20% of patients with Alzheimer's disease, are not predictive of that disease. PMID- 1732440 TI - Carbon-11-putrescine: back to the drawing board. PMID- 1732439 TI - Is [1-11C]putrescine useful as a brain tumor marker? AB - Our experience with 11C-putrescine underscores the difficulty of finding a selective brain tumor tracer, uniquely incorporated by neoplastic glia or metastatic cells within brain, but not by the proliferating, nontransformed cells which constitute a normal pathophysiological reaction to various disease processes. Thirty-three patients with 36 lesions were studied with 11C-putrescine to determine the specificity of labeled putrescine for tumor tissue. The uptake of 11C-putrescine was correlated with local cerebral glucose metabolic rate in various lesions, including different types of tumors, to assess the relationship between 11C-putrescine uptake and tumor biology. Carbon-11-putrescine uptake was similar in malignant tumor and benign, non-neoplastic lesions with blood-brain barrier breakdown, illustrating the lack of tumor specificity of this tracer. Carbon-11-putrescine was not well incorporated into poorly enhancing lesions, regardless of their pathology, emphasizing the requirement of a disrupted blood brain barrier for 11C-putrescine uptake. The ratio of 11C concentration within lesions, compared to that in a region of interest in the contralateral brain, weakly correlated with an analogous ratio for local cerebral glucose metabolic rate in various lesions. Physiological processes not unique to tumors are associated with polyamine active transport and metabolism and contribute to the lack of tumor specificity of 11C-putrescine. Carbon-11-putrescine appear to have less diagnostic utility than 18FDG in brain tumors. The potential of 11C putrescine for evaluating the effect of antineoplastic therapy and providing prognostic information on brain tumors remains to be investigated. PMID- 1732441 TI - Prognostic significance of late imaging results in technetium-99m-labeled red blood cell gastrointestinal bleeding studies with early negative images. AB - The prognostic significance of the results of late imaging in patients with early negative 99mTc-labeled red blood cell (RBC) gastrointestinal (GI) bleeding studies was examined in a retrospective review of studies performed on 48 patients. Twenty-two studies showed intraluminal accumulation of labeled RBCs only on late images acquired from 3-24 hr following RBC injection. Patients with late positive studies had larger transfusion requirements than those with negative late images (mean total units transfused: 7.3 versus 3.5 (p less than 0.05); mean units transfused following scan commencement: 4.5 versus 2.0 (p less than 0.005)). Patients with late positive studies more frequently required angiography (3/22 versus 0/26) and surgery (5/22 versus 2/26). Sites of bleeding were more commonly identified in the stomach or small bowel in patients with late positive studies, while colon bleeding sources were more commonly found in those with late negative studies. The location of intraluminal blood on late images did not reliably discriminate upper from lower tract hemorrhage. In patients with early negative GI bleeding studies, results of later imaging provide objective evidence of the presence or absence of continued intermittent hemorrhage, and suggest both the region of bowel responsible and the relative risk for requiring further invasive procedures. PMID- 1732442 TI - Detection of abnormal cardiac adrenergic neuron activity in adriamycin-induced cardiomyopathy with iodine-125-metaiodobenzylguanidine. AB - Radiolabeled metaiodobenzylguanidine (MIBG), an analog of norepinephrine (NE), serves as an index of adrenergic neuron integrity and function. Using a rat model of adriamycin-induced cardiomyopathy, we tested the hypothesis that abnormal cardiac adrenergic neuron activity may appear and be exacerbated dose-dependently in adriamycin cardiomyopathy. The degree of vacuolar degeneration of myocardial cells was analyzed in relation to the duration of adriamycin treatment (2 mg/kg, once a week). There were no abnormalities or only isolated degeneration in the 1- or 2-wk treatment groups, isolated or scattered degeneration in half of the 3-wk group, frequent scattered degeneration in the 4-wk group, scattered or focal degeneration in the 5-wk group, and extensive degeneration in the 8-wk group. Myocardial accumulation of [125I]MIBG 4 hr after intravenous injection did not differ between the controls and the groups treated 3 wk or less. However, the 4 wk group had a slightly lower accumulation in the right ventricular wall (82% of the control) and significantly lower accumulation in the left ventricular wall (about 66% of the control: p less than 0.05). In the 5-wk group, MIBG accumulation in the right and left ventricular wall was 35% and 27% of that in controls, respectively (p less than 0.001). In the 8-wk group, MIBG accumulation in the right and left ventricular wall was 18% and 14% of that in controls, respectively (p less than 0.001). Thus, MIBG accumulation in the myocardium decreased in an adriamycin dose-dependent manner. The appearance of impaired cardiac adrenergic neuron activity in the presence of slight myocardial impairment (scattered or focal vacuolar degeneration) indicates that MIBG scintigraphy may be a useful method for detection of adriamycin-induced cardiomyopathy. PMID- 1732443 TI - Adriamycin, congestive cardiomyopathy, and metaiodobenzylguanidine. PMID- 1732444 TI - Radioimmunolocalization of neuroblastoma xenografts with chimeric antibody chCE7. AB - This study was performed to evaluate the tumor targeting ability of chCE7 with a view to clinical applications in neuroblastoma imaging and therapy. A chimeric (mouse/human) monoclonal antibody (chCE7) of gamma 1/kappa isotype directed against a neuroblastoma-associated cell-surface glycoprotein is described. In vitro chCE7 binds with high affinity (KD approximately 1 x 10(-10) M) to SKN-AS human neuroblastoma cells. Binding studies with 125I-labeled chCE7 show temperature-dependent modulation of antigen binding and indicate that a proportion of the bound antibody is internalized due to rapid antigen turnover. In vivo biodistribution of radioiodinated chCE7 in nude mice bearing SKN-AS tumors shows optimal tumor uptake after 24 hr with about 30% of the injected dose per g. Optimal tumor/blood ratios (3.4:1) are reached after 4-5 days. Uptake in other organs including the reticuloendothelial system is low with tumor/organ ratios of 10 and more. Tumor uptake of chCE7 and the parent murine CE7 are found to be similar. Stability of chCE7 during and after radiolabeling is good with no loss of immunoreactivity in preparations labeled with 123I up to 100 mCi/mg and 80% immunoreactivity after labeling with 13 mCi/mg of 131I. Neuroblastoma xenografts can be imaged by radioimmunoscintigraphy with 123I- and and 131I labeled chCE7. PMID- 1732445 TI - Contrast material iodides: potential effects on radioactive iodine thyroid uptake. AB - The levels of contaminant, free inorganic iodide and iodine were determined in several commonly used ionic and nonionic intravenous contrast media to gain a better understanding of the roles of these compounds in radioactive iodine uptake inhibition. The method, which involved a reduction-oxidation reaction using sodium nitrite, yielded accurate and precise data for the iothalomate based ionic contrast media as well as the nonionic contrast media. There was no free iodine in any of the contrast media tested. There was considerable variation in free iodide levels, ranging from 1.38 microgram/ml to 20.84 microgram/ml among the different contrast media, although significant differences between the ionic and nonionic media were not found. These levels of contaminant iodide are thought to play a role in the short-term inhibition of radioactive iodine uptake. PMID- 1732446 TI - Ratio of hepatic arterial-to-portal venous blood flow--validation of radionuclide techniques in an animal model. AB - The ratio of hepatic arterial-to-portal venous blood flow can be determined from the analysis of a first-pass bolus through the liver by a number of techniques. This study examines the validity of four radiotracer techniques in an animal model. Thirty-four flow studies (3 mCi 99mTc-DTPA/study) were performed in seven anesthetized pigs. Images were acquired for 200 sec and time-activity curves were generated from lung, liver and kidney ROIs. These curves were analyzed using a slope-based (HPI), a height-based (mHAR) and two deconvolution-based methods employing exponential or gamma variate fits. There was an excellent correlation (r greater than 0.9) between results obtained with flow probes and the radiotracer techniques, with the exception of the HPI technique (r = 0.75). The mHAR and deconvolution techniques were inaccurate at very low and high arterial flows, due respectively to noise limitations and hemodynamic instability in the animal. Nevertheless, these techniques appear to be the most promising for routine clinical use. PMID- 1732448 TI - The current role of SPECT in imaging subdural hematoma. PMID- 1732447 TI - Cerebral blood flow imaging with technetium-99m-HMPAO SPECT in a patient with chronic subdural hematoma: relationship with neuropsychological test. AB - We report the relationship between cerebral blood flow (CBF) and neuropsychologic tests in a patient with a chronic subdural hematoma suffering from severe dementia and left hemiparesis. Regional CBF was quantified using 99mTc-HMPAO SPECT and 133Xe-CBF. CBF-SPECT could detect the hematoma which was isodense by CT scan and the neuropsychological test improved remarkably with the increase in CBF after surgery. We conclude that if there is a strong clinical suspicion of subdural hematoma and CT scan is not diagnostic then CBF-SPECT may be valuable in localizing the hematoma and monitoring the effect of operation. PMID- 1732449 TI - Spread of infectious complications of odontogenic abscess detected by technetium 99m-HMPAO-labeled WBC scan of occult sepsis in the intensive care unit. AB - We report a rare case of odontogenic abscess, detected while the patient was in the intensive care unit (ICU), which resulted in sepsis and the patient's death due to mediastinitis, skull osteomyelitis, and deep neck cellulitis. The detection of infectious focus in occult sepsis in ICUs is usually difficult because many diagnostic procedures cannot be conveniently performed. The use of 99mTc-hexamethylpropyleneamineoxime-labeled white blood cells scan allowed accurate diagnosis and appropriate surgical drainage. PMID- 1732450 TI - False-negative morphine-augmented cholescintigraphy: a case of subacute gallbladder perforation. AB - The gallbladder and an infected pericholecystic biloma secondary to subacute perforation were visualized during morphine-augmented cholescintigraphy. Perforation of the gallbladder may relieve cystic duct obstruction and contribute to false-negative visualization in the setting of acute cholecystitis. PMID- 1732451 TI - Large focal defect on liver/spleen scan caused by fatty liver and masquerading as neoplasm. AB - Focal fatty infiltration of the liver may be mistaken for metastatic disease, primary tumor or other space-occupying lesions on CT or ultrasound. Usually, a 99mTc-sulfur colloid scan is sensitive in documenting the presence of Kupffer's cell in such a process. We present a case that was suggestive of focal fatty infiltrate on a CT scan, nondiagnostic on ultrasound, and seen as a large focal defect on the 99mTc-sulfur colloid liver/spleen scan. A 133Xe inhalation study, however, did show uptake in the area of fatty infiltration. A needle biopsy confirmed the diagnosis. PMID- 1732452 TI - Embolization of hepatic arteriovenous malformations using radiolabeled and nonradiolabeled polyvinyl alcohol sponge in a patient with hereditary hemorrhagic telangiectasia: case report. AB - Polyvinyl alcohol sponge (PVA) radiolabeled with 99mTc-sulfur colloid was used to evaluate a large hepatic arteriovenous malformation (AVM) in a 71-yr-old white female prior to embolization. The patient had hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu) with severe left-to-right shunting through the hepatic AVM which resulted in high-output congestive heart failure. The patient also had severe pulmonary hypertension. Scintigraphic imaging of the embolized radiolabeled PVA particles allowed us to be certain that the particles did not flow through the liver and inadvertently embolize the lungs; with the patient's already poor pulmonary status, embolization could have been fatal. PMID- 1732453 TI - Segmental branch renal artery stenosis diagnosed with captopril renography. AB - There have been only a few reports of post-Captopril renography diagnosing segmental renal arterial stenosis. These previous case reports have involved an accessory renal artery when multiple renal arteries were present. This case report describes Captopril-induced segmental dysfunction due to an intrarenal branch stenosis of a single renal artery. PMID- 1732454 TI - Osteoid osteoma: the role of radionuclide bone imaging, conventional radiography and computed tomography in its management. PMID- 1732455 TI - Application of artificial neural network to computer-aided diagnosis of coronary artery disease in myocardial SPECT bull's-eye images. AB - We have developed a computerized system that can aid in the radiologist's diagnosis in the detection and classification of coronary artery diseases. The technique employs a neural network to analyze 201Tl myocardial SPECT bull's-eye images. This multi-layer feed-forward neural network with a backpropagation algorithm has 256 input units (pattern: compressed 16 x 16-matrix images), 5-140 units in a single hidden layer, and eight output units (diagnosis: one normal and seven different types of abnormalities). The neural network was taught using pairs of training (learning) input data (bull's-eye "EXTENT" image) and desired output data ("correct" diagnosis). The effects of the numbers of hidden units and learning iterations in the network on the recognition performance were examined. In our initial stage, the results show that the recognition performance of the neural network is better than that of the radiology resident but worse than that of the experienced radiologist. Our study also demonstrates that the result produced in the neural network depends on the variety of the training examples used. The preliminary study suggests that the neural network approach is useful for the computer-aided diagnosis of coronary artery diseases in myocardial SPECT bull's-eye images. PMID- 1732456 TI - Absorbed dose calculations for rapidly growing tumors. AB - One of the most promising areas for cancer therapy with administered radiopharmaceuticals is the treatment of very small tumors and micrometastases. Small tumors and micrometastases, however, may be rapidly growing at the time of treatment, resulting in a substantial change in mass during the period of irradiation. In this work, the formalism required to calculate the average absorbed dose to rapidly growing tumors is developed and applied to an in vitro tumor model. Further application to in vivo human myeloma tumors reveals that tumor growth may have a significant effect on the average dose delivered to the tumor from incorporated radionuclides. These considerations may assist in establishing dose-response relationships necessary for radiopharmaceutical cancer therapy. PMID- 1732458 TI - Don't confuse me with ethics: I already know what's right. PMID- 1732457 TI - Spatial low frequency pattern analysis in positron emission tomography: a study between normals and schizophrenics. AB - Using the two-dimensional Fourier transform and the brain's centroidal principal axis, a method is developed for the analysis of PET metabolic brain images without the use of predefined anatomic regions of interest. We applied the method to images from a group of 11 normal and 12 medicated schizophrenics tested under resting conditions and under a visual task. A cortical/subcortical spatial pattern was found to be significant in two directions; anterior/posterior and chiasmatic (left-anterior/right-posterior). The best individual clinical classification (Jackknife classification) occurred under visual task at two axial brain levels: at the basal ganglia with correct classification rates of 91% and 84%, while the cerebellum had rates of 82% and 92%. These high classification rates were obtained using only the four coefficients of the lowest spatial frequency. These results point to the generalized brain dysfunction of regional glucose metabolism in chronic medicated schizophrenics both at rest and at a visual image-tracking task. PMID- 1732459 TI - Thyroid uptake neck phantoms are not created equal. AB - The main purpose of this communication is to alert nuclear medicine departments to the fact that the earlier version of the water phantom grossly overestimates soft-tissue attenuation in the neck, resulting in calculated thyroid uptake values which are significantly overestimated (in hyperthyroid patients we noted uptake values approaching or exceeding 100%). We believe that the solid Lucite phantom (which is the one recommended by IAEA) better approximates the human neck soft tissue overlying the thyroid. Institutions that continue to use the water phantom should be aware that their thyroid uptakes will be relatively elevated and the normal range must be shifted accordingly. Our normal range is 10%-30% uptake of 123I at 24 hr for the Lucite phantom. For the water phantom, the estimated normal range would be 15%-45%. In addition, the phantom type should be considered when comparing uptake results with those from another institution for a particular patient. Also, treatment doses for Grave's disease could be significantly affected, if such doses are calculated by a formula that depends on uptake. PMID- 1732460 TI - Evaluation of heparin and anticoagulant citrate dextrose in the preparation of technetium-99m-red blood cells with UltraTag RBC kit. PMID- 1732461 TI - Correlation of chemotherapy-induced kidney disorder and antimyosin antibody uptake in kidneys. PMID- 1732462 TI - The scintigraphic appearance of Alzheimer's disease: a prospective study using technetium-99m-HMPAO SPECT. PMID- 1732463 TI - Ethanol-fed Sprague-Dawley rats maintain normal levels of insulin-like growth factor I. AB - Ethanol-fed rats do not gain weight as fast as their isoenergetically pair-fed controls; the reasons for this slower rate of growth remain uncertain. Insulin like growth factor I (IGF-I) is a major component of the growth-promoting endocrine system. To determine whether ethanol impairs growth by interfering with this component of the endocrine system, rats were pair-fed ethanol-containing and control liquid diets. When rats were meal-fed on the day before the experiment there were no differences in serum IGF-I concentrations or hepatic IGF-I mRNA levels between ethanol-fed and control animals after 2, 3 or 4 wk. These results indicate that ethanol consumption per se does not interfere with IGF-I production and that energy derived from ethanol sustains this component of the growth promoting endocrine system as well as carbohydrate energy. The schedule used to administer the diets, however, did have a significant effect on hepatic IGF-I mRNA levels. When after 4 wk of dietary treatment rats were not meal-fed but received their entire dietary ration in a single morning feeding on the day before the experiment, the ethanol-fed animals had significantly higher hepatic IGF-I mRNA levels than their pair-fed controls. This finding indicates that these animals are nutritionally dissimilar despite isoenergetic pair-feeding. PMID- 1732464 TI - Interaction of carnitine and propionate with pyruvate oxidation by hepatocytes from clofibrate-treated rats: importance of coenzyme A availability. AB - Propionate interferes with normal hepatic metabolic regulation secondary to accumulation of propionyl- and methylmalonyl-CoA. Clofibrate-treatment increases hepatic CoA content and carnitine acetyltransferase activity, both of which may modulate propionate toxicity. Therefore, inhibition of pyruvate oxidation by propionate was studied in hepatocytes isolated from rats maintained on a control or 0.5% clofibrate diet for 7-9 d. Propionate (10 mmol/L) inhibited 14CO2 formation from [1-14C]pyruvate (10 mmol/L) by 60 +/- 2% in hepatocytes from control rats, but by only 46 +/- 3% in cells from clofibrate-treated rats (P less than 0.05). The smaller inhibitory effect of propionate in hepatocytes from clofibrate-treated rats occurred despite increased cellular propionyl-CoA content as compared with controls, but was associated with increased CoASH and total CoA contents. Despite greater carnitine acetyltransferase activity (20-fold) and propionylcarnitine production (2.5-fold) in hepatocytes from clofibrate-treated rats, reversal of propionate's inhibition of pyruvate oxidation by 10 mmol/L carnitine was small (8.7 +/- 3.9%) and not different from that observed in cells from control animals (6.7 +/- 2.4%). Carnitine (10 mmol/L) decreased hepatocyte total acid-soluble CoA content by 20-30% in cells from both control and clofibrate-treated rats. This carnitine-induced decrease in CoA content may limit the efficacy of carnitine under conditions of acyl-CoA accumulation. Clofibrate induced increased CoA content provides partial protection against propionate toxicity. Metabolic toxicity of propionate is the result of both the increased cellular propionyl-CoA content and the depletion of cellular unesterified CoA. PMID- 1732465 TI - Changes in pig serum lipids, nutrient digestibility and sterol excretion during cecal infusion of propionate. AB - Dietary water-soluble fiber supplementation elicits hypolipidemic effects. Propionate, the 3-carbon short-chain fatty acid derived from colonic fiber fermentation, has previously exhibited inhibition of cholesterol synthesis in vitro and may contribute to serum lipid lowering. This study examines the effect of colonic propionate absorption on serum lipids, sterol excretion and nutrient digestibility. Nine barrows were surgically cannulated at the distal ileum and cecum. Pigs received for 16 d continuous cecal infusions of either propionate (36 mmol/kg 0.75 daily) or saline (control) in a crossover design. Propionate infusion did not lower serum lipids, but increased total serum cholesterol by 15% (P less than 0.05) and LDL cholesterol by 15% (P less than 0.05). Differences in sterol excretion, ileal and fecal nutrient digestibilities, and weight gain were not detected between infusion treatments. The results suggest that propionate absorption does not result in decreased serum lipids and is not responsible for the serum lipid-lowering effects of water-soluble fibers. PMID- 1732466 TI - Profile of short-chain fatty acids and rectal proliferation in rats fed sucrose or cornstarch diets. AB - Rats were fed high fat (231 g/kg diet), low calcium (1.3 g/kg diet), low cellulose (20 g/kg diet) diets in which carbohydrates were represented by sucrose or starch (460 g/kg diet). A subgroup of animals was treated with 1,2 dimethylhydrazine (DMH) twice, 4 and 8 d before the beginning of the dietary treatments. Animals fed the starch diet, compared with those fed the sucrose diet, had higher concentrations of cecal and fecal short-chain fatty acids and a significantly lower acetic acid:butyric acid ratio in the cecal contents at d 105. Ratios were 14.7 +/- 1.7 and 6.8 +/- 0.4 for rats fed the sucrose and starch diets, respectively (P less than 0.01). Cecal pH was significantly lower in animals fed the starch diet for 105 d. At d 105, rectal proliferation was lower in rats fed the starch diet (labeled cells/crypt were 7.89 +/- 0.56 and 3.57 +/- 0.40 for rats fed the sucrose and starch diets, respectively, P less than 0.01); at d 30 the effect of starch on proliferation was evident in controls but not in DMH-treated rats. Rectal proliferation data were negatively correlated with the concentration and percentage of butyric acid and positively correlated with the percentage of acetic acid, the acetic acid:butyric acid ratio and cecal pH. These results suggest that low rectal proliferation in animals fed a high fat, high starch diet might be associated with a lower relative concentration of butyric acid in the cecal contents. PMID- 1732467 TI - Evaluation of the preruminant calf as a model for the study of human carotenoid metabolism. AB - This study evaluated the preruminant calf as an animal model for the study of human carotenoid metabolism. Fifteen newborn male Holstein calves were fed a carotenoid-free milk replacer diet to maintain them in the preruminant state. After a 7-d adjustment period, three calves were killed and 12 calves received a single oral dose (20 mg) of beta-carotene in the form of water-soluble beadlets. Blood samples were collected periodically and samples of various tissues were collected when the calves were killed. Three animals each were killed by exsanguination at 1, 3, 6 and 11 d post-dosing. Serum beta-carotene concentrations peaked between 12 and 30 h post-dosing and declined slowly afterwards. Serum data were fitted to a two-compartment model and yielded an elimination constant (k2) that was similar to reported human values. Adrenal tissue showed significant concentrations of beta-carotene by 24 h post-dosing, and levels were still elevated at 264 h. Liver, spleen and lung beta-carotene concentrations were significantly elevated by 24 h and rapidly declined thereafter. Adipose and kidney peak beta-carotene concentrations were observed at 72 and 144 h, respectively. Heart and muscle did not display significant changes in beta-carotene concentrations. The preruminant calf shows promise as an animal model for the study of absorption and metabolism of carotenoids by humans. PMID- 1732468 TI - Rat colonic antioxidant status: interaction of dietary fats with 1,2 dimethylhydrazine challenge. AB - The polyunsaturated fatty acid composition of dietary fat has been associated with increased oxidant stress in tissues. We previously showed that antioxidant mechanisms in colon mucosa are affected by dietary fat. In this study, the colonic antioxidant response to treatment with the colon-specific carcinogen 1,2 dimethylhydrazine (DMH) was determined. Sprague-Dawley rats were fed one of four AIN-76A-based diets with varied fat content. The basal diet contained 5% corn oil, the menhaden oil diet contained 19% menhaden oil and 1% corn oil, the corn oil diet contained 20% corn oil and the beef tallow diet contained 19% beef tallow and 1% corn oil. Animals were given one or two intraperitoneal injections of either DMH (20 mg/kg) or 1 mmol/L EDTA. Homogenates of colon mucosa were assayed to determine activity of catalase, glutathione peroxidase, total superoxide dismutase, manganese superoxide dismutase, glutathione reductase and glutathione-S-transferase, as well as total glutathione concentration, thiobarbituric acid-reacting substances and nuclear aberrations. Total glutathione concentration was greater in rats fed all diets following two injections of DMH, whereas glutathione reductase activity remained unchanged relative to controls. Lipid peroxidation was highest in the group fed menhaden oil; nuclear aberrations were greatest in the corn oil-fed group. In conclusion, both type and amount of dietary fat modified the response of colon tissue to genotoxic challenge, but none of the diets seemed more protective than the others. PMID- 1732469 TI - Route of administration of tryptophan and tyrosine affects short-term food intake and plasma and brain amino acid concentrations in rats. AB - The effects of route of administration of tryptophan and tyrosine on food intake and diet selection and on plasma and brain tryptophan or tyrosine concentrations were studied. Tryptophan and tyrosine given intraperitoneally at 100 mg/kg body wt suppressed food intake by 33-45% over a 2-h feeding period beginning 30 min after injections. No preferential effect was shown for either the high carbohydrate or high protein diet choice. When given intragastrically at this dose, neither tryptophan nor tyrosine affected food intake. Tryptophan, but not tyrosine, at 200, 400 and 600 mg/kg body wt given intragastrically reduced food intake and carbohydrate diet intake by an average of 20 and 25%, respectively, in the first 2 h of feeding. Plasma and brain tryptophan were higher for 30 min following intraperitoneal tryptophan injections than after tryptophan given intragastrically at 100 mg/kg body wt. However, intraperitoneal tyrosine (100 mg/kg body wt) resulted in higher plasma tyrosine levels at 5-10 min but lower levels at 30 min than when tyrosine was given intragastrically. Brain tyrosine was higher after intraperitoneal treatment only at 10 min and was similar to intragastric treatment at other times. When these amino acids were given intragastrically at 400 mg/kg body wt, higher plasma tryptophan, plasma tyrosine and brain tyrosine were found than following intraperitoneal injection of the behaviorally effective dose (100 mg/kg body wt) at 30 min. Thus the reduced effect on food intake of tryptophan or tyrosine given intragastrically compared with intraperitoneally is not readily explained by their lower concentrations in plasma and brain preceding and at the time that the rats had access to food. PMID- 1732470 TI - Atherogenic diets enhance endotoxin-stimulated interleukin-1 and tumor necrosis factor gene expression in rabbit aortae. AB - Cytokine-induced vascular changes may contribute to the atherogenic process. We examined the effect of atherogenic diets on the aortic expression of genes for the cytokines interleukin-1 alpha and beta and tumor necrosis factor alpha. Male New Zealand White rabbits were fed one of the following diets for 10 wk: nonpurified diet, a semipurified diet with 10% corn oil, a semipurified diet with 1% corn oil and 9% partially hydrogenated coconut oil, or the 9% coconut oil diet supplemented with either 0.1, 0.3 or 0.9% added cholesterol (n = 6/group). At 10 wk, 3 rabbits per group received lipopolysaccharide (200 micrograms/kg) intravenously. After 1.5 h the rabbits were killed and their aortae removed and analyzed. Histologic examination showed that the 0.3% and 0.9% cholesterol-fed rabbits developed appreciable aortic lesions. Diet had no major effect on the basal levels of aortic cytokine mRNA as determined by polymerase chain reaction analysis of DNAs. However, aortic tissue from rabbits fed 0.3% and 0.9% cholesterol diets showed significantly enhanced lipopolysaccharide-evoked levels of mRNA encoding interleukin-1 alpha (392 +/- 91 for saturated fat vs. 759 +/- 191 and 800 +/- 120 fmoles/reaction for 0.3% and 0.9% cholesterol), interleukin-1 beta (99 +/- 10 vs. 353 +/- 80 and 355 +/- 86) and tumor necrosis factor alpha (195 +/- 15 vs. 594 +/- 78 and 667 +/- 97). Extracts of aortae from rabbits injected with lipopolysaccharide contained interleukin-1 and tumor necrosis factor alpha activity but differences in biological activity due to diet were not detectable at the 1.5 h time point (chosen for maximal mRNA expression). Increased local cytokine gene expression in response to acute stimulus might influence the evolution of the vascular response to diets rich in cholesterol and saturated fats. PMID- 1732471 TI - Dietary vitamin C intake and concentrations in the body fluids and cells of male smokers and nonsmokers. AB - Inhaled cigarette smoke releases a variety of oxidizing agents. Ascorbic acid is recognized as an important biological antioxidant. To better characterize the antioxidant protective role of ascorbic acid, a comparison of ascorbic acid concentrations in plasma, leukocytes, bronchoalveolar lavage fluid and alveolar macrophages from a homogeneous group of healthy male smokers (n = 10) and nonsmokers (n = 14) was investigated. The resulting ascorbic acid contents were (means +/- SD) 91 +/- 25 (n = 10) and 87 +/- 25 (n = 14) mumol/L in plasma, 2.09 +/- 0.62 (n = 7) and 2.12 +/- 0.77 (n = 11) mumol/10(9) cells in mononuclear leukocytes, 3.2 +/- 2.2 (n = 10) and 1.7 +/- 1.5 (n = 13) mumol/L in bronchoalveolar lavage fluid and 3.4 +/- 2.3 (n = 8) and 1.6 +/- 1.3 (n = 6) mumol/10(9) cells in alveolar macrophages from smokers and nonsmokers, respectively. Mean daily dietary vitamin C intake was 116 +/- 68 and 107 +/- 59 mg/d for smokers and nonsmokers, respectively. The ascorbic acid contents of bronchoalveolar lavage [3.9 +/- 1.9 mumol/L (n = 8)] and alveolar macrophages [4.1 +/- 2.1 mumol/10(9) cells (n = 6)] of smokers consuming 15 to 20 cigarettes/d were significantly higher (P less than 0.05) than those of nonsmokers. The increased content of ascorbic acid in bronchoalveolar lavage and in alveolar macrophages of smokers compared with nonsmokers may reflect a defensive mechanism against free radical species derived from cigarette smoke. PMID- 1732472 TI - Long-term wheat germ intake beneficially affects plasma lipids and lipoproteins in hypercholesterolemic human subjects. AB - In previous short-term studies in rats and humans, the ingestion of raw wheat germ lowered plasma triglycerides and cholesterol. Thus, the present study was designed to investigate the possible long-term effects of wheat germ intake. Diet supplementation with raw wheat germ or partially defatted wheat germ was tested in two separate groups of 10 and 9 free-living human subjects, respectively. They all exhibited hypercholesterolemia (6.14-9.67 mmol/L cholesterol) and 11 had hypertriglyceridemia. None was diabetic. Fasting blood samples were taken at the beginning of the study, after 4 wk of 20 g/d wheat germ intake, after 14 additional weeks of 30 g/d wheat germ intake and after 12 wk without any supplementation. Dietary records were kept for seven and three consecutive days, before and during the wheat germ intake periods, respectively. Raw wheat germ intake significantly decreased plasma cholesterol (-8.7%) and tended to reduce VLDL cholesterol (-19.6%) after 4 wk. After 14 additional weeks, plasma cholesterol (-7.2%) and LDL cholesterol (-15.4%) remained lower and plasma triglycerides (-11.3%) tended to be lower. The apo B:apo A1 ratio significantly decreased after both periods. Partially defatted wheat germ transiently decreased plasma triglycerides and cholesterol after a 4-wk intake. The present data indicate that wheat germ reduces cholesterolemia in the long term and could play a beneficial role in the dietary management of type IIa and IIb hyperlipidemia. PMID- 1732473 TI - Calcium carbonate depresses iron bioavailability in rats more than calcium sulfate or sodium carbonate. AB - Calcium carbonate supplements depress iron bioavailability when consumed with meals. Our objective was to determine whether this effect is due to the calcium, the carbonate or a combination of the two. A rat hemoglobin repletion assay and an in vitro digestion procedure were used to assess the effects of four salts (calcium carbonate, calcium sulfate, sodium carbonate and sodium sulfate) on iron bioavailability. The salts were added to purified rat diets (for the hemoglobin repletion study) or to iron-fortified infant formula (for the in vitro study) at three different levels. Calcium carbonate had the greatest depressive effect on iron bioavailability, depressing hemoglobin iron gain in a dose-related manner. Calcium sulfate and sodium carbonate also depressed hemoglobin iron gain but to a lesser extent and only in rats fed diets containing the highest level of these two salts. Sodium sulfate did not affect hemoglobin iron gain. Significant interactions between cation, anion and salt concentration were found, suggesting that both the cation and the anion in calcium carbonate contribute to the iron absorption-depressing action of this salt. Results from the in vitro experiments were similar to the in vivo results. PMID- 1732474 TI - A mixture of nucleosides and a nucleotide alters hepatic energy metabolism 24 hours after hepatectomy in rabbits. AB - The effect of administering a nucleoside-nucleotide mixture on hepatic energy metabolism was evaluated at 24 h after hepatectomy in rabbits that had had 70% of their livers removed. After hepatectomy, animals were administered continuous intravenous infusion of 2 mL/(kg body wt-h) of 9 g/L NaCl (Group S), 5.99 mmol/L nucleoside-nucleotide mixture (Group N1) or 11.98 mmol/L nucleoside-nucleotide mixture (Group N2). At 24 h after hepatectomy, the hepatic adenylate energy charge in Group S (0.83 +/- 0.01, mean +/- SEM) was significantly lower than that before hepatectomy (0.90 +/- 0.01). By contrast, the values in Groups N1 and N2 after hepatectomy (0.74 +/- 0.04 and 0.73 +/- 0.04, respectively) were significantly lower than that in Group S. The hepatic mitochondrial phosphorylation rate before hepatectomy was 46.40 +/- 4.88 nmol ATP/(mg mitochondrial protein-min). After hepatectomy, significantly greater values were observed in Groups N1 and N2 (69.53 +/- 7.20 and 63.31 +/- 6.11, respectively), yet those values were less than observed in Group S (109.14 +/- 4.80). These results suggest that the nucleoside-nucleotide mixture suppressed the enhancement of hepatic mitochondrial phosphorylative activity at a time when hepatic adenylate energy charge is compromised. Such enhancement is needed to compensate for the increased energy expenditure due to surgical intervention. PMID- 1732475 TI - Replacement of digestible wheat starch by resistant cornstarch alters splanchnic metabolism in rats. AB - Splanchnic metabolism was investigated in rats fed either a diet containing highly digestible wheat starch (DS diet) or amylase-resistant cornstarch (RS diet). In rats fed the latter diet, there was a considerable enlargement of the cecum and an increase in the production and absorption of volatile fatty acids (VFA), chiefly acetic and propionic acids. As a result, the major substrates absorbed from the digestive tract were glucose in rats fed the DS diet and both glucose and VFA in rats fed the RS diet. The liver removed about one-third of the absorbed glucose in rats fed the DS diet, whereas there was a slight release of glucose by the liver in rats fed the RS diet. Plasma insulin was higher in rats fed the DS diet, and there were smaller fluctuations of plasma insulin and liver glycogen between the fed and postabsorptive periods in rats adapted to the RS diet. In these animals, propionate was the major VFA taken up by the liver and approximately 50% of absorbed acetate was also removed by the liver. During the postabsorptive period, there was still a substantial contribution of VFA, especially propionate, to liver metabolism. A depressive effect of the RS diet on plasma triglycerides, cholesterol and free fatty acids was observed only during the postabsorptive period. Replacement of a large part of absorbed glucose by VFA apparently allows time for absorption of energy fuels to be extended and dampens the fluctuations of glucose metabolism during the light: dark cycle. PMID- 1732477 TI - Adipose tissue lipogenesis and fat deposition in leaner broiler chickens. AB - Rates of hepatic lipogenesis and secretion of plasma triglyceride-rich lipoproteins in 6- to 7-wk-old broiler chickens were similar to the overall rate of fat deposition in these birds, although approximately 20% of [14C]-labeled VLDL was oxidized to CO2 within 8 h. Only 6-7% of VLDL and portomicron triglyceride was taken up by the abdominal fat pad, but this proportion of total triglyceride flux could account for about 80-85% of the total fatty acids accumulating in that depot. The rate of lipogenesis in adipose tissue was much lower than that in the liver, but it could account for much of the remaining fatty acids. Lipogenesis from [14C]acetate in cultured chicken adipocytes was markedly inhibited by adding VLDL as an exogenous source of fatty acids. However, adipose tissue lipogenesis was not increased in vivo by reduction of plasma lipoprotein flux by genetic selection, by the feeding of a high protein diet or by immunological intervention. The results confirm that adipose tissue lipogenesis makes only a small contribution to adipose tissue growth in normal broilers. Its importance does not increase in response to the reductions in hepatic lipogenesis that accompany genetic or nutritional manipulation of body composition. PMID- 1732476 TI - Penicillamine: pharmacokinetics and differential effects on zinc and copper status in chicks. AB - The pharmacokinetics of D(-)-penicillamine (PA) and the effects of PA on the nutritional status of Cu and Zn in chicks were studied. When administered by subcutaneous or intravenous injection, PA was eliminated from the plasma with biphasic kinetics: i.e., a fast (halftime: 6-7 min) and a slow (halftime: 52-55 min) component. The elimination of PA from the liver or pancreas showed only the slow (halftime: 42-46 min) component. When PA was administered orally, plasma PA concentrations responded over time in bimodal pattern, but the maximal level was only one-fifth that achieved by injection. The subcutaneous injection of PA elicited differential, transient changes in the concentrations of Zn and Cu in the plasma: plasma Zn concentration was decreased, and plasma Cu concentration was increased. Analyses of urine collected from ureter-cannulated chicks showed that PA was rapidly excreted via that route, and that urinary PA excretion was associated with increased urinary excretion of both Zn and Cu. These results show that PA treatment can alter tissue distributions particularly of Zn and, to a lesser extent, of Cu. J. Nutr. PMID- 1732478 TI - Adrenalectomy attenuates the effect of chemical castration on energy balance in rats. AB - The individual and combined influences of castration and adrenalectomy on energy balance in rats were investigated in a 2 x 2 factorial design. Castration was chemically achieved by treating rats with Buserelin, a gonadotropin-releasing hormone (GnRH) agonist. During the treatment, the rats were weighed every 2 or 3 d, and the total amount of food consumed was determined. At the end of the 14-d treatment period, rats were killed and their carcasses were analyzed for protein, fat and energy content. In the sham-operated groups, body weight gain was greater in Buserelin-treated rats than in saline-infused animals. However, in the adrenalectomized rats, there was no difference in body weight gain between the Buserelin- and saline-infused animals. In sham-operated groups, body protein and body fat gains were significantly larger in Buserelinthan in saline-infused animals. In saline-infused groups, protein gain was greater in adrenalectomized than in sham-operated animals. On the other hand, in the Buserelin-infused rats, there was no difference in the protein gain between the adrenalectomized and sham operated rats. The adrenalectomized groups of rats ate less and deposited less fat and energy than their respective sham-operated counterparts. In the sham operated groups, both the intake and the gain of energy were larger in the Buserelin-treated than in saline-infused rats. The present study indicates that adrenalectomy can significantly attenuate the influence of castration on energy gain, providing evidence that adrenals and ovaries can interact in the regulation of energy balance. PMID- 1732479 TI - Amniotic fluid composition responds to changes in maternal dietary carbohydrate and is related to metabolic status in term fetal rats. AB - The objective of this study was twofold: 1) to determine whether amniotic fluid composition responded to differences in the level or source (glucose vs. fructose) of maternal dietary carbohydrate, and 2) to establish whether any dietary-induced changes in amniotic fluid composition correlated with maternal or fetal metabolic status at term. Pregnant rat dams were fed graded levels (0, 4, 12 and 60%) of glucose or fructose in a triglyceride-based diet (Experiment 1) or isoenergetic low carbohydrate diets having 4% glucose equivalents as glucose, fructose, or lipid-glycerol (Experiment 2) throughout pregnancy. Amniotic fluid and maternal and fetal samples were collected at term (d21). Results demonstrated a significant increase in amniotic fluid glucose and a significant decrease in amniotic fluid uric acid as the level of carbohydrate increased in the maternal diet. Pearson correlation coefficients showed amniotic fluid glucose to be positively associated with maternal and fetal liver glycogen and fetal weight; amniotic fluid uric acid and urea nitrogen were negatively correlated with these same measures. Regression analysis indicated that amniotic fluid glucose was predictive of fetal body weight and fetal liver glycogen at term. The findings show that amniotic fluid can be modified by maternal diet and suggest that composition of amniotic fluid might be used as an accessible nutritional indicator of carbohydrate status in the developing fetus. PMID- 1732481 TI - Implantology--1992: still more questions than answers. PMID- 1732480 TI - Metabolism of [14C]- and [32P]pyridoxal 5'-phosphate and [3H]pyridoxal administered intravenously to pigs and goats. AB - To gain more information about the kinetics of vitamin B-6 metabolism in vivo, the metabolism of tracer was examined after the simultaneous intravenous administration of [32P] and [14C]pyridoxal phosphate and [3H]pyridoxal in two 93 kg pigs and two 60-kg goats. In the pigs, [14C] removal was monophasic with T1/2 of 16 and 18 min and clearance of 165 and 248 mL/min. In the goats, [14C] removal was biphasic with T1/2 of 49 and 114 min for 0-30 min and 209 and 227 min for 0.5 6 h (clearance 20 and 17 mL/min). Uptake of pyridoxal phosphate by liver and resecretion into the plasma were too small to cause a detectable decrease in the [32P]:[14C] ratio. Pyridoxal removal from plasma was similar in both species, with a half-life of approximately 12 min from 0-30 min and approximately 50 min for 0.5-3 h. Clearance of [3H]pyridoxal in the four animals ranged from 412 to 2258 mL/min. Little [14C] entered the erythrocytes. The [3H] entered readily but was converted to pyridoxal phosphate faster in the pigs than in the goats. [14C] and [3H] were excreted as pyridoxic acid at the same rate. However, during the 54 h after injection the goats excreted approximately 60% of the [14C] doses in the urine compared with approximately 30% in the pigs. About 5-10% of the [14C] and [3H] doses were recovered in goat milk over 54 h. Pyridoxal kinase activity was higher in lactating mammary tissue than in liver, kidney or muscle of goats. PMID- 1732482 TI - Surgically assisted rapid palatal expansion: an outpatient technique with long term stability. AB - This study presents the results of surgically assisted rapid palatal expansion done on an outpatient basis in 19 patients with a mean age of 30 years. Postsurgical and postorthodontic evaluation (mean, 2.4 years) showed a mean relapse rate of 8.8% in the canine region, 1% in the premolar region, and 7.7% in the molar region. These results show that the surgical procedure is feasible on an outpatient basis and the technique, as outlined, yields a stable long-term result. PMID- 1732483 TI - A clinical study of 205 patients with oral lichen planus. AB - Two hundred and five patients with oral lichen planus were divided into two groups: those with only reticular lesions (group 1) and those with atrophic erosive lesions with or without concomitant reticular lesions (group 2). A comparative study of the two groups showed that the most commonly affected oral location in both was the buccal mucosa. Lesions of the tongue, gingiva, lip, and palate predominated in group 2. Likewise, chronic liver disease and diabetes were more common in the second group, as was extension of the oral lesions (P less than .001). PMID- 1732484 TI - A comparison of two methods for inserting lag screws. AB - This study compared the accuracy of two accepted methods of inserting lag screws. The ability of the two techniques to produce concentrically drilled holes between the inner and outer cortices, the ability to accurately overdrill a small hole, and the displacement of the cortices following application and tightening of the lag screw were investigated using an experimental model of bony cortices. The results indicate that when the gap between the cortices is large, both methods of lag screw insertion are equally inaccurate. For smaller gaps between the cortices, the drill guide technique is more accurate than the other. PMID- 1732485 TI - Postoperative pain prevention by a single-dose formulation of diclofenac producing a steady plasma concentration. AB - The preoperative single-dose oral administration of a combination of rapid- and slow-release diclofenac (Voltaren, Ciba-Geigy, Basel, Switzerland) preparation in a placebo-controlled trial proved to be more effective in postoperative pain prevention following third molar removal than the combination of intramuscular diclofenac and an oral depot formula. PMID- 1732486 TI - The effects of the Le Fort I osteotomy on the periodontium. AB - Two age-matched populations of equal size (n = 40), one having orthodontic therapy and the other combined orthodontic therapy and orthognathic surgery, were evaluated for their periodontal status 1 to 10 years posttherapy. The parameters investigated were plaque index, gingival index, tooth mobility, width of keratinized tissue, probing depth, gingival recession, and attachment level. No significant differences were found (P less than .05). Within the surgery group, patients with maxillary osteotomies segmentalized between the central incisors (n = 11) and between the canines and second premolars (n = 12) were evaluated using the same parameters and compared with their nonsegmental counterparts. No significant differences were found for patients with osteotomies segmentalized between the central incisors. However, a statistically significant increase in probe depth and loss of attachment level of up to 0.3 mm was found at the sites of osteotomies segmentalized between the canine and second premolar (P less than .05). This difference was not considered clinically significant. PMID- 1732487 TI - In vitro wear performance of Proplast TMJ disc implants. AB - This study investigates the in vitro wear performance of Proplast-Teflon Interpositional Implants (PTIPI; Vitek, Inc, Houston, TX), employing a mechanical TMJ simulator. Predictions of in vivo service life of PTIPIs are presented based on the in vitro wear testing data. Commonly employed laboratory testing methodologies are discussed in the development of alloplastic TMJ devices. Penetrative wear rates of the PTIPI at a 20-lb (9.1 kg) load were calculated to be 2.29 mm/100,000 cycles, yielding a predicted in vivo service life of PTIPIs of approximately 3 years. These results combined with reported clinical fate of this implant indicate that the intermediate- and long-term survival of this implant are uncertain. PMID- 1732488 TI - Accelerated endochondral osteoinduction in the absence of bone matrix particles in a rat model system. AB - Ethanol-precipitated proteins obtained from demineralized rat bone powder (DBP) by 4M guanidine-HCl extraction have been shown to reproducibly induce ectopic endochondral bone formation when subcutaneously implanted in rats in the absence of bone matrix particles. Histologic and biochemical analysis revealed a temporal sequence of chondrocyte differentiation, calcified cartilage formation, neovascularization, osteoblast differentiation, bone formation, osteoclastic bone remodeling, and hematopoietic marrow development that is complete by 21 days. In contrast to previous reports, these results clearly show an osteoinductive response independent of the presence of insoluble extracellular bone matrix. Compared with conventional DBP implants, the guanidine-extractable protein (GE) produces an accelerated and more robust osteoinductive response. Histologically, the initial chondrogenic response at days 6 to 9 is greatly amplified. Alkaline phosphatase specific activity peaks at day 9, several days earlier than for DBP, and is sixfold higher. Calcium accumulation in GE implants at day 12 is fivefold greater than with DBP, and all mineral is localized within the matrix of newly calcified cartilage and new bone. Osteoclasts are up to ninefold more abundant in the rapidly remodeling GE ossicle, making space for hematopoietic marrow. Delivery of GE coprecipitated with inert bone matrix particles was also more effective than DBP, although the response was somewhat attenuated compared with GE alone. Bony filling of 4-mm defects in rat mandibular rami was elicited by 10 mg of GE and followed an endochondral process with increased neovascularization compared with DBP and unimplanted controls. This guanidine-extractable protein fraction should prove useful for inducing quantities of chondrocytes and osteoclasts for in vitro study, and for analysis of osteoinductive requirements.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732489 TI - Mandibular growth and tooth eruption after localized x-radiation. AB - The effect of localized x-radiation on the growth of mandibular bone and molar eruption was evaluated by morphometric methods. A dose of 20 Gy of x-radiation was given to the molar zone of growing rats. The animals were then killed in groups at 30 and 60 days postirradiation. Two groups of nonirradiated, age matched rats were used as controls. Parameters related to molar eruption, mandibular length, and mandibular height were measured on lateral radiographs. The results obtained showed that the values of the biometric parameters were lower in experimental than in control animals. Odontoblastic atrophy, alveolodentary ankylosis, and meager or no root formation were the most conspicuous histologic findings. Osteodentin was found between canaliculary dentin and bone in cases of ankylosis. The morphometric data presented confirm the probability of alterations in mandibular growth and tooth eruption following x-radiation and suggest that this be considered in planning radiotherapy in children. PMID- 1732490 TI - Advances in cardiovascular pharmacological therapy. AB - Progress in the pharmacological management of cardiovascular disease has been accompanied by the appearance of drugs with never before seen capabilities to alter human physiological processes. The widespread use of these potent agents poses a challenge to the oral and maxillofacial surgeon attempting to safely manage patients in the ambulatory setting. This article reviews the field of contemporary cardiovascular pharmacotherapy from the aspect of how it affects the practice of oral and maxillofacial surgery. PMID- 1732491 TI - Keeping abreast of the medical/dental literature: a simplified approach. AB - Surveys report journal reading to be the most important continuing education activity in terms of practitioner preference. Clinical reading can be increased by perfecting the skills and habits that will permit selection of the most useful articles. In this article, an algorithm and basic methods are presented to enhance the practitioner's skill in reviewing the literature. PMID- 1732492 TI - Acanthosis nigricans with oral lesions and a malignant visceral tumor: a case report. PMID- 1732493 TI - Odontogenic infection of the orbit: report of a case. PMID- 1732494 TI - Bullet embolus to the heart following gunshot wound of the mandible: a case report. PMID- 1732495 TI - A demodectic eruption (demodicidosis) of the face: case report. PMID- 1732496 TI - Helminth infection of the parotid gland. PMID- 1732497 TI - Odontogenic cyst giving rise to an adenomatoid odontogenic tumor: report of a case with peculiar features. PMID- 1732498 TI - Facial lawn mower injury treated by a vascularized costochondral graft. PMID- 1732499 TI - The use of a semipermeable membrane for the handling of skin grafts in preprosthetic surgery. AB - Difficulties often arise in the transfer of skin grafts from the donor site to the working area and then to the surgical stent. A method using semipermeable membrane that improves manipulation of the graft is described. PMID- 1732500 TI - Jawing about correct terminology. PMID- 1732501 TI - Plasma viremia in human immunodeficiency virus infection: relationship to stage of disease and antiviral treatment. AB - Quantitative culture of human immunodeficiency virus (HIV) was performed on 121 plasma samples from 76 HIV-infected individuals to determine the sensitivity of the assay at different stages of disease and to measure the effect of antiviral therapy on plasma viremia. Plasma virus was detected in 49 of 76 (64%) of patients, primarily those with AIDS and AIDS-related complex (36 of 38) versus asymptomatic subjects (13 of 38) (p less than 0.001, chi 2). Similarly, plasma cultures were more often positive in patients with less than 250 CD4+ T cells per microliter (38 of 40) than in those with greater than 250 CD4+ T cells per microliter (11 of 36) (p less than 0.001, chi 2). Plasma virus cultures were also more likely to be positive in patients with detectable serum p24 antigen (24 of 26) than in those without detectable p24 antigen (25 of 50) (p = 0.0023, chi 2). An effect of zidovudine (ZDV) treatment on plasma viremia was seen in a comparison of treated and untreated patients with less than 250 CD4+ T cells per microliter. Geometric mean titers of plasma viremia from 16 patients treated with ZDV for more than 3 months were significantly lower than titers from 24 untreated patients (10(1.3) versus 10(2.1), p less than 0.05, Student's t test. A comparison of pre- and posttherapy titers in 33 patients receiving antiviral treatment showed that plasma virus was not detectable at either time in 17 patients; there was a fall in plasma virus titer in 12; and titers were unchanged or increased in 4. In patients with advanced disease, plasma viremia is a potential marker of antiviral drug activity. PMID- 1732502 TI - Diagnosis of vertical HIV-1 transmission using the polymerase chain reaction and dried blood spot specimens. AB - We have used the polymerase chain reaction (PCR) to detect HIV proviral sequences in minute amounts of peripheral blood collected onto newborn screening blotters. Forty-three newborns, infants, and children of HIV-infected mothers were serially studied: dried blood spot (DBS) specimens were processed for PCR; serum was assayed for HIV antibodies, p24 antigen, and immunoglobulins; mononuclear cells were cultured and CD4 cells were quantitated by immunofluorescence. There was excellent agreement between the results of blood spot PCR, viral culture, and clinical and immunological indicators of HIV infection. Eighteen of 19 infected children tested positive by both PCR and culture, including six asymptomatic infants who were less than 10 weeks of age. As expected, p24 antigen capture assays were insensitive, detecting only 13 of the 19 infected children. One infected infant tested positive by PCR, but negative by culture and antigen. This infant was seropositive at 27 months and had pronounced hypergammaglobulinemia in association with non-specific symptoms. Twenty-four of the 43 infants were asymptomatic with normal immune profiles, declining antibody levels and no evidence of infection. These children tested repeatedly negative by PCR, culture, and p24 antigen assays. Our results indicate that DBS PCR is a sensitive, specific, and cost-effective alternative to viral culture for the early diagnosis (or exclusion) of perinatal HIV infection. DBS sampling opens the way for large scale prospective studies to determine the exact rates of vertical HIV transmission in industrialized, as well as, nonindustrialized countries. PMID- 1732503 TI - The effect of antiviral therapy on the natural history of human immunodeficiency virus infection in a cohort of hemophiliacs. AB - The antiviral drug zidovudine (ZDV) slows progression to AIDS and improves survival after AIDS diagnosis. Although clinical trials have demonstrated early improvement in CD4 lymphocyte number with ZDV, long-term effects of ZDV on CD4 in advanced and asymptomatic disease are not well known. The purpose of this study was to quantitate the effect of ZDV on the natural history of HIV infection, specifically the type and frequency of new AIDS cases, AIDS-free survival, survival after AIDS, and long-term change in an immunologic marker, CD4 number, in hemophiliacs. A cohort of 84 HIV(+) hemophiliacs for whom seroconversion dates and clinical outcomes are known was prospectively observed for the time to AIDS, pattern of primary AIDS diagnosis, rate of fall in CD4 lymphocyte levels AIDS free survival, and survival after AIDS diagnosis. The frequency of new AIDS cases has slowed since 1989, with Pneumocystis carinii pneumonia (PCP) less common (15 vs. 52%, p less than 0.04) and non-PCP opportunistic infection more common (54 vs. 20%, p less than 0.07) than prior to 1989. Patients treated with ZDV before AIDS was diagnosed (n = 39) experienced a longer AIDS-free survival than untreated patients (n = 45), as 25% progressed to AIDS by 8.2 years compared with 4.5 years, respectively, p = 0.0013. Median survival after AIDS among those untreated was significantly shorter than among those treated with ZDV either before or after AIDS was diagnosed, 0.5, 2.8, and 2.1 years, respectively, p = 0.0005. Despite these clinical advantages, there was little difference in the rate of fall in CD4 lymphocyte number between ZDV-treated and untreated groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732504 TI - Autopsy findings in HIV-infected inner-city patients. AB - To assess the importance of the autopsy in HIV-1 infection, we retrospectively reviewed the autopsy reports of 70 HIV-1-seropositive patients at Howard University Hospital. Of the 58 patients with AIDS, the diagnosis of AIDS was made after autopsy in 24 (41%) cases. The lung was the most common site of AIDS diagnostic diseases, and was affected in 90% of patients. Pneumocystis carinii infection was the most common AIDS-diagnostic disease, and was present in 50% of the AIDS patients. Thirty-eight percent of AIDS diagnostic diseases were diagnosed antemortem, including 15 of the 29 Pneumocystis carinii infections. Most of the AIDS-diagnostic diseases were disseminated at autopsy and two or more diseases were found in some organs. Overall, Pneumocystis carinii pneumonia was the most common cause of death, accounting for a mortality of 43% among AIDS patients. Bacterial infections were common and contributed to the mortality and morbidity of both AIDS and non-AIDS patients. Bacterial infection was the cause of death in 15 AIDS and 9 non-AIDS patients. The clinical cause of death concurred with the pathological cause in 53% of our cases. PMID- 1732505 TI - Pyrimethamine alone as maintenance therapy for central nervous system toxoplasmosis in 38 patients with AIDS. AB - We retrospectively assessed the efficacy of maintenance therapy with pyrimethamine alone in 38 patients with AIDS and central nervous system (CNS) toxoplasmosis. The diagnosis was based on clinical presentation and compatible CT scan abnormalities with subsequent response to therapy. Survival analysis was performed by the product limit method of Kaplan-Meier. Fourteen patients received maintenance therapy with 25 mg pyrimethamine per day (group 1), and 24 patients were treated with 50 mg per day (group 2). The median survival from initiation of maintenance therapy until death or end of the study for the entire study population was 32 weeks. Median survival in group 1 was 28 weeks, as compared with 36 weeks in group 2 (p = 0.34). Relapses occurred in 12 patients, six in group 1 and six in group 2. There was no significant difference in failure-free survival between the two treatment groups (p = 0.09). One patient in group 1 and two patients in group 2 experienced severe toxicity, requiring discontinuation of therapy. All three patients relapsed and died. Two patients in group 2 who stopped treatment on their own initiative also had relapses. Thus, all five patients who discontinued therapy had relapses. Five of 13 patients in group 1 and two of 20 patients in group 2 relapsed during continuous therapy with pyrimethamine (p = 0.13); these seven patients responded to reintroduction of combination therapy (n = 6) or treatment with 50 mg pyrimethamine per day (n = 1). The results of our retrospective analysis suggest that maintenance therapy with oral pyrimethamine, 50 mg per day, in AIDS patients with CNS toxoplasmosis is effective. PMID- 1732506 TI - The relationship between AIDS and immunologic tolerance. AB - A hypothesis is presented in which the immunodeficiency and cell loss leading to acquired immune deficiency syndrome and the clonal deletion associated with immunologic tolerance occur through a common mechanism. In a previous publication we proposed that the interaction of human immunodeficiency virus (HIV) with CD4 delivers activation signals that disrupt immune system regulation. In this article, we compare the biology of HIV infection with recent discoveries concerning the "two-signal" molecular mechanism for thymic selection. We propose that two-signal activation is normally followed by clonal expansion and the programmed death of most or all daughter cells through mechanisms that are proportional to the strength and duration of the activation signals. In the thymus, self-reactive cells are trapped in the continuous presence of both antigen signals (signal 1) and costimulatory signals (signal 2), leading to clonal deletion. In mature lymphocytes, we propose that HIV infection contributes a chronic high-affinity signal 2, which shifts the equilibrium of antigen activated T-cell populations further toward programmed death. This leads to incremental memory cell deficits and gradual clonal deletion at a rate dependent on the frequency of antigen exposure and the ability of an HIV quasispecies to induce signal 2. PMID- 1732507 TI - Lack of association between maternal antibodies to V3 loop peptides and maternal infant HIV-1 transmission. AB - We investigated the association between maternal antibodies to HIV-1 peptides in pregnant women and the acquisition of HIV-1 infection by their offspring. Pregnant HIV-1-infected Haitian women were tested for the presence of antibodies against peptides of 14-17 amino acid length from the V3 loop region of strains IIIb and MN. Antibody testing was performed in two separate laboratories by enzyme-linked immunosorbent assay (ELISA). Peptides from four regions of the V3 loop were synthesized in several different laboratories and the purity confirmed by high performance liquid chromatography (HPLC). The mothers of infants who acquired HIV-1 infections did not differ significantly from the mothers of uninfected infants in the prevalence or concentration of antibodies to any of the 15 peptides evaluated. Additional studies are indicated to determine if neutralizing antibodies or other immunologic parameters are associated with maternal-infant HIV-1 transmission. PMID- 1732508 TI - HTLV-I among U.S. Marines stationed in a hyperendemic area: evidence for female to-male sexual transmission. AB - Among 5,255 active duty United States Marines on permanent tour in Okinawa, Japan, screened for human T-cell leukemia/lymphoma virus type I (HTLV-I) seropositivity, 3 (0.06%) were confirmed by Western blot analysis to have core and envelope reactivity. All three seropositive individuals have a history of prolonged sexual contact with Okinawan women, and two of the three individuals are married to seropositive Okinawan wives. Two gave a prior history of gonorrhea, while all three were negative for syphilis (MHA-TP) and hepatitis B. No other risk factors associated with HTLV-I seropositivity in the United States were identified. A banked sample from one individual, obtained 8 months after initial sexual relations with his HTLV-I-seropositive Okinawan spouse and 20 months before being retested in the survey, showed a pattern suggesting seroconversion. Although based on small numbers, these data suggest that female to-male transmission of HTLV-I occurs in the absence of other cofactors, e.g., ulcerative genital lesions. PMID- 1732509 TI - HIV prevalence among intravenous drug users: model-based estimates from New Haven's legal needle exchange. AB - The legal needle exchange in New Haven, Connecticut, was signed into law in July 1990. As part of a rigorous effort to evaluate this program, all distributed syringes receive unique tracking codes, and a sample of returned needles are tested for the presence of HIV proviral DNA via polymerase chain reaction. These data, in conjunction with "back-of-the-envelope" statistical models, allow the estimation of HIV prevalence among those intravenous drug users participating in the needle exchange. We present four new techniques for calculating prevalence estimates: the majority rule cutoff model, the random sharing model, the sharing network model, and a version of Kaplan's "needles that kill" model. To our knowledge, these estimates are the first that attempt to infer HIV prevalence among intravenous drug users via syringe tracking and testing. All four techniques suggest a prevalence of infection of approximately 60%. PMID- 1732510 TI - Alternative confirmatory strategies in HIV-1 antibody testing. AB - Alternatives to confirmation of human immunodeficiency virus (HIV)-1 seropositivity by Western blot analysis were evaluated retrospectively using combinations of six anti-HIV-1 screening assays, including four enzyme-linked immunosorbent assays (ELISA) and two simple tests (a rapid dot immunoassay and an agglutination assay), according to an algorithm where sera are first screened by one assay and those repeatedly reactive on this assay are tested repeatedly by a second assay. Two panels of sera collected in Dar es Salaam, Tanzania, were used. Panel 1 was composed of 1,465 consecutive blood donor sera of which 99 (6.8%) were confirmed HIV-1 antibody positive, and panel 2 was composed of sera from 396 consecutively admitted patients at two medical wards of which 116 (29.3%) were confirmed HIV-1 antibody positive. Sera reactive on any of the six screening assays were also tested by a confirmatory Western blot assay. The sensitivity of the assays at the initial valid testing were as follows: Abbott 99.5%, Behring 99.5%, Organon 97.7%, Wellcozyme 100%, HIV CHEK-1 95.8%, and Serodia 95.8%. After repeat testing of sera that initially gave false-negative results all assays showed 100% sensitivity except HIV CHEK-1 (98.6%). The specificities after repeat testing were between 99.6 and 99.9% for all assays except for the Behring ELISA (98.1%). Several combinations of screening assays were found to give the same diagnostic accuracy as the screening assay followed by Western blot analysis. We conclude that an alternative confirmatory strategy can be fully satisfactory for some testing purposes. PMID- 1732511 TI - Preactivated merocyanine 540 inactivates HIV-1 and SIV: potential therapeutic and blood banking applications. AB - A novel photodynamic procedure employing "preactivated" merocyanine 540 (P-MC 540) was assessed for its effectiveness in inactivating human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Merocyanine 540 was preactivated by exposure to laser light at 514 nm prior to addition to viruses or infected cells. Treatment of cell-free HIV-1 and SIV with P-MC 540 significantly reduced their ability to infect and kill MT-4 cells in vitro. Preactivated MC 540 treatment of in vitro HIV-1-infected human peripheral blood mononuclear cells also decreased viral infection as assessed by a reduction in the amounts of HIV-1 p24 antigen produced and in the number of HIV-1 antigen-positive cells. Indirect immunofluorescence assays of target cell binding showed that treatment of cell free HIV-1 and SIV with P-MC 540 interfered with their ability to bind to CD4+ target cells. Immunoprecipitation with a monoclonal anti-CD4 antibody of P-MC 540 treated and radiolabeled HIV-1 incubated with soluble recombinant CD4 (srCD4) resulted in coprecipitation of HIV-1 viral p17 and p24 core antigens with the envelope gp120/CD4 complex, suggesting cross-linking of viral components. However, no significant decrease in the binding of treated HIV-1 to srCD4 was observed. Because of the antitumor and antiviral properties of P-MC 540, this photopreactivation procedure may represent a promising therapeutic means for controlling systemic malignancies and viral infections, and for eliminating viral contaminants in biological fluids. Unlike conventional phototherapy, this procedure does not require the delivery of light energy at the target sites following binding of the photosensitizing compounds. PMID- 1732512 TI - Transgenic mice carrying intact HIV provirus: biological effects and organization of a transgene. AB - Twelve transgenic founder animals retaining intact copies of the infectious molecular clone of human immunodeficiency virus (HIV)-1 were obtained. All the founders appeared healthy during a 9- to 12-month observation period. However, transgenic offspring of one of the founders (female #13), died within the 1st month of life while manifesting several symptoms characteristic of human AIDS. To discover why only one transgenic lineage was affected and why the founder animal in the affected lineage remained healthy while all of her transgenic offspring were diseased, we compared the organization of the transgene in the transgenic lineages. Restriction enzyme analysis showed that the founder no. 13 was a mosaic carrying in each transgenic cell four tandemly arranged copies of the infectious molecular clone. All the units of the tandem repeat appeared to be correctly preserved with the exception of the 3'-most copy, which terminated near the start of human sequences that flank the 3' long terminal repeat (LTR). The unaffected founders and their transgenic litter usually carried a high number of copies of the provirus. The 5' terminus of the transgene in the unaffected animals appeared to be deleted or rearranged. None of the 12 transgenic founders carried a single copy of integrated provirus. We conclude that infectious molecular clone of HIV-1 can be expressed in transgenic mice, and that the mode of proviral integration similar to that seen during the retroviral infectious cycle (i.e., a single-copy provirus) may be incompatible with the postnatal survival.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732513 TI - Contribution of charged amino acids in the CDR2 region of CD4 to HIV-1 gp120 binding. AB - Several negatively charged amino acids of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have been implicated in binding to the CD4 viral receptor. The CD4 region implicated in binding gp120 consists of a hydrophobic ridge protruding from a positively charged surface. To examine whether any of the surface charges on CD4 might contribute to gp120 binding, several amino acids near the CD4 regions previously implicated in gp120 binding were altered. Of the charged amino acids in the C'' and D strands of the amino-terminal domain of CD4, alteration of only two, lysine 46 and arginine 59, dramatically disrupted ability to bind gp120. In the three-dimensional structure of CD4, these two basic amino acids are located proximal to phenylalanine 43, which we confirmed to be important for gp120 binding. By contrast, we could not confirm the observations that alteration of residues in the F strand (CDR3-like region) of CD4 significantly affected gp120-binding ability. These results support a model of the gp120-binding site that is dependent on discontinuous amino acids but spatially limited. PMID- 1732514 TI - The first report of HTLV-I infection associated with blood transfusion in a thalassemic patient from Sicily, Italy. PMID- 1732515 TI - Seroprevalence stable and seroconversions rare among injection drug users in Los Angeles. PMID- 1732516 TI - Synthesis, specificity, and antifungal activity of inhibitors of the Candida albicans delta 24-sterol methyltransferase. AB - A series of side chain modified analogues of cholesterol and lanosterol (1-10) have been synthesized and evaluated as inhibitors of the Candida albicans delta 24-sterol methyltransferase. Two sterol substrate analogues 1 and 2 which contained a 24-thia substituent were relatively modest inhibitors of the enzyme (Ki = 1.5-72 microM). Compounds which mimic the carbocation intermediates proposed for the methyltransferase reaction, including sulfonium salts 4-6, amidines 7 and 8, and imidazoles 9 and 10 were substantially more potent inhibitors (Ki = 5-500 nM). All of the sterol analogues examined displayed less than 10-fold selectivity for inhibition of the methyltransferase versus the rat liver delta 24-sterol reductase. The sterol analogues were tested for in vitro antifungal activity against C. albicans, Candida tropicalis, and Torulopsis glabrata. The minimum inhibitory concentrations versus C. albicans correlated well with the Ki values for methyltransferase inhibition, and the potency of several compounds approached that of amphotericin B, although only modest fungicidal activity was observed. PMID- 1732517 TI - Decorporation of aged americium deposits by oral administration of lipophilic polyamino carboxylic acids. AB - Several new powerful chelating agents, suitable for the removal of a variety of certain heavy-metal ions from the body by oral application, have been synthesized and tested. Structurally, these compounds are partially lipophilic polyamino carboxylic acids (PACA). They were synthesized in nonaqueous media from triethylenetetramine (TT) by monoalkylation of a primary amino group, followed by exhaustive carboxymethylation of the remaining amino groups using ethyl bromoacetate and subsequent alkaline hydrolysis of the ester. Compounds were characterized using IR, 1H NMR, 13C NMR, and mass spectrometry. Synthesis and testing of two of these compounds, C12- and C22-triethylenetetraminepentaacetic acid (CnTT), is described in detail. Gel permeation chromatography of a mixture of the PACA and actinide elements have shown these substances to be strong chelating agents similar to EDTA or DTPA. They were capable of removing plutonium from contaminated liver cytosol in vitro. In contrast to their nonlipophilic counterparts EDTA and DTPA, the model substances exhibited appreciable absorption from the intestine and, therefore, can be administered orally. With increasing length of the alkyl chain, the chelons can be directed primarily to the liver, one of the target organs for actinide contamination. In vivo, absorption from the ligated duodenum and jejunum of rats after 2 h was 27% of the amount introduced. Compared to untreated controls, daily feeding of 200 mumol of the chelons (C12TT or C22TT)/kg of body weight to rats for 10 days reduced the whole body Am by 29% and 44%, respectively. Am was eliminated most efficiently from the liver, with a reduction of 71% and 89% (p less than 0.001). However, the skeletal retention also was reduced by 17% and 32% from the femora (p less than 0.001) and 20% and 37% from the carcass for the C12TT and C22TT compounds, respectively. No weight loss or other obvious signs of blood, kidney, liver, or intestinal toxicity were observed after the 10-day treatment. These new chelators are promising as agents for oral chelation therapy to remove actinides and possibly other elements from body stores. PMID- 1732518 TI - Synthesis and biological activities of fatty acid conjugates of a cyclic lactam alpha-melanotropin. AB - Four fatty acid conjugates of a cyclic lactam-bridged alpha-MSH fragment analogue were synthesized and their potencies and biological activities compared in several melanotropin bioassays. Palmitoyl, myristoyl, decanoyl, and hexanoyl conjugates of H-Asp-His-D-Phe-Arg-Trp-Lys-NH2 were prepared. In the in vitro mouse melanoma cell assay, each of the conjugates was 10-100 times more potent than alpha-MSH or the substrate peptide in elevating tyrosinase activity. The shorter conjugates of hexanoic and decanoic acid were as potent as alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogues were about 100 times less potent. The potency of the myristoyl and palmitoyl conjugates increased with time in contact with the skins. These observations may be related to the more lipid-like nature of these peptide-fatty acid conjugates. Each of the conjugates exhibited prolonged melanotropic activity in the lizard skin bioassays and in the mouse S91 melanoma tyrosinase bioassay, since the biological response continued following removal of the conjugates from the incubation media. The prolonged residual melanotropic activity resulted from conjugation of the fatty acids to the MSH fragment analogue since the analogue itself did not exhibit prolonged activity. PMID- 1732519 TI - Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor. AB - Pravadoline (1) is an (aminoalkyl)indole analgesic agent which is an inhibitor of cyclooxygenase and, in contrast to other NSAIDs, inhibits neuronally stimulated contractions in mouse vas deferens (MVD) preparations (IC50 = 0.45 microM). A number of conformationally restrained heterocyclic analogues of pravadoline were synthesized in which the morpholinoethyl side chain was tethered to the indole nucleus. Restraining the morpholine diminished the ability of these pravadoline analogues to inhibit prostaglandin synthesis in vitro. In contrast, mouse vas deferens inhibitory activity was enhanced in [2,3-dihydro-5-methyl-3-[(4 morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(4 methoxyphenyl)methano ne (20). Only the R enantiomer of 20 was active (IC50 = 0.044 microM). An optimal orientation of the morpholine nitrogen for MVD inhibitory activity within the analogues studied was in the lower right quadrant, below the plane defined by the indole ring. A subseries of analogues of 20 and a radioligand of the most potent analogue, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4 morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1 naphthalenyl)methanone (21) were prepared. Inhibition of radioligand binding in rat cerebellar membranes was observed to correlate with functional activity in mouse vas deferens preparations. Binding studies with this ligand (Win 55212-2) have helped demonstrate that the (aminoalkyl)indole binding site is functionally equivalent with the CP-55,940 cannabinoid binding site. These compounds represent a new class of cannabinoid receptor agonists. PMID- 1732520 TI - 2 beta-substituted analogues of cocaine. Synthesis and inhibition of binding to the cocaine receptor. AB - The potencies of a series of 2 beta-substituted cocaine analogues to displace [3H]-3 beta-(p-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester binding in rat striatal membranes demonstrate the requirement for a 2 beta-substituent with two hydrogen-bond acceptors. The insensitivity of the ester moiety to steric and electronic factors suggests its modification to provide site-specific irreversible ligands. PMID- 1732521 TI - N-modified analogues of cocaine: synthesis and inhibition of binding to the cocaine receptor. AB - Cocaine methiodide (2), N-norcocaine (1b), N-benzyl-N-norcocaine (1c), and N-nor N-acetylcocaine (1d) were synthesized and evaluated for their ability to inhibit binding of [3H]-3 beta-(4-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester (WIN 35,428) to the cocaine receptor. The study showed that removal of the N-methyl group to give 1b, or replacement with the larger N-benzyl group to give 1c, has a relatively small effect on binding potency. In contrast, replacement of the N-methyl group by the acetyl moiety to give 1d, or the addition of a methyl group to give 2, reduces affinity for the receptor by a large factor. In order to gain preliminary information concerning the importance of the nitrogen location on the tropane ring system, the receptor binding affinity of 8-methyl-8 azabicyclo[3.2.1]octan-3 beta-ol benzoate (5, beta-tropacocaine) was compared to that of the isomeric 6-methyl-6-azabicyclo[3.2.1]octan-3 beta-ol benzoate (4d). The fact that both compounds have similar binding affinities for the cocaine receptor suggests that 3 beta-(benzoyloxy)-6-methyl-6-azabicyclo[3.2.1] octane-2 carboxylic acid methyl ester, which is isomeric with cocaine, may possess binding potency similar to cocaine. PMID- 1732522 TI - Muscarinic activity of the thiolactone, lactam, lactol, and thiolactol analogues of pilocarpine and a hypothetical model for the binding of agonists to the m1 receptor. AB - Pilocarpine isosteres have been synthesized and characterized with regard to their in vitro muscarinic properties. The results indicate that the carbonyl oxygen of the lactone function of pilocarpine is of primary importance for agonist activity with the ether oxygen being of lesser or secondary importance. An X-ray structure determination for the hydrogen O,O'-ditoluoyltartrate salt of thiolactone pilocarpine isostere 2a has been performed. This compound has an unusual pharmacological profile exhibiting M1-agonist selectivity as well ass presynaptic antagonism. As a result this compound is also viewed as having therapeutic potential for Alzheimer's disease. A model for the binding of pilocarpine and other muscarinic agonists to the third transmembrane helix of the human m1 muscarinic receptor has been developed. PMID- 1732523 TI - Preparation and biological activities of potential vasopressin photoaffinity labels. AB - Several potential photoaffinity analogues of the peptide hormone vasopressin (VP) were prepared by classical solid-phase peptide synthesis using two different pathways. Peptide sequences were built by introduction of (a) Nar-protected aminophenylalanine or (b) nitrophenylalanine in the photolabeling position. Conversion to the azido peptide was completed in pathway a after cleavage and before purification and in pathway b from small quantities of purified nitrophenylalanine-containing precursor peptides. V1 receptor binding properties were measured using membranes prepared from rat liver cells. The binding potential of agonistic VP structures was abolished by the introduction of an azido or a nitro group into the aromatic side chain at position 3. Cyclo desamino beta,beta-dialkyl-Cys1-type VP antagonist structures were prepared with the photoactivable moiety in position 2 and an iodination residue in position 9. One particular compound, [Dmpa1, Phe(N3)2, Val4, Lys8,D-Tyr9]VP (8), containing beta,beta-dimethyl-beta-mercaptopropionic acid in position 1, had excellent binding properties, both in the radioiodinated (Kd = 4.8 +/- 1.9 x 10(-10) M) and noniodinated form (Kd = 6.4 +/- 0.98 x 10(-10) M). The analogues with long-chain beta-alkylation (diethyl and pentamethylene) and the linear antagonist photolabel showed significantly less affinity. Optimal binding properties were obtained within a very narrow range of hydrophobicity; greater or lesser hydrophobicity was correlated to less potent binding. The precursor analogues, containing nitrophenylalanine, displayed a structure-activity relationship similar to that of the azido peptides. The most potent analogues will be used for receptor labeling studies. A linear antagonist structure having a photosensitive group in position 1, has also been prepared, but this compound displayed much less affinity than the cyclic antagonists. The most potent compounds were also highly selective for the V1 receptor and did not recognize the V2 receptor from other preparations. PMID- 1732524 TI - New conformationally restricted 99mTc N2S2 complexes as myocardial perfusion imaging agents. AB - In developing 99mTc complexes as potential myocardial imaging agents, a new series of ligands based on a conformationally restricted N2S2 system were investigated. Using piperazine or homopiperazine as the starting material, two N2S2 ligands (4a and 4b) with additional conformation restriction between the two nitrogen donor atoms were synthesized. The 99mTc complexes were prepared by a direct labeling method with tin(II) tartrate as the reducing agent for [99mTc]pertechnetate. The resulting 99mTc complexes were purified through a sulfonpropyl Sephadex column and further purified by HPLC with a reverse-phase column eluting with a solvent system of acetonitrile/buffer. Biodistribution studies in rats showed initial uptake in the heart (0.21%, 0.42% dose/order for [99mTc]4a and 4b at 2 min postinjection). Carrier-added preparation of [99mTc]4b was successful. NMR, IR, UV, crystallographic, and elemental analysis of the [99Tc]4b complex suggest that it contains a TcVO3+ center core and is 1+ charged. The results suggest that this series of 1+ charged 99mTc complexes may have potential as myocardial imaging agents, and further study of the complexes is warranted. PMID- 1732525 TI - Heteroatom analogues of bemoradan: chemistry and cardiotonic activity of 1,4 benzothiazinylpyridazinones. AB - A series of close analogues of the potent, long-acting cardiotonic bemoradan (2a) was synthesized and examined in both in vitro and in vivo test systems. Changing the oxygen heteroatom at the 1-position of the benzoxazine ring of bemoradan to sulfur gave 4a, a more potent enzyme inhibitor and in vivo cardiotonic compound by the iv route. Intraduodenal administration of bemoradan, however, showed a superior response compared to its sulfur analogue, possibly due to oxidation of sulfur followed by a facile Pummerer rearrangement. Model studies were performed to examine the effect of the oxidation state of sulfur. Lack of a heteroatom at the 1-position, 3a (Y-590), afforded a compound with activity and potency very similar to those of bemoradan while the 1-selena compound gave a much less potent analogue 5. Analogues having a methyl group on the 4-nitrogen (2b, 3b, and 4b) were less potent than the desmethyl compounds, but all of these compounds have potent PDE III inhibiting activity and the ability to increase cardiac force in an anesthetized dog preparation when given iv. PMID- 1732526 TI - Inhibitors of protein kinase C. 1. 2,3-Bisarylmaleimides. AB - The design and synthesis of a series of novel inhibitors of protein kinase C (PKC) is described. These 2,3-bisarylmaleimides were derived from the structural lead provided by the indolocarbazoles, staurosporine and K252a. Optimum activity required the imide NH, both carbonyl groups, and the olefinic bond of the maleimide ring. 2,3-Bisindolylmaleimides were the most active, and the potency of these was improved by a chloro substituent at the 5-position of one indole ring (compound 28, IC50 0.11 microM). In a series of (phenylindolyl)maleimides, nitro compound 74 was most active (IC50 0.67 microM). Naphthalene 19 and benzothiophene 21 showed greater than 100-fold selectivity for inhibition of PKC over the closely related cAMP-dependent protein kinase (PKA). PMID- 1732527 TI - Phenylalkyl isothiocyanate-cysteine conjugates as glutathione S-transferase stimulating agents. AB - To develop analogues of phenylalkyl isothiocyanate with less toxicity and better biological activity, two water-soluble phenylalkyl isothiocyanate-cysteine conjugates, S-[N-benzyl(thiocarbamoyl)]-L-cysteine (1) and S-[N-(3 phenylpropyl)(thiocarbamoyl)]-L-cysteine (2), were synthesized. The induction of increased activity of the detoxifying enzyme glutathione S-transferase by the conjugates and their parent compounds was determined and compared in several tissues of A/J mice. The biological evaluation revealed that the conjugates as GST enzyme inducers appeared to be less toxic and even more potent than the parent compounds in the mouse bladder. Compounds 1 was much more active than 2 in all the tissues examined, while their parent compounds showed an inverse order of activity. Thus, an increase in the alkyl chain length of the parent isothiocyanates or a decrease in the alkyl length of the conjugates could result in higher enzyme-inducing activity in the same compound series. Since a number of nitrosamines have been identified as prime bladder carcinogens and phenylalkyl isothiocyanates have been reported to inhibit a wide range of carcinogenic nitrosamines, the corresponding conjugates may serve as prodrugs to protect against nitrosamine-induced urinary bladder carcinogenesis once they are delivered to the target organ. PMID- 1732528 TI - Syntheses and 5-HT2 antagonist activity of bicyclic 1,2,4-triazol-3(2H)-one and 1,3,5-triazine-2,4(3H)-dione derivatives. AB - A series of bicyclic 1,2,4-triazol-3(2H)-one and 1,3,5-triazine-2,4(3H)-dione derivatives with a 4-[bis(4-fluoro-phenyl)methylene]piperidine or 4-(4 fluorobenzoyl)piperidine group has been prepared and tested for 5-HT2 and alpha 1 receptor antagonist activity. Among the compounds prepared, 2-[2-[4-[bis(4 fluorophenyl)methylene]-piperidin-1-yl]ethyl]- 5,6,7,8-tetrahydro-1,2,4 triazolo[4,3-a]pyridin-3(2H)-one (7b) had the most potent 5-HT2 antagonist activity, which was greater than ritanserin (2), while 7b did not show alpha 1 antagonist activity in vivo. The central 5-HT2 receptor antagonism was approximately 1/30 that of 2 when tested for the ability to block head twitches induced by 5-hydroxytryptophan. Compound 21b, 3-[2-[4-(4-fluorobenzoyl)piperidin 1-yl]ethyl]-6,7,8,9-tetrahydro- 2H- pyrido[1,2-a]-1,3,5-triazine-2,4(3H)-dione also displayed potent 5-HT2 antagonist activity. The compound had moderate alpha 1 receptor antagonism, and the potency inhibiting head twitches was about one third that of ketanserin (1). These results indicate that 5,6,7,8-tetrahydro 1,2,4-triazolo[4,3-a]pyrimidin-3(2H)-one and 6,7,8,9-tetrahydro-2H-pyrido-[1,2-a] 1,3,5-triazine-2,4(3H)-dione ring systems are useful components of 5-HT2 antagonists. PMID- 1732529 TI - Synthesis, stability, and biological evaluation of water-soluble prodrugs of a new echinocandin lipopeptide. Discovery of a potential clinical agent for the treatment of systemic candidiasis and Pneumocystis carinii pneumonia (PCP). PMID- 1732530 TI - Potent non-6-fluoro-substituted quinolone antibacterials: synthesis and biological activity. PMID- 1732531 TI - Design and synthesis of potent, selective, and orally active fluorine-containing renin inhibitors. AB - A series of primate renin inhibitors containing difluorocarbinol and difluoroketone groups at the P1-P1' position have been synthesized and studied both in vitro and in vivo. In vitro, the compounds were evaluated as inhibitors of monkey renin and the closely related aspartic proteinase, cathepsin D (bovine), as a measure of enzyme selectivity. Interestingly, the difluoroketone derivatives showed greatly reduced selectivity compared with the corresponding alcohols. However, selectivity could be enhanced by judicious choice of other substituents. Sites influencing selectivity, included not only P2, which is well known to strongly affect selectivity, but also the P4, P1-P1', and P2' sites. These results make possible the design of inhibitors with a greater selectivity for either renin versus cathepsin D. In vivo several of the compounds in the difluoroketone series have shown good oral activity in the salt depleted normotensive cynomolgus monkey model. PMID- 1732532 TI - Structure-antigastrin activity relationships of new (R)-4-benzamido-5 oxopentanoic acid derivatives. AB - New (R)-4-benzamido-5-oxopentanoic acid derivatives were synthesized by a stereoconservative procedure and evaluated in vitro for their capacity to inhibit the binding of [125I](BH)-CCK-8 to either rat peripheral (CCK-A) or central (CCK B) CCK receptors, or the binding of [3H]pentagastrin to rabbit gastric glands, as well as to inhibit, in vivo, the acid secretion induced by pentagastrin infusion in the perfused rat stomach. The parent compound of this series (lorglumide) is the first nonpeptidic, potent and selective antagonist of the CCK-A receptor. Chemical manipulations of the structure of lorglumide led to the discovery of selective antagonists of the CCK-B/gastrin receptors. Structure-activity relationships are discussed. Some of these new derivatives exhibit different affinities with rabbit gastric gland cells and rat cortex membranes, suggesting that the stomach gastrin receptor (arbitrarily termed CCK-B1 receptor) is not as closely related to the CCK central receptor (termed CCK-B2) as previously hypothesized. The antigastric activity of the most potent compound of the series, i.e. (R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan- 8-yl)-5-oxopentanoic acid (compound 28, CR 2194) was further evaluated in vivo: in the first hour after administration the compound inhibits acid secretion induced by pentagastrin infusion, in both cat and dog (in the cat with gastric fistula and in the dog with Heidenhain pouch), with ID50s (mg/kg) of 15.5 (iv) (cat), 8.7 (IV) (dog) and 24.2 (oral) (Heidenhain dog). The characteristics of CR 2194, that is, the selectivity for the gastrin receptor, the simple nonpeptidic molecular structure, and the activity after oral administration, indicate that this compound is a useful tool in the study of the biological effects of gastrin and a potential agent for diagnostic or therapeutic use. PMID- 1732533 TI - Di- and triester prodrugs of the varicella-zoster antiviral agent 6-methoxypurine arabinoside. AB - 6-Methoxypurine arabinoside (9-beta-D-arabinofuranosyl-6-methoxy-9H-purine, 1) has potent and selective activity against varicella-zoster virus in vitro. An unfavourable metabolic profile observed with oral dosing in the rat led to the preparation of a variety of 2',3',5'-triesters (2a-n) and several 2',3'-, 2',5'-, and 3',5'-diesters of this arabinoside (3a-n, 4a-f, and 5a-j, respectively). The compounds were evaluated as prodrugs by measuring the urinary levels of 1 in rat urine after oral dosing. With the exception of triacetate 2a, the triesters failed to significantly enhance bioavailability. Administration of compound 2a resulted in a 3-fold increase in systemic availability of 1, possibly because of its increased water solubility (1.6 times more soluble than 1) and only slightly increased relative log P value (1.93 vs 0.50 for 1). The longer chain aliphatic triesters and aromatic triesters had lower water solubilities and increased lipophilic partitioning. These factors might account for the lower systemic bioavailability of these compounds. In contrast, the diesters, especially the aliphatic diesters, showed significantly improved systemic availability. This might be a consequence of the higher aqueous solubilities and enhanced partition coefficients seen with these compounds. 2',3'-Diacetate 3a showed the best combination of high systemic availability and water solubility of all the prodrugs of 1. PMID- 1732534 TI - Total synthesis of uracil analogues of sinefungin. AB - Analogues of sinefungin derivatives 18a and 18b have been prepared from uridine and L-aspartic acid. The key step in the synthesis was the coupling of the radical derived from 14 with the unsaturated amide 13. The latter was produced from the known N-hydroxy-2-thiopyridone ester of L-aspartic acid 12 with the olefin 11. Thus, the essential carbon skeleton was constructed by way of two radical coupling reactions. These analogues as well as 1a and 1b synthesized previously were tested for their antileishmanial effect in vivo and for their inhibitory activity of protein carboxymethylase (protein methylase II). The replacement of the adenine moiety by uracil or dihydrouracil considerably decreases the antiparasitic activity and the affinity for protein methylase II. The synthetic (S)-sinefungin was as active as the natural one. Interestingly, the C-6' epimer 1b was 50% less active in vitro than the natural sinefungin, but both had identical affinities for the target enzyme. PMID- 1732535 TI - Synthesis and biological activity of new dimers in the 7H-pyrido[4,3-c] carbazole antitumor series. AB - Ditercalinium (NSC 366241) is a 7H-pyrido[4,3-c]carbazole dimer with a diethylbipiperidine rigid chain linking the two heterocyclic rings. Ditercalinium is characterized by a high DNA affinity and bisintercalating ability, associated with potent antitumor properties, involving an original mechanism of action. Unfortunately as ditercalinium is hepatotoxic, its clinical evaluation has been interrupted. In order to eliminate or at least minimize the serious drawbacks related to its toxic effects, several chemical modifications have been made to the structure of ditercalinium, and their influence has been evaluated by measuring the DNA affinities, intercalation properties, and toxicity toward leukemia cells of the newly synthesized dimers. Reduction of the pyridinic moieties of ditercalinium, in order to suppress the permanent charges provided by the quaternizing chain, led to an almost complete loss of activity, although the DNA bisintercalating property of the dimer was preserved. Dimerization of the 7H pyrido[4,3-c]carbazole rings by introduction of the rigid spacer on the N7- or C6 positions corresponding to the convex face of the pyridocarbazole, instead of the N2-position in ditercalinium, led to DNA bisintercalating dimers practically devoid of antitumor properties. However after quaternarization of the N2 atoms, the dimer linked by the N7 atoms exhibited a very high DNA affinity (greater than 10(9) M-1) and recovered antitumor activity, supporting the requirement of positive charges for the emergence of antitumor activity in these dimers. Introduction on the C6 of the 7H-pyridocarbazole ring of an aminomethyl or carboxyl group, a sugar residue, or C or N free amino acids such as Lys or Glu has also been carried out, in order to increase the hydrophilic properties of the molecules or to enable them to use amino acid transport systems. Although some of these compounds were active, none of them exhibited the pharmacological potency of ditercalinium. PMID- 1732536 TI - Oxidation chemistry and biochemistry of the central mammalian alkaloid 1-methyl-6 hydroxy-1,2,3,4-tetrahydro-beta-carboline. AB - The electrochemical oxidation of the central mammalian alkaloid 1-methyl-6 hydroxy-1,2,3,4-tetrahydro-beta-carboline (1) has been studied in neutral aqueous solution at a pyrolytic graphite electrode (PGE). Voltammograms of 1 show two closely spaced oxidation peaks, Ia and IIa. At potentials less positive than the peak potential (Ep) for peak Ia, 1 is oxidized to a radical intermediate which dimerizes to give two diastereomers of 5,5'-bi(1-methyl-6-hydroxy-1,2,3,4 tetrahydro-beta-carboline) (5 and 6). At potentials more positive than Ep for peak Ia the putative radical intermediate is further electrooxidized to a C(5) centered carbocation which reacts with 1 in an ion-substrate reaction to give 5 and 6 or with water to give, ultimately, 1-methyl-1,2,3,4-tetrahydro-beta carboline-5,6-dione (12). Dimers 5 and 6 give two reversible oxidation peaks at the PGE, the second of which corresponds to peak IIa observed in voltammograms of 1. Because 5 and 6 are easily oxidizable compounds they are only observed as products in the initial stages of the controlled potential electrooxidation of 1. Tyrosinase/O2, human ceruloplasmin/O2, and peroxidase/H2O2 also oxidize 1 to 5, 6, and 12 as the initial products. In the presence of glutathione the electrochemically driven and enzyme-mediated oxidations of 1 result in the formation of 5-S-glutathionyl-1-methyl-6-hydroxy-1,2,3,4-tetrahydro-beta carboline as a major product. Central administration of diastereomer 5 or 6 to mice evoked behavioral responses similar to those caused by the opioid analgesics. These behavioral effects, which include spatial disorientation and a characteristic ducklike walk, became most pronounced approximately 3 h after drug administration and continued for about 3 days. Neurotransmitter and related metabolite analyses of whole brain reveal that 5 and 6 cause a general increase in dopaminergic and serotonergic activity and a small but significant decrease in cholinergic activity. These transmitter/metabolite disturbances appear to parallel the time course of the observed behavioral effects. The possible roles of in vivo oxidations of 1, an alkaloid which is elevated in mammalian brain following ethanol consumption, in the addictive, behavioral, and neurodegenerative consequences of chronic alcoholism are discussed. PMID- 1732537 TI - Stereoselective LSD-like activity in d-lysergic acid amides of (R)- and (S)-2 aminobutane. AB - The (R)- and (S)-2-butylamides of d-lysergic acid were prepared and evaluated in behavioral and biochemical assays of 5-HT2 agonist activity. In rats trained to discriminate 0.08 mg/kg LSD tartrate from saline, both isomers completely substituted for the training stimulus. Similarly, both isomers were found to possess very high affinity in displacing [125I]-(R)-DOI ([125I]-(R)-1-(2,5 dimethoxy-4-iodophenyl)-2- aminopropane) from rat cortical homogenate 5-HT2 receptors and in displacing [3H]-8-OH-DPAT ([3H]-8-hydroxy-2-(di-n propylamino)tetralin) from rat hippocampal 5-HT1A receptors. The difference in activity between the two isomeric amides was significant in both the behavioral and binding assays, with the R isomer possessing greater potency. Molecular mechanics were used to predict the active geometries of the subject compounds. It was found that the (R)-2-butylamide has a conformation quite similar to LSD, while the (S)-2-butylamide does not. These results suggest that stereochemical properties of the amide substituent of hallucinogenic lysergamides may exert a critical influence on activity. It is concluded that the conformation of the amide function may directly affect binding through stereoselective interactions with a hydrophobic region on the receptor, indirectly by inducing conformational changes elsewhere in the molecule, or by a combination of these two mechanisms. PMID- 1732538 TI - The three binding domain model of adenosine receptors: molecular modeling aspects. AB - Using molecular modeling, adenosine receptor ligands were fitted together to maximize correlations between the three most important factors controlling binding to the receptor, namely steric, hydrophobic, and electrostatic complimentarily. Structure-activity relationships can be explained by three binding domains on the receptors. These are hydrophobic, aromatic, and ribose binding domains. We propose that the N6, C2, and C8 hydrophobic binding domains are not discreet but occupy the same region of the receptor. PMID- 1732539 TI - Inactivation of calpain by peptidyl fluoromethyl ketones. AB - The syntheses of Z-Leu-Leu-Tyr-CH2F (1) and Z-Tyr-Ala-CH2F (3) are described. The ability of Z-Leu-Leu-Tyr-CH2F (1) and Z-Leu-Tyr-CH2F (2) to inactivate in vitro calcium-activated proteinase from chicken gizzard are compared. Like the analogous diazomethyl ketones 4 and 5, these inhibitors were also found to inactivate cathepsin L in common with other inhibitors under current investigation. However, other specific inactivators for cathepsin L are available, for example, the fluoromethyl ketone 3 and diazomethyl ketone 6 of Z Tyr-Ala-OH, which have no effect on the calcium-activated proteinase and therefore provide control inhibitors for observations made with Z-Leu-Leu-Tyr CH2F (1). PMID- 1732540 TI - Structure-function studies in a series of carboxyl-terminal octapeptide analogues of anaphylatoxin C5a. AB - The synthesis and structure-activity relationships of C-terminal octapeptide analogues of anaphylatoxin C5a have been studied. The introduction of hydrophobic amino acids into the N-acetylated native octapeptide (N-Ac-His-Lys-Asp-Met-Gln Leu-Gly-Arg-OH) (1) has led to an analogue with 100 times more activity than the native octapeptide in inhibiting the binding of 125I-labeled anaphylatoxin C5a to human neutrophil membrane receptors. The observed apparent binding Ki's for the compounds (8-10) are in the range of 1-3 microM, and they possess nearly full agonist activity, despite the fact that these analogues are one-eighth or -ninth the size of the natural ligand anaphylatoxin C5a. PMID- 1732541 TI - Nucleosides and nucleotides. 103. 2-Alkynyladenosines: a novel class of selective adenosine A2 receptor agonists with potent antihypertensive effects. AB - The synthesis and receptor-binding activities at A1 and A2 adenosine receptors for a series of 2-alkynyladenosines are described. The palladium-catalyzed cross coupling reaction of 2-iodoadenosine (4a) with various terminal alkynes in the presence of bis(triphenylphosphine)palladium dichloride and cuprous iodide in N,N dimethylformamide containing triethylamine gives 2-alkynyladenosines (5a-r). An economical synthetic method for the preparation of 9-(2,3,5-tri-O-acetyl-1-beta-D ribofuranosyl)-6-chloro-2-iodopurine++ + (2), which is a precursor of 4a, is also included. Several transformation reactions of 2-(1-octyn-1-yl)adenosine (5e) and 2-(1-ethyn-1-yl)adenosine (9) and a similar cross-coupling reaction of 6 chloropurine derivative 11 and 8-bromoadenosine (13) with 1-octyne are also reported. Many of these 2-alkynyladenosines tested for A1 and A2 adenosine receptor binding activities in rat brain are selective for the A2 adenosine receptor. Among them, 2-(1-hexyn-1-yl)adenosine (5c) has the highest affinity for both A1 and A2 receptors with Ki values of 126.5 and 2.8 nM, respectively. The structure-activity relationship of this series of compounds including 6- or 8 alkynylpurine nucleosides and 2-alkyl- and 2-alkenyladenosines is discussed in terms of potency at both receptor subtypes. Additionally, we describe how hypotensive activity and heart rate decrease brought on by 5 and some other compounds with spontaneously hypertensive rats are proportional to the order of the potency to both A1 and A2 binding affinities. Thus, 2-alkynyladenosines are interesting and promising as antihypertensive agents that should be considered for further detailed preclinical evaluation. PMID- 1732542 TI - Synthesis and antitumor activity of tropolone derivatives. 7. Bistropolones containing connecting methylene chains. AB - Bistropolone derivatives (4-12) containing differing lengths of linkage between the two tropolone rings were prepared and examined for their antitumor activity in in vitro (KB cell) and in vivo (leukemia P388 in mice) systems. Parent compound 3, related compounds previously prepared, and the new compounds 4-12 were evaluated for inhibitory activity against ribonucleotide reductase by indirect means to measure their effects on the dNTP pool imbalance. Present structure-activity relationship results would suggest that potently active bistropolones in vivo inhibit intracellular ribonucleotide reductase through chelating with the two irons at the two active sites of the enzyme. PMID- 1732543 TI - A new, general synthetic route to multidentate N,S ligands for use in technetium 99m radiopharmaceuticals. Preparation of diamido disulfur, diamino dithiol, and tripodal N3S3 prototypes. Comparative biodistributions of [99mTcvO-DADS]- analogues which contain 5,5,5- and 5,7,5-membered chelate ring systems. AB - A new, versatile synthetic route to a variety of tetradentate N2S2 and hexadentate N3S3 ligands for use in technetium-99m radiopharmaceuticals has been developed. The key reaction employs 1-(ethoxycarbonyl)-2-ethoxy-1,2 dihydroquinoline (EEDQ) for coupling an appropriate di- or triamine with S protected thioglycolic acid. Selected DADS (diamido disulfur) and DADT (diamino dithiol) and analogues derived from ethanediamine-1,2 and butanediamine-1,4, as well as a tripodal N3S3 analogue, were synthesized in high yield. The labeling of DADS analogue ligands derived from butanediamine-1,4 (DADS-bn) with 99mTc results in a single radiochemical product, or the expected number of stereoisomeric products. FAB MS analysis, using 99Tc, indicates that DADS-bn ligands form a tetradentate 5,7,5-membered ring chelate system with the monooxo Tc(V) core: [TcO DAD-bn]-. In rat biodistribution studies the DADS-en and DADS-bn complexes show very similar biological activity. Phenyl substituents on the 7-membered ring give rise to a 99mTc complex of increased lipophilicity, increased liver uptake, and minimal hepatobillary clearance. Thus, new classes of DADS ligand analogues which contain a 5,7,5-membered chelate ring system are readily synthesized and labeled with 99mTc to form stable complexes. The presence of the 7-membered chelate ring allows for facile introduction of pendant groups into the ligand system, and thus for a ready route to control the biodistribution of the resulting 99mTc radiopharmaceutical. The 99mTc labeling of the DADT analogues derived from butanediamine-1,4 provided to be erratic, despite the use of a variety of labeling techniques; the larger ring system of DADT-bn apparently does not favor the monooxo Tc(V) core and appears to generate a mixture of mono- and dioxo Tc(V) cores. PMID- 1732544 TI - Synthesis and evaluation of radioiodinated 2-(2(RS)-aminopropyl)-5- iodothiophenes as brain imaging agents. AB - Methods have been developed for the preparation of 2-(2(RS)-aminopropyl)-5 iodothiophenes. The syntheses and physical properties of 2-(2(RS)-aminopropyl)-5 iodothiophene and N-isopropyl-2-(2(RS)-aminopropyl)-5-iodothiophene are described. The radioiodinated agents are of interest because of the high expected uptake and prolonged brain retention that may result from binding to high capacity, relatively nonspecific amine binding sites. Radioiodine was introduced into the 5-position of 2-(2(RS)-aminopropyl)-5-iodothiophene and N-isopropyl-2 (2(RS)-aminopropyl)-5-iodothiophene by radioiodination of the corresponding 5 boronic acid or 5-(trimethylstannyl) derivatives. Tissue distribution studies in rats with 2-(2(RS)-aminopropyl)-5-[125I]iodothiophene showed high brain uptake (5 min, 2.77% dose/g; 30 min, 2.51% dose/g) and good brain/blood (B/B) ratios (5 min, 6/1; 30 min 3.8/1. A comparison of the brain uptake of the N-isopropyl derivative with the 2(RS)-aminopropyl analogue demonstrated higher initial brain uptake and brain to blood ratios (5 min, 3.2% dose/g; 10.3/1) but more rapid washout (30 min, 1.37% dose; 2.8/1). These data suggest that radiolabeled 2 (2(RS)-aminopropyl)-5-iodothiophenes are potentially useful agents for cerebral perfusion imaging by single-photon-emission computerized tomography (SPECT). PMID- 1732545 TI - Phenyl-substituted analogues of oxotremorine as muscarinic antagonists. AB - A series of phenyl-substituted analogues of the muscarinic agent oxotremorine (1) have been prepared. The new compounds (3b-11b and 9c) were assayed for antimuscarinic activity on the isolated guinea pig ileum and in intact mice. They were also evaluated for ability to inhibit the binding of the muscarinic antagonist (-)-[3H]-N-methylscopolamine to homogenates of the rat cerebral cortex. The phenyl-substituted derivatives were devoid of intrinsic muscarinic activity. Instead, they behaved as competitive muscarinic antagonists in these assays with similar or lower affinity for muscarinic receptors than the corresponding methyl-substituted analogues. The succinimide (8b) and the pyrrolidone (3b) derivatives of 1 substituted with a phenyl group at position 1 of the butynyl chain showed the highest antimuscarinic potency with dissociation constants (KD) of 0.10 and 0.20 microM, respectively, in the ileum assay. The phenyl-substituted analogues showed an approximately 10-fold lower in vivo antimuscarinic potency than their corresponding methyl-substituted positional isomers. A correlation was observed between in vitro and in vivo potency within subsets consisting of methyl- and phenyl-substituted derivatives. PMID- 1732547 TI - Conformational studies of muscarone analogues: X-ray analysis and molecular mechanics calculations. AB - The X-ray structure of muscarone analogues 3 and 4 was determined and compared with that of muscarone (1, iodide and picrate salts), muscarine 2, dioxolane 5, oxathiolane 6, and tetrahydrofuran 7. In order to better define the pharmacological stereoselectivity of muscarone, the conformational profiles of compounds 1, 2, 3, and 5 were analyzed using Allinger's MM2(85) program or, in the case of 4, by 1H NMR spectroscopy. The conformation of the ring in 1 proved similar to that of the other derivatives. MM2 calculations predicted a preferred gauche arrangement of the side chain for 1 and its analogues; such an arrangement was also observed in the solid state of muscarone picrate. Thus, the antiperiplanar arrangement reported for crystalline muscarone iodide appears to be due to crystallographic packing forces. As a consequence, the rationalization of the pharmacological profile of 1 based on the antiperiplanar arrangement is now highly questionable. The lack of stereoselectivity of 4 can be attributed to the absence of a stereocenter at C-2 whereas, in our opinion, there are currently no sound explanations for the low values of eudismic ratios for the muscarone enantiomers. PMID- 1732546 TI - Synthesis and muscarinic activity of quinuclidinyl- and (1-azanorbornyl)pyrazine derivatives. AB - The synthesis and cortical muscarinic activity of a novel series of pyrazine based agonists is described. Quinuclidine and azanorbornane derivatives were prepared either by reaction of lithiated pyrazines with azabicyclic ketones, followed by chlorination and reduction, or by reaction of the lithium enolate of the azabicyclic ester with 2-chloropyrazines followed by ester hydrolysis and decarboxylation. Substitution at all three positions of the heteroaromatic ring has been explored. Optimal muscarinic agonist activity was observed for unsubstituted pyrazines in the azanorbornane series. The exo-1-azanorbornane 18a is one of the most efficacious and potent centrally active muscarinic agonists known. Studies on the 3-substituted derivatives have provided evidence of the preferred conformation of these ligands for optimal muscarinic activity. Substitution at C6 gave ligands with increased affinity and reduced efficacy. Moving the position of the diazine ring nitrogens to give pyrimidine and pyridazine derivatives resulted in a significant loss of muscarinic activity. PMID- 1732548 TI - Zatosetron, a potent, selective, and long-acting 5HT3 receptor antagonist: synthesis and structure-activity relationships. AB - Antagonists of 5HT3 receptors are clinically effective in treating nausea and emesis associated with certain oncolytic drugs, including cisplatin. Moreover, these agents may be useful in pharmacological management of several central nervous system disorders, including anxiety, schizophrenia, dementia, and substance abuse. Our studies on aroyltropanamides led to the discovery that dihydrobenzofuranyl esters and amides are potent 5HT3 receptor antagonists. Simple benzoyl derivatives of tropine and 3 alpha-aminotropane possessed weak 5HT3 receptor antagonist activity, as judged by blockade of bradycardia produced by iv injection of serotonin (5HT) to anesthetized rats. Within this series, use of benzofuran-7-carboxamide as the aroyl moiety led to a substantial increase of 5HT3 receptor affinity. The optimal 5HT3 receptor antagonist identified via extensive SAR studies was endo-5-chloro-2,3-dihydro-2,2-dimethyl-N-(8-methyl-8 azabicyclo[3.2.1]oc t- 3-yl)-7-benzofurancarboxamide (Z)-2-butenedioate (zatosetron maleate). The 7-carbamyl regiochemistry, dimethyl substitution, chloro substituent, and endo stereochemistry were all crucial elements of the SAR. Zatosetron maleate was a potent antagonist of 5HT-induced bradycardia in rats (ED50 = 0.86 micrograms/kg i.v.). Low oral doses of zatosetron (30 micrograms/kg) produced long-lasting antagonism of 5HT3 receptors, as evidenced by blockade of 5HT-induced bradycardia for longer than 6 h in rats. Moreover, this compound did not produce hemodynamic effects after i.v. administration to rats, nor did it block carbamylcholine-induced bradycardia in doses that markedly blocked 5HT3 receptors. Thus, zatosetron is a potent, selective, orally effective 5HT3 receptor antagonist with a long duration of action in rats. PMID- 1732549 TI - Synthesis and antifolate properties of 5,10-ethano-5,10-dideazaaminopterin. AB - 2-Carbomethoxy-4-(p-carbomethoxyphenyl)cyclohexanone was prepared in a four-step process and thermally condensed with 2,4,6-triaminopyrimidine to afford methyl 2,4-diamino-4-deoxy-7-hydroxy-5,10-ethano-5,10-dideazapteroate+ ++. Reduction of the 7-oxo function with borane gave the 7,8-dihydro pterin which was subsequently oxidized to the fully aromatic pteroate ester with dicyanodichlorobenzoquinone. Saponification of the benzoate ester, coupling with diethyl glutamate and final ester hydrolysis afforded the title compound. This novel deazaaminopterin analogue was approximately as potent as methotrexate in vitro in terms of DHFR and L1210 cell growth inhibition. There are indications of diastereomeric differences in the enzyme inhibition measurements. A significant transport advantage over MTX for influx into L1210 cells was observed. The compound was active against the E 0771 murine mammary solid tumor, but further investigation with individual diastereomers is required to define the ED50. PMID- 1732550 TI - New neplanocin analogues. 1. Synthesis of 6'-modified neplanocin A derivatives as broad-spectrum antiviral agents. AB - Novel neplanocin A analogues modified at the 6'-position, i.e., 6'-deoxy analogues (2, 3, 6, 9, 20), 6'-O-methylneplanocin A (15), and 6'-C methylneplanocin A's (22a and 22b) have been synthesized and evaluated for their antiviral activity in a wide variety of DNA and RNA virus systems. These compounds showed an activity spectrum that conforms to that of S adenosylhomocysteine hydrolase inhibitors. They were particularly active against pox- (vaccinia), paramyxo-(parainfluenza, measles, respiratory syncytial), arena- (Junin, Tacaribe), rhabdo- (vesicular stomatitis), reo-, and cytomegalovirus. In order of (increasing) antiviral activity, the compounds ranked as follows: 3 less than 15 approximately 20 less than 6 less than 9 approximately 2 less than 22a. Of the two diastereomeric forms of 22, only 22a was active; 22a surpassed neplanocin A both in antiviral potency and selectivity. Compound 22a appears to be a promising candidate drug for the treatment of pox-, paramyxo-, arena-, rhabdo-, reo-, and cytomegalovirus infections. PMID- 1732551 TI - Synthesis and antifolate evaluation of the 10-propargyl derivatives of 5 deazafolic acid, 5-deazaaminopterin, and 5-methyl-5-deazaaminopterin. AB - 5-Deaza-10-propargylfolic acid (4), an analogue of the thymidylate synthase (TS) inhibitor 10-propargyl-5,8-dideazafolic acid (PDDF, 1), was prepared via alkylation of diethyl N-[4-(propargylamino)benzoyl]-L-glutamate (7) by 2-amino-6 (bromomethyl)-4(3H)-pyrido[2,3-d]pyrimidinone (15). Bromomethyl intermediate 15 was prepared from the corresponding hydroxymethyl precursor 14 by treatment with 48% HBr. Hydroxymethyl compound 14 was obtained by deamination of reported 2,4 diaminopyrido[2,3-d]pyrimidine-6-methanol (12a) in refluxing 1 N NaOH. Both 12a and its 5-methyl-substituted analogue 12b were converted to versatile 6 bromomethyl intermediates 13a and 13b from which important antifolates may be readily derived. Alkylation of 7 by 13a,b led to 10-propargyl-5-deazaaminopterin (5) and 5-methyl-10-propargyl-5-deazaaminopterin (6). As an inhibitor of TS from H35F/F cells, 4 gave an IC50 value showing it to be approximately 6-fold less inhibitory than PDDF (90 nM for 4 vs 14 nM for PDDF). In in vitro studies, IC50 (microM) values obtained for 4 vs L1210 and S180 of 1.50 and 2.35, respectively, were similar to those obtained for PDDF (2.61 and 1.97). Against HL60 cells, 4 was about 7-fold more cytotoxic than PDDF (IC50 values 0.72 and 5.29 microM). Inclusion of thymidine did not establish TS as the site of cytotoxic action for either 4 or PDDF in the cell lines used. In in vivo tests against L1210 in mice, 4 failed to show therapeutic effect. The 2,4-diamino compounds 5 and 6 were as potent inhibitors of DHFR from L1210 cells as MTX and 7- and 35-fold, respectively, more inhibitory than MTX toward L1210 cell growth. In mediated influx into L1210 cells, 5 and 6 were transported 2.7- and 8.5-fold, respectively, more readily than MTX. Against the EO771 mammary adenocarcinoma in mice, 6 produced greater antitumor effect than MTX. A dose of 36 mg/kg per day for 5 days caused no toxic deaths while the average tumor volume among 10 mice was reduced to 8-9% of that of the control, and 20% of the test animals were rendered tumor free. PMID- 1732552 TI - Structure-activity relationships of 1-[(2-hydroxyethoxy)methyl]-6 (phenylthio)thymine analogues: effect of substitutions at the C-6 phenyl ring and at the C-5 position on anti-HIV-1 activity. AB - The effect of substitution on the pyrimidine moiety of 1-[(2 hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and 1-[(2 hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine (HEPT-S) on anti-HIV-1 activity was investigated by synthesizing a series of 5-methyl-6-(arylthio) and 5 substituted-6-(phenylthio) derivatives. Preparation of the 5-methyl-6-(arylthio) derivatives was carried out based on either LDA lithiation of 1-[[2-(tert butyldimethylsiloxy)ethoxy]methyl]thymine (3) and 1-[[2-(tert butyldimethylsiloxy)ethoxy]methyl]-2-thiothymine (4) followed by reaction with diaryl disulfides or an addition-elimination reaction of 1-[[2-(tert butyldimethylsiloxy)ethoxy]-methyl]-6- (phenylsulfinyl)thymine (31) with aromatic thiols. Preparation of the 5-substituted-6-(phenylthio) derivatives was carried out based on either C-5 lithiation of the 1-[[2-(tert butyldimethylsiloxy)ethoxy]methyl]-6-(phenylthio)uraci l (41) with LTMP or the LDA lithiation of 5-alkyl-1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2 thiouraci l derivatives 45-47. Substitution at the meta position of the C-6 (phenylthio) ring by the methyl group improved the original anti-HIV-1 activity of HEPT, and introduction of two m-methyl groups to the phenylthio ring further potentiated the activity [EC50: 6-[(3,5-dimethylphenyl)thio]-1-[(2 hydroxyethoxy)methyl]thymine (28), 0.26 microM; 6-[(3,5-dimethylphenyl)thio]-1 [(2-hydroxyethoxy)methyl]-2-thiothymin e (30), 0.22 microM]. When the 5-methyl group was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity of HEPT was also improved remarkably [EC50: 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6 (phenylthio)-2-thiouracil (48), 0.11 microM; 5-isopropyl-1-[(2-hydroxyethoxy) methyl]-6-(phenylthio)-2-thiouracil (50), 0.059 microM; 5-ethyl-1-[(2 hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (54), 0.12 microM; 5-isopropyl 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (56), 0.063 microM]. 6 [(3,5-Dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]thymine derivatives 51 and 57 and 6-[(3,5-dimethylphenyl)thio]-5-isopropyl-1-[(2- hydroxyethoxy)methyl]thymine derivatives 52 and 58 inhibited the replication of HIV-1 in the nanomolar concentration range. PMID- 1732553 TI - Bis basic substituted diaminobenzobisthiazoles as potential antiarthritic agents. AB - A series of benzobisthiazoles were screened for antiinflammatory activity in the carrageenan paw edema and adjuvant arthritis tests. Compound 26, 2,6-bis(N,N diethylamino)benzo[1,2-d:5,4-d']bisthiazole, was found to inhibit the swelling of the uninjected paw in the prophylactic adjuvant arthritis model with an ED50 of 2.3 mg/kg orally. As with most compounds of this series, 26 was inactive in acute model of inflammation, such as paw edema; like steroids, it showed activity in the granuloma pouch assay but did not inhibit cyclooxygenase, indicating a mode of action different from the classical nonsteroidal antiinflammatory drugs (NSAID's). At doses higher than those producing antiinflammatory activity, 26 had some immunoregulating properties. PMID- 1732554 TI - New 8-(trifluoromethyl)-substituted quinolones. The benefits of the 8-fluoro group with reduced phototoxic risk. AB - A series of 8-(trifluoromethyl)-substituted quinolones has been prepared and evaluated for in vitro and in vivo antibacterial activity, and phototolerance in a mouse phototolerance assay. These analogues were compared to the corresponding series of 6,8-difluoro- and 6-fluoro-8H-quinolones (ciprofloxacin type). Although their in vitro antibacterial activities are less than the 6,8-difluoro analogues, the 8-(trifluoromethyl)quinolones are generally equivalent to their 8H analogues. In vivo, they are comparable to the 6,8-difluoro series and show up to 10-fold improvement in efficacy when compared to their ciprofloxacin counterparts vs Streptococcus pyogenes and Streptococcus pneumonia. In the phototolerance model, the 8-(trifluoromethyl)quinolones are comparable to the 8H-quinolones. Both of these series display much higher no effect doses (greater tolerance) than the corresponding 6,8-difluoroquinolones. PMID- 1732555 TI - Chromophore-modified antineoplastic imidazoacridinones. Synthesis and activity against murine leukemias. AB - The synthesis of 8-hydroxy and 8-methoxy analogues of some substituted 5 aminoimidazoacridinones (4) is described. The synthesis was carried out by a three-step sequence from the corresponding 1-chloro-4-nitro-9(10H)-acridinone precursors (1). The annulation of the imidazolo ring onto the aminoacridinone chromophore was accomplished by heating the required aminoacridinone (3) with formic acid or, in the case of 1-methyl derivatives, with N,N-dimethylacetamide. Potent cytotoxic activity against L1210 leukemia, as well as antitumor activity against P388 leukemia in mice, was demonstrated for the 8-hydroxy analogues. The corresponding 8-methoxy derivatives were not cytotoxic. However, in some cases, they showed significant in vivo antileukemic activity. PMID- 1732556 TI - Synthesis and biologic activity of 2'-fluoro-2-halo derivatives of 9-beta-D arabinofuranosyladenine. AB - The synthesis of 2-halo-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenines (4b and 4d) by coupling the 2,6-dihalopurine with 3-acetyl-5-benzoyl-2-deoxy-2-fluoro D-arabinofuranosyl bromide (2) followed by replacement of the 6-halogen with concomitant removal of the acyl blocking groups is described. 2-Fluoroadenine derivative 4g had to be prepared by the diazotization-fluorination of 2 aminoadenine nucleoside 4e. All three nucleosides provided good increases in life span of mice inoculated with P388 leukemia. The best results were obtained when the compounds were administered q3h x 8 on days 1, 5, and 9 after implantation of the leukemia cells. The 2',3'-dideoxynucleoside 5b, prepared by deacetylation of 4f and deoxygenation of the resultant 4h followed by removal of the benzoyl group of 5a, was slightly active against HIV in cell culture. PMID- 1732557 TI - Improvements in the minimum binding sequence of C5a: examination of His-67. PMID- 1732558 TI - Trauma versus critical care: it is time to end the debate. PMID- 1732559 TI - Delayed traumatic superior mesenteric arteriovenous fistula after a stab wound: case report. AB - Eight cases of superior mesenteric arteriovenous fistula (SMAVF) occurring after an abdominal stab wound have been reported. Seven of these patients manifested symptoms within 1 month after the stab wound. We report the case of a 28-year-old man whose SMAVF occurred 31 months after an abdominal stab wound. A SMAVF must be considered even when considerable time has elapsed between penetrating abdominal trauma and symptoms of abdominal pain, unexplained diarrhea, or an abdominal bruit or thrill. PMID- 1732560 TI - Retropharyngeal abscess, airway obstruction, and tetraplegia after hyperextension injury of the cervical spine: case report. AB - A retropharyngeal abscess resulted from perforation of the posterior pharyngeal wall by an anterior marginal osteophyte at the time of hyperextension injury of the cervical spine. The abscess communicated with the epidural space via the disrupted intervertebral disk, leading to the delayed onset of upper airway obstruction and tetraparesis. PMID- 1732561 TI - Coronal fracture of the body of the hamate: case reports. AB - Coronal fracture of the body of the hamate with associated dorsal subluxation of the bases of the fourth and fifth metacarpals is rare. Two cases of this injury with and without disruption of the dorsal carpometacarpal ligaments treated with open reduction and internal fixation are reported. Oblique roentgenographic views with the hand pronated 30 degrees from the true lateral were helpful in the assessment of the fracture. PMID- 1732562 TI - Computed tomography of posterior fracture-dislocations of the shoulder: case reports. AB - Computed tomographic (CT) evaluation of seven posterior shoulder dislocations in five patients is reported. Computed tomography provided better visualization of the trough fracture in the humeral head than did conventional x-ray films or tomography. It also demonstrated fracture fragments not seen on conventional roentgenograms. The two cases of bilateral dislocation and one case of unilateral dislocation were caused by seizures. The two other cases of unilateral dislocation were caused by trauma. PMID- 1732563 TI - A complicated forearm greenstick fracture: case report. AB - Closed reduction of a greenstick fracture of the forearm in a child of 9 years was complicated by interposition of the deep flexor tendon of the third, fourth, and fifth fingers in the fracture gap. Undelayed release of the tendon resulted in a complete functional recovery. PMID- 1732564 TI - Transverse divergent dislocation of the elbow in a six-year-old boy: case report. AB - Simultaneous dislocation of the elbow and the proximal radioulnar joint is a rare occurrence. Closed reduction led to a cure in a 6-year-old boy with transverse divergent dislocation of the elbow. PMID- 1732565 TI - Mucormycosis in trauma patients. AB - Cutaneous mucormycosis is a rare but often fatal infection in trauma patients. We retrospectively reviewed a 9-year experience with mucormycosis among injured patients. Eleven patients had biopsy- or culture-proven mucormycosis. Nine patients were victims of blunt trauma, two patients had burns measuring greater than 50% TBSA. No patient was at increased risk because of underlying disease or immunosuppression prior to injury. All 11 patients had open wounds on admission. Four patients died of mucormycosis. All nonsurvivors had phycomycotic gangrenous cellulitis of the head, the trunk, or both. In contrast, survivors had involvement of only the extremities. Because of underlying disease, contaminating wounds, antibiotic use, or immunocompromise secondary to shock and sepsis, trauma patients are at risk of developing mucormycosis. To successfully treat mucormycosis, diagnosis must be prompt and accompanied by aggressive debridement and parenteral administration of amphotericin B. PMID- 1732566 TI - Hepatic portal venous gas identified by computed tomography in a patient with blunt abdominal trauma: a case report. PMID- 1732567 TI - Alternative approaches to abdominal wound closure in severely injured patients with massive visceral edema. AB - Excessive tension in an abdominal incision line may lead to fascial necrosis and wound sepsis. We utilized two alternative approaches to wound closure in 13 patients with severe abdominal trauma (2 blunt, 11 penetrating) whose midline incision could not be closed primarily without excessive tension at the initial operation because of massive visceral edema. In five patients synthetic mesh was used to bridge the fascial defect. Four patients survived the early postoperative period but had large open midline wounds that required one or more delayed procedures to close the wound or cover the visceral mass with skin graft. Two patients currently have large abdominal wall hernias. In the other eight patients the skin was reapproximated over the visceral mass utilizing towel clips at the initial operation. Six patients survived to be reexplored within 48-96 hours. Acute hemorrhage had stopped, the edema of the bowel and retroperitoneum had largely resolved, and the fascia could be closed primarily without excessive tension. All wounds went on to heal satisfactorily. When massive edema makes fascial closure at the initial operation difficult or impossible, closure of the skin over the visceral mass promotes resolution of the edema and often allows satisfactory primary closure within 48-96 hours. Synthetic mesh should be reserved for cases of abdominal wall tissue loss or dehiscence associated with wound sepsis. PMID- 1732568 TI - Thromboembolism following multiple trauma. AB - The true incidence of thromboembolic complications following multiple trauma is unknown, and no method of prophylaxis has been shown to be both safe and effective in managing seriously injured patients. In this prospective study, 113 trauma patients were assigned on admission to receive either low-dose heparin (LDH), (5,000 U subcutaneously every 12 hours) or to wear sequential compression devices (SCDs) as prophylaxis against the development of deep venous thrombosis (DVT). Both groups of patients were serially studied with duplex venous ultrasound imaging to detect thrombus in the veins of the thigh. Ventilation perfusion lung scans and pulmonary angiograms were performed when pulmonary embolism (PE) was suspected clinically. There were 12 patients who had thromboembolic complications, including 9 of 76 in the SCD group (12%) and 3 of 37 in the LDH group (8%). Five patients had DVT only, four had PE without detectable DVT, and three had both DVT and PE. None of the patients with PE died, and there were no major complications associated with either method of prophylaxis. Compared with the patients who did not develop DVT/PE, those with thromboembolic complications were older (49 +/- 23 vs. 36 +/- 17 years, p less than 0.02), spent more hospital days immobilized (24 +/- 15 vs. 10 +/- 13 days, p less than 0.001), received more transfusions (11 +/- 12 vs. 3 +/- 5 U, p less than 0.001) and had clotting abnormalities on admission, as demonstrated by prolonged PTT values (39 +/- 28 vs. 26 +/- 5 seconds, p less than 0.001). It appears that there is an identifiable subgroup of injured patients at highest risk for PE who warrant both prophylaxis and close surveillance for DVT. PMID- 1732569 TI - The pharmacokinetics of prophylactic antibiotics in trauma. AB - Despite prophylactic antibiotic use in abdominal trauma patients, infection rates remain high. A previous study from our institution indicated that higher doses of prophylactic antibiotics in trauma patients could significantly reduce subsequent infection rates. To determine if this resulted from altered pharmacokinetic profiles, we performed individualized pharmacokinetic analysis of the prophylactic amikacin regimens given to 28 trauma patients undergoing laparotomy. Patients were prospectively randomized to receive a standard regimen of 11 mg/kg of amikacin every 12 hours or to have their regimens adjusted based upon pharmacokinetic analysis. Repeated pharmacokinetic analyses were performed daily for the three-day prophylactic regimen. There was a significant expansion in the apparent volume of distribution for amikacin that correlated with fluid resuscitation. This, along with increased elimination rates, helps to explain the failure to achieve adequate amikacin levels using standard regimens in trauma patients. Such underdosing may contribute to relatively high infection rates following major abdominal injury. PMID- 1732570 TI - Neurologic consequences of traumatic asphyxia. AB - Patients with traumatic asphyxia treated at a single institution during a 10-year period were studied to determine the incidence and sequelae of neurologic impairment associated with this entity. Traumatic asphyxia was identified in 14 patients from 4 to 73 years old. Each had sustained thoracic crush injuries from objects weighing more than 1,000 pounds. The mechanism of injury was crush by farm implement in six patients, entrapment beneath a vehicle in five, compression by a large hay bale in one, crush by a farm animal in one, and a ditch cave-in in one. Craniocervical cyanosis and subconjunctival hemorrhage were apparent in all patients. Associated chest wall and intrathoracic injuries were present in 11 (79%) patients. Neurologic abnormalities included loss of consciousness in eight patients, prolonged confusion in five, seizures in two, and pronounced visual disturbances in two. There were no deaths in this series and no long-term neurologic sequelae were evident. However, careful serial neurologic assessment should be performed in these patients and other causes of neurologic symptoms excluded. PMID- 1732571 TI - Revision of TRISS for intubated patients. AB - The TRISS system is an important, widely used method for predicting survival in trauma patients. One significant shortcoming of TRISS is its inability to include intubated patients in survival analysis because a respiratory rate and a verbal response are not obtainable. This report describes one approach to this problem. Data from 994 patients with blunt trauma were examined. Like TRISS, survival probability was calculated using a logistic regression model that included age and Injury Severity Score (ISS); however, the best motor response and systolic blood pressure were used in place of the Revised Trauma Score (RTS). With this model, the sensitivity, specificity, and misclassification rate were 57%, 98.9%, and 3.6%, respectively. For TRISS, the sensitivity, specificity, and misclassification rate are 58.8%, 99.3%, and 3.0%, respectively. Thus, our model has predictive performance comparable with TRISS. More importantly, it is applicable to intubated patients who are not pharmacologically paralyzed. Further investigation with larger data bases is necessary. PMID- 1732572 TI - Autotransfusion of potentially culture-positive blood (CPB) in abdominal trauma: preliminary data from a prospective study. AB - Increased use of autotransfusion for traumatic hemorrhage may reduce amounts of banked blood needed for severe injuries. Autotransfusion is standard for traumatic hemothorax, but has been limited for abdominal injuries. This prospective study used microbiologic data from 152 patients with intestinal injuries. Where anticipated blood loss was greater than 1,000 mL, blood from the peritoneal cavity was cultured, washed, concentrated, and recultured before reinfusion. Infection rates were stratified using the Penetrating Abdominal Trauma Index (PATI). Fifty patients with PATI greater than 20 who received banked blood (group I) (mean: 1,800 mL) were compared with 20 patients (group II) who received autotransfused, potentially culture-positive blood (CPB) (mean: 3,900 mL). Wound infection rates were identical in both groups (25%). No statistically significant increase was found in site-specific infection risk when severity of injury was stratified according to PATI. Bacteremias, pulmonary infections, and urinary infections were not caused by bacteria cultured from autotransfused blood. We conclude that washed CPB may be autotransfused without significantly increased risk of infection in patients with severe abdominal injuries. PMID- 1732573 TI - Identification of preventable trauma deaths: confounded inquiries? AB - The published evaluation of methods for identifying preventable trauma deaths contains many unstudied confounding factors. To investigate the reliability of methods for identifying such preventable deaths, we compared three consensus systems using separate five-member general review panels assessing 20 non-central nervous system fatalities: panel A, independent judgments; panel B, discussion of all cases preceding individual judgments; and panel C, independent judgments followed by discussion and equivocal case reassignment. The Kappa concordance index was low for all methods (method A, 0.20; methods B and C, 0.40). Of the 11 deaths judged preventable by at least one panel, only one death was judged preventable by all three panels. Consensus agreement (four of five assessors) was 20% for panel A, 45% for panel B, and 10% for panel C (difference between panels B and C, p less than 0.03). In panel C, discussion affected the rate of equivocal case designation from 30% to 5%. Thus different consensus methods yielded different results. We conclude that individual case review can be severely flawed and therefore should not be used to measure institutional quality of patient care. We recommend that assessment of institutional performance should be based on objective evaluation methods, which require the study of patient population outcomes, rather than on subjective methods in which individual cases are reviewed. PMID- 1732574 TI - Diagnostic peritoneal lavage: integration with clinical information to improve diagnostic performance. AB - Management of abdominal trauma requires both the detection of injuries sustained and an ability to distinguish patients who require operative repair from those who do not. In this prospective study of 200 patients receiving diagnostic peritoneal lavage (DPL) following blunt trauma, relationships among DPL result, clinical features (information from initial patient assessment), and laparotomy outcome were investigated. The DPL result alone predicted requirement for laparotomy with an accuracy of 93%, a specificity of 96%, a sensitivity of 85%, a positive predictive value (PV-Positive) of 87%, and a negative predictive value (PV-Negative) of 95%. Combining clinical features with the DPL result reduced the number of unnecessary laparotomies (increased PV-Positive and specificity), but increased the number of missed necessary laparotomies (decreased PV-Negative and sensitivity). The best diagnostic performance was found by combining the DPL result with circulatory status, which, in this series of patients, predicted necessary laparotomy with an accuracy of 95%, a specificity of 99%, a sensitivity of 81%, a PV-Positive of 98%, and a PV-Negative of 94%. PMID- 1732575 TI - The validity of self-reported behavioral risk factors: seatbelt and alcohol use. AB - Data from state telephone surveys of self-reported seatbelt use, driving while intoxicated, and drinking five or more alcoholic drinks at one sitting were compared with objectively observed belt use in traffic and evidence of blood alcohol in fatally injured drivers. Self-reported belt use overstates actual use by more than 20 percentage points on average. Self-reported alcohol use is not predictive of the percentage of fatally injured drivers with evidence of blood alcohol among the states. PMID- 1732576 TI - Intoxication and injury. AB - The relationship between alcohol use and injury severity was investigated in trauma patients admitted to a tertiary referral hospital during a 23-month period. Admission blood alcohol levels (BALs) were obtained on 427 trauma patients, who were stratified into three groups: those with no measurable blood alcohol, those within the legal limit of 100 mg/dL, and those over the legal limit or intoxicated. The no-alcohol group had significantly lower injury severity than the other two groups (p less than 0.001). Even when the BAL was well within the legal limit, injuries suffered by those in the alcohol-positive groups were more severe than those in the no-alcohol group. Confirmatory evidence of the effect of alcohol on injury severity was reflected by a 2.3% mortality in alcohol-negative patients compared with a 13.3% death rate in alcohol-positive patients (p less than 0.0001). To assess the potentially confounding effect of alcohol on injury scoring accuracy, we examined the change in Glasgow Coma Scale (GCS) scores following admission. No significant differences were found when admission GCS values were compared with GCS determinations made 24 hours following admission by separate observers. To correct for any potential bias as a tertiary referral center, repeat analysis with exclusion of transferred patients was done with essentially no change in results. Our data revealed a highly significant relationship between alcohol use, degree of injury, and resource consumption. PMID- 1732577 TI - Examination of the pathologic anatomy of ankle fractures. AB - A prospective study of the translational and rotational displacement of the lateral malleolus in ankle fractures was carried out utilizing roentgenographic techniques. Twenty-six ankle fractures in 25 patients were studied using both routine plain films and CT scanning with two- and three-dimensional multiplanar reconstruction. Eighty-one percent were Lauge-Hansen supination-external rotation type injuries. Overall, 21 fractures did not involve the medial malleolus. Initial talar shift was less than or equal to 2 mm in 15 fractures. Although all patients exhibited external rotation deformities of the lateral malleolus on plain films, only one fracture was found to possess any degree of external rotation relative to the talus. The proximal fibula was seen on CT scans to have increased internal rotation with respect to the tibia in 19 cases. One patient had a slightly externally rotated proximal fibula; the remainder appeared normally aligned. The displacements measured by the CT scans at the talofibular articulation were compared with the standard plain film measurements. The displacements at the distal lateral malleolus were consistently overestimated by the plain roentgenograms, presumably because the capsular and ligamentous attachments to the distal fibula limit malleolar displacement. The talocrural angle, determined on both plain films and CT scans, was also not found to be a sensitive measure of fibular shortening nor of the severity of the fracture. The results of this study suggest that, in an isolated lateral malleolar ankle fracture, the apparent external rotation of the fracture fragment is relative only to the proximal fibula and is not associated with derangement of the talofibular articulation. Based on these mechanical considerations, surgical intervention for such fractures may not be necessary. This hypothesis is consistent with previous long-term clinical studies. PMID- 1732578 TI - A preliminary experience with the Russell-Taylor reconstruction nail for complex femoral fractures. AB - Eleven cases of complex femoral fractures were seen from November 1987 to November 1989; five ipsilateral femoral neck and shaft fractures and six comminuted subtrochanteric fractures. High-energy accidents accounted for most of these injuries. There were numerous associated injuries, many requiring operative procedures. All of the fractures were treated with Russell-Taylor reconstruction nails. All fractures united, but there were two delayed unions. There was no delay in diagnosis of the femoral neck fractures, and all healed without avascular necrosis. Malalignment occurred in one case, shortening of the femur occurred in two cases, and in two cases only one screw could be placed in the femoral head. In three patients technical errors related to nail insertion led to fracture complications. The use of the Russell-Taylor reconstruction nail is technically demanding. However, we conclude that in complex femoral fractures, this device offers superior stabilization over other currently used methods of internal fixation. PMID- 1732579 TI - Open tibial shaft fractures: a comparative analysis of different methods of fixation in southwestern Greece. AB - A change in the method of managing open-grade-III tibial shaft fractures provided a new opportunity for a comparative study. One series of patients was treated exclusively by internal fixation and compared with another series treated with external fixation solely as well as with a series treated initially by external skeletal fixation and later by "Sarmiento walking plaster." The latter method was found to be a successful treatment and a good alternative to internal fixation for open grade-II and grade-III tibial shaft fractures when soft-tissue healing was completed. The supplementary use of the Sarmiento walking plaster had dramatically decreased the duration of hospital stay, saving the patient from an additional operation. There were no nonunions in this series. In open grade-I-II tibial shaft fractures, the deep infection rate in the cases in which internal fixation was used was significantly higher (5.4%), than that observed in the cases treated with external fixation, in which there was no deep infection. The nonunion rate was higher (22%) in the external fixation group compared with the internal fixation group (9%). The functional impairment of the ankle joint of the affected limb was less (15%) by using internal fixation than that of either the external fixation group (20%) or the group where the external fixation was changed to a Sarmiento walking plaster (35%). PMID- 1732580 TI - Successful use of autologous fibrin gel in traumatic bronchopleural fistula: case report. AB - Bronchopleural fistula has been successfully treated by bronchoscopic application of fibrin glue. We report the use of intrathoracic fibrin gel pleurodesis in traumatic bronchopleural fistula. PMID- 1732581 TI - Magnetic resonance imaging in traumatic diaphragmatic rupture: case reports. AB - Acute traumatic diaphragmatic rupture is usually diagnosed by plain chest x-ray studies or at laparotomy. On occasion, ancillary diagnostic procedures such as computed tomography (CT) and fluoroscopy are necessary for diagnosis. Suspected acute traumatic diaphragmatic rupture was definitively diagnosed by magnetic resonance (MR) imaging in two patients. In another three patients, MR imaging was used to rule out diaphragmatic rupture. Magnetic resonance imaging may be the ancillary diagnostic procedure of choice following equivocal chest radiographs. PMID- 1732582 TI - Acute adrenal insufficiency presenting as shock after trauma and surgery: three cases and review of the literature. AB - Profound nonhemorrhagic shock developed in one postoperative and two trauma patients. Cardiovascular collapse was characterized by severe hypotension (systolic blood pressure less than 80 mm Hg), hyperdynamic cardiac indices (CI greater than 4 L/min/m2), low systemic vascular resistance (SVR less than 500 dyne.sec/cm5.m2), and multiple organ failure. Sepsis was not found by culturing of specimens or visual inspection at laparotomy. Screening cortisol levels were low (less than 2 micrograms/dL in two patients) and did not respond appropriately to synthetic ACTH (cosyntropin) challenge. Administration of exogenous glucocorticoids promptly and dramatically reversed shock and organ failure in two patients. Oral glucocorticoid and mineralocorticoid supplementation were required at hospital discharge. Acute adrenal insufficiency is rare after trauma, but may produce life-threatening cardiovascular collapse, mimicking the "septic" shock state. Cosyntropin stimulation testing confirms the diagnosis and is accurate in traumatized patients. Outcome is dependent upon early recognition and exogenous glucocorticoid administration. Appropriate endocrine evaluation prevents unnecessary use of steroids in a population of trauma patients who are already in a state of immunosuppression. PMID- 1732583 TI - The natural history of asymptomatic urolithiasis. AB - An inception cohort of 107 patients was reviewed to establish the natural history of asymptomatic urolithiasis. With an over-all mean followup of 31.6 months, 73 patients (68.2%) remained asymptomatic and were censored at the time of the last clinical visit. A symptomatic event developed in 34 patients (31.8%). Spontaneous passage occurred in 16 patients (15.0%), endoureteral removal was done in 6 (5.6%), percutaneous nephrostolithotomy was done in 3 (2.8%) and 9 (8.4%) were referred for therapeutic lithotripsy. Cumulative 5-year probability of a symptomatic event developing was 48.5%. A linear association was identified between the development of a symptomatic event and the number of previous stones as well as the number of asymptomatic stones at identification. A significant burden of illness is associated with an expectant strategy as an approach to asymptomatic urolithiasis. Of the patients who had a symptomatic event 47% had spontaneous stone passage, while 26.5% required urological intervention and 26.5% were referred for therapeutic lithotripsy. Prophylactic extracorporeal shock wave lithotripsy, although often advocated, has associated risks and is not always a benign procedure. A randomized controlled trial is required to evaluate properly the role of prophylactic lithotripsy versus an expectant strategy. PMID- 1732584 TI - Inferior pole collecting system anatomy: its probable role in extracorporeal shock wave lithotripsy. AB - In addition to the gravity-dependent position, we believe that other particular anatomical features may be important in the retention of stone debris in the lower calices after extracorporeal shock wave lithotripsy (ESWL). We analyzed the inferior pole collecting system anatomy in 146, 3-dimensional polyester resin corrosion endocasts of the pelviocaliceal system. The inferior pole was drained by multiple calices disposed in 2 rows in 56.8% of the cases and by 1 midline caliceal infundibulum in 43.2%. In 60.3% of the cases there was a lower infundibulum equal to or greater than 4 mm. in diameter and 39.7% had a lower infundibulum smaller than 4 mm. in diameter. In 74.0% of the cases an angle of greater than 90 degrees was formed between the lower infundibulum and the renal pelvis, and in 26.0% the angle was 90 degrees or smaller. We believe that the physician should consider these anatomical features when suggesting ESWL to treat calculi in the lower calices. PMID- 1732585 TI - Urinary acidification in renal stone patients from northeastern Thailand. AB - Hypokalemia, hypokaliuria and hypocitraturia are common findings in patients with renal stone disease in Northeastern Thailand. However, hyperchloremic metabolic acidosis seldom is seen. Therefore, we studied renal acidification in 29 renal stone disease patients who were living in rural Northeast Thailand. Baseline blood and average 24-hour urine biochemical parameters were measured. Hypokalemia, hypokaliuria and hypocitraturia were found in 10%, 83% and 93% of the patients, respectively. By multiple regression, urinary citrate excretion correlated positively with serum potassium and urinary potassium excretion, and negatively with urinary ammonium (r = 0.640, p = 0.005). An abnormal response to acid loading was found in only 1 patient. Thus, hypokaliuria and hypocitraturia in our renal stone disease subjects were infrequently due to distal renal tubular acidosis. Perhaps potassium depletion might be a contributing factor in these metabolic abnormalities. PMID- 1732586 TI - Clinical aspects of polycystic kidney disease. AB - A total of 316 patients (167 men and 149 women) with autosomal dominant polycystic kidney disease was studied retrospectively by a multi-institute group. With advancing patient age renal function decreased, and blood pressure, prevalence of liver cysts and probability of end stage renal failure increased. The probability of end stage renal failure was 39% in the patients in their sixties. Regression analysis indicated that polycystic kidney disease patients could expect to lose 1.1 ml. per minute of creatinine clearance per year, reaching a level of 10 ml. per minute, a point of end stage renal failure, by the age of 72.7 years. The better prognosis in our study than that reported previously in white patients might be due to the inclusion of more asymptomatic persons and/or milder genotypic expression of polycystic kidney disease in Japan. There was no sex difference in the prevalence of liver cysts (54.6%), pancreatic cysts (7.1%), intracranial aneurysms (8.0%) and hypertension (63.6%). The occurrence of pancreatic cysts was significantly associated with liver cysts. Our study clarifies several clinical characteristics of polycystic kidney disease in Japan. PMID- 1732588 TI - Renal function in unilateral nephrectomy subjects. AB - Renal function in 32 subjects who had undergone unilateral nephrectomy (17 transplant donors and 15 subjects with unilateral renal disease) was compared with that of 22 normal subjects. The age-adjusted glomerular filtration rate was lower in transplant donors (79 +/- 15% of normal) than in those whose nephrectomy was performed for unilateral renal disease (90 +/- 12% of normal). The donors were also significantly older at nephrectomy (48 +/- 10 years versus 24 +/- 13 years, p less than 0.001). This finding may represent less capacity for compensatory hypertrophy. Proximal tubular and medullary function as assessed by 15-minute phenolsulfonphthalein excretion, maximum urinary concentration in response to water deprivation plus exogenous vasopressin, and urinary acidification in response to an oral acid load were all within normal limits for glomerular filtration rate. Overall renal function was well preserved after nephrectomy. A small number of patients did have increased cast excretion, which may signify the presence of mild renal disease in these subjects. PMID- 1732587 TI - Xanthogranulomatous pyelonephritis: experience in 36 cases. AB - A retrospective study of 36 patients with xanthogranulomatous pyelonephritis who underwent nephrectomy at our hospital was performed. The disease occurred most frequently in middle-aged women with a history of recurrent urinary tract disorder. There were 2 cases of focal xanthogranulomatous pyelonephritis, 2 associated with emphysematous pyelonephritis, 2 that manifested as fistula formation between the colon or skin, and 1 with deep sinus formation into the hip joint that presented as septic arthritis. Flank pain and fever were the most frequent complaints. Escherichia coli (67%) and Proteus mirabilis (26%) were the most common organisms isolated from the voided urine, kidney and blood stream. Cephalothin plus gentamicin or tobramycin were the drugs of choice before surgical intervention. PMID- 1732589 TI - A survey on incidental renal cell carcinoma in Japan. AB - A survey of renal cell carcinoma detected incidentally (incidental renal cancer) in 116 collaborating institutions was conducted nationwide in Japan between 1980 and 1988. The analysis of 1,428 cases demonstrated a dramatic increase in the frequency of incidental renal cancer from 20 cases in 1980 to 338 cases in 1988. Detection occurred at routine health examinations in 32.8% of the cases and during examinations for unrelated diseases in the remainder. The chief method of detection was ultrasonography (68.2%), followed by computerized tomography (21.8%) and excretory urography (6.4%). Tumor extension was within the renal capsule in 76.5% of the cases and the mean tumor diameter was 5.4 cm. The 5-year survival rate was 76.5% but it was better in cases detected by ultrasonography compared with other methods. These results were compatible with previous reports and suggest that incidental renal cancer detection may improve the prognosis of renal cancer as a whole. PMID- 1732590 TI - Interleukin-2 by inhalation: local therapy for metastatic renal cell carcinoma. AB - We report the use of inhaled natural interleukin-2 in patients with metastatic disease. Six patients with metastatic renal cell carcinoma received 100,000 units natural interleukin-2, 5 times per day by inhalation in addition to a 4-day cycle of intravenous natural interleukin-2 every 2 weeks and subcutaneous interferon 5 days per week. Response was clearly correlated with metastatic site. Distinct tumor burden and the poor condition of the patient did not impair success. Pulmonary metastases responded in 5 of 5 patients. Metastases in the mediastinum, liver, abdomen and pelvis were stabilized in 4 patients. No response was noted in 3 solitary bone metastases, which were successfully removed surgically after several months of therapy, and a pleural metastasis progressed despite a clear response of the pulmonary disease in the same patient. New metastases did not develop in any of the patients during treatment (median followup 183 days of treatment, range 97 to 296 days). The over-all importance of the low toxicity of this novel route of administration (World Health Organization classification not exceeding grade 1) making long-term outpatient treatment possible must be emphasized because limitations of systemic interleukin-2 application are mainly caused by pulmonary side effects, for example pulmonary capillary leakage syndrome and edema. However, this new type of topical natural interleukin-2 application and combination with low dose intravenous interleukin-2 achieved considerable antitumor responses. PMID- 1732591 TI - Do upper ureteral stones need to be manipulated (push back) into the kidneys before extracorporeal shock wave lithotripsy? AB - The current practice for the management of upper ureteral stones is to push the stone back into the renal pelvis before extracorporeal shock wave lithotripsy (ESWL*). The results in 903 patients with an upper third ureteral stone pushed back before ESWL were compared to those of 815 with an upper third ureteral stone treated by ESWL in situ with a ureteral stent bypassing the stone. The stone size in the in situ group was larger than in the push back group. More shocks at a higher kilovoltage were required to treat the in situ group. The retreatment rate and post-ESWL secondary procedure rate for the push back group with single stones were 4% and 1.5%, respectively, compared to 5% and 7.5%, respectively, for the in situ group. The stone-free rate with single stones at 3 months was 73% in the push back group and 79% in the in situ group. There appears to be little advantage in manipulating a ureteral stone into the kidney (push back) before treatment by ESWL. PMID- 1732592 TI - Endopyeloureterotomy via a transpelvic extraureteral approach. AB - Endopyeloureterotomy has been accepted as a procedure to relieve obstruction of the ureteropelvic junction and upper ureteral stenosis. However, in patients with a long stenotic segment poor results are often obtained with the conventional technique. To resolve this problem we developed a new technique using a 22F urethrotome and a transpelvic extraureteral approach. In this technique the renal pelvis was incised for 1 to 1.5 cm. from the ureteropelvic junction in the direction of the parenchyma using the cold knife of the urethrotome under direct vision. For upper ureteral stenosis the dilated pelvic and ureteral posterolateral walls were incised 1 to 1.5 cm. from the stenotic segment toward the ureteropelvic junction. Then, the stenotic segment was treated with the urethrotome after it was advanced into the retroperitoneal space through the incision in the renal pelvis. We treated 21 patients with the new technique between August 1988 and August 1990. Our series included 3 patients with the high insertion type of ureteropelvic junction obstruction and 4 with a long stenotic segment. The success rate was 95% without any severe complication. These results indicate that our new technique could become a useful procedure for endopyeloureterotomy. PMID- 1732593 TI - Conversion from external appliance wearing or internal urinary diversion to a continent urinary reservoir (Florida pouch I and II): surgical technique, indications and complications. AB - A total of 20 patients with diversion requiring an external appliance or internal urinary diversion underwent conversion to a continent urinary reservoir (Florida pouch I or II). All patients subsequently reported an improvement in the quality of life and expressed satisfaction with the new urinary diversion procedure. Of the patients 15 (75%) previously had an ileal conduit, while 1 (5%) had undergone ureterosigmoidostomy, 1 (5%) had cutaneous ureterostomy, 1 (5%) had a suprapubic tube, 1 (5%) had a sigmoid conduit and 1 (5%) had a cecal conduit. After the original diversion 3 patients (15%) had recurrent urinary infections, 3 had complications related to the stoma and external appliance (stenosis and skin dermatitis) and 5 (25%) had ureteral obstruction in 7 ureters. A total of 17 patients with conduits (85%) underwent conversion via different surgical technical aspects depending on the status of the intestinal segment from the conduit and the function of the ureteral reimplantation: in 14 the conduit was discarded or was used only to patch the newly created Florida I colonic pouch, while in 6 the conduit was preserved and 9 ureterointestinal reimplantations were left undisturbed (Florida pouch II). Among 7 ureters preoperatively obstructed (original diversion), reimplanting them into the pouch failed to prevent further renal damage in 5 (71%). Three renal units required nephrectomy, 2 kidneys deteriorated and 2 recovered renal function after percutaneous balloon dilation and stenting. Among 31 preoperatively nonobstructed renoureteral units (original diversion), 22 were reimplanted into the colonic reservoir. One of these units (4.2%) became obstructed postoperatively and 3 (13.5%) presently have reflux. The 10 reimplantations left undisturbed in the detubularized conduit drain satisfactorily without postoperative obstruction and in 6 reflux has not been demonstrated. Renal function (serum creatinine) is preserved in all patients but 15 (75%) have hyperchloremia of mild degree. Two patients (10%) have acidosis and 1 (5%) of these had low red blood cell folic acid. Conversion of an external or internal diversion to a continent colonic urinary reservoir (Florida pouch I or II) can be successful and improve the quality of life of the patient. The functioning renal units that were preoperatively obstructed were associated with a high failure rate (71%) after reimplantation. Metabolic alterations will require long-term followup, and are particularly worrisome in children and young adults. PMID- 1732594 TI - Further experience with the urethral Kock pouch. AB - In 185 men a urethral Kock pouch was constructed as a bladder substitute after radical cystectomy for cancer. A total of 117 patients was followed for a minimum of 1 year and is fully evaluable. Of the patients 108 (92%) are completely continent during the day, while 85 (73%) are dry at night. Also, 8 patients had an excellent response to imipramine hydrochloride. Stability or improvement in the configuration of the upper tract was noted in 210 renal units (90%). A total of 24 renal units showed evidence of deterioration due to reflux (16) and an anastomotic stricture (8). Stability of the antireflux nipple valve was ensured by creation of a window in the mesentery of the corresponding bowel segment and by anchoring the valve to the wall of the pouch by an additional row of staples. On the basis of this favorable outcome the procedure is recommended for male patients for whom cystectomy is indicated and in whom the urethra can be preserved. PMID- 1732595 TI - Extraperitoneal pelvioscopy in lymph node staging of bladder and prostatic cancer. AB - In view of the inadequate accuracy of radiological investigations, surgical lymphadenectomy is generally the last resort to assess lymph node involvement in bladder and prostatic cancers. Extraperitoneal pelvioscopy is a simple and effective method to avoid such invasive surgery, which is always slightly regrettable to perform purely for staging purposes. The investigation is performed with the patient under low spinal anesthesia via a short iliac incision using an instrument derived from the mediastinoscope. It allows biopsies from the external iliac, internal iliac, common iliac and obturator lymph node chains. We analyzed our results of pelvioscopy in 101 patients (36 prostatic and 65 bladder cancers). Extraperitoneal pelvioscopy, unilateral in 78 and bilateral in 23 cases, corrected the conclusions of the radiological assessment in 39% of the prostatic cancer cases and in 28% of the bladder cancer cases. The specificity and positive predictive value is 100%, sensitivity 84%, negative predictive value 93% and over-all reliability 95%. On the basis of the quality of the results and the low morbidity (5 cases of rapidly resolving lymphorrhea, 1 injury to the external iliac vein and 1 obturator nerve lesion), extraperitoneal pelvioscopy can be considered as a useful complement to the preoperative staging of bladder and prostatic cancer. PMID- 1732596 TI - Intravesical instillations of 4-epi-doxorubicin (epirubicin) in the prophylactic treatment of superficial bladder cancer: results of a controlled prospective study. AB - A controlled prospective study in 65 patients was done to evaluate the efficacy of intravesical epirubicin administration as prophylactic treatment in regard to the pattern of tumor recurrences after complete endoscopic resection of superficial transitional cell carcinoma of the bladder. Intravesical instillations of the drug were given weekly for 6 consecutive weeks and to the responders an intermittent maintenance therapy was administered for the first 2 years after each followup examination. Of the patients treated prophylactically with epirubicin 37% had recurrence within a total of 1,136 patient-months compared to 55% of the controls who were followed for a total of 436 months, a difference that was not statistically significant (p greater than 0.05). However, examining the results in another manner, the control patients demonstrated a significantly shorter mean interval to recurrence and higher recurrent tumor rate per 100 patient-months. To clarify further the efficacy of epirubicin therapy, comparisons of the treatment outcome according to several tumor factors were done. These comparisons revealed a significant benefit for those who received epirubicin with respect to history of tumor recurrences and multiplicity at presentation. Drug-induced toxicity was acceptable. Our study suggests that epirubicin is safe and effective against the recurrence of superficial bladder cancer. PMID- 1732597 TI - Bacillus Calmette-Guerin therapy for high risk stage T1 superficial bladder cancer. AB - Numerous studies have shown bacillus Calmette-Guerin (BCG) to be an effective prophylactic and therapeutic agent for superficial transitional cell carcinoma of the bladder. The high grade stage T1 lesion treated by transurethral resection alone is reported to progress to muscle invasion in 30 to 50% of the patients. Therefore, some have recommended treatment with cystectomy. To evaluate BCG treatment of the stage T1 lesion we reviewed our results with a single or repeated 6-week course of the Armand-Frappier Pasteur strain BCG and compared them with those in the literature. We also compared these results with those of treatment of the stage TA lesion. We treated 30 stage T1 cancer patients who were described as at high risk based on the criteria of histology grade 3 in 24 and grade 2 in 6, carcinoma in situ present in 14 and positive urine cytology results 2 to 3 weeks after transurethral resection in 26. Followup ranged from 12 to 78 months, with a mean of 39 months. After a single 6-week course of BCG 14 patients (47%) had negative cytology and biopsy findings at 6 months. Also, 6 patients had conversion to negative cytology and biopsy results after a second 6-week course of treatment, for an over-all complete response rate of 66%. After the initial course of BCG 4 patients had progression to cystectomy: 1 for muscle invasion and 3 for a persistent stage T1 lesion. They had no evidence of disease 12 to 60 months postoperatively. One patient had progression to metastasis after a second course of BCG. Therefore, the over-all progression rate to cystectomy or metastasis was 17% (5 of 30 patients). All 5 patients were among the 16 who failed to achieve a complete response after the initial course of BCG. In conclusion, our experience and that of others demonstrate that BCG therapy is an effective initial treatment of stage T1 disease to prevent progression and recurrence, and to preserve bladder function. Close monitoring will identify those nonresponders who require surgical intervention. PMID- 1732598 TI - The artificial urinary sphincter (AS 800): experience in 166 consecutive patients. AB - The artificial urinary sphincter model AS 800 was implanted in 166 patients with incontinence of various etiologies. Followup ranged from 6 to 94 months, with a mean of 41.6 months. Patient age ranged from 5 to 84 years, with a mean age of 59.4 years. There were 10 female patients (6%) and 156 male patients (94%). The cuff was implanted around the bladder neck in 27 patients (16%) and around the bulbous urethra in 139 (84%). A total of 40 reoperations (27 revisions and 13 device removals) was performed in 32 of 166 patients (19.3%). There were 13 mechanical device failures (7.8%), 11 cuff erosions (6.6%) and 2 periprosthetic infections (1.2%). Total or near total continence was achieved in 125 patients (75.3%), while 25 (15.1%) had improved urinary control. PMID- 1732599 TI - Risk factors associated with penile prosthesis infection. AB - We reviewed all 269 patients who underwent penile prosthetic implantation during the last 10 years. The data were analyzed to determine the rate of penile prosthesis infection and to determine the risk factors associated with these infections in this group. We also examined the effect of strict surgical technique, intraoperative scrub and perioperative antibiotics on these implant infections. All patients had perioperative antibiotics, an intraoperative shave and a 15-minute intraoperative skin preparation. There were 162 semirigid and 107 inflatable prostheses inserted. Mean followup was 32 months (range 2 to 123 months). Only 6 patients had an infection, with 5 prostheses being removed. All 6 patients had a history of urinary tract infection. Furthermore, all 6 patients had either a neurogenic bladder (4), diabetes (1) or an ileal conduit (1), which increased the risk of a urinary tract infection. Cultures of the infected prostheses revealed only enteric organisms. No staphylococci were cultured. We conclude that perioperative antibiotics, intraoperative shave and scrub, and strict surgical technique resulted in a low prosthesis infection rate (1.9%) in these patients. However, a group of patients still exist that despite these precautions are at risk for infection due to conditions that may predispose them to urinary tract infections. PMID- 1732600 TI - Use of glycosylated hemoglobin to identify diabetics at high risk for penile periprosthetic infections. AB - We report an 18-month prospective study of 90 patients undergoing penile prosthesis implantation to evaluate a possible cause-and-effect relationship between degree of diabetic control and the risk of infection complicating the operation. Long-term diabetic control was objectively evaluated by measurement of the glycosylated hemoglobin of the patient, which is known to provide an objective value for degree of control for the preceding 60 to 90 days. Of 90 patients 5 (5.5%) had a periprosthetic infection requiring explantation and all infections occurred in the 32 diabetics (36%) in the population (p less than 0.009). Of the 32 diabetics 13 (41.1%) were poorly controlled with time as demonstrated by a glycosylated hemoglobin level of greater than 11.5% and 4 of the infections occurred in this group. Of the 19 remaining controlled diabetics (glycosylated hemoglobin level less than 11.5%) only 1 infection occurred. Therefore, infection occurred in 31% of the poorly controlled versus 5% of the adequately controlled patients (p less than 0.0003). Measurement of glycosylated hemoglobin values appears to be a useful tool to evaluate diabetic patients before implantation of a penile prosthesis. Patients with a glycosylated hemoglobin level of 11.5% or greater should be more optimally controlled before undergoing implantation in an effort to avoid infectious complications. PMID- 1732601 TI - Coagulase-negative staphylococcus in chronic prostatitis. AB - Three male patients with a clinical history of prostatitis with coagulase negative staphylococci localized to the expressed prostatic secretion and who did not respond to antibiotics were studied intensively 4 weeks after cessation of therapy with repeat culture of the prostatic fluid, as well as with culture, and histological and ultrastructural examination of multiple prostatic biopsies. Coagulase-negative staphylococci were cultured in the biopsied prostatic tissue, and gram-positive staphylococci were identified in sparse and focal microcolonies adherent to the prostatic ductal walls. Coagulase-negative staphylococci may be implicated in the pathogenesis of chronic bacterial prostatitis. PMID- 1732602 TI - Balloon ureteral occlusion: a new reversible technique in the management of ureteral fistulas. PMID- 1732603 TI - Meatal spreader. PMID- 1732604 TI - Application of argon beam coagulation in urological surgery. AB - Argon beam coagulation is a new form of electrocautery that has proved useful to control diffuse bleeding in other surgical specialties. We report its application to urology. Three cases are presented in which argon beam coagulation provided excellent hemostasis in situations that are often difficult to control, such as partial nephrectomy for penetrating trauma, hemorrhagic cystitis refractory to other forms of treatment and after anterior exenteration for bladder cancer. The basis, technique and advantages of argon beam coagulation are discussed, as well as other instances in urological surgery in which it may have application. Argon beam coagulation is an alternative to conventional methods of hemostasis whenever there is a diffusely bleeding operative site. PMID- 1732605 TI - Relevance of detrusor hyperreflexia, vesical compliance and urethral pressure to the occurrence of vesicoureteral reflux in myelodysplastic patients. AB - Bladder pressure in the storage phase is considered to be relevant to the changes in the upper urinary tract. We analyzed retrospectively detrusor hyperreflexia, vesical compliance and maximum urethral closing pressure to determine which is the most significant factor relevant to the incidence of vesicoureteral reflux in 91 myelodysplastic patients. Vesicoureteral reflux was demonstrated in 29 of 91 patients. Cystometry and urethral pressure profilometry were performed in 69 and 27 patients, respectively. Vesicoureteral reflux was observed in 43% of the female patients, which was significantly greater than in the male patients (20%). Detrusor hyperreflexia was noted in 43 patients. Average vesical compliance was 11.3 +/- 8.3 ml./cm. water in 58 evaluable patients. Maximum urethral closing pressure was 56.7 +/- 25.8 cm. water. Vesical compliance in the patients with vesicoureteral reflux was 10.2 +/- 7.5, which was not significantly lower than in those without vesicoureteral reflux (12.2 +/- 8.8). The incidences of vesicoureteral reflux were 38% in the patients with vesical compliance of less than 10, 40% in those with vesical compliance of greater than 10 but less than 20 and 36% in those with vesical compliance of more than 20. The differences were not significant among these patients. Urethral pressure in the patients with vesicoureteral reflux was significantly higher than in those without vesicoureteral reflux (73.8 +/- 23.5 versus 48.2 +/- 23.0, p less than 0.05). The incidence of vesicoureteral reflux was 53% in the patients with urethral pressure of greater than 50, while it was only 8%, significantly less (p less than 0.05), in the lower urethral pressure group. Vesicoureteral reflux was noted in 44% of the patients with detrusor hyperreflexia, which was not significantly greater compared to 31% in the patients without detrusor hyperreflexia. These results suggest that in myelodysplastic patients maximum urethral closing pressure is highly relevant to the incidence of vesicoureteral reflux, while vesical compliance and detrusor hyperreflexia are not. The incidence of vesicoureteral reflux was significantly greater in female patients (43%, p less than 0.05) than in male patients (20%), although urethral pressure values showed no difference between them, indicating that female patients may be another risk factor for vesicoureteral reflux. PMID- 1732606 TI - Risk factors for upper tract deterioration in chronic spinal cord injury patients. AB - A total of 140 patients with neurogenic voiding dysfunction secondary to chronic spinal cord injuries was assessed initially at a tertiary care urodynamic center an average of 8 years after the acute injury. As a result of testing patients were divided into 2 functional urodynamic groups. Group 1 included 40 patients with an areflexic bladder, of whom 33 maintained normal upper tracts and 7 had significant upper tract deterioration. Group 2 included 100 patients with a hyperreflexic bladder, of whom 84 maintained normal upper tracts and 16 had documented upper tract deterioration. Maximum detrusor pressure during urine storage in group 1 with abnormal upper tracts was significantly higher than in those with normal kidneys (p less than 0.0001). Maximum detrusor contraction pressure during voiding in group 2 was significantly higher in those with abnormal upper tracts secondary to neurogenic outflow obstruction (p less than 0.0001). The most common outflow problem in this group was type 3 detrusor sphincter dyssynergia. With guidelines thus developed for acceptable detrusor pressure in both types of bladder, silent upper tract damage can probably be prevented in most cases by proper and diligent followup and appropriate intervention, avoiding major morbidity and mortality in these high risk patients. PMID- 1732607 TI - Lower urinary tract dysfunction in cerebral palsy. AB - The urodynamic findings in 33 patients with cerebral palsy referred with lower urinary tract symptoms were reviewed. Difficulty urinating was the predominant symptom in approximately half of the patients and half of these also had hyperreflexia and urgency when full. Three patients had varying degrees of retention and the remaining 14 had difficulty initiating a urinary stream. The other half had urgency incontinence as a major presenting symptom and this was associated in nearly all cases with hyperreflexia. There were 10 adults: 5 with difficulty urinating and 5 with urgency. The more serious manifestations, such as retention, were found only in the adults, suggesting that difficulty urinating may progress in adult life. Classical detrusor-sphincter dyssynergia with bladder wall changes was seen only once, and the cause of difficulty urinating in the other patients seemed to be due to a lack of voluntary control over and the hypertonus of the pelvic floor. PMID- 1732608 TI - Use of transvaginal endosonography in the evaluation of women with stress urinary incontinence. AB - Hypermobility of the bladder neck in response to increased intra-abdominal pressure is the anatomical cause of uncomplicated stress urinary incontinence in women. Transvaginal endosonography is a reliable, minimally invasive technique for demonstrating bladder neck hypermobility in patients with genuine stress urinary incontinence. Of 279 patients with genuine stress urinary incontinence evaluated during a 24-month period 271 (97%) had bladder neck hypermobility demonstrated by transvaginal endosonography. Resolution of stress urinary incontinence after surgical bladder neck suspension correlated with stabilization of bladder neck mobility on ultrasound examination. The technique is painless and easily performed in the office setting. PMID- 1732609 TI - Unilateral post-obstructive diuresis in the neonate. AB - A total of 3 neonates exhibited severe post-obstructive diuresis after relief of unilateral ureteral obstruction. Of the neonates 2 had normal kidneys contralaterally while 1 had a contralateral kidney that was dysplastic. Urinary output exceeded oral intake initially in all 3 patients necessitating intravenous supplementation for up to 10 days. Stabilization resulted from an increase in oral intake rather than a decrease in diuresis. Unilateral post-obstructive diuresis is extremely uncommon but it may occur in the neonate because of the unique features of the renal physiology encountered in this age patient. PMID- 1732610 TI - Perforation and intravesical erosion of a ventriculoperitoneal shunt in a child with an augmentation cystoplasty. AB - Bladder augmentation has evolved into a common method of management in children with a low capacity and/or poorly compliant bladder secondary to a neuropathic condition. We report on a 4-year-old girl with myelodysplasia who presented with sepsis and who had a perforation of the augmented bladder, which was surgically repaired. She returned for evaluation 1 month after she was discharged from the hospital when the distal component of the ventriculoperitoneal shunt was noted to protrude per urethram after clean catheterization. Distal shunt replacement with prolonged bladder drainage successfully resolved this perforation of the augmented bladder. The patient has had no further difficulties. We discuss the diagnosis and management of this case with reference to the current literature regarding complications of augmentation cystoplasty. PMID- 1732611 TI - Omeprazole in post-gastrocystoplasty metabolic alkalosis and aciduria. AB - The use of segments of stomach for bladder augmentation is gaining popularity in pediatric urology due to favorable muscular and secretory properties. However, in a renal failure patient who underwent gastrocystoplasty a high level of acid production within the bladder associated with persistent hypergastrinemia was noted leading to severe systemic metabolic alkalosis. This condition was unresponsive to standard acid-inhibiting or neutralizing therapies but it was treated successfully with omeprazole, a proton-pump inhibitor recently introduced for treatment of peptic ulcer disease. PMID- 1732612 TI - Exstrophy of the bladder: primary closure after iliac osteotomies without external or internal fixation. AB - A total of 6 consecutive infants with exstrophy of the bladder underwent iliac osteotomy in association with primary closure of the bladder without internal or external fixation of the bony pelvis. Hospitalization time was short and there were no resulting problems with healing, bony deformities or subsequent ambulation. Fixation of the bony pelvis after iliac osteotomy does not appear to be necessary in nonambulatory patients undergoing primary closure for bladder exstrophy. PMID- 1732613 TI - Effectiveness of a handsewn nipple valve for reflux prevention in bladder reconstruction. AB - The intussuscepted nipple has proved to be a versatile mechanism to provide continence or prevent reflux in urological reconstructive surgery. Early in its use detussusception of the nipple was recognized as a common complication, which was usually prevented by using several rows of staples to stabilize the nipple. The use of staples has reduced the rate of reoperation for eversion or obstruction but it has led to a higher stone formation rate, ranging from 10 to 18% in recent series. Since 1983 we have used a handsewn intussuscepted ileal nipple stabilized without staples as our antireflux mechanism in bladder augmentations and continent diversions. This technique has been performed in 30 patients with an average followup of greater than 3 years. A small bladder stone developed in only 1 (3%) of the patients, who was completely dependent on intermittent catheterization, while 4 (13%) required reoperation due to eversion of the nipple. This incidence compares well with nipple reoperation rates in recent series, which range from 7 to 28%. We conclude that absorbable sutures are as effective as staples in stabilizing the antireflux nipple, and that they result in a lower incidence of subsequent stone formation. PMID- 1732614 TI - The effect of medium-fill and slow-fill saline cystometry on detrusor pressure in infants and children with myelodysplasia. AB - A total of 38 infants and children with myelodysplasia was selected for statistical comparison of the effects of medium-fill and slow-fill saline cystometry on detrusor pressure. Medium-fill cystometry was performed at 20% of estimated bladder capacity per minute (up to 25 cc per minute) and slow-fill cystometry at 2% (up to 2.5 cc per minute). In 26 cases the filling rate did not determine the change in detrusor filling pressure while in 12 the change in detrusor filling pressure was greater than 15 cm. water during medium-fill but not slow-fill cystometry (p = 0.001). In 24 cases the filling rate did not determine the occurrence of maximal detrusor pressure greater than 40 cm. water but in 14 such pressure was noted during medium-fill but not slow-fill cystometry (p = 0.0005). Excluding 10 children with coexisting vesicoureteral reflux, differences in the change of detrusor filling pressure greater than 15 cm. water and maximal detrusor pressure greater than 40 cm. water remained statistically significant (p = 0.01 and p = 0.005, respectively). It is concluded that detrusor pressure can be manipulated by varying bladder filling with saline solution. PMID- 1732615 TI - Extrarenal angiomyolipomas of the perinephric space. AB - We describe 2 cases of extrarenal angiomyolipoma of the perinephric space. All other cases of extrarenal angiomyolipoma of the retroperitoneum are reviewed and the clinical relevance of this unusual pathological entity is discussed. PMID- 1732616 TI - Histology of ureter after unsuccessful endoscopic intubated incision. AB - Endoscopic intubated ureterotomy is used increasingly for treatment of ureteral stricture. In 3 patients with recurrent stricture after this procedure the area of ureterotomy was excised after 69, 78 and 83 days, and cut in cross section to examine the scar for muscle regeneration. A segmental scar was found consisting of collagen-rich connective tissue with few fibroblasts. Scarce smooth muscle fibers were dispersed within the scar, mostly at the edge of incision but without a regular structure. PMID- 1732617 TI - Primary retroperitoneal plasmacytoma with tumor thrombus within the renal vein. AB - A case of primary retroperitoneal plasmacytoma causing a tumor thrombus within the renal vein is described. The use of radiographic techniques, including ultrasonography, computerized tomography and angiography, facilitated an accurate diagnosis. Tumor thrombi within the renal vein are rare except in cases of primary renal or adrenal neoplasms. In a patient with radiological evidence of retroperitoneal tumor and tumor thrombus within the renal vein plasmacytoma should be considered in the differential diagnosis of retroperitoneal neoplasms. PMID- 1732618 TI - Adenocarcinoma in an isolated rectosigmoid bladder: case report. AB - We report a case of primary adenocarcinoma of the rectum 11 years after a radical operation and construction of an isolated rectosigmoid bladder for squamous cell carcinoma of the bladder. The isolated rectosigmoid bladder, which is not exposed to the fecal stream, may be associated with adenocarcinoma as in ureterosigmoidostomy. PMID- 1732619 TI - Primary localized amyloidosis of the bladder: an immunohistochemical study of a case. AB - We report a case of primary localized amyloidosis of the bladder causing renal failure. Immunohistochemically, amyloid fibril protein originated from the lambda type light chain of immunoglobulin. PMID- 1732620 TI - Paravesical suture granuloma: a problem following herniorrhaphy. AB - We discuss the diagnosis and management of a paravesical suture granuloma and review 11 such cases reported in the literature. Granulomas are an unusual complication of surgery, which have been noted to occur from several months to 11 years postoperatively. Of the 11 patients reported on 10 had undergone previous inguinal herniorrhaphy and presented with urinary symptoms and a palpable mass, and 1 had undergone femoral herniorrhaphy. In 7 cases the clinical diagnosis was a malignancy. It is important to consider suture granulomas in the differential diagnosis of a suprapubic mass involving the bladder so that unnecessary major surgery can be avoided. PMID- 1732621 TI - Paraneoplastic neurological syndrome in transitional cell carcinoma of the bladder. AB - Paraneoplastic neurological syndromes are well known sequelae of some malignancies but they have never been reported in transitional cell carcinoma of the bladder. A paraneoplastic neurological syndrome characterized by visual changes, glossal spasm and dysphagia associated with an invasive high grade transitional cell carcinoma of the bladder is reported. Neurological symptoms resolved after extirpation confirming a paraneoplastic condition. Recurrent disease was associated with recurrent neurological symptoms and resolved after a complete response to combination chemotherapy. PMID- 1732622 TI - Endovaginal sonography: new diagnostic approach for urethral diverticula. AB - Too often a urethral diverticulum is a long-standing and unrecognized problem. Suprapubic sonography has been proposed for assessment of this pathological condition. We report on endovaginal sonography as a new improved imaging modality for the diagnosis of urethral diverticula. In positive cases additional morphological information can be obtained from positive pressure urethrography or from diverticulography via fine needle puncture under digital guidance or under sonographic control. Direct puncture of the lesion allows the injection of contrast material to facilitate the diverticulectomy. PMID- 1732623 TI - Penile fracture with urethral injury. AB - A case of penile fracture secondary to a mishap during sexual intercourse is reported. The injury involved both corpora cavernosa and complete transection of the urethra. The injury was repaired with plastic reanastomosis of the corpora cavernosa and urethra. The result with limited followup was satisfactory. The diagnosis and treatment of this unusual injury are discussed. PMID- 1732624 TI - Radiation therapy for classic Kaposi's sarcoma presenting only on the glans penis. AB - Kaposi's sarcoma is an endothelial neoplasm with a variety of clinical presentations that rarely involve only the glans penis. Although it is recognized as a radiosensitive lesion, most of the reported cases of penile Kaposi's sarcoma have been treated surgically. We describe 2 otherwise healthy men with this unusual presentation of Kaposi's sarcoma who were treated with radiation therapy. These cases and a review of the literature demonstrate the role of radiation therapy in the conservative management of classic Kaposi's sarcoma involving the penis. PMID- 1732625 TI - Asymmetrical autonomic dysfunction of the feet after retroperitoneal surgery in patients with testicular cancer: 2 case reports. AB - We report on 2 patients with hyperhidrosis and decreased temperature of the leg on the unoperated side after unilateral retroperitoneal lymph node dissection. Both patients had a 4 to 5C difference in skin temperature of the feet, with the operated side being warm and dry compared to the nonoperated side. This condition is most likely due to a lesion of sympathetic fibers or ganglia located in close proximity to the retroperitoneal lymph nodes, resembling a unilateral lumbar sympathectomy. In addition, both patients had profuse sweating and a subjective feeling of coldness of the leg on the nonoperated side, which caused considerable discomfort. This latter phenomenon most likely represents a compensatory sympathetic hyperfunction due to the decreased sympathetic function in the other leg. PMID- 1732626 TI - Cutaneous fistulas to the vas deferens. AB - Cutaneous fistulas to the vas deferens are a rare occurrence. A careful review of the literature revealed only 16 cases reported within the last 70 years. We present a case of such a fistula occurring after inguinal orchiectomy for suppurative epididymo-orchitis and review the etiologies of all reported cases to provide recommendations for management. PMID- 1732627 TI - Injury of rat renal vessels following extracorporeal shock wave treatment. AB - The locations of extracorporeal shock wave treatment induced renal vascular injury and the sources of significant renal hemorrhage were determined in a rat model by means of two different vascular casting procedures. Silicone-rubber injected vascular preparations for light microscopy or corrosion casts for scanning electron microscopy were made following gross examination of the treated organs and their contralateral controls. After 1000 shock waves at 18 kV, five out of 20 treated kidneys appeared to be normal or minimally affected, while 15 showed gross evidence of marked vascular injury. Gross interstitial hemorrhage (15/20), subcapsular hematomas (7/20), and hemorrhages into the renal pelvis (5/20) were confirmed by extravasations of casting materials. These could be traced back to their vascular sources in several instances. Disruptions of interlobar and arcuate veins gave rise to most significant interstitial, subcapsular, and renal pelvic extravasations. On a microscopic scale cortical venules were among the most frequently injured vessels. The arterial vasculature was not spared. Arterial injury ranged from complete arcuate occlusion to small afferent arteriolar and glomerular capillary extravasations. The significance of shock wave induced vascular injury is discussed with respect to potential clinical side effects of ESWL. PMID- 1732628 TI - Enhancement of bacillus Calmette-Guerin attachment to the urothelium by removal of the rabbit bladder mucin layer. AB - It has been established that the urothelial mucin layer functions as a bacterial anti-adherence factor. Intravesical Bacillus Calmette-Guerin is used to treat patients with superficial bladder cancer. The proposed mechanism of action of Bacillus Calmette-Guerin is adherence to the urothelium with induction of an immunologic and/or inflammatory response. The current study was designed to determine if rabbit bladder mucin removal results in increased Bacillus Calmette Guerin urothelial adherence. PAS and colloidal iron stains were used to demonstrate that intravesical instillation of 50% acetone renders rabbit bladder urothelium mucin deficient. The urothelium remains mucin deficient at two hours, but by 24 hours the mucin layer has been regenerated. Two hours following intravesical 3H-labeled Escherichia coli administration, bacterial adherence was 29-fold greater in mucin deficient than mucin intact rabbits (p = 0.05). By 12 hours, the difference in adherence was not significant. Two hours following intravesical administration of 3H-labeled Bacillus Calmette Guerin, mucosal adherence was 21-fold greater in mucin deficient compared to mucin intact rabbits (p = 0.002). After mucin removal, Bacillus Calmette Guerin urothelial adherence was significantly increased. The significant increase in Bacillus Calmette Guerin adherence after mucin removal may be clinically exploitable. PMID- 1732629 TI - Cellular effects of piezoelectric versus electrohydraulic high energy shock waves. AB - High energy shock waves produced by a piezoelectric lithotripter, (EDAP LT.01) and an electrohydraulic lithotripter, (Dornier HM3) were examined for their effects on Chinese hamster ovary cells in suspension. The EDAP caused acute lactate dehydrogenase release, consistent with severe membrane disruption in a proportion of cells, with the remaining proportion of cells replicating normally as measured by clonogenic assay. Similarly, the Dornier also caused lactate dehydrogenase release. However, a significant proportion of cells which remained "viable" after Dornier treatment, (intact to lactate dehydrogenase), did not replicate by clonogenic assay. The Dornier HM3 lithotripter has been reported to produce free radicals in aqueous solution. In the current investigation, we could not detect significant free radical formation from the EDAP LT.01. Chinese hamster ovary cell killing by the Dornier HM3 was significantly augmented by radiosensitizers, 5-iodo-2-deoxyuridine or buthionine sulfoximine, while radioprotectors cysteamine and WR-1065 had no protective effect. EDAP cell killing was not influenced by either radioprotectors or radiosensitizers. The mechanism of in vitro cytotoxicity differs between piezoelectric and electrohydraulic high energy shock wave delivery. PMID- 1732630 TI - Improved lymphocyte cytotoxicity against murine renal cell carcinoma. AB - In this paper we describe the generation of antibody dependent cellular cytotoxicity against a murine renal cell carcinoma. Using human recombinant interleukin-2 and in vitro adherence to plastic, we generated lymphokine activated killer and adherent lymphokine activated killer cells. Adherent lymphokine activated killer cells had significant (p less than 0.05) higher unrestricted cytotoxicity than LAK cells. Using a rabbit antibody against Renca developed in our laboratory, we induced significant (p less than 0.01) antibody dependent cellular cytotoxicity using fresh spleen, lymphokine activated killer and adherent lymphokine activated killer cells. The strongest antibody dependent cellular cytotoxicity killing was mediated by adherent lymphokine activated killer cells and was restricted only to the renal cell carcinoma target. Using FACS cell surface analysis and antibody and complement depletion of selected effector cell subsets, we also demonstrate that the antibody dependent cellular cytotoxicity effector cell population consists of asialoGM1+ Lyt 2.1- natural killer cells. This first description of antibody dependent cellular cytotoxicity against renal cell carcinoma by activated natural killer cells suggests a novel method for more efficient use of cytotoxic effector cells against this type of cancer. PMID- 1732631 TI - Inhibition of PAIII rat prostatic adenocarcinoma growth and metastasis by a new diarylsulfonylurea antitumor agent, LY181984. AB - LY181984 is a compound in a series of orally active diarylsulfonylureas with broad spectrum in vivo activity against syngeneic rodent and human xenograft tumor models. The PAIII rat prostatic adenocarcinoma model was used to evaluate the effects of this antitumor agent on the lymphatic and pulmonary metastasis of the tumor in male Lobund Wistar rats. LY181984 was inactive against the proliferation of PAIII cells in vitro. Following subcutaneous implantation of 10(6) PAIII cells in the tail, oral administration of LY181984 (25.0, 50.0, or 100.0 mg./kg./day) for 30 days had no significant effects on body weight gain. LY181984 treatment produced significant (p less than 0.05) dose-dependent inhibition of primary tumor growth in the tail (max. inhibition = 46% from untreated control levels). In these same animals, LY181984 administration produced significant (p less than 0.05) dose-dependent inhibiton of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximal responses = 79% and 80% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by oral LY181984 treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (p less than 0.05) reduced by LY181984 administration in a dose-dependent manner (maximal reduction = 78% from control values). While the non-toxic doses (less than 100.0 mg./kg./day for 28 days) of LY181984 produced marked decreases in tumor growth and metastasis, administration of the compound had no effect on the survival of PAIII-bearing rats. These data support the contention that LY181984 represents a new class of orally active antitumor and antimetastatic agents with potential efficacy in the treatment of hormone-insensitive prostatic cancer. Further studies are needed to define maximal efficacy of LY181984 and other sulfonylurea agents in urogenital solid tumor animal models. PMID- 1732632 TI - Modulation by cis-diamminedichloroplatinum (II) of the susceptibilities of human T24 lined and freshly separated autologous urinary bladder transitional carcinoma cells to peripheral blood lymphocytes and lymphokine activated killer cells. AB - The effects of cis-diamminedichloroplatinum (II) on the susceptibilities of human T24 lined and freshly separated autologous urinary bladder transitional carcinoma cells to lysis by peripheral blood lymphocytes of patients with urinary bladder cancer was analysed in a 12-hour 51Cr release assay. Treatment of T24 cells with cis-diamminedichloroplatinum (II) at 10 micrograms./ml. for three hours enhanced their susceptibility to peripheral blood lymphocytes and lymphokine activated killer cells. Kinetics studies demonstrated that the enhancement of their susceptibility became noticeable by three hours and continued until 12 hours. The susceptibilities of autologous tumor cells to both large granular lymphocytes and T lymphocytes were also enhanced by treatment of them with cis diamminedichloroplatinum (II). There was no significant difference in the number of peripheral blood lymphocytes binding to cis-diamminedichloroplatinum (II) treated T24 cells as compared with untreated T24 cells. Treatment of T24 cells with mitomycin C did not change their natural killer sensitivity. Pretreatment of T24 cells with cis-diamminedichloroplatinum (II) and lysosomotrophic agents (L leucin-methyl-ester or chloroquine) reduced the enhancement of their susceptibility to natural killer cells by cis-diamminedichloroplatinum (II) alone. On the other hand, pretreatment of peripheral blood lymphocytes with cis diamminedichloroplatinum (II) had no influence on the cytotoxicity against T24 cells. These results indicate that cis-diamminedichloroplatinum (II) may have an augmenting effect on the susceptibility of tumor cells to the cell-mediated cytotoxicity partly through a modification of cell membrane independently of its antimetabolic activity and this modification may be one of the possible mechanisms responsible for tumor regression after chemotherapy with cis diamminedichloroplatinum (II). PMID- 1732633 TI - The effect of pH on the in vitro colony forming ability of transitional cell carcinoma cells treated with various chemotherapeutic agents: implications for in vivo therapy. AB - Extracellular pH may affect the sensitivity of cancer cells to chemotherapy agents. In an attempt to maximize the conditions for chemotherapy treatment of transitional cell carcinoma we tested the effect of pH on sensitivity of MGH-U3 transitional cell carcinoma cell line to thiotepa, doxorubicin, and mitomycin c in vitro. The toxicity of each agent tested varied with pH. There was no variation in cell growth in response to pH alone. The cytotoxic activity of thiotepa was markedly enhanced when cells were treated with a diluent pH of 5.5. Significant differences were also observed after treatment with doxorubicin and mitomycin c with a diluent pH of 7.0. This in vitro assay may be useful for clinical application of pH modulation during intravesical chemotherapy. PMID- 1732634 TI - Prediction of metastatic potential by cancer cell motility in the Dunning R-3327 prostatic adenocarcinoma in vivo model. AB - Many grading systems for prostatic carcinoma exist; however, none allows pathologists to predict accurately the prognosis of individual patients. The Dunning R-3327 prostatic adenocarcinoma model consist of sublines of known metastatic potential which were indistinguishable until recently. A visual grading system of cancer cell motility distinguished the metastatic potentials of Dunning sublines maintained in vitro and was validated prospectively. We used this grading system to assess the metastatic potential of cells harvested directly from in vivo Dunning tumors. We graded the motility of cells from three Dunning sublines of low (less than 10%) (G, AT1, AT2) and three sublines of high (greater than 90%) AT3, (MAT-LyLu, PAT2) metastatic potential. Cells obtained from primary tumors were studied by time lapse videomicroscopy after passages 1, 3 and 5 in vivo and in vitro. Membrane ruffling, pseudopodal extension and cellular translation were graded 0-10. Serial analysis of mean and heterogeneity (coefficient of variation) of membrane ruffling, pseudopodal extension and cellular translation demonstrated that subline motility grades were assessed adequately by a sample of 10 cells. Motility did not depend upon whether cells were maintained in vitro or in vivo; however, motility increased with successive passages in four of six sublines. In 60 cells harvested directly from the fifth in vivo passage, three sublines of low metastatic potential were distinguished from three sublines of high metastatic potential (Student's t test, p less than 0.01). Individual cells from the sublines were identified correctly as high or low metastatic in 83, 78 and 70% of cases by membrane ruffling, pseudopodal extension and cellular translation respectively, and logistic regression analysis failed to improve classification accuracy. A visual grading system of cancer cell motility described the metastatic potential of in vivo neoplasms in the Dunning model and may warrant testing in human prostatic cancer. PMID- 1732636 TI - Head of 1992 AIDS conference outlines many innovations and new participation. PMID- 1732635 TI - Interferon-alpha can alter the lytic susceptibility of murine bladder transitional cell carcinoma (MBT-2) by their original poor specific cytotoxic tumor infiltrating lymphocytes. AB - Fresh isolated or short term in vitro cultured tumor infiltrating lymphocytes derived from MBT-2 tumor possess little specific cytotoxicity on MBT-2 cells despite the presence of low (10 units/ml.), moderate (500 units/ml.), or high (1000 units/ml.) dose interleukin-2 and irradiated tumor cells; however the lytic susceptibility of the MBT-2 cells and their expression of MHC class I antigen can be significantly enhanced if the tumor cells are preincubated with interferon alpha for 24 hours. But the specific antitumor activity of tumor infiltrating lymphocytes cannot be further augmented by coculturing them with irradiated interferon-alpha pretreated MBT-2 tumor cells. In vivo experiments by subcutaneous peritumor injection of interferon-alpha (500 units/ml.) combined with interleukin-2 (500 units/ml.) can suppress the tumor growth of those three day inoculated MBT-2 cells significantly but not on those already established tiny tumors. We therefore concluded that interferon-alpha can alter the specific antitumor activity of tumor infiltrating lymphocytes chiefly through its modulating effect on the target tumor cells instead of the tumor infiltrating lymphocytes. PMID- 1732637 TI - Harassment hinders women's care and careers. PMID- 1732638 TI - Harassment linked to fraud, lawsuit alleges. PMID- 1732639 TI - Multidrug-resistant tuberculosis poses challenge. PMID- 1732640 TI - Ultrasound may help detect breast implant leaks. PMID- 1732641 TI - From the Food and Drug Administration. PMID- 1732642 TI - From the Centers for Disease Control. The second 100,000 cases of acquired immunodeficiency syndrome--United States. PMID- 1732643 TI - A piece of my mind. That's politics. PMID- 1732644 TI - HIV-infected surgeons. PMID- 1732645 TI - HIV-infected surgeons. PMID- 1732646 TI - HIV-infected surgeons. PMID- 1732647 TI - HIV-infected surgeons. PMID- 1732649 TI - Medical ethics in review. PMID- 1732648 TI - HIV-infected surgeons. PMID- 1732650 TI - Morning distort: we are not amused. PMID- 1732651 TI - Academic boycott of South Africa. PMID- 1732652 TI - The effect of filtered-coffee consumption on plasma lipid levels. Results of a randomized clinical trial. AB - OBJECTIVE: --To determine the effect of filtered-coffee consumption on plasma lipoprotein cholesterol levels in healthy men. DESIGN: --Randomized controlled trial with an 8-week washout period followed by an 8-week intervention period during which men were randomly assigned to drink 720 mL/d of caffeinated coffee, 360 mL/d of caffeinated coffee, 720 mL/d of decaffeinated coffee, or no coffee. SETTING: --Outpatient clinical research center in a university medical center. PARTICIPANTS: --One hundred healthy male volunteers. OUTCOME MEASURE: --Changes in plasma lipoprotein cholesterol levels during the intervention period. RESULTS: --Men who consumed 720 mL of caffeinated coffee daily had mean increases in plasma levels of total cholesterol (0.24 mmol/L, P = .001), low-density lipoprotein cholesterol (0.17 mmol/L, P = .04), and high-density lipoprotein cholesterol (0.08 mmol/L, P = .03). No significant changes in these plasma lipoprotein levels occurred in the other groups. Compared with the group who drank no coffee the group who drank 720 mL/d of caffeinated coffee had increases in plasma levels of total cholesterol (0.25 mmol/L, P = .02), low-density lipoprotein cholesterol (0.15 mmol/L, P = .17), and high-density lipoprotein cholesterol (0.09 mmol/L, P = .12) after adjustment for changes in diet. CONCLUSION: --Consumption of 720 mL/d of filtered, caffeinated coffee leads to a statistically significant increase in the plasma level of total cholesterol, which appears to be due to increases of both low-density lipoprotein and high density lipoprotein cholesterol levels. PMID- 1732653 TI - The benefits of treating hyperlipidemia to prevent coronary heart disease. Estimating changes in life expectancy and morbidity. AB - OBJECTIVE: To evaluate the lifetime benefits of reducing total serum cholesterol levels to prevent coronary heart disease (CHD). DESIGN: We developed a CHD primary prevention computer model to estimate the benefits associated with lifelong risk factor modification. We validated the model by comparing the computer estimates with the observed results of three primary CHD prevention trials. PATIENTS: Men and women age 35 to 65 years who are free of CHD, with total serum cholesterol levels ranging from 5.2 to 7.8 mmol/L (200 to 300 mg/dL), with or without additional CHD risk factors. INTERVENTIONS: Serum cholesterol reduction through dietary modification or diet and medications. MAIN OUTCOME MEASURES: Changes in life expectancy and the delay of symptomatic CHD. RESULTS: The computer forecasts for CHD end points closely matched the observed results of the Lipid Research Clinics Trial, the Helsinki Heart Study, and MRFIT. We then applied the computer model to low-risk and high-risk men and women with total serum cholesterol levels between 5.2 and 7.8 mmol/L (200 and 300 mg/dL) and estimated that, after reducing serum cholesterol levels 5% to 33%, the average life expectancy would increase by 0.03 to 3.16 years. We also forecast that the average onset of symptomatic CHD would be delayed among these patient groups by 0.06 to 4.98 years. CONCLUSION: We conclude that this computer model accurately estimates the results of clinical trials and can be used to forecast the changes in life expectancy and morbidity (the development of CHD) associated with specific CHD risk reduction interventions. The wide variation surrounding these estimates underscores the need to better define which groups of individuals will gain the most from cholesterol reduction. PMID- 1732654 TI - Measles herd immunity. The association of attack rates with immunization rates in preschool children. AB - OBJECTIVE: To examine the association between incidence of measles and immunization coverage among preschool-age children. DESIGN: An ecological study in which measles incidence was compared with immunization coverage among census tracts. The independent effects of race and population density were controlled for. SETTING: A recent measles outbreak in Milwaukee, Wis. Immunization coverage data were estimated from a retrospective, school-based survey of Milwaukee grade school students. PATIENTS: One thousand eleven persons (less than or equal to 17 years) who had confirmed measles from September 1989 through June 1990. MAIN OUTCOME MEASURES: Confirmed measles cases grouped by census tract, corresponding census tract preoutbreak immunization coverage, racial breakdown, and population density. RESULTS: Census tracts stratified into four levels, with mean immunization rates of 50.4%, 60.2%, 69.9%, and 81.0%, had respective median attack rates of 11.6, 5.0, 1.7, and 0.0 cases per 1000 persons (P less than .01). The association between immunization coverage and measles attack rate remained significant even after controlling for race and population density. CONCLUSIONS: Modest improvements in low levels of immunization coverage among 2-year-olds confer substantial protection against measles outbreaks. Coverage of 80% or less may be sufficient to prevent sustained measles outbreaks in an urban community. PMID- 1732655 TI - Risk factors for delirium in hospitalized elderly. AB - OBJECTIVE: To determine risk factors for delirium in elderly hospitalized patients. DESIGN: Cohort analytic study. Using a reliable and valid instrument for detection of delirium, we prospectively followed up a cohort of elderly patients admitted to an acute care hospital. Using standardized criteria, we collected risk factor data from patient medical records. SETTING: General medical and surgical wards of a tertiary-care hospital. PATIENTS: Patients (n = 325) were 65 years of age or older, from either a geographically defined community or a long-term-care institution. We studied those patients (n = 291) not delirious on first evaluation. Fifty-seven patients or their families refused participation. MAIN OUTCOME MEASURES: Incidence of delirium and risk factors calculated as adjusted odds ratios (ORs). MAIN RESULTS: Delirium developed in 91 patients. By stepwise logistic regression, the independent risk factors for in-hospital delirium included prior cognitive impairment (OR, 8.97; 95% confidence interval [CI], 3.99 to 20.14), age over 80 years (OR, 5.22; 95% CI, 2.60 to 10.46), fracture on admission (OR, 6.57; 95% CI, 2.23 to 19.33), symptomatic infection (OR, 2.96; 95% CI, 1.42 to 6.15), and male sex (OR, 2.40; 95% CI, 1.19 to 4.84). Among medication groups, only neuroleptic use (OR, 4.48; 95% CI, 1.82 to 10.45) and narcotic use (OR, 2.54; 95% CI, 1.24 to 5.18) were independently associated with delirium. Anticholinergic use was not associated with delirium. CONCLUSIONS: Delirium in hospitalized patients is most closely associated with factors already present on admission such as prior cognitive impairment, advanced age, and fracture. In the hospital, use of neuroleptics and narcotics and the presence of infection are less strongly associated with this syndrome. PMID- 1732656 TI - Pneumocystis carinii pneumonia among patients without AIDS at a cancer hospital. AB - OBJECTIVES: To determine the predisposing factors, attack rate by underlying disease, and outcome of Pneumocystis carinii pneumonia among patients without the acquired immunodeficiency syndrome (AIDS) at a cancer center. DATA SOURCE: Twelve year retrospective review from a tertiary-care cancer center. STUDY SELECTION: One hundred forty patients, constituting 142 cases, with morphologically proved P carinii pneumonia. DATA SYNTHESIS: Hematologic malignancy (47%) (including lymphoma [27%] and leukemia [18%]), solid tumor (31%), or bone marrow transplantation (18%) was the underlying condition in the majority of cases. Twenty-five cases (18%) were diagnosed at autopsy. All but seven patients had previously established predisposing factors for P carinii pneumonia, including corticosteroid use in 87%. The attack rate for hospitalized patients with primary or metastatic brain tumor increased during the 12-year interval. The attack rates for hospitalized patients with hematologic neoplasm or bone marrow transplantation were stable. The overall mortality rate did not change during the period reviewed. CONCLUSIONS: Despite the availability of effective prophylaxis, P carinii pneumonia continues to occur among patients with neoplastic disease. In addition to patients with certain hematologic neoplasms, those with primary or metastatic brain neoplasm who receive corticosteroids are at risk for the development of P carinii pneumonia and should receive P carinii pneumonia prophylaxis. PMID- 1732657 TI - Work-related cumulative trauma disorders of the upper extremity. AB - Cumulative trauma disorders due to performance of repetitive tasks account for more than 50% of all occupational illnesses in the United States today. Employees affected by these disorders frequently experience substantial pain and functional impairment that may require a change in occupation. For the employer, these injuries result in loss of productivity and increased costs in the form of higher medical expenses and disability payments for injured workers. Successful treatment of work-related repetitive tissue injuries depends on early diagnosis and appropriate therapy. Prevention requires identifying sites and tasks that place employees at risk of injury and supporting efforts to develop safer work environments. PMID- 1732658 TI - Effect of HIV posttest counseling on STD incidence. AB - OBJECTIVE: To evaluate the effectiveness of human immunodeficiency virus (HIV) testing and posttest counseling in reducing subsequent high-risk behavior. METHODS: The incidence of sexually transmitted diseases (STDs) in the Baltimore, Md, public STD clinic population after HIV testing and counseling was determined by chart review and was compared in two groups, 868 HIV-seropositive patients and 1104 HIV-seronegative patients, matched by age, sex, and month in which HIV test was conducted. Patients were observed for incident STDs at intervals of 6 through 23 months. Patients with incident STDs were classified hierarchically after being notified of the HIV test result and receiving posttest counseling. RESULTS: Of HIV-seropositive patients, 615 (71%) returned for their test results and received posttest counseling; 694 HIV-seronegative patients (63%) returned. Of all those who returned for results and posttest counseling, 60 (9.7%) of 615 HIV seropositive patients and 61 (8.8%) of 694 HIV-seronegative patients were diagnosed at least once with definite STD (syphilis, gonorrhea, or trichomoniasis) (P, not significant). Twenty-four HIV-seropositive patients (3.9%) and 71 HIV-seronegative patients (10.2%) returned with probable STD (nongonococcal urethritis or pelvic inflammatory disease) (P less than .001). Nine HIV-seropositive patients (1.5%) and 23 HIV-seronegative patients (3.3%) returned having had an STD-infected sexual partner (P less than .03). Age, sexual orientation, and drug use behavior did not predict return with STD. CONCLUSIONS: Both HIV-seropositive and HIV-seronegative patients showed high rates of repeat STDs after posttest counseling, an important public health challenge in creating effective high-risk behavior prevention strategies. PMID- 1732659 TI - Hypertrophic cardiomyopathy. PMID- 1732660 TI - Sex testing in international athletics. A small step forward. PMID- 1732661 TI - The myelodysplastic syndrome. Meeting by the European School of Haematology and the Hematology Collaborating Group, Stockholm, 22-24 April 1991. PMID- 1732662 TI - The treatment of acute myeloid leukaemia preceded by the myelodysplastic syndrome. AB - Most cases of acute myeloid leukaemia in the elderly and about 15% of the cases in younger patients follow a myelodysplastic disease. This disease may differ biologically from de novo AML making cure more difficult. Understanding post-MDS AML is difficult because of its preponderance in the elderly and the many complicating factors of treating old people. However, it is likely that post-MDS AML is more resistant to chemotherapy than de novo AML, that periods of pancytopenia last longer following chemotherapy and remissions are shorter. In younger patients high complete remission rates are achievable with aggressive chemotherapy and good supportive care. Such patients are best served by subsequent allogeneic bone marrow transplantation if possible, and if not then bone marrow autograft or a maintained remission should be considered. For older patients cure is unlikely and the choice between low-dose cytarabine and aggressive chemotherapy must be made if treatment is to be contemplated at all. The former leads to less hospitalisation and fewer side effects, but the latter gives a greater chance of a complete remission. This will not be long lasting. PMID- 1732663 TI - Prognostic factors in the myelodysplastic syndromes. AB - The myelodysplastic syndromes are acquired clonal hematologic malignancies characterized by progressive cytopenia and an increased risk of evolution to acute myeloid leukemia. It mainly affects elderly people, but may also be found in younger patients and children. MDS represents a heterogeneous group of disorders. Some patients will experience prolonged survival, whereas a substantial number of patients will die within the first year after diagnosis. Treatment of patients should be based on life expectancy. Patients with pancytopenia, excess of bone marrow blasts, complex chromosome abnormalities, abnormal chromosome 7, older age and secondary MDS have a poor prognosis. Several simple scoring systems are available, based on peripheral counts, percent of bone marrow blasts and age, that provide significant prognostic information about life expectancy in patients with MDS. The most widely used is the Bournemouth scoring system. The prognostic factors and the scoring systems are reviewed. PMID- 1732664 TI - Complications and treatment of the myelodysplastic syndromes. AB - The major complications of the myelodysplastic syndromes (MDS) are related to cytopenia and evolution to acute myeloid leukemia (AML). Hematopoietic growth factors are only of limited benefit to alleviate the cytopenia. Therapy in MDS patients over the age of 50 should aim at prolonging survival while limiting the risk of toxicity. Those with stable disease should only receive supportive care; those with progressive cytopenia should have a trial with low-dose chemotherapy. Aggressive chemotherapy should only be reserved for those failing low-dose therapy. Therapy in MDS patients under the age of 50 should aim at cure of the disease. Although aggressive chemotherapy can induce complete remission in the majority of these patients, remission is usually short. Allogeneic bone marrow transplantation is probably the only curative option in these patients and should be the treatment of choice. PMID- 1732665 TI - Morphologic classification of the myelodysplastic syndromes (MDS): combined utilization of bone marrow aspirates and trephine biopsies. AB - In a retrospective and prospective follow-up study from 1975 to 1991, bone marrow biopsies, aspirates and clinical features of 495 patients with MDS were investigated. Sections of undecalcified plastic embedded biopsies and smears of bone marrow aspirates were stained according to Giemsa. Bone marrows with MDS were characterized by three main categories of morphologic alterations: (1) cellular abnormalities, (2) architectural disorganization in the bone marrow and (3) stromal changes; the combined use of aspirates and trephine biopsies enabled a more reliable and accurate diagnosis of MDS than either one alone. The bone marrow findings fell into one of 7 subtypes, with the frequencies and median survivals in brackets: (1) MDS sideroblastic (19%, 62 months), (2) MDS megaloblastoid (13%, 56 months), (3) MDS proliferative (22%, 31 months), (4) MDS blastic (15%, 9 months), (5) MDS hypoplastic (15%, 26 months), (6) MDS fibrotic (6%, 29 months), and (7) MDS inflammatory (10%, 42 months). In follow-up studies patients with secondary MDS were excluded and the prognosis and subsequent evolution for each of the morphologic subtypes were evaluated. The conclusion is drawn that aspirates and trephine biopsies are complementary procedures and both are required for diagnosis, classification and decisions on current treatment modalities of patients with MDS. PMID- 1732666 TI - Minimal diagnostic criteria for the myelodysplastic syndrome in clinical practice. PMID- 1732667 TI - Chromosomal deletions in the myelodysplastic syndrome. AB - Karyotypic abnormalities in primary myelodysplastic syndrome (P-MDS) are less frequent than in secondary myelodysplasia. A review of the literature involving over 3000 reported cases, shows the incidence of karyotypically abnormal clones at presentation in nearly 48% of cases. Approximately 50% of the abnormalities comprise of deletions of chromosomes 5, 7, 11, 12, 13 and 20. Localisation of a number of haemopoietic growth factors and their receptors to the deleted segments of the chromosomes, has invoked considerable interest in the molecular pathology of the interstitial deletions and their consequent role in the multistep pathogenesis of MDS. Present evidence suggests chromosome abnormalities are a later event in the multistep painogenesis, and it is suggested their occurrence may be restricted to a restricted myeloid progenitor cell, although the initial event(s) occur at the common lymphoid-myeloid progenitor. Much has been gleaned from the dominant modes of leukaemogenesis, such as the occurrence of missense mutations at specific positions of RAS and FMS mutations. It is suggested that a similar enquiry into the mechanisms of chromosomal deletions in P-MDS is required in order to delineate the role of these abnormalities in the clonal evolution of this group of diseases. PMID- 1732668 TI - Minimal diagnostic criteria for the myelodysplastic syndrome. PMID- 1732669 TI - Cytogenetic findings in primary and secondary MDS. AB - More than 1300 MDS cases with clonal cytogenetic abnormalities, 200 of them secondary MDS, have been reported. The most common aberrations in primary MDS are del(5q) (27%), trisomy 8 (19%), monosomy 7 (15%), der(11q) (7%), -5, der(12p) and -Y (5%), del(7q) (4%), and t(1;7), der(3q), del(13q), i(17q) and del(20q) in 2% or less. The 5q- is mostly, but not always, a del(5)(q13q33); it is the cytogenetic hall-mark of the "5q- syndrome" and is frequently found as the sole abnormality. The frequency of the aberrations varies among MDS subgroups: 5q- is most frequent in RA, -5, -7, and der(12p) are more common in CMML and especially in RAEB, and +8 and der(11q) are more often found in RARS. The most common aberrations in secondary MDS are -7 (41%), del(5q) (28%), -5 (11%), der(21q) (9%), 7q-, +8 and der(12p) (8%), t(1;7) and -12 (7%), der(17p) (6%), der(3p) and der(6p) (5%), and der(3q), der(11q), -17, -18 and der(19q) (4%). The average number of abnormalities per case is 5.3, compared with 2.9 in unspecified MDS. The frequency of cytogenetically unrelated clones is 5.7% in secondary and 4.3% in primary MDS. When the literature data are broken down by type of genotoxic exposure, it turns out that -5, -7, and der(17p) are over-represented in patients who have received chemotherapy, whereas 5q- is associated with no exposure or preceding radiotherapy only. The karyotypic profile is prognostically important: patients with -7 or complex karyotypes have a higher risk of progression to acute leukemia and shorter survival. PMID- 1732670 TI - Gene mutations in myelodysplasia. AB - The myelodysplastic syndrome is a paradigm of human preleukaemia. Normal haemopoiesis is progressively displaced by an abnormal clone derived from a mutated stem cell. The initial mutation is unknown but its occurrence may be related to the overall load of random mutations which are a consequence of both intrinsic DNA defects and external mutagens. Evolution of the pathological population is marked by an increasing load of genetic lesions at the molecular and cytogenetic levels. Ras mutations can be detected in the blood of about 50% of MDS patients. Fms mutations are less common but these lesions can be found both in patients and in haematologically normal subjects who have previously received cytotoxic therapy suggesting that they can occur early in the preleukaemic process. Clonal haemopoiesis in the absence of either ras or fms mutations can occur in these subjects. The data suggest the inability of mutant ras or fms genes alone to produce observable preleukaemic changes but that subjects with these mutations may be predisposed to future MDS. Ras mutations are a common accompaniment of a wide variety of malignancies and experimental transfection of the mutant gene can induce a malignant phenotype in cultured cells. There are many possible mechanisms for this transformation which may be relevant in a clinical context. Experimentally observed effects include a direct influence on the cell cycle, the induction of drug resistance and the stimulation of autocrine growth factor production. It may eventually be possible to define which gene mutations are important in conferring a malignant state, which determine phenotype and which are of incidental significance. PMID- 1732671 TI - Minimal diagnostic criteria for the myelodysplastic syndrome in clinical practice. PMID- 1732672 TI - Myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) with myelofibrosis. AB - Acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) enter rarely in the differential diagnosis of myelofibrosis (MF). MF of marked intensity, resulting in either "dry taps" or non-representative smears, is encountered in approximately 10% of cases. MF may be observed in any type of AML, most frequently in acute megakaryoblastic leukemia (M7). Apart from some typical cases of MDS, MF is associated with cases of acute myelodysplasia with myelofibrosis (and a major megakaryocytic component). This syndrome has been described under various headings: acute or malignant myelosclerosis, and acute MF. It should be distinguished from M7 and from myeloproliferative syndromes. PMID- 1732673 TI - The "n-1" model for myelodysplastic syndromes. AB - Current models of leukaemogenesis tend to visualize this process as consisting of several discrete steps. Since myelodysplastic syndromes (MDS) are, almost by definition, pre-leukaemic disorders, we expect that bone marrow from patients with MDS must contain cells which are one step short of full leukaemic transformation: we designate these cells, for convenience, as "n-1". It should be possible therefore to obtain leukaemic transformation in vitro by introducing into n-1 cells a gene with an appropriate leukaemogenic mutation. We have found, by using as a model system the retrovirus VSN-2 (which carries the neomycin phosphotransferase gene, neo, which confers resistance to the antibiotic G418), that human bone marrow cells can be successfully transfected in microcultures. Indeed, G418-resistant CFU-CM have been recovered from these cultures for a period of several weeks. PMID- 1732674 TI - Minimal diagnostic criteria for the myelodysplastic syndrome (MDS) in clinical practice. PMID- 1732675 TI - Radiotherapy- and chemotherapy-induced myelodysplasia and acute myeloid leukemia. A review. AB - A highly increased risk of myelodysplasia (MDS) and acute myeloid leukemia (AML) has been demonstrated following therapy with alkylating agents. The risk increases with cumulative dose and with the age of the patient. Most cases of MDS and AML following therapy with alkylating agents present chromosome aberrations, primarily loss of whole chromosomes No. 5 and/or No. 7 or various parts of the long arms of these chromosomes. The risk of MDS and AML following high-voltage radiotherapy is much lower. Recently an increased risk of AML has been demonstrated following therapy with the epipodophyllotoxins etoposide and teniposide. These leukemias typically present without preceding MDS and often show balanced aberrations of chromosome bands 11q23 and 21q22. PMID- 1732676 TI - Exposure to organic solvents and risk of haematological malignancies. AB - Epidemiological studies indicating that exposure to organic solvents is a risk factor for haematological malignancies are reviewed. Exposure to benzene is a risk factor for ANLL. A preleukaemic phase with pancytopenia is common and may be associated with a normo- or hypercellular marrow with morphological characteristics suggesting MDS. There are indications that other organic solvents than benzene may be leukaemogenic. Certain chromosome aberrations are characteristic in leukaemic cells from solvent exposed ANLL patients. The average latency time from start of occupational exposure until diagnosis is about 10-11 years. There is epidemiological evidence that exposure to organic solvents may also increase the risk of lymphoproliferative malignancies, i.e. ALL, NHL, HD and myeloma. PMID- 1732677 TI - Bone marrow dysfunctions preceding acute leukemia in children: a clinical study. AB - This is a review of preleukaemic states in children. In a prospective series of 109 children with AML the overt disease was preceded by MDS in 22 cases. Ten of these patients had Down's syndrome. Advanced FAB groups were represented in the series. An important subgroup is the bone marrow monosomy 7 syndrome. Cytogenetic anomalies are common in MDS, and multiple and complicated abnormalities develop in nearly all patients with progressing disease. Some children die before transformation to overt ANLL. Transformation usually occurs, few children survive. With cytostatic treatment the risk of irreversible aplasia is great. The choice of schedule should therefore be carefully considered. Bone marrow transplantation has proved beneficial in a number of cases, but these are still quite few. The dysfunction of the bone marrow preceding ALL is due to transient aplastic anaemia--spontaneous remission--overt ALL, often FAB type L1, immunophenotype CALLA. The ALL reacts to the same treatment as de novo ALL of the same type and the prognosis is the same. PMID- 1732678 TI - Prognostic factors in myelodysplastic syndromes. AB - The extreme variability in prognosis among patients with myelodysplastic syndromes (MDS) complicates decision-making regarding their therapy. Several studies carried out in recent years have recognized the prognostic value of some clinical and biological characteristics. The percentage of blast cells in bone marrow, cytopenias, age and chromosome abnormalities are the most relevant factors affecting outcome. More importantly, some of these studies have resulted in the development of prognostic regression formulas and scoring systems for accurately estimating the individual prognosis of patients. The major aim of this review is to offer the clinician useful tools for treating MDS patients on a risk fitted strategy. PMID- 1732679 TI - Minimal diagnostic criteria for the myelodysplastic syndrome in clinical practice. PMID- 1732680 TI - Clinical features of MDS. AB - MDS is primarily a disease of the elderly. Cases who give a history of exposure to X-rays, cytotoxic drugs or leukaemogenic chemicals may be younger. Many cases of MDS present because of an incidental blood count. The most prominent clinical features are those of anaemia, neutropenia, thrombocytopenia. Because haemopoietic tissue is also dysfunctional the pathological effect is often greater than the figures would suggest, even leading to infection of bleeding with normal neutrophil or platelet counts. Occult abscesses are a particular feature. Despite documented abnormalities of the lymphoid system, neither infections characteristic of T-cell immunodeficiency nor autoimmunity is a problem. The proliferation of monocytes in CMML leads to organomegaly, leukaemia cutis, serous effusions and vasculitic lesions caused by the mishandling of circulating immune complexes. Cancer is no commoner than in age-matched controls, but coincident lymphoid tumours do occur. Many patients require long-term blood transfusion and will run into problems of iron overload unless precautions are taken. PMID- 1732681 TI - Minimal diagnostic criteria for the myelodysplastic syndrome. PMID- 1732682 TI - Mechanisms and management of central sleep apnea. AB - Central sleep apnea is not a single disease but represents the final pathway in a large group of heterogeneous disorders. Control of normal breathing during sleep relies upon finely coordinated anatomical and physiological mechanisms and their destabilization leads to central apnea. The causes of central sleep apnea can be classified into 4 groups: neurologic disorders, periodic breathing, upper airway abnormalities, and idiopathic syndromes. Clinical features result from the interaction between the underlying disorder and control of respiration. Two different prototypes emerge: patients who are hypercapnic (central hypoventilation and/or impaired respiratory mechanics) and those who are eucapnic or hypocapnic (periodic breathing and idiopathic hyperventilation). The causes and severity of apnea can be determined by clinical assessment, pulmonary function testing, and overnight polysomnography. Further management involves specific treatment of the underlying condition and reducing the sequelae of recurrent apneas during sleep, namely cardiorespiratory dysfunction and sleep disruption. This review outlines an approach to the management of central sleep apnea based upon an understanding of its pathophysiology. PMID- 1732683 TI - Superoxide dismutase inhibits radiation-induced lung injury in hamsters. AB - An animal model of pulmonary radiation-induced lung injury was established in the hamster and the effects of pretreatment with recombinant human CuZn superoxide dismutase (SOD) on the development of the lesion were evaluated. Hamsters exposed to a single irradiation dose of 2000 cGy delivered to the thorax were treated with 150 mg/kg body weight of SOD or an equivalent volume of saline intraperitoneally 75 min and subcutaneously 5 min before receiving irradiation. At 4, 8, and 16 weeks following irradiation, pulmonary injury was evaluated by the grading of morphologic changes semiquantitatively, measurement of lung hydroxyproline content, and analysis of bronchoalveolar lavage fluid for total and differential cell counts and total protein concentration. Radiation-induced lung injury in saline-pretreated animals was documented at 16 weeks by histologic morphology and increased protein in bronchoalveolar lavage fluid. SOD protected against radiation-induced pulmonary injury as indicated by the absence of severe histopathologic changes and prevention of elevation in bronchoalveolar lavage protein levels. The beneficial effects of SOD in preventing radiation-induced pulmonary toxicity suggests that this recombinant enzyme may play a role in protection against radiation-induced pulmonary injury in humans. PMID- 1732685 TI - Outcome of noncardiac operations in patients with severe coronary artery disease successfully treated preoperatively with coronary angioplasty. AB - The risk of perioperative myocardial infarction and death was evaluated in 50 patients (mean age, 68 years) with severe coronary artery disease who underwent a noncardiac operation after revascularization had been achieved by successful percutaneous transluminal coronary angioplasty. Before angioplasty, all patients were thought to be at high risk for perioperative complications on the basis of assessment of clinical variables and findings on specialized diagnostic tests. Of the 50 patients, 31 had Canadian Heart Association class III or IV angina or unstable angina. All patients who underwent functional testing had positive results. At catheterization, 38 patients (76%) had multivessel disease. The 50 patients underwent 54 noncardiac operations at a median of 9 days after angioplasty. The overall frequency of perioperative myocardial infarction was 5.6%, and the mortality was 1.9%. Two nonfatal non-Q-wave infarctions and one fatal Q-wave infarction occurred. In patients who have undergone successful angioplasty for severe coronary artery disease, the risk of major cardiac complications associated with a noncardiac surgical procedure is low. PMID- 1732684 TI - Pulmonary responses of asthmatic and normal subjects to different temperature and humidity conditions in an environmental chamber. AB - Determining the possible adverse health effects of air pollutants can be complicated by differences in the environmental conditions of temperature and humidity. To evaluate the potentially confounding effects of differences in temperature and humidity, we exposed 8 normal male subjects and 8 male subjects with asthma to the extremes in temperature and humidity that could be maintained in an environmental chamber. We performed serial pulmonary function tests for these subjects before and during 6 hr exposure periods on 5 separate occasions: cold, dry (10 degrees C, 10% relative humidity); cold, humid (10 degrees C, 50% relative humidity); normal ambient (22 degrees C, 40% relative humidity); hot, dry (37 degrees C, 15% relative humidity); and hot, humid (37 degrees C, 60% relative humidity). The exposure period included a 12 min exercise on a cycle ergometer. We found no significant change in spirometry, airways resistance, or diffusing capacity for either group of subjects at rest alone over the 6 hr period of exposure for any exposure condition. However, there were changes in spirometry and airways resistance as a result of the 12 min period of exercise. The subjects with asthma had significant decreases in forced expiratory volume in 1 sec (FEV1) (20-21%) and increases in specific airways resistance when exercising in conditions of cold and dry, cold and humid, and hot and dry. The normal subjects had an average increase in FEV1 of approximately 6% when exercising in the hot and humid conditions. We found significant correlations for the changes in FEV1 with the water content of the exposure conditions for both groups of subjects. We also found that the work performance (expressed as the external work performed divided by the oxygen consumed) was decreased for the subjects in both groups at the conditions of the higher temperature (37 degrees C) compared with the lower temperature (10 degrees C). These results confirm that controlling for the conditions of temperature and humidity is essential in chamber studies, field studies, or epidemiologic evaluations determining the adverse effect of an air pollutant. PMID- 1732686 TI - Dr. Charles H. Mayo and the automobile. PMID- 1732687 TI - The pituitary gland in hyperthyroidism. AB - The pituitary glands of 33 patients (24 women and 9 men, 18 to 78 years old) who died in thyrotoxicosis (18 with Graves' disease and 15 with toxic multinodular goiter [Plummer's disease]) were examined by histologic and immunocytologic methods. Thirteen patients (39%) died in "thyroid storm." The avidin-biotin peroxidase complex immunostaining method was used to demonstrate the spectrum of pituitary hormones, including growth hormone, prolactin, adrenocorticotropic hormone, thyrotropin, follicle-stimulating hormone, luteinizing hormone, and alpha-subunit. The most striking finding was a pronounced decrease or loss of immunoreactivity to thyrotropin in all thyrotoxic cases, a consistent change that allowed ready distinction of thyrotoxic from euthyroid pituitary glands. When immunoreactive thyrotrophs were identified, they were sparse and small and demonstrated only faint thyrotropin reactivity. No morphologic differences were noted between the pituitary glands of patients with Graves' disease or Plummer's disease or between sexes. Loss of thyrotropin immunoreactivity was found to be reversible in that thyrotropic cells in the pituitary glands of 16 additional concurrently studied patients, who had thyrotoxicosis but were treated and subsequently had normal thyroid function or hypothyroidism, appeared normal or even hyperplastic. Other types of adenohypophysial cells in both the thyrotoxic and the successfully treated groups exhibited no abnormalities. Pituitary adenomas were incidental findings in 6 of the 33 patients (18%). Their immunotypic spectrum included three prolactin-immunoreactive tumors, two growth hormone-containing adenomas (one of which was plurihormonal), and one tumor with follicle-stimulating hormone and luteinizing hormone; no thyrotropin-containing adenomas were noted. No examples of pituitary hyperplasia were encountered in pituitary glands of thyrotoxic patients, and no hypophysitis or fibrosis was noted.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732688 TI - Severe reflux esophagitis after vertical banded gastroplasty for treatment of morbid obesity. AB - Herein we describe two patients with medically refractory, severe reflux esophagitis after vertical banded gastroplasty for morbid obesity. Neither patient had symptoms of reflux preoperatively. Both patients underwent conversion to a vertical Roux-en-Y gastric bypass, an operation that prevents acid and peptic reflux and maintains a weight-reducing anatomy. Symptoms of gastroesophageal reflux are common (they occur in approximately 38% of patients) after vertical banded gastroplasty has been performed. Patients with unusually severe reflux may require operative management. PMID- 1732689 TI - Medical mythology: Horus. PMID- 1732690 TI - Syphilis: a disease to exclude in diagnosing sarcoidosis. AB - A 37-year-old man was referred to our institution for assessment of possible sarcoidosis with involvement of the central nervous system. Before referral, he experienced a systemic illness that persisted for several months, during which time ocular and pulmonary noncaseating granulomas were identified. Sarcoidosis with involvement of the central nervous system was tentatively diagnosed. Because of several inconsistencies in the preliminary diagnosis of sarcoidosis, further assessment was pursued, and syphilis was diagnosed. Herein we emphasize the useful clinical features for distinguishing syphilis from sarcoidosis and review the clinical manifestations of pulmonary syphilis. PMID- 1732691 TI - The Mayo Clinic and carpal tunnel syndrome. AB - A review of the medical records at the Mayo Clinic revealed the evolution of a common clinical condition--carpal tunnel syndrome. Initially, treatment was limited to those patients with the most severe neuropathies and derangements of wrist anatomy. As confidence with the diagnosis and therapy increased, progressively less severe anatomic deformities and then progressively less severe neurologic impairments were considered for surgical management. Documentation shows that the first surgical division of the flexor retinaculum for treatment of distal median neuropathy was performed in 1924, almost a decade earlier than previously thought. PMID- 1732692 TI - The Olmsted County Benchmark Project: primary study findings and potential implications for corporate America. AB - Since 1965, expenditures for medical care in the United States have increased 10 fold. As a result, corporate outlays for health benefits have skyrocketed. Employers have instituted various cost-containment measures based in part on reports of wide variations in rates of utilization and the assumption that unnecessary or inappropriate utilization of medical care contributes to increasing costs. Frequently, however, employers lack adequate means for identifying sources of variation or for evaluating its appropriateness. In this article, we report on a project in which hospital utilization among several US corporate populations was compared with that for a geographically defined benchmark population to assist employers in the assessment of their rates of utilization and expenditures and to identify specific areas that merit further investigation. Our findings illuminate the difficulties in constructing valid rates from medical-care claims data and emphasize potential biases due to problems of comparability between populations. We also address the potential value of such comparison for helping corporations identify areas in which cost containment efforts may be most effective and yet not jeopardize the quality of medical care. PMID- 1732693 TI - Antibiotic therapy for severe infections in infants and children. AB - In infants and children, the absorption, distribution, metabolism, and excretion of drugs may differ considerably in comparison with these factors in adults; consequently, differences exist in therapeutic efficacy and toxicity of various antibiotic agents. Because of known toxicity, certain drugs--such as chloramphenicol in high doses, the sulfonamides, and tetracycline--should not be used in neonates. Antibiotic therapy should be modified in neonates because of biologic immaturity of organs important for the termination of drug action. Because of poor conjugation, inactivation, or excretion, the serum concentrations of many antibiotics may be higher and more prolonged in neonates than in older infants; thus, lower doses and longer intervals between administration may be necessary. In this article, we suggest dosages of antimicrobial agents for severe infections in children, older infants, and neonates. Included in the discussion are the cephalosporins, especially the third-generation cephalosporins that have assumed an important role in empiric treatment of bacterial meningitis in pediatric patients because of their ability to penetrate the central nervous system and their effectiveness against beta-lactamase-positive and negative strains of Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and many gram-negative bacteria in the Enterobacteriaceae group. In patients with congenital or acquired immunodeficiencies, antifungal, antiviral, or anti-Pneumocystis agents are often added to the antimicrobial regimen for severe infections. We review the agents available for such treatment in children, the drugs used for childhood tuberculosis, and certain new antibiotics (aztreonam, ticarcillin-clavulanate, ciprofloxacin, and imipenem-cilastatin) that have proved useful in select cases but whose precise role in pediatric practice will necessitate additional clinical experience. PMID- 1732694 TI - Antifungal agents used for deep-seated mycotic infections. AB - The increased use of immunosuppressive regimens in organ transplantation and in the treatment of malignant lesions and the epidemic of acquired immunodeficiency syndrome (AIDS) are major reasons for the greater prevalence of fungal infections seen in clinical practice during the past decade. The traditional cornerstone of antifungal treatment, amphotericin B, continues to play a major role in deep seated mycotic infections. The indications for intravenously administered miconazole have become limited. Orally administered flucytosine remains useful in certain infections, particularly cryptococcal meningitis. The new orally administered antifungal agents ketoconazole and fluconazole have been approved for clinical use and have supplanted amphotericin B in certain situations. Investigational antifungal agents, including liposomal amphotericin B, itraconazole, and saperconazole, hold promise for the future. Active investigation in the development of new antifungal agents is expected to continue. PMID- 1732695 TI - Benchmarks and business. PMID- 1732696 TI - Reducing the cardiac risk associated with vascular surgical procedures: balloons as prophylactics. PMID- 1732697 TI - Pituitary adenylate cyclase activating polypeptide stimulates insulin release from the isolated perfused rat pancreas. AB - Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide structurally related to vasoactive intestinal peptide (VIP) and glucagon like peptide-1(7-36) amide (tGLP-1) in its N-terminal portion. Therefore, their levels of insulinotropic potency were compared using an isolated rat pancreas perfusion. It was found that 0.1 nM PACAP (1-27) amide (PACAP27) significantly stimulated insulin release under a perfusate glucose concentration of 5.5 mM, whereas 1 nM PACAP27 did not under a perfusate glucose concentration of 2.8 mM. The potency was evaluated as tGLP-1 greater than PACAP27 greater than VIP. These results indicate that PACAP is a glucagon superfamily peptide which stimulates insulin release in a glucose dependent manner. PMID- 1732698 TI - Travel stress alters the intestinal migrating myoelectric complex in rats: antagonist effect of trimebutine. AB - A novel stress model was developed that may closely resemble a real-life situation. Intestinal motility was monitored in rats before and after a 12 hour train voyage (travel stress). Travel stress reduced the duration of phase III of the intestinal MMC by 30% (3.2 +/- 0.3 vs 4.7 +/- 0.6 min; p less than 0.001) while the durations of phase I and II were unaffected. This effect persisted for two days. Phase III duration returned to basal values after 3 days indicating a reversible alteration on intestinal migrating myoelectric complex (MMC). The infusion of trimebutine at a flow rate of 166 micrograms/kg/h during the stress exposure abolished the changes observed in the duration of phase III of the MMC; the infusion of diazepam (16.6 micrograms/kg/h) had no effect. These results indicate that the travel stress model may be similar to common life events that induce alterations of intestinal motility. Furthermore, trimebutine prevented the reduction of phase III duration induced by travel stress suggesting its possible action on mechanisms involved in the mediation of the stress-induced intestinal motility changes. PMID- 1732699 TI - Possible mediation of GABA induced growth hormone secretion by increased calcium flux in neonatal pituitaries. AB - Gamma-aminobutyric acid (GABA) stimulates growth hormone (GH) secretion from pituitaries of young (less than 20-day old) rats (1,2). Present work revealed that the GH stimulatory effect of GABA was abolished in the absence of calcium and response was attenuated by Nifedipine. The calcium efflux from 45CaCl2 preloaded neonatal pituitaries was enhanced by GABA or by muscimol, and this effect was antagonized by the GABA antagonist picrotoxin. In pituitaries of 21 day old or adult rats GABA stimulated neither GH secretion nor calcium efflux. These results indicate that in neonatal pituitaries GABA influences calcium transport and its GH releasing effect is linked to the presence of calcium. PMID- 1732700 TI - Protein glycation: effects upon protein recognition by the proximal tubule. AB - Reabsorption of 125I labeled unmodified and glycated albumin by the proximal tubule was studied using micropuncture technique in wistar rats of different ages. The data show a significant reabsorption of unmodified albumin while the glycated albumin was virtually excluded from reabsorption process. Among different explanations considered, we think molecular charge of the protein seems to be a key factor for the altered recognition of glycated albumin by the proximal tubule. PMID- 1732701 TI - Routine determination of [18F]-L-6-fluorodopa and its metabolites in blood plasma is essential for accurate positron emission tomography studies. AB - A batch-contact alumina-extraction method has been used to separate [18F]-L-6 fluorodopa (FD) from its principal metabolite, 3-O-methyl-[18F]-6-fluorodopa (3 OMe-FD), in arterial blood plasma samples collected from subjects pretreated with carbidopa during positron emission tomography (PET) scans. The time course of the metabolite-corrected blood plasma activity is then used as an input function for kinetic analysis of striatal FD uptake. Results obtained from using the batch contact alumina-extraction method were compared with those from high performance liquid chromatography, and also with those from a chromatographic alumina cartridge technique developed in this laboratory. In 60 human subjects including normal healthy volunteers and patients diagnosed as having a movement disorder, arterial blood plasma samples were collected after FD injection during a two-hour PET scan and analyzed by the batch-contact alumina-extraction method. The activity ratio (metabolites/FD) increased linearly with time for all subjects. However, there was a wide variation in the slope of the plot of the activity ratio (metabolites/FD) versus time among the subjects. No significant linear or curved relationship was observed between the slope and the age of the subject. Separation of FD from its metabolites is therefore necessary for each PET-FD study conducted. PMID- 1732702 TI - Serotonergic abnormalities in the central nervous system of seizure-naive genetically epilepsy-prone rats. AB - Seizure predisposition in Genetically Epilepsy-Prone Rats (GEPRs) is characterized by abnormal sensitivity to a number of seizure provoking stimuli. The GEPR model is composed of two independently derived colonies with each exhibiting a characteristic convulsive pattern. In response to a standardized sound stimulus, GEPR-3s exhibit moderate or clonic convulsions while GEPR-9s exhibit more severe tonic extensor convulsions. In order to further characterize the neurochemical abnormalities that underlie seizure predisposition in GEPRs, the current study examined serotonin concentrations in 14 discrete brain areas of controls, GEPR-3s and GEPR-9s. In all areas examined, serotonin concentrations were lower in either one or both GEPR types than in seizure resistant controls. In 6 of the 14 areas both GEPR-3s and GEPR-9s had levels significantly lower than controls. In an additional 7 areas GEPRs had serotonin concentrations of similar magnitude which were significantly lower than control when the GEPR values were combined. In cerebellum, GEPR-3s had significantly lower serotonin concentration than either controls of GEPR-9s while in the striatum, GEPR-9s had significantly lower serotonin levels than either GEPR-3s or controls. In summary, GEPRs have widespread deficits in serotonin concentration and that these abnormalities appear to contribute to the seizure predisposition that characterizes these animals. PMID- 1732703 TI - Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells. AB - The mechanism by which the inhibitory effect of d-ala2-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the Gi alpha subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through Gi. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with Gi that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase. PMID- 1732704 TI - Microtubule functions. AB - Microtubules, with intermediate filaments and microfilaments, are the components of the cell skeleton which determinates the shape of a cell. Microtubules are involved in different functions including the assembly of mitotic spindle, in dividing cells, or axon extension, in neurons. In the first case, microtubules are highly dynamic, while in the second case microtubules are quite stable, suggesting that microtubule with different physical properties (stability) are involved in different functions. Thus, to understand the mechanisms of microtubule functions it is very important to understand microtubule dynamics. Historically, tubulin, the main component of microtubules, was first characterized as the major component of the mitotic spindle that binds to colchicine. Afterwards, it was found that tubulin is particularly more abundant in brain than in other tissues. Therefore, the roles of microtubules in mitosis, and in neurons, have been more extensively analyzed and, in this review, these roles will be discussed. PMID- 1732705 TI - An assay to measure the simultaneous uptake of [3H]dopamine and [14C]serotonin by the human platelet reveals an imipramine-insensitive [3H]dopamine uptake mechanism. AB - To determine whether a specific dopamine uptake mechanism is present on the human platelet the simultaneous uptake of [3H]dopamine and [14C]serotonin by platelets was measured. Utilising a dual radiolabel uptake technique, platelets have been shown to take up serotonin more rapidly and to a greater extent than they take up dopamine. Furthermore, at high concentrations serotonin was able to reduce dopamine uptake by platelets by 60% whereas dopamine had no effect on serotonin uptake. Similarly, imipramine and reserpine reduced (97% and 74% respectively) serotonin uptake by platelets in a dose-dependent manner, but did not affect the uptake of dopamine. Our data show that platelets take up dopamine by a mechanism independent of the imipramine-sensitive serotonin uptake mechanism. Furthermore, the increased capacity of platelets to store serotonin is because serotonin, unlike dopamine, is transported into the dense granules of the platelet. PMID- 1732706 TI - Systemic administration of calcitonin through ocular route. AB - Calcitonin has been used clinically to treat hypercalcemia, Vitamin D intoxication, osteolytic bone metastases and increased skeletal remodeling in Paget's disease. In general calcitonin is given every 6 to 12 hrs intramuscularly or subcutaneously. It has been found in this study that the same results can be achieved by giving calcitonin through eyes as ophthalmic solutions. When 25 microliters of 0.05% calcitonin was given as eyedrops to New Zealand white rabbits, it did not reach the concentration achieved by i.v. administration at the same dose level. The systemic absorption of calcitonin did not reach the level achieved by i.v. administration even though the eyedrop concentrations were increased 2-fold (0.1%) to 10-fold (0.5%). When absorption enhancers such as BL-9 and Brij-78 were added to calcitonin eyedrops, however, the systemic absorption of calcitonin was enhanced markedly. BL-9 (0.5%) increased calcitonin (0.5%) absorption 16-20 fold and raised blood concentration of calcitonin above levels achieved by i.v. injection (25 microliters, 0.05%) with 0.5% calcitonin eyedrops instillation. Effects of Brij-78 (0.5%) were even more impressive. It increases calcitonin absorption 22-24 fold and raised the blood concentration of calcitonin above the levels achieved by i.v. injection (25 microliters 0.05%) with 0.15% and 0.5% calcitonin eyedrops instillation. These results indicate that the therapeutic level of calcitonin can be reached through the ocular route. PMID- 1732707 TI - Hypoglycemic activities of MTP-3631, a novel thiopyranopyrimidine derivative. AB - MTP-3631 is a novel thiopyranopyrimidine derivative structurally different from any existent hypoglycemic agents. MTP-3631 markedly decreased basal blood glucose and improved glucose intolerance in genetically diabetic C57BL/6J ob/ob mice, which was not affected by tolbutamide. MTP-3631 also caused hypoglycemic effects in normal rats, but no change was observed in plasma insulin level. Unlike buformin, MTP-3631 did not change plasma lactate level in ob/ob mice. These results suggest that the hypoglycemic mechanism of MTP-3631 may be essentially different from those of sulfonylureas and biguanides. PMID- 1732708 TI - Regulation of D1A and D2 dopamine receptor mRNA during ontogenesis, lesion and chronic antagonist treatment. AB - The developmental characteristics of D1A and D2 dopamine receptor mRNA levels were determined by Northern blot analyses. Striatal D1A and D2 dopamine receptor mRNAs of male Fischer 344 rats were about 60% of adult (day 120) levels at postnatal day 1 and reached their highest levels at day 30 (126 and 139% adult levels) and then decreased by day 120 (100%). D1 and D2 dopamine receptors showed much greater quantitative changes with densities at day 30 about 6- and 14-fold higher than at day 1, respectively, while mRNA levels showed only a 2-fold increase. The highest level of D2 dopamine receptor mRNA in the midbrain was reached at day 14 (195% of adult levels) while the level at day 1 was 31% higher than that at day 120. Striatal beta-actin mRNA levels decreased gradually as the rats developed with the level at postnatal day 1 almost twice that at day 120 postpartum. Treatment of adult rats with the selective D2 dopamine receptor antagonist, haloperidol (0.5 mg/kg/day, s.c., for 2 h, 7, 14, 21 days or 21 days + 3 days withdrawal) had no effect on striatal D2 dopamine receptor mRNA levels in spite of significant increases in dopamine receptor density at the later time points. However, 21 days following a 6-hydroxydopamine lesion of the nigrostriatal pathway, striatal D2 dopamine receptor mRNA levels were increased by 53%. PMID- 1732709 TI - Michigan State Medical Society 1992 membership directory. PMID- 1732710 TI - Drugs for tuberculosis. PMID- 1732711 TI - Safety of terfenadine and astemizole. PMID- 1732712 TI - Behavioral risk factor survey of Vietnamese--California, 1991. AB - Since 1975, an estimated 979,700 refugees from Vietnam and other Southeast Asian countries have immigrated to the United States (L. Bussert, Office of Refugee Resettlement, U.S. Department of Health and Human Services, personal communication, 1991). Although public health agencies have reported extensively on the occurrence of infectious diseases in these populations (1-4), the prevalence of risk factors for noninfectious health concerns (e.g., heart disease, cancer, and unintentional injuries) have not been well defined. To characterize risk factors for selected noninfectious diseases and injuries among the estimated 280,200 Vietnamese who have relocated to California, the University of California, San Francisco, and the California Department of Health Services developed a Vietnamese-language version of CDC's Behavioral Risk Factor Surveillance System (BRFSS) for use in a computer-assisted telephone interviewing (CATI) system. This report summarizes findings from the 1991 survey and compares them with data for the general California or U.S. population. PMID- 1732713 TI - Hazardous-waste sites: priority health conditions and research strategies--United States. AB - Uncontrolled disposal sites containing hazardous waste and other contaminants have created national environmental problems (1). Because of potential health problems associated with the more than 33,000 hazardous-waste sites in the United States, the Agency for Toxic Substances and Disease Registry (ATSDR)--as part of its federally legislated mandate--has developed a list of seven priority health conditions (PHCs) to 1) assist in evaluating potential health risks to persons living near these sites and 2) determine program and applied human health research activities involving hazardous substances identified at the sites. This report summarizes the development and intended applications of the seven PHCs. PMID- 1732714 TI - Prevalence of tuberculous infection among U.S. residents of Cuban descent--Dade County, Florida, 1982-84. AB - The strategic plan to eliminate tuberculosis (TB) in the United States emphasizes the need for improved understanding of the epidemiology of tuberculous infection among recent immigrants and other groups at increased risk for TB (1). Data regarding the prevalence of tuberculous infection are limited for groups such as U.S. residents of Cuban descent (i.e., Cubans) and other minority populations. To estimate the prevalence of tuberculous infection among self-reported Cubans, CDC's National Center for Health Statistics analyzed data from tuberculin skin testing performed on all examinees aged 6 months to 74 years in Dade County (which includes incorporated Miami), Florida (1990 population: 1.9 million; Hispanic population: 950,000), during the first Hispanic Health and Nutrition Examination Survey (HHANES), 1982-84 (2). PMID- 1732715 TI - Infant mortality--United States, 1989. AB - In 1989, the infant mortality rate for the United States--9.8 infant deaths per 1000 live births--was the lowest final rate ever recorded; the previous low (10.0 per 1000 live births) was recorded in 1988. However, the infant mortality rate in the United States remains higher than that in many other developed countries. This report summarizes 1989 infant mortality data based on information from death certificates compiled through the Vital Statistics System of CDC's National Center for Health Statistics (NCHS) (1) and compares findings with those for 1988. PMID- 1732716 TI - The rat 5-hydroxytryptamine1B receptor is the species homologue of the human 5 hydroxytryptamine1D beta receptor. AB - The relationship between the serotonin 5-hydroxytryptamine1B (5-HT1B) and 5-HT1D receptors has been the topic of much investigation and speculation since their complementary species distribution was first appreciated. The cloning of genes encoding 5-HT1D receptors has provided tools to investigate this relationship directly. In this study, a rat gene has been cloned that encodes the rat 5-HT1B receptor. Evaluation of the structure of this gene shows that it is a member of the guanine nucleotide-binding protein-coupled receptor superfamily. Comparison of the amino acid sequence of the rat gene with the human 5-HT1D beta gene showed a 93% overall identity and a 96% identity in the transmembrane regions. Comparison of the two sequences revealed zero to two amino acid changes in each of these transmembrane regions, as well as a striking conservation in the connecting loops, indicative of the relationship expected for species homologues of the same gene. The rat gene was expressed transiently in COS-7 cells, and membranes derived from these cells were shown to bind [125I]iodocyanopindolol. The pharmacological profile of this binding site closely matched that of the native rat 5-HT1B receptor (r = 0.95) but not the 5-HT1D receptor (r = 0.07). The cloned rat 5-HT1B receptor was found to couple to the inhibition of adenylate cyclase activity, as expected for a 5-HT1B receptor. These data indicate that, although the 5-HT1B and 5-HT1D receptors are pharmacologically distinct, they are species variants of the same receptor gene, the 5-HT1D beta gene. PMID- 1732717 TI - Antagonistic effect of a vasoactive intestinal peptide fragment, vasoactive intestinal peptide(1-11), on guinea pig trachea smooth muscle relaxation. AB - The conformation of various regions of vasoactive intestinal peptide (VIP) has been analyzed by semiempirical methods, CD, and NMR spectroscopy, indicating that residues 11-21 are most likely to be helical, whereas the amino-terminal portion VIP(1-11) could exhibit two beta-turn structures. VIP(1-11) inhibits 125I-VIP binding to intact guinea pig tracheal epithelial cells and the VIP-induced smooth muscle response. However, the endecapeptide exhibits no effect on the muscle tone. All these data suggest that VIP(1-11) may be a useful tool in studying VIP receptor recognition, its regulation, and cellular functions. PMID- 1732718 TI - Regulation of GDP and GTP binding in cardiac sarcolemma by muscarinic receptor agonists. AB - Regulation of GTP and GDP binding and GTPase activity of cardiac sarcolemmal guanine nucleotide-binding proteins was investigated. In purified sarcolemmal membranes, carbachol and a variety of other muscarinic receptor (MR) agonists induced increases in [3H]GTP, [gamma-32P]GTP, and [3H]GDP binding to relatively high affinity sites. Carbachol-dependent GTP and GDP binding changes were maximal within 5 sec at 30 degrees and thereafter remained at steady state. Carbachol increased GTP binding to two sites with apparent Kapp values of 50 nM and 250 nM and GDP binding to a single site with a Kapp of 100 nM. N-Ethylmaleimide attenuated carbachol-dependent GDP and GTP binding, tentatively identifying the binding sites as Gi and/or Go. Further studies showed that [3H]GDP and [3H]GTP bound to Gi/Go in the presence of carbachol rapidly exchanged with GTP and GDP in the medium. In membranes preincubated with carbachol and [gamma-32P]GTP or carbachol and [3H]GDP, postaddition of atropine resulted in complete hydrolysis of [gamma-32P]GTP bound to Gi/Go, to unlabeled GDP and 32Pi, by GTPase, within 10 sec, whereas [3H]GDP remained bound. This study also showed that bound [3H]GDP did not exchange with GDP or GTP in the absence of an MR agonist. Under identical conditions, atropine reversed adenylate cyclase (AC) inhibition by carbachol and GTP or GDP in 5-10 sec. MR agonists appear to increase the rate of dissociation of GDP from Gi/Go, which results in rapid GTP turnover on these sites by a combination of GTPase and GDP/GTP exchange reactions. Furthermore, MR-Gi/Go may be tightly coupled during AC inhibition, so that GTP hydrolysis as well as MR Gi/Go uncoupling may be required to reverse AC inhibition. PMID- 1732719 TI - Phase II metabolism of warfarin in primary culture of adult rat hepatocytes. AB - We used adult rat hepatocytes in primary culture (HPC) as a model system to study the hepatic phase II metabolism of the anticoagulant warfarin. Hepatocytes were isolated by a collagenase perfusion technique and maintained for 24 hr in Waymouth's medium containing 0.1 mM (R)-warfarin. When HPC medium was analyzed by reverse phase high performance liquid chromatography with diode-array detection, 4'-, 6-, and 7-hydroxywarfarin were identified. Several putative conjugates were observed eluting between 13 and 18 min. Treatment of hepatocyte medium with beta glucuronidase and sulfatase resulted in the loss of five putative conjugates and concomitant increases in 4'-, 6-, and 7-hydroxywarfarin and warfarin, suggesting that these metabolites and warfarin were conjugated. Use of the beta glucuronidase inhibitor saccharic acid 1,4-lactone enabled the determination of the relative extents of conjugation of each metabolite by glucuronic acid and sulfate. Glucuronidation was the predominant pathway for 4'-hydroxywarfarin, whereas 6-hydroxywarfarin and warfarin occurred mainly as sulfate conjugates. In contrast, 7-hydroxywarfarin was converted to both glucuronide and sulfate conjugates. Exposure of HPC to phenobarbital resulted in a decrease in cytochrome P-450-mediated production of hydroxylated warfarin metabolites; however, an increase in the production of 8-hydroxywarfarin was observed when HPC were exposed to beta-naphthoflavone. Unique conjugation patterns were found when hydroxylated warfarins were substituted for warfarin in HPC medium. Both 7- and 8 hydroxywarfarin were converted to one sulfate and two glucuronide conjugates, whereas 4'-hydroxywarfarin was converted to a single glucuronide conjugate. A spectral library of these conjugates was used to identify the major conjugates of warfarin formed by rat HPC. PMID- 1732720 TI - A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits and is up-regulated by chronic nicotine treatment. AB - The subunit composition and pharmacological regulation of rat neuronal nicotinic cholinergic receptors were assessed. Specific immunoprecipitation was determined in solubilized rat brain homogenates using [3H]cytisine, a high affinity agonist at nicotinic receptors, in conjunction with polyclonal antisera generated against nonhomologous domains of the various subunits comprising this receptor class. In all brain regions tested, only antisera generated against the alpha 4 and beta 2 subunits were able to immunoprecipitate specifically receptors labeled by [3H]cytisine. Thus, these sera were further characterized in order to validate and optimize their use in the immunoprecipitation protocol. Preincubation of solubilized receptors from rat forebrain with antisera generated against the alpha 2, alpha 3, alpha 5, beta 3, or beta 4 subunits did not decrease the amount of precipitable alpha 4 or beta 2 subunit. On the other hand, when either anti alpha 4 or anti-beta 2 serum was used to immunoprecipitate solubilized receptors from rat forebrain, the supernatants contained little if any remaining receptors that could be specifically precipitated by either antibody. Because these antisera do not cross-react, the data indicate that alpha 4 and beta 2 subunits are associated with each other in at least one neuronal nicotinic receptor subtype that has high affinity for agonists. Moreover, these results imply that all alpha 4 subunits that are labeled by [3H]cystisine are coupled to beta 2 subunits. We also present evidence that the alpha 4/beta 2 subtype characterized in this report is significantly increased in the cortex of rats chronically treated with nicotine. PMID- 1732721 TI - 2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl] methyl)hexanamide (NPC 15437): a novel inhibitor of protein kinase C interacting at the regulatory domain. AB - NPC 15437 is a prototype member of a new class of synthetically derived protein kinase C (PKC) inhibitors. PKC activity and binding of phorbol ester to the enzyme were inhibited by NPC 15437, with IC50 values of 19 +/- 2 microM and 23 +/ 4 microM, respectively. No inhibition of cAMP-dependent or calcium/calmodulin dependent protein kinases was observed at concentrations of NPC 15437 up to 300 microM. To investigate the mechanism by which NPC 15437 exerts its effects, a kinetic analysis of the inhibition with respect to three activators of the enzyme, phosphatidylserine, calcium, and phorbol ester, was performed. NPC 15437 was a competitive inhibitor of the activation of PKC by phorbol ester (Ki = 5 +/- 3 microM). Stimulation of PKC alpha by phosphatidylserine was competitively inhibited by NPC 15437 (Ki = 12 +/- 4 microM). The inhibition was mixed with respect to activation by calcium. These results suggest that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the enzyme. NPC 15437 inhibited PKC in intact cells, dose-dependently antagonizing the phorbol ester-induced phosphorylation of a 47-kDa protein in human platelets. PMID- 1732722 TI - Stable expression, secretion, and characterization of active human renin in mammalian cells. AB - Human renin is synthesized as a 406-amino acid preprorenin protein that is processed by a signal peptidase during secretion, to release prorenin as a 386 amino acid zymogen. The 46-amino acid "pro" domain is removed by a renin processing enzyme, to produce enzymatically active renin, by cleavage at an Arg Leu bond. The effects of the renin-processing enzyme can be mimicked by trypsin activation, where high concentrations of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolytic processing by trypsin. In order to study the role of the pro segment in the secretion, folding, and activity of human renin, we engineered a construct where the pro domain from the preprorenin cDNA was deleted. This construct was introduced into mammalian cells and its expression was assayed in transient and stable systems. In COS-1 cells transfected with the prerenin expression vector pREN3, active renin was secreted with a specific activity of 1360 micrograms of angiotensin l/min/mg, compared with trypsin-activated prorenin, which has a specific activity of 818 micrograms of angiotensin l/min/mg. The active renin secreted in this system had a significantly reduced potency for the renin inhibitor SQ 32,970. These results demonstrate that the pro segment is dispensable for the folding and secretion of renin. A permanent cell line expressing the active form of renin was obtained by co-transfection of NRP cells with pREN3 and pHyg. A colony designated B/1 was identified, subcloned, and shown to secrete active renin (110 pg of renin/10(6) cells) optimally when maintained in both G418 and hygromycin. PMID- 1732723 TI - Activation of the transcription factor kappa B in human KG-1 myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine. AB - The present studies have examined the effects of 1-beta-D arabinofuranosylcytosine (ara-C) on activation of the transcription factor kappa B (NF-kappa B). The results demonstrate that treatment of human KG-1 myeloid leukemia cells with ara-C is associated with induction of protein binding to the NF-kappa B consensus sequence. NF-kappa B binding was activated at 30 min and reached maximal levels of binding at 1-2 hr of ara-C treatment. The NF-kappa B consensus sequence was ligated to the heterologous thymidine kinase (TK) promoter and the human growth hormone (GH) reporter gene to determine whether ara-C induced NF-kappa B activity includes an enhancer function. Ara-C treatment had little effect on transient expression of pTKGH in KG-1 cells but increased transcription of the p (NF-kappa B) TKGH vector by 8-fold. The results also demonstrate that ara-C transiently increases NF-kappa B mRNA levels. However, the finding that ara-C-induced binding of NF-kappa B to DNA occurs in the presence of cycloheximide indicates that this agent activates preexisting NF-kappa B protein. These results suggest that ara-C induces a cytoplasmic pathway that transduces signals to the nucleus by activation of NF-kappa B. PMID- 1732724 TI - Transcriptional and posttranscriptional regulation of H1 histone gene expression by 1-beta-D-arabinofuranosylcytosine. AB - Recent studies have demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) activates the transcription of the jun/fos early response genes in human myeloid leukemia cells. The basis for ara-C-induced control of gene expression remains unclear. However, down-regulation of H1 histone mRNA levels has been reported as one of the earliest changes in specific gene expression associated with ara-C treatment. In this report, we describe the mechanisms responsible for H1 histone expression by this agent. Treatment of HL-60 cells with ara-C resulted in a decrease in H1 histone mRNA levels that was detectable by 15 min. In contrast, this down-regulation by ara-C was completely blocked by treatment of the cells with cycloheximide. Nuclear run-on analyses demonstrated that ara-C treatment is associated with inhibition of H1 histone gene transcription. The results also demonstrate that cycloheximide abrogates the transcriptional down-regulation by ara-C but alone has no detectable effect. We also show that ara-C treatment is associated with a decrease in stability of the H1 histone transcript and that this effect is also reversed by inhibition of protein synthesis. Taken together, these findings demonstrate that ara-C regulates H1 histone expression at both the transcriptional and posttranscriptional levels. The results also indicate that control of this gene by ara-C involves the activation of at least two signaling events that require de novo protein synthesis. PMID- 1732725 TI - In vitro evidence for direct complexation of ADR-529/ICRF-187 [(+)-1,2-bis-(3,5 dioxo-piperazin-1-yl)propane] onto an existing ferric-anthracycline complex. AB - ADR-529 protects against anthracycline cardiotoxicity, possibly by preventing free radical induction. We hypothesize that this occurs by ADR-529 forming a ternary anthracycline-iron-ADR-529 complex. This study used 200-MHz Fourier transformed NMR to demonstrate the ability of ADR-529 to do this. Peak assignments were by proton-correlated spectroscopy and proton-carbon heteronuclear-correlated spectroscopy. Ga3+ served as a probe for Fe3+, and D2O was the system solvent. Doxorubicin and epirubicin were the studied drugs. Proton spectra of multiple combinations (including pure standards as controls) were obtained. Both Ga3+ plus ADR-529 and Ga3+ plus doxorubicin showed evidence of complexation, as seen by appropriate peak shifts and changes in the associated coupling constants. Ga3+ plus ADR-529 plus epirubicin showed complexation different from that of Ga3+ plus ADR-529 or Ga3+ plus doxorubicin and consistent with the proposed structure. We conclude that ADR-529 would be able to form a ternary complex with an existing anthracycline-Fe3+ complex in an isolated aqueous environment. PMID- 1732726 TI - Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative. AB - The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes. PMID- 1732727 TI - Segments of the POU domain influence one another's DNA-binding specificity. AB - The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain. PMID- 1732728 TI - Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNA(Leu(UUR)) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. AB - Cytoplasts from two unrelated patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) harboring an A----G transition at nucleotide position 3243 in the tRNA(Leu(UUR)) gene of the mitochondrial genome were fused with human cells lacking endogenous mitochondrial DNA (mtDNA) (rho 0 cells). Selected cybrid lines, containing less than 15 or greater than or equal to 95% mutated genomes, were examined for differences in genetic, biochemical, and morphological characteristics. Cybrids containing greater than or equal to 95% mutant mtDNA, but not those containing normal mtDNA, exhibited decreases in the rates of synthesis and in the steady-state levels of the mitochondrial translation products. In addition, NADH dehydrogenase subunit 1 (ND 1) exhibited a slightly altered mobility on polyacrylamide gel electrophoresis. The mutation also correlated with a severe respiratory chain deficiency. A small but consistent increase in the steady-state levels of an RNA transcript corresponding to 16S rRNA + tRNA(Leu(UUR)) + ND 1 genes was detected. However, there was no evidence of major errors in processing of the heavy-strand encoded transcripts or of altered steady-state levels or ratios of mitochondrial rRNAs or mRNAs. These results provide evidence for a direct relationship between the tRNALeu(UUR) mutation and the pathogenesis of this mitochondrial disease. PMID- 1732729 TI - Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal. AB - Previous work has shown that the firefly (Photinus pyralis) luciferase contains a C-terminal peroxisomal targeting signal consisting of the tripeptide Ser-Lys-Leu. This report describes the microinjection of two proteins, (i) luciferase and (ii) albumin conjugated to a peptide ending in the sequence Ser-Lys-Leu, into mammalian cells grown in tissue culture. Following microinjection, incubation of the cells at 37 degrees C resulted in peroxisomal transport of these exogenous proteins into catalase-containing vesicles. The translocation was both time and temperature dependent. The transport could be inhibited by coinjection of synthetic peptides bearing various peroxisomal targeting signal motifs. These proteins could be transported into peroxisomes in normal human fibroblast cell lines but not in cell lines derived from patients with Zellweger syndrome. These results demonstrate that microinjection of peroxisomal proteins yields an authentic in vivo system with which to study peroxisomal transport. Furthermore, these results reveal that the process of peroxisomal transport does not involve irreversible modification of the protein, that artificial hybrid substrates can be transported and used as tools to study peroxisomal transport, and that the defect in Zellweger syndrome is indeed the inability to transport proteins containing the Ser-Lys-Leu targeting signal into the peroxisomal lumen. PMID- 1732730 TI - The restricted promoter activity of the liver transcription factor hepatocyte nuclear factor 3 beta involves a cell-specific factor and positive autoactivation. AB - The transcription factor hepatocyte nuclear factor 3 (HNF-3) is involved in the coordinate expression of several liver genes. HNF-3 DNA binding activity is composed of three different liver proteins which recognize the same DNA site. The HNF-3 proteins (designated alpha, beta, and gamma) possess homology in the DNA binding domain and in several additional regions. To understand the cell-type specific expression of HNF-3 beta, we have defined the regulatory sequences that elicit hepatoma-specific expression. Promoter activity requires -134 bp of HNF-3 beta proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites interacts with a novel cell-specific factor, LF-H3 beta, whose binding activity correlates with the HNF-3 beta tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is involved in the establishment of HNF-3 beta expression. We propose that both the LF-H3 beta and HNF-3 sites play an important role in the cell-type-specific expression of the HNF-3 beta transcription factor. PMID- 1732731 TI - Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation. AB - In the yeast Saccharomyces cerevisiae, a double-strand chromosome break created by the HO endonuclease is frequently repaired in mitotically growing cells by recombination between flanking homologous regions, producing a deletion. We showed that single-stranded regions were formed on both sides of the double strand break prior to the formation of the product. The kinetics of the single stranded DNA were monitored in strains with the recombination-deficient mutations rad52 and rad50 as well as in the wild-type strain. In rad50 mutants, single stranded DNA was generated at a slower rate than in the wild type, whereas rad52 mutants generated single-stranded DNA at a faster rate. Product formation was largely blocked in the rad52 mutant. In the rad50 rad52 double mutant, the effects were superimposed in that the exonucleolytic activity was slowed but product formation was blocked. rad50 appears to act before or at the same stage as rad52. We constructed strains containing two ura3 segments on one side of the HO cut site and one ura3 region on the other side to characterize how flanking repeats find each other. Deletions formed preterentially between the homologous regions closest to the double-strand break. By varying the size of the middle ura3 segment, we determined that recombination initiated by a double-strand break requires a minimum homologous length between 63 and 89 bp. In these competition experiments, the frequency of recombination was dependent on the length of homology in an approximately linear manner. PMID- 1732732 TI - Cell-type-specific expression of the rat thyroperoxidase promoter indicates common mechanisms for thyroid-specific gene expression. AB - A 420-bp fragment from the 5' end of the rat thyroperoxidase (TPO) gene was fused to a luciferase reporter and shown to direct cell-type-specific expression when transfected into rat thyroid FRTL-5 cells. Analysis of this DNA fragment revealed four regions of the promoter which interact with DNA-binding proteins present in FRTL-5 cells. Mutation of the DNA sequence within any of these regions reduced TPO promoter activity. The trans-acting factors binding to these sequences were compared with thyroid transcription factor 1 (TTF-1) and TTF-2, previously identified as transcriptional activators of another thyroid-specific gene, the thyroglobulin (Tg) gene. Purified TTF-1 binds to three regions of TPO which are protected by FRTL-5 proteins. Two of the binding sites overlap with recognition sites for other DNA-binding proteins. One TTF-1 site can also bind a protein (UFB) present in the nuclei of both expressing and nonexpressing cells. TTF-1 binding to the proximal region overlaps with that for a novel protein present in FRTL-5 cells which can also recognize the promoter-proximal region of Tg. Using a combination of techniques, the factor binding to the fourth TPO promoter site was shown to be TTF-2. We conclude, therefore, that the FRTL-5-specific expression of two thyroid restricted genes, encoding TPO and Tg, relies on a combination of the same trans-acting factors present in thyroid cells. PMID- 1732733 TI - Conserved DNA binding and self-association domains of the Drosophila zeste protein. AB - The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures. PMID- 1732734 TI - Both muscle-specific and ubiquitous nuclear factors are required for muscle specific expression of the myosin heavy-chain beta gene in cultured cells. AB - Expression of the myosin heavy-chain beta gene is controlled by multiple cis acting regulatory elements in the 5' flanking region; two of these, referred to as A (-276 to -263) and B (-207 to -180), are essential for conferring muscle specific activation on homologous and heterologous promoters. Here we report on the identification of nuclear protein factors that specifically bind to these two elements. By using the A element as a probe, as well as nuclear extracts from muscle cells, we found two protein-DNA complexes that displayed distinct bands in a gel mobility shift assay but had identical methylation interference patterns. One complex was present mainly in nuclear extracts from undifferentiated muscle and nonmuscle cells, whereas the other was observed mainly in nuclear extracts from differentiated muscle cells. Thus, the muscle-specific complex formation with the A element appears to be involved in determining tissue-specific expression. Furthermore, competition analysis demonstrated that the A-element binding factors also bind to the muscle-CAT motif in the cardiac troponin T gene. By using the B element as a probe, we saw similar patterns of gel-shifted bands and methylation interference in nonmuscle and muscle nuclear extracts. In addition, both elements A and B were found to be necessary for tissue-specific expression, suggesting that the muscle-specific activation of the myosin heavy chain beta gene may require interaction between a muscle-specific and a ubiquitous protein-DNA complex. PMID- 1732735 TI - A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity. AB - Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation. PMID- 1732736 TI - Sequential expression of multiple POU proteins during amphibian early development. AB - The octamer motif is a common cis-acting regulatory element that functions in the transcriptional control regions of diverse genes and in viral origins of replication. The ability of a consensus octamer motif to stimulate transcription of a histone H2B promoter in frog oocytes suggests that oocytes contain a transcriptionally active octamer-binding protein(s). We show here that frog oocytes and developing embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer binding-proteins were isolated from Xenopus cDNA libraries by virtue of their homology with the DNA binding (POU) domain of Oct-1. The predicted POU domains of these proteins were most highly related to mammalian Oct 3 (also termed Oct-4), a germ line-specific gene required for mouse early development. Transcripts from these amphibian POU-domain genes were most abundant during early embryogenesis and absent from most adult somatic tissues. One of the genes, termed Oct-60, was primarily expressed as a maternal transcript localized in the animal hemisphere in mature oocytes. The protein encoded by this gene was present in oocytes and early embryos until the gastrula stage of development. Transcripts from a second POU-domain gene, Oct-25, were present at low levels in oocytes and early embryos and were dramatically upregulated during early gastrulation. In contrast to the Oct-60 mRNA, translation of Oct-25 mRNA appeared to be developmentally regulated, since the corresponding protein was detected in embryos during gastrulation but not in oocytes or rapidly cleaving embryos. Transcripts from the third POU protein gene, Oct-91, were induced after the midblastula transition and reached their highest levels of accumulation during late gastrulation. The expression of all three genes decreased during late gastrulation and early neurulation. By analogy with other members of the POU domain gene family, the products of these genes may play critical roles in the determination of cell fate and the regulation of cell proliferation. PMID- 1732737 TI - Characterization of two developmentally regulated sea urchin U2 small nuclear RNA promoters: a common required TATA sequence and independent proximal and distal elements. AB - The promoters of two U2 small nuclear RNA genes isolated from the sea urchin Lytechinus variegatus were mapped by microinjection of genes into sea urchin zygotes. One gene, LvU2E, is expressed only in oocytes and embryos and is found in a tandemly repeated gene set, while the other gene, LvU2L, is a single-copy gene and is expressed in embryos and somatic cells. The promoters each contain a TATA sequence at -25 which is required for expression, a proximal sequence element (PSE) centered at -55 required for expression, a sequence at -100 which couples the core promoter (PSE plus TATA box) to the upstream element, and an upstream sequence which stimulates expression fourfold. The PSE together with the TATA sequence is sufficient to determine the transcription start site. There is no sequence similarity between the -100 and PSE sequences of the two genes. The 100 sequences can be interchanged between the two genes. The LvU2E PSE functions in the context of the LvU2L gene, but the LvU2L PSE functions poorly in the context of the LvU2E gene. PMID- 1732738 TI - The binding site of a steroid hormone receptor-like protein within the Drosophila Adh adult enhancer is required for high levels of tissue-specific alcohol dehydrogenase expression. AB - Developmental and tissue-specific transcription from the Adh distal promoter is regulated in part by the Adh adult enhancer, located 450 to 600 bp upstream from the distal RNA start site. We have characterized four proteins (DEP1 to DEP4), present in Drosophila tissue culture cell nuclear extracts, which bind to this enhancer. DEP1 and DEP2 bind to a positive cis-acting element (-492 to -481) and share nucleotide contacts. A small linker replacement deletion mutation, which disrupts the overlapping DEP1- and DEP2-binding sites, reduces Adh distal transcription in an alcohol dehydrogenase (ADH)-expressing cultured cell line, in the adult fat body (the major tissue of ADH expression), as well as in some but not all adult tissues where ADH is normally expressed. This enhancer element contains an imperfect palindromic sequence similar to steroid hormone receptor superfamily response elements. Binding-site screening of a lambda gt11 expression library has identified the steroid receptor superfamily member fushi tarazu factor 1 (FTZ-F1) as a protein that binds to this site. Anti-FTZ-F1 antibodies have identified DEP1 as FTZ-F1. DEP2 also binds to the FTZ-F1 site from the fushi tarazu zebra element, suggesting that DEP2 may also be a steroid receptor superfamily member. Our results raise the possibility that Adh regulation in certain adult tissues involves a hormone-mediated pathway. Because DEP1 (FTZ-F1) and DEP2 contact some of the same nucleotides within the positive cis element, it is unlikely that they can bind simultaneously. Such alternative binding may play a role in the tissue-specific and developmental transcription of Adh. PMID- 1732739 TI - RelB, a new Rel family transcription activator that can interact with p50-NF kappa B. AB - We have identified a serum-inducible gene, relB, which encodes a protein of 558 amino acids containing a region with high similarity to c-Rel and other members of the Rel family. Transcriptional activation analysis of GAL4-RelB fusion proteins in yeast cells reveals that RelB contains in its C-terminal 180 amino acids a transcriptional activation domain. The N-terminal part including the region of similarity with the Rel family shows no detectable transcriptional activity. RelB does not bind with high affinity to NF-kappa B sites, but heterodimers between RelB and p50-NF-kappa B do bind to different NF-kappa B binding sites with a similar affinity to that shown by p50-NF-kappa B homodimers. However, RelB/p50-NF-kappa B heterodimers, in contrast to p50-NF-kappa B homodimers, transactivate transcription of a promoter containing a kappa B binding site. PMID- 1732740 TI - Complex recognition site for the group I intron-encoded endonuclease I-SceII. AB - We have characterized features of the site recognized by a double-stranded DNA endonuclease, I-SceII, encoded by intron 4 alpha of the yeast mitochondrial COX1 gene. We determined the effects of 36 point mutations on the cleavage efficiency of natural and synthetic substrates containing the Saccharomyces capensis I-SceII site. Most mutations of the 18-bp I-SceII recognition site are tolerated by the enzyme, and those mutant sites are cleaved between 42 and 100% as well as the wild-type substrate is. Nine mutants blocked cleavage to less than or equal to 33% of the wild-type, whereas only three point mutations, G-4----C, G-12----T, and G-15----C, block cleavage completely. Competition experiments indicate that these three substrates are not cleaved, at least in part because of a marked reduction in the affinity of the enzyme for those mutant DNAs. About 90% of the DNAs derived from randomization of the nucleotide sequence of the 4-bp staggered I-SceII cleavage site are not cleaved by the enzyme. I-SceII cleaves cloned DNA derived from human chromosome 3 about once every 110 kbp. The I-SceII recognition sites in four randomly chosen human DNA clones have 56 to 78% identity with the 18-bp site in yeast mitochondrial DNA; they are cleaved at least 50% as well as the wild-type mitochondrial substrate despite the presence of some substitutions that individually compromise cleavage of the mitochondrial substrate. Analysis of these data suggests that the effect of a given base substitution in I-SceII cleavage may depend on the sequence at other positions. PMID- 1732741 TI - Zinc fingers and other domains cooperate in binding of Drosophila sry beta and delta proteins at specific chromosomal sites. AB - The closely related Drosophila serendipity (sry) beta and delta zinc finger proteins display consensus in vitro DNA recognition sequences differing by 4 of 13 nucleotide positions and bind in vivo to distinct sets of sites on polytene chromosomes. We compared the pattern of in vivo chromosomal binding of deleted forms of the sry delta protein fused to beta-galactosidase and expressed in Drosophila transgenic lines. Results show that the carboxy-terminal DNA-binding finger domain is required and sufficient for binding at specific chromosomal sites but that this binding does not nearly reproduce the wild-type pattern. An NH2-terminal domain of the sry delta protein is essential to its specificity of in vivo interaction with chromatin. In vitro and in vivo experiments using reciprocal finger swap between the sry beta and delta proteins suggest that the in vivo specificity is dependent on selective protein-protein contacts at defined chromosomal sites, in addition to DNA specific recognition. PMID- 1732742 TI - RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae. AB - The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector. PMID- 1732743 TI - Alternate use of divergent forms of an ancient exon in the fructose-1,6 bisphosphate aldolase gene of Drosophila melanogaster. AB - The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities. PMID- 1732744 TI - Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation. AB - Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide. PMID- 1732745 TI - Activation of a system for the joining of nonhomologous DNA ends during Xenopus egg maturation. AB - Mature Xenopus laevis eggs provide an elementary reaction system of illegitimate recombination which efficiently joins nonhomologous DNA ends (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). Here we show that stage VI oocytes, known to express a system for homologous recombination (D. Carroll, Proc. Natl. Acad. Sci. USA 80:6902-6906, 1983), are completely devoid of this joining system. Nonhomologous DNA end-to-end joining, however, attains full activity only at an extremely late stage of egg maturation. Cycloheximide inhibition patterns indicate that nonhomologous joining activity is regulated at the G2 restriction point of the cell cycle. Implications of homologous and nonhomologous recombination activities during egg maturation are discussed. PMID- 1732746 TI - mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization. AB - Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending. PMID- 1732747 TI - Molecular cloning of the alpha-globin transcription factor CP2. AB - CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1. PMID- 1732748 TI - Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13. AB - Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12 p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children. PMID- 1732749 TI - Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster. AB - Pre-mRNAs cotranscriptionally associate with a small group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. We have previously identified two genes in Drosophila melanogaster, Hrb98DE and Hrb87F (i.e., genes at 98DE and 87F encoding putative hnRNA binding proteins), which encode five protein species homologous to the mammalian A-B hnRNP proteins. The studies presented herein show that antibodies against the RNP domains of Hrb98DE reacted with 10 to 15 distinct spots of 38 to 40 kDa in the basic region of two dimensional gels. These nuclear proteins bound single-stranded nucleic acids and were extracted from Drosophila tissue culture cells as 40 to 80S hnRNP complexes in association with 300 to 800 nucleotide fragments of RNA. The peak of poly(A)+ RNA sequences was coincident with the peak of HRB proteins in sucrose gradients, strongly suggesting that the HRB complexes identified are Drosophila hnRNP complexes. The repertoire of HRB proteins did not change significantly during embryogenesis and was similar to that observed in Drosophila tissue culture cells. Analyses with peptide-specific antisera demonstrated that the major proteins in the hnRNP complex were encoded by the two genes previously identified. Although the Drosophila HRB proteins are only approximately 60% identical throughout the RNP domains to the mammalian A-B hnRNP proteins, features of the basic pre-mRNA packaging mechanism appear to be highly conserved between D. melanogaster and mammals. PMID- 1732750 TI - Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases. AB - tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3' extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system. PMID- 1732751 TI - An alternatively processed mRNA from the avian c-erbB gene encodes a soluble, truncated form of the receptor that can block ligand-dependent transformation. AB - At least four major transcripts are produced by the avian c-erbB/epidermal growth factor receptor gene. cDNAs corresponding to the smallest one, a 2.6-kb transcript, were isolated from an adult chicken liver cDNA library. Sequence analysis revealed that the 3' end of one cDNA clone diverged from the known sequence of the extracellular ligand-binding domain (LBD) of the full-length receptor. A genomic DNA subfragment that contained this unique 3' divergent end was isolated. Sequence analysis of this genomic DNA fragment revealed that the 2.6-kb c-erbB transcript is produced by alternative processing. Translation of this 2.6-kb transcript would produce a secreted, truncated receptor molecule which contains the amino-terminal three-fourths of the extracellular LBD of the native receptor. COS1 cells and primary chicken embryo fibroblast cells were transfected with expression vectors that contained the 2.6-kb c-erbB cDNA. Conditioned medium from these transfected cells contained a 70-kDa protein that was specifically immunoprecipitated by a polyclonal antiserum directed against the LBD of the avian c-erbB gene product. The 70-kDa truncated receptor could be coimmunoprecipitated from conditioned medium of transfected COS1 cells that was supplemented with recombinant human transforming growth factor alpha (TGF alpha) by a monoclonal antibody against human TGF alpha. Additionally, transfected chicken embryo fibroblast cells that overexpressed the 70-kDa truncated receptor were blocked in their ability to form TGF alpha-dependent colonies in soft agar. These data suggest that the secreted, truncated receptor encoded by the 2.6-kb c erbB transcript can bind to TGF alpha and may play an important growth-regulatory function in vitro. PMID- 1732752 TI - A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation. AB - Lymphotoxin (LT; also known as tumor necrosis factor-beta) is a pleiotropic cytokine whose expression is tightly regulated in most cells and is repressed prior to activation signals. In some early B cells and Abelson murine leukemia virus-transformed pre-B-cell lines, LT mRNA is constitutively expressed. To examine the molecular regulation of the LT gene in a constitutively expressing cell line, we studied the Abelson murine leukemia virus-transformed lines PD and PD31. As demonstrated by primer extension analysis, constitutively expressed pre B-cell-derived and inducibly expressed T-cell-derived LT mRNA were initiated at the same cap sites and predominant cap site utilization was conserved. Furthermore, we delineated an upstream activating sequence that was an important functional component of lymphotoxin transcriptional activation in PD and PD31 cells. The upstream activating sequence was localized to an essentially homopolymeric A + T-rich region (LT-612/-580), which was bound specifically by recombinant human high-mobility group I protein (HMG-I) and a PD/PD31 nuclear extract HMG-I (HMG-I-like) protein. The nuclear extract-derived HMG-I-like protein was recognized by anti-HMG-I antibody and bound to LT DNA to effect an electrophoretic mobility shift identical to that of bound recombinant human HMG I. These findings implicate HMG-I in the regulation of constitutive lymphotoxin gene expression in PD and PD31 cells. HMG-I and HMG-I-like proteins could facilitate the formation of active initiation complexes by altering chromatin structure and/or by creating recognition sites for other activator DNA-binding proteins, some of which may be unique to or uniquely modified in these constitutive LT mRNA producers. PMID- 1732753 TI - Far-field potentials in circular volumes: evidence to support the leading/trailing dipole model. AB - The leading/trailing dipole model explains the production of far-field potentials as an asymmetry in the leading and trailing dipole moments of a propagating action potential detected by a referential montage. This investigation documents the production of far-field potentials produced by a pure dipole generator in a circular volume conductor. Multiple equipotential waveforms are recorded in an adjoining circular volume conductor attached to the one in which the dipole generator is located. This finding substantiates the "wick electrode" effect that explains the equipotential and instantaneous distribution of far-field potentials over relatively large distances in volume conductors. The present findings support a number of the leading/trailing dipole model proposals which explain far field potential generation. PMID- 1732754 TI - Possible model for generation of P9 far-field potentials. AB - The distribution of P9 far-field somatosensory evoked potentials, after stimulation of the median nerve with a knee reference, was examined to determine the mechanism of the generation of P9 potentials. In addition to positive potentials (P9s), we found a negative potential (N9) recorded from the chest ipsilateral to the stimulation. The simulation of the distribution of these P9/N9 potentials by an electrical circuit diagram suggested the validity of this model for generation of the P9s. PMID- 1732755 TI - Case-of-the-month: autosomal recessive myotonia congenita: marked muscle weakness in a 16-year-old boy. AB - A 16-year-old boy had a 10-year history of stiffness in leg muscles. There was marked weakness of neck flexors, shoulder abductors, and ankle dorsiflexors, with hypertrophy of most muscle groups and both action and percussion myotonia. The parents were normal. Motor unit potential mean duration was reduced in the weakest muscle (tibialis anterior), and a biopsy of the same muscle showed only minimal abnormalities. Exercise and repetitive stimulation (30 Hz) of the tibialis anterior disclosed a decline in the compound muscle action potential that appeared to correlate with the muscular weakness. PMID- 1732756 TI - Indirect measurement of sensory conduction by sympathetic skin responses. PMID- 1732757 TI - Axonal degeneration in the Guillain-Barre syndrome and anti-GM1 ganglioside antibodies. PMID- 1732758 TI - Desmin and vimentin in regenerating muscles. AB - Desmin is a normal constituent of skeletal muscle fibers; vimentin is contained in myoblasts and connective tissue cells. The intracellular localization of both intermediate filament proteins in regenerating rat muscles was investigated by immunohisto- and immunocytochemistry. Necrosis was induced by hot Ringer solution. Desmin and vimentin were diffusely distributed in myoblasts and young myotubes. Both proteins became arranged in a sarcomeric fashion between the Z lines when the sarcomeres got into register. Desmin reactivity persisted, but vimentin disappeared after about 2 weeks. Traces were found for up to 4 weeks. This observation is in contrast to findings in fetal and cultured muscles in which several authors did not find expression of vimentin after myoblast fusion. The presence of vimentin may well be a useful marker for regenerated muscle fibers in muscle biopsies from patients with neuromuscular disorders. PMID- 1732759 TI - Myokymic discharges and enhanced facial nerve reflex responses after recovery from idiopathic facial palsy. AB - A functional disorder of facial muscle activity commonly occurs in patients after recovery from Bell's palsy with axonal degeneration. The postparalytic facial dysfunction is probably related to the aberrant growing of regenerating axons, although other theories such as ephaptic transmission, spontaneous generation of impulses, and enhancement of motoneuron excitability should also be considered. In this work, we have carried out a comparative electrophysiological study of both sides of the face in 23 patients who had recovered from a unilateral Bell's palsy with axonal degeneration. At rest, spontaneous firing of motor units was observed in muscles of the previously paralyzed side. Direct motor responses to facial nerve stimulation were smaller in the muscles of the previously paralyzed side, but reflex responses obtained in the same muscles by stimulation of either the facial or trigeminal nerve were larger when compared with those of the contralateral side. These data indicate that patients with "postparalytic facial dysfunction" may have an increased background muscle activity, as well as an enhanced recruitment of facial motoneurons to reflex activation in the side of the previous paralysis. These findings are compatible with an enhanced level of motoneuron excitability in the facial nucleus. PMID- 1732760 TI - Failure to detect HTLV-I by in situ hybridization in the biopsied muscles of viral carriers with polymyositis. AB - Direct infection of muscle fibers by human T-lymphotropic virus type I (HTLV-I) has recently been reported in a patient with polymyositis infected with both HTLV I and human immunodeficiency virus (HIV). Coinfections of these viruses are frequently found in the United States. In Kagoshima, Japan, patients with polymyositis have a significantly increased incidence of seropositivity to HTLV-I alone, when compared with the general population of Kagoshima. In this study, we examined muscle tissue from 6 HTLV-I-positive patients with polymyositis from Kagoshima. To detect HTLV-I products, sensitive immunohistochemistry and in situ hybridization analysis were performed. These were compared with muscle fibers from a well-characterized transgenic mouse model which expressed HTLV-I tax. No specific signals were detected in the biopsied muscles of patients with polymyositis infected with HTLV-I alone. HIV co-infection may, therefore, augment HTLV-I expression through either immunosuppression or direct viral interactions. PMID- 1732761 TI - Influence of peripheral afferents on cortical and spinal motoneuron excitability. AB - The objective of this study was to establish to what extent muscle, cutaneous, and joint afferents alter the excitability of spinal and cortical motor neurons. This question was examined by studying the impact of electrical stimulation of the second and third digits, the median nerve at the wrist, and the recurrent thenar motor branch on the F/H and magneto-electrical cortical motor responses (MEPs) of the thenar muscles. The firing frequencies of single F/H motor unit action potentials were unaltered by the foregoing conditioning peripheral stimuli. MEPs conditioned by motor threshold stimulation of the median nerve at the wrist or the recurrent motor branch were significantly increased in size at conditioning to test intervals of 50 to 80 milliseconds. No significant change in MEP size resulted from conditioning stimulation of the digital nerves. We conclude that muscle afferents were primarily responsible for the increase in MEP size. Conditioning stimuli may allow examiners to assess central motor conduction where it would otherwise be impossible. PMID- 1732762 TI - Absence of malignant hyperthermia contractures in Becker-Duchenne dystrophy at age 2. AB - Two 2-year-old males underwent muscle biopsy that established the histopathologic diagnosis of Becker dystrophy in one, and Duchenne dystrophy in the other. Concomitant contracture testing with caffeine or halothane was normal for malignant hyperthermia (MH). The results suggest that acute hypermetabolism or acute rhabdomyolysis during anesthesia, in patients with these disorders, is related to the X-linked myopathy and its associated muscle deterioration, rather than to the autosomal dominant MH. PMID- 1732763 TI - Electrophysiologic studies in the Guillain-Barre syndrome: effects of plasma exchange and antibody rebound. AB - Nerve conduction studies (NCS) and antiperipheral nerve myelin antibody (A-PNM Ab) titers were measured serially in 29 patients with Guillain-Barre syndrome (GBS), of whom 21 were treated with plasmapheresis. Data were obtained from 3 to 6 days until 1 to 2 years after onset of symptoms. Within 3 to 6 days, mean NCS were abnormal. They improved some by 1 week and became maximally abnormal by 4 to 8 weeks, during which time A-PNM Ab fell to low levels. In 5 patients plasmapheresed, A-PNM Ab fell and then increased at 4 to 8 weeks, followed by significant deterioration of NCS (P = 0.01) compared with those without antibody rebound at 18 weeks. These results suggest that, in monophasic GBS, there may be two mechanisms of conduction dysfunction such as early paranodal retraction and later demyelination. In some patients plasmapheresed, A-PNM Ab may rebound associated with further conduction dysfunction. These patients may benefit from further plasmapheresis. PMID- 1732764 TI - Deficiency of acetylcholine receptors in a case of end-plate acetylcholinesterase deficiency: a histochemical investigation. AB - A young boy is described who, since early infancy, suffered from weakness of predominantly proximal limb muscles. Electromyography revealed impairment of neuromuscular transmission. There were no antibodies against acetylcholine receptors. The amplitude of miniature end-plate potentials was reduced. Neuromuscular junctions showed somewhat coarse postsynaptic junctional folds and degeneration products in the synaptic clefts and in postsynaptic areas. Using qualitative and semiquantitative histochemical methods, at light- and electronmicroscopical levels, acetylcholinesterase (AChE) activity and acetylcholine receptors (AChRs) were demonstrated to be deficient. This patient demonstrates that the clinical picture of congenital myasthenia may closely resemble a congenital myopathy. The findings provide evidence that AChR deficiency offers protection from some of the effects of AChE deficiency. The clinical features of AChE deficiency depend, not only on the level of residual enzyme activity, but also on the degree of AChR reduction, if present. PMID- 1732765 TI - Peripheral sensory abnormalities in patients with multiple sclerosis. AB - Although multiple sclerosis primarily affects myelin within the central nervous system, both pathologic and physiological studies suggest that mild deficits in peripheral nervous system myelin may be common. To evaluate this question further, we performed near nerve studies on sural nerves of 14 patients with multiple sclerosis. Peak-to-peak amplitude and maximum conduction velocity were normal in 9 of 14 patients, while minimum conduction velocity, or the velocity of the slowest-conducting component of the sensory action potential, was abnormally reduced in 9 patients. In addition, the supernormal period was evaluated for patients and compared with a control sample; multiple sclerosis patients showed a significant reduction in the amplitude of supernormality. Both the reduction in minimum conduction velocity and the alteration in the supernormal period are consistent with a mild defect in peripheral myelin. PMID- 1732766 TI - Effect of low Ca2+ solution on muscle contraction of developing, preclinical dystrophic (dy2j) mice. AB - EDL muscles from normal and dystrophic (dy2j) mice aged 7 to 21 days of postnatal life were examined. Muscles were divided into 2 groups according to age, 7 to 14 days and 16 to 21 days postnatal, so as to assess age- and/or phenotype-related differences in the muscle response to low Ca2+ solution. Tension production was already much impaired in "predystrophic" muscles. At this stage, however, there was essentially no difference in twitch kinetics between normal and dystrophic muscles. Upon exposure to low Ca2+ solution, twitch responses of both normal and dystrophic muscles declined in a similar manner. In the youngest animals studied (7 to 14 days), the tetanic responses of both normal and dystrophic muscles to low Ca2+ solution were also similar. In animals 15 to 21 days old, however, the tetanic tension developed in low Ca2+ solution by dystrophic muscles, was significantly less than that of normal. Moreover, under these conditions (i.e., in low Ca2+ solution), and following tetanic stimulation, the membrane potential of dystrophic muscles in this age group was significantly more depolarized than that of normal muscles. Our results suggest that the ability of the cell to deal with extracellular Ca2+ is normal in predystrophic muscles up to 21 days of postnatal life. The results also clearly point to the fragility of the membrane in these muscles. PMID- 1732767 TI - Muscle excitation in elderly adults: the effects of training. AB - Muscle membrane excitability is thought to decline with aging; the extent of this decline may be noninvasively assessed by measurement of the electrically evoked compound muscle action potential (M-wave). The intent of this study was two-fold: (1) to compare the M-wave in the brachioradialis (BR), tibialis anterior (TA), and thenar (TH) muscles of elderly (mean age = 66.3 +/- 3.7 years) and young (mean age = 31.2 +/- 4.9 years) adults, and (2) to determine the effects of 12 weeks of resistance training on M-wave characteristics in elderly adults. Prior to training, the elderly subjects had significantly smaller (P less than 0.05) resting M-waves than the young adults in the BR (4.8 mV vs. 8.7 mV), TA (8.8 mV vs. 11.0 mV), and TH (5.2 mV vs. 10.2 mV) muscles. During a 2-minute voluntary fatigue paradigm (3 seconds MVC per 2 seconds rest for 2 minutes), there was no evidence of excitability failure in either group. Following training, there was a significant increase (P less than 0.05) in the size of the M-wave of the TH (pretraining: 5.2 mV; posttraining: 8.96 mV) and BR (pretraining: 4.8 mV; posttraining: 6.1 mV), and a nonsignificant increase in the M-wave of the TA, but there was no change in the relative behavior of the M-wave during the 2-minute voluntary fatigue paradigm. It is suggested that the decline in muscle membrane excitation with aging may be due, at least in part, to the effects of a decreased membrane potential on the muscle fiber action potential.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1732768 TI - Repetitive nerve stimulation vs. twitch tension in rats with EAMG. AB - Two methods were compared with regard to their ability to detect acetylcholine receptor-dependent neuromuscular dysfunction in rats with experimental autoimmune myasthenia gravis. In both cases, detection of AChR impairment required amplification of symptoms by administration of the AChR antagonist curare and appeared to be directly related to increasing levels of circulating anti-AChR antibodies. First, in vivo evaluations of decremental compound motor action potentials following repetitive nerve stimulation were performed by electromyography. Impaired neuromuscular function (i.e., greater than 10% decrement) was noted only after rats had been immunized twice with AChR, requiring levels of circulating anti-AChR antibody greater than about 200 micrograms/mL in serum. In contrast, the direct in vitro evaluation of stimulated isometric twitch tension appeared to be more sensitive in that impaired AChR dependent muscle contraction was clearly observed following a single AChR immunization, and, as shown previously, required anti-AChR antibody levels of about 50 micrograms/mL in serum. Further discussion is presented concerning the advantages and disadvantages associated with each method of monitoring disease. PMID- 1732769 TI - Safety and cost effectiveness of high-osmolality as compared with low-osmolality contrast material in patients undergoing cardiac angiography. AB - BACKGROUND AND METHODS: Low-osmolality contrast agents produce fewer hemodynamic and electrophysiologic alterations during cardiac angiography, but they are 20 times more expensive than high-osmolality contrast agents. In a randomized, double-blind trial comparing a nonionic low-osmolality contrast agent (Omnipaque 350) with a high-osmolality agent that does not avidly bind calcium (Hypaque 76) in 505 patients undergoing cardiac angiography, we determined the incidence of minor, mild, moderate, and severe adverse reactions, identified risk factors for such reactions, and evaluated the cost effectiveness of various strategies for the use of contrast material. RESULTS: The 253 patients who received a high osmolality contrast agent were three times more likely to have a moderate adverse reaction (95 percent confidence interval for the relative risk, 1.6 to 5.5) but no more likely to have a severe reaction (95 percent confidence interval, 0.2 to 2.3) than the 252 patients who received a low-osmolality agent. All 10 severe reactions occurred in patients who were older than 60 years or had unstable angina. Patients with these characteristics were also 3.5 times more likely (95 percent confidence interval, 1.8 to 6.8) to have a moderate reaction (44 of 310 patients, or 14 percent) than those without either characteristic (8 of 195 patients, or 4 percent). We estimated that the incremental cost of each moderate reaction avoided would be $1,698 with a strategy that involved giving a low osmolality contrast agent only to patients who were over 60 years of age or had unstable angina, instead of giving a high-osmolality agent to all patients. The incremental cost per moderate reaction avoided by giving a low-osmolality contrast agent to all patients rather than only to those over 60 or with unstable angina would be $5,842. CONCLUSIONS: The use of contrast agents with low rather than high osmolality during cardiac angiography reduces the risk of moderate, but not of severe, adverse reactions to the agent used. A strategy of reserving low osmolality contrast agents for use in patients at high risk for adverse reactions would be more cost effective than one requiring their use in all patients. PMID- 1732770 TI - A comparison of nonionic, low-osmolality radiocontrast agents with ionic, high osmolality agents during cardiac catheterization. AB - BACKGROUND: Nonionic, low-osmolality radiocontrast agents are used frequently because they are believed to be safer than ionic, high-osmolality agents, but they are also more expensive. We conducted a randomized trial to compare the incidence of adverse events after the administration of ionic, high-osmolality and of non-ionic, low-osmolality radiocontrast agents during cardiac angiography. METHODS: We compared the need to treat patients for adverse reactions and the frequency and severity of specific hemodynamic, systemic, and symptomatic side effects in two groups of patients randomly assigned to receive either ionic, high osmolality or nonionic, low-osmolality radiocontrast material, and also in 366 patients who could not be randomized. RESULTS: Treatment for adverse events was required in 213 of 737 patients who received high-osmolality contrast agents (29 percent) but in only 69 of 753 patients who received nonionic agents (9 percent) (95 percent confidence interval for the percent difference, 15.9 to 23.6 percent). Hemodynamic deterioration and symptoms also occurred more often in the high-osmolality group, as did severe or prolonged reactions (2.9 percent, as compared with 0.8 percent in the nonionic group; P = 0.035). The severe reactions were largely confined to patients with severe cardiac disease. Multivariate analysis showed that the presence of severe coronary disease and unstable angina were predictors of clinically important adverse reactions. If all the patients in our randomized trial had been given nonionic contrast material, the incremental cost per procedure would have been $89. CONCLUSIONS: Nonionic, low-osmolality contrast material is better tolerated during cardiac angiography than ionic, high osmolality contrast material. Since cost constraints may prevent the universal use of nonionic contrast material, its selective use in patients with severe cardiac disease could be considered. PMID- 1732771 TI - A controlled trial of a program for the active management of labor. AB - BACKGROUND: Over the past two decades, the rate of cesarean section in the United States has risen from 5 percent to 25 percent of deliveries, primarily because of the increased frequency of dystocia (arrest of labor). One strategy that has been proposed for increasing the rate of vaginal delivery is a program of active management of labor that encourages early amniotomy, early diagnosis of slow progress in labor, and the use of higher than usual doses of oxytocin; the efficacy and safety of this approach are uncertain, however. METHODS: We conducted a randomized trial in which nulliparous women in spontaneous labor at term were randomly assigned to either active management of labor or traditional management. With active management, amniotomy was performed within one hour of the diagnosis of labor, and when the rate of cervical dilation was less than 1 cm per hour, oxytocin was infused at an initial rate of 6 mU per minute. The dose was increased by 6 mU per minute every 15 minutes (to a maximum of 36 mU per minute) until there were seven contractions every 15 minutes. RESULTS: For the women assigned to active management (n = 351), the cesarean-section rate was 10.5 percent, as compared with 14.1 percent for those assigned to traditional management (n = 354, P = 0.18). The 26 percent reduction in the cesarean-section rate was due primarily to a decrease in dystocia. After we controlled for potential confounding variables, the reduction in the rate of delivery by cesarean section was statistically significant (odds ratio for women given active as compared with traditional management, 0.57; 95 percent confidence interval, 0.36 to 0.95). With active management, the average length of labor was shortened by 1.66 hours, principally because of earlier amniotomy and earlier use of oxytocin. There was no increase in maternal or neonatal morbidity, and there were significantly fewer infectious complications in the mothers. CONCLUSIONS: The program we studied for the active management of labor reduces the incidence of dystocia and increases the rate of vaginal delivery without increasing maternal or neonatal morbidity. PMID- 1732772 TI - Pancreatic carcinoma. PMID- 1732773 TI - Mullerian inhibiting substance as a marker for ovarian sex-cord tumor. PMID- 1732774 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 7-1992. A 57-year-old man with a 20-year history of episodic headache, Flushing, hypotension, and occasional syncope. PMID- 1732775 TI - Low-osmolality contrast agents--who needs them? PMID- 1732776 TI - Treatment of HIV infection--progress in perspective. PMID- 1732777 TI - An affair of the heart. PMID- 1732778 TI - Correction: Rice-based oral electrolyte solutions for infantile diarrhea. PMID- 1732779 TI - Bacterial and protozoal gastroenteritis. PMID- 1732780 TI - Bacterial and protozoal gastroenteritis. PMID- 1732781 TI - Bacterial and protozoal gastroenteritis. PMID- 1732782 TI - Cholesterol, apolipoproteins, and the risk of myocardial infarction. PMID- 1732783 TI - Cholesterol, apolipoproteins, and the risk of myocardial infarction. PMID- 1732784 TI - Cholesterol, apolipoproteins, and the risk of myocardial infarction. PMID- 1732785 TI - Chagas' heart disease. PMID- 1732786 TI - GM-1 ganglioside for spinal-cord injury. PMID- 1732787 TI - GM-1 ganglioside for spinal-cord injury. PMID- 1732788 TI - Ocular ultraviolet exposure from halogen lamps. PMID- 1732789 TI - What drives neonatal screening programs? PMID- 1732790 TI - New York's health care proxy law. PMID- 1732791 TI - Disappearance of thyrotropin-blocking antibodies and spontaneous recovery from hypothyroidism in autoimmune thyroiditis. AB - BACKGROUND: Hypothyroidism may result from the production of antibodies that block the actions of thyrotropin. How often these thyrotropin-blocking antibodies are a cause of hypothyroidism and whether their production may cease, causing hypothyroidism to disappear, have not been extensively studied. METHODS: We determined the frequency with which thyrotropin-blocking antibodies were present in 172 hypothyroid patients with goitrous autoimmune thyroiditis (Hashimoto's disease) and 64 hypothyroid patients with atrophic autoimmune thyroiditis (idiopathic primary hypothyroidism). For 6 to 11 years we then followed 21 of these patients who were found to have thyrotropin-blocking antibodies. They received levothyroxine therapy for 3.5 to 8 years, after which it was discontinued. At frequent intervals during this time we measured the patients' serum concentrations of thyroxine, triiodothyronine, thyrotropin, and thyrotropin blocking antibodies (measured as immunoglobulins that inhibit thyrotropin binding and immunoglobulins that inhibit thyrotropin bioactivity). RESULTS: Thyrotropin blocking antibodies were detected in 9 percent of the patients with goitrous autoimmune thyroiditis and in 25 percent of those with atrophic autoimmune thyroiditis. Among the 21 patients studied serially while receiving levothyroxine, thyrotropin-blocking antibodies disappeared in 15 (group 1), 7 of whom had goiter initially, and persisted in 6 (group 2), none of whom had goiter initially. Levothyroxine therapy was subsequently discontinued in these 21 patients. Six of those in group 1 (four with goiter) remained euthyroid (mean follow-up after discontinuation of therapy, 2.1 years), and nine became hypothyroid again within 3 months. All six patients in group 2 remained hypothyroid. CONCLUSIONS: Hypothyroidism in some patients with autoimmune thyroiditis may be due to thyrotropin-blocking antibodies. The production of thyrotropin-blocking antibodies may subside, producing remissions of hypothyroidism. Chronic autoimmune thyroiditis may therefore cause transient as well as permanent hypothyroidism. PMID- 1732792 TI - Aphasia. PMID- 1732793 TI - Seminars in medicine of the Beth Israel Hospital, Boston. Mutations in collagen genes as a cause of connective-tissue diseases. PMID- 1732794 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 8-1992. A 47-year-old man with recurrent fever and fulminant hepatic failure 31 days after a liver transplant. PMID- 1732795 TI - Vanishing hypothyroidism. PMID- 1732796 TI - Reforming the health insurance market for small businesses. PMID- 1732798 TI - Is there sex bias in the management of coronary artery disease? PMID- 1732797 TI - Is there sex bias in the management of coronary artery disease? PMID- 1732799 TI - Is there sex bias in the management of coronary artery disease? PMID- 1732800 TI - Pancreatic injury after cardiopulmonary bypass. PMID- 1732801 TI - Pancreatic injury after cardiopulmonary bypass. PMID- 1732802 TI - Pancreatic injury after cardiopulmonary bypass. PMID- 1732803 TI - Inflammatory bowel disease. PMID- 1732804 TI - Inflammatory bowel disease. PMID- 1732805 TI - Development of infants with iron deficiency. PMID- 1732806 TI - Allergic rhinitis. PMID- 1732807 TI - Guillain-Barre syndrome after Chlamydia pneumoniae infection. PMID- 1732808 TI - Patients do benefit from using additives, and you can reduce the risks. PMID- 1732809 TI - Quality control in the dialysis unit is key when prescribing concentrate additives. PMID- 1732810 TI - The aftermath from the RVS will be a shocker for nephrologists. PMID- 1732811 TI - Professional recognition and compensation for staff RNs: 1990 and 1995. AB - Hospitals are searching for means to attract and retain professional nurses. Professionally oriented recognition and compensation programs have been suggested. In 1990 programs relied heavily on basic, time-limited incentives such as educational leave and tuition reimbursement. Projections for 1995 suggest increased implementation of more motivational programs such as gain sharing and salaried compensation. PMID- 1732812 TI - Nursing, competition, and quality. AB - As the economic activity of the health care system becomes increasingly organized according to traditional market forces, administrators and those involved in quality assessment of nursing care must anticipate changing economic incentives and the challenges and opportunities they will present. PMID- 1732813 TI - Validated indexes: key to nursing acuity standardization. AB - Classification systems for nurse staffing could be standardized and used in reimbursement and outcome studies if validated health status and illness severity measures were used as proxies for nursing acuity. PMID- 1732814 TI - Calculating and updating nursing turnover costs. AB - A detailed study allowed FY 1988 nursing turnover costs to be calculated at four southeastern hospitals. These turnover costs were later updated using a procedure incorporating Consumer Price Index information. This procedure allows nurse administrators to determine and update turnover costs, to accurately evaluate the impact of nursing turnover, and to facilitate resource allocation. PMID- 1732815 TI - Searching for excellence. PMID- 1732816 TI - Is a baccalaureate in nursing worth it? AB - This study investigated economic costs and benefits of generic BSN and BS degrees from students' points of view. Economic benefits from the baccalaureate degree exceeded benefits from an RN-credential only, after a lifetime of employment. The net present value of a BSN degree discounted at 5% was four times greater than the net present value of a BSRN/diploma and three times greater than a BSN/associate degree. PMID- 1732817 TI - The intrapreneurial nursing department: nature and nurture. AB - By creating and promoting an "intrapreneurial climate" with appropriate "actions" within the department of nursing, nurses may exploit their creativity and commitment to health care without leaving the organization. This article discusses an approach to fostering nurse intrapreneurship and gives examples of success achieved at Stanford University Hospital. PMID- 1732818 TI - Forces influencing public policy. PMID- 1732819 TI - Does improving access mean rationing health care? PMID- 1732820 TI - Shoot the messenger. PMID- 1732821 TI - Cost and regulation of drugs. AB - Drug cost and regulation involves numerous complex issues, a few of which have been addressed here. Health care professionals, consumers, federal and state governments, and the pharmaceutical industry are among the key players in the continuing debate over drug cost and regulation. PMID- 1732822 TI - The techniques of developing self-managed teams: the nurse manager's role. PMID- 1732823 TI - The economic benefits to service and education of a critical care learning experience for baccalaureate students. PMID- 1732824 TI - Brainstorming: a process for cost reduction. PMID- 1732825 TI - An interview with Sharon Flynn Hollander. Interview by Connie R. Curan. PMID- 1732826 TI - [Too old to treat?]. PMID- 1732827 TI - [Neuromodulation; a new treatment method for incontinence and micturition disorders]. PMID- 1732828 TI - [Frequent side effects of cotrimoxazole in persons infected with HIV, possibly due to metabolites of sulfamethoxazole]. PMID- 1732829 TI - [Referral policy in unwanted delay of pregnancy]. PMID- 1732830 TI - [Revision of the trauma score]. PMID- 1732831 TI - [The cost of illness]. AB - This study is an essential prerequisite to gain more insight into the complex relationship between public health and the costs of medical care. It offers a first tentative but comprehensive description of the total direct costs generated by all diseases in the Dutch population. We classified estimated direct costs of illness (39.8 thousand million guilders) by type of care, sex, age and 48 diagnostic categories for 1988. In order to estimate the costs of health care in the year 2030, we linked this information with demographic development. We were able to allocate 75% of all costs to diseases. Ranking by major disease categories revealed that mental disorders account for the highest proportion of costs (20%), followed by diseases of the circulatory system (9%), and diseases of the digestive system (8%). Costs of medical care increased significantly with age and were presumably incurred for non fatal ailments. Health care costs for men and women were almost similar. In the year 2030 the costs of dementia and diseases of the circulatory system will increase most. A reliable estimate of the costs should also take epidemiological and economic considerations into account. PMID- 1732832 TI - [HIV infection in pregnant women in The Netherlands]. AB - A review is given of the pregnant women with HIV infection known to the co ordinating centre of the 'Dutch prospective study of HIV seropositive pregnant women and their children'. Fifty-five women with 57 pregnancies were discovered between 1 September 1985 and 1 January 1991. Of these women, 60% were not of Dutch origin and 56% were living in Amsterdam. Thirty-two women were (former) intravenous drug users. In 23 women heterosexual transmission was likely; two women were also blood transfusion recipients. Nearly half (11/23) of these women came from, or had a partner from, an AIDS-endemic area. In 16 women HIV seropositivity was known before pregnancy. Twenty-four women were specifically tested during pregnancy or within 1 week after parturition because of risk factors for HIV infection. Fifteen women were discovered in a HIV screening programme of pregnant women in Amsterdam (n = 13) and the University Hospital of Groningen (n = 2). At first, eight of these women did not mention risk factors. Thirty-five women could choose abortion (less than or equal to 20 weeks of gestation); the majority (69%) decided to continue pregnancy. Twelve pregnancies ended in an elective or spontaneous abortion, 39 in the birth of 40 children (one pair of twins) of whom 39 were viable, three women were still pregnant and of three the pregnancy outcome was unknown. Preterm delivery (8/39) and birth weight less than 2500 gram (7/39) were seen frequently, the latter particularly in women (formerly) using intravenous drugs (6/25). PMID- 1732833 TI - [Consensus in the therapy of acute otitis media]. AB - Treatment strategies in acute otitis media show important differences. To find agreement on the different therapeutic options a Dutch Consensus Conference was organized. An attitude of watchful waiting is the approach of choice for all children older than one year. Although myringotomy was advised for pain relief for many years, analgesics are the preferred symptomatic treatment at this moment. Antibiotics are useful in very young children up to age of one year, recurrences of otitis media in young children and in children in whom the infection recurs an irregular course. Myringotomy is limited to cases in which antibiotics fail to improve the middle ear condition, determination of the causative agent is mandatory or to confirm the middle ear infection if this cannot be done by mirror examination. Since the clinical course of acute otitis media has become milder in the last three decades adjustment of the present consensus will be necessary in the near future. PMID- 1732834 TI - [Initial experiences with neuromodulation as treatment for incontinence and micturition disorders in The Netherlands]. AB - Neuromodulation is a new treatment modality for disturbances of bladder function. By stimulation of a sacral nerve the pelvic floor is activated, thus affecting the reflex arcs which control micturition and continence. Along with 2 case histories the methods of the preoperative percutaneous test stimulation and the implantation of stimulator and electrode are described. PMID- 1732835 TI - [Medical activities under adverse conditions. The Directorate-General International Cooperation]. PMID- 1732836 TI - [Nipple secretion: a concern for the family physician?]. PMID- 1732837 TI - [Nipple secretion: a concern for the family physician?]. PMID- 1732838 TI - [Was nervous consumption a precursor of anorexia nervosa?]. PMID- 1732839 TI - [Always damp]. PMID- 1732840 TI - [Dry-bed training and diagnosis in nocturnal enuresis]. PMID- 1732841 TI - [Thymectomy as treatment of myasthenia gravis and of thymoma]. PMID- 1732842 TI - ["Spontaneous" bacterial peritonitis]. PMID- 1732843 TI - [Calcium pyrophosphate arthropathy: more than chondrocalcinosis and pseudogout]. PMID- 1732844 TI - [Developments in cancer mortality in The Netherlands since 1950]. PMID- 1732845 TI - [Bilateral lung transplantation in patients with cystic fibrosis]. AB - Bilateral lung transplantation in patients with cystic fibrosis is reported. Five were operated upon: one died six days after operation; during the operation extracorporeal circulation had been used in view of severe haemodynamic instability. One other patient died after 96 days due to invasive ulcerative aspergillosis. Three patients are alive and well. Selection of patients is shown to be the most important determinant of successful outcome. PMID- 1732846 TI - [The STING procedure: subureteral teflon application in vesico-ureteral reflux]. AB - Apart from open surgical correction of vesicoureteral reflux an endoscopic technique exists in which teflon paste is injected under the refluxing ureteral orifice; this is known as the STING procedure. We describe this technique, which if necessary may be repeated. The results in our patients are comparable with results from literature, with 21/23 (91%) of the ureters without reflux after treatment. The advantages and disadvantages of this technique are discussed. The good results, the short hospital stay, and the minimal perioperative morbidity favour the STING procedure when compared with the open surgical technique, and make it applicable as a good alternative to open surgical technique. PMID- 1732847 TI - [Hypomagnesemia and chondrocalcinosis]. AB - A female patient aged 45 years is described with a rare form of chondrocalcinosis caused by hypomagnesaemia due to excessive renal loss of magnesium. The patient also had impaired renal conservation of potassium leading to a hypokalemia. She most probably had an idiopathic renal tubular dysfunction. Magnesium supplementation prevented further symptoms. Therefore in young patients with chondrocalcinosis it can be of therapeutic importance to search for an underlying treatable metabolic disorder. PMID- 1732848 TI - [Chronic fatigue syndrome]. PMID- 1732849 TI - [Chronic fatigue syndrome]. PMID- 1732850 TI - AIDS: advocacy & activism. PMID- 1732851 TI - In 1992--a nurse in every school. PMID- 1732852 TI - Uncompensated care--the millstone around the neck of U.S. health care. PMID- 1732853 TI - Managing polarities--containing costs and improving quality in nursing education. PMID- 1732854 TI - New leaders needed. PMID- 1732855 TI - The long-term care dilemma--what nurses need to know about Medicare. PMID- 1732856 TI - Keep the options open. PMID- 1732857 TI - A few points. PMID- 1732858 TI - Drug calculation errors of baccalaureate nursing students. AB - Most researchers who study the dosage calculation skills of nursing students have looked at the mathematical or computational ability of the students. The authors report findings from a study that analyzed the dosage calculation errors of nursing students from conceptual, mathematical, and measurement perspectives. PMID- 1732859 TI - Veterinary surgical experiences for nursing students. PMID- 1732860 TI - Teaching for creativity. AB - Although nurse education programs state that creativity is a valued attribute of the nursing student, some programs do not stimulate creative nursing approaches in their students. The author applies the psychophysiological theory of brain hemisphericity to nurse education to identify teaching approaches that model and encourage the development of creativity. Teaching methods that encourage right hemisphere divergent thinking are emphasized to facilitate creative nursing care in nursing practice. PMID- 1732861 TI - Nursing education and service collaborate on graduate curriculum development. AB - Nursing and nursing education exist because of a need by society. Nursing education is translated into reality by those in nursing service. The authors present a systematic process that was used to determine the need in one community for a master's clinical specialization in pediatric critical care and analyze the relationship between nursing educational goals and the needs of nursing service. This method can be adapted and used by others in program development and evaluation. PMID- 1732862 TI - Sharing of time: using interviews with grandparents to introduce gerontology to nursing students. PMID- 1732863 TI - Faculty development and curriculum change in substance abuse. AB - What approaches can be used to upgrade nursing education and clinical skills in alcohol and drug abuse? The authors discuss the development of faculty and curricula in three schools of nursing. The programs described are part of a national initiative to ensure that health care professionals have basic knowledge and clinical skills in screening, assessment, intervention, and the appropriate use of referral systems for clients with substance abuse problems. PMID- 1732864 TI - Integrating gerontology into the baccalaureate nursing curriculum: does it make a difference? PMID- 1732865 TI - Intervention: a strategy to help chemically dependent students. AB - Chemical dependence in nursing students is not a pleasant reality to face. However, facing rather than denying this disease in our students may help to save a valuable professional resource. The author describes how one nurse educator used the chemical dependence tool of intervention as a strategy to help a student eventually enter the nursing profession. PMID- 1732866 TI - A rural health experience. PMID- 1732867 TI - Liability issue related to advising and counseling on health issues. PMID- 1732868 TI - Integrated science modules: preparing non-nurse MSN students in the basic sciences. AB - Many nursing programs are developing masters degree tracks for non-nurses. The author discusses the use of integrated science modules as an independent, successful, and less traditional approach to these students learning needs. PMID- 1732869 TI - Strategies and faculty roles for teaching RN students. AB - To help maximize the learning of RN students, the author discusses strategies and faculty roles that are particularly effective with these students. Methodologies from nursing practice, supervision, and management, combined with principles from adult education, provide the basis for this success-oriented approach. PMID- 1732870 TI - A cost-effective method of skills practice. PMID- 1732871 TI - Improving student competency via videotaping. PMID- 1732872 TI - Managing your academic career--feedback in clinical teaching. PMID- 1732873 TI - Orientation programs for nurse-adult learners: fostering a sense of community. AB - Are you searching for ways to create a sense of community among the students and faculty in your program? The authors find that an orientation that combines humor, warmth, empathy, and introduces the concept that students and faculty share the identity of "adult learner" is key to developing a community of scholars. Based on their experience, the authors describe three principles for developing an orientation program for nurse-adult learners that can be adapted to other educational settings. PMID- 1732875 TI - Are they fit for work? PMID- 1732874 TI - Crisis management--a guide to survival. PMID- 1732876 TI - Guide to EC directives: Part 3. PMID- 1732877 TI - Manual handling of loads at work. PMID- 1732878 TI - Recruiting competent staff: a general health warning. PMID- 1732879 TI - [Waldenstrm macroglobulinemia presenting in the form of cold urticaria and cold purpura]. AB - A Waldenstrom macroglobulinemia case, manifested in the form of non-specific skin symptoms (cold urticaria and cold purpura) is described in a 37 years old female patient. As new skin-symptoms progressive telangiectasia and naevi aranei could be observed. Involvement of skin may be explained by macroglobulins with kryoprotein properties, making early diagnosis possible at the same time. PMID- 1732880 TI - [The second half of our century and the future of hepatology]. PMID- 1732881 TI - [Remembering Lajos Bakay]. PMID- 1732882 TI - [Results of prenatal cytogenetic screening at the Prenatal Genetic Center of the Postgraduate Medical University between 1980-1990]. AB - Evaluation of prenatal cytogenetic diagnosis by Genetic Center of Postgraduate Medical University in 1980 and 1990. Between 1980 and 1990, 1039 amniocenteses (AC), 1263 chorionic villus samples (CVS), and 30 fetal blood sampling were performed for cytogenetic reasons. The rate of chromosome abnormalities were 5.5 per cent in the first trimester CVS, 5.2 per cent in the second trimester CVS, and 3.1 per cent in AC. The Down syndrome was the most frequent abnormality (46 fetuses) and the next was the Edwards syndrome (15 cases). It was established that though the case number is fourteen times more than the beginning of this decade, this was enough only for screening women 39 or over. During this period several new methods were introduced making possible the diagnosis from 9th week of pregnancy until term. Among these methods the CVS has not only become an alternative to the AC but now it is the most frequent procedure in our laboratory. Though most pregnants are still referred for prenatal cytogenetic investigation because of their advanced age, the authors search for other risk factors which would make possible screening in younger women, too. PMID- 1732883 TI - [Electron microscopic structure of mastocytes of the airways and changes in the clinical status during steroid inhalation therapy]. AB - The structure of epithelial mast cells obtained by bronchial biopsy from asthmatic patients reveals characteristic qualitative and quantitative features. Great amount of partly or completely depleted mast cells can be observed in active asthmatic patients, indicating the liberation of bronchoconstrictive mediators. After a two months treatment with inhalative steroid (budesonide) the process of degranulation slows down, the number of depleted mast cells decreases. Parallel with the above described phenomenon, the number of dyspneic attacks, as well as the necessity of other antiasthmatic drugs show a significant decrease. However, in this patient material, during the treatment period, airway hyperreactivity, detected by bronchial provocation, failed to show any remarkable change. PMID- 1732884 TI - [Mitochondrial myopathy (mono- and multisystem mitochondrial diseases)]. AB - Four mitochondrial myopathy cases are reported. In addition to complaints, clinical and laboratory findings the changes in number, size, shape and in structure of the mitochondria in skeletal muscles are detailed. In three of their cases the disease is monosystemic, one case seems to be multisystemic in character: beside the morphologically proven muscle and liver changes it is likely that cardiac muscle and the central nervous system is also affected on the base of the clinical symptoms. PMID- 1732885 TI - [Surgical treatment of ischemic heart disease and unstable angina pectoris caused by intramyocardial descending coronary artery in a young patient]. AB - Authors present a case report about the surgical management of a genetically predisposed ischemic heart disease occurred in young age with rare anatomical variation causing unstable angina pectoris. Besides the description the type of surgery, emphasis is made on the importance of whole scale cardiological investigations and--as in the described case--on the role of nearly ideal cooperation between the cardiologist and heart surgeon, which finally could prevent a larger size of myocardial infarction to occur. PMID- 1732886 TI - The angiosomes of the mammals and other vertebrates. AB - This is a comparative study of the vasculature of the integument and underlying deep tissues of a range of mammals and other vertebrates. The investigation was conducted in the pig, monkey, dog, cat, possum, guinea pig, rat, rabbit, duck, and toad. The results from each are compared not only to each other, but also to previously performed human studies. The arterial network of the fresh animal cadaver was injected with a mixture of lead oxide and gelatin. The vascular anatomy of the skin, deep tissues, and individual muscles was defined by dissection, cutaneous perforator counts, photography, and radiography. A similar pilot study of the venous framework was performed in the pig, dog, and rabbit that included maps of the sites and orientations of the valves. The vasculature of the integument and deep tissues was correlated, and we found that we were able to define angiosomes (composite blocks of tissue supplied by the same source vessel) in each animal. Results revealed a marked dissimilarity of the overlying cutaneous vessels in many cases, yet a striking resemblance of the vascular architecture of the deep tissues. The size and density of the cutaneous perforators bore a close relation to the degree of the skin mobility, being large and sparse where the skin was mobile and smaller and more densely grouped where the integument was tethered or fixed. The cutaneous vasculature of the human resembled that of the monkey closely, was similar to that of the dog, cat, and possum, and was dissimilar to that of the pig, rat, guinea pig, and rabbit. Studies of the amphibian and bird bore many resemblances to those of the mammals. They provided basic concepts regarding modification of the animals' vascular anatomy in response to the functional demands of the species. In each animal, the arteries formed an unbroken network throughout the body. This consisted of anatomic territories linked by anastomotic vessels that were usually of reduced caliber. The pattern of the venous system was almost identical. Valved venous territories were linked by avalvular (oscillating) veins. The common denominator in the vascular system is the capillary bed. Conceptually, the anatomic arrangement of the arteries and veins, reproduced in each species, appears to be a sophisticated mechanism to allow equilibration of flow and pressure arriving at and departing from the capillary bed. The angiosome concept is reinforced by the animal studies. Although this investigation is essentially a detailed pilot study, it embraces many animals commonly used for experimentation and provides a reference atlas of their vasculature.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732887 TI - The nasal tip: anatomy and aesthetics. AB - New anatomic observations and expanded aesthetics are presented based on an in depth analysis of 50 patients undergoing primary open rhinoplasty. The alar cartilages can be conceived of as three crura (medial, middle, and lateral), each composed of two segments, plus distinct intervening junction points of aesthetic importance. The classic four-dot tip aesthetics can be expanded and wrapped around the nasal lobule in a three-dimensional fashion. Three nasal tip angles are easily defined (angle of tip rotation, angle of domal definition, and angle of domal divergence) and can be created surgically. PMID- 1732888 TI - Anatomic variations of the nasolabial fold. AB - The nasolabial fold varies considerably from person to person. Three main groups may be distinguished: convex, concave, and straight. It is the muscles of smiling that are directly responsible for the shape and depth of the fold, and in their absence of function, as in facial palsy, the nasolabial fold disappears. Cadavers were selected in accordance with the nasolabial fold they presented and were dissected to analyze the difference in underlying anatomy between one fold shape in one cadaver and another fold shape in another. The study demonstrates that the nasolabial fold is the result of a conflict between soft and dynamic tissues of the middle face or an interaction between the skin and fat envelope on one side and the underlying muscles on the other. The greater this conflict, the more excess there is of cheek skin and the more pronounced a nasolabial fold. The mechanism that creates the nasolabial fold and the anatomy of the fold are described in this paper. PMID- 1732889 TI - A comparison of absorbable and nonabsorbable suture materials for skin repair. AB - This prospective clinical study was conducted to compare the outcome of elective surgical wound repair in the occipital region during rhytidectomy using absorbable and nonabsorbable suture materials. On an alternative basis, 6-0 polypropylene and 6-0 plain catgut were used to repair the incisions on the upper and lower half of the surgical wounds in 80 sites. These sites were then compared for stitch marks, erythema, hypertrophic scars, infection, and wound necrosis. This study revealed slightly visible stitch marks in 4 of 40 (10 percent) sites repaired with catgut and in 10 of 40 (25 percent) sites repaired with polypropylene material (p less than 0.10); however, this was not statistically significant. There were five incidences of suture-site erythema (12.5 percent) noted in the group of catgut repairs in comparison with three incidences (7.5 percent) in the group repaired using polypropylene. Furthermore, there was no statistically significant difference in hypertrophic scarring or infection rate between these groups. The incidence of erythema following repair with catgut was higher, but this was also not statistically significant. Considering these findings, coupled with the avoidance of patient discomfort, suture removal, and time spared for the surgeon and staff when absorbable suture material is used, the superiority of plain catgut over nonabsorbable material becomes evident. PMID- 1732890 TI - Is reversed venous flow safe in free-flap transfer? A dilemma with the radial forearm flap. AB - The distally based radial artery forearm flap has become our workhorse flap for hand and finger coverage, relying on reversed or retrograde venous outflow through the venae comitantes. Free-flap transfer, however, has been used by us only with antegrade venous anastomoses. This study was intended to determine if a single retrograde venous anastomosis would be adequate for flap viability. Six groups of saphenous flaps were developed in New Zealand White rabbits. In situ flaps compared antegrade with retrograde venous outflow in groups 1 and 2. Microvascular venous anastomoses with antegrade or retrograde outflow were compared in groups 3 and 4. Free-flap transfer with antegrade or retrograde venous outflow was compared in groups 5 and 6. No significant differences in survival was found between groups 1 and 2. A significant difference in survival (p = 0.025) was found between groups 3 and 4, but technical differences make these groups incomparable. Significantly better survival (p = 0.014, chi-squared test) was found in group 5 with antegrade outflow versus group 6 with retrograde outflow. PMID- 1732891 TI - Electron microscopic observations of degeneration of human Pacinian corpuscles in amputated fingers. AB - Amputated human fingers were used to observe the morphologic changes in degeneration of Pacinian corpuscles, and postoperative moving two-point discrimination of the replanted fingers was examined to analyze sensory recovery after replantation. Normal corpuscles are composed of an axon terminal and inner and outer cores, resembling a sliced onion. The inner core is composed of thin, multilayered lamellar cells, and the outer core consists of multiple layers of thin perineurial cells. Based on our morphologic findings, following mitochondrial degeneration in the axon terminal, the terminal and inner core cells disappeared within 9 to 16 hours, but the outer core did not lose its structure until more than 24 hours after amputation. Collagen fibrils in the corpuscles appeared from 5 hours after amputation and periodically increased their amount up to 27 hours after amputation. Postoperative sensory recovery of the replanted fingers was significantly poorer with 9 hours or more of cold ischemia. These findings suggest that the inner core cells originating from Schwann cells degenerate at over 9 hours after amputation, and this may be related to the poor sensory recovery of replanted fingers. It also appears that the outer core cells originating from the perineurial cells in the amputated fingers survive even up to 27 hours after amputation and produce collagen fibrils in the extramatrix spaces of the outer core cells. PMID- 1732892 TI - Walking track analysis: a long-term assessment of peripheral nerve recovery. AB - Functional recovery following sciatic, tibial, and peroneal nerve injury was assessed over a 1-year period using walking track analysis in the rat. Internal neurolysis did not affect nerve function. Crush injury induced a temporary, but complete, loss of function that recovered to control levels by 4 weeks. Nerve transection resulted in complete loss of function without any evidence of recovery. After nerve repair, functional recovery occurred, reaching near-optimal recovery by 12 weeks. The degree of functional recovery varied with the specific nerve involved. The sciatic nerve recovered 41 percent of function, whereas the tibial nerve recovered 54 percent of function. The peroneal nerve exhibited the highest degree of recovery, achieving functional levels similar to control values. Assessment of neural regeneration using walking track analysis appears to be a valuable addition to the traditional methods of histology and electrophysiology. PMID- 1732893 TI - An extended approach for the vascular pedicle of the lateral arm free flap. AB - We present an extension of the surgical approach for harvesting the lateral upper arm free flap by which an additional 6 to 8 cm of pedicle length may be gained. First, the flap is raised by the standard lateral approach. Then, by proceeding proximally and posteriorly, the triceps muscle is split between its lateral and long heads to expose the entire length of the profunda brachii vessels in the spiral groove. A tunnel is developed beneath the lateral head of the triceps, and the flap or its pedicle is delivered through this. We describe the surgical technique and present details of a dissection study on 25 fresh cadaver limbs. The nerve branches to the lateral head of the triceps, which are close to the vessels of the flap, are highly variable in number and location. When unusually short and distally placed, they are at risk of damage, but damage can be avoided if the tunnel is not unduly widened. We present our early clinical experience in 10 consecutive cases using the extended-pedicle lateral arm flap. The free pedicle length in this series ranged from 8 to 13 cm. The maximum flap size was 5 x 19 cm. All cases were successful, although one required reoperation for venous thrombosis. Although postoperative testing of upper arm muscle function showed some weakness and impaired endurance, this was found equally in the surgically disturbed triceps and in the untouched elbow flexors and thus could not be attributed to motor nerve damage to the triceps muscle. PMID- 1732894 TI - The effect of fibrin glue on skin grafts in infected sites. AB - Fibrin bonding of skin grafts to wounds is an essential part of the graft adherence process. Bacteria, in concentrations greater than 10(5)/gm of tissue, are associated with graft failure. Sixty-five rats were randomly divided into three groups, dorsal split-thickness skin grafts were harvested, and the sites were inoculated with Staphylococcus aureus. After incubation, each wound was quantitatively biopsied and treated with saline, fibrin glue with aprotinin, or fibrin glue alone. We found that the addition of commercially available fibrin glue with or without the antifibrinolytic agent aprotinin is capable of restoring graft adherence to normal levels in graft sites infected with greater than 10(5) bacteria/gm of tissue. Fibrin glue may have potential for increasing skin-graft take in the clinical situation where the graft bed is infected. PMID- 1732895 TI - Efficacy of operative cure in pressure sore patients. AB - The advent of air flotation-type beds and purified growth factors that may accelerate open wound contraction, coupled with very high recurrence rates and decreasing health resources, suggests that surgical reconstruction of pressure sores may not be indicated in all patients. In an effort to define which patients might benefit from operation, we reviewed the data from 40 consecutive patients with 68 pressure sores operated on under the direction of a single surgeon between 1981 and 1989. Patients were categorized on the basis of the presence or absence of paraplegia and its etiology. Sixty-six operations were performed, 55 muscle or fasciocutaneous flaps and 11 cutaneous flaps. There was a 36 percent operative complication rate, with no operative mortalities. Follow-up ranged from 1 to 71 months, with a mean of 21 months. Despite an 80% healed rate at the time of discharge, 61% of sores and 69% of patients had recurrent ulceration within a mean of 9.3 months. Analysis of these data indicates that surgical reconstruction of pressure sores does not appear to be efficacious in young posttraumatic paraplegics or cerebrally compromised elderly patients. Further review of the data failed to identify those patients likely to remain healed after operative repair of their pressure sores. PMID- 1732896 TI - Desmopressin decreases operative blood loss in spinal cord injury patients having flap reconstruction of pelvic pressure sores. AB - To test the effectiveness of desmopressin in decreasing operative blood loss in major flap reconstructions, 44 hemostatically normal patients with spinal cord injury and pelvic pressure sores participated in a randomized, prospective, double-blind clinical trial. Each patient received a single dose of desmopressin (0.3 micrograms/kg) or saline placebo intravenously at the initiation of a reconstructive surgical procedure. Preoperative and postoperative hemoglobin, hematocrit, von Willebrand factor, and factor VIII determinations and measurement of intraoperative blood loss and transfusions of packed red cells were recorded. Desmopressin-treated patients experienced a smaller decline in hemoglobin and hematocrit levels postoperatively. In those patients requiring major flap reconstructions, the use of desmopressin significantly decreased intraoperative blood loss and subsequent transfusion requirements. The levels of von Willebrand factor and factor VIII tended to be higher, although not significantly so, in subjects receiving desmopressin. No patient experienced an adverse reaction to the drug. We conclude that a single dose of desmopressin, given immediately preoperatively, is safe and effectively decreases blood loss and transfusion requirements in patients undergoing major flap reconstructive surgery. PMID- 1732897 TI - Muscle microcirculatory impairment following acute compartment syndrome in the dog. AB - Visualization of the intramuscular microcirculation during and after compartmental syndrome was studied by microangiograms and histologic cross sections. A marked reduction in the circulation of the endomysial capillary network was found during compartment tamponade, whereas the perimysium arteriolar system was patent. Revascularization took place by formation of distorted blood vessels accompanied by intramuscular hematomas in muscles 7 and 14 days after the compartment insult. The cross sections show massive fibroblastic activity around blood vessels that caused concealed intramuscular pressure-ischemic contracture resulting in the foci of myofibrillar necrosis seen within normal muscle tissue. The muscle located in the tamponaded compartment profusely bleeds when it is touched, even though its viability is in doubt. The explanation for this clinical observation might be the abnormal intramuscular revascularization that was found in this work. PMID- 1732898 TI - Parallel-fibered muscles transplanted with neurovascular repair into bipennate muscle sites in rabbits. AB - Experiments were performed on 20 New Zealand White male rabbits. Our hypotheses were that (1) latissimus dorsi (LTD) muscles transplanted into the site of a bipennate rectus femoris (RFM) muscle with neurovascular repair would retain their parallel-fibered structure and (2) the parallel-fibered structure of latissimus dorsi grafts would reduce their total fiber cross-sectional area and adversely affect force development relative to that of bipennate rectus femoris grafts and muscles. Compared with their respective donor muscles, 120 to 150 days after grafting, latissimus dorsi and rectus femoris grafts showed no change in the number of fibers and a decrease in the mean single-fiber cross-sectional area to approximately 70 percent. The latissimus dorsi grafts, which remained parallel fibered, developed maximum forces 34 and 23 percent of the values for fully activated rectus femoris grafts and muscles, respectively. The deficit in the maximum force of the latissimus dorsi grafts resulted primarily from the smaller total-fiber cross-sectional area as a result of the parallel-fibered structure. PMID- 1732899 TI - An absorbable catheter system for use in microvascular and peripheral vascular surgery: an experimental study in the canine. AB - An absorbable catheter for use in regional anticoagulation in microvascular and peripheral vascular surgery was studied in 20 sites in 10 adult beagle dogs to answer three questions: (1) Could the polyglycolic acid and trimethylene carbonate catheter withstand intraarterial pressures of infusion and completely absorb over a predictable time interval? (2) Could the catheter be filled with heparin and maintain patency for reuse after a 24-hour interval? (3) Could the catheter be placed in a side branch of a major artery and, after catheter dissolution, maintain long-term patency of the primary feeding artery? The catheters were completely absorbed from 24 to 34 weeks following implantation. The catheters were able to withstand intraarterial pressures, and no evidence of significant thrombosis of the primary feeding artery was seen in any animal studied. No complications of catheter leak, hematoma formation within the catheter placement sites, or sepsis were noted in any of the 20 catheter sites studied. PMID- 1732901 TI - Image and reality, words and deeds. PMID- 1732900 TI - The effect of low-energy laser on skin-flap survival in the rat and porcine animal models. AB - Low-energy lasers are currently being used in the therapy of rheumatoid arthritis, chronic pain, muscle strain, and the promotion of wound healing in human and veterinary medicine. This study examined the effects of low-energy laser on skin-flap survival in a controlled interspecies study using the rat and porcine models. Twenty dorsal skin flaps based caudally were performed in 20 rats (10 laser-treated and 10 control flaps). The wounds were closed, and the flaps were sutured over the skin. Forty dorsal pig skin flaps based medially were raised in five pigs. The flaps were treated once per day for 10 days: 4 days preoperatively, the day of surgery, and 5 days postoperatively (30 s/cm3 per day). The average surviving rat flap surface area for the laser-treated flaps was 653 +/- 112 mm (mean +/- SD) and 580 +/- 60 mm in the control flaps, which was not significant (p greater than 0.05). In the porcine model, the average surviving area for the 20 laser-treated flaps was 949 +/- 174 mm, and the control average (n = 20) was 969 +/- 147 mm, also not significant. No beneficial effect was seen with low-energy laser preoperative and postoperative treatment of skin flaps in the rat and porcine models. PMID- 1732902 TI - Scalp reconstruction with a prefabricated abdominal flap carried by the radial artery. AB - Custom prefabrication of free flaps provides an unlimited variety of applications, since flaps can be created with expendable tissues and without restriction to naturally occurring vascular territories. These principles also can be used to customize flaps that could not be completed by conventional means. We report a case of scalp reconstruction using a random-pattern abdominal flap in which a radial artery fascial flap was induced to serve as the vascular carrier. In addition to providing durable scalp coverage, the prefabricated free flap enabled salvage of an abdominal flap that would otherwise have been aborted after intermediate transfer to the forearm. PMID- 1732903 TI - Chemotherapy and surgical resection combined with immediate reconstruction in a 1 year-old child with rhabdomyosarcoma of the maxilla. AB - Parameningeal head and neck rhabdomyosarcomas of childhood are often considered unresectable and are treated with irradiation and chemotherapy. High-dose radiation therapy has a very long-term detrimental effect on the developing face and also results in many other significant long-range complications. With the availability of advanced craniofacial surgical and free-tissue-transfer techniques, one-stage resection and immediate reconstruction, along with the use of effective preoperative and postresection chemotherapy instead of local radiation, may be the logical approach to the treatment of selected patients with chemosensitive parameningeal head and neck rhabdomyosarcomas. PMID- 1732904 TI - A successful bypass operation for long-standing venous stasis ulcer of the leg. AB - A case of long-standing venous stasis ulcer after thrombophlebitis of the deep vein system is reported that was treated successfully by transferring a pedicled greater saphenous vein and its branches from the healthy side. PMID- 1732905 TI - Compound functioning free muscle flap transplantation (lateral half of soleus, fibula, and skin flap). AB - The functioning free-muscle transfer is a microneurovascular technique that has proven effective for patients who have a major muscle or muscle group loss for which no other less complicated procedures are available. A new functioning muscle (lateral half of the soleus) transfer was used for forearm reconstruction. This functioning muscle can be transferred alone, or it can be used with overlying skin or nearby fibular bone or both. It was used in a selective complicated case in which not only were major functional muscles lost, but a bone deficiency and skin loss also were seen. The operation was done in one stage with the composite flap (muscle plus bone plus skin). The recovered transferred muscle provided adequate strength and excursion for a functional hand and forearm. PMID- 1732906 TI - A stereotactic system for guiding complex craniofacial reconstruction. AB - A stereotactic system has been designed to address the problem of achieving symmetry in complex and extensive craniofacial defects. Preliminary testing suggests that such a system, which allows for the intraoperative application of preoperative CT planning, will be useful in guiding the reconstruction of congenital or acquired bony time, is being used to investigate the correlation of intraoperative globe position following enophthalmos correction with long-term outcome, particularly as it relates to the size and location of the orbital defect, and the timing of the procedure. PMID- 1732907 TI - Repair of scalp defects using a tissue expander and Marlex mesh. AB - A simple technique using Marlex mesh and a tissue expander to cover scalp defects is described and two patients are presented. This technique is suitable for medium-sized defects that cannot be closed primarily. Marlex mesh is sutured to the wound edges in lieu of a temporary skin graft and to prevent enlargement of the defect during tissue expansion. The tissue expander is placed under adjacent normal scalp in a subgaleal pocket developed through the scalp defect. The scalp defect is closed secondarily using the expanded scalp flap. This technique was performed in two patients with satisfactory results. Marlex mesh obviates the need for a temporary skin graft to cover the scalp defect. PMID- 1732908 TI - Evaluation of a technique designed to correct nasolabial folds. AB - This is a long-term follow-up of correction of nasolabial folds in conjunction with face lift that was first published in 1987. In the last 200 face lifts, nasolabial lipectomy has been carried out in 90 percent. Refinements and extensions of the procedure are also described with case illustrations. PMID- 1732909 TI - Ferris N. Smith: the man and his art. PMID- 1732910 TI - Pedicled osteomyocutaneous latissimus dorsi flap for large chest-wall full thickness reconstruction. PMID- 1732911 TI - Plastic surgeons as inventors. PMID- 1732912 TI - Retroauricular skin graft: preferred location of donor site. PMID- 1732913 TI - Basal cell carcinoma occurring in the perianal region. PMID- 1732914 TI - Capsule thickness and breast firmness. PMID- 1732915 TI - Does irradiation to the augmented breast produce scar contracture? PMID- 1732916 TI - Reconstruction of the scrotum. PMID- 1732917 TI - Treatment of exophthalmos. PMID- 1732918 TI - Low-tech plastic and reconstructive surgery in two war-affected developing countries: experience with 1500 operations. PMID- 1732919 TI - An instance of double publishing. PMID- 1732920 TI - The evolution of mammography. AB - The history of mammography can be arbitrarily subdivided into three periods: The Age of Pioneers highlights the work of Salomon, Kleinschmidt, Warren, Vogel, Seabold, Gershon-Cohen, Leborgne, Egan, Gallager, Martin, Dodd, Strax and their colleagues; The Age of Technical Progress adds the names of Gould, Wolfe, and Gros and their co-workers; The Modern Era reflects the contributions of Price, Butler, Ostrum, Becker, Isard, Moskowitz, Sickles, Kopans, Homer, Tabar, and their associates. The ultimate success of mammography, the preeminent method of breast cancer screening, could not have been achieved without the intense vision, idealism, and scientific skill of its creators and nurturers. These investigators deserve a debt of gratitude that society can never adequately repay. PMID- 1732921 TI - Breast imaging: current status and future directions. PMID- 1732922 TI - Evaluation of the postoperative breast. AB - With widespread use of mammography for breast cancer screening, the number of surgical procedures has also increased. Overlapping with radiographic signs of malignancy, including masses, areas of asymmetric density and architectural distortion, microcalcifications, and skin thickening, postsurgical changes may make mammographic evaluation difficult. After tumor excision and irradiation where breast alterations are more profound and prolonged, the task of distinguishing recurrent tumor from scarring or fat necrosis is even more challenging. Mammograms after breast conservation therapy for carcinoma or after cosmetic surgery require correlation with physical findings and the surgical procedures that were performed. Responses of tissue to lumpectomy and radiation, such as breast edema and skin thickening, are most pronounced 6 to 12 months after treatment, gradually resolving within 1 to 3 years. Carefully tailored mammographic studies will promote the dual goal of early detection of local tumor recurrence and avoidance of misinterpreting postoperative and irradiation changes as malignancy. Sequential examinations should begin with a postoperative preradiation mammogram for residual carcinoma, particularly when microcalcifications have been present, followed by the baseline postradiation examination at 6 months with the next study 6 months later (1 year after initial treatment). Mammograms of the treated breast may be performed at intervals of 6 months until radiographic stability has been recognized. Annual studies thereafter are suggested. The contralateral, unaffected breast should be evaluated mammographically according to screening guidelines or clinical concerns. Mammograms performed after cosmetic and reconstructive procedures should be correlated with the surgical techniques and clinical history. Modified views for silicone implants can maximize visualization of breast parenchyma. Ultrasonography is a useful complement to mammography in demonstrating the origin of a palpable mass either within the implant or the breast parenchyma. In reduction mammoplasty, distorted architecture, parenchymal bands, tissue redistribution, and fat necrosis should be recognized. After mastectomy, myocutaneous reconstruction may be performed. Masses that develop within flap reconstructions most frequently represent fat necrosis, which, when calcifying oil cysts are seen, may have a characteristic radiographic appearance. PMID- 1732923 TI - Prebiopsy needle localization. Methods, problems, and expected results. AB - The use of mammography to screen patients for the presence of early breast cancer results in the detection of nonpalpable lesions. As screening becomes more widespread, the number of needle localizations performed will continue to increase. This procedure requires involvement of the radiologist, surgeon, and pathologist. Close cooperation between these disciplines will ensure optimal patient care. PMID- 1732924 TI - The status of mammographically guided fine needle aspiration biopsy of nonpalpable breast lesions. AB - Mammographically guided needle biopsy is a promising technique for the evaluation of nonpalpable breast lesions. If proved accurate, it should lead to less discomfort and anxiety, fewer complications, less cosmetic disfigurement, and a significant decrease in cost when compared with conventional surgical biopsy. The use of FNAB should lead to surgery on a much more highly selected group of patients, thus producing higher true positive surgical biopsy rates for breast cancer than has been previously possible in this country. These incentives should serve to increase compliance with screening mammography guidelines by women and their physicians. Nevertheless, it is likely that stereotactic FNAB will be best suited to high volume practices in which a large number of biopsies are performed and in which experienced mammographers, cytopathologists, and breast surgeons use a team approach to manage each patient. PMID- 1732925 TI - Ultrasound-guided needle biopsy of the breast and other interventional procedures. AB - The techniques of and indications for ultrasound-guided needle biopsy of nonpalpable breast lesions are described. High-frequency ultrasonography offers several distinct advantages over sterotaxy and is the technique of choice for guiding the drainage of cysts and fluid collections and the biopsy of any solid mass that can be identified on sonograms. Ultrasonography also can be used for localizing such lesions preoperatively. PMID- 1732926 TI - Update of the Swedish two-county program of mammographic screening for breast cancer. AB - The results of the Swedish two-county trial of mammographic screening for breast cancer are presented, updated to December 31, 1990, which is an average of 10.8 years follow-up per person. The main result of the trial in terms of breast cancer mortality remained the same: compared with the control group, the group invited to screening had a relative breast cancer mortality of 0.70 (P = 0.0002) with 95% confidence interval (0.58, 0.85). Analysis of survival showed that relative to the control group, the cancers detected at prevalence screen, incidence screens, and in the interval between screens had a good prognosis, whereas cancers detected in those who had refused screening had a very poor prognosis. When adjusted for tumor size, lymph node status, and tumor grade (differentiation), the better survival associated with incidence screen and interval detection was largely accounted for, indicating that the benefit of incidence screening is largely achieved through the effect of screening on the three prognostic variables, notably size of the tumor. Results indicate that to achieve a substantial mortality reduction, 50% of screen-detected invasive cancers should be less than 15 mm in diameter, at least 30% of screen-detected grade 3 tumors should be less than 15 mm, and at least 70% of screen-detected tumors should not have lymph node metastases. The percentage of grade 3 tumors of a given size should be the same in screen-detected cancers as in clinically detected, and breast cancer prevalence at first screen should be at least three times the expected incidence rate in the absence of screening. This should be achieved without the recall rate for further examination exceeding 9%, and procedures including further imaging techniques and fine needle aspiration or core biopsy should be used before resorting to surgical biopsy. These aims can be achieved in specialist screening centers if particular attention is paid to resources for screening and diagnostic evaluation, specialist training of clinical and technologic screening staff, and ongoing monitoring of mammographic quality, recall rates, and the attributes of the tumors detected. PMID- 1732927 TI - The art of mammographic positioning. AB - The discovery of clinically occult breast cancer creates an exciting opportunity to alter the natural history of one of the major killers of women in our society. The skills required for this endeavor depend on high-quality images that provide the mammographer with sufficient information to construct three-dimensional perceptions recognizable as departures from normal architecture. Altering the natural course of breast cancer depends on early detection. Early detection of breast cancer depends on high-quality imaging techniques. Paramount among the imaging techniques for breast cancer detection is mammographic positioning. Optimal mammographic positioning is achieved by understanding the capabilities of available dedicated mammographic equipment and applying this understanding to take full advantage of natural breast mobility in overcoming various anatomic limitations. Compression of breast tissue, essential for proper parenchymal imaging, is achieved by moving one surface of the breast toward the other. The concept of moving the mobile surface of the breast toward the more fixed and immobile surface has been stressed as an important principle in optimizing the amount of tissue that can be imaged on standard mammographic views. Visualizing the fine details of a lesion or the margins of an area of clinical or perceived radiographic concern may be crucial to determining the need for biopsy. Visualization of such details is best achieved by projecting the suspected lesion into interface with adjacent radiolucent fat through separation of overlapping parenchyma by using spot compression or by tangential imaging against subcutaneous fat. Unique problems require creative, tailored solutions. Such tailoring is made less difficult by understanding and using equipment capability with breast anatomy and mobility. The very small, very large, or very dense breast can be imaged properly with modified techniques. Likewise, the augmented breast, mastectomy site, or axilla can be imaged with specialized techniques. Artistic application of these mammographic positioning principles will be rewarded with high-quality images, fewer missed breast cancers, and more lives saved. PMID- 1732928 TI - A screening mammography program. Staying alive and making it work. AB - The success of a mammography screening program requires thorough planning. A dependably high volume and a streamlined efficient operation are essential to survival of the program. Factors that warrant consideration prior to designing such a program include the following: Distinction between screening and diagnostic mammography examinations. Selection of a site that will meet the needs of the community and yet provide a consistently high volume. Low examination cost for screening mammography coupled with a detailed financial analysis and reappraisal on an ongoing basis. A customized marketing program that incorporates methods to increase awareness, compliance, and utilization by women and referring physicians. Well-trained, efficient, and dedicated personnel. An operation that is designed for rapid throughput and expeditious patient flow. An efficient plan for film handling, interpretation, reporting, and storage. Timely communication of examination results. A reliable mechanism for follow-up evaluation and outcome data collection. Establishment of a consistent and reliable quality assurance program and the production of high quality mammograms. PMID- 1732929 TI - Guidelines for screening for breast cancer. Is a revision in order? AB - Screening women over the age of 50 for breast cancer has clearly been proven to be capable of reducing population mortality from breast cancer. Population based screening for women aged 40 to 49 has not been shown to be effective, and in several trials there is an early excess breast cancer mortality in the screened population. Because tumors tend to grow more rapidly in younger women than in older women, those trials performed at biannual intervals will detect only the intermediate and slower growing lesions. The more rapidly growing tumors will occur between screens. Furthermore, maintaining a high positive predictive value will lead to a lower sensitivity rate. If the screening interval is too long or the positive predictive value too high, the reassuring effect of a false-negative screening examination may contribute to delay in diagnosis beyond even the usual clinical detection threshold. This may cause more deaths in the study population than in the control population. Guidelines that take the various factors into account have been proposed. PMID- 1732930 TI - The politics of mammography. AB - Mammography has had a major impact on the earlier detection, treatment options, and management decisions and survival and mortality rates of breast cancer. Consequences include overwhelming demand for mammography; problems with optimum response by radiology; limited availability of the examination, especially to the socioeconomically disadvantaged; self-referral for mammography by unqualified physicians for less than altruistic reasons; and unrealistic expectations of mammography by women, physicians, and lawyers. Responses to the overwhelming demand for high-quality mammography include ACR postgraduate and continuing education courses and its mammography accreditation program; a more comprehensive examination on mammography for certification by the ABR; and increasing state and federal government interest and legislation for reimbursement, quality assurance, and delivery of mammography. The precedents this sets for radiology if not all of medicine suggests that collaboration of the private and public sectors offers the greatest promise of an appropriate response, namely reproducible optimum mammography accurately interpreted with the lowest possible radiation dose for all eligible women in the United States. PMID- 1732931 TI - Quality assurance in mammography. Accreditation, legislation, and compliance with quality assurance standards. AB - The status of mammography quality assurance in the United States has been reviewed briefly. The history, goals, current status, and possible future directions of the ACR Mammography Accreditation Program have been described, and other ACR activities in mammography quality assurance have been discussed, including ACR Standards of Practice in Mammography, ACR Mammography Quality Control Manuals, and the ACR/CDC Cooperative Agreement on Quality Assurance in Mammography. The quality assurance provisions of recently adopted federal legislation on mammography have been reviewed, including the Medicare legislation on screening mammography, along with the proposed Women's Health Equity Act mammography quality assurance provisions. Finally, a simple plan has been proposed to fuse these activities into a coherent program for ensuring consistently high quality mammography at every site in the United States. PMID- 1732932 TI - Standardized mammography reporting. AB - The use of mammographic screening to detect breast cancer at a preclinical stage is increasing rapidly in the United States. High-quality imaging and accurate interpretation are critical elements for successful mortality reduction. The communication of the interpretation is being scrutinized in an effort to eliminate ambiguity and confusion. This can be accomplished by an organized approach to interpretation and a structured analysis of significant findings. These can be grouped into five major categories ranging from a negative examination to findings suggesting a very high probability of malignancy. A concisely organized reporting system is to be proposed by committees of the ACR that will provide clinically relevant information. The system will suggest the use of an approved vocabulary and urge that significant findings be described concisely in a standardized format. All studies should be categorized using the five clinically useful assessment groups. This combined with the maintenance of a data base for determining results will improve the accuracy of the screening effort, permit the monitoring of the screening results, and provide feedback for improving the program. PMID- 1732933 TI - Quality assurance. How to audit your own mammography practice. AB - The medical audit is an important component of a comprehensive mammography quality assurance program. It provides a direct assessment of one's ability to detect otherwise occult breast cancer, the ultimate indicator of mammography performance. Audits must be properly planned, executed, interpreted, and used. The audit that shows successful clinical results can be used to build and maintain a high state of morale among mammography personnel as well as to encourage new and repeat referrals to a mammography practice. The audit that uncovers a deficiency in clinical performance can be instrumental in helping to plan remedial measures that correct the problem. PMID- 1732934 TI - Medicolegal aspects of breast imaging. AB - The response to the public health challenge of screening women in this country for breast cancer is a goal to which all radiologists should direct their efforts. The decision to perform screening procedures, diagnostic procedures, or both will vary with the individual circumstances of one's practice. Careful planning and attention to certain parameters regarding such different types of practices will provide for a satisfying medical practice that is consistent with a sound risk-management profile. While no practice can be considered insulated from legal redress, physicians should not fear the legal consequences of appropriate and reasonable breast imaging service. PMID- 1732935 TI - New and future developments in screen-film mammography equipment and techniques. AB - Two improvements in mammography equipment during the last 4 years will greatly affect the practice of mammography during the next decade: dose reduction and improved testing equipment for monitoring the quality of mammography. The increased use of digital radiography has stimulated studies comparing digital mammography with screen-film mammography. Some digital algorithms for detecting clusters of calcifications may have application in screening mammography within the next decade. PMID- 1732936 TI - Breast masses. Mammographic and sonographic evaluation. AB - Asymmetric breast tissue can nearly always be distinguished from a true mass by means of mammographic evaluation. Stellate masses from early invasive breast cancer are often extremely subtle so that optimal technique and meticulous interpretation are essential. Benign stellate masses such as post-biopsy scarring and fat necrosis frequently have a characteristic appearance. A radial scar is usually indistinguishable from malignancy on the mammogram. Nearly all circumscribed masses are benign and are usually cysts, fibroadenomas, or intramammary lymph nodes. A few circumscribed masses represent in situ or invasive carcinoma or both. Characteristics that may allow a definitively benign diagnosis for a circumscribed mass include the presence of fat and certain calcification patterns on the mammogram and features of a simple cyst on the sonogram. Management decisions for other circumscribed masses will depend on characteristics such as shape, margins, calcification, multiplicity, size, stability, and sonographic features as well as patient age and risk factors. Most nonspecific circumscribed masses should be followed rather than biopsied as they are commonly present on mammograms and have a change of malignancy of less than 5%. Even when biopsied on the basis of interval change, most small circumscribed cancers will not have metastasized to the regional nodes. For palpable breast masses, selection of mammography or ultrasonography as the primary imaging modality will depend on patient's age and risk factors. PMID- 1732937 TI - Mammographic analysis of calcifications. AB - Because mammographically detected calcifications are frequently the only sign of breast cancer, the breast radiography equipment, screen-film imaging package, and film processing should be optimized to detect such calcifications. For this purpose, dedicated units with molybdenum targets, microfocal spot magnification capability, reciprocating grids, and high output x-ray tubes are required. With the greater use of state-of-the-art mammography, intraductal carcinoma, or DCIS, manifested only by calcifications is being detected more frequently than ever. DCIS can be of the comedo, cribriform, or micropapillary types. Comedocarcinoma, characterized by linear and branching (casting) calcifications, is the most aggressive type, and it has the highest rate of recurrence after breast conserving surgery. Cribriform and micropapillary calcifications are characteristically punctate and vary in size and shape. In addition to histologic type, the recurrence of DCIS is related to its extent at detection and whether adequate tissue was removed at the time of breast-conserving surgery. Biopsies for suspicious calcifications should be followed immediately by specimen radiography to verify their removal. If breast-conserving surgery is elected for DCIS, the resected segment of tissue should be examined with pathologic techniques designed to determine whether the margins are clear of tumor. For DCIS and invasive cancers with extensive intraductal component, microfocus magnification mammography over the surgical site is recommended prior to radiotherapy to identify any residual tumor calcifications. Although state-of-the art mammography is very sensitive in the detection of calcifications, it is low in specificity, thus resulting in a large number of false-positive mammograms and a relatively low true-positive biopsy rate. While some benign calcifications cannot be distinguished from those of malignancy, the number of biopsies for benign conditions can be decreased by careful analysis of the mammograms in a search for features indicating benignity. PMID- 1732938 TI - Neurogenic dysfunction of the bladder in infants and children: recent advances and the role of radiology. PMID- 1732939 TI - Renal lesions: great strides in imaging. PMID- 1732940 TI - Lung cancer staging: efficacy of CT. PMID- 1732941 TI - Use of low-osmolality contrast medium does not increase prevalence of medullary sponge kidney. AB - To assess the prevalence of intrapapillary linear collections of contrast medium as well as of homogeneous papillary blush on excretory urograms obtained with a low-osmolality contrast medium, iohexol was used in 300 patients. Intrapapillary linear collections of contrast medium (ie, three or more linear collections of contrast material within a papilla) were found in 10 (9.5%) of the 105 patients with renal stone disease and two (1.0%) of the 195 patients without nephrolithiasis (P less than .001). These prevalences are similar to those found in a previous study with use of a high-osmolality contrast medium (sodium amidotrizoate). The difference in the prevalence of homogeneous papillary blush between stone formers and non-stone formers was nonsignificant. The authors conclude that intrapapillary linear collections of contrast medium on excretory urograms obtained with use of a low-osmolality contrast medium should be considered to have the same clinical significance as those on excretory urograms obtained with use of a high-osmolality contrast medium, that is, as indicating the presence of medullary sponge kidney. PMID- 1732942 TI - Fostering research by radiologists: recommendations of the 1991 Summit meeting. AB - At the annual Radiology Summit Meeting sponsored by the Intersociety Commission of the American College of Radiology, leaders of U.S. and Canadian radiologic organizations met in part to discuss ways to improve radiology research. Support by radiology departments and radiologic organizations is currently not sufficient to improve the research of the specialty. Five issues of central importance to improving research are (a) the need for a definition of radiology research, (b) a lack of financial resources, (c) a lack of mentors, (d) hesitancy to embark on a research career, and (e) conflicts between clinicians and researchers. The adoption of 31 recommendations regarding these five issues was urged. PMID- 1732943 TI - Bronchogenic carcinoma: analysis of staging in the mediastinum with CT by correlative lymph node mapping and sampling. AB - One hundred forty-three patients with bronchogenic carcinoma were studied prospectively with computed tomography (CT) to determine the accuracy of CT in the evaluation of mediastinal nodal metastases. Mediastinal lymph nodes were localized according to the lymph node mapping scheme of the American Thoracic Society and were considered abnormal if they exceeded 1 cm in short-axis diameter. All patients underwent surgical staging, which consisted of either mediastinoscopy alone or mediastinoscopy and thoracotomy. At the time of surgical staging, all accessible nodes were either removed or sampled. The sensitivity of CT for mediastinal nodes on a per-patient basis was 64%, with a specificity of 62%. The sensitivity of CT for individual nodal stations involved with tumor was only 44%. The presence of obstructive pneumonitis did not appreciably alter the sensitivity of CT, but the specificity was lower (43%). The likelihood of metastases increased with lymph node size; however, seven of 19 (37%) lymph nodes that measured 2-4 cm in short-axis diameter were hyperplastic and did not contain metastases. The relative insensitivity of CT makes formal nodal sampling at the time of mediastinoscopy or thoracotomy essential to detect lymph node metastases. PMID- 1732944 TI - Cystic lung disease associated with eosinophilic granuloma and tuberous sclerosis: air trapping at dynamic ultrafast high-resolution CT. AB - Thin-walled lung cysts can occur in association with advanced pulmonary eosinophilic granuloma and lymphangioleimyomatosis (LAM). In LAM, cysts occur as an isolated abnormality or in association with tuberous sclerosis. The cause of these cysts is unclear, but some investigators have postulated that they result from air trapping. To determine if lung cysts in these two diseases are associated with air trapping, the authors performed dynamic ultrafast high resolution computed tomography (DUHRCT) during forced expiration in two patients (one with eosinophilic granuloma and one with tuberous sclerosis) with lung cysts and correlated the results with results of pulmonary function tests. DUHRCT demonstrated focal and diffuse air trapping; in some lung regions, a less than normal increase in lung attenuation during forced exhalation was evident. These studies do not allow a conclusion regarding the mechanism of cyst formation in eosinophilic granuloma and LAM-tuberous sclerosis, but they confirm the association between lung cysts and morphologic findings of air trapping. PMID- 1732945 TI - Pulmonary metastatic nodules: CT-pathologic correlation. AB - To elucidate the characteristics of pulmonary metastatic nodules on high resolution computed tomographic (HRCT) scans, a correlative computed tomographic (CT)-pathologic study was performed with five human lungs after autopsy. The relationship of metastatic nodules to pulmonary vessels was studied with HRCT scans, radiographs of the specimen, and stereomicroscopic study in 264 nodules 0.6-9.0 mm in diameter. On radiographs and stereomicroscopic images, 190 small nodules (less than 3 mm in diameter) were in contact with the pulmonary lobule on the central bronchovascular bundles (n = 33 [17.4%]), located between the central bronchovascular bundle and the perilobular structure (n = 127 [66.8%]), or attached to perilobular structures (n = 30 [15.8%]). On HRCT scans, 21 small nodules (11.1%) were located on the central bronchovascular bundle; 130 small nodules (68.4%), between the central bronchovascular bundle and the perilobular structure; and 39 small nodules (20.5%), on the perilobular structure. On radiographs and stereomicroscopic images, 43 of 74 large nodules (greater than 3 mm in diameter) (58%) compressed both bronchovascular bundles and perilobular structures. The central bronchovascular bundle was invaded in only 13 large nodules (18%). PMID- 1732946 TI - Usual interstitial pneumonia: histologic correlation with high-resolution CT. AB - The authors reviewed 46 cases of idiopathic pulmonary fibrosis with usual interstitial pneumonia (UIP), correlating findings on high-resolution computed tomographic (HRCT) scans with findings in specimens obtained at open lung biopsy and autopsy. The following HRCT findings were observed: (a) an accumulation of small cystic spaces with thick walls, (b) air bronchiolograms within areas of intense lung attenuation, (c) rugged pleural surfaces, (d) irregularly thickened pulmonary vessels, (e) bronchial wall thickening, and (f) slightly increased lung attenuation. Macroscopic honeycombing correlating with small cystic spaces was demonstrated at HRCT and pathologic examination. Air bronchiolograms in the areas of intense lung attenuation (ie, microscopic honeycombing) corresponded to dilated bronchioles (greater than 1 mm in diameter) with fibrosis. Irregularly thickened vessels and bronchial walls and irregular pleural surfaces were the result of fibrosis in the periphery of the secondary pulmonary lobules. Areas of slightly increased lung attenuation seen on the HRCT scans correlated with patchy alveolar septal fibrosis or inflammation. The authors conclude that microscopic honeycombing and a perilobular distribution in UIP may be clearly identified with HRCT. PMID- 1732947 TI - Solitary pulmonary nodule: CT evaluation of enhancement with iodinated contrast material--a preliminary report. AB - The authors hypothesized that the degree of contrast material enhancement of a pulmonary nodule, measured with computed tomography (CT), may indicate the likelihood of malignancy. Fifty-two patients with uncalcified solitary pulmonary nodules (diameter, 6-30 mm) were studied. Five single serial thin-section CT scans were obtained at 1-minute intervals after injection of 100 mL of nonionic contrast material. Twenty-two patients were excluded because the diagnosis was not clearly established: The observation period was less than 2 years, or the examination was technically inadequate. Malignant nodules were identified in 23 of the 30 remaining patients, and benign nodules were identified in seven. Within the first 2 minutes after the injection, all the malignant nodules had enhanced by 20 HU or greater (only one benign nodule had that degree of enhancement). The authors conclude that the degree of contrast material enhancement of pulmonary nodules as measured with CT may indicate the likelihood of malignancy. PMID- 1732948 TI - Pulmonary sarcoidosis: CT assessment of lesion reversibility. AB - By comparing serial computed tomographic (CT) scans obtained when sarcoidosis was clinically active and after the onset of remission, an attempt was made to differentiate inflammatory from fibrotic lesions in the lungs of patients with sarcoidosis. Twenty patients with pulmonary infiltration seen on their chest radiographs were studied. For each patient, lesions found on the first CT scan were assessed by two observers as being decreased or increased on the second CT scan. Nodules (n = 8), irregularly marginated nodules (n = 5), and alveolar or pseudoalveolar consolidation (n = 5) always disappeared or clearly decreased. Septal lines (n = 10), nonseptal lines (n = 9), and lung distortion (n = 7) remained unchanged or increased. Some findings varied among patients: Micronodules (n = 9) and subpleural thickening (n = 5) disappeared or decreased in sarcoidosis of recent origin. Many findings of pulmonary infiltration seen on the first CT scan can be considered expressions of either inflammatory (reversible CT findings) or fibrotic (irreversible CT findings) lesions. PMID- 1732949 TI - The computer-based cumulative report: improvement in quality and efficiency. AB - A cumulative radiology report, like clinical progress notes, contains a listing of examinations and findings in chronologic order. Word processing is the most efficient way to create such a report, with follow-up examinations handled as additions to an existing document. Since 1988, cumulative reports on 127 cases involving 483 examinations have been made for a long-term industrial chest radiology survey by using a personal computer and custom software that employs minimum keystrokes to manage files. The results were as follows: (a) Quality improved operationally owing to routine auditing of prior information. (b) Efficiency improved owing to computer speed and software functions that included a refined "last in-first out" index and user-defined macros. (c) The radiologist saved time by not having to reiterate previously reported findings that were unchanged. This method is ideal for long-term surveys and could be a useful option for follow-up examinations in general. PMID- 1732950 TI - Lymphoma: predictive value of Ga-67 scintigraphy after treatment. AB - The negative predictive value (PV-) and positive predictive value (PV+) of gallium-67 scintigraphy and computed tomography (CT) were compared after treatment in 43 patients with Hodgkin disease and in 56 patients with non-Hodgkin lymphoma. The usefulness of these studies in predicting survival was also evaluated. In patients with Hodgkin disease, the PV- of Ga-67 scintigraphy was 0.84 and of CT was 0.88. The PV+ was 0.80 for Ga-67 studies and only 0.29 for CT. In patients with non-Hodgkin lymphoma, the PV- of Ga-67 scintigraphy was 0.84 and of CT was 0.80. The PV+ was 0.73 and 0.35, respectively. For both groups, the differences in disease-free survival between patients with negative and positive Ga-67 studies were significant (P less than .05 in Hodgkin disease and P less than .001 in non-Hodgkin lymphoma), but the differences were not significant for CT. These data show that, after treatment of patients with lymphoma, Ga-67 scintigraphy is a good predictor of clinical outcome and can be used beneficially in patient treatment. PMID- 1732951 TI - How accurate was GMENAC?--A retrospective review of supply projections for diagnostic radiologists. AB - In 1982, the Graduate Medical Education National Advisory Committee (GMENAC), a prominent national panel, predicted there would be 25,650 full-time equivalent (FTE) diagnostic radiologists, a 34% oversupply, by 1990. The radiologists involved in GMENAC, however, using models developed by the American College of Radiology, projected 19,800 FTE diagnostic radiologists in 1990, which was similar to the GMENAC estimate of need. The disagreement arose principally from different assumptions about residents entering the specialty. Recent data show there actually were approximately 21,900 FTE diagnostic radiologists in 1990. The radiologists' projection was 10% below this figure; the GMENAC projection was 17% above it. GMENAC erred principally in assuming diagnostic radiology residencies would not replace general radiology residencies, but rather be an addition to them. The radiologists erred principally in their assumption about the effects of the financial problems of hospitals on the number of residency positions. Accurate long-term projection of physician supply in individual specialties may well not be feasible. PMID- 1732952 TI - Constrictive pericarditis and restrictive cardiomyopathy: evaluation with MR imaging. AB - Twenty-nine patients who were referred with the possible diagnosis of constrictive pericarditis underwent electrocardiographically gated transverse spin-echo magnetic resonance (MR) imaging to determine the accuracy of spin-echo MR imaging for the diagnosis of constrictive pericarditis and to compare the morphologic features of constrictive pericarditis with those of restrictive cardiomyopathy as seen on spin-echo MR images. Constrictive pericarditis was verified by means of surgery and/or catheterization in 17 patients. The sensitivity, specificity, and accuracy of MR imaging in the diagnosis of constrictive pericarditis were 88%, 100%, and 93%, respectively. Thickened pericardium was observed in 88% of patients with proved constrictive pericarditis. Pericardial thickening was not identified in patients with restrictive myocarditis (n = 4). The most frequent site of pericardial thickening was over the right ventricle. In constrictive pericarditis, the signal intensity of the thickened pericardium was similar or decreased compared with that of the myocardium. Indirect findings of impaired right ventricular diastolic filling (eg, dilatation of the inferior vena cava and right atrium) were identified in constrictive pericarditis and restrictive cardiomyopathy. MR imaging can serve as a noninvasive examination for the definitive diagnosis of constrictive pericarditis and can help distinguish between constrictive pericarditis and restrictive cardiomyopathy on the basis of pericardial thickness. PMID- 1732953 TI - Antimyosin-labeled monocrystalline iron oxide allows detection of myocardial infarct: MR antibody imaging. AB - The synthesis and in vivo antigen targeting of a novel iron oxide compound were studied. A monocrystalline iron oxide nanoparticle (MION) was synthesized that contains a small (mean diameter, 2.9 nm +/- 0.9) single crystal core, passes through capillary membranes, and exhibits superparamagnetism. The MION was attached to antimyosin Fab (R11D10) and used for immunospecific magnetic resonance (MR) imaging of cardiac infarcts One hour after intravenous administration of MION-R11D10 in rats (100 mumol/kg), a marked decrease in the signal intensity of infarcted myocardium was observed. Immunohistochemical correlation confirmed the specific binding of the immunoconjugate to infarcted, but not to normal, myocardium. No decrease in cardiac signal intensity was observed when unconjugated MION was administered intravenously. The results indicate the feasibility of immunospecific MR imaging in living organisms. PMID- 1732954 TI - Popliteal and tibioperoneal arteries: feasibility of two-dimensional time-of flight MR angiography and phase velocity mapping. AB - To assess the feasibility of using magnetic resonance (MR) angiography and velocity-encoded cine MR imaging to evaluate morphology and function in the popliteal and tibioperoneal arteries, the profiles of blood flow velocity measured with velocity-encoded cine MR were compared with those measured with color-coded sonography. Two-dimensional time-of-flight MR angiography was performed in the popliteal and tibioperoneal arteries of 10 healthy subjects; velocity-encoded cine MR and color-coded sonography were performed above and below the trifurcation. The velocity waveforms acquired with velocity-encoded cine MR and color-coded sonography correlated well and showed a typical triphasic pattern. At peak systole in the popliteal artery, spatial maximum and spatial mean velocities measured with velocity-encoded cine MR were 42.29 cm/sec +/- 9.55 (standard deviation) and 27.7 cm/sec +/- 5.8, respectively; the peak velocity measured with color-coded sonography was 44.2 cm/sec +/- 12.3. It is concluded that use of both MR angiography and velocity-encoded cine MR should be considered for identification of arterial stenoses and assessment of the hemodynamic importance of peripheral vascular stenoses. PMID- 1732955 TI - Chronic major-vessel thromboembolic pulmonary artery obstruction: appearance at angiography. AB - The pulmonary angiograms of 250 patients evaluated for chronic thromboembolic pulmonary hypertension were reviewed. Pulmonary thromboendarterectomy was performed in each of these individuals, and the surgical findings were correlated with abnormal angiographic patterns. The pulmonary angiographic findings suggestive of chronic thromboembolic disease included "pouching" defects, webs or bands, intimal irregularities, abrupt vascular narrowing, and complete vascular obstruction. Pouching is reported by the authors to be a previously undescribed angiographic feature of this disease. Carefully obtained and properly interpreted pulmonary angiograms are necessary to confirm the diagnosis of operable chronic thromboembolic disease. Differential diagnostic possibilities should be considered prior to a decision to perform surgical correction. PMID- 1732956 TI - Percutaneous drainage of tubo-ovarian abscesses. AB - The authors performed percutaneous drainage of 27 tubo-ovarian abscesses (TOAs) in 16 patients in whom medical therapy with triple antibiotics prior to catheter drainage had not been successful. Percutaneous drainage was successful in 15 of 16 patients (94%). One patient underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy 3 days after catheter placement because of persistent symptoms and lack of drainage from the catheter; at laparotomy, a large infected phlegmon was found. Two patients had recurrent disease at 3 and 4 months after catheter placement. Bilateral salpingectomy was performed in one patient and total abdominal hysterectomy and bilateral salpingo-oophorectomy in the other. One of these patients had cervical carcinoma, and the other had a long history of recurrent pelvic inflammatory disease and TOAs. The long-term avoidance of surgery was 81.2%. Access routes for catheter drainage were through the anterior abdominal wall for 10 abscesses, through the posterior transgluteal route for 11, and through the transvaginal route for six. Duration of drainage was 1-20 days (mean, 6 days). Complications consisted of transient sciatic pain in two patients and mild bacteremia in one. The results indicate that percutaneous drainage of TOAs is effective in patients in whom medical therapy is not successful. PMID- 1732957 TI - Acute embolic occlusions of the infrainguinal arteries: percutaneous aspiration embolectomy in 102 patients. AB - Percutaneous aspiration embolectomy (PAE) was performed on acute embolic occlusions of infrainguinal arteries unrelated to percutaneous transluminal angioplasty or chronic atherosclerotic arterial occlusive disease. Of 102 patients, most (62.7% [n = 64]) had limb-threatening ischemia (stages III and IV according to the Fontaine classification); 86.3% (n = 88) had cardiac disease that caused the embolic occlusion. The clinical success rate was 87.3% (n = 89). Major complications occurred in 8.8% (n = 9) of the cases. The 30-day mortality was 3.9% (n = 4). In comparison with Fogarty-catheter embolectomy, PAE has a higher success rate and a lower mortality. PAE has several advantages: It is a simple technique with reduced invasiveness, combines a diagnostic and a therapeutic procedure, enables treatment of tibial and pedal vessels, and can be combined with all other angioplastic methods. PMID- 1732958 TI - Nd:YAG laser-assisted angioplasty in femoropopliteal artery occlusions: "hot" versus "cold" recanalization with transparent contact probe. AB - Percutaneous recanalization of femoropopliteal artery occlusions (1-21 cm; median, 8 cm) was attempted in 50 patients. A 2.2-mm-diameter contact probe catheter connected to a continuous-wave neodymium yttrium aluminum garnet (Nd:YAG) laser was used. The laser was activated (15 W, 1-second pulses) only if too much resistance was met. Balloon angioplasty was performed after successful traversal of the occlusion. Primary success was achieved in 40 of 50 patients (80%). In 20 cases, recanalization was achieved mechanically (cold group). In the other 20 cases, recanalization was achieved with the help of laser irradiation (hot group: 15-405 J; median, 90 J). Except for the length of the obstruction (longer in the cold group), the two groups did not differ in baseline characteristics. Neither the length of the occlusion nor the duration of symptoms correlated with failure or success or with the delivered laser energy. Cold and hot groups did not differ with regard to functional improvement and angiographic patency at 3 and 12 months (94% +/- 4). Thus, brief laser activation doubled the cold primary success rate, but the major action of the laser contact probe is mechanical remodeling of the obstruction. PMID- 1732959 TI - Percutaneous embolectomy: in vitro investigations of the self-expanding tulip sheath. AB - A self-expanding sheath with a tulip-shaped distal end was designed for performance of percutaneous embolectomy. Its ability to retrieve clots was tested in an in vitro flow model; results were compared with those obtained with a conventional 10-F sheath. Simulated embolectomy of clots weighing 0.1-1.5 g was performed with a 0.75-mL Fogarty balloon on a 4-F catheter. The clot material that embolized distally during the procedure was sampled and quantified. No effective embolectomy was performed via a conventional sheath. With the tulip sheath, however, complete clot removal was achieved unless the clot size exceeded the tulip volume. On the basis of results of in vitro testing, the tulip design is promising for use in several applications during percutaneous interventions. PMID- 1732960 TI - Evaluation of renal function with delayed CT after injection of nonionic monomeric and dimeric contrast media in healthy volunteers. AB - A new nonionic dimeric contrast medium (CM), iodixanol, was intravenously administered to 40 healthy male volunteers in doses of 0.3-1.2 g of iodine per kilogram of body weight, nonionic monomeric iopamidol and iopentol were administered to 20 others, and the renal effects were studied up to 120 hours after administration. Computed tomography of the kidneys was performed up to 80 hours after injection. Creatinine clearance as an index of the glomerular filtration rate was unchanged with all CM. Urine volume and osmolar clearance increased most with the monomeric CM. The proximal tubular brush border enzyme alkaline phosphatase increased with all CM. The lysosomal enzyme N-acetyl-beta glucosaminidase increased more with the monomeric CM than with iodixanol. A persistent increased attenuation in the region of the cortex was observed with all CM. Attenuation returned to baseline within 80 hours, with the slowest decline with iodixanol. This delayed cortical enhancement did not correlate with the effects of the CM on the tubular enzyme excretion. PMID- 1732961 TI - Renal lesions: controlled comparison between CT and 1.5-T MR imaging with nonenhanced and gadolinium-enhanced fat-suppressed spin-echo and breath-hold FLASH techniques. AB - Nonenhanced and gadolinium-enhanced fat-suppressed spin-echo and breath-hold fast low-angle shot (FLASH) magnetic resonance (MR) imaging techniques were compared with iodine contrast material-enhanced computed tomography (CT) for the detection and characterization of renal masses. MR studies included T1-weighted fat suppressed spin-echo (T1FS) and FLASH images followed by rapid injection of gadopentetate dimeglumine and a repeated FLASH image obtained at 1 second, a T1FS image at 30 seconds, and a FLASH image at 10 minutes. Of 38 patients, 17 had renal cysts, 18 had solid tumors, two had cortical scarring, and one had a hypertrophied column of Bertin. With contrast-enhanced T1FS, contrast-enhanced FLASH, and CT images, 114, 110, and 109 lesions, respectively, were detected. With MR imaging and CT, cysts smaller than 5 mm in diameter and solid tumors as small as 1 cm in diameter were detected. With combined contrast-enhanced FLASH and T1FS images, 112 lesions were correctly characterized as cystic or solid; with nonenhanced T1FS images, 110; with nonenhanced FLASH images, 107; and with nonenhanced CT, 103. PMID- 1732962 TI - Suprascapular nerve entrapment: evaluation with MR imaging. AB - Entrapment of the suprascapular nerve is frequently overlooked in the differential diagnosis of shoulder pain. The diagnosis is typically not considered until patients develop severe weakness secondary to atrophy of the spinatus (spinous) musculature that the nerve supplies. Twenty-seven masses were identified adjacent to the suprascapular nerve on magnetic resonance (MR) images of the shoulder; there were 21 ganglion cysts, two synovial sarcomas, one Ewing sarcoma, one chondrosarcoma, one metastatic renal cell carcinoma, and one hematoma associated with a fracture. Atrophy of both the supraspinatus and infraspinatus muscles was seen in association with anteriorly located masses and proximal entrapment of the nerve in 11 cases (40%); isolated atrophy of the infraspinatus muscle was seen in association with posteriorly located masses and distal entrapment of the nerve in nine cases (33%). MR imaging may facilitate the diagnosis of suprascapular nerve entrapment in patients with shoulder pain of unclear origin when perineural masses and atrophy of the spinatus musculature are present. PMID- 1732963 TI - Avascular necrosis of the hip: comparison of contrast-enhanced and nonenhanced MR imaging with histologic correlation. Work in progress. AB - In 15 hips with typical signs of avascular necrosis of the femoral head on plain radiographs and magnetic resonance (MR) images, gadolinium-enhanced spin-echo and fat-suppressed MR images were obtained and compared with nonenhanced T1- and T2 weighted images. Both enhanced and nonenhanced areas were consistently detected in the abnormal femoral heads. Enhanced areas showed a low signal intensity (SI) on T1-weighted MR images obtained before contrast material was administered and an intermediate to high SI on T2-weighted images. Nonenhanced areas showed an SI either identical (pattern 1) or hypointense (pattern 2) to that of fat on both sequences. Histologic correlation (six resected femoral heads) helped confirm that enhanced and nonenhanced areas corresponded respectively to viable and necrotic tissue. In most cases, SI analysis of nonenhanced T1- and T2-weighted images allows the differentiation of hypervascularized viable tissue from hypovascularized necrotic tissue of the sequestrum. PMID- 1732964 TI - Quantitative chemical shift imaging of vertebral bone marrow in patients with Gaucher disease. AB - To evaluate extent of bone marrow involvement and disease severity in Gaucher patients, results of modified Dixon quantitative chemical shift imaging (QCSI) of the lumbar spine were correlated with quantitative analysis of marrow triglycerides and glucocerebrosides and with quantitative determination of splenic volume at magnetic resonance (MR) imaging. High-field-strength MR spectra of surgical marrow specimens were dominated by a single fat and a water peak, validating use of QCSI. QCSI showed average vertebral marrow fat fractions of 10% +/- 8 in Gaucher patients (normal adult averages, 29% +/- 6). Relaxation times for lipid and water approximated normal averages; bulk T1 values were significantly longer, reflecting decreased marrow fat. Glucocerebroside concentrations were higher in Gaucher marrow and inversely correlated with triglyceride concentrations. Extent of marrow infiltration determined by fat fraction measurements correlated with disease severity measured by splenic enlargement. These results show that as Gaucher cells infiltrate bone marrow and displace normal marrow adipocytes, bulk T1 increases due to the higher T1 of water compared with that of fat. QCSI provides a sensitive, noninvasive technique for evaluating bone marrow involvement in Gaucher disease. PMID- 1732965 TI - Carotid artery disease: evaluation with acetazolamide-enhanced Tc-99m HMPAO SPECT. AB - Sixty patients were studied for carotid artery disease and were further evaluated with hexamethyl-propyleneamine oxime (HMPAO) single photon emission computed tomography (SPECT) both at baseline (with the patient resting) and after administration of acetazolamide (ACZ). Of these 60 patients, 58 (97%) had symptoms and 49 (82%) had stenoses greater than 70% in at least one internal carotid vessel. Nine patients (15%) had symmetric findings on baseline examinations and at SPECT with ACZ. Thirty-two patients (53%) had asymmetric findings on baseline, but in 24 of these patients (75%) additional lesions were observed after ACZ administration. Nineteen patients (32%) had asymmetric findings only after ACZ was administered. HMPAO SPECT with ACZ allows detection of diminished cerebral perfusion reserve that is not found when HMPAO SPECT is performed with the patient at rest. This procedure helps provide an objective evaluation of the hemodynamic effects of carotid stenosis. PMID- 1732966 TI - Determination of cerebral blood flow with a phase-contrast cine MR imaging technique: evaluation of normal subjects and patients with arteriovenous malformations. AB - This study evaluated a phase-contrast cine magnetic resonance (MR) imaging technique capable of simultaneously allowing determination of velocity and volume flow rate (VFR) in both carotid arteries and the basilar artery. Forty patients were studied; 24 were neurologically normal, and 16 had intracerebral arteriovenous malformations (AVMs). In the normal group, mean basilar flow was significantly less than mean carotid flow. Mean velocity and VFR showed a significant decline with age in the basilar artery. Carotid artery flow and total cerebral blood flow did not decline with age. In the AVM patients, flow and velocity measurements were significantly elevated in all three arteries. Flow in the carotid artery ipsilateral to the AVM was significantly greater than flow in the contralateral carotid artery. VFR increased in all three arteries with increasing AVM volume. Four patients underwent partial embolization, and a corresponding decrease in flow was observed. Phase-contrast cine MR imaging provides rapid, simultaneous, noninvasive velocity and VFR measurement in the major intracranial arteries. PMID- 1732967 TI - Absence of the carotid canals at skull base CT. AB - Congenital absence of one or both internal carotid arteries (ICAs) has a high association with circle of Willis aneurysm formation. Since the carotid canals in the skull base form secondary to the presence of the embryonic ICA, absence or hypoplasia of a carotid canal on a computed tomographic (CT) scan through the skull base should suggest a congenital ICA abnormality and prompt a search for associated intracranial vascular abnormalities. Of four patients with carotid canal underdevelopment evident at CT, two had associated circle of Willis aneurysms and a third had an extensive skull base rete mirabile supplying an abnormal tangle of vessels in the basal cisterns. Only two patients (one with an aneurysm, one with the skull base rete mirabile) presented with subarachnoid hemorrhage. One patient presented with monocular decreasing vision due to an enlarging aneurysm, and one patient was essentially asymptomatic. If asymmetry or absence of the carotid canals is evident on CT scans of the head, further evaluation to rule out a potentially life-threatening intracranial vascular abnormality such as those found in these patients should be seriously considered, even in a young or asymptomatic patient. PMID- 1732968 TI - Experimental allergic encephalomyelitis and multiple sclerosis: lesion characterization with magnetization transfer imaging. AB - Magnetization transfer imaging (MTI) was initially performed in normal guinea pigs and human volunteers. A magnetization transfer ratio (MTR) was calculated in the normal white matter and was found to be 42%-44%, with less than 2.5% variation, which indicates the high reproducibility of the measurement. MTI was then applied to an animal model of white matter disease, acute experimental allergic encephalomyelitis (EAE). In this model of EAE, pathologically proved lesions were edematous with essentially no demyelination. MTRs decreased slightly but significantly (5%-8%) compared with the MTRs of the same tissue region measured before the onset of the lesion [corrected]. Fifteen patients with multiple sclerosis (MS) also underwent MTI. In the 15 patients with MS, all lesions (209 plaques) had a significantly decreased MTR (average, 26%). The authors believe that demyelination produced the lower MTR, and, thus, lesions varied in transfer ratio on the basis of the extent of myelin loss. In patients with MS, particularly those with chronic and/or progressive MS, the MTR of the normal-appearing white matter was significantly decreased. The data suggest that calculated MTR obtained with in vivo MTI may enable differentiation of edema from demyelination, and that MTI can demonstrate white matter abnormalities that cannot be seen with standard spin-echo or gradient-echo magnetic resonance imaging. PMID- 1732969 TI - Gray matter heterotopias: MR characteristics and correlation with developmental and neurologic manifestations. AB - Magnetic resonance (MR) images and clinical records of 20 patients with gray matter heterotopias were retrospectively reviewed to correlate MR characteristics of the heterotopias with clinical findings. On the basis of the MR images, patients were divided into three groups: those with subependymal heterotopias (eight patients), focal subcortical gray matter heterotopias (six patients), and diffuse subcortical heterotopias (six patients). Patients with subependymal heterotopias had a significantly higher prevalence of normal development than patients in the other two groups (P = .02). When all patients with gray matter heterotopias were considered, patients with thick heterotopias and those with overlying cortical gyral anomalies, which correlated with one another, had a significantly higher prevalence of developmental delay (P = .002). Patients with thick focal gray matter heterotopias had a substantially increased prevalence of motor dysfunction. In three cases, gray matter heterotopias were associated with infoldings of dysplastic cortex containing blood vessels or cerebrospinal fluid. If not properly analyzed, these anomalies can be mistaken for vascular or cystic tumors. PMID- 1732970 TI - Fetal crown-rump length: reevaluation of relation to menstrual age (5-18 weeks) with high-resolution real-time US. AB - Because of recent challenges in the literature regarding the validity of the older crown-rump length (CRL) data developed with conventional static-image ultrasound scanners, the authors evaluated the relationship between CRL and menstrual age of fetuses in a population of 416 patients with good menstrual dates. By using a variety of commercially available transabdominal and transvaginal real-time ultrasound probes, the authors demonstrated that measurements can be made successfully for CRLs varying in size from 2 mm to 12 cm. Regression analysis of the data resulted in development of a new table for predicting menstrual age of fetuses on the basis of CRL measurements obtained between 5 and 18 weeks gestation. Although the magnitude of the raw residuals increased over time, the variability in predicting menstrual age was demonstrated to be relatively constant at +/- 8% (2 standard deviations) when expressed as a percentage of the predicted value. The accuracy in predicting menstrual age from CRL after 14 weeks was equivalent to but not better than conventional measurements such as biparietal diameter and femur length. PMID- 1732971 TI - Autosomal recessive osteopetrosis: bone marrow imaging. AB - Technetium-99m sulfur colloid scintigraphy was performed prospectively in 12 infants and children with autosomal recessive osteopetrosis, to correlate the appearance of bone marrow stores with advancing age. Baseline images were obtained in all patients, and one to five follow-up images were obtained in eight patients after they began therapy with calcitriol, interferon-gamma, or both. Conventional radiography was performed along with the nuclear studies in all cases. Magnetic resonance (MR) images of the head or lower extremities were also obtained in six patients and were correlated with the scintigraphic findings. Patterns of abnormal distribution of bone marrow appeared to be age-dependent. In patients younger than 1 year, marrow stores were primarily in the skull base and at the ends of the long bones. In patients aged 3-5 years, marrow stores shifted to the diaphyseal regions of long bones and to the calvarium. In the appendicular skeleton, areas of greatest bone marrow activity corresponded to regions of relative decreased opacity on radiographs and areas of intermediate or high signal intensity on T2-weighted MR images. The skull base showed appreciable marrow activity in spite of densely sclerotic bone on radiographs. PMID- 1732972 TI - I-131 MIBG imaging after bone marrow transplantation for neuroblastoma. AB - Thirty-one children with stage III and IV neuroblastoma were studied with iodine 131 metaiodobenzyl-guanidine (MIBG) scintigraphy, technetium-99m medronate scintigraphy, skeletal plain radiography, and computed tomography (CT) before and after bone marrow transplantation (BMT). Twenty-six pre-BMT and 90 post-BMT studies were reviewed. Fourteen patients were alive without tumor, and one patient died at 4 months while free of tumor. I-131 MIBG scans obtained before and after BMT were negative in eight of the 15 patients. Seven had positive I-131 MIBG scans obtained before BMT that became negative after BMT. Six of 15 patients had small, questionable lesions on CT scans of the chest or abdomen that never increased in size, and the I-131 MIBG scans remained negative. Sixteen children had progressive disease or relapse, including four in whom I-131 MIBG scans showed new abnormalities, four with persistently positive I-131 MIBG scans, and three who had negative I-131 MIBG scans at relapse. Two of the 16 patients initially had small lesions seen on abdominal CT scans that were not seen on I 131 MIBG scans; mass lesions were later seen at CT in these locations. Detectable I-131 MIBG uptake in abdominal and thoracic lesions was related to the diameter of the lesion. Both I-131 MIBG scintigraphy and CT should be used to evaluate patients with neuroblastoma who have undergone BMT. PMID- 1732973 TI - Right common carotid artery reconstruction in neonates after extracorporeal membrane oxygenation: color Doppler imaging. AB - Thirty-three neonates treated for reversible respiratory failure underwent reconstruction of their previously ligated right common carotid arteries (RCCAs) immediately after extracorporeal membrane oxygenation (ECMO). Cerebral color Doppler imaging, performed during and repeatedly after ECMO, revealed antegrade flow in the right internal carotid artery (ICA) in all neonates within 6 days after successful RCCA reconstruction. Mean ICA velocity was significantly less in the right artery compared with the left during ECMO and within 1 hour of reconstruction, but there was no difference after 12 hours. In neonates with successful RCCA reconstruction, the flow in the proximal right anterior cerebral artery was antegrade in only 4% of examinations during ECMO but became antegrade in 94% after 6 days. Retrograde flow in the right posterior communicating artery persisted in 50% of examinations performed 1 day after reconstruction. ICA flow become antegrade with symmetric velocities shortly after successful RCCA reconstruction. Collateral flow persists longer but decreases rapidly. PMID- 1732974 TI - Cannula-induced vertebral steal in neonates during extracorporeal membrane oxygenation: detection with color Doppler US. AB - To determine whether flow through the subclavian artery might be affected during extracorporeal membrane oxygenation (ECMO), 40 neonates were examined with color Doppler ultrasound during and after ECMO. Retrograde flow in the right vertebral artery, noted in 12 of the 40 neonates (30%), was consistent with vertebral steal. Brachial systolic velocity was significantly less (P less than .01) on the right than on the left side in neonates both with and without vertebral steal. When the arterial cannula was removed after ECMO, vertebral artery flow became antegrade with symmetric velocity. Brachial velocities became symmetric in infants without vertebral steal, but mild asymmetry persisted in neonates who had had vertebral steal. Only one neonate had clinical signs of arm ischemia, which resolved promptly after removal of the cannula. No surviving neonates (n = 11) had neurologic findings related to the vertebrobasilar insufficiency over a 12-22 month period of observation. Vertebral steal appears to be common during ECMO and is resolved after removal of the cannula. PMID- 1732975 TI - MR imaging of the juvenile temporomandibular joint: preliminary report. AB - Thirty-two children were evaluated by means of medical history and physical examination for signs and symptoms of internal derangement (ID) of the temporomandibular joint (TMJ) and mandibular dysfunction. These children also underwent magnetic resonance (MR) imaging of the TMJs. The study was double blind. At clinical examination, 19 patients (59%) had at least one positive finding of ID of the TMJ and/or mandibular dysfunction. MR images of the TMJ obtained in 60 of the 64 TMJs demonstrated 57 normal joints (95%) and three abnormal joints (5%). Two of these three joints had a mild anterior-lateral disk displacement, and one joint had an anterior dislocated disk. There were no false positive MR examinations. MR imaging failed to depict abnormalities in 16 patients who had positive findings at history and/or physical examination. Although MR imaging may fail to depict ID of the TMJ in some patients, clinical techniques commonly used in population surveys probably overstate the prevalence of ID of the TMJ in children. PMID- 1732976 TI - Magnetization transfer of hepatic lesions: evaluation of a novel contrast technique in the abdomen. AB - The authors evaluated the technique of magnetization transfer to determine if it could enable distinction of benign from malignant liver lesions. Thirteen patients with 27 hemangiomas or cysts and 13 patients with 31 malignant liver lesions underwent magnetic resonance imaging. Hepatic malignancies demonstrated magnetization transfer similar to that of the liver. However, hemangiomas and cysts showed significantly less magnetization transfer than malignant liver lesions. Gradient-recalled-echo imaging with the off-resonance saturation pulse showed increased lesion contrast compared with the liver for hemangiomas and cysts but not for malignancies. On the basis of signal intensity measurements alone (ie, ignoring morphologic criteria), the magnetization transfer studies showed an area under the receiver operating characteristic curve of 0.96 for distinguishing benign from malignant liver lesions. Though the separation of these two groups was imperfect, it was comparable with that achieved with qualitative analysis of signal intensity ratios at spin-echo imaging. PMID- 1732977 TI - Purely cystic hydatid disease of the liver: treatment with percutaneous aspiration and injection of hypertonic saline. AB - Percutaneous aspiration of purely cystic liver lesions was performed in 15 patients aged 11-56 years. After aspiration under guidance with computed tomography (CT) in 12 patients, a membrane that is diagnostic for hydatid disease was visible in the lumen of the cyst on CT scans. Hypertonic saline was injected in the cystic cavities of these patients as a scolecidal agent. No major complications occurred during or after the procedures. In the follow-up period of 6-16 months, control CT and ultrasound scans revealed a progressive decrease in the size of the lesions and no evidence of peritoneal seeding. It is concluded that percutaneous aspiration and hypertonic saline injection for purely cystic hydatid disease of the liver seem to be an effective form of treatment and may eventually prove to be an alternative to surgical intervention. PMID- 1732978 TI - Hepatic cavernous hemangiomas: simple diagnostic sign with dynamic bolus CT. AB - Many hepatic hemangiomas are discovered incidentally during incremental dynamic bolus computed tomography (CT). To meet the established criteria for diagnosis with CT, however, a second CT examination with single-level dynamic bolus imaging is necessary. A prospective evaluation was performed to examine a simple sign that may be used to diagnose cavernous hemangiomas during incremental dynamic bolus CT. This sign is the visualization of foci of globular enhancement within the hemangioma, analogous to areas of puddling of contrast material seen at angiography. A total of 34 lesions in 21 patients demonstrated foci of globular enhancement. Of the 34 lesions, 32 (94%) proved to be hemangiomas. All 21 patients underwent confirmatory evaluation. Foci of globular enhancement seen during dynamic bolus CT are a strong indication that the lesion is a cavernous hemangioma. This diagnostic sign may obviate further, more expensive imaging studies. PMID- 1732979 TI - Recurrent rectal cancer and scar: differentiation with PET and MR imaging. AB - The value of positron emission tomography (PET) and magnetic resonance (MR) imaging in differentiating recurrent rectal cancer and scar was investigated. PET with fluorine-18 2-fluoro-2-deoxy-D-glucose (FDG) and MR imaging were performed in 15 patients with suspected recurrence. FDG accumulation in the mass was measured by means of the differential absorption ratio (DAR). All 11 patients with confirmed recurrent rectal cancer had increased accumulation of FDG in the mass (DAR = 4.73 +/- 2.28). Low FDG accumulation in the mass (DAR = 0.97 +/- 0.15) was noted in the remaining four patients, in whom the presence of a scar was proved by means of follow-up observation with or without biopsy. On the MR images, the recurrent tumor could be differentiated from scar in all but one case. The lesion-muscle signal intensity ratios on the T2-weighted images for the recurrent tumor and scar were 2.18 +/- 0.55 and 0.89 +/- 0.30, respectively. PET and MR imaging complement each other in the differential diagnosis between recurrent rectal cancer and scar. PET may also permit the evaluation of the effect of therapy. PMID- 1732980 TI - Peritoneal reflections of left perihepatic region: radiologic-anatomic study. AB - To clarify the anatomy of the peritoneal reflections of the left perihepatic region, the authors examined 95 cadavers. Thirty-eight were studied radiographically, 37 with sagittal dissection, and 20 with transverse dissection. In over 80% of the cadavers, the left triangular ligament of the liver separated the left suprahepatic space into anterior and posterior sections. The lesser omentum extended to the diaphragm, where its anterior layer reflected and continued as the posterior layer of the left triangular ligament. Thus, the posterior left suprahepatic space and the lesser sac were clearly separated by the lesser omentum and the stomach and over-lapped each other in three dimensions. The posterior left suprahepatic space was located anterosuperior to the lesser sac and in turn was continuous with the gastrohepatic space inferiorly. Carefully researched diagrams of both the midline sagittal and left parasagittal perihepatic spaces were developed. This information has clinical value when the radiologist is called on to drain a left perihepatic abscess. PMID- 1732981 TI - Regional lymph node metastasis in gastric cancer: evaluation with endoscopic US. AB - Endoscopic ultrasound (EUS) was performed in 83 patients with gastric cancer to evaluate regional lymph node metastasis. Histopathologic findings were compared with preoperative EUS findings in a total of 1,519 resected lymph nodes. In lymph node staging, the prevalence of metastatic adenopathy was 31.3% (26 of 83 patients); EUS had an accuracy of 83.1% (69 of 83 patients), sensitivity of 53.8% (14 of 26 patients), specificity of 96.5% (55 of 57 patients), positive predictive value of 87.5% (14 of 16 patients), and negative predictive value of 82.1% (55 of 67 patients). The greater the maximum diameter of the node with metastasis, or the larger the ratio of the metastatic area to the cross-sectional area of the node, the higher the detection rate. In tumors classified on the basis of depth of invasion according to the 1987 TNM system, the rate of detection of metastasis in individual nodes was 0% in pT1 tumors (none of five nodes), 20% in pT2 tumors (17 of 85 nodes), 29% in pT3 tumors (20 of 70 nodes), and 10% in pT4 tumors (three of 31 nodes). It is concluded that the most important use of EUS will be in diagnosis of regional lymph node metastasis. PMID- 1732982 TI - Receptor-directed contrast agents for MR imaging: preclinical evaluation with affinity assays. AB - In this study, the target-specific behavior of magnetic resonance (MR) imaging contrast agents directed at human hepatic asialoglycoprotein (ASG) receptors was evaluated in vitro with use of two novel assays: relaxation time measurements of incubated human cell membrane solutions and iron staining of biopsy samples. Specific uptake of ASG receptor-directed agents was demonstrated in human samples of normal liver tissue, areas of hepatitis, regenerating nodules, areas of focal nodular hyperplasia, and hepatic adenomas. A conventional iron oxide preparation not directed at ASG receptors failed to demonstrate specific uptake in these tissues. Attachment of the ASG receptor-directed agents was competitively blocked with a receptor agonist (D(+)-galactose) in these tissues. No attachment of conventional or receptor agents was seen in areas of hepatocellular carcinoma, cholangiocarcinoma, or liver metastases. The studies indicate that in vitro receptor assays are useful in predicting the affinity of new receptor-directed MR imaging contrast agents in human tissue prior to clinical trials. PMID- 1732983 TI - Postcatheterization femoral artery injuries: repair with color flow US guidance and C-clamp assistance. AB - Color flow ultrasound-guided compression repair of postcatheterization femoral artery injuries was attempted with the assistance of a C-clamp device in 10 patients. The C-clamp device was designed to hold the transducer in the optimal compression position and eliminate operator fatigue. In nine patients, the lesion was eliminated in a mean compression time of 59 minutes. The C-clamp maintained the transducer in an effective compression position in six procedures; in the other four, intervention to prevent slippage was required. PMID- 1732984 TI - Silicone drain mimicking a surgical sponge. AB - Because the appearances of a retained surgical sponge and the radiopaque portion of an in situ Snyder Hemovac flat silicone drain are similar at radiographic evaluation, confirmation should be obtained that a patient in whom a retained surgical sponge is suspected does not have this type of drain in place. The difference between the two items is especially difficult to discern in large patients or in patients with overlying dressings or a gas-distended abdomen. PMID- 1732985 TI - Lesions of the foramen ovale: CT-guided fine-needle aspiration. AB - To verify perineural spread of tumor along the mandibular division of the trigeminal nerve in four patients, the authors obtained cytologic specimens by means of a CT-guided transfacial fine-needle aspiration technique. Diagnoses were squamous cell carcinoma (n = 3) and meningioma (n = 1). The technique allows biopsy of deep lesions that would otherwise require open surgical biopsy. PMID- 1732986 TI - A simple method to lock large mushroom-tip catheters. AB - Dislodgment is a major drawback with large-bore Malecot catheters. A locking mechanism with a suture affixed to the distal portion of the mushroom tip is described. In a 32-month period, 17 Malecot catheters with locking mechanisms were placed in 15 patients. One catheter dislodged as a result of suture failure. A variation in design prevented subsequent failure of sutures. This simple locking mechanism prevents collapse of the catheter wings and thereby maintains catheter placement. PMID- 1732987 TI - Radiographic exposure calculator and mammographic dose calculator. AB - A set of dose calculators has been designed to facilitate quick and easy calculation of surface exposure (or skin dose) for routine radiography and average glandular dose in mammographic studies. Acceptance by technologists has been good, and in two inspections by the Joint Commission for Accreditation of Health Care Organizations, the calculators were deemed adequate to satisfy the diagnostic radiology standards that doses be monitored. PMID- 1732988 TI - Verification of lumbosacral segments on MR images: identification of transitional vertebrae. AB - To accurately identify lumbosacral transitional vertebrae and disease location, cervicothoracic sagittal scout images were obtained in addition to the standard images used in magnetic resonance imaging studies of the lumbar spine, and vertebrae were counted down from C-2 rather than up from L-5. In 200 patients, these techniques revealed 24 transitional vertebrae (15 cases of sacralization of L-5 and nine cases of lumbarization of S-1). PMID- 1732989 TI - Grading of splenic trauma. PMID- 1732990 TI - Brain death in the neonate: assessment with P-31 MR spectroscopy. PMID- 1732991 TI - Cranial MR imaging in HIV-seropositive individuals. PMID- 1732992 TI - The SCS/ARS/CES pesticide properties database for environmental decision-making. AB - The only thing all pesticides have in common is that they are used to control pests. Otherwise, they come from almost every imaginable class of chemical. Everyone associated with pesticide use--farmers, Extension, EPA, state regulatory agencies, manufacturers, and environmentalists--needs information that will allow them to distinguish between pesticides that may be a problem as pollutants in certain situations, and those which may not. There are five basic properties that, when combined with information about site and use, provide much information about the potential of a pesticide to be a pollutant. These five properties are solubility in water, volatility, soil sorption tendency, persistence, and ionization potential. We have compiled the most complete collection of these properties available, using others' compilations but verifying values from the primary literature in many cases. A complete primary literature search was not done. For each parameter we suggest a "Selected Value" which we believe to be the best available, recognizing, however, that persistence and soil sorption are sensitive to specific site conditions. These Selected Values are being incorporated into pesticide environmental-impact risk assessment procedures by state and federal agencies, and are considered to be consensus values. However, there is a serious potential for misuse of these data, particularly the error of using small differences between active ingredients to make regulatory distinctions between them. The ability to relate these data to environmental impact is an essential need and is improving, but is currently at a primitive level. PMID- 1732993 TI - Deltamethrin: uses and environmental safety. AB - Deltamethrin is a photostable pyrethroid providing valuable insecticidal activity against a large number of pests. Since it has potential uses for crop, cattle, and human health protection, extensive research work was done to evaluate its safety. Protection against insect pests in agriculture requires safety not only for applicators, but also for honey bees, nontarget insects and other arthorpods, mammals and birds in the wild and terrestrial fauna. Protection against vectors of harmful or endemic diseases involves direct treatment of waters with resulting risks for aquatic fauna. Extensive preharvest and postharvest use of a single pesticide demands thorough knowledge of residues on each crop and how they are affected by food processing. These various topics were considered in order to assess the safety of this new insecticide. PMID- 1732994 TI - 1,3-Butadiene: toxicity and carcinogenicity in laboratory animals and in humans. AB - 1,3-Butadiene is a high production volume chemical used largely in the manufacture of synthetic rubber. The production and use of 1,3-butadiene increased dramatically during World War II with the development of the synthetic rubber industry. Before the 1980s, 1,3-butadiene was not considered to be particularly hazardous to human health; therefore, OSHA established a permissible limit of 1,000 ppm for occupational exposure to this chemical. Results of recent inhalation carcinogenicity studies have demonstrated clearly that 1,3-butadiene is a multiple-organ carcinogen in Sprague-Dawley rats and in B6C3F1 mice. Particularly noteworthy in mice were the early occurrences and extensive development of lymphomas, the induction of uncommon hemangiosarcomas of the heart, and the development of malignant lung tumors at exposure concentrations as low as 6.25 ppm. Because 6.25 ppm was the lowest concentration ever used in a long-term carcinogenicity of this gas, it is likely that lower exposure levels would also cause cancers in laboratory animals. In addition, multiple organ site neoplasia was induced in mice after only 13 weeks of exposure. Two reactive epoxides, 1,2-epoxy-3-butene and diepoxybutane, have been identified as intermediates in the biotransformation of 1,3-butadiene in rats and mice. Metabolism is probably an important factor in the carcinogenicity of 1,3 butadiene, because in vitro mutagenicity of 1,3-butadiene requires metabolic activation, whereas these epoxide intermediates are direct acting mutagens in bacteria and are carcinogens in rats and mice. The metabolism of 1,3-butadiene in rats and mice is linear up to concentrations of at least 1000 ppm. Pharmacokinetic studies on 1,3-butadiene and on 1,2-epoxy-3-butene have revealed certain quantitative differences in metabolic rates between Sprague-Dawley rats and B6C3F1 mice; however, these differences were not of sufficient magnitude to account for the reported different target site carcinogenic responses in these two strains of animals. Thus, additional factors must be involved in distinguishing site specificity in the carcinogenicity of 1,3-butadiene between species. In addition to its carcinogenic effects, 1,3-butadiene is a potent in vivo genotoxic agent to mouse bone marrow cells. Hematologic changes indicative of a partially regenerative anemia were induced in mice at 62.5 and higher concentrations. 1,3-Butadiene is also a reproductive and developmental toxicant. Epidemiology studies of workers employed in the production of 1,3-butadiene or of styrene-butadiene rubber have consistently revealed associations between occupational exposure to 1,3-butadiene and excess mortality due to lymphatic and hematopoietic cancers.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1732995 TI - Microbial bioconversion of pollutants. AB - Microorganisms totally detoxicate xenobiotics of various chemical structures, which are serious and, in some cases, very hazardous pollutants. At present, the efforts of a number of researchers promoted the establishment in this country of a collection of microorganisms able to degrade volatile toxic pollutants- toluene, isomeric xylenes, styrene, alpha-methylstyrene, crotonaldehyde; widely distributed xenobiotics chlorobenzoic acids; isomeric aryldicarboxylic acids; and ecologically hazardous pollutants such as aromatic nitrocompounds. The active strains-destructors are mainly representatives of the genera Pseudomonas and Rhodococcus. Research into their physiological characteristics, key enzymes, pathways of xenobiotics degradation, genetic mechanisms determining the degradation of these foreign compounds, and behaviour of the strains in a real environment made it possible to develop the theoretical principles of using these microbial cultures to purify real industrial wastes and remediate polluted areas of soil and water. Improvement of the methods of immobilizing the active xenobiotics-degrading strains on cheap and efficient carriers made it possible to significantly intensify the cleanup process of industrial wastes and eliminate a number of problems during the development of the biotechnologies for industrial waste cleanup. Successfully operated at present are the biotechnologies of the local cleanup of waste waters of terephthalate production, microbial purification of industrial waste waters in nylon-66 production from hexamethylenediamine, purification of coke production wastes from phenols, waste waters of polyisocyanate production from aromatic amines, local purification of waste waters in synthetic rubber production from alpha-methylstyrene, acetaldehyde production wastes from crotonaldehyde and mercury. Microbial strains constructed by gene engineering methods for the cleanup of contaminated soils from dicofol and 3-chlorobenzoate were successfully applied (Golovleva et al. 1988). PMID- 1732996 TI - Environmental biochemistry of arsenic. AB - Microorganisms are involved in the redistribution and global cycling of arsenic. Arsenic can accumulate and can be subject to various biotransformations including reduction, oxidation, and methylation. Bacterial methylation of inorganic arsenic is coupled to the methane biosynthetic pathway in methanogenic bacteria under anaerobic conditions and may be a mechanism for arsenic detoxification. The pathway proceeds by reduction of arsenate to arsenite followed by methylation to dimethylarsine. Fungi are also able to transform inorganic and organic arsenic compounds into volatile methylarsines. The pathway proceeds aerobically by arsenate reduction to arsenite followed by several methylation steps producing trimethylarsine. Volatile arsine gases are very toxic to mammals because they destroy red blood cells (LD50 in rats; 3.0 mg kg-1). Further studies are needed on dimethylarsine and trimethylarsine toxicity tests through inhalation of target animals. Marine algae transform arsenate into non-volatile methylated arsenic compounds (methanearsonic and dimethylarsinic acids) in seawater. This is considered to be a beneficial step not only to the primary producers, but also to the higher trophic levels, since non-volatile methylated arsenic is much less toxic to marine invertebrates. Freshwater algae like marine algae synthesize lipid-soluble arsenic compounds and do not produce volatile methylarsines. Aquatic plants also synthesize similar lipid-soluble arsenic compounds. In terrestrial plants, arsenate is preferentially taken up 3 to 4 times the rate of arsenite. In the presence of phosphate, arsenate uptake is inhibited while in the presence of arsenate, phosphate uptake is only slightly inhibited. There is a competitive interaction between arsenate and phosphate for the same uptake system in terrestrial plants. The mode of toxicity of arsenate is to partially block protein synthesis and interfere with protein phosphorylation but the presence of phosphate prevents this mode of action. There appears to be a higher affinity for phosphate than arsenate with a discriminate ratio of 4:1. It is estimated that as much as 210 x 10(5) kg of arsenic is lost to the atmosphere in the vapor state annually from the land surface. The continental vapor flux is about 8 times that of the continental dust flux indicating that the biogenic contribution may play a significant role in cycling of arsenic. It has not been established whether volatile arsenic can be released by plants. Further studies are needed to determine mass balances in the rate of transfer (fluxes) of arsenic in the environment. PMID- 1732997 TI - [What is your diagnosis? Tolosa-Hunt syndrome]. PMID- 1732998 TI - [The clinical importance of cardiovascular risk factors]. PMID- 1732999 TI - [The treatment of arterial hypertension: which drug for which patient?]. AB - Hypertension is a multifactorial disease. Various antihypertensive drugs can lower arterial pressure in a given patient in a more or less efficient way. The sequential testing of several drugs is most promising for lowering blood pressure by monotherapy. If necessary a drug combination is preferable to dose adjustments of a single substance because of the risk for side effects growing with the dose. PMID- 1733000 TI - [Prognostic aspects in the treatment of chronic heart insufficiency]. AB - Treatment of patients with heart failure due to major ventricular systolic dysfunction should aim not only at symptomatic but also at prognostic improvement. If correction of the underlying problem is not possible, treatment should slow down the progression of cardiac failure and eliminate triggers for sudden cardiac death due to electromechanical dissociation or arrhythmias. In every patient with chronic congestive heart failure screening for myocardial ischemia and complete revascularization is mandatory, if possible. In patients with coronary artery disease and diminished systolic function, beta-blockade may improve prognosis by reducing ischemic events and sudden cardiac death. The incidence of life-threatening arrhythmias in patients with heart failure may be reduced by eliminating facilitating factors like electrolyte disturbances, altered autonomic tone and raised intracardiac pressure rather than by antiarrhythmic medical treatment itself. One of the most important prognostic aspects in treatment is the interference with the development of the cardiomyopathy of overload, uniformly observed in chronic congestive heart failure. Modification of mechanical and neuroendocrine stimuli may postpone myocardial hypertrophy and interstitial hyperplasia as a consequence of altered gene expression. Early treatment with ACE inhibitors and in certain patients with betablockers are the most promising strategies to delay the progression of the disease. In contrast, positive inotropic drugs, including digitalis and phosphodiesterase inhibitors, do not improve prognosis. Calcium antagonists should also be used with restriction, as Verapamil and Diltiazem, but also Nifedipine may adversely affect the outcome in congestive heart failure patients. PMID- 1733001 TI - [The effect of captopril on the progression of heart insufficiency]. AB - We conclude from our studies and data from the literature that ACE inhibitors belong to the proper basic therapy of cardiac failure because of their marked influence on progression and death by pump failure. The therapeutic gain per 100 patients treated for one year exceeds that of other medications used commonly for secondary prophylaxis. PMID- 1733002 TI - [Insulin resistance. A syndrome responsible for various cardiovascular diseases]. AB - The X-syndrome described by G. M. Reaven comprises several risk factors, among them obesity. Type II diabetes, hypertension and certain dyslipidemias. The frequent association of these conditions is due to insulin-resistance and hyperinsulinism. The pathophysiology of this syndrome and the possibilities for its treatment are reviewed. PMID- 1733003 TI - [Current strategies in the diagnosis and therapy of hyperlipoproteinemias]. AB - The determination of total cholesterol (and eventually triglycerides) is sufficient for screening purposes. Values below 5.2 mmol/l (200 mg/dl) may be considered ideal, where as higher values need a differentiated diagnostic evaluation which should include the measurement of triglycerides and of HDL cholesterol after 12 h fasting. The decision for a drug therapy should consider other risk factors such as hypertension, smoking, diabetes mellitus, male sex, positive family anamnesis for myocardial infarction already existing and coronary heart disease. The treatment of a dyslipoproteinemia should always start with dietary measures. If these are not successful, a lipid-regulating drug therapy should be started in addition to the diet. The choice of the drug depends on the type of dyslipoproteinemia and its genetic and metabolic cause and on the individual side effects. PMID- 1733004 TI - [Combination therapy of cardiovascular risk factors]. AB - Risk factors for cardiovascular diseases, which are the leading cause of mortality in the industrialized countries, are well investigated; however, the results of intervention studies on the therapy of single risk factors were disappointing in the past. Recently, there has been growing evidence that there might be a closer pathophysiological relation between arterial hypertension, hypercholesterolemia, obesity, impaired glucose tolerance and genetic disposition than previously thought. For the treatment of the individual patient, this concept requires a complete work-up and comprehensive therapy of all risk factors. The therapy of several mildly elevated risk factors may be more beneficial than a too vigorous reduction of the blood pressure alone. At the beginning of every therapeutic regimen, there has to be a nonpharmacological approach. Diet and weight reduction even in mild obesity are more efficient in influencing several risk factors at the same time than pharmacological therapy. Metabolic consequences of drug treatment have to be carefully monitored. PMID- 1733005 TI - [What is your diagnosis? Tarsal tunnel syndrome]. PMID- 1733006 TI - [Computer application in practical diabetology]. PMID- 1733008 TI - [M. von Ardenne's multi-step cancer therapy and variations--universally applicable in oncology? Documentation No.23]. AB - M. von Ardenne advances 2 concepts for the treatment of cancer: the Multistep Cancer-Therapy (MCT) and the O2-Multistep-Immunostimulation (O2-MIS). Since the properties of cancer tissues postulated by. M. von Ardenne are considered common to all malignant tumours, MCT and O2-MIS can be employed in any type of malignancy. These concepts are recommended for cancer therapy and prophylaxis. The 1989 concept consists of the combination of O2-MIS (thymus) for 18 days followed by MCT on the 19th day. O2-MIS can be done on an outpatient basis, whereas MCT must be carried out in hospital. An investment of 5000 to 10,000 Fr. is needed to provide O2-MIS in a medical practice. In 1959, Prof. Dr. h. c. mult. M. von Ardenne transferred from research in the field of physics to cancer research. He published the basic concept of MCT in 1965, from which the Oxygen Multistep-Therapy (SMT) and the O2-MIS were later derived. Most of the proponents are found in the former Federal Republic of Germany, where the SMT-Society is responsible for the dissemination of the ideas and apparatus. The mechanism of action of MCT consists of the irreversible occlusion of tumor blood vessels as a result of "elective over-acidification of the cancer cells and tumour tissue by stimulating the aerobic fermentation metabolism of cancer cells discovered by O. Warburg in 1924". O2-MIS, on the other hand, works by potentiating non-specific defences. Contrary to his claims, von Ardenne has so far been unable to demonstrate a reproducible efficacy either for the MCT or for O2-MIS against cancer in man. Despite a supposedly large number of patients treated, no clinical trials have yet been published.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733007 TI - [Ambulatory transesophageal echocardiography. Diagnostic possibilities of a novel study method]. AB - Transesophageal echocardiography has become now a routine outpatient method to augment the diagnostic power of transthoracic echocardiography in 10-15 minutes. Indications are native and prosthetic valvular heart disease and congenital heart disease, the evaluation of embolic events and suspicion of aortic dissection. PMID- 1733009 TI - [Gas gangrene and hyperbaric oxygen]. AB - From 1964-1987 we have examined 31 patients suspected to have gaseous gangrene. The diagnosis was confirmed 13 times on clinical and microbiologic grounds. 61% of the patients underwent amputation, 28% died. On the basis of this experience we have tried to define a therapeutic attitude. We recommend an adequate debridement, i.e. the removal of necrotic tissue and drainage of the wound, microbiological investigations and the administration of antibiotics before hyperbaric oxygen treatment is begun (2-3 treatments over 3 days). PMID- 1733010 TI - [Enduring loneliness. An interview survey of aged widows]. AB - Interviews with 32 widows have given a clearer picture of the different ways of adaptation to solitude. The following types are distinguished: 1. the tormented (6 cases), who are crushed by loneliness and depression, even though they have the support of friends and relations 2. the isolated (7 cases), who may go for several days without seeing anyone 3. the misanthropes (3 cases): 'The less I see of my friends, the better I feel' 4. those who have found the answer to the problem of solitude in outside activities and contacts (5 cases) 5. those who have found tranquility after a lifetime of struggle (4 cases) 6. those with a naturally harmonious temperament who have overcome solitude and demonstrate the way to face it with courage, good sense and optimism (7 cases). This attempt at typology, which describes six different ways of living with solitude, has made possible a better analysis of the problem, its causes and its effects. Though solitude is often considered a negative aspect of growing old, it can also be a source of happiness, even of fulfillment. The capacity to live alone is something that has to be learned. PMID- 1733011 TI - [A case from practice (235). 1. Hyperprolactinemia--idiopathic--due to microprolactinoma. 2. Depressive mood]. PMID- 1733012 TI - [Pneumocystis carinii pneumonia in HIV-negative immunosuppressed patients]. AB - During a period of 10 years 129 immunosuppressed HIV-negative patients were evaluated for pulmonary complications. A definite diagnosis could be established in 72 cases (56%): Pneumocystis carinii pneumonia (PCP) (25), pulmonary involvement of underlying disease (10), drug toxicity (8), mycobacterioses (6), bacterial pneumonias (5), aspergillosis (5), others (13). The underlying conditions in patients with PCP were: lymphatic neoplasias (11), immunosuppression after solid organ (9) and after bone marrow transplantation (3), cytotoxic therapy for lupus erythematodes (1) and carcinoma (1). In 8 of 9 transplant patients anti-rejection therapy preceded the episode of PCP. Six patients (24%) died from respiratory failure 1 to 25 days after diagnosis of PCP, despite mechanical ventilation in four. Two patients recovered completely after mechanical ventilation for 14 and 30 days respectively. The frequency of PCP has markedly increased during the last few years: 1981-1987: 2 cases (6%), 1988: 4 (14%), 1989: 8 (42%) and 1990: 11 (26%). This can hardly be explained by improved diagnostic sensitivity or an increased number of immunosuppressed patients. Apart from the use of more potent immunosuppressive agents, the increased prevalence of Pneumocystis carinii may play an important role. PMID- 1733013 TI - [Permanent cardiac stimulation: evaluation of a 10-year experience]. AB - This 10-year prospective follow-up study (1978-1988) covers 430 patients who received 463 permanent pacemakers in a regional hospital. One cardiologist established the indication for implantation and a general surgeons' team performed the operation. Mortality was very low (0.5%). Among the local complications (6.2%), infection was the most dangerous and often required repeated surgery or even removal of the entire pacing system. PMID- 1733014 TI - [Fibrinolysis in a regional hospital]. AB - From January 1, 1989 to September 30, 1990, 116 patients with acute myocardial infarction were hospitalized at the regional hospital of Langenthal. Of those 116 patients, 27 (23%) were treated with intravenous streptokinase; in 12 of them (44%) CPK reached its peak within 6 hours after starting lysis; all were admitted within 4 hours after the beginning of chest pain. Of the fibrinolyzed patients, 18 (67%) had arrhythmias which needed to be treated. In 10 of these 18 patients CPK reached its peak within 6 hours. Of the 89 patients not treated with streptokinase, 27 (30%) did not fulfill the entry criteria of the protocol, 16 (18%) had exclusion criteria, and 46 (52%) had exclusion criteria as well as absent inclusion criteria. In only 4 patients (4.5%) was lysis not possible because they entered hospital later than 6 hours after the beginning of pain, and 8 patients (9%) exceeded the upper age limit of 70 years. Of the 116 patients, 13 (11.2%) died; 12 were not treated with streptokinase. Our study shows that fibrinolytic treatment with streptokinase is a safe and effective therapy for patients with acute myocardial infarction and can easily be performed in a regional hospital. PMID- 1733015 TI - [Does isolated TSH elevation need treatment? Study of risk factors for the development of manifest hypothyroidism]. AB - Isolated elevations of basal TSH levels are frequently observed in the general population. In a prospective study we analyzed the spontaneous evolution of thyroid function over time in such patients. The mean observation period was 5.4 (0.5-12) years. During the follow-up period 20% of these patients developed overt hypothyroidism. The risk of developing hypothyroidism was determined primarily by the initial TSH value and an additive effect was found for the thyroid antibodies and the thyroidal reserve (delta-T3) after TRH stimulation (Cox proportional hazard model). The cumulative risk for overt hypothyroidism after 10 years was only 22% for a mean TSH level of 12 mU/l for patients with negative thyroid antibodies and a good thyroidal reserve (low-risk), but increased to 63% for patients with positive antibodies and impaired T3 reserve (high-risk). Therefore, patients with isolated elevation of TSH can be divided into two subgroups according to the results of TSH, antibody status and T3 reserve: (1) In the "low risk group" with good prognostic factors the patients should be followed up by periodic laboratory testing only (TSH, FT4, every 2-3 years). (2) In the "high risk group" with clearly abnormal parameters, however, frequent controls are mandatory (every 6-12 months) or treatment with thyroxine may be indicated. PMID- 1733016 TI - [Deglutition syncope]. AB - In cases with a history of swallow syncope, an ECG should immediately be taken during ingestion of solid or liquid food in order to determine the rhythm disturbances responsible for the syncopes. Both esophageal passage and cardiac diagnosis are indicated, since, as shown in the literature, pathologic findings are not uncommon. The triad syncope, bradyarrhythmia while swallowing is considered to be an indication for definitive electrostimulation (pacemaker implantation) unless, exceptionally, halting digitalis medication puts an end to the syncopes. PMID- 1733017 TI - Urgent dietary intervention required in urbanising communities to prevent allergy epidemic. PMID- 1733018 TI - Neutron therapy improves control of malignant salivary gland tumours. PMID- 1733019 TI - AIDS in South Africa--into the second decade. PMID- 1733020 TI - Statistical approaches to determine the current extent and future prevalence of HIV infection in South Africa. PMID- 1733021 TI - Patient-controlled analgesia. PMID- 1733022 TI - Should food labelling be made mandatory? PMID- 1733023 TI - Children with AIDS--can we afford to treat them? PMID- 1733024 TI - Estimations of the total size of the HIV and hepatitis B epidemics in South Africa. AB - The total number of people infected with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) in South Africa was estimated from a number of sources of seroprevalence for each of these viruses. A total figure of 122,951 HIV-infected individuals in South Africa was arrived at for January 1991; 69% (85,247) were from the urban black population and 20% (24,474) from the rural black population. The male homosexual population constituted some 7% (8,175) of the total, 94% of whom were white; however, this probably represented a substantial underestimation of the size of this population. A total of 1,475,223 carriers of HBV virus was calculated for South Africa from data obtained from 1986 to 1990. Of these, 88% (1,302,741) came from the rural black population and 8% (114,118) from the urban black population. Extrapolations from small sample numbers and often with broad assumptions are subject to considerable error. Nevertheless, estimated total figures do provide a vivid picture of the extent of these two epidemics. PMID- 1733025 TI - A stochastic model for medium-term estimation of the prevalence of HIV infection in a South African heterosexual population. AB - This paper describes the development of a micro-simulation model to estimate the extent of HIV infection among black heterosexual South Africans and attempts to predict the number of newly acquired cases of HIV infection for the period 1985 2000. Owing to the lack of key data inputs, many assumptions are made. The inputs used are estimated demographic transition, HIV-positive immigrants, probability of being HIV-positive, probability of acquisition of infection upon at-risk contact by gender, time to AIDS distribution, average number of new sexual partners over time by age and gender, average number of sexual contacts per partner by gender and age group, and probability of sexual contact by age group between genders. Paediatric, homosexual and intravenous HIV cases are excluded from the model. The results indicate a fairly rapid increase in HIV infection from 1988 until the year 2000, when 5,664,487 adults, i.e. approximately 27% of the total adult (15-60 years) black population, could have become HIV-positive. This first phase of the model does not include the effect of a change in behaviour or the possible effect of effective drugs or vaccines and is therefore a worst-case scenario, maximum-potential estimate. The results are very similar to previously published South African actuarial and macro-simulation models. PMID- 1733026 TI - Perceptions and knowledge about AIDS among family planning clinic attenders in Johannesburg. AB - Many studies assessing the impact of national AIDS prevention programmes on knowledge, attitudes and practices have been published world-wide. Most have found that, while general knowledge increased, there was little change in behaviour. A survey of a random sample of 50 women attending a family planning clinic in Johannesburg determined the current knowledge, attitudes and practices of these sexually active women, aged 20-29 years. All selected respondents agreed to participate and a pretested structured questionnaire was completed. Most individuals had in excess of 7 years' schooling (78.0%), were single (58.0%) and domestic workers (28.6%). The majority were aware of AIDS (88.0%), but only 13.7% felt their knowledge was adequate. Fifty-two per cent knew sexual intercourse was a mode of transmission. Many misconceptions existed, 64.0% cited toilet seats, 47.1% sharing of utensils and 70.1% donating blood as routes by which HIV infection could be acquired. Only 47.1% and 34.1%, respectively, knew that the contraceptive pill and intra-uterine contraceptive device did not protect against HIV infection. Eighty-six per cent of the women were sexually active and 8.0% admitted to currently having more than one sexual partner. None used condoms and, generally, feelings regarding condom use were negative. Most (88.6%) believed AIDS patients should be hospitalised, 68.2% said in isolation wards, and 27.3% felt that AIDS patients should not be allowed to stay in the community. The implications of these findings are discussed and certain recommendations are made. PMID- 1733027 TI - Patient-controlled analgesia. Its South African debut in a provincial hospital. AB - Patient-controlled analgesia (PCA) is a well-established technique for the relief of acute and chronic pain. It is widely used in Western hospitals. Patient and staff education is required to provide successful analgesia. This paper reports the successful introduction of PCA on a large scale in a provincial hospital. The considerable potential difficulties in communication and education appear to have been overcome. The widespread introduction of PCA in all our hospitals would appear to be feasible. PMID- 1733028 TI - Mortality from external causes in South African adolescents, 1984-1986. AB - The external causes of death in South African adolescents are described. Nationally registered mortality data for 1984-1986 were used to calculate proportional mortality. Mortality rates were also calculated, except in the case of black deaths, since these deaths are known to be under-registered and the estimated population figures are known to be inaccurate. Of the 16,348 adolescent deaths registered in 1984-1986, external causes accounted for 56.8% and symptoms, signs and ill-defined conditions for 10.0%. A greater proportion of girls died from symptoms, signs and ill-defined conditions whereas a greater proportion of boys died from external causes. A larger proportion of black adolescent deaths were categorised as symptoms, signs and ill-defined conditions. The risk of death by external cause for coloureds aged 15-19 years was 1.7 that of whites, while in the 10-14 year age group it was the same as that of whites. In the 15-19-year age group assault was the most common external cause of death in blacks and coloureds, compared with road accidents for whites. The highest number of deaths by external cause per day occurred over the Christmas period. The analysis indicated that mortality rates in South African adolescents are high and that many deaths may be the result of risk-taking behaviour. With the increasing urbanisation of blacks, the impact of external causes of death can be expected to increase further. PMID- 1733029 TI - Treatment of chronic granulomatous disease with recombinant gamma interferon. AB - Chronic granulomatous disease is a rare, primary immunodeficiency associated with serious bacterial and fungal infections caused by phagocytic defects of oxidative metabolism. To date the mainstay of management has been aggressive treatment of infections and the use of prophylactic antibiotics. Two patients, who showed remarkable clinical improvement when treated with recombinant gamma interferon, are reported. Both have been on treatment for at least 18 months and have continued to thrive and remain free of infections. PMID- 1733030 TI - Sexually transmitted disease surveillance in a child abuse clinic. AB - The sexually transmitted disease surveillance system instituted at the Child Abuse and Neglect (CAN) clinic of the Transvaal Memorial Institute for Child Health and Development was evaluated after 1 year. The presenting complaint of the vast majority of the 227 patients was sexual abuse. In more than half (52%), child abuse was medically proven, and it was highly suspected in another 18%. In only 6% did no abuse take place. About half the patients suffered non-penetrative sexual abuse, 40% penetrative abuse and 10% suffered non-sexual abuse. Smears for gonorrhoea were positive in 2 out of 152 patients; for Chlamydia in 1 out of 140; for Gardnerella and Trichomonas in 2 and 1 case, respectively. Syphilis serology yielded 3 positive results out of 162, and hepatitis B, 6 out of 143. No positive results were found in tests for HIV and herpes. With the exception of hepatitis B tests, all positive results occurred in children considered on clinical grounds to have medically proven or highly suspected sexual abuse. These results will allow modification of the surveillance system and testing of those children more likely to test positive, while doing fewer tests overall. PMID- 1733031 TI - Bacteraemia in children in the south-western Cape. A hospital-based survey. AB - During 1989, of the 8,524 children admitted to the paediatric wards of Tygerberg Hospital, 165 (1.96%) had bacteraemia. The incidence of community-acquired bacteraemias was 1.6% and that of nosocomial bacteraemias 0.5%. The most important community-acquired isolates were Streptococcus pneumoniae, Staphylococcus aureus and Neisseria meningitidis. The most important nosocomial isolates were Klebsiella and Salmonella spp. Both bacteraemia (relative risk (RR) = 2.08) and severe malnutrition (RR = 3.01) were more common in black patients. Overall, severe malnutrition was more common than mild malnutrition or a normal nutritional status in bacteraemic patients (odds radio (OR) = 3.17). Nineteen patients with bacteraemia died, there was a significantly higher case-fatality rate in patients with extreme malnutrition (P = 0.03; OR = 3.7). Gram-negative bacilli were found more commonly in patients with extreme malnutrition (OR = 5.4) and patients with nosocomial bacteraemia (OR = 4.6). Three of 39 patients (7.6%) with nosocomial bacteraemia had suppurative thrombophlebitis. PMID- 1733032 TI - Oesophageal cancer in three regions of South Africa. AB - Cancer of the oesophagus is the commonest cancer in South African black males. The highest incidence rates occur in the south of Transkei. The rate among urban blacks, especially in Soweto, is also high. This study determined risk factors for oesophageal cancer in patients in three different environments--urban Soweto, rural Ciskei and rural-urban Bophuthatswana. Males were affected more than females. The majority of patients in all three regions were smokers. With regard to alcohol consumption, most Sowetans (84%) and Ciskeians (91%), but only 57% of patients from Bophuthatswana, were drinkers. Home-brewed drinks were the main source of alcohol. Oesophageal cancer occurs in both rural and urban environments, affects people without regard to tribal ethnicity, and occurs mainly in the 6th decade; moreover, almost all patients present with advanced disease. PMID- 1733033 TI - [Pasteurella haemolytica serotypes in cattle]. AB - Eight-two bovine Pasteurella haemolytica strains were serotyped. The majority of the strains were isolated from calves which had died from fibrinous pneumonia and small numbers from cases of pleuritis, sepsis and abortion. A total of eight different serotypes were noticed. Serotype A1 was found to be the most prevalent 37.8 per cent, followed by serotype A2 with 20.7 per cent. Antibiotic resistance was found for sulfonamide, tetracycline and penicillin in about half of all strains. Conventional combination of biotype to serotype per strain was not confirmed by the results of arabinose and trehalose fermentation. PMID- 1733034 TI - [Ivermectin poisoning in a dog. The use of ivermectin in companion animals]. AB - In this paper the problems that may arise when ivermectin is used in small animal medicine are discussed. A case-report is presented concerning a Canadian-American White Shepherd showing symptoms of fatal ivermectin poisoning following a normal dosage of ivermectin (220 micrograms per kilogramme of body weight). The treatment of dogs with ivermectin poisoning is described and some guide lines concerning the use of ivermectin are given. PMID- 1733035 TI - [Logbook registration in slaughtering pig farms within the project Integrated Quality Control. II. Drug utilization in relation to clinical observation, farm conditions and prevalence of pathological findings]. AB - During an Integrated Quality Control (IQC) project for finishing pigs, the application of veterinary drugs was registered by means of a logbook on 75 pig finishing farms. Investigated is as to how far the application of veterinary drugs to animals is linked up with clinical observations, management and climatic conditions on the farm and to what extent the application of veterinary drugs is linked up with the occurrence of pathological findings in pigs at the slaughterhouse. The results indicate that more veterinary drugs are used as there are more clinical findings on a pig farm. This is particularly true for the autumn-winter period. Farms that use more veterinary drugs have a higher percentage of pigs with pathological findings at the slaughterhouse. As the point of time of treatment of respiratory disorders is closer to the slaughter date, there is a higher percentage of pigs suffering from pneumonia at the slaughterhouse. Farms treating pigs in the first thirty days of the finishing period against intestinal disorders have a distinct higher percentage of pigs suffering from pneumonia at the slaughterhouse. It appears that less veterinary drugs are used at farms having specific housing and management characteristics. PMID- 1733036 TI - [Profile of the veterinary assistant]. AB - From a survey covering 338 employees in veterinary practices the profile of the veterinary technician/receptionist in the Netherlands is drawn. Demographic, motivational and financial features of this profile are discussed. PMID- 1733037 TI - [The dyspneic parrot]. PMID- 1733039 TI - [Talking about solidarity]. PMID- 1733038 TI - [Alpha-chloralose]. PMID- 1733040 TI - [Veterinary disciplinary law. Direct legal and business administrative affairs]. PMID- 1733041 TI - Central activity of acetylcholinesterase oxime reactivators. AB - The ability of various oximes to antagonize the sarin-induced hypothermia and reactivate phosphorylated acetylcholinesterase was used as an indicator of the central activity of oximes. HI-6, but neither toxogonin nor PAM Cl, antagonized sarin-induced hypothermia and reactivated brain acetylcholinesterase, in particular hypothalamic acetylcholinesterase. The sarin-induced hypothermia appears to be a muscarinic cholinergic action since atropine was also an effective antagonist of sarin-induced hypothermia. Neither HI-6 nor toxogonin antagonized oxotremorine-induced hypothermia, indicating that these oximes do not possess central cholinolytic activity. The results demonstrated that HI-6 penetrated the blood-brain barrier in a sufficient concentration to produce a biochemical and physiological action against sarin poisoning. PMID- 1733042 TI - Triphenyl phosphite-induced ultrastructural changes in bovine adrenomedullary chromaffin cells. AB - Primary cultures of bovine adrenomedullary chromaffin cells were treated with the phosphorus acid ester triphenyl phosphite (TPP), a chemical capable of producing Type II organophosphorus compound-induced delayed neurotoxicity (OPIDN), and the morphological changes were assessed by transmission electron and scanning microscopy. Following a 24-hr incubation with 100 microM TPP nearly all mitochondria were either disrupted or swollen and glycogen buildup within the cytoplasm was evident. The viability of cells treated with TPP and cultured on coverslips for scanning electron microscopy was very low. By scanning electron microscopy, the filopodia of these cells appeared contracted. The surface texture was very irregular and giant globular bodies were evident. Parallel studies were carried out with the cholinergic compound O,O-diethyl 4-nitrophenyl phosphate (paraoxon) and the Type I delayed neurotoxicant O,O diisopropylphosphorofluoridate (DFP). Transmission and scanning electron microscopy revealed that treatment with these organophosphorus compounds did not produce the ultrastructural effects that were seen with TPP. The morphological data were confirmed biochemically by assessing the viability of the mitochondria via measurement of [3H]adenosine incorporation into ATP. Treatment with 100 microM TPP for 4 or 24 hr caused a marked inhibition (90% relative to controls) of adenosine incorporation. Neither 100 microM paraoxon nor 100 microM DFP had an inhibitory effect on incorporation. The effect of TPP was time-dependent with significant biochemical effects as early as 60 min. In contrast, ultrastructural changes were not seen until 24 hr. Morphologically, the 60-min incubations showed no perturbation in mitochondrial integrity. Our results support a specific effect of the triphenylphosphite, TPP, a Type II OPIDN compound, not a general toxic effect of organophosphorus compounds since the cholinergic agent paraoxon and the Type I delayed neurotoxic compound DFP did nto alter the cells ultrastructurally or compromise the mitochondria biochemically. The apparent target for TPP toxicity is the mitochondria. PMID- 1733043 TI - Effect of methyl bromide on regional brain glutathione, glutathione-S transferases, monoamines, and amino acids in F344 rats. AB - Both metabolic and neurotransmitter changes have been implicated in the pathogenesis of monohalomethane neurotoxicity in rodents. This study in male and female F344 rats examined the effects of methyl bromide (MeBr) on regional brain glutathione-S-transferase (GST) activities and concentrations of glutathione (GSH), monoamines, and amino acid. Inhalation exposure to 150 ppm MeBr (6 hr/day x 5 days) yielded no histologic evidence of brain lesions but resulted in a number of biochemical changes. GSH depletion and GST inhibition were detected in the frontal cortex, caudate nucleus, hippocampus (examined for GSH only), brain stem, and cerebellum from animals of both sexes. Differences between sexes were detected for GSH depletion. Simultaneous treatment of rats with the inhibitor of monohalomethane toxicity, BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2 pyrazoline; 10 mg/kg bw ip, 1 hr pre- and 1 hr postexposure) completely protected against GST inhibition in all brain regions of both sexes. Partial protection by BW 755C against GSH depletion was observed in the cerebral cortex and in the cerebellum only. In males, MeBr exposure had no effect on the regional concentrations of the monoamines dopamine and serotonin and the amino acids glutamate, glutamine, taurine, and gamma-aminobutyric acid. Regional increases of brain aspartate and glycine levels were observed after exposure of males to MeBr but BW 755C had no effect on these changes induced by MeBr. Thus, of all the parameters studied, only GST, and in some brain areas GSH, correlated with inhibition of toxicity. It is concluded that, in contrast to the monoamines and the amino acids, GST and GSH are sensitive and potentially relevant indicators of MeBr neurotoxicity which could explain sex and regional differences in response to the monohalomethanes. PMID- 1733044 TI - Effects of dietary aluminum excess and manganese deficiency on neurobehavioral endpoints in adult mice. AB - Studies in mice have suggested that both dietary Al excess and dietary Mn deficiency promote oxidative tissue damage. To determine if these factors can interact to produce functional nervous system damage, female mice (N = 10-12 per group) were fed diets with control or low Mn (35 or 3 micrograms Mn/g diet) and/or control or high Al (25 or 1000 micrograms Al/g diet, Al as Al lactate) content for a 90-day period. No overt signs of neurotoxicity were observed in any group. Excess Al produced a threefold Al accumulation in both liver and brain, a slight acceleration of growth, decreased motor activity, decreased grip strength, and decreased startle responsiveness. Manganese deprivation led to liver, brain, and femur Mn depletion and reduced liver MnSOD activity but no neurobehavioral changes. No interactive effects between Al excess and Mn deficiency were observed. Neither Al excess nor Mn deficiency altered brain or liver lipid peroxidation measures. This study suggests that (1) subchronic dietary Al at doses of 1000 micrograms Al/g diet produces elevated brain Al and altered neurobehavioral indices in adult mice; (2) brain lipid peroxidation is not altered by this treatment; (3) dietary Mn deficiency does not influence Al neurotoxicity in adult mice. PMID- 1733045 TI - Quinone chemistry and toxicity. PMID- 1733046 TI - Degradation and metal composition of hepatic isometallothioneins in rats. AB - Recently, our laboratory demonstrated that metallothionein-1 (MT-1) is degraded faster than metallothionein-2 (MT-2) in liver of Zn-treated adult rats; however, it is not clear whether this phenomenon is unique to Zn treatment or the age of the animal. Furthermore, many investigators maintain that the degradation of MT is regulated by its metal composition. The objective of this study was twofold: (1) to determine if MT-1 is more susceptible than MT-2 to proteolytic breakdown regardless of age or chemical pretreatment and (2) to examine the hypothesis that the amount and type of metals bound to MT influences its resistance to degradation. Pulse-labeling experiments were conducted to determine the half lives of MT-1 and MT-2 in liver of adult rats (75-day-old), immature rats (1-day old), and mature rats treated with single dosages of Zn (1 mmol/kg, sc), Cd (10 mumol/kg, sc), or ethanol (109 mmol/kg, po). Atomic absorption spectrometry was utilized to measure the Zn, Cu, and Cd contents of MT-1 and MT-2 obtained in selected experimental groups. MT-1 had a shorter half-life than MT-2 in Zn treated adults (21 vs 33 hr) and in nontreated immature rats (49 vs 73 hr). In contrast, the half-life values of MT-1 and MT-2 were identical in nontreated adults (4 hr) and ethanol-treated adults (9 hr) and nearly identical in Cd treated adults (58 and 61 hr, respectively). Both isoforms obtained from immature rats and adults treated with Zn or ethanol contained approximately 6.0 g atoms Zn/mol MT, trace levels of Cu, and nondetectable quantities of Cd. In Cd-treated rats, both isoforms contained approximately equal amounts of Zn and Cd (3.2 g atoms metal/mol MT) and trace levels of Cu. These results indicate that MT-1 is either as susceptible or more susceptible than MT-2 to intracellular degradation depending on age or chemical pretreatment. Furthermore, factors unrelated to the metal composition of MT appear to regulate the degradation of MT-1 and MT-2. PMID- 1733047 TI - Comparative study of the acute lung toxicity of pure cobalt powder and cobalt tungsten carbide mixture in rat. AB - Alveolitis progressing to lung fibrosis has been reported in workers exposed to cobalt containing dust (e.g., tungsten carbide-cobalt mixture as produced by the hard metal industry) but rarely following exposure to pure cobalt dust (e.g., in cobalt-producing factories). We have previously demonstrated that tungsten carbide-cobalt mixture is more toxic toward rat alveolar macrophages in vitro than pure cobalt metal powder. The present study was undertaken to compare in female rats the acute pulmonary response (lung weight, lung histology, cellular and biochemical analyses of bronchoalveolar lavage fluid, and mortality) following the intratracheal instillation of pure cobalt (Co) particles (median particle size, d50:4 microns), pure tungsten carbide (WC) particles (d50:2 microns), tungsten carbide-cobalt (WC-Co) powder (d50:2 microns; cobalt 6.3%, tungsten 84%, carbon 5.4%) and crystalline silica (d50 less than 5 micron) used as pneumotoxic reference material. WC alone (15.67 mg/100 g body wt) behaves as an inert dust producing only a mild accumulation of macrophages in the alveolar duct walls. Co alone (1.0 mg/100 g) only causes a moderate inflammatory response. An identical amount of Co given as WC-Co mixture (16.67 mg/100 g; corresponding to 1.0 mg Co/100 g) produces a severe alveolitis and fatal pulmonary edema. Cellular and biochemical characteristics of bronchoalveolar lavage fluid collected 24 hr after the intratracheal instillation of WC (1.0 mg/100 g) or Co (0.06 mg/100 g) are not significantly different from those of control animals instilled with sterile saline. On the contrary, bronchoalveolar lavage fluid changes following administration of the WC-Co mixture (1.0 mg/100 g; corresponding to 0.06 mg Co/100 g) are very similar to those induced by crystalline silica (1.0 mg/100 g). The amount of cobalt excreted in urine is significantly higher when the animals are exposed to WC-Co powder as compared to an equivalent amount of pure cobalt particles, suggesting an increased bioavailability of cobalt metal when combined with tungsten carbide. This study demonstrates that the acute lung toxicity of tungsten carbide-cobalt mixture is much higher than that of each individual component and may explain why lung fibrosis is rarely if ever induced by exposure to pure cobalt dust. PMID- 1733048 TI - Effect of cadmium chloride on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment cultures--a new model for toxicological investigations of the "blood-testis" barrier in vitro. AB - The effect of various doses (0.75-24 microM) of cadmium chloride (CdCl2) on the development of intercellular tight junctions by immature rat Sertoli cells (Sc) was investigated in vitro using the two-compartment culture system. The status of tight junctions was monitored by repeated measurements of the transepithelial electrical resistance (TER). For defining the specificity of CdCl2 effects, the TER changes were correlated with Sc secretory activity (immunoactive inhibin), the cell number (DNA content), and viability (MTT test). The effects of CdCl2 depended on the concentration of the toxicant as well as on the onset and duration of exposure (4 and 18 hr on Day 1 or 5 of culture). The observed effects could be divided into four categories: (1) At highest doses employed, the TER values decreased significantly and irreversibly during 13 days of culture, and the decrease was accompanied by a significant and irreversible drop in inhibin secretion, cell viability, and cell number. (2) Within a narrow range of doses, the irreversible, or only partially reversible, decrease of TER was accompanied by a transient decrease, or no change, of the secretory activity and no significant changes in Sc cell number and/or viability. (3) With still lower doses, the TER values rapidly decreased and then returned to control level within 3-4 days. In this group, no changes in either inhibin secretion or cell viability were observed. (4) Exposure to the lowest doses of CdCl2 caused a delayed, but significant increase in TER. This increase was not accompanied by noticeable changes in other parameters evaluated. These data suggest that CdCl2 may selectively compromise, at least in vitro, the development and maintenance of the inter-Sc tight junctions, without affecting the secretory activity or the cell number and viability. However, increasing cumulative doses of CdCl2 (concentration multiplied by the time of exposure) led to decreased inhibition secretion and cell viability and then, finally, to irreversible cell damage and death. We believe that the experimental model and approach reported in this paper should be very useful for investigating the mechanism of action of known or potential testicular toxicants, particularly those suspected to compromise the integrity of the "blood-testis" barrier. PMID- 1733049 TI - Pulmonary inflammation and epithelial injury in response to acute ozone exposure in the rat. AB - To document the time course of the inflammatory response and epithelial injury in the lung following an acute ozone exposure, rats were exposed to 1.0 ppm ozone for periods between 4 and 24 hr. Some of the exposures were followed by postexposure periods in filtered air for up to 20 hr. Bronchoalveolar lavage fluid (BALF) analysis and electron microscopic morphometry on centriacinar regions of lungs fixed by intravascular perfusion were used to assess the degree of pulmonary inflammation and epithelial cell necrosis. Total protein and numbers of neutrophils and epithelial cells in BALF increased as the duration of ozone exposure increased, while BALF macrophages decreased. Quantitation of the neutrophil response in centriacinar lung regions (capillary, interstitial, and epithelial/luminal compartments of the terminal bronchiole and proximal alveolar duct) by morphometry generally correlated with the BALF analysis, and revealed a greater volume per surface area epithelial basal lamina (Vs) of neutrophils in the terminal bronchiole compartments compared to proximal alveoli. Necrosis of epithelial cells in terminal bronchioles, primarily ciliated cells, occurred as early as 4 hr after initiation of ozone exposure, before marked neutrophil migration, and continued during periods of maximal neutrophil influx. We concluded that the early epithelial necrosis in terminal bronchioles during the first few hours of ozone exposure was primarily due to direct ozone toxicity, but could not rule out the possibility of neutrophils contributing to the injury at later time points, especially between 8 and 12 hr of exposure (during periods of maximal neutrophil migration). PMID- 1733050 TI - Concentration-response relationships of rat lungs to exposure to oxidant air pollutants: a critical test of Haber's Law for ozone and nitrogen dioxide. AB - Exposure protocols were designed to ask whether lung damage in rats exposed to either ozone or nitrogen dioxide is proportional to dose rate or to cumulative dose. Thus, the response of rats to a constant product of concentration of oxidant air pollutant and time of exposure (C x T) was evaluated for 3-day exposures over a fourfold range of concentrations of ozone (0.2-0.8 ppm) or of nitrogen dioxide (3.6-14.4 ppm) for exposure durations of 6-24 hr per day. The response of rat lungs was quantified by changes in total protein content of lung lavage supernatants or by changes in content of specific cell types in lung lavage pellets. The results of these experiments clearly demonstrate that acute lung damage is a function of cumulative dose (that is, C x T product) for the three highest dose rates tested. However, when exposure duration is extended to include the entire 24-hr period (the lowest dose rate tested), there is a marked attenuation of pulmonary response. Rats were also exposed to mixtures of ozone and nitrogen dioxide with the C x T product held constant. Our results clearly demonstrate that when rats are exposed to combinations of ozone and nitrogen dioxide, lung damage is a function of peak concentration rather than a function of cumulative dose. This deviation from Haber's Law is attributed to a concentration-dependent, synergistic (greater than additive) response to this specific mixture of oxidant air pollutants. PMID- 1733051 TI - Further examination of the selective toxicity of CCl4 in rat liver slices. AB - Lipid peroxidation and loss of enzymes located predominantly in either periportal or centrilobular hepatocytes were investigated in precision-cut liver slices from male Sprague-Dawley rats. Pretreatment of animals with 80 mg/kg phenobarbital for the site-specific enzyme studies enhanced and accelerated CCl4 toxicity in slices resulting from increased radical formation. Liver slices were exposed to 0.57 mM CCl4 by vaporization using a roller incubation system at 37 degrees C for a total of 9 hr. Conjugated diene formation, an index of lipid peroxidation, was detected 15 min following CCl4 administration and increased over time. Loss of cytochrome P450 occurred in a time-dependent manner relative to controls where levels in treated slices were 42% of controls at 9 hr. A 48-hr fast prior to termination increased intracellular K+ leakage relative to that present in slices from fed animals. Significant leakage of glucose-6-phosphate dehydrogenase and beta glucuronidase from centrilobular hepatocytes occurred 9 hr following CCl4 administration. The content of the periportal enzymes (lactate dehydrogenase and sorbitol dehydrogenase) was unchanged in the same slices over the duration of the experiment. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a mitochondrial selective dye and indicator of viability, was significantly lower in treated slices from phenobarbital-treated animals at 9 hr relative to controls. These studies demonstrate that precision-cut slices are an ideal in vitro system for mechanistic studies and the investigation of site specific toxicants since the integral architecture of the liver and cellular identity are maintained. PMID- 1733052 TI - 2-Methoxyethanol metabolism in pregnant CD-1 mice and embryos. AB - Upon oxidation to 2-methoxyacetic acid (2-MAA), 2-methoxyethanol (2-ME) causes malformations in all animal species that have been examined. Commonly, 2-MAA is thought to be the proximate toxicant. However, our previous studies with [1,2 14C]2-ME and the present data obtained with [1-14C]2-MAA, [2-14C]2-ME and [methoxy-14C]2-ME revealed that metabolism beyond 2-MAA occurs. Regardless of the 14C position, dams exhaled approximately 5% of the radioactivity administered as a single teratogenic oral dose (3.3 mmol/kg on Gestation Day [gd] 11) as 14CO2. With all isotopic variants urine contained 70-80% of the dose within 24 hr after administration and 13-18% in the next 24 hr. Three labeled products were resolved using HPLC: an unidentified Peak A (12-18% of dose), 2-MAA (approximately 50%), and the glycine conjugate of 2-MAA (approximately 25%). Short-term (4 hr) whole embryo culture on gd 11 with 3 mM 2-MAA and a tracer dose of [1-14C]2-MAA, [2 14C]2-MAA, or [methoxy-14C]2-MAA showed that 14CO2 evolved from the former two substrates, while there was none detectable from the latter. The data indicate that dams metabolized [methoxy-14]2-MAA to 14CO2, while embryos apparently did not. The production of labeled CO2 from [2-14C]2-ME suggests that 2-methoxyacetyl approximately CoA (the precursor for amino acid conjugation with glycine) entered into the tricarboxylic acid (TCA) cycle. This interpretation is supported by the inhibition of 14CO2 evolution elicited by fluoroacetate (0.1 or 1.0 mM) and sodium acetate (5 mM). It is not yet clear whether entry of 2-methoxyacetyl approximately CoA as a "false substrate" in the TCA cycle is of significance for the embryotoxic effects of 2-ME/2MAA. PMID- 1733053 TI - Prophylactic and therapeutic efficacy of memantine against seizures produced by soman in the rat. AB - Male Sprague-Dawley rats injected sc with a single sublethal dose of the organophosphate nerve agent, soman (100 micrograms/kg), had motor limbic seizures within 5-15 min. Pretreatment with a single dose of memantine HCl (MEM, 18 mg/kg, sc), alone or in combination with atropine sulfate (ATS, 16 mg/kg, sc), before soman prevented seizures without sedation or ataxia. Rats appeared normal or demonstrated increased exploratory activity. Excessive salivation, a peripheral manifestation of soman intoxication, was decreased by ATS, but pretreatment with ATS alone did not prevent seizures. After seizure onset, MEM +/- ATS, but not ATS, abolished seizures. Acetylcholinesterase (AChE) activity in several brain regions (cortex, stem, striatum, and hippocampus) was markedly reduced by soman, but not by MEM, ATS, or MEM + ATS. Preadministration of MEM + ATS in vivo significantly protected AChE from inhibition by soman. Memantine reduced inhibition of AChE activity in crude brain homogenates by soman, but not by edrophonium (anionic site inhibitor) or decamethonium (peripheral site inhibitor). Thus, MEM may bind to a different modulatory site, not yet characterized, to protect AChE. When given after onset of soman-induced seizures, treatment with MEM +/- ATS did not reactivate AChE although seizures were controlled, suggesting additional anticonvulsant mechanisms of action. At concentrations (10(-4) to 5 x 10(-4) M) which did not significantly alter the spontaneous firing of action potentials (APs), MEM limited sustained high frequency repetitive firing (SRF) induced by depolarization of spinal cord (mouse and rat) and neocortical (mouse) neurons in monolayer-dissociated cell culture. In the same range of concentrations, ATS both limited SRF and suppressed spontaneous activity, suggesting toxicity. In addition, MEM and ATS reversibly produced use-dependent block of depolarizing responses to acetylcholine (ACh) applied by pressure ejection to spinal cord neurons. Thus, the anticonvulsant efficacy of MEM, with or without ATS, may have resulted from a combination of actions, including protection of AChE from inhibition by soman, limitation of high frequency firing of APs, and blockade of excitatory postsynaptic responses to ACh. PMID- 1733054 TI - Improved survival of primary cadaveric renal allografts in blacks with quadruple immunosuppression. AB - Black recipients of cadaveric kidneys have been shown to have a lower rate of allograft survival than whites. Data were reviewed from 642 primary cadaveric transplants: results in 276 patients (163 white and 113 black) (group 1) who had received triple therapy (azathioprine-CsA-prednisone, 1985-87) were compared with those in 366 patients (180 white and 186 black) (group 2) receiving quadruple immunosuppression (MALG-azathioprine-CsA-prednisone, 1987-90). Blacks in group 2 had better patient (97% vs. 91%, P = 0.03) and graft (77% vs. 55%, P = 0.0002) survival at 1 year than in group 1. There was no difference in these parameters among whites in either group. Racial differences in graft survival noted in group 1 disappeared in group 2. While HLA BDR matching improved in group 2 patients (P = 0.0001), whites received better matched kidneys than blacks in both groups (P = 0.001). HLA matching was associated with improved graft survival only in white recipients of 4 BDR-matched kidneys. In group 1, more blacks than whites had at least one episode of acute rejection (76% vs. 57%, P = 0.001); blacks also lost more grafts to acute and chronic rejection. In group 2, there were no racial differences in the number of rejection episodes or immunologic graft losses. Of 14 potential variables examined by parametric analysis, only quadruple therapy significantly reduced risk of graft loss in blacks. Quadruple immunosuppression improved primary cadaveric renal allograft survival in black recipients, abrogating previously noted racial differences. PMID- 1733055 TI - Serum lipid abnormalities in pediatric liver transplant patients. AB - Little is known about serum lipid abnormalities in pediatric liver transplant recipients. We performed a longitudinal cohort review of 102 outpatient pediatric liver recipients surviving greater than 6 months and immunosuppressed with cyclosporine and prednisone (+/- azathioprine). The median age was 6 years, median months posttransplant 25, and male-to-female ratio 1:1.5. The average cholesterol (mean of individual means) was 177 +/- 45 mg/dl and average triglyceride level 158 +/- 71 mg/dl. The mean percent of cholesterol levels greater than 170 mg/dl and triglyceride levels greater than 140 mg/dl was 47% and 50%, respectively. Age, obesity, sex, and family history of risk factors had no significant effect on cholesterol or triglyceride levels. Bivariate regression analysis showed no meaningful association between cholesterol or triglyceride levels and cyclosporine levels, cyclosporine dose, prednisone dose, or diastolic blood pressure. Triglyceride and cholesterol neither increased nor decreased with time posttransplant. The rate of change of triglyceride or cholesterol could not be predicted by the rate of change of cyclosporine levels (or dose), or prednisone dose. We found no evidence that rises or falls in cholesterol or triglyceride levels coincided with rises or falls in either cyclosporine level or prednisone dose. Cholestasis was significantly associated with increased cholesterol and triglyceride levels (P = 0.05). A multivariate analysis was unable to predict cholesterol or triglyceride levels from three predictors: cyclosporine level, prednisone dose, and liver function. The mean dietary intake of fat and cholesterol was above RDA and exercise patterns were suboptimal in school-aged children. CONCLUSIONS: 50% of children had a mean cholesterol greater than 75th percentile (170 mg/dl); 20% were above the 95th percentile; 56% had a mean triglyceride level greater than 140 mg/dl. By these criteria the majority of pediatric liver transplant patients have lipid abnormalities that may predispose them to atherosclerosis in later life. PMID- 1733056 TI - An approach to ABO-incompatible liver transplantation in children. AB - Survival in ABO-incompatible (ABO-I) liver transplantation has been reported to be between 40% and 60%. Management techniques have included routine immunosuppression as well as prophylactic antilymphoblast globulin, pre- and posttransplant plasmapheresis (PLPH), and splenectomy. Over a 6-year period, 155 orthotopic liver transplants were performed in 139 pediatric patients. Seven children received an ABO-I allograft. In the latter transplants, immunosuppression consisted of triple-drug therapy (cyclosporine A, prednisone, and azathioprine) along with prospective double-volume PLPH for ABO titers (IgA and IgM) greater than or equal to 1:8. Splenectomy was not performed on any patient. One patient was refractory to PLPH and was treated with a hemofiltration system using an immunoadsorption cartridge with synthetic A group antigen. The overall survival for patients receiving ABO-I allografts was 57% (4/7), with a 67% (4/6) graft survival in those patients treated with PLPH. The graft survival for patients treated with prospective PLPH and MALG was 60% (3/5). There was a 60% incidence of rejection in those patients treated with prospective PLPH and these episodes were all mild (steroid bolus only). While ABO-I transplantation is a reasonable option in the emergency setting, further study is necessary before it should be routinely used to increase the general donor organ pool in pediatric liver transplantation. PMID- 1733057 TI - Mizoribine pharmacokinetics and pharmacodynamics in a canine renal allograft model of local immunosuppression. AB - We utilized a canine renal transplant model to estimate the first-pass extraction of mizoribine (MZB) during renal artery infusion and to compare the efficacy and toxicity of continuous intraarterial (i.a.) versus intravenous (i.v.) MZB delivery, with and without a background of oral cyclosporine. Five autotransplanted mongrel dogs with programmable, implantable pump/catheter systems were given MZB by both i.v. bolus (5 mg/kg) and i.a. infusion (5.0 mg/kg/day). Mean +/- SD elimination half-life was 3.02 +/- 0.81 hr, and the transplanted kidney removed as much as 47-59% (mean 56%) of locally infused MZB. With increasing local and systemic MZB delivery in a single autografted dog undergoing both i.a. and i.v. pump/catheter placement, renal extraction decreased from at least 47% (5.0 mg/kg/day) to 33% (7.5 mg/kg/day), finally to 18% (10.0 mg/kg/day). A dose of 3.0 mg/kg/day MZB did not significantly prolong survival of renal allograft recipients over that of untreated controls (median survival time [MST] = 8 days) when administered either locally (MST = 9 days) or systemically (MST = 12 days). All dogs receiving 4.0 mg/kg/day MZB i.a. died from rejection, and a survival advantage was still not realized (MST = 7 days). In contrast, 4.0 mg/kg/day i.v. prolonged survival over controls (MST = 14 days; P = 0.03) but not when directly compared with the i.a. group (P = 0.30), and produced death from severe debility in five of seven animals with significantly higher mean systemic MZB levels (P = 0.02). Four of six dogs receiving 5.0 mg/kg/day MZB i.a. (MST = 14 days) and two of four dogs receiving 5.0 mg/kg/day i.v. (MST = 14 days) died from severe debility, though survival in both groups was prolonged over control values (P = 0.01 and P = 0.05, respectively). Coadministration of a subtherapeutic dose of oral CsA (5 mg/kg/day) significantly prolonged the overall survival of dogs receiving MZB 4.0 mg/kg/day i.a. (MST = 23; P = 0.01) but not i.v. (MST = 11; P = 1.00), so that a significant difference in overall survival between the combined MZB i.a. + CSA and MZB i.v. + CSA groups was now realized in favor of the former (P = 0.04). We conclude that at local doses required to achieve immunosuppression, the transplanted kidney was not able to extract enough MZB to prevent death from systemic toxicity, presumably as a result of saturation of renal elimination mechanisms, so that an overall survival benefit was not realized.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1733058 TI - Preliminary experience with alpha-2b-interferon therapy of viral hepatitis in liver allograft recipients. AB - Currently, alpha interferon is the only recognized therapy for chronic viral hepatitis. As a result of its success in several multicenter trials, the agent was approved recently by the FDA for use in the clinical management of patients with chronic hepatitis C. FDA approval for its use in chronic hepatitis B is anticipated. Based upon this experience in nonimmunosuppressed individuals, the efficacy of alpha interferon therapy in patients who are recipients of liver allografts and are receiving chronic immunosuppression was assessed in a preliminary trial of the agent in 30 patients (13 with HBV, 11 with HCV, and 6 with hepatitis non A, non B, non C). Therapy was initiated at a dose of 3 X 10(6) units three times per week and continued for 6 months. Dose reduction in the amount of the alpha interferon administered was determined by a preestablished protocol. Nine percent of those with HCV and 18% of those with hepatitis non A, non B, non C experienced a full response to alpha interferon therapy. No full responses to alpha interferon therapy. No full responses were seen in those with HBV disease. Partial responses were common in all three groups but were most frequent in those with hepatitis non A, non B, non C and least frequent in those with HCV-related disease. This preliminary experience demonstrates the following: 1. Viral hepatitis following OLTx can be treated with alpha-2b-interferon. 2. The complications of alpha-2b-interferon therapy utilized prior to OLTx can be avoided by giving the therapy following successful OLTx. 3. The high rate of partial responses noted suggests that future studies should utilize either higher doses or longer durations of therapy or both. 4. The response rate was greatest for those having non A, non B, non C hepatitis and least for those with HCV hepatitis. 5. In this small preliminary series, no episodes of liver graft rejection could be ascribed to the use of alpha-2b-interferon in the patients so treated. PMID- 1733059 TI - The signal-averaged electrocardiogram in cardiac transplantation. A noninvasive marker of acute allograft rejection. AB - Cardiac allograft rejection represents a major cause of morbidity and mortality in transplanted patients. Noninvasive markers of rejection have been sought, though transvenous endomyocardial biopsy remains the "gold standard" for the diagnosis of rejection. Sixty-one signal-averaged electrocardiograms (five in patients with rejection and 56 in patients without rejection) were obtained on 41 patients and prospectively analyzed in frequency domain via fast Fourier transform (FFT). Patients with acute allograft rejection demonstrate a significant increase in the high-frequency components of the QRS complex upon FFT analysis (QRS area ratio 203 +/- 57 vs. 66 +/- 10, P = 0.0007) compared with patients without rejection. Thus, frequency domain analysis may be a useful noninvasive marker of acute cardiac allograft rejection. PMID- 1733060 TI - The impact of donor and recipient hepatitis B surface antigen status on liver disease and survival in renal transplant recipients. AB - Ninety-eight renal transplant recipients who had been followed for 1-6.5 years (median 3.2 years) were reviewed to determine the effect of donor and recipient hepatitis B surface antigen status on the incidence of hepatitis and on patient survival. The cumulative risk of developing hepatitis posttransplant was significantly higher in hepatitis B surface antigen-positive versus hepatitis B surface antigen-negative patients (P = 0.001). Nine (60%) of 15 patients who were hepatitis B surface antigen-positive prior to transplantation, 3 (75%) of 4 patients who became hepatitis B surface antigen-positive after transplantation, and 17 (22%) of 79 patients who were persistently hepatitis B surface antigen negative developed hepatitis posttransplant. Five hepatitis B surface antigen negative patients received allografts from hepatitis B surface antigen-positive donors. None of the five, including one who was initially seronegative, became hepatitis B surface antigen-positive posttransplant. Of the four patients who became HBsAg-positive posttransplant, three received kidneys from donors of unknown HBsAg status in China, while one was transplanted with a kidney from a HBsAg-negative donor. In summary, we found that the risk of developing hepatitis after renal transplantation was significantly higher in hepatitis B surface antigen-positive patients. However, both patient and graft survival were similar in hepatitis B surface antigen-positive and hepatitis B surface antigen-negative patients. The transplantation of kidneys from HBsAg-positive donors to HBsAg negative patients did not result in clinically significant hepatitis or chronic HBsAg carriage. De novo hepatitis B infection may arise from sources other than the kidney itself. PMID- 1733061 TI - Disease recurrence and rejection following liver transplantation for autoimmune chronic active liver disease. AB - Autoimmune chronic active liver disease (ACALD), a major indication for liver transplantation, is associated strongly with antigenic determinants HLA-B8 and DR3. A retrospective analysis of 43 patients who underwent OLTx for putative ACALD and who, as well as their tissue organ donors, were typed, was performed. Disease recurrence and graft rejection episodes were determined by chart review and histopathological review of all material available. Disease recurrence was histologically documented in 11 (25.6%) of these 43 cases. Graft rejection episodes occurred in 24 (55.8%). All recurrences were in recipients of HLA-DR3 negative grafts. Nine of the recurrences were in HLA-DR3-positive recipients (odds ratio: 6.14, P less than 0.03). Two of 11 cases of disease recurrence were in recipients who were HLA-DR3-negative. Nine of these 11 had received HLA-DR3 negative grafts. Rejection occurred in 13 HLA-B8-positive recipients, 12 of whom received HLA-B8-negative grafts. Eleven HLA-B8-negative recipients experienced at least one rejection episode and 9 of these had received HLA-B8-negative grafts. Based upon these data we conclude: 1) that recurrence of putative ACALD is more likely to occur in HLA-DR3-positive recipients of HLA-DR3-negative grafts; (2) that recurrences were not seen in recipients of HLA-DR3-positive grafts; (3) that HLA-B8 status does not affect disease recurrence; and (4) that neither the HLA-B8 nor the DR3 status of the graft or recipient has an effect on the observed frequency of rejection. PMID- 1733062 TI - Injection of xenogeneic endocrine pancreatic tissue into the portal vein--effects on coagulation, liver function, and hepatic hemodynamics. A study in the pig-to dog model. AB - The safety of injecting discordant xenogeneic fetal endocrine pancreatic tissue into the portal vein was studied in a pig-to-dog system. It was found that minced fetal porcine pancreas and fetal porcine isletlike cell clusters prepared by collagenase digestion and culture could be injected with only minor or no hepatic hemodynamic disturbances. Coagulation studies revealed a small increase in plasma fibrinopeptide A, but this increase could be prevented by heparinization of the recipient. There was no consumption of fibrinogen or platelets. In contrast, injection of minced adult porcine pancreas caused pronounced hepatic hemodynamic changes and marked coagulation abnormalities, indicating consumption coagulopathy. The present finding that fetal porcine pancreas can be injected intraportally without deleterious effects in dogs provides a foundation for the eventual clinical use of such material as treatment for insulin-dependent diabetes mellitus. PMID- 1733063 TI - Renal allograft scintigraphy with Tc-99M-DTPA--its role during cyclosporine therapy. AB - There are no accurate noninvasive methods to distinguish renal allograft dysfunction due to rejection or cyclosporine nephrotoxicity. We have studied the value of Tc-99M-DTPA renal scanning in 90 episodes of renal allograft dysfunction occurring in 44 patients subjected to 57 renal biopsies in whom a clear diagnosis could be established. Renal scintigrams were assessed qualitatively and quantitatively by a blinded observer. Rejection was diagnosed when deterioration in perfusion occurred in the presence of maintained or declining radionuclide excretion. The diagnosis of cyclosporine nephrotoxicity was made by exclusion. The final diagnosis was based on the clinical response to therapy and/or the findings on renal biopsy. The scintigraphic diagnosis of rejection had a specificity of 87.9% and significantly contributed to the exclusion of cyclosporine nephrotoxicity (negative predictive value of 90.6%). Furthermore, a scintigraphic diagnosis compatible with cyclosporine nephrotoxicity, in the presence of a drug level above the therapeutic range, indicated a 90.4% probability of true nephrotoxicity. We conclude that, even in cyclosporine treated renal transplant patients, Tc-99M-DTPA scintigraphy is of clinical value and can be incorporated into an effective diagnostic algorithm for allograft dysfunction. PMID- 1733064 TI - Cytokine and T cell receptor gene expression at the site of allograft rejection. AB - Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression. PMID- 1733065 TI - Macrophage subpopulations in normal and transplanted heart and kidney tissues in the rat. AB - The purpose of the present study was to investigate the phenotype of macrophages that infiltrate normal and transplanted rat tissues. The macrophage monoclonal antibodies ED1, ED2, ED3, 52-1D4, ER15, and OX43, together with antibodies against lymphocyte and class II MHC antigens, were used in an indirect immunofluorescence technique with sections of normal tissues and heart and renal grafts that experienced long-term survival or rejection. A small number of ED1- and ED3-positive interstitial cells were detected in normal heart and renal tissues and their number increased dramatically in rejection. Normal heart tissue contained a population of ED2-positive cells with dendritic morphology that was not detected in renal tissue. Following transplantation, a diffuse increase of rounded ED2-positive cells was observed in heart grafts; no ED2-positive cells were detected in grafts removed after 20-30 days from nonimmunosuppressed recipients. Grafts from CsA-treated animals or grafts that survived greater than 50 days in nonimmunosuppressed recipients exhibited the interstitial dendritic pattern of ED2-positive cells. Only very few rounded ED2-positive cells were observed in renal allografts; if present, they were mostly located in the medulla. OX43, which bound in normal tissues to vessel endothelium and a population of macrophages, stained in allografts an additional small population of graft-infiltrating cells, and in F344 renal allografts a population of multinucleated giant cells. We conclude that the posttransplant macrophage infiltration pattern of heart and renal allografts, defined by the monocyte/macrophage antibodies ED1, ED3, 52-1D4, and ER15, is very similar for both types of organs, although the antibody ED2 and the endothelial-macrophage antibody OX43 revealed remarkable differences between the two types of organ allografts. PMID- 1733066 TI - Down-regulation of the immune response to histocompatibility antigens and prevention of sensitization by skin allografts by orally administered alloantigen. AB - The effects of oral administration of major histocompatibility antigens on the alloimmune response have not been investigated. Lymphocytes from inbred LEW (RT1u) rats that were pre-fed allogeneic WF (RT1l) splenocytes exhibited significant antigen specific reduction of the mixed lymphocyte response in vitro and delayed-type hypersensitivity response in vivo, when compared with unfed controls. In an accelerated allograft rejection model, LEW rats were presensitized with BN (RT1n) skin allografts 7 days before challenging them with (LEW x BN)F1 or BN vascularized cardiac allografts. While sensitized control animals hyperacutely reject their cardiac allografts within 2 days, animals prefed with BN splenocytes maintained cardiac allograft survival to 7 days, a time similar to that observed in unsensitized control recipients. This phenomenon was antigen-specific, as third-party WF grafts were rejected within 2 days. Immunohistologic examination of cardiac allografts harvested on day 2 from the fed animals had markedly reduced deposition of IgG, IgM, C3, and fibrin. In addition, there were significantly fewer cellular infiltrates of total white blood cells, neutrophils, macrophages, T cells, IL-2 receptor-positive T cells, and mononuclear cells with positive staining for the activation cytokines IL-2 and IFN-g. On day 6 posttransplant, the grafts from fed animals showed immunohistologic changes typical of acute cellular rejection usually seen in unsensitized rejecting controls. Feeding allogeneic splenocytes prevents sensitization by skin grafts and transforms accelerated rejection of vascularized cardiac allografts to an acute form typical of unsensitized recipients. Oral administration of alloantigen provides a novel approach to down-regulate the specific systemic alloimmune response against histocompatibility antigens. PMID- 1733067 TI - Improved immunosuppression with aerosolized cyclosporine in experimental pulmonary transplantation. AB - Rejection remains a major obstacle to long-term success of pulmonary transplantation. Direct delivery of cyclosporine to lung allografts may produce better control of rejection by generating high intragraft concentrations of drug with decreased systemic delivery and toxicity. The efficacy of inhaled cyclosporine in preventing allograft rejection was compared with systemic delivery by intramuscular injections in a rat model of lung transplantation (Brown-Norway to Lewis). Group 1 animals were given no immunosuppression. Group 2 received a single i.m. injection of 25 mg/kg CsA on the day of operation while group 3 received daily doses on postoperative days 0-3. Groups 4-7 received aerosolized CsA daily for seven days. The aerosol generator produced an airborne concentration of CsA of 180 mg/m3 with a mean particle size of 0.7 mu and estimated pulmonary depositions of CsA of 0.98-3.6 mg/kg/day. Animals were killed on POD 7, and the transplanted lungs graded histologically in a blinded fashion. All control animals showed destructive grade 4 changes by POD 7. Animals receiving high-dose aerosolized CsA (groups 6 and 7) showed minimal changes with a mean rejection grade of 1.3. A single i.m. dose of CsA (group 2) failed to prevent rejection; the mean grade was 2.2. Animals given four i.m. doses of CsA had a mean grade of 1.8. Aerosolized CsA provided significantly better control of rejection than did systemic CsA (groups 6 and 7 vs. groups 2 and 3; P less than 0.0002 and less than 0.0054, respectively). Local delivery of CsA by aerosol inhalation is effective in limiting acute rejection of the rat lung allograft. PMID- 1733068 TI - Transplantation--who delivers the care in what setting? PMID- 1733069 TI - Renal transplantation from a donor over 60 years old. PMID- 1733070 TI - Improvement of early renal allograft function with initiation of ALG pretransplant. PMID- 1733071 TI - Coronary artery imaging with intravascular ultrasound in patients following cardiac transplantation. PMID- 1733072 TI - The relationship between cadaver donor interleukin 6 levels and delayed graft function in kidney transplantation. PMID- 1733073 TI - Treatment of hyperlipidemia in renal transplant patients with gemfibrozil and dietary modification. PMID- 1733074 TI - A positive T cell crossmatch and accelerated acute rejection of a pancreas-spleen allograft. PMID- 1733075 TI - Recovery of liver graft after initial poor function. PMID- 1733077 TI - Discordant lung xenograft rejection in the rat. PMID- 1733076 TI - Extrahilar mesenterico-left portal shunt to relieve extrahepatic portal hypertension after partial liver transplant. PMID- 1733078 TI - Infiltration of perforin-positive mononuclear cells into the rejected kidney allograft. PMID- 1733079 TI - Human islet function after automatic isolation and bovine serum albumin gradient purification. PMID- 1733080 TI - Characterization of immunoreactive trypsin as a means of differentiating graft pancreatitis and allograft rejection after porcine pancreatic transplantation. AB - Graft pancreatitis and allograft rejection were both accompanied by increased serum levels of immunoreactive anionic trypsin (irAT) in a porcine pancreatic allograft transplantation model. Characterization of this immunoreactivity by gel filtration revealed different elution profiles in these conditions that can be helpful in the differentiation between them. During graft pancreatitis, a major part of the immunoreactivity was found within the high-molecular-weight fraction corresponding to the formation of complexes between trypsin and protease inhibitors. During allograft rejection, virtually all serum irAT increase could be attributed to the release of anionic trypsinogen without any evidence of activation. Since this transplantation model includes urinary diversion of the exocrine secretions, irAT and immunoreactive cationic trypsin (irCT) can also be measured in the urine. Characterization of this immunoreactivity showed that most of both irAT and irCT was found as active trypsin but a minor part was probably complexed with some protease inhibitor (possibly pancreatic secretory trypsin inhibitor [PSTI]). PMID- 1733081 TI - Prolongation of xenograft survival after combination therapy with 15 deoxyspergualin and total-lymphoid irradiation in the hamster-to-rat cardiac xenograft model. AB - Adequate immunosuppression remains a major obstacle to successful xenotransplantation, with early xenograft rejection appearing to be mediated by humoral factors. Total-lymphoid irradiation (TLI) and 15-deoxyspergualin (DOSP) have been shown to be effective immunosuppressive agents in allografs. In this study, TLI alone and in combination with DOSP and cyclosporine were evaluated in the hamster-to-rat heterotopic cardiac xenograft model. The animals were divided into four groups: group 1--control (n = 9); group 2--TLI alone, administered pretransplant at 125 cGy/day, four days per week, for three weeks (n = 12); group 3--TLI plus CsA at 10 mg/kg/day (n = 17); and group 4--TLI plus DOSP at 2.5 mg/kg/day (n = 10). Tissue sections were taken from rejected xenografts in all treatment groups for histological examination. Complement-dependent cytotoxicity assays were performed on the control group and also the TLI-DOSP group. The control animals were found to have a mean graft survival of 3.2 +/- 0.4 days. TLI alone (5.8 +/- 0.7 days) did not significantly improve graft survival in comparison with the control group. Combination of TLI with DOSP (26.3 +/- 5.9 days) results in significantly improved survival (P less than 0.05) in comparison with the control, TLI alone, and combination of TLI and CsA (13.6 +/- 8.6 days). Complement-dependent cytotoxicity assays revealed that control groups have low rat antihamster lymphocytotoxic antibody titer (1/1-1/10) prior to xenografting, and that these antibody titers show a precipitous rise to a level of 1/640-1/1280 by day 3, the time at which rejection occurred. This correlates with the histological findings of the rejected hearts showing a severe humoral type of rejection and no evidence of cellular rejection. In contrast, animals in the TLI DOSP group had markedly lowered rat antihamster lymphocytotoxic antibody titers (1/20-1/40) on day 3, and these titers only increased to 1/160 at time of rejection. This correlates with the histological findings of a lesser degree of humoral rejection in the TLI-DOSP group. Combination therapy with TLI and DOSP results in a marked increase of survival in xenografts in this model not seen with any other drug combination studied in over 500 xenografts in our laboratory. This study indicates that TLI combined with DOSP results in prolonged suppression of the antixenograft antibody response. This combination of agents appears to have the potential to prevent early xenograft rejection. PMID- 1733082 TI - A multicenter study to evaluate a novel assay for quantitation of soluble interleukin 2 receptor in renal transplant recipients. AB - The clinical utility of monitoring soluble interleukin 2 receptor (sIL-2R) as an indicator of immune stimulation in renal transplant patients was evaluated in a retrospective study at 3 centers. Serum samples (n = 2360) were obtained from 86 (17 living related donor, 69 cadaver) transplant recipients. The patients had received either triple therapy (n = 35) or antilymphocyte antibody induction therapy followed by triple therapy (n = 51). The mean period of postoperative observation was 118 days (range, 6-349 days). Serum sIL-2R concentrations were quantitated by an automated microparticle enzyme immunoassay (MEIA) (Abbott Diagnostics) in which sIL-2R was captured by 7G7/B6 monoclonal antibody-coated microparticles and detected by an immunospecific rabbit antihuman sIL-2R-alkaline phosphatase conjugate. A distinct advantage of the technique was rapid turn around time: 1-24 results were obtained in less than 50 min. Cyclosporine trough concentrations were determined by radioimmunoassay or high-performance liquid chromatography. Diagnosis of rejection was established by clinical and histological criteria. The mean sIL-2R concentration in patients receiving antilymphocyte antibody induction therapy increased from 3486 +/- 1729 U/ml (+/- SD) at the time of transplant to a maximum of 7395 +/- 7101 U/ml on the third day posttransplant; this increase was not observed in patients receiving triple therapy (P less than 0.0001). By the sixth day of posttransplant, there were no differences in sIL-2R levels in the two groups. Fifty rejection episodes were observed in 29 patients on triple therapy. The mean sIL-2R concentration rose from 3022 U/ml at the data point prior to rejection to 3524 U/ml at the time of rejection. Thirty-four rejection episodes were observed in 26 patients receiving induction therapy. The mean sIL-2R concentration was 3015 U/ml at the data point prior to rejection and 4815 U/ml at the time of rejection. The sIL-2R concentrations began increasing earlier and rose higher in rejecting patients who received induction therapy than in those receiving triple therapy. Early posttransplant sIL-2R levels increased significantly more in cadaver recipients than in LRD recipients, reaching a maximum on day 2 posttransplant (P less than 0.001). Prerejection sIL-2R concentrations were significantly lower in LRD recipients than in cadaver recipients (2248 U/ml vs. 4290 U/ml, P less than 0.02), as were sIL-2R levels at the time of diagnosis of rejection (2800 U/ml vs. 4832 U/ml, P = 0.01). The mean sIL-2R level in stable long-term graft recipients was 2110 U/ml, with approximately 90% of values less than 3000 U/ml.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1733083 TI - Withdrawal of steroids after renal transplantation--clinical predictors of outcome. AB - Withdrawal of steroid therapy in renal transplant recipients is associated with a risk of acute allograft rejection. To define clinical risk factors for rejection associated with steroid withdrawal, we analyzed the clinical characteristics of 107 patients with drawn from steroid therapy at various times after transplantation. Both univariate and multivariate analyses suggested that the timing of steroid withdrawal is an important predictor of steroid withdrawal failure. Withdrawal of steroids was successful in only 13 of 32 patients (41%) in whom prednisone was discontinued shortly after transplantation. In contrast, steroid withdrawal has been successful in 59 of 75 patients (79%) in whom prednisone was discontinued at least 6 months after transplantation. Black race and donor-recipient racial mismatch also were significant predictors of rejection associated with steroid withdrawal. In patients undergoing steroid withdrawal at least 6 months posttransplant, serum creatinine concentration also correlated independently with the risk of rejection. Neither age, sex, HLA match, pretransplant PRA, source of the allograft (cadaver vs. living relative), acute tubular necrosis, nor the presence of diabetes was predictive of the outcome of steroid withdrawal. PMID- 1733084 TI - Pediatric renal transplants--results with sequential immunosuppression. AB - Cyclosporine has improved the results of renal transplantation. In 1984, we began using it as part of a sequential immunosuppression protocol (MALG, AZA, P, and delayed administration of CsA) in our pediatric renal transplant recipients. We studied the outcome of the 131 pediatric renal transplants (less than or equal to 18 years of age at transplant) performed at our institution between June 1984 and March 1991. We compared these results with the 144 similar transplants performed since January 1980 that did not involve CsA immunosuppression. In the sequential immunosuppression group, there were 97 primary (74%) (26 [27%] cadaver, 71 [73%] living donor [LD]) and 34 (26%) retransplant (23 [68%] CAD, 11 [32%]) recipients. Age at transplant (mean +/- SD) was 7.4 +/- 5.5. Overall, 1-year actuarial graft survival was 93%; 1-year patient survival was 100%. The mean number of hospital readmissions was 3.0 +/- 3.5; 26 (20%) were readmission-free. The mean number of rejection episodes was .87 +/- 1.3 per patient; 73 (56%) were rejection-free. Importantly, LD (vs. CAD) recipients had fewer rejection episodes (P = 0.06). In the first post-transplant year, the serum creatinine level was significantly lower in primary (vs. retransplant) recipients and in LD (vs. CAD) recipients (P less than 0.05). In the 144 patients not receiving CsA, there were 129 (90%) primary (27 CAD, 102 LD) and 15 (10%) retransplant (7 CAD, 8 LD) recipients. Age at transplant was 6.9 +/- 5.3 years. The 1-year actuarial graft survival rate was 82%; the 1-year patient survival rate was 94%. The mean number of hospital readmissions was 3.3 +/- 2.3; 5 (8%) were readmission-free. The mean number of rejection episodes was 1.2 +/- 1.5; 27 (45%) were rejection-free. There was no difference in the serum creatinine level based on donor source or transplant number. Sequential immunosuppression has significantly improved patient (P = 0.003) and graft survival (P = 0.004) rates. Comparing sequential vs. non-CsA immunosuppression, there was no difference in the number of readmissions (P = 0.47), number of rejection episodes (P = 0.17), or serum creatinine level. The number of rejection-free patients was significantly lower in LD (vs. CAD) recipients (P less than 0.05). There was no evidence of progressive deterioration in renal function in the sequential (vs. non-CsA) recipients. PMID- 1733085 TI - The influence of nephrectomy of the primary allograft on retransplant graft outcome in the cyclosporine era. AB - The present analysis was undertaken to evaluate the influence of primary allograft nephrectomies on the early function, incidence of rejection, and short term graft survival of subsequent renal retransplants. Among 95 consecutive cyclosporine treated retransplant recipients, 52 were retransplanted without primary allograft nephrectomy; 35 had removal of their primary grafts prior to retransplantation for fever and graft tenderness (30 patients) and persistent hematuria (5 patients); and 8 patients had an elective primary graft nephrectomy at the time of retransplantation. Demographic characteristics and immunosuppressive regimens were otherwise similar in all three groups. Nephrectomy of the primary allograft prior to retransplantation was associated with a significant subsequent rise in preformed cytotoxic antibody levels (57% having PRA greater than 30% compared with 33% in those with retention of primary grafts), a significantly higher incidence of delayed graft function among retransplants (63% compared with 30% in those who did not undergo primary allograft nephrectomy) and a trend toward decreased allograft survival in the subgroup who lost their primary allografts in the first year posttransplant. The incidence of acute rejection and 3-year posttransplant renal function in retransplants were not, however, influenced by nephrectomy of the primary allograft. PMID- 1733086 TI - The effect of donor age, recipient age, and HLA match on immunologic graft survival in cadaver renal transplant recipients. AB - We retrospectively analyzed 526 primary cadaver recipients transplanted at a single center to identify pretransplant variables that predict long-term survival with multivariate analysis. All recipients received at least three random blood transfusions and were treated under a quadruple-therapy protocol consisting of ALG, azathioprine, prednisone, and cyclosporine. Of 526 consecutive transplants, 86 grafts were lost from acute or chronic rejection. Thirteen grafts were lost for nonimmunologic reasons and 35 recipients died with a functioning graft. A total of 273 patients (52%) experienced at least one episode of acute rejection. Donor age ranged from 3 to 64 years, with 62% of donors less than 30 years of age and 9% of donors over 50 years of age. Donor age was not predictive of long-term graft survival and neither was the difference between donor and recipient age. Recipient age was predictive of subsequent immunologic graft less, with younger recipients at greater risk (P = 0.011). The rate of first rejection was also inversely related to recipient age, with younger recipients rejecting earlier (P = 0.0001). The degree of DR mismatch was the only other significant predictor of long-term graft success (P = 0.013). Transplant survival correlated with the degree of DR mismatch: 2 DR mismatch was the worst, 1 DR mismatch was intermediate and 0 DR mismatch was the best (P = 0.02). A, B, AB, and BDR did not influence long-term graft outcome. In our center, donor age does not predict graft failure. Younger recipients have a higher rate of early rejection and, combined with a poor DR match, are at higher risk for long-term graft failure. PMID- 1733087 TI - Analyses of the UNOS Scientific Renal Transplant Registry at three years--early events affecting transplant success. AB - The success of cadaveric renal transplants in the first year is determined largely by events that transpire during the transplant hospitalization. This conclusion is based upon analyses of data on 19,525 cadaver donor renal transplants performed since October 1987 and reported to the UNOS Scientific Renal Transplant Registry from more than 200 centers nationwide. Graft survival rates at 1 year differed by 20-30% depending upon whether or not the transplanted kidney functioned immediately and upon whether the patient required dialysis during the first week posttransplant, experienced rejection, or was discharged with a kidney that was functioning well. Recipients whose discharge serum creatinine level was less than 2.6 mg/dl or whose graft was functioning well at the time of discharge had 88% 1-year graft survival. A multistep logistic regression analysis showed cold ischemia time, transfusions, donor age and cause of death, HLA-DR mismatches, and peak sensitization to be significant factors in the first week. Prophylactic antilymphocyte antibodies (ALG/OKT3) reduced the incidence of rejection from 30% to 20% during the transplant hospitalization, but apparently only delayed rejection. By 6 months there was only a 3% reduction with ALG and a 5% reduction with OKT3 in the incidence of reported rejection and a 2% difference in 1-year graft survival. Although graft and patient survival are important measures of transplant success, graft survival is predicted upon both early and late events. The course of the transplant during the initial hospitalization and the quality of function at discharge were the strongest determinants of 1-year graft survival. PMID- 1733088 TI - Influence of race on crossmatch outcome and recipient eligibility for transplantation. AB - Data from the Office of the Inspector General and the United Network for Organ Sharing suggested that black end-stage renal disease patients had unequal access to organ transplantation, in that they waited twice as long as white ESRD patients for a renal transplant. We hypothesized that preTx histocompatibility factors were more influential in determining how quickly an ESRD patient was transplanted than had been realized by either the Office of the Inspector General or UNOS. To test this hypothesis we compared the crossmatch reactivity of 378 white and 227 black ESRD patients awaiting a primary renal Tx against 100 consecutive cadaveric donors (80 white, 10 black, and 10 Mexican-American). A positive XM frequency of 16% (179 positive XM/1121 total XM) was observed when white ESRD patients were crossmatched against white donors. A comparable frequency of 21% (39/186) (+) XM was observed when white ESRD patients were crossmatched against black donors. Similarly, black ESRD patients crossmatched against black donors showed a 17% (20/115) (+) XM frequency. In contrast, a significantly higher (+) XM frequency of 43% (283/655) was observed when black ESRD patients were crossmatched against white donors (P less than 0.001). When black ESRD patients with a (+) preTx blood transfusion history were crossmatched against white donors, a 55% (241/438) (+) XM frequency was observed. This (+) XM frequency was 3.2 times greater than when black ESRD patients were matched against black donors (P less than 0.001). However, when untransfused black ESRD patients were matched against white donors only a 19% (42/217) (+) XM frequency was observed. Finally, preoperatively transfused black ESRD patients displayed a higher PRA of 47 +/- 12% vs. 27 +/- 10%, P less than 0.01, and a longer median waiting time to transplant of 329 vs. 181 days, P less than 0.01, than comparable white patients. The data suggest that presentization of blacks, following preTx blood transfusions from nonblack donors, predisposes black ESRD patients to present as immunologically inappropriate recipients for predominantly white donor allografts and results in their spending a longer time on renal transplant waiting lists. PMID- 1733089 TI - The strong correlation of cytotoxic T lymphocyte-specific serine protease gene transcripts with renal allograft rejection. AB - Several immune mechanisms are likely to be responsible for renal allograft rejection. The relative importance of delayed-type hypersensitivity versus cytotoxic T lymphocytes is controversial. We analyzed human renal allografts biopsies for intragraft expression of IL-1 beta, IL-6, and TNF alpha genes- putative mediators of DTH--as well as IL-2, IL-2 receptor (R) beta, and a CTL specific serine protease gene. Total RNA was extracted from tissue samples and the mRNA fraction was converted to cDNA using oligo dT and reverse transcriptase. Then cDNA was amplified by the polymerase chain reaction (PCR) for 35 cycles using specific oligonucleotide primers. Each PCR analysis included beta-actin oligonucleotide primers to coamplify this constitutively expressed gene as an internal control. A total of 24 core allograft biopsies were studied and classified into a 3 histological categories: acute cellular rejection, equivocal components of rejection, and no evidence of rejection. There was no statistically significant difference in beta-actin expression among these histologic categories (P greater than 0.08). Interestingly, in this sample size, no significant difference was found between rejecting and nonrejecting samples for transcripts of any of the cytokines or IL-2R beta mRNAs. Apparently, DTH-like mechanisms are present in all allografts. However, detection of CTL-specific serine protease gene expression was almost exclusive to rejecting samples (P less than 0.003). These findings suggest that activation of CTLs play an active, but hardly exclusive, role as effectors of graft dysfunction in the rejection process. While this study does not define the relative importance of the genes examined, it does suggest that evidence of CTL-specific serine protease expression may provide a means of monitoring for rejection episodes or as a diagnostic aid when conventional diagnostic criteria are not conclusive. PMID- 1733090 TI - Prediction of successful allograft rejection retreatment with OKT3. AB - OKT3 is considered to be effective in the prophylaxis and treatment of rejection. However, anti-OKT3 immunization is still a major side-effect. Only the IgG antiidiotypic component of the response is responsible for neutralization of OKT3 therapeutic activity by inhibiting OKT3's binding to T cells (i.e., blocking antibodies). It has recently been reported that successful OKT3 retreatment could be performed in patients showing circulating anti-OKT3 antibodies assessed by ELISA, which does not distinguish between blocking and nonblocking antibodies. The aim of this study was to investigate the role of these two types of anti-OKT3 antibodies for their capacity to interfere with effective OKT3 retreatment. Twelve cadaver renal allograft recipients who received OKT3 inducing therapy, were given a second 10 day-course of OKT3 for treatment of rejection. Circulating CD3+ cells were sequentially monitored. Anti-OKT3 antibodies were detected by using both conventional ELISA and an immunofluorescence inhibition test specifically detecting blocking antibodies. OKT3 and the patient's serum were incubated for 30 min at 4 degrees C, and 2 x 10(5) normal T cells were added (30 min at 4 degrees C). After washing, the cells were incubated with FITC goat antimouse antiserum. Fluorescent cells were counted using a FACS analyzer. In 10 patients, at the end of the 10-day second course, less than 10% circulating CD3+ cells were detected. None of these patients had detectable antibodies in the IF inhibition assay at the beginning of retreatment, irrespective of anti-OKT3 antibody titers detected by ELISA. In contrast, in two patients, OKT3 therapy was ineffective: more than 50% circulating CD3+ cells were detected and OKT3 treatment had to be interrupted soon after it was initiated. In both of them, blocking antibodies were detected by the IF-inhibition assay. These results suggest that specific detection of blocking antiidiotypic antibodies by the IF inhibition assay is a reliable parameter for predicting the feasibility of OKT3 retreatment, avoiding misuse of this expensive therapy. PMID- 1733091 TI - The effect of indomethacin on the febrile response following OKT3 therapy. AB - Fifty renal transplant recipients with histologically documented acute allograft rejection were treated with OKT3 monoclonal antibody therapy. Group 1 (n = 25) received standard premedication with steroids, acetaminophen, and diphenhydramine. Group 2 (n = 25) received these agents plus indomethacin in an attempt to minimize the early adverse effects associated with OKT3. At 1 hr prior to the first dose of OKT3, 50 mg of indomethacin was administered orally followed by 25 mg every 6 hr for the next 48 hr. Demographics were similar in the two groups. Reversal of rejection occurred in 23 of 25 (92%) in group 1, and in 22 of 25 (88%) in group 2. Graft survival rates at six months after the rejection were 88% in group 1 and 80% in group 2. There was a single patient death in group 2, due to a suicide in a patient with a functioning kidney and pancreas graft. The maximum temperature was significantly diminished in the group receiving indomethacin during the first three days of OKT3 therapy. The percentage of patients with a maximum temperature less than 100 degrees F was significantly higher in group 2: day 1--16% vs. 36%, day 2--12% vs. 48%, day 3--52% vs. 68% for group 1 and group 2, respectively. No serious side effects occurred in either group--however, subjective side effects were less common in group 2. Serum creatinine levels were similar in the two groups prior to rejection, at the start of OKT3 therapy, at the peak during OKT3 therapy, at the end of OKT3 therapy, and 30 days and 180 days post OKT3. The data indicate that the concurrent use of indomethacin with OKT3 appears to significantly decrease the initial febrile response without compromising renal function or the efficacy of OKT3 therapy. PMID- 1733092 TI - The effects of pravastatin on hyperlipidemia in renal transplant recipients. AB - Hyperlipidemia may be one of the risk factors in the development of atherosclerotic disease in renal transplant recipients. In the present study, 24 kidney recipients with hyperlipidemia were treated with an HMG-CoA reductase inhibitor, pravastatin (10 mg/day). All recipients had been treated with cyclosporine (CsA), azathioprine (Az), and prednisolone (Pred). The mean total cholesterol (T-chol) level decreased from 323 +/- 7.4 to 261 +/- 7.9 mg/dl at one month after starting treatment (P less than 0.01) and this level did not change during treatment for further 6 months. The mean LDL cholesterol level was also decreased from 205.9 +/- 11.2 to 118.7 +/- 8.1 mg/dl at 3 months after starting treatment (P less than 0.01). On the other hand, pravastatin did not affect the levels of HDL-cholesterol and triglycerides. Pravastatin did not show any effects on the white blood cell, monocyte, and lymphocyte counts, or the hemoglobin concentration (NS). One patient displayed a slight elevation of aspartate aminotransferase and alanine aminotransferase levels, but this was not sufficient to cease treatment. Pravastatin did not adversely affect the renal function or creatinine phosphokinase (CPK) levels. Two recipients developed nausea and vomiting and their treatment was stopped. Pravastatin appears to be a safe and efficacious method of treating hyperlipidemia in renal transplant recipients. PMID- 1733093 TI - Tobamovirus-plant interactions. AB - It is clear that the genetic information responsible for the phenomenon we think of as TMV not only consists of the genes carried in the viral genome, but that numerous plant genes are equally important in viral gene functions. These gene products not only allow the virus to replicate, but may effect functions of evolution that determine what the virus is. Even the processes of pathogenesis and resistance appear to involve similarly precise plant interactions. The challenge of the future is to identify the plant genes involved in these precise interactions and to understand both components of genetic information that comprise plant viruses. PMID- 1733094 TI - Infectious entry pathway for canine parvovirus. AB - We have investigated whether canine parvovirus (CPV) infection involves a low pH dependent entry pathway. The effects of two lysosomotropic bases, NH4Cl and chloroquine, on CPV entry were studied by immunofluorescence and ultrastructural and biochemical methods. In the presence of these reagents, input virions appear to accumulate in large vacuoles. Ultrastructural studies indicated that uptake of virions takes place predominantly in small uncoated vesicles that appear to fuse with larger vesicles. In the presence of NH4Cl, virions accumulate in the latter structures and their uncoating appears to be prevented. Viral DNA as well as antigen synthesis were found to be significantly inhibited in the presence of these reagents. In addition, inhibition of viral DNA and antigen synthesis appeared to be most extensive when NH4Cl was present from 30 min preinfection, whereas no significant inhibition was observed when the cells were treated after 2 hr postinfection. Thus, the results indicate that CPV requires exposure to low pH in an endosomal compartment to initiate a productive infection. PMID- 1733095 TI - Identification of sequence elements containing signals for replication and encapsidation of the reovirus M1 genome segment. AB - In reovirus the genetic signals that control genome replication and encapsidation are unknown. Serial passage of reovirus results in the accumulation of deletion mutants that contain fragments of genome segments. The smallest fragments found in deletion mutants will consist of the minimum essential sequences for genome replication and assembly. T1 x T3 reassortants containing the L2 segment from T3 and the M3 segment derived from T1 generate deletions in segment M1 on serial passage. Fragments of M1 segments were produced by serial passage, characterized by PAGE and Northern blotting before amplification by PCR, cloning, and sequencing. Thirteen of the smallest deletion fragments were sequenced. All of the smallest fragments contained sequences from both termini of segment M1. The smallest fragment was 344 nucleotides long. The consensus sequences consisted of 132-135 nucleotides from the 5' end of the plus strand and 183-185 nucleotides from the 3' end of the plus strand. It is concluded that these regions contain all the signals necessary for the replication and assembly of the M1 genome segment. PMID- 1733096 TI - Biosynthesis of reovirus-specified polypeptides: ribosome pausing during the translation of reovirus S1 mRNA. AB - The steady-state distribution of translating ribosomes on the reovirus serotype 1 Lang strain polycistronic s1 mRNA and the monocistronic s4 mRNA was mapped using a sensitive primer extension assay and cDNA clones of the S1 and S4 genes. The distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was not uniform. Major positions of ribosome pausing were detected in both rabbit reticulocyte and wheat germ cell-free protein synthesizing systems programmed with wild-type and site-directed mutant mRNAs. Two of the ribosome pause sites represent initiation and termination, rate-limiting steps of translation. For the polycistronic s1 mRNA, analysis of mutants in which either the sigma 1 ORF1 initiation codon or the sigma 1NS ORF2 initiation codon was eliminated by site directed mutagenesis unequivocally established the identity of the specific ribosome pauses with specific ORF translational events. Ribosomes were far less uniformly distributed along the overlapping ORF regions of the polycistronic s1 mRNA than they were along the ORF of the monocistronic s4 mRNA. Furthermore, the rate-limiting initiation event at the sigma 1NS ORF2 AUG led to ribosome stacking and elongation arrest in the sigma 1 ORF1. These results begin to provide a conceptual framework for the dynamics of translation of complex as compared to simple viral mRNAs and suggest that ribosome activity may vary at multiple discrete regions on an mRNA. PMID- 1733097 TI - Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus. AB - Several biochemical properties and the sequence of the fusion glycoprotein (F) have indicated that turkey rhinotracheitis virus (TRTV) is a pneumovirus, subfamily Pneumovirinae of the Paramyxoviridae family. As TRTV was known to generate polycistronic mRNAs, cDNA was generated from TRTV strain UK/3BV/85 infected Vero cell mRNAs using an oligonucleotide primer corresponding to a region of the F gene. Sequencing of four cDNAs revealed that the gene adjacent to the beginning (3' end) of the F gene was that for the matrix (M) protein, i.e., that TRTV had the partial gene order 3'-M-F-5'. This was unexpected as human respiratory syncytial (RS) virus, the type species of the genus Pneumovirus, has the partial gene order 3'-M-SH-G-F-5', where SH and G are the small hydrophobic protein and attachment glycoprotein, respectively. Instead TRTV resembled the Morbillivirus and Paramyxovirus genera of the Paramyxoviridae (subfamily Paramyxovirinae) which have the partial gene order 3'-M-F-5'. Two further oligonucleotides, one corresponding to a sequence near the end of the M gene and the other (oligo B) to a sequence near the beginning of the F gene, with their 5' ends spaced 300 nucleotides apart on the basis of the cDNA sequence, were used in a polymerase chain reaction (PCR) using genomic RNA as template. Only a PCR product of 0.3 kb was obtained. The same sized product was also obtained using these oligonucleotides and genomic RNA from three other TRTV strains (SA/91/78, UK/8544/85, and SA/2381/88) which had been grown in chicken tracheal organ cultures. In addition PCR was performed using genomic RNA from TRTV-3BV and SA/2381/88 with oligo B and another oligonucleotide near the 5' end of the gene upstream from M, spaced 1141 nucleotides apart on the basis of the sequence data. Only a 1.14-kb PCR product was obtained. Larger products would have been expected if another gene had been situated between M and F. The absence of such larger products, plus the demonstration that infected cells contained M-F dicistronic mRNAs, supported the conclusion that in the TRTV genome the M gene is adjacent to the F gene in the order 3'-M-F-5'. The 5' termini of the M and F mRNAs were confirmed by mRNA mapping. The TRTV M gene encoded a protein of 254 amino acids, very similar to that of RS virus (256 residues; 37% amino acid identity) but very different from that of the morbilliviruses and paramyxoviruses (approximately 350 residues).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 1733098 TI - Characterization of RNAs specific to avocado sunblotch viroid synthesized in vitro by a cell-free system from infected avocado leaves. AB - Analysis by molecular hybridization of the RNAs transcribed by a cell-free fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated the presence of newly synthesized viroid-specific sequences, most of which were of the same polarity as the mature infectious viroid RNA. Treatment of the cell free fraction with DNase reduced the total synthesis of RNA considerably, but it did not influence that of the ASBV-specific RNAs, indicating that the latter were transcribed on an RNA template. Inhibition studies with alpha-amanitin showed that the synthesis of ASBV-specific RNAs was not affected by concentrations of 1 and 200 micrograms/ml of the drug, which typically inhibit RNA polymerase II and III, respectively, from most animal and plant systems. These results suggest that either RNA polymerase I or an unidentified RNA polymerase activity resistant to alpha-amanitin, acting on an RNA template, plays a role in the replication of ASBV, whereas for the rest of the viroids studied so far it appears that RNA polymerase II is involved. Analysis by polycrylamide gel electrophoresis under partially and fully denaturing conditions of the ASBV-specific RNAs synthesized in vitro showed that they contain unit and longer than unit length viroid strands, probably associated in complexes with single- and double-stranded regions. The structural properties of these complexes are similar to those of the RNAs accumulating in vivo in viroid-infected tissues, which are the postulated replicative intermediates of the rolling-circle mechanism proposed for viroid synthesis. PMID- 1733100 TI - Intracellular factors, but not virus receptor levels, influence the age-related outcome of DHBV infection of ducks. AB - Previous serological studies of experimental infection with duck hepatitis B virus (DHBV) have shown that the outcome of infection depends largely on the age of the duck at the time of inoculation. To examine the hypothesis that decreased susceptibility with increased age might be due to the loss of the virus receptor on hepatocyte membranes in adult ducks, we performed receptor binding studies using intact serum-derived DHBV virions and purified liver plasma membranes from both young ducklings and adult ducks. These studies showed that (1) DHBV was able to bind specifically to duck liver plasma membranes but not to internal membranes; (2) this binding could be inhibited by a monoclonal antibody to DHBV preS, a corresponding region in hepatitis B virus that binds to human hepatocytes; and (3) there was no significant difference in the receptor binding ability between plasma membranes from ducklings and from adult ducks. Since hepatocytes in the neonatal ducks are actively dividing, in contrast to the situation in adult ducks, we examined the effect of partial hepatectomy on DHBV carrier ducks. A sharp increase was noted in the level of DHBV in the serum after partial hepatectomy suggesting that DHBV replication was enhanced in dividing hepatocytes. Thus the age-related difference in susceptibility of ducks to DHBV infection is not due to loss of the receptor but may be related to an intracellular event associated with cell division. PMID- 1733099 TI - Mapping and molecular characterization of a functional thymidine kinase from Amsacta moorei entomopoxvirus. AB - A thymidine kinase (TK) gene from the entomopoxvirus of Amsacta moorei (AmEPV) has been identified, mapped, cloned, and sequenced. The AmEPV TK was shown to be biologically functional as cloning of the gene into a TK-derivative of the orthopoxvirus vaccinia creates a TK+ virus. The gene has been localized to a 1.5 kb EcoRI-Q DNA fragment which maps to the far left end of the viral genome. Sequence analysis reveals an open reading frame (ORF) of 182 amino acids potentially encoding a polypeptide of 21.2 kDa. Amino acid homology comparisons indicate that the gene is most closely related to the TKs of a variety of poxviruses (approximately 45%) and less so to the TKs of vertebrates (approximately 40%). The TK from African swine fever virus (ASF) showed the least homology (31.4%) to the AmEPV TK gene, suggesting that these two viruses are not closely related although ASF shares some biological features of poxviruses, and both ASF and AmEPV can replicate within arthropod hosts. PMID- 1733101 TI - Temperature-sensitivity of the replication of rabies virus (HEP-flury strain) in BHK-21 cells. I. Alteration of viral RNA synthesis at the elevated temperature. AB - We investigated the nature of temperature sensitivity of the HEP strain of rabies virus. After initial incubation for appropriate period (more than 12 hr) at the permissive temperature (36-37 degrees), incubation temperature of the rabies virus infected cultures was shifted to a nonpermissive temperature (39.5-40.5 degrees). Upon the upshift, virion production was ceased, but the rate of viral RNA synthesis was greatly increased and reached almost 10 times that of 36 degrees-infection within 8-10 hr, and then the activity quickly decreased together with the onset of accelerated CPE. Little or no 42S genome-sized RNA was produced at the elevated temperature, and almost all RNAs produced in large amounts seemed to be viral mRNAs and were shown to be functional in t he cell free translation system. Consistent with these observations, the viral ribonucleoprotein complex isolated from the temperature-upshifted culture was associated with relatively large amounts of small sized RNAs, which might reflect their increased transcriptive activity. These observations suggest that the viral RNA polymerase itself is not temperature-sensitive and the temperature-induced defect may reside in the regulatory factor which plays a role in turning on the synthesis of viral genome-sized RNA. PMID- 1733102 TI - Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27. AB - The herpes simplex virus (HSV) immediate-early protein ICP27 has been postulated to play an auxillary role in HSV. gene expression, augmenting or inhibiting the activation of different viral promoters by the other immediate-early proteins ICPO and ICP4. Here we show that ICP27 alone can up-regulate gene expression of a retroviral vector containing Moloney murine leukemia virus (MoMuLV) regulatory sequences. This is the first report of an effect of ICP27 on gene expression driven by heterologous virus regulatory sequences. The effect does not involve the region of ICP27 which inhibits gene activation of HSV early gene promoters by ICPO and ICP4, but rather is dependent on the same region of ICP27 as is required to augment the activation of HSV late gene promoters by ICPO and ICP4. This indicates that the two activation effects are likely to operate via the same mechanism. Activation by ICP27 is dependent on the 3' LTR and adjacent region of MoMuLV but is independent of the promoter in the 5' LTR which can be replaced by a heterologous promoter such as that of SV40 without affecting activation by ICP27. The significance of this effect for an understanding of the mechanism of action of ICP27 and its role in regulating the gene expression of HSV and potentially of other viruses is discussed. PMID- 1733103 TI - Immunization with tween-ether-treated SIV adsorbed onto aluminum hydroxide protects monkeys against experimental SIV infection. AB - In order to examine the efficiency of an AIDS vaccine potentially acceptable for human use we have investigated a split vaccine. Since such vaccines are safe and efficient, they have been in use for many years to protect man against enveloped RNA viruses, e.g., influenza and measles. Seven rhesus monkeys were immunized at Week 0, 4, 8, and 16 by im injection of 2 ml of vaccine containing 140 micrograms of Tween-ether-disrupted SIVmac251/32H adsorbed onto aluminum hydroxide. The immunized animals and three nonvaccinated control monkeys were challenged 2 weeks after the last immunization by iv injection of 10 to 50 minimal monkey infectious doses of SIVmac251/32H. Four of seven immunized animals did not show any signs of virus replication and therefore appeared to be protected. Nonvaccinated control animals and the vaccine failures showed a rise in their urinary neopterin concentrations 1 to 2 weeks after infection. At the end of the second week and thereafter, cocultures and polymerase chain reaction of their peripheral blood lymphocytes were positive. After the challenge, control animals and infected vaccinees showed a primary or secondary antibody response while antibody titers declined in virus-negative animals. Specific cytotoxic T-lymphocytes were not present prior to challenge, but were present in some animals thereafter. Therefore, these seem to reflect a response to viral replication rather than to immunization. Prior to challenge the CD4-positive lymphocytes of the peripheral blood of the four virus-negative animals only proliferated after exposure to the immunizing antigen. Thus, this reaction appears to predict protection. PMID- 1733104 TI - DNA sequence analysis of the gene encoding the HTLV-I p21e transmembrane protein reveals inter- and intraisolate genetic heterogeneity. AB - DNA sequence analysis was performed on a 235-bp region of the p21 e transmembrane protein gene of the human T-cell lymphoma/leukemia virus type I (HTLV-I) which encompassess the putative immunosuppressive peptide. Polymerase chain reaction based amplification was used to generate multiple molecular clones from isolates derived from fresh or cultured cells from 19 individuals. A dendrogram was constructed using the p21e DNA sequence information to compare the sequences among isolates in the current study and other previously published HTLV-I isolates including strains from Africa and Papua New Guinea. Examination of multiple clones from individual isolates revealed the presence of multiple genotypes in patients with tropical spastic paraparesis/HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma. These findings suggest that HTLV I, like HIV, may be present as a quasispecies in vivo. Our studies, however, failed to identify specific sequence motifs that segregated exclusively with the lymphoproliferative or neurological forms of the disease. PMID- 1733105 TI - The glycoprotein of Thogoto virus (a tick-borne orthomyxo-like virus) is related to the baculovirus glycoprotein GP64. AB - Thogoto (THO) virus is a tick-transmitted virus which shares morphological and biochemical characteristics with members of the Orthomyxoviridae. The genome of Thogoto virus comprises six segments of single-stranded, negative sense RNA. The complete nucleotide sequence of the fourth largest RNA segment of THO virus has been determined from cDNA analyses. This RNA segment is 1574 nt long and has a coding capacity for a glycoprotein of 512 amino acids with a predicted molecular weight of 57,550 Da. The sequence of this protein has extensive homology with the putative envelope protein of Dhori (DHO) virus, the only other recorded tick borne orthomyxo-like virus, but not with the envelope glycoproteins of the influenza viruses. A search of the translation of the available nucleotide database has produced evidence for a relationship between the glycoproteins of THO and DHO viruses and the gp64 glycoprotein of two DNA-containing insect baculoviruses, Autographa californica nuclear polyhedrosis virus and Orgyia pseudotsugata nuclear polyhedrosis virus. The baculovirus gp64 protein is a membrane protein implicated in endocytotic fusion events during infection. A multiple alignment between these four glycoproteins gave significance scores of greater than 28 standard deviations, indicating that the homologies between them are highly significant. The distribution of cysteine residues is conserved between all four proteins which also have similar hydropathy profiles, suggestive of a type I membrane protein topology. PMID- 1733106 TI - HIV1 cytopathogenicity-genetic difference between direct cytotoxic and fusogenic effect. AB - Formation of large syncytia, rapid cell killing, and early onset of replication are characteristics of the highly cytopathic Zairian virus strain HIV1 NDK compared with the HIV1 LAV prototype. Recombinant provirus molecules derived from cloned infectious DNAs of HIV1 LAV and NDK were constructed by reciprocal exchange of genetic material using conserved restriction sites. Different regions of the HIV1 genome were responsible for variability of the direct single-cell cytotoxic and fusogenic effects. A minimal, provisionally defined portion of genetic information responsible for the higher cytotoxicity of HIV1 NDK compared to the HIV1 LAV prototype was localized in the fragment Spel1042/EcoRl4183, containing the 3'-terminal half of gag and a majority of the pol gene. This region also determined the rapid replication properties of HIV1 NDK. The increased fusogenic potential of HIV1 NDK was associated with the simultaneous presence of HIV1 NDK fragments BssHll255/Spel1042 and EcoRl5278/Xhol8401 which contained the splicing donor, packaging sequence, p18 gag protein, and the HIV env gene. The increase in the direct killing effect but not in the syncytium forming ability of HIV1 NDK correlated with the early onset of replication and rapid spread of HIV1 NDK in cell cultures. The HIV1 NDK fragments BssHll/Spel and EcoRl/Xhol were by themselves necessary but not sufficient to induce formation of large syncytia. PMID- 1733108 TI - A frog virus 3 gene codes for a protein containing the motif characteristic of the INT family of integrases. AB - The integrase (INT) family of bacteriophage coded integrase-recombinase proteins are responsible for catalyzing strand exchange between DNA molecules and play an important role in the DNA replication of many bacteriophages. Within the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) of which the deduced amino acid sequence contains a motif characteristic of the INT family of integrases-recombinases. The ORF consists of 825 bp which codes for a protein of 275 amino acids with a predicted Mr of 29,945. RNA transcribed from this ORF during virus infection was detected by Northern blot analysis and it is a delayed early message of approximately 1100 bases. The 5' and 3' ends of the putative FV3 integrase-recombinase transcript were mapped. The transcriptional start site is preceded by a presumptive TATA box, and a region of hyphenated dyad symmetry is present at the 3' end of the message. A protein with an Mr of approximately 30,500 was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, indicating that the ORF can be transcribed into a message coding for a viral protein. In the FV3 life cycle, DNA replication occurs in a large complex formed through the recombination of small viral DNA molecules. Thus, at this stage, DNA replication and recombination are interlinked. Resolution of concatameric DNA is required for the packaging of genomes into virus particles. The putative FV3 INT gene may be involved in one or more of these functions. PMID- 1733107 TI - Cauliflower mosaic virus: a 420 subunit (T = 7), multilayer structure. AB - The structures of the Cabb-B and CM1841 strains of cauliflower mosaic virus (CaMV) have been solved to about 3 nm resolution from unstained, frozen-hydrated samples that were examined with low-irradiation cryo-electron microscopy and three-dimensional image reconstruction procedures. CaMV is highly susceptible to distortions. Spherical particles, with a maximum diameter of 53.8 nm, are composed of three concentric layers (I-III) of solvent-excluded density that surround a large, solvent-filled cavity (approximately 27 nm dia). The outermost layer (I) contains 72 capsomeric morphological units, with 12 pentavalent pentamers and 60 hexavalent hexamers for a total of 420 subunits (37-42 kDa each) arranged with T = 7 icosahedral symmetry. CaMV is the first example of a T = 7 virus that obeys the rules of stoichiometry proposed for isometric viruses by Caspar and Klug (1962, Cold Spring Harb. Symp. Quant. Biol. 27, 1-24), although the hexameric capsomers exhibit marked departure from the regular sixfold symmetry expected for a structure in which the capsid protein subunits are quasi equivalently related. The double-stranded DNA genome is distributed in layers II and III along with a portion of the viral protein. The CaMV reconstructions are consistent with the model based on neutron diffraction studies (Kruse et al., 1987, Virology 159, 166-168) and, together, these structural models are discussed in relation to a replication-assembly model (Hull et al., 1987, J. Cell Sci. (Suppl.) 7, 213-229). Remarkable agreement between the reconstructions of CaMV Cabb-B and CM1841 suggests that other strains of CaMV adopt the same basic structure. PMID- 1733109 TI - Characterization of a cis element required for packaging and replication of the human hepatitis B virus. AB - The hepatitis B virus (HBV) is a DNA virus; however, it replicates through a pregenomic RNA intermediate. Several HBV-specific mRNAs are transcribed, but only the pregenomic RNA transcript is encapsidated. The encapsidation of the HBV genome, therefore, is a highly specific and selective process. Using mutational analyses and complementation tests, we have defined a 99-nucleotide cis element located at the pre-C/C region of the HBV genome which is essential for encapsidation and the replication. Furthermore, HBV genome truncated down to 1.5 kb and HBV mutants carrying foreign DNA inserts could still replicate in our complementation system. PMID- 1733110 TI - Myb protein binds to multiple sites in the human T cell lymphotropic virus type 1 long terminal repeat and transactivates LTR-mediated expression. AB - The members of the c-myb proto-oncogene family encode sequence-specific transcriptional activators. In T cells, expression of c-myb and the related B-myb gene is induced following mitogenic stimulation. Using a purified recombinant protein, we report here that the human T cell lymphotropic virus type 1 (HTLV-1) LTR contains six specific binding sites for Myb. We also show that HTLV-1 LTR chloramphenicol acetyl transferase reporter plasmids are specifically transactivated by c-Myb. These data suggest a role for members of the Myb family as a link between transcriptional activation of the HTLV-1 LTR and T cell activation events. PMID- 1733111 TI - Frame-shift mutations within the vaccinia virus A-type inclusion protein gene. AB - The genetic basis for the failure of vaccinia virus (strain WR) to form a full length 150 kiloDalton (kDa) A-type inclusion protein was determined by sequencing a 4.1-kb pair segment of DNA and analyzing its transcription products. Open reading frames predicted to encode slightly overlapping 84.5- and 27.1-kDa proteins homologous to contiguous N-terminal segments of the A-type inclusion protein of cowpox virus were found. A putative deletion of two adjacent nucleotides occurring within several consecutive AG repeats and an insertion of 8 nucleotides accounted for the first and second reading frame shifts, respectively. Additional small mutations affecting reading frames were present in the C-terminal region of the gene. The vaccinia and cowpox virus mRNAs encoding the disparate size A-type inclusion proteins were similar in length, had equivalent 5' and 3' ends, and were expressed late in infection indicating the absence of mutations affecting transcriptional signals. PMID- 1733112 TI - SIV from stump-tailed macaques: molecular characterization of a highly transmissible primate lentivirus. AB - Over the past 6 years, simian immunodeficiency viruses (SIVs) have been isolated from four distinct species of macaques (Macaca mulatta, M. fascicularis, M. nemestrina, and M. arctoides) in captivity in the United States. However, the epidemiologic and genetic relationships among SIVs from the four species are not well understood. SIV from stump-tailed macaques (M. arctoides) (SIVstm) is unusual in that it has been associated with outbreaks of infection characterized by aggressive spread within stump-tailed macaque colonies at two separate primate centers in the United States. To characterize SIVstm at the molecular level, we have derived six biologically active viral DNA clones by polymerase chain reaction amplification of genomic DNA from infected cells. Nucleotide sequence analyses of one clone (SIVstm/37.16) showed that SIVstm was indeed a member of the previously defined group of simian lentiviruses that are closely related to the human immunodeficiency virus type 2 (HIV-2). However, our data indicate that SIVstm is equidistantly related to the other SIVs from macaques (SIVmac 251/142 and SIVmne) and SIV from African sooty mangabeys (SIVsmm). These findings suggest that SIV from captive macaques may have originated from several cross-species transmissions from imported sooty mangabeys and that additional spread has been fostered by the exchange of macaques among primate centers. PMID- 1733113 TI - Retroviral pseudotypes produced by rescue of a Moloney murine leukemia virus vector by C-type, but not D-type, retroviruses. AB - Human HOS cells containing a Moloney murine leukemia virus (Mo-MLV) recombinant genome were infected by a panel of retroviruses. The C-type viruses simian sarcoma associated virus, feline leukemia virus subgroup B, and the feline endogenous virus RD114 were able to form pseudotypes with the Mo-MLV genome, which transferred a selectable marker gene to target cells; however, Human T cell leukemia virus-1 and the D-type viruses Mason-Pfizer monkey virus and simian retrovirus-1 failed to rescue the Mo-MLV vector. Further characterization of the RD114 pseudotype demonstrated that it retained the receptor specificity of RD114 and will therefore prove useful in receptor characterization. PMID- 1733114 TI - Sequence changes in the live attenuated, cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus. AB - Nucleotide sequences were determined for the RNA segments coding for proteins other than the hemagglutinin and neuraminidase of the A/Leningrad/134/57 (H2N2) wild-type (A/Len/wt) virus and its two cold-adapted (ca) and attenuated variants, A/Leningrad/134/17/57 (A/Len/17/ca) and A/Leningrad/134/47/57 (A/Len/47/ca) that are used in the U.S.S.R. in the preparation of reassortant live attenuated vaccines. Ten nucleotide differences were detected between the sequences of the A/Len/wt and A/Len/17/ca viruses; of these, eight were deduced to encode amino acid (aa) substitutions. One aa substitution each was predicted for the PB2, M1, M2, and NS2 proteins, whereas two aa substitutions each were predicted for the PB1, and PA proteins of the A/Len/17/ca virus. Four additional nucleotide changes were found in the genome of the A/Len/47/ca virus; three of these were detected to code for one additional aa substitution each for the PB2, PB1, and NP proteins. PMID- 1733115 TI - Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses. PMID- 1733116 TI - Gastrointestinal surgery for severe obesity. Proceedings of a National Institutes of Health Consensus Development Conference. March 25-27, 1991, Bethesda, MD. PMID- 1733117 TI - Pathophysiology of obesity. AB - Individuals weighing greater than 100 kg represent a small fraction of the population and yet pose a major health risk to themselves. It is proposed that individuals be classified according to their body mass index (BMI). Class 0 individuals have a BMI of 20-25 kg/m2 and are not obese; Class I individuals have a BMI of 25-30 kg/m2 and are at low risk from their obesity; Class II individuals have a BMI of 30-35 kg/m2 and have moderate risk; Class III individuals have a BMI of 35-40 kg/m2 and have high risk associated with their obesity; Class IV individuals have a BMI of greater than 40 kg/m2 and are at very high risk for illness. Class IV is the primary group for surgical consideration. The pathophysiologic consequences of excess weight result in large part from increased food intake and/or decreased physical activity. Individuals in Class IV have additional problems related to their weight, including cardiomyopathy, Pickwickian/sleep apnea syndrome, pituitary/gonadal dysfunction, acanthosis nigricans, and significant osteoarthritis. PMID- 1733118 TI - Prevalence of overweight and weight gain in the United States. AB - Data from the Second National Health and Nutrition Examination Survey (NHANES II) indicated that in the period 1976-1980 approximately 34 million US adults (25.7%) were overweight, with more women (19 million) than men (15 million) affected. Selected demographic factors, eg, low educational attainment and low family income were associated with the prevalence of overweight and with the incidence of weight gain. Analyses of the NHANES I Epidemiologic Follow-up Survey indicated that in a 10-y period, women had a greater mean weight gain. The overall incidence of a major weight gain (ie, an increase of five or more body mass index units) was twice as great among females (5.3%) than males (2.3%). Data on overweight and weight gain in the United States by selected demographic characteristics are summarized. PMID- 1733119 TI - Economic costs of obesity. AB - Approximately 34 million US adults were obese in 1980. Obesity is associated with increased risk of noninsulin-dependent diabetes mellitus (NIDDM), hypertension, cardiovascular disease, gallbladder disease and cholecystectomy, and colon and postmenopausal breast cancer. Using a prevalence-based approach to cost of illness, we estimated the economic costs in 1986 attributable to obesity for these medical conditions. Indirect costs due to morbidity and mortality were discounted at 4%. Overall, the costs attributable to obesity were $11.3 billion for NIDDM, $22.2 billion for cardiovascular disease, $2.4 billion for gall bladder disease, $1.5 billion for hypertension, and $1.9 billion for breast and colon cancer. Thus a conservative estimate of the economic costs of obesity was $39.3 billion, or 5.5% of the costs of illness in 1986. Addition of costs due to musculoskeletal disorders could raise this estimate to 7.8%. The costs of treatment for severe obesity must be weighed against the improved health status and quality of life. PMID- 1733120 TI - Morbidity of severely obese subjects. AB - The prevalences of several risk factors and diseases are dramatically increased in obesity. In contrast, considerable inconsistencies have been reported for the relationship of obesity to the incidence of cardiovascular disease and total mortality. Suggested reasons for these inconsistencies have been confounders and surrogate risk factors, but the single most important cause is that far-reaching conclusions have been drawn from small short-term studies. Several large studies have recently proven that the incidence of cardiovascular disease is increased in obesity. Correct classification of obesity and its subgroups is also of great importance. Visceral obesity constitutes one subgroup at high risk. It seems possible to link diabetes, hypertriglyceridemia, reduced fibrinolysis, and hypertension to elevated portal free fatty acid concentrations because of an increased visceral adipose tissue depot. The quantitation of visceral adipose tissue has been improved by techniques based on computed tomography (CT) and by CT-calibrated anthropometric methods. Results from controlled intervention studies of obesity are entirely lacking but one such study has been started. PMID- 1733121 TI - Psychological aspects of severe obesity. AB - Studies of several overweight persons conducted before their undergoing antiobesity surgery have shown 1) that there is no single personality type that characterizes the severely obese; 2) that this population does not report greater levels of general psychopathology than do average-weight control subjects; and 3) that the complications specific to severe obesity include body image disparagement and binge eating. Studies conducted after surgical treatment and weight loss have shown 1) that self-esteem and positive emotions increase; 2) that body image disparagement decreases; 3) that marital satisfaction increases, but only if a measure of satisfaction existed before surgery; and 4) that eating behavior is improved dramatically. The results of surgical treatment are superior to those for dietary treatment alone. Practitioners should be aware that severely obese persons are subjected to prejudice and discrimination and should be treated with an extra measure of compassion and concern to help alleviate their feelings of rejection and shame. PMID- 1733122 TI - The thermogenic role of exercise in the treatment of morbid obesity: a critical evaluation. AB - Exercise induces negative energy balance either directly or by enhancing meal thermogenesis, increasing resting metabolic rate, and/or decreasing food intake. A quantitative evaluation of these effects in programs of weight control led to the following conclusions: 1) energy cost of exercise per se is minimal, 2) effects on thermic of food are negligible, and 3) exercise training may be advantageous in conjunction with low-calorie diet programs because it helps to maintain resting metabolic rate and fat-free mass. However, exercise may not prevent, and may even accentuate, the fall in metabolic rate in programs of severe calorie restriction, thus hampering weight reduction. Overall, exercise should not be envisioned as a sole agent to induce negative energy balance, but it is an essential element in comprehensive programs for morbidly obese patients due to its effects on lipids, carbohydrate metabolism, and cardiovascular system. PMID- 1733123 TI - Drug treatment of obesity. AB - The currently available drugs for treatment of obesity act on two pharmacologic systems in the central nervous system: the noradrenergic system and the serotonergic system. There are clear and convincing clinical data that these drugs are effective and safe. However, several types of barriers exist to their proper and effective use, including public perceptions that obesity is a disease resulting from lack of willpower, professional expectations that anorexiant drugs should cure obesity, hindrance by state licensing agencies, regulatory rigidity, limited research funding, and legislative inaction. In spite of these limitations, several new and potentially valuable drugs are under development, and given an appropriate clinical and therapeutic environment, the future is bright for treatment of obesity. PMID- 1733124 TI - Behavioral treatment of severe obesity. AB - Behavioral approaches to obesity are usually employed in the context of short term (10-20 wk) treatment interventions. These programs produce weight losses averaging 10 kg at the end of the program and 6.6 kg at 1-y follow-up. Improvements in long-term results may depend on a shift to a chronic disease model of obesity treatment, in which patients remain in ongoing care for extended periods of time. Highly structured diets and supervised exercise warrant further investigation as components of such a chronic disease approach to the treatment of severe obesity. PMID- 1733125 TI - Overview of surgical techniques for treating obesity. AB - Nonsurgical methods fail to maintain clinically significant weight loss greater than or equal to 5 y in severely obese patients. Vertical banded gastroplasty and Roux-Y gastric bypass are the main operations for obesity. Modifications of intestinal bypass reserved for special cases require particular expertise in long term management. Operations function by inducing satiety, nimiety, or aversion. Optimal weight loss or goal weights have not been defined and outcome predictors are inadequate. Results depend more on motivation and behavior than on metabolic, gastrointestinal, or technical factors. New approaches such as adding vagotomy or using inflatable cuffs to adjust outlet size in gastroplasty or modifying outlets or segment lengths in gastric bypass might improve long-term results. A staged approach to surgical treatment of obesity is proposed. Surgery will persist as a viable treatment alternative for severe obesity until effective preventive measures are taken to reduce the prevalence of this serious disease. PMID- 1733126 TI - Gastric restriction procedures for treating severe obesity. AB - Gastric restriction procedures are operations to decrease gastric volume. They are the most common, simple, and safe operations for the treatment of severe obesity. The original horizontal gastroplasties were unsuccessful but modern operations such as vertical banded gastroplasty, silastic ring gastroplasty, and gastric banding produce good weight loss with improvement in health. Although late weight gain does occur, satisfactory 5-y results are available and are presented. Patient selection, complications, mechanism of action, and future studies are also discussed. Long-term data with complete patient follow-up and randomized trials comparing modern operations with nonoperative treatment are still needed. PMID- 1733127 TI - Gastric bypass for treating severe obesity. AB - Gastric bypass (RY-GBP) has a very small gastric pouch with a 1-cm diameter Roux Y gastrojejunostomy. RY-GBP is associated with early satiety and an aversion to sweets secondary to dumping syndrome symptoms and has a significantly better weight loss than various gastroplasty procedures, including the vertical banded gastroplasty. However, it may be associated with vitamin B-12 deficiency and iron deficiency anemia in menstruating females, preventable with prophylactic oral iron and vitamin B-12. With an 80% 5 y follow-up, RY-GBP patients lose two-thirds of their excess weight within 2 y, 60% at 5 y, and greater than 50% at 9 y. The RY-GBP can be beaten by nibbling "junk foods" (potato or corn chips). Conversion to a malabsorptive procedure may cause severe malnutrition and fat-soluble vitamin deficiencies and should be used only for "superobese" patients who fail a standard RY-GBP and have severe comorbidity. RY-GBP is the most effective procedure for morbid obesity, especially in patients addicted to "sweets." PMID- 1733128 TI - Gastrointestinal malabsorptive procedures. AB - Morbid obesity is a complex disease, the etiology of which is clearly multifactorial. The weight loss produced by intestinal shunting procedures has been profound and the etiology of the weight loss is clearly more complex than rapid intestinal transit and gross malabsorption of foodstuffs. The best known surgically produced malabsorptive procedure for the treatment of morbid obesity is the jejunoileal bypass. This procedure produces substantial weight loss but has been associated with late postoperative complications that make its use problematical. Other procedures (biliary bypass, biliopancreatic diversion, and long limb Roux-en-Y gastric bypass) have not been associated with liver dysfunction. Varying degrees of malnutrition are frequently associated with these procedures. Careful study of the patients with these procedures is warranted. PMID- 1733129 TI - Physiological satiety implications of gastrointestinal antiobesity surgery. AB - This manuscript reviews the known satiety signals and the impact of antiobesity surgery on these physiological satiety mechanisms. Satiety signals originate from the stomach and small bowel to stop eating behavior. Stomach signals (gastric distension) produce early satiety by releasing hypothalamic cholecystokinin (CCK). The intermeal interval is probably mediated by peripheral CCK released by a threshold level of intraluminal calories. Anti-obesity operations probably rely little on these physiological satiety signals. Gastric balloons and gastroplasty produce nonphysiological gastric distension whereas intestinal bypass causes malabsorption. Gastric bypass combines supramaximal gastric distension with taste aversion from dumping. Future physiological manipulation of the satiety cascade will lead to improve obesity intervention. PMID- 1733130 TI - Perioperative risks and safety of surgery for severe obesity. AB - The National Bariatric Surgery Registry (NBSR) results reflect low perioperative risk for obesity surgery. Five deaths occurred within 40 d of operation in 5178 patients (0.1%). A subset of 3174 patients with complete information for complication and postoperative hospital stay was further studied. Females comprised 87% of the data set. Median values were determined for age, 37 y (18-70 y); operative weight, 121 kg (77-288 kg); and operative body mass index (BMI), 44 kg/m2 (29-91 kg/m2). Patients with no complications (89.7%) were reported to have a median postoperative stay of 4 d (2-23 d). The most severe complications were deep venous thrombosis (0.3%) and gastrointestinal leak (0.6%), with median postoperative hospital stay of 12 d (ranges 2-27 and 4-59 d, respectively). The most frequent complication reported was respiratory (4.5%), with median postoperative stay of 6 d (3-34 d). Median postoperative hospital stay for wound infection (1.6%) was 5 d. PMID- 1733131 TI - Critical analysis of results: weight loss and quality of data. AB - Difficulties associated with outcome assessment of operations performed for treatment of morbid obesity include lack of uniform standards for reporting results, failure to account for the response of related medical problems to weight loss, and lack of actuarial data for patients greater than or equal to 45 kg overweight. The purpose of this report is to critically analyze various methods of outcome assessment including the 5-y postoperative weight loss results of vertical banded gastroplasty and Roux-en-Y gastric bypass. Weight loss after these procedures usually reaches a nadir between 18 and 24 mo postoperatively. Mean percent excess weight loss at greater than or equal to 5 y ranged from 48% to 74% after gastric bypass and from 50% to 60% after vertical banded gastroplasty. Medical problems are almost invariably improved with satisfactory weight loss. Surgery remains the mainstay in treatment of morbid obesity because of the nearly 100% failure rate of nonoperative treatment in these patients. PMID- 1733132 TI - Surgical treatment of obesity and its effect on diabetes: 10-y follow-up. AB - Since 1980 we have performed the identical Greenville gastric bypass (GGB) procedure on 479 morbidly obese patients with an acceptable morbidity and a mortality rate of 1.2%. The weight loss in the series was well maintained over the follow-up period of 10 y. The GGB can control non-insulin-dependent diabetes mellitus (NIDDM) in most patients. The group of 479 patients included 101 (21%) with NIDDM and another 62 (13%) who were glucose impaired. Of these 163 individuals, 141 reverted to normal and only 22 (5%) remained with inadequate control of their carbohydrate metabolism. Those patients who were older or whose diabetes was of longer duration were less likely to revert to normal values. The gastric bypass operation is an effective approach for the treatment of morbid obesity. Along with its control of weight, the operation also controls the hyperglycemia, hyperinsulinemia, and insulin resistance of the majority of patients with either glucose impairment or frank NIDDM. PMID- 1733133 TI - Heart disease and hypertension in severe obesity: the benefits of weight reduction. AB - Severe obesity is associated with abnormalities of cardiac structure and function. These include an increased cardiac workload and ventricular hypertrophy. Hypertension in combination with severe obesity seriously burdens the heart because the increased preload and afterload compound cardiac work. Weight reduction induced by gastric operations for severe obesity is associated with resolution of hypertension, reduction in ventricular wall thickness and cardiac chamber size, as well as improved systolic function. Additional data are needed to predict when in the course of development of obese cardiomyopathy the changes in contractile function become irreversible. Additionally, the impact of coronary artery disease on the progression of obese cardiomyopathy and the effects of surgical weight reduction on cardiac structure and function need to be further clarified. Studies of the association between obesity, its treatment, and modification of cardiovascular risk are a major focus of preventive cardiology today. PMID- 1733134 TI - Results of surgery: long-term effects on hyperlipidemia. AB - To define frequency of lipid abnormalities and to monitor improvement or correction of those abnormalities postoperatively, 66 patients with chronic morbid obesity had total cholesterol, high-density-lipoprotein (HDL) cholesterol, and triglyceride determinations preoperatively and at staged intervals up to 5-7 y after Roux-Y gastric bypass. Preoperative abnormal HDL-cholesterol and triglyceride concentrations were frequent. Major improvements occurred in these lipid concentrations by 6 mo postoperatively, and some further improvements occurred with additional weight loss at 1 y. At 5-7 y, among 33 patients, raised concentrations of HDL cholesterol were upheld in women (P less than 0.01); reductions in triglycerides were maintained in men (P less than 0.025); and reduced total cholesterol:HDL-cholesterol, which was achieved by 6 mo, was sustained in both men and women (P less than 0.01). In comparing lipid profiles of gastric surgery through 5 y with recent data from the surgical arm of the Program on Surgical Control of the Hyperlipidemias (POSCH), postulates are made of anticipated reduction in morbid, and even fatal, cardiac events in the operated morbidly obese population. PMID- 1733135 TI - Bariatric surgery in morbidly obese sleep-apnea patients: short- and long-term follow-up. AB - Forty-seven obese sleep-apnea patients were investigated in the sleep laboratory before and after a massive weight reduction achieved by bariatric surgery. The first postoperative sleep investigations were performed approximately 1 y after surgery and revealed a highly significant decrease in the number of apneic episodes per hour of sleep and a significant improvement in all sleep-quality related measures. A second postoperative sleep study was performed approximately 7 y postoperatively and revealed that regaining of weight was associated with the reappearance of sleep apnea syndrome, although the great majority of the patients still felt, subjectively, that they were well and did not suffer from recurrence of the sleep apnea syndrome. PMID- 1733137 TI - Metabolic risk of obesity surgery and long-term follow-up. AB - Protein-calorie deprivation occurs after weight reduction operations and creates a potential for micronutrient deficiency syndromes. Whether clinical deficiency occurs depends primarily on the nature of the operation carried out. Current procedures include those that mainly diminish intake and those that shunt either food or digestive enzymes to minimize the absorption of ingested food. The metabolic price that some patients pay postoperatively makes the choice of operation and the quality of follow-up critical to the patient's well-being. Restrictive operations (gastroplasty) are devoid of long-term metabolic complications. Gastric bypass patients rarely have deficiency syndromes but often develop micronutrient deficiencies. Malabsorptive procedures carry the highest risk and malnutrition, with multiple micronutrient deficiencies, may supervene despite close medical follow-up. PMID- 1733136 TI - Long-term effects of gastric surgery for treating respiratory insufficiency of obesity. AB - The Pickwickian syndrome can be divided into two primary breathing disorders, which can affect patients alone or in combination: sleep apnea syndrome (SAS) and obesity hypoventilation syndrome (OHS). Between 1980 and 1990, 126 patients with respiratory insufficiency underwent gastric surgery for morbid obesity, 12.5% of the entire series. These patients weighed more (164 +/- 36 vs 135 +/- 25 kg, P less than 0.0001) and were more often men (62% vs 14%, P less than 0.001) than those without pulmonary dysfunction. Sixteen had OHS alone, 65 had SAS alone, and 45 had both. Of those with OHS, 38 have been followed for 5.8 +/- 2.4 y since surgery and 29 are currently asymptomatic. In the 12 patients in whom arterial blood gases were available greater than 5 y since surgery, the PaO2 increased from 54 +/- 10 to 68 +/- 20 mm Hg (P less than 0.0001) and PaCO2 fell from 53 +/- 9 to 47 +/- 11 mm Hg (P = 0.05). Of the 110 patients with SAS, 57 were available for follow-up an average of 4.5 +/- 2.3 y since surgery and 38 were completely asymptomatic, 15 had mild SAS, and 4 had both SAS and OHS. In 40 patients with pre- and post-weight reduction sleep polysomnograms, the sleep apnea index fell from 64 +/- 39 to 26 +/- 26 (P less than 0.0001). Although respiratory insufficiency of obesity patients had a higher operative mortality than did patients without pulmonary dysfunction (2.4% vs 0.2% after gastric bypass), weight loss was associated with significant improvements in sleep apnea, arterial blood gases, pulmonary hypertension, left ventricular dysfunction, lung volumes, and polycythemia. PMID- 1733138 TI - Reoperative surgery--indications, efficacy, and long-term follow-up. AB - The Roux-en-Y gastric bypass with fascial support (RYGBPF) has proved to be effective as a primary as well as a revision/conversion operation in the management of morbid obesity. Since May 1984, 361 primary and 100 revision RYGBPF operations have been reported with low morbidity and mortality although morbidity and mortality were higher in the revision than in the primary group. The most common late complications of gastric-reduction operations resulting in weight regain were staple line disruption and pouch or stoma enlargement. With the use of the RYGBPF as a primary as well as a revision operation, weight loss results at 5 y have been good and our revision operation rate has dropped to less than 1%. PMID- 1733139 TI - Assessment of quality of life before and after surgery for severe obesity. AB - Quality of life is poor in obese people because of poor physical health and mental well-being and impaired psychosocial functioning. Obese people perceive discrimination and prejudice against them as their heaviest burden. Reports of absence of psychopathology in obese people reflect adaptation to chronic disease or failure of assessment instruments to detect disturbances. We present information on the extraordinary suffering and perceived discrimination of obese people and discuss econometric assessment of quality of life. The Swedish national population study of obese subjects (SOS) is presented as well as studies of effects of surgical weight loss on quality of life. Most studies lack adequate controls and extrapolations from surgical populations are uncertain. Psychosocial factors are important predictors of outcome in terms of physical as well as mental health. Operated patients with significant weight loss after surgery demonstrate dramatic improvement in quality of life. This alone justifies treating severely obese patients surgically. PMID- 1733140 TI - Gastrointestinal surgery for severe obesity: National Institutes of Health Consensus Development Conference Statement. AB - The National Institutes of Health Consensus Development Conference on Gastrointestinal Surgery for Severe Obesity brought together surgeons, gastroenterologists, endocrinologists, psychiatrists, nutritionists, and other health care professionals as well as the public to address: the nonsurgical treatment options for severe obesity, the surgical treatments for severe obesity and the criteria for selection, the efficacy and risks of surgical treatments for severe obesity, and the need for future research on and epidemiological evaluation of these therapies. Following 2 days of presentations by experts and discussion by the audience, a consensus panel weighed the evidence and prepared their consensus statement. Among their findings, the panel recommended that (1) patients seeking therapy for severe obesity for the first time should be considered for treatment in a non-surgical program with integrated components of a dietary regimen, appropriate exercise, and behavioral modification and support, (2) gastric restrictive or bypass procedures could be considered for well informed and motivated patients with acceptable operative risks, (3) patients who are candidates for surgical procedures should be selected carefully after evaluation by a multidisciplinary team with medical, surgical, psychiatric, and nutritional expertise, (4) the operation be performed by a surgeon substantially experienced with the appropriate procedures and working in a clinical setting with adequate support for all aspects of management and assessment, and (5) lifelong medical surveillance after surgical therapy is a necessity. The full text of the consensus panel's statement follows. PMID- 1733141 TI - Intubation of newborns. PMID- 1733142 TI - Pubic hair in infancy. PMID- 1733143 TI - Neonatal presentation of Prader-Willi syndrome. PMID- 1733144 TI - Pertussis in adults. AB - A survey was conducted of 89 households in each of which at least one patient with culture-confirmed pertussis had been detected. The source of infection was found to be an adult in 10 (11.2%) of the 89 households, and the rate of secondary attack was 19 (10.3%) of 185. Furthermore, a laboratory study disclosed 17 adults with subclinical pertussis; the subclinical infection rate was 17 (25.0%) of 68. When compared with pertussis in young children, the adult illness was generally less severe and had different clinical features. Adult pertussis showed neither leukocytosis nor lymphocytosis, but it produced anti-pertussis toxin antibody more quickly and higher levels of anti-filamentous hemagglutinin and agglutinin antibodies, and showed stronger growth inhibition of Bordetella pertussis. Although adult pertussis is usually unrecognized because of its different clinical and laboratory features, it is a significant health threat that requires some measures for disease control. PMID- 1733145 TI - Pertussis antibodies, protection, and vaccine efficacy after household exposure. AB - During a randomized trial of a cellular pertussis vaccines, significantly fewer recipients of a two-component vaccine (Japanese National Institute of Health [JNIH]-6) were diagnosed as primary or coprimary cases in households than either placebo recipients or those who received a monocomponent pertussis toxoid vaccine (JNIH-7). After household exposure to a culture-confirmed primary case, efficacy for JNIH-6 was estimated to be 35% (95% confidence interval, -14% to 57%) against any culture-confirmed disease and 58% (95% confidence interval, -6% to 84%) against clinical disease with 21 days or more of coughing spasms. The corresponding efficacy estimates for JNIH-7 were 67% (95% confidence interval, 32% to 80%) and 82% (95% confidence interval, 41% to 96%). Differences between the JNIH-6 and JNIH-7 vaccines in efficacy after household exposure were not statistically significant. No association could be established between protection against pertussis after household exposure and serum levels of IgG antibody to pertussis toxin or filamentous hemagglutinin in vaccinated individuals, in either study children or other household members. PMID- 1733146 TI - Pertussis outbreaks in groups claiming religious exemptions to vaccinations. AB - Four recent outbreaks of pertussis in Massachusetts illustrate some features that contribute to the increased incidence of the disease. The outbreaks involved unimmunized groups of children with philosophical or religious exemptions from school or day-care immunization requirements and children and adults who were reluctant to undergo antibiotic prophylaxis or therapy. Parents and physicians should be aware that failure to immunize and to cooperate in follow-up preventive measures can have public health and potential medicolegal repercussions. PMID- 1733147 TI - Prevention of secondary transmission of pertussis in households with early use of erythromycin. AB - To examine the effectiveness of erythromycin therapy and prophylaxis for pertussis, 17 households with one secondary case or more were compared with 20 households without secondary cases following a community-wide pertussis outbreak in Maricopa County, Arizona, in 1988. There were no significant differences between the two household groups in age distribution of members, size, crowding, race, proportion of children aged 7 months to 18 years with three or more diphtheria and tetanus toxoids and pertussis vaccine doses, or in the age distribution, vaccination status, or medical care of patients with primary cases. However, median intervals from onset of illness in primary cases to initiation of erythromycin therapy (for cases) and prophylaxis (for contacts) were 11 and 16 days, respectively, in households without secondary spread, vs 21 and 22 days, respectively, in households with secondary spread. These results provide additional evidence that erythromycin is effective in the medical management of pertussis and should be initiated promptly to minimize secondary spread. PMID- 1733148 TI - Failure of thrombolytic therapy in four children with extensive thromboses. AB - In three children with central vein thromboses and a fourth with a pulmonary artery thrombosis, thrombolytic therapy failed to produce ultrasonographic evidence of clot lysis. Low-dose streptokinase (50 to 250 U/kg per hour) was infused directly into the clot in three children, followed by streptokinase and urokinase in systemic doses (streptokinase, 1000 to 1750 U/kg per hour; urokinase, 4400 to 5000 U/kg per hour). A fourth child treated sequentially with systemic doses of streptokinase, urokinase, and recombinant tissue-type plasminogen activator developed a significant retroperitoneal and intrapleural hemorrhage after 19 hours of recombinant tissue-type plasminogen activator infusion at a dose of 0.7 mg/kg per hour. All of the children survived. The most likely reason for treatment failure was that the clots (estimated to be between 2 and 3 weeks of age) were organized and thus resistant to lysis. Early diagnosis and prompt thrombolysis of significant lesions may contribute to the successful management of pediatric thrombosis. However, controlled studies are clearly needed to establish guidelines for the optimal use of thrombolytic agents in children. PMID- 1733149 TI - Neurodevelopmental outcome of term infants with intraventricular hemorrhage. AB - Intraventricular hemorrhage and its sequelae have been reported infrequently in term infants. We investigated the outcome of intraventricular hemorrhage in 15 term infants born between 1982 and 1988. One infant (7%) died. Complications of pregnancy were identified in seven mothers (47%). Age at diagnosis ranged from in utero to 28 days. Clinical presentation included feeding intolerance, fever, jaundice, irritability, and seizures. Severity of hemorrhage was of prognostic value. Of the four children with grade 4 hemorrhage, one died and the three survivors were severely handicapped. Overall, nine (64%) of 14 survivors had no or mild handicap. Perinatal alloimmune thrombocytopenia emerged as the single most important cause of severe hemorrhage and poor outcome. Identification and treatment of these infants must begin in utero if we are to prevent intraventricular hemorrhage and its complications in this group of patients. PMID- 1733150 TI - Outcome of nonoperative management of splenic injury with nuclear scanning. Clinical significance of persistent abnormalities. AB - Uncertainties remain about the frequency and need for diagnostic imaging following recovery from splenic injury with nonoperative management. To gain further understanding, the final appearance of the splenic roentgenographic image was evaluated in 20 consecutive children (mean age, 10.1 years) undergoing serial studies up to 70 weeks following injury. A total of 65 technetium 99m sulfur colloid scans, including 45 follow-up studies, were obtained and evaluated. By 20 weeks following injury, six patients (30%) were normal, four (20%) demonstrated minimal residual effects, and 10 (50%) had significantly improved, leaving some persistent abnormality. None of the patients in the last group showed any clinical problem. No distinctions could be made by comparing the severity of the initial injury with a persisting imaging defect. We conclude that clinical considerations alone should determine whether any follow-up imaging be performed in children recovering from splenic injury. PMID- 1733151 TI - Internal carotid artery blood flow velocities before, during, and after extracorporeal membrane oxygenation. AB - Blood flow velocities in the internal carotid arteries were studied with pulsed Doppler in 25 neonatal patients (birth weight range, 2600 to 4100 g) who had extracorporeal membrane oxygenation (ECMO). Time averaged mean systolic, mean diastolic, and mean blood flow velocities were calculated. Five infants had right common carotid artery reconstruction. Blood flow velocities measured in 15 healthy full-term infants were used as controls. Findings during ECMO included the following: (1) forward flow in the right internal carotid artery in 50% of the infants; (2) significant increase in the mean diastolic and the mean flow velocities (48% and 128%, respectively) in the left internal carotid artery when compared with pre-ECMO and control infants' values; (3) the elevation in the mean and the mean diastolic velocities was associated with changes in the PaCO2 and with an increase in the diastolic blood pressure; and (4) forward blood velocities in the right internal carotid artery were comparable with blood velocities in the left internal carotid artery and with the blood velocities of control infants. After ECMO, the mean diastolic velocity in the left internal carotid artery decreased significantly, but it remained elevated when compared with pre-ECMO values. Infants with right common carotid reconstruction had blood velocities in the right internal carotid artery comparable with the simultaneous blood velocities in the left internal carotid artery and to the blood velocities of control infants. Twenty-eight percent of the infants had major neuroanatomic lesions. Right or left preponderance was not noted. No association between blood velocity values in the internal carotid arteries or flow direction and the presence or the absence of brain lesions was noted. PMID- 1733152 TI - Coaches. A missing link in the health care system. AB - The number of children and adolescents who participate in interscholastic athletics demands attention to the quality of the coaching they receive and to the opportunities that the athlete-coach relationship provides for modification of high-risk behaviors, social skills training, and character formation. Although the need for coaches has increased due to the advent of girls' athletic programs, which was mandated by Title IX legislation, only a minority of states require certification for coaches who work in school systems. Four coaching curricula are summarized and contrasted: the American Coaching Effectiveness Program, the curriculum of the National Youth Sports Coaches Association, the Athletic Health Care System, and the Coach Effectiveness Training Program. Recommendations for coach certification by states, physician advocacy for coaching standards, and improved sports medicine services are discussed. PMID- 1733153 TI - Cognitive and motor development in infants at risk for human immunodeficiency virus. AB - To evaluate the natural course of cognitive and motor development among infants infected with human immunodeficiency virus from birth, the Bayley Scales of Infant Development were administered to 96 infants between 5.5 and 24 months of age. Infants were divided into three groups on the basis of subsequent assessment of human immunodeficiency virus serologic status: seronegative (N = 45), seropositive (N = 12), and seroreverter (N = 39). Groups did not differ in race, infant age at initial testing, maternal age, maternal education level, maternal history of intravenous drug abuse, or percentage of children in foster care placement. Significant group differences were found on the Mental Development Index and Psychomotor Development Index, with the seropositive infants scoring significantly lower than the seronegative or seroreverter infants. PMID- 1733154 TI - Asymmetric septal hypertrophy in infants of diabetic mothers. Fetal echocardiography and the impact of maternal diabetic control. AB - Maternal hyperglycemia may result in fetal hyperinsulinemia and asymmetric septal hypertrophy, macrosomia, and hypoglycemia in infants of diabetic mothers. We monitored glycosylated hemoglobin levels in 61 pregnant diabetic women each trimester as an index of maternal glycemic control and did serial fetal echocardiograms starting at 18 weeks of gestation. At delivery, cord blood C peptide levels were obtained as an index of fetal hyperinsulinemia. Infants were assessed for hypoglycemia, macrosomia and septal thickening by echocardiography. Nineteen of the 61 infants (31%) had septal hypertrophy, were heavier, and had higher cord blood C-peptide levels and lower serum glucose levels than unaffected infants. Maternal glycosylated hemoglobin levels were higher during the third trimester in mothers of affected infants. Our data support a possible relationship between third-trimester maternal hyperglycemia and neonatal asymmetric septal hypertrophy, macrosomia, and hypoglycemia. PMID- 1733155 TI - Ultrasound screening of high-risk infants. A method to increase early detection of congenital dysplasia of the hip. AB - Congenital dysplasia of the hip (CDH) continues to be missed by routine physical screening examinations in the early months when treatment is most effective. Real time ultrasonography (US) is valuable in the detection of CDH in the young infant. We performed a prospective study to evaluate one US screening strategy that targets a select "high-risk newborn" population at risk for CDH aiming to increase the early diagnosis of this condition. From 1772 consecutive births at one hospital, we identified 97 (5.5%) newborns with risk factors for CDH: breech delivery, 73 babies; family history, 26 babies; postural abnormalities, five babies; and oligohydramnios, four babies. Eleven newborns had two risk factors. We studied 69 of these newborns with US. There were four cases of CDH in this group. Three of these babies had completely normal pediatric physical examination results at the time of the US study (at 14, 75, and 100 days, respectively) despite dysplasia diagnosed by US. All were successfully treated with a harness as outpatients. We conclude that a screening program entailing identification and subsequent US of the hip of newborns with specific physical and historical risk factors for CDH increases early diagnosis. Further analysis suggests this approach is cost-effective. PMID- 1733156 TI - A clinical trial of fiberoptic phototherapy vs conventional phototherapy. AB - We conducted a randomized, controlled trial to compare fiberoptic phototherapy with conventional phototherapy in healthy jaundiced newborns with birth weights greater than 2500 g. Twelve patients received fiberoptic phototherapy and 14 patients received conventional phototherapy. There were no significant differences between the groups with respect to birth weight, gestational age, feeding method, presence of hemolytic disease, hematocrit, reticulocyte count, or initial serum bilirubin level. Measured irradiance at 425 to 475 nm for conventional phototherapy was greater than that of fiberoptic phototherapy (9.2 +/- 0.9 microW/cm2 per nanometer vs 8.2 +/- 1.2 microW/cm2 per nanometer). Both types of phototherapy lowered the level of serum bilirubin after 18 hours of therapy (fiberoptic group, from 231 +/- 29 to 210 +/- 24 mumol/L; conventional group, from 231 +/- 21 to 188 +/- 26 mumol/L), but the mean serum bilirubin level was lower after 18 hours of therapy in the conventional phototherapy group (188 +/- 26 vs 210 +/- 24 mumol/L). There were no side effects in either group of newborns. Both methods of phototherapy decreased the serum bilirubin level, but conventional phototherapy did so more effectively, probably because of its greater irradiance. PMID- 1733157 TI - A faculty-house staff retreat. AB - There has been much discussion regarding the causes of stress in residency training programs and solutions to alleviate the pressure. As a partial solution, we have found discussions of retreats for pediatric interns in the medical literature, but no discussion of department-wide retreats. Since the early 1970s, the University of Wisconsin (Madison) Department of Pediatrics has held a faculty house staff retreat every 2 years. The more recent retreats used process-oriented discussions in its goals of fostering understanding through group communication to reduce stress. The 1989 agenda was an expansion of previous efforts with extensive faculty and house staff involvement before, during, and after the retreat. The purposes of this article are to review the literature on the use of retreats in various settings, especially residency training programs; describe the past and present use of retreats by the University of Wisconsin Department of Pediatrics; describe the 1989 retreat; and describe the positive and negative aspects relating to retreats as we have used them. We believe our retreat is unique, serves many purposes, and has been a successful tool for relieving residency stress. PMID- 1733158 TI - Radiological case of the month. Brain-stem astrocytoma presenting with aspiration pneumonia. PMID- 1733159 TI - Picture of the month. Tuberous sclerosis. PMID- 1733160 TI - Pathological case of the month. Primary oxalosis (primary hyperoxaluria). PMID- 1733161 TI - Harold Francis Falls: eye geneticist and pioneer American medical geneticist. PMID- 1733162 TI - Scattered memorabilia: a living history autobiography of Harold F. Falls. PMID- 1733163 TI - Noninactivation of a portion of Xq28 in a balanced X-autosome translocation. AB - We present a balanced translocation (X;9) (q28;q21) in which the normal X chromosome is preferentially active. The derivative X chromosome is inactive in 93% of fibroblasts, but the X portion translocated onto chromosome 9 is not inactivated, as apparent from DNA methylation and chromosome replication patterns. Consequently, the patient is functionally disomic for the part of Xq28 distal to the locus LICAM. PMID- 1733164 TI - Functional disomies of the X chromosome influence the cell selection and hence the X inactivation pattern in females with balanced X-autosome translocations: a review of 122 cases. AB - We reviewed 122 cases of balanced X-autosome translocations in females, with respect to the X inactivation pattern, the position of the X break point and the resulting phenotype. In 77% of the patients the translocated X chromosome was early replicating in all cells analysed. The break points in these cases were distributed all along the X chromosome. Most of these patients were either phenotypically normal or had gonadal dysgenesis, some had single gene disorders, and less than 9% had multiple congenital anomalies and/or mental retardation. In the remaining 23% of the cases the translocated X chromosome was late replicating in a proportion of cells. In these cells only one of the translocation products was reported to replicate late, while the remaining portion of the X chromosome showed the same replication pattern as the homologous part of the active, structurally normal X chromosome. The analysis of DNA methylation in one of these cases confirmed noninactivation of the translocated segment. Consequently, these cells were functionally disomic for a part of the X chromosome. The presence of disomic cells was highly prevalent in translocations with break points at Xp22 and Xq28, even though spreading of X inactivation onto the adjacent autosomal segment was noted in most of these cases. This suggests that selection against cells with a late replicating translocated X is driven predominantly by a functional disomy X, and that the efficiency of this process depends primarily on the position of the X break point, and hence the size of the noninactivated region.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733165 TI - Tentative assignment of gene for oto-palato-digital syndrome to distal Xq (Xq26 q28). AB - Detailed physical mapping of oto-palato-digital (OPD) syndrome gene on the X chromosome was attempted on a family of 3 generations with 2 affected men. Although the result remains statistically non-significant, it indicates that the OPD-I gene might be located on the distal Xq. PMID- 1733166 TI - Acromelic frontonasal "dysplasia": further delineation of a subtype with brain malformation and polydactyly (Toriello syndrome). AB - We report on a stillborn boy with frontonasal malformation (Sedano-Jirasek type D DeMyer type I), associated with encephalocoele, occipital meningocele and preaxial polydactyly of the feet. This form of frontonasal dysplasia was documented previously in a few other cases with various combinations of postaxial polydactyly, tibial hypoplasia, epibulbar dermoid, occipital encephalocoele, corpus callosum agenesis and Dandy-Walker malformation. Most cases are sporadic. PMID- 1733167 TI - Study of color vision in fragile X syndrome. AB - Various theories have been postulated to account for the unusual inheritance pattern observed in the fragile X syndrome. The recent finding of a secondary amplification of the fragile X mutation in the offspring of carrier females [Oberle et al., 1991; Yu et al., 1991] is consistent with a maternal imprinting process. Laird [1987] has proposed that the fragile X mutation blocks complete reactivation of a previously inactivated fragile X chromosome. We have tested whether or not such a localized block extends as far distal as the red/green color-vision complex at Xq28. We found no evidence of color-vision defects among 25 male subjects with the fragile X syndrome. A fragile X positive woman also had normal color vision, despite being an obligate carrier of her father's gene for red/green color blindness. We conclude that the fragile X gene does not affect the function of neighboring color-vision genes, nor does it affect their ability to compensate adequately for inherited color deficiency on the homologous X chromosome in females. PMID- 1733168 TI - Genomics of human dysmorphogenesis. AB - A survey of Mendelian Inheritance in Man emphasizes the large Mendelian contribution to human dysmorphogenesis and contrasts single gene conditions with chromosomal disorders. There were 1761 conditions that involved altered morphogenesis (49% of disease entries), including 1040 multiple defect syndromes and 721 inherited single birth defects. Premature death (36-57%), mental retardation (20-59%), and growth retardation (37-59%) are more frequent in autosomal recessive or X-linked syndromes, while predisposition to tumorigenesis was more common in dominant (16%) than recessive (3.4%) syndromes. Comparison of the Mendelian conditions with 100 chromosomal disorders showed a strikingly similar spectrum of malformation, with skeletal, craniofacial, eye, epidermal, and neuromuscular systems being most frequently affected. Chromosomal syndromes average 10.6 systems affected per disorder, in contrast to 3.55 for Mendelian syndromes, and pleiotropy does correlate weakly with aneuploid segment length. Genomic understanding of these relationships is still primitive, with 74 of 1609 (4.6%) autosomal conditions and 43 of 152 (29%) X-linked conditions mapped to specific chromosomal regions. The societal toll of human dysmorphogenesis and the evident progress with X-linked disorders provide a powerful rationale for the Human Genome Project. PMID- 1733169 TI - Prevalence of nonclassical congenital adrenal hyperplasia among women self referred for electrolytic treatment of hirsutism. AB - Nonclassical congenital adrenal hyperplasia (NCCAH) is well recognized among women who seek medical attention for hirsutism. However, the prevalence of this disorder among women self-referred for electrolytic treatment of hirsutism is unknown. We hypothesized that the prevalence of NCCAH among women attending an electrolysis clinic might be high. By measuring the morning salivary 17 hydroxyprogesterone (17-OHP) as a screening test for NCCAH in 46 women in the follicular phase of their menstrual cycle, we identified 12 subjects with a high basal salivary 17-OHP. Eleven agreed to have a 60-minute Cosyntropin-stimulation test, as did an additional 6 of 9 women with normal basal salivary 17-OHP, but with a particularly high hirsutism score. One of the women with high basal salivary 17-OHP had a 60-minute Cosyntropin response, which was diagnostic of NCCAH. She was of the Ashkenazi Jewish decent, a group previously reported to have a high prevalence of NCCAH. A second woman with high salivary 17-OPH had a Cosyntropin-stimulation response consistent with heterozygosity for 21 hydroxylase deficiency. None of the Cosyntropin-stimulation responses in those chosen for a high hirsutism score were diagnostic. Thus, 1 of 46 (2.2%) of the women who entered our study had unrecognized NCCAH, a prevalence only about 2 fold greater than that reported in the general population. Therefore, we recommend that electrolysis clinics advise clients from ethnic groups known to have a high frequency of NCCAH of the advisability of having a formal medical evaluation for NCCAH. PMID- 1733170 TI - Familial occurrence of Duchenne dystrophy through paternal lines in four families. PMID- 1733171 TI - Duchenne muscular dystrophy inherited through paternal lines. PMID- 1733172 TI - 11q;22q translocation: third case of imbalance not due to 3:1 nondisjunction in first meiosis. PMID- 1733173 TI - Chromosome abnormalities in cell cultures derived from the leukoplakia of a female patient with dyskeratosis congenita. PMID- 1733174 TI - Conference report: First International Scientific Workshop on Prader-Willi Syndrome and Other Chromosome 15q Deletion Disorders. May 2-3, 1991, DeLeeuwenhorst, The Netherlands. Abstracts. PMID- 1733175 TI - Selection bias in in vitro fertilization programs. AB - Pregnancy rates per cycle reported from different in vitro fertilization-embryo transfer programs vary widely. While several programs have reported constant pregnancy rates per cycle, others report declining pregnancy rates. Selection biases at the point of entry and between cycles are discussed as possible explanations of these discrepancies. PMID- 1733176 TI - Incidence of pelvic inflammatory disease after first-trimester legal abortion in women with bacterial vaginosis after treatment with metronidazole: a double blind, randomized study. AB - OBJECTIVE: The purpose of this study was to evaluate the effect of metronidazole treatment on the incidence of postoperative pelvic inflammatory disease after first-trimester abortion in women with bacterial vaginosis. STUDY DESIGN: A double-blind, randomized, multicenter study was conducted on 231 women undergoing first-trimester legal abortion and fulfilling the criteria for bacterial vaginosis. The women were randomized to either metronidazole 500 mg three times daily for 10 days or placebo. Treatment was started at the outpatient visit the week before the operation. RESULTS: Among the 174 women who could be evaluated, pelvic inflammatory disease developed in 14 after the abortion. In the treatment group there were three infections (3.8%) compared with 11 (12.2%) in the placebo group (p less than 0.05). CONCLUSION: These data suggest that patients with bacterial vaginosis should be treated in conjunction with first-trimester abortion because treatment with metronidazole reduces the postoperative infection rate more than three times. PMID- 1733177 TI - Depressive symptoms in women in the six months after miscarriage. AB - This study, the first systematic investigation of the psychiatric impact of miscarriage, tests whether miscarriage markedly increases depressive symptoms in the 6 months after loss. We interviewed 382 miscarrying women entering the study at 2 weeks, 6 weeks, or 6 months after loss and, for comparison, 283 pregnant women and 318 community women not recently pregnant. Among women interviewed 2 weeks after miscarriage the proportion highly symptomatic on the Center for Epidemiologic Studies-Depression scale was 3.4 times that of pregnant women (95% confidence limits 2.0 and 5.0) and 4.3 times that of community women (95% confidence limits 3.0 and 5.8). Among women first interviewed 6 weeks and 6 months after miscarriage the proportion highly symptomatic was three times that of the community women. Women reinterviewed at 6 weeks and 6 months did not have elevated symptom levels, a result attributed to the unintended therapeutic and test effects of study interviews. Interviews were fully structured, readily administered by telephone by nonmedical personnel. The possibility that such interviews afford miscarrying women substantial psychologic benefits merits future investigation. PMID- 1733178 TI - Alloimmune thrombocytopenia may be associated with systemic disease. AB - Adverse outcome was encountered in a case of neonatal alloimmune thrombocytopenia after in utero platelet transfusion. This may have resulted from generalized systemic vascular endothelial damage, because several cell types, including umbilical endothelial cells, have been shown to contain surface molecules similar to the receptor containing the PlA1 antigen. PMID- 1733179 TI - Spontaneous abortion and subsequent adverse birth outcomes. AB - OBJECTIVE: Our purpose was to evaluate the association between spontaneous abortion and subsequent adverse birth outcomes. STUDY DESIGN: Washington State birth certificate records for 1984 to 1987 were used to examine the association between spontaneous abortion and adverse outcomes in the subsequent live birth. Adverse birth outcomes were examined for women with one spontaneous abortion before the index pregnancy (n = 2146) and for women with three or more prior spontaneous abortions and no other prior pregnancies (n = 638); compared with women with no prior spontaneous abortions (n = 3099). Logistic regression was used to estimate the relative risk associated with prior spontaneous abortion of each adverse outcome. RESULTS: Women with three or more prior spontaneous abortions were at higher risk for delivery at less than 37 weeks' gestation (relative risk 1.5, 95% confidence interval 1.1 to 2.1), placenta previa (relative risk 6.0, 95% confidence interval 1.6 to 22.2), having membranes ruptured greater than 24 hours (relative risk 1.8, 95% confidence interval 1.2 to 2.9), breech presentation (relative risk 2.4, 95% confidence interval 1.6 to 3.6), and having an infant with a congenital malformation (relative risk 1.8, 95% confidence interval 1.1 to 3.0). CONCLUSION: These findings suggest that common causes may underlie recurrent spontaneous abortion and certain adverse birth outcomes. They may also help guide clinical management of pregnancies in women with a history of recurrent spontaneous abortions. PMID- 1733180 TI - Prolonged effects of a novel, low-dosage continuous progestin-cyclic estrogen replacement program in postmenopausal women. AB - OBJECTIVE: Our objective was to ascertain long-term tolerability and effects of a novel, low-dosage continuous progestin-cyclic estrogen regimen on vasomotor flushing, vaginal bleeding patterns, bone density, and plasma lipoprotein lipids. STUDY DESIGN: Eighteen postmenopausal women with climacteric symptoms received low doses of dl-norgestrel (0.075 mg/day) continuously and 17 beta-estradiol (1 mg/day) intermittently (25 of 28 days) for 3 years. The results were compared with baseline values. RESULTS: The hormonal regimen was well tolerated. Vaginal bleeding, vaginal spotting, and vasomotor flushing occurred in only 0.4%, 4.2%, and 7.7% of cycles, respectively. Bone density was stable. Mean fasting plasma total cholesterol concentration fell 19%, low-density lipoprotein cholesterol 23%, high-density lipoprotein cholesterol 13%, and triglycerides 31% (p less than 0.01) over the 3-year period, while the ratios of low-density lipoprotein cholesterol/high-density lipoprotein cholesterol and total cholesterol/high density lipoprotein cholesterol decreased by 14% and 10%, respectively. CONCLUSION: Long-term compliance with female hormonal replacement is feasible once vaginal bleeding is essentially eliminated, allowing for potentially better prophylaxis against coronary heart disease, osteoporosis, endometrial cancer, and stroke. PMID- 1733181 TI - Attitudes toward reproduction in a nonpatient population. AB - OBJECTIVE: A basic question to be answered in an attempt to understand human reproductive behavior as it manifests itself in clinical practice is, why do people want to have children? STUDY DESIGN: A psychometric instrument for the assessment of reproductive attitudes was developed. A total of 746 men and women at 20, 30, and 40 years of age, who were randomly selected from a nonpatient population, answered. Data were analyzed by factor analysis. Reproductive profiles were constructed. RESULTS: The two most important factors in both sexes were "children as existential satisfaction" and "children as lack of freedom," indicating a basic conflict. The third most important factor in both sexes was "the importance of one's own parents as examples in parenthood," supporting earlier findings that reproductive conflicts are transmitted from one generation to another. Reproductive profiles were uniform in the different age groups. CONCLUSION: The element of ambivalence may be a clue to a deeper understanding of human reproductive behavior. PMID- 1733182 TI - Acute posttraumatic fetal anemia treated with fetal intravascular transfusion. AB - Fetal trauma resulting in acute anemia after maternal blunt abdominal trauma is a rare but potentially lethal condition. We recently managed such a case with the use of fetal intravascular transfusion. PMID- 1733183 TI - Serum levels of macrophage colony-stimulating factor in patients with ovarian cancer undergoing second-look laparotomy. AB - OBJECTIVE: The purpose of this study was to evaluate the prognostic significance of macrophage colony-stimulating factor serum levels in patients with ovarian cancer undergoing second-look laparotomy. STUDY DESIGN: The presurgical serum levels of macrophage colony-stimulating factor from 33 consecutive patients with ovarian cancer undergoing second-look laparotomy were determined and compared with those of 50 healthy control subjects. Mean differences in groups were evaluated with the Student t test. RESULTS: Sixteen of 33 patients had a positive result at second look and a mean serum macrophage colony-stimulating factor level of 2.31 +/- 1.45 ng/ml. Seventeen of 33 patients had a negative result at second look and a mean macrophage colony-stimulating factor level of 1.90 +/- 0.86 ng/ml (p greater than 0.05). The mean macrophage colony-stimulating factor level in the control group was 1.20 +/- 0.51 ng/ml. This was statistically lower than the mean level found in patients with a negative second-look result (p less than 0.05). CONCLUSION: Regardless of tumor status, serum macrophage colony-stimulating factor levels tend to be elevated at the time of second-look laparotomy. PMID- 1733184 TI - Population differences of fetal biophysical and behavioral characteristics. AB - Population differences in nonstress test reactivity have been reported with a threefold increase in the likelihood of nonreactive nonstress tests observed in black fetuses as compared with white fetuses. We analyzed fetal behavioral states and fetal heart rates in 14 black and 15 white fetuses at term to explain this observed difference in nonstress test reactivity. Two-hour Doppler and real-time ultrasonographic examination of each patient revealed no differences in percent time spent in the four behavioral states between the two populations. A 9.5 beats/min difference between black and white fetuses was found. The higher baseline heart rate of the black fetuses persisted in each behavioral state and may affect nonstress test reactivity because of rate-dependent decreases in short term variability and rate-dependent limitations of maximal acceleration amplitude. PMID- 1733185 TI - Abruptio placentae associated with perforated appendicitis and generalized peritonitis. AB - A primigravid woman at 35 weeks' gestation was admitted with abdominal pain, fever, and vomiting. Forceful contractions and signs of fetal distress suggested abruptio placentae. During caesarean section, seropurulent exudate and a perforated appendix were found; an appendectomy was performed. A mechanism linking appendicitis with abruptio placentae is suggested. PMID- 1733186 TI - May-Hegglin anomaly: a rare case of maternal thrombocytopenia in pregnancy. AB - The May-Hegglin anomaly, a rare cause of thrombocytopenia, is an autosomal dominant disorder that may have adverse maternal and fetal consequences. We present herein a case of May-Hegglin anomaly in pregnancy. The characteristic features of this anomaly, clinical presentation, and management options are discussed. PMID- 1733187 TI - Oral magnesium and the prevention of preterm labor in a high-risk group of patients. AB - OBJECTIVE: The null hypothesis of this study is that treatment with oral magnesium gluconate 1 gm four times daily will not decrease the rate of preterm labor and delivery in a high-risk group of pregnant women. STUDY DESIGN: Fifty four women at risk for preterm delivery were selected randomly to receive magnesium gluconate 1 gm orally four times daily or placebo. These women were monitored prospectively for signs and symptoms of preterm labor. A subgroup of 31 women also received a home uterine activity monitor. The serum magnesium level was measured initially and again 2 weeks after study enrollment. The data were analyzed with Fisher's exact test and analysis of variance. RESULTS: Preterm labor developed in 15 women in the placebo group and in 16 women in the magnesium group. There were no differences in birth weight or gestational age at delivery. The mean increase in serum magnesium level while the patients were taking magnesium gluconate was 0.10 mg/dl (p = not significant). Five women did not have an increase in serum magnesium level and preterm labor developed in all of them. CONCLUSION: Magnesium gluconate in a dose of 1 gm four times daily is not effective for preventing preterm delivery in a high-risk group of patients. PMID- 1733188 TI - Pelvic abscess complicating transcervical embryo transfer. AB - A severe pelvic infection resulting in a tuboovarian abscess after transcervical embryo transfer is reported. The case is unique in that the recipient was an agonadal woman who had not undergone prior transvaginal aspiration. Although rare, pelvic infection after embryo transfer may occur in spite of normal precautions. PMID- 1733189 TI - The effect of continuous subdermal levonorgestrel (Norplant) on carbohydrate metabolism. AB - Blood glucose and plasma insulin levels during an oral glucose tolerance test were measured in 20 women using continuous subdermal levonorgestrel (Norplant) for contraception over a 12-month period. Changes in carbohydrate metabolism occurred as early as 1 month but were most marked after 6 months. After 1 month the area under the glucose and insulin curves increased by 12.3% and 37.7%, respectively. This increase was more marked by 6 months, but by 12 months it was significantly less marked. Although changes in the blood glucose and plasma insulin levels at different times during the oral glucose tolerance test occurred, they were all within normal limits for normal women. The peak blood glucose and insulin levels after insertion of the implant occurred 60 minutes after the glucose load, as opposed to 30 minutes before insertion. Apart from this, there was a significant delay in the return of these levels to fasting values. We conclude that this product, like any other progesterone-containing contraceptive, alters carbohydrate metabolism, but these alterations are not clinically significant in normal women. It is, however, possible that in potential cases of diabetes it may predispose to frank diabetes. PMID- 1733190 TI - Urinary excretion of prostacyclin and thromboxane metabolites in threatened preterm labor: effect of indomethacin and nylidrin. AB - OBJECTIVE: We studied the role of smooth muscle-relaxing prostacyclin and its endogenous antagonist, thromboxane A2, in preterm labor by assessing the urinary output of the breakdown products of prostacyclin (6-keto-prostaglandin F1 alpha and 2,3-dinor-6-keto-prostaglandin F1 alpha) and those of thromboxane A2 (thromboxane B2, 2,3-dinor-thromboxane B2). STUDY DESIGN: Thirty-three women in preterm labor between 25 and 34 weeks of gestation were studied before, during, and after treatment with indomethacin (n = 16) or nylidrin (n = 17). Urinary prostanoid levels were determined by high-performance liquid chromatography followed by radioimmunoassay, and the excretion was expressed as nanograms of prostanoids per millimole of creatinine. Statistical analyses were done by paired and unpaired Student t test, by Spearman's correlation, and by Wilcoxon signed rank test. RESULTS: Preterm labor was accompanied by a median 32% higher output of prostacyclin and thromboxane A2 metabolites as compared with those in 25 controls. At 8 hours after the start of treatment indomethacin induced maximal drops in 6-keto-prostaglandin F1 alpha (70%), in dinor-6-keto-prostaglandin F1 alpha (60%), in thromboxane B2 (85%), and in dinor-thromboxane B2 (95%) excretion. Within 1 week after the cessation of indomethacin, output of prostacyclin metabolites had recovered to pretreatment values, whereas output of thromboxane A2 metabolites was yet lower than the pretreatment value. Nylidrin induced no change in the output of prostacyclin and thromboxane A2 metabolites. CONCLUSION: Threatened preterm labor is associated with a rise in prostacyclin and thromboxane A2 synthesis. Indomethacin inhibits more thromboxane A2 than does prostacyclin synthesis. These findings may explain the fetal vascular changes during maternal indomethacin treatment. PMID- 1733191 TI - Rapid growth of leiomyoma in patient receiving tamoxifen. AB - A 49-year-old patient with breast cancer had a leiomyoma that rapidly enlarged shortly after she started therapy with tamoxifen. Exploratory laparotomy was necessary to confirm the diagnosis. The rapid growth may have resulted from the estrogen agonist properties of tamoxifen or alternatively by ovarian stimulation resulting in endogenous estrogen production. PMID- 1733192 TI - Transient severe unilateral and subsequent bilateral primary fetal hydrothorax with spontaneous resolution at 34 weeks' gestation associated with normal neonatal outcome. AB - Unilateral primary fetal hydrothorax with spontaneous resolution is a rare occurrence. We present a case in which severe unilateral primary fetal hydrothorax was visualized at 28 weeks' gestation. Two weeks later bilateral primary fetal hydrothorax was documented. Ultrasonographic follow-up demonstrated further dynamic changes in the fetal condition, with an isolated unilateral hydrothorax 1 week later and subsequent complete resolution at 34 weeks' gestation. Delivery resulted in a normal neonate with no signs of pulmonary hypoplasia. PMID- 1733193 TI - Meconium aspiration syndrome: reflections on a murky subject. AB - Meconium-stained amniotic fluid occurs in approximately 12% of live births. In approximately one third of these infants meconium is present below the vocal cords. However, meconium aspiration syndrome develops in only 2 of every 1000 live-born infants. Ninety-five percent of infants with inhaled meconium clear the lungs spontaneously. Recent investigations have suggested that a reexamination of our assumptions about the etiology of meconium aspiration syndrome is in order. Several authors have provided evidence that support the hypothesis that it is not the inhaled meconium which produces the primary pathologic condition of meconium aspiration syndrome but rather it is fetal asphyxia that is the etiologic agent. Asphyxia in utero produces pulmonary vasospasm and hyperreactivity of the pulmonary vessels. With severe asphyxia the fetal lungs undergo pulmonary vascular damage with pulmonary hypertension. The damaged lungs are then unable to clear the meconium. In the most severe cases there is right-to-left shunting and persistent fetal circulation with subsequent fetal death. The incidence of meconium aspiration may thus be essentially unaffected by current obstetric and pediatric interventions at birth. For the asphyxiated or distressed infant we recommend suctioning at birth and tracheal intubation. In the healthy fetus observation may be sufficient. PMID- 1733194 TI - Transforming growth factor-alpha and epidermal growth factor messenger ribonucleic acid and protein levels in human placentas from early, mid, and late gestation. AB - OBJECTIVE: Human placenta expresses receptors for transforming growth factor alpha and epidermal growth factor throughout pregnancy. Experiments were performed to determine whether epidermal growth factor or transforming growth factor-alpha might be synthesized by placental cells and act through an autocrine mechanism to influence functioning of placental cells in vivo. STUDY DESIGN: Human placentas from early, mid, and late gestations were analyzed for transforming growth factor-alpha and epidermal growth factor messenger ribonucleic acid and proteins. Polyadenylic acid-positive ribonucleic acid was isolated from placentas from 10, 11, 13, 21, 32, 38, 39, and 40 weeks of gestation and analyzed by Northern analysis for hybridization with complementary deoxyribonucleic acid probes specific for epidermal growth factor or transforming growth factor-alpha. Levels of immunoreactive epidermal growth factor and transforming growth factor-alpha were measured by specific radioimmunoassays in pools of placentas from early, mid, and late gestations, and levels of epidermal growth factor and transforming growth factor-alpha receptor-active protein were measured by radioreceptor assay. RESULTS: All placentas had a strong transforming growth factor-alpha hybridization band at 4.5 kb and a weak epidermal growth factor hybridization band at 5.2 kb. High levels of transforming growth factor alpha immunoreactive protein (90 to 180 ng/mg protein) and low levels of immunoreactive epidermal growth factor (3 to 9 pg/mg protein) were detected in pools of placentas from early, mid, and late gestations. High levels of epidermal growth factor and transforming growth factor-alpha receptor-active protein (250 ng/mg protein) were also detected. CONCLUSION: Human placentas contain relatively high levels of immunoreactive and receptor-active transforming growth factor alpha, as well as transforming growth factor-alpha messenger ribonucleic acid, throughout gestation. This finding suggests that transforming growth factor-alpha may act by an autocrine system to influence human placental cell function in vivo. PMID- 1733195 TI - Familial occurrence of thrombotic thrombocytopenic purpura in two sisters during pregnancy. AB - The etiology of thrombotic thrombocytopenic purpura is not fully understood. A genetic predisposition and other inciting factors have been suggested by several studies to bring about the full-blown syndrome. We report two cases of thrombotic thrombocytopenic purpura in two sisters with sudden onset during late pregnancy. PMID- 1733196 TI - Both 17 beta-estradiol and tamoxifen induce c-fos messenger ribonucleic acid expression in human endometrial carcinoma grown in nude mice. AB - We previously demonstrated the estrogen-like effects of tamoxifen on the acceleration of growth and increased progesterone receptor concentrations of human endometrial carcinomas grown in the nude mouse experimental model. In our current study the modulation of protooncogene expression by 17 beta-estradiol and tamoxifen in human endometrial carcinomas was investigated. The protooncogenes investigated in this study were c-fos, c-jun, c-myc, N-myc, HER-2/neu, c-erbB, c fms, and c-Ha-ras. Among those we found that c-fos expression was induced by 17 beta-estradiol in the following 17 beta-estradiol-sensitive tumors: EnCa-101 and EnCa-X. The induction was apparent within 1 hour, reached peak level at 2 hours (16-fold), and remained constant up to 4 hours. The c-fos messenger ribonucleic acid returned to prestimulation level by 12 hours. Tamoxifen also stimulated c fos expression, the expression pattern being similar to that of 17 beta-estradiol albeit of a lesser degree. The messenger ribonucleic acid transcripts for other protooncogenes tested did not show significant changes during hormonal manipulation. The induction of c-fos expression by tamoxifen is consistent with its estrogen-like effect on endometrial carcinoma growth. PMID- 1733197 TI - The effect of preovulatory peritoneal fluid from cases of endometriosis on murine in vitro fertilization, embryo development, oviduct transport, and implantation. AB - OBJECTIVE: The null hypothesis of our study is that the success of in vitro and in vivo murine fertilization and embryo development is not decreased by gamete exposure to peritoneal fluid from superovulated patients with endometriosis. STUDY DESIGN: A murine in vitro fertilization model was used to test the effects of endometriosis versus nonendometriosis peritoneal fluid at concentrations of 1%, 5%, and 10% versus an unsupplemented control. Fertilization and blastocyst formation were compared by analysis of variance. In a second experiment superovulated mice were given intraperitoneal injections of endometriosis or nonendometriosis fluid or saline solution 8 hours after human chorionic gonadotropin and then mated. Some mice were killed 3 days after coitus to assess embryo number, cleavage stage, and uterine versus tubal position by means of analysis of variance and covariance with repeated measures. Others were killed 12 days after coitus with the mean number of implantations per animal between groups compared by Student's t test. RESULTS: In vitro fertilization rates decreased as peritoneal fluid concentration increased in both the endometriosis (65%, 43%, 33%) and nonendometriosis (65%, 52%, 35%) groups at 1%, 5%, and 10% peritoneal fluid concentration, respectively. Mice receiving intraperitoneal endometriosis or nonendometriosis fluid or saline solution injections showed no differences in embryo number, cleavage, uterine versus tubal position, or mean implantation number. CONCLUSION: Peritoneal fluid from superovulated patients had no differentially negative effect when compared with the effect of nonendometriosis peritoneal fluid on murine in vitro or in vivo fertilization and embryo development, tubal embryo transport, or implantation. PMID- 1733198 TI - Numeric analysis of heart rate variation in intrauterine growth-retarded fetuses: a longitudinal study. AB - OBJECTIVE: We attempted to determine changes occurring with time in fetal heart rate and its variation in fetuses with intrauterine growth retardation in whom late antepartum fetal heart rate decelerations eventually develop. STUDY DESIGN: Thirteen fetuses with intrauterine growth retardation were studied over a median period of 25 days. One-hour fetal heart rate records were made two to five times per week and were analyzed numerically. Fetal movements were recorded by the women. RESULTS: On average long-term fetal heart rate variation decreased gradually with time and fell below the norm (30 milliseconds) at about the same time decelerations appeared. Mean heart rate showed a slight but statistically significant increase after the occurrence of decelerations. There were large interfetal differences in all parameters studied. CONCLUSION: In fetuses with intrauterine growth retardation a decrease in long-term fetal heart rate variation is a rather late sign of impairment that coincides with the occurrence of late decelerations. In the surveillance of the fetus with intrauterine growth retardation it might be most appropriate to use each fetus as its own control. PMID- 1733199 TI - Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. AB - OBJECTIVE: We hypothesized that the endothelial-derived relaxing factor nitric oxide may contribute to low resting vascular tone and may attenuate vasoconstrictor action in the human fetal-placental circulation. STUDY DESIGN: Isolated human placental cotyledons were dually perfused in vitro, and the effects of N-monomethyl-L-arginine and N-nitro-L-arginine (3 x 10(-4) mol/L), which are nonmetabolizable analogs of L-arginine, the substrate for nitric oxide synthase, on resting perfusion pressure and on the fetal-placental circulation preconstricted with U46619 (10(-8) mol/L) or endothelin-1 (10(-8) mol/L) were established. Responses before and after inhibition were compared by paired t test. The effects of glyceryl trinitrate (10(-6) mol/L), acetylcholine (10(-4) mol/L), the calcium ionophore A23187 (10(-6) mol/L), and histamine (10(-8) to 10( 4) mol/L) were also determined in the preconstricted fetal-placental circulation. RESULTS: Both N-monomethyl-L-arginine and N-nitro-L-arginine (3 x 10(-4) mol/L) increased resting perfusion pressure (p less than 0.06), and N-nitro-L-arginine promptly and significantly increased perfusion pressure in the fetal-placental circulation preconstricted with U46619 (p less than 0.0004) or endothelin-1 (p less than 0.06). Nitric oxide generated by addition of glyceryl trinitrate (10( 6) mol/L) attenuated the vasoconstrictor effects of U46619 (p less than 0.026) or endothelin-1 (p less than 0.01). Neither acetylcholine nor the calcium ionophore A23187 had an effect on the fetal-placental circulation, whereas bradykinin further increased perfusion pressure. Histamine only relaxed the preconstricted preparations at concentrations (10(-6) to 10(-4) mol/L) above those shown to release nitric oxide in other systems. CONCLUSION: The stimulus to nitric oxide generation in the fetal-placental circulation may be hydrodynamic. Nitric oxide appears to contribute to maintenance of basal vascular tone and to attenuate the actions of vasoconstrictors in this circulation. PMID- 1733200 TI - Influence of terbutaline on ovine uterine response to prostaglandin E2 challenge. AB - OBJECTIVE: Our purpose was to test the effects of terbutaline on uterine electric and contractile responses to prostaglandin E2. STUDY DESIGN: In five late gestation ewes, prostaglandin E2 (22.9 +/- 2.3 micrograms/min for 3 minutes) was given twice at 30-minute intervals during control. Terbutaline sulfate (2 micrograms/min) was then infused for 30 minutes. Prostaglandin E2 challenge was repeated 10 minutes after the onset of terbutaline infusion and thereafter at 30 minute intervals. Two-way analysis of variance for repeated measures revealed a significant main effect for time (p less than or equal to 0.0001) and between time and response (p less than or equal to 0.05). RESULTS: Both electric (p less than or equal to 0.0001) and intrauterine pressure (p less than or equal to 0.0001) responses were suppressed during terbutaline. The influence on intrauterine pressure persisted 10 minutes after terbutaline (p less than or equal to 0.01) while the electric response was not different from control. CONCLUSIONS: Terbutaline initially diminishes both uterine contractile and electric activity, but electric recovery precedes contractile recovery. PMID- 1733201 TI - Circulating hyaluronic acid in nonpregnant, pregnant, and postpartum guinea pigs: elevated levels observed at parturition. AB - OBJECTIVE: Dilatation of the uterine cervix at parturition is associated with an increase in cervical hyaluronic acid content. The objective is to test the hypothesis that circulating hyaluronic acid is increased at parturition. STUDY DESIGN: Serum hyaluronic acid levels from nonpregnant (n = 5), pregnant (n = 13), and postpartum (n = 4) adult Hartley guinea pigs were determined with a radiometric assay that utilizes iodine 125-labeled hyaluronic acid-binding protein. Results were analyzed for statistical significance with Student's paired t test and regression analysis. RESULTS: The serum hyaluronic acid level in nonpregnant animals was 238 +/- 88 ng/ml (mean +/- SEM). During pregnancy, serum hyaluronic acid levels were 127 +/- 12 and 126 +/- 34 ng/ml at 25 and 50 to 63 days' gestation, respectively. At parturition, hyaluronic acid levels increased fivefold to 765 +/- 111 ng/ml (p less than 0.001). Hyaluronic acid levels returned to antepartum values 2 days post partum (153 +/- 27 ng/ml). There was no significant difference between arterial and venous levels. CONCLUSION: Circulating hyaluronic acid levels increase significantly at parturition in the guinea pig. PMID- 1733202 TI - Change in electrocardiogram T-wave amplitude during umbilical cord compression is predictive of fetal condition in sheep. AB - OBJECTIVE: The purpose of this study is to assess the usefulness of the dynamic change in T/QRS ratio in fetal electrocardiograms in predicting the fetal condition when repetitive variable decelerations are seen in intrapartum cardiotocograms. STUDY DESIGN: We investigated the relationship, using linear regression and Wilcoxon's test, between T/QRS and blood gas values, catecholamine concentrations, and blood pressure during repetitive cord compression in five chronically instrumented lamb fetuses. RESULTS: T/QRS during cord compression correlated significantly (p less than 0.01) with fetal arterial pH (r = -0.7711), norepinephrine concentration (r = 0.7551), and duration of elevated blood pressure during compression (r = -0.8619). Fetal arterial pH and base excess were lower, the duration of elevated blood pressure during compression was shorter, and carbon dioxide partial pressure and catecholamine concentrations were higher in the stage with higher (greater than 0.50) T/QRS during compression (p less than 0.005). CONCLUSION: We can estimate the severity of fetal distress by measuring T/QRS near the bottom of the decelerations. PMID- 1733203 TI - Changes in human platelet angiotensin II binding sites associated with early pregnancy. PMID- 1733204 TI - The maternal and fetal effects of milrinone and dopamine in normotensive pregnant ewes. AB - The safety of milrinone, administered during gestation, was evaluated in seven chronically instrumented pregnant ewes and their fetuses. Each of the following six intravenous regimens was administered in random order: milrinone, 75 micrograms.kg-1, over 1 minute, followed by 240-minute infusions of the drug at a rate of 1, 2, or 4 micrograms.kg-1.min-1; dopamine 5 or 10 micrograms.kg-1.min-1 over 60 minutes; or normal saline solution, 0.5 ml.min-1 for 240 minutes. Maternal and fetal acid-base parameters, heart rate, and blood pressure were monitored as were the ewe's venous and intraamniotic pressures and uterine blood flow. Milrinone concentrations determined in maternal arterial blood samples obtained during 1 and 2 micrograms.kg-1.min-1 infusions were found to be within the human therapeutic range. Bolus injection of milrinone, as well as the lowest drug infusion, resulted in no significant changes in uterine blood flow, whereas 2 micrograms.kg-1.min-1 milrinone infusion led to a 14% to 19% increase in uterine blood flow between 120 and 240 minutes. With the highest milrinone infusion, this increase was approximately 20%. In contrast, with both dopamine infusions, a dose-related decrease in uterine blood flow of 15% to 26% occurred between 15 and 60 minutes. No milrinone could be detected in any fetal plasma samples. Fetal arterial pH and blood gas tensions did not change during milrinone infusions. Dopamine 10 micrograms.kg-1.min-1 led to a progressive decrease in fetal arterial pH and an increase in PaCO2, which may have been related to similar changes in the ewe. It is concluded that milrinone has no adverse effects on uterine blood flow and fetal well-being when administered during ovine pregnancy. PMID- 1733205 TI - Periconceptional exposure to topical 5-fluorouracil. PMID- 1733206 TI - Criteria for the diagnosis of infertility. PMID- 1733207 TI - Anticancer drugs during pregnancy: are we able to discard them? PMID- 1733208 TI - The uterine stapling device for cesarean section: a positive view. PMID- 1733209 TI - The short umbilical cord. PMID- 1733210 TI - Crown-rump length dating of pregnancy at less than nine weeks' gestation. PMID- 1733211 TI - First successful cesarean section in the British empire. PMID- 1733212 TI - Eosinophilic cystitis during pregnancy. AB - The first report of a case of eosinophilic cystitis during pregnancy is presented. Premature cervical dilatation was associated with onset of the disease at 19 weeks' gestation. PMID- 1733213 TI - Upper vaginectomy for in situ and occult, superficially invasive carcinoma of the vagina. AB - Between Aug. 1, 1985, and July 31, 1990, 32 patients underwent upper vaginectomy for grade 3 vaginal intraepithelial neoplasia. Thirty-one of these patients had undergone hysterectomy, 25 because of cervical neoplasia. Fourteen patients had undergone treatment for vaginal intraepithelial neoplasia. Nine (28%) had invasive cancer on final pathologic examination. Among the remaining 23 patients, recurrence of vaginal neoplasia developed in four (17%), with a mean time to recurrence of 78 weeks, and one was found to have superficial invasion at the time of recurrence. The remaining 19 patients remain alive with no evidence of recurrent disease at a mean follow-up interval of 152 weeks. In our patients upper vaginectomy was efficacious for the diagnosis of occult invasive carcinoma of the vagina and for the treatment of in situ and superficially invasive carcinoma of the vagina. PMID- 1733214 TI - Transient symptomatic subendocardial ischemia during intravenous magnesium sulfate tocolytic therapy. AB - Although beta-sympathomimetic tocolytic therapy has been associated with transient subendocardial ischemia, magnesium sulfate has rarely been noted to cause acute chest pain and is, in fact, known to improve myocardial perfusion in patients with variant angina. We believe this report represents the first case in which intravenous magnesium sulfate administered as a tocolytic agent caused acute chest pain accompanied by transient electrocardiographic evidence of subendocardial ischemia. PMID- 1733215 TI - Main clinical types and subtypes of eclampsia. AB - Eclampsia has been traditionally divided in three types: antepartum, intrapartum, and postpartum. Several authors consider two more subtypes, early cases and intercurrent eclampsia. The clinical analysis of 990 patients with eclampsia divided according to such classification revealed numerous significant differences that could give grounds for the interpretation of conflicting results in medical research. Maternal and perinatal mortality, types and incidence of complications, obstetric and eclamptic profiles, and incidence of underlying diseases were strikingly higher in antepartum eclampsia, especially in early cases. The features of intrapartum eclampsia were closer to those of the postpartum group than to those of antepartum cases, and intercurrent eclampsia was oddly benign for the mother but not for the fetuses. These findings indicate that a more precise classification of eclampsia must consider seven differential facts: (1) timing of convulsions, (2) length of pregnancy, (3) complications , (4) underlying diseases, (5) maternal age, (6) number of index pregnancy, and (7) single or multiple gestation. PMID- 1733216 TI - Use of urinary luteinizing hormone immunoassays in the assessment of luteal function in infertile women. AB - It has been suggested that the chronologic date of an endometrial biopsy performed to evaluate luteal adequacy should be based on the date of the luteinizing hormone surge rather than the date of the next menstrual period. Sixty-four infertile women used a urinary luteinizing hormone immunoassay to identify the luteinizing hormone surge; timed serum progesterone level tests and an endometrial biopsy were then performed. An out-of-phase endometrium was identified in 26.6% of cycles dated traditionally and 28.1% of cycles dated from the luteinizing hormone surge. No relationship was identified between progesterone levels and endometrial biopsy results when the next menstrual period was used. When the luteinizing hormone surge was used no progesterone cutoff value could be identified that would reliably distinguish between in-phase and out-of-phase cycles. Use of a urinary luteinizing hormone immunoassay offers no advantage over the next menstrual period and does not lead to better agreement between histologic and chronologic dating. PMID- 1733217 TI - Intracerebral, aortic, and umbilical artery flow velocity waveforms in the late first-trimester fetus. AB - OBJECTIVES: Our objectives were to determine the success rate in obtaining flow velocity waveforms in the first-trimester fetal circulation and to establish possible preferential flow to the fetal cerebrum at this early stage of gestation. STUDY DESIGN: Flow velocity waveform recordings were made in the umbilical artery, fetal descending aorta, and fetal intracerebral arteries in 30 normal pregnancies between 11 and 13 weeks of gestation. RESULTS: Technically acceptable waveforms were obtained from the descending aorta in 15 fetuses, from the intracerebral circulation in 17 fetuses, and from the umbilical artery in all 30 fetuses. Absent end-diastolic velocities in the descending aorta and umbilical artery were associated with forward flow throughout the cardia cycle in intracerebral arteries. CONCLUSION: A relatively low cerebral vascular resistance in the late-first-trimester normal fetus is suggested. PMID- 1733218 TI - Ovarian metastases in stage IB carcinoma of the cervix: a Gynecologic Oncology Group study. AB - Surgical and pathologic findings at laparotomy for radical hysterectomy in 990 patients with clinical stage IB carcinoma of the cervix were analyzed to determine the frequency of metastases to the ovary. Ovarian spread was identified in 4 of 770 (0.5%) patients with squamous carcinoma and 2 of 121 (1.7%) with adenocarcinoma. No patients with adenosquamous carcinoma (n = 82) or other histologic types (n = 17) had ovarian metastases. Although the frequency of metastases was greater among patients with adenocarcinoma, this was not statistically significant (p = 0.19, Fisher's exact test). All 6 patients with ovarian metastases had other evidence of extracervical disease. Three underwent radical hysterectomy and bilateral salpingo-oophorectomy. Of these, one patient received extended field radiotherapy and died of disease 18 months after diagnosis. Two patients, one treated with combination chemotherapy and one with no adjunctive therapy, are alive without evidence of disease at 59 and 62 months, respectively. Three patients underwent exploratory laparotomy with salpingo oophorectomy and lymphadenectomy without hysterectomy. All three patients died of disease at 2, 3, and 30 months; the first and last patient received adjunctive radiotherapy. Not all patients underwent oophorectomy. Of 347 patients with at least unilateral ovarian preservation, no postoperative pelvic radiotherapy, and no gross extracervical disease or metastasis to the paraaortic nodes, pelvic recurrence developed in 16. There was no excess of pelvic recurrences in patients with adenocarcinoma (0/41) or adenosquamous carcinoma (1/29, 3.4%) when compared with those with squamous carcinoma (15/270, 5.6%). This suggests no excess of occult ovarian metastases in nonsquamous tumors of the cervix. There is no evidence in these data of an increased risk of ovarian preservation in patients with stage IB carcinoma of the cervix with no gross extracervical disease. PMID- 1733219 TI - Describing and interpreting 24-hour blood pressure patterns in physiologic pregnancy. AB - The time course of blood pressure in clinically healthy (pregnant and nonpregnant) women was followed by automatic ambulatory monitoring. Chronobiologic methods revealed the time course of dynamic rhythm characteristics as a function of gestational age. Differences were found between nonpregnant and pregnant women with an overall lowering during pregnancy of the rhythm-adjusted midline estimating statistic of rhythm (mesor). PMID- 1733220 TI - Derivation of new mathematic formulas for determining whether a D-positive father is heterozygous or homozygous for the D antigen. AB - OBJECTIVES: This article derives new mathematic formulas to calculate the probability that the husband of a D-negative woman is heterozygous for the D antigen. STUDY DESIGN: Current published formulas and tables that estimate this probability are biased in that they incorrectly assume that the father is randomly selected from the general population. With Bayes' theorem, new formulas were derived to eliminate this bias. RESULTS: The probability of the husband being homozygous or heterozygous for the D antigen can be expressed as a function of the number of previous D-positive children. If he has any D-negative children, he is an obligate heterozygote. CONCLUSION: Previously published formulas and tables that estimate the likelihood of the father being heterozygous or homozygous at the D locus should no longer be used in the obstetric setting. PMID- 1733221 TI - Impairment of counterregulatory hormone responses to hypoglycemia in pregnant women with insulin-dependent diabetes mellitus. AB - Intensive insulin therapy directed at elimination of hyperglycemia is advocated during pregnancy in women with insulin-dependent diabetes mellitus. Because such treatment is complicated by frequent hypoglycemic episodes, we evaluated maternal and fetal responses in nine intensively treated pregnant women with insulin dependent diabetes mellitus during an insulin-induced, gradual, controlled fall in plasma glucose levels. In contrast to values in nonpregnant control women, reductions in glucose to 44 +/- 2 mg/dl in pregnant diabetic patients failed to elicit an increase in glucagon levels. Epinephrine release during hypoglycemia was also markedly suppressed in the pregnant diabetic subjects (106 +/- 32 vs 327 +/- 52 pg/ml in controls, p less than 0.001). Furthermore, the plasma glucose level at which epinephrine and growth hormone were released was 5 to 10 mg/dl lower in the pregnant women with insulin-dependent diabetes mellitus (p less than 0.05). The basal fetal heart rate remained unchanged and continued to manifest accelerations during the hypoglycemic state. We conclude that the high frequency of hypoglycemia in intensively treated pregnant women with insulin-dependent diabetes mellitus may be due in part to impaired counterregulatory hormonal responses. PMID- 1733222 TI - Intrapartum amniotic fluid index and its relationship to fetal distress. AB - Amniotic fluid index was measured in 50 consecutive laboring women after membrane rupture. The 10th percentile of the normal range was 6.2 cm. Thirty-three women had a repeat measurement by a second observer. Although there was no systematic bias between the two observers, the limits of agreement were wide: 95% of the measurements by one observer were between 0.59 and 2.07 times those of the second. Closer agreement was observed when amniotic fluid index was low (less than 6.2 cm). The relationship between intrapartum amniotic fluid index and fetal distress was then investigated in a further 60 laboring women. When compared with women with a normal intrapartum amniotic fluid index, women with a low amniotic fluid index had higher incidences of fetal heart rate abnormalities during the first stage of labor (64% vs 20%, p less than 0.01), meconium (grade II or III) at delivery (64% vs 35%, p less than 0.05), and operative delivery for fetal distress (57% vs 17%, p less than 0.01). Umbilical artery pH and Apgar scores were, however, similar for the two groups. Measurement of intrapartum amniotic fluid index may be an appropriate method for selecting women suitable for intrapartum aminoinfusion. PMID- 1733223 TI - Colposcopy to establish physical findings in rape victims. AB - OBJECTIVE: Conventional rape examination protocols have been poor in yielding genital findings (10% to 30% typically). We report our experience with a revised protocol employing colposcopy to perform genital examinations of victims and to document findings. STUDY DESIGN: Physical examinations were performed on rape victims seen by San Luis Obispo County's Suspected Abuse Response Team between 1985 and 1990, and the results were reviewed for this study. RESULTS: Of 131 patients seen within 48 hours and who experienced penile penetration, 114 (87%) had positive findings. Colposcopic magnification allowed examiners to characterize these findings as acute mounting injuries, typically seen at 3, 6, and 9 o'clock on the posterior fourchette and consisting chiefly of lacerations, ecchymosis, and swelling. CONCLUSIONS: An examination protocol that includes colposcopy may be the most reliable means to document and characterize genital findings in rape victims and to evaluate whether findings may be linked to a reported sexual assault. PMID- 1733224 TI - Natural killer cell activity from pregnant subjects is modulated by RU 486. AB - Natural killer cells form an integral component of the body's innate immune system. Natural killer cell activity is reduced during pregnancy, especially in the latter half. To investigate the role progesterone may play in immunomodulating natural killer cell activity during pregnancy, we evaluated the effect of RU 486 on natural killer cells isolated from pregnant subjects. Natural killer cell activity was measured with an 18-hour, Chromium 51 release, microcytotoxicity assay with K-562 cells as target cells. We demonstrated that RU 486, in a concentration range from 5 to 40 mumol/L, augmented natural killer activity threefold to fivefold over baseline. This augmentation of activity was suppressed to baseline by the addition of excess progesterone. The addition of hydrocortisone resulted in an insignificant reduction in this augmented activity. This study suggests that progesterone may play a role as an immunomodulating factor in maternal acceptance of the fetal allograft. PMID- 1733225 TI - Prognosis in fetal diaphragmatic hernia. AB - The reported mortality for prenatally detected congenital diaphragmatic hernia is high. Polyhydramnios and presentation in early pregnancy have been suggested as high-risk factors adversely affecting outcome. We retrospectively reviewed 55 cases of congenital diaphragmatic hernia diagnosed prenatally in our unit. There was an overall mortality of 73%. The mortality in cases with presentation before 25 weeks' gestation was 74%, if the cases resulting in termination of pregnancy are excluded, compared with a mortality of 60% in those seen after this gestational age. Underdevelopment of left-sided cardiac structures was found to be a helpful prognostic factor. We were unable to confirm the predictive nature of hydramnios. Associated chromosomal anomalies were found in two fetuses and major congenital heart disease in nine. Although diagnosis before 25 weeks' gestation is associated with a higher mortality than in cases detected later, it is not universally fatal. If congenital heart disease and chromosomal anomalies are excluded and there is little or no evidence of left heart underdevelopment, the odds for survival will improve. This should be taken into account when the management of these cases is planned. PMID- 1733226 TI - Endothelin-1,2 levels are increased in the amniotic fluid of women with preterm labor and microbial invasion of the amniotic cavity. AB - The purpose of this study was to determine the effect of gestational age, labor, and microbial invasion of the amniotic cavity on amniotic fluid concentrations of endothelin-1,2. Amniotic fluid was retrieved by amniocentesis from 148 women: patients at term with and without labor, patients with preterm labor with and without intraamniotic infection, and women in the second trimester of pregnancy. Endothelin-1,2 was measured by a sensitive and specific radioimmunoassay. Immunoreactive endothelin-1,2 was detectable in all samples of human amniotic fluid. Advancing gestational age and spontaneous term labor did not result in changes in amniotic fluid concentrations of endothelin-1,2. Women with preterm labor and positive amniotic fluid cultures for microorganisms had higher amniotic fluid concentrations of endothelin-1,2 than did those without microbial invasion of the amniotic cavity (p less than 0.05). These results support a role for endothelins in the mechanisms responsible for preterm delivery associated with intraamniotic infection. PMID- 1733227 TI - Mechanisms for intracellular distribution of mRNA: in situ hybridization studies in muscle. AB - The intracellular distribution of mRNA in striated muscle fibers is highly ordered, as is the structural organization of the fibers' contractile apparatus. Results from in situ hybridization of muscle mRNA are reviewed in an attempt to discern the mechanisms involved in mRNA distribution and to determine its relationship to developmental, growth, and repair processes in muscle. Nonradioactively labeled complementary RNA probes allow anatomic localization of mRNA at the light and electron microscopic level. Myosin mRNA in striated muscle is concentrated around transcriptionally active nuclei, myosin mRNA is excluded by the myofibrillar mass, myosin mRNA distribution correlates with that of cytoskeletal elements, and myosin mRNA is concentrated in regions of rapid growth and repair. The even distribution of myosin mRNA along the length of myofibrils gives no indication of specific association with either the thick or thin filaments. Of the possible mechanisms directing mRNA distribution, results from in situ hybridization and other analyses support a restricted diffusion model. Diffusion of mRNA (and polysomes) is severely limited by the myofibrillar lattice. It is possible that myosin mRNA is also associated with a cytoskeletal element, which may direct the mRNA to specific intracellular locations and affect translational activity. PMID- 1733228 TI - Buthionine sulfoximine-induced cytostasis does not correlate with glutathione depletion. AB - The effects of L-buthionine-(S,R)-sulfoximine (BSO) on the proliferation of normal rat kidney fibroblasts (NRK-49F) were determined and compared with the effects of BSO on cellular glutathione (GSH) content. The proliferation rate of exponentially growing NRK-49F cells was found to be slowed in 0.01 and 0.1 mM BSO and arrested in 1.0 and 10 mM BSO. There is no retardation in the proliferation of cells cultured in 0.001 mM BSO. However, varying BSO concentrations at and above 0.1 mM did not result in concordant differences in the rate and extent of GSH depletion. A dose-dependent effect of BSO on GSH levels was observed at BSO concentrations less than or equal to 0.01 mM. BSO was found also to inhibit epidermal growth factor (EGF)-induced DNA synthesis in NRK-49F cells arrested by serum deprivation in a dose-dependent pattern dissimilar to that of BSO-induced cellular GSH depletion. Removal of BSO allowed cells to resume proliferation. Further, growth-arresting BSO treatments were found to affect neither cell viability nor colony-forming efficiency. Addition of exogenous GSH or cysteine overcame BSO inhibition of EGF-induced DNA synthesis but not BSO depletion of cellular GSH levels. BSO was further found to inhibit the uptake of cysteine, cystine, and alpha-[1-14C]-methylaminoisobutyric acid (MeAIB) by the EGF stimulated quiescent cells in a dose-dependent fashion. The results presented here thus demonstrate that BSO inhibits the proliferation of NRK-49F cells. This effect, however, does not correlate with BSO-induced cellular GSH depletion and is not due to an overt toxic effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733229 TI - Dual effects of a phorbol ester on calcium-dependent chloride secretion by T84 epithelial cells. AB - Ca(2+)-dependent secretagogues (e.g., carbachol, histamine, ionomycin, and 4 bromo-A23187) have relatively transient effects on chloride secretion, even if there is a sustained increase in cytosolic calcium ([Ca2+]i) (as for the ionophores). Because these agents increase both [Ca2+]i and protein kinase C (PKC) activity, chloride secretion might be stimulated by [Ca2+]i and terminated by PKC activity. We tested the effect of a PKC activator, phorbol 12-myristate 13 acetate (PMA), on Cl- secretion by T84 cell monolayers by measuring short-circuit current (Isc). PMA alone had no effect on Isc but potentiated increases in Isc when added 10 min or less before Ca(2+)-dependent secretagogues. Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited the increases both in [Ca2+]i and Isc induced by carbachol with or without brief PMA pretreatment. Longer preincubations with PMA inhibited Isc responses to Ca(2+)-dependent secretagogues, even when increased [Ca2+]i was sustained by ionophores. Inhibitors of PKC could reverse the inhibitory effect of PMA but did not reverse the potentiating effect. The effects of PMA on Cl- secretion were reproduced by 1,2-dioctanoyl-sn-glycerol and were mirrored by effects on K+ channel opening. Thus PMA has dual effects on chloride secretion. Initially, it exerts a stimulatory action and subsequently an inhibitory action. The stimulatory effect only occurs if Ca(2+)-dependent secretion is ongoing. The inhibitory effect of PMA is mediated by PKC and cannot be overcome by increasing [Ca2+]i. PMID- 1733230 TI - Corticosteroid receptors in cells derived from rat brain microvessels: mRNA identification and aldosterone binding. AB - B7 is a cell clone derived from rat brain microvessels. Expression of an amiloride-sensitive cationic channel has been recently established in these cells. In this study, the polymerase chain reaction (PCR) was used to amplify definite segments of mineralocorticoid and glucocorticoid receptor mRNA in B7 cells. Aldosterone binding was also characterized. Two classes of sites were detected. Aldosterone exhibited a high affinity for type I sites [dissociation constant (Kd) approximately 0.3 nM] and a lower one for type II sites (Kd approximately 20 nM). RU 28362, a highly specific glucocorticoid agonist, did not compete for type I sites. RU 28362 and dexamethasone were better competitors for type II sites than aldosterone. The sedimentation coefficients of aldosterone type I and type II complexes were approximately 9S. These characteristics are close to the one exhibited by aldosterone type I and type II receptors in rat kidney and other target tissues. In intact B7 cells, aldosterone binding expressed as number of acceptor sites per cell was higher (approximately 41,000 for type II and 8,800 for type I) than in the soluble cellular extract (approximately 18,000 for type II and 1,000 for type I). PMID- 1733231 TI - Changes in pHi associated with activation of ion secretion in avian nasal salt gland cells. AB - The fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF) was used to determine changes in intracellular pH (pHi) associated with activation of secretion in isolated cells from the salt secreting avian nasal gland. A correction procedure overcoming artifacts due to BCECF leakage is described. Resting pHi averaged 7.15 +/- 0.03 and was unaffected by the nominal removal of medium HCO3- or by the addition of the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was significantly reduced by amiloride (7.07 +/- 0.02). Muscarinic activation of secretion resulted in a rapid intracellular acidification that was compensated by mechanisms which raised pHi to restore approximately resting levels within 5 min. The principal mechanism involved was amiloride-sensitive and independent of any sustained intracellular Ca2+ concentration change. Recovery of pHi was also aided by HCO3(-)-dependent and DIDS-sensitive mechanisms not seen in the resting cell. The direction of the latter was pHi-dependent, with DIDS further decreasing pHi in acidified cells and increasing pHi in alkalinized cells. This suggests that the DIDS-sensitive pathways are activated under conditions where pHi has been shifted away from resting levels in either direction and act primarily to restore resting pHi. PMID- 1733232 TI - Na current density at and away from end plates on rat fast- and slow-twitch skeletal muscle fibers. AB - Na current density and membrane capacitance were studied with the loose patch voltage clamp technique on rat fast- and slow-twitch skeletal muscle fibers at three different regions on the fibers: 1) the end plate border, 2) greater than 200 microns from the end plate (extrajunctional), and 3) on the end plate postsynaptic membrane. Fibers were treated with collagenase to improve visualization of the end plate and to enzymatically remove the nerve terminal. The capacitance of membrane patches was similar on fast- and slow-twitch fibers and patches of membrane on the end plate had twice the capacitance of patches elsewhere. For fast- and slow-twitch fibers, the sizes of the Na current normalized to the area of the patch were as follows: end plate greater than end plate border greater than extrajunctional. For both types of fibers, the amplitudes of the Na current normalized to the capacitance of the membrane patch were as follows: end plate approximately end plate border greater than extrajunctional. At each of the three regions, the Na current densities were larger on fast-twitch fibers and fast-twitch fibers had a larger increase in Na current density at the end plate border compared with extrajunctional membrane. PMID- 1733233 TI - 1 alpha,25-(OH)2 vitamin D3 enhances expression of the genes encoding Ca(2+) binding proteins MRP-8 and MRP-14. AB - Two closely related Ca(2+)-binding proteins, migration inhibitory factor-related protein (MRP)-8 and MRP-14, are synthesized under specific conditions of myeloid cell differentiation. Because 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces myeloid cell differentiation and expression of other S-100 class calcium binding proteins, we examined the effects of 1,25-(OH)2D3 on MRP mRNA levels in human U-937 histiocytic lymphoma cells. 1,25-(OH)2D3 increased MRP-8 and MRP-14 mRNA levels in a time- and dose-dependent manner. MRP mRNA levels were maximal at 24 h and remained elevated for at least 96 h after exposure of the cells to 1,25 (OH)2D3. MRP-8 mRNA accumulation required 100- to 1,000-fold higher concentrations of 25-(OH)D3, which binds to the 1,25-(OH)2D3 intracellular receptor with 100- to 1,000-fold lower affinity. Other differentiating agents, dimethyl sulfoxide, retinoic acid, and dexamethasone, also increased levels of MRP-8 and MRP-14 mRNA. Phorbol myristate acetate enhanced MRP-14 mRNA levels to a greater extent than MRP-8 mRNA levels, suggesting differential regulation of MRP gene expression by protein kinase C. The 1,25-(OH)2D3-induced relative increase in MRP mRNA levels was not changed by a 1,000-fold reduction in extracellular [Ca2+]. Thus 1,25-(OH)2D3 is potentially a physiological modulator of MRP gene expression. Expression of the MRP-8 and MRP-14 genes may be important for differentiation of myeloid cells. PMID- 1733234 TI - Cystine uptake and glutathione level in endothelial cells exposed to oxidative stress. AB - The uptake of L-cystine into cultured human umbilical vein endothelial cells was Na+ independent and inhibited competitively by glutamate. It is concluded that the uptake of cystine in endothelial cells is mediated by system x-c. The contents of glutathione in endothelial cells decreased when the cells were cultured in cystine-free medium or in glutamate-enriched medium, suggesting that the glutathione level in endothelial cells is regulated by the cystine uptake via system x-c. The uptake rate of cystine increased three times the normal rate when the cells were exposed to H2O2 generated by glucose and glucose oxidase. This increase required protein synthesis. The cystine transport activity was also enhanced when the cells were cocultured with activated polymorphonuclear leukocytes. Intracellular glutathione levels were decreased when the cells were exposed to H2O2 despite an increase in the cystine uptake. The extracellular glutathione levels were increased at that time, suggesting the efflux of glutathione. In endothelial cells, cystine transport activity seems to be involved in the cell defense against oxidative stress. PMID- 1733235 TI - Modulation of the delayed rectifier, IK, by cadmium in cat ventricular myocytes. AB - The effects of cadmium on the delayed outward potassium current (IK) were investigated in isolated cat ventricular myocytes using the single suction pipette voltage-clamp technique. IK activation was examined using peak tail currents elicited after 750-ms voltage-clamp steps to selected membrane potentials from a holding potential of -40 mV. In the presence of Cd2+ (0.2 mM), peak tail currents increased from a control value of 85 +/- 12 to 125 +/- 18 pA (n = 4). Activation curves constructed from the average peak tail-current measurements in all experiments showed that Cd2+ shifted the voltage dependence of activation to more positive potentials by 16.4 +/- 2.0 mV and increased the slope factor of the activation curve from 6.1 +/- 0.2 to 6.9 +/- 0.2 mV. In the absence of Cd2+, increases in holding potential from -30 to -70 mV had no effect on the magnitude of the peak tail currents, suggesting that the Cd(2+)-induced increase was not the result of a voltage-dependent increase in the number of available K+ channels at the holding potential. Slow voltage ramps from -70 to +70 mV revealed that Cd2+ increased the outward current at membrane potentials positive to +20 mV and shifted the voltage range in which IK inwardly rectified to more positive potentials. The fully activated current-voltage relationship was also shifted to more positive potentials by Cd2+. Cd2+ did not alter channel selectivity for K+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733236 TI - Calcium-dependent inactivation of inwardly rectifying K+ channel in a tumor mast cell line. AB - Antigenic stimulation of rat basophilic leukemia (RBL-2H3) cells, a tumor mast cell line, is associated with an increase in intracellular free Ca2+ concentrations ([Ca2+]i) and membrane polarization. We recorded whole cell and single-channel currents through the inwardly rectifying K+ channel, a major resting conductance of cells, using the patch-clamp technique, and we examined interactions between channel activity and [Ca2+]i. With 10 microM Ca2+ in the pipette, the amplitude of whole cell currents gradually declined within 5 min to 48 +/- 13% of that immediately after rupture of the patch membrane, in the presence of 1 mM ATP which minimized intrinsic rundown. In inside-out patches, activity of the channel was reduced by increasing the concentration of Ca2+ in the internal medium, both in the presence and absence of 1 mM ATP, with no apparent change in single-channel conductance. Time-averaged mean current activity in inside-out patches in the presence of 5 microM Ca2+ was less than 50% of that with Ca2+ of 100 nM or less. These results suggest that a rise in [Ca2+]i leads to a closure of the inwardly rectifying K+ channel. In some inside-out patches, inward currents characterized by burst composed of rapid transitions between open and closed states were observed (flickering currents). Single channel properties of the flickering currents are similar to the inwardly rectifying K+ channel except for kinetics (single-channel conductance of 24.5 +/- 7.9 pS, inward rectification, and permeability to K+).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733237 TI - Exercise training, glucose transporters, and glucose transport in rat skeletal muscles. AB - It was previously found that voluntary wheel running induces an increase in the insulin-sensitive glucose transporter, i.e., the GLUT4 isoform, in rat plantaris muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). The present study was undertaken to determine whether 1) the increase in muscle GLUT4 protein is associated with an increase in maximally stimulated glucose transport activity, 2) a conversion of type IIb to type IIa or type I muscle fibers plays a role in the increase in GLUT4 protein, and 3) an increase in the GLUT1 isoform is a component of the adaptation of muscle to endurance exercise. Five weeks of voluntary wheel running that resulted in a 33% increase in citrate synthase activity induced a 50% increase in GLUT4 protein in epitrochlearis muscles of female Sprague-Dawley rats. The rate of 2-deoxy-glucose transport maximally stimulated with insulin or insulin plus contractions was increased approximately 40% (P less than 0.05). There was no change in muscle fiber type composition, evaluated by myosin ATPase staining, in the epitrochlearis. There was also no change in GLUT1 protein concentration. We conclude that an increase in GLUT4, but not of GLUT1 protein, is a component of the adaptive response of muscle to endurance exercise and that the increase in GLUT4 protein is associated with an increased capacity for glucose transport. PMID- 1733238 TI - Fluid transport across cultured bovine corneal endothelial cell monolayers. AB - The mammalian corneal endothelium is known to transport fluid from the stromal compartment to the aqueous humor, thereby maintaining corneal transparency. Corneal endothelial cells have been cultured for some years now, but whether they preserve their in vivo ability to actively transport fluid is not known. We have now grown bovine corneal endothelial cell monolayers (BCECM) on permeable substrates (Transwell) and report that, just like their counterparts in vivo, these cultured cells pump fluid from the basal to the apical compartment and display measurable electrical resistance and potential difference across the monolayer. BCECM were grown on collagen-treated permeable supports using Dulbecco's modified Eagle's medium (DMEM)/20% fetal bovine serum with antibiotics. Cells grew to confluence in 5-7 days and displayed polygonal shape. Only cells from passages 1-3 were utilized. Inserts were fitted directly into Lucite chambers specially built. The rate of fluid pumping by BCECM was 3.96 +/- 0.49 (SE) microliter.h-1.cm-2 (n = 13) and could be measured continuously for several hours; fluid pumping was inhibited by 0.2 mM amiloride. The specific electrical resistance of the monolayers was 180 +/- 22 omega.cm2 (n = 11). A mean electrical potential difference of 63.8 +/- 3.7 microV (n = 15, range 40-100 microV, apical side negative) was recorded across the monolayers in DMEM. The availability of the commercial inserts makes this procedure practical; as a consequence, the rate of fluid transport by cultured corneal endothelium has been quantitated for the first time. This method can now be extended to other cultured layers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733239 TI - Attenuated responses of muscle protein synthesis to fasting and insulin in adult female rats. AB - One-year-old adult female rats were fasted for 12 or 36 h followed by a 30-min infusion of insulin. The responses of the fractional rate of protein synthesis (Ks) in the individual muscles (measured in vivo) to fasting were small and mostly nonsignificant. After 12 h of fasting, only the epitrochlearis muscle (ET) showed a significant decrease in Ks, and, even after 36 h of fasting, a significant decrease in Ks was seen in only ET, extensor digitorum longus, and tensor fasciae latae (TFL). After the 36-h fast, infusion of insulin restored the fed Ks in all muscles except TFL. The fiber-type composition of the individual muscles appeared to influence the muscles' responsiveness to the fasting, since the highly glycolytic TFL was the most sensitive (particularly after 36 h of fasting), whereas the highly oxidative adductor longus and soleus muscles were unaffected by either fasting or insulin. In a second experiment, refeeding of fasted adult rats also had little effect on Ks, consistent with the low sensitivity to fasting shown by the first experiment. The parallel results in the two experiments confirmed that the low responsiveness to fasting and insulin infusion in these adult rats was not a result of failure to absorb in "fed" animals or insufficient levels of insulin during insulin infusions. In contrast, a third experiment showed that muscle protein synthesis in the gastrocnemius muscle from young adult (5-mo-old) female rats was significantly reduced after only 12 h of fasting. PMID- 1733240 TI - Downregulation of the renin-angiotensin system in streptozotocin-diabetic rats. AB - To determine if insulin has the ability to regulate components of the renin angiotensin system, renin and angiotensinogen mRNA and plasma concentrations were determined in 4-wk streptozotocin (STZ)-diabetic rats. In another group of STZ diabetic rats, replacement insulin therapy was given over the 4-wk period, and the above parameters were examined. In STZ-diabetic rats, there was a significant regression of white adipose tissue that was accompanied by an increase in the yield of RNA obtained. Changes in white adipose tissue were reversed by insulin replacement therapy in STZ-diabetic rats. There were no changes in brown adipose tissue weight or RNA yield in STZ-diabetic rats. Plasma renin activity (PRA) was significantly decreased in STZ-diabetic rats; however, plasma angiotensinogen concentration was not significantly affected by diabetes. PRA was restored to control levels in STZ-diabetic rats with insulin replacement. Kidney renin mRNA as well as liver, epididymal, and interscapular fat angiotensinogen mRNA were significantly decreased in STZ-diabetic rats. Renin and angiotensinogen mRNA were not significantly different from control in all tissues examined in STZ-diabetic rats with insulin replacement therapy. Results from this study suggest a downregulation of the renin-angiotensin system in 4-wk STZ-diabetic rats at the level of mRNA expression that is restored by replacement therapy with insulin; therefore, insulin may directly or indirectly regulate the renin-angiotensin system. PMID- 1733241 TI - Glucocorticoids in the CNS regulate BAT metabolism and plasma insulin in ob/ob mice. AB - Adrenalectomy stimulates depressed brown adipose tissue (BAT) metabolism and decreases hyperinsulinemia in ob/ob mice, with minimal effects in lean mice. A single intracerebroventricular injection of dexamethasone (250 ng) into adrenalectomized ob/ob mice completely reversed the effects of adrenalectomy on BAT thermogenesis as assessed by mitochondrial GDP binding, approximately doubled plasma insulin, lowered whole body metabolic rates by 17%, and increased food intake by 19%. These responses were rapid in onset, with changes in BAT metabolism and plasma insulin occurring within 30 min of dexamethasone injection. Adrenalectomized lean mice were much less responsive to dexamethasone than their ob/ob counterparts. The dexamethasone-induced decrease in BAT thermogenesis in adrenalectomized ob/ob mice was associated with an organ-specific decrease in BAT sympathetic nerve activity as assessed by norepinephrine turnover, whereas the dexamethasone-induced increase in plasma insulin was blocked by atropine, suggesting involvement of the parasympathetic nervous system. Intracerebroventricular injection of corticotropin-releasing hormone did not affect BAT thermogenesis in dexamethasone-injected adrenalectomized ob/ob mice but markedly lowered plasma insulin concentrations, possibly by suppression of the parasympathetic nervous system. In conclusion, dexamethasone alters regulation of the autonomic nervous system in ob/ob mice. PMID- 1733242 TI - Model equations for condensation biosynthesis using stable isotopes and radioisotopes. AB - Important syntheses in living systems occur by condensation reactions of the type nA----1B (where n is the number of A molecules needed to synthesize 1 molecule of B). Quantitative relationships for estimating the rate of synthesis of B from radioactive and stable isotope tracers are compared. With radioisotope tracers, only a single quantity is detected, the amount of radioactivity in B. In contrast, isotopes of varying mass produce multiple mass isotopomers B that are detected using mass spectrometry. The analysis demonstrates that the rate of synthesis of B is identifiable from stable isotope data but not from radioisotope data. This results because the isotopomer distribution of B at any time after tracer addition is a function of only the multinomial distribution representing the synthesis of B from n molecules of A and two parameters representing the fractional fluxes of isotopically enriched molecules to the sampled compartment of B. The model considers the possibility that the sampled compartment of B may not reach isotopic steady state during the experiment. A graphical method for obtaining initial estimates of the two parameters is presented. PMID- 1733243 TI - Normal plasma calcium, phosphate, and parathyroid hormone levels during 1,25(OH)2D3 infusions in rats. AB - The changes in plasma calcium, phosphate, and parathyroid hormone (PTH) levels that accompany 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administration to experimental animals represent major obstacles to determining the precise role that 1,25(OH)2D3 plays in cell function in vivo. These difficulties arise because calcium, phosphate, and PTH have major cellular effects independent of 1,25(OH)2D3. To circumvent this obstacle, we have developed an animal model in which plasma 1,25(OH)2D3 levels were raised from 20 +/- 3 to 96 +/- 19, 240 +/- 49, and 459 +/- 66 pg/ml in vitamin D-deficient rats without influencing plasma ionized calcium, total calcium, phosphate, or NH2-terminal immunoreactive PTH (irPTH) levels. The elevated 1,25(OH)2D3 levels were achieved by constant subcutaneous infusion of 1,25(OH)2D3 using osmotic minipumps. Progressive reduction in the calcium and phosphorus content of the diet as the 1,25(OH)2D3 infusion rate was increased prevented concomitant changes in plasma calcium, phosphate, and irPTH levels. This experimental model, in conjunction with our previously developed normocalcemic rat model of vitamin D deficiency, provides a powerful experimental tool for the investigation of 1,25(OH)2D3 effects in vivo in the absence of concomitant changes in other parameters of calcium homeostasis. PMID- 1733244 TI - Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism. AB - The metabolic effects of recombinant human insulin-like growth factor I (rhIGF-I) on glucose, amino acid, and free fatty acid (FFA) metabolism were examined in nine healthy nonobese subjects. Each received a 3-h primed continuous infusion of rhIGF-I (20 micrograms/kg bolus, 0.4 microgram.kg-1.min-1) while maintaining euglycemia using an exogenous glucose infusion. Total IGF-I levels increased from 125 +/- 11 to 444 +/- 22 ng/ml, and free IGF-I levels rose from undetectable to 73 +/- 5 ng/ml. Insulin levels fell from 95 +/- 9 to 64 +/- 8 pM, and C-peptide fell from 453 +/- 48 to 206 +/- 29 pM; circulating glucagon levels also declined from 72 +/- 9 to 42 +/- 4 pg/ml, rhIGF-I produced a two- to threefold increase in glucose uptake as measured by [3H] glucose (from 10.3 +/- 0.6 to 27.4 +/- 3 mumol.kg-1.m-1), and, despite the fall in insulin secretion, there was a marked 60-70% inhibition of hepatic glucose production. Furthermore, FFA and branched chain amino acids declined by 40-60% (411 +/- 58 to 165 +/- 36 and 406 +/- 23 to 219 +/- 14 microM, respectively). Our data demonstrate that rhIGF-I has potent effects on glucose (hepatic and peripheral), lipid, and amino acid metabolism in normal humans. The scope of the actions of rhIGF-I closely resemble those of insulin, despite a concomitant inhibitory effect on insulin secretion. PMID- 1733245 TI - Hypertension without peripheral insulin resistance in spontaneously hypertensive rats. AB - We performed euglycemic clamp studies with labeled glucose to measure insulin's effect on hepatic glucose output (HGO) and peripheral glucose clearance in eight conscious mobile spontaneously hypertensive rats (SHR) and eleven normotensive Wistar-Kyoto (WKY) rats age 9-10 wk. Systolic blood pressure was elevated in the SHR (P less than 0.001), whereas means of 12-h-fasted plasma insulin (P greater than 0.4), glucose (P greater than 0.07), HGO (P greater than 0.25), and glucose clearance (P greater than 0.2) did not differ significantly between groups. Infusions of human insulin into SHR and WKY rats (1 and 1.5 mU.min-1.kg-1, respectively) during concomitant somatostatin administration reestablished basal insulinemia in both groups. Neither HGO (P greater than 0.15) nor glucose clearance (P greater than 0.3) differed significantly between SHR and WKY rats under those conditions. Somatostatin plus higher-dose insulin infusions (4 mU.min 1.kg-1 in SHR and 3 or 6 mU.min-1.kg-1 in WKY rats) resulted in physiological hyperinsulinemia in all rats. Insulin sensitivity, calculated as the increase in glucose clearance effected by an increase in circulating insulin during higher dose insulin infusions, did not differ significantly between SHR and WKY groups (P greater than 0.3). HGO was completely suppressed in SHR and WKY rats during the higher-dose insulin infusions. Our data indicate that hypertension is present in SHR at an age when insulin-mediated glucose disposal is not different from age matched WKY rats. These findings do not support a role for peripheral insulin resistance in the genesis of hypertension in SHR. PMID- 1733246 TI - Increased glucose transport in nonexercising muscle. AB - Changes in blood flow and muscle glycogen in nonexercising muscle during exercise suggest that glucose transport may be increased in nonexercising muscles. Therefore, we compared 3-O-methyl-D-glucose (3-MG) transport in exercised and nonexercised perfused rat hindlimb muscles (soleus, plantaris, and red and white gastrocnemius), in the absence and presence of insulin (30 nM). Specifically, the following four treatments were used: 1) normal rest, 2) normal exercise animals (90 min running 15 m/min, 8% grade), 3) hindlimb-suspended animals at rest (90 min), and 4) hindlimb-suspended animals while exercising on the forelimbs (90 min running 15 m/min, 8% grade). In separate groups of animals, it was shown from the analyses of the electromyographic interference patterns that muscle activity was sharply reduced in hindlimb-suspended muscles both at rest and during exercise (soleus and plantaris). Glycogen decrements were also observed in nonexercising muscles during exercise (soleus, plantaris, and red gastrocnemius; P less than 0.05), although these decrements were less than in the exercised muscles (P less than 0.05). Glucose transport differed among muscles (soleus = plantaris greater than red gastrocnemius greater than white gastrocnemius), and typical increments were observed after exercise (P less than 0.05) and with insulin stimulation (P less than 0.05). An additive effect of insulin and exercise was also observed (P less than 0.05). In nonexercised muscles with no insulin in the perfusate, an increase in 3-MG transport occurred (P less than 0.05). In the presence of insulin, an increase in 3-MG transport was also observed in the nonexercised red and white gastrocnemius muscles (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733247 TI - Oxidation of leucine and alpha-ketoisocaproate to beta-hydroxy-beta methylbutyrate in vivo. AB - The oxidation of alpha-ketoisocaproate (KIC) to beta-hydroxy-beta-methylbutyrate (HMB) by the enzyme KIC dioxygenase has been previously described in rat and human liver; however, the importance of this pathway in normal leucine metabolism has not yet been assessed. A series of experiments was conducted in young lambs and pigs to determine whether HMB is produced from KIC in vivo and to estimate the importance of this pathway in leucine metabolism. In the first study, lambs were fed a bolus of KIC, and the change in plasma HMB concentration was monitored over a 24-h period. Administration of KIC increased plasma HMB from basal concentrations of 2 to approximately 7 microM 4 h after the supplementation. In the second experiment, lambs were infused with [6,6,6-2H3]KIC into the duodenum, and the appearance of labeled [2H3]HMB was measured. Under basal conditions, a minimum of 18% of the HMB was derived from KIC, but when unlabeled KIC was infused into the duodenum at a rate of 1.6 mumol.kg-1.min-1, plasma HMB concentration doubled, and essentially 100% of the HMB present was derived from KIC. In a third experiment, young pigs were infused with [6,6,6-2H3]leucine. At steady state, [2H3]-leucine and HMB enrichments were nearly identical, indicating that plasma HMB is derived solely from leucine. In a fourth experiment, both lambs and pigs were injected intravenously with 600 mg of HMB daily, and urinary HMB excretion was quantitated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733248 TI - Effect of adrenalectomy and high-fat diet on the fatty Zucker rat. AB - Lean and obese Zucker fatty rats were adrenalectomized or sham operated at 10 wk of age. At 15 wk one-half of each group was placed on a high-fat diet. At 32 wk of age the experiment was ended. Several conclusions can be drawn about the effects of adrenalectomy, high-fat diets, and their interaction in the Zucker fatty rat. First, adrenalectomy slowed the weight gain in both obese fatty rats and in the lean animals, although the effect was greater in the fatty rats. Second, weight gain was accelerated in intact lean and fatty rats eating a high fat diet. Third, adrenalectomy attenuated the weight gain associated with a high fat diet and reduced the body content of fat and protein in the lean animals and fatty rats fed the low-fat diet. Fifth, adrenalectomy significantly affected the retroperitoneal and subcutaneous fat depots but not the epididymal fat depot. Sixth, adrenalectomy decreased fat cell number in retroperitoneal and subcutaneous fat depots, but this was much less evident in the epididymal fat depot. Seventh, lipoprotein lipase activity expressed per milligram protein increased after adrenalectomy in the fatty rat but was reduced on the same basis in lean animals regardless of diet. Finally, the increase in retroperitoneal lipoprotein lipase activity expressed per fat cell observed in lean animals fed the high-fat diet was not observed in the fatty rat. These studies show that a high-fat diet and adrenalectomy interact in the development of obesity in both lean and fatty Zucker rats. PMID- 1733249 TI - Bidirectional ion transport in thyroid: secretion of anions by monolayer cultures that absorb sodium. AB - Cultured porcine thyroid cell monolayers transport Na+ in an apical-to-basal direction, resulting in the development of a basal-positive transepithelial potential difference (TEP) and the formation of domes (fluid-filled elevations of the cell layer above the culture dish substrate). Stimulation by prostaglandin E2 (PGE2) increases the magnitude of the TEP, the short-circuit current (Isc) measured in Transwell Ussing chambers, and the height of domes in cultures grown on impermeable substrates. A phenamil-resistant, PGE2-stimulated component of the Isc in Transwells and of the TEP in monolayers in conventional culture dishes was inhibitable by bumetanide, a diuretic drug that blocks NaKCl2 symporters, mediating active transport of Cl-. The rate of decrease in height of domes in cultures after addition of phenamil, presumably indicative of transport of fluid in a basal-to-apical direction, was also reduced by bumetanide. Studies with Transwells in Cl(-)-free, HCO(3-)-free or Cl(-)- and HCO(3-)-free media indicated that thyroid cells transported HCO3- as well as Cl- in a basal-to-apical direction. It was concluded that the thyroid epithelium is both sodium absorbing and anion secreting. PMID- 1733250 TI - Hepatic metabolism of glucose, galactose, and lactate after milk feeding in newborn lambs. AB - The purpose of this study was to test the ability of the liver to efficiently clear substrates absorbed from the gastrointestinal tract after a feeding before entry into the systemic circulation. We placed a hepatic vein (HV) catheter in utero at 135-140 days gestation. The lamb was then allowed to deliver spontaneously, and additional catheters were placed in the portal vein (PV) and femoral artery (FA) at 1-3 days postnatal age. After at least 2 days recovery, lambs were fasted overnight at 4-10 days of age and then allowed to nurse ad libitum. There was significant hepatic glucose release during fasting and after 80 min postprandially. No net hepatic uptake of glucose was observed before or after feeding. PV galactose was significantly greater than FA and HV from 40 to 160 min after feeding (P less than 0.005). Hepatic glucose extraction was negligible in the fasted state (1 +/- 3%) and increased after feeding to 75 +/- 2% when PV galactose was less than 1.0 mM. There was a significant hepatic arteriovenous concentration difference of lactate (0.23 +/- 0.03 mM) and of oxygen (0.27 +/- 0.01 mM), which did not change significantly after feeding. The metabolic quotient for galactose increased significantly after feeding, such that galactose was the largest carbon contributor for postprandial hepatic carbon accretion. After a milk feeding, the newborn liver efficiently extracts galactose, lactate, and oxygen, but not glucose. PMID- 1733251 TI - Transcriptional and posttranscriptional mechanisms in uncoupling protein mRNA response to cold. AB - Three mechanisms account for the rapid elevation and maintenance of uncoupling protein (UCP) mRNA levels in cold-exposed rats, namely, an increase in the rate of transcription initiation, an increase in the fraction of nascent UCP transcripts undergoing elongation, and stabilization of the mature UCP mRNA. The second mechanism precedes and outlasts the increase in the rate of UCP gene transcription, which is brisk but short lived. After 48 h of cold exposure, mature UCP mRNA levels are maintained elevated solely on the basis of stabilization, since the levels of both transcription initiation and fifth intron containing transcripts (precursors) have returned to basal. Results in hypothyroid rats given 3,5,3'-triiodothyronine (T3) and in dispersed brown adipocytes show that T3 is involved both in the increase in UCP mRNA precursor level and stabilization of mature UCP mRNA. These mechanisms are rapidly reversed when the rats are returned to thermoneutrality. These coordinated transcriptional and post-transcriptional mechanisms modulating UCP gene expression ensure a rapid increase in the concentration of UCP and prevent further accumulation of the protein as physiologically adequate levels are attained. PMID- 1733252 TI - Hepatic insulin and EGF receptor phosphorylation and dephosphorylation in fetal rats. AB - Hepatic insulin receptor and epidermal growth factor (EGF) receptor phosphorylation and dephosphorylation were studied in normal and growth-retarded fetal rats. Insulin receptor autophosphorylation at a subsaturating ATP concentration (0.5 microM) increased by 10-fold from day 17 to 21 of gestation and decreased by 50% in term growth-retarded fetuses of fasted mothers. In vitro kinase activation at 0.5 mM ATP did not change with gestation or maternal fasting. EGF receptor autophosphorylation increased in parallel with receptor number with advancing gestation and did not change with maternal fasting. Protein tyrosine phosphatases (PTPases), which might attenuate receptor signaling in livers from growth-retarded fetuses, were measured using polybasic and polyacidic artificial substrates as well as the insulin receptor kinase domain. Fetal membrane PTPase activities were twofold higher than in the adult and declined with advancing gestation. However, activities were similar in normal and growth retarded fetuses. We conclude that decreased hepatic growth in growth-retarded fetuses may involve decreased insulin receptor tyrosine kinase activation in vivo, as indicated by diminished receptor autophosphorylation at subsaturating ATP concentrations. Changes in EGF receptor kinase activity and PTPases could not be implicated based on our in vitro findings. PMID- 1733253 TI - Effect of osmolality on cytosolic free calcium and aldosterone secretion. AB - Alterations in extracellular osmolality have powerful inverse effects on basal and potassium- and angiotensin-stimulated aldosterone secretion. With the use of bovine glomerulosa cells grown in primary culture, the effects of alterations in osmolality on cytosolic calcium concentration ([Ca2+]c), efflux and uptake of 45Ca2+, and aldosterone secretion were determined. Alterations in osmolality, independent of sodium concentration, have inverse effects on aldosterone secretion, which are correlated with simultaneous changes in [Ca2+]c measured using fura-2. Reductions in osmolality cause dose-dependent biphasic increases in [Ca2+]c different from the monophasic increases in [Ca2+]c produced by increases in potassium concentration. Like potassium- and angiotensin-stimulated increases in [Ca2+]c, hypotonically induced increases in [Ca2+]c are associated with an increase in 45Ca2+ efflux. Reductions in osmolality also increased the uptake of 45Ca2+, an effect apparent at 2 min and persistent for at least 30 min. In the absence of extracellular calcium, reductions in osmolality, as increases in potassium concentration but not angiotensin, fail to increase [Ca2+]c, efflux of 45Ca2+, or aldosterone secretion. In conclusion, osmolality-induced alterations in aldosterone secretion are associated with parallel changes in [Ca2+]c, effects caused by alteration in the influx of extracellular calcium. On the basis of these and previous studies, we hypothesize that osmolality affects calcium influx by activating voltage-dependent or stretch-activated calcium channels. PMID- 1733254 TI - Effects of amiloride analogues on AVP binding and activation of V1-receptor expressing cells. AB - We tested the interactions between amiloride analogues and V1 vascular arginine vasopressin (AVP) receptors of human platelets, rat glomerular mesangial cells, and A7r5 smooth muscle cells by using radioligand binding techniques, intracellular calcium monitoring, platelet aggregation, and cell contraction techniques. Amiloride analogues were competitive inhibitors of both the agonist [3H]AVP and the tritiated V1 antagonist [1-(beta-mercapto-beta,beta cyclopentamethylenepropionic acid), 2-O-methyl)tyrosine]AVP ([3H]d(CH2)5Tyr(Me)AVP) binding to V1 AVP receptors in the three different cell types used. The order of potency was ethylisopropyl amiloride (EIPA) greater than Benzamil greater than amiloride. AVP mobilization of intracellular calcium was blocked by the V1 antagonist d(CH2)5Tyr(Me)AVP and was reduced by EIPA in a dose dependent manner. Moreover, EIPA also inhibited prostaglandin F2 alpha mobilization of intracellular calcium. Alkalinization of the intracellular pH with ammonium chloride reversed the inhibitory effect of EIPA but not that of the V1 antagonist on AVP-induced calcium mobilization. Both amiloride and EIPA blocked AVP-induced aggregation of human platelets and contraction of mesangial cells and glomeruli preparations independently of receptor site antagonism. In conclusion, amiloride analogues interfere with activation of V1 vascular receptors by AVP at different levels including binding to the receptor site, mobilization of intracellular calcium, cell contraction or aggregation, and presumably alteration of intracellular ion transports. PMID- 1733255 TI - Protein kinase C differentially modulates PTH- and PGE2-sensitive adenylate cyclase in osteoblast-like cells. AB - The effects of phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, on receptor-mediated stimulation of adenylate cyclase were evaluated in a rat osteosarcoma cell line (UMR-106) with the osteoblast phenotype. Pretreatment of UMR-106 cells with PMA increased parathyroid hormone (PTH)-stimulated adenylate cyclase activity and inhibited prostaglandin E2 (PGE2) responsive enzyme activity. In addition, PMA enhanced enzyme activation by forskolin, which is thought to exert a direct stimulatory action on the catalytic subunit of adenylate cyclase. The regulatory effects of PMA were concentration dependent and of rapid onset (less than or equal to 1 min). Treatment with PMA also resulted in translocation of protein kinase C activity from the cytosol to the particulate cell fraction. Pertussis toxin, which attenuates inhibition of adenylate cyclase mediated by the inhibitory guanine nucleotide-binding regulatory protein (Gi), augmented PTH-sensitive adenylate cyclase activity and reduced the incremental increase in PTH response produced by PMA. The results suggest that activation of protein kinase C increases PTH-stimulated adenylate cyclase activity by actions on Gi and/or the catalytic subunit and decreases PGE2 responsiveness by a mechanism involving the PGE2 receptor. PMID- 1733256 TI - Role of nitric oxide as a mediator of internal anal sphincter relaxation. AB - The studies were performed in in vitro to examine the role of nitric oxide (NO) in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of the internal anal sphincter (IAS) smooth muscle strips of opossums. NO caused a concentration dependent fall in the resting tension of the IAS. The inhibitory action of NO may be exerted directly on the IAS smooth muscle since it was not modified by the neurotoxin tetrodotoxin (1 x 10(-6) M), which abolished the neurally mediated fall in the IAS tension. The inhibitor of NO synthesis NG-nitro-L-arginine (L NNA) produced concentration-dependent suppression of the neurally mediated fall in the IAS tension. The suppression of the neurally mediated IAS relaxation was stereoselective because D-NNA had no effect on the control responses. The suppressant action of L-NNA was selectively reversed by L-arginine in a concentration-dependent manner. The reversal was complete with 3 x 10(-4) M L arginine. D-Arginine on the other hand, at the same concentration had no effect on L-NNA-suppressed IAS relaxation. Interestingly, the fall in the IAS tension caused by vasoactive intestinal polypeptide (VIP) (an inhibitory neurotransmitter in the IAS) was also inhibited by L-NNA (3 x 10(-5) M). From these data we conclude that NO or NO-like substances serve as important inhibitory mediators for the NANC nerve-mediated IAS relaxation. A part of the inhibitory action of VIP on the IAS involves NO-synthase pathway. The exact site of formation and release of NO or NO-like substances in response to NANC nerve stimulation remain to be investigated. PMID- 1733257 TI - Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance. AB - The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation. PMID- 1733258 TI - Acetylcholine release from colonic submucous neurons associated with chloride secretion in the guinea pig. AB - Electrical field stimulation of submucous neurons in the guinea pig distal colon evokes an increase in chloride secretion sensitive to cholinergic blockade. This study was undertaken in the guinea pig to determine the feasibility of measuring acetylcholine (ACh) release simultaneously with ion transport in sheets of colonic submucosa/mucosa set up in flux chambers modified for perfusion of the submucosal surface. Release of [3H]ACh was determined in the absence of cholinesterase inhibitors as the stimulus-evoked outflow of 3H from preparations preloaded with [3H]choline. [3H]ACh released in response to electrical stimulation correlated with short-circuit current at frequencies from 0.5 to 10 Hz. At 5 and 10 Hz, the stimulus-evoked release of [3H]ACh decreased during subsequent stimulation periods. The stimulus-evoked increase in [3H]ACh was attenuated by tetrodotoxin. [3H]ACh release evoked at stimulus frequencies of 0.5 10 Hz was not altered by atropine despite a reduction in short-circuit current. This study illustrates the feasibility of measuring ACh release simultaneously with ion transport in flux chambers. The results provide new information on the response characteristics of colonic submucous neurons and provide direct evidence for regulation of chloride secretion by ACh. PMID- 1733259 TI - CRF in the paraventricular nucleus mediates gastric and colonic motor response to restraint stress. AB - The role of corticotropin-releasing factor (CRF) in the paraventricular nucleus of the hypothalamus (PVN) in the control of gastric emptying of a nonnutrient meal and colonic transit was investigated in conscious fasted rats chronically implanted with hypothalamic cannulas and catheters in both the stomach and proximal colon. CRF (0.06-0.6 nmol) microinfused unilaterally into the PVN resulted in a dose-dependent inhibition of gastric emptying (0-51%) and stimulation of colonic transit (0-93%). CRF (0.6 nmol)-induced delay in gastric emptying was inhibited by 50% by subdiaphragmatic vagotomy or atropine methyl nitrate (1 mg/kg ip), whereas the stimulation of colonic transit was completely abolished by atropine methyl nitrate and reduced by 19% by vagotomy. Microinfusion of CRF (0.6 nmol) into the lateral hypothalamus or central amygdala had no effect. Restraint exposure for 1 h delayed gastric emptying and stimulated colonic transit by 28 and 78%, respectively. Bilateral microinfusion of the CRF antagonist alpha-helical CRF-(9-41) (13 nmol) into the PVN before restraint abolished stress-induced alterations of gastric and colonic transit. The CRF antagonist did not alter basal gastric and colonic transit under basal conditions. These data indicate that the PVN is a specific responsive site for central CRF-induced alterations of gastric and colonic transit and suggest that endogenous CRF in the PVN plays a role in mediating restraint stress-related alterations of gastrointestinal transit. PMID- 1733260 TI - Contribution of the large hepatic veins to postsinusoidal vascular resistance. AB - We tested the hypothesis that the larger (greater than 2 mm ID) hepatic veins are the primary site of the portal-to-caval venous pressure gradient in the dog. Double-lumen catheters were inserted through the caval wall into hepatic veins of pentobarbital sodium-anesthetized dogs. One lumen opened at the end, and the other to the side. Each catheter was advanced until stopped and then it was withdrawn. The pressure at either port dropped from 87 +/- 31 to 13 +/- 11% of the portal-to-caval pressure difference as each moved past a transition point (TP). The location of the TP depended on the catheter diameter. Intraportal histamine or norepinephrine, 4 and 2.6 micrograms.min-1.kg body wt-1 respectively, augmented only the pressure measured upstream to the TP. A mathematical model of flow through a vessel with a catheter inside predicted a marked increase in resistance when the ratio of catheter OD to vessel ID exceeded approximately 0.6. Autopsy revealed ratios greater than 0.6 upstream to the TP. A hydraulic model confirmed that this effect caused the appearance of the TP. Given the depth (11.7 cm) at which near caval pressures could be found, even during histamine administration, we conclude that the major pressure gradients in the canine liver must lie upstream to the large hepatic veins. PMID- 1733261 TI - Characterization of the antigen-induced contraction of colonic smooth muscle from sensitized guinea pigs. AB - To study the potential of inflammatory mediators to alter colonic motility, we characterized the response of distal colonic smooth muscle to antigen challenge. Addition of ovalbumin to isolated segments of circular smooth muscle obtained from sensitized guinea pigs produced a biphasic contraction. The initial response consisted of a rapid contraction followed by a late response, which was a more sustained but smaller increase in tone and phasic activity. Interestingly, these two responses could be antagonized differentially. Pretreatment with mepyramine (10 microM) inhibited the initial response, whereas the leukotriene antagonist WY 48252 (10 microM) inhibited the late response. The mast cell stabilizer doxantrazole (0.1 microM) reduced only the late response. Inhibition of cyclooxygenase with meclofenamic acid (1 microM) potentiated both responses, whereas blocking neuronal activity with tetrodotoxin (1 microM) only enhanced the initial response. These data indicate clear differences between the inflammatory mediators important in the initial vs. the late response. The initial response is probably mediated by the release of histamine, with enteric neuronal interactions important in attenuating the magnitude of this response. In contrast, the late response appears to be mediated by the release of peptidyl leukotrienes. In this system, cyclooxygenase products apparently function to decrease the response of the smooth muscle to these mediators. These results suggest that release of mediators during an inflammatory response could profoundly alter colonic motility and that these alterations may be important in the pathophysiological manifestations associated with colonic inflammation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733262 TI - Transport of 5-methyltetrahydrofolate in basolateral membrane vesicles of rat liver. AB - We examined 5-methyltetrahydrofolate transport across the basolateral membrane (BLM) of rat liver using isolated membrane vesicles. Uptake of 5 methyltetrahydrofolate by BLM vesicles (BLMV) was found by osmolarity and initial uptake studies to be mostly the result of transport of the substrate into an active, intravesicular space with some binding (approximately 25%) to membrane surfaces. Uptake was similar in the presence of an inwardly directed Na+ or K+ gradient, indicating that transport is not dependent on Na+. Uptake of 5 methyltetrahydrofolate was linear for the first 30 s and was more rapid when an initial pH gradient was imposed across the vesicular membrane [extravesicular pH (pHo) = 5.0, intravesicular pH (pHi) = 7.5]. Under pH gradient conditions, uptake at 3-5 min was about twofold higher than at equilibrium (60 min). The initial rate of uptake at pHo = pHi = 5.0 was more rapid than at pHo = pHi = 7.5, but in neither case was uptake above equilibrium observed. Uptake under pH gradient conditions and at pH 5.0 (no pH gradient) was saturable (apparent Michaelis constants of 0.58 and 0.36 microM, respectively; maximum uptake rates of 1.25 and 0.79 pmol.10 s-1.mg protein-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733263 TI - Surface hydrophobicity of the intestinal tract. AB - To quantitate surface hydrophobicity of the intestine, we measured contact angles formed with water droplets in multiple regions of rabbit intestine at varying ages (suckling, weanling, and adult) and after dinitrochlorobenzene-induced colitis. Contact angles were measured using novel methods: axisymmetric drop shape analysis-contact diameter for contact angles less than 90 degrees and axisymmetric dropshape analysis-maximum diameter for contact angles greater than 90 degrees. To determine whether mucus was present on the surface of intestine used, indirect immunofluorescence was performed using antibody specific to goblet cell mucin. To confirm that intestinal mucus could be responsible for the physical properties of surface mucosa, surface tensions of mucus prepared from distal ileum, distal colon, and inflamed distal colon of adult rabbits were measured by axisymmetric drop-shape analysis on pendant drops. Contact angles of adult small intestine [duodenum, 38.0 +/- 11.2 degrees (SD); jejunum, 44.0 +/- 22.9 degrees; ileum, 56.4 +/- 23.3 degrees] were less than proximal colon (93.2 +/- 6.7 degrees; P less than 0.05) and distal colon (86.4 +/- 24.2 degrees; P less than 0.05). Contact angles on proximal colon from suckling rabbits (53.2 +/- 8.4 degrees) were less than both weanling (93.2 +/- 23.3 degrees; P less than 0.05) and adult rabbits (93.2 +/- 6.7 degrees; P less than 0.05). Contact angles on inflamed adult distal colon (54.7 +/- 20.6 degrees) were decreased from values on normal distal colons (86.4 +/- 24.2 degrees). Indirect immunofluorescence demonstrated that mucin was present in both vacuoles of goblet cells and on the colonic surface.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733264 TI - Ontogeny of membrane and soluble amino-oligopeptidases in rat intestine. AB - Intestinal amino-oligopeptidase (AOP) plays an essential role in protein digestion. To characterize its postnatal development, we measured AOP activity in intestinal membrane and cytosolic fractions in suckling and weaned rats, compared the subunit structures of the membrane and soluble enzymes, and assessed the biochemical relationship of these peptidases. At weaning, jejunal membrane AOP activity doubled while soluble AOP activity in the ileum fell abruptly. The maturational increase in the molecular mass of ileal membrane AOP was due to alterations in the N-linked glycosylation of this protein. Ileal membrane and soluble AOP exhibited similar substrate affinities, pH optima, inhibition characteristics, and antigenic epitopes. However, soluble AOP was 25-35 kDa smaller than the membrane enzyme. Peak incorporation of [35S]methionine into ileal brush-border AOP preceded maximal radioactivity in soluble AOP, suggesting that the membrane peptidase is a precursor of the soluble enzyme. We conclude that membrane and soluble AOP are closely related proteins with distinct developmental profiles and that the soluble peptidase may be derived from endocytosis of the membrane enzyme. PMID- 1733265 TI - Leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. AB - Leuprolide acetate, a gonadotropin-releasing hormone (GnRH) analogue, is currently being proposed to control debilitating symptoms in women with functional bowel disease. Whether leuprolide alters gastrointestinal motility as part of its actions is unknown. This study was designed to assess, using myoelectric techniques in an animal model, the effects of leuprolide on potential mechanisms of neuromuscular function of small intestine. Female rats with (n = 6) or without (n = 8) bilateral ovariectomy were used to study jejunal motility before and after leuprolide therapy. Throughout the study, daily leuprolide dosages of 0.02, 0.2, or 0.4 micrograms/kg were injected into intact rats and 0.02, 0.2, 0.4, 1.0, or 2.5 micrograms/kg into ovariectomized rats. Recordings were made while the rats were fasted and postprandial and before and after leuprolide administration. Under control conditions, migrating myoelectric complexes (MMCs) were found in intact female rats, whether fasted or postprandial. After ovariectomy, postprandial controls and those treated with low dose leuprolide (0.02, 0.2, and 0.4 micrograms) had typical fed-state patterns and no MMCs, but at 1.0 and 2.5 micrograms the fed state was inhibited and cycling MMCs occurred at a frequency similar to that of fasted controls. Reproductive hormones thus have a significant effect on gastrointestinal motility. PMID- 1733266 TI - The ins and outs of albumin-dependent hepatic uptake. PMID- 1733267 TI - Studies in vivo and in vitro on effects of PGE2 on colonic motility in rabbits. AB - Prostaglandins (PG) of the E series are synthesized throughout the gastrointestinal tract, and their elevated levels have been reported in many diarrheal states, including inflammatory bowel disease. It is already known that PGE2 has region-specific and muscle layer-specific effects in different areas of the intestine. The aim of this study was to evaluate possible dose-related motor effects of PGE2 on rabbit proximal and distal colon both in vivo and in vitro. We found that, in the proximal colon in vivo, PGE2 caused inhibition of myoelectric and mechanical activity at low doses but at higher doses caused marked excitation. Under the same experimental conditions, PGE2 caused only excitation in the distal colon, a phenomenon associated with an increase in antegrade contractions and diarrhea. In vitro, PGE2 caused excitation of both proximal and distal colonic longitudinal muscle and relaxation of the circular muscle. Its actions, however, were much more pronounced in the distal region. It is concluded that PGE2 has profound effects on colonic motility that are concentration dependent and that differ with the region of the colon under study. Furthermore, the evidence also suggests that elevated PGE2 levels in disease states may play a significant role in abnormal colonic motility and may facilitate the onset of diarrhea. PMID- 1733268 TI - Release of cholecystokinin and secretin by sodium oleate in dogs: molecular form and bioactivity. AB - The release of cholecystokinin (CCK) and secretin into both circulation and duodenal lumen, after intraduodenal perfusion with sodium oleate or oral ingestion of fat, was studied in anesthetized and conscious dogs, respectively. Intraduodenal infusion with sodium oleate (4 mmol.kg.-1.h-1, pH 9.5) in anesthetized dogs with diversion of bile and pancreatic juice stimulated the release of both CCK and secretin not only into the circulation but also into the duodenal lumen. The concentration of CCK and secretin in the luminal perfusate increased from 0.2 +/- 0.1 to 2.1 +/- 0.4 nM and 0.34 +/- 0.16 to 2.59 +/- 0.63 nM, respectively. Intraduodenal infusion of NaHCO3 solution at pH 9.5 did not result in release of either hormone. Luminal release of both hormones was also observed by intraduodenal infusion of sodium oleate in the dogs without diversion of bile and pancreatic juice, albeit at lower concentrations than those released in the dogs with diversion. Analysis of the molecular form of luminal secretin by gel filtration, ion-exchange chromatography, and high-performance liquid chromatography showed only a single form of secretin with molecular size, hydrophobicity, and charge similar to those of natural porcine secretin. In contrast, multiple forms of CCK were released into both circulation and duodenal lumen with CCK-58 as the predominant form. In conscious dogs, CCK-58 was also found to be the predominant form of CCK released into the circulation after oral ingestion of fat.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733269 TI - Prostaglandin stimulates Cl(-)-HCO3- exchange in amphibian oxynticopeptic cells. AB - Prostaglandins, shown to stimulate Cl- transport in epithelial cells of several different tissues, protect gastric mucosa against physiological injury induced by luminal acid. To clarify the relationship between the stimulation of Cl(-) transport and the protection of gastric mucosa, the effect of prostaglandin on Cl(-)-HCO3- exchange in oxynticopeptic cells (OPC) was examined in intact sheets of in vitro frog gastric mucosa, in which OPC were selectively loaded with the pH sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF). In omeprazole (0.3 mM)-pretreated frog fundic mucosae, in which H+ secretion was totally inhibited, 16,16-dimethyl prostaglandin E2 (dmPGE2) induced a significant decrease in intracellular pH (pHi) in OPC simultaneously with a significant increase in pHi in adjacent muscularis mucosae, an effect abolished by removal of ambient Cl- or addition of 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DIDS) (0.5 mM). dmPGE2 accentuated the rates of alkalinization of OPC after either removal of ambient Cl- or addition of serosal H2DIDS. During exposure to luminal or serosal acid, dmPGE2 significantly attenuated acidification of OPC induced by the exogenous H+, effects abolished either by removal of ambient Cl- or by addition of H2DIDS (0.5 mM). These results suggest that 1) dmPGE2 stimulates extrusion of HCO3- through the basolateral Cl(-)-HCO3- exchanger in resting OPC (H+ secretion inhibited) and that 2) relatively high extracellular [HCO3-] on the basolateral surface afforded by dmPGE2 protects OPC from acidification during exposure to luminal or serosal acid. PMID- 1733270 TI - Erythromycin contracts rabbit colon myocytes via occupation of motilin receptors. AB - Erythromycin stimulates gastroduodenal motility via action on motilin receptors. We evaluated erythromycin as a colonic muscle motilin agonist using in vitro rabbit colon studies. Isolated myocytes contracted to erythromycin with a half maximal effective concentration of 2 pM and peak shortening of 22.4 +/- 2.5% at 1 nM, which was superimposable with the response to motilin. 125I-labeled motilin binding to colon muscle homogenates was saturable and specific with a dissociation constant (Kd) of 0.39 nM and maximal binding (Bmax) of 41 +/- 3 fmol/mg protein. Motilin displaced specifically bound 125I-motilin, with a Kd of 0.31 nM. Erythromycin displaced 125I-motilin but was less potent, with an inhibitory constant of 84.0 nM. Bmax values from displacement studies were similar to the Scatchard data. Motilin receptor protection from alkylation by N ethylmaleimide preserved contraction to motilin and erythromycin but not acetylcholine or cholecystokinin, whereas protection with erythromycin preserved contraction to motilin but not other agonists. In conclusion, erythromycin binds to colon muscle motilin receptors present in densities similar to reported values for the upper gut. Furthermore, erythromycin contracts colonic myocytes via specific action on motilin receptors. Thus erythromycin may have colonic motor stimulating properties by action on motilin receptors. PMID- 1733271 TI - Effect of fluid perfusion and cleansing on canine colonic motor activity. AB - We investigated the effect of absorbable and nonabsorbable fluid perfusion and cleansing on colonic motor activity in eight intact conscious dogs. Each dog was instrumented with an indwelling catheter in the proximal colon and seven strain gauge transducers on the entire colon. After an overnight fast, a control recording was made for 3 h, followed by 3 h of perfusion and 3 additional h of postperfusion recording. Next day, a 3-h recording was made when the colon was empty. The colon exhibited normal migrating and nonmigrating motor complexes in the control uncleansed state. The perfusion of absorbable electrolyte or nonabsorbable Colyte solution immediately disrupted the migrating motor complexes and replaced them with almost continuous but irregular contractions at all recording sites. Both solutions significantly prolonged the mean and total duration per hour of contractile states in the proximal, middle, and distal colon. The dogs began to leak fluid stools in squirts approximately 40-80 min after the start of perfusion. This type of incontinence was not associated with any specific type of motor activity. Infrequently, giant migrating contractions occurred during perfusion and caused explosive diarrhea. The migrating motor complexes remained disrupted during the 3-h postperfusion period. However, on the next day, the empty colon exhibited normal migrating motor complexes. The frequency of giant migrating contractions during perfusion and in the empty colon was significantly greater than that in the normal uncleansed colon. The total duration per hour of colonic motor activity in the empty colon was also greater than that in the normal uncleansed colon. We conclude that excessive fluid in the colon significantly alters its motor pattern.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733272 TI - Hypothalamic unitary responses to gastric vagal input from the proximal stomach. AB - Gastric vagally evoked extracellular unitary responses were recorded in the hypothalamus of anesthetized cats. The evoked unitary responses were localized in the paraventricular dorsomedial region, ventromedial nucleus, and lateral hypothalamus. The mean latency of the gastric vagally evoked hypothalamic neuronal responses in these three areas ranged from 368 +/- 39.8 to 371 +/- 45.2 (SD) ms. The majority (82%) of the gastric vagally evoked hypothalamic responses consisted of one to five spikes, while the remaining 18% were tonically active units. The vagal effect was inhibitory in 78% of the tonically active hypothalamic units responding to gastric vagal input. Convergence of gastric vagal input on single hypothalamic units from afferents in the dorsal and ventral vagal trunks was observed. Units were identified in the hypothalamus that responded to activation of mechanoreceptors in the proximal stomach by an intragastric balloon. This study provided new direct evidence of the density, localization, and characteristics of neuronal processing of gastric vagal input from the proximal stomach in the hypothalamus. The reciprocal connections between these areas of the hypothalamus and nucleus tractus solitarius in the caudal brain stem suggest that the hypothalamus may serve an important role in modulating the input of primary vagal afferent input from the proximal stomach. PMID- 1733273 TI - Measurement of hepatic blood flow after severe hemorrhage: lack of restoration despite adequate resuscitation. AB - Although Ringer lactate (RL) is routinely used for resuscitation, it is not known whether the volume of RL that restores cardiac output after severe hemorrhagic shock also restores the depressed effective hepatic blood flow (EHBF). To study this, a 5-cm midline laparotomy was performed in rats (i.e., trauma induced), and the animals were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of RL. Animals were then resuscitated with four or five times the volume of maximum bleedout with RL. EHBF was determined during hemorrhage and at various intervals thereafter by an in vivo indocyanine green (ICG) clearance technique and corrected by the appropriate hepatic extraction ratio for ICG. Cardiac output was determined by ICG dilution, and hepatic microvascular blood flow (HMBF) was measured with laser Doppler flowmetry. In addition, hepatic blood flow was assessed by using radioactive microspheres. Results indicate that resuscitation markedly improved but did not restore the depressed EHBF after trauma and hemorrhagic shock despite the fact that cardiac output was restored. Similar changes in EHBF, HMBF, and hepatic blood flow as determined by microspheres were observed, suggesting that the in vivo ICG clearance is a reliable method to assess effective hepatic perfusion. Thus the lack of restoration of EHBF may be responsible for the subsequent hepatocellular dysfunction after trauma and severe hemorrhage. PMID- 1733274 TI - Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. AB - The absorption and lymphatic transport of peroxidized MaxEPA fish oil was studied using the lymph fistula rat to determine the role of mucosal glutathione (GSH) in intestinal metabolism of luminal lipid hydroperoxides. Decreasing intestinal GSH concentrations with buthionine sulfoximine (BSO, 1.15 +/- 0.20 nmol/g), diethyl maleate (DEM, 0.93 +/- 0.26 nmol/g), phorone (1.46 +/- 0.14 nmol/g), or 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU, 1.54 +/- 0.18 nmol/g) compared with control (2.60 +/- 0.38 nmol/g) resulted in higher luminal recovery of the infused lipid hydroperoxide (% of infused dose): BSO (87.8 +/- 4.8%), DEM (86.1 +/- 1.3%), phorone (78.1 +/- 2.1%), and BCNU (71.7 +/- 4.8%) compared with control (52.8 +/- 4.3%). These results suggest that decreased elimination of luminal peroxidized lipids is associated with decreased tissue GSH. Treatment of rats with BSO, DEM, phorone, or BCNU resulted in dramatic increases in appearance of peroxidized lipids in lymph over 6-h lipid infusion (54.7 +/- 3.7, 57.7 +/- 4.6, 46.4 +/- 2.7, and 42.1 +/- 3.9 nmol, respectively) compared with control (20.5 +/- 3.4 nmol). The results are consistent with decreased intracellular metabolism of absorbed hydroperoxides and enhanced transport into lymph under GSH-deficient conditions. The current findings suggest that the function of the mucosal GSH peroxidase/oxidized glutathione (GSSG) reductase system may play an important role in intestinal handling of luminal lipid hydroperoxides. A compromised function of this detoxication mechanism in GSH-deficient states can significantly alter the metabolic fate of dietary peroxidized lipids. PMID- 1733275 TI - Long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions. AB - Current concepts of the pathogenesis of lung injury and repair are derived from in vitro cellular and in vivo investigations. Studies with viable ex vivo models may offer additional insights into disease processes, since essential cellular interactions would be maintained. However, a major limiting factor has been the availability of a model that maintains normal parenchymal structure, viability, and homeostasis beyond 4 wk in serum-free conditions. We have succeeded in establishing an ex vivo lung culture system which reproducibly maintains parenchymal architecture for up to 9 wk. Our method is a simple, modified version of previously utilized techniques. Thin slices of mature murine lung were inflated with agar-defined medium and cultured on Gelfoam saturated with serum free medium. Normal pulmonary parenchyma, with the exception of endothelial cells, was maintained for up to 60 days as assessed chronologically by light and electron microscopy. The integrity of the microvasculature and endothelial cells was lost beyond 7 days. The adult lung ex vivo culture system maintained necessary epithelial and interstitial cellular interactions in the alveolar wall without systemic circulatory influences. Future studies with this model may provide important insights in assessing the pathogenesis of many acute and chronic lung diseases and clarify existing controversies raised from in vitro and in vivo studies. PMID- 1733276 TI - Mechanisms for enduring biological change. PMID- 1733277 TI - Autonomic innervation to feline tracheal submucosal glands for mucus glycoprotein secretion. AB - We examined the effect of electrical field stimulation (FS) on radiolabeled glycoconjugate released from feline tracheal isolated glands. Trichloroacetic acid precipitable [3H]-glycoconjugates released into the culture medium were counted, divided by the dry weight of sample, and data were expressed as percent of the release from the same animal that was unstimulated but otherwise received identical treatment. FS produced an increase in the glycoconjugate release that was dependent on the duration of stimulation, reaching a maximum response of 215% of nonstimulated sample for 30 min stimulation (10 Hz, 10 V). FS-evoked secretion was abolished by 10(-7) M tetrodotoxin but not altered by 10(-5) M hexamethonium. Atropine (10(-6) M) alone abolished the response to FS for 3 min or less. By contrast, a mixture of 10(-6) M atropine, 10(-5) M propranolol, and 10(-5) M phentolamine blocked only part of the response to FS for 15 min or more. The mixture of three antagonists reduced the response to FS for 30 min to 159% of control, which was significantly higher than control. Furthermore, 10(-6) M atropine, 10(-5) M propranolol, or 10(-5) M phentolamine significantly reduced the response to FS for 30 min to 162, 193, and 195%, respectively, from 215% of sample in the absence of blockers. Serotonin (10(-5) M) augmented FS-evoked response, which was abolished by 10(-6) M atropine or by 10(-5) M methysergide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733278 TI - Hyperoxia increases plasminogen activator activity of cultured endothelial cells. AB - Confluent calf pulmonary artery endothelial monolayers exposed to 95% oxygen for 1, 2, or 3 days exhibit a time-dependent increase in adherence to substratum, which closely parallels changes in actin cytoarchitecture and the distribution of focal contact proteins vinculin and talin. Oxygen exposure also resulted in elevated plasminogen activator (PA) activity in conditioned media (CM) and in cytoskeletal protein- and focal contact protein-enriched fractions, with highest levels achieved in the latter two fractions at 48 h after oxygen exposure. PAs have been shown to participate in dismantling of extracellular matrix in a number of physiological and pathological situations. Immunocytochemical studies demonstrated extensive restructuring of matrix proteins collagen IV, laminin, and fibronectin, which correlated temporally with elevated PA levels. Further, when protease-containing cell fractions were used to study degradation of isolated matrices, those obtained from hyperoxia-exposed cells were substantially more active than those from normoxia-exposed cells. Our data suggest that hyperoxia induced production of PA (and perhaps other proteases) may be partly responsible for degradation of the extracellular matrix of endothelial cells. PMID- 1733279 TI - Distinct effects of oxygen on surfactant protein B expression in bronchiolar and alveolar epithelium. AB - Hyperoxia causes severe lung injury in association with altered expression of surfactant proteins and lipids. To test whether oxygen induces surfactant protein B (SP-B) expression in specific respiratory epithelial cells, adult B6C3F1 and FVB/N mice were exposed to room air or 95% oxygen for 1-5 days. Northern blot analysis demonstrated an 8- to 10-fold increase in SP-B mRNA after 3 days that was maintained thereafter. In situ hybridization localized SP-B mRNA to bronchial, bronchiolar, and alveolar epithelial cells. Hyperoxia was associated with increased SP-B mRNA, noted primarily in the bronchiolar epithelium and decreased SP-B mRNA in the alveolar epithelium. After 5 days, central regions of lung parenchyma were nearly devoid of SP-B mRNA, while SP-B mRNA was maintained in alveolar cell populations close to vascular structures. To determine whether increased bronchiolar expression of SP-B mRNA during hyperoxia was a specific response, the abundance of CC10 mRNA (a Clara cell protein) was assessed. CC10 mRNA was detected in tracheal, bronchial, and bronchiolar, but not alveolar epithelium and was decreased upon exposure to hyperoxia. Immunocytochemistry demonstrated that SP-B proprotein was detected in bronchial, bronchiolar, and alveolar epithelial cells with staining increased in the bronchial and bronchiolar epithelium upon exposure to hyperoxia. SP-B gene expression in the respiratory epithelium is regulated at a pretranslational level and occurs in a cell specific manner during hyperoxic injury in the mouse. PMID- 1733280 TI - Sequence, ontogeny, and cellular localization of murine surfactant protein B mRNA. AB - We have isolated and characterized murine surfactant protein (SP-B) cDNAs and determined both the cellular distribution and developmental expression of SP-B mRNA. The nucleotide sequence of the composite SP-B cDNAs and cloned genomic SP-B DNA predicted a primary translation product of 377 amino acids (molecular mass = 41.7 kDa) that contained a single asparagine linked glycosylation site. The 410 base 3' untranslated region contained a novel sequence consisting of a (CCA)21 repeat. Tissue specificity and developmental expression of SP-B mRNA was assessed by Northern blot analysis. A single 1.6-kb mRNA species was detected specifically in lung tissue. In fetal mouse lung SP-B mRNA was detected at low levels on day 15 of gestation and increased markedly between days 16 and 17, reaching adult levels by birth. In situ hybridization analysis of SP-B mRNA demonstrated SP-B expression primarily in bronchiolar and alveolar epithelium, although discrete bronchial cells also contained the SP-B mRNA. The murine SP-B mRNA was expressed in proximal and distal epithelial cells within the respiratory tract and shares a close structural relationship to SP-B from other species. PMID- 1733281 TI - Bombesin stimulates human nasal mucous and serous cell secretion in vivo. AB - Bombesin, gastrin-related peptide (GRP), and related peptides sharing the common carboxyterminal sequence stimulate lactoferrin (serous cell marker) and glycoconjugate (mucous cell and goblet cell marker) release from human nasal mucosal explants in vitro. In vivo, GRP released from trigeminal sensory nerves may act upon GRP-bombesin binding sites on respiratory epithelial cells and submucosal glands. To determine whether GRP-bombesin can stimulate nasal secretion in vivo, bombesin was administered to eight normal subjects by unilateral, topical administration. Secretions from both nostrils were collected for measurement of total protein, lysozyme, hexose-containing glycoconjugates, and albumin (marker of vascular permeability). Baseline secretions contained 72.0 +/- 17.3 micrograms/ml of total protein, 14 +/- 2 micrograms/ml of lysozyme, 113 +/- 44 micrograms/ml of hexose-containing glycoconjugates, and 7.8 +/- 3.4 micrograms/ml of albumin. Hexose-containing glycoconjugate secretion was significantly increased after 1 nmol (385 +/- 63 micrograms/ml, P less than 0.001 by analysis of variance), 10, 100, and 1,000 nmol of bombesin, but the secretion was not dose dependent. Significant lysozyme (24 +/- 3 micrograms/ml, P less than 0.05) and total protein (155 +/- 23 micrograms/ml, P less than 0.01) secretion occurred after 1,000 nmol. No statistically significant changes in albumin secretion occurred at any dose. Saline had no significant effects on secretion. Therefore, bombesin stimulated secretion from submucosal glands and possibly epithelial cells in the human nose without affecting vascular permeability. PMID- 1733282 TI - Agonist-induced myosin phosphorylation during isometric contraction and unloaded shortening in airway smooth muscle. AB - We measured myosin phosphorylation during isometric contraction at optimal length (Lo) and unloaded shortening induced by K(+)-depolarization, electrical stimulation, carbachol, histamine, and phorbol dibutyrate (PDB) in bovine trachealis. Peak myosin phosphorylation during unloaded shortening was lower than that during isometric contraction in response to all stimuli. The lower peak myosin phosphorylation during unloaded shortening appeared to be a stretch sensitive response because myosin phosphorylation was either equally low or further reduced during the second unloaded shortening of preshortened tissues. Similar to peak myosin phosphorylation, steady-state myosin phosphorylation was also lower during unloaded shortening in carbachol-induced contractions. However, steady-state phosphorylation during unloaded shortening and isometric contraction were not significantly different in histamine- and PDB-induced contractions. Since the coupling between Ca2+ and myosin phosphorylation was not stretch sensitive, these results suggest the coexistence of stretch-sensitive and stretch insensitive signal transduction mechanisms in the airway smooth muscle cell membrane, and the stretch-insensitive signal transduction mechanism might involve protein phosphorylation by protein kinase C. PMID- 1733283 TI - Oxidants and antioxidants in alveolar epithelial type II cells: in situ, freshly isolated, and cultured cells. AB - Antioxidant enzyme activities, H2O2 clearance, and H2O2 generation by rat alveolar epithelial type II cells were compared between in situ, freshly isolated (6 h ex vivo), and cultured cells (48 h ex vivo). Immunocytochemical studies did not show changes in catalase, Mn superoxide dismutase, or CuZn superoxide dismutase labeling density in cytoplasm, peroxisomes, or mitochondria. Numbers of peroxisomes and mitochondria per cell decreased in cultured cells. Biochemical studies showed that cell culture resulted in a significant decrease in activities of catalase (49%), glutathione reductase (50%), glutathione peroxidase (74%), and in the capacity of the cells to scavenge extracellular H2O2. Addition of the specific catalase inhibitor, aminotriazole, decreased the rate of consumption of exogenously added H2O2 in freshly isolated cells but not in cultured cells. Neither aminotriazole nor 1,3-bis (2-chloroethyl)-1-nitrosourea, which inactivates glutathione reductase, altered H2O2 consumption by cultured cells. The rate of extracellular H2O2 release in both freshly isolated and cultured cells was 0.71 nmol.min-1.mg protein-1. It can be concluded that levels of some antioxidant enzymes fall in cultured alveolar epithelial type II cells, and that, although catalase likely plays a significant role in protection of freshly isolated cells against oxidant stress, this pathway may be less important after culture. PMID- 1733284 TI - Lipopolysaccharide stimulates de novo synthesis of PGH synthase in human alveolar macrophages. AB - Lipopolysaccharide (LPS)-stimulated human alveolar macrophages (HAMs) produce large amounts of prostaglandin (PG) E2 for up to 72 h. The mechanism of this enhanced and prolonged metabolism of arachidonic acid to PGE2 is unknown. To determine whether LPS-stimulated HAM PGE2 production is due in part to an increase in the new synthesis of the first committed enzyme, PGH synthase, we measured PGE2 formation, PGH synthase activity, and newly synthesized PGH synthase at 2, 6, and 24 h after LPS-stimulation. PGE2, measured by radioimmunoassay, was not increased at 2 h but was increased at 6 and 24 h after stimulating HAMs with LPS. Unstimulated HAMs did not produce PGE2. Likewise, the activity of PGH synthase extracted from HAMs was not increased at 2 h but was increased at 6 and 24 h after LPS stimulation. Cycloheximide and actinomycin D markedly inhibited PGE2 production in LPS-stimulated HAMs. Newly synthesized PGH synthase measured by immunoprecipitating 35S-labeled PGH synthase was not detected at 2 h but was detected at 6 and 24 h after stimulation. The parallel increases in PGE2 production, PGH synthase activity, and newly synthesized PGH synthase coupled with the dependence of PGE2 on the ability of the alveolar macrophage to synthesize protein suggest that LPS-stimulated HAM PGE2 production is in part regulated by the de novo synthesis of PGH synthase. PMID- 1733285 TI - Ammonium and bicarbonate transport in rat outer medullary collecting ducts. AB - Previous in vitro studies have demonstrated spontaneous bicarbonate absorption in the outer stripe portion of the rat outer medullary collecting duct (OMCD) and inner medullary collecting duct, but net acid transport has not been studied in the inner stripe of the rat OMCD (OMCDIS). When we perfused isolated OMCDIS segments with identical bath and perfusate solutions containing HCO-3 and NH4Cl, HCO-3 was spontaneously absorbed, and total ammonia was spontaneously secreted at rapid rates in tubules from both deoxycorticosterone (DOC)-treated and untreated rats. We next measured the NH3 flux due to imposed NH3 concentration gradients. Carbonic anhydrase (CA), when added to the lumen, enhanced the NH3 flux, implying an absence of endogenous CA. The NH3 permeability was 0.0042 +/- 0.0007 cm/s. By measuring the luminal pH in perfused OMCDIS segments with an imposed lumen-to bath NH3 gradient, we determined the pH at the end of the lumen to be 0.23 units below the equilibrium pH calculated from the simultaneously measured total CO2 concentration in collected fluid, confirming the lack of luminal CA. These results are consistent with the view that ammonium secretion in the OMCDIS occurs predominantly by H+ secretion and parallel NH3 diffusion. A luminal disequilibrium pH due to H+ secretion in the absence of endogenous luminal CA enhances the NH3 entry rate. Spontaneous net acid secretion appears to occur more rapidly in the OMCD than in other parts of the rat collecting duct system. PMID- 1733286 TI - Secretory renal proximal tubules in seawater- and freshwater-adapted killifish. AB - A population of proximal tubules when isolated from the glomerular kidneys of seawater-adapted (SW) and freshwater-adapted (FW) killifish (Fundulus heteroclitus) spontaneously secrete fluid. Regardless of SW or FW adaptation, Na and Cl are the dominant electrolytes in secreted fluid. Mg concentrations in fluid secreted by both tubules are significantly greater than those in the peritubular bath, and Mg concentrations are inversely related to Na concentrations. Proximal tubules from either SW or FW fish exhibit low transepithelial voltage (-1 to -2 mV) and low transepithelial resistances (20-30 omega.cm2) typical of other vertebrate proximal tubules. Transepithelial diffusion potentials for Na, Cl, Mg, and SO4 suggest that the paracellular pathway is Na selective and impermeable to divalent ions. Consideration of transepithelial electrochemical potential differences for Na, Cl, Mg, and SO4 suggests active transport of Mg, SO4, and Cl in proximal tubules isolated from SW and FW-adapted fish. The similarities in the functional properties of secretory proximal tubules isolated from SW- and FW-adapted killifish are striking and raise questions about the in vivo role of these tubules in the renal adaptations to seawater and freshwater. PMID- 1733287 TI - Taurine attenuates renal disease in chronic puromycin aminonucleoside nephropathy. AB - Repeated administration of low doses of puromycin aminonucleoside (PAMN) to rats induces a proteinuric renal disease that resembles focal segmental glomerulosclerosis (FSGS). Reactive oxygen molecules may be involved in the progressive course of this nephropathy. Therefore we evaluated whether taurine, an endogenous antioxidant, could limit the extent of renal injury. Sprague-Dawley rats received low-dose injections of PAMN, 2 mg/100 g body wt, over a 12-wk period. Two groups were studied: 1) controls given tap water (n = 23), and 2) an experimental group that drank 1% taurine-supplemented water (n = 22). Taurine treated nephrotic rats had a reduction in albuminuria, as assessed by the urinary albumin-to-creatinine ratio (26 +/- 4 vs. 44 +/- 4, P less than 0.0001). After 12 wk, creatinine clearance was 0.33 +/- 0.03 (experimental) vs. 0.17 +/- 0.03 ml.min-1.100 g body wt-1 (control) (P less than 0.001), and inulin clearance (n = 6 pairs) was 0.26 +/- 0.04 (experimental) vs. 0.13 +/- 0.02 ml.min-1.100 g body wt-1 (control) (P less than 0.025). Administration of taurine reduced the percentage of segmentally sclerosed glomeruli (9.8 +/- 1.7 vs. 16.2 +/- 1.8%, P less than 0.02) and the tubulointerstitial injury score (1.36 +/- 0.19 vs. 2.61 +/- 0.25, P less than 0.0025) in experimental vs. control rats. Taurine treatment normalized the elevated renal cortical malondialdehyde level in rats with PAMN nephropathy (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733288 TI - Renal vascular reactivity to vasodilator prostaglandins in genetically hypertensive rats. AB - The objective of this study was to test the hypothesis that the vasodilator prostaglandins E2 (PGE2) and PGI2) participate in the mechanisms involved in the increased renal vascular reactivity (RVR) observed in genetic hypertension. Studies were performed on anesthetized young and adult spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY). Renal blood flow (RBF) was measured during bolus injections into the renal artery of different doses of viprostol and iloprost (stable receptor agonists of PGE2 and PGI2, respectively) before and during inhibition of prostaglandin synthesis by indomethacin. Under control conditions, PGE2 increased RBF equally in young SHR and WKY. However, after cyclooxygenase inhibition the PGE2-induced increase in RBF was larger in young SHR than in WKY. Adult SHR displayed reduced reactivity to PGE2 relative to age matched WKY under control conditions. This strain difference was abolished after indomethacin administration. PGI2 increased RBF slightly in young rats before and after indomethacin administration. In contrast, both strains of older animals displayed significant increases in RBF in response to PGI2 injections. Indomethacin administration enhanced this PGI2-induced relaxation in adult SHR but not WKY. We propose that the action of vasodilator PGs in the renal vasculature of rats developing hypertension may be limited by low density of their renal receptors and/or the opposing action of vasoconstrictor PGs. As age advances, PGI2 seems to be activated, possibly as part of a regulatory response to counterbalance the increased renal vascular resistance following the establishment of the disease. PMID- 1733289 TI - Production and polarized secretion of basement membrane components by glomerular epithelial cells. AB - To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side. PMID- 1733290 TI - Role of cytoskeleton in volume regulation of rabbit proximal tubule in dilute medium. AB - When proximal tubules are immersed in hypotonic medium, they quickly swell to a peak volume. In a second, slower phase, termed volume regulatory decrease (VRD), they shrink as K, anion, and water leave the cells. We investigated the role of the cytoskeleton during this biphasic hypotonic volume regulatory response. Isolated, collapsed rabbit proximal convoluted tubules (PCT) were crimped tightly between two pipettes, and their volume was assessed optically. PCT volume increased to a peak 70-80% above baseline on sudden immersion in dilute medium (150 mosmol/kgH2O). After completing VRD, control tubules had regulated their volume 73 +/- 2% back toward baseline. Tubules exposed to the microtubule inhibitor vincristine (5 microM) regulated 75 +/- 2%. Tubules exposed to the microfilament inhibitor cytochalasin B (50 microM) regulated less (57 +/- 5%), and tubules exposed to both inhibitors regulated only 39 +/- 3% (P less than 0.01 vs. control). Hypotonic VRD was unimpaired in PCT loaded with NaCl by prior exposure to ouabain but was significantly reduced by cytochalasin B. We conclude that VRD is not cation specific and that intact microtubules and microfilaments play a synergistic role in the VRD of rabbit PCT in hyposmotic medium. PMID- 1733291 TI - Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells. AB - Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport. PMID- 1733292 TI - Release of angiotensins in paraventricular nucleus of rat in response to physiological and chemical stimuli. AB - The brain angiotensin (ANG II and III) system is known to play an important role in the central control of cardiovascular function and body water homeostasis. A number of components of the angiotensin system including active peptides, precursors, synthetic enzymes, and receptors have been localized to specific brain nuclei including the paraventricular nucleus (PVN) of the hypothalamus. We and others have hypothesized that the PVN is a major integrative hub of the central angiotensin system receiving angiotensinergic input from central detectors (circumventricular organs) and sending efferents to higher brain and spinal cord centers. Implicit in this idea is that angiotensins, like all neurotransmitters, should be releasable with appropriate chemical and physiological stimuli. Therefore we examined the ability of water deprivation or direct infusion of either 65 mM K+ or 80 microM veratridine to stimulate the release of angiotensins from the PVN of the rat. Using push-pull cannulas to perfuse the PVN and radioimmunoassay (RIA) to analyze the superfusate for immunoreactive angiotensins, we established that 24 h of water deprivation resulted in an approximate 5-fold increase in the angiotensin release rate, whereas 48-h deprivation produced a dramatic 492-fold increase in release. Direct infusion of 65 mM K+ into the PVN was unable to stimulate angiotensin release, but 80 microM veratridine elicited a sevenfold increase in the angiotensin release rate. High-performance liquid chromatographic separation and RIA analysis of veratridine- and water deprivation-stimulated angiotensin release demonstrated that 93.4% of the releasable angiotensin coeluted with ANG III, whereas only 6.8% eluted with authentic ANG II.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733293 TI - Control of kidney size by sex hormones: possible involvement of glucosylceramide. AB - Because androgenic hormones produce an increase in kidney size and the production of kidney glycosphingolipids and because the level of tissue glucosylceramide (GlcCer) has been implicated as a modulator of organ size, we measured the activities of two enzymes in mouse kidney that (in part) determine the level of this glycolipid. GlcCer synthase was found to rise in specific activity with age and increased kidney size, whereas GlcCer glucosidase was found to decrease with age; both enzyme changes act to elevate the level of kidney GlcCer and point to the level of this lipid as a determinant of kidney size. The increase in synthase was especially rapid in male kidney, consistent with the more rapid growth of male kidney. Testosterone injection into both males and females produced elevated levels of GlcCer synthase, decreased levels of the hydrolase, and more rapid growth. Injection of 17 beta-estradiol, which produced slow or negative kidney growth, caused the synthase activity to decrease in female mouse kidney and the glucosidase activity to rise in both sexes. Thus the changes in kidney size produced by the two hormones paralleled the changes in GlcCer level predicted by the hormone effects on the two enzymes. In liver, estradiol produced increases in both enzymes but only the male liver increased in size. Testosterone did not affect the liver. The testes grew with age but the activities of both enzymes decreased. PMID- 1733294 TI - Effect of insulin on potassium secretion in rabbit cortical collecting ducts. AB - Insulin is known to play an important role in the regulation of extrarenal K homeostasis. Previous clearance studies have shown that insulin decreases urinary K excretion, but the responsible nephron segments have not been identified. In this microperfusion study, in vitro, the effect of insulin on K transport in the cortical collecting duct (CCD), which is thought to be an important segment for regulation of the final urinary K excretion, was investigated. Basolateral insulin (10(-6) M) significantly inhibited net K secretion by 20% (mean JK = 26.2 +/- 4.2 peq.mm-1.min-1 for controls compared with -21.1 +/- 3.4 with insulin, P less than 0.001) and depolarized the transepithelial voltage (VT, from -14.6 +/- 3.5 to -10.8 +/- 3.5 mV, P less than 0.005), recovery did not occur over 60 min. Insulin (10(-11)-10(-5) M) depressed K secretion and depolarized the VT in a concentration-dependent manner. The half-maximal concentration was 5 x 10(-10) M, which is within the physiological range of plasma insulin concentration. In tubules of deoxycorticosterone acetate-treated rabbits, insulin also produced a significant fall in K secretion (from -43.4 +/- 7.5 to -36.1 +/- 5.7 peq.mm-1.min-1, P less than 0.05). Although luminal Ba (2 mM) decreased K secretion (from -14.4 +/- 2.9 to -7.0 +/- 1.7 peq.mm-1.min-1), basolateral insulin (10(-6) M) inhibited K secretion further (to -4.7 +/- 1.3 peq.mm-1.min-1, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733295 TI - Effects of barium and 5-(N-ethyl-N-isopropyl)-amiloride on proximal tubule ammonia transport. AB - Ionic NH+4 transport is an important mode of ammonia transport in the proximal convoluted tubule (PCT). NH+4 transport via the Na-H exchanger has been previously demonstrated. Potassium channels are present in the proximal tubule, but their role in ammonia transport has not been evaluated. We studied rat PCTs perfused in situ at 30 nl/min with solutions containing 5 mM HCO3; ammonia was measured with a fluorometric method. When perfused with the potent amiloride analogue 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 500 microM) or with BaCl2 (10 mM), ammonia entry (14.3 +/- 1.7 and 11.8 +/- 1.5 pmol.min-1.mm-1, respectively) was unaffected compared with control (12.8 +/- 1.0 pmol.min-1.mm-1). However, the combination of EIPA and barium inhibited entry (7.4 +/- 1.0 pmol.min-1.mm-1, P less than 0.02 vs. other groups). Also, when perfused with 10 mM ammonia, neither EIPA nor BaCl alone blocked ammonia loss (70.5 +/- 9.1 and 60.5 +/- 5.9 pmol.min 1.mm-1, respectively) compared with control (53.4 +/- 6.0 pmol.min-1.mm-1). However, the combination inhibited ammonia loss (29.2 +/- 6.3 pmol.min-1.mm-1, P less than 0.025 vs. other groups). Thus blocking both the Na-H exchanger and potassium channels decreases PCT ammonia transport. As the combination was required, this implies that multiple pathways exist for NH+4 transport in the PCT. This is the first demonstration that a mode of NH+4 transport other than via the Na-H exchanger is important in this segment, and the data are most consistent with transport of ammonia via potassium channels. PMID- 1733296 TI - Suppression of blood flow autoregulation plateau during nitric oxide blockade in canine kidney. AB - We examined the autoregulation of renal blood flow (RBF) and renal function in anesthetized dogs during nitro-L-arginine (NLA)-induced blockade of endothelium derived nitric oxide (EDNO). Intrarenal infusion of NLA (50 micrograms.kg-1.min 1) increased systemic arterial pressure (AP) and renal vascular resistance (RVR). RBF decreased by 27 +/- 3%, but glomerular filtration rate remained unchanged. There were reductions in urine flow (24 +/- 5%), urinary sodium excretion (42 +/- 10%), and fractional excretion of sodium (40 +/- 11%). The vasodilatory responses to intrarenal injections of ATP (1, 5, 10 microM) were reversed, whereas such responses to doses (10, 50, 100 ng) of acetylcholine (ACh) were attenuated during NLA infusion. Indomethacin (5 mg/kg iv) treatment further reduced but did not completely abolish ACh-induced vasodilation, suggesting that factor(s) other than EDNO and prostaglandins may also mediate ACh-induced vasodilation in the kidney. Although there was a suppression of the plateau of the AP-RBF relationship with a rightward shift in the slope of the linear portion of the curve during EDNO blockade, the normal autoregulatory pattern remained intact. Similar responses were seen in dogs treated with the angiotensin-converting enzyme inhibitor, MK 422. These data indicate that EDNO contributes to the normally low renal vascular tone by influencing an autoregulation-independent component of RVR. However, the basic capability to adjust RVR (autoregulation-responsive component) in response to changes in AP is essentially autonomous from EDNO activity. PMID- 1733297 TI - Assembly of distinctive coated pit and microvillar microdomains in the renal brush border. AB - The plasma membrane of the kidney brush border is composed of two compositionally distinct microdomains, microvilli and clathrin-coated pits. To study their assembly we have immunolocalized brush border marker proteins in the developing proximal tubule epithelium of the neonatal rat and compared their time and site of appearance with those of basolateral markers, Na-K-ATPase and fodrin. The proteins studied were dipeptidyl peptidase IV (DPPIV) (microvilli), actin and villin (microvillar cytoskeletal proteins), glycoprotein 330 (gp330) (coated pits), and clathrin (coated pit cytoskeleton). Although apical microvilli and coated pits were first seen in the stage III nephron, many brush border markers including DPPIV, actin, and clathrin appeared earlier in the development and initially were not polarized. Only during stage III did they become concentrated at the apical membrane. Villin first appeared in the stage III proximal tubule where it was located diffusely in the cytoplasm and in lysosomes as well as along the apical membrane. It did not completely colocalize with actin until stage IV. Gp330 first appeared during stage III and from the beginning was restricted to the apical clathrin-coated membrane domains and endosomes. The results demonstrate that 1) the expression of renal brush border proteins during development is asynchronous, and 2) unlike the basolateral plasmalemmal domain, which is established early in nephrogenesis, brush border assembly occurs later, approximately coinciding with the onset of glomerular filtration. PMID- 1733298 TI - Prostaglandin interactions with angiotensin, norepinephrine, and thromboxane in rat renal vasculature. AB - The objective of this study was to investigate the ability of the vasodilator prostaglandins E2 (PGE2) and I2 (PGI2) to interact with the vasoconstrictor action of angiotensin II (ANG II), norepinephrine (NE), and thromboxane A2 (TxA2) in the renal vasculature. In 12-wk-old anesthetized Munich-Wistar rats pretreated with indomethacin, renal blood flow (RBF) was measured during bolus injection of ANG II, NE, U-46619 (TxA2 agonist), PGE2, viprostol (PGE2 agonist), PGI2 or iloprost (PGI2 agonist) into the renal artery. Agents were injected separately and in a mixture of one constrictor with one dilator. ANG II, NE, and U-46619 induced large decreases in RBF, whereas PGs and their analogues produced slight, but significant, vasodilatation. To evaluate possible interactions in the vasomotor mechanisms between the dilators and the constrictors, curves of transient responses of separate injections were added (one constrictor plus one dilator) to create a predicted response of a mixture. NE exhibited additive effects with all PGs, as evidenced by the similarity of measured and predicted responses. In contrast, the TxA2 agonist interacted in a nonlinear fashion with all PGs; the measured maximum vasoconstriction was less than that predicted, and all kinetic parameters for the measured response were shorter than those predicted. The measured response to mixtures of ANG II and all PGs had a faster recovery than that predicted. We propose that the similarity between measured and predicted responses is due to independent actions of these agents via distinct mechanisms. In contrast, nonadditive responses suggest that the mechanisms mediating vasomotor effects of these agents interact in some cellular event. PMID- 1733299 TI - Regulation of glycolytic metabolism during long-term primary culture of renal proximal tubule cells. AB - Adequate oxygenation was a major factor regulating the induction of glycolytic metabolism in primary cultures of rabbit renal proximal tubule cells during short term (less than 1 day) and long-term (1-7 day) culture. As measured by cellular lactate content, glucose consumption, and lactate dehydrogenase activity, less glycolytic metabolism was induced in cultured cells that were constantly aerated than in cells that were held stationary. When oxidative metabolism is supported by providing 2-10 mM heptanoate (HEP) as a metabolic substrate glycolytic metabolism further decreased during short-term, but not long-term culture. Cellular proliferation did not play a major role in regulating the induction of glycolytic metabolism, since glycolytic metabolism increased before cell growth had occurred, did not decline once logarithmic cell growth had ceased, and was stimulated less by cell growth than by inadequate oxygenation. Fructose-1,6 bisphosphatase and alkaline phosphatase, representative markers of gluconeogenic and brush-border membrane enzyme activities, respectively, declined during culture regardless of culture conditions or the presence of HEP. Therefore, glycolytic metabolism can be effectively minimized by constantly aerating cultured proximal tubule cells and can be further reduced by the addition of HEP during short-term culture. PMID- 1733300 TI - Age-related changes in renal cytochrome P-450 arachidonic acid metabolism in spontaneously hypertensive rats. AB - We recently demonstrated that renal synthesis of cytochrome P-450-dependent arachidonic acid (AA) metabolites is increased in spontaneously hypertensive rats (SHR) during the rapid elevation of blood pressure. In this study, the chemical identity of these metabolites is described, and the structural analysis together with differential susceptibility to antibodies suggested that they are derived from at least two different cytochrome P-450 isozymes: 1) the epoxygenase that metabolizes AA mainly to 11,12-epoxyeicosatrienoic acid (EET), which is further hydrolyzed to 11,12-dihydroxyeicosatrienoic acid (DHT) and 2) omega/omega-1 hydroxylase(s) that generate the 20-hydroxyeicosatetraenoic acid (HETE) and 19 HETE, respectively. Their production and release from the isolated kidney was activated by arginine vasopressin and inhibited by cytochrome P-450 enzyme inhibitors. The formation of these metabolites in SHR or WKY cortical microsomes was age dependent. The production rates of EET, DHT, and 19-HETE increased from fetal to 9 wk of age by 3-, 6- and 4-fold, respectively, whereas that of 20-HETE increased by 27-fold. The omega/omega-1 hydroxylase activities were significantly higher in SHR, whereas epoxygenase activity (sum of EET and DHT production) demonstrated no differences between the two strains at any age group tested, although the amount of EET vs. DHT in a given age was significantly different. Since these metabolites have a wide and contrasting spectrum of biological and renal effects (vasodilation and vasoconstriction, inhibition and stimulation of Na(+)-K(+)-ATPase), their relative production rates at a given age may influence not only renal hemodynamics and salt and water balance but also pro- and antihypertensive mechanisms in SHR. PMID- 1733301 TI - Periventricular lesions block natriuresis to hypertonic but not isotonic NaCl loads. AB - The renal excretion of Na and water after an intravenous load of hypertonic or isotonic saline was studied in conscious sheep in which periventricular tissue in the region of the lamina terminalis had been ablated. Hypertonic saline (3.4-4.2 mmol/l) was infused at 0.06 mmol.kg-1.min-1 for 40 min into the jugular vein. Plasma Na concentration increased 5 mmol/l, and in normal sheep a natriuresis and increase in glomerular filtration rate ensued during the next hour. Such a natriuretic effect did not occur in sheep with periventricular lesions. By contrast, intravenous infusion of isotonic saline (30 ml/kg body wt, i.e., 0.23 mmol.kg-1.min-1 for 20 min) caused similar increase in renal Na excretion in normal sheep and sheep with periventricular lesions. When the same intravenous load of NaCl (0.23 mmol.kg-1.min-1 for 20 min) was administered as hypertonic 20% NaCl, ablation of periventricular tissue greatly impaired the excretion of this Na load. We suggest that the periventricular tissue in the region of the lamina terminalis has a role in regulation of renal Na excretion in conditions where the plasma Na concentration increases. This tissue is also involved in osmoregulatory thirst and vasopressin secretion. We further propose that increased renal Na excretion in response to hypernatremia is another cerebrally mediated osmoregulatory response. PMID- 1733302 TI - Contribution of nitric oxide to dilation of resistance coronary vessels in conscious dogs. AB - Endothelium-dependent relaxation of conductance coronary vessels involves nitric oxide formation from L-arginine. The present study examines whether a similar mechanism intervenes in the vasomotor control of resistance coronary vessels. In conscious dogs, the excess of coronary blood flow (CBF) created by intracoronary acetylcholine (3.0 ng/kg) averaged 7.2 +/- 1.1 ml. Intracoronary adenosine (100 ng/kg) increased CBF by 12.4 +/- 1.4 ml. Intracoronary nitroglycerin (175 ng/kg) increased CBF by 7.4 +/- 1.2 ml. CBF repayment-to-debt ratio after a 15-s coronary arterial occlusion averaged 2.8 +/- 0.2. After an intracoronary N omega nitro-L-arginine dose (10 micrograms.kg-1.min-1 x 12 min) was given to inhibit nitric oxide formation, baseline CBF was not altered. CBF increases with acetylcholine averaged 2.4 +/- 0.5 and 6.4 +/- 0.7 ml with adenosine, both less (P less than 0.01) than responses before the arginine analogue. CBF increases with nitroglycerin averaged 7.2 +/- 1.1 ml, similar to control responses. CBF repayment-to-debt ratio during reactive hyperemic responses fell (P less than 0.01) to 1.7 +/- 0.1. L-Arginine (1.0 mg.kg-1.min-1 x 12 min) partially reversed the inhibitory effect of the arginine analogue on CBF responses to acetylcholine. Thus nitric oxide formed in resistance coronary vessels is a major mediator of coronary vasodilation to acetylcholine, adenosine and transient ischemia. PMID- 1733303 TI - Hypoxia inhibits calcium influx in rabbit basilar and carotid arteries. AB - We examined the hypothesis that hypoxia inhibits Ca2+ influx in isolated rabbit common carotid, internal carotid, and basilar arteries. In arteries mounted for measurement of isometric tension and exposed to 122 mM K+ in Ca(2+)-free Krebs, cumulative addition of Ca2+ produced Ca(2+)-force relations that were right shifted by hypoxia (PO2 approximately 15 Torr) with no decrease in maximum force attained. In arteries precontracted with 122 mM K+, exposure to hypoxia produced relaxations whose rates and magnitudes were enhanced by reductions in bath Ca2+ from 8.0 to 0.8 mM. Using an ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N' tetraacetic acid method for 3-min 45Ca influx measurements, modified for use in rabbit basilar and carotid arteries, we found that resting levels of Ca2+ influx (mumol.min-1.kg dry wt-1) were significantly higher in basilar (67 +/- 1, n = 10) than in internal carotid (27 +/- 1, n = 12) or common carotid (33 +/- 1, n = 12) arteries. K+ stimulation increased Ca2+ influx more than two-fold compared with control in all three artery types, and hypoxia inhibited this increase by 74% in basilar, 49% in internal carotid, and 33% in common carotid arteries. Exposure to 10 microM serotonin and 100 microM uridine 5'-triphosphate (UTP) also increased Ca2+ influx, but these increases were less than observed during K+ contractions and averaged 10 (basilar), 31 (internal carotid), and 82% (common carotid) above control. Hypoxia completely inhibited serotonin- and/or UTP-induced increases in Ca2+ influx in basilar and internal carotid segments and inhibited 47% of this increase in the common carotid segments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733304 TI - Control of capillary hydraulic conductivity via membrane potential-dependent changes in Ca2+ influx. AB - Capillary permeability has been shown to be sensitive to the levels of intracellular calcium. We examined the role of membrane potential in the regulation of capillary water permeability by a Ca2+ leak mechanism. Repeated measures of Lp were taken in situ on individually perfused mesenteric capillaries of cerebrally pithed frogs (Rana pipiens). A rise in extracellular potassium ([K+]o) to 24 mM induced a 45% decrease in Lp (n = 20), whereas lowering [K+]o to 0.24 mM elevated Lp by twofold (n = 9). To investigate whether these changes in Lp were due specifically to changes in membrane potential and consequent changes in the driving force for Ca2+ influx, we performed the following experiments: 1) [K+]o was elevated while the product of [K+]o and extracellular chloride concentration [Cl-]o was kept constant, 2) [K+]o was elevated under nominally Ca(2+)-free conditions, 3) K+ leak was induced by addition of 10 microM valinomycin, and 4) Na(+)-K+ pump was blocked by 10 microM ouabain. A constant [K+]o [Cl-]o product did not prevent high K+ from lowering Lp. Nominally Ca(2+) free conditions abolished the effect of high K+. Valinomycin mimicked the response to low K+, and ouabain failed to change Lp. The data from this study conform to the hypothesis that membrane potential is an important regulator of capillary barrier properties via changes in Ca2+ influx through leak channels. PMID- 1733305 TI - Rhythmicity of urinary sodium excretion, mean arterial blood pressure, and heart rate in conscious dogs. AB - The existence of urinary excretion rhythms in dogs, which is a matter of controversy, was investigated under strictly controlled intake and environmental conditions. In seven conscious dogs, 14.5 mmol Na, 3.55 mmol K, and 91 ml H2O.kg body wt-1.24 h-1 were either administered with food at 8:30 A.M. or were continuously infused at 2 consecutive days. During these 3 days, automatized 20 min urine collections, mean arterial blood pressure (MABP), and heart rate (HR) recordings were performed without disturbing the dogs. Fundamental and partial periodicities, the noise component of urinary sodium excretion (UNaV), MABP, and HR were analyzed using a method derived from Fourier and Cosinor analysis. Oral intake (OI) leads to powerful 24-h periodicities in all dogs and seems to synchronize UNaV. UNaV on OI peaked between 1 and 3 P.M. Under the infusion regimen, signs of nonstationary rhythms and desynchronization predominated. UNaV under the infusion regimen could be separated into two components: a rather constant component continuously excreted and superimposed to this an oscillating component. No direct coupling between UNaV and MABP periodicities could be demonstrated. On OI, an increase in HR seems to advance the peak UNaV in the postprandial period. HR and MABP signals were both superimposed with noise. We conclude that UNaV rhythms are present in dogs. They are considerably more pronounced on OI. PMID- 1733306 TI - Leukocyte rolling: a prominent feature of venules in intact skin of anesthetized hairless mice. AB - Leukocyte (white blood cell; WBC) rolling in postcapillary venules is a frequently reported phenomenon in the microvasculature of experimental preparations. In most reports where this phenomenon has been systematically studied the confounding effects of various procedures associated with tissue preparation have been present. Thus there is sparse information on the extent of WBC rolling under fairly normal conditions. Here observations and features of this phenomenon in venules in the intact skin microvasculature of the homozygous hairless mouse ear are described. One venule in each of 10 mice was observed and continuously video recorded for 90 min. The parameters determined (mean +/- SD) were diameter, 15.9 +/- 3.1 microns; red blood cell velocity, 359 +/- 227 micron/s; flux of rolling WBCs, 3.2 +/- 2.6/min; velocity of rolling WBCs, 9.6 +/ 1.1 micron/s; systemic WBC count (CWBC), 3,220 +/- 1,072/microliters; and total WBC flux, estimated as the product of CWBC and calculated venule blood flow, 8.5 +/- 3.0/min. Overall, 44.8 +/- 13.8% of the total WBC flux exhibited rolling with a velocity that was 3.6 +/- 2.9% of the red blood cell velocity. During the total 15-h combined observation time, no WBCs were seen to be adherent. These findings establish that in small venules of normal skin, WBC rolling is common, since on the average nearly one of two WBCs delivered to the venule exhibits rolling. Furthermore, because the translational rolling speed is very low, they contribute to the marginated pool, which, according to the present data, might be better termed the "rolling" pool. PMID- 1733307 TI - Effect of naloxone on hypertension in Dahl salt-sensitive rats. AB - Experiments were conducted to test the hypothesis that chronic administration of an opioid receptor antagonist, naloxone, would affect the outcome of the developmental phase of hypertension in Dahl salt-sensitive (S/JR strain) rats. Accordingly, S/JR rats were maintained on either a low-salt (0.45% NaCl) or a high-salt (7% NaCl) diet for 4 wk. Half of the animals of each dietary group were treated with naloxone (100-130 micrograms/h) by osmotic minipump. Food and water intakes of the high-salt animals were measured for the first 25 days, and blood pressure was measured at the end of the 4 wk via an indwelling femoral arterial catheter. Naloxone treatment slightly but significantly reduced the level of hypertension attained in the high-salt animals (158 +/- 2 mmHg in naloxone treated animals vs. 168 +/- 3 mmHg in control animals; P less than 0.05) and also attenuated food (and hence salt) and water intakes. Naloxone did not affect the blood pressure of the low-salt animals. To determine whether the slight attenuation of hypertension might be secondary to a reduction of salt intake, a group of control S/JR animals were fed a moderately high-salt diet (2% NaCl), and naloxone-treated S/JR animals were salt-intake matched to this group by daily adjustment of the dietary salt content. Blood pressures after 4 wk of treatment were not different between these two groups. Finally, acute administration of 1 and 30 mg/kg of naloxone failed to lower blood pressure of animals with established hypertension.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733308 TI - Positive end-expiratory pressure potentiates the severity of canine right ventricular ischemia-reperfusion injury. AB - Positive end-expiratory pressure (PEEP) increases right ventricular (RV) afterload and oxygen demands. However, whether increased RV oxygen demands with high levels of PEEP can potentiate the severity of RV ischemic damage is unknown. In 20 anesthetized, closed-chest dogs randomly assigned to 0 cmH2O PEEP (ZEEP; n = 10) or 15 cmH2O PEEP (PEEP; n = 10), RV blood flow (radioactive microspheres) and segmental shortening (ultrasonic crystals) were determined during 90 min ischemia and 120 min reperfusion while mean aortic pressure was maintained above 90 mmHg. The in vivo RV area at risk (gentian violet) and area of necrosis (triphenyltetrazolium chloride) were assessed. After application of 15 cmH2O PEEP, pulmonary vascular resistance increased by 75% (P less than 0.05). During ischemia, the RV rate-pressure product remained greater with PEEP (2,403 +/- 174 mmHg.beat.min-1) than with ZEEP (1,909 +/- 94 mmHg.beat.min-1; P less than 0.05), indicating higher oxygen demands with PEEP. The area at risk from ischemia relative to RV free wall tended to be greater with PEEP (68.5 +/- 2.4%) than with ZEEP (60.0 +/- 3.9%; P = 0.08), and collateral blood flow in this risk zone was significantly lower during ischemia with PEEP (9.0 +/- 1.7 ml.min-1 x 100 g-1) than with ZEEP (18.3 +/- 3.6 ml.min-1 x 100 g-1; P less than 0.05). Accordingly, PEEP extended RV necrosis in the area at risk from 21.8 +/- 5.3% (ZEEP) to 58.1 +/- 8.4% (PEEP; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733309 TI - Favorable effects of hyperosmotic reperfusion on myocardial edema and infarct size. AB - Myocardial water content and infarct size were studied in 39 pigs randomly assigned to a nonintervention group, a group with an intracoronary infusion of a control solution, and a group with a hyperosmotic infusion to 450 mosM by the addition of D-mannitol. The intracoronary solutions were selectively infused into the left anterior descending coronary artery just distal to the occlusion site starting 48 min after occlusion. Reperfusion was performed 3 min later and the infusion rate progressively tapered off over the following 33 min. Multiple myocardial fragments were then obtained in nine pigs, from endocardial, mesocardial, and epicardial regions of the ischemic and control myocardium. Water content measured after 48 h of dessication was significantly greater in the reperfused [530 +/- 7 ml/100 (mean +/- SE) g dry wt] compared with control myocardium (374 +/- 3; P less than 0.0001) and similar in reperfused control and isotonic infusion groups (556 +/- 7 and 543 +/- 8 ml/100 g dry wt); it was 491 +/ 11 with intracoronary D-mannitol infusion, representing 35% less increase (P less than 0.001). In the 30 remaining pigs, area at risk and infarct size were measured 24 h later by in vivo fluorescein and in vitro triphenyltetrazolium chloride. Infarct size was similar in control and in the isotonic reperfused hearts, 6.80 +/- 1.05 and 6.22 +/- 0.76% of ventricular weight, and smaller with D-mannitol, 4.46 +/- 0.46 (P less than 0.05). The ratio of infarct size to area at risk was also smaller [0.415 +/- 0.029 vs. 0.543 +/- 0.052 and 0.547 +/- 0.045 (P less than 0.02)].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733310 TI - Is there a shortening-heat component in mammalian cardiac muscle contraction? AB - It has been suggested that there is a shortening-heat component that is an extra liberation of heat on shortening above that due to the external work, which contributes to the total energy expenditure of the beating heart. The presence of a shortening heat component was studied in isolated papillary muscles from the right ventricle of rabbits killed by cervical dislocation. At the onset of a contraction, muscles were shortened from various initial lengths through fixed distances at near maximum velocity before being allowed to develop force at the new length; the heat production accompanying such contractions was measured. The measured heat was compared with heat values predicted from previously established heat-stress curves obtained by using either preshortening or latency release methods. There was no shortening-related increment in heat output per contraction when comparison was made to a control heat-stress curve, obtained using the latency release method. An increase in heat production of 10% was observed with long shortening distances when comparison was made to a control heat-stress curve obtained by preshortening the muscles; however, this difference is most likely due to an underestimate of the magnitude of the activation heat component in these control heat-stress curves. An increase in isometric heat production due to maintained stretch per se was observed. The present data indicate that it is unlikely that there is a significant shortening heat component when cardiac muscle shortens. The absence of such a metabolic component may account for the rapid fall off in total enthalpy output in isotonic contractions at low to medium afterloads when compared with the skeletal muscle data. PMID- 1733311 TI - Acute volume expansion enhances baroreceptor discharge in dogs. AB - By recording single-unit discharge from the carotid sinus nerve of normal dogs in the control and in the volume-expanded state, both with the sinus open to the circulation and with it isolated, the effects of acute volume expansion on baroreceptor discharge sensitivity were investigated. The carotid sinus was vascularly isolated except for the common carotid and external carotid arteries. Inflation of hydraulic occluders on these vessels completely isolated the sinus from the systemic circulation. Flow and diameter changes of the carotid sinus were measured. Plasma volume was expanded with isotonic, isoncotic dextran in normal saline until the left ventricular end-diastolic pressure reached a value of 20-25 mmHg. In the group with the sinus open to the circulation, volume expansion did not depress baroreceptor discharge sensitivity; in fact, it augmented baroreceptor activity (peak discharge = 39.2 +/- 1.6 vs. 49.9 +/- 3.3 spikes/s, P less than 0.05). There was no such effect in the group with the sinus isolated from the circulation during volume expansion. In the group with the sinus open to the circulation, volume expansion significantly increased flow through the common carotid artery and shifted the carotid sinus pressure-diameter curves upward without a change in compliance. These data suggest that acute volume expansion augments baroreceptor discharge, and this phenomenon may be mediated either by some circulating substance(s) or by an increase in flow through the carotid sinus during volume expansion. PMID- 1733312 TI - Adaptation to sodium restriction in renin-immunized spontaneously hypertensive and normotensive rats. AB - The influence of active immunization against renin on the systemic and renal adaptation to abrupt suppression of dietary sodium was assessed in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Very low level of plasma renin activity and no response of circulating renin to sodium restriction were observed in immunized rats. The final systolic arterial pressure achieved in immunized SHR (144 +/- 3 mmHg) was similar to that observed in nonimmunized WKY (137 +/- 4 mmHg), and no influence of renin immunization on renal blood flow was observed in both strains maintained on ad libitum sodium intake. Dietary sodium restriction was associated with a consistent decrease in arterial pressure in immunized rats, whereas no change occurred in sham-immunized rats. No impairment in the renal adaptation was detected in immunized rats (6-day cumulative sodium excretion of 1.67 +/- 0.2 and 1.65 +/- 0.3 mmol in immunized SHR and WKY, respectively, and 1.50 +/- 0.2 and 1.21 +/- 0.2 mmol in sham-immunized SHR and WKY rats). These studies indicate that renin immunization decreases arterial pressure but does not affect renal adaptation to dietary sodium restriction in hypertensive and normotensive rats. PMID- 1733313 TI - Reflex control of fetal arterial pressure and hormonal responses to slow hemorrhage. AB - Late-gestation fetal sheep respond to slow hemorrhage with increases in plasma concentrations of adrenocorticotropic hormone (ACTH), hydrocortisone, arginine vasopressin (AVP), and plasma renin activity (PRA) that correlate to the acidemia and hypercapnia also produced by hemorrhage. This study was designed to investigate the role of peripheral chemoreceptors in the mediation of these responses. Chronically catheterized fetal sheep were left intact or were subjected to bilateral section of cervical vagosympathetic trunks and carotid sinus nerves. At least 5 days after surgical preparation (between 121 and 138 days of gestation) fetuses were bled at a rate of 11 ml/10 min for 2 h. Denervated fetuses were studied with or without simultaneous infusion of phenylephrine. Denervation exaggerated the decrease in mean arterial pressure and arterial pH and the increase in arterial PCO2 during hemorrhage. Infusion of phenylephrine in the denervated fetuses prevented the decrease in blood pressure and reduced the magnitudes of changes in blood gases. Fetal plasma ACTH, hydrocortisone, and PRA responses to the hemorrhage were exaggerated in the denervated fetuses (not infused with phenylephrine) compared with the intact fetuses. Phenylephrine infusion attenuated the ACTH response and inhibited the AVP response but did not alter the PRA response. We conclude that the sectioned fibers are important for the maintenance of blood pressure and blood gases during hemorrhage and that the PRA, AVP, and ACTH responses to slow hemorrhage are not mediated by peripheral chemoreceptors. PMID- 1733314 TI - Effects of ischemia and reperfusion on cardiac tolerance to oxidative stress. AB - Oxidative stress may affect cardiac function and metabolism. Oxidants are normally inactivated by reacting with reduced glutathione (GSH), with resulting formation and release of oxidized glutathione (GSSG). However, ischemia might affect glutathione metabolism. This might render ischemic hearts less resistant against subsequent oxidant injury during reperfusion, and it might also affect the reliability of GSSG measurements as a means to investigate oxidative stress in reperfused hearts. We compared the metabolic and functional consequences of an oxidant load in control rabbit hearts and in hearts reperfused after 30 min of normothermic total ischemia. In controls, H2O2 infusion (H2O2; 5-30 microM) induced a dose-dependent stimulation of GSSG release and a progressive impairment of cardiac function. At these doses, H2O2 challenge of postischemic hearts resulted in biochemical and functional changes identical to those observed in controls. Release of lactate dehydrogenase (LDH) and of GSH was negligible, similar in both groups. In additional experiments, infusion of H2O2 at a much higher dose (200 microM) elicited a further increase in GSSG release from both groups, although GSSG concentrations were lower in postischemic hearts. The functional effects of the 200 microM H2O2 infusion were similar in both groups, all hearts showing rapid and irreversible deterioration of function. Occurrence of irreversible cell injury was also manifested by a large release of LDH and GSH to a similar extent in both groups. These data demonstrate that cardiac tolerance toward oxidants is largely unaffected by a relatively brief episode of severe ischemia and indicate that GSSG release can be reliably used to investigate oxidative stress in reperfused hearts. PMID- 1733315 TI - Temperature and hydrostatic pressure-dependent pathways of low-density lipoprotein transport across microvascular barrier. AB - To further investigate the chemical and physical nature of low-density lipoprotein (LDL) transport pathways across intact microvessels, the effect of changes in temperature and microvessel hydrostatic pressure were measured in individually perfused postcapillary vessels within frog mesenteric vascular beds. LDL microvessel transport was measured at two microvessel temperature ranges (18 21 degrees C and 4-6 degrees C) and compared with transport of fluorescein, a small solute. Also, LDL transport was measured at a series of hydrostatic pressures (3-20 cmH2O) at microvessel temperatures of 18-21 degrees C and 4-6 degrees C to determine whether LDL transport was coupled to water flow, which would be evidence for hydraulic pathways of solute transport across the microvascular barrier. Quantitative fluorescence microscopy was employed to determine apparent solute permeability coefficients (Ps) under the various temperature and hydrostatic pressure conditions studied. The ratio of Ps fluorescein 18-21 degrees C/4-6 degrees C [1.6 +/- 0.3 (SD)] indicated that fluorescein was freely diffusible across the microvascular barrier through water filled pathways as transport was inversely proportional to temperature-dependent changes in viscosity. The larger ratio for LDL (Ps LDL 18-21/4-6 degrees C = 9.5 +/- 8.1) than for fluorescein cannot be explained by LDL transport through fixed hydraulic pathways alone and suggests additional or alternate LDL transport mechanisms. In addition, Ps LDL increased as microvessel hydrostatic pressure increased at microvessel temperatures of 18-21 degrees C but not at 4-6 degrees C. Coupling of LDL transport to water flow at the high microvessel temperature range, but not at the low range, indicated the presence of a hydraulic transport pathway that was effectively absent when the microvessel was cooled. These results demonstrated a highly temperature and hydrostatic pressure-dependent LDL pathway that is consistent with a dynamic porous extracellular or transcellular mechanism of LDL transport. PMID- 1733316 TI - gp60 is an albumin-binding glycoprotein expressed by continuous endothelium involved in albumin transcytosis. AB - Albumin reduces capillary permeability and acts as a carrier for various small molecules. Recently, we identified a 60-kDa sialoglycoprotein (gp60) on the surface of cultured rat microvascular endothelial cells (MEC) that binds albumin and antiglycophorin serum (alpha-gp). We verified that alpha-gp recognizes the albumin-binding gp60 by affinity, purifying proteins from MEC extracts using immobilized albumin. gp60 was immunoblotted with alpha-gp only when the MEC extract was reacted with albumin and not in controls. We immunoprecipitated gp60 from biosynthetically radiolabeled MEC lysates and from extracts containing endothelial surface proteins of isolated rat hearts that were radioiodinated in situ. gp60 was immunoblotted selectively in rat tissue microvascular beds lined with continuous endothelium (heart, lung, diaphragm, fat, skeletal muscle, mesentery, and duodenal muscularis but not cortical brain) and not those exclusively lined with fenestrated or sinusoidal endothelium (adrenal, pancreas, liver, and small intestinal mucosa). MEC isolated from rat heart, lung, and epididymal fat pad expressed gp60 and bound albumin, whereas various nonendothelial cells and brain-derived MEC did not. gp60 is an albumin-binding glycoprotein expressed specifically on the surface of continuous endothelium that binds albumin apparently not only to initiate its transcytosis via plasmalemmal vesicles but also to increase capillary permselectivity. PMID- 1733317 TI - An evaluation of pulsus alternans in closed-chest dogs. AB - Pulsus alternans is a condition in which the arterial pressure generated by the heart oscillates between two levels on a beat-to-beat basis. We evaluated the onset of pulsus alternans in chronically instrumented dogs subjected to tachycardia and inferior vena caval occlusion. During pulsus alternans, the left ventricular (LV) end-diastolic volume (EDV) was larger before the strong beats (28.7 +/- 5.3 vs. 25.9 +/- 4.5 ml, P less than 0.001 by paired t test), suggesting that the Frank-Starling mechanism participates in the alternating difference in end-systolic pressure. In addition, however, the ratio of pressure to volume at end systole was greater in the strong beats (2.01 +/- 0.36 vs. 1.46 +/- 0.45, P less than 0.005 by paired t test), a difference that cannot be explained by the Frank-Starling mechanism alone. This indicates that there is also a difference in end-systolic inotropic states between strong and weak beats. These changes occurred without significant alterations in beat-to-beat levels of coronary flow. The time constant of isovolumic pressure fall (T) was faster for the strong beats (37.5 +/- 4.2 vs 61.1 +/- 12.7 ms, P less than 0.002 by paired t test). The onset of oscillation in T preceded the onset of changes in LVEDV and LV systolic pressure in every case by an average of seven beats (range 3-11), suggesting that abnormalities of intracellular calcium handling led to the occurrence of pulsus alternans.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733318 TI - Bromcresol green assay is nonspecific for rat plasma albumin. AB - Bromcresol green (BCG) assay has been used in microvascular studies to determine albumin in rat plasma. The purpose of this study was to demonstrate that BCG overestimates rat plasma albumin partly because BCG binds to transferrin, a beta globulin. The light absorbance of a transferrin-BCG reagent solution is shown to increase with time; appreciable binding occurs within a few seconds. Pure transferrin produced BCG assay results (P less than 0.001) that could be expressed as pseudoalbumin concentrations. Albumin and transferrin solutions of equal concentrations were mixed in equal parts; a plot of albumin concentration determined by BCG vs. actual albumin concentration had a slope greater than the expected value of 1.0 (P less than 0.001). Plasma samples were obtained from six rats and assayed for albumin. Electrophoresis yielded a plasma albumin of 2.97 +/ 0.11 g/dl, whereas BCG assay yielded a significantly (P less than 0.01) greater value of 3.58 +/- 0.07 g/dl. We conclude that BCG assay estimates of albumin-to globulin ratio (A/G), in which G is determined from the difference between total protein and albumin, are especially subject to error. PMID- 1733319 TI - Onset of exercise shifts operating point of arterial baroreflex to higher pressures. AB - This study was designed to test the hypothesis that the increase in sympathetic nerve activity (SNA) and mean arterial pressure (MAP) at the onset of exercise is dependent on a rapid upward shift of the operating point of the arterial baroreflex. To test this hypothesis, we recorded renal sympathetic nerve activity (RSNA) in 16 New Zealand White rabbits during treadmill running (12.6 m/min, 20% grade) under control conditions and during concomitant intravenous infusions of nitroglycerin (NTG) to attenuate the exercise pressor response. In the control condition, MAP increased 18 +/- 2 mmHg. This was associated with an increase in heart rate (HR) (104 +/- 4 beats/min) and RSNA (414 +/- 20%). The increases in RSNA (848 +/- 32%) and HR (155 +/- 5 beats/min) at the onset of exercise were significantly augmented when the rate of development of the exercise pressor response (0.3 +/- 0.03 to 0.12 +/- 0.01 mmHg/s) and the magnitudes of the pressor response (91 +/- 2 to 79 +/- 1 mmHg) were attenuated by infusions of NTG. These data suggest that at the onset of exercise the operating point of the arterial baroreflex is reset toward higher pressures. The MAP, RSNA, and HR responses to exercise were also determined in eight sinoaortic-denervated (SAD) rabbits. In the absence of a functional baroreflex, MAP (-46 +/- 2 mmHg), RSNA (-19 +/- 1%), and HR (-62 +/- 3 beats/min) decreased at the onset of exercise and recovered 1 min to -42 +/- 2, +13 +/- 1, and +9 +/- 1% of control, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733320 TI - Distribution of blood flow in normal and arthritic joints. Role of arteriovenous shunting studied in growing dogs. AB - Juvenile dog knee with chronic carrageenin-induced arthritis was studied under general anesthesia with 15-microns and 50-microns-sized microspheres (MS) to compare the distribution of absolute and weight-standardized blood flow in normal and arthritic limbs and to localize possible sites of arteriovenous (AV) shunting. Arthritic joints had severe synovial and capsular hyperemia. Absolute as well as standardized blood flow was increased in juxta-articular epiphyses and patella. Shafts were atrophic and had decreased absolute flow but normal standardized flow. However, redistribution of blood flow occurred among regions within the shafts, e.g., in metaphyses away from growth plates. The mean nonentrapment of 15-microns MS was 13.8% in arthritic limbs and 4.2% in control limbs. The uptake of 50-microns MS was lower than that of 15-microns MS in all bony flow compartments due to differences in their rheologic behavior in larger arteries. The relative distribution of 50-microns MS and 15-microns MS varied considerably among regions within bone. Arthritis caused a net shift in the uptake of 50-microns MS relative to that of 15-microns MS from central to subchondral epiphyseal bone, evidencing precapillary vasodilation, but the relationship was strictly unchanged when bones were examined in toto. This result militates against the hypothesis of AV shunting in arthritic bone. PMID- 1733321 TI - Vulnerability of rabbit atrium to reentry by hypoxia. Role of inhomogeneity in conduction and wavelength. AB - In isolated superfused left atria of the rabbit, the inducibility of tachyarrhythmias by single early premature stimuli was highly increased by hypoxia. High-resolution mapping showed that these arrhythmias were caused by circus movement around a functional arc of conduction block (leading circle reentry). To determine the electrophysiological changes by hypoxia responsible for the higher vulnerability to intra-atrial reentry, the wavelength of the atrial impulse and spatial inhomogeneities in refractory periods and local conduction delays were measured. Hypoxia caused a transient increase in refractory periods during the first 10-15 min of hypoxia. After this period, refractory periods shortened again to values slightly lower than during control. During the whole period of hypoxia, local differences in refractory periods were enlarged. Conduction velocity was significantly depressed by hypoxia. As a result, the wavelength of the atrial impulse gradually shortened during hypoxia to approximately 80% of control. Inhomogeneity in conduction was quantified by phase maps in which the maximal local delays in conduction are plotted. Hypoxia caused a marked increase in inhomogeneity in conduction both during slow rhythm (inhomogeneity index increased from 2.3 to 3.4) and premature activation (from 3.1 to 4.7). We conclude that the higher vulnerability of the atrium for reentrant arrhythmias by hypoxia is based on a combination of a moderate shortening of the wavelength and an increase in inhomogeneity in conduction of premature wavefronts. PMID- 1733322 TI - Mechanical properties of the canine mitral valve: effects of autonomic stimulation. AB - Experiments were designed to determine whether the canine mitral valve actively contracts and, if so, if its mechanical activity can be modulated by the autonomic nervous system. A miniature displacement gauge was sutured to the atrial (muscular) surface of the septal leaflet in 65 dogs under pentothal sodium anesthesia during total cardiopulmonary bypass. During bypass, with blood constantly drained from the left ventricle (LV), the leaflet deflected into the LV during ventricular systole as would be expected with active contraction. With blood present in the LV, leaflet deflection was reversed, indicating a passive displacement. After application of 85% phenol to the atrial surface, ventricular displacement of the leaflet was abolished, whereas the atrial displacement was significantly augmented. There was a positive inotropic effect during sympathetic stimulation and a negative inotropic effect with parasympathetic stimulation, i.e., increased and decreased ventricular deflection, respectively. These effects of autonomic stimulation were abolished by application of phenol to the leaflet muscle. During simultaneous electrical and mechanical recordings from the valve, activation appeared to originate from the atrium, before ventricular depolarization. It was concluded that the valvular muscle actively contracts to assist in bringing the valve leaflets into early apposition. This contraction imparts a measurable level of active "stiffness" to the valve, reducing atrial displacement during ventricular systole. This "stiffness" can be modified by autonomic input and may contribute to dysfunction of morphologically normal mitral valves. PMID- 1733323 TI - Myocardial depression produced by sustained tachycardia in rabbits. AB - Much recent attention has been focused on the tachycardia-induced heart failure model. We hypothesized that sustained tachycardia would lead to myocardial depression in rabbits, as it does in dogs and swine. We evaluated the passive and active length-tension relations and postrest contraction behavior in right ventricular papillary muscles from 22 New Zealand White rabbits, 11 controls, and 11 subjected to ventricular pacing at a rate of 400 beats/min for 29.4 +/- 10.6 days. Studies were performed in oxygenated buffer at 22 degrees C. Active tension was significantly reduced at muscle lengths of 0.95.Lmax and above; at Lmax it was 4.7 +/- 0.2 g/mm2 for the control group and 3.3 +/- 0.2 g/mm2 for the paced group (P less than 0.005). Both groups showed increased force development when the concentration of calcium in the buffer was increased. There were no differences between the groups in the passive length-tension relations. Of note, postrest contraction data showed that the second postrest beat was smaller for the paced animals for rest intervals up to 2 min, suggesting that beat-to-beat trans-sarcolemmal calcium handling may differ from normal in this model. We conclude that sustained tachycardia will lead to myocardial depression in rabbits; the extension of this model to a small animal species may offer new ways to explore its causative mechanisms. PMID- 1733324 TI - Contribution of extravascular compression to reduction of maximal coronary blood flow. AB - Effects of ventricular compression on maximally dilated left circumflex coronary blood flow were investigated in seven mongrel dogs under pentobarbital anesthesia. The left circumflex artery was perfused with the animals' own blood at a constant pressure (63 mmHg) while left ventricular pressure was experimentally altered. Adenosine was infused to produce maximal vasodilation, verified by the hyperemic response to coronary occlusion. Alterations of peak left ventricular pressure from 50 to 250 mmHg resulted in a linear decrease in total circumflex flow of 1.10 ml.min-1 x 100 g heart wt-1 for each 10 mmHg of peak ventricular to coronary perfusion pressure gradient; a 2.6% decrease from control levels. Similar slopes were obtained for systolic and diastolic flows as for total mean flow, implying equal compressive forces in systole as in diastole. Increases in left ventricular end-diastolic pressure accounted for 29% of the flow changes associated with an increase in peak ventricular pressure. Doubling circumferential wall tension had a minimal effect on total circumflex flow. When the slopes were extrapolated to zero, assuming linearity, a peak left ventricular pressure of 385 mmHg greater than coronary perfusion pressure would be required to reduce coronary flow to zero. The experiments were repeated in five additional animals but at different perfusion pressures from 40 to 160 mmHg. Higher perfusion pressures gave similar results but with even less effect of ventricular pressure on coronary flow or coronary conductance. These results argue for an active storage site for systolic arterial flow in the dilated coronary system. PMID- 1733325 TI - Oxygen free radicals affect cardiac and skeletal cell membrane potential during hemorrhagic shock in rats. AB - Oxygen free radical (OFR) damage of excitable cell membranes (heart and skeletal muscle) during hemorrhagic shock and after resuscitation was studied in control rats and in rats pretreated with superoxide dismutase (SOD) and catalase (CAT; 6,000 U each) before hemorrhage. Their mean arterial pressure (MAP) was lowered to and maintained at 45 mmHg until 30% of the shed blood was spontaneously reinfused. The remaining blood and twice that volume of lactated Ringer solution were then infused. Cardiac output and organ blood flow were measured by the microsphere technique. The resting membrane potential (Em) and tissue ATP content in the heart and skeletal muscle were determined. There was no significant difference between the control and SOD + CAT groups in shock duration, maximal shed blood, hemodynamics, regional blood flow, or in ATP content in both heart and skeletal muscle, both during shock and after resuscitation. Radical scavenger treatment did not prevent muscle depolarization during shock. After resuscitation, however, significant repolarization in hearts and skeletal muscle of the SOD + CAT group (heart, -70.0 +/- 1.1; muscle, -87.0 +/- 0.6 mV) was noted when compared with the controls (heart, -62.5 +/- 1.2; muscle, -82.7 +/- 1.1 mV; P less than 0.05). This implicates OFRs as mediators of excitable cell membrane injury following resuscitation. PMID- 1733326 TI - Acute myocardial ischemia causes a transmural gradient in glucose extraction but not glucose uptake. AB - We assessed the relationship between myocardial glucose metabolism and blood flow during ischemia in eight open-chest swine. Coronary flow was controlled by an extracorporeal perfusion circuit. Left anterior descending coronary arterial (LAD) flow was reduced by 60%, while left circumflex flow was normally perfused. The rate of glucose uptake (Rg) was measured with a coronary infusion of 2-deoxy D-[14C]glucose and myocardial blood flow with radiolabeled microspheres. Myocardial biopsies were taken after 50 min of ischemia. Regional arterial-venous glucose difference was calculated as Rg per myocardial blood flow. Subendocardial blood flow decreased from 1.27 +/- 0.19 to 0.25 +/- 0.11 ml.g-1.min-1 (P less than 0.0001). The subendocardial arterial-venous glucose difference was greater in the LAD bed (1.38 +/- 0.35 mumol/ml) than the left circumflex coronary arterial perfusion bed (0.10 +/- 03; P less than 0.01); however, there was no statistically significant difference in the rate of glucose uptake between the two beds. Subendocardial glycogen concentration in the LAD perfusion bed was reduced to 26% of circumflex bed values. In conclusion, acute ischemia stimulated a dramatic increase in glucose extraction; however, this did not compensate for the decrease in blood flow, and thus the rate of glucose uptake did not increase significantly. The high rate of glycolysis is primarily supported by accelerated net glycogen breakdown rather than increased glucose uptake. PMID- 1733327 TI - Effects of physiological aging on cardiac electrophysiology in perfused Fischer 344 rat hearts. AB - Aging effects on heart rate and atrioventricular (AV) conduction were studied in Langendorff-perfused hearts from 18 mature (4-6 mo), 12 middle-aged (12-14 mo), and 18 senescent (24-26 mo) Fischer 344 rats. Heart rate decreased with increasing age from 218 +/- 18 in mature to 196 +/- 27 (mean +/- SD) beats/min in middle-aged rats to 183 +/- 22 beats/min in senescent rats (analysis of variance, P less than 0.001). Spontaneous AV conduction time increased from 43 +/- 7 to 49 +/- 5 to 62 +/- 9 ms with aging (P less than 0.0001). Paced AV conduction time also lengthened with aging, and AV Wenckebach block cycle length increased from 122 +/- 10 to 133 +/- 9 to 152 +/- 16 ms (P less than 0.005). Intra-atrial conduction time was unaffected by age. Age differences in heart rate and AV conduction responses to isoproterenol (0.5 x 10(-9) to 1 x 10(-7) M) were noted with greater sensitivity at lower doses in hearts from younger rats. In separate experiments, 18 mature and 19 senescent Fischer 344 rats received reserpine (0.25 mg.kg-1.day-1 ip) for 6 days before study. Age differences in heart rate and AV conduction persisted (P less than 0.0001). Histopathological examination of AV nodal and His-bundle tissues in three hearts from each age group showed increased intercellular collagen with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733328 TI - Spontaneously hypertensive rat: lymphoid depression is age dependent and mediated via a mononuclear cell subpopulation. AB - Immune dysfunction has been reported in spontaneously hypertensive rats (SHR), particularly in mature animals with established hypertension. The current study examined the time course of development of immune dysfunction and defined its cellular basis in male SHR and control normotensive Wistar-Kyoto rats (WKY). Mitogen-induced proliferative responses in lymphoid cells obtained from induced proliferative responses in lymphoid cells obtained from SHR thymus and spleen before (age 4 wk) and during the development of (ages 8 and 12 wk) hypertension and in age-matched normotensive WKY were monitored. A 50% reduction in concanavalin A (Con A)-induced proliferative responses was seen in SHR thymocytes compared with those of WKY at 12 wk only, suggesting differences in immature T cell populations. Con A-induced T-cell proliferative responses in splenocytes also differed between strains: greatest (as much as 8-fold) decreases were found in 12-wk-old SHR. Similar findings were obtained in splenocytes stimulated with lipopolysaccharide (LPS), indicating differences in B-cell function. Mononuclear cells depleted of their adherent cell population were prepared from SHR and WKY at 12+ wk of age and assayed for their proliferative responses to LPS and Con A. The remaining nonadherent mononuclear cells of SHR had proliferative responses equal to or greater than those of WKY. Further, when SHR splenic mononuclear cells were allowed to adhere to plastic, and the adherent fraction was co cultured with either SHR G-10 nonadherent or unfractionated SHR splenic mononuclear cells, proliferative responses were suppressed by as much as 88%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733329 TI - Carbachol induces granular cell exocytosis and serotonin release in rabbit cerebral arteries. AB - Previously, we reported that rabbit cerebral arteries contain mast cells that frequently establish close contacts with parasympathetic-like nerve fibers. Here we have examined the possible function of this link by comparing the effects of carbachol and compound 48/80 on mast cell morphology and on the serotonin (5-HT) and histamine content of these arteries. In vivo, 2 micrograms/min of compound 48/80 or 1 micrograms/min of carbachol was infused for 30 min into one internal carotid artery of pentobarbital anesthetized rabbits, the contralateral artery being infused with vehicle. In vitro, the action of 10(-6) M carbachol was tested on isolated middle cerebral artery trees (MCAs) in the presence or absence of 10( 7) M atropine. The effects of carbachol were also tested in vitro on sympathectomized arteries. The 5-HT and histamine contents of all MCAs were measured by radioenzymatic assay, and fragments were prepared for electron microscopy. No histamine was detectable in any artery studied. The 5-HT content of arteries from control animals and those perfused with vehicle (in vivo) or incubated in the physiological solution (in vitro) was 250-300 pmol/mg protein. Both compound 48/80 and carbachol reduced this amount by approximately 50% and induced a marked degranulation of mast cells. Both secretion and degranulation were dramatically blocked in vitro by atropine. No difference in the 5-HT content was observed between intact and sympathectomized arteries under any condition. We conclude that a large proportion of rabbit cerebrovascular 5-HT is stored in mast cells and that cholinergic nerve activation could theoretically release this pool by acting on mast cell muscarinic receptors. PMID- 1733330 TI - Modulation of bradykinin-induced gastric-cardiovascular reflexes by histamine. AB - Both histamine and bradykinin induce gastric-cardiovascular reflexes and are released during several pathophysiological conditions. This study examined the possibility that histamine modulates the magnitude of the reflex response to stimulation by bradykinin. Thus in chloralose anesthetized cats, the cardiovascular response to stimulation of the gastric serosa with 1 microgram/ml bradykinin was monitored before and after topical application of 100 micrograms/ml histamine (n = 6) or 1 mg/ml diphenhydramine (H1-receptor antagonist) and histamine (n = 5). After application of histamine, bradykinin induced increases in mean arterial pressure and left ventricular pressure were attenuated by 23 and 27%, respectively. Conversely, when the H1-receptors on the serosal surface of the stomach were blocked (n = 5) before application of histamine, the pressor response to bradykinin was augmented by 26%. To determine the afferents that might contribute to the attenuating effect of histamine, we recorded single unit activity in 14 A delta and 21 C visceral afferent fibers in response to bradykinin stimulation before and after histamine stimulation. We observed that the impulse activity of 10 of the A delta and 14 of the C fibers to bradykinin stimulation was reduced after treatment with histamine. These results suggest that histamine induces an inhibitory effect on the nerve endings of visceral A delta and C fibers to the action of bradykinin through an H1-receptor mechanism. This inhibitory effect attenuates the magnitude of the consequent cardiovascular reflex response. PMID- 1733331 TI - Blood ion regulation during repeated maximal exercise and recovery in humans. AB - We investigated the ionic changes in arterial (a) and femoral venous (fv) blood that accompany muscle fatigue with repeated maximal exercise. Measurements were made on separated plasma and hemolysed whole blood to quantify the relative contributions of plasma and erythrocytes to this acid-base challenge. Five healthy males performed four 30-s bouts of maximal isokinetic cycling exercise, with 4 min of rest between bouts, and recovery was followed for 90 min. In whole blood, maximal increases in [K+]a amounted to 10 +/- 2.0 meq/l and in [K+]fv to 7 +/- 4.3 meq/l and occurred at the end of bout 2. Whole blood lactate concentration ([Lac-]) peaked at 15.3 +/- 1.39 ([Lac-]a) and 16.7 +/- 1.59 meq/l ([Lac-]fv) at the end of bout 4. In plasma, peak [Lac-]a and [Lac-]fv were both 21 meq/l at the end of bout 4. Plasma [H+]a increased from 36 +/- 1.0 neq/l at rest to 44 +/- 2.9 neq/l at the end of the first bout of exercise; 80% of this increase was due to a 2.9 meq/l decrease in arterial strong ion difference ([SID]), and 20% was due to an increase in plasma protein ([Atot]a); a reduction in arterial PCO2 to 29 mmHg had an alkalinizing effect. In contrast, plasma [H+]fv increased from 39 +/- 0.5 neq/l at rest to 93 +/- 4.1 neq/l, with an increase in PfvCO2 to 97 +/- 7 mmHg contributing 75%, a decrease in [SID]fv 15%, and an increase in [Atot]fv 10% to the increase in [H+]fv. In later exercise bouts, the relative contributions of [SID]a, [Atot]a, and arterial PCO2 to plasma [H+]a were similar, but the contribution of [SID]fv to [H+]fv increased and that of femoral venous PCO2 decreased, with the contribution of [Atot]fv remaining unchanged (8-12%). During exercise and recovery, the changes in both arterial and femoral venous PCO2 and [K+] were more rapid than changes in [Lac-], and the time course of whole blood [K+] was slower than that of plasma [K+]. Erythrocytes may play an important role in regulating plasma [Lac-] and [K+] with intense exercise. PMID- 1733332 TI - Pregnancy-induced changes in ovine cerebral arteries. AB - We examined the effects of pregnancy on the ovine cerebral vasculature by comparing several characteristics of isolated endothelium-intact segments of three intracranial arteries including the middle cerebral (MCA), posterior communicating (PC), and basilar (BAS) arteries taken from pregnant sheep (138-143 days gestation, term approximately 145 days) and nonpregnant controls. For comparison, segments of the extracranial common carotid (COM) artery were also studied. With pregnancy, vessel water content increased (5.4-5.8%) in all arteries except the PC. Additionally, cellular protein content increased in all arteries (4.4-50.0%). Arterial stiffness, as determined by passive stress-strain determinations, was significantly decreased during pregnancy in the MCA but not in the larger arteries. Maximum contractile responses, when normalized to vessel wall cross-sectional area, were consistently greater in arteries from pregnant than in those from nonpregnant animals (10.1-49.7%). Relaxation to the endothelium-independent guanylate cyclase stimulator S-nitroso-N-acetyl penicillamine (SNAP) increased with pregnancy only in the distal MCA (approximately 17%). Endothelium-dependent relaxation to the calcium ionophore A23187 decreased only in the larger and more proximal COM (-39%). Thus pregnancy was associated with an increase in production of contractile force, a decrease in peripheral vascular stiffness, a decrease in the relaxant response to A23187 in the COM, and an increase in the relaxant response to SNAP in the MCA. Together, these findings indicate that pregnancy has widespread and important vessel specific cerebrovascular consequences that affect not only arterial composition, but also contractility and endothelial reactivity. PMID- 1733333 TI - Quantitative changes in long-chain fatty acids during fetal and early postnatal development in rats. AB - The quantitative importance of triacylglycerol as a source of total essential fatty acids during early postnatal development is reported in the accompanying article. Our objective here was to measure the quantitative changes in individual long-chain fatty acids in specific lipid classes of the carcass, liver, and brain of the developing rat mainly to describe the relative accumulation of long-chain vs. precursor fatty acids. Fatty acids in carcass phosphatidylcholine (micrograms/g) were lower at fetal days 18-21 than at either fetal day 15 or postnatal days +3 to +9. Individual long-chain fatty acids in liver phosphatidylcholine and phosphatidylethanolamine increased markedly by day +3 postnatally, whereas in brain phosphatidylethanolamine, the postnatal increase was delayed to between days +6 and +9. Fatty acids in carcass and liver triacylglycerols increased quantitatively by 10- to 300-fold from fetal day 21 to postnatal day +3 with amounts of both arachidonic and docosahexaenoic acid equaling linoleic acid. The ratios of linoleic and alpha-linolenic acids to respective long-chain products were significantly higher in triacylglycerols, whereas that of stearic to oleic acid was higher in phospholipids. We conclude that, during early postnatal life, oleic, linoleic, and alpha-linolenic acids are required in quantitatively greater amounts in triacylglycerols, whereas stearic acid and long-chain essential fatty acids are required in phospholipids. PMID- 1733334 TI - Sucrose-induced lipid, glucose, and insulin elevations, microvascular injury, and selenium. AB - Injury to microvessels caused by the chronic consumption of sucrose can be prevented by selenium (Se). The objective of this study was to determine the temporal sequence of changes in serum triglycerides, total cholesterol, glucose, and insulin induced by sucrose and their relationship to Se status and microvascular injury. Two groups of 24 Wistar rats were fed ad libitum diets in which the entire carbohydrate was either corn starch or sucrose. Two other groups were fed identical diets supplemented with 0.1 micrograms Se/g. At 6, 8, and 10 mo, eight rats from each group were fasted for 12 h and had blood taken. Rats were then given a glucose tolerance test and killed, and their retinal microvessels were evaluated for injury. After 6 mo, sucrose-fed rats had elevated triglycerides and total cholesterol. Abnormal glucose clearance and hyperinsulemia developed after 8 mo. Evidence of microvascular injury became apparent after 10 mo. These changes did not occur in rats provided the starch based diets, and microvascular injury did not develop in the sucrose-fed rats provided supplemental Se. Glutathione peroxidase activity was normal in all groups throughout the 10-mo experiment. These results chronicle the sucrose induced systemic insult and show that the protective effect of Se does not occur by diminishing this insult to the microvessels. PMID- 1733335 TI - Circadian variation in human cerebrospinal fluid production measured by magnetic resonance imaging. AB - Recent advances in magnetic resonance imaging have made it possible to visualize and quantify flow of cerebrospinal fluid (CSF) in the brain. The net flow of CSF through the cerebral aqueduct was used to measure CSF production in six normal volunteers at different times during a 24-h period. CSF production varied greatly both intra- and interindividually. The average CSF production in each time interval showed a clear tendency to circadian variation, with a minimum production 30% of maximum values (12 +/- 7 ml/h) approximately 1800 h and a nightly peak production approximately 0200 h of 42 +/- 2 ml/h. The total CSF production during the whole 24-h period, calculated as an average of all measurements, was 650 ml for the whole group and 630 ml for repeated measurements in each time interval in one of the volunteers. PMID- 1733336 TI - Role of vasopressin in cardiovascular responses to acute and chronic hyperosmolality. AB - Experiments were performed in conscious chronically instrumented rats to determine the role of arginine vasopressin (AVP) in the cardiovascular adjustments to acute and chronic increases in plasma osmolality. Animals were implanted with pulsed Doppler flow probes and arterial and venous catheters for the determination of cardiac output, mean arterial blood pressure (MABP), and heart rate and for the calculation of total peripheral resistance and baroreflex sensitivity (BRS). Before and after raising plasma osmolality by either 48-h water deprivation or acute hypertonic saline infusion, specific V1- or V2 vasopressinergic receptor antagonists or vehicle were administered to the animals, and the cardiovascular responses were noted. MABP was significantly elevated in water-deprived animals. These animals also exhibited significantly increased BRS, which was further increased by administration of the V1-receptor antagonist. Animals subjected to acute hypertonic saline infusion also demonstrated increased MABP, although the infusion, unlike water deprivation, did not affect BRS. We observed no significant effects on any other variable measured. We conclude that AVP plays a relatively minor role in the cardiovascular adjustments to acute and chronic hyperosmolality. PMID- 1733337 TI - Relationship of adipocyte size to hyperphagia in developing male obese Zucker rats. AB - In growing male obese Zucker rats, hyperphagia reaches a maximum or "breakpoint" and declines at an earlier age with high fat than with chow-type diets. A serial adipose tissue biopsy technique was used to correlate changes of retroperitoneal adipocyte size and feeding behavior in 5- to 7-wk-old male lean and obese rats fed laboratory chow or a 35% fat diet until 30 wk of age. Although chow-fed groups had significantly greater cumulative intake, fat-fed groups had significantly greater body weight gain, retroperitoneal depot weight, and adipocyte number. Mean adipocyte size increased continuously in chow-fed groups but decreased over weeks 20-30 in fat-fed groups, reflecting increased adipocyte number. In fat-fed obese rats, hyperphagia reached a breakpoint at 11 wk and disappeared by 13 wk. In chow-fed obese rats, hyperphagia reached a breakpoint at 15-16 wk and disappeared by 19 wk. Biopsy samples revealed that adipocyte size of fat-fed obese rats was already close to maximal at 10 wk (1.12 micrograms lipid), while that of chow-fed obese rats only approached maximal at 20 wk (0.81 microgram lipid). At these time points, lipoprotein lipase activity paralleled adipocyte size. These data indicate that the duration of the growing obese rat's hyperphagia coincides with adipocyte filling and suggest the existence of feeding stimulatory and inhibitory signals from adipose tissue. PMID- 1733338 TI - Caudal brain stem systems mediate effects of bombesin-like peptides on intake in rats. AB - Intraoral intakes of sucrose (SI; 0.1 M) and distilled water (WI) were measured in tube-fed neurologically intact and tube-fed chronic supracollicular decerebrate (CD) rats after intraperitoneal injections of saline and bombesin (BN) and gastrin-releasing peptide (GRP) in concentrations of 1, 2.5, 5, and 10 micrograms/kg. Also, to determine whether BN and GRP administration interacted with taste processing, oral motor responses during the 1st min of the intraoral infusion were videotaped and subsequently analyzed to determine the number of ingestive and aversive taste reactivity (TR) responses. Injections of greater than or equal to 2.5 micrograms/kg BN reliably suppressed SI similarly in both control and CD rats. In response to GRP, the only group difference was that injections of 1 microgram/kg GRP reliably suppressed SI in control but not CD rats. Administration of greater than or equal to 2.5 micrograms/kg GRP suppressed SI similarly in both control and CD rats. Although SI was suppressed by peripheral administration of BN and GRP, these treatments had no effect on the TR responses elicited by sucrose in either group. To determine whether the effects of BN and GRP were selective for sweet nutritive stimuli, intraoral WI was also measured in control rats after the same treatments. Injections of 5 and 10 micrograms/kg BN and 10 micrograms/kg GRP reliably suppressed WI, but lower doses had no effect. BN and GRP had no reliable effect on the pattern of TR responses elicited by water. Intraoral WI by CD rats was minimal (less than 1 ml) after injections of saline, BN, and GRP, and hence peptide effects could not be reliably assessed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733339 TI - Blockade of type A, not type B, CCK receptors attenuates satiety actions of exogenous and endogenous CCK. AB - Recent work has suggested a role for an endogenous release of cholecystokinin (CCK) acting at either type A or type B CCK receptors in the control of food intake. In an effort to investigate whether the mechanisms by which exogenously administered and endogenously released CCK inhibits food intake are similar and depend upon interactions with either type A or type B CCK receptors, we examined in rats the ability of the type A (L 364718) and type B (L 365260) CCK receptor antagonists to 1) block the inhibition of glucose consumption produced by an intraperitoneal injection of 4 micrograms/kg of CCK and 2) increase glucose consumption in the absence of exogenous CCK after a 6-h daytime deprivation. Increasing dosages (10-100 micrograms/kg) of the type A CCK antagonist resulted in a dose-related blockade of the inhibition of intake produced by CCK, and the 100 micrograms/kg dose of the A antagonist significantly increased glucose intake above baseline levels. In contrast, no dose (10-1,000 micrograms/kg) of the B antagonist blocked the inhibitory action of exogenous CCK at any time point. In the absence of exogenous CCK, the 32 and 100 micrograms/kg doses of L 364718 increased intake above baseline levels. No dose (3.2-320 micrograms/kg) of the type B antagonist, L 365260, affected intake in this paradigm. These results suggest that the mediation of the feeding-inhibitory effects of exogenous and endogenous CCK are similar and depend upon activation of type A CCK receptors. PMID- 1733340 TI - Vasopressin responses to unloading arterial baroreceptors during cardiac nerve blockade in conscious dogs. AB - We examined the relative contributions of afferent input from the heart and from arterial baroreceptors in the stimulation of arginine vasopressin (AVP) secretion in response to hypotension caused by thoracic inferior vena caval constriction (TIVCC). Afferent input from cardiac receptors was reversibly blocked by infusing 2% procaine into the pericardial space to anesthetize the cardiac nerves. Acute cardiac nerve blockade (CNB) alone caused a rise in mean arterial pressure (MAP) of 24 +/- 3 mmHg but no change in plasma AVP. If the rise in MAP was prevented by TIVCC, plasma AVP increased by 39 +/- 15 pg/ml, and if MAP was allowed to increase and then was forced back to control by TIVCC, plasma AVP increased by 34 +/- 15 pg/ml. Thus the rise in MAP during CNB stimulated arterial baroreceptors, which in turn compensated for the loss of inhibitory input from cardiac receptors on AVP secretion. These results indicate that the maximum secretory response resulting from complete unloading of cardiac receptors at a normal MAP results in a mean increase in plasma AVP of 39 pg/ml in this group of dogs. When MAP was reduced 25% below control levels (from 95 +/- 5 to 69 +/- 3 mmHg) by TIVCC during pericardial saline infusion, plasma AVP increased by 79 +/- 42 pg/ml. However, the same degree of hypotension during CNB (MAP was reduced from 120 +/- 5 to 71 +/- 3 mmHg) led to a greater (P less than 0.05) increase in plasma AVP of 130 +/- 33 pg/ml. Because completely unloading cardiac receptors can account for an increase of only 39 pg/ml on average in this group of dogs, the remainder of the increase in plasma AVP must be due to other sources of stimulation. We suggest that the principal stimulus to AVP secretion after acute CNB in these studies arises from unloading the arterial baroreceptors. PMID- 1733341 TI - Pressure natriuresis and angiotensin II in reduced kidney mass, salt-induced hypertension. AB - In normal subjects, high sodium intake causes little change in mean arterial pressure (MAP). However, MAP is sodium sensitive after reduction of kidney mass. The present study examined the role of increased renal artery pressure and decreased angiotensin II (ANG II) formation in maintaining sodium balance during high sodium intake in dogs with reduced kidney mass. In seven dogs with pressure natriuresis intact, increasing sodium intake from 36 to 466 meq/day for 7 days raised MAP from 91 +/- 2 to 106 +/- 2 mmHg. Sodium excretion increased promptly and cumulative sodium balance increased by only 80 +/- 26 meq after 7 days of high sodium intake. When renal perfusion pressure was servo-controlled to prevent pressure natriuresis, comparable increases in sodium intake raised MAP from 88 +/ 2 to 128 +/- 4 mmHg after 7 days. Sodium excretion rose to match intake, but cumulative sodium balance increased by 226 +/- 34 meq after 7 days. In dogs in which ANG II levels were held constant by converting enzyme inhibition and constant ANG II infusion (2 ng.kg-1.min-1 iv), raising sodium intake for 7 days elevated MAP from 126 +/- 2 to 146 +/- 4 mmHg after 7 days while increasing cumulative sodium balance by 212 +/- 29 meq. When renal perfusion pressure was servo-controlled and ANG II levels held constant, raising sodium intake elevated MAP from 125 +/- 3 to 166 +/- 11 mmHg and increased cumulative sodium balance by 399 +/- 128 meq. These data indicate that pressure natriuresis and decreased ANG II formation are important in minimizing sodium retention and hypertension during high sodium intake. However, other mechanisms can increase sodium excretion independent of pressure natriuresis and suppression of ANG II during salt-induced hypertension. PMID- 1733342 TI - Cardiovascular and renal effects of gamma-MSH in spontaneously hypertensive and normotensive Wistar Kyoto rats. AB - The objective of this study was to compare the cardiovascular and renal effects of the two gamma-melanocyte-stimulating hormone (MSH) peptide sequences in the pro-opiomelanocortin prohormone structure in conscious, anesthetized, and pithed spontaneous hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) controls. In the conscious but not in the pithed rats, intravenous injection of gamma 2- and gamma 1-MSH induced a rapid and dose-dependent increase in mean arterial pressure (MAP), and gamma 2-MSH was more potent than gamma 1-MSH. The pressor response was more pronounced and more sustained in the SHR compared with the WKY. There were dose-dependent and significant increases in heart rate (HR) after gamma 2- and gamma 1-MSH in the SHR. At intravenous infusions of low doses of gamma 2-MSH, which did not significantly influence MAP or HR, urinary sodium excretion was significantly increased in both SHR and WKY. In conscious, but not in anesthetized rats, intracerebroventricular administration of the gamma 2-MSH peptide induced sustained increases in MAP in both SHR and WKY. After intrathecal administration, there were transient pressor effects of gamma 2-MSH. We conclude that the pro-opiomelanocortin-derived gamma 2- and gamma 1-MSH peptide sequences possess potent rapid pressor actions. The pressor effects, which require an intact sympathetic nervous system, are more pronounced in the SHR strain. Moreover, gamma 2-MSH induces natriuresis when administered in nonpressor doses in WKY and SHR. PMID- 1733343 TI - Triacylglycerol: an important pool of essential fatty acids during early postnatal development in rats. AB - Developmental changes in the content and composition of major organ lipid pools are not well known. Our objective was to assess quantitatively the changes in lipids, particularly those containing long-chain fatty acids, in the placenta and the brain, liver, and carcass of the fetal and suckling rat. Pregnant dams were killed at days 15, 18, and 21 (term) of pregnancy and the placentas and fetuses removed and analyzed; suckling rats were killed at days +3, +6, and +9 of lactation. Whereas the long-chain fatty acid content of the phospholipids (mg/g) of the fetal or suckling rat remained relatively constant from day 18 of pregnancy to day +9 of lactation, long-chain fatty acids in triacylglycerols increased from prenatal values by 10- to 12-fold during the first 9 postnatal days. Prenatally, triacylglycerol accounted for no more than 32% of total whole body essential fatty acids (day 21), but postnatally this increased to 81-88%. From day 21 to day +9, the proportion of n-6 and n-3 essential fatty acids within the total triacylglycerol pool of the suckling rat increased 71 and 317%, respectively. We conclude that in the suckling rat, triacylglycerol is quantitatively the most important source of essential fatty acids during at least the first 9 days of life. PMID- 1733344 TI - Use of a thermal pulse decay system to assess avian renal blood flow during reduced renal arterial perfusion pressure. AB - Using a simplified avian kidney model, renal arterial perfusion pressure (RAPP) was reduced from 120 (control) to 70 mmHg (near the glomerular filtration rate autoregulatory limit) and then to 46 mmHg (below the glomerular filtration rate autoregulatory range) in kidneys with ambient or partially restricted renal portal flow. Renal blood flow (RBF) was measured with a thermal pulse decay (TPD) system, using TPD thermistor probes inserted at three locations to evaluate regional differences in RBF. The clearance (CPAH) and extraction of p aminohippuric acid were used to calculate renal plasma flow (RPF). CPAH, RPF, and RBF values were consistently lower for kidneys with restricted portal flow than for kidneys with ambient portal flow. Reducing RAPP to 46 mmHg did not significantly reduce CPAH, RPF, or RBF in the ambient group but did significantly reduce CPAH and RPF (regressed on RAPP) in the restricted group. RBF was not significantly affected when RAPP was reduced in the restricted group, although significant regional differences in blood flow were recorded. Renal vascular resistance decreased significantly as RAPP was reduced to 46 mmHg in the ambient group, confirming the renal autoregulatory response. In separate validation studies, significant reductions in RBF were detected by the TPD system during acute obstructions of portal and/or arterial flow. Overall, the results support previous evidence that avian RBF remains constant over a wide range of RAPPs. Observations of nonuniform intrarenal distributions of portal blood flow suggest that the portal system maintains the constancy of RBF in regions with proportionately high portal-to-arterial flow ratios. PMID- 1733345 TI - Quantitative analysis on odor intensity and quality of optical isomers in turtle olfactory system. AB - No systematic electrophysiological study on differences in odor intensity and quality between optical isomers has been carried out. In the present study, we measured the turtle olfactory bulbar responses to six pairs of highly pure optical isomers and compared the differences in odor intensity and quality between the optical isomers. The results obtained indicated that with all odorants tested, there was no difference in odor threshold and intensity in the whole concentration range examined between optical isomers. The difference in odor quality of optical isomers was evaluated by a quantitative cross-adaptation method in which odorant concentration was varied. The degree of cross adaptation between optical isomers greatly varied with species of odorants. The rank order of the magnitude of the differences in odor quality between optical isomers was carvone greater than beta-citronellol greater than menthol greater than hydroxycitronellal greater than citronellal greater than limonene. PMID- 1733346 TI - A novel biotinylated probe specific for hyaluronate. Its diagnostic value in diffuse malignant mesothelioma. AB - Diffuse malignant mesotheliomas are known to secrete a large amount of hyaluronate, whereas adenocarcinomas produce predominantly neutral mucins. In the present study, we assessed the diagnostic usefulness of a new, highly specific and sensitive hyaluronate binding probe to discriminate between mesotheliomas and adenocarcinomas. We studied 33 mesotheliomas and 37 adenocarcinomas in order to establish specific diagnostic criteria for using the hyaluronate binding probe. Of the adenocarcinomas, only three showed significant positive staining for hyaluronate (8%). By contrast, all the mesotheliomas exhibited positive staining for hyaluronate. Furthermore, the staining reaction was classed as moderate or greater in 26 mesotheliomas (79%), thus suggesting the utility of this probe in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. We conclude that strong cytoplasmic or membranous staining for hyaluronate is highly predictive of malignant mesothelioma. The hyaluronate binding probe should therefore be considered an important adjunct to be used in combination with electron microscopy and immunohistochemistry in the histologic diagnosis of diffuse malignant mesothelioma. PMID- 1733347 TI - Rosai-Dorfman disease of soft tissue. AB - Whereas Rosai-Dorfman disease (RDD) or sinus histiocytosis with massive lymphadenopathy is well described in lymph nodes and other organs, it is frequently not recognized in soft tissue. We studied the clinical and histologic features of 23 previously unreported soft tissue lesions from 17 patients (13 females, 4 males) who were 24 to 66 years of age (mean, 46 years). These lesions involved the extremities (12, 52%), trunk (6, 26%), head and neck (3, 13%), and the retroperitoneum (2, 9%). Associated lymph node involvement was present in four cases; most patients were asymptomatic. RDD of soft tissue had more subtle histologic features than its lymph node counterpart. Emperipolesis was less conspicuous and proliferating histiocytes were frequently spindled, associated with collagen deposition, and arranged in a vague storiform pattern with scattered lymphoplasmacytic aggregates. These features led to a variety of diagnoses, including benign inflammatory and fibrohistiocytic lesions (13 cases) as well as lymphoma and malignant fibrous histiocytoma (three cases). RDD was correctly diagnosed in only one case. Diagnosis was confirmed in 16 of 18 lesions by detection of S-100 protein and histiocytic markers KP1 (12 of 13) and lysozyme (eight of 11) in the characteristic histiocytes. Recognition that RDD of soft tissue occurs in an older patient population than does nodal RDD and that it mimics fibrous and inflammatory lesions of soft tissue is important. PMID- 1733348 TI - Thymomas presenting as pleural tumors. Report of eight cases. AB - Eight cases of thymomas presenting as pleural-based lesions are presented. The patients are five men and three women between the ages of 38 and 71 years (mean 54.5). Histologically, six tumors showed morphologic features indistinguishable from classical mediastinal thymomas, namely lobulation produced by fibrous bands and a biphasic cell population composed of epithelial cells admixed with small lymphocytes. One case showed prominent spindle cell configuration and marked vascularization resulting in a hemangiopericytomalike appearance; in another case, the epithelial component was associated with cystic structures. The radiographic findings were diffuse pleural thickening with encasement of the lung in four cases; an ill defined mass involving the right diaphragmatic pleura with engulfment of the left lower lobe in one case; diffuse pleural thickening along the mid lateral axillary line in one case; and left-sided pleural masses involving the diaphragmatic and chest wall pleural surfaces in another. In the remaining case, a massive unilateral left pleural effusion obscured the underlying lesion. Clinically, the patients presented with varied symptoms, including shortness of breath, fever, and weight loss. Follow-up information was obtained in four patients. One patient died 1 month after initial diagnosis, but no details of the cause of death were available. Another had metastasis to the groin 1 year after diagnosis. The other two patients were alive and well 2-10 years after the initial diagnosis. PMID- 1733349 TI - A flow cytometric, clinical, and histological study of stromal neoplasms of the gastrointestinal tract. AB - Histological sections of 102 stromal neoplasms of the gastrointestinal tract occurring in 100 patients have been assessed for 23 clinical and histological parameters and the corresponding paraffin embedded (archival) material processed for flow cytometry. Where possible, information as to clinical presentation and survival was obtained. The only absolute criterion for malignancy was the presence of spread of tumour beyond the organ of origin at the time of diagnosis. Of the remaining tumours (i.e., tumours locally confined at diagnosis), those found incidentally at operation and those of a small size (less than 60 mm diameter) behaved in a generally benign fashion. Of the histological parameters, six correlated with malignant behaviour: high mitotic count, high cellularity, marked nuclear pleomorphism, rounded as opposed to spindle cell shape, bizarre mitoses, and vascular invasion. The presence of DNA aneuploidy as shown by flow cytometry correlated strongly with a poor prognosis (p less than 0.0005). Tumours with a high mitotic count [greater than 9 per 10 high-power fields (hpf) (1.59 mm2)] behaved in an almost uniformly malignant fashion. Those with a low mitotic count [less than 3/10hpf (1.59 mm2)], behaved in a benign fashion apart from one case where no mitoses were discernible yet the tumour metastasised and killed the patient. The intermediate group of tumours (3-9 mitoses per 10 hpf inclusive) were difficult to predict, although the majority behaved in a malignant fashion. Within this group the presence of DNA aneuploidy appeared most useful in predicting prognosis. PMID- 1733350 TI - Lipogranulomas and gold in the liver in rheumatoid arthritis. AB - Liver biopsies have been performed routinely as part of a protocol to evaluate methotrexate therapy in severe rheumatoid arthritis. All patients in the study had failed standard medical therapy, including gold treatment. Twenty-three of 41 patients (56%) had well-formed lipogranulomas (LGs) in the lobules, compared with an incidence of approximately 5% in our general biopsy population. Twenty-seven of 41 patients (66%) had a unique pigment in their livers. In 20 of these, the pigment was in LGs; in the seven patients with pigment not associated with lobular LG, it was found in lipid droplets in portal triads. The pigment varied from irregular pale brown granules slightly larger than those of hemosiderin, to smaller black round granules. Lipogranuloma-associated pigment of this type is an unusual finding, reminiscent of argyria. There was a variable appearance upon polarization, the black granules at times being strikingly refractile. There was a positive correlation between the prominence of LG and the quantity of pigment. The pigment resembled that described with gold deposition in other tissues. Radiographic microanalysis of both brown and black granules was performed in three cases. Characteristic spectra (energy-dispersive spectroscopy) demonstrated the presence of gold in each case. Silver was not identified. The high incidence of LG may reflect the frequent administration of gold in an oily vehicle. Gold may remain trapped in the liver for a prolonged time. Thus far, we have not detected any adverse effect from the presence of LG-associated gold. PMID- 1733351 TI - An abbreviated standard procedure for accurate tumor volume estimation in prostate cancer. AB - Quantitated tumor volume is reported to be the single most important morphologic predictor for lymph node metastasis and distant spread in the individual patient with adenocarcinoma of the prostate. There is a practical need for a system of tumor volume estimation that avoids the serial blocking of prostatectomy specimens required by standard techniques. Accordingly, in 145 prostatectomy specimens, cancer volumes obtained from computer planimetry of the full set of slides were compared with cancer volumes estimated visually in samples with fewer than half the standard number of slides selected according to three abbreviated protocols. Two of these abbreviated protocols gave cancer volume estimates that were within +/- 20% of the computer value on the full slide set in 96% and 89% of cases. These findings define a simple and relatively inexpensive procedure for deriving important prognostic information in a systematic fashion from the morphologic data obtained in prostatectomy specimens for adenocarcinoma of the prostate. PMID- 1733352 TI - Granular cell tumor of the biliary tree. PMID- 1733354 TI - Papers of the Society for Surgery of the Alimentary Tract. 32nd annual meeting, New Orleans, Louisiana, May 20-22, 1991. PMID- 1733353 TI - Alveolar soft part sarcoma. Is there a definite diagnostic criterion for this entity? PMID- 1733355 TI - Influence of levamisole on pancreatic infection in acute pancreatitis. AB - We investigated the effect of levamisole on pancreatic infection in a model of acute pancreatitis (AP) in cats. Animals with and without AP received Escherichia coli intravenously. Blood was then taken at intervals for culture. AP reduced phagocytic function by 28% as measured by the rate of bacterial disappearance from the blood (p less than 0.03). In other cats, AP was induced, and E. coli were placed into the pancreatic duct. Levamisole was given orally in some cats; the remainder were untreated. Control cats (neither AP nor levamisole) also received E. coli. Seven days later, pancreases from all control cats were sterile. In AP cats, the pancreatic infection rate was 73%. Levamisole reduced the rate of infection to 22% (p less than 0.03). We concluded that phagocytic function was impaired in cats with AP. Levamisole reduced the rate of pancreatic infection. PMID- 1733356 TI - Early surgical debridement of symptomatic pancreatic necrosis is beneficial irrespective of infection. AB - In order to assess the recent trend of nonoperative management of pancreatic necrosis, we reviewed 82 variables in 73 consecutive patients with symptomatic necrotizing pancreatitis. The mortality rate for the series was 25% (18 of 73). The only preintervention variables that correlated with mortality were APACHE II score greater than 15 (p = 0.01), preintervention blood transfusion (p less than 0.001), respiratory failure (p less than 0.001), and shock (p less than 0.01). Patients who developed recurrent sepsis following the initial intervention had a significantly higher mortality rate (17 of 34) than those who did not (1 of 39) (p less than 0.001). The rate of recurrent sepsis varied widely among individual surgeons and correlated with APACHE II score. The presence of infected versus noninfected necrosis did not correlate significantly with outcome. When percutaneous radiologically guided drainage was the initial therapeutic modality (n = 6), recurrent sepsis requiring surgical drainage inevitably occurred. Patients treated with percutaneous drainage (often in combination with surgical drainage) had a longer hospital stay (82 versus 42 days, p less than 0.001), spent more days in the intensive care unit (31 versus 6 days, p less than 0.001), and required more days of total parenteral nutrition (57 versus 27 days, p less than 0.001) than those treated solely by surgical means. We conclude that aggressive initial surgical debridement should be the first step in managing symptomatic pancreatic necrosis and that the presence of infection should not be the sole determinant of intervention. PMID- 1733357 TI - An endoscopic retrograde cholangiopancreatography (ERCP)-based algorithm for the management of pancreatic pseudocysts. AB - In the treatment of pancreatic pseudocysts, percutaneous and endoscopic drainage have, in certain cases, become alternatives to surgery. However, each treatment modality carries risks of complications and recurrences that may be minimized by the appropriate allocation of therapy. This article proposes the use of an endoscopic retrograde cholangiopancreatography (ERCP)-based algorithm as a means to allocate pseudocyst therapy based on the findings of pancreatic duct obstruction or pseudocyst communication. To evaluate this algorithm, the records of a series of patients with pancreatic pseudocysts seen at Duke University Medical Center from 1984 to 1990 were reviewed. Of 102 patients, 73 had symptomatic pseudocysts that required treatment. Forty of the 69 elective interventions were preceded by ERCPs and retrospectively applied to the algorithm. The number of adverse outcomes (treatment failures + complications) of the group that followed the algorithm was 3 of 26 (12%), while the number of adverse outcomes of the group that did not follow the algorithm was 6 of 14 (43%) (p less than 0.04 by Fisher's exact test). These two subgroups were similar in all other characteristics examined. Therefore, this ERCP-based algorithm may be used to allocate pseudocyst treatment; however, a prospective trial is necessary to prove its efficacy. PMID- 1733358 TI - Spectrum of cystic tumors of the pancreas. AB - The pathologic and clinical classification, as well as the behavior, of cystic tumors of the pancreas has been the subject of controversy. We retrospectively reviewed 50 patients with a diagnosis of cystic tumor of the pancreas observed at The Johns Hopkins Hospital from 1984 to 1991. These tumors were classified into three broad groups: I, cystadenoma; II, cystadenocarcinoma; and III, adenocarcinoma with mucin production or an associated cyst. The three groups did not differ with respect to age or sex. The most common clinical presentation was abdominal pain. Symptoms and signs among the three groups were similar except that patients with cystadenomas were less likely (p less than 0.05) to be jaundiced and more likely (p less than 0.05) to be asymptomatic. Radiologic findings on computerized tomography, cholangiography, and arteriography also overlapped, making precise preoperative determination of tumor type difficult. Operative classification was also often not possible. The resectability rate (Group I, 91%; Group II, 67%; Group III, 53%) and 5-year survival rate (Group I, 90%; Group II, 72%; Group III, 14%) correlated with careful pathologic determination. Cystic tumors of the pancreas represent a spectrum of disease ranging from benign cystadenoma to adenocarcinoma masquerading as cystadenocarcinoma. We recommend resection whenever possible, even when preoperative evaluation suggests benign disease. PMID- 1733359 TI - Do sensory neurons mediate adaptive cytoprotection of gastric mucosa against bile acid injury? AB - Pretreatment with the mild irritant 1 mmol acidified taurocholate protects the gastric mucosa from the injury induced by the subsequent application of 5 mmol acidified taurocholate, a phenomenon referred to as "adaptive cytoprotection." How this occurs remains an enigma. The purpose of this study was to investigate the role of sensory neurons and mucus secretion in this phenomenon. Prior to injury with 5 mmol acidified taurocholate (pH 1.2), the stomachs of six groups of rats were subjected to the following protocol. Two groups were topically pretreated with either saline or the mild irritant 1 mmol acidified taurocholate. Two other groups received the topical anesthetic 1% lidocaine prior to pretreatment with either saline or 1 mmol acidified taurocholate. The last two groups got the mucolytic agent 10% N-acetylcysteine (NAC) after pretreatment with either saline or 1 mmol acidified taurocholate. Injury was assessed by measuring net transmucosal ion fluxes, luminal appearance of deoxyribonucleic acid (DNA), and gross and histologic injury. Pretreatment with the mild irritant 1 mmol acidified taurocholate significantly decreased bile acid-induced luminal ion fluxes and DNA accumulation, suggesting mucosal protection (corroborated by gross and histologic injury analysis). This effect was negated by lidocaine but not by NAC. Thus, it appears that sensory neurons, and not increased mucus secretion, play a critical role in adaptive cytoprotection. PMID- 1733360 TI - Role of octreotide in the prevention of postoperative complications following pancreatic resection. AB - Though morbidity and mortality rates following pancreatic resection have improved in recent years, they are still around 35% and 5%, respectively. Typical complications, such as pancreatic fistula, abscess, and subsequent sepsis, are chiefly associated with exocrine pancreatic secretion. In order to clarify whether the perioperative inhibition of exocrine pancreatic secretion prevents complications, we assessed the efficacy of octreotide, a long-acting somatostatin analogue. We conducted a randomized, double-blind, placebo-controlled, multicenter trial in 246 patients undergoing major elective pancreatic surgery. Patients were stratified into a high-risk stratum (limited to patients with pancreatic and periampullary tumors) or low-risk stratum (patients with chronic pancreatitis). Patients received octreotide (3 x 100 micrograms) or placebo subcutaneously for 7 days perioperatively. Eleven complications were defined: death, leakage of anastomosis, pancreatic fistula, abscess, fluid collection, shock, sepsis, bleeding, pulmonary insufficiency, renal insufficiency, and postoperative pancreatitis. Two hundred patients underwent pancreatic head resection, 31 patients underwent left resection, and 15 patients had other procedures. The overall mortality rate within 90 days was 4.5%, with 3.2% in the octreotide group and 5.8% in the placebo group. The complication rate was 32% in the patients receiving octreotide (40 of 125 patients) and 55% in patients receiving placebo (67 of 121 patients) (p less than 0.005). In the patients in the high-risk stratum, complications were observed in 26 of the 68 (38%) patients treated with octreotide and in 46 of 71 (65%) patients given placebo (p less than 0.01). Whereas in patients in the low-risk stratum, the complication rate was 25% (14 of 57 patients) in those treated with octreotide and 42% (21 of 50 patients) in patients given placebo (p = NS). The perioperative application of octreotide reduces the occurrence of typical postoperative complications after pancreatic resection, particularly in patients with tumors. PMID- 1733361 TI - A selective approach to preexisting portal vein thrombosis in patients undergoing liver transplantation. AB - Splanchnic venous inflow is considered mandatory to ensure graft survival after liver transplantation. Over a 68-month period, we performed 570 liver transplants in 495 patients. Portal vein thrombosis was present in 16 patients. At transplant, the extent of the occlusion included portal vein alone (n = 4), portal including confluence of the splenic and superior mesenteric veins (n = 8), portal, splenic, and distal superior mesenteric veins (n = 2), and the entire portal vein, splenic vein, and superior mesenteric vein (n = 2). The operative approach included thrombectomy alone (n = 5), anastomosis at the confluence of the splenic and superior mesenteric splenic veins (n = 8), and extra-anatomic venous reconstruction (n = 3). The mean operative blood loss was 22 +/- 22 units, and the mean operative time was 9.7 +/- 4.8 hours. The 1-year actuarial survival rate was 81%, with a mean follow-up of 12.5 months. In summary, with a selective approach and the use of innovative forms of splanchnic venous inflow, portal vein thrombosis is no longer a contraindication to liver transplantation. PMID- 1733363 TI - Morphine effects on human colonic myoelectric activity in the postoperative period. AB - Colonic myoelectrical activity was studied in 25 patients, 18 of whom received morphine sulfate, using bipolar electrodes placed in the ascending and descending colon during laparotomy. Baseline myoelectrical activity was recorded daily, then morphine (3 to 15 mg) was administered intravenously, intramuscularly, or epidurally, and recordings continued. Seven activity patterns were observed during recovery from postoperative ileus. During the first 2 postoperative days, morphine at any dose did not affect colon myoelectrical activity. From the third postoperative day on, morphine given intravenously or intramuscularly initiated clusters of short, nonmigrating, phasic spike bursts occurring on each successive slow wave in 14 of 18 patients, which lasted for 30 to 45 minutes. When morphine was administered epidurally, there was no colonic response in any patient. These findings suggest that: (1) morphine intravenously or intramuscularly induces predominantly nonmigrating colonic spike bursts; (2) morphine-induced activity alters the normal pattern of colonic motility during recovery from postoperative ileus; and (3) these phenomena are not due to direct action of morphine on the spinal cord since epidural morphine had no effect. PMID- 1733362 TI - Treatment of Budd-Chiari syndrome due to inferior vena cava occlusion by combined portal and vena caval decompression. AB - This study concerns Budd-Chiari syndrome (BCS) caused by occlusion of the subdiaphragmatic inferior vena cava (IVC). It describes the experimental and clinical evaluation of the treatment of this disorder by one-stage combined portal and vena caval decompression with a direct side-to-side portacaval shunt (PCS) and a caval-atrial shunt (CAS) graft. BCS was produced in rats by gradual occlusion of the suprahepatic IVC with an ameroid constrictor. When ascites and portal hypertension were established, 12 control rats survived a sham thoracolaparotomy, 16 rats survived a mesoatrial shunt, and 16 rats survived combined PCS and CAS graft. All control rats re-formed ascites and died within 2 months. Nine of 16 rats with mesoatrial shunt developed graft thrombosis, re formed ascites, and died within 2 months. In contrast, only 2 of 16 rats that underwent combined PCS and CAS developed graft thrombosis, re-formed ascites, and died. Liver biopsies showed reversal of severe pathologic changes in rats with patent grafts. Clinical evaluation of combined PCS and CAS using a 20-mm ring reinforced Gore-Tex graft has been undertaken in five patients with BCS and ascites, hepatosplenomegaly, intense hepatic congestion on biopsy, and angiography showing occlusion of both the IVC and hepatic veins. All five patients are alive and well 6 months to 7.5 years postoperatively with patent grafts, no ascites or need for diuretics, no encephalopathy, normal liver function, and reversal of liver pathology. It is concluded that combined PCS and CAS create a high-flow shunt that decompresses both the portal system and IVC, has a low incidence of graft thrombosis, has been consistently effective in relieving BCS caused by IVC occlusion, and appears to be superior to mesoatrial shunt. PMID- 1733364 TI - Burn-induced transcriptional regulation of small intestinal ornithine decarboxylase. AB - The mechanisms responsible for gut repair after burn injury have not been established. Polyamines are required for eukaryotic cell growth and differentiation. The enzyme ornithine decarboxylase (ODC) catalyzes the rate limiting step in polyamine biosynthesis. The role of ODC activity in repair of injured small bowel mucosa after burns has not been investigated. This study examined the effects of burn injury on gut mucosal mass and regulation of ODC gene expression and ODC activity in small bowel mucosa. After an overnight fast, 18 male Sprague-Dawley rats (250 to 300 g) were randomized into sham, 20% burn, or 60% burn groups. We measured ODC activity, mucosal weight, deoxyribonucleic acid (DNA) content, and protein content in proximal and distal small bowel mucosa at postburn intervals of 0, 3, 12, 24, and 48 hours. Gut mucosal ODC messenger ribonucleic acid (mRNA) levels were determined. Burn injury caused significant atrophy of the gut mucosa by 12 hours postburn; restoration was evident by 48 hours after burn. ODC activity was increased in the proximal small bowel at 12 and 24 hours after burn in the rats in both the 20% burn and 60% burn groups; by contrast, only rats in the 60% burn group had increased ODC activity in the distal small bowel. ODC mRNA levels increased in the proximal gut mucosa as early as 3 hours after the burn and returned to control values after 24 hours. These data show that mucosal restoration begins soon after burn injury and that the induction of ODC mRNA and ODC activity are important events. PMID- 1733365 TI - Beneficial effects of prewarming the donor liver for rat liver transplantation. AB - Cold preservation of donor liver can injure the microcirculation enough to impair the function and survival of the transplanted liver. In this study, prewarming the donor liver to 10 degrees C to 12 degrees C after cold storage prior to implantation significantly increased survival rates in rats 1 week after surgery. Following 6, 8, or 9 hours of cold storage in chilled normal saline, prewarming increased survival rates from 40% to 78.6%, from 0% to 35.7%, and from 0% to 14.3%, respectively. The results suggest that pretreatment of the donor liver with warmth following cold storage and before implantation will improve the survival rate of liver transplanted rats. PMID- 1733366 TI - Cytokine regulation of intestinal glutamine utilization. AB - The effects of cytokines on intestinal glutamine metabolism were studied to gain further insight into the regulation of altered glutamine metabolism that occurs during severe infection. One hundred thirteen adult rats were given a single dose of interleukin-1 (IL-1, 50 micrograms/kg), tumor necrosis factor (TNF, 50 micrograms/kg or 150 micrograms/kg), or saline (controls), and flux studies were performed 4 or 12 hours later. Intestinal blood flow was not different between control and cytokine-treated animals at either time point. At the 4-hour time point, arterial glutamine fell by 16% to 21% in the cytokine-treated animals (p less than 0.05); at the 12-hour time point, the arterial glutamine concentration had returned to normal. Intestinal glutamine extraction decreased in the animals treated with IL-1 at both time points (4 hours: 13% +/- 1.3% in IL-1 versus 20% +/- 1.6% in controls, p less than 0.05; and 12 hours: 9% +/- 2% in IL-1 versus 17% +/- 2% in controls, p less than 0.05). Consequently, net intestinal glutamine uptake fell in the animals treated with IL-1 at both time points (p less than 0.05). Similarly, the activity of mucosal glutaminase, the principal enzyme of glutamine hydrolysis in the gut, fell by 50% in the 4-hour study (6.1 +/- 0.6 mumol/h/mg protein in IL-1 versus 9.6 +/- 0.8 mumol/h/mg protein in controls, p less than 0.01) and by 40% in the 12-hour study (5.4 +/- 0.5 mumol/h/mg protein in IL-1 versus 8.8 +/- 0.4 mumol/h/mg protein in controls, p less than 0.05). Concomitant with the aforementioned decrease in gut glutamine metabolism was a 25% incidence of positive blood cultures for gram-negative organisms in IL-1 treated rats studied at the 12-hour time point (p = 0.05 versus controls). In the doses administered and at the time points studied, TNF had no effects on the parameters of gut glutamine metabolism examined. The results indicate that IL-1 is a potential mediator of the alterations in gut glutamine metabolism observed in sepsis and endotoxemia. PMID- 1733367 TI - Effect of sepsis or cytokine administration on release of gut peptides. AB - The effect of sepsis on plasma levels of various gut peptides was studied in rats. Sepsis was induced by cecal ligation and puncture (CLP); control animals underwent sham operation. Sixteen hours after CLP or sham operation, portal and systemic blood was drawn, and plasma levels of gastrin, vasoactive intestinal peptide (VIP), secretin, peptide YY (PYY), gastrin-releasing peptide (GRP), and substance P were determined by radioimmunoassay. Plasma levels of gastrin, VIP, PYY, and secretin were elevated in septic rats compared with nonseptic animals, with the highest levels noted in portal blood. There was no effect of sepsis on GRP or substance P levels. In other experiments, human recombinant interleukin 1 alpha (IL-1 alpha) or recombinant tumor necrosis factor alpha (TNF alpha) was injected intraperitoneally (300 micrograms/kg body weight in 3 divided doses over 16 hours). There was no change in plasma levels of gut peptides after IL-1 alpha injection. TNF alpha induced elevation of PYY levels in portal plasma with no change in other gut peptide levels. The results suggest that sepsis stimulates release of certain gut peptides and that TNF, but not IL-1, may be partly responsible for this response. The mechanism of the release of gut peptides and its significance in the pathophysiologic changes induced by sepsis remain to be determined. PMID- 1733368 TI - Hypercarbia during carbon dioxide pneumoperitoneum. AB - Patients with cardiopulmonary insufficiency undergoing laparoscopic surgery with carbon dioxide (CO2) pneumoperitoneum may retain CO2 resulting in clinically significant respiratory acidosis. A canine model of pulmonary emphysema induced by papain inhalation was utilized to evaluate the respiratory effects of both CO2 and helium pneumoperitoneum. Prior to papain inhalation and 5 and 8 weeks after initial treatment under general anesthesia, mechanical ventilation was adjusted to maintain the end-tidal CO2 (ETCO2) at 40 mm Hg during baseline and pneumoperitoneum physiologic monitoring periods. Utilizing an analysis of variance, hemodynamic and respiratory physiologic parameters were compared. In this canine model, all dogs demonstrated consistent hypercarbia during CO2 pneumoperitoneum prior to papain treatments, but CO2 retention was significantly increased in the emphysematous state. The occurrence of hypercarbia during CO2 pneumoperitoneum may be underestimated by ETCO2 monitoring as was revealed by an increased PaCO2 (arterial carbon dioxide pressure)-ETCO2 gradient with an increasing time interval between papain exposure and period of physiologic monitoring. Irrespective of the pulmonary condition of the dog, helium pneumoperitoneum did not produce any hypercarbic or acidic changes when compared with the concomitant baseline period of dogs prior to the induction of pneumoperitoneum, thus suggesting that helium pneumoperitoneum may be a reasonable alternative in patients at risk for CO2 retention. PMID- 1733369 TI - Circadian rhythm in prostacyclin activity in gastric tissue of the fasting rat. AB - Gastroduodenal ulcer disease may result from the desynchronization of the circadian rhythms of gastric protective and destructive factors. The purpose of this study was to evaluate whether gastric tissue 6-keto prostaglandin F1 alpha (PGF1 alpha), a catabolic derivative of the putative protective factor prostacyclin, is produced in a circadian fashion in the rat model. Forty-eight male Sprague-Dawley rats were acclimatized in sound-attenuated, lightproof chambers for 3 weeks on a 12:12 hour light/dark entrainment schedule. After an 18 hour fast, six rats were killed at each of eight sampling times. The stomachs were exposed, removed, and assayed for total 6-keto PGF1 alpha content by radioimmunoassay. Cosinor analysis of the data showed significant (p = 0.0262) circadian rhythmicity in 6-keto PGF1 alpha content with an acrophase (peak time) value of 0503 HALO (hours after lights on) or in the middle of the lights-on inactive period for the rats. Hypothetically, the circadian rhythm in some gastric protective factors may render the gastric mucosa vulnerable to injury in a circadian fashion. PMID- 1733370 TI - Where have the general surgeons (doctors) gone? PMID- 1733371 TI - Postoperative T-tube tract choledochoscopy. AB - One hundred twenty-six patients underwent postoperative fiberoptic T-tube tract choledochoscopy for the management of retained biliary calculi as demonstrated by T-tube cholangiography. Extraction was successful in 94% of patients with retained stones. Thirty-nine patients had more than 1 stone, 20 patients had heptic duct stones, and 14 patients had large stones requiring electrohydraulic lithotripsy or laser fragmentation. Stone removal was not possible in six patients, secondary to either slippage of the T-tube with obliteration of the tract, inability to remove the stones with available instruments, a tortuous tract, or choledochoscope malfunction. Minor complications, most commonly transient fever, occurred in 12 patients. No serious complications or deaths occurred. The advantages of T-tube tract choledochoscopy include direct visualization of the biliary tree, avoidance of radiation exposure, and easy access to hepatic duct stones. This is the preferred method for treating retained biliary calculi in patients with a T-tube in situ. PMID- 1733372 TI - Does selective vagotomy prevent delayed gastric emptying and altered myoelectric activity following Roux-en-Y gastrojejunostomy? AB - Delayed gastric emptying occurs frequently following Roux-en-Y gastrojejunostomy. The role of vagal denervation in the etiology of this "Roux-stasis syndrome" is controversial. This study evaluates the effect of selective vagotomy on gastric emptying and motility following Roux-en-Y. Four dogs underwent control gastric emptying studies. The animals then underwent selective vagotomy, antrectomy, and Billroth II gastrojejunostomy, with placement of serosal electrodes. Gastric emptying was assessed with simultaneous myoelectric recordings, and the animals were converted to Roux-en-Y, followed by repeat studies. Gastric emptying was unchanged following selective vagotomy, antrectomy, and Billroth II gastrojejunostomy (T 1/2: 132 +/- 18 min [SEM] versus 118 +/- 14 min control) but was markedly delayed following Roux-en-Y diversion (T 1/2: 286 +/- 44 min; p less than 0.01). All animals went into the fed pattern following Billroth II gastrojejunostomy (migrating myoelectric complex [MMC] interval: 326 +/- 6 min postprandial versus 92 +/- 5 min fasting; p less than 0.01), but no fed pattern was recognized in three of four animals following Roux-en-Y diversion (MMC interval: 68 +/- 12 min postprandial versus 62 +/- 1.5 min fasting; p = NS). In a canine model, selective vagotomy does not prevent delayed gastric emptying or myoelectric alterations following Roux-en-Y. PMID- 1733373 TI - Effect of duodenal switch procedure on gastric acid production, intragastric pH, gastric emptying, and gastrointestinal hormones. AB - The duodenal switch operation preserves the pylorus and the proximal 3 to 7 cm of duodenum in continuity with the stomach while diverting pancreaticobiliary secretions. We compared it with the Roux-en-Y without vagotomy or antrectomy in 12 dogs with innervated gastric pouches. Acid secretion was inhibited between tests using ranitidine in the Roux-en-Y group only, but two of the six dogs still developed stomal ulcers and the remainder showed stomal hyperemia. This may be due to a significant increase in gastric acid output after Roux-en-Y, but gastric emptying and plasma gastrin, cholecystokinin, secretin, gastric inhibitory polypeptide, peptide YY, and neurotensin were similar after both procedures. In 12 patients and a further 6 dogs, the duodenal switch caused no significant change in the intragastric pH environment as assessed by intragastric pH monitoring. The duodenal switch is a suitable procedure for pancreaticobiliary diversion. PMID- 1733374 TI - Anal sphincter-saving operations for chronic ulcerative colitis. AB - Three anal sphincter-saving operations--ileorectostomy, ileal pouch-anal anastomosis, and ileal pouch-distal rectal anastomosis--are currently being used in the surgical treatment of chronic ulcerative colitis. All three operations remove the disease, or most of it, and yet they maintain transanal defecation, reasonable fecal continence, and a satisfactory quality of life. All three avoid permanent abdominal ileostomy. Ileorectostomy is the easiest to perform, but it leaves residual disease in the remaining rectum and proximal anal canal that may cause symptoms and that may predispose the patient to cancer. In contrast, ileal pouch-anal anastomosis, although a more technically demanding procedure, totally eradicates the colitis. Its main drawbacks--frequent stooling, nocturnal fecal spotting, and pouchitis--are usually satisfactorily treated with loperamide hydrochloride and metronidazole. Ileal pouch-distal rectal anastomosis is somewhat easier to perform than ileal pouch-anal anastomosis and may result in less nocturnal fecal spotting. Like ileorectostomy, however, the operation leaves residual disease in the distal rectum and proximal anal canal. Considering all of these factors, the ileal pouch-anal operation is preferred today for most patients who require surgery for chronic ulcerative colitis. PMID- 1733375 TI - Technique and results of transanal endoscopic microsurgery in early rectal cancer. AB - The anatomy of the pelvis makes it difficult to perform local excisions in the rectum when the tumor is some distance from the anal verge. We have, therefore, developed a new minimally invasive technique for tumor resection. A rectoscope with a 40-mm diameter permits tumor resection under stereoscopic control in the gas-dilated rectal cavity. Excisions in full-thickness technique up to segmental resections with end-to-end anastomosis can be performed. In selected cases, local excision of a small rectal cancer can be regarded as appropriate treatment. However, most local resections of carcinomas are performed when removal of an adenoma is planned, and the postoperative histology shows a carcinoma. Since 1983, we have operated on 326 patients, 274 who have been enrolled in a prospective clinical trial. Definitive histologic examination proved that 74 of these tumors were carcinomas. The rate of severe complications in patients with carcinomas was 9%, and the mortality rate was 0%. The advantages of this new technique are: The stereoscopic magnified view in the gas-dilated rectum allows precise surgery in an operative field that is otherwise difficult to reach. During the postoperative period, minimal discomfort and pain result in a short hospitalization. PMID- 1733376 TI - Effects of chronic corticosteroids and vitamin A on the healing of intestinal anastomoses. AB - The ability of vitamin A to reverse the inhibitory effects of chronic corticosteroids on cutaneous and fascial wound healing is well established. To investigate this in the unique low-collagen environment of the intestinal anastomosis, 35 rabbits received twice-daily injections of either saline (control), dexamethasone (0.1 mg/kg/day), dexamethasone plus low-dose vitamin A (1,000 IU/kg/day), or dexamethasone plus high-dose vitamin A (10,000 IU/kg/day) for a 2-week period. Animals then underwent creation of single-layer, inverting small and large intestine anastomoses. All injections were continued postoperatively. A fifth group received only dexamethasone preoperatively and dexamethasone plus high-dose vitamin A postoperatively. On postoperative day 7, animals underwent in situ assessment of anastomotic bursting pressure and subsequent histologic examination using a modified Ehrlich/Hunt scale. Corticosteroids significantly impaired the healing of small and large intestine anastomoses, with decreased bursting pressures and histologic parameters at 1 week. Only high-dose vitamin A significantly reversed this inhibitory effect, whether given preoperatively or only postoperatively. PMID- 1733377 TI - Protein C activity, stage of disease, and vascular thrombosis in colon carcinoma. AB - To determine the etiology of the increased incidence of postoperative deep venous thrombosis (DVT) in patients with carcinoma of the colon, serum levels of protein C were measured preoperatively in 65 patients with colorectal adenocarcinoma. Noninvasive lower-extremity Doppler studies were performed on all patients prior to discharge to assess patency of the deep veins. Six patients (9%) were found to have DVT. The protein C level was considered elevated if it was greater than 125% of control values and reduced if less than 75% of control values. The development of DVT was found to be independent of the serum carcinoembryonic antigen, albumin, total protein, hemoglobin, hematocrit, platelet count, prothrombin time, partial thromboplastin time, and the patient's age and percentage of ideal body weight. There was an inverse relationship between the protein C level (p less than 0.001), Dukes stage of the tumor (p less than 0.001), and the development of DVT. Linear regression analysis revealed that only the tumor stage and the protein C level could be used to predict the development of DVT. The data show that for these patients with colorectal malignancy, the development of DVT may be related to decreased levels of protein C. PMID- 1733378 TI - Preservation of continence after ileoanal anastomosis by the coordination of ileal pouch and anal canal motor activity. AB - Nocturnal incontinence may occur after ileoanal anastomosis and may be related to loss of an effective anal canal pressure barrier during sleep; how pressure and contractions in the proximal bowel influence this barrier is unknown. Our aim was to evaluate the relationship between anal canal pressure and contractions and contractile activity of the pouch in continent subjects after ileal pouch-anal anastomosis (IPAA) and of the rectum in normal controls. A fully ambulatory system for 24-hour pressure recording was used. A flexible transducer catheter was introduced endoscopically so that sensors were at 2, 3, 8, 12, 16, and 24 cm from the anal orifice in 12 healthy controls (7 men, 5 women, mean age: 35 years) and 7 fully continent IPAA patients (4 men, 3 women, mean age: 34 years) more than 12 months postoperatively. Twenty-four hour spontaneous motor activity was stored in a 2.5 megabyte (MB) digital portable recorder. Mean anal canal pressure was calculated, and rectal motor complexes and ileal pouch large pressure waves were characterized. During sleep, resting anal canal pressures were similar in the two groups (72 +/- 12 mm Hg in controls versus 66 +/- 9 mm Hg in IPAA patients [mean +/- standard deviation (SD)], p = NS), but anal canal pressure showed cyclic relaxations (periodicity: 95 +/- 11 min in controls, 54 +/- 18 min in IPAA patients, p less than 0.05), during which the mean pressure trough was 15 +/- 4 mm Hg in controls and 14 +/- 5 mm Hg in IPAA patients (p = NS). In the control patients, during sleep, a mean of six rectal motor complexes were identified (range: 3 to 9). In patients with IPAA, during sleep, a mean of eight large pressure waves per hour were identified (range: 2 to 20). Importantly, in both controls and patients, rectal motor complexes or large pressure waves were always accompanied by rapid return of anal canal pressure from trough to basal values and increased contractile activity. We concluded that, in healthy patients and in continent patients after IPAA, motor activity of the rectum and of the ileal pouch was associated with changes in pressure and contractile activity of the anal canal so that rectal- and neorectal-anal canal pressure gradient, and, in turn, fecal continence were preserved. PMID- 1733379 TI - Surgical approach to occult gastrointestinal bleeding. AB - In 5% of patients with gastrointestinal bleeding, standard evaluation fails to reveal the source of the bleeding. We describe the management of 71 patients treated for obscure gastrointestinal bleeding at the Mount Sinai Medical Center, New York, New York, from 1985 to 1991. There were 38 men (54%) and 33 women (46%). The mean age was 60 years. The patients had bleeding episodes for a mean period of 26 months and required an average of 20 units of blood prior to surgical treatment. All had undergone an extensive diagnostic workup including barium contrast studies, endoscopies, and angiographies. Some had multiple bleeding scans, Meckel scans, and surgical explorations. Three patients were found to have "watermelon stomach" on endoscopy and had an antrectomy. Sixty eight (96%) patients underwent a preoperative small bowel enteroscopy, which revealed the precise diagnosis in 50 (70%) of the patients. All patients underwent surgery. In 30 (42%) patients in whom the bleeding site was not apparent at exploration, intraoperative enteroscopy was performed. Two actively bleeding patients had intraoperative enteroscopy, which failed to localize the bleeding site, and intraoperative scintigraphy was utilized. The bleeding was found to originate in small bowel arteriovenous malformation (AVM) (28 patients), leiomyoma (8 patients), primary small bowel malignancies (11 patients), and other causes (24 patients). Fifty-six patients (80%) had no further bleeding; 9 with multiple small bowel AVM have experienced rebleeding and are alive. Six patients died of recurrent bleeding, and six died of metastatic cancer. An aggressive approach should be applied in patients in whom standard evaluation fails to localize the source of gastrointestinal bleeding. Enteroscopy, surgical exploration with additional intraoperative enteroscopy, and occasional intraoperative scintigraphy can achieve an excellent yield and allow resection and potential cure. PMID- 1733380 TI - Efficacy of intraoperative enteroscopy in diagnosis and prevention of recurrent, occult gastrointestinal bleeding. AB - The role and effectiveness of intraoperative enteroscopy in the evaluation of gastrointestinal (GI) bleeding of obscure origin is not clearly defined. Our aim was to determine if intraoperative enteroscopy is effective in identifying a source, which would lead to therapy and prevent recurrent gastrointestinal hemorrhage. Forty-four patients (median age: 64 years) underwent intraoperative enteroscopy. Median number of preoperative blood transfusions, duration of bleeding (months), and prior hospitalizations for GI hemorrhage were 19, 15, and 2, respectively. Many patients had risk factors associated with bleeding. All had undergone an extensive preoperative evaluation. Intraoperative enteroscopy was completely negative in 13 (30%). A site-specific source was seen in the small bowel in 31 patients (70%); 27 patients had lesions amenable to segmental resection with or without other means of definitive management. Only 6 of 31 patients (19%) had lesions that were actively bleeding. Twenty-three (52%) patients have had recurrent bleeding requiring transfusion (median follow-up: 21 months). Although intraoperative enteroscopy identified specific mucosal abnormalities in 70% of patients, the therapeutic efficacy in preventing recurrent hemorrhage was only 41%. Intraoperative enteroscopy is an effective tool in selected patients with occult GI bleeding and correctly identifies a treatable source and prevents recurrent bleeding in 41% of patients. PMID- 1733381 TI - Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. AB - OBJECTIVE: To investigate in the prospective Helsinki Heart Study, whether chronic Chlamydia pneumoniae infection, indicated by elevated antibody titers against the pathogen, chlamydial lipopolysaccharide-containing immune complexes, or both, is a risk factor for coronary heart disease. DESIGN AND SETTING: The Helsinki Heart Study was a randomized, double-blind, 5-year clinical trial to test the efficacy of gemfibrozil in reducing the risk for coronary heart disease. Participants were randomized to receive either gemfibrozil (2046 patients) or placebo (2035 patients). Fatal and nonfatal myocardial infarction and sudden cardiac death were the main study end points. Serum samples were collected at 3 month intervals from all patients. PATIENTS: One hundred forty cardiac events occurred during the follow-up period. Serum samples from 103 case patients obtained 3 to 6 months before a cardiac end point were matched with those from controls for time point, locality, and treatment. Samples were tested for markers of chronic chlamydial infection. MEASUREMENTS: Immunoglobulin A (IgA) and G (IgG) antibodies to C. pneumoniae were measured using the microimmunofluorescence method. Lipopolysaccharide-containing immune complexes were measured using two antigen-specific enzyme immunoassays, the lipopolysaccharide-capture and immunoglobulin M (IgM)-capture methods. MAIN RESULTS: Using a conditional logistic regression model, odds ratios for the development of coronary heart disease were 2.7 (95% CI, 1.1 to 6.5) for elevated IgA titers, 2.1 (CI, 1.1 to 3.9) for the presence of immune complexes, and 2.9 (CI, 1.5 to 5.4) for the presence of both factors. If we adjusted for other coronary heart disease risk factors such as age, hypertension, and smoking, the corresponding values would be 2.3 (CI, 0.9 to 6.2), 1.8 (CI, 0.9 to 3.6), and 2.6 (CI, 1.3 to 5.2), respectively. CONCLUSION: The results suggest that chronic C. pneumoniae infection may be a significant risk factor for the development of coronary heart disease. PMID- 1733382 TI - Infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis. AB - OBJECTIVE: To investigate the risk factors, clinical features, molecular epidemiology, and treatment outcomes associated with an outbreak of infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis. DESIGN: Case-control and molecular genetics study. SETTING: Tertiary care Veterans Affairs hospital. PATIENTS: Sixty-five patients infected or colonized with beta-lactamase-producing high-level gentamicin-resistant E. faecalis (case patients) were matched and compared with 65 randomly selected patients infected or colonized with beta-lactamase-negative, gentamicin susceptible E. faecalis. MEASUREMENTS AND MAIN RESULTS: During the 20-month study period, 124 of 1506 isolates (8.2%) of E. faecalis from 70 patients were found to produce beta-lactamase. Univariate analysis showed older age, higher APACHE II score, nosocomial acquisition, recent surgical procedure, total parenteral nutrition, and antibiotic treatment to be significantly associated with the acquisition of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis. A multivariate analysis, done using stepwise multiple logistic regressions, showed that only two variables remained significant: an APACHE II score greater than 6 (odds ratio, 9.5; 95% CI, 4.4 to 20.3) and antibiotic treatment (odds ratio, 10.2; CI, 4.5 to 23.2). The mortality rate for case patients was 7.7% (5 of 65 patients; CI, 1.2% to 14.2%); no control patient died. Of 23 infections occurring in case patients, 13 were treated with "inappropriate" antibiotics (regimens that included a beta-lactamase-unstable antibiotic); 2 patients improved and 11 had complete resolution of disease. "Appropriate" treatments (regimens that included a beta-lactamase-stable antibiotic) were used in 10 patients; 5 of 10 infections were fatal. Restriction enzyme digests of total chromosomal DNA showed nearly identical patterns for selected isolates of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis, suggesting dissemination through the hospital of a single strain of E. faecalis. CONCLUSIONS: Fatal infections were observed despite treatment with beta-lactamase stable antibiotics. The risk for infection or colonization with beta-lactamase producing, high-level gentamicin-resistant E. faecalis was strongly associated with severe underlying disease (acute physiology and chronic health evaluation [APACHE] II score, greater than 6) and previous antibiotic treatment. These results may be useful in targeting high-risk patients for infection-control interventions. PMID- 1733383 TI - Valvular heart disease in the primary antiphospholipid syndrome. AB - OBJECTIVE: To determine the prevalence of cardiac valvular involvement in patients with the primary antiphospholipid syndrome. DESIGN: Cross-sectional study with evaluation of case patients and control patients by Doppler echocardiography. The mean follow-up for case patients was 22 months. SETTING: University-based tertiary medical center. PATIENTS: Twenty-eight consecutive patients who were diagnosed with the primary antiphospholipid syndrome during a 10-year period; 28 age- and sex-matched healthy controls. MEASUREMENTS AND MAIN RESULTS: Ten patients (36%; 95% Cl, 19% to 56%) with the primary antiphospholipid syndrome had cardiac valvular involvement: Four patients had mitral valve involvement; four patients, aortic valve involvement; and two patients, both mitral and aortic valvular involvement; no patients had tricuspid or pulmonary valve disease. Eight of 10 patients had a regurgitant murmur. None of the control patients had valvular disease. The mean mitral valve thickness in patients with mitral valve involvement was 7.0 +/- 1.6 mm, compared with 2.7 +/- 0.8 mm in patients with normal valves and 3.2 +/- 0.9 mm in the control group. The mean aortic valve thickness in patients with aortic valve involvement was 3.8 +/- 0.5 mm compared with 1.4 +/- 0.3 mm in patients with normal valves and 1.4 +/- 0.5 mm in the control group. Stenotic lesions were not found. Regurgitation was severe in two patients (one required surgery), moderate in three patients, and mild in three patients. CONCLUSIONS: Valvular involvement is frequently found in patients with the primary antiphospholipid syndrome. The lesions are left-sided, causing regurgitation that may be clinically important. PMID- 1733384 TI - Hospitalization in an urban homeless population: the Honolulu Urban Homeless Project. AB - OBJECTIVE: To determine the rate and estimate the cost of hospitalization in a defined urban homeless population. DESIGN: Retrospective chart review. SETTING: Kalihi-Palama Health Clinic Health Care for the Homeless Project, Hawaii State Hospital and seven acute care hospitals in Honolulu, Hawaii. PATIENTS: A total of 1751 homeless clients contacted between 1 December 1988 and 30 November 1990. MEASUREMENTS AND MAIN RESULTS: A total of 1751 individuals were studied for an aggregate of 871.3 person-years. Five hundred sixty-four hospitalizations were identified: ninety-two to the state psychiatric hospital and 472 to acute care hospitals. The age- and sex-adjusted hospitalization rate for acute care hospitals was 542/1000 person-years (compared with the state rate of 96/1000 person-years). Homeless persons were admitted to acute care hospitals for 4766 days compared with a predicted 640 days. The age- and sex-adjusted rate of admission to the state psychiatric hospital was 105/1000 person-years (compared with the state rate of 0.8/1000 person-years). Homeless persons were admitted to the state psychiatric hospital for 3837 days compared with a predicted 139 days. CONCLUSIONS: Homeless individuals in this study were hospitalized in acute care and psychiatric hospitals far more frequently than were members of the general population. PMID- 1733386 TI - Low-dose splenic irradiation in the treatment of autoimmune thrombocytopenia in HIV-infected patients. PMID- 1733385 TI - Using transjugular intrahepatic portosystemic shunts to control variceal bleeding before liver transplantation. AB - OBJECTIVE: To determine the safety and efficacy of transjugular intrahepatic portosystemic shunts (TIPS) in controlling bleeding from esophageal varices in patients awaiting liver transplantation. DESIGN: Prospective, uncontrolled trial. SETTING: University medical center with an active liver transplant program. PATIENTS: Thirteen patients referred for liver transplantation with either active variceal hemorrhage or recurrent variceal hemorrhage despite sclerotherapy; four patients had been previously treated with surgical portosystemic shunts. INTERVENTION: An intrahepatic portosystemic shunt created via a transjugular approach to the hepatic veins using expandable, flexible metallic stents. MEASUREMENTS: Portal pressures before and after the creation of the shunt, the direction of portal blood flow at differing diameters of the shunts, procedure related complications, and outcome in terms of survival, liver transplantation, and recurrent variceal bleeding. MAIN RESULTS: The transjugular intrahepatic portosystemic shunt was placed successfully in 13 patients, and bleeding was controlled acutely in all 13. After the procedure, the mean portal pressure decreased from 34 +/- 8.9 cm H2O to 22.4 +/- 5.4 cm H2O (P less than 0.001). No complications were associated with the procedure; however, two patients died of causes unrelated to the procedure. Seven patients subsequently underwent liver transplantation and are doing well, and three patients are being managed conservatively. Bleeding recurred in one patient 102 days after the procedure secondary to shunt occlusion caused by neointimal proliferation. CONCLUSION: Placement of a transjugular intrahepatic portosystemic shunt is apparently safe and effective therapy for variceal hemorrhage in patients referred for liver transplantation. PMID- 1733387 TI - Cardiomyopathy associated with antiretroviral therapy in patients with HIV infection: a report of six cases. PMID- 1733388 TI - Gastric syphilis: five recent cases and a review of the literature. AB - OBJECTIVE: To describe five cases of early syphilis with gastric involvement and to review the literature pertaining to this disorder. DATA IDENTIFICATION: Five patients were diagnosed with gastric syphilis at Kings County Hospital and the Brooklyn Veterans Affairs Hospital between 1987 and 1990. English-language articles pertaining to gastric syphilis were identified by searching MEDLINE and by manually reviewing bibliographies of retrieved articles. STUDY SELECTION: Sources containing information pertinent to the clinical manifestations and diagnosis of gastric syphilis were selected. DATA SYNTHESIS: The most common clinical manifestations of gastric syphilis are abdominal pain, vomiting, and weight loss. Endoscopic findings in the stomach range from minimal nodularity and erythema to deep ulceration. Complications of gastric syphilis include hemorrhage, gastric outlet obstruction, and perforation. The diagnosis can be confirmed by serologic testing and by demonstration of spirochetes on silver and immunofluorescent stains of gastric mucosal biopsy specimens. Response to treatment is usually prompt and complete. CONCLUSIONS: The current syphilis epidemic will likely result in an increased incidence of gastric syphilis. Unless syphilis is considered as a cause of gastric mucosal inflammation and ulceration, misdiagnosis may delay appropriate treatment, and serious complications can occur. PMID- 1733390 TI - Syphilis and AIDS in Belle Glade, Florida, 1942 and 1992. PMID- 1733389 TI - Predictors of mortality among HIV-infected women in Kigali, Rwanda. AB - OBJECTIVE: To better characterize the natural history of disease due to human immunodeficiency virus (HIV) infection in African women. DESIGN: Prospective cohort study over a 2-year follow-up period. PARTICIPANTS: A total of 460 HIV seropositive women and a comparison cohort of HIV-seronegative women recruited from prenatal and pediatric clinics in Kigali, Rwanda in 1988. MEASUREMENTS: Clinical signs and symptoms of HIV disease, AIDS, and mortality. MAIN RESULTS: Follow-up data at 2 years were available for 93% of women who were still alive. At enrollment, many seropositive women reported symptoms listed in the World Health Organization (WHO) clinical case definition of AIDS, but these were nonspecific and often improved over time. The 2-year mortality among HIV-infected women by Kaplan-Meier survival analysis was 7% (95% CI, 5% to 10%) overall, and 21% (CI, 8% to 34%) for the 40 women who fulfilled the WHO case definition of AIDS at entry. In comparison, the 2-year mortality in women not infected with HIV was only 0.3% (CI, 0% to 7%). Independent baseline predictors of mortality in seropositive women by Cox proportional hazards modeling were, in order of descending risk factor prevalence: a body mass index of 21 kg/m2 or less (relative hazard, 2.3; CI, 1.1 to 4.8), low income (relative hazard, 2.3; CI, 1.1 to 4.5), an erythrocyte sedimentation rate exceeding 60 mm/h (relative hazard, 4.9; CI, 2.2 to 10.9), chronic diarrhea (relative hazard, 2.6; CI, 1.1 to 5.7), a history of herpes zoster (relative hazard 5.3; CI, 2.5 to 11.4), and oral candida (relative hazard, 7.3; CI, 1.6 to 33.3). Human immunodeficiency virus disease was the cause of death in 38 of the 39 HIV-positive women who died, but only 25 met the WHO definition of AIDS before death. CONCLUSIONS: Human immunodeficiency virus disease now accounts for 90% of all deaths among child-bearing urban Rwandan women. Many symptomatic seropositive patients may show some clinical improvement and should not be denied routine medical care. Easily diagnosed signs and symptoms and inexpensive laboratory tests can be used in Africa to identify those patients with a particularly good or bad prognosis. PMID- 1733391 TI - HIV in Africa: what is the future? PMID- 1733392 TI - The lessons of Belle Glade. PMID- 1733393 TI - Hepatitis C after needlestick injuries. PMID- 1733394 TI - Hilar adenopathy in aspergillosis. PMID- 1733395 TI - Hilar adenopathy in aspergillosis. PMID- 1733396 TI - Crimson crescents--a possible association with the chronic fatigue syndrome. PMID- 1733397 TI - Nephrotic range of proteinuria and interferon therapy. PMID- 1733398 TI - Improving cardiac resuscitation in the community. PMID- 1733399 TI - Advance directives. PMID- 1733400 TI - Advance directives. PMID- 1733401 TI - Emergency department stat test turnaround times. A College of American Pathologists' Q-Probes study for potassium and hemoglobin. AB - We report aggregate turnaround times (TATs) from phlebotomy to result reporting of emergency department patients' hemoglobin and potassium results for 722 subscribers to the College of American Pathologists Q-Probes program. Approximately 40,000 specimens were obtained for each analyte. Median interinstitutional TAT time of 25 minutes for hemoglobin and 36 minutes for potassium varied little by shift, weekdays, or weekends. The type of personnel collecting the specimen and the method of specimen transport were the most important factors affecting TATs. Specimen transit times accounted for approximately one third of the total TATs, but when couriers transported hemoglobin specimens, the median transit time was equivalent to the median intralaboratory test TAT. The influence of various measures used to improve test transit and TATs is presented. PMID- 1733402 TI - Pulmonary microvascular cytology in the dyspneic cancer patient. Merits and caution. PMID- 1733403 TI - Subacute cor pulmonale due to tumor embolization. AB - Pulmonary tumor embolism is a rare but well-documented cause of respiratory failure in patients with cancer. This entity is probably clinically underrecognized and may represent an important cause of mortality and morbidity in patients with cancer. Pulmonary tumor embolization may present at any stage of the patient's illness and indeed may be the first presentation of an occult carcinoma. In a review of 1069 nonmedicolegal autopsy protocols, we recently encountered three cases in which death had occurred from subacute cor pulmonale due to tumor embolization from breast, lung, and ovarian carcinoma. Recent advances in cytologic examination of blood samples obtained from Swan-Ganz catheters may prove useful in the diagnosis of this entity. PMID- 1733404 TI - Endothelial papillary fibroelastomas arising in and around the aortic sinus, filling the ostium of the right coronary artery. AB - Endothelial papillary fibroelastomas are rare endocardial and usually valvular lesions, most often found incidentally at autopsy. Occasionally these lesions cause clinical sequelae by embolizing to the heart and brain, or (in the case of valvular ones) by intermittent prolapse into a coronary artery. Papillary fibroelastomas arising in the right coronary ostium, and filling the origin of the right coronary artery are described. Other similar but smaller lesions were found on the surrounding aortic intima. To our knowledge, these are the first such lesions to be described. The case is reviewed in the context of previously published literature on the subject. PMID- 1733405 TI - Heterotopia of gastric mucosa and liver involving the gallbladder. Report of two cases with literature review. AB - Two cases of heterotopic tissue involving the gallbladder are presented (one gastric mucosa, the other liver), and the relevant literature is reviewed highlighting problems in differential diagnosis and complications, particularly with regard to gastric mucosal heterotopia. A firm diagnosis of gastric heterotopia is based on the presence of fundic mucosa replete with parietal and chief cells; a clear distinction from intestinal metaplasia should be made, but at times may be difficult. Potential complications include mucosal ulceration, obstruction, and hemorrhage. Of 39 patients presented, about half were 30 years of age or younger, and only nine have had calculi noted in the cholecystectomy specimen. Heightened awareness of gastric heterotopia with separation of smooth muscle bundles may help to avoid a malignant diagnosis. Hepatic tissue involving the gallbladder that is completely separate from the main part of the liver is an even rarer form of heterotopia that should be distinguished from an accessory lobe. PMID- 1733406 TI - Elastin in adenomatoid tumor. Light microscopic and electron microscopic study. AB - Elastin distribution was examined in adenomatoid tumors of the human genital tract. Two distinct patterns were identified: strongly positive or completely negative for elastin in the stroma, according to the case. Even in cases appearing to have very similar histologic features, the stroma was rich in elastin in some cases and was almost devoid of elastin in others. Electron microscopic examination with tannic acid staining revealed that the elastin in the stroma was composed mainly of amorphous material surrounded by a small amount of microfibrils and abundant collagen fibers. Fibroblasts were sparsely distributed in the stroma. Tumor cells displayed mesothelial cell-like features, such as abundant microvilli on the surface, numerous cytoplasmic organelles, several well-developed basal lamina. In some areas, elastin seemed to be formed by mesothelial cells. In some adenomatoid tumor cases, elastogenesis would be enhanced by activated mesothelial tumor cells as well as stromal fibroblasts. PMID- 1733407 TI - Bilateral adrenocortical adenomas causing Cushing's syndrome. Report of two cases with enzyme histochemical and ultrastructural studies and a review of the literature. AB - Patient 1 (a 50-year-old woman) with bilateral adrenocortical adenomas causing Cushing's syndrome underwent right-sided adrenalectomy. After an interval of 4 years 5 months, she underwent left-sided tumorectomy. Patient 2 (a 46-year-old woman) underwent bilateral tumorectomy after a frozen-section diagnosis. Each adrenal contained a single macronodule that consisted of compact cells, clear cells, and oxyphilic cells, which had characteristic enzyme histochemical and ultrastructural features. In addition, the adrenals had a so-called pigmented nodule and extracapsular small focus of compact and clear cell proliferation in case 2. The nonnodular part of the cortex was atrophic in both cases. Fourteen patients (mean +/- SD age, 42.5 +/- 5.55 years) with bilateral adrenocortical adenomas associated with Cushing's syndrome have been described in the literature. Thirteen (93%) of the patients were female. Three patients had unilateral or bilateral multiple adenomas. While the earlier described patients underwent bilateral total adrenalectomy, those in recent reports had part of the adrenal resected unilaterally or bilaterally (ie, tumorectomy). The above features are useful in differentiating bilateral adrenocortical adenoma from other types of bilateral adrenocortical lesions. PMID- 1733408 TI - HMB-45 reactivity in adrenal pheochromocytomas. AB - Melanoma-specific antibody (HMB-45) is highly specific for junctional nevi, malignant melanomas, and related lesions. Rarely have other benign or neoplastic tissues demonstrated positivity with the monoclonal antibody following purification. In the present study, four of 12 pheochromocytomas contained chief cells that reacted with HMB-45. We discuss the clinical and pathologic implications of this finding. PMID- 1733409 TI - DNA ploidy of oncocytic-granular renal cell carcinomas and renal oncocytomas by image analysis. AB - DNA ploidy analysis of five renal oncocytomas, six pure oncocytic-granular renal cell carcinomas, 15 pure clear cell renal carcinomas, and two cases of mixed oncocytic-granular and clear cell heterogenous renal cell carcinomas were determined on Feulgen-stained paraffin sections using the (CAS-200 image Analyzer). All five renal oncocytomas were diploid, as were six oncocytic granular renal cell carcinomas. Three of the 15 clear renal cell carcinomas were aneuploid. In one heterogeneous renal cell carcinoma, the oncocytic-granular foci were diploid and the clear cell focus was aneuploid. The other heterogeneous renal cell carcinoma was uniformly diploid. In 21 renal cell carcinomas, one of 14 stage I tumors was aneuploid, all four stages II and III tumors were diploid, and two of three stage IV cases were aneuploid. All stages I, II, and III patients were free of disease 3 to 48 months after surgery. All three stage IV cases were dead of their disease within 36 months of surgery. We conclude that DNA analysis by image cytometry best identifies the heterogeneity of ploidy patterns in mixed cell type carcinomas, generally correlates with the stage of disease, and may be of value in predicting overall prognosis of renal epithelial neoplasms. DNA analysis by image cytometry, however, did not reveal significant differences between oncocytic granular cell carcinoma and oncocytoma, since both lesions were generally diploid. The distinction between the two tumors is best made on morphologic grounds. PMID- 1733410 TI - Ruptured saccular pulmonary artery aneurysm associated with persistent ductus arteriosus. AB - We report a case of rupture of a saccular pulmonary artery aneurysm with fatal hemopericardium in a 29-year-old man who also had persistent ductus arteriosus with severe pulmonary hypertension. Histologic examination of the pulmonary artery showed cystic medionecrosis similar to that observed in patients with ruptured dissecting pulmonary artery aneurysms. The two types of ruptured aneurysms probably only represent different morphological expressions in the same underlying process in which the main contributory factor is pulmonary hypertension. PMID- 1733411 TI - Pathology of pentamidine-induced pancreatitis. AB - Extensive acute necrotizing pancreatitis occurred in three patients with the acquired immunodeficiency syndrome who had received both aerosolized pentamidine as prophylaxis and intravenous pentamidine for Pneumocystis carinii pneumonia. The clinical manifestations along with gross and microscopic pathologic findings at autopsy are presented. PMID- 1733412 TI - Nasopharyngeal teratomas. AB - Teratomas are the most common congenital tumors, but neoplasms of the nasopharynx are rare in neonates and children. Four histologic types of nasopharyngeal teratomas occur-dermoids, teratoids, true teratomas, and epignathi-of which dermoids comprise the vast majority. A case is presented of a neonate born at term exhibiting signs of respiratory difficulty, which were found to be caused by a true teratoma of the nasopharynx. A review of several large case series of pediatric teratomas confirmed the rarity of occurrence at this site. An extensive review of case reports identified those meeting the criteria of true teratomas of the nasopharynx; the characteristics of these cases are delineated. PMID- 1733413 TI - Unusual biclonal plasma cell dyscrasia with crystallike inclusions producing colonic tumors (plasma cell polyposis) and terminating in T-cell lymphoma. AB - A 62-year-old man underwent a partial colectomy for persistent rectal bleeding. Multiple submucosal lesions up to 7 cm in diameter were identified and were composed of plasmacytoid and histiocytoid cells with crystallike inclusions. Ultrastructurally, inclusions were bounded by rough endoplasmic reticulum. Immunohistochemical studies demonstrated a monoclonal population of IgA kappa positive plasma cells. Immunofixation using a saline extract of the tumor revealed two distinct bands: one for IgG kappa and another for IgA kappa. Southern blotting revealed a germline configuration for the immunoglobulin heavy chain gene and the T-cell antigen receptor beta-chain gene. A bone marrow aspirate contained 12% plasma cells, some of which had cytoplasmic inclusions. Eight months after surgery, the patient developed a large mediastinal mass. Histologic and immunohistochemical studies indicated that this mass was an undifferentiated neoplasm. However, gene rearrangement studies indicated a T-cell lymphoma. This patient illustrates a rare association between a plasma cell neoplasm and a T-cell lymphoma. PMID- 1733414 TI - Varicella embryopathy. AB - Varicella embryopathy is a rare entity afflicting infants born to mothers who have contracted varicella during the first 20 weeks of pregnancy. The teratogenicity of varicella has not been established from epidemiologic studies, but isolated case reports describe characteristic malformations following early maternal infection. We describe a male neonate delivered at 40 weeks' gestation to a 26-year-old grava 2, para 2 mother who developed varicella during the first trimester. The infant lived 7 days and died of bronchopneumonia. At postmortem examination there was growth retardation, multiple cicatricial skin lesions, flexion contractures of all major joints, hypoplastic right diaphragm, bilateral hydroureters and mucosal fibrosis of the trachea, as well as intestinal fibrosis and colonic stricture. The brain contained areas of cystic necrosis involving the frontal, parietal, temporal, and occipital lobes, with generalized ventriculomegaly. The midbrain, pons, and medulla were hypoplastic. There was denervation atrophy of muscles of the lower limbs and loss of dorsal root ganglia as well as of neurons of the anterior horn of the spinal cord. The cerebral white matter was degenerated, with proliferation of reactive astrocytes. Chorioretinitis was not observed. Immunocytochemical stains using two commercially available antivaricella antibodies were negative in all tissues examined. The sporadic nature and pathogenesis of the varicella embryopathy, which may have been caused by focal defects in the fetal T-cell immune response, are discussed. PMID- 1733415 TI - Pulmonary tumor embolism to alveolar septal capillaries. An unusual cause of sudden cor pulmonale. AB - Although metastatic spread of tumor to the lungs is common, subsequent production of cor pulmonale is not. The involvement of pulmonary alveolar capillaries causing sudden cor pulmonale is very rare. We describe a patient who presented with chest pain and sudden shortness of breath. Autopsy disclosed diffuse pulmonary microembolism to septal capillaries caused by tumor cells from a squamous cell carcinoma of the cervix. To our knowledge, this is the second report of this kind of pulmonary tumor embolism. PMID- 1733416 TI - Benign glandular schwannoma. AB - The vast majority of reported glandular schwannomas, the rarest type of divergent differentiation in nerve sheath neoplasms, have been malignant tumors. We describe a benign glandular schwannoma, less than 1 cm in diameter, that developed in the deep subcutaneous region of the left flank of a 36-year-old woman. The glandular component occurred as a single large cyst with an undulating lining occupying the central one third of the lesion and was lined in large part by a well-oriented, flattened, single cell layer of cuboidal to low columnar cells with a basement membrane. This extremely unusual lesion, apparently the fourth benign instance reported, is important for several reasons: (1) it does not appear to be a result of inclusion of previously postulated dermal adnexal glands; (2) it further establishes the existence of a true benign counterpart of the glandular schwannoma, of which pathologists should be aware; (3) it can be distinguished from the recently reported pseudoglandular schwannoma; and (4) it lends additional support to the concept of a direct metaplastic origin of the epithelial element from the schwannian component because of the focal presence of a maloriented pseudoglandular element. PMID- 1733417 TI - Cytologic diagnosis of metastatic penile carcinoma in pleural effusion. AB - A 66-year-old man presented with unilateral pleural effusion. Cytologic examination of the fluid revealed squamous carcinoma cells. The patient, who died 3 days after this diagnosis was rendered, had a history of surgically-treated squamous cell carcinoma of the penis. To our knowledge, metastatic penile carcinoma in pleural effusion has not previously been described. PMID- 1733418 TI - Idiopathic multifocal calcification of the ovarian stroma. AB - We report an apparently unique case of extensive, bilateral, multifocal calcification of the ovarian stroma. The lesion was an incidental finding in a 50 year-old gravida 5, para 5 woman who underwent a hysterectomy and a bilateral salpingo-oophorectomy. Gross inspection revealed ovaries that were of normal size but stony hard. On microscopic examination, the ovarian stroma was extensively replaced by uniformly distributed, spherical foci of calcification that were focally psammomatous. No underlying cause for the calcification, which probably represents an unusual form of dystrophic calcification, could be identified. The differential diagnosis of ovarian calcification, which may arise in diverse conditions, is discussed. PMID- 1733419 TI - Malignant granular cell tumor. AB - We report a case of malignant granular cell tumor of the chest wall that recurred in the right breast with axillary lymph node metastases. The recurrent tumor clinically and grossly mimicked a breast carcinoma. Electron microscopy and immunohistochemical techniques were used to confirm the cytologic and histologic diagnosis of granular cell tumor. The importance of true metastases in the diagnosis of malignant granular cell tumor and their differential diagnosis are discussed. PMID- 1733420 TI - Respiratory epithelial cyst ventral to the brain stem. Report of a case and review of the literature. AB - We present the case of a 30-year-old woman who suddenly developed intense occipital headaches. Computed tomography and magnetic resonance imaging demonstrated a cyst located ventrally to the brain stem at the pontomedullary junction. The lesion was resected. Histologically, it was lined by a pseudostratified, ciliated, columnar epithelium indistinguishable from respiratory epithelium. Immunohistochemistry and electron microscopy confirmed the epithelial differentiation of the lining. This case is briefly compared with the few other brain-stem cysts presented in the literature and the concept of their "origin" is discussed. PMID- 1733421 TI - Historical factors in the development of ETOH-conditioned place preference. AB - Three groups of male Sprague-Dawley rats were conditioned with ethanol (ETOH) and water in a Conditioned Place Preference task. To assess the contribution of prior drug/behavioral history on the relative hedonic valence of ETOH, the three groups differed in the task demands and degree of prior ETOH exposure. One group was trained to self-administer 20% w/v ETOH in a home-cage self-administration task using a "Samson sucrose-fading" procedure prior to place conditioning trials (Group SA/CPP). A second group was initially trained in a two-choice ETOH-Saline drug discrimination (DD) task and subsequently conditioned in the place preference paradigm (Group DD/CPP). The third group of rats had no history of drug exposure and were experimentally naive prior to the place learning task (Group NH/CPP). Group SA/CPP developed a significant conditioned place preference; Group DD/CPP failed to demonstrate either a preference or aversion, whereas the Group NH/CPP demonstrated a significant place aversion. Data suggest that both present and past contingencies contribute to the algebraic summation of ETOH's hedonic valences. PMID- 1733422 TI - Neurotensin modulates K(+)-stimulated dopamine release from the caudate-putamen but not the nucleus accumbens of mice with differential sensitivity to ethanol. AB - Slices of caudate-putamen (CP) and nucleus accumbens (NA) prepared from Long Sleep (LS) and Short-Sleep (SS) mice were used to determine the effects of neurotensin (NT) and ethanol on K(+)-stimulated 3H-dopamine (3H-DA) release and to test the hypothesis that ethanol acts, in part, via NT receptor-mediated processes. Slices prepared from either LS or SS CP or NA did not differ in submaximal (25 mM) K(+)-stimulated 3H-DA release but 60 mM K+ induced significantly greater 3H-DA release from LS CP slices compared with SS CP slices. NT had no effect on unstimulated 3H-DA overflow but enhanced 25 mM K(+) stimulated 3H-DA release from slices of the CP of both lines of mice. Augmentation of DA release by NT from caudate slices was concentration dependent and tetrodotoxin (TTX) insensitive, implicating a role of presynaptic neurotensin receptors located on nigrostriatal DA neurones. In contrast, NT did not enhance K(+)-stimulated 3H-DA release from NA slices from either line of mice. The absence of an NT effect in NA slices was not due to a rapid desensitization of NT receptors but the data were consistent with the absence of presynaptic NT receptors on dopaminergic terminals in the NA. Between-line differences were observed in the effect of ethanol on NT enhancement of 25 mM K(+)-stimulated 3H DA release from CP slices. Ethanol (100 mM) applied concomitantly with NT blocked the NT enhancement of 3H-DA release from CP slices of LS but not SS mice.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733423 TI - 1-Methyl-tetrahydro-beta-carboline-3-carboxylic acid is present in the rat brain and is not increased after acute ethanol injection with cyanamide treatment. AB - We conducted analyses of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1Me3C-THBC) by gas chromatography-mass spectrometry (negative chemical ionization mode) to investigate its presence and the in vivo condensation between tryptophan and AcH. 1Me3C-THBC was found in the cerebellum and the cerebrum of normal rat [117.0 +/- 41.7 and 46.5 +/- 13.9 pmol/g tissue (mean +/- SEM), respectively]. The concentrations of 1Me3C-THBC and tryptophan were higher in the cerebellum than those in the cerebrum. The level of 1Me3C-THBC in both regions remained unchanged following a single oral ethanol administration alone or with cyanamide pretreatment. These data suggest that acetaldehyde is an unlike precursor of 1Me3C-THBC as a result of ethanol ingestion. 1Me3C-THBC also existed in the rat chow (282.0 +/- 24.2 pmol/g), so that most of brain 1Me3C-THBC detected in the rat brain might have originated from dietary sources. However, the possibility of a biosynthesis from tryptophan and alpha-keto acid still remained, especially after long-term ethanol treatment. PMID- 1733424 TI - Acute and chronic ethanol alter somatosensory-evoked potentials in conscious rats. AB - This study examined the effects of acute and chronic ethanol on cortical somatosensory-evoked potentials (SEPs) of laboratory rats. Evoked potentials were recorded following stimulation of the left hindpaw before and after injection of either saline or ethanol. Animals were then chronically exposed to ethanol in vapor inhalation chambers for five weeks. Recordings of SEPs before and after acute ethanol injection were then obtained 24 h and again seven days after withdrawal from ethanol exposure. The results indicate that acute ethanol produced a dose-dependent reduction in SEP amplitude, but did not alter peak latencies. Chronic ethanol exposure and withdrawal resulted in a significant increase in preinjection baseline response amplitudes when measured at 24 h after withdrawal, but not at seven days, and this treatment did not alter response latency or the effects of acute ethanol administration. PMID- 1733425 TI - Comparison of salsolinol excretion in alcoholics and nonalcoholic controls. AB - Salsolinol was measured in urine of alcoholics after one through four and after 21 days of withdrawal, in nonalcoholic controls with and without ingestion of alcohol and in young children. HPLC equipped with fluorescence detection was employed. There were no significant differences in salsolinol excretion rate between alcoholics and controls one day after alcohol deprivation. A significant difference in salsolinol output in urine of alcoholics between the first and the twenty-first day of withdrawal was found, but salsolinol output expressed as creatinine-related output did not change. There was no trend of urinary salsolinol excretion rate by alcoholics during the first 4 days of abstinence. The results indicate that the urinary salsolinol excretion rate and the creatinine-related salsolinol output are insufficient to distinguish between alcoholics and nonalcoholics. PMID- 1733427 TI - Sham ingestion of alcohol in rats. AB - Sprague-Dawley rats were surgically fitted with gastric fistulas and their intakes of 6% ethanol measured first with the fistula closed (normal drinking) and then on three test sessions with the fistula open (sham drinking). The rats were water deprived prior to the 1-h test sessions. On the second and third sham drinking sessions, intakes of alcohol were significantly increased above those in both the closed and first open fistula session. The effectiveness of the sham ingestion preparation to minimize absorption was shown by the much lower blood alcohol levels in sham compared with real drinking rats. Thus, reduction of the postingestive effects of alcohol leads to an acquisition of increased oral consumption. The similarities and differences between sham intakes of water and alcohol are discussed. PMID- 1733426 TI - HAD and LAD rats respond differently to stimulating effect but not discriminative effects of ethanol. AB - The drug discrimination paradigm (DD) was used to evaluate behavioral differences of rats selectively bred for differential ethanol drinking preferences. Seventh generation high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats were trained to discriminate between ethanol (0.5 g/kg, IP) and saline vehicle, following a 2-min presession interval (PI), using an FR-10 schedule of reinforcement. The HAD line was more responsive than the LAD line to the stimulating effect of ethanol as measured by total response rates. ED50 values of 0.239 and 0.244 g/kg for the HAD and LAD lines, respectively, do not reflect any difference in the discriminative effects of ethanol. Response rates during DD indicated a dissociation of rate-increasing effects and discriminative performance following ethanol. In addition to differential drinking preference, these data suggest that selective breeding for the HAD and LAD animals also involves the stimulant action of ethanol but not on the discriminative effects. PMID- 1733428 TI - Ribosomal RNA activity and protein in skeletal muscles of chronic ethanol-fed rats. AB - The effect of prolonged ethanol exposure on ribosomal RNA activity and the content of RNA and protein in skeletal muscles of 15- and 22-25-month-old rats was evaluated. Experimental rats were fed a liquid diet containing 6.7% ethanol for 2, 4 and 6 months, and control rats were pair-fed an isocaloric diet. The in vivo incorporation of [3H]puromycin into nascent peptides on messenger RNA ribosome complexes was determined to assess muscle ribosomal RNA activity. This activity was significantly reduced in extensor digitorum longus and soleus muscles of rats fed ethanol for 2 months. While the total RNA content of these muscles was unchanged after feeding ethanol for 2, 4 and 6 months, their messenger RNA content was decreased from 26-34%. The total protein content was reduced after ethanol was consumed for 6 months. Taken together, the results suggest that alterations in the transcriptional or posttranscriptional control of messenger RNA may contribute toward the development of alcoholic myopathy after prolonged ethanol consumption. PMID- 1733429 TI - Effect of chronic ethanol on D-galactose absorption by the rat whole intestinal surface. AB - The in vivo absorption of D-galactose by rat whole intestinal surface after 4 weeks of 30% ethanol ingestion in drinking water has been studied, and the results were compared with ad lib-fed control rats. The total serosal intestinal area was determined by integration obtaining similar values between control and alcohol-treated groups. In the caecum surface of ethanol-fed rats slight but not significant increases were found, while the jejunum area decreased with respect to control rats. Total galactose absorption during 10 min of perfusion was slightly increased in ethanol-fed rats but these results were not significant with the substrate concentrations tested. When absorption data were referred to serosal surface, the absorption/cm2 values in ethanol-fed rats were increased at the studied galactose concentrations although these results were only statistically significant at 10 mM. In conclusion, the present data indicates a slight increase in D-galactose absorptive capacity by the whole intestine in ethanol-fed rats which suggest that the tissue traditionally not evaluated such as caecum and colon could modify the functional response to the absorption nutrients. PMID- 1733430 TI - Alcoholic and nonalcoholic beer drinking during gestation: offspring growth and glucose metabolism. AB - Female Long-Evans rats were allowed voluntary access to beer, food and water for 52 days prior to mating, throughout mating and throughout gestation, and were compared to animals pair-fed nonalcoholic beer and to regular controls. Alcoholic beer drinkers gained more weight during gestation than drinkers of nonalcoholic beer. Significant hypoglycemia was observed in the newborn male offspring of alcoholic beer drinkers. At 20 days of age, all animals responded normally to glucose tolerance tests. At 20 days of age, liver weights of offspring of beer drinkers (alcoholic and nonalcoholic) were enlarged; pancreas weights of alcoholic beer drinkers were increased. At 65 days of age, body weights of male offspring of alcoholic beer drinkers were low. These results indicate sex differences in response to maternal beer drinking, and suggest some of the observed alterations in development were due to components in beer other than alcohol. PMID- 1733431 TI - Nucleolar organiser regions in pathology. PMID- 1733432 TI - Time to loco-regional recurrence after resection of Dukes' B and C colorectal cancer with or without adjuvant postoperative radiotherapy. A multivariate regression analysis. AB - Factors influencing time to loco-regional recurrence were identified in a multivariate regression analysis of data from a series of 468 radically operated patients (260 Dukes' B and 208 Dukes' C) with carcinoma of the rectum and the rectosigmoid. A number of clinical and pathological characteristics were prospectively collected and recorded. In addition, carcinoembryonic antigen (CEA) was measured within 1 week before surgery. The endpoint used was recurrence below the level of the umbilicus. All patients were followed for at least 5 years or until time of death. The two Dukes' stages B and C were analysed in two separate analyses using the Cox proportional hazards model. In patients with Dukes' B tumours, an increased risk of loco-regional recurrence was associated with perineural invasion, tumour located less than 10 cm from the anal verge, patient aged above 70 years, and small tumour size. In patients with Dukes' C tumours, the necessity to resect neighbour organs, perineural and venous invasion, tumour located less than 10 cm from the anal verge, and large tumour size were all associated with a poor loco-regional outcome. Postoperative radiotherapy was not a significant prognosticator for loco-regional control. An update of the 5-year results of the randomised study of post-operative radiotherapy (50 Gy with 2 Gy per fraction in an overall treatment time of 7 weeks) showed no survival benefit from adjuvant radiotherapy in either Dukes' category and no statistically significant improvement in the 5-year loco-regional control rate. However, when the comparison was restricted to a group of high-risk patients there was a statistically significant benefit from radiotherapy with respect to loco-regional control (P = 0.03) but not with respect to survival (P = 0.23). The potential advantage, in terms of the required number of patients, of restricting clinical trials of intensified loco-regional therapies to the high-risk patients, is illustrated. PMID- 1733433 TI - Breast cancer and specific types of combined oral contraceptives. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives. AB - Data on 2,754 cases and 18,565 controls from a multinational hospital-based, case control study were analysed to determine whether observed associations between combined oral contraceptives and breast cancer are similar for oral contraceptives with varying types and doses of oestrogens and progestins. After stratifying on duration of use, risk was found to be increased in current and recent users, and to decline with time since last use. These associations, of similar strength, were observed for users of products that contain mestranol and ethinyl estradiol, for women who used preparations with progestins derived from 19-nortestosterone and 17-alpha-hydroxyprogesterone, and for those who took preparations with relatively higher and lower doses of oestrogen. When products with equal doses of the same oestrogen or progestin and varying doses of the other hormonal constituent were considered, slightly higher relative risks per year of use were estimated for users of products with relatively higher than lower doses of either the constituent oestrogen or progestin, but the differences in relative risk could readily have occurred by chance. This study provides no evidence that risk of breast cancer in users of oral contraceptives varies by the type of oestrogen or progestin consumed. PMID- 1733434 TI - Maternal and foetal outcome following Hodgkin's disease in pregnancy. AB - The peak incidence of Hodgkin's disease occurs during the reproductive age, and its association with pregnancy is at a rate of between 1:1,000-1:6,000. We studied the effects of Hodgkin's Disease on the course and survival of 48 women who had Hodgkin's Disease and who were pregnant, and compared their outcome with non-pregnant matched women who were of similar stage of disease, age at diagnosis, and calenderic year of treatment. Twenty-year survival of pregnant women with Hodgkin's Disease was not different from that of their matched controls. Pregnant women with Hodgkin's Disease had similar distribution of stages to the controls. PMID- 1733435 TI - The effect of systemic adjuvant chemotherapy on local breast recurrence in node positive breast cancer patients treated by lumpectomy without radiation. AB - A randomised trial has previously been repeated in which 437 women with node positive breast cancer received either a 12-week chemohormonal regimen consisting of cyclophosphamide, methotrexate, fluorouracil, vincristine, prednisone, adriamycin and tamoxifen or 36 weeks of CMFVP. The present analysis concerns the local recurrence rates for the 122 lumpectomy patients who did not receive breast irradiation. The cumulative rate of local breast recurrence was greater in the 12 week than the 36-week group, P = 0.02. Similarly, in the lumpectomy patients, the cumulative rate of distant recurrence was greater in the 12-week than the 36-week group, P = 0.04. In conclusion, our results suggest that adjuvant chemotherapy impacts on local breast recurrence in a similar manner to other sites in Stage II breast cancer patients treated by lumpectomy without radiation. Despite the use of a conventional 36-week adjuvant chemotherapy regimen, the local breast recurrence rate was substantial. PMID- 1733436 TI - A pharmacokinetic comparison of intravenous versus intra-arterial folinic acid. AB - Recent clinical trials have suggested that a combination of folinic acid and 5 fluorouracil (5-FU) may improve response rates and survival in patients with advanced colorectal cancer. However, this regimen has been complicated by potentially life threatening toxicity. Regional delivery of folinic acid via a hepatic artery catheter might be expected to reduce systemic exposure and subsequent adverse effects. The present study compared the pharmacokinetic profiles of intravenous and intra-hepatic arterial infusions of folinic acid in patients with colorectal liver metastases (n = 6) who were being treated with weekly regional infusions of 5-FU. The mean area under the plasma concentration- time curve, the peak plasma concentration and the steady state volume of distribution were 163 micrograms ml-1 h-1 (SD 41), 18.5 micrograms ml-1 (SD 1.2) and 7.41 m-2 (SD 0.44) respectively following intravenous administration of folinic acid compared with 142 micrograms ml-1 h-1 (SD 45), 14.8 micrograms ml-1 (SD 2.4) and 11.21 m-2 (SD 1.22) following intra-hepatic arterial administration (P less than 0.05). Regional folinic acid was therefore associated with a statistically significant reduction in systemic exposure compared with the intravenous route. PMID- 1733437 TI - British Psychological Oncology Group: seventh annual conference. PMID- 1733438 TI - Isolation of a lactoferrin cDNA clone and its expression in human breast cancer. AB - A cDNA library constructed from mRNA from a human breast carcinoma metastasis was screened with a polyclonal antibody to deglycosylated human milk fat globule membrane, resulting in the isolation of eight clones from a total of 10(5) plaques. One of these (J16) was identified as lactoferrin. It was highly expressed (as a 2.5 Kb mRNA) in lactating breast and in both normal resting tissue taken from adjacent to carcinoma or from reduction mammoplasties. Immunoreactive lactoferrin was localised to ductal cells and their secretions in both normal and mildly hyperplastic ducts. In a normal tissue screen J16 was highly expressed in stomach, poorly in skin and lymphocytes and absent from other organs examined. It was variably expressed in 33/59 invasive primary breast tumours; lactoferrin protein in these was heterogeneously distributed in epithelial tumour foci. Presence of J16 was inversely related to expression of oestrogen receptor protein (P = 0.0001). There was no significant relationship to other clinical parameters. We also found immunoreactivity in 20/41 (49%) cases of ductal carcinoma in situ. Expression was not observed in any breast or gastric cell line examined. Thus lactoferrin appears to be down regulated in some forms of cancer. The presence of lactoferrin could be a contraindication for effective endocrine therapy. PMID- 1733439 TI - Effects on tumour microcirculation in mice of misonidazole and tumour necrosis factor plus hyperthermia. AB - We examined the effects of misonidazole (MISO) and recombinant human tumour necrosis factor (rh-TNF) on tumour blood flow in mice given hyperthermic treatments. MISO (500 mg kg-1) or rh-TNF (6 x 10(4) unit kg-1) was administered intraperitoneally (i.p.) prior to hyperthermia to nude mice bearing a xenoplanted human gastric cancer and tumour blood flow was measured by a hydrogen diffusion method based on polarographic determinations. MISO plus hyperthermia produced a temperature-dependent decrease in blood flow and, at 43.5 degrees C, the flow decreased to 15-30% of control and remained low for up to 24 h. Blood flow following rh-TNF plus hyperthermia was less than that at the same temperatures following MISO plus hyperthermia, and, at 43.5 degrees C, the flow decreased to 10-20% of control and remained low for up to 48 h. Tumour growth delay was closely related to the duration of the decrease in blood flow. Thus, the profound decrease in tumour blood flow following hyperthermia plus MISO or rh-TNF and the consequential tumour regression may well be of potential clinical significance. PMID- 1733440 TI - The combination of degradable starch microspheres and angiotensin II in the manipulation of drug delivery in an animal model of colorectal metastasis. AB - Both biodegradable emboli and pharmacological agents can enhance regional therapy for hepatic targeting. Using a rat model with similar haemodynamic characteristics to human colorectal liver tumour and a radio-labelled marker of similar molecular weight to Adriamycin, we have combined the two approaches to see if the effect was addictive. Following induction of liver tumour in male hooded rats by intrahepatic injection of HSN sarcoma cells, the relative distribution of marker, 99mTc methylene diphosphonate (MDP), was studied in three groups given the following by injection into the hepatic artery. (1) Saline (Control) + MDP; (2) Degradable Starch Microspheres (DSM) + MDP; and (3) Angiotensin II + DSM + MDP. Both Degradable Starch Microspheres alone (P less than 0.001) and Degradable Starch Microspheres + Angiotensin II (P = 0.003) significantly increased the retention of marker in liver and tumour at 1 min following injection, with a 12-fold improvement over controls, but the tumour:liver ratio was unaltered. By 90 min the MDP levels in normal hepatic parenchyma had returned to control values. There was relatively less washout with significant retention in tumour tissue in both DSM (P = 0.03) and combination treated animals (P = 0.001), with a significantly improved (P = 0.001) tumour to liver ratio (5.22:1) in combination treated animal relative to those treated with DSM alone. PMID- 1733442 TI - Use of photosensitive, antibody directed liposomes to destroy target populations of cells in bone marrow: a potential purging method for autologous bone marrow transplantation. AB - Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry. PMID- 1733441 TI - Ras p21 in breast tissue: associations with pathology and cellular localisation. AB - Immunocytochemistry with monoclonal antibody Y13-259 demonstrated p21 ras in paraffin sections of breast tissue from 171 women: 85 with invasive breast carcinoma, 14 with non-invasive carcinoma and 72 with benign changes only. Many different tissue elements contributed to ras expression. Semiquantitative assessment showed that intensity of immunostaining in the normal epithelium of large ducts, small extralobular ducts and terminal duct lobular units (TDLU) was usually exceeded by that of myoepithelial cells. Vascular smooth muscle and apocrine epithelium also stained strongly, but the flat epithelial cells lining cysts did not express detectable p21 ras. There was a progressive increase from normal epithelium through epithelial hyperplasia of usual type and atypical hyperplasia to carcinoma in situ, without further increase in invasive carcinoma. Expression in carcinomas was inversely related to oestrogen receptor content but independent of the prognosis-associated variables of size, histological type, vascular invasion or lymph node metastasis. PMID- 1733443 TI - Oestrone sulphatase activity in mammary tumours and the liver of N nitrosomethylurea treated rats. AB - Oestrone sulphatase may be an important means of production of intra-tumoural oestrogens in breast cancer cells. The N-nitrosomethylurea induced rat mammary tumours, which is a good model of human breast carcinoma, was utilised to examine the significance of intra-tumoural oestrone sulphatase levels. The particular fraction (100,000 g pellet) was prepared from both the liver and the tumour of NMU treated rats and assayed for sulphatase activity. The tumour enzyme had an optimum pH of 7.2, Km value of 14.8 microM and Vmax of 0.90 nmoles min-1 mg-1, while the hepatic enzyme was optimal at pH 7.4, Km of 10.8 microM and Vmax of 3.71 nmoles min-1 mg-1. The relationship of intra-tumoural sulphatase levels with tumour regression and progression in endocrine responsive tumours was investigated. Tumour regression as a result of oophorectomy was associated with a significantly decreased intra-tumoural sulphatase level (mean level = 0.165 nmoles min-1 mg-1) in comparison to a control group (mean level = 0.319 nmoles min-1 mg-1, P less than 0.05) in which the tumours remained stable. This significant difference was not observed in the corresponding hepatic samples suggesting that it is the intra-tumoural rather than the peripheral production of oestrogens by oestrone sulphatase that is important in supporting growth of endocrine responsive tumours. PMID- 1733444 TI - Stromal influences on breast cancer cell growth. AB - Paracrine influences from fibroblasts derived from different sources of breast tissue on epithelial breast cancer cell growth in vitro were investigated. Medium conditioned (CM) by fibroblasts derived from tumours, adjacent normal breast tissue, and normal breast tissue obtained from reduction mammoplasty or from skin tissue significantly stimulated the growth of the steroid-receptor positive cell lines MCF-7 and ZR 75.1. The proliferation index (PI) on MCF-7 cells with CM from fibroblasts derived from breast tumour tissue was significantly higher than that obtained with fibroblasts derived from adjacent normal breast tissue (2p less than 0.05, n = 8). The PI obtained with CM from normal fibroblast cultures from reduction mammoplasty tissue, like normal tissue adjacent to the tumour, fell in the lower range of values. Skin fibroblast, like tumour tissue derived fibroblast, CM caused a high range PI. MDA-MB-231 and Evsa-T, two steroid receptor negative cell lines, showed only a minor growth stimulatory responses with some of the fibroblast CM's. Evsa-T was occasionally inhibited by CM's. In conclusion, stromal factors play a role in the growth regulation of human breast cancer cells. The effects on cancer cell growth are, however, varying depending on the source of the stroma and the characteristics of the epithelial tumour cells. PMID- 1733445 TI - An investigation of different methods of cell cycle analysis by flow cytometry in rectal cancer. PMID- 1733446 TI - Visualisation of doxorubicin in human and animal tissues and in cell cultures by immunogold-silver staining. AB - In previous pharmacologic studies, the native fluorescent properties of doxorubicin (DOX) have been utilised to visualise tissue and cellular drug distribution. Such distribution studies provide valuable additional information to that obtained by measuring tissue drug concentration alone. An alternative immunocytochemical method of drug localisation using a rabbit immunoadsorbed antiserum to DOX and silver-enhanced gold-labelled second antibodies has been used to achieve visualisation of DOX in normal and malignant tissues from drug treated animals and patients, and in human tumour cell lines treated in vitro. Non-specific staining in untreated tissues or in controls stained without primary antibody was minimal. Widespread dark brown to black specific immunostaining was observed in the normal tissues of drug-treated animals and in rat sarcoma and in the mouse EMT6 mammary tumour. In human breast tumour biopsy samples obtained at surgery 1 h following a 25 mg intravenous dose of DOX, considerable variation in drug distribution was observed which appeared to be related to drug concentration. Both nuclear and membrane staining was apparent; the latter was especially noticeable in human tumour cells grown in the presence of DOX at concentrations greater than 0.92 microM. Immunolocalisation using silver enhanced gold-labelled reagents provides an additional technique to study cell and organ specific differences in drug uptake and distribution. PMID- 1733447 TI - Evaluation of bcl-2 protein expression and 14;18 translocation as prognostic markers in follicular lymphoma. AB - Conflicting results have been published on the prognostic significance of t(14;18) in follicular lymphoma: Yunis et al. (1989) reported that its presence indicated poor response to therapy and short survival, whereas Levine et al. (1988) showed no difference in prognosis between cases with and without the translocation. However these results were based on small series of cases and on follow-up periods (no longer than 7 years) which are relatively short for a disease with such a slow clinical evolution. Here we report an investigation of 70 cases of follicular lymphoma with long term follow-up data (up to 17 years). This series has been studied for the presence of the 14;18 translocation and for the expression of bcl-2 protein. Our results show that there are no grounds for considering either the 14;18 translocation or the expression of the bcl-2 protein to be useful prognostic markers in clinical practice. PMID- 1733448 TI - The activation of polymorphonuclear neutrophils and the complement system during immunotherapy with recombinant interleukin-2. AB - The toxicity due to interleukin-2 (IL-2) strongly resembles the clinical picture seen during septic shock. In septic shock activation of polymorphonuclear neutrophils (PMN) and the complement system contribute significantly to the pathophysiology of the condition. We therefore investigated whether similar events contributed to the toxicity observed with IL-2. Four patients received seven cycles of escalating dose IL-2 (18.0 to 72.0 X 10(6) IU m-2 day-1) and 16 were treated with 20 cycles of fixed dose IL-2 (12.0 or 18.0 X 10(6) IU m-2 day 1). Toxicity, as judged by hypotension (P = less than 0.005) and capillary leakage (fall in serum albumin 18.2 vs 4.0 gm l-1; P = less than 0.0005 and weight gain 4.0 vs 1.2 kg; P = less than 0.025) were worse with the esc. dose protocol. PMN became activated following IL-2 with mean peak elastase/alpha 1 antitrypsin (E alpha 1 A) and lactoferrin values of 212 (SEM = 37) and 534 (SEM = 92) ng ml-1 respectively occurring 6 h after the IL-2. Peak values for the esc. dose IL-2 group being generally higher than 500 ng ml-1. Activation of the complement cascade was evidenced by a dose dependent elevation of peak C3a values (fixed dose 9.1 (SEM = 0.6); esc. dose 25.7 (SEM = 6.33); P = less than 0.005) on day 5 of IL-2. There was a significant correlation between C3a levels and the degree of hypotention during the first 24 h after IL-2 (r = 0.91) and parameters of capillary leakage such as weight gain and fall in serum albumin (r = 0.71). These data suggest that activation of PMN initiates endothelial cell damage which subsequently leads to activation of the complement cascade. This latter system then contributes to the haemodynamic changes and capillary leakage seen in IL-2 treated patients. PMID- 1733449 TI - Detection of agents causing genetic or reproductive damage. PMID- 1733450 TI - Postpartum changes in maternal blood lead concentrations. AB - Studies of lead concentrations in blood during pregnancy are of interest because of the possibility of adverse effects on the fetus. One report of a single case suggested that blood lead concentrations are raised during pregnancy. This is consistent with the hypothesis of a pregnancy induced mobilisation of lead from bone. Data presented herein, however, indicate that blood lead measures are appreciably lower at delivery than they are at six months post partum. Other factors including but not limited to transmission to the fetus, may be influencing lead concentrations during pregnancy. PMID- 1733451 TI - Repeated measurements of tibia lead concentrations by in vivo x ray fluorescence in occupational exposure. AB - A group of workers occupationally exposed to lead have had measurements of their tibia lead concentrations made on two occasions separated by five years; on the second occasion calcaneus lead concentrations were also measured. The results serve to confirm the reliability of the measurement technique and to illustrate the improved precision achieved through technical improvements. More importantly, the relation between tibia lead concentration and cumulative blood lead found in this longitudinal study was entirely consistent with that previously reported, which had been based on cross sectional studies. Furthermore, the relation between lead concentrations in the tibia and in calcaneus found here was similar to that previously found in a larger cross sectional survey. It is concluded that this technique of measuring bone lead concentrations non-invasively is likely to be used increasingly as a biological monitor of cumulative exposure to lead. PMID- 1733452 TI - Clinical findings among hard metal workers. AB - In 1940, the first report appeared describing a pulmonary disorder associated with occupational exposures in the cemented tungsten carbide industry. The disease, known as "hard metal disease," has subsequently been characterised in detail and comprises a wide range of clinical signs and symptoms. In this report, clinical findings in a group of 41 hard metal workers employed until recently are described. A high prevalence of respiratory symptoms was found. Thirteen workers (31%) had abnormal chest radiographs indicative of interstitial lung disease. Fifty per cent of these had been employed in hard metal manufacturing for less than 10 years. Abnormalities of pulmonary function were also frequent and included a restrictive pattern of impairment and decrease in diffusing capacity (27%). Associations were found between diffusing capacity, chest radiographic abnormalities and right ventricular ejection fraction at exercise indicating cardiopulmonary effects. The findings show the continuous need to control excessive occupational exposures to prevent hard metal disease, the history of which now enters its sixth decade. PMID- 1733453 TI - Assessment of the permissible exposure level to manganese in workers exposed to manganese dioxide dust. AB - The prevalence of neuropsychological and respiratory symptoms, lung ventilatory parameters, neurofunctional performances (visual reaction time, eye-hand coordination, hand steadiness, audioverbal short term memory), and several biological parameters (calcium, iron, luteinising hormone (LH), follicle stimulating hormone (FSH), and prolactin concentrations in serum, blood counts, manganese (Mn) concentration in blood and in urine) were examined in a group of workers (n = 92) exposed to MnO2 dust in a dry alkaline battery factory and a matched control group (n = 101). In the battery plant, the current exposure of the workers to airborne Mn was measured with personal samplers and amounted on average (geometric mean) to 215 and 948 micrograms Mn/m3 for respirable and total dust respectively. For each worker, the lifetime integrated exposure to respirable and total airborne Mn dust was also assessed. The geometric means of the Mn concentrations in blood (MnB) and in urine (MnU) were significantly higher in the Mn exposed group than in the control group (MnB 0.81 v 0.68 microgram/100 ml; MnU 0.84 v 0.09 microgram/g creatinine). On an individual basis, MnU and MnB were not related to various external exposure parameters (duration of exposure, current exposure, or lifetime integrated exposure to airborne Mn). On a group basis, a statistically significant association was found between MnU and current Mn concentrations in air. No appreciable difference between the exposed and the control workers was found with regard to the other biological measurements (calcium, LH, FSH, and prolactin in serum). Although the erythropoietic parameters and serum iron concentration were in the normal range for both groups, there was a statistically significant trend towards lower values in the Mn exposed workers. The prevalences of reported neuropsychological and respiratory symptoms, the lung function parameters, and the audioverbal short term memory scores did not differ between the control and exposed groups. The Mn workers, however, performed the other neurofunctional tests (visual reaction time, eye hand coordination, hand steadiness) less satisfactorily than the control workers. For these tests, the prevalences of abnormal results were related to the lifetime integrated exposure to total and respirable Mn dust. On the basis of logistic regression analysis it may be inferred that an increased risk of peripheral tremor exists when the lifetime integrated exposure to Mn dust exceeds 3575 or 730 micrograms Mn/m3 x year for total and respirable dust respectively. The results clearly support a previous proposal by the authors to decrease the current time weighted average exposure to Mn dust. PMID- 1733454 TI - Allergy to laboratory animals: an epidemiological study. AB - A large cross sectional survey was carried out using a self administered questionnaire to examine the prevalence of laboratory animal allergy (LAA) and the factors associated with its development. Out of 5641 workers who were exposed to animals at 137 laboratory animal facilities in Japan, 23.1% had one or more allergic symptoms related to laboratory animals. The commonest symptom as rhinitis. About 70% of LAA subjects developed symptoms during their first three years of exposure. Atopy (past and family history), the number of animal species handled, and the time spent in handling correlated significantly with the development of LAA as did some types of job. A close relation between nasal symptoms and exposure to rabbits and between skin symptoms and exposure to rats were found. LAA subjects developed symptoms most quickly to rabbits. PMID- 1733455 TI - A mortality study of Danish stokers. AB - This study was set up to investigate whether work as a stoker is associated with an increased risk of specific malignant neoplasms. For this purpose, a cohort of 2777 male stokers was followed up through a 10 year period with regard to cause specific mortality. Comparisons were made with another cohort of unskilled male workers in physically demanding jobs. The mortality of the stokers was significantly increased for lung cancer (standardised mortality ratio (SMR) 145, 95% confidence interval (95% CI) 110-186) and for multiple myeloma (SMR 388, 95% CI 106-994). Also, increases were seen for cancer of the urinary organs and cancer of the mouth and throat. The combustion products to which the stokers have been exposed comprise several carcinogenic agents including polycyclic aromatic hydrocarbons, benzene, arsenic, and radionuclides. It seems likely that the occupational exposure of stokers has contributed to their excess cancer mortality. PMID- 1733456 TI - Semen quality in welders exposed to radiant heat. AB - Several studies suggest that welding is detrimental to the male reproductive system. Welding fume and radiant heat are of interest as possible causal factors. This study investigates semen quality and sex hormone concentrations among 17 manual metal arc alloyed steel welders with a moderate exposure to radiant heat (globe temperature ranging from 31.1 degrees to 44.8 degrees C), but without substantial exposure to welding fume toxicants. During exposure to heat the skin temperature in the groin increased on average by 1.4 degrees C (SE +/- 0.72 degrees C). Sperm count and motile sperm count were non-significantly reduced among welders in comparison with two different reference groups. Within the group of welders the proportion of sperm with normal shape declined significantly after six weeks of exposure to heat and increased after a break in exposure. Sperm count and sperm concentration had the same pattern of intraindividual change in relation to exposure to radiant heat, but the changes were not statistically significant. No consistent changes in concentrations of sex hormones were found. The welders investigated were more exposed to radiant heat than welders in general. The results suggest that the study group of welders experienced a reversible decrease in semen quality, most likely caused by a moderate exposure to radiant heat (about five hours a day through several weeks). It remains to be established if even lower levels of exposure to radiant heat in the general population of welders has any impact on semen quality and fertility. PMID- 1733457 TI - A cross sectional epidemiological survey of shipyard workers exposed to hand-arm vibration. AB - The hand-arm vibration syndrome, widely known as vibration white finger, is a disorder of nerves and blood vessels that occurs in workers exposed to segmental vibration. A cross sectional symptom survey was performed on a sample of workers employed by a large shipyard in the north eastern United States. Random samples were drawn from departments composed of full time dedicated pneumatic grinders, workers with part time exposure to vibration, and other workers not exposed to vibratory tools. Of the 375 workers sampled, 79% responded. The prevalence of white finger symptoms was 71%, 33%, and 6% among the three exposure groups respectively. Similarly, the prevalence of numbness and tingling in the hands and fingers in the three exposure groups was 84%, 50%, and 17%. Workers were classified according to the Stockholm Workshop staging systems for vascular and sensorineural symptom severity. Exposure-response analyses of both vascular and sensorineural stage showed monotonically increasing prevalence of higher disease stages with increasing duration of exposure. Logistic regression analysis, performed to control for potential confounding factors including age and current smoking state, produced highly significant (p less than 0.001) associations between cumulative duration of exposure and prevalence of symptoms. In these analyses smoking state was significantly related to vascular and sensorineural symptoms and age was not. Average latency to onset of symptoms was less than five years of full time equivalent work with vibratory tools. Logistic regression analyses were performed to assess the effect of use of particular work practices on reported symptoms. Further study of this workforce with objective, quantitative measures of peripheral neurological and vascular function is required to characterise the clinical and subclinical effects of vibration exposure. PMID- 1733458 TI - Occupational noise induced vestibular malfunction? AB - This paper comprises a review of the evidence for the possibility that exposure to noise may damage the vestibular receptors in the internal ear as well as those in the cochlea. The review covers lay and medical publications, observations on patients, experimental studies, and compensation claims. It concludes that the verdict must be "not proven"--that is, although such damage is possible, the evidence is not strong enough to regard it as probable. PMID- 1733459 TI - A report of two cases of chronic serious manganese poisoning treated with sodium para-aminosalicylic acid. AB - Two cases of chronic manganese poisoning were treated with sodium para aminosalicylic acid (PAS-Na; 6 g/day in 500 ml of 10% glucose solution by intravenous drip). The results indicated that one had been clinically cured and that the other had obviously improved in clinical symptoms and signs. Thus PAS-Na appears to be an effective drug for treatment of serious chronic manganese poisoning. PMID- 1733460 TI - A critical review of the effect of factory closures on health. PMID- 1733461 TI - Asbestos and cancer: history and public policy. PMID- 1733462 TI - Biological monitoring of MDA. PMID- 1733463 TI - Polymyositis and a thalamic mass. PMID- 1733464 TI - A winning indigent care program. PMID- 1733465 TI - Public safety: Virginia's impaired drivers. AB - Through its licensing procedure, the Department of Motor Vehicles plays a key role in the preservation of personal and public safety. Restriction of license to drive is limited to that necessary to assure this safety. Most people can drive safely and should be allowed to do so. Constant vigilance and the cooperation of physicians, family, police, and other drivers is needed to identify those persons whose ability to drive is questionable for medical reasons. Each case receives individual consideration by the Board. Adequate medical information is critical to the decision. The DMV's Medical Control Unit is available by telephone at 804 367-6639 or by mail at PO Box 27412, Richmond VA 23269-0001. PMID- 1733466 TI - [The task of the radiologist in the diagnosis and differential diagnosis of acute appendicitis]. PMID- 1733467 TI - [Dysplasia epiphysialis multiplex tarda. The differential evidence of MRT and the x-ray picture]. PMID- 1733468 TI - [Permeability disorders of the blood-brain barrier following intravascular contrast medium administration in kidney failure]. PMID- 1733469 TI - [X-ray measurement of the level of rectal carcinomas and its dependence on the functional status of the pelvic floor]. AB - The influence of the various functional states of the pelvic floor on the radiological assessment of the tumour levels and the distant tumour-free distance in rectum neoplasms was investigated. The parameters "anal canal length", "anorectal angle" and "impression of the puborectalis muscle" were measured in lateral distant views of the rectum in a series of healthy controls (n = 160). In addition, these parameters and the "distant tumour-free distance" were measured in patients with rectal cancer (n = 40). For each patient the lateral distant view at rest, during contraction and during maximal relaxation (straining) of the pelvic floor, were available for retrospective analysis. Depending on the various functional states of the pelvic floor, the differences between the same parameters were statistically significant (p less than 0.001). The average difference between the distal tumour-free distances during contraction and straining was 1.5 cm. Therefore, measurements of this distance in one lateral distant view exclusively may result in an inaccurate assessment of the tumour level. For the individual planning of extreme sphincter-saving surgery in low rectal cancer based on lateral distant view, the view at rest appears to be the most suitable. However, additional x-rays during contraction and maximal relaxation of the pelvic floor, respectively, should be available to identify the view at rest for an accurate assessment of the tumour level and to avoid misinterpretations which would have falsely influenced the planning of rectum surgery in 20% of our cases. PMID- 1733470 TI - [Oral contrast media for the magnetic resonance tomography of the abdomen. A clinical trial of the tolerance for gadolinium-DTPA]. AB - Safety and efficacy of gadopentetate-dimeglumine (Gd-DTPA) as a MR bowel contrast agent were determined in 133 patients with CT-proved abdominal and retroperitoneal mass lesions using a buffered formulation (1 mmol/l Gd-DTPA, 15 g/l mannitol, 25 mmol/l sodium-citrate, 6-17 ml/kg). Short-lived gastrointestinal side effects were noted in 32% of patients. Gd-DTPA provided uniform, hyperintense bowel labelling and contrast enhancement in the region of interest in 81% of patients. Among 78 patients with pre- and postcontrast images lesion delineation was improved in 62%. In 55 studies with postcontrast images only, Gd DTPA proved useful in 65%. In 105 of 109 cases IV injection of scopolamine or glucagon eliminated image artifacts arising from peristalsis of opacified bowel. The authors conclude that Gd-DTPA is a safe and effective MR bowel contrast agent. PMID- 1733471 TI - [MR angiography of occlusive changes in the superior thoracic inlet veins]. AB - We used MR angiography to assess the thoracic inlet veins in 5 normal volunteers and 14 patients with thrombosis of these veins. The results were compared with conventional venography and contrast-enhanced computed tomography (CT). The normal anatomy was readily depicted by MR angiography in all normal volunteers. Pathology was assessed correctly by MR angiography in all patients. Thrombosis of the internal jugular vein was seen on MR angiography in all cases whereas conventional venography failed to demonstrate this region adequately in 3 cases. The best overview of the thoracic inlet veins was achieved in the coronal plane; however, for detailed demonstration of pathology, imaging in additional planes was necessary. We conclude that MR angiography is a promising method for non invasive assessment of thoracic veins. PMID- 1733472 TI - [The value of radioimmunoscintigraphy compared to computed tomography in the diagnosis and relapse diagnosis of colorectal tumors]. AB - Radioimmunoscintigraphy (= RIS, scintigraphic "specific" imaging of benign and malignant diseases by means of radioactively marked monoclonal antibodies) has been performed in Germany in clinical studies since 1985 in patients suffering from colorectal cancer. After having been successfully proven in primary studies, RIS is now being used in the early diagnosis of recurrences and metastases. In the prospective study presented here the clinical usefulness of RIS was assessed in comparison against well-tried diagnostic methods including computed tomography in patients suffering from colorectal cancer. It was shown that RIS in SPECT technique (= single photon emission computed tomography) with 99mTc-labelled monoclonal CEA antibodies can visualise local recurrences if diagnostic findings are doubtful, with a sensitivity of 78% versus 50% for CT findings. PMID- 1733473 TI - [Clinical experiences with the Gianturco-Z stent in endotracheal and -bronchial stenoses]. AB - We inserted Gianturco-Z stents in 17 patients with tracheobronchial stenoses on 18 occasions. Three patients had benign, all others malignant stenoses due to compression of the tracheobronchial tree by metastasising malignancies. In all cases the stents could be easily positioned and the patients experienced relief of their severe dyspnea. In one case there was fatigue breakage of the stent. The patient was therefore given a silicone tracheal stent. PMID- 1733474 TI - [Percutaneous CT-guided catheter drainage of intrathoracic fluid accumulations]. AB - To verify the value of percutaneous CT-guided drainage of thoracic fluid collections we studied the outcome in 39 patients retrospectively. 24 (61.5%) of the fluid collections were located in the pleural space, 10 (25.6%) in the lungs and 5 (12.8%) in the mediastinum. 11 CT-guided drainages after a previous attempt were necessary in 9 patients, because of recurrent (n = 6) or residual (n = 5) fluid collections. 70% of the drainage procedures were done using Seldinger's, 30% using trocar technique, mainly with 8.3-12 F catheters. The mean duration of drainage was 10.7 days. In 28 patients (71.8%) the percutaneous CT-guided drainage was curative. In 9 cases (23.1%) the patient's course was stabilised and surgery could be applied electively. 2 patients (5.1%) died because of their underlying end-stage malignancy. None of the drainage procedures changed the patient's course to the worse. There was only 1 pneumothorax with no need of any treatment; no other complication occurred. Our results suggest that percutaneous CT-guided drainage of thoracic fluid collections is a safe and straightforward alternative to surgical treatment. PMID- 1733475 TI - [The HR computed tomography of interstitial lung diseases]. AB - In 50 patients with pulmonary disease of varying aetiology and involvement of the pulmonary interstitium, high resolution CT with 1 mm sections have been performed. Various morphological changes within the secondary lobule could be demonstrated which helped in reducing the differential diagnosis. Compared with conventional CT with 10 mm cuts it was also possible to improve the demonstration of pleural lesions by changing window levels. PMID- 1733476 TI - [Interstitial lung diseases. A comparative study between a film-screen combination and a digital storage phosphor technic]. AB - Chest examinations were carried out in 60 individuals using film-screen and digital storage phosphor techniques and identical radiation doses. 29 of these individuals were patients with interstitial pulmonary disease; 31 were normals. Postprocessing of the digital images was carried out with filtering using an unsharp mask and identical parameters for all patients. An ROC analysis using 10 observers showed no significant difference in the evaluation of the interstitial lung disease. Correlation analysis showed a significant positive correlation (p less than 0.001) for the findings in both techniques. Under the present circumstances, film-screen and digital storage phosphor technique are of equal value in the diagnosis of interstitial pulmonary disease. PMID- 1733477 TI - [The visibility of a central venous catheter using digital luminescence radiography in intensive care radiology]. AB - The aim of the following study was to assess the impact of dose alterations on the detection of catheters. We compared the performance of well-exposed conventional and digital portable chest radiographs in the detection of thin catheters and tested the influences of dose alterations. Portable chest radiographs of 20 patients were obtained with conventional film/screen (FR) and with storage phosphors at 50% (SRL), 100% (SRN), and 250% (SRH) of the conventionally required exposure dose. The region of the mediastinum was subdivided into an average of 18 fields, 50% of which were superimposed with thin catheter segments. ROC analysis of 11,600 observations by 8 readers found only SRH equivalent to FR in catheter visualisation. Performance decreased significantly with SRN and SRL. Detection of low contrast catheters was found to be significantly decreased in storage phosphor radiographs obtained with standard exposure dose. A dose reduction is not feasible with current equipment if performance equivalent to conventional radiography is to be achieved. PMID- 1733478 TI - [The pathophysiological etiological mechanism of destructive wrist joint arthropathy in pseudogout]. AB - The pathogenesis of destructive joint lesions of the wrist in pseudogout is explained using the concept of compartments of the radiocarpal joint and bearing in mind the biomechanical situation. The progressive destruction of the joints in the wrist begins with degenerative changes in the radial styloid, then zigzags through the scapho-lunate joint into the medio-carpal joints and ultimately leads to collapse of the carpus. In its final stage there is a characteristic extensive defect in the radial styloid process. Following cartilage degeneration there is subchondral sclerosis, bone destruction, sclerosing cysts and occasionally loose bodies in the joint. Chondrocalcinosis of the triangular cartilage is a common finding which assists in making the diagnosis. PMID- 1733479 TI - [Legg-Calve-Perthes disease. The value of MRT in its early diagnosis and the assessment of its course]. AB - We report on the value of MRT in diagnosis and follow-up of Perthes' disease. 38 children who were clinically suspect of suffering from Perthes' disease were examined by conventional x-ray and MRT. 25 children were proven to have Perthes' disease, 12 of them in an early stage. During the onset of the disease MRT showed a higher sensitivity (58% vs 50%) and accuracy (74% vs 71%) than conventional x ray while specificity was equal (83% for both). Additionally, MRT in combination with the still obligatory x-ray gave no false-positive results. In some cases, the diagnosis may be found up to 6 weeks earlier using MRT. Conservative therapeutic regimens obviously will not profit from this. Although excluding other diseases is certainly helpful, the value of MRT as a primary diagnostic procedure is limited. During follow-up, MRT helps to reduce the number of x-ray examinations since it can easily assess the containment of the hip and the bone marrow revitalisation. Scintigraphy, another valuable method to judge revitalisation, should remain limited to selected cases, as it requires significant amounts of radiation. PMID- 1733480 TI - [NMR tomography in plasmacytoma]. AB - In 20 patients with multiple myeloma, the results of MR, x-ray, bone scintigraphy, bone marrow scintigraphy and bone marrow biopsy were compared. MR proved the most sensitive imaging method for the detection of bone marrow infiltration followed by x-ray examinations. Bone marrow scintigraphy and especially bone scintigraphy revealed false-negative results more often. These findings were more impressive by the direct comparison of several investigated regions showing different findings of each imaging method. False-positive results were not found. In 4 patients false-negative results of bone marrow biopsy had to be assumed because of definitely pathological findings by x-ray and MRI. PMID- 1733481 TI - [Residual and reconverted hematopoietic bone marrow in the distal femur. Spin echo and opposed-phase gradient-echo MRT]. AB - The distal femur contains usually only fatty marrow in the adult. We investigated the incidence of areas of residual and reconverted hematopoietic marrow in the distal femur in a series of 50 adult patients using conventional spin-echo and opposed-phase gradient-echo MR images. Zones with intermediate to low signal intensity in both sequences representing hematopoietic marrow within high signal intensity fatty marrow were observed in 17 of the 50 patients. Opposed-phase gradient-echo sequences demonstrated significantly greater red-yellow marrow contrast than conventional spin-echo sequences. Residual red marrow may represent a biologic variation of the normal adult pattern of red-yellow marrow distribution in women of menstruating age. Reconverted red marrow appears to be related to increased erythrocyte demand. It should not be mistaken for bone marrow malignancy. Absence of epiphyseal involvement, cortical destruction and soft-tissue mass in residual and reconverted hematopoietic marrow are helpful differential criteria. PMID- 1733482 TI - [Fluorosis following the uncontrolled administration of sodium fluorophosphate]. PMID- 1733483 TI - [Pulmonary alveolar proteinosis in CT and HR-CT]. PMID- 1733484 TI - Current status of magnetic resonance imaging of the ankle and the hindfoot. AB - With the increasing use of magnetic resonance imaging (MRI) for examining the musculoskeletal system, numerous applications have been found in the evaluation of ankle and hindfoot problems. For example, MRI is ideal for assessing tendon disorders, which are common in this region. In addition, the technique allows accurate staging of osteochondral fractures, as well as both soft-tissue and bony neoplasms. Tarsal coalition can be readily evaluated and the type determined. Increasingly, arthritis and synovial abnormalities are coming under scrutiny with MRI; this is currently an area of avid interest. In this article the authors review the current status of MRI of the ankle and the hindfoot and illustrate a variety of these disorders. PMID- 1733485 TI - Wegener's granulomatosis: findings from computed tomography of the chest in 10 patients. AB - Radiographs and computed tomography (CT) scans of the chest were reviewed for 10 patients with pathologically proven Wegener's granulomatosis. The CT scans revealed multiple pulmonary nodules in seven patients and a single nodule in one. The nodules ranged in diameter from 2 mm to 7 cm, and most had irregular margins. All of the nodules larger than 2 cm in diameter showed evidence of cavitation in the CT scans. Additional CT findings included associated areas of consolidation (in two patients), pleural thickening (in two) and pleural effusion (in two). Chest radiographs were available for eight patients, and the CT scans contributed information additional to that available from the radiographs for seven of these. In one patient lung nodules were visible in the CT scans but could not be distinguished from surrounding areas of consolidation in the chest radiographs. CT revealed additional nodules in five of the six patients in whom multiple nodules were seen in chest radiographs and in one of these also revealed cavitation tht was not visible in plain radiographs. CT excluded the possibility of a nodule that was suspected from the chest radiographs in a patient who had been treated previously for Wegener's granulomatosis. The authors conclude that Wegener's granulomatosis is characterized in CT scans by multiple nodules with irregular margins and by cavitation in nodules larger than 2 cm in diameter. CT may also demonstrate nodules and cavitation not apparent in radiographs. PMID- 1733486 TI - Incorrect positioning of nasogastric feeding tubes and the development of pneumothorax. AB - The authors describe 12 patients in whom feeding tubes were inadvertently placed in the bronchial tree a total of 14 times. All but four of the misplacements were complicated by pneumothorax. No deaths were directly attributable to the misplacements, although one cardiac arrest occurred as a late complication of intrapleural feeding. Careful, controlled insertion of feeding tubes and radiographic confirmation of their placement are essential to reduce morbidity and mortality. PMID- 1733487 TI - Gastric ulceration after fundoplication. AB - Gastric ulceration after fundoplication for gastroesophageal reflux is relatively uncommon, occurring in 1% to 3% of cases. During the period 1974 to 1979, approximately 100 modified Belsey fundoplications were performed at McMaster University Medical Centre. In four patients gastric ulceration developed after the surgery. In all cases the ulcers were located in the proximal stomach, an unusual site. Published reports of gastric ulceration after fundoplication were reviewed, special attention being given to the cause. The authors conclude that local ischemia and mechanical trauma are important in the development of ulceration, which can occur as early as one week after fundoplication. The detection of ulcers requires awareness of the condition and special attention to the symptoms. Because the gastric anatomy is altered by the fundal wrap, the area can be visualized more easily by double-contrast barium studies than by endoscopy. PMID- 1733488 TI - Gaucher's disease involving the spleen. AB - Gaucher's disease is a rare inherited disorder that results from progressive accumulation of glucocerebrosides within the reticuloendothelial system and affects the liver, the spleen, the bone marrow and the lymph nodes. Ultrasonography of the spleen typically demonstrates hypoechoic focal masses; however, the lesions may be hyperechoic if they contain extensive fibrosis or infarction. The authors describe a young Ashkenazi man who presented with splenomegaly as the single clinical manifestation and in whom were found splenic masses of mixed echogenicity, none of which demonstrated fibrosis or infarction when studied pathologically. PMID- 1733489 TI - Duodenal duplication cysts: case report and review of the current literature. AB - Duodenal duplication cysts constitute a rare congenital anomaly of the gastrointestinal system, and a preoperative diagnosis of the condition is even less common. The authors present a case of a duodenal duplication cyst in a 3 year-old child in whom they were able to make the correct diagnosis preoperatively by means of ultrasonography and computed tomography. PMID- 1733490 TI - Calcified herniations of the thoracic disk: role of magnetic resonance imaging and computed tomography in surgical planning. AB - Herniations of thoracic disks are uncommon, and their surgical management can be challenging. Magnetic resonance imaging (MRI) is rapidly becoming the method of choice for assessing degenerative disease in thoracic disks. However, calcification may be difficult to detect with MRI and plain films alone. The authors report a case in which MRI and myelography underestimated the true extent of disk calcification, the detection of which would have altered the initial surgical approach. PMID- 1733491 TI - Hydrocephalus and prominence of the choroid plexus: an unusual computed tomographic presentation of cerebral toxoplasmosis in AIDS. AB - Toxoplasmosis is a frequent cause of infection of the central nervous system (CNS) in patients with acquired immunodeficiency syndrome. Computed tomography (CT) usually shows solitary or multiple parenchymal lesions, which are most often located in the cortex, the juxtacortical white matter and the basal ganglia. The authors describe a 30-year-old immunocompromised Haitian woman with pathologically proven CNS toxoplasmosis who presented with hydrocephalus and prominence of the choroid plexus; there was no evidence of focal parenchymal lesions in contrast-enhanced CT scans. An autopsy revealed diffuse destruction of the ependyma of the lateral, the third and the fourth ventricles. Necrosis was evident, and the periventricular tissues and the choroid plexus were infiltrated with neutrophils and macrophages. Pseudocysts of Toxoplasma were identified near the ventricular surface. PMID- 1733492 TI - Critical evaluation of radiologic technologies. PMID- 1733493 TI - Reversible cerebral, hepatic and renal lesions in severe pre-eclampsia. AB - Several authors have reported the appearance of reversible hypoattenuated cerebral lesions, representing ischemia, in computed tomography scans of patients with severe pre-eclampsia. Hepatic hemorrhage and sometimes rupture have also been reported in this setting, but these problems have apparently never occurred in a patient with reversible ischemia. The authors describe a 34-year-old patient with severe pre-eclampsia in whom reversible cerebral ischemia developed in combination with hepatic and renal hematomas, which subsequently partially resolved. PMID- 1733494 TI - Residents' corner. Answer to case of the month #11. Typhlitis. PMID- 1733495 TI - Current status of magnetic resonance imaging of the wrist. AB - Conventional imaging of the wrist has relied heavily on plain radiography, tomography, fluoroscopy and arthrography. More recently, computed tomography and magnetic resonance imaging (MRI) have been added to this armamentarium. In this article the authors review the normal anatomy of the wrist and demonstrate a variety of pathologic conditions that can be assessed with MRI, including avascular necrosis and tears of the intrinsic and the extrinsic ligaments and the triangular fibrocartilage. MRI of the wrist is still evolving rapidly, and its place in the work-up of wrist disorders is only now being defined. PMID- 1733496 TI - Cap recap: the involvement of eIF-4F in regulating gene expression. PMID- 1733497 TI - A family of proteins involved in intracellular transport. PMID- 1733498 TI - The generation of diversity and pattern in animal development. PMID- 1733499 TI - The origin of pattern and polarity in the Drosophila embryo. PMID- 1733500 TI - Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question. PMID- 1733501 TI - Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons. PMID- 1733502 TI - A position effect on the time of replication origin activation in yeast. AB - The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late. This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times. The basis of this temporal regulation in the yeast S. cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501). We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function. For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere. PMID- 1733503 TI - Integrin modulating factor-1: a lipid that alters the function of leukocyte integrins. AB - The avidity of integrin CR3 (also known as alpha M beta 2, Mac-1, Mo-1, and CD11b/CD18) may be reversibly altered without changes in the number of cell surface receptors. Here we describe a molecule termed integrin modulating factor (IMF-1), which controls CR3 avidity. Addition of IMF-1 to polymorphonuclear leukocytes (PMNs) or to purified CR3 causes enhanced binding of ligand. IMF-1 is not present in resting PMNs, but stimulation of cells results in a transient rise in IMF-1 content that parallels a transient rise in CR3 activity. We suggest that PMNs control adhesivity by controlling synthesis of IMF-1, which then acts as an allosteric activator of leukocyte integrins. IMF-1 is an acidic, amphiphilic molecule of Mr340 +/- 16 that does not contain ester, phosphate, amide, sialic acid, or glycosidic or vicinal hydroxyl functionalities, but does contain a carbon-carbon double bond. These results suggest that IMF-1 is an unsaturated fatty acid or an isoprenoid acid. PMID- 1733504 TI - Inhibition of experimental autoimmune uveitis by retinal photoreceptor antigens coupled to spleen cells. AB - Experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are CD4+ T cell-mediated inflammatory diseases of the uveal tract and retina of the eye and of the pineal gland. EAU and EAP can be induced by several retinal autoantigens including S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP). In this study we investigated the effect of intravenous administration of S-Ag and IRBP coupled to syngeneic spleen cells on the development of EAU and EAP. Injection of S-Ag or IRBP coupled to spleen cells 5 days prior to immunization with native S-Ag or IRBP, respectively, was effective in preventing the induction of EAU and EAP in LEW rats. Conversely, LEW rats receiving S-Ag-coupled spleen cells and challenged with IRBP or LEW rats receiving IRBP-coupled spleen cells and challenged with S-Ag developed a severe EAU within 10 days to 2 weeks following immunization, as did all control animals receiving sham-coupled spleen cells and challenged with the two retinal antigens. The results show that the administration of retinal autoantigens coupled to spleen cells effectively protects against the development of EAU when animals are subsequently challenged with the tolerizing antigen but not when challenged with another unrelated pathogenic retinal autoantigen. PMID- 1733505 TI - Abnormal expression of IL-2R beta (p70)-binding polypeptide on HIV-infected patients' cells. AB - The constitutively expressed IL-2R beta (p70) chain participates in the formation of high-affinity (h.a.) IL-2R and transduces IL-2-mediated signals to normal cells. Its expression on HIV-infected patients' PBMC was evaluated and was found to be decreased in both nonstimulated CD4+ and CD8+ cells. Mitogenic cell stimulation induced IL-2R beta chain expression on both CD4+ and CD8+ cells from asymptomatic and persistent generalized lymphadenopathy patients but not on those from AIDS patients. Comparison of mean fluorescence intensity of IL-2R beta positively stained cells from normals and patients did not reveal significant differences. Cross-linking of 125I-rIL-2 on patients' PHA-blasts revealed decreased signals corresponding to both IL-2-binding chains and, in some cases, neither IL-2R alpha nor IL-2R beta chains could be detected. Decreased expression of IL-2R beta polypeptide was associated with impaired accumulation of the corresponding mRNA transcripts. Binding experiments with 125I-rIL-2 under h.a. conditions showed a decreased number of IL-2-binding sites/cell which was more pronounced in patients with AIDS than in patients with less advanced clinical stages. In vitro HIV infection of normal PHA-blasts also resulted in a decreased number of h.a. IL-2R/cell. High concentrations of rIL-2 in the absence of other mitogenic stimuli induced a decreased cell proliferation and expression of the IL 2R alpha chain and did not enhance the constitutive NK and the generation of LAK activity in several patients, suggesting an impaired IL-2R beta chain-mediated cell activation. PMID- 1733506 TI - Function of dendritic cells and changes in T cell proliferation in antigen induced nonresponsiveness. AB - The ability of dendritic cells (DC) to acquire and present antigen to T cells during antigen-induced nonresponsiveness (AINR) in contact sensitivity was examined by studying cells from lymph nodes draining the sites of antigen challenge. Mice were pretreated on the right flank with either vehicle (AOO), oxazolone (Ox), or fluorescein isothiocyanate (FITC) and challenged 5, 10, or 20 days later with FITC on the left flank. At 5, 10, and 20 days, compared with animals pretreated with vehicle and challenged with FITC, those pretreated and challenged with FITC showed reduced acquisition of antigen by DC and the DC showed a reduced ability to stimulate naive T cells in vitro. Proliferation of T cells immediately on isolation (reflecting in vivo activity) was also reduced. When the time between pretreatment and challenge was extended to 40 days, the proliferative responses and antigen acquisition returned to normal. Animals sensitized with Ox and challenged with FITC showed nonspecific inhibition of T cell proliferation at 5 days only and not at later times and antigen levels on the DC from these animals were normal. The results show that low T cell proliferation during specific AINR in contact sensitivity may be a consequence of reduced acquisition and presentation of antigen by DC. PMID- 1733507 TI - Production and differentiation of NK lineage cells in long-term bone marrow cultures in the absence of exogenous growth factors. AB - Neither lytic NK cells nor IL-2-responsive NK precursors were produced in myeloid (Dexter) long-term bone marrow cultures (LTBMC). However, when myeloid LTBMC were switched to lymphoid (Whitlock-Witte) conditions and reseeded ("recharged") with fresh bone marrow cells (BMC), nonadherent cells with NK lytic activity and NK 1.1+ phenotype were produced within 1-2 weeks without the addition of exogenous IL-2 to the cultures. NK- and T cell-depleted BMC proliferated extensively in switched cultures and in 2 weeks generated cells that lysed the NK target YAC-1 but not the LAK target P815. The presence of NK precursors in the cultures was confirmed by reculturing nonadherent cells harvested from recharged LTBMC in fresh medium containing 50 U rIL-2/ml. High levels of NK lytic activity were generated. Sequential expression of NK 1.1 and IL-2 responsiveness followed by lytic activity was demonstrated by harvesting cells early after recharge, prior to the appearance of lytic cells. Elimination of NK 1.1+ cells depleted the ability to respond to IL-2 in secondary culture. Our studies demonstrate that myeloid-to-lymphoid switched LTBMC support the proliferation and differentiation of NK lineage cells from their NK 1.1-, nonlytic progenitors in the absence of an exogenous source of growth factors. PMID- 1733508 TI - Evidence for involvement of TNF-alpha in the induction phase and IFN-beta in the effector phase of antiproliferative activity of splenic macrophages. AB - Splenic macrophages play a key role in regulating cell proliferation during a variety of chronic perturbations of the hematopoietic system. This regulatory activity is in sharp contrast to the activities of inflammatory monocytes/macrophages in that it is not dominated by the secretion of prostaglandins or toxic metabolites such as peroxides. A productive model for studying these nontoxic regulatory activities of splenic macrophages has been provided by macrophages generated in vitro (M phi-c) during autologous spleen cell culture. The M phi-c effectively inhibit (greater than 90%) lymphocyte proliferation by inhibiting G1----S phase progression without inhibiting the production of interleukins by the lymphocytes. Conditioned medium from M phi-c activated with LPS + rIFN-gamma effected a similar G1 arrest of activated lymphocytes. The involvement of IFN-beta in effecting the antiproliferative activity is suggested by (1) the ability of monospecific anti-IFN-beta mAB, but not anti-TGF-beta, anti-IL-1, anti-TNF-alpha, or anti-IFN-gamma, to neutralize the antiproliferative activity in the M phi-c supernatants and (2) the ability of purified IFN-beta to effect a similar inhibition of cell proliferation (i.e., G1 arrest without inhibition of interleukin production). rTNF-alpha and rIFN-gamma could not effect such an inhibition of cell proliferation and did not synergize with IFN-beta in producing such an antiproliferative effect. The M phi-c could be activated to effector function by a combination of LPS + rIFN-gamma or rTNF-alpha + rIFN-gamma, but not by any one of those reagents alone. LPS alone was sufficient to stimulate TNF-alpha production by the M phi-c. Activation of the M phi-c by LPS + rIFN-gamma could be completely blocked by anti-TNF-alpha antibodies. These data suggest that the M phi-c can be induced to produce inhibitory levels of cytostatic cytokines by a TNF-alpha autocrine loop that is IFN-gamma dependent. The in vivo relevance of this effector mechanism is suggested by, and discussed in the context of, the recent reports of "spontaneous" production of IFN-beta during immunological disorders. PMID- 1733509 TI - Characterization of alpha beta+ CD4- CD8- CTL lines isolated from mixed lymphocyte cultures of adult mouse spleen cells. AB - Several CD4- CD8- and CD4- CD8+ T cell lines and clones have been isolated from the same mixed lymphocyte cultures. They express the alpha beta T cell receptor, are CD3+, V beta 8- and heat stable antigen-, and exhibit highly specific cytolytic activity mapping to H2Db. However, monoclonal antibody against H2Db failed to block lysis by CD4- CD8- cytotoxic T lymphocytes (CTL). One possible explanation is that the "double negative" cytotoxic T lymphocytes may recognize H2Db in the context of other class I molecules (Qa/Tla). CD4 and CD8 could be detected on some lines cloned in high concentrations of EL4 supernatant and the cell surface expression was influenced by growth conditions. The alpha beta+ CD4- CD8- CTL described in this report are clearly different from the so-called "autoimmune double negatives" which suggests a function for them in normal immune responses. PMID- 1733510 TI - Functional role of self IA molecules in polyclonal B cell activation using an autoreactive B cell clone derived from (NZB X NZW) F1 mice. AB - The mechanism of polyclonal B cell activation in autoimmune diseases was investigated by using an autoreactive B cell clone established by somatic hybridization with B cells derived from NZB X NZW (B/W) F1 mice. Briefly, splenic B cells from B/W F1 mice were fused with M12.4.1, a mutant of a B cell line, in the presence of polyethylene glycol and DMSO. NW47.7, a subclone of a resulting hybridoma, expresses B cell surface antigens on the cell membrane, namely IAd, IgM, B220, the receptors for the C3 fragment of complement (C3R), and the Fc portion of IgG (Fc gamma R). It also possesses a receptor molecule for mouse red blood cells treated with bromelain (Br-MRBC) on its surface, by rosette-forming assay with Br-MRBC. In contrast, parental M12.4.1 does not express IAd and IgM on the cell membrane, and does not bind to Br-MRBC under the same conditions. Thus, it is likely that NW47.7 may be an autoreactive B cell clone specific for Br MRBC. Interestingly, NW47.7 was induced to generate a significant number of IgM secreting cells when treated with Br-MRBC and rIL-5. Furthermore, mAb against IAd molecules, but not IAk and KdDd, markedly inhibited the differentiative effect of polyclonal activators such as LPS and rIL-5. Also, when MHC identical irradiated B cells were added to the culture of NW47.7 as a stimulator, the induction of IgM producing cells was greatly augmented, but this augmenting effect was lost by interfering with direct contact of NW47.7 cells with stimulator B cells using a semipermeable membrane, as well as by the addition of mAb against IAd molecules. In addition, irradiated NW47.7, but not M12.4.1, by itself could enhance the secretion of IgM by NW47.7 as a stimulator, but this enhancing effect markedly decreased in the presence of anti-IAd mAb. The present results suggest that surface IA molecules on B cells are involved during the differentiative response to polyclonal activators, and may directly provide a differentiative signal for maturation of B cells into IgM-secreting cells. PMID- 1733511 TI - Motility of IL-2-stimulated lymphocytes in neutral and acidified extracellular matrix. AB - The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit. PMID- 1733512 TI - Induction of CD4+ cytotoxic T cells by sensitization with allogeneic melanomas bearing shared or cross-reactive HLA-A. AB - Human melanoma is an immunogenic neoplasm whereby enhancement of specific cell mediated immunity can alter tumor progression. HLA-A2-restricted CTL have been demonstrated to kill allogeneic HLA-A2-matched melanoma. We investigated the ability of allogeneic melanoma cells sharing HLA-A antigens to sensitize melanoma patients' lymphocytes to induce HLA-A-restricted CTL to autologous melanoma. PBL from melanoma patients were cocultured with autologous melanoma cells in defined "cocktail medium" to generate melanoma-specific HLA-A-restricted CTL lines. CTL generated by sensitization with allogeneic melanoma bearing shared HLA-A2, A11, A24, or "cross-reactive" HLA-A antigens could kill almost as many autologous melanoma cells as CTL sensitized with autologous melanoma. There are HLA-A antigens that are immunogenically cross-reactive because they share determinant epitopes. CTL were not activated NK or LAK cells. The HLA restriction and melanoma cell specificity of the CTL were demonstrated by cold target inhibition with autologous and allogeneic melanoma and B lymphoblasts. Anti-CD3 and anti-HLA AB inhibited CTL killing of melanoma. The CTL were predominantly CD3+CD4+ TCR alpha/beta+. These studies demonstrate that melanomas being shared or cross reactive HLA-A can be used for in vitro generation of HLA-restricted CTL that recognize melanoma-associated antigens. The findings have very important implications in human tumor immunotherapy. PMID- 1733513 TI - In vivo modulation of lymphokine-activated killer cell activity by cell wall components of Candida albicans. AB - We have previously reported that inoculating CD2F1 mice intraperitoneally with five doses of 2 x 10(7) inactivated Candida albicans (CA) cells was associated with the induction of lymphokine-activated killer (LAK)-like effectors. In this study we investigated the ability of some purified cell wall components of CA (CA CW) to induce LAK-like cells in vivo. Multiple administrations of glucan ghost (GG), a mannoprotein mixture (MP) and a low-protein mannan fraction (M) at variance with whole CA did not induce LAK-like cells in the peritoneal cavity. However, the broad-spectrum antitumor cytotoxicity induced by CA could be recalled to a high level by a booster dose of MP and M, but not GG, given up to 70 days after the multiple CA-treatment. This induced cytotoxicity was maximum when the booster was given on Day +14 after CA-treatment and minimum on Day +70. In CA-treated mice, inoculated on Day +30 with CA or MP, LAK-like cytotoxicity was already significantly increased 4 hr after the booster, but the maximum value was reached at 24 hr. Anti-mannan antibodies did not interfere with LAK-like cells induction by CA because splenectomy before CA-treatment or passive administration of anti-mannan antibodies had no effect on the rapid activation of cytotoxicity by CA or a booster dose of MP. Administration of recombinant human interleukin-2 (rhIL-2) to CA-treated mice induced a higher level of NK activity than that induced by the same dose in untreated control mice, but did not activate LAK-like effectors. The results indicate that LAK-like effectors are easily generated in the peritoneal cavity by a booster with a defined antigenic constituent of CA cell wall for a long period in CA-sensitized mice. PMID- 1733514 TI - Arthritis by autoreactive T cell lines obtained from rats after injection of intestinal bacterial cell wall fragments. AB - T cell lines (B13, B19) were isolated from the lymph nodes of Lewis rats 12 days after an arthritogenic injection of cell wall fragments of Eubacterium aerofaciens (ECW), a major resident of the human intestinal flora. These cell wall fragments consist of peptidoglycan polysaccharide complexes (PPC). The cell lines that bear the helper phenotype were arthritogenic in knee or ankle joints upon intravenous injection into irradiated Lewis recipients. B13 was, however, not arthritogenic in irradiated F344 recipients that are largely RT1 identical. The arthritis induced in the knee joints of the irradiated Lewis rats was clearly shown by a 99mtechnetium-pertechnetate scanning technique and was confirmed histologically. In vitro the cell lines showed a proliferative response after stimulation with syngeneic spleen cells alone. The proliferation was significantly higher when bacterial PPC, isolated in soluble form from normal feces or ileostomy fluid were added. Recognition by B13 appeared to be MHC class II restricted. These results show that autoreactive T cell lines can be isolated from rats after injection of bacterial cell wall antigens and that these cell lines can be arthritogenic. This suggests a role for autoreactive T cells in the induction of bacterial cell wall arthritis and might give a clue for the arthritogenic properties of the normal human intestinal flora. PMID- 1733515 TI - The hu-PBL-SCID mouse model. Long-term human serologic evolution associated with the xenogeneic transfer of human peripheral blood leukocytes into SCID mice. AB - We present a 2-year serologic analysis of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL, hu-PBL-SCID mice). After 10-20 x 10(6) PBL transfer, human IgG serum levels generally increased in the SCID mouse recipient for 2 months, and thereafter decreased without returning to zero for at least 2 years. Great variability existed between different hu-PBL-SCID mice with regard to Ig serum levels even when derived from the same donor's PBL aliquot. The ratio of IgM to IgG serum levels was lower in hu-PBL-SCID mice than in the donors. The half-life of human IgG in the SCID mouse is shorter than in the human (8 days vs 23 days), suggesting a much higher production of IgG than expected from serum levels. The majority of hu-PBL-SCID mouse sera analyzed by high resolution electrophoresis had a smear appearance suggestive of diverse human Ig, generally with superimposed multiple faint mIg. Few mice developed strong human mIg, associated with lymphoproliferative diseases. In the hu-PBL-SCID mouse model, the transfer of cells from donors making antibody with defined specificity against TT and nuclear antigen resulted in the appearance of these antibodies in only a minority of the recipients. PMID- 1733516 TI - The birth of immunology. III. The fate of the phagocytosis theory. AB - During the period of 1895-1910, immunology was preoccupied with defining the cellular (Elie Metchnikoff's phagocytosis theory) as opposed to the humoral basis of bactericidal defense. Although initial discovery of immunopathologic phenomena had been made (e.g., relating to transplantation, autoimmunity, allergy), focus on microbicidal therapy and diagnosis of infectious diseases remained the major stimuli of inquiry. The debate concerning the relative roles of phagocytes, complement, amboceptors (sensibilizing factors, antibody, antitoxins), various lysins (e.g., bacteriolysins, spermatolysins, hemolysins), agglutinins, stimulines, and then Almoth Wright's opsonins reflects the ambiguity of a scientific language being created in an era still struggling with a poorly defined experimental system, for the language, both its vocabulary (newly studied phenomena) and grammar (operational mechanisms) was yet to be codified. The joint award of the Nobel Prize to Metchnikoff and Paul Ehrlich in 1908 for their respective contributions to the "theory of immunity" appeared to proclaim a consensus, but the secret Nobel Committee reports that evaluated Metchnikoff's contributions reveal only a grudging acceptance of his position, and the award was clearly made on the basis of an apparent complementarity between the theoretical views of the humoralists and those elements of the phagocytosis theory that fit the then current discussion of immunity. In this regard, opsonins played an especially important role as both an experimental and conceptual bridge between the competing schools. What was no longer under consideration (and in fact never was explicitly debated) concerned the intellectual foundation of Metchnikoff's original concept of immunity as those activities that defined organismal identity, (developed from Metchnikoff's research in developmental biology) and which regarded host defense mechanisms as only subordinate to this primary function. Immunology in the first half of the 20th century pursued issues pertinent to chemically characterizing immune specificity and only later returned to the Metchnikovian question of how the immune identity was established. This latter venture has achieved molecular sophistication, but even such a formulation may be an inadequate answer to the Metchnikovian postulate. The theoretical discussion between cellularists and humoralists continues in new guises, for the essential debate remains unresolved. PMID- 1733517 TI - Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line. AB - Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated. The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA). However, the surface-bound EA is not internalized. The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3. alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative. A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced. The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers. The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis. PMID- 1733518 TI - Inhibition of NK cell-mediated cytotoxicity by oxysterols. AB - Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols. PMID- 1733519 TI - Spoilage yeasts. AB - Yeasts are best known for their beneficial contributions to society, and the literature abounds with discussions of their role in the fermentation of alcoholic beverages, bread, and other products. Yeasts also cause spoilage, but, with a few exceptions, this unwanted activity often goes unrecognized and underestimated as a major problem in the food and beverage industries. In some cases, there is only a fine line between what is perceived as either a spoilage or beneficial activity. This review examines the occurrence and growth of yeasts in foods and beverages with respect to their spoilage activities, the biochemistry of this spoilage, and technologies for the enumeration and identification of spoilage yeasts. PMID- 1733520 TI - Strategies for the genetic manipulation of Saccharomyces cerevisiae. AB - The budding yeast Saccharomyces cerevisiae is now widely used as a model organism in the study of gene structure, function, and regulation in addition to its more traditional use as a workhorse of the brewing and baking industries. In this article the plethora of methods available for manipulating the genome of S. cerevisiae are reviewed. This will include a discussion of methods for manipulating individual genes and whole chromosomes, and will address both classic genetic and recombinant DNA-based methods. Furthermore, a critical evaluation of the various genetic strategies for genetically manipulating this simple eukaryote will be included, highlighting the requirements of both the new and the more traditional biotechnology industries. PMID- 1733521 TI - Biochemistry and genetics of actinomycete cellulases. AB - The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source. PMID- 1733522 TI - Production of food and fodder yeasts. AB - A decade or so ago, there was considerable interest in developing single cell protein production from raw materials. Many factors have influenced the development of fodder yeast technology, notably the biochemistry and physiology of the yeast. It is shown that those considerations have led to the choice of a continuous fermentation technology. PMID- 1733523 TI - The production of organic acids. AB - The production of organic acids covers two aspects: first, the metabolic pathways involved in the biosynthesis, and, second, the industrial process strategy adopted. The review seeks to show the underlying biochemical similarities in the biosynthesis of organic acids and the resulting similarities in the commercial processes. Two groups of acids are defined, those with a "long" biosynthetic path from glucose, involving much of the glycolytic pathway and the tricarboxylic acid cycle, and those acids with a "short pathway", essentially a biotransformation of glucose. The regulation of the pathways and the future developments in metabolic control theory and genetic manipulations relating to them are considered. The organisms used industrially are also limited, Aspergillus sp. and Candida yeasts; again the underlying metabolic similarities lead to similar strategies for all the acids discussed. PMID- 1733524 TI - Role of the laryngeal mask in airway management. PMID- 1733525 TI - Dual end-tidal CO2 monitoring and double-lumen tubes. PMID- 1733526 TI - Understanding anaesthesia. PMID- 1733527 TI - But yet, I'll make assurance double sure. PMID- 1733528 TI - Chest wall motion during halothane anaesthesia in infants and young children. AB - Chest wall motion during anaesthesia may differ from the awake state because of the effect of anaesthetic agents on the muscles of respiration. The purpose of this study was twofold (1) to describe the pattern of chest wall motion in infants and children during halothane anaesthesia (HA) using respiratory inductive plethysmography (RIP) and (2) to calibrate the voltage output of RIP in units of volume. Seven infants (2.3 +/- 1.7 mo, 5.9 +/- 0.7 kg) and five children (2.9 +/- 1.1 yr, 15.5 +/- 1.5 kg) were studied. Since results in both age groups were qualitatively similar they are presented as a single group. Respiratory excursions of the rib cage (RC) and abdomen (ABD) were measured using RIP. Airflow was measured with a pneumotachograph. During spontaneous breathing the analogue signals of airflow, pressure, RC and ABD were recorded. Measurements were taken during (1) halothane anaesthesia and (2) during emergence from anaesthesia. The XY plots of the RC and ABD signals were plotted for each period. In addition the voltage output of the respiratory excursions of the RC and ABD signals was converted to units of volume using the simultaneous solution of equation method. The accuracy of conversion factors was validated by regression analysis of the predicted and measured tidal volume using breaths sampled at random throughout the entire period of study. Regression analysis of this relationship gave a slope between 0.85 and 1.15 (r2 value greater than 0.7) in five of the twelve patients. The pattern of chest wall motion in the XY plots showed synchronous motion between RC and ABD signals during HA in nine of the twelve patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733529 TI - Continuous oxygen saturation monitoring following rectal methohexitone induction in paediatric patients. AB - Rectal methohexitone has been used to induce anaesthesia in paediatric patients for a number of years. This study was conducted in order to confirm the safety of this method of induction for uncomplicated routine paediatric patients. Children between the ages of six months and six years were considered candidates for induction with methohexitone (10%, 25-30 mg.kg-1). Patients were monitored with a continuous oxygen saturation recording. Forty-nine patients participated in this study and anaesthesia was induced successfully in 44. The mean age of the patients was 2.7 +/- 1.6 yr. The mean weight was 13.8 +/- 4.3 kg and the mean dose of methohexitone for successful induction was 27.0 +/- 3.0 mg.kg-1. Continuous oximeter recordings were available in 31 of the 42 patients who allowed oximeter placement prior to administration of methohexitone. No major desaturation events were noted in any patient. Two brief episodes of desaturation occurred. One with a nadir of 90% which lasted for 45 sec and another with a nadir of 86% which lasted for 26 sec. Both children had their heads flexed over their parents' shoulders at the time of the event resulting in partial airway obstruction. Both of these episodes were the result of upper airway obstruction which was clinically diagnosed by the anesthetist and readily corrected by repositioning the head. This study confirms the efficacy and safety of rectal methohexitone for induction of general anaesthesia in children. Mechanical obstruction of the airway following induction seems to be the most likely cause for oxygen desaturation. Monitoring of pulse oximetry does not appear necessary provided the child is carefully observed for adequacy of air exchange. PMID- 1733530 TI - A comparison of transcutaneous end-tidal and arterial measurements of carbon dioxide during general anaesthesia. AB - A randomized, prospective study was performed to evaluate the accuracy of a new transcutaneous carbon dioxide (CO2) monitor (Fastrac) during general anaesthesia. Twenty-two adult patients undergoing elective surgery were subjected to three different levels of minute ventilation by varying their respiratory rates in a randomized cross-over design. Simultaneous measurements of transcutaneous CO2 (PTCCO2) and arterial CO2 (PaCO2) were obtained at three levels of minute ventilation (low, medium and high). End-tidal CO2 (PETCO2) values were also recorded from a mass spectrometer (SARA) at each time period. A total of 66 data sets with PaCO2 ranging from 28-62 mmHg were analyzed. The PTCCO2 values demonstrated a high degree of correlation with PaCO2 over the range of minute ventilation (y = 0.904x + 6.36, r = 0.92, P less than 0.001). The PETCO2 measurement also demonstrated a generally good correlation with PaCO2 (y = 0.62x + 9.21, r = 0.89, and P less than 0.01). However, the PETCO2-PaCO2 gradients (mean 7.0 +/- 3.1 mmHg) were greater than the PTCCO2-PaCO2 gradients (mean 2.3 +/ 2.4 mmHg) at all three levels of minute ventilation (P less than 0.05). These differences were greatest when PaCO2 was in the high range (48-60 mmHg). We conclude that the new Fastrac CO2 monitor is accurate for monitoring carbon dioxide levels during general anaesthesia. The new transcutaneous devices provide an effective method for non-invasive monitoring of CO2 in situations where continuous, precise control of CO2 levels is desired. PMID- 1733531 TI - Curare 1942-1992. PMID- 1733532 TI - Does propranolol alter the vascular response to phenylephrine before or during halothane anaesthesia in patients with coronary artery disease? AB - Preoperative beta-adrenergic blockade with propranolol, by allowing unopposed alpha-adrenergic stimulation in response to stress, has been suggested as a factor contributing to hypertension following coronary artery bypass surgery (CABG). Thus, one might expect to find an exaggerated haemodynamic response to phenylephrine (PHE), an alpha 1 agonist. To study this, the cardiovascular response to PHE infusion at 30, 40, and 50 microgram.min-1 prior to and during halothane anaesthesia was measured before surgical stimulation during elective CABG in patients taking chronic propranolol therapy and compared with that of patients not taking any cardiovascular medications. Chronic propranolol therapy did not alter the haemodynamic response to PHE, before or during halothane anaesthesia, and the incidence of postoperative hypertension requiring vasodilator therapy was the same for both groups. PMID- 1733533 TI - Carboxypeptidase N concentration during cardiopulmonary bypass in humans. AB - Carboxypeptidase N (CPN) is an inactivator of anaphylatoxins and kinins, peptides implicated in the pathogenesis of complications in extracorporeal circulation. To investigate whether the level of CPN is altered during cardiopulmonary bypass (CPB) we studied 15 patients undergoing cardiac surgery utilizing CPB. The concentration of CPN decreased to about 48% of the initial value upon initiation of CPB and remained low throughout the procedure. A similar decrease was observed in the level of alkaline phosphatase, an enzyme that was measured to assess the degree of haemodilution. When the data were normalized for dilution, no difference in the concentration of CPN was observed during CPB. Moreover, no changes in the concentration of CPN were observed when protamine was given to neutralize heparin and none of the 15 patients experienced any side-effects of protamine administration. We conclude that the decrease in CPN during CPB was due primarily to dilution and not to changes in CPN synthesis or catabolism. Protamine administration is not associated with significant changes in the level of CPN in patients who have an asymptomatic reversal of heparin anticoagulation. PMID- 1733535 TI - Images. PMID- 1733534 TI - Induction, maintenance and recovery characteristics of desflurane in infants and children. AB - To determine the induction and recovery characteristics of the new poly fluorinated anaesthetic desflurane, 78 fasting and unpremedicated neonates, infants and children up to 12 yr of age were studied. Patients were stratified according to age: full-term neonates less than 28 days of age (n = 12), infants 1 6 mth (n = 12) infants 6-12 mth (n = 15), children 1-3 yr (n = 15), 3-5 yr (n = 12), and 5-12 yr (n = 12). After preoxygenation for two minutes and an awake tracheal intubation, neonates were anaesthetized with stepwise increases in the inspired concentration of desflurane in an air/oxygen mixture. Infants 1-12 mth of age and children were anaesthetized with stepwise increases in the inspired concentration of desflurane in oxygen. Their tracheas were intubated under deep desflurane anaesthesia without muscle relaxation. The incidence of airway reflex responses (including breathholding, coughing, laryngospasm, bronchospasm and oropharyngeal secretions), incidence of excitement, minimum arterial oxygen saturation, and times to loss of eyelash reflex and tracheal intubation during induction were recorded. After skin incision, anaesthesia was maintained with desflurane (approximately 1 MAC) in 60% nitrous oxide and oxygen. Heart rate and systolic arterial pressure were recorded awake, at approximately 1 MAC before and after skin incision and throughout surgery. At the completion of surgery, all anaesthetics were discontinued and the lungs were ventilated with 100% oxygen. During emergence, the end-tidal concentration of desflurane was recorded until extubation. The incidence of airway reflex responses and the times to eye opening and extubation after the discontinuation of desflurane were recorded.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 1733536 TI - Neuromuscular contributions in eight anaesthesia journals. AB - Entries in the index issues of eight English-language anaesthesia journals were examined for the years 1950-1990, noting the number of original articles, reviews and editorials. Those which included topics involving neuromuscular blockade were examined in greater detail; these averaged almost one-tenth of the annual totals, despite its eightfold increase over the four decades. During the eighties the strongest research interest involved atracurium, pancuronium, succinylcholine, tubocurarine, and vecuronium, but the share for the long-acting agents was waning by 1990. PMID- 1733537 TI - Vomiting and aspiration pneumonitis with the laryngeal mask airway. AB - We report a case of severe aspiration pneumonitis in the dependent lung of a 74 yr-old man following Austin-Moore arthroplasty. A laryngeal mask airway provided a clear airway until anaesthesia became too light during manipulation of the fractured femoral head. Active vomiting occurred and gastric contents were "reflected" back into the trachea. Tracheal intubation and suction were immediately performed but the patient required postoperative ventilatory and inotropic support for three days. PMID- 1733539 TI - Continuous lumbosacral block using a Tuohy needle and catheter technique. AB - Three cases are reported where continuous lumbosacral block was performed using a catheter through an epidural needle technique. Good unilateral lower limb surgical anaesthesia was achieved in all three cases with successful blockade of the lumbar and sacral plexuses. A 17-gauge Tuohy needle was positioned between the transverse processes of L4 and L5 and an epidural catheter inserted into the space between the quadratus lumborum and psoas muscles. Forty to seventy millilitres of local anaesthetic were injected and resulted in good surgical anaesthesia within 12-20 min. Radiographic studies in these patients confirmed placement of the catheter in close proximity to the lumbosacral plexus. Experience in a further 12 cases is also reported. There were no side-effects. The technique is successful and is recommended when unilateral lower limb anaesthetic is required and when spinal and epidural anaesthesia are contraindicated. PMID- 1733538 TI - Early respiratory depression during caesarean section following epidural meperidine. AB - A case of respiratory depression which occurred following administration of epidural meperidine during Caesarean section is described. Epidural meperidine, 75 mg (10 mg.ml-1) was given after delivery of the infant to provide postoperative analgesia. Oxygen desaturation (SaO2 90%) and a decrease in respiratory rate (4.min-1) were noted 30 min after epidural meperidine was administered. Naloxone, 0.1 mg, was given iv which resulted in prompt improvement in both respiratory rate and oxygen saturation. Vascular absorption of meperidine from the epidural venous plexus is the most probable explanation for this case of early respiratory depression. We recommend a maximum bolus dose of 50 mg of epidural meperidine for pain management after Caesarean section. It is also important to monitor oxygen saturation continuously during the intraoperative period, and to monitor the patient closely in the recovery room for at least one hour for evidence of respiratory depression. PMID- 1733540 TI - Flow resistance of coaxial breathing systems: investigation of a circuit disconnect. AB - A clinical incident involving an undetected disconnection occurred during the use of a CPRAM coaxial breathing circuit. The flow resistance of this circuit was evaluated and compared with that of a Bain circuit to determine the factors involved. A differential pressure transducer was used to monitor the pressure drop across each circuit during simulation of controlled ventilation with a fresh gas flow of 6 L.min-1. An Ohio V5 anaesthesia ventilator was adjusted to different flow rates and tidal volumes and a test lung simulated an airway resistance of 2.7 cm H2O.L-1.sec and a compliance of 0.05 L.cmH2O-1. Absolute pressure at the ventilator was also monitored when the circuit was disconnected from the test lung. The CPRAM circuit displayed a pressure drop from the ventilator to the endotracheal tube of 6.3 cm H2O at 60 L.min-1, about twice that found with the Bain. A disconnection at the tracheal connector produced an absolute pressure at the ventilator of 9.6 cm H2O with the CPRAM and 5.5 cm H2O with the Bain. Since the ventilator low pressure alarm was preset to 9.2 cm H2O, the alarm provided a warning with the Bain but not the CPRAM. The elevated flow resistance of the CPRAM circuit was attributed to a restriction in the flow area at the patient end of the circuit. Capnographs or adjustable low-pressure alarms provide more reliable monitoring for breathing circuit disconnects. PMID- 1733541 TI - Anaphylactic reactions. PMID- 1733542 TI - Intrapleural block.